Distribution of human papillomavirus genotypes by severity of cervical lesions in HPV screened positive women from the ESTAMPA study in Latin America

The proportion of HPV16 and 18-associated cervical cancer (CC) appears rather constant worldwide (≥70%), but the relative importance of the other HR-HPV differs slightly by geographical region. Here, we studied the HPV genotype distribution of HPV positive Latin American (LA) women by histological grade, in a sub-cohort from the ESTAMPA study; we also explored the association of age-specific HPV genotypes in severe lesions. Cervical samples from 1,252 participants (854 ≤CIN1, 121 CIN2, 194 CIN3 and 83 CC) were genotyped by two PCRs-Reverse Blotting Hybridization strategies: i) Broad-Spectrum General Primers 5+/6+ and ii) PGMY9/11 PCRs. HPV16 was the most frequently found genotype in all histological grades, and increased with the severity of lesions from 14.5% in ≤ CIN1, 19.8% in CIN2, 51.5% in CIN3 to 65.1% in CC (p < 0.001). For the remaining HR-HPVs their frequency in CC did not increase when compared to less severe categories. The nonavalent vaccine HR-types ranked at the top in CC, the dominant ones being HPV16 and HPV45. HR-HPV single infection occurs, respectively, in 57.1% and 57.0% of ≤CIN1 and CIN2, increasing to 72.2% and 91.6% in CIN3 and CC (p<0.001). No association between age and HPV type was observed in CC, although the risk of HPV16 infection in CIN3 cases increased with age. Results confirm the relevance of HPV16 in the whole clinical spectrum, with a strong rise of its proportion in CIN3 and cancer. This information will be relevant in evaluating the impact of HPV vaccination, as a baseline against which to compare genotype changes in HPV type-specific distribution as vaccinated women participate in screening in LA region. Likewise, these data may help select the best HPV testing system for HPV-based efficient, affordable, and sustainable screening programmes.

Therefore, in RESULTS, a new sentence was added: Lines 210-212: "No substantial differences were observed in the prevalence of HPV genotype risk-based groups when distinguishing between colposcopy/biopsy negative and CIN1 diagnoses (Table S1)" 2 -Table 2 includes 5 groups, HPV16/18, other HR-HPV, Possibly HR-HPV, LR-HPV, negative.Many cases showed multiple HPV infections.The last row shows the numbers: 854, 121, 194, 83, all with 100%.I assume HPV16/18 group includes the cases with some other HPV infection (multiple); other HR-HPV group with no HPV 16/18; LR-HPV with no any other HR-HPV et al.Suggest the authors to explain details.
The reviewer interpreted correctly.To further clarify the description already included in MATERIAL AND METHODS (Statistical analysis), we added a phrase (highlighted in yellow in the revised manuscript) as follows: Lines 175-181: "The five groups were mutually exclusive; participants were included in a single risk-based group following a hierarchical rule described as follows: (i) HPV16/18 included the participants positive for HPV16 and/or HPV18 (both in single and multiple infections) (ii) other HR-HPV included those positive for any genotype in the group without HPV16/18, (iii) possibly HR-HPV included those positive for any genotype in the group without HPV16/18 or other HR-HPV infections (iv) LR-HPV included those participants positive for any genotype in the group without any of the previous infections." 3-Based on the study design all the precipitants were HPV positive.I do not understand why these 138 women (126, 6, 6) had negative HPV testing in table 2.
As the Reviewer says, this study included women who had been HPV positive by a screening test (HC2 or Cobas HPV) performed on the first PreservCyt Vial 1 (first cytobrush obtained).In a second cytobrush, obtained subsequent to the previous one (PreservCyt Vial 2), genotyping was performed by PCR-RBH, which allows the identification of 37 HPV genotypes (alone or coinfecting).In 138 samples, no HPV genotype was detected.This has been previously described by other authors (Leinonen MK et al, Br J Cancer. 2013;Hesselink AT et al., J Clin Microbiol. 2006).
The DISCUSSION presents the possible reasons that justify this result, as follows: Lines 388-396: "We found 11% of samples (mostly in women diagnosed with ≤CIN1) that were HPV-positive at screening (predominantly by HC2 test), but not in HPV genotyping.It should be considered that the PCR-RBH genotyping was performed in vial 2, which could have had fewer cells than vial 1 in which the HPV screening test was performed.However, it cannot be ruled out that these discrepant results could be false positives or represent cross-reactions with non-carcinogenic HPV types.Others also reported this drawback of the HC2 test [44,62].Moreover, two studies have found that increasing the cut-off level from the FDA-approved of 1.0 to 2 or 3 relative light units (RLU/Co ratio) greatly decreases the HPV prevalence among women with normal cytology or mild cytological abnormalities and reduces screening's false positive results [44,63].

4-
Overall, more than 20% cases had multiple HPV infections.I assume that Table 3 showed the prevalence of any HR-HPV Genotype (single or multiple).It is great if the authors list also the prevalence of single HR-HPV genotype, which can demonstrate individual HPV effect on cervical lesions.
We have accepted the Reviewer´s suggestion.We added a new phrase in MATERIAL AND METHODS (Statistical analysis) and RESULTS as follows: Line 185-187: "In addition, the frequency and prevalence of HR-HPV genotypes within each histologic group for single infections were also computed, thus restricting the analysis to those women whose sample was positive for only one HPV genotype (666 out of 1,252 participants)" Lines 238-239: "Similar results were found when considering the participants with a single infection".
We also added a supplementary table (Table S3) in the support information section, which shows individual HPV effect on the analyzed groups.