|Year : 2015 | Volume
| Issue : 1 | Page : 36-39
|Comparison of four diagnostic techniques for detection of Trichomonas vaginalis infection in females attending tertiary care hospital of North India
Razia Khatoon1, Noor Jahan1, Siraj Ahmad2, Haris Manzoor Khan3, Tamkin Rabbani4
1 Department of Microbiology, Era's Lucknow Medical College and Hospital, Lucknow; Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, A.M.U., Aligarh, Uttar Pradesh, India
2 Department of Community Medicine, Teerthanker Mahaveer Medical College and Research Center, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India
3 Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, A.M.U., Aligarh, Uttar Pradesh, India
4 Department of Obstetrics and Gynecology, Jawaharlal Nehru Medical College and Hospital, A.M.U., Aligarh, Uttar Pradesh, India
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|Date of Web Publication||11-Feb-2015|
| Abstract|| |
Background: Trichomonas vaginalis causes a common sexually transmitted disease trichomoniasis, which may lead to increased risk of transmission of human immunodeficiency virus infection and other pelvic inflammatory diseases. Wet mount examination is the most common test for diagnosis, but it has low sensitivity. Acridine orange staining can be used for diagnosis, but it requires special microscopic facility. Culture is considered as the gold standard, but it takes a long time for diagnosis. OSOM Trichomonas Rapid Test is a recently introduced rapid method based on immunochromatographic assay of trichomonal protein antigens. Hence, the present study was done to compare these four diagnostic techniques for detection of trichomoniasis in females with vaginal discharge. Materials and Methods: Vaginal swabs were taken from 835 female patients and wet mount examination, acridine orange staining, culture in Kupferberg medium, and OSOM Trichomonas Rapid Test, were performed. Results: Out of 835 patients included in our study, 68 (8.1%) positive cases of trichomoniasis were detected by culture. OSOM Trichomonas Rapid Test detected 63 (7.5%) cases, acridine orange staining detected 53 (6.3%) cases, whereas, wet mount examination detected only 45 (5.4%) positive cases. OSOM Trichomonas Rapid Test performed well and showed high sensitivity and specificity of 88.2% and 99.6%, respectively. Conclusion: As OSOM Trichomonas Rapid Test is a point of care test and gave better results than both wet mount examination and acridine orange staining; it can be used as a routine test in peripheral areas lacking laboratory facilities.
Keywords: Acridine orange staining, culture in Kupferberg medium, OSOM Trichomonas Rapid Test, Trichomonas vaginalis, wet mount examination
|How to cite this article:|
Khatoon R, Jahan N, Ahmad S, Khan HM, Rabbani T. Comparison of four diagnostic techniques for detection of Trichomonas vaginalis infection in females attending tertiary care hospital of North India. Indian J Pathol Microbiol 2015;58:36-9
|How to cite this URL:|
Khatoon R, Jahan N, Ahmad S, Khan HM, Rabbani T. Comparison of four diagnostic techniques for detection of Trichomonas vaginalis infection in females attending tertiary care hospital of North India. Indian J Pathol Microbiol [serial online] 2015 [cited 2018 Mar 23];58:36-9. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/36/151172
| Introduction|| |
Vaginal trichomoniasis is a sexually transmitted disease (STD) caused by a parasitic protozoan Trichomonas vaginalis. It is estimated by the World Health Organization to account for almost half of all curable sexually transmitted infections worldwide, with an annual incidence exceeding 180 million cases per year. , It is mostly a disease of reproductive age group and has a cosmopolitan distribution. Clinical syndrome in females varies from asymptomatic presentations to vaginitis with copious discharge and sometimes associated with punctate hemorrhagic lesion of the cervix known as "strawberry" cervix. 
Multiple studies have linked T. vaginalis infection to significant and hazardous health outcome, such as, pelvic inflammatory diseases.  There is also an increased risk of transmission of other sexually transmitted infections, including human immunodeficiency virus infection. , In addition, a relationship between T. vaginalis infection and cervical cancer has also been suggested. 
