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.

Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.

Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion.

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.

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.

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.

Toxicity and activity of purified trichosanthin.

Trichosanthin was purified from fresh Chinese root tubers of Trichosanthes kirilowii and evaluated for anti-HIV activity. Trichosanthin inhibited syncytium formation between infected H9 cells and uninfected Sup-T1 cells from 0.5 to 4 micrograms/ml. Trichosanthin also inhibited HIV replication in H9 and CEM-SS cells at 1 microgram/ml, but was toxic for MT-4 cells (HTLV-I-positive), at doses greater than 0.25 microgram/ml. This new purification procedure confirms the anti-HIV activity of trichosanthin on some cell lines in different biological assays.

Comparison between direct binding, competition and agglutination assays in the characterization of polyclonal anti-idiotypes against anti-HBs human monoclonal antibodies.

Polyclonal anti-idiotypic antibodies to human monoclonal anti-HBs antibodies (MoAb1) were raised in rabbits and designated Ab2-H1 and Ab2-H2. These Ab2 were characterized using three assays. A direct binding ELISA evaluated specificity towards a panel of human monoclonal antibodies and gamma globulins. Competition radioimmunoassay (CRIA) revealed Ab2 specificities towards Ab1 antigen binding sites by inhibition of HBsAg/Ab1 binding. Ab2-H1 and Ab2-H2 had comparable reactivities in ELISA and CRIA, whereas, using affinity purified Ab2, a fast (10 min) agglutination test (Spherotest) revealed different Ab1/Ab2 binding properties. Ab2-H1 reacted in this Spherotest with the Ab1 against which it was known to be specific (Ab1-H1), whereas in the same assay Ab2-H2 showed no activity towards the variable regions of the Ab1 used for its production (Ab1-H2). When injected into rabbits Ab2-H1 induced anti-HBs Ab3 antibodies but Ab2-H2 did not, thereby confirming the assay results.

Human monoclonal antibodies with a protective activity against tetanus toxin.

The antibodies and their protective activity in response to tetanus toxoid in man were studied by producing human antitetanus monoclonal antibodies after transformation of peripheral blood lymphocytes with Epstein Barr virus. Two human monoclonal IgG that reacted with the heavy chain of the toxin were obtained. One of them binds the COOH-terminal moiety and the other the NH2-terminal moiety. Only the NH2-terminal specific monoclonal antibody neutralized toxin in mouse, but in doses approximately 100-fold higher than those of a polyclonal antiserum. However, the association of these 2 antibodies was protective with doses lower than necessary for the monoclonal antibodies alone. To replace the polyclonal antibodies used, a good protection could be achieved by mixed human monoclonal antibodies against different epitopes of tetanus toxin.

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.

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.

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.

[Antitetanus vaccination by Polan T in the elderly: followed of the immune response by Vaccitest T after vaccination and booster]. / Vaccination antitétanique par Polan T dans une population âgée: suivi de la réponse humorale par le Vaccitest T après vaccination et rappel.

Tetanus vaccine activity without mineral adjuvant (Polan T) has been measured in a population of aged people (36 females and 11 males-mean age = 80.18 years). Titrations by hemagglutination of sensitized turkey red cells are performed before and after primo-immunization and first booster. This research gives knowledges regarding to the antitetanus response in elderly, regarding to the efficiency of the Polan T vaccine in this population uneasy to immunize and regarding to the quality of the simple and cheap assay for antitetanus antibodies titration.

Combined tetanus-rabies vaccination.

Studies in mice have shown that Calcium Phosphate adsorbed tetanus toxoid (IPADT) can be used as a vehicle for freezedried rabies vaccine. Trials were undertaken in human volunteers and patients receiving a post-exposure treatment using the same vaccines to evaluate tolerance and antibody response to both vaccines.

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.

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.