acid-fast

**Mechanism:** Mycobacterial cell walls are rich in mycolic acids (very long-chain, 60–90 carbon fatty acids) linked to arabinogalactan and peptidoglycan, forming a waxy, hydrophobic barrier. Gram stain penetrates poorly, so these organisms are weakly Gram-positive or "ghost-like." Carbol fuchsin (with heat in Ziehl-Neelsen, or high phenol concentration in cold Kinyoun) penetrates the wall. Once stained, the dye is retained despite decolorization with acid-alcohol (HCl + ethanol). Counterstain (methylene blue or malachite green) colors non-acid-fast material blue/green. Acid-fast organisms appear bright red.

**Stain types:** - Ziehl-Neelsen (hot): carbol fuchsin + heat. - Kinyoun (cold): higher phenol concentration, no heat. - Auramine-rhodamine: fluorescent screening stain, with yellow-orange organisms on a dark background. It is more sensitive and used for screening. - Modified (weak) acid-fast: uses weaker decolorizer (e.g., 1% H2SO4) for partially acid-fast organisms.

**Acid-fast organisms (high-yield):** - *Mycobacterium tuberculosis*: strongly acid-fast. Cord factor (trehalose dimycolate) is a virulence factor. Sputum AFB smear shows red rods, and culture uses Löwenstein-Jensen or Middlebrook media. - *M. leprae*: acid-fast, but it can't be cultured in vitro (grows in armadillos and mouse footpads). It is diagnosed by skin biopsy. Lepromatous leprosy shows many bacilli. Tuberculoid leprosy shows few. - *M. avium-intracellulare* (MAC): disseminated disease in AIDS with CD4 <50. Prophylaxis is azithromycin. - *M. kansasii*: TB-like lung disease. *M. marinum*: "fish tank granuloma." - *Nocardia*: weakly (partially) acid-fast, Gram-positive branching filaments, aerobic. Found in soil, causes pulmonary disease in immunocompromised hosts and can spread to the brain. Treat with TMP-SMX. - Protozoa (modified acid-fast oocysts): *Cryptosporidium* (watery diarrhea in AIDS), *Cyclospora* (traveler's diarrhea, imported produce), *Isospora (Cystoisospora) belli*. - Others: *Rhodococcus equi* (variably acid-fast), and spores/cysts of certain organisms.

**Clinical pearls:** - A negative smear does not rule out TB. Sensitivity of smear is about 50–70%, so culture and NAAT (e.g., GeneXpert MTB/RIF, which also detects rifampin resistance) are used. - Histology: caseating granulomas with Langhans giant cells. Acid-fast bacilli may be seen in tissue. - Because of the lipid-rich wall, mycobacteria grow slowly (long generation time, around 15–20 hours for M. tuberculosis), resist many disinfectants, and need prolonged multidrug therapy (RIPE: rifampin, isoniazid, pyrazinamide, ethambutol). - Differentiate: Nocardia (weakly acid-fast, modified AFB stain) vs. Actinomyces (not acid-fast, sulfur granules, anaerobic, treated with penicillin). - Gram stain is not useful for mycobacteria. Use an acid-fast stain.