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Background Men who have sex with men are increasingly diagnosed with sexually transmitted infections (STI) in France. To address this situation, quarterly screening for HIV combined with hepatitis B (HBV) and hepatitis C (HCV), as well as annual screening for C.trachomatis (CT) and N.gonorrhoeae (NG) are recommended. The MemoDepistages program offered an at-home screening solution for these infections. This study describes the feasibility of this screening process, the rate of positive test results, and the factors associated with positivity. Methods Participants were recruited online. Laboratories verified the quantity and quality of the samples. Logistic regression was used to determine the associated factors for infection. Results Overall, 1556 out of 1908 (81.6%) blood samples were tested for at least HIV. A total of eight participants (0.5%) were newly diagnosed with HIV and four with HCV (0.3%). No new infection was confirmed for HBV. Overall positivity was 9.3% for CT and 9.6% for NG. The highest positivity was reported in rectal swabs for CT (7.3%) and in pharyngeal swabs for NG (7.2%). Factors associated with extragenital CT/NG were age under 30 years (for pharyngeal and rectal infections) and having at least 10 partners in the past 6 months ( pConclusions The self-sampling kit for multiple STIs can perform comprehensive tests and identify new infections in young people, especially in extragenital sites.

Background & aim We investigated the combination of rapid antigen detection (RAD) and RT-qPCR assays in a stepwise procedure to optimize the detection of COVID-19. Methods From August 2020 to November 2020, 43,399 patients were screened in our laboratory for COVID-19 diagnostic by RT-qPCR using nasopharyngeal swab. Overall, 4,691 of the 43,399 were found to be positive, and 200 were retrieved for RAD testing allowing comparison of diagnostic accuracy between RAD and RT-qPCR. Cycle threshold (Ct) and time from symptoms onset (TSO) were included as covariates. Results The overall sensitivity, specificity, PPV, NPV, LR-, and LR+ of RAD compared with RT-qPCR were 72% (95%CI 62%–81%), 99% (95% CI95%–100%), 99% (95%CI 93%–100%), and 78% (95%CI 70%–85%), 0.28 (95%CI 0.21–0.39), and 72 (95%CI 10–208) respectively. Sensitivity was higher for patients with Ct ≤ 25 regardless of TSO: TSO ≤ 4 days 92% (95%CI 75%–99%), TSO > 4 days 100% (95%CI 54%–100%), and asymptomatic 100% (95%CI 78–100%). Overall, combining RAD and RT-qPCR would allow reducing from only 4% the number of RT-qPCR needed. Conclusions This study highlights the risk of misdiagnosing COVID-19 in 28% of patients if RAD is used alone. A stepwise analysis that combines RAD and RT-qPCR would be an efficient screening procedure for COVID-19 detection and may facilitate the control of the outbreak.

BackgroundDiagnosing SARS CoV-2 infection with certainty is essential for appropriate case management. We investigated the combination of rapid antigen detection (RAD) and RT-qPCR assays in a stepwise procedure to optimize the detection of COVID-19.MethodsFrom August 2020 to November 2020, 43,399 patients were screened in our laboratory for COVID-19 diagnostic by RT-qPCR using nasopharyngeal swab. Overall, 4,691 of the 43,399 were found to be positive, and 200 were retrieved for RAD testing allowing comparison of diagnostic accuracy between RAD and RT-qPCR. Cycle threshold (Ct) and time from symptoms onset (TSO) were included as covariates.ResultsThe overall sensitivity, specificity, PPV, NPV, LR-, and LR+ of RAD compared with RT- qPCR were 72% (95%CI 62%–81%), 99% (95% CI95%–100%), 99% (95%CI 93%–100%), and 78% (95%CI 70%–85%), 0.28 (95%CI 0.21-0.39), and 72 (95%CI 10-208) respectively. Sensitivity was higher for patients with Ct ≤ 25 regardless of TSO: TSO ≤ 4 days 92% (95%CI 75%–99%), TSO > 4 days 100% (95%CI 54%–100%), and asymptomatic 100% (95%CI 78-100%). Overall, combining RAD and RT-qPCR would allow reducing from only 4% the number of RT-qPCR needed.ConclusionThis study highlights the risk of misdiagnosing COVID-19 in 28% of patients if RAD is used alone. Thus, negative results from RAD needs to be confirmed by RT-qPCR prior to making treatment decisions. A stepwise analysis that combines RAD and RT-qPCR would be an efficient screening procedure for COVID-19 detection and may facilitate the control of the outbreak.

Background & aim Screening for genital infection (GI) such as bacterial vaginosis (BV) and yeast infection, for sexually transmitted infection (STI), and for asymptomatic carriage of group B streptococcus (GBS) in pregnant women are common reason for medical appointments. The diagnosis and control of GIs, STIs, and GBS are major issues, for fertility and overall well-being of affected women. Conventional testing is performed using vaginal/cervical classical sampling (VCS); this procedure requires pelvic examination performed by health care professionals which raises concerns among women. Vaginal-self-sampling (VSS), as an alternative to VCS, might capture more women. The aim was first to show non-inferiority of VSS compared with VCS to screen for GIs, STIs, and GBS; second to determine the feasibility of VSS. Methods VSS and VCS from 1027 women were collected by health care professionals and simultaneously carried out on each patient. GIs, STIs, and GBS were systematically screened in both paired VSS and VCS samples. Non-inferiority of VSS compared with VCS was assessed using z statistic for binomial proportions. Results Prevalence of GIs were 39.7% using VSS and 38.1% using VCS (p = 0.0016). Prevalence of STIs was 8.5% (VSS) vs 8.1% (VCS) (p = 0.0087). Prevalence of GBS was 13.4% (VSS) and 11.5% (VCS) (p = 0.0001). Most participants (84%) recommended the use of VSS. Conclusions This study shows that VSS was not inferior to VCS for the detection of GIs, STIs, and GBS. This study provides evidence that VSS can be used as a universal specimen for detection of lower genital tract infections in women. Study identification number ID-RCB 2014-A01250-4.

