Trichomonas vaginalis infection is associated with increased risk of cervical carcinogenesis: A systematic review and meta‐analysis of 470 000 patients

Trichomonas vaginalis infection is the most prevalent non‐viral sexually transmitted infection (STI) in women and has been suggested as a risk factor for developing cervical cancer.


| INTRODUC TI ON
Cervical cancer is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer-related death in women. 1 Currently, there are effective ways of fighting cervical cancer through immunization, screening, and oncologic treatment. 2HPV vaccination provides a high level of protection against oncogenic HPV strains and can reduce the burden of cervical cancer.Moreover, cervical smear and HPV tests improve the reliability of cervical cancer screening significantly. 3In the treatment line of cervical cancer, immunotherapy and target therapy can have an increasing role besides the classical chemotherapeutic regimens. 4Despite all of these, cervical cancer is still the most frequently diagnosed cancer in developing countries, and the leading cause of cancer-related mortality in emergent nations. 1,2e main risk factor of cervical cancer is infection with high-risk HPV types, responsible for various cancer types.Most notably, HPV 16 and HPV 18 types are accountable for 70% of cervical cancers worldwide. 5After being incorporated into the host cell genome, the virus leads to overexpression of proto-oncogene proteins. 6,7A persistent HPV infection and the inability of the immune system to clear out the infection in the cervix are key elements of the carcinogenesis. 5The disruption of the vaginal microbiota performs an essential role in persistent HPV infection, as vaginal dysbiosis occurs, proinflammatory cytokines are increased and immunclearance is reduced. 8[11] Trichomonas vaginalis, a common cause of STI, causes around 170-190 million infections annually. 12Infection of the genital tract with these anaerobic protozoa can lead to discomfort by causing odorous discharge, dysuria, itching, and vulvar irritation.
However, up to 85% of trichomoniases can be symptomless in women.Moreover, 5%-35% of women can also be reinfected. 13ichomonas vaginalis can contribute to the development of cervical cancer by causing inflammation, abruption of the cervical epithelium, and influencing the immune system to eliminate HPV.
Current evidence on the relationship between T. vaginalis infection, cervical dysplasia, and cervical cancer is conflicting.5][16][17] Two meta-analyses have been conducted on this topic.
9][20] The other meta-analysis focused on cervical dysplasia without differentiating between the different states of cervical lesions and did not investigate the relationship between T. vaginalis and HPV. 21nce, on the basis of the available literature, this study aimed to investigate the association between T. vaginalis and HPV, cervical dysplasia, and carcinogenesis.We hypothesized that T. vaginalis was a risk factor for developing cervical cancer.

| MATERIAL SANDME THODS
We conducted our systematic review and meta-analysis according to the PRISMA 2020 and MOOSE guidelines (see Figure 1; 3][24] The pre-study protocol was registered in PROSPERO (CRD42021286097), and we fully adhered to it.

| Literaturesearchandeligibilitycriteria
The systematic search was conducted using five major databases on October 20, 2021: MEDLINE (via PubMed), Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus, and Web of Science.We accepted only peer-reviewed articles; therefore, we did not search on Clini calTr ials.gov,nor did our preliminary search find any suitable studies.No filters or restrictions were applied during the search.We used two population, exposure, and outcome (PEO) frameworks to define the eligibility criteria for the articles. 25l studies reporting sexually active (P 1 ) or HPV-positive (P 2 ) women who were screened for T. vaginalis infection (E) were deemed eligible.The outcomes of interest (O 1 ) were HPV positivity, cervical dysplasia, and cervical cancer.In HPV-positive women (P 2 ), the investigated outcomes (O 2 ) were cervical dysplasia and cervical cancer.The articles had to include a population of T. vaginalis-negative women forming the control group.
Articles were considered where T. vaginalis was detected with cytology, wet-mount, culture, or polymerase chain reaction (PCR) methods.Articles were excluded in which T. vaginalis was diagnosed on the basis of clinical features or medical history.Studies were suitable if HPV exposure was diagnosed with any nuclear amplification method.
Articles where HPV was detected only by cytology were excluded because of the low sensitivity of the method. 26Cytologic and histopathologic diagnoses were acceptable for cervical intraepithelial neoplasia (CIN) and cancer confirmation.We evaluated the following outcomes in the dysplasia group: atypical squamous cells of undetermined significance (ASCUS), atypical glandular cells, atypical squamous cells for which one could not rule out high-grade squamous intraepithelial lesions (ASC-H), low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL).For cytologic samples, the Bethesda classification was required.Articles that proclaimed CIN1-3 diagnoses were divided into LSIL (CIN1) and HSIL (CIN2-3) groups for a more straightforward interpretation.
Observational studies, such as cross-sectional, case-control, and cohort analyses, were accepted.Abstracts were excluded in our review.Non-English language articles were translated for possible evaluation.

