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Mycobacteriology
Update 2024
Margie Morgan, PhD, D(ABMM)
Mycobacteria
 Acid Fast Bacilli (AFB)
 Termed Acid Fast for once organisms stained, resist de-colorization with
acid alcohol (HCl)
 Thick outer cell wall made of complex mycolic acids (mycolates) and
free lipids contribute to staining traits and the hardiness of the genus
 How does AFB staining compare to partial acid fast (PAF) staining ?
 AFB stain uses HCl to decolorize Mycobacteria stain (+) Nocardia (-)
 PAF stain H2SO4 to decolorize Mycobacteria stain (+) Nocardia (+)
 Faintly stain on Gram stain as beaded Gram-positive rods
 Aerobic, no spores produced, and the rods rarely branch
AFB stain Gram stain
AFB Laboratory Safety
Level 3 biosafety precautions required in AFB laboratories that process, identify and perform
susceptibility testing of mycobacteria. BSL-3 requirements include:
 Restricted access to laboratory
 Anteroom for donning personal protective equipment (PPE) and exit hand washing
 PPE include:
 N95 mask (yearly mask fit) or PAPR (powered air purifying respiratory mask)
 Disposable surgical gowns and gloves
 Negative air flow that pulls air into the BSL-3 laboratory (6-12 air exchanges per hour)
 Level 2 HEPA- filtered biosafety cabinet with return air vented to outside
 Cabinet certified at least yearly to insure proper operation
 Autoclave within the BSL-3 laboratory for decontamination of all waste
Level 2 HEPA filtered Biosafety Cabinet
PAPR
Return air
vented to the
outside
N95 Mask
PPD (Purified Protein Derivative)
Mantoux test or TB skin test
 Detects latent or current TB complex exposure and/or infection by detecting
delayed hypersensitivity (T cell reactivity) to TB complex antigens
 False positive reactions occur in patients immunized with BCG
 BCG = Bacillus of Calmette-Guerin (live attenuated Mycobacterium bovis)
 False negative reactions (25%) can occur due to patient having low T cell
numbers or T cell reactivity
 Technical problems with PPD administration and subjective test interpretation
can lead to both false negative and false positive results
 Measure (mm) area of induration at injection site
 >=15mm positive result
 >=10mm positive in immune suppressed or early detection in an outbreak investigation
Positive = measure
the area of induration
Cell Mitogen Assays –
Interferon Gamma Release Assays (IGRAs)
 QuantiFERON-TB-Gold Plus (QFTP) most used
 CD4 and CD8 T cells in patient whole blood are stimulated in-tube with TB
specific antigens
 If patient has active or latent exposure to TB, the stimulated T cells will be
induced to produce a measurable amount of gamma interferon
 Of note: Overwhelming active TB infection may suppress the cellular immune response
(T cells) and result in a negative IGRA assay
 Enzyme immunoassay measures the amount of gamma interferon produced
 A quantitative endpoint determines a positive or negative reaction
 Indeterminate reactions can occur if T cell numbers are low and/or inactive due to
medications or immune system disease
 Specific for M.TB, subjects with BCG immunization will test negative
 Sensitivity >=80%,
 Should NOT replace culture for disease diagnosis
 Similar sensitivity to PPD/ but much improved specificity for TB diagnosis
Mycobacteria Taxonomy (2 groups @ 200 species)
 Group 1 - TB complex organisms
 Mycobacterium tuberculosis
 M. bovis
 Includes the Bacillus Calmette-Guerin (BCG) strain
 Attenuated strain of M. bovis used for vaccination against TB
 M. africanum
 Rare species of mycobacteria
 Mycobacterium microti
 Mycobacterium canetti
 Mycobacterium caprae
 Mycobacterium pinnipedii
 Mycobacterium suricattae
 Mycobacterium mungi
 Group 2 - Mycobacteria other than TB complex (“MOTT”)
also known as the Non-Tuberculous Mycobacteria
Non-Tuberculous Mycobacteria (MOTT) that cause disease
(1) Slow growing Mycobacteria (> 7 days)
 M. avium-intracellulare complex
 M. genavense
 M. haemophilum
 M. kansasii (3) Mycobacterium leprae
 M. malmoense
 M. marinum
 M. simiae
 M. szulgai
 M. ulcerans
 M. xenopi
 M. smegmatis
(2) Rapid growers (growth in < = 7 days)
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum
Mycobacteria that rarely if ever cause disease!
If so, only in the severely immunocompromised!
Slowly growers
 Mycobacterium gordonae
 M. gastri
 M. celatum
 M. scrofulaceum
 Mycobacterium (Mycolicibacter) terrae complex
Mycobacteria Taxonomy Rearrangement
 Historically: One genera Mycobacterium, but what was one is now five!
 Mycobacterium genera remains and includes the major human pathogens
New taxonomic genera:
 Mycolicibacterium: type species M. fortuitum
 Mycolicibacter: type species M. terrae
 Mycolicibacillus: type species M. trivale
 Mycobacteroides: type species M. abscessus and M. chelonae
 The division of mycobacterial species into five distinct groups can focus on
studying of unique genetic characteristics that differentiate members
 Updated name(s) format: Mycolicibacterium (Mycobacterium) fortuitum
 This shakes up the taxonomy and may add confusion before bringing clarity
Mycobacteria Identification over the decades
 For decades, Runyon classification used to place mycobacteria into select
groups. Testing determined the ability of a mycobacteria species to form
a yellow carotenoid pigment when exposed to light or in the dark, followed
with select biochemical reactions, determining growth rate and optimum
temperature for growth. Obsolete
 With expanding taxonomy, biochemical reactions were unable to identify
newly recognized species, so High Performance Liquid Chromatography
(HPLC) became useful. Obsolete
 DNA/RNA hybridization probes – no longer available. Obsolete
Current Mycobacteria Identification Methods
 MALDI-TOF (Matrix Assisted Laser
Desorption/Ionization Time of Flight)
 Mass Spectrometry analyzes cellular proteins,
producing spectral peaks that are computer analyzed,
providing Genus and Species identification.
 MALDI-TOF is rapid, precise, easy to perform, and relatively
inexpensive following instrument acquisition.
 Sequencing - 16S rRNA gene sequencing of
organism grown in culture for difficult to identify
species and resistance marker information
 PCR & 16 S rRNA sequencing methods developed for
direct patient specimen testing or positive cultures
Direct detection of TB complex from respiratory
specimens using molecular amplification (PCR)
 FDA cleared assays for rapid detection of TB complex in sputum.
 Detects TB complex gene sequence and Rifampin resistance gene (rpoB)
 Sensitivity of assays
 99% for AFB specimens with a positive concentrated AFB smear
 <=75% for AFB specimens with a negative concentrated AFB smear
 Test of diagnosis, not cure
 Diagnosis: One positive TB PCR can support diagnosis of TB in highly suspicious patient
 Residual rRNA and DNA can be present for up to 6 months after initial diagnosis and
start of appropriate therapy, so this test cannot be used for evidence of cure
 Two negative TB PCR assays and 3 negative AFB smears exclude the diagnosis of TB
 AFB culture and sensitivity must always be performed in addition to PCR, to
confirm the results of the amplification assay and provide additional
information about the organism.
