Acid-Fast and Partially Acid-Fast Gram-Positive Bacilli Study Guide

General Overview of Genus Mycobacteria

• General Characteristics:

  • Obligate aerobes, non-motile, non-spore-forming bacilli.
  • Slow-growing organisms requiring whole egg media for growth.
  • Difficult to stain with Gram stain due to high lipid content; once stained, they resist decolorization with acid-alcohol owing to the presence of hydroxymethoxy acid (mycolic acid) in their cell wall.

• Three Major Taxonomic Groups:

  • Mycobacterium tuberculosis Complex
  • Mycobacterium Other Than Tuberculosis (MOTT) / Runyon's Classification
  • Mycobacterium leprae

Mycobacterium leprae

• General Properties & Synonyms:

  • Commonly known as Hansen's Bacillus.
  • Etiologic agent of leprosy (Hansen's disease).

• Biochemical Signature:

  • Hydrolyzes 3,4-dihydroxy-phenylalanine (DOPA) via DOPA oxidase / Hydrolysis test.
  • M. leprae possesses the unique metabolic capability to synthesize and oxidize DOPA.

• Cultivation & Maintenance:

  • Non-culturable (non-cultivable) in vitro on synthetic, artificial culture media.
  • Viability and propagation are maintained in vivo using mouse foot pads or the foot pads of the nine-banded armadillo.

• Microscopic Morphology:

  • Under microscopic examination, bacilli arrange in parallel bundles termed the cigar packet arrangement or picket fence appearance.

• Clinical Features & Transmission:

  • Diagnosis is primarily established via clinical manifestations and physical findings.
  • Characterized as infectious, but NOT highly contagious.
  • Mode of Transmission: Requires prolonged, close physical contact with an infected individual (unlike M. tuberculosis, which is transmitted via airborne droplets).

• Three Cardinal Signs of Leprosy (Definitive Diagnostic Criteria):

  • Skin lesion with a definite loss of sensation.
  • Thickened peripheral nerve accompanied by sensory or motor impairment.
  • Demonstration of Acid-Fast Bacilli (AFB) in a skin smear or biopsy specimen.
  • Note: All three features must be evaluated comprehensively to confirm the diagnosis of leprosy.

Specimen Processing and Handling for Mycobacteria

• Step 1: Classification of Specimen Type:

  • Sterile Specimens: Pleural fluid, Cerebrospinal Fluid (CSF aspirated via syringe).
  • Non-Sterile Specimens: Sputum (considered contaminated due to passing through the lower respiratory tract and oropharynx containing normal flora).

• Step 2: Processing Protocol Based on Contamination Risk:

  • Sterile Specimens: Centrifuge directly to concentrate.
  • Non-Sterile Specimens: Must undergo Digestion and Decontamination prior to centrifugation.

• Step 3: Sediment Processing:

  • The sediment obtained after centrifugation is used to prepare smears for Direct Sputum Smear Microscopy (DSSM) and to inoculate culture media.

• Step 4: Screening Test:

  • Confirmed via Acid-Fast Staining techniques.

• Sputum Collection Guidelines:

  • Specimen of Choice: Two sputum samples collected early morning on two consecutive days (or a first-morning sample followed by a second sample taken at a 1-hour interval), per Department of Health (DOH) guidelines for optimal sensitivity.
  • Culture Acceptability Criteria: Sputum smears must display $\mathbf{< 10}$ squamous epithelial cells and $\mathbf{> 25}$ polymorphonuclear neutrophils (PMNs) per low power field.
  • Patient Preparation Prior to Expectoration (to prevent salivary contamination):
  • Water gargle (DOH standard: gargle with water only).
  • Toothbrushing (acceptable per Bailey & Scott).
  • Removal of dentures if present.

• Methods of Decontamination and Digestion:

  • N-acetyl-L-cysteine + Sodium Hydroxide ($\mathbf{\text{NALC} + \text{NaOH}}$): Considered the BEST METHOD for simultaneous decontamination and digestion.
  • NALC: Mucolytic agent that digests and liquefies sputum mucus.
  • 2--4% NaOH: Decontaminating agent that eliminates normal flora while preserving acid-fast bacilli.
  • Trisodium Phosphate + Benzalkonium Chloride (Zephiran) with 6% Oxalic Acid: Used specifically to decontaminate specimens heavily contaminated with Pseudomonas aeruginosa.
  • Dithiothreitol (Sputolysin) + NaOH.

