Overview
This lecture covers Mycobacterium tuberculosis (TB) end to end: its history and current global burden, the organism’s distinctive cell wall, how it transmits and establishes infection, the innate and adaptive host response that leads to granuloma formation and either containment or disease, how active and latent infection are diagnosed in the lab, and how drug-susceptible and drug-resistant TB are treated.
History and epidemiology
- TB (“consumption”) is an ancient human pathogen; in 17th-18th century Europe it caused 25% of adult deaths.
- Robert Koch identified the causative bacillus in 1882 (“Koch’s bacillus”).
- Mortality fell through decreased transmission (improved socioeconomic conditions: less overcrowding, better nutrition; public health measures: isolation) and the availability of anti-mycobacterial drugs: streptomycin (1946), isoniazid (1952), rifampicin (1970).
- WHO Global TB Report 2024: an estimated 10.8 million new cases and 1.25 million deaths in 2023.
- Incidence is highest in sub-Saharan Africa, South/Southeast Asia and parts of the Pacific.
- Case notifications dipped around 2020 (COVID-era disruption) and have since risen back toward pre-pandemic levels, approaching but not reaching the WHO’s 2025 milestone.
- Countries with the largest absolute case numbers (≥100,000 incident cases) include India, Indonesia, China, Bangladesh, Philippines, Pakistan, Nigeria and the Democratic Republic of the Congo.
- HIV prevalence among new/relapse TB cases is highest in sub-Saharan and southern Africa, with elevated levels also in parts of Russia and Central Asia.
Organism and transmission
- M. tuberculosis is an acid-fast bacillus: its cell wall is rich in lipids, particularly mycolic acids, making it hydrophobic.
- It is slow growing (doubling time 15-20 hours) and a facultative intracellular pathogen.
- It is neither Gram positive nor Gram negative: its envelope is a distinct lipid-rich structure containing mycolic acid, lipoarabinomannan (LAM), mannophosphoinositide and galactan alongside peptidoglycan, unlike the lipoteichoic acid/peptidoglycan wall of Gram-positive bacteria or the LPS/porin/peptidoglycan wall of Gram-negative bacteria.
- M. tuberculosis itself spreads person to person via respiratory droplets/aerosols; Mycobacterium bovis (part of the M. tuberculosis complex) can instead be transmitted via unpasteurised milk.
- Talking, tachypnoea, exercising and coughing all generate aerosols. M. tuberculosis survives for prolonged periods in evaporated “droplet nuclei” in air.
- Aerosols (<5 µm) can float for hours, be inhaled, and travel beyond 1 metre.
- Droplets (5-100 µm) travel less than 1 metre and fall within 5 seconds; they cannot be inhaled.
- Particles >100 µm fall as fomites onto surfaces.
- Close proximity and poorly ventilated spaces increase transmission risk by allowing infectious aerosols to accumulate.
Natural history of infection
- Whether exposure leads to infection depends on environmental factors (crowding, occupation, poor ventilation), bacterial factors (infectious dose, duration, proximity) and host factors (age, immune status, malnutrition, diabetes, smoking).
- Of those exposed, roughly 70% remain uninfected and roughly 30% become infected.
- Of those infected, 5-10% develop primary tuberculosis; 90-95% achieve containment and persistence (latent TB infection, LTBI).
- From containment/LTBI, reactivation occurs in roughly 10% of healthy adults, 20% of children under 5, 30% of HIV-positive patients, and 40% of children under 2, as well as in other high-risk clinical settings; reactivation can present as post-primary pulmonary, extra-pulmonary, or miliary tuberculosis.
- Public health interventions act at three points on this pathway: (1) airborne infection control, (2) intensive case finding and treatment of active TB, and (3) diagnosis and treatment of LTBI.
Innate response: phagocytosis and the phagolysosome
- Inhaled droplet nuclei reach the alveoli and are phagocytosed by alveolar macrophages (bacterium bound by macrophage receptors, engulfed into a phagosome, which fuses with a lysosome to form a phagolysosome, followed by exocytosis of soluble debris).
- The phagolysosome is a hostile environment for the bacterium:
- Acidic pH (4.0-5.0), achieved by vacuolar ATPase pumping H+ in.
- Reactive oxygen species (ROS, via NADPH oxidase, producing superoxide that combines with Fe/H2O2 to form damaging hydroxyl radicals) and reactive nitrogen species (RNS, via nitric oxide synthetase using arginine to produce citrulline and NO/·O2-/ONOO-/NO2-).
- Hydrolytic enzymes (roughly 50 types of lysosomal acid hydrolases, including lysozyme).
- Antimicrobial peptides (defensins) that permeabilise the bacterial cell membrane.
