ABSTRACT
We performed autopsies and serologic tests in 189 subjects (152 men and 37 women) between 20 and 50 years of age with no history of immunosuppression who died unexpectedly and whose bodies were referred to the San Francisco coroner's office. Forty-eight of the 88 single men for whom addresses were available lived in areas of the city with a high incidence of the acquired immunodeficiency syndrome (AIDS). In addition, 36 of the subjects (30 men) were intravenous drug abusers. Antibody to the retrovirus associated with AIDS was present in 23 (18%) of the 121 subjects whose sera were tested. However, neither pathologic nor laboratory manifestations of AIDS were present in any of the 189 subjects who underwent autopsy. These results suggest that antibody to the retrovirus is common but subclinical manifestations of AIDS are uncommon in San Francisco, a city where the incidence of clinical AIDS is high.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Antibodies, Viral/analysis , Death, Sudden/etiology , Deltaretrovirus/immunology , Adult , Cytomegalovirus/immunology , Death, Sudden/pathology , Female , Hepatitis B Core Antigens/immunology , Humans , Lung/pathology , Male , Middle Aged , Pneumocystis/immunologyABSTRACT
AIM: To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. METHOD: Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. RESULTS: In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. CONCLUSION: In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.
Subject(s)
HIV Infections/microbiology , HIV-1/genetics , HIV-1/isolation & purification , RNA, Viral/blood , RNA, Viral/genetics , Virology/methods , DNA, Viral/genetics , Gene Amplification , HIV Infections/blood , Humans , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Viremia/blood , Viremia/microbiology , Virology/statistics & numerical dataABSTRACT
OBJECTIVE: To define parameters determined before and after 4 weeks of interferon therapy (3 MU three times per week for 24 weeks) which could be reliable predictors of a response to therapy. PATIENTS: Thirty-four patients with chronic hepatitis C virus (HCV) infection were investigated prospectively. METHODS: A complete response was defined as the normalization of serum alanine aminotransferase levels (ALT) at the end of treatment. The genotype of HCV was determined and the level of HCV-RNA was quantitated both before and after 4 weeks of treatment. RESULTS: After 4 weeks, 16 out of 20 responders [95% confidence interval (CI) 54-94%] and two out of 14 non-responders (95% CI 2-44%) normalized their ALT levels (P = 0.0002). The prevalence of genotype 1b was significantly (P < 0.04) higher among non-responders (eight out of 10; 95% CI 44-92%) than in responders (four out of 18; 95% CI 4-40%). Before treatment, the viraemia determined by branched DNA was significantly lower in responders than in non-responders (46.4 versus 116 x 10(5) eq virus/ml). After 4 weeks of treatment, the level of viraemia in responders was still significantly lower than that in non-responders (22.8 versus 66 x 10(5) eq virus/ml). In responders, a significant decrease in the level of viraemia was observed after 4 weeks of treatment. CONCLUSION: In a stepwise regression analysis only age and the normalization of ALT levels after 4 weeks of treatment were predictive of response to interferon at the end of treatment.
Subject(s)
Hepatitis C/therapy , Interferons/therapeutic use , Adult , Age Factors , Aged , Alanine Transaminase/blood , Chronic Disease , Female , Hepacivirus/genetics , Hepacivirus/isolation & purification , Hepatitis C/enzymology , Hepatitis C/virology , Humans , Interferons/administration & dosage , Male , Middle Aged , Prospective Studies , RNA, Viral/analysis , Treatment OutcomeABSTRACT
This article describes laboratory methods available for testing serum to determine whether a patient is infected with human immunodeficiency virus (HIV). Included is discussion of uses and limitations of the assays, patient counseling, laboratory safety, and interpretation of the test results.
