Culture-independent prediction of isoniazid resistance in Mycobacterium tuberculosis by katG gene analysis directly from sputum samples

Background: The molecular prediction of isoniazid (INH) resistance in Mycob acterium tuberculosis is hampered by the need for specialized equipment, ex pertise, high costs, a limited range of detectable mutations, or several of these factors. The rationale for the study was to find a practical alterna tive and to demonstrate generally valid problems. Methods and Results: DNA extracted from decontaminated sputum pellets was u sed to amplify a 0.26 kb target sequence within the katG gene. Mutations of codon 315, frequently found in isoniazid-resistant isolates, could be disc riminated in a simple agarose minigel format following an AciI digest of th e nested polymerase chain reaction (PCR) product. Within a panel of 22 sput um samples, INH resistance could be predicted in 5 of 10 samples containing isoniazid-resistant M. tuberculosis. The protocol is robust, requires litt le expertise and no specialized equipment, and provides the test results wi thin 2 days. Conclusion: The results show the feasibility to rapidly and easily detect m utations highly predictive of isoniazid resistance. Nevertheless, this, lik e any other molecular resistance prediction test, is affected by often negl ected factors, including mutation prevalences, the phenomenon of heteroresi stance, and a possible bias toward one's own method.