Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction.

Serum components inhibit DNA polymerase, thereby obviating direct detection of serum viral DNA sequences by the polymerase chain reaction (PCR). This has necessitated extraction of nucleic acid from sera before performing PCR and has resulted in loss of sensitivity. By adsorbing virus to a solid surface (microcentrifuge tubes or antibody coated microparticles) followed by proteinase K digestion, as little as three viruses per 200 microliters serum may be directly detected by PCR without nucleic acid extraction. The sensitivity is dependent on the surface area of the adsorptive surface and is increased by having antibodies on the adsorptive surface. The nucleic acid sequence of the amplified DNA fragments may be directly determined by the dideoxy method. Of 24 plasma samples from HBsAg+ volunteer blood donors, HBV DNA was detected in 7 by dot blot assay, 7 by liquid hybridization, and 9 by PCR. PCR detected DNA in every sample that was positive by another assay. Analysis of serial samples of two patients with acute self-limited hepatitis B found detectable HBsAg and pre-S2 antigenemia before HBV DNA by the PCR method. These results suggest that surface antigenemia may precede viremia during acute hepatitis.

Variation in antibody reactivity to the hepatitis C virus by comparative immunoscreening and enzyme immunoassay.

The detection of antibody to the hepatitis C virus C100-3 antigen from the nonstructural region (NS3/NS4) of the viral genome was the first useful marker developed to detect past or potentially active infection with the hepatitis C virus. A systematic epitope survey of the nonstructural region has uncovered other immunogenic antigens. In order to assess the possible diagnostic utility of these antigens, their reactivity against a limited panel of sera from patients with chronic liver disease due to hepatitis C virus and other etiologies was tested. Antibody assays were performed using an immunoblot plaque assay and an enzyme-linked immunosorbent assay (ELISA). In a study of 16 C100-3-reactive individuals, all 16 patients were reactive using the plaque assay for the NS3 3' (409-1-1) and NS3 5' (C33u). In this same group of patients, antibodies by ELISA were reactive to NS3 3' in 12 of 16 patients (75%), NS3 5' in 15 of 16 patients (93%), and a capsid antigen (NC450) in 14 of 16 patients. In a group of five patients who were diagnosed with cryptogenic liver disease (C100-3 negative), 4 of 5 patients were reactive for antibody to all of the above epitopes. In a survey of 23 patients with other forms of chronic liver disease (nonviral liver disease, hepatitis B, alcoholic liver disease, cholestatic liver disease, and autoimmune hepatitis), only 1 of 23 patients was reactive for antibody to the C100-3 and 4 of 23 patients were reactive for antibodies to structural and nonstructural regions of the virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of hepatitis C after liver transplantation. Four serologic tests compared.

To determine the best method for detecting HCV infection in immunosuppressed patients, stored frozen serum from 101 liver transplant recipients was tested for hepatitis C virus. Each sample was tested by four assays. HCV RNA was detected by both polymerase chain reaction (PCR) and branched DNA signal amplification. Antibody to HCV was determined using second-generation enzyme-linked immunoassay (EIA) and recombinant immunoblot assay. Forty one transplant recipients met the working definition for true positives of HCV infection. Of these "true positives," 98% were positive by HCV RNA PCR assay, 88% by b-DNA signal amplification assay, 88% by anti-HCV EIA, and 63% demonstrated two or more reactive bands on recombinant immunoblot. Five of 57 (9%) HCV-antibody negative recipients had HCV RNA detected by both methods. Of 44 HCV enzyme-linked immunoassay (EIA) repeatedly reactive samples, the recombinant immunoblot was negative in 2 and indeterminate in 13. HCV RNA was present in 9 of 13 recombinant immunoblot indeterminate sera. Nine EIA repeatedly reactive sera were negative by both tests for HCV RNA. In liver transplant recipients, HCV infection is best determined by measurement of HCV RNA. Antibody formation may be delayed or suppressed in a minority of patients despite > 10(9) equivalents/L (> 10(6)/mL) of HCV RNA in serum. Recombinant immunoblots with a single reactive band pattern often indicate HCV infection in immunosuppressed patients.

Serological detection of HBeAg and anti-HBe using automated microparticle enzyme immunoassays.

