|Year : 2023 | Volume
| Issue : 1 | Page : 40-44
Prevalence of Trichomonas vaginalis by polymerase chain reaction-based molecular method among symptomatic women from Northern India
Rajneesh Dadwal1, Nandita Sharma2, Rimjhim Kanaujia2, Sakshi Malhotra3, Hemant Chaudhry2, Shivali Rathore2, Aastha Saini4, Rashmi Bagga5, Abhishek Mewara6, Sumeeta Khurana6, Rakesh Yadav2, Sunil Sethi2
1 Clinical Microbiology and Bioactive Screening Laboratory, CSIR-IMTECH, Chandigarh, India
2 Department of Medical Microbiology, PGIMER, New Delhi, India
3 AIIMS, New Delhi, India
4 Department of Pulmonary Medicine, PGIMER, New Delhi, India
5 Department of OBS and Gynaecology, PGIMER, New Delhi, India
6 Department of Medical Parasitology, PGIMER, New Delhi, India
|Date of Submission||21-Feb-2022|
|Date of Decision||21-Jul-2022|
|Date of Acceptance||04-Mar-2023|
|Date of Web Publication||06-Jun-2023|
Dr. Sunil Sethi
Department of Medical Microbiology, PGIMER, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Trichomoniasis remains one of the most common sexually transmitted infections, which is curable. To prevent complications and transmission, prompt and correct diagnosis is essential to treat Trichomonas vaginalis. The present study was done to evaluate polymerase chain reaction (PCR) with other conventional techniques for the diagnosis of T. vaginalis infection and determine the prevalence of T. vaginalis in women with vaginal discharge based on PCR assay. Methods: Vaginal swabs were collected by the trained health-care professional using FLOQSwabs™ (Copan, Italy) during routine pelvic examinations among 1974 symptomatic females. The wet microscopy, culture, and PCR were performed. Results: The sensitivity of wet mount and culture in comparison to PCR was 60.87% and 56.52%, respectively. The kappa inter-rater agreement of T. vaginalis PCR showed substantial agreement with wet mount microscopy (κ = 0.742) and culture (κ = 0.707). The PCR detected an additional 17 cases that were missed by conventional techniques. Discussion: The study highlights the importance of PCR for T. vaginalis screening among symptomatic females.
Keywords: Diagnosis, polymerase chain reaction, Trichomonas vaginalis, trichomoniasis
|How to cite this article:|
Dadwal R, Sharma N, Kanaujia R, Malhotra S, Chaudhry H, Rathore S, Saini A, Bagga R, Mewara A, Khurana S, Yadav R, Sethi S. Prevalence of Trichomonas vaginalis by polymerase chain reaction-based molecular method among symptomatic women from Northern India. Indian J Sex Transm Dis 2023;44:40-4
|How to cite this URL:|
Dadwal R, Sharma N, Kanaujia R, Malhotra S, Chaudhry H, Rathore S, Saini A, Bagga R, Mewara A, Khurana S, Yadav R, Sethi S. Prevalence of Trichomonas vaginalis by polymerase chain reaction-based molecular method among symptomatic women from Northern India. Indian J Sex Transm Dis [serial online] 2023 [cited 2023 Sep 30];44:40-4. Available from: https://ijstd.org/text.asp?2023/44/1/40/378137
| Introduction|| |
Among common sexually transmitted infections (STI), trichomoniasis which is caused by the parasitic protozoan Trichomonas vaginalis remains one of the curable STIs. The estimates of the World Health Organization account for nearly half of all curable STIs as trichomoniasis. Worldwide, the prevalence ranging from 1.3% to 16.5% has been reported., According to the 2016 global prevalence data of T. vaginalis, an incidence of 156.0 million cases was reported. The adults in the age group of 15–49 years were most affected, with an incidence of 5.3% and 0.6% in females and males, respectively.
T. vaginalis infection is asymptomatic in 25%–50% of the cases. However, symptomatic females present with clinical features such as copious purulent vaginal discharge, pruritis, burning micturition, dyspareunia, lower abdominal pain, and rarely with a punctate hemorrhagic lesion of the cervix known as “strawberry cervix.” In males symptoms such as urethritis, prostatitis, balanitis, and epididymitis are seen. If untreated, it leads to complications such as infertility and pelvic inflammatory disease., In pregnant females, adverse outcomes such as preterm rupture of membranes, preterm birth, and low birth weight babies are also described. The association of T. vaginalis with other STIs and cervical neoplasia has been implicated. T. vaginalis infection causes changes in the local vaginal and cervical microbiome causing the accumulation of HIV-infected cells such as lymphocytes and monocytes which leads to increased transmission of HIV.
To prevent complications and transmission, prompt and correct diagnosis is essential to treat T. vaginalis. The syndromic approach to treating STIs is limited by the fact that most of the patients are asymptomatic. Furthermore, the overlapping of clinical symptoms of T. vaginalis and other STIs makes a diagnosis based on clinical symptoms unreliable. Traditionally, wet mount microscopy is used to diagnose trichomoniasis. The delay in sample transportation leads to low sensitivity and nearly 50% of the cases can be missed. The culture techniques are more sensitive but are time-consuming, cumbersome, and require expensive culture media. In comparison to wet microscopy, nucleic acid amplification tests have shown higher sensitivity for T. vaginalis detection.
