Advertisement

Antonie van Leeuwenhoek

, Volume 112, Issue 5, pp 679–685 | Cite as

A new colorimetric method for rapid detection of ethambutol and streptomycin resistance in Mycobacterium tuberculosis: crystal violet decolorization assay (CVDA)

  • Ahmet Yilmaz CobanEmail author
  • Ahmet Ugur Akbal
  • Ismail Ceyhan
  • Meltem Uzun
  • Deniz Sertel Selale
  • Gonul Aslan
  • Nuran Delialioglu
  • Mustafa Ozyurt
  • Beyhan Bektore
  • Can Bicmen
  • Ahmet Aslanturk
  • Nilay Ucarman
  • Ali Albay
  • Ali Korhan Sig
  • Nuri Ozkutuk
  • Suheyla Surucuoglu
Original Paper
  • 129 Downloads

Abstract

Streptomycin (STR) and ethambutol (EMB) are important drugs used for the treatment of tuberculosis. There is a need for fast, reliable and inexpensive methods for detecting resistance to these drugs. The aim of this study was to evaluate the performance of the crystal violet decolorization assay (CVDA) for the detection of STR and EMB resistance that is important drugs in tuberculosis treatment. In this study, drug susceptibility testing was performed on 140 Mycobacterium tuberculosis isolates provided from nine centers. Three tubes were used for each isolate. One of the tubes had a concentration of 2 mg/L STR and the other 5 mg/L EMB. The third was drug-free control tube. Sensitivity, specificity, positive predictive value (PPD), negative predictive value (NPD) and agreement for STR were found to be 81.8%, 94.6%, 87.8%, 91.5% and 90.57%, respectively. For EMB, sensitivity, specificity, PPD, NPD, and agreement were found to be 76%, 98.23%, 90.47%, 94.87% and 94.2%, respectively. The results were obtained in 11.3 ± 2.7 days (8–21 days). CVDA is rapid, reliable, inexpensive, and easy to perform for rapid detection of STR and EMB resistance, and it could be adapted for drug susceptibility testing.

Keywords

Mycobacterium tuberculosis CVDA STR EMB Susceptibility testing 

Notes

Authors’ contribution

AYC planned the experiment and prepared the draft manuscript. AYC and AUA performed the laboratory tests in the Center 1. Other authors tested the isolates with reference method before sending Center 1 and read the manuscript.

Funding

There is no any fund for this study.

Compliance with ethical standards

Ethical approval

None needed.

