Catherine Tamalet

Significance of HPV 16 and 18 viral load quantitation in women referred for colposcopy

The clinical utility of HPV 16 and 18 viral loads remains debated. The aim of this study was to assess the clinical significance of HPV 16 and 18 viral load and to determine a cut‐off for optimal prediction of grade 2 or higher cervical intraepithelial neoplasia among patients referred to colposcopy. A total of 186 cervico‐vaginal specimens harboring HPV 16 and/or 18 obtained at the time of colposcopy from patients without previous cervical neoplasia were tested for HPV 16 and 18 detection and quantitation using quantitative duplex real‐time PCR method. Grade 2 or higher cervical intraepithelial neoplasia was diagnosed in 87 (46.8%) cases. Only HPV 16 median viral load increased significantly with the lesion grade: 9.1 × 104 in normal cervix or grade 1 cervical intraepithelial lesion versus 4.0 × 106 copies per million cells in grade 2 or higher cervical intraepithelial lesion (P < 0.001). The highest predictive value for grade 2 or higher cervical intraepithelial lesion was observed with a HPV 16 viral load cut‐off of 3.0 × 106 copies per million cells (91% specificity, 58.2% sensitivity). Using this cut‐off, the highest predictive value of HPV 16 viral load was observed among those referred for previous low‐grade abnormal cervical cytology (96.4% specificity, 88% sensitivity). HPV 18 quantitation showed very poor predictive value. Specific attention should be given when performing colposcopic examination of women with an HPV 16 viral load higher than 3.0 × 106 copies per million cells, especially among those referred after a low‐grade abnormal cytology. J. Med. Virol. 84:306–313, 2012.

Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism

OBJECTIVES To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. PATIENTS AND METHODS The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay. RESULTS Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%. CONCLUSIONS The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism.

Lack of correlation between the size of HIV proviral DNA reservoir and the level of immune activation in HIV-infected patients with a sustained undetectable HIV viral load for 10 years

BACKGROUND The persistence of HIV residual replication in patients with an undetectable plasma viral load (pVL) may limit immune recovery and facilitate inflammation-induced comorbidities. OBJECTIVE The objective was to evaluate any correlation between immune restoration and intracellular [IC] HIV-DNA in cART-treated patients with a sustained undetectable pVL. STUDY DESIGN This retrospective cross sectional study included 62 patients with a median duration of undetectable pVL of 10.3 years. IC HIV DNA in peripheral mononuclear blood cells (PBMCs) and T cell subsets were measured at the last visit. pVL, CD4(+) and CD8(+) T cell counts were retrospectively collected from the onset of long-term inhibition by antiretroviral treatment. The patients were separated into two groups: 27 non-blippers (sustained pVL< threshold value during all the visits) and 35 blippers ( ≥ 1 episodes of pVL> threshold but < 1000 copies/ml). The median pVL in blippers was 115 copies/ml. RESULTS The median IC HIV DNA rate was 34 copies/10(6) PBMCs (71% ≥ 20 copies/10(6) PBMCs) with no significant difference between the groups. The proportion of CD8(+)CD38(+) and CD8(+)DR(+) T cells was higher in blipper patients, but the difference was only significant for the CD8(+)DR(+) marker (p = 0.036). No correlation was found between markers of immune activation on CD4(+) and CD8(+) T cells and the IC HIV-DNA level. CONCLUSION No relation was found between the size of HIV reservoirs and immune activation in patients with sustained undetectable pVL. Mechanisms of immune activation have to be better understood in order to define specific therapeutic interventions.

HIV infection en route to endogenization: two cases.

The long-term spontaneous evolution of humans and the human immunodeficiency virus (HIV) is not well characterized; many vertebrate species, including humans, exhibit remnants of other retroviruses in their genomes that question such possible endogenization of HIV. We investigated two HIV-infected patients with no HIV-related disease and no detection with routine tests of plasma HIV RNA or cell-associated HIV DNA. We used Sanger and deep sequencing to retrieve HIV DNA sequences integrated in the human genome and tested the host humoral and cellular immune responses. We noticed that viruses from both patients were inactivated by the high prevalence of the transformation of tryptophan codons into stop codons (25% overall (3–100% per gene) and 24% overall (0–50% per gene)). In contrast, the humoral and/or cellular responses were strong for one patient and moderate for the other, indicating that a productive infection occurred at one stage of the infection. We speculate that the stimulation of APOBEC, the enzyme group that exchanges G for A in viral nucleic acids and is usually inhibited by the HIV protein Vif, has been amplified and made effective from the initial stage of the infection. Furthermore, we propose that a cure for HIV may occur through HIV endogenization in humans, as observed for many other retroviruses in mammals, rather than clearance of all traces of HIV from human cells, which defines viral eradication.

