Introduction

Trichomonas vaginalis is a sexually transmitted protozoan that colonizes the epithelium of human urogenital organs. Based on the World Health Organization (WHO) estimates from 2008 to 2016 (WHO 2012), trichomoniasis is considered the most prevalent non-viral sexually transmitted disease (STD) worldwide, affecting more than 276 million people every year (Leitsch 2016). In women, infection can persist for many years, with higher ratio of cases manifested by pruritus, vaginitis, and odorous vaginal discharge (Iqbal et al. 2016). In men, 50–75% of cases of trichomoniasis are asymptomatic and may not seek any treatment, while symptomatic subjects present with mild urethritis that is cleared by the host immune system within 3 weeks (Kissinger 2015; Leitsch 2016).

In long-term asymptomatic infections in men, T. vaginalis parasite can ascend the urethra to the prostate and infect prostatic epithelium (Seo et al. 2014). The parasite is generally an extracellular organism, but its presence in endo-epithelial cells and in sub-epithelial tissues suggests that it has the ability to invade cells and tissues (Gardner et al. 1986; Lopez et al. 2000; Iqbal et al. 2016). It is worth mentioning that T. vaginalis provokes an inflammatory response in prostate epithelial cells and secretes macrophage inhibitory factor that increases prostate cell proliferation. Thus, persistent infection in the prostate may result in a tumor promoting pro-inflammatory microenvironment (Twu et al. 2014).

Interestingly, T. vaginalis has been recognized as a major cause of chronic prostatitis and its sequelae as benign prostatic hyperplasia and prostate cancer (Iqbal et al. 2016; Kim et al. 2019). Despite that several researches reported a positive correlation between trichomoniasis and prostate cancer (Sutcliffe et al. 2006; Stark et al. 2009; Kim et al. 2019), others denied such association (Sutcliffe et al. 2009; Groom et al. 2012; Fowke et al. 2016). In Egypt, a former study detected T. vaginalis in 28.8% of male patients with urethral discharge and in 8.2% of those suffering from impotence and infertility (el Seoud et al. 1998). However, no previous study assessed the relationship between T. vaginalis infection and prostate cancer risk among Egyptians, which necessitates paying more efforts in exploring such link and filling the current gap induced by the conflicting results from other countries. Hence, this study is conducted to clarify whether there is an association between trichomoniasis vaginalis seropositivity and prostate cancer incidence in our locality. Also, this study aims to detect any correlation between different prognostic factors [tumor stage, nodal stage, metastasis status, Gleason score used for grading prostate cancer, and prostate specific antigen (PSA)] and overall survival (time from diagnosis to death or last follow-up) for prostate cancer patients in relationship to T. vaginalis seropositivity.

Subjects, materials, and methods

Ethical consideration

The current study was approved by the Institutional Review Board (IRB No. R.18.03.96) and performed following the ethical standards of the Helsinki declaration. An informed written consent was obtained from each participant after explaining the purpose and procedures of this study. The enrollment of human cases and use of their data and samples in the study were in compliance with the guidance of the Institutional Committee of Research Ethics, Faculty of Medicine, Mansoura University, Egypt.

Study population and design

A case-control study has been conducted on male patients referred to the Department of Clinical Oncology and Nuclear Medicine, Mansoura University Hospital, Egypt, between November 2017 and February 2019. A total of 325 patients aged 40–75 years; 126 with pathologically confirmed prostate cancer adenocarcinoma (all stages and grades whatever the treatment options, such as surgery, radiotherapy, hormonal or chemotherapy), 108 with bladder cancer, and 91 with different types of cancers other than urogenital neoplasms (head and neck cancer, n = 35; colorectal cancer, n = 28; pancreas cancer, n = 14; esophagus cancer, n = 14) were recruited in the study. In addition, 120 age-matched males from the attendants of Mansoura University Hospital outpatient clinics with no history of cancer were enrolled as a negative control group.

Cases with history of recent or past urinary tract or genital tract complaints or treatment for STDs were excluded from the study (considering the fact that trichomoniasis in male is usually asymptomatic, while genitourinary complaints are usually related to bacterial and viral infections).

Intervention

Full medical sheets were taken from all cases. The following data were reviewed: age, demographics, lifestyle, history of smoking, medical history, and diagnosis. For prostate cancer patients, data concerning the disease details (tumor stage, nodal stage, metastasis status, Gleason score, and PSA level) and overall survival were collected.