Identification of Non-Tuberculus
Mycobacteria, The old way…..
 Runyon Classification System
 (1) Establish if an organism can produce a yellow pigment when
incubated in the dark, or only when exposed to 8 hours of incandescent
light (The Light Test), or no pigment is produced in light or dark
 (2) Is the growth rate of mycobacteria <=7 days
 Light test categorizes into Four Runyon groups:
 Photochromogen Pigment produced only when exposed to light
 Scotochromogen Pigment produced in both light and dark
 Non-photochromogen No pigment produced in light or dark
 Rapid Grower Growth rate <= 7 days
Runyon Classification System / Organisms
 Runyon Group I – Photochromogen – bright yellow
pigment produced only when exposed to light
 M. kansasii
 M. simiae
 M. szulgai is photochromagen when incubated at 25˚C*
 M. marinum
 Runyon Group II – Scotochromogen – bright yellow
pigment produced in light or dark
 M. gordonae
 M. scrofulaceum
 M. xenopi (most strains)
 M. szulgai is scotochromogen when incubated at 37°C*
Runyon Classification continued
 Runyon Group III – Non-photochromogen – No
pigment produced in light or dark
 M. avium-intracellulare complex
 M. haemophilum
 M. genovense
 Runyon Group IV – Rapid growers – Growth
within 7 days
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum
Specimens for AFB Culture
 Respiratory specimens
 Expectorated Sputum – 3 early morning collections (old way), to save time now
1 early morning, plus 2 collected at least 8 hours apart (3-5 ml of sputum)
 Also, can test induced sputa, bronchial lavage or endotracheal aspirate
 Non-Respiratory specimens
 Tissues, lesions, stool
 Sterile body fluids including CSF
 Urine – 3 to 5 early morning collections
 Gastric – collected in children, due to difficulty in collecting sputum, must
neutralize with sodium bicarbonate to pH of 7.0 after collection
 Blood and bone marrow
 Automated systems for detection using culture bottles
manufactured specifically for AFB detection
 If Transport to a reference laboratory is necessary must use Category B
packaging, carefully sealed transport containers to protect all those in the
transport process
Processing for potentially contaminated specimens
3 - 5 ml of specimen in conical thick plastic Falcon tube
 (1) Decontaminate and liquify specimen for 15 minutes with:
 5 ml of 2% NaOH which increases the specimen pH to 9 and
kills contaminating bacteria in the specimen
 0.5% N-acetyl-L-Cysteine added to liquify the sputum
 (2) Neutralize specimen following decontamination by adding
phosphate buffer to the top of the tube to adjust pH back to 7.0
 (3) Centrifuge for 30 minutes
 (4) Pour off supernatant
 (5) Remaining Pellet used to prepare a concentrated slide prep
 Dilute the pellet with small amount of sterile saline for culture prep
Oxalic acid used for decontamination of CF specimen to
eliminate mucoid strains of Pseudomonas aeruginosa
 2% NaOH will not kill mucoid strains of Pseudomonas
 Oxalic acid should not be used routinely for processing all
specimens for it would decrease yield of AFB in cultures
 Oxalic acid will kill AFB if left on specimen for > 15
minutes.
Specimen decontamination of respiratory specimen
collected from cystic fibrosis patient
Specimen centrifugation
 Thick plastic tube used for specimen processing to withstand rapid
centrifugation speeds without cracking
 Centrifugation at 3000 X g (high speed) using tightly sealed safety cups
to protect from specimen leakage into the centrifuge from cracked tubes
 Speed of centrifugation is important
 AFB cell wall contains high % lipid, will float if not centrifuged at high speed
 Must firmly pellet so AFB are not decanted with supernatant
 Sensitivity of the AFB stain and culture dependent upon pelleting of AFB
Media types used for cultures
 Middlebrook – Synthetic media with optimized
and defined chemical ingredients. Many
formulations (7H9, 7H10, 7H11 with supplements)
 Clear colored solid and liquid media
 Used for both culture and susceptibility testing
 Sterilize by autoclaving
 Lowenstein-Jensen – Hen’s egg with glycerol and flour
 Solid agar, green due to addition of malachite green
 Used only for culture
 Sterilize by inspissation – drying
 Solid agars incubated at 37˚C , 5-10% C0₂, for 8 weeks
AFB automated detection
BACTEC MGIT 960 for automated growth detection of AFB
 Middlebrook 7H9 liquid medium with a growth indicator disc in the bottom
of the culture tube (observe arrow)
 Detection method
 As AFB grow in the 7H9 medium, the AFB respire CO₂ and the amount
of O₂ decreases. The decreasing amount of O₂ causes fluorescence of the
indicator disc at the bottom of the tube which indicates growth. The
development of fluorescence triggers a positive alarm on the instrument.
 Incubation in instrument for 6 weeks at 37˚C for “No Growth” final report
 BACTEC NAP test for identification of TB complex using MGIT 960
NAP = chemical (p-nitro-α-acetylamino-B-hydroxypropiophenone)
Inoculate AFB in tube containing NAP, incubate in instrument for 5 days.
 TB complex does not grow in the tube containing NAP
 All non-tuberculous mycobacteria species grow in the NAP solution
MGIT 960
Acid Fast Stains for Mycobacteria
 Carbol fuchsin based stains
 Carbol fuchsin is a red colored primary stain with
affinity to the lipid mycolic acids in AFB cell wall
 Potassium permanganate or methylene blue is
the blue-colored counterstain
 Two carbol fuchsin stain methods:
 Ziehl-Neelsen (ZN) – heat used to drive carbol
fuchsin into lipid laden mycobacteria (AFB)
 Kinyoun – high % of phenol drives stain into lipid
layer of mycobacteria (AFB)
 Read numerous microscopic fields for 5 minutes,
using light microscopy and 100x oil objective,
observe for pink to reddish stained organisms
Fluorochrome stain
 Auramine Rhodamine
 Mycobacteria stain fluorescent yellow with black background
 Nonspecific fluorochrome binds to the lipid mycolic acids
present in the mycobacteria cell wall
 Read using 25X or 40X lens for 2 min, viewing numerous
fields using a fluorescence microscope
 Considered more sensitive than carbol fuchsin based stains
for concentrated patient specimen slide examination, mostly
due to the vivid fluorescence of the stained organism.
Acid Fast Mycobacteria morphology
M. avium complex
Short rods / tend to
randomly clump.
M. tuberculosis - Organisms
are long, often beaded, and can
appear as if they are sticking
together [due to cord factor = lipid]
In broth
cultures of M. TB
ropes of AFB
can form due to
cord factor
M. kansasii – organisms
are large, beaded, and tend to
randomly clump, occasionally
bend in what is known as
Shepherd’s crook morphology.
