To make a firm diagnosis of NTM disease in an individual patient, culture of representative clinical specimens and histological examination of tissue biopsy specimens are generally necessary. Given the importance of culture in the diagnosis of NTM disease, it is important to realize for microbiologists and clinicians that good communication between both parties may actually help to optimize culture conditions according to the particularities of the patient and therewith increase the sensitivity of culture and laboratory diagnosis of NTM disease.
Microbiology: Sample Decontamination and Smear Microscopy
Smear microscopy is mostly done in a two-step procedure, where samples are screened by fluorochrome (auramine) staining and positives are confirmed by classical Ziehl-Neelsen staining. Fluorochrome staining is the screening method because of its high sensitivity but low specificity. NTM are as likely as M. tuberculosis to be detected by fluorochrome staining.
To lower the loads of commensal flora associated with the human airways and digestive tract and thus overgrowth of cultures, sputum and bronchoalveolar lavage fluid as well as feces samples are decontaminated prior to inoculating them on selective media for Mycobacterium culture. Several different protocols for respiratory sample decontamination have been tried. Decontamination by 1% n-acetyl-L-cysteine (NaLC)-NaOH is most commonly used; An increase of NaLC-NaOH concentrations from 1% to 1.25% lowers contamination rates but also leads to a 10% decrease in detection of mycobacteria in culture and is not recommended. Sulfuric acid (final concentration 3%) was recently shown to improve detection rates of NTM specifically (no influence on M. tuberculosis), compared with 1% NaLC-NaOH, by liquid culture.
For samples frompatients with cystic fibrosis (CF), which differ in their chemical composition as well their commensal flora, different approaches have been tested. In sputum samples of CF patients, decontamination by 0.25%/1% NALC-NaOH, followed by 5% oxalic acid treatment, reduced the contamination rate from 74% of Lowenstein-Jensen slants or 36% of BacTec vials (BDBiosciences, Sparks, MD) (for NaLC-NaOH alone) to only 5% and 3%; yet, in a multisite reproducibility study, this method performed well only in acid-fast bacillus (AFB) smear positive samples. In a study comparing 1% chlorhexidine alone to 0.25%/1% NaLC-NaOH followed by 5% oxalic acid in 827 sputum samples of CF patients, the former yielded twice as many NTM-positive cultures (6.50 vs 3.25%), despite a higher contamination rate after chlorhexidine treatment (20 vs 14.2%). Hence, it is important for mycobacteriology laboratories to know if a specimen comes from a CF patient or not—another example of a setting in which good communication can significantly improve clinical diagnostics.
The choice of media for primary isolation largely determines the sensitivity. Liquid media are, in general, more sensitive than solid media such as Lowenstein-Jensen, Ogawa, Coletsos, and Middlebrook 7H10/7H11.[36,37] Several studies have revealed that the widely used automated nonradiometric Mycobacteria Growth Indicator Tube (MGIT) method and its predecessor the radiometric BacTec460 method (both: BD Biosciences, Sparks, MD), both applying liquid media, were of about equal sensitivity; the slightly higher sensitivity of BacTec460(BD) observed in some studies was largely outweighed by its labor-intensive handling and use of radioactive materials with inherent safety concerns.[38,39] Both these liquid culture systems apply an antibiotic supplement to suppress overgrowth of other bacteria and fungi, labeled PANTA, the acronym for polymyxin B (50 U/mL), amphotericin B (5 μg/mL), nalidixic acid (20 μg/mL), trimethoprim (5 μg/mL), and azlocillin (10 μg/mL).
There are more automated nonradiometric liquid culture systems available, including the MB BacT (bioMérieux, Durham, NC) and the recently renewed VersaTREK system (formerly known as ESP culture system II; Trek Diagnostics, Cleveland, OH). The MB BacT system (bioMérieux) has been shown to be as sensitive as the MGIT (BD Biosciences) method.[37,40] The time to detection and percentage contaminated cultures were lower in MGIT (BD Biosciences).[37,40] For the VersaTREK system (Trek Diagnostics), no comparative studies of primary culture are currently available.
The comparative studies outlined here have offered important insights in the performance of the various culture methods. Nonetheless, they may not reveal the full sensitivity that can be obtained in the routine clinical setting because both the solid and the liquid media were generally incubated at a single temperature, 35°C. This temperature optimum is relevant to M. tuberculosis, but it limits the recovery of NTM, many of which have a growth optimum at 30°C.[1,3,5] Thus optimal recovery of NTM is likely to be reached if both solid and liquid media are used and incubated at both 35 and 30°C. Whether a liquid and solid medium should be incubated at both temperatures, or a hybrid of liquid at 35°C and solid at 30°C, or liquid media only but at both 30°C and 35°C, as well as cost-effectiveness of these approaches have not yet been studied, despite the increasing clinical importance of NTM in many settings.
