Parasitologists United Journal

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 9  |  Issue : 2  |  Page : 80--86

The endosymbiotic relationship between Trichomonas vaginalis and Mycoplasma hominis in Egyptian Women and its correlation with pathogenicity


Eman K El-Gayar1, Amira B Mokhtar1, Soha I Awad2, Rasha H Soliman1, Wael A Hassan3,  
1 Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
2 Department of Parasitology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence Address:
Eman K El-Gayar
Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia
Egypt

Abstract

Background Trichomonas vaginalis is the etiological parasite of trichomoniasis. Endosymbiotic Mycoplasma hominis can exist in T. vaginalis populations. However, its consequences are not yet known. Recently, T. vaginalis isolates positive for M. hominis as proven by PCR had greater cytopathic effects on human vaginal epithelial cells and also on Madin–Darby canine kidney cells in vitro. Objective This study aimed to detect the presence of M. hominis infecting Egyptian T. vaginalis isolates and to evaluate the pathogenicity of this association in vivo. Patients and methods Forty-five symptomatic and asymptomatic T. vaginalis isolates were obtained from Suez Canal and General Hospitals, Ismailia city, Egypt. All isolates were axenically cultivated in Diamond’s TYM medium, followed by DNA extraction and PCR using primer pair targeting 16S rRNA gene to detect M. hominis-infected isolates. Positive M. hominis PCR products were subjected to sequencing analysis. All isolates were experimentally inoculated intravaginally in female albino mice to assess the pathogenicity of different isolates. Results The detection rate of M. hominis-positive T. vaginalis isolates was 20% as determined with PCR. No statistically significant association was recorded between M. hominis-infected T. vaginalis among symptomatic and asymptomatic isolates. Experimental mice infection showed varying degrees of inflammation by the different isolates. Conclusion To our knowledge, this study is the first report of T. vaginalis infection by M. hominis among Egyptian isolates and it was deduced that the association of M. hominis and T. vaginalis does not affect the clinical presentation of vaginal trichomoniasis and does not cause enhanced pathological changes in infected mice.



How to cite this article:
El-Gayar EK, Mokhtar AB, Awad SI, Soliman RH, Hassan WA. The endosymbiotic relationship between Trichomonas vaginalis and Mycoplasma hominis in Egyptian Women and its correlation with pathogenicity.Parasitol United J 2016;9:80-86


How to cite this URL:
El-Gayar EK, Mokhtar AB, Awad SI, Soliman RH, Hassan WA. The endosymbiotic relationship between Trichomonas vaginalis and Mycoplasma hominis in Egyptian Women and its correlation with pathogenicity. Parasitol United J [serial online] 2016 [cited 2017 Jun 28 ];9:80-86
Available from: http://www.new.puj.eg.net/text.asp?2016/9/2/80/205169


Full Text

 Introduction



Trichomonas vaginalis is a worldwide flagellated protozoan that is considered as one of the most common causes of sexually transmitted disease [1]. Clinically, there is a great variation in disease presentation ranging from no symptoms to severe vaginitis in women or urethritis and prostatitis in men. Symptomatic women with vaginal trichomoniasis usually complain of vaginal discharge, vulvovaginal soreness, pruritus vulvae, dysuria, and dyspareunia [2]. In pregnant women, trichomoniasis may lead to preterm delivery, low birth weight, and increased infant mortality [3],[4]. Moreover, trichomoniasis predisposes individuals to HIV/AIDS and cervical and prostatic cancers [5],[6]. In immunocompromised patients, T. vaginalis can induce pneumonia, bronchitis, and oral lesions [7],[8],[9].

For infection to be established in humans, cytoadherence occurs to vaginal and cervical epithelial cells, and then binds and degrades the vaginal mucus [10]. Upon contact with vaginal epithelial cells, T. vaginalis changes its shape from pear shape to an ameboid form with the release of adhesion molecules as chemoattractants, which lead to attraction of more parasites to the infection site. The morphological changes lead to the formation of areas where tight association between parasite and target cell become evident [11].