Mycobacterium tuberculosis
 Optimal growth temp 37˚ C in 12 –25 days
 Buff colored, dry cauliflower-like colony
 Manual biochemical tests for identification – old way
 Positive Niacin accumulation test
 Niacin produced from growth of TB on egg containing medium (LJ)
 Positive Nitrate reduction test
 Currently used identification methods:
 MALDI-TOF mass spectrometry
 16S rRNA sequencing
Mycobacterium tuberculosis
Cord factor – Due to the high lipid
content in the cell wall, organisms stick
together and develop long ropes of
organisms when grown in broth media –
this staining appearance is unique to M.
tuberculosis
Long beaded AFB / stick together
Susceptibility testing of TB
Liquid 7H9 medium containing anti-TB antibiotic solutions, analyzed on the
automated BACTEC MGIT 960 system
 Primary TB drug panel consists of 5 antibiotics:
 Isoniazid Ethambutol Pyrazinamide
 Rifampin Streptomycin
 If resistant to at least Isoniazid and Rifampin, considered a “multi-drug
resistant TB” and second line drugs should be tested
 2nd line: Fluoroquinolones, Kanamycin, Amikacin, and Capreomycin
 If resistant to 2nd line agents, it is an “Extensive drug resistant TB”
 Whole–genome sequencing may disclose additional resistance mechanisms
 If patient culture remains positive after four months of treatment, the isolated
organism must be retested for possible drug resistance
Tuberculosis
 Primary tuberculosis is a slowly progressive pulmonary infection with
cough, weight loss, and low-grade fever.
 Tuberculosis presentation can vary in the immune suppressed:
 Ghon complex: Primary lesion in lung (tuberculoma) with associated hilar lymph
node involvement
 Miliary TB: Wide-spread dissemination of infection via the bloodstream, occurs
most often in AIDS, elderly, children, immunosuppression and with some
medications (Remicade-infliximab)
 Secondary TB: occurs mostly in adults as a reactivation infection
 Granulomatous inflammation is more florid and widespread than in primary disease.
 Upper lung lobes are most affected, and cavitation is common
 Tuberculosis is spread by respiratory droplets, so all patients suspicious for TB require
respiratory isolation when hospitalized
TB infection with
lung nodules
Pathology of Mycobacterium tuberculosis
• M. tuberculosis infected tissue usually presents with a necrotizing
granulomatous inflammation, composed of epithelioid histiocytes
surrounding a central necrotic zone with variable number of
multinucleated giant cells and lymphocytes.
• Of note: non-necrotizing granulomas can also be present in TB
Nodule
with
cavitation
Necrotizing
granuloma
Non-necrotizing
granuloma
Normal lung tissue
TB in HIV/AIDS patients
 TB is a common opportunistic infection in HIV/AIDS
 Trend toward being multi-drug resistance (INH and Rifampin)
 Progressive decline of cell mediated immunity (low CD4 count) makes
for greater risk of extra pulmonary (miliary) dissemination
 Granulomas with and without caseation can be seen
Miliary TB
M. tuberculosis outside the lung
 Scrofula –Unilateral lymphadenitis (cervical lymph node)
most often seen in immunocompromised adult or child
 Fine needle aspiration to obtain diagnostic specimen
 Caseous necrosis (coagulative and cheese-like) often present
 M. tuberculosis most common agent in adults
 M. avium complex and other MOTT (2-10%) in children
 Pott’s disease - Infection of the spine
 Usually, secondary infection from progressive pulmonary infection
 Manifests as a combination of osteomyelitis and arthritis
that usually involves more than 1 vertebra.
Pott’s disease
Scrofula
Mycobacterium bovis
 Produces disease in warm blooded animals, cattle most common
 Human infection by inhalation in nature or ingesting raw milk products
 Disease states in humans closely resemble those of TB
 Biochemical differentiation of M. bovis and M. tuberculosis:
M. bovis M. TB
Nitrate/ Niacin accumulation Negative Positive
Growth in T2H* No growth Growth
Pyrazinamidase enzyme produced Negative Positive
Pyrazinamide susceptibility Resistant Susceptible
*(Thiophene-2-carboxylic hydrazide)
 M. bovis BCG (Bacillus Calmette-Guerin)
 BCG is a live attenuated strain of M. bovis
 BCG used for TB vaccination in countries with high prevalence of TB
 Intravesical administration of BCG into bladder used for immuno-therapy to treat bladder
cancer / occasionally can replicate and cause a urinary/bladder infection with possibly
disseminated infection
Mycobacterium ulcerans
 Infection known as Buruli ulcer in Africa and Bairnsdale ulcer in Australia
 Endemic in tropical areas with limited medical care
 Infection begins following mild skin trauma exposed to contaminated stagnant water
(containing M. ulcerans), and this leads to formation of a skin nodule
 Skin nodules progress to form destructive/debilitating ulcers, peak age group 5-15yrs
 Ulcerations develop due to production of mycolactone (cell cytotoxin)
 Optimum growth temp @ 30˚ C* / Does not grow well or at all at 37*C
 Note: All skin lesions suspicious for AFB should be cultured at both 30˚ & 37˚C
 Non-photochromogen, genetically related to M. marinum (another water borne AFB)
 Culture, AFB stain, molecular testing (PCR) and histology can assist with diagnosis
Mycobacterium kansasii
 Clinical disease mimics pulmonary TB
 Disease usually acquired from contaminated tap water
 Predisposition for diseased lung (COPD, pneumoconiosis)
 More likely seen in immune suppressed, alcohol abuse, and HIV
 Produces granulomatous inflammation in lung and unlikely
to disseminate to bloodstream or other organs
 Culture 37* C, growth in10-20 days
 Photochromogen
 Niacin accumulation test negative
 Nitrate reduction positive
 Tween 80 positive / for lipase enzyme
 68*C catalase positive
 AFB larger than those of TB, rectangular shaped and very beaded
sometimes with Shepherd’s crook morphology
No light
Light
exposure
Mycobacterium marinum
 Source is contaminated fresh and salt water due to
diseased fish living in water
 Infection associated with skin trauma occurring in
infected waters
 Swimming pools (swimming pool granuloma)
 Cleaning fish tanks with bare hand and arm
 Ocean (surfing)
 Punctures from fish fins
 Lesion culture - optimum temp for growth is 30˚ C
with growth in 5-14 day
 No unique biochemical reactions of note
 Identify using MALDI-TOF or Sequencing
Mycobacterium marinum
 Tender, red or blue/red subcutaneous nodules develop at the site of
trauma/infection develops 2-3 weeks following injury
 Lesions classically spread along lymphatics, extending up the arm
 M. marinum infection can resemble those caused by other organisms, such as
Sporotrichosis, Nocardiosis, and rapid growing Mycobacteria
 Diagnostic biopsy of skin nodule: histopathology and culture to
establish diagnosis
Pseudoepitheliomatous
downgrowth into the dermis into a
region of necrosis and
inflammation.
