Urogenital Tract Infection with Chlamydia trachomatis among Women Attended at Different Units in a Referral Hospital in Spain

Aims: The objective of this study was to estimate the prevalence and characteristics of Chlamydia trachomatis infections in a group of women visiting different Units in a referral Hospital from Spain Study Design: This was a hospital retrospective and descriptive study for the presence of C. trachomatis in endocervical, vaginal and urine swabs obtained from consecutive sexually active women attendees at different Units. Also their medical records were reviewed. Retrospective ethical approval was granted by the Ethical Committee of Clinical Investigation of Principality of Asturias. Methodology: We included 1997 symptomatic and asymptomatic unselected women (mean age 29.1 range 18 to 45 years) who were evaluated for urogenital chlamydial infection. Results: The overall prevalence of C. trachomatis was 6.3%. The C. trachomatis infection had the highest prevalence among the age group below 25 years of age (n=30, 7.5%). Genotypes E, G and D constitute 89.4% of the genotyped strains. Infections with genotypes G and F were more often (n=31, 42%) associated with clinical manifestations that suggest cervical infection and genotype E was observed more frequently (n=17, 85%) in asymptomatic women. Conclusions: In our study, similar prevalence rates between both symptomatic and asymptomatic women, under 25 years, were found. Self-collected vaginal swabs are an appropriate alternative for routine diagnosis of C. trachomatis infection. The findings of this work highlighted the need for a possible Chlamydia screening program, offered especially in younger women. Delays in seeking a diagnosis and treatment among asymptomatic females can result in increased transmission of this bacterium and its serious consequences for women reproductive health.


INTRODUCTION
C. trachomatis is currently, the major cause of bacterial sexually transmitted infections (STIs) in Europe as well as many other countries around the world [1]. An important characteristic of these infections is their ability to cause long-term sequels in the upper genital tract. In women, chlamydial urogenital infections, having a clinical course varying for asymptomatic to clinical manifestations, which include urethritis, cervicitis and pelvic inflammatory disease (PID) may lead to serious sequelae such as ectopic pregnancy, infertility and chronic lower abdomen pain [2,3].
Due to their frequent asymptomatic nature and the prevalence among young women, this infection is a public health problem to resolve [4]. Despite major advances in the diagnosis and management of chlamydial infections, in Spain there is no a general cost-effective plan of screening programs that would guarantee a decrease of the aftermath caused by untreated infections.
Undiagnosed and untreated chlamydial infections are, thus, not only creating major health problems and consequences for individuals, but also result in major social and economical problems. The aim of this study was to determine the prevalence and characteristics of Chlamydia trachomatis infections in a group of women attended to at different Units from a referral Hospital in Spain. Symptomatic women were defined as those suffereing from one genitourinary clinical symptom (abnormal vaginal discharge, cervicitis, PID, lower abdominal pain, dyspareunia, dysuria or vulvar erythema). Asymptomatic women were defined as those who contacted a clinician for pregnancy control, routine Pap smear annual examination or infertility, and they did not have symptoms or signs of infection at the time the specimen was taken.

Patients and Clinical Samples
On the other hand, STI microbiologic testing: culture of bacteria, VHS-2, Candida spp, serological testing (HBV, syphilis and HIV) or NAAT for VPH were routinely performed considering clinical signs or symptoms of patients.
To compare vaginal samples and endocervical samples as better detection specimen, 129 vaginal and endocervical swabs belonging to 129 women were acquired simultaneously.
Furthermore, a possible association between particular genotypes and the occurrence of clinical manifestations was studied in 85 C. trachomatis single infected-women.

Chlamydia trachomatis Detection
C. trachomatis DNA was extracted and detected using AMPLICOR CT/NG Specimen Preparation Kit (Roche Diagnosis System) and COBAS TaqMan CT Test v2.0 (Roche Diagnosis) respectively. Positive C. trachomatis specimens were kept at -70ºC in 2SP medium until retrospective ompA genotyping.

Genotyping
C. trachomatis DNA was extracted using the Nuclisens Easy Mag system (bioMerièux, Marcy l`Etoile France). To genotype C. trachomatis specimens, a 990pb fragment of the ompA gene was amplified according to a nested-PCR methodology previously described [5]. PCR amplified ompA gene fragments were purified from agarose gels using Montage DNA Gel Extraction Kit (Millipore, Bedford, MA) and sequenced with BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA) using inner primers (MOMP87-RSV1059).
The amplicon sequences were aligned to analogous sequences from reference strains of each genotype by using Clustall-W2 program.

