1887

Abstract

Purpose. This study was undertaken to evaluate the efficiency of the pyrosequencing (PSQ) assay for the rapid detection of resistance to rifampicin (RIF), fluoroquinolones (FQs) and second-line injectables (SLIs) such as capreomycin (CAP) and kanamycin (KAN) in Mycobacterium tuberculosis (Mtb) clinical isolates.

Methodology. Pyrosequencing is a simple and accurate short read DNA sequencing method for genome analysis. DNA extraction from Mtb clinical isolates was performed using Tris-HCl buffer and chloroform. The rpoB (RIF), gyrA (FQs) and rrs (aminoglycosides) genes were amplified, followed by sequencing using the PyroMark Q24 ID system. The PSQ results were compared with the results from the conventional drug susceptibility testing performed in the laboratory.

Results. The sensitivity of the PSQ assay for the detection of resistance to RIF, FQ, CAP and KAN was 100 %, 100 %, 40 % and 50 %, respectively. The specificity of the PSQ assay was 100 %.

Conclusion. The PSQ assay is a rapid and effective method for detecting drug resistance mutations from Mtb clinical isolates in a short period of time.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000669
2018-07-20
2020-01-26
Loading full text...

Full text loading...

/deliver/fulltext/jmm/67/9/1212.html?itemId=/content/journal/jmm/10.1099/jmm.0.000669&mimeType=html&fmt=ahah

References

  1. Dheda K, Gumbo T, Maartens G, Dooley KE, McNerney R et al. The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis. Lancet Respir Med 2017;291–360 [CrossRef][PubMed]
    [Google Scholar]
  2. Bending the curve – ending TB: Annual report 2017.pdf [Internet]. cited 31 July 2017 Available fromhttp://apps.who.int/iris/bitstream/10665/254762/1/978929022584-eng.pdf
  3. WHO MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB) 2016 UPDATE.pdf [Internet]. cited 31 July 2017 Available fromwww.who.int/tb/challenges/mdr/mdr_tb_factsheet.pdf
  4. SEARO | Revised National Tuberculosis Control Programme (RNTCP) – Guidelines for TB Control in India [Internet]. [cited 25 May 2016] Available fromwww.searo.who.int/india/tuberculosis/topic/tb_rntcpguidelines/en/
  5. World Health Organization, Global Tuberculosis Programme. WHO treatment guidelines for drug-resistant tuberculosis: 2016 update. [Internet]. [cited 10 July 2017] Available fromwww.ncbi.nlm.nih.gov/books/NBK390455/
  6. Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: advances in technology and drug susceptibility testing. Indian J Med Microbiol 2017;35:323 [CrossRef][PubMed]
    [Google Scholar]
  7. Calligaro GL, Moodley L, Symons G, Dheda K. The medical and surgical treatment of drug-resistant tuberculosis. J Thorac Dis 2014;6:186–195 [CrossRef][PubMed]
    [Google Scholar]
  8. Caulfield AJ, Wengenack NL. Diagnosis of active tuberculosis disease: From microscopy to molecular techniques. J Clin Tuberc Other Mycobact Dis 2016;4:33–43 [CrossRef]
    [Google Scholar]
  9. Revised National TB Control Programme Manual of Standard Operating Procedures (SOPs) [Internet]. cited 30 July 2017 Available fromhttp://tbcindia.nic.in/WriteReadData/l892s/7293794058standard%20operating%20procedures%20for%20C&DST%20labs.pdf
  10. Jureen P, Engstrand L, Eriksson S, Alderborn A, Krabbe M et al. Rapid detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing technology. J Clin Microbiol 2006;44:1925–1929 [CrossRef][PubMed]
    [Google Scholar]
  11. Lin SY, Rodwell TC, Victor TC, Rider EC, Pham L et al. Pyrosequencing for rapid detection of extensively drug-resistant Mycobacterium tuberculosis in clinical isolates and clinical specimens. J Clin Microbiol 2014;52:475–482 [CrossRef][PubMed]
    [Google Scholar]
  12. Engström A, Morcillo N, Imperiale B, Hoffner SE, Juréen P. Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing. J Clin Microbiol 2012;50:2026–2033 [CrossRef][PubMed]
    [Google Scholar]
  13. Hamze M, Ismail MB, Rahmo AK, Dabboussi F. Pyrosequencing for rapid detection of tuberculosis resistance to Rifampicin and Isoniazid in Syrian and Lebanese clinical isolates. Int J Mycobacteriol 2015;4:228–232 [CrossRef][PubMed]
    [Google Scholar]
  14. Ajbani K, Lin SY, Rodrigues C, Nguyen D, Arroyo F et al. Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries. Antimicrob Agents Chemother 2015;59:414–420 [CrossRef][PubMed]
    [Google Scholar]
  15. Guo Q, Zheng RJ, Zhu CT, Zou LL, Xiu JF et al. Pyrosequencing for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis. Int J Tuberc Lung Dis 2013;17:1008–1013 [CrossRef][PubMed]
    [Google Scholar]
  16. Zetola NM, Shin SS, Tumedi KA, Moeti K, Ncube R et al. Mixed Mycobacterium tuberculosis complex infections and false-negative results for rifampin resistance by GeneXpert MTB/RIF are associated with poor clinical outcomes. J Clin Microbiol 2014;52:2422–2429 [CrossRef][PubMed]
    [Google Scholar]
  17. Wang S, Zhao B, Song Y, Zhou Y, Pang Y et al. Molecular characterization of the rpoB gene mutations of Mycobacterium tuberculosis isolated from China. J Tuberc Res 2013;01:1–8 [CrossRef]
    [Google Scholar]
  18. Patra SK, Jain A, Sherwal BL, Khanna A. Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing. Indian J Clin Biochem 2010;25:315–318 [CrossRef][PubMed]
    [Google Scholar]
  19. van Deun A, Aung KJ, Bola V, Lebeke R, Hossain MA et al. Rifampin drug resistance tests for tuberculosis: challenging the gold standard. J Clin Microbiol 2013;51:2633–2640 [CrossRef][PubMed]
    [Google Scholar]
  20. van Deun A, Barrera L, Bastian I, Fattorini L, Hoffmann H et al. Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. J Clin Microbiol 2009;47:3501–3506 [CrossRef][PubMed]
    [Google Scholar]
  21. Bravo LT, Tuohy MJ, Ang C, Destura RV, Mendoza M et al. Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones. J Clin Microbiol 2009;47:3985–3990 [CrossRef][PubMed]
    [Google Scholar]
  22. Hillemann D, Rüsch-Gerdes S, Richter E. Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2009;47:1767–1772 [CrossRef][PubMed]
    [Google Scholar]
  23. Georghiou SB, Seifert M, Lin SY, Catanzaro D, Garfein RS et al. Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis. BMC Infect Dis 2016;16:458 [CrossRef][PubMed]
    [Google Scholar]
  24. Kambli P, Ajbani K, Nikam C, Sadani M, Shetty A et al. Correlating rrs and eis promoter mutations in clinical isolates of Mycobacterium tuberculosis with phenotypic susceptibility levels to the second-line injectables. Int J Mycobacteriol 2016;5:1–6 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000669
Loading
/content/journal/jmm/10.1099/jmm.0.000669
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error