[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998] Update International Editors Update ------------------------------------------------------------------------ In this new section, we welcome commentary and updates from our newly formed Board of International Editors. (See inside front cover for a list of names.) Emerging Infectious Diseases-Brazil Hooman Momen Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil [Picture of Hooman Momen] Dr. Momen is a senior research scientist; head of the Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ; and Editor of Memorias do Instituto Oswaldo Cruz. His research interests include biochemical systematics of pathogenic microorganisms. Dr. Momen is temporary adviser to both WHO and PAHO at several technical consultations and scientific working groups. Brazil's large size (more than 8.5 million km[sup 2] and 150 million population) and inadequate public health infrastructure pose a considerable challenge in assessing the status of emerging infectious diseases. Under the current system, most infectious diseases are not notifiable. In diseases for which notification is required, underreporting is common and varies widely by region and disease, and notification is often delayed, which causes the data to be revised frequently. Moreover, in hospital and clinical settings, the etiologic agent of an infectious disease is often not identified. For example, more than a million hospital admissions are recorded per year by the public health system under parasitic and infectious diseases (the category excludes AIDS and respiratory illnesses); of these diseases, more than 70% are diagnosed as ill-defined intestinal infections and a further 10% as food poisoning and septicemia, both without identification of the etiologic agents. For these reasons, the numbers reported here (1996 data, unless otherwise stated) may not reflect the true numbers of cases, and only diseases whose prevalence has changed markedly in recent years will be included in the review. Parasitic Diseases Malaria Among parasitic diseases, malaria causes the most illness, approximately half a million cases annually, nearly all from the northern (Amazon) region. The disease has been controlled in the remainder of the country for years. Where systematic and sustained efforts have been made in the Amazon, control has also been successful. Due to the different epidemiologic situations in the Amazon, no single strategy is effective. Worrying developments in this region include the establishment of the disease in the periphery of the principal cities and the increase in drug- resistant parasites. American Trypanosomiasis American trypanosomiasis (Chagas disease) is the most lethal parasitic disease in Brazil, causing more than 5,000 deaths per year. Several million people are chronically infected carriers of this disease, for which there is still no effective treatment. A very effective control program against the principal insect vector, Triatoma infestans, and improved control of the blood supply have reduced the incidence of new cases to a very low level. Factors that continue to make the disease a re-emerging threat include secondary vectors, forest clearance, and congenital transmission. Leishmaniasis Leishmaniasis (cutaneous and visceral) has increased in incidence in recent years. This increase is modified by apparent cyclic variations of undetermined cause (cyclic duration for the visceral form has been estimated at 10 years). The disease has increased not only in the usual areas of forest and recent clearance, but also in areas of traditional colonization as well as in the urban areas of the northeast. One factor involved in the spread of this disease, uncontrolled urban growth due principally to rural migration, has led to pockets (normally on the periphery of the cities) of extreme poverty that favor the spread of many diseases. Viral Diseases Dengue Among the notifiable viral diseases, dengue causes the most illness. Its mosquito vector, Aedes aegypti, was reintroduced into Brazil in the 1970s; large outbreaks followed in the next decade. Since the vector's reintroduction, more than 700,000 cases have been reported (180,000 cases in 1996). The presence of dengue-1 and -2 indicates that cases of hemorrhagic dengue are also occurring. Ae. aegypti is now present in all states, and a plan has been formulated by the federal ministry of health to eradicate the vector, although plan implementation has met with operational difficulties. The widespread distribution of Ae. aegypti, also the vector of yellow fever, in areas where this disease was considered endemic poses a serious risk for the reemergence of the epidemic form of yellow fever. Vaccination and sanitation campaigns earlier in the century considerably reduced the incidence of the disease (fewer than 100 cases per year-the last urban cases were reported in 1942). Measles Measles-a viral disease that was controlled and nearly eradicated-reemerged with a vengeance in 1997. The outbreak began in the state of São Paulo, where only 15 cases of measles were confirmed in 1996, and has spread to other states. As of October 1997, 61,000 cases (48,000 in São Paulo) have been reported, and 17,000 (13,000 in São Paulo) have been confirmed. Most cases have occurred in young adults under 30 years of age. The vaccination campaign has been reformulated in response to the outbreak, and a national campaign has been launched. Given Brazil's history of success in organizing such campaigns, it is likely that the outbreak will be rapidly contained and that the effort for measles eradication will be resumed. Other Viral Diseases Other viral diseases include AIDS, with more than 17,000 new cases registered in the last year. The number of deaths due to AIDS was 30% lower in the first half of 1997 than in the first half of 1996. This decrease is attributed to the introduction of new antiviral drug combinations. The spread of the AIDS epidemic, particularly into rural areas, has resulted in coinfections with different infectious agents, producing a variety of novel pathologies. Several subtypes of HIV, as well as novel recombinations, have been reported. Enteric transmission of hepatitis continues; hepatitis A and D comprise most of the cases, although the peak incidence is now in young adults rather than in young children. The incidence of hepatitis B is declining as a result of vaccination, while that of hepatitis C is increasing. A high prevalence of hepatitis D exists in some regions of the Amazon, where in conjunction with hepatitis B, it is believed to be the principal cause of Labrea black fever. Influenza is a major concern. Imported vaccine is expensive and not generally available, and the proportion of elderly people (and the threat for a large-scale epidemic) is increasing. The vast forests that still cover large areas of Brazil are home to many known and unknown viruses. The Evandro Chagas Institute in Belem alone has isolated more than 183 new arboviruses. The increasing exploitation of sylvatic resources put humans in direct contact with these viruses, but because of inadequate or nonexistent medical facilities (entire municipalities still lack a single medical doctor), fevers and even deaths often are not diagnosed or are misattributed. Among new viruses causing fatal infections recently discovered in Brazil are Rocio virus, which causes encephalitis, and Sabiá virus, which causes hemorrhagic fever. Several cases of hantavirus infection have also been reported, but on these and other occasions, the lack of a biosafety level 4 laboratory in the country impeded further work. Bacterial Diseases Enteric bacterial infections are an important cause of illness in Brazil. National figures on prevalence are not available, except for cholera. However, an alarming increase has been reported in antibiotic-resistant strains. Cholera The cholera epidemic in Brazil started with the reemergence of the disease in Latin America in 1991, reached a peak of more than 60,000 confirmed cases and 670 deaths in 1993, and then declined to 1,000 cases in 1996. In 1997, the disease resurged with more than 5,000 reported cases and 2,600 confirmed cases. The reawakening of epidemiologic research in cholera caused by its resurgence led to the discovery of a new biotype of Vibrio cholerae in the Amazon region. This biotype is of the O1 serotype; it has distinct multilocus enzyme electrophoresis and RAPD profiles from other pathogenic O1 V. cholerae. About 50 isolates have been made from cases of diarrhea in the upper Amazon (Solimoes) River. The microbe apparently lacks the principal known virulence factors (e.g., the toxin gene cassette and the major colonization factor, TCP); however, some isolates present a cytotoxic effect for Y-1 cells. Mycotic Diseases Fungal infections, including histoplasmosis, paracoccidioidomycosis, and cryptococcosis, occur; however, their cause is often unknown and even if a diagnosis is made, the diseases are not reported. Public Health Infrastructure The emerging infectious disease picture in Brazil will not change markedly without a sustained and determined effort to improve the country's public health infrastructure. The existing, generally passive epidemiologic surveillance system produces information that arrives too late to be effective; however, a number of measures, if implemented immediately, can mitigate the impact of any future epidemic: a containment laboratory (biosafety level 4) that can handle known and unknown microbes of high virulence; at least one infirmary, properly designed and fully equipped, to treat highly contagious and virulent diseases. The current lack of this facility poses a great danger to the population should an outbreak of such a disease occur. The financial, technical, and human resources needed to activate these measures already exist. In infectious diseases, Brazil has a long tradition of fruitful international collaboration that can be tapped for additional support. The success of national vaccination and sanitation campaigns in the eradication and control of some infectious diseases at the beginning of this century and in more recent times demonstrates that much can be accomplished. Brazil can become a successful model for other developing countries. Acknowledgments I thank the Fundação Nacional de Saude for providing data on different diseases; Drs. H. Schatzmayr, J.R. Coura, and P. Mason for interesting discussions and criticisms of the manuscript; and Drs. Ana Gaspar, Clara Yoshida, Ana Carolina Vicente, and Marilda Siqueira for useful information. Address for correspondence: Hooman Momen, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brazil 4365, Rio de Janeiro 21045-900, Brazil; fax: 55-21-590-3545; e-mail: hmomen@gene.dbbm.fiocruz.br. ------------------------------------------------------------------------ Perspectives Risk for Transfusion-Transmitted Infectious Diseases in Central and South America Gabriel A. Schmunis, Fabio Zicker, Francisco Pinheiro, and David Brandling-Bennett Pan American Health Organization, Washington, D.C., USA ------------------------------------------------------------------------ We report the potential risk for an infectious disease through tainted transfusion in 10 countries of South and Central America in 1993 and in two countries of South America in 1994, as well as the cost of reagents as partial estimation of screening costs. Of the 12 countries included in the study, nine screened all donors for HIV; three screened all donors for hepatitis B virus (HBV); two screened all donors for _Trypanosoma cruzi_; none screened all donors for hepatitis C virus (HCV); and six screened some donors for syphilis. Estimates of the risk of acquiring HIV through blood transfusion were much lower than for acquiring HBV, HCV, or _T. cruzi_ because of significantly higher screening and lower prevalence rates for HIV. An index of infectious disease spread through blood transfusion was calculated for each country. The highest value was obtained for Bolivia (233 infections per 10,000 transfusions); in five other countries, it was 68 to 103 infections per 10,000. The risks were lower in Honduras (nine per 10,000), Ecuador (16 per 10,000), and Paraguay (19 per 10,000). While the real number of potentially infected units or infected persons is probably lower than our estimates because of false positives and already infected recipients, the data reinforce the need for an information system to assess the level of screening for infectious diseases in the blood supply. Since this information was collected, Chile, Colombia, Costa Rica, and Venezuela have made HCV screening mandatory; serologic testing for HCV has increased in those countries, as well as in El Salvador and Honduras. _T. cruzi_ screening is now mandatory in Colombia, and the percentage of screened donors increased not only in Colombia, but also in Ecuador, El Salvador, and Paraguay. Laws to regulate blood transfusion practices have been enacted in Bolivia, Guatemala, and Peru. However, donor screening still needs to improve for one or more diseases in most countries. Preventing the transmission of infectious diseases through blood transfusion in developing countries is difficult given that the resources needed are not always available, even when policies and strategies are in place (1). Avoiding paid donors, selecting blood donors through questionnaires, and limiting the number of transfusions can prevent the transmission of infections. Testing for specific antibodies is the final measure for eliminating unsafe blood. The risk for transfusion-transmitted infectious diseases can be estimated on the basis of screening level for each infectious agent and the prevalence rate of the infection in the donor population. Estimates may also take into account the sensitivity, specificity, and window period of the testing assays. We report here an estimate of such a risk in 12 Central and South American countries and the cost of reagents required for the screening of these infectious diseases as a proxy of resources needed to reduce the risk. Source of Information This report analyzes data from 1993 on screening of blood donors from five countries (Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua) of Central America. Data were also analyzed for 1993 from five countries (Bolivia, Chile, Colombia, Peru, and Venezuela) and for 1994 from two countries (Ecuador and Paraguay) of South America. Information was obtained from Ministry of Health reports during technical meetings in which the situation of each country was reviewed (2-5) or from an official report (6). In addition, data are presented on the least expensive reagents for detecting antibodies for HIV, hepatitis C virus (HCV), _Trypanosoma cruzi _, and _Treponema pallidum_, and for detecting hepatitis B virus (HBV) antigen (HBsAg) (2,3). All data are national except for Peru, where the information was for the city of Lima only (3). Population data were from the Pan American Health Organization's publication Health Conditions in the Americas (7). Estimates are based on reported results of donor screening activities (2-6). Assumptions For the best possible scenario, the following assumptions were made: 1) Because the laboratory procedures and brands of reagents used in the 12 countries may differ in sensitivity and specificity, comparisons between them are not straightforward. In addition, results of the screening are influenced by the existence of an organized system of quality control and proficiency testing for the serology and for the evaluation of the diagnostic kits, which most countries lacked from 1993 to 1994. Most countries reported the use of different brands of second, third, and fourth generation immunologic assays for screenings of HCV, HIV, and HBV, respectively. Therefore, we assumed that the specificity of the tests for viral diagnosis was 100%, but the sensitivity was 90.00% for HCV, 99.99% for HIV, and 99.90% for HBV. These specificity and sensitivity estimates fit well with those reported for second, third, and fourth generations of assays for HCV, HIV, and HBV, respectively, as mentioned in the package insert by two of the manufacturers of reagents used in the countries. Average window periods for those assays were 20 to 25 days (8,9), 82 to 84 days (9,10), and 51 days (9) for HIV, HBV, and HCV, respectively. In the case of _T. cruzi_ serology, we selected the upper range of reported sensitivity and specificity (90% and 95%, respectively) (11,12). For _T. cruzi_, the probability that a person may become a donor during the window period is low because infection is usually acquired in childhood and in rural areas. 2) We assumed that prevalence of infection in unscreened donors was the same as the national average prevalence for each infectious disease. 3) Chile (6) and Peru (3) were the only countries that reported a fractionation index, 1.85 and 1.5, respectively. As no other country provided data on the fractionation index or data allowing one to be calculated, to put the countries in the same category, it was assumed that every blood donation corresponded to a single transfusion to one recipient. Screening Coverage and Prevalence Rates Table 1 shows coverage of screening and prevalence rates of seropositive tests for specific infectious agents among blood donors reported by each of the 12 countries. For HIV, 100% of the donors were screened in all countries, except Bolivia (36.20%), Ecuador (89.50%), and Colombia (98.80%). Prevalence rates for HIV varied from 3.90 per 1,000 in Honduras to 0.04 per 1,000 in Nicaragua. For HBV, only Costa Rica, Peru, and Venezuela screened 100% of donors. The highest values of HBV prevalence estimated were 14.40 per 1,000 for Venezuela and 13.00 per 1,000 for Paraguay. Bolivia, Costa Rica, and Paraguay did not screen for HCV at all, and all other countries screened fewer than 58% of donors; prevalence rates varied from 0.50 to 9.40 per 1,000. Screening for syphilis was not complete in Bolivia, Chile, Colombia, Ecuador, Nicaragua, and Paraguay; prevalence rates were 5.00 to 28.00 per 1,000. For _T. cruzi_ infection, only Venezuela and Honduras screened 100% of donors; prevalence rates were 2.00 per 1,000 in Ecuador to 147.90 per 1,000 in Bolivia. In 1993, Peru and Costa Rica had not yet introduced screening for _T. cruzi_. Table 1. Coverage of screening (sup a) of blood donors and seroprevalence rates (per 1,000) of infectious diseases, by country (sup b) --------------------------------------------------------------------------- HIV HBV (sup c) HCV Syphilis _T. cruzi_ Prev. Cov. (sup Prev. Prev. Prev. Country (sup e) Cov. (/10 Cov. (/10 Cov. (/10 Cov. Prev. d) (/10 (%) (sup (%) (sup (%) (sup (%) (/10 (%) (sup 3)) 3)) 3)) (sup 3)) 3)) --------------------------------------------------------------------------- Bolivia 36.2 0.10 14.5 2.00 0 ? (sup 37.9 18.10 29.4 147.90 f) Chile 100 3.40 98.7 2.00 34.0 6.40 95.2 11.40 76.7 12.00 Colombia 98.8 2.00 98.3 7.00 24.7 9.00 87.3 13.00 1.4 12.00 Costa Rica 100 0.34 100 4.50 0 ? 100 5.00 0 ? Ecuador 89.5 1.00 88.2 3.80 32.9 1.40 86.7 11.50 51.0 2.00 El Salvador 100 1.30 96.0 8.00 31.4 2.50 100 19.00 42.5 14.70 Guatemala 100 3.00 79.8 7.00 37.2 8.00 100 19.00 75.0 14.00 Honduras 100 3.90 83.5 2.70 27.8 0.50 100 7.00 100 12.40 Nicaragua 100 0.04 53.1 4.00 53.1 4.40 88.4 16.00 58.4 2.40 Paraguay 100 0.70 92.9 13.00 0 ? 66.9 28.00 86.8 45.00 Peru 100 2.80 100 8.60 57.4 4.40 100 9.60 0 23.60(sup g) Venezuela 100 2.10 100 14.40 31.0 9.40 100 10.70 100 13.20 --------------------------------------------------------------------------- (sup a)Coverage of screening = (number of screened donors ÷ total number of donors) x 100. (sup b)Data as reported by the countries from 1993, except for Ecuador and Paraguay, which were for 1994. (sup c)HBsAg only. (sup d)Coverage. (sup e)Prevalence. (sup f)Screening not performed and prevalence not known. (sup g)Data from a survey of 2,237 samples. --------------------------------------------------------------------------- Estimating Potential Infectivity of the Blood Supply The probability of receiving an infected transfusion unit _P(R)_ in each country was estimated by multiplying the prevalence of a specific infection by 1-level of screening (Table 1). For those estimates, the sensitivity and specificity of the different tests were taken into account. As the overall assumed sensitivity of HIV screening was 99.99%, the adjustment of prevalence rates makes no material difference to the precision of the figures in Table 1. The probability of getting a transfusion-transmitted infection _P(I)_ was calculated as the result of the probability of receiving an infected transfusion _P(R)_ multiplied by the infectivity risk. For countries reporting 100% of screening coverage for a specific disease, a residual _P(R)_ was estimated as prevalence x 1-screening sensitivity (Table 2). Infectivity risk (defined as the likelihood of being infected when receiving an infected transfusion unit) was assumed to be 90% for HIV (13), 75% for HBV (14), 90% for HCV (15), and 20% for _T. cruzi_ (16) (Table 2). Estimates for transfusion-acquired syphilis are not presented because the infectivity risk depends on length of refrigeration (17). Table 2. Probability of receiving an infected transfusion _P(R)_(sup a) and probability of getting a transfusion-transmitted infection _P(I)_ (sup b), by country(sup c) --------------------------------------------------------------------------- HIV (x10(sup HBV (x10(sup HCV (x10(sup _T. cruzi_ 4)) 4)) 4)) (x10(sup 4)) Country _P(R)_ _P(I)_ _P(R)_ _P(I)_ _P(R)_ _P(I)_ _P(R)_ _P(I)_ --------------------------------------------------------------------------- Bolivia 0.64 0.57 17.27 12.95 NSP(sup NSP 1096.38 219.28 d) Chile 0.00 0.00 0.26 0.20 46.46 41.82 29.36 5.87 Colombia 0.24 0.22 1.20 0.90 74.55 67.09 124.24 24.85 Costa 0.45(sup Rica 0.00 0.00 x) 0.34 NSP NSP NSP NSP Ecuador 1.05 0.95 4.52 3.39 10.33 9.38 10.29 2.06 El Salvador 0.00 0.00 3.23 2.42 18.87 16.97 88.75 17.75 Guatemala 0.00 0.00 14.28 10.71 55.26 49.74 36.75 7.35 Honduras 0.00 0.00 4.49 3.37 3.97 3.57 13.02(sup 2.60 x) Nicaragua 0.00 0.00 18.95 14.21 22.70 20.43 10.48 2.10 Paraguay 0.00 0.00 9.32 6.99 NSP NSP 62.37 12.47 Peru 0.00 0.00 0.87(sup 0.65 20.62 18.56 247.80 49.56 x) Venezuela 0.00 0.00 1.45(sup 1.09 71.35 64.21 13.86 (sup 2.77 x) x) --------------------------------------------------------------------------- (sup a)_P(R)_ = probability of receiving an infected transfusion = prevalence of infection x 1- level of screening; (sup x)for countries in which reported screening level was 100%, a residual _P(R)_ was estimated as prevalence x 1- screening sensitivity rate x 10,000. (sup b)_P(I)_= probability of getting a transfusion-transmitted infection = _P(R)_ (sup x) infectivity index (infectivity indexes used were HIV=90%; HBV=75%; HCV=90%; _T.cruzi_=20%). For calculations of _P(R)_ and _P(I)_ the prevalence was corrected taking into account the sensitivity of the screening. (sup c)Data from 1993, except for Ecuador and Paraguay, which were for 1994. (sup d)No screening performed, so _P(R)_ and _P(I)_ not known. --------------------------------------------------------------------------- Considering the low prevalence rates and the incompleteness of HIV screening, only Bolivia, Colombia, and Ecuador could have missed detecting an HIV-infected transfusion unit; the probability of getting an infection in these countries was estimated at 0.57, 0.22, and 0.95 per 10,000 transfusions, respectively. For HBV and HCV this risk is higher. Up to 14.21 HBV infections (Nicaragua) and 67.09 HCV infections (Colombia) per 10,000 transfusions may have occurred. The highest risk for transfusion-transmitted infection was estimated for _T. cruzi:_ 219.28 per 10,000 and 49.56 per 10,000 for Bolivia and Peru, respectively, and approximately 2 to 24 per 10,000 for the other seven countries (Table 2). Table 3 shows estimates of the absolute number of infections that may have been induced by transfusion, calculated as [no. of donors x _P(I)_], for each country. Because Chile (6) and Peru (3) reported fractionation of blood units by 1.85 and 1.5, respectively, the estimated number of infected units transfused in those countries was multiplied by these factors. For the remaining countries, it was assumed that each donated unit was given to only one recipient. An index of infectious disease spread through blood transfusion was calculated by dividing the estimated total number of transfusion-related infections (for any one of the infectious agents considered) by the total number of donors. This index indicates the health risks associated with blood transfusion and can be used as an outcome indicator to assess the cost-effectiveness of screening programs. Table 3. Estimates of transfusion-transmitted infectious diseases, by country (sup a) --------------------------------------------------------------------------- Infection spreading Absolute no. of transfusion- index(sup transmitted infectious c) Ratio of No. of diseases (sup b) /10 (sup infections: Country donors -------------------------------4) donations _T. HIV HBV HCV cruzi_ Total --------------------------------------------------------------------------- NA Bolivia 37,9 48 2 49 (sup 832 883 233 1:43 e) Chile (sup d) 217,312 0 8 1681 236 1925 88 1:113 Colombia 352,316 8 32 2364 875 3279 93 1:107 Costa 2 Rica 50,692 0 (sup NA NA NA NA NA f) Ecuador 98,473 9 33 92 20 154 16 1:639 El Salvador 48,048 0 12 82 85 179 37 1:268 Guatemala 45,426 0 49 226 33 308 68 1:147 Honduras 27,885 0 9 10 7 (sup 26 9 1:1072 f) Nicaragua 46,001 0 65 94 10 169 37 1:272 Paraguay 32,893 0 23 NA 41 64 19 1:514 Peru (sup 4 g) 52,909 0 (sup 147 393 544 103 1:97 f) 22 Venezuela 204,316 0 (sup 1312 57 (sup 1391 68 1:147 f) f) --------------------------------------------------------------------------- (sup a)Data from 1993 except for Ecuador and Paraguay, which were for 1994. (sup b)Number of cases transmitted by blood transfusion = [number of donors x _P(I)_ ]. For calculations of number of infections, the prevalence was corrected taking into account the sensitivity of the screening. (sup c)Infection spreading index = (total number of infections transmitted ÷ number of donors) x 10,000. (sup d)Fractionation index = 1.85. (sup e)Data not available. (sup f)Residual infectivity considering that sensitivity of diagnostic tests is not 100%. (sup g)Fractionation index = 1.5. --------------------------------------------------------------------------- The highest value for the infection spreading index was obtained for Bolivia, where 233 transfusion-related infections may have occurred per 10,000 donations. This was a result of a very high prevalence rate of antibodies to _T. cruzi_ and a lower level of screening. For most other countries considered, the index was 68 to 103 infections per 10,000 donations. Due to low seroprevalence rates and good screening levels in some cases, the risk for transfusion-related infections was relatively low in Honduras (nine per 10,000), Ecuador (16 per 10,000), and Paraguay (19 per 10,000). Table 3 also shows the ratio of number of infections per donation by country. One infection (HIV, HBV, HCV, or _T. cruzi_) might have been transmitted in every 43 (Bolivia) to 1,072 (Honduras) donated units. Screening Costs The unitary cost for serologic screening, estimated solely from expenditures on the least expensive laboratory reagents in each country and considering the prevalence rates reported by the countries was US$0.9 to US$2.4 for an HIV enzyme-linked immunosorbent assay (ELISA), US$0.5 to US$3.5 for HBV screening (enzyme immunoassay, radioimmunoassay, or passive reverse hemagglutination), US$3.5 to US$10.0 for HCV ELISA, US$0.25 to US$1.0 for a _T. cruzi_ test (ELISA, radioimmunoassay, or indirect hemagglutination) (Table 4), and US$0.09 to US$0.60 for syphilis serology (RPR or VDRL). Using other tests might have increased the costs significantly for some of the infections. For example, the rapid agglutination test for HIV is usually more expensive than ELISA. Table 4. Estimated unitary cost of preventing transfusion-transmitted infections, by country, 1993 (sup a) --------------------------------------------------------------------------- Cost (US$) ---------------------------------------------------------------------- HIV HBV (sup b) HCV _T. cruzi_ ----------------------------------- ---------------------------------- Preventing Preventing Preventing Preventing one one one one Single infected Single infected Single infected Single infected Country test unit test unit test unit test unit -------------------------------------------------------------------------------- 1.2 3.5 NA Chile 2.3 676 (sup 599 (sup d 547 (sup NA c) ) e) Costa 0.5 Rica 1.1 3,280 (sup 111 NA NA NA NA c) Ecuador 1.0 0.35 (sup f) 1.7 1,708 (sup 263 10.0 7,136 (sup 175 c) g) El 2.0 1,550 1.9 238 4.5 1,802 1.0 68 Salvador (sup (sup c) g,h) 1.0 1.7 0.9 Guatemala 1.8 601 (sup 243 3.5 438 (sup 65 c) g) Honduras 0.9 232 0.9 334 3.5 6,971 0.45 36 (sup 186 (sup c) h) 0.5 (sup h) 0.5 0.5 Nicaragua 1.0 23,000 (sup 125 3.5 797 (sup 209 h) h) Peru 2.4 858 3.5 407 8.2 1,862 0.25 11 (Lima) (sup 279 (sup c) g) 2.4 (sup i) Venezuela 1.3 619 1.3 90 4.5 479 0.5 38 (sup 0.3 23 c) (sup g) -------------------------------------------------------------------------------- (sup a)Cost of preventing (=detecting) one infected unit was calculated as [(number of donors x test cost) ÷ (total number of positive donors detected)]. All costs refer to enzyme-linked immunosorbent assay, unless otherwise indicated. (sup b)HBsAg only (sup c)Enzyme immunoassay. (sup d)Estimated cost based on cost of test in other countries. (sup e)Data not available. (sup f)Donors and prevalence for 1994, costs for 1993. (sup g)Indirect hemagglutination. (sup h)Radioimmunoassay. (sup i)Passive reverse hemagglutination. -------------------------------------------------------------------------------- The cost of preventing the transfusion of one infected unit was estimated as [(no. of donors x cost of each test)/ total number of positive donors] as reported by each country. This value represents the cost of detecting one unit positive for any one of the infections studied in each country by using one diagnostic test for each infectious disease. For example, using two tests, one for antibody detection and one for antigen detection of HIV, increases costs. Detection of _T. cruzi_ was the least expensive (US$11-$209 per positive unit), followed by HBV (US$90-US$599 per unit), HCV (US$438-$7,136 per unit), and HIV (US$232-$23,000 per unit) (Table 4). The wide variation of cost primarily reflects differences in the prevalence of each infection and in the cost of each test in the countries. The costs per capita to carry out a complete screening of blood donors in each country was US$0.008 to US$0.04 for HIV, US$0.008 to US$0.02 for HBV, US$0.01 to US$0.08 for HCV, US$0.0008 to US$0.003 for syphilis, and US$0.0025 to US$0.009 for _T. cruzi._ Limitations of the Data Difficulties and limitations of the use of public health data for policy decisions, even in industralized countries, are well recognized. Figures presented here were generated to establish an approximation of the problem by providing an overview of the risk of receiving tainted blood in different countries in Central and South America. One potential cause of underestimation of viral infections transmitted by blood is the residual risk because of the window period, even when 100% of donors are screened by serology (8,9). However, this residual risk would be difficult to ascertain in most countries of Central and South America. Investigation of clinically identified cases after a transfusion, follow-up of recipients for seroconversion, and special laboratory studies detecting seronegative donors for missed infections are laborious and expensive and could seldom be undertaken in those countries. Another possibility would be studies that combine estimates of incidence rates of infection among repeated and first-time donors (who seroconvert) with estimates for the duration of the preseroconversion period for a specific infectious agent (22). Excellent results were obtained by this method in the United States, where more than 80% of donations come from repeat donors. Those studies involved hundreds of thousands of donors and millions of donations. To have incidence data on repeat donors, it is necessary to have a significant number of voluntary donors who will repeat donations. Therefore, it is unlikely that studies of that sort could be carried out in the countries mentioned here. First, the population of the countries is much smaller; therefore, the number of donations is smaller. For example, in all Central America the number of donations is approximately 210,000 per year. Second, the number of repeat donations from voluntary donors is small. Voluntary donors accounted for 30% and 40% of all donations in Colombia and Costa Rica and 4% to 10% of all donors in Chile, Bolivia, Peru, and Venezuela (2-6). The number of voluntary donors was also small in the remaining countries. In all countries of Central and South America, most donations come from directed donors, relatives or friends of patients. In addition, there is no national registry of donors to allow for follow-up. Using incidence rates for first-time donors instead of repeat donors is not a solution because official incidence rates for HIV or other viruses were not available at the time of this study. The risk for transfusion-related infection could also be overestimated. Recipients may already be infected. This is especially likely for _T. cruzi _ infection in Bolivia, where the seroprevalence in the general population could be higher than 20% (18,19). Another source of overestimation is that only some of the cases detected by screening would be confirmed. In several countries, a confirmatory test is mandatory for HIV, syphilis, and HCV. However, as the primary function of blood banks is donor screening, seropositive donors for any of the diseases mentioned here are usually referred to specialized services or reference laboratories for confirmation of the results of the screening, and if results are confirmed, for treatment and counseling. Results of this confirmatory serology are not often sent back to the blood bank, even when privacy concerns allow for it. Chile was the only country that reported results of confirmatory tests for HIV: results indicated that only 9% of those found positive by the screening were confirmed positive (6). With _T. cruzi_, as there is no confirmatory test, it is assumed that a true positive is a unit that is positive on more than one test. By these criteria, a recent study in Brazil suggested that only one out of five donors positive for _T. cruzi_ could be considered a true positive (23). Those facts, however, do not reduce the public health relevance of the problem presented here, although the real numbers of potentially infected units/infected persons may be still lower than our estimates. Establishment of a screening process in every country will depend on balancing the benefits and costs. Although costs for preventing transfusion of one tainted unit or preventing one infection seem high for some etiologic agents, they are not so. Even in the case of Nicaragua, the country with the lowest HIV prevalence, the cost to prevent the transfusion of one potentially HIV-infected unit (by testing all donors with ELISA) was estimated at US$23,000, while treatment costs (drugs only) for an AIDS patient would be approximately US$12,000 per year. In general, the risk for an infectious disease through tainted transfusion is not as high as that reported from some countries of Africa (24). Since 1993, donor screening has improved in several countries. Chile, Colombia, Costa Rica, and Venezuela, for example, have made screening for HCV mandatory, and coverage for serology for that infection has increased in those countries, as well as in El Salvador and Honduras. _T. cruzi_ screening is now mandatory in Colombia, and the percentage of screened donors not only increased in Colombia but also in Ecuador, El Salvador, and Paraguay. Laws to regulate blood transfusion practices have been enacted in Bolivia, Guatemala, and Peru. The figures presented, however, underline the need for improvement and stress the importance of an information system that allows assessing the level of screening for infectious diseases in the blood supply. Universal screening of donors for HCV is still a priority in most countries, and increased donor screening for _T. cruzi_ is a priority for Bolivia and possibly for Peru. Continuous collection of the type of information shown here, which has only been partially available (1), provides a baseline against which future achievements can be measured and is essential for obtaining the support needed to maintain or expand the screening of blood donors. Address for correspondence: Gabriel A. Schmunis, Pan American Health Organization-PAHO, 525 23th Street NW, Washington, D.C. 20037, USA; fax: 202-974-3688; e-mail: schmunig@paho.org. References 1. Linares J, Vinelli E, editors. Taller Latinoamericano de servicios de transfusión sanguínea y óptimo uso de los recursos. Cruz Roja Finlandesa; 1994. p. 167. 2. Organización Panamericana de la Salud. Taller para el control de calidad de sangre en transfusiones: serología para la detección de Chagas, hepatitis B y C, sífilis y HIV/SIDA. Document OPS/HPC/HCT/94.42. 3. Organización Panamericana de la Salud. Países andinos. Taller sobre control de calidad de sangre en transfusiones: serología para la detección de hepatitis B y C, sífilis, tripanosomiasis americana y VIH/SIDA. Document OPS/HCP/HCT/95-61. 4. Organización Panamericana de la Salud. Simposio internacional sobre control de calidad en bancos de sangre del Cono Sur y de Brasil. Informe final OPS/HCP/HCT/95.55. 5. Organización Panamericana de la Salud. Taller sobre control de calidad en serología de bancos de sangre. OPS/HCP/HCT/96/79. 6. Ministerio de Salud, Chile. Diagnóstico de la situación de los bancos de sangre y medicina transfusionál en Chile 1993. Santiago, Chile: La Ministerio; 1995. Ser Inf Téc No.14. 7. Pan American Health Organization. Health conditions in the Americas. Washington (DC): The Organization; 1994. PAHO Sci Pub No. 549. 8. Lackritz EM, Satten GA, Aberle-Grasse J, Dodd RY, Raimondi VP, Janssen RS, et al. Estimated risk of transmission of the human immunodeficiency virus by screened blood in the United States. N Eng J Med 1995;333:1721-5. 9. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of transfusion-transmitted viral infections. N Eng J Med 1996;334:1685-90. 10. Van de Poel CL, Cuypers HT, Reesink HW, Hepatitis C virus six years on. Lancet 1994;344:1475-9. 11. Andrade ANS, Martelli CMT, Luquetti AO, Oliveira OS, Almeida e Silva S, Zicker F. Triagem sorologica ra o Trypanosoma cruzi entre doadores de sangue do Brasil central. Bol Oficina Sanit Panam 1992;113:19-27. 12. Lorca MH, Child RB, Garcia AC, Silva MG, Osorio JS, Atias M. Evaluación de reactivos comerciales empleados en el diagnóstico de la enfermedad de Chagas en bancos de sangre de Chile. I Selección de reactivos. Rev Med Chile. 1992;120:420-6. 13. Donegan E, Stuart M, Niland JC, Sacks HS, Azen SP, Dietrich SL, et al. Infection with human immunodeficiency virus type 1 (HIV-1) among recipients of antibody-positive blood donations. Ann Intern Med 1990;113:733-9. 14. Gocke DJ. A prospective study of post-transfusional hepatitis. JAMA 1972;219:1165-70. 15. Aach RD, Stevens CE, Hollinger FB, Mosley JW, Peterson DA, Taylor PE, et al. Hepatitis C virus infection in post-transfusion hepatitis. An analysis with first- and second-generation assays. N Engl J Med 1991;325:1325-9. 16. World Health Organization. The control of Chagas disease. Geneva: WHO Tech Rep Ser No.811:32;990. 17. National Institutes of Health. Infectious disease testing for blood transfusion. NIH Consens Statement 1995;13:13-4. 18. Schmunis GA. _Trypanosoma cruzi_, the etiologic agent of Chagas disease: status in the blood supply in endemic and non endemic countries. Transfusion 1991;31:547-55. 19. Schmunis GA. American trypanosomiasis as a public health problem. Chagas disease and the nervous system. PAHO Sci Pub 994;547:3-29. 20. Pedersen C, Lindhardt BO, Jensen BL, Lavritzen E, Gerstoft J, Dickmeiss E, et al. Clinical course of primary HIV infection: consequences for subsequent course of infection. BMJ 1989;299:154-7. 21. Alter M. Residual risk of transfusion associated hepatitis. In: Program and Abstracts of the National Institutes of Health Development Conference on Infectious Diseases Testing for Blood Transfusions. 1995 Jan 9-11; Bethesda (MD): National Institutes of Health;1995. p. 23-7. 22. Busch MP. Incidence of infectious disease markers in blood donors, implications for residual risk of viral transmission by transfusion. In: Program and Abstracts of the National Institutes of Health Development Conference; 1995. Jan 9-11; Bethesda (MD): National Institutes of Health; 1995. p. 29-30. 23. Hamerschlak N, Pasternak J, Amato Neto V, Carvalho MB, Guerra CS, Coscina AL, et al. Chagas' disease, an algorithm for donor screening and positive donor counseling. Rev Soc Bras Med Trop 1997;30:205-9. 24. McFarland W, Mvere D, Shandera W, Reingold A. Epidemiology and prevention of transfusion-associated human immunodeficiency virus transmission in sub-Saharan Africa. Vox Sang 1997;72:85-92. ------------------------------------------------------------------------ Perspectives Calicivirus Emergence from Ocean Reservoirs: Zoonotic and Interspecies Movements Alvin W. Smith,* Douglas E. Skilling,* Neil Cherry,† Jay H. Mead,‡ and David O. Matson§ *Oregon State University, Corvallis, Oregon, USA; †Lincoln University, New Zealand; ‡Red Cross, Portland, Oregon, USA; and §Children's Hospital of the King's Daughters, Eastern Virginia Medical School, Norfolk, Virginia, USA --------------------------------------------------------------------------- Caliciviral infections in humans, among the most common causes of viral-induced vomiting and diarrhea, are caused by the Norwalk group of small round structured viruses, the Sapporo caliciviruses, and the hepatitis E agent. Human caliciviruses have been resistant to in vitro cultivation, and direct study of their origins and reservoirs outside infected humans or water and foods (such as shellfish contaminated with human sewage) has been difficult. Modes of transmission, other than direct fecal-oral routes, are not well understood. In contrast, animal viruses found in ocean reservoirs, which make up a second calicivirus group, can be cultivated in vitro. These viruses can emerge and infect terrestrial hosts, including humans. This article reviews the history of animal caliciviruses, their eventual recognition as zoonotic agents, and their potential usefulness as a predictive model for noncultivatable human and other animal caliciviruses (e.g., those seen in association with rabbit hemorrhagic disease). In vitro cultivation of caliciviruses indicates that these pathogens have been emerging periodically from ocean sources for 65 years (1). The best-documented example of ocean caliciviruses causing disease in terrestrial species is the animal disease vesicular exanthema of swine (VES) (1). Feline calicivirus (the only member of the group with a seemingly ubiquitous and continuous terrestrial presence) also appears to have ocean reservoirs (2). The source of caliciviruses causing gastroenteritis in humans is frequently shellfish, which do not always come from beds contaminated with human waste (3,4). The origins of hepatitis E are often obscure, but water is one suspected source (5). The most recent emerging calicivirus is associated with rabbit hemorrhagic disease (RHD), and although an ocean association has not been reported, the agent readily moves between continents and crosses ocean channels (6). Finally, the only reported in vitro isolation and sequential propagation of a calicivirus pathogenic for humans is a virus residing in the sea (7). The Caliciviridae are divided into five groups, tentatively designated distinct genera, on the basis of sequence relatedness and genomic organization (8). Four are known human pathogens-Sapporo, Norwalk-like small round structured viruses, hepatitis E, and the marine (animal) caliciviruses-while the fifth group, which includes RHD virus, is not yet proven to be a human pathogen. The human Sapporo viruses are more closely related to the marine caliciviruses than to the other human group causing gastroenteritis, the Norwalk-like viruses. On the basis of homology and genomic organization, RHD virus falls between these two groups. In addition, the genomic organization of hepatitis E is most closely related to that of the only other hepatotropic calicivirus currently described, RHD virus (8). Many marine calicivirus strains in the tentative genus of VES virus-like caliciviruses have been passaged in vitro; their characterization has facilitated understanding of calicivirus geographic distribution and host versatility (1). Dozens of serotypes were described on the basis of serum neutralization tests; this antigenic complexity complicated serodiagnosis and hampered studies of effects on host species. The illnesses associated with two recently discovered viruses classified as Caliciviridae, hepatitis E virus and RHD virus, have altered the notion that caliciviruses produce only transient clinical disease but not death (1,9,10). Hepatitis E virus is fatal for 25% of the pregnant women in developing countries who contract hepatitis E (11); RHD can kill 95% of infected rabbits within 24 to 48 hours of exposure (6). The oceans are reservoirs in which caliciviruses are exposed in a water substrate to life forms from zooplankton to whales and in which they, like other RNA viruses, can amplify to very high numbers with variants occurring in every replicative cycle (12). Such a varied replicative setting has served this parasite well. With the right tools, evidence of previous infection with caliciviruses can often be shown in fish, avian, and many mammalian species, including humans (1). It is not known why some caliciviruses have become potential hemorrhagic agents associated with purpura hemorrhagica in aborted piglets (13), neonatal hemorrhagic syndrome in pinnipeds (A.W. Smith, D.E. Skilling, unpub. data), hemorrhagic disease in fatal hepatitis E in humans (5), and RHD (6). However, it is known that caliciviral diseases can be difficult or impossible to contain and eradicate. Pathogenic caliciviruses can be expected to continue emerging from the sea in unexpected forms at unexpected times in unexpected places. Studying those that have emerged and are compliant to in vitro propagation can provide insights into those that cannot be cell-culture adapted and those yet to be discovered. History and Its Lessons The 66-year history of the caliciviruses with ocean reservoirs can be divided into three periods: 1932 to 1972, the species-specific era (14); 1972 to 1982, the new era of virology, during which oceans were first found to be reservoirs of viral disease infecting domestic animals (7,9); and 1976 to the present. The first evidence of infection with caliciviruses of marine origin can be traced to 1932. A large herd of swine in Orange County, California, was being fed raw garbage collected from restaurants and institutions in the Los Angeles area. When some animals became sick with vesicular lesions on the feet and nose, regulatory veterinarians were notified because vesicular diseases of livestock were reportable. The farm with sick swine and adjacent farms were quarantined for foot-and-mouth disease, and more than 19,000 head of exposed cattle and swine were destroyed and buried in quicklime (14). The outbreak was contained. One year later and 100 miles to the south in San Diego, California, the second known outbreak occurred and was contained (14). This time the disease was found not to be foot-and-mouth disease, because the virus would not infect cattle, but instead was described as a new disease of swine and was called VES (14). In 1934, a third outbreak occurred in San Francisco, California, and VES was again contained (14). In 1935, the events repeated themselves, but from 1936 through mid-December 1939, the disease disappeared and then abruptly reappeared, at times involving 40% of California swine herds. All of these outbreaks in the 1930s and 1940s were shown by cross-infectivity studies to be caused by many distinct but related VES virus strains. The embargoes placed on raw California pork were successful in containing VES within California until 1952. That year, a passenger train between San Francisco and Chicago served California pork and discarded the raw pork trimmings into the garbage in Cheyenne, Wyoming; the garbage was fed to swine subsequently redistributed by auction sale yard. Within 14 months, all major swine-growing areas in the United States (41 states) had reported VES. For the first time, the federal government, rather than just the California state government, activated eradication and quarantine measures against VES, including enforcement of federal laws requiring garbage to be cooked before it was fed to swine. By 1956, the last reported outbreak of VES had been contained, and the disease was said to have been eradicated. In 1959, VES was declared a foreign animal disease, even though it had never been reported outside the United States (14). Forty years later, the natural history of this calicivirus still contained few details. Its origins were not known but were said to have been de novo or from some unknown wild animal reservoir, which was extensively sought but not found (14,15). Swine were the only naturally infected host species; no evidence of human infection had been observed (14). Control of VES had been a notable success story for regulatory veterinary medicine in the United States; within 24 years of its discovery as an entirely new disease, it was said to have been eradicated (14). Internationally accepted animal disease diagnostic tests using swine, horse, and bovine infectivity profiles for vesicular stomatitis virus, foot-and-mouth virus, and VES virus were used routinely; VES virus did not infect cattle, whereas the other viruses did (14,15). The discovery of a new paradigm in "viral traffic" (16) began the second period of calicivirus history. The movement of a member of the Caliciviridae from ocean reservoirs to terrestrial hosts changed the understanding of the natural history of a virus thought to be host specific and eradicated (1,15,17). The first virus isolate from a pinniped occurred in 1972. The agent, named San Miguel sea lion virus type 1 (SMSV-1), was a calicivirus that caused classic VES in swine (17). Thus began a series of isolating and characterizing viruses in ocean species that were officially designated as "viruses indistinguishable from VES virus" because they were additional VES virus types. They could not be called VES virus (18) since VES had been officially eradicated. Should the VES virus reappear, its status as a foreign animal disease would mandate immediate implementation of eradication measures; eradication was viewed as an impossibility because of the wide range of reservoirs for VES virus, both migratory and ocean species. The 13 marine caliciviruses serotypes (100 TCID50 vs. 20 units of neutralizing antibody) isolated from swine before 1956 were called VES viruses; those isolated after 1972 have been designated SMSV-1 through 17 or given more proper nomenclature, e.g., the pygmy chimpanzee isolate (primate calicivirus Pan paniscus type 1) (1,19). By 1982, 11 species of pinnipeds and cetaceans of the North Pacific Ocean and Bering Sea (monk seals, California sea lions, northern sea lions, northern elephant seals, northern fur seals, walrus, gray whales, sei whales, sperm whales, bowhead whales, and Pacific bottlenosed dolphins) were known to be susceptible to calicivirus infection, as was an ocean fish, the opaleye perch (Girella nigricans) (20). Furthermore, in many instances, the virus had crossed the intertidal zone to infect terrestrial species (18). On the basis of these data and the established ocean ranges of known calicivirus host species, the shores of Mexico, the United States, Canada, Russia, Korea, Japan, China, and perhaps others bordering the North Pacific Ocean had been regularly exposed to large numbers of marine caliciviruses with unknown host ranges and tissue trophisms (21). By this time, type-specific neutralizing antibodies to two of four serotypes tested were reported in human patients in the United States (22). Cumulatively, these findings lead to the conclusion that fish and perhaps other ocean products provide a vehicle for transmission of these marine caliciviruses to terrestrial animals. The magnitude of potential exposure to marine caliciviruses from the sea is substantial. For example, a 35-ton gray whale, shown by electron microscopy to have more than 106 caliciviruses per gram of feces, can eat 5% or more of its body weight per day and eliminate an equivalent quantity of feces containing an estimated 1013 caliciviruses daily. Marine caliciviruses remain viable more than 14 days in 15°C seawater (20). Although marine mammals were often infected, fish and fish products were more likely to transport the virus from sea to land (23). In contrast to the 1932 to 1936 introductions of VES from raw fish, the rapid and uncontrolled spread of VES virus throughout California after 1939 and then across the United States in 1952 was a pig-to-pig cycle through raw garbage feed. However, new virus serotypes were also introduced through feeding raw fish scraps to swine (1,15). That VES was not a species-specific disease became accepted, but the possibility of human infection, although suspected (22), was largely untested. During the third historical period (1976 to present), which overlaps with the second, viral traffic across the land/sea interface has been observed repeatedly, as the following examples show. A calicivirus isolated from an opaleye perch and designated SMSV-7 produced fulminating