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Clinical Pathology Department
Project numbers
Analytical Methodology: Development and Interpretative Application
Magnesium Metabolism in Humans and Biological Systems
Assessment of the Clinical Performance of Lab Tests
Evaluation of Techniques for the Analysis of Lipoproteins and Apolipoproteins
Development and Diagnostic Use of Techniques for the Determination of
Genes Involved in Lipid/Lipoprotein Disorders
Histidine-rich Glycoprotein: Characterization and Clinically Significant Studies
Assessment of Hydrogen Peroxide Generation in Neutrophils
Clinical Utility of IV Catheter Tip Cultures for Short-term Lines
Development of Methods for Determining Strain-relatedness of Clinical Isolates
Methods for Rapid Detection of CMV
In Vitro Culture and Susceptibility Testing of Microsporidia
Detection and Identification of Microsporidia in Stool by PCR
Identification of Molecular Defects in Patients with von Willebrand's Disease
Survival of Microsporidia After Exposure to Disinfectants and Environmental Conditions
Use of PCR and RFLP Analysis for Identification of Mycobacteria and Nocardia
Investigation of the Molecular Mechanisms of Heparin-induced Thrombocytopenia
Treatment of Symptomatic, Venous Access Device-related Deep Venous Thrombosis
with Recombinant Tissue Plasminogen Activator
Molecular Defect in the Hereditary Resistance to Activated Protein C
Telomerase TestingMolecular Screening for Early-stage Tumors
CP Susceptibility Testing Procedures for Rapidly Growing Mycobacteria and Nocardia
Development of PCR for CMV, HSV, and VZV from Spinal Fluid
Can Oral Washes be Used to Detect Pneumosystis Pneumonia?
Development of a PCR Assay for Diagnosis of Legionella Pneumonia
Does Removal of Catheters Help Clear Catheter-related Gram-negative Bacteremias?
Development of a PCR Procedure for Quantitative Measurement of CMV in Blood
Methods for Detection of BK Virus in Allegeneic Bone Marrow Transplant Patients
Detection of Microsporidia in Stool by Modified Trichrome Stain
Markers of Disease Activity in Idiopathic Inflammatory Myopathy
Use of Neural Networks in Monitoring Quality Control of Laboratory Data
Platelet Aggregation and von Willebrand Factor Defects in Hermansky-Pudlak Syndrome
Development of a Molecular-based Assay to Determine Susceptibility of Viruses to Antiviral Agents
Quantitative Detection of CMV by Using a Commercially Developed Research Assay
PCR-SSCP for the Detection and Speciation of Micro-sporidia in Clinical Specimens
Comparison of Microbiologic and Cytologic Results for Bronchoalveolar Lavages
DNA Sequence Variation in the Helicobacter Species Urease Gene
Detection and Identification of Mycobacteria in Clinical Specimens
Molecular Characterization of a Novel Cell Envelope Biosynthetic Gene with a
Distinctive Sequence Feature of Sugar Acylation in Mycobacterium Tuberculosis
Platelet-associated Antibodies in Patients with Autoimmune Thrombocytopenic Purpura
Follow-up Study of Patients with Large Granular Lymphocytic Leukemia
Assessment of Peripheral Blood Eosinophils
Assessment of Peripheral Blood Monocytes in Patients with Recurrent Mycobacterial Infection
Assessment of Lymphocytes in Patients with Autoimmune Lymphoproliferative Syndrome
Flow Cytometric Intracellular Protein Evaluation in CGD Granulocytes
Genetic Defect in Bernard Soulier Syndrome
Genetic Abnormalities in Patients with Thrombophilia
Evaluation of an Alternative Pathway for Class I MHC Product-dependent
Presentation of Polypeptides to CD8 T Cells
Development and Diagnostic Use of Rapid Immunoassays
The Experimental Treatment of Transfusion-dependent 5Q Minus Syndrome
Heparin Cofactor II Levels in Patients with Paroxysmal Nocturnal Hemoglobinuria
Platelet Glycoprotein IIIa Polymorphisms in Young Patients with Coronary Artery Disease
INTRAMURAL RESEARCH PROJECT Z01 CL-10001-23 CP
October 1, 1997 to September 30, 1998
Title of Project: Analytical Methodology: Development and Interpretive Application
Principal Investigator: N.N. Rehak, Ph.D. (Senior Staff) CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: G. Csako, M.D., CCS, CPD
S.A. Cecco, M.T., CCS, CPD
Collaborating Units: None
Staff-Years: 0.1
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: We investigated saliva as a possible contaminant causing unexplained increases in serum potassium (K) concentration in our laboratory. Random saliva samples were collected from healthy adult volunteers and assayed for K and sodium (Na). Most samples were also analyzed for 25 general chemistry tests. Concentrations of some biochemical analytes were much lower in saliva than in serum [alkaline phosphatase, alanine aminotransferase, Na, chloride (Cl), bicarbonate (HCO3), pH (based on concentration)] and some were comparable with serum [aspartate aminotransferase, total magnesium (Mg), total and ionized calcium (Ca), uric acid, urea nitrogen]. Glucose, ionized Mg, albumin, and total protein were unmeasurable with the routine laboratory methods. Amylase, K, and inorganic phosphorus (Pi) were much higher in saliva than in serum. The salivary pH (mean 7.01) was close to the acid dissociation constant of H2PO4- is less than or greater than HPO4(2-) + H+ (pKa is approximately 7.2) indicating the concentrations of these two phosphate anions are approximately equimolar, that is 1 mmol of Pi carries 1.5 negative charge (equal to 1.5 meq). The calculated mean difference between the measured equivalents of cations and anions (anion gap, AG) was similar to serum (4.2 vs. 6.6 meq/L) only when phosphate anion was included in the calculation.
The positive AG values indicate that unmeasured anions are commonly present in the saliva (e.g., thiocynate in smokers and vegetarians, fluoride from drinking water and toothpaste). The value of the salivary AG was not dependent on the pH (p equals 0.067). Significant relationship was found between phosphate anion and several cations: K (p is less than 0.0001), total Ca (p=0.0033), bound Ca (p is less than 0.0001), total Mg (p = 0.020) and between Cl anion and K cation (p = 0.034). The concentrations of phosphate anion, K, total Mg, total and bound Ca decreased with increasing pH (p is less than 0.05), but Na and Cl were not affected by pH changes (p equals 0.45). The relation between Pi and ionized Ca was not significant (p=0.853). The decrease of ionized Ca with increasing pH (p=0.047) was most likely due to increased protein binding of Ca. Thus, phosphate is the main complexing anion in saliva and Ca-K-Mg-P complexes are deposited during the remineralization process that occurs at higher salivary pH. Further, contamination of blood or any other body fluid (such as urine) with saliva may cause not only erroneously high amylase results but also falsely high K and Pi results. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10010-22 CP
October 1, 1997 to September 30, 1998
Title of Project: Magnesium Metabolism in Humans and Biological Systems
Principal Investigator: N.N. Rehak, Ph.D. (Senior Staff)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: S.A. Cecco, M.T., CCS, CPD
Collaborating Units: NHLBI, IR, CE (R.S. Balaban, Ph.D.; K.M. Ward, Ph.D.)
Academy Medical Center (Amsterdam, Netherlands;
G.T. Sanders, M.D.; H.J. Huijgen, Ph.D.)
Univ. of Louisville, Medical Center (Louisville, KY;
R.J. Elin, M.D., Ph.D.)
Staff-Years: 0.9
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The secretion of parathyroid hormone (PTH) by the parathyroid gland is directly regulated by extracellular calcium ions (Ca2+) via the Ca2+-sensing receptor. However, there are reports of magnesium (Mg) effect on PTH secretion: acute hypermagnesemia can suppress PTH. It is suggested that the mechanism of suppression is similar to that of hypercalcemia. We investigated the relationship between Ca, Mg, and inorganic phosphorus (Pi) in patients with hyperparathyroidism (HrPT, n=62) and hypoparathyrodism (HoPT, n=27). We measured the serum concentrations of total Ca (TCa), ionized Ca (iCa), total Mg (TMg) and ionized Mg (iMg) and, by calculating the ionized fraction (i.e., ionized/total), indirectly determined the bound fraction (i.e., bound to albumin and complexed with anions) of these cations.
The relationship between the concentrations of TCa and iCa, and of TMg and iMg, for both groups of patients was significant (p is less than 0.0001). However, there was a major difference in the relationship of Ca and Mg forms (i.e., total, ionized, and ionized fraction) and Pi. For HrPT patients there was a positive relationship between iCa and iMg (p is less than 0.0001) indicating simultaneous increase of both ions. The iCa fraction was not affected by TMg (p is greater than 0.1) but the iMg fraction decreased as the TCa increased (p=0.04). The bound fractions of Ca and Mg were not affected by albumin (p is greater than 0.1). However, the bound fraction of Ca increased (p equals 0.004), and that of Mg decreased (p equals 0.009), with the increasing concentrations of Pi, (i.e., the concentration of Ca-phosphate increased and of Mg-phospate decreased). This indicates that for HrPT patients the equilibrium between the free and bound Ca and Mg is mainly determined by the concentration of Pi. In contrast, for HoPT patients, no significant relationship was found between Ca and Mg forms (p is greater than 0.1), and the bound fractions of Ca and Mg were not affected by Pi (p equals 0.8). Additional studies need to be performed to establish the full clinical usefulness of iMg determinations for HrPT patients.
