# Prevalence and bio-clinical characterisation of Trichomonas vaginalis infection in a large cohort of French patients screened using a molecular syndromic panel for sexually transmitted infections

Lya Hamet, Florence Robert-Gangneux, Clément Cazala, Gabriel Auger, Jean-Pierre Gangneux

► To cite this version:

Lya Hamet, Florence Robert-Gangneux, Clément Cazala, Gabriel Auger, Jean-Pierre Gangneux. Prevalence and bio-clinical characterisation of Trichomonas vaginalis infection in a large cohort of French patients screened using a molecular syndromic panel for sexually transmitted infections. Journal of Infection, 2025, 91 (4), pp.106610. 10.1016/j.jinf.2025.106610. hal-05296129

HAL Id: hal-05296129

https://hal.science/hal-05296129v1

Submitted on 15 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
performance is influenced by pre-analytical criteria (type of sample, shipment duration) and is operator-dependent. The fresh mount must be performed rapidly after sampling, within a maximum of 2 h, in order to avoid false-negative results, because the presence of motile forms on direct examination makes it easier to identify the protozoa. Fixing the sample followed by staining is also possible, using May Grünwald Giemsa staining, for example, and increases the sensitivity of detection. Sample culture has long been considered the reference technique for diagnosing urogenital trichomoniasis because of its greater sensitivity than microscopy, which can be as high as 95% with several culture media described, the most common being Diamond, modified Diamond or Roiron® media.¹⁰,¹¹

In recent years, molecular detection of parasite DNA using commercial or in-house PCR assays has been developed to improve the diagnosis of trichomoniasis. Nucleic acid amplification tests (NAATs) were shown to be much more sensitive than microscopy or culture methods.¹⁰,¹¹ Of note, molecular techniques may also detect DNA from non-viable organisms, which raises the question of specificity. The number of false-negative results was reduced overall by NAAT techniques, which are now routine methods in many laboratories for diagnosing *T. vaginalis* infection. In addition, the use of syndromic multiplex PCR allows screening a wide range of patients and co-infections with other STIs (*Chlamydia trachomatis*, *Neisseria gonorrhoeae*, *Mycoplasma genitalium...*) on a single sample. However, these methods require equipment and reagents that are often expensive, which limits their use in low-income countries where the estimated incidence of trichomoniasis is the highest.

One hundred fifty-six million new cases per year were estimated by the World Health Organisation (WHO) in 2020 in people aged between 15 and 49, with around a third of infections occurring in Africa and the Americas.¹³ *Trichomonas vaginalis* has a higher prevalence than the combined prevalence of the 3 other curable STIs (C. trachomatis, N. gonorrhoeae and Treponema pallidum) in women, making it the leading agent responsible for non-viral STIs worldwide (5.3% in women and 0.6% in men).¹⁴ An average prevalence of trichomoniasis of 1.7% (CI95% = 1.3 - 2.4) was reported in a French multicentre study with a positivity rate ranging from 0 to 4.4%.¹⁵ However, these epidemiological data on trichomoniasis are probably underestimated, because at that time the diagnosis mainly relied on microscopy and because of the high proportion of asymptomatic cases.

## Objectives

This work aimed to prospectively study the epidemiology of urogenital trichomoniasis at the Rennes University Hospital, as well as associated bacterial STIs. In addition, the specificity of the Allplex® STI Essential syndromic panel (Seegene) for *T. vaginalis* detection was assessed compared to in-house PCR. Confirmation of infection by microscopic examination (fresh mount) was not possible because of pre-analytical delay between sampling and PCR results.

## Materials and methods

### *Study population*

We prospectively analysed 41,507 samples from 30,190 patients sent for STI screening between January 2020 and March 2024 at the Rennes University Hospital. When a sample was positive for *T. vaginalis*, additional data were collected. Demographic (date of birth, age, sex), clinical (clinical signs and/or reason for consultation, anti-infective treatment prescribed), and biological data (type of sample, HIV status, co-infections with other microorganisms, PCR C~T~) were extracted from the laboratory's computer system and the patient's record. The study was carried out following the Declaration of Helsinki and was approved by the local ethics committee

(institutional review board/institutional ethics committee [Comité d'éthique CHU Rennes N° 24-155]).

### *Syndromic molecular PCR panel*

The Allplex® STI Essential molecular panel (Seegene Inc., Seoul, South Korea) used at Rennes University Hospital targets *T. vaginalis*, *C. trachomatis*, *N. gonorrhoeae*, *M. genitalium*, *M. hominis*, *Ureaplasma parvum*, and *U. urealyticum*. The samples analysed were swab samples discharged in transport medium (vaginal swabs, endocervical swabs, throat swabs, anorectal swabs) or urine samples taken in a sterile tube without additives. The samples collected were extracted using the MICROLAB® STARlet automated system (Hamilton Company, Reno, NV, USA) and amplified by real-time PCR on a CFX96 automated system (Bio-Rad, Marnes-la-Coquette, France). The raw PCR curves obtained were analysed using Seegene® viewer software.

### *DNA Extraction and in-house simplex PCR assays*

A hundred and sixty positive samples for *T. vaginalis* with the syndromic molecular PCR panel were stored at -20 °C for further confirmation using simplex in-house qPCR assays targeting *T. vaginalis*.

### *Extraction*

Briefly, 200 µL of swab suspension was extracted on the EZ1® Advanced XL device (Qiagen, Courtaboeuf, France) using the EZ1® DSP Virus Kit (Qiagen), and 3 µL of internal control (IC) (Virocult, Eurogentec qPCR control, Eurogentec, Angers, France) were also added before the extraction process. DNA was then stored at -20 °C until amplification using two real-time quantitative PCR assays.

### *TaqMan real-time PCR*

Real-time PCR (qPCR) was performed using BTUB9/2 primers and a probe targeting *T. vaginalis* beta-tubulin gene to amplify a 112 base-pair region, as previously described.¹⁶ The primer and probe sequences were as follows: TV-BTUB 9: 5'-CAT-TGA-TAA-CGA-AGC-TCT-TTA-CGA-T-3'; TV-BTUB 2: 5'-GCA-TGT-TGT-GCC-GGA-CAT-AAC-CAT-3' and TV-Betatub-P, probe: 5'-6-FAM-ACG-GCG-ATC-TTA-ACC-ACC-TTG-TTT- C-BHQ1-3'.

Amplification was performed on 5 µL extracted DNA in a 25 µL reaction volume containing: TaqMan Universal PCR Master Mix 2X (Applied Biosystems, ThermoFisher, Waltham, Massachusetts, USA), forward and reverse primers and probe at a final 0.5 µM and 0.2 µM concentrations, respectively, IC Mix (Eurogentec). The amplification reaction was performed using an Applied Biosystems QuantStudio 5 device (ThermoFisher) and the following program: 2 min at 50 °C, then 10 min at 95 °C, followed by 45 cycles at 95 °C for 15 s, and 60 °C for 1 min.

### *SYBR Green RT-qPCR*

A second qPCR was carried out on samples testing negative with the simplex TaqMan qPCR. This second qPCR targeted a conserved region of the *T. vaginalis* 18S rDNA, using primers TVK3 and TVK7 (5'-AAA-GAT-GGG-TGT-TTT-AAG-CTA-GAT-AAG-G-3' and 5'-TCT-GTG-CCG-TCT-TCA-AGT-ATG-G-3'), as previously described.¹⁷

Real-time PCR was performed in a 25 µL reaction volume of the following composition: Power up SYBR Green PCR Master Mix 2X (Applied Biosystems), forward and reverse primers at a final 0.5 µM concentration, and 5 µL of extracted DNA. The amplification reaction was performed using an Applied Biosystems QuantStudio 5 device (ThermoFisher) using the same cycling program as above, followed by a dissociation program. Melting curves were analysed to verify the specificity of amplification (T~M~ = 77.8 °C).
<table><caption>**Table 1**
Distribution of *T. vaginalis* positive samples according to sampling site and sex.</caption><thead><tr><th>Sample type</th><th>Women</th><th>Men</th><th>Unknown</th><th>Total</th></tr></thead><tbody><tr><td>Vaginal</td><td>156</td><td>-</td><td>9</td><td>165</td></tr><tr><td>Endocervix</td><td>121</td><td>-</td><td>4</td><td>125</td></tr><tr><td>Urine</td><td>20</td><td>10</td><td>5</td><td>35</td></tr><tr><td>Anorectal</td><td>4</td><td>-</td><td>-</td><td>4</td></tr><tr><td>Throat</td><td>1</td><td>2</td><td>1</td><td>4</td></tr><tr><td>Urethral</td><td>-</td><td>1</td><td>-</td><td>1</td></tr><tr><td>Ocular</td><td>1</td><td>-</td><td>-</td><td>1</td></tr><tr><td>Surgical</td><td>1</td><td>-</td><td>-</td><td>1</td></tr><tr><td>Pus</td><td>1</td><td>-</td><td>-</td><td>1</td></tr><tr><td>Total</td><td>305</td><td>13</td><td>19</td><td>337</td></tr></tbody></table>

*T. vaginalis* DNA extracted from a microscopic fresh-mount positive sample was used as a positive control for the amplification reaction with both methods (TaqMan and SYBR Green).

## Results

### Prevalence and age

From January 2020 to March 2024, 41,507 samples for STIs screening (45.8% from women, 28% from men and 26.2% anonymised samples) were analysed using the AllPlex STI Essential panel. Almost half (44.8%, n=18,586) of the samples were positive for at least one of the microorganisms included in the panel. With regard to the *T. vaginalis* target, 0.8% of all samples analysed were positive, representing a total of 337 samples (282 patients; 0.93%), of which 305 samples were from women (90.5%), 13 from men (3.9%), and 19 were anonymised (5.6%). Various sample types were used, mostly vaginal and endocervical swabs (Table 1). In the majority of cases, the parasite load was high, with a $C_T$ below 30 in 85% of samples.

Young adults between 20 and 40 years old represented the vast majority of positive patients in this cohort (n=261/318). The highest rate of positivity was observed in patients between 20 and 24, i.e. 1.6% (n=89/5489), whereas the number of cases after 45 years was anecdotal (0,4%; n=16/3687). Only one *T. vaginalis*-positive patient was over 60(67) at the time of screening. The median age of infection was 28 in women and 33 in men. Prevalence was ten times lower in men than in women on average, regardless of age (Table 2).

### Prevalence of co-infection with other STIs

Among positive samples, a minority (n= 44/337, 13%) were positive for *T. vaginalis* alone, of which 6/44 (13.6%) patients were HIV-positive. Besides, 87% of samples were positive with at least one other bacteria targeted by the molecular panel, mainly saprophytic *Mycoplasma* of the genitourinary tract with a clear predominance of both *U. parvum* (n= 208/337, 62%) and *M. hominis* (n= 196/337, 58%). A total of 82/337 samples (24%) were co-infected with at least one pathogen such as *C. trachomatis* (n= 57, 17%) and/or *M. genitalium* (n= 31, 9.2%) and/or *N. gonorrhoeae* (n= 12, 3.6%) (Fig. 1).

<table><caption>**Table 2**
Proportion of *T. vaginalis* positivity by age and sex. Anonymised samples were not included in these data.</caption><thead><tr><th rowspan="2">Sex</th><th colspan="7">Age group (years)</th></tr><tr><th>&lt; 20</th><th>20 - 24</th><th>25 - 29</th><th>30 - 34</th><th>35 - 39</th><th>40 - 44</th><th>45 - 49</th><th>≥ 50</th></tr></thead><tbody><tr><td>Women % (n/N)</td><td>0.9 (22/2392)</td><td>2.0 (88/4420)</td><td>1.8 (67/3750)</td><td>1.1 (35/3323)</td><td>1.9 (50/2634)</td><td>2.1 (29/1411)</td><td>1.9 (10/513)</td><td>0.7 (4/559)</td></tr><tr><td>Men % (n/N)</td><td>0.11 (1/874)</td><td>0.09 (1/1075)</td><td>0.12 (2/1617)</td><td>0.14 (3/2129)</td><td>0.17 (3/1813)</td><td>0.08 (1/1307)</td><td>0.13 (1/743)</td><td>0.05 (1/1872)</td></tr><tr><td>Total % (n/N)</td><td>0.7 (23/3266)</td><td>1.6 (89/5495)</td><td>1.3 (69/5367)</td><td>0.7 (38/5452)</td><td>1.2 (53/4447)</td><td>1.1 (30/2718)</td><td>0.9 (11/1256)</td><td>0.2 (5/2431)</td></tr></tbody></table>

n: Number of positive for *T. vaginalis*; N: Number of patients tested

## Symptoms and treatment

Of the 282 patients (337 samples), 239 (85%) cases could be clinically documented: 46% (n=111/239) were symptomatic, of which 46% (n=51/111) had typical symptoms suggestive of trichomoniasis (abnormal leucorrhoea, vulvar pruritus and/or dysuria). In a further 50/111 cases, patients presented pelvic pain and/or metrorrhagia only, and in 10/111 cases other symptoms were present (dyspareunia, cervicitis only, abdominal or lumbar pain, obstetric problems). Of the 128 asymptomatic cases, 107 patients were screened at a pre-abortion consultation or following unprotected sex. Two patients experienced obstetric complications, corresponding to a spontaneous miscarriage and a premature rupture of the membranes, leading to a late miscarriage.

Over 65% (n=186/282) of patients were treated, mostly with secnidazole (2 gr a day as a single dose) or metronidazole (2 gr a day as a single dose or 500 mg twice a day for 7 or 14 days). Twenty-one patients were not treated at all. No information on treatment was available for the other 75 cases.

Among clinically documented patients, 94% (47/50) of those with clinical signs suggestive of trichomoniasis, 92% (47/51) of those with non-specific signs, and 88% (89/101) of asymptomatic patients were treated (Table 3).

### Specificity of the syndromic multiplex PCR panel for *T. vaginalis* detection

One hundred and sixty samples (½ of positive samples) were collected to analyse the specificity of the Allplex® multiplex PCR versus simplex PCR using specific primer pairs for the diagnosis of trichomoniasis. These samples corresponded to 100 vaginal swabs, 35 endocervical swabs, 19 urine samples, 3 anorectal swabs, 2 throat swabs and 1 genital swab without more information.

Sample number 29 (vaginal swab) was positive for *T. vaginalis* on direct examination and was therefore used as a positive control for the amplification reactions.

The qPCR targeting the beta-tubulin gene confirmed the Tv detection in 135 out of 160 (84.4%) samples. The remaining 25 samples (2 doubtful samples with late CTs at 39 and 38 after 1:10 dilution, and 23 negative ones) were further tested with the SYBR Green qPCR targeting Tv 18S rDNA. Seventeen of the 25 samples displayed specific amplification using this technique. The two doubtful results using the TaqMan method were amplified with the SYBR Green method with $C_T$ values of 34 and 30, respectively. Finally, amplification of the samples by simplex PCRs showed an excellent concordance rate of 95% (n= 152/160) with the multiplex PCR (Fig. 2).

## Discussion

Sexually transmitted infections due to *C. trachomatis*, *N. gonorrhoeae*, *Treponema pallidum* and *T. vaginalis* are responsible for one million new cases a day worldwide, corresponding to an estimated prevalence of 376.4 million new cases in 2016. Re-infections and co-infections are frequent, and these four STIs are curable; thus, access to diagnosis is a cornerstone of patient management.¹⁴ In the meta-analysis carried out by the WHO between 2009 and 2016, the global
<figure><img src="image_2.png" /><figcaption>Fig. 1. Flow chart of *T. vaginalis* positive samples and associated co-infections, using the Allplex STI Essential panel for STI screening.</figcaption></figure>

prevalence of urogenital trichomoniasis was 5.3% in women and 0.6% in men, compared with 1.6% and 0.2% in Europe, respectively.¹⁴

In France, a decree dated 2024 provides for free no-prescription screening in medical laboratories for 3 out of 4 curable STIs (chlamydia, gonorrhoea, syphilis, but not trichomoniasis) and hepatitis B, for patients under the age of 26.¹⁸ Until now, such free anonymous diagnosis has been available only for HIV screening.¹⁹ The possibility of screening on request and without direct payment makes it possible to improve the clinical and therapeutic management of these infections, to prevent their transmission and to reduce their incidence. Here, our objective was to estimate the prevalence of *T. vaginalis* in a French cohort of more than 30,000 individuals (40,000 samples) at risk for STIs.

In our study, the observed prevalence of *T. vaginalis* was 0.93% which confirms the low prevalence reported in a previous French study performed 10 years ago (1.7%).¹⁵ In Europe, the reported prevalences are also in a low range, as reported in Germany (0.19%) and Spain (0.57%),²⁰ the Netherlands (0.6–1.5%),²¹,²² Portugal (1.0%),²³ Belgium (1.03%) and Wales (1.64%),²⁰ In contrast, prevalence is higher in the United States (8.7%),²⁴ Iran (4.30%)²⁵ and Turkey (5.94%).²⁶

*T. vaginalis*-positive patients from our cohort were young adults aged below 40 in 89% of cases, with the highest general prevalence (1.6%) in young adults (20–24 years). Women represented more than 90% of the cases, with a mean prevalence from 20 to 49 years of 1.7 ± 0.2%. A slight increase in prevalence was observed in women

<table><caption>**Table 3**
Treatment of patients who tested positive for *T. vaginalis*, depending on whether or not the symptoms were suggestive of trichomoniasis at the time of sampling (n=207).</caption><thead><tr><th></th><th>Treated patient</th><th>Non treated</th><th>Unknown</th><th>Total</th></tr></thead><tbody><tr><td><strong>Typical symptoms:</strong> Leucorrhea, pruritus, dysuria</td><td>47</td><td>3</td><td>1</td><td>51</td></tr><tr><td><strong>Non-specific symptoms:</strong> Abdomino-pelvic pain, metrorrhagia, dyspareunia, cervicitis, obstetrical issue</td><td>47</td><td>4</td><td>9</td><td>60</td></tr><tr><td><strong>Asymptomatic</strong></td><td>89</td><td>12</td><td>27</td><td>128</td></tr><tr><td><strong>Unknown</strong></td><td>3</td><td>2</td><td>38</td><td>43</td></tr><tr><td><strong>Total</strong></td><td>186</td><td>21</td><td>75</td><td>282</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Flow chart of simplex PCR performed on *T. vaginalis* positive samples with the Allplex STI Essential (Seegene) panel. Two different pairs of primers were used, depending on the method (TaqMan and SYBR Green).</figcaption></figure>

between 35 to 39 (1.9%) and 40 to 44 (2.1%). This increase in prevalence for women after 35 was also observed in the study of Li and al. (2025), who reported the highest trichomoniasis prevalence in women aged 30 to 50,²⁷ and by Ginocchio and al. (2012), trichomoniasis reaching a maximum prevalence in women after 40.²⁴ In men, we observed a peak at 0.17% in the [35–39] age group. In the literature, it has been reported that *T. vaginalis* was more prevalent in men over the age of 30,²⁸ and Pereyre and al. showed in their study a more marked increase in the proportion of *T. vaginalis* in men, peaking at 4.3% in the [35–44] age group.¹⁵ Altogether, we can therefore assume that *T. vaginalis* infection is the lowest among the four pathogenic STIs, with a peak in positivity between 20 and 40, and a probable further decrease in older age groups in general, probably linked to a lower risk-taking sexual practices. Another explanation for the decrease in general prevalence observed in our study may be that middle-aged individuals are less likely to be screened, even if STIs risk remains similar.

However, this disease must not be underestimated, as more than half of the cases were asymptomatic (54%) in our cohort. Among symptomatic patients, almost half had signs suggestive of urogenital trichomoniasis, such as the presence of pathological leucorrhoea, genital pruritus and/or functional urinary signs. In the previous French study, nearly 80% of symptomatic patients had typical signs of trichomoniasis, while 61.5% of cases were asymptomatic.¹⁵ In the present study, a positive multiplex PCR for *T. vaginalis* led to prescription in almost 90% of cases (n=186/207). Asymptomatic carriers were treated in 88% of cases, showing the added value of multiplex PCR screening in detecting and treating the many asymptomatic cases of *T. vaginalis* infection in order to stop the dissemination. Furthermore, treatment of all positive cases, including non-sympathetic, helps to avoid complications that have an impact on sexual and reproductive health, particularly for women.²⁹ Most patients of

this series would not have benefited from treatment if they had not been screened with a multiplex PCR, as *T. vaginalis* is very fragile and frequently missed if swabs are not transmitted quickly to the lab.

*Trichomonas vaginalis* is the only known parasite responsible for STIs, but is frequently associated with other microorganisms.¹⁴,¹⁵,²⁴,³⁰,³¹ We observed in this work a high rate of co-infections (n=293/337, 87%), of which 28% were co-infections with pathogenic bacteria (17% *C. trachomatis*, 9.2% *M. genitalium* and 3.6% *N. gonorrhoeae*). Co-infection rate reached 100% (n=17/17) in a small series of patients positive for *T. vaginalis* using the same Allplex® STI Essential multiplex PCR, mainly with saprophytic *Mycoplasma*,³⁰ whereas this proportion was 46.2% (n=18/39, 3.2% *Chlamydia* and 5.1% *Gonococcus*) for Pereyre and al.,¹⁵ and 24.2% (n=161/663, 17.3% *Chlamydia* and 9.6% *Gonococcus*) for Ginocchio and al. using the Aptima TV Assay multiplex PCR which does not detect *M. genitalium*.²⁴ The proportion of co-infections is therefore dependent on the panel used, since not all existing micro-organisms are detected with all molecular panels. However, the presence of any STI is a sign of high-risk sexual behaviour and should encourage a broader search for other STIs, particularly using syndromic multiplex PCR panels. Of note, *Mycoplasma hominis* is reported as an endobiont of *T. vaginalis*. However, our molecular approach with a multiplex PCR cannot differentiate co-infections from symbiosis.³²,³³

The remaining question was to check the specificity of *T. vaginalis* detection using newly developed syndromic multiplex PCR panels. Here, we showed an excellent concordance rate of 95% with simplex-specific PCRs. Eight discordant results were found to be positive by Allplex STI and negative by specific PCR. Most of these samples had late CTs in multiplex PCR and could correspond to either false positives with multiplex PCR or to false negatives in simplex PCR. Although unlikely, such false negativity of specific PCR could be linked to genetic polymorphisms at the primer and/or probe binding
sites preventing hybridisation or to parasitic DNA degradation by the freezing/thawing cycle during storage.

A surprising result was observed with one throat swab positive with the TVK3/7 primer pair and after a tenfold dilution of the DNA extract. The specificity of *T. vaginalis* in this sample is debatable. The Allplex° STI method found a relatively late CT at 33, and the specific beta-tubulin primers amplified poorly. This could suggest the presence of PCR inhibitors or another *Trichomonas* species such as *T. tenax*, a parasite of the oral cavity.³⁴ In a recent publication, Chesnay and al. showed that sequencing the 5.8S rRNA or ITS1/2 regions increases the specificity of the *Trichomonas* species identification and may be relevant for throat and anorectal swabs to understand this difference.³⁵ Here, we did not perform sequencing. Another limitation of this study is the use of a syndromic panel limited to bacteria and *T. vaginalis*. It is of interest to also estimate co-infections with viruses, for example with the newly marketed Vivalytic® STI Assay.³⁶ This new panel developed by Randox Laboratories Ltd includes 10 microorganisms with two bacteria (*Treponema pallidum* and *Hae-mophilus ducreyi*) and two viruses (*Herpes simplex virus* 1 and 2), which are not included in the Seegene Allplex° STI Essential panel.³⁶

## Conclusion

STIs are a major public health problem due to their worldwide prevalence, their contagiousness and their long-term impact on fertility. Trichomoniasis is the fourth prevalent STI for which a curative treatment is available and non-costly. In our study, nearly 300 patients were diagnosed over 3 years among more than 30,000 patients screened for STIs. More than half of them were asymptomatic, and trichomoniasis was not raised as a first-line diagnosis by clinicians. Therefore, patients were diagnosed by chance because of the use of syndromic PCR. Syndromic multiplex PCR assays such as the Allplex° STI Essential panel dramatically improve the detection of the fragile parasite *T. vaginalis*, in symptomatic and asymptomatic cases, with an excellent specificity. This syndromic approach provides a much better description of the epidemiology of trichomoniasis than the historical microscopic technique, and also emphasises the high rate of co-infections with other micro-organisms responsible for STIs.

## Research support

This research received no external financial or non-financial support.

## Relationships

There are no additional relationships to disclose.

## Patents and intellectual property

There are no patents to disclose.

## Other activities

There are no additional activities to disclose.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jinf.2025.106610.

## References

1. Petrin D, Delgaty K, Bhatt R, Garber G. *Clinical and microbiological aspects of Trichomonas vaginalis*. *Clin Microbiol Rev* 1998;11(2):300-17.

2. Schwebke JR, Burgess D. *Trichomoniasis*. *Clin Microbiol Rev* 2004;17(4):794-803.

3. Van Gerwen OT, Craig-Kuhn MC, Jones AT, Schroeder JA, Deaver J, Buekens P, et al. *Trichomoniasis and adverse birth outcomes: a systematic review and meta-analysis*. *BJOG* 2021;128(12):1907-15.

4. Silver BJ, Guy RJ, Kaldor JM, Jamil MS, Rumbold AR. *Trichomonas vaginalis* as a cause of perinatal morbidity: a systematic review and meta-analysis. *Sex Transm Dis* 2014;41(6):369-76.

5. Moodley P, Wilkinson D, Connolly C, Moodley J, Sturm AW. *Trichomonas vaginalis* is associated with pelvic inflammatory disease in women infected with human immunodeficiency virus. *Clin Infect Dis* 2002;34(4):519-22.

6. Wiringa AE, Ness RB, Darville T, Beigi RH, Haggerty CL. *Trichomonas vaginalis*, endometritis and sequelae among women with clinically suspected pelvic inflammatory disease. *Sex Transm Infect* 2020;96(6):436-8.

7. Haute Autorité de Santé (HAS). Prise en charge du patient atteint d'infection à Trichomonas vaginalis; 2024. https://www.has-sante.fr/jcms/p_3562494/fr/prise-en-charge-du-patient-atteint-d-infection-a-trichomonas-vaginalis.

8. World Health Organisation. Recommendations for the treatment of Trichomonas vaginalis, Mycoplasma genitalium, Candida albicans, bacterial vaginosis and human papillomavirus (nongenital warts) [Internet]. Geneva: World Health Organization; 2024 (WHO Guidelines Approved by the Guidelines Review Committee). Available on: [http://www.ncbi.nlm.nih.gov/books/NBK605758/].

9. Desoubeaux G, Chesnay A. *Diagnostic de la trichomonose à Trichomonas vaginalis*. *Rev Francoph Lab* 2021;2021(530):71-5.

10. Ibáñez-Escribano A, Nogal-Ruiz JJ. The past, present, and future in the diagnosis of a neglected sexually transmitted infection: trichomoniasis. *Pathogens* 2024;13(2):126.

11. Hobbs MM, Seña AC. Modern diagnosis of Trichomonas vaginalis infection. *Sex Transm Infect* 2013;89(6):434-8.

12. Herbst de Cortina S, Bristow CC, Joseph Davey D, Klausner JD. A systematic review of point of care testing for Chlamydia trachomatis, Neisseria gonorrhoeae, and *Trichomonas vaginalis*. *Infect Dis Obstet Gynecol* 2016;**2016**:4386127.

13. World Health Organisation. *Trichomoniasis* [Internet]. 2023. Available on: [https://www.who.int/fr/news-room/fact-sheets/detail/trichomoniasis].

14. Rowley J, Vander Hoorn S, Korenromp E, Low N, Unemo M, Abu-Raddad LJ, et al. *Chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates*, 2016. *Bull World Health Organ* 2019;97(8):548-562P.

15. Pereyre S, Laurier Nadalié C, Bébéar C, investigator group. *Mycoplasma genitalium and Trichomonas vaginalis* in France: a point prevalence study in people screened for sexually transmitted diseases. *Clin Microbiol Infect* 2017;23(2):122.e1-7.

16. Madico G, Quinn TC, Rompalo A, McKee KT, Gaydos CA. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. *J Clin Microbiol* 1998;36(11):3205-10.

17. Kengne P, Veas F, Vidal N, Rey JL, Cuny G. *Trichomonas vaginalis*: repeated DNA target for highly sensitive and specific polymerase chain reaction diagnosis. *Cell Mol Biol* 1994;40(6):819-31.

18. Vautrin Catherine, Le Maire Bruno, Valletoux Frédéric. Arrêté du 8 juillet 2024 fixant la liste des infections sexuellement transmissibles dépistées à la demande du patient en laboratoire de biologie médicale et les modalités de ces dépistages [Internet]; 2024. TSSP2417443A juill 9, 2024 p. Texte n°53. Available on: [https://www.legifrance.gouv.fr/eli/arrete/2024/7/8/TSSP2417443A/jo/texte].

19. Article L162-13-2, Modifié par LOI n°2022-1616 du 23 décembre 2022 - art. 30. Code de la sécurité sociale. Sect. Section 3: Directeurs de laboratoires; 2022.

20. Perry MD, Jones S, Bertram A, de Salazar A, Barrientos-Durán A, Schiettekatte G, et al. The prevalence of Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) at testing centers in Belgium, Germany, Spain, and the UK using the cobas TV/MG molecular assay. *Eur J Clin Microbiol Infect Dis* 2023;42(1):43-52.

21. Geelen TH, Hoebe CJPA, Dirks A, Dukers-Muijers NHTM, van Bergen JEAM, Wolffs PFG. Low positivity rate after systematic screening for Trichomonas vaginalis in three patient cohorts from general practitioners, STI clinics and a national population-based chlamydia screening study. *Sex Transm Infect* 2013;89(6):532-4.

22. de Jong AS, Rahamat-Langendoen JC, van Alphen P, Hilt N, van Herk C, Pont S, et al. Large two-centre study into the prevalence of Mycoplasma genitalium and *Trichomonas vaginalis* in the Netherlands. *Int J STD AIDS* 2016;27(10):856-60.

23. Silva J, Cerqueira F, Teixeira AL, Campainha R, Amorim J, Medeiros R. Prevalence of Neisseria gonorrhoeae and *Trichomonas vaginalis* in Portuguese women of child-bearing age. *J Obstet Gynaecol* 2021;**41**(2):254-8.

24. Ginocchio CC, Chapin K, Smith JS, Aslanzadeh J, Snook J, Hill CS, et al. Prevalence of *Trichomonas vaginalis* and coinfection with *Chlamydia trachomatis* and Neisseria gonorrhoeae in the United States as determined by the Aptima Trichomonas vaginalis nucleic acid amplification assay. *J Clin Microbiol* 2012;50(8):2601-8.

25. Moghadamizad Z, Khalili JY, Olfatifar M, Badri M, Khazaei S. The prevalence of *Trichomonas vaginalis* infection among the female population of Iran: a systematic review and meta-analysis. *Int Health 2024*;16(3):240-51.

26. Beyhan YE. A systematic review of *Trichomonas vaginalis* in Turkey from 2002 to 2020. *Acta Trop* 2021;**221**:105995.
27. Li S, Xu J, Ru S, Hu C, Liu C, Sun X, et al. Prevalence of Trichomonas vaginalis among women in the Chinese population: a systematic review and meta-analysis. *Trop Med Infect Dis* 2025; **10**(4):113.

28. Horner P, Blee K, O'Mahony C, Muir P, Evans C, Radcliffe K, et al. 2015 UK National Guideline on the management of non-gonococcal urethritis. *Int J STD AIDS* 2016; **27**(2):85-96.

29. Van Gerwen OT, Muzny CA, Marrazzo JM. Sexually transmitted infections and female reproductive health. *Nat Microbiol* 2022; **7**(8):1116-26.

30. Moodley C, Tootla H, Amien I, Engel ME. Evaluating the utility of the Allplex STI Essential Assay to determine the occurrence of urogenital sexually transmitted infections among symptomatic and asymptomatic patients in Cape Town, South Africa. *PLoS One* 2023; **18**(11):e0292534.

31. Occhipinti S, Incognito GG, Palumbo M. The influence of the vaginal ecosystem on vaginitis, bacterial vaginosis, and sexually transmitted diseases: an epidemiological study and literature review. *Arch Gynecol Obstet* 2024; **311**:347-53.

32. Ibáñez-Escribano A, Nogal-Ruiz JJ, Escario JA, Ponce-Gordo F. Biomolecular characterization of Trichomonas vaginalis from Spain: evaluating genetic correlation with drug resistance and endobionts. *Biology* 2025; **14**(6):618.

33. Fichorova R, Fraga J, Rappelli P, Fiori PL. Trichomonas vaginalis infection in syphilis with Trichomonasvirus and Mycoplasma. *Res Microbiol* 2017; **168**(9-10):882-91.

34. Maritz JM, Land KM, Carlton JM, Hirt RP. What is the importance of zoonotic trichomonads for human health? *Trends Parasitol* 2014; **30**(7):333-41.

35. Chesnay A, Pastuszka A, Richard L, Beby-Defaux A, Cateau E, Le Brun C, et al. Multiplex PCR assay targeting Trichomonas vaginalis: need for biological evaluation and interpretation. *Diagn Microbiol Infect Dis* 2022; **104**(4):115808.

36. Bolaric B, Begovac J, Planinic A. Evaluation of a multiplex real-time PCR assay for the diagnosis of sexually-transmitted infections. *Mol Exp Biol Med* 2022; **4**(1):43-6.