Mycobacteria Spp. PDF

Mycobacterium spp. General Characteristics: Slender, slightly curved or straight, rod-shaped Non-motile, non-spore forming EXCEPT M. marinum, difficult to stain with basic aniline dyes at room temperature but once stained difficult to decolorize due to MYCOLIC ACID (aka N-gly...

Mycobacterium spp. General Characteristics: Slender, slightly curved or straight, rod-shaped Non-motile, non-spore forming EXCEPT M. marinum, difficult to stain with basic aniline dyes at room temperature but once stained difficult to decolorize due to MYCOLIC ACID (aka N-glycolylmuramic acid = cell wall with extremely high lipid contents) It takes up dye with increased staining time or application of heat but resist decolorization with ethanol: ACID FAST. Strictly aerobic but increased CO2 concentration will enhance the growth of some species. Pathogenic mycobacteria grow more slowly (2-6 weeks) than most other species and requires WHOLE EGG for growth EXCEPT for M. leprae which fails to grow in vitro. Mycobacterium groups Mycobacterium tuberculosis complex MOTT (Runyon’s Classification) Mycobacterium leprae - Agents of Tuberculosis - according to pigment production; rapid growers Non-culturable - Slow growers Causative agent of Leprosy M. tuberculosis – TB in man M. bovis – TB in cattles; Intestinal TB in man; colonies appearaing WATER DROPLETS M. africanum – TB in man in Africa M. microti M. canetti Culture media for Mycobacterium Non-Selective Agar Based Selective Liquid Egg-based medium with malachite Clear media for easy examination of With antibiotics Non-conventional media and for green (6-10 weeks) colonies (10-12 days) easier detection Common disadvantage: PROLONGED TIME OF GROWTH Lowenstein Jensen Media Middlebrook 7H10 with dextrose Gruft Modified Lowenstein Jensen Bactec 12B - Commonly used Middlebrook 7H11 with casein Selective Middlebrook 7H!1 Bactec 13A hydrolysis Mitchinson Selective 7H11 Septi-chek Petragnani medium Mitchinson 7H11 Middlebrook 7H9 - For non-sterile spx. Dorset Egg media Wallenstein medium - Recommended for isolation of MAI complex Media Criteria: 1) It must always contain malachite green 2) It must always be egg-based or protein-based 3) Media must not be sterilized through autoclaving. Use of pasteurization or Arnold’s sterilization is important. For sputum samples in growing mycobacteria: o Perform DIGESTION-DECONTAMINATION PROCEDURE Purpose: To eliminate large debris from specimen that may contaminate and outgrow Mycobacteria Decontamination – to remove contaminants and normal flora Digestion – to liquify mucus to free any trapped microorganisms o Specimen that contains mucus require digestion-decontamination, while fluid specimen require decontamination only o For sputum: 2 SPUTUM SAMPLES EVERY MORINIG FOR 2 CONSECUTIVE DAYS (DOH) 1) N-ALC (Na-acetyl-L-cysteine) or dithotreitol (aka sputolysin) & 2% NaOH – gold standard; can be used for sample with Pseudomonas aeruginosa from patients with cystic fibrosis 2) NaOH (2%, 3%, or 4%) – commonly used 3) Zephiran (benzalkonium chloride) and Trisodium phosphate (Z-TSP) 4) 1% Cetylpyridium chloride – can prolong shelf life of sputum for 8 days 5) 5% Oxalic acid – for specimen that contains Pseudomonas aeruginosa Killing of Mycobacteria: 1) Boiling = 10 mins 3) Dried sputum = 6 – 8 months 2) Direct sunlight: 20 – 30 hours 4) 5% Phenol = 24 hours Culture + Sunlight = 2 hours Sputum + sunlight = 8 – 10 hours Identification Specimen: Expectorated sputum, CSF, gastric lavage, synovial fluid, pleural fluid, biopsy material, urine Rules in specimen processing Specimen from sterile body sites can be concentrated by centrifugation and inoculated Non-sterile must be decontaminated and concentrated 1) Acid Fast Staining – screening test for Mycobacteria spp. Purpose Ziehl-Neelsen (Hot Method) Modified Kinyoun’s (Cold Method) Primary Stain Carbol Fuchsin Mordant Heat Tergitol Decolorizer Acid Alcohol (3% HCl in 95% ETOH) Acid Alcohol (1% Sulfuric acid in 70% ETOH) Counterstain Methylene blue Result (+) Red bacilli in a blue background (-) Blue bacilli in a blue background Important note: DSSM – Direct Sputum Smear Microscopy