VES in exposed swine and spread from pig to pig by contact transmission (23). A reptilian calicivirus Crotalus-1 was isolated from three species of snakes and one species of amphibian (24) and from three species of marine mammals whose population distributions spanned the North Pacific from Mexico to the Bering Sea (1). Mink fed a diet of ground-up calicivirus-infected seal meat became infected with VES virus (25). Parasitic nematode larvae from California sea lions in San Diego, California, were used to infect opaleye fish with calicivirus (SMSV-5); when the fish were killed and fed to Northern fur seals on the Pribilof Islands in the Bering Sea 30 days later, the seals developed vesicular disease, and the virus was recovered from the lesions (1). Shellfish resident to the tidal zone were exposed to marine caliciviruses and held at less than 10°C in a continuous flow of sterile seawater. The caliciviruses were reisolated 60 days later in mammalian cell lines (26). When tested with a cDNA calicivirus group-specific hybridization probe from a marine calicivirus (SMSV-5), some shellfish beds on U.S. coasts were positive for caliciviruses of unknown type (26). Feline calicivirus was shown to cause disease not only in dogs, but also in seals (on the basis of 17 of 20 adult sea lions having neutralizing antibody to FCV-F9 with titers of 1:15 to 1:220). Only 11 of 20 of these sea lions had neutralizing antibody to a sea lion isolate, SMSV-13 (titer 1:10). This demonstrates a probable feline calicivirus ocean presence in California sea lions (2). In swine, a so-called mystery pig disease (porcine respiratory reproductive syndrome) was reproduced in pregnant sows in 1992 with a three-plaque passage purified cytolytic calicivirus isolate from stillborn piglets with mystery pig disease (13). A second calicivirus serotype isolated from the same piglets was the same as that isolated from walruses in 1976 (1). A white tern (Gygis alba rothschildi), a migratory sea bird sampled in the mid-Pacific (French Frigate Shoals), had a blistering disease caused by a calicivirus (27). In the first fully documented human case of clinical disease caused by a marine calicivirus, SMSV-5 was isolated from blisters on the hands and feet of a patient (7). A second, less well-documented, case involved a field biologist who was handling sea lions and developed severe facial blistering. An untypable calicivirus was isolated in tissue culture (Vero cells) from throat washings (7). Extent of Exposure The extent of human disease is not known because test reagents are not readily available and diagnosticians are not alerted to caliciviral causes of human disease, except for diarrhea and occasionally hepatitis. However, evidence of human exposure was shown when 150 serum specimens from normal blood destined for donor use were tested. The samples were antibody-negative for hepatitis B surface and core antigen, HIV-1 and -2, HIV P-24 antigen, human T-cell lymphotropic virus Type 2, and hepatitis C virus. Approximately 19% had antibodies reactive to a polyvalent antigen made up of equal quantities of cesium chloride-banded SMSV-5, 13, and 17. (Figure 1A). To demonstrate that these reactions were not cross-reactions to the human Norwalk calicivirus antibody, serum samples from eight persons with Norwalk virus-induced diarrhea were also tested. Both acute- and convalescent-phase serum specimens were tested by enzyme-linked immunosorbent assay with the same SMSV antigens and Norwalk capsid protein (Figure 1B, C, and D). Although cross-reactivity was not detected, the serum samples may still have been able to cross-react with the calicivirus causing hepatitis E or Sapporo calicivirus, which were not tested. However, sera typed to all 40 marine caliciviruses reacted negatively when tested against Sapporo antigen (data not shown). These results suggest possible human exposure and antigenic response to marine caliciviruses. If that is not the case, such results present a confusing diagnostic picture of calicivirus exposure and diagnosis of human disease. [fig] Figure 1. A) 150 blood donor sera tested against a polyvalent antigen containing San Miguel sea lion viruses (SMSVs) 5, 13, and 17 purified by CsCl; B) Eight acute- and eight convalescent-phase sera from a confirmed outbreak of Norwalk gastroenteritis tested against the polyvalent SMSVs 5, 13, 17 antigen; C) The eight acute-phase sera from the same outbreak of Norwalk gastroenteritis tested in B also tested against the baculovirus expressed Norwalk virus capsid protein; D) The eight convalescent-phase sera paired with the acute-phase sera (See C) tested as in C. Organism Characteristics The history of marine caliciviruses demonstrates that their biologic properties have great plasticity. The VES virus-like caliciviruses can replicate at temperatures of 15°C to 39°C, have diverse tissue trophisms, and travel by land (terrestrial reptiles, amphibians, and mammals), sea (pinnipeds, cetaceans, teleosts, and perhaps filter-feeding mollusks), and air (pelagic birds, e.g., the white tern). They can persist in nonlytic cycles in many reservoir hosts, and they have a wide diversity of successful antigenic types (1) (more than 40 serotypes on the basis of virus neutralization, e.g., no cross-protection between types). Their cup-like surface morphology is characteristic (Figure 2). Finally, they are zoonotic: this is a paradigm shift (7). No other virus has been shown to have its origins and primary reservoirs in the sea yet emerge to cause disease in humans. To measure calicivirus adaptivity and preclude strong presumptions of host specificity on the basis of calicivirus type or species of origin, the following list of 16 hosts is given for a single virus serotype, SMSV-5: known natural hosts-five genera of seals, cattle, three genera of whales, donkeys, fox, and humans-and susceptible hostso-paleye fish, horses, domestic swine, and primates (1). The lists are still growing. SMSV-5 can also persist for 60 days in shellfish, but infectivity has not been measured (26). Feline calicivirus (FCV-F9) has an apparent ocean presence among California sea lions and is not host-specific (2). All members of the family Felidae are susceptible to infection, not just domestic cats. In addition, cheetahs are susceptible; the agent has naturally infected and caused disease in dogs and experimentally infected coyotes (28,29). Reports of human antibody against the feline virus suggest zoonotic potential for the feline calicivirus (30). [Fig] Figure 2. Electron photomicrograph of Cetacean Calicivirus Tursiops - 1 (CCVTur-1). Negative staining using phosphotungstic acid on a carbon-coated grid showing typical surface cup morphologic features as commonly seen by electron microscopy. Bar = 100 nm. Tissue Trophisms The broad host range and diverse mechanisms of transmission and survival of marine caliciviruses are expected of an RNA virus quasispecies (12). If structural simplicity associated with a capsid made up of a single protein species and replicative strategies conserved across rather broad tissue and phylogenetic distances is a measure, caliciviruses are primitive RNA viruses. Caliciviral RNA replicative mechanisms are thus expected to generate numerous mutants (perhaps as high as one to 10 per template copy (12), which will come in contact with many pelagic and terrestrial biota. Opportunity exists to form clusters of virus adapted across a diversity of life forms. Actual mutation rates have not been demonstrated for the Caliciviridae, but plaque-size reversion studies have found that the mutation rate for this phenomenon is one per 106 replicates (14,15). In addition, the expected versatility from RNA virus replicative infidelity and the resulting successful adaptive mechanisms are manifested in the wide spectrum of calicivirus tissue trophisms. Disease conditions involving calicivirus tissue trophisms include blistering of the skin (particularly on the appendages and around the mouth), pneumonia, abortion, encephalitis, myocarditis, myositis, hepatitis, diarrhea, and coagulation/hemorrhage (1,3,5,7; Table 1). Caliciviruses have the inherent potential and adaptive mechanisms to successfully parasitize essentially all organ systems of the many animal species that have been examined in detail. The Future Calicivirus disease manifestations in animals will likely continue but will only become well defined with improved diagnostic means. With cultivatable marine caliciviruses as models, the role of disease-causing caliciviruses can be further defined. Now caliciviruses infecting humans can only be visualized by electron microscopy or histochemistry but cannot be propagated in vitro. Thus, miscarriage and birth defects in human patients, hepatitis other than types A through G, hand-foot-and-mouth-like diseases, viral myocarditis, viral encephalitis of unknown etiology, and joint and muscle disease, for example, should be examined for caliciviruses when other causes of disease are not found. Table 1. Calicivirus tissue trophisms --------------------------------------------------------------------------- Disease conditions Species affected Calicivirus groupa --------------------------------------------------------------------------- Skin blistering Cattle, cats, dogs, VESV, SMSV, FCV, CCV humans, primates, seals, swine Pneumonia Cats, cattle, swine FCV, SMSV Abortion Seals, swine VESV, SMSV Encephalitis Cats, primates, seals, VESV, SMSV swine Myocarditis Seals, swine VESV, SMSV Hepatitis Humans, rabbits, swine VESV, RHDV, HEV Diarrhea Cattle, dogs, humans, VESV, SMSV, CCV, SRSV, reptiles, swine Sapporo Coagulation/hemorrhage Humans, rabbits, seals, RHDV, VESV, HEV swine --------------------------------------------------------------------------- aThe family Caliciviridae has been tentatively divided into five groups, each proposed to be a genus. Group 1: Vesicular exanthema of swine (VESV), San Miguel sea lion virus (SMSV), Feline calicivirus (FCV), Canine calicivirus (CCV); Group 2: Sapporo calicivirus (Sapporo); Group 3: Rabbit hemorrhagic disease virus (RHDV); Group 4: Hepatitis E virus (HEV); Group 5: Small round structured virus (SRSV), which includes Norwalk virus. In the absence of data, extrapolating from cultivatable caliciviruses to predict future effects of poorly characterized caliciviruses should be useful, particularly when there is an urgent need to assess possible human risk. The calicivirus implicated in RHD is a case in point for it might be expected to infect humans. Additional evidence exists. An anecdotal account mentions a Mexican worker who developed antibodies to RHD while eradicating the disease in Mexico (31). An Australian study designed to assess the risk for illness after RHD escaped from Wardang Island (32) examined a group of 269 persons (153 reporting exposure to rabbits or samples infected with RHD virus and 116 reporting no known RHD virus contact) from two Australian states with the greatest amount of RHD virus activity in rabbits. Exposure was categorized by degree of skin exposure to infected materials. Date of first exposure was noted, but no cumulative exposure index was developed. A "high" exposure category could derive from one exposure, and "low" exposure categories could include multiple exposures, each with low exposure. Symptoms were assessed by recall of illness over the previous 13 months. Because the RHD agent was in high security containment facilities for the first 3 months of the recall period and geographically confined for the following 3 months, that period was considered a low exposure period. Because of the rapid spread of the virus in the two states, the last 6 months of the recall period were considered the high exposure period. The data (Table 2) show the rate ratios for the occurrence of different illness in the two periods. All rate ratios were considerably greater than 1.00, and the rate ratios for any illness, diarrhea/gastroenteritis, flu/fever, and neurologic illness are significant (p < 0.005). Because each group contained health histories for 3 spring months or 3 autumn months, 1 summer month, and 2 winter months, the data are seasonally adjusted; hence, winter illness does not explain the excess symptoms observed in the high exposure group, and RHD virus exposure remains a plausible explanation for increased disease incidence. It is difficult to produce pure cultures of noncultivatable caliciviruses to carry out Koch's postulates and establish cause and effect for a single pathogen strain or species. For RHD, both a calicivirus and a parvovirus have been identified in ill rabbits, and a parvovirus has been isolated in vitro and reported to fulfill Koch's postulates (33-35). Yet, caliciviruses have been purified from infected organs to the limits of purity by physical means, and those preparations also cause RHD (35). The caliciviruses purified by physical means cannot be proven to be free of contaminating agents, such as parvovirus (35). If RHD is parvovirus-driven, extrapolation from what is known of other small DNA viruses suggests a rather stable genome and a reduced host range with less likelihood of new host relationships (12). On the other hand, if calicivirus is the primary pathogen, the genomic infidelity that occurs during small RNA virus replication and the documented cross-species transmission of the cultivatable caliciviruses suggest that RHD might also move across species barriers (1,12). Table 2. Population incidence of rabbit hemorrhagic disease (RHD) virus for seasonally equalized periods (July-December and February-July), derived from Mead et al. (32) --------------------------------------------------------------------------- Jul-Dec Feb-Jul Rate 95% Confidence 1995 1996 Ratio Interval --------------------------------------------------------------------------- Exposure to Low High RHD virus Any illness 112 210 1.88 1.49-2.36 Flu/fever 94 189 2.01 1.57-2.57 Diarrhea/ 41 73 1.78 1.21-2.61 gastroenteritis Neurologic 18 49 2.72 1.58-4.67 symptoms Rashes/skin 3 10 3.33 0.92-12.1 Bleeding/hepatitis 2 4 2.00 0.18-22.1 --------------------------------------------------------------------------- Adequate diagnostic reagents for epidemiologic studies need to be made available; they include antigens, monoclonal antibodies, polymerase chain reaction primer sets, and cDNA probes based on group epitopes. In addition, biotype- or pathotype-specific reagents are needed to differentiate pathogenic from nonpathogenic infections. The future also holds the confounding problem of vaccines. Although vaccines can be produced, because of calicivirus antigenic diversity, their efficacy would be predictably short-lived and marginal. Other approaches will need to be sought. Conserved traits that render the Caliciviridae viable as a virus with certain predictable genomic expressions must be sought, and if they exist, targeted for immune attack. Conclusions Only one of the five known calicivirus groups can be grown in vitro and subjected to the full range of host-parasite tests and conditions necessary to more fully define a virus in nature. Therefore, extrapolations developed from this group, the cultivatable marine caliciviruses, should provide insights as a predictive model to help answer questions for the noncultivatable caliciviruses such as small round structured virus, Sapporo virus, hepatitis E virus, and rabbit caliciviruses. From the replicative strategy of the Caliciviridae (as RNA viruses), one would predict considerable diversity. In vitro cultivation has shown that caliciviruses exhibit survivability and plasticity in nature. Many of the factors regarding host spectrum, zoonotic potential, disease conditions, transport, intermediate hosts, and abrupt appearance or disappearance, which may be unknown in newly emerging calicivirus diseases (e.g., RHD), may be more reliably predicted with an established model such as the cultivatable marine caliciviruses. New and better biologic tools for diagnostic and epidemiologic assessments must be developed. This should be augmented by recognizing the zoonotic potential of the cultivatable caliciviruses of ocean origin and then examining them as possible models to help solve many unanswered questions for pathogenic Caliciviridae. Acknowledgments We thank Ms. Christine Robinette for manuscript preparation. This study was funded by the Oregon Agricultural Experiment Station through the College of Veterinary Medicine. Alvin W. Smith is professor of veterinary virology and head, Laboratory for Calicivirus Studies at the College of Veterinary Medicine, Oregon State University, Corvallis, Oregon. Dr. Smith is interested in mechanisms for the preservation and movement of pathogenic viruses in nature, particularly those contained in ocean reservoirs. His research has focused on the marine caliciviruses. References 1. Smith AW, Boyt PM. Caliciviruses of ocean origin: a review. The Journal of Zoo Wildlife Medicine 1990;21:3-23. 2. Smith AW, Skilling DE. Comparison of antibody titers for 13 calicivirus serotypes between aborting and full-term adult Zalophus californianus. Special report submitted to the National Marine Mammal Laboratory, National Marine Fisheries Service. Corvallis (OR): Laboratory for Calicivirus Studies, Oregon State University; 1994. 3. Gunn RA, Janowski HT, Lieb S, Prather EC, Greenberg HB. Norwalk virus gastroenteritis following raw oyster consumption. Am J Epidemiol 1982;115:348-51. 4. Morse DL, Grabau JJ, Hanrahan R, Stricof M, Shaegani M, Diebal R, et al. Widespread outbreaks of clam- and oyster-associated gastroenteritis: role of Norwalk virus. N Engl J Med 1986;314:678-81. 5. Bradley DW. Hepatitis E virus: a brief review of the biology, molecular virology, and immunology of a novel virus. J Hepatol 1995;22:140-5. 6. Chasey D. Possible origins of rabbit haemorrhagic disease in the United Kingdom. Vet Rec 1994;135:496-9. 7. Smith AW, Berry ES, Skilling DE, Barlough JE, Poet SE, Berke T, et al. In vitro isolation and characterization of a calicivirus causing a vesicular disease of the hands and feet. Clin Infect Dis. In press 1998. 8. Berke T, Golding B, Jiang K, Cubitt DW, Wolfaardt M, Smith AW, Matson DO. A phylogenetic analysis of the caliciviruses. J Med Virol. In press 1997. 9. Barlough JE, Berry ES, Skilling DE, Smith AW. The marine calicivirus story-part I. Compendium on Continuing Education for the Practicing Veterinarian 1986;8:F5-F14. 10. Barlough JE, Berry ES, Skilling DE, Smith AW. The marine calicivirus story-part II. Compendium on Continuing Education for the Practicing Veterinarian 1986;8:F75-F82. 11. Bradley DW. Enterically transmitted non A non B hepatitis. Br Med Bull 1990;46:442-61. 12. Holland J. Replication error, quasispecies populations, and extreme evolution rates of RNA viruses. In: Morse SS, editor. Emerging Viruses. Oxford: Oxford University Press; 1993. p. 203-17. 13. Smith AW, Skilling DE, Applegate GL, Trayor TR, Lola TJ, Poet SE. Calicivirus isolation from stillborn piglets in two outbreaks of swine infertility and respiratory syndrome (SIRS). Proceedings of the World Association of Veterinary Microbiologists, Immunologists, and Specialists in Infectious Disease. Davis (CA): University of California, Davis; 1992. 14. Smith AW, Madin SH. Vesicular exanthema of swine. In: Leman AD, Straw B, Glock RD, Mengeling WL, Penny RHC, Scholl E, editors. Diseases of swine. 6th edition. Ames (IA): Iowa State Press; 1986. p. 358-68. 15. Bankowski RA. Vesicular exanthema. In: Bankowski RA, editor. Advances in veterinary science. Academic Press; 1965. p. 23-64. 16. Morse SS. Examining the origins of emerging viruses. In: Morse SS, editor. Emerging viruses. Oxford: Oxford University Press; 1993. p. 10-28. 17. Smith AW, Akers TG, Madin SD, Vedros NA. San Miguel sea lion virus isolation, preliminary characterization and relationship to vesicular exanthema of swine virus. Nature 1973;244:108-10. 18. Smith AW, Akers TG. Vesicular exanthema of swine. J Am Vet Med Assoc 1976;169:700-3. 19. Smith AW, Skilling DE, Ensley PK, Benirschke K, Lester TL. Calicivirus isolation and persistence in a pygmy chimpanzee (Pan paniscus). Science 1983;221:79-81. 20. Smith AW, Skilling DE, Prato CM, Bray HL. Calicivirus (SMSV-5) infection in experimentally inoculated opaleye fish (Girella nigricans). Arch Virol 1981;67:165-8. 21. Smith AW, Skilling DE, Barlough JE, Berry ES. Distribution in the North Pacific Ocean, Bering Sea, and Arctic Ocean of animal populations known to carry pathogenic caliciviruses. Diseases of Aquatic Organisms 1986;2:73-80. 22. Smith AW, Prato CM, Skilling DE. Caliciviruses infecting monkeys and possibly man. Am J Vet Res 1978;39:287-9. 23. Smith AW, Skilling DE, Dardiri AH, Latham AB. Calicivirus pathogenic for swine: a new serotype isolated from opaleye Girella nigricans, an ocean fish. Science 1980;209:940-1. 24. Smith AW, Anderson MP, Skilling DE, Barlough JE, Ensley PK. First isolation of calicivirus from reptiles and amphibians. J Am Vet Med Assoc 1986;47:1718-21. 25. Sawyer JC. Vesicular exanthema of swine and San Miguel sea lion virus. J Am Vet Med Assoc 1976;169:707-9. 26. Smith AW, Reno P, Poet SE, Skilling DE, Stafford C. Retention of ocean-origin caliciviruses in bivalve mollusks maintained under experimental depuration conditions. In: Fenwick B, editor. Proceedings of the 25th Annual Meeting of the International Association of Aquatic Animals; 1994; Vallejo, California. Baltimore: International Association of Aquatic Animal Medicine; 1994. 27. Poet SE, Skilling DE, Megyesi JL, Gilmartin WG, Smith AW. Detection of a non-cultivatable calicivirus from the white tern (Gygis alba rothschildi). J Wildl Dis 1996;32:461-7. 28. Evermann JF, Bryan CM, McKiernan AJ. Isolation of a calicivirus from a case of canine glossitis. Canine Practice 1981;8:36-9. 29. Evermann JF, McKiernan AJ, Smith AW, Skilling DE, Ott RL. Isolation of a calicivirus from dogs with enteric infections. Am J Vet Res 1985;46:218-20. 30. Cubitt DW. Proceedings of the European Society of Veterinary Virology. Readings, United Kingdom: Reading University; 1996. 31. Bureau of Resource Studies. Rabbit calicivirus disease. Canberra, Australia: Australian Government Printing Office; August 1996. p. 20-56. 32. Mead C, Kaldor J, Canton M, Gamer G, Crerar S, Thomas S. Rabbit calicivirus and human health. Canberra, Australia: Department of Primary Industries and Energy, Australian Government (Released under the Official Information Act). Report of the Rabbit Calicivirus Human Health Study Group; 1996. 33. Xu WY. Viral haemorrhagic disease of rabbits in the People's Republic of China: epidemiology and virus characterization. Revue Scientifique et Technique, Office International des Epizooties 1991;10:2393-408. 34. Gregg DA, House C, Meyer R, Berninger M. Viral haemorrhagic disease of rabbits in Mexico: epidemiology and viral characterization. Revue Scientifique et Technique, Office International des Epizooties 1991;10:2435-51. 35. Ohlinger VF, Thiel HJ. Identification of the viral haemorrhagic disease in rabbits as a calicivirus. Revue Scientifique et Technique, Office International des Epizooties 1991;10:2311-23. --------------------------------------------------------------------------- Perspectives Outbreak Investigations-A Perspective Arthur L. Reingold University of California, Berkeley, California, USA --------------------------------------------------------------------------- Outbreak investigations, an important and challenging component of epidemiology and public health, can help identify the source of ongoing outbreaks and prevent additional cases. Even when an outbreak is over, a thorough epidemiologic and environmental investigation often can increase our knowledge of a given disease and prevent future outbreaks. Finally, outbreak investigations provide epidemiologic training and foster cooperation between the clinical and public health communities. Investigations of acute infectious disease outbreaks are very common, and the results of such investigations are often published; however, surprisingly little has been written about the actual procedures followed during such investigations (1,2). Most epidemiologists and public health officials learn the procedures by conducting investigations with the initial assistance of more experienced colleagues. This article outlines the general approach to conducting an outbreak investigation. The approach applies not only to infectious disease outbreaks but also to outbreaks due to noninfectious causes (e.g., toxic exposure). How Outbreaks Are Recognized Possible outbreaks of disease come to the attention of public health officials in various ways. Often, an astute clinician, infection control nurse, or clinical laboratory worker first notices an unusual disease or an unusual number of cases of a disease and alerts public health officials. For example, staphylococcal toxic shock syndrome and eosinophilia myalgia syndrome were first noted by clinicians (3,4). Frequently, it is the patient (or someone close to the patient) who first suspects a problem, as is often the case in foodborne outbreaks after a shared meal and as was the case in the investigation of a cluster of cases of apparent juvenile rheumatoid arthritis near Lyme, Connecticut, which led to the discovery of Lyme disease (5). Review of routinely collected surveillance data can also detect outbreaks of known diseases, as in the case of hepatitis B infection among the patients of an oral surgeon in Connecticut and patients at a weight reduction clinic (6,7). The former outbreak was first suspected when routinely submitted communicable disease report forms for several patients from one small town indicated that all of the patients had recently had oral surgery. However, it is relatively uncommon for outbreaks to be detected in this way and even more uncommon for them to be detected in this way while they are still in progress. Finally, sometimes public health officials learn about outbreaks of disease from the local newspaper or television news. Reasons for Investigating Outbreaks The most compelling reason to investigate a recognized outbreak of disease is that exposure to the source(s) of infection may be continuing; by identifying and eliminating the source of infection, we can prevent additional cases. For example, if cans of mushrooms containing botulinum toxin are still on store shelves or in homes or restaurants, their recall and destruction can prevent further cases of botulism. However, even if an outbreak is essentially over by the time the epidemiologic investigation begins-that is, if no one is being further exposed to the source of infection-investigating the outbreak may still be indicated for many reasons. Foremost is that the results of the investigation may lead to recommendations or strategies for preventing similar future outbreaks. For example, a Legionnaires' disease outbreak investigation may produce recommendations for grocery store misting machine use that may prevent other outbreaks (8). Other reasons for investigating outbreaks are the opportunity to 1) describe new diseases and learn more about known diseases; 2) evaluate existing prevention strategies, e.g., vaccines; 3) teach (and learn) epidemiology; and 4) address public concern about the outbreak. Once a decision is made to investigate an outbreak, three types of activities are generally involved-the epidemiologic investigation; the environmental investigation; and the interaction with the public, the press, and, in many instances, the legal system. While these activities often occur simultaneously throughout the investigation, it is conceptually easier to consider each of them separately. Epidemiologic Investigation Outbreak investigations are, in theory, indistinguishable from other epidemiologic investigations; however, outbreak investigations encounter more constraints. 1) If the outbreak is ongoing at the time of the investigation, there is great urgency to find the source and prevent additional cases. 2) Because outbreak investigations frequently are public, there is substantial pressure to conclude them rapidly, particularly if the outbreak is ongoing. 3) In many outbreaks, the number of cases available for study is limited; therefore, the statistical power of the investigation is limited. 4) Early media reports concerning the outbreak may bias the responses of persons subsequently interviewed. 5) Because of legal liability and the financial interests of persons and institutions involved, there is pressure to conclude the investigation quickly, which may lead to hasty decisions regarding the source of the outbreak. 6) If detection of the outbreak is delayed, useful clinical and environmental samples may be very difficult or impossible to obtain. Outbreak investigations have essential components as follows: 1) establish case definition(s); 2) confirm that cases are "real"; 3) establish the background rate of disease; 4) find cases, decide if there is an outbreak, define scope of the outbreak; 5) examine the descriptive epidemiologic features of the cases; 6) generate hypotheses; 7) test hypotheses; 8) collect and test environmental samples; 9) implement control measures; and 10) interact with the press, inform the public. While the first seven components are listed in logical order, in most outbreak investigations, many occur more or less simultaneously. The importance of these components may vary depending on the circumstances of a specific outbreak. Case Definition In some outbreaks, formulating the case definition(s) and exclusion criteria is straightforward; for example, in an outbreak of gastroenteritis caused by Salmonella infection, a laboratory-confirmed case would be defined as a culture-confirmed infection with Salmonella or perhaps with Salmonella of the particular serotype causing the outbreak, while a clinical case definition might be new onset of diarrhea. In other outbreaks, the case definition and exclusion criteria are complex, particularly if the disease is new and the range of clinical manifestations is unknown (e.g., in a putative outbreak of chronic fatigue syndrome). In many outbreak investigations, multiple case definitions are used (e.g., laboratory-confirmed case vs. clinical case; definite vs. probable vs. possible case; outbreak-associated case vs. nonoutbreak-associated case, primary case vs. secondary case) and the resulting data are analyzed by using different case definitions. When the number of cases available for study is not a limiting factor and a case-control study is being used to examine risk factors for becoming a case, a strict case definition is often preferable to increase specificity and reduce misclassification of disease status (i.e., reduce the chance of including cases of unrelated illness or no illness as outbreak-related cases). Case Confirmation In certain outbreaks, clinical findings in reported cases should be reviewed closely, either directly, by examining the patients, or indirectly, by detailed review of the medical records and discussion with the attending health-care provider(s), especially when a new disease appears to be emerging (e.g., in the early investigations of Legionnaires' disease, AIDS, eosinophilia myalgia syndrome, and hantavirus pulmonary syndrome) (4,9-11). Clinical findings should also be examined closely when some or all of the observed cases may be factitious, perhaps because of laboratory error (12); a discrepancy between the clinical and laboratory findings generally exists, which may be discernible only by a detailed review of the clinical findings. Establishing the Background Rate of Disease and Finding Cases Once it is clear that a suspected outbreak is not the result of laboratory error, a set of activities should be undertaken to establish the background rate of the disease in the affected population and to find all the cases in a given population in a certain period. This set of activities should prove that the observed number of cases truly is in excess of the "usual" number (i.e., that an outbreak has occurred), define the scope of the outbreak geographically and temporally, find cases to describe the epidemiologic features of those affected and to include them in analytic epidemiologic studies (see below) or, most often, accomplish a combination of these goals. When hundreds of acute onset diarrhea cases are suddenly seen daily in a single outpatient setting (10), an outbreak is clearly occurring. On the other hand, when too many hospitalized patients are dying unexpectedly of cardiac arrest (13) or the number of cases of listeriosis in a given county in recent months is moderately elevated, it may be necessary to establish the background rates in the population to determine whether an outbreak is occurring. In such situations, the period and geographic areas involved would provide the most useful baseline data, keeping in mind that the labor and time required to collect such information is often directly proportional to the length of the period and the size of the geographic area selected. Because disease incidence normally fluctuates by season, data from comparable seasons in earlier years should be included. Establishing the background rate of a disease is generally more straightforward if confirmatory tests are available than if laboratory tests are unavailable or infrequently used. The rate of certain invasive bacterial infections (e.g., listeriosis and meningococcal infections) in a given area can be easily documented by reviewing the records of hospital clinical microbiology laboratories; however, cases for which specimens were not submitted to these laboratories for testing will go undetected. When a disease is less frequently laboratory-confirmed because health-care providers may not have considered the diagnosis or ordered the appropriate laboratory tests (e.g., for Legionnaires' disease), establishing the background rate of disease in a community or a hospital suspected of having an outbreak generally requires alternative case-finding strategies and is almost invariably more labor intensive. In an outbreak of a new disease, substantial effort is often necessary to determine whether or not cases of that disease had been occurring but had gone unrecognized. Once data concerning the background rate of a disease (including case-finding for the current period) have been collected, it is generally possible to determine whether or not an outbreak is occurring or has occurred, although in some situations it may remain unclear whether or not the number of cases observed exceeds the background rate. In part, the problem may relate to how an outbreak is defined. To paraphrase a U.S. Supreme Court justice speaking about pornography, "I can't define an outbreak, but I know one when I see one." Thus, it may be difficult to detect and prove the existence of small outbreaks, but large ones are self-evident. An outbreak can also be difficult to identify when during the period under study changes occur in the care-seeking behavior and access to care of patients; the level of suspicion, referral patterns, and test-ordering practices of health-care providers; the diagnostic tests and other procedures used by laboratories; and the prevalence of underlying immunosuppressive conditions or other host factors in the population. All these factors, which can affect the apparent incidence of a disease and produce artifactual changes perceived as increases (or decreases) in the actual incidence, need to be considered when interpreting the findings. Descriptive Epidemiology By collecting patient data, the case-finding activities provide extremely important information concerning the descriptive epidemiologic features of the outbreak. By reviewing and plotting on an "epidemic curve" the times of onset of the cases and by examining the characteristics (e.g., age, sex, race/ethnicity, residence, occupation, recent travel, or attendance at events) of the ill persons, investigators can often generate hypotheses concerning the cause(s)/source(s) of the outbreak. While linking the sudden onset of gastroenteritis among scores of persons who attended a church supper to the single common meal they shared is generally not a challenge, an otherwise cryptic source can be at least hinted at by the descriptive epidemiologic features of the cases involved. For example, in a particularly perplexing outbreak of _Salmonella_ Muenchen infections ultimately traced to contaminated marijuana, the age distribution of the affected persons and of their households was markedly different from that typically seen for salmonellosis (14). Or, similarly, in the outbreak of legionellosis due to contaminated misting machines in the produce section of a grocery store, before the link to this exposure was even suspected, it was noted that women constituted a substantially higher proportion of the cases usually seen with this disease (5). The shape of the epidemic curve can also be very instructive, suggesting a point-source epidemic, ongoing transmission, or a combination of the two. Generating a Hypothesis The source(s) and route(s) of exposure must be determined to understand why an outbreak occurred, how to prevent similar outbreaks in the future, and, if the outbreak is ongoing, how to prevent others from being exposed to the source(s) of infection. In some outbreaks, the source and route are obvious to those involved in the outbreak and to the investigators. However, even when the source of exposure appears obvious at the outset, a modicum of skepticism should be retained because the obvious answer is not invariably correct. For example, in an outbreak of nosocomial legionellosis in Rhode Island, the results of an earlier investigation into a small number of hospital-acquired cases at the same hospital had demonstrated that _Legionella pneumophila_ was in the hospital potable water supply, and a sudden increase in new cases was strongly believed to be related to the potable water (15). However, a detailed epidemiologic investigation implicated a new cooling tower at the hospital as the source of the second outbreak. While the true source of exposure, or at least a relatively short list of possibilities, is apparent in many outbreaks, this is not the case in the more challenging outbreaks. In these instances, hypotheses concerning the source/route of exposure can be generated in a number of ways beyond a detailed review of the descriptive epidemiologic findings. A review of existing epidemiologic, microbiologic, and veterinary data is very useful for learning about known and suspected sources of previous outbreaks or sporadic cases of a given infection or disease, as well as the ecologic niche of an infectious agent. Thus, in an outbreak of invasive _Streptococcus zooepidemicus_ infections in New Mexico due to consumption of soft cheese made from contaminated raw milk, the investigation focused on exposure to dairy products and animals because of previous microbiologic and veterinary studies (16). A review of existing data generally only helps confirm what is already known about a particular disease and is far less helpful in identifying totally new and unsuspected sources or routes of infection (i.e., marijuana as a source of Salmonella). When neither review of the descriptive epidemiologic features of the cases nor review of existing scientific information yields the correct hypothesis, other methods can be used to generate hypotheses about what the patients have in common. Open-ended interviews of those infected (or their surrogates) are one such method in which investigators try to identify all possibly relevant exposures (e.g., a list of all foods consumed) during a given period. For example, in an investigation of _Yersinia enterocolitica_ infections in young children in Belgium, open-ended interviews of the mothers of some of the ill children showed that many gave their children raw pork sausage as a weaning food, providing the first clue as to the source of these infections (17). Similarly, in two outbreaks of foodborne listeriosis, a variant of this process led to the identification of the source of the outbreak. In one of these outbreaks, a search of the refrigerator of one of the case-patients who, as a visitor to the area, had had very limited exposure to foods there, suggested cole slaw as a possible vehicle of infection (18). In the other outbreak, an initial case-control study found no differences between cases and controls regarding exposure to a number of specific food items but showed that case households were more likely than control households to buy their food at a particular foodstore chain. To generate a list of other possible food sources of infection, investigators shopped with persons who did the shopping for case households and compiled a list of foods purchased at that foodstore chain that had not been reported in the previous study. This approach implicated pasteurized milk from that chain as the source of the outbreak (19). In some particularly perplexing outbreaks, bringing together a subset of the patients to discuss their experiences and exposures in a way that may reveal unidentified links can be useful. Testing the Hypothesis Whether a hypothesis explaining the occurrence of an outbreak is easy or difficult to generate, an analytic epidemiologic study to test the proposed hypothesis should be considered. While in many instances a case-control study is used, other designs, including retrospective cohort and cross-sectional studies, can be equally or more appropriate. The goal of all these studies is to assess the relationship between a given exposure and the disease under study. Thus, each exposure of interest (e.g., each of the meals eaten together by passengers on a cruise ship and each of the foods and beverages served at those meals) constitutes a separate hypothesis to be tested in the analytic study. In outbreaks where generating the correct hypothesis is difficult, multiple analytic studies, with additional hypothesis-generating activities in between, are sometimes needed before the correct hypothesis is formed and tested (19). In interpreting the results of such analytic studies, one must consider the possibility that "statistically significant" associations between one or more exposures and the disease may be chance findings, not indicative of a true relationship. By definition, any "statistically significant" association may have occurred by chance. (When the standard cut point of p < 0.05 is used, this occurs 5% of the time.) Because many analytic epidemiologic studies of outbreaks involve testing many hypotheses, the problem of "multiple comparisons" arises often. While there are statistical methods for adjusting for multiple comparisons, when and even whether to use them is controversial. At a minimum, it is important to go beyond the statistical tests and examine the magnitude of the effect observed between exposure and disease (e.g., the odds ratio, relative risk) and the 95% confidence intervals, as well as biologic plausibility in deciding whether or not a given "statistically significant" relationship is likely to be biologically meaningful. Evidence of a dose-response effect between a given exposure and illness (i.e., the greater the exposure, the greater the risk for illness) makes a causal relationship between exposure and disease more likely. Whether the time interval between a given exposure and onset of illness is consistent with what is known about the incubation period of the disease under study must also be assessed. When illness is "statistically significantly" related to more than one exposure (e.g., to eating each of several foods at a common meal), it is important to determine whether multiple sources of infection (perhaps due to cross-contamination) are plausible and whether some of the noted associations are due to confounding (e.g., exposure to one potential source is linked to exposure to other sources) or to chance. When trying to decide if a "statistically significant" exposure is the source of an outbreak, it is important to consider what proportion of the cases can be accounted for by that exposure. One or more of the patients may be classified as "nonexposed" for various reasons: incorrect information concerning exposure status (due to poor memory, language barriers); multiple sources of exposure or routes of transmission (perhaps due to cross-contamination); secondary person-to-person transmission that followed a common source exposure; or patients without the suspected exposure, representing background cases of the disease unrelated to the outbreak. The plausibility of each of these explanations varies by outbreak. While there is no cutoff point above or below which the proportion of exposed case-patients should fall before an exposure is thought to account for an outbreak, the lower this proportion, the less likely the exposure is, by itself, the source. Other possibilities need to be considered when the analytic epidemiologic study finds no association between the hypothesized exposures and risk for disease. The most obvious possibility is that the real exposure was not among those examined, and additional hypotheses should be generated. However, other possibilities should also be considered, particularly when the setting of the outbreak makes this first explanation unlikely (e.g., when it is known that those involved in the outbreak shared only a single exposure or set of exposures, such as eating a single common meal). Two other explanations for failing to find a "statistically significant" link between one or more exposures and risk for illness also need to be considered-the number of persons available for study and the accuracy of the available information concerning the exposures. Thus, if the outbreak involves only a small number of cases (and non-ill persons), the statistical power of the analytic study to find a true difference in exposure between the ill and the non-ill (or a difference in the rate of disease among the exposed and the unexposed) is very limited. If the persons involved in the outbreak do not provide accurate information about their exposure to suspected sources or vehicles of infection because of lack of knowledge, poor memory, language difficulty, mental impairment, or other reasons, the resulting misclassification of exposure status also can prevent the epidemiologic study from implicating the source of infection. Studies have documented that even under ideal circumstances, memory concerning such exposures is faulty (20). However, given the usually enormous differences in rates of disease between those exposed and those not exposed to the source of the outbreak, even small studies or studies with substantial misclassification of exposure can still correctly identify the source. Environmental Investigation Samples of foods and beverages served at a common meal believed to be the source of an outbreak of gastroenteritis or samples of the water or drift from a cooling tower believed to be the source of an outbreak of Legionnaires' disease can support epidemiologic findings. In the best scenario, the findings of the epidemiologic investigation would guide the collection and testing of environmental samples. However, environmental specimens often need to be obtained as soon as possible, either before they are no longer available, as in the case of residual food from a common meal, or before environmental interventions are implemented, as in the case of treating a cooling tower to eradicate Legionella. Because laboratory testing of environmental samples is often expensive and labor-intensive, it is sometimes reasonable to collect and store many samples but test only a limited number. Collaborating with a sanitarian, environmental engineer, or other professional during an environmental inspection or collection of specimens is always beneficial. While finding or not finding the causative organism in environmental samples is often perceived by the public, the media, and the courts as powerful evidence implicating or exonerating an environmental source, either positive or negative findings can be misleading for several reasons. For example, finding Legionella in a hospital potable water system does not prove that the potable water (rather than a cooling tower or some other source) is responsible for an outbreak of Legionnaires' disease (21). Similarly, not finding the causative organism in an environmental sample does not conclusively rule out a source as the cause of the problem, in part because the samples obtained and tested may not represent the source (e.g., because of error in collecting the specimens, intervening changes in the environmental source) and in part because the samples may have been mishandled. Furthermore, in some outbreaks caused by well-characterized etiologic agents, laboratory methods of detecting the agent in environmental samples are insensitive, technically difficult, or not available, as in the case of recent outbreaks of _Cyclospora_ infections associated with eating imported berries (22,23). Control Measures Central to any outbreak investigation is the timely implementation of appropriate control measures to minimize further illness and death. At best, the implementation of control measures would be guided by the results of the epidemiologic investigation and possibly (when appropriate) the testing of environmental specimens. However, this approach may delay prevention of further exposure to a suspected source of the outbreak and is, therefore, unacceptable from a public health perspective. Because the recall of a food product, the closing of a restaurant, or similar interventions can have profound economic and legal implications for an institution, a manufacturer or owner, and the employees of the establishments involved, acting precipitously can also have substantial negative effects. The recent attribution of an outbreak of _Cyclospora_ infections to strawberries from California demonstrates the economic impact that can result from releasing and acting on incorrect information (22,23). Thus, the timing and nature of control measures are difficult. Balancing the responsibility to prevent further disease with the need to protect the credibility and reputation of an institution is very challenging. Interactions with the Public and Press While the public and the press are not aware of most outbreak investigations, media attention and public concern become part of some investigations. Throughout the course of an outbreak investigation, the need to share information with public officials, the press, the public, and the population affected by the outbreak must be assessed. While press, radio, and television reports can at times be inaccurate, overall the media can be a powerful means of sharing information about an investigation with the public and disseminating timely information about product recalls. Dr. Reingold worked as an epidemiologist at the Centers for Disease Control and Prevention for 8 years before joining the faculty of the School of Public Health at the University of California, Berkeley. He is currently professor of epidemiology and head of the Division of Public Health Biology and Epidemiology. Address for correspondence: Arthur L. Reingold, Division of Public Health Biology and Epidemiology, School of Public Health, University of California, Berkeley, 140 Warren Hall, Berkeley, CA 94720-7360, USA; fax: 510-643-5163; e-mail: reingold@uclink3.berkeley.edu. References 1. Goodman RA, Buehler JW, Koplan JP. The epidemiologic field investigation: science and judgment in public health practice. Am J Epidemiol 1990;132:9-16. 2. MacKenzie WR, Goodman RA. The public health response to an outbreak. Current Issues in Public Health1996;2:1-4. 3. Chesney PJ, Chesney RW, Purdy W, Nelson D, McPherson T, Wand P, et al. Epidemiologic notes and reports: toxic-shock syndrome-United States. MMWR Morb Mortal Wkly Rep 1980;29:229-30. 4. Hertzman PA, Blevins WL, Mayer J, Greenfield B, Ting M, Gleich GJ, et al. Association of eosinophilia-myalgia syndrome with the ingestion of tryptophan. N Engl J Med 1980;322:871. 5. Steere AC, Malawista SE, Syndman DR, Shope RF, Andman WA, Ross MR, Steele FM. Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis Rheum 1977;20:7. 6. Reingold AL, Kane MA, Murphy BL, Checko P, Francis DP, Maynard JE. Transmission of Hepatitis B by an oral surgeon. J Infect Dis 1982;145:262. 7. Canter J, Mackey K, Good LS, Roberto RR, Chin J, Bond WW, et al. An outbreak of hepatitis B associated with jet injections in a weight reduction clinic. Arch Intern Med 1990;150:1923-7. 8. Mahoney FJ, Hoge CW, Farley TA, Barbaree JM, Breiman RF, Benson RF, McFarland LM. Communitywide outbreak of Legionnaires' disease associated with a grocery store mist machine. J Infect Dis 1992;165:736. 9. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Sharrar RG, et al. Legionnaires' disease: description of an epidemic of pneumonia. N Engl J Med 1977;297:1189-97. 10. Kriedman-Kien A, Laubenstein L, Marmor M, Hymes K, Green J, Ragaz A, et al. Kaposi's sarcoma and _Pneumocystis_ pneumonia among homosexual men-New York City and California. MMWR Morb Mortal Wkly Rep 1981;30:305-8. 11. Koster F, Levy H, Mertz G, Young S, Foucar K, McLaughlin J, et al. Outbreak of acute illness-southwestern United States. MMWR Morb Mortal Wkly Rep 1993;42:421-4. 12. Weinstein RA, Bauer FW, Hoffman RD, Tyler PG, Anderson RL, Stamm WE. Factitious meningitis: diagnostic error due to nonviable bacteria in commercial lumbar puncture trays. JAMA 1975;233:878. 13. Buehler JW, Smith LF, Wallace EM, Heath CW, Rusiak R, Herndon JL. Unexplained deaths in a children's hospital: an epidemiologic assessment. N Engl J Med 1985;313:211. 14. Taylor DN, Wachsmuth IK, Shangkuan Y-H, Schmidt EV, Barrett TJ, Schrader JS, et al. Salmonellosis associated with marijuana: a multistate outbreak traced by plasmid fingerprinting. N Engl J Med 1982;306:1249. 15. Garbe PL, Davis BJ, Weisfeld JS, Markowitz L, Miner P, Garrity F, et al. Nosocomial Legionnaires' disease: epidemiologic demonstration of cooling towers as a source. JAMA 1985;254:521. 16. Espinosa FH, Ryan WM, Vigil PL, Gregory DF, Hilley RB, Romig DA, et al. Group C streptococcal infections associated with eating homemade cheese: New Mexico. MMWR Morb Mortal Wkly Rep 1983;32:514. 