Trichomonas  Vaginalis  Genotyping in Upper Egypt

doi:10.21203/rs.3.rs-4811368/v1

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Trichomonas Vaginalis Genotyping in Upper Egypt

https://doi.org/10.21203/rs.3.rs-4811368/v1

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Background: Egypt is a developing country with high population density and a high proportion of people with moderate or low socioeconomic levels. The threat of sexually transmitted diseases, such as trichomoniasis, forms an important public health concern. Trichomoniasis, especially in asymptomatic individuals, remains neglected and poorly addressed .Trichomonas Vaginalis, a parasitic protozoa that causes the sexually transmitted infection trichomoniasis, is the sexually transmitted infection with the largest annual incidence and considered as one of the neglected tropical parasitic infections. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection, we conducted analysis of the actin gene of T. vaginalis by applying the Restriction fragment length polymorphism (RFLP) and nucleotide sequencing method.

Aim and objectives: To investigate the genetic variability of T.Vaginalis in infecting Egyptian women in Upper Egypt.

Patient & methods: This cross-sectional research was conducted during the period from 2019 to 2022 on 150 female patients attending the outpatient clinics outpatient clinics of gynecology and obstetrics at Sohag General Hospital with complaints suggestive of trichomoniasis. Vaginal washout samples were collected, and subjected to direct wet mount microscopic examination, and culture on TYM Diamond's medium, then analysis of the actin gene of T. vaginalis by PCR-RFLP( PCR amplification, restriction enzyme digestion) and detection of DNA fragments by electrophoresis in 2.5–3% (w/v) agarose gels

Results: By using direct wet-mount microscopic examination, out of 150 collected wash-out vaginal samples, 12 cases (8%) were positive for T. vaginalis trophozoites, and 16 samples (10.6%) were positive for culture , (13) samples applied to PCR-RFLP ,The restriction enzyme HindII digested the amplified products into three DNA fragments of 60, 213, and 827 bp. RsaI produced 5 DNA fragments of 87, 103\ 106, 236, & 568 bp, & MseI produced 3 different fragments of 204, 315, & 581 bp ,and detection of DNA fragments by electrophoresis in agarose gels .

Conclusion: According to our study Genotype E of T. vaginilais is the most prevalent in upper Egypt.

T.Vaginalis

Nested PCR

Genotyping

Trichomoniasis

Restriction fragment length polymorphism (RFLP)

Trichomoniasis, one of the most common non-viral sexually transmitted disease caused by T. Vaginalis parasite (Menezes et al ,2016). Though it is a curable infection, it can lead to adverse health consequences. According to WHO, there are about 143 million new cases annually(Dyab et al.,,2021). Worldwide, the estimated prevalence rates vary with geographic location, age, race, community and the method used for diagnosis. In 2016, the WHO global estimate for trichomoniasis in women was estimated at 5.3% and 0.6% in men with a total incidence of 156 million cases (Rowley et al., 2019). In Egypt, a prevalence rate of 6.0% was documented among symptomatic women in Beni Suef Governorate (Hamdy and Hamdy,2018); in addition to 11% in Benha, Qalyubia Governorate (Hussein et al., 2015), 3% in Cairo (Mahmoud et al., 2015) .

Female patients infected with T. vaginalis have a wide range of clinical symptoms, ranging from asymptomatic to acute or chronic inflammatory disease. Trichomoniasis is accompanied by serious complications of the reproductive tract, including infertility, ectopic pregnancy, pelvic inflammatory diseases, premature rupture of membranes, preterm delivery, and low birth weight (Hamouda et al., 2022). Additionally, it plays a significant role in human immunodeficiency virus (HIV) transmission and acquisition (Alves et al., 2020) and Human papillomavirus (Heikal et al., 2023). Such a difference in clinical traits as virulence, pathogenicity and drug resistance, signifies the need to link phenotypic variation to genotype (Hawksworth et al.,2015).

In Egypt, former attempts to investigate the diversity of T. vaginalis included isoenzyme patterns (Salem et al.,1992)., serotyping (Azab et al,. 1992), immunoblotting (el-Okbi et al,. 2005)., biological variability (Boulos et al., 2012), HSP70-RFLP (Hussien et al., 2005)., multilocus sequence typing (Mohamed et al,. 2019). These studies concluded that the different clinical isolates have different and common patterns at the levels 8 of antigens, immunogens, pathogenicity and MTZ resistance.

T. Vaginalis has a large genome of 160 Mb with approximately 60000 protein-coding genes. Most of its genome has repeats and transposable elements (Carlton et al ,2007). Actin protein of T. vaginalis is the main component of its cytoskeleton and has an essential role in cellular mobility and cell interaction (Crucitti et al. ,2008). This structural protein has a well- conserved ubiquitous nature, making it a feasible choice for intra-species molecular identification (Drouin et al. ,1994)

Commonly, PCR and its related methods were widely used for genetic studies in organisms. Random amplified polymorphic DNA (RAPD) method was used to reveal an association between T. vaginalis genotype and metronidazole resistance (Meade and Carlton, 2013). Also, restriction fragment length polymorphism (RFLP) and PCR-RFLP methods were applied to demonstrate the genetic diversity in clinical isolates of T. vaginalis (Meade et al., (2009)

Currently, the PCR-RFLP technique based on the actin gene amplification provides a sensitive and reliable method for typing of T. vaginalis isolates (Oliaee ,.et al 2017)

Using PCR-RFLP targeting actin gene were done in Turkey (Demirağ et al . 2017), China (Zhang et al.,2018) and Iran (Orujzadeh et al., 2019). Among twenty T. vaginalis isolates from symptomatic females, genotype E was the most common, followed by G, with only one isolate assigned for H, and two mixed isolates of genotypes E and H (Demirağ et al. 2017). In another report, out of 68 T. vaginalis isolates, two E and H genotypes were found in addition to mixed genotypes in three isolates (Zhang et al., 2018). Recently, investigators detected three genotypes (E, G and I)

The purpose of this research was to investigate the genetic diversity of T. Vaginalis in women living in upper Egypt by means of PCR-REFLP DNA sequencing. Acquiring an understanding of the genetic features of T. vaginalis populations is helpful for the prevention & management of trichomoniasis in human beings.

This cross-sectional research was conducted at Medical Parasitology Department, Faculty of Medicine, Assuit University during the period from 2019 to 2022

Study population

We carried out an observational study included 150 female suspected of trichomoniasis selected from those attending the Outpatient Clinics of Gynecology and Obstetrics at Sohag General Hospital.

Eligibility Criteria

We included all non-menstruating female patients with gynecological manifestations suggestive of trichomoniasis, including vaginal discharges with or without pruritis vulvae, dyspareunia, and dysuria.

Exclusion Criteria

History of intercourse or douching for the last 2 days, use of antiprotozoal or antibiotics for the last 2 weeks. Menstruating females or virgins. Each participant in the study provided a complete demographic and clinical history. A written informed consent was taken from each patient after brief description of the research idea.

Samples Collection

Vaginal washouts collection samples was obtained from each participant as follows (McMillan et al., 1979).

: Sterile vaginal Cusco speculum was inserted into the patient’s vagina then rotated 90° until an optimal view of cervix was seen.

– 5 ml of the sterile isotonic PBS solution was introduced into the vagina using sterile syringe without its needle.
– Fluid in the posterior fornix of the vagina was aspirated by the plastic pipette and put into sterile tubes.
– Vaginal washouts were examined for T. Vaginalis by direct wet mount, modified Diamond’s culture medium, and Part of the sample was divided into micro-centrifuge tubes (1.5 ml) and sent to the laboratory (kept at -20°C) for further PCR examination.

Examination of vaginal washout was done through:

Direct Wet Mount Microscopic Examination

Microscopic examination was done immediately after sample collection to detect motile flagellate T. vaginalis trophozoites using ×10, ×40 and ×100 objectives of a light microscope(Radonjic et al .,2006).

In Vitro Culture on Diamond’s Tripticase-Yeast-Maltose (TYM) Medium

The inoculation of culture media was performed according to Garcia and Bruckner. The vaginal washout was shacked then one drop was inoculated on the culture tube containing Diamond TYM medium and incubated at 37°C with daily microscopic examination for the presence of T. vaginalis trophozoites. Negative specimens were continually incubated under the same conditions until the seventh day following the initial inoculation, at which point samples with no trophozoites upon microscopic examination were considered negative for T. vaginalis using the culture method (Garcia and Alderete.,2007).

DNA extraction

The T. vaginalis DNA lysates were prepared from vaginal washout (100 µl) using MagMAX™ CORE Nucleic Acid Purification Kit (Cat. No. A32700, ThermoFischer Scientific, USA).

Nested PCR was conducted utilizing the outer primers Tv8S and Tv9R, as well as the inner primers Tv10S & Tv11R, which were selected in accordance with the actin gene of T. vaginalis (GenBank: AF237734). At approximately 1260 & 1100 base pairs, the outer & inner primers amplify two fragments of the actin gene, respectively.

