1.18 Mycobacterium

MYCOBACTERIUM

TRANS NO.

Summary: Manual , Lecture (Dr. Santos) Date of lecture: September 22, 2012 Transcriptionist: Xanne

MYCOBACTERIUM

Group IV

rod-shaped aerobic bacilli neither Gram-positive or negative acid fast  due to mycolic acid do not stain readily  once stained  resistant to decolorization with acid or alcohol  ACID FAST - decolorizer is acid alcohol (95% ethyl alcohol + 3% HCl) - Cell wall: waxy, hydrophobic and high lipid contents 60% of dry weight - Adjuvant properties responsible for development of delayed type hypersensitivity - ALL mycobacterial pathogens are intracellular pathogens  major pathogens: o M. tuberculosis (Koch’s bacillus) o M. leprae (Hansen’s bacillus) o M. bovis Potentially pathogenic: o M. avium complex o M. kasasii o M. fortuitum  MOTT (Mycobacterium Other Than Tuberculosis) - Anonymous, atypical, unclassified Mycobacteria, NMT (Non-tuberculose Mycobacteria)  Ranyoun Classification Representative Growth Pigment Niacin Species TB M. tuberculosis slow Pigmented (+) Complex M. africanum M. bovis -

Group I

M. asiaticum M. kasasii M. simiae M. marinum

Slow

Photochromogens (1)

(-)

Group II

M. flavescens M. scrofulaceum M. gordonae M. szulgai

Slow

Scotochromogens (2)

(-)

Group III

M. avium M.haemophilium M. ulcerans M. celatum M. gastri M. xenopi

Slow

Nonchromogens (3)

(-)

(1) (2) (3) (4)

M. fortuitum M. chelonei M. smegmatis M. flavescens

Rapid (4)

(-)

Pigment formed in the presence of light only Pigment formed in the presence or absence of light Non-pigmented Growth < 1 week

The Global Emergency     

8-12M new infections per year 2-3M people die from TB per year Emergence of MDR M. Tuberculosis th Philippines is the 9 out of the 22 highly burdened countries th Philippines is the 8 out of the 25 high MDRTB cases Mycobacterium tuberculosis

Morphology - acid fast bacilli - non-motile, non-sporogenous, non-encapsulated - arranged singly or in groups - long, slender rods, beaded - much granules in gram’s staining - easily destroyed by pasteurization - Cord formation when obtained in liquid culture medium - Requires glycerolated agar or potato medium - Stained by acid-fast stains (Ziehl-Neelsen, Kinyoun) or flourochrome stain - Culture: Non-pigmented, rough, irregular, wrinkled, may show cauliflower-like appearance Physiology - obligate aerobes - growth enhanced by O2 tension - Lowenstein-Jensen or BACTEC - Small, dry, scaly colonies with corrugated surfaces - champion slow grower o generation time: 13 – 15 hours o doubling time: 18 hours o 9 – 8 weeks before reported as negative culture

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resistant to drying  survive in the environment for long periods of time one of the most resistant among non-sporeformers may result in familial infection

Constituents of the Cell Wall A. Lipids a. Mycolic acid b. Waxes c. Phosphatides o mycolic acid + muramyl dipeptide (from peptidoglycan)  granuloma formation o phospholipids  induce caseation necrosis B. Proteins a. elicit the tuberculin reaction b. elicit formation of antibodies C. Polysaccharides a. induce immediate type of hypersensitivity b. serve as antigens in reaction with sera of infected persons Virulence Factors - NO toxins A. Cord Factor o trehalose-6,6’-dimycolate o responsible for “serpentine cord” formation (bacilli arranged in parallel chains) o inhibits migration of PMNs o elicits granuloma formation o serve as immunologic adjuvant B. Sulfatides o synergistically potentiate toxicity of cord factor o promote survival of bacilli inside macrophages C. Antibacterial resistance Pathogenicity and Pathogenesis - among the top 10 disease killers in the Philippines - predisposing factors: o chronic fatigue o sedentary life o malnutrition o poor housing conditions o occupational Modes of Transmission - inhalation of droplet nuclei (coughing or sneezing) o 1 org enough to establish infection - ingestion of unpasteurized milk or milk products - kissing

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fomites skin contact

Pathogenesis  Inhalation of droplet nuclei -> deposit in alveolar space of lungs -> engulfed by macrophages 

Portion: resist intracellular destruction -> persist -> multiply and kill the macrophages stimulate and inflammatory focus -> mature into a granulomatous lesion (tubercle) -> CASEOUS NECROSIS -> erosion of tubercle into an adjacent airway -> cavitation -> release of massive numbers of bacilli into the sputum

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Resistant host: tubercle -> calcified Early infection: o Spread distally o Indirectly thru lymphatics -> hilar or mediastinal LN o Directly into the circulation by erosion into pulmonary vessel *Infection with the tubercle bacillus usually involves the lungs, but any area of the body can be involved.

