Mycobacterium Flashcards

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1
Q

Mycobacteria General Characteristics

A
  • Acid fast
  • Aerobic
  • Non spore forming
  • Non motile
  • Have a high lipid cell wall content
  • Temperature Requirements
    • Most mycobacteria can be isolated at 36 o C with 7 10% CO 2
    • There are several species which require a significantly lower primary recovery temperature
    • 30 to 32 o C.
    • There are also some which require a higher temperature for recovery as well.
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2
Q

Group I Photochromogens

A
  • They are defined as colonies which develop a yellow orange pigment on exposure to the light of a 60 watt bulb placed 6 inches above the tube for 60 minutes. They take longer than 7 days to appear on solid media.
  • Members include:
    • M. kansasii , M. simiae , M. marinum , M. asiaticum
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3
Q

Group II Scotochromogens

A
  • They are defined as colonies that develop a yellow-orange pigment in the dark or light and take longer than 7 days to appear on solid media.
  • Members of this group include
    • M. gordonae
    • M. scrofulaceum
    • M. szulgai (at 37 o C)
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4
Q

Group III Nonphotochromogens

A
  • They are defined as colonies that are non-pigmented regardless of whether they are grown in the dark or light and take longer than 7 days to appear on solid media.
  • Members include:
    • M. avium complex
    • M. intracellulare
    • M. xenopi
    • M. ulcerans
    • M. malmoense
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5
Q

Group IV Rapid Growers

A
  • They are defined as colonies that grow on solid media in less than 7 days.
  • Mycobacteria forming colonies within 7 days are termed rapid growers, those requiring longer periods are termed, slow growers
  • Inoculate well isolated colony of organism to 7H9 broth containing Tween 80
  • Incubate broth for several days until medium faintly turbid
  • Dilute broth 1:100, streak inoculate to Middlebrook 7H10 agar plate. Observe cultures at 5 to 7 days and (if no growth) weekly thereafter for visible colonies
  • Members of this group include:
    • M. fortuitum
    • M. chelonae
    • M. peregrinum
    • M. abscessus
    • M. thermoresistibile
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6
Q

M. leprae General

A
  • Cause of Hansen’s Disease or Leprosy
  • Discovered by the Norwegian G. H. Armauer Hansen in 1873
  • 2 3 million cases worldwide
  • WHO listed 91 countries where it is endemic
  • In 2004 there were 407,791 new cases world wide
  • In 2015 the CDC reported 178 cases in the United States
  • There are 4 strains of M. leprae
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7
Q

M. leprae Clinical Picture

A
  • Causes severe, disfiguring skin sores and nerve damage in the arms, legs and skin areas around the body.
  • It can also attack the eyes and the thin mucus lining of the nose.
  • The main symptom is disfiguring skin sores, lumps or bumps that do not go away after several weeks or months.
  • Skin sores are pale colored.
  • Nerve damage is also common.
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8
Q

M. leprae Laboratory Testing

A
  • There are no serological or skin tests available.
  • Skin biopsy needed for the definitive diagnosis.
  • PCR for M. leprae DNA may be needed in special circumstances.
  • Cardinal signs along with skin biopsy is the most common way to diagnosis leprosy.
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9
Q

Leprosy Treatment

A
  • Treatment is done as a multi drug regimen.
  • Treatment for paucibacillary leprosy:
  • 6 month regimen
  • Adult: 100 mg Dapsone/daily & 600 mg
  • Rifampicin/monthly
  • Treatment for multibacillary leprosy:
  • 12 month regimen
  • Adult: Dapsone 100 mg/daily & Clofazimine 300 mg/monthly & Rifampicin 600 mg/monthly or 50 mg/daily
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10
Q

M. Tuberculosis Complex

A
  • M. tuberculosis
  • M. africanum
  • M. bovis & the Bacillus Calmette Guerin strain (humans +)
  • M. microti
  • M. canetti
  • M. caprae
  • M. pinnipedii
  • M. suricattae
  • M. mungi
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11
Q

M. Tuberculosis Complex Symptoms & Transmission

A
  • Symptoms:
    • Early symptoms include
      • Weight loss
      • Fever
      • Night sweats
      • Loss of appetite
  • Transmission:
    • Aerosol from one person to another usually thru coughing or sneezing even singing!
  • Extrapulmonary TB
    • M. tuberculosis can infect any organ of the body
    • Symptoms vary by site of disease
  • Pulmonary TB
    • Cough >2 weeks
    • often productive (sputum), can be bloody
    • Fever
    • Night sweats
    • Weight loss
    • Chest pain
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12
Q

