Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 152-157

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 152-157

Original Article

The presence of dsRNA virus in Trichomonas vaginalis isolates from symptomatic and asymptomatic Indian women and its correlation with in vitro metronidazole sensitivity

N Malla¹, P Kaul¹, R Sehgal¹, I Gupta²

¹ Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh - 160012, India
² Department Obstetrics and Gynaecology, Postgraduate Institute of Medical Education & Research, Chandigarh - 160012, India

Correspondence Address: N Malla Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh - 160012 India drmallanancy@gmail.com

Code Number: mb11035

PMID: 21654110

DOI: 10.4103/0255-0857.81801

Abstract

Keywords: Double stranded RNA virus, metronidazole, trichomoniasis, Trichomonas vaginalis, Trichomonas vaginalis virus

Introduction

Trichomonas vaginalis, the aetiological agent of human trichomoniasis, is a parasitic protozoan with 170 million infected cases worldwide. The disease encompasses a broad range of symptoms ranging from a state of severe inflammation and irritation to a relatively asymptomatic carrier state. In India, the prevalence rate varies between 6 and 10%. ^[1],[2],[3] The disease has important medical, social and economical implications. Even though the parasite is one of the major non-viral causes of sexually transmitted disease, the virulence markers of the parasite have not been clearly delineated. Putative factors include adhesion proteins, β haemolytic activity, cysteine proteinases and presence of double stranded (ds) RNA virus.^[4],[5] Humoral and local antibody responses, isoenzyme analysis ^[6] and Restriction Fragment Length Polymorphism (RFLP) pattern ^[7] are not able to differentiate clearly between symptomatic and asymptomatic isolates. It is suggested that the Random Amplified polymorphic DNA (RAPD) technique might be helpful to delineate the pathogenic mechanism(s) for its virulence. ^[8]

Specific viruses have been identified among parasitic protozoa, and Trichomonas vaginalis virus (TVV) is believed to be the first virus positively identified in a protozoan. ^[9] The genome of TVV consists of a 5.5-kb dsRNA. Infection of trichomonads by dsRNA viruses causes upregulation of synthesis and surface expression of a highly immunogenic protein, P270. ^[10] The presence of dsRNA viral infection of T. vaginalis is associated with differential qualitative and quantitative expression of cysteine proteinases, ^[11] which are linked with cytoadherence, cytotoxicity, and degradation of basement membrane components. ^[12] It is reported that dsRNA viruses induce various phenotypic changes that may impact T. vaginalis virulence. ^[11],[13] TVV has no known lytic cycle, and attempts to infect uninfected isolates have been unsuccessful. ^[14] Therefore, it is probable that the virus is acquired exclusively through vertical transmission, making its presence or absence a useful genetic marker.

Metronidazole is the drug currently licensed for treatment of human trichomoniasis in the United States ^[15] and is the main drug of choice for treatment of this infection in India. Parasite resistance to metronidazole has been reported. Further, it has been demonstrated that T. vaginalis isolates harbouring virus are significantly more closely related to each other than to the isolates without virus, and high level of relatedness is indicated among isolates with in vitro metronidazole resistance. ^[15] The present study was undertaken to assess the presence of TVV in fresh and long-term cultivated Indian isolates from symptomatic and asymptomatic women and to correlate its presence with symptomatology/virulence and in vitro drug sensitivity.

Materials and Methods

Subjects

One thousand women in the childbearing age group attending the Obstetrics and Gynaecology out-patient department were selected at random for the study after taking their due consent. The patients attending the clinic with the complaints of vaginal discharge, itching, dysuria and dyspareunia were considered as symptomatic (S), and patients attending for antenatal/postnatal checkup and family planning advice without any symptoms suggestive of trichomoniasis were considered as asymptomatic (AS). ^[8] Vaginal swabs and urine samples were collected from each subject.

Parasite detection and isolation

Vaginal swabs and urine samples were subjected to wet smear examination and culture techniques for detection and isolation of parasites. ^[16]

Axenisation of isolates: Axenisation of isolates was achieved by adding antibiotics, penicillin (1000 units/ml)and streptomycin (1000 μg/ml), in the first three to five subcultures.

Long-term cultivation: The isolates from 15 symptomatic and 15 asymptomatic women maintained in culture by serial subculturing for 6 months-2 years were labelled as long-term cultivated isolates.

