Review article
Diagnosis of nontuberculous mycobacterial infections

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Isolation procedures

The first step toward recognition and confirmation of NTM disease is the detection and recovery of the NTM from clinical specimens. Specimens obtained from normally nonsterile body sites require processing, including a procedure that induces homogenization, decontamination, and concentration of the sample to prevent overgrowth of bacteria while preserving and concentrating the mycobacteria in the specimen before inoculation of culture media [2]. The laboratory technician must be aware, however,

Microscopy

Following decontamination procedures, acid fast smears are prepared from clinical specimens. The reliability of smear microscopy of a sample containing NTM is dependent on the experience of the microscopist, the quality of the specimen, and the number of acid fast bacilli (AFB) in the sample. Because of the large amount of lipids present in their cell walls, which generally make them impermeable to crystal violet, NTM do not stain well using routine Gram stain procedure. An experienced

Selection of media for culture

The recently published (December 2000) National Committee for Clinical Laboratory Standards (NCCLS) recommendation for culture of M tuberculosis and the NTM includes the use of both solid and liquid media to optimize recovery of all mycobacterial species [7]. Solid media are necessary to support growth of NTM that may not grow well in broth and also helps to detect mixed cultures [7]. Solid media also allows for organism quantitation, which is an important aspect of the diagnostic criteria of

Solid culture media

Solid media for mycobacterial growth is either egg-based, or agar-based. Of the egg-based media, such as Löwenstein–Jensen (L–J) media, Petragnani, and American Trudeau Society media, L–J medium is the one most commonly used in clinical laboratories. Of the agar-based media, the most commonly used is Middlebrook 7H10 or 7H11 medium.

Each medium has specific advantages and disadvantages. For example, although egg-based media has a good buffer capacity and long shelf life when refrigerated, it

Liquid (broth) culture media

The use of liquid based culture media has significantly impacted the rate and the recovery of mycobacteria, including the NTM [9]. A number of commercial broth culture systems are currently available and in use. These include the MGIT and BACTEC 460TB Systems (both manufactured by Becton Dickinson Microbiology Systems, Sparks, MD), ESP Culture System II (Trek Diagnostic Systems Inc., Westlake, OH), and MB/BacT System (Organon Teknika, Durham, NC).

The MGIT system contains a modified Middlebrook

Incubation temperatures

In general, mycobacterial cultures for NTM are incubated at 35°C except for skin and soft tissue specimens, where the RGM and fastidious species such as M ulcerans, M haemophilum, M chelonae, and M marinum are common pathogens. In this setting, solid media cultures should be incubated at 28°C to 30°C [2], [8]. In these species growth is poor, especially on primary isolation, at the higher temperatures that are optimal for respiratory pathogens such as M tuberculosis. One species, M xenopi, has

Commercially available genetic probes for identification

An acridinium-ester-labeled non-radioactive DNA probe based on the detection of rRNA (AccuProbe; GeneProbe Inc., San Diego, CA) is commercially available and FDA approved for identification of M tuberculosis as well as some common species of NTM, including M kansasii, M avium complex (M avium and M intracellulare), and M gordonae [10], [11]. No commercial probes are available for identification of other NTM species including the RGM [12].

Testing with these DNA probes can be performed using

Conventional phenotypic tests

These methods are listed last because the current emphasis in the laboratory is on the rapid growth and detection methods. Although conventional biochemical testing is well standardized and reproducible, it is unfortunately slow and limited in enabling the laboratory to distinguish many of the newly described species of NTM. For example, Springer and colleagues recently compared identification by standard biochemical methods with molecular techniques and found that biochemical testing is often

Diagnosis of NTM disease: laboratory results in a clinical context

The major problems diagnosing NTM disease clinically stem from a critical difference between diagnosing disease caused NTM and disease caused by M tuberculosis (and most other microbial pathogens). Under most circumstances a single specimen that is culture positive for M tuberculosis is adequate to diagnose clinical tuberculosis. However, a single positive NTM culture is frequently not sufficient for diagnosing NTM disease. NTM can be found in the environment and can contaminate laboratory

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      Historically, descriptions of NTM infection in patients with CF were relatively uncommon,10,21,22 in part due to the lack of laboratory methodology specific to CF. Recovery of NTM from CF sputum samples was difficult because of culture overgrowth by P aeruginosa and other CF microbes before the slower-growing mycobacteria could be detected.50–52 Development of effective sample decontamination protocols to remove conventional bacteria and fungi has allowed for improved culture-based detection of mycobacteria in CF samples.53,54 Currently, the standard approach to decontamination involves 2 steps51 to avoid excessive decontamination, which can reduce NTM viability in samples.55

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