- M. tuberculosis survives this environment through several mechanisms:
- Impairs phagosome maturation via protein kinase G. [slide flagged as lower priority for exam]
- Its lipid-rich cell wall protects it against H+ and antimicrobial peptides.
- Secretes urease, converting urea to NH3 to raise the local pH. [slide flagged as lower priority for exam]
- Produces enzymes that protect against RNS and ROS, e.g. catalase peroxidase (2H2O2 → 2H2O + O2). [slide flagged as lower priority for exam]
Adaptive response and granuloma formation
- Dendritic cells acquire mycobacterial antigen either by cross-priming (taking up apoptotic bodies containing mycobacterial peptides from infected macrophages, presented via MHC I) or by direct infection (direct presentation via MHC I/MHC II), then traffic from the lung to the lymph node to prime T cells.
- Primed CD8+ T cells traffic back to the site of infection, produce IFNγ and TNFα to activate macrophages, and directly kill infected macrophages.
- Primed CD4+ T cells differentiate into:
- Th1 cells, producing IFNγ and TNFα, which activate macrophages.
- Th17 cells, producing IL-17, which activates neutrophils.
- IFNγ (with TNFα also required) activates macrophages for enhanced killing by: more efficient lysosome-phagosome fusion (increasing delivery of the bacterium to the phagolysosome), increased ROS/RNS, and increased antimicrobial peptides. Despite this enhancement, M. tuberculosis can still remain viable within the macrophage.
- Chronic inflammation dependent on TNFα and IFNγ produces a granuloma, which restricts spread of the bacterium. A solid granuloma has a fibrous wall surrounding a rim of T cells, dendritic cells, epithelioid cells, macrophages and B cells around a central hypoxic/necrotic core.
- Hypoxic and nutrient-limited conditions within the granuloma drive the bacterium into dormancy: low metabolic activity, altered metabolic pathway use, and non- to low-replicating persistence.
- Risk factors including immunosuppression (e.g. steroids, TNFα inhibitors), diabetes, HIV, alcoholism and poor nutrition can cause granuloma breakdown: the solid granuloma progresses to a necrotic granuloma (small central necrotic area) and then a caseous granuloma (large central area of bacterial proliferation), overall marking the combined outcome of activated macrophages controlling M. tuberculosis versus granuloma breakdown allowing bacterial escape.
Laboratory diagnosis
- Direct detection
- Microscopy: Ziehl-Neelsen-type acid-fast staining. Sequence: primary stain carbol fuchsin, decoloriser acid alcohol, counterstain methylene blue. [reagent-level detail flagged as lower priority for exam] Acid-fast organisms retain the red primary stain through decolourisation; non-acid-fast organisms are decolourised and take up the blue counterstain. Detection in sputum requires about 10,000 organisms/ml; sensitivity is only ~67%.
- Nucleic acid amplification test (NAAT), e.g. GeneXpert MTB/RIF: sputum is liquefied and inactivated, loaded into a cartridge, automatically filtered/washed, organisms lysed ultrasonically to release DNA, mixed with dry PCR reagents, and amplified/detected by seminested real-time PCR, giving a result (including rifampicin resistance status) in about 1 hour 45 minutes. Sensitivity is 98% for smear-positive samples but only 73% for smear-negative samples.
- Culture: solid media is slow (mean 4-6 weeks for visible colonies). Automated liquid culture (BACTEC MGIT 960, Mycobacterial Growth Indicator Tube) is faster (mean 10-14 days) and more sensitive (roughly 20% higher yield than solid media).
- Susceptibility testing: requires a culture isolate, tested by growth in the BACTEC MGIT 960 in the presence or absence of a drug. First-line drugs tested: isoniazid, rifampin, ethambutol, pyrazinamide. Second-line drugs (e.g. streptomycin, moxifloxacin) can also be tested. Culture is therefore essential, especially given increasing rates of drug-resistant TB.
Treatment of drug-susceptible TB
- Multiple drugs are used together to prevent resistance: a single drug selects out a resistant population, which two drugs can prevent.
- Typical regimen: initial treatment with 4 drugs, reduced to 2 drugs at 2 months once susceptibility results are available.
- Treatment continues for at least 6-9 months, because dormant M. tuberculosis necessitates prolonged treatment to achieve cure.
- New Zealand practice is guided by the Guidelines for Tuberculosis Control in New Zealand (2019, Manatū Hauora/Ministry of Health).
- Barriers to therapy: long treatment duration (leading to poor adherence, treatment failure, drug resistance, and loss to follow-up), complex and toxic/expensive drug regimens, toxicity when combined with antiretrovirals, and drug-resistant TB itself.
Drug-resistant TB
- Rifampicin-resistant (RR) TB: resistant to rifampicin.
- Multidrug-resistant (MDR) TB: resistant to both isoniazid and rifampicin.
