Special Lab - Mycobacteriology

Question 1

M. leprae

Answer 1

leprosy/ Hansen’s disease

Question 2

Mycobacteria is divided into two groups

Answer 2

• M. tuberculosis complex (MTBC)
• Nontuberculous Mycobacteria (NTM) = environmental organisms that arely cause human illness

Question 3

Mycobacterium sp are …

Answer 3

• aerobic, non-spore forming bacilli that vary in length from cb to long, slender bacilli
• distinctive staining property due to extremely high lipid content in their cell walls (mycolic acids)

Question 4

how to improve dye uptake of mycobacteria

Answer 4

• stains combined with phenol (carbolfuchsin) and heat is applied
• once stained = mycobacteria resist decolourization with acid-alc bc of mycolic acids = ACID FAST BACILLI

Question 5

T or F. Mycobacteria are strict aerobes but some show enhanced growth when incubated in CO2

Answer 5

T

Question 6

T or F. Non Pathogenic mycobacteria grow more slowly than other bacteria

Answer 6

F! Pathogenic mycobacteria ; may take 2-6 wks of incubation

Question 7

slow growth of mycobacteria is due to,,,

Answer 7

hydrophobic nature of cell wall (LIPIDS) which decreases uptake of nutrients

Question 8

This mycobacteria fails to grow in vitro

Answer 8

M. leprae

Question 9

MTBC three main organisms

Answer 9

M. tuberculosis
M. bovis
M. bovis (BCG)

Question 10

M. tuberculosis

Answer 10

• highly contagious, higher incidence of infection in certain races in poor socioeconomic conditions
• high risk of lab acquired infection
• grows slowly only at 37C on enriched media
• colonies are not pigmented, rough, dry, and crumbly
• mature colonies = niacin pos, nitrate pos, aerobic, grow in presence of thiophen-2-carboxylic acid hydrazide (TCH)

Question 11

M. bovis

Answer 11

• seldom encountered in lab (bovine origin)
• rough and pale buff or colourless colonies
• growth only occurs at 37C; enhanced on media containing pyruvic acid
• niacin neg and nitrate neg

Question 12

M. bovis, consistent with BCG

Answer 12

• rarely causes human infections, second most often isolated member of the M. tuberculosis complex
• isolated from vax sites or form urinary/bladder specimens as a result of superficial bladder treatment w BCG
• culture similarities to both M. tub and M. bovis
• both M. bovis and BCG are intrinsically resistant to pyrazinamide (PZA)

Question 13

NTM

Answer 13

pigmented and non-pigmented acid-fast bacilliwhich are not formally recognized as potential human pathogens until 40-50 yrs ago

Question 14

characteristics of NTM

Answer 14

• less restrictive temps for growth
• growth rates tend to be more variable - some grow in less than 7 days, others = generation time similar tp M. tuberculosis
• many are pigmented yellow to orange
• varying degrees of susceptibility to first line antitubercular agents
• considered non-infectious; human infections are only associated with pre-existing disease or trauma

Question 15

first line antitubercular agents

Answer 15

Rifampin
Isoniazid
Ethambutol

Question 16

what is the greatest problem in culturing specimens for mycobacteria

Answer 16

presence of rapidly growing miroorganisms
(ex: generation time of M. tub is 24 hrs vs. E. coli is 20 mins)
- specimens should be processed as quickly as possible and refrigerated if delay occurs (CSF and blood always held at RT only)

Question 17

gastric washings for mycobacteria

Answer 17

• purpose is to obtain specimens of swallowed sputum and may be allowable in young children
• gastric contents are toxic to tubercle bacilli = specimen processed within 4 hrs; if not possible = phosphate buffer added to container to prevent acid destruction of mycobacteria

Question 18

CSf for mycobacteria

Answer 18

after collection, CSF from tuberculosis meningitis may produce a spider web clot
if this occurs = portion of membranous material should be used for smear prep

Question 19

TB meningitis is often associated with

Answer 19

increase in lymphs or mononuclear cells

Question 20

blood cultures for mycobacteria

Answer 20

SPS or citrate and heparin collection tubes
then inoculated into a recommended blood culture media system (ex: Becton Dickinson, MycoF/lytic media)

Question 21

specimens with high amounts of contaminating flora such as sputum, require …

Answer 21

digetion and decontamination procedures to destroy contaminating organisms that would quickly overgrown any mycobacteria

Question 22

The purpose of digestion:

Answer 22

• decontmaination = AFB rsistant to acid and alkali; NaOH added to specimen to eliminate organisms other than mycobacteria ; acid is then added as aa neutralizer before AFB are destroyed
• liquefication = mucolytic agent such as N-acetyl-L-cystine added to break down mucus; allows for homogenization as well as decreased time with NaOH; also allows for concentration by centrifugation
• homogenization = allows organisms to spread throughout the specimen so present in DS and culture

Question 23

T or F. ZN is better for visualizing mycobacteria than Auramine=Rhodamine

Answer 23

F! fluoresecnt stain more sensitive bc mycolic acids appear to have greater binding capacity with auramine than carbol fuchsin
smears positive by fluorescent techniques are confirmed by ZN; rule out non-viable organisms or non-specific fluor material
after DS => PCR to ID organism

Question 24

four types of culture media for mycobacteria

Answer 24

egg base = Lowenstein-Jensen (malachite green for selectivity)
agar-base = Middlebroookk serum albumin based media
Liquid media = quicker growth seen in liquidd media; enriched and/or selective by addition of antimicrobial agents ; aids in recovery of mycobacteria present in low numbers
selective media

Question 25

ID of mycobacteriaa

Answer 25

• after intitial isolate, differentiate whether MTBC or NTM
• then final ID =

if MTBC = niacin and nitrate pos = M. tuberculosis; both neg = M. bovis; variable = other

if NTM = DNA sequencing or MALDI-TOF MS for ID

Question 26

first-line anti-tuberculosis drugs recommended for testing used against MTBC

Answer 26

isoniazid
rifampin
ethambutol
pyrazinamide
streptomycin

slow growing so E-test and KB unsuitable