We compared the analytical performance of the three currently available Mg ion-selective electrodes. We also determined the effect of freezing and of pH change on the iMg results. Fresh and frozen (-20 degrees C) serum samples from volunteers and patients were analyzed at NIH (AVL and NOVA) and at the Academic Medical Center (AMC) in Amsterdam, Netherlands (AVL, KONE). The best correlation was found between the AVL and KONE analyzers (slope 0.964, intercept -0.01). Freezing affected only the KONE and the NOVA results. There was a significant decrease in NOVA results and the extent of this decrease was related to the pH change that occurred during storage of samples. For KONE, some results were increased while some were decreased, and these changes were not pH dependent. Theoretically, such pattern would be observed if interfering factors (e.g., drugs) were themselves affected by freezing. This difference in the analytical response of the three Mg electrodes is important because freezing is often used, especially in research, to preserve the samples for later batch-analysis.
The apparent dissociation constant of the magnesium-ATP complex, KDMgATP, is used for determination of intracellular free magnesium with 31P NMR spectroscopy. We determined the constant experimentally by measuring the chemical shift difference between a- and b-ATP in the phosphorus (P) spectrum with 31P NMR spectroscopy and by determining the concentration of free magnesium using the fluorescence indicator, Mag-fura-2. The value of KDMgATP = 8.2 ± 0.5 mM at 37 degrees C, pH=7.2, and ionic strength 0.15 were verified by calculation using the concentration of total magnesium that was determined with the atomic absorption spectroscopy. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10153-12 CP
October 1, 1998 to September 30, 1999
Title of Project: Assessment of the Clinical Performance of Lab Tests
Principal Investigator: A.T. Remaley, M.D., Ph.D. (Senior Staff)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: None
Staff-Years: 0.1
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This study has been terminated. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10206-07 CP
October 1, 1997 to September 30, 1998
Title of Project: Evaluation of Techniques for the Analysis of Lipoproteins and Apolipoproteins
Principal Investigator: G. Csako, M.D. (Senior Staff Advisor)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: R. Costello, M.T., CCS, CPD
Collaborating Units: NHLBI, Cardiology Branch (R.O. Cannon, M.D.)
Staff-Years: 0.9
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Since plasma high-density lipoprotein (HDL) is known to be protective against coronary artery disease and plasma low-density lipoprotein (LDL) is atherogenic, measurement of the cholesterol (C) content of these lipoproteins is important for assessing cardiovascular risk, diagnosing lipid disorders, and monitoring lipid-modifying interventions. First a variety of heterogeneous assays (requiring manual pretreatment/separation step), then a variety of homogeneous assays (requiring no off-line pretreatment step) have recently become available for measuring HDL-C and LDL-C. We assessed the analytical performance of four homogeneous HDL-C and two homogeneous LDL-C methods in 120 human sera. For comparison, HDL-C and LDL-C were also determined by electrophoretic and heterogeneous methods. LDL-C was also estimated from two different formulas. Except for one of the homogeneous methods for HDL-C, the electrophoretic ("reference") method for HDL-C and LDL-C generally correlated highly with the respective heterogeneous and homogeneous methods, and with the two LDL-C formulas. However, biases between the old and some of the new methods suggested unsatisfactory standardization. Given their simplicity and adaptability to automation, properly standardized new homogeneous methods may provide cost-effective alternatives to earlier manual and semi-automated methods measuring for HDL-C and LDL-C.
Because analysis of serum lipid, lipoprotein, and apolipoprotein constituents is not always possible in freshly collected specimens, and published data often are conflicting or not available for newer methods, we examined the effect of various storage conditions (+4 degrees C, -10 degrees C, and -70 degrees C) on these analytes, often by measuring with a variety of methods. Statistically significant (p is less than 0.05) differences were common with both parametric and nonparametric statistics between the initial and subsequently measured lipid (total cholesterol and glycerol-blanked triglycerides), lipoprotein-cholesterol (HDL-C: two methods, LDL-C: four methods, and VLDL-C), and apolipoprotein A-I, B, and E concentrations. The differences, however, were generally small (rarely up to 10 percent) and inconsistent. Only the apoE concentration tended to decrease (up to 40 percent) under all storage conditions, and, out of all analytes, only the differences for storing apoE at -70 degrees C were significant with ANOVA analysis. Thus, in contrast to some earlier observations, most serum lipids, lipoprotein-cholesterol fractions, and apolipoproteins can be reliably measured by a variety of currently available methods in refrigerated or frozen specimens.
In collaborative work, the clinical utility of various lipid, lipoprotein, and apolipoprotein measurements was assessed in patients with cardiovascular disease and postmenopausal women. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10207-07 CP
October 1, 1997 to September 30, 1998
Title of Project: Development and Diagnostic Use of Techniques for the Determination of Genes Involved in Lipid/Lipoprotein Disorders
Principal Investigator: G. Csako, M.D. (Senior Staff Advisor)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: Y. Wu, M.D., Ph.D., CCS, CPD
R. Costello, M.T., CCS, CPD
R. Delgado, M.T., CCS, CPD
Collaborating Units: Pharmacy Department, CC (F. Pucino, Pharm.D.)
Geriatric Psychiatry Branch, NIMH (T. Sunderland, M.D.)
Staff-Years: 1.0
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Because plasma lipoprotein(a) (Lp(a)) is atherogenic and apolipoprotein(a) (apo(a)) isoform size may affect plasma Lp(a) concentrations, we evaluated a new, lectin affinity-based method for serum Lp(a)-cholesterol (Lp(a)-C), then studied the relationship between apo(a) isoforms, total Lp(a) mass, Lp(a)-C, and other serum lipid parameters. For apo(a) phenotyping, the isoforms were classified according to low (less than or equal to 20), medium (21-29), and high (greater than or equal to 30) number of kringle 4 repeats. While no detectable loss of Lp(a) occurred in the initial filtrate and wash buffer of the LP(a)-C method, an Lp(a) concentration-dependent fraction (less than or equal to 10 percent of total Lp(a) mass, less than or equal to 25 percent of total Lp(a)-C] was often obtained by repeating the elution step, requiring addition to the Lp(a)-C results. Serum Lp(a) only moderately correlated with Lp(a)-C (r-squared=0.73). The regression equation (Lp(a)-C = 0.14 * Lp(a) + 24.2) indicated that Lp(a)-C as a fraction of total Lp(a) mass decreases with increasing Lp(a) concentrations. Further, decreasing apo(a) isoform sizes tended to increase the Lp(a)-C and, to a different degree, the total Lp(a) mass concentrations, indicating that Lp(a)-C is, at least partially, an Lp(a) mass-independent measure of Lp(a) particles. No other lipid parameters (glycerol-blanked triglycerides, total cholesterol, high-density lipoprotein-cholesterol, apolipoproteins A-I and B, low-density lipoprotein-cholesterol) showed a similar segregation with apo(a) phenotypes.
A new, quantitative approach based on image analysis of restriction endonuclease digested polymerase chain reactionamplified genomic DNA products was developed for apolipoprotein E genotyping. Quantitation of band intensity considerably improved the diagnostic reliability of apo E genotyping for both lipid disorders and Alzheimer's disease.
In collaborative clinical studies, we continued to study the association between apo(a) isoforms and premature thromboembolic events in patients with systemic lupus erythematosus and confirmed that knowledge of apo(a) phenotypes improves prediction. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10220-06 CP
October 1, 1997 to September 30, 1998
Title of Project: Histidine-rich Glycoprotein: Characterization and Clinically Significant Studies
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: None
Staff-Years: 0.5
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Histidine-rich glycoprotein (HRGP), a plasma protein with antiheparin and antifibrinolytic activity, has been associated with thrombophilia in three families. Therefore, it may promote hypercoagulability. The following objectives have been achieved in the laboratory: purification of the HRGP, using a combination of cobalt-sepharose, and development of an enzyme-linked immunosorbent assay for its quantitation. We have confirmed HRGP's antiheparin activity.
In addition, we have demonstrated that HRGP binds to activated human platelets. The amount of binding is increased by several divalent cations, but not calcium or magnesium. It is also enhanced significantly by plasminogen, although HRGP does not have the reciprocal effect of increasing plasminogen binding to platelets. These observations suggest that platelet-bound plasminogen may serve as a binding site for HRGP, although there appear to be other sites as well. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10222-06 CP
October 1, 1997 to September 30, 1998
Title of Project: Assessment of Hydrogen Peroxide Generation in Neutrophils
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: LHD, DIR, NIAID (H. Malech, M.D.)
DTM (S. Leitman, M.D.)
Staff-Years: 0.2
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minor
(a2) Interviews
Summary of Work: Previous work established that the flow cytometric dihydrorhodamine (DHR)-based assay for evaluation of oxidative burst is a sensitive method for evaluating phagocytes in patients with possible chronic granulomatous disease (CGD). It has also been useful as an accurate test for screening possible X-linked CGD carriers. This assay also is a good screen for the genotype of the disease. The method was optimized and is sensitive to one normal cell in less than or equal to 10,000 abnormal cells. A project was continued to track granulocytes post-transfusion in CGD patients following allogeneic leukocyte transfusions. In addition, a second phase of gene therapy of CGD was undertaken directed at patients with X-linked CGD. The DHR assay is being applied as a critical method for establishing the presence of gene-corrected granulocytes in the circulation and monitoring the duration of the gene-corrected cells. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10227-05 CP
October 1, 1997 to September 30, 1998
Title of Project: Clinical Utility of IV Catheter Tip Cultures for Short-term Lines
Principal Investigator: V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: F. Stock, B.S., MS, CPD
Collaborating Units: CCM, CC (R. Cunnion, M.D.; D. Landucci, M.D.)
Staff-Years: 0.05
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Culture of intravenous catheter tips has been a routine practice for the past 10 years. Based on the findings of Maki et al. (Maki, D.G., C.E. Weiss, and H.W. Sarafin. 1977. A Semiquantitative Culture Method for Identifying Intravenous Catheter-related Infection. N Engl J Med 296:1305-1309), a quantitative technique of rolling the tip on a blood agar plate and counting the resulting number of colonies has been a standard practice in most laboratories. The validity and value of these cultures has come into increasing debate, but little well-documented data have been published to clarify this issue.
