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Abstract
Objectives The main objective of this study is to determine the prevalence of trichomoniasis in men with and without symptoms of urethritis, with concomitant analysis of sociodemographic and behavioural specificities of both groups. Also, the objective is to evaluate laboratory methods used in the diagnostics of this parasitic disease.
Methods A total of 500 men with and 200 without urethritis symptoms were included in the study. Every respondent filled out a questionnaire asking for some general data, specific information about habits, sexual behaviour and symptoms. Sediment of first void urine was analysed by wet mount microscopy, cultivation in Diamond's medium and real-time PCR.
Results In the symptomatic group, Trichomonas vaginalis infection was documented in 2.4% of respondents by wet mount microscopy, in 4.8% by cultivation and in 8.2% by real-time PCR. In the asymptomatic group, infection was proven using the same methods in 1.0%, 1.5% and 2.0% of the respondents, respectively. Trichomoniasis prevalence was statistically significantly higher in the respondents manifesting urethritis symptoms when cultivation (χ2=4.20, p=0.041) and real-time PCR (χ2=9.20, p=0.002) were used. Several epidemiological risk factors were identified, and greater sensitivity of real-time PCR was found in comparison with microscopy and culture.
Conclusions Trichomonas infection was statistically more frequent in men with urethritis syndrome. Assuming that the samples found positive by any laboratory technique are truly positive, it can be concluded that the real-time PCR showed the greatest sensitivity of all the methods used in this study.
- TRICHOMONAS
- URETHRITIS
- PARASITOLOGY
- SEXUAL HEALTH
- MOLECULAR TECHNIQUES
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Introduction
Trichomonas vaginalis is sexually transmitted flagellated parasite from the protist kingdom which can be found in lower female reproductive tract and male urethra. It has a simple life cycle and is transmitted from person to person via close contact of genital organ mucosa. According to estimates of the WHO, with an annual incidence of almost 250 million cases, trichomoniasis can be considered the most common non-viral sexually transmitted infection in the world.1 That number exceeds the estimates of annual global incidence of Chlamydia trachomatis infections (101 million cases), as well as infections with Neisseria gonorrhoeae (88 million) and Treponema pallidum (11 million).1
However, these data on the frequency of trichomoniasis are based on visual detection of T. vaginalis under the microscope—diagnostic method with a limited sensitivity range. Newer studies indicate that the detection of trichomoniasis could be higher if patients are screened with more sensitive methods.2–5 Although it is not currently used that often for routine diagnostics, the results suggest that PCR is the most sensitive method for diagnosing this parasitosis. In addition to higher sensitivity, one of the advantages of PCR is also the ability to detect non-viable organisms in tested clinical specimens.6 ,7
Male patients with Trichomonas infection can be divided into three groups: those with acute symptomatic disease, mildly symptomatic and with asymptomatic disease.8 The most common symptoms encountered in symptomatic infections are clear or purulent discharge and dysuria, often accompanied by itching and burning sensation within the urethra. The percentage of asymptomatic male patients is higher when compared with women, exceeding 50% of infected men.9 Given that the clinical signs and symptoms are neither specific nor reliable indicators for trichomoniasis, accurate diagnosis depends on laboratory confirmation of the pathogen.
The lack of information on the epidemiology of trichomoniasis in men is often attributed to asymptomatic infections in the vast majority of affected individuals, as well as physician's assessment that it is an insignificant, self-limiting infection.10 ,11 Because targeted research on trichomoniasis prevalence in Croatia has been never done and the spread of this infection in the male population is unknown, the objective of this study is to determine the prevalence of trichomoniasis in adult men, with and without symptoms of urethritis. We also assessed the value of real-time PCR by comparing results acquired by this molecular method with the results from more routine microbiological diagnostic procedures (ie, microscopy and cultivation).
Materials and methods
A total of 700 respondents aged 18–66 years were included in this case–control study and classified depending on the presence or absence of symptoms of urethritis into two groups. The symptomatic group consisted of 500 adult men referred to the Croatian National Institute of Public Health for routine aetiological diagnostic work-up of urethritis. Criteria used for determining clinically overt urethritis was the presence of at least one of the following symptoms: dysuria, burning sensation in the urethra, urethral discharge and redness of the urethral orifice.12 The control group consisted of 200 adult men without symptoms of urethritis, referred to laboratories of the Croatian National Institute of Public Health as a part of a preventive medical screening.
Before sampling, each respondent who agreed to participate in the study signed an informed consent statement and completed a survey that (among others) contained the following variables: place of residence, year of birth, education level, marital, intimate and family status, sexual orientation, sexual history (sexual intercourses outside of Croatia, number of sexual partners in the past year, age of first sexual intercourse, usage of condoms during sexual intercourse, paying for sexual services, charging for sexual favours and acquired sexually transmitted diseases in the past), the use of drugs/alcohol before sexual activity, HIV antibody testing and information about symptoms. No patients were excluded from the study after they agreed to participate. Materials and methods can be found in online supplementary file.
