Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.     

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu,South Korea

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

A quantitative resazurin assay to determinate the viability of Trichomonas vaginalis and the cytotoxicity of organic solvents and surfactant agents

Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted disease. To test anti-Trichomonas agents, usually many with low water solubility, organic solvents and surfactant agents should be used. Therefore, the minimal inhibitory concentration (MIC) of acetone, methanol, ethanol, isopropanol, DMSO, Tween 20, Tween 80, and Triton X-100 was determined against T. vaginalis isolates using the quantitative resazurin method. Our results showed that solvents and surfactant agents can be employed as vehicles to test bioactive compounds at lower concentrations than MIC values and we suggest acetone and DMSO as preferential. Moreover, a new methodology is established to substitute or to complement the counting of viable trophozoites. The amount of resazurin reduced by T. vaginalis can be quantified by fluorescence spectroscopy, making the test a quantitative determination of cell viability. These results contribute for pharmacological investigations of bioactive compounds that need the use of solvents as solubilization vehicles to test anti-Trichomonas activity.     

A serum-free,lipid-supplemented medium for the growth of Trichomonas vaginalis

A serum-free medium for the primary culture and sub-culture of Trichomonas vaginalis is presented. It consists of DIFCO Bacto AC Medium plus l-cysteine hydrochloride and a lipid supplement of cholesterol and oleic acid in BSA. Compared to the popular Diamond's plus fetal bovine serum, it gives an identical yield of organisms and supports primary grown from urethral/vaginal swab specimens at lower initial cell densities of at least 2 × 10³ motile trichomonads/ml. Mass culturing of T. vaginalis for metabolic studies can be performed without interference from naturally occurring enzyme inhibitors in serum.     

Prevalence of Trichomonas vaginalis by PCR in Men Attending a Primary Care Urology Clinic in South Korea

Trichomonas vaginalis, a causative agent of trichomoniasis, may trigger symptomatic or asymptomatic nongonococcal urethritis and chronic prostatitis in men. Despite the availability of highly sensitive diagnostic tests, such as nucleic acid amplification tests, including PCR, few prospective studies present data on male T. vaginalis infection in South Korea. In the present study, the prevalence of T. vaginalis and associated clinical conditions were evaluated in 201 male patients from a primary care urology clinic in South Korea. The prevalence of T. vaginalis infection in our cohort was 4% (8/201) by PCR. T. vaginalis infection was common in men older than 40 years (median age, 52 years). Among the 8 Trichomonas-positive patients, 87.5% (7/8) had prostatic diseases, such as prostatitis and benign prostatic hyperplasia, and 25.0% (2/8) and 12.5% (1/8) were coinfected with Chlamydia trachomatis and Mycoplasma genitalium, respectively. Our results suggest that T. vaginalis infection is not rare in men attending primary care urology clinics in South Korea, especially in those older than 40 years, in whom it may explain the presence of prostatic disease. The possibility of T. vaginalis infection should be routinely considered in older male patients with prostatic diseases in South Korea.     

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.     

Dolichyl-phosphate-glucose is used to make O-glycans on glycoproteins of Trichomonas vaginalis

Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.     

Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity

Trichomoniasis is the most common non-viral sexually transmitted infection caused by the vaginotropic extracellular protozoan parasite Trichomonas vaginalis. The infection is recurrent, with no lasting immunity, often asymptomatic, and linked to pregnancy complications and risk of viral infection. The molecular mechanisms of immune evasion by the parasite are poorly understood. We demonstrate that galectin-1 and -3 are expressed by the human cervical and vaginal epithelial cells and act as pathogen-recognition receptors for the ceramide phosphoinositol glycan core (CPI-GC) of the dominant surface protozoan lipophosphoglycan (LPG). We used an in vitro model with siRNA galectin knockdown epithelial clones, recombinant galectins, clinical Trichomonas isolates, and mutant protozoan derivatives to dissect the function of galectin-1 and -3 in the context of Trichomonas infection. Galectin-1 suppressed chemokines that facilitate recruitment of phagocytes, which can eliminate extracellular protozoa (IL-8) or bridge innate to adaptive immunity (MIP-3α and RANTES (regulated on activation normal T cell expressed and secreted)). Silencing galectin-1 increased and adding exogenous galectin-1 suppressed chemokine responses to Trichomonas or CPI-GC/LPG. In contrast, silencing galectin-3 reduced IL-8 response to LPG. Live Trichomonas depleted the extracellular levels of galectin-3. Clinical isolates and mutant Trichomonas CPI-GC that had reduced affinity to galectin-3 but maintained affinity to galectin-1 suppressed chemokine expression. Thus via CPI-GC binding, Trichomonas is capable of regulating galectin bioavailability and function to the benefit of its parasitic survival. These findings suggest novel approaches to control trichomoniasis and warrant further studies of galectin-binding diversity among clinical isolates as a possible source for symptom disparity in parasitic infections.     

Trichomonas vaginalis: Investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.     

Unexpected Presence of Blastocystis Subtype 1–3 DNA in Human Vaginal and Sperm Samples Coinfected with Trichomonas vaginalis

There have been few reports on extra-enteric infections by Blastocystis STs and none have been molecularly identified in samples from human reproductive organs. We report for the first time the identification of 3 different subtypes of Blastocystis (ST1–3) in vaginal and sperm samples, from patients infected with Trichomonas vaginalis. Blastocystis STs were identified by PCR-sequencing and by phylogenetic inferences using 28 vaginal swab samples and 7 sperm samples from patients trichomoniasis. Blastocystis STs were identified in 6 of 28 vaginal swabs (21.4%) and in 3 of 7 sperm samples (42.8%). In both biological samples, STs 1–3 were found; one vaginal sample showed subtype co-infection with ST1 and ST3. High genetic variation was observed in the sequences obtained and no specific clustering in the phylogenetic trees was detected. Most of the haplotypes identified were placed far from the main dispersal centers. Our finding suggested that incorrect cleaning of the genital area or a contamination by combination of anal and vaginal intercourse.     

Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.     

Designed Chemical Intervention with Thiols for Prophylactic Contraception

Unlike somatic cells, sperm have several-fold more available-thiols that are susceptible to redox-active agents. The present study explains the mechanism behind the instant sperm-immobilizing and trichomonacidal activities of pyrrolidinium pyrrolidine-1-carbodithioate (PPC), a novel thiol agent rationally created for prophylactic contraception by minor chemical modifications of some known thiol drugs. PPC, and its three derivatives (with potential active-site blocked by alkylation), were synthesized and evaluated against live human sperm and metronidazole-susceptible and resistant Trichomonas vaginalis, in vitro. Sperm hexokinase activity was evaluated by coupled enzyme assay. PPC irreversibly immobilized 100% human sperm in ∼30 seconds and totally eliminated Trichomonas vaginalis more efficiently than nonoxynol-9 and metronidazole. It significantly inhibited (P<0.001) thiol-sensitive sperm hexokinase. However, the molecule completely lost all its biological activities once its thiol group was blocked by alkylation. PPC was subsequently formulated into a mucoadhesive vaginal film using GRaS excipients and evaluated for spermicidal and microbicidal activities (in vitro), and contraceptive efficacy in rabbits. PPC remained fully active in quick-dissolving, mucoadhesive vaginal-film formulation, and these PPC-films significantly reduced pregnancy and fertility rates in rabbits. The films released ∼90% of PPC in simulated vaginal fluid (pH 4.2) at 37°C in 5 minutes, in vitro. We have thus discovered a common target (reactive thiols) on chiefly-anaerobic, redox-sensitive cells like sperm and Trichomonas, which is susceptible to designed chemical interference for prophylactic contraception. The active thiol in PPC inactivates sperm and Trichomonas via interference with crucial sulfhydryl-disulfide based reactions, e.g. hexokinase activation in human sperm. In comparison to non-specific surfactant action of OTC spermicide nonoxynol-9, the action of thiol-active PPC is apparently much more specific, potent and safe. PPC presents a proof-of-concept for prophylactic contraception via manipulation of thiols in vagina for selective targeting of sperm and Trichomonas, and qualifies as a promising lead for the development of dually protective vaginal-contraceptive.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 × 10⁰ cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Dissimilarity in the occurrence of Bifidobacteriaceae in vaginal and perianal microbiota in women with bacterial vaginosis

