Tropical Medicine and Health Vol. 38 No. 1, 2010, pp.

29-34
Copyright! 2010 by The Japanese Society of Tropical Medicine ２
９

Original article

Prevalence of Trichomonas vaginalis in vaginal swabs from

sex workers in Angeles City, Pampanga, Philippines as detected by PCR

Vanissa A. Ong1,2 and Windell L. Rivera1,2,＊

Received 17 August, 2009 Accepted 19 December, 2009 Published online 6 February, 2010

Abstract: A prevalence study was conducted on Trichomonas vaginalis infection among female sex workers at-
tending the Reproductive Health and Wellness Center of Angeles City, Pampanga in the Central Luzon region of
the Philippines. Polymerase chain reaction (PCR) using T. vaginalis-specific primers TV3/7 was utilized to detect
T. vaginalis in vaginal swabs from the study population. The lower limit sensitivity of T. vaginalis detection by the
PCR assay was found to be one trichomonad. The overall prevalence in 377 women was 9.55%. More than half of
the study subjects are 23-27 years old. However, the largest proportion of positive cases was found among subjects
18-22 years old, making it the age group with the highest T. vaginalis prevalence (12.84%).
Keywords: Trichomonas vaginalis, female sex workers, PCR, Philippines

high rates of infection with T. vaginalis. Using the medium

INTRODUCTION
Trichomoniasis, caused by the protozoan parasite
Trichomonas vaginalis, is one of the most common nonviral
sexually transmitted infections (STI) in the world [1]. T.
vaginalis infection may lead to vaginitis, urethritis, cervici-
tis, infertility, post-hysterectomy infection and pregnancy
complications such as premature labor and low-weight off-
spring [2, 3]. The parasite also causes nongonococcal ure-
thritis, prostatitis, and perhaps other lower genitourinary
tract syndromes in infected men [4]. Trichomoniasis has
also been shown to be a risk factor for pelvic inflammatory
disease [5] as well as transmission and infection with hu-
man immunodeficiency virus (HIV) [6]. Concomitant in-
fection with other urethral pathogens such as Neisseria gon-
orrhea and/or Chlamydia trachomatis has also been found
to be common in men and women with trichomoniasis [7].
The estimated global incidence of T. vaginalis infec-
tions is over 170 million cases per year [6]. An estimated
7.4 million new cases of T. vaginalis infection occurred in
2000 in the United States, compared to 2.8 million cases of
C. trachomatis infection and 718,000 cases of N. gonorrhea
infection [8]. Although trichomoniasis and other STIs have
also been reported from Southeast Asia [9], data on tricho-
moniasis cases in most of the countries in the region are
lacking. In the Philippines, female sex workers are prone to
described by Feinberg and Whittington in 1957 [10], it was
established in a study in 1986 that after incubation of cul-
tures of vaginal swabs for 3 to 5 days, 42% of the female
sex workers in Angeles City were positive for T. vaginalis
infection [11]. More recently, Queza (2008) reported
6.81% of trichomoniasis cases among 969 women from dif-
ferent areas in the Philippines [12]. Various socio-
demographic factors such as race, age, histories of previous
STI, access to care, and personal health practices have been
correlated with the presence of T. vaginalis and may be
used to predict infection. The lack of specific guidelines for
T. vaginalis screening in women may be due to the absence
of epidemiologic data on T. vaginalis infection in many ar-
eas in the Philippines. Therefore, knowledge of T. vaginalis
infection, distribution and prevalence in high-risk popula-
tions is necessary to evaluate the need for preventive meas-
ures and more sensitive diagnostic methods. In recent years,
no population-based prevalence study on T. vaginalis infec-
tion has been conducted in Angeles City, Pampanga, Philip-
pines. For this reason, we conducted the present study to
estimate the prevalence of T. vaginalis infection in the city.
T. vaginalis infection was detected by polymerase chain re-
action (PCR), a method which has been found to be rela-
tively sensitive and is now readily used for the detection and
screening of other STI-causing pathogens such as N. gonor-