Mycobacterium szulgai
 Unique fact: Scotochromogen at 37˚C /
Photochromogen at 25˚C
 Only AFB species that has a different light test result
based on temperature of incubation
 Growth at 37 ˚C in 12 - 25 days
 Causes pulmonary disease in adults, similar to
TB, associated with alcohol abuse, smoking,
COPD, AIDS and immune suppression
25˚ C - Photochromogen
37˚ C - Scotochromogen
Mycobacterium xenopi
 Thermophilic AFB, grows at 42˚C (high temp for AFB)
 Capable of growing in hot water systems, aerosolization of water from
systems, is a common sources of infection
 Can occur as colonization or cause of infection, need to investigate
patient factors to establish diagnosis
 Growth in 14 - 28 days
 Egg nest–like colony produced on solid media
 Scotochromogen (pigment produced in light and dark)
 Rare cause of pulmonary disease:
 Clinical disease like TB
 Occurs in patients with preexisting lung disease (chronic obstructive
pulmonary disease or bronchiectasis) and HIV/AIDS
Mycobacterium genavense
• Fastidious, thermophilic, slowly growing AFB species
(growth in 3-12wks)
• Better recovery in Middlebrook liquid medium with
acidic pH (5) with iron supplementation
• Disease is usually associated with immunosuppression.
• Sites of infection include:
• Lymphadenitis, bone marrow, liver, spleen, bowel,
blood, genital, and soft tissue infections.
• Appearance in fixed tissue is very similar to M. avium
complex, short rods with random clumping
• One of the most common mycobacteriosis in pet birds
Bone marrow; Kinyoun stain
Mycobacterium avium complex (MAC)
 Complex includes eight AFB species of environmental and animal origin
 M. avium, M. intracellulare, M indicus pranii, M chimaera, M arosiense, M vulneris, M
bouchedurhonense, M colombiense, M marseillense, M yongonense, and M
timonense (M. avium and M. intracellular most common species)
 Non-photochromogen
 Species biochemically inert and genetically very similar
 Growth at 37 ˚C in 7 – 21 days
 Smooth / creamy colony / buff colored
 Short rods, not beaded, irregular clumping of AFB in stains
 Identification:
 MALDI-TOF mass spectrometry- method of choice to identify to species
 16s rRNA sequencing (identify to species)
M. avium complex clinical correlation
 Disease in the somewhat normal host:
 Adult: chronic pulmonary disease, fibro-cavitary or nodular
bronchiectasis presentation
 Produces chronic cough in elderly with prior lung damage (COPD)
 Children: Scrofula with chronic granulomatous inflammation and
lymph node involvement
 M. chimaera (emerging species within the M. avium complex)
 Environmental (airborne transmission) and nosocomial pathogen
 Pulmonary and extra-pulmonary infections
 Established as a nosocomial contaminate (2016) during the
manufacturing of heater–cooler units used during cardiac surgery.
Aerosolized water sprayed from the instrument contaminated the
surgical field with M. chimera leading to post surgical infections
M. avium complex disease in HIV/AIDS
 Common opportunistic infection, symptoms include:
 Nonspecific low-grade fever, weakness, weight loss,
picture of fever of unknown origin rather than pulmonary
infection
 Diagnosis: Isolation in culture from respiratory, blood or
bone marrow, AFB staining of bone marrow
 Abdominal pain and/or diarrhea with malabsorption
 Diagnosis: positive stool AFB smear and culture
 In tissue:
 Pathology: non-necrotizing granulomas
 High organism load can be present in infected tissue
 AFB are small (short) rods and not beaded and occur in
irregular clumps
Bowel -Lamina propria expanded from predominately
Lymphohistocytic infiltration
M. avium complex in the bowel
Mycobacterium haemophilum
 Fastidious slow growing, requiring the addition of
hemoglobin or hemin to culture media for growth
 Will not grow on LJ media, Middlebrook, or in automated
system media (Middlebrook 12B media) without the
addition of hemin supplement
 Will grow on chocolate agar which contains hemin
 Grows best at 30*C, poor growth at 37*C
 Diseases:
 Painful subcutaneous nodules and ulcers isolated
primarily in patient with AIDS patients or
immunosuppressed
 Lymphadenitis in children
Mycobacterium gordonae
Rarely if ever causes disease
 Commonly found in tap water and is the most common
AFB culture contaminant.
 Contamination can occur during specimen collection or
laboratory processing.
 Must use sterile distilled water for processing to prevent such
contamination.
 Scotochromogen
 Need to accurately identify so not confused with a true
pathogen
 MALDI-TOF
 Sequencing
 Growth in <=7 days at 35*/37*C
 Routine bacteriologic cultures can isolate due to rapid growth
 20 species, environmental organisms found in soil and
water and can be transmitted from contaminated
medical supplies (nosocomial transmission)
 The 3 most common human pathogens:
 M. fortuitum skin and surgical wound infections
and catheter sites
 M. chelonae skin infections in immune
suppressed and catheter sites
 M. abscessus chronic lung infection (cystic
fibrosis) and skin infection in immune
suppressed
 In fixed tissue, elicit a mixed response of
granulomatous and/or pyogenic reaction
Rapidly Growing Mycobacteria
Intraepithelial abscess
M. abscessus
Rapid growers
 Growth on 5% Sheep’s blood agar and AFB media as a
non pigmented dry gray colony
 Less beaded on Gram stain than most AFB species and have weak
AFB staining due to low cell wall lipid content
 Biochemical reactions:
 All species Arylsulfatase positive
 M. fortuitum: Nitrate reduction positive
(Mycolicibacterium) Iron uptake positive
 M. chelonae: Nitrate negative No growth 6.5% salt
(Mycobacteroides)
 M. abscessus: Nitrate negative Growth in 6.5% salt
(Mycobacteroides)
 Identification to species using MALDI-TOF and sequencing
Gram stain
Rapid growing (MGM) susceptibility testing
 Antibiotic therapy is assisted by directed
susceptibility testing
 Clarithromycin is susceptible to most MGM
species and is primary therapy for infections
 Clarithromycin and other appropriate antibiotics can be
tested by broth multi-dilution (MIC) testing or Etest
 Molecular genetic testing for the erm gene can
assist with the rapid and precise detection of
clarithromycin resistance
 M. abscessus and M. fortuitum isolates should be tested
for erm gene activity, to more sensitively detect
inducible resistance of the organism to clarithromycin
Etest
Microdilution
Mycobacterium leprae complex
 Leprosy also known as Hansen’s Disease
 Global health concern with @ 200,000 cases/year. Endemic in India, Brazil
and Indonesia, @ 100 cases in US per year
 Affects skin, mucous membranes, and peripheral nerves,
 Infection spread via inhalation of infectious droplets from humans
 Armadillo is zoonotic reservoir of M. leprae complex
 Begins with skin lesion (hypopigmented or red lesion) and/or enlarged
nerve(s) and sensory loss.
 Untreated, can progress to a peripheral neuropathy with nerve thickening,
numbness in earlobes or nose, and loss of eyebrows
 Curable with early diagnosis and appropriate therapy (dapsone plus
rifampin or clofazimine). BCG vaccine is partially protective (50%)
 Non-culturable
 Diagnosis: AFB staining of infected tissue and PCR assays (Public Health)
Tuberculoid leprosy/Paucibacillary
Less severe lesions
Less AFB seen in stains of lesions
Lepromatous leprosy/Multibacillary
Severe disfiguring lesions, low immune response
Large numbers of AFB in lesions
Skin biopsy - AFB seen in nerve fiber (FITE stain)
M. leprae has less mycolic acid
in the cell wall than other
Mycobacteria spp and stain
more reliably on Fite stain than
ZN/Kinyoun type AFB stains.