In India, the prevalence has been evaluated based on only wet mount microscopy. The present study was done to evaluate polymerase chain reaction (PCR) with other conventional techniques for the diagnosis of T. vaginalis infection and determine the prevalence of T. vaginalis in women with vaginal discharge based on PCR assay.
| Methods|| |
Study design and recruitment
The present prospective study was conducted from January 2017 to December 2018 at the regional STI reference, research, and training center which caters to 14 peripheral centers in Chandigarh. A total of 1974 clinical samples were collected from females (16–65 years of age) with vaginitis. Females presenting with chief complaints of genitourinary symptoms such as discharge, burning micturition, itching, lower abdominal pain, and infertility were enrolled in this study. Pregnant and menstruating females were excluded from the study.
Vaginal swabs were collected by the trained health-care professional using FLOQSwabs™ (Copan, Italy) during routine pelvic examinations. Two swabs were obtained from each patient. The study was approved by the Institutional Ethics Committees, PGIMER, Chandigarh (Ethics approval no. INT/IEC/2019/00222,2), and informed consent from all participants was obtained.
Extraction of genomic DNA
One vaginal swab was used to prepare wet mount and culture inoculation. The wet mount was reported within 4 h of sample collection for the presence of motile trichomonads by the trained microbiologist. Additionally, a Gram stain was also prepared from the same swab for the presence of yeast and bacterial vaginosis (BV) (Nugent's scoring). The second swab was squeezed in 1 ml normal saline, and aliquots were made. These samples were used for DNA extraction, and the remaining aliquots were stored at −20°C.
DNA extraction and Trichomonas vaginalis conventional polymerase chain reaction
The DNA extraction was performed according to the previously published protocol by Ong and Rivera using Chelex 100 (Sigma-Aldrich, St Louis, MO, USA). The tvk3 and tvk7 primers were used as the targets specifically to amplify a 261 bp sequence of the 18S SS-rRNA gene segments of T. vaginalis. The conventional PCR was performed as per the published protocol.
Trichomonas vaginalis culture
Clinical samples of T. vaginalis were cultured in the Diamond's trypticase-yeast extract-maltose medium which was supplemented with 10% horse serum (Gibco, Grand Island, NY, USA), streptomycin (250 μg/mL), penicillin (250 U/mL), and amphotericin B (50 μg/mL) (Thermo Fisher Scientific, Waltham, MA, USA).
Data analysis and statistics
The data were entered into the Statistical Package for the Social Sciences (SPSS) 26.0 for Mac OS (SPSS, Inc., Chicago, IL, USA). Continuous variables were compared using the Mann–Whitney U-test or Student's t-test as appropriate. Categorical variables were compared using the Chi-square test or Fisher's exact test, as appropriate. P < 0.05 was used to analyze epidemiological data.
| Results|| |
Epidemiological characteristics of Trichomonas vaginalis
In total, 1974 samples from female patients were included in this study, with 65.2% (1325) and 31.9% (647) in the years 2017 and 2018, respectively. The mean age of the study population was 31.58 years (range: 16–65). The age distribution is described in [Figure 1]. The highest number of positive cases belonged to the age group of 31–40 years (n = 23) followed by 21–30 years (n = 14). Among all samples, 46 were T. vaginalis-positive samples, yielding a positive rate of 2.34%.
|Figure 1: Age distribution of Trichomonas vaginalis infection among all patients|
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Overall, vaginal discharge (68.1%, n = 1384) was the most common clinical presentation. The presence of infertility, itching, and lower abdominal pain was present in 13.9% (n = 282), 3.6% (n = 73), 2.33% (n = 47), and 0.7% (n = 15) of the patients, respectively. Nearly 11.1% (n = 225) of the females were asymptomatic. Among 46 T. vaginalis PCR-positive patients, vaginal discharge was the most common clinical presentation (87%, n = 40). The presence of infertility and itching was seen in 2.2% (n = 1) of the patients. The presence of vaginal discharge was observed to be significantly associated with T. vaginalis infection [P = 0.013, [Table 1]] Concomitant Neisseria gonorrhoeae was present in 4.3% (2, P = 0.026) while BV was associated in 30.4% (14, P = 0.000). Concomitant syphilis was present in 6.5% (n = 3), and candidiasis was observed in 8.6% (n = 4) of the patients.
|Table 1: Clinical complaints used in relation to polymerase chain reaction results|
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The sensitivity of wet mount and culture in comparison to PCR was 60.87% and 56.52%, respectively. The specificity of both wet mount and culture was >99%. The results of 28 microscopy-positive cases were observed to be nonconflicting to/with the results of PCR. In the 1946 microscopy-negative cases, 18 were found to be positive by PCR [Table 2]. The kappa inter-rater agreement of T. vaginalis PCR showed substantial agreement with wet mount microscopy (κ = 0.742) [Table 2]. The kappa inter-rater agreement of T. vaginalis PCR with culture was observed to be 0.707. The nomogram shown in [Figure 2] displays the probability that a patient has a T. vaginalis infection after diagnosis by all three methods. The blue line indicates the positive likelihood ratio (LR+) for the positive test which was 0.39, 0.44, and 0.00 for wet mount, culture, and PCR, respectively. The red line indicates the negative LR for negative test results which was: >1000 for all methods.
