MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted virus known to be a causative agent of cervical cancer (CC), one of the most frequent cancers in women worldwide. HPV is a double stranded DNA virus of approximately 7,900 bp; belonging to Papillomaviridae family. To date, about 202 low risk (LR) and high risk (HR) HPV genotypes have been identified. However, available vaccines against HPV infection are designed based on the most common known genotypes. Therefore, it is critical to understand the scope and diversity of HPV genotypes in all geographical locations which can help to inform the design and development of future vaccines. OBJECTIVE: The objective of this study was to describe the burden and diversity of HPV genotypes in a cohort of women in routine care in northern South Africa. METHODS: Eighty seven women consented to participate in the study and each provided a specimen for analysis. With the help of qualified health care practitioners, Aptima Cervical Specimen Collection and Transport Kit (Hologic, San Diego, CA) was used to collect cervical specimens from each study participant following the manufacturer’s procedure. Total DNA was purified from the cervical pellet using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified DNA was then subjected to a single round conventional PCR in a reaction volume of 100 μl to amplify HPV L1 gene comprising of approximately 450 bp. A portion of each PCR amplicon from each participant was denatured, hybridized and genotyped using the Linear Array HPV genotyping Test Kit (Roche Molecular Systems, Inc. Branchburg, NJ USA). The kit is designed to detect 37 HPV genotypes (genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108). To detect the HPV genotypes, the Linear Array (LA) reference guide was used for results interpretation following the manufacturer’s instructions. The other portion of each of the amplicons was subjected to next generation sequencing (NGS) using the Illumina MiniSeq platform. Using the Nextera XT DNA Library preparation kit, an initial input of 1ng genomic DNA was tagmented, cleaned up, normalized and pooled. The pooled library was then denatured with 0.1 N NaOH and diluted into a final volume of 500 μl at 1.8 pM then sequenced using the Local Run Manager option following the manufacturer’s instructions. The generated sequence data was downloaded into fastaQ format and analysed using Genious 11.0.5 software. RESULTS: Of the 87 participants, the overall proportion of women harbouring HPV DNA by linear array (LA) PCR was 23% (n=20). Of the 20, 16 (80%) were living with HIV. However, this difference was not significant (p=0.077). Genotyping data generated by Roche LA method was successful for all the 20 positive amplicons. In this study, 27 (73%) of the 37 HPV genotypes incorporated in the Roche Linear Array method were detected. The detected genotypes include: types 84, 83, 81, 73, 72, 71, 70, 69, 68, 66, 62, 61, 59, 54, 53, 52, 51, 45, 42, 39, 35, 26, 18, 16, 6, IS39 and CP6108. Most women (15/20;75%) harboured multiple infections compared to single infection. In terms of genotypes distribution, the most frequent genotypes detected LR HPV types in increasing order of frequency included HPV type 61 and 83 (12%), 62 (36%) and 81 (43%). On the other hand, HPV type 66, 53, 52, 51, 18 and 16 were the most common genotypes detected HR HPV types. In contrast, although genotyping data was successfully generated from 15 of 20 women (75%), NGS technology was seen to be more sensitive compared to Roche LA method. Nearly all the detected genotypes identified by the commercial kit were detected by NGS. In addition, NGS detected 10 namely: HPV types 11, 31, 33, 40, 55, 56, 58, 64, 67, and 82 that were not detected by the LA yet incorporated in the kit. Moreover, it was observed that NGS identified additional 6 HPV types including HPV types 2, 27, 30, 35, 85 and 102 not incorporated in the Roche LA kit. A similar distribution of HPV multiple infections was observed in the study population, however, high frequency of 93% (14 of 15) was detected by NGS. The proportion of women harbouring one or more of the 22 LR HPV types was 100% (n=15).The most frequent LR genotypes in increasing order of frequency was HPV type 62 and 70 (27%), 6 (40%) and 11 (47%). HPV types 40, 42, 54, 72, 64, and 81 were the least detected genotypes with n=1 (7%) each. Furthermore, the common combination observed among the participants was type 6 and 11. In contrast, the most frequent detected genotypes in the study population by NGS under the HR HPV types in increasing order of frequency include type 35 (21%), 39, 56 and 82 (29%), 68 (36%) and 51 (50%). In addition, HPV types 26, 31, 45, 53, 56, 58 and 66 were the least detected genotypes n=1 (7%) in the study population. HPV 39 and 68 were observed as the common combination detected under HR HPV types. Following genotyping by LA and NGS, the demographic and clinical data of all the 20 positive subjects by PCR were subjected to statistical analysis to determine the association between HPV positive DNA status and associated risk factors. Smoking status (p=0.000), age at first sexual intercourse (p=0.011), vaccination status (p=0.000), gender of sexual partner (p=0.000), highest level of education (p=0.004), marital status (p=0.008) and number of sexual partners (p=0.000) were found to be having a positive statistical association. CONCLUSION: Amplification of targeted HPV DNA from cervical specimens demonstrated the presence of HPV infection in the study cohort, with a proportion of 23%. The findings illustrate that there is a diversity of HPV genotypes prevalent in the study population as shown by Roche LA and NGS methods. However, the NGS method was observed to be more sensitive than Roche LA in detecting HPV genotypes. Furthermore, NGS identified 6 additional HPV types not incorporated in the Roche LA. Thus, there are genotypes that may be present in the study population that the Roche commercial kit may fail to detect. Therefore, is it imperative to use both genotyping methods to confirm HPV genotypes. / NRF