- Extensively drug-resistant (XDR) TB: resistant to rifampicin, any fluoroquinolone, and at least one of bedaquiline or linezolid. Pre-XDR TB is resistant to rifampicin plus any fluoroquinolone.
- 2023: approximately 400,000 MDR/RR TB cases globally, with the largest numbers in India, and other concentrations in Russia, China, the Philippines and Indonesia; 28,982 XDR or pre-XDR cases were identified, though not all cases are tested for this.
- Globally, the percentage of TB patients with MDR/RR TB has been declining: 3.2% among patients with no previous TB history, versus 16% among previously treated patients (higher because prior treatment selects for resistance).
- Treatment of MDR/RR, pre-XDR and XDR TB traditionally required 6-18 months or longer using second- and third-line drugs (more expensive, more toxic, less effective). The newer 6-month all-oral BPaLM regimen (bedaquiline + pretomanid + linezolid + moxifloxacin) achieves ~90% success and is now recommended first-line for MDR/RR TB; BPaL (without moxifloxacin) is used for pre-XDR TB.
Latent TB infection: testing and treatment
- LTBI testing looks for evidence of a cellular immune response to M. tuberculosis:
- Tuberculin skin test (TST, Mantoux technique): intradermal injection of purified protein derivative (tuberculin), read as a delayed-type hypersensitivity reaction at 48-72 hours. Limitation: cross-reactivity with BCG vaccination and non-tuberculous mycobacteria.
- Interferon-gamma release assay (IGRA, e.g. QuantiFERON Gold): an in vitro blood test in which an antigen-presenting cell processes M. tuberculosis-specific recombinant antigens (not present in BCG or most non-tuberculous mycobacteria) and presents them to an antigen-specific T cell, which produces IFNγ that is then measured.
- Neither TST nor IGRA can distinguish LTBI from active TB disease.
- Testing goal: identify individuals at increased risk of developing active TB, and test only those who would benefit from treatment (e.g. likely recent infection, or decreased capacity to contain latent infection such as immunosuppression or HIV).
- LTBI treatment options: isoniazid + rifapentine weekly for 3 months (rifapentine not currently available in NZ), isoniazid + rifampicin daily for 3 months, rifampicin alone for 4 months, or isoniazid alone for 9 months.
Latent TB infection vs TB disease
| Feature | Latent TB infection | TB disease |
|---|---|---|
| M. tuberculosis present | Yes | Yes |
| TST/IGRA | Positive | Positive |
| Chest X-ray | Normal | Abnormal |
| Sputum smears/cultures | Negative | Cultures positive, smears +/- |
| Symptoms | None | Symptomatic (e.g. cough, fever, weight loss) |
| Infectious | No | Infectious prior to treatment |
| Defined as a case of TB | No | Yes |
Self-test
- Describe the mycobacterial cell wall and explain why M. tuberculosis is neither Gram positive nor Gram negative.
- Distinguish transmission of M. tuberculosis from that of M. bovis.
- Explain why aerosols but not larger droplets or fomite particles can transmit TB infection over distances beyond 1 metre.
- Following exposure to M. tuberculosis, describe the approximate proportions of outcomes: uninfected, primary tuberculosis, and containment/persistence, and what happens from containment.
- List the host groups with the highest rates of reactivation from latent infection.
- Describe the components of the phagolysosome environment that are hostile to M. tuberculosis.
- Describe the mechanisms M. tuberculosis uses to survive within the phagolysosome.
- Describe the two ways dendritic cells acquire mycobacterial antigen to prime T cells.
- Distinguish the roles of CD8+ T cells, Th1 CD4+ T cells and Th17 CD4+ T cells in the immune response to TB.
- Explain how IFNγ and TNFα enhance macrophage killing of M. tuberculosis, and why the bacterium can still survive.
- Describe the structure of a solid granuloma and how it can progress to a caseous granuloma.
- List the risk factors for granuloma breakdown.
- Distinguish microscopy, NAAT and culture as methods of TB diagnosis, including their relative speed and sensitivity.
- Why is culture essential even though it is the slowest diagnostic method?
- Explain the rationale for using multiple drugs in TB treatment and describe a typical treatment course.
- Define rifampicin-resistant, multidrug-resistant, and extensively drug-resistant TB.
- Describe the current first-line regimen for MDR/RR TB and its success rate.
- Distinguish the tuberculin skin test from the IGRA as tests for latent TB infection, including a limitation of each.
- A patient has a positive IGRA, a normal chest X-ray, negative sputum cultures, and no symptoms. Distinguish whether this represents latent TB infection or TB disease, and explain your reasoning.
Answers
Reveal answers
- The mycobacterial cell wall contains mycolic acids (making it hydrophobic and acid-fast), lipoarabinomannan (LAM), mannophosphoinositide, galactan and peptidoglycan. This lipid-rich structure differs from both the lipoteichoic acid/peptidoglycan wall of Gram-positive bacteria and the LPS/porin/peptidoglycan wall of Gram-negative bacteria, so it is neither.
- M. tuberculosis is transmitted person to person via respiratory droplets/aerosols; M. bovis (part of the M. tuberculosis complex) is transmitted via unpasteurised milk.
- Aerosols are <5 µm, can float in air for hours and be inhaled, so they can travel beyond 1 metre. Droplets (5-100 µm) fall within 5 seconds and travel under 1 metre, and cannot be inhaled. Particles >100 µm fall onto surfaces as fomites and do not remain airborne.
- Roughly 70% remain uninfected and 30% become infected. Of those infected, 5-10% develop primary tuberculosis and 90-95% achieve containment and persistence (LTBI); from containment, reactivation can occur later.
- Reactivation is most common in children under 2 years (~40%), HIV-positive patients (~30%), children under 5 years (~20%), and healthy adults (~10%), plus other high-risk clinical settings.
- Acidic pH (4.0-5.0) from vacuolar ATPase; reactive oxygen species from NADPH oxidase; reactive nitrogen species from NO synthetase; hydrolytic enzymes (lysosomal acid hydrolases); and antimicrobial peptides (defensins) that permeabilise the bacterial membrane.
- It impairs phagosome maturation via protein kinase G, uses its lipid-rich cell wall to resist H+ and antimicrobial peptides, secretes urease to convert urea to NH3 and raise pH, and produces enzymes such as catalase peroxidase to neutralise ROS/RNS.
- Cross-priming: dendritic cells take up apoptotic bodies containing mycobacterial peptides from infected macrophages, presenting via MHC I. Direct presentation: dendritic cells are themselves infected and present antigen via MHC I/MHC II.
- CD8+ T cells produce IFNγ and TNFα to activate macrophages and directly kill infected macrophages. Th1 CD4+ cells produce IFNγ and TNFα, which activate macrophages. Th17 CD4+ cells produce IL-17, which activates neutrophils.
- IFNγ (with required TNFα) increases lysosome-phagosome fusion, ROS/RNS production, and antimicrobial peptide levels, enhancing bactericidal activity. However, M. tuberculosis can still remain viable despite this enhanced killing.
- A solid granuloma has a fibrous wall surrounding a rim of T cells, dendritic cells, epithelioid cells, macrophages and B cells around a central hypoxic/necrotic core. Under risk factors such as immunosuppression, diabetes, HIV, alcoholism or poor nutrition, it progresses to a necrotic granuloma (small central necrosis) and then a caseous granuloma (large central area of bacterial proliferation) as the structure breaks down.
- Immunosuppression (e.g. steroids, TNFα inhibitors), diabetes, HIV, alcoholism, and poor nutrition.
- Microscopy (acid-fast stain) is fast but has only ~67% sensitivity and requires ~10,000 organisms/ml. NAAT (e.g. GeneXpert) gives a result in under 2 hours with 98% sensitivity in smear-positive and 73% in smear-negative samples. Culture is slowest (4-6 weeks on solid media, 10-14 days with automated liquid culture) but most sensitive, and is the only method that yields an isolate.
- Only a culture isolate allows susceptibility testing, which is essential to detect drug resistance, particularly with increasing rates of MDR and XDR TB.
- Using a single drug selects out a resistant population; multiple drugs prevent this. A typical course starts with 4 drugs, reduces to 2 at 2 months once susceptibility is known, and continues for at least 6-9 months because dormant bacteria require prolonged treatment.
- RR TB is resistant to rifampicin. MDR TB is resistant to both isoniazid and rifampicin. XDR TB is resistant to rifampicin, any fluoroquinolone, and at least one of bedaquiline or linezolid (pre-XDR is resistant to rifampicin plus any fluoroquinolone, without the additional resistance).
- BPaLM (bedaquiline + pretomanid + linezolid + moxifloxacin), an all-oral 6-month regimen, is now first-line for MDR/RR TB, with a success rate of about 90%.
- TST (Mantoux) is an intradermal injection of tuberculin read as a delayed-type hypersensitivity reaction at 48-72 hours, but cross-reacts with BCG vaccination and non-tuberculous mycobacteria. IGRA measures IFNγ release from T cells against M. tuberculosis-specific antigens not present in BCG or most non-tuberculous mycobacteria, avoiding that cross-reactivity.
- This represents latent TB infection: M. tuberculosis is present and TST/IGRA is positive in both LTBI and TB disease, but a normal chest X-ray, negative sputum cultures and absence of symptoms are all features of LTBI rather than TB disease (which shows an abnormal X-ray, positive cultures, and symptoms).