Mycoplasma hominis is a type of bacteria, a member of the Mollicutes, frequently affecting human genitalia. The bacterium could be isolated from symptomatic and asymptomatic individuals, and, despite its unclear pathogenic mechanisms, it has been associated with pelvic inflammatory diseases, preterm labor, and low birth weight infants [12],[13]. It was shown that M. hominis could naturally infect T. vaginalis isolates and human epithelial cells [14],[15]. Several studies reported that M. hominis is a natural endosymbiotic bacteria of clinical T. vaginalis isolates regardless of geographic origin; it has been recorded in 75% [16] and 50% [17] of clinical T. vaginalis isolates. Vancini et al. [18] recorded an increase in the cytopathic effect of cultured human epithelial cells and Madin–Darby canine kidney cells infected by T. vaginalis harboring M. hominis.

Dessì et al. [19] described the cellular location of M. hominis in T. vaginalis using confocal microscopy, gentamicin protection assays, and double immunofluorescence. They showed that M. hominis can invade T. vaginalis and persist in its cytoplasm in the apical, basal, and equatorial sections. In contrast, Vancini and Benchimol [20] showed that M. hominis can enter T. vaginalis by endocytosis and anchor themselves by a polar tip to the trichomonad plasma membrane. Evidence of M. hominis replication within T. vaginalis cells was noted, and it was observed that the flagellate might provide the bacteria with resistance during human infection to environmental stresses such as host immunity and pharmacological therapies [19].

However, the exact nature of this intimate relationship is still unknown, but it has been proposed to be a mutually beneficial endosymbiotic relationship [21]. The influence of this association on pathogenicity has so far received little attention. It has been suggested that the symbiosis of M. hominis in T. vaginalis may be linked to metronidazole resistance in vitro [17],[22]. In an in vitro study, Vancini et al. [18] described the cytopathic effect on infected epithelial cells as increased intercellular spaces, decreased viability, and elevated destruction rate. There was also a higher ameboid transformation rate of the trichomonads and extreme phagocytic activity, indicating higher virulence behavior. Thus, the aim of our present study was to detect the association of M. hominis among T. vaignalis Egyptian isolates and to evaluate the pathogenic effects of M. hominis-infected T. vaginalis and M. hominis-free T. vaginalis infection on experimentally infected mice.

 Patients and methods



Type of study

An animal experimental study was performed between July 2015 and January 2016 at the Medical Parasitology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt.

Patient information and collection of clinical data

A thorough medical history was obtained from 260 women attending Obstetrics and Gynecology outpatient clinics of Suez Canal University and Ismailia General Hospitals, Ismailia, Egypt. Isolates obtained from patients complaining of one or more symptoms such as vaginal discharge, vulval pruritus, dysuria, and/or dyspareunia were considered as symptomatic, whereas isolates obtained from women attending the outpatient clinic for routine checkup, infertility, or family planning and having no suggestive symptoms were considered as asymptomatic. Each patient was subjected to complete history taking, pervaginal examination, and nonlubricated sterile speculum examination. All women who were in the childbearing period (18–45 years) denied a history of douching for at least previous 2–3 days, or use of antibiotics, antiprotozoal, or steroid treatments for the past 15 days.

T. vaginalis isolate collection

Two sterile vaginal cotton swabs were obtained from each participant in the study. The first swab was for wet smear examination, which was immediately examined using a clean glass slide with cover for motile T. vaginalis under ×10 and ×40 objectives. The second swab was immediately inoculated in Diamond’s TYM medium, pH 6.2, supplemented with 10% heat-inactivated horse serum, 100 U/ml penicillin, and 100 µg/mg streptomycin and subcultured every 2–3 days until an axenic culture was obtained [23]. The culture was examined daily for the presence of T. vaginalis trophozoites. If the parasite was not seen, the culture was incubated again for up to 1 week, with daily examination. If no trichomonads were seen, it was considered negative. In the exponential growth phase (4×106) parasites were placed in −70°C freezer for subsequent DNA extraction.

