Unit 2: Mycobacteria, Acid-fast Bacteria Flashcards
How do bacilli strains differ in their morphology?
to grow as solitary rods or long strands serpentine cords
What causes bacteria to be acid-fast?
mycolic acid
lipid-rich cell wall resistant to digesting agents such
as strong acids and bases
can be strong or weak acid-fast
Acid -fast bacteria do not stain readily, but once stained with ____________ dyes, resist decolorization with tenacity.
arylmethane
Should it be reported if only one acid-fast bacteria is seen?
Yes! Report if you seen ANY.
What cell wall component makes the classification of bacteria?
peptidoglycan
What group of people are more likely to get active TB?
HIV/AIDS patients
What are the four clinical significant Mycobacteria?***
-Mycobacterium tuberculosis complex
-Mycobacterium ulcerans
-Mycobacterium leprae
-Nontuberculosis mycobacteria
What is part of the Mycobacterium tuberculosis complex?**
-M. tuberculosis
-M. bovis
-M. microti
-M. africanum
What are the nontuberculosis mycobacteria that fall under the Runyon classification?***
-Runyon I: M. Kansasii & M. marinum
-Runyon II: M. scrofulaceum
-Runyon III: M. avium complex
-Runyon IV: M. chelonei and M. fortuitum
Mycobacterium tuberculosis (MTB) is a pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases of tuberculosis (TB) First discovered in 1882 by ____________.
Robert Koch
TB is a pandemic disease, tuberculosis is especially common in the developing world owing to HIV infection (_______% of individuals with HIV disease may have tuberculosis).
15-20
a chronic, granulomatous, slowly progressive infection, usually of the lungs; eventually, many other organs and tissues may be affected.
Mycobacterium tuberculosis
Microbiologic characteristics of TB?
Aerobic and nonmotile.
No endospores or capsules
Gram-positive.
Cell wall, thicker, hydrophobic, waxy, and rich in mycolic acids/mycolates. Some species are fastidious.
TB adapts readily to growth on very simple substrates, using ___________ or amino acids as nitrogen sources and __________as a carbon source in the presence of mineral salts.
ammonia, glycerol
What is the optimum growth for TB?
25 to over 50 degrees Celsius
General symptoms of TB?
Fever, fatigue, night sweats, weight loss & malaise
Septicemia, multiple organ failure
Weight loss despite increased appetite – “consumption”
Symptoms of pulmonary tuberculosis?
pneumonia, chronic cough and coughing up blood
Symptoms of digestive tuberculosis?
abdominal pain and diarrhea, fever, weight loss. Bowel obstruction or bleeding may occur.
Symptoms of renal tuberculosis?
dysuria, hematuria and flank pain. WBCs in urine.
What are the virulence factors of TB?
-Waxy cell wall.
-Major factor is ability to invade and survive within macrophages* as surface protein called “exported repetitive protein” prevents phagosome from joining with lysosome.
-It produces no exotoxins or no LPS.
How are TB mycobacterial antigens classified as?
1- Soluble (cytoplasmic) and insoluble (cell wall lipid bound).
2- Carbohydrates or proteins
3- by their distribution within the genus
How is TB transmitted? and how does the disease progress?
by droplets from person with active case of tuberculosis.
The microorganism is very stable in sputum droplets and can remain viable in very even dry sputum for up 6 days.
M. tuberculosis in droplets is then inhaled and reach the highly aerobic environment of the lung where it produces non-specific pneumonitis
-inflammation —> “exudative lesion” and end up with granulomas
In TB, Granuloma becomes surrounded by fibrin which calcifies witch is called a ___________, it can be seen by chest X – ray. The infection may stop at this point and the individual may have no more symptoms.
tubercle
What are granulomas composed of?
collection of macrophages
What is a tubercle bacillus?
-macrophage bacteria complex.
-calcified
-enclosed —> no infection unless it bursts
How can TB become reactivated?
What is the mortality rate?
-The tubercle can disseminate throughout the body – becomes systemic infection
-Tubercle can be coughed up and swallowed, becoming systemic via the gastrointestinal tract
-Tubercle can burst years after primary infection
50%
What are the two ways TB can be diagnosed?
- AFB in sputum smear: indicative of tuberculosis.