PCR-RFLP

In this method, the effects of endonucleases (restriction enzymes) are used to differentiate Trichomonas strains. The actin gene was amplified by nested PCR, using two pairs of specific primers. External primer pair included: Tv8s (5’-TCT GGA ATG GCT GAA GAA GAC G -3 ‘) and Tv9R (5’- CAG GGT ACA TCG TAT TGG TC-3) and a pair of internal specific primers: Tv10s (5’ -CAG ACA CTC GTT ATC G-3’) and Tv11R (5’- CGG TGA ACG ATG GAT G-3’). The target gene had a length of 1100 bp. Since nested PCR was used, the first stage product was a template for the second PCR. The second stage was similar to the one conducted and only primers of F2 and R2 should have been used instead of primers of F1 and R1, and also instead of the original DNA, the product of the first step with a volume of 1 µL should have been added.

The First Stage of PCR

The PCR components present in each microtube with a volume of 0.2 mL for the reaction volume of 25µL were as follows: master mix 12.5 µL, forward primer (F1) 1 µL reverse primer (R1) 1 µL, ddw 9.5 µL, and DNA 1 µL. PCR was performed as follows: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 3 minutes; and final extension at 72°C for 7 minutes.

Second Stage of PCR

The PCR components present in each microtube with a volume of 0.2 mL and a reaction volume of 25 µL were: master mix 12.5 µL, forward primer (F2) 1 µL, reverse primer (R2) 1 µL, and DNA (diluted) 1 µL.

In the second stage of nested PCR, after Master Mix preparation, the samples were placed in the thermal cycler. This stage was different from the first stage only in that annealing temperature was set at 55°C instead of 61°C. After completing the second stage, the PCR product was electrophoresed on 1% agarose gel and the desired band was compared to a 100 bp DNA ladder (the target band was about 1100 bp in size).

In the second stage, PCR protocol was followed as: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 61.3°C for 30 seconds, extension at 72°C for 3 minutes; and final extension at 72°C for 7 minutes.

Genotyping by PCR-RFLP

PCR-RFLP was used to identify polymorphisms for the loci B-actin gene. PCR amplification, restriction enzyme digestion, and detection of DNA fragments by electrophoresis in 2.5–3% (w/v) agarose gels

Statistical analysis

The data that was gathered was systematically organized and evaluated utilizing SPSS version 16 (SPSS Inc., Chicago, ILL Company). As percentages and numbers, the data were presented. Fisher's exact test, or the chi-square test (X2), is utilized to determine significance. In order to ascertain confidence intervals for the detection rate, the Student's t-test was employed. A two-sided P value below 0.05 was deemed to be statistically significant.

Ethical considerations

The study design was reviewed and approved by the Research Ethics Committee,Faculty of Medicine Assiut University, according to the regulation of Egyptian Ministry of Higher Education in accordance to declaration of Helsinki. Informed

consent was obtained from each participant in the study after clear explanation of the study objectives. Cases proved positive for trichomoniasis were prescribed

appropriate therapy by the gynecologist ( Ethical no 1720024 /27-8-2018).

The attempted evaluation of infection by T. vaginalis in 150 female patients with suggestive clinical picture using direct wet-mount microscopic examination revealed 12 (8%) positive cases. Attempted isolation of trophozoites by cultivating the wash out samples succeeded in 16 samples (10.6%).

Table

showed that the restriction enzyme HindII fragmented the amplified products of DNA into three 827-bp, 213-bp, and 60-bp segments. The amplified product was digested with RsaI to yield five DNA fragments measuring 87, 103\106, 236, & 568 base pairs. Upon digestion of the amplified product with MseI, three fragments measuring 204, 315, & 581 base pairs were generated. The DNA fragment patterns of all isolates exhibit actin genotype E. None of the actin genotypes A, G, I, M, N, or P were detected in the isolates.

Figure 1 shows T. vaginalis trophozoites revealed after culture on TYM Diamond's media and detected by the wet mount microscopic examination using an oil immersion lens (100x). The trophozoite's distinctive motility was the main indicator of its presence. It was noticed that after culture, T. vaginalis trophozoite exhibited spherical form; arrow points to round up T. vaginalis trophozoite (Figure A). In Figure B arrow points to T. vaginalis trophozoite, which has lost its typical pear shape. The flagellae were also invisible in both Figures (A and B).

