Mycobacterium

• Briefly describe Mycobacteria
  • Mycobacteria belong to the Order Actinomycetales, Family Mycobacteriaceae, and Genus Mycobacterium. There are more than 80 species of mycobacteria, the two major pathogens being:
    • Mycobacterium tuberculosis (Koch, 1882)
    • Mycobacterium leprae (Hansen, 1874).
  • The rest are environmental organisms collectively known as Non-tuberculous mycobacteria (NTM). NTM are responsible for opportunistic infections especially in immunosuppressed patients.
• List the components of the mycobacterium cell wall from inside outward
  • Peptidoglycan layer – rigidity and shape
  • Arabinogalactan layer
  • Mycolic acid layer
  • Mycosides – phenolic glycolipid 1 is the major component here
• List the characteristics of Mycobacteria
  • Mycobacteria are slim, rod-like, obligate anaerobes that are non-motile, unencapsulated, and non-sporing.
  • They are sensitive to UV light, sunlight, and heat at 60-80*C.
  • They have a modified gram positive cell wall that makes them hardy, impenetrable and hydrophobic.
  • Human strains show heavy growth on culture (eugonic) while bovine strains show poor growth on glycerol containing media (dysgonic).
  • Mycobacteria are acid fast, they resist decolourisation with acid and alcohol once stained with arylmethane dyes. All are intracellular pathogens and the primary host cell infected in humans are macrophages.
  • The mycobacterium cell wall is unique as it is waxy, hydrophobic and has a high lipid content (upto 60% of the dry weight is mycolic acid, a long chain branched fatty acids)
  • Mycolic acids and short chain fatty acids form a pseudo outer membrane that has unusual staining characteristics.
  • Cord factor (Trehalose dimycolate) is essential for growth and adherance, and it is correlated with virulence (virulent strains grow in a characteristic **“serpentine”**cordlike pattern, avirulent strains lack cord factor)
  • Wax-D (glycolipids) have adjuvant properties (in vaccines) and phosphatides play a role in caseation necrosis.
  • It also has several proteins (antigens in the purified protein derivative, PPD), which when combined with waxes produce a delayed type hypersensitivity reaction in PPD.
  • The mycobacterium cell wall is resistant to alkalis, acids and most disinfectants. The lipid-rich cell wall is responsible for: Acid-fastness, slow growth, resistance to detergents, resistance to common antibacterial antibiotics, antigenicity, and host immune response.
  • Mycobacterial disease process is largely due to reaction to mycobacterial lipids (cord factor, wax-D) Mycobacteria do not produce endotoxins, or exotoxins and instead elicit a granulomatous response that has a chronic course.
• Briefly describe Mycobacterium tuberculosis (MTB)
  • Mycobacterium tuberculosis is an obligate aerobe (MTB – add CO2, M.bovis – reduce O2 tension), slow growing (divides every 18-24 hours, compared to E.coli which divides every 30min), sensitive to heat (pasteurization) and UV light.
  • It is susceptible to alcohol, formaldehyde and glutaraldehyde.
