Genotypes Diversity and Virulence Factor screening of Trichomonas vaginalis Isolated from Pregnant Women in Mosul (North of Iraq)

: Trichomonas vaginalis is a causative agent of trichomoniasis , one of the most common non-viral sexually transmitted disease (STD) over all the world, especially in immunocompromised women such as pregnant. Wet smear and Giemsa stain are the current methods used in hospital to diagnosis trichomoniasis. DNA based diagnosis is still to be validated to diagnose the local isolates, the objective of the present study was to compare the conventional methods of disease diagnosis with the DNA-based method to diagnose Trichomonas incidence in local isolates. In the present study, 105 samples were collected from outpatient women (18-45 years) of Maternity hospital in Mosul who showed a classical presentation of Trichomonas infection including foul-smelling vaginal discharge with a pH exceeding 4.5. Samples underwent microscopic examination and nucleic acid detection of AP65-1 gene, the wet smear test showed that 16.18% of the collected specimen were positive while the parasite appeared only in 8.9% of the samples stained with Giemsa dye, the molecular screening for AP65-1 gene that encodes for the surface attachment protein showed high sensitivity level of 100 at 50 specificity level in compare with other routine methods, the algorithm was evaluated according to specificity, sensitivity, and predictive values. Random Amplification Polymorphic DNA (RAPD) was achieved to estimate genetic indices among isolated strains. Phylogenetic analysis was performed using PAST (Paleontological statistics software) and dendrogram with neighboring clusters was constructed. According to the outcomes of research, we recommended to utilize a probiotic vaccines and molecular silencing vaccine (like miRNA sense and antisense oligonucleotides) as a treatment in.


Introduction:
Sexually transmitted diseases (STDs) are spreading across the world, especially among immunocompromised women and women in their reproductive years who have sexual partners. Sexual activity continues to be a major source of infection in heterosexuals, with potentially dangerous bacterial, viral, fungal, and protozoal causative agents 1 . Recently, it has confirmed that uterus tissues are usually contaminated with unfavorable microorganisms (MOs) categories, like Gardenerlla sp., Mycoplasma sp., uncultivated bacteria, Candida sp., in addition to T. vaginalis as an irritant protozoan. Previous MOs were required to alter the vaginal milieu physiologically in order to produce a favorable setting for viral infection, such as HIV, HPV, and other causative agents [2][3][4][5][6][7] .
T. vaginalis is one of the most pathogenic species, causing lysis of the genital tract's mucoid layer and irritating living tissue, particularly epithelial cells. This causes invasion of neighboring cells with certain causatives, which leads to increased complications, such as duct thickness and the development of papilloma and neoplasia. Asymptomatic infection is more common in men, and it's associated with urethral infection and dysuria. Several symptoms in an infected woman include a yellowish-green foamy discharge, purities, dysuria, and the strawberry cervix 8,9 . P65 is refers to surface protein of T. vaginalis, it is a prominent functional protein that's also bound to both the surface and its ligand to influences host cell binding 10,11 , In comparison to other adhesion proteins, Garcia et al. 12 , found that T. vaginalis AP65 is still a crucial adherent protein of trichomonads 12 .
RAPD markers are a non-directed technique for generating polymorphic PCR products from genomic templates utilizing monomer oligonucleotides that anneals with numerous sites in diverse genomic regions and simultaneously amplifies several genetic loci for each sample. These markers have also been shown to be effective genetic markers for a variety of eukaryotic organisms 13 . Many studies were carried out in this field for diagnose and classification of T. vaginalis based on classical and molecular techniques [14][15][16][17] . Our objective was included detection of T. vaginalis in pregnant women samples, then diagnosed it by routine methods and confirmed this investigation by conventional PCR technique and classification via molecular utilizing RAPD-PCR application to determine the genetic variation among the detected genera of this protozoan.

Material and Methods: Subjects
Duplicated high vaginal swabs (HVS) were collected by gynecologist from 105 pregnant women admitted to Mosul maternity hospitals during July-December 2019.