Principal Types of Lesions A. Exudative Type o consists of inflammatory reaction, PMN leukocytes & later monocytes around the tubercle bacilli o particularly seen in lung tissue o may be absorbed or lead to massive necrosis of tissue or may develop into productive type o Tuberculin test (+) B. Productive Type o chronic granuloma o consists of 3 zones a. central area of multinucleated giant cells containing tubercle bacilli b. midzone of epithelioid cells c. peripheral zone of fibroblasts, lymphocytes & monocytes o peripheral zone develops fibrous tissue o central area is site of caseation necrosis  tubercle  break into bronchus & empty contents  cavitation o healing by fibrosis or cavitation o the production, development, and healing or progression are determined by:  number of bacilli in the inoculum  resistance & hypersensitivity of the host

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Extrapulmonary Tuberculosis - 42% - lymphatics - 21% - pleural - 11% - bone / joint - 10% - others 7% - genitourinary 5% - meningeal 4% - peritoneal Once infected by the tubercle bacillus, one of the two things will occur: 1. Latently Infection o the individual’s immune system contains the spread of infection 2. Active Tuberculosis o the individual’s immune system is unable to contain the infection

Individuals or Groups at Risk of Tuberculosis 1. Close contacts of persons or known suspected to have TB 2. People born outside of the US, from areas that have a high incidence of TB 3. Residents & employees of high-risk congregate settings 4. Health care workers who serve high-risk clients 5. Some medically underserved, low income populations, defined locally as having an increased prevalence of TB 6. Infants, children, & adolescents exposed to adults in high-risk categories 7. Persons who inject illicit drugs & other locally identified high-risk substance abusers like crack cocaine users Individuals with Higher Probability of Progressing to Active Disease 1. Persons with HIV infection 2. Persons who were recently infected with M. tuberculosis (within the past two years), particularly infants and very young children 3. Persons who have certain medical conditions known to increase the risk of disease if infection occurs 4. Persons who inject illicit drugs and other locally identified high-risk substance abusers like crack cocaine users 5. Persons with history of inadequately treated TB Primary Infection st - 1 contact with tubercle bacilli - acute exudative lesion develops  lymphatics & regional lymph nodes  often heals rapidly

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lymph node undergoes massive caseation  calcifies (+) Tuberculin test most often involve the base of the lung

Reactivation - tubercle bacilli survive the primary lesion - characterized by chronic tissue lesions, tubercle formation, caseation & fibrosis - regional lymph nodes are only slightly involved without caseation - located at the apex of the lung Immunity 1. Humoral Immunity o antibodies  non-protective 2. Cellular Immunity o activated CD4 & TH1  release large amounts of gamma interferon  activate macrophage  destroy intracellular bacilli o growth and spread of tubercle bacilli is interfered with 3. BCG Vaccination o no absolute immunity, only relative immunity o there is less incidence or tuberculosis among BCG vaccinated individuals Tuberculin Test 1. Antigens a. Old Tuberculin (OT) b. Purified Protein Derivative (PPD) 2. Older Antigens a. Old Filtrate (BF) b. Bacillary Emulsion (BE) c. Tuberculin Residuum (TR) d. Synthetic Old Tuberculin Trichloroacetic Precipitate (SOTT) 3. Methods a. Mantoux – intradermal b. Tine – disposable kit c. Koch – subcutaneous d. Volmer – patch e. Von Pirquet – cutaneous f. Moro – percutaneous Reactions to Tuberculin - skin test must be read in 24 – 72 hours - (+) skin test  induration of > 10 mm, erythema & edema - (+) test persists for several days - (+) test does not indicate active disease or immunity to the disease

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within 4 – 6 weeks after infection  (+) test after BCG vaccination  (+) test that lasts for 3 – 7 years false negative results o general anergy o Hodgkin’s disease o sarcoidosis o AIDS o immunosupression

Clinical Findings - afternoon rises in body temperature - fatigue - weakness - loss of appetite - weight loss - chronic cough of more than 2 weeks with or without hemoptysis  usually associated with far-advanced TB Diagnosis - specimens o fresh sputum o gastric washings o urine o CSF o blood o biopsy material Specimen Selection & Collection - volume - number of specimens o one specimen per day for 3 – 5 days - timing o early morning specimens Culture 1. Lowenstein Jensen medium o egg-based 2. Middlebrook medium o agar-based or broth o

*Incubate at 35 – 37 C in 5 – 10% CO2 for up to 8 weeks Staining 1. Acid Fast Stain Two Methods a. Ziehl Neelsen method  hot method  conterstain  methylene blue b. Kinyoun method

 cold method  counterstain  malachite green 2.

