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To meet the testing demands and overcome supply chain issues during the SARS-CoV-2 pandemic, many clinical laboratories validated multiple SARS-CoV-2 molecular testing platforms. Here, we compare three different molecular assays for SARS-CoV-2 that received emergency use authorization (EUA) from the U.S. Food and Drug Administration. In order to determine the agreement among Roche cobas® SARS-CoV-2 Test (Cobas), Abbott RealTime SARS-CoV-2 assay (ART), and Mayo Clinic Laboratory SARS-CoV-2 Molecular Detection Assay (Mayo LDT), 100 each of anterior nares (AN), nasopharyngeal (NP), oropharyngeal (OP), and NP+OP swabs were tested on each platform. The consensus result was defined as agreement by 2 or more methods. Furthermore, 30 positive NP swabs from each molecular platform (n = 90 total) were tested on the three platforms to determine the PPA among positive samples. ART platform called more specimens positive than the other two platforms. All three assays performed with greater than 90% agreement for NP specimens throughout the study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cytomegalovirus (CMV) viral loads overall were 0.29 log IU/mL higher with cobas CMV for use on the cobas 6800/8800 System (cobas CMV) compared with Cobas AmpliPrep/Cobas TaqMan CMV Test (CAP/CTM CMV). Cytomegalovirus DNAemia was detected 11.5 days earlier by cobas CMV, whereas clearance was delayed by 12.8 days. Cytomegalovirus remained detectable by cobas CMV in 44.2% of patients at the time of viral clearance as determined by CAP/CTM CMV. Undetectable viral load by cobas CMV at end of treatment was associated with reduced risk for retreatment (odds ratio, 0.26; 95% confidence interval, 0.04-0.99; P = .05). The use of different quantitative cytomegalovirus nucleic acid tests may affect direct patient care as a result of significant differences in reporting the degree of CMV DNAemia and the time to first detection and clearance of CMV DNAemia., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Hepatitis virus C (HCV) genotype (GT) determination and subtype (ST) differentiation (1a versus 1b) remain important for the selection of appropriate direct-acting antiviral (DAA) therapy., Objectives: This study is a retrospective comparison of HCV GT and ST result distribution when using the Abbott RealTime HCV Genotype II assay (HCVGT II) alone and in combination with the Abbott RealTime HCV Genotype Plus RUO assay (HCVGT Plus) for routine testing of clinical serum specimens at a reference laboratory., Study Design: HCVGT II results of specimens tested from June 2014 through January 2016 (period 1) were compared with combined results from HCVGT II and HCVGT Plus (HCVGT II/Plus) performed from January 2016 through January 2017 (period 2)., Results: A total of 44,127 and 25,361 specimens were tested during periods 1 and 2, respectively. Use of HCVGT II/Plus significantly reduced the frequency of GT 1 results without ST (0.4%) when compared to preliminary HCVGT II results during period 2 (5.3%; p < 0.01) and final HCVGT II results in period 1 (5.5%; p < 0.01). HCVGT II/Plus also resulted in GT 6 reactivity in 38 specimens with results of "HCV detected" (n = 17) or GT 1 (n = 21) following initial HCVGT II testing during period 2., Conclusions: When compared to the use of HCVGT II alone, HCVGT II/Plus significantly reduced the frequency of GT 1 without ST results observed in a large reference laboratory, while also enabling the identification of HCV GT 6., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cytomegalovirus (CMV) pneumonia causes major morbidity and mortality. Its diagnosis requires demonstration of viral cytopathic changes in tissue, entailing risks of lung biopsy. This study aimed to determine CMV viral load (VL) thresholds in bronchoalveolar lavage fluid (BALF) for diagnosis of CMV pneumonia in immunocompromised patients. CMV VL in BALF was studied in 17 patients (83% transplant recipients) and 21 control subjects with and without CMV pneumonia, respectively, using an FDA-approved PCR assay (Cobas ® AmpliPrep/Cobas TaqMan ® CMV Test, Roche Molecular Systems, Inc.) calibrated to the WHO International Standard for CMV DNA (NIBSC: 09/162). Receiver operating characteristic curve analysis produced a BALF CMV VL threshold of 34 800, IU/mL with 91.7% sensitivity and 100.0% specificity for diagnosis of possible, probable, and proven CMV pneumonia in transplant patients, while a threshold of 656 000 IU/mL yielded 100% sensitivity and specificity among biopsy-proven cases. For all immunocompromised patients, a VL threshold of 274 IU/mL was selected. VL thresholds also were normalized to BALF cell count yielding a threshold of 0.32 IU/10 6 cells with 91.7% sensitivity and 90.5% specificity for possible, probable, and proven CMV pneumonia in transplant recipients. Monitoring CMV VL in BALF may be a less invasive method for diagnosing CMV pneumonia in immunocompromised patients., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Quantification of cytomegalovirus (CMV) deoxyribonucleic acid (DNA) has important diagnostic, prognostic, and therapeutic implications in the management of transplant recipients. We aimed to assess a viral load in plasma and whole blood that distinguishes CMV disease from asymptomatic infection in a cohort of solid organ and hematopoietic stem cell transplantation., Methods: We prospectively measured and compared CMV viral load in paired plasma and whole blood samples collected from transplant recipients with CMV infection and disease. Cytomegalovirus viral loads were determined by a commercially available US Food and Drug Administration-approved quantitative assay (COBAS AmpliPrep/COBAS TaqMan CMV Test [CAP/CTM CMV]) calibrated to the first World Health Organization International Standard for CMV DNA quantification., Results: Moderate agreement of CMV viral load was observed between plasma and whole blood, with 31% of samples having discordant findings, particularly among samples with low DNA levels. Among the subset of samples where both paired samples had quantifiable levels, we observed a systematic bias that reflected higher viral load in whole blood compared with plasma. Based on receiver operating curve analysis, an initial plasma CMV viral load threshold of 1700 IU/mL in solid organ transplant recipients (sensitivity 80%, specificity 74%) and 1350 IU/mL in allogeneic hematopoietic stem cell transplant recipients (sensitivity 87%, specificity 87%) distinguished CMV disease and asymptomatic infection., Conclusions: This study identifies standardized viral load thresholds that distinguish CMV disease from asymptomatic infection using CAP/CTM CMV assay. We propose these thresholds as potential triggers to be evaluated in prospective studies of preemptive therapy. Plasma was better than whole blood for measuring viral load using the CAP/CTM CMV assay.

Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA., (Copyright © 2017 American Society for Microbiology.)

Diversity of human immunodeficiency virus type 1 (HIV-1) has important implications for the diagnosis, treatment, and management of HIV-1-infected individuals. HIV-1 pol sequences from 3895 clinical plasma specimens collected in the United States over a 1-year period and submitted for routine HIV-1 genotypic drug resistance testing were retrospectively analyzed for HIV-1 subtype. Of these 3895 HIV-1 sequences, 207 (5.31%) were determined to be non-B subtypes (including recombinant forms). Among individual states, the percentage of non-B subtypes ranged from 0% (12 states) to 28.57% in South Dakota, with 7 states having percentages of >10%. All 4 states with the highest percentages of non-B subtypes were located within the US West North Central region: Minnesota, 11.82%; Iowa, 15.38%; North Dakota, 25.00%; and South Dakota, 28.57%. Reasons for the unexpectedly wide diversity of HIV-1 subtypes present in multiple states located in the West North Central region of the United States remain to be determined., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Testing of clinical plasma specimens by the COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV), COBAS AMPLICOR CMV MONITOR Test (CAM CMV), and a laboratory-developed assay using analyte-specific reagents (LC CMV) demonstrated substantial result bias for CAP/CTM CMV versus CAM CMV (r = 0.436) and CAP/CTM CMV versus LC CMV (r = 0.773)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Abbott HBV RUO Sequencing assay (Abbott Molecular Inc., Des Plaines, IL), which combines automated sample processing, real-time PCR, and bidirectional DNA sequencing, was evaluated for detection of nucleos(t)ide analogue (NA) resistance-associated mutations located in the hepatitis B virus (HBV) polymerase (Pol) gene. Interpretive software from the assay manufacturer was modified to allow interrogation of the overlapping HBV surface (S) gene sequence for HBV genotype determination and detection of immune escape mutations. Analytical sensitivity (detection and sequencing) of the assay was determined to be 103.9 IU/ml (95% confidence interval [CI], 80.0 to 173.3) for HBV genotype A. Testing of commercially available HBV genotype panels consisting of 23 individual members yielded complete agreement between expected results and results obtained from the laboratory-developed HBV genotype library. Excellent specificity was observed among clinical specimens with serologic or molecular markers for various unrelated blood-borne viruses (n = 6) and sera obtained from healthy, HBV-negative blood donors (n = 20). Retrospectively selected clinical specimens tested by a commercial reference laboratory HBV sequencing assay (n = 54) or the Trugene HBV Genotyping kit (n = 7) and the Abbott HBV RUO Sequencing assay showed minor differences in detection and reporting of NA resistance-associated mutations in 7 of 61 (11.5%) specimens but complete agreement of genotype results. The Abbott HBV RUO Sequencing assay provided a convenient and efficient assay workflow suitable for routine clinical laboratory use, with the flexibility to be modified for customized detection of NA resistance-associated mutations, HBV genotype determination, and detection of immune escape mutations from a single contiguous HBV sequence.

Hepatitis C virus (HCV) genotype (GT) distribution and frequency were studied among 22,407 unique specimens tested at a national reference testing laboratory. Subjects with HCV GT 3 were younger (P < 0.0001) than those with GT 1, 2, or 4, and the regional frequencies of HCV GT 2 and 3 ranged from 19.9% to 29.2%.

No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

A prototype, real-time reverse-transcription PCR assay, based on MultiCode-RTx technology, quantifying hepatitis C virus (HCV) RNA by targeting the HCV 3' untranslated region demonstrated linearity over 7 logs, with a good correlation between the quantitative results of this assay and the results of two commercially available comparator assays for 466 clinical specimens comprising all six HCV genotypes.

