Determination of HCV genotype using two antibody assays and genome typing.

Two serotyping assays for hepatitis C virus (Serotyping 1-6 assay; Murex, UK and RIBA Serotyping SI; Chiron, USA) were compared to a standardized genotyping assay (Inno-LiPA HCV II; Innogenetics, Belgium) using serum samples collected from 126 patients chronically infected with hepatitis C virus. Serotyping was positive in 87% and 80% of the samples tested with Murex and Chiron, respectively, and concordant with genotyping in 93% and 88%, respectively. Sequence analysis of the NS5b region of 15 samples with discrepant typing results confirmed the Inno-LiPA finding in all instances. The Murex enzyme immunoassay serotyping method was less sensitive for identifying genotype 2 (P<0.05). The concordance with genotyping of the RIBA serotyping method was lower for genotype 2 than for the other genotypes (P<0.05).

Nosocomial outbreak of influenza virus A (H3N2) infection in a solid organ transplant department.

BACKGROUND: There is a strong body of evidence in favor of influenza virus immunization in solid organ recipients. However, little attention has been devoted to other reservoirs, such as the patients' relatives and, at the time of hospital admission, to the healthcare workers. METHODS: Analysis of the epidemiology of an outbreak of nosocomial influenza A in a solid organ transplant unit. RESULTS: Four cases of influenza A virus infection were reported during a short 4-day outbreak in a 12 single-room transplant unit. None of the patients had been immunized against influenza. Three patients had not been visited by their relatives between admission and influenza infection. Three nurses, among the 27 healthcare workers, presented with clinical flu symptoms at times consistent with nosocomial transmission. CONCLUSIONS: Because the prevention of influenza infection by vaccination warrants a global strategy to target the different reservoirs, we suggest that the modern policy of vaccinating solid organ patients should be extended both to their relatives and to the healthcare workers of transplant units.

HIV-1 subtyping using phylogenetic analysis of pol gene sequences.

HIV-1 pol gene sequencing is now used routinely in France to identify mutations associated with resistance to reverse transcriptase (RT) or protease (PR) inhibitors. These sequences may also provide other information, such as the HIV-1 subtype. HIV-1 subtyping was compared using the RT and PR gene sequences to heteroduplex mobility assay (HMA) of the envelope gene. The RT and PR genes of 51 samples that had been subtyped earlier by HMA were sequenced. Sequences were aligned and subtypes were determined by phylogenetic analysis with reference HIV sequences. HMA gave the following subtypes: A (20), B (19), C (1), D (3), F (1), G (3) and CRF01-AE (4). Phylogenetic analysis of the RT gene gave: A (5), B (19), C (2), D (3), F (1), G (6), J (2), CRF01_AE (4), CFR02_AG (7) and undetermined (2). PR gene analysis did not infer subtypes with sufficient confidence. HMA and RT subtyping was not in agreement in nine cases. RT subtyping can identify CFR02_AG and CRF01_AE variants from A subtype RT. It was shown that phylogenetic analysis of the RT gene could provide a useful method for HIV-1 subtyping. The length of the amplicon and the relative performance of each primer pair used in this study favoured RT sequences as a subtyping tool. One potential advantage over env subtyping HMA is the ability to identify some circulating recombinant forms (CRFs).

Shift in HIV resistance genotype after treatment interruption and short-term antiviral effect following a new salvage regimen.

OBJECTIVE: To investigate the changes in genotypic drug-resistance pattern, plasma HIV RNA and CD4 cell count after treatment interruption and assess the short-term antiviral effect of a new salvage regimen. DESIGN: Prospective study of 38 patients with multiple failing regimens who had completely stopped all medication for 3 months before a three to five-drug regimen was reintroduced according to clinical guidelines. METHODS: Patients were tested for HIV resistance before and after treatment interruption by population-based sequencing and clonal analysis of selected patients. RESULTS: Discontinuation of therapy for 3 months was associated with a median increase in HIV RNA of 0.4 log10 and a median decrease in CD4 cell count of 43 x 10(6)/l. Sixty-one per cent of patients had a shift from the drug-resistant genotype to a predominantly wild-type genotype. The patients significantly likely to show genotype reversion were those in Centers for Disease Control groups A or B, who had been exposed to few drugs, had a low plasma HIV RNA, or a high CD4 cell count. The only independent factor predicting genotype reversion was the clinical stage. The median change in plasma HIV RNA at month 3 after treatment reintroduction was -2.3 log10 copies/ml in patients who had genotype reversion compared with -0.6 log10 copies/ml in patients without genotype reversion (P = 0.004). CONCLUSION: Suspending treatment for 3 months after multiple failures could be a suitable strategy for optimizing salvage therapy provided it is instituted early, before the HIV disease becomes too advanced.