Background Recent data have suggested that failure to achieve sustained virological response with direct-acting antiviral therapy is usually due to relapse and is primarily associated with the emergence of resistance-associated substitutions. The aim of this study was to investigate the prevalence and characterization of non-structural-5A resistance-associated substitutions in patients infected with HCV genotypes 1, 3 and 4 treated by direct-acting antiviral therapy, including anti-non-structural-5A, and to characterize the pre-existing resistance-associated substitutions in subjects treated with anti-non-structural-5A inhibitors. Methods From January 2014 to March 2016, 2,995 patients infected with HCV genotypes 1, 3 and 4 were exposed to non-structural-5A inhibitors. Sequencing results at the time of virological failure were available for 61 patients; sequencing at baseline was available for 35 of these patients. Results Among the 35 patients with sequencing results available at baseline, 15 had no resistance-associated substitution, 16 had only one resistance-associated substitution, and 4 had more than one resistance-associated substitution. Resistance-associated substitutions were harbored in 57% of the sequences in the non-structural-5A region. Among the 61 patients sequenced at virological failure, 50 (82%) patients presented at least one resistance-associated substitutions inducing a high level of resistance to non-structural-5A inhibitors (>10-fold resistance). Conclusions This pooled analysis suggests that non-structural-5A resistance-associated substitutions screening should be recommended when considering retreatment with a non-structural-5A inhibitor regimen in patients who have previously experienced failed non-structural-5A treatment.

High-risk human papillomaviruses (HPVs) have been identified as the main contributors to cervical cancer. Despite various diagnostic tools available, including the predominant Papanicolaou test (Pap test), technical limitations affect the efficiency of cervical cancer screening. The aim of this study was to evaluate the diagnostic performance of spliced HPV16 E6/E7 mRNA viral loads (VL) for grade 2 or higher cervical intraepithelial neoplasia diagnosis. A new dedicated (quantitative reverse transcription polymerase chain reaction) qRT-PCR assay was developed, allowing selective quantification of several HPV16 E6/E7 mRNA: Full length (FL) with or without all or selected spliced forms (total E6/E7 mRNA corresponding to SP + E6^E7 mRNA (T), + spliced E6/E7 mRNA containing intact E7 ORF (SP), and E6/E7 mRNA containing disrupted E6 and E7 ORFs calculated by the following subtraction T-SP (E6^E7)). Twenty HPV16 DNA and mRNA positive uterine cervical smears representative of all cytological and histological stages of severity were tested. We have shown that all E6/E7 mRNA isoforms expression levels were significantly increased in high grade cervical lesions. Statistical analysis demonstrated that the SP-E6/E7 VL assay exhibited: (i) The best diagnostic performance for identification of both cervical intraepithelial neoplasia (CIN)2+ (90% (56&ndash, 100) sensitivity and specificity) and CIN3+ (100% (72&ndash, 100) sensitivity and 79% (49&ndash, 95) specificity) lesions, (ii) a greater sensitivity compared to the Pap test for CIN2+ lesions detection (80% (44&ndash, 97)), (iii) a predictive value of the histological grade of cervical lesions in 67% of atypical squamous cells of unknown significance (ASC-US) and 100% of low-grade (LSIL) patients. Overall, these results highlight the value of SP-E6/E7 mRNA VL as an innovative tool for improving cervical cancer screening.

We describe the first case of a patient presenting Kaposi's sarcoma with human herpes virus 8 (HHV8) viremia after switching from a protease inhibitor to an integrase inhibitor-based combination antiretroviral therapy, followed by a rapid remission when resuming protease inhibitor. We suggest that the recent recommendations to switch all HIV patients to protease inhibitor-free regimens should be carefully re-evaluated especially in MSM HIV patients which are at higher risks of HHV8 infections and associated malignancies.

Summary Background The impact of the IL28B genotype on the real-life treatment decisions for patients infected with the hepatitis C virus (HCV) is unknown. Objective To prospectively analyze the impact of IL28B genotype in HCV genotype 1 (G1)- or 4 (G4)-infected patients using buccal epithelial cell samples in real-life clinical practices. Patients and methods From October 2011 to March 2013, 1007 CHC patients were included among 127 French clinical centers. Results The IL28B CC, CT, and TT genotype distribution was 252 (25%), 576 (57%), and 177 (18%), respectively. The treatment decisions were recorded and matched with the initial intentions for 433 patients. Multivariate analysis on intention to start treatment showed that patients with HCV G4 were less likely to be intended to be treated than HCV G1 patients (odds ratio [OR] = 0.43 [95% CI 0.19–0.97], P = 0.04); similarly HIV-HCV coinfected patients were less likely to be intended to be treated than HCV monoinfected patients (OR = 0.20 [0.09–0.41], P P = 0.004). Multivariate analysis on final decision to treat showed that Patients with F3–F4 were more likely to be treated than others (OR = 2.06 [1.26–3.38], P = 0.004). Conversely, although P -values are not significant, patients recruited in public hospitals tended to be less treated (OR = 0.65 [0.40–1.04], P = 0.069), similarly to HIV-HCV coinfected patients (OR = 0.55 [0.28–1.11], P = 0.095). Conclusion Our study showed that the IL28B genotype is used for the management of HCV-infected patients. In the context of future treatments, IL28B genotyping may remain useful if it can be used to develop individualized treatment strategies, identifying patients who can be successfully treated with shorter, simpler, or cheaper regimens.

