Mouna Lazrek

Evaluation of the Genotypic Prediction of HIV-1 Coreceptor Use versus a Phenotypic Assay and Correlation with the Virological Response to Maraviroc: the ANRS GenoTropism Study

ABSTRACT Genotypic algorithms for prediction of HIV-1 coreceptor usage need to be evaluated in a clinical setting. We aimed at studying (i) the correlation of genotypic prediction of coreceptor use in comparison with a phenotypic assay and (ii) the relationship between genotypic prediction of coreceptor use at baseline and the virological response (VR) to a therapy including maraviroc (MVC). Antiretroviral-experienced patients were included in the MVC Expanded Access Program if they had an R5 screening result with Trofile (Monogram Biosciences). V3 loop sequences were determined at screening, and coreceptor use was predicted using 13 genotypic algorithms or combinations of algorithms. Genotypic predictions were compared to Trofile; dual or mixed (D/M) variants were considered as X4 variants. Both genotypic and phenotypic results were obtained for 189 patients at screening, with 54 isolates scored as X4 or D/M and 135 scored as R5 with Trofile. The highest sensitivity (59.3%) for detection of X4 was obtained with the Geno2pheno algorithm, with a false-positive rate set up at 10% (Geno2pheno10). In the 112 patients receiving MVC, a plasma viral RNA load of <50 copies/ml was obtained in 68% of cases at month 6. In multivariate analysis, the prediction of the X4 genotype at baseline with the Geno2pheno10 algorithm including baseline viral load and CD4 nadir was independently associated with a worse VR at months 1 and 3. The baseline weighted genotypic sensitivity score was associated with VR at month 6. There were strong arguments in favor of using genotypic coreceptor use assays for determining which patients would respond to CCR5 antagonist.

A part of the VP4 capsid protein exhibited by coxsackievirus B4 E2 is the target of antibodies contained in plasma from patients with type 1 diabetes.

The capsid protein VP4 was identified previously as the target of antibodies contained in plasma enhancing the coxscakievirus B4 (CV‐B4) E2‐induced production of IFN‐α by peripheral blood mononuclear cells (PBMCs). The sequence of VP4 recognized by these antibodies was investigated. This sequence was identified as amino acids 11 to 30 by using synthetic overlapping peptides spanning VP4CV‐B4 E2 in competition experiments for antibodies enhancing the CVB4 E2 induced production of IFN‐α by PBMCs. This amino acid sequence was the major target of anti‐VP4 antibodies according to enzyme‐linked immunosorbent assays (ELISA). There was a positive correlation between the levels of anti‐VP4 and anti‐VP411–30 peptide antibodies detected by ELISA. The levels and the prevalences of these antibodies were significantly higher in patients with type 1 diabetes than in healthy controls. The proportions and the levels of those antibodies in patients were independent of HLA‐DR alleles, age, or presence of ketosis in blood and were not associated with newly or previously diagnosed disease. The VP4CV‐B4 E2 amino acid sequence was submitted to the Swiss‐model in project mode to visualize the possible shape of the sequence of VP4 corresponding to amino acids 11–30 which appeared to be constituted principally by an non‐structured loop. In conclusion, the sequence of VP4 corresponding to amino acids 11–30, or a part of it plays a role in the plasma‐dependent enhancement of CV‐B4 E2‐induced production of IFN‐α by PBMCs, suggesting that at 37°C the virus exhibits that region of VP4 to antibodies. J. Med. Virol. 80:866–878, 2008.

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.

A cohort study of treatment-experienced HIV-1-infected patients treated with raltegravir: factors associated with virological response and mutations selected at failure.

This study aimed to identify factors associated with virological response (VR) to raltegravir (RAL)-containing regimens in 468 treatment-experienced but integrase inhibitor-naive HIV-1 patients receiving a RAL-containing regimen. VR was defined at Month 6 (M6) as HIV-1 RNA viral load (VL) <50 copies/mL. The impacts on VR of baseline integrase mutations, VL, CD4 count, genotypic sensitivity score for nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors, and the number of new antiretrovirals used for the first time associated with RAL were investigated. For patients with VL >50 copies/mL at M6, integrase mutations selected were characterised. Median baseline VL was 4.2 log(10)copies/mL (IQR 3.3-4.9 log(10) copies/mL) and CD4 count was 219 cells/mm(3) (IQR 96-368 cells/mm(3)). At M6, 71% of patients were responders. In multivariate analysis, baseline VL and CD4 count and ≥ 2 new antiretrovirals among darunavir, etravirine, maraviroc and enfuvirtide were associated with VR to RAL. Neither HIV-1 subtype nor baseline integrase polymorphisms were associated with VR to RAL. Among 63 failing patients at M6, selection of ≥ 1 change in the integrase gene was observed in 49 (77.8%), and 27/63 (42.9%) were considered as RAL-associated resistance mutations. Factors independently associated with the occurrence of ≥ 1 RAL-associated resistance mutation were VL at failure >3 log(10) and having no new drugs associated with RAL. RAL showed great potency in treatment-experienced patients. The number of new drugs associated with RAL was an important factor associated with VR. HIV-1 subtype and baseline integrase polymorphisms do not influence the RAL VR.

