Performance of genotypic algorithms for predicting tropism for HIV-1 CRF01_AE recombinant.

OBJECTIVES: There is no consensus about the performances of genotypic rules for predicting HIV-1 non-B subtype tropism. Three genotypic methods were compared for CRF01_AE HIV-1 tropism determination. METHODS: The V3 env region of 207 HIV-1 CRF01_AE and 178 B subtypes from 17 centers in France and 1 center in Switzerland was sequenced. Tropism was determined by Geno2Pheno algorithm with false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25, net charge rule and NXT/S mutations. RESULTS: Overall, 72.5%, 59.4%, 86.0%, 90.8% of the 207 HIV-1 CRF01_AE were R5-tropic viruses determined by Geno2pheno FPR5%, Geno2pheno FPR10%, the combined criteria and the 11/25 rule, respectively. A concordance of 82.6% was observed between Geno2pheno FPR5% and the combined criteria for CRF01_AE. The results were nearly similar for the comparison between Geno2pheno FPR5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR10%. Neither HIV viral load, nor current or nadir CD4 was associated with the discordance rate between the different algorithms. CONCLUSION: Geno2pheno predicted more X4-tropic viruses for this set of CRF01_AE sequences than the combined criteria or the 11/25 rule alone. For a conservative approach, Geno2pheno FPR5% seems to be a good compromise to predict CRF01_AE tropism.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Recent evidence of underestimated circulation of hepatitis C virus intergenotypic recombinant strain RF2k/1b in the Rhône-Alpes region, France, January to August 2014: implications for antiviral treatment.

Since the beginning of 2014, hepatitis C virus (HCV) recombinant forms RF2k/1b have been detected in the Rhône-Alpes French region in 10 patients originating from the Caucasus area. Circulation of this particular HCV strain is very likely to be underestimated. It is also prone to be misgenotyped when using genotyping methods based on the 5' region of the viral genome, which may lead to suboptimal treatment.

Proficiency of transient elastography compared to liver biopsy for the assessment of fibrosis in HIV/HBV-coinfected patients.

Transient elastography (TE) is a noninvasive technique to evaluate liver fibrosis. We compared the performance of TE with liver biopsy (LB) in patients with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection. Patients prospectively underwent TE and LB. The diagnosis accuracy of TE was calculated using receiver operating characteristic (ROC) curves for different stages of fibrosis, and optimal cut-off values were defined. A sequential algorithm combining TE with biochemical score (Fibrotest) is proposed. Fifty-seven patients had both TE and LB (median time: 3 days) and two with proven cirrhosis, only TE. Forty-six (78%) were under antiretroviral therapy with anti-HBV drugs in 98%, and 19 (32%) had elevated alanine aminotransferase (ALT). A significant correlation was observed between liver stiffness measurement (LSM) and METAVIR fibrosis stages (P < 0.0001). Patients with elevated ALT tended to have higher LSM than those with normal ALT. The areas under the ROC curves were 0.85 for significant fibrosis (≥ F2), 0.92 for advanced fibrosis (≥ F3) and 0.96 for cirrhosis. Using a cut-off of 5.9 kPa for F ≥ 2 and 7.6 kPa for F ≥ 3, the diagnosis accuracy was 83% and 86%, respectively. With an algorithm combining TE and Fibrotest, 97% of patients were well classified for significant fibrosis. Using this algorithm, the need for LB can be reduced by 67%. In HIV/HBV-coinfected patients, most of them with normal ALT under antiretroviral treatment including HBV active drugs, TE was proficient in discriminating moderate to severe fibrosis from minimal liver disease.

Sexually transmitted HCV infection and reinfection in HIV-infected homosexual men.

Multiple, concomitant or successive hepatitis C virus (HCV) infections have been described in injection drug users and following organ transplantation and blood transfusion. However, data on sexual HCV reinfection is scarce. We report sexual HCV reinfection following viral eradication of a first HCV infection in two homosexual HIV-infected men. The first patient acquired HCV genotype 4 infection after resolution of an initial acute HCV genotype 1a infection. The second patient was infected with genotype 1a HCV following remission of an initial acute HCV genotype 4c/d infection. The two subjects were successfully treated with peginterferon alpha-2a and ribavirin for their first and second infection and achieved a sustained virological response on both occasions. Unprotected anal intercourse with multiple partners known to be HIV-positive (serosorting) was the only risk factor for HCV transmission reported by both patients. Therefore, sexual HCV reinfection can occur in homosexual men having unprotected sex and "serosorting" should be considered a risk factor for the sexual transmission of HCV.