1
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines
2
Molecular Protozoology Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City 1101, Philip-
pines
*Corresponding author:
E-mail: wlrivera@science.upd.edu.ph
Fax: +63-2-9205471
３
０
rhea and C. trachomatis. In this study, T. vaginalis-
specific primers TV3/7 were used. The results of this study
are essential to the development of preventive strategies.
MATERIALS AND METHODS
Study area
This study was conducted in Angeles City, Pampanga,
located in the Central Luzon region of the Philippines (Fig-
ure 1). Angeles City is locally classified as a first-class
highly urbanized city. The northwestern part of the city is
the well-known Clark Freeport Zone, the former Clark Air
Base and site of the largest United States (US) air base out-
side the continental United States of America. During the
American colonial period, a large number of Filipino mesti-
zos (people of mixed parentage, mainly American and Fili-
pino) were born in the city. Today, Angeles City and Clark
form a center for business, industry, aviation and tourism as
well as a popular entertainment and gaming area in Central
Luzon. However, since the early days of the Clark Air Base,
prostitution has also been prevalent in many areas in the
city especially those frequented by American servicemen
and foreign tourists.
Philippine
South Sea
Chino
Sea
LUZON
CENTRAL LUZON
PAMPANGA
VISAYAS
Sulu
Sea
MINDANAO
MALAYSIA
Figure 1. Map of the Philippines showing the location of the study area, Angeles City, Pampanga
Angeles City is one of the foremost cities in the coun-
try serving as an HIV/AIDS sentinel site. The city has a
Reproductive Health and Wellness Center (RHWC) which
provides care and support programs for people with HIV/
AIDS and serves as a resource center for STI/HIV/AIDS-
related concerns.
Collection of specimens
Sample size estimation was determined using the sta-
tistical formula of Jones et al. (2003) [13], that is, assuming
0.05 level of significance α and 37% trichomoniasis preva-
lence in the Philippines [11]. The minimum calculated
sample size for the study was 377. Therefore, vaginal
swabs were acquired consecutively from 400 female sex
workers undergoing weekly medical check-ups at RHWC.
Informed consent was obtained from all study participants,
and the study was approved by the Ethics Review Commit-
tee of the Office of the City Health Officer, Angeles City,
Pampanga. Due to cross-contamination during collection
and transport, only 377 of the 400 samples collected were
processed. Each vaginal swab was placed in a sterile screw-
capped tube containing 2 mL phosphate-buffered saline
(PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1 mM
TARLAC
ANGELES
Angeles City
PAMPANGA
ZAMBALES
BULACAN
BATAAN
Manila Bay

KH2PO4, pH 7.2) for DNA extraction.
DNA extraction and PCR
The vaginal swab samples in PBS were transported on
ice to the Molecular Protozoology Laboratory of the Natu-
ral Sciences Research Institute, University of the Philip-
pines. Approximately 1.5 mL aliquot of each sample was
centrifuged at 10,000 rpm for 2 minutes, and the superna-
tant was discarded. The pellet was suspended in 75 µL ster-
ile distilled water and subjected to genomic DNA extraction
by the Chelex method [14]. Briefly, each cell suspension
was supplemented with 200 µL of a 5% suspension of
chelating resin, Chelex (Sigma) in PCR-grade water and in-
cubated at 56° C for 20 minutes. The preparation was then
mixed vigorously and boiled for 8 minutes. DNA was sepa-
rated from Chelex beads by centrifugation at 10,000 rpm
for 2 minutes after which the supernatant was transferred to
a different microfuge tube and stored at -20° C until use.
PCR assays were performed by batches in an auto-
mated thermocycler (Techne). PCR using oligonucleotide
primers, TV3 and TV7 was performed on all samples. This
primer set specifically amplifies a 312-bp sequence from re-
petitive DNA in the T. vaginalis genome [15, 16]. Reac-
tions consisted of 2U Taq DNA polymerase, 1x PCR buffer
(MgCl2 free), 2 mM MgCl2, 0.2 mM of each dNTP, 10 µL
of DNA template, and 1 µM of each primer (TV3 and TV7)
with PCR-grade water to make up a final volume of 25 µL.
Positive and negative controls were included in all PCR
runs. The positive control consisted of DNA of T. vaginalis
isolate from a previous vaginal swab sample collection from
patients attending the Social Hygiene Clinic in Davao City,
Philippines [12], while the negative control consisted of
PCR mix with primers but no DNA template. PCR was
performed with 1 cycle of 95° C for 5 minutes; 35 cycles of
90° C for 1 minute, 60° C for 30 seconds, and 70°C for 2 min-
utes, and a final extension step of 72°C for 7 minutes.
Dilutions were made from a live culture of a clinical
isolate of T. vaginalis and tested to detect the lower limit
sensitivity of the PCR assay. Dilutions corresponding to 1,
5, 10, and 100 trichomonads were made in deionized water,
and then DNA was extracted and processed as described
above.
Agarose gel electrophoresis
An aliquot (7 µL) of each of the PCR products was run
in 1.5% agarose gels in 1x TAE (Tris-acetate-EDTA, 0.04
M Tris-acetate, 0.001 M EDTA) buffer at 100V. The gels
were then stained with 0.5 µg/mL ethidium bromide,
viewed under UV illumination, and photographed. The
sizes of the amplified products were assessed by compari-
son with a commercial 100-bp weight marker (Roche Diag-
３
１
nostics, Inc.). Samples containing 300-bp fragments were
considered positive for T. vaginalis.
RESULTS
In the present study, all of the 400 study subjects will-
ingly participated in the study. The median age of the study
population was 24.56 (range, 18-44). The largest portion of
the study population was comprised of patients 23-27 years
of age (51.59%), followed by ages 18-22 (28.84%), 28-32
(16.40%), 33-37 (2.65%), and 43-47 (0.27%). There were
no study participants in the 38-42 age range (Figure 2).
All personal identifiers had been removed from sam-
ples and new, unique identifiers (sample codes 1-400) were
assigned. A total of 377 samples were processed for PCR
using primers TV3/7. Of the 377 clinical samples tested, 36
(9.55%) were positive for T. vaginalis infection. Figure 3
represents the agarose gel electrophoresis of some of the T.
vaginalis-positive samples detected by the PCR. The lower
limit sensitivity of T. vaginalis detection by the PCR assay
was found to be one trichomonad (Figure 4).
Figure 5 shows the prevalence of trichomoniasis for
each age group. To calculate the trichomoniasis prevalence,
the number of positive cases within each age group was di-
vided by the total number of participants corresponding to
the group. As a result, the highest prevalence of T. vagina-
lis infection was found in the age group 18-22 (12.84%),
60 51.59％
50
Study population(％)
40
28.84％
30
16.4％
20
10 2.65％
0.27％
0
18-22 23-27 28-32 33-37 43-47
Age range(Years)
Figure 2. Age distribution of study participants
Figure 3. Gel profile of PCR analysis of representative T.
vaginalis-positive samples. Lanes: MW ―
molecular weight marker; 1 ― sample 1; 2 ―
sample 11; 3 ― sample 88; 4 ― sample 216
３
２