Acid-Fast and Fluorochrome Staining Techniques

• Screening Methods (Acid-Fast Staining):

  • Ziehl-Neelsen Method: Classic hot staining method requiring heat to facilitate carbolfuchsin penetration.
  • Kinyoun's Method: Cold staining method utilizing high phenol concentration instead of heat.
  • Fite-Faraco Method: Specialized stain for M. leprae; utilizes hematoxylin as the secondary stain for tissue biopsy sections.
  • Microscopic Appearance: Red, slender rod-shaped bacilli against a light blue background.
  • Truant's Method: Uses Auramine-Rhodamine fluorochrome dye.
  • Spengler's Method: Specialized acid-fast stain for color-blind laboratory personnel; positive result displays black bacilli.

• Fluorochrome Staining Protocol:

  • Primary Stain: Auramine-Rhodamine dye.
  • Mordant: None required.
  • Decolorizer: $0.5\%$ acid alcohol.
  • Secondary Stain / Counterstain: $0.5\%$ Potassium Permanganate ($\text{KMnO}_4$).
  • Microscopic Result: Bright yellow fluorescing rods against a dark/black background.

AFB Smear Quantification and Reporting Standards

• National Standard Reporting Scale (AFB Grading):

  • Grade 0: No AFB seen in 300 fields.
  • Grade +n: 1 to 9 AFB seen in 100 viewing fields (report exact count).
  • Grade 1: 10 to 99 AFB seen in 100 viewing fields.
  • Grade 2: 1 to 10 AFB per Oil Immersion Field (OIF) in at least 50 fields.
  • Grade 3: More than 10 AFB per OIF in at least 20 fields.

• CDC Method of Reporting AFB:

  • NAFBS: No Acid-Fast Bacilli Seen.
  • Doubtful: 1 to 2 AFB per 300 fields.
  • +1: 1 to 9 AFB per 100 fields.
  • +2: 1 to 9 AFB per 10 fields.
  • +3: 1 to 9 AFB per single field.
  • +4: $> 9$ AFB per single field.

Culture Media for Mycobacteria

• Lowenstein-Jensen (LJ) Medium:

  • Egg-potato-based medium containing malachite green dye to inhibit contaminating bacteria.

• Classification of Culture Media:

  • Non-Selective Egg-Based Media:
  • Lowenstein-Jensen, Petragnani, American Thoracic Society (ATS), Dorset Egg.
  • Mnemonic: LPAD
  • Colony growth visible in 6 to 10 weeks.
  • Non-Selective Agar-Based Media:
  • Middlebrook 7H10 (with dextrose), Middlebrook 7H11 (with casein hydrolysate), Mitchison 7H11.
  • Mnemonic: 3M (Mid Dextrose 7H10, Mid Casein Hydrolysate 7H11, Mitch 7H11).
  • Clear media allowing rapid examination of colonies; growth visible in 10 to 12 days.
  • Selective Media:
  • Gruft Modified Lowenstein-Jensen, Selective Middlebrook 7H11, Mitchison Selective 7H11C.
  • Formulated with added antibiotics.
  • Mnemonic: GLuck Sa Mama Mo
  • Liquid Media (Non-Conventional):
  • BACTEC 12B, BACTEC 13A, Septi-Chek, Middlebrook 7H9.
  • Mnemonic: 2 BB sa SM

• Antimicrobial Susceptibility Testing:

  • Middlebrook 7H10 and Middlebrook 7H11 are specifically utilized for performing mycobacterial antimicrobial susceptibility testing.

Biochemical Tests for Species Identification

• 1. Niacin Test (Kunyoun / Kuno-Runyon Test):

  • Required Medium: Lowenstein-Jensen.
  • Reagents: Cyanogen bromide + Niacin + Aniline dye.
  • Positive Result: Canary yellow color.
  • Significance: Most important diagnostic biochemical test for identifying M. tuberculosis.

