International Journal of Microbiology and Mycology | IJMM

pISSN: 2309-4796
http://www.innspub.net
Vol. 4, No. 1, p. 1-8, 2016

Open Access RESEARCH PAPER

RESEARCH ARTICLE
Evaluation of pcr in the molecular diagnosis of trichomonas
vaginalis infection in comparison with other conventional
methods

Dr. Kiranmai*, Dr. A. Neelima

Department of Microbiology, MediCiti Institute of Medical Sciences, Ghanpur, Hyderabad, India

Keywords: Polymerase chain reaction (PCR), Trichomonas vaginalis, Trichomoniasis, Female sexual workers.

Publication date: August 21, 2016

Abstract
Trichomonas vaginalis (T. vaginalis) is a common pathogen with worldwide distribution. It is estimated that
worldwide 180 million people are infected annually. Trichomoniasis is associated with vaginitis, cervicitis, low
birth weight, and preterm delivery. PCR has the advantage of high sensitivity, shorter time for diagnosis and the
ability to detect nonviable or defective organism. In this study we used these three methods for evaluation of
PCR in comparison with conventional methods like wet mount and culture in the detection of T. vaginalis in
vaginal discharge. Three vaginal swab specimens were obtained from each of 200 cases, of the age group 18-
40years, both symptomatic and asymptomatic females attending Gynaecology OPD(50) and Family planning
OPD(50) at Gandhi hospital, Secunderabadand two FSW(Female sex workers) clinics (100) in highly
concentrated areas of them in Hyderabad, for validation of various forms of Trichomonas vaginalis diagnostic
procedures. One swab was immediately examined by wetmount microscopy, a second swab was placed in
Wittington’s medium for cultivation, and other swab is placed in 2SP transport medium for PCR for T.vaginalis.
A total of 58 samples positive in one or more tests were identified: 11 (5.5%) infections were detected by wet
mount microscopy, and 30 (15%) positives in culture respectively. PCR was positive in 50 (25%) samples. PCR
appears to be the most sensitive method with high detection rate and method of choice for detection of genital
infections with T. vaginalis.
* Corresponding Author: Dr. Kiranmai  [email protected]

1 Kiranmai and Neelima

Introduction To date, numerous T. vaginalis specific PCR assays
Worldwide, Trichomonas vaginalis causes approx- have been described. Examples of targets include the
imately 180 million new infections per year, making it ferridoxin gene, beta tubulin gene, highly repeated
the most prevalent non-viral sexually transmitted DNA sequencing and 18s ribosomal genes. (Riley et

disease (STD) agent. (Petrin et al., 1998; Kengue et al., 1992; Kengue et al., 1994; Madico et al., 1998;
Mayta et al., 2000).
al., 1994; Madico et al., 1998).

Marcia M. Hobbs et al., compared culture and PCR

Infections in women can cause vaginitis, urethritis, ELISA in urethral swabs, urine and semen for TV
and cervicitis, and complications include premature detection in male sexual partners of women with
labor, lowbirth-weight offspring, and post abortion or trichomoniasis identified by wet mount and culture.
post hysterectomy infection (Shaio, et al., 1997). It TV was detected more often in men with wet mount
has been estimated that 10 to 50% of T. vaginalis positive partners emphasizing the importance of
infections in women are asymptomatic and in men partner evaluation and treatment. Even with a
the proportion may even be higher. (Burstein, G. R et sensitive PCR assay, reliable detection of TV in male
partners required multiple specimens.
al., 1999) This parasite has also been implicated as a
cofactor in the transmission of the human Charlotte Gaydos et al., (2006), compared Gene
immunodeficiency virus and other nonulcerative STD Probe transcription mediated amplification TV
agents. However, since the incidence of T. vaginalis research assay and real time PCR for TV detection
infection is highest for groups with a high prevalence using a Roche Light Cycler instrument with female
of other STDs, this latter hypothesis remains to be self-obtained vaginal swab samples and male urine
confirmed (Madico et al., 1998). In addition, a samples. The Gen Probe TMA assay is commercially
relationship between T. vaginalis infec-tion and available as an analyte specific reagent and offers
cervical cancer has recently been suggested (Zhang et laboratories a highly sensitive and specific assay for

al., 1995). use clinically.

