Chlamydia trachomatis in the gingival sulcus and pharynx in patients of Northeast Mexico

Abstract Background The oral microenvironment provides the conditions for the establishment of microorganisms not usually considered residents of the normal oral microbiota. Sexually transmitted microorganisms such as Chlamydia trachomatis can adhere to any mucosal surface and ascend to reach appropriate locations to survive and develop symptomatic infections. Materials and methods To determine the presence of C. trachomatis, direct immunofluorescence of this microorganism was carried out in 76 randomly selected patients attending a periodontal clinic during a period of 1 year. Samples from the gingival sulcus and the pharynx were collected for detection of C. trachomatis. Patients who attended the periodontal clinic were divided into two groups: those without periodontitis and those with periodontitis. For the purpose of performing other statistical analyses, all patients were also divided by gender and age. Results From the total of 76 patients, in the group without periodontitis, 61% were positive for C. trachomatis in the gingival sulcus and 63.4% in the pharynx; in the periodontitis group, 45.7% were positive in the sulcus and 40% in the pharynx. When we compared patients by gender or age, no statistical difference was found. Conclusions The prevalence of C. trachomatis in this group was 53.9% in the gingival sulcus and pharynx of the studied patients.


| INTRODUCTION
Normal microbiota of the human oral cavity includes more than 500 different bacterial species including Bacteroidetes, Actinobacteria, Proteobacteria, Euryarchaeota, and others. These microorganisms have the ability to colonize gingival crevices and the outer surface of the tooth (Dewhirst et al., 2010;Reynolds-Campbell, Nicholson, & Thoms-Rodriguez, 2017). The oral microenvironment provides conditions for the establishment of microorganisms that are not usually considered normal oral microbiota and that are associated with periodontitis, poor hygiene, and/or a compromised immune system. These microorganisms may or not have a role in periodontal health or disease but the periodontal biofilm may be a source of dissemination and development of systemic infections by these pathogenic microorganisms (Vieira Colombo, Magalhães, Hartenbach, Martins do Souto, & Maciel da Silva-Boghossian, 2016), including cardiovascular disease, adverse pregnancy outcomes, rheumatoid arthritis, inflammatory bowel disease and colorectal cancer, respiratory tract infections, and abscesses (Chan et al., 2016).
Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium with a propensity for the mucous membranes of their host.
C. trachomatis is a common pathogen of the genital tract; however, it can live in any mucous membrane to which it adheres, including the mucosa of the oral cavity (Reed, Lopatin, Foxman, & Burt, 2000), pharynx, and other extra genital areas (Chan et al., 2016).
Although C. trachomatis extragenital infections are not considered risk factors because they are asymptomatic, it is important to know their prevalence and incidence because they serve as a reservoir and promote the propagation of antibiotic-resistant strains (Chan et al., 2016;Danby et al., 2016).
For all these reasons, we focused on detecting the presence of C.
trachomatis in the gingival sulcus and pharynx of patients with or without periodontal disease.

| MATERIALS AND METHODS
This was a cross-sectional observational study of patients who attended the periodontal unit of the School of Dentistry of the Universidad Autonoma de Nuevo Leon in Monterrey, Mexico. A total of 76 patients were randomly selected from 6,127 patients who attended the clinic during 1 year. Patients were divided into two groups: (a) periodontitis group, patients with active periodontitis, regardless of their stage of disease (mild, moderate, or severe) and (2) patients without periodontitis or gingivitis.
The inclusion criteria were as follows: men or women between 18 and 80 years of age at the time of the dental clinic visit; patients who had or have any of these diseases or symptoms such as conjunctivitis, xerostomia, otitis, rhinosinusitis, urinary infection, polyuria, urine sand, abortions, preeclampsia, or premature birth. The exclusion criteria were patients with a history of antibiotic treatment for C.  Index," where plaque on the vestibular and lingual surfaces of the entire dentition is ranked on a scale of 0-5 after using a revealing agent. After all the surfaces are evaluated an average has to be calculated (Turesky, Gilmore, & Glickman, 1970); and the gingival bleeding index (Bessa Rebelo & de Queiroz, 2011).

| Presence of C. trachomatis in the gingival sulcus and pharynx
Gingival sulcus samples were collected from each patient using sterile periodontal Gracey curettes (5/6); after that gingival curettage was done. Pharyngeal swabs were collected from each lateral posterior wall, including tonsillar crypts. Samples was transferred separately into a microtube with sterile saline solution and preserved at room temperature before extending on a slide and fixing with 90% alcohol.  infection, is also characteristic of seminal infection (Gallegos et al., 2008;Gallegos-Avila, Ancer-Rodríguez, Ortega-Martínez, & Jaramillo-Rangel, 2010). The high prevalence in this group may be due to the lack of symptoms in chronic infections, multiple sex partners, poor hygiene, and oral sex (Chan et al., 2016).

