Prevalence and Genotype of Trichomonas vaginalis among Men in Xinxiang City, Henan Province, China

Trichomonas vaginalis (T. vaginalis) could cause trichomoniasis through sexual transmission, which was globally distributed. In this study, the prevalence and phylogenetic analyses of T. vaginalis among men in Xinxiang were conducted. From October 2018 to December 2019, a total of 634 male clinical samples were collected, including 254 samples of semen, 43 samples of prostate fluid, and 337 samples of urine. These samples were examined by nested PCR and a total of 32 (5.05%) T. vaginalis-positive samples were detected. Among these samples, the positive rates of T. vaginalis in semen, prostate fluid, and urine were 7.87% (20/254), 4.65% (2/43), and 2.97% (10/337), respectively. Three actin genes were successfully isolated and sequenced from the 32 positive DNA samples, and the analysis of the sequence and phylogenetic tree showed that the three actin gene sequences exhibited 99.7%–100% homology to the published actin gene sequence (EU076580) in NCBI, and the T. vaginalis strains in the three positive samples were identified as genotype E. Our results demonstrate a notable genotype of T. vaginalis in the male population and provide insight into the performance of these genetic markers in the molecular epidemiology of trichomoniasis. However, further studies are needed to research the association between the genotype and the pathogenicity of T. vaginalis.


Introduction
T. vaginalis is a fagellated protozoan that parasitizes the urogenital tract of humans, which causes nonviral sexually transmitted disease. Te World Health Organization (WHO) estimated that there were about 156 million cases of trichomoniasis worldwide in 2016, accounting for almost half of the incidence of globally transmitted infections that year [1]. T. vaginalis parasitizes the vagina and urethra of women, causing Trichomonas vaginitis, cervicitis, and urethritis, increasing the risk of pregnancy complications, preterm delivery, and cervical cancer [2][3][4], and can also lead to infertility [5,6]. Moreover, T. vaginalis is also parasitic on the male urethra and prostate gland, causing urethritis, prostatitis, and epididymitis [5]. Te infection of T. vaginalis increases the risk of male infertility and prostate cancer [7][8][9]. In addition, trichomoniasis can also increase the risk of HIV transmission by two or three times [5,10,11]. However, 85% of women and 77% of men with T. vaginalis are asymptomatic [12].
Despite the public health importance and global distribution of trichomoniasis, there are still many unanswered questions about the various features of the T. vaginalis infection. Understanding the genetic characteristics of T. vaginalis is valuable for the prevention and control of human trichomoniasis [13]. Currently, a number of methods have been developed to study gene polymorphism of T. vaginalis isolates, which include multilocus sequence typing (MLST), microsatellite (MS) genotyping, and internal transcribed spacer (ITS) typing [14,15]. Phylogenies have already witnessed applications in numerous practical domains, such as in epidemiology [16], multiple sequence alignment [17], and prediction of gene and protein function [18,19]. Homology analysis of genomic sequences can be used to construct phylogenetic trees to evaluate the genetic diversity and evolutionary relationships of T. vaginalis [20,21]. High levels of sequence similarity can consistently produce accurate evolutionary trees [22].
So far, there are few studies on the genetic diversity and molecular epidemiology of T. vaginalis in China, and the infection data of male trichomoniasis has been lacking. In order to understand the infection status and genetic diversity of T. vaginalis in Xinxiang City, Henan Province, China, we evaluated the prevalence of T. vaginalis in men by detecting the trophozoites of T. vaginalis in male semen, prostate fuid, and urine with nested PCR. Te products amplifed by nested PCR were sequenced to analyze the genotype of T. vaginalis prevalent in men.

Ethics Statement.
Te study was reviewed and approved by the Ethics Review Committee of Xinxiang Medical University (Reference No. XYLL-2018S006). October 2018 to December  2019, we collected 634 samples, including 254 semen, 43 prostatic fuid, and 337 urine samples. Te prostatic fuid, urine, and semen were collected from men who went to the hospital outpatient clinic for routine examination.

DNA Extraction.
Te samples of semen and prostatic fuid were centrifuged at 2000 rpm for 5 min. Te collected urine was allowed to stand at room temperature for 1 hour, discarded the supernatant, and then centrifuged at 2000 rpm for 5 min. Te precipitation was used to extract DNA with Universal Genomic DNA Kit (CWBIO, Beijing, China) according to the manufacturer's instructions. Te DNA sample was preserved by freezing at −20°C before analyses.

Nested PCR.
Te actin gene (GenBank: AF237734) was selected as the target gene of nested PCR [23][24][25]. According to the primer sequences reported in the literature [24,26], as shown in Table 1, the external primers (Tv8S and Tv9R) and internal primers (Tv10S and Tv11R) were synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd. Te length of the amplifed target gene fragment was 1100 bp. PCR amplifcation was performed in two steps by a thermocycler (Bio-Rad, USA). Te frst stage of the PCR master mixture consisted of 1 μl DNA template, 12.5 μl 2 × Taq Plus Master Mix (Vazyme, Nanjing, China), 2 μl external primers (20 pmol each of Tv8S and Tv9R), and distilled water to adjust the fnal volume to in 25 μl. Te second stage of the PCR master mixture was composed of 2 μl frst stage PCR products, 25 μl 2 × Taq Plus Master Mix, 4 μl internal primers (20 pmol each of Tv10S and Tv11R), and distilled water in 50 μl of the fnal volume. Te frst amplifcation procedure consists of 35 cycles, and each cycle is composed of denaturation (95°C for 15 s), annealing (55°C for 15 s), and extension (72°C for 1 min). Te denaturation at 95°C was conducted for 3 min before the frst cycle, and the fnal extension was conducted at 72°C for 5 min after the last cycle. Tere were also 35 cycles in the second step. Te denaturation and extension are the same as the frst step, but the annealing temperature is 50°C. After PCR, we analyzed 10 μl PCR product through electrophoresis on 1% agarose gel in Tris-borate-EDTA bufer (TBE and pH 8.3) and then visualized it under UV light with GoldView I Nuclear Staining Dyes (Solarbio, Beijing, China).
Te length of the target gene was 1100 bp, which is only 28 bp shorter than the size of the actin gene's open reading frame. Te PCR products of positive samples were separated by 1% agarose gel electrophoresis, and the target DNAs were recovered by E.Z.N.A. ® Gel Extraction Kit (OMEGA, USA).

Sequence Analysis.
All recovered DNA products were sequenced by Sanger technology (GeneCreate, Wuhan, China) to determine the base sequence of the target gene. Clustal W was used to edit and align the sequence (https:// www.ebi.ac.uk/Tools/msa/clustalo/), which was compared with the reference sequences (EU076580, AF237734, EU076582, EU076584, EU076579, EU076578, EU076585, EU076583, EU076586, and AB468096) [23] obtained from GenBank by basic local alignment search tool (BLAST). To analyze the genotype of T. vaginalis, we constructed a phylogenetic tree with MEGA software (version 7.0) [27] using the neighbor-joining (NJ) algorithm, containing sequences representing T. vaginalis isolates in GenBank.

Statistical Analysis.
Diferences in T. vaginalis prevalence for diferent variables were analyzed using a chi-square test. Statistical analysis was performed using SPSS 20 software for Windows (SPSS Inc., Chicago, Illinois, USA). Te diferences were considered statistically signifcant if p < 0.05.

Nested PCR.
Te actin gene of T. vaginalis was amplifed by nested PCR, and agarose gel electrophoresis indicated that 1100 bp fragment was visualized among the T. vaginalis positive sample. Tere was no diference in the length of the positive amplicons ( Figure 1). All the positive DNA samples were recovered for sequencing. Table 2, a total of 634 samples were tested in this present study, and the total positive rate of T. vaginalis was 5.05%. Moreover, the positive rates were diferent in the male genitourinary tract samples of semen (7.87%), prostatic fuid (4.65%), and urine (2.97%), respectively. Tis result indicated that the positive rate of T. vaginalis in semen was signifcantly higher than that in prostatic fuid and urine (p < 0.05).

