High prevalence of sexual Chlamydia trachomatis infection in young women from Marajó Island, in the Brazilian Amazon

Background Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) in the world. Approximately 80% of infected women are asymptomatic, although this infection can lead to serious complications in the female reproductive tract. Few data on Chlamydia infection are available in rural Amazonian communities. Objectives To evaluate the prevalence of sexual C. trachomatis infection in women from Marajó Archipelago communities in the Amazon region of Brazil and to identify associated factors and genotypes. Methods We utilized amplification of the ompA gene by nested PCR. Positive samples were genotyped by sequencing. Study participants completed a questionnaire on social, epidemiological, and reproductive health variables. A Poisson regression was used to evaluate the degree of association of these variables with the infection. Results The sexual infection by C. trachomatis was observed in 4% (16/393) of the subjects, and was more often found in women aged ≤25 (14.3%; 95% CI = 2.83–35.47; p <0.001), and in women with a household income of less than one Brazilian monthly minimum wage (5.2%; 95% CI = 1.33–11.37; p = 0.014). The ompA gene was sequenced in 13 samples, revealing F genotypes (38.4%, n = 5), D (23%, n = 3), E (15.3%, n = 2), Ia (7.6%, N = 1), J (7.6%, n = 1) and B (7.6%, n = 1). Conclusions We recorded a high prevalence of sexual infection by C. trachomatis in young and poor women from the interior of the Brazilian Amazon. This high prevalence and the frequencies of the main genotypes were similar to those found in major Brazilian urban centers. Our results reinforce the importance of the screening of this neglected infection, and the prevention of later sequelae in young women from rural and urban areas of Brazil.


Objectives
To evaluate the prevalence of sexual C. trachomatis infection in women from Marajó Archipelago communities in the Amazon region of Brazil and to identify associated factors and genotypes.

Methods
We utilized amplification of the ompA gene by nested PCR. Positive samples were genotyped by sequencing. Study participants completed a questionnaire on social, epidemiological, and reproductive health variables. A Poisson regression was used to evaluate the degree of association of these variables with the infection. PLOS

Introduction
Chlamydia trachomatis is the most prevalent bacterial Sexually Transmitted Infection (STI) in the world [1], with about 128 million cases being diagnosed annually [2]. The infection is asymptomatic in approximately 80% of the infected women, going completely unnoticed by most women. The lack of an early diagnosis may make it impossible to treat the disorder adequately, with serious consequences for the female reproductive tract, such as salpingitis, Pelvic Inflammatory Disease (PID), ectopic pregnancy and tubal infertility, with an annual cost of up to four billion dollars [3][4][5][6]. This infection is the most common cause of preventable infertility in sexually active women and is a risk factor for other STIs [7-10].
The ompA gene of C. trachomatis has 19 genotypes, which are associated with a range of pathologies in humans [11]. The A, B, Ba and C genotypes are associated with trachoma [12], while the D, Da, E, F, G, Ga, H, I, Ia, J and K genotypes are associated with non-invasive urogenital infections [13], and the L1, L2, L2a and L3 genotypes cause lymphogranuloma venereum [14]. In Brazil, the actual prevalence of this infection and the genotypes present in different populations are still unclear, making it difficult to understand the dynamics of infection and preventing the creation of effective C. trachomatis screening programs by Brazilian public health agencies. Previous studies using nucleic acid amplification techniques have demonstrated that C. trachomatis infection rates range from 5% to 31% in Brazilian youths and adults, and that the most frequent genotypes in the country are the E, F and D types [15][16][17][18][19].
In the Brazilian Amazon region, many communities are located far from major urban centers, and have little access to public healthcare services or other resources [19,20]. Understanding the diversity and distribution of the genotypes is essential for the monitoring of the infection, although few studies are available on the genotypes of sexual C. trachomatis infections in Brazil, and only one has focused on an urban Amazonian population [18]. The vast Marajó Archipelago is located at the mouth of the Amazon River, in the Brazilian state of Pará, and is inhabited by a largely rural population, living in small communities with few public healthcare services or even basic programs focusing on reproductive health [21,22]. The present study aimed to determine the prevalence of C. trachomatis and its genotypes in the endocervical infections of women that have limited access to public health services, resident in four communities of the Marajó Archipelago, and to describe the potential. Study participants completed a questionnaire on social, epidemiological, and reproductive health variables associated with the prevalence of this disease.

