Can Mycobacterium avium (M. avium) cultures remain negative if identified by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) and if so, how can drug susceptibilities be checked?

MALDI-TOF Identification with Negative Cultures and Susceptibility Testing

Yes, Mycobacterium avium can be identified by MALDI-TOF even when subsequent cultures remain negative, and in such cases, molecular detection methods targeting resistance genes should be used for susceptibility determination rather than traditional culture-based testing. 1

Understanding the MALDI-TOF Limitation

MALDI-TOF requires viable organisms from culture for identification—it cannot identify organisms directly from clinical specimens without growth. 1 The scenario you describe likely involves one of two situations:

• Initial positive liquid culture identified by MALDI-TOF, but subsequent subcultures fail to grow: When MALDI-TOF is applied to newly positive liquid cultures, only 50% of isolates can be immediately identified, and the remainder require subculture on solid media until visual growth occurs to obtain reliable results 1

• Low organism burden with transient culture positivity: A single positive culture (especially broth-only) with subsequent negative cultures may represent low colony counts, contamination, or fastidious growth requirements 1

How to Obtain Susceptibility Data Without Viable Cultures

Molecular Susceptibility Testing (Primary Approach)

For M. avium complex, molecular detection of resistance mutations is the preferred method when cultures are unavailable or fail to grow: 1

• Macrolide resistance testing: Perform molecular detection of point mutations in the 23S rRNA (rrl) gene, which confer acquired macrolide resistance in M. avium complex 1

• Amikacin resistance testing: Detect mutations in the 16S rRNA (rrs) gene, which cause acquired amikacin resistance 1

• Technical approach: Use nucleic acid sequencing directly on the original positive specimen or stored isolate material, targeting the rrl and rrs genes 1

Repeat Culture Attempts with Optimized Methods

If high clinical suspicion exists but cultures remain negative, review decontamination procedures and use supplemented media with molecular detection methods: 1

• Optimize culture conditions: M. avium complex grows optimally at 36±1°C for slow growers, and cultures should be incubated for at least 2-3 weeks, though some fastidious strains may require up to 8-12 weeks 1

• Use both solid and liquid media: Broth media alone may be inadequate due to bacterial overgrowth; solid media (Löwenstein-Jensen or Middlebrook 7H10/7H11) should be used concurrently 1

• Consider supplemented media: While rarely necessary for M. avium complex (unlike M. haemophilum or M. genavense which require special supplementation), review whether appropriate media were used 1

Molecular Species Confirmation

All clinically relevant NTM isolates should be confirmed by molecular methods, not MALDI-TOF alone: 1

• Gene sequencing is the preferred identification method: Use 16S rRNA sequencing complemented with additional targets (hsp65, rpoB, or 16S-23S ITS) for optimal discriminatory power 1

• MALDI-TOF has limitations: While discriminatory power has improved with enhanced databases and protein extraction protocols, not all species and subspecies can be differentiated, and pure cultures work best 1

Clinical Decision Algorithm

When MALDI-TOF identifies M. avium but cultures subsequently fail:

1. Retrieve and freeze the original positive isolate material for future molecular testing and to distinguish reinfection from relapse if recurrence occurs 1

2. Perform molecular confirmation of species identification using gene sequencing (16S rRNA plus hsp65, rpoB, or ITS) on stored material 1

3. Conduct molecular resistance testing for macrolides (23S rRNA mutations) and amikacin (16S rRNA mutations) on the original positive specimen or stored isolate 1

4. Repeat cultures with optimized techniques if clinical suspicion remains high and treatment decisions depend on susceptibility results 1

5. If molecular testing is unavailable and clinical disease is confirmed: Initiate empiric treatment with a macrolide-ethambutol-rifampin regimen, as baseline macrolide susceptibility correlates with treatment outcomes 1

Common Pitfalls to Avoid

• Do not rely solely on MALDI-TOF for species identification: Molecular methods provide superior discriminatory power and are the preferred standard 1

• Do not assume all subsequent negative cultures mean the organism is gone: Low colony counts (≤10 colonies on solid media or broth-only positivity) may represent true infection with low organism burden rather than contamination 1

• Do not delay treatment waiting for traditional susceptibility testing: If molecular testing is unavailable and clinical disease is present, empiric treatment should be initiated, as acquired macrolide resistance is the primary concern and can be detected molecularly 1

• Do not discard the original positive culture material: Freeze and save isolates for future molecular testing and to distinguish reinfection from relapse 1