| Statistical analysis
It is well known that oral sex is common in men and women and urogenital organisms are increasingly transmitted from the male urethra to the female pharynx and vice versa by fellatio. Bacteria reach the uterus during vaginal intercourse, suggesting that the presence of C. trachomatis in the pharynx, and others sites in the oral cavity reflect an important reservoir of infection (Danby et al., 2016).
The aim of this study was to detect the presence of C. trachomatis in the gingival sulcus and pharynx in dental patients and to determine if periodontal disease, gender, or age were determinants for their presence. Although it could be considered that the number of individuals evaluated for this study is low, the presence of C. trachomatis in the pharynx and gingival sulcus in 53% of these patients indicates that it is necessary to routinely search for these microorganisms in the community. Our results compared with other studies are contradictory, since others report a lower percentage of C. trachomatis in the sulcus and the pharynx (Chan et al., 2016;Danby et al., 2016;Dukers-Muijrers et al., 2015;Reed et al., 2000). More information is necessary to identify these discrepancies. Nevertheless, as we mentioned before, when we take into account the high percentage found in infertile couples in this region of Mexico (≥90%) (Gallegos et al., 2008;Gallegos-Avila et al., 2009), our figure of 53% of C. trachomatis in the pharynx and gingival sulcus is reliable and indicates the regional conditions of this high prevalence.
Recently, more attention has been given to the presence of C.
trachomatis in extra genital areas, such as the pharynx, colon-rectum, and others (Chan et al., 2016;Danby et al., 2016;Dukers-Muijrers et al., 2015). Only one study has attempted to relate the presence of C.
trachomatis with the development of periodontal disease. These authors detected C. trachomatis using a direct fluorescent monoclonal antibody (DFA). Only 7% (6/87) of the patients were positive and they concluded that C. trachomatis is not a periopathogenic agent. These data differ from ours, where we found that 40% of patients were positive for C. trachomatis in the periodontal sulcus (Reed et al., 2000).
In our patients, there was no statistical difference between the presence of C. trachomatis in the sulcus and the pharynx: 58% of men versus 50% (p > .05). These data are not in accordance with other similar research. Danby et al. (2016) reported positivity of C. trachomatis in 2.2% in men and in 1.7% women from pharyngeal swabs. In the same year, Chan et al. (2016) found a mean between women and men of 1.7 and 1.6%, respectively. More clinical information and an analysis of applied diagnosis techniques are necessary. We need more information in order to clear this discrepancy. Although we did not find a statistical difference between periodontitis and no periodontitis groups, ages or gender, it is important to note the presence of a genital microorganism in the oral cavity and to elucidate the population with a higher risk.
In the opinion of some authors, oral infections and poor oral health can provoke the introduction of oral microorganisms into the bloodstream or the lymphatic system. The subsequent attachment and multiplication of these bacteria on tissues or organs can lead to focal oral infections. Pathogenic agents may also remain at their primary oral site but released toxins can reach an organ or tissue via the bloodstream and cause metastatic infection that causes bacteremia, and heart, head, neck, respiratory system, and gastrointestinal system infections (Dukers-Muijrers et al., 2015).
Among the limitations of this study is the use of DIF for the detection of C. trachomatis. Molecular techniques, such as polymerase chain reaction, are more commonly used for detection; however, DIF was chosen because of its rapid result and low cost since patients were attending the clinic for dental treatment.
Our group is considering the implementation of an extragenital test in the public health setting that may lead to an increase in case detection and optimal patient management of C. trachomatis infections and its sequela (Nall, Barr, McNeil, & Bachmann, 2016). Regional climate differences, migration of people, sexual education, and poor health care programs would be taken into account to consider C.
trachomatis infection as a public health problem that requires major attention.
We conclude that the oral cavity it is an important reservoir for C.
trachomatis. It is also important to identify its presence in order to improve clinical management to avoid reinfection in the community. A future study of a less diverse age group with a more thorough periodontal investigation and microbiological evaluation of the periodontal pockets to classify the severity of periodontal disease could be useful.

ACKNOWLEDGMENT
We thank Sergio Lozano-Rodriguez, M.D., for his help in translating and editing the manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest and that all have seen and approved the manuscript. We also declare that this manuscript is the authors' original work.