Fresh and respective long-term culture maintained 30 isolates (15 from symptomatic and 15 from asymptomatic women) were subjected to assess the presence or absence of dsRNA virus. The same fresh isolates were earlier subjected to RAPD analysis. ^[8]

Evaluation of dsRNA viral infection

Presence of dsRNA virus was detected as detailed earlier, ^[14] with slight modifications. Briefly, parasites were harvested at late-logarithmic or stationary phase of growth and were washed twice in ice-cold phosphate buffered saline (PBS). Pellets were suspended in 500 μl of PBS containing 10% sodium dodecyl sulphate (SDS). Total nucleic acids of Trichomonas were obtained and mixed with an equal volume of phenol. The preparation was microfuged at maximum speed for 3 min. The aqueous phase was transferred to microfuge tubes containing 500 μl of phenol and mixed, and this procedure was repeated. The aqueous phase was then mixed with an equal volume of chloroform, following which the new aqueous phase was mixed with 50 μl of 3 M sodium acetate and 1 ml of 100% ethanol. The nucleic acids were precipitated at -70°C for 30 min and pelleted by centrifugation for 20 min. The pellet was washed with 7% ethanol in diethylene pyrocarbonate (DEPC) treated water. After drying, the pellet was dissolved in 20 μl of DEPC treated water containing electrophoresis loading dye. Nucleic acids were stained with ethidium bromide after electrophoresis in 1% agarose gel and visualised under gel documentation system.

To determine whether the dsRNAs were possibly protected within viral capsids, cytoplasmic fraction of lysed parasites was treated with RNase A (100 μg/ml) or with proteinase K (50 μg/ml) followed by RNase A. The preparations were then extracted with phenol chloroform and precipitated with ethanol before agarose gel electrophoresis of nucleic acids. ^[17]

Drug sensitivity

The susceptibility of T. vaginalis isolates to metronidazole was determined in vitro, as detailed earlier. ^[18] Trichomonads from log phase of growth (5 × 10 ⁴ /ml) were washed in PBS (pH 7.2) twice. Metronidazole (Sigma-Aldrich, India) stock solution was prepared (2 mg/ml) fresh for each experiment. Standard round bottom well microtitre plates were used and 100 μl media was put into each well, followed by addition of 100 μl of drug suspension. Twofold serial dilutions were made. To each well, 100 μl of parasite suspension was added and plates were incubated at 37°C for 48 hours. The minimum lethal concentration (MLC) was determined as the lowest dilution of metronidzole in which no motile organisms were observed microscopically. Each assay was performed twice for each isolate and mean reading was considered as the reading for that sample. Dimethyl sulfoxide was used as drug carrier control.

Ethical clearance

The study was granted clearance by the Institute Ethical Committee.

Results

Out of the 1000 women, 537 were symptomatic and 463 asymptomatic. T. vaginalis was detected in 38 subjects, of whom 22 (57.9%) were symptomatic and 16 (42.1%) were asymptomatic. Out of total 537 symptomatic subjects, 57.3% were having vaginal discharge; 20.8% had vaginal discharge and itching; 15.8% had vaginal discharge, itching and dysuria; and 5.95% had vaginal discharge, itching and dyspareunia. On per speculum examination, out of 22 symptomatic patients, 12 (54.5%) showed vaginal discharge as the main presenting symptom, while all the 16 asymptomatic subjects had normal appearance of vagina and cervix. ^[8]

Double stranded RNA virus

Electrophoresis of RNA extracted from 30 isolates (15 fresh and 15 long-term cultivated) was performed in agarose gels with extended separation time and low voltage. Under these improved analytical conditions, the apparent single species of dsRNA was resolved into three segments of RNA with sizes of 1.5, 2.5 and 5.5 kb. The three bands were present in all the 30 fresh isolates irrespective of symptomatology of subjects. After long-term cultivation, three bands (1.5, 2.5 and 5.5 kb) were observed only in 7 (3 from symptomatic and 4 from asymptomatic women) out of 30 isolates [Figure - 1].

No statistically significant association was found between isolates from symptomatic and asymptomatic women and presence of TVV (P > 0.05), while significant difference was found for the presence of TVV in fresh Versus long-term cultivated isolates (P < 0.05).

