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: 2.35). 02/2009; 81(2):217-23. DOI: 10.1002/jmv.21390
Source: PubMed


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.

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    • "The evaluation of HIV replication capacity was determined by measuring the HIV-RNA copy numbers in cell culture supernatants or, for entry assay, in the cell lysate after elimination of cell debris. In detail, viral HIV-1 RNA was extracted from supernatants or cell extracts using the automatic Easy Mag extractor (Biomerieux, Lyon, France) and quantified by real time PCR targeting a conserved region in the long terminal repeat (LTR) as previously reported [62]. "
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    ABSTRACT: Sialic acid-binding Ig-like lectin-7 (Siglec-7) expression is strongly reduced on natural killer (NK) cells from HIV-1 infected viremic patients. To investigate the mechanism(s) underlying this phenomenon, we hypothesized that Siglec-7 could contribute to the infection of CD4pos target cells following its interaction with HIV-1 envelope (Env) glycoprotein 120 (gp120). The ability of Siglec-7 to bind gp120 Env in a sialic acid-dependent manner facilitates the infection of both T cells and monocyte-derived macrophages (MDMs). Indeed, pre-incubation of HIV-1 with soluble Siglec-7 (sSiglec-7) increases the infection rate of CD4pos T cells, which do not constitutively express Siglec-7. Conversely, selective blockade of Siglec-7 markedly reduces the degree of HIV-1 infection in Siglec-7pos MDMs. Finally, the sSiglec-7 amount is increased in the serum of AIDS patients with high levels of HIV-1 viremia and inversely correlates with CD4pos T cell counts. Our results show that Siglec-7 binds HIV-1 and contributes to enhance the susceptibility to infection of CD4pos T cells and MDMs. This phenomenon plays a role in HIV-1 pathogenesis and in disease progression, as suggested by the inverse correlation between high serum level of sSiglec-7 and the low CD4pos T cell count observed in AIDS patients in the presence of chronic viral replication.
    Full-text · Article · Dec 2013 · Retrovirology
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    • "HIV-DNA was extracted from 200 µL of blood and 1 mL of CVL, using the Nucleospin blood kit (Macherey-Nagel). Total cell-associated HIV-DNA was quantified in these extracts by using the ANRS ultrasensitive real-time PCR method (Biocentric, Bandol, France), as previously described (amplification of the LTR region), in quadruplicate [14]. Results were expressed as log10 copies per million PBMCs and as log10 copies per million vaginal cells. "
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    ABSTRACT: To assess the impact of long-term combined antiretroviral therapy (cART) on HIV-RNA and HIV-DNA levels in cervicovaginal secretions of HIV-1-infected women with sustained undetectable plasma RNA viral load (PVL); to explore factors predictive of residual viral shedding; and to evaluate the risk of heterosexual transmission. Women with undetectable PVL (<50 copies/mL) for >6 months were included in this cross-sectional study. HIV-RNA and HIV-DNA were measured in blood and cervicovaginal lavage fluid (CVL). Women were systematically tested for genital infections. The risk of transmission to male partners during unprotected intercourse was estimated. Eighty-one women composed the study population: all had HIV-RNA <40 copies/mL in CVL. HIV-DNA was detectable in CVL of 29/78 patients (37%). There was a weak positive correlation between HIV-DNA levels in PBMCs and CVL (r = 0.20; p = 0.08). In multivariate analysis, two factors were associated with HIV-DNA detection in CVL: previous AIDS-defining illnesses (OR = 11; 95%CI = 2-61) and current residual viremia (20<PVL<50 cp/mL) (OR = 3.4; 95%CI = 1.1-10.9). Neither the classes of cART regimen nor the presence of genital bacterial or fungal colonization were associated with HIV-DNA detection in CVL. Twenty-eight percent of the women had unprotected intercourse with their regular HIV-seronegative male partner, for between 8 and 158 months. None of their male partners became infected, after a total of 14 000 exposures. In our experience, HIV-RNA was undetectable in the genital tract of women with sustained control of PVL on cART. HIV-DNA shedding persisted in about one third of cases, with no substantial evidence of residual infectiousness.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "3. Proviral HIV-1 group M DNA PCR (Biocentric) which targets the LTR gene of HIV [6]. "
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    ABSTRACT: Introduction Replication of the human immunodeficiency virus involves an obligatory step of reverse transcription of the viral ribonucleic acid genome into a double-stranded deoxyribonucleic acid, and subsequent integration of the deoxyribonucleic acid into the human chromatin to form the proviral deoxyribonucleic acid. This proviral human immunodeficiency virus deoxyribonucleic acid is a critical marker for the diagnosis of acute infections, mother-to-child transmissions and for the confirmation of indeterminate serological reactions. We describe a case of a human immunodeficiency virus positive woman, naïve to antiretroviral treatment, who was persistently negative for human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction. This observation, to the best of our knowledge, is the first time that it has been described in Africa. Case presentation A 28-year-old Gabonese woman living in Cameroon requested a human immunodeficiency virus diagnosis in our laboratory. She had an unprotected heterosexual contact 6 months earlier while on vacation in Gabon. The request for a human immunodeficiency virus test was as a result of apprehensions developed after the exposure episode. Human immunodeficiency virus serological examinations were ambiguous and confirmatory tests (including human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction) were carried out. Apart from the human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction that was persistently negative, all other polymerase chain reactions carried out were positive. The deoxyribonucleic acid sequences have been submitted to the GenBank database with accession numbers: KC626022, KC626023 and KC626024 for the protease, reverse transcriptase and gp41 genes respectively. Conclusion The persistently negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reaction in a person with a confirmed human immunodeficiency virus infection is of immense importance in the human immunodeficiency virus diagnostic field. This could highlight the fact that cases of false negative human immunodeficiency virus proviral deoxyribonucleic acid polymerase chain reactions exist especially with the high genetic variations observed with human immunodeficiency virus. The challenges presented by such false negative tests in the identification of acute infections, mother-to-child transmissions and the confirmation of indeterminate serological reactions are daunting. These data therefore would be invaluable especially to clinicians in Africa where non-B human immunodeficiency virus subtypes circulate.
    Full-text · Article · Jun 2013 · Journal of Medical Case Reports
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