AFB in cigar
packet
arrangement

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Mycobacteriology update 2024 Margie Morgan.ppt

  • 2. Mycobacteria  Acid Fast Bacilli (AFB)  Termed Acid Fast for once organisms stained, resist de-colorization with acid alcohol (HCl)  Thick outer cell wall made of complex mycolic acids (mycolates) and free lipids contribute to staining traits and the hardiness of the genus  How does AFB staining compare to partial acid fast (PAF) staining ?  AFB stain uses HCl to decolorize Mycobacteria stain (+) Nocardia (-)  PAF stain H2SO4 to decolorize Mycobacteria stain (+) Nocardia (+)  Faintly stain on Gram stain as beaded Gram-positive rods  Aerobic, no spores produced, and the rods rarely branch AFB stain Gram stain
  • 3. AFB Laboratory Safety Level 3 biosafety precautions required in AFB laboratories that process, identify and perform susceptibility testing of mycobacteria. BSL-3 requirements include:  Restricted access to laboratory  Anteroom for donning personal protective equipment (PPE) and exit hand washing  PPE include:  N95 mask (yearly mask fit) or PAPR (powered air purifying respiratory mask)  Disposable surgical gowns and gloves  Negative air flow that pulls air into the BSL-3 laboratory (6-12 air exchanges per hour)  Level 2 HEPA- filtered biosafety cabinet with return air vented to outside  Cabinet certified at least yearly to insure proper operation  Autoclave within the BSL-3 laboratory for decontamination of all waste Level 2 HEPA filtered Biosafety Cabinet PAPR Return air vented to the outside N95 Mask
  • 4. PPD (Purified Protein Derivative) Mantoux test or TB skin test  Detects latent or current TB complex exposure and/or infection by detecting delayed hypersensitivity (T cell reactivity) to TB complex antigens  False positive reactions occur in patients immunized with BCG  BCG = Bacillus of Calmette-Guerin (live attenuated Mycobacterium bovis)  False negative reactions (25%) can occur due to patient having low T cell numbers or T cell reactivity  Technical problems with PPD administration and subjective test interpretation can lead to both false negative and false positive results  Measure (mm) area of induration at injection site  >=15mm positive result  >=10mm positive in immune suppressed or early detection in an outbreak investigation Positive = measure the area of induration
  • 5. Cell Mitogen Assays – Interferon Gamma Release Assays (IGRAs)  QuantiFERON-TB-Gold Plus (QFTP) most used  CD4 and CD8 T cells in patient whole blood are stimulated in-tube with TB specific antigens  If patient has active or latent exposure to TB, the stimulated T cells will be induced to produce a measurable amount of gamma interferon  Of note: Overwhelming active TB infection may suppress the cellular immune response (T cells) and result in a negative IGRA assay  Enzyme immunoassay measures the amount of gamma interferon produced  A quantitative endpoint determines a positive or negative reaction  Indeterminate reactions can occur if T cell numbers are low and/or inactive due to medications or immune system disease  Specific for M.TB, subjects with BCG immunization will test negative  Sensitivity >=80%,  Should NOT replace culture for disease diagnosis  Similar sensitivity to PPD/ but much improved specificity for TB diagnosis
  • 6. Mycobacteria Taxonomy (2 groups @ 200 species)  Group 1 - TB complex organisms  Mycobacterium tuberculosis  M. bovis  Includes the Bacillus Calmette-Guerin (BCG) strain  Attenuated strain of M. bovis used for vaccination against TB  M. africanum  Rare species of mycobacteria  Mycobacterium microti  Mycobacterium canetti  Mycobacterium caprae  Mycobacterium pinnipedii  Mycobacterium suricattae  Mycobacterium mungi  Group 2 - Mycobacteria other than TB complex (“MOTT”) also known as the Non-Tuberculous Mycobacteria
  • 7. Non-Tuberculous Mycobacteria (MOTT) that cause disease (1) Slow growing Mycobacteria (> 7 days)  M. avium-intracellulare complex  M. genavense  M. haemophilum  M. kansasii (3) Mycobacterium leprae  M. malmoense  M. marinum  M. simiae  M. szulgai  M. ulcerans  M. xenopi  M. smegmatis (2) Rapid growers (growth in < = 7 days)  M. fortuitum group  M. abscessus  M. chelonae  M. mucogenicum
  • 8. Mycobacteria that rarely if ever cause disease! If so, only in the severely immunocompromised! Slowly growers  Mycobacterium gordonae  M. gastri  M. celatum  M. scrofulaceum  Mycobacterium (Mycolicibacter) terrae complex
  • 9. Mycobacteria Taxonomy Rearrangement  Historically: One genera Mycobacterium, but what was one is now five!  Mycobacterium genera remains and includes the major human pathogens New taxonomic genera:  Mycolicibacterium: type species M. fortuitum  Mycolicibacter: type species M. terrae  Mycolicibacillus: type species M. trivale  Mycobacteroides: type species M. abscessus and M. chelonae  The division of mycobacterial species into five distinct groups can focus on studying of unique genetic characteristics that differentiate members  Updated name(s) format: Mycolicibacterium (Mycobacterium) fortuitum  This shakes up the taxonomy and may add confusion before bringing clarity
  • 10. Mycobacteria Identification over the decades  For decades, Runyon classification used to place mycobacteria into select groups. Testing determined the ability of a mycobacteria species to form a yellow carotenoid pigment when exposed to light or in the dark, followed with select biochemical reactions, determining growth rate and optimum temperature for growth. Obsolete  With expanding taxonomy, biochemical reactions were unable to identify newly recognized species, so High Performance Liquid Chromatography (HPLC) became useful. Obsolete  DNA/RNA hybridization probes – no longer available. Obsolete
  • 11. Current Mycobacteria Identification Methods  MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight)  Mass Spectrometry analyzes cellular proteins, producing spectral peaks that are computer analyzed, providing Genus and Species identification.  MALDI-TOF is rapid, precise, easy to perform, and relatively inexpensive following instrument acquisition.  Sequencing - 16S rRNA gene sequencing of organism grown in culture for difficult to identify species and resistance marker information  PCR & 16 S rRNA sequencing methods developed for direct patient specimen testing or positive cultures
  • 12. Direct detection of TB complex from respiratory specimens using molecular amplification (PCR)  FDA cleared assays for rapid detection of TB complex in sputum.  Detects TB complex gene sequence and Rifampin resistance gene (rpoB)  Sensitivity of assays  99% for AFB specimens with a positive concentrated AFB smear  <=75% for AFB specimens with a negative concentrated AFB smear  Test of diagnosis, not cure  Diagnosis: One positive TB PCR can support diagnosis of TB in highly suspicious patient  Residual rRNA and DNA can be present for up to 6 months after initial diagnosis and start of appropriate therapy, so this test cannot be used for evidence of cure  Two negative TB PCR assays and 3 negative AFB smears exclude the diagnosis of TB  AFB culture and sensitivity must always be performed in addition to PCR, to confirm the results of the amplification assay and provide additional information about the organism.