Fully automated microparticle enzyme immunoassays (EIA) were developed for the detection of HBeAg (IMx HBe) and antibodies against HBeAg (IMx anti-HBe), respectively. Specimens from blood donors, diagnostic and hospital patients and individuals with a variety of infectious and immune diseases were tested both in house and at four clinical sites. The overall agreement between IMx HBe and Abbott HBe RIA/EIA was 99.7% (2985 of 2994) and between IMx anti-HBe and anti-HBe RIA/EIA was 95.8% (2330 of 2432). Almost all anti-HBe discordant specimens (94.1%, 96 of 102) were reactive by IMx anti-HBe but negative by anti-HBe RIA/EIA. off anti-HBe discordant specimens were also reactive for anti-HBc. The IMx anti-HBe assay was 2- to 4-fold more sensitive than the current RIA as determined by serial dilution of anti-HBe reactive specimens. The ability of these IMx assays to detect HBeAg and anti-HBe in 199 HBsAg reactive specimens was also evaluated. 43.7% (87 of 199) and 66.3% (132 of 199) specimens were reactive for HBeAg and anti-HBe by IMx, respectively. Only one specimen was negative for both IMx assays compared to 14 (7.0%) non-reactive for both HBe and anti-HBe RIA. There were 24 specimens (12.1%) positive for both HBeAg and anti-HBe by IMx compared to 1 (0.5%) positive by the corresponding RIAs. This increased detectability of anti-HBe in HBsAg carriers using IMx anti-HBe may result from increased sensitivity for 'free' anti-HBe and/or increased ability to detect anti-HBe in immune complex. IMx anti-HBe also detected more reactives among volunteer blood donor specimens reactive for anti-HBc but negative for HBsAg (55.5%, 86 of 155), compared to RIA (38.7%, 60 of 155). IMx anti-HBe may be useful in confirming prior exposure to HBV in blood screened positive by Corzyme.

Hepatitis C in liver transplant recipients.

HCV infection is commonly found in patients with chronic liver disease undergoing liver transplantation. However, the presence of antibody to HCV does not appear to be associated with the development of hepatitis posttransplant. No other risk factors were identified that appear to predispose patients to development of hepatitis in the posttransplant period, including amount of blood product exposure. The role of immunosuppression in the acquisition and expression of liver disease caused by HCV remains to be determined.

A ribonuclease protection assay for the direct detection and quantitation of hepatitis C virus RNA.

A ribonuclease protection assay (RPA) was developed for the direct detection and quantitation of HCV RNA in infected patients' sera or plasma using HCV [(32)P]RNA from the conserved 5'-untranslated region (5'-UTR) as a probe. We were able to directly detect the presence of HCV RNA by RPA in several infected patients' samples. The viremic status of HCV infected patients with indeterminate recombinant immunoblot assay (RIBA II) was also determined by this assay. Polymerase chain reaction (PCR) was also performed on all these samples and were found to be positive with a concordance of 100% between the results of PCR and RPA. The RPA was able to detect approximately 1 pg of HCV RNA. A limited sequence heterogeneity among HCV isolates was also observed by this assay, suggesting that this may be a faster method of detecting heterogeneous HCV sequences in patients' samples. This simple and specific method could be used to quantitate HCV RNA in order to better determine viremia and follow the course of HCV infection especially when RIBA II results are indeterminate.

Fluctuation of serum HCV-RNA levels in untreated blood donors with chronic hepatitis C virus infection.

The hepatitis C virus (HCV)-RNA levels were measured in 281 serum samples from 32 untreated volunteer blood donors prospectively collected over a period of 14-73 months. The HCV-RNA levels were tested by the branched DNA signal amplification assay. The mean HCV-RNA levels of each donor ranged from 4.92 log10-6.36 log10 gene equivalents/mL (25%, median, 75% percentile; 5.51, 5.79, 6.12 log10 gene equivalents/mL). The fluctuations of HCV-RNA levels in individuals, represented by the ratio of the maximum value divided by the minimum value, ranged from a 1.7- to a 141-fold change. Fluctuations with more than a 10-fold change were observed in five subjects: 11-, 15-, 17-, 96- and 141-fold changes. Eleven subjects were followed for at least 5 years; all subjects had fluctuations of HCV-RNA levels greater than 3-fold during the observation period. No blood donor was observed whose HCV levels changed from a high-level phase to a low-level phase or from low to high. No subjects cleared HCV during follow-up, although two had undetectable HCV-RNA levels transiently. These findings reveal that changes in HCV-RNA levels occur which are unrelated to treatment with interferon and ribavirin.

Prevalence of hepatitis C virus antibodies among patients infected with human immunodeficiency virus.