| Searchstrategy
During the systematic search, we used the following main concepts: "trichomonas", "human papillomavirus", "cervical intraepithelial neoplasia", and "cervical cancer".The whole search key can be found in Supporting Information.

| Studyselectionanddatacollection
A reference management program (EndnotE X9) was used to select the articles.First, a duplicate removal was performed, then, two independent reviewers (BH, EH) carried out a title and abstract selection and then full-text selection.Cohen's κ coefficient measured the degree of agreement. 27 centers, the detection method of T. vaginalis, HPV, and cytologic/ histologic lesions.Where possible, data regarding the outcomes were extracted in two-by-two tables.Otherwise, we collected the unadjusted odds ratios (ORs).In order to handle confounding factors, when possible, we collected adjusted ORs, and the variables for these results were adjusted.In case of any disagreement, a consensus was reached involving a third investigator (ZSH).

| Riskofbiasandqualityassessmentofthe included articles
To critically assess the outcome data, we performed a risk of bias assessment with the help of the Quality in Prognostic Studies (QUIPS) tool. 28The QUIPS tool includes six domains: study participation, study attrition, prognostic factor measurement, outcome measurement, study confounding, and statistical analysis reporting.In each domain, four classifications can be given: not applicable, low risk, moderate risk, and high risk of bias.To grade the level of evidence of our findings, we implemented the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) approach.The Summary of Findings table was prepared by using the GradEPro tool. 29Both QUIPS and GRADE were performed by two independent reviewers (BH, EH), and in case of disagreement, a third investigator resolved the dispute (ZSH).

| Synthesismethods
During data synthesis, both qualitative and quantitative assessments were carried out.The R programming language was used for statistical data analysis (R Core Team, 2022; R version 4.2).The minimum number of studies for performing the quantitative synthesis was three.Forest plots were used to visualize individual studies and overall results.Subgroup analyses were performed based on the detection method of T. vaginalis and the country of origin of the article.Sensitivity analyses were carried out for four outcomes.
1][32] Statistical significance was substantiated for a result when P < 0.05.I 2 and Cochran's Q tests were used to measure statistical heterogeneity, where P < 0.1 indicated significant heterogeneity. 28general interpretation of the heterogeneity values is as follows: 0%-40% possibly not important heterogeneity; 30%-60% moderate heterogeneity; 50%-90% substantial heterogeneity; and 75%-100% considerable heterogeneity.Beside I 2 , we also reported the prediction intervals (i.e. the expected range of effects of future studies) of the pooled estimates if the minimum study number was reached. 33e inspection of funnel plots and an Egger's test were used to assess publication bias when a minimum of 10 articles were available for one outcome.

| Basiccharacteristicsofincludedstudies
The eligible articles were published between 2009 and 2021, with 11 publications from Asia, five from Europe, seven from South America, five from Africa, and one from North America.According to study type, we found 22 cross-sectional, five case-control, and one prospective cohort study.
As for demographics, the mean age of women was 37.57 years.
In 15 articles, T. vaginalis was detected with PCR, in eight with wetmount, in four with cytology and in two with cultures and wetmount.All the studies assessed the exposure and the outcome at the same time.
Altogether 473 740 women were included in our meta-analysis.
Of them, 8518 patients had T. vaginalis infection in the exposure group.Baseline characteristics of the eligible studies are detailed in Table 1.

| Quantitativeandqualitativeanalysis
The association between T. vaginalis and HPV infections Twenty-four studies including 7291 women in the T. vaginalisinfected group and 452 161 in the control group reported an association between T. vaginalis and HPV infections. 14,16,17, Our esults showed that T. vaginalis-positive women were 1.79 times more likely to be diagnosed with an HPV co-infection (95% CI 1.27-2.53;I 2 95%; Figure 2) compared with T. vaginalis-negative women.When a T. vaginalis infection was confirmed with the wet-mount method, the odds of detecting a co-infection with HPV were slightly higher, by the odds of 2.29 (95% CI 1.23-4.28;I 2 97%).The results from the subgroups based on region showed that T. vaginalis-positive women from Asia had the highest chance for HPV co-infection (OR 2.05, 95% CI 1.08-3.88;I 2 97%; see Figure S1).A sensitivity analysis (leave-one-out method) did not recognize any influential study (see Figure S2).