  • 13. Identification of Non-Tuberculus Mycobacteria, The old way…..  Runyon Classification System  (1) Establish if an organism can produce a yellow pigment when incubated in the dark, or only when exposed to 8 hours of incandescent light (The Light Test), or no pigment is produced in light or dark  (2) Is the growth rate of mycobacteria <=7 days  Light test categorizes into Four Runyon groups:  Photochromogen Pigment produced only when exposed to light  Scotochromogen Pigment produced in both light and dark  Non-photochromogen No pigment produced in light or dark  Rapid Grower Growth rate <= 7 days
  • 14. Runyon Classification System / Organisms  Runyon Group I – Photochromogen – bright yellow pigment produced only when exposed to light  M. kansasii  M. simiae  M. szulgai is photochromagen when incubated at 25˚C*  M. marinum  Runyon Group II – Scotochromogen – bright yellow pigment produced in light or dark  M. gordonae  M. scrofulaceum  M. xenopi (most strains)  M. szulgai is scotochromogen when incubated at 37°C*
  • 15. Runyon Classification continued  Runyon Group III – Non-photochromogen – No pigment produced in light or dark  M. avium-intracellulare complex  M. haemophilum  M. genovense  Runyon Group IV – Rapid growers – Growth within 7 days  M. fortuitum group  M. abscessus  M. chelonae  M. mucogenicum
  • 16. Specimens for AFB Culture  Respiratory specimens  Expectorated Sputum – 3 early morning collections (old way), to save time now 1 early morning, plus 2 collected at least 8 hours apart (3-5 ml of sputum)  Also, can test induced sputa, bronchial lavage or endotracheal aspirate  Non-Respiratory specimens  Tissues, lesions, stool  Sterile body fluids including CSF  Urine – 3 to 5 early morning collections  Gastric – collected in children, due to difficulty in collecting sputum, must neutralize with sodium bicarbonate to pH of 7.0 after collection  Blood and bone marrow  Automated systems for detection using culture bottles manufactured specifically for AFB detection  If Transport to a reference laboratory is necessary must use Category B packaging, carefully sealed transport containers to protect all those in the transport process
  • 17. Processing for potentially contaminated specimens 3 - 5 ml of specimen in conical thick plastic Falcon tube  (1) Decontaminate and liquify specimen for 15 minutes with:  5 ml of 2% NaOH which increases the specimen pH to 9 and kills contaminating bacteria in the specimen  0.5% N-acetyl-L-Cysteine added to liquify the sputum  (2) Neutralize specimen following decontamination by adding phosphate buffer to the top of the tube to adjust pH back to 7.0  (3) Centrifuge for 30 minutes  (4) Pour off supernatant  (5) Remaining Pellet used to prepare a concentrated slide prep  Dilute the pellet with small amount of sterile saline for culture prep
  • 18. Oxalic acid used for decontamination of CF specimen to eliminate mucoid strains of Pseudomonas aeruginosa  2% NaOH will not kill mucoid strains of Pseudomonas  Oxalic acid should not be used routinely for processing all specimens for it would decrease yield of AFB in cultures  Oxalic acid will kill AFB if left on specimen for > 15 minutes. Specimen decontamination of respiratory specimen collected from cystic fibrosis patient
  • 19. Specimen centrifugation  Thick plastic tube used for specimen processing to withstand rapid centrifugation speeds without cracking  Centrifugation at 3000 X g (high speed) using tightly sealed safety cups to protect from specimen leakage into the centrifuge from cracked tubes  Speed of centrifugation is important  AFB cell wall contains high % lipid, will float if not centrifuged at high speed  Must firmly pellet so AFB are not decanted with supernatant  Sensitivity of the AFB stain and culture dependent upon pelleting of AFB
  • 20. Media types used for cultures  Middlebrook – Synthetic media with optimized and defined chemical ingredients. Many formulations (7H9, 7H10, 7H11 with supplements)  Clear colored solid and liquid media  Used for both culture and susceptibility testing  Sterilize by autoclaving  Lowenstein-Jensen – Hen’s egg with glycerol and flour  Solid agar, green due to addition of malachite green  Used only for culture  Sterilize by inspissation – drying  Solid agars incubated at 37˚C , 5-10% C0₂, for 8 weeks
  • 21. AFB automated detection BACTEC MGIT 960 for automated growth detection of AFB  Middlebrook 7H9 liquid medium with a growth indicator disc in the bottom of the culture tube (observe arrow)  Detection method  As AFB grow in the 7H9 medium, the AFB respire CO₂ and the amount of O₂ decreases. The decreasing amount of O₂ causes fluorescence of the indicator disc at the bottom of the tube which indicates growth. The development of fluorescence triggers a positive alarm on the instrument.  Incubation in instrument for 6 weeks at 37˚C for “No Growth” final report  BACTEC NAP test for identification of TB complex using MGIT 960 NAP = chemical (p-nitro-α-acetylamino-B-hydroxypropiophenone) Inoculate AFB in tube containing NAP, incubate in instrument for 5 days.  TB complex does not grow in the tube containing NAP  All non-tuberculous mycobacteria species grow in the NAP solution MGIT 960
  • 22. Acid Fast Stains for Mycobacteria  Carbol fuchsin based stains  Carbol fuchsin is a red colored primary stain with affinity to the lipid mycolic acids in AFB cell wall  Potassium permanganate or methylene blue is the blue-colored counterstain  Two carbol fuchsin stain methods:  Ziehl-Neelsen (ZN) – heat used to drive carbol fuchsin into lipid laden mycobacteria (AFB)  Kinyoun – high % of phenol drives stain into lipid layer of mycobacteria (AFB)  Read numerous microscopic fields for 5 minutes, using light microscopy and 100x oil objective, observe for pink to reddish stained organisms
  • 23. Fluorochrome stain  Auramine Rhodamine  Mycobacteria stain fluorescent yellow with black background  Nonspecific fluorochrome binds to the lipid mycolic acids present in the mycobacteria cell wall  Read using 25X or 40X lens for 2 min, viewing numerous fields using a fluorescence microscope  Considered more sensitive than carbol fuchsin based stains for concentrated patient specimen slide examination, mostly due to the vivid fluorescence of the stained organism.
  • 24. Acid Fast Mycobacteria morphology M. avium complex Short rods / tend to randomly clump. M. tuberculosis - Organisms are long, often beaded, and can appear as if they are sticking together [due to cord factor = lipid] In broth cultures of M. TB ropes of AFB can form due to cord factor M. kansasii – organisms are large, beaded, and tend to randomly clump, occasionally bend in what is known as Shepherd’s crook morphology.
