Study of Trichomonas Vaginalis and Bacterial Vaginosis in Pap smear at a Tertiary Health Care Centre of Nepal

Cervical-vaginal infections are common problems in women of reproductive age presented with clinical features like white vaginal discharge, foul smelling odor and pruritis. Trichomonas vaginalis, Candidia and Gardnerella vaginalis are responsible for infectious vaginitis in creating anaerobic environment favoring Bacterial vaginosis caused by organisms like Gardnerella vaginalis, Chlamydia Trachomatis. Papanicolaou (Pap) smear examination is the simple, quick, painless routine screening test used for identification and detection of precancerous changes of uterine cervix as well as the cervico-vaginal infections resulting from bacterial, fungal and candida infections. The prime objective of the study was to identify the age wise distribution of cervical-vaginal infections and determine the frequency of Trichomonas vaginalis (TV) and Bacterial vaginosis (BV). A descriptive cross-sectional study was carried out in the Department of Pathology, Nepal Medical College and Teaching Hospital, Kathmandu. A total of 163 cases of TV and BV diagnosed on Pap smear were taken for the study. However, 564 Pap smears were received in the Department of Pathology for cytological evaluation during the study period. Once the smears were stained, the presence of clue cells, cannon balls and Trichomonas were observed. Out of 564 Pap smears received during the study period, 163 cases were of BV, TV and combined BV and TV. These 163 cases were included in our study. The age ranged from 20 to 65 years with majority (19.1%) of the women belonging to 35-39 years and the mean age was 38.18 ± 10.18 years. There were 97 cases (17.2%) of BV, 50 cases (8.9%) of TV and 16 cases (2.8%) of TV and BV co-infection. Bacterial vaginosis was the most predominant type of cervicovaginal infection followed by TV infection and TV and BV co-infections. Trichomonas vaginalis may create an environment favoring the development of BV. Pap screening could be the beneficial tool in early disease diagnosis and helps in preventing further complications by starting appropriate treatment. 1Department of Pathology, 2Department of Obstetrics and Gynecology, Nepal Medical College Teaching Hospital, Attarkhel, Gokarneshower-8, 3National Ayurveda Research and Training Center, Kirtipur, Kathmandu, Nepal Corresponding author Dr. Rakesh Pathak Assistant Professor, Department of Pathology, Nepal Medical College Teaching Hospital, Attarkhel, Gokarneshower-8, Kathmandu, Nepal Email: dr_rakesh@hotmail.com Orcid ID: 0000-0003-0780-7416 DOI: https://doi.org/10.3126/nmcj.v22i1-2.29926


INTRODUCTION
Cervical infections are the most commonly observed health issues in women of reproductive age group  years) that usually present with white vaginal discharge, foul smelling odor and pruritis. 1 Trichomonas vaginalis (TV), Candidia and Gardnerella vaginalis are responsible for nearly 90% of diseases with infectious vaginitis. 2 A flagellate protozoan, TV is sexually transmitted and helps in creating anaerobic environment favoring Bacterial vaginosis (BV) caused by organisms like Gardnerella vaginalis, Chlamydia trachomatis. 3 Anaerobic bacteria are the most common agents that cause vaginitis/ vaginosis which lead to vulvo-vaginal candidiasis, BV and TV infection. 4 Most of the bacteria-induced infections can easily be diagnosed by the Papanicolaou (Pap) smear examination. It is a simple, quick, painless routine screening test that is mostly used for identification and detection of precancerous dysplastic changes of uterine cervix as well as the cervico-vaginal infections resulting from bacterial, fungal and candida infections. 5 In the case of Nepal, women living in mountainous and rural areas are mostly illiterate and are married at early age having limited access to health care and they are at particular risk of late diagnosis of cervical cancer and other cervico-vaginal infections with the peak incidence in the age group 45 to 65 years. 6 The wide variety of pathogens cause cervical, vaginal and other infections resulting the identification of infectious agent more challenging. Physical examination of the patient, history and the appropriate tests like Pap smears are suggested for the effective diagnosis and treatment of such infections. 7 The objective of this study was to identify the spectrum of causative agents responsible for cervico-vaginal infections and determine the frequency of TV and BV in Pap smears received in the Department of Pathology at Nepal Medical College Teaching Hospital (NMCTH), Attarkhel, Gokarneshwor-8, Kathmandu.