Occult HBV infection may represent a major risk factor of non-response to antiviral therapy of chronic hepatitis C.

Occult hepatitis B virus (HBV) infection is common in chronic hepatitis C patient. However, its significance and consequences are still unclear. The aim of this study was to evaluate the prevalence of occult HBV among HCV chronic carriers in France and to assess its impact on liver histology and response to antiviral therapy. To this end a cohort of 203 patients with chronic hepatitis C without hepatitis B surface antigen (HBsAg) has been examined. Serum HBV-DNA was detected using a highly sensitive PCR with primers located in the S and X genes. HBV viraemia levels were further determined by real-time PCR. Results showed that 47 of 203 (23%) patients had occult HBV infection with a low HBV load (10(2)-10(4) copies/ml) but significantly higher HCV-RNA titers (P < 0.05). No significant difference in age, gender, serum ALT level, HCV genotypes, and the presence of anti-HBc was observed between patients with or without HBV-DNA. When compared histologically, patients with occult HBV infection had higher activity (A2-A3 in 53% vs. 38%, P < 0.01) and more advanced fibrosis (60% vs. 33%, P < 0.001) than HBV-DNA negative cases. Sustained response to combination therapy against Chronic hepatitis C was achieved in 11 (28%) of 40 HBV-DNA positive cases, compared with 65 (45%) of the 144 HBV-DNA negative cases (P < 0.05). Among the 144 HBV-DNA negative HCV patients those with genotype 1 responded less frequently to therapy as compared to other genotypes infected patients (38% vs. 55%, P < 0.05). Surprisingly, when considering all patients studied, irrespective to the HBV-DNA status no significant difference was observed in response to combination therapy regarding HCV genotypes (39% vs. 44%, P > 0.05). In conclusion, HBV-DNA is found in 1/4 of French chronic hepatitis C patients regardless of the presence of anti-HBc. Such an occult HBV co-infection is associated with more severe liver disease, higher HCV viral load and decreased response to antiviral therapy irrespective of HCV genotypes.

The risk of hospital-acquired GB virus C infection: a pilot case-control study.

The risk of hospital-acquired infection with GB virus C (GBV-C) was explored among 42 patients. The factors independently associated with detection of GBV-C RNA in serum were bronchoscopic examination [adjusted odds ratio (OR)=18.1 (95% confidence interval 1.3-255.3), P=0.03] and a history of illicit drug use [OR=14.5 (1.0-218.7), P=0.05]. In this cohort of patients, invasive procedures appear to be associated with GBV-C infection but not with hepatitis C virus (HCV) infection.

Prevalence of primary resistance to zidovudine and lamivudine in drug-naive human immunodeficiency virus type-1 infected patients: high proportion of reverse transcriptase codon 215 mutant in circulating lymphocytes and free virus.

The presence of primary zidovudine (AZT)-resistance (mutation T215Y/F) or lamivudine (3TC)-resistance (mutation M184V) was evaluated in 90 drug-naive patients infected with human immunodeficiency virus type-1 (HIV-1) between 1987 and 1997. The proportion of mutant strains in proviral samples or plasma viral samples was determined using a differential hybridization assay. Mutation T215Y/F was found in five (5.6%) patients infected between 1994 and 1997, whereas none of these patients harbored the mutation M184V. The T215Y/F mutation was present in the virus and/or provirus and persisted for at least two years. In one patient, the mutant provirus was associated with only wild-type free virus. Four of these patients were followed, and two were treated subsequently to a regimen containing AZT but with low response. The persistence of primary resistance mutations might depend on the proportion of these mutations at the time of infection, although mutant provirus might not give rise to replicating variants.