  • 25. Mycobacterium tuberculosis  Optimal growth temp 37˚ C in 12 –25 days  Buff colored, dry cauliflower-like colony  Manual biochemical tests for identification – old way  Positive Niacin accumulation test  Niacin produced from growth of TB on egg containing medium (LJ)  Positive Nitrate reduction test  Currently used identification methods:  MALDI-TOF mass spectrometry  16S rRNA sequencing
  • 26. Mycobacterium tuberculosis Cord factor – Due to the high lipid content in the cell wall, organisms stick together and develop long ropes of organisms when grown in broth media – this staining appearance is unique to M. tuberculosis Long beaded AFB / stick together
  • 27. Susceptibility testing of TB Liquid 7H9 medium containing anti-TB antibiotic solutions, analyzed on the automated BACTEC MGIT 960 system  Primary TB drug panel consists of 5 antibiotics:  Isoniazid Ethambutol Pyrazinamide  Rifampin Streptomycin  If resistant to at least Isoniazid and Rifampin, considered a “multi-drug resistant TB” and second line drugs should be tested  2nd line: Fluoroquinolones, Kanamycin, Amikacin, and Capreomycin  If resistant to 2nd line agents, it is an “Extensive drug resistant TB”  Whole–genome sequencing may disclose additional resistance mechanisms  If patient culture remains positive after four months of treatment, the isolated organism must be retested for possible drug resistance
  • 28. Tuberculosis  Primary tuberculosis is a slowly progressive pulmonary infection with cough, weight loss, and low-grade fever.  Tuberculosis presentation can vary in the immune suppressed:  Ghon complex: Primary lesion in lung (tuberculoma) with associated hilar lymph node involvement  Miliary TB: Wide-spread dissemination of infection via the bloodstream, occurs most often in AIDS, elderly, children, immunosuppression and with some medications (Remicade-infliximab)  Secondary TB: occurs mostly in adults as a reactivation infection  Granulomatous inflammation is more florid and widespread than in primary disease.  Upper lung lobes are most affected, and cavitation is common  Tuberculosis is spread by respiratory droplets, so all patients suspicious for TB require respiratory isolation when hospitalized TB infection with lung nodules
  • 29. Pathology of Mycobacterium tuberculosis • M. tuberculosis infected tissue usually presents with a necrotizing granulomatous inflammation, composed of epithelioid histiocytes surrounding a central necrotic zone with variable number of multinucleated giant cells and lymphocytes. • Of note: non-necrotizing granulomas can also be present in TB Nodule with cavitation Necrotizing granuloma Non-necrotizing granuloma Normal lung tissue
  • 30. TB in HIV/AIDS patients  TB is a common opportunistic infection in HIV/AIDS  Trend toward being multi-drug resistance (INH and Rifampin)  Progressive decline of cell mediated immunity (low CD4 count) makes for greater risk of extra pulmonary (miliary) dissemination  Granulomas with and without caseation can be seen Miliary TB
  • 31. M. tuberculosis outside the lung  Scrofula –Unilateral lymphadenitis (cervical lymph node) most often seen in immunocompromised adult or child  Fine needle aspiration to obtain diagnostic specimen  Caseous necrosis (coagulative and cheese-like) often present  M. tuberculosis most common agent in adults  M. avium complex and other MOTT (2-10%) in children  Pott’s disease - Infection of the spine  Usually, secondary infection from progressive pulmonary infection  Manifests as a combination of osteomyelitis and arthritis that usually involves more than 1 vertebra. Pott’s disease Scrofula
  • 32. Mycobacterium bovis  Produces disease in warm blooded animals, cattle most common  Human infection by inhalation in nature or ingesting raw milk products  Disease states in humans closely resemble those of TB  Biochemical differentiation of M. bovis and M. tuberculosis: M. bovis M. TB Nitrate/ Niacin accumulation Negative Positive Growth in T2H* No growth Growth Pyrazinamidase enzyme produced Negative Positive Pyrazinamide susceptibility Resistant Susceptible *(Thiophene-2-carboxylic hydrazide)  M. bovis BCG (Bacillus Calmette-Guerin)  BCG is a live attenuated strain of M. bovis  BCG used for TB vaccination in countries with high prevalence of TB  Intravesical administration of BCG into bladder used for immuno-therapy to treat bladder cancer / occasionally can replicate and cause a urinary/bladder infection with possibly disseminated infection
  • 33. Mycobacterium ulcerans  Infection known as Buruli ulcer in Africa and Bairnsdale ulcer in Australia  Endemic in tropical areas with limited medical care  Infection begins following mild skin trauma exposed to contaminated stagnant water (containing M. ulcerans), and this leads to formation of a skin nodule  Skin nodules progress to form destructive/debilitating ulcers, peak age group 5-15yrs  Ulcerations develop due to production of mycolactone (cell cytotoxin)  Optimum growth temp @ 30˚ C* / Does not grow well or at all at 37*C  Note: All skin lesions suspicious for AFB should be cultured at both 30˚ & 37˚C  Non-photochromogen, genetically related to M. marinum (another water borne AFB)  Culture, AFB stain, molecular testing (PCR) and histology can assist with diagnosis
  • 34. Mycobacterium kansasii  Clinical disease mimics pulmonary TB  Disease usually acquired from contaminated tap water  Predisposition for diseased lung (COPD, pneumoconiosis)  More likely seen in immune suppressed, alcohol abuse, and HIV  Produces granulomatous inflammation in lung and unlikely to disseminate to bloodstream or other organs  Culture 37* C, growth in10-20 days  Photochromogen  Niacin accumulation test negative  Nitrate reduction positive  Tween 80 positive / for lipase enzyme  68*C catalase positive  AFB larger than those of TB, rectangular shaped and very beaded sometimes with Shepherd’s crook morphology No light Light exposure
  • 35. Mycobacterium marinum  Source is contaminated fresh and salt water due to diseased fish living in water  Infection associated with skin trauma occurring in infected waters  Swimming pools (swimming pool granuloma)  Cleaning fish tanks with bare hand and arm  Ocean (surfing)  Punctures from fish fins  Lesion culture - optimum temp for growth is 30˚ C with growth in 5-14 day  No unique biochemical reactions of note  Identify using MALDI-TOF or Sequencing
  • 36. Mycobacterium marinum  Tender, red or blue/red subcutaneous nodules develop at the site of trauma/infection develops 2-3 weeks following injury  Lesions classically spread along lymphatics, extending up the arm  M. marinum infection can resemble those caused by other organisms, such as Sporotrichosis, Nocardiosis, and rapid growing Mycobacteria  Diagnostic biopsy of skin nodule: histopathology and culture to establish diagnosis Pseudoepitheliomatous downgrowth into the dermis into a region of necrosis and inflammation.
