Abstract:

Objective To evaluate the application value of fluorescent PCR probe melting curve method (probe melting curve) in detecting the resistance of MTB clinical isolated from elderly tuberculosis patients to first-line anti-tuberculosis drugs. Methods A retrospective study was conducted on 159 elderly pulmonary tuberculosis patients aged ≥65 years and admitted to Hebei Chest Hospital from September 2018 to October 2020. All the patients were definitely diagnosed of tuberculosis, and with positive signs of imaging examination, positive culture of BACTEC MGIT 960, and preliminary identification of Mycobacterium tuberculosis (MTB) by growth test of p-nitrobenzoic acid (PNB) differential medium.MGIT 960 drug sensitivity test and probe melting curve were used to detect the drug resistance to rifampicin, isoniazid, ethambutol and streptomycin,and mutation of MTB clinical isolates.The results of MGIT 960 drug sensitivity test were selected as the reference standard, the detection efficiency of the probe fusion curve method was evaluated, and the mutation sites of drug-resistance genes were sequenced in the strains with inconsistent results of the two drug sensitivity detection methods. Results Using the results of MGIT 960 drug sensitivity test as the standard, the sensitivities of MTB to rifampicin, isoniazid, ethambutol and streptomycin by probe fusion curve method were 98.31% (58/59), 96.72% (59/61), 87.50% (35/40) and 81.40% (35/43), respectively, the specificities were 97.00% (97/100), 95.92% (94/98), 81.51% (97/119) and 80.17% (93/116), respectively; the coincidence rates were 97.48% (155/159), 96.23% (153/159), 83.02% (132/159) and 80.50% (128/159), respectively; and the Kappa values were 0.973, 0.974, 0.751 and 0.785, respectively. There were 4, 6, 27 and 31 strains of MTB with different resistance to rifampicin, isoniazid, ethambutol and streptomycin detected by the two detection methods. Among them, there were 3, 4, 22 and 23 strains of MTB with drug resistance detected by the probe melting curve method, and drug resistance mutation sites were found in all the resistant strains by drug resistance gene sequencing. Conclusion The probe fusion curve method could quickly detect MTB drug-resistant mutations with high sensitivity and specificity, with good consistency to the results of MGIT 960 drug sensitivity test; it could be used as the basis for the early anti-tuberculosis programs in elderly tuberculosis patients.

Key words: Tuberculosis, multidrug-resistant, Polymerase chain reaction, Microbial susceptibility tests, DNA probes, Comparative study