Background: Men who have sex with men (MSM) are disproportionately affected by sexually transmitted infection. The aim of this cross-sectional study is to prospectively detect the prevalence of chlamydia trachomatis (CT), neisseria gonorrhoeae (NG), mycoplasma genitalium (MG), and high risk human papillomavirus (HR-HPV), and syphilis in a population of asymptomatic sexually active MSM. Methods: Rectal, pharyngeal, and urine samples for CT, NG, MG, and HR-HPV were analyzed in 116 MSM patients attending the clinic for their routine follow-up during the period the study was conducted: 99 patients were issued from the clinic routine follow-up for their HIV infection, and 17 attended the clinic because they were sexual partners of an HIV infected male. Results: An STI was found in 16% of the patients (19/116), with at least one bacterial strain (CT, NG, or MG) found in one site (the pharynx, rectum, or urine). Conclusions: In this study, 16% of the MSM reporting recent RAI were asymptomatic carriers of rectal CT, NG, or MG. According to the high prevalence of asymptomatic STIs found in our MSM population and in other studies, prevention efforts in the form of counseling about the risk of STI need to be done in the population of MSM.

Background and objective The aim of this study was to prospectively evaluate whether the addition of peg-IFN to a stable NA regimen leads to loss of HBsAg in HBeAg-negative patients with chronic hepatitis and HBV DNA fully suppressed by long-term NA treatment. Study design We analyzed HBsAg levels in 10 HBsAg-positive, HBeAg-negative patients who received peg-IFN alpha-2a in addition to a NA regimen. Treatment lasted a maximum of 96 weeks, according to changes in the HBsAg titer. Before peg-IFN therapy, HBV DNA levels had been below the limit of detection for at least three years. Results HBsAg levels declined in nine patients. Among these nine, four became HBsAg-negative after 48 weeks of peg-IFN treatment; these patients received peg-IFN for only 48 weeks. NAs were stopped in these four patients, and these levels remained stable for at least 18 months (loss of HBsAg; HBV-DNA negative). HBs seroconversion was observed in two patients. The remaining five patients received 96 weeks of peg-IFN therapy. One patient became HBsAg-negative at the end of peg-IFN therapy; another became HBsAg-negative six months later. Three patients did not become HBsAg-negative. NAs were stopped in the two patients who became HBsAg-negative with no relapse during 12 months of follow up. Conclusions In HBsAg-positive, HBeAg-negative patients with HBV DNA were fully suppressed by long-term NA treatment, the addition of peg-INF for a maximum of 96 weeks based on HBsAg-titer monitoring led to a loss of HBsAg and cessation of NA therapy in six out of ten patients, with no relapse for 12–18 months of follow up. HBs seroconversion was observed in two patients.

The objective of this study was to assess the analytical and clinical relevance of minority variants using a new pyrosequencing (PSQ) assay and to detect minor variants with frequencies below the current 20% clinical setting limit. A PSQ approach for detecting and quantifying mutations was developed for the analysis of 14 codons of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene below the limit of conventional sequencing. Ten patients who experienced virological failure (VF) after a first-line regimen of lamivudine, tenofovir, and either efavirenz, nevirapine, or etravirine, as well as 10 controls patients without VF, were included in this retrospective study. Baseline plasma and plasma from the time of VF were assessed using Sanger sequencing and PSQ methods. The analytical sensitivity for the detection of minor sequence variants is 5%. At baseline, no minority variant was detected in 10/10 patient controls using both the Sanger sequencing and PSQ assays, whereas, two patients who failed therapy had baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations that were not detected by the standard genotyping. At the time of VF, standard genotyping detected mutations in four out of the 10 VF patients, whereas, PSQ detected mutations in five out of the 10 VF patients. Clinically, minority mutations at a 10% level of detection can be assessed efficiently by pyrosequencing and used as a suitable predictor of the evolution of viral populations. These traits allow for a better interpretation of data analysis, which can help clinicians in providing a suitable treatment for HIV. J. Med. Virol. 86:394–403, 2014. © 2013 Wiley Periodicals, Inc.

Preemptive therapy in hematopoietic cell transplantation is initiated by a diagnostic technique at first detection of cytomegalovirus (CMV). The aim of this study was to use the viral dynamics of CMV DNA viral to start preemptive therapy, and to prospectively compare the CMV viral load kinetics to pp65 antigenemia. Two hundred sixty-three blood samples were collected prospectively from 93 patients. All clinical decisions regarding use of preemptive therapy were based on CMV antigenemia. Based on the positivity of the antigen assay and clinical CMV outcome in allotransplant patients, an optimal threshold of 3.05 log 10 (1,130 copies/ml) was found to discriminate patients who required preemptive therapy and those who did not (sensitivity, 71%; specificity, 65%). A DNAemia level increase of 2.24 log 10 (174 copies/ml) per day was the optimal threshold to discriminate between patients who required preemptive therapy and those who did not (sensitivity, 93%; specificity, 43%). Sensitivity of PCR assay was 92.4% compared with 39% for the antigen assay (P

BACKGROUND The detection of high-risk human papillomavirus (HPV) DNA provides higher sensitivity but lower specificity than cytology for the identification of high-grade cervical intraepithelial neoplasia (CIN). OBJECTIVES The aim of this study was to combine DNA- and RNA-based assays in order to improve the detection of advanced disease (CIN2+). METHODS 107 ASCUS+ women were included in Marseilles (France) between March 2007 and June 2008. Up to five tests were carried out on a liquid PreservCyt sample: Hybrid Capture 2 (HCII) (Digene), Papillocheck (Greiner), Abbott RealTime HR HPV (RT HR HPV) (Abbott), Linear Array (Roche), and EasyQ HPV (Biomerieux). RESULTS 36 (34%) women had CIN2+ histology; among them 6 (6%) had CIN3+ histology. For CIN2+ detection, all tests had comparable sensitivities except EasyQ HPV test: HCII 94%, Papillocheck and LA 92%, RT HR HPV 89%, and NucliSENS EasyQ HPV 75% (P not significant). On the other hand, EasyQ HPV had a higher specificity than the other assays (except RT HR HPV assay). Combining DNA-based and RNA-based assay would allow to reduce the need of colposcopies to be performed among patients HPV positive with DNA-based assay: 32% (7/22) colposcopies not needed in < 30 years old patients, and 29% (14/49) colposcopies not needed in ≥ 30 years patients. CONCLUSIONS All tests had comparable diagnostic values for CIN2+ detection, but DNA-based tests seemed to be more sensitive and RNA-based assay more specific. The combined use of DNA- and RNA-based assays considerably reduces the number of colposcopies to be performed, and provides better diagnostic accuracy for CIN2+ disease.