Diagnosis of TB Disease

A
  • Signs and Symptoms consistent with TB
  • Chest X ray
  • Clinical Judgment
  • Bacteriology
    • AFB smear microscopy
    • Nucleic Acid Amplification Testing
    • Culture and Identification
    • Drug susceptibility testing
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13
Q

TB Specimens for Testing

A
  • Sputum 3 first morning collections
  • Bronchial washes
  • Stool
  • Biopsies
  • CSF
  • Gastric washings
  • Urine
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14
Q

TB Primary Isolation Medias

A
  • Lowenstein Jensen (LJ’s) slants
  • Lowenstein Jensen Gruft’s
  • Middlebrook 7H9 broth
  • Middlebrook 7H10 plates
  • Middlebrook 7H11 plates
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15
Q

Digestion Methods

A
  • 3 most common methods for sputum are;
    • NAOH method (sodium hydroxide)
    • Zephiran trisodium phosphate method
    • NALC NaOH method (N-Acetyl L-cysteine)
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16
Q

Identification by Biochemical Procedures

A
  • The following biochemical procedures are run when identifying unknown mycobacteria:
    • Growth rate
    • Colony morphology
    • Photochromogenicity
    • Nitrate Reduction
    • Niacin Accumulation
    • Arylsulfatase
    • 68 o C Catalase
    • Semi quantitative catalase
    • Tween 80 hydrolysis
    • Tellurite Reduction
17
Q

Treatment of TB

A
  • Active TB
    • Isoniazid (INH)
    • Rifampin (RIF)
    • Ethambutol (EMB)
    • Pyrazinamide (PZA)
18
Q

Drug-Resistant TB

A
  • Defined as TB bacteria that are resistant to at least one first line anti TB drug.
  • Multidrug resistant TB (MDRTB) is resistant to more than one anti TB drug and at least Isoniazid (INH and Rifampin (RIF).
  • Extensively drug resistant TB (XDRTB) is a rare type of MDRTB that is resistant to Isoniazid and Rifampin, plus any fluoroquinolone and at least one of the three
  • injectable second line drugs.
    • Second line drugs include: Amikacin, Kanamycin or Capreomycin.
19
Q

CCR 5199 f (4)(F) Safe Procedures

A
  • Establish safe handling and prohibit practices, such as sniffing in vitro cultures, that myincrease employee exposure to infectious agents”
  • CCR 5199 f (4)(F) Decon
    • “Establish effective decontamination anddisinfection procedures
20
Q

Respiratory (Pulmonary) Specimens

A
  • Sputum
    • Most frequent specimen submitted for Mycobacteriological testing
      • Expectorated
      • Induced
  • Bronchoalveolar lavage
  • Bronchial wash
  • Bronchial brush
  • Transtracheal aspirate
21
Q

Extrapulmonary Specimens

A
  • Non Respiratory Samples
    • Tissue, Body fluids, Stool, Gastric Lavage
  • Two groups
    • Aseptically collected specimens
    • Specimens known to contain contaminating flora
22
Q

Culture of acid-fast bacilli

A
  • Egg based medium (Lowenstein Jensen)
  • Agar and broth based medium (Middlebrook 7H10, Dubos Tween Albumen Broth)
23
Q

Lowenstein-Jensen Egg Base Medium

A
  • Coagulated whole eggs
  • Potato flour
  • Glycerol
  • Defined salts
  • Malachite Green (0.025 g/100 mL) (Petragnani 0.052 g/100 mL) (ATS 0.020 g/100 mL)
24
Q

Middlebrook Agar Base 7H10 Medium

A
  • Defined salts
  • Vitamins and Cofactors
  • Oleic acid
  • Albumin
  • Catalase
  • Glycerol
  • Dextrose
  • Malachite Green (0.0025g/100 mL)
25
Q

Middlebrook Agar Base 7H11 Medium

A
  • The same composition as Middlebrook 7H10 except for 0.1% casein hydrolysate added for enhanced recovery of fastidious isoniazid-resistant Mycobacterium tuberculosis
  • Selective 7H11 contains carbenicillin, amphotericin B, polymixin B, and trimethoprim to inhibit oropharyngeal commensals
26
Q