PCR amplification of M. hominis 16S rRNA

According to Mayta et al. [24], axenic cultures containing 4×106 trophozoites were gradually thawed on ice and then pelleted by means of centrifugation at 300 g for 5 min at 4°C. The cellular pellet was washed twice with PBS pH 7.2 at 4°C, and then DNA was extracted using a commercial extraction kit (Gentra Systems, Minneapolis, Minnesota, USA) following the manufacturer’s protocol. PCR primers for the specific amplification of a 334 bp DNA fragment of the 16S rRNA gene of M. hominis were used on T. vaginalis isolates as previously described by Blanchard et al. [25]. The sequences of the oligonucleotides were 5′-CAATGGCTAATGCCGGATACGC-3′ (RNAH1, forward) and 5′-GGTACCGTCA GTCTGCAAT-3′ (RNAH2, reverse). PCR reagents were used to make a 50 µl PCR reaction with 2 µl template DNA. The PCR cycle conditions for the M. hominis gene amplification were 40 cycles each at 95°C for 25 s, 62°C for 1 min, and 72°C for 1 min. Parallel reactions performed with distilled water without DNA were included as negative controls. Amplicons were analyzed on a 1% agarose gel in Tris–borate–EDTA buffer containing ethidium bromide and photographed under ultraviolet visualization.

Identification of M. hominis 16S rRNA by sequencing

The PCR products for the suspected M. hominis samples were purified and sequenced in an automated DNA sequencer ABI3730XL (Solgent Co. Ltd., Daejeon, South Korea).

Experimental mice infection

A total of 184 female albino balb/c mice, 4–6 weeks old, weighing 20–24 g, purchased from Veterinary Medicine Animal Lab, Suez Canal University, were used for in vivo study. Each isolate was inoculated intravaginally into four mice, and an additional control group (four mice) was inoculated with parasite-free culture medium. All mice in the test and control groups were under estrogen treatment throughout the experiment to favor the establishment of the intravaginal T. vaginalis infection. Estradiol treatment was initiated 2 days before the inoculation with the parasite [26], where each mouse was injected intraperitoneally with 0.1 ml of 500 μg estrogen on day 1 and every 2 days throughout the study. On day 3 (2 days after estradiol treatment), each mouse received 2×106 T. vaginalis trophozoites in a 15 μl volume intravaginally. On fourth, fifth, and sixth day (24, 48, and 72 h after T. vaginalis inoculation), the vagina of each infected mouse was washed repeatedly with 30 μl of room temperature Diamond’s TYM medium without antibiotics. The wash was pipetted into 12 ml TYM medium additionally supplemented with antibiotics and incubated at 37°C for a week after inoculation and checked daily for the presence of T. vaginalis trophozoites [26],[27]. All mice were euthanized on the seventh day after infection for histopathology.

Detection of pathogenicity of T. vaginalis isolates

The upper part of the vagina of each scarified mouse was fixed in 10% phosphate-buffered formalin and routinely processed and embedded in paraffin to form the blocks. Serial sections of 4 µm thickness were cut and stained with haematoxylin and eosin for histopathological examination [28]. According to Teras and Roigas [29], histopathological changes were scored from 0 to 4: no, mild, moderate, and severe.

Statistical analysis

The χ2-test was used to test statistical significance for categorical data. A P value less than 0.05 was defined as statistically significant.

Ethical considerations

Informed consent was obtained from patients to use their vaginal discharge samples in the study. The experimental animal studies were conducted in accordance with the international valid guidelines and they were housed independently in well-ventilated, filter-top cages and provided sterile rodent chow and water ad libitum. The animals were maintained in animal house at the Faculty of Medicine, Suez Canal University, at 25°C and with a relative humidity of 40–60%. The research was approved from the Scientific Research Ethical Committee, Faculty of Medicine, Suez Canal University.

 Results



The isolation rate of T. vaginalis in the study population

Of the 260 female participants recruited in the study, 188 (72.3%) manifested symptoms consistent with vaginal trichomoniasis and 72 (27.7%) were asymptomatic. T. vaginalis infection was found in 98 (37.7%) participants, 67 (68.4%) from the symptomatic group and 31 (31.6%) from the asymptomatic group. Fifty-three of them were excluded from the study due to infection with other concomitant microorganisms and/or fungal or bacterial overgrowth during cultivation. Forty-five isolates were successfully obtained and axenized. The main complaint in the symptomatic group was vaginal discharge (100%), followed by soreness (66%), pruritus vulvae (60%), dysuria (50%), and dyspareunia (46%). The minimum and maximum age among participants with trichomoniasis was 20 and 42 years, and the mean age was 31.4±5.75 years. The highest infection rate occurred among women above 25 years of age (83.2%).

