1. Introduction
Sexually transmitted infections (STIs) remain a significant global health concern, with Chlamydia trachomatis (CT) standing out as one of the most prevalent bacterial pathogens. Global reports from the World Health Organization (WHO) highlight a concerning rise in STI cases, placing Chlamydia trachomatis, alongside Neisseria gonorrhoeae, at the forefront, each estimated to cause approximately 106 million new infections annually [1]. In the United States alone, reported chlamydial infections reached a staggering 1,441,789 cases in 2014, marking the highest number since record-keeping began in 1984. The period between 2004 and 2014 witnessed a substantial increase in the reported chlamydial infection rate, escalating from 316.5 to 456.1 cases per 100,000 population [2]. Data from the European Centre for Disease Prevention and Control (ECDC) also corroborate this alarming trend, indicating a surge in notified CT infections across Europe, from 191,000 in 2004 to 385,000 in 2013, corresponding to incidence rates of 162.8 and 181.8 per 100,000 inhabitants, respectively [3]. It is crucial to acknowledge that the actual burden of new infections is likely even greater, as many chlamydial infections are asymptomatic and, therefore, go undetected.
Several factors contribute to the observed increase in diagnosed CT infections. These include shifts in sexual behavior, gaps in prevention and education efforts, and, importantly, the advancements in diagnostic capabilities leading to more frequent and effective testing. The introduction of molecular techniques, specifically nucleic acid amplification tests (NAATs), has significantly enhanced both the sensitivity and specificity of STI testing. This review aims to provide a comprehensive overview of the laboratory tests currently employed for the detection of CT infections. The selection of appropriate diagnostic tests and their subsequent diagnostic value are intrinsically linked to the unique biological characteristics of the pathogen and the diverse clinical presentations of CT infection, which will be briefly discussed initially.
2. Pathogenesis, Genotypes, and Clinical Manifestations of Chlamydia
Chlamydia are obligate intracellular bacteria, distinguished by their unique intracellular lifestyle and replication mechanisms within host cells [4]. Their life cycle involves two distinct forms: the replicative reticulate bodies (RB) and the infectious elementary bodies (EB). Extracellular EBs initiate infection by targeting host cells, mediated by interactions between bacterial outer membrane proteins (MOMP, OmcB, PmpD) and host cell receptors, such as heparan sulfate proteoglycans, mannose-6-phosphate receptor, and growth-factor receptors. Upon internalization, EBs reside within membrane-bound inclusions, which express bacterial inclusion proteins that prevent fusion with lysosomes. These inclusions are then transported along microtubules to microtubule organizing centers (MTOC), where EBs differentiate into RBs. RBs multiply through binary fission and subsequently re-differentiate into EBs. The cycle concludes with the liberation of EBs from inclusion bodies through cell lysis or extrusion, enabling infection of new host cells [5].
The host immune system recognizes the presence of chlamydia through pattern recognition receptors (PRRs) of the innate immune system. PRRs identify pathogen-associated molecular patterns (PAMPs), triggering both innate and adaptive immune responses aimed at pathogen elimination. However, the inflammatory response to chlamydia is often subdued, leading to many infections remaining asymptomatic. Chlamydia employs various mechanisms to modulate host defenses, including reduced antigen presentation and inhibition of genes involved in cellular immunity, as well as inducing anti-apoptotic effects in infected host cells. These strategies contribute to the potential for persistent infections. During cellular immune responses, interferon-gamma (IFNy)-induced indoleamine 2,3-dioxygenase leads to a reduction in intracellular tryptophan levels. As chlamydia are tryptophan auxotrophs, this stress response triggers the development of morphologically aberrant, non-replicative, persistent forms, which are believed to revert to replicative forms when environmental conditions become favorable again [4].
C. trachomatis is further categorized into different serovars or genotypes, each associated with distinct clinical manifestations. Genotypes A–C are predominantly linked to ocular infections. Acute infections manifest as conjunctivitis, which, if untreated, can become chronic and progress to trachoma. While less common in Europe and North America, these infections remain a significant cause of blindness in Africa and Asia [6]. Genotypes D–K are primarily responsible for infections of the urogenital tract, rectum, pharynx, and conjunctiva [7]. Perinatal transmission can also result in conjunctivitis, pharyngitis, and pneumonia in newborns [8]. Many rectal and pharyngeal infections, as well as a substantial proportion of lower genital tract infections, are asymptomatic. However, both symptomatic and asymptomatic infections can lead to serious sequelae, particularly in women. Ascending infections can cause chronic inflammation in the upper genital tract, resulting in pelvic inflammatory disease (PID), which is associated with increased risks of ectopic pregnancy and tubal factor infertility (TFI) [9]. To mitigate the incidence of CT infections and their complications, numerous countries have implemented screening programs to proactively identify and treat asymptomatic infections in affected individuals and their partners [10].