- M.tuberculosis culture: egg yolk or oleic acid albumin containing media agar; 2-8 wks (Löwenstein–Jensen medium, Liquid BACTEC?)
- Chest X – ray
- Tuberculin skin test (Mantoux Test)
What is the special media for M. tuberculosis?
What agar does the bacillus not grown on?
TheLöwenstein–Jensen medium*
blood agar
medium used for TB that contains radioactive metabolites and growth can be detected by the presence of radioactive carbon dioxide production in about 2 weeks
Liquid BACTEC medium*
How does TB appear when grown on Löwenstein–Jensen medium?
brown, granular colonies (sometimes called “buff, rough and tough”)*
How long must TB be incubated onLöwenstein–Jensen medium?
incubated for a significant length of time, usually four weeks, due to the slow doubling time ofM. tuberculosis(15–20 hours) compared with other bacteria.
Purified protein derivative (PPD), part of cell wall is injected under the skin, 24 – 48 hours measure the size of the welt that forms. Cell have been primed and recognize the PPD.
Tuberculin skin test (Mantoux Test)
What does a positive Mantoux test mean?
the pt. has been exposed to the organism
- Could have active infection
- Could have been infected but were one of the 90% who are Asymptomatic.
- You have been immunized
Mycobacterium leprae staining morphology is virtually indistinguishable from __________.
M. tuberculosis
Mycobacterium leprae has not been cultured on routine mycobacteria media, what media is used instead?
-mouse footpad
-Nine-banded armadillo
-Chronic disease of the skin, mucous membranes, and nerves
-Infectivity very low (requires close contact)
Leprosy (Hansen’s Disease)
What bacteria causes Leprosy (Hansen’s Disease)?
Mycobacterium leprae
Leprosy:
Most pathology is associated with organism’s tropism for…
peripheral nerves
Leprosy:
localized; confined to skin and nerves.
Tuberculoid
Leprosy:
disseminated. Associated with a impaired cell-mediated immunity
Lepromatous
How is Nontuberculous Mycobacteria transmitted?
not usually person to person. Frequently environmental
Isolated from culture: may not indicate disease
What are other names for nontuberculous mycobacteria?
MOTT (Mycobacteria Other Than Tubercle Bacilli), Atypical or Opportunistic
Runyon Classification:
Group I?
Photochromogens (pigment
on exposure to light)
Runyon Classification:
Group II?
Scotochromogens (pigment in dark)
Runyon Classification:
Group III:?
Non-photochromogens
Runyon Classification:
Group IV:?
Rapid Growers growth at 4-6 days
What are two examples in Runyon Group I?
-M. kansasii
-M. marinum
Where is M. kansasii (group I) found?
water
What temp does M. marinum (group I) grow better at?
at 30 degrees Celsius
Where is M. marinum (group I) found?
Found in salt water and fresh water—contamination from infected fish and other wildlife
What diseases does M. kansasii (group I) cause?
Causes chronic pulmonary disease and extrapulmonary diseases such as cervical lymphadenitis and cutaneous disease
What diseases does M. marinum (group I) cause?
Causes cutaneous disease (usually enters through open wounds or traumatic injury)
What are the Runyon Group II examples?
*rarely isolated
-M. gordonae (biosafety level I)
-M. scrofulaceum
-M. szulgai
Runyon Group II:
Water contaminant. Rarely causes disease
M. gordonae (biosafety level I)
Runyon Group II:
Raw milk, soil, water, and other dairy products. Cervical adenitis in children, pulmonary disease, skin infections and bacteremia
M. scrofulaceum
Runyon Group II:
Water and soil. Pulmonary disease, cervical adenitis, and bursitis
M. szulgai
Nonphotochromogens
-Non-pigmented
->7 days to appear on solid media
-Frequently isolated in laboratory
Runyon Group III
Examples of Runyon group III
-M. avium complex
-M. xenopi
-M. ulcerans
-M. haemophilum
-Most common NTM in the USA
-Ubiquitous: water, soil, animals
-Includes many species and subspecies
M. avium Complex
M. avium and ___________ are different organisms but closely resemble each other.
M. intracellulare
-Distinction cannot be made by routine laboratory testing or on clinical grounds
M. avium Complex is frequently called _______.
MAI
What diseases does M. avium complex cause?
