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Tuberculosis (TB) - Microbiology

1. The Organism

• Slender, slightly curved, rod-shaped bacillus: 0.5 µm × 3 µm
• Non-motile, non-encapsulated, non-spore-forming
• Strictly aerobic (high G+C content: 65.6%, reflecting aerobic metabolism)
• Weakly Gram-positive (Gram stain unreliable due to lipid-rich wall)
• Harrison's Principles of Internal Medicine 22E, p. 1424
• Fishman's Pulmonary Diseases and Disorders, p. 2290

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2. The Cell Wall - The Defining Feature

Cell Wall Structure (outside → in):
Mycolic acids (long-chain fatty acids, C60–C90)
    ↓ linked to
Arabinogalactan (complex polysaccharide)
    ↓ linked to
Peptidoglycan
    ↓
Plasma membrane

1. Acid-fastness - Once stained with carbolfuchsin (Ziehl-Neelsen), bacilli resist decolorization even by acid-alcohol. This is the basis of the AFB smear.
2. Antibiotic resistance - Very low permeability of cell wall renders most standard antibiotics ineffective.
3. Resistance to drying - Allows MTB to survive in dried droplet nuclei for hours, enabling airborne spread.
4. Slow growth - The metabolic cost of producing mycolic acids slows replication to a doubling time of ~15-20 hours (vs. 20 minutes for E. coli), hence colonies appear in 4-6 weeks on solid media.
5. Immune evasion - Wall lipids help MTB avoid killing by non-activated macrophages.

• Harrison's, p. 1424; Fishman's, p. 2290; Sherris & Ryan's Medical Microbiology 8th Ed.

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3. The Mycobacterium tuberculosis Complex (MTBC)

• Harrison's 22E, p. 1424; Fishman's, p. 2290

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4. Staining & Microscopy

Ziehl-Neelsen (ZN) Stain - Hot Method

1. Flood smear with carbolfuchsin (red dye + phenol)
2. Heat to drive dye into waxy wall
3. Decolorize with acid-alcohol (3% HCl in 95% ethanol)
4. Counterstain with methylene blue
5. Result: MTB stains red/pink on blue background (acid-FAST = retains red dye)

Auramine-Rhodamine Fluorochrome Stain

• Faster screening method; bacilli appear bright yellow-green under fluorescent microscopy
• More sensitive than ZN for screening, but ZN used for confirmation

• Fishman's, block 25; Tintinalli's Emergency Medicine

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5. Culture

• Lowenstein-Jensen (LJ) medium - egg-based, enriched; colonies appear in 4-6 weeks
• Colonies: rough, buff-colored, "breadcrumb" or "cauliflower" appearance
• In liquid media, characteristic serpentine cords form (due to cord factor)

• BACTEC MGIT 960 (Mycobacteria Growth Indicator Tube) - detects growth in 9-16 days by fluorescence quenching as O2 is consumed
• Time to detection depends on initial bacterial load in specimen

• Strictly aerobic
• Optimal temperature: 37°C (hence preferential lung apex colonization - high O2 tension)
• Non-pigmented (photochromogen or scotochromogen classification does NOT apply - MTB is non-chromogenic)
• Fishman's, p. 2290

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6. Genome

• 4.4 million base pairs
• 4043 protein-coding genes; 50 genes encoding stable RNAs
• High G+C content: 65.6%
• Large proportion of genes devoted to cell wall lipid metabolism
• Substantial genetic variability among strains - used for epidemiologic typing

• IS6110 RFLP typing (classical)
• Spoligotyping
• MIRU-VNTR (mycobacterial interspersed repetitive unit - variable number tandem repeats)
• Whole genome sequencing (WGS) - current gold standard for strain typing
• Harrison's 22E, p. 1424

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7. Transmission

• Droplet nuclei = particles <5 µm generated by coughing, sneezing, singing, or procedures (bronchoscopy, sputum induction, autopsy)
• These particles have an extremely slow settling rate (~0.5 mm/sec) and can remain suspended in air for hours
• Mycolic acid wall protects MTB within dried droplet nuclei - infectiousness is retained
• A single inhaled droplet nucleus may be sufficient for infection
• Procedures that generate aerosols (bronchoscopy, autopsy, abscess irrigation) are also sources

• Fishman's, p. 2290; Sherris & Ryan's

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8. Pathogenesis - From Infection to Disease

Stage 1: Inhalation and Alveolar Macrophage Uptake

• Inhaled droplet nuclei reach terminal alveoli
• Alveolar macrophages phagocytose the bacilli
• MTB subverts phagosome maturation - blocks phagolysosome fusion (via LAM and other lipids)
• Bacilli survive and replicate inside macrophages

Stage 2: Primary Complex

• Local multiplication in alveolar macrophages
• Bacilli spread via lymphatics to hilar lymph nodes
• Ghon focus (parenchymal lesion) + hilar lymphadenopathy = Ghon/Ranke complex

Stage 3: Hematogenous Dissemination (occurs in ALL primary infections)

