Increased levels of HIV-1-infected cells in endocervical secretions after the luteinizing hormone surge.

Levels of HIV-1 RNA in endocervical specimens fluctuate with the menstrual cycle, suggesting that cell-free HIV-1 levels may vary during the cycle, which could influence infectivity. Here, we examined daily changes in endocervical HIV-1-infected cells during 1 cycle. There were significant positive associations between the number of days from the luteinizing hormone surge and the number of HIV-1 DNA copies/swab (P = 0.001) and the number of total cells/swab (P < 0.001) in endocervical specimens. These data suggest that sampling of cell-associated endocervical HIV-1 increases after the periovulatory period, which could result in increased exposure to HIV-1-infected cells during sexual contact.

Hormonal contraceptive use, herpes simplex virus infection, and risk of HIV-1 acquisition among Kenyan women.

BACKGROUND: Studies of the effect of hormonal contraceptive use on the risk of HIV-1 acquisition have generated conflicting results. A recent study from Uganda and Zimbabwe found that women using hormonal contraception were at increased risk for HIV-1 if they were seronegative for herpes simplex virus type 2 (HSV-2), but not if they were HSV-2 seropositive. OBJECTIVE: To explore the effect of HSV-2 infection on the relationship between hormonal contraception and HIV-1 in a high-risk population. Hormonal contraception has previously been associated with increased HIV-1 risk in this population. METHODS: Data were from a prospective cohort study of 1206 HIV-1 seronegative sex workers from Mombasa, Kenya who were followed monthly. Multivariate Cox proportional hazards analyses were used to adjust for demographic and behavioral measures and incident sexually transmitted diseases. RESULTS: : Two hundred and thirty-three women acquired HIV-1 (8.7/100 person-years). HSV-2 prevalence (81%) and incidence (25.4/100 person-years) were high. In multivariate analysis, including adjustment for HSV-2, HIV-1 acquisition was associated with use of oral contraceptive pills [adjusted hazard ratio (HR), 1.46; 95% confidence interval (CI), 1.00-2.13] and depot medroxyprogesterone acetate (adjusted HR, 1.73; 95% CI, 1.28-2.34). The effect of contraception on HIV-1 susceptibility did not differ significantly between HSV-2 seronegative versus seropositive women. HSV-2 infection was associated with elevated HIV-1 risk (adjusted HR, 3.58; 95% CI, 1.64-7.82). CONCLUSIONS: In this group of high-risk African women, hormonal contraception and HSV-2 infection were both associated with increased risk for HIV-1 acquisition. HIV-1 risk associated with hormonal contraceptive use was not related to HSV-2 serostatus.

Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests.

Quantitative polymerase chain reaction (PCR)-based tests are used in several different scientific fields to determine levels of a target DNA sequence of interest (the target molecule). The accuracy of quantitative PCR-based tests can be assessed by using the assay to determine the number of copies of the target molecule in a sample with a known concentration of the target molecule. For example, a sample with a known concentration of a target DNA sequence is serially diluted into replicate aliquots and these are tested to determine if the observed quantity of the target is close to the expected quantity (given the concentration in the original sample and the dilution). Statistical methods that are conventionally used to assess the accuracy of these assays do not take into account the variability in the number of target molecules in each aliquot from the original sample. We develop methods that take into account this extra variation and which determine the accuracy of quantitative PCR-based tests in estimating the number of target molecules based on a set of assays of serial dilutions from an original sample with a known concentration of target molecules. These methods are applied to data from an experiment to test the accuracy of a real-time PCR assay at low HIV-1 DNA copy levels.

Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters.

Studies of human immunodeficiency virus type 1 (HIV-1) transmission suggest that genital HIV-1 RNA and DNA may both be determinants of HIV-1 infectivity. Despite its potential role in HIV-1 transmission, there are limited quantitative data on genital HIV-1 DNA. Here we validated an in-house real-time PCR method for quantification of HIV-1 DNA in genital specimens. In reactions with 100 genomes to 1 genome isolated from a cell line containing one HIV-1 provirus/cell, this real-time PCR assay is linear and agrees closely with a commercially available real-time PCR assay specific for a cellular housekeeping gene. In mock genital samples spiked with low numbers of HIV-1-infected cells such that the expected HIV-1 DNA copy number/reaction was 100, 10, or 5, the average copy number/reaction was 80.2 (standard deviation [SD], 28.3), 9.1 (SD, 5.4), or 3.1 (SD, 2.1), respectively. We used this method to examine genital HIV-1 DNA levels in specimens from women whose low plasma HIV-1 RNA levels are typical of HIV-1 nontransmitters. The median HIV-1 DNA copy number in endocervical secretions from these women (1.8 HIV-1 DNA copies/10,000 cells) was lower than that for women with higher plasma HIV-1 RNA levels (16.6 HIV-1 DNA copies/10,000 cells) (P=0.04), as was the median HIV-1 DNA copy number in vaginal secretions (undetectable versus 1.0 HIV-1 DNA copies/10,000 cells). These data suggest that women with low plasma HIV-1 RNA and thus a predicted low risk of HIV-1 transmission have low levels of genital HIV-1 cell-associated virus. The assay described here can be utilized in future efforts to examine the role of cell-associated HIV-1 in transmission.

Risk factors for human immunodeficiency virus type-1 acquisition in women in Africa.

Over 65% of individuals infected with human immunodeficiency virus type 1 (HIV-1) reside in sub-Saharan Africa, and thus efforts to change the course of the HIV-1 pandemic should focus on this geographic region. Here, we discuss factors, including sexually transmitted diseases and hormonal contraceptive use, that impact susceptibility to HIV-1 in women in sub-Saharan Africa. This population is of special interest, given that women in this region are more likely to be infected than men. In addition, they are a potential source of transmission to their infants. A better understanding of the factors that influence HIV-1 susceptibility is key to developing interventions to control the epidemic.

Cyclic shedding of HIV-1 RNA in cervical secretions during the menstrual cycle.

The association between hormone fluctuations during the menstrual cycle and human immunodeficiency virus type 1 (HIV-1) RNA shedding in cervical and vaginal secretions was examined daily for 17 HIV-1-seropositive women, for the duration of 1 cycle. Serum levels of RNA were evaluated 3 times/week. A marginally significant positive correlation between serum levels of progesterone and serum levels of HIV-1 RNA (P=.04) was observed. Cervical virus levels were significantly correlated with the number of days from the midcycle surge in luteinizing hormone (LH) (P=.008). The lowest levels of cervical HIV-1 RNA were present at the LH surge, and this nadir was followed by an increase in virus levels that reached a maximum before the start of menses. In contrast, there was no significant association between the number of days from the LH surge and the level of HIV-1 RNA in vaginal secretions (P=.4). These data support the hypothesis that the level of HIV-1 RNA in cervical secretions is influenced by the menstrual cycle, and they suggest that the risk of heterosexual transmission of HIV-1 may increase as menses is approached.

Identification of a human papillomavirus type 16-specific epitope on the C-terminal arm of the major capsid protein L1.

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and -52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.

Validation of performance of the gen-probe human immunodeficiency virus type 1 viral load assay with genital swabs and breast milk samples.

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in > or =77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load.