Mycobacterial Susceptibility and Latent Infection Testing

Test the initial isolate from every patient against isoniazid, rifampin, ethambutol, and pyrazinamide. Repeat testing if the culture remains positive after 3 months of therapy, or sooner if there is clinical evidence of failure, because resistance can emerge during treatment. In the agar-proportion reference method, an isolate is resistant when 1% or more of the inoculated population grows at the critical drug concentration. Results take about 3 to 4 weeks. Commercial liquid systems such as the Mycobacteria Growth Indicator Tube (MGIT) 960 are preferred where available because they shorten turnaround to roughly 1 to 2 weeks for most drugs. Pyrazinamide testing has its own requirements. MGIT is the only platform with a standardized method. The acidic medium is prone to false-resistant results when the inoculum is not prepared correctly. A resistant result is confirmed against the pncA gene before it is released. Combined MGIT plus pncA sequencing is now the accepted reference for pyrazinamide.^{22 World Health Organization. WHO operational handbook on tuberculosis. 2025.}

Molecular assays complement phenotypic testing, which continues alongside them. Probe-based and line-probe assays detect a predefined set of mutations, or infer resistance when a wild-type probe drops out. Sequencing reads the base sequence of each target and is interpreted against the World Health Organization (WHO) catalog of mutations, which grades each variant for its association with resistance to a specific drug. The loci in routine use are:

A rifampin-resistance result is a practical screen for multidrug-resistant tuberculosis because isoniazid resistance often accompanies it. The two are not equivalent, because isoniazid can remain susceptible. Borderline rpoB variants such as Leu430Pro, Asp435Tyr, and Leu452Pro raise the minimum inhibitory concentration (MIC) only modestly, so isolates carrying them can test susceptible at the standard MGIT critical concentration. Ile491Phe is the classic resistance mutation that RRDR-based assays miss. It lies outside the region most cartridge and line-probe assays cover, and in the 2009 Eswatini drug-resistance survey it accounted for 38 of 125 multidrug-resistant isolates that Xpert MTB/RIF reported as rifampin-susceptible.^{1818 Sanchez-Padilla E, et al. Detection of drug-resistant tuberculosis by Xpert MTB/RIF in… 2015.} Silent rpoB changes are not resistance markers. On a line-probe assay, probe dropout caused by a synonymous mutation is interpreted as resistance, and sequencing does not support that result. Heteroresistance, a resistant subpopulation below an assay's detection threshold, can cause discordant molecular and phenotypic results in both directions.^{2,32 World Health Organization. WHO operational handbook on tuberculosis. 2025.3 World Health Organization. Catalogue of mutations in Mycobacterium tuberculosis complex and… 2023.}

Second-line testing is triggered by demonstrated or suspected rifampin resistance, resistance to two or more first-line drugs, prior anti-tuberculosis therapy, contact with a drug-resistant case, or cultures still positive after 3 months of treatment. The panel reflects current treatment regimens. Bedaquiline and linezolid are central drugs for rifampin-resistant tuberculosis, so WHO prioritizes susceptibility testing for them and for the fluoroquinolones, pyrazinamide, and clofazimine. The panel also includes amikacin and streptomycin when those drugs will be used. Kanamycin and capreomycin were dropped from recommended regimens from 2019 to 2021 because of toxicity and outcomes. Ethionamide and p-aminosalicylic acid have no reliable growth-based method, so molecular testing (inhA promoter, ethA) or no testing is preferred for them. CLSI M24 retains critical concentrations for older injectable drugs that laboratories may still be asked to test. Rifabutin is included for patients who need a rifamycin during antiretroviral therapy. The Molecular Detection of Drug Resistance service of the Centers for Disease Control and Prevention (CDC) accepts isolates and, for some mutations, direct specimens, and sequences the loci above plus others. A negative molecular result still requires phenotypic confirmation because not every mechanism is characterized.^{2,32 World Health Organization. WHO operational handbook on tuberculosis. 2025.3 World Health Organization. Catalogue of mutations in Mycobacterium tuberculosis complex and… 2023.}