Background HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66 are considered carcinogenic for human beings. DNA-chip technology, Papillocheck ® HPV-screening (Greiner) and reverse dot blot, Linear Array (LA) (Roche) are tools to assess the distribution of HPV genotypes. Objectives The aim of the study was to compare the clinical performance of Papillocheck and LA assays using a clinical cut-off of CIN2+. The secondary aim was to comparatively assess the distribution of HPV types using these two assays. Study design The study population comprised 239 women referred for colposcopy and histology. Papillocheck, LA, and Hybrid Capture II (HCII) tests were done on all samples. Results All tests showed good sensitivity and NPV (greater than 90%). None of the comparisons of sensitivities, specificities, PPVs, and NPVs showed statistically relevant differences between tests. High-risk HPV positivity rate was similar for all tests (Papillocheck 75%, LA 77%, and HCII 73%). Agreement between tests was good. The concordance levels between HCII and Papillocheck and between HCII and LA were 93% ( k =0.82) and 92% ( k =0.80), respectively. Papillocheck and LA tests showed a high overall concordance rate of 96% ( k =0.90). HPV16 was the most detected type (45% with Papillocheck, and 47% with LA), and HPV31 was the second most detected type (13% with Papillocheck, and 14% with LA). Conclusions The Papillocheck HPV-screening test and LA test have a good clinical sensitivity to detect HPV types in CIN2+ patients. These assays allow, in the same experiment, to detect and determine the virus type. Our study showed that HPV types 16 and 31/33 are the most prevalent.

Background/Aims Homocysteine metabolism is linked to DNA methylation, a mechanism potentially involved in the course of hepatitis B virus (HBV) infection. We evaluated the association of determinants of homocysteine metabolism with the outcome of HBV infection. Methods Four hundred and fifty-five healthy adults from Togo and Benin were tested for HBV serologic markers, HLA DR alleles, folate, vitamin B12, methylenetetrahydrofolate reductase (MTHFR) 677 C→T, 1298 A→C and methionine synthase 2756 A→G polymorphisms. Results Seventy-eight percent of the study population was anti-HBc positive. Among them, 202 (56.9%) were anti-HBs positive and 58 (16.3%) were HBsAg positive. After stepwise logistic regression, the MTHFR 677 T allele was independently associated with persistence of detectable anti-HBs antibodies (OR: 2.47; 95% CI: 1.29–4.71; p =0.006). The mean HBV DNA level was significantly lower in HBsAg positive subjects carrying the 677 T allele than in those with the 677 CC genotype (1000±1406 vs. 2,400,000±214,000 copies/ml, p =0.005). Beninese origin and HLA-DRB1*09 allele were the other determinants independently associated with favorable outcome of HBV infection. Conclusions The methylenetetrahydrofolate reductase 677 T allele seems to protect against chronic HBV infection in young African adults.

Background The detection of low frequency mutants in patients with hepatitis C virus (HCV) receiving direct-acting antivirals (DAAs) is still debated. The clinical relevance of the mutant viral load has not yet been evaluated. Objectives To assess the viral load of resistance associated variants (RAVs) in patients at different time points, including the baseline, virological failure and one year after the cessation of therapy. Study design The study included 22 patients who were previously treated with protease inhibitors (PI) (with telaprevir and boceprevir). For each patient, three time points were assessed using ultra-deep pyrosequencing (UDPS). Results Baseline mutations were observed in 14/22 patients (64%). At virological failure, RAVs were detected in 18/22 patients (82%). Persistent RAVs were observed in four HCV GT 1a patients (18%). Persistence mutations were found only in HCV GT 1a patients. The baseline relative V36M, R155K, R155T and A156T mutation load of patients with persistent RAVs was significantly higher ( P Conclusion The UDPS follow-up analysis demonstrated that the presence of BOC or TLP-RAVs persist one year after therapy cessation only in HCV GT 1a patients. The relative mutant viral load should be considered prior to any PI based re-treatment. This concept of the baseline mutation viral load must be validated using current therapy and must be validated on a larger cohort.

SUMMARY. The clinical significance of hepatitis B virus (HBV) genotypes is still under debate. The aims of this study were to assess the distribution of HBV genotypes in France and to identify the associations between HBV genotypes and patient demographics, severity of liver disease and HBeAg status in patients referred to tertiary care centres. This was a French, multicentre, retrospective study on 262 patients with chronic HBV infection. HBV genotypes were determined using INNO-LiPA. Liver fibrosis damage was evaluated by histological analysis of biopsy samples. Patients were mainly male (74%), of Caucasian (65%), Asian (17%) or African (18%) ethnicity and 36% were HBeAg positive. All A‐G genotypes were found, the most frequent being genotypes D (27%) and A (24%), followed by E (13%) and C (12%), and B (7%). Mixed genotypes were detected in 16% of the cases. Genotype A was associated with sexual contact (P < 0.001) and genotype D with transfusion (P < 0.001) and HBe antibody positivity (P ¼ 0.03).The distribution of HBV genotypes differed with regard to the ethnicity, and may reflect migration patterns. Genotypes A and D were the most frequent in France. Genotype A was associated with HBeAg positivity and genotype D with HBe antibody positivity. In our European patients, we find no clear association between a given HBV genotype and liver disease severity.

