|Year : 2019 | Volume
| Issue : 2 | Page : 157-161
Laboratory diagnosis of nontuberculous mycobacteria in a Belgium Hospital
Anandi Martin1, Alexandre Colmant2, Alexia Verroken2, Hector Rodriguez-Villalobos2
1 Department of Medical Microbiology, Institute of Experimental and Clinical Research, Université Catholique de Louvain, Brussels, Belgium
2 Department of Microbiology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Brussels, Belgium
|Date of Web Publication||14-Jun-2019|
Department of Medical Microbiology, Institute of Experimental and Clinical Research, Université Catholique de Louvain, Avenue Hippocrate, 54, 1200-Brussels
Source of Support: None, Conflict of Interest: None
Background: Nontuberculous mycobacteria (NTM) have been identified in human pulmonary and extrapulmonary infections and are increasing globally, which makes it challenging to identify them. This article reports our experience with the laboratory identification of NTM in clinical practice among pulmonary and extrapulmonary samples received in our routine work. Methods: The study was conducted at the Université Catholique de Louvain at the Cliniques Universitaires Saint-Luc, Brussels, Belgium, from 2015 to 2018. A total of 386 clinical samples were collected from patients suspected of having pulmonary or extrapulmonary mycobacterial infections. Routine laboratory methods phenotypic and molecular tests were performed. Results: The majority of NTM species were isolated from pulmonary samples (68%). The most prevalent species identified were Mycobacterium chimaera_intracellulare group (32%), followed by Mycobacterium avium complex (21%), Mycobacterium abscessus complex (18%), Mycobacterium gordonae (9%), and Mycobacterium chelonae (4%). In extrapulmonary samples, M. avium and M. chimaera_intracellulare were the most frequently isolated. Conclusion: The species diversity of NTM found in our setting suggests the importance of the use of new modern methods for accurate identification of NTM at species level and in some case at subspecies level for the proper treatment and management of patients.
Keywords: Culture, diagnosis, matrix-assisted laser desorption ionization-time of flight, mycobacteria, sequencing
|How to cite this article:|
Martin A, Colmant A, Verroken A, Rodriguez-Villalobos H. Laboratory diagnosis of nontuberculous mycobacteria in a Belgium Hospital. Int J Mycobacteriol 2019;8:157-61
|How to cite this URL:|
Martin A, Colmant A, Verroken A, Rodriguez-Villalobos H. Laboratory diagnosis of nontuberculous mycobacteria in a Belgium Hospital. Int J Mycobacteriol [serial online] 2019 [cited 2020 Feb 18];8:157-61. Available from: http://www.ijmyco.org/text.asp?2019/8/2/157/260375
| Introduction|| |
Nontuberculous mycobacteria (NTM) refer to species of the genus Mycobacterium other than Mycobacterium tuberculosis complex and Mycobacterium leprae. The increasing importance of NTM in the clinical laboratory is now well recognized, and there is an increased interest in NTM identification. NTM are considered as new emerging pathogens that affect both immunocompromised and immunocompetent patients leading to an emerging public health problem. The rise in NTM isolation stresses the need for faster methods for their identification and the selection of appropriate therapy. However, NTM remain difficult to diagnose and very problematic to treat., The magnitude of the public health challenge associated with NTM is probably largely under-recognized because the disease caused by NTM is usually nonnotifiable. Laboratory methods play an important role in the NTM diagnosis. The NTM are grouped into slow and rapid growers. Of clinical importance is the slowly growing mycobacteria, namely the Mycobacterium avium complex (MAC), Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium xenopi, Mycobacterium simiae, and the rapidly growing mycobacteria, namely, Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum. In the present study, we report our experience with the laboratory identification of NTM in our routine work and give an overview of the NTM isolated in our setting.
| Methods|| |
Study site and data collection
Retrospective analysis of suspected patients of mycobacterial infections, with clinical suspicion of pulmonary or extrapulmonary mycobacterial infection, attending Saint-Luc University Hospital in Brussels, Belgium, between 2015 and 2018. Data were collected retrospectively from the laboratory database for the pulmonary samples (sputum, bronchoalveolar lavage fluid, and lung tissue) and extrapulmonary samples (pleural fluid, ascitic fluid, articular liquid, gastric aspirate, bone marrow, urine, and lymph node aspirate) collected consecutively.
