“Hidden” sexually transmitted infections among women in primary care health services,Amazonas,Brazil

Abstract

Summary

This study describes the prevalence of infection by Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in a female population in Amazonas, Brazil. We collected cervical samples from 361 women examined at 10 primary care health services in the city of Coari, Amazonas, Brazil. The women were interviewed about socio-economic data, clinical history and sexual behaviour. Pelvic examinations were performed and cervical specimens were collected for detection of pathogens by PCR. The prevalence of infection was: 12.7% for Trichomonas vaginalis, 6.4% for Chlamydia trachomatis and 1.4% for Neisseria gonorrhoeae. There were no statistically significant associations between infections by any of the pathogens nor by any pathogen alone with any clinical variable, socio-demographic data or sexual behaviour. This study draws attention to the need for surveillance and possible need for screening for Chlamydia trachomatis, which often progresses asymptomatically. For the significant prevalence found, attention should also be given to asymptomatic infection by Trichomonas vaginalis, since this pathogen has recently been implicated as a risk factor for HIV infection.

Keywords

Sexually transmitted infection prevalence epidemiology Brazil PCR

Introduction

Most sexually transmitted infections (STIs) are asymptomatic or subclinical, and these “hidden infections” are partly responsible for the spread and persistence of them in a population. The lack of correspondence between the complaint of cervical or vaginal discharge and a positive diagnosis of Chlamydia trachomatis or Neisseria gonorrhoeae, making many of these patients truly “ silent pathogen carriers,” highlights the need for improvement of diagnostic and screening coverage.^1,2

Untreated chlamydial and gonococcal infections cause late effects in women, mainly to their reproductive health. Chlamydia infection became the most commonly reported bacterial infection in Europe and North America over the past decade. Therefore, strategies for Chlamydia screening were established in several developed countries, and information about the prevalence of Chlamydia infection has become better known in these places. The same did not occur in the same magnitude regarding information about gonococcal infection.^1–4 Vaginal trichomoniasis, by contrast, is an infection that results in greater clinical symptomatology. However, in about 30% of cases, women are asymptomatic.^5,6 Only more recently, infection with T. vaginalis has been associated with adverse effects on women's reproductive health, and one of the most serious associations is the possibility that T. vaginalis infection increases about 2–3 times the risk of transmission of HIV.^7–10

In Brazil, the epidemiological pattern of distribution of these infections is not precisely known. This is due to the fact that in Brazil chlamydial urethrithis and cervicitis are not reportable diseases. Also, most public health services do not provide specific diagnostic test routinely, nor are data available from local investigations. The same happens with gonococcal and trichomonal infections. Additionally, there are no organized screening programmes for these infections.^11–15

Because the Amazon region's territory is so large, there are many isolated communities in rural and indigenous areas to which access is very limited, only possible by river. These communities do not have easy access to preventive health care. Similarly, small urban areas have a very precarious health system, making the access to primary and secondary preventions difficult. The aims of this study were to estimate the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis infections in a sample of women living within the city of Coari, Amazonas, and compare the data between infected and uninfected women.

Material and methods

Context of the study

This study was conducted in the city of Coari, in the state of Amazonas, Brazil. Coari is a small town with an urban population of about 68,000 inhabitants, whose access is via river and air. It was a typical city in the Amazon until a few years ago. However, with the discovery and exploitation of petroleum in the city, there was the arrival of male workers coming from various other regions of Brazil in the past 10 years. In this context, changes in sexual behaviour in this population happened, especially among women.

The prevalence of STI in a previous Brazilian study of 42.0%¹⁶ with margin of error of 5% and confidence of 95% was considered for this study. Our sample consisted of 361 women registered for health care in 10 Basic Healthcare Units, during their routine exams, according to spontaneous demand. The samples were collected during the months of May through November of 2010. The inclusion criteria were: sexually active women, over 18 years of age, who agreed to participate in this study and signed a Statement of Informed Consent (IC), whether or not were symptomatic and whether or not were pregnant. The exclusion criteria were: hysterectomized women, women undergoing recent antibiotic treatment or women treated for any recent genital infection in the last 3 months. The women were interviewed by two previously trained nursing students. A questionnaire with open and closed questions was applied, which contained questions about the main clinical, socio-economic and behavioural factors. All women underwent clinical examination, including pelvic examination. The samples were collected with the aid of vaginal speculum and using endocervical brush, packed in a 1.5 mL microtube containing 400 µL of TRIS-EDTA buffer (TRIS-HCl 10 mM e EDTA 1 mM ph 8.0) and placed on ice immediately.