A study was undertaken to determine the prevalence and risk factors for serological evidence of hepatitis C virus (HCV) infection in patients infected with the human immunodeficiency virus (HIV). Tests for anti-HCV antibody were carried out by enzyme-linked immunoassay (EIA) on 101 HIV-infected patients from two university-based outpatient clinics. Anti-HCV antibody reactive samples were tested by using a recombinant immunoblot assay (RIBA) for HCV antibodies. Fourteen of 101 (13.9%) HIV-infected patients were anti-HCV reactive by EIA. Of these 14, only seven were reactive by RIBA: four were intravenous drug users as a sole risk factor for HIV infection; and the remaining three acquired HIV by blood transfusion, contaminated instrument exposure or IV drug use and sexual contact. Acquisition of HIV by sexual activity alone was not associated with HCV infection. It is concluded that HCV infection is found in approximately 7% of a university HIV clinic population. False-positive anti-HCV antibody serology may lead to overestimation of the prevalence of HCV infection. Female sex and intravenous drug use are significantly associated with HCV infection among HIV-infected individuals.

Detection of antibody to immunodeficiency viruses by dot immunobinding assay.

The dot immunobinding assay (DIA), a modified enzyme immunoassay (EIA), has been demonstrated to be a highly sensitive and specific assay for the detection of antibody to a number of viruses. Different laboratory procedures are available for detecting antibody to the immunodeficiency viruses; however, these procedures require a certain amount of sophisticated equipment and trained personnel. Further, commercial kits for detecting antibody to human immunodeficiency virus, as now available, are not easy to use in the nonlaboratory setting. The DIA, as described herein, may be formatted to test up to 30 serum samples and is designed to be used in the absence of laboratory equipment. To determine the effectiveness of the DIA as a test kit for the detection of HIV and human T-cell leukemia virus type I (HTLV-I) antibodies, the kit was compared with commercial EIA and Western blot (WB; immunoblot) kits. Testing approximately 1,000 human serum samples for HIV antibody by DIA and EIA revealed a total agreement of 98.1%, a specificity of 99.0%, and a sensitivity of 95.9%. For 804 serum samples tested (200 were tested independently in two laboratories), eight results were discrepant: four DIA negatives which were EIA borderline positive and four DIA positives which were EIA negative. Testing the eight discrepant sera by immunofluorescence assay and WB resulted in their being either negative or indeterminate. The four DIA positives were indeterminate by WB. Close agreement was obtained when the remaining sera were compared by DIA, EIA, and WB. Of interest was finding that the DIA results compared favorably with those obtained by WB. Twenty-six suspect HTLV-I-positive serum samples tested by DIA also gave results comparable to those obtained by EIA and WB.

Hepatitis C virus in blood samples from volunteer donors.

The specificities of four assays for hepatitis C virus (HCV) were compared by using units from volunteer blood donors. Upon Food and Drug Administration licensure of the first immunoassay for anti-HCV, EIA-1, units previously deemed acceptable for transfusion and all subsequent blood donations were screened. EIA-1 repeat-reactive (RR) units were tested for HCV by a second-generation enzyme-linked immunoassay (EIA-2) and by a four-antigen recombinant immunoblot assay (RIBA II) and for HCV RNA by reverse transcriptase polymerase chain reaction. All HCV RNA-positive samples were reactive by both RIBA II and EIA-2. All RIBA II-reactive sera were EIA-2 RR. In EIA-1, 0.45% of the prescreened units and 0.59% of the prospectively screened donors were RR. Of these, 52.5 and 54%, respectively, were EIA-2 RR, 71.4 and 69% of the EIA-2 RR units were reactive on RIBA II, and 93 and 88% of the RIBA II-reactive samples were HCV RNA positive. When the sample/cutoff ratio for EIA-2 was greater than 5, 91% of the samples were RIBA II reactive and 82% of the samples were HCV RNA positive. None of EIA-2 RR units with a sample/cutoff ratio of < 5 was RIBA II reactive or HCV RNA positive. In conclusion, RIBA II and RT PCR results are highly concordant. A sample/cutoff ratio of > 5 in EIA-2 is a good discriminator for the likelihood of a true HCV infection on the basis of RT PCR and RIBA II assays.

Detection of antibodies to human immunodeficiency virus type 2 (HIV-2) in blood donor sera using United States assay methods for anti-HIV type 1.

Twelve serum samples from French blood donors that were uniformly reactive in tests for antibody to human immunodeficiency virus type 2 (anti-HIV-2) also were reactive in 92 to 100 percent of tests with three anti-HIV type 1 (anti-HIV-1) enzyme-linked immunoassays currently in widespread use for donor screening in the United States. Supplemental tests for anti-HIV-1 on these anti-HIV-2-reactive samples differed in their responses. All samples reacted in a licensed anti-HIV-1 Western blot, but there was an atypical band near the p41 position, which could be a clue to the fact that this result was a cross-reaction with anti-HIV-2. A recombinant immunoblot gave an indeterminate result for anti-HIV-1 in all 12 samples. A local immunofluorescence assay for anti-HIV-1 reacted with 92 percent of the samples, but a commercial one detected only 58 percent.

The prevalence of hepatitis C virus (HCV) in infants and children after liver transplantation.

Hepatitis C virus (HCV) is an important cause of liver injury following liver transplantation in adults. We hypothesized that the prevalence of HCV infection in children following liver transplantation would be lower than the prevalence in adults after liver transplantation because HCV-related liver disease leading to liver transplantation in children is low and children require less blood products than adults during transplantation. We therefore performed a cross-sectional study to determine the prevalence of HCV infection in children who had undergone liver transplantation. Serum samples were obtained from 62 of 65 (95.4%) consecutive patients surviving for more than six months after transplantation. Using a second-generation enzyme-linked, immunosorbent assay (ELISA-2) and a second-generation recombinant immunoblot assay (RIBA-II), antibodies to HCV were detected in 5.1% (3 of 59) of the subjects. Using a single-step, polymerase chain reaction (PCR), HCV RNA was detected in 6.2% (4 of 62). All HCV-positive children had undergone liver transplantation before the initiation of routine screening for HCV in blood donors; overall 30 patients were transplanted prior to routine screening of blood products for HCV. The prevalence of HCV in infants and children after liver transplantation in our study is substantially less than the rates reported in adults. This difference may be due, in part, to the lower volume of blood product exposure and to the fact that children, as opposed to adults, rarely have chronic HCV infection as a cause of end-stage liver disease.

Comparison of two second-generation anti-hepatitis C virus ELISA on 21431 US blood donor samples.

We have compared two different second-generation (2.0) enzyme-linked immunosorbent assays (ELISA) for the presence of antibodies to hepatitis C virus (anti-HCV) in blood from volunteer, unpaid donors. At two separate blood centres, a total of 21,431 donor samples were tested with Abbott Anti-HCV 2.0 ELISA and Ortho Anti-HCV 2.0 ELISA. Samples found to be repeatedly reactive were tested by supplemental/investigational assays. MATRIX HCV (Abbott) and anti-HCV RIBA II (Ortho/Chiron), to 'confirm' the presence of anti-HCV. Discordant ELISA samples were additionally tested by the polymerase chain reaction (PCR) for the presence of HCV RNA. The Abbott anti-HCV assay had a repeatedly reactive rate of 0.59% (127/21,431) and the Ortho anti-HCV assay 0.51% (110/21,431). Overall agreement between assays was 99.76%, 72/127 (56.7%) of Abbott repeatedly reactive samples confirmed on MATRIX and 61/127 (48.0%) on RIBAII; 70/110 (63.6%) of Ortho repeatedly reactivate samples confirmed on MATRIX and 61/110 (55.5%) on RIBA II. Discordant ELISA samples tested by PCR yielded negative results. Hence the two ELISA had equal sensitivity, as defined by detection of true positive samples; the slightly lower specificity of the Abbott Anti-HCV 2.0 ELISA may be owing to culling of donors with a false positive test by Ortho's Anti-HCV 1.0 and 2.0 ELISA tests (the routine tests in place at each blood centre). A sample found to be repeatedly reactive by two different ELISA tests for anti-HCV is likely to be a true positive and may not require further 'confirmatory' testing.

Significance of antibody to hepatitis B core antigen in blood donors as determined by their serologic response to hepatitis B vaccine.

