الفهرس 
Trichomonas vaginalisOnly 14 pages are availabe for public view
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Abstract
Trichomoniasis caused by the parasite T. vaginalis is considered the most frequent cause of non-viral sexually transmitted infections in the world and is responsible for more than 180 million new cases each year worldwide. Current knowledge of T. vaginalis population genetics has been limited by a lack of appropriate tools. Crude genotyping markers such as RAPD and RFLP indicated genetic variation among T. vaginalis isolates and inconclusively detected evidence of population structure. These methods, however, are highly sensitive to contaminating DNA or to slight variation in conditions, which may influence the interpretation of data collected with these techniques. Moreover, these studies have yielded discordant results for a number of different phenotypes including metronidazole resistance, geographical distribution, the presence of a linear double-stranded RNA virus known as T. vaginalis virus and clinical manifestation of infection in patients.
The aim of the present study was to use multilocus sequence typing (MLST) to identify genotypes among Egyptian isolates of T. vaginalis. In part the study aimed to evaluate possible relationships between these genotypes and clinical features of trichomoniasis and determination of phylogenetic relationships of the Egyptian isolates in comparison with reference strains.The study was done during the period from May 2015 to July 2017 on 300 vaginal washout samples collected from women aged 20-45 years old suspected for trichomoniasis, attending the Early Cancer Detection Unit of Gynecology Obstetrics Hospital Ain Shams University, Outpatient Clinics of Cairo University and Ministry of Health Hospitals. The collected washouts were examined by direct wet mount technique and in vitro culture on TYM media was done for positive isolates. Determination of in vitro metronidazole resistance was done by estimation of the MLC for the different isolates. DNA was extraction from the samples and PCR amplification of the target three house-keeping genes (P1, P8, P13) was done followed by sequencing of the amplification products. Determination of genetic diversity by multilocus sequence typing and sequence analysis with reference strains retrieved from GenBank was done.
The results showed that, out of the 300 collected washout vaginal samples, 12 (4.0%) proved microscopically positive for T. vaginalis trophozoites. All positive patients complained of vaginal discharge (100%). Additional complaints were pruritus 9/12(75%), burning micturition 3/12 (25%) and dyspareunia 2/12(16.6%).
The 12 isolates exhibited different growth characteristics in terms of duration of log phase and growth peaks reached. Isolates 1, 2, 6, 8 and 10 had a log phase of 48 hours. Isolates 4, 5, 7, 11 and 12 had a log phase of 72 hours. Isolates 3 and 9 had a log phase of 96 hours. Regarding the mean count of the parasite, isolates 9, 10, 7, 5, 3, 6 had greater highly significant counts than the other isolates (p value ≤ 0.001) at 24, 48, 72, 96, 120 and 144 hours, respectively. The means of growth peaks of the 12 isolates at the end of the cultures’ log phase revealed that isolate 10 had the highest highly significant growth peak of 150.25±3.13 at 48 hours, and isolate 3 showed the least growth peak of 40.5±21 at 96 hours (p value ≤ 0.001).
In total, among the 12 positive T. vaginalis samples, PCR gave positive amplification in 5 (41.7 %) at the levels of P1, P8 and P13. Three (25%) gave positive result at the levels of P1and P13 and one (8.3%) gave positive at the levels of P1 and P8. Two samples (16.7%) gave positive result at the level of P8 alone. It is noteworthy that one sample (8.3%) gave negative results at the 3 loci, isolate 7, although it was successfully amplified by TVK3/7 PCR at 300bp.
After purification of PCR products, sequencing revealed percentage of identity that ranged between 99-100% between reference strains found on Genbank and our obtained sequences at the 3 loci.
Sequence diversity of the MLST scheme revealed twenty-six polymorphic nucleotide sites among the 10 (as 2 excluded) isolates. The number of polymorphic sites for each locus ranged from 4 to 14. The calculated nucleotide diversity for these loci ranged from 0.00473 to 0.00706 differences /site. Each distinct nucleotide sequence in a locus was considered as a different allele. The number of different alleles for each locus ranged from 3 to 5. Each isolate was assigned an allelic profile or sequence type (ST) based on the alleles located at each of the 3 loci in that isolate. Isolate 1 sequenced at the 3 loci with ST (1, 1, 1) for the 3 genes. Five isolates (2, 5, 8, 9, 10) sequenced at 2 loci with different STs: isolate 2 with STs (1, 4) at p8 and P13, isolate 5 with STs (1, 3) at P1 and P13; isolate 8 with STs (2, 3) at P1 and P8; isolate 9 with STs (3, 3) at P1 and P13; isolate 10 with STs (2, 2) at P1 and P13. Four isolates (2, 4, 11, 12) sequenced at a single locus: isolate 3 had ST 1 at P8; isolate 4 had ST 2 at P8; isolate 11 had ST 4 at P8; and isolate 12 had ST 5 at P8.
Sequencing of the amplification products was successful in 10 out of 12 isolates (83.3%). Two isolates were discarded from sequence analysis; number 6 due to failure of PCR amplification and number 7 due to short sequences. In total at the level of P1, 4 isolates (20 %) belonged to type I (20 %) and one belonged to type II. At the level of P8, 5 (71.5 %) belonged to type I and 2 (28.5 %) belonged to type II. At the level of P13, 4 (80 %) belonged to type I and one (20 %) belonged to type II. At the level of one or more genes, combined level, 70 % belonged to type I, 20% belonged to type II and one isolate No. 8 (10 %) exhibited mixed type.Clinical data of positive cases was correlated with T. vaginalis genotypes. Although patients infected with genotype I were older than patients infected with genotype II, no statistical significant difference was found between genotype and age of patients. Regarding the character of vaginal discharge, the present study revealed that patients infected with genotype I presented mostly with mild watery yellow to white discharge (71.5 %). One patient of genotype II presented with mucoid bloody discharge. In the present study vaginal examination revealed variable clinical findings with congested cervix the commonest finding in genotype I. Severe pathology in the form of bleeding on vaginal examination was detected in both genotypes. Again, these findings were statistically insignificant between both genotypes.
No significantly statistical difference was found between growth rates of parasites belonging to genotype I and genotype II, although the latter grew slowly. Isolate 3 and 9 typed as genotype II had the longest log phase of 96 hours. Comparison of the growth peaks of the isolates and number of parasites at the end of log phase, revealed that parasites belonging to genotype I had significantly higher numbers (p = 0.08). Isolates 3, 9 typed as genotype II had significantly the least numbers of parasites at the end of log phase; 40.5+2.3, 63.5+1.7, respectively. This characteristic is likely to provide an advantage in transmission. However, faster growth rates are also frequently associated with increased damage to the host.The present study, as with other studies, proved the genetic diversity of the Egyptian isolates at the level of single and combined loci analyzed by MLST. Genotyping of the isolates has also shown that they comprised two types, genotype I, constituting 70 % of the isolates and genotype II, 20 %. Mixed infection was detected in 10 % of cases. A limitation of the present study is that the sample size of patients with available clinical and demographic data was small and consequently the number of isolates was small. However, the study has supplemented the importance of MLST in investigating the genetic diversity of the Egyptian isolates of parasites for better understanding their molecular epidemiology. It is recommended that a future larger multicenter study should be done, whereby a larger number of isolates should be obtained from both females and males.