Blood samples were collected from all cancer patients and normal controls. Sera were separated and kept at − 80 °C in accordance with safety regulations until used (Campbell et al. 2008). T. vaginalis serostatus was examined by detection of anti-Trichomonas IgG antibody. Also, PSA was measured in all enrolled individuals. For prostate cancer patients, PSA levels were reported at time of initial diagnosis and follow-up, but we only considered its level before any therapeutic intervention. In all other enrolled participants, level of PSA was measured to exclude undiagnosed or secondary prostate malignancy and to confirm cancer-free status of the control subjects.

Assessment of Trichomonas vaginalis IgG antibody

Human T. vaginalis IgG antibody was detected in serum using ELISA kits (SinoGeneClon Biotech, HangZhou, China, Catalog number: SG-16001). The kits use purified antigen to coat microtiter plate wells to make solid phase antigen, then T. vaginalis antibody was added to wells. If antibody is present in sample, it will combine first with the coated antigen then with another antigen labeled with horseradish peroxidase (HRP) to make sandwich ELISA. Briefly, 10 μl of serum (brought to room temperature) was mixed with 40 μl of sample diluent, added to the designated wells and incubated for 30 min at 37 °C, then washed. A total of 50 μl of the supplied HRP-conjugate reagent was added to wells (except the blank) and incubated for 30 min at 37 °C. According to manufacturer’s instructions, TMB substrate solution was then added after washing of ELISA plates to give blue color, then the reaction was terminated by addition of sulfuric acid solution, and the absorbance value of the obtained color was measured spectrophotometrically at a wavelength of 450 nm. All samples and controls were tested randomly and blindly in duplicate, then their mean optical density (OD) readings were calculated.

The calculated mean OD of negative control samples was 0.23. According to manufacturer’s instructions, the cutoff value of the test was determined: 0.23 + 0.15 = 0.38, samples with OD < 0.38 were considered negative, and those with OD ≥ 0.38 were considered positive.

To ensure quality of the used kit, the OD of the included positive control sample was measured and serial dilutions (50%, 25%, 12.5%, and 6.3%) of this sample were prepared using the provided sample diluent.

The OD values of the positive and negative controls and the cutoff value of the test were all used to provide a scoring system for Trichomonas IgG serostatus. Score 0: samples with OD < negative control OD; score 1: samples with OD > negative control but < cutoff; score 2: samples with OD > cutoff and < 12.5% diluted positive sample; score 3: samples with OD between 12.5 and 25% diluted positive samples; score 4: samples with OD between 25 and 50% diluted positive samples; score 5: samples with OD > 50% diluted positive sample.

Assessment of prostate-specific antigen

The level of PSA in serum was assessed using enzyme immunoassay test kit (Chemux Bioscience, San Francisco, USA, Catalog number: 10109). The test used is a solid phase two-site immunoassay. Following manufacturer’s instructions, rabbit anti-PSA was coated on the surface of the microtiter plate wells and the other anti-PSA monoclonal antibody labeled with HRP was used as the tracer. A total of 50 μl of the tested serum, standards, and controls were added to the designated wells and incubated with 100 μl of the zero buffer at room temperature for 60 min. Then, PSA molecules found in the standard solution or serum were sandwiched between the two used antibodies. After formation of the antibody-antigen-antibody-enzyme complex, the unbound tracers were removed by plate washing. The HRP activity bound in the wells was then assayed by a colorimetric reaction using a microtiter plate reader at 450 nm within 15 min, and the intensity of color formed was proportional to the concentration of PSA in the sample assayed. All samples, standards, and controls were tested blindly, in duplicate, then their mean ODs were calculated. By using the mean ODs of all reference standards, a standard curve was plotted and the PSA value (ng/ml) of each sample was determined using its mean OD.

Statistical analysis

Data were entered and statistically analyzed using the Statistical Package for Social Sciences (SPSS) version 16. Qualitative data were described as numbers and percentages. The Chi-square test and the Monte Carlo test were used for comparison between groups, as appropriate. Quantitative data were described as mean ± SD or median, as appropriate, and they were tested for normality by Kolmogorov-Smirnov test. In the normally distributed variables, one-way ANOVA with LSD post hoc multiple comparisons was used for comparison between groups. In the non-normally distributed variables, Kruskal-Wallis test was used for comparison between groups.

Overall survival of prostate cancer patients was studied using Kaplan-Meier survival analysis, and Log-rank test was used to compare survival distribution across groups. Correlation between two continuous variables was performed using Pearson’s correlation, while non-parametric correlations were conducted using Spearman’s rank correlation. Odds ratio and their 95% confidence interval (CI) were calculated. P values ≤ 0.05 were considered statistically significant.

