One hundred (100) consecutive, consenting, non-pregnant, HIV sero-positive females (18) years or older were recruited from the Infectious Disease Clinic at the Princess Margaret Hospital in Nassau, Bahamas. The criteria for eligibility required all of the following: Females 18 years and older, a positive HIV via ELISA and Western Blot methods and HIV infectivity via sexual intercourse. All participants were interviewed, provided informed written consent and had a liquid based pap smeared performed after receiving consent. The study received ethical approval from the Ethics Committee of the Public Hospital Authority and the University of the West Indies – Bahamas Campus.
Sample and data collection
Each participant had a vaginal examination performed by an Obstetrics and Gynaecology Resident. Cervical samples were collected using a cytobrush (cytobroom) and stored in Sure Path vials. A cytotechnologist screened the prepared slides. All positive results and fifteen percent (15%) of all negatives results were reviewed by a board certified cytopathologist. The cytology was reported as either negative, atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesion (LGSIL) or high-grade squamous intraepithelial lesion (HGSIL). Additionally co-infections as well as presence of HPV changes were noted. The Obstetrics and Gynaecology Oncologist referred all patients with abnormal cytology results to Colposcopy Clinic for further evaluation, treatment and follow-up as determined.
Each patient was interviewed and a questionnaire completed by the interviewer. Information on associated HPV risk factors such as parity, number of sexual partners, and birth control method, risky behavior such as drug use, sex under the influence of drugs and condom usage as well as demographic were obtained via the questionnaire. The medical records were reviewed to obtain patient's date of HIV testing and confirm sero-positivity, most recent CD4+ lymphocyte counts and viral load, and use of high active anti-retroviral therapy.
CD4+ and viral load
Samples collected from patients were stained with FlowCARE PLG CD4 Reagent. CD4+ lymphocytes counts were determined by flow cytometry. White blood cell counts were obtained with a COULTER STKS Instrument. The values were expressed in terms of percentage (%) of total lymphocytes and absolute count (cells/μL).
The DNA HIV viral load was tested using a quantiplex HIV-1 RNA 3.0 Assay. This assay is a nucleic acid hydridization procedure to directly quantify HIV-1 RNA in human plasma. The HIV-1 is concentrated from the plasma by centrifugation then the genomic RRNA is liberated from the virions and captured on a microwell by specific synthetic oligonucleotide capture probes. The probes bind to different regions of the pol gene of the viral RNA which is them amplified.
Multiple copies of the alkaline phosphatase-labelled probe are then hybridized to an immobilized complex to amplify the signal. Detection is achieved by incubating the complex with a chemiluminescent substrate. The light emission generated by the bound alkaline phosphatase reacting with the chemiluminescent substrate is recorded as relative light units (RLUs). The light emission is directly proportional to the amount of HIV-1 present in each sample. A standard curve is defined by light emission from standards containing known concentrations of beta proiolactone (BPL)-treated virus. Concentrations of HIV-1 RNA in plasma specimens are determined form this standard curve.
HPV was detected using the HPV Hybrid Capture II assay which is a signal amplification nucleic acid method for the qualitative detection of eighteen types of human papillomavirus (HPV) DNA in cervical specimens. The assay differentiates between high risk and low risk groups. High HPV types detected are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.
Clinical and laboratory risk factors for the study population were imported to a statistical software package for analysis. Statistical analyses were performed using the Intercooled STATA (version SE 10.0) software (StataCorp. LP, College Station TX). Factors associated with high-risk HPV status were selected based on cross-tabulations and univariate logistic regression analyses. Cross-tabulations were analyzed using the Fisher's exact test. Logistic regression was used to perform the analyses of associations between demographic, behavioral and clinical variables with high-risk HPV status. Crude odds ratios were first calculated. Potential confounders were identified based on p-values = 0.05. Logistic regression was used to calculate the relative odds of high-risk HPV status after adjusting for the potential confounders (age (continuous), marital status, number of pregnancies, Tobacco use, duration of HAART therapy, CD4 counts, viral load and cervical cytology).