A retrospective examination of quantitation standard growth curves associated with 1,000 unique clinical serum specimens tested by a laboratory-developed TaqMan hepatitis C virus analyte-specific reagent-based assay revealed anomalous growth curves associated with 0.40% (95% confidence interval, 0.11% to 1.00%) of these specimens.

The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay (ART) and the Cobas AmpliPrep/Cobas TaqMan HIV-1 test (CTM) are commercially available assays for quantification of HIV-1 RNA in plasma. We evaluated performance characteristics, workflow, throughput, reliability, and direct costs of these assays. Both assays yielded good correlation of quantitative results (r = 0.95) among clinical specimens, with a mean difference of -0.34 log(10) copies/ml. Testing of healthy donor plasma specimens yielded "target not detected" results by ART, with "HIV-1 RNA detected, <40 copies/ml" results for 3.3% (3 of 90 samples) of these specimens by CTM. Both the m2000sp/m2000rt (ART) and docked CAP/CTM96 (CTM) instrument systems were capable of operating with continuous, uninterrupted workflow. When daily maintenance and cleaning were included, ART and CTM run durations (5 h 52 min and 6 h 4 min, respectively) and hands-on times (53 min and 46 min, respectively) were similar for a run batch size of 24. While ART was more flexible in terms of run batch size, CTM required fewer user interventions and consistently produced higher specimen throughput rates at 8, 16, and 24 h. Assay run failure rates were 6.3% (1 of 16 runs) and 4.2% (1 of 24 runs) for ART and CTM, respectively (P = 1.000), with invalid specimen result rates of 1.0% (5 of 495 specimens) and 2.8% (11 of 399 specimens), respectively (P = 0.073). Direct reagent and consumable costs for each assay were comparable (difference of <10%). In selecting an assay for implementation, laboratories should consider how various assay and instrument features might impact laboratory operation and patient care.

The COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5 (CAP/CA), and the COBAS AMPLICOR HIV-1 MONITOR Test, version 1.5, were compared. CAP/CA reduced and consolidated labor while modestly increasing assay throughput without increased failure rates or direct costs, regardless of batch size and assay format.

Background: The AMPLICOR HIV-1 DNA Test, version 1.5 (AMP HIV-1 DNA 1.5), is a new commercially available PCR assay for the detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in human whole blood., Objective: This study evaluates the performance characteristics of the assay following automated sample processing by the MagNA Pure LC instrument (MP)., Study Design: Analytical sensitivity and reproducibility were assessed by testing replicate HIV-1 DNA dilution panels over 5 days. Clinical sensitivity and specificity were studied among 28 HIV-1 DNA-positive clinical specimens, 60 specimens from healthy blood donors, and 63 specimens from HIV-1-seropositive patients with HIV-1 RNA plasma levels ranging from <50 to >100,000 copies/mL., Results: Following MP sample processing, the assay yielded an analytical sensitivity (95% detection rate) of 66.3 copies/mL (95% CI, 50.7-106.8), with clinical sensitivity and specificity of 100%., Conclusions: MP is a reliable, labor-saving platform capable of processing specimens for AMP HIV-1 DNA 1.5. When combined with MP sample processing, AMP HIV-1 DNA 1.5 is a sensitive and reproducible assay for the detection of HIV-1 DNA in clinical whole blood specimens. However, the current AMP HIV-1 DNA 1.5 kit configuration may result in inefficient utilization of reagents.

Background: INNO-LiPA HBV Genotyping (LiPA HBV GT) and INNO-LiPA HBV PreCore (LiPA HBV PC) are commercially available assays for hepatitis B virus (HBV) characterization. These assays are labor-intensive and may be prone to exogenous DNA contamination due to their use of nested PCR amplification procedures and lack of contamination control measures., Objective: Standardized, single-round INNO-LiPA PCR amplification protocols incorporating uracil N-glycosylase and automated sample processing by the MagNA Pure LC instrument were evaluated., Study Design: HBV standards containing 10,000, 1000, 100, 10, and 0 IU/mL were analyzed to determine the analytical sensitivity and reproducibility of these modified procedures. One hundred clinical serum specimens with viral titers ranging from 390 to 16,900,000 IU/mL were tested by modified LiPA HBV GT, while 34 specimens with viral titers ranging from 378 to 11,600,000 IU/mL were tested by modified LiPA HBV PC., Results: Modified LiPA HBV GT and LiPA HBV PC each yielded analytical sensitivities of 100% at an HBV DNA level of 1000 IU/mL and 90% at a level of 100 IU/mL. Among clinical specimens, success rates for both INNO-LiPA procedures were > or =94%., Conclusions: Both modified INNO-LiPA procedures were sensitive and reproducible, with improved efficiency and suitability for routine laboratory use.