Subject(s)
Acquired Immunodeficiency Syndrome/diagnosis , Antibodies, Viral/analysis , Antigens, Viral/analysis , HIV/immunology , Humans , Serologic TestsABSTRACT
The diagnosis and monitoring of hepatitis C virus (HCV) infection have been aided by the development of HCV RNA quantification assays A direct measure of viral load, HCV RNA quantification has the advantage of providing information on viral kinetics and provides unique insight into the disease process. Branched DNA (bDNA) signal amplification technology provides a novel approach for the direct quantification of HCV RNA in patient specimens. The bDNA assay measures HCV RNA at physiological levels by boosting the reporter signal, rather than by replicating target sequences as the means of detection, and thus avoids the errors inherent in the extraction and amplification of target sequences. Inherently quantitative and nonradioactive, the bDNA assay is amenable to routine use in a clinical research setting, and has been used by several groups to explore the natural history, pathogenesis, and treatment of HCV infection.
ABSTRACT
With this statement, Sherlock and Dooley have described two of the three major challenges involved in quantitatively measuring any analyte in tissue samples: the distribution of the analyte in the tissue; and the standard of reference, or denominator, with which to make comparisons between tissue samples. The third challenge for quantitative measurement of an analyte in tissue is to ensure reproducible and quantitative recovery of the analyte on extraction from tissue samples. This chapter describes a method that can be used to measure HCV RNA quantitatively in liver biopsy and tissue samples using the bDNA assay. All three of these challenges-distribution, denominator, and recovery-apply to the measurement of HCV RNA in liver biopsies.
ABSTRACT
Indirect immunofluorescence antibody testing is a mainstay of retrovirus serodiagnosis in the public health community, affording quick, inexpensive, and clear results with significant advantages over Western blot testing. Limitations regarding training of personnel and availability of reagents will probably continue and limit indirect immunofluorescence antibody testing to the present users.
Subject(s)
Fluorescent Antibody Technique , Retroviridae Infections/diagnosis , Serologic Tests , Bibliographies as Topic , Fluorescent Antibody Technique/history , History, 20th Century , Humans , Retroviridae Infections/history , Serologic Tests/historySubject(s)
Hepatitis Antibodies/analysis , Hepatitis C/transmission , Kidney Transplantation , Liver Diseases/etiology , Tissue Donors , Enzyme-Linked Immunosorbent Assay , Hepatitis C/epidemiology , Hepatitis C Antibodies , Humans , Liver Diseases/epidemiology , New England , Prevalence , Tissue BanksSubject(s)
Heart Transplantation/physiology , Hepatitis Antibodies/blood , Hepatitis C/transmission , Kidney Transplantation/physiology , Liver Transplantation/physiology , Tissue Donors , Antigens, Viral/analysis , Antigens, Viral/immunology , Cadaver , Enzyme-Linked Immunosorbent Assay , Hepatitis C Antibodies , Humans , Polymerase Chain Reaction/methods , Recombinant Proteins/analysis , Recombinant Proteins/immunology , Retrospective StudiesABSTRACT
This is a summary of a presentation made at the 13th International Convocation on Immunology. Nucleic acids in patient samples can be quantified directly using a solid phase nucleic acid hybridization assay based on branched DNA (bDNA) signal amplification technology. For example, HIV RNA is detected in a plasma sample by hybridization of multiple specific synthetic oligonucleotides to the target, 10 of which capture the target onto the surface of a microwell plate and 39 of which mediate hybridization of branched DNA molecules to the pol region of each HIV RNA molecule. Alkaline phosphatase-labeled probes bind to each arm of the branched DNA molecules. Detection is achieved by incubating the complex with a chemiluminescent substrate and measuring the light emission. The signal is directly proportional to the level of target nucleic acid, and the quantity of HIV RNA in a sample is determined by comparison with a 4-point standard curve. In order to ensure that different subtypes of HIV-1 were detected and quantified equally, in vitro RNA transcripts of the pol region of HIV subtypes A-F were purified and quantified by OD 260, phosphate analysis, and hyperchromicity. These characterized transcripts were then quantified using the bDNA assay. Comparisons were made using a ratio of signal per attomole for each transcript. Genetic subtypes A-F quantified within a factor of 1.5, indicating that the bDNA assay can be used to measure viral load in clinical samples regardless of genotype. Accuracy is important because several studies indicate that there may be a threshold level of virus which predicts progression of HIV disease. Detection of change in viral load is important in determining the efficacy of therapy. The bDNA assay for HCV RNA can be used to determine level of virus in HCV-infected individuals and assist in establishing prognosis prior to initiation of alpha-interferon therapy. Patients with lower levels of virus are more likely to have a sustained response to therapy. Patients who respond to treatment typically have a rapid decline in virus load within one to four weeks of the start of therapy. Many patients relapse when therapy is discontinued as evidenced by a rise in virus load to near pre-treatment levels. Sustained response is most often seen with patients who have lower pre-treatment levels of RNA.