As part of a larger line-maintenance protocol in the Critical Care Medicine Department, our study will use these patients to look at colony counts and their predictive value in determining line sepsis from short-term central lines. Approximately 200 patients have been enrolled in this study. Organisms from patients who have positive blood cultures and concomitantly positive catheter tip cultures will be examined by molecular typing methods to determine whether the catheter tip isolates are identical to the blood culture isolates, thereby supporting catheter-related bacteremia.
Patient accrual has been completed and molecular analysis of the strains of bacteria (strain typing) isolated from these patients is almost complete. The rate of infection following placement of these lines was low, so the number of episodes to assess is limited. Preliminary review of the data, however, suggests that, although patients with positive blood cultures commonly had greater than 15 colonies on the rolled catheter culture, there were instances of positive blood cultures when the colony counts were less than 15. In addition, there were cases where the organism cultured from the catheter tip was not the same as that cultured from the blood, and instances where catheter tip cultures yielded greater than 15 colonies but were not associated with positive blood cultures. Data collection has been completed, and results are being prepared for publication. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10230-05 CP
October 1, 1997 to September 30, 1998
Title of Project: Development of Methods for Determining Strain-relatedness of Clinical Isolates
Principal Investigator: V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: F. Stock, B.S., MS, CPD
S. Weir, Ph.D., MS, CPD
Collaborating Units: None
Staff-Years: 0.25
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The continued prevalence of nosocomially acquired infections and concomitant clinical and epidemiological interest in typing isolates have resulted in many large clinical laboratories using molecular epidemiological methods to investigate the relatedness of clinically isolated microorganisms. In the Microbiology Service, we are currently evaluating different approaches to molecular strain typing, including plasmid analysis, restriction endonuclease analysis of genomic DNA, pulsed-field gel electrophoresis (PFGE) of genomic DNA, and typing methods based on the polymerase chain reaction. The goal of this project is to develop a coordinated battery of typing methods that can be used to provide information on a wide variety of clinically significant microorganisms. This would permit us to respond promptly in potential outbreak or nosocomial spread situations. Our technical development of these methods includes simplifying procedures so they can be done rapidly, and thereby be of greater epidemiological use. The use of molecular typing methods greatly expands the species of organisms for which we will be able to offer typing. Additional uses of typing availability will be to look at the relatedness of species common to particular patient populations.
Thus far, the use of plasmid analysis and PFGE has proven reliable and sufficiently discriminatory for staphylococci. PFGE will also be suitable for a variety of gram-negative rods, such as Serratia. PFGE, however, is slow and labor intensive, often needing to be repeated. We have found that, for some organisms, getting an adequate number of bands for sufficient discrimination is difficult. Our recent work with random amplified polymorphic (RAPD) DNA assays may prove to be of greater use for these organisms, such as has been the case with Roseomonas and Burkholderia species. We have already used our molecular typing procedures to investigate several questions of either patient-to-patient spread, or common source acquisition of pathogens. By working in conjunction with the Hospital Epidemiology Service, we have been able to quickly resolve these issues.
Our current developmental concerns are to define optimal procedures for each group of significant pathogens so we can respond reliably when needed. At present we can reliably type strains of staphylococci, enterococci, Pseudomonas, Roseomonas, Serratia, and Burkholderia by either PFGE or RAPD. We are planning to enhance our ability to utilize RAPD in place of PFGE for preliminary results, because RAPD can be done much more rapidly and often more reliably and definitively than PFGE. We completed a large-scale investigation of a lethal Klebsiella outbreak that was seriously compromising the colony of marmosets at the Poolesville veterinary facility. We were able to timely and accurately identify the infecting strain by RAPD's rapid results. This allowed us to recognize the infected marmosets promptly, define a program to reduce or eradicate carriage, and, thereby, bring the outbreak under control. The ease and speed of RAPD was critical to the success of this project, as PFGE was too slow and labor intensive for evaluating hundreds of strains.
We are also working on setting up a data repository for PFGE and RAPD patterns to be able to store, retrieve, and manipulate the strain-typing data that we generate. We are using a new software program (Molecular Analyst, Bio-Rad Labs) to do this. Our early evaluation of this program is very encouraging. It will allow us to retrieve hospital pattern data (e.g., for detecting trends in the distribution of antibiotic resistant strains) as well as to follow a particular patient or set of patients in a longitudinal fashion, looking at their colonization/infection patterns. We are particularly interested at looking at specific patient populations that have recurrent infections, to determine the patterns of colonization, infection, and relapse/reinfection that can now be more accurately assessed by these methods. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10233-05 CP
October 1, 1997 to March 1, 1998
Title of Project: Methods for Rapid Detection of CMV
Principal Investigator: H.D. Engler, Ph.D. (Senior Staff Microbiologist)|
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: L. Calhoun, MS, CPD
J. Preuss, MS, CPD
Collaborating Units: None
Staff-Years: 0.35
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This ongoing project involves several studies. Efforts to increase the sensitivity of the shell vial assay in detecting cytomegalovirus (CMV) continue. We are in the process of examining the effect of thymidine analogues, such as indo- or bromo-deoxyuridine, on CMV growth to determine if they can be added to our shell vial cultures to enhance the sensitivity of the shell vial culture to detect CMV in clinical specimens. Hexadimethrine bromide, or polybrene, has been demonstrated to help HHV-6 penetrate the cells in culture and therefore increase the sensitivity of the assay. We will examine the effect of polybrene on CMV in shell vial cultures to see if it can be a means of increasing the sensitivity of the shell vial assay.
The detection of pp65 early structural protein of CMV has been found to be a rapid and sensitive means of predicting progression to disease caused by CMV. We are examining and evaluating a monoclonal antibody pool to pp65 that recognizes the epitopes expressed in the nuclei of the infected peripheral polymorphonuclear leukocytes and monocytes that are different from our currently used antibodies to pp65. It is hoped that this new antibody pool will allow an increased detection sensitivity of this CMV antigen in clinical blood specimens.
CMV can be a cause of pneumonitis in the patient with human immunodeficiency virus. Detection of CMV in smears consisting of cells found in bronchoalveolar lavages can be a rapid means of diagnosing CMV pulmonary disease. We will examine such smears using immunofluorescent reagents for the detection of the CMV pp65 early structural antigen and the CMV 72dk immediate early antigen to determine their clinical utility as a rapid diagnostic method.
Dr. Engler has left NIH and this study has been terminated. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10234-05 CP
October 1, 1997 to September 30, 1998
Title of Project: In Vitro Culture and Susceptibility Testing of Microsporidia
Principal Investigator: D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: N.A. Nelson, M.S., CPD
S.C. Weir, Ph.D., MS, CPD
Collaborating Units: None
Staff-Years: 0.15
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: In the past few years, a considerable number of case reports describing predominantly gastrointestinal, urinogenital, and ophthalmic manifestations of microsporidial infections in acquired immune deficiency syndrome patients have been published. Although anecdotal evidence of the therapeutic efficacy of a number of antimicrobial agents has appeared in the literature, there has been little or no in vitro data to substantiate these claims. Of the principal species of microsporidia that are human pathogens, all but Enterocytozoon bienusi (E. bienusi) have been propagated in cell culture.
We continue to look for ways to enhance the propagation of microsporidia in vitro. The current cell culture systems are inefficient. We have evaluated a number of cell lines and culture conditions to optimize growth of microsporidia in our assay. Efforts will continue to find a cell culture system for the propagation of E. bienusi. Once these objectives are achieved, we intend to screen potentially useful antimicrobial agents to determine which, if any, have deleterious effects on microsporidial replication, and if this inhibition is species specific. Compounds will also be examined for their ability to prevent infection of cell culture monolayers by microsporidia.
Various laboratory methods will be used to evaluate the effectiveness of antimicrosporidial agents. Quantitation of released spores from established cultures will provide information concerning the effect of agents on microsporidial replication and spore production. A monoclonal antibody produced by Dr. Nash (NIAID, LPD) will be used to stain and count infected cells to determine inhibition of infection by antimicrobial agents. We have developed a reliable assay for quantitation that uses polymerase chain reaction and a europium-labeled DNA probe. Quantitation of microsporidial DNA will provide information about the ability of agents to prevent microsporidial replication within the host cell and the ability of agents to prevent infection of cell host cells. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10235-05 CP
October 1, 1997 to September 30, 1998
Title of Project: Detection and Identification of Microsporidia in Stool by PCR
Principal Investigator: D.P. Fedorko, Ph.D. MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: N.A. Nelson, MS, CPD
Collaborating Units: None
Staff-Years: 0.20
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Several reports have appeared recently in the literature detailing cases in which gastrointestinal disease, often with significant associated morbidity, occurred in acquired immune deficiency syndrome patients infected with microsporidial parasites. As a consequence of these reports, there has been an increased awareness among clinicians of the potential importance of microsporidia as gastrointestinal (GI) pathogens. Attention is now being focused on methods for both diagnosis and treatment. There is considerable interest in determining the diagnostic value of stool specimens for detecting GI disease due to microsporidia. A variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is clearly required.
We have published a report that describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. The PCR primers used in the assay will amplify all of the currently identified microsporidial human pathogens. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project will be to determine the sensitivity of our assay and the length of time specimens can be stored in formalin and still allow detection of the microsporidia. We have developed a shorter, cheaper, and less rigorous method for extraction of microsporidial DNA from stool. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10241-05 CP
October 1, 1997 to September 30, 1998
Title of Project: Identification of Molecular Defects in Patients with von Willebrand's Disease
Principal Investigator: M.E. Rick, M.D. (Senior Investigator) HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: D. Krizek, CPD
Collaborating Units: None
Staff-Years: 1.0
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: One family that has an abnormal von Willebrand factor (vWf) with a defective binding site for factor VIII has been studied, and the genetic defect has been identified. The binding defect was initially evaluated by assessing the ability of the patient's vWf to bind purified factor VIII. Subsequently, DNA was purified from peripheral blood leukocytes, specific regions of the vWf gene were amplified by polymerase chain reaction (PCR), and direct sequencing of the DNA was carried out. A transition of nucleotide 2451 (T to A) was found, which results in the substitution of GLN for HIS at amino acid 54 in the mature vWf subunit. We recently used a PCR mutagenesis technique to insert the mutation into cloned DNA and have expressed the abnormal protein. The latter was tested in an assay for binding factor VIII and was shown to manifest decreased binding of factor VIII.
