Mycobacteriology Flashcards
Why do acid-fast bacteria stain acid fast?
Cell walls contain mycolic acid (long-chain fatty acids)
- When stained w/ special dye, cells can’t be decolorized w/ weak-acid-alcohol solution
- Acid-fast organisms stain red
What is the “ideal” contamination rate that mycobacteria labs should strive for when assessing their decontamination rate?
Mycobacterial culture contamination rate of 3-5% is tolerable
- < 3% → overly harsh decontamination
- > 5% → insufficient contamination
Which mycobacterium species are identified on the GeneXpert (Xpert MTB/RIF) system?
M. tuberculosis complex
What antimicrobial susceptiblity are identified on the GeneXpert (Xpert MTB/RIF) system?
Rifampin resistance
What two categories of solid media are used for the isolation of mycobacteria
Egg and agar
Give examples of egg-based media
- Lowentstein-Jensen
- American Trudeau Society
Give examples of agar-based media
- Middlebrook 7H10
- Middlebrook 7H11
What two types of broth media are used for the isolation of mycobacteria?
- MGIT
- Bactec Alert
Which type media, solid or broth, is able to recover organisms more quickly?
Broth
How do you set up a skin culture for mycobacteria, including temperature, media, etc.
Set up 7H10 @ 37C and 30C and set up MGIT @ 37C
Scotochromogen
Produces pigments independent of light exposure
Photochromogen
Produces pigments only after exposure to light
Nonchromogen
Never produces a pigment
Rapid grower
Requires < 7 days to produce colonies when subculutred onto certain media
Slow grower
Requires > 7 days to produce colonies on solid media under ideal culture conditions
What broth medium is used in MGIT, Bact/ALERT, and BACTEC Myco/F Lytic system?
Middlebrook 7H9
Which mycobacteria are nonpathogenic and may be isolated from tap water?
- M. avium
- M. genavense
- M. kansasii
- M. gordonae (common contaminant)
Which mycobacteria are acid-fast and modified acid-fast?
Acid-fast
- Gordonia spp (weakly-positive)
- Nocardia spp (positive, beaded appearance)
- Rhodococcus spp (weakly-positive)
- Segniliparus spp (strongly-positive)
- Tsukamurella spp (weakly-positive)
What do mycobacteria and the background look like when using the Ziehl-Neelsen or Kinyoun methods?
Mycobacteria are stained red; background is blue
List the strain of M. bovis which is used to produce the M. tuberculosis vaccine
BCG
List the species of Mycobacteria which cannot be cultured in the lab routinely (requires living cells)
- M. tuberculosis
- M. leporae
Differentiate the temperature(s) at which plates are incubated for skin cultures and sputum (respiratory) cultures
- Skin cultures are incubated at 30C
ALL cultures are set up on what media?
Solid and broth media
Outline the specimen collection recommendations for respiratory mycobacteria cultures
3 separate specimens must be collected at least 8 hours apart
What biosafety level and lab containment mechanisms are required for working w/ mycobacterial cultures?
BSL-3
- Floor, wall, and ceiling surfaces are sealed and coated w/ an epoxy for ease of disinfection
- Air is drawn from the outside of the lab INTO the lab (negative air pressure)
- All work w/ clinical specimens and cultures is conducted in biological safety cabinets
- Separated from BSL-2 lab space by at least 2 doors
____ stain is the best choice for screening patients and is specific for acid-fast organisms
Auramine-Rhodamine
What is the most common submitted specimen for TB diagnosis?
Sputum
Why must one of the sputum specimens be collected in the morning?
Crap concentrates in the lungs throughout the night
Define incubation times and conditions for mycobacterial cultures
- Broths are incubated w/in a continuous monitoring system
- Agar- and egg-based media are incubated in 5-10% CO2
- MTB and most other mycobacteria incubated at 35-37C
- Skin specimens incubated at 30C
How long should cultures be incubated before being reported as negative?
6-8 weeks
If specimens are smear-positive but don’t grow in 6-8 weeks, add how many more weeks onto the incubation duration?
4 weeks
Specimens from skin should be incubated for how many weeks if M. ulcerans is suspected?
8-12 weeks
Cultures should be examined how many days after inoculation?
3-5 days
How often should cultures be examined for the first 4 weeks?
Twice per week
After 4 weeks, cultures should be examined how often?
Once per week until 6-8 weeks
Why does the DNA probe used to identify M. tuberculosis target rRNA and not chromosomal DNA?
This makes the assay more sensitive since there are 10,000 copies of RNA per bacterium and only 1-2 copies of DNA; if the DNA probe binds to target RNA molecules, the DNA-RNA hybrid can be detected by chemilumescence
Which mycobacteria are inhibied by T2H?
