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. 2024 Jul 23;48(3):624–629. doi: 10.1007/s12639-024-01703-0

Detection of Trichomonas vaginalis by microscopy and molecular methods in women referred to health centers in Tabriz, Northwest Iran

Gholamreza Barzgar 1,#, Ehsan Ahmadpour 2,#, Reza Ahmadi 3,✉, Roghayeh Norouzi 4, Abolghasem Siyadatpanah 5, Mohammad Hasan Kohansal 6,✉
PMCID: PMC11319573  PMID: 39145365

Abstract

Trichomoniasis is a parasitic disease that affects the human reproductive and urinary systems, representing a substantial non-viral sexually transmitted infection worldwide. Given its impact on reproductive health, and the limited available information on the prevalence of Trichomonas vaginalis, this study aimed to evaluate the prevalence of T. vaginalis among women referred to health centers in Tabriz, Northwest Iran. Study was conducted on 448 suspicious women who attended to 29Bahman hospital in Tabriz, Northwest Iran, during September 2020 to September 2021. Demographic data were collected according to the study protocol. Vaginal discharges were obtained using sterile swabs, and the prevalence of T. vaginalis was determined using Papanicolauo staining and PCR method. Among the 448 cases studied, 48 (10.7%) samples were suspected as a T. vaginalis infection, while 4 (0.89%) confirmed using the PCR method. The mean age of infected individuals was 41.7 ± 9.4 years. No statistical correlation was observed between inflammation, method of contraception and infection (p = 0.8). The present study revealed a relatively low prevalence of T. vaginalis infection within the study population. Additionally, the utilization of the PCR method can be beneficial in confirming suspected samples.

Keywords: Trichomoniasis, PCR, Papanicolauo stain, Prevalence

Introduction

Trichomoniasis is a parasitic infection caused by the flagellated protozoan Trichomonas vaginalis (T. vaginalis). It is one of the most common non-viral sexually transmitted infections (STIs) worldwide among women and men. While men are often asymptomatic carriers, some may experience dysuria and discharge. In women, the infection varies from asymptomatic to severe and reported during the reproductive years (Newman et al. 2015; Aquino et al. 2020; Zhang et al. 2022). Recent studies have shown that approximately 6.5% of women have asymptomatic infection. However, symptoms include a malodorous, purulent discharge, with the serious symptoms such as infertility, premature rupture of placental membranes, premature delivery, low-birth-weight infants, and neonatal death have been reported (Meites et al. 2015). Furthermore, numerous studies have linked T. vaginalis infection to important and costly health outcomes such as human immunodeficiency virus (HIV). To mitigate the complications of HIV, particularly in regions where T. vaginalis is prevalent, it is essential to control T. vaginalis (Yang et al. 2018; Masha et al. 2019; Rowley et al. 2019; Ebrahimi et al. 2023). The World Health Organization (WHO) estimated 156 million cases of Trichomonas vaginalis infections worldwide, constituting almost half of the total global incidence of STIs among adults aged 15 to 49 years (Rowley et al. 2019). Several studies also have been conducted in Iran and the prevalence rate of infection in different age groups of women reported 0.5% to about 39% (Taghavi et al. 2014; Hezarjaribi et al. 2015; Dehghan Haghighi et al. 2019).

High prevalence of trichomoniasis has been associated with population density, migration to suburban areas with poor socio-economic and cultural conditions, and high-risk behaviours among adolescents (Hezarjaribi et al. 2015; Siyadatpanah et al. 2023). Diagnosing trichomoniasis based solely on symptoms can lead to misdiagnosis, as some individuals may be asymptomatic or their symptoms may be confused with other STDs (Huppert et al. 2007). Hence, laboratory investigation is important for accurate diagnosis and proper treatment of trichomoniasis (Al-Ardi 2021). The diagnosis of T. vaginalis conducted with different methods including wet mount, staining, parasite culture, polymerase chain reaction (PCR), and antigen–antibody rapid screening (Matini et al. 2014; Momeni et al. 2015; Spotin et al. 2016; Van Der Pol 2016). Despite the numerous studies conducted in different regions of Iran, little information about the prevalence of T. vaginalis in northwest of Iran has been reported. The current study aimed to evaluate the prevalence of T. vaginalis among women referred to 29Bahman hospital in Tabriz, Northwest Iran.

Materials and methods

Sample collection

In the present study, 448 vaginal swabs were collected during September 2020 to September 2021 from women showing suspicious signs of T. vaginalis at the 29Bahman Hospital of Tabriz, Northwest Iran (Fig. 1). The swabs were obtained with the aid of a disposable vaginal speculum and sterile swabs. Written consent was provided to each participant for sample collection. Demographic information was self-reported by the participants. All patients provided an informed consent, and the study design was approved by the Ethics Committee of Tabriz University of Medical Sciences, Tabriz, Iran (IR.TBZMED.REC.1398.359).

Fig. 1.

Fig. 1

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Tabriz province location on the map of Iran

Papanicolauo staining and microscopy examination

The collected specimens were transferred to the department of parasitology and examined using a Pap-prep cytology kit (Ilia Tak Kimia Sahand, Iran). A prepared smear was initially fixed, followed by staining according to the Papanicolaou staining procedure. Subsequently, an experienced laboratory technician conducted a microscopic examination, thoroughly scrutinizing the prepared slide (Lara-Torre et al. 2003).

DNA extraction and PCR

The total DNA of T. vaginalis was directly extracted from positive slides using the YTA genomic DNA extraction tissue kit (Cat No. YT9030, YTA, Iran) according to the manufacturer’s instructions. A PCR test was performed using a T. vaginalis-specific primer set (TVK3-TVK7). Each PCR reaction tube contained 5 µl DNA, 1 µl forward primer, 1 µl reverse primer, 10 µl PCR mastermix, and 3 µl DDW. The PCR conditions were as follows: an initiation step at 94˚C for 5 min, followed by 30 cycles at 94˚C for 30 s, 52˚C for 30 s, and 72˚C for 30 s. A final extension step at 72˚C was then conducted for 5 min. The PCR primer set F: 5-ATTGTCGAACATTGGTCTTACCCTC-3 and R: 5-TCTGTGCCGTCTTCAAGTATGC-3 amplified the 261 bp (bp) products. Positive control consisted of DNA extracted from a clinical isolate of T. vaginalis grown in vitro, and negative control included nuclease-free water. Additionally, a 100 kb ladder was used as a size marker (Spotin et al. 2016).

Statistical analysis

Data was recorded and analyzed with the SPSS v.18 software (SPSS Inc., Chicago, ILL, USA) using Mann-Whitney and Chi-square tests. The p value < 0.05 was considered as significant.

Results

Out of the symptomatic women referred to the health center, 48 cases were initially diagnosed with suspected T. vaginalis infection through staining (Fig. 2). The infection was confirmed in 4 cases after utilizing the molecular method (Fig. 3; Table 1). All of the infected individuals were married, with a mean age of 41.7 ± 9.4 years. The findings regarding contraceptive methods indicated that 11.6%, 11.8%, and 6.47% of the subjects used condoms, oral tablets, and intrauterine devices (IUDs), respectively. Additionally, 11.6% of the referred women showed a high rate of inflammation (Table 2). A statistical analysis revealed no significant correlation between inflammation, method of contraception, and parasitological results (p = 0.8).

Fig. 2.

Fig. 2

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(A) T. vaginalis in direct microscopy 40X in urine sample; (B) T. vaginalis in papanicolauo staining 100X in Pap smear microscopic

Fig. 3.

Fig. 3

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Agarose gel electrophoresis of TVK3-TVK7 PCR products; Line1 100 bp ladder, Line 2 positive control, line 3 negative control and Line 4–7 amplification product of patients

Table 1.

The results of the methods used in the diagnosis of individuals suspected of T. Vaginalis

Method Positive (%) Negative (%) Total
Papanicolauo staining 48 (10.71) 400 (89.29) 448
PCR 4 (0.89) 444 (99.11) 448
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Table 2.

The results of the investigated factors in individuals suspected of T. Vaginalis

Contraception methods Inflammation Factor responsible for inflammation Age groups
None Contraceptive drugs Natural Condom IUD Tubal ligation None Mild Moderate High Trichomoniasis Bacteria Fungus None-infectious agent
- 19 94 24 5 2 17 85 77 22 14 63 32 62 23–34
- 26 57 27 18 33 22 61 57 17 16 35 49 78 35–46
76 8 29 1 6 23 20 31 26 13 18 32 15 34 47–59
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Discussion

There are various microbial agents associated with women’s vaginal discharge, with trichomoniasis being the most common sexually transmitted disease (STD) worldwide. The global prevalence of trichomoniasis has been estimated to range between 5% and 74% (Johnston and Mabey 2008; Korycińska et al. 2017). In Iran, in a systematic review and meta-analysis study, the prevalence rate of trichomoniasis in different age groups has been estimated to be between 8% and 38.8% (Hezarjaribi et al. 2015). In the present study, the mean age of infected individuals was 41.7 ± 9.4 years. The age-specific distribution of Trichomonas infection is important, and in the current study, women aged 47–59 years had the highest infection rate. This finding is consistent with the results of several previous studies (Matini et al. 2012; Nazari et al. 2015; Patel et al. 2018). Additionally, factors associated with aging have been suggested to influence older adults’ susceptibility to STIs, potentially due to biological changes in older women serving as predisposing factors for parasite growth (Nazari et al. 2015).

The study conducted by Haghighi et al. in Zahedan, was in line with our study and reported a 23.3% (21/90) infection rate based on wet mount microscopy (Dehghan Haghighi et al. 2019). Ambrozio et al. in Brazil revealed that 9% of patients tested positive for T. Vaginalis infection through direct examination (Ambrozio et al. 2016). In another study in Brazil, the presence of T. vaginalis was identified in 16% of samples using the TYM culture medium (von Glehn et al. 2017). Sutton et al. in the USA, conducted a study on women aged 14–49 years, which found a 3.1% infection rate (Sutton et al. 2007). Additionally, Keşli et al. in Turkey, surveyed the incidence of T. vaginalis in women with vaginal discharge complaints using wet-mount and Giemsa staining methods, and reported a 9% infection rate (Keşli et al. 2012).

In addition, the detection of infection is another important factor affecting the prevalence rate of trichomoniasis, and involves a variety of methods, ranging from clinical diagnosis to laboratory-based techniques. Microscopic examination of a vaginal wet mount is one of the most important methods for diagnosing T. vaginalis infection (Van Der Pol 2016). The sensitivity range of wet mount microscopic examination is estimated to be 44–68%. While the wet mount method is rapid and inexpensive, the quality of this diagnostic test largely depends on the expertise and experience of the person operating the microscope (Hobbs and Seña 2013; Van Der Pol 2016). Liquid-based preparations have been shown to facilitate the diagnosis of T. vaginalis. Women noted to carry the organism are recommended to undergo confirmatory testing. Recent studies have reported that molecular-based techniques such as PCR are highly specific and sensitive, providing more reliable estimates (Ginocchio et al. 2012; Momeni et al. 2015; Schwebke et al. 2018). The results of a study by Blavo-Kouaméet al., were in line with our study and confirmed a higher sensitivity (100%) and specificity (97.40%) of the PCR method compared to the staining method, with 3.61% of the 194 specimens testing positive(Be et al. 2017). In our study, 48 samples that were considered suspicious for infection through Papanicolaou staining were also analyzed using PCR. Molecular amplification revealed that only 4 samples were related to T. vaginalis infection. Therefore, it should be noted that there is a possibility of reporting pseudo-positive cases. This result is consistent with a study conducted by Haghighi et al., in Zahedan, Iran, where it was expected that the molecular method would produce better results (Dehghan Haghighi et al. 2019).

The prevalence of T. vaginalis varies in different studies, which may be attributed to various factors such as socio-demographic characteristics and personal hygiene (Poole and McClelland 2013). According to the Hezarjaribi et al., study, trichomoniasis is more common in central provinces of Iran, with the lowest rates in the eastern provinces, and a mean prevalence rate of 6.9% and 6.7% in the west and north, respectively. It is likely that the low prevalence of trichomoniasis in our study could be due to better access to primary health and diagnostic services, allowing for the early detection of T. vaginalis infections among individuals (Hezarjaribi et al. 2015). The findings regarding contraceptive methods indicate that 11.6%, 11.8%, and 6.47% of the subjects used condoms, oral tablets, and IUDs, respectively. It appears that the majority of cases reported using natural contraceptive methods were women who were preventing pregnancy naturally (Azambakhtiar et al. 2018). The study has several limitations that should be considered. Firstly, the study had a small number of women diagnosed with trichomoniasis, which may limit the generalizability of the findings. Additionally, the study population consisted of individuals who routinely attended STD clinics, which may not accurately represent the entire female population in Iran in terms of their sexual behaviours and knowledge about STDs. Another limitation is the lack of follow-up of patients after trichomoniasis diagnosis, which could have provided valuable information on treatment outcomes and the long-term effects of the infection.

Conclusion

In summary, our study revealed a relatively low prevalence of T. vaginalis infection among the female population in this area. The PCR method proved valuable in confirming the diagnosis of suspected cases. Further studies, incorporating larger clinical sample sizes and utilizing at least two diagnostic methods, are recommended for the accurate diagnosis of T. vaginalis infection.

Acknowledgements

This study was supported by the Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran (Grant number 62666 and 63162). We would like to express our gratitude to all the staff of 29Bahman Health Center in Tabriz who helped us in collecting and preparing the samples.

Author contributions

GB carried out the experiment. MHK wrote the manuscript. RA, RN, AS processed the experimental data, performed the analysis. EA conceived the original idea and supervised the project. All authors discussed the results and contributed to the final manuscript.

Funding

This study was supported by the Infectious and Tropical Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran (Grant No. 63162).

Declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Gholamreza Barzgar and Ehsan Ahmadpour contributed equally to this work.

Contributor Information

Reza Ahmadi, Email: drrezaahmadi17@gmail.com.

Mohammad Hasan Kohansal, Email: kohansalhasan@gmail.com.

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