Transmissions of hepatitis C virus during the ancillary procedures for assisted conception.

Since mother to child transmissions of hepatitis C virus (HCV) have been reported to be low, teams involved in assisted reproductive technologies have accepted HCV positive patients into their programmes. We report in the present paper two cases of undoubted patient to patient HCV transmission while patients were attending for assisted conception. In both cases, HCV genotyping and sequencing of the first hypervariable region of the HCV genome provided molecular evidence for nosocomial transmission. Investigations made to elucidate the route of contamination have shown that the most likely route of contamination is through healthcare workers. Such nosocomial HCV infection has been reported in other healthcare situations, mainly in dialysis units, and physical proximity was also suspected to be at the origin of the infection. We conclude that assisted reproduction teams must be very prudent when including such patients in their programmes.

Hepatitis C virus infection in French hemodialysis units: a multicenter study.

The aims of the study were: (i) to evaluate the prevalence of hepatitis C virus (HCV) antibodies (third generation tests) and RNA (standardized ultrasensitive RT-PCR assay) in a large cohort of hemodialysis patients, and (ii) to correlate HCV markers with bioclinical features and alanine-aminotransferase (ALT) activity. Antibodies were assayed by two methods in 1,323 patients (60% men, median age 65 years, median hemodialysis duration 3 years) attending 25 French hemodialysis centers including 9 self-care units. RNA was assayed using the Cobas Amplicor 2.0 method in pooled samples from 10 anti-HCV(-/-) patients and on individual samples from the other patients. Of the 16.3% patients (range 0-44%) tested (+/+) for HCV antibodies (anti-HCV), 2.3% tested (+/-) and 81.4% tested (-/-). 70% of the anti-HCV(+/+) patients and 3% of the HCV(+/-) patients were RNA(+). Pooled analysis revealed that 5/1077 anti-HCV(-/-) patients (0.5%) were RNA(+); all 5 displayed subsequently an increase in ALT and became anti-HCV(+/+). Mean ALT was higher (multiple of normal) in anti-HCV(+/+) RNA(+) patients than in anti-HCV(+/+) RNA(-) patients (0.46 +/- 0.08 vs. 0.22 +/- 0.07, P < 0.0001) and similar in all the RNA(-) patients, whatever their HCV antibody status. Multivariate analysis demonstrated that HCV status was linked to hemodialysis duration, previous kidney transplantation and positive anti-HBc. To summarize, the determination of the RNA status of anti-HCV(+/-) patients may have clinical relevance if a policy of isolation is contemplated. Standardized ultrasensitive RT-PCR assay combined with a pooling strategy is a promising method for use in epidemiological studies.

Longitudinal analysis of hepatitis C virus replication and liver fibrosis progression in renal transplant recipients.

The pathogenesis of hepatitis C virus (HCV) infection was investigated by analysis of changes in viral and histologic parameters in 36 renal transplant recipients who were infected with HCV before transplantation. Each patient was classified according to development of liver fibrosis as assessed by 2 liver biopsies done 45 and 81 months after transplantation: 13 had progressing liver fibrosis (fibrosers) and 23 did not (nonfibrosers). All developed high-titer posttransplant viremia with a significant increase of 1.2 log RNA copies/mL. There were no significant differences in the increases in serum HCV RNA or genotype distributions in fibrosers and nonfibrosers. The hypervariable region (HVR)-1 of the HCV genome was analyzed by cloning and sequencing 20 clones per sample from 5 fibrosers and 5 nonfibrosers. Comparison of samples revealed that liver fibrosis progression was significantly associated with slower HVR-1 quasispecies diversification, suggesting the selection of more aggressive variants in fibrosers.

Genetic heterogeneity of hypervariable region 1 of the hepatitis C virus (HCV) genome and sensitivity of HCV to alpha interferon therapy.