In contrast to genotypes A–K, which typically remain confined to the mucosal epithelium, genotypes L1, L2, and L3 possess the ability to penetrate the epithelium, disseminate via the lymphatic system, and cause invasive infections known as lymphogranuloma venereum (LGV). LGV is endemic in regions of Africa, Asia, South America, and the Caribbean. However, outbreaks of genotype L2 have been reported among men who have sex with men (MSM) in Europe, North America, and Australia since 2003 [11]. These patients predominantly experience anorectal symptoms, differing from the classical inguinal syndrome seen in sporadic LGV cases [12]. In 2013, over 1000 cases were reported to the European Centre for Disease Prevention and Control (ECDC), with approximately 50% originating from the UK [3]. The actual number is likely higher, as LGV is not routinely reported in many European countries. Furthermore, LGV infections are not always symptomatic, contributing to persistently high incidence rates [13].
3. Diagnostic Procedures for Chlamydia Detection
Chlamydia testing is indicated for individuals presenting with urogenital, anorectal, and ocular symptoms, those diagnosed with other STIs, sexual contacts of individuals with STIs, and individuals targeted for chlamydia screening programs [7]. Diagnostic procedures for detecting CT infections encompass both direct and indirect methods. Generally, localized infections are assessed using assays for direct pathogen detection, including culture, antigen tests (EIA, direct fluorescent antibody (DFA), and immune chromatographic RDTs), nucleic acid hybridization, and amplification tests. Indirect methods rely on the detection of antibodies against C. trachomatis, which are useful in the diagnostic evaluation of chronic/invasive infections (PID, LGV) and post-infectious complications, such as sexually acquired reactive arthritis (SARA). In these conditions, pathogens may have crossed the epithelial barrier and may no longer be detectable in swabs. Conversely, serology is not appropriate for diagnosing acute infections of the lower genital and anal tracts because the antibody response typically becomes detectable only weeks to months after infection and is often less pronounced.
4. Isolation of C. trachomatis in Cell Culture
Established cell lines for C. trachomatis isolation include McCoy, HeLa 229, and Buffalo Green Monkey Kidney cells. Suitable specimens for culture include swabs from various anatomical sites (endocervix, urethra, anal canal, conjunctivae), but these must be collected using specific devices and transport media [14]. Specimens are centrifuged onto confluent cell monolayers and analyzed for the development of characteristic intracytoplasmic inclusions after 48–72 hours, typically by staining with Giemsa, iodine, or fluorescence-labeled antibodies targeting chlamydial antigens (LPS or MOMP). Using MOMP-specific antibodies for staining cell culture enhances detection specificity [15], making it long considered the reference test for CT detection. However, culture relies on viable organisms, and its detection rate is, at best, 60%–80%, even in experienced laboratories [16]. Sensitivity can be compromised by inadequate specimen collection, storage, and transport, the presence of toxic substances in clinical specimens, and overgrowth of cell cultures by commensal microbes. Additional drawbacks include the extended turnaround time, labor intensity, and challenges in standardization. Consequently, cell culture is now rarely used in routine diagnostic laboratories. However, the methodology remains essential, particularly in reference laboratories, for monitoring antibiotic susceptibility, virulence changes, or when the highest specificity is required, such as in cases of suspected sexual assault.
5. Nucleic Acid Amplification Tests (NAATs): The Gold Standard for Chlamydia Diagnosis
Nucleic acid amplification tests (NAATs) are recognized as the most sensitive methods available for chlamydia diagnosis. These tests offer high specificity, comparable to cell culture, but crucially, they do not require viable pathogens, simplifying specimen transport and handling. Consequently, NAATs are widely considered the gold standard for chlamydia detection, having replaced culture as the primary diagnostic method [14,17]. Antigen tests, including EIA, DFA, and RDTs, are no longer recommended for routine chlamydia testing due to their insufficient diagnostic accuracy [14,17].