Study population and data collection
This cross-sectional study was conducted between March 2013 and May 2015. The target population included autochthonous women (n = 393), from four small communities [São Sebastião da Boa Vista (n = 113), Anajás (n = 96), Portel (n = 60) and Chaves (n = 124)] of the Marajó Archipelago in Pará, in the Brazilian Amazon region. This archipelago is considered to be one of Pará's main tourist destinations. Its principal islands are Marajó, Caviana, Mexiana and Gurupá, with 16 municipalities and a large network of rivers, creeks, lakes, and marshlands that criss-cross the islands and impede terrestrial access to most communities.
During the study period, the multidisciplinary team travelled from Belém (Pará state capital) to the communities for the collection of data. The sexually-active women in each community were informed of the periodicity of the health exams and asked to seek gynecological care during the home visits of the designated municipal health agents.
We investigated a non-probabilistic, intentional, and conventional sample, composed of women 18 to 79 years old, who had either never had a Pap smear or who had last had one more than one year previously. Exclusion criteria were pregnancy, menstruation, and not wishing to participate in the study or not signing the informed consent form. The participants were asked to answer a questionnaire, and were made aware of the importance of providing reliable answers, in order to minimize possible bias. The following variables were investigated: age, conjugal status, occupation/education, household income, age at fist sexual intercourse, lifetime number of sexual partners, number of sexual partners in the past year, use of condoms, use of contraceptives, previous pregnancies, natural childbirth, miscarriage, and whether the Pap test was taken.
Cervical secretions were collected during routine pelvic examinations using an endocervical brush, and the samples were stored in cryogenic tubes containing 1 ml Tris-EDTA buffer (TE) [10 mM Tris-HCl pH 8.5; 1 mM EDTA] at a temperature of -20˚C for testing.

Ethics statement
This study was part of the "Epidemiological Markers in the Healthcare of the Marajó Archipelago" project, which was approved by the Ethics Committee for Research of the State Foundation for Hemotherapy, with authorization number 0003.0.324.000-10. All participants signed up to be adults, with no participants below the age of 18. Free and informed consent for participation in the study was obtained in writing before the collection of samples and epidemiological data. All the data were analyzed with complete anonymity.

Extraction of the DNA
The DNA was extracted using a pureLink Genomic DNA Purificationkit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and stored at -20˚C until analysis. A Polymerase Chain Reaction (PCR) of the human β globin gene was conducted prior to the detection of C. trachomatis to confirm the suitability of the samples [23].

Detection of the ompA gene of C. trachomatis
Detection of C. trachomatis was performed using a nested PCR protocol modified by Jalal et al. 2007 [24], which amplified 394bp of the ompA gene of C. trachomatis. The first reaction used 6.0 μL of GoTaqGreen Master Mix (Promega, Madison, WI, USA), 0.5 μL (containing 20 pmol/μL of each primer) of the primers P1 (A) (5'GACTTTGTTTTCGACCGTGTT-3 ') and P2 (5'AGCRTATTGGAAAGAAGCBCCTAA-3 '), 2 μL genomic DNA, and 3 μL sterile water for a final volume of 12 μL. The second reaction used 0.5 μL of the solution of the first reaction, 6.0 μL Go Taq Green Master Mix (Promega, Madison, WI, USA), 4.5 μL of sterile water, 0.5 μL (20 pmol/μL) of the primers P3 (5'-AAACWGATGTGAATAAAGARTT-3') and P4 (5'-TCC CASARAGCTGCDCGAGC-3'). In the two steps of the nested PCR, a negative and a positive control was used to optimize the result. In the first and second stages of the nested PCR, the initial activation was at 95˚C for 5 min and 1 min, respectively, followed by 35 cycles of denaturation at 94˚C for 40 s, annealing at 54˚C for 30 s, and elongation at 72˚C for 90 s, with a final extension step at 72˚C for 7 min. The amplified products were visualized after electrophoresis in 1% agarose gel with ethidium bromide (0.5mg/mL) staining.