Therefore, the detection of T. vaginalis infection is of utmost clinical importance in order to safeguard women against these complications. However, it is difficult to diagnose trichomoniasis clinically, due to its heterogeneous presentation and nonspecific symptoms. Hence, the diagnosis has to be based on laboratory procedures. The conventional laboratory methods for diagnosis of T. vaginalis include direct microscopic examination of wet mount preparation and culture. Although wet mount microscopy is commonly done and provides immediate results, it is a subjective test that requires technical experience.
The present "gold standard" for the diagnosis of T. vaginalis is culture. Various culture media that have been described for growth of T. vaginalis include Diamond's, Trichosel, Kupferberg and InPouch TV. ,,
Staining techniques can also be used to detect T. vaginalis. Acridine orange staining is a nonspecific nucleic acid staining procedure, which can be applied for fluorescence-based detection of T. vaginalis. 
OSOM Trichomonas Rapid Test is a newer diagnostic method, which has recently been approved by Food and Drug Administration for use on direct vaginal swabs or wet mount saline. It is an immunochromatographic monoclonal-antibody based detection system that is highly sensitive and specific.
Hence, the present study was done to compare wet mount examination, acridine orange staining, culture in Kupferberg medium and OSOM Trichomonas Rapid Test in detecting vaginal trichomoniasis.
| Materials and Methods|| |
The present study was a hospital based prospective study done over a period of 1.5 years (January 2009 to June 2010). The study group comprised of 835 females of reproductive age group (15-45 years) attending gynecological out-patient department with complaints of foul smelling vaginal discharge, pruritus, and pain in the lower abdomen.
Pregnant females, those who had recently douched or used spermicidal agents within 72 h prior to testing, and those who were menstruating at the time of examination were excluded from the study.
The study was approved by the Institutional Ethics Committee. An oral informed consent was taken from all the patients, and vaginal fluid was collected from posterior fornix of the vagina using sterile swabs.
Wet mount examination
The vaginal swab was used to prepare wet mount by mixing it with 0.85% physiological saline and examined with a light microscope at ×10 and ×40. The trophozoites of T. vaginalis were identified by their size (10-20 μm), oval shape and characteristic twitching motility. 
Acridine orange staining
A smear was prepared from the vaginal swab and air dried. It was then fixed with methanol for 2-3 min and covered with freshly prepared acridine orange dye (Hi Media Laboratories, India) in a concentration of 5 mg/ml and left at room temperature for 2 min. It was then rinsed with distilled water and examined under a fluorescence microscope (with a 470-490 nm filter) at a magnification of ×40. The trophozoites of T. vaginalis were identified by their characteristic brick red color with a yellowish green nucleus. 
Culture in Kupferberg medium
To prepare the medium, 23.5 g of Kupferberg Trichomonas base (Hi Media, India) was dissolved in 950 ml distilled water and sterilized by autoclaving at 121°C for 15 min. Then 50 ml of sterile bovine serum was added aseptically after cooling. The medium was then supplemented with rehydrated contents of Trichomonas selective supplement I (Hi Media, India) containing Streptomycin 500 mg and Penicillin G 125,000 units.
The vaginal swabs were placed into the culture medium and incubated at 37°C. The cultures were examined microscopically on days 3, 5 and 7 for the presence of motile T. vaginalis. Negative results were defined as the absence of motile trichomonads after 7 days of incubation. 
OSOM Trichomonas Rapid Test
The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, UK) is an immunochromatographic, capillary flow, dipstick technology based on the detection of trichomonas proteins present in vaginal fluid. To perform this test, vaginal swab was placed in a tube containing 0.5 ml of sample buffer, mixed vigorously and allowed to stand for 1 min after which OSOM Trichomonas Rapid Test stick was placed in the tube and result was read at 10 min. In a positive sample, the trichomonal protein will form a complex with the primary anti-trichomonas antibody conjugated to colored particles. The complex then binds to second anti-trichomonas antibody coated on the nitrocellulose membrane resulting in the appearance of a visible blue test line along with the red control line [Figure 1].
|Figure 1: OSOM Trichomonas Rapid Test showing positive result with blue line along with red control line|
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The collected data were analyzed using SPSS Data Editor Software, version 16 (SPSS Inc., United States). Pearson's Chi-square test was performed, and P < 0.05 was considered statistically significant.
