**4. Clinical manifestation and diagnosis of** *C. trachomatis* **infection**

Chlamydial infection of the lower genital tract, which causes endocervicitis in women, can be asymptomatic or may the patients complain of mucopurulent, odorless vaginal discharge, or postcoital bleeding. Additionally, edema and congestion of the cervix can also be observed. Urethritis can be concomitant with cervicitis. In such cases, a culture-negative leukocyturia is commonly suggestive for *C. trachomatis*. Furthermore, chlamydial infection in the lower genital tract does not cause vaginitis. Therefore, in the presence of vaginal findings, a different diagnosis should be considered. An ascending infection can lead to pelvic inflammatory disease (PID) [4, 13]. Endometritis is commonly related to an ascending infection and may cause irregular uterine bleeding. On the other hand, salpingitis and PID are usually asymptomatic. It has been shown that *C. trachomatis* is the cause of at least 60% of cases of acute PID [4, 14]. Some of the very well-known symptoms of PID include absent to severe abdominal pain with high fever, dyspareunia, prolonged menses, and intramenstrual bleeding. It has been shown that about 20% of women who developed PID become infertile, while 18% develop chronic pain and 9% eventually experience a tubal pregnancy (Ref. 13). Studies suggested that infertile women should be routinely tested for chlamydial infection. Moreover, specific anti-chlamydial antibodies are considered as valuable, noninvasive diagnostic methods [15, 16].

More recently, it has been reported that the estimated risk of post-chlamydial PID varies between 0.5 and 72%, depending on the study population and the definition criteria used in these investigations [16]. Subfertility and ectopic pregnancy occurred in 0.1–6% and 0–1% of women, respectively, after chlamydial infection [16]. Furthermore, studies have revealed that repeated diagnoses of *C. trachomatis* infections increased the risk of PID by 22% [16]. In another study from the UK, it has been reported that 20% of PIDs, 5% of ectopic pregnancies, and 30% of tubal factor subfertility pathologies are associated with post-chlamydial infections in women between the age of 16 to 44 years of age [16, 17].

In summary, studies suggest that several factors, including clinical symptoms, coinfections, reinfections, and sexual risk habits, probably affect the development of post-chlamydial complications in the female population. However, to definitely determine the risk and predisposing risk factors of the mentioned late complications, prospective studies are needed to provide robust data for prevention strategies related to *C. trachomatis* infections and complications [18, 19].

Nowadays, specific anti-chlamydial antibodies have been accepted as a valuable, noninvasive diagnostic tool. On the other hand, because *C. trachomatis* is an obligate intracellular bacteria, cell culture is considered as a reference method. However, various commercial non-culture-based diagnostic techniques are available [4]. Specimens for diagnosis can be obtained either by invasive or noninvasive approaches. While invasive approaches include endocervical and urethral swabs; self-collected specimens, such as first-void urine (FVU) and vulvovaginal swabs (VVS), are considered as noninvasive techniques [4].

Although cell culture has almost 100% specificity, it is not recommended for routine use, due to its pretty low sensitivity and its technical difficulty. Transport and storage difficulties of the specimens are additional drawbacks associated with the cell culture method. This technique should only be considered for medico-legal issues and for antibiotic susceptibility testing purposes [4].

While direct fluorescent staining with monoclonal antibodies (DFA) is a rapidly performed and specific test; it is subjective and not suitable for a large number of specimens [4]. Enzyme immunoassay (EIA) is an automated test and more usable than DFA, and the sensitivity is comparable to that of cell culture.

Nucleic acid hybridization tests, including DNA probing, have been described as the first molecular DNA test for *C. trachomatis*, which was widely used for a certain period. Studies showed that the performance of these tests is comparable to that of the DFA/EIA and cell culture [4].

Recently, nucleic acid amplification tests (NAATs) are suggested as the "Gold Standart" technique because of their high specificity and sensitivity, and their availability for a large range of sample types such as VVS and FVU in cases with chlamydial infections. There are various NAATs that use different technologies, including PCR and real-PCR, strand displacement amplification, transcription-mediated amplification, and nucleic acid sequence-based amplification. These measurement techniques are automated and can be used for screening programs and for the detection of *C. trachomatis* as well as *Neisseria gonorrhoeae* in the same sample [4].