References

  1. Aziz MA, Wright A, Laszlo A, De Muynck A, Portaels F, Van Deun A, Wells C, Nunn P, Blanc L, Raviglione M (2006) Epidemiology of antituberculosis drug resistance (the Global Project on Anti-tuberculosis Drug Resistance Surveillance): an updated analysis. Lancet 368:2142–2154CrossRefGoogle Scholar
  2. Clinical and Laboratory Standards Institute (CLSI) (2011) Susceptibility testing of Mycobacteria, Nocardia, and other aerobic actinomycetes. Approved standard M24-A2, 2nd ed. Clinical and Laboratory Standard Institute, Wayne, PAGoogle Scholar
  3. Coban AY (2014) A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay. Mem Inst Oswaldo Cruz 109:246–249CrossRefGoogle Scholar
  4. Coban AY, Uzun M (2013) Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay. Mem Inst Oswaldo Cruz 108:1021–1023CrossRefGoogle Scholar
  5. Coban AY, Deveci A, Sunter AT, Martin A (2014a) Nitrate reductase assay for rapid detection of isoniazid, rifampin, ethambutol, and streptomycin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Clin Microbiol 52:15–19CrossRefGoogle Scholar
  6. Coban AY, Deveci A, Sunter AT, Palomino JC, Martin A (2014b) Resazurin microtiter assay for isoniazid, rifampicin, ethambutol and streptomycin resistance detection in Mycobacterium tuberculosis: updated meta-analysis. Int J Mycobacteriol 3:230–241CrossRefGoogle Scholar
  7. Coban AY, Akbal AU, Uzun M, Cayci YT, Birinci A, Durupinar B (2016a) Evaluation of crystal violet decolarization assay for minimal inhibitory concentration detection of primary antituberculosis drugs against Mycobacterium tuberculosis isolates. Mem Inst Oswaldo Cruz 111:454–459CrossRefGoogle Scholar
  8. Coban AY, Akbal AU, Bicmen C, Albay A, Sig AK, Uzun M, Selale DS, Ozkutuk N, Surucuoglu S, Albayrak N, Ucarman N, Ozkutuk A, Esen N, Ceyhan I, Ozyurt M, Bektore B, Aslan G, Delialioğlu N, Alp A (2016b) Multicenter evaluation of crystal violet decolorization assay (CVDA) for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis. Sci Rep 6:39050CrossRefGoogle Scholar
  9. De la Iglesia AI, Stella EJ, Morbidoni HR (2009) Comparison of the performances of two in-house rapid methods for antitubercular drug susceptibility testing. Antimicrob Agents Chemother 53:808–810CrossRefGoogle Scholar
  10. Dixit P, Singh U, Sharma P, Jain A (2012) Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis. J Microbiol Methods 88:122–126CrossRefGoogle Scholar
  11. Farnia P, Masjedi MR, Mohammadi F, Tabarsei P, Farnia P, Mohammadzadeh AR, Baghei P, Varahram M, Hoffner S, Velayati AA (2008) Colorimetric detection of multidrug-resistant or extensively drug-resistant tuberculosis by use of malachite green indicator dye. J Clin Microbiol 46:796–799CrossRefGoogle Scholar
  12. Jadaun GP, Agarwal C, Sharma H, Ahmed Z, Upadhyay P, Faujdar J, Gupta AK, Das R, Gupta P, Chauhan DS, Sharma VD, Katoch VM (2007) Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay. J Antimicrob Chemother 60:152–155CrossRefGoogle Scholar
  13. Lemus D, Montoro E, Echemendía M, Martin A, Portaels F, Palomino JC (2006) Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories. J Med Microbiol 55:861–863CrossRefGoogle Scholar
  14. López-Roa P, Ruiz-Serrano MJ, Alcalá L, García-Escribano Ráez N, García de Viedma D, Bouza E (2012) Susceptibility testing to second-line drugs and ethambutol by Geno Type MTBDRsl and Bactec MGIT 960 comparing with agar proportion method. Tuberculosis (Edinb) 92:417–421CrossRefGoogle Scholar
  15. Martin A, Montoro E, Lemus D, Simboli N, Morcillo N, Velasco M, Chauca J, Barrera L, Ritacco V, Portaels F, Palomino JC (2005a) Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis. J Microbiol Methods 63:145–150CrossRefGoogle Scholar
  16. Martin A, Morcillo N, Lemus D, Montoro E, Telles MA, Simboli N, Pontino M, Porras T, León C, Velasco M, Chacon L, Barrera L, Ritacco V, Portaels F, Palomino JC (2005b) Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs. Int J Tuberc Lung Dis 9:901–906Google Scholar
  17. Martin A, Panaiotov S, Portaels F, Hoffner S, Palomino JC, Angeby K (2008) The nitrate reductase assay for the rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis. J Antimicrob Chemother 62:56–64CrossRefGoogle Scholar
  18. Mengatto L, Chiani Y, Imaz MS (2006) Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis. Mem Inst Oswaldo Cruz 101:535–542CrossRefGoogle Scholar
  19. Miyata M, Pavan FR, Sato DN, Marino LB, Hirata MH, Cardoso RF, de Melo FA, Leite CQ (2013) Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs. Braz J Microbiol 44:281–285CrossRefGoogle Scholar
  20. Montoro E, Lemus D, Echemendia M, Martin A, Portaels F, Palomino JC (2005) Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis. J Antimicrob Chemother 55:500–505CrossRefGoogle Scholar
  21. Said HM, Kock MM, Ismail NA, Baba K, Omar SV, Osman AG, Hoosen AA, Ehlers MM (2012) Between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa. BMC Infect Dis 12:369CrossRefGoogle Scholar
  22. Shah NS, Wright A, Bai GH, Barrera L, Boulahbal F, Martín-Casabona N, Drobniewski F, Gilpin C, Havelková M, Lepe R, Lumb R, Metchock B, Portaels F, Rodrigues MF, Rüsch-Gerdes S, Van Deun A, Vincent V, Laserson K, Wells C, Cegielski JP (2007) Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis 13:380–387CrossRefGoogle Scholar
  23. Watson PF, Petrie A (2010) Method agreement analysis: a review of correct methodology. Theriogenology 73:1167–1179CrossRefGoogle Scholar
  24. Zhao P, Fang F, Yu Q, Guo J, Zhang JH, Qu J, Liu Y (2014) Evaluation of BACTEC MGIT 960 system for testing susceptibility of Mycobacterium tuberculosis to first-line drugs in China. PLoS ONE 9:e99659CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Ahmet Yilmaz Coban
    • 1
    Email author
  • Ahmet Ugur Akbal
    • 2
  • Ismail Ceyhan
    • 3
  • Meltem Uzun
    • 4
  • Deniz Sertel Selale
    • 4
  • Gonul Aslan
    • 5
  • Nuran Delialioglu
    • 5
  • Mustafa Ozyurt
    • 6
  • Beyhan Bektore
    • 6
  • Can Bicmen
    • 7
  • Ahmet Aslanturk
    • 8
  • Nilay Ucarman
    • 8
  • Ali Albay
    • 9
  • Ali Korhan Sig
    • 9
  • Nuri Ozkutuk
    • 10
  • Suheyla Surucuoglu
    • 10
  1. 1.Department of Nutrition and Dietetics, Faculty of Health SciencesAkdeniz UniversityAntalyaTurkey
  2. 2.Department of Medical MicrobiologyOndokuz Mayis University Medical SchoolSamsunTurkey
  3. 3.Medical Microbiology LaboratoryAtaturk Chest Disease and Chest Surgery Training and Research HospitalAnkaraTurkey
  4. 4.Department of Medical Microbiology, Istanbul Medical SchoolIstanbul UniversityIstanbulTurkey
  5. 5.Department of Medical MicrobiologyMersin University Medical SchoolMersinTurkey
  6. 6.Department of Medical Microbiology, Sultan Abdulhamit Han Training and Research Hospital, Gulhane Medical SchoolHealth Sciences UniversityIstanbulTurkey
  7. 7.Medical Microbiology LaboratoryUniversity of Health Sciences, Dr. Suat Seren Chest Diseases and Surgery Training and Research HospitalIzmirTurkey
  8. 8.Tuberculosis Reference LaboratoryPublic Health Agency of TurkeyAnkaraTurkey
  9. 9.Department of Medical Microbiology, Gulhane Medical SchoolHealth Sciences UniversityAnkaraTurkey
  10. 10.Department of Medical MicrobiologyCelal Bayar University Medical SchoolManisaTurkey

Personalised recommendations