Clinical evaluation of the branched DNA assay for hepatitis B virus DNA detection in patients with chronic hepatitis B lacking hepatitis B e antigen and treated with interferon-alpha.

The aim of this study was to evaluate the Chiron branched DNA (bDNA) assay for detection of serum hepatitis B virus (HBV) DNA in patients with chronic hepatitis B lacking hepatitis B e antigen (HBeAg) and undergoing interferon (IFN) therapy. Results obtained with the bDNA assay were compared with those obtained using the Abbott liquid hybridization (LH) assay and the polymerase chain reaction (PCR). Serial samples (274) from 34 patients were analysed. Analysis of variance results indicated that bDNA values were more significantly correlated than LH values with both PCR positive/negative results (probability of artifact (Prob > F) = 0.7 and 0.09 for LH and bDNA assays, respectively) and presence/absence of precore mutations (Prob > F = 0.21 and 0.001 for LH and bDNA assays, respectively). Both bDNA and LH results correlated highly with alanine aminotransferase (ALT) values (both had Prob > F values of 0.0) while PCR was not correlated with ALT (Prob > F = 0.05). In 26 evaluable patients, a model based on a generalized Knodell score was used to predict response to IFN therapy, as defined by normalization of ALT values during therapy. This model discriminated well between non-responders and responders. The bDNA results correlated well with the generalized Knodell score, while the LH results did not (Prob > F = 0.04 and 0.19 for the bDNA and LH assays, respectively). In conclusion, the bDNA assay appears to be useful for quantification of HBV DNA levels in HBeAg-negative chronic hepatitis as it correlates with biochemical and histological indications of disease severity as well as with response to IFN therapy.

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: characterization of major epitopes useful for antigen detection.

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells: influence of viral load, viral genotype, and cell phenotype.

Extrahepatic sites capable of supporting hepatitis C virus (HCV) replication have been suggested. We analyzed the influence of virological factors such as viral genotype and viral load, and cellular factors such as cell phenotype, on the detection rate of HCV sequences in hematopoietic cells of infected patients. Thirty-eight chronically infected patients were included in the study: 19 infected by genotype 1 isolates (1a and 1b), 13 by nongenotype 1 isolates (including genotypes 2 a/c, 3a, and 4), and 6 coinfected by genotype 1 and 6 isolates. Polymerase chain reaction (PCR) detection efficiency of viral genomic sequences, both the positive and negative strand RNA, was evaluated using RNA transcripts derived from genotype 1, 2, 3, and 4 cloned sequences and found to be equivalent within one log unit. The serum viral load, ranging from less than 2 x 10(5) Eq/mL to 161 x 10(5) Eq/mL, did not influence the detection rate of either strand of RNA in patients' peripheral blood mononuclear cells (PBMCs). Positive and negative strand RNA were found in PBMCs of all 3 cohorts of patients with a detection rate ranging from 15% to 100% and from 8% to 83.3% for the positive and negative strand RNA, respectively. Coinfected patients showed a detection rate in all cases greater than 80%. Patients infected with genotype 1 isolates showed a higher detection rate of either strands of RNA when compared with patients infected with other genotypes (P <.001 and P <.04). Both strands were found restricted to polymorphonuclear leukocytes, monocytes/macrophages, and B (but not T) lymphocytes. These data show that HCV genomic sequences, possibly reflecting viral replication, can be detected in PBMCs of chronically infected patients independent of the viral load and that specific associated cell subsets are implicated in the harboring of such sequences.

Investigations into the cross-reactivity of rabbit antibodies raised against nonhomologous pairs of synthetic peptides derived from HIV-1 gp120 proteins.