M. bovis
List the mycobacteria that grow optimally at 30°C (6)
- M. abscessus/chelonae
- M. fortuitum
- M. haemophilum
- M. ulcerans
- M. marinum
List the mycobacteria that grow optimally at 35-37°C (5)
- M. tuberculosis complex (M. tuberculosis, bovis, africanum)
- M. avium
- M. fortuitum
- M. kansasii
- M. szulgai
- M. scrofulaceum
List the mycobacteria that grow at 42C and 45C
- M. xenopi (42°C)
- M. thermoresistable (45°C)
What are the special requirements for isolation of M. haemophilum?
M. haemophilum requires hemin (X factor in CHOC agar)
- Fastidious
What are the special requirements for isolation of M. genavense?
M. genavense requires mycobactin J
- VERY fastidious
List the slow growing mycobacteria
- M. avium
- M. genavense
- M. gordonae
- M. haemophilum
- M. kansasii
- M. marinum
- M. scrofulcaneum
- M. szulgai
- M. tuberculosis complex
- M. ulcerans
- M. xenopi
Describe the appearance of M. tuberculosis on agar
ROUGH, wrinkled, waxy, BUFF-colored
Describe the appearance of M. kansasii on agar
Rough, wrinkled
Describe the appearance of M. xenopi on agar
Smooth and yellow-colored
Describe the appearance of M. fortuitum on agar
Umbonate (raised center) colonies surrounded by a flat, dry skirt of growth
DNA probe assays that are currently available for which 4 Mycobacteria species?
- M. tuberculosis complex
- M. avium-intracellulare complex
- M. gordonae
- M. kansasii
Which species of mycobacteria are commonly implicated in scorula?
- M. avium complex (#1)
- M. scrofulaceum (until 1980s)
Which species of mycobacteria are historically associated w/ AIDS patients and that are isolatable from stool and blood?
M. avium complex
- M. avium
- M. intracellulare
- M. chimaera
Which tests distinguish M. tuberculosis and M. bovis
- Nitrate reduction
- T2H
- Pyrazinminidase (PZA)
List examples of scotochromogens
- M. gordonae
- M. scrofulaceum
- M. szulgai (@ 37C)
- M. xenopi
List examples of photochromogens
- M. kansasii
- M. marinum
- M. szulgai (@ 25°C)
List examples of nonchromogens
- M. abcessus/chelonae
- M. avium
- M. fortuitum
- M. haemophilum
- M. tuberculosis
List the rapid growing mycobacteria
- M. abscessus/chelonae
- M. fortuitum
- M. smegmatis
Which biochemical test which allows differentiation of rapid growers from slow-growing nonchromogenic mycobacteria?
Arylsulfatase
Which biochemical test is used to distinguish M. scrofulaceum from M. gordonae?
Urease
Which test is used to distinguish M. tuberculosis from M. bovis and M. marinum from M. kansasii
Pyrazinamidase
- M. tuberculosis (+)
- M. bovis (-)
- M. marinum (+)
- M. kansasii (-)
Describe the utility of the pyrazinamidase test
Pyrazinamidase is an enzyme that converts the drug pyrazinamide to pyrazinoic acid
Which biochemical test helps distinguish M. fortuitum from M. abscessus/chelonae?
Iron uptake test
Which rapid-growing mycobacteria can grow in the presence of 5% NaCl?
M. abscessus
Which organisms are detected by the Xpert MTB/RIF real time PCR test
M. tuberculosis complex
- M. tuberculosis
- M. bovis
- M. africanum
Which antibiotic resistance gene is detected w/ the Xpert MTB/RIF real time PCR test?
Rifampin resistance (and isoniazid resistance is predicted from this)
Is the Xpert MTB/RIF real time PCR test performed on culture or direct patient specimen?
Direct patient sputum
What component of mycobacteria is analyzed using high performance liquid chromatography (HPLC)?
Mycolic acid from mycobacterial cell wall
List the two most common causes of NTM lung disease
M. avium complex (#1 cause) - M. avium - M. intracellulare - M. chimaera M. kansasii (#2 cause)
What is the common route of infection associated w/ M. marinum?
- Fresh-water fish tanks
- Salt water
What disease is caused by M. ulcerans
Buruli ulcer (aka Brainsdale ulcer)
What disease is caused by M. leprae?
