Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis

Abstract

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for diagnosis and management of multidrug-resistant tuberculosis (MDR-TB). Resistance of M. tuberculosis to rifampicin is mainly due to mutations in hot-spot region of rpoB gene (HSR-rpoB). The prevalence of disputed (generally missed by rapid phenotypic DST methods) rpoB mutations, which mainly include L511P, D516Y, H526N, H526L, H526S, and L533P in HSR-rpoB and I572F in cluster II region of rpoB gene, is largely unknown. This study determined the occurrence of all disputed mutations in HSR-rpoB and at rpoB codon 572 in M. tuberculosis strains phenotypically susceptible to rifampicin in Kuwait.MethodsA total of 242 M. tuberculosis isolates phenotypically susceptible to rifampicin were used. The DST against first-line drugs was performed by Mycobacteria growth indicator tube (MGIT) 960 system. Mutations in HSR-rpoB (and katG codon 315 and inhA-regulatory region for isoniazid resistance) were detected by GenoType MDBDRplus assay. The I572F mutation in cluster II region of rpoB was detected by developing a multiplex allele-specific (MAS)-PCR assay. Results were confirmed by PCR-sequencing of respective loci. Molecular detection of resistance for ethambutol and pyrazinamide and fingerprinting by spoligotyping were also performed for isolates with an rpoB mutation.ResultsAmong 242 rifampicin-susceptible isolates, 0 of 130 pansusceptible/monodrug-resistant isolates but 4 of 112 polydrug-resistant isolates contained a disputed rpoB mutation. All 4 isolates were also resistant to isoniazid and molecular screening identified additional resistance to pyrazinamide and ethambutol in one isolate each. In final analysis, 2 of 4 isolates were resistant to all 4 first-line drugs. Spoligotyping showed that the isolates belonged to different M. tuberculosis lineages.ConclusionsFour of 242 (1.7%) rifampicin-susceptible M. tuberculosis isolates contained a disputed rpoB mutation including 2 isolates resistant to all four first-line drugs. The occurrence of a disputed rpoB mutation in polydrug-resistant M. tuberculosis isolates resistant at least to isoniazid (MDR-TB) suggests that polydrug-resistant strains should be checked for genotypic rifampicin resistance for optimal patient management since the failure/relapse rates are nearly same in isolates with a canonical or disputed rpoB mutation.

Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China

“…Another important finding was that most (77.4%) of the isolates with disputed mutations were of the Beijing lineage, although a possible association between disputed mutations and a specific genotype has not been observed in other studies (22,43). Of note, four of the five isolates with disputed mutations reported by Ho et al (22) were of the Beijing lineage, and interestingly, three of the four Beijing lineage isolates were isolated from patients who were born in China.…”

Discordant line probe genotypic testing vs culture-based drug susceptibility phenotypic testing in TB endemic KwaZulu-Natal: Impact on bedside clinical decision making

“…High rates of Mtb transmission in high endemicity populations increase the prevalence of mixed infections, and therefore in majority of cases, the standard rule remains to treat patients with mixed populations for both DS-TB and DR-TB. Furthermore polydrug-resistant strains should be analyzed for RIF resistance as studies have demonstrated that the failure and relapse rates are almost similar in isolates with a recognized or disputed rpoB mutation [53] . Patients with disputed rpoB mutations should be treated for MDR-TB ± high dose rifampicin as the clinical outcome is worse with standard treatment [54] , [55] .…”

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India

“…GeneXpert and LPA are the two WHO approved molecular diagnostic tests that detect mutations in the RRDR of the rpoB gene for the earlier diagnosis of rifampicin resistance in clinical specimens and culture isolates. However, these tests are not specific and they do not detect mutations outside of the 81bp RRDR of the rpoB gene of Mtb, which contains 95% of known rifampicin resistance-associated codons, leading to false-negative rifampicin resistance [13,39,40]. In addition, MGIT 960 culture, which is the current standard resistance test endorsed by the WHO to validate novel molecular assays may miss occult rifampicin resistance due to 'disputed' mutations [39].…”

“…However, these tests are not specific and they do not detect mutations outside of the 81bp RRDR of the rpoB gene of Mtb, which contains 95% of known rifampicin resistance-associated codons, leading to false-negative rifampicin resistance [13,39,40]. In addition, MGIT 960 culture, which is the current standard resistance test endorsed by the WHO to validate novel molecular assays may miss occult rifampicin resistance due to 'disputed' mutations [39]. This is due to the pre-set standard conditions of rifampicin critical concentration of 1 μg/ml.…”