A cross-sectional study was performed on 152 consecutive patients (88 females; 64 males) attending OIC at Parirenyatwa Group of Hospitals and Harare Central Hospital between November 2014 and June 2015. Eleven patients opted not to be included in this study with the main reason given for refusal being lack of time. A total of 9000 adult patients are enrolled at the two OIC, with a male:female ratio of 1:1.5. The OIC serve a low to middle-income urban population of Harare, Zimbabwe. Patients visit the OIC for HIV testing and counselling services, routine follow up of HIV disease, and fulfilment of prescribed antiretroviral drugs. 85% of patients enrolled at the OIC are on highly active antiretroviral therapy (HAART). Ethical approval for this study was obtained from the University of Zimbabwe, College of Health Sciences Joint Research Ethics Committee and the Harare Central Hospital Ethics Committee. The study was registered with the Medical Research Council of Zimbabwe.
Males and females age ≥ 18 years were included in the study after providing informed signed consent. The source test document was checked to verify that each participant was HIV-positive. Demographic data and behavioural characteristics were collected through a face-to-face structured interview. CD4 counts and viral loads were verified by inspection of the participant’s clinical record.
Anal sample collection
The participant was positioned on a standard examination couch in the left lateral position with knees drawn up to the chest and buttocks at the edge of the bed. The perianal region was inspectedafter gently parting the buttocks. A Dacron-tipped swab was moistened in 0.9% normal saline, then introduced blindly into the anal canal as far as possible [5]. Using the external anal sphincter as a fulcrum, the swab was firmly rotated against the anal canal wall for 30 s while being withdrawn from the anal canal. Clean scissors were used to cut off the Dacron tip of the swab into a labelled 2 ml cryotube containing 0.5 ml 10% guanidine thiocyanate and frozen at − 70 °C until analysis.
HPV genotyping
HPV testing was done using primer pair MY09/MY11 polymerase chain reaction (PCR), followed by typing using the dot blot method, as described previously [5]. For internal quality control beta-globin was co-amplified during each PCR run and was probed for using a human beta-globin gene probe. Samples that tested negative for beta-globin were excluded from analysis.
Samples were thawed to room temperature. HPV DNA was extracted using the conventional ammonium acetate method. AmpliTaq Gold Polymerase enzyme was added to the samples to amplify target DNA. The PCR cycles were at: 95 °C for 9 min, 40 cycles of (95 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min), 72 °C for 5 min and then held at 4 °C. After PCR, the DNA amplification mixture was denatured using an alkaline buffer then, applied to a Biodyne B membrane. The DNA was fixed to the Biodyne B membrane by baking at 80 °C for 1 h and then exposed to an HPV L1 consensus probe mixture. A sample was defined as HPV-positive if it reacted positively to the consensus probe mixture. Hybridisation was then carried out between the sample DNA and specific biotin-labelled HPV probes for 14 high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and 15 low-risk HPV genotypes (6, 11, 26, 32, 40, 53, 54, 55, 61, 69, 70, 73, 82, 83 and 84). The hybridization products were then detected using enhanced chemiluminescence (ECL). Commercial HPV standards were blotted onto each membrane as positive controls for their respective genotype.
Statistical analysis
Data collection forms were inspected for completeness and accuracy by trained study personnel. Patients with missing data for a particular analysis were excluded only from that analysis. Data was analysed using IBM SPSS Statistics for Windows, version 22 (IBM Corp., Armonk, N.Y., USA). Comparison between groups of frequencies was performed with Chi-square test or Fisher’s exact test of association. All results were evaluated at a 95% confidence interval; significance was set at p < 0.05.