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Mycobacterial susceptibility and Nocardia

17 min

Skills you'll build4 skills
  • Pair a TB rifampin-resistance NAAT with growth-based testing for the other drugs
  • Keep a rapid-grower clarithromycin result pending until the day-14 read
  • Tell Nocardia from other branching rods with a modified acid-fast stain
  • Extend culture incubation when Nocardia is suspected

At the bench: Select specimen preparation, media, and incubation for a culture · Review susceptibility results for method validity and expected resistance

Try first

Try first

A sputum NAAT reports Mycobacterium tuberculosis complex (MTBC) detected, rifampin resistance not detected. What does this result say about isoniazid?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Rapid rifampin resistance testing

A tuberculosis NAAT detects MTBC and mutations in rpoB, the gene behind most rifampin resistance. It does not detect resistance to isoniazid, ethambutol or pyrazinamide.1,2 Every patient's first isolate is tested against all four first-line drugs, usually in a liquid system that gives results in about 1 to 2 weeks.1 A rifampin-resistance-detected result is called at once to the clinician and the public health authority and confirmed by another rapid method.2 Multidrug-resistant tuberculosis (MDR-TB) is resistance to at least isoniazid and rifampin.1

Clarithromycin testing of rapid growers

M. abscessus subspecies abscessus and bolletii carry erm(41), a gene for inducible macrolide resistance that is expressed only after days of exposure to the drug. The M. fortuitum group carries erm(39), and some M. chelonae carry erm(55).3,4 Clinical and Laboratory Standards Institute (CLSI) document M24 therefore specifies a clarithromycin read at day 3 to 5 and again at day 14 for rapid growers. A day-3 susceptible result remains pending until the day-14 read.3,5

Staining Nocardia

Nocardia appears on Gram stain as beaded, branching, weakly gram-positive rods. It is only partially acid-fast. The acid-alcohol of a conventional acid-fast stain decolorizes it. The modified Kinyoun stain uses a weak acid, often 0.5% to 1% sulfuric acid, and shows the partial acid-fastness. Partial acid-fastness varies, so a negative modified stain does not exclude Nocardia. It grows aerobically, which separates it from Actinomyces.6

Nocardia culture

Colonies usually appear in 2 to 7 days and sometimes take weeks. When nocardiosis is suspected, plates are kept at least 14 days. NALC-NaOH decontamination for mycobacterial culture can kill Nocardia, so a shared specimen may need a separate aliquot that is not decontaminated.6,7 Identification uses MALDI-TOF with a Nocardia library or gene sequencing.6

Follow the bench demonstration

Sources for this step
References
  1. World Health Organization. WHO Operational Handbook on Tuberculosis. Module 3: Diagnosis. WHO; 2025. Accessed September 27, 2026. https://www.who.int/publications/i/item/9789240110991
  2. Centers for Disease Control and Prevention. Clinical and laboratory diagnosis for tuberculosis. Updated January 31, 2025. Accessed September 27, 2026. https://www.cdc.gov/tb/hcp/testing-diagnosis/clinical-and-laboratory-diagnosis.html
  3. Clinical and Laboratory Standards Institute. Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes. 3rd ed. CLSI standard M24. CLSI; 2018. Accessed September 27, 2026. https://clsi.org/shop/standards/m24/
  4. Brown-Elliott BA, Wallace RJ Jr, Wengenack NL, et al. Emergence of inducible macrolide resistance in Mycobacterium chelonae due to broad-host-range plasmid and chromosomal variants of the novel 23S rRNA methylase gene, erm(55). J Clin Microbiol. 2023;61(7):e0042823. doi:10.1128/jcm.00428-23
  5. Clinical and Laboratory Standards Institute. Mycobacterium chelonae: extended incubation needed for clarithromycin antimicrobial susceptibility testing. CLSI Insights blog. May 1, 2025. Accessed September 27, 2026. https://clsi.org/resources/insights-blog/mycobacterium-chelonae-extended-incubation-needed-for-clarithromycin-antimicrobial-susceptibility-testing/
  6. Traxler RM, Bell ME, Lasker BA, Headd B, Shieh WJ, McQuiston JR. Updated review on Nocardia species: 2006-2021. Clin Microbiol Rev. 2022;35(4):e00027-21. doi:10.1128/cmr.00027-21
  7. Centers for Disease Control and Prevention. Clinical overview of nocardiosis. Reviewed January 31, 2025. Accessed September 27, 2026. https://www.cdc.gov/nocardiosis/hcp/clinical-overview/index.html

Watch one

Read each decision and its explanation.

M. abscessus grows from the sputum of a woman with bronchiectasis. The subspecies has not been determined. The broth microdilution panel is read on day 4. The clarithromycin MIC is 0.5 µg/mL, which falls in the susceptible category. What happens to the clarithromycin result?

  1. Classify the organism: M. abscessus is a rapid grower, so the CLSI M24 rule for rapid growers applies.

    The extended read applies to rapidly growing mycobacteria.

  2. Check the day of the read: day 4 is the first read, before inducible erm(41) resistance can appear.

    Inducible resistance appears only after days of exposure.

  3. Consider the subspecies: it is unknown, so the isolate may have a functional gene. A known subspecies would not replace the day-14 read.

    Subspecies abscessus and bolletii usually have a functional erm(41).

  4. Keep the clarithromycin result pending, and read the panel again on day 14.

    A final result is released only after the last required read.

  5. Read day 14: the clarithromycin MIC is now more than 16 µg/mL. That is interpreted as resistant, the pattern of inducible resistance.

    The day-14 MIC is the one that shows inducible resistance.