study contrasts sharply with the results reported by the sen-

Figure 4. Sensitivity of PCR with primers TV3/7 in de-
tecting T. vaginalis. Lanes: MW ― molecular
weight marker; 1 ― 100 trichomonads; 2 ― 10
trichomonads; 3 ― 5 trichomonads; 4 ― 1
trichomonad
14 12.84％
T.vaginalis prevalence(％)
tinel STI etiologic surveillance systems (SSESS) in 2004.
This sentinel surveillance system was created to provide re-
gional and national STI programs with current and readily
available data on the distribution and frequency of STIs in
the country [18]. Groups monitored by SSESS are regis-
tered female sex workers (RFSW), freelance sex workers
(FLSW), male sex workers (MSW) and injecting drug users
(IDUs) in different sentinel STI surveillance sites around
the country [19]. SSESS reported no cases of trichomonia-
sis among these respondents in Angeles City. However, the
data on STIs in the Philippines as reported by SSESS are
currently limited since STI surveillance has traditionally re-
lied on passive reports from social hygiene clinics (SHCs).
Moreover, this is likely to be an underestimate of true

12
9.60％
10 8.20％
8 The prevalence in the present study was also higher
6 than that reported by Wi and co-workers in a cross-sectional
4
2 0％
0 women with symptoms of abnormal vaginal discharge [20].
18-22 23-27 28-32 33-37
Age group
Figure 5. Age distribution of T. vaginalis-positive individuals as
determined by PCR
followed by 28-32 (9.6%) and 23-27 years old (8.2%). No
positive cases were identified in the 33-37 and 43-47 age
groups.
DISCUSSION
Constant screening of high-frequency transmitter
groups such as female sex workers is an important approach
in controlling the spread of STIs such as trichomoniasis.
Female sex workers and their male clients are thought to act
as core and bridging populations in the epidemiology of
STIs [17]. The RHWC functions under the City Health Of-
fice of Angeles City as the only reproductive health clinic
regularly monitoring the city’s entertainment workers. Sex
workers from different parts of the city visit the clinic for a
weekly smear check-up which is required to continue work-
ing or to renew health licenses in entertainment establish-
ments. Therefore, the sample size obtained from the clinic
in the present study represented the female sex workers
throughout the city.
The prevalence of trichomoniasis among 377 women
attending the RHWC in Angeles City was 9.55% using PCR
primers TV3/7. The prevalence determined in the current
prevalence as wet mount miscroscopy alone was used for
the identification of trichomoniasis.
study in 2002. They detected trichomoniasis in 3.18% of
selected female respondents from different parts of the
0％ country, and a 3.38% occurrence rate of the parasite among
43-47
This study used culture techniques and microscopy to detect
T. vaginalis.
In direct microscopy or the wet mount method, the
most frequently used diagnostic tool for trichomoniasis, the
presence of motile trichomonads signifies a positive test.
Although direct visualization through microscopy is both
rapid and inexpensive, low sensitivity reduces its reliability
as a method for T. vaginalis detection. The culture method
is the current “gold standard” for the diagnosis of trichomo-
niasis, but it is performed by relatively few laboratories due
to the cost of the essential reagents and the laborious nature
of techniques such as frequent microscopic observations for
positive samples for up to 7 days [21]. The large number of
specimens being tested in the present study also limits its
use as a detection tool for T. vaginalis in vaginal exudates
of women. Another potential drawback is that the culture
technique may sometimes generate false-negative or false-
positive results due to the failure of T. vaginalis to thrive in
cultures with bacterial and fungal overgrowth over time, as
previously observed in our laboratory. It has also been
proven that dead T. vaginalis cells, which may also be en-
countered on dried vaginal smears during routine check up,
appear dense and granulated and may resemble the nucleus
of vaginal epithelial cells.
Alternatively, PCR of vaginal swabs may be advanta-
geous over the latter method of T. vaginalis detection espe-
cially in settings where incubation of cultures is not possi-
ble and shipment of specimens to a reference laboratory is

