Study population

All adolescents seen at the family planning clinic were invited to participate in the study. This clinic is a resource for adolescents who wish to plan their contraception. A total of 196 adolescents aged 14–19 years were selected for the study. Patients with a confirmed diagnosis of any of the STIs investigated in this study were excluded for that specific pathology but were included in the screening for the other STIs. Patients who had used antibiotic therapy or any intravaginal chemical substance in the last two weeks preceding the day of the examination were excluded, as were indigenous and foreign adolescents who were unable to communicate. All patients underwent anamnesis, general physical and gynecological examinations, and sample collection. A questionnaire was administered to assess the epidemiological-sociodemographic profiles of the patients. All adolescents and their guardians were informed of the results of the tests performed and were instructed to undergo appropriate treatment if necessary.

Endocervical samples were collected during the gynecological examination to be tested for four STIs (C. trachomatis, N. gonorrhoeae, T. vaginalis, herpes simplex). After this step, in the last stage of care, blood samples were collected by finger prick with a retractable lancet, and blood was drawn with a sterile capillary tube; this sample was used for the detection of HIV, syphilis, and hepatitis B and C.

STI detection of sexually transmitted pathogens by multiplex PCR

The samples that were collected from the endocervix with a cytobrush were transferred to Eppendorf tubes (Eppendorf, Hamburg, Germany) containing 1.8 mL of sterile saline solution. Genomic DNA was extracted with Biopur Mini Spin Plus (Mobius Life Science, Paraná, Brazil) following the manufacturer’s instructions. DNA was quantified in a NanoDrop 2000 spectrophotometer (Thermo Scientific, Epsom, UK). TaqMan multiplex real-time polymerase chain reaction (qRT‒PCR) was performed using the RUO Multiplex Sexually Transmitted Diseases 9 FTD kit (Fast Track Diagnostics, Junglinster, Luxembourg) for the detection of C. trachomatis, N. gonorrhoeae, T. vaginalis, and herpes simplex viruses 1 and 2. The reactions were performed according to the manufacturer’s instructions using the 7500 Fast Real-Time PCR system (Applied Biosystems, MA, USA) as previously described [17]. The total volume used for the assay was 25 μL for each dilution, with 15 μL of amplification mixture and 10 μL of the sample. The thermocycling program was 50°C for 15 minutes for reverse transcription, followed by activation of PCR at 94°C for 1 minute, then 40 cycles of 8 seconds at 94°C and 1 minute at 60°C. Amplification was carried out on 96-well plates (Optical 96-Well Reaction Plate, MicroAmp®) using the thermocycler 7500 Real-time PCR System (Applied Biosystems, CA, USA). The molecular procedures were performed at the Laboratory of Molecular Biology of the Central Laboratory of Public Health of Rondônia.

Immunological rapid detection tests for sexually transmitted pathogens

Blood samples were collected using the digital pulse technique with the aid of a retractable lancet into sterile capillary tubes. The samples were dispensed on a specific slide for each test for the detection of HIV, syphilis, and hepatitis B and C. For HIV detection, we used the HIV 1/2/O Tri-line Human Immunodeficiency Virus Rapid Test (ABON Biopharm, Zhejiang, China). For syphilis detection, we used the Immuno-Rapid Syphilis kit (Wama Diagnóstica, Sao Paulo, Brazil), hepatitis B was detected with the rapid immunochromatographic VIKIA^® HBs Ag test (bioMérieux, Marcy-l’Étoile, France), and for hepatitis C detection, we used the Alere HCV Kit (Alere, Massachusetts, USA). The protocol established by each manufacturer was carefully followed.

Statistical analysis

The sample size was calculated before study initiation using the program WinPepi version 11.65. Due to the few studies on the prevalence of STIs in Brazil, with especially few studies on female adolescents, the sample size calculation was based on the study carried out by the Brazilian Ministry of Health on the prevalence and relative frequencies of sexually transmitted diseases (STDs) in selected populations of six Brazilian capitals [16]. To this end, the prevalence of syphilis, gonorrhea and chlamydia was used in pregnant women in six Brazilian capitals, which included Manaus, located in the northern region of Brazil, in the western Amazon. Therefore, based on the prevalence of syphilis (average of 2.6% and maximum of 4.4%), the required sample size was 51 (if the rate was 26 per 1000, with confidence level = 95% and acceptable diff. = 44 per 1000). Based on the prevalence of gonorrhea (average 1.5% and maximum 4.3%), the required sample size was 73 (if the rate is 15 per 1000, confidence level = 95%. and acceptable diff. = 28 per 1000), and based on the prevalence of chlamydia (average of 9.4% and maximum of 14.8%), the required sample size was 113 (if the rate is 94 per 1000 with a confidence level = 95% and acceptable diff. = 54 per 1000). Then, based on the need for the largest n, estimating a C.I. of up to 108 per 1000, the final required sample size was at least 189.