Subject(s)
DNA, Viral/analysis , Viral Load/methods , Humans , Nucleic Acid Hybridization/methodsABSTRACT
Five commercial assays were compared to a standardized complement fixation (CF) test for the detection of antibody to cytomegalovirus. Two hundred and thirty serum specimens were analyzed. In addition, nine pairs of acute- and convalescent-phase sera were tested by two of the commercial assays. The assays were compared as to sensitivity, specificity, and positive and negative predictive value, as well as incidence of false-positive and -negative results. Samples which did not agree in all the assays were retested and tested with an indirect fluorescent-antibody assay. Of 228 specimens, 103 (45.2%) were positive by CF. Of the 230 samples, 2 (0.9%) were inconclusive by CF and readable in the other assays. Of the 230 specimens, 97 (42.2%) were positive by an enzyme immunoassay (EIA; Litton Bionetics), 100 (43.5%) were positive by a second EIA (Abbott Laboratories), 104 (45.2%) were positive by a third EIA (M. A. Bioproducts). One hundred and eight (47.0%) were positive by indirect hemagglutination (IHA; Cetus Corporation), and 110 (47.8%) were positive by latex agglutination (LA; Hynson, Westcott and Dunning). Sensitivity and specificity were similar with all the assays (93 to 100%). The greater numbers of positive results by IHA and LA were confirmed by repeat CF testing at less than 1:8 dilution, and by indirect fluorescent-antibody assay. Acute- and convalescent-phase serum pairs showed a significant rise in antibody titer when tested by anticomplement immunofluorescence, IHA, and LA. There was good agreement among the assays, with LA having the highest sensitivity.
Subject(s)
Antibodies, Viral/analysis , Cytomegalovirus/immunology , Immunoassay/methods , Complement Fixation Tests , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/immunology , Evaluation Studies as Topic , False Negative Reactions , False Positive Reactions , Fluorescent Antibody Technique , Hemagglutination Tests , Humans , Immunoenzyme Techniques , Latex Fixation TestsABSTRACT
Direct detection of hepatitis C virus (HCV) RNA in serum or plasma is useful for validating the performance of anti-HCV assays and for the discrimination of persons with persistent HCV infections from those with resolved infections. Quantitation of HCV RNA may also be useful for disease prognosis and therapeutic monitoring. Previous studies have reported detection of HCV RNA in 50 to 70 percent of blood donors who were positive on anti-HCV supplemental tests. There is concern that specimen processing and storage conditions might influence the stability, and hence the detectability, of HCV RNA. To address this concern, the rate of detection of HCV RNA by the polymerase chain reaction (PCR) using donor pilot tube sera (PTS) previously subjected to routine donor screening and supplemental testing was compared with HCV PCR results obtained with fresh-frozen plasma (FFP) derived from the same donations. All 16 anti-HCV supplemental test-positive donations evaluated were HCV RNA positive with FFP, whereas only 10 (62.5%) were positive with PTS (p = 0.024). None of 11 FFP or PTS samples from HCV enzyme immunoassay-reactive donations not confirmed by supplemental anti-HCV assays tested positive for HCV RNA. Direct comparison of sample type (serum vs. plasma) and various storage conditions using specimens from two seropositive donors showed that room-temperature storage results in marked reduction in HCV RNA signal, while replicate freezing and thawing caused a moderate reduction. These data indicate that well-controlled sample processing and storage conditions are critical to the sensitive and potentially quantitative analysis of HCV RNA.