An unrelated patient with von Willebrand's disease and an abnormal distribution of vWf multimers has been studied, and a new mutation in the A1 region of the vWf gene has been identified. The mutation was cloned into an expression vector and the expressed abnormal vWf is being characterized. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10244-04 CP
October 1, 1997 to September 30, 1998
Title of Project: Survival of Microsporidia After Exposure to Disinfectants and Environmental Conditions
Principal Investigator: D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: S.C. Weir, Ph.D., MS, CPD
N.A. Nelson, MS, CPD
Collaborating Units: None
Staff-Years: 0.15
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Microsporidia are ubiquitous parasites causing infections in insects, fish, and mammals. Recently microsporidia have been demonstrated to infect humans. These organisms cause ophthalmic and gastrointestinal infections, primarily in patients with acquired immune deficiency syndrome. Several genera of human pathogens have been cultivated in cell culture. Currently, there are no data regarding the ability of the human pathogens Encephalitozoon intestinalis and Encephalitozoon hellem to survive under various environmental conditions. Also, there are no reports regarding the effects of disinfectants on spores of these two species.
The survival of microsporidial spores after exposure to disinfectants such as chlorine, alcohol, and quaternary ammonium compounds, and environmental conditions such as elevated temperature and dessication will be studied. Cultivation of microsporidia in the shell vial system using various cell lines has been investigated and microsporidia appear to replicate in both fibroblast and epithelial cell lines. Staining methods are being evaluated to detect microsporidial inclusions in infected cell culture after exposure to disinfectants and environmental conditions. A monoclonal antibody produced by Dr. Theodore Nash (NIAID, LPD) has replaced giemsa staining for detection of infected cells. Although the monoclonal antibody makes detection of infected cells easier, the assay is still cumbersome and tedious. In an effort to replace staining of infected cell cultures, a method for quantitation of microsporidial DNA has been developed. This assay utilizes polymerase chain reaction and a europium-labeled DNA probe and is semi-quantitative. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10247-04 CP
October 1, 1997 to September 30, 1998
Title of Project: Use of PCR and RFLP Analysis for Identification of Mycobacteria and Nocardia
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: P.S. Conville, M.S., MS, CPD
S.H. Fischer, M.D., Ph.D., MS, CPD
Collaborating Units: None
Staff-Years: 0.40
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Polymerase chain reaction (PCR) amplification of a portion of the genome of both rapidly growing mycobacteria and nocardia, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, is being evaluated to assess the utility of these procedures for use in the diagnostic laboratory. The technique has already proven useful in providing preliminary identification of these organisms within a few days of organism isolation, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures seem generally to allow more accurate discrimination among species and subspecies than is possible with biochemical testing. Our work with a portion of the genome for 16S ribosomal RNA has suggested the existence of a hitherto unrecognized Nocardia species. Some of this work is currently being prepared for publication.
For the rapidly growing mycobacteria, results of the studies with this procedure have stimulated us to reassess the salt tolerance test, which is widely used to help with the phenotypic identification of these organisms. We have defined the optimal conditions for performing the salt tolerance test, and have shown that even under the best of circumstances it does not have the discriminating power of the molecular methods. A manuscript reporting these results has been published. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10249-04 CP
October 1, 1997 to September 30, 1998
Title of Project: Investigation of the Molecular Mechanisms of Heparin induced Thrombocytopenia
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: None
Staff-Years: 0.3
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Immunoglobulin G (IgG) from patients with heparin-induced thrombocytopenia binds to platelets in an environment that contains approximately equimolar concentrates of platelet factor 4 (PF4) and heparin. The binding requires the F(ab1)2 end of the IgG but not the F end. The specific IgG binding to platelets can be isolated by binding to the PF4 that is attached to heparin-sepharose.
We have more recently elucidated a detail about how complexes of heparin, PF4, and IgG bind to the platelet surface. Binding of the termolecular complexes occurs via the heparin component. When heparin is in excess, free heparin competes with the complexes and displaces them from the platelet membrane. This explains why the molar ratio of heparin to PF4 is critical in determining the in vitro and in vivo reactivity of the complexes with platelets. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10250-04 CP
October 1, 1997 to September 30, 1998
Title of Project: Treatment of Symptomatic, Venous Access Device-related Deep Venous Thrombosis with Recombinant Tissue Plasminogen Activator
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: Radiology Dept., CC (R. Chang, M.D.; J. Doppmann, M.D.)
Staff-Years: 0.1
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Thirty-two patients have been treated with intraclot, pulse-sprayed tissue plasminogen activator, 31 of whom were available. The veins of 12 patients failed to reopen. Three patients could not be adequately heparinized; their veins rapidly reoccluded. The remaining 16 patients were observed on warfarin for a least 2 months. The offending venous access device (VAD) was left in place in four of these patients; three suffered venous reocclusion. VADs were removed from the other ten patients; none of their veins reoccluded. Only two patients developed hypofibrinogenemia; neither hemorrhaged. Five patients had minor bleeding episodes. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10254-03 CP
October 1, 1996 to September 30, 1997
Title of Project: Molecular Defect in the Hereditary Resistance to Activated Protein C
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: D. Krizek, R.M.T., CPD
O. Wilson, R.M.T., CPD
K. Hansmann, R.M.T., CPD
Collaborating Units: None
Staff-Years: 0.25
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Activated protein C (APC) is an important regulatory protein that helps maintain the balance of procoagulant and anticoagulant properties within the circulation. It acts by cleaving activated factor V and activated factor VIII, limiting clot formation at the appropriate level. An abnormal factor V that is resistant to cleavage by APC has been described recently in patients with venous thrombosis. We have set up an assay using DNA amplification and restriction enzyme analysis of the portion of the factor V gene responsible for its resistance to APC. Seven patients have been assessed and appropriate clinical treatment instituted as a result of these test results.
This study is now melded into a broader study entitled genetic abnormalities in patients with thrombophilia. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10255-03 CP
October 1, 1998 to September 30, 1999
Title of Project: Telomerase TestingMolecular Screening for Early-stage Tumors
Principal Investigator: A.T. Remaley, M.D., Ph.D. (Senior Staff)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: Y. Wu, M.D., Ph.D., CPD
R. Delgado, R.M.T., CPD
Staff-Years: 1.7
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Telomerase activation occurs in the majority of primary human cancers, thus making telomerase one of the most widespread cancer markers discovered. While the mechanism linking telomerase activation to cancer is still under investigation, it has become evident that telomerase stabilizes tumor cells' chromosomes by replenishing TTAGGG repeats at their telomeric ends. With the advent of the Telomeric Repeat Amplification Protocol (TRAP), it is now possible to develop and standardize a highly sensitive test for telomerase.
We compared two different formats for the TRAP assay (ELISA and electrophoresis) and found that both assays had a limited linear range of up to 5,000 tumor cells, in the presence or absence of normal cells, with a similar precision (C.V. 11 to 12 percent). Both assays were susceptible to interfering substances present in patient specimens. We also tested different assay parameters that were likely to affect the TRAP assays and made the following improvements in the assay: 1.) Add three cycles of freeze-thaw to increase the extraction efficiency. 2.) Add a 2-hour incubation for the telomerase extension reaction to increase assay sensitivity. 3.) Incorporate dUTP-UNG system to the TRAP procedures to prevent polymerase chain reaction contamination. 4.) Screen specimens using an electrophoretic assay at several cell extract concentrations within the range of 500 to 5,000 nucleated cells, and confirm any positive results with the more quantitative and specific ELISA assay. Using the modified TRAP procedure on microdissected glioma specimens, we found a 100 percent positive rate for telomerase activation compared to a 55 to 78 percent positive rate, using the current recommended procedure.
Even with the above modifications, the telomerase assay is not optimal for routine use on clinical specimens. The current assay has limited linearity, is susceptible to interference, thus requiring that the assay be performed at several cell extract concentrations. In addition, the assay is very tedious and time consuming, and requires highly trained personnel. Our future efforts will be aimed at developing and evaluating new telomerase assays that are suitable for routine clinical use. We will also evaluate the use of laser capture microdissection for preparing specimens for measuring telomerase activity. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10257-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Susceptibility Testing Procedures for Rapidly Growing Mycobacteria and Nocardia
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: G.A. Fahle, B.S., MS, CPD
Collaborating Units: None
Staff-Years: 0.05
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: No standardized susceptibility testing procedures exist for rapidly growing mycobacteria or Nocardia species. Several different procedures have been suggested for use, including broth microdilution and the Etest (manufactured by AB Biodisk, Culver City, CA). We are comparing both a microdilution procedure (JustOne, manufactured by Radiometer America, Inc., Westlake, OH) and the Etest procedure, for their comparability and reproducibility, looking simultaneously at a number of other variables such as inoculum concentration, incubation time, and testing medium.