TaqMan hepatitis B virus (HBV) analyte-specific reagent (ASR; Roche Molecular Systems, Inc., Branchburg, NJ) is designed for the quantification of HBV DNA in serum or plasma. The performance characteristics of TaqMan HBV ASR following automated sample processing with the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, IN) were evaluated in this study. Analytical sensitivity and precision were assessed with commercially available HBV standards, while clinical serum specimens from HBsAg-seropositive patients and healthy blood donors were used to determine clinical sensitivity, specificity, and correlation with other commercially available assays. Analytical studies yielded a limit of detection of 2.4 IU/ml, with good linearity and correlation (R(2) = 0.9958) with expected HBV DNA titers over a wide range (6.0 x 10(0) to 2.1 x 10(8) IU/ml). Clinical sensitivity and specificity of the assay combined with automated sample processing were both 100%. Comparison of TaqMan HBV ASR and VERSANT HBV DNA 3.0 assay (bDNA; Bayer HealthCare LLC, Tarrytown, NY) results among clinical specimens yielded good correlation (R(2) = 0.9237), with a mean difference in titer of -0.213 log(10) IU/ml (95% confidence interval, -0.678 to 1.10 log(10) IU/ml). The overall test failure rate was 2.0% among 204 clinical serum specimens tested. Total time required for MP sample processing and automated postelution handling of 24 samples was 224 min, with 57 min of actual hands-on time. MP is a reliable, labor-saving platform suitable for use with TaqMan HBV ASR, providing sensitive and accurate quantification of HBV DNA levels over a range of 8 log(10) IU/ml.

The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5' noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (>/=90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 x 10(7) IU/ml) showed 98% concordance with results obtained from the TRUGENE HCV 5' NC Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5' NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.

Background: The current manual sample processing method recommended for use with the TRUGENE HBV Genotyping Kit (TRUGENE HBV; Bayer HealthCare LLC, Tarrytown, NY) is labor-intensive and may be prone to specimen cross-contamination. Recent evaluations of the MagNA Pure LC (MP; Roche Applied Science, Indianapolis, IN) suggest that it is suitable for automated, contamination-free extraction and purification of viral nucleic acids from large-volume (1.0 mL) serum or plasma specimens., Objectives: We evaluated the MP Total Nucleic Acid Isolation Kit--Large Volume (Roche Applied Science) in conjunction with TRUGENE HBV to establish the analytical sensitivity (threshold titer) of the assay, in HBV DNA International Units (IU)/mL, for obtaining consistent, interpretable sequence data from TRUGENE HBV., Study Design: HBV analytical standards, prepared as 10 replicates (1.0 mL each) at each of the following concentrations: 200, 1000, 5000, and 10,000 IU/mL, were processed by MP and analyzed by TRUGENE HBV according to manufacturer's instructions. Performance of TRUGENE HBV used in conjunction with MP sample processing was evaluated further using 22 clinical serum specimens containing low titers of HBV DNA., Results: All replicates of HBV analytical standards at 1000, 5000, and 10,000 IU/mL yielded interpretable TRUGENE HBV sequences, whereas interpretable sequences were obtained in 90% (9 of 10) of the replicates at 200 IU/mL. TRUGENE HBV sequences were interpretable in 86% (19 of 22) of the clinical specimens studied., Conclusions: MP sample processing is efficient and suitable for use with TRUGENE HBV. When combined with MP sample processing, TRUGENE HBV yielded interpretable sequences from HBV analytical standards and clinical serum specimens with HBV DNA titers of > or =200 IU/mL.

The COBAS TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.) for hepatitis C virus (HCV) performed on the COBAS TaqMan 48 Analyzer (Roche Molecular Systems) currently relies on a manual sample processing method. Implementation of an automated sample processing method would facilitate the clinical use of this test. In this study, we evaluated the performance characteristics of TaqMan HCV following automated sample processing by the MagNA Pure LC instrument (MP; Roche Applied Science, Indianapolis, Ind.). The analytical sensitivity of TaqMan HCV following sample processing by MP was 8.1 IU/ml (95% confidence interval, 6.1 to 15.2). The assay showed good linearity (R(2) = 0.99) across a wide range of HCV RNA levels (25 to 5 x 10(6) IU/ml), with coefficients of variation ranging from 10% to 46%. Among 83 clinical specimens, the sensitivity and specificity of TaqMan HCV were 100% and 95%, respectively, when compared to the COBAS AMPLICOR hepatitis C virus test, version 2.0 (COBAS AMPLICOR; Roche Molecular Systems), with TaqMan HCV detecting two more HCV RNA-positive specimens than COBAS AMPLICOR. Both specimens were confirmed to be HCV RNA positive by the VERSANT HCV RNA qualitative test (Bayer HealthCare LLC, Tarrytown, N.Y.). There was also strong correlation (R(2) = 0.95) and good agreement between the results from TaqMan HCV and the VERSANT HCV RNA 3.0 assay (bDNA) (Bayer HealthCare LLC) among a group of 93 clinical specimens. The MP is a versatile, labor-saving sample processing platform suitable for reliable performance of TaqMan HCV.