Subject(s)
Blood Preservation , Hepacivirus/genetics , RNA, Viral/analysis , Specimen Handling , Cold Temperature , Humans , Polymerase Chain ReactionABSTRACT
Cloning of the hepatitis C virus (HCV) genome was a tremendous advance in the development of tests for diagnosis and monitoring of HCV-infected patients. Serological tests, including enzyme-linked immunoassays and RIBA strip immunoblot assays, are primarily used to screen blood donations and to diagnose and confirm HCV infection. Tests for HCV RNA, including polymerase chain reaction (PCR)-based assays and the branched-DNA (bDNA) assay, are used for therapeutic monitoring and prognostics. Here, we present the development and future potential of these diagnostic tests. We also provide examples of how these tests are used to follow the progression of disease, select and adjust treatment protocols, and evaluate the efficacy of therapeutic regimens.
Subject(s)
Hepacivirus/genetics , Hepatitis C/diagnosis , Cloning, Molecular , Disease Progression , Enzyme-Linked Immunosorbent Assay , Genome, Viral , Hepacivirus/isolation & purification , Hepatitis C/therapy , Humans , Monitoring, Physiologic , Polymerase Chain Reaction , Prognosis , RNA, Viral/analysisABSTRACT
The aim of this study was to determine the stability of viral load over an extended period in patients with chronic hepatitis C virus (HCV). Sequential serum specimens collected from fourteen non-alcoholic adult patients with chronic HCV between 1990 and 1997 were tested retrospectively for HCV RNA levels by branched DNA assay (Quantiplex HCV RNA 2.0 [Chiron Diagnostics, Emeryville, CA]). A minimum of three serum samples was obtained at various intervals from each patient. None of the patients received antiviral therapy. Liver biopsies, available for 10 of 14 patients, showed mild or moderate hepatitis in seven and cirrhosis in three (one developed cirrhosis during follow-up). RIBA strip immunoassay showed that 7, 3, and 4 patients had viral genotypes 1, 2, and 3, respectively. The follow-up time averaged 5.3 years (range, 3.7 to 6.6 years). Eight patients (57.2%) showed increased viral levels from baseline to follow-up, the remaining six patients (42.8%) showed decreased viral levels. The three cirrhotic patients had the highest viral levels over time. The mean change was a 0.29-fold decrease (median, +1.14 [corrected]; range, -17.49 to +7.32). A less than twofold change in either direction was demonstrated for six patients (42.8%), and a less than threefold change was demonstrated for 10 patients (71.4%). Variation from baseline to last follow-up as calculated by log determination showed that the viremic load varied less than one log10 in all but one individual. These results show that viral load remains relatively stable over prolonged periods in most untreated patients with chronic hepatitis C.
Subject(s)
DNA, Viral/blood , Hepacivirus/genetics , RNA, Viral/blood , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Genotype , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/genetics , Hepatitis C, Chronic/virology , Humans , Liver Cirrhosis/etiology , Liver Cirrhosis/virology , Longitudinal Studies , Male , Retrospective Studies , Viral LoadABSTRACT
When hepatitis C virus antibody (anti-HCV) enzyme immunoassay (EIA1) testing became available in 1990, we tested samples from previously transfused blood units, traced the recipients of reactive units, and evaluated the recipients for HCV infection during the 12 months after transfusion. Ten of 42 recipients of EIA1-reactive blood were anti-HCV reactive on follow-up by EIA1 and 12 were reactive by a second-generation assay (EIA2). Reverse transcriptase-polymerase chain reaction (RT-PCR) detected HCV RNA in 5 seronegative recipients. In all, 17 of 42 recipients (40%) of EIA1-reactive blood had evidence of HCV infection. In comparison, 54 surgery patients, who received either no transfusion or autologous EIA1-nonreactive blood, remained EIA1 nonreactive and RT-PCR negative for 1 year; 1 patient (1.8%) became EIA2 reactive (P < or = .01). Of the recipients of anti-HVC reactive blood transfusions (reactive by both EIA1 and a supplemental 4-antigen strip immunoblot assay [RIBA2]), 14 (93%) of the recipients had evidence of HCV infection compared with only 3 of 27 recipients (11%) of EIA1-reactive, RIBA2-nonreactive blood (P < or = .01). Thus, blood components reactive for anti-HCV EIA1 may have transmitted HCV up to 40% of the time, but blood components found reactive by both EIA1 and RIBA2 may transmit HCV with a frequency of greater than 90%.
Subject(s)
Hepatitis Antibodies/analysis , Hepatitis C/transmission , Transfusion Reaction , Adult , Aged , Blood Donors , Female , Hepacivirus/chemistry , Hepatitis C/immunology , Hepatitis C/microbiology , Hepatitis C Antibodies , Humans , Male , Middle Aged , Polymerase Chain Reaction , RNA, Viral/analysisABSTRACT
Current criteria for a reactive (positive) interpretation on hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) require > or = 1+ reactivity to at least two of the four HCV antigens present in the assay. Given that 5-1-1 is a subcomponent of c100-3, there is concern that donor samples reacting only with these two antigens (and not with c22-3 or c33c) could be incorrectly classified as positive on the basis of limited reactivity to only one HCV gene product. It is determined that 0.23 to 0.44 percent of HCV enzyme immunoassay-repeatably reactive donor sera demonstrate a pattern of 5-1-1 and c100-3 only on RIBA. Evaluation of six such donor sera using peptide enzyme immunoassays spanning the c100-3 antigen showed highly restricted reactivity to the 5-1-1 N-terminal region of c100-3, in contrast to broad 5-1-1 and c100-3 C-terminal peptide reactivity observed in the majority of donor sera with other positive RIBA patterns. HCV polymerase chain reaction and follow-up serologic evaluations of four of these donors indicated the absence of viremia or evolving seroconversion in all cases. It is concluded that, in the blood donor setting, a pattern of only 5-1-1 and c100-3 reactivity is typically not indicative of HCV infection. To avoid overinterpretation, it is recommended that RIBA grading criteria be revised to require reactivity to two or more HCV-encoded gene products.
Subject(s)
Antigens, Viral/immunology , Blood Donors , Hepacivirus/isolation & purification , Hepatitis Antibodies/blood , Hepatitis C/blood , Recombinant Proteins/immunology , Viral Nonstructural Proteins/immunology , Amino Acid Sequence , Hepacivirus/genetics , Hepatitis Antibodies/analysis , Hepatitis C/diagnosis , Hepatitis C/transmission , Hepatitis C Antibodies , Humans , Immunoblotting/methods , Peptides/chemical synthesis , Peptides/immunology , Polymerase Chain ReactionABSTRACT
The polymerase chain reaction (PCR) for human immunodeficiency virus type 1 (HIV-1) DNA was performed on specimens from 197 homosexual and bisexual men enrolled in studies of HIV-1 infection. Thirty cycles of amplification were conducted, followed by detection with probes corresponding to two gag primer pairs (SK 38/39 and SK 101/145). Of 107 men who were HIV-1 antibody-negative, 105 (98%) were PCR-negative. Two who were initially PCR-positive antibody-negative were PCR- and antibody-negative on repeat testing of both the same specimen and specimens drawn 8-10 months later; this suggests that the first PCR results were false-positive. Of 90 men who were antibody-positive, PCR was positive in 87 (97%), including all 13 with AIDS, all 22 with AIDS-related conditions, all 11 with generalized lymphadenopathy only, and 41 (93%) of 44 without signs or symptoms of HIV-1 infection. On repeat testing, all 3 PCR-negative, antibody-positive men were PCR-positive. In this population and with this technique, PCR had excellent agreement with the HIV-1 antibody test.
Subject(s)
DNA, Viral/analysis , HIV Infections/diagnosis , HIV-1/isolation & purification , Base Sequence , Bisexuality , DNA Probes , DNA, Viral/genetics , HIV Antibodies/analysis , HIV-1/genetics , HIV-1/immunology , Homosexuality , Humans , Male , Molecular Sequence Data , Polymerase Chain Reaction , Predictive Value of TestsABSTRACT
BACKGROUND: The clinical and pathologic significance of quantitative serum hepatitis C virus (HCV) RNA levels in patients with chronic type C hepatitis is unknown. The aim of this study was to determine whether serum levels of HCV RNA were associated with mode of viral transmission or with histological severity of liver disease. METHODS: A branched DNA signal amplification assay for HCV RNA was done on the sera of 127 patients with well-defined risk factors for viral hepatitis. Seventy persons acquired HCV infection by blood transfusion and 57 via tattoo application or former intravenous drug use. Group I included 42 patients with chronic persistent hepatitis, group II consisted of 39 patients with chronic active hepatitis, and group III included 40 individuals whose liver biopsies showed both chronic active hepatitis and cirrhosis, as well as six patients with clinically decompensated cirrhosis. RESULTS: The median HCV RNA level [equivalents/ml (eq/ml) x 10(5)] for patients who acquired infection from transfusion [73.5 x 10(5) (eq/ml)] was not significantly different from that of patients who reported prior intravenous drug use [50 x 10(5) eq/ml] (p = 0.283). The median HCV RNA level for groups I, II, and III was 29.5, 76, and 71, respectively. Group I differed significantly from groups II and III combined (median = 73) (p = 0.02). No difference was noted between group II and group III (p = 0.947). Age did not correlate with level of viremia (r2 = 0.01). No relationship was found between serum alanine aminotransferase and the level of viremia (p = 0.52). Multivariate analysis showed that only the histological severity of the disease proved to be predictive of HCV RNA level (p = 0.04). CONCLUSION: The lowest levels of hepatitis C viremia are, in general, associated with minimal liver disease. Overall, histological severity of chronic hepatitis C infection best predicts HCV RNA levels.
Subject(s)
Hepacivirus/isolation & purification , Hepatitis C/diagnosis , Hepatitis, Chronic/microbiology , Liver/pathology , RNA, Viral/blood , Biopsy , Hepatitis C/epidemiology , Hepatitis C/transmission , Hepatitis, Chronic/diagnosis , Hepatitis, Chronic/epidemiology , Humans , Liver Cirrhosis/microbiology , Liver Cirrhosis/pathology , Multivariate Analysis , Risk Factors , Substance Abuse, Intravenous/complications , Tattooing/adverse effects , Transfusion ReactionABSTRACT
BACKGROUND: There is a high prevalence of liver disease among the recipients of organs from donors with antibodies to hepatitis C virus (HCV). We undertook a study to determine the frequency of persistent HCV infection, as indicated by the presence of HCV RNA, among both cadaveric organ donors positive for antibodies to HCV (anti-HCV) and the recipients or organs from these donors. METHODS: Serum samples from donors and recipients were tested for HCV RNA with the reverse transcriptase polymerase chain reaction, with use of primers from the 5' untranslated region of the HCV genome, and for anti-HCV with the first-generation enzyme-linked immunosorbent assay (ELISA) and two second-generation tests. RESULTS: HCV RNA was detected in 9 of the 11 organ donors (82 percent) with a positive first-generation ELISA for anti-HCV. Among the organ recipients, the prevalence of HCV RNA increased after transplantation: 7 of 26 patients (27 percent) had positive samples before transplantation, as compared with 23 of 24 patients (96 percent) after transplantation (P less than 0.001). Among 13 recipients who were HCV RNA-negative before receiving organs from the nine HCV RNA-positive donors, HCV infection was detected in all 13 after transplantation, and anti-HCV developed in 8 (62 percent). On the basis of a positive test for HCV RNA, the maximal sensitivity of the three anti-HCV tests was 57 percent (positive in 4 of 7 patients with end-stage organ failure) before transplantation and 70 percent (positive in 16 of 23 patients) after transplantation. CONCLUSIONS: Nearly all the recipients of organs from anti-HCV-positive donors become infected with HCV. The current tests for anti-HCV antibodies underestimate the incidence of transmission and the prevalence of HCV infection among immunosuppressed organ recipients.