Drug sensitivity

Fresh isolates: Drug sensitivity pattern of fresh isolates from 15 symptomatic and 15 asymptomatic women indicated that MLC range varied from 16 to 31 μg/ml (Mean = symptomatic 28.0 ± 6.21; asymptomatic 26.0 ± 7.32). MLC ≥ 50 μg/ml was considered resistant to metronidazole ^[15] . All fresh isolates were found to be sensitive to metronidazole.

Long-term culture maintained isolates: The drug sensitivity pattern of 30 long-term culture maintained (6 months-2 years) isolates from 15 asymptomatic and 15 symptomatic women indicated that the MLC range varied from 31 to 250 μg/ml. Out of 30 isolates, 24 were found to be resistant to the drug (MLC values ≥50 μg/ml). Out of the six sensitive isolates, two were from symptomatic and four were from asymptomatic women following in vitro culture maintenance for 2 years [Table - 1].

No significant difference in drug sensitivity pattern was found in both fresh and long-term maintained isolates between symptomatic and asymptomatic women. Significant difference was found among fresh versus long-term culture maintained isolates both for isolates from symptomatic and asymptomatic subjects (P < 0.05).

Correlation of presence of Trichomonas vaginalis virus and drug sensitivity pattern

Fresh T. vaginalis isolates: All 30 fresh isolates, whether from symptomatic or asymptomatic women, were harbouring TVV and were also found to be sensitive to metronidazole (MLC ≤ 50 μg/ml). This correlation was found to be statistically significant (P < 0.05).

Long-term culture maintained isolates: TVV was found in 7 out of 30 isolates. Out of these seven isolates harbouring virus even after 2 years (till the time isolates were followed up), six were sensitive to metronidazole and one isolate showed borderline drug resistance. Out of these six drug sensitive isolates, two were from symptomatic and four were from asymptomatic women. The association between the absence of TVV and drug resistance in long-term culture maintained isolates was found to be statistically significant (P < 0.05) [Table - 2].

Discussion

The relationship between the presence of a dsRNA virus and phenotypic variation in T. vaginalis shows that the virus-parasite interaction may have consequences for the host during infection. Virus harbouring trichomonads synthesise an increased amount of a highly immunogenic phenotypically varying outer membrane protein compared with viral uninfected T. vaginalis isolates. This observation is corroborated by finding upregulated levels of phenotypically variable immunogen mRNA. ^[10],[19] The upregulation of major immunogen expression, along with its surface localisation, appears to be aborted when the dsRNA virus is lost through in vitro cultivation of the parasite. ^[13] In the present study, TVV was found in fresh isolates both from symptomatic and asymptomatic women. These results are in agreement with an earlier report ^[4] whereby dsRNA viral infection of T. vaginalis was not associated with the presence of discharge, dysuria, genital pruritis, genital irritation or odour.

Trichomonads harbouring viruses have been reported to contain at least three dsRNA segments (5.5, 4.8 and 4.3 kb) and the reports suggest segmented nature of the TVV. ^[17] Different studies from different locations indicate different sizes of the segments of TVV. ^[20],[21] It is suggested that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously. ^[22] In the present study, three unique segments (5.5, 2.5 and 1.5 kb) of dsRNA were detected. Importantly, the resolution of these segments was best achieved with dsRNA obtained during stationary phase of parasite growth, when replication of these dsRNA elements appears to be complete. ^[17] During the logarithmic phase of growth, three dsRNA segments extracted from these cells appeared as a broad smear in agarose gels, thus making it difficult to visualise three different segments. This latter result may be due to detection of viral replicate intermediately of many sizes, which are indicative of virus replication during the rapid phase of trichomonal growth. T. vaginalis encounters a highly complex and constantly changing host environment during infection and different growth conditions may affect synthesis of these segments or their induction. The difference in the band sizes found in present study (1.5, 2.5 and 5.5 kb) and earlier study ^[17] may be due to the differences in strains in different geographical areas and the parasite growth under different cultivation conditions.

