Use of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates

Abstract

A commercially available DNA strip assay (Genotype MTBDR; Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect mutations conferring resistance to rifampin (RMP) and isoniazid (INH) in clinical Mycobacterium tuberculosis complex isolates. A total of 103 multidrug-resistant (MDR; i.e., at least resistant to RMP and INH) and 40 fully susceptible strains isolated in Germany in 2001 in which resistance mutations have been previously defined by DNA sequencing and real-time PCR analysis were investigated. The Genotype MTBDR assay identified 102 of the 103 MDR strains with mutations in the rpoB gene (99%) and 91 strains (88.4%) with mutations in codon 315 of katG. All 40 susceptible strains showed a wild-type MTBDR hybridization pattern. The concordance between the MTBDR assay and the DNA sequencing results was 100%. Compared to conventional drug susceptibility testing, the sensitivity and specificity were 99 and 100% for RMP resistance and 88.4 and 100% for INH resistance, respectively. In conclusion, the MTBDR assay is a rapid and easy-to-perform test for the detection of the most common mutations found in MDR M. tuberculosis strains that can readily be included in a routine laboratory work flow.The rapid determination of drug resistance in clinical isolates of Mycobacterium tuberculosis is the prerequisite for the initiation of effective chemotherapy ensuring successful treatment of the patient and preventing further spread of drugresistant isolates (17). The development of drug resistance in M. tuberculosis isolates is the result of random genetic mutations in particular genes conferring resistance (30). Based on this knowledge, molecular assays have been established that allow the prediction of drug resistance in clinical M. tuberculosis isolates within one working day and potentially are the most rapid methods for the detection of drug resistance (16). The utility of such assays depends on different aspects, such as the technical requirements of each method and the ability of different tests to detect the most common drug resistance mutations in a given area.The mutations that predominate in rifampin (RMP)-resistant M. tuberculosis isolates are located in an 81-bp "core region" of the rpoB gene (ca. 95% of all RMP-resistant strains (1, 27). In contrast, the mutations causing isoniazid (INH) resistance are located in several genes and regions (24). Approximately 50 to 95% of INH-resistant strains have been found to contain mutations in codon 315 of the katG gene (14,15,26), 20 to 35% contain mutations in the inhA regulatory region (15,18,26), and an additional 10 to 15% have mutations in the ahpC-oxyR intergenic region (8,18,26), often in conjunction with katG mutations outside of codon 315 (25).In order to allow the detection of the majority of mutations causing RMP resistance and also a high proportion of mutations causing INH resistance in M. tuberculosis isolates, a DNA strip assay was developed (Genotype MTBDR; Hain Lifescience, Nehren, Germany). It is based on a multiplex PCR in combinat...

Tuberculosis ethambutol resistance: Concordance between phenotypic and genotypic test results

“…based on DNA sequencing, Real Time PCR, or stripe technologies) for the detection of mutations conferring resistance have been increasingly used and have the potential to shorten the time to detection of resistance to one working day. [5][6][7] However, while molecular mechanisms involved in resistance to INH and RMP have been identified with clear cut associations between mutations in particular genes such as katG and rpoB with phenotypic resistance to INH and RMP, respectively, 8 associations between mutations and phenotypic ethambutol (EMB) resistance are less clear. Ethambutol is a key component of the first-line antituberculosis treatment regimen, and is also an important drug to be included in second-line regimens for MDR-TB, where susceptibility can be demonstrated.…”

Detección de mutaciones asociadas a cepas multidrogo resistente de Mycobacterium tuberculosis en Chile

“…Estudios previos demuestran que la mutación S531L fue la más frecuente 18,27 . En ninguna de las cepas resistentes a RIF se detectaron mutaciones simultaneas en más de un codón y tampoco se encontraron inserciones en los codones 514 y 521 como se ha descrito 27,29 . En 3 cepas resistentes a RIF no se detectaron mutaciones en la región del gen rpoβ que se estudió.…”

“…Estudios previos han identificados otros genes responsables de la resistencia a INH, como los genes ahpC, kasA y ndh 13,29 . Las mutaciones en inhA o su promotor pueden presentarse por sí solas o en combinación con las mutaciones en katG, lo cual fue observado en una de las cepas resistentes a INH 11 .…”

Evaluation of Genotype® MTBDRplus Assay for Rapid Detection of Resistance to Isoniazid and Rifampin in Mycobacterium tuberculosis Isolates Collected in Tunisia

“…With respect to culture isolates, the sensitivities of the MTBDR assay for the detection of RIF resistance were recently reported to be in the range of 95% to 99% [9][10][11][12]. Both the rarity of RMP-resistance-associated mutations in codons other than the rpoB 81-bp hot spot region and the rarity of silent mutations in the hot spot region are responsible for the high rate of detection of RMP resistance by investigation of this region [12][13][14].…”