  • However, it is resistant to dehydration (survives in dried expectorated sputum), and alkalis (NaOH is used to concentrate clinical specimens, destroy unwanted bacteria, human cell, and mucus but not the organism), acids and quartenary ammonium compounds
  • Does not grow below 25 C or above 40 C
• Describe the pathophysiology of Tuberculosis
  • Primary infection
    • Inhaled bacilli settle in the terminal airways
    • Alveolar macrophages and dendritic cells engulf the bacteria
    • Evasion of immune system: inhibition of phagolysosome fusion via exported repetitive protein
    • Bacilli multiply rapidly, rupture and are released to infect more macrophages
    • Circulating macrophages are attracted to the site to engulf MTB cells
    • Infected macrophages spread MTB to Bone Marrow, spleen, kidneys and CNS
    • Cell-Mediated Immunity (adaptive) sets in after 2-3 weeks
    • Caseating Granulomas form: Antigen presentation → IFN-y, IL-12 Th1 response, TNF-a, PDGF, TGF-B, FGF → neutrophil and phagocyte chemoattraction → transformation of macrophages to epitheloid cells → fusion of macrophages to form multinucleate giant cells → peripheral lymphocytes surround tubercles
    • Mycobacterium tuberculosis infects the lungs (pulmonary tuberculosis)
  • Localization in the lungs
    • Ghon focus: Primary granulomatous reaction in the lung (primary complex for bovis is tonsils, or cervical LNs, intestinal LNs, or Mesenteric LNs)
    • Ghon complex: Ghon focus + hilar lymph node due to dissemination from primary site
    • Tubercles: Granulomas that heal but contain viable MTB in calcified areas (Non-incoming O2 makes the organism enter a dormant state)
  • Spread of disease (disseminated TB)
    • Local spread through macrophages to surrounding tissue
    • Lymphatic spread to LNs in pharynx, bronchi, GI or regional LNs
    • Hematogenous spread, causing seeding and development of distant infections (meningitis, pleurisy, renal disease, spine disease, Miliary TB)
    • By natural passage: lungs to pleura, transbronchial to adjacent segments, TB salpingitis to peritoneum, sputum in larynx swallowed into GIT, renal lesions to ureter and bladder
  • Progressive primary TB
    • Develops directly from primary lesions without latent phase
    • Occurs in immunocompromised and young children, usually in disseminated disease
  • Post-primary TB
    • Ghon complex resolves in many leaving dormant foci bacilli (Evidence of infection – tuberculin reactivity)
    • Exogenous re-infection occurs in enedmic areas
    • Reactivation of dormant bacilli occurs spontaneously or during intercurrent illness, or immunosuppression. At risk individuals include old age, degenerative disease, immunosuppressive conditions or therapy, alcoholism, malnutrition etc. Often occurs in the upper lobes of the lungs + other well oxygenated sites (Kidney, brain, bone). Granuloma formation recurs with tissue destruction as proteases from activated macrophages casue softening and liquefaction (caseation, tuberculoma). TNF (cachectin) and other cytokines cause fever and wasting. Continuous tissue destruction leads to leakage of caseous material into the bronchi with cavity formation (bacilli thrive within the walls of these cavities, caseous centres do not favor proliferation) 20% of cavitating lesions resolve without treatment. Once cavity is formed, easy access to spurum by the bacilli facilitates spread (open/infectious case)
    • Post primary cutaneous TB (Lupus vulgaris) is a chronic condition with scarring and deformity.
    • Sclofuroderma: Secondary to sinus formation between TB lymph nodes and skin
    • Spread of bacilli from cavities
      • Through bronchus to other parts (lung, trachea, larynx)
      • Swallowed (if unable to cough IE. children – intestinal tract ulcers and fistulas)
      • Hematogenously (Kidney, adrenal gland, bone marrow, spleen)
      • Spread through lymphatics is unusual

In a tabular format, list the difference between primary and post-primary TB lesions

• Describe the relationship between Mycobacterium tuberculosis and HIV infections
  • Most cases are due to reactivation rather than reinfection, and can occur at any stage.
  • Pathology and clinical manifestations are dependent on the disease stage
  • Alterations linked to immunosuppression (AIDS)
    • Low CD4 count and atypical TB on CXR
    • Negative PPD and more smear negative cases
    • Lack of granuloma formation
    • More cases of disseminated TB and more mortality
    • Fewer cases of lung cavitations
• Describe the Laboratory features of Mycobacterium Tuberculosis
  • Definitive diagnosis: Demonstration of MTB in a clinical specimen
  • Specimen: Sputum (Induced in children), Gastric washing, Pleural Biopsy, Broncho-alveolar washing (Fiber-optic bronchoscopy), Biopsy specimen of lungs and other anatomical sites, Aspirates (Pleural, Pericardial, Peritoneal), CSF, Urine. Describe their collection frequency, indications, and techniques
  • Smear Microscopy
  • Molecular Techniques
    • Loop-mediated isothermal amplification for the detection of MTB (TB-LAMP
  • Culture Diagnosis
