Chapter 22 – Mycobacterium tuberculosis & Other Nontuberculous Mycobacteria MLAB 2434 – Clinical Microbiology Cecile Sanders & Keri Brophy-Martinez.
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Chapter 22 – Mycobacterium tuberculosis & Other Nontuberculous MycobacteriaMLAB 2434 – Clinical MicrobiologyCecile Sanders & Keri Brophy-Martinez
General Characteristics
Slender, slightly curved or straight rod-shaped organisms
Non-motile Do not form spores Cell wall with extremely high lipid
content Staining requires longer time or
application of heatOnce stained, resist decolorization
with acid-alcohol (acid-fast)
General Characteristics (cont’d) Strictly aerobic Grow more slowly than most bacteria Traditional characteristics used to
identify Mycobacterium Rate of growth Colony morphology Pigment production Nutritional requirements Optimum incubation temperature Biochemical test results
General Characteristics (cont’d) Newer techniques
Automated culture system, such as BACTEC
Nucleic acid probes with PCRThin-layer chromatographyGLCHigh-performance liquid
chromatography
Safety Considerations
Mycobacteriology workers are three times more likely to seroconvert (develop positive skin test) Adequate safety equipment Safe laboratory procedures training Information on hazards Preparations for unexpected
accidents Staff must be monitored regularly by
medical personnel
Safety Considerations (cont’d) Skin test
Also called “Mantoux test” and PPDThose with positive skin test must
be advised to have chest X-ray Proper Ventilation
Separate from other parts of labNegative air pressure (6 to 12
room air changes/hour)
Safety Considerations (cont’d) Biological Safety Cabinet Use of Proper Disinfectant
Bactericidal for mycobacteriaAlso called “tuberculocidal”
Other precautionsDisposablesProtective clothing, face masks
Specimen Collection and Processing Variety of clinical specimens,
including respiratory, urine, feces, blood, CSF, tissues, and aspirations
Should be collected before antibiotic therapy and processed ASAP
Sputum is most common; should be collected in a wide-mouth container to avoid aerosols
Specimen Collection and Processing (cont’d) Sputum
Number of specimens needed is inversely related to the frequency of smear positivity
Should be from a deep cough or expectorated sputum induced by neubulization
Bronchial washings or lavages may be collected
Specimen Collection and Processing (cont’d) Gastric aspirates
Used to recover mycobacterium that may have been swallowed during the night
Only used when patient is unable to produce a good quality sputum specimen
Urine – First morning preferred
Specimen Collection and Processing (cont’d) Stools – primarily collected from AIDS
patients to determine Mycobacterium avium complex (MAC)
Blood – most commonly from AIDS and other immunosuppressed patients
Tissues and other body fluids – need a fairly large volume of CSF, since number of organisms in that site are rare
Digestion & Decontamination of Specimens Because Mycobacterium grow so
slowly and are often collected from non-sterile body sites, they are easily overgrown by other bacteria
Specimens from non-sterile sites, therefore, must be “decontaminated”
Sputums or other viscous specimens also must be “digested”
Specimens from sterile sites (CSF, etc.) do not need decontamination
Digestion & Decontamination of Specimens (cont’d) Decontamination
Specimen from non-sterile site is mixed with an agent that will kill non-mycobacterium bacteria
Common decontamination agents• NaOH is most common• Benzalkonium chloride (Zephiran)• Oxalic acid
After decontamination, the agent must be neutralized so that it will not eventually kill the Mycobacterium
Digestion & Decontamination of Specimens Digestion
Liquefying mucus enables the mycobacterium to contact and use the nutrients in the agar medium
Common digestion agents• N-acetyl-L-cysteine – most common• Trisodium phosphate (Z-TSP) – used
with Zephiran
Concentration
After decontamination and digestion, the specimen is centrifuged in a closed, vented centrifuge to concentrate the organisms
Acid Fast Stains
After centrifugation, the button at the bottom of the tube is used to make a smear and to inoculate media
Acid Fast Stains Ziehl-Neelsen – uses heat to drive the color
