Evaluation Of Phenotypic and Genotypic Methods for Detection of Multidrug Resistant Tuberculosis in Pakistan

Abstract

Objective: To evaluate the phenotypic and genotypic approaches for the diagnosis of drug-resistant Mycobacterium Tuberculosis.
Study Design: A cross-sectional study.
Place and Duration of Study: This multicenter study was conducted over a period of six months, from April 01, 2025, to September 30, 2025, in collaboration with several healthcare and academic institutions across Pakistan. The study activities were carried out in the Biosafety Level 3 (BSL-3) Laboratory at the Department of Microbiology, Health and Population Department, Lahore; Tehsil Headquarters (THQ) Hospital Jand, district Attock; Health Services Academy, Islamabad; Islamabad Medical and Dental College, Barakaho; Metropole Laboratories, Islamabad; and Sindh Medical College, Karachi.
Materials and Methods: Eighty-Seven culture-positive samples of Mycobacterium Tuberculosis (MTB) isolates were evaluated for susceptibility to first-line anti-tuberculosis medications such streptomycin, isoniazid, ethambutol, and rifampicin, by using the Mycobacteria Growth Indicator Tube (MGIT) 960 system. The GeneXpert for MTB was used for its rapid detection and rifampicin resistance. Multiplex polymerase chain reaction (PCR) assays were optimized to amplify mutations in the rpoB gene associated with rifampicin
resistance, the katG gene associated with isoniazid resistance, and the embB gene associated with ethambutol resistance. The amplified PCR products were analyzed by agarose gel electrophoresis to determine the banding patterns associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) MTB isolates.
Results: Among the 84 MTB isolates, 30 samples were single-drug resistance (SDR), while 4 samples were identified as MDR. Among the SDR isolates, resistance to isoniazid was the most common (n = 24), followed by7 ethambutol (n = 3) and rifampicin (n = 2). Three non-MTB samples (negative, positive, and normal controls) were included as controls. Additionally, 50 samples were identified as Mycobacteria Other Than Tuberculosis (MOTT).
Multiplex PCR analysis included the following diagnostic performance parameters for MDR: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy (DA) and it demonstrated 66.67%, 100%, 33.33%, 71.43%, and 80% for multidrug resistance; 66.67%, 100%, 50%, 75%, and 85.71% for isoniazid resistance; 80%, 96%, 50%, 84.50%, and 100% for rifampicin resistance; and 50%, 75%, 100%, 80%, and 80% for ethambutol resistance, respectively.
Conclusion: Genotypic methods for diagnosing drug-resistant MTB were faster than phenotypic methods to address important public health challenges in countries like Pakistan.