**3. Molecular techniques used for diagnosing** *Chlamydia*

Chlamydia, obligate intracellular bacteria, requires tissue culture techniques to isolate and propagate. Culture in permanent cell lines or embryonated chicken eggs is still widely accepted as the gold standard for chlamydial diagnosis, as it is necessary to

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

demonstrate the viability of a strain of a field and facilitate detailed characterization by molecular and biochemical methods [12].

Culture methods were first allowed as standards in the 1970s and were developed using McCoy cells. This method, originally developed for trachoma and LGV, was approximately 100% specific, but laboratory sensitivity was estimated to be only 70–85%, and overall sensitivity was less than 40–85% due to sampling errors and varying laboratory standards. There are two main approaches to diagnose *Chlamydia* and *Chlamydophila* spp. infections in mammals and birds. The first involves directly detecting its agent in the tissue, and the second includes serological screening of blood samples with anti-chlamydial antibodies [13]. Individual methods of detecting antigen characteristics in sample types sent to the diagnostic laboratory may affect the performance. Tests such as the use of DNA-based PCR offer particular advantages. In the 1980s, there was a return to the fashion of microscopic identification without culturing using a technique known as direct fluorescence testing, where the test was performed by binding specially produced antibodies to specific sites on the outer membrane of *Chlamydia*. Compared to other methods, the results were more variable, and this method was used because it was fast and relatively inexpensive, although it was observed to be influenced by many factors, including the recognition of three strains or serotypes of *C. trachomatis*.

Advances in molecular biology have revolutionized the laboratory diagnosis of all diseases, many of which have been developed by new biotechnology companies and monoclonal antibodies. There are new tests for antibodies (complement fixation and microimmunofluorescence) and new antigen tests (direct fluorescence test and enzyme immunoassay). All were compared to the gold standard of cell culture, but there was a growing suspicion that it was better than any of the newer tests. In the 1990s, new assays using new DNA technologies, particularly the polymerase chain reaction (PCR) technologies, became available to develop nucleic acid amplification assays (NAATs). In these tests, fragments of *Chlamydia* DNA extracted from clinical samples were amplified in repeated cycles to produce samples large enough for colorimetric evaluations. The first test of this type was introduced by Roche in 1993. Abbott Laboratories from Illinois mediated ligase chain reaction and transcription and Gen-Probe, whereas La Jolla from California mediated amplification. A review of the new tests found that although they were based on different molecular strategies, they had equivalent specificity and sensitivity to Roche Amplicor [14].

In recent years, there has been a revolution in diagnostic methodology with the introduction of nucleic acid amplification tests (NAATs). These tests are much more sensitive than the previous non-culture tests [15].

For the first time, diagnostic laboratories have a more sensitive technology in tissue culture than in isolation (TC). While TC, which has long been considered the gold standard for the diagnosis of *C. trachomatis*, is considered to have a specificity approaching 100%, NAAT offers more sensitive tests than culture [14].

In a study published in 1997 where the old enzyme immunoassay methods found a prevalence of 1.6% (0.8–2.7%), 60% sensitivity and 100% specificity and the prevalence of ligase chain reaction was found to be 2.5 (1.5–3.9%) in relation to the improvement offered by new DNA-based techniques; a sensitivity of 90% and specificity of 99.8% were indicated. New technologies not only offer greater specificity, sensitivity, and accuracy but also are cheaper and easier [16].

Therefore, it has become possible to go beyond diagnostic testing to screen for asymptomatic chlamydial infection in both men and women. Paradoxically, NAAT technologies may be responsible for the continued increase in new cases reported, as they enable more testing and greater precision [4].

*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 collection of urethral swab specimens is unacceptable for many asymptomatic men. PCR applications using various gene targets, such as various cryptic plasmids, omp 1 (the gene encoding major outer membrane protein-MOMP), and rRNA genes, are more sensitive than culture [3, 4, 6, 9–11, 17–19]. Conventional PCR enables real-time PCR quantification, while it identifies most chlamydial species for the presence and absence of a particular pathogen with the ompB gene, a gene specific to the chlamydia family.

