Coinfection of High-Risk Human Papillomavirus and Lower Genital Tract Pathogens in the Development of High-Grade Cervical Lesions

Purpose This study investigated the infection status and relationship between other common lower genital tract infectious pathogens and high-risk human papillomavirus (HR-HPV) in the high-grade cervical lesions. Methods Overall, 882 patients were enrolled in this retrospective study, of which 339 patients (≥HSIL group) were confirmed with high-grade squamous intraepithelial lesions (HSIL) or cervical squamous cell carcinoma (SCC), while 543 patients (≤LSIL group) were diagnosed with low-grade squamous intraepithelial lesions (LSIL) or normal cervical pathology diagnosis. Cervical swab specimens were tested for HPV, pathogenic bacteria (PB), U. urealyticum (UU), M. hominis (MH), and C. trachomatis (CT) in both groups. Results The infection rates of HR-HPV, PB, UU (at high density), and CT were higher in the ≥HSIL group than in the ≤LSIL group (P < 0.001); however, higher infection rates with MH were not observed (P > 0.05). PB, UU, and CT were associated with HR-HPV infection (P < 0.001). The PB and UU infection rates in the ≥HSIL group were significantly different from those in the ≤LSIL group, regardless of whether there was an HR-HPV infection at the same time (P < 0.05). However, this was not the case for the CT (P > 0.05). Furthermore, 259 pathogenic bacterial strains were detected in 882 cases. The difference in the distribution of pathogenic bacterial flora in the different grades of cervical lesions had no statistical significance, which was prioritized over Escherichia coli (P > 0.05). Conclusion PB, UU, and CT infection is associated with susceptibility to HR-HPV, HR-HPV coinfection with these pathogens might increase the risk of high-grade cervical lesions, and PB and UU might be independent risk factors for cervical lesions.


Introduction
Cervical squamous cell carcinoma (SCC) is one of the most common malignant tumors in gynecology [1]. High-grade squamous intraepithelial lesions (HSIL) are a precancerous lesion closely related to SCC. Biological and epidemiological studies have confirmed that persistent infection of high-risk human papillomavirus (HR-HPV) is the leading cause of cervical precancerous lesions and SCC [2]. Most HPV infections are transient and cleared by host immune responses, while only a small percentage of infections persist and progress to HSIL and invasive SCC [3], which suggests that there are other synergistic factors in the development of cervical epithelial cells into malignant lesions after HPV infection.
Recent studies have found that certain HPV types and HPV viral loads may be related to HPV infection persistence and cancer progression [4]. In addition, several possible risk factors lead to the development of cervical lesions, such as first sexual intercourse at an early age [5], smoking [6], multiple sexual partners [7], impaired immune function [8], and hormonal contraceptives [9], and more importantly, studies have found that patients with lower genital tract infection (LGTI) other than HPV have an increased risk of developing SCC [10]. e incidence of lower genital tract infections has been increasing in recent years. An epidemiological survey of women in Beijing, China, showed that 11.4% of women had LGTIs [11]. Previous studies have found that genital pathogen infection may affect the susceptibility and clearance of HPV infection, and chronic inflammation caused by nonspecific genital infection is associated with SCC [12][13][14][15]. However, there is no consensus on the role of lower genital tract infection in high-grade cervical lesions. e aim of this study was to evaluate the prevalence of HR-HPV and other lower genital tract pathogens in different grades of cervical lesions in Fujian, China, and investigate the correlations of coinfection of HR-HPV and lower genital tract pathogens on the risk of high-grade cervical disease.

Study Population.
e study population was from Fujian provincial lesion screening cohorts (n > 140,000) and had undergone cervical secretion testing, which involved 2 cohorts, one consisting of healthy patients undergoing routine physical examination and the other consisting of patients visiting the outpatient clinic for any gynecologic conditions. All women received cervical secretion testing between January 2012 and January 2016 (n = 1178). e participants were required to meet the following criteria: (1) women in nonmenstrual period; (2) nongestational period, nonlactation period; (3) no acute and chronic illness; (4) no hysterectomy history; (5) no vaginal washing three days before sampling; (6) no use of contraceptives and other vaginal drugs; (7) no previous experience of radiotherapy, chemotherapy, or surgery. e study was approved by the Ethics Committee of Fujian Maternity and Child Health Hospital, Affiliated Hospital of Fujian Medical University (2016-002) and obtained the exemption of informed consent. e study flowchart is shown in Figure 1.

Specimen Collection.
Gynecologists performed routine gynecological examinations. Cervical cytology samples were collected from all subjects using plastic brushes, which were then immersed in a preservation solution for HPV DNA testing, and stored at −20°C. At the same time, cervical samples were collected using sterile cotton swabs for the detection of pathogens U. urealyticum (UU), M. hominis (MH), and C. trachomatis (CT).

