PyroTyping, a novel pyrosequencing-based assay for Mycobacterium tuberculosis genotyping

Molina-Moya, Bárbara; Lacoma, Alícia; García-Sierra, Nerea; Blanco, Silvia; Haba, L.; Samper, Sofía; Ruiz-Manzano, Joan; Prat, Cristina; Arnold, Catherine; Domínguez, J.

doi:10.1038/s41598-017-06760-5

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Whereas molecular beacon-based assays use real-time PCR, dual-labeled probes that form a quenched, stem-loop structure in native state and fluoresce upon hybridization to the target nucleotide sequence (Lawn and Nicol, 2011 ), line probe assays involve PCR and reverse hybridization with specific oligonucleotide probed fixed to a nitrocellulose strip in parallel lines (World Health Organization, 2008 ). Sequence-based methods include pyrosequencing (Molina-Moya et al, 2017 ), Sanger sequencing (Schleusener et al, 2017 ), and next-generation sequencing (Jagielski et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan

et al. 2018

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Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

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Section: Introductionmentioning

confidence: 99%

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan

et al. 2018

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“…DR-TB is a se rious public health problem globally [68]. In order to improve TB control, it is important to track the spread of MTB, identify index cases, and detect outbreak cases [69]. Monitoring and proper management of patients infected with drug-resistant MTB strains is key to eff ective control of drug-resistant TB globally [70].…”

Section: Tackling the Global Drug-resistant Tuberculosis Crisismentioning

confidence: 99%

Drug-Resistant Tuberculosis Types and Their Treatment Regimens Using First-Line, Second-Line Injectable, Third-Line, Fluoroquinolones, Aminoglycosides, Cyclic Polypeptides, Novel and Repurposed Anti-Tuberculosis Drugs

Mumena¹,

Kwenda²,

Ngugi³

et al. 2022

J Biomed Res Environ Sci

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Drug-Resistant Tuberculosis (DR-TB) causes high mortality and morbidity rates globally. DR-TB and COVID-19 pandemic are posing a major risk to global public health and economic security, and are jeopardizing efforts in the control, prevention and elimination of TB globally. Mycobacterium tuberculosis (MTB) has continued to evolve resistance to anti-TB drugs. Different types of DR-TB have been defined and they include; mono drug-resistant TB, Multi Drug-Resistant TB (MDR-TB), poly drug-resistant TB, pre-Extensively Drug-Resistant TB (pre-XDR TB), Extensively Drug-Resistant TB (XDR-TB), Extremely Drug-Resistant TB (XXDR-TB), and Totally Drug-Resistant TB (TDR-TB). DR-TB is caused by several factors which include: non-adherence, poor compliance, low efficacy anti-TB drugs, delayed diagnosis, interrupted supply, stock-outs, inadequate infection control, HIV co-infection, spontaneous mutations, and chromosomal replication errors. Global TB targets have gone off-track and years of progress reversed due to DR-TB and the COVID-19 pandemic. Treatment failure, death and costs incurred are higher among patients suffering from DR-TB than among those with susceptible TB. For this reason, susceptible TB needs to be diagnosed quickly and treated effectively to prevent its progression to DR-TB. Treatment for susceptible TB requires the use of first-line anti-TB drugs; rifampicin, isoniazid, pyrazinamide, and ethambutol. While DR-TB is treated using the second- and third-line anti-TB drugs. Effective treatment of TB is dependent on: prompt and accurate diagnosis of TB and recognition of drug-resistance; adherence to treatment; robust contact tracing and prophylactic treatment of TB contacts; and screening for TB infection in high-risk groups.

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“…IS6110-RFLP әдісі 1993 ж. ұсынылды және қазіргі таңда M. tuberculosis генотиптеуінің «алтын стандарты» болып табылады, ӛйткені бұл әдістің дискриминациялау қабілеті ӛте жоғары [5]. Бірақ, IS6110-RFLP анализінің әдістемелік күрделілігі, алынған мәліметтерді интерпретациялау және әдістің кӛп еңбекті талап етуі сияқты кемшіліктері IS6110-RFLP әдісінің дүниежүзінде аса кең пайдаланылмайтындығына септігін тигізді [4][5][6][7][8][9]. Сполиготиптеу әдісі M. tuberculosis-ң DR (direct repeat) локусында 43 бірегей спейсерлік аймақтардың болуын немесе болмауын анықтауға негізделген [10] және бұл әдіс 1 мен 34 спейсерлік аймақтардың арасында сигналдардың болмауымен сипатталатын Beijing тұқымдасының штаммдарын идентификациялауда жиі қолданылады [11].…”

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Генотипирование Клинических Изолятов M. Tuberculosis Среди Повторных Случаев Туберкулеза В Казахстане

