Claire Camus

Platelets alter gene expression profile in human brain endothelial cells in an in vitro model of cerebral malaria.

Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM) in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC) and human brain microvascular endothelial cells (HBEC) and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF) and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

Comparison of ultra-deep versus Sanger sequencing detection of minority mutations on the HIV-1 drug resistance interpretations after virological failure.

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 all of the mutations found by population sequencing and identified additional 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 drug-resistance 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.

Dried Blood Spot Sampling for Hepatitis B Virus Serology and Molecular Testing

Background & Aims Dried 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. Methods Paired 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. Results The 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. Conclusion This 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.

Clinical and analytical relevance of NNRTIs minority mutations on viral failure in HIV-1 infected 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.

Étude des performances et de la stabilité des principaux milieux de conservation et/ou de transport de cellules du col de l’utérus utilisés en France par le test Cobas ® 4800 HPV (Roche Diagnostics)

Analytical and stability performances of ten media were compared to PreservCyt medium using the Cobas 4800 HPV test: 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). Most of media show a good correlation for all the performance characteristics studied. Cyt-All and NovaPrep HQ+ media are perfectly concordant with PreservCyt all parameters and genotypes considered. CMDH and M4RT have a reduced stability at +25°C (3 and 2 days respectively) and would not be conformed to current shipping practices. Most of media tested show analytical and stability performances equivalent with the reference medium. The prospects of such study are interesting because in the near future, providers would make available media adapted to the problem of cervical smear but also to the conservation, transport of virus or bacteria for performing simultaneous searches of HPV, Chlamydia trachomatis or Neisseria gonorrhea.

High Prevalence of Asymptomatic Sexually Transmitted Infections among Men Who Have Sex with Men

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.

Clinical impact of ultra deep versus Sanger sequencing detection of minority mutations on HIV-1 drug resistance genotype interpretation after virological failure

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.

Clinical relevance of the HCV protease inhibitor-resistant mutant viral load assessed by ultra-deep pyrosequencing in treatment failure

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<0.001) than those of patients without persistent RAVs. 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.

Nationwide large survey on hepatitis B surface antigen quantification use in real-life clinical practice.

Background and aimHepatitis 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. MethodsFrom 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. ResultsSeventy-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. ConclusionThis 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.