Multidrug-resistant tuberculosis (MDR-TB) is resistance to at least isoniazid and rifampin. Rifampin-resistant tuberculosis (RR-TB) is resistance to rifampin alone, with or without isoniazid resistance. WHO groups the two as MDR/RR-TB because management is the same. Pre-XDR-TB is MDR/RR-TB with additional resistance to any fluoroquinolone. Extensively drug-resistant tuberculosis (XDR-TB), in the WHO definitions effective January 2021, is MDR/RR-TB with resistance to any fluoroquinolone plus at least one other Group A drug (bedaquiline or linezolid). Group A drugs for regimen-building are levofloxacin, moxifloxacin, bedaquiline, and linezolid. Older texts and case series use an earlier XDR-TB definition based on resistance to a second-line injectable. The injectable criterion was removed from the definition because injectables were removed from recommended regimens. In a pre-2021 source, XDR-TB refers to that earlier definition. Under the current definition, susceptibility testing for bedaquiline and linezolid is required to classify a case as XDR.^{44 World Health Organization. WHO announces updated definitions of extensively drug-resistant… 2021.}

Clinicians start therapy before susceptibility results are available, so report each result as soon as it is final. The first-line panel tests isoniazid, rifampin, ethambutol, and pyrazinamide. It does not include rifapentine or moxifloxacin, the drugs of the 4-month regimen used since 2022, so moxifloxacin needs a second-line request.

Test only clinically significant isolates. For Mycobacterium avium complex (MAC), in vitro susceptibility correlates with clinical response only for the macrolides and amikacin. Ethambutol and rifamycin MICs do not predict outcome. The 2020 joint guideline of the American Thoracic Society, European Respiratory Society, European Society of Clinical Microbiology and Infectious Diseases, and Infectious Diseases Society of America (ATS/ERS/ESCMID/IDSA) bases this on a randomized trial that found no association between susceptibility to either drug and treatment failure or relapse. For M. kansasii, the most important drug to test is rifampin. Broth microdilution per CLSI M24 is the recommended method for both rapid and slow growers. Results are reported as an MIC plus a categorical interpretation, except where CLSI assigns no interpretive criteria and only the MIC is reported: tigecycline against rapid growers, and clofazimine, bedaquiline, and tedizolid against any nontuberculous mycobacterium (NTM).^{1,51 Clinical and Laboratory Standards Institute. M24, Susceptibility Testing of Mycobacteria, Nocardia spp., and…5 Daley CL, et al. Treatment of nontuberculous mycobacterial pulmonary disease. 2020.}

Amikacin results for MAC are read against separate thresholds for the intravenous drug and for the inhaled liposomal suspension, because lung exposure differs enough to require a different breakpoint. Clarithromycin is tested as the class representative for the macrolides. Resistance in MAC and in M. abscessus comes from rrl 23S rRNA mutations at positions 2058 and 2059, and these mutations predict resistance to both azithromycin and clarithromycin.

Inducible macrolide resistance is essential knowledge on the rapid-grower bench. M. abscessus subsp. abscessus and subsp. bolletii carry erm(41), an inducible methylase gene. A thymine at position 28 predicts a functional enzyme, and a T-to-C change at that position inactivates it. Subsp. massiliense carries a truncated, nonfunctional erm(41) and is macrolide-susceptible unless it has acquired an rrl mutation. Because induced resistance takes days to appear, CLSI M24 requires clarithromycin to be read at day 3 to 5 and again at day 14 for rapid growers. A day-3 susceptible result cannot be finalized until the day-14 read. Inducible macrolide resistance also occurs outside M. abscessus. The M. fortuitum group carries the related gene erm(39), and most M. fortuitum, M. porcinum, and M. septicum isolates are inducibly clarithromycin-resistant. M. chelonae lacks erm(41). M. chelonae was long taught to have no erm gene, but plasmid-borne and chromosomal variants of a novel methylase gene, erm(55), were described in clinical isolates in 2023. The plasmid form confers high-level inducible clarithromycin resistance that appears only at the day-14 read, so CLSI now advises the extended incubation for M. chelonae as well.^{16,1716 Brown-Elliott BA, et al. Emergence of inducible macrolide resistance in Mycobacterium… 2023.17 CLSI. Mycobacterium chelonae. 2025.} Species identification therefore does not exclude inducible resistance. Genotype is useful when the subspecies is known. The extended-incubation read detects inducible resistance regardless of species, subspecies, or genotype.^{1,51 Clinical and Laboratory Standards Institute. M24, Susceptibility Testing of Mycobacteria, Nocardia spp., and…5 Daley CL, et al. Treatment of nontuberculous mycobacterial pulmonary disease. 2020.},^{1414 Nash KA, et al. A novel gene, erm(41), confers inducible macrolide resistance to… 2009.},^{1515 Kim SY, et al. Species distribution and macrolide susceptibility of… 2019.}