Rasoul Jamali et al., (2006), compared diagnosis of

The most common tool for diagnosis of T. vaginalis Trichomonas vaginalis infection using PCR method to
infection is still microscopic examination of wet mount culture and wet mount microscopy and concluded
preparations, which has a sensitivity of approximately that PCR had high sensitivity and slightly less
38-60%. (Fouts et al., 1980) Microscopic examination specificity than wet mount taking culture as the
of cultures of the parasite in specialized mediaimproves standard.
the sensitivity to 70-85% (Gelbart et al., 1990; Schmid
This study was done to diagnose both symptomatic
et al., 1989). However, the quality of these diagnostic
and asymptomatic female patients with Trichomonas
tests is strongly dependent on theskills and experience
vaginalis infection by Microscopy, Culture and PCR
of the microscopist and also on the quality of the and to compare the efficacy of each of the above
sample. Diagnosis traditionally depends on the diagnostic modalities in terms of rapidity, sensitivity
microscopic observation of motile protozoa in vaginal and specificity.
discharge. Culture requires a special medium and the
result takes up to 7 days. Diagnostic improvements Material and methods
have been suggested since years. Finally, molecular Sample size: A total of 200 females, of the age

techniques such as fluorescent in situ hybridization, group 18-40 years, both symptomatic and
asymptomatic females attending Gynaecology OPD
oligonucleotide probing, and PCR have been
(50) and Family planning (FP) OPD (50) at Gandhi
developed. (Petrin et al., 1998; Kengue et al., 1994;
hospital, Secunderabad and two FSW (Female sex
Zhang et al., 1995; Muresu et al., 1994; Philips Heine et
workers) clinics (100) in highly concentrated areas of
al 1997; Riley et al., 1992; Rubino et al., 1991).
them in Hyderabad from November 2008 to February
2010.

2 Kiranmai and Neelima

Relevant data from the subjects are recorded. All the To produce the axenic isolates the medium is
subjects were informed about the study and they supplemented with the antibiotics (Penicillin and
agreed to participate in the study. Streptomycin). The culture medium was dispersed in
a screw capped tubes in the volume of 5ml each and

Specimen collection stored at 4oC.

Females with complaints of vaginal discharge, itching, Cultivation

dysuria and dyspareunia were considered as Before inoculation of medium, the culture tubes were
symptomatic and those without symptoms of warmed up to 37oC for 15min. The vaginal swabs were
trichomoniasis were considered as asymptomatic in placed into the medium and left to incubate at 37 oC
the study. At the time of per-speculum examination for 7days. The cultures were examined
three vaginal swabs from the posterior fornix and also microscopically on days 2, 5 and 7 after inoculation. A
touching both lateral fornices and middle third of positive result is defined as the presence of motile T.

vaginal wall were taken, using sterile Dacron swabs. Vaginalis at any time, a negative result was defined as
absence of motile T. vaginalis at all readings (Fig.2).
Specimens collected prior to disinfection or local
antibiotic used for routine microscopic examination,
the second one was used to inoculate the culture
medium and third swab was placed in 0.5ml of 2SP
transport medium and stored at -20oC.

Wet mount preparation

The swab inoculated with vaginal discharge for each
patient was gently agitated in one drop of normal
saline on a clean slide and then covered with a cover
slip. The wet mount was examined with 40 objective
and the presence of motile T. vaginalis was detected
by the characteristic twitching motility (Fig.1).
Fig. 2. Trichomonas in culture.

Trichomonas vaginalis Polymerase chain reaction

(TVPCR) (Rosche NG/CT kit modification)
DNA Extraction: 2ml screw cap tube is centrifuged for
10min after transferring 250μl of sample suspension
after vortexing thoroughly for 20 seconds. 250μl LYS
is added to the supernatant and mixed by vortexing
and incubated for 10min. 250μl DIL is added to each
tube and mixed by vortexing and incubated for
10min. processed specimens are kept at room
temperature for up to 2 hrs before transferring
aliquots to the PCR reaction tubes.