Bacterial Testing.
After obtaining the cervical swabs, they were sent to the laboratory immediately. Culture was performed on blood agar plates, chocolate agar plates, and Sabouraud's dextrose agar plates (Beiruite, Zhengzhou, China) and incubated for up to 48 h in 5-10% CO 2 at 37°C. Individual colonies with large numbers of bacteria were selected, and clinical isolates were identified using standard microbiological methods. VITEK 2 Gram-positive-GP ID and Gram-negative-GN ID cards (BioMérieux, Balmes-les-Grottes, France), based on colorimetric detection, were used for the identification of bacteria according to the manufacturer's instructions. Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Enterobacter cloacae ATCC 700323 strains were used as controls.

UU/MH/CT Testing.
Cervical specimens were collected for UU, MH, and CT testing. UU and MH were isolated by Mycoplasma IST 2 (BioMérieux), using the liquid culture method. Diagnostic criteria were based on the presence or absence of MH and UU and an estimate of concentration variation (cut-off 104-color-changing units-CCU/mL). CT was detected using an immunochromatography assay (Clearview; Unipath Ltd., Bedford, UK).

Statistical
Analysis. Data were analyzed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). e associations between other lower genital tract pathogens and HR-HPV infection were determined using chi-square tests. To compare the LGTIs positive rates based on pathological diagnosis, chisquare tests or Fisher exact tests were performed. Multivariate logistic regression analysis was used to assess the simultaneous effect of more than one variable on the risk of high-grade cervical lesions and to identify possible confounding factors. Data are reported as numbers (%) or odds ratios (OR) with the corresponding 95% CI. P < 0.05 were considered statistically significant.
In our study, a total of 259 PB strains were detected, and the distribution is shown in Figure 3. Among 458 samples in the normal group, 73 (15.9%) women presented with PB infection; of the 85 samples in the LSIL group, 20 (23.5%) were infected with PB; of the 139 samples in the HSIL group, 59 (42.4%) were infected with PB; and of the 200 samples in SCC group, 78 (39.0%) women presented with PB infection. 12 women were infected with two cases of bacteria in ≤LSIL group, and 17 had two cases of bacteria in ≥HSIL group. e difference in the distribution of PB flora in the normal, LSIL, HSIL, and SCC groups was not significant, which was prioritized over Escherichia coli (E. coli) (P > 0.05, Figure 3).

Coinfection with HR-HPV and Other Pathogens.
Infection with other pathogens was frequently detected in HPV positive women. In fact, PB were more likely to be diagnosed in HR-HPV positive women than women who were HR-HPV negative (   HPV and PB infection was observed (P < 0.001). Similarly, UU was detected in 27.2% (125/460) of HR-HPV positive patients and CT in 16.5% (76/460) of HR-HPV positive patients. UU and CT were more likely to be diagnosed in HR-HPV positive women than in those who were HR-HPV negative (P < 0.001, P < 0.001, respectively). e prevalence of MH was 6.7% (31/460) among HR-HPV positive women and 7.1% (30/422) among HR-HPV negative women. Statistical analysis did not reveal any association between the presence of HR-HPV and MH (P > 0.05, Table 2). Table 2 shows the risk of coinfection of HR-HPV with PB, UU, CT, and different grades of cervical lesions. Coinfection of HR-HPV and pathogens showed a higher increased risk for ≥HSIL (OR: 2.250, 95% CI: 1.423-3.557, P < 0.001). Coinfection of HR-HPV and UU increased risk of ≥HSIL (OR: 1.791, 95% CI: 1.096-2.926, P � 0.020). Coinfections of CT plus HR-HPV increased the risk of ≥HSIL (OR: 3.070, 95% CI: 1.540-6.120, P � 0.001). No similar trends were observed in MH.

