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An Official ATS/IDSA Statement: Diagnosis, Treatment, and Prevention of Nontuberculous Mycobacterial Diseases

THIS OFFICIAL STATEMENT OF THE AMERICAN THORACIC SOCIETY (ATS) AND THE INFECTIOUS DISEASES SOCIETY OF AMERICA (IDSA) WAS ADOPTED BY THE ATS BOARD OF DIRECTORS, SEPTEMBER 2006, AND BY THE IDSA BOARD OF DIRECTORS, JANUARY 2007

	CONTENTS

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
Summary

Diagnostic Criteria of Nontuberculous Mycobacterial

Lung Disease

Key Laboratory Features of NTM

Health Care- and Hygiene-associated

Disease Prevention

Prophylaxis and Treatment of NTM Disease

Introduction

Methods

Taxonomy

Epidemiology

Pathogenesis

Host Defense and Immune Defects

Pulmonary Disease

Body Morphotype

Tumor Necrosis Factor Inhibition

Laboratory Procedures

Collection, Digestion, Decontamination, and Staining of Specimens

Respiratory Specimens

Body Fluids, Abscesses, and Tissues

Blood

Specimen Processing

Smear Microscopy

Culture Techniques

Incubation of NTM Cultures

NTM Identification

Antimicrobial Susceptibility Testing for NTM

Molecular Typing Methods of NTM

Clinical Presentations and Diagnostic Criteria

Pulmonary Disease

Cystic Fibrosis

Hypersensitivity-like Disease

Transplant Recipients

Disseminated Disease

Lymphatic Disease

Skin, Soft Tissue, and Bone Disease

Health Care– and Hygiene-associated Disease and Disease Prevention

NTM Species: Clinical Aspects and Treatment Guidelines

M. avium Complex (MAC)

M. kansasii

M. abscessus

M. chelonae

M. fortuitum

M. genavense

M. gordonae

M. haemophilum

M. immunogenum

M. malmoense

M. marinum

M. mucogenicum

M. nonchromogenicum

M. scrofulaceum

M. simiae

M. smegmatis

M. szulgai

M. terrae complex

M. ulcerans

M. xenopi

Other NTM Species/Pathogens

Research Agenda for NTM Disease

Summary of Recommendations

	SUMMARY

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
Diagnostic Criteria of Nontuberculous Mycobacterial Lung Disease
The minimum evaluation of a patient suspected of nontuberculous mycobacterial (NTM) lung disease should include the following: (1) chest radiograph or, in the absence of cavitation, chest high-resolution computed tomography (HRCT) scan; (2) three or more sputum specimens for acid-fast bacilli (AFB) analysis; and (3) exclusion of other disorders, such as tuberculosis (TB). Clinical, radiographic, and microbiologic criteria are equally important and all must be met to make a diagnosis of NTM lung disease. The following criteria apply to symptomatic patients with radiographic opacities, nodular or cavitary, or an HRCT scan that shows multifocal bronchiectasis with multiple small nodules. These criteria fit best with Mycobacterium avium complex (MAC), M. kansasii, and M. abscessus. There is not enough known about most other NTM to be certain that these diagnostic criteria are universally applicable for all NTM respiratory pathogens.

Clinical.

1. Pulmonary symptoms, nodular or cavitary opacities on chest radiograph, or an HRCT scan that shows multifocal bronchiectasis with multiple small nodules.
   and
   
2. Appropriate exclusion of other diagnoses.
   

Microbiologic.

1. Positive culture results from at least two separate expectorated sputum samples. (If the results from the initial sputum samples are nondiagnostic, consider repeat sputum AFB smears and cultures.)
   or
   
2. Positive culture results from at least one bronchial wash or lavage.
   or
   
3. Transbronchial or other lung biopsy with mycobacterial histopathologic features (granulomatous inflammation or AFB) and positive culture for NTM or biopsy showing mycobacterial histopathologic features (granulomatous inflammation or AFB) and one or more sputum or bronchial washings that are culture positive for NTM.
   
4. Expert consultation should be obtained when NTM are recovered that are either infrequently encountered or that usually represent environmental contamination.
   
5. Patients who are suspected of having NTM lung disease but who do not meet the diagnostic criteria should be followed until the diagnosis is firmly established or excluded.
   
6. Making the diagnosis of NTM lung disease does not, per se, necessitate the institution of therapy, which is a decision based on potential risks and benefits of therapy for individual patients.
   

Key Laboratory Features of NTM

1. The preferred staining procedure is the fluorochrome method. Specimens should be cultured on both liquid and solid media. Species that require special growth conditions and/or lower incubation temperatures include M. haemophilum, M. genavense, and M. conspicuum. These species can cause cutaneous and lymph node disease.
   
2. In general, NTM should be identified to the species level. Methods of rapid species identification include commercial DNA probes (MAC, M. kansasii, and M. gordonae) and high-performance liquid chromatography (HPLC). For some NTM isolates, especially rapidly growing mycobacterial (RGM) isolates (M. fortuitum, M abscessus, and M. chelonae), other identification techniques may be necessary including extended antibiotic in vitro susceptibility testing, DNA sequencing or polymerase chain reaction (PCR) restriction endonuclease assay (PRA).
   
3. Routine susceptibility testing of MAC isolates is recommended for clarithromycin only.
   
4. Routine susceptibility testing of M. kansasii isolates is recommended for rifampin only.
   
5. Routine susceptibility testing, for both taxonomic identification and treatment of RGM (M. fortuitum, M abscessus, and M. chelonae) should be with amikacin, imipenem (M. fortuitum only), doxycycline, the fluorinated quinolones, a sulfonamide or trimethoprim-sulfamethoxazole, cefoxitin, clarithromycin, linezolid, and tobramycin (M. chelonae only).
   

Health Care– and Hygiene-associated Disease Prevention
Prevention of health care–related NTM infections requires that surgical wounds, injection sites, and intravenous catheters not be exposed to tap water or tap water–derived fluids. Endoscopes cleaned in tap water and clinical specimens contaminated with tap water or ice are also not acceptable.

Prophylaxis and Treatment of NTM Disease

1. Treatment of MAC pulmonary disease. For most patients with nodular/bronchiectatic disease, a three-times-weekly regimen of clarithromycin (1,000 mg) or azithromycin (500 mg), rifampin (600 mg), and ethambutol (25 mg/kg) is recommended. For patients with fibrocavitary MAC lung disease or severe nodular/bronchiectatic disease, a daily regimen of clarithromycin (500–1,000 mg) or azithromycin (250 mg), rifampin (600 mg) or rifabutin (150–300 mg), and ethambutol (15 mg/kg) with consideration of three-times-weekly amikacin or streptomycin early in therapy is recommended. Patients should be treated until culture negative on therapy for 1 year.
   
2. Treatment of disseminated MAC disease. Therapy should include clarithromycin (1,000 mg/d) or azithromycin (250 mg/d) and ethambutol (15 mg/kg/d) with or without rifabutin (150–350 mg/d). Therapy can be discontinued with resolution of symptoms and reconstitution of cell-mediated immune function.
   
