Mycobacteria, Tuberculosis & Emerging Respiratory Pathogens

Mycobacteria & Pulmonary Tuberculosis

Taxonomy & Major Human Pathogens

• Rod-shaped, strict aerobes, non-spore-forming, classically called acid-fast bacilli (AFB) because after staining they resist de-colorisation by acids/alcohols.
• > $200$ recognised species.
• Species always considered pathogenic to humans:
– Mycobacterium tuberculosis → pulmonary & disseminated tuberculosis (TB).
– M. leprae → leprosy.
– M. bovis → bovine & human TB-like disease (rare in developed nations).
• Species potentially pathogenic, especially in immuno-suppressed:
– M. avium complex (MAC) → disseminated pulmonary disease, very common in AIDS.
– M. kansasii → pulmonary & extra-pulmonary lesions.

Causative Agents of Pulmonary TB

• M. tuberculosis (predominant).
• M. bovis.
• M. africanum.

Morphology & Staining

• In tissue: thin, straight rods; on artificial media: coccoid & filamentous forms.
• Acid-fastness depends on intact waxy envelope rich in mycolic acids.
• Ziehl–Neelsen stain → red AFB.
• Fluorochrome stains (auramine, rhodamine) → yellow-orange fluorescence under UV.

Culture Media & Growth

• Strict aerobes, optimum $37^\circ\text{C}$ , require enriched media, slow growth (doubling time $\approx 18\text{ h}$ ).
(a) Middlebrook 7H10/7H11 agar – growth in weeks if inoculum large.
(b) Lowenstein–Jensen (L-J) or Ogawa egg medium (malachite green inhibits flora) – small clinical inocula grow in $3–6$ weeks.
(c) Middlebrook 7H9/7H12 broth – faster growth.
(d) Radiometric BACTEC 460 – detects $\text{CO}_2$ release, positivity $\approx 2$ weeks.
(e) Non-radiometric MGIT (Mycobacterial Growth Indicator Tube) – automated fluorescence detection.

Physical & Chemical Resistance

• Hydrophobic cell surface confers resistance to many dyes (e.g. malachite green) & antibiotics (e.g. penicillin).
• Survive acids/alkalis → these agents used during specimen decontamination.
• Survive drying in sputum; but killed by UV, pasteurisation, $5\%$ cresol.

Colony Variation & Virulence

• “Serpentine cords” on smear correlate with virulence (due to cord factor).
• Lipid wall components:
– Mycolic acids & muramyl-dipeptide complex → granuloma formation.
– Phospholipids → caseous necrosis.
• Virulence attributes:
– Intracellular survival in macrophages.
– Induction of host sensitisation by tuberculoprotein & wax D.
– Blocking of phagocyte killing.
– Environmental hardiness.

Pathogenesis of Tuberculosis

Source & Mode of Infection

• Human “open” cases emit droplets $< 25\,\mu\text{m}$ that dry to droplet nuclei capable of alveolar deposition.
• Bovine strain transmitted via ingestion of unpasteurised milk.

Host Response & Lesions

(A) Two basic lesions

Exudative (Acute) Lesion – oedema, PMNs → resembles lobar pneumonia; tuberculin conversion occurs during this phase.
Productive (Granulomatous) Lesion – concentric zones: central caseation, surrounding epithelioid & Langhans giant cells, outer fibroblasts; called a tubercle. Caseous focus may evacuate into bronchus → cavity.

(B) Spread – direct extension, lymphatics, blood ( → miliary TB), bronchogenic, GI tract (swallowed sputum).

(C) Intracellular Niche – bacilli live in monocytes, reticulo-endothelial & giant cells; intracellular persistence complicates chemotherapy.

Clinical Forms

Primary TB – usually childhood but now also adults; lesion often at lung base with draining hilar nodes → Ghon focus/complex.
Reactivation (Post-primary) TB – latent bacilli re-emerge, classically at lung apex (high $PO_2$ ); marked caseation, fibrosis, cavitation; minimal nodal disease.

Clinical Findings

• Constitutional: fatigue, weight loss, fever, night sweats.
• Pulmonary: chronic cough, haemoptysis (advanced).
• Meningitis or urinary tract disease may occur without lung findings.
• Miliary spread → multiple organ lesions, high mortality.