Wet mount microscopy, cultivation and real-time PCR were the methods used for the detection of trichomoniasis in this study. The first voided morning urine was used as a specimen, collected in a sterile container in the Institute. Ten millilitre of urine from the cup has been added to the test tube with a conical bottom and centrifuged at 1000 rpm for 5 min. After centrifugation, the liquid part has been discarded, and the sediment was divided into three equivalent parts: the first part was used for a wet mount preparation, the second part was inoculated to the Diamond's medium, while the third part was submitted to PCR testing.
In wet mount microscopy, a drop of urine sediment from first void morning urine was used as a sample and observed with a dry light microscope magnification of ×400. Prepared slide was completely examined. Positive result was a finding of T. vaginalis trophozoites with characteristic motion and morphology.13 Slides prepared from cultures using Diamond's medium incubated at 37°C were examined in the same way for up to 5 days.
Real-time PCR was performed in the 7500 Real Time PCR System Machine from Applied Biosystems manufacturer. TaqMan Universal PCR Master Mix Kit (Applied Biosystems, USA) was used. The presence of inhibitors of PCR reaction was controlled with internal control—TaqMan exogenous Internal Positive Control Reagents from the same manufacturer (Applied Biosystems, USA). Every real-time PCR test included positive (trichomonal DNA) and negative control (ddH2O). T. vaginalis DNA was isolated from urine sediment samples according to the manufacturer's recommendations (Qiagen GmbH, Germany) using a commercial kit QIAamp DNA Mini Kit. Sixty-seven-base repeating sequence of T. vaginalis genome sequence has been used as amplicon with the following base sequences14—forward primer: 5′-CATTGACCACACGGACAAAAAG-3′, reverse primer: 5′-CGAAGTGCTCGAATGCGA-3′ and the probe with the following sequence of bases: 5′-FAM-TCATTTCGGATGGTCAAGC AGCCA-TAMRA-3′.
This study was conducted entirely in the Microbiology Department of the Croatian National Institute of Public Health for a 3-year period. Standardised protocols for data collection and trained laboratory study personnel were important factors in addressing and minimising potential sources of bias. Every respondent was informed about the aims and objectives of this study and has given their written consent. Those who declined to participate because of their personal reasons were not included in the sample.
Sample size estimation was used to establish an optimal number of cases to draw statistically significant conclusions. α Value of 0.05 and β error 0.1 (power of 90%) were used for that purpose. For the expected values in symptomatic and asymptomatic group, the PCR prevalence of 8% and 2% has been chosen, respectively. We choose the ratio symptomatic/asymptomatic 2 and that led us to minimal sample size of 195 asymptomatic and 390 symptomatic patients (one-sided test).
Obtained data were analysed using the SPSS V.13.0 program. The χ2 test was used to determine whether there is an association between a categorical risk factor and T. vaginalis infection. Fisher's exact test was used to measure the association between two variables in a 2×2 contingency table when sample sizes were small. The non-parametric Mann–Whitney test was used to compare the medians of ordinal, non-parametric data between the two groups to determine whether they are statistically different.
Results
The prevalence of TV infection in men with urethritis symptoms ranged from 2.4% to 8.2%, depending on the diagnostic method used. Using wet mount microscopy, Trichomonas was found in 12 (2.4%) of 500 respondents. By means of cultivation in Diamond's medium, trichomoniasis has been found in twice as more examinees, precisely 24 (4.8%) of them. The highest prevalence of trichomoniasis was demonstrated using real-time PCR, which detected this parasitosis in 41 (8.2%) of the respondents.
The prevalence of trichomoniasis in asymptomatic men was 1% by wet mount microscopy (2/200), 1.5% when cultivation was used (3/200), whereas real-time PCR yielded the highest result of 2% (4/200).
The frequency of trichomoniasis in respondents with urethritis syndrome was statistically significantly higher than the prevalence of this parasitic disease in control subjects when cultivation methods and real-time PCR were used for diagnosis (χ2=4.20, p=0.041; χ2=9.20, p=0.002; table 1).