Leprosy
Nitrate reduction
- Media
Potassium nitrate
Nitrate reduction
- Reagents
- Alpha-naphthylamine or dimethyl-alpha-napthylamine
- Sulfonilic acid
- Zinc dust
Nitrate reduction
- Positive test
- Negative test
- Pos: Red
- Neg: Colorless
Niacin
- Positive test
- Negative test
- Pos: yellow
- Neg: white/clear
Tween 80 hydrolysis
- Positive test
- Negative test
- Pos: red (nonpathogenic)
- Neg: yellow/orange (pathogenic)
Heat-stable catalase
- Positive test
- Negative test
- Pos: bubbles
- Neg: no bubbles
Arylsulfatase
- Positive test
- Negative test
- Pos: red
- Neg: clear
Urease
- Positive test
- Negative test
- Pos: pink
- Neg: yellow
Pyrazinaminidase
- Positive test
- Negative test
- Pos: pink
- Neg: no pink top layer
Iron uptake
- Positive test
- Negative test
- Pos: brownish slant
- Neg: not brown
T2H
- Positive test
- Negative test
- Pos: growth
- Neg: no growth
5% NaCl
- Positive test
- Negative test
- Pos: growth > 50 colonies
- Neg: no growth < 50 colonies
MAC w/o Crystal Violet
- Positive test
- Negative test
- Pos: growth
- Neg: no growth
Tellurite reduction
- Positive test
- Negative test
- Pos: gray/clear
- Neg: yellow
List the “first-line” antibiotics used to treat infections caused by MTB complex organisms
- Rifampin
- Isoniazid
- Pyrazinamide
- Ethambutol
- Streptomycin
List infections associated w/ rapidly-growing mycobacteria
- Post-traumatic wound infections
Define drug-resistant TB
Resistant to rifampin and isoniazid
Define multi-drug resistant TB
Resistant to rifampin, isoniazid, quinolones (e.g., ciprofloxacin), and other drugs
What standard method is used for testing MTB against almost all antimicrobials (except pyrazinamide)?
Method of Proportion (MOP)
What 2 testing methods are used for antimicrobial susceptibility testing of NTM mycobacteria?
- Broth microdilution
- Disk or strip diffusion (Etest)
Describe the administration method of the TB skin test (TST)
Purified proteins (tuberculin) from MTB cells are injected intradermally on the inner forearm
If a person has been exposed to TB bacteria, a ____ ____ reaction occurs
Delayed-type hypersensitivity
TB skin test
- False positives
- Previous BCG vaccination (vaccination w/ M. bovis BCG)
- Incorrect method of TST administration
- Infection w/ some NTM
TB skin test
- False negatives
- Very recent TB infection (w/in 8-10 weeks of exposure)
- Very old TB infection (many years ago)
- Recent vaccination against measles or smallpox
- Incorrect administration or interpretation of the TST
How many tubes are collected for the QuantiFERON Gold In-Tube test?
3 tubes are collected
- Red, purple, grey
Which QuantiFERON tube is the negative control and does NOT contain ____
Grey; Ags
Which QuantiFERON tube is the “test” tube?
Red
Which QuantiFERON tube is the positive control and contains a substance that elicits ____
Purple; interferon-gamma release
QuantiFERON Gold In-Tube test
- False positive
Infection w/ some NTM
QuantiFERON Gold In-Tube test
- False negative
- Dysfunctional immune ells of lymphocytopenia
- Positive control will also be negative
List the secondary antibiotics used to treat infections caused by MTB complex organisms
- Capreomycin
- Ciprofloxacin (Cipro)
- Cycloserine
- Doxycycline or minocycline
- Kanamycin
- Rifabutin
- Trimethoprim-sulfamethoxazole
Which mycobacteria are POSITIVE for arylsulfatase?
- M. abscessus
- M. africanum
- M. fortuitum
Which mycobacteria are POSITIVE for niacin?
M. tuberculosis
Which mycobacteria are POSITIVE for nitrate?
- M. fortuitum
- M. kansasii
- M. szulgai
- M. tuberculosis
Which mycobacteria are NEGATIVE for urease?
- M. africanum
- M. avium
- M. gordonae
- M. haemophilum
- M. ulcerans
- M. xenopi
Which mycobacteria are NEGATIVE for PZA?
- M. africanum
- M. bovis
- M. gordonae
- M. kansasii
- M. scrofulaceum
- M. szulgai
- M. ulcerans
Which mycobacteria are NEGATIVE T2H?
M. bovis
Which mycobacteria are POSITIVE for Tween 80 hydrolysis?
- M. genavense
- M. gordonae
- M. kansasii
- M. marinum
Which mycobacteria are POSITIVE for 68°C catalase?
- M. abscessus
- M. chelonae
- M. fortuitum
- M. genavense
- M. gordonae
- M. kansasii
- M. marinum
Which mycobacteria grow in the presence of 5% NaCl?
- M. abscessus
- M. fortuitum
Which mycobacterium is POSITIVE for iron uptake?
M. fortuitum
Which mycobacteria grow on MAC w/o crystal violet?
- M. abscessus
- M. fortuitum
Which mycobacteria are associated with soil?
Rapid growers
- M. gordonae
Describe microscopic examination of fluorescent stains
A minimum of 30 fields using 250x magnification