3. Prophylaxis of disseminated MAC disease. Prophylaxis should be given to adults with acquired immunodeficiency syndrome (AIDS) with CD4⁺ T-lymphocyte counts less than 50 cells/µl. Azithromycin 1,200 mg/week or clarithromycin 1,000 mg/day have proven efficacy. Rifabutin 300 mg/day is also effective but less well tolerated.
   
4. Treatment of M. kansasii pulmonary disease. A regimen of daily isoniazid (300 mg/d), rifampin (600 mg/d), and ethambutol (15 mg/kg/d). Patients should be treated until culture negative on therapy for 1 year.
   
5. Treatment of M. abscessus pulmonary disease. There are no drug regimens of proven or predictable efficacy for treatment of M. abscessus lung disease. Multidrug regimens that include clarithromycin 1,000 mg/day may cause symptomatic improvement and disease regression. Surgical resection of localized disease combined with multidrug clarithromycin-based therapy offers the best chance for cure of this disease.
   
6. Treatment of nonpulmonary disease caused by RGM (M. abscessus, M. chelonae, M. fortuitum). The treatment regimen for these organisms is based on in vitro susceptibilities. For M. abscessus disease, a macrolide-based regimen is frequently used. Surgical debridement may also be an important element of successful therapy.
   
7. Treatment of NTM cervical lymphadenitis. NTM cervical lymphadenitis is due to MAC in the majority of cases and treated primarily by surgical excision, with a greater than 90% cure rate. A macrolide-based regimen should be considered for patients with extensive MAC lymphadenitis or poor response to surgical therapy.
   

	INTRODUCTION

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
This is the third statement in the last 15 years dedicated entirely to disease caused by NTM (1, 2). The current unprecedented high level of interest in NTM disease is the result of two major recent trends: the association of NTM infection with AIDS and recognition that NTM lung disease is encountered with increasing frequency in the non-AIDS population. Furthermore, NTM infections are emerging in previously unrecognized settings, with new clinical manifestations. Another major factor contributing to increased awareness of the importance of NTM as human pathogens is improvement in methodology in the mycobacteriology laboratory, resulting in enhanced isolation and more rapid and accurate identification of NTM from clinical specimens. Consistent with the advances in the mycobacteriology laboratory, this statement has an emphasis on individual NTM species and the clinical disease–specific syndromes they produce. A major goal is facilitating the analysis of NTM isolates by the health care provider, including determination of the clinical and prognostic significance of NTM isolates and therapeutic options.

There are controversies in essentially all aspects of this very broad field and, whenever possible, these controversies are highlighted. Hence, an attempt is made to provide enough information so that the clinician understands the recommendations in their appropriate context, especially those made with inadequate or imperfect supporting information. Also, when there is not compelling evidence for one recommendation, alternative recommendations or options are presented.

This statement also includes new topics not addressed in previous statements, including advances in the understanding of the pathogenesis of NTM disease, descriptions of new NTM pathogens, clinical areas of emerging NTM disease such as cystic fibrosis, new NTM disease manifestations such as hypersensitivity-like lung disease, and public health implications of NTM disease such as prevention and surveillance. This statement will also take advantage of web-based resources through the American Thoracic Society (ATS) website for illustration and amplification of selected topics, discussion of additional topics not included in this document, as well as the capacity for updating information in this rapidly changing field.

Large gaps still exist in our knowledge. Limitations in systematic data have made it necessary for many of the recommendations in this document to be based on expert opinion rather than on empirically derived data. Unquestionably, more and better studies of NTM diseases must be done. The committee organized to create this document is composed of scientists and physicians residing in the United States. This document, therefore, represents a United States' perspective of NTM diseases that may not be appropriate for NTM diseases in other parts of the world.

	METHODS

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
Review of the available literature and subsequent grading of recommendations were accomplished by the members of the writing committee. The search for evidence included handsearching journals, reviewing previous guidelines, and searching electronic databases including MEDLINE and PubMed. Only articles written in English were considered. Final decisions for formulating recommendations were made by voting among committee members. The recommendations are rated on the basis of a system developed by the U.S. Public Health Service and the Infectious Diseases Society of America (IDSA) (3). The rating system includes a letter indicating the strength of the recommendation, and a roman numeral indicating the quality of the evidence supporting the recommendation (3) (Table 1). Thus, clinicians can use the rating to differentiate among recommendations based on data from clinical trials and those based on the opinions of the experts comprising the writing committee, who are familiar with the relevant clinical practice and scientific rationale for such practice when clinical trial data are not available. Ratings following each numbered recommendation pertain to all points within the numbered recommendation. Each member of the writing committee has declared any conflict of interest. These guidelines were developed with funding provided by the ATS.

	TAXONOMY

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

Early taxonomic studies compared up to 100 growth and biochemical tests of large numbers of strains in multiple collaborative laboratories. Work focused around the International Working Group on Mycobacterial Taxonomy. New species were defined on the ability to phenotypically separate the new taxon from established species. This work was time and labor intensive and not adequate for separating many NTM species. Subsequently, species were identified by comparisons of genomic DNA; new species had similarity (homology) of less than 70% on DNA–DNA pairing experiments with established species. This type of comparison was highly technical, highly labor intensive, and required comparison of possible new species to all established related species. By its very nature, this technique limited identification of new species.

The dramatic change in mycobacterial taxonomy came with the ready availability and reliability of DNA sequencing. Investigators recognized that the mycobacterial 16S rRNA gene was highly conserved, and that differences in the sequence of 1% or greater generally defined a new species (4, 5). Also critical to the dramatic change was the appearance of publicly available databases that stored the 16S rRNA gene sequences of established mycobacterial species. Recognition of a novel NTM species is now relatively simple: perform 16S rRNA gene sequence analysis of a suspected new species and compare the results with those in the databases. Numerous new species are appearing from laboratories all over the world rather than from a small number of mycobacterial taxonomists. It is likely that the number of new species will continue to expand rapidly as 16S rRNA gene sequencing analysis is performed on increasing numbers of isolates of clinical disease that cannot be identified with commercial nucleic acid probes. It is possible that the number of NTM species will increase to more than 150 before the publication of the next NTM disease statement.

This expansion in new NTM species is, therefore, largely a consequence of newer identification techniques that are capable of separating closely related NTM species, as opposed to the sudden appearance of new NTM species. For instance, M. triplex, M. lentiflavum, M. celatum, and M. conspicuum (among others) might previously have been identified as MAC based on traditional biochemical and/or phenotypic analyses. The clinical significance of these species separations may be subtle or negligible, but the clinician inevitably will continue to be confronted by new NTM species designations.