    • Gold standard TB diagnosis. Requires incubation for 6-8 weeks before declared negative. Requires processing of sputum prior (Digestion of mucus, decontamination f normal flora, concentration by centrifugation – look up the methods and reagents, also how would you differentiate MTB from Mbovis on culture
    • Conditions: 35-37*C, Aerobic conditions
    • Solid Culture:
      • Lowenstein Jensen (Egg yolk, Potato starch, Animal serum, Citrate, Glycerol): Eugonic dry bread crumbs heaped up on each other, earliest colonies 2 weeks to grow, 4-8 weeks for usable colonies
      • Middlebrook agar 7H 10 or 7H11(7H10, 7H11 – agar based):
    • Liquid Media:
      • Middlebrook 7H 12
    • Semi-automated Liquid Culture System:
      • BACTEC 460 (Can be used for drug susceptibility testing, Faster growth detection, Higher sensitivity than solid media) – Middlebrook broth, Fluorescent sensor, Positive 4-14 days, 8 weeks for negative cultures, Requested if microscopy or Xpert is negative but picture show TB and patient is seropositive (HIV)
    • Automated Liquid Culture System:
      • MGIT – Mycobacterial Growth Indicatory Tube
    • Confirm MTB culture
      • Slow Growth
      • Morphology: rough, thick, wrinkled, Irregular margin, faintly-buff coloured, Canetti has smooth white and glossy colonies
      • Catalase positive: at 68*C
      • Nitrate reductase positive
      • Niacin test Positive
    • Advantages of culture
      • Detects small numbers of organisms (as few as 10 bacilli)
      • Confirms diagnosis of TB in HIV+ patients
      • Allow species Id
      • Allows drug susceptibility testing and Resistance surveillance
      • Allows epidemiological studies
    • Limitations of TB culture
      • Slow growth of MTB (Long turnaround time)
      • Expensive, Limited number of labs
      • Essential needs (Biosafety, Adequate infrastructure, Qualified staff, Equipments, Continuous supply of media and reagents, Transport systems, Quality management)
  • Immunological tests
    • Tuberculin Skin Test: Does not distinguish vaccination and disease. Negative in patients with advanced AIDS. Look up Principles, Causes of false positives and negatives
    • QuantiFERON, T-SPOT TB: Detects IFN-y. Used for active and latent TB but cannot differentiate between the two.
    • Lateral Flow urine Lipoarabinomannan assay (LF-LAM): diagnosis and screening of active TB in patient with HIV
    • Loop-mediated
  • Drug Sensitivity Tests
    • Conventional methods – Resistance ratio, Absolute concentration, Proportion method
    • Molecular method: Identify katG & inhA genes (INH), rpoB gene (Rifampicin), embA and embB (For ethambutol) gene mutations
    • Microscopic Observation Drug Susceptibility Assays (MODS): Culture using plate, incorporate drugs, use microscope
    • Disadvantages of conventional DST
      • Slow, generally takes 4-6 weeks for results
      • Delays diagnosis of MDR-TB (Increased mortality, Patients improperly treated until results are ready, Further resistance can occur, Patient continues spreading resistant strains)
• Briefly describe the demonstration of Mycobacterium Tuberculosis infection
  • Specimen: Sputum, Gastric Lavage (Children), Pus, Bronchoscopy with Bronchoalveolar lavage, Biopsy
  • Microscopy
    • AAFB (Z-N) – flood with carbolfusschin, heat, destain with 3% acid alcohol, counterstain with malachite green or giemsa
    • Auramine-rhodamine stain (Fluorescence Microscopy)
  • Culture: Rough, thick, wrinkled, irregular margin, faintly-buff coloured
    • Solid culture: Lowenstein Jensen agar (6 WEEKS), Middlebrook Agar 7H10 or 7H11
    • Liquid media: Middle brook 7H 12
    • Semi-automated Liquid Culture system: BACTEC 460 (7-10 day culture)
    • Automated Liquid Culture System: Mycobacterial Growth Indicatory Tube (MGIT)
  • Biochemical: Catalase positive at 68*C, Nitrate reductase positive, Niacin test positive
  • Immunological tests: Lateral Flow Urine Lipoarabinomannan assay (LF-LAM), Immunochromatographic tests, ELISA tuberculosis (IgA antibody to MTP Kp90 immuno-cross reactive antigen compun ImCRAC)
  • Molecular Techniques: NAA (DNA/RNA) – PCR GeneXpert (6 HOURS)
• Briefly describe the treatment MTB infection
  • Anti-TB drugs: for drug sensitive new patients, relapse, previous failures return after default depending on DST results
    • First-line
      • Rifampicin
      • Isoniazid
      • Pyrazinamide
      • Ethambutol
    • Second-line
      • Para-aminosalicylic acid
      • Cycloserine
      • Capreomycin
      • Ethionamide
      • FQs: Ciprofloxacin, Levofloxacin, Oxafloxacin
      • AGs: Amikacin, Kanamycin
    • Regimen
    • Directly Observed Therapy (DOT): supervised to improve adherence
    • Treatment for at least 6 months ensures sterilization of the lesion
    • Treatemnt can be upto 9 months in severe TB

• Classify drug resistant Tuberculosis (TB)
  • Monoresistant: Resistant to one first-line anti-TB drug only
  • Polydrug resistant (PDR TB): Resistance to more than one first-line anti-TB drug other than both INH and Rifampicin
  • Rifampicin Resistant TB (RR TB):
    • RR TB without MDR TB
      • Resistant to Rifampicin, with or without resistance to other RIG; Injectables or Quinolones (RR TB without MDR TB).