DNA sequencing
The Sanger method of nucleotide sequencing was used. An approximately 990bp fragment of the ompA gene was amplified by nested PCR using primers P1(B) (5 0 -ATGAAAAAACTCTT GAAATCGG-3 0 ) and OMP2 (5 0 -ACTGTAACTGCGTATTTGTCTG-3 0 ), and whenever reamplification was necessary, the inner primers MOMP87 (5 0 -TGA ACC AAG CCT TAT GAT CGA CGG A-3 0 ) and RVS1059 (5 0 -GCA ATA CCG CAA GAT TTT CTA GAT TTC ATC-3 0 ) were used [25]. The first step of the nested PCR was run in a 0.5 μL volume containing 20 pmol/μL of each primer P1(B) and OMP2 and 5.0 μL of the DNA extracted from the endocervical secretion, 14 μL of sterile water, 1.0 μL of MgCl 2 , 1.0 μL deoxynucleoside triphosphate (10mM), 2.5 μL of 10x buffer, and 0.5 μL of Hotstar Taq DNA Polymerase 1.5U (Qiagen). Amplification was run in a final reaction volume of 25 μl [25]. In the two steps of nested PCR a negative and a positive control was used to optimize the result, but these controls were not used in the sequencing.
In the initial step of the nested PCR, amplification conditions were 95˚C for 5 min, followed by 40 cycles of 94˚C for 30 s, 55˚C for 30 s, and 72˚C for 90 s, and a final extension at 72˚C for 7 min. In the nested PCR, the MOMP87-RVS 1059 primer pair was used with 1.5 μl of the product of the first stage of the nested PCR, which was added to a final volume of 25 μl. The conditions of the second step of the nested PCR were the same as those described above, except for the annealing temperature which was 60˚C, and the addition of 17.5 μl of sterile water [25].
The amplified products were visualized by ethidium bromide (0.5 mg/mL) staining after electrophoresis in 1% agarose gel. The products obtained by the nested PCR were purified using a BigDye Xterminator Purification kit (Applied Biosystems, Foster City, CA, USA) to sequence both strands. A BigDyeTerminator Cycle kit (Foster City, CA, USA) was used for the sequencing reaction, according to the manufacturer's instructions. The reaction mixtures were sequenced in an ABI 3130 (Applied Biosystems, Foster City, CA, USA).

Phylogenetic analysis and Genotyping
The sequences were assembled using the CAP3 software, aligned in MAFFT v 7.221 and edited in Bioinformatic Geneious v 8.1.7. The consensus sequences were compared with known C. trachomatis lineages [26] using the BLAST search tool in the National Center for Biotechnological Information (www.ncbi.nlm.nih.gov). The residues that correspond to the flanking primers were excluded from the analyses.
The phylogenetic analysis was run in three stages. The first stage involved the use of IQ-TREE v 1.

Statistical analysis
The data were analyzed using the Statistical Package for Social Sciences (SPSS) version 21.0 (SPSS, Chicago, illinois, EUA). A Poisson regression was used to examine the unadjusted and adjusted associations between the Prevalence Ratio (PR) and the different variables. A 95% confidence interval (CI) was estimated and a critical p value of 0.05 was considered in all analyses.

Results
The ages of the 393 women investigated in the present study ranged from 18 to 79 years. The median (interquartile range) age was 40.0 (29.0-52.0) years, and 82.2% (n = 323) of the participants were older than 25 years. Most of the women (n = 68.7%) were married, 75.9% (n = 259) were employed informally, and 55.7% (n = 219) had a family income of no more than the Brazilian minimum wage (U$250-300/month). Just over half 51.1% of the women (n = 201) were not using oral contraceptives, 90.6% (n = 356) were already mothers, and 90.4% (n = 355) of these mothers reported having had a vaginal delivery. Around one third, 32.6% (n = 128), reported having suffered at least one miscarriage, and 81.4% regularly have Pap tests.
The total prevalence of sexual infection by C. trachomatis was 4.1% (16/393). The infection was detected in 14.3% of the women that were 25 years old or younger, with an adjusted prevalence 10 times higher (95% CI = 2.83-35.47; p <0.001) than in women older than 25 years. The prevalence of infection was also 3.88 times higher in women from low-income households (below minimum wage) in comparison with those from higher income households (95% CI = 1.33-11.37; p = 0.014). No other variables were associated statistically with C. trachomatis infection ( Table 1).
The ompA gene was sequenced in 13 of the 16 positive samples. Six genotypes were detected F (38.4%, n = 5), D (23%, n = 3), and E (15.3%, n = 2), with one sample each (7.6%) of the Ia, J, and B genotypes. The phylogenetic analysis revealed the evolutionary relationship among the C. trachomatis genotypes from the present study, with 99-100% similarity among the lineages, and 87.9% of similarity with the C. trachomatis reference lineage (Fig 1). The nucleotide sequences of the different C. trachomatis lineages identified in the present study were deposited in GenBank (NCBI) under access codes KU295204-KU295216. (Figs 1 and S1)

