Table 1.

Detection, identification and drug sensitivity testing procedures for non-tuberculous mycobacteria (NTM) in 10 European Union countries

Country	Detection NTM	Identification NTM	Drug sensitivity testing
Croatia	Smear microscopy and culture on solid and liquid media	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany) supplemented with phenotypic analysis including examination of growth characteristics, temperature tolerance, pigmentation and biochemical properties	For rapidly growing species* by E-test (AB Biodisk, Solna, Sweden) strips or minimum inhibitory concentration (MIC) testing.
Denmark	Smear microscopy, culture on solid (Löwenstein-Jensen) and liquid media (BD Bactec MGIT 960) and PCR	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany) supplemented by 16S rDNA sequencing and/or other methods	For rapid growing species: MIC determination on TREK microwell plates (TREK Diagnostic Systems, Cleveland, Ohio, United States of America). For slow growing species only at specific request of clinician
Estonia	Smear microscopy, culture on solid media (e.g. Löwenstein-Jensen) and liquid media (BD Bactec MGIT 960)	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany)	Not performed
Finland	Culture on solid media (e.g. Löwenstein-Jensen or Middlebrook 7H11) and liquid media (BD Bactec MGIT 960)	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany) and, if needed, 16S rDNA sequencing	For species belonging to M. avium complex (MAC): narrow range MIC method for azithromycin, clarithromycin, ethambutol, clofatzimin, and rifabutin. For rapid growing species belonging to the M. chelonae-fortuitum complex: E-test (BioMerieux, Lyon, France)
Germany	Smear microscopy and culture on solid and liquid media, and for certain specimens (skin, lymph nodes, sputum from CF patients) incubation on specific media at lower temperatures (31°C)	Line probe assays or sequence analysis(16S rDNA, ITS, rpoB)	Slowly growing species: modified proportion method using MGIT liquid media (BACTEC MGIT 960 or manual system). Drugs tested comprise first line TB drugs as well as clarithromycin or on special request fluoroquinolones. For other slow growing species, i.e. M. avium/intracellulare: A combination of drugs is applied for DST testing
Greece	Smear microscopy and culture on solid and liquid media	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany) and when requested by the clinicians, 16S rDNA and hsp65 gene sequencing	For slow growing species, the modified proportion method using BACTEC MGIT 960 or LJ is applied. First line anti-TB drugs and clarithromycin are tested according to ATS/ IDSA guidelines [5]. On special request FQs are also tested. For rapid growing species, MIC testing on microwell plates is performed (TREK Diagnostic Systems, Cleveland, Ohio, United States of America).
Italy	Culture on solid (Löwenstein-Jensen) and liquid media (BD Bactec MGIT 960)	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany) and sequencing of genetic regions (16S rRNA and/or hsp65 genes for slow growers, rpoB gene for rapid growers, ITS1 for members of MAC) whenever the commercially available molecular probes (Genotype CM and AS) produced an identification known to be not 100% free from cross reactions with other species	For rapid growing species: MIC determination (Rapidly growing mycobacteria panel, TREK Diagnostic Systems (Cleveland, Ohio, United States of America). For slow growing species: susceptibility to chlarithromycin is determined for some cases.
Luxembourg	Culture on solid (Lowenstein Jensen, Coletsos and Middlebrook 7H11) and liquid media (BD Bactec MGIT 960)	To distinguish MTB complex from NTM isolates the Anyplex MTB/NTM Real Time detection kit (Seegene) is used. In a second stage, NTM specimens are sequenced (sequencing of the hsp65 gene)	Not performed in country. On explicit request of the doctor, the isolate is sent to the Belgian Scientific Institute for Public Health
Netherlands	Culture on liquid media (BD Bactec MGIT 960)	Commercially available reverse line blot assays	7H10 agar dilution method [23] for both slow and rapid growing species
Slovenia	Culture on solid and liquid media. Some clinical samples are incubated at 30°C and on media with special supplements.	GenoType Mycobacterium CM/AS (Hain Lifescience, Nehren, Germany), colony morphology on transparent medium Middlebrook 7H10, and some phenotypic tests	At specific request of clinician for rapid growing species and some slow growing species: E-test (AB Biodisk, Solna, Sweden)

*For definition of slow growing and rapidly growing species see reference [24].