Clarithromycin remains pending until day 14. The day-14 MIC of more than 16 µg/mL is reported as resistant, the sign of inducible macrolide resistance.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A sputum NAAT reports MTBC detected with rifampin resistance detected. What does the laboratory do with the result?

Correct. MTBC with rifampin resistance detected indicates a high probability of rifampin resistance and requires immediate notification of the clinician and the public health authority. Where rifampin resistance is uncommon, a larger share of resistance calls are false, some from silent rpoB mutations. Additional rapid confirmation follows, and growth-based testing continues because NAAT does not replace it.

Incorrect. NAAT does not replace culture, growth-based susceptibility testing, or genotyping, and a resistance call needs rapid confirmation.

Incorrect. Inhibitor retesting applies when a smear-positive specimen gives a negative NAAT. A positive result with rifampin resistance is reported immediately.

Hint
  1. The NAAT result is available weeks before culture. Consider who needs it now and what testing follows.
  2. Consider what the NAAT does not show about the other drugs.

Review Identification methods

Problem 2 of 3 · Hints available

An M. chelonae isolate from a skin abscess is clarithromycin-susceptible at the day-3 read. A colleague says M. chelonae lacks erm(41), so the result can be finalized. What do you do?

CLSI M24 applies the day-14 read to rapid growers, and CLSI now advises it for M. chelonae as well. The species alone does not rule out inducible resistance.

M. chelonae lacks erm(41), and some isolates carry erm(55), which shows resistance only at the day-14 read.

Finalized a day-3 clarithromycin result for a rapid grower

Inducible erm(41) or erm(39) resistance takes days to appear, so CLSI M24 requires a second clarithromycin read at day 14 for rapid growers. Finalizing a day-3 susceptible result can report an inducibly resistant M. abscessus as susceptible, and subspecies identity alone cannot replace the day-14 read.

The panel shows a susceptible MIC at day 3. The day-14 read determines the interpretation, and the result remains pending until then.

Hint
  1. Check which organisms the day-14 rule covers.
  2. erm(41) is one of several inducible methylase genes.

Review Nontuberculous mycobacteria

Problem 3 of 3 · On your own

A brain abscess aspirate shows beaded, branching, weakly gram-positive rods. A conventional acid-fast stain with acid-alcohol decolorizer is negative. What does the negative stain mean?

Nocardia loses carbol fuchsin under acid-alcohol and appears non-acid-fast on a conventional stain.

Excluded Nocardia after a negative conventional AFB stain

Nocardia is only partially acid-fast and loses carbol fuchsin under the acid-alcohol of a conventional stain, so it looks non-acid-fast there. The modified Kinyoun stain, with a weak acid decolorizer, shows its partial acid-fastness, although results vary even then. A negative conventional stain leaves Nocardia possible.

Mycobacteria keep carbol fuchsin under acid-alcohol and rarely branch. Beaded branching rods suggest aerobic actinomycetes such as Nocardia, or to Actinomyces.

The conventional stain worked as designed. Its strong decolorizer is the reason it misses Nocardia.

The weak-acid decolorizer of the modified stain shows the partial acid-fastness of Nocardia. Aerobic growth and colony identification complete the workup.

Review Nocardia

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Delphine Piper, 46, keeps a rose garden and runs a pottery studio. She takes long-term corticosteroids after a kidney transplant.
  • A clinician calls: nocardiosis is suspected, and a percutaneous lung nodule aspirate is on its way for bacterial culture and AFB culture.
  • The Gram stain shows beaded, branching, weakly gram-positive rods.
Decision 1 of 3

How long are the bacterial culture plates kept?

Nocardia often needs 2 to 7 days and can take weeks. Discarding the plates at 48 hours loses it.

Discarded a suspected-Nocardia culture at the routine cutoff

Nocardia usually becomes visible in 2 to 7 days and sometimes takes weeks, so a culture discarded at the routine cutoff can miss it. Plates from a specimen with suspected nocardiosis are kept at least 14 days. An aliquot of a shared specimen may need to skip NALC-NaOH decontamination, which can kill Nocardia.

A negative modified acid-fast stain never excludes Nocardia, so it cannot shorten the incubation.

Because nocardiosis is suspected, the plates are incubated at least 14 days.

Review Nocardia

Decision 2 of 3

The aspirate is small, and AFB culture was also ordered. How is it divided?

The aspirate comes from a sterile site, so an untreated aliquot is plated directly for Nocardia. The AFB bench prepares the rest under its own procedure.

NALC-NaOH can kill Nocardia. Media for Nocardia are inoculated with material that was not decontaminated.

Discarded a suspected-Nocardia culture at the routine cutoff

Nocardia usually becomes visible in 2 to 7 days and sometimes takes weeks, so a culture discarded at the routine cutoff can miss it. Plates from a specimen with suspected nocardiosis are kept at least 14 days. An aliquot of a shared specimen may need to skip NALC-NaOH decontamination, which can kill Nocardia.

Mycobacterial media can grow Nocardia, and decontamination can kill it first. The bacterial plates are the intended media for Nocardia.

Review Nocardia

Decision 3 of 3

On day 5, dry, chalky white colonies grow aerobically on blood agar. A modified Kinyoun stain of the colonies shows partially acid-fast branching rods. What is the next step?

Actinomyces is non-acid-fast and grows anaerobically. Aerobic growth and partial acid-fastness argue against it.

That name described an old phenotypic group and is obsolete. Species are named after molecular identification.

Aerobic growth, chalky colonies and partial acid-fastness support Nocardia. Other aerobic actinomycetes can also be partially acid-fast, so a species-level method completes the identification.

Review Nocardia

The workup follows from the clinician's call about suspected nocardiosis. Because of it, the plates were incubated for 14 days, an aliquot was plated without decontamination and the modified Kinyoun stain was used.

For reference

Sources checked