	EPIDEMIOLOGY

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
NTM are widely distributed in the environment with high isolation rates worldwide (6, 7). Organisms can be found in soil and water, including both natural and treated water sources (M. kansasii, M. xenopi, and M. simiae are recovered almost exclusively from municipal water sources and rarely, if ever, from other environmental sources). When identical methods are used, isolation rates of NTM from the environment are remarkably similar in diverse geographic areas (6, 7). There is no evidence of animal-to-human or human-to-human transmission of NTM (7–11). Even in patients with cystic fibrosis (CF), an apparently highly susceptible population and a population in which other opportunistic organisms are clearly passed between patients, there has been no documentation of human-to-human transmission of NTM (12). Human disease is suspected to be acquired from environmental exposures, although the specific source of infection usually cannot be identified (13).

NTM may cause both asymptomatic infection and symptomatic disease in humans. Rates of asymptomatic infection have been inferred from both antibody and skin test studies. In areas where infection with M. tuberculosis is uncommon, antibody to a common mycobacterial antigen, lipoarabinomannin (LAM), can be assumed to be due predominantly to NTM infection. Antibody to LAM is detectable among children in the United States, rising rapidly between the ages of 1 and 12 years, then appearing to plateau (14). Skin test studies in adults indicate that a substantial proportion have had prior and presumably asymptomatic infection with NTM (15, 16). Skin test studies with an M. intracellulare purified protein derivative (PPD-B) conducted among U.S. Navy recruits in the 1960s showed that reactions of greater than 4 mm induration were more common in the southeastern than northern United States, suggesting higher background rates of NTM infection in these areas (16). Reactions to skin tests derived from NTM are not sufficiently species specific to indicate which nontuberculous mycobacterium might have been responsible for these asymptomatic infections, and it is possible that cross-reactivity with M. tuberculosis infection contributed to some of these reactions. However, because MAC organisms are the most common cause of NTM disease in the United States, it is likely that MAC was also the most common cause of infection. In these patients, asymptomatic infection with NTM has not been shown to lead to latent infection, so that in contrast to TB, there is currently no evidence that NTM are associated with reactivation disease.

NTM diseases have been seen in most industrialized countries; incidence rates vary from 1.0 to 1.8 cases per 100,000 persons (17). These overall incidence rates are estimates based on numbers of NTM isolates reported. MAC is the most common NTM species causing disease in most series, but many species have been implicated (Table 2). Rates appear to be similar in most developed countries, but surveillance information is limited. Because NTM diseases are not communicable, they are not reportable in the United States. Although several reports have suggested that the incidence of NTM diseases has increased over the past several decades, this observation has not been conclusively established due to the lack of comprehensive surveillance efforts. The most common clinical manifestation of NTM disease is lung disease, but lymphatic, skin/soft tissue, and disseminated disease are also important (18, 19). In the United States between 1981 and 1983, 94% of the NTM isolates reported to the Centers for Disease Control and Prevention (CDC) laboratory were pulmonary, whereas 3% were lymph node and 3% were skin/soft tissue isolates (19). In the late 1980s and early 1990s, NTM isolates associated with disseminated disease in patients with AIDS were reported almost as frequently as pulmonary isolates (20, 21). More recently, the CDC published the results of NTM isolates reported by state public health laboratories to the Public Health Laboratory System (PHLIS) database for the period 1993 through 1996 (22). During this period, of the NTM isolated, 75% were pulmonary, whereas 5% were from blood, 2% from skin/soft tissue, and 0.4% from lymph node isolates. The most frequently reported potentially pathogenic species and corresponding report rates over the 4-year period (per 1,000,000 population) were as follows: MAC, 29 to 36 isolates; M. fortuitum, 4.6 to 6 isolates; and M. kansasii, 2 to 3.1 isolates. Regionally, all three NTM species were reported most often from the southeastern United States. There are, however, significant limitations interpreting and extrapolating these data. Reporting of NTM species suspected to be involved in disease to PHLIS was voluntary, and not all states participated. Therefore, the data do not represent the absolute occurrence and distribution of NTM species in the United States. In addition, the numbers of isolates are presented as "report rates," "to reflect a lack of verification of clinical significance of the report" (emphasis in report). Because the reports were not verified, interpreting the association of these isolates with clinical data is problematic.

More epidemiologic information is provided in the discussions of specific disease syndromes and for individual NTM species, and in Table 2.

	PATHOGENESIS

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Over the past two decades, three important observations have been made regarding the pathogenesis of NTM infections:

1. In patients infected with HIV, disseminated NTM infections typically occurred only after the CD4⁺ T-lymphocyte number had fallen below 50/µl, suggesting that specific T-cell products or activities are required for mycobacterial resistance (23, 24).
   
2. In the HIV-uninfected patient group, genetic syndromes of disseminated NTM infection have been associated with specific mutations in interferon (IFN)- and interleukin (IL)-12 synthesis and response pathways (25, 26) (IFN- receptor 1 [IFNR1], IFN- receptor 2 [IFNR2], IL-12 receptor 1 subunit [IL12R1], the IL-12 subunit p40 [IL12p40], the signal transducer and activator of transcription 1 [STAT1], and the nuclear factor- essential modulator [NEMO]).
   
3. There is also an association between bronchiectasis, nodular pulmonary NTM infections and a particular body habitus, predominantly in postmenopausal women (e.g., pectus excavatum, scoliosis, mitral valve prolapse) (27).
   

Host Defense and Immune Defects
Mycobacteria are initially phagocytosed by macrophages, which respond with production of IL-12, which in turn up-regulates IFN- (28). IFN- activates neutrophils and macrophages to kill intracellular pathogens, including mycobacteria. There is a positive feedback loop between IFN- and IL-12, which is critical for the control of mycobacteria, as well as certain other intracellular infections. Disseminated NTM disease is a definite manifestation of immunologic defect, either acquired, such as HIV and iatrogenic factors, or genetic, caused by defects in the above IFN-/IL-12 pathway genes. However, these genetic factors only predispose to disseminated disease.

Pulmonary Disease
Lung disease due to NTM occurs commonly in structural lung disease, such as chronic obstructive pulmonary disease (COPD), bronchiectasis, CF, pneumoconiosis, prior TB, pulmonary alveolar proteinosis, and esophageal motility disorders (12, 19, 29–32). Abnormal CF genotypes and ₁-antitrypsin (AAT) phenotypes may predispose some patients to NTM infection (33–35). NTM lung disease also occurs in women without clearly recognized predisposing factors (32, 36–38). Bronchiectasis and NTM infection, usually MAC, often coexist, making causality difficult to determine. These patients may carry multiple MAC strains over time, suggesting either polyclonal infection or recurrent infection with distinct strains (38). It is unclear whether this problem is due to local abnormalities (e.g., bronchiectasis) or to immune defects. In one study from Japan, 170 patients with MAC lung infection were studied: of 622 siblings of those patients, 3 had MAC lung disease. The implication is that the sibling risk for MAC infection is much higher than previously estimated population prevalence (11).