The JustOne strips are useful because they allow selection of dilutions of individual antimicrobial agents that may be particularly relevant clinically, rather than the use of a whole array of agents as found in the usual microdilution plate format. We hope to be able to select a procedure that will provide rapid and reproducible results, and not be so technically demanding as to be unsuitable for use in the routine diagnostic laboratory. Work to date indicates that the Etest produces results that are difficult to interpret, and we intend to concentrate our further efforts on the microdilution procedure. We have also been involved in a multi-institution collaborative study to determine the inter- and intra-institution reproducibility of both the Etest and a microdilution procedure for susceptibility testing of rapidly growing mycobacteria. The inter-institution results have been collected and are currently being analyzed. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10260-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Development of PCR for CMV, HSV, and VZV from Spinal Fluid
Principal Investigator: S.H. Fischer, M.D., Ph.D. (Senior Staff)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: G. Fahle, M.T., MS
X. Qin, Ph.D., MS
V.J. Gill, Ph.D., MS
Collaborating Units: None
Staff-Years: 0.3
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Diagnosis of meningitis in immunocompromised hosts, such as those with AIDS or cancer, is difficult because of the unavailability of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), Herpes simplex virus (HSV) and Varicella-Zoster virus (VZV), are difficult to grow from cerebrospinal fluid (CSF), and no good antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we have undertaken development of a battery of in-house PCR assays for CMV, HSV, and VZV so they will be available as routine service tests for CSF from NIH patients.
In 1997 development and clinical validation of the PCR assay for CMV in spinal fluid was completed and the test began to be offered as a routine molecular diagnostic assay. During 1998 final development and validation of the PCR assay for HSV in spinal fluid was completed and the test is now also offered as a routine molecular diagnostic assay. The HSV assay on spinal fluid will specifically determine the presence of either HSV1, HSV2, or both viral DNAs. Development of a VZV assay with the same performance characteristics as the existing CMV and HSV assays will continue. All three assays are designed to utilize internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition, in order to eliminate this source of false negative results. Endpoint detection by europium-labeled fluorimetric hybridization probes allows a lower limit of detection of three to five viral genome equivalents. Evaluations of the CMV and HSV assays show excellent specificity. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10262-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Can Oral Washes be Used to Detect Pneumocystis Pneumonia?
Principal Investigator: V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: S.N. Huang, M.D., Ph.D., CCM
E. O'Shaughnessy, M.D., MS, CPD
S. Fischer, M.D., Ph.D., MS, CPD
F. Stock, B.S., MS, CPD
Collaborating Units: CCM, CC (S.N. Huang, M.D., Ph.D.; J. Kovacs, M.D.;
H. Masur, M.D.)
Staff-Years: 0.10
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Our earlier work showed that the laboratory diagnosis of Pneumocystis pneumonia could be done reliably with bronchoalveolar lavage (BAL) specimens and often as well with induced sputa, particularly in conjunction with a fluorescent monoclonal antibody stain. In addition, we reported that Pneumocystis polymerase chain reaction (PCR) can increase the sensitivity of detection when testing induced sputa. More recent work from Denmark suggests that PCR can successfully detect Pneumocystis even from simple oral washes. The use of oral washes would greatly benefit patients since sputum induction is often difficult and unpleasant. We have started a prospective study to look at the comparative sensitivity of induced sputa and oral washes to detect Pneumocystis. For this study, every patient scheduled to undergo sputum induction will simultaneously submit an oral wash specimen by gargling first with a salt solution. Both the oral wash specimen and the induced sputum will be stained for Pneumocystis using our standard fluorescent monoclonal antibody procedure. All specimens will then be tested, in a blinded fashion by PCR assays for Pneumocystis. We have designed two separate assays: one utilizing primers for the major surface glycoprotein (MSG) developed here by J. Kovacs, and another using primers described by Wakefield et al. (Lancet 336:451-453, 1990) based on mitochondrial rRNA genes. Patients who have induced sputum stains negative for Pneumocystis will proceed to have a BAL performed, when clinically indicated. Results of stains of the BAL specimens have generally been regarded as the "gold standard" and thus will be used to determine which of these patients would traditionally be regarded as negative for Pneumocystis. Preliminary studies comparing the two assays using a dilution series of known positive control material indicated that both PCR assays were similar in sensitivity. Similarly, evaluation of the patient specimens thus far suggests that both assays have similar sensitivity, although the MSG assay has detected a few additional positive reactions not positive by the mitochondrial assay. We have also detected patients that were negative by FA stain, but positive by PCR on the oral wash, Our results may reflect the identification of patients harboring Pneumocystis without apparent infection, although this remains to be substantiated by additional patients. The occurrence of false negative and possible false positive reactions are currently being investigated by looking at the clinical information available on each of these patients. More patients will be needed to more definitively establish the usefulness of this technique for patient-care use, so the study is now being expanded to include patients at a second site (Johns Hopkins). Preliminary results will be presented at the next IDSA annual meeting, and are also being prepared for publication. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10263-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Development of a PCR Assay for Diagnosis of Legionella Pneumonia
Principal Investigator: S. Weir, Ph.D. (Visiting Research Fellow)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: V.J. Gill, Ph.D., MS, CPD
F. Stock, B.S., MS, CPD
D. Williams, B.S., MS, CPD
S. Fischer, M.D., Ph.D., MS, CPD
Collaborating Units: None
Staff-Years: 0.10
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Laboratory tests to diagnose Legionella pneumonia are currently very insensitive, relying mainly on the culture of the organism, which may require up to 2 weeks, or on serologic conversion, which may take 2 to 4 weeks. In recent years, a urinary antigen assay has become commercially available, first as a radiometric assay but more recently in an enzyme immunoassay format. We have evaluated this assay using control organisms and random patient specimens and have found it to be highly specific, detecting only Legionella pneumophila serotype 1. This assay is a welcome addition to diagnostic tests for Legionella but may be insufficient because of its high specificity, detecting only serotype 1.
Recent published reports suggest that polymerase chain reaction (PCR) assay may provide a more sensitive technique for diagnosis. We have used a commercially available PCR assay for detection of Legionella from environmental samples as a starting point for developing a test for clinical specimens, particularly for sputum and bronchoalveolar lavage (BAL). The advantage of this procedure is its ability to detect almost all species of the genus Legionella, as well as to identify by specific probe the presence of L. pneumophila. Identification of other species would need further development. Another significant advantage is the inclusion of a simple dot-blot assay, which we found sensitive enough to detect less than 100 colony-forming units per ml (using spiked specimens), yet yield no false negative reactions. Inhibition of PCR by blood in the specimens was removed by washing pelleted specimens in distilled water.
For this study we screened all induced sputa and BAL specimens submitted for routine culture to determine the incidence of specimens that are positive by this assay. For all PCR positive assays, we looked at patient history, urinary antigen assay, culture, and serologic conversion to assess the validity of the PCR results. Of 126 patient specimens screened by PCR, 1 induced sputum and 3 BALs were positive by PCR. All four cases were validated as true positives either by culture of the organism or by serologic conversion. The entire PCR with hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 10 to 12 days, serologic conversion, 2 to 4 weeks.
These studies have provided validation for in-house use of this procedure as an assay for the diagnosis of Legionella pneumonia; the assay will be offered by the Microbiology Service as a new molecular diagnostic test. A manuscript describing our studies and findings has been accepted for publication. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10264-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Does Removal of Catheters Help Clear Catheter-related Gram-negative Bacteremias?
Principal Investigator: V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: NCI, DCS (A. Freifeld, M.D.; D. Fisher; L. Shay)
Staff-Years: 0.05
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Line-related bacteremias associated with long-term catheters (Hickman/Broviac, Groshong, pulmonary artery catheter, and peripherally inserted central catheter) are being encountered regularly now that these catheters are used so commonly. Earlier studies indicated that these catheter-related episodes did not necessitate removing the offending line if the infecting organism was a coagulase-negative Staphylococcus, most commonly S. epidermidis. Antibiotic treatment with the line left in place generally eradicated the organism. For other organisms, however, particularly gram-negative rods, this issue has not yet been resolved.
We would like to examine whether removal of the catheter results in more reliable initial clearance of the gram-negative rod than nonremoval, and also whether the incidence of recurrent infection with the same organism increases in patients in whom the catheter was left in place. Approximately 70 episodes of catheter-related gram-negative bacteremia seen over the past 2.5 years are being examined to determine whether there are any significant differences between patients with catheters removed and those with catheters left in place.
Patient data on these patients is currently being reviewed. In addition, more patients will need to be accrued in order to have statistically evaluable findings. This work is still in progress. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10265-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Development of a PCR Procedure for Quantitative Measurement of CMV in Blood
Principal Investigator: S.H. Fischer, M.D., Ph.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: G.A. Fahle, M.T., MS, CPD
J.M. Parker, B.S., MS, CPD
X. Qin, Ph.D., MS, CPD
J. Canosa, M.T., MS, CPD
Collaborating Units: CCM, CC (H. Masur, M.D.)
DIR, NIAID (M. Polis, M.D.)
Staff-Years: 0.3
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Cytomegalovirus (CMV) disease is a relatively frequent and often serious complication in immunocompromised CMV-infected patients. In the last few years it has become apparent that in order to differentiate between subclinical viral shedding and large scale viral replication occurring during the prodrome before the onset of active disease it is necessary to utilize sequential monitoring with a quantitative assay. Several studies have shown that CMV quantitative polymerase chain reaction (PCR) assays are more sensitive than buffy coat CMV antigen detection assays. This extra sensitivity can in some cases give an additional week of warning before the onset of CMV disease in a patient. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance that the patient will go on to develop active CMV disease.
We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. A standard amount of mimic of the DNA target sequence is included in the reaction mixture of each PCR tube to detect and account for variations in tube-to-tube PCR efficiency in the calculations of viral copy number made from the measured signal strength. The assay is capable of detecting as few as three to five viral genome equivalents in an amplification reaction. Preliminary comparisons of the quantitative PCR protocol with p65 antigenemia determinations in a series of patient samples demonstrates that the PCR assay has greater sensitivity and permits an earlier detection of the CMV prodrome before the onset of CMV disease. The coefficient of variance (CV) of our assay is about 40 percent, in line with other published descriptions of assays of this type.