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P

Changes in hepatitis C virus RNA viremia concentrations in long-term renal transplant patients after introduction of mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) is a potent immunosuppressive agent and might inhibit chronic rejection, at least in primates. The prevalence of chronic hepatitis C virus (HCV) infection is high in renal transplant (RT) patients. To date, it has not been demonstrated whether MMF has any effect upon HCV viremia. METHODS: Fourteen long-term HCV(+) RT patients with chronic allograft dysfunction whose maintenance immunosuppression was based on cyclosporine, were given MMF therapy either in place of azathioprine (n=11) or in addition to baseline therapy (n=3). HCV viremia levels were measured by the Amplicor HCV-Monitor RT-PCR assay (Roche Diagnostic Systems) on two separate occasions before the introduction of MMF, and 1 year after changing to MMF or at the last follow-up visit. RESULTS: MMF therapy was associated with a significant rise in HCV viremia, i.e., 5.8+/-0.5 vs. 5.2+/-0.7 log copies/ml (P=0.01), although there were no significant changes in liver enzymes. The increase in HCV viremia was not related to HCV genotypes either. At the patient level, HCV RNA concentrations changed in only seven patients (group B), i.e. >1 log copies/ml, whereas it remained stable in the others (group A). Before conversion, the only significant difference between group A and B was the level of HCV RNA, i.e., 5.5+/-0.4 log copies/ml in group A and 4.9+/-0.7 log copies/ml in group B (P=0.05). CONCLUSION: Our study suggests that MMF should be used with caution in stable HCV RT patients whose maintenance immunosuppressive therapy is based on cyclosporine, at least in the case of patients with a low HCV RNA titer.

Mutations conferring resistance to zidovudine diminish the antiviral effect of stavudine plus didanosine.

This study evaluated the influence of zidovudine (ZDV) resistance mutations on the antiviral effect of the combination of stavudine (D4T) plus didanosine (ddI) in patients treated previously with ZDV plus zalcitabine (ddC). Twenty patients who had been treated with ZDV plus ddC for a median duration of 11 months (range, 7-42 months) were switched to D4T (40 mg twice a day [BID]) + ddI (200 mg BID) in an open pilot study lasting 6 months. The CDC classes were A (n = 10) and B (n = 10). The median baseline CD4 count was 285/mm(3) and the median baseline plasma virus RNA (Amplicor HIV Monitor RT-PCR assay) was 4.6 log copies/ml. Population-based sequence analysis detected mutations associated with resistance to reverse transcriptase (RT) inhibitors in the RT domain of virus RNA from baseline plasma samples in 13/20 (65%) patients. Twelve patients had mutations associated with zidovudine resistance (3 T215Y - M41L - L210W; 3 T215Y - M41L; 2 T215Y - L210W; 3 T215Y; 1 K70R) and 1 patient had a multi-dideoxynucleoside resistance mutation (QI5IM). Patients with a resistance mutation had a significantly lower RNA suppression after 3 and 6 months (median RNA reduction -0.5 log and -0.1 log) than the remaining patients (-1.6 log and -2 log). Fifty percent of patients with wild-type viruses had undetectable plasma RNA after 24 weeks of D4T plus ddI therapy, whereas all those with mutated viruses had HIV RNA concentration > 3 log copies/ml at week 24 (P <.05). Our finding may have implications when deciding on a second line therapy with three or four drugs that includes two new nucleoside analogues. Cross-resistance between nucleoside analogues deserves maximal attention to ensure optimal antiretroviral therapy and design algorithms for antiretroviral management based on genotypic antiretroviral resistance testing.

Using RT-PCR and bDNA assays to measure non-clade B HIV-1 subtype RNA.

The performance of the new version of RT-PCR assay (Amplicor HIV-1 Monitor v1.5) was assessed. The quantification of non-B subtype HIV-1 plasma RNA (30A, 1C, 1D, 3E, 2F, 3G) obtained using Monitor v1.5 was compared to the former version of this assay (Monitor v1.0) and to the Quantiplex v2.0 bDNA assay. The new primers used in Monitor v1.5 were similar to the former version in both specificity and sensitivity. The new primers corrected the detection and quantification defect observed previously for HIV-1 non-B subtypes and gave slightly higher RNA concentrations than those measured using the bDNA assay (+0.39 log copies/ml).

Study of the association between major histocompatibility complex class II genes and the response to interferon alpha in patients with chronic hepatitis C infection.

OBJECTIVE: The aim of the study was to determine the influence of HLA class II genes on the response to interferon-alpha (IFN-alpha) in patients with chronic hepatitis C. METHODS: The distribution of HLA DRB1 and DQB1 alleles was assessed in 170 caucasoïd patients treated with IFN-alpha for chronic hepatitis C. 50 patients had a long term sustained response to treatment whereas 120 patients were nonresponders. RESULTS: Female sex, non-1 HCV genotype particularly genotype 2 and pretreatment low serum HCV RNA level were associated with long-term sustained response to IFN-alpha. A trend towards a higher prevalence of DRB1*07 allele in non responders than in patients with sustained response (45% vs. 28%, odds ratio 2.1; P < 0.05) on the one hand and of DQB1*06 allele in HCV genotype 1 patients with sustained response than in HCV genotype 1 nonresponders (75% vs 27.3%, odds ration 7.9; P < 0.02) on the other hand, were observed. However, none of these two differences remained significant after Bonferroni's correction. CONCLUSION: Accordingly, we conclude that the response to IFN-alpha therapy is more tightly related to virus factors than to host's HLA class II genes.