17. Tauxe RV, Walters G, Goossen V, VanNoyer R, Vandepitte J, Martin SM, et al. _Yersinia enterocolitica_ infections and pork: the missing link. Lancet 1987;5:1129. 18. Schlech WF, Lavigne PM, Bortolussi RA, Allen AC, Haldane EV, Wort AJ, et al. Epidemic listeriosis: evidence for transmission by food. N Engl J Med 1983;308:203. 19. Fleming DW, Cochi SL, MacDonald KL, Brondum J, Hayes PS, Plikaytis BD, et al. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N Engl J Med 1985;312:404. 20. Decker MD, Booth AL, Dewey MJ, Fricker RS, Hutcheson RH, Schaffner W. Validity of food consumption histories in a foodborne outbreak investigation. Am J Epidemiol 1986;124:859. 21. Hayes EB, Matte TD, O'Brien TR, McKinley TW, Logsdon GS, Rose JB, et al. Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply. N Engl J Med 1989;390:1372. 22. Chambers J, Somerfieldt S, Mackey L, Nichols S, Ball R, Roberts D, et al. Outbreaks of _Cyclospora cayetanensis_ infection-United States, 1996. MMWR Morb Mortal Wkly Rep 1996;45:549-51. 23. Hofman J, Liu Z, Genese C, Wolf G, Manley W, Pilot K, et al. Update: outbreaks of _Cyclospora cayetanensis_ infection-United States and Canada, 1996. MMWR Morb Mortal Wkly Rep 1996;45:611-2. --------------------------------------------------------------------------- Synopses Genetic Diversity of Wild-Type Measles Viruses: Implications for Global Measles Elimination Programs William J. Bellini and Paul A. Rota Centers for Disease Control and Prevention, Atlanta, Georgia, USA --------------------------------------------------------------------------- Text Version Wild-type measles viruses have been divided into distinct genetic groups according to the nucleotide sequences of their hemagglutinin and nucleoprotein genes. Most genetic groups have worldwide distribution; however, at least two of the groups appear to have a more limited circulation. To monitor the transmission pathways of measles virus, we observed the geographic distribution of genetic groups, as well as changes in them in a particular region over time. We found evidence of interruption of indigenous transmission of measles in the United States after 1993 and identified the sources of imported virus associated with cases and outbreaks after 1993. The pattern of measles genetic groups provided a means to describe measles outbreaks and assess the extent of virus circulation in a given area. We expect that molecular epidemiologic studies will become a powerful tool for evaluating strategies to control, eliminate, and eventually eradicate measles. Until the advent of a live-attenuated vaccine in the early 1960s, measles was an epidemic disease worldwide. Today many countries have controlled measles, but the disease remains endemic on most continents. Development of a live-attenuated measles vaccine and implementation of laws that required proof of vaccination upon school entry dramatically reduced the incidence of measles in the United States. The number of reported cases plummeted from approximately 500,000 before vaccine introduction in 1963 to fewer than 1,500 in 1983. Despite these measures, a reemergence or resurgence of measles in the United States from 1989 to 1991 resulted in more than 55,000 cases of measles and approximately 120 measles-associated deaths (Figure 1; 1). In exploring the reasons for the resurgence, our laboratory genetically characterized measles viruses isolated from wild-type virus-infected persons from the same outbreak or temporally and geographically distinct outbreaks in the United States; in the regions examined, all measles viruses isolated during the period of resurgence were almost identical in nucleotide sequence and genetically distinct from vaccine strains (2). Measles isolates from many regions of the world have been characterized in parallel studies by our laboratory and by others. In conjunction with classic epidemiologic investigations, these well-characterized viruses have formed a picture of the distribution of wild-type measles viruses in most areas of the world. Eight distinct genotypes have been identified, and undoubtedly more will be added. Some are localized to specific regions, while most are widely distributed. The assembly of these sequences into a large database, which includes their geographic distribution, has become a new means by which measles transmission pathways can be traced and control measures can be assessed. This "molecular epidemiology" has affected the U.S. measles elimination program, and if used appropriately with standard epidemiologic methods, it will affect global measles elimination and eradication. This article summarizes the status of the known measles genotype distribution throughout the world and describes how molecular epidemiologic information has been used to assess the effectiveness of measles elimination in the United States. [graph.gif (6842 bytes)] Figure 1. Incidence of U.S. measles cases and measles deaths between 1980 and 1997. Total number of measles cases for years 1993-1997 (week 35) are indicated above each bar. Global Distribution of Measles Genotypes In most cases, genetic characterization of wild-type measles viruses has been conducted by sequencing the genes coding for the hemagglutinin (H) protein or the nucleoprotein (N). Of the six genes on the viral genome, the H and N genes are the most variable. Over their protein coding regions, the H and N genes vary by up to 7% at the nucleotide level. The single most variable part of the measles genome is the 450 nucleotides that code for the COOH-terminus of the N protein, where nucleotide variability between various wild-type viruses can approach 12%. Several laboratories have analyzed the sequences of wild-type measles viruses and assigned the viruses to various genetic groups(2, 14). Many of our studies have focused on the genetic characterization of measles viruses associated with cases and outbreaks in the United States during the last 10 years (2, 11). These viruses can be separated into at least eight distinct genetic groups (Table; Figure 2). Phylogenetic analyses using various computer programs (15, 17) indicated good statistical support for each of the groups described below. Actually, more than eight genetic groups are listed when viruses from groups not yet found in the United States are included (e.g., Zambia: 1993, Germany: 1992) (Figure 2). The number of genetic groups is likely to increase since the true extent of genetic heterogeneity among wild-type measles viruses is still unknown, and virologic surveillance has not been conducted or has only just begun in many areas of the world. Table. Sources of genotypes isolated in the United States, 1995-1997 No. of Imports Also Group isolates from circulating in ------------------------------------------------------------------- 4 13 Germany, Spain, Central Europe, United Kingdom, Canada, Brazil, Brazil, Austria, United Kingdom Italy,Greece, Ukraine 3 9 Japan Japan, Thailand 5 8 Italy, Germany Central Europe, United Kingdom, Brazil 7 3 Paskistan, Kenya South Africa,Canada 8 2 China, Vietnam China 2 1 Philippines United States 1989-1992, Micronesia 1 1 Unknown United Kingdom, Russia, China, Argentina 6 1 Kenya The Gambia, Cameroon, Gabon, Zambia ------------------------------------------------------------------------ Group 1 contains the prototype, Edmonston strain, which was isolated in 1954. This group also contains all vaccine viruses sequenced regardless of whether they were derived from Edmonston (Attenuvax, Edmonston-Zagreb, AIKC, Schwarz) or from temporally and geographically independent wild-type isolates (Shanghai-191: China, Changchun-47: China, CAM-70: Japan, Leningrad-16: Russia) (18). Relatively few wild-type viruses from the prevaccine era are available for molecular characterization. These viruses, which were isolated in Japan, Russia, Finland, Romania, and the United States during the 1950s and 1960s, are in group 1 (9). Therefore, while viruses belonging to the other genetic groups may have been present, group 1 viruses must have had widespread distribution during the prevaccine era. Group 1 viruses continue to circulate, and viruses from group 1 were isolated from patients with clinical measles in the United States, United Kingdom, Russia, China, and Argentina during the last 7 years (5, 11, 19, 20, and unpub. observations). These recent group 1 wild-type viruses have several nucleotide substitutions that distinguish them from vaccine viruses. In contrast, measles vaccine viruses reisolated from immunosuppressed patients with giant cell pneumonia had nucleotide sequences nearly identical to those of the vaccine virus found in the vaccine vial (unpub. observations). This suggests that vaccine viruses are very stable even after prolonged replication in a human host. Therefore, it is unlikely that the group 1 wild-type viruses represent laboratory contamination of cultures with vaccine virus or reisolation of vaccine virus from recently vaccinated persons. Sequence studies have failed to identify a distinct set of genetic markers that consistently differentiate wild-type and presumably virulent viruses from attenuated viruses. Current studies are focusing on the analysis of the noncoding regions of the viral genome. More studies are needed to compare attenuated strains with their more virulent or reactogenic precursors. The recent development of an infectious clone for measles (21) will, no doubt, contribute to those studies. [fig] Figure 2. Phylogenetic tree showing genetic relationships between the eight genetic groups of measles virus associated with U.S. outbreaks and cases since 1988. The location and year of isolation is given for each virus. Viruses not assigned to one of the eight groups are labeled in brown. The unrooted tree is based on the sequence of the protein coding region of the H gene (1854 nt). Wt-Edmonston = low passage seed of the original Edmonston isolate. SSPE = sequences obtained from cases of subacute sclerosing panencephalitis. Group 2 viruses were associated with the resurgence of measles in the United States between 1989 to 1991, an epidemic that had an unusually high incidence of deaths and hospitalizations(Figure 1). The circulation of group 2 viruses within the United States was interrupted in 1993, and this will be described in more detail below. Among these viruses, the Illinois-1 (Chicago-1) strain has become a representative of recent wild-type viruses, and almost the entire genome has been sequenced. Group 2 viruses were first isolated in Japan during the early 1980s; more recently, they were isolated in Japan, the Philippines, and Micronesia (2,11-13,22). The group referred to as group 3 viruses can actually be divided into two distinct groups with a common geographic distribution. These viruses have been isolated from outbreaks in Japan and Thailand and from sporadic cases following importation into North America and Europe (2,11). Although virus from groups 2 and 3 cocirculated in Japan during the late 1980s and early 1990s, the group 3 viruses have recently become the predominant genotype (23). Groups 4 and 5 appear to be circulating widely in many countries in western Europe, particularly Germany, Spain, and the United Kingdom, where virologic surveillance has been conducted (6,7,24). Viruses from this group are also circulating in France, Italy, Austria, and Greece since they have been associated with multiple importations from these areas into the United States (2,11; Table). All representatives of the group 6 viruses have been isolated in the African countries of The Gambia (4), Cameroon, Gabon, and Zambia or associated with importations into the United States from Kenya. There is more genetic variability within the group 6 viruses than among most of the other genetic groups, yet all group 6 viruses contain a subset of nucleotide substitutions that places them on this African lineage. Relatively few viruses from central Africa, where most measles infections are occurring, have been isolated for genetic analysis, and it will be interesting to determine if other genotypes are also present in this area. Group 7 viruses were first isolated during an epidemic in Montreal, Canada, in 1988 (2,11). Group 7 was the predominant group among a number of recently isolated viruses from Johannesburg, South Africa (9,25,26). The identification of a group 7 virus in association with an importation to the United States from Pakistan suggests (11) that the viruses in this group may be circulating widely in Africa and Asia. The group 8 viruses form a highly distinct group isolated in four provinces within the People's Republic of China during the early 1990s (19). Like group 6, group 8 viruses have more nucleotide variability within the group (up to 3%) than the other groups. Recent evidence also suggests that group 8 viruses are circulating in other parts of China (Hong Kong) and in Vietnam. Several recently isolated viruses do not fit into the eight genetic groups that, so far, contain most recent isolates (Figure 2). Some outliers represent single isolations of a unique genetic type. However, preliminary analysis of a number of wild-type viruses from Zambia indicates that these viruses belong to a genetic group that is distinct from the eight groups described thus far (unpub. observations). If viruses isolated during the early to mid-1980s were included into the genetic analysis (not shown), it would be apparent that more genetic groups exist. However, viruses representing these groups have not been isolated in the last 10 to 15 years, and it must be assumed that these groups are circulating in restricted geographic regions, are circulating at such a low frequency as to escape surveillance, or are extinct. A summary of genetic groups (Figure 3) represents a static picture that simply identifies where particular genetic groups have been isolated, with no accounting for frequency of isolation or source of the virus. Certain regions of the world (including much of Africa and most of southern Asia) are still vastly underrepresented. A survey of recent Australian isolates is in progress. The pattern of genotypes in the United Kingdom and the United States is very complex because of relatively good strain surveillance and the frequency of international travel to these locations. [fig] Figure 3. Global distribution of measles genetic groups. Colored circles indicate areas where measles viruses from various genetic groups have been isolated. Viruses not assigned to one of the eight groups are labeled in brown. Molecular Epidemiology Measles has long been considered one of the most communicable of diseases. The resurgence of disease from 1989 to 1991 in the United States (Figure 1) provides a good example of the rapid transmissibility of the virus. During this resurgence only group 2 viruses were isolated, and the sequences from these viruses were highly related (2). With continued molecular surveillance, we were able to document the interruption of transmission of the group 2 viruses and monitor the change in measles genotypes associated with outbreaks and sporadic cases from 1994 to the present. Molecular surveillance of measles viruses was most useful when the change in genotypes was observed over time. Without that information, it would not have been possible to describe the transition from an apparently "indigenous" lineage to importation of multiple lineages (Figure 4). This is in contrast to the situation in South and Central America. In these areas, viral surveillance was not conducted before mass vaccination campaigns were initiated, so the identity of the prevailing genotype could not be determined. Therefore, it is difficult to interpret the genetic data obtained from viruses currently causing outbreaks in these regions. The molecular surveillance of wild-type viruses in the United States between 1989 and 1997 provides the best example of dynamic molecular surveillance (Figure 4). Viruses isolated over a 4-year period from major outbreaks in New York, Philadelphia, Chicago, Los Angeles, Houston, and southern Texas varied by less than 0.4% at the nucleotide level in the H and N genes (2). Analysis of the few wild-type measles viruses isolated in the United States before 1988 indicates lineages other than group 2. This suggests that the group 2 viruses were probably imported during the late 1980s and were rapidly transmitted to the entire country. While numerous importations occurred during the resurgence, apparently these viruses did not circulate widely enough to be detected by molecular surveillance. Perhaps the number of measles-susceptible persons in the U.S. population during the resurgence was high enough to sustain continuous transmission without accumulation of variants or displacement by other imported viruses. [fig] Figure 4. Change in genetic groups of measles viruses associated with U.S. cases and outbreaks between 1988 and September 1997. Arrows indicate sources of virus, if known. More aggressive childhood vaccination programs, the introduction of a two-dose schedule, and successful mass vaccination campaigns conducted by the Pan American Health Organization in South and Central America greatly reduced the number of reported measles cases in the United States in 1993 (Figure 1). During a 6-week period at the end of 1993, no indigenous cases of measles were reported (27). Molecular surveillance of measles viruses associated with cases and outbreaks in the United States during 1994, 1995, 1996, and 1997 documented this interruption of transmission of what had been the indigenous genotype (2,11). Only one group 2 virus was detected in the United States after 1993, and this was directly linked to importation from the Philippines (11). Molecular surveillance data allow us to draw several conclusions about the transmission of measles virus in the United States. The first is that increasing the level of population immunity by vaccination can interrupt the transmission of measles virus. This is hardly new information, and interruption of transmission was described for The Gambia in 1983 and more recently in Finland. However, our studies are the first in which genetic analysis of measles strains has been used to document interruption of transmission. Secondly, long-term asymptomatic transmission of virus is unlikely since no group 2 viruses were detected in the United States after 1993 that were not directly linked to importation. Finally, measles will not be fully controlled anywhere until it is controlled globally. Virus introduced by importation will continue to fuel sporadic outbreaks and epidemics even in areas with relatively good control measures. These observations should strengthen our resolve to accelerate measles control activities on a global level. The molecular data imply that under conditions of continuous indigenous transmission of measles virus, the number of circulating genotypes is limited. As population immunity increases, the pattern of genotypes becomes more complex to reflect the multiple sources of imported virus. We hope to test this model further by conducting molecular surveillance of wild-type measles viruses circulating in areas that still have endemic measles. Conclusions Genetic characterization of wild-type measles viruses provides a valuable means to measure the level of virus circulation in areas just beginning to implement measles control plans. In areas that already achieved good measles control, molecular epidemiologic studies provide a means to describe outbreaks and cases. Identifying the source of the virus can lead to improved control measures. To be maximally effective, molecular epidemiologic studies must include surveys of viral genetic groups from all areas of the world. Specimens for viral isolations should be obtained from as many chains of transmission as possible. Obtaining specimens must become an integral part of measles surveillance and be included in the standard operating procedures for investigating measles cases. If we can establish a large database to describe the indigenous genetic groups before large-scale control measures are enacted, we can closely monitor the ability of these control measures to reduce or interrupt transmission of measles. Molecular epidemiology will greatly enhance measles elimination and eradication efforts. Dr. Bellini is chief of the Measles Section, Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, CDC. Dr. Rota is a microbiologist in the Measles Section, CDC. The authors work closely with national and international public health agencies to support global measles eradication activities. They provide expertise and training on the genetic characterization of measles viruses and laboratory diagnosis of measles infections. Research interests include molecular virology, viral pathogenesis, and immune response to viral infections. References 1. Atkinson WL, Orenstein WA, Krugman S. The resurgence of measles in the United States, 1989-1990. Annu Rev Med 1992;43:451-63 2. Rota JS, Heath JL, Rota PA, King GE, Celma ML, Carabaña J, et al. Molecular epidemiology of measles virus: identification of pathways of transmission and implications for measles elimination. J Infect Dis 1996;173:32-7. 3. Kreiss S, Whistler T. Rapid identification of measles virus strains by the heteroduplex mobility assay. Virus Res 1997;47:197-203. 4. Outlaw MC, Jaye A, Whittle H, Pringle C. Clustering of hemagglutinin sequences of measles viruses isolated in the Gambia. Virus Res 197;48:125-31. 5. Outlaw MC, Pringle C. Sequence variation within an outbreak of measles virus in the Coventry area during spring/summer 1993. Virus Res 1995;39:3-11. 6. Rima BK, Earle JAP, Yeo RP, Herlihy L, Baczko K, ter Meulen V, et al. Temporal and geographical distribution of measles virus genotypes. J Gen Virol 1995;76:1173-80. 7. Rima BK, Earle JAP, Baczko K, ter Meulen V, Liebert U, Carstens C, et al. Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 1997;78:97-106. 8. Rima B, Earle JAP, Baczko K, Rota PA, Bellini WJ. Measles virus strain variations. Current Topics Microbiol Immunol 1995;191:65-84. 9. Rota PA, Bloom AE, Vanchiere JA, Bellini WJ. Evolution of the nucleoprotein and matrix genes of wild-type strains of measles virus isolated from recent epidemics. Virology 1994;198:724-30. 10. Rota PA, Rota JS, Bellini WJ. Molecular epidemiology of measles virus. Seminars in Virology 1995;6:379-86. 11. Rota JS, Rota PA, Redd SC, Pattamadilok S, Bellini WJ. Phylogenetic analysis of measles viruses isolated in the United States 1995-1996. J Infect Dis. In press 1997. 12. Saito H, Sato H, Abe M, Harata S, Amano K, Suto T, et al. Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain. Virus Genes 1994;8;2:107-13. 13. Sakata H, Kobune F, Sato TA, Tanabayashi K, Yamada A, Sugiura A. Variation in field isolates of measles virus during an 8-year period in Japan. Microbiol Immunol 1993;37:233-7. 14. Taylor MJ, Godfrey E, Baczko K, ter Meulen V, Wild TF, Rima BK. Identification of several different lineages of measles virus. J Gen Virol 1991;72:83-8 15. Devereaux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984;12:387-95. 16. Felsenstein J. Phylogenies from molecular sequences; inferences and reliability. American Review of Genetics 1988;22:512-65. 17. Swofford DL. PAUP: phylogenetic analysis using parsimony [computer program]. Version 3.1.1. Champaign (IL): Illinois Natural History Survey; 1991. 18. Rota JS, Wang ZD, Rota PA, Bellini WJ. Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 1994;31:317-30. 19. Xu WB, Tamin A, Rota JS, LiBi J, Bellini WJ, Rota PA. New genetic group of measles virus isolated in the People's Republic of China. Virus Res. In press 1998. 20. Heider A, Santibanez S, Tischer A, Gerike E, Tikhonova N, Ignatyev G, et al. Comparative investigation of the long non-coding M-F genome region of wild-type and vaccine measles viruses. Arch Virol. In press 1998. 21. Radecke F, Speilhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, et al. Rescue of measles virus from cloned DNA. EMBO J 1995;14:5773-84. 22. Guris D, Auerbach S, Vitek C, Maes E, McCready J, Durand M, et al. Measles outbreaks in Micronesia, 1991-1994. J Pediatr. In press 1997. 23. Katayama Y, Shibahara K, Kohama T, Homma M, Hotta H. Molecular epidemiology and changing distribution of genotypes of measles virus field strains in Japan. J Clin Microbiol 1997;35:2651-3. 24. Jin L, Brown DWG, Ramsay MEB, Rota PA, Bellini WJ. The diversity of measles virus in the UK, 1992-1995. J Gen Virol 1997;78:1287-94. 25. Kreis S, Whistler T. Rapid identification of measles virus strains by the heteroduplex mobility assay. Virus Res 1997;47:197-203. 26. Kreis S, Vardas E, Whistler T. Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype. J Gen Virol 1997;78:1581-7. 27. Centers for Disease Control and Prevention. Absence of reported measlesUnited States, November 1993. MMWR Morb Mortal Wkly Rep 1993;42(48):925-6. --------------------------------------------------------------------------- Synopses Diversity among Multidrug-Resistant Enterococci Barbara E. Murray, M.D. University of Texas Houston-Medical School, Houston, Texas, USA ------------------------------------------------------------------------ Enterococci are associated with both community- and hospital-acquired infections. Even though they do not cause severe systemic inflammatory responses, such as septic shock, enterococci present a therapeutic challenge because of their resistance to a vast array of antimicrobial drugs, including cell-wall active agents, all commercially available aminoglycosides, penicillin and ampicillin, and vancomycin. The combination of the latter two occurs disproportionately in strains resistant to many other antimicrobial drugs. The propensity of enterococci to acquire resistance may relate to their ability to participate in various forms of conjugation, which can result in the spread of genes as part of conjugative transposons, pheromone-responsive plasmids, or broad host-range plasmids. Enterococcal hardiness likely adds to resistance by facilitating survival in the environment of a multidrug-resistant clone, thus enhancing potential spread from person to person. The combination of these attributes within the genus Enterococcus suggests that these bacteria and their resistance to antimicrobial drugs will continue to pose a challenge. Enterococci, which have been known as a cause of infective endocarditis for close to a century, more recently have been recognized as a cause of nosocomial infection and "superinfection" in patients receiving antimicrobial agents (1). The enterococcus is now receiving increased attention because of its resistance to multiple antimicrobial drugs, which probably explains a large part of its prominence in nosocomial infections. The most common enterococci-associated nosocomial infections are infections of the urinary tract, followed by surgical wound infections and bacteremia (1-3). Enterococci are often present in intraabdominal and pelvic infections, although not all patients with such infections require specific antienterococcal therapy. Other enterococcal infections include infections (including meningitis and bacteremia) in very ill neonates; central nervous system infections in adults, typically with a history of central nervous system surgery or intrathecal chemotherapy; and rarely, osteomyelitis and pulmonary infections. Enterococci frequently arise from colonization of indwelling T tubes, causing liver or biliary infection in liver transplant patients (1). Antimicrobial Resistance Most enterococci have naturally occurring or inherent resistance to various drugs, including cephalosporins and the semisynthetic penicillinase-resistant penicillins (e.g., oxacillin) and clinically achievable concentrations of clindamycin and aminoglycosides. Compared with streptococci, most enterococci are relatively resistant to penicillin, ampicillin, and the ureidopenicillins, with MICs of 1 µg/ml to 8 µg/ml for most Enterococcus faecalis and even higher for most E. faecium. Many enterococci are also tolerant to the killing effects of cell-wall active agents, including ampicillin and vancomycin; recent data suggest that this property may not be inherent, but rather acquired after exposure to antibiotics (4). Inherent in vivo resistance of E. faecalis to trimethoprim-sulfamethoxazole may explain the lack of efficacy in animal models. In vitro, trimethoprim-sulfamethoxazole readily inhibits most enterococci at low concentrations, but this activity is lessened by exogenous folates (5). Moreover, bactericidal activity against E. faecalis seems unreliable and very method dependent (6). In animal models, this combination has not shown good activity and is not generally accepted as an effective antienterococcal therapy, especially for systemic infections (7,8). In addition to natural resistance to many agents, enterococci have also developed plasmid- and transposon-mediated resistance to tetracycline (as well as minocycline and doxycycline), erythromycin (plus the newer compounds azithromycin and clarithromycin), chloramphenicol, high levels of trimethoprim, and high levels of clindamycin. The propensity of E. faecalis to acquire multiple antibiotic-resistance traits may result from a variety of distinctly different mechanisms for conjugation, i.e., bacterial mating. The best studied system of conjugation involves oligopeptides called pheromones and pheromone-responsive plasmids (9;Figure 1). Briefly, strains of E. faecalis typically secrete into the culture medium a number of different small peptide sex pheromones specific for different types of plasmids. When a cell containing a pheromone-responsive plasmid (the potential donor cell) comes into contact with its corresponding pheromone, transcription of a gene on the plasmid is turned on, resulting in the synthesis of a sticky substance (called aggregation substance) on its surface. When the donor cell bumps into another E. faecalis, aggregation substance, which contains two Arg-Gly-Asp motifs, sticks to the binding substance on the surface of most E. faecalis cells, causing them to clump together. In the test tube, clumps of cells actually fall to the bottom of the tube, resulting in a visible aggregate. By a process not yet well understood, the pheromone-responsive plasmid can then transfer from the donor bacterium to the other (recipient) bacterium. Once the recipient cell has acquired this particular plasmid, the synthesis of the corresponding sex pheromone is shut off to prevent self-clumping. This system of conjugation, which occurs primarily in E. faecalis, is highly efficient and results in transfer of plasmids in both filter and broth matings. Another system of conjugation, also not well understood, involves broad host-range plasmids that can transfer among species of enterococci and other gram-positive organisms such as streptococci and staphylococci (10). The transfer frequency is generally much lower than with the pheromone system and is much more efficient with filter than with broth matings. Since staphylococci, streptococci, and enterococci share a number of resistance genes, these broad host-range plasmids may be a mechanism by which some of these resistance genes have spread among different genera. [Fig] Figure 1. Enterococcus faecalis pheromone-responsive conjugative system. Pheromone A released from the potential recipient cell (right) interacts with plasmid A in the potential donor cell (left) to induce synthesis of aggregation substance. Attachment of aggregation substance to binding substance causes the cells to clump into visible aggregates. Once the pheromone-responsive plasmid A has transferred from donor to recipient cell, synthesis of pheromone A is shut off. A third type of conjugation, which involves conjugative transposons, may also explain the spread of resistance genes to many different species (11). As opposed to ordinary transposons, which can jump within a cell from one DNA location to another, conjugative transposons also encode the ability to bring about conjugation between different bacterial cells. Since plasmids typically require rather complex machinery for replication (often depending on successful interactions with host proteins) and must face additional problems of surface exclusion and incompatibility, conjugative transposons (which do not replicate, but instead insert into the chromosome or into a plasmid of the new host) appear to be an even more efficient and far-reaching way of disseminating a resistance gene. This may explain why the tetM gene of the conjugative transposon Tn916 has spread beyond the gram-positive species into gram-negative organisms, including gonococci, meningococci, and Haemophilus ducreyi, as well as into mycoplasma and ureaplasma, among others (12,13). Other resistance genes, including those encoding resistance to erythromycin and kanamycin, are also found on conjugative transposons; these frequently contain or are related to Tn916. Such transposons may have evolved from a Tn916 ancestor; their emergence suggests the possibility of further dissemination of resistance among gram-positive organisms. Particularly ominous are reports of the vanB gene cluster within large conjugative chromosomal elements that appear similar, at least in function, to conjugative transposons (14). High-Level Aminoglycoside Resistance Although some acquired resistance of enterococci is not clinically important because the agents involved are not commonly used, other resistance greatly affects enterococcal therapy; high-level resistance (HLR) to aminoglycosides is an example. This resistance is added onto the normal low-level resistance of enterococci to aminoglycosides and typically results in MICs of > 2,000 >/= [mu]g/ml. This degree of resistance predicts, without exception, resistance to synergism between cell-wall active agents and the aminoglycoside to which the organism is highly resistant (1). High-level aminoglycoside resistance is most often due to aminoglycoside-modifying enzymes; HLR to streptomycin can also be ribosomal, that is, due to a mutation that results in ribosomes resistant to streptomycin inhibition. HLR to kanamycin (without gentamicin) is a fairly common trait and is due to the production of a 3'-phosphotransferase, APH(3')-III. This enzyme is important because it also eliminates synergism between cell-wall active agents and amikacin (through phosphorylation of the 3'-hydroxyl group), although it does not necessarily confer HLR to amikacin. HLR to gentamicin results from the bifunctional protein (AAC(6')-I/APH(2")-I), encoded by a single gene with two active sites, one with 6'-acetyltransferase activity and the other, 2"-phosphotransferase activity (15). The combination of these activities results in HLR or resistance to synergism for all commercially available aminoglycosides except streptomycin, which is not modified by this enzyme. However, HLR to streptomycin (due to either ribosomal resistance or a streptomycin adenylyltransferase) is also common and can coexist with the gene(s) for HLR to other aminoglycosides. Spectinomycin is also not modified by the bifunctional enzyme, but this agent, which is not a true aminoglycoside, is not generally bactericidal against enterococci and does not appear to show synergism with cell-wall active agents. Strains of enterococci from patients with endocarditis and other serious infections for whom combination therapy is desired should be screened for HLR to streptomycin and gentamicin. HLR screening for tobramycin is not generally performed or advisable It could, in principle, be used for E. faecalis, but E. faecium isolates have a chromosomally encoded, naturally occurring gene for a 6'-acetyltransferase that eliminates synergism with tobramycin, although it does not cause HLR (MICs are typically 128-500 g/ml); a probe for this gene has been used to confirm the identification of E. faecium isolates to species. In addition, HLR of an E. faecium isolate (without HLR to gentamicin) to tobramycin, due to an adenylyltransferase, was recently described (16). Therefore, the use of tobramycin for possible synergism in serious enterococcal infections would need to be preceded by screening for HLR to tobramycin (a test that is not commonly available), as well as for identification to species, neither of which is practical. More recently, veterinary and human isolates resistant to moderate levels of gentamicin (256 µg/ml) were found to have a new gentamicin-modifying enzyme encoded by a gene designated aph(2")-Ic (17). This gene conferred resistance to synergism between gentamicin and cell-wall active agents and may be less easily detected than strains producing the bifunctional enzyme. Beta-Lactamase-- and non-Beta--Lactamase--Associated Penicillin Resistance The first known penicillinase-producing isolate of enterococcus was an isolate of E. faecalis recovered from a patient in Houston, Texas, in 1981 (18). Although rare, these isolates have been reported from the United States (Texas, Florida, North Carolina, Delaware, Pennsylvania, New York, Massachusetts, and Connecticut), Lebanon, Canada, and Argentina (19). Like other enterococci, beta-lactamase-producing strains have been found as colonizers, as in the large "outbreak" of colonization in a Boston children's hospital, but they have also been associated with true infections, as was demonstrated by cases at a Virginia Veterans Administration hospital, by isolates from Argentina, and in other reports (20,21). The enterococcal penicillinase gene, identical to the gene encoding staphylococcal type A penicillinase, almost always occurs in strains with HLR to gentamicin and is often found on a transferable plasmid that also contains aph(2")-Ia/aac(6')-Ie . The relatively low levels of beta-lactamase produced by enterococci result in a marked inoculum effect with these strains so that at low and even moderate inocula (10[sup]3-10[sup]5 CFU/ml), penicillinase-producing enterococci usually appear no more resistant than other enterococci, while at high inocula (> 10[sup 7] CFU/ml), these organisms are usually highly resistant to penicillin, ampicillin, and ureidopenicillins. The activity of the penicillinase is reversed by the beta-lactamase inhibitors clavulanate, sulbactam, and tazobactam; in animal models of endocarditis, beta-lactamase inhibitors have been shown to markedly enhance the therapeutic efficacy of ampicillin or penicillin. In the clinical laboratory, penicillinase-producing enterococci are generally not detected by routine laboratory susceptibility testing, such as MICs or disk diffusion. For this reason, if a penicillin is to be used for therapy, enterococcal isolates from patients with endocarditis or other serious infections should be tested for penicillinase production by using a specific beta-lactamase test such as the chromogenic cephalosporin nitrocefin. Nonpenicillinase-producing, penicillin-resistant enterococci have been reported for decades and usually are E. faecium. Until recently, MICs of penicillin typically ranged from 8 µg/ml to 64 µg/ml, with an occasional isolate having higher levels of resistance. However, increasingly, strains with much higher levels of penicillin resistance have been reported (22). Whether a large number of strains have converted from low-level to high-level resistance or a more limited number of strains have been disseminated is unclear. The mechanisms involved in this resistance are overproduction of a low-affinity penicillin-binding protein (a cell-wall synthesis enzyme) and a further decrease in the affinity of one of these enzymes for penicillin (23). As a possible explanation for why many vancomycin-resistant E. faecium also have very high levels of resistance to ampicillin, Rice and colleagues (24) showed that transfer of vancomycin resistance from one strain to another was linked to transfer of ampicillin resistance. Vancomycin Resistance Most surprising in recent years has been the emergence among enterococci of acquired resistance to vancomycin. Vancomycin had been in clinical use since the 1950s, although it was not heavily used until the late 1970s and particularly the 1980s. Because multiple genes are involved in generating vancomycin resistance, the development of resistance was neither easy nor recent. Three phenotypes of vancomycin resistance (types A, B, and C) are now well described; a fourth, type D, has been recently reported (25). VanA-type strains are typically highly resistant to vancomycin and moderately to highly resistant to teicoplanin. This phenotype is often plasmid or transposon mediated and is inducible (i.e., exposure of bacteria to vancomycin results in the induction of the synthesis of several proteins that together confer resistance) (26). In vancomycin-susceptible enterococci, D-alanyl-D-alanine (formed by an endogenous D-alanine-D-alanine ligase) is added to a tripeptide precursor to form a pentapeptide precursor. The D-Ala-D-Ala terminus is the target of vancomycin; once vancomycin has bound, the use of this pentapeptide precursor for further cell-wall synthesis is prevented. In the VanA phenotype, one of the proteins whose synthesis is induced by exposure of bacterial cells to vancomycin is called VanA; VanA is a ligase and resembles the D-alanine-D-alanine ligase from Escherichia coli and other organisms, including vancomycin-susceptible enterococci (27). VanA generates D-Ala-D-X, where X is usually lactate; the formation of D-lactate is due to the presence of VanH, a dehydrogenase encoded by vanH. The depsipeptide moiety, D-Ala-D-Lac, is then added to a tripeptide precursor, resulting in a depsipentapeptide precursor. Vancomycin does not bind to the D-Ala-D-Lac terminus, so this depsipentapeptide can be used in the remaining steps of cell-wall synthesis. However, when the normal pentapeptide precursor ending in D-Ala-D-Ala is also present, cells are not fully vancomycin resistant, despite the presence of D-Ala-D-Lac containing precursors. This apparent problem is taken care of in large part by vanX, which encodes a dipeptidase, VanX, that cleaves D-Ala-D-Ala, preventing its addition to the tripeptide precursor. Should any D-Ala-D-Ala escape cleavage and result in a normal pentapeptide precursor, vanY encodes an ancillary or back-up function. That is, it codes for a carboxypeptidase, VanY, which cleaves D-alanine and D-lactate from D-Ala-D-Ala and D-Ala-D-Lac termini, respectively, resulting in tetrapeptide precursors, to which vancomycin does not bind. The other genes involved in the VanA resistance complex include vanR and vanS, whose encoded proteins are involved in somehow sensing the presence of extracellular vancomycin or its effect and signaling intracellularly to activate transcription of vanH, vanA, and vanX (27). A final gene in the vanA cluster is vanZ, which encodes VanZ, the role of which is not known. VanB, encoded by vanB in the vanB gene cluster, is also a ligase that stimulates the formation of D-Ala-D-Lac. The VanB phenotype is typically associated with moderate to high levels of vancomycin resistance but is without resistance to teicoplanin. This is explained by the observation that vancomycin, but not teicoplanin, can induce the synthesis of VanB and of VanH[sub beta] and VanX[sub beta]. However, because mutants resistant to teicoplanin can readily be selected from VanB strains on teicoplanin-containing agar, clinical resistance would likely occur among VanB strains if teicoplanin were widely used. Most of the proteins encoded by the vanA gene cluster have homologues encoded by the vanB gene cluster, except for VanZ. The vanB gene cluster has an additional gene, vanW, of unknown function. The VanC phenotype (low-level resistance to vancomycin, susceptible to teicoplanin) is an inherent (naturally occurring) property of E. gallinarum and E. casseliflavus. This property is not transferable and is related to the presence of species-specific genes vanC-1 and vanC-2, respectively (28); a third possible species, E. flavescens and its gene vanC-3, are so closely related to E. casseliflavus and vanC-2 that different names are probably not warranted (29). These species appear to have two ligases; the cell-wall pentapeptide, at least in E. gallinarum, ends in a mix of D-Ala-D-Ala and D-Ala-D-Ser (29,30). The genes vanC-1 and vanC-2 apparently lead to the formation of D-Ala-D-Ser containing cell-wall precursors, while D-Ala-D-Ala ligases, also present in these organisms, result in D-Ala-D-Ala. The presence of both D-Ala-D-Ala and D-Ala-D-Ser precursors may explain why many isolates of these species test susceptible to vancomycin and why even those isolates with decreased susceptibility display only low-level resistance. VanD-type glycopeptide resistance has been recently described in an E. faecium isolate from the United States (25). The organism was constitutively resistant to vancomycin (MIC > 64 µg/ml) and to low levels (4 µg/ml) of teicoplanin. Following polymerase chain reaction amplification with primers that amplify many D-Ala-D-Ala ligases, a 605-bp fragment was identified whose deduced amino acid sequence showed 69% identity to VanA and VanB and 43% identify to VanC. Molecular Epidemiology of Newer Resistance Traits High-Level Gentamicin Resistance The DNA sequence of the gene encoding HLR to gentamicin in E. faecalis is the same as the sequence of the gentamicin resistance gene of staphylococci (15). Since this gene was well established in staphylococci by the 1970s but HLR to gentamicin was not reported in enterococci until 1979, the seemingly obvious conclusion is that this gene spread from staphylococci to enterococci rather than vice versa or, at least, staphylococci acquired it first. However, the disk diffusion method used in the 1970s and microtiter dilution MICs done later are capable of detecting gentamicin resistance in staphylococci but do not distinguish enterococci with high-level gentamicin resistance from those with low-level, inherent resistance. Therefore, since laboratories were not screening enterococci by special techniques for high-level gentamicin resistance, it cannot be definitively stated that this resistance did not appear in enterococci earlier or coincident with its emergence in staphylococci. However, several observations support the likelihood that gentamicin resistance appeared and disseminated in staphylococci before it did in enterococci. In 1971, Moellering et al. reported the lack of high-level gentamicin resistance among enterococci (31). Watanakunakorn reported the absence of high-level gentamicin resistance among 126 enterococci from 1980 to 1984, with HLR subsequently appearing in 1985 (32). Phillips et al. from the United Kingdom reported no highly gentamicin-resistant strains in 1969 to 1979 or 1980 to 1985 and appearance of strains in 1986 (33). Zervos et al. reported that only one (0.04%) of 269 isolates of E. faecalis had high-level gentamicin resistance in 1981; this figure gradually increased to 7.7% in 1984 (34). High-level gentamicin resistance in E. faecium appears to have occurred after its appearance in E. faecalis, with the first report occurring in 1988 (35). Delineation of the molecular epidemiology of strains of enterococci was limited in the past by the lack of an easy, reliable, and widely accessible method for subspecies strain differentiation. Zervos and Schaberg reported the use of plasmid patterns in enterococci to suggest the intrahospital spread of strains with high-level gentamicin resistance. Pulsed-field gel electrophoresis (PFGE) of E. faecalis with HLR to gentamicin found that different isolates from both the same and different locations had markedly different restriction endonuclease digestion patterns. That is, it found no evidence of a common strain or strains that predominated among gentamicin-resistant organisms (36). Strains isolated between 1981 and 1984 at the University of Michigan demonstrated that plasmids encoding high-level gentamicin resistance were heterogeneous, which again argues against clonal dissemination of a limited number of strains or plasmids to account for spread of this property (34,37). We have subsequently shown that gentamicin resistance in enterococci can be encoded on a transposon identical to that in staphylococci (38). In addition, the enterococcal gentamicin-resistance gene has been found in other genetic settings, one of which has also been found in North American Staphylococcus aureus with gentamicin resistance (39). Since all enterococci with HLR to gentamicin (MIC >/= 2,000 µg/ml) that have been tested have hybridized with the same gene probe, this property could be termed a "gene epidemic." However, by the time gentamicin resistance was discovered in enterococci, this gene was already widespread with no evidence of either a common plasmid or a common or predominant strain. Other genetic elements encoding HLR to gentamicin have also been described. Thal et al. have described a 27-kb element designated Tn924 that encodes HLR to gentamicin and could be mobilized from the chromosome of an E. faecalis by a coresident plasmid (40). Rice et al. have described a large (ca. 60 kb) transferable element, tentatively named Tn5385, which appears to contain within it an 18-kb conjugative transposon (Tn5381) encoding tetracycline resistance and a 26-kb IS256-based composite transposon (Tn5384) encoding resistance to gentamicin and to erythromycin (41). Penicillin-Resistant Penicillinase-Producing Enterococci PFGE analyses of penicillinase-producing enterococci have shown that a common penicillinase-producing strain (or "clone"), defined as having an identical or related chromosomal digestion pattern, was present in Texas, Florida, North Carolina (unpub. observation), Delaware, Pennsylvania, and Virginia, which had a large outbreak with numerous infections (42,43). Moreover, at each of these locations, all isolates of penicillinase-producing E. faecalis examined were derivatives of this strain; in the hospital in which this strain has been endemic for many years, a single penicillinase-producing isolate of E. faecium, the only such isolate ever reported, was also found (44). Among numerous non-Bla+ E. faecalis, a PFGE pattern similar to that of Bla+ E. faecalis has been found only once from an isolate from a Philadelphia hospital that had one of the Bla+ isolates (43 and unpub. obs.). Penicillinase-producing isolates from Connecticut, Boston, Canada (unpub. observations), Lebanon, and Argentina represent different strains (19). However, clonal relatedness of three isolates with an almost identical pattern was demonstrated in Connecticut (despite one of these lacking HLR to gentamicin) (45), and all six isolates in Argentina appeared to be a single strain. The Boston isolates have not been studied by PFGE, but a number of isolates from the same hospital had a single shared plasmid, suggesting that these also represent clonal dissemination of a single strain. While all of these penicillinase-producing E. faecalis could be detected by nitrocefin, a vancomycin-resistant E. faecalis that tested negative for penicillinase by nitrocefin but showed a marked inoculum effect with penicillin and destroyed penicillin in a bioassay has been reported (46). Thus, in contrast to high-level gentamicin resistance in enterococci, which appeared to be widely disseminated on different plasmids and in different strains by the time this phenotype was studied, penicillinase production in enterococci is still largely associated with a limited number of strains; moreover, in locations known to have more than one isolate, oligoclonal spread within each setting remains the rule. Nonpenicillinase­Producing Penicillin-Resistant Strains PFGE analyses of highly penicillin-resistant E. faecium from the Medical Colleges of Virginia and Pennsylvania have shown that within each location, most highly resistant strains represented a single clone (47). Analysis of the PFGE patterns also raised the possibility, on the basis of similarities between patterns of isolates in these two different locations, that a strain may have spread from one institution to the other (47); this conclusion was supported by the finding that these isolates belonged to the same multilocus enzyme cluster (unpub. observations). Circumstantial evidence of intrahospital spread of highly penicillin-resistant E. faecium also comes from the study of Grayson et al. (22), who reported a sudden increase in more highly penicillin-resistant isolates of E. faecium at the Massachusetts General Hospital in 1988. Although no genetic analysis was done, the fact that gentamicin resistance also simultaneously appeared and most of the E. faecium highly resistant to penicillin were also highly resistant to gentamicin suggests clonal dissemination of one or a few strains within that hospital (22). Vancomycin Resistance Vancomycin resistance in enterococci is heterogeneous on many levels. For example, three different, well-described types of vancomycin resistance are known, each associated with different ligase genes, (vanA, vanB, vanC1, and vanC2), and a fourth type, VanD, has been reported recently (25). VanA and VanB type resistance is encoded by gene clusters that are acquired (i.e., not part of the normal genome of enterococci) and are often transferable. In contrast, vanC1 and vanC2 are normally occurring genes that are endogenous species characteristics of E. gallinarum and E. casseliflavus, respectively, and are not transferable. The acquired gene clusters associated with vanA and vanB are found in different genetic surroundings. The vanA gene cluster has been found in a small Tn3-like transposon, Tn1546, and in elements that appear to be closely related (e.g., Tn5488, which has an insertion sequence [IS1251] within Tn1546 [48,49]) or lacking vanZ (50). These elements have in turn been found on both transferable and nontransferable plasmids, as well as on the chromosome of the host strain. VanB type resistance was initially not found to be transferable, but at least in some instances, the vanB gene cluster has been found on large (90 kb to 250 kb) chromosomally located transferable elements, one of which contains within it a 64-kb composite transposon (Tn1547) ((Figure 2;14). More recently, vanB has been found as part of plasmids. In addition to being found in different genetic surroundings, the vanA and vanB gene clusters have also been found in a number of different bacterial species. vanA has been found in multiple enterococcal species as well as in lactococci, Orskovia, and Arcanobacteria (51). The distribution of the vanB gene cluster seems somewhat more restricted, having been found primarily in E. faecium and E. faecalis, although it has recently been found in Streptococcus bovis (51). [Fig] Figure 2. Potential modes of spread of vancomycin-resistant genes. Adapted in part from Quintiliani and Courvalin (14). The vanB gene cluster (shown on the left) on a 64-kb transposon is part of a 250-kb mobile element shown to move from the chromosome of one enterococcus and insert into the chromosome of another. Although not demonstrated, circularization of the vanB containing large mobile elements resembles the mechanism described for conjugative transposons that can excise from the chromosome of one strain, circularize, transfer from one enterococcus to another, and reinsert into the chromosome of the recipient (such as the one on the right). The 64-kb transposon shown on the left can also jump to another plasmid within the host enterococcus. If it is a conjugative (Tra+) plasmid, that plasmid can then transfer by conjugation to other bacteria, taking the van resistance genes with it. In one instance, the vancomycin resistance transposon was shown to transpose to a plasmid encoding the virulence factor hemolysin (Hly). When vancomycin-resistant enterococci (VRE) from patients in a given hospital have been examined, particularly after the first recovery of VRE, evidence is often found of a single or predominant strain (53-58). Finding isolates with identical or highly related PFGE patterns in different hospitals indicates interhospital clonal transmission (59-61). Some reports do not find a single or a predominant strain, especially when VRE have been present in a hospital or area for some time (62-63). This was also true of two reports from France in which all of 16 and all of 24 vancomycin-resistant E. faecium were different (50,64,65). The reports from France likely reflect another observation regarding the diversity of vancomycin resistance. vanA has also been shown to be present in normal fecal enterococci of healthy, nonhospitalized persons in different parts of Europe (68); in one study, 20 different strains (identified by PFGE) of vancomycin-resistant E. faecium were found in the fecal flora of 17 persons in two areas in Belgium (68). vanA containing E. faecium have also been found in the feces of healthy animals as well as from animal products in Europe (50,67,69-71); in one study, VRE were found in the feces of healthy meat-eaters but not vegetarians (72). VRE have not, however, been found as part of the normal fecal flora in the United States (73) possibly because glycopeptides, often used in animal feed in Europe, are not used in the United States. For example, 24,000 kg annually of the glycopeptide avoparcin were reportedly used in recent years in Denmark (74). Reports of VRE in the feces of animals on farms using glycopeptides, but not in those without such use, support this hypothesis (71,75). While oral glycopeptide use markedly increases the numbers of VRE per gram of stool in humans (76) and by analogy, presumably does so in animals, glycopeptide use does not explain the origin of these gene clusters. The problem of multidrug-resistant enterococci promises to be with us for the foreseeable future. The enterococcus has likely emerged as a major nosocomial pathogen in part because of its resistance to multiple antibiotics, which allows it to survive and subsequently infect patients. With its propensity to acquire new traits, such as high-level gentamicin, penicillin, and vancomycin resistance, the enterococcus continues to create new therapeutic problems and dilemmas; its ability to transfer some of its plasmids to streptococci and staphylococci and the implications of a possible spread of penicillin and vancomycin resistance to these and other gram-positive species are also of concern. Dr. Murray is professor and director, Division of Infectious Diseases, and codirector, Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Houston-Medical School. Her main interests are in the areas of antibiotic resistance, molecular epidemiology, and bacterial pathogenesis. Recent work has been focused on possible mechanisms of virulence of enterococci. Address for correspondence: Barbara E. Murray, Division of Infectious Diseases, University of Texas Houston-Medical School, 6431 Fannin, 1.728 JFB, Houston, TX 77030, USA; fax: 713-500-6761; e-mail: iminfdis@heart.med.uth.tmc.edu. References 1. Murray BE. The life and times of the enterococcus. Clin Microbiol Rev 1990;3:46-65. 