Background/Aims This study aimed at correlating the presence of extrahepatic manifestations with hepatitis B virus (HBV) genotypes in patients with chronic HBV infection. Methods This was a national (France), multicenter, retrospective, cross-sectional study. HBV genotypes were determined in 190 patients HBsAg-positive for at least 6 months and documented before any treatment. Results Patients were aged 42±15 years and mainly male (77%). Alcohol intake was high in 6% of them, ALT elevated in 73%; 27% were cirrhotic. All HBV genotypes were found, mainly A (24%), D (29%), C (11%), and E (10%). Thirty (16%) patients had clinical extrahepatic manifestations, mainly sensory-motor deficiency, sicca syndrome, myalgia, glomerulonephritis, and arthralgia-arthritis. Their presence was not related to any epidemiologic, viral (including genotypes) or hepatic factor, but to a higher platelet count ( P =0.004). Twenty-nine (15%) patients had biological extrahepatic manifestations, mainly anti-smooth muscle, antinuclear, and anti-nucleosome antibodies. Their presence was related only to anti-HBe antibodies positivity ( P =0.007) or elevated platelet count ( P =0.003). Carrying precore mutant HBV increased by 2.8 folds the risk to have at least one extrahepatic biological manifestation. Conclusions No relationships between HBV genotypes and the presence of extrahepatic manifestations were evidenced in patients with chronic HBV infection.

Development of hepatitis B virus (HBV)-resistant strains following nucleos(t)ide analogue treatment is a major concern. The A181V mutation within the reverse transcriptase (RT) of HBV has been shown to be associated with HBV resistance to adefovir dipivoxil (ADV), and its level of sensitivity to other nucleos(t)ide analogues is an important issue. This article reports two cases of chronically HBV infected patients who developed rtA181V HBV mutants following lamivudine (LAM) monotherapy. This was subsequently associated with virological breakthrough under LAM monotherapy or LAM/ADV bi-therapy, which were rescued by tenofovir disoproxil fumarate treatment. These observations suggest that rtA181V mutation may unusually emerge under LAM monotherapy, and may be associated with cross resistance to LAM and ADV, but remains sensitive to tenofovir disoproxil fumarate. Moreover, they highlight that HBV sequence analysis is an essential tool to optimize therapeutic management of HBV chronic infection in clinical practice in order to choose the appropriate nucleos(t)ide analogues.

WHO International Standards for nucleic acid tests are used widely to compare the different assays used in HCV RNA quantitation. The aim of the study was to assess the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. Twenty-seven naive patients infected chronically by HCV were treated with ribavirin plus PEG-interferon-alfa-2b for 48 weeks. SVR was obtained for 16 patients (the other were non-responders). For HCV RNA quantitation, four assays were undertaken: Versant HCV RNA 3.0 (Bayer), Real time PCR (TaqMan, Roche), LCx HCV RNA (Abbott), and Cobas Amplicor-Monitor v2 (Roche). Considering a 2-log decline at Week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non-response. At Week 4 and Week 12, significant differences were observed between Versant HCV RNA 3.0 versus PCR HCV Taqman, Versant HCV RNA 3.0 versus LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 versus LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 versus PCR HCV Taqman (P < 0.001). The HCV RNA cutoff, given a 100% negative predictive value at Week 4 and Week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. Eighty-nine percent of serum values for HCV RNA were concordant by the IU standard. All assays, however, failed to detect HCV RNA in some cases. Despite the use of the IU standard HCV-infected patients might be monitored with only one assay.

International audience; OBJECTIVE:To assess the prevalence of TT virus (TMV) infection in a series of patients with chronic hepatitis C virus (HCV) infection, with or without benign (mixed cryoglobulinemia) or malignant (B-cell non-Hodgkin lymphoma (B-NHL)) lymphoproliferative disease.METHODS:Sixty-six HCV patients were studied, including patients with mixed cryoglobulinemia (n=30), B-NHL (n=15), and no mixed cryoglobulinemia or B-NHL (n=21). All HCV patients had increased transaminase levels and were HCV RNA positive. Patients were considered to have mixed cryoglobulinemia if two successive determinations of their serum cryoglobulin level were above 0.05 g/L. Mixed cryoglobulinemia-negative patients never had mixed cryoglobulins in their serum on multiple determinations. Subjects without HCV infection included 79 patients with histologically proven B-NHL, and 50 healthy blood donors. Serum samples were analyzed for TTV DNA by nested polymerase chain reaction, with two couples of primers in different regions of the genome, in two independent laboratories.RESULTS:In the group of HCV-positive patients, TTV DNA was found in one of 15 (6.7%) patients with B-NHL, and in nine of 51 (17.6%, P = 0.43) of those without B-NHL. Among HCV-positive patients without B-NHL, TTV DNA was more frequently found in those with type II mixed cryoglobulinemia vasculitis than in those without it (six of 16 (37.5%) versus two of 21 (9.5%), P = 0.05). In subjects without HCV infection, TTV DNA was present in 10 of 79 (12.7%) patients with B-NHL and in seven of 50 (14.0%, P = 0.82) blood donors.CONCLUSION:In patients chronically infected with HCV, TTV co-infection: (1) is not associated with the presence of B-NHL; and (2) is more frequently found in patients presenting a type II mixed cryoglobulinemia vasculitis.

Hepatitis B surface antigen quantification (qHBsAg) is a relevant biomarker assay in the therapeutic management of hepatitis B virus-infected patients; however, little is known about its use in France. The aim of this study was to describe the knowledge of qHBsAg use and the indications for the prescription of qHBsAg in France.From March 2014 to May 2014, 135 questionnaires were sent to hepatologists and gastroenterologists from several health centers (private practice, public practice, and outlying health centers). There were 20 items in each questionnaire on the use of qHBsAg.Seventy-six percent of the practitioners had previously used qHBsAg, among whom 88% had prescribed the use of qHBsAg before treatment, 73% had prescribed the use of qHBsAg in combination with hepatitis B virus viral load, 64% had prescribed the use of qHBsAg at week 12 or week 24 of treatment, 62% had prescribed the use of qHBsAg for stopping rules, and 49% had prescribed the use of qHBsAg to identify inactive carriers. The reason for nonprescription of qHBsAg was mainly because of difficulty accessing the test (50% of the practitioners), followed by nonreimbursement of the test (27%); 97% of the practitioners who did not prescribe qHBsAg indicated an interest in accessing the test.This survey describes the characteristics of the prescription of qHBsAg in France. More than three out of four practitioners have previously used qHBsAg. The use of the qHBsAg just before treatment was the main reason for prescription. The main reason for nonprescription was because of difficulty in accessing the test.

