A hospital-based study on the prevalence of trichomoniasis and evaluation of accuracy of various diagnostic techniques.

BACKGROUND: The sexually transmitted infections (STIs) caused by Trichomonas vaginalis have been associated with adverse pregnancy outcomes and increased risk of HIV transmission. Trichomoniasis remains underreported despite being easy to diagnose and treat. Moreover, availability of battery of diagnostic tools causes dilemma on the most appropriate techniques to be used. AIMS AND
OBJECTIVES: The purpose of this study was to determine the prevalence of T. vaginalis and its diagnostic accuracy employing various diagnostic techniques in women presenting with vaginal discharge in gynecological outpatient department (GOPD) of our tertiary care hospital.
MATERIALS AND METHODS: Five vaginal swabs were collected from 204 patients with symptomatic vaginal discharge attending GOPD. Wet mount microscopy, Giemsa and acridine orange staining, culture in Kupferberg media and InPouch™ TV culture system, and polymerase chain reaction (PCR) were performed and compared.
RESULTS: The prevalence of T. vaginalis was 1.96% in the present study. Wet mount microscopy, staining method, and culture detected 1.96% of cases, whereas PCR detected 2.45% of cases.
CONCLUSION: The prevalence of T. vaginalis was <3% among symptomatic vaginal discharge patients from GOPD. Although PCR had a higher detection rate, there was no significant difference in sensitivity and specificity between other diagnostic methods (direct wet mount, Giemsa/acridine orange staining, and InPouch™ TV culture system). Hence, the availability in a particular setting would determine the methods of choice to be used for the diagnosis of T. vaginalis. Copyright:

INTRODUCTION

The sexually transmitted parasitic agent Trichomonas vaginalis has been responsible for an estimated 156 million new cases of trichomoniasis in 2016.[1] The World Health Organization estimates trichomoniasis to account for almost half of all curable sexually transmitted infections (STIs).[23] Worldwide, researchers have reported a prevalence of 1.3%–16.5% of T. vaginalis in reproductive tract infections.[45]

In symptomatic females, the disease is characterized by copious vaginal discharge, severe pruritus, dysuria, dyspareunia, or lower abdominal pain and sometimes with punctate hemorrhagic lesion of the cervix known as “strawberry cervix.” In 25%–50% of women, it can be asymptomatic.[6]

Trichomoniasis is associated with adverse pregnancy outcomes such as preterm rupture of membranes, preterm birth, and low birth weight babies.[2] It can also lead to infertility and pelvic inflammatory disease. An association between T. vaginalis and cervical neoplasia has also been reported.[7] T. vaginalis also increases viral load and transmission of HIV by local accumulation of HIV-infected cells or HIV-susceptible cells such as lymphocytes and monocytes.[2] In some circumstances, it may have a major impact on the epidemiologic dynamics of HIV and other STIs. Hence, it is important to diagnose correctly and treat it.

The prevalence study based on only wet mount microscopy has been done in this part of the state more than a decade back. Limited studies have compared the available laboratory tool for the diagnosis of T. vaginalis in India. Hence, the purpose of this study was to determine the prevalence of T. vaginalis and evaluate its diagnostic accuracy employing various diagnostic techniques in women with vaginal discharge.

MATERIALS AND METHODS

This cross-sectional study was conducted after clearance from the institutional ethics committee and written informed consent form from patients. The study group comprised 204 sexually active women of age group ≥18 years attending the gynecological outpatient department (GOPD) of our tertiary care hospital with self-reported symptoms of vaginal discharge between January 2018 and April 2019.

Women who have recently douched or used spermicidal agent within 72 h before testing, menstruating women at the time of examination, and women who had taken a course of antibiotics within the last 3 weeks were excluded from the study.

Five vaginal swabs were taken from the posterior vaginal fornix under aseptic condition. The first swab was used for direct microscopy. The second swab was used for the preparation of smear for staining procedure. Third and fourth swabs were used for culture in Kupferberg and InPouch™ TV, respectively. The fifth swab was used for polymerase chain reaction (PCR) analysis that was stored in extraction buffer at −20°C till DNA extraction followed by PCR.

