Hepatitis C virus transmission between eight high-income countries among men who have sex with men: a whole-genome analysis.

BACKGROUND: Microelimination of the hepatitis C virus (HCV) among men who have sex with men (MSM) could be complicated by continuous external introductions and the emergence of phylogenetic clusters harbouring clinically significant resistance-associated substitutions (RAS). To investigate international clustering and the prevalence and transmission of RAS, we aimed to analyse whole-genome HCV sequences from MSM with a recently acquired infection who participated in a large, international HCV treatment trial. METHODS: For this whole-genome analysis, we obtained HCV sequences from 128 MSM who had acquired HCV within the past 12 months and were participating in the REACT trial. The participants from whom sequences were obtained were recruited at 24 sites in eight countries. We inferred maximum-likelihood phylogenies and identified transmission clusters for HCV genotypes separately. We constructed time-scaled phylogenies to estimate cluster introduction dates and used a Bayesian Skygrid approach to estimate the effective population size over the past 50 years. We calculated the prevalence of RAS and the extent of RAS transmission in the study population. FINDINGS: The majority of recent HCV infections were part of international networks that arose in the late 1990s and early 2000s. Sequences obtained in the same country clustered frequently, and in 36% of subclusters since 2015 we found evidence of international transmission. European MSM were more likely than non-European MSM to be in a cluster (odds ratio 11·9 [95% CI 3·6-43·4], p<0·0001). The effective population size decreased rapidly since around 2015 in Europe. RAS associated with substantially diminished cure rates were infrequently detected and transmission of highly resistant viruses was not observed. INTERPRETATION: Despite antiviral treatment becoming widely available, international transmission of HCV among MSM has still occurred over the past 8 years, which could complicate microelimination of the virus in this population. RAS-enriched clusters and widespread RAS transmission are currently not a threat to elimination goals. These findings support an international approach for HCV microelimination among MSM. FUNDING: National Institutes of Health and Dr. C.J. Vaillant Fonds.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection. Importance: With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

Epidemiologic and Genomic Analysis of SARS-CoV-2 Delta Variant Superspreading Event in Nightclub, the Netherlands, June 2021.

We report a severe acute respiratory syndrome coronavirus 2 superspreading event in the Netherlands after distancing rules were lifted in nightclubs, despite requiring a negative test or vaccination. This occurrence illustrates the potential for rapid dissemination of variants in largely unvaccinated populations under such conditions. We detected subsequent community transmission of this strain.

Erratum to: Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries: A Dutch Nationwide Cohort Study.

[This corrects the article DOI: 10.1093/ofid/ofab006.].

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries: A Dutch Nationwide Cohort Study.

BACKGROUND: The majority of hepatitis C virus (HCV) infections are found in low- and middle-income countries, which harbor many region-specific HCV subtypes. Nevertheless, direct-acting antiviral (DAA) trials have almost exclusively been conducted in high-income countries, where mainly epidemically spread HCV subtypes are present. Recently, several studies have demonstrated suboptimal DAA efficacy for certain nonepidemic subtypes, which could hamper global HCV elimination. Therefore, we aimed to evaluate DAA efficacy in patients treated for a nonepidemic HCV genotype infection in the Netherlands. METHODS: We performed a nationwide retrospective study including patients treated with interferon-free DAAs for an HCV genotype other than 1a/1b/2a/2b/3a/4a/4d. The genotype was determined by NS5B region phylogenetic analysis. The primary end point was SVR-12. If stored samples were available, NS5A and NS5B sequences were obtained for resistance-associated substitutions (RAS) evaluation. RESULTS: We included 160 patients, mainly infected with nonepidemic genotype 2 (41%) and 4 (31%) subtypes. Most patients were from Africa (45%) or South America (24%); 51 (32%) were cirrhotic. SVR-12 was achieved in 92% (140/152) of patients with available SVR-12 data. Only 73% (8/11) genotype 3-infected patients achieved SVR-12, the majority being genotype 3b patients with 63% (5/8) SVR. Regardless of SVR, all genotype 3b patients had 30K and 31M RAS. CONCLUSIONS: The DAA efficacy we observed in most nonepidemic genotypes in the Netherlands seems reassuring. However, the low SVR-12 rate in subtype 3b infections is alarming, especially as it is common in several HCV-endemic countries. Alongside earlier results, our results indicate that a remaining challenge for global HCV elimination is confirming and monitoring DAA efficacy in nonepidemic genotypes.

Dried blood spot self-sampling at home is a feasible technique for hepatitis C RNA detection.