Use of the MagNA Pure LC Total Nucleic Acid Isolation Kit-Large Volume in conjunction with the TRUGENE HIV-1 Genotyping Kit yielded consistent, interpretable sequence data from 1-mL plasma preparations containing HIV-1 RNA concentrations of > or =400 copies/mL. This finding was confirmed in 18 of 24 low-titer clinical specimens.

Ethnic differences in the outcome of hepatitis C have been described. Our aim was to investigate ethnic differences in the distribution of genotypes associated with polymorphisms of the tumor necrosis factor-alpha promoter, interleukin-10 promoter, and transforming growth factor-beta1 leader sequence in patients with hepatitis C. Genomic DNA was obtained from 71 Egyptians and 67 Caucasians (hepatitis C and control patients). Amplification of appropriate gene segments was followed by direct sequencing. Infrequently occurring polymorphisms were identified at positions -244 and -77 of the tumor necrosis factor-alpha promoter and at positions -851 and -657 of the interleukin-10 promoter. The G/A genotype associated with tumor necrosis factor-alpha promoter positions -376 and -244 was more frequent in Egyptians (P =0.001 and P =0.004, respectively). The -244 G/A genotype occurred only in healthy Egyptians (P =0.024). Thus, ethnic differences in the distribution of genotypes of the tumor necrosis factor-alpha promoter exist, which may have clinical implications on the outcome of hepatitis C.

Hepatitis C virus (HCV)-infected patients were tested for the presence of HCV RNA using two qualitative assays at various time points during interferon-ribavirin therapy. Among patients treated for 48 weeks, transcription-mediated amplification and the COBAS AMPLICOR Hepatitis C Virus Test results at Week 24 predicted subsequent virologic non-response or virologic relapse in 12/15 (80%) and 8/15 (53%) patients, respectively.

The TRUGENE HCV 5'NC genotyping kit (GeneLibrarian modules 3.1.1 and 3.1.2) and VERSANT HCV genotyping assay were compared by using 96 hepatitis C virus (HCV) RNA-positive patient specimens, including HCV genotypes 1, 2, 3, 4, 5, 6, and 10. The TRUGENE HCV 5'NC genotyping kit (GeneLibrarian module 3.1.2) yielded the most accurate genotyping results.

The COBAS AMPLICOR system has played a major role in the transition of molecular diagnostics from research to routine clinical laboratory use by automating the nucleic acid amplification and detection processes. However, sample preparation remains a labor-intensive portion of the procedure. In this study, we evaluated the performance of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, N.J.) following manual hepatitis C virus (HCV) RNA extraction versus automated extraction with the MagNA Pure LC instrument (Roche Applied Science, Indianapolis, Ind.). Parallel replicate testing was performed with standard dilutions of 100, 75, 60, and 0 HCV IU/ml and 153 clinical specimens. An analytical sensitivity of 75 IU/ml was achieved with either the manual or the standard-volume (200 microl) automated extraction methodologies (25 of 26 [96.2%]; 95% confidence interval [95% CI], 80.4 to 99.9), whereas the clinical sensitivity and specificity were both 100% with either extraction method. A large-volume (1 ml) automated extraction method was also evaluated with standard dilutions of 40, 25, 10, and 0 IU/ml and the same 153 clinical specimens. The analytical sensitivity of the COBAS AMPLICOR assay with the large-volume extraction method was 25 HCV IU/ml (26 of 26 [100%]; 95% CI, 86.8 to 100), whereas the clinical sensitivity and specificity were both 100%. The MagNA Pure LC instrument is a versatile, labor-saving platform capable of integration with minimal modification of the existing assay procedure. The increased sensitivity of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 performed in conjunction with large-volume HCV RNA extraction may be important in HCV diagnostic testing as new therapeutic strategies evolve.

We evaluated serum samples from 18 chronic hepatitis B virus (HBV) patients who underwent liver transplantation for the presence of HBV polymerase and S gene mutations and HBV genotype using a new commercially available sequencing assay. All three patients with hepatitis B immune globulin (HBIG) treatment failure followed by nucleoside analogue treatment failure were infected with HBV genotype C; a pre-existing HBV S antigen (HBsAg) mutation (sD144A) was identified in one patient pretransplant, while sG145R mutations emerged in the other two patients post-transplant. These HBsAg mutations persisted for the duration of the study (5-6 years), despite the absence of HBIG administration for a 4-5-year period. Significant viral polymerase mutations (rtL180M and rtM204I/V) also emerged in all of these patients following treatment with lamivudine and/or famciclovir. Four of six patients with HBIG breakthrough without nucleoside analogue treatment failure yielded potentially significant HBsAg mutations post transplant. These data do not support previous reports highlighting the disappearance of HBsAg mutants in liver transplant recipients after discontinuation of HBIG. Determination of HBV genotype, as well as identification of HBV polymerase and S gene mutations in liver transplant candidates may be warranted to optimize HBV management strategies post transplant.

