16SrRNA高通量测序研究持续性高危型HPV感染患者阴道菌群特征

Study on the characteristics of vaginal flora in patients with persistent HPV infection by 16S rRNA high-throughput sequencing

目的 通过16Sr RNA高通量测序研究高危型人乳头瘤病毒(HR-HPV)感染患者在不同型别和不同宫颈病变组间的阴道菌群特征。方法 选择2020年6月至2021年7月在粤北人民医院就诊的36例持续HR-HPV感染者为HR-HPV阳性组,10例无HR-HPV感染者为HR-HPV阴性组。HR-HPV阳性组按单一和多重HR-HPV感染分为单一HR-HPV阳性和多重HR-HPV阳性两个亚组。HR-HPV阳性组中共有23例接受宫颈活检,按宫颈病变级别分为三组:慢性炎症组、低级别鳞状上皮内病变(LSIL)组、高级别鳞状上皮内病变(HSIL)组。采用16Sr RNA高通量测序结合β多样性分析、微生物组成等生物信息学结果,对单一和多重HR-HPV阳性组与HR-HPV阴性组间、慢性炎症组与LSIL和HSIL组间的阴道菌群特征进行分析。结果 HR-HPV阴性组、单一HR-HPV阳性组、多重HR-HPV阳性组的阴道菌群β多样性分析显示,差异有统计学意义(H=12.872,P <0.001);单一HR-HPV阳性组和多重HR-HPV阳性组的阴道菌群结构,差异有统计学意义(Z=3.214,P <0.001)。关键物种分析显示Lactobacillus菌属在多重HR-HPV阳性组出现了明显的丰度下调,差异有统计学意义(Z=4.647,P <0.05)。Dialister菌属在多重HR-HPV阳性组出现了丰度上调,差异有统计学意义(Z=3.479,P <0.05)。慢性炎症组、LSIL组和HSIL组的阴道菌群结构,差异有统计学意义(H=11.653,P <0.05);慢性炎症组与LSIL组、LSIL组与HSIL组的阴道菌群结构,差异有统计学意义(Z=2.782,2.649,P <0.05)。关键物种分析显示Ureaplasma菌属在慢性炎症组和LSIL组间比较,差异有统计学意义(Z=2.816,P <0.05)。Enterococcus菌属占比在慢性炎症组和HSIL组间差异有统计学意义(Z=3.673,P <0.05)。结论 阴道菌群的多样性增加和物种的丰度变化与HR-HPV的持续性感染和宫颈高级别病变相关。

Objective To study the characteristics of vaginal flora in patients with high-risk human papillomavirus(HR-HPV) infection of different types and different cervical lesions by 16Sr RNA highthroughput sequencing. Methods In this study, 36 patients with persistent HR-HPV infection who visited Yuebei People’s Hospital from June 2020 to July 2021 were included into the HR-HPV positive group;10 patients without HR-HPV infection were included into the HR-HPV negative group. The HR-HPV positive group was divided into two subgroups according to single and multiple HR-HPV infections, the single HR-HPV positive group and the multiple HR-HPV positive group. A total of 23 patients in the HR-HPV positive group received cervical biopsy. They were divided into three subgroups according to the level of cervical lesions, i.e., the chronic inflammation group, the low-grade squamous intraepithelial lesion(LSIL) group, and the highgrade squamous intraepithelial lesion(HSIL) group. The 16Sr RNA high-throughput sequencing, β diversity analysis, microbial composition and other bioinformatics results were integrated to analyze the characteristics of vaginal flora among the single and multiple HR-HPV positive subgroups and the HR-HPV negative groups as well as among the chronic inflammation group and the LSIL and HSIL groups. Results The β diversity analysis on vaginal flora showed that there was statistically significant difference among the HR-HPV negative group, the single HR-HPV positive group and the multiple HR-HPV positive group(H=12.872, P < 0.001);there was statistically significant difference in vaginal flora structure between the single HR-HPV positive group and the multiple HR-HPV positive group(Z=3.214, P < 0.001). The analysis of key species showed that the abundance of Lactobacillus genus was significantly decreased in the multiple HR-HPV positive group, with statistical significance(Z=4.647, P < 0.05);the abundance of Dialister genus was increased in the multiple HR-HPV positive group, with statistical significance(Z=3.479, P < 0.05). There was statistically significant difference in vaginal flora structure among the chronic inflammation group, the LSIL group and the HSIL group(H=11.653, P < 0.05). There was statistically significant difference in vaginal flora structure between the chronic inflammation group and the LSIL group as well as between the LSIL group and the HSIL group(Z=2.782/2.649, P < 0.05). The analysis of key species showed that there was statistically significant difference in Ureaplasma genus between the chronic inflammation group and the LSIL group(Z=2.816, P < 0.05);there was statistically significant difference in the proportion of Enterococcus genus between the chronic inflammation group and the HSIL group(Z=3.673, P < 0.05). Conclusion The increase of vaginal flora diversity and the change of species abundance are related to the persistent infection of HR-HPV and high grade cervical lesions.