Recent taxonomic advances based on 16S and 23S rRNA gene sequences have divided the Chlamydiaceae family into two genera and nine species, five of which have been found to infect humans. There are several simple methods for detecting and identifying all species precisely and specifically. In this study, the omp2 gene was demonstrated as a target for the molecular identification of suitable Chlamydiaceae. Phylogenetic analysis accepts partial omp2 gene sequences from all nine species based on recently published taxonomic changes. The use of a family-specific PCR primer pair capable of amplifying the 5-end on ribosomal genes is described for the omp2 gene from all *Chlamydiaceae*, except for some strains of *Chlamydophila pecorum*. The identification of all nine species was obtained using restriction fragment length polymorphism analysis with the Alu I enzyme, which was confirmed by DNA sequencing. A PCR enzyme-linked oligonucleotide assay can be developed that can identify mixed human chlamydial infections or a lone chlamydial genome analysis [6].

Another method used for rapid detection of pathogenic bacteria is the DNA microarray method, which is based on DNA hybridization. There are studies examining nine species belonging to the *Chlamydiaceae* family with the help of probes developed to identify species-specific regions such as the ribosomal RNA opreon region. Identification was made both in culture and direct clinical tissue bacteria samples [5].

Isothermal amplification assays such as loop-mediated isothermal amplification (LAMP) are of great utility in the development of rapid diagnostics for infectious diseases, as they have high sensitivity, pathogen specificity, and application potential. However, eliminating nonspecific amplification remains a major challenge for the optimization of LAMP assays [20].

Ocular infections are more difficult to diagnose and confirm than systemic infections or infections of other organs due to their delicate anatomy and fewer sample types and volumes that can be safely collected from the eye. Next-generation sequencing (NGS)-based approaches are revolutionary molecular diagnostics, also called highthroughput. NGS-based approaches can amplify tens to hundreds of samples containing limited genomic content and transcriptomes, allowing characterization of all of them. This technique is of particular interest in clinical diagnosis in ophthalmology, as the sample quantity is limited and difficult to obtain. NGS needs to be developed for clinical applications in ophthalmology by making its use more convenient [14, 17].

The third widely used nucleic acid target amplification method in the United States is isothermal TMA (commercial nucleic acid amplification) based on *C. trachomatis*. Within two hours, the RNA amplification rate increased 109 -fold. Within four hours, the DNA amplification rate increased 106 -fold. Nucleic acid amplification assays have been used for verification in previous years because of their high sensitivity, but in recent years, they have been used for screening various samples. Consequently, as nucleic acid-based diagnostic assays continue to evolve, such tests need to be established in both small- and large-scale clinical laboratory settings. Commercial nucleic acid hybridization is nucleic acid hybridization using

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

oligonucleotide sequences designed to bind to the complementary sequence in the target nucleic acid. It is used in conjunction with cell culture methods to provide optimal conditions due to its low sensitivity. Target and oligonucleotide probe nucleic acid concentrations do not change. It contains target-specific chromosomal and cryptic plasmid sequences for detecting *C. trachomatis* [19].

Due to the lack of a genetic transformation system, studying the molecular biology of Chlamydia, an obligate intracellular bacterium, has been difficult. With genome sequencing, knowledge of the biology of these pathogens has greatly increased. Comparing the seven sequenced genomes of the Chlamydia genome provides an overview of gene content and gene diversity. Genome sequences have allowed general investigations in terms of both transcript and protein content throughout the evolution of the Chlamydial cycle. Chlamydiae form chlamydial inclusions, and the proteins released from this inclusion can interact with host cell proteins and produce changes in the host cell's response to infection. The identification of these proteins is difficult because the cytoplasm of the host cell infected with Chlamydia cannot be purified. This problem has been overcome by comparative proteomics [21].

Ligase chain reactions (LCVs) are among the noninvasive nucleic acid amplification tests and are among the amplification tests that can be used in low-prevalence populations because they are likely to give false positives and need confirmation [22].