## 3 Methods for _C. trachomatis_ Antimicrobial Susceptibility Testing  
Antimicrobial susceptibility testing for _C. trachomatis_ deviates from standard bacterriology, requiring assessment of its replication within cells via detection of intracellular inclusions [56; 57; 58]. Here, cell culture systems are used, predominantly with McCoy cells, although HeLa, HEp-2, and HL (and less commonly BGMK or Vero cells) are also used [56; 57]. Immunofluorescence staining identifies these inclusions, with optimal yields influenced by factors like high glucose, neutral pH, as well as elevated centrifugation temperatures (33-35 \({}^{\circ}\)C). Variables such as inoculum size and timing of antibiotic administration and removal alongside laboratory conditions like temperature and pH may affect results, though their impact is underexplored; however, the quality control of cell lines is essential [57]. Unlike natural infections where antibiotics act post infection and inflammation, in vitro testing applies drugs concurrently or soon after infection, potentially missing viable but non-infectious states. Determining minimal inhibitory concentrations (or MICs) in such cell cultures is complicated by subjective interpretation of inclusions, with MIC\({}_{\text{TP}}\) (transition point MIC) serving as a marker for the concentration at which 90% or more of the inclusions exhibit abnormal morphology [56; 58]. This process can be further enhanced by employing a secondary passage of the culture onto fresh cells without the presence of the antimicrobial, aiming to ascertain the minimal chlamicidal concentration (MCC), indicative of complete organism eradication. Such culture-based approaches are instrumental not only in quantifying the count of infectious EBs, known as recoverable inclusion-forming units (IFU), but also in evaluating the antimicrobial's efficacy in preventing the formation of inclusions. Still, it has to be noted that the interpretation of elevated MICs in _C. trachomatis_ is very cumbersome without breakpoints or MIC distribution data for wild-type strains and those after treatment failure [56]. And although there were early attempts to standardize the process, the approach can vary substantially between different laboratories [59]. Alongside this classic approach in McCoy cells and MIC determination, a research team from China recently introduced the fractional inhibitory concentration (FIC) method, using a checkerboard assay [60]. This was pursued to evaluate the interaction of antibiotic combinations (i.e., azithromycin plus moxifloxacin, azithromycin plus minocycline, or minocycline plus moxifloxacin) against C. _trachomatis_ by serially diluting one antibiotic along the ordinate and the other along the abscissa of 96-well plates, overlaying inoculated wells with both drugs in two-fold dilutions. In their approach, FIC was calculated as the sum of FIC A and FIC B, where the latter two can be seen as the MICs of each drug in combination divided by their MICs alone, with synergy defined as FIC \(\leq\) 0.5, indifference as 0.5 \(<\) FIC \(<\) 2, and antagonism as FIC \(\geq\) 2 [60]. However, evidence points to the fact that identifying resistance genes may better account for the high treatment failure rates in urogenital _C. trachomatis_ infections compared to MIC results, emphasizing the importance of genetic testing for antimicrobial resistance in affected patients [47]. This is why, for some time now, there has been an emphasis on molecular methods for the purposes of _C. trachomatis_ susceptibility testing. And one of the pioneering studies in using molecular approaches for antimicrobial susceptibility testing of _C. trachomatis_ was the work performed by [PERSON] and co-authors [61], who introduced and evaluated a reverse transcriptase-PCR (RT-PCR)-based method for comparing it to the conventional method of cell culture and immunofluorescence (IF). The RT-PCR method aimed to improve sensitivity and reduce subjectivity in determining MICs. _C.