A direct wet mount was prepared from the first vaginal swab using 0.85% physiological saline and was examined in the GOPD under ×40 objective of light microscope.

Of the two smears subsequently prepared from the second swab, one was fixed with methanol and subjected to Giemsa stain (HiMedia Laboratory, India) diluted 1:20 with phosphate buffer saline, pH 7.2 for 20 min, and examined under ×100 for trophozoite. The second smear after fixing with methanol was stained with freshly prepared (0.5% in DW) acridine orange stain (HiMedia Laboratory, India) for 2 min and examined under a fluorescent microscope with a 470–490 nm filter with ×40 objective.

The third vaginal swab was placed into the Kupferberg culture medium (HiMedia, India) in a screw-capped test tube and incubated under anaerobic condition at 37°C. The cultures were examined microscopically till 1 week for the presence of motile T. vaginalis.

The fourth vaginal swab was used to inoculate InPouch™ TV culture media, which was incubated at 37°C and examined for motile T. vaginalis similar to previous culture test but directly through the pouch.

DNA was extracted from the fifth vaginal swab using extraction kit (AmpliSens®, Russia) by following the manufacturer's instruction and subsequently amplified using oligonucleotide primers TVK3 and TVK7, which independently targets T. vaginalis genome.[8] Culture-positive T. vaginalis and DNA-free distilled water were used as positive and negative controls, respectively.

Forward primer – TVK3, 5'AT TGT CGA ACA TTG GTC TTA CCC TC3'

Reverse primer – TVK7, 5'TCT GTG CCG TCT TCA AGT ATG C3'.

The amplification program included initial one cycle of predenaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 90°C for 60 s, annealing at 60°C for 30 s, and extension at 70°C for 120 s, followed by one cycle of final extension at 72°C for 7 min.[8]

1.5% agarose gel electrophoresis with ethidium bromide staining was used to visualize the 300 bp amplicon of PCR products using ultraviolet illumination.

RESULTS

In wet mount microscopy, the trophozoites of T. vaginalis were identified by their size (7–23 μm × 5–12 μm), oval shape, and characteristic twitching motility under ×40 objective of light microscope [Figure 1].

Figure 1

Arrow shows pear shaped trophozoite of Trichomonas vaginalis with four anterior flagella and fifth one incorporated in undulating membrane on direct wet mount preparation under ×40 objective of light microscope

In Giemsa staining, T. vaginalis trophozoite was identified by its pyriform shape, size, and internal structure under ×100 objective of light microscope [Figure 2].

Figure 2

Giemsa staining of Trichomonas vaginalis (arrow) on ×100 objective of light microscope

The trophozoites of T. vaginalis were characteristic brick red color with a yellowish green nucleus in acridine orange-stained slide under ×40 objective of fluorescent microscope [Figure 3].

Figure 3

Arrow shows brick red colored trophozoite of T. vaginalis with yellow colored nucleus on ×40 objective of fluorescent microscope

Figure 4 shows the presence of 300 bp band on gel after PCR amplification indicated positive result for T. vaginalis.

Figure 4

Polymerase chain reaction for Trichomonas vaginalis.Lane 1, 2 and 3: 300 base pair position of Trichomonas vaginalis-positive sample. Lane 4: 100–3000 bp ladder. Lane 5: Negative control. Lane 6: Sample negative for Trichomonas vaginalis

Total four different patients' samples (4/204, 1.96%) were positive by microscopy (direct wet mount, Giemsa, and acridine orange), culture method (Kupferberg media and InPouch™ TV), and PCR. However, one more patient's sample detected T. vaginalis by PCR only (5/204), which was negative by microscopy and culture techniques, thus increasing the positivity to 2.45%. The rest all 199 samples were negative by all detection techniques. Table 1 details the total number of samples which were positive for T. vaginalis by different detection techniques.

Table 1

Positivity of cases detected by different diagnostic methods

Methods	Number of positive cases	Number of negative cases
Microscopy
Wet mount microscopy	4	200
Giemsa staining	4	200
Acridine orange staining	4	200
Culture
Kupferberg media	4	200
InPouch™ TV	4	200
Molecular
PCR	5	199

PCR: Polymerase chain reaction

Taking culture isolation in Kupferberg media as gold standard, the burden of T. vaginalis in sexually active women in the present study was 1.96%.

For the calculation of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), on 204 samples, all the tests (direct wet mount, Giemsa staining, acridine orange staining, InPouch™ TV, and PCR) were compared with gold standard (Kupferberg media). True positive, true negative, false positive, and false negative were calculated as per Table 2. Table 3 shows the sensitivity, specificity, PPV, and NPVof different detection techniques.

Table 2

True positive, true negative, false positive, and false negative of different techniques for the calculation of sensitivity, specificity, positive predictive value, and negative predictive value

Diagnostic Tests	Results	Culture in Kupferberg media (Gold standard)
		Positive	Negative
Wet mount	Positive	4	0
Giemsa staining	Negative	0	200
Acridine orange staining
In PouchTM TV
PCR	Positive	4	1
	Negative	0	199

Table 3

Sensitivity, specificity, positive predictive value, and negative predictive value of different diagnostic techniques for Trichomonas vaginalis keeping culture in Kupferberg media as gold standard

Diagnistic methods	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
Wet mount microscopy	100	100	100	100
Giemsa staining
Acridine orange staining
InPouch™ TV
PCR	100	99.5	80	100

PPV: Positive predictive value, NPV: Negative predictive value, PCR: Polymerase chain reaction

The McNemar's test was used for the comparison of microscopy (direct wet mount, Giemsa staining, and acridine orange staining), InPouch™ TV culture system, and PCR with culture in Kupferberg media (gold standard), where no significant difference between the methods for the detection of T. vaginalis was noted (P = 1).

DISCUSSION

In the present study, the burden of T. vaginalis in symptomatic sexually active females presenting to GOPD was 1.96% as compared to higher prevalence (4.7%) reported from the STI clinic of the same institute eight years back.[9] Similar higher prevalence was also reported from Chandigarh (4.28%) 12 years back.[10] Even higher prevalence between 6.1% and 8.6% has been reported from Punjab[11] and some parts of South India (Tamil Nadu and Karnataka) between 2005 and 2013.[121314] Lower prevalence comparable to the present study has also been reported from Bangaluru, Karnataka(2.1%),[15] Gujarat (2.8%),[16] and Maharashtra (3.3%).[17]

Studies from the Southeast Asia, Europe, Africa, and America done in 2008 show the prevalence between 5.6% and 22%.[18]

All these data show a varying prevalence in both developing and developed countries. In countries where the occurrence has decreased, an enhanced awareness concerning STIs with added condom use may be accountable for the drop in cases.

Out of total five positive cases of T. vaginalis, the current study detects 1.96% (4/204) by culture (Kupferberg media and InPouch™ TV) and microscopy (direct wet mount, Giemsa, and acridine orange stain). However, PCR (using TVK3 and TVK7) detected additional one case increasing the positivity to 2.45%.

Although a study from Aligarh[19] reported similar positivity rate by acridine orange staining, nonetheless, they could detect only 66% positive cases by wet mount microscopy as in a study from Karnataka[20] with a resultant lower sensitivity (60%). Studies from Uganda (25%)[21] and Egypt (33.3%)[22] have even lower sensitivity of wet mount microscopy. A delay in transport of the sample with resultant reduced motility of the organism can compromise with sensitivity of this technique. The fact that direct wet mount microscopy was done in the GOPD room itself by trained personnel could account for the increased sensitivity in the present study. Currently, wet mount microscopy is the most rapid and widely used method of diagnosis from vaginal secretions, especially in resource-constraint settings. However, a negative test cannot rule out trichomoniasis as sensitivity may vary from as low as 25% to even 100%.

Dry vaginal smear stained with Giemsa or acridine orange can compensate for the delay in the transport without loss of reliability in the diagnosis, provided that the smear has been adequately fixed to preserve the morphology of the parasite. The sensitivity and specificity of both the staining techniques in the current study are comparable to culture. However, the cost of fluorescent microscope and loss of fluorescence with the passage of time are its major limiting factor. Giemsa stain overcomes this pitfall. In contrast to 100% sensitivity of Giemsa staining in the present study, reports from Egypt (33.33%)[22] and Aligarh (80%)[23] give a lower sensitivity in their account. The possibility of timely fixation of smear preserving the morphology of T. vaginalis could be the reason for the elevated sensitivity in the current study.

Gold standard for the diagnosis of trichomoniasis is isolation in culture as it is a reliable and sensitive method. The culture requires an incubator with constant electric supply, anaerobic condition of incubation, and expensive culture media. Moreover, preparation of the media is cumbersome with a large number of ingredients and has a short shelf life. Furthermore, culture cannot detect nonviable organism and one may have to wait 3–7 days for the results. All these issues limit its use in the resource-poor settings. InPouch™ TV culture system is available readymade and has unique advantages of ease of inoculation by gynecologist, thus alleviating the need for the transportation of the specimen along with longer shelf life of 1 year at room temperature. Moreover, there is no need to provide separate anaerobic condition of incubation and can be directly visualized under a microscope to detect the growth of T. vaginalis with sensitivity of <10 organism/ml. However, the major limiting factor is its cost. In the present study, InPouch™ TV culture system had diagnostic accuracy similar to the isolation in Kupferberg media. Hence, this system may be a better alternative in setting where the cost is not a constraint. Although sensitivity of InPouch™ TV culture system as reported from Karnataka was 73.33%, a higher sensitivity of the same in the present study could be due to inoculation of the system at sample collection site room itself, thus avoiding delay in transport leading to loss of viability.[20]

A study reported in 2003 from Belgium, comparing different PCR assays for detection of T. vaginalis from vaginal swabs, showed sensitivity and specificity of PCR using the TVK3/TVK7 primer set as 88% and 97.3%, respectively, similar to the present study. However, in their same study, sensitivity and specificity of PCR with primer set TVA5/TVA6 were 63.9% and 99.7%, respectively.[8] The oligonucleotide primers TVK3 and TVK7 used for PCR amplification help in specially amplifying a 300 bp sequence from the repetitive DNA in T. vaginalis genome. Hence, the amplification is highly specific and does not amplify human DNA, other organism found in genitourinary tract or other Trichomonas species. In addition, PCR has the ability to detect nonviable or defective T. vaginalis. Low level of extracted DNA and presence of nonspecific inhibitors in sample that could inhibit the amplification process resulting in 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%). Similar results have been reported by Patil et al.,[20] Wendel et al.,[24] and Caliendo et al.[25] The ability of PCR to amplify nonviable or defective T. vaginalis could account for increased positivity in the current study as compared to culture or microscopy.

The present study determined the prevalence of T. vaginalis only in symptomatic patients presenting to GOPD. This does not estimate the true burden of T. vaginalis as approximately 25%–50% of T. vaginalis patients are known to be asymptomatic.[5] Besides, other patients (both males and females) presenting to STI clinic were not taken into consideration in the present study. Moreover, high-risk cases (such as sex workers) were also not investigated in the present study.

CONCLUSION

A higher prevalence has been detected by PCR and hence would be the diagnosis of choice where feasible. However, time taken by PCR would exceed that by wet mount microscopy which is a rapid, inexpensive screening technique with good sensitivity and specificity when performed in GOPD room itself. The InPouch™ TV culture system is expensive and not easily available. The present study also states that there was no significant difference in Giemsa and acridine orange staining when compared with Kupferberg media for the detection of T. vaginalis. Hence, the availability in a particular setting would determine the methods of choice to be used for diagnosis of T. vaginalis.

Financial support and sponsorship

Intramural research grant of University College of Medical Sciences and Guru Teg Bahadur Hospital.

Conflicts of interest

There are no conflicts of interest.