Study setting
The study was conducted by the University of Geneva in collaboration with Madagascar’s Health and Family Planning Ministry and the Saint-Damien Healthcare Centre in Ambanja, a mainly rural area with 125,056 inhabitants.
All participants signed an informed consent form before inclusion. The study was conducted from February to March 2015 in the Saint-Damien Healthcare Centre. Ethical approval of the study was obtained from the Malgasch National Commission for the Ethics of Science and Technology, as well as from the Ethical Cantonal Board of Geneva, Switzerland (CER: 14–071).
Study participants and procedures
We recruited 449 women aged between 30 and 65 years. Exclusion criteria were former conisation or hysterectomy and pregnancy beyond 20 weeks.
Participants received educational intervention on cancer and HPV infection. It was followed by information on how to use the self-HPV, written in the local dialect language. At the same time the participants received a sterile, cotton-tipped swab in a dry, labelled tube. Self-HPV was always performed without supervision. Then participants were invited to answer a questionnaire regarding socio-demographic information. HPV tests were analysed in Switzerland. HPV test analysis was done in a minimum time of 15 days after the sampling.
HPV-negative women were informed that they were not at risk for CC and that they would not require a test within the next 5 years. HrHPV-positive women were invited for colposcopic examination. A biopsy on acetowhite lesions coupled with endocervical curettage was performed, or a biopsy at the 6 o’clock position and endocervical curettage if colposcopy was normal. Treatment and follow-up was proposed according to the histological diagnosis. In case of an invalid result, women were asked to repeat their self-sampling. Histology was also analysed in Switzerland.
Laboratory methods
Upon arrival to the laboratory, the swabs were suspended in 4.3 mL of cobas® PCR media and were pulse vortexed for 3 × 10 s. A volume of 400 μL of each sample was used for DNA extraction and the rest of the sample was stored at 4 °C. DNA extraction was carried out using the cobas® HPV Test (Roche Molecular Systems). Nucleic extracts were then stored at −20 °C.
HPV analysis was accomplished by two different real-time PCR tests: the cobas® HPV Test (cobas) and the Anyplex™ II HPV28 (H28) test (Seegene, Seoul, South Korea), using the same extracted DNA from samples.
Amplification and detection were first carried out with cobas (at Biopath Lab, Lausanne), which detects 12 pooled hrHPV genotypes (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and concurrently provides separate results for HPV-16 and HPV-18. The detection is based on amplification of the L1 gene and TaqMan probes [16]. The human reference gene ß-globin s also detected.
Amplification and detection using the stored DNA extracts were then performed with the H28 test using the CFX96™ real-time thermocycler (at Buhlmann Laboratories AG Schönenbuch, Switzerland). Data recording and interpretation were automated. Details of the procedure and evaluation were described by Estrade et al. [17], Kim et al. [18] and Kwon et al. [19].
H28 is a semi-quantitative real-time multiplex PCR assay for screening and HPV genotyping. This test uses Dual Priming Oligonucleotides (DPO™) and Tagging Oligonucleotide Cleavage and Extension (TOCE™) technologies and allows to simultaneously detect 19 high-risk HPVs (including types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73 and 82) and 9 low-risk HPVs (including types 6, 11, 40, 42, 43, 44, 54, 61 and 70). ß-globin is also detected for internal control of assay validity.
Knowledge of the step at which the melting curve becomes positive allows for semi-quantification of the DNA load of ß-globin gene and HPV genomes, from low (+; positive after 40 PCR cycles, <102 copies/reaction), to intermediate (++; positive within 31 to 39 PCR cycles, ≥102 and < 105 copies/reaction), to high (+++; positive before 31 PCR cycles, ≥ 105 copies/reaction) (17).
Data analysis and statistics
Data were analysed with STATA 13 software package (StataCorp, Texas, USA).
Inter-rater agreement statistics and kappa coefficient with 95 % confidence intervals, percent total agreement and percent positive agreement were calculated for the paired results obtained by the cobas and H28 tests. The calculation was restricted to 14 hrHPV.
The trend of association between HPV results and DNA load was evaluated with the chi-square test. We also evaluated the effect of transport time on sample degradation by applying a Kaplan–Meier failure estimate.
The HPV prevalence was calculated from the number of positive cases divided from the number of tested specimens by both PCR methods. HPV positivity was distributed by age-group.
HPV type distribution in single and multiple infections of 19 hrHPVs and 9 lrHPVs was explored using the H28 test.