Objective To assess the rates of co-infections between human papillomavirus (HPV) and 13 key markers of bacterial vaginosis in cervical samples by multiplex polymerase chain reaction in a population with a high rate of abnormal cytology and a positive HPV test.
Methods The study included a total of 213 women aged 18–72 years screened using Papanicolaou smears for determining cervical abnormalities and for HPV and bacterial vaginosis by single-target and multiplex polymerase chain reaction.
Results A total of 83 (39%) women were negative for intraepithelial lesion or malignancy cytology and 130 (61%) had abnormal cytology. HPV-DNA prevalence was 69.9% and bacterial vaginosis was 72.7 %. Co-infections between bacterial vaginosis with HPV-DNA and high-risk HPV were associated with an increased risk for squamous intraepithelial lesions of low-grade cytology and high-grade squamous intraepithelial lesions plus cervical cancer. The most frequent bacterial vaginosis agent was Gardnerella vaginalis (33.8%), and co-infection with HPV-DNA and high-risk HPV increased the risk for squamous intraepithelial lesions of low grade cytology and high-grade squamous intraepithelial lesions plus cervical cancer. Co-infection between Megasphaera type I and high-risk HPV increased the risk for high-grade squamous intraepithelial lesions plus cervical cancer.
Conclusions Our results reinforce the hypothesis that some bacterial vaginosis agents may play a role as co-factors in HPV-mediated cervical carcinogenesis, at least in some populations.
- squamous cervical cancer
- bacterial vaginosis
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Co-infections of high-risk HPV with key markers of bacterial vaginosis increased the risk of cervical lesions/cancer.
Gardnerella vaginalis and Megasphaera type I were associated with high-risk HPV in cervical lesions/cancer.
The results reinforce the hypothesis that some key markers of bacterial vaginosis may play a role as co-factors in HPV-mediated carcinogenesis.
Squamous cervical cancer is currently the fourth leading cause of female mortality by cancer in general, despite the efficient prevention and screening methods currently applied.1 Genital human papillomavirus (HPV) infection is usual among the female population and it is established that persistent infection with high-risk HPV is fundamental for cervical cancer development.2 Despite this, only a few women have an increased risk of developing cervical lesions and cervical cancer.3 4 However, little is known about the reasons for this shifting natural history, but it is accepted that other causes or co-factors are complementary for the development of neoplasia in HPV-infected women.3 4
A number of risk factors have been associated with persistent high-risk HPV in the past two decades contributing to cervical carcinogenesis.5–9 Co-factors associated with an individual’s behavior, such as smoking and the use of hormonal contraceptives, multiple sex partners, early sexual activity, and immunodeficiency, are associated with cervical cancer.5 6 Furthermore, different conditions such as intra-type HPV variations, multiple type HPV infections, co-infection with other sexually transmitted agents, and host genetic variability are associated with the persistence and progression of a pre-malignant status to cervical cancer.7–9 Recently, several studies have pointed out the role of vaginal microbioma in the process of acquisition and persistence of HPV and the risk of cervical cancer development.10 Despite this, there is no consensus on these topics.
Over the past decades, the concept of microbiome has been used to explain the co-existence between the human body and a large number of microrganisms, which has multiple implications in a range of multifactorial diseases including obesity, Crohn’s disease, asthma, and infertility.11 12 In this context, studies of the female reproductive tract in both healthy and diseased subjects have been carried out and Lactobacillus spp was established as a prevalent group controlling the overgrowth of other bacteria.13 In fact, changes in the microbiome are associated with bacterial vaginosis, the most prevalent genital tract syndrome in women.14 Bacterial vaginosis is considered an ecological disorder of the vaginal microbiome. The main characteristics of bacterial vaginosis are a reduction primarily in Lactobacillus and an increase in the number and diversity of facultative and strictly anaerobic bacteria, primarily Gardnerella vaginalis.14–16 However, studies on vaginal specimens using modern molecular techniques have shown a dramatic increase in the number of bacterial species inhabiting the vaginal environment, many of which have been shown to be highly associated and/or to be key markers of bacterial vaginosis.17–19 Despite these advances in the knowledge of bacterial vaginosis and the suggestion that some agents are potential risk factors for cervical lesions and squamous cervical cancer, there is no consensus as to whether it may be a co-factor associated with high-risk HPV in cervical carcinogenesis.
