Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation ؊15C3T in the promoter of the mabA-inhA operon.Isoniazid (INH) together with rifampin (RIF) form the cornerstone of a short chemotherapy course for tuberculosis, and resistance to either drug hampers seriously the complete cure of patients (17). Mutations at several chromosome loci of Mycobacterium tuberculosis (fur-katG, mabA-inhA, oxyR-aphC, kasA, and ndh) are associated with resistance to INH (6,7,11,13). Mutations both at the Ser315 codon of katG, encoding catalase-peroxidase, the enzyme oxidizing INH (4), and at the regulatory region of the mabA-inhA operon, encoding a target of activated prodrug, enoyl-acyl carrier protein reductase (2), occur most frequently in INH-resistant isolates (9,11,13). In this study we searched for the most common mutations in katG and the mabA-inhA regulon of INH-resistant isolates to evaluate their significance for rapid drug resistance prediction in Lithuania.In total, 364 INH-resistant and 78 INH-susceptible M. tuberculosis isolates collected from different patients (93 female, 349 male; age range, 19 to 94 years) in the National Tuberculosis Reference Laboratory from 1998 to May 2002 were studied. New and previously treated tuberculosis cases were defined according to World Health Organization recommendations (19). Among INH-resistant isolates 157 were from new cases and 207 were from previously treated cases. Among INH-susceptible isolates 66 and 12 were from new and previously treated cases, respectively. Analysis of M. tuberculosis was carried out by standard procedures (5, 18). Drug susceptibility testing was done either by the proportion method on Löwenstein-Jensen medium or with BACTEC 460 (Becton Dickinson, Sparks, Md.). Critical drug concentrations were 0.2 g/ml for INH, 4 g/ml for streptomycin (STR), 40 g/ml for RIF, and 2 g/ml for ethambutol (EMB) in Löwenstein-Jensen medium and 0.1, 2, 2, and 2.5 g/ml for INH, STR, RIF, and EMB, respectively, with the BACTEC 460. DNA from M. tuberculosis was extracted as described elsewhere (15). The 264-bp katG fragment targeting codon 315 and the 248-bp fragment spanning the mabA-inhA regulon were amplified by using primers 5Ј-TGGAGCAGATGGGCTTGG and 5Ј-CAG TGGCCAGCATCGTCG (12) and primers 5Ј-CCTCGCTGC CCAGAAAGGGA and 5Ј-ATCCCCCGGTTTCCTCCGG (14), respectively. PCR products purified with silica powder were subjected to manual sequencing by using the CycleReader DNA sequencing kit (Fermentas, Vilnius, Lithuania). For the restriction fragment length polymorphism (RFLP) assay, katG-specific PCR products were digested with TauI or SatI (Fermentas) and analyzed in a 1.5% agarose gel. Both endonucleases possess the overlapping GC sequence at a single recognition site of the 315 triplet AGC, and mutations at these positions eliminate cle...

The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi-and extensively drug-resistant tuberculosis.

The INNO-LiPA Rif.TB assay is designed for the detection of rpo〉 gene mutations causing rifampin resistance in isolates. We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly. Results were obtained in 78.3% of clinical specimens, and all were concordant with those obtained by BACTEC 460. The assay could have major impact on the management of multidrug-resistant tuberculosis.

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