pmmv week 5

mycobacterium

170 rec and propsed spp, m. tuberculosis MTB, M. leprae- hansen disease/ leprosy, nonTB mycobacteria NTM atypical or MOTT

mycobacterium morph

slender, slightly curved or straight, rod, high lipid cell wall (mycolic acid), resist basic aniline dyes like Gram stain at rt, stain with heat or more time, acid fastness- cannot decolorize AFB

mycobacterium growth

nonmotile, no spores, strict aerobe, glow grower, 2-6 weeks to grow if disease, enhance MTB with CO2, M. leprae NG in vitro

runyon group III nonchromogen

nonphotoreactive in light or dark, smooth or rough, buff (light and dark), color intensify with age

Runyon group I photochromogen

smooth or rough buff (dark), addition of light= cartene pig (pale yellow orange), m. kansasii

runyon group II scotochromogen

produce pig in light or dark, pale yellow-orange, smooth or rough, M. gordonae, m. scrufulacem

M. TB complex

rough, buff light and dark, m. Tb, M. bovis, m. africanum

Runyon group IV rapid growers

under 7 days, smooth or rough, buff or orange (light and dark)

incidence of pos TB skin test

3x higher amoung mycobacteriology lab workers

BSL-3

prevents aerosols and inhalation, tested yearly, only process and transfer specimen inside, disinfectant cleaning and UV light, neg air pressure

noncirculating ventilation system

6-12 room air changes per hr, effectively remove 99% or more of airborne particles within 30-45 min

disinfect

cover surface in towel soaked in mycobacteria bactericidal disinfectant to reduce aersols for 10-30 min, sodium hypochlorite 0.05%-0.5% (1:50-10 household bleach), make fresh daily

collection containers

1, sterile, wide mouth cup, tight lid, sputum- 50 mL centrif tube

sputum collection

early morn, 3 consec days, if 2 pos dx, 0-1 additional specimens needed, 5-10 mL from deep cough or induced inhalation of hypertonic saline aerosol (better)

unable to obtain sputum

bronchoscopy- washing, bronchoalveolar lavage BAL, transbronchial biopsy, less dx due to inhib of lidpcaine or saline dilution, pt produce sputum after examine

gastric aspirates

mycobacteria swallowed at night (only if nonambulatory, cannot produce by induction or under 12), in morn after night fasting within 3 days, instill 30-60 mL of sterile water via nasogastric tube aspiration

gastric washing

lavage, dx alternative only if unable to make sputum and BAL can be contraindicated

Gastric aspiration/washing care

pprolonged exposure to gastric acid kills, process asap or neutralize with sodium carbonate or buffer to pH 7

urine exam

first morn midstrem on 3 successive days, 15mL-entire vol, catheter, refrigerate if delay, process promptly, no 12-24 hr pool speciemens (contaimination)

stool exam

AFB presence in AIDS- can develop disseminated mycobacterial disease from MAC, 68% neg, no preservatives, asap, freeze to -20 C if over 3 hrs

blood exam

mycobacteremia often with AIDS less common in other immunocomp, caused by MAC

blood collection

isolator lysis centrif (quantitative), direct inoculation of blood into MYCO/F, BacT/Alert MB medium

tissue and bodily fluids

larger vol increases yield, 2 mL for CSF, 3-5 for exudates and pericardial and synovial fluids, 10-15 abdominal and chest fluid, place in 10-15 mL sterile saline to prevent dehydration

digestion (liquefaction)

breaks down thick mucus and proteinaceous material in sputum to release trapped mycobacteria allowing contact with agar nutrients for growth

decontamination

eliminated fast growing norm flora and bactera with harsh chemical agents (sodium hydroxide), mycobacteria is very resistant to chem injust

concentration

centrifuge to pellet the mycobacteria, increasing sensitivity of smear and culture

sodium hydroxide NaOH

2-4%, decontaminating and digestant agent

N-acetyl-L-cysteine NALC

liquifying digestion or mucolytic agent

benzalkonium chloride

liquifies sputum to help digest and decontaiminate

oxalic acid

decontaiminates P. aeruginosa from cystic fibrosis CF pt

after decontaimination

use neutralizing agent so mycobacteria is not eventually killed

concentration procedure

treat sample with mucolytic agents to split mucoprotein allowing greater sedimentation of AFB, low sg- need 3000xg centrif to concentrate (slow speed takes longer and expose bacteria to toxic chem longer)

ziehl-neelsen stain

carbolfuchsin- primary while heating, acid alc decolor, methylene blue counterstain

kinyoun stain

same as ziehl but no heat

auramine or auramine-rhodamine fluorochrome stain

more senstitive than carbolfuchsin, mercury vapor lamp with strong blue filtered light, pos= bright yellow-orange

acid fast smear interpretation

1-2/300 carbolfuchsin 1-2/70 fluor resub another specimen, 1-9/100 and 1-2/70 1+, 1-9/10 and 2-18/50 2+, 1-9 and 4-26 3+, >9 and >36 4+

generation time

over 12 hrs, M. TB 20-22, 2-6 weeks of incub, enhance MTB in 5-10% CO2

culture req

strict aerobe, pH 6.5-6.8, humidity

egg based culture

malachite green suppresses GP orgs, lowenstein-jensen LJ, american thoracic society ATS, petragnani, whole eggs, potato flour, glycerol, salts milk

gruft mod of LJ medium and mycobactosel

antimicrobial agents (selective) with nonselective media (shelf life 1 yr, hard to distinguish growth from debris) to increase isolation when contaminated

agar based media

chem defines, OADC enrichment (oleic acid, alb, dextrose, catalase), malachite green, glycerol (pri carb/energy), casein hydrolysate in 7H11, inorg salts in 7H10

opaque egg based media, clear agar based media

exam using dissecting microscope for early detection of growth and colony morph, drug sus tests without altering drug concentrations

CHOC agar

primary isolation for skin and other body surface specimens, M. haemophilum, ferric ammonium or hemin

middlebrook 7H10 agar

supplemented with hemolyzed sheep blood cells or another source of hemin

25-33 C optimal temp, poorly at 35-37

m. haemophilum, m. marinum, m. ulcerans, and m. chelonae

42-45 C

M. xenopi

liquid media

mycobacterium grows more rapidly, primary isolation and subculture, stock strains, sicking single colonies, preping inoculum, middlebrook 7H9, duboys tween alb

inhibits nonmycobacteria growth in liquid medium

plymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocillin PANTA

mycobacterial growth indicator tube MGIT

BD dx system, modified middlebrook 7H9 broth with a fluorescence quenching based oxygen sensor, growth consumes oxygen allow fluorescence with UV

disadvantages of liquid media

no colony morph or pig to diff mycobacteria from comtaminant, spp with lower optimal temp may not grow (m. haemophilum, m. marinum, m. ulcerans, m. chelonae)

isolator lysis centrifuge system ILCS

blood collection system containing saponin to liberate intracellular orgs, inoculate in liquid and solid after centrif, quantifies mycobacteria, additional selective medium if heavy contamination

rough friable (egg) colony morph

m. TB- cording- patterned texture, curved strands of bacilli

smooth and soft colony morph

m. avium-intracellular complex, glossy, white, with smaller translucent colonies

growth rate

3-60 days, rapid growers under 7 days, slow over

niacin

most make free niacin to niacin ribonucleotide, 95% of m. TB fre niacin (nicotinic acid) lack connecting enzyme, nicotinic acid with cyanogen bromide and amine = yellow

niacin results

neg on young culture with few colonies, done on egg agar 3-4 weeks old with 50+ colonies, if neg repeat several weeks later

do not perform niacin on

scotochromogenic or rapid spp, m. simiae, BCG stain of m. bovis, m. africanum, m. marium, m. chelonae, and m. bovis, may be pos rarely

nitrate reduction

incub with 2mL of sodium nitrate at 37 C 2hr, add hydrochloric acid, sulfonamide, and N-napthulenediamine dihydrochloride, red= pos, confirm neg with zinc turning it pink

catalase

mycobacteria split hydrogen peroxide into water and oxygen, if heated to 68 C for 20 min neg bc not heat stable unless isoniazid resistant

semi-quantitation of catalase

add tween 80 (detergent) and hydrogen peroxide to a 2 week old culture in agar deep, after 5 min measure column of bubble < or > 45mm

hydrolysis of tween 80 detergent

lipases split detergent into oleic acid and polyoxyethylated alc sorbitol, pH indicator is neutral red, record as pos after 24 hr 5 day or 10 day

hydrolysis of tween 80 detergent distinguish

m. kansasii rapidly pos, scotochromogenic- m. gordonae pos, scrofulaceum neg

iron uptake

20% aqueous solution of ferric ammonium citrate to iron oxide, rusty brown= pos, neg= m. chelonae

arylsulfatase

hydrolyze sulfate group and aromatic ring bond with R-OSO3 H, tripotassium phenolphthalein→ phenolphthalein pH change with sodium bicarb= pink

arylsulfatase pos orgs

M. fortuitum comples, M. chelonae, M. xenopi, M. trivale= rapid in 3 days, M. marinum and M. szulgai = 14 days

pyrazinamidase

PZA hydrolized to pyrazinole acid and ammonium in 4 days red with ferrous ammonium sulfate, MTB, M. marinum

tellurite reduction

potassium tellurate to black tellurium 3-4 days, pos = MAC, neg= other nonchromogenic spp

urease

org in 4 mL of urea broth 37 C 3 days, pink= pos, M. scrofulaceum

inhibitory test- Thiophene-2-carboxylic acid hydrazide T2H

M. bovis S to lower concentrations of T2H than MTB, depends on concentration of inhib agent and incub temp

inhibitory test- NaCl tolerance 5%

egg based media inhib most mycobacteria, M. flavescens, M. triviale and most rapid growers survive

inhibitory test- growth on MAC

M. fortuitum- chelonae complex grows w/o crystal violet, most mycobacteria cannot grow here

chromatography

detects mycolic acid (long chain fatty acid), basic saponification, acidification and extraction, ID most spp, HPLC IDs agree with nucleic acid probe and biochem IDs, replaced by MALDI-TOF and molecular

hybridization and nucleic acid amplification tests NAAT for MTB

several specific probes, auto DNA sequencing, direct nucleic acid ampli (bacilli directly from specimen 6-8 hrs), MALDI-TOF

automated dna sequencing

gene coding is 16S rRNA, PCR amplification ID and comparison of nucleotide sequence of ref)