Discussion
As expected, we found a significant association between PB, UU, CT, and HR-HPV infection, and HR-HPV coinfection with these pathogens increased the risk of high-grade cervical disease. Among them, PB or UU infections may contribute to development of high-grade cervical lesions as an independent risk factor, regardless of whether there is HR-HPV infection. In contrast, we did not find a correlation between MH and HR-HPV or high-grade cervical lesions. In addition, in this study, the distribution of PB flora was also not statistically different between different grades of cervical lesions group.
A survey of cervical HPV prevalence in China showed that, among the cervical intraepithelial neoplasia (CIN)2+ patients, the HPV infection rate was 84.97% [16], which was slightly lower than the infection rate of HPV in our ≥HSIL group (93.8%). Local inflammation caused by lower reproductive tract infection leads to local metaplasia, which may increase the chance of HR-HPV infection and HR-HPV viral load. In this study, we found that women infected with pathogens had an increased risk of coinfection with HR-HPV, and a similar trend was found in UU and CT. is supports the hypothesis that these pathogens are closely related to persistent HR-HPV infection. e healthy cervical bacterial community usually consists of Lactobacillus spp. It contains about 90% of the cervical bacterial flora and serves as the first line of defense against pathogenic bacteria [17]. More and more evidence shows that the microflora environment of the lower   reproductive tract may be related to HPV infection, persistent infection, local mucosal immunity, and cervical lesions [18]. Vaginal flora imbalance, especially lactic acid bacteria, may lead to cervical cytology abnormalities, leading to an increased risk of cervical cancer precancerous lesions and cervical cancer [19][20][21]. In our study, the lower genital pathogen was the most common non-HPV pathogen, HR-HPV infection is prone to coinfection with pathogens, and coinfection with HPV increases the risk of high-grade cervical diseases, which was essentially in agreement with another study [22]. In addition, our study did not find that the increase in vaginal microbiota diversity is associated with the occurrence of cervical lesions and may be related to the different detection of pathogenic microorganisms (for example, conventional microscopy and/or culture methods and PCR-based methods). CT is one of the most widely studied sexually transmitted pathogens. Most epidemiological studies have shown that Chlamydia trachomatis infection rates are higher in patients with cervical cancer [23][24][25][26]. Discacciati MG et al. found that CT induces MMP-9/RECK imbalance during cervical inflammation, and this imbalance plays an important role in HPV-mediated cervical cancer [27]. CT infection can also reduce the host's ability to resolve HPV infection by increasing free radical production and reducing host cell-mediated immunity, resulting in persistent HPV infection [28]. CTmay play a specific role in the history of cervical lesions through the above mechanisms. is is consistent with our findings. In our study, CT and HR-HPV coinfection showed a 3.288-fold increase in the risk of high-grade cervical lesions, but there was no significant difference in patients without HR-HPV infection. erefore, we speculate that CT and HR-HPV act synergistically as one of the risk factors for high-grade cervical lesions. Our findings emphasize the need for further studies to assess the actual impact of coinfection on the risk of different grades of cervical lesions.
In addition, Lukic et al. [29] found that the higher the level of cervical lesions, the higher the UU infection rate (35% for LSIL and 45% for HSIL). Xiao et al. [12] found that the significant combination of HPV infection and UU can strengthen the development of the disease and lead to the pathogenesis of cervical cancer. All the above studies are consistent with our study, UU infection was significantly associated with HR-HPV infection, and UU might be independent risk factor for cervical lesions. Further research is needed to identify the problem and the potential mechanisms associated with these pathogens.
In our research population, the prevalence of mixed infections deserves the attention of the public health services. Our study further demonstrates the efficacy of coinfection with HR-HPV and other lower genital pathogens, supporting the high coinfection rate of pathogens, CT, UU, and HR-HPV, and their possible risk in high-grade cervical lesions. Whether it is HR-HPV, pathogens, CT, or UU, the mode of transmission is closely related to sexual intercourse. erefore, advocating healthy sexual behavior is essential for the prevention and treatment of cervical lesions. It is suggested that, in the screening of clinical SCC and precancerous lesions, in addition to detecting HR-HPV, vaginal microecological detection should be carried out, attention should be paid to the balance of pathogens in the lower genital tract pathogens, and the coinfection of pathogens, CT, UU, and HR-HPV should be screened to block its ability to affect the host's ability to clear HPV infection to determine appropriate diagnostic and therapeutic methods. e advantages of our research include a relatively large number of patients, and the percentage of data missing in patients who participated in the study and met the inclusion and exclusion criteria was small. It is worth noting that our results are based on a summary analysis of rough epidemiological data, with moderate heterogeneity in the study, and without considering risk factors associated with sexual behavior in the study population. In addition, our study is mainly a cross-sectional study, lacking longitudinal observation of HR-HPV and other cervical pathogen infection processes and therefore it is difficult to study the relationship between persistent HR-HPV infection, genital pathogen infections, and cervical lesions. Future prospective cohort studies need to explore their relationship from a vertical perspective.
In conclusion, this study provided an opportunity to detect HPV simultaneous detection of pathogens and determine the rate of coinfection between HR-HPV and other lower genital tract pathogens in different grades of cervical lesions. Our results reinforce the hypothesis that some lower genital tract pathogens may be associated with HR-HPV infection and may be a risk factor for high-grade cervical lesions. is study was considered critical to further elucidate the pathogenesis of high-grade cervical disease and to improve early screening and intervention strategies.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.