International Journal of Integrated Health Sciences. 2019;7(1) 9
Original Article
Prevalence of Trichomonas vaginalis Based on Clinical Manifestation
and Polymerase Chain Reaction among Reproductive Women
Correspondence:
Huriyah Hafizhotul ‘Ummah, Faculty of Medicine,
Universitas Padjadjaran
Jl. Raya Bandung-Sumedang KM 21 Jatinangor,
Indonesia
e-mail: huriyah.hafizhotul@gmail.com
Abstract Objective: To measure the prevalence of Trichomonas vaginalis (T. vaginalis)
based on clinical manifestations and polymerase chain reaction (PCR) among
reproductive women.
Methods: Subjects of the study were the vaginal swab obtained from
reproductive women who attended the gynecology examination at
Kandanghaur and Sindang primary health care of Indramayu District, West
Java in 2016. This study was a descriptive study with cross-sectional method.
Sampling was performed with total sampling method and 76 of vaginal swabs
were included in this study. The prevalence of T. vaginalis was measured
using PCR. The vaginal specimens were collected and then processed for PCR
analysis using TVK3/TVK7.
Results: Prevalence of T. vaginalis among reproductive women in Indramayu
District, West Java that analyzed using PCR was 0%. This result could be
affected by the study setting in community, presence or absence of symptoms,
and population studied.
Conclusions: There were no positive results of T. vaginalis, suggested by the
samples that obtained from community-based of a low-risked population.
Keywords: Trichomonas vaginalis, polymerase chain reaction, prevalence,
reproductive women
pISSN: 2302-1381; eISSN: 2338-4506; http://doi.org/10.15850/ijihs.v7n1.1490
IJIHS. 2019;7(1):9–15
Introduction
Trichomonas vaginalis (T. vaginalis) is a
flagellated protozoa known as the most
common human parasite that responsible
for sexually transmitted infection (STI) in the
world.1–5 World Health Organization (WHO)
estimated that there were 170–190 million
cases of T. vaginalis infection worldwide
each year and nearly 90% of these infections
occur in resource-limited settings country.1,2,4
According to WHO estimation, the prevalence
of T. vaginalis infection in women between the
ages of 15 and 49 in 2008 were 22% in America,
20.2% in Africa, 8% in Eastern Mediterranean,
5.8% in Europe, 5.7% in Western Pacific, and
5.6% in Southeast Asia.6 The prevalence of the
T. vaginalis infection in Indonesia had not been
clearly studied. However, a study showed that
the prevalence of T. vaginalis among female
workers in Kupang, Nusa Tenggara Timur
Province was 5%.7
The trichomonas vaginalis infection or
trichomoniasis among women has been
associated with mild to severe reproductive
health outcomes including vaginitis, cervicitis,
urethritis, low birth weight, premature
rupture of membranes, pre-term delivery, and
pelvic inflammatory disease.3,4 Trichomoniasis
also have several serious complications
Received:
November 26, 2018
Revised:
February 24, 2019
Accepted:
March 19, 2019
:9–15
Huriyah Hafizhotul ‘Ummah,1 Gita Widya Pradini,2 Reiva Farah Dwiyana,3 Muhammad
Ersyad Hamda2
1Faculty of Medicine, Universitas Padjadjaran
2Department of Microbiology and Parasitology, Faculty of Medicine, Universitas Padjadjaran
3Department of Dermatology and Venereology, Faculty of Medicine, Universitas Padjadjaran-Dr. Hasan Sadikin
General Hospital, Bandung
10 International Journal of Integrated Health Sciences. 2019;7(1)
including increased risk of HIV acquisition,
increased risk of cervical cancer, and shedding
of herpes simplex virus-2 (HSV-2) in the
genital tract of women, which could result in
increased transmission of other STIs.1,3,4 While
73% of women infected with T. vaginalis are
asymptomatic, one-third of them became
symptomatic within six months.3 In women
who have symptoms, usually the symptoms are
similar to other STIs, include vaginal discharge
which is often malodorous and yellow-green
colour, dysuria, itching, vulvar irritation, and
abdominal pain.1,8 However, trichomoniasis
has specific symptoms that only occurred
in 2–5% of women, known as “strawberry
cervix”, as well as frothy vaginal discharge in
12% of women with trichomoniasis.8
There are several methods to identify T.