|Figure 2: Nomogram of (a) wet mount; (b) culture; and (c) polymerase chain reaction|
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| Discussion|| |
In the current study, T. vaginalis was detected more in the adult age group (21–40 years). This was similar to a study by Muthusamy et al., where T. vaginalis were detected more frequently in females in the age group of 20–39 years. The prevalence of trichomoniasis was found to be 2.34% using the PCR method as the gold standard. A similar burden (1.96%) of T. vaginalis was reported among sexually active symptomatic females. Lower prevalence (1.96%–2.8%) comparable to the present study has also been reported from Bengaluru, Karnataka, and Gujarat.,, However, higher prevalence ranging from 4.28% to 8.6% has also been reported in a few studies from Chandigarh, Punjab, Tamil Nadu, and Karnataka.,,, All these data display varying prevalence in both developing and developed countries. The use of different patient selection criteria and methods of trichomoniasis diagnosis can be the key difference in defining the prevalence in various studies. In countries where the incidence has decreased, increased awareness about STIs among people and prompt management in case of syndromic approaches may be accountable.
The presence of discharge was observed in 87.0% of the T. vaginalis-positive population and was found to be significantly associated with T. vaginalis patients (P < 0.05). Madhivanan et al. also reported abnormal vaginal discharge (37%) in most of the patients followed by genital itching and burning sensation in 18% and 16% of the females, respectively.
The diagnosis of trichomoniasis should not be performed solely based on clinical symptoms due to overlapping presentation with other STIs. Furthermore, many patients with infection are asymptomatic and classical symptoms such as the strawberry cervix and discharge are present in 2% and 12% of the patients, respectively. A wet mount is a simple and rapid method for trichomoniasis diagnosis. A minimum concentration of 104 organisms/ml in the sample is required for microscopic diagnosis. However, it should be performed within 10 min of the sample collection as the motility of the organism reduces with time. In the present study, the microscopy (direct wet mount) and culture were positive in 1.46% (29/1974) and 1.31% (26/1974) of the patients, respectively. However, PCR detected additional 17 cases, increasing the positivity to 2.34%. The sensitivity of wet mount was observed to be 60% in the current study, which is comparable to other studies.,,,
Culture requires 300–500 viable organisms/ml in the sample and anaerobic condition for incubation for positivity. Media available for the cultivation of T. vaginalis are expensive and have a short shelf-life, and the technique itself is cumbersome. It requires days to weeks for a positive test result by culture. In a study by Cevahir et al., T. vaginalis infection was diagnosed in 10.5% and 11% of the females by culture in Diamond's medium and PCR, respectively. Furthermore, the culture results require a minimum of 3–7 days. All these issues limit its use in resource-poor settings.
Thus, molecular techniques play a role in the diagnosis. A study from Belgium revealed comparable sensitivity and specificity of 88% and 97.3%, respectively, by PCR using the TVK3/TVK7 primer set as in the present study. However, a decrease in the sensitivity, i.e., 63.9%, was noted when TVA5/TVA6 primer sets were used. The TVK3 and TVK7 primers especially amplify a 300 bp sequence from the repetitive DNA in the T. vaginalis genome. A low level of extracted DNA and the presence of nonspecific inhibitors in the sample that could inhibit the amplification process resulting in a false-negative test are the limiting factors for PCR. In the current study, PCR positivity was higher (2.45%) as compared to culture or microscopy (1.96%). The ability of PCR to amplify nonviable or defective T. vaginalis could account for increased positivity as compared to culture or microscopy.,,
We observed a significant association of T. vaginalis infection in females with BV. This is crucial as females with BV are ignored in treatment guidelines. Madhivanan et al. also observed a significant association of BV (19.4% vs. 2.7%; P < 0.0001) with T. vaginalis infection. In a recent meta-analysis, a nearly two-fold higher risk for acquiring T. vaginalis infection was observed in patients with BV infection. Lactobacillus species produce substances with antimicrobial properties (hydrogen peroxide, lactic acid, and bacteriocin-like substances) that may play a key role in inhibiting the growth of cervicovaginal pathogens. Bacteria frequently associated with BV produce sialidases and mucinases, damage genital epithelia, and disrupt innate immunity, compromising the physical and immune barriers to infection.
Our study had few limitations. The present study determined the prevalence of T. vaginalis only in symptomatic patients. This does not estimate the true burden of T. vaginalis as approximately two-thirds of patients are known to be asymptomatic.
The public health implication of T. vaginalis should be reconsidered as it is an easily treatable STI. The high-risk population should be focused, and strategies to test and treat these populations should be tailored. There is a need for further evaluation and implementation of molecular techniques to diagnose trichomoniasis, particularly in settings where young females seek health care.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]