The immunological cross-reactivity of several peptides with specific pattern-property characteristics related to the epitopes of human immunodeficiency virus type 1 (HIV-1) gp160/ 120 envelope proteins has been investigated. Proteins with similar primary structures can be expected to show functional or topographic similarities, such as specific epitopes which may cross-react with antibodies derived from the immunisation of animals with other members of the same protein family. These structure-function characteristics may be revealed as periodicities derived from presentations based on the discrete Fourier transformation of the distributions of various physico-chemical amino acid descriptors, constituting the polypeptide backbone and amino acid side-chains of the protein molecule. Such approaches, for example, have permitted prediction of periodicities corresponding to secondary structural motifs, including amphipathic alpha-helices and beta-sheets, within protein sequences, and have helped to clarify potential binding sites for ligands, substrates or cofactors with interacting macromolecules. Based on this approach, characteristic periodicities have been identified which represent common Fourier transform spectral properties of the envelope (ENV) gp160/120 glycoproteins from a range of HIV-1 isolates. In addition, similar periodicities have been detected as components of the discrete Fourier transform representation of the corresponding amino acid descriptors of the CD4 binding domain of gp120. Accordingly, we have synthesised several peptides having periodic characteristics in their discrete Fourier transform representations similar to these HIV-1 proteins. These nonhomologous synthetic peptides induced cross-reactive antibodies in New Zealand White rabbits. Polyclonal antibodies raised to one of these peptides reacted with HIV-1 ENV gp120-related proteins, as determined by enzyme-linked immunosorbent assay and Western blotting techniques. These findings provide further evidence for a role of immunological cross-reactivity and molecular biomimicry in the development of peptide-based vaccines directed against viral or bacterial pathogens.

Correlation between antiretroviral resistance mutations, biological parameters, and clinical evolution in zidovudine-treated patients infected with human immunodeficiency virus type 1.

To evaluate the correlation between zidovudine (ZDV) resistance mutations of human immunodeficiency virus type 1 (HIV-1), biological parameters, and clinical evolution, 111 HIV-1-infected patients treated with ZDV were studied. Specific mutations at codons 70, 215, and 41 in the HIV-1 reverse transcriptase coding region conferring resistance to ZDV were detected using a selective polymerase chain reaction. The appearance of ZDV resistance mutations was significantly correlated with baseline clinical stage, CD4+ cell count, and viral load, but not with duration of ZDV therapy or p24 antigen level. In univariate analysis, results showed a prognostic role of mutations at codons 215 and 41 for clinical progression to the acquired immune deficiency syndrome (AIDS) or death. In multivariate analysis after controlling for viral load, CD4+ cell count, and clinical stage, the presence of the mutation at codon 215 (but not at codon 41) remained an independent predictor of subsequent clinical evolution.

Comparison of HCV RNA assays for the detection and quantification of hepatitis C virus RNA levels in serum of patients with chronic hepatitis C treated with interferon.

Detection and quantification of hepatitis C virus (HCV) RNA levels by using the standardized qualitative Amplicor HCV and quantitative Amplicor HCV Monitor assays (Roche Molecular Systems) were evaluated in 48 patients with chronic hepatitis C treated with interferon. Results were compared with an in-house reverse transcription and polymerase chain reaction (RT-PCR) assay and the branched DNA (bDNA) assay (Quantiplex, version 1.0, Chiron Diagnostics). Concordance of the qualitative results with the Amplicor HCV and in-house RT-PCR assays occurred in 82% of the samples. All but one of the discrepant specimens were found positive by the Amplicor HCV assay and negative by the in-house RT-PCR. Among the samples with HCV RNA levels measurable with the Amplicor HCV Monitor assay, 22% had HCV RNA titers below the detection limit of the Quantiplex assay. A statistically significant correlation was found between the 2 quantitative assays, although lower titers were obtained with the Amplicor HCV Monitor assay. More important, a good correlation was observed in the evolution of viremia as measured by the 2 assays during interferon therapy. During follow-up of interferon treatments, with the Amplicor HCV Monitor assay, persisting viremia was still detected in 27% of the patients who normalised alanine aminotransferase (ALT), emphasizing the bioclinical relevance of the assay. Pre-treatment serum HCV RNA levels above 10(5) copies/ml were found more frequently in nonresponders than in responders (76% vs. 44%; P < 0.05). Given their great sensitivity and the significant correlations, the Amplicor HCV qualitative and quantitative assays appear useful for the diagnosis and management of hepatitis C infection, and especially for monitoring of therapy.

Development of a reverse transcriptase PCR-enzyme-linked immunosorbent assay for quantification of human immunodeficiency virus type 1 RNA in plasma: comparison with commercial quantitative assays.

