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Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

ABSTRACT: With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P<0.05 for rifampicin or isoniazid or streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (?) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (?, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (?, 0.97) while targeted sequencing yielded lower agreement (? range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (? range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be more useful when accurate ethambutol susceptibility results are warranted.

SUBMITTER: Ahmad S

PROVIDER: S-EPMC4838278 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

Ahmad Suhail S Mokaddas Eiman E Al-Mutairi Noura N Eldeen Hanaa S HS Mohammadi Shirin S

PloS one 20160420 4

With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) ...[more]

PMID: 27096759

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Project description:BackgroundIncreasing incidence of multidrug-resistant Mycobacterium tuberculosis infections is hampering global tuberculosis control efforts. Kuwait is a low-tuberculosis-incidence country, and ~ 1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait.MethodsNinety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460 TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, pyrazinamide, ethambutol and streptomycin were detected by genotype MTBDRplus assay and/or PCR sequencing of three rpoB regions, katG codon 315 (katG315) + inhA regulatory region, pncA, three embB regions and rpsL + rrs-500-900 regions. Spoligotyping kit was used, spoligotypes were identified by SITVIT2, and phylogenetic tree was constructed by using MIRU-VNTRplus software. Phylogenetic tree was also constructed from concatenated sequences by MEGA7 software. Additional PCR sequencing of gidB and rpsA was performed for cluster isolates.ResultsPansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG and/or inhA were detected in 93/93 and 92/93 MDR-TB strains, respectively. Mutations were also detected for pyrazinamide resistance, ethambutol resistance and streptomycin resistance in MDR-TB isolates in pncA, embB and rpsL + rrs, respectively. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns while 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93), and 11 isolates showed nine orphan patterns. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Interestingly, 22 isolates in eight clusters by both methods were isolated from TB patients typically within a span of 2 years. Five of eight clusters were confirmed by additional gidB and rpsA sequence data.ConclusionsOur study provides the first insight into molecular epidemiology of MDR-TB in Kuwait and identified several potential clusters of local transmission of MDR-TB involving 2-6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.

Dataset Information

Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

Publications

Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country.

Similar Datasets

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