Fluorochrome Stain

Biochemical Tests - Niacin test: (+) - Nitrate reduction: (+) - Catalase test: ( - ) PCR (Polymerase Chain Reaction) - 55 – 90% sensitive - 99% specific Antibiotic Susceptibility Testing - very important due to increasing number of multi-drug resistant strains Other Tests - gas chromatography - mycodot – rapid method o 70% sensitivity o 96% specificity - nucleic acid hybridization o excellent specificity & sensitivity o very expensive o requires technical expertise Treatment st - 1 Line Anti-TB Drugs o Isoniazid, Rifampicin, Ethambutol, Pyrazinamide o used for 6 months to 1 year o DOTS (Direct Observation Therapy Shortterm)  useful to ensure patient compliance -

nd

2 Line Anti-TB Drugs o Cycloserine, Ethionamide, Paraaminosalicylic acid, Vancomycin, Capreomycin, Fluoroquinolones, Kanamycin, Rifambutin o Streptomycin  not used anymore because of CN VIII deafness

Drug-Resistant Tuberculosis - in the 1900’s, multi-drug resistant tuberculosis (MDRTB), defined as resistant to Isoniazid & Rifampicin began to emerge both in the US & worldwide - today  400,000 cases of MDR-TB globally - outbreak of extensively drug-resistant tuberculosis (XDR-TB)  MDR-TB with additional resistance of

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Fluroquinolones & at least one of the following injectibles: Capreomycin, Amikacin, or Kanamycin in South Africa & in over 40 countries estimated nearly 30,000 XDR-TB cases globally

Why is there so much drug-resistant tuberculosis? Drug resistance is created when patients with tuberculosis are not treated adequately either because: a. The patient does not take the drug treatment regimen as prescribed (nonadherent) b. The treating physician or program does not treat the patient with the appropriate drug regimen Treatment Regimen - patients with drug susceptible TB  four-drug regimen st that is administered for 6 months  1 line drugs Isoniazid, Rifampin, Ethambutol & Pyrazinamide st - after 1 2 months  D/C Pyrazinamide & Ethambutol  continue Isoniazid & Rifampicin for another 4 months to complete the 6-month regimen - medications can be administered daily, twice a week, or three times a week, with similar results - the expected outcomes with this treatment regimen are excellent with few treatment failures & relapse rate of only 2 – 3%

Mycobacterium leprae -

acid-fast bacilli singly, parallel bundles, or globular masses regularly found in scrapings from skin or mucous membranes (nasal septum) of leper

LEPROSY (Hansen’s disease) - incubation period is short (few days) up to 40 years - onset is insidious - involves the cooler tissue of the body: o skin o superficial nerves o nose o pharynx o larynx o eyes o testicles - mode of transmission is through prolonged contact and exposure Skin Lesions - pale, anesthetic macular lesions (1 – 10 cm in diameter)

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discrete erythematous, infiltrated nodules 1 – 5 cm in diameter diffuse skin infiltration

Neurologic Disturbances - nerve infiltration and thickening  anesthesia, neuritis, paresthesia, trophic ulcers, bone - resorption and shortening of digits - disfigurement (leonine facies) due to skin infiltration and nerve involvement - saddle nose – due to bone resorption

Two Major Types Features

Tuberculoid Leprosy

Lepromatous Leprosy

Lesion

macular, hyperpigmentated

erythematous, infiltrated nodules, diffuse

Amount of bacteria

paucibacillary

multibacillary

Patchy anesthesia

localized

diffuse

Disfigurement

absent

present

Lepromin test

positive

negative

Appearance of Disease            

Lepromatous Deficient CMI Progressive disease Nodular skin lesions Abundant bacilli in lesions Symmetric nerve involvement Slow onset IL-4 and IL-10 Skin is infiltrated with suppressor T cells Ab levels are high Malignant Continuous bacteremia Negative lepromin, extract of lepromatous tissue, skin test

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Tuberculoid Intact CMI Non-progressive disease Macular skin lesions Few bacilli in lesions

 Severe asymmetric nerve involvement  Sudden onset  IL2 IFN gamma and IL12  Skin infiltrated with Th1 cells  Benign  Positive lepromin skin test

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Diagnosis / Clinical Findings - acid fast staining of skin lesions or nasal scrapings - Leromin test for tuberculoid leprosy o Fernandez reaction – early reaction o Mitsuda reaction – late reaction - no serologic test is useful - cannot be grown in any artificial laboratory medium Treatment - Dapsone (Diaminophenylsulfone) o mainstay of therapy - Dapsone + Rifampicin + Clofazimine o drug combination is used in case of an emerging drug resistance - 2 years duration Prevention - isolation of patients - for exposed children and household members  chemoprophylaxis  Dapsone

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