A quantitative reverse transcriptase PCR assay with automated detection by nonradioactive hybridization was developed for the determination of human immunodeficiency virus (HIV) type 1 RNA levels. This assay is based on the use of an external standard curve with an internal standard. The accuracy of quantification was verified by comparison with reference commercial tests, the Chiron branched-DNA and Roche AMPLICOR HIV MONITOR assays. This assay was able to quantify viremia in patients with CD4 cell numbers below and above 500/mm3 and to quantify some HIV strains which could not be titrated by the MONITOR assay.

Predictive value of HIV-1 RNA detection in plasma by branched DNA assay during long-term zidovudine therapy.

The predictive value of human immunodeficiency virus type 1 (HIV-1) RNA detection in plasma using branched DNA assay was studied in a subgroup of 36 asymptomatic HIV-1-infected patients enrolled in a multicenter, double-blind, randomized study. Patients were randomized to receive either zidovudine (AZT) 1 g/day or placebo and were monitored for a mean time of 61 months. HIV-1 RNA was detected in plasma using branched DNA assay at months 0, 6, 12, 24, and 36. HIV-1 RNA was detected at levels of > or = 10(4) RNA eq/ml (eq/ml) in 8.3% of the patients at baseline, and this percentage increased during the first two years in the placebo group only. The detection rate of HIV-1 RNA at three years was 50% in both the AZT and the placebo groups. HIV-1 RNA levels ranged from 10(4) to 478 x 10(3) RNA eq/ml. HIV-1 RNA was detected at levels of > or = 10(4) eq/ml a mean time of 19 +/- 13 months before progression to AIDS in 76.5% of progressing patients. In a multivariate analysis including baseline CD4+ count, the initial randomization group, HIV-1 RNA detection in plasma, and detection of p24 antigenemia during the first three years of follow-up, the best independent predictors of progression to AIDS at five years and the best independent predictors of death at five years were HIV-1 RNA detection in plasma and p24 antigenemia.

Stability of hepatitis C virus RNA in serum from samples collected in a closed-tube system for serum separation and transport, as measured by a quantitative competitive PCR assay.

Recovery of hepatitis C virus (HCV) RNA, after variable time intervals from collection, was assessed using a closed-tube system for collection, separation and transport (SST tubes). Blood from four hepatitis C-infected patients was collected in 12 SST tubes and centrifuged within 1 to 3 h of collection. Tubes were then left 0, 8, 12, 24, 48 and 72 h at room temperature and at 4 degrees C before removing serum. Hepatitis C virus RNA levels were measured by quantitative polymerase chain reaction (PCR) using the AMPLICOR HVC MONITOR assay. Hepatitis C virus RNA levels in these samples were stable for at least three days at both temperatures. Polymerase chain reaction signals never decreased by more than 0.5 log. The reproducibility of the assays showed that the quantitative PCR method can be used with the storage conditions tested here. Our data suggests that processing blood in SST tubes may be very useful in following hepatitis C virus RNA titres in infected patients, especially those receiving treatment.

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n = 20) and extrahepatic (sera, n = 32; PBMC, n = 26 and fresh bone marrow cells, n = 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5' noncoding region, with or without a tag selected to display different viral loads (10(5)-3 x 10(7) genomic equivalent/ml or gram) and viral genotypes (n = 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, even either 5' noncoding region primer pair was used, whereas both artifacts were dramatically reduced (mispriming) or eliminated (selfpriming) using CAP-based RT-PCR assay. Mispriming artifacts were directly correlated to the titer of positive strand RNA present in the sample. Using the CAP-PCR assay, the presence of HCV negative strand RNA was found in 75% of livers (16:20) and only 8% of PBMC, independent of the genotype involved, but could not be documented in sera (0:32) and fresh bone marrow cells (0:6). These findings suggest that caution regarding the type of RT-PCR assay used and the level of HCV positive strand RNA present in the biological sample analyzed has to be taken to avoid false identification of viral reservoirs. The findings suggest that hematopoietic peripheral cells can support HCV replication, although in a very limited number of carriers.