Combination ART Reduces the Risk of HPV in HIV+ Women
Combination ART Reduces the Risk of HPV in HIV+ Women
In this prospective cohort study, women known with HIV infection were approached for enrolment provided they were cART-naive. Patients known with any cancer (including cervical) and pregnant women were excluded. The study was approved by the Stellenbosch University Human Research Ethics Committee (N09/04/1065).
Follow-up visits were conducted twice a year. At each visit, the clinical, obstetric and sexual behaviour data were recorded and a cervical Pap test was performed. Referral for colposcopy evaluation occurred after a single abnormal cytology result.
The decision to initiate ART was not determined by the study protocol, but was based on the South African Government Protocol for the Initiation of ART. Adherence to the prescribed regimen was ascertained by a combination of the pharmacy pick-up and patient self-report methods. Although interruptions were recorded, distinction was not made between those that were physician-initiated and those that were patient-initiated.
Response to ART was measured by testing the CD4 T-cell count and plasma HIV-RNA viral load determination at study enrolment and at each six monthly follow-up visits. The viral load determination done at baseline is referred to as the viral set point.
Cervical HPV samples were collected with a Cervexbrush at baseline and 6 monthly intervals, placed in PreservCyt (Hologic, Bedford, Massachusetts, USA) solution and stored at -80C for later analysis by Roche linear array, as previously described. Test strip results were read independently by two molecular scientists. Both were blinded as to the ART and cytological status detected in any of the study participants. Proviral DNA was isolated from sample specimens using the Qiagen DNA Blood and Tissue Kit (Qiagen, Hilden, Germany). HIV proviral DNA copy numbers were measured by the real-time quantitative PCR method (qPCR) on the DNA extracted from the cervical specimens for the HPV analysis, as previously described. Primers and probes were obtained from Integrated DNA Technologies (South Africa) and targets part of the HIV-1 LTR-gag fragment for amplification. The cell numbers were calculated by using pc-CCR5 (obtained from the NIH AIDS reference reagent laboratory) as a reference gene for cellular copy number, while pMJ4 was used as a positive control to determine HIV-1 copy number. Negative and positive-control DNAs were included in all reactions. Samples were run in duplicate on a CFX96 Touch Real-Time PCR Detection system (Bio-Rad Ltd., Hercules, California, USA) and analyses completed using CFX Manager software.
A SQL database (Microsoft SQL Server 2008) was created to capture, store and retrieve study data. Once extracted, the data were analysed using Microsoft Excel 2010, XLSTAT (version 2013.3.01) and Stata (version 11; Stata Corp., College Station, Texas, USA). Baseline demographic variables were assessed using Pearson's chi-square test or Fisher's exact test to compare proportions, and Student's t test to compare means between groups.
For the purpose of this analysis, we defined ART in two ways. The first variable, 'cART treatment status', was used as a binary variable to indicate if the patient was using cART at a visit. A patient who had as yet not commenced treatment, or had an interruption of more than 1 month, was labelled 'not on treatment' at that visit. For the other variable, 'time on cART', the cART start date was recorded and the time that had elapsed since the first date of administration was calculated at each visit. For women not initiated to cART, the value remained 0 for the time until they started taking antiretrovirals. This variable would not be influenced by adherence to the cART regimen.
We modelled the probability of positive detection of HPV using mixed-effects logistic regression as a function of cART treatment status or time on cART. Mixed models incorporate a random effect to account for within-participant correlation due to the repeated measurements. Generalized estimating equation (GEE) procedure, assuming an exchangeable correlation structure, was used to estimate the population average effect of ART expressed as an odds ratio (OR) and the associated 95% CIs. All models were adjusted for 'excision treatment of cervical neoplasia sexual activity' and the CD4 T-cell count as time-dependent variables. Age was included as a non-time-dependent variable due to the relatively short follow-up time. We compared the effect of cART on the overall risk of detection of each HPV genotype to the effect of cART on HPV-16 genotype by estimating the ratio of OR for each genotype to the OR for HPV-16 genotype using the GEE models. Thereafter, using meta-analysis techniques of combining results across subgroups, we estimated the overall effect of cART on hrHPV genotypes (HPV-16, 18, 31, 33, 35, 39, 45, 52, 58) and all others as low-risk HPV (lrHPV) genotypes compared to the effect of cART on HPV-16 genotype alone. These results were expressed as the ratio of ORs and the associated 95% CIs. For all analyses, P values reported were two-tailed at the 5% alpha level.
