Thomas Bourlet

Evaluation of the Roche Cobas TaqMan and Abbott RealTime extraction-quantification systems for HIV-1 subtypes.

Objectives:We conducted a comparison of the Abbott Molecular RealTime (Rungis, France) and Roche Diagnostics Cobas Taqman (Meylan, France) automated nucleic acid extraction and real-time polymerase chain reaction (PCR) amplification systems for their capacity to quantify HIV RNA of various subtypes. The systems were tested on culture supernatants belonging to HIV-1 group M (n = 29), HIV-1 group O (n = 8), and HIV-2 (n = 7). We also tested 88 plasma samples from patients infected with HIV-1 group M (B-D [n = 7], A-CRF01 [n = 16], CRF02 [n = 49], and other strains [n = 16]). Results:The Abbott RealTime system quantified all 29 HIV-1 group M supernatants. One of these samples was not detected by the Roche Cobas TaqMan system. The Abbott RealTime system quantified 7 HIV-1 group O strains. Neither technique cross-reacted with HIV-2. The 79% intraclass correlation coefficient for the 88 plasma samples was barely acceptable, but 4 plasma samples were underestimated by more than 1 log by the Roche Cobas TaqMan system. Similar values were obtained for subtype B and D strains with the tests, indicating that the primers and probes are suitable for these strains. In contrast, the large differences observed with other subtypes, particularly CRF02, show the importance of primer and probe selection. Conclusion:The limitation of real-time PCR to span the entire diversity of HIV must be taken into account during treatment monitoring, resistance studies, and clinical trials.

Enteroviral Capsid Protein VP1 Is Present in Myocardial Tissues From Some Patients With Myocarditis or Dilated Cardiomyopathy

BACKGROUND There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Enteroviruses Can Persist with or without Active Viral Replication in Cardiac Tissue of Patients with End-Stage Ischemic or Dilated Cardiomyopathy

To investigate enterovirus replication versus persistence in end-stage cardiac diseases, endomyocardial biopsies from explanted hearts of 70 patients with idiopathic dilated cardiomyopathy (IDCM), 64 patients with chronic coronary disease (CCD), and 45 donors of healthy hearts (controls) were examined by reverse transcriptase-polymerase chain reaction for genomic and antigenomic enterovirus RNA and by VP1 antigen immunohistochemistry. Enterovirus genome was detected in 25 of 70 patients with IDCM and in 21 of 64 patients with CCDs (35.7 vs. 32.8%, respectively; P=.12). Of the 46 patients positive for genomic RNA, only 3 exhibited antigenomic RNA and VP1 antigen that demonstrated active viral replication, whereas 43 had latent infection characterized by the absence of antigenomic RNA associated with or not with VP1 antigen expression. No viral component was detected in control subjects. The findings demonstrate that a small percentage of patients with end-stage chronic cardiac diseases had active enterovirus replication in their myocardium.

HIV-1 Load Comparison Using Four Commercial Real-Time Assays

ABSTRACT The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated “true” viral load and by the coefficient of reliability, were significantly different (P < 10−4) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients.

Basic rationale, current methods and future directions for molecular typing of human enterovirus

Enterovirus is a genus of the Picornaviridae family including more than 80 serotypes belonging to four species designed Human enterovirus A to D. The antigens of the structural proteins support the subdivision of enteroviruses into multiple serotypes. Comparative phylogeny based on molecular typing methods has been of great help to classify former and new types of enterovirus, and to investigate the diversity of enteroviruses and the evolutionary mechanisms involved in their diversity. By now, molecular typing methods of enterovirus rely mainly on the sequencing of an amplicon targeting a variable part of the region coding for the capsid proteins (VP1 and, alternatively, VP2 or VP4), either from a strain recovered by cell culture or, more recently, by direct amplification of a clinical or environmental specimen. In the future, microarrays are thought to play a major role in enterovirus typing and in the analysis of the determinants of virulence that support the puzzling diversity of the pathological conditions associated with human infection by these viruses.