Sequence and Phylogenetic Analyses.
To evaluate the genetic diversity among T. vaginalis isolates from men, we sequenced the 32 positive DNA samples, but only 3 sample DNAs were sequenced successfully, which were submitted to GenBank (Accession Number: MZ014497, MZ014498, and MZ014499). We conducted a multiple alignments of the various T. vaginalis actin genotypes with the sequences found in this study and reference isolates and constructed a tree with the NJ algorithm ( Figure 2). Sequence analysis indicated that the percentages of the similarity between the three sequences and the reference sequence in GenBank (Accession Number: EU076580) were 99.7%, 99.8%, and 100%, respectively, which showed high sequence homology. According to the genetic distance between sequences, we draw the branch length in proportion. To assess the reliability of this tree, bootstrap analysis was carried out with 1000 replicates. Phylogenetic diversity analysis showed that the 451, 476, and 692 strains of T. vaginalis were sequenced successfully in the study with a dark dot belonged to an independent branch, and the three strains belong to genotype E.

Discussion
In 2016, WHO estimated that the global infection rate of trichomoniasis was 0.6% in males and 5.3% in females [1]. It has been reported that more than 276.4 million people worldwide have been infected with T. vaginalis [28,29]. In 2013, the prevalence of trichomoniasis was estimated to be about 58 million cases, and women were twice as likely to be infected as men [7]. Te latest epidemiological data on the prevalence of trichomoniasis in women and men in the United States were released in 2018 [30]. Te results showed that the infection rate among American adults (ages 18-59) from 2013 to 2014 was 0.5% for males and 1.8% for females. On the other hand, there are signifcant ethnic diferences in T. vaginalis between African-American men and women, with an estimated prevalence of 6.8% in the black population and 0.4% in the rest of the population. Seike et al. [31] found that the infection rate of T. vaginalis was 1.4% in Japanese male patients with urethritis and 1.0% in men without urethritis. Seo et al. [32] found that the infection rate of T. vaginalis was 4% in Korean men, 2.4%-8.2% in Croatian men with urethritis, and 1% in asymptomatic men with trichomoniasis [23]. In Iran, various studies have determined that the prevalence of trichomoniasis is between 2% and 8% and can reach more than 30% depending on culture and social status [25].
Te results of this study showed that the infection rate of T. vaginalis was 5.05% in males, which is higher than that of the United States, Japan, and South Korea, lower than that of African Americans, and similar to that of Iran. Patel et al. conducted an epidemiological study on T. vaginalis infection in the United States, indicating that the number of sexual partners was positively correlated with the rate of male T. vaginalis infection [30]. In addition, some studies showed that the prevalence of T. vaginalis was correlated with seasons to some extent, and the prevalence rate of T. vaginalis in the autumn and winter was higher than that in spring and summer [23,33]. Samples collected in this study were also concentrated in autumn and winter. Besides, patients with low immunity could also lead to a high rate of   T. vaginalis infection. It is impossible for us to determine the number of sexual partners of these men and the time of infection, so a long-term investigation is needed to understand the overall natural history of Trichomonas infection.
In an epidemiological study on T. vaginalis infection in the United States, the rate of T. vaginalis infection in males was lower than that in females [30]. Not so in Xinxiang city, the infection rate of male was higher than that of female. Due to the limited number of samples collected in this experiment, the data may be limited to some extent. Furthermore, we failed to obtain the subjects' age, occupation, ethnicity, educational background, sexual partner status, and whether there are other sexually transmitted diseases and failed to discuss from relevant aspects, which is also one of the limitations of this experiment.
In this study, we found that T. vaginalis infection was more common among men in Xinxiang, and our results showed that the positive detection rate of T. vaginalis was diferent in diferent parts of the urogenital tract. Te positive rate of the semen sample was the highest, while the positive rate of the urine sample was the lowest. Moreover, in a global epidemiological study of T. vaginalis, a zinc-rich environment in the prostate may prevent T. vaginalis from continuing infection, and the detection rate of T. vaginalis should be low [34]. Te low positive detection rate of urine samples may be due to the dilution of T. vaginalis by a large amount of urine. It may also be that the patient has discharged urine before collecting urine samples, resulting in the loss of T. vaginalis in subsequent urine sample collection. In addition, because the subjects might sufer from other urogenital diseases and the parasitic position of T. vaginalis might change with the time of infection, the type of positive specimens may not fully refect the anatomical site of infection [35]. Terefore, the precise location of T. vaginalis in the male genitourinary tract is worth further study to determine a sample type with a high detection rate.