Objectif : Les traitements répétés des lésions précancéreuses du col utérin (HSIL), nécessaires en cas d’échecs de traitement, sont associés à des issues obstétriques négatives, telle qu’une augmentation de la mortalité néonatale. Nous avons investigué l’association entre un grand nombre de facteurs de risque potentiels pour l’échec de traitement des HSIL dans le but d’identifier des prédicteurs potentiellement modifiables de l’échec de traitement. Méthodes : La population source était constituée de 1 548 femmes canadiennes qui ont subi un premier traitement pour HSIL. L’échec de traitement a été défini comme étant un diagnostic histologique de HSIL ou cancer au cours des deux années suivant le traitement. Nous avons mené une étude cas-témoins nichée incluant les 101 cas d’échec de traitement ainsi que les témoins appariés 1 :1 par centre de traitement et par date d’échec. Nous avons calculé des rapports de cotes (OR) et intervalles de confiance (CI) à 95% à l’aide de régressions logistiques conditionnelles, pour les associations entre l’échec de traitement et l’âge, le nombre d’accouchements, le statut tabagique, le nombre de partenaires sexuels, l’utilisation du condom, la méthode de contraception, les marges, le nombre de passages, le diagnostic sur le spécimen de traitement, le génotype du VPH, et le nombre de types. Nous avons aussi estimé l’association entre la charge virale et les variants du VPH16 et du VPH18 et l’échec de traitement. Résultats : Les marges positives vs négatives (OR ajusté=4.05, 95% CI 1.57-10.48), la positivité pour le VPH16/18 vs autres types (OR ajusté=2.69, 95% CI 1.32-5.49), et avoir un variant similaire au prototype du VPH16 vs le prototype (OR ajusté=2.49, 95% CI 1.07-5.83) étaient des prédicteurs de l’échec de traitement des HSIL. Être plus âgé, avoir des lésions plus sévères, avoir une infection monotype, et avoir une variation à la position 7521 chez celles avec le VPH16 pourraient augmenter le risque d’échec de traitement, mais les associations n’étaient pas statistiquement significatives. Les estimations pour les autres facteurs étaient proches de la valeur nulle. Nous n’avons pas observé de modification d’effet du génotype sur le risque de l’échec de traitement par le tabagisme, ni par les marges. Conclusion : Seules les marges positives, la positivité pour le VPH16/18 et avoir un variant similaire au prototype étaient des prédicteurs d’un échec de traitement au cours des deux années suivant le traitement. Malgré l’aspect non-modifiable des prédicteurs identifiés, ils sont informatifs et pourront éclairer la prise en charge et le suivi clinique. / Objective: Repeated treatments for high-grade squamous intraepithelial lesions (HSIL), which are necessary in the case of treatment failure, are associated with negative obstetric outcomes, such as an increased risk of neonatal death. We investigated the association between a large number of potential risk factors and HSIL treatment failure in an effort to identify potentially modifiable predictors of treatment failure. Methods: The source population included 1,548 Canadian women who received a first treatment for HSIL. Treatment failure was defined as the histological diagnosis of HSIL or cancer within the two years following treatment. We conducted a nested case-control study that included all 101 cases of treatment failure and controls that were matched 1:1 on treatment center and date of failure. We used conditional logistic regression to calculate the odds ratios (OR) and 95% confidence intervals (CI) between treatment failure and age, parity, smoking status, number of sexual partners, condom use, method of contraception, margins, number of passes, diagnosis on the treatment specimen, HPV genotype and number of types. We also estimated the association between HPV16 and HPV18 viral loads and variants and HSIL treatment failure. Results: Having positive vs. negative margins (adjusted OR=4.05, 95% CI 1.57-10.48), being positive for HPV16 and/or HPV18 vs. any other type (adjusted OR=2.69, 95% CI 1.32-5.49), and having a prototype-like variant of HPV16 vs. the prototype (adjusted OR=2.49, 95% CI 1.07-5.83) were predictors of HSIL treatment failure. Older age, more severe lesions, single-type infections and a variation at the 7521 position of the HPV16 genetic sequence may lead to a higher risk of treatment failure but were not statistically significant. Estimates for all other factors were near the null value. The effect of genotype on the risk of treatment failure was not modified by smoking status, nor by margin status. Conclusion: Only positive margins, HPV16/18 positivity, and having a prototype-like variant of HPV16 were predictors for HSIL treatment failure within two years of treatment. Despite being non-modifiable, the identified predictors are clinically significant in regards to management and follow-up of patients.