Molecular Approaches to the Diagnosis of Chlamydia

Abstract

Chlamydia trachomatis is known as the most common bacterial infection agent to pass with sexual transition. This microorganism is an obligatory intracellular parasite. A variety of infections are caused by C. trachomatis, including trachoma, pneumonias in newborns, genital and urinary tract infections, and lymphogranuloma venereum (LGV), which is caused by LGV strains. The diagnosis of Chlamydia trachomatis can be made by cultures and isolations, antigens and antibodies (direct fluorescence, enzyme immunoassays), hybridization, or polymerase chain reaction (PCR). Each year, infection and diagnosis rates increase in the developed world. Since Chlamydia is mostly asymptomatic, screening, and treatment are a key to detecting cases. Polymerase chain reaction (PCR), ligase chain reaction (LCR), and nucleic acid sequence-based amplification (NASBAa) molecular methods can be used for the detection, low concentration, quantification, and identification of organisms. While the traditional PCR method confirms its existence, it can quantify real-time PCR (RT-PCR). This method (RT-PCR) may have low sensitivity among variants of the same species. Also, PCR scans, which receive urine service, offer great advantages. PCR from initial void urine (FVU) samples is highly sensitive in detecting the organism. Urine Chlamydia screenings are more acceptable in large populations and asymptomatic detections.

1. Introduction

The Chlamydiaceae family is an obligate intracellular bacterial member. Only the genus Chlamydia has 9 species distributed in vertebrates that cause serious effects on human health. The history of Chlamydia infection in humankind dates back to 4000 years [1]. Chlamydia trachomatis is one of the most common sexually transmitted pathogens worldwide. A C. trachomatis infection can cause urethritis, cervicitis, proctitis, and conjunctivitis, depending on the anatomical site of the infection. 50% of men and 70% of women are asymptomatic [2]. When CT infection is not treated, it can cause infertility in men and women, such as epididymitis and pelvic inflammatory disease [3]. If C. trachomatis infection increases, it can cause endometriosis of the female upper genital tract, pelvic inflammatory disease and tissue scarring, infertility, ectopic pregnancies, and fibrotic disorder and is also potentially associated with cervical and uterine cancers [4].

Culture methods were first allowed as standards in the 1970s and were developed using McCoy cells. The specificity of this method, originally developed for trachoma and lymphogranuloma venereum (LGV), was 100%, but the overall sensitivity was estimated to be 40–85% lower due to laboratory sensitivity, sampling errors, and variable laboratory standards. In the 1980s, there was a return to the fashion for microscopic identification without culturing using a technique known as direct fluorescence testing, and testing was performed by binding specially produced antibodies to specific sites on the outer membrane of Chlamydia. It was used because it was quick and relatively inexpensive, with results being more variable compared to other methods, influenced by many factors, including the recognition of three strains or serotypes of C. trachomatis. Because of the revolutionization of the laboratory diagnosis of all diseases using monoclonal antibodies, thanks to many of the advances in molecular biology developed by new biotechnology companies, 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 growing doubt that this 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 such test was introduced by Roche in 1993. AMPLICOR C. trachomatis. An evaluation by doctors in Bordeaux the following year found it to have a sensitivity of 95.3% and a specificity of 100%, concluding that it was superior to culture methods. This was followed by other tests from other companies [1, 5].

Recent taxonomic developments based on 16S and 23S rRNA gene sequences, along with the development of molecular tests, have divided the Chlamydiaceae family into two genera and nine species, including 5 species found to infect humans [6]. Over time, new variants of C. trachomatis have emerged, and new test techniques that are inexpensive and easily applicable may emerge to identify them [7]. Chlamydia is the most commonly diagnosed bacterium among sexually transmitted diseases [8].

Infection diagnosis rates continue to increase in the developed world. Because Chlamydia is largely asymptomatic, screening is the most basic way to detect cases and reduce transmission [9].

2. Target genes used in the identification of chlamydia

Recent methods for typing Chlamydia have used the Chlamydiaceae 16S and 23S rRNA gene sequences. The gene targets most frequently used in the identification of Chlamydia are omp 1 (the gene encoding the major outer membrane protein-MOMP), cryptic plasmid, and 16S and 23S rRNA genes. The omp2 gene has been identified in all Chlamydiaceae except C. pecorum strains [6, 10, 11].

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 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, 10, 11, 17, 18, 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 high-throughput. 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 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].

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 target-specific 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].