LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01).

Assistance Publique - Hôpitaux de Paris, CHU Necker-Enfants Malades, Service de Virologie, Paris, France.
Journal of Medical Virology (Impact Factor: 2.22). 02/2009; 81(2):217-23. DOI: 10.1002/jmv.21390
Source: PubMed

ABSTRACT HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.
    eLife Sciences 09/2014; · 8.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed.
    PLoS ONE 11/2014; 9(11):e111919. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Un traitement antirétroviral efficace aboutit généralement à une charge VIH-1 indétectable dans le plasma. Néanmoins, le virus persiste dans certaines cellules du sujet infecté sous diverses formes d’ADN intégré ou non intégré. Ce réservoir représente le plus grand défi à la guérison totale de l’infection à VIH-1 et ses caractéristiques influent fortement sur le cours de la maladie. La quantification de l’ADN du VIH-1 dans le sang constitue à l’heure actuelle l’approche la plus pratique pour mesurer cette infection résiduelle. La PCR quantitative en temps réel (qPCR) est la méthode la plus utilisée pour la quantification de l’ADN du VIH-1 et plusieurs techniques ont été développées pour mesurer les différentes formes de l’ADN du VIH-1. Dans la littérature, plusieurs techniques « maison » ont été utilisées, et il existe un besoin de standardisation pour avoir des résultats comparables. De plus, la qPCR est limitée dans la quantification précise des taux faibles par le bruit de fond. Parmi les nouvelles techniques en développement, la PCR « numérique » s’est révélée comme une méthode précise de quantification de l’ADN du VIH-1. L’ADN total est le plus souvent mesuré en pratique clinique. La quantification absolue des provirus et des formes non intégrées est plus souvent utilisée à des fins de recherche.
    Pathologie Biologie 09/2014; 63(1). · 1.07 Impact Factor