Network Analysis of Ct-Derived Molecular Resistance Marker Pathways in Mycobacterium tuberculosis: Deciphering Drug-Resistance Architectures in Rural Eastern Cape, South Africa

Background Drug-resistant tuberculosis remains a major public health challenge, particularly in high-burden settings where rapid characterization of resistance patterns is important for surveillance. Molecular diagnostics routinely detect resistance-associated targets, but these are commonly evaluated individually. This study investigated whether cycle threshold-derived molecular marker-detection profiles form reproducible resistance-associated networks among culture-positive Mycobacterium tuberculosis specimens from the Eastern Cape, South Africa. Methods A retrospective laboratory-based molecular epidemiology study analysed 2,430 unique culture-positive M. tuberculosis specimens tested between January 2021 and December 2024. Binary detection indicators were generated for inhA , katG , gyrA1 , gyrA2 , gyrA3 , and rrs. Marker co-detection and resistance relationships were examined using phi correlation, association-rule mining, resistance co-occurrence analysis, and integrated network modelling with centrality metrics. Results Simultaneous detection of all six markers occurred in 95.8% (2,327/2,430) of specimens. Strong correlations were observed between gyrA1–gyrA2 (φ = 0.84), gyrA1–gyrA3 (φ = 0.84), gyrA2–gyrA3 (φ = 0.81), and inhA–rrs (φ = 1.00). The integrated network comprised 17 nodes and 88 statistically supported edges, with a density of 0.647. Second-line resistance had the highest degree and betweenness centrality, while amikacin, capreomycin, kanamycin, and composite injectable resistance formed a tightly interconnected module. Association-rule analysis identified recurrent multidimensional marker combinations associated with injectable resistance. Conclusions Cycle threshold-derived molecular markers and resistance outcomes formed interconnected co-detection and co-occurrence architectures rather than isolated patterns. Network analysis identified coordinated isoniazid/ethionamide, fluoroquinolone, injectable, and second-line resistance modules. These findings demonstrate the potential utility of network-based approaches for interpreting routinely generated molecular tuberculosis surveillance data, though validation with sequence-confirmed and longitudinal data is required.