2. Moellering RC Jr. Emergence of enterococcus as a significant pathogen. Clin Infect Dis 1992;14:1173-8. 3. Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbial etiology of nosocomial infection. Am J Med 1991;91(Suppl 3B):72S-75S. 4. Hodges TL, Zighelboim-Daum S, Eliopoulos GM, Wennersten C, Moellering RC Jr. Antimicrobial susceptibility changes in Enterococcus faecalis following various penicillin exposure regimens. Antimicrob Agents Chemother 1992;36:121-5. 5. Zervos MJ, Schaberg DS. Reversal of the in vitro susceptibility of enterococci to trimethoprim-sulfamethoxazole by folinic acid. Antimicrob Agents Chemother 1985;28:446-8. 6. Najjar A, Murray BE. 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Arthur M, Molinas C, Bugg TDH, Wright GD, Walsh CT, Courvalin P. Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci. Antimicrob Agents Chemother 1992;36:867-9. 28. Dutka-Malen S, Molinas C, Arthur M, Courvalin P. Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance. Gene 1992;112:53-8. 29. Navarro F, Courvalin P. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrob Agents Chemother 1994;38:1788-93. 30. Billot-Klein D, Gutmann L, Sable S, Guittet E, van Heijenoort J. Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J Bacteriol 1994;176:2398-405. 31. Moellering RC Jr, Wennersten C, Medrek T, Weinberg AN. 1971. Prevalence of high-level resistance to aminoglycosides in clinical isolates of enterococci. Antimicrobial agents and chemotherapy--1970. Washington: American Society for Microbiology; 1971. p. 335-40. 32. Watanakunakorn C. The prevalence of high-level aminoglycoside resistance among enterococci isolated from blood cultures during 1980-1988. J Antimicrob Chemother 1989;24:63-8. 33. Phillips I, King A, Gransden WR, Eykyn SJ. The antibiotic sensitivity of bacteria isolated from the blood of patients in St. Thomas' Hospital, 1969-1988. J Antimicrob Chemother 1990;25:59-80. 34. Zervos MJ, Dembinski S, Mikesell T, Schaberg DR. High-level resistance to gentamicin in Streptococcus faecalis: risk factors and evidence for exogenous acquisition of infection. J Infect Dis 1986;153:1075-83. 35. Eliopoulos GM, Wennersten C, Zighelboim-Daum S, Reiszner E, Goldmann D, Moellering RC Jr. High-level resistance to gentamicin in clinical isolates of Streptococcus (Enterococcus) faecium. Antimicrob Agents Chemother 1988;32:1528-32. 36. Murray BE, Singh KV, Heath JD, Sharma BR, Weinstock GM. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J Clin Microbiol 1990;28:2059-63. 37. Zervos MJ, Mikesell TS, Schaberg DR. Heterogeneity of plasmids determining high-level resistance to gentamicin in clinical isolates of Streptococcus faecalis. Antimicrob Agents Chemother 1986;30:78-81. 38. Hodel-Christian SL, Murray BE. Characterization of the gentamicin resistance transposon Tn5281 from Enterococcus faecalis and comparison to staphylococcal transposons Tn4001 and Tn4031. Antimicrob Agents Chemother 1991;35:1147-52. 39. Hodel-Christian SL, Smith M, Zscheck KZ, Murray BE. 1991. Comparison of a gentamicin resistance transposon and a beta-lactamase gene from enterococci to those from staphylococci. In: Dunny GM, Cleary PP, McKay LL, editors. Genetics and molecular biology of streptococci, lactococci, and enterococci. Washington: American Society for Microbiology; 1991. p. 54-8. 40. Thal LA, Chow JW, Clewell DB, Zervos MJ. Tn924, a chromosome-borne transposon encoding high-level gentamicin resistance in Enterococcus faecalis. Antimicrob Agents Chemother 1994;38:1152-6. 41. Rice LB, Carias LL, Marshall SH, Bonafede ME. 1996. Sequences found on staphylococcal ß-lactamase plasmids integrated into the chromosome of Enterococcus faecalis CH116. Plasmid 1996;35:81-90. 42. Seetulsingh PS, Tomayko JF, Coudron PE, Markowitz SM, Skinner C, Singh KV, Murray BE. Chromosomal DNA restriction endonuclease digestion patterns of ß-lactamase-producing Enterococcus faecalis isolates collected from a single hospital over a 7-year period. J Clin Microbiol 1996;34:1892-6. 43. Tomayko JF, Murray BE. Analysis of Enterococcus faecalis isolates from intercontinental sources by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis. J Clin Microbiol 1995;33:2903-7. 44. Coudron PE, Markowitz SM, Wong ES. Isolation of a ß-lactamase-producing, aminoglycoside-resistant strain of Enterococcus faecium. Antimicrob Agents Chemother 1992;36:1125-6.44 45. Patterson JE, Singh KV, Murray BE. Epidemiology of an endemic strain of ß-lactamase-producing Enterococcus faecalis. J Clin Microbiol 1991;29:2513-6.45 46. Handwerger S, Perlman DC, Altarac D, McAuliffe V. Concomitant high-level vancomycin and penicillin resistance in clinical isolates of enterococci. Clin Infect Dis 1992;14:655-61.46 47. Miranda AG, Singh KV, Murray BE. DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool. J Clin Microbiol 1991;29:2752-7.47 48. Handwerger S, Skoble J. Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1995;39:2446-53. 49. Handwerger S, Skoble J, Discotto LF, Pucci MJ. Heterogeneity of the vanA gene cluster in clinical isolates of enterococci from the Northeastern United States. Antimicrob Agents Chemother 1995;39:362-8. 50. van den Bogaard AE, Jensen LB, Stobberingh EE. Vancomycin-resistant enterococci in turkeys and farmers. N Engl J Med 1997;337:1558-9. 51. Power EGM, Abdulla YH, Talsania HG, Spice W, Aathithan S, French GL. vanA genes in vancomycin-resistant clinical isolates of Oerskovia turbata and Arcanobacterium (Corynebacterium) haemolyticum. J Antimicrob Chemother 1995;36:595-606. 52. Poyart C, Pierre C, Quesne G, Pron B, Berche P, Trieu-Cuot P. Emergence of vancomycin resistance in the genus Streptococcus: characterization of a vanB transferable determinant in Streptococcus bovis. Antimicrob Agents Chemother 1997;41:24-9. 53. Biavasco F, Miele A, Vignaroli C, Manso E, Lupid R, Varaldo PE. Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis. Microb Drug Resist 1996;2:231-7. 54. Boyce JM, Opal SM, Chow JW, Zervos MJ, Potter-Bynoe G, Sherman CB, et al. Outbreak of multidrug-resistant Enterococcus faecium with transferable vanB class vancomycin resistance. J Clin Microbiol 1994;32:1148-53. 55. Handwerger S, Raucher B, Altarac D, Monka J, Marchione S, Singh KV, et al. Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin and gentamicin. Clin Infect Dis 1993;16:750-5. 56. Livornese LL Jr, Dias S, Samel C, Romanowski B, Taylor S, May P, et al. Hospital-acquired infection with vancomycin-resistant Enterococcus faecium transmitted by electronic thermometers. Ann Intern Med 1992;117:112-6. 57. Moreno F, Grota P, Crisp C, Magnon K, Melcher GP, Jorgensen JH, Patterson JE. Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium during its emergence in a city in southern Texas. Clin Infect Dis 1995;21:1234-7. 58. Perlada DE, Smulian AG, Cushion MT. Molecular epidemiology and antibiotic susceptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey. J Clin Microbiol 1997;35:2342-7. 59. Chow JW, Kuritza A, Shlaes DM, Green M, Sahm DF, Zervos MJ. Clonal spread of vancomycin-resistant Enterococcus faecium between patients in three hosptials in two states. J Clin Microbiol 1993;31:1609-11. 60. Clark NC, Cooksey RC, Hill BC, Swenson JM, Tenover FC. Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Antimicrob Agents Chemother 1993;37:2311-7. 61. Sader HS, Pfaller MA, Tenover FC, Hollis RJ, Jones RN. Evaluation and characterization of multiresistant Enterococcus faecium from 12 U.S. medical centers. J Clin Microbiol 1994;32:2840-2.61 62. Boyle JF, Soumakis SA, Rendo A, Herrington JA, Gianarkis DG, Thurberg BE, Painter BG. Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycin-resistant enterococci. J Clin Microbiol 1993;31:1280-5. 63. Morris JG Jr, Shay DK, Hebden JN, McCarter RJ Jr, Perdue BE, Jarvis W, et al. Enterococci resistant to multiple antimicrobial agents, including vancomycin. Ann Intern Med 1995;123:250-9. 64. Bingen EH, Denamur E, Lambert-Zechovsky NY, Elion J. Evidence for the genetic unrelatedness of nosocomial vancomycin-resistant Enterococcus faecium strains in a pediatric hospital. J Clin Microbiol 1991;29:1888-92. 65. Plessis P, Lamy T, Donnio PY, Autuly F, Grulois I, LePrise PY, Avril JL. Epidemiologic analysis of glycopeptide-resistant Enterococcus strains in neutropenic patients receiving prolonged vancomycin administration. Eur J Clin Microbiol Infect Dis 1995;14:959-63. 66. Jordens JZ, Bates J, Griffiths DT. Faecal carriage and nosocomial spread of vancomycin-resistant Enterococcus faecium. J Antimicrob Chemother 1994;34:515-28. 67. Klare I, Heier H, Claus H, Bohme G, Marin S, Seltmann G, et al. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microbial Drug Resist 1995;3:265-72. 68. Van der Auwera P, Pensart N, Korten V, Murray BE, Leclercq R. Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly-glycopeptide-resistant enterococci. J Infect Dis 1995;173:1129-36. 69. Aarestrup FM, Ahrens P, Madsen M, Pallesen LV, Poulsen RL, Westh H. Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster. Antimicrob Agents Chemother 1996;40:1938-40. 70. Bates J, Jordens Z, Selkon JB. Evidence for an animal origin of vancomycin-resistant enterococci [letter]. Lancet 1993;342:490. 71. Klare I, Heier H, Claus H, Reissbrodt R, Witte W. vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol Lett 1995;125:165-72. 72. Schouten MA, Voss A, Hoogkamp-Korstanje JAA. VRE and meat. Lancet 1997;349:1258. 73. Coque TM, Tomayko JF, Ricke SC, Okhuysen PO, Murray BE. Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States. Antimicrob Agents Chemother 1996;40:2605-9. 74. Kjerulf A, Pallesen L, Westh H. Vancomycin-resistant enterococci at a large university hospital in Denmark. APMIS 1996;104:475-9. 75. Klare I, Heier H, Claus H, Witte W. Environmental strains of Enterococcus faecium with inducible high-level resistance to glycopeptides. FEMS Microbiol Lett 1993;106:23-30. ------------------------------------------------------------------------ Synopses Proteases of Malaria Parasites: New Targets for Chemotherapy Philip J. Rosenthal San Francisco General Hospital and University of California, San Francisco, California, USA --------------------------------------------------------------------------- The increasing resistance of malaria parasites to antimalarial drugs is a major contributor to the reemergence of the disease as a major public health problem and its spread in new locations and populations. Among potential targets for new modes of chemotherapy are malarial proteases, which appear to mediate processes within the erythrocytic malarial life cycle, including the rupture and invasion of infected erythrocytes and the degradation of hemoglobin by trophozoites. Cysteine and aspartic protease inhibitors are now under study as potential antimalarials. Lead compounds have blocked in vitro parasite development at nanomolar concentrations and cured malaria-infected mice. This review discusses available antimalarial agents and summarizes experimental results that support development of protease inhibitors as antimalarial drugs. Hundreds of millions of cases of malaria occur annually, and infections with _Plasmodium falciparum_, the most virulent human malaria parasite, cause more than one million deaths per year (1). Despite extensive control efforts, the incidence of the disease is not decreasing in most malaria-endemic areas of the world, and in some it is clearly increasing (2). Malaria also remains a major risk to travelers from industrialized to developing countries. Because malaria parasites are increasingly resistant to antimalarial drugs, appropriately counseled travelers to malaria-endemic regions are more likely to contract malaria now than they were 40 years ago. Malaria control efforts include attempts to develop an effective vaccine, eradicate mosquito vectors, and develop new drugs (2,3). However, the development of a vaccine has proven very difficult, and a highly effective vaccine will probably not be available in the near future (4). Efforts to control _Anopheles_ mosquitoes have had limited success, although the use of insecticide-impregnated bed nets does appear to reduce malaria-related death rates (5). In addition, methods to replace natural vector populations with mosquitoes unable to support parasite development are under study and may contribute to malaria control in the long term (6). However, the current limitations of vaccine and vector control, as well as the increasing resistance of malaria parasites to existing drugs, highlight the continued need for new antimalarial agents. Established Antimalarial Drugs Antimalarial drugs have been used for centuries. Early natural products, including the bark of the cinchona tree in South America and extracts of the wormwood plant in China, were among the first effective antimicrobial agents to be used. Cinchona bark was used in Europe beginning in the 17th century, and upon its isolation from bark in 1820, quinine became widely used. In the last 50 years, extensive efforts, including the screening of hundreds of thousands of compounds, have led to the development of a number of effective synthetic antimalarial drugs. The most important of these, chloroquine, has been the mainstay of antimalarial chemotherapy for the last 50 years. The compound eradicates parasites rapidly, has minimal toxicity, is widely available at low cost throughout the world, and needs to be taken only once a week for chemoprophylaxis. However, resistance to chloroquine has been steadily increasing since the drug's initial use in South America and Southeast Asia in the late 1950s. Chloroquine resistance is now widespread in most _P. falciparum_ -endemic areas of the world (3). Thus, the use of chloroquine for presumptive treatment of falciparum malaria or for chemoprophylaxis is usually no longer appropriate (7). Moreover, resistance to chloroquine of _P. vivax_, the second most lethal human malaria parasite, is increasing in South Asia (8). No other antimalarial drug (9-12) is as efficacious and safe as chloroquine (Table 1). The best antimalarial drug for treating chloroquine-resistant falciparum malaria remains quinine (or intravenous quinidine), which is fairly toxic; quinine resistance is increasing in Southeast Asia, particularly in the border areas of Thailand (9). Amodiaquine, used to treat chloroquine-resistant malaria in developing countries, is also quite toxic, and resistance to it is also common (13). Mefloquine (14) is widely used for chemoprophylaxis against chloroquine-resistant _P. falciparum_, but its use is limited by toxicity (15) and (in the developing world) high cost. Mefloquine is not approved for treatment of malaria in the United States because of the neurotoxicity of doses required for the treatment. Fansidar, a combination of sulfadoxine and pyrimethamine, is no longer recommended for chemoprophylaxis because of its dermatologic toxicity (15). Fansidar is also not an ideal drug for treatment because it is slow acting, but it is increasingly important in treating chloroquine-resistant malaria in developing countries because economic constraints limit the use of other agents (16). The use of both mefloquine and Fansidar will increasingly be limited by drug resistance, already widespread in parts of Southeast Asia (9,17). Table 1. Established antimalarial drugs (sup a) --------------------------------------------------------------------------- Drug Role Best Feature(s) Limitations --------------------------------------------------------------------------- Chloroquine TX of and CP Very safe; low Widespread R against non- Pf cost; long and sensitive Pf half-life parasites Quinine/quinidine Best TX for Pf Limited R; Fairly toxic malaria; low cost rapidly acting (cinchonism, cardiac) Amodiaquine (sup TX of R Pf malaria Low cost Toxicity (bone b) marrow, liver); R common Mefloquine CP against R Pf Relatively little Moderately toxic malaria; not R, though (mostly CNS); approved for TX in increasing; long high cost; R in United States half-life SE Asia Fansidar TX of Pf malaria; Relatively low Skin toxicity (can no longer cost; long be fatal); recommended for CP half-life increasing R Primaquine Eradication of Only drug for Hemolysis with chronic liver this indication G6PD deficiency; stage Pv, Po increasing R malaria Proguanil (sup b) CP only (often Low cost; R common with chloroquine) nontoxic Maloprim (sup b) CP only (often Low cost R common; skin with chloroquine) rashes Tetracyclines CP; TX of Pf Low cost Skin and malaria in gastrointestinal combination with toxicity quinine --------------------------------------------------------------------------- (sup a) TX, therapy; CP, chemoprophylaxis; R, resistance/resistant; Pf, _Plasmodium falciparum_; Pv, _P. vivax_; Po, _P. ovale_; CNS, central nervous system; G6PD, glucose 6-phosphate dehydrogenase. (sup b) Not available in the United States. --------------------------------------------------------------------------- Other antimalarial drugs have specialized uses. Tetracyclines and some other antibiotics (clindamycin, sulfas) are slow acting and generally best used as an adjunct to quinine therapy in treating falciparum malaria (9). Doxycycline is also used for chemoprophylaxis in regions with high levels of drug resistance, especially Southeast Asia (10,17). Other drugs for chemoprophylaxis include proguanil, which remains effective in combination with chloroquine in many areas other than Southeast Asia, and Maloprim, a combination of dapsone and pyrimethamine (10,17). Resistance to these drugs is fairly common, however. Primaquine has a well-defined specific role: eradicating chronic liver stages of _P. vivax_ and _P. ovale_ after treating the acute blood infection with chloroquine. New Antimalarial Drugs Relatively few new antimalarial drugs are undergoing clinical testing (Table 2). Halofantrine, identified in the 1940s, was not developed until the 1980s; its use has been limited by variable oral absorption and cardiac toxicity (12,18). The drug is approved in the United States for treatment of chloroquine-resistant _P. falciparum_ infection, although in most cases quinine (or intravenous quinidine) is preferable. The most effective new drugs are artemisinin and related compounds. Artemisinin was isolated in 1972 from _Artemisia annua_, a plant used in China for centuries to treat fever (19). Artemisinin derivatives (artesunate, artelinate, artemether, arteether, dihydroartemisinin) have been synthesized and are undergoing extensive clinical testing. These compounds, which are already widely used in some areas, are potent, rapidly acting antimalarials that are effective against chloroquine-resistant _P. falciparum_ (20). Because recrudescences of infection after treatment are common, however, artemisinin and related compounds might best be used in combination with another drug. Table 2. New antimalarial drugs --------------------------------------------------------------------------- Drug Role Best Feature(s) Limitations -------------------------------------------------------------------------- Halofantrine TX of Pf malaria; Usually effective Variable not approved for against R Pf bioavailability, CP malaria cardiac toxicity Artemisinin and TX of Pf malaria Rapidly acting; Recurrence after related compounds effective against TX fairly common (sup a) multidrug-R strains Atovaquone ? TX of Pf Limited toxicity Limited studies so malaria; ? CP far show frequent (probably in recurrence after combination with TX proguanil) Pyronaridine (sup ? TX of Pf Effective against Studies limited to a) malaria R strains date Desferrioxamine ? TX of severe Pf Well tolerated Studies limited to malaria when used for date iron overload Azithromycin ? CP Limited toxicity Studies limited to date -------------------------------------------------------------------------- For abbreviations, see Table 1, footnote a. (sup a) Not available in the United States. Other compounds are under evaluation. Atovaqone (21), which is approved for treating patients with _Pneumocystis_ infections, appears to be effective against malaria in combination with proguanil (22), but its use has been limited by recrudescence after treatment. Pyronaridine, an acridine derivative used to treat malaria in China, has shown efficacy against falciparum malaria (23). The iron chelator desferrioxamine enhances the clearance of parasites in mild malaria (24) and, in conjunction with quinine and Fansidar, hastens recovery from deep coma in severe falciparum malaria (25). Azithromycin, a quinolone antibiotic, appears efficacious in malaria chemoprophylaxis (26). Malarial Proteases: New Targets for Chemotherapy The limitations of antimalarial chemotherapy underscore the need for new drugs, ideally directed against new targets. Potential targets for chemotherapy include malarial proteases (27). The erythrocytic life cycle, which is responsible for all clinical manifestations of malaria, begins when free merozoites invade erythrocytes. The intraerythrocytic parasites develop from small ring-stage organisms to larger, more metabolically active trophozoites and then to multinucleated schizonts. The erythrocytic cycle is completed when mature schizonts rupture erythrocytes, releasing numerous invasive merozoites. Proteases appear to be required for the rupture and subsequent reinvasion of erythrocytes by merozoite-stage parasites and for the degradation of hemoglobin by intraerythrocytic trophozoites figure. [fig] Figure. Protease targets in erythrocytic malaria parasites. The _Plasmodium falciparum_ erythrocytic life cycle is shown schematically, and data supporting cysteine (CP), serine (SP), and aspartic (AP) proteases of the different parasite stages as chemotherapeutic targets are provided in italics. Proteases and Erythrocyte Rupture and Invasion The rupture of erythrocytes by mature schizonts and the subsequent invasion of erythrocytes by free merozoites appear to require malarial protease activity, possibly to breach the erythrocyte cytoskeleton, a complex network of proteins. In addition, a number of malarial proteins are proteolytically processed during the late schizont and merozoite life-cycle stages; for example, merozoite surface protein-1 is processed in a manner inhibited by serine protease inhibitors (28), presumably to facilitate the complex series of events involved in erythrocyte rupture and invasion (29). Although the specific roles of different classes of proteases are not completely clear, inhibitors of cysteine and serine proteases have consistently blocked erythrocyte rupture and invasion (27). Candidate _P. falciparum_ rupture/invasion proteases have been identified, but none has been fully characterized biochemically or molecularly: 1) a 68 kD cysteine protease was identified in schizonts and merozoites and localized to the merozoite apex, suggesting that it may be released from the rhoptry organelle during invasion (30); 2) a cysteine protease of mature schizonts and a serine protease of merozoites were identified in highly synchronized parasites (31); 3) a serine protease was shown to be bound to the schizont/merozoite membrane by a glycosyl-phosphatidylinositol anchor, to be activated by phosphatidylinositol-specific phospholipase C during the merozoite stage, and to be capable of cleaving the erythrocyte cytoskeletal protein band 3 (32,33); 4) another protease, inhibited by both cysteine and serine protease inhibitors, hydrolyzed the erythrocyte cytoskeletal proteins spectrin and band 4.1 (34); and 5) the serine repeat antigen (35,36) and the related protein SERP H (37), both expressed in mature schizonts, have important similarities in their sequences with cysteine proteases. Further research should identify the specific biologic roles of the proteases mentioned and better characterize these enzymes, thus fostering the development of specific inhibitors. Host proteases may also play a role in erythrocyte rupture by _P. falciparum_. In recent studies, host urokinase was shown to bind to the surface of _P. falciparum_-infected erythrocytes, and the depletion of urokinase from parasite culture medium inhibited erythrocyte rupture by mature schizonts (38). This inhibition was reversed by exogenous urokinase. Drug Development Efforts Synthetic peptide inhibitors of the _P. falciparum_ schizont cysteine protease Pf 68 inhibited erythrocyte invasion by cultured parasites (39,40). The most effective peptide, GlcA-Val -Leu-Gly-Lys-NHC (sub 2)H(sub 5), inhibited the protease and blocked parasite development at high micromolar concentrations (40; Table 3). Although these results do not demonstrate levels of inhibition expected to be therapeutically relevant, they suggest that a specific protease activity is required for erythrocyte invasion by malaria parasites and thus is a potential target for antimalarial drugs. Proteases and Malarial Hemoglobin Degradation Extensive evidence suggests that the degradation of hemoglobin is necessary for the growth of erythrocytic malaria parasites, apparently to provide free amino acids for parasite protein synthesis (27,50). In _P. falciparum_, hemoglobin degradation occurs predominantly in trophozoites and early schizonts, the stages at which the parasites are most metabolically active. Trophozoites ingest erythrocyte cytoplasm and transport it to a large central food vacuole. In the food vacuole, hemoglobin is broken down into heme, a major component of malarial pigment (51), and globin, which is hydrolyzed to its constituent amino acids. The food vacuole is an acidic organelle analogous to lysosomes. Several lysosomal proteases are well characterized, including cysteine (cathepsins B, H, and L) and aspartic (cathepsin D) proteases (52), and malaria parasites contain analogous food vacuole proteases that degrade hemoglobin. At least two aspartic proteases and one cysteine protease have been isolated from purified _P. falciparum_ food vacuoles (53). Malarial aspartic protease activities have been identified (54-60). Two recently characterized aspartic proteases (plasmepsin I and plasmepsin II) are located in the food vacuole, have acid pH optima, and share sequence homology with other aspartic proteases (41,53,61,62). Furthermore, the aspartic proteases can cleave hemoglobin. One of the enzymes, plasmepsin I, cleaves native hemoglobin (53,59). Plasmepsin II appears to prefer denatured globin as a substrate (53). On the basis of these data, plasmepsin I is thought to be responsible for initial cleavages of hemoglobin after the molecule is transported to the food vacuole (53). Incubation of cultured _P. falciparum_ parasites with the protease inhibitor leupeptin caused trophozoite food vacuoles to fill with apparently undegraded erythrocyte cytoplasm (63-65). Analysis of the leupeptin-treated parasites showed that they contained large quantities of undegraded globin, while minimal globin was detectable in control parasites (64,66). Leupeptin inhibits both cysteine and some serine proteases, but the highly specific cysteine protease inhibitor E-64 also caused undegraded globin to accumulate. After parasites were incubated with inhibitors of other classes of proteases including the aspartic protease inhibitor pepstatin (63-67), globin did not accumulate. More recent studies that used nondenaturing electrophoretic methods demonstrated that cysteine protease inhibitors not only blocked malarial globin hydrolysis, but also inhibited earlier steps in hemoglobin degradation, including denaturation of the hemoglobin tetramer and the release of heme from globin (68). Another study showed that E-64, but not pepstatin, inhibited the production of hemozoin (the malarial end product of heme) by cultured parasites (69). These results suggest that a cysteine protease is required for initial steps in hemoglobin degradation by _P. falciparum_. A _P. falciparum trophozoite cysteine protease with biochemical features expected for a food vacuole hemoglobinase has been identified (31) and biochemically (70-72) and molecularly (73) characterized. This protease, called falcipain, degraded denatured and native hemoglobin in vitro; its acid pH optimum, substrate specificity, and inhibitor sensitivity indicated that it was a papain family cysteine protease (64,70,71). Specific inhibitors of falcipain blocked hemoglobin degradation and prevented parasite development. The degree of inhibition of falcipain by fluoromethyl ketones (44) and vinyl sulfones (46) correlated with their inhibition of hemoglobin degradation and parasite development, supporting the hypothesis that falcipain is the cysteine protease required for hemoglobin degradation. The specific mechanism for hemoglobin degradation in the malarial food vacuole remains unclear. As noted above, both the aspartic protease plasmepsin I and the cysteine protease falcipain have been identified in parasite food vacuoles and shown to cleave denatured and native hemoglobin in vitro (53,71). Results showing that only cysteine protease inhibitors block hemoglobin processing and globin hydrolysis in cultured parasites suggest that falcipain is required for initial steps of hemoglobin degradation (66-68,74). However, other studies have shown that native hemoglobin is cleaved by plasmepsin I, but not falcipain, in nonreducing conditions that may be present in the food vacuole (53,59,72). In any event, regardless of the exact sequence of hemoglobin processing, multiple enzymes, including at least the three proteases already identified, appear to participate in the degradation of hemoglobin. These proteases are thus logical targets for antimalarial drug development. Aminopeptidase activity has also been described in malaria parasites (75-77). This activity, with a neutral pH optimum, was not found in food vacuole lysates (77). When these lysates were incubated with hemoglobin, discrete peptide fragments, but not free amino acids, were identified (77). These results suggest that hemoglobin is degraded to small peptides in the food vacuole, that these peptides are transported to the parasite cytosol, and that additional processing of hemoglobin peptides is mediated by cytosolic aminopeptidase activity (77). Drug Development Efforts Both the cysteine protease inhibitor E-64 and the aspartic protease inhibitor pepstatin blocked _P. falciparum_ development (63-67). Administered together, the two inhibitors acted synergistically (67). However, only E-64 blocked globin hydrolysis (64-67). Numerous peptide-based cysteine protease inhibitors, including fluoromethyl ketones (44,70,78) and vinyl sulfones (46), inhibited falcipain at low nanomolar concentrations and inhibited _P. falciparum_ development and hemoglobin degradation at concentrations below 100 nanomolar (Table 3). In a malaria animal model, a fluoromethyl ketone that inhibited falcipain at low nanomolar concentrations blocked _P. vinckei_ protease activity in vivo after a single subcutaneous dose, and, when administered for 4 days, cured 80% of murine malaria infections (45). Thus, despite the theoretical limitations of potentially rapid degradation in vivo and inhibition of host proteases, peptide protease inhibitors show promise as candidate antimalarial drugs. Fluoromethyl ketones have subsequently shown toxicity in animal studies, but evaluations of related, apparently nontoxic inhibitors of falcipain as antimalarial drugs are under way. Table 3. Protease targets for chemotherapy --------------------------------------------------------------------------- Effective inhibitors (sup a) ------------------------------------------------------------------------ Protease Biologic Compound (Reference) In vitro (sup In vivo (sup role b) c) (mg/kg/day) (IC(sub 50); (Microgram M) ------------------------------------------------------------------------------------------------- Pf68 Erythrocyte GlcA-Val-Leu-Gly-Lys-NHC(sub 900 invasion 2)H(sub 5) (40) Plasmepsin Hemoglobin SC-50083 (41) 2-5 I degradation 0.25 Ro 40-4388 (42) Plasmepsin Hemoglobin Compound 7 (43) 20 II degradation Falcipain Hemoglobin Z-Phe-Arg-CH2F (44) 0.064 degradation Mu-Phe-HPh-CH2F (45) ~0.03 400 Mu-Leu-HPh-VSPh (46) 0.01 Oxalic bis ((2-hydroxy-1-naph- 7 thylmethylene)hydrazide) (47) 1-(2,5-dichlorophenyl)-3- 0.23 (4-quinolinyl)-2-propen-1-one (48) 7-chloro-1,2-dihydro-2-(2,3-di- 2 methoxy-phenyl)-5,5-dioxide- 4-(1H,10H)-phenothiazinone (49) ------------------------------------------------------------------------------------------------- (sup a) The structures of these compounds and details of the described studies are in the references noted. (sup b) Assays compared the development of new ring-form parasites or the uptake of [ (sup 3) H]hypoxanthine by treated and control parasites. (sup c) Cure of _Plasmodium vinckei_-infected mice. A computer model for the structure of falcipain was used to identify nonpeptide inhibitors (47). Screening of potential nonpeptide inhibitors identified a low micromolar lead compound (47; Table 3). Subsequent synthesis and testing of small molecules based on the structure of the lead compound have identified biologically active falcipain inhibitors, including chalcones that block parasite metabolism at submicromolar concentrations (48) and phenothiazines that block parasite metabolism and development at low micromolar concentrations (49). Peptidelike aspartic protease inhibitors are potent inhibitors of plasmepsins I and II. In independent studies SC-50083 (41), Ro 40-4388 (42), and "compound 7" (43) inhibited plasmepsin I or II at nanomolar concentrations and blocked parasite development at high nanomolar to micromolar concentrations (Table 3). Drug development efforts should be assisted by the recent determination of the structure of plasmepsin II (43). Inhibitors of aspartic and cysteine proteases have synergistic effects in inhibiting the growth of cultured malaria parasites (67), and these proteases also act synergistically to degrade hemoglobin in vitro (41). Therefore, the combination of inhibitors of malarial cysteine and aspartic proteases may provide the most effective chemotherapeutic regimen and best limit the development of parasite resistance to protease inhibitors. Ultimately, a better understanding of the biochemical properties and biologic roles of malarial proteases will foster the development of protease inhibitors that specifically inhibit parasite enzymes and thus are the most suitable candidates for chemotherapy. Acknowledgments Work in the author's laboratory was supported by grants from the National Institutes of Health, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the American Heart Association. Dr. Rosenthal is associate professor, Department of Medicine, Division of Infectious Diseases, San Francisco General Hospital and the University of California, San Francisco. His research interests include the evaluation of proteases of malaria parasites as chemotherapeutic targets. Address for correspondence: Philip Rosenthal, Box 0811, University of California, San Francisco, CA 94143-0811, USA; fax: 415-206-6015; e-mail: rosnthl@itsa.ucsf.edu. References 1. Walsh JA. 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FEBS Lett 1990;259:257-9. --------------------------------------------------------------------------- Synopses Zoonotic Tuberculosis due to Mycobacterium bovis in Developing Countries O. Cosivi,* J.M. Grange,† C.J. Daborn,‡ M.C. Raviglione,* T. Fujikura,¶ D. Cousins,§ R.A. Robinson,** H.F.A.K. Huchzermeyer,†† I. de Kantor,‡‡ and F.-X. Meslin* *World Health Organization, Geneva, Switzerland; †Imperial College School of Medicine, National Heart and Lung Institute, London, United Kingdom; ‡Edinburgh University, Edinburgh, Scotland; ¶The University of Zambia, Lusaka, Zambia; §Department of Agriculture, South Perth, Australia; **College of Veterinary Medicine, Veterinary Teaching Hospital, --------------------------------------------------------------------------- The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals. Tuberculosis (TB), one of the most widespread infectious diseases, is the leading cause of death due to a single infectious agent among adults in the world. Mycobacterium tuberculosis is the most common cause of human TB, but an unknown proportion of cases are due to M. bovis (1). In industrialized countries, animal TB control and elimination programs, together with milk pasteurization, have drastically reduced the incidence of disease caused by M. bovis in both cattle and humans. In developing countries, however, animal TB is widely distributed, control measures are not applied or are applied sporadically, and pasteurization is rarely practiced. The direct correlation between M. bovis infection in cattle and disease in the human population has been well documented in industrialized countries. Whereas little information is available from developing countries (2,3), risk factors for M. bovis in both animals and humans are present in the tropics. TB is a major opportunistic infection in HIV-infected persons (4). The vast majority of people carrying this dual infection live in developing countries; however, dual HIV and M. bovis infection has been reported in industrialized countries (5-11). The epidemic of HIV infection in developing countries, particularly countries in which M. bovis infection is present in animals and the conditions favor zoonotic transmission, could make zoonotic TB a serious public health threat to persons at risk (3,12-14). We summarize available epidemiologic information on TB and zoonotic TB, examine risk factors that can influence the occurrence of zoonotic TB in developing countries, and describe the most recent TB activities of the World Health Organization (WHO) (15-18). Human TB: Global Situation and Trends The global incidence of TB is greatly underestimated. In 1995, 3.3 million cases were reported to the Global Tuberculosis Programme of WHO, whereas a more likely number is 8.8 million. Of the reported cases, 62% occurred in the Southeast Asian and Western Pacific regions, 16% in sub-Saharan Africa, and 7% to 8% in each of the regions of the Americas, Eastern Mediterranean, and Europe. Many countries, especially those with few resources, are unable to report all TB cases because of difficulties in identifying suspected cases, establishing a diagnosis, and recording and reporting cases. In 1995, an estimated 8.8 million new TB cases occurred-5.5 million (62%) in the Southeast Asian and Western Pacific regions and 1.5 million (17%) in sub-Saharan Africa. The annual global incidence is predicted to increase to 10.2 million by the year 2000, an increase of 36% from 1990. Southeast Asia, Western Pacific regions, and sub-Saharan Africa will account for 81% of these new cases (Table 1). For 1990 to 1999, in the absence of effective control, global TB incidence and deaths will reach 88 million and 30 million, respectively (19); 70% of the new cases will occur in patients 15 to 59 years of age, the most economically productive segment of the population. As a result of the HIV epidemic, the crude incidence rate of TB is expected to increase in sub-Saharan Africa from 191 cases per 100,000 in 1990 to 293 in 2000. However, the total number of new cases will double by the year 2000. Because of the HIV epidemic, the decline of the crude incidence rate in the Southeast Asian and Central and South American regions is expected to be slower than in previous years. In industrialized countries, a small increase in crude incidence rate and total cases is expected as the result of immigration from countries with a high prevalence of dual HIV and TB infection. The worldwide incidence of HIV-attributable TB cases is estimated to increase from 315,000 (4% of the total TB cases) in 1990 to 1.4 million (14% of the total TB cases) by the year 2000. In 2000, approximately 40% of these HIV-attributable cases will occur in sub-Saharan Africa and 40% in Southeast Asia. Ten percent of the total number of TB cases expected during 1990 to 1999 are estimated to be attributable to HIV infection. While demographic factors, such as population growth and changes in population structure, will largely account for the expected increase in TB incidence worldwide, the HIV epidemic in sub-Saharan Africa will have a greater role than demographic factors. By the year 2000, 3.5 million persons will be dying of TB annually, an increase of 39% from 1990. In Southeast Asia alone, 1.4 million deaths will occur annually. During 1990 to 1999, an estimated 30 million will die of TB, with 9.7% of the cases attributable to HIV infection. M. tuberculosis will be largely responsible for the new TB cases and deaths, but an unknown, and potentially important, proportion will be caused by M. bovis. Table 1. Estimated human tuberculosis and HIV-attributable tuberculosis cases in 1990, 1995, and 2000, by region (19) 1990 1995 2000 --------------------------- ------------------------- -------------------------- TB HIV- TB HIV- TB HIV- Region cases Rate(supa) attributed cases Rate attributed cases Rate attribut ----------------------------------------------------------------------------------------------- Southeast Asia 3,106,000 237 66,000 3,499,000 241 251,000 3,952,000 247 571,000 Western Pacific (supb) 1,839,000 136 19,000 22,045,000 140 31,000 2,255,000 144 68,000 Africa 992,000 191 194,000 1,467,000 242 380,000 2,079,000 293 604,000 Eastern Mediter- ranean 641,000 165 9,000 745,000 168 16,000 870,000 168 38,000 Americas(supc)569,000 127 20,000 606,000 123 45,000 645,000 120 97,000 Eastern Europed 194,000 47 1,000 202,000 47 2,000 210,000 48 6,000 Industr- ialized countries (supe) 196,000 23 6,000 204,000 23 13,000 211,000 24 26,000 Total TB cases 7,537,000 143 315,000 8,768,000 152 738,000 10,222,000 163 1,410,000 Attributed to HIV 4.2% 8.4% 13.8% Increase since 1990 16.3% 35.6% ------------------------------------------------------------------------------------------------- (supa)Rate: incidence of new cases per 100,000 population. (supb)Western Pacific Region of WHO except Japan, Australia, and New Zealand. (supc)American Region of WHO, except USA and Canada. (supd)Eastern European countries and independent states of the former USSR. (supe)Western Europe, USA, Canada, Japan, Australia, and New Zealand. Bovine TB in Developing Countries Although prevalence data on animal TB in developing countries are generally scarce, information on bovine TB occurrence and control measures exists (20,21). Africa Of 55 African countries, 25 reported sporadic/low occurrence of bovine TB; six reported enzootic disease; two, Malawi and Mali, were described as having a high occurrence; four did not report the disease; and the remaining 18 countries did not have data (Figure 1). Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all (Figure 2). Almost 15% of the cattle population are found in countries where bovine TB is notifiable and a test-and-slaughter policy is used. Thus, approximately 85% of the cattle and 82% of the human population of Africa are in areas where bovine TB is either partly controlled or not controlled at all. Asia Of 36 Asian nations, 16 reported a sporadic/low occurrence of bovine TB, and one (Bahrain) described the disease as enzootic; ten did not report bovine TB; and the remaining nine did not have data (Figure 3). Within the Asian region, seven countries apply disease control measures as part of a test-and-slaughter policy and consider bovine TB notifiable. In the remaining 29 countries, bovine TB is partly controlled or not controlled at all (Figure 4). Of the total Asian cattle and buffalo populations, 6% and less than 1%, respectively, are found in countries where bovine TB is notifiable and a test-and-slaughter policy is used; 94% of the cattle and more than 99% of the buffalo populations in Asia are either only partly controlled for bovine TB or not controlled at all. Thus, 94% of the human population lives in countries where cattle and buffaloes undergo no control or only limited control for bovine TB. Latin American and Caribbean Countries Of 34 Latin American and Caribbean countries, 12 reported bovine TB as sporadic/low occurrence, seven reported it as enzootic, and one (Dominican Republic) described occurrence as high. Twelve countries did not report bovine TB. No data were available for the remaining two countries (Figure 5). In the entire region, 12 countries apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease. In the remaining 22 nations, the disease is partly controlled or not controlled at all (Figure 6). The regional prevalence of bovine TB has been estimated at 1% and higher in 67% of the total cattle population and 0.1% to 0.9% in a further 7%; the remaining 26% are free of the disease or are approaching the point of elimination (22). Of the total Latin American and Caribbean cattle population, almost 76% is in countries where bovine TB is notifiable and a test-and-slaughter policy is used. Thus, approximately 24% of the cattle population in this region is either only partly controlled for bovine TB or not controlled at all. It is also estimated that 60% of the human population live in countries where cattle undergo no control or only limited control for bovine TB. [Fig] [Fig] Figure 1. Bovine tuberculosis Figure 2. Control measures for occurrence, Africa (21). bovine tuberculosis based on test-and-slaughter policy and disease notification, Africa (21). [Fig] [Fig] Figure 3. Bovine tuberculosis Figure 4. Control measures for bovine occurrence, Asia (21). test-and-slaughter tuberculosis based on policy and disease notification, Asia (21). [Fig] [Fig] Figure 5. Bovine tuberculosis Figure 6. Control measures for occurrence, Latin America and bovine tuberculosis based on the Caribbean (21). test-and-slaughter policy and disease notification, Latin America and the Caribbean (21). Zoonotic TB in Humans TB caused by M. bovis is clinically indistinguishable from TB caused by M. tuberculosis. In countries where bovine TB is uncontrolled, most human cases occur in young persons and result from drinking or handling contaminated milk; cervical lymphadenopathy, intestinal lesions, chronic skin TB (lupus vulgaris), and other nonpulmonary forms are particularly common. Such cases may, however, also be caused by M. tuberculosis. Little is known of the relative frequency with which M. bovis causes nonpulmonary TB in developing nations because of limited laboratory facilities for the culture and typing of tubercle bacilli. Agricultural workers may acquire the disease by inhaling cough spray from infected cattle; they develop typical pulmonary TB. Such patients may infect cattle, but evidence for human-to-human transmission is limited and anecdotal. In regions where bovine TB has been largely eliminated, a few residual cases occur among elderly persons as a result of the reactivation of dormant lesions. These are fewer than 1% of all TB cases. Surveys in the United States, Scandinavia, and South England have shown that approximately half of these postprimary cases are pulmonary, a quarter involve the genitourinary tract (a rare occurrence in primary disease), and the remainder involve other nonpulmonary sites, notably cervical lymph nodes (23). In the same regions, approximately 10% of cases caused by M. tuberculosis are nonpulmonary, although, for reasons that are not clear, the incidence is higher, approximately 20%, in ethnic minority populations. Information on human disease due to M. bovis in developed and developing countries is scarce. From a review of a number of zoonotic tuberculosis studies, published between 1954 and 1970 and carried out in various countries around the world, it was estimated that the proportion of human cases due to M. bovis acccounted for 3.1% of all forms of tuberculosis: 2.1% of pulmonary forms and 9.4% of extrapulmonary forms (24). Table 2 summarizes the findings of more recent reports of TB caused by M. bovis in industrialized countries. Table 2. Human tuberculosis due to Mycobacterium bovis, industrialized countries Cases ------------------------------------ % of Pulmonary total (% of total Country(ref.) Years No. TB M. bovis) -------------------------------------------------------- Australia(25) 1970-94 240 0.43-3.1 71.69(supa) England (23) 1977-90 232 1.2 40.0 Germany (26) 1975-80 236 4.5 73.7 Ireland Rural (27) 1986-90 17 6.4 70.6 Urban (28) 1982-85 9 0.9 88.8 New Zealand(29) 1983-90 22 7.2 31.8 Spain (30) 1986-90 10 0.9 50.0 Sweden (17) 1983-92 96 2.0 - Switzerland(31) 1994 18 2.6 - U.S.