PCR primers: The primers based on T. vaginalis β-

tubulin gene for PCR identification were used. The
Fig.1. Wet mount of T. Vaginalis, T. vaginalis
sequences of primers were as follows:
culture β-tubulin 9/2 primer 1: Biotin- 5’ GCA TGT TGT GCC
To prepare Whittington medium, Trichomonas agar GGA CAT AAC CAT.
base is added to distilled water and sterilized by β-tubulin 9/2 primer 2: Biotin- 5’ CAT TGA TAA CGA
autoclave for 15min at 15lbs pressure (121oC). It is AGC TCT TTA CGAT.
cooled to 50oC and aseptically horse serum is added.

3 Kiranmai and Neelima

PCR protocol: PCR reactions were performed with an Detection
automated the rmocycler. The total volume of PCR All reagents and samples were brought to room
reactions was 50μl of the master mix and 50μl of the
temperature. Working wash solution is prepared.
DNA extracted were added to the PCR tube. The
25μlof denatured amplicon is pippeted to micro well
amplification was performed in the PCR tubes and
the procedure is as follows: plate and incubated for 1hr at 37oC. After washing the
Hold Program: 50°C- 2 min plate 5 times AV-HRP and SUB-A were added as
Hold Program: 95°C- 5 min given in the insert of the ROCHE NG/CT kit. At last,
CYCLE: Denaturation: 95°C- 45sec
stop solution is added and read under Elisa reader.
Annealing: 62-52°C- 45sec 35 cycles
Amplification: 72°C- 60sec Results were interpretated as shown in table 1.

Hold Program: 72°C- 7 min

Hold Program: 72°C- Not to exceed 24 hours

Table 1. Interpretation of PCR results.

Result A450 Interpretation
 0.3 DNA not detected. Negative.
 0.8 DNA detected. Specimen is presumptive positive for T. vaginalis.
 0.3,  0.8 Equivocal. Results are inconclusive. Repeat PCR testing on specimen in duplicate. Two of
three results over 0.5 shall report the specimen as positive.

Results finding have shown that signs of trichomoniasis are

Clinical Examination present in 82(82%) of FSWs, 50(100%) of females
Total vaginal swab specimens collected from women attending the gynaecology OPD and in 23(46%) of the
attending OPD and FSW clinics were examined. females attending the family planning OPD i.e.,
Females attending the family planning OPD were controls. The most prevalent clinical symptom was
considered as control as they were asymptomatic at vaginal discharge. (Table 2) The majority were in the
the time of presentation. During per speculum age group of 21-30 years.
examination of patients,

Table 2. Symptom wise distribution among FSW, GYNAEC OPD AND FP.
Symptoms FSW GYNAEC FP
Vaginal discharge 78(78%) 50(100%) 23(46%)
Pruritus 02(2%) 04(8%) 02(4%)
Odour 10(10%) 08(16%) 01(2%)
Ulcer 01(1%) (0%) (0%)
Burning micturition 12(12%) 03(6%) 01(2%)

Microscopic examination and culture The growth in Whittington’s medium was observed in
The incidence of positivity for Trichomonas vaginalis 30 out of 200. Totally 30 were positive both by
by wet mount was high (10%) in the female sex culture and wetmount examination. Of these 19
workers than in females attending Gynaecology OPD
samples were not detected by direct microscopic
(2%). Positivity for TV by wet mount was negative in
examination but were positive by cultivation method,
all control cases.
and all wetmount positives were also shown positive

Culture showed 27% positives for Trichomonas by culture. (Table3) Considering the culture as the

vaginalis in the high risk group i.e., female sex gold standard, the sensitivity of direct microscopy was
workers and 6% positives in the females attending the 37% and specificity was 81%.
gynaecology OPD.

4 Kiranmai and Neelima

Table 3. Comparison of Wet mount positivity with culture positivity of T.vaginalis.
culture positive (n=30) culture negative (n=170)
wet mount positive (n=10) 11 00
wet mount negative (n=90) 19 170

All the 30 patients shown positive by culture and T. vaginalis was detected in 50(25%) of 200 samples.
microscopy were all symptomatic with vaginal PCR could detect 28 positives which could not be
discharge on correlating with the examination. All the detected by culture. 8 cases which were positive by
controls (family planning cases) were negative both
culture were not detected by PCR. This may be due to
by culture and also by wetmount examination.
the absence of β-tubulin gene in the trichomonas
PCR results which were detected by culture. (Table4). The
PCR assay was performed on all the 200 samples. β- sensitivity of PCR was 88.37% and specificity was
tubul in primers were used for PCR assay. Detection 83.5% taking culture as the gold standard.
was made with ELISA reader and results interpreted.