Sample processing and microbiological identification
Acid-fast bacilli (AFB) staining and culture for mycobacteria remain the core of our diagnostic process. Samples received are processed for AFB staining and then processed for digestion and concentration following the standard protocol. The concentrated sample was inoculated in the BACTEC MGIT 960 tube following manufacturer's instructions (Becton Dickinson) and incubated at 37°C. In parallel, three Lowenstein–Jensen medium were incubated at 37°C, 30°C, and 42°C. From the MGIT-positive tube, a smear was prepared for Ziehl–Neelsen/auramine fluorescence staining and a drop inoculated into a blood agar plates for checking any contamination (defined as growth after 48 h of incubation at 37°C). If positive for AFB, it was then subject to immunochromatographic assay (TBcID, Becton Dickinson) for MPT64 antigen detection. Those found negative for MPT64 antigen (meaning the presence of NTM) were further identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS according to the manufacturer's instructions (Bruker Daltonics GmbH Germany) and if needed by the sequencing of the rpoB gene. Conventional PCR was performed for partial amplification of rpoB gene according to Adékambi et al.
| Results|| |
A total of 386 samples were enrolled during the study 2015–2018. Among them, 264 (68%) were pulmonary samples and 122 (32%) were extrapulmonary samples. [Figure 1] shows the overall proportion of the different NTM species identified. The most prevalent species were Mycobacterium chimaera_intracellulare group (32%), followed by MAC (21%), M. abscessus complex (18%), Mycobacterium gordonae (9%), M. chelonae (4%), M. xenopi (3%), M. kansasii (2%), M. neoaurum (2%), M. paragordonae, and 1% or less of M. colombiense, M. peregrinum, M. septicum, M. sentence, M. marinum, and M. marseillense. The majority of NTM species were isolated from pulmonary samples mostly coming from sputum samples (193), bronchial lavage (61), as well as from endotracheal aspirate (9), and others from strains and BACTEC myco tubes received (4) for identification. In extrapulmonary samples, the majority of NTM were identified from biopsies (79), articular liquid (14), some from urine (9), heat cooler device (5), hemoculture (5), stools (4), smears (4), bone marrow (3), ascitic puncture (3), gastric aspirate (2), or catheter (1). There was a diversity of NTM species isolated in our hospital in both pulmonary and extrapulmonary samples. M. chimaera_intracellulare, M. avium, M. abscessus, and M. chelonae were the majority species reported in pulmonary samples [Table 1]. M. avium and M. chimaera_intracellulare were most frequently isolated in extrapulmonary samples [Table 2]. Furthermore, a nonnegligible number of biopsy samples evaluated only by rpoB sequencing gave negative results.
|Table 1: Nontuberculous mycobacteria species distribution on pulmonary mycobacterial isolates|
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|Table 2: Nontuberculous mycobacteria species distribution on extrapulmonary mycobacterial isolates|
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| Discussion|| |
NTM are important pathogens due to their high level of drug resistance. Thus, the correct identification of NTM is essential, as their treatment varies widely between species. Nevertheless, identification is challenging due to the slow and fastidious growth of these mycobacteria. Methods used for the laboratory diagnosis of mycobacteria are continually evolving to achieve more rapid and accurate results. We present here the first retrospective laboratory-based study in the identification of clinical NTM species in a Belgium hospital in Brussels attending patients suspected of mycobacterial disease. We found that the majority of NTM were collected from respiratory samples. Furthermore, the results underline the importance of identifying NTM in suspected nonpulmonary TB patients.
