Molecular Detection of Multidrug-Resistant Strains: Isoniazid Resistance

The escalation of drug-resistant strains (e.g., MDR-, XDR-, TDR-TB) combined with fear of global out-of-control transmission has prompted the investigation into faster molecular methods to detect resistance other than the methods of laboratory diagnosis and susceptibility testing approved and standardized in the United States since the 1960s. Developing countries, desperate for technologies to identify the increasing number of resistant strains of MTB, are employing methods that detect rifampin (RIF) and isoniazid (INH) resistance.

• During the 1990s, mutations of genes associated with resistance in INH and RIF were identified.
• INH resistance appears more elusive because of its complex group of related gene mutations (katG, inhA, ahpC, and ndh) and 80% to 90% involvement.
• RIF resistance, on the other hand, has been found to have a single gene region, rpoB, which is associated with 95% of its resistance.

In 2003, researchers from the California Department of Public Health Services published a paper to verify previous studies detecting both INH and RIF with molecular beacons (MB). Results proved the sensitivity of the MB assay for INH (target sequence katG and inhA) was unacceptably low at 82.6% (105 out of 127 isolates) and showed no mutations in 22 of the isolates. However, the RIF sensitivity of the method was an acceptable 97.5%. Tests for both drugs were highly specific (for MTBC) at 100%.³⁰

In 2011, techniques using multiplex allele-specific polymerase chain reaction (MAS-PCR) assays to target katG and inhA regions were described by workers in two separate studies (University of Oxford Clinical Research Unit, Ho Chi Minh City, Viet Nam, and Queen Mary Hospital, the University of Hong Kong, China) for rapidly screening INH resistance, with sensitivity and specificity of 90% and 100% respectively. The first small study included 100 INH-resistant and 50 susceptible isolates, with the sensitivity and specificity of cultured isolates recorded as 90% and 100%, respectively. In the second MAS-PCR study, 203 MTB isolates and 487 respiratory specimens had less striking results for sensitivity—83% for the cultured strains and 79% for the direct respiratory specimens.