Molecular typing can provide information about the genetic diversity, population structure, and epidemiological links of T. vaginalis in the population [36,37]. In addition, the investigation of genetic diversity helps to identify the source of pathogenicity, drug resistance, and recurrent infection of the parasite [24,38,39]. PCR-RFLP based on the actin gene has been extensively studied for the genotyping of T. vaginalis strains. In Turkey, the majority of T. vaginalis isolates were the actin genotype E (45%), the remaining  isolates were genotype G (7.5%), genotype N (1.5%), and genotype H (1.5%), two of which were mixed genotypes E and H (10%) [40]. In Kenya, fve actin genotypes were revealed by RFLP analysis, and the researchers used the latter method to detect fve genotypes: E (50%), G (13.6%), I (4.5%), N (27.3%), and P (4.5%) [41]. However, the overall prevalence of trichomoniasis in northern Iran was low. Te major genotypes were E (0.2%), G (0.2%), and I (0.08%) [42]. Based on the sequence of the 18S rRNA gene, the genetic variation of T. vaginalis was found in Anyang, Zhengzhou, Shangqiu, Luoyang, Pingdingshan, Zhumadian, and Xinyang of Henan Province. Te results indicated that T. vaginalis isolated from central China could be considered as a single population [43]. Genotype E (58.82%), H (17.65%), mixed 1 (17.65%), and mixed 2 (5.88%) were identifed as the T. vaginalis genotypes of the female infected in Xinxiang [23]. In this study, 3 out of 32 positive DNA samples were sequenced successfully, and these sequences were used to analyze genotypes. Te low success rate of sequencing may be caused by the low content of positive DNA. In our previous study [23], T. vaginalis was isolated and cultured, and then the trophozoites were harvested and extracted DNA. Phylogenetic analysis showed that T. vaginalis 451, 476, and 692 belonged to genotype E and existed in the same clade. Tis indicates that the infection of T. vaginalis between males and females in Xinxiang City has a high correlation. According to sequence alignment and phylogenetic analysis, the level of gene diversity was observed. We observed the nucleotide changes between the genotype E sequence and the reference genome. Sequence alignment showed that the "C" nucleotide was replaced by the "G" nucleotide ( Some studies have shown that the genotype of T. vaginalis originally comes from two diferent evolutionary branches [15,44,45]. However, only genotype E was found in our study, and no other actin genotypes of T. vaginalis (A, G, H, I, M, N, and P) were found. Due to the limitations of research methods, we could not obtain the gene sequences of all positive samples. In addition, the sample size was limited. Terefore, it is necessary to further study whether there are other genotypes of T. vaginalis in male infection in Xinxiang. More studies are needed in the future to clarify the association between certain genotypes and clinical manifestations. Previous research has established that metronidazole resistance is related to the mutation of the actin gene [45]. Bradic et al. found that two metronidazole-resistant single nucleotide changes occurred in the actin-like gene of Trichomonas foetus [46]. Terefore, regular genotyping of Trichomonas in diferent areas to master the latest information on the relationship between Trichomonas genotypes and drug resistance will help us better understand the drug resistance process of T. vaginalis, which is conducive to the prevention and control of trichomoniasis.

Conclusion
Tis is the frst report on the prevalence and molecular characterization of T. vaginalis in men from Xinxiang. Te results showed that the infection of T. vaginalis was common in men in Xinxiang. Phylogeny showed that the prevalent genotypes of T. vaginalis in men in the Xinxiang area were mainly type E, and all of them existed in the same evolutionary branch. Tese fndings provide insights for evaluating the performance of genetic markers in the molecular epidemiology of trichomoniasis. Further research is needed to improve our understanding of the epidemiology of T. vaginalis and to explore the relationship between genotypes and clinical manifestations.

Data Availability
Te data supporting the conclusions of this article are included within the article. Te sequences generated in the present study were submitted to the GenBank database under the accession numbers MZ014497, MZ014498, and MZ014499.

Ethical Approval
Te study was reviewed and approved by the Ethics Review Committee of Xinxiang Medical University (Reference No. XYLL-2018S006).

Conflicts of Interest
Te authors declare that they have no conficts of interest.