Reverse transcriptase PCR quantitation of hepatitis C virus.

OBJECTIVE: To present a brief review of the diagnostic benefits of quantitating viral load for hepatitis C and how the reverse transcriptase polymerase chain reaction is being used as an aid to better diagnose and manage the disease. DATA SOURCE: Research articles about hepatitis C and the reverse transcriptase polymerase chain reaction, as well as data gathered by the authors. STUDY SELECTION: Performed by the authors. DATA EXTRACTION: Performed by the authors. DATA SYNTHESIS: Hepatitis C viral infection is a worldwide health problem, affecting about 100 million people worldwide. Numerous serological tests exist to detect antibodies to hepatitis C antigens, but some affected people fail to generate an immune response. Reactivity in the reverse transcriptase polymerase chain reaction is definitive proof of hepatitis C infection. The titer of RNA indicates patient response to antiviral therapy. Measuring the presence and quantity of RNA by the reverse transcriptase polymerase chain reaction has become an important aid for diagnosis and monitoring of hepatitis C infection. CONCLUSION: The reverse transcriptase polymerase chain reaction method is a highly sensitive and accurate aid in diagnosing or confirming diagnosis of hepatitis C viral infection. This method is widely used to assess likelihood of patient response to therapy, and to monitor efficacy during therapy.

Comparison of virus culture and the polymerase chain reaction for diagnosis of mucocutaneous herpes simplex virus infection.

BACKGROUND AND OBJECTIVES: The diagnosis of mucocutaneous herpes simplex virus (HSV) is hampered by suboptimal sensitivity of virus culture and atypical clinical morphology. GOALS: To compare the diagnostic usefulness of the polymerase chain reaction (PCR) and virus culture. STUDY DESIGN: Consecutive samples from 246 patients at an urban sexually transmitted diseases clinic were tested for HSV by both PCR and virus culture. RESULTS: Only 59% of HSV-positive samples were correctly diagnosed by the clinician; 11% had an atypical appearance. HSV-positive lesions were more often vesiculoulcerative or crusted than HSV-negative lesions, and of shorter median duration. Thirty-one samples were PCR positive and virus culture negative; these were often from crusted or older lesions. However, PCR was negative in 27 instances in which HSV was diagnosed clinically, of which 2 were vesicular and 15 ulcerative. CONCLUSIONS: HSV PCR is more rapid and sensitive than virus culture for diagnosis of mucocutaneous lesions. The data suggesting that PCR may be suboptimally sensitive need to be further investigated.

Ecocardiografía Doppler en la fibrosis endomiocardica / Echo-Doppler studies of endomyocardial fibrosis

Doce pacientes con fibrosis endomiócardica, con comprobación angiográfica y/o histológica, fueron estudiados mediante ecocardiografía Doppler con el propósito de describir las características ecocardiográficas e identificar los sitios de afección. La edad promedio de los pacientes fue de 41 años (límites de 16 a 59 años); 2 hombres y 10 mujeres. Tres paciente (25 por ciento) tuvieron afección aislada del ventrículo derecho; 1 paciente (8 por ciento), del ventrículo izquierdo y, en 8 pacientes (66 por ciento), el compromiso fue biventricular. Los hallazgos ecocardiográficos Doppler fueron: dilatación de la aurícula derecha (91 por ciento), dilatación del tracto de salida del ventrículo derecho (83 por ciento), movimiento septal paradójico (83 por ciento), dilatación de la aurícula izquierda (33 por ciento), prolapso valvular mitral y tricuspídeo (50 por ciento), derrame pericárdico (41 por ciento), insuficiencia mitral (75 por ciento), insuficiencia tricuspídea (100 por ciento), obliteración de ápex (50 por ciento) y patrón de llenado tipo restrictivo (50 por ciento). La ecocardiografía Doppler es un método útil en el diagnóstico de la fibrosis endomiocárdica, los hallazgos de ventrículos normales o pequeños con obliteración del ápex, que contrasta con aurículas muy dilatadas, de insuficiencia mitral y/o tricuspídea con un patrón de llenado de tipo restrictivo son característicos de esta enfermead. En nuestro medio, la localización aislada o predominante del ventrículo derecho fue la más frecuente: respresentó el 83 por ciento de los casos.

Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies.

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.

Hemoglobin A1C by isoelectric focusing.

We separated the hemoglobins in an unwashed erythrocyte hemolysate according to their isoelectric points on a thin-layer horizontal polyacrylamide gel containing ampholyte (40 g/L) over a pH gradient of 6-8. We scanned the fixed, unstained gels by microdensitometry and calculated the percentage of hemoglobin A1C. The overall CV (between-run imprecision) for a normal hemolysate (mean, 5.2% of total hemoglobin) stored at -70 degrees C was 12.6%. An above-normal pooled specimen (mean 8.5%) showed an overall CV of 8.4%. We confirmed that hemoglobins S, C, and F do not interfere; acetylated F co-migrates with A1C. The mean A1C percentage in non-diabetic adults was 4.9%, the reference interval 3.9-6.4%. The mean values for diabetics in various degrees of control were 7.6% in the group rated "good," 9.9% in the "fair" group, and 12.2% in the "poor" group. Results for patients' samples (y) were compared with results by cation-exchange chromatography (x). The slope was 1.0 and the intercept was 2.2%. The percentage of hemoglobin A1C in erythrocytes remains constant for seven days in samples stored at 30 degrees C.

C-terminal parathyrin (parathyroid hormone) radioimmunoassay in serum with commercially available reagents.

We describe a sequential double-antibody assay for measuring C-terminal parathyrin in serum with commercially available reagents. Intact bovine hormone, used as a working standard, is iodinated by conventional Chloramine T procedures. The antibody affinity is characteristic of high affinity binding (1.4 to 1.6 x 10(10) L/mol of intact parathyrin). The antibody also cross reacts with a C-terminal parathyrin fragment (amino acids 53-84) but not with a synthetic N-terminal parathyrin fragment (amino acids 1-34). The assay thus also measures both a C-terminal fragment of parathyrin and the intact hormone. The detection limit (250 ng/L; 500 int. units/L) is below the reference interval for healthy adults (430-1860 ng/L; 860-3720 int. units/L). Several commonly recognized problems with iodinated parathyrin are eliminated and accuracy and precision of the procedures for standard preparation and calibration are improved. Overall CV (between-run imprecision) is 10-17%. Analytical recovery is 80-90%, and C-terminal parathyrin measured in fresh sera and in sera stored for seven days at 30 degrees C is equivalent.

Diagnostic utility of C-terminal parathyrin measurement as compared with measurements of N-terminal parathyrin and calcium in serum.

We compared results obtained from two parathyrin (parathyroid hormone) assays with differing specificities, using sera from 172 normal donors and from 98 patients with disorders of calcium regulation. Intact parathyrin was measured in both assays; the C-parathyrin assay also measured the 53-84 amino acid C-terminal hormone fragment; the N-parathyrin assay also measured the 1-34 N-terminal fragment. The reference interval for the C-parathyrin assay (860-3720 int. units/L; 430-1860 ng/L) was markedly higher than for the N-parathyrin assay (460-1260 int. units/L; 230-630 ng/L), a finding consistent with the longer half-life of C-parathyrin fragments in human circulation. Mean C-parathyrin in primary hyperparathyroid sera--5720 (SD 2760) int. units/L or 2860 (SD 1380) ng/L--clearly exceeded the reference interval and values for sera from patients with non-parathyroid malignancy [1740 (SD 760) int. units/L; 870 (SD 380) ng/L]. Secondary hyperparathyroid patients also had supranormal C-parathyrin values: 6100 (SD 2720) int. units/L; 3050 (SD 1360) ng/L. We found no consistent correlation between parathyrin and serum calcium in any clinical group. The two parathyrin assays showed about equal diagnostic power, but their results could not be used interchangeably in sequential monitoring of patients.