into the lipids of the cell wall; decolorized with acid-alcohol
Kinyoun – cold stain Auramine or auramine-rhodamine
fluorochrome stain – more sensitive After staining, a minimum of 300 oif are
examined
Culture Media and Isolation Methods Mycobacterium are strictly aerobic Slow growers; cultures held for 6
weeks before calling negative Media
Lowenstein-Jensen (LJ) media – egg based
Middlebrook 7H10 and 7H11 agar – serum based
Middlebrook 7H9 broth
Culture Media and Isolation Methods (cont’d) Labs with large volumes of
Mycobacterium cultures use an automated reader (BACTEC)BACTEC broth contains 14C-
labeled substrateWhen organisms grow, 14C in the
form of 14CO2 is released and detected radiometrically
Laboratory Levels or Extents of Service for Mycobacterium American Thoracic Society levels
1. Specimen collection only2. Acid-fast stains and/or
inoculation only3. Isolation and presumptive
identification of Mycobacterium species
4. Definitive identification and antibiotic sensitivity testing
Identification of Mycobacterium Colony Morphology
Either smooth and soft or rough and friable
Growth rateRapid growers – colonies in < 7
daysSlow growers – colonies in > 7 days
Temperature
Identification of Mycobacterium (cont’d) Photoreactivity
Photochromogens – produce carotene pigment upon exposure to light
Scotochromogens – produce pigment in light or dark
Nonchromogenic – no pigment; these colonies are a buff color
Identification of Mycobacterium (cont’d) Biochemical Identification
Most labs now use nucleic acid probes with or without PCR
Older tests• Niacin accumulation• Nitrate reduction• Catalase• Hydrolysis of Tween 80• Iron uptake• Arylsulfatase
Identification of Mycobacterium (cont’d)
Older tests (cont’d)• Pyrainamidase• Urease• Inhibitory tests
• NAP• TCH• Growth in 6.5% NaCl• Tellurite reduction• Growth on MacConkey
Antibiotic Sensitivity Testing for Mycobacterium These tests must be performed with great
attention to detail, because Mycobacterium is fairly resistant and only a few organisms left can cause reinfection
Development of drug-resistance Common antibiotics (usually two or more are
given) Isoniazid Rifampin Ethambutol Streptomycin Pyrazinamide
Mycobacterium Infections Truly pathogenic
M. tuberculosis M. bovis M. ulcerans
Potential pathogens M. kansasii M. marinum
Other possible pathogens and rare pathogens listed on p. 670
Mycobacterium tuberculosis Primarily a pathogen of the
respiratory tract (“TB”) One of the oldest communicable
diseases Over 1 billion cases worldwide,
with 8 to 10 new cases each year and 3 million deaths per year
Once called “consumption”
Mycobacterium tuberculosis (cont’d) Primary tuberculosis
Spread by coughing, sneezing, or talking Inhaled into alveoli, where the organisms
are phagocytized If the organism does not cause
immediate infection, the organism can be “walled off” in a granuloma
Granulomas can break down in future and the organisms can cause infection later
Mycobacterium tuberculosis (cont’d) PPD Test
Mycobacterium tuberculosis (cont’d) PPD Test (cont’d) Positive Test
Mycobacterium tuberculosis (cont’d) Extrapulmonary tuberculosis
SpleenLiverLungsBone marrowKidneyAdrenal glandEyes
Mycobacterium tuberculosis (cont’d) Identification
Slow growerColonies are thin, flat, spreading
and friable with a rough appearance
May exhibit characteristic “cord” formation
Grows best at 35 to 37° CColonies are NOT photoreactive
Mycobacterium tuberculosis (cont’d)
Other Mycobacteria
Mycobacterium bovis – primarily in cattle, dogs, cats, swine, parrots and human; disease in humans closely resembles M. tuberculosis
MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous mycobacteria) Most found in soil and water Chronic pulmonary disease resembling
TB
Other Mycobacteria (cont’d) NTM (cont’d)
M. avium Complex (MAC)M. kansasiiMycobacterium fortiutum-
chelonei ComplexM. marinumEtc., etc.
Mycobacterium leprae
Causes leprosy or Hansen’s Disease Infection of the skin, mucous membranes
and peripheral nerves Most cases are from warm climates Bacteria infect the cooler areas of the
body (ears, nose, eyebrows, fingers, toes) Diagnosis made from finding acid-fast
bacilli in scrapings from lesions Not culturable, except in mouse foot pads
Mycobacterium leprae (cont’d)
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