Table 4. Comparison of PCR with culture positivity of T. vaginalis.

culture positive (n=30) culture negative (n=170)
PCR positive (n=50) 22 28
PCR negative (n=150) 08 92

Discussion Wet mount was positive in 10% females in the high

Accurate diagnosis of Trichomoniasis in sexually risk group i.e., female sex workers and 2% in the
active women is extremely important since T.
study group attending Gynaecology OPD Similar
vaginalis may be the cause of high morbidity and, in
observations were made in the studies conducted by
common with other non-ulcerative sexually
transmitted diseases, may be regarded as a risk factor Charles Beal et al (1992) 5.8%,Alex Van.
for contraction of HIV infection.
A Pillay et al (2004)- 6%, Angelika Stary et al.
The most common tool for diagnosis of T. vaginalis
(2002)- 6.5%, Rasoul Jamali et al. (2006)-3.46%. The
infection is still microscopic examination of wet
mount preparations and culture. Diagnostic low positivity by wet mount may be due to delay in
improvements have been suggested in past years. reaching the microscope after preparation of wet
Finally, molecular techniques such as probing and mount or due to low number of organisms.
PCR have been developed. These molecular
techniques could assist clinicians in achieving the
Culture, positivity by Wittington medium for
correct diagnosis, leading to prompt, sensitive and
specific treatment under syndromic management. Trichomoniasis was 27% in female sex workers and
6% of females attending Gynaecology OPD and none
This study describes two prospective clinical studies in the control group attending Family Planning OPD.
performed to evaluate various diagnostic methods for
Studies supporting this result in females attending
the detection of Trichomoniasis in vaginal swab
gynaecology OPD are-Charlotte Gaydos et al. (1997)-
samples obtained from females.
6.6%, Alex Van Belkum et al. (1999)- 4.9-5.7%.
In this study, most common affected age group was Studies supporting culture positivity in high risk
21-30yrs (57.5%). This correlates well with other group (FSWs) as observed in this study are Khan et
studies conducted by BM Agarwal et al. (2000) and al. (1991)- 28.4%; Schwebke et al. (1999)- 26% and
Marcia M. Hobbs et al. (2006); Swygard et al. (2004).
Adu Sarkode et al. (2004)- 27.5%.

5 Kiranmai and Neelima

The Molecular methods adopted for the detection of Culture should be used when the wet mount is
Trichomonas vaginalis in this study was Roche PCR negative. Here we used culture as a gold standard to
which is the modification of the Roche PCR kit for NG, determine the sensitivity and specificity of wet
CT. According to T. Crucitti et al (2003) detection of preparation and PCR. The reference study to calculate

Trichomonas vaginalis by the Roche NG, CT PCR kit is the sensitivity and specificity of culture method in
some papers is the culture medium itself, which may
equal to other PCR kits for Trichomonias vaginalis
yield higher estimate of sensitivity for the culture.
detection. In this study, PCR was positive in 45% of
(Patel et al., 2000).
the female sex workers. Other studies correlating well
with the results are-Marcia M. Hobbset al (2002) - Conclusion
38.4%, Schwebke et al. (2002)- 52%. We analysed a number of clinical samples by culture,
direct microscopy and PCR, none of the diagnostic
In the study group attending Gynaecology, the PCR assays could detect all positive samples, but PCR
positivity was 10%, this compares well with the showed a higher detection rate than others in
A.Pillay et al (2004)- 12.6% of females are positive by detection of T. vaginalis in vaginal swab samples.
PCR for Trichomoniasis. Overall, the sensitivity of the PCR assay resulting
from this study was lower than those previously
described. These findings could be the result of the
PCR was found to be superior among the three
nature of the specimen population and suggests a
diagnostic modalities for the detection of
strain variability.
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