required [22]. Several groups of investigators have reported
findings on the development of a PCR technique for tricho-
monads. It has been found that the detection of T. vaginalis
from vaginal swabs by PCR employing primers TV3/7, the
primer set utilized in this present study, was equivalent to
culture with a sensitivity and specificity of 88.7% and
97.1%, respectively [22]. Also, PCR-based detection of T.
vaginalis using vaginal specimens may provide an alterna-
tive to culture. This is consistent with the findings of Queza
(2008) showing that the sensitivity of PCR using primers
TV3/7 was comparable to that of culture at 96.97% [12].
One strategy for increasing the diagnosis and treatment
of trichomoniasis is the use of a screening test with higher
sensitivity compared to the traditional wet prep of vaginal
fluid commonly used in social hygiene clinics around the
country. In the present study, PCR using primers TV3/7 ap-
pears to be highly sensitive, being able to amplify DNA
from one T. vaginalis cell. This result is consistent with
that of Lawing and co-workers who determined that the
lower limit sensitivity of PCR with primers TV3/7 was one
organism [22]. Therefore, PCR using vaginal swabs and
urine specimens, compared to diagnosis by wet mount mi-
croscopy, culture, and fluorescent staining, appeared to be
the method of choice for the detection of genital infections
with T. vaginalis [23].
In this study, DNA extractions from samples were per-
formed in batches using the Chelex method. As a chelating
resin, Chelex 100 has been used to extract heavy metals
from solutions and, when used for DNA extraction, may
also eliminate PCR inhibitors present in the samples and/or
prevent the disruption of the genomic DNA [14]. The ab-
sence of PCR inhibitors and DNA contaminants is essential
to the performance of PCR in detecting T. vaginalis in vagi-
nal swabs.
Angeles City is a priority area for STI control and HIV
prevention. The city has the highest cumulative number of
HIV seropositive individuals among the ten HIV surveil-
lance sites in the country [20]. Trichomoniasis has been
shown to be a risk factor for transmission and infection with
HIV. T. vaginalis disruption of urogenital epithelial mono-
layers could facilitate passage of HIV-1 to underlying cells,
and activation of local immune cells by T. vaginalis in the
presence of infectious HIV-1 might lead to increased viral
replication [24]. Therefore, to effectively direct HIV pre-
vention and control programs, a test for T. vaginalis infec-
tion should be included in routine clinical check-ups among
high-risk individuals, just as adequate information on the
prevalence and distribution of trichomoniasis should be
made readily available. PCR has allowed a greater under-
standing of the global epidemiology of T. vaginalis and
raised concerns about the potential impact on HIV transmis-
３
３
sion and the reproductive health of females [17]. Moreover,
significant reductions in the prevalence of a nuisance STI
such as trichomoniasis are possible when effective interven-
tions reach core groups such as female sex workers. The
correlation of age of female respondents and known risk
factors of trichomoniasis is also an important topic for fur-
ther investigation.
ACKNOWLEDGEMENT
This work was supported by a research grant from the
Office of the Vice-Chancellor for Research and Develop-
ment of the University of the Philippines-Diliman (Grant
No. 080809 PNSE) to W.L.R.
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