Akhmetova¹,

ЧАМОЙЕВА²,

ҚҰРМАНҒАЛИ³

et al. 2022

Vestnik

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Қазақстанда туберкулез мәселесі әлі күнге дейін өзекті болып қалуда. Жылдан жылға дәріге-төзімді туберкулез формаларымен ауру деңгейінің жоғарылауы – аталған аурумен күресте негізгі мәселелердің бірі. Қазақстан дүниежүзінде туберкулезге қарсы қолданылатын ең тиімді бірінші қатардағы препараттар – изониазид пен рифампицинмен ассоциацияланған көптік дәріге төзімді туберкулез көрсеткіштері жоғары отыз мемлекеттер қатарына кіреді. Жұмыстың мақсаты MIRU-VNTR әдісін пайдалана отырып, Қазақстанда таралған туберкулездің қайталанған жағдайларының арасында M. tuberculosis клиникалық изоляттарының биологиялық әртүрлілігін бағалау болып табылады. Аталған зерттеу жұмысында рецидивтердің арасында 79 M. tuberculosis клиникалық изоляттары жиналды. M. tuberculosis клиникалық үлгілерін генотиптеу 12 MIRU локустары бойынша өткізілді. Барлық 12 локустың ПТР-амплификациясы реакцияға 5М бетаин қосу арқылы жүзеге асырылды. ПТР-өнімдері бромистік этидиймен боялған 1,5% агароздық гельде бөлінді. Содан кейін MIRU локустарындағы қайталанулар саны есептелді және әрбір M. tuberculosis клиникалық изолятына 12 саннан тұратын сандық профиль алынды. Жиналған M. tuberculosis клиникалық изоляттарының тұқымдастары MIRU-VNTRplus.org мәліметтер базасы көмегімен анықталды. Генотиптеу нәтижелері қайталанылған туберкулез жағдайларының арасында 88,6% (n=70) изоляттар Beijing тұқымдасына жататынын көрсетті. В Казахстане проблема туберкулеза остается актуальной. С каждым годом увеличивается уровень заболеваемости лекарственно-устойчивыми формами туберкулеза, что является главной преградой в борьбе с данным заболеванием. Казахстан является одной из тридцати стран в мире с высоким показателем туберкулеза с множественной лекарственной устойчивостью ассоциированного с лекарственной устойчивостью к самым эффективным противотуберкулезным препаратам первого ряда – изониазиду и рифампицину. Цель данной работы заключается в оценке биологического разнообразия клинических изолятов M. tuberculosis среди рецидивов в Казахстане с использованием MIRU-VNTR метода. В данном исследовании было собрано 79 клинических образцов M. tuberculosis среди повторных случаев туберкулеза из разных областей Казахстана. Генотипирование собранных клинических изолятов было проведено по 12 MIRUлокусам. ПЦР-амплификация всех 12 локусов была выполнена с добавлением в реакцию 5М бетаина. Разделение ПЦР-продуктов было проведено на 1,5% агарозном геле. Далее проводился подсчет тандемных повторов в MIRU локусах и был получен 12-ти значный цифровой профиль для каждого образца. M. tuberculosisсемейства собранных клинических изолятов были определены с помощью базы данных MIRU-VNTRplus.org. Результаты генотипирования показали, что 88,6% (n=70) изолятов среди рецидивов были отнесены к семейству Beijing. Tuberculosis still remains one of the important health issues in Kazakhstan. Extension of the incidence of drug-resistant forms every year is the main obstacle in the fight against the disease. Kazakhstan is one of the thirty countries on the planet with high rates of mulridrug-resistant tuberculosis associated with resistance to the most effective first-line antituberculosis drugs – isoniazid and rifampicin. Aim of this research is to evaluate biological diversity of clinical isolates of M. tuberculosis among recurrent tuberculosis cases in Kazakhstan by MIRU-VNTR method. 79 clinical isolates of M. tuberculosis were gathered from the patients with recurrent tuberculosis. Genotyping of the clinical samples was conducted by 12 MIRU loci. PCR amplification of 12 loci was done by addition of 5M betaine solution. PCR products were visualized on 1,5% agarose gel. Tandem repeats in the loci were calculated and the digital profile for every sample was obtained. M. tuberculosis families of the gathered clinical isolates were determined using MIRU-VNTRplus.org database. Genotyping results revealed that 88,6% of isolates (n=70) among recurrent cases were identified as the isolates of Beijing family.

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PyroTyping, a novel pyrosequencing-based assay for Mycobacterium tuberculosis genotyping

Cited by 4 publications

References 21 publications

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan

Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan

Drug-Resistant Tuberculosis Types and Their Treatment Regimens Using First-Line, Second-Line Injectable, Third-Line, Fluoroquinolones, Aminoglycosides, Cyclic Polypeptides, Novel and Repurposed Anti-Tuberculosis Drugs

Генотипирование Клинических Изолятов M. Tuberculosis Среди Повторных Случаев Туберкулеза В Казахстане

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