Current disease guidelines explain why MAC susceptibility reporting centers on macrolides and distinguishes intravenous from inhaled liposomal amikacin breakpoints. The laboratory reports the organism identification, MICs, method-specific interpretations, and inducible macrolide-resistance finding. The infectious diseases or pulmonary service selects the regimen, route, and dosing schedule.^{55 Daley CL, et al. Treatment of nontuberculous mycobacterial pulmonary disease. 2020.}

A positive tuberculosis blood test or skin test usually indicates infection, but neither test distinguishes latent infection from active tuberculosis disease. Latent infection is diagnosed only after the clinical evaluation excludes active disease. A positive acid-fast smear alone cannot establish tuberculosis because other mycobacteria can produce that finding. Laboratory results are interpreted with clinical and radiographic evidence.^{66 Centers for Disease Control and Prevention. Clinical and laboratory diagnosis for tuberculosis. 2025.} Selection of the immune-response test and interpretation of its cutoffs are covered in Tuberculosis.

Tuberculin skin test (TST, Mantoux method). The test uses 0.1 mL of purified protein derivative (5 tuberculin units), injected intradermally. Induration is measured 48 to 72 hours later. False-positive reactions can result from NTM infection and bacille Calmette-Guérin (BCG) vaccination. False-negative reactions can result from poor technique, reagent storage failure, or anergy. A second test placed 1 to 3 weeks after a negative first test can restore a waned reaction (the booster effect). Two-step baseline testing is used when serial testing will follow, so that a boosted reaction is not misread as a new conversion and counted as recent infection.

Interferon-γ release assay (IGRA). The whole-blood QuantiFERON assay measures T-cell interferon-γ release in response to M. tuberculosis-specific antigens (ESAT-6 and CFP-10 in current QuantiFERON-TB Gold Plus; the older QuantiFERON-TB Gold In-Tube also included TB7.7) that are absent from BCG and from most NTM. The classically cited exceptions are M. kansasii, M. szulgai, and M. marinum. A single blood draw is collected into the manufacturer's antigen tubes. The laboratory incubates the tubes and reports interferon-γ release. Prior BCG vaccination does not affect IGRA results. T-SPOT.TB, the other IGRA approved by the Food and Drug Administration (FDA), uses the same ESAT-6 and CFP-10 antigens in an enzyme-linked immunospot (ELISPOT) format on separated peripheral blood mononuclear cells.^{66 Centers for Disease Control and Prevention. Clinical and laboratory diagnosis for tuberculosis. 2025.}

Neither test predicts which infected person will progress to disease, so both are used only as screening tools.

Containment for M. tuberculosis complex is tiered by activity, per Biosafety in Microbiological and Biomedical Laboratories (BMBL). Manipulating clinical specimens suspected of containing MTBC can be done at biosafety level 2 (BSL-2) when the procedures do not generate aerosols. Propagating or manipulating any culture of MTBC requires BSL-3 practices, containment equipment, and facilities. In practice, direct-smear and initial-setup work in a mycobacteriology laboratory may be done at BSL-2 after a procedure-specific risk assessment. All work downstream of a positive culture, including susceptibility testing, is done at BSL-3.^{77 Centers for Disease Control and Prevention, et al. Biosafety in Microbiological and Biomedical Laboratories, 6th ed. 2020.}

