Evaluation of hepatitis C virus RNA RT/PCR qualitative and quantitative second generation assays.

Hepatitis C virus (HCV) RNA qualitative and quantitative second generation assays (Amplicor HCV v2.0 and Amplicor HCV Monitor v2.0, respectively) were evaluated by testing serum samples from 132 blood donors anti-HCV positive HCV RNA negative by first generation qualitative assay and 326 viremic patients. An HCV RNA transcript was synthesized and ten-fold dilutions were used to assess sensitivity. Second generation assays were one log more sensitive than their respective first generation tests (10(2) copies per ml vs. 10(3) for the qualitative tests; 10(3) copies per ml vs. 10(4) for the quantitative tests). From the 132 anti-HCV positive RNA negative subjects, 6 (5%) were positive by Amplicor v2.0. Quantification figures by Monitor v2.0 were similar in genotypes 1, 2 and 3, whereas Monitor 1.0 values were higher in genotype 1 than in genotype 2 or 3. In 114 patients, branched-DNA v2.0 obtained higher values than Monitor v2.0 and Monitor v1.0 (6.6+/-0.6 log RNA copies per ml, 6.4+/-0.6, and 5.3+/-0.7, respectively, P<0.001). HCV RNA qualitative and quantitative second generation assays are more sensitive and genotype independent than first generation assays.

Blood screening by nucleic acid amplification technology: current issues, future challenges.

BACKGROUND: Nucleic acid amplification technology (NAT) is presently being evaluated in US clinical trials to determine the safety and efficacy of mini-pool testing for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA in the blood-donor population. Although the risk for transfusion-transmitted HIV and HCV infection is extremely low, there is still a small chance that blood donated by infected individuals before seroconversion can escape detection by current antibody-based assays. METHODS: This report describes the amplification technologies being used and reviews several issues surrounding NAT-based blood screening. The performance features of NAT and current enzyme immunoassay technologies are compared, and the benefits of NAT in reducing transfusion-transmitted infections are discussed. CONCLUSIONS: The current US clinical trials of mini-pool NAT testing for HIV and HCV RNA have successfully identified preseroconversion infectious blood units. Although the current NAT-based screening systems are semiautomated, mini-pool testing represents an unprecedented innovation among government and nongovernment agencies in the highly regulated blood transfusion industry. Despite cost-effectiveness issues, based on the public perception of infectious diseases acquired through blood transfusion, NAT-based screening of the blood supply is expected to become a standard in transfusion medicine.

Utility of early testing for HCV viremia as predictive factor of sustained response during interferon or interferon plus ribavirin treatment.

BACKGROUND/AIM: To evaluate the utility of early testing for hepatitis C viremia as a predictor of treatment outcome during interferon or combination therapy. METHODS: We studied 184 patients with chronic hepatitis C who received interferon and were monitored for HCV RNA. Sixty-two patients received interferon alone for 12 months and 122 patients, who were still HCV RNA positive at 2 months, received an additional 12-month course of interferon and ribavirin combination therapy. RESULTS: Using this strategy, sustained response occurred in a total of 34 patients (18.5%). Independent variables associated with sustained response were HCV genotype (p=0.06), viral load < or = 5.1 logs/ml (p= 0.005) and negative HCV RNA at 1 month (p<0.0001) in the interferon group, and female sex (p=0.04), genotype (p=0.03), viral load < or = 5.5 logs/ml (p=0.01), normal ALT (p=0.001) and decline in viral load > or = 1.2 logs/ml after 2 months of interferon monotherapy (p<0.001) and negative viremia at 5 months of ribavirin onset (p<0.0001) in the combination therapy group. Persistence of viremia at 1 month of interferon monotherapy and at 5 months of combination therapy were the strongest predictors of non-response (negative predictive value of 100% and 99%, respectively). CONCLUSIONS: Qualitative assessment of HCV RNA during treatment is the strongest predictor of sustained response during interferon or combination therapy for chronic hepatitis C.

Experience with PCR screening.

The COBAS AmpliScreen Hepatitis C (HCV) Test, Version 2.0, which is designed for screening pools composed of samples from individual units of blood or plasma, employs a MultiPrep sample processing procedure that simultaneously extracts and concentrates HCV, HIV-1 and Hepatitis B virus particles from plasma. An HCV Internal Control (IC) RNA serves as an extraction and amplification control for each independently processed sample. Processed samples are amplified by RT-PCR using HCV-specific complementary primers and detected by hybridization of the amplified products to HCV- and IC-specific oligonucleotide probes. The analytical sensitivity of the test is 25 International Units (IU) of HCV per mL of pooled plasma; all HCV genotypes are detected with similar efficiency. The test detected HCV RNA 23 to 32 days prior to anti-HCV antibody seroconversion for four of the five seroconversion panels tested. The test had sufficient sensitivity to reproducibly detect a single infected unit containing 2.4 x 10(3) copies of HCV per mL in a pool with 23 uninfected units. COBAS AmpliScreen tests for HIV-1 and HBV now being validated by Roche Molecular Systems also incorporate the MultiPrep specimen processing method, thereby making it possible to use a single processed specimen to screen for all three viruses.

