Application of Replicate Organism Detection and Counting Method (RODAC) in Measuring Mycobacterium Tuberculosis Contamination in High Burden Laboratories

Lidya Chaidir, Neng Rina Susilawati, Mandala Ajie, Jessi Annisa, Muti'ah Nurul Jihadah

Abstract


Background: Technicians working in high burden tuberculosis (TB) laboratories pose a higher risk of being infected by Mycobacterium tuberculosis from clinical samples. Contamination control is mandatory to detect the release of bacteria into the working environment and to minimize the risk of exposure to the workers. The contamination measurement is rarely performed due to the lack of standard methodology. This study optimized and applied a unique culture-based method named Replicate Organism Detection and Counting (RODAC) plates to assess the presence of M. tuberculosis contaminant in the laboratory with high burden of clinical samples.

Methods: RODAC was applied on twenty working surfaces in the Mycobacteriology Laboratory of Universitas Padjadjaran. The results of RODAC were compared with DNA-based detection from the same working surfaces using in-house IS6110 real-time PCR (IS6110-qPCR). The detection limit of the RODAC plate was 19.6 CFU mL-1.

Results: From all working surfaces tested, two distinct colonies were found on RODAC plate stamped on the Ziehl-Neelsen staining basin. Those colonies were identified as M. tuberculosis and non-tuberculous mycobacteria (NTM), as confirmed by the MPT64 antigen test and the presence of acid-fast bacilli. IS6110-qPCR detected the presence of M. tuberculosis DNA in ten sampling points, including the ZN staining basin, incubators, and microscopy areas. IS6110-qPCR detected more working surface contamination versus RODAC. However, it was noted that RODAC, which was a culture-based method, detected live bacteria, while PCR could not distinguish between live and dead bacteria.

Conclusion: The application of the RODAC plate is more suitable for monitoring the contamination of live bacteria in the working environment and to inform a proper corrective action.


Keywords


Contamination control, IS6110, Mycobacterium tuberculosis, qPCR, RODAC

Full Text:

PDF

References


  1. Garnett J, Jones D, Chin G, Spiegel JM, Yassi A, Naicker N. Occupational tuberculosis among laboratory workers in South Africa: applying a surveillance system to strengthen prevention and control. Int J Environ Res Public Health. 2020;17(5):1462.
  2. World Health Organization. Tuberculosis laboratory biosafety manual. Geneva: World Health Organization; 2012.
  3. Shinnick TM, Glipin C. A risk assessment-based approach to defining minimum biosafety precautions for tuberculosis laboratories in resource-limited settings. Applied Biosafety. 2012;17(1):6–10.
  4. Van Soolingen D, Wisselink HJ, Lumb R, Anthony R, van der Zanden A, Gilpin C. Practical biosafety in the tuberculosis laboratory: containment at the source is what truly counts. Int J Tuberc Lung Dis. 2014;18(8):885–9.
  5. FIND. TB laboratory quality management systems towards accreditation harmonized checklist (Incorporating SLIPTA and GLI stepwise process towards TB laboratory accreditation) [Internet]. 2016. [cited 2022 April 17]. Available from: https://www.finddx.org/wp-content/uploads/2016/07/NEW-TB-Harmonized-Checklist-v2.1-2-2016.pdf.
  6. European Centre for Disease Prevention and Control. Handbook on tuberculosis laboratory diagnostic methods in the European Union: technical report. Stockholm: ECDC; 2018.
  7. Ancona N, Bernardo J, Desmond E, Etter M, Gaynor A, Jamieson F, et al. Mycobacterium tuberculosis: assessing your laboratory [Internet]. Silver Spring, MD: Association of Public Health Laboratories; 2019. [cited 2022 April 17]. Available from: https://www.aphl.org/aboutAPHL/publications/Documents/ID-2019Apr-TB-Toolkit.pdf.
  8. Kim CK, Chang CL. Quality assurance of laboratory tests for tuberculosis. Korean J Clin Microbiol. 2009;12(4):147–53.
  9. Shiferaw MB, Hailu HA, Fola AA, Derebe MM, Kebede AT, Kebede AA, et al. Tuberculosis laboratory diagnosis quality assurance among public health facilities in West Amhara Region, Ethiopia. PLoS One. 2015;10(9):e0138488.
  10. Johnson MG, Lindsey PH, Harvey CF, Bradley KK. Recognizing laboratory cross-contamination: two false-positive cultures of Mycobacterium tuberculosis—Oklahoma, 2011. Chest. 2013;144(1):319–22.
  11. Daneau G, Nduwamahoro E, Fissette K, Rudelsheim P, van Soolingen D, de Jong BC, et al. Use of RODAC plates to measure containment of Mycobacterium tuberculosis in a Class IIB biosafety cabinet during routine operations. Int J Mycobacteriol. 2016;5(2):148–54.
  12. Arora J, Kumar G, Verma AK, Bhalla M, Sarin R, and Myneedu VP. Utility of MPT64 antigen detection for rapid confirmation of Mycobacterium tuberculosis complex. J Glob Infect Dis. 2015;7(2):66–9.
  13. Chaidir L, Ganiem AR, Vander Zanden A, Muhsinin S, Kusumaningrum T, Kusumadewi I, et al. Comparison of real time IS6110-PCR, microscopy, and culture for diagnosis of tuberculous meningitis in a cohort of adult patients in Indonesia. PLoS One. 2012;7(12):e52001.
  14. Forbes BA, Hall GS, Miller MB, Novak SM, Rowlinson MC, Salfinger M, et al. Practical guidance for clinical microbiology laboratories: mycobacteria. Clin Microbiol Rev. 2018;31(2):e00038–17.
  15. van Zyl-Smit RN, Binder A, Meldau R, Mishra H, Semple PL, Theron G, et al. Comparison of quantitative techniques including Xpert MTB/RIF to evaluate mycobacterial burden. PLoS One. 2011;6(12):e28815.
  16. Nambiar R, Chatellier S, Bereksi N, Van Belkum A, Singh N, Barua B, et.al. Evaluation of Mycotube, a modified version of Lowenstein–Jensen (LJ) Medium, for efficient recovery of Mycobacterium tuberculosis (Mtb). Eur J Clin Microbiol Infect Dis. 2017;36(10):1981–8.
  17. Mickymaray S, Alfaiz FA, Paramasivam A. Efficacy and mechanisms of flavonoids against the emerging opportunistic nontuberculous Mycobacteria. Antibiotics (Basel). 2020;9(8):450.
  18. Kyaw SP, Hanthamrongwit J, Jangpatarapongsa K, Khaenam P, Leepiyasakulchai C. Sensitive detection of the IS 6110 sequence of Mycobacterium tuberculosis complex based on PCR-magnetic bead ELISA. RSC Adv. 2018;8(59):33674–80.
  19. Raj A, Singh N, Gupta KB, Chaudhary D, Yadav A, Chaudhary A, et al. Comparative evaluation of several gene targets for designing a multiplex-PCR for an early diagnosis of extrapulmonary tuberculosis. Yonsei Med J. 2016;57(1):88–96.
  20. Hanscheid T, Grobusch MP. Biosafety and tuberculosis laboratories in Africa. Lancet Infect Dis. 2010;10(9):582–3.
  21. Obenza A, Cruz P, Buttner M, Woodard D. Microbial contamination on ambulance surfaces: a systematic literature review. J Hosp Infect. 2022;122:44–59.
  22. Okamoto K, Rhee Y, Schoeny M, Lolans K, Cheng J, Reddy S, et al. Centers for Disease Control and Prevention Epicenters Program. Flocked nylon swabs versus RODAC plates for detection of multidrug-resistant organisms on environmental surfaces in intensive care units. J Hosp Infect. 2018;98(1):105–8.




DOI: https://doi.org/10.15850/amj.v9n4.2789

Article Metrics

Abstract view : 523 times
PDF - 390 times


 This Journal indexed by

                  

          

 

Creative Commons License
AMJ is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License

 


View My Stats