Sexually transmitted diseases in symptomatic and asymptomatic Thai women and girls: a study from Bangkok and nearby

Participants included 284 Thai women and girls aged 15-54 years attending the Buddhachinaraj Phitsanulok Hospital, Bangrak STD clinic, which is part of the Office of Disease Prevention and Control 1 Bangkok, and Office of Disease Prevention and Control 3 of Chonburi province. All participants were HIV-negative, and had neither cervical cancer nor previous cervical cancer treatment. Participants were grouped as (i) upper genital tract (UGT), (ii) lower genital tract (LGT), and (iii) undefined UGT or LGT infection; based on U.S. Centers for Disease Control and Prevention definitions [2]. Samples were obtained by clinicians, following a standard cervical swab sample collection method and a medium using M4RT (Illinois, USA). For the symptomatic group, 100 mg of doxycycline, twice daily for 14 days, was prescribed, and refrain from sexual contact during treatment until follow-up sample collection 14 days after treatment was completed [2, 7, 11, 12]. All samples were collected during September 2011–2012 for Buddhachinaraj Phitsanulok Hospital, and March 2013–2014 for the other sites.

The study was approved by the Institutional Review Board of Buddhachinaraj Phitsanulok Hospital (101/54), and the Ethics Committee for Research in Human Subjects of the Department of Disease Control, Bangkok (FWA00013622). Written informed consent was obtained from all subjects for their participation in the study. Clinical samples were identified by a unique identification number with no link to patient names.

DNA was extracted from a 100 μL aliquot of each clinical sample was according to High Pure Template Preparation Kit instructions (Roche Diagnostics Corporation, Indianapolis, IN, USA). Concentration and quality of the extracted DNA were measured by A_{260 nm} and A₂₆₀/A_{280 nm} spectrophotometry. The extracted DNA had an average A₂₆₀/A₂₈₀ between 1.80 and 2. C. trachomatis, N. gonorrhoeae, HPV, and human β-globin were detected by PCRs following established protocols [12, 16-18]. Primers and PCR annealing temperatures were listed in Table 1: Ct.ompA.60UF and Ct.ompA.VB3 were primers for C. trachomatis ompA first-half; Ct.ompA.MVF3 and Ct.ompA.220DR for ompA second-half; Ng.opa.F and Ng.opa.R to Ng.opa.R2 for primary N. gonorrhoeae; Ng.porA.F and Ng.porA.R for confirmed positive N. gonorrhoeae; PGMY11-A to PGMY11-D, and PGMY09-F to HMB01 as forward and reverse primer cocktails for HPV L1 gene; and globinF and globinR for human β-globin. Sizes and semi-quantitation of the PCR products were determined from agarose gel electrophoresis. All negative PCR results were repeated to confirm the negative results.

trpA PCR were performed using F.Ct.trpA and R.Ct.trpA primers (Table 1). The ompA and trpA PCR products were DNA purified, and sequenced at Macrogen Inc (Seoul, Korea). All sequences were validated using an electropherogram (BioEdit version 7.0.0, Carlsbad, CA, USA). The sequences were deposited in GenBank under accession numbers KM369928 to KM369961. The ompA sequences were identified as C. trachomatis serovars by BLASTN against the GenBank nucleotide nonredundant database [19]. Multiple sequences were aligned using MEGA 5 software (Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park, PA, USA), and compared with 19 reference C. trachomatis serovars A-L₃: A/Har-13 (NC007429), B/TW-5 (DQ064281 ompA), Ba/ Apache-2 (DQ064282 ompA, AY096806 trpA), C/ TW-3 (NC023060), D/UW-3 (NC000117), Da/TW-448 (X62921 ompA, KM369947 trpA), E/Bour (NC020971), F/IC-Cal3 (DQ064287 ompA, AY096810 trpA), G/UW-57 for ompA (AF304326), G/UW-524 trpA (AY096811), H/UW-4 (AE304328 ompA, AY096812 trpA), I/UW-12 (DQ064290 ompA, AY096813 trpA), Ia/IU-4168 (AF063201 ompA, KM369958 trpA), J/UW-36 (DQ064292 ompA, AY096814 trpA), Ja/IU-37538 ompA (AF202458), Ja/UW-92 trpA (KM369959), K/UW-31 (DQ064293 ompA, AY096815 trpA), L₁/440 (DQ064294 ompA, AY096816 trpA), L₂/434 (DQ064295 ompA, AY096817 trpA), L₂a/TW-396 (AF304858 ompA, KM369961 trpA), and L₃/404 (DQ064296 ompA, AY096818 trpA). A variant was defined as having ≥ 1 nucleotide (nt) difference from the sequence of the reference strain. The p-distance and neighbor-joining tree with 1,000 bootstrap replicates were constructed using MEGA 5.

