Prevalence and risk factors for Trichomonas vaginalis infection in pregnant women in South Africa

Abstract

There are insufficient data on the prevalence of Trichomonas vaginalis infection in pregnant women in South Africa. The present study aims to determine the prevalence of T. vaginalis (TV) in pregnant women and identify the risk factors associated with this infection in pregnancy. This was a cross-sectional study which included 362 pregnant women attending the antenatal clinic at the King Edward VIII Hospital in Durban, South Africa. A self-collected vaginal swab was provided by the enrolled women for laboratory diagnosis by real-time polymerase chain reaction. The protozoan load was determined as the logarithm of gene copies per milliliter. The prevalence of TV infection was 12.9% and 59.6% of the women who tested positive were asymptomatic (p = 0.011). Women who presented with abnormal vaginal discharge had a 4.6 times likelihood of testing positive for TV infection (odds ratio 4.62; 95% confidence interval 1.83 to 11.70; p = 0.001). TV infection was high amongst pregnant women. Therefore, routine testing for this sexually transmitted infection is encouraged for proper management.

Keywords

women sexual behaviour

Introduction

Trichomonas vaginalis is an anaerobic protozoan that causes the sexually transmitted infection (STI) trichomoniasis. Trichomoniasis is the most common curable STI, with a reported prevalence of 276.4 million infections annually worldwide.^1–3 In the African region, 42.8 million infections occur annually.⁴ In South Africa, the estimated T. vaginalis (TV) infection prevalence ranges between 3.1% and 20.0%.⁵ Studies that have been conducted in South African pregnant women in South Africa have reported TV infection prevalence rates of 15.3%⁶ and 20.2%.⁷

Infection with TV is often asymptomatic in 25 to 50% of women.^8,9 However, when symptoms arise, women experience vaginal discharge, pruritus and/or dysuria, vaginitis and cervicitis.^8,10 Trichomoniasis has been found to be associated with various health complications including pelvic inflammatory disease, significant pregnancy complications, cervical cancer prostatitis and infertility.^11,12 Significant pregnancy complications include pre-term labour, low birth weight and premature rupture of membranes.^13,14

Several risk factors associated with TV infection have been reported including older age, co-infection with other STIs,^15,16 sexual behaviour, socio-economic status, phase of menstrual cycle.¹⁵ In African women, older age, marital status, multiple sex partners, poor hygiene and socio-economic status have been shown to be the risk factors associated with TV infection.¹

TV infection in pregnancy has been associated with preterm delivery, low birth weight,^13,14,17 neonatal morbidity and mortality.¹⁷ It has been reported that 2 to 17% of female infants acquire TV infection through direct vulvo-vaginal infection during delivery through passage in an infected birth canal.^2,17 TV infection can cause respiratory and vaginal infections in new-borns.¹⁶

The aim of this study was to determine the prevalence of TV infections by real-time polymerase chain reaction (PCR) and identify the risk factors associated with infection in the studied population.

Methods

Study design and population

This study was a cross-sectional study which included 362 pregnant women. The study population was recruited during October 2018–March 2019. The women who were enrolled in the study were 18 years and older, willing to provide written informed consent and willing to provide a self-collected vaginal swab sample to be tested for TV infection. The swab was a sterile individually-wrapped Dacron swab. The study population was recruited from the King Edward VIII Hospital in Durban. At enrolment, all women provided data on their socio-demographic statuses, sexual behaviour and clinical history by use of a structured questionnaire which was administered by the study team. The laboratory testing was conducted at the School of Clinical Medicine Research Laboratory at the Nelson R Mandela School of Medicine, University of KwaZulu-Natal.

Ethical statement

Ethics approval for this study (BE296/18) was obtained from the Biomedical Research Ethics Committee (BREC), University of KwaZulu-Natal.

Laboratory testing

DNA extraction

Each vaginal swab was suspended in a 2 mL volume of phosphate buffered saline (PBS). Following agitation and rinsing in PBS, the swabs were discarded. The remaining suspension was centrifuged at 14,000 r/min for 10 min and the supernatant was discarded. Recovered pellets were then subjected to a DNA extraction method using the PureLink Microbiome Kit (ThermoFisher Scientific, Waltham, Massachusetts,US), according to the manufacturer’s instructions. The concentration of extracted DNA was determined using the Nanodrop Spectrophotometer (ThermoFisher Scientific, Waltham, Massachusetts,US). Resulting total DNA concentrations occurred in the range of 0.3–68.5 ng/µL, with accompanying A260/A280 ratios in the range of 0.57–2.21.