    • All RR TB
      • Includes any resistance to Rifampicin whether monoresistant, MDR, or polydrug resistant or extensive drug resistance )AA RR TB)
  • Multidrug Resistant TB (MDR TB): Resistance to at least both INH and Rifampicin
• Briefly describe Immune reconstitution inflammatory syndrome (IRIS)
  • Exacerbation of symptoms in AIDS patients with TB due to HAART
  • HAART increases CD4 cells → Increased inflammatory response
  • Prevention: Treat underlying infection before starting HAART
• Briefly describe the prevention of MTB infection
  • Prompt detection of cases and effective treatment
  • Isolation of cases on treatment until non-infections
  • Follow up contacts of cases
  • Reducing overcrowding
  • INH prophylaxis – look up indications and duration
  • Vaccination with BCG
    • A Live attenuated vaccine derived from M. bovis. Given to infants and children in high-prevalence areas, results in 60-80% reduced incidence. Does not prevent infection, reduces risk for disseminated disease in children, does little to prevent adult disease.
    • Contraindication: HIV infected, may cause disseminated BCG infection in immunosuppressed patient
    • Efficacy wanes over a number of years

Regarding Mycobacterium leprae, describe the following:

• Modes of transmission
  • Prolonged skin-to-skin contact
  • Contact with respiratory secretions or wound exudates
• Portals of entry and exit
  • Skin
  • URT-nasal mucosa
• Average incubation period
  • 3-5 years, can be upto 30 years
  • Peak onset in young adults
• Antigenic structure
  • Peptidoglycan: Rigidity and shape, Innermost
  • Lipoarabinomannan-B (LAM-B) – Major antigen, Induces Ab
  • Mycolic acid – LCFA, 3rd layer
  • Mycoside: Phenolic Glycolypid (PGL-1) is major component, outermost layer
  • Acid-fast bacilli with a dense lipid capsule
  • Very slow doubling time: 12- 13 days
  • Obligate intracellular organism
• Virulence factors
  • Phenolic Glycolipid 1 (PGL-1): Specific to Mycobacterium leprae, Binds C3 → Phagocytosis → Removes OH- and superoxide anions to prevent oxidative killing
  • Intracellular location: Resistance to killing by macrophages
  • Lipoarabinomannan: inflammation
• Briefly describe the pathogenesis of leprosy
  • The pathogen once inoculated into the human attacks the peripheral nerves
  • It binds to the axon of the Schwann cells which are the principle target cells
  • Leads to demyelination of the nerves and therefore loss of axonal conductance
  • This results in deformity and loss of pain, temperature and touch sensations
  • Infiltration of the skin by bacilli causes pigmentary changes
  • Destruction of the nasal bones causes collapse, and large numbers of bacilli are discharged through nasal secretions
  • Additional damage is caused by immune responses
• Briefly describe the Ridley-Jopling classification of leprosy
  • Hyper-reactive tuberculoid (TT)
  • Borderline tuberculoid (BT)
  • Mid-borderline (BB): most patients
  • Borderline lepromatous (BL)
  • Anergic lepromatous leprosy (LL)
• Briefly describe the WHO classification of leprosy
  • Based on number of lesions and presence of bacilli on skin smear
    1. Paucibacillary leprosy (PB): 5 or fewer lesions without any bacilli on skin smear. Include TT and BT.
    2. Multibacillary leprosy (MB): 6 or more lesions with bacilli on skin smear. Includes BB, BL and LL.