We found that the most commonly isolated NTM were M. chimaera_intracellulare group, MAC, and M. abscessus complex. MAC is by far the most frequently encountered NTM pathogen in pulmonary disease in both European and worldwide countries.,, In an inventory study of NTM in the European Union performed by van der Werf et al. in 2014, it was shown that 99 different NTM species were identified. M. avium, M. gordonae, M. xenopi, M. intracellulare, and M. fortuitum were the most frequently identified. M. gordonae is also well known to be the most frequently recovered NTM from environmental samples and mostly considered not to be clinically relevant. However, global NTM studies are limited. The frequency of M. chimaera among Belgian patients has been reported in 2015; however, no cardiac infection caused by M. chimaera has been reported until today. Nevertheless, as also shown in our study, an important number of pulmonary infections caused by M. chimaera_ intracellulare group are diagnosed each year in Belgium. M. chimaera and M. intracellulare are genetically very close but recently appeared to the present different epidemiological and clinical significance. Therefore, it has become important for laboratories to use adequate techniques allowing precise species identification. In our study, of the 47 M. chimaera_intracellulare group identified in sputum samples, 17 were M. chimaera, 2 were M. intracellulare, 1 was M. malmoense, and 1 was M. xenopi classified by rpoB sequencing. However, 24 were not performed to sequencing level, which is a limitation of our study. We were also limited for some samples to species results as reported in the laboratory database, which frequently grouped M. abscessus complex despite their clinical subspecies differences.
In our algorithm, all clinical samples are first processed for AFB smear microscopy. AFB staining smear microscopy is important in alerting the clinician at an early stage for the presence of mycobacteria. Cultures are prone to overgrowth with other respiratory flora and copathogens which hide the presence of slow growth NTM and therefore reduce the diagnosis, particularly in cystic fibrosis (CF). To overcome this issue, different possibilities have been recommended, such as the decontamination of sputum samples with 1% or 2% N-acetyl-L-cysteine-NaOH, with a second-step employing additional agents, such as 5% oxalic acid or 1% chlorhexidine for more heavily contaminated samples., A novel selective agar rapidly growing mycobacteria (RGM) medium (Newcastle upon Tyne, United Kingdom) has been proposed for the isolation of RGM from the sputum of CF patients. Furthermore, extended incubation of RGM for 28 days facilitates the isolation of slow-growing species including MAC. We have included the RGM medium in our algorithm for the samples coming from CF patients. Processing the sample soon after collection also reduces bacterial overgrowth and delays in sample processing may potentially decrease yield, which may be ameliorated by refrigeration if delay is unavoidable. Liquid culture media such as the BACTEC 960 MGIT system (Becton Dickinson) are more sensitive for the detection of NTM and offer faster time to detection than solid L-J culture.
In our experience, MALDI-TOF MS procedure involves little handling and few working hours and has definitively replaced the identification by biochemical tests, which were time-consuming and restricted to a limited number of species. However, phenotypic characteristic on solid medium such as morphology, pigmentation, and grow (rapid or slow growers) can add valuable information. The Bruker library, unfortunately, groups the complex level for the M. tuberculosis complex, MAC complex, M. chimaera_intracellulare group, and the M. abscessus group (M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense). The differentiation of these species is important since virulence differences have been observed for example between M. avium and M. intracellulare or M. abscessus subsp. bolletii that has been reported to show resistance to clarithromycin. Additional tests are thus required to be performed in these special cases. Molecular line probe assays such as the GenoType CM/AS (Hain Lifescience, Nehren, Germany) identify common NTM species although they are expensive. Although reliable, they are less used in our laboratory due to their cost and the labor involved. Furthermore, line probe assays can detect only a limited number of specified targets against which the probes are directed. However, the GenoType NTM-DR enables M. abscessus subspecies identification and the simultaneous determination of antibiotic resistance to macrolides and aminoglycosides, which is very useful for the adequate therapy regimen.
DNA sequencing of the 16S rRNA gene has been extensively used for the identification of NTM; however, it cannot distinguish between closely related species such as those within the M. abscessus complex. It is recommended to use other targets such as rpoB or hsp65 genes for the identification of both rapid and slow grower mycobacteria. Whole genome sequencing is not yet currently used in routine mainly due to the costs but also for the complexity of the data analysis and the missing platform analysis. We believe that it will become soon accessible for most laboratories.
| Conclusion|| |
The identification of NTM at species and subspecies level is required, given differences in treatment options and implications for patient outcome. Modern molecular tests such as MALDI-TOF and sequencing are accurate for NTM identification and have significantly improved turnaround time in our routine work.
