Dataset Information

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

ABSTRACT: Understanding why some multidrug-resistant tuberculosis cases are not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostic assays and advance toward personalized tuberculosis treatment. Here, we combine whole-genome sequencing with single-colony phenotyping to identify a multidrug-resistant strain that had infected a patient for 9 years. Our investigation revealed the failure of rapid testing and genome-based prediction tools to identify the multidrug-resistant strain. The false-negative findings were caused by uncommon rifampicin and isoniazid resistance mutations. Although whole-genome sequencing data helped to personalize treatment, the patient developed extensively drug-resistant tuberculosis, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens.

SUBMITTER: Cancino-Munoz I

PROVIDER: S-EPMC6581888 | biostudies-other | 2019 Jun

REPOSITORIES: biostudies-other

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Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

Cancino-Muñoz Irving I Moreno-Molina Miguel M Furió Victoria V Goig Galo A GA Torres-Puente Manuela M Chiner-Oms Álvaro Á Villamayor Luis M LM Sanz Francisco F Guna-Serrano María Remedio MR Comas Iñaki I

The Journal of infectious diseases 20190601 2

Understanding why some multidrug-resistant tuberculosis cases are not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostic assays and advance toward personalized tuberculosis treatment. Here, we combine whole-genome sequencing with single-colony phenotyping to identify a multidrug-resistant strain that had infected a patient for 9 years. Our investigation revealed the failure of rapid testing and genome-based prediction tools to identify the multid ...[more]

PMID: 30875421

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Project description:BackgroundNearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait.FindingsDirect DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence.ConclusionsAlthough fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.

Project description:ObjectivePyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA + upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis.MethodsDrug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA (Rv2044c-pncA-Rv2042c) and PZase assay were performed for 448 M. tuberculosis clinical isolates.ResultsOur data showed that among 448 M. tuberculosis clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drug-resistance. A total of 60.04% (269/448) M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZAR isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZAR. Compared with phenotypic data, 84.38% (227/269) PZAR strains with mutations in pncA + upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C-114del) in upstream of pncAwt nullified the effect of A-11G mutation and induced positive PZase activity, divergent from five PZase negative A-11G PZAR mutants. Twenty-six PZAR strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms.ConclusionOur study revealed that PZAR rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA + UFR, rpsA, panD, Rv2783c, and Rv2044c provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.

Dataset Information

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

Publications

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

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