Body Morphotype
Women with nodular NTM pulmonary infections associated with bronchiectasis have similar clinical characteristics and body type, sometimes including scoliosis, pectus excavatum, mitral valve prolapse, and joint hypermobility (27). These phenotypic characteristics may represent markers for specific genotypes that affect both body morphotype and NTM infection susceptibility. Alternatively, the morphotype itself may influence mycobacterial infection susceptibility, through such features as poor tracheobronchial secretion drainage or ineffective mucociliary clearance.

Tumor Necrosis Factor Inhibition
IFN- and IL-12 control mycobacteria in large part through the up-regulation of tumor necrosis factor (TNF)-, made predominantly by monocytes/macrophages. The critical role of TNF- in controlling intracellular infections is made clear through the use of TNF- blocking agents. The potent TNF- blocking antibodies infliximab and adalimumab and the soluble receptor etanercept are effective antiinflammatory agents and lead to relatively high rates of development of active TB in those who are latently infected (39, 40). The onset of TB after administration of infliximab ranges from weeks to months. In addition to TB, the TNF- blocking agents predispose to invasive fungal infections, such as aspergillus, histoplasmosis, and coccidioidomycosis (41). Infections with mycobacteria and fungi are seen with all three agents, but significantly more with infliximab than etanercept. Adalimumab should be regarded as having similar risks. The risk posed by TNF- blocking agents for predisposing to NTM infections or promoting progression of active NTM infection is unknown. Until more information is available, expert opinion is that patients with active NTM disease should receive TNF- blocking agents only if they are also receiving adequate therapy for the NTM disease.

	LABORATORY PROCEDURES

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
Since the publication of the last ATS statement on NTM, the Clinical and Laboratory Standards Institute (CLSI), formerly known as the National Committee for Clinical Laboratory Standards (NCCLS), has published an approved standard for NTM susceptibility testing by mycobacteriology laboratories (42, 43). The institute provides a global forum for the development of standards and guidelines. All proposed standards from the institute are subjected to an accredited consensus process before being published as "accepted standards." The institute suggests that to maintain efficiency, effectiveness, and consistency in the interpretation of test results, it is important that other health care–associated organizations ascribe to the same standards and practices as approved by the CLSI. Unless noted in the text, recommendations in this document are consistent with CLSI published standards.

Collection, Digestion, Decontamination, and Staining of Specimens
Specimens for mycobacterial identification and susceptibility testing may be collected from almost any area of the body. Collection of all specimens should avoid potential sources of contamination, especially tap water, because environmental mycobacteria are often present. Specimens should be submitted without fixatives. Observing routine safety precautions by collecting samples in sterile, leak-proof, disposable, labeled, laboratory-approved containers is important. Transport media and preservatives are not usually recommended, although refrigeration of samples at 4°C is preferred if transportation to the laboratory is delayed more than 1 hour. For diagnostic purposes, it may be necessary to collect multiple respiratory specimens on separate days from outpatients. Specimens for mycobacterial analysis can be shipped or mailed. Overnight shipping with refrigerants such as cold packs is optimal, although mycobacteria can still be recovered several days after collection even without these measures. The longer the delay between collection and processing, however, the greater is the risk of bacterial overgrowth. Treatment with commonly used antibiotics such as macrolides and quinolones might adversely affect the yield of NTM recovery. Therefore, if possible, antibiotic use should be limited during diagnostic evaluation of NTM diseases.

Respiratory Specimens
To establish the diagnosis of NTM lung disease, the collection of three early-morning specimens on different days is preferred. For patients unable to produce sputum, sputum can also be induced. Induced sputum is an effective method for diagnosing TB; however, similar data establishing the effectiveness of sputum induction for diagnosing NTM lung disease are not available (44). In addition, the optimal methodology for sputum induction in this setting has not been determined. If sputum cannot be obtained, bronchoscopy with or without lung biopsy may be necessary. Because of clinical similarities between NTM lung disease and TB, appropriate precautions to prevent the nosocomial transmission of TB should be followed when performing these procedures. It is also important to perform appropriate cleaning procedures for bronchoscopes that include the avoidance of tap water, which may contain environmental mycobacteria.

Body Fluids, Abscesses, and Tissues
Aseptic collection of as much body fluid or abscess fluid as possible by needle aspiration or surgical procedures is recommended. Swabs are not recommended for sample collection because they often are not aggressively applied, resulting in limited culture material, and are also subject to dessication, thus decreasing chances for recovery of NTM. If a swab is used, the swab should be saturated with the sampled fluid to assure an adequate quantity of material for culture. When submitting tissue, the specimen should not be wrapped in gauze or diluted in liquid material. If only a minute amount if tissue is available, however, it may be immersed in a small amount of sterile saline to avoid excessive drying.

Blood
Several commercial mycobacterial blood culture systems for NTM are available (see online supplement). Coagulated blood or blood collected in ethylenediaminetetraacetic acid (EDTA) is unacceptable. For rapidly growing mycobacteria (RGM), these special mycobacterial systems are not required as most RGM species grow well in routine blood culture systems.

Specimen Processing
To minimize contamination or overgrowth of cultures with bacteria and fungi, digestion and decontamination procedures should be performed on specimens collected from nonsterile body sites. Samples from contaminated sites contain other organisms that may grow more rapidly than NTM and interfere with the recovery of mycobacteria. Because NTM, especially RGM, are much more susceptible to decontamination than M. tuberculosis, these procedures should not be so severe as to eliminate the mycobacteria potentially present. Tissue samples or fluids from normally sterile sites do not require decontamination. Tissues should be ground aseptically in sterile physiological saline or bovine albumin and then directly inoculated onto the media.

The most widely used digestion–decontamination method uses N-acetyl-L-cysteine–sodium hydroxide (NALC-NaOH). This method is often used in conjunction with a 5% oxalic acid procedure ("double processing") for specimens from patients with CF or bronchiectasis whose sputa are known to be contaminated with aerobic gram-negative rods, especially Pseudomonas aeruginosa (42, 45). Instructions for commonly used digestion–decontamination methods are described elsewhere (46–48). Because NTM may be sensitive to oxalic acid decontamination, with reduced yield on culture, another option is to use a two-step decontamination approach reserving oxalic acid only for those specimens overgrown by bacteria other than NTM (47–49).

Smear Microscopy
The recommended method for staining clinical specimens for AFB, including both M. tuberculosis and NTM, is the fluorochrome technique, although the Ziehl-Neelsen method or Kinyoun stain are acceptable but less sensitive alternatives. The Gram stain is not adequate for detection of mycobacteria. In many cases, the NTM, especially the RGM, may be more sensitive to the AFB decolorization procedure and may not stain at all with fluorochrome stains. Therefore, if RGM are suspected, it may be prudent to use a weaker decolorizing process. It is also noteworthy that negative smears do not necessarily mean that NTM, especially RGM, are not present in a clinical sample.