To facilitate HCV diagnosis, we developed an HCV-RNA testing service, which involved home-sampled dried blood spots (DBS). The main objective of this study was to evaluate the feasibility of self-sampling at home. Furthermore, to optimise the processing of DBS samples for RNA detection, we evaluated two elution buffers: phosphate-buffered saline (PBS) and L6-buffer. 27 HCV-RNA and 12 HIV-1 RNA positive patients were included. Laboratory spotted DBS (LabDBS) were made by a technician from blood samples drawn at inclusion. Patients received a DBS home-sampling kit and were requested to return their self-sampled DBS (ssDBS) by mail. We compared the RNA load of PBS and L6-eluted labDBS, and of L6-eluted ssDBS, L6-eluted labDBS and plasma. LabDBS load measurements were repeated after 7-13 and 14-21 days to evaluate RNA stability. All 39 plasma samples provided quantifiable RNA loads. In 1/39 labDBS sample, RNA could not be detected (plasma HCV load: 2.98 log10 IU/ml). L6-eluted samples gave a 0.7 log10 and 0.6 log10 higher viral load for HCV and HIV-1 respectively, compared to PBS-eluted samples. Strong correlations were found between labDBS and ssDBS HCV RNA (r = 0.833; mean difference 0.3 log10 IU/mL) and HIV-1 RNA results (r = 0.857; mean difference 0.1 log10 copies/mL). Correlations between labDBS and plasma values were high for HCV (r = 0.958) and HIV-1 (r = 0.844). RNA loads in DBS remained stable over 21 days. Our study demonstrates that self-sampling dried blood spots at home is a feasible strategy for the detection of HCV and HIV-1 RNA. This could facilitate one-step diagnostics and treatment monitoring in communities with high HCV prevalence.

High frequency and diversity of parechovirus A in a cohort of Malawian children.

Parechoviruses (PeVs) are highly prevalent viruses worldwide. Over the last decades, several studies have been published on PeV epidemiology in Europe, Asia and North America, while information on other continents is lacking. The aim of this study was to describe PeV circulation in a cohort of children in Malawi, Africa. A total of 749 stool samples obtained from Malawian children aged 6 to 60 months were tested for the presence of PeV by real-time PCR. We performed typing by phylogenetic and Basic Local Alignment Search Tool (BLAST) analysis. PeV was found in 57% of stool samples. Age was significantly associated with PeV positivity (p = 0.01). Typing by phylogenetic analysis resulted in 15 different types, while BLAST typing resulted in 14 different types and several indeterminate strains. In total, six strains showed inconsistencies in typing between the two methods. One strain, P02-4058, remained untypable by all methods, but appeared to belong to the recently reclassified PeV-A19 genotype. PeV-A1, -A2 and -A3 were the most prevalent types (26.8%, 13.8% and 9.8%, respectively). Both the prevalence and genetic diversity found in our study were remarkably high. Our data provide an important contribution to the scarce data available on PeV epidemiology in Africa.

Carriage of Blastocystis spp. in travellers - A prospective longitudinal study.

INTRODUCTION: A lack of prospective and longitudinal data on pre- and post-travel carriage of Blastocystis spp. complicates interpretation of a positive test post-travel. Therefore we studied dynamics of Blastocystis carriage in a cohort of Dutch travellers. METHODS: From the prospective, multicentre COMBAT study among 2001 Dutch travellers, a subset of 491 travellers was selected based on travel destination to 7 subregions (70 or 71 travellers each). Faecal samples taken directly before and after travel were screened for Blastocystis with qPCR, followed, when positive, by sequence analysis to determine subtypes. RESULTS: After exclusion of 12 samples with missing samples or inhibited qPCR-reactions, stool samples of 479 travellers were analysed. Before travel, 174 of them (36.3%) carried Blastocystis and in most of these, the same subtype was persistently carried. However, in 48/174 of those travellers (27.6%; CI95 20.8-36.6%) no Blastocystis or a different subtype was detected in the post-travel sample, indicating loss of Blastocystis during travel. Only 26 (5.4%; CI95 3.7%-8.0%) of all travellers acquired Blastocystis, including two individuals that were already positive for Blastocystis before travel but acquired a different subtype during travel. DISCUSSION: This study shows that Blastocystis carriage in travellers is highly dynamic. The observed acquisition and loss of Blastocystis could either be travel-related or reflect the natural course of Blastocystis carriage. We demonstrate that the majority of Blastocystis detected in post-travel samples were already carried before travel.

Human Parechovirus 1, 3 and 4 Neutralizing Antibodies in Dutch Mothers and Infants and Their Role in Protection Against Disease.