Different isolates of hepatitis C virus (HCV) show nucleotide sequence variability throughout the genome. Detection of antibodies to recombinant proteins derived from hepatitis C virus genotype 1, the prototype HCV clone HCV-PT, constitutes the main method for screening HCV infection. The influence of the genomic variability on the serological diagnosis of HCV by enzyme immunoassay remains poorly defined. The aim of this study was to assess the serological reactivity of a panel of well characterized French HCV isolates typed by sequence analysis from patients with chronic hepatitis. The 73 sera samples were tested in three third generation EIA tests and three confirmatory assays. HCV isolates were determined by RT-PCR and sequencing in NS5B region of the genome. The 73 sera were positive in the three EIA tests. The three confirmatory tests showed a weaker reactivity with NS5 protein whatever the genotype, and a lower reactivity in NS4 antigens of non-type 1 sequences, particularly for genotype 3. Even though the reactivity of the antigens differed among the HCV isolates, the 73 isolates from genotype 1-6 were reactive with the three commercial screening assays. These results demonstrate that using a single test is adequate in the routine diagnosis of HCV infection in clinical laboratory, as recommended by the last French and European consensus conference.

We assessed the performance of a new assay (VERSANT HCV RNA 3.0 [bDNA 3.0] assay [Bayer Diagnostics]) to quantitate HCV RNA levels and compared the results of the bDNA 3.0 assay to results of the Quantiplex HCV RNA 2.0 (bDNA 2.0) assay. Samples used in this study included 211 serum specimens from hepatitis C virus (HCV)-infected persons from two sites (Bordeaux and Marseille, France) with different genotypes; 383 serum specimens from HCV antibody-negative, HCV RNA-negative persons; and serial dilutions of World Health Organization (WHO) HCV RNA standard at a titer of 100,000 IU/ml. The specificity of the bDNA 3.0 assay was 98.2%. A high correlation was observed between expected and observed values in all dilutions of WHO standard ( r = 0.9982), in serial dilutions of pooled samples ( r = 0.9996), and in diluted sera from different HCV genotypes ( r = 0.9930 to 0.9995). The standard deviations (SD) for the within-run and between-run reproducibility of the bDNA 3.0 assay were ≤0.2 and ≤0.14, respectively. The intersite SD ranged from 0.03 to 0.14. The bDNA 3.0 assay results were positively correlated with the bDNA 2.0 assay results ( r = 0.9533). Taking in account the overall performance, this assay could be used as a routine tool for the HCV RNA quantification.

Background The COBAS® AmpliPrep®/COBAS® TaqMan® HCV Test, v2.0 (CAP/CTM2) is used for HCV RNA viral load monitoring. Objectives The performance of the CAP/CTM2 was compared to other widely used tests, including a manual version of the assay (the COBAS® TaqMan® HCV Test, v2.0 for use with the High Pure System, HPS/CTM2) predominantly used during phase III clinical trials for the new direct acting antiviral therapies. Study design Low HCV RNA level comparisons were performed across tests (Abbott Realtime HCV Test, ART; COBAS® AmpliPrep®/COBAS® TaqMan® HCV Test, v1.0, CAP/CTM1; CAP/CTM2; and HPS/CTM2) using dilutions of the 2nd HCV WHO International Standard. Additionally, the clinical performance of the CAP/CTM2 was evaluated with 421 leftover HCV RNA-positive routine clinical samples. Results All quantifiable WHO dilutions were within ±0.3 log10 IU/mL of the expected results across tests and the analytical sensitivity resulted in a limit of detection of 12 IU/mL (95% confidence interval, 10, 15). When clinical samples were tested the results for 87% (367 of 421) of all sample comparisons were within ±0.5 log10 IU/mL. When low viral load results (25–3500 IU/mL) were compared, values obtained by the ART assay were significantly lower (p Conclusions The new CAP/CTM2 showed good accuracy with comparable sensitivity to comparator assays. The new kit is well-suited for use in the routine diagnostic laboratory, especially for accurate monitoring of patients receiving triple therapy or interferone-free regimens.

Les performances analytiques et de stabilite de dix milieux ont ete comparees au milieu PreservCyt en utilisant la methode Cobas 4800® HPV : Easyfix (Labonord SAS, France), Qualicyt (Qualicyt, France), NovaPrep HQ+(NovaCyt, France), CMDH (SARL Alphapath France), Cyt-All (Cytomega, France), Digene Cervical Sampler (Qiagen, USA), Aptima (Gen-Probe, USA), Multi-Collect (Abbott, Allemagne), M4RT Micro Test (Remel, USA), et PCR-Media (Roche, Suisse). La plupart des milieux presentent une bonne correlation sur l’ensemble des parametres etudies. Les milieux Cyt-All et NovaPrep HQ+ presentent une parfaite conformite de tous les parametres avec ceux du milieu de reference PreservCyt, cela pour tous les genotypes detectables. Notons que deux milieux, le CMDH et le M4RT, presentent une duree de stabilite a +25 °C relativement reduite de 3 et 2 jours respectivement, incompatible avec les pratiques courantes de transport. La plupart des milieux testes presentent des performances analytiques et de stabilite sensiblement equivalentes a celles du milieu de reference. Les resultats de cette etude ouvrent des perspectives importantes non seulement pour les fabricants de milieux polyvalents adaptes a la problematique des frottis cervico-uterin, mais egalement a celle de la conservation, du transport de virus ou de bacteries pour la recherche simultanee des HPV, de Chlamydia trachomatis, ou de Neisseria gonorrhea.