Semiquantitative analysis of smears can be useful for diagnostic purposes. Fluorochrome smears are graded from 1+ (1–9 organisms per 10 high-power fields) to 4+ (> 90 organisms per high-power field) (47). The burden of organisms in clinical material is usually reflected by the number of organisms seen on microscopic examination of stained smears. Environmental contamination, which usually involves small numbers of organisms, rarely results in a positive smear examination. Previous studies have indicated that specimens with a high number of mycobacteria isolated by culture are associated with positive smears and, conversely, specimens with a low number of mycobacteria isolated by culture are less likely to have positive smears (50).

Culture Techniques
All cultures for mycobacteria should include both solid and broth (liquid) media for the detection and enhancement of growth (43). However, broth media cultures alone may not be satisfactory because of bacterial overgrowth. Cultures in broth media have a higher yield of mycobacteria and produce more rapid results than those on solid media. The advantages of solid media over broth media are that they allow the observation of colony morphology, growth rates, recognition of mixed (more than one mycobacterial species) infections, and quantitation of the infecting organism, and serve as a backup when liquid media cultures are contaminated.

Broth media.
One of the most widely used broth systems is the nonradiometric mycobacteria growth indicator tube (MGIT) (Becton Dickinson, Sparks, MD), which contains a modified Middlebrook 7H9 broth in conjunction with a fluorescence quenching–based oxygen sensor to detect mycobacterial growth. As the mycobacteria grow and deplete the oxygen present, the indicator fluoresces when subjected to ultraviolet light. For detailed discussion of broth (liquid) media culture techniques, see the online supplement.

Solid media.
Recommended solid media include either egg-based media, such as Löwenstein-Jensen agar or agar-based media such as Middlebrook 7H10 and 7H11 media. The agar-based media may also be used for susceptibility testing. Biphasic media, such as the Septi-Chek System (Becton Dickinson), provide enhanced recovery of most NTM in one system, but these are not rapid detection systems.

Semiquantitative (0–4+) reporting of NTM colony counts on solid media is recommended by the CLSI. A single positive respiratory sample with a low colony count (e.g., broth culture positive only) is less likely to be clinically significant than a sample with a high colony count (e.g., growth on both solid and broth media). This approach also helps in the assessment of response to therapy. After successful therapy of MAC lung disease and at least 10 months of negative cultures during therapy, a single positive culture that is AFB smear negative and of low culture positivity (< 10 colonies on solid media and/or positive in the broth media only) generally represents either contamination (false-positive culture) or transient new infections and not relapse of the original infecting strain (38, 51).

M. haemophilum, M. genavense, M. avium subsp. paratuberculosis (formerly M. paratuberculosis), and M. ulcerans are examples of fastidious NTM that require special supplementation for recovery on culture. M. haemophilum grows only on media supplemented with iron-containing compounds such as ferric ammonium citrate, hemin, or hemoglobin. Because M. haemophilum has a predilection for skin and the body's extremeties, all specimens from skin lesions, joints, or bones should be cultured in a manner suitable for recovery of this species. M. genavense and M. avium subsp. paratuberculosis require mycobactin J, and M. ulcerans may be optimally recovered with egg yolk supplementation.

Incubation of NTM Cultures
The optimal temperature for most cultures for NTM is between 28° and 37°C. Most clinically significant slowly growing mycobacteria grow well on primary isolation at 35° to 37°C with the exception of the following: the newly described M. conspicuum, which requires temperatures from 22° to 30°C for several weeks and only grows at 37°C in liquid media, M. haemophilum, which prefers temperatures from 28° to 30°C, M. ulcerans, which grows slowly at 25° to 33°C, and some strains of M. chelonae, which require temperatures between 28° and 33°C (5). Cultures for RGM and M. marinum should be incubated at 28° to 30°C. All skin, joint fluid, and bone specimens should be cultured at 28° to 30°C and at 35° to 37°C. Optimal recovery of all species may require duplicate sets of media at two incubation temperatures.

Most NTM grow within 2 to 3 weeks on subculture. To detect M. ulcerans or M. genavense, cultures should be incubated for at least 8 to 12 weeks. Rapidly growing mycobacteria usually grow within 7 days of subculture. Earlier detection of NTM can be expected with liquid-based systems. When stated on the laboratory report, the time in days to the detection of mycobacterial growth can be helpful to clinicians as an indication of isolation of a rapidly growing species.

Recommendations:  1. As much material as possible for NTM culture should be provided with clear instructions to the laboratory to culture for mycobacteria (C, III).  2. All cultures for NTM should include both a rapid detection broth (liquid) media technique and solid media cultures (C, III).  3. Quantitation of the number of colonies on plated culture media should be performed to aid clinical diagnosis (C, III).  4. Supplemented culture media and special culture conditions (lower incubation temperatures) should be used for material cultured from skin lesions, joints, and bone (A, II).  5. The time (in days) to detection of mycobacterial growth should be stated on the laboratory report (C, III).

NTM Identification
Because of differences in antimicrobial susceptibility that determine treatment options, species-level identification of the NTM is becoming increasingly clinically important (43). Several factors increase the likelihood of clinical significance of NTM isolates, including the recovery from multiple specimens or sites, recovery of the organism in large quantities (AFB smear–positive specimens), or recovery of an NTM isolate from a normally sterile site such as blood. For initial clinical mycobacterial isolates, however, it is sometimes difficult to determine the clinical significance of the isolate without species identification. Therefore, identification of most mycobacterial isolates to the species level and not merely as groups, such as "M. chelonae/abscessus group" is strongly recommended. If, after consultation between the clinician and the laboratorian and in the event that a specific laboratory does not have the necessary technology for species identification of an NTM isolate, the isolate could be sent to a reference laboratory for further analysis.

It is equally important to recognize that not all clinically obtained NTM isolates, especially from sputum, will need extensive identification efforts. For instance, a pigmented rapidly growing mycobacterium recovered in low numbers from only one of multiple sputum specimens from a patient undergoing therapy for MAC lung disease would not need an extensive effort for identification of that isolate as it would not likely be clinically significant. Awareness of the context from which an NTM isolate is obtained can be critically important in determining the need for speciation of that isolate. Again, communication between the clinician and laboratorian is critical for making this type of determination.

Phenotypic testing.
Preliminarily, NTM can be categorized by growth rate and pigmentation, which will help guide in the selection of proper testing procedures, including appropriate media selection and incubation temperatures. Recent studies have shown, however, that identification using only conventional biochemical analysis is both time consuming and increases turnaround time, leading to significant delays in diagnosis (52).

Detailed descriptions of methods, procedures, and quality control measures have been published (47, 48). Isolates of NTM, which form colonies on subculture in 7 days or fewer, are referred to as "rapidly growing mycobacteria" or RGM. Conversely, those isolates of NTM that require more than 7 days to form mature colonies on subculture are termed "slowly growing mycobacteria." Although many of the conventional and traditional laboratory tests for mycobacterial evaluation are not routinely used, the rate of growth is still useful for preliminary broad classification of a nontuberculous mycobacterium.