Enterovirus D68 detection in respiratory specimens: Association with severe disease

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)‐D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV‐D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT‐PCR. This multiplex RT‐PCR does not detect EV‐D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV‐specific‐RT‐PCR. EV‐positive specimens were tested with an EV‐D68‐specific‐RT‐PCR and genotyped. Eleven specimens of 2014 tested positive in the EV‐specific‐RT‐PCR and of these seven were positive in the EV‐D68‐specific‐RT‐PCR. Typing confirmed these as EV‐D68. Median age of EV‐D68‐positive patients was 3 years (1 month‐91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV‐D68‐positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV‐specific‐RT‐PCR but negative in the EV‐D68‐specific‐RT‐PCR. In conclusion, use of an EV‐specific‐RT‐PCR allowed us to detect EV‐D68 circulation in autumn 2014 that was not detected by the multiplex RT‐PCR and was associated with severe disease.

Necessity to critically review the automatic results of the Xpert Flu assay

Abstract While using the Xpert Flu assay we became aware of false-negative results. The study aimed to analyze the causes of these false-negative results. One hundred fifty-nine respiratory specimens were tested in the Xpert Flu assay and in multiplex reverse transcription–polymerase chain reactions (RT-PCRs) for respiratory viruses. Discordant specimens were tested in the Influenza A/B r-gene assay. One hundred fifty-two (96%) and 151 (95%) specimens yielded concordant results for influenza A and B, respectively. Fifteen specimens tested negative in the Xpert Flu assay and positive in a multiplex RT-PCR. Positive results were confirmed for 12 of these specimens in the Influenza A/B r-gene assay. Xpert Flu assay amplification curves and endpoints suggested that the false-negative results were mainly due to erroneous automatic result interpretation. We report false-negative results of the Xpert Flu assay due to erroneous automatic result interpretation. Careful analysis of amplification curves and endpoints is needed to avoid reporting of false-negative results.

Persistent viral DNA detection in blood after primary herpes simplex 1 infection revealed by hepatitis with hemophagocytic syndrome.

We here report the case of a 52-year old man who presented hepatitis with hemophagocytic syndrome triggered by herpes simplex 1 (HSV-1) primary infection. A high-level viremia was observed, and HSV-1 DNA remained detectable in blood for a long time after patients recovery.

Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV

Comparability between CMV assays could be facilitated by the first WHO International Standard for human CMV (standard). Standard dilutions were submitted to nucleic acid extraction with Versant kPCR Molecular systems SP or MagNA Pure LC System followed by the kPCR PLX™ CMV DNA (kPCR) or the CMV R-gene™ assay (R-gene), respectively; 139 clinical specimens were tested. Both assays correlated well with the standard (R2 > 0.96) and a matrix effect was observed. Quantitative results correlated reasonably between both assays for whole blood (R2 = 0.79) and well for other specimen types (R2 = 0.93). Quantification differences were within one log10 of the averaged log10 results for 25/27 blood specimens and for 32/33 other specimens. Calibration to the standard did not increase this percentage. In conclusion, results of both assays showed reasonable correlation with each other and good correlation with the standard. Calibration to the standard did not improve comparability of quantitative results.

Relapsing Pityriasis Rosea With HHV-7 Reactivation in an 11-Year-Old Girl

We describe an 11-year-old girl with recurrent episodes of PR over several years that was associated with HHV-7 DNA detection. Pityriasis rosea (PR) usually presents as acute exanthema with oval erythematous-squamous lesions localized on the trunk, arms, and legs with spontaneous remission. We present an unusual case of PR with frequent relapses during a period of 7 years. An 11-year-old white female patient presented with many pruritic erythematous oval lesions on her trunk. A second episode followed 2 years later with several pruritic erythematous lesions on her lower limbs. During the following 5 years, the patient had several relapses per year, with 1 to 3 lesions on changing localizations. PR was diagnosed on the basis of the clinical presentation and detection of human herpesvirus 7 DNA. Spontaneous remission occurred without treatment in each episode. Relapsing PR is a rare form of PR characterized by a lower number of lesions and smaller sized lesions compared with the classic form of PR. Pediatricians should consider the diagnosis of relapsing PR even if only a single or few erythematous lesions are present.

In Vivo Persistence of Human Rhinoviruses in Immunosuppressed Patients

Several species of the genus Enterovirus cause persistent infections in humans. Human rhinovirus (HRV) infections are generally self-limiting but occasionally persistent infections have been described. This study aimed to identify persistent HRV infections and investigate the clinical and virologic characteristics of patients with persistent infections. From January 2012 to March 2015, 3714 respiratory specimens from 2608 patients were tested for respiratory viruses by using a multiplex reverse transcription–polymerase chain reaction. A retrospective study was performed. Patients with at least two specimens positive for HRV/enterovirus taken 45 days or longer apart were identified and the HRV/enteroviruses were typed. Patients with persistent infection were compared to patients with reinfection and patients with cleared infection. Phylogenetic analysis of the viral protein(VP)4/VP2 region was performed. 18 patients with persistent HRV/enterovirus infection were identified. Minimum median duration of persistence was 92 days (range 50–455 days). All but one patients with persistence were immunosuppressed. Immunosuppression and hematologic disorders were more frequent in patients with persistence (n = 18) than in patients with reinfection (n = 33) and with cleared infection (n = 25) (p = 0.003 and p = 0.001, respectively). In conclusion, this retrospective study identified HRV persistence in vivo which occurred mainly in immunosuppressed patients.