Prevalence of Trichomonas vaginalis in the groups of symptomatic and asymptomatic male respondents (χ2 test was used in statistical analysis)
According to the completed questionnaire, risk factors for trichomoniasis in men demonstrated in this study were engaging in sexual relations outside Croatia (p=0.035), purchasing sexual services at least once (p<0.001), as well as relatively older age during first sexual intercourse (p=0.049). Although heterosexual orientation could also be considered as a significant risk factor according to the statistical analysis (p=0.036), it must be noted that no homosexual patients infected with TV were found in this study. No statistically relevant association was found between trichomoniasis and the place of residence (p=0.213), employment (p=0.673) and the use of condoms during the last sexual intercourse (p=0.718) or sexual intercourse in general (p=0.573). Likewise, marital status (p=0.330), parenthood (p=0.652) and HIV antibody test (p=0.297) did not prove to be risk factors for trichomoniasis. Number of sexual partners in the last year (p=0.109), drugs or alcohol usage (p=0.611) and education (p=0.968) were also not shown to be significant risk factors for this disease (table 2).
Overview of characteristics for symptomatic group of men with and without proven Trichomonas vaginalis infection by real-time PCR
We also analysed methods used in the diagnosis of T. vaginalis infection and compared current gold standard in the diagnosis of trichomoniasis (cultivation) with wet mount microscopy and real-time PCR. Comparison of the results yielded by cultivation and microscopy revealed a sensitivity of 50.0% and specificity of 99.3% for microscopy (table 3). The positive predictive value of microscopy was 64.3% and negative predictive value 97.4%. Total percentage of overlap between the two methods was 96.7%.
Contingency table comparing wet mount microscopy with cultivation in detection of Trichomonas vaginalis infection
Comparison of the results yielded by cultivation and real-time PCR indicated a greater likelihood of finding T. vaginalis using PCR method. While using culture, the prevalence of TV was found to be 3.9% (1.5%–4.8%), and the use of PCR method showed a trichomoniasis prevalence of 6.4% (2.0%–8.2%; table 4). Calculated measures of validity demonstrated that the sensitivity and specificity of PCR were 100.0% and 97.3%, respectively. The positive predictive value of T. vaginalis detection by PCR was 60.0% and negative predictive value 100.0%. Total percentage of overlap between the two methods was 97.4%.
Contingency table comparing real-time PCR with cultivation in detection of Trichomonas vaginalis infection
Assuming that the real-time PCR results are truly positive, the sensitivity of culture is modest at 60% (table 4), with a 95% CI of 44% to 72%. If real-time PCR is correct, the specificity of culture is 100% (table 4). The sensitivity of wet mount microscopy is even lower at 31%, with a 95% CI of 20% to 46%.
Discussion
This is the first prospective study of T. vaginalis prevalence in adult men conducted in Croatia. The aim of our research is to create a more objective picture of the spread of this infection in the male population. Using a combination of methods, the prevalence of T. vaginalis infection was statistically significantly higher in the respondents manifesting urethritis symptoms when cultivation and real-time PCR were used. The prevalence of T. vaginalis was highest when using real-time PCR. Assuming that the samples found positive by any technique are truly positive, it can be inferred that real-time PCR has the highest sensitivity.
Primary strengths of this study are the large number of patients included and the use of PCR to detect T. vaginalis. A parallel diagnostic testing by wet mount microscopy and cultivation ascertained the performance of the PCR used in this study. Also, all the testing has been conducted in single national reference laboratory for human parasitoses with rigorously controlled conditions, which ensured uniformity and comparability of results. In addition, criteria to determine clinically overt urethritis were strictly defined.
Our study has several limitations. This research studied male population consisting of men predominantly from the city of Zagreb, Croatia. Although the sample was big enough to result in sufficient power to analyse multivariate models between behavioural and demographic characteristics and T. vaginalis, the results may not be generalisable to other male populations. Response bias also always represents concern, although the use of standardised interviews by a trained staff aimed to minimise any kind of information bias. Since all epidemiological information was retrieved prior to testing, there was no apparent reason to suspect differential bias linked to trichomonal infection.
Depending on the method used for the diagnosis, there was a statistically significant difference in the frequency of trichomoniasis between groups of subjects when cultivation and real-time PCR have been used, due to a higher number of detected TV infections by these methods. Based on these data, trichomoniasis was significantly higher in the group of symptomatic men; however, comparing the data on the trichomoniasis frequency obtained by wet mount microscopy, the difference between the patient group and the control group has not been found.
Previous research on TV prevalence in men has shown different results. In 2002, Schwebke and Lawing15 described much higher prevalence of TV infections (17%) in men with urethritis symptoms by PCR. When culture was used in the same research, prevalence of trichomoniasis was almost equal to ours (5%). One other group of American investigators demonstrated the prevalence of trichomoniasis of 13% using PCR in a group of men with symptoms of urethritis.16 The highest prevalence (21%) was found by Hobbs et al,17 in 1999, after testing the patients with urethritis using PCR method as well. It must be noted that some of the aforementioned studies and our research mutually differ in primer sets used for PCR, which may account for some differences in the prevalence.