Methods
Study Design, Population, Setting, Inclusion Criteria and Outcomes Definitions
In this prospective cohort study, women known with HIV infection were approached for enrolment provided they were cART-naive. Patients known with any cancer (including cervical) and pregnant women were excluded. The study was approved by the Stellenbosch University Human Research Ethics Committee (N09/04/1065).
Follow-up visits were conducted twice a year. At each visit, the clinical, obstetric and sexual behaviour data were recorded and a cervical Pap test was performed. Referral for colposcopy evaluation occurred after a single abnormal cytology result.
The decision to initiate ART was not determined by the study protocol, but was based on the South African Government Protocol for the Initiation of ART. Adherence to the prescribed regimen was ascertained by a combination of the pharmacy pick-up and patient self-report methods. Although interruptions were recorded, distinction was not made between those that were physician-initiated and those that were patient-initiated.
Response to ART was measured by testing the CD4 T-cell count and plasma HIV-RNA viral load determination at study enrolment and at each six monthly follow-up visits. The viral load determination done at baseline is referred to as the viral set point.
Cervical HPV samples were collected with a Cervexbrush at baseline and 6 monthly intervals, placed in PreservCyt (Hologic, Bedford, Massachusetts, USA) solution and stored at -80C for later analysis by Roche linear array, as previously described. Test strip results were read independently by two molecular scientists. Both were blinded as to the ART and cytological status detected in any of the study participants. Proviral DNA was isolated from sample specimens using the Qiagen DNA Blood and Tissue Kit (Qiagen, Hilden, Germany). HIV proviral DNA copy numbers were measured by the real-time quantitative PCR method (qPCR) on the DNA extracted from the cervical specimens for the HPV analysis, as previously described. Primers and probes were obtained from Integrated DNA Technologies (South Africa) and targets part of the HIV-1 LTR-gag fragment for amplification. The cell numbers were calculated by using pc-CCR5 (obtained from the NIH AIDS reference reagent laboratory) as a reference gene for cellular copy number, while pMJ4 was used as a positive control to determine HIV-1 copy number. Negative and positive-control DNAs were included in all reactions. Samples were run in duplicate on a CFX96 Touch Real-Time PCR Detection system (Bio-Rad Ltd., Hercules, California, USA) and analyses completed using CFX Manager software.
Statistical Analysis
A SQL database (Microsoft SQL Server 2008) was created to capture, store and retrieve study data. Once extracted, the data were analysed using Microsoft Excel 2010, XLSTAT (version 2013.3.01) and Stata (version 11; Stata Corp., College Station, Texas, USA). Baseline demographic variables were assessed using Pearson's chi-square test or Fisher's exact test to compare proportions, and Student's t test to compare means between groups.
For the purpose of this analysis, we defined ART in two ways. The first variable, 'cART treatment status', was used as a binary variable to indicate if the patient was using cART at a visit. A patient who had as yet not commenced treatment, or had an interruption of more than 1 month, was labelled 'not on treatment' at that visit. For the other variable, 'time on cART', the cART start date was recorded and the time that had elapsed since the first date of administration was calculated at each visit. For women not initiated to cART, the value remained 0 for the time until they started taking antiretrovirals. This variable would not be influenced by adherence to the cART regimen.
We modelled the probability of positive detection of HPV using mixed-effects logistic regression as a function of cART treatment status or time on cART. Mixed models incorporate a random effect to account for within-participant correlation due to the repeated measurements. Generalized estimating equation (GEE) procedure, assuming an exchangeable correlation structure, was used to estimate the population average effect of ART expressed as an odds ratio (OR) and the associated 95% CIs. All models were adjusted for 'excision treatment of cervical neoplasia sexual activity' and the CD4 T-cell count as time-dependent variables. Age was included as a non-time-dependent variable due to the relatively short follow-up time. We compared the effect of cART on the overall risk of detection of each HPV genotype to the effect of cART on HPV-16 genotype by estimating the ratio of OR for each genotype to the OR for HPV-16 genotype using the GEE models. Thereafter, using meta-analysis techniques of combining results across subgroups, we estimated the overall effect of cART on hrHPV genotypes (HPV-16, 18, 31, 33, 35, 39, 45, 52, 58) and all others as low-risk HPV (lrHPV) genotypes compared to the effect of cART on HPV-16 genotype alone. These results were expressed as the ratio of ORs and the associated 95% CIs. For all analyses, P values reported were two-tailed at the 5% alpha level.
Source...