For a microbiology laboratory, CDC's ventilation table lists negative-pressure rooms exhausted to the outside, single-pass nonrecirculating air, directional inward airflow, and a minimum of 6 mechanical air changes per hour (ACH) with direct outdoor exhaust. The 12 ACH figure often cited elsewhere applies to airborne-infection isolation rooms. CDC sets 12 ACH for newly constructed or renovated isolation rooms and autopsy suites and permits 6 ACH for existing isolation rooms where 12 is infeasible. These are patient-care and autopsy requirements and do not apply to the laboratory.^{88 Centers for Disease Control and Prevention. Guidelines for preventing the transmission of Mycobacterium… 2005.}

Use a certified Class I or Class II biological safety cabinet for all specimen and culture handling, with certification at least annually and after relocation, repair, or filter change. Perform aerosol-generating procedures, including inoculating media and opening centrifuge cups, inside the cabinet. Load, unload, and open sealed rotors or gasketed safety cups there as well. Respiratory protection is the last layer of protection, used when aerosol-generating work occurs outside the cabinet. An N95 is certified against a near-worst-case aerosol centered around 0.3 µm, the most penetrating particle size, and must show at least 95% filtration efficiency. The infectious droplet nuclei the respirator is meant to stop measure 1 to 5 µm, larger than the 0.3 µm test aerosol used for certification. A respiratory-protection program under the Occupational Safety and Health Administration (OSHA) standard 29 CFR 1910.134 requires medical evaluation, fit testing before first use and at least annually, refitting on model or facial change, and a user seal check with each donning.^{8,98 Centers for Disease Control and Prevention. Guidelines for preventing the transmission of Mycobacterium… 2005.9 Occupational Safety and Health… 29 CFR 1910.134, Respiratory protection. 1910.}

Surface disinfection uses a tuberculocidal product registered by the Environmental Protection Agency (EPA) (List B), applied at the label dilution and wet-contact time. Alcohol alone evaporates too quickly to keep the surface wet for the contact time. High-efficiency particulate air (HEPA) filtration and upper-room ultraviolet germicidal irradiation (UVGI) can supplement ventilation where recirculation cannot be avoided. Neither one replaces outdoor air. Ship under the infectious-substance rules. Cultures of MTBC ship as Category A (UN 2814) and patient specimens as Category B (UN 3373). NTM cultures are classified by risk assessment and are not automatically Category A.^{1010 US Environmental Protection Agency. Registered antimicrobial products effective against…}

Since 2019, US guidance no longer recommends routine annual TST or IGRA screening of health-care and laboratory personnel. Baseline testing at hire and post-exposure testing are still recommended, and annual TB education continues for all personnel. Annual symptom screening is specified for personnel with untreated latent TB infection, and a facility may add it for higher-risk groups, after transmission in the setting, or where state or local law requires it. The change was made because serial testing of US personnel produced few conversions and many false positives and reversions.^{1111 Sosa LE, et al. Tuberculosis screening, testing, and treatment of U.S. health… 2019.}

Nocardia is an environmental saprophyte. The laboratory usually encounters it in specimens from pneumonia, brain abscess, or a nonhealing wound. Pulmonary infection follows inhalation of the organism. Pulmonary disease predominates, so lower respiratory specimens (sputum, bronchoalveolar lavage) are the most common source, with aspirate or tissue from the involved site preferred where it can be obtained. Submit brain-abscess aspirate or tissue, and cerebrospinal fluid (CSF) when central nervous system (CNS) involvement is suspected. An abscess aspirate is the better specimen. CSF culture is often negative even in CNS disease. Blood culture is indicated when dissemination or catheter infection is suspected, but a negative result does not exclude it. Skin and soft-tissue aspirates or biopsies are the specimens for cutaneous and lymphocutaneous disease, which also occurs in immunocompetent hosts after traumatic inoculation. Host risk factors for invasive disease include long-term corticosteroids, solid-organ or hematopoietic stem-cell transplantation, hematologic malignancy, chronic lung disease, and advanced HIV. Pulmonary disease mimics tuberculosis or malignancy with fever, cough, and nodular or cavitary infiltrates, and hematogenous spread to brain and skin is common.^{1212 Centers for Disease Control and Prevention. Clinical overview of nocardiosis. 2025.}

