**4. Use of molecular techniques in chlamydia diagnosis and screening**

Since the late 1990s, chlamydia has been the most commonly reported sexually transmitted infection (STI) in Europe and the United States. The infection is caused by the bacterium *C. trachomatis* (*C. trachomatis*), and its common name was given in the late nineteenth century after an infection-causing pathogen. In 2017, there were just over 203,116 new diagnoses in the UK. In contrast, 7137 syphilis and 44,676 gonorrhea and more than 3,126,000 chlamydia diagnoses were made by the National Chlamydia screening program. The disease was equally prevalent in men, and although similar effects on the seminal vesicles are expected, there is as yet no evidence of a strong association between infection and male infertility. The program began in 2004, and the reported incidence of chlamydia has skyrocketed, with journalists warning of "a time bomb for fertility" from these reports. Chlamydia was first recognized as a specific sexually transmitted infection in the 1970s, but in 1988, it became reportable.

There are different types of screening options for chlamydial infections. It has the highest rate of spread on the scale of infectious diseases among youth and late adults. Chlamydia is easily transmitted sexually and can also be transmitted to newborns. A large number of infectious individuals may be overlooked in screening because they do not have symptoms. Although chlamydia can cause serious infections, it can be easily diagnosed with various new tests with high sensitivity. The new urine tests are rapid and noninvasive and give rapid results. It has been shown that scans reduce the rates of pelvic inflammatory disease (PID) and improve birth outcomes when pregnant women are screened and treated. Cost studies, on the other hand, show that screening for women is more cost-effective than no screening. The evidence for screening in men is quite limited. Little is known about how often any of these groups will be screened. Worse health outcomes, such as recurrent infections, PID in women, and ectopic pregnancies, should be followed by conventions, which are currently largely in the domain of public health programs, not clinical practice. As the responsibility for these tasks shifts to public health organizations, clinicians may become more involved

in these secondary preventive measures [23]. Increasing evidence suggests that the rate of progression of endocervical chlamydia to pelvic inflammatory disease is lower than previously thought. Population-based studies consistently estimate the incidence rates of pelvic inflammatory disease to be lower than that by clinical-based studies. Therefore, infections detected by screening asymptomatic individuals may have a better prognosis than that of symptomatic infections due to differences in the burden of the organism. However, descriptions of chlamydial infection and its consequences and models of the impact of screening almost always refer to higher estimates [14].

Compared to nucleic acid amplification tests, the sensitivity of conventional methods is very low [19].

Commercial polymerase chain reaction (PCR) uses multiple amplifier product line-based methods to target DNA. This is a cryptic plasmid DNA of 207 nucleotides, which is highly conserved among serotypes for *C. trachomatis.*

Chlamydia species are the leading cause of bacterial STDs, important respiratory pathogens, and the etiologic agent of endemic blinding trachoma, a zoonotic threat. In the last decade, molecular genetic analyses of Chlamydia species have advanced rapidly [24]. *C. trachomatis* (Ct) is the most common sexually transmitted disease worldwide. A Ct infection can cause urethritis, cervicitis, proctitis, and conjunctivitis depending on the anatomical site of the infection. It is asymptomatic in 50% of men and 70% of women. When *C. trachomatis* infection is not treated, it can cause infertility in men and women, such as epididymitis and pelvic inflammatory disease [3].

In the study using DNA enzyme immunoassay (DEAI) and reverse hybridization assay techniques, 19 serovar typing was performed by distinguishing it from other bacteria and chlamydia species or commensal microorganisms in the genital tract with very high sensitivity as a result of the identification of *C. trachomatis*. First, PCR products were hybridized with the probe mixes for the cryptic, plasmid, and omp 1 genes, and *C. trachomatis* was detected [18]. *C. trachomatis* is the most common sexually transmitted bacterial infection in the United States, usually by asymptomatic individuals. FDA-approved molecular methods for diagnosing urogenital *C. trachomatis* include nucleic acid hybridization, signal amplification, polymerase chain reaction, chain displacement amplification, and transcription-mediated amplification [25].