Because large numbers of volunteer blood donors may be disqualified for "false-positive" results on tests for antibody to hepatitis B core antigen (anti-HBc), a more specific definition of anti-HBc enzyme immunoassay (EIA)-reactive was evaluated, including only those donor samples that were "strongly" reactive (sample-to-cutoff absorbance ratio, < 0.45). Results using this definition and other anti-HBc test methods were compared to the serologic response (antibody to hepatitis B surface antigen [anti-HBsAg]) to hepatitis B vaccination. Fifty-eight volunteer blood donors who had previously been deferred as donors, because of reactive anti-HBc tests (all other blood screening tests were negative, including those for HBsAg and anti-HBsAg) on two occasions, were vaccinated for hepatitis B. It was assumed that an anamnestic response to vaccine indicated past infection with hepatitis B, while a primary response to vaccine indicated lack of past infection. One (2%) of 43 donors with a historically "weak" anti-HBc (reactive absorbance ratio, > or = 0.45) had an anamnestic response to vaccine, compared to 8 (53%) of 15 with historically "strong" anti-HBc (reactive absorbance ratio, < 0.45) (p < 0.005). Anti-HBc testing using the microparticle EIA method also correlated well with hepatitis B vaccination results. The use of a narrower definition of "reactive" for anti-HBc EIA testing yielded much more specific, but slightly less sensitive, results.

Evidence of hepatitis in patients receiving transfusions of blood components containing antibody to hepatitis C.

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%.

Absence of hepatitis B and C viruses in pediatric idiopathic membranoproliferative glomerulonephritis.

The blood-borne hepatitis viruses, hepatitis B virus (HBV) and hepatitis C virus (HCV), have similar epidemiological features. The association of chronic HBV infection and glomerulonephritis is well established, particularly in children. Recent reports have shown an association between HCV infection and glomerulonephritis in adults. In order to assess the role of these hepatotropic viruses in membranoproliferative glomerulonephritis (MPGN) we screened 34 children with idiopathic MPGN for the presence of HBV and HCV infection using highly sensitive polymerase chain reaction techniques for the detection of HBV DNA and HCV RNA. Also, enzyme-linked immunosorbent assays were used to detect the presence of antibody to hepatitis B surface antigen and antibody to HCV. No evidence of HBV or HCV infection was demonstrated in any of the patients. We conclude that HBV and HCV are not significant causes of idiopathic MPGN in children in the United States.

Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.

Seroepidemiology of viral infections among intravenous drug users in northern California.

Intravenous drug users are frequently exposed to parenterally transmitted viral infections, and these infections can spread to the general population through sexual activity. We investigated the prevalence of serologic markers for human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type I/II (HTLV-I/II), hepatitis B virus (HBV), and hepatitis C virus (HCV) in intravenous drug users and their sexual contacts. Of 585 drug users from northern California tested for these serologic markers, 72% were reactive for the antibody to HCV, 71% for the antibody to hepatitis B core antigen, 12% for HTLV-I/II antibodies, and 1% for the HIV-1 antibody. The prevalence of serologic markers for these four viruses correlated with the duration of intravenous drug use, the ethnic group, and the drug of choice. More than 85% of subjects infected with either HCV or HBV were coinfected with the other virus. All persons reactive to HTLV-I/II antibodies had antibodies for either HBV or HCV. Of 81 sexual contacts tested, 17% had evidence of HBV infection while only 6% were reactive for HTLV-I/II antibodies and 4% for the antibody to HCV. None of this group was infected with HIV-1. We conclude that HTLV-I/II and HCV are inefficiently transmitted to sexual contacts while HBV is spread more readily. Programs designed to discourage the sharing of drug paraphernalia, such as needle and syringe exchanges, should decrease the risk of parenterally spread viral infections in intravenous drug users and thus slow the spread of these infections to the general population.

Prevalence of and risk factors for antibody to hepatitis E virus seroreactivity among blood donors in Northern California.

To evaluate antibody to hepatitis E virus (anti-HEV) seroreactivity, 5000 US blood donors were tested for anti-HEV by two EIAs: a mosaic protein assay (MPr-EIA) and a recombinant protein assay (RPr-EIA). Overall, 59 (1.2%) were seroreactive by MPr-EIA and 70 (1.4%) were seroreactive by RPr-EIA. The overall concordance between tests was 98.5% (4925/5000); the concordance among reactive sera by either test was only 27% (27/102). In a case-control study, seroreactive persons were more likely than seronegative persons to have traveled to countries in which HEV is endemic (odds ratio [OR] for MPr-EIA = 4.3, P < .001; OR for RPr-EIA = 2.5, P = .005), but 31% of MPr-EIA anti-HEV-reactive persons and 38% of RPr-EIA anti-HEV-reactive persons had no history of international travel. These findings suggest that travelers to regions in which HEV is endemic can acquire subclinical HEV infection. The significance of anti-HEV seroreactivity among persons without an international travel history needs to be determined.