(32) 1954-68 6 0.3 33.3 U.S. (9) 1980-91 73 3.0 52.0(supb) 12.0(supc) --------------------------------------------------------- (supa) Overall percentage includes 80.6 % males and 51.2% females (supb)Adults. (supc)Children Human disease caused by M. bovis has been confirmed in African countries. In an investigation by two Egyptian health centers, the proportions of sputum-positive TB patients infected with M. bovis, recorded during three observations, were 0.4%, 6.4%, and 5.4% (33). In another study in Egypt, nine of 20 randomly selected patients with TB peritonitis were infected with M. bovis, and the remaining with M. tuberculosis (34). Isolation of M. bovis from sputum samples of patients with pulmonary TB has also been reported from Nigeria. Of 102 M. tuberculosis complex isolates, 4 (3.9%) were M. bovis (35). Another study in Nigeria reported that one of 10 mycobacteria isolated from sputum-positive cultures was M. bovis (36). In a Zaire study, M. bovis was isolated from gastric secretions in two of five patients with pulmonary TB (37). In the same study, the prevalence of the disease in local cattle was approximately 8% by tuberculin testing and isolation of M. bovis. In a recent investigation in Tanzania, seven of 19 lymph node biopsies from suspected extrapulmonary TB patients were infected with M. tuberculosis and four with M. bovis (14). No mycobacteria were cultured from the remaining eight (Table 3). Although the number of samples was low, the high proportion (36%) of M. bovis isolates is of serious concern. Table 3. Isolates from suspected extrapulmonary tuberculosis patients, Tanzania, 1994 (14) No. of M. tuber- M. Occupation samples culosis bovis Neg ------------------------------------------------- Livestock 4 0 2 2 keeper Farmers 6 2 1 3 Children 3 2 1 0 Unknown 6 3 0 3 Total 19 7 4 8 ------------------------------------------------- In an epidemiologic study in Zambia (38), an association between tuberculin-positive cattle and human TB was found. Households that reported a TB case within the previous 12 months were approximately seven times more likely to own herds containing tuberculin-positive cattle (odds ratio = 7.6; p = 0.004). Although this could be explained by zoonotic TB transmission, other factors such as transient sensitivity to tuberculin of cattle exposed to TB patients and coincidental environmental factors favoring both human clinical TB and sensitivity to bovine tuberculin should also be considered. In Latin America, a conservative estimate would be that 2% of the total pulmonary TB cases and 8% of extrapulmonary TB cases are caused by M. bovis. These cases would therefore account for 7,000 new TB cases per year, a rate of nearly 2 per 100,000 inhabitants. From a nationwide study in Argentina during 1982 to 1984, 36 (0.47%) of 7,672 mycobacteria cultured from sputum samples were M. bovis (39). However, in another study in Santa Fe province (where most of the dairy cattle industry is concentrated) during 1984 to 1989, M. bovis caused 0.7% to 6.2% of TB cases (40). Very limited data on the zoonotic aspects of M. bovis are available from Asian countries. However, cases of TB caused by M. bovis were not reported in early investigations in India (41). Epidemiology Much information on the epidemiologic patterns of zoonotic TB has been obtained in this century from industrialized countries. However, some striking epidemiologic differences related to both animal and human populations in developing countries require particular attention. Risk Factors: Animal Population Animal reservoirs. The widespread distribution of M. bovis in farm and wild animal populations represents a large reservoir of this microorganism. The spread of the infection from affected to susceptible animals in both industrialized and developing countries is most likely to occur when wild and domesticated animals share pasture or territory (42). Well-documented examples of such spread include infection in badgers (Meles meles) in the United Kingdom and possums (Trichosurus vulpecula) in New Zealand. Wild animal TB represents a permanent reservoir of infection and poses a serious threat to control and elimination programs. Milk production and animal husbandry. Milk production has increased in most developing countries as a consequence of greater demand for milk for human consumption (43; Figure 7). This increased demand for milkestimated at 2.5% per year for 1970 to 1988 for sub-Saharan Africa (44) led to increases in the number of productive animals and milk imports and intensification of animal production through the introduction of more productive exotic breeds. Although the prevalence of the disease within a country varies from area to area, the highest incidence of bovine TB is generally observed where intensive dairy production is most common, notably in the milksheds of larger cities (1). This problem is exacerbated where there is inadequate veterinary supervision, as is the case in most developing countries. In addition, in some industrialized countries such as the United States, where bovine TB is close to elimination, large dairy herds (i.e., 5,000 or more cows) that are crowded together represent the main source of infection (45). [Fig] Figure 7. Cow milk production by region (43). In developing countries, bovine TB infects a higher proportion of exotic dairy breeds (Bos taurus) than indigenous zebu cattle (Bos indicus) and crossbred beef cattle (1). However, under intensive feedlot conditions, a death rate of 60% and depression of growth have been found in tuberculous zebu cattle (46). In those areas where extensive management is more common, animal crowding (e.g., near watering ponds, dip tanks, markets, and corrals) still plays a major role in the spread of the disease. Control measures and programs. The basic strategies required for control and elimination of bovine TB are well known and well defined (47). However, because of financial constraints, scarcity of trained professionals, lack of political will, as well as the underestimation of the importance of zoonotic TB in both the animal and public health sectors by national governments and donor agencies, control measures are not applied or are applied inadequately in most developing countries. Successful conduct of a test-and-slaughter policy requires sustained cooperation of national and private veterinary services, meat inspectors, and farmers, as well as adequate compensation for services rendered. Only a few developing countries can adhere to these requirements. In addition, bovine TB does not often justify the emergency measures required for other zoonotic diseases (e.g., Rinderpest, East Coast fever, and foot and mouth disease). The full economic implications of zoonotic TB are, however, overlooked in many developing nations where the overall impact of the disease on human health and animal production needs to be assessed. According to recent estimates, annual economic loss to bovine TB in Argentina is approximately 63 million US dollars (48). In a study recently conducted in Turkey, the estimated socioeconomic impact of bovine TB to both the agriculture and health sectors was approximately 15 to 59 million US dollars per year (49). Several Latin American countries, through agreements between governments and cattle owners associations, have made the decision to control and eliminate bovine TB. Where foot and mouth disease has been eliminated, bovine TB and other existing infections such as brucellosis become important because of their impact on the meat and live animal export trade. Bovine TB and brucellosis also limit the development of the dairy industry and its expansion at the regional level. Risk Factors: Human Population Close physical contact. Close physical contact between humans and potentially infected animals is present in some communities, especially in developing regions. For example, in many African countries cattle are an integral part of human social life; they represent wealth and are at the center of many events and, therefore, gatherings. In addition, with 65% of African, 70% of Asian, and 26% of Latin American and Caribbean populations working in agriculture, a significant proportion of the population of these regions may be at risk for bovine TB. Food hygiene practices. Consumption of milk contaminated by M. bovis has long been regarded as the principal mode of TB transmission from animals to humans (1). In regions where bovine TB is common and uncontrolled, milkborne infection is the principal cause of cervical lymphadenopathy (scrofula) and abdominal and other forms of nonpulmonary TB. Although proper food hygiene practices could play a major role in controlling these forms of TB, such practices are often difficult to institute in developing countries. In all countries of sub-Saharan Africa, there is active competition between large-scale, often state-run, processing and marketing enterprises and the informal sector. The informal sector can ignore standards of hygiene and quality, and producers often sell directly to the final consumers. In addition, an estimated 90% of the total milk produced is consumed fresh or soured (44). Although it has been stated that Africans generally boil milk and that the souring process destroys M. bovis (44), other sources strongly contradict these statements (39). M. bovis was isolated from seven (2.9%) of 241 samples of raw milk in Ethiopia (17). Both M. bovis and M. tuberculosis have also been found in milk samples in Nigeria (36) and Egypt (34). Thus, serious public health implications of potentially contaminated milk and milk products should not be underestimated. HIV/AIDS. According to recent WHO global estimates, of the 9.4 million people infected with both HIV and TB in mid-1996, 6.6 million (70%) live in sub-Saharan Africa (4). The greatest impact of HIV infection on TB is in populations with a high prevalence of TB infection among young adults. The occurrence of both infections in one person makes TB infection very likely to progress to active disease. In many developing countries, TB is the most frequent opportunistic disease associated with HIV infection. HIV seroprevalence rates greater than 60% have been found in TB patients in various African countries (4). Persons infected with both pathogens have an annual risk of progression to active TB of 5% to 15%, depending on their level of immunosuppression; approximately 10% of non-HIV infected persons newly infected with TB become ill at some time during their lives. In the remaining 90%, effective host defenses prevent progression from infection to disease. TB cases due to M. bovis in HIV-positive persons also resemble disease caused by M. tuberculosis. Thus, they manifest as pulmonary disease, lymphadenopathy, or, in the more profoundly immunosuppressed, disseminated disease. M. bovis has been isolated from HIV-infected persons in industrialized countries. In France, M. bovis infection accounted for 1.6% of TB cases in HIV-positive patients. All isolated strains were resistant to isoniazid (7). Taking into consideration the intrinsic resistance of M. bovis to pyrazinamide, two of the first-line anti-TB drugs were not effective. WHO-recommended standard treatment for new TB cases includes, in the initial phase, isoniazid, rifampicin, pyrazinamide, and streptomycin or ethambutol. In situations of high primary resistance to isoniazid and streptomycin, the intrinsic resistance of M. bovis to pyrazinamide may severely limit the efficacy of treatment of TB caused by M. bovis. In a Paris hospital, a source patient with pulmonary TB due to a multidrug-resistant strain of M. bovis led to active disease in five patients. Disease occurred 3 to 10 months after infection (10). This observation led to three concerns: 1) human-to-human M. bovis transmission leading to overt disease, 2) a short interval between infection and overt disease, and 3) disseminated multidrug-resistant M. bovis. In another study, conducted in San Diego, California, one of 24 adults with pulmonary TB and 11 of 24 adults with nonpulmonary TB due to M. bovis had AIDS. One of 25 children, a 16-year-old boy with abdominal TB, was also HIV-positive (9). It is commonly believed that M. bovis is less virulent than M. tuberculosis in humans and therefore less likely to lead to overt postprimary disease and that human-to-human transmission leading to infectious disease is rare. However, if the apparent difference in virulence is the result of differences in responsiveness of the host defense mechanisms, HIV-induced immunosuppression could well lower host defenses leading to overt disease after infection. Surveillance of TB due to M. bovis The use of direct smear microscopy as the only method for diagnosis of suspected TB, although an essential requirement of any national TB program, could partly explain the relatively low notification rate of disease caused by M. bovis in developing countries. Direct smear microscopy does not permit differentiation between species of the M. tuberculosis complex; in addition, culture and speciation are often not carried out, and even when culture facilities are available, M. bovis grows poorly in standard Löwenstein-Jensen medium, one of the most widely used culture media (50). In some countries, human disease caused by M. bovis is merely reported as TB to avoid inquiries from disease control agencies, which might generate problems of patient confidentiality (2). The collection of representative data on the incidence of TB due to M. bovis from most laboratories in developing countries has additional problems. For example, the location and coverage of laboratories are often biased towards city populations; sputum specimens may predominate, with relatively few specimens from extrapulmonary lesions, particularly among children. Specimens from children with TB are frequently negative on culture, and biopsies are difficult to take from lesions. Recent outbreaks of multidrug-resistant TB in some parts of the world underscore the need for surveillance through wider application of reliable culture and drug susceptibility tests. Control Measures and Programs in Developing Countries Bovine TB can be eliminated from a country or region by implementing a test-and-slaughter policy, if no other reservoir host of infection exists. While the test-and-slaughter policy is likely to remain the backbone of national elimination bovine TB programs, the policy has numerous constraints in developing countries. Alternative strategies (e.g., programs based on slaughterhouse surveillance and traceback of tuberculous animals to herds of origin) may be technically and economically more appropriate in these countries. Measures to prevent transmission of infection should be the primary objective to be achieved with trained public health personnel, public education, and proper hygienic practices. Test-and-slaughter programs may be feasible and appropriate in areas with low bovine TB prevalence and effective control of animal movement. Animal Vaccination and Research Developments Although not usually considered relevant to elimination programs in livestock (47), vaccination of animals against TB would be a viable strategy in two disease control situations: in domesticated animals in developing countries and in wildlife and feral reservoirs of disease in industrialized countries where test-and-slaughter programs have failed to achieve elimination of the disease. Many issues need to be addressed before vaccination becomes a realistic option for control of disease in cattle and other animals. First, a highly effective vaccine needs to be developed. The results obtained globally with bacillus Calmette-Guérin (BCG) have been suboptimal, and efficacy has varied considerably from region to region (42,51). Secondly, the delivery of the vaccine poses few problems in domesticated animals, but it is fraught with difficulties in wild animals. Thirdly, vaccination may compromise diagnostic tests. A vaccine that induces tuberculin reactivity would invalidate the key diagnostic tool used in control programs. Fourthly, short of performing lengthy and expansive field studies, evaluation of the protective efficacy of a new vaccine will pose serious difficulties. Traditionally, the guinea pig and mouse have been used for this purpose, but the information gained has been of little value. Recent work has, however, indicated that deer may well prove a suitable mammal for evaluating new vaccines and optimum delivery systems (52). Enzyme-linked immunosorbent assay and gamma-interferon tests may prove to be more sensitive and specific than the tuberculin test and may facilitate diagnostic procedures. Nucleic acid-based technology, notably polymerase chain reaction and related methods, may provide more rapid, sensitive, and specific diagnostic tools. Multicenter studies of the applicability of these techniques to the diagnosis of human TB have, however, shown that their sensitivity and specificity are not as high as originally expected and that many problems need to be solved before the techniques are introduced into routine laboratory practice (53). Restriction fragment length polymorphism analysis (DNA fingerprinting) could be useful in epidemiologic studies that trace the spread of disease between cattle, other animals, and humans (54) or in the rapid differentiation of M. bovis within the M. tuberculosis complex (55). The use of these techniques is limited by resources in most developing countries. WHO and Zoonotic TB The public health importance of animal TB was recognized early by WHO, which in its 1950 report of the Expert Committee on Tuberculosis (56) stated: "The committee recognizes the seriousness of human infection with bovine tuberculosis in countries where the disease in cattle is prevalent. There is the danger of transmission of infection by direct contact between diseased cattle and farm workers and their families, as well as from infected food products." Since then, TB in animals has been controlled and almost eliminated in several industrialized countries but in very few developing countries. More recently, WHO has been involved in zoonotic TB through the activities of the Division of Emerging and other Communicable Diseases Surveillance and Control at WHO in Geneva (WHO/EMC) and the Veterinary Public Health program of the WHO Regional Office for the Americas, Pan American Health Organization (PAHO/HCV). WHO/EMC has organized and coordinated a working group of experts from countries worldwide (15-17). Their subjects are epidemiology, public health aspects, control, and research on zoonotic TB. In addition, a joint WHO, Food and Agriculture Organization of the United Nations (FAO), and Office International des Epizooties (OIE) Consultation on Animal Tuberculosis Vaccines was held to review current knowledge on TB vaccine development for humans and animals and make recommendations for animal TB vaccine research and development (57). Promising results of cattle vaccination with low doses of BCG were reported. It is also planned for field trial cattle vaccination to commence early in 1998 in Madagascar in collaboration with national and international research institutions, OIE and WHO. In the framework of the working group activities, the guidelines for speciation within the Mycobacterium tuberculosis complex (50) have been prepared to respond to the growing need for reliable differentiation between M. tuberculosis, M. africanum, and M. bovis and to promote and strengthen surveillance. A Plan of Action for the Eradication of Bovine Tuberculosis in the Americas (18) has been developed by PAHO in collaboration with member countries of the region. PAHO/HCV, in cooperation with the Pan American Institute for Food Protection and Zoonosis (INPPAZ), Buenos Aires, Argentina, and other technical institutions (e.g., FAO), provides technical support to the regional plan. PAHO/HCV activities train specialists in diagnosis, reporting, surveillance systems, and quality control of reagents, as well as supporting the planning and implementation of national programs. INPPAZ acts as a reference center for these activities. The first phase of the regional plan is expected to lead, in the next 10 years, to the elimination of bovine TB from countries with more advanced national programs. In the remaining countries, the objectives will be to strengthen epidemiologic surveillance, defining areas at risk and setting up control and elimination programs. Conclusions Although the epidemiology of bovine TB is well understood and effective control and elimination strategies have been known for a long time, the disease is still widely distributed and often neglected in most developing countries. Its public health consequences, although well documented from the past experiences of industrialized countries, have scarcely been investigated and are still largely ignored in these regions. Because of the animal and public health consequences of M. bovis, disease surveillance programs in humans should be considered a priority, especially in areas where risk factors are present. The increase of TB in such areas calls for stronger intersectoral collaboration between the medical and veterinary professions to assess and evaluate the scale of the problem, mostly when zoonotic TB could represent a significant risk, for example, in rural communities and in the workplace. Industrialized countries, where the test-and-slaughter policies have not completely eliminated infection in cattle because of wild animal reservoirs, are now reconsidering wild animal vaccination. Any vaccination research and development program should therefore also take into account the possible application of vaccines to cattle, particularly in developing countries. In developing countries, where HIV and bovine TB are likely to be common, particularly in young persons, the ability of HIV infection to abrogate any host factors that prevent the progression of infection by M. bovis to overt disease may lead to higher incidence and case-fatality rates for human TB caused by this species and increased human-to-human transmission of this disease. This should be of great concern in those developing countries where bovine TB is present and measures to control spread of infection are not applied or are applied inadequately. 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Unpub. document WHO/CDS/VPH/94.138. --------------------------------------------------------------------------- Synopses What Makes Cryptococcus neoformans a Pathogen? Kent L. Buchanan and Juneann W. Murphy University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA ------------------------------------------------------------------------ Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases. Cryptococcus neoformans is an encapsulated fungal organism (Figure 1) that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts (1,2). Most susceptible to infection are patients with T-cell deficiencies (1,2). C. neoformans var. neoformans causes most cryptococcal infections in humans, so this review will focus on information from the neoformans variety of this basidiomycetous fungus. C. neoformans var. neoformans is found worldwide; its main habitats are debris around pigeon roosts and soil contaminated with decaying pigeon or chicken droppings (1,3). Not part of the normal microbial flora of humans, C. neoformans is only transiently isolated from persons with no pathologic features (2,4). It is generally accepted that the organism enters the host by the respiratory route in the form of a dehydrated haploid yeast or as basidiospores. After some time in the lungs, the organism hematogenously spreads to extrapulmonary tissues; since it has a predilection for the brain, infected persons usually contract meningoencephalitis (1). If untreated, cryptococcal meningoencephalitis is 100% fatal, and even when treated with the most effective antifungal drugs, cryptococcal infections can be fatal if the host does not have adequate T-cell­dependent immune function (2). To be classified as a pathogen, an organism must be able to cause infection under certain conditions. By this definition, C. neoformans can certainly be classified as a pathogen. Because the immunodeficient are more susceptible than the immunocompetent to infection with this yeast-like organism, C. neoformans is frequently referred to as an opportunistic pathogen. The factors that make C. neoformans a pathogen can be divided into two major groups. The first comprises the basic characteristics needed to establish an infection and survive in the human host; the second comprises the virulence factors that affect the degree of pathogenicity. [Fig] Figure 1. Transmission electron micrograph of budding C. neoformans showing the characteristic polysaccharide capsule. Basic Characteristics for Pathogenicity The Infectious Particle To enter the alveolar spaces of the lungs and establish pulmonary infection, an organism must produce viable forms smaller than 4 µm in diameter. The typical vegetative form of C. neoformans is the yeast form with a cell diameter of 2.5 µm to 10 µm. The organism can also undergo sexual reproduction, and since it is a basidiomycete (Filobasidiella neoformans), it forms basidiospores. Sexual reproduction appears to occur much less frequently in nature than asexual or vegetative reproduction. The sexual spores (basidiospores) are approximately 1.8 µm to 3 µm in diameter and result from crosses of the and µ a-mating types on an appropriate medium (1). Although the exact nature of the infectious particles of C. neoformans is not known, they are presumed to be the dehydrated yeast cells or basidiospores of the appropriate size range to get into the lungs. Once inside the lungs, the yeast cells become rehydrated and acquire the characteristic polysaccharide capsule (Figure 2). In the case of basidiospores, they would convert to encapsulated blastoconidia. [Fig] Figure 2. Proposed means of infection by C. neoformans. Recently, Wickes et al. (5) reported that the [Alpha]-mating type of C. neoformans can produce monokaryotic hyphae on a solid medium without a nitrogen source or water. The monokaryotic hyphae contain clamp connections and basidia with short chains of basidiospores and are similar in all respects except in nuclei number to the dikaryotic hyphae of the sexual forms (5). The haploid fruiting bodies formed by nitrogen-starved [Alpha]-mating type isolates also produce abundant amounts of blastoconidia, which on the appropriate medium can produce haploid hyphae (5). None of the a-mating type strains tested produced the haploid fruiting bodies. Backcross studies indicated that the ability to undergo haploid fruiting lies within (or is tightly linked to) the MAT[Alpha] locus, which is responsible for the [Alpha]-mating type phenotype (5). These results suggest basidiospores from the monokaryotic hyphae may be another source of the infectious particle in nature. Regardless of the nature of the infectious particle (yeast cell or basidiospore), if a cryptococcal isolate is not able to produce small infectious particles, it cannot be pathogenic under the usual conditions for establishing the disease. Mating Types Kwon-Chung and Bennett (6) surveyed the mating types of C. neoformans isolates from environmental and clinical sources and found a 30- to 40-fold higher frequency of [Alpha]-mating type than [Alpha]-mating type. The skewed ratio of [Alpha]-mating type to a-mating type was not thought to be due to a genetic preponderance towards [Alpha]-mating type progeny because crosses between two test strains resulted in a 1:1 ratio of [Alpha]-mating and [Alpha]-mating type progeny (7). To examine the reason for the disparate ratio, Kwon-Chung and colleagues (8) constructed a pair of congenic strains of C. neoformans that differed only in their mating type. Survival studies with these congenic strains demonstrated that the [Alpha]-mating type (B-4500) was significantly more virulent than the a-mating strain (B-4476) when injected intravenously into mice (8). Eighty percent of mice infected with 10 [sup6] B-4500 ([Alpha]-mating type) were dead within 36 days; whereas, it took 93 days for 80% of mice infected with 10 [sup6] B-4476 (a-mating type) to die (8). Although the mating type locus of C. neoformans has been cloned and partially characterized, the genes or gene products that contribute to the increased virulence of [Alpha]-mating type isolates are not known. The mating type locus (MAT[Alpha]) cloned from an [Alpha]-mating type C. neoformans isolate is 35 kb to 45 kb long (9). Moore and Edman (9) demonstrated that introducing a 2.1-kb fragment of the MAT[Alpha] locus into an a-mating strain isolate stimulated filament formation on starvation medium. Basidia and basidiospores were not produced on the filaments. In contrast, electroporation of an [Alpha]-mating strain with the same MAT [Alpha] DNA fragment was ineffective in stimulating filament formation under the same growth conditions (9). Sequence analysis of the 2.1-kb fragment from the MAT[Alpha] locus identified a 114-bp open reading frame that encodes a 38-amino acid propheromone peptide (9). The nature and the effects of the active pheromone peptide on mating and possibly virulence of C. neoformans are unknown; consequently, how the mating type relates to virulence at the genetic level is also unknown. However, the observation of haploid fruiting by [Alpha]-mating type isolates of C. neoformans on nitrogen- and water-depleted medium (9) may help explain why the [Alpha]-mating type isolates from clinical specimens are more frequent than the a-mating type strains (6). Growth In Vivo To cause infection in humans, a C. neoformans isolate must grow at 37 degrees C in an atmosphere of approximately 5% CO2 and at a pH of 7.3 to 7.4. To survive at 37 degrees C, the organism must have an intact gene that encodes the C. neoformans calcineurin A catalytic subunit (10). Calcineurin is a serine-threonine specific phosphatase that is activated by Ca2+-calmodulin and is involved in stress responses in yeasts (10). Although calcineurin A mutant strains of C. neoformans can grow at 24 degrees C, they cannot survive in vitro at 37 degrees C, in 5% CO2, or at alkaline pH (10). Since these are similar to conditions in the host, one would predict that the calcineurin A mutant would not survive in the human host. In support of that prediction, Odom et al. have shown that such mutants are not pathogenic for immunosuppressed rabbits (10). Calcineurin A appears to be a basic requirement for C. neoformans survival in the host and consequently is a necessary factor for the pathogenicity of the organism. Virulence Factors Virulence factors increase the degree of pathogenicity of a microbe. C. neoformans has a number of virulence factors; generally, the virulence of an isolate cannot be attributed to any single factor, but rather it is attributed to many working in unison to cause progressive disease. As virulence factors go, those of C. neoformans would be considered low-grade. We will discuss each virulence factor separately; however, the severity of the host's disease results from a combination of several virulence factors superimposed on the host's innate and immune resistance status. The virulence factors that will be discussed are capsule, cryptococcal products, melanin production, mannitol production, and potential factors such as superoxide dismutase, proteases, phospholipase B, and lysophospholipase. The polysaccharide capsule and the soluble extracellular constituents of C. neoformans (referred to here as cryptococcal products) are probably the dominant virulence factors. Capsule C. neoformans has a capsule composed primarily of a high molecular weight polysaccharide that has a backbone of [Alpha]-1,3-D-mannopyranose units with single residues of ß-D-xylopyranosyl and ß-D-glucuronopyranosyl attached (11-15). This polysaccharide is referred to as glucuronoxylomannan (GXM) (11) and has four serotypes: A and D, produced by C. neoformans var. neoformans, and B and C, produced by C. neoformans var. gattii. The evidence indicates that the capsule is a key virulence factor for C. neoformans; acapsular mutants are typically avirulent, whereas encapsulated isolates have varying degrees of virulence (20). Similar observations have been made with the CAP64 gene (21,22). Although their biochemical functions have not been defined, it appears that the two genes are essential for capsule formation and virulence. Chemotactic Effects on Leukocytes Some properties of the C. neoformans capsule enable the host to more effectively clear the organism from tissues; however, others protect the organism from host defenses. The capsules of serotype A and D isolates are chemotactic for neutrophils (23). Moreover, complement is fixed by cryptococcal capsules by the alternative pathway (24), and this process produces chemotactic peptides such as C5a (23). Chemotaxis of leukocytes induced by either of these mechanisms would be advantageous to the host. Effects of Complement Interactions Complement fixing by C. neoformans in tissue would result in chemotactic factor production and attraction of leukocytes into the infection site. Once in the infected tissue, the leukocytes would interact with and kill the organism. As complement is fixed, C3b and C3bi are deposited on the surface of the cryptococci (24). The capsule can mask the C3b and C3bi deposits (24-26); however, if they are not completely masked, the deposited complement components facilitate binding of the cryptococci to CR3 receptors on leukocytes (27). Such binding interactions are advantageous to the host; they enhance the opportunity for the leukocytes to kill the cryptococci either extracellularly or after phagocytosis. The organism can also be opsonized by antibodies to GXM, but the capsule may block the Fc portion of the antibody and prevent it from binding to Fc receptors on the phagocytic host cells (28). Some of these functions of the capsule favor the host; however, if the capsule is very large, the organism is protected (24-26). The cryptococci could deplete complement in the host, creating an environment that favors the cryptococci (29). Effects on Phagocytosis All considered, the capsule is more beneficial to the organism than to the host. Encapsulated C. neoformans cells are not phagocytized or killed by neutrophils, monocytes, or macrophages to the same degree as acapsular mutants (25,30-33). Encapsulated C. neoformans have a stronger negatively charged surface than acapsular cells or Saccharomyces cerevisiae cells (34). The high negative charge could cause electrostatic repulsion between the organism and the negatively charged host effector cells and reduce intimate cell-cell interactions required for clearance of the cryptococci (34). Altered Antigen Presentation The inability of macrophages to ingest the encapsulated organisms could also diminish antigen presentation to T cells and consequently reduce immune responses. This speculation has been experimentally demonstrated by Collins and Bancroft (35). Other studies with human alveolar macrophages have confirmed that antigen presentation is not as effective with encapsulated cryptococci as with acapsular strains (33,36). Unlike acapsular cells, encapsulated isolates cannot stimulate proliferative responses in T cells because of the reduced secretion of interleukin-1 (IL-1) by the antigen-presenting cells stimulated with the encapsulated yeasts (36). Effects on Cytokine Production In addition to being antiphagocytic, more resistant to killing, and less able to stimulate T-cell proliferation (25,30-33,35,36), highly encapsulated isolates of C. neoformans opsonized with a heat-labile serum component, presumably complement, do not stimulate monocytes and macrophages to produce proinflammatory cytokines such as TNF[Alpha], IL-1ß, and IL-6 as effectively as similarly opsonized nonencapsulated or weakly encapsulated isolates (37-41). The signal for induction of cytokine production can be a direct result of the attachment of the monocyte or macrophage to the acapsular cryptococci or can be an outcome of the phagocytic process, induced by acapsular cryptococci. Since the capsule blocks phagocytosis, any cytokines induced by the phagocytic process would not be induced by the encapsulated C. neoformans cells. If the cryptococcal cell wall materials must be exposed to induce cytokine production, the capsule would block the direct induction of cytokine production. Regardless of the mechanism of stimulation, the lack of production of proinflammatory cytokines could have a bearing on protection. TNF[Alpha] is necessary for the induction of the protective immune response against C. neoformans (42). Consequently, the lack of or reduced production of TNF[Alpha] in infections with highly encapsulated isolates of C. neoformans would prevent the induction of protective immunity, resulting in progressive disease. The roles of IL-1ß and IL-6 in protecting against C. neoformans have not been defined, but it is highly probable that the lack of these two cytokines could compromise the protective responses of the host and give cryptococci the advantage. In contrast to the reduced TNF[Alpha], IL-1ß, and IL-6 produced by stimulating monocytes and macrophages with highly encapsulated cryptococci, IL-10 produced by these host cells increases after interaction with encapsulated strains (43). Neutralization of IL-10 with anti-IL-10 in cocultures of human peripheral blood mononuclear cells and encapsulated cryptococci increased the amounts of TNF[Alpha] and IL-1ß produced, which indicates that the induction of IL-10 production by stimulating macrophages with encapsulated C. neoformans downregulates the production of the proinflammatory cytokines TNF[Alpha] and IL-1ß (43). One might predict that the induction of high levels of IL-10 would also preferentially stimulate the induction of a T-helper 2 (Th2) response rather than a Th1 response in the T cells (44). Since the Th1 response is associated with protection against C. neoformans (45), increased levels of IL-10 would dampen induction of the protective immune response. Encapsulated cryptococcal cells do not affect the different types of leukocytes in the same manner. Although encapsulated isolates do not stimulate macrophages to produce proinflammatory cytokines, they do stimulate neutrophils to produce proinflammatory cytokines and the chemotactic factor IL-8 more effectively than weakly encapsulated or acapsular organisms (46). As with the stimulation of macrophages by acapsular cryptococci to produce proinflammatory cytokines, serum is required for the encapsulated organisms to induce neutrophils to produce cytokines (46). In the case of cytokine production by neutrophils in response to encapsulated C. neoformans, it appears that the opsonization process releases a factor into the supernatant that induces the neutrophils to produce the cytokines (46). How these opposing in vitro observations with macrophages and neutrophils relate to the in vivo system or pathogenicity is yet to be determined. Cryptococcal Products In disseminated cryptococcosis, measurable levels of cryptococcal products are present in the body fluids of the patients (47). Although GXM is the major cryptococcal component in body fluids, it is highly probable that the organism also sheds galactoxylomannan (GalXM) and mannoproteins (MP) in vivo. This speculation is based on indirect evidence from in vivo studies and on the fact that GalXM and MP are in culture medium when the organism is grown in vitro (15,48). Cryptococcal antigens in serum or spinal fluid are diagnostic for cryptococcosis (47). Furthermore, if disseminated cryptococcosis patients have high cryptococcal antigen titers at the onset of therapy, they are less likely to respond to therapy or more likely to die before therapy is completed than patients with low cryptococcal antigen titers (49). The direct relationship of cryptococcal antigen levels in body fluids and the severity of disease suggests that the cryptococcal antigens in the host's circulatory system or spinal fluid may have adverse effects on host defenses. Effects on Leukocyte Migration It has been recently demonstrated in the mouse model that intravascular cryptococcal antigens inhibit the migration of leukocytes from the bloodstream into an inflammatory site (50). Intravascular cryptococcal antigens significantly reduce leukocyte infiltration into a site of acute inflammation induced by such stimuli as cryptococcal culture filtrate antigen, TNFa, or the chemotactic peptide FMLP (formylmethionyl leucyl phenylalanine) or into a delayed-type hypersensitivity reaction site induced by purified protein derivative of Mycobacterium tuberculosis or by C. neoformans antigen(s) (50). Each of the identified cryptococcal products, GXM, GalXM, and MP, when given intravenously to mice, can inhibit leukocyte migration (50). Considering that GXM is the dominant cryptococcal component in the host's circulation, one might predict that GXM is mainly responsible for the reduced leukocyte migration into inflammatory sites (50). These observations imply that pulmonary cryptococcosis patients, who have low to no cryptococcal antigen concentrations in their serum, would have a normal influx of leukocytes into pulmonary sites of infections. On the other hand, in severe pulmonary infections or in disseminated cryptococcosis patients who have high levels of circulating cryptococcal antigens, minimal inflammation would be expected in the infected tissues. In fact, for years investigators have commented on the minimal host tissue responses observed in patients with disseminated cryptococcosis (3). These recent data demonstrate that the circulating cryptococcal antigens are responsible, at least in part, for the lack of host tissue response. Cryptococcal antigen(s) can potentially prevent leukocytes from migrating into an inflammatory site in two nonexclusive ways (51,52). First, GXM can stimulate neutrophils to shed L-selectin, a surface molecule necessary for the first step in neutrophil movement into tissues (51). Without L-selectin the neutrophils do not slow down and roll along the inflamed endothelial cells lining the blood vessels. With this first step in extravasation blocked, the numbers of neutrophils in infected tissues would be greatly reduced. Second, GXM and GalXM can bind to CD18, the beta chain of the adhesion molecule LFA-1, and prevent the binding of anti-CD18 to LFA-1 (52). Consequently, this binding of GXM and GalXM could prevent LFA-1 on the neutrophils from binding to the LFA-1 ligand, ICAM-1 on the inflamed endothelial cell surface. If leukocytes are inhibited from entering tissues by either or both of these mechanisms, the organism is not effectively eliminated, and the disease is more severe. Induction of Immunomodulatory Cells Cryptococcal antigens injected into the bloodstream of experimental animals can induce regulatory T cells, which dampen or ablate the anticryptococcal humoral as well as cell-mediated immune responses (53-71). Some clinical correlates support the concept that the antigenemia seen in disseminated cryptococcosis downmodulates the immune responses (72,73). A long-lasting, specific immunologic unresponsiveness has been reported in persons cured of cryptococcal meningitis (72-74). Although they made antipneumococcal polysaccharide antibodies to the same degree as control volunteers after vaccination with pneumococcal polysaccharide, cured patients could not make antibodies to cryptococcal polysaccharides after vaccination with cryptococcal antigens (72,73). Henderson et al. (72,73) speculated that the intense, prolonged antigenemia associated with the cryptococcosis may account for the observed unresponsiveness. It is not unusual for patients with disseminated cryptococcosis to have depressed cell-mediated immune (CMI) responses to cryptococcal antigens (75,76). However, sufficient information is not available to determine whether the patients had a generalized defect in CMI function or had a specifically depressed CMI response because of the cryptococcal antigenemia. Experimental animal models demonstrate convincingly that cryptococcal antigens given intravenously can induce immunoregulatory T cells that downmodulate the anticryptococcal CMI response (56-71). Serum from C. neoformans-infected mice with a cryptococcal antigen titer of 10-4 when transferred intravenously to naive mice induces regulatory T cells that specifically depress the anticryptococcal CMI response (67). Similar immunoregulatory T cells are induced by simulating the antigenemia with intravenous injection of cryptococcal culture filtrate antigen (56,57,60-63,66-69). The immunoregulatory CD4+ T cells, which appear in the lymph nodes of the mice 7 days after antigen injection, diminish the induction of T cells responsible for the anticryptococcal delayed-type hypersensitivity response and reduce the ability of the mice to clear cryptococci from tissues (56,57,60,68). A second immunoregulatory T cell, a CD8+ cell, has been found in spleens of mice (57). The CD4+ immunoregulatory cells do not alter the numbers or types of leukocytes that infiltrate an anticryptococcal delayed-type hypersensitivity reaction; however, they do have a downregulating effect on IL-2 and IFN[Alpha] production at the site (77). In contrast, the CD8+ immunoregulatory cells appear to affect the numbers of neutrophils that infiltrate the delayed-type hypersensitivity reaction site (Murphy, unpub. data). The details of the characteristics and functions of these immunoregulatory T cells have been reviewed (45). The available data strongly support the notion that cryptococcal products in the circulation induce an array of immunoregulatory T cells, which depress the anticryptococcal immune responses and protection. More consideration should be given to the impact of high levels of cryptococcal antigen in the cerebrospinal fluid (CSF) on progression of disease. As suggested by Denning et al. (78), high cryptococcal antigen concentrations could change the osmolality of the CSF, thereby affecting its outflow and adsorption and increasing intracranial pressure. The increased pressure may cause headaches, visual loss, and early death (78). It is also possible that release of mannitol by C. neoformans contributes to increased intracranial pressure in cryptococcal meningitis patients (78). Melanin Synthesis One characteristic that differentiates pathogenic isolates of C. neoformans from nonpathogenic isolates and other Cryptococcus species is the organism's ability to form a brown to black pigment on a medium (such as birdseed or caffeic acid agar) that contains diphenolic compounds (1). This pigment, first described by Staib (79), is a melaninlike compound produced by C. neoformans isolates with phenoloxidase activity (80). The importance of melanin production in C. neoformans virulence was first demonstrated in the early 1980s. Rhodes, Polacheck, and Kwon-Chung (81) reported that naturally occurring C. neoformans mutants lacking melanin (Mel-) were less virulent in mice than melanin-producing strains. Others (82-84), using chemically induced mutants or isogenic pairs of C. neoformans, have confirmed and extended this observation. Biochemical analyses suggest that in C. neoformans melanogenesis is accomplished by conversion of dihydroxyphenols such as 3,4-dihydroxyphenylalanine (DOPA) to dopaquinone (Figure 3). This conversion is catalyzed by a phenoloxidase and is the rate-limiting step, presumably because subsequent steps in the pathway, such as dopaquinone rearranging to dopachrome and ultimately autoxidation to melanin, are spontaneous (85). C. neoformans lacks a tyrosinase enzyme required for endogenous production of dihydroxyphenols (83); thus to produce melanin, a C. neoformans isolate must be able to acquire diphenolic compounds from its growth environment, and it must have the enzyme phenoloxidase to catalyze conversion of these compounds into the subsequent melanin intermediates. The brain is a tissue rich in catecholamines such as DOPA and is a favorite target for infection by C. neoformans. However, the organism cannot use catecholamines as a sole carbon source, which suggests that the brain is not a preferred nutritional niche for growth of C. neoformans (86); rather, it may serve as a survival niche as described below. [Fig] Figure 3. Proposed pathway for melanin synthesis by C. neoformans. Polacheck et al. (86) reported that melanin-producing isolates of C. neoformans were resistant to damage by an in vitro epinephrine oxidative system, whereas mutants lacking phenoloxidase activity were highly susceptible, as evidenced by decreased survival. Jacobson and Tinnell (87) found that melanin protected C. neoformans from damage by hypochlorite and permanganate but not by hydrogen peroxide. In these experiments, hypochlorite was 100 times more fungicidal than hydrogen peroxide, and C. neoformans could produce sufficient levels of melanin to effectively protect the organism from oxidative compounds produced by macrophages (87). Wang and Casadevall (88) extended these findings by examining survival of C. neoformans in the presence of nitric oxide and reactive nitrogen intermediates as well as in the epinephrine oxidative system described by Polacheck et al. (86). Wang and Casadevall cultured C. neoformans cells with L-DOPA to melanize the yeast cells (88). Melanized cryptococci survived damage by both nitrogen- and oxygen-derived oxidants significantly better than nonmelanized organisms of the same strain. These results support the hypothesis that C. neoformans uses catecholamines in the brain to make melanin, thereby protecting the organism from oxidative damage by scavenging free radicals (86). Recently, cryptococcal diphenoloxidase has been purified, and the gene CNLAC1 encoding this enzyme was cloned (89). The glycosylated protein has a molecular weight of 75 kDa and contains copper. The substrate specificity of the enzyme indicates that it is a laccase. CNLAC1 contains 14 introns, a 624 amino acid open reading frame; transcriptional activity is derepressed in the absence of glucose (89), which confirms an earlier report of low glucose requirements for melanin formation (90). Disruption of CNLAC1 resulted in loss of virulence of C. neoformans, whereas complementation with CNLAC1 increased virulence of Mel- mutants in mice (84); these results suggest that the laccase (phenoloxidase) encoded by CNLAC1 is a potential virulence factor of C. neoformans. Furthermore, transcripts of the CNLAC1 gene have been detected by reverse transcription-polymerase chain reaction (RT-PCR) in C. neoformans yeast cells isolated from CSF in a rabbit model of cryptococcal meningitis (84). Besides acting as an antioxidant, melanin production may help C. neoformans survive in the host in other ways. Melanized yeast cells are less susceptible to amphotericin B than nonmelanized yeast cells, and this may contribute to the inability to effectively treat infections in immunocompromised hosts (91). Furthermore, phagocytosis of melanized C. neoformans by a macrophage cell line in the presence of an anti-GXM antibody was decreased, which suggests that melanin deposition in the cell wall may inhibit opsonization by specific antibodies (92). Recently, Huffnagle and coinvestigators (93) reported that melanized heat-killed C. neoformans strain 145 yeast cells stimulated less TNF[Alpha] production by alveolar macrophages and less antigen-specific T-cell proliferation than nonmelanized heat-killed 145 strain cells. The authors suggest that melanin "cloaks" C. neoformans from recognition by host effector cells and inhibits induction of a protective T-cell-mediated immune response (93); however, it is possible that scavenging of host-produced oxidants and inhibition of phagocytosis by melanin may contribute to the decreased TNF[Alpha] production and lymphoproliferation observed. Although melanin production is important in the virulence of C. neoformans, there is very little evidence demonstrating the presence of melanin in vivo. One report indicates that phenoloxidase activity in C. neoformans is greatly diminished at 37 degrees C compared with activity at 25 degrees C, which suggests that melanin production may be limited in vivo (94). Detection of melanin in vivo is hampered by the lack of specific antibodies or stains. A modified Fontana-Masson stain has been used to detect a brown pigment in the cell wall of C. neoformans cells in histologic brain sections; however, the stain is not specific because Cryptococcus laurentii, which is Mel-, also stains with Fontana-Masson (95). In summary, accumulating evidence indicates that melanin production is an effective survival (virulence) factor of C. neoformans, and melanin may serve multiple roles in protecting this medically important fungus from host defenses. Two caveats should be noted in labeling melanin as a major virulence factor for C. neoformans. The C. neoformans isolate must be able to internalize the melanin precursors, and the phenoloxidase enzyme must make a sufficient amount of melanin precursor at 37 degrees C. Consequently, much more work is needed to delineate the role of melanin production in virulence of C. neoformans, especially in light of the apparent temperature sensitivity of the cryptococcal phenoloxidase enzyme. Mannitol Production Accumulating evidence suggests that production of the hexitol D-mannitol may contribute to survival of C. neoformans in the host. Wong et al. (96) reported that of the 12 human isolates of C. neoformans examined, all produced and secreted D-mannitol into growth medium. Further, by using a rabbit cryptococcal meningitis model, they showed that C. neoformans can produce D-mannitol in vivo. CSF from rabbits treated with cortisone and infected intracisternally with C. neoformans contained more D-mannitol than CSF from controls, cortisone-treated uninfected rabbits, or rabbits infected with C. neoformans but not treated with cortisone (96). In the latter group, cryptococcal infection was limited. The levels of D-mannitol in infected rabbit CSF correlated well with both the numbers of culturable C. neoformans and the cryptococcal antigen titer of the CSF, which suggests that levels of D-mannitol in CSF may be prognostic (96); however, it is not known whether different isolates of C. neoformans vary in mannitol production. These authors suggested two means by which mannitol production may contribute to C. neoformans pathogenesis. First, high concentrations of D-mannitol in the CNS may contribute to brain edema. Second, mannitol is a potent scavenger of hydroxyl radicals, and cryptococcal-produced D-mannitol may help protect the organism from oxidative damage (96). To investigate the role of mannitol in cryptococcosis, Chaturvedi and co-workers (97) isolated a low mannitol producing mutant after UV irradiation of C. neoformans strain H99. The mutant, mannitol low producer (MLP), was similar to the parent strain H99 in many growth and morphology characteristics and in known virulence factors such as capsule production and phenoloxidase activity (97). However, the mutant MLP strain was more susceptible to growth inhibition and killing by heat and high salt than the parent strain. In addition, the mutant MLP strain was significantly less virulent than the parent strain (MLP LD50 = 3.7 X 106 CFU, H99 LD50 = 6.9 X 102) (97). Further comparisons of H99 and MLP strains showed that polymorphonuclear leukocytes (PMNL) killed MLP significantly better than H99 at several effector-to-target ratios (98). Addition of superoxide dismutase, mannitol, or dimethyl sulfoxide inhibited PMNL killing of both strains, but addition of catalase did not alter killing, which suggests that reactive oxygen intermediates such as the hydroxyl radical and hypochlorous acid are potent effector molecules against C. neoformans and mannitol may protect against oxidative killing by scavenging such compounds (98). The results above indicate that mannitol production by C. neoformans correlates with increased resistance to heat stress, osmotic stress, and damage by reactive oxygen intermediates, as well as increased pathogenicity of this fungal agent. Additional studies are required to determine the role of mannitol production in virulence of C. neoformans. Isogenic strains lacking the enzyme required for mannitol production, namely mannitol dehydrogenase, are not available because the gene for this enzyme has not been found. However, a gene (Mtl) has been isolated from C. neoformans that can induce expression of the S. cerevisiae mannitol dehydrogenase gene (99). The 346 amino acid product encoded by the Mtl gene is thought to be involved in regulating mannitol production in C. neoformans and may be beneficial in future studies of mannitol production (99). Other Potential Virulence Factors Superoxide Dismutase Jacobson and coinvestigators (100) examined superoxide dismutase (SOD) production by C. neoformans to determine whether SOD levels increased at 37 degrees C to compensate for possible decreases in melanin production at this temperature. These investigators observed an increase in SOD levels at 37 degrees C, which suggests that SOD may participate in free radical scavenging at this higher temperature in vivo (100); however, there is no evidence that SOD production serves as a virulence factor for C. neoformans. Proteases Muller and Sethi (101) reported that C. neoformans grown in culture produced a protease that could digest human plasma proteins, and Brueske (102) reported that C. neoformans culture supernatants contained a protease capable of digesting casein. However, neither of these studies determined whether the proteases were manufactured in vivo. Limited evidence indicating in vivo production of proteases has been presented by Salkowski and Balish (103). These investigators observed skin lesions on T-cell-deficient mice after intravenous infections with C. neoformans strain SLHA (103). Microscopic examination of the lesions suggested that the cryptococcal yeast cells were degrading collagen bundles in the dermis (103). Supernatants of SLHA strain cultures liquefied gelatin in vitro indicating that this cryptococcal strain secreted a collagenaselike protein (103). Thus, C. neoformans proteases possibly serve as virulence mechanisms by initiating invasion of host tissues; however, more studies with isogenic strains of C. neoformans are required before proteases can be listed as virulence factors. Phospholipases Recently, Chen and co-workers (104,105) reported phospholipase, lysophospholipase, and lysophospholipase-transacylase activity of C. neoformans grown on egg yolk agar. Of 50 strains tested, 49 had phospholipase activity (104), due to phospholipase B secreted into cultures (105). Analysis of four strains with varying levels of phospholipase activity indicated a correlation between phospholipase activity and virulence in BALB/c mice (104). The authors suggest that extracellular phospholipase activity produced by C. neoformans may disrupt mammalian cell membranes and allow the yeast cells to penetrate into host tissues (104,105); however, further investigations are necessary to establish the role, if any, of these types of products in the virulence of C. neoformans. Regulation of Virulence Regulation of virulence factors such as capsule production and melanin formation is not well understood. However, the gene GPA1, which encodes a G-protein [Alpha]-subunit, is involved in the regulation of these virulence factors as well as in C. neoformans mating (106). Disruption of GPA1 resulted in defects in mating in response to nitrogen starvation, capsule production in response to iron limitation, and melanin synthesis in response to glucose starvation (106). Furthermore, gpa1 mutants were much less virulent in a rabbit model of cryptococcal meningitis (106). Reconstitution of the gpa1 mutant with wild-type GPA1 restored mating, capsule production, melanin synthesis, and virulence. Addition of cyclic AMP also restored these phenotypes, which suggests that C. neoformans GPA1 regulates these factors by sensing the nutritional signals of the environment and regulating cyclic AMP metabolism in the organism (106). These findings along with results of other molecular studies are intriguing and represent the initial steps in defining at the molecular level the factors controlling virulence in C. neoformans. Conclusions Virulent isolates of C. neoformans must be able to produce small particles that can get into the alveolar spaces, must be able to grow at 37 degrees C at a pH of 7.3 to 7.4 in an atmosphere of approximately 5% CO2, must have an intact calcineurin pathway, and (possibly) must be an [Alpha] -mating type. The ability to produce a large capsule and shed great amounts of capsular material into the body fluids makes the organism highly virulent. Other factors, such as melanin, mannitol, superoxide dismutase, protease, and phospholipase production, may enhance the pathogenicity of C. neoformans. The effectiveness of many of these cryptococcal virulence factors depends on the status of the host's defensive mechanisms. Although we have learned much about the pathogenicity and virulence of C. neoformans, many gaps still remain in our knowledge. Acknowledgments We thank Drs. J.A. Alspaugh, J.R. Perfect, and J. Heitman for making their manuscript available before publication. Dr. Buchanan is a research assistant professor in the Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA. Dr. Murphy is a George Lynn Cross Research Professor of Microbiology and Immunology in the same department. Address for correspondence: Juneann W. 