MATERIALS AND METHODS
The present hospital based cross sectional study was carried out in the Department of Pathology, NMCTH, Kathmandu from September 2019 to December 2019. With the reference of study done by Vandana et al, 8 the calculated sample size for the study was 95. However, 163 Pap smears with TV, BV and combined TV and BV coinfection were included out of 564 Pap smears received during the study period. Symptomatic, asymptomatic and pregnant women visiting Gynecology Department were enrolled and screened with Pap smears. The Pap smear slides were fixed in 95% ethanol for minimum of 15 minutes and sent to the Department of Pathology for cytopathological examination. The consent was taken in the Gynecology OPD before taking the Pap smear. Once the Pap smears were received in the Department of Pathology, they were checked for fixation and slides were stained by conventional method.

Papanicolaou Method:
After alcohol fixation, the smears were removed from fixative and rinsed in water. Then the smears were stained with Harris's Hematoxylin for 5 minutes, rinsed in water for 2 minutes and differentiated in 0.5% aqueous HCl for 10 seconds. These smears were rinsed in water, blued in Scott's water for 2 minutes and again rinsed in water for 2 minutes. Then the smears were dehydrated in 70% and 95% ethanol for 2 minutes each. Then the slides were stained in OG6 for 2 minutes and rinsed in 95% alcohol for 2 minutes and stained in EA 50 for 3 minutes. Finally it was rinsed in 95% alcohol for 1 minute before mounting.
These stained slides were examined under binocular microscope (Nikon Eclipse E200). The Bethesda system for reporting cervical pathology 2014 was followed for reporting. Besides diagnosing the intraepithelial lesions and carcinoma, presence of clue cells, cannon balls (pushballs polyballs) and filmy hazy background were noted. The diagnosis of shift in vaginal flora suggestive of BV was given if there were presence of clue cells (more than 20%) and hazy background due to proliferation of coccobacilli. The coccobacilli were visualized as clumps of round cocci organism in Pap stain. Squamous cells covered by a layer of coccobacilli obscuring the cell membrane known as clue cells were observed. Filmy background was seen due to large numbers of coccobacilli.
Similarly, identification of one or more of the following morphological characters was considered conclusive for TV, pear-shaped, oval to round cyanophilic organism ranging in size from 15-30 microns ; pale nucleus, vesicular, and centrally located with eosinophilic granular cytoplasmic. Few associated background changes like mature squamous cells with small perinuclear halo and 3-dimensional clusters of neutrophils (polyballs) were included.

Statistical analysis:
The data were entered in MS Excel 2016 and then analyzed statistically. The mean age and age wise distribution of TV, BV and combined TV and BV cases were found out.

RESULTS
Out of 564 Pap smear samples received in the Department of Pathology, 163 (28.9%) Pap smears were diagnosed with BV ( Fig. 1), TV (Fig. 2) and TV and BV co-infection (Fig. 3)    The important complication of BV as cervical dysplasia was explained by Tokyol et al 17 and it was hypothesized that BV could be important in developing cervical neoplasia. In this condition, abnormal microflora produce carcinogenic nitrosamins which can lead to carinoma.
In conclusion, cervicovaginal infections was found to be highest in the sexually active women of reproductive age group (15-49) years. Bacterial vaginosis was the most predominant type of infections observed. Although hygiene behaviors, socio-demographic and clinical status play the important role in such infections, routine Pap smear examination plays a significant role in identification of TV and BV in majority of women. The Pap screening could be beneficial in early disease diagnosis and also helps in preventing further complications by commencing appropriate treatment.