BACKGROUND: Human parechoviruses (HPeVs) are common pathogens in young children, and in the Netherlands, HPeV1, HPeV3 and HPeV4 are the most frequently detected genotypes. HPeV3 in particular has been associated with severe disease in young infants below 3 months of age while the other genotypes more often infect older children and elicit mild symptoms. We investigated if maternal neutralizing antibodies (nAbs) against HPeV1, HPeV3 and HPeV4 protect young Dutch infants from severe disease related to HPeV infection. METHODS: We conducted a prospective case-control study of Dutch mother-infant pairs. Thirty-eight HPeV-infected infants and their mothers were included as cases, and 65 HPeV-negative children and their mothers as controls. RESULTS: In control infants, we observed nAb seropositivity rates of 41.4%, 33.3% and 27.6%, with median nAb titers of 1:16, 1:12 and 1:8, against HPeV1, HPeV3 and HPeV4, respectively. In control mothers, nAb seropositivity rates were 84.6%, 55.4% and 60.0% with median nAb titers of 1:128, 1:32 and 1:45 against HPeV1, HPeV3 and HPeV4, respectively. The HPeV3 nAb seroprevalence was significantly lower in HPeV3-infected infants and their mothers (0.0% with P < 0.05 and 10.0% with P < 0.001, respectively). In contrast, no differences in nAb seroprevalence against HPeV1 or HPeV4 could be detected between case and control infants or mothers. CONCLUSIONS: Our results suggest that young Dutch infants are protected against severe disease related to HPeV1 and HPeV4 by maternal nAbs, but less so against HPeV3 explaining the distinct age distributions and disease severity profiles of children infected with these HPeV genotypes.

High frequency of Polio-like Enterovirus C strains with differential clustering of CVA-13 and EV-C99 subgenotypes in a cohort of Malawian children.

Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of the samples was EV positive (89.9%). 12.9% of the typed samples belonged to EV species A (EV-A), 48.6% to species B (EV-B) and 38.5% to species C (EV-C). More than half of the EV-C strains (53%) belonged to subgroup C containing, among others, Poliovirus (PV) 1-3. The serotype most frequently isolated in our study was CVA-13, followed by EV-C99. The strains of CVA-13 showed a vast genetic diversity, possibly representing a new cluster, 'F'. The majority of the EV-C99 strains grouped together as cluster B. In conclusion, this study showed a vast circulation of EVs among Malawian children, with an EV prevalence of 89.9%. Identification of prevalences for species EV-C comparable to our study (38.5%) have only previously been reported in sub-Saharan Africa, and EV-C is rarely found outside of this region. The data found in this study are an important contribution to our current knowledge of EV epidemiology within sub-Saharan Africa.

Respiratory Viruses in Invasively Ventilated Critically Ill Patients-A Prospective Multicenter Observational Study.

OBJECTIVES: The presence of respiratory viruses and the association with outcomes were assessed in invasively ventilated ICU patients, stratified by admission diagnosis. DESIGN: Prospective observational study. SETTING: Five ICUs in the Netherlands. PATIENTS: Between September 1, 2013, and April 30, 2014, 1,407 acutely admitted and invasively ventilated patients were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Nasopharyngeal swabs and tracheobronchial aspirates were collected upon intubation and tested for 14 respiratory viruses. Out of 1,407 patients, 156 were admitted because of a severe acute respiratory infection and 1,251 for other reasons (non-severe acute respiratory infection). Respiratory viruses were detected in 28.8% of severe acute respiratory infection patients and 17.0% in non-severe acute respiratory infection (p < 0.001). In one third, viruses were exclusively detected in tracheobronchial aspirates. Rhinovirus and human metapneumovirus were more prevalent in severe acute respiratory infection patients (9.6% and 2.6% vs 4.5 and 0.2%; p = 0.006 and p < 0.001). In both groups, there were no associations between the presence of viruses and the number of ICU-free days at day 28, crude mortality, and mortality in multivariate regression analyses. CONCLUSIONS: Respiratory viruses are frequently detected in acutely admitted and invasively ventilated patients. Rhinovirus and human metapneumovirus are more frequently found in severe acute respiratory infection patients. Detection of respiratory viruses is not associated with worse clinically relevant outcomes in the studied cohort of patients.

The hepatitis C virus nonstructural protein 3 Q80K polymorphism is frequently detected and transmitted among HIV-infected MSM in the Netherlands.