Morphological diagnosis
Swabs were impregnated in 1ml of sterile saline solution and then used for wet mount and Giemsa staining technique to diagnose T. vaginalis. A drop of the sample was placed on the slide and covered with a coverslip. Prepared smears were examined at 40x lenses to detect the protozoan motility, which was diagnosed by its Jerky movement as a wavelike actions. In addition, wet mount is often used to show the presence of clue cells in vaginal secretions, these epithelial cells were surrounded by masses of bacteria. Concerning Giemsa staining method, the dried smears were fixed by methanol for 5minutes, and stained for 20 minutes at dilution 1:20. For parasite identification, scanning has been performed at 100x magni-fication level, pear shaped trophozoites have been recognized 18,19 . For conduct whiff test, vaginal discharge was collected by cotton swab, then a drop of vaginal discharge was mixed with a drop of KOH(10%) and placed onto a strip of pH paper (Qualigen, India), the outcome was compared with a standardized colorimetric reference chart to estimate the actual pH 20 .

Molecular profile
Genomic(g) DNA was extracted from the parasite using Wizard gDNA purification kit according to manufacturer instruction Promega, USA 21 , gDNA concentration (34-39ng/ml.) and purity were achieved by nano-spectrophotometer and their template integrity was evaluated by electrophoresis for conventional and RAPD-PCR application. The primer set AP65-1-Forward (5'-3') and Reverse (5'-3'), was designed in this study (AccessionNo.U18346), more information was detailed in Tab.1. Amplification program for detection of AP65-1 gene included; 95C as DNA unwinding for 3min and 35cycles of each of denaturation 94C, annealing temperature 62C, extension 72C, the periods were 30sec for each step, in addition to final extension 72C for 5min. The molecular diversity of T. vaginalis was investigated using the following random primers; OPAX-01(GTGTGCCGTT), OP AV-08 (GGACGGCGTT) and OPV-10 (GGACCTGCTG) which were designed and supplied by (Bioneer, S. Korea). Amplification tubes were transported to Applied Bio-system gradient thermo cycler (USA) for achieve following program: initial incubation 95C for 3min, and 35 cycles of 95C for 30s as initial denaturation, 34-39C for 30sec as gradient annealing temperature and 72C for 30sec as an extension and final extension 72C for 180sec. Amplification premix (Bioneer, S. Korea) consist of lyophilized Taq-DNA polymerase 1U, dNTPs 250Μm (dATP, dCTP, dGTP, dTTP), 1.5mM. of MgCl, 30mM of KCl, 10mM of Tris-HCl (pH 9.0), in addition to methylene blue as a loading dye. Whereas the amplification mixture was included 1μl for each primer, 5μl as gDNA (template) and 13μl represent nuclease free water (total volume 20μl). DNA marker was 100bps, Bioneer, S. Korea 22 .

Outcomes documentation and analysis
All amplification products were detected and photographed using Gel Documentation System (ATTA, Japan). By each primer, the banding pattern size of each isolates was scored (bp) in comparison with the size of DNA marker for AP65-1 gene (Bioneer, S. Korea). All random bands were classified to polymorphic and single, depending on the DNA marker (100bp.). The bands size was calculated using CS analyzer software (ATTA, Japan), then the bands were converted into two digital data (1,refere to band; 0,absence). After that, a simple matching (SM) coefficient was investigated via Jaccard algorithm to determine a dendrogram and using PAST software 23 . The sensitivity of all results were also compared and calculated by applying the following equations: sensitivity = positive result/total positive result X 100 24 .

Ethical approval
The current study was carried out in accordance with the Human Rights Health Protection Guidelines governed by the ethics committee at Mosul maternity facilities, Iraq, and in accordance with the Helsinki Declaration on Ethical Principles.

Results and Discussion:
Of all 105 (100%) samples, 16(15%) was diagnosed morphologically as T. vaginalis via wet smear, 11(10.5%) was detected by Giemsa stain and 8(7%) by molecular screening depending PCR technique. Our findings, were nearly to those of Rajonic et al. 25 , who diagnosed the parasite by wet mount in 14 (7.0%) cases and Giemsa stain 11(5.5%) in 21(10.5%) women. Pointof-care testing, such as vaginal pH assessment, microscopic investigation of vaginal fluid, and the wiff test, is routinely performed. According to this study, low rates of assessment for prior testing were seen among women admitted to maternity hospitals 26 .
All phenotypically suspect samples 16 of T. vaginalis were genetically verified using PCR method, with eight of them giving a negative result for the presence of the AP65-1 gene, and the other eight yielding a positive result. The AP65-1 gene (Fig. 1, Tab.2) was screened for two purposes. The first was to validate the presence of T. vaginalis, the second aim was to evaluate the existence of an essential virulence factors that help this protozoan attach to the surface of host cells, which is an extremely specialized process mediated by adhesion proteins and is an early and critical phase in colonization 27 . The proteins of AP65 could be secreted out of trophozoite of T. vaginalis and attach to the surface of trophozoites and host cells 12 .   28 , who used PCR in first-catch urine.