The association between T. vaginalis and cervical dysplasia
For the ASCUS outcome, five studies evaluated 1493 women in the exposure group and 75 135 women in the control group. 15,16,43,54,551,[54][55][56] When examining the association between T. vaginalis and LSIL, we found that women who were infected with T. vaginalis had almost twofold odds of having LSIL (OR 1.92, 95% CI 0.78-4.77;I 2 91%; see Figure S6), compared with women who were not T. vaginalis-infected.
However, the findings were statistically not significant.When T. vaginalis was detected with PCR, women had higher odds of having an LSIL diagnosis (OR 3.66, 95% CI 1.51-8.86;I 2 69%).Regarding the analysis of regional differences, we detected a ninefold chance for LSIL when T. vaginalis was present in women from South America (OR 9.36, 95% CI 2.34-37.36;I 2 63%; Figure S7).When the leave-oneout analysis was carried out, the exclusion of the study by Al-Awadhi et al. 15 resulted in a higher association between T. vaginalis and LSIL detection (OR 2.79; 95% CI 1.61-4.82;I 2 65%).Furthermore, when the article by Amorim et al. 56 was excluded, we found an OR of 1.51 (95% CI 0.65-3.55;I 2 95%; see Figure S8).

Regarding a relationship between T. vaginalis and ASC-H, we
could not find any association (OR 1.78, 95% CI 0.21-15.12;see Figure S9). 16,43,54even studies assessed 1796 women in the exposure group and 80 276 women in the control group for the association between T.
One paper 54 performed a multivariate analysis (see Table S3).

The association between T. vaginalis and cervical cancer
Three articles were quantitatively analyzed, with 219 women in the T. vaginalis-positive group and 397 women in the control group. 17,51,58men who were T. vaginalis-positive had 5.24 times higher odds of having cervical cancer (OR 5.23, 95% CI 3.03-9.04;I 2 3%; Figure 4) compared with T. vaginalis-negative women.

Association between T. vaginalis, cervical lesions, and cervical cancer in the HPV-positive population
We found four articles for the quantitative synthesis regarding the HPV-positive population when evaluating the association between T. vaginalis infection and cervical lesions. 17,50,51,57r LSIL, three articles were analyzed, assessing 1932 women in the exposure group and 20 033 in the control group. 17,50,51ichomonas vaginalis-positive women had 2.81 higher odds for LSIL diagnosis than women who were not T. vaginalis infected (95% CI 2.37-3.33;I 2 0%; see Figure 5).
In total, there were 1921 women in the exposure group and 20 750 women in the control group for HSIL. 17,50,51,57Patients who were diagnosed with T. vaginalis had more than twofold odds of having HSIL compared with women who were not diagnosed with T. vaginalis (OR 2.36, 95% CI 1.79-3.11;I 2 10%; see Figure 5).
Three studies examined cervical cancer, with 1811 women in the exposure group and 19 331 in the control group. 17,50,51We found that women who were T. vaginalis-positive had increased odds of being diagnosed with cervical cancer compared with women who were not T. vaginalis-positive (OR 3.09, 95% CI 1.66-5.77;I 2 45%; see Figure 5).

| Riskofbiasassessmentand qualityofevidence
The results of the risk of bias assessment are presented for every outcome.For the T. vaginalis-HPV co-infection outcome, seven articles demonstrated "a moderate risk for study confounding bias", and three articles demonstrated "a high risk of bias".In the study participation domain we detected a high risk of bias in three articles.In the cervical dysplasia groups, the risk of bias was low.In the study confounding domain, we found one article to be at a high risk of bias in ASCUS and two articles of a high risk of bias in the LSIL and HSIL groups.In the cervical cancer group, we found one article in the confounding domain of the study at a high risk of bias.(see Our Summary of Findings included six outcomes for the first PEO and three for the second PEO (see Tables S3 and S4).The quality of evidence was "low" for six outcomes and "very low" for three outcomes.

F I G U R E 3
Forest plot of studies representing that Trichomonas vaginalis infection was associated with high-grade squamous intraepithelial lesions.CI, confidence interval; HSIL, high-grade squamous intraepithelial lesions; OR, odds ratio; PCR, polymerase chain reaction; TV, Trichomonas vaginalis.

| Publicationbiasandheterogeneity
We performed the Egger test and a funnel plot to assess publication bias in the T. vaginalis-HPV co-infection, T. vaginalis-LSIL, and T. vaginalis-HSIL groups.In all three cases, the funnel plots showed some asymmetry.Even on the basis of Egger test, we did not find a significant publication bias as the P values were greater than 0.1 (see Figures S23-S25).