Traditionally, NTM have also been divided into three groups based on the production of pigment. These growth characteristics are rarely detailed in modern mycobacterial laboratories, but the presence of pigmentation and smooth colony morphology quickly exclude the isolate as belonging to the M. tuberculosis complex that forms nonpigmented and rough colonies.

Biochemical testing of NTM uses a battery of tests, again based on the growth rate of the NTM. The use of conventional testing alone does not allow identification of many of the newly described NTM and thus newer methods, including HPLC and molecular methods, must be used.

Chemotaxonomic testing: HPLC.
HPLC is a practical, rapid, and reliable method for identifying many slowly growing species of NTM. HPLC can also be used in the direct analysis of primary cultures of mycobacteria grown in BACTEC 7H12B medium (Becton Dickinson) and the identification of MAC directly from samples with AFB smear–positive results (53). However, HPLC has limitations. Recognition of some newer species and species within the M. simiae complex is difficult. It has also been reported that some species within the M. fortuitum group and the M. smegmatis group are difficult to separate, and distinction between M. abscessus and M. chelonae may not be possible (54). HPLC analysis will be less useful in the future, as identification of NTM species will be accomplished by molecular methods.

Genotypic methods for identification of NTM.
COMMERCIALLY AVAILABLE MOLECULAR PROBES. Acridium ester–labeled DNA probes specific for MAC (or separately for M. avium and M. intracellulare), M. kansasii, and M. gordonae have been approved by the U.S. Food and Drug Administration (FDA) and are currently used in many clinical laboratories (AccuProbe; GenProbe, Inc., San Diego, CA) for the rapid identification of NTM. This technique is based on the release of target 16S rRNA from the organism. Testing can be performed using isolates from solid or liquid culture media and identification of these species can be achieved within 2 hours. Studies have shown 100% specificity with a sensitivity between 85 and 100%. Only a few NTM species have available probes, representing a major limitation. In addition, there is the potential for cross-reaction of the probe for M. tuberculosis with M. celatum (55).

PRA.
The current PRA method widely adopted for the identification of NTM is based on the coupling of the PCR of a 441-bp sequence of the gene encoding the 65-kD heat shock protein (hsp65) followed by restriction enzyme digestion. The size of the restriction fragments is generally species specific (56–59). In one study, 100% of 129 nonpigmented RGM were identified using PRA (60). However, some taxa may require additional endonucleases for species identification (60).

The PRA method is relatively rapid, does not require viable organisms, and identifies many NTM species that are not identifiable by phenotypic or chemotaxonomic techniques alone. However, this system is not commercially available; therefore, the clinician may need to work closely with a public health or reference laboratory to determine the best method for species identification for a particular NTM isolate.

DNA SEQUENCE ANALYSIS.
The 16S rRNA is an approximately 1,500 nucleotide sequence encoded by the 16S ribosomal DNA (rDNA), which is a highly conserved gene with regions common to all organisms (conserved regions) and also areas where nucleotide variations occur (variable regions). For purposes of mycobacterial identification, sequence analysis focuses on two hypervariable sequences known as regions A and B. The sequence of region A is usually adequate to identify most NTM species, although sequencing of region B may be necessary, especially in the identification of undescribed species or those species which cannot be differentiated by sequence of the region A alone (5). Examples include M. kansasii/M. gastri, as well as M. ulcerans and M. marinum, and M. shimoidei and M. triviale. Isolates of M. chelonae and M. abscessus cannot be differentiated within the regions A and B, although they do vary at other 16S rRNA gene sites (although only by a total of 4 bp) (60, 61).

Problems with this method are that species of recent divergence may contain highly similar 16S rRNA gene sequences. For example, the difference between M. szulgai and M. malmoense is two nucleotides, although it is well established that these are two distinct species. In addition, no interstrain nucleotide sequence difference value that unequivocally defines different species has been established for mycobacteria (48).

The automation of sequence analysis by the introduction of commercial systems like the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems, Foster City, CA), in which a 500-bp sequence of the NTM is analyzed and compared with a commercially prepared database, has made sequencing more efficient for use in the clinical laboratory. One of the major limitations of this system, however, is that the MicroSeq database has only one entry per species (generally the type strain) (61). This is particularly problematic when the unknown isolate does not have an exact match in the databases. Currently, isolates may be reported as "most closely related to" a species depending on the sequence difference between the unknown isolate and the database entry (62, 63).

Recommendations:  1. Clinically significant NTM isolates should be routinely identified to the species level. An important exception is MAC because the differentiation between M. avium and M. intracellulare is not yet clinically significant. Although not routinely recommended, this differentiation may be important epidemiologically and, in the future, therapeutically (C, III).  2. The RGM (especially M. chelonae, M. abscessus, and M. fortuitum) should be identified to species level using a recognized acceptable methodology, such as PRA or biochemical testing, not HPLC alone (A, II).  3. Susceptibility of RGM for eight agents, including amikacin, cefoxitin, clarithromycin, ciprofloxacin, doxycycline, linezolid, sulfamethoxazole, and tobramycin, can also be used to facilitate identification of M. abscessus, M. chelonae, and M. fortuitum (C, III).  4. Communication between the clinician and laboratorian is essential for determining the importance and extent of the identification analysis for a clinical NTM isolate (C, III).

Antimicrobial Susceptibility Testing for NTM

Context: There is ongoing debate about the role of in vitro susceptibility testing for managing patients with NTM disease. The controversy primarily stems from the observation that, unlike M. tuberculosis, MAC response to anti-TB drugs such as rifampin and ethambutol may not be reliably predicted on the basis of current in vitro susceptibility test methods. NTM such as M. kansasii, M. marinum, or M. fortuitum are susceptible in vitro to multiple antimicrobial drugs and the clinical response to therapeutic agents appears to closely parallel the in vitro susceptibility pattern, although this observation has not been evaluated by randomized controlled trials. In contrast, MAC has limited in vitro susceptibility, and clinical response has been shown to correlate only with the macrolides. Last, organisms such as M. abscessus and M. simiae have limited in vitro susceptibility, with limited evidence for a correlation between in vitro susceptibility to any agent and clinical response in the treatment of pulmonary disease caused by these agents. Interestingly, for skin and soft tissue infections caused by M. abscessus, there does appear to be a correlation between in vitro susceptibility and clinical response, although this observation has not been prospectively evaluated. Susceptibility breakpoints have been defined in the laboratory to distinguish populations of mycobacteria that are labeled susceptible and resistant. For many NTM, however, these laboratory cutoffs have not been confirmed to be clinically meaningful, so that there are few data to validate susceptibility testing for many NTM species as a guide for choosing antibiotics. Until the relationship between in vitro susceptibility of many NTM and their clinical response to antimicrobial drugs is better understood and clarified, the clinician should use in vitro susceptibility data with an appreciation of its limitations and with the awareness that, unlike TB, some NTM disease may not be eradicated in a given patient with therapy based on in vitro susceptibility results. The use of in vitro susceptibility testing for NTM is outlined below and consistent with CLSI recommendations.