Increased severity of disease and persistence of symptoms have been recently reported in some patients with simultaneous infection of Borrelia burgdorferi and Babesia microti in the northeastern and northern midwest United States. This study used a murine model to examine whether defined disease conditions such as arthritis and carditis differed in severity in mice infected solely with B. burgdorferi and in mice coinfected with B. microti and B. burgdorferi. C3H.HeJ and BALB/c mice cohorts were coinfected or singly infected and then monitored experimentally for 15 and 30 days after inoculation. Carditis and arthritis was determined by blinded histopathologic evaluation of myocardium and tibiotarsal joints. Cytokine measurements were made on lymph node and spleen supernatants for interferon-gamma, interleukin (IL)-4, IL-10, and IL-13. No differences were observed for C3H.HeJ mice cohorts; however, coinfected BALB/c mice had a significant increase in arthritis severity at day 30. This clinical observation was correlated with a significant reduction in expression of the cytokines IL-10 and IL-13.

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (P = 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (P < or = 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (P < or = 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays.

Background/aims: In hepatitis C infection, the production of inappropriate cytokine levels appears to contribute to viral persistence and to affect the response to antiviral therapy. Additionally, polymorphisms in the cytokine genes may affect the production of the cytokines. In this study, we determined the frequency of the genotypes associated with polymorphisms of the interleukin-10 and tumor necrosis factor-alpha gene promoters, and transforming growth factor-beta 1 gene leader sequence, and investigated their association with clinical features and the response to interferon-alpha and ribavirin therapy in chronic hepatitis C infection., Methods: Genomic DNA from 80 patients and 37 racially matched healthy controls was studied by polymerase chain reaction and direct automated sequencing., Results: The interleukin-10 -1082 G/G genotype was identified more frequently in patients than in controls (P=0.048). The transforming-growth factor-beta 1 +29 (codon 10) C/C genotype was associated with resistance to the therapy (P=0.029). After adjusting for potential confounding variables, patients exhibiting the C/C genotype were less likely to respond to treatment than patients with the T/T or T/C genotypes., Conclusions: These results suggest that inheritance of the interleukin-10 -1082 G/G and the transforming growth factor-beta 1 +29 C/C genotypes, which appear to affect the cytokine production, may be associated with susceptibility to chronic hepatitis C infection and resistance to combined antiviral therapy.

Serologic assays for diagnosis of hepatitis C infection may yield indeterminate results despite improvements in sensitivity and specificity through second- and third-generation assays. Direct detection of hepatitis C virus (HCV) RNA based on qualitative reverse transcription-polymerase chain reaction or transcription-mediated amplification allows diagnosis in the early stages of acute infection and in patients unable to mount an antibody response. Quantitative HCV RNA assays are useful for selecting appropriate antiviral therapies, but until recently they have lacked comparability between tests. More sensitive qualitative assays should be used for determining duration of treatment or recognizing a sustained virologic response to therapy. Hepatitis C virus genotyping can be performed from a limited sequence analysis of the viral genome by using various techniques. Although newer genotyping methods are relatively practicable and are satisfactory for the discrimination of the majority of genotypes, discrimination between subtypes can be challenging. Serologic typing of HCV lacks sensitivity and specificity compared with molecular-based techniques. Recent advances in serologic assays and nucleic acid detection techniques allow physicians to make accurate diagnoses, and these assays serve as important tools in treatment planning.

Infection of different strains of laboratory mice with the agent of Lyme disease, Borrelia burgdorferi, results in arthritis, the severity of which has been correlated with the dominance of Th1 cytokines. In this study, we demonstrate that changes in B. burgdorferi-specific immunologic responses associated with pregnancy can alter the outcome of Lyme arthritis in mice. Whereas nonpregnant female C3H mice consistently developed severe Lyme arthritis, pregnant mice had a marked reduction in arthritis severity that was associated with a slight reduction in IFN-gamma and markedly increased levels of IL-4 production by B. burgdorferi-specific T cells. Similar reductions in arthritis severity and patterns of cytokine production were observed in nonpregnant, progesterone-implanted mice. Ab neutralization of IL-4 in progesterone-implanted mice resulted in severe arthritis. Our results are consistent with the known shift toward Th2 cytokine expression at the maternal-fetal interface, and are the first to show a pregnancy-related therapeutic effect in an infectious model.

Objective: Because many patients with chronic viral hepatitis do not progress to end-stage liver disease, it is possible that host factors such as human leukocyte antigen (HLA) differences are important. Our aims were to determine HLA marker-specific rates of progression to liver transplantation among patients with chronic hepatitis C; and to determine if polymerase chain reaction (PCR)-based HLA DRB1 typing can be performed on stored serum samples., Methods: Forty-two hepatitis C virus RNA-positive liver transplant patients and 87 untransplanted patients were included in a Cox proportional hazards model to test whether the occurrence of certain HLA DRB1 markers were associated with progression to liver transplantation. HLA DRB1 typing was performed on stored serum samples using a PCR method., Results: There were no differences among the HLA DRB1 markers with regard to the HLA marker-specific rate of progression to transplantation among patients with chronic hepatitis C., Conclusions: HLA DRB1 markers do not appear to be associated with progression of disease in chronic viral hepatitis C. It is possible to perform PCR-based HLA DRB1 typing on stored frozen serum samples.