Real-time quantitative polymerase chain reaction

PCR amplification was performed on the Quant Studio 5 real-time PCR detection system (ThermoFisher Scientific, Waltham, Massachusetts,US), in a 384-well microtiter reaction plate. We screened for TV using the Applied Biosystems™ TaqMan® Assays. Commercial primers and probes (Pr04646256_S1, ThermoFisher Scientific, Waltham, Massachusetts,US) which target the alpha tubulin 1 gene of TV were used.

Briefly, each reaction was performed in a final volume of 5 µL that comprised of 0.5 µL FAM-labelled probe/primer mix for individual targets, 2.5 µL Fast Start 4× probe master mix (Pr04646256_s1), 1.5 µL template DNA and nuclease-free water. We also included non-template control reactions. Amplification was performed under the following conditions: 1 cycle at 95°C for 30 s followed by 45 cycles of denaturation at 95°C for 3 s and annealing at 60°C for 30 s. Detection of fluorescent products was performed at the end of the annealing period.

The determination of the gene copies/mL for each positive sample was carried out according to the provided formula by the manufacturer that was based on the amplification dynamics of positive control dilution series with a linear relationship between log10 gene copy numbers and Ct values. A serial dilution of plasmids for each organism was used to determine the accurate quantification from the patient samples.

Statistical data analyses

Descriptive characteristics of study participants are presented, by TV status, as frequencies and percentages of the categorical variables. Comparisons by TV in the descriptive characteristics were done using Chi square tests with a 5% significance level. With respect to trimester of pregnancy, women between gestational ages 4 and 12 weeks were categorised as first trimester, 13 and 27 weeks as second trimester and 28 and 40 as third trimester. Participants were further categorised as asymptomatic or symptomatic. Women were characterised as symptomatic if an abnormal vaginal discharge (AVD) was present (foul-smelling and yellowish-green). In addition, women who presented with symptoms of vaginal itching were characterised as symptomatic. Women were characterised as asymptomatic if they did not present with any of the above-mentioned symptoms.

The socio-demographic, behavioural and clinical history factors were then assessed for their likelihood to predict testing TV positive in univariate and multivariate logistic regression analyses, with p-values less than 0.05 considered as statistically significant. Variables were included one by one in the model with TV and were considered as potential confounders if they caused the model coefficient for TV to change by 10% or higher. The TV protozoan load for symptomatic and asymptomatic pregnant women as well as for those experiencing a current AVD were assessed and are presented in means and medians. T tests, with a 5% level of significance, were used to compare the means of the TV protozoan loads. All the analyses reported in this manuscript were conducted using Stata v15 (Stata Corp, College Station, TX, USA).

Results

Baseline characteristics and prevalence of TV infection

A total of 362 pregnant women were screened for TV infection. The mean ± SD (Coefficient of variation [CV], %) age of the study participants was 28.3 ± 7.10 years (25.1). The median (Q1–Q3) age of the women was 28.0 years (24.0–32.5). The minimum and maximum ages were 18.0–39.0. The majority of the study women had completed high school (69.8%), were not employed (69.9%) and were unmarried (88.4%). Baseline characteristics stratified by infection status are summarised in Table 1. A total of 47/362 (12.9%; 95% confidence interval [CI] 9.89 to 16.87) women tested positive for TV infection. A total of 59.6% of the women who tested positive for TV in this study did not show symptoms of a current abnormal discharge (foul-smelling and yellowish-green discharge) or vaginal itching, i.e. were asymptomatic.

Table 1.

Baseline characteristics for pregnant women who were screened for T. vaginalis infection.

