Can Molecular Methods Detect 1% Isoniazid Resistance in Mycobacterium tuberculosis?

Abstract

Patients may harbor both drug-susceptible and -resistant bacteria, representing heteroresistance. We studied mixtures of isoniazid-resistant and -susceptible Mycobacterium tuberculosis strains. Conventional drug susceptibility testing was the most sensitive method of detection, whereas the line probe assay and sequencing were not able to detect the clinically relevant 1% proportion of resistant bacteria. Patients with tuberculosis (TB) usually harbor susceptible Mycobacterium tuberculosis but may also have a small proportion of drug-resistant isogenic variants that develop spontaneously during replication. For isoniazid (Inh) resistance, mutations are introduced at rates of approximately 1 mutation per 10 8 to 10 10 divisions (1). The mutations are found in several genes but mainly in the katG gene and in the inhA promoter (2). An increased proportion of resistant variants in a susceptible main population of isogenic or nonisogenic bacteria is called "heteroresistance" in this paper. Standard short-course chemotherapy with combinations of several drugs prevents the selection of drug-resistant bacteria during treatment. The proportion of patients infected with heteroresistant tuberculosis is largely unknown. In the 1960s, the criteria for defining clinically relevant drug resistance were set from clinical and bacteriological studies. By consensus, a cutoff of 1% for Inh was decided. This is the proportion of resistant bacteria above which therapeutic success had been demonstrated to be unlikely (3, 4). Therefore, phenotypic drug susceptibility testing (DST) aims to determine if 1% or more of the bacterial population in clinical specimens is drug resistant.In recent years, commercial genotypic methods have become available for detection of mutations that may confer resistance. Among these, it is only with the line probe assay (LPA) GenoType MTBDRplus that resistance to Inh can be detected by analysis of the katG and inhA genes (5). The LPA uses PCR covering areas where mutations associated with resistance are common. It is followed by hybridization of target sequences to a membrane strip; the hybrids are visualized as bands. The test detects specific mutations as well as unspecified mutations, the latter by the absence of at least one wild-type band.In June 2008, a WHO Expert Consultation concluded that sufficient information was available to recommend the use of LPAs for detection of multidrug resistance (MDR) in M. tuberculosis isolates and smear-positive sputum specimens (http://www.who .int/tb/features_archive/policy_statement.pdf). The newly published European Union Standards for Tuberculosis Care recommends also that rapid testing for Inh and rifampin should be performed on primary specimens when MDR TB is suspected (6).These assays are important for the global scaling up of detection of MDR and extensively drug-resistant (XDR) M. tuberculosis. However, little is known of the sensitivity of these methods for detection of small proportions of drug-resistant bacteria. Furthermore, to facilitate the DSTs, labor...

Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

“…When testing the Haarlem strains (WT and the H526Y mutant), we were able to find the targeted 1% Rif resistance with MGIT DST but not with the molecular methods. We could not find the same sensitivity for the Beijing strains (WT and the S531L mutant), in contrast to our study on isoniazid heteroresistance that did not show a difference among the test strains (13). One possible explanation is a loss of fitness in the mutated Beijing strain.…”

“…For phenotypic DST, we used Bactec MGIT960 (BD) (11) with a 1/100 diluted control and 1.0 mg/ml Rif. All analyses were interpreted as unknown samples as in routine situations (12,13).…”

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

“…TB-SPRINT, like the Xpert MTB/RIF assay, covers about 95% of all rifampin-resistant isolates; therefore, 100% correlation with phenotypic DST results is not to be expected in routine practice. We must also mention that many NAATs currently are unable to detect heteroresistance, an important issue for the clinical efficiency of antibiotics (42).…”

Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing

“…A previ-ous study reported a detection limit of 15% for pyrosequencing and 30% for Sanger sequencing for detection of lamivudine-resistant hepatitis B virus (14). Low sensitivity encompasses not only sequencing but also other molecular methods, including both the GenoType MTBDRplus and GeneXpert MTB/RIF tests (15,16). This exemplifies the notion that sensitivities varies between different assays and methods.…”