Identification of M. hominis associated with T. vaginalis

M. hominis was detected in nine of the 45 (20%) T. vaginalis isolates, with no significant association between M. hominis-infected T. vaginalis isolates among symptomatic and asymptomatic patients ([Table 1]). Association of M. hominis-infected T. vaginalis isolated from symptomatic patients showed significant occurrence among patients suffering from dysuria, vaginal edema, and vaginal erythema ([Table 2]). M. hominis infection was detected in 20% (6/30) of symptomatic female patients and 20% (3/15) of the asymptomatic group. M. hominis infection was identified by the presence of a 334 bp band ([Figure 1]).{Table 1}{Table 2}{Figure 1}

Nucleotide sequence accession number

The 16S rRNA sequence of M. hominis was inserted in the GenBank NCBI/EMBL/DDBJ nucleotide sequence data libraries under the following accession no.: KU891983. All nine positive isolates for 16S rRNA M. hominis in the PCR reactions were also correctly identified as M. hominis by 16S rRNA gene sequencing when searched through NCBI blast, revealing 100% homology. The sequence of one M. hominis-positive T. vaginalis isolate is shown in [Figure 2].{Figure 2}

Experimental mice infection

The axenically cultured vaginal wash obtained from all mice inoculated intravaginally with T. vaginalis trophozoites showed positive cultures 24 h after inoculation and remained positive for up to 48 and 72 h.

Histopathology results

No statistically significant difference was recorded between pathological changes in vaginal tissue of mice experimentally infected by M. hominis-infected T. vaginalis isolates and noninfected ones. The histopathological scoring proved that 33.3% of M. hominis-infected isolates and 41.7% of noninfected isolates had severe inflammatory changes. However, 66.6 and 58.3% showed mild to moderate changes in the two groups, respectively ([Table 3]). The inflammatory changes revealed diffuse infiltrates of chronic inflammatory cells (lymphocytes, plasma cells, and macrophages, with scattered neutrophils) and congested blood vessels. In severe vaginal inflammation, nodular collections of chronic inflammaotry cells were also seen in the lamina propria ([Figure 3]a–[Figure 3]c).{Table 3}{Figure 3}

 Discussion



T. vaginalis is one of the most common protozoan parasites that can induce a considerable morbidity in infected patients. The spectrum of clinical trichomoniasis in women ranges from the asymptomatic carrier state to flagrant vaginitis [2]. Research studies have shown that the difference in surface carbohydrates and proteins lead to the emergence of different strains of T. vaginalis [30],[31],[32]. However, it is not clear whether the variable clinical outcome is influenced more by host factors or by phenotypic expression differences of individual T. vaginalis isolates [33].

It was shown that M. hominis has the ability to assault, survive, and replicate within T. vaginalis cytoplasm where it can get its food, shelter, and long-term survival [13],[19]. It was also noted that transmission of Mycoplasma spp. between individuals may be enhanced by its capability to live inside T. vaginalis isolates [17]. Our study is the first report in Egypt that detected M. hominis-infecting T. vaginalis isolates, and we used sequencing methodology to confirm this analysis. We found that 20% of T. vaginalis isolates were infected with M. homins, which is in agreement with two previous studies that detected similar lower prevalence of infected isolates (20 and 25%, respectively) [34],[35]. Reports on the prevalence of M. hominis-infecting T. vaginalis vary widely according to the geographical location. In China, 50% of the parasite isolates were infected by these bacteria [17], and in South Brazil it was 56.7% [36]. Furthermore, a study performed on 40 T. vaginalis isolates collected from Italy, Angola, and Mozambique showed that 37 (92.5%) isolates were infected with M. hominis. Moreover, the latter authors reported that T. vaginalis isolates [15] exhibit different susceptibilities to M. hominis as shown by variability in the number of M. hominis per cell.