DNA extraction

For extraction of DNA, 400 µL of proteolytic TPK buffer (TE [TRIS HCl 50 mM + EDTA 50 mM pH = 8.0], Tween 20% e Proteinase K 10 mg/mL) were added to 400 µL of each sample, and the samples were incubated for 60 min at 56°C and for 10 min at 95°C in dry bath. Then, DNA was extracted for phenol/chloroform method, precipitated with absolute ethanol and resuspended in 50 µL of ultra pure water (ph = 7.6).¹⁷ Quantification of DNA was performed in equipment NanoDrop (Thermo, Brazil) to check the extraction efficiency.

PCR for C. trachomatis DNA detection

The primer pair KL1/KL2¹⁸ was used, which amplifies a fragment of 241 bp of plasmid DNA. The reaction final volume was 25 µL, containing 5 U of Platinum Taq DNA polymerase high fidelity (Invitrogen, Brazil), 5 pmol of each primer, 2.5 µL of reaction buffer × 10, 50 mM of MgCl₂, 10 mM of dNTP, 2.5 µL of sample and water. The reactions obeyed the following thermocycler: 94°C for 30 s, 40 cycles of 94°C for 30 s, 54°C for 30 s and 68°C for 2 min, ending with 68°C for 5 min.

PCR for N. gonorrhoeae DNA detection

The primer pair PapR/ParF¹⁹ was used, which targets the gene porA and amplifies a fragment of 132 bp. The reaction final volume was 25 µL, containing 5 U of Platinum Taq DNA Polymerase (Invitrogen, Brazil), 5 pmol of each primer, 2.5 µL of reaction buffer × 10, 50 mM of MgCl₂, 10 mM of dNTP, 2.5 µL of sample and water. The reactions obeyed the following thermocycler: 95°C for 30 s, 40 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 2 min, ending with 72°C for 5 min.

PCR for T. vaginalis DNA detection

A Touchdown method for thermal cycling was used, and the pair of primers used was BTUB2/BTUB9.⁷ Theses primers target a well-conserved region of the beta-tubulin gene of TV and amplify a fragment with 112 pb. The reaction final volume was 25 µL, containing 5 U of Platinum Taq DNA Polymerase (Invitrogen, Brazil), 5 pmol of each primer, 2.5 µL of reaction buffer × 10, 50 mM of MgCl₂, 10 mM of dNTP, 2.5 µL of sample and water. Cycling times were 75 s at 95°C followed by 60 cycles of denaturation temperature 95°C for 45 s, annealing temperature beginning at 62°C and ending at 52°C for 45 s and extension temperature of 72°C for 1 min. The annealing temperature was lowered one degree every four cycles until reaching 52°C, and this annealing temperature was then kept until the end of the cycling process.

In the reactions in this study, water was included as a negative control, and a sample previously tested as positive control. The reactions were performed in a thermocycler Veriti (Applied Biosystems). The amplifications products were subjected to electrophoresis on agarose gel 2.0% stained with ethidium bromide (0.5 mg/mL), and then visualized with the aid of a transilluminator. The images were then captured by digital camera OLYMPUS SP-500uz.

Statistical analysis

The data were analyzed through software Epi-Info 3.5.3 for Windows (Center of Disease Control and Prevention (CDC) (www.cdc.gov.br). In the analysis of the quantitative variables, when the data presented normal distribution, the mean and the standard deviation were calculated; when there was rejection of the normality hypothesis, the no parametric test of Mann-Whitney was used. During the analysis of the quantitative variables, the mean and the standard deviation were calculated whenever the data presented normal distribution. The non-parametric Mann-Whitney test was used whenever there was rejection of the normality hypothesis.