High pre-treatment serum hepatitis B virus titre predicts failure of lamivudine prophylaxis and graft re-infection after liver transplantation.

BACKGROUND/AIMS: Orthotopic liver transplantation has an established role for the treatment of patients with chronic liver failure secondary to hepatitis B virus (HBV) infection. Unfortunately, recurrent infection of the graft can be associated with aggressive disease, and with diminished graft and patient survival. Currently, the role of nucleoside analogues for prevention of graft re-infection is being evaluated. Preliminary results are encouraging, but treatment failure has been associated with emergence of drug-resistant virus. METHODS: We have studied ten consecutive patients who received lamivudine prophylaxis for prevention of HBV graft reinfection. Sequential sera, collected prelamivudine then during treatment before and after liver transplantation, were examined. Conventional serological markers were measured, as were serum viral DNA levels with a sensitive quantitative polymerase chain reaction assay. RESULTS: Lamivudine treatment effected a reduction in serum HBV levels, but six patients still had measurable viral DNA at the time of transplantation. Five patients developed graft re-infection with lamivudine-resistant virus. Resistant virus emerged 8 to 15 months post-transplant. The likelihood of emergence of resistant virus was related to the pre-treatment serum HBV titre. Persistent serum viral DNA positivity and evidence of graft re-infection during the early post-transplant period did not predict the subsequent emergence of resistant virus. CONCLUSIONS: Our observations suggest that the resistant species may be present in the viral quasispecies in the serum and liver of patients with high-level replication prior to lamivudine exposure. The resistant species can persist during lamivudine treatment prior to transplantation, and emerge following transplantation. These observations suggest strategies which might prevent the emergence of drug-resistant species, and imply that graft re-infection may be a preventable phenomenon.

The AMPLICOR® HCV Tests for the Detection and Quantitation of Serum or Plasma HCV RNA.

Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.

Quality control of the polymerase chain reaction.

The polymerase chain reaction (PCR) has revolutionized both the basic and the applied aspects of the biomedical field with more than 40,000 papers having been published employing this technique. PCR has become an indispensable tool for basic research applications, such as cloning (1), sequencing (2), mRNA analysis (3), DNA and RNA quantitation (4-6), mutagenesis (7), and gene detection (8). Because of its ability to amplify minute quantities of nucleic acid specifically, PCR has also been applied with great success in clinical diagnostics (9-11), genetic testing (12-15), forensics (16,17), and environmental testing (18,19).

Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B.

BACKGROUND: The Amplicor HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates. OBJECTIVE: The performance of the Amplicor HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor HBV Monoitor assay was compared to the Digene Hybrid Capture System HBV DNA assay for the quantitation of HBV in patient sera. STUDY DESIGN: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays. RESULTS: The detection limit was found to be 10(3) copies/ml with the Amplicor PCR assay compared to 10(6) to 10(7) copies/ml with the Digene hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10(3) and 10(7) copies/ml and all of them tested below the detection limit with the hybridization assay. CONCLUSION: The Amplicor HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.

Identification of a linked set of genes in Serpulina hyodysenteriae (B204) predicted to encode closely related 39-kilodalton extracytoplasmic proteins.

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein).

Performance of the automated COBAS AMPLICOR system for the detection of hepatitis C virus RNA.

BACKGROUND: The COBAS AMPLICOR (CA) instrument for the amplification and detection steps of the AMPLICOR molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator. OBJECTIVE: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test). STUDY DESIGN: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR Test. RESULTS: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number. CONCLUSION: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.

The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.

A 32-nucleotide deletion (delta 32) within the beta-chemokine receptor 5 (CCR5) gene has been described in subjects who remain uninfected despite extensive exposure to HIV-1. This allele was found to be common in the Caucasian population with a frequency of 0.0808, but was not found in people of African or Asian ancestry. To determine its role in HIV-1 transmission and disease progression, we analyzed the CCRS genotype of 1252 homosexual men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). No infected participant was found to be homozygous for the delta 32 allele, whereas 3.6% of at-risk but uninfected Caucasian participants were homozygous, showing the highly protective role of this genotype against sexual acquisition of HIV-1. No evidence was found to suggest that heterozygotes were protected against HIV-1 infection, but a limited protective role against disease progression was noted. The delta 32 allele of CCR5 is therefore an important host factor in HIV-1 transmission and pathogenesis.

Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction.