SLOWLY GROWING MYCOBACTERIA.
For the slowly growing NTM, no single susceptibility method is recommended for all species. For isolates of MAC, the CLSI has recommended a broth-based method with both microdilution or macrodilution methods being acceptable (43). Until further multicenter studies have been performed with the other slowly growing mycobacteria, broth and solid methods of susceptibility testing may be performed with the caveat that each laboratory must validate each method for each species tested, and quality control and proficiency testing requirements should be enforced.

MAC.
Initial isolates from patients with previously untreated MAC lung disease should be tested to clarithromycin to establish baseline values. The isolate may also be saved for future testing, especially to determine if future isolates represent relapse or new infections by comparative DNA fingerprinting of baseline and subsequent isolates (51). Other isolates to be tested include the following:

1. Isolates from patients who previously received macrolide therapy to determine whether or not the isolates are still macrolide susceptible.
   
2. Isolates from patients with MAC pulmonary disease on macrolide-containing regimens who relapse or fail after 6 months of macrolide-containing therapy.
   
3. Isolates from patients with AIDS who develop bacteremia on macrolide prophylaxis.
   
4. Positive blood cultures after 3 months of treatment with macrolide-containing regimens for patients with disseminated MAC.
   

Strains that appear intermediate in susceptibility to clarithromycin rarely occur and should be confirmed by another testing event. Patients with these intermediate MICs should be followed closely for possible emergence of macrolide resistance. Macrolides should be included in treatment regimens for these patients unless the isolate is found on subsequent testing to be macrolide resistant.

Because no correlation between in vitro susceptibility results for MAC and clinical response for agents other than macrolides has been established, the 2003 CLSI document states that clarithromycin is the only drug for which susceptibility testing for MAC isolates is recommended (43).

The role of extended in vitro susceptibility testing for macrolide-resistant MAC isolates is unproven. However, some experts suggest that it may be reasonable to test other antimicrobials such as the 8-methoxy fluoroquinolone moxifloxacin and linezolid for patients who fail initial macrolide-based therapy (43). Although there is a paucity of published data, some experts feel that moxifloxacin has in vitro activity against MAC at clinically achievable serum levels. It was recently reported that out of 189 isolates of MAC, only 13% of the isolates studied had MICs of 8 µg/ml or less for linezolid; however, there have been reports of successful multidrug therapy with linezolid in the treatment of MAC (66, 67). Testing of anti-TB medications does not provide useful clinical information.

M. kansasii.
The 2003 CLSI document states that only susceptibility to rifampin should be routinely performed for isolates of M. kansasii (43). MICs of all agents in untreated (wild) strains fall in a narrow range and treatment failure is usually associated with resistance to rifampin. Resistance to isoniazid and ethambutol acquired on therapy may also occur, but resistance to these agents is usually associated with resistance to rifampin (68). Isolates that are susceptible to rifampin are also susceptible to rifabutin, which may be substituted for rifampin in HIV-infected patients being treated with highly active antiretroviral therapy (HAART), including some protease inhibitors and nonnucleoside reverse transcriptase inhibitors (see DISSEMINATED MAC DISEASE). If the isolate proves to be rifampin resistant, susceptibility to secondary agents, including amikacin, ciprofloxacin, clarithromycin, ethambutol, rifabutin, streptomycin, sulfonamides, and isoniazid, should be tested. Susceptibility to the new 8-methoxy fluoroquinolone, moxifloxacin, should be performed separately because ciprofloxacin is the class representative for ciprofloxacin, ofloxacin, and levofloxacin only. In addition, the standard critical concentrations of 0.2 and 1 µg/ml for isoniazid used for testing strains of M. tuberculosis should not be tested as MICs for untreated strains between 0.5 and 5 µg/ml. Thus, the 0.2-µg/ml standard concentration appears resistant and the 1.0-µg/ml standard concentration may yield variable results, even with multiple cultures of the same strain (43).

M. marinum.
Routine susceptibility testing of this species is not recommended (43). There is no documentation of significant risk of mutational resistance to the antimycobacterial agents and there is no appreciable variability in susceptibility patterns to clinically useful agents (69). Disease involving M. marinum is typically localized and the number of organisms present is low; 95% of tissue biopsy cultures are AFB smear negative (2). Isolates of M. marinum are susceptible to clarithromycin, as well as the sulfonamides, the tetracyclines, rifampin, and ethambutol. Ciprofloxacin is not recommended because some strains are resistant and monotherapy carries the risk of mutational resistance (70). However, some experts report anecdotally that the newer 8-methoxy fluoroquinolone, moxifloxacin, is more active in vitro and could be considered for multidrug therapy. Susceptibility testing should be considered for patients who remain culture positive after more than 3 months of therapy.

MISCELLANEOUS SLOWLY GROWING NTM (M. SIMIAE COMPLEX, M. TERRAE/NONCHROMOGENICUM COMPLEX, M. MALMOENSE, M. XENOPI).
Because too few isolates of each species have been studied, no specific susceptibility method can be recommended at this time for the less commonly isolated NTM including several newly described species (5, 43). Until further data are available, testing should be performed as for rifampin-resistant M. kansasii (i.e., rifampin and secondary agents should be tested) (43).

RGM.
The CLSI has recommended the broth microdilution MIC determination for susceptibility testing of RGM. Agar tests, including the E-test (epsilometer test), cannot be recommended due to inconsistency of results (71). The broth microdilution technique is described in the 2003 NCCLS M24A-approved document (40). MICs for imipenem are problematic with isolates of M. chelonae, M. abscessus, and M. immunogenum because of the lack of reproducibility (43, 72). For the majority of M. abscessus and M. chelonae isolates, imipenem is the preferred carbapenem over meropenem and ertapenem (73). Imipenem may still be useful clinically in treatment regimens for these organisms. In contrast, MICs for imipenem with isolates of the M. fortuitum group, M. smegmatis group, and M. mucogenicum are reproducible.

True aminoglycoside resistance with M. abscessus is unusual but does occur, especially in patients on long-term treatment with aminoglycosides, such as patients with CF or chronic otitis media. In vitro susceptibility studies suggest that tobramycin is the most active aminoglycoside for M. chelonae; therefore, it is recommended to report tobramycin MICs only for this species (43).

In vitro testing of clarithromycin may present interpretation problems with RGM. Thus, the CLSI has recommended that isolates of the M. fortuitum group with indeterminate or unclear susceptibility endpoints with clarithromycin should be reported as resistant until further data are available with these isolates (43). Recent studies have shown that all isolates of M. fortuitum contain an inducible erythromycin methylase gene erm (39), which confers macrolide resistance (74). The presence of this gene with variable expression is likely responsible for this phenomenon. Similar erm genes have been reported in other RGM species that are macrolide resistant (e.g., M. smegmatis) but not in M. abscessus despite its general poor response to macrolide therapy (75).

FASTIDIOUS SPECIES OF NTM.
As for the previous group of less commonly recovered NTM, no current standardized method can be recommended at this time due to lack of experience and available data concerning methods and results.

