Mycobacterium Nontuberculosis Species

The growing importance of nontuberculous mycobacteria (NTM) in the past 2 decades prompted development of sophisticated microbiologic techniques that enable more rapid and accurate identification of older species and those that are emerging as human pathogens. More than 180 species of mycobacteria are currently recognized.

Epidemiology

NTM species are ubiquitous in soil, water, foodstuffs, and domestic and wild animals. Human strains of Mycobacterium avium and M. intracellulare (collectively called M. avium complex [MAC]) are found in fresh and brackish waters in warm climates and in some animals, particularly swine. Bird strains of M. avium are molecularly different from human strains. Tap water has emerged as the major reservoir for several NTM species, including M. kansasii, M. xenopi, M. gordonae, M. simiae, M. mucogenicum, M. fortuitum, M. chelonae, and M. abscessus. ^, Fish and fish tank water are a major reservoir for M. marinum . Rapidly growing mycobacteria (i.e., M. fortuitum, M. mucogenicum, M. immunogenum, and M. abscessus) are associated with healthcare-associated infections, most likely from environmental sources. When identical methods are used, isolation rates of NTM are remarkably similar in diverse geographic areas.

There are particular geographic regions in which pathogenic species are found more commonly in the local environment and as etiologic agents of disease. ^, These include M. simiae (e.g., southwestern US, Europe, Israel), M. malmoense (e.g., northern and central Europe), M. xenopi (e.g., Canada, US, Europe), M. ulcerans (e.g., Africa, Australia), and M. kansasii (e.g., Europe, areas of the central and southern US).

The incidence of human disease due to NTM increased significantly in the 1980s and 1990s, in parallel with increasing numbers of AIDS cases. ^, Epidemiologic data suggest that the incidence of infections due to NTM among immunocompetent individuals has also risen in the past few decades. ^, ^, The changes in incidence may be real or could reflect increased awareness and enhanced laboratory detection. ^,

One study in children found seasonal variation in the incidence of NTM disease in temperate climates. Peaks occurred in late winter and spring, and troughs occurred in autumn, analogous to the reported seasonality for tuberculosis, which could be related to seasonal fluctuations in vitamin D levels. This may have an impact on the susceptibility to mycobacterial infections.

Pathogenesis

The pathogenesis of NTM infection in humans is not well understood. Humans are thought to acquire these organisms primarily from environmental sources. Although it was previously thought that there was no human-to-human transmission, ^, ^, data from cases with cystic fibrosis (CF) suggest that M. abscessus can be transmitted between patients. Reports of healthcare-associated outbreaks of M. abscessus within CF centers have been a growing concern, but the existence of person-to-person transmission remains controversial. Pulmonary NTM infection results from inhalation of airborne organisms, whereas skin and soft tissue infections result from direct inoculation. Ingestion of NTM can result in gastrointestinal disease or cervical lymphadenitis. Although the pathogenesis of disseminated disease, especially for rapidly growing mycobacteria, is not understood completely, colonization of central venous catheters (CVCs) is likely to be important.

Many host factors that increase the risk of NTM infection have been identified. ^, ^, ^, Each species has a characteristic level of virulence and propensity to cause infection in particular organs or settings ( Table 135.1 ). This includes contaminated wounds and open fractures (e.g., M. fortuitum, M. goodii ) , prosthetic devices such as indwelling CVCs (e.g., M. fortuitum, M. mucogenicum ) and prosthetic heart valves (e.g., M. fortuitum, M. abscessus) , ^, ^, tympanostomy tubes (e.g., M. abscessus, MAC), ^, immune suppression with organ transplantation (e.g., M. chelonae, M. abscessus ) , and bronchiectasis in the setting of CF (e.g., MAC, M. abscessus ). Another iatrogenic factor predisposing to mycobacterial disease is treatment with tumor necrosis factor (TNF)-inhibiting agents (e.g., infliximab, etanercept), usually in the context of rheumatoid arthritis or inflammatory bowel disease (see Chapter 107 ).

TABLE 135.1

Clinical Manifestations of Infection Caused by Nontuberculous Mycobacteria

Site	Predominant Mycobacterium Species	Typical Lesions	Setting
Skin and soft tissue	M. marinum	Papular, nodular, or plaque-like; less commonly ulcerative	Usually associated with aquatic trauma (aquarium granuloma, swimmer’s granuloma)
	M. fortuitum	Granuloma	Postoperative, central venous catheter associated, after trauma or penetrating injury
	M. abscessus	Granuloma
	M. chelonae	Granuloma
	M. ulcerans ^a	Ulcer	Mode of transmission is uncertain
Lymphadenitis	M. avium complex	Submandibular or anterior cervical mass	Typically unilateral; subacute; 1- to 5-year-old children
	M. haemophilum
	M. malmoense ^a
Otitis media and mastoiditis	M. abscessus	Chronic otorrhea; unresponsive to conventional antibiotics	Almost exclusively in patients with tympanostomy tubes in situ
Otitis media and mastoiditis	M. avium complex	Chronic otorrhea; unresponsive to conventional antibiotics
Pulmonary infection	M. avium complex	Cavitation or nodular disease and bronchiectasis	Typically in patients with preexisting lung disease; cystic fibrosis and bronchiectasis in children and adolescents; chronic obstructive pulmonary disease in adults
	M. kansasii
	M. xenopi
	M. abscessus
	M. fortuitum
	M. simiae ^a
Disseminated infection	M. avium complex	Organism recovered from blood, bone marrow, liver, and lymph nodes	Immunocompromised host (malignancy, impaired cell-mediated immunity, HIV infection, immunosuppressive treatment or chemotherapy)
	M. kansasii
	M. abscessus
	M. chelonae
	M. haemophilum

a Common only in certain geographic areas.