After orthotopic liver transplantation (OLT), patients with chronic hepatitis C virus (HCV) infection show nearly universal persistence of viremia and reinfection of the liver, but identifying the point at which the liver is reinfected morphologically can be difficult. One tool that may potentially be useful to detect reinfection is reverse transcriptase-polymerase chain reaction (RT-PCR), which has proven to be highly sensitive for detecting HCV RNA in formalin-fixed paraffin-embedded liver tissue. Our purpose was to gain insight into the time frame of HCV reinfection by assaying for HCV RNA in serial posttransplant liver biopsy specimens. Our study population consisted of 14 patients who underwent liver transplantation for hepatitis C and had confirmed HCV RNA in pretransplant serum, absence of HCV RNA in donor livers, and available consecutive posttransplant liver allograft specimens. We performed RT-PCR for HCV RNA in serial posttransplant liver biopsy specimens, beginning at 1 week until at least one biopsy from each tested positive. HCV RNA was detected in liver tissue by RT-PCR in 1-week post-OLT liver samples in 6 of 14 (42.8%) patients, the earliest being 5 days post-OLT. Eventually, each of the remaining eight samples became RT-PCR positive as well; the first detections occurred in these at 3 weeks (three cases), 4 weeks (three cases), 48 weeks (one case), and 144 weeks (one case). Histologic identification of hepatitis C recurrence was relatively insensitive in relation to these molecular data. These data suggest that (1) HCV RNA reinfection is nearly universal after liver transplantation in patients with chronic hepatitis C infection, (2) molecular reinfection by HCV occurs at a variable interval post-OLT, with the majority of allograft livers reinfected as early as 1 week, and (3) morphologic features of hepatitis C are usually appreciable at the time of "molecular" recurrence.

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Consistent with other members of the family Flaviviridae, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5' untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5'UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5'UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2. 0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5'UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5'UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.

Background: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV., Methods: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells., Results: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells., Conclusion: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

Objectives: Hepatitis G virus (HGV) is a recently discovered member of the flavivirus family that has been associated with acute and chronic hepatitis. HGV infection has been reported to coexist in 10-20% of patients with chronic hepatitis C. The significance of simultaneous infection with HGV and hepatitis C virus (HCV) remains to be clarified, as do the effects on HGV of therapeutic interventions such as interferon treatment or liver transplantation., The Aims of Our Study Were: 1) to examine the frequency of HGV infection in the settings of liver transplantation and interferon therapy for hepatitis C; and 2) to compare HGV RNA levels before and after liver transplantation or interferon treatment., Methods: Pre-treatment sera were available in 65 patients with chronic hepatitis C treated with interferon; pretransplant sera were available in 49 patients transplanted for end stage liver disease associated with chronic hepatitis C. Information collected included age, sex, risk factors for hepatitis, concurrent liver disease, patient and allograft survival, biochemical response to interferon, histological activity index, and degree of fibrosis/cirrhosis. HCV genotyping was performed by sequencing the NS-5 region. HGV quantitation was performed using a research-based branched DNA (bDNA) assay with a set of probes directed at the 5' untranslated region., Results: HGV was detected in 10 of 49 patients (20%) before transplant and in 13 of 65 patients (20%) treated with interferon. There was a female predominance among HGV-positive compared with HGV-negative transplant patients (80% vs 20%; p < 0.01), but such a difference was not observed in the interferon-treated group. Hepatic iron concentration was lower in hepatic explants from patients who were HGV-positive than in those who were HGV-negative (318 +/- 145 microg/g dry weight vs 1497 +/- 2202 microg/g dry weight; p = 0.02). HCV exposure after 1980 was more common in the HGV-positive patients than in those who were HGV-negative for the entire study population (10 of 20 [50%] vs 16 of 66 [24%]; p = 0.03), as well as for the nontransplant subgroup (8 of 12 [67%] vs 12 of 39 [31%]; p = 0.03). HGV RNA levels declined at 1 yr after transplant in seven of eight patients. Among nine patients tested during or after interferon treatment, HGV RNA levels declined from pretreatment levels in all and disappeared in three., Conclusions: Among patients with chronic hepatitis C treated with either interferon or liver transplantation, the frequency of coinfection with HGV is about 20%. HGV may be a more recent virus in the US than HCV. Coinfection with HGV does not appear to affect the likelihood of response to interferon in patients with hepatitis C. Finally, HGV RNA levels appear to decline after both liver transplantation and interferon therapy, suggesting possible suppression by increased HCV replication in the former case, and a possible drug treatment effect in the latter.