Molecular methods are both rapid and reliable for screening genital species in areas with high disease prevalence. The clinical and analytical sensitivity of some tests is seriously reduced when testing from urine samples. In vitro experiments have shown that transcriptional origin amplification is more sensitive than other molecular-based assays [19].

In the detection of *C. trachomatis*, screening tests without amplification include the direct fluorescent antibody test (DFA), optical immunostaining (OIA), and rapid solid-phase enzyme immunoassay (EIA). The accuracy in the diagnosis of *C. trachomatis* using nonmolecular methods confirms the quality of the reference tests [26].

Commercial nucleic acid hybridization is nucleic acid hybridization using oligonucleotide sequences designed to bind to the complementary sequence in the target nucleic acid. It can be used in conjunction with cell culture methods that do not provide optimal conditions due to low sensitivity. In commercial signal amplification, neither target nor oligonucleotide probe nucleic acid concentrations change. It contains targetspecific chromosomal and cryptic plasmid sequences for detecting *C. trachomatis* [19].

*C. trachomatis* infections can be detected using cell culture, immunofluorescence (IF), enzyme immunoassay, direct DNA hybridization, and PCR (identifiable). Laboratory diagnosis of chlamydial infection by culture is limited by the fact that the collection of urethral swab specimens is unacceptable for many asymptomatic men.

PCR applications using various gene targets, such as cryptic plasmid, omp 1 (the gene encoding major outer membrane protein-MOMP), and rRNA genes, are more sensitive than EIR, IA, and culture [10].

*C. trachomatis* is the most common sexually transmitted bacterial infection in the United States, usually by asymptomatic individuals. FDA-approved molecular methods for diagnosing urogenital *C. trachomatis* include nucleic acid hybridization, signal amplification, polymerase chain reaction, chain displacement amplification, and transcription-mediated amplification. Molecular methods are both rapid and reliable for screening genital species in areas with high disease prevalence [27]. The clinical and analytical sensitivity of some tests is seriously reduced when testing from urine samples. In vitro experiments have shown that transcriptional origin amplification is more sensitive than other molecular-based assays [19].

Commercial nucleic acid hybridization is nucleic acid hybridization using oligonucleotide sequences designed to bind to the complementary sequence in the target nucleic acid. It can be used in conjunction with cell culture methods that do not provide optimal conditions due to low sensitivity [19]. Samples isolated from 40 patients with *C. trachomatis* infection were transferred to the laboratory, and the methods obtained from the culture method with chlamydial nucleic acids were compared. By PCR, either the characteristic 7.5 kb plasmid DNA or the 16 Cyclic r RNA gene segment was used for identification. All PCR results were validated in Sothern or dot blot format. As a result, 5 *C. trachomatis* were isolated from 6 samples that were PCR positive. More samples (9) were found to be positive, as nucleic acid sequencing showed rRNA-PCR-amplified products in variants. These data showed that *C. trachomatis* infections in patients were either unrecognized or detected variants carrying the *C. trachomatis* plasmid [28].

Epidemiological studies on molecular typing have been conducted since ancient times. Molecular typing and serotyping of 150 *C. trachomatous* specimens isolated from genital sources of 10 different serovars were compared. The most common omp 1 genotypes, E (51.7%), F (17.3%), D (8.8%), and G (8.4%), were determined from the samples collected over 29 months. Molecular biology methods that require molecular biology techniques and equipment allow typing as well as immunology techniques [29].

A different study used rapid antigen identification to identify *C. trachomatis* infections and compared it with direct fluorescent antibody staining and tissue culture. In a study conducted on 507 patients, the sensitivity was found to be 75%, whereas the specificity was 99% [30].

Chlamydia detection and screening methods made from urine samples are commonly used. Sample supply is easier as noninvasive methods are used for sample collection.

However, PCR can also be used because urine specimens are more convenient to collect and more acceptable to patients. Studies have shown that the application of restriction fragment length polymorphism (RFLP), which is another technique in addition to PCR, facilitates the identification of many serovars that are difficult to identify using PCR [31].