Relapse of hepatitis C virus after 14 months of sustained virological response following pegylated-interferon alpha plus ribavirin therapy in a human immunodeficiency virus type 1 infected patient

It has been demonstrated that sustained virological response (SVR) in patients with chronic hepatitis C indicates resolution of infection. We describe a late hepatitis C virus (HCV) relapse with nearly identical HCV genotype 1a RNA, 14 months after a SVR achievement following a 12-month pegylated-interferon plus ribavirin treatment in a human immunodeficiency virus (HIV) infected patient. This virological relapse occurred concomitantly with interruption of highly active antiretroviral therapy and subsequent increased immunosuppression. HCV retreatment was successful and HCV RNA was undetectable at 50 months of follow-up. This case suggests that late relapse of HCV infection in HIV-positive patients with SVR is possible in case of increased immunodeficiency related to highly active antiretroviral therapy interruption. In such circumstances, a close monitoring of HCV viremia and aminotransferases should be performed.

Hepatitis B virus reactivation in HBsAg-negative patients is associated with emergence of viral strains with mutated HBsAg and reverse transcriptase

BACKGROUND/AIMS Virological factors associated with hepatitis B virus reactivation (HBV-R), following chemotherapy for cancer in hepatitis B surface antigen (HBsAg)-negative patients, are not well known. MATERIALS AND METHODS HBV strains from 16 patients presenting HBV-R following chemotherapy were studied and compared to those obtained from 51 HBV chronically-infected patients. RESULTS HBsAg variability was significantly increased within the major hydrophilic region, the a determinant and the C-terminal region. Amino acid substitutions were more frequently found in HBV-R patients as compared to controls at 17 and 11 positions within HBsAg and HBV-RT, respectively. This resulted in atypical serological testing in 56% of patients and detection of resistance mutation to nucleoside analogs in 12.5%. CONCLUSION HBsAg and HBV-RT mutations are frequently encountered in patients with HBV-R, resulting in atypical serological testing and emergence of HBV strains resistant to nucleos(t)ides analogs.

Two years outcome of women infected with high risk HPV having normal colposcopy following low-grade or equivocal cytological abnormalities: are HPV16 and 18 viral load clinically useful predictive markers?

Management of patients infected with high‐risk HPV (hrHPV) despite normal colposcopy following abnormal cytology remains a clinical challenge. The aim of this study was to evaluate if, in that specific population, initial HPV 16 and HPV 18 viral loads are predictive of infection clearance over a 24‐month follow‐up. A total of 67 women infected with hrHPV having normal colposcopy following equivocal or low‐grade cytological abnormalities were recruited and attended regular follow‐ups based on repeat colposcopies and HPV testing. HPV16 and HPV18 infection were diagnosed in 36 (53.7%) and 7 (10.4%) cases, respectively. Viral load was quantified using the quantitative duplex real‐time PCR method. Although this was not observed for HPV 18, initial HPV 16 viral load was highly associated to HPV 16 infection outcome (receiver operating characteristic curve analysis, area under curve: 0.90). Thus, women who had cleared their HPV 16 infection had significantly lower median initial HPV 16 viral load than those with persistent HPV 16 infection: 1.5 × 103 copies per million cells (CPMC) versus 3.8 × 106 CPMC, respectively (P = 0.006). The best prediction of HPV 16 clearance was obtained with an initial HPV 16 viral load of <7.5 × 104 CPMC: 86.7% specificity and 85.7% sensitivity. Finally, six patients were diagnosed with grade 2 or 3 cervical or vaginal intraepithelial neoplasia. Although all had a persistent hrHPV infection, neither HPV 16 nor 18 viral loads were found to be predictive of the risk of cervical or vaginal intraepithelial neoplasia. HPV16 viral load quantitation could represent a clinically useful marker in that very specific population. J. Med. Virol. 84:964–972, 2012.

