Background: The aim of the study was to investigate the risk of human papillomavirus (HPV) genotyping particularly vaccine genotypes and multiple infections for cervical precancer and cancer, which might contribute to developing genotype-specific screening strategy and assessing potential effects of HPV vaccine. Methods: The HPV genotypes were identified using the Seq HPV assay on self-collected samples. Hierarchical ranking of each genotype was performed according to positive predictive value (PPV) for cervical intraepithelial neoplasia 2/3 or worse (CIN2+/CIN3+). Multivariate logistic regression model was used to estimate the odds ratios (ORs) with 95% confidence interval (CI) of CIN2+ according to multiplicity of types and vaccine types. Results: A total of 2811 HPV-positive women were analyzed. The five dominant HPV genotypes in high-grade lesions were 16/58/52/33/18. The overall ranking orders were HPV16/33/35/58/31/68/18/ 56/52/66/51/59/45/39 for CIN2+ and HPV16/33/31/58/45/66/52/18/35/56/51/68/59/39 for CIN3+. The risks of single infection versus co-infections with other types lower in the hierarchy having CIN2+ were not statistically significant for HPV16 (multiple infection vs. single infection: OR = 0.8, 95%CI = 0.6-1.1, P = 0.144) or other genotypes (P > 0.0036) after conservative Bonferroni correction. Whether HPV16 was present or not, the risks of single infection versus multiple infection with any number (2, ≥2, or ≥ 3) of types for CIN2+ were not significantly different. In addition, HPV31/33/45/52/58 covered by nonavalent vaccine added 27.5% of CIN2, 23.0% of CIN3, and 12.5% of cancer to the HPV16/18 genotyping. These genotype-groups were at significantly higher risks than genotypes not covered by nonavalent vaccine. Moreover, genotypes covered by nonavalent vaccine contributed to 85.2% of CIN2 lesions, 97.9% of CIN3 and 93.8% of cancers. Conclusions: Partial extended genotyping such as HPV33/31/58 but not multiplicity of HPV infections could serve as a promising triage for HPV-positive self-samples. Moreover, incidence rates of cervical cancer and precancer were substantial attributable to HPV genotypes covered by current nonavalent vaccination.

Lower female genital tract is colonized by a variety of microbes (cervicovaginal microbiota, CVM) which associate with the risk of genital infection. This study characterized CVM for 149 Chinese women with different status of human papillomavirus (HPV) infection and squamous intraepithelial lesion (SIL): no HPV infection (HPV-), HPV infection without significant SIL (HPV+NoSIL), HPV infection with low-grade SIL (HPV+LSIL) and HPV infection with high-grade SIL (HPV+HSIL). Analysis results showed CVM has dramatically changed in HPV+HSIL group when compared to HPV+LSIL group, but it exhibited no significant differences between HPV- and HPV+NoSIL groups as well as between HPV+NoSIL and HPV+LSIL groups. In consistence, random forest analysis found more notable differences in HPV+HSIL vs HPV+LSIL comparison than in other comparisons. In addition, depletion of Lactobacillus in CVM was more to be frequently identified in SIL-positive women as compared to SIL-negative individuals. Our findings suggested that significant CVM differences occurred when SIL developed to HSIL which was caused by persistent HPV infection.

Background: Self-sampling for human papillomavirus (HPV) testing is a feasible option to improve the cervical screening coverage. However, an ideal triage method for HPV-positive self-samples does not yet exist. The aim of this study was to explore the utility of HPV genotyping and p16^INK4a immunostaining (p16) in triaging HPV-positive self-samples, focusing on HPV-positive, cytology-negative (HPCN) women. Methods: A total of 73,699 women were screened in a cervical screening project in China via SeqHPV assay on self-samples. HPV-positive women were called-back and collected cervical sample for p16 immunostaining and liquid-based cytology, those who met any result of HPV16/18+ or visual inspection with acetic acid (VIA) + or p16+ were referred for colposcopy, and HPCN women with adequate data on p16 and pathology were analyzed. A triage strategy was considered acceptable if the negative predictive value (NPV) for cervical intraepithelial neoplasia 3 or worse (CIN3+) was 98% or more, combined with an improvement of sensitivity and specificity for CIN2+/CIN3+ in reference to the comparator, being HPV16/18 + . Results: A total of 2731 HPCN women aged 30–64 years were enrolled, 136 (5.0%) CIN2+ and 53 (1.9%) CIN3+ were detected. Five triage strategies met the criteria: p16+; HPV16/33+; ‘HPV16+ or HPV33/58/31/35+&p16+’; ‘HPV16/33+ or HPV58/31/35+&p16+’; HPV16/18/31/33/45/52/58 + & p16+. These strategies required less or similar colposcopy referrals, and less colposcopies to detected one case of CIN2+/CIN3+, achieving favorable false positive (negative) rates to the comparator. Among them, p16 staining detected 83.1% (79.2%) of underlying CIN2 + (CIN3+) in HPCN women. Moreover, three triage strategies were favorable in sensitivity and/or specificity to the ‘HPV16/33+’ strategy: p16+; ‘HPV16+ or HPV33/58/31/35 + &p16+’; HPV16/18/31/33/45/52/58 + &p16 + . Conclusions: Genotyping for HPV16/33 could be utilized to optimize the management of HPCN women. Moreover, p16 immunostaining, either alone or combined with extended genotypes, is more effective than HPV genotypes alone in the triage of HPCN women.

