Abstract
BackgroundThe Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. However, the assay is limited to a restricted number of HPV, and sequence variations in the detection region of the HPV probes could give false negatives results. Recently, 454 Next-Generation sequencing (NGS) technology has been efficiently used also for HPV genotyping; this methodology is based on massive sequencing of HPV fragments and is expected to be highly specific and sensitive. In this work, we studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS.MethodsTwo hundred thirty three cervical samples from women Without cervical lesions (WCL, n = 48), with Cervical intraepithelial neoplasia grade 1 (CIN I, n = 98), or with Cervical cancer (CC, n = 87) were recruited, DNA was extracted, and HPV positivity was determined by PCR amplification using PGMY09/11 primers. All HPV- positive samples were genotyped individually by LA. Additionally, pools of amplicons from the PGMY-PCR products were sequenced using 454 NGS technology. Results obtained by NGS were compared with those of LA for each group of samples.ResultsWe identified 35 HPV genotypes, among which 30 were identified by both technologies; in addition, the HPV genotypes 32, 44, 74, 102 and 114 were detected by NGS. These latter genotypes, to our knowledge, have not been previously reported in Mexican population. Furthermore, we found that LA did not detect, in some diagnosis groups, certain HPV genotypes included in the test, such as 6, 11, 16, 26, 35, 51, 58, 68, 73, and 89, which indicates possible variations at the species level.ConclusionsThere are HPV genotypes in Mexican population that cannot be detected by LA, which is, at present, the most complete commercial genotyping test. More studies are necessary to determine the impact of HPV-44, 74, 102 and 114 on the risk of developing CC. A greater number of samples must be analyzed by NGS for the most accurate determination of Mexican HPV variants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0391-4) contains supplementary material, which is available to authorized users.
Highlights
The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample
HPV genotyping by Linear Array test From the 48 Without cervical lesion (WCL) samples, 12 (25 %) were positive for HPV
Calculating the frequency of HPV genotypes detected by LA in the different groups analyzed (Table 1), we observed that the most frequent HPV genotypes found in WCL samples were 16, 51, and 84, followed by 56, 59, and 66 (16.7 % each), and last, HPV genotypes 6, 11, 31, 39, 52, 68, 81, and 89 (8.3 % frequency)
Summary
The Linear Array® (LA) genotyping test is one of the most used methodologies for Human papillomavirus (HPV) genotyping, in that it is able to detect 37 HPV genotypes and co-infections in the same sample. We studied HPV prevalence in cervixes of women in Western Mexico by LA and confirmed the genotypes found by NGS. Regarding Cervical intraepithelial neoplasia grade 1 (CIN I), HPV-42 and 84 were reported as more prevalent in Sweden [10], while in Chile, HPV-61 and 89 [11] are most prevalent, and in Asia, HPV-52 [12] and in the USA (in rural American Indian women), HPV61 [13]. In Mexico, a recent study of our research group, using genotyping by Linear Array, show that the HPV genotypes most frequently found in cervical cancers were 16 (62.8 %), 18 (11.6 %), 45 (8.3 %), 52/58 (6.6 %), and 39 (5.8 %). A high percentage (58–64 %) of coinfections were found in CIN I—CIN III, suggesting that coinfections could contribute to regression or progression of the cervical lesions [14]
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