Assessment of human papillomavirus detection using different methods and clinical samples

Abstract

Human papillomavirus (HPV) is the etiological agent implicated cancers include those of the cervix, vulva, vagina, penis, anus, rectum and oropharynx. Currently, the Papanicolaou (Pap) smear is the recommended for cervical cancer screening but only a small percentage of women follow this screening. Overall, this thesis aimed to develop the technique and type of specimen to detect HPV DNA in Thai population and to characterize of the HPV genotypes in Thai patients with anogenital warts. Cervical swab and urine sample of 116 women and 100 men were collected before the Pap smear test. The histological examination of women can classified into 4 group: normal cytology in 52, ASCUS in 9, LSIL in 24, and HSIL in 31. In male patients, there were 45 heterosexual males who visited hospital for health check-up and 55 were documented as homosexuals or MSM. HPV DNA was genotyping by electrochemical DNA chip and PCR sequencing showed HPV16 was the most prevalence in this study. The HPV detection rate was generally lower in urine samples compared with cervical samples. There was good agreement for detection of carcinogenic HPV from female cervical samples between the DNA chip and PCR/sequencing, with 88.8% agreement. In male urine samples, the level of agreement was higher in heterosexuals compared with homosexuals. Moreover, the 164 pair of urine and cervical specimens were tested with Genoarray assay. The result showed overall concordance percentage for HPV detection was 65.2%. In addition, the comparison of 101 self-collected vaginal and physician-collected cervical swabs of known cytology was performed by DNA chip assay. The result showed the concordance was relatively high between self-collected and physician-collected samples. The most common HPV genotype detected was HPV 51. Also, 58 cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, DNA chip, and NGS. These findings show that 7 HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or DNA chip. This findings further showed the alternative method to detected HPV genotyping. Finally, the study was investigate HPV-associated anogenital warts in the Thai population and whether genotypes found are represented in the vaccine. A total of 206 anogenital swab samples were analyzed by PCR/sequencing. HPV positive was identified in 88.3% (182/206) of the samples. The majority of HPV (75.2%) were low-risk genotypes HPV6 and HPV11. This finding conclusion, this study demonstrated urine and self-collected swab is a promising technique for HPV screening. NGS may serve as an alternative for diagnostic HPV genotyping in certain situations. Moreover, quadrivalent vaccine could potentially prevent the genital warts in the Thai population.