Discussion
This preliminary study presented a molecular approach for the diagnosis of C. trachomatis infection in women who are socially deprived, residents in communities with little or no access to health care and laboratory procedures, which not only identified infection rates, but also the genotypes involved. The study participants completed a questionnaire on social, epidemiological, and reproductive health variables associated with the prevalence of this disease.
The low prevalence (4.1%) of C. trachomatis sexual infection identified in these communities may be related to the maturity of the women surveyed (mean age of 41 years and 82.2% more than 25 years old). On the other hand, we identified a significantly higher prevalence (14.3%) of C. trachomatis infection in young women (� 25 years old). High prevalence (11%) [18] and (18%) [19] were also identified in young women from the region's largest city (Belém). Access to health services is precarious in some parts of northern Brazil, such as Marajó Island, mainly due to its geographical characteristics [20,21]. In addition to being more common in younger women, the infection was also associated with a low family income.
In urban areas of other Brazil regions, infection rates vary from 9.6% to 31.0% [17,[27][28][29][30][31][32]. Even at such high prevalence rates, the association with risk variables is not easily identified, but there is evidence linking prevalence to young age and sexual conduct [29], as well as low income [30]. Precarious social conditions, a lack of economic opportunities, and risky sexual behavior are all closely associated with the incidence of STIs in young people [32].
High rates of sexual infection by C. trachomatis were recorded in Spain (8.5%) [33] and Great Britain (12.3%) [34]. Monitoring urogenital C. trachomatis infection by DNA amplification in women of less than 25 years of age should be a priority in most of these countries [35][36][37][38].
Although three samples could not be sequenced, six genotypes were identified in the remaining 13 samples. In general, our findings confirmed a higher prevalence of C. trachomatis genotypes F, D, E in sexual infections, as already described in an urban study in northern Brazil, together with genotype J [18]. Previous serological studies in the Amazon have shown that the distribution of C. trachomatis serotypes is similar to that observed in the present study [22]. Similar C. trachomatis genotypes have also been found in other regions of the Brazil [15][16] and the world [39][40][41][42].
The present study identified the occurrence of the B genotype of C. trachomatis in sexual infections of women from rural communities in the Brazilian Amazon region. The presence of the B genotype of C. trachomatis, an ocular genotype, is not unusual in the genital samples and has been reported in other studies [43][44][45]. Trachoma infection is another prominent clinical feature of C. trachomatis infection that has been reported from the Marajó Archipelago [46,47]. However, it is still too early to confirm that this finding is related to cross-infection or gene recombination, which were not investigated specifically in the present study.
The lack of a public health program for the screening of this STI in Brazil is a major obstacle to the understanding of the epidemiology of C. trachomatis in this country. This favors the onset of reproductive problems, which can develop slowly and silently [48,49].
The molecular screening for C. trachomatis infection is not yet included in the National Strategic Plan for STDs and AIDS in Brazil [50]. However, it would be important to include techniques with high sensitivity and specificity for the primary screening of active infection by C. trachomatis. More studies on the molecular prevalence, cross-infection, gene recombination, and genotypes of C. trachomatis will also be important, especially in young women, to elucidate the epidemiological network of this neglected STI.
The principal limitations of this study may be related to the relatively reduced sample size in comparison with the vast universe of communities in the Brazilian Amazon region, together with possible social biases in the responses of the women 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 effective strategies for the improvement of the quality of life of the populations of the Marajó Archipelago. While it should be possible to extrapolate these findings to other, similar Amazonian communities, further research will be important to elucidate the exact nature of this infection and the distribution of the sexual and ocular genotypes of C. trachomatis in the region.

Conclusions
We report a high prevalence of sexual infection by C. trachomatis in young and poor women from the interior of the Brazilian Amazon region. This prevalence and the diversity of C.