• 2. Nitrate Reduction Test:

  • Principle: Measures the presence of nitrate reductase enzyme reducing nitrate ($\text{NO}_3$) to nitrite ($\text{NO}_2$).
  • Required Medium: Sodium nitrate broth.
  • Reagents: Hydrochloric acid (HCl) + Sulfanilamide + n-naphthylethylenediamine.
  • Initial Result: Positive = Red color; Negative = No color change.
  • Re-confirmatory Protocol for Negative Result: Add powdered zinc / zinc dust to uncolored tubes. Zinc chemically reduces unreduced nitrate to nitrite.
  • Post-Zinc Result: Positive = No color change (nitrate was already reduced by organism); Negative = Red color (zinc reduced remaining nitrate).
  • Positive Control: Mycobacterium tuberculosis.

• 3. Iron Uptake (Iron Cyclase) Test:

  • Required Medium: Lowenstein-Jensen.
  • Reagent: 20% Ferric citrate.
  • Positive Result: Rust-brown color change.
  • Positive Control: Mycobacterium fortuitum.

• 4. Arylsulfatase Test:

  • Required Medium: Phenolphthalein media.
  • Principle: Tripotassium phenolphthalein disulfate/sulfate is hydrolyzed by arylsulfatase enzyme to free phenolphthalein.
  • Application: Presumptive identification test for Group IV Rapid Growers.
  • Positive Control: Mycobacterium fortuitum-chelonae complex.

• 5. Heat-Stable Catalase Test ($\mathbf{68^\circ\text{C}}$):

  • Procedure: Inoculate slant and heat at $68^\circ\text{C}$ for 20 minutes prior to reagent addition.
  • Required Medium: Tween 80 Medium.
  • Reagent: 30% $\text{H}_2\text{O}_2$ (Superoxol).
  • Positive Result: Bubbling column height $\mathbf{> 45\,\text{mm}}$.
  • Positive Species: M. kansasii, M. fortuitum, M. gordonae.

• 6. Pyrazinamidase Test:

  • Required Medium: Pyrazinamidase agar.
  • Reagent: 1% ferrous ammonium sulfate.
  • Positive Result: Formation of a pink-ferrous complex band.
  • Positive Control: Mycobacterium marinum.

• 7. Tween 80 Hydrolysis Test:

  • Significance: Essential test for confirming Mycobacterium kansasii.
  • Positive Result: Pink to red color shift within 5 days.
  • Positive Species: M. kansasii.

• 8. Tellurite Reduction Test:

  • Significance: Primary identification test for Mycobacterium avium complex.
  • Positive Result: Black metallic tellurium precipitate.
  • Positive Species: M. avium.

• 9. Salt Tolerance Test ($\mathbf{5\%}$ or $\mathbf{6.5\%\,\text{NaCl}}$):

  • Positive Species: Mycobacterium triviale.

• 10. TCH (Thiophene-2-Carboxylic Acid Hydrazide) Susceptibility Test:

  • Purpose: Differentiates M. bovis from M. tuberculosis.
  • Susceptible (Positive / Growth Inhibition): M. bovis.
  • Resistant (Negative / Growth Uninhibited): M. tuberculosis.

• 11. Urease Test:

  • Positive Result: Red/pink color development.
  • Positive Species: M. bovis, M. scrofulaceum, M. gastri.

• 12. Growth on MacConkey Agar without Crystal Violet:

  • Positive Species: Mycobacterium fortuitum-chelonae complex.

Comparative Biochemical Reaction Matrices

• Table 1: Differential Reactions for M. tuberculosis Complex

• Table 2: Differential Reactions for Group I Photochromogens

• Table 3: Differential Reactions for Group III Non-Photochromogens

• Table 4: Differential Reactions for Group IV Rapid Growers

Mycobacterium tuberculosis Complex

• Taxonomic Group Members:

  • M. tuberculosis
  • M. bovis
  • M. africanum
  • M. caprae
  • M. microti
  • M. canetti

• General Properties & Synonyms:

  • Common Names: Koch's Bacillus (discovered by Robert Koch), Tubercle Bacillus.
  • Causative agent of Pulmonary Tuberculosis (PTB) and extrapulmonary TB.

• Virulence Factors:

  • Cord Factor (Trehalose-6,6'-dimycolate):
  • Glycolipid present in the mycobacterial cell wall.
  • Produces the characteristic serpentine/cord-like architecture of virulent strains.
  • Pathogenic Mechanisms: Inhibits neutrophil migration and disrupts host cell mitochondrial membrane integrity (leads to energy depletion, cellular apoptosis, and pulmonary tissue necrosis).
  • Sulfatides:
  • Sulfated glycolipids on the cell surface.
  • Pathogenic Mechanisms: Prevents phagosome-lysosome fusion inside host macrophages and inhibits oxidative burst killing mechanisms.