Typing of Human Enterovirus by Partial Sequencing of VP2

ABSTRACT The sequencing of the VP1 hypervariable region of the human enterovirus (HEV) genome has become the reference test for typing field isolates. This study describes a new strategy for typing HEV at the serotype level that uses a reverse transcription-PCR assay targeting the central part of the VP2 capsid protein. Two pairs of primers were used to amplify a fragment of 584 bp (with reference to the PV-1 sequence) or a part of it (368 bp) for typing. For a few strains not amplified by the first PCR, seminested primers enhanced the sensitivity (which was found to be approximately 10−1 and 10−4 50% tissue culture infective dose per reaction tube for the first and seminested assay, respectively). The typing method was then applied to 116 clinical and environmental strains of HEV. Sixty-one typeable isolates were correctly identified at the serotype level by comparison to seroneutralization. Forty-eight of 55 “untypeable” strains (87.3%) exhibited the same serotype using VP1 and VP2 sequencing methods. For six strains (four identified as EV-71, one as E-9, and one as E-30 by the VP2 method), no amplification was obtained by the VP1 method. The last strain, typed as CV-B4 by VP1 and CV-B3 by VP2 and monovalent antiserum, could exhibit recombination within the capsid region. Although the VP2 method was tested on only 36 of the 68 HEV serotypes, it appears to be a promising strategy for typing HEV strains isolated on a routine basis. The good sensitivity of the seminested technique could avoid cell culture and allow HEV typing directly from PCR products.

Detection and Characterization of Hepatitis C Virus RNA in Seminal Plasma and Spermatozoon Fractions of Semen from Patients Attempting Medically Assisted Conception

ABSTRACT To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.

In Vitro Evaluation of Viability, Integrity, and Inflammation in Genital Epithelia upon Exposure to Pharmaceutical Excipients and Candidate Microbicides

ABSTRACT The use of microbicides is a promising approach for the prevention of HIV-1 transmission. Unfortunately, various candidates failed in clinical trials. In some cases, the candidate microbicide even resulted in enhanced virus transmission. Therefore, there is an urgent need to develop more predictive preclinical strategies to anticipate the in vivo efficiency/toxicity rate, including in vitro assays that evaluate effects on epithelial integrity and inflammation. The present study aims to identify potential safety issues concerning the use of microbicides and excipients commonly used in vaginal microbicide preparations. The toxicities of various active pharmaceutical ingredients (APIs; TMC-120, UC-781, tenofovir [PMPA], PRO-2000, and glycerol monolaurate [GML]) and excipients (preservatives, cosolvents, surfactants, and cyclodextrins) were evaluated using an in vitro dual-chamber model and uterine cervical explants. Epithelial viability and permeation of fluorescent virus-sized beads, as well as induction of interleukin-8 (IL-8; as a sensitive marker of an inflammatory response), were assessed. Surprisingly, cell viability and epithelial layer integrity were compromised by most excipients at concentrations near the typical concentration used in vaginal gels, and a significant increase in the production of IL-8 was observed at subtoxic concentrations. Within the APIs, TMC-120, UC-781, and PMPA showed higher selectivity indices than PRO-2000 and GML. In conclusion, identification of safety issues concerning the use of pharmaceutical excipients could help to formulate less toxic vaginal microbicide preparations.

Measure of viral load by using the abbott real-time HIV-1 assay on dried blood and plasma spot specimens collected in 2 rural dispensaries in Cameroon.

Background:This study aimed to evaluate the use of dried blood spots (DBSs) and dried plasma spots (DPSs) locally collected in 2 rural dispensaries in Cameroon for the quantification of HIV-1 RNA. Methods:Forty-one subjects were sampled and spots of whole blood and plasma were deposited onto Whatman 903 cards and dried at ambient temperature under local conditions. Two sets of DBS and DPS cards were done per patient. The rest of the liquid plasma (LP) was frozen until use. LPs were tested at the “Chantal Biya” International Reference Centre (Yaoundé, Cameroon) by the Abbott Real-Time HIV-1 assay (Abbott Molecular Diagnostics, Wiesbaden, Germany). One series of DBS and DPS was transported and tested between 2 and 6 weeks later at the Virology Laboratory of Saint-Etienne (France). The second series was routed by mail and tested after up to 3 months of storage at ambient temperature. Results:From the first series, the correlation rate between viral loads obtained from LP and DBS, and from LP and DPS, was 0.98 and 0.99, respectively; specificity of DBS and DPS results was 100%. The results obtained from the second series indicate a great stability of DBS after long-term storage. Conclusion:This study demonstrates that DBSs collected under local conditions in resource-limited settings are suitable for the differed quantification of HIV-1 RNA.

Natural Recombination Event within the Capsid Genomic Region Leading to a Chimeric Strain of Human Enterovirus B

ABSTRACT Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.