Smear size: 2 x 3 cm Examination of 300 fields before reporting NEGATIVE RESULT Drying of smear prior to heat fixation = to prevent aerosol 2) Fluorochrome Staining – for better visualization Primary Stain Auramine rhodamine / TRUANT (+) Result: Yellow fluoroscence Mordant - Decolorizer 0.5% Acid alcohol Counterstain 0.5% potassium manganate Other stains: Fite Faraco – for staining tissues obtained from biopsies Spergler’s – AFS for color blinded person (+) result: BLACK BACILLI Manner of Reporting (compiled) Mahon and RITM/DOH Fluorochrome Fluorochrome National Report CDC (x1000) (x1000) (x450) (x250) Tuberculosis Association 0 0 0 0 0 No AFB seen in 300 visual fields 1-2/300 fields 1-9/100 fields 1-2/70 fields 1-2/30 fields 1-2/slide Doubtful / n+ 1-9/100 fields 10-99/100 field 1-2/70 fields 1-9/10 fields 3-9/slide 1+ 1-9/10 fields 1-10/OIF/50 2-18/50 fields 1-9/field >10/slide 2+ fields 1-9/field >10/OIF/20 4-36/field 10-90/field >1/OIF 3+ fields >9/fields >36/field >90/fied 4+ 3) Pigment Production – for MOTT 5) Nitrate Reduction Test = MTB (+) 4) Niacin Test = MTB (+) Media: Sodium Nitrate Broth Reagent: Cyanogen bromide + Niacin + Aniline Dye Reagents: Sulfanilic acid + N-napthylethylene diamine Media: Lowenstein Jensen (+) PINK/RED (-) NONE Principle: All Mycobacterium spp. produce enzyme to convert niacin to If negative, confirm using ZINC DUST to detect unreduced nitrate niacin ribonucleotide EXCEPT MTB. (NO3) 6) Catalase and Heat Stable Catalase = MTB complex (-) M. kansasii 10) Pyrazinamidase Test = MTB, M. marinum (+) vs. M. bovis, M. (+) kansasii (-) Medium: Tween 80 Agar Reagent: Ferrous ammonium sulfate Reagent: 30% H2O2 (+) RED (-) YELLOW (+) result: >45mm height of effervescence 11) Tellurite Reduction Test = MAI Complex (+) vs. M. kansasii (-) 7) Iron Uptake Test = Rapid growers EXCEPT M. chelonae (+) BLACK (-) NO REACTION Reagent: Ferric ammonium citrate 12) Urease Test = M. scrofulaceum (+) vs. M. gordonae (-) 8) Arylsulfatase Test = M. fortuitum and M. chelonae (+) from M. phlei Media: Urease (-) Indicator: Phenol Red Principle: Tripotassium phenolphthalein acted upon by arysulfatase (+) RED (-) YELLOW converted to free phenolphthalein Media: Phenolphthalein media Inhibitory Tests: Reagent: Sodium bicarbonate 13) NaCl tolerance test = M. triviale (+) vs. MTB (-) (+) PINK 14) TCH/T2H Susceptibility Test = M. bovis (S) vs. MTB (R) Ab used: Thiozene-2-carboxylic acid-hydrazide) 9) Tween 80 Hydrolysis Test = M. kansasii (+) for 6 hours to 3 days 15) Growth on Mac Conkey w/o Crystal Violet = M. fortuitum, M and MTB (+) in 10-20 days vs. M. avium (-) chelonei complex (+) Substrate: Tween 80 (polyxyethylenesorbitan monooleate) (+) PINK/RED (-) NONE Mycobacterium tuberculosis Agent of Tuberculosis Mimics other disease such as pneumonia, neoplasm, and fungal infections Common name: Koch’s Bacillus, Tubercle Bacillus, Captain of All Men of Death In culture media: “cauliflower-like” growth at 37-37C exhibiting cording (curved strands) for 8 weeks to 2 months. MUCH’S GRANULES = resistance to drying Virulence factors: 1) Cord Factor – destroys the mitochondria of host cells 2) Sulfatides – prevents phagocytosis Clinical Infection Primary Tuberculosis Pathogenesis: Airborne droplets → Alveoli → Phagocytosis by macrophages → Intracellular multiplication of bacteria → Secretion of IL-12 and TNF- a for T cells and NK cells recruitment and enhance of inflammation → Th1 differentiation and release of IFN-y → DESTROY INTRACELLULAR MYCOBACTERIA → Fibroid cells → Granuloma Important clinical picture: ↑ MONOCYTES, Granulomatous lesion seen on X-RAY If bacteria not eliminated, pathologic features are as follows in terms of antigen load and hypersensitivity reaction: 1) If there is little antigen and strong hypersensitivity reaction = HARD TUBERCLE or GRANULOMA (Calcification or Ghon’s complexes) 2) If both are high =TISSUE NECROSIS (due to enzymes of degenerating macrophages) Diagnosis: 1) Signs and symptoms 2) (+) PPD Skin Test = redness after 48 hours Different methods of PPD Skin Test 1) Tuberculin Test 2) Mantoux Test = (+) wheal reaction >10 mm = past infection 3) Mendel’s Test >5 mm but