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Project numbers
Analytical Methodology: Development and
Interpretive Application
Magnesium Metabolism in Humans and Biological Systems
Investigation of Hemorrhagic and Thrombotic
Disorders in Humans
Chemical and Structural Studies on Factor VIII/von
Willebrand Factor Protein
Platelet Antigens and Platelet Activation
Assessment of the Clinical Performance of Lab Tests
Thrombolytic Therapy and Anticoagulant Therapy in
Catheter-Related Thrombosis
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
Urokinase to Treat Occlusions of Venous Access Devices
(Protocol # 93-CC-0135)
Histidine-Rich Glycoprotein: Characterized
Clinical Significant Studies
Assessment of Hydrogen Peroxide Generation in
Neutrophils by Flow Cytometry
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 Polymerase Chain Reaction
Clinical Relevance of Yeast Susceptibility Testing
Treatment of Severe Aplastic Anemia With Cyclosporin
and Antithymocyte Globulin
Identification of Molecular Diseases 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 (Protocol # 95-CC-0053)
Trial of Low Molecular Weight Heparin to Prevent
Thrombosis (Protocol # 94-CC-0136)
Molecular Defect in the Hereditary Resistance
to Activated Protein C
Telomerase Testing Molecular Screening for
Early-Stage Tumors 153
Susceptibility Testing Procedures for Rapidly-Growing
Mycobacteria and Nocardia
Optimum Inoculum for BACTEC Susceptibility Testing
of Mycobacterium tuberculosis 155Z01 CL-10259-02 CP Detection and Identification of Mycobacteria in
Clinical Specimens
Development of PCR for CMV, HSV, and VZV From
Spinal Fluid
Clinical and Microbiological Features of
Roseomonas gilardii
Can Oral Washes be Used to Detect Pneumocystis
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 Allogeneic Bone
Marrow Transplant Patients 163
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
Acquired Coagulation-Fibrinolytic-Platelet Defects
in HIV and Other Viruses
Carbohydrate Differences in Plasma and Platelet
von Willebrand Factor
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 Microsporidia
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 Search for Biosynthetic Genes of Mycobacterium
tuberculosis Cord Factor
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
October 1, 1996 to September 30, 1997
Title of Project: Analytical Methodology: Development and Interpretive Application
Principal Investigator: R.J. Elin, M.D., Ph.D. (Chief)
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: G. Csako, M.D., Senior Staff Physician, CCS,
CP
N.N. Rehak, Ph.D., Senior Staff Chemist, CCS, CP
Collaborating Unit: None
Staff-Years:
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Three so-called direct and two calculation-based
methods were assessed for possible interference of hemolysis, hyperbilirubinemia,
and lipemia with the measurement of low-density lipoprotein cholesterol
(LDL-C). While neither hemolysis nor hyperbiliru-binemia influenced the
accuracy of LDL-C measurements, the presence of lipemia variably affected
the different methods. In general, calculation-based, indirect methods showed
more interference due to lipemia than methods based on "direct"
measurements. Further,
the analytical interference usually was more related to the type of hyperlipidemia
than to
the cholesterol and/or triglyceride concentration. On the other hand, even
the method based on electrophoretic separation of lipoprotein fractions
failed when the triglyceride concentration was extremely high. Recognition
of likely interference patterns is important for correct clinical interpretation
of LDL-C results obtained by various techniques.
Thiocyanate (SCN-) found in serum ordinarily is the metabolite of cyanide that is inhaled with tobacco smoke, ingested with cyanogenic foods, and forms after the administration of certain drugs. We investigated the effect of SCN- on the ionized magnesium and ionized calcium results determined with the AVL and NOVA ion-selective electrodes. We measured SCN- concentration in serum from nonsmokers and smokers and found that ionized magnesium results obtained with the NOVA, but not with the AVL, electrode decreased with increasing SCN- concentration. The interference by SCN- appeared to be equimolar for the NOVA electrode. On the other hand, the ionized calcium results obtained by both methods were unaffected by changes in the SCN- concentration. Thus, the serum SCN- commonly found in smokers and, possibly, after ingesting cyanogenic foods and drugs, is expected to cause a clinically significant spurious decrease in the NOVA ionized magnesium results. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Magnesium Metabolism in Humans and Biological Systems
Principal Investigator: R.J. Elin, M.D., Ph.D.
CCS, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: N.N. Rehak, Ph.D., Senior Staff Chemist, CP,
CC
G.A. Csako, M.D., Senior Staff Physician, CP, CC
M.J. Eckardt, M.D., Acting Chief, DICBR, NIAAA
M. Linnoila, M.D., Acting Chief, DICBR, NIAAA
Collaborating Unit: None
Staff-Years: 0.5
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Chronic alcoholism is associated with a marked
deficit in total magnesium,
but little is known about the status of the physiologically active form,
ionized magnesium.. We assessed serum ionized magnesium (measured with AVL
and Nova ion-selective electrodes) and total magnesium concentrations in
chronic alcoholics at admission and after abstinence, and compared these
results with those for a control group. At admission, the total and Nova-ionized
magnesium concentrations in alcoholics were significantly lower than in
controls. After 3 weeks of abstinence there was a significant increase in
total magnesium concentration and both the AVL- and Nova-ionized magnesium
concentrations. Total magnesium concentrations positively correlated with
the AVL results in both alcoholics and controls, but only in controls with
the Nova results. The altered status of ionized magnesium in alcoholics
was clearly instrument-dependent.
In another study we compared abnormal low and high AVL- and Nova-ionized
magnesium results in serum samples from randomly selected patients. Major
intermethod differences were
found for samples with normal total magnesium concentration: most had abnormally
low Nova results and normal AVL results. The agreement for the clinical
interpretation of ionized magnesium results based on the reference interval
for each method was only 32%. The extent of the intermethod difference was
associated with a particular patient rather than with a specific diagnosis.
Changes in serum total and ionized magnesium (measured with Nova ion-selective
electrode) were monitored in 3 patients who transiently developed severe
to profound hypomagnesemia due
to cisplatin or interleukin-2 therapies. Independent of the etiology, the
ionized magnesium fraction increased as the concentration of total magnesium
decreased. When total magnesium was 0.35 mmol/L or less, the ionized magnesium
concentration exceeded the total magnesium concentration. Similar overestimation
of serum ionized magnesium was found for other randomly selected patients
who
had abnormally low concentrations of total magnesium, suggesting an error
in the measurement
of ionized magnesium with the Nova method.
We assessed the effect of smoking on serum ionized magnesium concentration
as measured
with AVL and Nova ion-selective electrodes. A significant intra-method difference
between smokers and non-smokers was found only for Nova results. A dose-dependent
decrease in Nova results was observed with an increase in cigarettes/day.
Nova results inversely correlated with white blood cell (WBC) counts. Nicotine,
cotinine, arachidonic acid, and related metabolites did not affect the per-formance
of either ion-selective electrode. Smoking may induce a serum factor, possibly
related to WBCs, that negatively interferes with the response of the Nova
electrode. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10012-29 CP
October 1, 1996 to September 30, 1997
Title of Project: Investigation of Hemorrhagic and Thrombotic
Disorders in Humans
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: L. McKeown, Research Medical Technologist, CP
O.J. Wilson, Research Medical Technologist, CP
K.E. Hansmann, Research Medical Technologist, CP
S. Williams, Research Medical Technologist, CP
Collaborating Unit: DTM
Staff-Years: 3.0
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
x (a2) Interviews
Summary of Work: Congenital and acquired hemorrhagic and thrombotic
disorders
in humans were investigated to identify the clinical conditions and the
molecular basis
for the clinical defects. This investigation has involved purification of
coagulation
proteins and inhibitors, and definition of the roles of these proteins in
normal and
pathologic hemostasis. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10013-29 CP
October 1, 1996 to September 30, 1997
Title of Project: Chemical and Structural Studies on Factor VIII/von
Willebrand Factor Protein
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S. Williams, Research Medical Technologist, CP
L. McKeown, Research Medical Technologist, CP
L. Garfinkel, Ph.D., Biotech Gen., Israel
Collaborating Unit: None
Staff-Years: 2.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The long-range purpose of this project is to
study the immunologic, biochemical, and molecular genetic aspects of von
Willebrand factor (vWf) protein. We
have initiated studies to identify fragments of vWf that can act as antithrombotic
agents. These vWf fragments are in the Val449-Ser728 region of the plasma
protein. Identification
of a recombinant fragment of vWf Met504-Lys728 inhibits plasma and platelet
vWf binding to GPIb, heparin, and GPIIb/IIIa. Topics of present interest
are (1) the defects of the vWf protein in von Willebrand's disease; (2)
the importance of carbohydrate content in biological functions; (3) the
relationship of carbohydrate content to atherosclerosis; (4) the mechanism(s)
of thrombin activation; and (5) the biochemical characterization and carbohydrate
content
of vWf. (Back to the project list)
October 1, 1996 to July 1, 1997
Title of Project: Platelet Antigens and Platelet Activation
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator)
HS, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: M. Vail, Medical Technologist, CP, CC
N. Murray, Medical Technologist, CP, CC
Collaborating Unit: None
Staff-Years: 2.5
Human Subjects: x (a) Human subjects x (b) Human Tissue (c) Neither
x (a1) Minors
(a2) Interviews
Summary of Work: We have developed several methods to study platelet
activation ex
vivo and in vitro. These studies were made possible by the development and
implement-
ation of techniques to isolate platelets from human blood without inducing
platelet acti-
vation or secretion. We have developed nine monoclonal antibodies that react
only with activated platelets. Our results have shown that in some forms
of von Willebrand's disease, platelet activation is present and causes thrombocytopenia.
In patients with arteriosclerotic heart disease, platelet activation is
increased after exercise. This finding suggests that some endotoxin, spontaneous
platelet activation or enhances agonist-induced platelet activation
is not observed. Exercise in individuals with minor or mild atherosclerosis
does not induce platelet activation.
We have identified patients with paroxysmal nocturnal hemoglobinuria
(PNH) platelet activation. The activation was shown by release of alpha
granule protein and increased fibrinogen binding to the platelet surface.
New evidence of coagulation activation was
observed. These findings suggest that platelet activation may be the primary
mechanism
of venous thrombosis in PNH. Studies to test this hypothesis are being implemented.
This study was terminated in July 1997. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Assessment of the Clinical Performance of Lab Tests
Principal Investigator: A.T. Remaley, M.D (Senior Investigator)
CCS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: J. DeLeo, DCRT
Collaborating Unit: 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 focused on factors that impact on
the clinical efficacy
of diagnostic laboratory tests. A new graphical method (cost-error plot)
was developed
for incorporating prevalence and the costs of misclassification on test
performance, two important factors that affect the practicality of diagnostic
tests. Unlike receiver operating characteristic plots, the clinical efficacy
of laboratory tests can be readily compared by
cost-error plots over a range of possible prevalence and misclassification
costs. Future
work will be done developing software to facilitate production of cost-error
plots. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Thrombolytic Therapy and Anticoagulant Therapy
in
Catheter-Related Thrombosis
Principal Investigator: H.R. Gralnick, M.D. (Chief, Hematology
Service)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: None
Staff-Years: 1.2
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This study duplicates projects Z01 CL-10250-03
CP and
Z01 CL-10251-03 CP and therefore has been terminated. (Back
to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Evaluation of Techniques for the Analysis of
Lipoproteins
and Apolipoproteins
Principal Investigator: G. Csako, M.D. (Senior Staff Advisor/Acting
Chief)
CCS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: R. Costello, M.T., Research Technologist, CCS,
CP
R.J Elin, M.D., Ph.D., CCS, CP
M.N. Bui, M.D., Dept. of Intern. Med., Georgetown University
R.O. Cannon, M.D., NHLBI
A.A. Quyyumi, M.D., NHLBI
Collaborating Unit: None
Staff-Years: 0.9
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: We evaluated two methods of estimating low-density
lipoprotein cholesterol (LDL-C) in human sera: a recently reformulated "direct"
method (Magnetic LDL-C, Polymedco)
and a new, calculation-based indirect method. The new indirect method is
based on the use of total cholesterol, triglycerides, and apolipoprotein
B and has been claimed to be superior over the Friedewald formula for the
estimation of LDL-C in non-chylomicronemic human sera. Both of
these new methods were compared with two well-established "direct"
LDL-C techniques (Sigma Direct LDL-C and Helena LipidProfile) and with conventionally
calculated LDL-C (the Friedewald formula), based on the measurement of total
cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C).
In the Sigma Direct LDL-C method, HDL and very low-density lipoprotein (VLDL)
are removed by immunoabsorption and the remaining (total) cholesterol is
measured as being equal to LDL-C in the absorbed serum. The Polymedco method
removes LDL and calculates LDL-C as the difference between total cholesterol
and the cholesterol content of the remaining lipoproteins. The Helena LipidProfile
cholesterol method involves quantitative assessment of cholesterol by a
coupled enzyme reaction following electrophoretic separation of various
lipoprotein fractions in agarose gel. Hence, this method concomitantly measures
cholesterol in all major lipoprotein frac-
tions such as HDL, VLDL, LDL and chylomicrons. In addition to being measured
with lipoprotein cholesterol electrophoresis, HDL-C was also assessed by
a solid-phase dextran sulfate-Mg2+ method (Polymedco) in which non-HDL lipoproteins
are magnetically removed by dextran sulfate-coated particles from the serum.
The re-formulated Polymedco LDL-C method, which has been used to analyze
over 100 normal and abnormal human sera, showed improved analytical performance
over the earlier version and compared well with the Sigma Direct method
in nonturbid sera. On the other hand, the new formula showed no clear advantage
over the traditional Friedewald formula for calculation of LDL-C.
There has been growing interest in the possible pathogenetic role and diagnostic importance of circulating anti-oxidized LDL autoantibodies in atherosclerosis. In collaborative studies, we studied the analytical and diagnostic performance of an in-house and a commercial enzyme-linked immunosorbent assay (ELISA) for the quantification of these antibodies in the sera of various groups of human subjects. Only the serum anti-oxidized LDL autoantibody titer obtained with the in-house ELISA appeared to correlate with the development of atherosclerosis. The lack of similar correlations with the commercial assay and the lack of correlation between the two ELISA methods indicate that diagnostically important differences may exist among various assays for the measurement of these autoantibodies. (Back to the project list)
October 1, 1996 to September 30, 1997
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/Acting
Chief)
CCS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: R. Costello, MT, CCS, CP
F. Pucino, Pharm.D., PHAR
Collaborating Unit: None
Staff-Years: 0.5
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: With technical improvements in both the detection
and classification
of various apo(a) isoforms, we continued to study the possible relationship
between apo(a) isoforms and the serum lipoprotein(a) (Lp[a]) concentration.
After implementation of a recently developed technique for quantitative
determination of Lp(a)-cholesterol (Lp[a]-C) concentration, we investigated
associations among apo(a) isoforms, Lp(a) concentration
and Lp(a)-C concentration in a variety of human sera.
In collaborative clinical studies, we further studied the association
between apo(a) isoforms and premature thromboembolic events in patients
with systemic lupus erythe-
matosus (SLE). We investigated the relative value of apo(a) isoform detection
and the
conventional risk assessment of measuring the type and titer of anti-cardiolipin
antibodies (ACA). Time to premature thromboembolic events from the diagnosis
of SLE (Kaplan-
Meier method) was significantly less in the medium vs. non-medium apo(a)
isoform
subsets (p = 0.007, log-rank test) and in the IgG and/or IgM ACA
positive vs. negative subsets (p = 0.014). Backward stepwise proportional
hazard regression analysis yielded a model in which age at SLE diagnosis,
medium-sized apo(a) isoforms, and IgG and/or IgM ACA positivity all were
significant predictors of the time from SLE diagnosis to premature thromboembolic
event (all p < 0.03). Thus, the genetically determined apo(a)
isoforms improved the prediction of premature thromboembolism in patients
with SLE.
In other collaborative studies, we continued to explore the relationship between various apo(a) isoforms and various parameters of fibrinolysis in patients with extreme changes in their clinical thyroid status (from hyperthyroid through euthyroid to severely hypothyroid). (Back to the project list)
October 1, 1996 to May 27, 1997
Title of Project: Urokinase to Treat Occlusions of Venous Access
Devices
(Protocol # 93-CC-0135)
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: None
Staff-Years: 0.1
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Forty-two patients received urokinase (40,000u/hr)
through their
venous access devices (VADs) to relieve obstruction caused by a fibrin sheath.
Twenty-one
of these also received heparin (320u/hr) with the urokinase. In each group
16 VADs opened within 12 hours of treatment. By actual analysis the probability
was only 0.28 that a reopened catheter would reocclude within 6 months.
The results have been published (Journal of Clinical Oncology 1997;15:2709-2714)
and the study terminated. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Histidine-Rich Glycoprotein: Characterized Clinical
Significant Studies
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: None
Staff-Years: 0.6
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 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 and demonstrated that it binds to plasminogen. An ongoing project is the study of HRGP binding to proteins that contain a "kringle" structure. By identifying binding sites for HRGP, we hope to discover potential functions for this protein. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Assessment of Hydrogen Peroxide Generation in
Neutrophils
by Flow Cytometry
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology
Service)
IS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Units: LHD, DIR, NIAID (H. Malech, M.D.); DTM (S. Leitman, M.D.)
Staff-Years: 0.2
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(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 1 normal cell in 10,000 abnormal cells. Because of this
sensitivity, this method has been extended to track granulocytes post-transfusion
in CGD patients following allogeneic leukocyte transfusions. Pretransfusion
and 1-hr, 6-hr, and 24-hr post-transfusion DHR tests are being performed
in these patients, and data are being compared to other measures
of cell survival, clinical status, and circulating cell numbers.(Back to the project list)
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: F. Stock, B.S., MS, CPD, CC
D. Landucci, M.D., CCM, CC
R. Cunnion, M.D., CCM, CC
Collaborating Unit: None
Staff-Years: 0.05
Human Subjects: (a) Human subjects (b) Human Tissue 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 based on 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 has 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 if 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 still in progress.
The rate of infection follow-
ing 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 have > 15 colonies on
the rolled catheter culture, there were instances when the colony counts
were < 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 > 15 colonies but were not associated
with positive blood cultures. Typing of strains and analysis of the data
is ongoing. (Back to the project list)
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: F. Stock, B.S., MS, CPD, CC
S. Weir, Ph.D., MS, CPD, CC
Collaborating Unit: 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 hospital-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 will permit us to respond
promptly in potential outbreak or nosocomial spread situations. Our technical
development of these methods includes simplifying procedures so that 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 DNA (RAPD)
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,
and by working in conjunction with the Hospital Epidemiology Service have
been able to quickly resolve those issues.
Our current developmental concerns are to define optimal procedures for
each group of significant pathogens so that 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, since RAPD can be done much more
rapidly and often more reliably and definitively than PFGE. We are also
working on setting up a data repository for PFGE and RAPD patterns in order
to be able to store, retrieve, and manipulate the strain typing data that
we generate. This will allow us to retrieve hospital pattern data, e.g.,
for detecting trends in the distribution of antibiotic resistant strains,
as well as be able
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 determined by these methods. (Back to the
project list)
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: L. Calhoun, Medical Technologist, Research, MS,
CPD, CC
J. Preuss, Medical Technologist, MS, CPD, CC
Collaborating Unit: 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 culture to detect CMV in clinical specimens. Hexadimethrine bromide, or polybrene, has been demonstrated to help human herpes virus 6 penetrate 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 increase the sensitivity of the shell vial assay.
The detection of pp65, the early structural protein of CMV, has been
found to be a
rapid and sensitive means of predicting CMV disease progression. We are
examining and evaluating a pool of monoclonal antibodies to pp65 that recognizes
the epitopes expressed
in the nuclei of infected peripheral polymorphonuclear leukocytes and monocytes
and that are different from our currently used antibodies to pp65. It is
hoped that these new anti-
bodies 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 HIV. 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 CMV pp65 and the CMV 72kd
immediate early antigen to determine their clinical utility as rapid diagnostic
methods. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: In Vitro Culture and Susceptibility Testing of Microsporidia
Principal Investigator: D.P. Fedorko, Ph.D. MS, CPD, CC, NIH,
Bethesda, MD 20892-1508
Other Personnel: N.A. Nelson, MS, CPD, CC
S.C. Weir, Ph.D., MS, CPD, CC
Collaborating Unit: 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 AIDS 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 bieneusi
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. Efforts to find a cell
culture system for the propagation of E. bieneusi will continue.
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. The ultimate goal of this study
is to be able to correlate in vitro susceptibility results with in vivo
therapeutic responses in order to help document the effectiveness,
or lack thereof, of different pharmacologic modalities for treating microsporidial
disease.
Various laboratory methods will be used to evaluate the effectiveness of
anti-microsporidial agents. Quantitation of released spores from established
cultures will provide information concerning the effect of agents on microsporidial
replication and spore production. 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 host cells. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Detection and Identification of Microsporidia
in Stool by Polymerase Chain Reaction
Principal Investigator: D.P. Fedorko, Ph.D. MS, CP, CC, NIH, Bethesda,
MD 20892-1508
Other Personnel: N.A. Nelson, MS, CP
Collaborating Unit: 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 (GI) disease, often
with significant associated morbidity, occurred in AIDS 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 GI pathogens, and 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, and 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. Since 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, a more widely
available approach to identification is clearly required.
We have published a report describing 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. The assay is undergoing modification to allow detection of microsporidia in formalin-fixed stool specimens. A dot-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. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Clinical Relevance of Yeast Susceptibility Testing
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: P.S. Conville, M.S., Microbiology Service, CPD,
CC
Collaborating Unit: None
Staff-Years: 0.10
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The number of available antifungal agents is
now increasing, as is the incidence of significant yeast infections. The
National Committee for Clinical Laboratory Standards has recently developed
a standard for yeast susceptibility testing. While there is increasing clinical
interest in the results of in vitro tests to help guide clinical decision-making
in selecting agents to treat yeast infections, it remains to be determined
how well in vitro results correlate with clinical responsiveness. In this
study, we intend to (1) compare commercially-available testing procedures
such as the E-test and a colorimetric method (AccuMed International, Inc.)
with results obtained by the proposed reference method,
(2) determine the clinical relevance of in vitro susceptibility results
by correlating in vitro
and clinical results, and (3) collect clinical yeast isolates and assess
any changes over time
of susceptibility results. Our work to date suggests that the E-test results
are difficult to interpret accurately and that the colorimetric test offers
the most promise for a rapid and accurate testing procedure that is feasible
to perform in a routine diagnostic laboratory. The manufacturer of the plates
has resumed production of the plates, and we now think that results with
these plates correlate well enough with results obtained by the reference
method to use this procedure in lieu of the much more labor-intensive reference
method. Given the difficulties we have had with interpreting the results
obtained by the E-test, we are not planning any further work with that procedure
for fungal susceptibility testing. The present rate of accrual
of significant clinical isolates of yeasts has been so low that we have
decided to terminate
the data collection portion of this study, but we will continue to offer
yeast susceptibility testing on clinical isolates as a research tool for
clinical investigators. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Treatment of Severe Aplastic Anemia With Cyclosporin
and Antithymocyte Globulin
Principal Investigator: N. Young, M.D. (Senior Investigator) IR,
NHLBI, NIH, Bethesda, MD 20892
Other Personnel: S. Rosenfeld, M.D., Senior Staff Physician, CP
J. Kimball, Research Nurse, IR, NHLBI
Collaborating Unit: IR, NHLBI
Staff-Years: 0.7
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
x (a2) Interviews
Summary of Work: Immunosuppressive therapy can produce hematologic improvement in a large proportion of patients with severe aplastic anemia. We have established antithymocyte globulin and cyclosporin as the current treatment of choice for patients who do not have histocompatible sibling donors or who are otherwise ineligible for allogeneic bone marrow transplant. About 70% of patients respond to this therapy. We are currently following 112 patients and continuing to accrue patients onto this protocol to determine the durability of response, the incidence of late complications, and the long-term prognosis. Interim results have been published. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Identification of Molecular Diseases in Patients
With von Willebrand's Disease
Principal Investigator: M.E. Rick, M.D. (Senior Investigator) HS,
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: D. Krizek, Research Medical Technologist, CP
Collaborating Unit: None
Staff-Years: 1.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: A 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 Gin 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 will be used in our assay for factor VIII to
assess its binding capacity.
Two related families with different variants of von Willebrand's disease
and a vWf with an abnormal affinity for binding to platelets have been identified.
Studies of the
binding region of the vWf for platelets have not yielded any abnormalities
to date,
and further sequencing studies are being carried out. (Back
to the project list)
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: S.C. Weir, Ph.D., MS, CPD, CC
N.A. Nelson, MS, CPD, CC
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. Microsporidia have recently been
demonstrated to infect man. These organisms cause ophthalmic and gastrointestinal
infections, primarily in patients with AIDS. Several genera of human pathogens
have been cultivated in cell culture. At present there are no data regarding
the ability of the human pathogens Encephalitozoon intestinalis
and Encephalitozoon hellem to survive under various environmental
conditions, nor are
there 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 or to environmental
conditions such as elevated temperature and desiccation will be studied.
Cultivation of microsporidia in the shell vial system using various cell
lines has been investigated. Microsporidia appear to replicate in both fibroblast
and epithelial cell lines. Staining methods are being evaluated
to detect microsporidial inclusions in infected cell cultures after exposure
to disinfectants
and environmental conditions. 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. (Back to the project list)
October 1, 1996 to September 30, 1997
Title of Project: Use of PCR and RFLP Analysis for Identification
of Mycobacteria and Nocardia
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: P.S. Conville, M.S., Microbiology Service, CPD,
CC
S. Fischer, M.D., Ph.D., Microbiology Service, CPD, CC
Collaborating Unit: 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 to allow more accurate discrimination among
species and subspecies than
is possible with biochemical testing. We are currently correlating results
obtained with phenotypic methods with molecular methods. For the nocardia
group, we are investigating the use of other primer pairs to assure that
amplification of the portion of the genome with which we are working can
indeed be obtained from all species in the genus Nocardia.
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 we have shown that even under
the best of
circumstances it does not have the discriminating power of the molecular
methods. (Back to the project list)
October 1, 1996 to May 31, 1997
Title of Project: Investigation of the Molecular Mechanisms of
Heparin-Induced Thrombocytopenia
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: None
Staff-Years: 0.2
Human Subjects: x (a) Human subjects (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 PF4 that is attached to heparin-sepharose. This
study has been concluded and the results published (Journal of Laboratory
and Clinical Medicine 1996;127:435-442). (Back to the
project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10250-03 CP
October 1, 1996 to September 30, 1997
Title of Project: Treatment of Symptomatic, Venous Access Device-Related
Deep Venous Thrombosis With Recombinant Tissue Plasminogen Activator (Protocol
# 95-CC-0053)
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: R. Chang, M.D., DR
J. Doppmann, M.D., DR
Collaborating Unit: None
Staff-Years: 0.1
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Twenty-eight patients have been treated with intraclot, pulse-sprayed tissue plasminogen activator, 27 of whom were available. The veins of 8 patients failed to reopen. Three patients could not be adequately heparinized; their veins rapidly reoccluded. The remaining 16 patients were observed on warfarin for at least 2 months. The offending venous access device (VAD) was left in place in 4 of these patients; 3 suffered venous reocclusion. VADs were removed from the other 12 patients; none of their veins reoccluded. Only 2 patients developed hypofibrinogenemia; neither hemorrhaged. Five patients had minor bleeding episodes. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10251-03 CP
October 1, 1996 to December 19, 1996
Title of Project: Trial of Low Molecular Weight Heparin to Prevent
Thrombosis (Protocol # 94-CC-0136)
Principal Investigator: M.K. Horne, M.D. (Senior Clinical Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: D.J. Mayo, R.N., NURS
Collaborating Unit: None
Staff-Years: 0.1
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Thirty patients were accrued over 21/2
years. The study was terminated in December 1996 because of an inadequate
supply of patients. The number
of patients studied was too low to yield meaningful results. (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 20892-1508
Other Personnel: D. Krizek, Research Medical Technologist, CP
O. Wilson, Research Medical Technologist, CP
Collaborating Unit: None
Staff-Years: 0.25
Human Subjects: x (a) Human subjects x (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. In acts by cleaving activated factor V and activated factor VIII, thereby 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 has been instituted as a result of these test results. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10255-03 CP
October 1, 1996 to September 30, 1997
Title of Project: Telomerase TestingMolecular Screening for Early-Stage Tumors
Principal Investigator: A.M. Hruszkewycz, M.D., Ph.D. (Senior
Staff Fellow) CCS, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: Y. Wu, M.D., Ph.D., Senior Staff Fellow, CP, CC
R. Delgado, Research Medical Technologist, CP, CC
M.M. Walther, M.D., Attending Surgeon, SB, NCI
W.P. Bennett, M.D., Senior Staff Fellow, DCE, NCI, DCE
D. Fedorko, Ph.D., Senior Staff, CP, CC
Collaborating Unit: Beckman Instruments, Inc., Fullerton, CA
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 about 90% of
primary human cancers, making telomerase one of the most widespread cancer
markers discovered. Activated telo- merase is found in 95% of late-stage
breast cancers, 95% of colorectal carcinoma, and 90% of bladder carcinomas.
While the mechanism linking telomerase activation to cancer is under intense
investigation, it has become evident that telomerase stabilizes tumor cell
chromosomes by replenishing TTAGGG repeats at their telomeric ends. With
the advent of the Telomeric Repeat Amplification Protocol (TRAP), it is
now possible to standardize a
highly sensitive test for telomerase.
Our objective is to develop a molecular diagnostic test for detecting cancer
cells in body fluids. To do this, we have modified the TRAP assay to make
it compatible with the clinical laboratory. In the first step of the TRAP
assay, the telomerase from cell extracts adds telomeric repeats to the 3'
ends of substrate oligonucleotides. The telomerase-generated products are
then amplified by a polymerase chain reaction (PCR) which includes dUTP.
The products generated vary by six base increments and are resolved by electrophoresis
or by hybridization to complimentary sequences.
Our TRAP assay can detect fewer than 50 tumor cells, and quantification
of the telomerase signal with fluoroimage analyses shows that our TRAP assay
is linear, with extracts derived from between 500 and 5000 cells. The inter-assay
coefficient of variation
is 12%. Telomerase from tumor cells is readily detected in cell mixtures
containing fewer than one 1% of telomerase-positive cells. Having demonstrated
that our assay detects telomerase activity in clinical specimens that contain
cancer cells, we are now develop-
ing approaches that will further improve test resolution with clinical specimens,
such
as urine and bladder washings.
In addition, we continue to develop new PCR-based modalities for detecting
cancer-related DNA mutations in the human K-ras oncogene. Adjunctively,
in collaboration with the Microbiology Service, we are applying our non-isotopic
PCR-SSCP (single-strand conformation polymorphism) methods toward the identification
and characterization of
encephalitozoon species. We anticipate that, once validated, our
protocols will become
routine molecular diagnostic tests. (Back to the project
list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10257-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Susceptibility Testing Procedures for Rapidly-Growing
Mycobacteria and Nocardia
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: G.A. Fahle, B.S., Microbiology Service, CPD, CC
Collaborating Unit: None
Staff-Years: 0.10
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, including broth microdilution
and the E-test (manufactured by AB Biodisk,
Culver City, CA). We are comparing a microdilution procedure (JustOne, manufactured
by Radiometer America, Inc., Westlake, OH) with the E-test procedure for
their comparability and reproducibility. We are simultaneously looking 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 E-test produces results that are difficult to interpret,
and we intend to concentrate our further efforts
on the microdilution procedure. We are also involved in a multi-institution
collaborative study to determine the inter- and intra-institution reproducibility
of both the E-test and
a microdilution procedure for susceptibility testing of rapidly-growing
mycobacteria. (Back to the project list)
October 1, 1996 to December 31, 1996
Title of Project: Optimum Inoculum for BACTEC Susceptibility Testing
of Mycobacterium tuberculosis
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: P.S. Conville, M.S., Microbiology Service, CPD,
CC
Collaborating Unit: None
Staff-Years: 0.03
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The BACTEC 460 TB System (Becton Dickinson Diagnostic
Instrument Systems, Sparks, MD) is widely used to perform susceptibility
testing of clinical isolates of Mycobacterium tuberculosis. This
system currently provides more rapid susceptibility results than other available
methodologies, and its use is currently recommended for the earliest possible
detection by nonmolecular methods of multidrug-resistant M. tuberculosis
(MDR-TB) isolates. Preliminary studies done in our laboratory suggested
that some of the organism inoculum recommendations provided by the manufacturer
may not in fact be optimal and may require repetition of the test to obtain
interpretable results. We evaluated the use of different inoculum sizes
with this system. We found that while the higher inoculum concentration
(which we suspected would generally be more useful) was in fact satisfactory,
it showed no consistent superiority to the lower inoculum concentration
recommended
by the manufacturer. This project has been concluded. (Back
to the project list)
October 1, 1996 to March 31, 1997
Title of Project: Detection and Identification of Mycobacteria
in Clinical Specimens
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: P.S. Conville, M.S., Microbiology Service, CPD,
CC
J. Parker, B.S., Microbiology Service, CPD, CC
Collaborating Unit: None
Staff-Years: 0.05
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Conventional detection and identification procedures for mycobacteria require growing the organisms from patient specimens and then determining various phenotypic characteristics of the isolated organisms. These techniques may take from a minimum of several days to a month or more. Molecular methods offer the hope of drastically reducing these time periods, potentially allowing much more rapid diagnosis of mycobacterial infections. We have been working on methods to extract mycobacterial DNA directly from patient specimens (we have been using a freeze-boil technique) and then using the polymerase chain reaction (PCR) procedure to amplify a portion of the mycobacterial 16S rRNA gene. Work in this area has continued but now involves the use of different, newly-designed primers and another amplicon-detection mechanism. For details on that project, see project number Z01 CL-10279-01 CPD. (Back to the project list)
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: G. Fahle, M.T., MS
X. Qin, Ph.D., MS
V.J. Gill, Ph.D., MS
Collaborating Unit: 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 lack 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, 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 are developing a battery of in-house PCR assays for CMV,
HSV, and VZV so that they will be available as routine service tests for
CSF from NIH patients. The PCR assay for CMV in spinal fluid has been completely
developed, and validated and is being offered as a routine molecular diagnostic
assay. The HSV and VZV assays are being developed with the same performance
characteristics as the existing CMV protocol so that simultaneous detection
of all three viruses can be accomplished in a single assay run. All three
assays utilize internal mimics to evaluate the efficiency of amplification
within individual PCR tubes and to detect PCR inhibition, which will eliminate
this source of false negative results. Endpoint detection by europium-labeled
fluorimetric hybridization probes allows
a lower limit of detection of 3 to 5 viral genome equivalents. Evaluations
of the CMV and
HSV assays show excellent specificity. (Back to the project
list)
October 1, 1996 to September 30, 1997
Title of Project: Clinical and Microbiological Features of Roseomonas gilardii
Principal Investigator: V.J. Gill, Ph.D. (Chief) MS, CPD, CC,
NIH, Bethesda, MD 20892-1508
Other Personnel: F. Stock, B.S., MS, CPD, CC
D. Williams, B.S., MS, CPD, CC
S. Weir, Ph.D., MS, CPD, CC
Collaborating Unit: PO, NCI (L. Lewis, M.D.)
Staff-Years: 0.04
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Roseomonas, a recently described genus
of gram-negative coccobacilli, was formerly designated as "pink coccoid
group" by the National Communicable Disease Center (NCDC) because of
the organism's characteristic pink colonies. Since 1991 we have isolated
Roseomonas from 8 patients; 7 from blood cultures and 1 from a skin
lesion. The 7 blood isolates were from patients with significant underlying
diseases and central venous catheters. Our findings indicated that this
organism causes central venous catheter-associated bacteremia in immunocompromised
patients. Patients were predominantly female (5/7) and had solid tumors
(5/7); 1 patient was in end-stage HIV disease. Five had fever as the primary
basis for obtaining blood cultures, while another had catheter site tenderness.
Four of the 7 had mixed-organism infections. The isolates had originally
been identified by us as NCDC pink coccoid Group III. Using the newly proposed
criteria for this genus, we determined that all of our 8 isolates were consistent
with Roseomonas gilardii. To determine whether we were dealing with
the same strain of organism, indicative of a common source, we attempted
to type these isolates using molecular typing procedures. Although pulsed-field
gel electrophore-sis (PFGE) patterns could be obtained using restriction
enzymes SpeI + Xba1, this method was quite labor-intensive and not sufficiently
discriminatory. More definitive results were obtained using a random amplified
polymorphic DNA (RAPD) technique, which showed
that each of the 8 isolates had distinctly different RAPD patterns. RAPD
is faster and simpler to perform than PFGE and is the method of choice for
Roseomonas strain typing.
This work was recently published as "Infections with Roseomonas
gilardii and Review of Characteristics Used for Biochemical Identification
and Molecular Typing" (Am J Clin Pathol 1997;108:210-216). This
study is completed. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10262-02 CP
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: S.N. Huang, M.D., Ph.D., CCM, CC
E. O'Shaughnessy, M.D., MS, CPD, CC
S. Fischer, M.D., Ph.D., MS, CPD, CC
F. Stock, B.S., MS, CPD, CC
J. Kovacs, M.D., CCM, CC
H. Masur, M.D., CCM, CC
Collaborating Unit: None
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 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 the BAL specimen stains 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 suggest that
both assays have similar sensitivity (4/7 MSG, 3/7 Mito). The occurrence
of false-negative and possible false-positive reactions is currently being
investigated by looking at the clinical information available on each of
these patients. Further studies are needed to more
definitively establish the clinical usefulness of this technique. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10263-02 CP
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: V.J. Gill, Ph.D., MS, CPD, CC
F. Stock, B.S., MS, CPD, CC
D. Williams, B.S., MS, CPD, CC
S. Fischer, M.D., Ph.D., MS, CPD, CC
Collaborating Unit: 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 24 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. We found it to be highly specific, detecting only Legionella pneumophila serotype 1. This assay is a welcome addition to diagnostic tests for Legionella, but it may be insufficient because of its high specificity, detecting only serotype 1.
Recent published reports suggest that polymerase chain reaction (PCR) assays
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 yielded 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 deemed 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 4 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 1012
days, and serologic conversion, 24 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
submitted for publication. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10264-02 CP
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: None
Collaborating Unit: DCS, NCI (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 so commonly used. 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. epider-midis. 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 does, and also whether the incidence of recurrent infection with the same organism increases in patients in whom the catheter is 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.
Data on these patients is still being organized and reviewed. It is likely that 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-10265-02 CP
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel:
G.A. Fahle, M.T., MS, CPD, CC
J.M. Parker, B.S., MS, CPD, CC
X. Qin, Ph.D., MS, CPD, CC
J. Canosa, M.T., MS, CPD, CC
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 anti-viral therapy
at an earlier time point in the prodromal stage may decrease a patient's
risk
of developing active CMV disease.We are developing a competitive quantitative
PCR assay to detect 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 so that variations in tube-to-tube PCR efficiency are detected and
accounted for in the calculations of viral copy number made from the measured
signal strength. The assay has the capability of detecting as few as 3 to
5 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. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10267-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Methods for Detection of BK Virus in Allogeneic
BoneMarrow Transplant Patients
Principal Investigator: H.D. Engler, Ph.D. (Senior Staff Microbiologist)
MS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: 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 under way, 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. This project is continuing for another year. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10268-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Detection of Microsporidia in Stool by Modified
Trichrome Stain
Principal Investigator: D.P. Fedorko, Ph.D. MS, CP, CC, NIH, Bethesda,
MD 20892-1508
Other Personnel: N.A. Nelson, MS, CP
Collaborating Unit: 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.
The currently accepted standard protocol for modified trichrome staining
of microsporidia includes a 1-hour incubation in the modified trichrome
stain. We hope to demonstrate that a rapid staining method, which includes
shorter exposure to the modified trichrome stain, will provide comparable
or superior results. A device that allows stain to be maintained at 50°
to 60°C will be used in this effort to provide a rapid and easily interpretable
staining method.