Our PCR results indicated no significant association between M. hominis-infected T. vaginalis isolates among symptomatic and asymptomatic patients despite the significant occurrence among patients suffering from dysuria, vaginal edema, and erythema. The prevalence rate of M. hominis-infected T. vaginalis among symptomatic isolates was the same as in asymptomatic ones. In accordance, a previous study also did not record any enhanced pathological lesions or increase in symptomatic presentations. It was concluded that there is no obligatory association between the two microbes, suggesting that their presence together in the female genital tract, is not essential for the pathogenicity of either of the two pathogens [16]. Moreover, this result was also reached for men suffering from urethritis-like syndromes [37].

Several authors have focused on the initial events required to establish infection by T. vaginalis. Multiple mechanisms are incriminated, such as cellular adhesion, hemolysis, extracellular proteinases and cell detaching factor excretion, and the host inflammatory response [30],[38],[39],[40],[41],[42]. Adhesion of T. vaginalis to vaginal epithelium and release of cytotoxins that cause host cell lysis are extensively studied. However, the role of T. vaginalis in induction of inflammation as a part of its pathogenesis remains elusive. Fichorova et al. [43] described proinflammatory and anti-inflammatory responses to T. vaginalis through the activation of nuclear factor- kappa B (NF- κB) which induces production of proinflammatory cytokines such as interleukin (IL)-1β, IL-12, and TNF-α that are involved in innate and adaptive immunity. Other authors showed that T. vaginalis may inhibit the NF- κB activity of macrophages and induce neutrophil apoptosis through the activation of caspase 3 and reduced expression of the neutrophil antiapoptotic protein [44],[45]. Fiori et al. [46] hypothesized that one possible explanation for this variability could be related to the symbiotic relationship with M. hominis.

Trichomoniasis is one example of diseases in which host–parasite interaction affects the outcome of the infection [6]. To evaluate the pathogenicity of the parasite, a suitable model is needed. Mice have been considered as the most common animal used in experimental trichomoniasis. To prompt and establish infection successfully, pre-estrogenization must be carried out. This could induce estrus cycle, increase glycogen levels, and cause a slight drop in vaginal pH, which potentiate infection by T. vaginalis [47]. Several studies reported that there was an increase in parasite load, an elevated number of vaginal epithelial cells in vaginal discharge, and an increased ability to cause exfoliation of these cells in mice inoculated with symptomatic T. vaginalis isolates as compared with asymptomatic ones [48],[49],[50]. However, Honigberg [51] reported that symptomatic T. vaginalis isolates were able to stimulate strong chemotactic responses toward polymorphonuclear leukocytes, leading to higher inflammatory response manifested as leukorrhea as compared with those isolated from asymptomatic cases.

In the present work, there was no significant difference between pathological changes in vaginal tissue of mice experimentally infected by T. vaginalis isolates harboring M. hominis and T. vaginalis isolates not infected with M. hominis. In 2013, an in vitro study carried out by Fiori et al. [46] showed that symbiotically associated T. vaginalis and M. hominis elicited a synergistic effect over the proinflammatory response of human macrophages. T. vaginalis and M. hominis proved to be more potent inducers of inflammatory mediators such as IL-8, TNF-α, IL-1β, and IL-23. The researchers suggested that this happens in vivo as well, thus affecting the severity of the disease, and that this synergistic upregulation of inflammatory cytokines may have impact on several risks associated with trichomoniasis (HIV and cancer) believed to be linked to chronic inflammatory phenomena. Our results contrast these findings as we showed that there is no statistically significant difference between infected T. vaginalis isolates and noninfected ones when studied experimentally in vivo.

Conclusion

This is the first report studying the association between T. vaginalis infection and M. hominis among Egyptian T. vaginalis isolates, wherein no association was detected as regards the symptomatic status. Our study was limited by the small sample size because of the significant loss of isolates secondary to poor growth kinetics. Further research is needed on a larger sample size to investigate infection by T. vaginalis when symbiotically associated with M. hominis and to further elucidate the impact of M. hominis-infected isolates on patient clinical presentation.

Authors’ contribution: S. Awad suggested the research idea, shared in manuscript writing; E. El-Gayar planned the study design, shared in the experimental studies and manuscript writing; A.B. Mokhtar shared in the experimental studies and manuscript writing; R.H. Soliman shared in manuscript writing; W.A. Hassan assessed the histopathological results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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