Molecular evidence for nosocomial transmission of hepatitis C virus in a French hemodialysis unit.

A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.

Intermittent selection pressure with zidovudine plus zalcitabine treatment reduces the emergence in vivo of zidovudine resistance HIV mutations.

The development of mutations conferring drug resistance was investigated in 49 antiretroviral-naive asymptomatic HIV-1 subjects with CD4+ cell counts of 250-500/mm3 given intermittent (6-week courses, 6 weeks apart) or continuous treatment with zidovudine (AZT) plus zalcitabine (ddC) over 54 weeks. The concentration of human immunodeficiency virus type 1 RNA in the plasma and the CD4 cell counts were measured every 6 weeks. The rate of decrease of HIV-1 RNA concentration in plasma after a 6-week course of AZT + ddC was similar for each treatment cycle (approximately 1-log reduction). The plasma HIV-1 RNA concentration returned to its initial level at each treatment interruption. The mean CD4 cell counts after 54 weeks in the two treatment groups were similar. Genotype analysis by sequencing the reverse transcriptase coding region from plasma viral RNA on treatment showed a lower frequency of AZT resistance mutations after 54 weeks in patients given intermittent treatment (18%) than in those treated continuously (79 %, P < 0.001). No mutations conferring ddC resistance or multidideoxynucleoside resistance were observed in either group. These findings may have clinical implications for long-term treatment strategies.

Decay of HIV-1 DNA in patients receiving suppressive antiretroviral therapy.

We have examined the effect of potent antiretroviral regimens on the latent reservoirs of HIV-1. The HIV-1 DNA in the peripheral blood mononuclear cells (PBMC) of 10 patients with undetectable plasma HIV-1 RNA (<20 copies/ml) who had received combination antiretroviral therapy was assayed every 12 weeks. No evidence of residual viral replication was found in the PBMC after 24 weeks of treatment. Although HIV-1 DNA remained detectable in all patients, it decreased significantly from 3.5 log copies/10(6) cells (range, 1.8-4.7 log copies/10(6) cells) to 2.3 log copies/10(6) cells (range, 0.6-3.1 log copies/10(6) cells) after 60 weeks of suppressive therapy. Analysis based on 6 patients who reached 60 weeks showed a slow decline with an estimated half-life of 40 weeks (range, 26-163 weeks). Genotypic analysis by sequencing the HIV-1 pol gene revealed no changes in the reverse transcriptase or protease coding regions after 48 to 60 weeks of therapy. The findings suggest that, in addition to potent antiretroviral regimens, new strategies must be developed to ensure eradication of the latent reservoir of provirus, and hence of the virus itself.

Baseline level and early suppression of serum HCV RNA for predicting sustained complete response to alpha-interferon therapy.

The relationship between serum hepatitis C virus (HCV) RNA and the outcome of alpha-interferon (alpha-IFN) therapy in patients with chronic hepatitis C has important implications for therapeutic research and clinical care. Serum HCV RNA was tested for HCV genotype and quantified by a standardized reverse transcriptase-polymerase chain reaction assay as a measure of viral load in a cohort of 130 patients with chronic hepatitis C treated with alpha-IFN at a standard dose of 3 million units three times a week scheduled for 6 (n = 50) or 12 months (n = 76). Twenty-one of 126 evaluable patients (16.7%) developed a sustained complete response to alpha-IFN according to biochemical and virological criteria. The 3 pretreatment independent factors associated with a sustained complete response were a low baseline serum HCV RNA concentration, non-1 HCV genotype, and female sex. A multivariate logistic regression model, with pretreatment and month 1 variables, showed that a lower baseline serum HCV RNA concentration, female sex, and a greater suppression of RNA were the significant predictors of sustained complete response. The lowest baseline serum HCV RNA concentration was observed in patients with genotype 2 infection and the greatest decrease in HCV RNA from baseline to month 1 in those with genotype 3. The findings suggest that measuring HCV RNA in serum before and soon after beginning treatment can be helpful for selecting patients who are most likely to have a sustained complete response to standard schedule of alpha-IFN and for identifying patients in whom alternative strategies should be examined.

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay.

A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10(6) cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11-4.7 log copies/10(6) cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21-58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus.

Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens.

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.