Objective: Drug-resistance mutations are routinely detected using standard Sanger sequencing, which does not detect minor variants with a frequency below 20%. The impact of detecting minor variants generated by ultra-deep sequencing (UDS) on HIV drug-resistance interpretations has not yet been studied. Design: Fifty HIV-1 patients who experienced virological failure were included in this retrospective study. Methods: The HIV-1 UDS protocol allowed the detection and quantification of HIV-1 protease and reverse transcriptase variants related to genotypes A, B, C, F and G. DeepChek-HIV simplified drug-resistance interpretation software was used to compare Sanger sequencing and UDS. Results: The total time required for the UDS protocol was found to be approximately three times longer than Sanger sequencing with equivalent reagent costs. UDS detected allof themutations foundby population sequencingand identifiedadditional resistance variants in all patients. An analysis of drug resistance revealed a total of 643 and 224 clinically relevant mutations by UDS and Sanger sequencing, respectively. Three resistance mutations with more than 20% prevalence were detected solely by UDS: A98S (23%), E138A (21%) and V179I (25%). A significant difference in the drugresistance interpretations for 19 antiretroviral drugs was observed between the UDS and Sanger sequencing methods. Y181C and T215Y were the most frequent mutations associated with interpretation differences. Conclusion: A combination of UDS and DeepChek software for the interpretation of drug resistance results would help clinicians provide suitable treatments. A cut-off of 1% allowed a better characterization of the viral population by identifying additional resistance mutations and improving the drug-resistance interpretation. 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2014, 28:000‐000

Objective To analyse the presence of genotypic and phenotypic resistance in lymph node mononuclear cells from patients with sustained undetectable plasma HIV-1 RNA with highly active antiretroviral therapy. Design Cross-sectional study on 27 HIV-infected patients receiving triple therapy for a mean period of 232.8 ± 22.1 weeks. Methods HIV-1 RNA was measured in plasma and lymph node cells using PCR. Reverse transcriptase and protease genes were sequenced from HIV-1 RNA obtained from lymph node cells and from peripheral blood mononuclear cell proviral DNA using a commercially available kit (TruGene). Phenotypic resistance was assessed by using a recombinant virus assay (AntiVirogram). Results Mutations were not found in lymph node mononuclear cell RNA in six out of nine patients on first-line regimens although they were detected in 15 out of 18 who received prior suboptimal combinations. Phenotypic resistance was confirmed in most of these cases. These patterns of resistance were closely related to patients’ history of antiretroviral therapy and genotypic analysis of plasma HIV-1 RNA taken just before initiation of the current regimen. In half the patients analysed, resistance mutations found in lymph nodes were not always detected in archival proviral DNA from blood cells. Mean levels of HIV-1 RNA in lymph node cells were not different in patients exhibiting resistance compared with those harbouring wild-type viruses. Conclusion These data demonstrate that resistant HIV-1 is produced in lymphoid tissues for prolonged periods despite effective therapy. The mechanism could represent a release from previously infected cells rather than new cycles of cellular infection.

Occult hepatitis C infection is regarded as a new entity that should be considered when diagnosing patients with a liver disease of unknown origin. Using an ultrasensitive real-time PCR assay, we demonstrated that occult hepatitis C virus (HCV) infection cannot be found in peripheral blood mononuclear cells of patients with cryptogenic liver diseases, HCV-associated systemic vasculitis, or connective tissue diseases. The significance of such occult infection must be elucidated.

Hepatitis C virus (HCV) is of major concern in the management of patients on maintenance haemodialysis. Many studies have reported a high prevalence of HCV infection in dialysis centres. The objective of our study was first, to perform a prospective follow-up of the evolution of HCV infection in a haemodialysis centre, and second, to assess the rate of viral clearance in patients on dialysis. For this, genotypes, HCV antibodies (anti-HCV) and HCV RNA were evaluated initially and 9 months later. HCV RNA quantification was also performed. Of 136 patients, 62 (45.6%) were anti-HCV positive by third-generation enzyme immunoassay (EIA 3) in the first survey and 64 of 136 (47.1%) were anti-HCV positive by EIA 3 in the second survey. The rate of new HCV infection, estimated from the two seroconversions between the surveys, was 1.9% per year. One of the two patients was initially HCV RNA positive, with a titre of 0.6 x 10(6) eq ml-1. The viral load measured in the dialysis patients was low and does not seem to be influenced by dialysis. No significant difference was observed in viral load between the two periods nor were there any gender-related differences in viral load. In conclusion, detection of antibodies to HCV, together with HCV RNA, seems to be relevant in haemodialysis patients, but this strategy is not suitable for use in all haemodialysis centres because of its high cost.

Methods Fifty HIV-1 patients who experienced virological failure were included in this retrospective study. The HIV-1 UDS protocol was performed using the GS Junior (Roche 454 Life Sciences Branford, CT). This UDS protocol allowed the detection and quantification of minor and major HIV-1 protease and reverse transcriptase variants related to genotypes A, B, C, E, F and G. DeepChekHIV (ABL, SA and TherapyEdgeTM, USA) simplified drug resistance (DR) interpretation software was used to compare Sanger sequencing and UDS at two different thresholds (≥1% and ≥20%). DeepChek-HIV utilizes the ANRS, HIVdb and Rega algorithms.