-Pulmonary infections in patients with preexisting pulmonary disease
-Cervical lymphadenitis
-Disseminated disease in HIV & AIDS
Runyon Group III –> M. avium complex:
-Buruli ulcer, a neglected tropical disease reported in >30 countries.
-Causes scarring and can progress to bone.
-Grows at lower temperatures.
M. ulcerans
Runyon Group III –> M. avium complex:
-Associated with water and can survive high temperature.
-May cause slowly progressing pulmonary infection
M. xenopi
Runyon Group III –> M. avium complex:
-Skin infections but can disseminate.
-Requires hemin.
-Sub chocolate plate
M. haemophilum
Runyon IV characteristics?
-Grow in <7 days.
-Grow on routine media
-Stain weakly on Gram-stain. -Ubiquitous
-Cause disease in immunocompromised persons
Examples of Runyon IV (account for 90% of clinical diseased caused by RGM)
-M. fortuitum
-M. chelonae
-M. abscessus
Causes post-operative infections of the chest, skin and soft tissue. Localized, pulmonary, and CNS diseases
M. fortuitum (Runyon IV)
Causes skin and soft tissue, postoperative wound infections, and keratitis
M. chelonae (Runyon IV)
causes skin and soft tissue, pulmonary, postoperative infections
M. abscessus (Runyon IV)
__________ ranks high among laboratory-acquired infections.
Tuberculosis
Tuberculosis has a low infective dose, ____% chance of infection with fewer than 10 AFB.
50
What safety measures are made for TB specimen processing?
(Level 2), minimum:
BSCI or II, gloves, limited access, slides heat-fixed within BSC.
What safety measures are made for positive TB cultures?
(Level 3): Lab separated by interlock anteroom and has negative pressure, Type II BSC with UV/HEPA, materials for disposal decontaminated before removal (usually autoclaved)
PPE: gloves, gown, respirator
What type of mycobacteria specimens are acceptable and why?
almost any specimen is acceptable because mycobacteria present with a wide rang of infections.
swabs should not be accepted
Gastric aspirate mycobacteria samples should be processed within ____ hours of collection or neutralized with ____________.
4, 10% sodium carbonate
All mycobacteria samples except blood should be stored how?
refrigerated within 1 hour of collection if not immediately processed
Some specimens can be directly inoculated to media; others must be processed. Processing means?
mucus digestion and decontamination.
What are three ways specimens are processed?
-NaOH
-(N-acetyl-L-cysteine) NALC
-Zephiran-trisodium phosphate & Oxalic acid
________ acid is sometimes used for specimens contaminated with Pseudomonas or Proteus.
Oxalic
digestant (liquefies the sample)
(N-acetyl-L-cysteine) NALC
-NALC-NaOH (most
common)
Used for decontamination (mucolytic as well)
NaOH
-too much NaOH can also kill Mycobacteria
What is the disadvantage of the strong alkaline reagent usually used for the decontamination step?
it is also toxic to mycobacteria
Steps of specimen processing?
-Add NaOH/NALC-digestion and decontamination
-Vortex, let sit for 15 min
-Add buffer to stop the reaction; centrifuge at high speed
-Pour off the supernatant and make smears and inoculate solid and liquid media.
-monitor
What are the steps for quality control for Decontamination Process?
-Prepare a simulated sputum sample with 102, 103, and 104 CFU/mL.
-Add to sterile sputum.
-Process as usual and inoculate media.
-Inoculate a set of media without processing.
unprocessed samples would also show __________ growth
reduced
What does the MGIT™ Mycobacteria Growth Indicator Tubes contain?
4 ml of Middlebrook 7H9 Broth base with a fluorescent indicator.
Modified Middlebrook 7H9 broth Growth & Antibiotic supplement _________ sponges provide unique growth matrix
cellulose
Modified Middlebrook 7H9 broth:
broth is contained in a plastic tube with screw-top and is supplemented with _____________ enrichment and _____________ antibiotic mixture.
BD MGIT™ OADC
BD MGIT™ PANTA™
What does the Löwenstein Jensen agar contain?
Contains eggs and
malachite green (inhibits most other bacteria)
How should tubed solid media be incubated?
Slanted position with screw caps loose for 5–7 days, after 7 days caps should be tightened and tubes may be positioned upright. 6-8wks. Liquid media: 6wks
How should plated solid media be incubated?
CO2-permeable plastic bags, medium-side down (5–10% CO2)
What is the optimum temperature for MTBC?
35–37°C. Some NTM grow best at 25–33°C. M. xenopi requires 40–42°C.
How should culture be incubated for skin, bone, and joint biopsies?
inoculate two sets of media including chocolate, one at 35-37°C and the other at the lower optimal temperature (usually 30°C)
-M. marinum, M. ulcerans, M. haemophilium
What are two preliminary identificators on culture?
- rate of growth on solid media
- colonial pigmentation
What is the appearance of M. tuberculosis colonies?
crumbly, and buff colored
What are the microscopy detection methods for AFB?
A. Light microscopy stains
Ziehl-Nielson
Kinyon
B. Fluorescent stain
Fluorochrome
(Auramine-Rhodamine)
Microscopically, Mycobacterium avium-intracellulare infection is marked by numerous acid fast organisms growing within ____________.
macrophages
What are the identification tests based off of?
-Growth rate
-Pigment
-Colony morphology (do acid fast stain to make sure it is an AFB).
-Complex media requirements
The three examples of identification tests?
-The Niacin Test: Mycobacterium tuberculosis produces large amounts of niacin.
-The Catalase Test: M. tuberculosis produces a heat sensitive catalase.
-The Nitrate Test: M. tuberculosis can reduce nitrate.
The niacin test is detected by the addition of what to a suspension of the culture?
10% cyanogen bromide and 4% aniline in 96% ethanol
What color is a positive niacin test?
canary yellow
An example that would be positive for the niacin test? negative?
M. tuberculosis
M. bovis
Is the catalase test quantitative?
semiquantitative
Most mycobacteria have a lipase that can split Tween 80. What are the exceptions?
Mtb, M. avium, M. bovis
What color is a positive Tween 80 test? negative?
positive- pink
negative-orange
What distinguish M. tuberculosis from M. bovis?
TCH (Thiopene-2-carboxylic acid hydrazide)
-M. bovis is susceptible
_______ reduces tellurite in 3 to 4 days, most other nonchromogenic species do not.
MAC
Do most nonchromogenic species reduce tellurite?
No
All rapid growers reduce tellurite in ____ days.
3
What does a positive and negative Tellurite reduction test look like?
positive- cloudy
negative- clear
What organism is strongly positive for the Arylsulfatase test?
What is negative?
M. fortuitum-chelonei
M-avium
What does a positive arylsulfatase test look like?
red/pink
What type of bacteria does the arylsulfatase test detect?
fast growers
What is the principle of the Arylsulfatase test?
Tripotasium phenolphthaleindisulfide/sulfate producing free phenolphthalein by arylsulfatase enzyme
What can have a positive or negative result for Iron uptake?
Fortuitum/chelonae
-Identifies mycobacteria by analysis of mycolic acids
-test is based on separation of mycolic acids
-It is the primary means of ID in health dept/CDC for non-TB mycobacteria
HPLC
-Range of speciation and sensitivity greater than DNA probe (without amplification) and biochemical testing.
-Can be performed directly on smear-positive specimen or on cultures.
HPLC
identification method:
-Limited number of species (MTB complex and MAV complex)
-Uses single-strand DNA probe
-Probe is labeled – often with a chemiluminescent label.
-DNA:RNA hybrid
-A selection reagent removes unbound probe
-Read in luminometer
DNA probe
Most laboratories that identify mycobacteria use automated instruments to detect growth and for susceptibility testing.
Two examples?
BacTec and the mycobacteria growth indicator tube (MGIT).
Automated detection systems:
detects CO2 production produced by viable bacteria.
BacTec
Automated detection systems:
a fluorescence-based system relying on oxygen consumption rather than carbon dioxide production.
MGIT (mycobacteria growth indicator tube)
Bactec NAP test:
if growth is detected in the control bottle but not in the NAP bottle, it can be identified as…
M. tuberculosis
A rapid method of identification, that inhibits Mtb complex
Bactec NAP test
Identification method:
Requires extraction (special sample processing)
-Extract proteins and
inactivate sample
MALDI-TOF
ex: Bruker
What are the advantages of the MALDI-TOF method?
-Rapid identification
-Good identification to species level for rapid growers and most other groups
-MALDI-TOF instrumentation is becoming more commonly available
What are the disadvantages of the MALDI-TOF method?
-Time-consuming extraction protocol
-Cannot differentiation species in MTB complex
-Must have culture growth on solid media
-Many groups currently working on methods for detection directly from liquid media
If results are inconclusive by TB/MAC PCR and MALDI-TOF what should done next?
DNA sequencing
Both Bruker BioTyper and bioMerieux VITEK MS
take ______ hours to process prior to loading on the MALDI-TOF instrument
2-2.5
Bruker BioTyper OR bioMerieux VITEK MS?
-30 minute heat lysis inactivation
-Ethanol wash
-Silica bead disruption with acetonitrile/formic acid extraction
Bruker BioTyper
Bruker BioTyper OR bioMerieux VITEK MS?
-Kit based
-Ethanol/bead inactivation/disruption
-No heat lysis
-Formic acid/acetonitrile extraction
bioMerieux VITEK MS
-Multiplex real-time PCR
-Detection directly from patient sample: Sputum
-2 hour TAT
-Sample-to-answer
-Moderate complexity
GeneXpert – MTB/RIF
GeneXpert – MTB/RIF:
Simultaneous detection of both MTB and _________ resistance mutations, which are markers for MDR-TB strain.
rifampin
A single Xpert MTB/RIF Assay result detected approximately ___% of patients who were acid-fast bacilli (AFB) smear–positive and culture-confirmed as infected with Mycobacterium tuberculosis complex (MTBC)
97
Two serial Xpert MTB/RIF Assay results detected ____ % of AFB smear–positive/MTBC culture-positive patients.
100
One or two Xpert MTB/RIF Assay tests detected ____% and ____%, respectively, of sputum specimens that were AFB smear–negative but culture-positive for MTBC.
55, 69
What is the treatment for acid-fast bacteria?
Long-term therapy (6–9 months) with antituberculous drugs (isoniazid, rifampicin, pyrazinamide, ethambutol).
-Combination therapy should always be given*
What is a growing and a persistent problem with treatment?
Drug Resistance
What is a prevention method for TB?
bacille Calmette–Guérin (BCG) vaccination containing live attenuated organisms, in childhood.
What does MDR-TB stand for?
Multi-drug Resistance tuberculosis
A global program that manages regimentation of drug delivery as a cornerstone of managing TB disease
directly observed therapy (DOT) ?
Some cases of human contracting ______ are from vaccine, or from drinking unpasteurized milk from infected cow.
M. bovis
What is the vaccine for TB?
BCG (Bacilli Calmete Guerin) vacine
The BCG vaccine uses live attenuated ________, causes TB in cows.
M. bovis
How does the BCG vaccine work?
-Made to stimulate the cell-mediated immune response as M. tuberculosis is largely an intracellular pathogen.
-Most effective in children, given as part of early childhood regime in endemic regions.
What is used for prevention of TB?
Chemoprophylaxis
-Isoniazid given to people who were in contact
What is used for treatment of latent TB?
Isoniazid and/or Rifampin (used together, 12 dose min)
What is the first “new” TB antibiotic in 40 years?
Diarylquinolines
-Inhibit ATP production
-Novel mechanism
-Shorter course of therapy
What does DOTS stand for?
direct observation treatment system
What is the proposed reference method for MTB susceptibility testing with pyrazinamide?
radiometric BACTEC TB 460 (BD Biosciences, Sparks, MD)
What is the reference MTB susceptibility testing method for all drugs except pyrazinamide?
Agar proportion
What is the TAT for MTB susceptibility testing?
3 weeks
Resistance definition…
therapy is less likely to be clinically successful when >1% of the bacterial population being tested in vitro is resistant.
What are the two FDA-cleared systems for MTB susceptibility testing?
-Radiometric BACTEC TB 460
-ESP Culture System II (Trek Diagnostics, Westlake, OH)
How is susceptibility testing done for resistant MTB strains?
Molecular testing
What is the most frequently used method for susceptibility testing of MTBC in the U.S.?
Radiometric BACTEC TB 460
Susceptibility testing:
What are the primary drugs that are recommended by the NCCLS subcommittee for testing against isolates of MTBC?
isoniazid (at 2 concentrations)
and rifampin, ethambutol, and pyrazinamide (1 concentration of each).
What is the TAT for susceptibility testing of MTBC?
7-10 days