M. HAEMOPHILUM.
Isolates are generally susceptible to the first-line anti-TB agents (except ethambutol), clarithromycin, and the sulfonamides (76).

M. GENAVENSE, M. AVIUM SUBSP. PARATUBERCULOSIS, M. ULCERANS.
Susceptibility testing of these species is difficult since they do not grow in standard susceptibility media without supplementation and extended incubation; therefore, standardized guidelines for in vitro susceptibility procedures are not available for testing these species (77–82).

Recommendations:  1. Clarithromycin susceptibility testing is recommended for new, previously untreated MAC isolates. Clarithromycin is recommended as the "class agent" for testing of the newer macrolides because clarithromycin and azithromycin share cross-resistance and susceptibility. No other drugs are recommended for susceptibility testing of new, previously untreated MAC isolates. There is no recognized value for testing of first-line antituberculous agents with MAC using current methodology (A, II)  2. Clarithromycin susceptibility testing should be performed for MAC isolates from patients who fail macrolide treatment or prophylaxis regimens (A, II).  3. Previously untreated M. kansasii strains should be tested in vitro only to rifampin. Isolates of M. kansasii that show susceptibility to rifampin will also be susceptible to rifabutin (A, II).  4. M. kansasii isolates resistant to rifampin, should be tested against a panel of secondary agents, including rifabutin, ethambutol, isoniazid, clarithromycin, fluoroquinolones, amikacin, and sulfonamides (A, II).  5. M. marinum isolates do not require susceptibility testing unless the patient fails treatment after several months (A, II).  6. There are no current recommendations for one specific method of in vitro susceptibility testing for fastidious NTM species and some less commonly isolated NTM species (C, III).  7. Validation and quality control should be in place for susceptibility testing of antimicrobial agents with all species of NTM (C, III).

Molecular Typing Methods of NTM
Molecular typing methods have become valuable epidemiologic tools in the investigation of outbreaks, pseudo-outbreaks, and epidemics involving NTM. If a laboratory that performs molecular typing methods of NTM isolates cannot be readily identified, the clinician or investigator should contact the state's Department of Health or the CDC for advice about laboratories that can assist with this analysis.

Pulsed-field gel electrophoresis.
Pulsed-field gel electrophoresis (PFGE) is one of the most widely used and valuable methods of molecular typing of NTM. This technique involves embedding the isolates in agarose gels, lysing the DNA, and digesting chromosomal DNA with specific restriction endonucleases (79–81). This is a time-consuming procedure because the organisms must be actively grown such that a sufficient biomass is available to yield accurate results. Furthermore, more than one-half of the DNA from strains of M. abscessus will spontaneously lyse or be digested during the procedure, although this can be corrected with the addition of thiourea to stabilize the running buffer (79–81).

Despite its limitations, however, PFGE is currently the most common typing method for strain differentiation of RGM and other NTM. Unrelated strains of most species of RGM are highly heterogeneous and restriction fragment length polymorphism (RFLP) patterns for the same strain are identical or indistinguishable (83–86).

Other typing methods.
Other methods have been used for strain comparison, including random amplified polymorphic DNA PCR, multilocus enzyme electrophoresis using multiple housekeeping cellular enzymes, and hybridization with multicopy insertional elements in M. avium but not M. intracellulare.

	CLINICAL PRESENTATIONS AND DIAGNOSTIC CRITERIA

TOP CONTENTS SUMMARY INTRODUCTION METHODS TAXONOMY EPIDEMIOLOGY PATHOGENESIS LABORATORY PROCEDURES CLINICAL PRESENTATIONS AND... NTM SPECIES: CLINICAL ASPECTS... RESEARCH AGENDA FOR NTM... SUMMARY OF RECOMMENDATIONS REFERENCES

 
Pulmonary Disease
Epidemiology.
Chronic pulmonary disease is the most common clinical manifestation of NTM (19, 87, 88). MAC, M. kansasii, and M. abscessus, in that order, were the most frequent NTM pulmonary pathogens in the United States between 1981 and 1983 (19). In that study, there was a predominance of males with NTM pulmonary disease in general and in disease caused by all species except M. chelonae (probably M. abscessus) and M. simiae (19). The mean age of patients with NTM pulmonary disease was 57 years (19). In the CDC report from the mid-1990s, MAC, M. kansasii, and M. fortuitum were the most frequent pulmonary pathogens in the United States between 1993 and 1996 (22). Males predominated in disease caused by all species except M. abscessus and the majority of isolates were from patients 50 years of age or older. In light of more recent information that reflects a postmenopausal female patient predominance for MAC lung disease, it is likely that these epidemiologic estimates are not currently valid (36, 37).

Symptoms and signs.
The symptoms of NTM pulmonary disease are variable and nonspecific. However, virtually all patients have chronic or recurring cough. Other symptoms variably include sputum production, fatigue, malaise, dyspnea, fever, hemoptysis, chest pain, and weight loss. Constitutional symptoms are progressively more prevalent with advancing NTM lung disease. Evaluation is often complicated by symptoms caused by coexisting lung diseases, such as bronchiectasis, chronic obstructive airway disease associated with smoking, CF, and pneumoconiosis.

Physical findings are nonspecific and reflect underlying pulmonary pathology, such as bronchiectasis and chronic obstructive lung disease. On chest auscultation, findings may include rhonchi, crackles, wheezes, and squeaks. Patients with nodular/bronchiectatic MAC disease tend to be postmenopausal women, many of whom also have a characteristic morphotype with a thin body habitus and may also have scoliosis, pectus excavatum, and mitral valve prolapse (27).

Radiographic studies.
Radiographic features of NTM lung disease depend on whether the lung disease is primarily fibrocavitary (similar to TB) or characterized by nodules and bronchiectasis (nodular/bronchiectatic disease) (see the online supplement). Compared with the radiographic findings in TB, patients with NTM disease and predominantly fibrocavitary radiographic changes tend to have the following characteristics: (1) thin-walled cavities with less surrounding parenchymal opacity, (2) less bronchogenic but more contiguous spread of disease, and (3) to produce more marked involvement of pleura over the involved areas of the lungs (2, 88, 89). None of these differences, however, is sufficiently specific to exclude the diagnosis of TB on the basis of the radiographic appearance. NTM may produce dense airspace disease or a solitary pulmonary nodule without cavitation. Basal pleural disease is not often found, and pleural effusion is rare.

For patients with predominantly noncavitary disease, the abnormalities on chest radiograph are primarily found in the mid- and lower lung field. Studies with HRCT of the chest have shown that up to 90% of patients with mid- and lower lung field noncavitary disease with MAC have associated multifocal bronchiectasis, with many patients having clusters of small (< 5 mm) nodules in associated areas of the lung (90–94). These findings correspond histopathologically to bronchiectasis, bronchiolar and peribronchiolar inflammation, and granuloma formation (94). Cavitatio