Statistical Analyses
Statistical analyses of the data were performed using the Χ 2 or Fisher´s exact test. A p-value < .05 was considered to be statistically significant. Absolute and relative frequencies and 95% accuracy were given. Also, the kappa index was used to measure the correlation in the diagnostic yield of different samples.
The overall prevalence of C. trachomatis was 6.3% (6.9% in symptomatic and 4.5% in asymptomatic females) (p= .06). In women with an age between 25 and 40 years, the highest prevalence (7.1%) was found in those with some genitourinary clinical symptom. We observed similar prevalence rates (7.7% and 7%) among the age group younger than 25 years in both symptomatic and asymptomatic women, respectively (Table 1).
Prevalence rates observed in women attending a Gynecology Unit, were higher (6.9%) in patients with clinical manifestations than in those visiting the Unit for an annual routine Pap smear examination (3.8%) (p= .04). Prevalence rates found in evaluated pregnant women and in asymptomatic women with infertility were 5% and 10%, respectively (Table 1).  Table 3).
The phylogenetic analysis of the ompA gene from 94 positive specimens showed that the most frequent genotypes were G (n=35, 37.2%) followed by D (n=26, 27.7%), E (n=23, 24.5%), F (n=8, 8.5%) and K (n=2, 2.1%). In the 74 positive specimens belonging to symptomatic women infected only with C. trachomatis, genotype G (47.3%) was the most prevalent, followed by D (31%), F (10.8%), E (8.1%) and K (2.7%). On the other hand, 20 asymptomatic females presented E (85%) and D (15%) genotypes. The most relevant difference between symptomatic and asymptomatic women was the detection of genotype E in 8.1% of the symptomatic patients vs 85% in the asymptomatic females (p< .001). Infections with genotypes G and F were more often (n=31, 42%) associated with clinical manifestations that suggest cervical infection (cervicitis, lower abdominal pain and PID). Genotype G was found only in symptomatically infected patients (p< .001) and genotype D was associated with abnormal vaginal discharge (p<0.0001) (Fig. 4).

DISCUSION
Infections caused by C. trachomatis, have showed a progressive increase in the past decade in Europe and others parts of the developed world [1].
In our research, a clear age dependency was observed. The highest C. trachomatis prevalence rate was found among women younger 25 years of age (7.5%), in both symptomatic (7.7%) and asymptomatic patients (7%). The C. trachomatis prevalence rate decreased significantly in women older than 40 years. This is in accordance with previous publications reporting higher percentage of patients with asymptomatic infections [4]. It is therefore of importance to not forget the silent nature of this infection. The microbiology laboratory, by using sensitive and specific techniques, plays a significant role allowing a rapid confirmation of the diagnosis.
Young age is the factor that is most strongly associated with the infection (relative risk among women younger than 25 years as compared with older women, 2.0 to 3.5) [6]. This association is largely attributed to the higher level of sexual activity among younger women. Also, in younger women, the squamocolumnar junction of the cervix often lies well out on the ectocervix, forming a bright red central zone of ectopic columnar epithelium called ectropion [7].  Furthermore, it has been shown that screening is cost effective at prevalence of 3.1-10% and cost saving (over testing symptomatic women) at prevalence as low as 1.1%, if age was chosen as a selection factor and DNA based test were used. These studies reveal a significant reduction in the number of cases of PID and ectopic pregnancies, after the introduction of C. trachomatis screening [23][24][25][26][27][28]. However, due to the low number of pregnant and infertile women analyzed as well as to the unavailability of demographic data relating to them, further studies and strategies for suitable control are needed in our hospital.
There are several studies that defend the increased sensitivity of endocervical specimens [29,30]. Other authors, point out that vaginal samples have better sensitivity because collecting more DNA from the two potential sites for C. trachomatis infection in women, the urethra and endocervix [31,33]. This work, showed that both vaginal and endocervical swabs were optimal at detecting C. trachomatis infection, in women. We showed that self-collection of vaginal specimens is an appropriate alternative for C trachomatis infection screening.
On the other hand, we observed that the genotypes E, G and D constitute 89.4% of the genotyped strains. Sequence analysis showed that the most prevalent ompA sequence corresponded to genotype G. The genotypes D, E and F are the most common genotypes in Europe [34,35] as well as in Spain [36], where the most frequent genotypes are also E, D, G and F. Genotype E is the most prevalent genotype in both men and women.
The E genotype was observed more frequently in samples from women without clinical symptoms or signs of infection (85%) compared to females with clinical manifestations, which are a reservoir able to transmit the infection. In symptomatic females (the majority of tested women), infections with genotypes G and F were more often (41.9%) associated with clinical manifestations that suggest cervical infection (cervicitis, lower abdominal pain and PID). These results are in agreement with two previously published studies in which an association was found between genotype G and developing cervical cancer and genotype F and lower abdominal pain and dyspareunia [37,38]. Perhaps, the highest number of women with cervical pathology explains the high frequency of G genotype in our study but, it is also likely that patient characteristics are a key component to understanding the genotype distribution. Unfortunately data such us sexual behaviors, history of previous Chlamydia trachomatis infection or prior history of gynecological pathology were not made available to the present study.
Considering all the data presented in this research and knowing that many infections are asymptomatic, it is essential to investigate in detail the infection and carefully design the most appropriate methods for detection. Thus, with the implementation of cost-effective screening we could increase our knowledge about the epidemiology and transmission of infections caused by this bacterium.

CONCLUSION
Chlamydia trachomatis infections are an important public health problem due to their frequent asymptomatic nature, the prevalence among people younger than 25 years and their severe reproductive complications. Thus, interrupting their route of transmission by identifying and treating patients with Chlamydia trachomatis is essential in the prevention of this STI. In Spain, there is a need for effective strategies for an early detection of Chlamydia trachomatis infection especially in asymptomatic women of childbearing age.

ETHICAL APPROVAL
Retrospective ethical approval was granted by the Ethical Committee of Clinical Investigation of Principality of Asturias (registration number 128/2012).

CONSENT
It is not applicable.