PCR is more sensitive and accurate than other methods in chlamydia samples isolated from endocervical swabs. Cell culture PCR results obtained from urine samples as well as the samples collected from the cervix were compared, and the sensitivity was 87% in culture, 92% in cervix PCR, and 95% in urine PCR. Culture from endocervical and urethral swabs is the gold standard for diagnosing chlamydia. However, its sensitivity is affected by many factors, such as conditions during transportation. Recently, PCR amplification methods and ligase chain reactions have been shown to be more sensitive. In recent years, PCR scans from the urine cervix have offered great advantages. Urine

PCR culture is more sensitive than specific culture. Urine chlamydia screenings are more acceptable in large populations and for asymptomatic detection [32].

*C. trachomatis* (Ct) is an atypical agent for developing acute, subclinical, and chronic conjunctivitis. The chain reaction (PCR) procedure was used for the conjunctivitis test and enzyme-linked fluorescence assay (IFA and ELFA) and molecular analysis of Ct DNA search (Ct DNA) with polymerase among a total of 3520 patients who visited the examination room of the G. d'Annunzio University Eye Clinic between 2006 and 2008 in the study formed with the records of 171 patients with occasional mild, moderate, or severe illness from Chieti, Italy, in a prospective open three-arm study using conventional assays such as immunofluorescence in order to evaluate the presence of *C. trachomatis* against trachoma.

Molecular tests such as the GeneXpert CT/NG test are highly sensitive. However, cost constraints prevent these technologies from being implemented in environments with limited resources. Pooled testing is a strategy to reduce the cost per sample, but the extent of the savings depends on the prevalence of the disease. One study used a pooling strategy based on the identification of sociodemographic and laboratory factors associated with the prevalence of CT/NG in a high-risk area of Zambian female sex workers. Factors associated with positive testing for CT/NG through single mothers' logistic regression modeling conducted from 2016 to 2019 included city, young age, low education, and long-acting reversible contraception. Based on these factors, the study population was divided into high-, medium-, and low-prevalence subgroups. *Trichomonas vaginalis* infection was tested in pools of 3 or 4, respectively, according to bacterial vaginosis and syphilis infection. The fee was reduced from \$18 to \$9.43 per sample in the low-prevalence subgroup. The described checklist tool and pooling approach can be used in a variety of ways. This is especially valuable in areas where resources are limited. It is also important in treating asymptomatic CT/NG infections missed by traditional syndromic management.

The most attractive DNA amplification methods can be recommended for screening trachomatis infections because of their excellent sensitivity and good performance. This scan has been shown before in the FVU (first-void urine) samples. PCR testing with FVU is cost-effective in a low-prevalence population. A side risk factor for *C. trachomatis* infection is infection exceeding 3.9%. Recent research has shown that neoplasia is a risk factor for pregnancy outcomes other than ectopic pregnancy and possibly for cervical development. The cost-effectiveness of screening the sexually active population with DNA amplification methods can be reassessed.

Pathogens were compared using multiplex real-time polymerase chain reaction (PCR) in men with acute urethritis. Test results were compared in 83 patients using urethral swab samples, multiple real-time PCR, and A.F. Genital System tests in men diagnosed with acute urethritis. The pathogen of urethritis was detected in 69 patients with PCR and in 15 patients with AF. Compared with AF genital tract multiplex PCR, its sensitivity is low in male patients with previous acute urethritis [33]. Urethritis in men is one of the most common sexually transmitted diseases, and although there have been important developments in treatment and diagnosis in recent years, the most common method is the use of polymerase chain reactions [34].

Highly conserved Chlamydial proteins can be used as specific markers in the diagnosis of chlamydial and constitute new targets of drugs specific to these bacteria. In total, 59 Chlamydia proteins, 79 Chlamydiaceae proteins, 20 both Chlamydia and Chlamydophila, and 445 ORFs were found to be specific to Protochlamydia [18].

Studies have been conducted using tandem mass spectrometry and affinity chromatography methods for Chlamydia infections, which do not have an effective vaccine yet and have resulted in significant deaths worldwide [35].

*Molecular Approaches to the Diagnosis of* Chlamydia *DOI: http://dx.doi.org/10.5772/intechopen.109746*