We will participate in an international multicenter diagnostic study to compare the sensitivity and specificity of the modified trichrome stain with the polymerase chain reaction. We will receive 50 specimens (both positive and negative) to stain with the modified trichrome stain and examine them for the presence of microsporidial spores. The data from the 12 participating laboratories will be combined and a manuscript prepared for publication. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10269-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Markers of Disease Activity in Idiopathic Inflammatory Myopathy
Principal Investigator: Margaret E. Rick, M.D. (Senior Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: P. Merryman, Medical Technologist, CP
A. Cullinane, Medical Technologist, CP
Collaborating Units: IRP, 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 includes 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 patient 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 CC-10272-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Use of Neural Networks in Monitoring Quality
Control of Laboratory Data
Principal Investigator: A.T. Remaley, M.D (Senior Investigator) CCS,
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: J. DeLeo, DCRT
Collaborating Unit: None
Staff-Years: 0.1
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This project focused on the usefulness of neural
networks in monitoring patient laboratory data for quality control. We developed
a neural network by training
it with patient data on total serum cholesterol. We also trained the neural
network with mathematically simulated data that failed our standard quality
control procedures. After training, the neural network was able to recognize
the usual pattern of cholesterol results
in our laboratory and to differentiate it from incorrect laboratory results
produced by a
problem with the chemistry analyzer. The neural network was able to recognize
problems
in bias, as well as precision in the laboratory data, using a sample size
as small as 20 patients. Receiver operating characteristic analysis showed
that the neural network approach is superior in sensitivity and specificity
in error detection compared with other approaches. Future studies will be
aimed at extending the neural network approach to other analytes and to
inter-face a neural network with a chemistry analyzer to test the practicality
of this approach in monitoring quality control. (Back to
the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10273-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Acquired Coagulation-Fibrinolytic-Platelet Defectsin HIV and Other Viruses
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator),
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S. Williams, Research Medical Technologist
Collaborating Unit: None
Staff-Years: 2.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors
x (a2) Interviews
Summary of Work: Patients with HIV commonly have coagulation deficiencies.
Most of the deficiencies are related to reduced amounts of protein S or
to thrombocytopenia.
We have identified therapies that may improve coagulation by increasing
protein S and
C levels. We have developed an assay to identify glycocalicin in plasma
that can differ-
entiate between thrombocytopenia caused by increased destruction of platelets
and by
decreased production of platelets. This assay allows the clinician to identify
the potential cause of the platelet defect and to treat the platelet abnormality
by specific therapies;
e.g., platelet transfusion, intravenous gamma globulin, and the use of vincristine
and
recombinant thrombopoietin.
To date we have assayed blood from 50 HIV-positive adults and 49 HIV-positive
children. We have found that some individuals have high glycocalicin and
low platelet counts, while others have high glycocalicin and normal platelet
counts. This test may
be able to detect thrombocytopenia before the platelet count is clinically
low. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10274-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Carbohydrate Differences in Plasma and Platelet
von Willebrand Factor
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator),HS,
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S. Williams, Research Medical Technologist, CP
Collaborating Unit: None
Staff-Years: 2.0
Human Subjects: x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Von Willebrand factor (vWf) protein is synthesized
in endothelial cells and megakaryocytes. We identified differences in the
carbohydrate content between plasma and platelet vWf. Studies are now in
progress to specify the role(s) of carbohydrates in vWf function, survival,
molecular size, and bioactivity. O-linked carbohydrates appear
to be important in ristocetin-medicated binding. Plasma vWf binds more with
ristocetin
than platelet vWf. We plan to further study the differences in carbohydrate
content of
the two proteins. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10275-02 CP
October 1, 1996 to September 30, 1997
Title of Project: Platelet Aggregation and von Willebrand Factor
Defects in Hermansky-Pudlak Syndrome
Principal Investigator: H.R. Gralnick, M.D. (Senior Investigator),HS,
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: L. McKeown, Research Medical Technologist, CP
O.J. Wilson, Research Medical Technologist, CP
K.E. Hansmann, Research Medical Technologist, CP
Collaborating Unit: 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 plateletaggregation 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 other platelet a-granule constituents
such as PF-4 and b-TG in platelet lysates of HPS patients.
We are also in the process of correlating bleeding times and clinical bleeding
histories
with platelet and plasma vWf activity and antigen levels. In the future,
we plan to examine
a-granule contents in HPS patients, employing flow cytometry. (Back
to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL- 10276-01 CP
July 1, 1997 to September 30, 1997
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, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S. Fischer, M.D., Ph.D., MS, CP, CC
L. Calhoun, MT (ASCP), MS, CP, CC
Collaborating Unit: 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-agent 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 analyze the contents of the shell vials for growth inhibition by quantitative polymerase chain reaction. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10277-01 CP
August 1, 1997 to September 30, 1997
Title of Project: Quantitative Detection of CMV by Using a Commercially
Developed Research Assay
Principal Investigator: H. Engler, Ph.D. (Senior Investigator) MS,
CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S. Fischer, M.D., Ph.D., MS, CPD, CC
D. Fedorko, Ph.D., MS, CPD, CC
L. Calhoun, MT (ASCP), MS, CPD, CC
Collaborating Unit: None
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 that allows 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 blood specimens collected from allogeneic bone marrow transplant patients, and comparing its results with a quantitative PCR assay developed in-house, with shell vial culture of CMV, and with the CMV antigenemia quantitative assay 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-10278-01 CP
October 1, 1996 to September 30, 1997
Title of Project: PCR-SSCP for the Detection and Speciation of
Microsporidiain Clinical Specimens
Principal Investigator: D.P. Fedorko, Ph.D. MS, CPD, CC, NIH, Bethesda,
MD 20892-1508
Other Personnel: N.A. Nelson, MS, CPD, CC
A.M. Hruszkewycz, M.D., CS, CPD, CC
R.M. Delgado, CS, CPD, CC
Collaborating Unit: Tulane University Medical Center (E.S. Didier,
Ph.D.)
