HIV-1 detection in cervicovaginal secretions during pregnancy.

OBJECTIVE: To assess the reproducibility of and factors associated with HIV detection in cervicovaginal secretions (CVS). DESIGN: Longitudinal study of 43 HIV-1-infected pregnant women in Paris. METHODS: HIV DNA was detected in peripheral blood mononuclear cells (PBMC) by Amplicor and gag nested polymerase chain reaction (PCR) assays. The HIV genotype was determined by heteroduplex mobility assay. Amplicor and gag nested PCR assays were performed on serial CVS samples for HIV DNA detection, and the HIV Monitor test was used for HIV RNA detection in plasma and CVS. RESULTS: A total of 144 CVS samples were collected from the women included in the study. HIV-1 DNA was detected in 36 (25%) of the 144 samples, from 16 (37.2%) of the 43 women. Results of HIV-1 DNA detection were concordant in the first two samples in 27 (84.4%) of the 32 women with at least two CVS samples. The last CVS sample collected in each woman was HIV-1 DNA-positive in 13 (30.2%) of the 43 women. Three factors were found to be independently associated with HIV-1 DNA detection in CVS: HIV-1 subtype B, absence of zidovudine therapy, and microbial cervicovaginal infection. HIV RNA was detected in CVS from 10 (23.3%) out of 43 women and correlated with DNA detection in the same sample and HIV RNA detection in plasma. CONCLUSIONS: DNA and RNA PCR can be used to detect HIV in cells and supernatants of CVS. These techniques may be useful in cohort studies to investigate HIV transmission and to evaluate the efficacy of antiretroviral drugs to reduce HIV excretion.

Polymorphism of the human immunodeficiency virus type 2 (HIV-2) protease gene and selection of drug resistance mutations in HIV-2-infected patients treated with protease inhibitors.

We described the baseline polymorphism of the human immunodeficiency virus type 2 (HIV-2) protease gene from 94 treatment-naive patients and the longitudinal follow-up of 17 protease inhibitor-treated patients. Compared to the HIV-2 consensus sequences, baseline polymorphism involved 47 positions. Substitutions selected under treatment were observed at positions corresponding to HIV-1 resistance mutations as well as at positions of currently unknown impact on HIV-1.

Detection of circulating human immunodeficiency virus type 2 in plasma by reverse transcription polymerase chain reaction.

Genomic RNA was detected using a reverse transcription (RT) nested polymerase chain reaction (PCR) method on plasma from 24 HIV2-infected patients. Results were interpreted based on immune and clinical status and results of plasma and cellular viraemia assays. Amplification of RNA extracted from plasma was positive in 13 of the 24 cases studied (54%). There was a negative correlation between the detection of RNA in plasma and the patients' CD4+ cell counts: all 5 patients with counts below 200 x 10(6)/l were RT-PCR RNA-positive, compared to only 4 of the 12 patients with counts above 500 x 10(6)/l. Cellular viraemia was positive in 12 of the 24 patients, and the results correlated with the CD4+ cell count. HIV2 was isolated from the plasma of 3 of the 24 patients, all of whom had CD4+ cell counts below 200 x 10(6)/l. The small viral load in HIV2-infected patients before the onset of immunodeficiency appeared to be a major limiting factor in the detection of the virus with current tests. The low percentage of RNA-positive plasma samples contrasts with the high rate of positivity in HIV1-infected patients. Differences in viral load and replication between HIV1 and HIV2 correlate with differences in the epidemiology and pathogenicity of the two viruses.

Comparative study of single and nested PCR for the detection of proviral HIV2 DNA.

We have tested seven pairs of primers for the detection of HIV2 DNA by single PCR in positive cultures from 21 infected patients. Four of these primer pairs were then used in a comparative study of single and nested PCR for the detection of HIV2 in fresh lymphocytes from 33 patients infected by the virus. HIV2 DNA was detected in 17 of the 33 patients (51.5%) by single PCR and 29/33 (88%) by nested PCR. All the patients negative in both nested and single PCR were asymptomatic and had CD4+ lymphocyte counts of at least 500 x 10(6)/l. This lack of PCR sensitivity for the detection of proviral HIV2 DNA in fresh lymphocytes cannot be totally attributed to genetic variability and may be related to a low viral load in asymptomatic HIV2-infected patients.