C. trachomatis TrpA three-dimensional structure was constructed using consensus TrpA sequences from reference serovars D to K, by MODWEB (http://salilab.org/modweb) [20]. MODWEB uses PSI-BLAST [19] and IMPALA [21] to obtain representative three-dimensional structures from the Protein Data Bank (PDB) (http://www.rcsb.org), and then uses MODELLER to align our query sequence with the representative PDB structure [22]. Every modeled structure was given a GA341 model score. The GA341 of >0.7 means reliable structure prediction, with >95% probability of the correct fold [20-22], and all of our modeled structures had GA341 of 1.0. The structures were visualized using the UCSF Chimera tool (http://www.cgl.ucsf.edu/chimera) [23]. Critical sites, including active sites, binding sites and TrpB communication (COMM) domain, were annotated based on the structural alignment to the representative PDB structures (1qopA for TrpA and 1qopB for TrpB, from Salmonella typhimurium) [24], and literature reviews [25, 26]. The structural alignments were accomplished using the MultAlign Viewer program [23].

Of 284 total participants, HPV (86 individuals: 30.3%) was detected most frequently, followed by C. trachomatis (37 individuals: 13.0%) and N. gonorrhoeae (30 individuals: 10.6%), respectively. After categorizing based on symptomatic versus healthy or asymptomatic groups, the ranking remained valid for symptomatic urogenital group (HPV 48%, C. trachomatis 22%, and N. gonorrhoeae 6%), but asymptomatic group (N. gonorrhoeae 16%, HPV 10%, and C. trachomatis 3%) (Table 2). For the symptomatic group, coinfections with C. trachomatis and HPV were found commonly, compared with C. trachomatis and N. gonorrhoeae coinfections (Tables 2 and 3). Samples with no C. trachomatis, N. gonorrhoeae, or HPV were positive for the human -globin gene (data not shown). This was to confirm the quality of the DNA, because all samples must be at least positive for this human housekeeping gene.

Characterization of symptoms based on the progression of the diseases (UGT or LGT) showed that the pathogens were more frequently detected in the UGT and LGT than in the undefined, and the asymptomatic groups; except for N. gonorrhoeae (Table 2). HPV and C. trachomatis were found even more frequently when symptoms reached the UGT than LGT (23% HPV, and 12% C. trachomatis). Although samples were collected at the cervix and the number of participants with UGT was smaller than the number of participants with LGT. Additionally, categorization of symptomatic women and girls by age showed that those in the 15–34 age range had more than 70% of infections with C. trachomatis (76%), N. gonorrhoeae (78%), and HPV (72%) (Table 2).

For symptomatic women and girls, those not engaged in commercial sex (non-CSWs) had a lower prevalence of C. trachomatis (82% CSW compared to 18% in non-CSW), N. gonorrhoeae (67% CSW, 33% non-CSW), and HPV (68% CSW, 32% non-CSW) infections. In those women and girls who appeared healthy, C. trachomatis was found relatively more commonly in CSWs (Table 2).

Nineteen random symptomatic patients with C. trachomatis infections were ompA sequenced to identify serovars: 6 belonging to D, 4 E, 4 F, 2 G, 2 H, and 1 K. These patients underwent a complete course of doxycycline treatment. While the majority of patients did not come back for C. trachomatis follow-up examination after treatment, all patients who came back had persistence of C. trachomatis of the same serovar found before treatment (Table 3).

Full-length ompA sequences of 14 clinical isolates from Thai women and girls, compared with respective reference serovars, demonstrated that all clinical Ds had 2-4 ompA nt polymorphisms compared to reference D, and D/U14bf and D/L11bf had 0.003– 0.006 ± 0.002–0.003 p-distance from D (Table 4). Note that D/U1bf and D/U9bf harbored the same ompA polymorphisms that caused 0.000 p-distance against D (Table 4). ompA variants were also observed in 1 clinical F and 2 Hs. Most polymorphisms were of different nt positions, and many encoded for amino acid changes and thus high dN/dS. Bidirectional sequencing indicated H/U20bf and H/ L23bf containing a substitution that caused a stop codon (W295). However, these ompA variants remained phylogenetically clustered with their respective reference strains (Figure 1).

Figure 1

Clinical D, Gs, and H demonstrated trpA polymorphisms; many of which encoded for nonsynonymous amino acid changes, causing high dN/ dS. Moreover, the trpA polymorphisms were at the positions that affected the phylogenetic relatedness among serovars. These clinical trpA variants became grouped with the different reference serovars: D/L11bf instead clustering with H, Ia, J, and Ja; and G/U18bf, G/U31bf, and H/U20bf clustered with F and I (Table 4 and Figure 2).

Figure 2

To investigate the possible effects of clinical trpA variants, three-dimensional structures of TrpA and active TrpA-TrpB complex were computed. The computed structures yielded the GA341 model scores of 1.0, which represented perfect modeling scores. This indicated that greater than 95% of the modeling structures were correct [20]. Figure 3A demonstrates the clinical trpA polymorphisms at around TrpA critical sites. For instance, C177Y was one of the active TrpA residues, and Q37R/R37Q was located close to another active TrpA residue. Further, for tryptophan synthesis to proceed, TrpA and TrpB subunits must form an active, planar TrpA–TrpB–TrpB–TrpA complex (Fig. 3B). During the process, the COMM domain of TrpB joined and lifted up TrpA to broaden the tunnel lining TrpA and TrpB active residues, allow indole substrate to be converted to tryptophan. In Figure 3B, C177Y is also positioned close to the interacting COMM domain. By contrast, when tryptophan is available, TrpA and TrpB do not interact and the COMM domain of TrpB shifts downward [24-26].

Figure 3