## 3 Methods for _C. trachomatis_ Antimicrobial Susceptibility Testing
trachomatis_ still has to be cultured in McCoy cells with varying concentrations of antimicrobials, and after 48 h, RNA can be extracted, so RT-PCR targeting the _DnaK_ gene can be performed [61]. The MIC is then determined as the lowest concentration of antibiotic that inhibited the appearance of a PCR product. [PERSON] et al. showed that the RT-PCR method consistentlyyielded higher MIC values compared to IF, indicating greater sensitivity, and suggested that RT-PCR detects viable Chlamydia at lower levels of replication than IF [61]. Furthermore, it may even detect aberrant forms of Chlamydia that IF misses. Hence, it can be seen as a more sensitive and objective method for assessing _C. trachomatis_ susceptibility, particularly for detecting low-level replication and potential persistence. The application of PCR methods extends beyond mere detection, encompassing the direct quantification of _C. trachomatis_ from clinical specimens and offering a more nuanced understanding of the bacterial load. This is exemplified in the work of a Hungarian research group that pioneered a direct qPCR-based methodology [58; 62]. Their innovative approach bypasses the conventional nucleic acid purification step, instead opting for a straightforward lysis of infected host cells through freeze-thaw cycles, followed by the direct application of EvaGreen-based qPCR on these lysates. This method has proven effective in assessing the impact of various antibiotics, antiseptic, and growth-promoting compounds on _Chlamydia_, showcasing its versatility [58; 62]. A multicentric research team from the Netherlands, U.S., and India developed a specific _235 rRNA_ gene PCR assay to detect known macrolide resistance-associated mutations (RAMs) directly from urogenital and rectal samples, bypassing the need for prior culture and thus offering a high-throughput, less technically demanding alternative to _C. trachomatis_ culture for genotypic resistance surveillance [63]. The assay's suitability for clinical samples was validated through systematic in silico analysis and specificity testing, showing no false positives in 40 _C. trachomatis_-negative swabs (20 vaginal and 20 rectal) or with a specificity panel of bacteria except for _C. suis_ and _Chlamydia muridarum_; however, clinical sample testing confirmed detection was specific to _C. trachomatis_ sequences [63]. With an analytical sensitivity consistently detecting the _C. trachomatis_ _235 rRNA_ gene at \(10^{3}\) IFU/mL, the assay demonstrates strong performance for direct susceptibility screening. Very recently, a team from Portugal used a multiplex NGS-based approach, focusing on detecting resistance markers in the _235 rRNA_ (mutations at A2057, A2058, and A2059 for macrolide resistance) and _gyrA/parC_ genes (QDRR substitutions for fluoroquinolone resistance) [64]. The amplification is carried out via nested and multiplex PCR and further sequenced with Sanger and Illumina NextSeq 550 platforms before tools like INSaFLU and MEGA11 can be used against reference strains. The benefit of their approach is also the possibility to survey genomic diversity through pmpH, CT105, and CT442 loci, identifying strain-specific insertions and phylogenetic relationships and in turn providing very detailed insights into resistance mechanisms and genomic backgrounds of _C. trachomatis_[64]. It is already evident that, in the future, whole-genome sequencing may offer insights into _C. trachomatis_ strain evolution and the pertaining AMR patterns. [PERSON] et al. [65] evaluated four methods using positive clinical samples and showed how target enrichment with custom SureSelect probes targeting _C. trachomatis_, followed by Illumina sequencing, can successfully produce whole genomes from 64% of positive samples. By enabling the analysis of circulating strains and genotypic resistance, it may indeed be a promising tool for susceptibility testing, surveillance, and subsequent public health strategies. There are also some alternative approaches, but their use has not fully materialized in quotiian practice.