| DISCUSS ION
Our study, which included nearly half a million women from population-based studies, showed a positive association between T.
vaginalis and cervical carcinogenesis.First, we investigated the association between T. vaginalis and HPV.We found that women with a T. vaginalis infection had higher odds of being diagnosed with a concomitant HPV infection than women who were T. vaginalis-negative.
In the relation between T. vaginalis and cervical dysplasia, a significant association was found.When we evaluated the relationship between T. vaginalis and cervical cancer, we also found a statistically significant association resulting in higher odds of developing cervical cancer among women infected with T. vaginalis.Regarding our second clinical question in the HPV-positive population, we found a positive association between T. vaginalis, LSIL, HSIL, and cervical cancer.
As for strengths, our study is the first meta-analysis to investigate the relationship between T. vaginalis, HPV, and cervical lesions in detail.As a result of the large sample size in the assessed articles, we could include nearly half a million patients in the analysis.
Moreover, most of the studies carried a low risk of bias.
However, the results need to be interpreted together with the limitations.First, as none of the studies followed up the T. vaginalisinfected population, and all participants were screened for T. vaginalis, HPV, and cervical carcinogenesis simultaneously, we do not know how T. vaginalis can contribute to the development of the outcomes.
Second, as many studies did not perform multivariate analyses, we could not calculate pooled adjusted ORs.The inadequate control of confounders may lead to an underestimation or overestimation of the analyzed associations.Third, it is not clear whether T. vaginalis infection causes the cervical environment to be more susceptible to HPV infection and to the subsequent CIN, or whether cervical dysplasia makes the environment more attractive to T. vaginalis infection. 59Fourth, in the diagnosis of cervical lesions, some studies used cytology, which is subjective, and it is a diagnostic method that is difficult to replicate. 60Fifth, according to the GRADE assessment, the quality of evidence was low in six and very low in three outcomes.Sixth, not all HPVs are oncogenic though in the T. vaginalis-HPV association, 10 studies included non-oncogenic HPV strains in their investigation too.
The association between T. vaginalis and HPV showed that STIs often coexist because of similar behavioral risk factors such as young age, a high number of sexual partners, and unprotected intercourse. 16,61Therefore, we cannot conclude that T. vaginalis infection affects HPV acquisition because both infections can be concomitantly present.The etiology of cervical cancer and most CIN are attributable to high-risk HPV types. 5Therefore, HPV could be a confounding factor for our cervix-related outcomes.However, if we only investigate the HPV-positive population, we could observe an even more increased association between T. vaginalis cervical dysplasia and cancer.
In contrast, not all HPV types carry the same oncogenic risk as HPV 16 and HPV 18, which cause around 70% of all cervical cancers worldwide. 5Therefore, HPV positivity in women does not represent a homogeneous population from an oncogenic point of view.Some prospective studies suggest that the likelihood of a persistent HPV infection increases in the presence of concomitant T. vaginalis infection. 45,62Behind this observation there is a pre- of different genital infections, Chlamydia trachomatis, and bacterial vaginosis can also induce persistent HPV infection, resulting in cervical dysplasia progression. 66,67en assessing ASCUS, we found a significant association with T. vaginalis infection.A Belgian study also found that women diagnosed with ASCUS had been HPV-negative but T. vaginalispositive in a few cases, suggesting that T. vaginalis could also lead to ASCUS. 16In the cervical dysplasia group, we found the highest odds for cervical lesions when T. vaginalis was detected with PCR, probably because this method was the most sensitive for T. vaginalis detection. 19In the T. vaginalis and cervical dysplasia group, we found higher odds in the ASCUS and LSIL groups in South America, although we had only one article in the ASCUS group.The prevalence of T. vaginalis is deeply connected to socioeconomic variables, sexual behaviors, and access to health care.
Without surveillance programs, the actual epidemiologic state of T. vaginalis is unknown.However, countries where the populations have higher incomes generally have a lower prevalence of T. vaginalis, and countries where the populations have lower incomes generally have a higher prevalence. 13In the sensitivity analysis, two articles could have altered our results.One of the outliers 15 led to a lower association between T. vaginalis and cervical dysplasia.In this study, T. vaginalis was diagnosed with cytology, which is not a reference standard detection of T. vaginalis. 19The other article 56 came from an area of Brazil where poverty rate was high, and cervical cancer was the second most common cancer. 3These findings can explain the high ORs we experienced in the T. vaginalis and cervical dysplasia group.
Lipophosphoglycan (LPG), a virulence factor found on the surface of T. vaginalis, can induce immunologic reactions depending on the type of LPG.In reaction to these LPG particles, the host epithelial cells can secrete proinflammatory cytokines, interleukin-8 and macrophage inflammatory protein 3α, which induce the inflammation of the cervix and the vagina.At the same time, other LPGs found on T. vaginalis can decrease the level of proinflammatory cytokines and evade immune reactions.This is in line with the clinical finding that T. vaginalis can often be asymptomatic or can cause persistent infection. 64flammation of the cervix has been associated with an increased risk of CIN in one study. 7Another article found elevated levels of interleukin-6 and interleukin-8 in CIN and cervical cancer. 68Generally, inflammation is considered a risk factor for developing many cancer types. 69One study investigated the microbial component of the vagina in cervical cancer patients and non-cervical cancer patients, assuming that cervical cancer disrupts the vaginal microbiota and makes it attractive to infectious diseases.Trichomonas vaginalis is possibly less of a cofactor than a consequence of cervical cancer. 70e intact state of the vaginal microbiome with Lactobacillus species is essential for protection against STIs.The abruption of this complex microbiome increases the probability of genital infections due to decreased defensive barriers. 71One study proved the proinflammatory synergism between vaginal dysbiosis and T. vaginalis; moreover, it suggested a surface biofilm that makes them more resistant to antibiotic treatment. 72Overall, STIs and vaginal infections have been considered possible cofactors in the development of CIN and cervical cancer.In one meta-analysis, Chlamydia trachomatis was found to be associated with cervical cancer, whereas another metaanalysis also found an association between bacterial vaginosis and cervical lesions. 61,67Our findings also support the idea that STIs and vaginal infections might act as cofactors in the development of cervical cancer.
We believe that more studies are needed to control the confounding factors; therefore, the true effect of T. vaginalis on cervical carcinogenesis could be estimated in a more reliable way.Second, we recommend that clinicians who treat women in their practice always consider HPV infection and cervical lesions when diagnosing T. vaginalis infection.Even though we cannot conclude a causative relationship between T. vaginalis and cervical carcinogenesis, T. vaginalis is associated with HPV infection, cervical lesions, and cervical cancer, so a follow up of patients after the T. vaginalis diagnosis might be beneficial.Many countries have implemented HPV-based cervical cancer screening programs, which means a greater detection rate in HPV strains. 73According to our study, T. vaginalis and HPV are associated; therefore, in the case of an HPV diagnosis, the screening and treatment of T. vaginalis are advisable because of its potential carcinogenic effect on the cervix.
In conclusion, our results showed that T. vaginalis infection might increase the odds of cervical lesions and cancer development in sexually active women.We advise clinicians to evaluate HPV and cervical dysplasia in the case of a T. vaginalis diagnosis.
A third independent investigator (ZSH) agreed on debated articles.If we could not find an article, or data were missing, we contacted the authors.Two independent reviewers (BH, EH) extracted variables from the eligible studies into a pre-defined Microsoft ExcEl spreadsheet (Windows 11 Pro).The following variables were collected from each article: first author, publication year, digital object identifier, study design, study type, demography (age, sample size), country, F I G U R E 1 PRISMA 2020 flowchart representing the study selection process.