HIV-1 Infection and First Line ART Induced Differential Responses in Mitochondria from Blood Lymphocytes and Monocytes: The ANRS EP45 “Aging” Study

Background The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence. Methods 49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999). In more than 88% of treated patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. ROS (reactive oxygen species) production and ΔΨm (inner membrane potential) were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters). Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively) were analysed by western blotting. Results Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm) or morphological (network volume density, fragmentation and branching) parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria. Conclusions In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes), or from first line ART (monocytes). Together with other tissue impairments, these changes may contribute to global aging. Trial Registration ClinicalTrials.gov NCT01038999 NCT01038999

Hepatitis C virus NS3 protease genotyping and drug concentration determination during triple therapy with telaprevir or boceprevir for chronic infection with genotype 1 viruses, southeastern France

Telaprevir and boceprevir, the two first hepatitis C virus (HCV) NS3 protease inhibitors (PIs), considerably increase rates of sustained virologic response in association with pegylated interferon and ribavirin in chronic HCV genotype 1 infections. The 30 first patients treated by telaprevir or boceprevir including anti‐HCV therapies since 2011 in Marseille University hospitals, France, were monitored. HCV loads and plasmatic concentrations of telaprevir and boceprevir were determined on sequential blood samples. HCV NS3 protease gene population sequencing was performed at baseline of treatment and in case of treatment failure. Fifteen patients (including 7 co‐infected with HIV) received telaprevir and the other 15 patients (including 4 co‐infected with HIV) received boceprevir. At baseline, HCV NS3 protease from six patients harbored amino acid substitutions associated with PI‐resistance. Treatment failure occurred at week 12 for 7 patients. Amino acid substitutions associated with PI‐resistance were observed in six of these cases. HCV NS3 R155K and T54A/S mutants, all of genotype 1a, were found from four patients. Median (interquartile range) plasma concentrations were 3,092 ng/ml (2,320–3,525) for telaprevir and 486 ng/ml (265–619) for boceprevir. For HIV–HCV co‐infected patients, median concentrations were 3,162 ng/ml (2,270–4,232) for telaprevir and 374 ng/ml (229–519) for boceprevir. Plasma drug concentration monitoring revealed undetectable concentrations for two patients at week 4, and probable non‐adherence to therapy for another patient. These findings indicate that routine HCV NS3 protease sequencing and plasma PI concentration monitoring might be helpful to characterize cases of therapy failure, at a cost dramatically low compared to that of anti‐HCV therapy. J. Med. Virol. 86:1868–1876, 2014.

Prevalence and Risk Factors for Anal Human Papillomavirus Infection in Human Immunodeficiency Virus–Positive Men Who Have Sex with Men

Background We assessed prevalence and risk factors for anal human papillomavirus (HPV) in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), who are at high-risk of HPV-related anal cancer. Methods APACHES is a multicentric, prospective study of anal HPV infection and lesions in HIV-positive MSM aged ≥35 years. At baseline, participants underwent anal swabs for HPV and cytology, plus high-resolution anoscopy. High-risk HPV (HR-HPV) was tested by Cobas4800, with genotyping of HR-HPV positives by PapilloCheck. Results Among 490 participants, prevalence of HPV16 and HR-HPV was 29% and 70%, respectively, and did not differ significantly by age, sexual behavior, or markers of HIV or immune deficiency. Smoking was the only, albeit weak (odds ratio, 1.8; 95% confidence interval, 1.2-2.7), predictor of HR-HPV. High-risk HPV and HPV16 prevalence increased strongly with anal diagnosis severity, both by worse cytological/histological (composite) diagnosis at APACHES baseline and worse historical diagnosis. HPV16 rose from 19% among participants who were negative for lesions to 63% among participants with high-grade lesions. In contrast, non-HPV16 HR-HPVs were less prevalent in high-grade (37%) than negative (64%) composite diagnosis, and their causal attribution was further challenged by multiple HPV infections. Conclusions Human papillomavirus 16 is ubiquitously frequent among human immunodeficiency virus -positive men having sex with men, and more strongly associated with high-grade anal lesions than other high-risk types, confirming it as a target for anal cancer prevention.