Recent data point at the similarity between the perianal and vaginal microflora in terms of Lactobacillus species involved. Bacterial vaginosis, the most common perturbation of the vaginal microflora involving primarily overgrowth of Gardnerella vaginalis, has also been suggested to involve a recto-vaginal pathway. We addressed this issue with regard to bacteria of the Bifidobacteriaceae family. In particular, we investigated the putative concordance of the presence of G. vaginalis and a series of Bifidobacteria between the perianal and vaginal microflora in 10 patients with bacterial vaginosis through multicolor fluorescence in situ hybridization analysis of desquamated epithelial cells.G. vaginalis was found in a biofilm mode of growth at the perianal and vaginal sites. In most women at least one of the following species was detected perianally: Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium breves, Bifidobacterium bifidum and Bifidobacterium catenulatum. At the vaginal site, none of these Bifidobacteria was found.We conclude that bacterial vaginosis does not occur as a result of simple growth per continuum of perianal bacteria. Only some species originating from the intestinal tract do display pronounced vaginotropism, like G. vaginalis, whereas many other species do not.     

Genetic identity and differential gene expression between Trichomonas vaginalis and Trichomonas tenax

Background  

Trichomonas vaginalis is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while T. tenax is a commensal of the human oral cavity, found particularly in patients with poor oral hygiene and advanced periodontal disease. The extent of genetic identity between T. vaginalis and its oral commensal counterpart is unknown.     

Anti-trichomonal IgA antibodies in trichomoniasis before and after treatment

Anti-trichomonal IgA antibodies were estimated by enzyme-linked immunosorbent assay (ELISA) in serum and vaginal secretions of 25 symptomatic and 25 asymptomaticTrichomonas vaginalis positive patients before and after treatment and in 25 age-matched controls. Significantly higher levels of antitrichomonad IgA antibodies were found inT. vaginalis positive patients when compared to control subjects, especially in vaginal secretions. In addition, a significant decrease in these antibodies was observed after treatment, which was more pronounced in vaginal secretions. It seems that anti-trichomonal IgA antibodies in serum and more so in vaginal secretions are directly related to and specific to the presence ofT. vaginalis in the urogenital tract.     

Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation

Background

Bacterial vaginosis (BV) is an enigmatic disease of unknown origin that affects a large percentage of women. The vaginal microbiota of women with BV is associated with serious sequelae, including abnormal pregnancies. The etiology of BV is not fully understood, however, it has been suggested that it is transmissible, and that G. vaginalis may be an etiological agent. Studies using enzymatic assays to define G. vaginalis biotypes, as well as more recent genomic comparisons of G. vaginalis isolates from symptomatic and asymptomatic women, suggest that particular G. vaginalis strains may play a key role in the pathogenesis of BV.

Methodology/Principal Findings

To explore G. vaginalis diversity, distribution and sexual transmission, we developed a Shannon entropy-based method to analyze low-level sequence variation in 65,710 G. vaginalis 16S rRNA gene segments that were PCR-amplified from vaginal samples of 53 monogamous women and from urethral and penile skin samples of their male partners. We observed a high degree of low-level diversity among G. vaginalis sequences with a total of 46 unique sequence variants (oligotypes), and also found strong correlations of these oligotypes between sexual partners. Even though Gram stain-defined normal and some Gram stain-defined intermediate oligotype profiles clustered together in UniFrac analysis, no single G. vaginalis oligotype was found to be specific to BV or normal vaginal samples.

Conclusions

This study describes a novel method for investigating G. vaginalis diversity at a low level of taxonomic discrimination. The findings support cultivation-based studies that indicate sexual partners harbor the same strains of G. vaginalis. This study also highlights the fact that a few, reproducible nucleotide variations within the 16S rRNA gene can reveal clinical or epidemiological associations that would be missed by genus-level or species-level categorization of 16S rRNA data.     

Trichonomas vaginalis Metalloproteinase Induces Apoptosis of SiHa Cells through Disrupting the Mcl-1/Bim and Bcl-xL/Bim Complexes

To elucidate the roles of metalloproteinases and the Bcl-2 family of proteins in Trichovaginalis. vaginalis-induced apoptosis in human cervical cancer cells (SiHa cells) and vaginal epithelial cells (MS74 cells), SiHa cells and MS74 cells were incubated with live T. vaginalis, T. vaginalis excretory and secretory products (ESP), and T. vaginalis lysates, either with or without the specific metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT), and examined apoptotic events and Bcl-2 signaling. The live T. vaginalis and the T. vaginalis ESP induced the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP. Additionally, the live T. vaginalis, but not the T. vaginalis lysate, induced the cleavage of the proapoptotic Bim protein. The live T. vaginalis and the T. vaginalis ESP, but not the T. vaginalis lysate, induced the dose-dependent cleavage of the antiapoptotic Bcl-xL and Mcl-1 proteins and decreased the association levels of Bcl-xL/Bim and Mcl-1/Bim complexes. We performed gelatin zymography and casein-hydrolysis assays on the live T. vaginalis and the T. vaginalis ESP to identify the apoptosis-inducing factor. Both the live T. vaginalis and the ESP contained high levels of metalloproteinases, of which activities were significantly inhibited by 1,10-PT treatment. Furthermore, the 1,10-PT blocked the cleavage of Bcl-xL, Mcl-1, PARP, caspase-3, and caspase-9, as well as the release of cytochrome c into the cytosol, and it significantly increased the association levels of the Bcl-xL/Bim and Mcl-1/Bim protein complexes, returning them to normal levels. Our results demonstrate that T. vaginalis induces mitochondria-dependent apoptosis in SiHa cells through the dissociation of Bcl-xL/Bim and Mcl-1/Bim complexes and that the apoptosis is blocked by the metalloproteinase inhibitor 1,10-PT. These results expand our understanding of the role of metalloproteinases in T. vaginalis-induced apoptosis and the signaling pathway in trichomoniasis of the cervicovaginal epithelial cells.     

Identification of Trichomonas Vaginalis Genotypes Using by Actin Gene and Molecular Based Methods in Southwest of Iran

Background:Trichomonas vaginalis (T. vaginalis) is a sexually transmitted protozoan parasite and the causative agent of trichomoniasis. The genetic characterization of T. vaginalis isolates shows notable genetic variation in this parasite. In the present study, we aimed to identify the T. vaginalis genotypes based on analyzing of actin gene in women specimens referred to health centers of Ilam city, southwest Iran.Methods:A total of 1765 female samples were collected from gynecology clinics in the city of Ilam. DNA was extracted from positive samples and nested polymerase chain reaction (Nested PCR) was used to amplify the actin gene. Then, partial sequencing and genotyping of the actin gene was performed. A phylogenetic tree was drawn using the detected genotypes of T. vaginalis and reference sequences.Results:Twenty-one of the 1765 urine and vaginal samples were positive for T. vaginalis. All infected individuals were married and their age in years was between 25 to 34. Further, the majority of infected women had cervical lesions, patchy erythema, and white color discharge. According to sequencing analysis, the isolates were identified as genotype G (n= 8) and genotype E (n= 2).Conclusion:From the collected samples, we were able to distinguish at least two genotypes (G and E) of T. vaginalis. However, lesser is known about these genotypes in the city of Ilam. Further studies with a higher number of isolates should be performed in order to understand the implications of these results in this region.Key Words: Actin gene, Genotypes, Ilam, Iran, Trichomonas vaginalis