Immunity & Hypersensitivity

• First infection induces partial resistance (better localisation, slower spread) and delayed-type hypersensitivity (DTH).
• Tuberculin sensitivity arises $4–6$ weeks post-infection; mediated by CD4 T cells reacting to tuberculoprotein + wax D.

Laboratory Diagnosis of TB

Specimen Collection

• Sputum (early-morning ×3 on $2$ consecutive days), gastric lavage (children/ non-productive cough), laryngeal swab, urine, CSF, pleural fluid, biopsies, blood (esp. MAC).

Decontamination & Concentration

• Liquefy with N-acetyl-L-cysteine, decontaminate with $\text{NaOH}$ , neutralise, centrifuge.

Microscopy

• Ziehl–Neelsen (20 % $\text{H}<em>2\text{SO}</em>4$ decolouriser) → red rods.
– M. tuberculosis: slender/beaded.
– M. bovis: shorter, stubbier.
• Fluorescence (auramine–rhodamine) – yellow rods on dark field; more sensitive but requires confirmatory ZN.

Culture & Identification

• Inoculate processed sample onto L-J or Middlebrook 7H10/7H11 and into 7H9/7H12 broth.
• Incubate $35–37^\circ\text{C}$ , $5–10\%$ $\text{CO}_2$ up to $8$ weeks.
• If smear+ / culture− or NTM suspected → also incubate $24–33^\circ\text{C}$ for $12$ weeks.
• Colony morphology on L-J:
– M. tuberculosis: dry, rough, buff-coloured, wrinkled.
– M. bovis: moist, smooth, colourless/flat.
• Niacin test: positive for M. tuberculosis, negative for M. bovis.
• Rapid systems: BACTEC 460, MGIT 960.
• Molecular probes for rRNA; HPLC of mycolic acids for speciation.
• PCR / NAAT – detects DNA directly; multiplex assays target rifampin resistance genes.
• Interferon-γ Release Assays (IGRA) – measure $\text{IFN-}\gamma$ from sensitised CD4 T cells; unaffected by BCG.

Drug Susceptibility Testing (DST)

• Conventional culture-based DST; rapid methods: line-probe assay, molecular beacons, phage assays, MODS, BACTEC, MGIT.

Diagnosis of Tuberculous Meningitis

• CSF collected by lumbar puncture (three bottles).
• Macroscopy: clear/slightly turbid, forms cobweb clot on standing.
• Cytology: $\approx 20–500$ cells/mm $^3$ , lymphocyte predominance.
• Protein ↑, glucose ↓.
• Smear from clot or centrifuged deposit → ZN.
• Culture on L-J, niacin, DST.

Tuberculin Skin Test (TST)

• Material: PPD (purified protein derivative), standardised in Tuberculin Units (TU): $1\,\text{TU}$ (first-strength), $5\,\text{TU}$ (intermediate), $250\,\text{TU}$ (second).
• Read at $48–72$ h:
– $\ge 5\,\text{mm}$ induration positive in HIV/exposed high-risk.
– $\ge 10\,\text{mm}$ in moderate-risk.
– $\ge 15\,\text{mm}$ in low-risk.
• False-negative in anergy (overwhelming TB, measles, sarcoid, AIDS, Hodgkin, immunosuppression).

Treatment of TB

• Standard short-course chemotherapy: $2$ months INH + RIF + PZA + EMB followed by $4–6$ months INH + RIF (alt. regimens exist).
• Cure rates $> 95\%$ with adherence.
• Drug resistance:
– MDR-TB = resistance to INH + RIF.
– XDR-TB = MDR + resistance to any fluoroquinolone + $\ge 1$ injectable second-line drug (amikacin, kanamycin, capreomycin).
• Rapid DST essential for regimen choice.

Epidemiology, Prevention & Control

• Human respiratory spread; enhanced by close contact, under-nutrition, extremes of age, comorbidities (silicosis, diabetes), immunosuppression (HIV).
• BCG vaccine (attenuated M. bovis) confers partial, time-limited protection against severe childhood TB & meningitis.
• Public health: early case detection, effective therapy, contact tracing, prophylaxis (INH $6–9$ mo or RIF $4$ mo; alternative regimens for drug-resistant exposure), pasteurisation of milk, cattle eradication programmes.

Emerging & Re-Emerging Respiratory Pathogens

Highly Pathogenic Avian Influenza (H5N1), MERS-CoV, SARS-CoV, SARS-CoV-2, Legionella spp., etc.