Results

In a trial to clarify the relationship between T. vaginalis infection and prostate cancer among Egyptians, the current study included a total of 325 male cancer patients; 126 with prostate cancer (mean age = 64 ± 6.9 years), 108 with bladder cancer (mean age = 65.2 ± 8.1 years), and 91 with other types of cancers (mean age = 64.1 ± 6.1 years). Additionally, 120 age-matched normal males were included as a control group (mean age = 64 ± 3.2 years). All demographic and life-style data of the study participants are depicted in Table 1.

Table 1 Characteristics of the study participants
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Depending on T. vaginalis-IgG OD scores for case classification, 81% (102/120) of prostate cancer patients were seronegative (score 1), while the 19% (26/120) seropositive subjects were distributed among scores 2–5 (7.1%, 7.1%, 2.4%, and 2.4%, respectively). For bladder cancer group, 88.9% of patients (96/108) were seronegative and 11.1% (12/108) were seropositive; score 2. In the group of other types of cancer, 76.9% (70/91) were seronegative, while 23.1% (21/91) of patients were seropositive; designated to scores 2 and 3 (7.7% and 15.4%, respectively). Concerning the control group, 91.7% of cases (110/120) were seronegative and 8.3% (10/120) were seropositive (score 2), with statistically significant differences compared to prostate cancer group and other types of cancer group (P = 0.001, P < 0.0001, respectively), as described in Table 2.

Table 2 Trichomonas vaginalis-IgG optical density score distribution among the study participants
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Further in-depth analysis showed significant differences in the seroprevalence of trichomoniasis in prostate cancer group and the group of other types of cancer compared to the normal controls (P = 0.015 and 0.003, respectively). However, there was no significant difference in T. vaginalis seropositivity in prostate cancer group vs. bladder cancer group (P = 0.09) or other cancers group (P = 0.47), as shown in Table 3.

Table 3 Trichomonas vaginalis serostatus in the recruited cancer groups and normal controls
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The characteristics of prostate cancer patients were detailed in Table 4. No significant relationships between T. vaginalis seropositivity rates and tumour stage, nodal status, presence of metastasis, or Gleason score were detected. However, T. vaginalis-IgG OD scores among trichomoniasis-seropositive prostate cancer patients (n = 24) showed significant associations with PSA level and tumour stage (P < 0.0001 and P = 0.02, respectively), as shown in Table 5.

Table 4 Disease characteristics of prostate cancer patients in relationship to Trichomonas vaginalis serostatus
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Table 5 Correlation between some cancer prognostic factors and Trichomonas vaginalis-IgG optical density scores among trichomoniasis-positive prostate cancer cases (n = 24)
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Analysis of the overall survival of prostate cancer patients showed a mean duration of 53.6 ± 2.0 months; about 54.6 ± 4.5 and 53.4 ± 2.3 months for seropositive and seronegative patients, respectively, with no statistically significant difference (P = 0.86) (Fig. 1).

Fig. 1
figure 1

Overall survival analysis by Kaplan-Meier survival analysis to compare between Trichomonas vaginalis-seropositive (blue line) and Trichomonas vaginalis-seronegative (green line) prostate cancer patients

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Discussion

Despite the worldwide distribution of T. vaginalis infection, its prevalence is thought to be underestimated, mostly because of asymptomatic presentation in many populations. Some previous studies have linked trichomoniasis with increased risk of human immunodeficiency virus (HIV) (McClelland et al. 2007), cervical cancer, and prostate cancer as well as advanced and metastatic prostate cancer (Zhang and Begg 1994; Stark et al. 2009). In fact, trichomoniasis is a curable disease; however, it may indirectly be a life-threatening malady.

Epidemiologic studies demonstrated conflicting results concerning association of T. vaginalis and prostate cancer risk among different populations (Table 6). In the current study, statistically higher T. vaginalis seropositivity rates were recorded in prostate cancer patients compared to normal controls (19% vs. 8.3%, P < 0.05), raising the possibility of an association between trichomoniasis and prostate cancer. Our findings corroborate with the Health Professionals Follow-up Study in America (Sutcliffe et al. 2006), the local cross-sectional study in Radiation and Nuclear Medicine Hospital, and Teaching Hospitals in Iraq (Al-Mayah et al. 2013), and the local study on males with prostate tumors in Korea (Kim et al. 2019), which recorded higher seropositivity for trichomoniasis among men with prostatic cancers. However, the current results differ from the Prostate Cancer Prevention Trial (PCPT) study conducted on Caucasian men (Sutcliffe et al. 2009), the Southern Community Cohort Study (SCCS) on African American men (Fowke et al. 2016), and the PLCO (prostate, lung, colorectal, and ovarian) Cancer Screening Trial on Caucasian and African American men (Marous et al. 2017), which did not support any association between T. vaginalis infection and prostate cancer. The discrepancy between results may be due to studying different population (varied races, ethnicities, and genetic characteristics) from various localities and in turn the possibility of different infecting T. vaginalis genotype strains and subsequent pathology. Moreover, differences in study design, number and demographics of the enrolled participants, methods evaluating T. vaginalis seropositive status as well as the diagnostic procedures for prostate cancer cannot be ruled out.