• Physiological Requirements & Cultivation:

  • Obligate aerobe; requires $5\%\,\text{CO}_2$ for growth.
  • Incubation time: Requires 8 weeks (2 months) for visible culture growth.
  • Produces characteristic rough, buff, warty "cauliflower-like" growth at $35\text{--}37^\circ\text{C}$.

• Resistance & Survival:

  • Highly resistant to desiccation/drying due to metachromatic storage granules (Much's granules).
  • Resistant to chemical disinfectants including $5\%$ phenol.

• Transmission, Anatomic Habitat & Symptoms:

  • Transmission: Airborne respiratory droplets (requires N95 particulate respirator protection).
  • Anatomic Habitat: Lungs, meninges, long bones, and spinal column (causative agent of Pott's disease / skeletal TB).
  • Clinical Features of PTB: Productive cough, hemoptysis (coughing up blood), low-grade fever, night sweats, anorexia, and weight loss. Historically termed Consumption Disease.
  • Dissemination: Disseminates via blood and lymphatic system causing meningeal and miliary/disseminated TB.

• Diagnostic Immunologic Tests (Delayed Hypersensitivity):

  • Tuberculin Skin Test / PPD (Purified Protein Derivative): Mantoux test, Mendel's test, Vollmer's patch test, Von Pirquet test.
  • Positive Result: Erythema and induration evaluated after 48 hours.

• Immunization:

  • BCG Vaccine (Bacillus Calmette-Guérin): Administered to infants; prepared from a live-attenuated strain of Mycobacterium bovis.

• Anti-TB Chemotherapy:

  • Primary First-Line Anti-TB Drugs (Mnemonic: RIPES):
  • Rifampin
  • Isoniazid
  • Pyrazinamide
  • Ethambutol
  • Streptomycin
  • Secondary Second-Line Anti-TB Drugs (Mnemonic: CARE COCK):
  • Capreomycin
  • Amikacin
  • Rifabutin
  • Ethionamide
  • Cycloserine
  • Ofloxacin
  • Ciprofloxacin
  • Kanamycin
  • Drug Resistance Definitions:
  • MDR-TB (Multi-Drug Resistant TB): Resistant to at least the two core first-line drugs: Isoniazid and Rifampin.
  • XDR-TB (Extensively Drug-Resistant TB): Resistant to Isoniazid, Rifampin, any fluoroquinolone, and at least one injectable second-line drug.

• Advanced Diagnostic Assays:

  • Interferon Gamma Release Assay (IGRA)
  • QuantiFERON-TB Gold Plus
  • LipoArabinomannan (LAM): Point-of-care (POCT) urine assay kit for intrathoracic and latent tuberculosis.
  • GeneXpert (MTB/RIF): Automated molecular nucleic acid amplification assay for rapid simultaneous detection of M. tuberculosis DNA and Rifampin resistance.

Mycobacterium bovis

• Etiology: Causative agent of Bovine Tuberculosis (tuberculosis in cattle).
• Human Pathogenicity: Causes intestinal tuberculosis in humans, acquired via ingestion of unpasteurized infected milk.
• Key Biochemical Differentiation:
  • Niacin Test: Negative ($-$)
  • Nitrate Reduction Test: Negative ($-$)
  • TCH Susceptibility Test: Susceptible (+\n)

Mycobacterium Other Than Tuberculosis (MOTT) / Runyon's Classification

• Also designated as Atypical Mycobacteria or Non-Tuberculous Mycobacteria (NTM).

• Classification Criteria: Identified based on biochemical reactions, pigment production, photosensitivity (photoreactivity), and growth rate.

• GROUP I: PHOTOCHROMOGENS:

  • Produce non-pigmented colonies in the dark; produce bright yellow-orange carotenoid pigments only upon light exposure.
  • Growth Rate: Slow growers (visible colonies in 10 to 21 days).
  • M. kansasii: Known as the Cross-Barred Bacillus. Regarded as the #2 NTM causing TB in AIDS patients.
  • M. marinum: Etiologic agent of Fish Tank Granuloma or Swimming Pool Granuloma.
  • M. asiatica
  • M. simiae

• GROUP II: SCOTOCHROMOGENS:

• Produce yellow-orange pigments in both light and dark conditions.