Figure 1

T. vaginalis growth on TYM Diamond’s medium detected by wet mount examination using an oil immersion lens (100x).

Figure 1: Incidence of T. vaginalis infection between the studied cases using wet mount microscopic examination

Table

The fragment sizes acquired subsequent to the restriction enzyme digestion of the actin gene with HindII, MseI, and RsaI, in addition to the genotype assignment of T. vaginalis determined by integrating the patterns across the three enzyme profiles

Sample name	HindII fragment sizes	RseI fragment sizes	MsaI fragment sizes	Genotype
TV1	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV2	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 3	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 10	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 15	827, 213, 60	568, 236, 106/103, 87	581, 315, 204	E
TV13	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 21
TV24	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 30	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 32	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 33
TV35	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 36	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV 41
TV 65	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E
TV98	827, 213, 60	568, 236, 106/ 103, 87	581, 315, 204	E

In the current research, T. vaginalis infection was positive in 12 cases (8%) using wet mount preparation of vaginal washout. Our result agrees with a study done by Heikal et al.(2023), and 8 samples out of 96 samples (8.3%) were microscopically positive for T. vaginalis trophozoites, and we use the same technique in taking vaginal swabs as Heikal et al.(2023),

Hassan et al. (2019) reported 12.7% positive cases by wet microscopical examination of vaginal swabs among examined females. FAROUK et al.,(2021) In a study was carried out in the outpatient clinic of Aswan University Hospital, wet mount detected 5% (10 positive cases out of total 200 suspected women). Among Egyptian researchers, a lower percent were recorded for positive T. vaginalis infection using a direct wet mount in selected symptomatic females. One of these percents was 1% by Mahmoud et al. (2015). These researchers depended on a vaginal swab or wooden spatula for taking the vaginal samples.

In our study, T. vaginalis culture by TYM Diamond's medium, 22 cases of tichomoniasis were identified out of 150. So, the percent of positivity of T. vaginalis increased from 12 cases (8%) by wet mount to 16 cases (10.6%) by culture. The improvement of trichomonas detection by TYM Diamond's medium culture in the current research is in accordance with Al Saeedet al.(2011), who recorded 5.4% using culture of T. vaginalis compared to 2.4% by wet mount. Also, the corresponding percents were 2.1% and 1.7% by Matini et al. (2012). FAROUKet al.,(2021) In a study was carried out in the outpatient clinic of AswanUniversity Hospital trichomonas detection by TYM Diamond‘s medium culture was 13.5% (27 positive cases out of total 200 suspected women). So FAROUKet al.,(2021) recorded that The sensitivity of wet mount was 40% as it detected 10 positive cases out of total 27 positive cases detected by culture method.,Nagat.,( 2015) who reported that wet mount microscopy detected about half of the culture positive samples 13 (56%) cases positive by wet mount out of 22 total positive cases and 13 cases positive (56%) by wet mount out of total 22 positive cases respectively

It is worthy to mention that in the current study, the revealed Trichomonas trophzoite, after being cultured, possessed some morphological and motility alteration in comparison to the original generation detected previously by wet mount preparation. This alteration included the rounding of the trophozoite and the disappearance of its flagella, along with sluggish motility, which was hardly observed.

In our study, only 13 samples gave a result, and in all the analyzed samples, the amplified products were digested by the restriction enzyme HindII into three DNA fragments with lengths of 60, 213, & 827 bp. The amplified product was digested with RsaI, which resulted in the production of five DNA fragments with lengths of 87, 103\ 106, 236, & 568 bp. The amplified product was digested by MseI, which resulted in the production of three different fragments with lengths of 204, 315, & 581 bp. The actin genotype E may be seen in the patterns of DNA fragments from each individual isolate. In these particular isolates, neither the A nor the G nor the I nor the M nor the P actin genotypes were discovered.

In a study by R. Chetty et al. (2020), the actin gene's PCR-RFLP analysis helped to distinguish between three different T. vaginalis genotypes. G represented the most prevalent genotype. Additional genetic investigations of T. vaginalis carried out in Africa have consistently demonstrated the high prevalence of genotypes G and E. Masha and others (Masha et al ., 2017) The actin gene revealed the existence of eight distinct genotypes in a study on female gender workers in Zambia and the Democratic Republic of the Congo (DRC). The genotype E was the most predominant in the DRC, while the genotype G was most prevalent in Zambia (Crucitti et al ., 2008)

We concluded that the prevalence rate was estimated 8%. In diagnosing the disease, we found culture method more sensitive than the direct method ,and specimen collected by vaginal washout using phosphate buffer solution better than other swabs. Further, our results demonstrated a genetic variation of the parasite the most common genotype of T. vaginalis in upper Egypt is genotype E.

Conflict of interest:

The authors of this paper have no conflict of interest regarding the commercial, academic, political, financial and personal aspects.

Ethical approval:

It has been informed that there is no need of taking approval from the ethical committee to carry out the work on this purpose.

Contributions

All the authors have equally contributed to the successful writing of this paper and the works associated with it.

Funding:

The authors have not disclosed any funding.

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Trichomonas Vaginalis Genotyping in Upper Egypt

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