Genetic diversity of T. vaginalis
The eight samples that tested positive for the AP65-1 gene were chosen for random amplification to explore genetic diversity across T. vaginalis strains (Fig. 2), which was represented by a similarity and dissimilarity chart and a dendrogram. Due to the use of the same random primers with many species or genera, RAPD is an essential confirmatory technique at the molecular level 13 . Random primers are made up of ten nucleotides that anneal to various locations on the targent gDNA choromosome based on repeating nucleotides. As a result, a variety of random bands emerged 29 .

Similarity and distance indices
The observative data, involeved the highest similarity percent between 2&6 (0.38462), and the lowest similarity percent between 2&7(0.071429), whereas the other relationships were founded between those values, more detailes in Tab. 4, our results agree with that reported data 30 who discovered significance difference among T. vaginalis at molecular level using RAPD application, the outcomes were reported in the Tab. 4. On the other hand, phylogenetic tree categorized the parasites under research into two major classes (F&E), E line was represented by no.8, F was split into two neighbouring clusters (G&D), D subdivision included two isolates (5&6), while G subclass revealed C and A, in turn C has two subbranches 4 and 7, and finally A was clustered into subbranches B (2&3) and isolate no.1 line (Fig. 3). There are a close genetic relationship and certain gene polymorphism among the T. vaginalis isolates, thus geographical origin plays little role to the genetic characteristics 15 .

Future directions:
The development of a common framework for genotyping T. vaginalis is essential to provide the necessary tools for addressing potential correlations between T. vaginalis the organism, and trichomoniasis the disease. The DNA-based techniques, MLST and MS genotyping, are most suitable for these investigations, but the relationship between the two population types identified in each of those approaches needs to be clarified. Analysis by both MLST and MS genotyping of a common set of clinically annotated T. vaginalis isolates has recently been initiated by the authors. This will facilitate investigations into the relationships between mixed infections, T. vaginalis genetic identity, the unique two-type population structure, and clinical manifestations of the disease. The interaction between the vaginal microbiome, patient immune response, and T. vaginalis genetic diversity can then be investigated in order to obtain a comprehensive understanding of trichomoniasis and its associated disease sequelae. The extensive genetic diversity and two clade population structure in T. vaginalis will also need to be carefully considered in other investigations of trichomoniasis. The use of proteomic and genomic-based techniques to elucidate genetic factors controlling clinical manifestations and severity, association with other disease entities, and drug resistance will require T. vaginalis isolates which, other than the relevant trait of interest, are as genetically similar as possible in order to maximize the discovery of the relevant genetic elements and avoid differences rooted in genetic diversity. By contrast, future vaccine design and development of antigen-based rapid diagnostic tests for trichomoniasis will need to focus on antigenic determinants which are broadly representative of the entire T. vaginalis population. The existence of a two-type population structure, and the association of particular clinical and phenotypic traits with genetic identity also highlight the need for genome sequencing of additional T. vaginalis strains. To date, the sequence of only a single T. vaginalis strain, G3 (American Type Culture Collection reference number Pra98), has been generated, and it is imperative that the genome sequence of additional clinically relevant isolates representative of both T. vaginalis population types be determined.

Conclusion:
Despite the fact that T. vaginalis is one of the most common STIs, it remains an orphan disease that relies on outdated technology, clinical suspicion, and empirical care. The disease appears to have a wide-ranging effect on the transmission of other sexually transmitted infections as well as the unborn child. The accuracy of a trichomoniasis diagnosis based solely on complaints of vaginal discharge is low. It's a crucial diagnostic test that's especially useful for detecting women who don't have clinically obvious disease. To date, screening approaches such as molecular analysis at a qualitative level using polymerase chain reaction play an essential role in reducing the complications associated with asymptomatic infection and progression of prevalent STIs among reproductiveage women.