A clear trend for a positive correlation between pathogenicity of T. vaginalis strains for natural and experimental hosts and their generation times in vitro is reported.^[23] It is concluded that low growth rates or energy metabolism in vitro of pathogenic strains of T. vaginalis could be due to their greater dependence upon the host environment for some nutritional factors. It is suggested that trichomonads which had lived for prolonged periods in a nutrient-rich environment became dependent on some factors present in such a milieu. Therefore, in the present study, it was desired to assess the presence of TVV in fresh and long-term culture maintained isolates. However, till date, there have been limited studies regarding the presence or absence of TVV in fresh versus long-term culture maintained isolates. ^[13],[24] The data indicated that phenotypic variation generates negative phenotype trichomonads with enhanced virulence exhibited by their potential for immune evasion, capabilities and rates of contact dependent disruption and killing of cells in monolayer cultures. Two strains, IR 78 and RU 375, did not undergo phenotype variation and have remained without any variation for more than 4 years. ^[24] This supports the results of the present study whereby seven isolates showed the presence of TVV till 2 years (till the time isolates were maintained) following in vitro culture and it appears that it may take more than 2 years to lose TVV during in vitro cultivation. Trichomonal isolates capable of phenotypic variation possess dsRNA, whereas the absence of T. vaginalis surface expression of the major immunogen corresponds with the loss of viral dsRNA. The study showed that 11 out of 15 fresh isolates initially comprised heterogenous populations for monoclonal antibody reactivity and all 11 isolates possessed the dsRNA. After serial in vitro cultivation for several months, the remaining eight isolates lost the dsRNA upon long-term growth and comprised totally negative phenotype parasites. ^[13] Shaio et al., in 1993, had shown that among 12 isolates of T. vaginalis, 9 were infected with TVV, and upon long-term cultivation (>9 months), 3 lost the virus during the passage process. ^[20]

In the present study, fresh isolates of T. vaginalis, both from symptomatic and asymptomatic women, were found to be sensitive to metronidazole in vitro (MLC range 16-31 μg/ml). It is suggested that the assays must be performed under aerobic conditions as the strains responsible for clinical resistance reported so far have all displayed the aerobic type of resistance. ^[25] Therefore, in the present study, metronidazole sensitivity assay was carried out under aerobic conditions. The in vitro results of present study are also corroborated by the results obtained on in vivo treatment of patients from whom the isolates were obtained. All the patients got cured after treating both partners with metronidazole, as indicated by repeat examination of vaginal swabs and urine samples following treatment.

Another interesting finding of the present study is that all the 30 isolates were found to be sensitive to metronidazole when freshly isolated, while after long-term cultivation only six were found to be sensitive to the drug and these six isolates sensitive to drug were still showing the presence of TVV. In other words, it was found that out of seven isolates harbouring TVV after long-term cultivation (maximum 2 years), six were sensitive to metronidazole and only one had shown borderline resistance, which can be justified on the basis that the remaining one isolate might be ready to lose the virus and had become resistant recently as it showed only borderline resistance. This correlation of absence of TVV and drug resistance in the present study is supported by an earlier study. ^[23] The results of the present study also corroborate another report ^[15] which stated that isolates with TVV were significantly more likely to be susceptible to metronidazole, whereas isolates with the ITS C66T mutation and without TVV were significantly less likely to be susceptible. It was concluded that metronidazole resistance exists in a closely related group of isolates, indicating that only one or few mutations have occurred which result in resistance. The present study suggests that some genealogical lines of T. vaginalis may be genetically predisposed for the development of metronidazole resistance and it might be possible to identify a marker for resistance that could lead to improved treatment strategies.

To conclude, the study showed that TVV was present in all the fresh Indian isolates both from symptomatic and asymptomatic women and suggests that the presence of TVV alone may not be associated with the establishment of symptomatic infection and/or may not serve as a virulence marker. Further studies are desired for molecular characterisation of viral RNA found in T. vaginalis and establishment of conditions which will allow for infection of isolates without detectable virus and presence of viral dsRNAs may eventually elucidate the relationship between the virus and virulence properties of this sexually transmitted protozoan. All the fresh isolates were sensitive to metronidazole in vitro and all the patients were also cured of infection following treatment, thereby also suggesting in vivo sensitivity. The positive correlation of presence of TVV and drug sensitivity in all the fresh Indian isolates and absence of TVV and drug resistance in few isolates following long-term cultivation further supports the earlier reports from other countries.

References

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