To develop an assay with improved performance, and, therefore, better potential predictive value for disease onset or progression in patients, we have worked on a "real-time" PCR version of our assay. Assays of this design often have CVs of 10 percent or less. Development of one version of a real-time PCR assay using our existing validated primers and probe sequences is complete. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10267-03 CP
October 1, 1997 to March 1, 1998
Title of Project: Methods for Detection of BK Virus in Allogeneic Bone Marrow Transplant Patients
Principal Investigator: H.D. Engler, Ph.D. (Senior Staff Microbiologist)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: None
Staff-Years: 0.2
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The human polyomavirus BK virus infects a high proportion of the general population and remains latent in the kidney after primary infection. Reactivation can occur when T-cell functions are deficient (e.g., in recipients of bone marrow or organ transplants). BK viruria has been implicated as a cause of hemorrhagic cystitis in bone marrow transplant patients. Rapid detection of active BK virus may help in the medical management of these patients. A 72-hour shell vial culture assay for BK virus was developed to evaluate the incidence and significance of BK virus in the bone marrow transplant patient population. Development of a polymerase chain reaction (PCR) assay for detection of BK virus is underway, and a clinical comparison will be performed to determine whether shell vial culture, PCR, or cytological examination of exfoliated urinary epithelial cells is the most sensitive and rapid method for the detection of BK virus.
Dr. Engler has left NIH and this study has been terminated. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10268-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Detection of Microsporidia in Stool by Modified Trichrome Stain
Principal Investigator: D.P. Fedorko, Ph.D. MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: N.A. Nelson, MS, CPD
Collaborating Units: None
Staff-Years: 0.20
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Definitive diagnosis of microsporidial infection relies on observing microsporidia in biopsy tissue, bodily fluid specimens, or stool examined by transmission electron microscopy. This method lacks sensitivity when performed on stool and is a very laborious and specialized procedure. Detection of microsporidia by light microscopic examination of stained stool specimens from patients with gastrointestinal infection allows rapid diagnosis in a routine clinical microbiology laboratory. Because microsporidia are not detected in traditional ova and parasite examinations of stool specimens, modifications of the trichrome stain are now available for the detection of microsporidia in stool specimens.
We participated in an international diagnostic multicenter study to compare the sensitivity and specificity of the modified trichrome stain compared to the polymerase chain reaction. We received 50 specimens (both positive and negative) to stain with the modified trichrome stain and examine for the presence of microsporidial spores. The data from the 12 participating laboratories have been combined and the results of the study have been published as "Blinded, Externally Controlled Multicenter Evaluation of Light Microscopy and PCR for Detection of Microsporidia in Stool Specimens" (J Clin Microbiol 1998;36:1814-1818).
This study is completed. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10269-03 CP
October 1, 1997 to September 30, 1998
Title of Project: Markers of Disease Activity in Idiopathic Inflammatory Myopathy
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: P. Merryman, M.T., CPD
A. Cullinane, M.T., CPD
Collaborating Units: Intramural Research Program, NIAMS (P. Plotz, M.D.)
CEBR/FDA (L. Rider, M.D.)
Staff-Years: 0.15
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
(a2) Interviews
Summary of Work: This study is designed to aid in the evaluation of clinical disease activity in patients with idiopathic inflammatory myopathies, a diverse group of diseases that include inflammation in skeletal muscle. Since the pathology includes primary muscle capillary endothelial cell damage, we have assessed markers of activation and injury to endothelial cells and activation of coagulation factors, including complexes of thrombin-antithrombin, plasmin-antiplasmin, tPa, and thrombomodulin. We have studied 38 patients and are currently analyzing the data to determine clinical correlations with disease activity. A subset of patients shows an increase in thrombin-antithrombin complexes which is a sensitive assay for activation of coagulation factors. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10272-03 CP
October 1, 1998 to September 30, 1999
Title of Project: Use of Neural Networks in Monitoring Quality Control of Laboratory Data
Principal Investigator: A.T. Remaley, M.D., Ph.D. (Senior Staff)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: DCRT, NIH (J. DeLeo)
Staff-Years: 0.1
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This study is terminated. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10275-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Platelet Aggregation and von Willebrand Factor Defects in Hermansky-Pudlak Syndrome
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892
Other Personnel: L. McKeown, R.M.T., CPD
O. J. Wilson, R.M.T., CPD
K.E. Hansmann, R.M.T., CPD
H.R. Gralnick, M.D.
M.K. Horne, M.D.
S. Rosenfeld, M.D.
K. Rosenfeld, M.D.
Collaborating Units: None
Staff-Years: 0.5
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Patients with Hermansky-Pudlak Syndrome (HPS) have minor to moderate bleeding histories. We have examined platelet function, in particular platelet aggregation and release reaction, in these patients and found that they have deficient dense granule contents. We have measured plasma and platelet von Willebrand factor, and have measured other platelet a- granule constituents such as PF-4 and b-TG in platelet lysates of HPS patients. There is significant decrease in platelet von Willebrand factor activity compared with non-HPS patients and control subjects. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10276-02 CP
October 1, 1997 to March 1, 1998
Title of Project: Development of a Molecular-based Assay to Determine Susceptibility of Viruses to Antiviral Agents
Principal Investigator: H. Engler, Ph.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: S. Fischer, M.D., Ph.D. MS, CPD
L. Calhoun, M.T. (ASCP), MS, CPD
Collaborating Units: None
Staff-Years: 0.07
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The increasing incidence of antiviral resistance by viruses that cause human disease indicates the need for a rapid method to determine the susceptibility of virus isolates to antiviral agents. Current methods available are laborious, tedious, and nonstandardized. Our approach is to grow the virus, initially Herpes simplex virus, in replicate shell vials containing different dilutions of an antiviral agent, and then analyzing the contents of the shell vials by quantitative polymerase chain reaction for growth inhibition.
Dr. Engler has left NIH and this study has been terminated. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10277-02 CL
October 1, 1997 to September 30, 1998
Title of Project: Quantitative Detection of CMV by Using a Commercially Developed Research Assay
Principal Investigator: D.P. Fedorko, Ph.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: H.D. Engler, Ph.D., MS, CPD
L. Calhoun, M.T. (ASCP), MS, CPD
Collaborating Units: American Medical Laboratories (R. Clark)
Staff-Years: 0.2
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Cytomegalovirus (CMV) can be devastating in the patient who has undergone an allogeneic bone marrow transplant. Rapid detection of CMV infection in these patients can be life-saving, allowing the prompt administration of anti-CMV drugs. There is also a clinical need for a quantitative measure of the CMV present in the clinical specimen to allow for monitoring therapy and predicting emergence of drug-resistant variants. A research assay has been developed by a commercial vendor allowing the use of quantitative polymerase chain reaction (PCR) to measure the CMV DNA extracted from whole blood. We will evaluate this PCR assay by using peripheral blood leukocytes collected by American Medical Laboratories and compare the results with the CMV antigenemia quantitative assay (performed at American Medical Laboratories) that detects the presence of CMV pp65 early structural protein in polymorphonuclear leukocytes and monocytes. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10278-02 CP
October 1, 1997 to September 30, 1998
Title of Project: PCR-SSCP for the Detection and Speciation of Microsporidia in Clinical Specimens
Principal Investigator: D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: N.A. Nelson, MS, CPD
A.M. Hruszkewycz, M.D., CS, CPD
R.M. Delgado, CS, CPD
Collaborating Units: Tulane Univ. Medical Center (E.S. Didier, Ph.D.)
Staff-Years: 0.20
Human Subjects: (a) Human subjects x (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: There are many options for the detection and speciation of microsporidia in clinical specimens. Light microscopy allows for detection of the parasites, but does not allow speciation. Electron microscopy is the gold standard for speciation, but is insensitive as a method of detection. The polymerase chain reaction (PCR) is a sensitive technique with many different methods for confirming a positive result and determining the genus and species causing an infection. Speciation can be achieved by using species-specific primers in the PCR assay, or by using DNA probes or restriction endonucleases to determine the species after the PCR assay is performed.
Single strand confirmation polymorphism (SSCP) combined with PCR (PCR-SSCP) has been used to identify bacteria, fungi, and viruses. We will use our PCR assay for the detection of microsporidia in stool specimens and apply SSCP to determine the specific genus and species of the parasite. Organisms from cell culture will also be used to validate the method. This portion of the project is near completion and will soon be submitted for publication.
The project has been expanded to apply SSCP to the detection of microsporidial genotypes for use in epidemiological studies. Newly published primers allow speciation when the PCR products are digested with restriction enzymes. SSCP anaylsis will make this task easier and more sensitive than restriction enzyme analysis. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10279-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Comparison of Microbiologic and Cytologic Results for Bronchoalveolar Lavages
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: F. Stock, B.S., MS, CPD
V.J. Gill, Ph.D., CPD
Collaborating Units: Cytopathology Section, LP, DCS, NCI (A.D. Abati-Scott, M.D.)
