An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains.

The objective was to develop a method of HCV genome sequencing that allowed simultaneous genotyping and NS5A inhibitor resistance profiling. In order to validate the use of a unique RT-PCR for genotypes 1-5, 142 plasma samples from patients infected with HCV were analysed. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. In parallel, partial NS3 sequences were analyzed obtained for genotyping. Phylogenetic analysis showed concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and compared to the list of known Resistant Associated Substitutions recently published. In conclusion, this tool allows determination of HCV genotypes, subtypes and identification of NS5A resistance mutations. This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5) in the EU and the US.

Emergence of HCV resistance-associated variants in patients failing sofosbuvir-based regimens: an observational cohort.

BACKGROUND: Real-life effectiveness data of new hepatitis C direct-acting antivirals are now required. The present study aims to assess the rate of sustained viral response (SVR) and virological failure (VF) in patients infected with chronic HCV treated with sofosbuvir (SOF)-based regimens in routine medical practice. METHODS: This observational study included a total of 106 patients infected with HCV genotypes (G)1-4, who initiated SOF-based regimens in 2014. Viral load was followed at baseline, week (W)2, W4, W12 (or W24) and W12 post-treatment. For all VFs, resistance-associated variants (RAVs) were determined at baseline and failure by sequencing of NS5A, NS5B and/or NS3 genes, using the Sanger method. RESULTS: SVR rate was 85% for the whole cohort, 91% for the patients who underwent the full treatment course. The distribution of HCV genotypes was as follows: G1 n=66 (1a=33, 1b=29; 62%), G2 n=8 (8%), G3a n=20 (19%) and G4 n=12 (11%). The main regimens used were SOF+daclatasvir (37%), SOF+ribavirin (33%), SOF+simeprevir (26%) and SOF+ledipasvir (3%). Twenty-five (23%) patients were HIV-coinfected and 1 was HBV-coinfected. Seventy (65%) patients had a prior treatment experience. All VF were relapses (n=9): 3 G1a, 1 G2, 4 G3a and 1 G4, and mutations conferring resistance to NS5A inhibitors were found but none for NS5B polymerase inhibitors. CONCLUSIONS: In a real-life context, the rate of SVR in DAA-treated HCV-infected patients is close to clinical Phase III trial results. RAVs emerged for all patients treated by the anti-NS5A daclatasvir, and persisted several weeks after the end of treatment.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin.

The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.

Quantification of two viral transcripts by real time PCR to investigate human herpesvirus type 6 active infection.

BACKGROUND: Human herpesvirus 6 (HHV-6) causes exanthema subitum and is associated with symptomatic reactivations in immunocompromised patients, particularly after hematopoietic stem cell transplantation. The detection of viral mRNA can help to make the difference between latent, chromosomally integrated and true replicating virus. It can also be a useful tool to investigate viral multiplication in different cell types. OBJECTIVES: To develop molecular tools for the detection and quantification HHV-6 transcripts that can be used in a clinical setting. STUDY-DESIGN: Two one-step reverse-transcriptase quantitative real-time PCR (RT-qPCR) were developed for the quantification of the immediate early U90 and the late U100 mRNAs. Viral mRNA loads were compared to viral DNA loads during infection in vitro and in blood samples collected from stem cell transplanted patients. RESULTS: Analytical performances of the two quantitative real-time PCR were good. In vitro, kinetics of both transcripts was well correlated with DNA levels. Sixty blood samples from patients with active HHV-6 infection were analyzed. Overall agreement of qualitative results for HHV-6 DNA, U90 RNA and U100 RNA was good. HHV-6 DNA loads were significantly higher than mRNA loads. In clinical samples, the amounts of U100 and U90 mRNAs were low and their detection was mainly associated to viral DNA loads upper than 1000 copies/ml of blood. CONCLUSION: The new assays are sensitive and reliable methods for the monitoring of viral transcription in vitro and in vivo. As their detection is associated to high DNA loads in vivo, they can be helpful tools for the diagnosis of active infection.

A FUT2 gene common polymorphism determines resistance to rotavirus A of the P[8] genotype.

Attachment to carbohydrates of the histo-blood group type of several human Rotavirus strains (RVA) has recently been described. Synthesis of these ligands requires a functional FUT2 enzyme, suggesting that FUT2 null homozygote (ie, nonsecretor) individuals may not be recognized by most human RVA strains. Whereas such individuals represent 20% of the control population, this retrospective study determined that none of 51 patients infected by P[8] rotavirus strains were nonsecretors. The lack of α1,2fucosylated carbohydrate motifs in the gut surface mucosa is thus associated with resistance to symptomatic infection and virus attachment to such motifs is essential to the infection process.

Genotyping and resistance profile of hepatitis C (HCV) genotypes 1-6 by sequencing the NS3 protease region using a single optimized sensitive method.

The objective was to develop a method of NS3 gene sequencing that allowed simultaneous genotyping and protease inhibitor (PI) resistance profiling of HCV genotypes 1-6. To validate the use of a unique RT-PCR for genotypes 1-6 and evaluate its sensitivity, the NS3 protease region was amplified from 140 plasma samples from patients infected with HCV without previous PI therapy. In parallel, NS5b sequences were obtained. Amplification of NS3 was successful in 139/140 samples (99%). For the 135 samples with both NS5b and NS3 sequencing results, phylogenetic analysis showed concordance of genotypes with a bootstrap >90% for each cluster. PI resistance mutations were analyzed using the Geno2pheno [hcv] v1.0 tool. For the 63 genotype 1 (G1) Nantes clinical strains, 12 (19%) presented a natural resistance mutation. This proportion was higher (p<0.05) than that observed in a sample of 374 G1 reference sequences. This significant difference was observed only in subtype 1b (n=7; 25% against n=19; 8%). In conclusion, this tool allows determination of both HCV genotype and identification of PI-resistance mutations. It can be used to detect pre-existing resistance mutations in NS3 before treatment and follow the emergence of resistant viruses during therapy.

Influenza virus A subtype H1N1 is inhibited by methylated ß-lactoglobulin.

Addition of methylated ß-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 µg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO2. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 µg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 µg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.

Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature.

For several years, azole antifungal drugs have been a treatment option for potentially life-threatening Candida infections. However, azole resistance can occur through various mechanisms such as alterations in ERG11, encoding lanosterol 14alpha-demethylase (CYP51). In this study, we investigated the antifungal susceptibility to fluconazole, itraconazole, and voriconazole of 73 clinical isolates of Candida albicans. Screening for amino acid substitutions in Erg11 was performed on each of the 73 isolates. Twenty isolates displayed a marked decrease in azole susceptibility. Amino acid substitutions were detected in more than two-thirds of the strains. In all, 23 distinct substitutions were identified. Four have not been described previously, among which N136Y and Y447H are suspected to be involved in azole resistance. We suggest that the high genetic polymorphism of ERG11 must be considered in the rationale design of new azole compounds targeting lanosterol 14alpha-demethylase. A review of all Erg11 amino acid polymorphisms described to date is given.

Antiviral Action of Methylated ß-Lactoglobulin on the Human Influenza Virus A Subtype H3N2.

Antiviral activity of methylated ß-lactoglobulin (Met-BLG) against H3N2 infected into MDCK cell lines depended on concentration of Met-BLG, viral load, and duration of infection. IC50% of the hemagglutination activity for 1 and 0.2 MOI (multiplicity of infection) after 24 h of incubation at 37 °C in the presence of 5% CO2 were 20 ± 0.8 and 17 ± 0.7 µg mL(-1) Met-BLG, respectively. Longer incubation period (4 days) was associated with low IC50% of the hemagglutination activity (7.1 ± 0.3 µg mL(-1) Met-BLG) and low IC50% of immuno-fluorescence of viral nucleoproteins (9.7 ± 0.4 µg mL(-1) Met-BLG) when using 0.2 and 0.1 MOI, respectively. A concentration of 25 µg mL(-1) of Met-BLG reduced the amount of replicating virus by about 2 and 1.3 logs when the viral load was 0.01 and 0.1 MOI, respectively, while higher concentrations reduced it by about 5-6 logs. Antiviral action of Met-BLG was coupled with a cellular protective action, which reached 100% when using 0.01 and 0.1 MOI and 83% when using 1.0 MOI. The time of Met-BLG addition after the viral infection was determinant for its antiviral efficacy and for its protection of the infected MDCK cell lines. Anti-hemagglutination action and cell protective action decreased gradually and in parallel with the delay in the time of Met-BLG addition to disappear totally after 10 h delay.

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR).

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test. EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p<0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs. There was a significant difference (p<0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing. In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy.

Two quantitative duplex real-time PCR assays were developed for co-amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 10(6) peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 x 10(9) PBLs/L. Albumin co-amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre-emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article.

Molecular study of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among human immunodeficiency virus-infected patients from two geographical areas: Niamey, Niger, and Hanoi, Vietnam.

Microsporidiosis cases due to Enterocytozoon bieneusi and Encephalitozoon intestinalis are emerging opportunistic infections associated with a wide range of clinical syndromes in humans. The aim of this study was to specify microsporidial epidemiology in two different geographical areas. From November 2004 to August 2005, 228 and 42 stool samples were collected in Niamey, Niger, and Hanoi, Vietnam, respectively. Screening for microsporidia was performed using UV-light microscopy. Detection was confirmed by molecular biology using two methods specific for E. bieneusi and E. intestinalis. All samples positive for E. bieneusi were subjected to genotyping. In this study, we found high prevalences of microsporidiosis among human immunodeficiency virus-infected patients, 10.5% and 9.5%, respectively, in Niamey and Hanoi. These levels of prevalence are similar to those recorded in European countries before highly active antiretroviral therapy was introduced. In the samples positive for E. bieneusi, we found seven distinct genotypes, including two genotypes not previously described. The E. bieneusi genotype distributions in the two geographical areas suggest different routes of infection transmission, person-to-person in Niger and zoonotic in Vietnam.