• Before immunity develops (~2-8 weeks), bacilli spread hematogenously to multiple organs
• Preferential seeding of sites with HIGH O2 tension: lung apices, renal cortex, vertebral bodies, meninges, metaphyses of long bones
• Most infections are contained at this stage once specific immunity develops

Stage 4: Granuloma Formation (Immune Response)

• T-cell activation (CD4+ Th1 cells) → production of IFN-γ
• IFN-γ activates macrophages → increased killing ability
• Activated macrophages and epithelioid cells wall off the bacilli → granuloma (tubercle)
• Granuloma structure: central caseous necrosis + epithelioid macrophages + Langhans giant cells + lymphocytes + fibroblasts
• In ~90% of immunocompetent individuals: granulomas contain infection → Latent TB infection (LTBI)
• In ~10%: granulomas break down → active disease

Stage 5: Latency and Reactivation

• MTB enters a non-replicating latent state within granulomas
• Can persist for decades
• Reactivation risk triggered by: HIV, TNF-α inhibitors, malnutrition, diabetes, silicosis, aging, corticosteroids
• HIV increases annual reactivation risk from 5% lifetime to 10-16% per year

• Harrison's 22E; Sherris & Ryan's 8th Ed.; Murray & Nadel's Respiratory Medicine

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9. Virulence Factors Summary

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10. Drug Resistance - Microbiological Basis

• In 2023: ~400,000 MDR/RR-TB cases globally; highest burden in India, Russia, Indonesia, China, Philippines
• Resistance arises from spontaneous chromosomal mutations (MTB has no plasmid-mediated resistance) and is selected by inadequate treatment or non-adherence
• MTB does NOT transfer resistance genes via horizontal gene transfer
• Harrison's 22E, p. 1425; Murray & Nadel's

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11. Diagnostic Methods Summary

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• MTB = acid-fast, aerobic, non-spore-forming, strictly human pathogen
• Mycolic acids = acid-fastness + slow growth + antibiotic resistance + immune evasion
• Disease is caused by host immune response (DTH), not bacterial toxins
• Cord factor (trehalose dimycolate) = serpentine cords in culture
• BACTEC detects growth in 9-16 days vs. 4-6 weeks on LJ
• MDR-TB = resistant to INH + rifampin; XDR-TB adds fluoroquinolone + Group A drug resistance

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TB - Laboratory Diagnosis & Drug Resistance Treatment

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PART 1: LABORATORY DIAGNOSIS

A. Specimen Collection

• Tintinalli's Emergency Medicine; Harrison's 22E

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B. AFB Smear Microscopy

• Fishman's Pulmonary Diseases and Disorders; Tintinalli's

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C. Culture - The Gold Standard

• Definitively identifies the species
• Allows drug susceptibility testing (DST)
• Detects as few as 10 bacteria/mL (vs. 5,000/mL for smear)

• Fishman's, p. 2290; Tintinalli's Emergency Medicine

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D. Drug Susceptibility Testing (DST)

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E. Nucleic Acid Amplification Tests (NAAT)

• PCR-based, fully automated, results in ~2 hours
• Detects M. tuberculosis DNA AND rifampin resistance (rpoB mutations) simultaneously
• Can detect 1-10 organisms/mL
• Sensitivity: >90-95% in smear-positive cases; 50-80% in smear-negative cases
• False positives: detects dead and live organisms - stays positive even after adequate treatment; not ideal for monitoring treatment response
• A negative NAAT does NOT exclude TB
• WHO recommends as the initial diagnostic test in all adults and children with signs/symptoms of TB

• More sensitive version, particularly in smear-negative and extrapulmonary TB
• Also detects additional rpoB mutations

• Detects resistance to isoniazid (katG, inhA) and rifampin (rpoB) rapidly
• Also MTBDRsl for second-line drugs (fluoroquinolones, aminoglycosides)
• Direct from smear-positive sputum; results in 1-2 days
• Used as first-line DST for MDR-TB detection
• Tintinalli's Emergency Medicine; Harrison's 22E

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F. Tests for TB Infection (LTBI)

1. Tuberculin Skin Test (TST / Mantoux / PPD)

• Cross-reacts with NTM and BCG vaccination (false positives)
• False negatives in: severe immunosuppression (HIV, malnutrition), overwhelming TB, sarcoidosis, recent viral infection, very early infection
• "Boosting phenomenon": a second TST 1-5 weeks after the first can artificially increase reaction size (boosting of a previously waned response - must distinguish from true conversion)
• Cannot distinguish latent infection from active TB
• Cannot distinguish M. tuberculosis from prior BCG or NTM

2. Interferon-Gamma Release Assays (IGRAs)

• Not affected by BCG vaccination
• Not affected by most NTM (except M. kansasii, M. szulgai, M. marinum which share ESAT-6/CFP-10)
• Single visit (no need to return in 48-72 hours)
• More specific

