PMNV 2.1 intro to mycobacteria and lab safety

most familiar species of mycobacterium

M. tuberculosis (MTB) → causative agent of tuberculosis; M. leprae → causative agent of Hansen disease (leprosy)

nontuberculous mycobacteria (NTM)

atypical mycobacteria or mycobacteria other than tubercle bacillus (MOTT)

Microscopic morphiolgy

slender, slightly curved or straight, rod-shaped organisms

cell wall characterisitics

has high lipid (mycolic acid) content

-resistant staining with basic aniline dyes

Mycobacteria dye troublshooting

increase staining time or application of heat but resists decolorization with acid-ethanol

-organisms know as AFB

distinguishing characteristic of mycobacterium

acid- fastness

growth characteristics

nonmotile; non spore former; strictly aerobic; slow growers (could take 2-6 weeks to produce growth); temp requirements

M, tuberculosis growth requirements

enhanced by increase CO2, often requires complex media

M. leprae growth requirements

fails to grow in vitro

Mycobacterium tuberculosis complex types (need to know)

M. tuberculosis

Non-cultivable nontuberculous mycobacteria

M. leprae

slow-growing photochromogens nontuberculous mycobacteria

M. kansasii, M. marinum

slow growing scotochromogens nontuberculous mycobacteria

M. gordonae, M. xenopi

rapid growing nontuberculous mycobacteria

M. chelonae, M. abscessus

nonchromogen photoreactivity

nonphotoreactive in light or dark conditions

photochromogen photoreactivity

nonpigmented until addition of light = photoreactive; carotene pigment upon exposure to light= pale yellow to orange

scotochromogen photoreactivity

produce pigment in light or dark conditions; produce a pale yellow to orange color

M. tuberculosis complex time for growth on solid media

>7 days

M. tuberculosis complex colony appearance

rough, buff (light and dark)

M. tuberculosis complex organsims

M. tuberculosis

Runyon Group I photochromgens time for growth on media

>7 days

Runyon Group I photochromgens colony appearance

smooth or rough, buff (dark), becoming lemon yellow or orange when exposed to light

Runyon Group I photochromgens organisms

M. marinum, M. kansasii

Runyon group II scotochromogens time for growth on plate

>7 days

Runyon group II scotochromogens colony appearance

smooth or rough, yellow to orange (light and dark)

Runyon group II scotochromogens organisms

M. gordonae, M. scrofulaceum

Runyon group III nonchromogens growth time on plate

>7 days

Runyon group III nonchromogens colony appearance

smooth or rough, nonpigmented, buff (light and dark), color may intensify with age

Runyon group III nonchromogens organisms

M. ulcerans

Runyon group IV rapid growers time for growth on plate

< 7 days

Runyon group IV rapid growers colony appearance

smooth or rough, buff or orange (light and dark)

Runyon group IV rapid growers organisms

M. abscessus, M. chelonae,

incidence of positive TB skin positivity among mycobacteriology laboratory workers

3x higher

each employee much have for working in a mycobacteria lab

adequate safety equipment; trained in safe laboratory procedures; informed of hazards associated with procedures; prepared for action following an unexpected accident; monitored regularly by medical personnel with skin test; appropriate PPE

BSL-3

prevents aerosols and proper installation of maintenance; processing clinical specimens or transferring viable cultures outside a safety cabinet should not be permitted; disinfectant cleaning and use of ultraviolet light require

when should testing for maintenance on the BSL-3 be done?

testing for performance at least yearly for trained personnel

outcome of ventilation with negative air pressure

physically separated from the rest of the laboratory

noncirculating ventilation system

6-12 room air change per hour to effectively remove 99% or more of airborne particles within 30-45 minutes

use of proper disinfectant

cover surface in towel or absorbance pad soaked in disinfectant, must be bactericidal for mycobacteria

-should be made fresh daily and contact time should be 10-30 minutes

bactericidal for mycobacteria disinfectant

sodium hypochlorite at conc 0.05- 0.5%

specimen collection guidelines

specimen should be confined to a single collection; sterile cu; with all specimens for microbiological examination, aseptic collection is important

sputum and other respiratory secretion collection

early morning sputum should be collection on 3 consecutive days

-3 collection is not needed if the first 2 direct smear are both positive

sputum and other respiratory volume

5-10 mL of sputum produced by deep coughing and expectoration of sputum

what happens is sputum is not obtainable?

bronchoscopy to obtains samples like bronch washing, BAL, or transbronchial biopsy specimen

how can you induce sputum in a patient?

inhalation or an aerosol of hypertonic saline

-increases the chances of detection and yield of mycobacteria

when are respiratory brushing appear to be more commonly diagnostic?

washing or biopsy specimen could possibly cause an inhibitory effect of lidocaine used during bronchoscopy or dilution by saline

gastric aspirates

used to recover mycobacteria that may have been swallowed during the night

when should gastric aspirates be used?

patients who do not produce sputum by aerosol induction; children younger than 12 years old; and nonambulatory individual

Gastric lavage

offers a diagnostic alternative only for those unable to expectorate sputum and in whom BAL might be contraindicated

obtaining gastric aspirates

the morning after overnight fasting

-3 specimens within 3 days

-sterile water, 30-60mL is instilled orally or nasogastric tube aspiration

prolonged exposure to gastric acid _______ and diminishes culture yield

kills mycobacteria

when should specimen processes be done in gastric aspirates and washings?

expeditiously, or the specimen should be neutralized with sodium carbonate or another buffer to pH 7.0 as soon as possible after specimen collection

urine processing

first morning midstream on 3 successive days; minimum of 15 mL in a sterile container; should be refrigerated during the interval between collection and processing; over 12-24 hours are not recommended

urine catheter

collected with a sterile needle and syringe

stool collection

clean containers without any preservative and sent directly to lab; should be frozen if not processed right away

when is testing stool for mycobacterium useful for?

in identifying patients, such as those with AIDS, who may be at risk for developed disseminated mycobacterial disease resulting from MAC

when is mycobacteria often seen in patients?

patients with AIDs but less frequently in other immunocompromised hosts

-most infection are caused by MAC

-recovery of the organism form blood is associated with clincial disease

Recovery/collection systems for mycobacteria in blood

isolator lysis-centrifugation system; direct inoculation of blood into a MYCO/F bottle; bacT/Alert MB blood medium

Blood isolator system of mycobacterium in blood

allows quantitative analysis, which may be used to monitor therapy and evaluate prognosis

general guidelines for body fluid

2mL of CSF, 3-5 mL for exudates and pericardial and synovial fluids; 10-15mL for abdominal and chest fluids

-collected aseptically and placed in 10-15 mL of sterile saline to prevent dehydration

digestion (liquefaction) purpose

breaks down thick mucus and proteinaceous material in sputum to release trapped mycobacteria

-allows mycobacterium to contact nutrients in agar to grow

decontamination purpose

eliminates fast-growing normal flora/bacteria using a harsh chemical agent (like sodium hydroxide) because mycobacteria are more resistant to chemical injury than other bacteria

concentration purpose

uses centrifugation to pellet the mycobacteria, increasing the sensitivity of smear microscopy and culture recovery

sodium hydroxide (NaOH), 2%, 3%, 4%

serves as decontaminating agent and digestant

N-acetyl-L-cysteine (NALC)

serves as a liquefying (digestion) or mucolytic agent

Benzalkonium chloride

liquefies sputum to help digest and decontaminate

oxalic acid

used to decontaminate P. aeruginosa from cystic fibrosis patients

after decontaminating, a ______ must be used so it doesn’t eventually kill the mycobacterium

neutralizing agent

treatment of sample with mucolytic agents

splits mucoprotein, allowing greater sedimentation of AFB

low specific gravity of concentration procedures

need at least 3000x g centrifugation to concentrate

-slow speeds takes longer and expose bacteria to toxic chemicals longers

-important in large volumes