We would like to thank the technical support of Laetitia Toussaint, Florian Bressant, Ali Zitouni, and Marie-Christine Vermeiren for their daily contribution in the mycobacterial diagnostic laboratory.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Porvaznik I, Solovič I, Mokrý J. Non-tuberculous mycobacteria: Classification, diagnostics, and therapy. Adv Exp Med Biol 2017;944:19-25.
Tortoli E, Mariottini A, Mazzarelli G. Evaluation of INNO-LiPA MYCOBACTERIA v2: Improved reverse hybridization multiple DNA probe assay for mycobacterial identification. J Clin Microbiol 2003;41:4418-20.
Prevots DR, Marras TK. Epidemiology of human pulmonary infection with nontuberculous mycobacteria: A review. Clin Chest Med 2015;36:13-34.
Roux AL, Catherinot E, Ripoll F, Soismier N, Macheras E, Ravilly S, et al.
Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France. J Clin Microbiol 2009;47:4124-8.
van Ingen J, Bendien SA, de Lange WC, Hoefsloot W, Dekhuijzen PN, Boeree MJ, et al.
Clinical relevance of non-tuberculous mycobacteria isolated in the Nijmegen-Arnhem region, the Netherlands. Thorax 2009;64:502-6.
Kent PT, Kubica GP. Public Health Mycobacteriology: A Guide for the Level III Laboratory. Atlanta, Georgia: Centers for Disease Control, US Department of Health and Human Services; 1985.
Adékambi T, Colson P, Drancourt M. RpoB-based identification of nonpigmented and late-pigmenting rapidly growing mycobacteria. J Clin Microbiol 2003;41:5699-708.
Hoefsloot W, van Ingen J, Andrejak C, Angeby K, Bauriaud R, Bemer P, et al.
The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: An NTM-NET collaborative study. Eur Respir J 2013;42:1604-13.
Johnson MM, Odell JA. Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 2014;6:210-20.
van der Werf MJ, Ködmön C, Katalinić-Janković V, Kummik T, Soini H, Richter E, et al.
Inventory study of non-tuberculous mycobacteria in the European union. BMC Infect Dis 2014;14:62.
Soetaert K, Vluggen C, André E, Vanhoof R, Vanfleteren B, Mathys V, et al.
Frequency of Mycobacterium chimaera
among Belgian patients, 2015. J Med Microbiol 2016;65:1307-10.
Haworth CS, Banks J, Capstick T, Fisher AJ, Gorsuch T, Laurenson IF, et al.
British thoracic society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD). Thorax 2017;72:ii1-ii64.
Floto RA, Olivier KN, Saiman L, Daley CL, Herrmann JL, Nick JA, 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. Thora×2016;71 Suppl 1:i1-22.
Preece CL, Perry A, Gray B, Kenna DT, Jones AL, Cummings SP, et al.
Anovel culture medium for isolation of rapidly-growing mycobacteria from the sputum of patients with cystic fibrosis. J Cyst Fibros 2016;15:186-91.
Plongla R, Preece CL, Perry JD, Gilligan PH. Evaluation of RGM medium for isolation of nontuberculous mycobacteria from respiratory samples from patients with cystic fibrosis in the United States. J Clin Microbiol 2017;55:1469-77.
van Ingen J. Microbiological diagnosis of nontuberculous mycobacterial pulmonary disease. Clin Chest Med 2015;36:43-54.
Brown-Elliott BA, Nash KA, Wallace RJ Jr. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev 2012;25:545-82.
Mougari F, Bouziane F, Crockett F, Nessar R, Chau F, Veziris N, et al.
Selection of resistance to clarithromycin in Mycobacterium abscessus
subspecies. Antimicrob Agents Chemother 2017;61. pii: e00943-16.
Monteserin J, Paul R, Lopez B, Cnockaert M, Tortoli E, Menéndez C, et al.
Combined approach to the identification of clinically infrequent non-tuberculous mycobacteria in Argentina. Int J Tuberc Lung Dis 2016;20:1257-62.
[Table 1], [Table 2]