	TV negative	TV positive
Characteristics	n (%)	n (%)	p
Socio-demographic status
Age (years)			0.172
18–20	25 (7.9)	2 (4.3)
21–25	77 (24.4)	18 (38.3)
26–30	89 (28.3)	9 (19.1)
>30	124 (39.4)	18 (38.3)
Education level			0.580
Did not attend school	6 (1.9)	0 (0.0)
Primary school	6 (1.9)	2 (4.3)
High school	220 (69.8)	32 (68.1)
College or university	83 (26.3)	13 (27.7)
Employment			0.332
No	223 (70.8)	30 (63.8)
Yes	92 (29.2)	17 (36.2)
Married			0.478
No	277 (87.9)	43 (91.5)
Yes	38 (12.1)	4 (8.5)
Cohabiting			0.677
No	191 (60.6)	27 (57.4)
Yes	124 (39.4)	20 (42.6)
Risk behaviour
Regular sexual partner			0.531
No	49 (15.6)	9 (19.1)
Yes	266 (84.4)	38 (80.9)
Age at first sex (years)			0.589
<15	4 (1.3)	0 (0.0)
15–20	237 (75.2)	33 (70.2)
21–25	64 (20.3)	13 (27.7)
>25	10 (3.2)	1 (2.1)
Sexual partners in lifetime			0.365
1	75 (23.8)	11 (23.4)
2–4	193 (61.3)	31 (66.0)
>4	47 (14.9)	5 (10.6)
Partner has other partners			0.406
Do not know	179 (56.8)	29 (61.7)
No	74 (23.5)	7 (14.9)
Yes	62 (19.7)	11 (23.4)
Condom use			0.646
Always	2 (0.6)	0 (0.0)
Never	92 (29.2)	10 (21.3)
Rarely	5 (1.6)	1 (2.1)
Sometimes	216 (68.6)	36 (76.6)
Clinical history
Trimester of pregnancy			0.608
1st	43 (13.7)	6 (12.8)
2nd	108 (34.3)	13 (27.7)
3rd	164 (52.1)	28 (59.6)
Current abnormal vaginal discharge			0.011
No	242 (76.8)	28 (59.6)
Yes	73 (23.2)	19 (40.4)
Previous STI treatment			0.558
No	195 (61.9)	27 (57.4)
Yes	120 (38.1)	20 (42.6)
STI symptoms in the past three months			0.488
Abnormal vaginal discharge	114 (36.2)	16 (34.0)
Genital itching	44 (14.0)	4 (8.5)
None	157 (49.8)	27 (57.4)

TV: Trichomonas vaginalis.

Socio-demographic status

For the women who tested TV positive, the highest proportions fell in the age groups 20–25 (38.3%) and above 30 (38.3%). However, age was not significantly associated with infection (p = 0.172). There was also no statistical significance with regard to the level of education and employment status (p = 0.580 and p = 0.332, respectively) for the TV-positive women.

Risk behaviour

There was high prevalence of TV infection among pregnant women who were not married (91.5%), who had a regular sexual partner (80.9%), who were not living with their partner (57.4%), who had their first sexual encounter between the age of 15 and 20 (70.2%), who had two to four life-time sexual partners (66%), who did not know if their partners had other partners (61.7%) and those who reported that they sometimes use condoms (76.6%). However, these behavioural factors were not statistically significant (p > 0.05).

Clinical history

Women who were in their third trimester of pregnancy and those who reported no STI symptoms (AVD, abnormal vaginal odour and genital itching or sores or warts) in the past three months had a high prevalence of TV infection (59.6% and 57.4%, respectively), However, these factors were not statistically significant (p > 0.05). There was high prevalence of TV infection in women that were asymptomatic (no AVD at the time of the study enrolment; prevalence = 59.6%, p = 0.011).

Risk factors associated with TV infection

In univariate and multivariate analyses (Table 2), there was no statistically significant association between demographic and behavioural factors with TV infection (p > 0.05). With respect to clinical symptoms, there was a significant association between AVD and TV infection. In the multivariate analysis, pregnant women who reported no previous history of STI symptoms had an increased likelihood of testing positive for TV (odds ratio [OR] 3.85; 95% CI 1.42 to 10.43; p = 0.008). In the univariate analysis, pregnant women who reported AVD had an increased likelihood of testing positive for TV (OR 2.25; 95% CI 1.19 to 4.26; p = 0.013). This association was also shown to be significant in the multivariate analysis. Women who presented with AVD had a 4.6 times likelihood of testing TV positive (OR 4.62; 95% CI 1.83 to 11.70; p = 0.001).

Table 2.

Univariate and multivariate analysis of factors associated with T. vaginalis infection in pregnant women.

	Univariate		Multivariate
Characteristics	OR (95% CI)	p	OR (95% CI)	p
Age groups (years)
15–19	1.00		1.00
20–24	2.77 (0.34 to 22.75)	0.342	2.17 (0.43 to 10.86)	0.347
25–29	1.75 (0.21 to 14.62)	0.604	0.99 (0.18 to 5.46)	0.988
30–34	1.63 (0.19 to 13.93)	0.658	1.15 (0.22 to 6.12)	0.872
35–39	1.35 (0.15 to 12.63)	0.790	1.59 (0.77 to 3.29)	0.208
40–44	2.89 (0.29 to 28.94)	0.367	0.78 (0.23 to 2.68)	0.699
Education level
Primary or less	1.00		1.00
High school	0.87 (0.19 to 4.08)	0.863	1.26 (0.23 to 6.78)	0.790
College/university	0.94 (0.19 to 4.69)	0.940	1.15 (0.19 to 6.83)	0.876
Employment status
No	1.00		1.00
Yes	1.37 (0.72 to 2.61)	0.333	1.68 (0.80 to 3.53)	0.167
Marital status
No	1.00		1.00
Yes	0.68 (0.23 to 2.00)	0.480	0.81 (0.23 to 2.86)	0.740
Has regular sexual partner
No	1.00		1.00
Yes	0.78 (0.35 to 1.71)	0.532	0.69 (0.29 to 1.65)	0.406
Cohabiting
No	1.00		1.00
Yes	1.14 (0.61 to 2.12)	0.677	1.43 (0.66 to 3.08)	0.367
Age at first sex (years)
<21	1.00		1.00
≥21	1.38 (0.70 to 2.72)	0.349	1.54 (0.71 to 3.36)	0.276
Sexual partners in lifetime
1	1.00		1.00
2–4	1.19 (0.56 to 2.55)	0.657	1.15 (0.48 to 2.76)	0.748
>4	0.79 (0.25 to 2.45)	0.679	0.86 (0.24 to 3.06)	0.820
Partner has other partners
Do not know	1.00		1.00
No	0.58 (0.24 to 1.39)	0.225	0.56 (0.21 to 1.44)	0.228
Yes	1.10 (0.52 to 2.32)	0.813	1.47 (0.64 to 3.40)	0.368
Condom use
No	1.00		1.00
Yes	1.53 (0.73 to 3.20)	0.262	1.39 (0.62 to 3.08)	0.423
Trimester of pregnancy
1st trimester	1.00		1.00
2nd trimester	0.86 (0.31 to 2.42)	0.779	0.90 (0.29 to 2.72)	0.846
3rd trimester	1.22 (0.48 to 3.14)	0.675	1.22 (0.43 to 3.42)	0.707
Current AVD
No	1.00		1.00
Yes	2.25 (1.19 to 4.26)	0.013	4.62 (1.83 to 11.70)	0.001
Previous STI treatment
No	1.00		1.00
Yes	1.20 (0.65 to 2.24)	0.559	1.44 (0.71 to 2.90)	0.309
STI symptoms in the past three months
AVD	1.00		1.00
Genital itching/sores	0.65 (0.21 to 2.04)	0.459	1.37 (0.36 to 5.19)	0.647
None	1.23 (0.63 to 2.38)	0.548	3.85 (1.42 to 10.43)	0.008

AVD: abnormal vaginal discharge; OR: odds ratio; CI: confidence interval.

TV protozoan load in relation to clinical symptoms

The load of TV was compared across women who reported having any previous symptoms of STIs and women who reported a current AVD (Table 3). There was no significant difference in the load of the pathogen in women who reported previous STI symptoms versus women who did not have previous symptoms (p = 0.304). Similarly, there was no significant difference in the mean and median protozoan loads of TV between women who reported current AVD when compared to those who were asymptomatic (p = 0.190).

Table 3.

Trichomonas vaginalis protozoan load in relation to clinical symptoms.

Characteristic	Mean PL	Median PL	n	p
Clinical symptoms				0.304
Symptomatic^a	32.92383	34.10841	16
Asymptomatic	32.39298	31.04182	31
Current AVD				0.190
No	31.79429	30.5795	28
Yes	33.72228	35.03839	19

PL: protozoan load; AVD: abnormal vaginal discharge.

^aSymptomatic – AVD, abnormal vaginal odour, genital itching/sores/warts.

Discussion

In this study, we describe the prevalence, risk factors and quantification of TV in South African pregnant women. The prevalence of TV infection varies greatly depending on the study population, type of sample collected as well as laboratory diagnostic techniques used. TV infection prevalence has been studied in different populations such as women and men attending STI clinics, sex workers and pregnant women. Studies that have been conducted in pregnant women report prevalence rates ranging from 0% to approximately 20%.^{2,6,7,17–20} The prevalence of TV infection in this study was 12.9%. This result is similar to the prevalence found in a study that was conducted in Umlazi, South Africa, that showed a prevalence of 15.3% in pregnant women.⁶ In a study that was conducted in three primary healthcare clinics in the Tshwane District, South Africa, found a prevalence of 20.2% in pregnant women.⁷ High prevalence rates of TV infection among pregnant women can be attributed to the hormonal changes, cessation of contraception use and increased vaginal pH observed during pregnancy (approximately 5.5 to 5.8). During pregnancy, changes in hormonal levels result in physiological changes. Such physiological changes cause vaginal mucosa congestion and hypertrophy, which benefit the growth of pathogenic microorganisms within the vagina.²¹ The increase in vaginal pH promotes colonisation of the urogenital tract by TV.¹⁵

AVD, pruritus and/or dysuria, vaginitis and cervicitis are the most common symptoms reported in TV infection.^8,10 Among women who tested positive for TV infection in this study, 34% reported AVD, 8.5% reported genital itching and sores and 57.4% reported no symptoms in the past three months. AVD is the most common symptom and it is reported in more than 50% of TV cases.¹¹ In this study, 40.4% of women who tested positive for TV reported AVD at the time of the study.

There are minimal data on prevalence of asymptomatic trichomoniasis among pregnant women. In the present study, 40.4% of the women who tested positive for TV reported AVD. We also observed high prevalence of TV infection among asymptomatic participants. The estimated prevalence of asymptomatic TV infections among women at STD clinics is approximately 50%.⁹ Price et al. also reported a high prevalence of asymptomatic trichomoniasis among pregnant women (49%) in South Africa.²² In a study that was conducted in Vietnam, in non-pregnant women, found an overall prevalence of 6.6%, with significant differences between symptomatic and asymptomatic women (19.3% and 0.7%, respectively). However, these results were obtained by microscopic examination.²³

Several demographic and behavioural factors have been associated with TV infection. In African women, older age, low socio-economic status, multiple sex partners, being unmarried and poor hygiene practices have been associated with trichomoniasis.¹ In this study, there were no demographic and behavioural factors that were statistically associated with trichomoniasis. However, we did observe high percentages in certain groups of study participants who tested positive for TV. A high percentage of women in the age groups of 21 to 25 and those who were above 30 tested positive for TV infection. Women who were unemployed, not married, had a regular sexual partner and those who reported inconsistent condom use had a high percentage of TV positivity. In another study conducted in Durban (South Africa) found being unmarried and non-cohabitating, having more than three lifetime sexual partners and having an infection with any STI were the risk factors associated with trichomoniasis among non-pregnant women.⁸ De Waaij et al. observed that single status, use of hormonal contraceptives, a positive human immunodeficiency virus (HIV) status and visiting bars as factors associated with trichomoniasis.⁴

It has been described that the microbial load of infectious organisms affects or influences the clinical manifestations of an infection.⁴ In the present study, we compared the protozoan load between the symptomatic versus asymptomatic groups. There were no significant differences in the mean protozoan load of TV in these groups. This result is similar to that observed by de Waaij et al., where they did not find any significant association between the protozoan load and reported symptoms of TV infection. We also compared the protozoan load between women who reported AVD versus those who did not. We found no significant differences between AVD and the protozoan load. This finding contrasts with that reported by de Waaij et al. They observed a significantly higher protozoan load within women who presented with AVD during examination. These differences in results may be due to different study populations.

This study had several limitations. The study participants were recruited from one clinic. However, King Edward VIII Hospital is a central tertiary hospital and it represents the wider population of Durban. Another limitation is that the clinical symptoms were self-reported by study participants, and thus this might have introduced reporting biases. Also, data on STI treatment three weeks prior to sample collection from study participants were not collected. Therefore, nucleic acids from non-viable TV may have been detected by PCR. The study did not collect information on HIV status which is also a risk factor for STIs. The strength of this study is that it fills a gap in the literature on the prevalence of asymptomatic TV infection among pregnant women in Durban, South Africa, as well as identifies the risk factors associated with this infection.

Conclusion

This study observed a high prevalence of TV infections in an antenatal population. Therefore, routine testing for this STI is encouraged to aid in the identification and management of TV since this infection can lead to severe maternal and neonatal complications. The identification of the risk factors associated with this infection will act as a cornerstone for future management efforts.

Footnotes

Acknowledgements

We acknowledge the women who participated in this study and the King Edward VIII Hospital antenatal clinic management.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the College of Health Science, University of KwaZulu-Natal (grant number 636713).

ORCID iD

Nonkululeko Mabaso

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