Table 6 Summary of some previous studies on the association between trichomoniasis seropositivity and prostate cancer
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The association of T. vaginalis infection and prostatic cancer has been proposed for several reasons, including parasite´s known prostatic tropism, its ability to elicit inflammation and damage in prostate epithelium, identification near foci of inflammation and hyperplastic prostate lesions, and its tendency to cause chronic and subclinical infections (Sutcliffe et al. 2009). T. vaginalis has also been shown to produce large amounts of polyamines (Sutcliffe 2010) and up regulate expression of anti-apoptotic and other proto-oncogenes that have been linked to prostate cancer (Sutcliffe et al. 2012), and to increase the growth and invasiveness of benign and malignant prostate cells (Han et al. 2016; Zhu et al. 2016). A recent in vitro study declared that prostate epithelial cells stimulated by T. vaginalis produce cytokines such as IL-6, CCL2, and CXCL8, some of which stimulate the migration of THP-1 monocytes, and M2 polarization of macrophages which in turn promote proliferation of prostate cancer cells. Therefore, they suggested that T. vaginalis infection of prostate could create an inflammatory microenvironment by recruiting other immune cells and producing inflammatory cytokines (Han et al. 2020).

In our study, no significant relationship (P > 0.1 for all variables) was detected between T. vaginalis seropositivity rate and prognostic factors (i.e., tumor stage, lymph node affection, metastasis, and Gleason score) in prostate cancer patients. This is coping with results of Shui et al. (2016) that did not support the possibility of increased risk of advanced or metastatic prostate cancer in Americans with T. vaginalis seropositivity, and those of Marous et al. (2017) that did not link T. vaginalis serostatus with aggressive prostate cancer in Caucasian men. The current findings also corroborate those of Tsang et al. (2019) and Vicier et al. (2019), which declared no association between T. vaginalis seropositivity and likelihood of overall or prostate cancer mortality, or risk of intermediate, high grade, or fatal prostate cancer, respectively. However, our results differ from those of two previous studies, which revealed positive association between T. vaginalis seropositive status and risk of aggressive prostate cancer, including risk of advanced-stage prostate cancer (Sutcliffe et al. 2006), extra-prostatic extension, metastasis, and fatal disease (Stark et al. 2009).

Interestingly, positive correlations between high levels of PSA and high-grade cancer with increased T. vaginalis-IgG OD scores among prostate cancer patients were detected. Hence, we speculate that heavy burden of trichomoniasis may indicate a poor prognosis for prostate cancer. A recent study conducted on young American military members did not strongly support prostate involvement during T. vaginalis infection; however, authors declared non-significant association between high T. vaginalis serostatus and greater PSA concentrations (≥ 0.70 ng/ml). This may be relevant to chronic prostate involvement, as higher early life to midlife PSA concentrations have been found to predict greater prostate cancer risk later in life (Langston et al. 2019).

To the best of our knowledge, this is the first study testing T. vaginalis serostatus among Egyptian patients with prostate cancer compared to those with different types of cancer. It is worth mentioning that T. vaginalis seropositivity rate was 23.1% among patients with different types of neoplasms other than urogenital cancers, which could be linked to the general condition and immunosuppression status of cancer patients.

Conclusively, our findings support a role for T. vaginalis infection in the development of prostate cancer and suggest that prostate cancer might be a parasite-burden dependent.

The reliance on measuring serum anti-T. vaginalis IgG cannot help differentiating recent from old infections, or determining duration or frequency of infection. Moreover, the inability to indicate time between infection and cancer incidence are other points of study limitations that need to be considered in further researches. Nonetheless, large-scale multicenter prospective studies with detailed trichomoniasis and cancer history presentations, and using different epidemiological and laboratory approaches are in demand to explore the causes of population differences in seropositivity to T. vaginalis and to clarify the possible mechanisms for the association between trichomoniasis and malignancies.