Staff-Years: 0.10
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Bronchoalveolar lavage (BAL) specimens are usually split between cytology and microbiology. As different methodologies are used by these two laboratories in the work-up of these specimens, we thought it would be useful to review the results obtained on these specimens by each laboratory. Such a review might help define the relative sensitivities of the different procedures employed, suggest areas of redundancy that might be candidates for elimination, and help identify the procedures most likely to produce clinically significant results. Preliminary results suggest that cytology preparations are more sensitive for the direct detection of significant fungal pathogens than the smears prepared in microbiology. This is presumably because of the larger volume of material used for preparation of smears in cytology. The data for approximately 7 years have been collected and are currently being analyzed to assess the relative sensitivities of the procedures performed in the two laboratories for the detection of other pathogens such as Mycobacterium tuberculosis and Pneumocystis carinii. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10280-02 CP
October 1, 1997 to September 30, 1998
Title of Project: DNA Sequence Variation in the Helicobacter Species Urease Gene
Principal Investigator: S. Weir, Ph.D. (Visiting Research Fellow)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: V.J. Gill, Ph.D., MS, CPD
S. Fischer, M.D., Ph.D., MS, CPD
F. Stock, B.S., MS, CPD
Collaborating Units: None
Staff-Years: 0.2
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: There are currently more than 18 Helicobacter species described and the numbers are increasing. Many of these bacteria are not culturable by standard laboratory methods, because they require microaerophilic conditions and also the presence of hydrogen gas for growth. Many laboratories classify gram-negative, urease-positive spiral bacteria isolated from gastric specimens as H. pylori without further work-up. Consequently, the incidence and pathogenicity of Helicobacter spp. other than H. pylori are relatively unclear. Nonculturable Helicobacter-like organisms have been reported in human gastric biopsies and there are reports of patients with a positive urease breath test with negative gastric biopsy cultures for H. pylori. It is, at present, unknown whether or not these cases are due to a Helicobacter spp. other than H. pylori. There are several reports of Helicobacter-like organisms in blood.
Most Helicobacter spp. are closely related by 16S rRNA sequencing and possess a rapid urease activity similar to H. pylori. The urease enzyme is strongly expressed and has been considered as a target for vaccine development against H. pylori. However, the database of Helicobacter spp urease DNA sequences is limited to three species other than H. pylori. Using polymerase chain reaction (PCR) with degenerate primers, we were able to amplify a region of the ureB gene from H. hepaticus and F. rappini. These PCR products were cloned and sequenced. Analysis of these sequences revealed considerable variability when compared with available sequences. The ureB gene should prove to be a useful PCR amplification target for identification and differentiation between fastidious or nonculturable Helicobacter spp. We have also improved our PCR assay for rapid identification of fastidious Helicobacter-like organisms in patient specimens. We have identified two F. rappini isolates in blood using PCR of the urease gene. This information should be useful in studies of the prevalence and clinical significance of Helicobacter species other than H. pylori. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10281-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Detection and Identification of Mycobacteria in Clinical Specimens
Principal Investigator: S.H. Fischer, M.D., Ph.D. (Senior Staff)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: E. O'Shaughnessy, M.D., MS, CPD
P. Conville, M.S., MS, CPD
F. G. Witebsky, M.D., MS, CPD
J.M. Parker, B.S., MS, CPD
Collaborating Units: Beckman Corp. (J. Rampal, Ph.D.)
Staff-Years: 0.3
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Detection and identification of acid-fast bacilli of the genus Mycobacterium by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may require days to 1 or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of individual species of mycobacteria in clinical samples. At present, there are no commercially available amplification assay systems that are capable of detecting all members of the genus Mycobacterium while excluding cross-reactive signals from other bacteria commonly present in clinical samples.
We have continued development of a PCR assay using primers, targeting a segment of the 16S ribosomal RNA gene, which are capable of amplifying DNA sequences from all members of the genus Mycobacterium while excluding cross-reactive signals from most other bacteria commonly present in clinical samples. Individual mycobacterial species are then identified using specific europium-labeled fluorescent probes. We are currently optimizing the assay conditions to increase sensitivity. We are also examining several new nucleic acid extraction methodologies that may greatly simplify sample preparation for mycobacterial nucleic acid detection assays. A highly sensitive, genus-wide mycobacterial nucleic acid detection system, coupled with a simple, reliable sample preparation technique, could greatly reduce the time needed to detect mycobacterial infections in patients.
A new collaboration has been started with a scientist at Beckman Corporation to develop a better endpoint detection method for the 20-odd most commonly encountered species of mycobacteria from human samples. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10282-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Molecular Characterization of a Novel Cell Envelope Biosynthetic Gene with a Distinctive Sequence Feature of Sugar Acylation in Mycobacterium tuberculosis
Principal Investigator: X. Qin, Ph.D. (Clinical Microbiology Fellow)
MS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: S.H. Fischer, M.D., Ph.D., MS. CPD
Collaborating Units: None
Staff-Years: 0.1
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The principal focus of this work has been to search for biosynthetic genes of cord factor unique to Mycobacterium tuberculosis (Mtb). Because the virulence factor trehalose dimycolate (TDM), the molecular element responsible for "cord" formation in virulent Mtb (cord factor), is found exclusively in the lipid constituents of the cell walls of pathogenic Mtb, study of the synthetic enzyme encoding genes will increase our knowledge of mycobacterial cell-wall biosynthesis and function, hence permitting the design of a tailor-made drug to inhibit key catalytic enzyme. Furthermore, because the cell wall mycolic glycoconjugates are large and complex molecules, the biosynthetic enzymes are believed to be membrane-bound proteins that carry out some of the major condensation steps outside of the cytoplasm to avoid employing complex transport systems. Therefore, these membrane-bound proteins could, in theory, be used for vaccine targets.
The current chemical knowledge of the mycobacterial cellular envelope greatly outweighs our understanding of its genetic and molecular synthetic processes. Nevertheless, experience in well-studied cell envelope features of gram-positive and gram-negative bacteria makes it possible to speculate about principal catalytic steps involved in formation of distinctive molecules such as TDM. The biosynthesis of gram-negative outer membrane component lipid A has been elucidated, and several of the relevant genes are known. By homology search in Sanger MycDB genome database, I have found lpx-analogous genes in the Mtb genome that have not been given definitive functional assignment. One of the corresponding Mtb sequence--z79701/e264134--exhibited profound hexapeptid tandem-repeats resembling the E. coli and B. subtilis counterparts. The unique hexapeptide repeat feature of the UDP-GlcNAc O-acyltransferases in bacteria was further confirmed by searching and excluding its counterpart in two eukaryotic genome databases: the Saccharomyces cerevisiae and the human genome databases.
In conclusion, a possible sugar acylation enzyme-encoding gene (z79701/e264134) has been found in the genome of the Mtb H37Rv strain. Characterization of the gene will be carried out in three steps. First, the gene is going to be cloned and in-frame fused to a fluorescent reporter gene. Therefore, the cell-surface location of the gene product will be determined by fluorescence microscopy. The second step will involve over-expression and crystallization of the protein to compare it to the crystallographic model of the E. coli LpxA. The final step will be the collaborative tests performed with experts in computational biology to design drugs to inhibit the UDP-GlcNAc O-acyltransferases-like catalytic activities, and with immunologists to identify unique antigenic epitops for vaccine development. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10283-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Platelet-associated Antibodies in Patients with Autoimmune Thrombocytopenic Purpura
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
C. Rivera, M.D.
Other Personnel: M. Vail, M.T., CPD
M. Riordan, M.T., CPD
N. Barney, M.T., CPD
K. Hansmann, M.T., CPD
Collaborating Units: NHLBI (C. Dunbar, M.D.; R. Huhn, M.D.)
Staff Years: 0.25
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Autoimmune (Idiopathic) Thrombocytopenic Purpura (ITP) is a disease caused by autoantibodies directed against platelets. Their demonstration has been difficult for a variety of reasons. In general, when the antibodies can be demonstrated, there is an inverse correlation with the platelet count. We have set up two assays, one screening assay and one specific assay for specific platelet glycoproteins, to aid in the diagnosis and progress of treatment for patients with ITP. We will utilize the tests particularly for following patients pre- and post-treatment in a pilot study with NHLBI in the treatment setting of T-cell-depleted auto-stem cell transplantation in patients with severe ITP. Ten patients have been studied. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10284-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Follow-up Study of Patients with Large Granular Lymphocytic Leukemia
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
C. Rivera, M.D.
Other Personnel: M. Riordan, M.T., CPD
N. Barney, M.T., CPD
Collaborating Units: NHLBI (J. Barrett, M.D.)
Staff-Years: 0.12
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Flow cytometry is used in the diagnosis and follow-up of lymphoproliferative diseases, including Large Granular Lymphocytic Leukemia. A unique treatment trial utilizing cyclosporine has been undertaken by the Hematology Branch of NHLBI and has been successful in decreasing some of the cytopenias associated with this disease. Since June, 1997, the Hematology Service of CPD has begun performing follow-up flow cytometry of these patients during the course of their treatment and will be correlated with their responses. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10285-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Assessment of Peripheral Blood Eosinophils
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: LPD, DIR, NIAID (T. Nutman, M.D.)
Staff-Years: 0.2
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: An extensive panel of monoclonal antibodies is being used to evaluate and characterize eosinophils in normal subjects and patients with eosinophilia resulting from infection or other causes. The data are being generated using a novel gating approach that was developed by Dr. Nutman's lab. These data are continuing to be collected for evidence of changes in peripheral eosinophils as a consequence of eosinophil mobilization and disease, in particular the hyper eosinophilia. They will be used to evaluate an extended pedigree with familial hyper eosinophilia. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10286-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Assessment of Peripheral Blood Monocytes in Patients with Recurrent Mycobacterial Infection
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: LHD, DIR, NIAID (S. Holland, M.D.)
Staff-Years: 0.3
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
(a2) Interviews
Summary of Work: Monocytes are being characterized for the expression levels of CD40, CD80, CD86, CD120b, and the gamma interferon receptor alpha chain. These studies have identified two patients with gamma interferon receptor alpha chain deficiency, and the molecular level of this defect is being actively examined. In addition, one patient with interferon gamma receptor beta chain deficiency also has been characterized at the molecular level. As a consequence of these studies, an intracellular flow cytometric method for evaluating STAT protein phosphorylation has been developed and validated. This approach can be adapted for use for a wide variety of activation associated protein alterations. This line of investigation currently is being pursued. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10287-02 CP
October 1, 1997 to September 30, 1998
Title of Project: Assessment of Lymphocytes in Patients with Autoimmune Lymphoproliferative Syndrome
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: LCI, DIR, NIAID (S. Straus, M.D.)