OBJECTIVES: The Q80K polymorphism is a naturally occurring resistance-associated variant in the hepatitis C virus (HCV) nonstructural protein 3 (NS3) region and is likely transmissible between hosts. This study describes the Q80K origin and prevalence among HCV risk groups in the Netherlands and examines whether Q80K is linked to specific transmission networks. DESIGN AND METHODS: Stored blood samples from HCV genotype 1a-infected patients were used for PCR and sequencing to reconstruct the NS3 maximum likelihood phylogeny. The most recent common ancestor was estimated with a coalescent-based model within a Bayesian statistical framework. RESULTS: Study participants (n = 150) were either MSM (39%), people who inject drugs (17%), or patients with other (15%) or unknown/unreported (29%) risk behavior. Overall 45% was coinfected with HIV. Q80K was present in 36% (95% confidence interval 28-44%) of patients throughout the sample collection period (2000-2015) and was most prevalent in MSM (52%, 95% confidence interval 38-65%). Five MSM-specific transmission clusters were identified, of which three exclusively contained sequences with Q80K. The HCV-1a most recent common ancestor in the Netherlands was estimated in 1914 (95% higher posterior density 1879-1944) and Q80K originated in 1957 (95% higher posterior density 1942-1970) within HCV-1a clade I. All Q80K lineages could be traced back to this single origin. CONCLUSION: Q80K is a highly stable and transmissible resistance-associated variant and was present in a large part of Dutch HIV-coinfected MSM. The introduction and expansion of Q80K variants in this key population suggest a founder effect, potentially jeopardizing future treatment with simeprevir.

Genetic characterization of multiple hepatitis C virus infections following acute infection in HIV-infected men who have sex with men.

OBJECTIVE: High rates of hepatitis C virus (HCV) reinfections among HIV-infected men who have sex with men (MSM) following clearance of a primary infection suggest absence of protective immunity. Here, we investigated the incidence of HCV super and reinfections in 85 HIV-infected MSM with incident HCV infection. DESIGN AND METHODS: Serial sequencing of a fragment of NS5B and the HCV envelope was used to longitudinally characterize the virus. If the primary genotype was still present at the most recent viremic time point, as indicated by the NS5B sequence analysis, serial envelope 2/hypervariable region 1 (E2/HRV1) sequence analysis was performed to distinguish a new infection with the same genotype (clade switch) from intrahost evolution. Incidence rate and cumulative incidence of secondary infections were estimated, and the effect of the primary genotype (1a versus non1) on the risk of acquiring a second infection with the same genotype was determined using Cox proportional-hazards analysis. RESULTS: Among 85 patients with a median follow-up of 4.8 years, incidence rate of secondary infections was 5.39 cases/100 person-years (95% confidence interval 3.34-8.26). Cumulative incidence of genotype switches was markedly higher than the cumulative incidence of clade switches (26.7 versus 4.8% at 5 years, respectively). In patients with HCV-1a as primary infection, the risk for acquiring another HCV-1a infection was reduced compared to those with a primary non-HCV-1a subsequently acquiring HCV-1a (hazard ratio 0.25, 95% confidence interval 0.07-0.93). CONCLUSION: Risk of acquiring a secondary infection with the primary genotype was strikingly reduced compared with the risk of acquiring a secondary infection with a different genotype.

A comparison of 454 sequencing and clonal sequencing for the characterization of hepatitis C virus NS3 variants.

We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing.

Unexplained diarrhoea in HIV-1 infected individuals.

BACKGROUND: Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. METHODS: In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). RESULTS: HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. CONCLUSION: Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea.

Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir.

BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare.

Simplified PCR protocols for INNO-LiPA HBV Genotyping and INNO-LiPA HBV PreCore assays.

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.

Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay.

We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0 bDNA assay has a linear dynamic range of 2.5 x 10(3) to 4.0 x 10(7) HCV RNA copies per ml (c/ml). The performance of the HCV 3.0 bDNA assay was evaluated using three different test panels. An overall specificity of 96.8% relative to the detection limit of the HCV 3.0 bDNA assay was found. The intra- and interrun reproducibilities for both the dilution panel and the NAP (AcroMetrix, Benicia, Calif.) panel were consistent with coefficients of variation of less than 9%. Quantitation with the HCV 3.0 bDNA assay was linear over the entire range of both panels (ranges of 4.4 x 10(3) to 3.5 x 10(6) c/ml and 5 x 10(3) to 2 x 10(6) IU/ml, respectively), with correlation coefficients of 0.999, slopes close to one, and intercepts close to zero. The regression equation indicated that 1 IU corresponded to about 4.8 copies of HCV RNA. A correlation coefficient of 0.941 was found for HCV RNA values (in international units per milliliter) obtained from the HCV 3.0 bDNA assay and the HCV Monitor version 2.0 assay (HCV Monitor 2.0 assay) (Roche Diagnostic Systems, Branchburg, N.J.). Quantitative results obtained close to the lower limit of the HCV 3.0 bDNA assay might imply that its lower limit should be reconsidered and raised, if necessary. It appeared that quantitation values obtained from the HCV Monitor 2.0 assay of between 5 x 10(2) and 10(5) IU/ml were in general higher than those obtained from the HCV 3.0 bDNA assay, whereas values obtained from the HCV Monitor 2.0 assay were underestimated for samples with HCV RNA levels above 10(5) IU/ml.