Culture Conditions—Medium Enrichment
As typically environmental organisms, the NTM are quite versatile in their metabolic needs. Yet, for a small number of species, enrichment of culture media is needed to allow growth of the organism. For human medicine, M. genavense and M. haemophilum are the most prominent examples. For M. haemophilum, an iron source (ferric ammonium citrate or hemin) has to be added to the medium, and the media are best incubated at 30°C. For M. genavense, some success has been reported for media composed of blood, charcoal, caseine, and yeast extracts, acidified to pH 6.0. Yet this species remains very difficult to culture. Perhaps the most intriguing example is M. tilburgii, which has not been successfully cultured to date, despite the presence of large numbers of bacteria in clinical samples. Local epidemiology can guide the use of supplemented media.
Correct species identification is very important because NTM species differ in their clinical relevance.[6,43] Isolation of M. kansasii and (in northwestern Europe) M. malmoense from pulmonary specimens indicates disease in >70% of all patients.[5,6,44]Mycobacterium gordonae and, to a lesser extent, M. simiae or M. chelonae, are typically contaminants rather than causative agents of true disease[5,6] and MAC, M. xenopi, and M. abscessus form an intermediate category between these two extremes.[5,6,43]
The methods for identification of mycobacteria in clinical laboratories have changed dramatically over the past 2 decades. Molecular methods have now surpassed biochemical tests and high-performance liquid chromatography of cell wall mycolic acid content as the method of choice for NTM identification. Among molecular methods, two approaches are commonly used. The first are line probe assays, which are easy to perform, albeit costly, assays that allow a reasonable level of discrimination and will allow identification of the most frequently encountered species. Second is (partial) gene sequencing which allows a higher level of discrimination, often up to subspecies level, but is only feasible for laboratories with access to sequencing facilities. The target(s) selected for sequencing determine the discriminatory power: the hsp65 and rpoB genes and the 16S-23S internal transcribed spacer (ITS) offer high discriminatory power and can identify up to the subspecies level,[46–48] whereas 16S rRNA gene sequencing allows discrimination to the species level for most species, or at least to the complex level, particularly among the rapid growers (M. fortuitum complex, M. chelonae-M. abscessus complex).[45,48]
A new tool for species identification of NTM is matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The optimal method for protein extraction from mycobacteria and the exact discriminatory power of this method have not yet been established.
Impact of Diagnostic Criteria
Currently, both the British Thoracic Society (BTS) and the American Thoracic Society (ATS) have issued diagnostic criteria for nontuberculous mycobacterial (lung) disease.[5,50] Both state that isolation of NTM from normally sterile body sites is proof of true NTM disease, after exclusion of sample or laboratory contamination. Histological evidence of granulomatous inflammation strengthens the diagnosis. Diagnostic criteria for pulmonary NTM disease are less straightforward. Both BTS and ATS criteria emphasize the importance of symptoms and radiographic features suggestive of mycobacterial disease but differ in the level of detail of their microbiological criteria. The BTS criteria state that "multiple isolates are needed from nonsterile sites to establish disease. The clinical presentation and any predisposing factors are also helpful." The ATS criteria state that three separate respiratory specimens, produced over several days or weeks, should be analyzed and that two positive cultures with the same species are required for diagnosis. Only in the setting of nodular bronchiectatic disease with little or no sputum production, a single positive sputum from a BAL specimen may suffice to diagnose NTM lung disease. The previous 1997 ATS diagnostic criteria had less stringent radiological criteria but more stringent microbiological criteria requiring three positive cultures with the same species. Multiple studies have revealed that more patients meet the 2007 than the 1997 ATS diagnostic criteria, and this could lead to overdiagnosis and overtreatment of NTM lung disease in some.[51,52] On the other hand, less strict criteria may decrease diagnostic delay. The sensitivity of the different diagnostic criteria has not been tested in clinical studies; this is particularly problematic in select patient populations, such as CF patients, whose underlying disease resembles NTM pulmonary disease in terms of both the symptoms and the radiological features.
Semin Respir Crit Care Med. 2013;34(1):103-109. © 2013 Thieme Medical Publishers