https://orcid.org/0000-0002-0153-2231
Figures
Abstract
In Thailand, sexually transmitted infections (STIs) persist as a significant public health issue, notwithstanding the affordability of treatments. The primary challenge lies in diagnostic methodologies. According to the Thai National Treatment Guidelines for abnormal vaginal discharge, wet preparation using proportion of white blood cell (WBC) counts and epithelial cell (EC) guides presumptive STI treatment. This study investigated the prevalence of common STI pathogens in sexually active women presenting with abnormal vaginal discharge and WBC > EC under microscopy; and the cost-minimization analysis of this approach. A cross-sectional study was done during July 2021–March 2023 at the Siriraj Female STI Clinic, Bangkok, Thailand. The eligible participants were non-pregnant Thai women aged 18–50 years with the following conditions; being sexually active in prior one year, presenting with abnormal vaginal discharge, having WBC > EC under microscopy, and no allergy to cefixime and azithromycin which were the presumptive treatment in the study. The endocervical swabs were sent for molecular diagnosis of STI pathogens (polymerase chain reaction; PCR). Cost-minimization analysis comparing two approaches, PCR and wet preparation, was done. From an initial 199 participants, 186 were eligible. The average age was 31.1 ± 9.4 years and their sex debut was at 19.7 ± 3.8 years. Around 10% of them, sex partners had STIs. Prevalent STI pathogens included C. trachomatis(20.4%), N. gonorrhoeae(7.0%), M. genitalium(5.9%) and T. vaginalis(3.8%). Presumptive treatment yielded no severe immediate or delayed adverse effects. Using wet preparation as a primary test with presumptive treatment, cost per one case cured was almost three times lower than that of using PCR as a primary test (1250.0 vs 3454.4 Thai Baht). In summary, a quarter of the sexually-active Thai women with abnormal vaginal discharge and WBC > EC under microscopy had either C. trachomatis or N. gonorrhoeae. The use of wet preparation-guided presumptive STI treatment is practical and cost-saving, compared with the PCR approach. Trial registration: Thai Clinical Trial Registry (TCTR20210702001, 2 July 2021).
Citation: Pichailuck C, Sakunborrirak P, Kittiyaowamarn R, Neungton C, Taunwaena P, Girdthep N, et al. (2025) Prevalence of common bacterial STI pathogens and the microscopic diagnostic approach to abnormal vaginal discharge in a tertiary care hospital in Bangkok, Thailand. PLoS One 20(9): e0331668. https://doi.org/10.1371/journal.pone.0331668
Editor: Caroline Watts, The University of Sydney, AUSTRALIA
Received: February 23, 2025; Accepted: August 19, 2025; Published: September 8, 2025
Copyright: © 2025 Pichailuck et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This work was funded by the Research Development, Faculty of Medicine Siriraj Hospital, Mahidol University Grant number R016433015, Fund 3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Abnormal vaginal discharge is a common presenting symptom in gynaecological clinics and often results from vaginal dysbiosis or sexually transmitted infections (STIs) [1–2]. While dysbiosis is more prevalent, STIs exert greater healthcare strain [1]. The World Health Organization (WHO) reported an upsurge in STIs, especially among young people. In 2020, 374 million new STI cases were estimated, with trichomoniasis (41.7%), chlamydial cervicitis (34.5%) and gonorrhoea (21.9%) predominating. This trend incurs significant economic costs due to its subclinical nature, expanding sexual networks, vertical transmission and long-term sequelae [3]. Thailand has witnessed a similar increase in STIs, particularly among 15–24-year-olds [4]. Untreated STIs can cause severe health issues such as pelvic inflammatory disease, chronic pain, infertility and ectopic pregnancy [5]. During pregnancy, they heighten the risk of preterm birth and labour. Many infected individuals are asymptomatic, complicating detection and treatment [6].
Prompt diagnosis and treatment are pivotal for STI control [2]. However, in resource-scarce areas, high-cost molecular diagnostic methods, such as nucleic acid amplification tests, are often prohibitive and lack the ability to produce immediate results. Studies indicate that Gram staining is a viable diagnostic method, linking a white blood cell (WBC) count ≥30/1000x with C. trachomatis infections [7–8], and with an even lower threshold of ≥5/1000x found in a Thai female sex worker cohort [9]. Nevertheless, the limited availability and lack of standardization of Gram staining hinder its broader use [10].
Wet preparation is quicker, more cost-effective, and more suited to all Thai healthcare levels than Gram staining. The method is also covered in medical education and supported by online resources [11]. Wet preparation contains a comparable diagnostic yield for common female reproductive tract infections [2] and, like Gram staining, no STI pathogen grading system [12]. However, wet preparation may appear more influenced by sample preparation technique, subjectivity and durable record. In 2022, the Royal Thai College of Obstetricians and Gynaecologists introduced guidelines for managing abnormal vaginal discharge in reproductive-aged women, recommending same-day nucleic acid amplification testing, bedside wet preparation and a syndromic approach [2]. A higher WBC-to-epithelial cell (EC) ratio, especially WBC ≥ 30/400x, indicates the presence of STI pathogens in sexually active women. The syndromic approach is limited to mobile units due to concerns about antibiotic overprescription.
In general, young people or those with multiple sex partners are considered the population at risk for STI [10]. The present study demonstrated the prevalence of common bacterial STIs in all reproductive-aged women who presented with abnormal vaginal discharge and WBC > EC under microscopy. Additionally, as Thailand has limited resource of molecular diagnostic tool, we assessed the cost-minimization of wet preparation as the primary test for STI treatment initiation, focusing on the prevalence of four common pathogens—C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis.
Materials and methods
Study design and setting
This cross-sectional study was conducted at the Siriraj Female STI Clinic from July 2021 to March 2023. The study was approved by the Siriraj Institutional Review Board (reference Si-1064/2020) and registered with the Thai Clinical Trial Registry (TCTR20210702001, 2 July 2021).
Population
The inclusion criteria were age 18–50 years, having unprotected sexual intercourse in prior one year; no cervical lesions, and WBC > EC under microscopy. Patients with allergy to cefixime and azithromycin were excluded. Those with inconclusive or invalid polymerase chain reaction (PCR) results were withdrawn from the study.
Study protocol and data collection
All eligible participants were explained about the study. Those who agreed to participate filled up the written consent form. They underwent sexual risk behaviour assessments, pelvic examinations with a dry speculum and specimen collection. The specimens were examined using vaginal pH measurement, wet preparation and PCR, which was the gold standard for diagnosing each STI in this study. The PCR detected seven pathogens, including C. trachomatis, N. gonorrhoeae, M. genitalium, M. hominis, U. urealyticum, U. parvum and T. vaginalis. However, current consensus has shown that M. hominis, U. urealyticum and U. parvum are not clinically relevant STIs and should not be treated [13]. However, these pathogens are included in the commercial test package being used in the study. After providing consent, the participants were placed in the lithotomy position, and a vaginal speculum was reinserted for endocervical swab collection. The swab, rotated three times in the cervical os, was stored in modified Minimum Essential Medium at 4–8°C for weekly PCR testing.
In accordance with the Centers for Disease Control and Prevention (CDC) 2021 guidelines [10], presumptive treatment for N. gonorrhoeae and C. trachomatis began immediately in women with likelihood of STI acquisition. Participants with WBC count ≥30/400x were given azithromycin (1 g) and cefixime (800 mg) and monitored for at least 15 minutes for side effects. Those with concurrent trichomoniasis, candidiasis, or bacterial vaginosis received metronidazole (2 g) or fluconazole (200 mg) at enrolment [2].
To prevent reinfection and protect the vaginal ecosystem, the patients were advised against vaginal intercourse until PCR results were available. Delayed side effects were assessed at the two-week follow-up. PCR results guided tailored treatment for non-gonococcal/non-chlamydial infections: metronidazole 2 g for 1 day for trichomoniasis; and for M. genitalium, a 7-day course of doxycycline 100 mg twice daily plus azithromycin 1 g for 1 day, followed by 500 mg for 3 days [2]. STI management included partner notification and treatment for C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium as routine practice [2].
Outcome measurement
Pelvic examination and vaginal pH.
The diagnosis of pelvic inflammatory disease involved assessing cervical motion tenderness, uterine tenderness during palpation, or adnexal tenderness by pressing areas adjacent to the uterus, with any of these signs indicative of pelvic inflammatory disease. Vaginal pH was measured using a pH paper with the scale of 0.5 (MQuant, Germany). It was measured by applying pH paper to discharge on a dry speculum, with a pH ≥ 5 indicating abnormal.
Wet preparation.
In the wet preparation procedure, a cotton swab was used to collect vaginal discharge from the posterior and lateral fornix of participants in the lithotomy position. The swab was then immersed in 1 mL of saline solution for immediate light microscope. At 100x magnification, the presence of T. vaginalis was discerned by a size comparable to that of a WBC and its characteristic jerky motion. The application of 10% potassium hydroxide revealed pseudohyphae, signifying C. albicans, and thus indicative of vaginal candidiasis.
At 400x, the specimen was evaluated for WBC count for at least 3 random fields to retrieve the average number of WBC count as previous evidence linking higher WBC counts with chlamydial infection [7]. (S1 File. Method of wet preparation)
PCR analysis for seven organisms.
This study investigated seven STIs, including C. trachomatis, N. gonorrhoeae, M. genitalium, T. vaginalis, M, hominis, U. urealyticum and U. parvum. Endocervical swabs and their 2 mL of transfer media were stored at 4–8°C before PCR. The swabs were equilibrated to ambient temperature and vortexed. A 200 µL aliquot from each sample was processed for DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) per the manufacturer’s protocol.
The AnyplexTM II STI-7e Detection Kit (Seegene, Seoul, Korea) facilitated multiplex real-time PCR for the pathogens, which was conducted according to the manufacturer’s instructions in a CFX96 thermocycler (Bio-Rad; Hercules, CA, USA). The PCR mixture included 5 μL of DNA, 4x STI-7 TOM and AnyplexTM PCR Mix, giving a total of 20 μL. Thermal cycling involved 4 minutes of UDG activation at 50°C and 15 minutes of pre-denaturation at 95°C. This was followed by 50 cycles of 95°C for 30 seconds, 60°C for 1 minute and 72°C for 30 seconds, with melting temperature analysis from 55°C to 85°C (5 seconds/0.5°C). Process controls were added pre-extraction to ensure DNA extraction and PCR efficacy.
The AnyPlexTM II STI-7e had good agreement for the detection of C. trachomatis and M. genitalium with the standard-of-care diagnostic methods, Cohen’s kappa of 0.87 [95% confidence interval; CI 0.82–0.92] and 0.80 [95%CI 0.74–0.87], respectively. There was lower agreement for the detection of N. gonorrhoeae and T. vaginalis, at 0.37 [95%CI 0.19–0.55] and 0.52 [95%CI 0.25–0.80], respectively [14].
Presumptive treatment and side effects.
All eligible participants received a single dose of azithromycin (250 mg, 4 tablets) and cefixime (100 mg, 8 tablets). The participants were monitored for immediate side effects for at least 15 minutes at the clinic. Delayed side effects were assessed at the 2-week follow-up.
Cost-minimization analysis.
The cost minimization analysis was done after all PCR results were retrieved. All eligible participants had WBC count ≥10/400x; and the prevalence of STI pathogens was not different between WBC count 10–29/400x and ≥30/400x. Therefore, to address the variability in WBC thresholds across guidelines, we calculated the cost per cured patient with a conservative WBC cut-off ≥10/400x, aiming to minimize misdiagnosis risks associated with higher WBC counts. Analysing from a societal perspective, we compared two approaches: (1) ‘presumptive treatment’ (initiating therapy based on WBC > EC under microscopy) and (2) ‘PCR-first’ (delaying treatment until PCR confirmation). Drawing on the 97–98% effectiveness rates reported in other studies, we assumed a 100% efficacy rate [15–16].
The standard presumptive treatment involved the oral administration of cefixime 800 mg for N. gonorrhoeae and azithromycin 1 g for C. trachomatis. Other STI pathogens that must be treated are M. genitalium (doxycycline 100 mg PO BID for 7 days, followed by azithromycin 1 g PO OD for 1 day, then azithromycin 500 mg PO OD for 3 days) and T. vaginalis (metronidazole 2 g PO single dose) [2]. As treatment of M. hominis, U. urealyticum and U. parvum is not recommended, it is excluded from the analysis [13].
The analysis incorporated direct medical costs (diagnostic tests, medications, healthcare services) and direct non-medical expenses (food, travel). Costs were derived from the Standard Cost List for Health Technology Assessment [17], and adjusted to 2022 Thai Baht using the consumer price index. Median reference drug prices were obtained from Thailand’s Ministry of Public Health’s Drug and Medical Supply Information Center (Table 1) [18].
The economic outcomes are presented as total costs and costs per one case cured. Additionally, cost of over-medical prescription and direct cost saved (excluding medication) in presumptive treatment were calculated. The cost of over-medical prescription equals to total drug cost of presumptive treatment minus that of PCR-first approach, and the direct cost saved was calculated using the same manner.
Sample size calculation and statistical analysis.
Descriptive statistics were used to summarize the data as frequencies (n, %), 95% CI, means (SD) and medians (range). Categorical variables were compared using Fisher’s exact test or the chi-squared test, and Student’s t-test or the Wilcoxon rank-sum test was applied to parametric and non-parametric data, respectively. Analyses were performed using Stata Statistical Software, version 12 (StataCorp LLC, College Station, TX, USA).
The sample size was estimated from a previous study of American women, in which 21.8% of those with a gram-stained WBC ≥ 30/400x had chlamydial cervicitis [7]. With an alpha of 0.05 and a margin of error of 0.07, the required sample size for patients with a WBC ≥ 30/400x was 139. All consecutive participants with WBC > EC under microscopy were enrolled during the study period.
Results
Among the 199 potential participants, 10 were excluded due to later report of no sexual activity in the last year, and three were withdrawn due to invalid PCR results. All eligible participants had ≥ 10 WBCs/400x, including 44 patients with WBC 10–29/400x (23.7%) and 142 with WBC ≥ 30/400x (76.3%). (Fig 1) Prevalent STI pathogens included C. trachomatis 38/186 (20.4; 95%CI 14.9–26.9), N. gonorrhoeae 13/186 (7.0; 95%CI 3.8–11.7), M. genitalium 11/186 (5.9; 95%CI 3.0–10.3) and T. vaginalis 7/186 (3.8; 95%CI 1.5–7.6). Prevalence of each detected organism divided by groups of WBC count was demonstrated in Fig 2. Pathogen prevalence did not significantly differ between groups of WBC counts.
From 199 potential participants, ten were excluded due to later report of no sexual activities in the last year and three were withdrawn due to invalid polymerase chain reaction results.
There was no statistical difference between WBC 10-29/400x and WBC ≥ 30/400x except for M. hominis (P 0.007).
The characteristics of the participants were shown in Table 2. The mean age was 31.1 ± 9.4 years, the body mass index was 22.1 ± 4.0 kg/m2, and the age at sexual debut was 19.7 ± 3.8 years. Over half had a bachelor’s degree. Approximately 20% had a history of miscarriage, and a quarter had a history of STIs. Of them, 10% had multiple partners in the last 6–3 months. Of their partners, 17 reported a history of STIs. The characteristics of participants with or without C. trachomatis were comparable whereas those with N. gonorrhoeae were older, started sexual activities at later age and contained a higher prevalence of trichomoniasis. Co-occurrence of C. trachomatis and N. gonorrhoeae with vaginal candidiasis was high, at 15.8% and 23.1%, respectively. (Table 2)
Table 3 compares the costs per cured patient between the presumptive treatment and PCR-first methods. With all treatments assumed to be 100% effective, the cost per cured patient was 2.76 times lower for the microscope-approach with presumptive treatment. The cost of over-medical prescription in presumptive treatment was 20 825.20 Thai Baht. However, the direct cost that could be saved by implementing presumptive treatment was 430 835.31 Thai Baht, compared to the PCR-first approach. Table 4 shows the detailed cost incurred by presumptive treatment and PCR approach.
After the presumptive treatment, two participants experienced immediate nausea without vomiting. Delayed side effects included nausea without vomiting in eleven patients, diarrhoea in three patients, and one headache, with no patient seeking medical advice. The clinical cure and microscopic findings are shown in Table 5.
Discussion
Wet preparation, a simple method requiring only a microscope, glass slide and normal saline solution; can be advocated as an initial diagnostic tool for women with abnormal vaginal discharge in resource-limited settings. Despite the fact that wet preparation appears more influenced by sample preparation technique, subjectivity and durable record, it appears much more practical than other bedside diagnostic tools like Gram staining. The WBC > EC under microscopy resembles WBC count ≥10/400x. Of them, the specimens reveal that 20% had C. trachomatis and 7% had N. gonorrhoeae. Consistent with prior research in Norwegian women [19], this study supports that WBC > EC under microscopy bolsters the possibility of starting presumptive treatment. Importantly, the present study demonstrates its cost-minimization far surpasses that of the PCR-first method, highlighting its value as a primary test in settings with limited resources. Additionally, the clinical cure following this approach was up to 72.2%. This study advocates the use of the Thai National Guidelines [2] to endorse microscope as a presumptive treatment-guided tool when same-day PCR result is not applicable.
Point-of-care STI test (POCT) is the ideal strategy to mitigate STI problems worldwide [20]. Wet preparation is a promising POCT as it fulfills the definition of appropriate POCT by the World Health Organization that includes REASSURED (Real time connectivity; Ease of sample collection; Affordability; Sensitive; Specific; User-friendly; Rapid, Equipment-free; Deliverable) [21]. Rapid real-time PCR or antigen-based tests have been continuously developed [22,23]; however, the diagnostic accuracy compared with the conventional PCR in real-life practice remains far from the expectation [24]. Albeit lack of standardization for diagnosing bacterial STI-related vaginitis [12], WBC > EC under microscopy suggests vaginal and pelvic inflammatory disease-related inflammation. Moreover, wet preparation detects bacterial vaginosis and vaginal candidiasis which are the two most common causes of abnormal vaginal discharge in reproductive-aged women [1].
The current investigation supports previous studies showing that mixed vaginal infections are very common [25]. Vaginal candidiasis, often encountered in gynaecological clinics [1], is diagnosed by clinical symptoms and detected pseudohyphae under microscopy, leading to frequent antifungal use. According to the Thai Guidelines [2], the microscopic picture of acute vaginal candidiasis is EC > WBC whereas WBC > EC ratios indicate bacterial, protozoan or chronic fungal infections [2]. In our study, among those treated for candidiasis, 15.4% (8/52) had C. trachomatis, and 3.8% (2/52) had N. gonorrhoeae. Without molecular tests, such STIs are at risk of being transmitted. Implementing wet preparation as a routine bedside test could promptly address them. On top of that, a two-week posttreatment microscopic review may aid in managing the co-infections [26].
The presumptive treatment aims to cover the common infections and to stop further onward transmission. The present study shows that a quarter of sexually-active Thai women with abnormal vaginal discharge and WBC > EC under microscopy acquiring either C. trachomatis or N. gonorrhoeae. Therefore, when the diagnosis using PCR is not available, in practice, oral medication suits better as the presumptive treatment. Cefixime, an oral third-generation cephalosporin, has been used as an alternative treatment of N. gonorrhoeae with high efficacy at 98% [27], alongside with azithromycin for C. trachomatis at 97% [16]. However, the present study shows much lower clinical cure, at 72.2%. The effectiveness of this presumptive treatment regimen as well as the long-term consequences such as drug resistance should be further studied.
The prevailing presumptive treatment mainly addresses C. trachomatis and N. gonorrhoeae, yet STI panels also cover Mycoplasma spp. and Ureaplasma spp. M. genitalium, now acknowledged as an STI pathogen, is symptomatic in only 10–30% of cases [28]. Although M. hominis, U. urealyticum and U. parvum are often seen as commensals [29,30] and require no treatment [13], they have been associated with bacterial vaginosis [31], which is a dysbiosis connecting to infections in the upper genital tract [32] and to vaginal candidiasis [25]. Moreover, the present study demonstrates a statistically significant association between M. hominis and elevated WBC count. This raises a caution about its inflammation potential. Further research is needed to clarify the association between these infections and vaginal dysbiosis, as well as the consequences of such inflammatory process.
The debate around initiating presumptive treatment has been significant among healthcare providers. This study supports the benefits of presumptive treatment, showing that its costs are significantly lower (by 2.76 times) than those of precise diagnostic tests. Our direct costs, based on the Standard Cost List for Health Technology Assessment [17], indicate public sector averages, with private settings often being higher. In line with the recommendations by the Centers for Disease Control and Prevention [10], the study also noted minimal adverse events from presumptive treatment. In contrast, untreated or delayed treatment in STI patients increases transmission risks due to extensive, unregulated sexual networks. While overuse of antibiotics resulting from a syndromic approach may risk drug resistance and not reduce long-term STI prevalence [33], wet preparation offers a mitigation strategy. Nonetheless, the higher standard of STI care remains our goal by means of enhancing STI surveillance; expanding high-risk group screenings; improving clinical management for symptomatic cases; and promoting affordable, molecular point-of-care tests in Thailand.
A key strength of the study is the introduction of evidence supporting the Thai guideline to use the microscope-guided presumptive treatment. This insight is invaluable for shaping policies in resource-limited nations. The study also highlights the notable rate of co-infections at both initial and follow-up visits, underscoring the need for heightened vigilance in managing vaginitis. The single-centre nature of the study and a small representative group are our limitations, given that STI prevalence can differ regionally. However, the diversity of participants from suburban areas partly compensates for this difference. Another limitation is that there is no control arm to demonstrate the prevalence of STI pathogens in WBC < 10/400x. The baseline prevalence of the two STI pathogens in low-risk population may be required to complement the national policy development.
Conclusion
Among sexually-active Thai women presenting with abnormal vaginal discharge, wet preparation can be an acceptable method to guide presumptive treatment, with almost three- time lower expense than using PCR-based approach. Nonetheless, this approach has pragmatic rather than diagnostic role and may be taken into consideration in resource-constraint settings. Its long-term effects, particularly drug resistance, and real-life effectiveness need to be further studied.
Supporting information
S1 File. QR code for techniques of wet preparation.
https://doi.org/10.1371/journal.pone.0331668.s001
(TIF)
Acknowledgments
The authors express sincere gratitude to the Faculty of Medicine Siriraj Hospital, Mahidol University and the Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital, Mahidol University for facilitating the study.
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