Mycobacterial Susceptibility, Latent Infection Testing, Biosafety, and Nocardia

Susceptibility testing for mycobacteria and Nocardia follows CLSI M24, 3rd edition, with interpretive tables in CLSI M24S, 2nd edition, including the June 10, 2024 correction. That correction changes a slowly growing NTM clarithromycin quality-control range; it does not change Nocardia breakpoints. This module does not quote paywalled M24S numeric breakpoints.¹

Mycobacterium tuberculosis complex susceptibility

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. The agar-proportion reference method calls an isolate resistant if 1% or more of the population grows at a critical drug concentration; it averages about 28 days. An FDA-approved liquid system that gives equivalent results in 5-7 days is preferred where available. Secondary-drug testing is triggered by rifampin resistance alone, resistance to any two primary drugs, or isoniazid monoresistance when a fluoroquinolone is planned.

Molecular assays complement phenotypic testing. Probe-based and line-probe methods detect known mutations; sequencing reads the actual sequence. Commonly interrogated loci include rpoB (rifampin), embB (ethambutol), gyrA (fluoroquinolones), the inhA and eis promoters and katG (isoniazid; ahpC and ndh account for a smaller share outside those loci), rrs (aminoglycosides), and tlyA/pncA (capreomycin/pyrazinamide). Because isoniazid and rifampin resistance frequently co-occur, a rifampin-resistance result is a practical surrogate for multidrug-resistant tuberculosis screening. Molecular testing is especially useful after prior TB treatment, origin in a high-resistance area, treatment failure, or known MDR-TB exposure. A negative molecular result still requires phenotypic confirmation because not every mechanism is characterized.

Multidrug-resistant tuberculosis (MDR-TB) is resistance to at least isoniazid and rifampin. Pre-XDR-TB is MDR or rifampin-resistant TB that is also resistant to any fluoroquinolone. Extensively drug-resistant tuberculosis (XDR-TB), WHO January 2021, is MDR/RR-TB that is also resistant to any fluoroquinolone and at least one additional Group A drug. Group A currently comprises levofloxacin, moxifloxacin, bedaquiline, and linezolid, so the additional agent after fluoroquinolone resistance is bedaquiline or linezolid. The laboratory secondary-drug panel may still include injectable aminoglycosides and capreomycin, along with ethionamide, fluoroquinolones, linezolid, p-aminosalicylic acid, and rifabutin. Current treatment regimens are selected separately according to resistance results and clinical guidance.²

Therapy is begun before susceptibility results are available and adjusted afterward. First-line agents for MTBC remain isoniazid, rifampin, ethambutol, and pyrazinamide.

Nontuberculous mycobacteria

Test clinically significant isolates. For some species, notably MAC against ethambutol and the rifamycins, in vitro susceptibility correlates poorly with clinical response. Broth microdilution per CLSI M24 is the recommended method for both rapid and slow growers. Results are reported as an MIC and as a categorical interpretation.

Latent infection testing

Two tests detect latent MTBC infection. A positive result indicates infection; active disease is established by smear, culture, or NAAT of an appropriate specimen. Clinical selection of which test to order, and how to act on a result, is owned by Immunology.

Tuberculin skin test (TST, Mantoux method). 0.1 mL of intermediate-strength (5 tuberculin units) purified protein derivative is injected intracutaneously; induration is measured at 48-72 hours. False-positive reactions occur after NTM infection and after BCG vaccination. False-negative reactions follow poor technique, reagent storage failure, or anergy. A second test placed as early as 1 week after the first can restore a waned reaction (the booster effect), which matters when a baseline is being established.

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; older QuantiFERON-TB Gold In-Tube also included TB7.7) that are absent from BCG and from most NTM (exceptions: M. kansasii, M. szulgai, 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 leaves IGRA results unaffected. T-SPOT.TB, the other FDA-approved IGRA, uses the same ESAT-6 and CFP-10 antigens in an ELISPOT format on separated peripheral blood mononuclear cells rather than whole-blood antigen tubes.³

Biosafety

Mycobacteriology laboratories limit airborne transmission with negative-pressure rooms exhausted to the outside; single-pass ventilation (supply at the ceiling, exhaust near the floor); a certified Class I or Class II biological safety cabinet for all specimen and culture handling; and a fit-tested particulate respirator that filters 1-5 μm particles. CDC 2005 health-care TB guidance sets a minimum of 6 mechanical air changes per hour for existing airborne-infection isolation rooms, with 12 ACH recommended where feasible and required for new construction or renovation; autopsy suites are commonly specified at 12 ACH. Aerosol-generating procedures, including inoculating media and opening centrifuge cups, stay inside the cabinet.⁴

Nocardia

Lower respiratory specimens (sputum, bronchoalveolar lavage) are the most common source because pulmonary disease predominates. Submit brain-abscess aspirate and CSF when central-nervous-system involvement is suspected. Skin and soft-tissue aspirates or biopsies cover cutaneous and lymphocutaneous disease. Blood culture is required when disseminated disease is suspected. Notify the laboratory because bottles may need extended incubation. Hold solid-media plates from a suspect specimen 2-3 weeks, beyond the routine 48-hour bacteriology cutoff.

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. Cutaneous disease after direct inoculation also occurs in immunocompetent hosts. Pulmonary nocardiosis presents with fever, cough, and nodular or cavitary infiltrates that mimic tuberculosis or malignancy. Hematogenous CNS spread is common enough that brain-abscess aspirate and CSF are appropriate whenever CNS disease is in the differential.

On Gram stain, Nocardia appears as beaded, branching, filamentous gram-positive rods. This morphology overlaps with Actinomyces and can resemble fungal hyphae at low power. The modified Kinyoun stain uses a weak 0.5-1% sulfuric-acid decolorizer, while the standard mycobacterial stain uses 3% hydrochloric acid-alcohol. Under the gentler decolorization, Nocardia is partially (weakly) acid-fast; standard acid-alcohol strips the dye. Partial acid-fastness plus aerobic growth distinguishes Nocardia from Actinomyces, which is acid-fast negative and grows anaerobically.

Nocardia grows aerobically on blood and chocolate agar, buffered charcoal yeast extract, mycobacterial media, and Löwenstein-Jensen. Colonies typically appear in 3-5 days but sometimes take 2-3 weeks. They are dry, chalky, heaped or wrinkled, white to orange, and often have a musty, moldy-basement odor.

Phenotypic panels cannot reliably resolve species. Species-level identification is required because susceptibility is species-dependent. It relies on MALDI-TOF with a Nocardia-adequate database (many commercial libraries need supplementation), 16S rRNA sequencing, and secA1 or hsp65 sequencing for finer resolution. Isolates that cannot be resolved in-house are referred. Broad-range 16S PCR with amplicon sequencing on sterile-site specimens (CSF, tissue, abscess aspirate) can establish a diagnosis when culture is negative or too slow.

Trimethoprim-sulfamethoxazole is first-line. Amikacin, imipenem, linezolid, and ceftriaxone are alternative or adjunctive agents, chosen by species and susceptibility. Each isolate requires its own susceptibility test. N. farcinica is characteristically resistant to third-generation cephalosporins such as ceftriaxone. CLSI M24 defines broth microdilution as the reference method; testing is generally performed at a reference laboratory.

Nocardia is an environmental saprophyte acquired by inhalation or traumatic inoculation. Catalase and superoxide dismutase neutralize the phagocyte oxidative burst, and the organism blocks phagosome-lysosome fusion. A surface cord factor (trehalose dimycolate) also contributes. 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 module; Actinomyces is separated here by modified acid-fast stain and anaerobic growth.