vaginalis include wet mount microscopy,
culture, and nucleic acid amplifications Tests
(NAATs).9 Wet mount microscopy method
has low sensitivity, which ranges from 44%
to 68% and requires trained microscopist.10
The sensitivity can dramatically reduce
if there was a delay as short as 10–30
minutes between collection and microscopic
examination.10 Meanwhile, culture method
provides sensitivity that ranges from 44–75%
but needs daily examination by a trained
microscopist and the final result may take up
to a week.10 Both wet mount microscopy and
culture method, require specimen handling,
processing, and transport conditions to
preserve viable, motile organism.10 In contrast
with NAATs method, polymerase chain
reaction (PCR) is one of the NAATs method
using replication and amplification technique
from specific individual deoxyribonucleic acid
(DNA) target sequences. Thus, the sensitivity
of NAATs is inherently greater than other
methods ranges from 76–100%.10 Primer
sets TVK3/TVK7 known as the most sensitive
primer among others primer sets to identify
T. vaginalis because the target of this primer
is a repetitive DNA fragment that improve the
detection level and produce positive results on
specimens from asymptomatic women.11
The prevalence of T. vaginalis has been
found to vary according to geographical
location, study setting (sexual health clinic or
community setting), the presence or absence of
symptoms, population studied (ethnic group,
age, and sex), and the diagnostic techniques
used.7 This study identified T. vaginalis from
reproductive women using vaginal swab as the
specimen and PCR method with TVK3/TVK7
as the primer sets.
Methods
This was a descriptive study with cross-
sectional method. Subjects of the study were
the vaginal swab obtained from reproductive
women who attended gynecology examination
at Kandanghaur and Sindang primary
health care of Indramayu District, West Java,
Indonesia, in 2016 by total sampling.
Inclusion criteria for this study were
vaginal swab from women with positive
visual inspection with acetic acid (VIA) test,
or women with vaginal discharge, or women
with vaginal discharge with itching, or women
with malodorous vaginal discharge. Exclusion
criteria was the volume of the vaginal swab
which less than 5μl.
The vaginal swab had already been obtained
in year 2016 and stored in -20 OC for long-term
examination. Therefore, samples used in this
study were stored biological material, thus
the informed consent was unnecessary. The
extracted genomic DNA samples underwent
PCR and electrophoresis at the Microbiology
and Molecular Laboratory of Faculty of
Medicine, Universitas Padjadjaran, Bandung,
Indonesia. The PCR kit consist of, primer sets
TVK3/TVK7 and KAPA Taq Extra HotStart
ReadyMix PCR kit®. The electrophoresis kit
consist of peqGREEN DNA and RNA Dye®.
The PCR master mix was made according to
the procedures provided by manufacturer. The
initial reaction for PCR was performed at 95 OC
for 5 minutes and then repeated 35 cycles of
30 sec at 95 OC, 30 sec at 57.4 OC, 2 minutes at
72 OC, finally additional extensions were done
at 72 OC for 7 minutes and hold at 4 OC.
Upon completion of PCR, an aliquot was
analyzed by electrophoresis in a 3% agarose
gel in TAE buffer. The gel was stained with
peqGREEN DNA and RNA dye® and was
photographed under short ultraviolet light.
The primer sets TVK3/TVK7 specifically
amplify a 300 bp fragment of T. vaginalis
genome.11 The size of amplified products was
assessed by comparison with a commercial
weight marker (1Kb ladder).
