Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

Daum, Luke T.; Rodriguez, J. D.; Worthy, Sue A.; Ismail, Nazir; Omar, Shaheed V.; Dreyer, Andries; Fourie, P. Bernard; Hoosen, Anwar A.; Chambers, James P.; Fischer, Gerald W.

doi:10.1128/jcm.01893-12

Cited by 72 publications

(58 citation statements)

References 20 publications

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“…The high-throughput bead format and the cost provide potential advantages for the application of TB-SPRINT in centralized laboratories and the MagPix in smaller laboratories. Next-generation sequencing of drug-resistant mutations is an attractive and promising strategy; however, the current cost remains out of reach for low-resource countries (43). TB-SPRINT is currently available as an assay for research use only, which will be applied for research in specialized clinical microbiology and reference laboratories.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2013

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e As a follow-up of the "spoligoriftyping" development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n ‫؍‬ 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n ‫؍‬ 162 for rpoB gene sequencing and n ‫؍‬ 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n ‫؍‬ 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2013

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“…Orange crystals were grown by slow evaporation of a MeOH solution of [Mn(atc-Et) 2 ] at room temperature. The data collection was performed using Mo-Kα radiation (λ = 71.073 pm) on a BRUKER APEX II Duo diffractometer.…”

Section: Crystal Structure Determinationmentioning

confidence: 99%

“…In addition, a number of anti-TB drugs may be discontinued in a few years due to the expanding number of multidrug resistant TB (MDR-TB) and extensively drug-resistant (XDR-TB) cases [2]. Therefore, there is a serious need for new anti-MTB agents that target new biochemical pathways and treat drug resistant forms of the disease [3,4].…”

Section: Introductionmentioning

confidence: 99%

Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

Oliveira

Maia

Souza

et al. 2014

Journal of Inorganic Biochemistry

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“…The pncA forward/reverse primers included pncAF1 (5=-CGGATTTGTCGCT CACTAC-3=) and pncAR1 (5=-GCCGGAGACGATATCCAGAT-3=), comprising the full gene and also the pncA promoter region. The expected size of the pncA amplicon was 960 bases (14). PCRs were performed in a total volume of 50 l, and the PCR mixture consisted of 5 l of 10ϫ buffer plus MgCl 2 (Longhorn Vaccines & Diagnostics, San Antonio, TX, USA), 2 l of 20 M forward and reverse pncA primers (Integrated DNA Technologies, Coralville, IA, USA), 0.5 l Platinum Taq enzyme (Life Technologies, Grand Island, NY, USA), 35.5 l nuclease-free water (Integrated DNA Technologies), and 5 l extracted DNA.…”

Section: Description Of Isolatesmentioning

confidence: 99%

“…Whole-gene next-generation Ion Torrent sequencing was performed using a novel but standardized M. tuberculosis protocol (14). All PCR products were subjected to full-gene pncA sequencing.…”

Section: Description Of Isolatesmentioning

confidence: 99%

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

Maningi

Daum²,

Rodriguez³

et al. 2015

J Clin Microbiol

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eThe technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n ‫؍‬ 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n ‫؍‬ 88), 83% (n ‫؍‬ 88), and 89% (n ‫؍‬ 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies. P yrazinamide (PZA) is a cornerstone first-line antituberculosis compound that is also commonly used in the second-line therapeutic treatment of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection in humans. The drug is a structural analogue of nicotinamide, requiring conversion into its active form, i.e., pyrazinoic acid, by the enzyme pyrazinamidase/nicotinamidase (PZase) (1, 2). PZase is encoded by the M. tuberculosis pncA gene (561 bp) (2). It has been postulated that the mechanism of action of PZA is through pyrazinoic acid, causing disruption of bacterial membrane-mediated energetics and ultimately causing inhibition of membrane transport (3). The contribution of PZA to the killing of M. tuberculosis as part of a multidrug regimen for the treatment of tuberculosis (TB) is considerable, and its inclusion with rifampin in anti-TB therapeutic regimens has resulted in the significant shortening of treatment duration from 18 to 6 months (3). The drug is known to specifically target semidormant bacteria that are not killed by other antituberculosis drugs (4, 5).Mutations in the pncA gene are associated with phenotypically pyrazinamide-resistant isolates of M. tuberculosis (2, 6, 7). Such mutations can occur in the promoter or coding regions and result in amino acid substitutions, frameshifts, or stop codon mutations. Furtherm...

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Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

Cited by 72 publications

References 20 publications

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

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

Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

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