Comparison of Mycobacterial Growth Indicator Tube with Culture on RGM Selective Agar for Detection of Mycobacteria in Sputum Samples from Patients with Cystic Fibrosis

Abstract

dNontuberculous mycobacteria (NTM) are an important cause of pulmonary disease in patients with cystic fibrosis (CF). A new culture medium (RGM medium) for the isolation of rapidly growing mycobacteria from the sputum of cystic fibrosis patients has recently been reported. The aim of this study was to compare culture of sputum samples on RGM medium with culture using a standard automated liquid culture method. Sputum samples were obtained from 187 distinct patients with CF attending King's College Hospital, London, United Kingdom. Each sample was decontaminated with 3% oxalic acid and inoculated into a mycobacterial growth indicator tube (MGIT) that was monitored for 42 days using the Bactec MGIT 960 instrument. Each sample was also cultured, without decontamination, onto RGM medium, which was incubated for 10 days at 30°C. Mycobacteria were isolated from 28 patients (prevalence, 15%). Mycobacteria were detected in 24 samples (86%) using the MGIT and in 23 samples (82%) using RGM medium (P ‫؍‬ 1.00). In this setting, RGM medium showed sensitivity equivalent to that of the MGIT for isolation of NTM from the sputum of patients with CF. RGM medium offers a simple, convenient tool that can be embedded into routine culture methods, allowing the culture of all sputum samples that are submitted from patients with CF. N ontuberculous mycobacteria (NTM) are an important cause of pulmonary disease in patients with cystic fibrosis (CF) (1). In the largest studies, the prevalence of NTM in sputum has been estimated to be 6 to 13% for patients with CF (2), and there is convincing evidence that prevalence is increasing in the CF population (3-6). NTM are detected by culture of sputum using methods that were designed primarily for the isolation of Mycobacterium tuberculosis. Consensus guidelines state that culture should be performed in liquid media using an automated growth detection system (such as the mycobacterial growth indicator tube [MGIT]) and note that concomitant culture on solid media (e.g., Lowenstein-Jensen medium) may increase the diagnostic yield (1).Detection of NTM is frequently challenging due to the presence of other bacterial and fungal species in the lungs of CF patients. Competing species typically show a higher growth rate than mycobacteria and are frequently resistant to multiple antibiotics that might be used to select for growth of mycobacteria. To resolve this, sputum samples are subjected to treatment with chemicals in order to eliminate or reduce the burden of nonmycobacterial species. Chemical treatments include N-acetyl-L-cysteine (0.5%) plus sodium hydroxide (2%), 5% oxalic acid, or 1% chlorhexidine (1). However, such decontamination protocols are labor intensive, they may reduce the viability of NTM, and they may be ultimately unsuccessful if there is a high burden of nonmycobacterial species (7-9).Culture on solid agar-based media is an attractive option as this can potentially allow recovery of NTM even in specimens that contain other viable species. For example, Esther et al. (10) demonstrat...

Performance of RGM Medium for Isolation of Nontuberculous Mycobacteria from Respiratory Specimens from Non-Cystic Fibrosis Patients

“…Although the sensitivity of RGM30 with a 28-day incubation period was comparable to that of the MGIT system (P ϭ 0.6171) and Middlebrook 7H11 agar (P ϭ 0.3711) for the recovery of MABSC, RGM30 performed better for the recovery of all rapidly growing mycobacteria isolated in this study (P ϭ 0.0001). This finding was consistent with previous studies (14,15), demonstrating that RGM medium provided performance equivalent to that of the MGIT system or even better for the recovery of MABSC and other rapidly growing mycobacteria. Notably, the time to recovery of MABSC on RGM medium was comparable to the average time to positivity of MABSC on the MGIT system (ϳ4 days).…”

“…For slowly growing mycobacteria, RGM30 and RGM35 failed to recover 13 and 10 isolates of MAC, respectively, compared to the MGIT system (P Ͻ 0.05). The difference between RGM30 and the MGIT system for the recovery of MAC from clinical specimens had not been shown in previous studies (14,15). RGM medium showed potential as an alternative medium for recovery of mycobacteria, especially rapidly growing mycobacteria.…”

“…Recently, a new selective medium (RGM medium), which is composed of Middlebrook 7H9-based agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) and four antimicrobial agents (12,13), has been shown to have high performance for the recovery of rapidly growing mycobacteria from respiratory specimens from CF patients (12)(13)(14)(15). RGM medium was superior to Burkholderia cepacia complex selective medium and showed performance equivalent to or higher than that of the MGIT system.…”

“…RGM medium was superior to Burkholderia cepacia complex selective medium and showed performance equivalent to or higher than that of the MGIT system. An added advantage was the ability of RGM medium to inhibit growth of other nonmycobacterial organisms in CF patients' respiratory specimens without requirement for a decontamination step (12)(13)(14)(15). The use of an extended incubation period of 28 days allows RGM medium to be used for the isolation of all NTM, rather than simply rapidly growing species, but there are only limited data on the recovery of slowly growing species (15).…”

Cell Growth Measurement

“…BACTEC MGIT [50,51] measures microbial growth by oxygen depletion which requires anaerobic conditions, so the bacteria must be grown in a sealed tube or compartment. This method has been widely applied in medical diagnosis but is not suitable for high-throughput plate-based AST assays.…”