A rapid and sensitive polymerase chain reaction (PCR)-based assay for detection of Chlamydia trachomatis in cervical specimens is described. This assay consists of (i) sample preparation which avoids the use of heat, centrifugation, or organic extractions; (ii) rapid, two-temperature PCR amplification of C. trachomatis cryptic plasmid sequences; and (iii) capture and colorimetric detection of amplified DNA in microwell plates. PCR was compared with culture by using 503 cervical specimens. After resolution of discrepant specimens with a confirmatory PCR assay directed against the chlamydial major outer membrane protein gene, PCR had a sensitivity of 97% and a specificity of 99.7% while culture had a sensitivity of 85.7% and a specificity of 100%. In a separate study, PCR was compared with a direct specimen enzyme immunoassay (Chlamydiazyme; Abbott Diagnostics) by using 375 cervical specimens. After resolution of discrepant specimens, PCR had a sensitivity and specificity of 100%, while the enzyme immunoassay had a sensitivity of 58.8% and a specificity of 100%.

Comparison of three nonradioisotopic polymerase chain reaction-based methods for detection of human immunodeficiency virus type 1.

Three nonradioisotopic polymerase chain reaction (PCR)-based detection techniques were evaluated for sensitivity and specificity in detecting human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. The Roche prototype HIV-1 PCR assay, the Du Pont enzyme-linked oligonucleotide sandwich assay (ELOSA), and the Gen-Probe hybridization protection assay (HPA) were compared with a standard radioisotopic oligonucleotide solution hybridization (OSH) technique. A panel of 111 well-characterized clinical samples that included peripheral blood mononuclear cells from 48 healthy, low-risk, HIV-1 antibody-negative subjects, 24 antibody-positive subjects with stable CD4 counts of less than 200/mm3, and 39 antibody-positive subjects with stable CD4 counts of greater than 800/mm3 were studied. Each method demonstrated good specificity, ranging between 96 and 100%; those of the OSH and ELOSA (Du Pont) were 100%, those of the HPA (Gen-Probe) were 100% with one probe and 96% with the other probe, and that of the HIV-1 PCR assay (Roche) was 96%. Sensitivities ranged from 96 to 100% for the low-CD4-count group, with the OSH, the HIV-1 PCR assay (Roche), and the HPA (Gen-Probe) all attaining a sensitivity of 100%. For the high-CD4-count group, sensitivities ranged from 69 to 97%, with the OSH attaining a sensitivity of 97% and the HPA attaining sensitivities of 97% with one probe and 95% with the other probe. These data indicate that the nonradioisotopic techniques are sensitive and specific for the detection of HIV-1 proviral DNA in clinical samples.

Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants.

Two non-isotopic polymerase chain reaction (PCR) methods were evaluated by testing blood from 41 HIV-1-seropositive and 16 HIV-1-seronegative Ugandan mothers and 56 of their children (aged 0.5-15.0 months). Amplification of HIV-1 sequences was performed in duplicate using a biotinylated primer pair to the gag region (SK 462-431) and nested primer pairs (JA 17-20) to the pol region of HIV-1. gag sequences were hybridized using a microtiter plate coated with the SK 102 probe followed by colorimetric detection using an avidin-horseradish peroxidase conjugate and tetramethylbenzidine/peroxide substrate. pol sequences were detected on agarose gel stained with ethidium bromide. Results of HIV-1 PCR analysis showed that 40 out of 41 (98%) seropositive mothers and 10 out of 29 (34%) seropositive children had detectable HIV-1 gag and pol sequences. None of the 16 seronegative mothers nor 27 seronegative or Western blot-indeterminate children had detectable HIV-1 sequences. Our results suggest that non-isotopic PCR methods are sensitive, specific, and potentially useful in the early diagnosis of HIV-1 infection in developed and developing countries.

Human humoral immunity to hsp70 during Trypanosoma cruzi infection.

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli.

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.

A Mr 90 000 surface polypeptide of Trypanosoma cruzi as a candidate for a Chagas' disease diagnostic antigen.

Trypanosoma cruzi (Peru strain) trypomastigotes and epimastigotes were biosynthetically labeled with [35S]methionine, and the proteins were analyzed by two dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE analysis of the trypomastigotes showed a complex array of polypeptides with distinct clusters at Mr 88 000-92 000, isoelectric point (pI) 5.6-6.0, and Mr 72 000-76 000, pI 5.6-5.8. 2D-PAGE analysis of the epimastigotes did not show the cluster of polypeptides at Mr 90 000. When the trypomastigote lysate was reacted with sera from either mice or humans chronically infected with T. cruzi, 10-50 polypeptides were immunoprecipitated. Five of these polypeptides were recognized by all sera tested. However, of these polypeptides, only three, two of Mr 90 000 and one of Mr 150 000, can be identified by immunoreaction of [35S]methionine-labeled live parasites as surface proteins of T. cruzi trypomastigotes. 125I-iminobiotinylated surface proteins isolated from T. cruzi trypomastigotes were immunoprecipitated with the same series of sera as described above. Chagasic sera immunoprecipitated an antigen of Mr 90 000. The [35S]methionine and 125I-labeled Mr 90 000 polypeptides were not immunoprecipitated with sera from individuals infected with Leishmania donovani, Leishmania braziliensis, Leishmania tropica or Leishmania mexicana. These data indicate that a surface polypeptide of Mr 90000, pI 5.8-5.9 is a viable candidate for a Chagas' disease diagnostic antigen.