Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results

Abstract

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST).We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay.Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation.Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays.

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

“…rpoB encodes for DNA-dependent RNA polymerase, and mutations within rpoB can result in rifampicin resistance (44,45). dnaK encodes heat shock protein that is the most abundant protein within Mtb and plays a crucial role in preventing partially misfolded proteins from aggregating by binding to hydrophobic residues, which then allows these proteins to refold (46).…”

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…Confirmation of these lower critical concentrations have come from clinical studies [209][210][211][212][213] and several investigations [214][215][216][217][218][219][220][221] of drug resistance-associated mutations in isolates with MICs below the standard breakpoint. Clinical studies using assumption-free methods such as machine learning have confirmed that these values (table 3) are in fact the MICs above which tuberculosis patients fail combination therapy.…”

“…Additionally, studies have been done in which treatment failed in patients who were infected with M tuberculosis who had drug target site mutations, but MICs were lower than current breakpoints, and were more consistent with the newly proposed breakpoints. [214][215][216][217][218][219][220][221] Therefore, the proposed concentrations have higher sensitivity for clinical decision making, and should be used to estimate the global burden of MDR tuberculosis.…”

Diagnosing Drug-Resistant Tuberculosis with the Xpert<sup>®</sup>MTB/RIF. The Risk for Rifampin Susceptible Cases

“…For example, in a report from Haiti, 86.9% of patients with rifampin resistance detected by the Xpert®MTB/RIF were later proved to have MDR-TB due to simultaneous resistance to isoniazid (INH) [8], while a report from Zimbabwe found that 85.2% of patients detected with the Xpert®MTB/RIF as resistant to rifampin were actually MDR [9].…”