Notify the laboratory of suspected nocardiosis, because routine cultures are otherwise discarded too early. Hold solid-media plates at least 14 days, and some protocols hold longer. Nocardia typically becomes visible within 2 to 7 days but can take weeks, so plates discarded at the routine 48-hour cutoff miss it. The harsh N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) decontamination used for mycobacterial cultures can also kill Nocardia, so a specimen shared with mycobacterial culture may need a separate aliquot processed more gently.^{12,1312 Centers for Disease Control and Prevention. Clinical overview of nocardiosis. 2025.13 Traxler RM, et al. Updated review on Nocardia species. 2022.}

The old umbrella name N. asteroides complex is obsolete and is no longer reported as an identification. It described a phenotypic group that molecular taxonomy has divided into separate species, and isolates once reported under it now have species names such as N. cyriacigeorgica. Identify isolates to species level because susceptibility varies by species and no single regimen fits every infection.^{1313 Traxler RM, et al. Updated review on Nocardia species. 2022.}

On Gram stain, Nocardia appears as beaded, branching, filamentous, weakly gram-positive rods. This morphology overlaps with Actinomyces and can resemble fungal hyphae at low power. The modified Kinyoun stain uses a weak-acid decolorizer (0.5 to 1% sulfuric acid in common protocols). The standard mycobacterial stain uses acid-alcohol. Under the gentler decolorization Nocardia is partially acid-fast. The exact decolorizer formulation varies by method, and partial acid-fastness is variable, so a negative modified acid-fast stain does not exclude Nocardia. Aerobic growth distinguishes it from Actinomyces, which is non-acid-fast and anaerobic or aerotolerant. These differences support only a presumptive identification. Several other aerobic actinomycetes are also partially acid-fast, so identification requires further testing.^{1313 Traxler RM, et al. Updated review on Nocardia species. 2022.}

Nocardia grows aerobically on blood and chocolate agar, buffered charcoal yeast extract, and mycobacterial media including Löwenstein-Jensen. Colonies are dry and chalky, heaped or wrinkled, white to orange, with aerial hyphae. N. farcinica is the usual exception to the chalky appearance. A musty odor is sometimes noticed but is not a dependable identification feature.

Phenotypic panels cannot reliably identify species. Identification relies on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with an adequate Nocardia database and extraction method, 16S rRNA sequencing, and secA1, hsp65, gyrB, or rpoB sequencing for finer resolution when 16S alone will not separate close species. Use whole-genome sequencing or refer to a reference laboratory for isolates that remain unresolved. Broad-range 16S polymerase chain reaction (PCR) with amplicon sequencing on sterile-site specimens can detect Nocardia when culture is negative, but no isolate is then available for susceptibility testing. Test every clinically significant isolate by CLSI M24 broth microdilution, generally at a reference laboratory. Trimethoprim-sulfamethoxazole is the usual first-line agent, but it is not universally active. N. farcinica can be resistant to it and to other drugs, so each significant isolate needs its own susceptibility result. Linezolid is active against nearly all species, and amikacin, imipenem, and ceftriaxone are the other agents commonly tested. Sulfonamide endpoints trail. Read the well at the standard 80% inhibition point after full incubation to avoid overcalling resistance. Handling Nocardia cultures requires BSL-2 practices and a biological safety cabinet for aerosol-generating procedures.^{1,12,131 Clinical and Laboratory Standards Institute. M24, Susceptibility Testing of Mycobacteria, Nocardia spp., and…12 Centers for Disease Control and Prevention. Clinical overview of nocardiosis. 2025.13 Traxler RM, et al. Updated review on Nocardia species. 2022.}

Catalase and superoxide dismutase are proposed contributors to survival of the phagocyte oxidative burst, and inhibition of phagosome-lysosome fusion and a surface cord factor (trehalose dimycolate) are described from experimental models. These mechanisms come largely from older N. asteroides studies. They are supported by models and remain unconfirmed across the more than 40 clinically relevant species. Disease is suppurative and abscess-forming and often disseminates from lung to brain and skin. Aerobic look-alikes (Streptomyces, Tsukamurella, Gordonia, Rhodococcus) are differentiated in the wound and genital-tract lesson. Actinomyces is distinguished from Nocardia by the modified acid-fast stain and anaerobic growth. When Nocardia is suspected, hold cultures for the extended incubation, because colonies may not appear until after the routine cutoff.