Objectives: To link local proinflammatory cytokines with HIV related nucleic acids in cervico-vaginal secretions and the factors associated with them. Methods: An observational study on 60 HIV ...positive women attending the department of obstetrics and gynaecology, University of Pavia, Italy. HIV-1 RNA in plasma, proviral HIV-1-DNA, cell associated and cell free HIV-1 RNA in cervico-vaginal secretions were evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). IL-1β, IL-6, and TNF-α were measured by ELISA in cervico-vaginal lavages. Multiple regression analysis on ordinal categorical variables was used to test for the simultaneous associations of clinical and microbiological variables on quartiles of cytokine concentrations in lavage samples. Results: Proviral HIV-1 DNA, cell associated and cell free HIV-1 RNA were detected in 76.7% (46/60), 70% (42/60), and 71.7% (43/60) of the patients, respectively. IL-1β concentration was directly correlated with proviral HIV-DNA (Spearman rho = 0.35, p = 0.01) and cell associated HIV-RNA levels (Spearman rho = 0.263, p = 0.05). IL-1β concentration (153.9 pg/ml) was higher (p<0.05) among women with cytological squamous intraepithelial lesion (SIL) than negative controls (73.4 pg/ml). In women with vaginal infection both IL-1β (41.7 pg/ml) and IL-6 (10.2 pg/ml) were lower (p<0.05) in comparison to negative controls (144.9 pg/ml and 23.7 pg/ml, respectively). Women receiving stable antiretroviral therapy had significantly lower TNF-α (34.4 pg/ml versus 44.4 pg/ml, p = 0.04) and higher IL-6 (24.0 pg/ml versus 1.4 pg/ml, p = 0.004) levels in lavage samples compared to untreated women. The associations between the presence of SIL, antiretroviral treatment, vaginal infection and cytokine concentrations in cervico-vaginal secretions were confirmed in multiple regression analysis. Conclusions: Local immune activation may modulate HIV-1 shedding in cervico-vaginal secretion with possible influence on vaginal physiology and host defence. Pharmacological agents lowering HIV-1 replication cause a shift to a pattern of cytokine production which seems less favourable to the transmission of the disease.
Four human cytomegalovirus (HCMV) isolates from four different AIDS patients treated with both ganciclovir and foscarnet and not responding clinically to antiviral treatment, were studied in order to ...verify the occurrence of double resistance to both drugs, and to define whether single or multiple HCMV strains could be responsible for the double resistance. Peripheral blood leukocytes (PBL), the relevant conventional viral isolates, and plaque-purified strains from all four patients were examined by antiviral drug susceptibility testing by an immediate-early antigen plaque reduction assay and by restriction fragment length polymorphism (RFLP) analysis using polymerase chain reaction (PCR)-amplified multiple genome regions and endonucleases. All four HCMV strains had a high level of resistance to both ganciclovir and foscarnet. A single HCMV strain was shown to be responsible for the dual resistance in each patient. HCMV strain identity and uniqueness were shown for each of the four patients in blood samples, viral isolates, and plaque-purified strains. In addition, in two patients the same single HCMV strain shifted progressively from drug sensitivity to ganciclovir and then to ganciclovir-foscarnet resistance. These findings document that resistance to both ganciclovir and foscarnet of HCMV strains recovered from blood of AIDS patients represents an emerging problem. Although it is known that multiple HCMV strains may cocirculate in the blood of AIDS patients, single strains appear to be responsible for the dual resistance. Molecular mechanisms responsible for the double resistance of the four reported strains are under study.
To examine the amount of cell-free and cell-associated virus in cervicovaginal secretions (CVS) of HIV-infected women.
Paired cervicovaginal and blood samples from 61 seropositive women were ...quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcription-PCR (cRT-PCR) for: (1) genomic RNA from plasma and cell-free CVS, and (2) unspliced (u/s) RNA transcripts and proviral DNA in cells from secretions.
HIV DNA was detected in 42.6%, u/s transcripts in 32.7% and cell-free HIV RNA in 31.1% of 61 cervicovaginal samples. The median copy numbers of HIV DNA, u/s transcripts, and cell-free RNA were 125 copies/105 cells, 40 copies/105 cells, and 300 copies/mL of secretion, respectively. Nineteen of 26 (73.1%) and 17 of 26 (65.3%) women positive for DNA were also positive for RNA transcripts and cell-free RNA, respectively (P<0.001). A significant correlation between the amounts of cell-free and u/s transcripts was also found (Spearman Rho 0.618, P=0.014). The prevalences of u/s transcripts and cell-free RNA were 42.6% and 53.8% respectively among patients with detectable blood RNA, and 22.9% (P=0.09) and 14.3% (P=0.0017) among patients with undetectable blood RNA. In stepwise logistic regression, cell-free RNA was independently associated with the presence of detectable blood viremia. The amount of HIV DNA was lower among subjects currently under treatment (50 copies/105 cells) than in untreated subjects (250 copies/105 cells) (P=0.037).
Both cell-free and cell-associated HIV could be detected and quantitated in CVS, providing a means to examine the level of viral activity in the female genital tract.
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