Roles of vaginal flora in human papillomavirus infection, virus persistence and clearance

Abstract

Vaginal flora plays a vital role in human papillomavirus (HPV) infection and progression to cancer. To reveal a role of the vaginal flora in HPV persistence and clearance, 90 patients with HPV infection and 45 healthy individuals were enrolled in this study and their vaginal flora were analyzed. Women with HPV infection were treated with Lactobacillus in the vaginal environment as a supplement to interferon therapy. Our results indicated that patients with high risk HPV (Hr-HPV) 16/18 infection had a significantly higher alpha diversity compared with the healthy control (p < 0.01), while there was no significant difference between the non-Hr-HPV16/18 group and the controls (p > 0.05). Patients with multiple HPV infection had insignificantly higher alpha diversity compared with single HPV infection (p > 0.05). The vaginal flora of patients with HPV infection exhibited different compositions when compared to the healthy controls. The dominant bacteria with the highest prevalence in HPV-positive group were Lactobacillus iners (n = 49, 54.44%), and the top 3 dominant bacteria in the HPV-persistent group were Lactobacillus iners (n = 34, 53.13%), Sneathia amnii (n = 9, 14.06%), and Lactobacillus delbrueckii (n = 3, 4.69%). Patients with HPV clearance had significantly lower alpha diversity, and the flora pattern was also different between groups displaying HPV clearance vs. persistence. The patients with persistent HPV infection had significantly higher levels of Bacteroidaceae, Erysipelotrichaceae, Helicobacteraceae, Neisseriaceae, Streptococcaceae (family level), and Fusobacterium, Bacteroides, Neisseria, and Helicobacter (genus level) than patients who had cleared HPV (p < 0.05).

Importance: Our study revealed differences in vaginal flora patterns are associated with HPV persistence and its clearance. Interferon plus probiotics can greatly improve virus clearance in some patients. Distinguishing bacterial features associated with HPV clearance in patients would be helpful for early intervention and reverse persistent infection.

Keywords: 16S ribosomal DNA sequencing; human papillomavirus; lactobacillus; probiotics; vaginal microbiota.

Figure 1

Prevalence of different HPV subtypes in infected groups. Hr-HPV16/18 (n = 28): HPV16/18 high-risk infection group; non-Hr-HPV16/18 (n = 62): non-HPV16/18 high-risk infection group; HPV: HPV-infected group (n = 90).

Figure 2

Comparison of vaginal microbial alpha diversity index (observed - species, Shannon, Simpson, Chao1, ACE, good - coverage) in infected and healthy individuals. The p-value on the top indicates the overall difference among three groups calculated using the Kruskal-Wallis nonparametric test method, and the asterisks on the top indicate a statistically significant difference between the two groups calculated using Dunn’s test (*p < 0.05, * *p < 0.01).

Figure 3

Principal coordinates analysis (PCoA) of variation in beta diversity of human vaginal bacterial communities in infected and healthy individuals based on unweighted UniFrac phylogenetic distance. Hr-HPV16/18 (n = 28): HPV16/18 high-risk infection group; Non-Hr-HPV16/18 (n = 62): non-HPV16/18 high-risk infection group; controls (n = 45): non-HPV infection group.

Figure 4

Vaginal flora at the genus level in infected and healthy individuals.

Figure 5

Comparison of relative abundance of the top 10 vaginal microflora genera between the Hr-HPV16/18 (n = 28), non-Hr-HPV16/18 (n = 62), and healthy (n = 45) individuals. p - values of 0.05 indicate a statistically significant difference, **p < 0.01, ***p < 0.001.

Figure 6

Vaginal flora at the species level of Hr-HPV16/18 (n = 28), non-Hr-HPV16/18 (n = 62), and controls (n = 45).

Figure 7

Comparison between alpha diversity index (observed - species, Shannon, Simpson, Chao1, ACE, good-coverage) of infected and healthy individuals. The p-value on the top indicates the overall difference among three groups calculated using the Kruskal-Wallis nonparametric test method, and the asterisks on the top indicate a statistically significant difference between the two groups calculated using Dunn’s test (* p < 0.05, ** p < 0.01). Single (n = 56): infected with a single HPV subtype; dual (n = 26): infected with two HPV subtypes; Multiple (n = 8): infected with three or more HPV subtypes.

Figure 8

Principal-coordinates analysis (PCoA) of variation in beta diversity of human vaginal bacterial communities in infected and healthy individuals, based on unweighted UniFrac phylogenetic distance.

Figure 9

LEfSe analysis comparing microbial variations at the genus level in patients infected with one, two, or multiple HPV subtypes. LEfSe cladogram representing differentially abundant taxa (p < 0.05). LDA scores as calculated by LEfSe of taxa are differentially abundant among groups. Only taxa with LDA scores of >2 were presented.

Figure 10

Alpha diversity index (observed - species, Shannon, Simpson, Chao1, ACE, good-coverage) in the HPV-cleared and HPV-persistent groups. The p-value on the top indicates the overall difference among three groups calculated using the Kruskal-Wallis nonparametric test method, and the asterisks on the top indicate a statistically significant difference between the two groups calculated using Dunn’s test (* p < 0.05, ** p < 0.01).

Figure 11

Heat map analysis of bacterial species found in the vaginal flora of 90 women. Each vertical line represents one sample. Different colors indicate relative abundance: red represents a high proportion and blue represents a low proportion. HPV-clearance: patients who cleared HPV; HPV-persistent: patients who did not clear HPV.

Figure 12

Vaginal flora at the genus level of HPV-cleared (n = 26) and HPV-persistent (n = 64) patients.

Figure 13

LEfSe analysis comparing microbial variations at the genus level in infected and healthy individuals. LEfSe cladogram representing differentially abundant taxa (p < 0.05). LDA scores as calculated by LEfSe of taxa are differentially abundant among groups. Only taxa with LDA scores of >2 are presented.

Figure 14

Chord diagram shows the relationship between HPV subtypes and dominant vaginal bacteria. The width of the strings (connecting lines) in the chord diagram shows the extent and proportion of the association between different HPV subtypes and dominant vaginal bacteria. The wider the width of the connecting lines, the higher the proportion. Different colors distinguish between different relationships.