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.

HPV genotype distribution according to severity of cervical neoplasia using the Digene HPV genotyping LQ test.

A new genotyping-based DNA assay (Digene LQ(®)) was developed recently. The primary aim was to assess the distribution of HPV types using this new assay in atypical squamous cells of undeterminate significance (ASCUS). The secondary aim was to correlate the HPV types with the severity of the disease. The study population comprised 376 ASCUS women. The women were all Hybrid Capture II (HCII) positive and were admitted in three European referral gynecology clinics between 2007 and 2010. A colposcopy with histological examination was performed in all these patients. HPV 16 was typed in 40 % of patients, HPV 18 in 7 %, and HPV 31 in 17 %, and 18 % of patients had mixed genotypes. Patients aged over 30 more often had the HPV 16 genotype than patients aged under 30 (29 % vs. 11 %, chi-square test p < 0.001). The risk of cervical intra-epithelial neoplasia of grade 2 or more (CIN2 +) when HPV 18 positive is lower than the probability associated with HPV 16 or HPV 31: 28 % vs. 58 % and 52 %, respectively (chi-square test, p = 0.005 and p = 0.05, respectively). The Digene LQ(®), a new sequence-specific hybrid capture sample preparation, is fast and efficient and allows high-throughput genotyping of 18 HR HPV types by PCR compared to traditional non-sequence-specific sample preparation methods.

Comparison of the performance of carcinogenic HPV typing of the Roche Linear Array and Qiagen LiquiChip® HPV assays.

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.

A cohort analysis of sexually transmitted infections among different groups of men who have sex with men in the early era of HIV pre-exposure prophylaxis in France.

BACKGROUND: MSM are at particular risk of STIs due to sexual behavior and substance use. HIV PrEP use may increase this risk. DESIGN: Our aim was to comparatively assess incident STIs among different at-risk groups-PLWHIV, HIV-negative PrEP and no-PrEP users-seen at our center early after PrEP implementation. METHODS: Clinical data were retrospectively collected on 636 MSM seen at the Infectious Diseases Department between September 2016 and October 2018. STI incidence rate was assessed among groups for the whole period, as well as separately for each year of the study. RESULTS: Overall STI incidence rate ratio was higher in HIV-neg when compared to PLWHIV. In multivariate analysis, STI risk was significantly higher among HIV-neg no-PrEP users compared to PLWHIV, while not different between PLWHIV and PrEP users.STI incidence globally increased during the first 2 years after PrEP approval among PLWHIV and no-PrEP users, stated by odds ratio (OR = 1.77 [1.23-2.55], p = 0.0020 and OR = 2.29 [0.91-5.73], p = 0.0774 respectively) while it remained rather stable for HIV-neg PrEP users (OR = 1.19 [0.60-2.38], p = 0.6181). The HIV-neg no-PrEP group remained at higher risk of STI than PLWHIV and PrEP users during the two periods. CONCLUSION: These results suggest that a proactive approach of an efficient follow-up of MSM participants since PrEP approval may have prevented an increase of the incidence of STIs among PrEP users.

Online self-sampling kits for human immunodeficiency virus and other sexually transmitted infections: Feasibility, positivity rates, and factors associated with infections in France.

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 (p<0.001) (for pharyngeal infections only). CONCLUSIONS: The self-sampling kit for multiple STIs can perform comprehensive tests and identify new infections in young people, especially in extragenital sites.

Algorithm based on CMV kinetics DNA viral load for preemptive therapy initiation after hematopoietic cell transplantation.

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 < 0.001). A standardized real-time PCR assay is more appropriate than the antigen assay for detecting CMV. It allowed earlier diagnosis of active CMV infection and monitoring of the response to anti-CMV treatment.

Acceptability and efficacy of vaginal self-sampling for genital infection and bacterial vaginosis: A cross-sectional study.

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.

An optimized stepwise algorithm combining rapid antigen and RT-qPCR for screening of COVID-19 patients.

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.

Occult hepatitis C virus infection revisited with ultrasensitive real-time PCR assay.

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.