Positive and negative controls were
included in all PCR runs. The DNA extract from
motile T. vaginalis in wet mount examination
was used as a positive control. The negative
control consisted of PCR master mix without
DNA. This study was approved through the
ethical clearance number 595/UN6.KEP/
EC/2018 by the Health Research Ethic
Committee, Faculty of Medicine, Universitas
Padjadjaran, Bandung.
:9–15
Prevalence of Trichomonas vaginalis Based on Clinical Manifestation and
Polymerase Chain Reaction among Reproductive Women
International Journal of Integrated Health Sciences. 2019;7(1) 11
Table 1 Characteristics of the Subjects
Characteristics Frequency (n)
PCR Positive of T. vaginalis
Frequency (n) Percent (%)
Age
20–29 years old 9 0 0
30–39 years old 42 0 0
40–49 years old 24 0 0
50–59 years old 1 0 0
Marital status
Yes 76 0 0
No 0 0 0
Obstetrical status
P0A0 5 0 0
P1A0 23 0 0
P1A1 3 0 0
P1A3 1 0 0
P2A0 17 0 0
P2A1 7 0 0
P3A0 9 0 0
P3A2 2 0 0
P4A0 3 0 0
P4A1 1 0 0
P4A2 1 0 0
P4A4 1 0 0
P5A0 1 0 0
P5A1 1 0 0
Education level
Uneducated 2 0 0
Elementary school 28 0 0
Junior high school 24 0 0
Senior high school 16 0 0
Diploma 2 0 0
Bachelor 4 0 0
Occupation
Housewife 68 0 0
Farmer 5 0 0
Government officer 1 0 0
Midwifery 1 0 0
Nurse 1 0 0
Huriyah Hafizhotul ‘Ummah, Gita Widya Pradini, et al.
:9–15
12 International Journal of Integrated Health Sciences. 2019;7(1)
Prevalence of Trichomonas vaginalis Based on Clinical Manifestation and
Polymerase Chain Reaction among Reproductive Women
Results
During the gynecology examination at
Kandanghaur and Sindang primary healthcare
in 2016, from 147 women attended, 143
vaginal swabs were obtained, and 76 samples
were stored in laboratory and underwent PCR
examination for this study, while the rest of
the samples could not be extracted because of
the volume less than 5μl. The characteristics of
subjects which vaginal swab underwent PCR
were described (Table 1). The prevalence of T.
vaginalis based on PCR were shown. Based on
these data, the PCR results of T. vaginalis were
negative in all samples.
The prevalence of T. vaginalis based on
clinical manifestation was shown (Table
2). From this data, in some subjects who
had one symptom or more, the PCR results
were negative for T. vaginalis.
Result of electrophoresis was revealed
(Fig. a–b). In all of the electrophoresis
results, there was the positive control that
has length 300kbp appropriate for the size
of the DNA that amplified by TVK/TVK7
primer sets, but none of the samples have a
positive result because there was no band
appeared in all of the results.
Discussion
The prevalence of trichomoniasis reported
in this study was 0% using PCR method (Fig.
a–b). The socio-demographic characteristics
and clinical manifestation of the study
population may affect this finding.12 The socio-
demographic characteristics of the study
population are portrayed (Table 1). Among
76 women who examined for T. vaginalis,
:9–15
Electrophoresis Sample 1–12 (a), Electrophoresis Sample 13–24 (b),
Electrophoresis Sample 25–28 (c), Electrophoresis Sample 65–76
Fig.