Identification of Mycobacterium tuberculosis Complex by AccuP robe

A
  • Acridinium ester labeled DNA probes utilized that hybridize to Mycobacterium tuberculosis complex specific 16S rRNA (AccuProbe, GenProbe Inc., San Diego,
  • Target 16S rRNA released by sonication of organisms recovered by culture
  • Acridinium ester is chemiluminescent, and DNA probe 16S rRNA hybrids emit light when acridinium ester hydrolyzed to ground state by alkaline peroxidation
  • Chemiluminescence measured in a luminometer
  • Amount of light emitted proportional to amount of DNA RNA hybrids formed
  • Total time for AccuProbe test is 2 hours
  • Available for TB, M. avium cplx, M. gordonae, M. kansasii
  • Positive identification for all members of the Mycobacterium tuberculosis complex, including Mycobacterium tuberculosis and Mycobacterium bovis (including attenuated BCG)
  • In clinical setting where invasive BCG infection possible (adjuvant therapy for bladder cancer) biochemical identification of BCG required
27
Q

Biochemial identification of Mycobacterium Bovis BCG

A
  • Growth on T2H
  • Niacin accumulation
  • Nitrate reduction
  • Pyrazinamidase
  • BCG is Neg for all
  • TB is POS for all
28
Q

Biochemical Tests for the Identification of Mycobacteria

A
  • Niacin accumulation
  • Nitrate reduction
  • Pyrazinamidase
  • Tween 80 hydrolysis
  • Urease
  • Arylsulfatase
  • Iron uptake
29
Q

Niacin Accumulation

A
  • Niacin (nicotinic acid) is a precursor in the biosynthesis of NAD and NADP
  • All mycobacteria produce nicotinic acid, but some species have a block in the NAD scavenging pathway and excrete niacin
  • Free niacin was detected with a cyanogen halide in the presence of a primary amine producing a yellow-colored product
  • Niacin paper strip test is commercially available (BD and Remel)
30
Q

Nitrate Reduction

A
  • Species of mycobacteria differ quantitatively in the ability to reduce nitrate to nitrite
  • Nitrate substrate broth heavily inoculated with organism
  • After 2 hr at 37 o C sulfanilamide and N-napthylethylenediamine added
  • Nitrite forms pink-red product (no color add zinc powder to confirm negative)
31
Q

Pyrazinamidase

A
  • Enzyme pyrazinamidase hydrolyzes pyrazinamide (PZA) to ammonia and pyrazinoic acid
  • Agar with Dubos broth containing PZA heavily inoculated with organism and incubated at 37 o C for 4 days
  • 1% ferrous ammonium sulfate added and agar observed after 4 hr
  • Pink band forms in agar from reaction of ferrous ammonium sulfate with pyrazinoic acid in a positive reaction
32
Q

Tween 80 hydrolysis

A
  • Lipases produced by several mycobacterial species hydrolyze the detergent polyoxyethylene sorbitan monooleate (Tween 80) into oleic acid and polyoxyethylene sorbitol
  • Tween 80 substrate solution inoculated with organism contains neutral red which is bound to Tween 80 producing an amber color
  • With hydrolysis of Tween 80 neutral red is released and produces a red color
  • A change in the color of Tween 80 from amber to pink or red after 24 h, 5 days, or 10 days is a positive result for Tween 80 hydrolysis
33
Q

Urease

A
  • Urease production useful in identification of scotochromogens and nonchromogens
  • Christensen’s urea agar with phenol red pH indicator inoculated with organisms visually read after 1, 3, and 5 days of incubation
  • Urease hydrolyzes urea to ammonia and CO 2 increasing agar pH to alkaline values
  • Positive reaction is a pink to red color of phenol red at an alkaline pH
34
Q

Arylsulfatase

A
  • Arylsulfatase hydrolyzes the sulfur ester bond linking the aromatic rings of tripotassium phenolphthalein disulfate, releasing free phenolphthalein
  • Although arylsulfatase activity can be detected with all mycobacteria with prolonged incubation, a 3 day incubation identifies several species. We also do the 14 day arylsulfatase test
  • Dubos liquid medium containing tripotassium phenolphthalein disulfate is inoculated, incubated 3 days, and Na 2 CO 3 added to alkalinize the medium
  • Free phenolphthalein turns a pink color at alkaline pH, and development of a pink color with addition of Na 2 CO 3 is a positive reaction
35
Q

Iron uptake

A
  • Iron uptake test utilized to identify rapidly growing mycobacteria capable of converting ferric ammonium citrate to an iron oxide
  • LJ slant inoculated with the organism incubated until visible growth develops, aqueous ferric ammonium citrate added, and the slant incubated for up to 21 days at 37 o C
  • Development of reddish-brown color in the colonies indicates production of iron oxide and is a positive result
  • Rapid Growers
  • Fortuitum = POS
  • Chelonae = NEG