Quantification of HPV16 E6/E7 mRNA Spliced Isoforms Viral Load as a Novel Diagnostic Tool for Improving Cervical Cancer Screening.

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% (56⁻100) sensitivity and specificity) and CIN3+ (100% (72⁻100) sensitivity and 79% (49⁻95) specificity) lesions; (ii) a greater sensitivity compared to the Pap test for CIN2+ lesions detection (80% (44⁻97)); (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.

Baseline and post-treatment hepatitis C NS5A resistance in relapsed patients from a multicentric real-life cohort.

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.

HHV8 and Kaposi's sarcoma: should we really give up protease inhibitors in all HIV-infected patients?

: 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.

Medically assisted procreation and transmission of hepatitis C virus: absence of HCV RNA in purified sperm fraction in HIV co-infected patients.

OBJECTIVE: The risk of hepatitis C virus (HCV) transmission in medically assisted procreation (MAP) is debated and some researchers have proposed to exclude MAP for HCV-positive infertile patients. The objectives of this study were to assess the presence of viral RNA in the final preparation of density gradient semen fractions collected from men with chronic HCV and HIV co-infection participating in a MAP program, and to assess whether HIV co-infection influences the rate of the presence of HCV RNA in the semen. DESIGN AND METHODS: The study was based on a cohort of 170 HCV-infected male patients (93 HIV co-infected) participating in a MAP program in a French center. Semen samples were subjected to standard MAP sperm preparation, using density-gradient centrifugation with 40 and 90% layers. All aliquots were tested with a commercially available HCV RNA assay (Roche Monitor), adapted for use with semen after a nucleic HCV RNA extraction modification (Organon Technika). RESULTS: Seminal plasma samples from 19 (11%) patients were HCV RNA positive. The positive HCV viral load in semen was less than 600 IU/ml. None of the 90% fractions from HCV-infected patients were HCV RNA positive. Among the 93 co-infected patients, 10 were positive for HCV RNA in semen and three were HIV/HCV RNA positive in semen. CONCLUSIONS: Although HCV RNA was found in the semen of 11% of patients, no purified sperm fraction, or spermatozoa used in MAP were HCV RNA positive. The 90% purified sperm fraction discards the virus and must be used with care in MAP.

Unusual selection of rtA181V HBV mutants cross-resistant to adefovir following prolonged lamivudine monotherapy: report of two cases.

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.

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.

Kinetics of hepatitis C virus RNA load during pegylated interferon alpha-2a and ribavirin treatment in naïve genotype 1 patients.

BACKGROUND: Pegylated interferon given for 24 or 48 weeks constitutes the most effective initial therapy for the treatment of chronic hepatitis C. It has been shown that viral load at week 2 appears the best time for predicting response to treatment. The objectives of this study were to assess whether the hepatitis C virus (HCV) RNA viral decline is predictive of sustained virological response (SVR) and to determine the best time for predicting complete response in our cohort of naïve patients treated with pegylated interferon alpha-2a (Peg-IFN alpha-2a) and ribavirin. RESULTS: Twenty patients treated with Peg-IFN alpha-2a and ribavirin for 48 weeks were studied. Six months after the end of treatment, a SVR (negative HCV RNA measured by PCR six months after the end of therapy) was obtained in 9 patients. Samples were obtained before and at week 2, 4, 8, and 12. At the end of week 2, viral load decreased more than 1.39 log in 8 out of the 9 patients with SVR and in 1 out of the 11 other patients. When we considered the viral load reduction from baseline to each week of treatment, week 2 appeared to be the best point time for predicting SVR, with a sensitivity of 91% (95%CI: 59;99), a specificity of 89% (52;98), a positive predictive value of 91% (59;99) and a negative predictive value of 89% (57;98). CONCLUSION: During treatment with Peg-IFN alpha-2a plus ribavirin in genotype 1 patients, when the main objective of the treatment is viral eradication, viral kinetics showed that week 2 appeared to be the best time point for predicting SVR. Our results must be further confirmed on a larger cohort.

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

BACKGROUND AND AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.