In South Africa, the prevalence of T. vaginalis infection in men with genital discharge syndrome is decreasing over the years (from 13.4% in 2007 to 4.8% in 2012).18 Seike et al19 found a low prevalence of uretheral T. vaginalis infection in Japanese men with (1.4%) and without urethritis (1.0%); in this study, possible relationship between the detection of T. vaginalis and clinical symptoms was not confirmed. The overall prevalence of trichomonal infection was 2% in a recent study conducted in Iran, although only wet mount microscopy and cultivation were used.20 No statistical correlation was observed between clinical presentation and parasitological results in that research.
In our study, the prevalence of trichomoniasis in asymptomatic men (2%) was significantly lower than the occurrence of this parasitosis in the group of symptomatic men (8.2%). Similar results were shown by a group of researchers using PCR methodology who found almost twice as low prevalence of trichomoniasis in the group of asymptomatic male subjects.17 In 2003, Schwebke and Hook21 found no statistically significant difference in the frequency of detecting this parasite by PCR between men with urethritis syndrome and those without any symptoms at all.
Diagnosing T. vaginalis infection is still mostly based on wet mount microscopy. Although numerous studies have demonstrated relative insensitivity of this method, only a small fraction of laboratories implement other, more sensitive methods in the routine diagnostic algorithm. Cultivation of organisms in Diamond's medium proved to be more sensitive for the diagnosis of trichomoniasis; however, the time required for the detection of organisms delays diagnosis considerably. The latter method also requires preparation or purchase of selective media for cultivation of this organism.
Several years have passed since the first description of PCR for the diagnosis of trichomoniasis,6 but this technique has not become entrenched in the routine diagnosis of this parasitic disease. Using the real-time PCR method, we diagnosed nearly three times more patients when compared with the results acquired by wet mount microscopy of the urine sediment. Twice as many patients were detected by real-time PCR than by cultivation.
Although wet mount proved to be the least sensitive, the results are also highly dependent on the proficiency level of the technician or microbiologist examining the slide. Wet mount microscopy is still the most commonly used method because of its ease, cost and rapidity. In our study, five positive results were found on wet mount and that culture determined as negative. This can imply that the inoculum was not heavy enough in those cases and that possibly not enough time was spent examining the specimens from Diamond’s medium.
The results of our study are comparable with other similar studies in the world, showing greater sensitivity of PCR when compared with conventional methods. Schwebke and Lawing15 found T. vaginalis in 15 (5%) of total 300 men with urethritis when using cultivation of the urine sediment, while PCR was positive in 52 (17%) of them. In the study by Hobbs et al,17 which included 293 men presenting with urethritis symptoms, trichomoniasis has been demonstrated in 38 (12.9%) of the cases using wet mount microscopy and cultivation of the urine sediment and in 51 (17.4%) patients using PCR. Lee et al22 found seven cases of trichomoniasis (21.2%) in 33 samples from men with chronic prostatitis and urethritis, whereas culture failed to detect any of it.
Although T. vaginalis is known as one of the possible causes of urethritis in men for a long time, this infection is rarely considered by clinicians; hence in many instances the disease is neither recognised nor adequately treated.23 Untreated trichomoniasis can result with a myriad of possible conditions. More recently, it has been associated with prostatitis, prostate hyperplasia, prostate cancer and infertility.24–26 Bearing in mind how prevalent it is in a population worldwide, we believe that the current, often ignorant approach to trichomoniasis will have to change—particularly with the increasing availability of new non-invasive, rapid and highly sensitive methods in our diagnostic arsenal.
Key messages
The prevalence of trichomoniasis was higher in men with urethritis syndrome than in men in the asymptomatic group.
Assuming the true positivity of positive samples, real-time PCR showed significantly higher sensitivity in detecting Trichomonas vaginalis when compared with wet mount microscopy and culture.
Hence, there is a need to incorporate PCR in standard diagnostic algorithm to adequately recognise and treat this infection.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online Croatian-questionnaire
- Data supplement 2 - Online English-questionnaire
Abstract in Crotian
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Abstract in Crotian - Online abstract
Footnotes
Handling editor Jackie A Cassell
Contributors MS substantially contributed to the study conception and design, acquisition, analysis and interpretation of data, drafting and writing the article and gave the final approval of the manuscript. EMM participated in the study conception and design, in the acquisition of data, drafting the article and gave the final approval of the manuscript. TM participated in the interpretation of results, structuring, writing and revising the article and gave the final approval of the manuscript. GV contributed to the acquisition of the data, critically revised intellectual content and gave the final approval of the manuscript. GMG chose the directions for data analysis, gave substantial intellectual contribution, participated in the revision of the article and gave the final approval of the manuscript.
Competing interests None.
Ethics approval This study received an ethical approval from the Ethics Committee of the Croatian National Institute of Public Health.
Provenance and peer review Not commissioned; externally peer reviewed.