荧光PCR熔解曲线法直接检测标本中结核分枝杆菌对氟喹诺酮类药物耐药性的研究OA

This study evaluated the feasibility and clinical utility of fluorescence PCR melting curve analysis for direct detection of fluoroquinolone(FQ)resistance in Mycobacterium tuberculosis(MTB)from clinical specimens.Clinical specimens and relevant clinical data from patients at our hospital and county-level hospitals were collected in 2024.Each specimen was divided into two parts:one was used for MGIT liquid culture followed by phenotypic drug susceptibility testing(DST)with the broth microdilution method,whereas the other was used for detection of FQ resistance-associated mutations with fluorescence PCR melting curve analysis.Among 211 MTB isolates,17(8.06%)were resistant to ofloxacin and moxifloxacin according to phenotypic DST,and fluorescence PCR melting curve analysis indicated that 18(8.53%)were mutants(resistant).The sensitivity,specificity,accuracy,and Kappa value of the fluorescence PCR melting curve analysis were 82.35%,97.94%,96.68%and 0.78,respectively,with phenotypic DST as the reference standard.Regardless of the bacterial load in the specimen,the sensitivity of the fluorescence PCR melting curve analysis exceeded 70.00%,the specificity and accuracy exceeded 90.00%,and the Kappa values exceeded 0.60.With increasing bacterial load,the Kappa value increased.Clinically,FQ-resistant patients had elevated likelihood of a history of prolonged FQ exposure(P<0.01)and retreatment(P<0.01),as well as severe disease manifestations,including high rates of multidrug-resistant tuberculosis(MDR-TB)and cavitary lesions on chest CT(P=0.012).The fluorescence PCR melting curve method directly and rapidly detected FQ resistance in MTB from clinical specimens,with excellent diagnostic performance.Combination of this method with phenotypic DST increased the accuracy of detection and may aid in the clinical formulation of reasonable individualized treatment plans.