A new selective medium for rapidly growing mycobacteria (RGM medium) was evaluated on respiratory specimens from non-cystic fibrosis patients and compared to the mycobacterial growth indicator tube (MGIT) system and Middlebrook 7H11 agar for the isolation of all nontuberculous mycobacteria (NTM). A total of 203 mucolyzed respiratory specimens collected from 163 patients were inoculated on RGM medium and incubated at both 30°C (RGM30) and 35°C (RGM35) over a 28day period. N-Acetyl-L-cysteine-sodium hydroxide (NALC-NaOH)-decontaminated specimens were inoculated into MGIT and Middlebrook 7H11 agar and incubated at 35°C for 42 days. NTM were identified by matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) or gene sequencing. A total of 133 NTM isolates were recovered overall from 101 (49.8%) specimens collected from 85 (52.1%) patients by a combination of all culture methods. The sensitivity of RGM30 for the recovery of NTM was significantly higher than that of either the MGIT system (76.7% versus 59.4%; P ϭ 0.01) or Middlebrook 7H11 agar (76.7% versus 47.4%; P ϭ 0.0001) alone, but it was not significantly different from that of an acidfast bacillus culture (AFC) which includes both MGIT and Middlebrook 7H11 agar (76.7% versus 63.9%; P ϭ 0.0647). RGM35 had significantly lower sensitivity than the MGIT system (49.6% versus 59.4%; P ϭ 0.0367) and AFC (49.6% versus 63.9%; P ϭ 0.0023). RGM medium was highly effective at inhibiting the growth of nonmycobacterial organisms in the respiratory specimens, with breakthrough contamination rates of 5.4% and 4.4% for RGM30 and RGM35, respectively.
Pulmonary disease arising from slow‐growing mycobacterial infections has emerged as an increasingly prevalent clinical concern over the past two to three decades. Proteins belonging to the family of ESAT‐6 secretion (Esx) systems play critical roles in the virulence of most pathogenic mycobacterial species and are associated with drug resistance. However, no clinical applications can detect and discriminate the expression of species‐specific variants of these proteins in clinical samples, such as early growth cultures, for rapid diagnosis of specific mycobacterial infections, which may require distinct interventions. Conventional immunoassay approaches are not suitable for this purpose due to the significant degree of conservation of Esx proteins among species. Herein we describe the development of a novel immunoprecipitation‐coupled mass spectrometry assay that can distinguish Esx proteins that are expressed by slow‐growing mycobacterial species commonly detected in clinical isolates. This approach uses custom antibodies raised against single semi‐conserved peptide regions in M. tuberculosis (Mtb) EsxB and EsxN to capture corresponding peptides from protein orthologs of mycobacteria associated with human respiratory infections, including Mtb, M. avium, M. intracellulare, M. kansasii, M. gordonae, and M. marinum, to detect these species in standard clinical cultures at the first sign mycobacterial growth to allow rapid disease diagnosis.
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