Investigation of genetic polymorphisms that increase host susceptibility to NTM is ongoing. The interferon-γ (IFN-γ), TNF, and interleukin-12 (IL-12) pathways play an important role in the host immune response to mycobacterial infections. Isolated immune defects identified in patients with disseminated mycobacterial infections include mutations in the IFN-γ receptor gene and IL-12 receptor deficiency. ^,

Knowledge on virulence factors that enhance the invasiveness of NTM species is still limited. ^, The complete genome of M. ulcerans was published in 2007, followed by the genomes of M. avium, M. marinum, and M. abscessus . ^, Additional NTM species have been sequenced since then. M. ulcerans produces mycolactone, a polyketide toxin that induces tissue necrosis and has immunosuppressive properties. The genes required for mycolactone production are plasmid encoded. Recent data suggest that certain glycolipids may play an important role in the pathogenicity of M. abscessus .

Laboratory Diagnosis

Stain, Culture, and Molecular Techniques

The mycobacterial species (as well as the subspecies in some instances), the site of specimen origin, and the quantity of growth in culture are important factors in assessing the potential pathogenic role of an NTM isolate. ^, Heavy growth of a single organism usually indicates infection, whereas light growth in a single sputum sample may indicate colonization or contamination. ^, This does not apply to tissue samples, normally sterile body fluids or bronchoalveolar lavage (BAL) fluid, in which specimens NTM isolation confirms infection. ^, Recommendations for isolation and interpretation of culture results are published. ^, Because some NTM have unusual requirements for nutrition and incubation (e.g., M. haemophilum, M. ulcerans, M. genavense, M. marinum ), ^, ^, ^, laboratory staff should be consulted to optimize specimen collection, transport, and culture conditions.

Conventional methods used for M. tuberculosis remain useful for processing and staining of specimens for NTM before culture. Typically, for sputum samples, N -acetyl- l -cysteine is used as a mucolytic agent to free acid-fast bacilli from proteinaceous material, combined with 1% sodium hydroxide to kill non-acid-fast organisms. However, some NTM species, especially rapidly growing mycobacteria such as M. abscessus, are highly susceptible to sodium hydroxide and can lose viability. Chlorhexidine decontamination may be preferable when M. abscessus is suspected. For tissue samples and fluids from normally sterile sites, decontamination procedures are not required.

Fluorochrome, Kinyoun, and Ziehl-Neelsen stains are used for direct staining. ^, Because the appearance of mycobacteria in stained specimens varies, experience is required for accurate interpretation. Colony morphology, growth rates, and pigmentation are superior to microscopic morphology as an indication of mycobacterial group; however, neither should be used to identify species.

Standard culture methods involve a combination of broth and solid media. Although most NTM species grow well with standard incubation at 37°C, some species, including M. ulcerans and M. marinum , grow poorly or not at all at 37°C. If these organisms are suspected, additional cultures should be incubated in parallel at 30°C–32°C. ^, ^,

Historically, isolation on solid media required 2–4 weeks, and identification based on biochemical tests took another 4–6 weeks. However, the use of broth culture incubation systems, such as the BioFM (Bio-Rad) and the Bactec MGIT 960 (Becton Dickinson), has reduced the time for detection of growth to an average of 1–2 weeks, depending on species and positivity of direct smear. ^, A more rapid method for mycobacterial species identification is cell wall mycolic acid analysis by thin-layer chromatography, gas-liquid chromatography, or high-performance liquid chromatography (HPLC). ^,

Use of matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry has recently been optimized for NTM species identification by updating the Mycobacterium databases and improving sample processing procedures. A variety of molecular methods also can be used for species identification, including commercial DNA probes (e.g., M. tuberculosis complex, MAC, M. gordonae, M. kansasii ) (AccuProbe, Gen-Probe) and commercial hybridization strips (i.e., line probe assays) for multiple species (INNO-LiPA Mycobacteria v2, Fuji-Rebio; GenoType Mycobacterium CM/AS, Hain Lifescience; MolecuTech REBA Myco-ID, YD Diagnostics). ^, Other methods include a commercial 16S rRNA gene sequencing kit (MicroSeq, Applied Biosystems), ^, noncommercial (in-house) sequencing, and polymerase chain reaction (PCR), plus restriction fragment length polymorphism (RFLP) analysis (PRA) of the hsp 65 gene.