  • 37. Mycobacterium szulgai  Unique fact: Scotochromogen at 37˚C / Photochromogen at 25˚C  Only AFB species that has a different light test result based on temperature of incubation  Growth at 37 ˚C in 12 - 25 days  Causes pulmonary disease in adults, similar to TB, associated with alcohol abuse, smoking, COPD, AIDS and immune suppression 25˚ C - Photochromogen 37˚ C - Scotochromogen
  • 38. Mycobacterium xenopi  Thermophilic AFB, grows at 42˚C (high temp for AFB)  Capable of growing in hot water systems, aerosolization of water from systems, is a common sources of infection  Can occur as colonization or cause of infection, need to investigate patient factors to establish diagnosis  Growth in 14 - 28 days  Egg nest–like colony produced on solid media  Scotochromogen (pigment produced in light and dark)  Rare cause of pulmonary disease:  Clinical disease like TB  Occurs in patients with preexisting lung disease (chronic obstructive pulmonary disease or bronchiectasis) and HIV/AIDS
  • 39. Mycobacterium genavense • Fastidious, thermophilic, slowly growing AFB species (growth in 3-12wks) • Better recovery in Middlebrook liquid medium with acidic pH (5) with iron supplementation • Disease is usually associated with immunosuppression. • Sites of infection include: • Lymphadenitis, bone marrow, liver, spleen, bowel, blood, genital, and soft tissue infections. • Appearance in fixed tissue is very similar to M. avium complex, short rods with random clumping • One of the most common mycobacteriosis in pet birds Bone marrow; Kinyoun stain
  • 40. Mycobacterium avium complex (MAC)  Complex includes eight AFB species of environmental and animal origin  M. avium, M. intracellulare, M indicus pranii, M chimaera, M arosiense, M vulneris, M bouchedurhonense, M colombiense, M marseillense, M yongonense, and M timonense (M. avium and M. intracellular most common species)  Non-photochromogen  Species biochemically inert and genetically very similar  Growth at 37 ˚C in 7 – 21 days  Smooth / creamy colony / buff colored  Short rods, not beaded, irregular clumping of AFB in stains  Identification:  MALDI-TOF mass spectrometry- method of choice to identify to species  16s rRNA sequencing (identify to species)
  • 41. M. avium complex clinical correlation  Disease in the somewhat normal host:  Adult: chronic pulmonary disease, fibro-cavitary or nodular bronchiectasis presentation  Produces chronic cough in elderly with prior lung damage (COPD)  Children: Scrofula with chronic granulomatous inflammation and lymph node involvement  M. chimaera (emerging species within the M. avium complex)  Environmental (airborne transmission) and nosocomial pathogen  Pulmonary and extra-pulmonary infections  Established as a nosocomial contaminate (2016) during the manufacturing of heater–cooler units used during cardiac surgery. Aerosolized water sprayed from the instrument contaminated the surgical field with M. chimera leading to post surgical infections
  • 42. M. avium complex disease in HIV/AIDS  Common opportunistic infection, symptoms include:  Nonspecific low-grade fever, weakness, weight loss, picture of fever of unknown origin rather than pulmonary infection  Diagnosis: Isolation in culture from respiratory, blood or bone marrow, AFB staining of bone marrow  Abdominal pain and/or diarrhea with malabsorption  Diagnosis: positive stool AFB smear and culture  In tissue:  Pathology: non-necrotizing granulomas  High organism load can be present in infected tissue  AFB are small (short) rods and not beaded and occur in irregular clumps
  • 43. Bowel -Lamina propria expanded from predominately Lymphohistocytic infiltration M. avium complex in the bowel
  • 44. Mycobacterium haemophilum  Fastidious slow growing, requiring the addition of hemoglobin or hemin to culture media for growth  Will not grow on LJ media, Middlebrook, or in automated system media (Middlebrook 12B media) without the addition of hemin supplement  Will grow on chocolate agar which contains hemin  Grows best at 30*C, poor growth at 37*C  Diseases:  Painful subcutaneous nodules and ulcers isolated primarily in patient with AIDS patients or immunosuppressed  Lymphadenitis in children
  • 45. Mycobacterium gordonae Rarely if ever causes disease  Commonly found in tap water and is the most common AFB culture contaminant.  Contamination can occur during specimen collection or laboratory processing.  Must use sterile distilled water for processing to prevent such contamination.  Scotochromogen  Need to accurately identify so not confused with a true pathogen  MALDI-TOF  Sequencing
  • 46.  Growth in <=7 days at 35*/37*C  Routine bacteriologic cultures can isolate due to rapid growth  20 species, environmental organisms found in soil and water and can be transmitted from contaminated medical supplies (nosocomial transmission)  The 3 most common human pathogens:  M. fortuitum skin and surgical wound infections and catheter sites  M. chelonae skin infections in immune suppressed and catheter sites  M. abscessus chronic lung infection (cystic fibrosis) and skin infection in immune suppressed  In fixed tissue, elicit a mixed response of granulomatous and/or pyogenic reaction Rapidly Growing Mycobacteria Intraepithelial abscess M. abscessus
  • 47. Rapid growers  Growth on 5% Sheep’s blood agar and AFB media as a non pigmented dry gray colony  Less beaded on Gram stain than most AFB species and have weak AFB staining due to low cell wall lipid content  Biochemical reactions:  All species Arylsulfatase positive  M. fortuitum: Nitrate reduction positive (Mycolicibacterium) Iron uptake positive  M. chelonae: Nitrate negative No growth 6.5% salt (Mycobacteroides)  M. abscessus: Nitrate negative Growth in 6.5% salt (Mycobacteroides)  Identification to species using MALDI-TOF and sequencing Gram stain
  • 48. Rapid growing (MGM) susceptibility testing  Antibiotic therapy is assisted by directed susceptibility testing  Clarithromycin is susceptible to most MGM species and is primary therapy for infections  Clarithromycin and other appropriate antibiotics can be tested by broth multi-dilution (MIC) testing or Etest  Molecular genetic testing for the erm gene can assist with the rapid and precise detection of clarithromycin resistance  M. abscessus and M. fortuitum isolates should be tested for erm gene activity, to more sensitively detect inducible resistance of the organism to clarithromycin Etest Microdilution
  • 49. Mycobacterium leprae complex  Leprosy also known as Hansen’s Disease  Global health concern with @ 200,000 cases/year. Endemic in India, Brazil and Indonesia, @ 100 cases in US per year  Affects skin, mucous membranes, and peripheral nerves,  Infection spread via inhalation of infectious droplets from humans  Armadillo is zoonotic reservoir of M. leprae complex  Begins with skin lesion (hypopigmented or red lesion) and/or enlarged nerve(s) and sensory loss.  Untreated, can progress to a peripheral neuropathy with nerve thickening, numbness in earlobes or nose, and loss of eyebrows  Curable with early diagnosis and appropriate therapy (dapsone plus rifampin or clofazimine). BCG vaccine is partially protective (50%)  Non-culturable  Diagnosis: AFB staining of infected tissue and PCR assays (Public Health)
  • 50. Tuberculoid leprosy/Paucibacillary Less severe lesions Less AFB seen in stains of lesions Lepromatous leprosy/Multibacillary Severe disfiguring lesions, low immune response Large numbers of AFB in lesions Skin biopsy - AFB seen in nerve fiber (FITE stain) M. leprae has less mycolic acid in the cell wall than other Mycobacteria spp and stain more reliably on Fite stain than ZN/Kinyoun type AFB stains. AFB in cigar packet arrangement