Most NAATs are based on polymerase chain reaction (PCR) technology and utilize fluorescence-labeled probes for real-time detection of amplification products, significantly reducing test duration. When combined with automated nucleic acid extraction, results can be generated within a few hours. Numerous studies have demonstrated high concordance between different NAATs [18,19]. Occasional discordant results may arise from variations in analytical sensitivity, nucleic acid isolation efficiency, and genetic variability within chlamydia genomes [20]. The importance of genetic variation was highlighted by the emergence of the Swedish variant (C. trachomatis strain E/SW2), which went undetected by some commercial CT NAATs due to a deletion in their target region [21]. Furthermore, gene regions can be exchanged through recombination when host cells are co-infected with multiple CT strains [22,23]. While the intracellular lifestyle of chlamydia presents a barrier to genetic recombination, genome sequencing of various CT strains indicates that horizontal gene transfer is not uncommon. Recombination primarily reflects adaptation to changing environmental conditions but can also lead to the development of new variants with increased virulence [23]. Recombination is also relevant to laboratory diagnostics, especially for nucleic acid-based tests. The incorporation of a second target region in NAATs (dual-target assays) represents a significant advancement, enabling the detection of new variants with deletions or recombination in one of the target regions [24].
Diagnostic sensitivity has also been enhanced by improved pre-analytical steps. The use of coated magnetic beads has improved nucleic acid isolation, resulting in higher quantity and quality [20]. These bead-based extraction systems can be automated and are integrated into high-throughput systems that allow for simultaneous, highly sensitive and specific testing for both chlamydia and gonococci [25].
In populations with low chlamydia prevalence, the positive predictive value of even highly specific tests decreases. Consequently, in 2002, the Centers for Disease Control and Prevention (CDC) recommended confirming positive NAAT results with a second test to avoid unnecessary treatment and potential psychosocial distress. However, subsequent studies with improved NAATs revealed that non-confirmed positive NAAT results are not necessarily false positives but may represent false negative results from confirmatory tests, particularly if the latter has lower analytical sensitivity [26]. In samples with low chlamydia loads, stochastic distribution of target DNA can lead to some aliquots having concentrations below the limit of detection (LOD). Generally, confirmation of positive NAAT results is no longer routinely required or recommended by the CDC, except in legal investigations, such as cases of sexual assault. In such scenarios, the serious consequences of false positive results necessitate tests with the highest possible specificity. While NAATs have been shown to be more sensitive than culture in detecting CT infections in sexual assault victims [27], positive results used as evidence of sexual abuse must be confirmed with another NAAT targeting a different genomic region [28].
6. Clinical Specimens for Chlamydia Testing
NAATs are versatile and can be used to analyze a wide range of clinical specimens, including urethral, cervical, vulvo-vaginal, anorectal, and ocular swabs, first-void urine (FVU), sperm, and tissue samples. FDA-cleared commercial NAATs are approved for first-void urine, urethral and cervical swabs, and most also include vaginal swabs in their approvals [14]. Non-invasive specimens are particularly preferred for screening asymptomatic individuals. For male patients, FVU and urethral swabs demonstrate equivalent performance in CT NAATs. However, urine collection is generally more acceptable to patients, making it the recommended sample type for men [17,29,30]. It is crucial to collect the first portion of micturition (approximately 20 mL) as chlamydia concentration decreases significantly during urination [31]. In contrast, women with urogenital infections tend to have higher chlamydia concentrations in genital swabs compared to urine [32]. A study comparing urine, vaginal, and cervical swabs collected simultaneously from asymptomatic women found that NAAT detection rates were highest in self-collected vaginal swabs [33]. Therefore, vaginal swabs (self-collected or clinician-collected) are the recommended sample type for women. Endocervical swabs are also suitable, particularly when a pelvic examination is clinically indicated. FVU is less sensitive for CT testing in women, and its use for NAAT screening should consider a detection rate up to 10% lower than vaginal and endocervical swabs [14].