Chronic infection with the hepatitis C virus (HCV) is the most common reason for liver transplantation. We examined the results of laboratory tests for HCV on a cohort of patients who received a liver transplant between 1990 and 1994 at three large centers. Seven hundred twenty-two recipients and 604 donors were tested for antibody to HCV (anti-HCV) using a second-generation enzyme-linked immunoassay (EIA-2), followed by recombinant immunoblot (RIBA-2) and HCV RNA confirmation by reverse-transcription polymerase chain reaction (RT-PCR) (with genotyping and viral quantification). Diagnosis of posttransplantation infection required detection of serum HCV RNA that could be genotyped by sequencing or was repeatedly positive despite being unsequenceable. Twenty-five percent of transplantation candidates were seropositive for anti-HCV. Approximately 86% of anti-HCV-positive, 93% of RIBA-positive, and 97% of HCV RNA-positive candidates developed infection after transplantation. Pretransplantation HCV RNA was superior to RIBA-2 for predicting posttransplantation infection. Whereas HCV genotype was identified in nearly all candidates and changed little after transplantation, serum viral levels rose markedly after transplantation. Fifteen donors were either anti-HCV- or HCV RNA-positive. Recipients of grafts from donors with HCV RNA all developed infection, whereas infection was not detected in recipients of grafts from donors with anti-HCV but without detectable HCV RNA. The rate of new infection fell significantly (P =.02) after the introduction of EIA-2 screening of blood. Donor and candidate markers for HCV predict posttransplantation infection.

Currently, one of the major indications for liver transplantation is infection with hepatitis C virus (HCV). Many studies have suggested that recurrent infection with HCV is universal after transplantation. Fastidious techniques, such as reverse transcriptase-polymerase chain reaction (RT-PCR), have proved to be highly sensitive for detecting HCV RNA in serum and in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) liver tissue. In this study, we wanted to determine whether the identification of HCV RNA in liver tissue by RT-PCR might reflect the detection of circulating HCV RNA in blood within the tissue, rather than implying true tissue infection. We performed RT-PCR for HCV RNA in FFPE liver biopsy specimens taken from 14 donor allografts shortly before and immediately after implantation into recipients. The recipients were known to have HCV RNA in serum and explanted liver tissue, as determined by RT-PCR. We were unable to detect HCV RNA in any of the study samples, either before or after transplantation. In a related study, qualitative and quantitative HCV RNA analyses were performed by RT-PCR and branched DNA (bDNA) amplification, respectively, on serum samples collected pretransplantation and immediately posttransplantation from 10 other patients who underwent transplantation for hepatitis C. HCV RNA was detected in all serum samples before and after transplantation by RT-PCR; however, the bDNA assay detected HCV RNA in only 6 of 10 samples pre-orthotopic liver transplantation (OLT) and in none of the immediately post-OLT samples. In our system, despite the RT-PCR detection of HCV RNA in serum before and after the transplantation, HCV RNA is not detectable in the peripheral blood that accompanies formalin-fixed liver tissue. This implies that RT-PCR detection of HCV RNA in tissue reflects true liver infection, rather than contamination by HCV RNA in accompanying peripheral blood.

High prevalence of hepatitis C (HCV) and hepatitis G (HGV) viruses has been reported among hemodialysis patients with substantial heterogeneity of HCV genotypes throughout the world. We studied HCV prevalence, clinical significance, genotype distribution, and HGV coinfection in hemodialysis patients from Syria. Ninety (75%) of 120 screened patients were HCV antibody positive. Forty-nine (87.5%) of 56 HCV antibody-positive patients had HCV RNA detected by the polymerase chain reaction. The HCV genotyping was possible in 37 of 49 patients (76%). The HCV genotype distribution was genotype 1a, seven (19%); genotype 1b, 10 (27%); genotype 4a, 11 (30%); unmatched sequences, nine (24%). Phylogenetic analysis of unmatched sequences indicated that they represent two distinct and novel subtypes of HCV genotype 4. Hepatitis G virus RNA was detected in 29 (59%) of the HCV RNA-positive patients. No differences were identified between patients infected with HCV alone and those coinfected with HGV. These data demonstrate that HCV infection is common in this population with a genotype distribution predominantly made up of types 1 and 4. Coinfection with HGV had no effect on the outcome of HCV infection.

It has been suggested that prolonged formalin fixation and block storage adversely affect hepatitis C virus (HCV) ribonucleic acid (RNA) detection in tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). We attempted to determine whether short-term perfusion fixation (3-5 days) or prolonged formalin storage adversely affects the detection of HCV RNA in paraffin-embedded tissue in comparison with 24-h fixation. Also, we examined the effects of prolonged storage of paraffin blocks on the sensitivity for HCV detection. We performed RT-PCR in formalin-fixed explanted livers from 20 liver allograft recipients known to be HCV positive (10 with specimens stored for 2-4 years and 10 with specimens stored for > 4 years). We compared the results of perioperative needle liver biopsy specimens fixed overnight with liver sections fixed by perfusion for 3-5 days and bulk liver tissue stored in formalin for years (mean, 6.25 years; range, 2-11 years). HCV RNA was detected in 100%, 85%, and 0% of specimens fixed for 24 h, 3-4 days, and years, respectively. We conclude that HCV can be readily detected in tissue fixed by formalin overnight, sensitivity decreases slightly with intermediate-length fixation, and HCV is rendered undetectable by prolonged fixation. In addition, retention of formalin-fixed tissue in paraffin blocks does not affect the sensitivity of HCV detection.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.