Methods to detect and identify NTM directly from clinical specimens are being studied, including DNA probes, target amplification methods (e.g., PCR), signal amplification methods, and gene amplification. ^, The most effective of these has been amplification, followed by identification of the 16S rRNA, hsp65 , rpoB , gyrA, or the 16S-23S internal transcribed spacer (ITS) genes. ^, ^, Multiplex PCR-based targeted gene sequencing platforms are being developed.

NTM have been identified by means of pyrosequencing of the hypervariable regions of the 16S rRNA gene. This method allows higher throughput, is usually less expensive than traditional chain-termination (Sanger) sequencing, and has a high level of accuracy for differentiating among mycobacterial species. The automated, PCR-based GeneXpert system (Cepheid) allows rapid detection of M. tuberculosis, combined with testing for rifampicin resistance genes with the Xpert MTB/RIF assay. Although this system is useful for M. tuberculosis and has become widely available across North America and Europe, it has not yet been developed to test for NTM. The Xpert MTB/RIF assay and most other PCR-based assays target genes within the MTB complex and, therefore, detect all eight species within the MTB complex, including M. africanum and M. bovis (including bacille Calmette-Guérin [BCG]). Most assays, including the Xpert MTB/RIF, target the 1610 gene, which is shared also by M. smegmatis . GeneXpert can be falsely positive when some NTM species (i.e. M. marinum , M. abscessus , M. semgmatis , M. phlei , M. aurum ) are present at a high bacterial load (10 ⁶ CFU/mL). However, the abnormal amplification plot and high C(t) values in these cases should raise suspicion of NTM species. M. abscessus and M. smegmatis can be flagged as RIF-resistant MTB due to the high C(t) value of Probe E. ^,

Standardized procedures for susceptibility testing of NTM in broth systems have been published. Susceptibility or resistance of NTM species in vitro often does not correlate well with clinical efficacy, with the exception of clarithromycin and amikacin resistance for treatment of disseminated or pulmonary MAC and M. abscessus infections, clarithromycin resistance for M. chelonae , and rifampin and clarithromycin resistance for pulmonary M. kansasii . ^, Susceptibility results for other drugs should guide, but not dictate, the choice of the antibiotic regimen. ^, ^, Moreover, a diversity of subclones with differing antimicrobial resistance profiles can be present in patients chronically infected with M. abscessus , and isolates from sputum samples may not reflect this.

Recently, enzyme immunoassays detecting anti-glycopeptidolipid-core IgA antibodies have shown some promise for differentiating MAC or M. abscessus pulmonary infections from NTM colonization or other lung diseases, including tuberculosis. ^, The antibody level may also reflect disease severity and, therefore, help predict treatment response and monitor disease activity. However, further trials are needed to clarify whether measurement of these antibodies can truly help to optimize patient care.

Interferon γ Release Assays

IFN-γ release assays (IGRAs) are established tools for the diagnosis Mycobacterium tuberculosis infection or disease (see Chapter 134 ). ^, Currently, several commercial IGRAs are available globally, but the most widely used assays in North America, Europe, and most parts of Asia are the QuantiFERON-TB Gold Plus (QFT-Plus) assay (Cellestis/Qiagen), having replaced the previous version of the assay QuantiFERON-TB Gold in-Tube (QFT-GIT) and the T-SPOT. TB assay (Oxford Immunotec). Both incorporate early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) as stimulatory antigens, which are absent from most NTM, with the notable exceptions of M. kansasii , M. marinum, and M. szulgai . IGRAs overall have greater specificity for M. tuberculosis infections compared with the Mantoux tuberculin skin test (TST). However, IGRAs have several limitations, particularly in children: (1) less robust performance in young children, (2) high proportions of indeterminate (i.e., uninterpretable) results in some pediatric studies, ^, ^, ^, and (3) incomplete understanding of the mechanisms underlying discordance with TST results, which is frequently attributed (without definitive evidence) to prior BCG vaccination or exposure to NTM. ^,

In one study of children with mycobacterial lymphadenitis (mainly caused by M. avium ), the QuantiFERON-TB Gold assay and the T-SPOT.TB assay showed a greater ability to discriminate between M. tuberculosis and NTM lymphadenitis than the tuberculin skin test (TST). Other reports support their usefulness to differentiate pulmonary infections due to M. tuberculosis from certain NTM infections, including MAC, M. malmoense , M. genavense, and M. gordonae . A negative IGRA result does not exclude M. tuberculosis infection. The pooled sensitivity of the QFT-GIT assay and T-SPOT. TB assay for patients with active tuberculosis in a meta-analysis was only 81% and 88%, respectively. To date, the published data on the performance of the QFT-Plus assay in children with tuberculosis remain limited, but data suggest that QFT-Plus does not have higher sensitivity than the previous generation assay. A positive IGRA result does not prove M. tuberculosis infection because most patients with NTM disease caused by M. kansasii , M. marinum, or M. szulgai have positive IGRA results. ^, ^, ^, ^, Case reports suggest that IGRAs can be useful to support a suspected diagnosis of M. marinum skin infection when PCR and culture results are pending.

In-house IL-2 or INF-γ enzyme-linked immunospot assays using a range of stimulatory antigens have been reported to potentially improve the diagnosis of NTM lymphadenitis.

Clinical Manifestations

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