Detection of extragenital CT infections (conjunctivitis, anorectal or pharyngeal infections, including LGV) requires testing of corresponding swabs or tissue samples. CT infections in MSM are frequently localized in the rectum or pharynx and are often asymptomatic. Screening urine samples alone would miss the majority of these infections [34,35], highlighting the necessity for testing oral and anal swabs for accurate diagnosis. Commercial NAATs are not always approved for testing non-genital sample types. However, numerous studies have demonstrated that NAAT detection of CT in these specimens is superior to culture or antigen tests [36,37,38,39]. Testing specimens outside the approved uses of commercial tests requires validation of test performance characteristics according to quality assurance guidelines for microbiological diagnostics, such as CLIA (Clinical Laboratory Improvement Amendments) in the US. This also applies to NAAT testing of tissue samples, such as endometrial samples or lymph node biopsies, which may be considered in patients with PID or LGV, respectively. Furthermore, LGV confirmation necessitates identification of genotypes L1, L2, or L3. LGV typing is clinically important as the recommended antibiotic treatment is longer (Doxycycline 200mg/day for at least 3 weeks versus one week for non-LGV cases) [40]. Typing methods include genotype-specific PCRs and RFLP or sequence analysis of relevant omp1-gene regions [11,41,42]. LGV and non-LGV strains can also be differentiated using PCR tests based on the pmpH gene, which contains a 30bp deletion in all LGV strains [42,43]. While the pmpH PCR assay does not identify specific genotypes, its sensitivity for LGV is high. However, it misses approximately 25% of non-LGV infections [44]. Therefore, the assay is a useful adjunct test for identifying LGV in CT-positive samples but should not be used as a primary diagnostic test [44].
7. Rapid Diagnostic Tests (RDTs) for Point-of-Care Chlamydia Diagnosis
In addition to diagnostic accuracy, the turnaround time for chlamydia test results is crucial for timely treatment initiation. Traditional NAATs are often performed in centralized laboratories, requiring specimen transportation and result transmission to clinicians. This process can necessitate a second patient visit, potentially delaying treatment or leading to no treatment if patients do not return, contributing to persistent high infection rates. Rapid diagnostic tests (RDTs) offer a solution by enabling near-patient (point-of-care) testing, providing results within minutes. This allows for immediate antibiotic therapy for positive patients. Most RDTs are immune chromatographic tests based on lateral-flow technology, detecting chlamydia LPS antigen in genital swabs or urine. However, compared to culture and PCR, these antigen-based RDTs exhibit significantly lower sensitivity and specificity. A study conducted in Maastricht (The Netherlands) using self-collected vaginal swabs from 772 patients reported sensitivities of 11.6%–27.3% for three chlamydia RDTs, using PCR as the reference test [45]. The low sensitivity of RDTs might be attributed to lower bacterial loads in asymptomatic patients. Even when testing endocervical swabs from symptomatic patients, RDT sensitivities were only 22.7%–37.7% [46]. One exception is an RDT developed at the University of Cambridge (CRT, Diagnostics for the Real World), which showed better performance. Based on combined data from four studies, its sensitivity for first-void urine and vaginal swabs was reported as 77% and 80%, respectively, each with a specificity of 99% [47]. However, subsequent studies have not consistently replicated these findings, reporting sensitivities of 41.2% and 74.2% for vaginal swabs [48,49] and 41.4% and 20% for male FVUs [49,50]. Consequently, antigen-based RDTs are generally not recommended for CT testing in either asymptomatic (screening) or symptomatic patients.
In contrast to antigen-based RDTs, molecular RDTs employing nucleic acid amplification techniques demonstrate high diagnostic accuracy, comparable to standard NAATs [51]. The Xpert assay from Cepheid was the first commercially available rapid NAAT, providing point-of-care testing for CT and gonococci in approximately 90 minutes. This assay utilizes real-time PCR within a closed system. After sample application to a cartridge, subsequent steps, including nucleic acid isolation, amplification, and PCR product detection, are fully automated. To maximize immediate treatment initiation, patients need to be willing to wait for the 90-minute result turnaround [51]. Newer molecular RDTs are emerging with even faster turnaround times. The Io POC-test Chlamydia (Atlas Genetics), based on PCR in microfluidic systems and electrochemical detection of PCR products, provides results in about 30 minutes [52,53] and has been approved in Europe since February 2016. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA), are even faster than PCR and may further enhance the utility of NAATs at the point of care [54]. It is important to note that NAAT-based rapid diagnostic tests are subject to quality assurance requirements for microbiological diagnostics, typically met by certified laboratories but more challenging to implement in point-of-care settings.
8. Serology in Chlamydia Diagnosis: Limited Role
Serological testing for chlamydia antibodies is not useful for diagnosing localized epithelial infections of the lower genital tract. This is due to several limitations, including a delay of several weeks before antibodies become detectable, potentially low antibody titers, and the inability of many serological tests to differentiate between antibodies against different chlamydia species. However, serology can be valuable in diagnosing chronic and invasive infections (PID, LGV, SARA). In many of these cases, bacteria may be undetectable in anogenital swabs or urine, and serological data can support the diagnosis of chlamydial infection. Persistent CT infections and complications of ascending infections are typically associated with a positive antibody response, making negative serology helpful in ruling out chlamydial involvement. Conversely, positive serology alone does not definitively prove active chlamydial infection.
Microimmunofluorescence (MIF) testing was historically considered the reference method for chlamydia antibody detection and has been used to diagnose neonatal C. trachomatis pneumonia. A serum IgM titer of 1:32 or greater is considered diagnostic of infection. IgG testing is less useful as it may reflect passively transferred maternal antibodies. However, current guidelines from the Centers for Disease Control and Prevention (USA), the British Association of Sexual Health and HIV (BASHH), and the International Union against STI (IUSTI) do not recommend serological testing for diagnosing infant pneumonia. Furthermore, MIF testing is time-consuming, labor-intensive, and prone to subjective interpretation of fluorescence signals [55]. Consequently, enzyme immunoassays (EIAs) and immunoblots or line assays are now more frequently used for detecting chlamydia antibodies [55,56]. Interpretation of serological results is complicated by cross-reactivity among chlamydia species pathogenic to humans and non-pathogenic environmental species. Some EIAs based on LPS antigen detection do not differentiate between chlamydia species. While chlamydial LPS is generally considered a genus-specific antigen, cross-reactivity with antibodies against LPS from other Gram-negative bacteria has been reported [57,58]. The diagnostic performance of chlamydia antibody testing has been improved by using species-specific proteins or peptides. Immunogenic proteins of C. trachomatis identified by 2D PAGE [56] have been incorporated, along with analogous proteins from C. pneumoniae and C. psittaci, into commercial line assays, enabling differential evaluation of Chlamydia antibody reactivity.
Recent advancements include the development of a proteome array containing GST fusion proteins representing 908 of the 918 known ORFs of the CT genome. Using this array, 27 immunodominant proteins reactive with >50% of human sera from confirmed CT infection cases were identified [59]. In another study, the proteome array was used to compare antibody profiles of patients with tubal factor infertility (TFI) and normal fertility [60]. Five proteins were found to be reactive exclusively with sera from TFI patients. Reactivity against these proteins was also detected in some sera from acute infection patients, but these sera showed additional reactivity against other proteins not observed in sera from chronic infection patients. These findings are promising, suggesting that distinct antibody reactivity patterns in acute and chronic CT infection may provide markers for different stages of infection [60].
9. Conclusions: NAATs Revolutionize Chlamydia Diagnosis
Chlamydia trachomatis is an obligate intracellular bacterium that infects epithelial cells and fibroblasts. Its interactions with host factors and commensal microbes lead to variable replication rates within infected host cells, often very low in asymptomatic and persistent infections. Consequently, direct detection of C. trachomatis necessitates highly sensitive diagnostic tests. Among available techniques, nucleic acid amplification tests (NAATs) stand out as the most sensitive. Their high specificity, comparable to culture, positions NAATs as the method of choice for C. trachomatis detection. A wide array of NAATs, including commercial assays and in-house protocols, are available for laboratory use, and selection should be guided by sample volume, automation requirements, and cost considerations. Regardless of the chosen NAAT, performance characteristics must be rigorously evaluated according to quality assurance standards for microbiological diagnostics. As NAATs are typically performed in centralized laboratories, sample transport and result reporting can delay treatment initiation, often requiring a second patient visit. To address this, rapid diagnostic tests (RDTs) have been developed to provide quick, easy-to-perform, point-of-care testing. However, antigen-based rapid tests have demonstrated insufficient sensitivity and specificity. Emerging rapid tests based on NAAT technology offer significantly improved performance, comparable to standard NAATs. These fully automated systems, independent of centralized laboratories, hold great promise for enhancing point-of-care testing for Chlamydia trachomatis infections in the future, facilitating immediate diagnosis and treatment.
Conflicts of Interest
The author declares no conflict of interest.
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