Vaginal microbiota (VM) links to the risk of persistent HPV infection and the progression of cervical intraepithelial neoplasia (CIN). However, a comprehensive understanding of concurrent alterations in VM and metabolome associated with that risk remains elusive in population-based studies, particularly among Chinese women. This study conducted an extensive analysis of VM and metabolome profiles in a cohort of 56 Chinese women, classified into HPV_C (natural clearance of HPV, n = 18) and HPV_PH(persistent HPV infection accompanied by high-grade CIN, n = 38), based on the result of 3–6 months follow-up visits. Our analysis revealed a higher prevalence of Lactobacillus-dominated samples in the HPV_C cohort. Notably, the vaginal metabolome exhibited a significant interaction with VM, with Lactobacillus emerging as a pivotal influencer. We identified 386 metabolites that significantly differentiated between HPV_C and HPV_PH groups, of which 364 were associated with VM components such as Lactobacillus, Hoylesella, Fannyhessea and Megasphaera. Further examination showed that 66 of these 364 metabolites positively correlated with Lactobacillus, including citric acid, DL-beta-Leucine, Xanoic acid and Norcholic acid. Conversely, 19 metabolites, including HPV_PH enriched maltotriose and N-Acetyl-L-aspartic acid, negatively correlated with Lactobacillus. Further analysis suggested potential bi-directional modulation between VM and persistent HPV infection accompanied by high-grade, being partially mediated by vaginal metabolites. This study provides additional insights into the correlations between concurrent alterations in VM and metabolome associated with persistent HPV infection accompanied by high-grade CIN.

Purpose: When used for cervical cancer primary screening, liquid-based cytology (LBC) has a high specificity but a low sensitivity. For histological diagnosis of high-grade lesions, p16^INK4a immunostaining has proven to be useful. Therefore, our objective was to evaluate the use of p16^INK4a immuno-cytology as a primary screen and a secondary screen after primary high-risk human papillomavirus (hrHPV) screening or LBC screening. Methods: A total of 1197 cytology slides were immuno-stained using automatic p16^INK4a staining system (PathCIN^®p16^INK4a) in two studies from cervical screening programs. In the primary screening study, 875 slides were randomly selected and analyzed for p16^INK4a. In the secondary screening study, 322 of the remaining slides were chosen by virtue of being HPV 16/18+, other hrHPV+/LBC≥ASC-US, or HPV-negative/LBC ≥LSIL. The sensitivity and specificity for detection of cervical intraepithelial neoplasia 2/3 or worse (CIN2+/CIN3+) were compared based on p16^INK4a, LBC and HPV test results. Results: In combining two studies, there were 431 cases with biopsy pathology. They included 83 cases with CIN2+ and 41 cases with CIN3+. The p16 positivity rate increased with pathologic and cytologic severity (P< 0.0001). For primary screening: p16 immuno-cytology was more specific than HPV testing and was similar in sensitivity. Also, p16 immuno-cytology compared favorably with routine LBC (≥ASC-US or ≥LSIL) in sensitivity and specificity. For secondary screening: after LBC screening, “Triaging ASC-US with p16” gave a higher specificity and a similar sensitivity as compared to the “Triaging ASC-US with hrHPV” algorithm. After HPV primary screening, p16 immuno-cytology was more specific than LBC (≥ASC-US); the calculated colposcopy referral rate was also decreased by using p16 immuno-cytology as triage. Triage of “HPV16/18 and p16” had higher specificity and similar sensitivity as compared to triage of “HPV16/18 and LBC ≥ASC-US”. Conclusion: For primary screening, p16^INK4a immuno-cytology compares favorably to routine LBC and HPV testing. p16^INK4a immunostaining could be an efficient triage to reduce the colposcopy referral rate after primary hrHPV screening or LBC screening. Therefore, p16^INK4a immuno-cytology may be applicable as a favorable technology for cervical cancer screening.

Objectives: This study aimed to describe the study design, and to analyze the type-specific distribution of cervical high-risk human papillomavirus (hrHPV) infection and its association with cytological and histological results in a large population-based screening program in Buji Street, Shenzhen, China.