Crucitti, Tania PHD*; Jespers, Vicky MD*; Mulenga, Chanda MSc†; Khondowe, Shepherd MPH†; Vandepitte, Judith MD*; Buvé, Anne PHD*
Trichomoniasis is the most common curable sexually transmitted infection (STI) worldwide.1 The infection is frequently asymptomatic in both women and men. When symptomatic, it can cause vaginitis, urethritis, and cervicitis in women and urethritis in men.2,3 There is growing evidence that Trichomonas vaginalis infection of the vagina is associated with an increased risk of acquisition and transmission of HIV infection.4–10 Moreover, trichomoniasis has been implicated in adverse pregnancy outcome.11
Although infection rates in sub-Saharan African women are estimated to be among the highest in the world, clinical research on T. vaginalis infection in Africa has not received the same attention as other STI such as gonorrhoea and syphilis. Published data on the prevalence and incidence of trichomoniasis in Africa are limited, but it appears that there are large variations in prevalence between different regions in sub-Saharan Africa. For instance, in Benin, the prevalence of trichomoniasis in women in the general population was 3.2%, whereas it was 47% in Uganda.12,13
In the multicentre study on factors determining the differential spread of HIV in 4 African cities, including Cotonou (Benin), Yaoundé (Cameroon), Kisumu (Kenya), and Ndola (Zambia), a high prevalence (34.3%) of T. vaginalis infection was found among women in the general population in Ndola.12,14 This finding was in line with the findings of a study performed 20 years earlier in Zambia, where 38.5% of antenatal clinic attendees and 31.4% of women at a gynecology clinic were infected with T. vaginalis.15 In addition, both studies found a high prevalence of T. vaginalis infection in young girls. Hira found prevalence rates of 4.7% to 8.5% in girls younger than 15 years old, and Buvé et al found that 40% of young women who denied that they had ever had sexual intercourse had trichomoniasis, as assessed by culture.14,15 This latter finding suggests that a certain proportion of women may have acquired T. vaginalis infection through transmission routes that do not involve penetrative sexual intercourse. Another possible explanation is that the intestinal flagellated parasite Pentatrichomonas hominis, which is morphologically very close to T. vaginalis, infects the vagina by cross contamination from the rectum.14,15
The aim of the current study was to assess the prevalence of infection with T. vaginalis and human trichomonads in 3 female populations with different sexual behavior patterns, in Ndola, Zambia.
MATERIALS AND METHODS
Study Design and Procedures
The study took place in Ndola in selected administrative areas that represent the different socioeconomic strata of town. We included in the study adolescent girls attending school, pregnant women attending antenatal clinic, and commercial sex workers (CSW). Adolescent girls aged between 13 and 16 years were recruited at school, and were not menstruating at the time of specimen collection. Informed consent was provided by a parent or legal guardian and assent was given by the girl herself. CSWs were self acknowledged sex workers and were recruited at their places of work or another convenient location. They took part in the study after giving their informed consent, and provided they did not menstruate at the time of specimen collection. Pregnant women were recruited at antenatal clinics in Ndola.
All women were interviewed about their sociodemographic characteristics, sexual behavior, and genital hygiene practices. Interviews were conducted face-to-face by trained interviewers. After the interview, the women were instructed on how to collect a sample of the vagina, the rectum, and the mouth. EZ culturette swabs (Becton Dickinson BBL, Sparks, MD) were used and after sampling were kept in a cooler box until transport to the laboratory of Tropical Diseases Research Centre (TDRC).
The self-administered samples were stored at the TDRC, at −20°C until shipment on dry ice to the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. The specimens were tested for T. vaginalis, P. hominis, and Trichomonas tenax by polymerase chain reaction assay (PCR). DNA was extracted from the clinical specimens using the QIAamp DNA minikit (Qiagen, Hilden, Germany). We used the TVK3/7 primer set, which targets a repetitive DNA fragment of T. vaginalis, and the PT3/7 and Th3/5 primers sets, to amplify a sequence of the 18SrRNA gene of T. tenax and P. hominis, respectively.16–18 Positive results for T. vaginalis were confirmed using the IP1/IP2 primer set targeting an E 650 family repeat.19 Positive results for T. tenax and P. hominis were confirmed through amplification of the internal transcribed spacer region of the 5.8S rRNA gene using the TFR1/2 primer set.20
T. vaginalis amplicons were detected using an enzyme immunoassay (EIA); the amplicons of T. tenax and P. hominis were detected using agarose gelelectrophoresis. The PCR testing procedures for T. vaginalis and P. hominis were described previously.18,21 For the detection of T. tenax the assay was performed as described by Kikuta et al. 17 The specificity of the above listed primer sets, with the exception of the TFR1/2 primer set, was evaluated using reference strains summarized in Table 1. The TFR1/2 primer set amplifies T. vaginalis, T. tenax, and P. hominis. The amplicons of both T. vaginalis and T. tenax have the same size of 368 bp, but they differ in nucleotide sequence with 25 different nucleotides. The amplicon of P. hominis is shorter, 339 bp. The other primer sets were species specific.
Inhibition and adequacy of the vaginal and oral specimens was assessed by amplification of the β2 microglobulin gene.22,23 Adequacy of the rectal specimens was assessed by amplification of Escherichia coli.24
Strain typing of T. vaginalis was performed on clinical specimens with a positive TVK3/7 amplification result. A previously published PCR-restriction fragment length polymorphism method was applied.25 Briefly, the actin gene target was amplified by a nested PCR, with outer primers Tv8S/Tv9R and inner primers Tv10S/Tv11R. The primers were synthesized by Eurogentec, Seraing, Belgium. Upon completion of PCR, 15 μL of each amplified specimen was analyzed by electrophoresis in a 2% agarose gel in Tris-acetate-EDTA buffer (pH 8.5). After visualization of the amplified product, 5 μL was digested for 4 hours at 37°C with restriction endonucleases Hind II, Mse I, and Rsa I, respectively. The restriction endonucleases Hind II and Rsa I were purchased from Roche Molecular Biochemicals, Mannheim, Germany. The restriction endonuclease Mse I was purchased from New England BioLabs Inc., Ipswich, MA.
Separation of the fragments was performed using 3% agarose gel in Tris-acetate-EDTA buffer (pH 8.5). The gel was stained with ethidium bromide 0.5 μg/mL (Sigma, Bornem, Belgium) and was photographed under short ultraviolet light. The sizes of the amplified products were assessed by comparing with a 100 bp commercial weight marker Smartladder SF (Eurogentec, Seraing, Belgium). The digestion of the amplified product with the restriction enzymes yielded distinct DNA fragments. Actin genotypes were assigned by combining the DNA fragments.
Each test run included 1 negative and 1 positive control. The negative control consisted of the reaction mixture with water instead of DNA extract, the positive control was a previously typed T. vaginalis ATCC reference strain, randomly selected from the ATCC reference strains at each run. Testing was considered valid if after nested PCR no amplified product was detected in the negative control and if after digestion the reference ATCC strain gave the previously determined actin genotype.
All assays were performed according to standard quality assurance guidelines for molecular diagnosis.26,27
A sample was considered positive for DNA of T. vaginalis, T. tenax, or P. hominis if at least 2 different PCR assays with different targets were positive for respectively T. vaginalis, T. tenax, or P. hominis.
Prevalence rates are presented with 95% confidence intervals. Differences in prevalence were tested on their statistical significance using the chi-squared test for proportions.
The study was approved by the Ethics Committees of the TDRC in Ndola and the ITM in Antwerp.
A total of 460 adolescent girls, 307 pregnant women, and 197 CSW gave their informed consent and were included in the study. The adolescent girls were 13 to 16 years old, one-third of them being 13 years old. The age range for the pregnant women was 15 to 42 years and for the CSW 14 to 45 years. Of the pregnant women, 34.2% were aged 20 to 24 years; among the CSW this percentage was 45.7%. Laboratory results were available for 936 (97%) of the 964 participating women. For the remaining 28 women, the laboratory results were invalid, mostly due to inadequate or missing specimen.
T. vaginalis was found in vaginal specimens of 108 of 439 adolescent girls (24.6%, 95% confidence interval [CI]: 20.6–28.6); 99 of 307 pregnant women (32.2%, 95% CI: 27.0–37.4) and 63 of 190 CSW (33.2%, 95% CI: 26.5–39.9) (Table 2). Prevalence was lower in adolescent girls than in pregnant women and CSW and this difference was statistically significant (χ2 = 7.31 on 2 df, P = 0.026). Among adolescent girls who reported that they had ever had penetrative sex the prevalence of T. vaginalis infection was 35.6% (21/59, 95% CI: 23.6–49.1); among adolescents who denied that they ever had sexual intercourse the prevalence was 22.9% (87/380, 95% CI: 18.8–27.5). Table 3 summarizes the prevalence rates of vaginal trichomoniasis found in adolescents by age and reported sexual activity.
T. vaginalis was also found in the mouth and the rectum (Table 2). The presence of T. vaginalis DNA in the rectum was strongly associated with the presence of T. vaginalis DNA in the vagina (odds ratio: 18.2, 95% CI: 4.0–82.5, P < 0.001).
We had specimens from the 3 collection sites from 223 pregnant women. Of the 65 women who had T. vaginalis DNA in the vagina, 1 also had T. vaginalis DNA in the mouth. None of the women had T. vaginalis DNA in the mouth alone.
T. tenax DNA was detected in samples from the vagina and from the mouth. Two of the 4 women who harbored T. tenax DNA in the vagina also had T. vaginalis DNA in the vagina. Among 24 women with detectable T. tenax DNA in the mouth, none had T. vaginalis DNA in the mouth, but 6 had T. vaginalis DNA in the vagina. P. hominis DNA was detected in rectal specimens of 3 participants, 1 of whom also had T. vaginalis DNA in the vagina.
Out of 270 vaginal samples with T. vaginalis DNA, 231 (85.6%) were successfully genotyped, but in only 2 out of 15 positive samples from the mouth and the rectum genotyping was successful. Nine different actin genotypes of T. vaginalis in the vagina were identified in the 3 study groups. The distribution of the genotypes identified in the vagina was very similar in the 3 groups of women (Table 4). The most common genotypes were G, H, and P, making up 74.5% of trichomonas infections. Genotype B was detected in 1 adolescent girl and genotype K in 1 pregnant woman. Genotype R was not detected in the adolescents and genotype I was not identified in the CSW.
Using PCR assays, we found high prevalence rates of T. vaginalis infection in 3 groups of women in Ndola, Zambia, with different levels of sexual activity. We found no difference in prevalence between CSW and pregnant women. The prevalence in adolescent girls was lower, but was still substantial as nearly 1 in 4 girls aged 13 to 16 years was found to have T. vaginalis infection.
In the multicentre study, on factors determining the differential spread of HIV in 4 African cities, which was conducted in 1997–1998, the prevalence of trichomoniasis was 42% among CSW in Ndola, compared to 33.2% in this study.28 A decrease in prevalence of T. vaginalis infection in CSWs was also observed in Kinshasa (Democratic Republic of Congo), Cotonou (Benin), Abidjan (Ivory Coast), and in Bobo-Dioulasso (Burkina Faso).29–32 These changes in prevalence may be attributed to changes in sexual behavior of the study populations, in particular increases in condom use, and improved STI case management.29
We had postulated that a certain proportion of cases of vaginal trichomoniasis, especially in adolescent girls who denied that they had ever had sexual intercourse, are in fact caused by P. hominis, a human trichomonad that is found in the large intestine and that is morphologically similar to T. vaginalis.14 We did not find evidence for the colonization of the vagina by P. hominis and our study confirms the conclusions of Adu-Sarkodie et al that P. hominis is not involved in the etiology of vaginal trichomoniasis.33
Inoculation studies conducted in the 1940s concluded that the trichomonads are body site specific and can not survive outside their natural habitat.34,35 We detected DNA of T. tenax, a trichomonad that is normally found in the oral cavity, in 4 vaginal specimens. As we did not culture T. tenax, we are unsure whether the DNA that was extracted was from viable organisms. We postulate that the vagina was infected with this oral trichomonad through autoinoculation via hands or through oral sex.
We also found T. vaginalis DNA outside the vagina. The presence of T. vaginalis in the rectum was associated with the presence of T. vaginalis in the vagina. The rectum could have been infected through cross contamination with T. vaginalis present in the vagina. Besides, the rectum could act as a source for T. vaginalis, from where the vagina could almost constantly be reinfected. In order to test the latter hypothesis, we analyzed 100 stool samples from girls aged 5 to 8 years. The samples were analyzed by PCR as described above. None of the specimens harbored T. vaginalis, T. tenax, or P. hominis (data not shown). We concluded that it is very unlikely that T. vaginalis present in the intestine colonizes the vagina and contributes to the high rates of vaginal trichomoniasis we have found in Ndola. The presence of T. vaginalis in the mouth was associated with the presence of T. vaginalis in the vagina in pregnant women. As with T. tenax, this finding suggests autoinoculation and/or oral sexual activity.
In our study populations, we identified 9 different T. vaginalis actin genotypes. In the 3 groups of women most infections were caused by 3 genotypes, including G, H, and P. There was no evidence for differences in the distribution of genotypes between the different groups of women. This suggests that women with different sexual behavior patterns and different sexual networks are infected with the same strains of T. vaginalis. Further research is needed to investigate the usefulness of other markers or combination of markers.
In this study, we used a genotyping method applied on clinical specimens. Although we achieved a sensitivity of 85% on the self collected vaginal specimens, the method was not sensitive on the self collected specimens from the mouth and rectum. The method could be improved by applying it on T. vaginalis isolates. This implies the use of culture medium and additional logistical requirements, and costs. In addition, the culture of T. vaginalis may lead to selective growth and selection of isolates. However, further improvement of the sensitivity of the method is required.
In conclusion, we confirmed that the prevalence of T. vaginalis infection is very high among adolescent girls, pregnant women, and CSWs in Ndola, Zambia. T. tenax and P. hominis, however, were relatively uncommon and were rarely found in the vagina. The data on trichomoniasis in adolescent girls who denied that they had ever had penetrative sexual intercourse, suggest that nonsexual routes of transmission may be more important in certain populations than generally acknowledged. The high rates of T. vaginalis infection found in this study are worrisome considering the association of trichomoniasis with poor pregnancy outcome and with increased risk for HIV acquisition. Screening and treatment of vaginal Trichomonas infection can easily be included in antenatal care programmes, at relatively low cost. In addition, control programmes with screening and treatment should be put in place for adolescent girls who are highly vulnerable to HIV infection.
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