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Cryptococcus neoformans mating and virulence are regulated by the G-protein [Alpha] subunit GPA1 and cAMP. Genes Dev 1997;11:3206-17. ----------------------------------------------------------------------- [Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998] Dispatches Hantavirus Infection in Children in Argentina Noemí C. Pini,* Amanda Resa,† Gladys del Jesús Laime,‡ Gustavo Lecot,¶ Thomas G. Ksiazek,§ Silvana Levis,* and Delia A. Enria* *Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I Maiztegui," Pergamino, Argentina; †Hospital de El Bolsón, Río Negro, Argentina; ‡Hospital de Orán, Salta, Argentina; ¶Hospital de Olavarría, Buenos Aires, Argentina; and §Centers for Disease Control and Prevention, Atlanta, Georgia, USA --------------------------------------------------------------------------- Text Version Clinical hantavirus infection was diagnosed in five Argentine children ages 5 to 11 years by immunoglobulin M (IgM)- capture enzyme-linked immunosorbent assay using Sin Nombre virus (SNV) antigens. Death in three of the children was associated with absence of detectable IgG to SNV antigens. An additional two cases in healthy children were studied: one, a breast-fed 15-month-old whose mother died of suspected hantavirus pulmonary syndrome (HPS) 8 months previously, had hantavirus IgG(>/= 1:6400); a second, whose mother survived HPS during month three of pregnancy, apparently had maternal antibodies no longer detectable 1 year after birth. In May 1993, a new hantaviral illness, hantavirus pulmonary syndrome (HPS), was recognized in the southwestern region of the United States (1). HPS is a viral zoonosis characterized by a febrile prodrome in young, healthy adults; the disease progresses to respiratory failure with the clinical picture of adult respiratory distress syndrome (ARDS). The striking pulmonary involvement differentiates HPS from a previously described hantaviral disease known as hemorrhagic fever with renal syndrome. In the first 100 HPS cases in the United States, the average age was 34.9 years (range 11 to 69); eight cases were in children or adolescents under 16 years of age (2). In Argentina, from 1987 to July 1997, 114 cases were diagnosed in three areas of the country where several strains of new world hantaviruses are known to cause HPS diseases (3,4). Before 1995, no cases were detected in Argentine children under 12 years of age. Ten cases were reported among adolescents (13 to 19 years) with a case-fatality rate of 30% (Instituto Nacional de Enfermedades Virales Humanas, [INEVH], unpub. data). The initial case definition referred to ARDS and included adults and young adults (5) as the affected population. The lack of HPS cases among children in the original outbreaks led to a circulating hypothesis that children were not at risk or were at a very low risk for HPS. Another hypothesis was that children were protected from pulmonary involvement, perhaps by immune system immaturity or a lack of other risk factors (such as cigarette smoking) for lung injury. In this report we describe five cases in children; in all of them the etiologic diagnosis was established by the presence of immunoglobulin M (IgM) antibody to Sin Nombre virus (SNV) antigens. Serologic results for two of the children were also positive for SNV IgG antibody. Serum samples were tested for IgM and IgG antibodies to SNV by enzyme-linked immunosorbent assay (ELISA) (6). An ELISA titer greater than or equal to 1:400 was considered positive (Table 1) Table 1. Hantavirus infection in children, Argentina, 1995-1997 --------------------------------------------------------------------------- Serology(supa) Date Date of IgM Case Sex Age (yrs) onset IgG Area(supb) Outcome --------------------------------------------------------------------------- 1 M 9 4-19-95 5-3-95 South Alive >6400 1600 2 F 5 3-21-97 3-23-97 North Dead >6400 Neg 3 M 9 3-30-97 4-23-97 North Alive 1600 400 4 F 11 4-14-97 4-16-97 North Dead 1600 Neg 5 M 5 4-27-97 4-28-97 Central Dead 1600 Neg --------------------------------------------------------------------------- (supa)Titer expressed as the reciprocal of the serum dilution reactive in enzyme-linked immunosorbent assay. (supb)Area of origin in Argentina. Patient 1 was identified during the study of the first outbreak in southern Argentina in 1995 (5). Four patients in this outbreak were from the same family. During interviews of the family members, we found that a 9-year-old boy had a febrile disease without respiratory involvement, beginning on April 19. Serology performed on May 3, 14 days after the onset of symptoms, demonstrated IgM and IgG antibodies to SNV antigens. The other four cases in children were identified during routine surveillance. From 1995 to 1997, samples from 25 children (ages 3 months to 12 years; mean = 5.8 years) were sent to INEVH for diagnosis. Table 2 summarizes the main clinical and laboratory findings. None of the children had renal failure; patient 2 had uremia of 0.30 g/l, and patient 4 had a serum creatinine level of 1.40 g/l. All patients who later died received supplemental oxygen as part of their treatment. Two special situations involving children arose during the study of the first cases of HPS in El Bolsón in 1995. 1) A woman belonging to the family of patient 1 contracted HPS during the first quarter of pregnancy. She had a febrile syndrome, without respiratory failure; chest X-rays showed bilateral interstitial infiltrates. Serologic tests showed both SNV IgM (>/=1:6400) and IgG (>/=:6400) on April 22, 1995, 8 days after the onset of symptoms. She delivered a healthy infant in October 1995. A sample of the newborn's cord blood was positive for SNV IgG (>/=1:6400) and negative for SNV IgM. A serum sample drawn from the mother at the same time had a SNV IgG titer >/=1:6400 and was negative for SNV IgM. A second serum sample, taken from the baby a year later during November 1996, had no detectable SNV IgG or IgM. 2) During a retrospective search for cases fulfilling the HPS case definition, a woman who died of ARDS in September 1994 was considered to have a possible case. No serum samples or autopsy tissues were available to make an etiologic diagnosis. Before dying, this woman breast-fed a 7-month-old baby; when tested for antibodies 8 months later in May 1995 at 15 months of age, the baby had SNV IgG >/=1:6400) and no detectable SNV IgM antibodies. A second serologic sample, collected 18 months later in November 1996, still had SNV IgG antibodies, with a similar titer. Both babies have continued to develop normally as of October 1997. Table 2. Laboratory results and clinical features of children with hantavirus infection, Argentina, 1995-1997 --------------------------------------------------------------------------- Case Tests and -------------------------------------------------------------- features 1 2 3 4 5 --------------------------------------------------------------------------- Leukocytes 9,200 27,000 12,800 10,600 69,200 [/mm(sup3)] Hematocrit 44 66 43 55 53 (%) Thrombocytes ND(supa)266,000 200,000 97,000 ND [/mm(sup3)] Sedimentation ND 4 28 8 1 rate (mm/hr) GOT/GPT(supb) ND Increased Increased Increased Increased (mild) Chest X-ray HI(supc) DII(supd) DII DII DII Respiratory None Distress Slight Tachypnea, Acute symptoms dyspnea clinical and respiratory X-ray insufficiency disassociation, hypoventilation --------------------------------------------------------------------------- (supa)ND: Not done. (supb)GOT/GPT: Glutamic oxalacetic transaminase/Glutamic pyruvic transaminase. (supc)HI: Hilar indistinctness. (supd)DII: Diffuse interstitial infiltrate. A case similar to that of patient 1 was detected in New Mexico in June 1993 (7) in the course of the investigation of a fatal HPS case. In this case, the patient also had a mild clinical course that did not meet the surveillance case definition for HPS. This case definition (revised 9/96) is as follows: "a febrile illness characterized by bilateral diffuse interstitial edema that may radiographically resemble ARDS, with respiratory compromise requiring supplemental oxygen, developing within 72 hours of hospitalization, and occurring in a previously healthy person; or an unexplained respiratory illness resulting in death, with an autopsy examination demonstrating noncardiogenic pulmonary edema without an identifiable cause" (8). Our remaining four cases were sporadic, in persons without previous contact with other HPS patients, and were suspected because their clinical symptoms were typical of HPS. Results of serologic testing with SNV antigens of the household contacts in cases 2 and 5 (five persons each) were negative. The clinical, radiologic, and laboratory findings were similar in children and in adults; severely ill patients had greater variation in laboratory values than mild cases, and in fatal cases, only SNV IgM was present. The case-fatality rate in this series was 60%, but the small number of cases does not permit conclusions. In previously reported cases in adolescents 13 to 19 years of age, the case-fatality rate was 30%. These cases originated in the three areas where the illness is endemic in Argentina. This is an important point because an unusual case of HPS in southern Argentina, with the possibility of person-to-person transmission, had been reported (9,10). Patient 1 and the baby that was breast-feeding when the mother died of suspected HPS could be further instances of person-to-person transmission. A case of hemorrhagic fever with renal syndrome and pregnancy was reported in 1992 (11); the dynamics of serum antibody persistence were similar to those found in the one instance where we believe antibody was passively transferred from mother to baby. These results indicate that HPS should be considered in the differential diagnosis of respiratory distress or atypical bilateral pneumonia in children, at least in areas where these diseases have been confirmed. Mild disease should be considered too, especially in contacts of HPS patients and in younger age groups. Our findings also suggest the transfer of passive antibodies from mother to fetus (without fetal infection) and the possibility of transmission of infection by maternal breast feeding. References 1. Centers for Disease Control and Prevention. Outbreak of acute illness: southwestern United States, 1993. MMWR Morb Mortal Wkly Rep 1993;42:421-4. 2. Khan AS, Khabbaz RF, Armstrong LR, Holman RC, Bauer SP, Graber J, et al. Hantavirus pulmonary syndrome: the first 100 US Cases. J Infect Dis 1996;173:1297-303. 3. Parisi MN, Enria DA, Pini NC, Sabattini M. Detección retrospectiva de infecciones clínicas por hantavirus en la Argentina. Medicina (B Aires) 1996;56:1-13. 4. Levis SC, Briggiler AM, Cacase M, Peters CJ, Ksiazek TG, Cortés J, et al. Emergence of hantavirus pulmonary syndrome in Argentina [abstract]. Am J Trop Med Hyg 1995;53:233. 5. Hughes JM, Peters CJ, Cohen ML, Mahy BWJ. Hantavirus pulmonary syndrome: an emerging infectious disease. Science 1993;262:850-1. 6. Ksiazek TG, Peters CJ, Rollin PE, Zaki S, Nichol S, Spiropoulou C, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995;52:117-23. 7. Armstrong LR, Bryan RT, Sarisky J, Khan AS, Rowe T, Ettestad PJ, et al. Mild hantavirus disease caused by Sin Nombre virus in a four-year-old-child. Pediatr Infect Dis J 1995;12:1108-10. 8. Centers for Disease Control and Prevention. Case definitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 1997;46:16. 9. Enria D, Padula P, Segura EL, Pini N, Edelstein A, Riva Posse C, Weissenbacher MC. Hantavirus pulmonary syndrome in Argentina. Possibility of person-to-person transmission. Medicina (B Aires) 1996;56:709-11. 10. Wells RM, Sosa Estani S, Yadon ZE, Enria DA, Padula P, Pini N, et al. An unusual hantavirus outbreak in southern Argentina: person-to-person transmission? Emerg Infect Dis 1997;2:171-4. 11. Silberberg L, Rollin PE, Keourani G, Courdrier D. Haemorrhagic fever with renal syndrome and pregnancy: a case report. Trans R Soc Trop Med Hyg 1993;87:65. --------------------------------------------------------------------------- Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: http://www.cdc.gov/ncidod/EID/vol4no1/pini.htm ------------------------------------------------------------------------ Dispatches Reemergence of Dengue in Cuba: A 1997 Epidemic in Santiago de Cuba Gustavo Kourí,* María Guadalupe Guzmán,† Luis Valdés,‡ Isabel Carbonel,‡ Delfina del Rosario, * Susana Vazquez, *José Laferté,* Jorge Delgado,§ and María V. Cabrera‡ *Instituto de Medicina Tropical "Pedro Kourí" (IPK), Marianao, Havana, Cuba; †WHO/PAHO Collaborating Center for the Study of Viral Diseases, Havana, Cuba; ‡Provincial Center for Hygiene, Epidemiology and Microbiology, Santiago de Cuba, Cuba; and §Ministry of Public Health, Cuba --------------------------------------------------------------------------- Text Version After 15 years of absence, dengue reemerged in the municipality of Santiago de Cuba because of increasing migration to the area by people from disease-endemic regions, a high level of vector infestation, and the breakdown of eradication measures. The 1997 epidemic was detected early through an active surveillance system. Of 2,946 laboratory-confirmed cases, 205 were dengue hemorrhagic fever, and 12 were fatal. No deaths were reported in persons under 16 years of age. Now the epidemic is fully controlled. Cuba had its first dengue epidemic of modern times in 1977; transmission continued probably until 1981, and more than 500,000 mild cases were reported. A 1978 serologic survey for flavivirus antibody indicated that 44.6% of the Cuban population had been infected with dengue-1 virus, whereas before 1977 only 2.6% had antibodies (1,2). A second dengue epidemic in 1981, caused by dengue-2 virus (2), was unusually severe and widespread. Of 344,203 cases, 10,312 were clinically classified as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and 158 persons (101 children and 57 adults) died (3). Before 1981, only 60 suspected or confirmed DHF sporadic cases had been reported in the region (4). Dengue-2 virus isolated during the 1981 epidemic was classified in the same genotype as New Guinea 1944 (5). Not previously known to circulate in the Americas, this genotype was not isolated again in the region until 1994 in Venezuela and in 1995 in Mexico (6). Retrospective studies show that although the 1981 epidemic was detected in May, the first cases occurred in December 1980. After the epidemic ended on October 10, 1981, a campaign to improve mosquito control and eradicate _Aedes aegypti_ was immediately launched. Eradication was not achieved, but most of the 169 Cuban municipalities were free of the vector. Passive Surveillance-1981 A passive dengue surveillance system was established at the end of the 1981 epidemic. Of 9,543 paired sera (acute- and convalescent-phase) from all suspected dengue patients, only 14 showed seroconversion to immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) (7); none developed IgM antibodies to dengue virus by capture IgM ELISA (8). Dengue virus infection was excluded on the basis of clinical and epidemiologic investigation. No _Ae. aegypti_ mosquitoes were found in the residence localities of these patients. The surveillance system detected cases, imported from other Latin American countries, that had no evidence of indigenous transmission. Since 1987, 4,983 samples received through the surveillance system for measles and rubella, as well as paired sera of patients with rash, were studied for dengue antibodies [María Guzmán, World Health Organization (WHO)/Pan American Health Organization (PAHO) Collaborating Center for the Study of Viral Diseases, unpub. info.). No dengue cases were identified. The low _Ae. aegypti_ premise indexes and the results of the passive surveillance system indicate no dengue transmission in Cuba between 1981 and the end of 1996. However, reinfestation has occurred in some areas; the municipality of Santiago de Cuba was reinfested in 1992 by _Ae. aegypti_ transported in imported tires (9). Active Surveillance-1997 In January 1997, the Institute of Tropical Medicine "Pedro Kourí" of the Cuban Ministry of Health (a WHO/PAHO Collaborating Center for the Study of Viral Diseases) established an active surveillance system for dengue in Santiago de Cuba municipality. The municipality is located in Santiago de Cuba province, in the eastern part of the country, and has several risk factors for the reemergence of dengue: limited water supply, inadequate eradication efforts, high vector infestation, and increasing migration of people from Latin American and Caribbean disease-endemic countries to the municipality. Following the Guidelines for the Prevention and Control of Dengue and Dengue Hemorrhagic Fever in the Americas (4), this surveillance system actively searched for febrile patients in the primary health-care subsystem whose clinical picture was compatible with dengue fever and whose sera collected 5 to 6 days after onset of the disease contained dengue IgM antibories. As a result of this system, dengue cases were detected on January 28, 1997, in one area of the municipality. In three of the first seven cases, dengue-2 virus was detected by polymerase chain reaction (10) and was confirmed by viral isolation and identification using C6/36 cell line and monoclonal antibodies to the four dengue serotypes. Although retrospective seroepidemiologic studies indicated that the initial transmission occurred during the second half of December 1996, it is highly probable that the cases detected on January 28 were the first. Of 60,000 cases reported from the emergency rooms of Santiago de Cuba hospitals from November 1 to January 28, 592 were clinically compatible with dengue fever. Home interviews of these 592 patients reduced the figure to 154. Blood samples from 143 of 154 patients were examined for IgM antibodies, but no positive cases were detected. The breakdown of the vector control campaign in this municipality interfered with our efforts to abort the epidemic, despite the early detection of the first dengue cases; however, the partial vector control measures implemented once the outbreak was detected prevented its extension to the other 30 Cuban municipalities infested with the _Ae. aegypti_ mosquito. Active surveillance continued from January to July 1997. Serologic confirmation of cases was carried out by IgM capture ELISA, confirming recent infection. The serologic diagnosis was decentralized to the Provincial Laboratory in Santiago de Cuba, which used an ultramicro-ELISA for dengue IgM detection (11). The Institute of Tropical Medicine served as the national reference laboratory for serology, viral isolation, and strain identification and characterization. During the epidemic, 17,114 febrile patients were initially considered to have dengue, but serologic testing of 10,024 of these patients confirmed dengue in only 2,946; 46 dengue-2 isolates from 160 serum samples were obtained. The nucleotide sequence of the E\NS1 gene junction of the first isolated strain (12) indicated that it belonged to the Jamaica genotype, which during recent years is being transmitted extensively throughout Latin American and Caribbean countries and is associated with DHF/DSS in some countries (6,13). Epidemiology After the end of the 1981 Cuban DHF epidemic, seroepidemiologic studies in Palmira, Cienfuegos, and Cerro municipalities examined dengue-1 and dengue-2 seroprevalence in these populations (14,15). Taking into consideration these data and the total population of the Santiago de Cuba municipality, we estimated the prevalence of dengue-1 and dengue-2 antibodies. The estimated total population at risk for dengue-2 infection was 301,986 adults and children susceptible to a primary infection by any dengue virus serotype (63.5% of the population) and 88,108 adults with antibodies to dengue-1 virus acquired during the epidemic of 1977 to 1980, now susceptible to a secondary infection with dengue-2 and at increased risk for DHF/DSS (18.5% of the population). The earlier Cuban experience (3) confirms other reports of secondary infection (dengue-1 and dengue-2) as the main risk factor for DHF/DSS. During the 1997 dengue outbreak, secondary infection was again confirmed as a risk factor for DHF/DSS. Of the 2,946 confirmed cases, 205 (including 12 fatal adult cases) were classified as DHF/DSS cases according to the criteria established by PAHO (4). DHF/DSS was observed mostly in adults, the only age group in whom secondary infection was possible. DHF/DSS-compatible symptoms were seen only in one child with primary infection. Preliminary studies indicated that secondary infection was present in 100 (98%) of 102 DHF/DSS cases. In fatal cases, secondary infection could be documented in 11 (92%) of 12 cases. In Thailand the greatest risk appeared when the secondary infection occurred 6 months to 5 years after the primary one (16). For that reason, an epidemic of DHF/DSS was not expected in Santiago de Cuba, perhaps only sporadic cases. However, DHF/DSS in adults who contracted a secondary infection at least 16 years after the primary infection was not previously reported. Because in Cuba dengue-1 circulated from 1977 to 1980-81, the youngest patients expected to contract secondary infection should be older than 16 years of age; the youngest DHF/DSS patient with confirmed secondary infection was a 17-year-old, which indicates that the "enhancing" antibodies can circulate and be effective for at least 16 years and maybe for life. A significant number of febrile patients with suspected dengue had respiratory signs and symptoms; therefore, simultaneous circulation of respiratory or other pathogens was considered. Serologic screening for respiratory viruses using hemagglutination-inhibition and ELISA confirmed that 29.3% of 41 nonconfirmed dengue cases were influenza A, influenza B, or adenovirus infections. Additionally, some children had fever and rash clinically compatible with herpangina, and some had diarrheal disease with fever, as is common in Cuba during the summer. These febrile syndromes contributed to the high number of patients whose infections were provisionally considered suspect dengue cases. Suspect dengue cases were broadly defined to maximize sensitivity of detection and retain all possible dengue cases. This active surveillance excluded other febrile syndromes but recorded them as suspected cases. In practice, the risk perception by the population was very high, especially when the epidemic was officially declared and deaths were noted. Both the patients and the health providers appeared to think of dengue as the first diagnostic possibility. For this reason, the figure of 17,114 cases was considered the magnitude of the epidemic from the clinical management perspective. Since most cases were tested serologically, the incidence of clinical cases was probably close to the 2,946 serologically or virologically confirmed cases. Because asymptomatic and subclinical dengue cases are frequent, especially in children, the true rate of infection may be higher. In a separate and limited study on asymptomatic contacts of dengue cases, for every clinical case, 13.9 asymptomatic or subclinical cases were produced. Serologic studies of contacts in Santiago de Cuba are planned for a more in-depth study of this question. Clinical Management The health authorities established a liberal policy of hospitalization that varied with the availability of beds. Hospitalization permitted vector control of the human reservoir, more precise case classification, and close clinical surveillance. When beds were available, all patients with suspected cases were hospitalized. When the numbers of patients surpassed the availability of beds, patients were treated at home under the supervision of the family doctor. The family doctor transferred the patient to the hospital if any medical complication appeared. Wards with specialized personnel were established where the patients were protected from vectors, and observation wards were organized for patients with complications. Intensive and intermediate care units, as well as an emergency subsystem for the transfer of patients from one unit to another, were available. As in 1981, some patients rapidly developed hypovolemic shock and died within hours of admission to the hospital (17). An ad hoc task force followed the case definitions for dengue and DHF/DSS established by PAHO (4) for classifying the cases at the closure of the medical record. The accumulated experience of the Cuban scientists and doctors and the increased international knowledge about dengue and DHF/DSS in the last 15 years permitted a much deeper and more comprehensive study of this outbreak with more accurate classification and management of cases than in 1981. Nevertheless, the case-fatality rate was three times higher, mainly because of a much better classification of DHF/DSS cases. Other countries in the region with a very accurate case classification, such as Puerto Rico (13), also have a high case-fatality rate. Vector Control The campaign to control the vector started before the beginning of the 1997 dengue outbreak and is well established. Although the campaign required the mobilization of scarce financial resources and experts from all over the country, early intervention prevented spread of the outbreak to other potentially vulnerable municipalities. Of 169 municipalities in Cuba, 30 had _Ae. aegypti_ mosquitoes. The epidemic was limited to the municipality of Santiago de Cuba; no autochthonous transmission to other municipalities of the province or country was detected. An active search for cases detected transmission very early, before "fever alert" signaled an outbreak. In the Provincial Center for Hygiene, Epidemiology, and Microbiology of Santiago de Cuba, a special Unit for Analysis and Trends maintains a permanent fever alert system. For several years, this system has provided a weekly tabulation of febrile patients for every population. The tabulation allows us to evaluate fever alert (4) as applied to an active surveillance system. Because the fever alert did not appear in the epidemic area until May 1997, after the epidemic was already occurring, we consider fever alert an indicator with low sensitivity for the early and timely detection of dengue transmission, at least under the conditions of this study. As a result of the 1997 epidemic, an epidemiologic alert was established, and antivector intervention, as well as active seroepidemiologic surveillance, was reinforced in the entire country. The epidemiologic characterization of the outbreak (now fully controlled) is in the final phase. Although mosquitoes persisted at a low level after the 1981 DHF/DSS epidemic, the campaign was successful in eradicating dengue from Cuba for more than 15 years, precisely when the disease was reemerging in nearly all the other tropical regions of the Americas. According to PAHO, 250,707 cases of dengue fever and 4,440 cases of DHF/DSS were reported in 1996 alone; 29 countries reported dengue in 1996, and 10 of these reported DHF/DSS. Overall, from 1981 to 1996, 25 countries reported 41,000 cases of DHF/DSS (F. Pinheiro, pers. comm.). The 1997 Cuban dengue outbreak demonstrated once again that dengue reappears where _Ae. aegypti_ control is relaxed. Taking into account these facts, Cuba maintains its policy of vector eradication and recommends an exerted effort in the American region to prevent a recurrence of dengue similar to the one in Southeast Asia, where DHF/DSS is the leading cause of hospitalization and death among children (18). References 1. Cantelar N, Fernández A, Albert L, Pérez E. Circulación de dengue en Cuba 1978-1979. Rev Cubana Med Trop 1981;33:72-8. 2. Kourí G, Mas P, Guzmán MG, Soler M, Goyenechea A, Morier L. Dengue hemorrhagic fever in Cuba, 1981: rapid diagnosis of the etiologic agent. Bull Pan Am Health Org 1983;17:126-32. 3. Kourí G, Guzmán MG, Bravo J, Triana C. Dengue hemorrhagic fever/dengue shock syndrome: lessons from the Cuban epidemic. Bull World Health Organ 1989;67:375-80. 4. Dengue and dengue hemorraghic fever in the Americas: guidelines for prevention and control. Washington: Pan American Health Organization; 1994. Scientific publication No. 548. 5. Guzmán MG, Deubel V, Pelegrino JL, Rosario D, Sariol C, Kourí G. Partial nucleotide and amino-acid sequences of the envelope and the envelope/nonstructural protein-1 gene junction of four dengue 2 virus strains isolated during the 1981 Cuban epidemic. Am J Trop Med Hyg 1995:52:241-6. 6. Ricco-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, et al. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 1997;230:244-51. 7. Fernández R, Vázquez S. Serological diagnosis of dengue by an ELISA inhibition method (EIM). Mem Inst Oswaldo Cruz 1990;85:347-51. 8. Vázquez S, Saenz E, Huelva G, González A, Kourí G, Guzmán MG. Detección de IgM contra el virus del dengue en sangre entera absorbida en papel de filtro. Rev Panamericana de Salud Pública. In press 1998. 9. Ministerio de Salud Pública de Cuba. Dengue en Cuba. Julio 1997. Boletín Epidemiológico Organización Panamericana de la Salud 1997;18:7. 10. Lanciotti RS, Calisher CH, Gubler DG, Chang G, Vordam V. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992;30:545-51. 11. Laferte J, Pelegrino JL, Guzmán MG, González G, Vázquez S, Hermida C. Rapid diagnosis of dengue virus infection using a novel 10µl IgM antibody capture ultramicroELISA assay (MAC UMELISA Dengue). Advances in Modern Biotechnology 1992;1:19.4. 12. Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 1990;174:479-93. 13. División de Prevención y Control de Enfermedades, Programa de Enfermedades, Programa de Enfermedades Transmisibles, HCP/HCT, OPS. Resurgimiento del dengue en las Américas. Boletín Epidemiológico. Organización Panamericana de la Salud 1997;18:1-6. 14. Guzmán MG, Kourí G, Bravo J, Hoz de la F, Soler M, Hernández B. Encuesta seroepidemiológica retrospectiva a virus dengue en los municipios Cienfuegos y Palmira. Rev Cubana Med Trop 1989;41:321-32. 15. Guzmán MG, Kourí G, Bravo J, Soler M, Vázquez S, Morier L. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study. Am J Trop Med Hyg 1990;42:179-84. 16. Halstead SB. Observations related to pathogenesis of dengue hemorrhagic fever. Yale J Biol Med 1970;42:350-60. 17. Díaz A, Kourí G, Guzmán MG, Lobaina L, Bravo J, Ruiz A, et al. Description of the clinical picture of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in adults. Bull Pan Am Health Organ 1988;22:133-44. 18. Gubler DJ, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis 1995;1:55-7. --------------------------------------------------------------------------- Dispatches Hantavirus Pulmonary Syndrome in a Chilean Patient with Recent Travel in Bolivia Ricardo Espinoza,* Pablo Vial,*† Luis M. Noriega,*† Angela Johnson,‡ Stuart T. Nichol,‡ Pierre E. Rollin,‡ Rachel Wells,‡ Sherif Zaki,‡ Enrique Reynolds,* and Thomas G. Ksiazek‡ *Clínica Alemana de Santiago, Santiago, Chile; †Escuela de Medicina, Universidad Católica de Chile, Santiago, Chile; ‡Centers for Disease Control and Prevention, Atlanta, Georgia, USA. --------------------------------------------------------------------------- A case of hantavirus pulmonary syndrome (HPS) was serologically confirmed in a critically ill patient in Santiago, Chile. The patient's clinical course had many similarities to that of other HPS patients in North and South America but was complicated by acute severe renal failure. The patient's history included self-reported urban and probable rural rodent exposure during travel in Bolivia. Comparison of a viral sequence from an acute-phase serum sample with other known hantaviruses showed that the hantavirus nucleic acid sequence from the patient was very similar to a virus recently isolated from rodents associated with HPS cases in Paraguay. Since its discovery in 1993 (1) and its association with Sin Nombre virus in North America (2), hantavirus pulmonary syndrome (HPS) has been identified in several countries in South America and is associated with Juquitiba virus in Brazil (3), Andes virus in Argentina and Chile (4-6), and Laguna Negra virus in Paraguay (7,8). Hantaviruses are rodent-borne, and each is associated with a specific primary rodent reservoir. Sigmodontine rodents are the vectors of hantaviruses associated with HPS (3). Case Report A 20-year-old male resident of Santiago, Chile, who had no prior history of medical problems, became ill after he backpacked (from February 4 to March 9, 1997) as a tourist in Bolivia. His travel itinerary included Barrio, Oruro, La Paz, Cochabamba, Villa Tunari, Santa Cruz, Vallegrande, Higueras, and Sucre. The tourist and a fellow traveler stayed in hotel in Santa Cruz where they saw black rats running through the bathroom they used. While in Higueras, they stayed in a rustic adobe house with no floor and joined local villagers in agricultural jobs, primarily harvesting hay. A diagnosis of HPS was considered. On March 26, 3 weeks after returning to Chile, the young man became ill with fever and cough. On March 28, he was admitted to the emergency room of a private hospital in Santiago with high fever (39°C), cough, and chest pain. Chest X-rays showed interstitial infiltrates in the left lower lobe. Laboratory results were as follows: hemoglobin 15.3 gm/dl, white blood cells 5,900, platelets 130,000, erythrocyte sedimentation rate 6, and a C-reactive protein 2.8 mg/dl. He was sent home on clarithromycin 250 mg, twice a day. Three days later (March 31), he returned to the emergency room with persistent high fever, myalgia, and shortness of breath; distal cyanosis was noted. Vital signs were as follows: blood pressure 115/80, pulse 120, temperature 38.5°C, and respiratory rate 32. Physical examination found few petechiae on the forearms, diffuse bilateral rales, regular cardiac rhythm, no murmurs, and no hepatomegaly or splenomegaly. Chest X-rays showed diffuse bilateral alveolar infiltrates. Oxygen saturation was 90%. Laboratory values included white blood cells 11,700 with a left shift, platelets 150,000, hemoglobin 19.4, erythrocyte sedimentation rate 2, C-reactive protein 8.18, international normalized ratio 1.6, activated partial thromboplastin 43s, blood urea nitrogen 38.9 mg/dl, and liver function tests normal limits. The patient was transferred to the intensive care unit with a diagnosis of bilateral pneumonia of unknown etiology and secondary respiratory failure. In 3 to 4 hours, acute respiratory distress developed; arterial blood gases (on 50% oxygen) were PaO(sub 2) 61, PaCO(sub 2) 22.7, pH 7.49, and HCO(sub 3) 17.1. The patient was connected to a ventilator and was started on imipenen, erythromycin, amantadine, dopamine, dobutamine, fluids, and nitric oxide. Two 500-mg doses of methylprednisolone were administered, and a Swan-Ganz catheter was installed. The pulse wedge pressure was 7, and the cardiac output 6.1. During the first 24 hours, acute renal failure developed, and hemodialysis was started. Hypotension was quite refractory to vasoactive drugs (mean BP 30 to 40), and critical hypoxemia (O(sub 2) saturation 70% to 80%) and hypotension persisted for 48 hours. Echocardiography showed a mild pericardial effusion. Blood cultures were negative for bacteria, as well as for influenza, adenovirus, respiratory syncytial virus, and parainfluenza virus. Serologic results for _Mycoplasma_, _Legionella_, and HIV were also negative. Blood gases started to improve on day 3, and the patient's clinical condition also slowly improved. On day 10, hemolytic anemia (Coombs negative) developed, and a bone marrow aspirate showed hemophagocytosis. On day 11, an episode of bleeding from the respiratory tract occurred. However, the patient's ventilatory function continued to improve. On day 15 after admission, his chest X-ray was clearing, but he was still on a mechanical ventilator (O(sub 2) saturation 98% on 40% O(sub 2)). However, he was anuric (BUN 100). On day 17, he had a second episode of bronchial bleeding, and bronchoscopy showed only a 4-mm tracheal ulcer. Platelets were 72,000, and hematocrit fell to 24%. Hantavirus serology was reported positive [Centers for Disease Control and Prevention (CDC), April 18], and intravenous ribavirin was started on April 22. A central venous catheter-related infection with _Pseudomonas_ and _Staphylococcus aureus_ was documented. The patient's condition deteriorated progressively, with further bronchial bleeding and markedly unstable ventilatory function, and required constant administration of vasoactive drugs; he died of massive pulmonary hemorrhage and shock on April 28. A postmortem lung sample was taken with a needle. The patient's serum, collected on April 1, was tested for immunoglobulin G (IgG) and IgM antibodies to Sin Nombre virus at CDC. Both IgG and IgM antibodies were found, which suggested recent infection with a hantavirus associated with Sigmodontine rodents. Subsequent testing of sera collected on April 21 and April 25 confirmed the initial findings. Hematoxylin and eosin stained sections of the lung sample showed diffuse alveolar damage with extensive hyaline membrane formation, proliferation of type II pneumocytes, and fibroblastic and edematous thickening of alveolar walls. Abundant fibrin, necrotic debris, and acute inflammatory cellular infiltrates were also observed in the alveolar spaces. Rare endothelial cells and macrophages were hantavirus-antigen positive by previously described immunohistochemical procedures (9). The destructive changes seen by histopathology and a small amount of antigen found in the patient's tissues are compatible with the long course of the patient's illness (9). Viral RNA was extracted from the earliest serum sample, and reverse transcriptase-polymerase chain reaction amplification with primers designed specifically for hantaviruses associated with Sigmodontine rodents (8) yielded polymerase chain reaction fragments for the S and M segments. These cDNA fragments were sequenced and compared with those of other American hantaviruses. These comparisons show that the virus is closely related to other South American hantaviruses, and most closely related to Laguna Negra virus detected in patients and _Calomys laucha_ rodents (vesper mice) in the Chaco region of Paraguay (7,8). The nucleotide sequence identity was 84% for the G1 protein encoding fragment and 87% for the N protein encoding fragment. However, the deduced amino acid sequences for both fragments were identical to Laguna Negra virus. This shows that the virus associated with this HPS case is a Laguna Negra virus variant and suggests that the virus is probably associated with the same or a very closely related species of rodent host. _C. laucha_, the apparent primary rodent reservoir for Laguna Negra virus, is common in savanna and grassland areas as far north as southern Brazil and Bolivia, throughout much of Paraguay and Uruguay, and in Argentina as far south as Rio Negro province, but not in Chile (10). A number of the lowland rural locations that the patient visited in Bolivia are within the range of _C. laucha_. The clinical course and travel history of the patient and the laboratory serology and molecular characterization of the viral RNA are compatible with infection in Bolivia with a Laguna Negra virus variant. This report and evolving information concerning hantaviruses associated with clinical HPS in Argentina and Paraguay strongly suggest that a diagnosis of HPS should be considered in patients with febrile respiratory distress syndrome throughout Latin America. References 1. Duchin JS, Koster F, Peters CJ, Simpson G, Tempest B, Zaki S, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. N Engl J Med 1994;330:949-55. 2. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, et al. Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science 1993;262:914-7. 3. Nichol ST, Ksiazek TG, Rollin PE, Peters CJ. Hantavirus pulmonary syndrome and newly described hantaviruses in the United States. In: Elliott RM, editor. The Bunyaviridae. New York: Plenum Press, 1996:269-80. 4. Lopez N, Padula P, Rossi C, Lazaro ME, Franze-Fernandez MT. Genetic identification of a new hantavirus causing severe pulmonary syndrome in Argentina. Virology 1996;220:223-6. 5. Centers for Disease Control and Prevention. Hantavirus pulmonary syndrome in Chile-1997. MMWR Morb Mortal Wkly Rep 1997;46:949-51. 6. Lopez N, Padula P, Rossi C, Miguel S, Edelstein A, Ramirez E, et al. Genetic characterization and phylogeny of Andes virus and variants from Argentina and Chile. Virus Research 1997;50:77-84. 7. Williams RJ, Bryan RT, Mills JN, Palma RE, Vera I, de Velasquez F, et al. An outbreak of hantavirus pulmonary syndrome in Western Paraguay. Am J Trop Med Hyg 1997;57:274-82. 8. Johnson AM, Bowen MD, Ksiazek TG, Williams RJ, Bryan RT, Mills JN, et al. Laguna Negra virus associated with HPS in western Paraguay and Bolivia. Virology. In press 1997. 9. Zaki S, Greer PW, Coffield LM, Goldsmith CS, Nolte KB, Foucar K, et al. Hantavirus pulmonary syndrome: pathogenesis of an emerging infectious disease. American Journal of Pathology 1995;146:552-79. 10. Redford KH, Eisenberg JF. Mammals of the neotropics, the southern cone. Chicago: The University of Chicago Press, 1992:279-80. --------------------------------------------------------------------------- Dispatches Prevalence of Tick-Borne Pathogens in _Ixodes scapularis_ in a Rural New Jersey County Shobha Varde,* John Beckley,† and Ira Schwartz* *New York Medical College, Valhalla, New York, USA; and †Department of Health, County of Hunterdon, Flemington, New Jersey, USA --------------------------------------------------------------------------- To assess the potential risk for other tick-borne diseases, we collected 100 adult _Ixodes scapularis_ in Hunterdon County, a rapidly developing rural county in Lyme disease-endemic western New Jersey. We tested the ticks by polymerase chain reaction for _Borrelia burgdorferi_, _Babesia microti_, and the rickettsial agent of human granulocytic ehrlichiosis (HGE). Fifty-five ticks were infected with at least one of the three pathogens: 43 with _B. burgdorferi_, five with _B. microti_, and 17 with the HGE agent. Ten ticks were coinfected with two of the pathogens. The results suggest that county residents are at considerable risk for infection by a tick-borne pathogen after an _I. scapularis_ bite. The vector-borne diseases Lyme disease, human babesiosis, and human granulocytic ehrlichiosis (HGE) are emerging in the Northeast and upper Midwest regions of the United States (1,2). The etiologic agents for these diseases (_Borrelia burgdorferi_, _Babesia microti_, and the HGE agent, respectively) appear to share the same vertebrate reservoir host (_Peromyscus leucopus_) and tick vector (_Ixodes scapularis_) (3-6). Immunologic evidence of human coinfection with these pathogens has been reported (7-10), and a culture-confirmed case of coinfection with _B. burgdorferi_ and the HGE agent has recently been described (11). Thus, in Lyme disease-endemic areas, there may be a substantial risk for coinfection with _B. burgdorferi_ and either _B. microti_ or the HGE-causing rickettsia. Hunterdon County (population 118,000) is a rural, but rapidly developing, county in western New Jersey (Figure), with many homes in wooded settings. In 1996, the county had the third highest case rate of Lyme disease (524 per 100,000) of all counties in the United States (Centers for Disease Control and Prevention case reports). The county has also had many suspected, but no serologically confirmed, cases of HGE. [fig] Figure. Map of New Jersey showing Hunterdon County. Black dots indicate tick collection sites. The risk of acquiring a tick-borne pathogen in a specific geographic location depends largely on tick density and the prevalence rate of the agent in the tick population. Although cultivation of a microbial agent is considered optimal, molecular detection is more practical since it permits the rapid assay of large numbers of individual specimens. We report here the prevalence rate of _B. burgdorferi_, _B. microti_, and the agent of HGE in _I. scapularis_ in Hunterdon County by species-specific polymerase chain reaction (PCR). One hundred adult _I. scapularis_ were collected by drag cloth or from personal clothing at 10 sites in the county during the fall of 1996 (Figure). Ticks were stored at room temperature in 70% ethanol until analysis. DNA was isolated from individual ticks with the Isoquick DNA extraction kit (ORCA Research, Bothell, WA) (12). The final DNA pellets were resuspended in 50 µl of sterile water, and a 10-µl aliquot was used for each PCR test. _B. burgdorferi_-specific PCR used primers IS1 and IS2 (13). The HGE agent was detected by PCR using primers GER3 and GER4, as described by Munderloh et al. (14), and _B. microti_ DNA was detected according to methods used by Persing et al. (15). PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide. For _B. burgdorferi_ and _B. microti_, hybridization with specific probes was carried out after transfer to nylon membranes (13). In each PCR experiment appropriate negative controls were used; these included master mix controls lacking template and reactions containing _B. burgdorferi_ DNA, HGE agent DNA, or _Escherichia coli_ DNA as additional controls for HGE agent, _B. burgdorferi_, or _B. microti_ PCR, respectively. To prevent cross-contamination, specimen preparation, PCR amplification, and post-PCR analysis were performed in separate laboratories. Results of PCR analysis for each of the pathogens on individual ticks are presented in the Table. At least one of the three pathogens was present in 55% of the ticks. The highest prevalence rate was that for _B. burgdorferi_ (43/100), followed by the agent of HGE (17/100) and _B. microti_ (5/100). Furthermore, these pathogens were widely distributed. At least one tick at each site was infected with _B. burgdorferi_, nine of 10 sites had HGE agent-positive ticks, and _B. microti_-positive ticks were found at five different collection sites. Ten ticks were coinfected with two of the three agents; no ticks were infected with all three pathogens. The _B. microti_ PCR amplification products were gel purified and subjected to automated DNA sequencing. The 238 bp products yielded sequences identical to that reported for _B. microti_ 18S ribosomal DNA (15). Table. Prevalence rate of _Borrelia burgdorferi_, human granulocytic ehrlichiosis (HGE) agent, and _Babesia microti_ in 100 adult _Ixodes scapularis_ ------------------------------------------------------------------ Pathogen No. infected ticks ------------------------------------------------------------------ _B. burgdorferi_ 43 HGE agent 17 _B. microti_ 5 _B. burgdorferi_ and HGE agent 6 _B. burgdorferi_ and _B. microti_ 2 HGE agent and _B. microti_ 2 _B. burgdorferi_, HGE agent, and _B. microti_ 0 ------------------------------------------------------------------ The prevalence rates for the three pathogens reported here are consistent with earlier studies at selected sites in the northeastern United States. Telford et al. reported prevalence rates of 36%, 11%, and 9% for _B. burgdorferi_, the HGE agent, and _B. microti_, respectively, in adult _I. scapularis_ on Nantucket Island, Massachusetts; coprevalence of _B. burgdorferi_ and the HGE agent was 4%, and no simultaneous infection with _B. burgdorferi_ and _B. microti_ was observed (4). We have reported prevalence rates of 52% and 53% for _B. burgdorferi_ and the HGE pathogen, and coprevalence of 26% at a site in Westchester County, New York (5). Given the simultaneous infection of _I. scapularis_ with these pathogens, it is not surprising that numerous serosurveys of Lyme disease patients indicate the coincident presence of antibodies to _B. burgdorferi_ and _B. microti_ and human granulocytic ehrlichia (7-10). These findings do not establish concurrent, active infection, nor do they demonstrate simultaneous transmission by a single tick bite; however, they suggest that persons living in disease-endemic areas are exposed to these agents, presumably as a result of tick bites. Human infection by any of the three tick-borne agents alone generally results in similar acute manifestations, including fever, headache, and myalgia (1,10,16). Reported cases of Lyme disease greatly outnumber those of babesiosis or HGE. This, in conjunction with seroepidemiologic data, suggests that many infections with _B. microti_ and granulocytic ehrlichia are subclinical. Dual infection with _B. burgdorferi_ and _B. microti_ often results in more severe illness (10); whether this is also true for concurrent infection by the agents of Lyme disease and HGE remains to be established. Human coinfection by multiple tick-borne agents may account for the variable nature of the clinical manifestations of Lyme disease. This study demonstrates that the agents of Lyme disease, human babesiosis, and HGE coexist in the tick population of a previously identified Lyme disease-endemic region of New Jersey. The study provides further evidence that these three pathogens may be prevalent throughout the range of _I. scapularis._ Infection with any of these three tick-borne pathogens should be considered for residents or visitors of a disease-endemic area who have flulike symptoms and a history of a tick bite. Acknowledgments We thank Gary Wormser and Dennis White for helpful comments, Chris Kolbert and David Persing for providing advice and positive controls for _B. microti_ PCR, and the Hunterdon County Tickborne Disease Research Group and Hunterdon Medical Center for their support. This work was supported in part by NIH grant AR41511. References 1. Walker DH, Barbour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, et al. Emerging bacterial zoonotic and vector-borne diseases: ecological and epidemiological factors. JAMA 1996;275:463-9. 