Severe Acute Respiratory Syndrome (SARS-CoV)

Pathogenesis & Clinical Picture

• Infects respiratory epithelium & salivary ducts; disseminates to kidney, liver, intestine.
• Incubation $\approx 6$ days.
• Early: fever, malaise, chills, headache, cough → dyspnoea.
• Chest radiographs abnormal; $\approx 10\%$ mortality (higher in elderly).

Diagnosis

• Virus isolation in Vero cells, RT-PCR on blood/respiratory/stool, ELISA for antibodies.

Epidemiology & Control

• Airborne & fomite transmission in health-care settings.
• 2002–2003 outbreak: $> 8000$ cases, $> 800$ deaths, spread via international travel; “super-spreaders” infected $> 10$ contacts each.
• Control: isolation, quarantine, PPE, travel restrictions; vaccines in development.

COVID-19 (SARS-CoV-2)

Virology

• RNA virus closely related to bat coronaviruses; pangolin may be intermediate host.
• Spike (S) protein binds ACE-2 receptor on respiratory epithelial cells; cleavage by TMPRSS-2 exposes fusion peptide → viral entry.
• Neuropilin-1 (NRP-1) acts as additional entry receptor.
• Neutralising antibody blocks S–ACE-2 interaction.

Variants of Concern (WHO)

• Alpha (B.1.1.7) – UK, Dec 2020.
• Beta (B.1.351) – South Africa.
• Gamma (P.1) – Brazil/Japan.
• Delta (B.1.617.2) – India.
• Omicron (B.1.1.529) – South Africa, Nov 2021.

Transmission

• Primarily inhalation of droplets/aerosols; aerosols remain suspended > $6$ ft & $>$ minutes.
• Shedding begins $2–3$ days pre-symptom, lasts $\approx 7$ days.
• Fomites via hand–face contact; vertical transmission rare.
• “Super-spreader” indoor events without masks/distancing noted.

Pathology & Pathophysiology

• Gross: diffuse alveolar damage, oedema, T-cell infiltrates.
• Microvascular thrombosis, endothelial injury, angiogenesis.
• Mechanisms:

Viral cytolysis of alveolar cells → impaired $\text{O}_2$ diffusion.
Endothelial death → microthrombi + cytokine storm → ARDS.
• Elevated IL-1, IL-6, TNF, bradykinin.

Clinical Features

• Incubation $2–14$ days (mean $5$ ).
• Common: fever, dry cough, dyspnoea.
• Systemic: fatigue, chills, headache, myalgia.
• Unique: anosmia, dysgeusia (often herald illness).
• Multi-organ: myocarditis, encephalopathy, GI symptoms, hypercoagulability → stroke/embolism.

Laboratory Diagnosis

Molecular (RT-PCR) on nasopharyngeal swabs: high sensitivity $\ge 2$ days after symptom onset; early collection → false-negatives. Rapid isothermal assays available.
Antigen EIA/LFA: rapid, cheap, lower sensitivity; negative does not exclude infection.
Serology: IgM from $\approx 5$ days, IgG $\approx 14$ days post-symptom; detection of past infection, $>95\%$ specificity.

Treatment Categories

• Antivirals: molnupiravir, paxlovid, remdesivir.
• Anti-SARS-CoV-2 monoclonal antibodies (therapeutic & prophylactic).
• Anti-inflammatory: dexamethasone.
• Immunomodulators: baricitinib, tocilizumab.
• Supportive: supplemental $\text{O}_2$ , mechanical ventilation, prone positioning.

Prevention & Control

• Non-pharmacological: masks, $\ge 6$ ft distancing, hand hygiene, cough etiquette, stay-at-home when ill, isolation of cases, quarantine of contacts, contact tracing, adequate PPE in hospitals.
• Vaccines (WHO-EUL & US-licensed):
– mRNA (Pfizer, Moderna).
– Recombinant spike protein (Novavax).
– Adenoviral-vectored (Johnson & Johnson).
• Mass vaccination began Dec 2020; doses continually updated.
• Monoclonal antibodies for pre-/post-exposure prophylaxis in high-risk.

These notes consolidate essential morphology, culture, pathogenic mechanisms, clinical features, diagnosis, treatment, epidemiology, and preventive strategies for Mycobacterium tuberculosis and major emerging respiratory viruses (SARS-CoV & SARS-CoV-2), providing an integrated reference for exam revision.