The present report investigated the rates of co-infection between HPV and important bacterial vaginosis agents in different cytological cervical samples by multiplex polymerase chain reaction to screen 13 key markers of bacterial vaginosis in a population with a high rate of abnormal cytology and a positive HPV test. We found that G. vaginalis and Megasphaera type I were the primary pathogens associated with high-risk HPV for increasing the risk of cervical abnormalities in this population, primarily for high-grade squamous intraepithelial lesions plus squamous cervical cancer, suggesting a possible synergistic action in cervical lesion progression.
This cross-sectional study enrolled women referred to a colposcopy clinic with a history of abnormal cytology residing in Maringá City/Paraná State/Brazil attending the reference service for colposcopy at the Zona Sul Clinic, according to the Public Health System, from February 2013 to March 2014. Women with any of the following factors were excluded: pregnancy, post-partum, previous hysterectomy, vaginal bleeding, previous history of cancer, sexual inactivity, recent treatment for any pathological condition of the urogenital tract, ablative or excisional therapy for the cervix within the previous 12 months, not receiving treatment with antimicrobials (oral or topical) within the previous 4 weeks, and those who had not been using an intrauterine device or contraceptives delivered directly to the vaginal mucosa. All participants voluntarily agreed to provide a sample for Papanicolaou (Pap) screening and DNA detection of HPV and bacterial vaginosis and signed an informed consent before enrollment. This study was approved by the Committee for Ethics in Research Involving Humans at the State University of Maringá/UEM/Brazil and registered at the National Commission for Research Ethics (CONEP)/Health Ministry of Brazil (No 085/2011 and No 104/2012).
Demographic and baseline characteristics were obtained from analysis of the data from the standard registration form of each woman. Cytological samples were obtained from women referred for colposcopy because of previous abnormal cytology findings. Cervico-vaginal samples were collected using Ayre’s spatula and cytobrush and were used for Pap smear preparation. For HPV and bacterial vaginosis DNA analyses, samples were immediately suspended in 1 mL sterile 0.9% NaCl solution and stored at −80°C until analysis.
Cytology and histology
Cytological examination was conducted without the knowledge of HPV status at an accredited clinical laboratory (Clinical Cytology Laboratory of UEM) and reported according to the Bethesda 2001 nomenclature.20 The threshold of abnormal cytology was used to define all abnormal cytological findings including low-grade and high-grade squamous intraepithelial lesions and cervical cancer. Negative for intraepithelial lesion or malignancy was used to define normal cytological findings. According to the Health Ministry of Brazil, all cases with cytological findings of high-grade squamous intraepithelial lesions and squamous cervical cancer should be analyzed by colposcopy and histology, properly treated, and followed up. Histology results were determined by a panel of three pathologists blinded to all participants.
Genomic DNA extraction
DNA was extracted using the AxyPrep Body Fluid Miniprep Kit (Axygen, California, USA) according to the manufacturer’s instructions. The quality and quantity of purified DNA were determined by spectrophotometry on a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Massachusetts, USA).
HPV detection and typing
HPV was detected by single-target polymerase chain reaction using the primers MY09 (5′-CGTCCMAARGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAYAATGG-3′).21 HPV-positive samples were typed by polymerase chain reaction–restriction fragment length polymorphism analysis, in which the amplified DNA was cleaved with restriction enzymes to generate DNA fragments of different molecular sizes. Aliquots of each amplified product were subjected to digestion with the restriction enzyme HpyCH4V (New England Biolabs, Ipswich, Massachusetts, USA).22 23 Typing was performed by comparing the molecular weights of fragments for each HPV type.22 A total of 40 individual HPV types can be determined by polymerase chain reaction–restriction fragment length polymorphism: 17 types are considered to be either high-risk or potentially high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82); 22 low-risk types that are not associated with carcinogenesis (6, 11, 30, 34, 40, 42, 43, 44, 54, 55, 61, 62, 64, 67, 69, 70, 72, 74, 81, 83, 84, and 91); and the carcinogenic risk for one type (22) has not yet been determined.21 22
Multiplex polymerase chain reaction for the detection of 13 key markers of bacterial vaginosis
Multiplex polymerase chain reaction assays were performed according to the method described by Malaguti et al.19 Briefly, to optimize the detection of the 13 bacterial vaginosis agents, multiplex polymerase chain reaction was split into three different reactions: assay 1 to detect six bacteria (G. vaginalis, Mobiluncus curtisii, Bacteroides fragilis, Mycoplasma hominis, Ureaplasma urealyticum, and Megasphaera type I); assay 2 to detect three bacteria ( Atopobium vaginae, Clostridia-like bacteria vaginosis-associated bacteria 1 and 2); and assay 3 to detect four bacteria (Mobiluncus mulieris mulieris, Clostridia-like bacteria vaginosis-associated bacteria 3, Sneathia sanguinegens, and Mycoplasma genitalium). The multiplex polymerase chain reaction products were electrophoresed on 8% polyacrylamide gel stained with 1 µg/mL ethidium bromide.
Different variables were evaluated for comparisons between normal cytology and abnormal cytology (low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions plus cervical cancer) for analytical calculations. A two-sided Fisher’s exact test for 2×2 contingency table was used to evaluate the statistical significance between different groups. Crude odds ratios (RR, relative risk) with 95% confidence intervals (CI) were calculated to estimate the association of HPV and bacterial vaginosis positive findings with different cervical cytological findings. Statistical significance was defined as p<0.05. All statistical analyses were performed using GraphPad Prism 6.0 (San Diego, California, USA).
Table 1 outlines the baseline features of the study cohort. A total of 213 women were included in the study with a mean (SD) age of 33.54 (11.93) years (range 18–72). A total of 83 (39%) women had normal cytology and 130 (61%) women had the following abnormal cytology: 37 low-grade squamous intraepithelial lesions (28.5%), 87 high-grade squamous intraepithelial lesions (66.9%), and six squamous cervical cancer (4.6%). Women with normal and abnormal cytology had similar ages (32.56 (11.6) and 34.15 (12.1) years, respectively; p=0.19) but more women with abnormal cytology were in the age group 35–44 years compared with those with normal cytology (26.1% and 9.6%, respectively; p=0.01). The age of first sexual intercourse, sexual partners during life, pregnancies to term, and use of hormonal contraceptives and other contraceptive methods were also similar among women with normal and abnormal cytology (Table 1).
HPV types in the overall population and by cytological status
The prevalence of HPV-DNA was very high (69.9%) since it is a special at-risk population referred to a clinical colposcopy for having abnormal cytology. A total of 39 HPV types were detected: 17 high-risk HPV, 21 low-risk HPV, and one of undetermined risk HPV. HPV-16 was the most prevalent in the overall population (28.64%) and in women with low-grade or high-grade squamous intraepithelial lesions/cervical cancer cytology (21.6% and 47.3%, respectively; Table 2, Figure 1). The HPV types with the next highest prevalence were HPV-31 (high-risk; 8.0%) and HPV-72 (low-risk; 6.6%). The other types had a prevalence of <5%. Low-risk HPV were detected in 25.3% of the women studied and were associated with low-grade squamous intraepithelial lesions (p=0.0045). Unknown risk HPV was detected in 0.94% of the women, with all exhibiting negative for intraepithelial lesion or malignancy. HPV multiple infections were detected in 21.2% of the women (Table 2). Thirty-one (68.8%) of these women had two HPV types, nine (20%) women had three HPV types, and five women (11%) had four HPV types. High-risk HPV was involved in 93.3% of HPV multiple infections.
Normal, low-grade squamous intraepithelial lesions or high-grade squamous intraepithelial lesions/cervical cancer cytology
HPV-72 was the most frequent type in women with normal cytology (4.8%), followed by HPV-54 and 62 (3.6% for both), and 16, 66, and 70 (2.4% for all). HPV-31 (12.3%) was the second most common type in women with abnormal cytology, followed by types 72, 18, 33, and 58 (10% for 72% and 6.1% for others).
HPV and relative risk of abnormal cytology
HPV-DNA, high-risk HPV, HPV-16, and HPV multiple infections were associated with low-grade and high-grade squamous intraepithelial lesions/cervical cancer cytology (Table 2). In relation to RR, women with HPV-DNA exhibited an increased risk of approximately two and four times for low-grade and high-grade squamous intraepithelial lesions/cervical cancer cytology (RR=2.04 and 4.3, respectively). Similar trends were observed in women with HPV multiple infections (RR=1.84 for low-grade squamous intraepithelial lesions and RR=4.3 for high-grade squamous intraepithelial lesions/cervical cancer. The RR increased even more in women with high-risk HPV mainly for high-grade squamous intraepithelial lesions plus squamous cervical cancer (RR=9.1). The highest risk for low-grade squamous intraepithelial lesions and for high-grade squamous intraepithelial lesions/cervical cancer cytology was observed in women with HPV-16 (RR=3.45 and 19.6, respectively). Women with low-risk HPV were only associated with an increased risk for low-grade squamous intraepithelial cytology (RR=1.68) (Table 2).
Bacterial vaginosis agents frequency in all samples analyzed
Bacteria were detected in 155 of the 213 samples (72.7%) as a single agent (34.7%) or as two or more agents simultaneously (38%). The prevalence of bacterial vaginosis agents was very high in this special at-risk population referred to a clinical colposcopy for having abnormal cytology (Table 2). Despite the high frequency, none of the bacterial vaginosis agents as single or multiple infections were associated with abnormal cytology, which was expected due to the fact that some of these microorganisms are part of the vaginal microbiota as saprophytes. The most frequent bacteria was G. vaginalis (33.8%), followed by M egasphaera type I (22.5%), Clostridia-like bacteria vaginosis-associated bacteria 1 (15.5%), Ureaplasma urealyticum and Clostridia-like bacteria vaginosis-associated bacteria 3 (12.7% each), Mobiluncus curtisii (9.4%), Clostridia-like bacteria vaginosis-associated bacteria 2 (8.9%), Atopobium vaginae (6.6%), Mycoplasma hominis and Sneathia sanguinegens (4.2% each), Mobiluncus mulieris (2.8%), Bacteroides fragilis (1.9%), and Mycoplasma genitalium (0.9%) (Table 3).
Co-infections between HPV and bacterial vaginosis agents and association with abnormal cytology
In this special at-risk population referred to a clinical colposcopy for having abnormal cytology, it was observed that co-infections between a single bacterial agent with HPV-DNA and high-risk HPV were associated with an increased risk for low-grade squamous intraepithelial lesion cytology (RR=1.3, and RR=1.8, respectively) and high-grade squamous intraepithelial lesions/cervical cancer (RR=2.27 and RR=4.28, respectively). Co-infections with simultaneous bacteria (two or more) with HPV-DNA were associated with an increased risk for low-grade squamous intraepithelial lesions (RR=1.7) and co-infections with high-risk HPV were associated with an increased risk for low-grade and high-grade squamous intraepithelial lesions/cervical cancer (RR=2.0 and 2.75, respectively). Considering individual bacteria, co-infections of G. vaginalis with HPV-DNA and high-risk HPV were associated with an increased risk for low-grade squamous intraepithelial lesions (RR=2.5 for both) and high-grade squamous intraepithelial lesions/cervical cancer (RR=2.98 and RR=4.24, respectively). Additionally, co-infections of Megasphaera type I with high-risk HPV were associated with an increased risk for high-grade squamous intraepithelial lesions/cervical cancer (RR=4.71). Finally, co-infections of Clostridia-like bacteria vaginosis-associated bacteria 1 and HPV-DNA were associated with an increased risk only for low-grade squamous intraepithelial lesions (RR=2.0) (Table 4).
This study investigated the rates of co-infections between HPV and 13 bacterial vaginosis agents in different cervical samples by multiplex polymerase chain reaction in a population with a high rate of abnormal cytology (61%) and a positive HPV test (69.9%). We found that G. vaginalis and Megasphaera type I were the primary pathogens associated with high-risk HPV in all grades of cervical abnormalities, particularly for high-grade squamous intraepithelial lesions/cervical cancer.
