1887

Abstract

. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the complex (MAC) and complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics.

. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment.

. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex.

. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of subsp. (MAM).

. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between subsp. /subsp. (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology.

. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients’ sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.

Funding
This study was supported by the:
  • Národní Agentura pro Zemědělský Výzkum (Award MZe-RO0518)
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001611
2022-12-13
2024-07-25
Loading full text...

Full text loading...

References

  1. Goetz D, Ren CL. Review of cystic fibrosis. Pediatr Ann 2019; 48:e154–e161 [View Article]
    [Google Scholar]
  2. De Boeck K. Cystic fibrosis in the year 2020: a disease with a new face. Acta Paediatr 2020; 109:893–899 [View Article]
    [Google Scholar]
  3. Bar-On O, Mussaffi H, Mei-Zahav M, Prais D, Steuer G et al. Increasing nontuberculous mycobacteria infection in cystic fibrosis. J Cyst Fibros 2015; 14:53–62 [View Article] [PubMed]
    [Google Scholar]
  4. Adjemian J, Olivier KN, Prevots DR. Epidemiology of pulmonary nontuberculous Mycobacterial sputum positivity in patients with Cystic fibrosis in the United States, 2010-2014. Ann Am Thorac Soc 2018; 15:817–826 [View Article]
    [Google Scholar]
  5. Falkinham JO 3rd, Iseman MD, de Haas P, van Soolingen D. Mycobacterium avium in a shower linked to pulmonary disease. J Water Health 2008; 6:209–213 [View Article] [PubMed]
    [Google Scholar]
  6. Williamson D, Howden B, Stinear T. Mycobacterium chimaera spread from heating and cooling units in heart surgery. N Engl J Med 2017; 376:600–602 [View Article]
    [Google Scholar]
  7. Bryant JM, Grogono DM, Greaves D, Foweraker J, Roddick I et al. Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study. Lancet 2013; 381:1551–1560 [View Article] [PubMed]
    [Google Scholar]
  8. Gupta RS, Lo B, Son J. Phylogenomics and comparative genomic studies robustly support division of the genus Mycobacterium into an emended genus Mycobacterium and four novel genera. Front Microbiol 2018; 9:67 [View Article] [PubMed]
    [Google Scholar]
  9. Qvist T, Gilljam M, Jönsson B, Taylor-Robinson D, Jensen-Fangel S et al. Epidemiology of nontuberculous mycobacteria among patients with cystic fibrosis in Scandinavia. J Cyst Fibros 2015; 14:46–52 [View Article] [PubMed]
    [Google Scholar]
  10. Floto RA, Olivier KN, Saiman L, Daley CL, Herrmann J-L et al. US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis: executive summary. Thorax 2016; 71:88–90 [View Article] [PubMed]
    [Google Scholar]
  11. Roux A-L, Catherinot E, Ripoll F, Soismier N, Macheras E et al. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in france. J Clin Microbiol 2009; 47:4124–4128 [View Article] [PubMed]
    [Google Scholar]
  12. Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: a new antibiotic nightmare. J Antimicrob Chemother 2012; 67:810–818 [View Article] [PubMed]
    [Google Scholar]
  13. Nash KA, Brown-Elliott BA, Wallace RJ. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother 2009; 53:1367–1376 [View Article] [PubMed]
    [Google Scholar]
  14. Cho EH, Huh HJ, Song DJ, Lee SH, Kim CK et al. Drug susceptibility patterns of Mycobacterium abscessus and Mycobacterium massiliense isolated from respiratory specimens. Diagn Microbiol Infect Dis 2019; 93:107–111 [View Article] [PubMed]
    [Google Scholar]
  15. Choi G-E, Shin SJ, Won C-J, Min K-N, Oh T et al. Macrolide treatment for Mycobacterium abscessus and Mycobacterium massiliense infection and inducible resistance. Am J Respir Crit Care Med 2012; 186:917–925 [View Article] [PubMed]
    [Google Scholar]
  16. Bastian S, Veziris N, Roux A-L, Brossier F, Gaillard J-L et al. Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing. Antimicrob Agents Chemother 2011; 55:775–781 [View Article] [PubMed]
    [Google Scholar]
  17. Maurer FP, Rüegger V, Ritter C, Bloemberg GV, Böttger EC. Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41). J Antimicrob Chemother 2012; 67:2606–2611 [View Article] [PubMed]
    [Google Scholar]
  18. Roux A-L, Catherinot E, Soismier N, Heym B, Bellis G et al. Comparing Mycobacterium massiliense and Mycobacterium abscessus lung infections in cystic fibrosis patients. J Cyst Fibros 2015; 14:63–69 [View Article] [PubMed]
    [Google Scholar]
  19. Martin A, Colmant A, Verroken A, Rodriguez-Villalobos H. Laboratory diagnosis of nontuberculous mycobacteria in a Belgium Hospital. Int J Mycobacteriol 2019; 8:157–161 [View Article] [PubMed]
    [Google Scholar]
  20. Moravkova M, Hlozek P, Beran V, Pavlik I, Preziuso S et al. Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues. Res Vet Sci 2008; 85:257–264 [View Article] [PubMed]
    [Google Scholar]
  21. Wilton S, Cousins D. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl 1992; 1:269–273 [View Article] [PubMed]
    [Google Scholar]
  22. Kim HY, Lee SY, Kim BJ, Kook YH. Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates. Indian J Med Microbiol 2016; 34:369–374 [View Article] [PubMed]
    [Google Scholar]
  23. Sevilla IA, Molina E, Elguezabal N, Pérez V, Garrido JM et al. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay. J Clin Microbiol 2015; 53:930–940 [View Article] [PubMed]
    [Google Scholar]
  24. Slana I, Kralik P, Kralova A, Pavlik I. On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol 2008; 128:250–257 [View Article] [PubMed]
    [Google Scholar]
  25. Kralik P, Slana I, Kralova A, Babak V, Whitlock RH et al. Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Vet Microbiol 2011; 149:133–138 [View Article] [PubMed]
    [Google Scholar]
  26. Huh HJ, Kim S-Y, Shim HJ, Kim DH, Yoo IY et al. GenoType NTM-DR performance evaluation for identification of Mycobacterium avium complex and Mycobacterium abscessus and determination of clarithromycin and amikacin resistance. J Clin Microbiol 2019; 57:e00516-19 [View Article]
    [Google Scholar]
  27. Jones RS, Shier KL, Master RN, Bao JR, Clark RB. Current significance of the Mycobacterium chelonae-abscessus group. Diagn Microbiol Infect Dis 2019; 94:248–254 [View Article] [PubMed]
    [Google Scholar]
  28. Balada-Llasat JM, Kamboj K, Pancholi P. Identification of mycobacteria from solid and liquid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry in the clinical laboratory. J Clin Microbiol 2013; 51:2875–2879 [View Article] [PubMed]
    [Google Scholar]
  29. Mather CA, Rivera SF, Butler-Wu SM. Comparison of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of mycobacteria using simplified protein extraction protocols. J Clin Microbiol 2014; 52:130–138 [View Article] [PubMed]
    [Google Scholar]
  30. Degiacomi G, Sammartino JC, Chiarelli LR, Riabova O, Makarov V et al. Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients. Int J Mol Sci 2019; 20:23 [View Article] [PubMed]
    [Google Scholar]
  31. Salsgiver EL, Fink AK, Knapp EA, LiPuma JJ, Olivier KN et al. Changing epidemiology of the respiratory bacteriology of patients with cystic fibrosis. Chest 2016; 149:390–400 [View Article] [PubMed]
    [Google Scholar]
  32. Caverly LJ, Carmody LA, Haig S-J, Kotlarz N, Kalikin LM et al. Culture-independent identification of nontuberculous Mycobacteria in cystic fibrosis respiratory samples. PLoS One 2016; 11:e0153876 [View Article]
    [Google Scholar]
  33. Caverly LJ, Zimbric M, Azar M, Opron K, LiPuma JJ. Cystic fibrosis airway microbiota associated with outcomes of nontuberculous mycobacterial infection. ERJ Open Res 2021; 7:00578-2020 [View Article]
    [Google Scholar]
  34. Kim K, Kim BJ, Shim TS, Hong SH, Kook YH et al. Development of a peptide nucleic acid-based multiprobe real-time PCR method targeting the hsp65 gene for differentiation among Mycobacterium abscessus strains. J Clin Microbiol 2015; 53:1403–1405 [View Article] [PubMed]
    [Google Scholar]
  35. Kim K, Hong S-H, Kim B-J, Kim B-R, Lee S-Y et al. Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples. BMC Infect Dis 2015; 15:325 [View Article]
    [Google Scholar]
  36. Syrmis MW, Pandey S, Tolson C, Carter R, Congdon J et al. Identification of Mycobacterium abscessus complex and M. abscessus subsp. massiliense culture isolates by real-time assays. J Med Microbiol 2015; 64:790–794 [View Article] [PubMed]
    [Google Scholar]
  37. Marras SAE, Chen L, Shashkina E, Davidson RM, Strong M et al. A molecular-beacon-based multiplex real-time PCR assay to distinguish Mycobacterium abscessus subspecies and determine macrolide susceptibility. J Clin Microbiol 2021; 59:e0045521 [View Article]
    [Google Scholar]
  38. Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997; 63:3741–3751 [View Article] [PubMed]
    [Google Scholar]
  39. Falkinham JO. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996; 9:177–215 [View Article]
    [Google Scholar]
  40. Thorel MF, Krichevsky M, Lévy-Frébault VV. Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. Paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov. Int J Syst Bacteriol 1990; 40:254–260 [View Article]
    [Google Scholar]
  41. Lee JS, Lee JH, Yoon SH, Kim TS, Seong M-W et al. Implication of species change of nontuberculous Mycobacteria during or after treatment. BMC Pulm Med 2017; 17:213 [View Article]
    [Google Scholar]
  42. Carvalho NFG de, Pavan F, Sato DN, Leite CQF, Arbeit RD et al. Genetic correlates of clarithromycin susceptibility among isolates of the Mycobacterium abscessus group and the potential clinical applicability of a PCR-based analysis of erm(41). J Antimicrob Chemother 2018; 73:862–866 [View Article] [PubMed]
    [Google Scholar]
  43. Chew KL, Cheng JWS, Hudaa Osman N, Lin RTP, Teo JWP. Predominance of clarithromycin-susceptible Mycobacterium massiliense subspecies: Characterization of the Mycobacterium abscessus complex at a tertiary acute care hospital. J Med Microbiol 2017; 66:1443–1447 [View Article] [PubMed]
    [Google Scholar]
  44. Lee SH, Yoo HK, Kim SH, Koh W-J, Kim CK et al. The drug resistance profile of Mycobacterium abscessus group strains from Korea. Ann Lab Med 2014; 34:31–37 [View Article] [PubMed]
    [Google Scholar]
  45. Maurer FP, Castelberg C, Quiblier C, Böttger EC, Somoskövi A. Erm(41)-dependent inducible resistance to azithromycin and clarithromycin in clinical isolates of Mycobacterium abscessus. J Antimicrob Chemother 2014; 69:1559–1563 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001611
Loading
/content/journal/jmm/10.1099/jmm.0.001611
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