3 | RE SULTS 3 . 1 |
Searchandselection Our comprehensive search identified 1707 articles.After duplicate removal, 1259 publications were screened based on title and abstract.During the full-text selection, 355 articles were screened, resulting in 29 eligible studies for the quantitative and qualitative data syntheses.Cohen's κ was 0.9 for title and abstract selection; and 0.85 for full-text selection.Despite contacting authors for non-retriveble articles, we received only a few responses.
Abbreviations: ASC-H, atypical squamous cells for which one cannot rule out high-grade squamous intraepithelial lesions; ASCUS, atypical squamous cells of undetermined significance; CA, cervical cancer; HCII, Hybrid capture II; HSIL, high-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesions; NA, not available; PCR, polymerase chain reaction.a Minimum-maximum age values.b Median age value.

F I G U R E 4
sumption of how T. vaginalis can alter HPV clearance.Trichomonas vaginalis can cause micro-lesions in the cervical epithelium, Forest plot of studies representing that Trichomonas vaginalis infection was associated with cervical cancer.CI, confidence interval; OR, odds ratio; TV, Trichomonas vaginalis.decreasethe protective mucus layer of the vagina, and induce proinflammatory cytokines through immune response, which can facilitate the spread of an HPV infection into the basal layer of the cervical epithelium and induce persistent HPV infection.7,63,64As persistent HPV infection occurs, the probability of cervical dysplasia increases, promoting cervical carcinogenesis.65Coexistence F I G U R E 5 Forest plot of studies representing that Trichomonas vaginalis was associated with low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer in the HPV-positive population.CI, confidence interval; HSIL, high-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesions.