Genetic Diversity and Host Immune Response of Trichomoniasis Among WomenKeanekaragaman Genetik dan Respons Imun Inang pada Trikomoniasis pada WanitaHraijaBaraa Abdulsalambabdulsalam@uowasit.edu.iqDepartment of Microbiology, College of Medicine, University of Wasit, WasitIraq150320261403202611
<bold id="_bold-12">Introduction</bold>
Trichomonas vaginalis is anaerobic, flagellated protozoan parasite and the causative agent of a sexually transmitted disease called trichomoniasis that infects human in industrialized countries (Mirzadeh et al., 2021). The parasite, belongs to the Trichomonadida order in the Metamonada phylum of Eukaryota domain, was first described in 1836 by Alfred Francois Donné who observed it as a motile protozoa in vaginal or cervical secretions (Gharban, 2023; Eanzi and Al-Kaabi, 2024). In 1916, Hohne declared trichomoniasis as a clinical entity with a wide range of genitourinary symptoms in affected women which could lead to more severe reproductive health complications including adverse pregnancy outcomes, and increased susceptibility to other sexually transmitted diseases including human immunodeficiency virus (HIV) disease (Van Gerwen et al., 2023; Joy et al., 2025). Although, the intricate life cycle of parasite involves several distinct stages that are crucial for its survival, proliferation, and transmission, it exists solely as a trophozoite, meaning it lacks a cyst stage and replicates by binary fission with directly contributing to its high infectivity and rapid onset of symptoms (Matti, 2022; Murkute et al., 2025).
The global prevalence of T. vaginalis in women is approximately 5.3%, yet it can be as high as 14.6% in specific cohorts, underscoring its significant epidemiological burden (Li et al., 2025). Also, the adverse clinical manifestations and the potential for asymptomatic carriage highlight the necessity of understanding the varied diagnostic approaches including microscopy, culture and nucleic acid amplification tests to effectively manage this widespread infection (Azeez et al., 2024; Shankar et al., 2025). While, wet mount microscopy remains a commonly utilized point-of-care test, its sensitivity for T. vaginalis detection is notably low often ranging from 31% to 60%, limiting its utility in identifying asymptomatic or low-parasite-burden infections (Cardoso et al., 2024). Conversely, molecular assays such as nucleic acid amplification tests, offer superior sensitivity and specificity for T. vaginalis detection, enabling more precise diagnosis and guiding targeted therapeutic interventions (Gharban, 2023; Borges et al., 2024).
In Iraq, the earliest reports of T. vaginalis were from Al-Shabandar (1979) in Baghdad, Al-Mallan and Al-Janabi (1983) in Mosul, Kadir et al. (1988) in Erbil, Al-Saeed (1995) in Dohok, and Mahdi (1995) in Basra. Then, several studies have been conducted in Iraq (AL-Marjan and Sadeq, 2022; Al-Hasnawy and Rabee, 2023; Hansh, 2024) as well as in Wasit province (Rahi et al., 2014a, b; Rahi and Jaleel, 2022a, b) to estimate the incidence rate of T. vaginalis infection; however, data concerned to genotypic diversity remain underscoring in Iraq (Merdaw et al., 2018; Al-Rubaye and Alkhashab, 2022; Nasir et al., 2022), with complete lack of such information in Wasit province. Therefore, this study identifies the prevalence rate of human trichomoniasis in women of Wasit province (Iraq) directly by molecular PCR assay through targeting ITS1, and phylogenetic analysis of study T. vaginalis isolates. Association of positivity to immune response was aimed, also.
<bold id="_bold-13">Materials and methods</bold>
Ethical approval
Scientific Committee in College of Medicine (University of Wasit) was licensed this study.
Samples
In total, 291 women with different socio-demographic characteristics and reproductive diseases were hospitalized with the private gynecological clinics in Wasit province (Iraq) in March-June (2025) and were picked to participate in the present study. Sampling of vaginal swabs was done on all of the study women that maintained frozen state of -20°C before being subjected to molecular examination. Additionally, 5ml of venous blood was drained from each study individual into free-anticoagulant glass-gel tube that centrifuged (5000rpm/5 min), and the obtained sera were kept into 1.5ml Eppendorf tubes and saved frozen (-20°C) until be tested serologically.
Molecular conventional PCR assaying
DNAs were purified out of the vaginal swabs after thawing in the water bath at 37° C with the g SYNCTM DNA extraction kit (Genaid, Taiwan), analyzed by the Nandrop spectrophotometer, and they were used to prepare MasterMix tubes at a final quantity of 25µl. One of the primers was specific to this study (ITS1): (IQF: 5ʹ-CCT GCC GTT GGA TCA GTT CT-3ʹ and IQR: 5ʹ-TTC CAG TTC AGC GGG TCT TC-3ʹ) using NCBI-GenBank T. vaginalis isolates (https://www.ncbi.nlm.nih.gov/nuccore/PX147461.1). Subsequent post amplification was carried out in Thermal Cycler system after modified conditions (Table 1), PCR products electrophoresis was conducted in 1.5% agarose-gel, with 90 minutes of Amperometric input of 80 Am and Voltage of 100 V and the positive samples of T. vaginalis isolates were visualized at an approximate position within the UV illuminator at 364 bp.
<bold id="_bold-18">Table (1): Thermal cycler parameters of traditional PCR reaction.</bold>
Tool
Step
Temperature / Time
Cycle No.
Qualitative PCR
Initial denaturation
94°C / 5 min.
1
Denaturation
94°C / 30 sec.
30
Annealing
56°C / 30 sec.
Extension
72°C / 1 min.
Final extension
72°C / 5 min.
1
Sequencing and phylogeny
All the positive T. vaginalis isolates DNA were sequenced at the Macogen Company (Korea). The information was submitted to the NCBI-GenBank database with accession numbers assigned, and it was analyzed by phylogenetic software with the NCBI-Viewer (version 1.26) and MEGA software (version 11). Homology sequence identity, multiple sequence alignment, and phylogenetic tree analysis were conducted to prove the strong correlation between the study local and NCBI-BLAST T. vaginalis isolates/strains.
Immunology
According to manufacturer instructions (SunLong Biotech, China) of quantitative ELISAs’ kits, the concentrations of CysLTs (Cat. No: SL0576Hu), IL-8 (Cat. No: SL1004Hu), and LTB4 (Cat. No: SL1068Hu) were measured in the sera of study population. For each immune marker, the kit’s contents and the sera were prepared, processed, and the absorbance was read by the ELISA Reader at an optical density (OD) of 450nm. Then, the ODs and concentrations of Standards in addition to ODs of the samples were plotted on the Standard Curve to calculate the concentration of immune marker tested samples.
Statistical analysis
The t-test in GraphPad Prism software was applied to detect significant differences between the values of positively and negatively infected individuals at p<0.05 with calculation of 95% confidence interval (95%CI), (Al-Gharban, 2017).
<bold id="_bold-26">Results</bold>
Molecular examination of 7.56% (22/291) of study women were positively infected by T. vaginalis using the conventional PCR and targeting the ITS1 region (Figure 1). The sequence data of all the positive T. vaginalis isolates (total=22) were entered in the NCBI-GenBank database, named (IQ-BARHAS 1, IQ-BARHAS 2, IQ-BARHAS 3, IQ-BARHAS 4, IQ-BARHAS 5, IQ-BARHAS 6, IQ-BARHAS 7, IQ-BARHAS 8, IQ-BARHAS 9, IQ-BARHAS 10, IQ-BARHAS 11, IQ-BARHAS 12, IQ-BARHAS 13, IQ-BARHAS 14, IQ-BARHAS 15, IQ-BARHAS 16, IQ-BARHAS 17, IQ-BARHAS 18, IQ-BARHAS 19, IQ-BARHAS 20, IQ-BARHAS 21, and IQ-BARHAS 22) and got respectively specific access numbers (PX920213.1, PX920214.1, PX920215.1, PX920216.1, PX920217.1, PX920218.1, PX920219.1, PX920220.1, PX920221.1, PX920222.1, PX920223.1, PX920224.1, PX920225.1, PX920226.1, PX920227.1, PX920228.1, PX920229.1, PX920230.1, PX920231.1, PX920232.1, PX920233.1, and PX920234.1). Phylogenetic analysis with the NCBI-BLAST T. vaginalis isolates / strains revealed that all study T. vaginalis isolates having a similarity (*) ranged from 98.10% to 99.46% and mutation / changes ranged from 0.003-0.02% with the NCBI-BLAST T. vaginalis Iraqi strain (ID: PQ403668.1), (Table 2, Figures 2-4).
<bold id="_bold-27">Figure (</bold>
<bold id="_bold-28">1</bold>
<bold id="_bold-29">): Total molecular results for testing 291 vaginal swabs by PCR assay</bold>
<bold id="_bold-30">targeting the </bold>
<bold id="_bold-31">
<italic id="_italic-48">ITS1</italic>
</bold>
<bold id="_bold-32">region for </bold>
<bold id="_bold-33">
<italic id="_italic-49">T. vaginalis</italic>
</bold>
<bold id="_bold-34">Table (2): Identity percentage (%) of the study and global NCBI-GenBank </bold>
<bold id="_bold-35">
<italic id="_italic-50">T. vaginalis</italic>
</bold>
<bold id="_bold-36">isolates / strains against the </bold>
<bold id="_bold-37">
<italic id="_italic-51">ITS1</italic>
</bold>
<bold id="_bold-38">region homology.</bold>
Local isolate
NCBI-BLAST isolate
Identity (%)
Name
Access. No.
Strain
Source
Country
Access. No.
IQ-BARHAS 1
PX920213.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.10
IQ-BARHAS 2
PX920214.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.83
IQ-BARHAS 3
PX920215.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.32
IQ-BARHAS 4
PX920216.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.31
IQ-BARHAS 5
PX920217.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.05
IQ-BARHAS 6
PX920218.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.79
IQ-BARHAS 7
PX920219.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.42
IQ-BARHAS 8
PX920220.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.51
IQ-BARHAS 9
PX920221.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.30
IQ-BARHAS 10
PX920222.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.87
IQ-BARHAS 11
PX920223.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.31
IQ-BARHAS 12
PX920224.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.33
IQ-BARHAS 13
PX920225.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.86
IQ-BARHAS 14
PX920226.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.47
IQ-BARHAS 15
PX920227.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.48
IQ-BARHAS 16
PX920228.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.61
IQ-BARHAS 17
PX920229.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.71
IQ-BARHAS 18
PX920230.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.19
IQ-BARHAS 19
PX920231.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.68
IQ-BARHAS 20
PX920232.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.49
IQ-BARHAS 21
PX920233.1
ZhSe2
Endocervix
Iraq
PQ403668.1
98.57
IQ-BARHAS 22
PX920234.1
ZhSe2
Endocervix
Iraq
PQ403668.1
99.16
<bold id="_bold-50">Figure (</bold>
<bold id="_bold-51">2</bold>
<bold id="_bold-52">): MSA</bold>
<bold id="_bold-53">of study and NCBI-GenBank </bold>
<italic id="_italic-52">
<bold id="_bold-54">T. vaginalis</bold>
</italic>
<bold id="_bold-55">isolates / strains </bold>
<bold id="_bold-56">targeting the </bold>
<italic id="_italic-53">
<bold id="_bold-57">ITS1</bold>
</italic>
<bold id="_bold-58">region </bold>
<bold id="_bold-59">by the MEGA software</bold>
<bold id="_bold-60">showing nucleic acids similarity and differences</bold>
<bold id="_bold-61">Figure (</bold>
<bold id="_bold-62">3</bold>
<bold id="_bold-63">): </bold>
<bold id="_bold-64">MSA of study and NCBI-GenBank </bold>
<bold id="_bold-65">
<italic id="_italic-54">T. vaginalis</italic>
</bold>
<bold id="_bold-66">isolates / strains </bold>
<bold id="_bold-67">targeting the </bold>
<bold id="_bold-68">
<italic id="_italic-55">ITS1</italic>
</bold>
<bold id="_bold-69">region </bold>
<bold id="_bold-70">by the NCBI-Viewer</bold>
<bold id="_bold-71">software</bold>
<bold id="_bold-72">showing nucleic acids </bold>
<bold id="_bold-73">similarity and </bold>
<bold id="_bold-74">differences</bold>
<bold id="_bold-75">Figure (</bold>
<bold id="_bold-76">4</bold>
<bold id="_bold-77">): Phylogenetic tree analysis of study and NCBI-GenBank </bold>
<bold id="_bold-78">
<italic id="_italic-56">T. vaginalis</italic>
</bold>
<bold id="_bold-79">isolates / strains</bold>
<bold id="_bold-80">targeting the </bold>
<bold id="_bold-81">
<italic id="_italic-57">ITS1</italic>
</bold>
<bold id="_bold-82">region</bold>
The findings of immunological markers have reported a significant variation (p<0.0001) in their values among the positively (Figures 5-7). Significantly, the positively infected women were reported an elevation (95%CI: 1105 to 1456) in values of CysLTs (276.04±12.58pg/ml) compared to those of negatively infected individuals (74.48±2.77pg/ml). For IL-8, there was a significant increase (95%CI: 518.7 to 685.4) in values of positively T. vaginalis infected women (130.75±4.59pg/ml) in comparison with those of negatively result (35.98±3.48pg/ml). For LTB4, the positively infected T. vaginalis women (1496.5±70.06pg/ml) were shown the significant higher values (95%CI: 6115 to 7997) than negative values (385.93±10.92pg/ml).
<bold id="_bold-95">Figure (</bold>
<bold id="_bold-96">6</bold>
<bold id="_bold-97">): </bold>
<bold id="_bold-98">Levels of serum </bold>
<bold id="_bold-99">IL-8 in positively and negatively study </bold>
<bold id="_bold-100">population</bold>
<bold id="_bold-101">to </bold>
<bold id="_bold-102">
<italic id="_italic-61">T. vaginalis</italic>
</bold>
<bold id="_bold-103">Figure (</bold>
<bold id="_bold-104">7</bold>
<bold id="_bold-105">): </bold>
<bold id="_bold-106">Levels of serum </bold>
<bold id="_bold-107">LTB4 in positively and negatively study </bold>
<bold id="_bold-108">population</bold>
<bold id="_bold-109">to </bold>
<bold id="_bold-110">
<italic id="_italic-62">T. vaginalis</italic>
</bold>
<bold id="_bold-111">Discussion</bold>
The findings of molecular PCR recorded that the incidence rates of human T. vaginalis in women of Wasit province were 7.56%. In comparison to other local studies, the occurrence of human trichomoniasis was 38.1% in Al-Anbar using the microscopic examination of vaginal swab (Al-Ani et al., 2001), 1.66% in Sulaimania by direct microscopic examination and culture (Fattah and Kadir, 2010), 5.23% in Thi-Qar by wet method (Al-Abady and Al-Khazrajee, 2014), 8.33-21.67 % in Wasit by wet mount microscopy, Whif test, and RT-PCR (Rahi et al., 2014a,b), 12.41% in Diyala by wet mount examination (Hussein and Shaker, 2017), 62% in Tikrit by direct wet mount and liquid cultivate (Al-Ammash, 2017), 27.90% in Al-Najaf by wet mount examination (Al-Abbas and Radhi, 2019), 26-32% in Al-Muthana by wet smear and PCR (Al-Abodi et al., 2019), 2.73-3.18% in Erbil by direct wet method and culture respectively (Nouraddin and Alsakee, 2019), 28.10% in Kirkuk by microscopic examination of endocervical and vaginal swabs as well as urine (Salman et al., 2019), 26.27% in Babylon by wet mount (Al-Kahfaji and Seher, 2020), 75.22% in Maysan by microscopic examination and cultivation (Al-Majidii and Alsaady, 2020), 25.5% and 29.5% in Al-Qadisiyah by wet smear and RT-PCR, respectively (AL-Khalidy and Al-abodi, 2020), 8.7-40.35% by microscopic examination of vaginal swabs and urine as well as indirect ELISA (Bedair and Ali, 2020), 0.5-1.6% in Basra by direct microscopic examination of vaginal swabs and urine (Kadhum et al., 2020), 5.5-8.62% in Karbala by wet mount, gram staining and PCR (Alhusseini and Alquraishi, 2021), 3.3% in Mosul by wet mount (AL-TAEI, 2022), 5.5% in Baghdad by PCR (Ghaima, 2022), and 9% in Duhok by microscopically wet mount examination (Murad et al., 2024). However, variations between the findings of the current study and the above mentioned studies could be attributed differences in diagnostic methods, study population (socio-demographic factors), geographical location, screening practices, sample size and type, and the presence of various T. vaginalis strains or other bacterial or viral infections that could interfere with the examination and interpretation of results.
Phylogenetically, high genetic identity was shown between the current study T. vaginalis isolates and the NCBI-BLAST Iraqi T. vaginalis strain that isolated from endocervix of non-married and married women in Duhok province suggesting that this strain might be circulated in Iraqi population women, and its responsible for infections across different locations in Iraq. However, ITS1 region that situated between the 18S rRNA and 5.8S rRNA genes is offered substantial variability that facilitates the taxonomic identification and subtyping of closely related protozoan parasites (Choudhary et al., 2015; Somasundaram and Yu, 2025). This genetic locus is present in multiple copies within the genome, which enhances the sensitivity of PCR amplification compared to single-copy targets (Ellingham et al., 2019). Consequently, this multi-copy nature not only increases diagnostic sensitivity but also provides sufficient sequence divergence to discriminate between distinct strains and subspecies during phylogenetic characterization (Mochizuki et al., 2017; Jarquín-Díaz, 2021).
In comparison with the negatively infected T. vaginalis population, the positively infected women were reported a significant elevation in values of studied immune markers including CysLTs, IL-8, and LTB4. These results in agreement with observed by many researchers (Nam et al., 2012; Lee and Shin, 2024; Farhan, 2025). Nonetheless, the precise mechanisms by which T. vaginalis influences the synthesis and modulation of CysLTs remain an active area of investigation, but it is hypothesized that parasitic factors directly or indirectly trigger host inflammatory cascades that likely involving the activation of specific immune cells and releasing of various pro-inflammatory mediators, and subsequently affect the arachidonic acid pathway to produce leukotrienes (Jafarzadeh et al., 2023; Kou et al., 2024; Zhang and He, 2024). This intricate interplay between the parasite and host immune response could lead to an up-regulation of enzymes involved in leukotriene synthesis thereby contributing to the inflammatory pathogenesis observed in trichomoniasis (Rashidi et al., 2022). Also, T. vaginalis can induce the production of IL-8, a potent chemokine that attracts neutrophils and other immune cells to the site of infection, contributing to the inflammatory response and potentially exacerbating tissue damage (Bhakta et al., 2020; Zhang and He, 2024). This sustained pro-inflammatory cytokine secretion can contribute to chronic cervicitis and create a microenvironment conducive to persistence and progression of infections thereby increasing the risk of cervical intraepithelial neoplasia (Gargiulo Isacco et al., 2023). Moreover, this chronic inflammatory state and altered immune cell recruitment, partly mediated by IL-8, can promote genomic instability and activate oncogenic pathways, facilitating viral integration and subsequently leading to infertility due to damaging of reproductive tissues and impairment of normal physiological processes (Jarrett et al., 2015; Avitabile et al., 2024). LTB4, a potent lipid mediator derived from arachidonic acid metabolism, is implicated also in the inflammatory response orchestrated by T. vaginalis (Maddipati et al., 2016; Cheng et al., 2025). This chemotactic agent contributes to the recruitment of neutrophils and the perpetuation of localized inflammation within the cervicovaginal epithelium to further exacerbating tissue damage and modulating the immune response (Bhakta et al., 2020; Obeagu, 2024).
<bold id="_bold-112">Conclusion</bold>
This study indicates that rapid test and/or molecular PCR can provide important data about the prevalence of T. vaginals in Iraqi population of various socio-demographic aspects who appeared with or without clinical symptoms. Phylogenetically, the local T. vaginalis isolates were markedly identical to another local T. vaginals strain indicating the importance of this strain and the possible its role in women infections. Also, targeting of ITS1 region offered a high sensitivity and specificity in molecular detection of infection and phylogenetic detection of local isolates. Therefore, additional molecular sequencing data based on ITS1 regions appears greatly necessary as such data can be utilized in epidemiology of parasitic infection. Serological elevation of immune markers in positively infected women might further complicates the pathological landscape and enhance the inflammatory cascade with promoting the proliferation of pathogenic bacteria.