• Growth Rate: Slow growers (visible colonies in 10 to 21 days).

• M. scrofulaceum: Causes cervical lymphadenitis (scrofula - inflammation of cervical lymph nodes).

• M. xenopi: Associated with hot and cold water taps.

• M. szulgai

• M. gordonae: Commonly known as the Tap Water Bacillus.

• M. thermoresistible

  • GROUP III: NON-PHOTOCHROMOGENS:

• Non-pigmented / non-photochromogenic colonies regardless of light exposure.

• Growth Rate: Slow growers (visible colonies in 10 to 21 days).

• M. avium-intracellulare complex (MAC): #1 NTM causing TB in AIDS patients. Comprises M. avium (causes chicken/bird tuberculosis) and M. intracellulare.

• M. terrae complex: Includes M. terrae (Radish Bacillus), M. triviale, and M. chromogenicum; rare etiology of septic arthritis and osteomyelitis.

• M. xenopi: Produces bird's-nest/branching colonies on Cornmeal agar.

• M. ulcerans: Known as the Inert Bacillus; causes Buruli ulcers.

• M. haemophilum

• M. gastri: Known as the J Bacillus; non-pathogenic, isolated from gastric lavage.

• M. genavense: Causes disseminated infections in patients with AIDS.

• M. avium subsp. paratuberculosis: Causative agent of Johne's disease (a form of inflammatory bowel disease characterized by chronic diarrhea in cattle and sheep); implicated in human Crohn's disease.

  • GROUP IV: RAPID GROWERS:

• Produce mature visible colonies in 3 to 5 days.

• M. fortuitum-chelonae complex: Positive for Arylsulfatase (+\n); capable of growth on MacConkey agar without crystal violet.

  • M. fortuitum: Positive for Iron Uptake (+\n) and Nitrate Reduction (+\n).
  • M. chelonae: Negative for Iron Uptake (-\n) and Nitrate Reduction (-\n).

• M. smegmatis: Normal flora of smegma; can be confused with M. tuberculosis in urine specimens.

• M. phlei: Provides \text{CO}_2$.\n * *M. mucogenicum*\n * *M. abscessus*\n\n# Human Immunodeficiency Virus (HIV) and AIDS\n\n* **Pathophysiology:** Retrovirus that attacks host \text{CD4}^+ T-lymphocytes.\n* **AIDS Diagnostic Threshold:** \mathbf{< 200\,\text{CD4}^+\text{ cell count/}\mu\text{L}} in blood.\n* **Modes of Transmission:**\n * Blood transmission / sharing contaminated needles.\n * Breastfeeding.\n * Transplacental / perinatal transmission.\n * Sexual intercourse.\n* **HIV Prevention Strategy (ABCDE Rule):**\n * **A:** Abstinence.\n * **B:** Be mutually faithful to your partner.\n * **C:** Correct and consistent use of condoms.\n * **D:** Do not use illegal drugs / share needles.\n * **E:** Education and early diagnosis/detection.\n* **Prophylaxis & Treatment Regimens:**\n * **PrEP (Pre-Exposure Prophylaxis):** Oral antiretroviral medication taken by HIV-negative, sexually active individuals to prevent infection. Follows the **2-1-1 dosing schedule**:\n * 2 tablets taken 2 to 24 hours prior to sexual intercourse.\n * 1 tablet taken 24 hours after the initial dose.\n * 1 tablet taken 48 hours after the initial dose.\n * **ARV (Antiretroviral Therapy):** Lifelong suppressive therapy prescribed for People Living with HIV (PLHIV).\n\n# Genus Nocardia and Related Organisms\n\n* **General Properties:**\n * Partially acid-fast / modified acid-fast (requires weak decolorizer: 1\%\,\text{H}_2\text{SO}_4).\n * Urease positive (+ $$), Gram-positive branching-rod morphology; causes pulmonary infections (pneumonia) and cutaneous nocardiosis.

• Cell wall peptidoglycan contains meso-diaminopimelic acid (DAP) alongside arabinose and galactose sugars.

• Capable of growth on standard culture media lacking antibiotics.

  • Hydrolysis Reaction Matrix for Aerobic Actinomycetes: