1.1 Mycobacteria infection (Tb)

Question 1

MYCOBACTERIA
Mycobacteria are ___________________ organisms (may also survive extracellularly but prefers an intracellular environment):
• __________, non-spore forming, non-motile bacilli (spores are implicated in resistance to heat and chemical sterilisation and facilitates survival in various environments) → M. tuberculosis can be managed with ______________
• Possess lipid-rich cell walls (containing _____________________) which protect the bacteria from lysosomal attack within macrophages

Mycobacteria takes a while to grow under incubation, and due to the difficulty in staining, it is important to alert the microbiology lab when suspecting TB:
• Proper incubation and plating must be used to identify the pathogens
• Standard bacterial cultures are only incubated for 2 – 3 days, and will not yield any Mycobacterium tuberculosis pathogens

Answer 1

facultative intracellular;

Aerobic;

proper sterilisation;

mycolic acid and long-chain glycolipids;

Question 2

M. tuberculosis is an _______________ (must have oxygen for survival) and a facultatively intracellular pathogen which usually infects ________________:
• Slow-growing bacterium with a generation time of 12 – 18 hours (contrasted to 20 – 30 minutes for E. coli) → visible growth in 3 – 8 weeks on solid media incubation
• Tends to grow in characteristic _________________
• Hydrophobic with a high ___________ content in the cell wall → tends to clump together (impermeable to usual stains like the Gram stain)
• Special stains (acid-fast stain/Ziehl-Neelsen stain) must be used

Answer 2

obligate aerobe;

mononuclear phagocytes (e.g. macrophages);

parallel groups (serpentine cording) ;

lipid

Question 3

Acid-fast bacilli (AFB) like M. tuberculosis are impermeable to various basic dyes unless they are combined with _________.

Ziehl-Neelsen stain: Slide is flooded with ______________ and phenol (causing retention) → resist decolourisation by acidified organic solvents (acid-fast) → identified with ________

Fluorescent auramine stain: Using a phenolic auramine or auramine-rhodamine stain and potassium permanganate (fluoresces) → more sensitive than ZN stain but less specific

Answer 3

phenol;

carbol fuchsin; LM

Question 4

[Tests for TB: AFB stain]

Advantages & Disadvantages?

Answer 4

Advantages

• same day results
• simple
• cheap

Disadvantages

• less sensitive (60%) then culture
• need >= 10 000 organisms/ ml (small samle inaccurate
• cannot distinguish between M Tb vs NTM

Question 5

[Test for Tb: Culture on solid media]

Advantages

• Gold standard (must use in conjunction with other methods)
• detects 10-100 organisms/mL
• colony morphology
• speciation & strain ID
• Quantification
• Susceptibility testing

Disadvantages?

Answer 5

Take 3-8 weeks for growth, usually 6 weeks +

Question 6

[TB Test: Liquid Broth]

Advantages

• sens and spec similar to solid media
• less workload
• speciation & strain ID
• susceptibility testing
• quicker than solid media (7-21d)

disadvantages?

Answer 6

cannot do colony morphology, cannot quantify growth

Question 7

[TB test: BATEC (liquid) ]

Advantages

• preliminary ID
• drug susceptibility results in 5-14 days

Disadvantages?

Answer 7

• must use in conjunction with solid media –> costly

- cannot quantify % of drug resistance

Question 8

[TB test: NAAT/PCR ]

• same day results
• sensitivity between AAFB and culture
• Identifies MTB complex

Disadvantages?

Answer 8

• cannot distinguish between dead & viable organisms

- must use in conjunction with culture for speciation, susceptibility etc

Question 9

[TB test: HPLC]

Advantages

• sensitivity and specificity close to 100%
• can identify _______________

Disadvantages: must use in conjunction with culture

Answer 9

M. bovis bcg

Question 10

[Tb test: molecular tests for c/s mutations]
- same day results, especially for _____________ (conferred by rpoB gene)

disadvantages

• validation in different clinical settings
• only for RIF resistance (not for any antibiotics)

Answer 10

RIF-resistance mutations

Question 11

IMMUNE RESPONSE
The immune system has 3 levels of defence (surface barriers, innate and adaptive immunity):
- Surface barriers: Enzymes, mucus, skin
- Innate immunity: Lacks immunological memory; involves monocytes (DCs/macrophages in tissues/skin), neutrophils, eosinophils, complement etc.
- Adaptive immunity: Involves B and T cells (mature T cells leaving thymus are CD4/8+)

During the first encounter with an antigen, naïve lymphocytes proliferate as part of the primary immune response, generating effector and memory B and T cells:
• Effector T cells: CD4+ helper T cells, CD8+ cytotoxic T cells
• Effector B cells: antibody-secreting plasma cells

During the adaptive immune response, lymphocytes are activated by antigen-presenting cells (DCs or macrophages):
• Macrophage: presents antigen to the __________, which secretes ___________________ (activates both the ______________________ to kill the intracellular pathogen)
• Dendritic cell: presents antigen to the ____________, which secretes ____________ which helps B cells secrete antibodies (B cells recognise antigen directly via MHC presentation or indirectly via immune complexes)

Answer 11

Th1 cell;

IFN-γ and IL-2;

macrophage and CD8+ cytotoxic T cells;

Th2 cell;

IL-4, 5, 6

Question 12

CD4+ T helper cells are responsible for regulating the immune response to tuberculosis:
• Macrophages phagocytose M. tuberculosis, then presents it to the CD4+ cells, and secretes cytokines which stimulate or suppress CD4 response
• If the macrophage produces more ______________, the CD4+ T cell produces IFN-γ, which induces the macrophage to kill the intracellular pathogen

Answer 12

IL-12 (or IL-1/TNF-α/ IL-6)

Question 13

Mycobacteria have developed several strategies to survive within macrophages: - Mycobacterial urease: Preventing _________________ (reduces efficacy of host bactericidal enzymes)

• Reduction of CD8+ cytotoxic response
• ROS detoxification: ____________________ helps to detoxify the ROS of phagocytes
• Direct protection from oxidants: Protection from host oxidants
• Suppression of early oxygen-mediated immune responses: Immune responses needed for efficient antigen presentation (including macrophage activation and apoptosis)

Answer 13

acidification of phagosome;

Superoxide dismutase, catalase, thioredoxin

Question 14

PATHOGENESIS
TB infection is a spectrum, ranging from asymptomatic (immediately cleared) to the clinical disease state; latent infections refer to replicating organisms with no clinical signs/symptoms:
• Initial exposure to TB: airborne droplets containing TB bacilli deposit in the terminal alveoli (initial focus usually in the ________________ (lower part of upper lobe and upper parts of middle/lower lobes) due to better airflow)
• ______________ (primary lesion): initially there is only 1 focus of infection (with a small area of granulomatous inflammation) developing about 4 weeks after infection → minimal or no symptoms as infection is contained to a point
• Progression: to overt pulmonary TB (with/without dissemination) or to ___________________ (with potential to reactivate at later stage)
o Overt infection: bacilli are ingested by macrophages, leading to macrophage death → more lymphocytes and monocytes migrate to site of infection in an attempt to control the infection → more inflammation and pneumonitis
• If left unchecked: infected macrophages enter bloodstream (in immunocompromised patients) or to regional lymph nodes (favouring _____________________ → but may spread anywhere (e.g. pericardium causing TB pericarditis)
• Tuberculin reactivity (sensitive skin): marks development of microbial growth inhibition by the body (adaptive immunity) 3 – 9 weeks after infection
o Abundant antibodies are produced (but minimal role in host defence)

Answer 14

subpleural mid-lung zone;

Ghon focus;

calcified nodule/subpleural plug;

lung apices, lymph nodes, kidneys, epiphyses of long bones, vertebrae and meninges

Question 15

Granulomas (chronic process) consist of ____, ___________, ___________ (fusion of several cells; usually macrophages) with some central necrosis:
• Protects host from a persistent irritant (causative agent is walled off and sequestered by cells of the macrophage lineage → containment/destroyed)
• May lead to tissue necrosis (caseating/cavitating) as the cells in the centre of the lesion die and release their toxic contents:
o Caseating necrosis: _____________ material ringed by the inflammatory component (hallmark of TB → until proven otherwise)
o Cavitating necrosis: associated with ___________________

• Langerhans giant cells (fusions of about 20 macrophages) have nuclei lined up along one edge of the cell → epithelioid macrophages are elongated with long pale nuclei and pink cytoplasm
• Ghon complexes are Ghon foci associated with ________________

Answer 15

macrophages, epithelioid cells and Langerhans giant cells;

amorphous pink cheese-like;

strong delayed-type hypersensitivity;

hilar or mediastinal lymphadenopathy

Question 16

TB re-infection refers to a latent infection becoming reactivated (endogenous) or patient becoming exposed to another TB infection (exogenous):
• Manifests as ____________ infiltrates (on CXR), cavitation but no _______________
• Risk factors: extremes of age (leading to local progressive disease +/- dissemination), immunosuppression, stress, malnutrition, pregnancy, malignancy, gastrectomy, jejunal-ileal bypass, ESRF, destructive local pulmonary processes (e.g. lung abscess)

Answer 16

apical or sub-apical ;

hilar lymph node enlargement

Question 17

[TB Spread]
TB is transmitted by airborne transmission of very small (1 - 5μm) respiratory droplets (with a very low infectious dose of _____________ → highly contagious):
• Adapted to intracellular survival within human macrophages → persist for a long time and cause lifelong infections
• Most contacts with an infected person do not get infected (only 30% are infected with _________________)
• Of those infected, 90 – 95% develop latent infection (mostly do not ever cause disease), and the remaining develop active TB (mostly develop pulmonary TB; only some develop non-infectious extra-pulmonary disease)

The clinical signs and symptoms of overt clinical disease include:

Symptoms (at least 2 – 3 weeks)

• General: ___, __________, ________, _________
• Organ-specific: pulmonary (cough – at least 3 weeks), extrapulmonary (CNS, bone, lymph nodes)

Signs

• General: fever, weight loss
• Organ-specific: pulmonary, extrapulmonary (cervical lymphadenopathy – __________)

Answer 17

1 – 10 bacilli;

positive tuberculin test;

fever, weight loss, anorexia, night sweats, malaise

scrofula

Question 18

Pulmonary TB is usually asymptomatic and is thus often undetected:
• M. tuberculosis is phagocytosed by _______________ but is resistant to alveolar killing, allowing it to slowly proliferate and spread locally (causing pneumonia) and to local lymph nodes
o Most patients recover without treatment; a few develop symptomatic disease
• Occurs mainly in the ___________ (due to reactivation of latent infection) with ___________ in immunocompetent patients (aggressive immune response attempting to control the infection) → healing via _______________________
• Standard course of treatment: lasts 6 months (HREZ2-HR4 regimen); given to patients with drug-susceptible localised pulmonary infection
o Duration of therapy is extended for disseminated infections or drug resistance

Answer 18

alveolar macrophages;

upper lobes;

cavitation;

fibrosis and calcification

Question 19

Miliary TB occurs due to ________________ to within the lungs or elsewhere:
• Presence of ______________ (resembling millet seeds)
• High risk of fatality
• Standard course of treatment: 9 – 12 months

Answer 19

massive haematogenous spread;

numerous small nodular lesions

Question 20

Tuberculous meningitis occurs due to haematogenous spread from a distant focus (usually the lungs) which lodges immediately deep to the pia mater (forms rich focus):
• May rupture into the ______________ to form exudates (purulent liquid) near the basal cisterns (including the floor of the 4th ventricle) → site of CSF drainage
• Provides infection with a route to spread around the brain and cause _____________(mass-like regions of caseous necrosis) or ___________ (causing ischaemic infarcts)
• Spread of infection may cause _________________ by blocking CSF outflow

The symptoms of tuberculous meningitis are variable depending on which neuroanatomical structure is affected:
- Non-specific: Weight loss, fever, night sweats, subacute presentation (for several weeks)
- Neurological: Personality change, focal neurological deficit, drowsiness/loss of consciousness (low GCS), cerebral oedema, brain herniation
• Oedema related to the tuberculoma could _____________________
• If oedema worsens, the brain could ____________________

A ___________________ is used to diagnose tuberculous meningitis, and presents with lymphocytosis in the CSF, low glucose (bacterial consumption), raised inflammatory protein levels:
• Standard treatment: at least 12 months of _______________________

Answer 20

subarachnoid space;

choroid plexitis;

arteritis;

obstructive hydrocephalus;

push against the ventricles and the midline of the brain;

herniate through the foramen magnum (causing death)

lumbar puncture (draining CSF);

anti-TB therapy and steroids

Question 21

Vertebral TB (Pott’s disease) occurs due to haematogenous spread (causing initial discitis), and further local spread of inflammation to vertebral bodies causes vertebral end-plate destruction and collapse:
• May extend anteriorly to cause a _____________, or posteriorly to ____________________ (medical emergency → immediate surgery indicated to prevent spinal cord damage and paralysis)
• Symptoms: fever, night sweats, weight loss, back pain
• Investigations: MRI/CT (with/without biopsy/aspirate)
• Treatment: 9 – 12 months of anti-TB therapy + surgery (if indicated)

Answer 21

iliopsoas abscess;

compress on the spinal cord

Question 22

DIAGNOSIS OF TB
The mainstays of traditional diagnosis of tuberculosis include:
1. Clinical history and epidemiological risk (general clinical picture)
2. CXR and other imaging (e.g. CT/MRI for spinal TB)
3. Smear microscopy (look for AFB using the ZN stain)
4. ___________ (gold standard) + sensitivity + identification

The tuberculin skin test and the IFN-γ release assays (IGRA) can be used to test for previous TB exposure or latent TB infection:

• Tuberculin/PPD skin test (Mantoux test): Tuberculin (purified protein derivative of TB) pricked into skin → positive test would be _______________________
• Not very sensitive (false negatives)
• IFN-γ release assays (IGRA): Done on whole blood → IFN-γ production by _____________________ indicates previous exposure to TB
• Involves quantiferon or T-spot

*These tests may have false negatives in immunocompromised patients (cannot mount proper immune response despite previous exposure) and cannot distinguish latent TB infection from overt clinical/active TB.

Answer 22

Solid culture;

localised skin inflammation (Type IV delayed hypersensitivity);

previously primed T cells

Question 23

BIOMEDICAL PREVENTION
Prevention of overt clinical tuberculosis can be done by giving the Bacillus Calmette-Guerin (BCG) vaccine or treating the latent TB infection (after detection above):
• BCG is a live attenuated vaccine created from ________________, which does not protect against primary TB infection or reactivation against latent TB (main reasons for TB transmission → limited efficacy against transmission)
• Prevents ________, ______ , and also the prevention of ____________
• No added benefits for giving a booster jab (may even create more side effects)
• Limitations: different strains of BCG, exposure to environmental Mycobacteria (affects patient immune response to vaccination → blocking: no response; masking: reduced response), cannot administer to immunocompromised or pregnant patients (risk of disseminated disease)

Answer 23

Mycobacterium bovis;

disseminated TB and TB meningitis;

leprosy

Question 24

Since tuberculosis is a slowly replicating pathogen (and thus develops resistance easily), 4 drugs are given as a first-line combination therapy, with other drugs as second-line therapy:

First-line medications : __________________

Second-line medications

• Quinolones
• Injectables (kana, ami)
• Ethionamide/Prothionamide
• Cycloserine
• Para-amino salicylic acid (PAS)
• Linezolid
• Clofazamine

Answer 24

Rifampicin (R), Isoniazid (H), Ethambutol (E), Pyrazinamide (Z)

Question 25

[Drug resistance]
Adherence to treatment is vital to achieve the therapeutic drug concentrations for bactericidal activity (especially in the early phase to maintain killing activity in the continuation phase):
• Poor adherence may result in drug-resistant TB or treatment failure → ________________ is used for improving adherence and safety monitoring
• Resistance to rifampicin may arise from changes in the _________________ (chromosomal rpoB gene)
- MDR-TB: Resistant to rifampicin and isoniazid
- XDR-TB: Resistant to rifampicin and isoniazid + _____________________ → at least 4 different types of drugs

Extremely difficult to treat → patient ends up using less effective drugs + longer treatment regimen (__________ months) with chance of treatment failure

Answer 25

directly observed therapy (DOTs);

RNA polymerase target ;

quinolones (e.g. ciprofloxacin) and aminoglycosides (e.g. kanamycin);

12 – 18

Question 26

TB-infected cells are killed biphasically, as _____________ are killed in the early bactericidal phase (first 2 months), while others are killed in the late sterilising phase (next 4 months):
• All 4 first line drugs are used in the early phase, followed by only ________________
• ______________: used to treat latent TB infections (prevents reactivation) → short-term measure which does not prevent re-exposure (H for 6 months or HR for 3 months)
• Clinical active TB must be excluded before starting IPT to reduce risk of treatment failure and development of drug-resistant

Answer 26

rapid multipliers;

rifampicin and isoniazid;

Isoniazid preventive therapy

Question 27

Side effects of RIfampicin

• ______________ urine, sputum, sweat, tears
• Rash, pruritus
• Anorexia, nausea, abdominal pain, _____________
• Rare (important): ____, _________, ___________, ___________

Answer 27

Orange coloured;

hepatitis;

thrombocytopenia, liver failure, renal failure, agranulocytosis

Question 28

Side effects of Isoniazid

• Peripheral neuropathy (prevent with ____________)
• Hepatotoxicity (rarely fulminant liver failure)
• Hypersensitivity reactions, rash
• Thrombocytopenia, agranulocytosis
• Optic neuritis

Answer 28

pyridoxine

Question 29

Side effects of Pyrazinamide (Z)

• ________________ (leading to gout)
• Anorexia, nausea, hepatitis
• Anaemia, thrombocytopenia
• Rash

Answer 29

Hyperuricaemia;

Question 30

Ethambutol (E)

• _____________ (full ophthalmology review upon treatment commencement)
• Renal impairment (________)
• Anaemia, thrombocytopenia
• Anorexia, nausea, abdominal pain, hepatitis

Answer 30

Optic neuritis;

interstitial nephritis

Question 31

HIV & TUBERCULOSIS
There is a high prevalence of HIV in newly diagnosed TB cases in Sub-Saharan Africa (>50%):
• HIV infections cause severe immunocompromise (due to ________________), increasing the risk of disseminated TB infections
• HIV-negative patients with latent TB have 5 – 10% lifetime risk of active TB, while HIV-positive patients with latent TB have ________________

Due to the immunocompromised state, the signs and symptoms present are less likely to be classical (immune system too weak to mount a response):
• HIV-TB is more likely to be ____________ (dissemination) with ______________ (variable)
• Deficient/malfunctioning T cells cause negative _________________
• Smear microscopy (of sputum) is also less sensitive due to reduced load of __________________________

Answer 31

destruction of CD4+ T cells;

5 – 10% risk per year of active TB;

extrapulmonary;

non-specific X-ray changes;

tuberculin and quantiferon tests ;

bacilli per mL of sputum

Question 32

MANAGEMENT
There are many challenges in the management of HIV-TB patients due to their severely immunocompromised state:
• Starting the patients on antiretroviral therapy could cause a ________________________ (but ARVs must still be used to suppress HIV and clear TB → increases CD4+ count)
• Prevention of HIV-TB may be achieved by reduction of HIV transmission (methods of safe sex, counselling), earlier initiation of ARVs, treatment of latent TB (IPT), infection control (particularly in healthcare settings)

Challenges

• Drug interactions: Most ARVs are metabolised in the liver by CYP450 (especially CYP3A4) – rifampicin is a ________________ → reduces [ARV] → HIV treatment failure + drug resistance
• Use ____________ (less potent CYP3A4 inducer) at varying doses → equally efficacious (but more expensive)
• Overlapping toxicity: Between ARVs and anti-TB drugs
• Duration of treatment: Important for adherence

Answer 32

paradoxical immune response and severe inflammatory reaction;

potent CYP3A4 inducer;

rifabutin;

Question 33

NON-TUBERCULOUS MYCOBACTERIA (NTM)
Non-tuberculous Mycobacteria are mainly found in ___________________, and include all Mycobacteria species except ____________________
• Classified as rapid (grow in 7 days on agar), slow or intermediate growth, or based on colony morphology or pigmentation
o Slow growing NTMs: _____________________
o Rapid growing NTMs: _______________
• Cannot be transmitted between people (environmental)

CLINICAL MANIFESTATIONS
The spectrum of disease caused by NTMs is highly variable (some patients present with fibro-nodular lesions in the lung, while others have cavitatory lesions):
• All people diagnosed with HIV should be screened for TB and vice versa

Answer 33

water and soil (environmental pathogens);

M. tuberculosis, M. bovis, M. africanum, M. microti, M. caprae, M. leprae:

M. avium-intracellulare complex (MAC);

M. fortuitum complex;

Question 34

Which species of NTMs cause chronic bronchopulmonary disease?

Answer 34

M.avium, M Kansasii, M, abscessus

Question 35

Which species of NTMs cause lymphadenitis (especially childeren)- cervical, other LN

Answer 35

M avium complex

Question 36

Which species of NTMs cause skin and soft tissue?

Answer 36

M. fortuitum, M. Chelonae, M abscessus, M marinum, M. ulcerans

Question 37

Which species of NTMs cause skeletal infections (bone, joint, tendon)

Answer 37

M. fortuitum, M. Chelonae, M abscessus, M marinum, M. ulcerans

Question 38

Which species of NTMs cause catheter related infections?

Answer 38

M. fortuitum, M. Chelonae, M abscessus

Question 39

Which species of NTMs cause disseminated disease (immunocompromised)

• HIV positive
• HIV negative
• M. marinum has been implicated in infections from ______________ and cause ulcers and possibly deep infections.

Answer 39

• HIV positive: MAC, M, Kansasii, M genavense, M haeomophilum, M.xenopi
• HIV negative: M abscessus, M chelonae

spas, whirlpools and fish tanks,

Question 40

DISSEMINATED MAC INFECTIONS
Disseminated MAC occurs in immunocompromised individuals (e.g. inherited defects in IFN-γ signalling pathway, HIV infections):
• MAC is normally acquired by ________________ (80 – 90% in HIV patients)
• Local invasion then entry into macrophages and other reticuloendothelial cells, allowing migration via blood to distant organs (e.g. liver, spleen, BM)

Histopathology
• Adenopathy
• Thickening of bowel mucosa
• GI haemorrhage (sometimes)
• Poorly formed abscesses
• Poorly formed granulomas
• Caseating necrosis (rare) 

Clinical presentation
• Fever, night sweats
• ____________ (important in considering MAC), weight loss
• Abdominal pain, diarrhoea, hepatosplenomegaly, intra-abdominal lymphadenopathy, raised serum ALP
• Pulmonary disease

Diagnosis
- Culture (blood, tissue etc.) → slow grower (takes 10 – 21 days on solid media) so start __________________

Treatment

• Combination therapy (__+_____+________) + address immune function (e.g. give ARVs for HIV patients)
• Duration is prolonged due to slow growth (further prolonged in disseminated MAC)

Answer 40

inhalation or ingestion;

Anaemia;

empiric therapy ;

macrolide + rifamycin + ethambutol