Evaluation of the GenoType MTBDRplus Assay for Rifampin and Isoniazid Susceptibility Testing of Mycobacterium tuberculosis Strains and Clinical Specimens

Abstract

The new GenoType MTBDRplus assay (Hain LifescienceGmbHThe worldwide increase in the rates of multidrug-resistant (MDR) tuberculosis (resistance to at least rifampin [RMP] and isoniazid [INH]) has made the timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains to achieve effective disease management and to prevent their spread extremely important. INH and RMP are the most important first-line antituberculosis drugs, and resistance to these drugs often results in treatment failures and fatal clinical outcomes (6, 7).Recently, nonradiometric fully automated systems that are used to screen for resistance and that have technical and safety advantages have been introduced (23). However, the time for resistance testing still is about 7 to 10 days, beginning from the time that a positive culture is obtained (23). The most rapid results could be achieved by direct testing of patient specimens by fast molecular methods (11, 25). These methods are based on the knowledge that resistance to RMP and INH in M. tuberculosis is most often attributed to mutations in the rpoB, katG, and inhA genes. By targeting mutations in the 81-bp "core region" of the rpoB gene, more than 95% of all RMPresistant strains can be detected (28). On the contrary, the mutations that cause INH resistance are located in several genes and regions. Between 50% and 95% of INH-resistant strains have been found to contain mutations in codon 315 of the katG gene (18,20,28), between 20 and 35% of INHresistant strains have been found to contain mutations in the inhA regulatory region (20,22,28), and an additional 10 to 15% of INH-resistant strains had mutations in the ahpC-oxyR intergenic region (13,22,28), often in conjunction with katG mutations outside of codon 315 (26). In a recent study, the strong statistical association between specific mutations in the katG, inhA, and ahpC genes and INH resistance could be confirmed (8). The authors estimated that a simple test for five molecular markers is able to detect 74% of INH-resistant (INH r ) isolates; 0 to 5% of the INH r M. tuberculosis isolates had mutations in the inhA open reading frame and 8 to 20% had mutations in the inhA promoter region (8,20,31).DNA strip assays targeting rpoB (INNO-LiPA Rif; Innogenetics N.V., Ghent, Belgium) or rpoB plus katG (GenoType MTBDR; Hain Lifescience GmbH, Nehren, Germany) were developed and evaluated for use with M. tuberculosis cultures and smear-positive specimens (1,11,16,17,29). The DNA strip assays are based on a multiplex PCR in combination with reverse hybridization. Either the omission of a wild-type band or the appearance of bands of DNA signals representing specific mutations indicates the existence of a resistant strain.In order to enlarge the capacity for the detection of drug resistance, the new GenoType MTBDRplus assay was developed. The assay has the ability to detect a broader variety of rpoB gene mutations and inhA gene mutations. By covering mutations in the regulatory region of inhA, it can be expected that additional INH-...

Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR

“…These isolates had another mutation in the same region and had mutations in other INH resistance-associated regions, and were therefore determined to be MDR by the REBA MTB-MDR assay. Although it is not easy to make a simple recommendation in such cases of mixed infection with resistant and susceptible M. tuberculosis strains as observed in this study, or in mixed infections containing M. tuberculosis and a non-tuberculous mycobacterium (Hillemann et al, 2007), we favour a conservative approach of reporting such strains as likely to be resistant after exclusion of involvement of a non-tuberculous mycobacterium.…”

“…Among the most successful of these molecular diagnostic methods is the GenoType MTBDRplus assay (Hain LifeScience), which is based on reverse hybridization of amplicons containing rpoB, katG and inhA from test strains to probes carrying specific mutations in these genes. This DNA line probe assay performs well and offers substantial promise as a rapid molecular DST methodology (Hillemann et al, 2007;Mäkinen et al, 2006). This kit was designed to probe mutations only in the katG gene and inhA promoter region in determining INH resistance; however, approximately 10-15 % of INH-resistant M. tuberculosis strains have mutations in the oxyR-ahpC intergenic region with additional mutation of katG outside codon 315.…”

Predictive Value of Molecular Drug Resistance Testing of Mycobacterium tuberculosis Isolates in Valle del Cauca, Colombia

“…Previous evaluations of the GenoType kits have documented their performance in detecting resistance to first-and second-line antituberculosis (anti-TB) drugs in different continents, like Africa, Asia, and Europe (2,3,4,5). From these studies, it became clear that there are geographic variations in the mutations that are associated with drug resistance in Mycobacterium tuberculosis (6).…”

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]. However, in our study sensitivity of the MTBDRplus assay measured for resistance to RIF was lower than those reported.…”