| Results|| |
A total of 835 female patients of reproductive age group were included in the study. The mean age of the patients was 30.56 ± 0.34 years. Out of 835 patients, maximum cases of vaginal trichomoniasis were detected by culture in Kupferberg medium (8.1%), followed by OSOM Trichomonas Rapid Test (7.5%) and acridine orange staining (6.3%), whereas, least number of cases (5.4%) was detected by wet mount examination [Figure 2]. Distribution of patients having T. vaginalis infection according to age group is shown in [Table 1]. Total number of patients positive for T. vaginalis infection were 68 (8.1%), out of which maximum number of patients (3.8%) belonged to 21-30 years age group, whereas, least number of cases (0.5%) belonged to 11-20 years age group. However, the association between T. vaginalis infection and age group of the patients was found to be statistically insignificant (P > 0.05).
|Figure 2: Distribution of patients suffering from vaginal trichomoniasis as detected by different diagnostic tests|
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|Table 1: Distribution of patients having T. vaginalis infection according to age group|
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A comparison of the efficacy of the test procedures was done by taking culture as the gold standard and the sensitivity, specificity, positive predictive value and negative predictive value of wet mount examination, acridine orange staining and OSOM Trichomonas Rapid Test were calculated [Table 2]. By putting Pearson's Chi-square test it was found that the difference between the sensitivity of OSOM Trichomonas Rapid Test and wet mount examination was statistically significant (P < 0.001).
|Table 2: Comparison of various diagnostic tests used for detecting T. vaginalis infection taking culture as gold standard|
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| Discussion|| |
In the present study, out of the 835 patients tested, 45 (5.4%) were found to be positive for T. vaginalis infection by wet mount examination. When compared with culture, the sensitivity of wet mount examination was found to be 58.8%. Although this technique is inexpensive and provides immediate results, it is a subjective test that requires clinical experience and access to a microscope. Even in the hands of trained observers, the wet mount is only 36-75% sensitive compared to culture.  Sluggish motility or low number of trichomonas can be easily missed. Also, douching prior to physical examination can lower the detection of motile trichomonas. 
Acridine orange staining detected 53 (6.3%) positive cases with sensitivity and specificity of 73.5% and 99.6% respectively. This is comparable to another study that showed the sensitivity and specificity of acridine orange staining as 71.43% and 99.44% respectively.  Although this staining technique is easy to perform and screening of vaginal smears is possible in less time, its major disadvantage is that it requires special microscopic facilities and trained personnel. Also, permanent record is not possible because of the loss of fluorescence with the passage of time.
In the present study, 68 (8.1%) out of 835 patients were found to be positive for trichomoniasis by culture in Kupferberg medium. Although, culture is the most accurate method with high sensitivity and is considered gold standard for the diagnosis of T. vaginalis infection, but laboratory facilities for culture are not widely available. Also, it takes 2-7 days to give positive results, thereby resulting in loss of patients in follow-up. This problem of delayed diagnosis can be overcome by the use of rapid tests.
OSOM Trichomonas Rapid Test detected 63 (7.5%) positive cases of trichomoniasis out of 835 patients included in our study, which is almost comparable to that of culture. It performed well and showed high sensitivity and specificity of 88.2% and 99.6% respectively. Our findings are supported by studies done by different workers who have also shown good performance of this rapid test with high sensitivity and specificity in comparison to wet mount examination. ,,
| Conclusion|| |
Trichomonas vaginalis causes STD in females of reproductive age group which needs to be timely diagnosed to prevent its adverse sequelae. Although wet mount examination and culture are commonly used methods for diagnosis of T. vaginalis infection, but, they are time consuming and needs technical expertise. Acridine orange staining could also be done in addition to wet mount examination, but it requires fluorescent microscopic facility and trained personnel. This problem can be overcome using rapid tests. OSOM Trichomonas Rapid Test had a good sensitivity and being a point of care test it is useful in remote areas lacking microscopic and culture facilities. As it is more sensitive than both wet mount examination and acridine orange staining, and requires less time (only ten minutes) than culture, it can be used as a routine test for diagnosing trichomoniasis.
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Dr. Noor Jahan
Department of Microbiology, Era's Lucknow Medical College and Hospital, Lucknow - 226 003
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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