a b
c d
International Journal of Integrated Health Sciences. 2019;7(1) 13
42 women in this study belonged to the age
group of 30–39 years, but none of them were
positive for T. vaginalis infection. This result
was similar to several previous studies which
majority of infected women were between the
ages of 35–40 years, 26–35 years (37.93%),
and 21–45 years (76%), respectively.6,8,12
Unlike other nonviral STIs, trichomoniasis
does not primarily reach young women
(15–25 years old). It affects women during
reproductive years, and high rates of infection
are found in women between the ages of 35
and 40. Predisposing factors comprise of
older age, use of oral contraceptives, trading
sex, smoking, single marital status, and low
socioeconomic class.6
Other socio-demographic factors for
trichomoniasis are marital status and
obstetrical status. All women in this study
have the same marital status, while obstetrical
status varied and the result was 17 women
(22.36%) had history of abortion. This result
was in contrast to a study which reported
women who have abortion previously will have
a higher prevalence of T. vaginalis infection.13
Both occupation and education level are
predisposing factors for trichomoniasis in
published literature. In the previous study,
majority of those with trichomoniasis were
unemployed and has less education level.12
However, in this study, there was no difference
between the occupation and education level
with T. vaginalis infection.
The overall prevalence of T. vaginalis
identification using PCR method was 0%. A
previous study identified T. vaginalis with the
same method as in this study which found
the prevalence of the infection was 30%.8 The
negative findings of T. vaginalis in this study
could be affected by samples that obtained
from low-risked women and the study setting
was community-based study. In contrary,
in clinic-based studies there was a higher
prevalence than community-based studies. In
community-based study, the prevalence was
1% and 0.6% respectively. While in clinic-
based study, the prevalence was 4.4% and 4.2%
respectively.14 Another risk factor associated
with trichomoniasis was the last sexual
intercourse. Therefore, the normal incubation
period for trichomoniasis is 4–28 days. If
the patients are infected by trichomoniasis,
the patients are expected to be symptomatic
within this period or immediately after. This
may explain the lower risk of finding in those
who had sexual intercourse over 30 days in
:9–15
Huriyah Hafizhotul ‘Ummah, Gita Widya Pradini, et al.
Table 2 Prevalence of T. vaginalis Based on Clinical Manifestation
Characteristics Frequency (n)
PCR Positive of T. vaginalis
Frequency (n) Percent (%)
Vaginal bleeding
Yes 1 0 0
No 75 0 0
Vaginal discharge
Yes 24 0 0
No 52 0 0
Vaginal discharge with itching
Yes 19 0 0
No 57 0 0
Malodorous vaginal discharge
Yes 10 0 0
No 66 0 0
VIA test
Positive 15 0 0
Negative 61 0 0
14 International Journal of Integrated Health Sciences. 2019;7(1)
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about last sexual intercourse.
The clinical manifestations varied among
the 76 women, 52 women had no vaginal
discharge, 57 women had no vaginal discharge
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This study was used PCR method to detect
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nonviable organisms.8 Since most cases of
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STIs.1,3,4 Thus, it is important to screen T.
vaginalis infection in low-risked women to
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Moreover, diagnosis of trichomoniasis
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presentation because the clinical symptom
may be synonymous with those of other STIs,
the specific symptom like strawberry cervix is
seen approximately 2% of patients, and frothy
discharge is seen only in 12% of women with
trichomoniasis. It has been demonstrated
that if these classical features are used alone
in the diagnosis of trichomoniasis, 88% of
these cases will not be diagnosed and 29%
of uninfected women will be falsely indicated
as having T. vaginalis infection. This suggests
that clinical manifestation are not reliable
diagnostic parameters and hence laboratory
diagnosis is necessary for early and accurate
diagnosis.8
This study had limitations such as the
examination only covered the low-risked
women and the study settings was a
community-based study. Therefore, a further
study can be conducted by using a broader
population including high-risk subject.
In conclusion, the prevalence of T. vaginalis
based on clinical manifestation and PCR among
reproductive women in this study was 0%.
However, the samples that obtained and stored
from 2 years ago have a limited possibility to
become contaminated because the samples
already been extracted into DNA and stored in
-20 OC. It was proven from the positive control
that appeared in each electrophoresis results.
The positive control used in this study was also
obtained 2 years ago from motile T. vaginalis
that underwent DNA extraction and stored
together with other samples.
:9–15
International Journal of Integrated Health Sciences. 2019;7(1) 15
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:9–15