2. Persing DH, Conrad PA. Babesiosis: new insights from phylogenetic analysis. Infect Agents Dis 1995;4:182-95. 3. Anderson J, Mintz E, Gadbaw J, Magnarelli L._ Babesia microti_, human babesiosis and _B. burgdorferi_ in Connecticut. J Clin Microbiol 1991;29:2779-83. 4. Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proc Natl Acad Sci U S A 1996;93:6209-14. 5. Schwartz I, Fish D, Daniels TJ. Prevalence of the rickettsial agent of human granulocytic ehrlichiosis in ticks from a hyperendemic focus of Lyme disease [letter]. N Engl J Med 1997;337:49-50. 6. Piesman J, Mather TN, Telford SR III, Spielman A. Concurrent _Borrelia burgdorferi_ and _Babesia microti_ infection in nymphal _Ixodes dammini_. J Clin Microbiol 1986;24:446-7. 7. Benach JL, Coleman JL, Habicht GS, MacDonald A, Grunwaldt E, Giron JA. Serological evidence for simultaneous occurrences of Lyme disease and babesiosis. J Infect Dis 1985;152:473-7. 8. Magnarelli LA, Dumler JS, Anderson JF, Johnson RC, Fikrig E. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol 1995;33:3054-7. 9. Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of coinfection with _Borrelia burgdorferi, Babesia microti_, and human granulocytic _Ehrlichia_ species in residents of Wisconsin and Minnesota. J Clin Microbiol 1996;34:724-7. 10. Krause PJ, Telford SR III, Spielman A, Sikand V, Ryan R, Christianson D, et al. Concurrent Lyme disease and babesiosis. Evidence for increased severity and duration of illness. JAMA 1996;275:1657-60. 11. Nadelman RB, Horowitz HW, Hsieh TC, Wu JM, Aguero-Rosenfeld ME, Schwartz I, et al. Simultaneous human granulocytic ehrlichiosis and Lyme borreliosis. N Engl J Med 1997;337:27-30. 12. Schwartz I, Varde S, Nadelman RB, Wormser GP, Fish D. Inhibition of efficient polymerase chain reaction amplification of _Borrelia burgdorferi_ DNA in blood-fed ticks. Am J Trop Med Hyg 1997;56:339-42. 13. Schwartz I, Wormser GP, Schwartz JJ, Cooper D, Weissensee P, Gazumyan A, et al. Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. J Clin Microbiol 1992;30:3082-8. 14. Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough JE, et al. Isolation of the equine granulocytic ehrlichiosis agent, _Ehrlichia equi_, in tick cell culture. J Clin Microbiol 1996;34:664-70. 15. Persing D, Mathiesen D, Marshall W, Telford S, Spielman A, Thomford J, Conrad P. Detection of _Babesia microti_ by polymerase chain reaction. J Clin Microbiol 1992;30:2097-103. 16. Boustani MR, Gelfand JA. Babesiosis. Clin Infect Dis 1996;22:611-5. --------------------------------------------------------------------------- Dispatches Plague, a Reemerging Disease in Madagascar Suzanne Chanteau,* Lala Ratsifasoamanana,† Bruno Rasoamanana,*† Lila Rahalison,* Jean Randriambelosoa,† Jean Roux,* and Dieudonné Rabeson† *Institut Pasteur, Antananarivo, Madagascar; and †Ministère Santé, Antananarivo, Madagascar --------------------------------------------------------------------------- [download.gif (1218 bytes)] Human cases of plague, which had virtually disappeared in Madagascar after the 1930s, reappeared in 1990 with more than 200 confirmed or presumptive cases reported each year since. In the port of Mahajanga, plague has been reintroduced, and epidemics occur every year. In Antananarivo, the capital, the number of new cases has increased, and many rodents are infected with Yersinia pestis. Despite surveillance for the sensitivity of Y. pestis and fleas to drugs and insecticides and control measures to prevent the spread of sporadic cases, the elimination of plague has been difficult because the host and reservoir of the bacillus, Rattus rattus, is both a domestic and a sylvatic rat. In the last 15 years, Madagascar (population 13 million) has accounted for 45% of the cases of plague in Africa (1). Epidemiology Plague was brought to the island of Madagascar in 1898 by steamboats from India and has never disappeared. As a result of vaccination campaigns, improved housing and public hygiene, and the discovery of streptomycin and insecticides, plague was controlled in the 1950s. During the next 30 years, only 20 to 50 cases per year were reported in the entire country. However, since 1989, the number of suspected cases has increased steadily (Figure 1). Since 1990, 800 to 1,500 cases of suspected plague have been reported, of which 150 to 230 were smear-positive (presumptive cases) or confirmed by the isolation of Y. pestis (2,3). The population exposed to plague in the plateaus is approximately 5 million; the mean annual rate of known human cases (except in Mahajanga) is 3 to 4 per 100,000 inhabitants. The mean annual death rate is 20% of the confirmed or presumptive cases. Bubonic plague is the main clinical form of the disease (approximately 95% of cases). [Fig] Figure 1. Human plague, Madagascar, 1982-1996. With the exception of the west coast port of Mahajanga, plague is endemic in areas more than 800 m high. The major focus area is a central triangle, and the minor focus is a northern diamond (Figure 2). The main plague foci (in order of importance) are Mahajanga, the district of Ambositra, the town of Antananarivo, and the districts of Fianarantsoa II, Miarinarivo, Betafo, and Soavinandriana. In the plateaus, the human plague season is September to April, while in Mahajanga it is July to November. [Fig] Figure 2. Plague foci in Madagascar. The plague-endemic zones are in the gray areas and in the port of Mahajanga. In 1996, 1,644 suspected plague cases were reported; biologic samples were available for 1,316. A total of 173 confirmed and 56 presumptive cases were officially reported. In 1997, 2,127 suspected cases were reported from January to October, and more than 2,500 total cases were expected by the end of 1997. So far, 260 are confirmed and 33 are presumptive cases. The number of confirmed or presumptive plague cases is underestimated because of underreporting in remote areas and the lack of sensitivity of the bacteriologic techniques used for diagnosis. Our preliminary results, obtained by immunodiagnostic tests, such as anti-F1 antibody detection (4) and F1 antigen tests (tests supplied by the Naval Medical Research Institute, Bethesda, MD), suggest a number of plague cases two to three times higher than that obtained by conventional methods (S. Chanteau and J. Burans, unpub. data). Whether the significant increase between 1996 and 1997 results from better notification of the cases following educational campaigns or a real increase in disease incidence is unknown. In Madagascar, the reservoirs of Y. pestis are two species of rats, Rattus norvegicus and R. rattus. The sylvatic reservoir of Y. pestis is not well documented. The urban domestic rat R. norvegicus, introduced to Madagascar during the 20th century, can only be found (in larger numbers than R. rattus) in coastal cities and Antananarivo. In contrast, R. rattus, probably imported into Madagascar with the first human migrations, has invaded every ecosystem of the island. Thus, it is both a domestic and a wild rodent. It is generally the only type of rodent found in small towns, villages, rice fields, and grasslands. The reservoirs of Y. pestis during the interseason period are the R. rattus and R. norvegicus that have resisted infection; therefore, it is almost impossible to eliminate plague from the island (2). Two species of rat fleas are vectors of plague in Madagascar. The most effective are the classic oriental rat flea, Xenopsylla cheopis, and the endemic flea, Synopsyllus fonquerniei (2); both are found on R. rattus, although X. cheopis is almost exclusively on urban and indoor rodents, while S. fonquerniei is found on outdoor and wild rodents (S. Laventure and J.M. Duplantier, unpub. data). Ongoing research is examining the interrelationships between the two species of rats and the two species of fleas in the epidemiologic cycle of plague in Madagascar. The reemergence of plague in Madagascar probably reflects the general breakdown of traditional measures of plague control. Prolonged maintenance of the domestic rat-to-flea cycle that accounted for the historic plague pandemics will almost surely provide an opportunity for selection of new traits that could make the organism even more virulent. In fact, three new variants of Y. pestis have recently emerged in the region of Ambositra and Ambohimahasoa, one of the most active plague foci of the island. These new ribotypes tend to spread to new geographic areas; whether or not they have acquired selective advantages is being explored (5). Furthermore, the opportunity for gene exchange with enteric bacteria is greatly enhanced. It may be important that the first naturally occurring antibiotic-resistant strain of Y. pestis was recently isolated in Madagascar (6,7). Plague in Mahajanga In Mahajanga (population 150,000), after two epidemics in the beginning of the 20th century, plague remained under control between 1928 and 1990 (2). In July 1991, the disease suddenly reappeared in a shantytown near the marketplace of Marolaka; of the 170 suspected cases, 41 were confirmed or presumptive. After 3 years without reported cases, three successive outbreaks occurred: July 1995 (8,9), July 1996, and July 1997. During the 1995 and 1996 outbreaks, a total of 1,058 suspected cases were reported; only 109 cases were confirmed, and 30 cases were smear-positive. However, serologic testing, mainly F1 antibody detection by enzyme-linked immunosorbent assay (ELISA) (4), allowed the diagnosis of 93 additional cases (10). Thus, the mean annual rate of known human cases is 77 per 100,000 inhabitants. During the ongoing epidemic, from July to October 1997, 376 suspected cases (120 bacteriologically confirmed) have been reported and tended to spread in new quarters of the town. Every human outbreak has been preceded by a high number of rat deaths. During and after epidemics, all shrews captured were infected with many X. cheopis. Ongoing studies are examining the role of the shrew Suncus murinus in the maintenance of plague between epidemics. Plague in Antananarivo Since plague was introduced in the highlands in 1921, it has never disappeared from remote villages; however, in Antananarivo, it has been controlled; no human cases were reported between 1953 and 1978. In 1979, the first confirmed case was found in one of the ancient foci of the town (11). In the 1990s, a growing number of cases have been reported; 10 to 25 each year have been confirmed or are presumptive (Figure 3). In 1996, confirmed plague cases were found in 17 quarters of the capital. The mean annual rate of human cases is 1.4 per 100,000 inhabitants in 1995 and 1996. [Fig] Figure 3. Human plague, Antananarivo, 1976-1996. In 1995, 10% of 625 rats trapped near a marketplace were infected with Y. pestis; 80% were anti-F1 seropositive (S. Chanteau, J.A. Drominy, and B. Rasoamanana, unpub. data). The monthly flea index (mean number of X. cheopis per rat) in this quarter was more than 4 year-round. Such a high index represents a serious threat, especially since most of these fleas are resistant to deltamethrin (12). Other families of insecticides, such as the carbamates, can be proposed. Since 1996, the Ministry of Health and local government have made serious efforts to educate the population and improve public hygiene. A preliminary study of rats trapped in eight other quarters of the capital in 1997 showed a mean seroprevalence of 14% by ELISA (J.A. Drominy and S. Chanteau, unpub. data). The circulation of Y. pestis in the rat population was significantly larger than it was in 1965 (4.5%) (2). National Plague Control Program The national plague control program is financially supported by the World Bank and the French Ministry of Cooperation. The surveillance system used is based on immediate compulsory notification of every suspected case of plague and its biologic confirmation by the Central Laboratory. All patients with suspected cases are treated with streptomycin, and their contacts are treated with sulfonamides to prevent disease spread. Insecticides are used for flea control. Every strain of Y. pestis isolated from humans, rats, and fleas is tested for drug sensitivity, and the susceptibility of fleas to insecticides is determined. The isolation of the first multidrug-resistant strain of Y. pestis in 1995 (6,7) and the increasing resistance of fleas to insecticides have caused much concern (12). This national program, implemented in Madagascar for several decades, has been hampered by economic and operational difficulties and urgently needs to be strengthened. References 1. World Health Organization. Human plague in 1995. Wkly Epidemiol Rec 1997;46:344-8. 2. Brygoo ER. Epidemiologie de la peste à Madagascar. Arch Inst Pasteur Madagascar 1966;35:7-147. 3. Blanchy S, Ranaivoson G, Rakotonjanabelo A. Epidémiologie clinique de la peste à Madagascar. Arch Inst Pasteur Madagascar 1993;60:27-34. 4. Rasoamanana B, Leroy F, Boisier P, Rasolomaharo M, Buchy P, Carniel E, Chanteau S. Field evaluation of an IgG anti-F1 ELISA test for the serodiagnosis of human plague in Madagascar. Clin Diagn Lab Immunol 1997;4:587-91. 5. Guiyoule A, Rasoamanana B, Buchreiser C, Michel P, Chanteau S, Carniel E. Recent emergence of new variants of Yersinia pestis in Madagascar. J Clin Microbiol 1997;35:2826-33. 6. Rasoamanana B, Leroy F, Raharimanana C, Chanteau S. Surveillance de la sensibilité aux antibiotiques des souches de Y. pestis à Madagascar de 1989 à 1995. Arch Inst Pasteur Madagascar 1995;62:108-10. 7. Galimand M, Guiyoule A, Gerbaud G, Rasoamanana B, Chanteau S, Carniel E, Courvalin P. Multiple antibiotic resistance in Yersinia pestis mediated by a self-transferable plasmid. N Engl J Med 1997;337:677-80. 8. Rasolomaharo M, Rasoamanana B, Andrianirina Z, Buchy P, Rakotoarimanana N, Chanteau S. Plague in Mahajanga, Madagascar. Lancet 1995;346:1234. 9. Boisier P, Rasolomaharo M, Ranaivoson G, Rasoamanana B, Rakoto L, Andriamahefazafy B, Chanteau S. Urban epidemic of bubonic plague in Mahajanga, Madagascar. Epidemiological aspects. Trop Med Int Health 1997;5:422-7. 10. Chanteau S, Rasoamanana B, Rasolomaharo M, Leroy F, Rahalison L, Buchy P, et al. Apport de la détection des anticorps anti-F1 et de l'antigénémie F1 dans l'analyse des épidémies de peste de la ville de Mahajanga. Proceedings of Colloque Scientifique de la Réunion du Conseil des Directeurs des Instituts Pasteurs et Instituts Associés; 1997; Paris. 11. Coulanges P. Situation de la peste à Tananarive, de son apparition en 1921 à sa résurgence en 1979. Arch Inst Pasteur Madagascar 1989;56:9-35. 12. Laventure S, Ratovonjato J, Rajaonarivelo E, Rasoamanana B, Rabarison P, Chanteau S, Roux J. Résistance aux insecticides des puces pestigènes à Madagascar et implications pour la lutte antivectorielle. Proceedings of the 5ème Congrès International de Médecine Tropicale de Langue Française; 1996; Mauritius. --------------------------------------------------------------------------- Dispatches Bayou Virus-Associated Hantavirus Pulmonary Syndrome in Eastern Texas: Identification of the Rice Rat, _Oryzomys palustris_, as Reservoir Host Norah Torrez-Martinez,* Mausumi Bharadwaj,* Diane Goade,* John Delury,† Peggy Moran,* Bradley Hicks,† Beverlee Nix,‡ James L. Davis,¶ and Brian Hjelle* *University of New Mexico School of Medicine, Albuquerque, New Mexico, USA; †Texas Department of Health, Austin, Texas, USA; ‡Texas Department of Health, Houston, Texas, USA; and ¶St. Elizabeth's Hospital, Beaumont, Texas, USA. --------------------------------------------------------------------------- We describe the third known case of hantavirus pulmonary syndrome (HPS) due to Bayou virus, from Jefferson County, Texas. By using molecular epidemiologic methods, we show that rice rats (_Oryzomys palustris_) are frequently infected with Bayou virus and that viral RNA sequences from HPS patients are similar to those from nearby rice rats. Bayou virus is associated with _O. palustris_; this rodent appears to be its predominant reservoir host. The 1993 discovery of a clinically distinct form of hantavirus disease now known as hantavirus pulmonary syndrome (HPS) highlighted the existence of a previously unrecognized complex of New World hantaviruses, each of which is associated with a specific rodent of the subfamily Sigmodontinae, family Muridae. The prototype of this complex is the Sin Nombre (SN) virus of the deer mouse, _Peromyscus maniculatus_. SN virus, which occurs most frequently in the western United States and Canada, is responsible for more than 95% of cases of HPS in North America. The case-fatality rate of HPS is nearly 50% (1). Three viruses other than SN have been associated with HPS in North America. All cases associated with viruses other than SN have occurred outside the range of the deer mouse. Two cases have been linked to New York virus in the northeastern United States; the white-footed mouse, _P. leucopus_, was the only hantavirus carrier in the area where the infections were contracted. The cotton rat (_Sigmodon hispidus_)-associated Black Creek Canal virus is believed to have caused a single case in southern Florida, albeit without molecular confirmation. Bayou (BAY) virus has been identified from cDNA sequences amplified from the necropsied tissues or the blood of patients from Louisiana and eastern Texas (1-6). Evidence implicates the rice rat, _Oryzomys palustris_, as the carrier of BAY virus. In a survey of archived rodent tissue samples obtained from species indigenous to Louisiana, only _O. palustris_ samples were positive for hantavirus antibodies, and cDNAs of a BAY-like virus were amplified from two of those samples (7). A few additional BAY virus-RNA-positive rice rat samples have appeared in subsequent studies (6). However, no substantial rodent collections have been conducted in association with BAY virus HPS cases, and no direct molecular association has been found between human BAY virus genomic sequences and those obtained from samples from rodents trapped at possible sites of human infection. The purpose of this study was to identify the carrier rodents associated with two human cases of BAY virus infections and to further characterize the clinical consequences of human infection with BAY virus. Diagnostic Studies Initial serologic investigations to detect antibodies to hantavirus used a sandwich µ-capture enzyme-linked immunosorbent assay (ELISA) as well as an immunoglobulin (Ig) G ELISA, with recombinant SN virus nucleocapsid (N) protein as the target antigen (8). Confirmatory testing with a larger array of recombinant-expressed viral N antigens was conducted by using strip immunoblot and Western blot formats (9-11). The strip immunoblot assay incorporates five membrane-bound antigens (recombinant-expressed SN virus N and G1, recombinant-expressed Seoul virus N, and synthetic peptides of SN N and G1)(11). Antibodies reactive to these antigens are detected with an anti-human immunoglobulin heavy+light chain conjugate, and the assay thus has some sensitivity for IgM but greater sensitivity for IgG. The Western blot uses both anti-IgG and anti-IgM conjugates in separate assays (9). Western blot studies used a panel of affinity-purified, full-length N antigens produced in _Escherichia coli_ in fusion with the phage T7 gene 10 protein in the pET23b vector (10). Recombinant N antigens were purified over metal chelation affinity columns through a polyhistidine tag on the C-terminus of each protein. The recombinant proteins were produced in an isogenic background and loaded at 500ng/lane before sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and electrophoretic transfer. The antigens were derived from the following hantaviruses: SN (3H226); BAY (OP-LA-475); Rio Mamoré (OM-556), Muleshoe (SH-Tx-339), Puumala (P360), and Seoul (80/39). Rodent Collection and Processing Since the two BAY virus-HPS cases from Texas occurred in neighboring cities, this study includes rodent samples collected in the investigation of both case P/Tx (6) and the case described here, T/Tx. Sherman live-traps were used to collect rodents in Jefferson County and neighboring Orange County. Heart blood samples were screened by a recombinant SN virus N antigen ELISA (8). Viral Sequencing and Phylogenetic Analyses Human peripheral blood mononuclear cells (2 x 106) and rodent lung, kidney, and spleen samples (~200 mg total) were used to make RNA (6,10). A standard set of partially nested primers in the viral S segment was used in reverse transcription-polymerase chain reaction (RT-PCR) analyses of the RNA (6,10). This procedure produces a 442-nucleotide (nt) amplification product; 397 nt of that sequence is internal to the primers. The PCR products were subjected to direct DNA sequencing with an ABI 377 automatic sequencer. Phylogenetic trees were constructed from the 397 nt of informative sequence by using maximum parsimony with PAUP 3.1 software (12). A Third Case of HPS Due to BAY Virus Patient T/Tx is a 54-year-old African-American man from Jefferson County, Texas. A heavy tobacco user, he has a more than 100 pack-year smoking history and chronic shortness of breath. Approximately 2 weeks before admission, his spouse noted that he had increased fatigue and somnolence. By August 20, 1996, his symptoms worsened, and he sought medical care. He visited a local emergency room with complaints of increasing shortness of breath and low-grade fever for 2 days. Hospital personnel believed that he had probable chronic obstructive pulmonary disease and chronic bronchitis. Results of a physical examination of the patient at that time were unremarkable, and a chest radiograph was interpreted as hyperinflated but without infiltrates (Figure 1A). He was afebrile (37°C) in the emergency room. Laboratory analysis showed a white blood cell count of 4,080/µl and a platelet count of 122,000/µl. He was given amoxicillin and bronchodilators and was discharged. [Fig] Figure 1. Chest radiographs of patient T/Tx at hospital admission on August 20, 1996 (Panel A), and during a period of increasing respiratory distress on August 22, 1996 (Panel B). Note the diffuse interstitial infiltrate and peribronchial cuffing that developed over that interval. On August 21, the patient returned to the emergency room with persistent and worsening dyspnea. He also had flulike symptoms, including myalgias, and had had a temperature of 39.4°C. Physical examination revealed bilateral rhonchi. He had a blood pressure of 90/60, pulse of 96/min, respiratory rate of 16/min, and an oral temperature of 37°C. Laboratory analysis noted an increase in his white blood cell count to 7,700/µl with a left shift and a decrease in platelet count to 65,000/µl. The patient was admitted to the hospital with a preliminary diagnosis of chronic obstructive pulmonary disease exacerbation and possible pneumonia. He was given broad-spectrum antibiotics, methylprednisolone, aminophylline, and ipatronium nebulizer treatments. The patient's condition deteriorated rapidly over the next 24 hours, and he was severely short of breath by the second hospital day. He was also febrile and was transferred to the intensive care unit to manage his worsening shortness of breath. His oxygen demand increased, and on the third hospital day, the patient required endotracheal intubation. A physical examination foundbilateral expiratory wheezing and bibasilar rales. A chest radiograph showed increasing bilateral interstitial and alveolar infiltrates. By the fourth hospital day, the patient required 100% oxygen and positive end-expiratory pressure support. A chest radiograph again showed interstitial and alveolar infiltrates with peribronchial cuffing and pleural effusion. His fluid intake was restricted to manage his pulmonary edema; he briefly required dopamine for hypotension. Blood and urine cultures remained negative. Sputum Gram stain showed a moderate number of white blood cells and few budding yeast; culture showed no pathogens. Serologic tests for mycoplasma and _Legionella_ were negative. By the fifth hospital day, the patient's condition had begun to improve. Over the next 5 days the fever diminished, the pulmonary edema improved, and requirement for supplemental oxygen decreased. The endotracheal tube was removed on the 10th day of hospitalization; by the 14th day, the patient was discharged-ambulatory, afebrile, and no longer requiring supplemental oxygen. The hematologic laboratory findings were generally most abnormal when the patient was most acutely ill clinically; the peak serum chemistry abnormalities trailed the clinical illness by several days. The maximum white blood cell count was 31,700/µl on August 23, with a differential count of 7% lymphocytes (including the characteristic plasmacytoid forms; [13]), 44% mature neutrophils, 35% band neutrophils, 3% metamyelocytes, and 2% myelocytes. The platelet count reached a nadir of 19,000/µl on that day, and the hemoglobin reached a maximum of 17.2 g/dL on August 22. The serum creatinine was 0.6 mg/dL on August 24 but peaked at 1.9 mg/dL on August 25. This mild azotemia was associated with proteinuria (300 mg/dL in a spot sample obtained on August 22). The serum enzymes creatine kinase and lactic dehydrogenase peaked on August 27 at 917 units/L and 933 units/L, respectively. Fractionation of the creatine kinase and lactic dehydrogenase isoenzyme forms were not supportive of myocardial injury as a cause for their elevation. Serum levels of the liver transaminases were modestly elevated. Environmental Assessment and Rodent Collection The patient worked as a laborer at a railroad construction company. For 2 months before his illness, he replaced old rail lines at three industrial plants (Beaumont, Neches River, and Orange) in Orange and Jefferson Counties and cleaned the yard and garage at work headquarters. He manually removed and replaced tracks and ties serving warehouses, transport pipes, and scrap metal piles. The Beaumont and Orange work sites were chemical plants. All rail work occurred within the confines of the mowed plant complex. Some of the track sites were next to drainage ditches and canals; others coursed by warehouses reported by plant employees to have rats. The patient had not seen any rodents in the months before becoming ill, but he remembered rodent droppings at a wooden crew trailer at the Beaumont plant. All rodents trapped at these plants tested negative for hantavirus antibodies. The Neches River work site was a recycling plant situated for commerce by rail and ship. The rails were within several meters of permanent bayous and swamps, piles of scrap metal, and the Neches River. The vegetation near the water was dense with trees and undergrowth. Truck and train traffic at the plant created a substantial dust problem. The site contained stray dogs and cats as well as a variety of wildlife, including snakes and alligators. Although trapped heavily, few rodents were collected from near the tracks and adjacent swamps. All rodents trapped at this facility were negative for hantavirus antibodies. Although the patient lived in an older pier-and-beam home, he and his wife saw no evidence of rodent infestation. A Texas Department of Health investigator verified the absence of rodents and rodent excreta. Although traps were set, no rodents were collected at the residence. During the 2 months before he became ill, the patient visited a casino boat in Louisiana and fished three times from the Pleasure Island jetty in southern Jefferson County. The jetty had large rock boulders at the water's edge and an asphalt road along its length. Sand and dense 6-foot high grasses covered the remainder of the jetty. Household trash and fishing debris were scattered throughout the area. Most rodents collected by the Texas Department of Health investigation team during this investigation were from this jetty; and most were _O. palustris_, including three seropositive specimens (Area 1, Figure 2). [Fig] Figure 2. Location of rodents trapped during investigations related to patient P/Tx (March 1996; [6]) and patient T/Tx (October 1996; this report). The locations in which rodents were collected are broadly classified into four areas, designated 1 through 4. Other sites were chosen between the patient's work sites and home because their habitats were likely to support rodents. Rice rat habitats (permanent water and grasses) were among those targeted, because the first Jefferson County hantavirus investigation documented the Bayou strain of hantavirus in _O. palustris_ (6). The Table and Figure 2 show the location and species of each rodent collected and the number of seropositive specimens in each of four targeted areas. Four hantavirus antibody-positive rodents were trapped at these miscellaneous locations during the investigation of the current HPS case. Two of the seropositive samples were from Area 1 of Figure 2 (not the jetty), and two were in Area 2. Area 2 yielded two seropositive rodents during the first Texas BAY virus case investigation (6). Table. Rodents collected in association with two cases of Bayou virus-hantavirus pulmonary syndrome in Jefferson and Orange Counties, Texas, March and October 1996 --------------------------------------------------------------------------- No. specimens collected (no. seropositive) ----------------------------------- No. PCR (sup b) Area Area Area Area Total tested Species 1(sup a) 2 3 4 (no. pos.) --------------------------------------------------------------------------- _Baiomys taylori_ 0 0 1(0) 3 (0) 4 (0) 0 _Mus musculus_ 30 (0) 21 (0)12 (1) 26 (0)88 (1) 1 (0) _Ochrotomys nuttali_ 0 0 0 1 (0) 1 (0) 0 _Oryzomys palustris_ 45 (7) 20 (5)3 (0) 8 (3) 76 (15) 15 (14) _Peromyscus leucopus_ 0 3 (0) 0 0 3 (0) 0 _Rattus norvegicus_ 1 (0) 1 (0) 0 1 (0) 3 (0) 0 _Rattus rattus_ 21 (0) 8 (0) 0 1 (0) 30 (0) 0 _Reithrodontomys fulvescens _ 0 7 (0) 0 7 (0) 14 (0) 0 _Reithrodontomys humulis_ 1(0) 0 0 2 (0) 3 (0) 0 _Sigmodon hispidus_ 95 (0) 5 (0) 11 (0) 4 (1) 114 (1) 1 (1) --------------------------------------------------------------------------- (sup a) Refer to Figure 2 for map areas. (sup b) PCR=polymerase chain reaction. --------------------------------------------------------------------------- Diagnostic Studies The IgG ELISA assay for SN virus antibodies in patient T/Tx's serum was negative, but the IgM test was positive at a 1:6400 dilution (8). The strip immunoblot assay show ed antibody reactivities typically seen with HPS caused by a hantavirus other than SN virus, with 2+ reactivity to the immunodominant SN virus N peptide and 4+ reactivity to the full-length recombinant N protein (11). There was no reactivity to the SN virus G1 antigen, either in peptide or recombinant form, or to Seoul virus recombinant N protein (data not shown). Antibody reactivity to the SN virus G1 antigen is specific for infection with SN virus. Western blot analysis of patient T/Tx's serum showed IgG antibodies to hantavirus N proteins (Figure 3, Panel A). Although the most intense staining was observed with the homologous (BAY virus) antigen, varying cross-reactivity was present against the N antigens of Muleshoe, SN, Rio Mamoré, and Puumala viruses but not of Seoul virus. A similar pattern was observed when another blot membrane was probed with an anti-human IgM conjugate, except that reactivities were somewhat more intense (data not shown). Serum samples of seropositive rice rats and deer mice showed similar cross-reactivities, although the intensity of reactivity to a particular N antigen was related to the sequence similarity between the membrane-bound antigen and the virus against which the antibodies were directed (Figure 3, Panels B and C). [Fig] Figure 3. Western blot assay for detecting IgG antibodies in patient and rodent blood samples. A 1:500 dilution of serum was used to probe Western blots containing equimolar amounts of recombinant-expressed N antigens of various hantaviruses: 1) Bayou; 2) Muleshoe; 3) Puumala; 4) Rio Mamoré; 5) Seoul; and 6) Sin Nombre. Serum samples are directed against specific hantaviruses as verified by reverse transcription-PCR and sequence analyses: (A) patient T/Tx, Bayou virus; (B) Bayou virus-seropositive _Oryzomys palustris_ specimen #505, collected in Jefferson County, Texas; (C) a Sin Nombre-seropositive deer mouse, collected in California; (D), a negative control (hantavirus-seronegative) deer mouse. Antibodies bound to the solid-phase antigens were detected with alkaline phosphatase-conjugated anti-human IgG (A) or with anti-_Peromyscus leucopus_ IgG (B-D). The arrow indicates the migration of the full-length T7-viral N antigen, ~55kDa. RT-PCR analyses and sequencing of the cDNA product of the patient's blood sample identified definitively BAY virus as the etiologic agent. When the 442 nt viral S segment amplification product from patient T/Tx was compared with the homologous sequence of other hantaviruses, it was closely similar to previously described BAY viruses from Louisiana and Texas (Figure 4). Nine viral sequences from seropositive _O. palustris_ and one from a seropositive _S. hispidus_ from Jefferson County, Texas, and Cameron and Terrebonne Parishes, Louisiana, were compared with those of patients T/Tx and P/Tx and with that of a 1993 case-patient from northern Louisiana (Case-LA-93, Figure 4). [Fig] Figure 4. Unweighted maximum parsimony tree produced by PAUP 3.1 software comparing a 397 nt portion of the hantavirus S genomic segments (residues 207 to 603) of patients and rodents infected with Bayou virus. A selection of other prototypical hantavirus sequences, each of which was compared antigenically in Figure 2, was included for comparison. Other hantaviruses are abbreviated as follows: RM=Rio Mamoré; MULE=Muleshoe; SN= Sin Nombre; PUU=Puumala; and SEO=Seoul. Human-derived Bayou virus sequences are indicated by Case, and rodent sequences by OP (_Oryzomys palustris_) or SH (_Sigmodon hispidus_). TX=Texas, LA=Louisiana. The sequences of all the Jefferson County rodents and patient T/Tx were closely related to each other, differing by 1 to 10 nt (0.25% to 2.5% of 397 evaluable residues) in pairwise comparisons. Four rodent-derived viral sequences differed from that of the patient T/Tx virus by 4 nt (1%), and all were from rodents collected in Jefferson County. Except for a brief visit to a Louisiana casino, patient T/Tx reported that he had not traveled outside east Texas in the 2 months before his illness. The generally close genetic similarity among the viral sequences from east Texas supports the hypothesis that patient T/Tx became infected in that region (14). Potential exposure sites, as defined by his travel history, were too numerous to allow us to identify a more precise site of infection. No rodent sequence was completely identical to that of patient T/Tx. The sequence obtained from patient P/Tx (6), who was thought to have been infected in Jefferson County, was not closely aligned with any eastern Texas rodent sequence or with that of patient T/Tx. The closest of the 11 eastern Texas viral sequences differed from that of patient P/Tx at 13 residues (3.3%). One sequence available from previous studies was far closer to that of patient P/Tx, differing at only four residues (1%). This sequence was obtained from a rice rat (OP-LA-5) collected in Cameron Parish, Louisiana, approximately 30 km from the former residence of patient P/Tx. Although patient P/Tx initially reported no travel back to his former residence (where his mother still lived) in the 6 weeks before his illness (6), he later was not certain about his travel history in the weeks before his illness, stating that he might have visited his mother in Louisiana in the weeks before his illness. In previous studies, _O. palustris_ was tentatively implicated as the predominant rodent reservoir for BAY virus, but the sample sizes were small, and specific cases of HPS were not linked to the occurrence of BAY virus-infected rodents at the presumed sites of infection (6,7). For this study, analysis of a substantial collection of rodents collected in the vicinity of sites of human infection showed that _O. palustris_ is the species with the highest hantavirus seroprevalence. Furthermore, the hantavirus genotypes in circulation in the eastern Texas/western Louisiana area were related to those of two human case-patients from the area, and all but one of those rodent-derived sequences came from _O. palustris_. Rice rats are found in wetlands and marshes from Texas throughout the southeastern United States, extending north as far as New Jersey on the eastern seaboard. Like all carriers of viruses associated with HPS, _O. palustris_ is a member of the subfamily Sigmodontinae, family Muridae. Although only a single species exists in the United States, the tribe Oryzomyini has scores of species in Latin America, where HPS is increasingly recognized as an important zoonotic disease. Viruses similar to BAY virus have been recognized recently in members of the Oryzomyini from Bolivia and Argentina, and in some cases these rodent-borne viruses have been linked to HPS in humans (1,15). The possibility that the genetically distinct clade of viruses associated with oryzomine and _Sigmodon_ rodents produces a disease that is qualitatively different from SN virus-associated HPS (16) has been raised repeatedly as new cases have been identified (5,6,17). Although patient T/Tx had a relatively mild course of HPS, his serum creatinine, urine protein concentration, and creatine kinase were nevertheless slightly elevated. Both the elevation of creatine kinase and chemical evidence of myositis have been observed in HPS caused by viruses of the oryzomine and _Sigmodon_ clade, but much less commonly in SN virus infection (1). Further studies are needed to determine the basis for the variant clinical symptoms of HPS in these patients. Acknowledgments We thank G. Haigh, McNeese State College, for donating the rice rat specimen OP-LA-5; J. Griffith for technical assistance with sequencing; and W. George, St. Elizabeth Hospital, for first recognizing HPS as a possible cause of patient T/Tx's illness. This study was supported by DHHS grant RO1 AI36336 (to B.H.). References 1. Schmaljohn C, Hjelle B. Hantaviruses: a global disease problem. Emerg Infect Dis 1997;3:95-104. 2. Song J-W, Baek L-J, Gajdusek DC, Yanagihara R, Gavrilovskaya I, Luft BJ, et al. Isolation of pathogenic hantavirus from white footed mouse (_Peromyscus leucopus_). Lancet 1994;344:1637. 3. Rollin PE, Ksiazek TG, Elliott LH, Ravkov EV, Martin ML, Morzunov S, et al. Isolation of Black Creek Canal virus, a new hantavirus from _Sigmodon hispidus_ in Florida. J Med Virol 1995;46:35-9. 4. Morzunov SP, Feldmann H, Spiropoulou CF, Semenova VA, Rollin PE, Ksiazek TG, et al. A newly recognized virus associated with a fatal case of hantavirus pulmonary syndrome in Louisiana. J Virol 1995;69:1980-3. 5. Khan AS, Spiropoulou CF, Morzunov S, Zaki SR, Kohn MA, Nawas SR, et al. Fatal illness associated with a new hantavirus in Louisiana. J Med Virol 1995;46:281-6. 6. Hjelle B, Goade D, Torrez-Martinez N, Lang-Williams M, Kim J, Harris RL, Rawlings JA. Hantavirus pulmonary syndrome, renal insufficiency and myositis associated with infection by Bayou hantavirus. Clin Infect Dis 1996;23:495-500. 7. Torrez-Martinez N, Hjelle B. Enzootic of Bayou hantavirus in rice rats (_Oryzomys palustris_) in 1983. Lancet 1995;346:780-1. 8. Ksiazek TG, Peters CJ, Rollin PE, Zaki S, Nichol S, Spiropoulou C, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995;52:117-23. 9. Jenison S, Yamada T, Morris C, Anderson B, Torrez-Martinez N, Keller N, Hjelle B. Characterization of human antibody responses to Four Corners hantavirus infections among patients with hantavirus pulmonary syndrome. J Virol 1994;68:3000-6. 10. Rawlings JA, Torrez-Martinez N, Neill SU, Moore GM, Hicks BN, Pichuantes S, et al. Cocirculation of multiple hantaviruses in Texas, with characterization of the S genome of a previously-undescribed virus of cotton rats (_Sigmodon hispidus_). Am J Trop Med Hyg 1996;55:672-9. 11. Hjelle B, Jenison S, Torrez-Martinez N, Herring B, Quan S, Polito A, et al. Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis. J Clin Microbiol 1997;35:600-8. 12. Swofford DL. PAUP: phylogenetic analysis using parsimony [computer program]. Version 3.1.1. Champaign (IL): Illinois Natural History Survey;1991. 13. Nolte KB, Feddersen RM, Foucar K, Zaki SR, Koster FT, Madar D, et al. Hantavirus pulmonary syndrome in the United States: a pathological description of a disease caused by a new agent. Hum Pathol 1995;26:110-20. 14. Hjelle B, Torrez-Martinez N, Koster FT, Jay M, Ascher MS, Brown T, et al. Epidemiologic linkage of rodent and human hantavirus genomic sequences in case investigations of hantavirus pulmonary syndrome. J Infect Dis 1996;173:781-6. 15. Bharadwaj M, Botten J, Torrez-Martinez N, Hjelle B. Rio Mamoré virus: genetic characteristics of a newly-recognized hantavirus of the pygmy rice rat _Oligoryzomys microtis_ from Bolivia. Am J Trop Med Hyg. In press 1997. 16. Duchin JS, Koster FT, Peters CJ, Simpson GL, Tempest B, Zaki SR, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. N Engl J Med 1994;330:949-55. 17. Khan AS, Gaviria JM, Rollin PE, Hlady WG, Ksiazek TG, Armstrong LR, et al. Hantavirus pulmonary syndrome in Florida: association with the newly identified Black Creek Canal virus. Am J Med 1996;100:46-8. --------------------------------------------------------------------------- Dispatches Laboratory Survey of Drug-Resistant _Streptococcus pneumoniae_ in New York City, 1993-1995 Richard Heffernan, Kelly Henning, Anne Labowitz, Annette Hjelte, and Marcelle Layton New York City Department of Health, New York, New York, USA --------------------------------------------------------------------------- Wide geographic variation in the prevalence of drug-resistant _Streptococcus pneumoniae_ demonstrates the importance of tracking antimicrobial resistance locally. This survey of hospital microbiology laboratories in New York City found that penicillin resistance (MIC >/= 2.0 mg/ml) increased from 1.5% of _S. pneumoniae_ isolates in 1993 to 6.3% in 1995 and that in 1995, one-third of isolates nonsusceptible to penicillin (MIC >/= 0.1 µg/ml) were also nonsusceptible to an extended-spectrum cephalosporin (MIC >/= 1 µg/ml). The emergence of drug-resistant _Streptococcus pneumoniae_ underscores the need for timely, local, population-based surveillance of antimicrobial resistance. The prevalence of resistance in U.S. communities varies widely, with 2% to 53% of _S. pneumoniae_ isolates found to have reduced susceptibility to penicillin (1-4). The Centers for Disease Control and Prevention recommends that empiric antibiotic therapy for pneumococcal infections be based upon local susceptibility patterns (2,5). However, few communities track drug-resistant _S. pneumoniae_. The Survey To estimate the prevalence of drug-resistant _S. pneumoniae_ in New York City, we surveyed hospital-based clinical microbiology laboratories from 1993 to 1995. A standardized questionnaire was mailed annually to each laboratory, and those that did not respond were contacted by telephone or were visited. To evaluate compliance with _S. pneumoniae_ penicillin susceptibility testing guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS) (6), we asked about criteria for selecting specimens and techniques for oxacillin disk diffusion screening and determination of penicillin MICs. To determine the prevalence of penicillin resistance, we asked for the number of _S. pneumoniae_ isolates identified during the year, the number tested for susceptibility to penicillin, and the number found to be possibly resistant by the oxacillin disk diffusion test and penicillin-intermediate or -resistant by MIC testing. We also asked that information be provided separately for isolates from normally sterile sites (e.g., blood, cerebrospinal fluid) and from nonsterile sites (e.g., sputum, nasopharyngeal swab). In 1995, we added questions regarding the MIC test results for extended-spectrum cephalosporins (ESCs), including the number of penicillin-nonsusceptible isolates that were also nonsusceptible to an ESC. No individual patient information was obtained. A report summarizing the results of the survey and describing NCCLS guidelines was mailed annually to microbiology laboratories, hospital infection control departments, and local infectious disease physicians and pediatricians. Analysis Of 67 hospital-based clinical microbiology laboratories in New York City, 100% completed the survey in 1993, 98% in 1994, and 100% in 1995. Overall, more than 5,000 _S. pneumoniae_ isolates were reported annually. Data were analyzed by using EpiInfo Version 6.0 (CDC, Atlanta, GA, USA). Drug-susceptibility results are presented for laboratories that conformed with NCCLS guidelines and provided complete data on all _S. pneumoniae_ isolates identified (Table 1). Table 1. Penicillin resistance among _Streptococcus pneumoniae_ isolates at New York City hospital laboratories --------------------------------------------------------------------------- No. of isolates 1993 1994 1995 No. (%) No. (%) No. (%) ------------------------------------------------------------------ Screened with oxacillin disk (sup 3,227 4,133 4,912 a) zone size /= 0.1 and /= 2.0 µg/ml). (sup e)Number of laboratories reporting sterile site isolate results in 1993, 1994, 1995 was 8, 12, 32, respectively. (sup f)Number of laboratories reporting nonsterile site isolate results in 1993, 1994, 1995 was 5, 9, 28, respectively. --------------------------------------------------------------------------- Susceptibility Criteria The NCCLS recommends routine screening by the oxacillin disk diffusion test of clinically important _S. pneumoniae_ isolates for susceptibility to penicillin. Isolates with a zone size /= 0.1 and /= 2 µg/ml) should also have MICs determined for susceptibility to an ESC such as cefotaxime or ceftriaxone (ESC-intermediate MIC = 1 µg/ml; ESC-resistant MIC >/= 2 µg/ml) (6). We will use the term "nonsusceptible" to refer to both intermediate and resistant isolates. Findings The proportion of laboratories conforming with NCCLS guidelines for penicillin susceptibility testing of _S. pneumoniae_ increased from 22% in 1993 to 69% in 1995. This was due to an increase in the number of laboratories that screened all isolates, a sharp decrease in the use of automated MIC tests, and a fourfold rise in the use of antibiotic gradient strips for determining MICs (Table 2). Overall, the proportion of isolates with oxacillin disk diffusion test zone size 100°F (37.8°C, oral or equivalent) and cough or sore throat of 72 hours duration. Other symptoms, such as streptococcal pharyngitis, were excluded. No age or gender restrictions were included. Volunteers had to have been in Nepal for the 5 days preceding illness. Only the first patient in any single household with similar symptoms within days of other household members was asked to participate. The AFRIMS field station in Kathmandu (locally known as the Walter Reed/AFRIMS Research Unit-Nepal or WARUN) was responsible for shipping specimens collected by the CIWEC Clinic to AFRIMS, Thailand. Since dry ice was not available in Kathmandu, dry ice and shipping containers were sent by AFRIMS, Thailand for use by WARUN. Shipments from WARUN were then sent back to AFRIMS, where specimens were repacked in dry ice and sent for testing at the central laboratory of the U.S. Air Force's Project Gargle (6) in San Antonio, Texas. Project Gargle has been testing viral respiratory specimens from distant Air Force installations for more than 20 years. Each specimen was tested for influenza A and B; parainfluenza virus 1, 2, and 3; adenovirus, enterovirus; and herpesvirus. Characterization of selected influenza A and B isolates by hemagglutination-inhibition testing was performed by the Centers for Disease Control and Prevention (CDC). Between December 1996 and February 1997, the CIWEC staff collected specimens from 18 patients. Samples were collected from 11 (61%) residents and seven (39%) tourists, who were evenly distributed by gender and had a median age of 35 years. Influenza B/Beijing/184/93-like viruses were isolated from five (28%) of the 18 specimens. All patients from whom influenza viruses were obtained had mild illnesses with fever and upper respiratory syndromes. Herpes virus type 1 and adenovirus type 6 were each identified in one other specimen. No respiratory viruses were identified in the remaining 11 specimens. Because of the importance of China in the emergence of new strains of influenza, CDC's WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza has worked with colleagues in China to establish a national Chinese network of influenza surveillance sites. Analysis of viruses isolated in China between 1988 and 1997 in comparison with other viruses obtained through WHO's global influenza surveillance network has shown that influenza variants are frequently identified in China before becoming prevalent in other regions of the world. Nepal is another especially valuable surveillance site, given its location between China and India (at the crossroads between northern and southern Asia) and its historic importance as a trans-Himalayan trade route. Especially relevant are data from China demonstrating that the two antigenically and genetically distinct lineages of influenza B viruses represented by B/Victoria/02/87 and B/Yamagata/16/88 (7) have continued to circulate and evolve in China, while only viruses related to B/Yamagata have been detected elsewhere in the world and are represented in the current trivalent vaccine by the B/Bejing/184/93-like component. Virologic surveillance in surrounding countries (8) such as Nepal is necessary to detect geographic spread of B/Victoria-like virus in the region. Our data suggest that these viruses have not yet spread to Kathmandu. Our unique international partnership between several civilian and military organizations (e.g., CIWEC Clinic, CDC, U.S. Air Force, and U.S. Army) demonstrates the feasibility of such partnerships as well as the usefulness of influenza surveillance data at both the local and global levels. Despite the small number of isolates obtained during this study, we were able to determine that the influenza B component of the trivalent vaccine prepared for the 1996-1997 influenza season would likely have offered protection for travelers and the local population against the influenza B strains isolated in Kathmandu. Ongoing surveillance data will establish geographic and temporal patterns of circulation of influenza viruses and thus provide valuable information for guiding public health policies for influenza vaccination. On a global level, these data are useful for annual vaccine strain selection. Advances in communication, laboratory, and specimen transport technologies contributed greatly to the identification of viral pathogens from a new sentinel surveillance site in Nepal. In evaluating future collaborative sites, prior surveillance experience and reliable specimen shipping should be prime considerations. Approaches that use existing resources might foster greater international cooperation toward improved global detection and reporting of infectious diseases. Acknowledgment Financial support was given by the United States Department of Defense Emerging Infectious Disease Program. Jeffrey M. Gambel,* David R. Shlim,† Linda C. Canas,‡ Nancy J. Cox,¶ Helen L. Regnery,§ Robert M. Scott,§ David W. Vaughn,§ Charles H. Hoke Jr.,* and Patrick W. Kelley* *Walter Reed Army Institute of Research, Washington, D.C., USA; †CIWEC Clinic, Kathmandu, Nepal; ‡Armstrong Laboratory - Diagnostic Virology, Brooks Air Force Base, Texas, USA; ¶Centers for Disease Control & Prevention, Atlanta, Georgia, USA; and §Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand References 1. Gross PA. Preparing for the next influenza pandemic: a reemerging infection. Ann Internal Med 1996;124:682-5. 2. Glezen WP. Emerging infections: pandemic influenza. Epidemiol Rev 1996;18:64-76. 