Staff-Years: 0.2
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
(a2) Interviews
Summary of Work: An extensive evaluation flow cytometric evaluation continues of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended-family members. The evaluation is based on characterization of the expanded double negative T-cell and B-cell populations. To date, the double negative T-cells have been demonstrated to be alpha beta TcR, CD57+, HLA-DR+, and CD45RA+. The phenotypic findings in the peripheral blood have been correlated with the histologic and phenotypic findings in the lymph tissue. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10288-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Flow Cytometric Intracellular Protein Evaluation in CGD Granulocytes
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: LHD, DIR, NIAID (H. Malech, M.D.)
Staff-Years: 0.25
Human Subjects: (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The development of a flow cytometric intracellular assay for the presence of the proteins gp91phox and p47phox has been accomplished using specific cell fixation and permeabilization procedures. This method has been applied to evaluate granulocytes from chronic granulomatous disease (CGD) patients and a pattern has been observed that distinguishes the X-linked form of CGD (gp91 phox deficient) from the most common autosomal recessive form of CGD (p47phox deficient). In addition, this method confirms that the maternal carriers of the X-linked form have two populations of circulating granulocytes, one normal (normal gp91phox expression) and one abnormal (absent gp91phox expression). The combination of this method with the DHR assay allows for a rapid and accurate genotype assignment in the majority of CGD patients. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10289-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Genetic Defect in Bernard Soulier Syndrome
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: C. Rivera, M.D., CPD
D. Krizek, R.M.T., CPD
Collaborating Units: None
Staff-Years: 0.25
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
(a2) Interviews
Summary of Work: Using flow cytometry, we discovered that a patient with thrombocytopenia and large platelets had absent platelet membrane glycoprotein Ib-IX complex. DNA was isolated from his peripheral blood leukocytes. Glycoprotein IX gene sequences were amplified and sequenced, revealing a new mutation. His parents' DNA was also isolated and sequenced, and both are heterozygotes for this mutation. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10290-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Genetic Abnormalities in Patients with Thrombophilia
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CPD, DD, NIH, Bethesda, MD 20892
Other Personnel: D. Krizek, R.M.T., CPD
O. Wilson, R.M.T., CPD
K. Hansmann, R.M.T., CPD
Collaborating Units: None
Staff-Years: 0.25
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Patients with recurrent venous thromboembolic disease, disease at an early age, or a family history of this disease are at higher risk for recurrences of thrombosis; their family members are also potentially at higher risk than normal subjects. These patients are being studied for genetic abnormalities that may predispose them to thrombosis. These include abnormalities in the factor V gene (factor V Leiden), prothrombin gene abnormality 20210, and the mutation leading to labile 5, 10 methylenetetrahydrofolate reductase (which increases plasma levels of homocysteine, leading to thrombosis). DNA is isolated from peripheral blood leukocytes, and the DNA is analyzed using polymerase chain reaction and restriction enzyme techniques. Five patients have been studied thus far for these abnormalities.(Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10291-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Evaluation of an Alternative Pathway for Class I MHC Product-dependent Presentation of Polypeptides to CD8 T Cells
Principal Investigator: R.J. Kurlander, M.D. (Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: E. Chao, Technologist, HS, CPD
J. Fields, Technologist, HS, CPD
Collaborating Units: None
Staff-Years: 2.5
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: These studies are designed to examine how antigen presenting cells (APC) are able to efficiently process lemA, a bacterial polypeptide produced by Listeria monocytogenes (LM), and present it to CD8 T cells. We also will test the feasibility of using functionally important elements borrowed from lemA to enhance the antigenicity of other class I-restricted peptide antigens.
LemA, unlike most peptide antigens that are presented to murine CD8 cells by the MHC molecules H2K, D, and L, is presented by a novel class Ib MHC molecule designated H2M3. LemA, a hydrophobic 185-amino-acid polypeptide of unknown function, and its 6 N-terminal amino acids have been identified as the immunogenic component recognized by H2M3-restricted CD8 effectors. Mice infected with LM regularly generate a significant lemA-specific CD8 response, and these cells can protect mice against infection confirming the physiologic relevance of this antigen in vivo.
In prior studies, we have documented that native lemA has several highly unusual properties. The immunogenic core of the molecule is profoundly resistant to degradation by a variety of proteolytic enzymes including proteinase K. Yet, unlike most class I-dependent antigens, which can not be processed and presented to CD8 as exogenous antigens, lemA is processed and presented by a range of APC via a novel brefeldin- and pepstatin-sensitive, TAP1/2-independent pathway.
To gain insight into the mechanisms underlying processing, we have, during the past year, constructed and expressed a truncated lemA product, containing only the immunogenic amino-terminus and adjacent transmembrane portion of lemA (31 amino acids) linked to a histidine tag to facilitate purification. Our studies to date have established that the resulting small (46 amino acid) construct retains all the distinctive functional properties of native lemA described above. Using site-directed mutagenesis, we have placed other amino acid sequences in the N-terminal location, and demonstrated that these chimeric molecules retain the specificity conferred by their aminoterminal peptide. They are processed and presented, however, via the distinctive pepstatin and brefeldin-sensitive pathway described above. Preliminary studies suggest that lemA constructs are highly effective in generating a CD8 immune response in mice, even in the absence of exogenous adjuvants.
During the coming year, we hope: 1.) to define with more precision the cellular biology of lemA processing and presentation; and 2.) to test in greater detail the efficacy of these products in inducing immunity in mice against CD8 epitopes. We are particularly interested in assessing the relative efficacy of lemA-chimer products in stimulating mucosal immunity at sites such as the GI tract, where local proteases would be expected to rapidly degrade more conventional peptide antigens. If these products can be shown to have high immunogenicity in vivo, they may ultimately have a variety of practical applications in enhancing host CD8 immunity. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10292-01 CP
October 1, 1998 to September 30, 1999
Title of Project: Development and Diagnostic Use of Rapid Immunoassays
Principal Investigator: A.T. Remaley, M.D., Ph.D. (Senior Staff)
CCS, CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: J. Glickman, Immunoassay Medical Technologist, CPD
Collaborating Units: None
Staff-Years: 0.25
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: There are several endocrine tumor markers that are routinely used in the diagnosis and management of various cancers. The current assays, however, typically take several hours to perform, which precludes their use in the intraoperative management of patients. The majority of the endocrine tumor markers have a half-life in the circulation of less than 5 minutes, thus making it feasible, if a rapid assay was available, to monitor the concentration of the hormones during surgery or during localization procedures. The primary indication for such assays would be in assessing the extent of residual tumor after surgery and in the localization of the tumors by selective venous or arterial sampling. A commercial rapid assay for monitoring parathyroid hormone (PTH) has recently become available. The goal of this study will be to assess the clinical utility of the rapid PTH assay in the management of patients with hyperparathyroidism. In addition, rapid immunoassays for ACTH and gastrin will be developed and their clinical utility will be determined for the treatment of patients with Cushing's disease and gastrinoma, respectively. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10293-01 CP
October 1, 1997 to September 30, 1998
Title of Project: The Experimental Treatment of Transfusion-dependent 5Q Minus Syndrome
Principal Investigator: C.E. Rivera, M.D. (Senior Staff Physician)
CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: NURS, CC (D.J. Mayo, R.N., B.S.N.) NCI, NHLBI
Staff-Years: 2.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The objective of this protocol is to determine whether an experimental drug can normalize hematopoietic cell growth and differentiation in patients with 5q minus syndrome The patients will be treated with an experimental drug for 3 months and will be monitored for improvements in their counts. In addition, bone marrow colony assays will be performed in the presence and absence of the experimental drug. Several genes in the 5q minus region will be screened for mutations by direct DNA sequencing. FISH analysis for these genes will also be performed in the cytogenetic material to assess the presence of the corresponding gene studied. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10294-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Heparin Cofactor II Levels in Patients with Paroxysmal Nocturnal Hemoglobinuria
Principal Investigator: C.E. Rivera, M.D. (Senior Staff Physician)
CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: O. Wilson, R.M.T., CPD
K. Hansmann, R. M.T., CPD
Collaborating Units: NHLBI
Staff-Years: 3.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Thromboembolic events are the most common cause of mortality in patients with paroxysmal nocturnal hemoglobinuria (PNH). Low plasma heparin cofactor II (HCII) levels have been shown to occur in a variety of hemolytic conditions including thalassemia intermedia and sickle cell disease. The level of HCII is related to the degree of hemolysis. A correlation between low HCII levels and thrombosis has been demonstrated in some of these patients. Because of the association between PNH and thrombosis we are exploring the possible association between HCII levels and PNH. We have already demonstrated that PNH patients have low normal baseline level of HCII (abstract submitted to American Society of Hematology). We will now focus our efforts towards measuring HCII in patients with significant history of thrombosis. Other coagulation parameters including ATIII, Prot C, Prot S, APCR, Prothrombin 20210, and Methylenetetrahydrofolate polymorphisms will also be measured.(Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10295-01 CP
October 1, 1997 to September 30, 1998
Title of Project: Platelet Glycoprotein IIIa Polymorphisms in Young Patients with Coronary Artery Disease
Principal Investigator: C.E. Rivera, M.D. (Senior Staff Physician)
CPD, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Units: NHLBI (J. Villagra, M.D.)
Staff-Years: 2.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The platelet glycoprotein IIb/IIIa functions as a receptor for fibrinogen and von Willebrand factor during platelet aggregation. Recent literature suggests that there is an association between the platelet glycoprotein polymorphisms IIIa PlA1/A2 gene polymorphisms and coronary artery disease. We are screening young patients with coronary artery disease through direct DNA sequencing for other mutations in the platelet glycoprotein genes which may have an association with coronay artery disease. (Back to the project list)