**7.3 Molecular techniques for detecting antigen, DNA, or RNA/rapid tests**

Since *C.trachomatis* only grows within columnar cells, it is essential to gather a specimen directly from the cervix or urethra that will involve cells. When trying to obtain cells from vaginal or urethral discharge, it should be tried to apply pressure to the inside of the cervix or urethra. In males, after the urethra is milked down for secretions, collection swabs should be inserted 1–2 cm inside to urethra or "kissing slide" method can be used for sample collection [59]. Transport and kit's manufacturer instructions should always be fulfilled.

Enzyme-linked immunosorbent assay (ELISA) is the most preferred test for Chlamydia in outpatient clinics and emergency departments for large number of patients since it is cost-effective and mostly automatized. It has 40–60% sensitivity and a 99% specificity.

Direct fluorescent antibody (DFA) testing for *C. trachomatis* has a sensitivity of 50–80% and a specificity of 99%. It is often preferred to confirm other assays but, labor and skilled personnel are needed to perform.

#### **7.4 Nucleic acid amplification tests**

Non-culture tests for the detection of *C. trachomatis* have been substantially replaced by higher-performing NAATs. These tests show high performance even in noninvasive samples. NAATs have recently become the test of choice to effectively screen and diagnose infection because of their high sensitivity and their ability to perform noninvasive testing that does not require pelvic examination or urethral swab [60–62]. NAATs target and amplify nucleic acid sequences found in almost

every clinical strain of *C. trachomatis*, including genital, LGV, and ocular serovars. The APTIMA Combo 2 Assay used for ribosomal RNA can be used on liquid-based Pap smear samples [63]. The gold-standard method for bacterium genotyping is DNA sequencing of the ompA gene, encoding the major outer membrane protein (MOMP) [64]. This method is relatively simple and inexpensive. Most studies have reported sensitivity of more than 70% and specificity of 97–99% in populations where the prevalence of infection in men and women is 5% or more. The FDA-approved NAATs are recommended for the detection of infections caused by *C.trachomatis* and *N.gonorrhoeae* in men and women with or without symptoms [56]. Multiplex polymerase chain reaction assays are now started to be used widely, especially for polymicrobial infections and simultaneously testing the possible STDs from a single specimen [65, 66]. Older non-culture or non-NAAT tests with lower sensitivity are not recommended anymore. The Centers for Disease Control and Prevention (CDC) recommend NAATs for extragenital sites such as rectal and oropharyngeal infections because of their higher sensitivity and ease of sample handling and processing. Routine repeat testing is not recommended for NAAT-positive genital tract infections because repeated testing does not increase the positive predictive value of the test. Cultures of *C. trachomatis* and *N. gonorrhoeae* may still be necessary for the detection of sexual abuse in boys and extragenital infections in girls [56].

While NAATs are sensitive, they also have some drawbacks. Primarily, they are expensive, and so healthcare units may not be able to use them for comprehensive screening due to cost [67]. Another disadvantage of FDA-approved NAATs is that they cannot distinguish LGV strains from others. This is an important point because the duration of treatment in LGV infections needs to be extended. In addition, NAATs detect the DNA or RNA of the bacterium rather than the live microorganism, and it is common for control tests 3 weeks after the end of treatment to still show positivity [68]. For this reason, NAATs should not be used as a cure-determination test except in pregnant women, who must be shown to have a cure 3–4 weeks after the end of treatment to prevent infection in the infant.

In studies to shorten test result times, a prototype developed by TwistDx (prototype TwistDx RPA assay, Cambridge, UK) with an isothermal recombinant polymerase amplification approach shows promise. CT detection with this method takes 15 minutes. Validation studies of this prototype are still ongoing. But if this test is approved and made commercially available, it will mark a milestone in CT infection control, allowing doctors to diagnose and treat it in the same session [69, 70].

#### **7.5 Serology**

Antichlamydia immunoglobulin M (IgM) positivity is not common in adults with genital tract infections. Antichlamydial immunoglobulin G (IgG) positivity is high in sexually active adults, even if there is no active infection, and this most likely indicates a previous infection. Although there is a statistically significant relationship between chlamydia-specific serum immunoglobulin A (IgA) and active infection, none of the serological tests are sufficient to detect active disease in terms of clinical sensitivity, specificity, and predictive values. Therefore, it is not recommended for the diagnosis of genital tract infections. Nevertheless, they are still being used for research purposes and appear to be useful, especially for the detection of past infections using IgG.

#### **7.6 CT, radiography, and ultrasonography**

Imaging techniques are usually not necessary for uncomplicated genital chlamydia infections. However, CT and ultrasonography may be useful in complicated upper genital tract infections. For example, Fitz-Hugh-Curtis syndrome (perihepatitis) can be diagnosed with CT, and ultrasound can be used to investigate the presence of a tubo-ovarian abscess.