Quantification of proviral load of human immunodeficiency virus type 2 subtypes A and B using real-time PCR.

We have developed and evaluated a new method to quantify human immunodeficiency virus type 2 (HIV-2) proviral DNA based on LightCycler real-time PCR. The assay has a detection limit of 5 copies/10(5) peripheral blood mononuclear cells (PBMC) and is insensitive to HIV-2 strain variability: HIV-2 subtypes A and B are both recognized and quantified. The intra- and interassay coefficients of variation range from 16 to 40% for high provirus concentrations (5 x 10(5) copies) and from 41 to 39% for low concentrations (5 copies). We used this method to compare the proviral DNA load and viral RNA load in plasma with clinical and immunological status for 29 patients infected by HIV-2 (subtype A in 17 and subtype B in 12). The proviral load (median, 201 copies/10(5) PBMC) was similar to that reported for HIV-1 infection. The median proviral loads did not correlate with the CD4(+) cell count categories and were as follows for CD4(+) cell counts of >400, 200 to 400, and <200 cells/mm(3), respectively: 121 copies/10(5) PBMC (n = 8; range, <5 to 712 copies/10(5) PBMC); 114 copies/10(5) PBMC (n = 9; range, <5 to 1,907 copies/10(5) PBMC); and 285 copies/10(5) PBMC (n = 12; range, 53 to 2,524 copies/10(5) PBMC). Proviral load did not correlate with plasma HIV-2 RNA positivity. As HIV-2 is considered to replicate less efficiently than HIV-1, these high proviral loads might be explained by the proliferation of infected cells.

[The isolation and subtyping of the human immunodeficiency virus (HIV) from children in Moldova. The evidence for subtype F]. / Izolarea si subtiparea virusului imunodeficientei umane (HIV) la copii din Moldova. Evidentierea subtipului F.

HIV epidemic related to nosocomial transmission in Romanian nursed children represents up to now more than 50% of paediatric AIDS cases in Europe. Although the sources of this epidemic are obscure, HIV-1 subtype F, a minor form in Brasil and Africa, realised a founder effect in this risk group, thus becoming the major form in Romanian epidemic. This study presents the results of the virus isolation from HIV-infected children in Northeast Romania. Heteroduplex mobility assay (HMA) was performed in order to establish HIV-1 viral subtype for 6 isolates. All tested HIV-1 strains were proved to belong to F subtype. So, our study confirm that HIV-1 subtype F is responsible for the epidemic in the risk group of Romanian nosocomially infected children.

Phylogenetic analysis of 49 newly derived HIV-1 group O strains: high viral diversity but no group M-like subtype structure.

We assess the genetic relationships between 49 HIV-1 group O strains from 24 and 25 patients living in Cameroon and France, respectively. Strains were sequenced in four genomic regions: gag (p24) and three env regions (C2-V3, gp41, and for 22 C2-gp41). In each of the genomic regions analyzed, the genetic diversity among the group O strains was higher than that exhibited by group M. We characterize three major group O phylogenetic clusters (O:A, O:B, and O:C) that comprised the same virus strains in each of the genomic regions analyzed. The majority of strains cluster in O:A, a cluster previously identified by analysis of pol and env sequences. Group O recombinants were also identified. Importantly, the distinction between these three major group O clades was weak compared to the strong clustering apparent in the global group M phylogenetic tree that led to the identification of subtypes. Thus, these clusters of group O viruses should not be considered as equivalent to the group M subtypes. This difference between the pattern of group O and the global group M diversity, both taking into account the pandemic status of the group M subtypes and the comparatively small number of group O-infected individuals (the majority being from Cameroon), indicates that the group O phylogeny primarily represents viral divergence in the Cameroon region, analogous to group M viral diversity present in the Democratic Republic of Congo.