During the analysis of the categorical variables, the absolute frequencies and relative simple were calculated. In some cases the confidence interval was 95%.

The Chi-square test was calculated for the analysis of categorical data. Whenever it was not possible to calculate the Pearson Correlation Test, the Fisher’s exact test was used instead. The level of significance determined during these testings was 5%.

Ethics

This research was conducted in compliance with all ethical issues listed to research with human beings, and the research project was approved by the Federal University of Amazonas' Ethical Research Committee.

Results

Characteristics of the sample

All women enrolled in this study affirmed they were sexually active. Their age range was from 18 to 78 years, with a mean age of 36.4 years old (SD = 13.4). Five of the women were pregnant. Two of them reported being HIV-positive. The data about clinical history and sexual behaviour of the women are summarized in Table 1.

Table 1.

Distribution by frequency of demographic variables of women sampled in the city of Coari, Amazonas.

Variables (n = 361)	f_i	%
Age
18–25	70	19.3
25–35	128	35.4
35–45	71	19.7
45–55	50	14.9
55–65	26	7.2
≥65	16	4.4
Mean ± SD	36.4 ± 13.4
Range	18–78
Marital status
Single	45	12.5
Married / Stable union	279	77.3
Divorced / Separated / Widowed	37	10.2
Age at first sexual intercourse
Mean± SD	15.9 ± 2.5
Range	11–29
Number of sexual partners over a lifetime
1 to 5	284	78.6
6 to 10	49	13.6
More than 10	28	7.8
Numbers of regular sex partners in last 12 months
None	34	9.4
1 to 3	326	90.3
More than 3	1	0.3
Number of casual partners in last 12 months
None	329	91.1
1 to 3	28	7.7
More than 3	4	1.1
Condom use with regular partner (n = 327)
Always	62	19.0
Not always/Rarely	78	23.8
Never	187	57.2
Condom use with casual partner (n = 32)
Always	25	78.1
Not always/Rarely	5	15.6
Never	2	6.3
Reason for consultation
Screening	187	51.8
Presentation of clinical complaints	172	47.6
Clinical complaints (n = 172)^a
Pelvic pain	105	61.0
Vaginal discharge	95	55.2
Pruritus	48	27.9
Difficulty urinating	38	22.1
Others	8	4.7

f_i: simple absolute frequency; SD: standard deviation.

^aIs not added, it can occur more than a clinical complaint.

The most given reasons for non-consistent condom use with steady partners were “trusting the partner,” “partner does not like it” and “use of another contraceptive method.” The most given reason for not using condoms consistently was “often I do not have it.”

Prevalence of STIs

The reactions for detection of pathogens revealed that the prevalence of infection with T. vaginalis was 12.7% (CI 95% = 9.6–16.7); 6.4% were infected with C. trachomatis (CI 95% = 4.2–9.5) and 1.4% were infected with N. gonorrhoeae (CI 95% = 0.5–3.4).

C. trachomatis infection

We found a greater prevalence for C. trachomatis in women over 64 years of age (12.5%), followed by women aged 55–64 years (11.5%). Comparing the mean age between infected and uninfected groups (38.7 ± 15.8 and 36.3 ± 13.2, respectively), a higher average among those infected was found, but without statistical significance (p = 0.394). The majority (54.5%) of the 23 infected women were completely asymptomatic, while 45.5% presented clinical complaints. The complaints reported were: pelvic pain, vaginal discharge and pruritus. The variables related to sexual behaviour of infected and uninfected women are compared in Table 2.

Table 2.

Distribution by frequency of clinical history regarding the infection for C. trachomatis and T. vaginalis in women sampled in the city of Coari, Amazonas.

Variables (n = 361)	Positive		Negative		Total	p
Chlamydia trachomatis	f_i	%	f_i	%	Total	p
Maritual status						0.153^a
Single / Divorced / Widowed	8	9.8	74	90.2	82
Married / Stable union	15	5.4	264	94.6	279
Number of sexual partners over a lifetime						0,605^a
1 to 5	16	5.6	268	94.4	284
6 to 10	4	8.2	45	91.8	49
More than 10	3	10.7	25	89.3	28
Condom use with regular partner (n = 327)						0.411^b
Not always	18	6.8	247	93.2	265
Always	3	4.8	59	95.2	62
Condom use with casual partner (n = 32)						0.648^b
Not always	1	14.3	6	85.7	7
Always	3	12.0	22	88.0	26
Reason for consultation						0.660^a
Screening	13	7.0	174	93.0	187
Presentation of clinical complaints	10	5.8	162	94.2	172
Trichomonas vaginalis
Marital status						0.866^a
Single / Divorced / Widowed	10	12.2	72	87.8	82
Married / Stable union	36	12.9	243	87.1	279
Number of sexual partners over a lifetime						0.357^a
1 to 5	34	12.0	250	88.0	284
6 to 10	6	12.2	43	87.8	49
More than 10	6	21.4	22	78.6	28
Condom use with regular partners (n = 327)						0.445^a
Not always	35	13.2	230	86.8	265
Always	6	9.7	56	90.3	62
Condom use with casual partner (n = 32)						0.605^b
Not always	–	–	7	100.0	7
Always	2	8.0	23	92.0	25
Reason for consultation						0.858^a
Screening	24	12.8	163	87.2	187
Presentation of clinical complaints	21	12.2	151	87.8	172

f_i: simple absolute frequency; SD: standard deviation.

^aChi square test.

^bFisher's exact test.

T. vaginalis infection

The group of women infected by T. vaginalis had a mean of age higher than the uninfected group (38.9 ± 15.2 years and 36.1 ± 13.1 years, respectively), but the difference was not statistically significant (p = 0.175). The two HIV-positive women in our study were negative for T. vaginalis. Of the 46 infected women, only 21 of them (45.6%) reported clinical symptoms, which were: pelvic pain (n = 14), vaginal discharge (n = 10), pruritus (n = 6) and painful urination (n = 3). Some women had more than one symptom. The remaining 54.4% were completely asymptomatic. When we analyzed the prior history of STI of these asymptomatic women, we found that only three of them reported previous infection by T. vaginalis, and 22 did not report. Only one asymptomatic woman reported a previous relationship with a partner who had been infected by T. vaginalis. The variables related to sexual behaviour of infected and uninfected women are compared in Table 2.

N. gonorrhoeae infection

Due to the finding of low absolute frequency of infected women (n = 5), little can be inferred about the characteristics of the infected group. Regarding the presentations of clinical complaints, of the five positive women, three had complaints, which were: difficulty urinating, vaginal discharge, itching and pelvic pain. One of the women displayed all four symptoms, one showed three symptoms and one showed just discharge. Two infected women were completely asymptomatic. We observed a lower mean age in infected women (33.8 ± 21.1 years) compared with uninfected (36.5 ± 13.3%), without, however, statistically significant difference (p = 0.660) between groups. The age of first sexual intercourse in infected women was 15.6 (SD = 3.7) years and the uninfected group was 15.9 (SD = 2.5) years (p = 0.783). With respect to sexual behaviour, four infected women reported having 1 to 5 sexual partners throughout life, and one reported to have had more than 10. No association with others risk factors cited in the literature (number of steady or occasional partner, inconsistent condom use, etc) was found (p ≥ 0.05).

Association with other variables

Data about infection by any of the sexually transmitted pathogens as well as each pathogen alone were crossed with some socio-demographic and sexual behaviour factors, such as education, age, numbers of partners and condom use. We found no statistically significant association with any of these variables and infection by any of the sexually transmitted pathogens (p ≥ 0.05).

Discussion

In Brazil, except for mandatory reporting STI (AIDS, syphilis and hepatitis B), there are very few data about other STI's incidence and prevalence at a national level. Knowledge about the prevalence of these infections come from regional studies, which although individually are of limited reach, taken together provide a good overview of the Brazilian situation.

In this study, we found 6.4% prevalence of infection by C. trachomatis in the sample of Amazonian women. Comparing this prevalence with other Brazilian studies carried out on samples similar to ours, that is, women undergoing routine gynecologic exam, we observed a lower prevalence in our study in relation to others held in Porto Alegre/RS (12.6%),²⁰ Vitória/ES (7.4%)¹⁴ and Curitiba/PR (10.4%),²¹ all of these studies using PCR also.

There are few studies on the prevalence of Chlamydia in the Amazon. Our study showed low prevalence compared to studies conducted in Amazonas state, specifically in the city of Manaus. This result was expected, since the samples of these studies came from special groups (pregnant women and women seen at STI or infertility clinics). Studies in Manaus found 20.7% positivity in women attending in STI clinic (using PCR as a detection method)²² and 10.0% and 13.0% using hybrid capture.^2,11 The multicenter study of the Brazilian Health Department revealed 7.8% of infection in women attending in STI clinic and 11.6% in pregnant women.¹⁶ In Manaus, the study by Freitas et al.²³ detected by PCR the highest prevalence of 52.8% in infertile women, corroborating with the literature regarding the relationship between infection with C. trachomatis and secondary infertility.

In this study, we found higher prevalence of infection in women over 64 years of age, followed by women aged 55–64 years. We attribute this difference to the wide age range of women participants in this study, noting that adolescents did not participate, a group considered with high prevalence of Chlamydia.^13,24 Regarding the variables of sexual behaviour, although there were no statistically significant differences between infected and uninfected women, in the infected group we found some agreement with the profile of sexual behaviour of women infected with chlamydia according to the literature, which reports: higher prevalence in women who had more than 10 sexual partners throughout life and those who reported not consistent condom use with both steady partners and with casual partners.

It is known that chlamydial infection can often present asymptomatically in women, who then remain silent reservoir of the pathogen (“silent epidemic”). In this study, we sought to identify this trait in women during a routine pelvic examination (opportunistic screening). In this study, most of the infected women were completely asymptomatic. The screening of asymptomatic women in order to control the transmission of STI and prevent future sequelae of an unknown infection is still under debate. Developed countries like USA, UK, Sweden and the Netherlands have held annual screening for Chlamydia in women less than 24 years of age, with positive cost-effectiveness tests when using nucleic acid amplification and self-collection with either vaginal swab or self-collectable urinalysis. In Brazil, the only STI screening in a systematic way has been the detection of syphilis and HIV conducted antenatally and/or during childbirth.^15,25

For the detection of C. trachomatis, nucleic acid amplifications tests (NAATs) use primers that can anneal in bacterial plasmid, chromosomal DNA and in the 16S ribosomal gene. The detection of a gene that is present in the plasmid DNA appears to confer advantage, since there are 7 to 10 copies of the plasmid in bacteria.^18,26 However, studies suggest that plasmid-free variants can, even on rare occasions, be present in clinical samples, and the infection would not be detected in these patients.^3,27 This is a limitation of this study, since the par of primers KL1-Kl2 (which anneal in plasmidial DNA) was used.

The prevalence of infection by N. gonorrhoeae in our study was 1.4%, much resembling the Brazilian studies performed on samples from asymptomatic women examined during routine gynecological care, which showed 1.5% prevalence of 1.9% and 2.0% in the cities of Curitiba/PR,¹⁴ São Paulo/SP²⁸ and Vitória/ES,²¹ respectively, using culture or PCR for diagnosis.

When analyzing studies in women attending STI clinics, the numbers are significantly higher because, although some cases may be asymptomatic, gonococcal infection usually produces troublesome symptoms, such as itching and vaginal discharge, causing the patient to seek the health service.^29,30 In a multicentre study of the Brazilian Health Department was observed prevalence of 3.3% in women attending STI clinics in referral hospitals in six Brazilian cities, using the hybrid capture detection method. In the city of Manaus the prevalence was 6.1% in women attending an STI clinic, being above the national average (3.3%).¹⁶ In the same STI clinic in the city of Manaus, Benzaken et al.² reported the high prevalence of 7.1%, this time using cell culture for detection of bacteria.

The clinical symptoms presented by symptomatic women were classically reported in the literature: difficulty in urination, vaginal discharge, pelvic pain and itching. Two women positive for N. gonorrhoeae were completely asymptomatic. These data are consistent with the literature, both with respect to the classic symptoms of gonococcal infection and to the possibility that women are asymptomatic. In these women, the silent and untreated infection may occur between 10% and 20% of cases and can result in pelvic inflammatory disease, which can manifest itself as salpingitis, endometritis, tubo-ovarian abscess, and can lead to ectopic pregnancy, infertility and chronic pain.^4,29,30 It is also interesting to analyze that, of the 19 women in our study who reported having had sex in the last 12 months with partners who had a history of STI, 57.8% reported having had gonorrhoea. The fact that all women were negative for N. gonorrhoeae in this study may be due to a very early prior history of disease in the partner and/or having been treated with efficiency, probably having been symptomatic. No association with other risk factors cited in the literature (early age at first intercourse, number of partners and any fixed, inconsistent condom use with steady partners or potential, etc.) was found (p ≥ 0.05).

For the detection of N. gonorrhoeae, we used a primer to target the porA pseudogene. This gene exists only in N. meningitidis and N. gonorrhoeae and not in the saprophytic species. Even though this gene has homologous regions among the pathogenic species, the authors affirm that specific primers for non-homologous regions were carefully designed.¹⁹ However, according to subsequent publication of these authors,³¹ even if the tests are compared with other methods of detection and are validated and tested in different populations, the peculiar genetic characteristics of the genus Neisseria, molecular diagnosis of this bacterium based in nucleic acid amplification testing is an ongoing challenge. More recently, the same authors published a false-negative case using porA pseudogene as target caused by sequence variation.³² This is a limitation of this study, since we only used primers for this gene.

Although the literature also shows some degree of co-infection of both pathogens (CT and NG) in our sample we did not find any case. If here in Brazil screening for Chlamydia is a controversial subject, screening for Chlamydia and the gonococcus is even more controversial. The fact of gonococcal infection is rarer and has more obvious clinical manifestations (not so silent infection such as chlamydia), screening is usually not recommended.

Studies about prevalence of infection by T. vaginalis are very rare in Brazil. Besides not being subject to mandatory notification, the less serious sequelae of this infection, as was believed until recently, did not make it subject to control and care by the health systems. Only now, however, with the knowledge of its relationship to HIV infection, more attention has been paid to this pathogen.

In our study, T. vaginalis was the most common pathogen found, with a prevalence of 12.7%. The few Brazilian studies make it difficult to compare this finding with other national data, both in terms of populations represented in studies and in relation to the diagnostic methods used. In our literature search, we found only one Brazilian study which used a molecular method (PCR) for diagnosis.²⁸ The authors, studying a sample similar to ours, found 3.2% of infection among women in routine pelvic examination in São Paulo/SP, far below the prevalence found in our study. Considering other methods commonly employed in studies and in clinical practice (culture, cytology and fresh exam), Brazil prevalence data vary from 2.0% to 8.9% in women in routine examination.

Less than half of women examined complained of clinical symptoms, which were: pelvic pain, vaginal discharge, pruritus and painful urination, and some women had more than one complaint. These symptoms are in agreement with the literature.^5,6 The large number of infected women who reported no symptoms (54.3%) is intriguing, because this is a pathogen that usually causes pronounced symptoms. But this finding is also consistent with the literature, as some authors claim that this infection may be asymptomatic in about half of the cases.^9,33,34 We could think that the infected women who did not exhibit symptoms could had been treated previously but did not complete the treatment regimen (in which cases women stop using the prescribed vaginal ointment after the symptoms disappear). However, when we analyze the history of STI in these asymptomatic women, we found that only three of them reported previous infection by T. vaginalis and 22 not reported, and only one asymptomatic women reported a history of relationship with a partner who reported past infection by T. vaginalis.

This study draws attention to the importance of surveillance and possible need for screening of some sexually transmitted pathogens, especially C. trachomatis, which often progresses asymptomatically, causing further damage to the reproductive health of women. Due to the high prevalence, it should also be given some attention to asymptomatic infection by T. vaginalis, since this pathogen has recently been implicated in reproductive health problems, such as pre-term birth, infection after hysterectomy, infertility and especially HIV transmission.³³

Footnotes

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors thank the Foundation for Research of the State of Amazonas (Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM) for financial support.

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