Staff-Years: 0.20
Human Subjects: (a) Human subjects (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 for the detection of microsporidia. 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 conformation polymorphism (SSCP) analysis combined with PCR (PCR-SSCP) has been used to identify bacteria, fungi, and viruses. We will use our PCR assay to detect microsporidia in stool specimens and apply SSCP analysis to determine the genus and species of the parasite. Organisms from cell culture will also be used to validate the method. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10279-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Comparison of Microbiologic and Cytologic Results
for Bronchoalveolar Lavages
Principal Investigator: F.G. Witebsky, M.D. (Senior Investigator)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: A.D. Abati-Scott, M.D., Cytopathology Section, LP,
DCS, NCI
F. Stock, B.S., Microbiology Service, CPD, CC
V.J. Gill, Ph.D., Microbiology Service, CPD, CC
Collaborating Unit: Cytopathology Section, LP, DCS, NCI
Staff-Years: 0.10
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Bronchoalveolar lavage 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 of the data analyzed
thus far suggest that cytology preparations are more sensitive for the direct
detection of significant fungal pathogens than the smears prepared in microbiology,
presumably because of the larger volume of material used for preparation
of smears in cytology. More data will need to be analyzed before any conclusions
can be reached on 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-10280-01 CP
October 1, 1996 to September 30, 1997
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 20892-1508
Other Personnel: V.J. Gill, Ph.D., MS, CPD, CC
S. Fischer, M.D., Ph.D., MS, CPD, CC
D. Williams, B.S., MS, CPD, CC
F. Stock, B.S., MS, CPD, CC
Collaborating Unit: 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, since 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 species other than H.
pylori is 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 not known whether or not these cases
are due to a Helicobacter species other than H. pylori. Additionally,
we isolated a strain of Flexispira rappini, a spe-cies that is closely
related to Helicobacter, in blood.
Most Helicobacter species 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
species 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 species. Furthermore,
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-10281-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Detection and Identification of Mycobacteria
in Clinical Specimens
Principal Investigator: S.H. Fischer, M.D., Ph.D. (Senior Staff)
Microbiology Service, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: E. O'Shaughnessy, M.D., MS
P. Conville, M.S., MS
F. G. Witebsky, M.D., MS
J.M. Parker, B.S., MS
Collaborating Unit: None
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 as long as one 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 mycobacterium species in
clinical samples. At present, there are no commercially available amplification
assay systems capable of detecting all members of the genus Mycobacterium
while excluding cross-reactive signals from other bacteria commonly
present in clinical samples.
We have developed a PCR assay using primers targeting a segment of the 16S ribosomal RNA gene. The assay is 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, which 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 to detect mycobacterial infections in patients. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10282-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Molecular Search for Biosynthetic Genes of Mycobacterium
tuberculosis Cord Factor
Principal Investigator: X. Qin, Ph.D. (Clinical Microbiology Fellow)
MS, CPD, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: S.H. Fischer, M.D., Ph.D., MS
Collaborating Unit: 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). Since 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 genes encoding the synthesizing enzymes
will increase our knowledge of mycobacterial cell-wall biosynthesis and
function, hence permitting the design of a tailor-made drug(s) to inhibit
key catalytic enzymes. Furthermore, since 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.
Though biosynthetic pathway(s) of Mtb wall lipid constituents are not known, experience in well-studied cell envelope features of gram-positive and gram-negative bacteria enables one to speculate on some principal catalytic steps involved in the formation of distinctive molecules such as TDM. The biosynthesis of Escherichia coli lipid A has been elucidated and several of the relevant genes are known. Among the known genes are lpxA and lpxD, which encode UDP-GlcNAc O-acyltransferases. Based on the primary sequence alignment of a number of lpxA/lpxD genes from various organisms (E. coli, Salmonella typhimurium, Yersinia enterocolitica, and Rickettsia rickettsii), degenerate oligonucleotide primers were designed to amplify the analogs in Mtb. A polymerase chain reaction (PCR) procedure amplified a 1Kb fragment from the chromosomal DNA of a virulent Mtb reference strain, H37Rv, while no comparable amplification product was observed from DNA controls of Bacillus subtilis or M. smegmatis. The length of the possible interdomain sequence amplified appeared to be about 2 times longer than those in the other species. A large open-reading frame in the sequence was deduced to contain hexad repeats, similar to those observed in LpxA/LpxD protein sequences. A Southern blot of chromosomal materials from H37Rv, H37Ra, M. smegmatis, and B. subtilis probed with the 1Kb fragment suggested that this PCR product was Mtb-specific not present in any other species tested. The DNA sequence obtained from the 1Kb PCR product produced a perfect match to a Mtb gene proposed to encode a potential membrane-associated protein with an ATPase motif. Further investigations of the location and the biological function of this gene will be carried out in the next 12 months. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10283-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Platelet-Associated Antibodies in Patients With
AutoimmuneThrombocytopenic Purpura
Principal Investigator: M.E. Rick, M.D. (Senior Investigator)
HS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: M. Vail, Medical Technologist, CP
Collaborating Unit: 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), a disease caused by autoantibodies directed against platelets, has
been difficult to demonstrate for a variety of reasons. In general, when
the antibodies can be demonstrated, there is an inverse correlation with
the platelet count. We are setting up two assays, one screening assay and
one more specific assay, to aid in the diagnosis and progress of treatment
for patients with ITP. We will particularly utilize the tests for following
patients pre- and posttreatment in
a pilot study with NHLBI in the treatment setting of T-cell-depleted auto-stem
cell transplantation in patients with severe ITP. (Back
to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10284-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Followup Study of Patients With Large Granular
Lymphocytic Leukemia
Principal Investigator: M.E. Rick, M.D. (Senior Investigator) HS,
CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: M. Riordan, Medical Technologist, CP; N. Barney,
Medical Technologist, CP
Collaborating Unit: 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 followup
of lympho-proliferative diseases, including large granular lymphocytic leukemia.
A unique treatment trial utilizing cyclosporine has been conducted 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 been performing followup
flow cytometry of these patients during the course of their treatment The
results will be
correlated with their responses. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10285-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Assessment of Peripheral Blood Eosinophils
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology
Service) IS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: 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 toevaluate 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 being reviewed for evidence of changes
in peripheral eosinophils as a consequence of eosinophil mobilization
and disease. (Back to the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10286-01 CP
October 1, 1996 to September 30, 1997
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, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: 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 2 patients with gamma interferon receptor alpha
chain deficiency,
and the molecular level of this defect is being actively examined. There
are emerging data that CD120b expression may also be abnormal in some patients
with recurrent mycobac-terial infection. Specific expression of the gamma
interferon receptor alpha chain is being evaluated on amniocytes as a possible
approach to prenatal diagnosis of this disorder. (Back to
the project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-10287-01 CP
October 1, 1996 to September 30, 1997
Title of Project: Assessment of Lymphocytes in Patients With AutoimmuneLymphoproliferative
Syndrome
Principal Investigator: T.A. Fleisher, M.D. (Chief, Immunology Service)
IS, CP, CC, NIH, Bethesda, MD 20892-1508
Other Personnel: None
Collaborating Unit: 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 has been undertaken of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended-family members, 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+. In addition, a number of asymptomatic family members appear to have an increased level of double negative T-cells. (Back to the project list)
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Questions about the Clinical Center? OCCC@nih.gov Or call: (301) 496-2563 National Institutes of Health, Warren Grant Magnuson Clinical Center, Bethesda, Maryland 20892. Last Modified 3/98 |