3. New Mycobacterium tuberculosis Antigen-Based Skin Tests (TBSTs)

• Combine simplicity of TST with IGRA specificity
• Use ESAT-6 and CFP-10 antigens (same as IGRA) in skin test format
• WHO 2022: accuracy similar to IGRAs, superior to TST
• Useful in HIV+ patients, children, and BCG-vaccinated persons
• Harrison's 22E, p. 1437

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G. Chest X-Ray (Radiology)

• Tintinalli's Emergency Medicine

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H. Additional Tests

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PART 2: TREATMENT - DRUG-SUSCEPTIBLE TB

First-Line Drug Regimens (Drug-Susceptible TB)

Intensive Phase (2 months):    H + R + Z + E  (daily)
                                    ↓
Continuation Phase (4 months): H + R  (daily)

• H = Isoniazid (INH)
• R = Rifampin (Rifampicin)
• Z = Pyrazinamide
• E = Ethambutol

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First-Line Drug Profiles

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Treatment Monitoring

• Sputum smear and culture at 2 months: If still positive at 3 months → suspect drug resistance
• Cavitary disease + culture still positive at 2 months: Consider extending continuation phase to 7 months (total 9 months)
• LFTs: Baseline and monthly (or if symptoms arise) - all four first-line drugs can cause hepatotoxicity
• Treatment success definition: Culture-negative status maintained at 5-6 months

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PART 3: DRUG RESISTANCE - MECHANISMS & TREATMENT

Mechanism of Drug Resistance in MTB

1. Monotherapy - prescribing only one effective drug (biggest cause)
2. Sequential addition of single drugs to a failing regimen - each addition selects resistant mutants
3. Poor adherence / treatment interruptions
4. Inadequate drug bioavailability (crushed tablets, poor absorption, subtherapeutic dosing)
5. Primary resistance - transmitted directly from a source case already harboring resistant strain

• Harrison's 22E, p. 1441

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Drug Resistance Classification

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Treatment of Drug-Resistant TB (WHO 2022 Framework)

MDR-TB Drug Groups (WHO 2022):

1. Levofloxacin or Moxifloxacin (fluoroquinolone)
2. Bedaquiline (diarylquinoline - inhibits mycobacterial ATP synthase)
3. Linezolid (oxazolidinone - inhibits 23S rRNA protein synthesis)

• Ethambutol
• Delamanid (nitroimidazole - inhibits mycolic acid synthesis)
• Pyrazinamide
• Imipenem-cilastatin or Meropenem (carbapenems)
• Amikacin (or streptomycin if amikacin unavailable)
• Ethionamide or Prothionamide
• p-Aminosalicylic acid (PAS)

Note: Kanamycin and capreomycin are no longer recommended in MDR-TB regimens.

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MDR-TB Regimen Options (Harrison's 22E, Table 183-4):

• BPaLM = Bedaquiline + Pretomanid + Linezolid + Moxifloxacin (6 months, all oral)
• For patients with no previous exposure to bedaquiline, delamanid, or linezolid
• BPaL = Bedaquiline + Pretomanid + Linezolid (for XDR-TB or treatment-intolerant MDR-TB)

• BLMZ or BLLfCfZ or BDLLfxZ
• For patients where fluoroquinolone resistance has been excluded

• Individually designed using Groups A, B, C
• At least 4 likely effective agents at start
• At least 3 effective agents for the rest of treatment

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Key Management Principles in MDR-TB

• Rapid molecular DST first (Xpert, LPA) - do not wait for culture DST
• Never add a single drug to a failing regimen - always add 2-3 drugs simultaneously
• Directly observed therapy (DOT) throughout
• Monthly bacteriologic monitoring (smear + culture)
• For localized disease with adequate pulmonary reserve: surgical resection (lobectomy/wedge) may be considered alongside drugs
• Preferably managed at specialized centers with adequate resources
• Ambulatory care preferred over hospitalization when clinically stable (better adherence, less transmission)
• Respiratory infection-control precautions throughout

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Treatment Failure and Relapse

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LTBI (Latent TB) Treatment

• Recent contacts of infectious TB cases
• HIV-positive individuals
• Recent TST converters
• Immunosuppressed patients starting TNF-α inhibitors

• Tintinalli's Emergency Medicine; Harrison's 22E

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HIV-TB Co-infection Treatment Points

• Start anti-TB drugs first, then ART as soon as possible (within 2 weeks if CD4 <50/µL)
• Exception: TB meningitis - delay ART for 8 weeks (regardless of CD4)
• Rifampin is a potent CYP450 inducer - reduces levels of most HIV protease inhibitors and some NNRTIs
• Preferred ART with rifampin-based TB therapy: dolutegravir or raltegravir (at doubled dose)
• Rifabutin can substitute for rifampin to reduce drug interactions
• Watch for IRIS (immune reconstitution inflammatory syndrome) - paradoxical worsening of TB symptoms after starting ART

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• Harrison's Principles of Internal Medicine 22E, p. 1437-1441
• Tintinalli's Emergency Medicine, p. 493-495
• Fishman's Pulmonary Diseases and Disorders, p. 2290