Hepatitis C virus (HCV) genotypes can be established by methods based on PCR typing and serological typing. The accuracy of these methods depends on their sensitivity and specificity. These should be compared with the reference method, direct sequencing, and analysis of viral genomes. Among the serologic methods recently developed, the performance of a new serotyping assay (RIBA HCV 3.0 SIA, Chiron corporation, Emeryville) was assessed using a panel of 147 well-characterized French isolates from chronic hepatitis C patients. Definitive genotypes of the isolates were established by direct sequencing in 5' NC and in some cases in NS-5B. HCV serotypes 1, 2, and 3 were determined by measuring type specific antibodies to core and NS-4 derived peptide antigens. Of the 147 sera, serotypic-specific antibodies were detected in 136 (sensitivity, 92.5%). The specificity of the RIBA SIA HCV serotyping assay was 92.6% (including samples with mixed results); without these, the specificity was 80.1%. Analysis of the 28 discrepant samples showed that (1) a different serotype was found in 18 samples including five for genotype 1, three for genotype 2, two for genotype 3, five for genotype 4, and three for genotype 5, and that (2) ten patients showed a reactivity with mixed serotypes, one had circulating antibodies to type 1 or 2, and nine had circulating antibodies to type 1 or 3. In summary, except for genotypes 4 and 5, the results of the test were well correlated (85.7%) with those of direct sequence genotyping. The former test is rapid and does not require the strict HCV RNA storage and preservation conditions of the latter. This new method may thus be considered as an alternative for HCV typing. However, although it is convenient, its lower sensitivity compared to the molecular typing method and the discrepant results limit its routine use in a clinical context.

The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.

The objective of this study was to assess the analytical and clinical relevance of minority variants using a new pyrosequencing (PSQ) assay and to detect minor variants with frequencies below the current 20% clinical setting limit. A PSQ approach for detecting and quantifying mutations was developed for the analysis of 14 codons of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene below the limit of conventional sequencing. Ten patients who experienced virological failure (VF) after a first-line regimen of lamivudine, tenofovir, and either efavirenz, nevirapine, or etravirine, as well as 10 controls patients without VF, were included in this retrospective study. Baseline plasma and plasma from the time of VF were assessed using Sanger sequencing and PSQ methods. The analytical sensitivity for the detection of minor sequence variants is 5%. At baseline, no minority variant was detected in 10/10 patient controls using both the Sanger sequencing and PSQ assays, whereas, two patients who failed therapy had baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations that were not detected by the standard genotyping. At the time of VF, standard genotyping detected mutations in four out of the 10 VF patients, whereas, PSQ detected mutations in five out of the 10 VF patients. Clinically, minority mutations at a 10% level of detection can be assessed efficiently by pyrosequencing and used as a suitable predictor of the evolution of viral populations. These traits allow for a better interpretation of data analysis, which can help clinicians in providing a suitable treatment for HIV.

Background Cervical cancer is caused by high-risk types of human papillomavirus (HPV). DNA testing of such high-risk types of HPV could improve cervical screening.The aim of the study was to compare the sensitivities and positive predictive values of two commercially available typing assays (Qiagen LQ and Roche LA) and to comparatively assess the distribution of HPV types with these two assays. Methods The study population comprised 311 ASCUS + women with abnormal pap tests who were HCII positive and who were admitted to three European referral gynecology clinics between 2007 and 2010 (Madrid, Marseille and Milan). All patients underwent LQ and LA tests. Results The sensitivity of the two assays for HPV typing was 94% for LQ and 99% for LA (compared with HCII). The overall concordance between LQ and LA was 93%. The three prevalent genotypes, HPV16, HPV18, and HPV31, were identified with a high concordance using the two assays: kappa 0.93, 0.83, and 0.91, respectively. Mixed genotypes were more frequently detected by LA than by LQ: 52% vs. 18%, respectively (p < .0001). Conclusions These assays have a good clinical sensitivity for detecting HPV types in CIN2+ patients and allow the virus type to be detected in the same experiment. Our study revealed no significant difference between LQ and LA for CIN2+ or CIN3+ diagnosis, indicating similar distributions of HPV types and a mixed genotype detection that is higher for LA than for LQ.

Background aimsDried blood spots (DBS) on filter paper have been successfully used to diagnose and monitor several infectious diseases. The aim was to investigate the performance of DBS in hepatitis B virus (HBV) diagnosis using commercial tests in comparison to standard methods.MethodsPaired DBS and plasma samples were collected from 200 patients: 100 patients with HBsAg negative status and 100 patients with HBsAg positive status. In the latter patient, HBeAg reactivity was tested. Ten samples of anti-HBs were collected from people vaccinated against HBV. We also studied 50 patients with positive HBV DNA viral load in plasma and 10 HBV DNA negative patients. HBV genotypes and gene polymerase mutations were determined in 10 randomly selected HBV-infected patients. The DBS sample consisted of 50 µL of whole blood, i.e. a 12-mm paper card.ResultsThe sensitivity thresholds of HBsAg and anti-HBs antibody were 0.30 ± 0.08 IU/mL and 18.11 ± 6.05 IU/mL, respectively, for DBS with 98% sensitivity and 100% specificity. Sensitivity was 98% and specificity 100% for the detection of HBV DNA on a blotter, considering an HBV DNA threshold of 914.1 ± 157.8 IU/ml. Ten patients had an HBeAg positive status in plasma, all were detected positive using DBS. HBV genotyping and mutation detection were successfully performed on DBS, with full concordance between the 10 paired DBS and plasma samples.ConclusionThis study shows DBS is a reliable alternative to plasma specimens for quantifying and detecting HBsAg, anti-HBs, HBeAg and genotyping. DBS may increase the opportunities for HBV testing and treatment follow-up in hard-to-reach individuals.

Patients infected with hepatitis C virus genotype 4 (HCV-4) respond to interferon alpha (IFN) as poorly as those infected with genotype 1. However, there is no information on the viral dynamics of HCV-4. Interferon-ribavirin treatment was administered to untreated patients infected with HCV-4. Viral load was assessed with Versant 3.0. Viral dynamics parameters were estimated based on the bi-phasic model for HCV during IFN treatment. Viral kinetics of HCV-4 follow a bi-phasic decline pattern also. The mean effectiveness of IFN in blocking production of HCV-4 was a decline of 77.8% during the first day of treatment. The half-life of free virions was estimated at 3.5 h and that of infected cells from 1.9 to over 70 days. The viral dynamics parameters of HCV-4 appear similar to those of HCV-1 and slower than those of HCV-2. HCV-4 infected patients should be grouped with those with HCV-1 when therapeutic schemes are considered in relation to genotype.