In this study, G. vaginalis was the most frequent bacterial vaginosis agent detected among the 13 bacteria examined. Our results are in agreement with several other authors who have reported that anaerobic bacteria species of Gardnerella, Prevotella, and Peptostreptococcus genera and/or aerobic bacteria of the Enterobacteriaceae family usually populate the vaginal environment in cases with depletion of Lactobacillus population, thereby causing bacterial vaginosis.24 25
Epidemiological studies have indicated that variations in the cervical-vaginal microbiota followed by Lactobacillus depletion and a rise in the microbial variety leads to bacterial vaginosis, increases the possibility of HPV infection, and may be associated with viral persistence and cancer development.26 Our data reinforce this evidence as co-infections between a single bacterial vaginosis with HPV-DNA, high-risk HPV, and HPV-16 increased the risk for low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions/cervical cancer. Similar results were observed for co-infections between multiple bacterial vaginosis agents with HPV-DNA and high-risk HPV, which increased the risk for low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions/cervical cancer. Considering that bacterial vaginosis is the most common vaginal bacterial dysbiosis in women of reproductive age,10 these are important findings.
With respect to the individual bacteria studied, co-infections of high-risk HPV and G. vaginalis increased the risk of low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions/cervical cancer. These data are consistent with previous studies,27 in which G. vaginalis was detected in 91.9% of women with cervical lesions in addition to a positive association with the presence of injury, indicating a higher detection in women with high-grade squamous intraepithelial lesions. In another study, a positive association was found between the presence of G. vaginalis and cervical injury.28 Therefore, our results and these studies reinforce the hypothesis discussed below. Epidemiological studies have suggested that concomitant infections with pathogens that are also transmitted through sexual activity contribute to an increased risk of squamous cervical cancer.7–9 Neisseria gonorrhoeae, Chlamydia trachomatis, herpes virus simplex, and Trichomonas vaginalis may primarily cause infectious cervicitis.29 30 Similarly, other microorganisms of gynecological importance such G. vaginalis and the class Mollicutes (bacteria without a cell wall), along with the genera Mycoplasma and Ureaplasma, are included in this context. Mollicutes have been described for over 30 years as being related to urogenital disorders. Some species are the main cause of non-gonococcal and non-chlamydial urethritis, as in the case of bacterial vaginosis.31 Mollicutes may induce the release of cytokines and chemokines and may cause intense inflammation. This response has been suggested as a possible risk factor for the development of pre-cancerous lesions.31–33 A recent study showed the microbial Community State Types IV-BV as a risk factor for HPV persistence and proposed Atopobium spp and the sialidase gene from G. vaginalis as microbial markers of HPV persistence.26
Conversely, another study showed that virtually all samples contained sequences of both Atopium vaginae and G. vaginalis but were not associated with HPV.33 Further, we did not observe any association between A. vaginae and Mollicutes with abnormal cytology different from that described above. It is possible that epidemiological variations in the studied population, differences in the methodologies used, and the criteria of patient selection may be the cause of the contradictory results between the studies.
Notably, co-infections of M egasphaera type I with high-risk HPV increased the risk of high-grade squamous intraepithelial lesions/cervical cancer. As far as we know, this is the first study to report this association. The possibility that M egasphaera type I and high-risk HPV act synergistically to influence the progression of cervical lesions is plausible because this bacterium has recently been considered as one of the bacterial vaginosis agents which possibly interacts with HPV in a manner similar to that of G. vaginalis in the progression of cervical lesions. However, our results highlight the need for further studies to evaluate these aspects.
The present study had no data on the history of HPV and vaginal infections as well as bacterial vaginosis agents in the women prior to enrollment in the study or during follow-up, which limits our interpretations of the influence on HPV persistence. Some other bacterial vaginosis agents were not included in the study, so we cannot extend our results to all bacterial vaginosis agents. It is important to note that our study did not aim to assess the carcinogenicity mechanisms of the synergistic action between high-risk HPV and bacterial vaginosis agents, but assessed the possible occurrence of this synergistic action.
In conclusion, this study provided an opportunity to determine the rates of co-infection between HPV and important bacterial vaginosis agents in different cytological cervical samples. We found that G. vaginalis and Megasphaera type I were the primary pathogens associated with high-risk HPV for the increased risk of cervical abnormalities, particularly for high-grade squamous intraepithelial lesions/cervical cancer, in a population with a high rate of abnormal cytology and a positive HPV test. Our results reinforce the hypothesis that some bacterial vaginosis agents may play a role as co-factors in HPV-mediated cervical carcinogenesis. Further cross-sectional studies are needed in different populations.
TTS and NálM contributed equally.
Funding This study was supported by by the Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico, Paraná State Government, and Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES), Brazilian Government.
Competing interests None declared.
Provenance and peer review Not commissioned, externally peer reviewed.