3. Patriarca PA, Cox NJ. Influenza pandemic preparedness plan for the United States. J Infect Dis 1997;176(Suppl 1):S4-S7. 4. Hampson AW. Surveillance for pandemic influenza. J Infect Dis 1997;176(Suppl 1):S8-S13. 5. Gambel JM, Hibbs RG. U.S. military overseas medical research laboratories. Mil Med 1996;161:638-45. 6. Williams RJ, Cox NJ, Regnery HL, Noah DL, Khan AS, Miller JM, ET AL. Meeting the challenge of emerging pathogens: the role of the United States Air Force in global influenza surveillance. Mil Med 1997;162:82-6. 7. Rota PA, Hemphill ML, Whistler T, Regnery HL, Kendal AP. Antigenic and genetic characterization of the hemagglutinins of recent circulating strains on influenza type B virus. J Gen Virol 1992:73:2737-42. 8. Centers for Disease Control and Prevention. Update influenza activity-United States and worldwide, 1996-97 season, and composition of the 1997-98 influenza vaccine. MMWR Morb Mortal Wkly Rep 1997;46:325-30. --------------------------------------------------------------------------- HIV-2 Infection and HIV-1/HIV-2 Dual Reactivity in Patients With and Without AIDS-Related Symptoms in Gabon To the Editor: Between 1996 and 1997, we evaluated the incidence of HIV-2 infection at the Fondation Jeanne Ebori, the second largest hospital in Libreville, capital of Gabon; we found an unexpected high prevalence of HIV-2-infected or HIV-1/HIV-2-dually reactive patients. During a 10-month period, 147 (14.3%) of 1,029 sera from inpatients and outpatients were found HIV-positive by the type III method recommended by the World Health Organization (two enzyme-linked immunosorbent assays are used to screen anti-HIV antibodies) (1). Further discrimination between HIV-1 and HIV-2 infections was assessed by using synthetic peptides specific for the gp41 and the gp120 of HIV-1 and the gp36 of HIV-2 (ImmunoComb II, PBS Orgenics, Illkirch, France). Of the 147 HIV-positive sera, 141 (96.0%) were exclusively HIV-1-positive; four were exclusively HIV-2-positive; and two were both HIV-1- and HIV-2-positive. Of the six sera with anti-gp36/HIV-2 reactivities, two (from patients A and B) were positive on HIV-2 Western blot, with marked anti-gag HIV-1 cross-reactivity and a discrimination assay positive only for HIV-2; two (from patients D and E) were positive on HIV-2 Western blot, with anti-gag and pol reactivities markedly lower than anti-env reactivities and a discrimination assay positive only for HIV-2; the two remaining sera (from patients C and F) showed typical dual reactivities for HIV-1 and HIV-2 infections, with positive patterns of HIV-1 and HIV-2 Western blots and a discrimination test positive for both viruses. As a whole, six (4.1%) of 147 HIV-positive sera showed either HIV-2 infection alone (n = 4) or dual reactivity. Of those, four were from Gabonese patients B, C, D, and E, and two were from immigrants from West Africa (patient A from Mali and patient F from Nigeria); two were female patients B and E. Among Gabonese patients, only one (patient E) had traveled to West Africa; the remaining three had never visited any neighboring country. However, one Gabonese man (patient C) lived in Port-Gentil, which has many West African immigrants. For all patients, the most likely risk factor for HIV was a heterosexual relationship with an unknown HIV-infected person. In three asymptomatic patients (A, B, and C) the HIV-2-serostatus was unexpected; in contrast, the three other patients had AIDS-related symptoms. Patients D and E had an HIV-2 Western blot pattern showing a marked decrease in anti-gag and pol reactivities compatible with their advanced stage of HIV-2 disease. The case of a 55-year-old exclusively heterosexual asymptomatic woman (patient B) suggests the possibility of a specific variant of HIV-2 in Central Africa (2). The high frequency in primates in Gabon of natural infection with simian immunodeficiency retroviruses, which show a high degree of genetic relatedness to HIV-2 (3), could support such a hypothesis. Two patients had typical dual reactivities to HIV-1 and HIV-2 antigens. To our knowledge, such dual reactivities have never been reported in Gabon (4). In the patient from Nigeria (patient F), the serologic pattern was typical of that usually observed in West Africa (5). Dual reactivity can result from genuine mixed infections and from serologic cross-reactivity in HIV-1 and HIV-2 infection alone; theoretically, it could also represent infection with a different, cross-reacting recombinant strain (5). HIV-2 infection in Gabon is epidemiologically related to West Africa, because of cultural and, above all, economic ties. However, HIV-2 is not limited to immigrant populations from West Africa or to Gabonese citizens traveling in this area; it has also reached the indigenous Gabonese population. The possibility of rare cases of HIV-1 and HIV-2 coinfections, recombinant HIV-1 and HIV-2 strains, and also peculiar HIV-2 variants from Central Africa, should be considered in Gabon. A possible entry of HIV-2 infection into Central Africa from Gabon in the near future could have major public health implications. Acknowledgments We thank Dr. Jean Wickings for reviewing the manuscript. CIRMF is funded by the Gabonese government, ELF Gabon, and the French Ministry of Cooperation. Carol Tevi-Benissan,* Madeleine Okome,† Maria Makuwa,* Moise Ndong Nkoume,† Joseph Lansoud-Soukate,* Alain Georges,* Marie-Claude Georges-Courbot,* and Laurent Belec‡ *Centre International de Recherches Medicales, Franceville, Gabon; †Fondation Jeanne Ebori, Libreville, Gabon; and ‡Centre Hospitalo-Universitaire Broussais, Paris, France. References 1. World Health Organization. Global programme on AIDS. Recommendations for the selection and use of HIV antibody tests. Wkly Epidemiol Rec 1992;67:145-9. 2. Belec L, Martin PMV, Georges-Courbot MC, Brogan T, Gresenguet G, Mathiot CC, et al. Dementia as the primary manifestation of HIV-2 infection in a Central African patient. Ann Inst Pasteur Virol 1988;139:291-4. 3. Georges-Courbot MC, Moisson P, Leroy E, Pingard AM, Nerrienet E, Dubreuil G, et al. Occurrence and frequency of transmission of naturally occurring simian retroviral infections (SIV, STLV, and SRV) at the CIRMF Primate Center, Gabon. J Med Primatol 1996;25:313-26. 4. Delaporte E, Janssens W, Peeters M, Buve A, Dibanga G, Perret J-L, et al. Epidemiological and molecular characteristics of HIV infection in Gabon, 1986-1994. AIDS 1996;10:903-10. 5. Peeters M, Gershy-Damet G-M, Fransen K, Koffi K, Coulibaly M, Delaporte E, et al. Virological and polymerase chain reactions studies of HIV-1/HIV-2 dual infection in Côte d'Ivoire. Lancet 1992;340:339-40. --------------------------------------------------------------------------- Q Fever in French Guiana: New Trends To the Editor: Q fever, the endemic disease caused by the rickettsial organism Coxiella burnetii, was first described in French Guiana in 1955 (1). Only sporadic cases were reported until 1996 when three patients were hospitalized in the intensive care unit of the Cayenne Hospital for acute respiratory distress syndrome. One of the patients died. Many cases of Q fever were diagnosed in the general population at the same time. A seroepidemiologic study was performed to determine whether the increase in cases was due to an increase in incidence or to an improvement in diagnosis. All paired samples of sera (acute-phase and convalescent-phase) from patients sent to the arbovirus laboratory for diagnosis of dengue infection from January 1, 1992, to December 31, 1996, were tested for antibodies to C. burnetii by immunofluorescence. All positive samples were also tested for immunoglobulin (IgM) by the same method; the IgG and IgM titers were determined by using a serial twofold dilution. A diagnosis of Q fever was made when there was a seroconversion from negative to positive or a twofold increase in IgG titer associated with the presence of IgM in the second sample. One hundred and fifty-one of 426 paired sera collected between 1992 and 1996 were from patients recently infected with dengue fever. Twenty-five (9.1%) of 275 remaining sera were from Q fever patients. Significant differences were observed in the rates of Q fever in different years (p < 0.01); one (1.9%) of 53 was positive in 1992, five (9.1%) of 55 in 1993, five (8.6%) of 58 in 1994, three (4.8%) of 63 in 1995; a large increase was observed in 1996 (11 [23.9%] of 46). Differences by residence were also assessed. Rates of infection were higher in Cayenne (21 [13.0%] of 161) than in rural areas (4 [3.5%] of 114) (p < 0.01). This study shows that cases of Q fever have occurred in French Guiana in recent years and that a significant increase in the incidence rate occurred in 1996. The reasons for this increase are unclear, and further studies of the epidemiology of Q fever in French Guiana are necessary. The epidemiology of Q fever is unusual in French Guiana because the rates of infection are much higher in Cayenne, the capital city, than in rural areas. No link with classical sources of infection (cattle, sheep, or goat birth products, or work in a slaughterhouse) was found. Indeed, Cayenne, with 80,000 inhabitants, is located near the Atlantic Ocean, and the prevailing winds blow from the sea. Airborne contamination from rural areas is therefore impossible. Furthermore, no large farm is in the immediate vicinity of the city. For identical reasons, contamination from the abattoirs is not likely; they are located on the west side of the city, near the Cayenne River, and the winds blow from the east. In our study, cases were almost equally distributed throughout the city, although many patients came from the same area. A seroepidemiologic study to determine possible new sources of infection (e.g., dogs, cats) and estimate rates of seropositivity in cattle and sheep and a case-control study on new cases are being conducted. F. Pfaff,* A. François,* D. Hommel,† I. Jeanne,* J. Margery,‡ G. Guillot,† Y. Couratte-Arnaude,‡ A. Hulin,† and A. Talarmin* *Pasteur Institute of French Guiana, Cayenne, French Guiana; †Cayenne Hospital, Cayenne, French Guiana; and ‡Army Health Care Service, Cayenne, French Guiana Reference 1. Floch H. La pathologie vétérinaire en Guyane française (les affections des porcins, des caprins et des ovins). Revue Elev Méd Vét Pays Trop 1955;8:11-3. --------------------------------------------------------------------------- Ixodes dammini: A Junior Synonym for Ixodes scapularis To the Editor: The authors of "A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini" (1) provide useful information regarding a possibly new tick-borne encephalitis-like virus. However, the use of the name Ixodes dammini is not accurate for describing this species. I. dammini (Spielman, Clifford, Piesman, and Corwin) was synonymized with Ixodes scapularis (Say) in 1993 by Oliver et al. (2) and was redescribed in 1996 (3) to reduce confusion regarding identification. Keirans and colleagues summarize a wide array of rigorous studies involving hybridization, assortative mating, isozymes, and morphometrics, all of which provide evidence supporting the synonymization of the two tick species (3). The synonymization of I. dammini with I. scapularis has been widely accepted. "I. scapularis (= I. dammini)" is still often used, but the use of I. scapularis as the sole nomen for this species is becoming more common (4). Oliver et al. (2) have established I. dammini as a junior subjective synonym of I. scapularis. If scientifically rigorous evidence exists justifying the reestablishment of the species name I. dammini, it must be published according to proper procedure. The proper nomenclature of any species, let alone one of such widespread notoriety and public health importance, is too important to be relegated to a footnote. Until such evidence is presented, the continued misuse of I. dammini serves only to confuse health-care providers, public health professionals, and lay persons. On a secondary matter, on page 167 of the dispatch, the authors state that "I. (Pholeoixodes) cookei is a one-host tick that is only distantly related to I. dammini and only rarely feeds on humans or mice" (1). I. cookei is a three-host tick (D.E. Sonenshine, pers. comm.), as are all the members of the genus Ixodes. Martin Sanders Maryland Department of Health and Mental Hygiene, Baltimore, Maryland, USA References 1. Telford SR III, Armstrong PM, Katavolos P, Foppa I, Garcia ASO, Wilson ML, Spielman A. A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini. Emerg Infect Dis 1997;3:165-70. 2. Oliver JH, Owsley MR, Hutcheson AM, James C, Chen W, Irby S, et al. Conspecificity of the ticks Ixodes scapularis and Ixodes dammini (Acari: Ixodidae). J Med Entomol 1993;30:54-63. 3. Keirans JE, Hutcheson HJ, Durden LA, Klompen JSH. Ixodes scapularis (Acari: Ixodidae): redescription of all active stages, distribution, hosts, geographical variation, and medical and veterinary importance. J Med Entomol 1996;33:297-318. 4. Centers for Disease Control and Prevention. Lyme disease-United States, 1996. MMWR Morb Mortal Wkly Rep 1997;46:531-5. --------------------------------------------------------------------------- The Name Ixodes dammini Epidemiologically Justified To the Editor: Although a large body of evidence has been interpreted as supporting conspecificity of the deer tick (Ixodes dammini) and the blacklegged tick (Ixodes scapularis), according to Chapter VI, Article 23 L of the International Code of Zoological Nomenclature (1), "A name that has been treated as a junior synonym may be used as the valid name of a taxon by an author who considers the synonymy to be erroneous...." Current use of I. scapularis to refer to the vector of Lyme disease obscures important epidemiologic issues. One of the reasons for "sinking" I. dammini was to make it easier to diagnose Lyme disease in areas where the disease was thought to be nonendemic: "The belief that I. dammini does not occur south of Maryland and that I. scapularis is a separate and distinct species yet unproven as a natural vector of Lyme disease has caused delays in Lyme disease surveillance in the South. The general attitude among physicians and veterinarians has been that Lyme disease is not a problem in that area, although patients present clinical symptoms of it" (2). Recognizing and reporting Lyme disease in southern and southcentral states should not, however, depend on whether the two ticks are conspecific. Only peer-reviewed descriptions of human cases of Lyme disease, with appropriate documentation of the diagnoses, should be accepted as evidence. Few such reports exist, and the evidence does not convincingly support a conclusion that Lyme disease exists as an epidemic zoonosis in southern states (3). This is not to say that residents outside the well-established eastern United States zoonotic sites (the Northeast and upper Midwest) do not have symptoms that fit one or more aspects of the current Centers for Disease Control and Prevention/Council of State and Territorial Epidemiologists/Association of State and Territorial Public Health Laboratory Directors surveillance definition for Lyme disease. Lyme disease-like infections, mainly manifesting as erythema migrans and strongly associated with Lone Star tick (Amblyomma americanum) bites are commonly seen in southern and southcentral states, but Borrelia burgdorferi does not seem to be the etiologic agent (4). Enzootic transmission of Lyme disease spirochetes among rodents and ticks had been documented in southern and southcentral states by the late 1980s (5-7). The question, however, is whether there is frequent zoonotic transmission. There are widespread southern U.S. enzootic cycles of Trypanosoma cruzi, but few autochthonous human Chagas disease cases seem to occur because the vectors (such as Triatoma sanguisuga) have behavioral traits that reduce their capacity to serve as zoonotic vectors (8). Nymphal I. scapularis apparently do not frequently bite humans (7,9), although adult ticks do. The major feature of Lyme disease epidemiology in the Northeast and in the upper Midwest, however, is transmission by nymphal I. dammini (10). Whether the predilection of nymphal I. dammini to feed on humans is environmentally determined or is a heritable trait with undescribed genetic markers remains unexplored. Particular mitochondrial DNA haplotypes seem to be more characteristic of I. dammini (11,12), and the use of such typing methods may enhance future analyses of the vectorial capacity of these ticks. For example, one might test the hypothesis that nymphal ticks removed from residents of sites in coastal North Carolina through Georgia, where both kinds of ticks have been collected, represent only I. dammini. But, if it is "widely accepted" that no differences exist between the two ticks, such studies may never be done. Similarly, many may wrongly assume that Lyme disease, human babesiosis, and human granulocytic ehrlichiosis are, or will become, epidemic throughout virtually all of the eastern United States. An equally likely scenario is that these zoonoses may never become public health problems for more southerly states. For the moment, then, distinguishing tick populations that frequently bite humans from those that rarely do seems to be a rational use of nomenclature, particularly for public health officials. Dr. Sanders is correct in pointing out that all Ixodes spp. are "three-host" ticks, although my intent in using the term "one-host" was to indicate that all stages of I. cookei tend to feed on the same kind of animal (sometimes a single animal, within burrows), usually woodchucks, skunks, or raccoons. I regret the confusion from my use of the acarologic term in a descriptive context. Sam R. Telford III Harvard School of Public Health, Boston, Massachusetts, USA References 1. International code of zoological nomenclature. 3rd ed. London: International Trust for Zoological Nomenclature; 1985. 2. Oliver JH Jr, Owsley MR, Hutcheson HJ, James AM, Chen C, Irby WS, et al. Conspecificity of the ticks Ixodes scapularis and I. dammini (Acari: Ixodidae). J Med Entomol 1993;30:54-63. 3. Barbour AG. Does Lyme disease occur in the South? A survey of emerging tickborne infections in the region. Am J Med Sci 1996;311:34-40. 4. Campbell GL, Paul WS, Schriefer ME, Craven RB, Robbins KE, Dennis DT, et al. Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993. J Infect Dis 1995;172:470-80. 5. Levine JF, Apperson CS, Nicholson WL. The occurrence of spirochetes in ixodid ticks in North Carolina. Journal of Entomological Science1989;24:594-602. 6. Kocan AA, Mukolwe SW, Murphy GL, Barker RW, Kocan KM. Isolation of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) from Ixodes scapularis and Dermacentor albopictus ticks (Acari: Ixodidae) in Oklahoma. J Med Entomol 1992;29:630-3. 7. Luckhart S, Mullen GR, Wright JC. Etiologic agent of Lyme disease, Borrelia burgdorferi, detected in ticks (Acari: Ixodidae) collected at a focus in Alabama. J Med Entomol 1991;28:652-7. 8. Pung OJ, Banks CW, Jones DN, Krissinger MW. Trypanosoma cruzi in wild raccoons, opossums, and triatomine bugs in southeast Georgia, USA. J Parasitol 1995;81:324-6. 9. Lavender DR, Oliver JH Jr. Ticks (Acari:Ixodidae) in Bulloch County, Georgia. J Med Entomol 1996;33:224-31. 10. Spielman A, Wilson ML, Levine JF, Piesman J. Ecology of Ixodes dammini borne human babesiosis and Lyme disease. Annu Rev Entomol 1985;30:439-60. 11. Rich SM, Caporale DA, Telford SR III, Kocher TD, Hartl DL, Spielman A, et al. Distribution of the Ixodes ricinus-like ticks of eastern North America. Proc Natl Acad Sci U S A 1995;92:6284-8. 12. Norris DE, Klompen JSH, Keirans JE, Black WC IV. Population genetics of Ixodes scapularis (Acari:Ixodidae) based on mitochondrial 16S and 12S genes. J Med Entomol 1996;33:78-89. 996;33:78-89. --------------------------------------------------------------------------- Ebola/Athens Revisited To the Editor: After our hypothesis that the plague of Athens (430 B.C.- 425 B.C.) could have been caused by Ebola virus was published in this journal (1996;2:155-6), it was brought to our attention that this hypothesis had been previously entertained. Gayle D. Scarrow had published a paper entitled "The Athenian Plague: A Possible Diagnosis" in The Ancient History Bulletin 2.1 (1988). Unfortunately, this had not come to our attention in our literature search, and therefore we assumed that we were the first to recognize the possibility. Clearly, Ms. Scarrow deserves credit for suggesting this first. Her arguments are compelling, even without the support of more recently available information and the observations advanced in our publication. We believe an evolving knowledge base (e.g., the information about the Côte d'Ivoire outbreak where a protracted epidemic has been meticulously documented) will serve to enhance the credibility of the Ebola/Athens hypothesis. P.E. Olson,* A.S. Benenson,† and E.N. Genovese† *U.S. Navy Balboa Hospital, San Diego, California, USA; †San Diego State University, San Diego, California, USA --------------------------------------------------------------------------- [EID Home Page] Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: http://www.cdc.gov/ncidod/EID/vol4no1/letters.htm ---------------------------------------------------------------------------- [Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998] News and Notes ------------------------------------------------------------------------ Meeting Summaries ------------------------------------------------------------------------ The Hot Zone--1997: Conference on Emerging Infectious Diseases On June 27-28, 1997, the University of Kentucky College of Medicine, University of Cincinnati College of Medicine, and Kentucky AIDS Consortium held a conference for clinicians and researchers in Lexington, Kentucky. Participants presented the latest findings from worldwide epidemiologic studies, basic science, and clinical research on emerging infectious diseases. The findings indicate that the war against infectious diseases is far from over. In the United States, between 1980 and 1992, deaths from infectious diseases increased by 58%, making serious infections the third leading cause of death; HIV infection is the leading cause of death among 25- to 44-year-olds; and antibiotic resistance costs the health-care system an estimated $100 million to $30 billion each year. With the global population growing from 2.5 to 5.8 billion over the last 25 years, large urban centers throughout the developing world are overcrowded and have inadequate sanitation, ideal for the emergence of infectious diseases. By 2025, the global population will reach 8.6 billion. In developing countries, this represents an 84% increase, which will intensify overcrowding in these areas. In industrialized countries, an aging population base, the advent of immunosuppressive medications, and the emergence of HIV are combining to increase the risk for opportunistic infection. Moreover, with increased travel, clinicians see increasing numbers of patients with exotic diseases acquired abroad. Recent migration of epidemic diphtheria from the former Soviet Union to Europe and the emergence of multidrug-resistant tuberculosis (TB) in the United States and elsewhere are but two examples of infections resulting from international travel; in addition, nearly 70% of the fruits and vegetables consumed in the United States originate in developing countries; disease outbreaks related to imported food frequently go unreported. Extensive cross-species contact among humans and certain domestic animals can dictate antigenic shifts in influenza viruses. The likelihood of the emergence of a new influenza virus in the near future increases with the growth of the hog population in China. The emergence of new viruses, such as HIV and filoviruses, indicates the virtually unlimited capacity of pathogenic organisms to mutate and rapidly adapt to environmental changes and selective pressures. HIV/AIDS Research Recent data from long-term survivors support the concept that HIV replication occurs when the number of CD4+ cells drops below the minimum level required to maintain CD8+ cell control of HIV. CD4+ cell production of IL-2 is needed for strong cell-mediated immunity. Without CD8+ cell responses, a more virulent, highly cytotoxic viral strain emerges, killing greater numbers of CD4+ cells and leading to AIDS. Because CD8+ cell loss appears to be related to a shift from a TH-1- to a TH-2-type cytokine response, therapeutic approaches that maintain TH-1 cell response, or enhance CD8+ cell anti-HIV activity through factors yet to be fully defined, are being actively investigated. Vaccines relying exclusively on antibody responses will almost certainly prove to be of limited value, while those using CD8+ cell antiviral activities hold substantially greater promise. A basic science research forum described the use of a murine model of immunodeficiency induced by a type C retrovirus. Because models of this type reproduce a number of the clinical pathophysiologic manifestations associated with human AIDS, they can significantly enhance our understanding of retrovirus-induced immunodeficiency. The results of recent research involving host immune responses to Pneumocystis carinii infection indicated that, compared with healthy controls, HIV patients with less than 200 CD4+ cells have similar IL-4 levels but significantly lower peripheral blood mononuclear cell proliferative responses and IFN-[gamma] levels to P. carinii major surface class glycoprotein (MSG). Centers for Disease Control and Prevention (CDC) Class 3 patients with previous P. carinii pneumonia (PcP) have significantly higher IL-4 (but not IFN-[gamma]) levels than Class 3 patients with no history of PcP. HIV+ patients who have recovered from PcP have sufficient memory cells to recognize MSG, but demonstrate a shift from a TH-1- to a TH-2-type antigen recall response. HIV Therapy The current state of CD4 and viral load testing and the 11 drugs available for HIV treatment were reviewed. Although combination drug therapy has consistently proved more effective than monotherapy in maintaining reduced viral load, the results of a recently completed follow-up study confirmed the effectiveness of zidovudine (AZT) in preventing neonatal HIV transmission; in a large cohort of pregnant women, AZT plus high titer immunoglobulin G was no more effective than AZT plus placebo, with both regimens producing results comparable with those achieved with AZT alone. A number of issues in HIV treatment remain unresolved, including when treatment should begin, how to manage inadequate response to therapy, whether to use HIV resistance genotyping to direct therapy, and how to best deal with prophylaxis for opportunistic infections in patients showing dramatic reductions in viral load. Dengue and Dengue Hemorrhagic Fever (DHF) Tens of millions of cases of dengue and hundreds of thousands of cases of DHF are reported annually, and more than 2.5 billion people are at risk for infection. Factors contributing to the emergence of dengue include unplanned and uncontrolled population growth associated with urbanization in tropical regions, lack of effective mosquito control, deteriorating water systems that increase densities of Aedes aegypti, and viral migration among tropical urban centers due to increasing international air travel. More than 50% of all air travel from the United States is to tropical destinations, and from 1977 to 1994, 2,248 suspected cases of imported dengue were reported in the United States. Because their clinical symptoms are initially nonspecific, dengue and other arboviral infections can be difficult to distinguish from other viral, bacterial, and parasitic infections. Correct diagnosis requires a detailed clinical summary, thorough epidemiologic information (including recent travel history), and a diagnostic laboratory test. The severe hemorrhagic form, DHF/dengue shock syndrome (DSS), has an average incubation period of 4 to 6 days before sudden onset of fever and nonspecific signs and symptoms. Because the major pathophysiologic abnormality observed in DHF/DSS is increased vascular permeability and leakage of plasma from the vascular compartment, early fluid replacement is effective. The geographic distribution of DHF/DSS has been expanding and now can be found in tropical areas of Asia, the Pacific, and the Americas, including Central America and Mexico. This expansion is associated with increased movement of dengue viruses by airplane travelers and the development of hyperendemicity in the Pacific Region and the Americas. A similar scenario, generated largely from human encroachment into new environments, may also emerge for other Aedes-transmitted illnesses such as yellow fever. The most cost-effective approach to control dengue and DHF is larval source reduction in disease-endemic areas. Programs should use both government and community resources to integrate environmental sanitation with the use of insecticides and biologic controls, targeted to breeding grounds such as tire dumps. Other Viral Diseases The exponential increase in ecologic change, both environmental and behavioral, was cited as the major driving force for the increasing human risk for viral infection. Microbial variability can play a causal role in disease emergence, but it more often enables viruses to adapt to new circumstances. Travel of infected humans and international transport of microbes and vectors help provide the maximum possible microbial evolutionary opportunities in the minimum amount of time. Viruses have emerged in the past, with measles providing a good example of the worrisome potential for future emerging RNA viruses. The emergence of cities in the Mesopotamian basin, resulting largely from the advent of irrigated agriculture, provided a populated substrate for interhuman transmission of short-incubation, nonlatent viruses. Domestication of livestock likely brought measles progenitors into close proximity with humans; a precursor of rinderpest/peste de petit ruminants then made an interspecies leap; much later measles spread to Europe, and, in the post-Columbian interchange, to the Americas. Viral hemorrhagic fevers include those caused by Ebola filovirus and hantaviruses. Ebola hemorrhagic fever is characterized by extensive and disseminated infection and necrosis in major organs, and lymphoid depletion. Aerosol transmission of Ebola virus has occurred between nonhuman primates and guinea pigs, but no evidence exists for interhuman transmission by airborne infection. Barrier nursing precautions generally prevent the spread to humans, but in areas having inadequate medical care facilities, the virus can amplify in humans and cause epidemics. Hantaviruses belong to a single genus in the family Bunyaviridae, and each virus infects a limited or unique rodent species with no apparent disease. Although hemorrhagic fever with renal syndrome has rarely been diagnosed in the Americas, hantaviruses from sigmodontine rodents cause hantavirus pulmonary syndrome, characterized by large bilateral pleural effusions and heavy, edematous lungs, interstitial pneumonitis, and extensive infection of endothelial cells in the pulmonary microvasculature. The first documented interhuman transmission of a hantavirus was an outbreak of 20 cases in Patagonia, with evidence overwhelmingly indicating spread between patients and physicians. The reasons for, and mechanism of, the spread are unknown, but the registry of U.S. cases was revised to ensure that this phenomenon was adequately monitored. Although early diagnosis and supportive care are potentially lifesaving in cases of hantavirus pulmonary syndrome, such efforts are of limited value in Ebola hemorrhagic fever. Prion Illnesses In prion illnesses such as Creutzfeldt-Jakob disease (CJD), risk factors (family history of CJD or dementia, history of poliomyelitis, exposure to sheep or cows) and iatrogenic factors (dura or corneal grafts and exposure to pooled human growth hormone) are important. At onset, CJD is commonly misdiagnosed as psychotic illness, Alzheimer's dementia, paraneoplastic syndrome, vascular brain disease, parkinsonism, or even drug-induced delirium. A variant of CJD, caused by a prion with an altered protein configuration, is bovine spongiform encephalopathy (BSE or mad cow disease). Although the precise mechanism of infection originally reported in U.K. cattle is unclear, BSE has been exported to other countries by feeding cattle inadequately processed bone meal. The potential for the emergence of BSE in the United States exists because similar reservoirs of infection are present. Casual handling of beef and beef products in processing plants and uncontrolled disposal of sick cattle may increase that risk. Several cases of CJD have been reported in Kentucky patients who consumed squirrel brains; however, a causal link has not been established. The most recently identified prion illness is a new variant of CJD reported in England that, unlike sporadic CJD, occurs in much younger patients (16 to 50 years of age), can last longer than 1 year, and is characterized by the presence of psychiatric and sensory symptoms and the absence of kuru plaques and electroencephalogram periodic complexes. The illness is thought to be linked to BSE. Mathematical modeling suggests that 75,000 to 80,000 cases will occur in the foreseeable future. Tick-Borne Diseases As with many other emerging infectious diseases, concern about tick-borne zoonotic diseases in the United States has increased because of environmental changes brought on by human settlement and socioeconomic factors that place humans at greater risk for tick exposure, such as the development of suburban housing in disturbed natural settings. Current conditions appear favorable for continued increases in vector tick populations and their geographic expansion and for increasing interaction between ticks and humans. In the United States, the prevalence of Rocky Mountain spotted fever may continue to increase as the urbanization of the western and southern regions expands opportunities for human exposure to tick-borne pathogens. The epidemiology of Lyme disease and tularemia was reviewed, and the concept of a southern (U.S.) tick-associated rash illness (STARI), the epidemiology of which is still being defined, was introduced. STARI is characterized by an expanding erythematous rash resembling that of Lyme disease, mild or absent constitutional symptoms, and no well-described sequelae. The rash responds well to antibiotic treatment. STARI is seen in the range of the human-biting Lone Star tick (Amblyomma americanum) and is frequently associated with a Lone Star tick bite. Studies indicate that this infection is not caused by Borrelia burgdorferi or other known tick-borne agents. Amplified segments of the genome of this spirochete have recently been described, and some investigators have proposed that it represents a new species, B. lonestari. It has been suggested that STARI may be closely related or identical to a commensal spirochete of deer, B. theileri. The need for early diagnosis and treatment of Rocky Mountain spotted fever was stressed by presenting data from 94 infected patients in North Carolina. Death rates were significantly lower (6.5% vs. 22.9%, p < 0.03) in patients receiving appropriate treatment within 5 days of onset of illness than in patients who received delayed or no treatment. Predictors of death included renal failure; elevated serum creatinine, aspartate transaminase, and bilirubin; decreased serum sodium; thrombocytopenia; neurologic involvement; and male gender. Parasitic Diseases The role of the cell surface glycoconjugate lipophosphoglycan (LPG) in the survival of Leishmania parasites whose life cycle alternates between intracellular parasitism and extracellular life in sand fly vectors was explored. Data indicating that LPG is a multifactorial molecule may lead to a biochemical rationale for LPG-targeted chemotherapeutic regimens. Isolation of a full-length genomic clone of the acid [alpha]-mannosidase from an epimastigote genomic library of Trypanosoma cruzi was reported. Sequence analysis showed a single open reading frame encoding the [alpha]-mannosidase gene. Because the size of this frame is consistent with that of lysosomal mannosidases in humans, these results could lead to the exploration of new chemotherapeutic options in Chagas disease. A bank of 5,200 insertion mutants has been used to characterize the parasitic mechanisms used by Legionella pneumophila. Results from transmission electronmicroscopy indicate that L. pneumophila has acquired genetic loci specific for survival and replication within mammalian cells, allowing evolution from a protozoan parasite into its present disease-causing agent. Alternatively, ecologic coevolution of L. pneumophila to parasite protozoa has led to the development of multiple redundant mechanisms, some of which do not function within mammalian macrophages. TB Poverty, changing immigration patterns, and the emergence of HIV disease were cited as factors contributing to an attenuated rate of decrease of TB incidence in the United States since the late 1980s and its increase as a major global problem. Poorly managed TB control programs, suboptimal access to health care, an inadequate physician knowledge base, and poor patient compliance have combined to increase the incidence of TB and, especially, multidrug-resistant TB; sensitivity testing is critical in the management of resistant TB. Updated CDC guidelines for interpreting the purified protein derivative skin test call for responses to be considered positive if induration is greater than 5 mm in those with HIV or who have recent TB contact, and greater than 10 mm in foreign-born patients, intravenous drug users, the homeless, and immunocompromised patients. Because of the "boosting" phenomenon, two-step purified protein derivative skin testing is now recommended for health-care workers and nursing home patients who are retested periodically. Treatment of active TB should use multiple drugs, avoid adding single agents, and include compliance monitoring, preferably directly observed therapy. Workplace prevention measures should be driven by a consistently high index of suspicion and should include appropriate isolation of suspected cases, work-ups following exposure, rigorous reporting to health departments, and locating recalcitrant patients. Effective control of TB will require social, political, and cultural changes, as well as medical innovation. Plague The epidemiology, pathophysiology, and treatment of plague, as well as the improvement of diagnostic techniques for infections caused by Yersinia pestis, were reviewed. Such new tests will permit the public health laboratory to quickly identify a plague outbreak and apply the appropriate control measures to limit its spread. The pathogenesis of plague was explored, and two separate, but essential, iron transport systems were identified in Y. pestis. The first, the yersinabactin (Ybt) system, enables the organism to proliferate from the site of an open wound or bite, while the second, identified as Yfe, is used by Ybt mutants to obtain iron during infection of internal organs. The Ybt iron transport system appears to be essential for growth in the early stages of bubonic plague infection, while the Yfe system functions to allow growth during, or after, infection of internal organs. Both systems are required for Y. pestis to be fully virulent. A novel mechanism by which a Y. pestis virulence protein is sequestered in, and transported within, host cells was described. When yersiniae contact a eukaryotic cell, a signaling event activates the expression and secretion of a set of four toxins (Yops) and causes their vectorial translocation into the cell cytoplasm. Three of the Yops derange cell signaling and cytoskeletal functions by kinase, tyrosine phosphatase, and actin depolymerization activities. Although no activity or intracellular target has been identified for the fourth known translocated Yop, YopM, immunoblot analysis and laser scanning confocal microscopy demonstrated that most YopM is vectorially translocated into HeLa cells or the macrophagelike cell line J774 by adherent Y. pestis and travels to the eukaryotic cell nucleus. Because a growing number of important human pathogens (e.g., Salmonella, Shigella, and Pseudomonas) have similar, but less well-studied, secretion/translocation mechanisms and putative secreted toxins, these findings will facilitate studies that ultimately could lead to novel therapies for these agents. Antibiotic Resistance The current development of staphylococci resistant to methicillin or fluoroquinolones and gram-negative bacilli resistant to extended-spectrum beta-lactams are but the most recently recognized patterns of antibiotic resistance. General approaches for modifying these trends include 1) source control, particularly handwashing, and the need to wear gloves during contacts with all patients, 2) improved antibiotic use and control, 3) improved infection control devices, and 4) better use of pathophysiology and immunologic modulation. The growing problem of vancomycin-resistant enterococci (VRE), with an incidence of 20% to 40% in some groups of U.S. hospitalized patients, necessitates maximal use of all these approaches. Skin proliferation of VRE produces extensive environmental contamination that may require universal use of gloves to control outbreaks or hyperendemic disease. In addition, vancomycin should be limited to treatment of beta-lactam resistant gram-positive bacteria (such as methicillin-resistant Staphylococcus aureus and gram-positive bacteria in beta-lactam allergic patients) and Clostridium difficile (only after metronidazole failure) and to endocarditis prophylaxis. Vancomycin should be avoided in routine surgical prophylaxis, empiric treatment of febrile neutropenia with negative cultures, and pneumonia prophylaxis in the intensive care unit. A number of experimental peptides and other agents (such as quinupristin-dalfopristin) under investigation as treatments for VRE infections were identified. Future trends in resistance may include further spread of vancomycin-resistant staphylococci (already reported in Japan), quinolone- or carbapenem-resistant gram-negative bacilli, and treatment-resistant viruses. Seventeen isolates of methicillin-resistant S. aureus with unique genotypes were studied to determine rates of resistance to the fluoroquinolones ciprofloxacin and levofloxacin. The mean single-step resistance to 4 x MIC ciprofloxacin was 1.05 x 10(sup-5) and to levofloxacin was 4.03 x 10(sup -6). When serially passaged in increasing antibiotic concentrations, the geometric mean MICs for ciprofloxacin and levofloxacin increased 3.0 +/- 1.5 times and 1.8 +/- 1.4 times, respectively (p < 0.0005). Only four strains became resistant to levofloxacin, but eight became resistant to ciprofloxacin, indicating that ciprofloxacin selects methicillin-resistant S. aureus more frequently than levofloxacin. Other Topics The laboratory evidence supporting the role of Chlamydia pneumoniae in the development of atherosclerosis was reviewed. Recent data indicate that infection of vascular endothelial cells with C. pneumoniae is associated with the production of chemokines and adhesion molecules that promote transendothelial migration of neutrophils and monocytes. These findings suggest that immunopathogenic responses to C. pneumoniae infection may contribute to the development of clogging deposits. Data were presented demonstrating the proliferation of human CD4+ T cells from unexposed persons in response to in vitro exposure to Toxoplasma gondii. Further studies showed that this proliferative response depends on HLA-DR molecules and requires processing of Tg antigens. In contrast to typical exogenous superantigens, analysis of TCR V[beta] expression after stimulation with Tg did not show a pattern of preferential increase of a specific TCR V[beta]-bearing subpopulation. [alpha][beta]T cells secreted significant amounts of IFN-[gamma] after incubation with Tg-infected monocytes. This process may play an important role in the early events of the immune response to T. gondii. Richard N. Greenberg,* Judith E. Feinberg,† and Claire Pomeroy* *University of Kentucky Medical Center, Lexington, Kentucky, USA; †University of Cincinnati College of Medicine, Cincinnati, Ohio, USA ------------------------------------------------------------------------ International Meeting on Borreliosis, Prague, Czech Republic Approximately 150 participants from 10 countries gathered in Prague, the Czech Republic, August 27-29, 1997, to discuss research topics related to the theme of the meeting, "Lyme Borreliosis-Basic Science and Clinical Approaches." The meeting was organized by the National Institute of Public Health (Centre of Epidemiology and Microbiology); the World Health Organization Collaborating Center for Reference and Research on Borreliosis; the Second School of Medicine, Charles University (Prague); and the Czech Medical Association J.E. Purkynê;. Meeting sessions focused on topics including epidemiology, clinical treatment, dermatology, diagnosis and treatment, neurology, and laboratory diagnosis. The session on epidemiology presented surveillance data on the incidence of Lyme borreliosis (LB) in the Czech Republic (incidence rates were 61.8/100,000 in 1995 and 41.2/100,000 in 1996) and in Slovakia, Austria, and Slovenia. Data underscored the high risk for transmission of LB in central and eastern Europe. The results of vaccine trials using the recombinant outer surface protein (Osp)A antigen of Borrelia burgdorferi were also presented; more detailed studies are needed to examine intraspecies variability of OspA antigens in Europe. The session on clinical approaches and treatment reviewed research conducted in the United States and discussed the diagnostic importance of organism-specific biologic markers, e.g., Borrelia-specific antigens or DNA, as well as pleocytosis in cerebrospinal or synovial fluid. Experience with the diagnosis and treatment of LB in the hyperendemic-disease regions of west Bohemia underscored the importance of accurate diagnosis in avoiding overtreatment. The use of nonhuman primates as models for studying neuroborreliosis was examined in the session on neurology. Problems related to the diagnosis and treatment of chronic disease, and their economic consequences, were identified. Several methods to assist clinicians in making a correct diagnosis were presented and discussed. The persistence of B. burgdorferi DNA in patients with Lyme arthritis was considered in the rheumatology session. Ultrastructural evidence for the intracellular location of B. burgdorferi in synovium also was presented. The session on laboratory diagnosis focused on the genomic sequence of the linear chromosome of B. burgdorferi (B31 strain) and the crystal structure of OspA; both apply to the laboratory diagnosis of LB. Other studies affirmed the importance of standardizing diagnostic methods to ensure reproducibility and uniformity of the results from different laboratories. The influence of certain in vivo-expressed antigens (virulence antigens) on invasiveness and the ability of B. burgdorferi to adapt to the host environment were noted. Other topics were the sensitivity and reproducibility of polymerase chain reaction and the importance of the primers selected for the assay. The studies presented in the poster session addressed a wide array of themes: among them, epidemiology and population awareness, reactivity of B. burgdorferi antigens in immunoblot procedures when specimens derived from humans or animals are used, and incidence of ticks and their association with disease in different regions. The importance of apoptosis in the morphology of LB, the role of Langerhans cells in the skin reactions, and the role of integrin CR3 in the interaction of B. burgdorferi with host cells were discussed. The sensitivity and the selection of the primers used for polymerase chain reaction to detect B. burgdorferi in ticks were considered. Aspects of vector biology and ecology were investigated (e.g., habitats, the tick as LB's major vector, vector capacity). Other diseases transmitted by Ixodes ricinus ticks in Europe (e.g., tick-borne encephalitis, babesiosis, ehrlichiosis) as well as human ehrlichiosis in Europe were reviewed. Dagmar Hulínská and Jirí Bašta National Institute of Public Health, Prague, Czech Republic ------------------------------------------------------------------------ Workshop on Climate Change and Vector-Borne and Other Infectious Diseases Climate changes may affect human health through a myriad of pathways; of particular interest are pathways affecting the geographic ranges and incidence of vector- and water-borne diseases. As society chooses how to deal with projections of long-term climate change, decisions must be based on scientific knowledge. A 2-day workshop(ft1) was convened in September 1997 to discuss what is known about the relationship between projected climate changes and the incidence of water-borne diseases (e.g., cholera) and vector-borne diseases, including those typically considered tropical (malaria, dengue fever, yellow fever, and schistosomiasis), plus subtropical or temperate-zone diseases whose vectors are likely to be affected by projected climate changes. The workshop participants discussed the systems involved in potential climate changes, from the global ocean-atmosphere-landmass system that drives climate to the regional ecologic and human socioeconomic systems where disease dynamics occur. These systems are extremely complex, as are the interactions among them, which underscores the need for more research before accurate projections can be made. Major research gaps were identified, and an agenda was framed for a sound scientific basis for public policy debates and decisions. The proposed agenda included the following items: climate modeling; ecosystem and habitat dynamics; disease surveillance; technologies for disease prevention and mitigation; disease transmission dynamics; data sets for empirical studies; integrated assessments; and detecting, understanding, and responding to unexpected events. Further discussion and implementation of this research agenda is encouraged. A summary of the workshop is available from the Electric Power Research Institute, TR-109516, EPRI Distribution Center, 207 Coggins Drive, P.O. Box 23205, Pleasant Hill, CA 94523; Telephone: 510-934-4212. (sup1)The workshop was commissioned by the Electric Power Research Institute, with additional sponsorship from the Department of Energy, the National Institute of Allergy and Infectious Disease, the National Institute of Environmental Health Sciences, and the National Aeronautics and Space Administration. The workshop was organized and conducted by the Washington Advisory Group. The 28 participants included representatives from agencies and institutions that conduct or fund research and experts in the fields of climatology and global climate modeling, public health, and the biology and ecology of vectors, pathogens, and the ecosystems they inhabit. ------------------------------------------------------------------------ The Fourth International Conference on HFRS and Hantaviruses Atlanta, Georgia, USA, March 5-7, 1998 The Centers for Disease Control and Prevention and other cosponsors will host the Fourth International Conference on HFRS and Hantaviruses to facilitate the exchange of scientific information in the following areas: 1) clinical aspects, 2) laboratory diagnostics, 3) pathogenesis and immune response, 4) hantavirus ecology, 5) hantavirus epidemiology, 6) molecular biology and cell interactions, 7) health education and prevention, and 8) antiviral and vaccine development. The meeting will offer plenary sessions with invited speakers, as well as oral and poster sessions based on accepted abstracts. For further information, contact Amy Corneli, Centers for Disease Control and Prevention, 1600 Clifton Road, MS A26, Atlanta, GA 30333, USA; fax: 404-639-1509; e-mail: akc8@cdc.gov; URL: http://www.cdc.gov/ncidod/diseases/hanta/hantconf.htm. ------------------------------------------------------------------------ Third International Congress on Tropical Neurology November 30-December 2, 1998 Organized by the Groupe Francophone d'Etude et de Recherche en Neurologie Tropicale, the Third International Congress on Tropical Neurology will convene in Fort de France, Martinique, from November 30 to December 2, 1998. The four main themes of the congress are central nervous system inflammatory, neurodegenerative, epileptic, and cerebrovascular disorders in tropical environments; however, presentations on other themes are welcome. A symposium on epilepsy in tropical zones will be held during the congress. For additional information, contact Professor M. Dumas (phone: 33-5-55-43-58-20, fax: 33-5-55-43-58-21) or Professor J.C. Vernant (phone: 33-5-96-55-22-61, fax: 33-5-96-75-45-90). ------------------------------------------------------------------------ International Conference on Emerging Infectious Diseases March 8-11, 1998 Atlanta Marriott Marquis Hotel Late-breaker abstract submission deadline: January 30, 1998 Information on abstract submission, conference registration, and exhibits can be obtained at www.asmusa.org, by sending an e-mail message to meetinginfo@asmusa.org, or by calling 202-942-9248. Proceedings of the conference will be published in the journal Emerging Infectious Diseases. Registration: limited to 2,500 - register NOW! Preliminary program information is available at http://www.cdc.gov/ncidod/EID/98conf.htm. ------------------------------------------------------------------------ Emerging Infectious Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA URL: http://www.cdc.gov/ncidod/EID/vol4no1/ascii/newsnote.txt Please note that figures and equations are not available in ASCII format; their placement within the text is noted by [fig] and [eq], respectively. Greek symbols are spelled out. The following codes are used: (ft) for footnote; (sup) for superscript; (sub) for subscript; /= for greater than or equal to.