Concordance Between Phenotypic Resistance to Fluoroquinolones and gyrA Mutations among Rifampin- Resistant Isolates of Mycobacterium Tuberculosis Complex from Pakistan

Abstract

Background: Fluoroquinolones (FQ) are the cornerstone of treatment for Rifampicin Resistant (RR TB). Here we investigate whether FQ resistance detected by line probe assay (LPA) shows good concordance with phenotypic susceptibility testing.
Methods: Mycobacterium tuberculosis isolates were collected from clinical samples received in clinical Microbiology Laboratory, Aga Khan University, Karachi between January 2016 and February 2017. RR TB isolates were grown in culture and those who
were resistant to ofloxacin (OFX) were selected for the present study. Minimum inhibitory concentrations (MICs) were performed for levofloxacin (LEV), moxifloxacin (MXF) and OFX using pre-prepared frozen plates (Thermo Fisher Scientific Inc., 194 Waltham, MA, USA). DNA was extracted using Genolyse® extraction kit (HAIN life sciences, Germany). For the detection of mutations, line probe assay Genotype MTBDRsl version 2 (HAIN Lifesciences, Germany) was used.
Results: From the total of 51 MDR TB stains that were included, majority of patients (n=29, 56.9%) were from Punjab province, 35.3% from Sindh province (n=18), and 5.9 %( n=3), and 1.9% (n=1) from Khyber Pakhtunkhwa and Baluchistan respectively.
Concordance between genotypic resistance detection by LPA, and the phenotypic resistance detection by MICs to FQ (any
one of OFX, LEV, or MXF) was observed in 84.3% of the isolates (n=43). The most common mutation identified was D94G in the gyrA gene in 50.9% of isolates (n=26). No gyrB mutations were detected. MIC testing showed high level of cross resistance between LEV/OFX and MFX MICS, with only 11/51 (21.6%) LEV/OFX resistant strains demonstrating MFX MICS of <1 μg/ml.
Conclusion: LPA method is a rapid and reliable method to identify resistan to FQ in MTB. However, for determination of susceptibility to individual FQs, further testing should be performed via phenotypic methods for confirmation.
Keywords: Rifampin resistant, Mycobacterium tuberculosis, Line probeassay, gyrA, Fluoroquinolones