The data were entered into and analyzed using SPSS v.25 (IBM Corp., New York, USA) and EPI-INFO 7.2 (Centers for Disease 168 Control and Prevention, GeorgiaGA, USA) for Windows. The results were reported using the mean, median, minimum, maximum, standard deviation, and absolute and relative frequencies (percentages) for the quantitative variables. To compare the qualitative variables, the chi-squared test or Fisher’s exact test was performed. Statistical significance was established as P < 0.05.

Discussion

In this study, we evaluated the prevalence of the main STIs in female adolescents in the western Amazon population. The results of this study are of paramount importance for public health measures to prevent and treat STIs because no studies have been conducted in this region on the prevalence of STIs, especially in adolescents. We determined the prevalence of the four most common curable STIs (C. trachomatis, N. gonorrhoeae, T. vaginalis, and syphilis) and four viruses, HIV, hepatitis B and C, and herpes viruses 1 and 2, and found that 32% of the adolescents treated at the family planning outpatient clinic had at least one STI.

The most prevalent infectious agent was C. trachomatis at a rate of 23%, which is substantially higher than rates found in other regions of the Amazon, namely, 18% in the city of Belém, state of Pará based on two studies in the eastern Brazilian Amazon [18], 14.3% in Marajó Island, state of Pará [19], 18% in the city of Coari in the state of Amazonas based on two studies in the western Brazilian Amazon, and 15.1% in the city of Manaus in the state of Amazonas [16]. Even at such high prevalence rates, the association with risk variables are not easily identified; however, there is evidence linking prevalence to young age and sexual conduct, early sexarche [19–21], and low income in the Amazon region, which is inhabited by a largely rural population living in small communities with few public healthcare services or even basic programs focusing on reproductive health [19,22].

Precarious social conditions, a lack of economic opportunities, and risky sexual behavior are all closely associated with the incidence of STIs in young people [19,23]. The high prevalence of chlamydia found in our study is very worrisome, especially due to the potential risks associated with this disease. It is estimated that 80% of infected women are asymptomatic but may develop complications and sequelae in the long term, while others will have some symptoms involving urethritis, cervicitis, and pelvic inflammatory disease, the main cause of infertility, chronic pelvic pain, and ectopic pregnancy [13,24]. Other complications associated with chlamydia may come from maternal infection of the birth canal causing complications in the newborn, such as pneumonia and conjunctivitis [25]. Chlamydia infection also brings obstetric complications such as miscarriage, premature birth, low birth weight, fetal growth restriction, premature rupture of membranes, perinatal mortality, and endometritis [26].

Furthermore, our results also demonstrated a high prevalence of pathogens responsible for other curable STIs: T. vaginalis (5.6%) and N. gonorrhoeae (3.1%). Thus, we found that 32% of the adolescents evaluated had at least one STI. The WHO classifies a rate of 5% as an STI prevalence that is under control for a given population, while the rate in our study was six times higher than this limit. This result indicates that we are facing a serious public health problem in this community [9]. Comparing our rates with other published rates, adolescents from Pittsburgh, Pennsylvania, USA, also showed higher rates of C. trachomatis and N. gonorrhoeae at 16% and 12.5%, respectively [27]. Compared to other Brazilian findings, the prevalence of N. gonorrhoeae in our study (3.1%) was almost double that found by Britto et al. (1.6%) [28] and higher than that found by Jalil et al. (2.1%) [29]. A review by Kissinger et al. evaluated the worldwide prevalence of T. vaginalis infection and showed heterogeneity among different countries (25). In the USA, the prevalence rates were 2.3% among adolescents and 3.1% among adult women, while in Zimbabwe and Tanzania, the prevalence ranged from 9.5% to 11% among the two sexes. Belgium had the lowest prevalence among the countries included in the study at 0.37% [30].

The fact that we found higher rates of STIs among the adolescents in our study when compared, especially to other Brazilian regions, may be due to several factors. Analyzing the data in detail, we found that 54.6% of the girls in the study had their first sexual intercourse when they were 14 years old or younger. The age at sexarche is decreasing among girls [31]. This finding has important implications for the health of adolescent girls because of the association of young age at sexarche with subsequent health problems and subsequent risky sexual behavior. There are many factors associated with young age at sexarche, such as low socioeconomic status, limited education, divorced parents, living with a partner, not practicing a religion, smoking, and drug use [32–34].

Girls who have first sexual intercourse at 14 years of age or younger are less likely to use contraception such as condoms on this occasion, take longer before starting to use contraception in sexual intercourse, are more likely to have multiple sexual partners, have a higher risk of depression, have lower self-esteem and more episodes of regret, and have a higher risk of contracting STIs and cervical cancer [35]. Our data confirm this association between early sexarche and an increased risk of STIs. We found that early sexarche increases the risk of having a T. vaginalis infection by 1.5 times and by N. gonorrhoeae by 1.9 times; therefore, public measures that promote responsible and healthy sexuality should be provided in homes, schools, medical and community centers [36].

Another factor that is probably associated with the high prevalence of STIs found in our study is that almost 60% of adolescents had more than one sexual partner, and approximately 20% had casual partners in the last year. Early sexarche appears to have a significant correlation with multiple sexual partners in the last 12 months, which in turn may put these adolescents at higher risk of contracting an STI [37]. It is believed that the type of partner influences the STI risk of women because the prevalence is higher in single subjects [38,39]. However, researchers generally restrict the type of partner to labels of “casual” versus “fixed” or “living in union” versus “single”, which can mask the variability of risk [40].

Only 1% of the adolescents used condoms in all sexual reactions. Behavioral and social factors can lead adolescents to riskier sexual relationships, which favors constant exposure to and a higher risk of STIs [41]. These situations are closely related to the high morbidity of STIs in this population, and it is estimated that adolescents are responsible for approximately 50% of new STI cases each year, while approximately 24% of adolescents between 14 and 19 years of age have 1 out of 5 STIs commonly reported [5,9]. These infections may have important and irreversible health consequences for these girls and their unborn babies [5,17].

There were no records of patients with hepatitis B, hepatitis C, or HIV in our study. We believe that this extremely low prevalence was due to the flow of referrals of adolescents to the family planning outpatient clinic, as most of these patients were referred from a risk maternity unit where seropositive patients are not admitted for treatment because they are referred to reference maternity hospitals. In terms of viral hepatitis, since 1996, the National Immunization Program of the Ministry of Health of Brazil has recommended universal vaccination of children against hepatitis B at birth, i.e., all patients included in the study would have already had the vaccination and therefore should be immunized against hepatitis B [42].

The high frequency of STIs found in our study, especially in association with an absence of symptoms, is very worrisome. The low symptomatology can perpetuate the transmissibility of these diseases in a vulnerable group, with potential sequelae in the medium and long term. Clinical diagnosis based on symptoms of infections caused by C. trachomatis and N. gonorrhoeae has low sensitivity, and up to 70% of infected women may be asymptomatic [43]. Therefore, the importance of this study from an epidemiological point of view is fundamental, as there has been no study on the prevalence of STIs in this part of the western Amazon. These results can serve as a basis for more effective public measures in the region.

Brazil is already planning to implement measures to track STIs. For example, the Ministry of Health recommends annual screening for HIV and syphilis for adolescents and sexually active adolescents up to 30 years of age and for pregnant women in the first prenatal visit, beginning of the 3rd trimester and at the time of delivery [44]. In addition, chlamydia and gonococcus screening is recommended for the general population according to risk and for all pregnant women under 30 years of age [44]. Despite those recommendations, considering the data from Brazilian studies on the prevalence of STIs, we can say that the implementation of these recommendations is far from realized. If these STIs are not diagnosed and treated, they can lead to important obstetric and gynecological complications that considerably increase the costs of treatment and hospitalization and negatively impact female morbidity rates, especially in adolescents [45].

The high prevalence detected in our study, which is higher than the average rate for the rest of Brazil, is astonishing. Although our study was conducted in only one family planning outpatient clinic with a specific group of patients—girls between 14 and 19 years old—the findings demonstrate that improving control measures is crucial to prevent STIs among adolescents. Reducing the prevalence of STIs requires the implementation of public policies that disseminate knowledge about STIs and their forms of transmission and prevention, including consistent condom use, vaccination, improved screening, appropriate diagnosis, and timely treatment according to the best practices recommended by the WHO.

Although we performed the sample size calculation before the study, and we evaluated a specific group, adolescent girls, one of the limitations of this study may be related to the sample size. The sample size may be relatively small compared to the vast universe of communities in the Brazilian Amazon region, which have different difficulties in accessing social and health care, together with possible social biases in the adolescents’ responses to the questionnaire. In the context of these considerations, this preliminary study is part of a larger project that aims to identify potential indicators that will support more effective health care strategies aimed at improving the quality of life of populations in this region of the western Amazon. Although it should be possible to extrapolate these findings to other similar Amazonian communities, we must keep in mind that more research will be important, especially with other groups, to elucidate the prevalence of STIs and, therefore, use these data to improve public policies.