Mycobacteriology Review 2017

1. Mycobacteriology
Review
Update 2017
Margie Morgan, PhD., D(ABMM)

2. PPD (Purified Protein Derivative)
aka the TB skin test
 Determined exposure to TB for decades
 Detects past or current TB exposure
 X-reacts with BCG immunization
 @ 25% false negative reactions
 Measures delayed hypersensitivity response to TB Ags
 T cells react to TB and related antigens
 Lymphokine is released
 Forms area of induration at the injection site
 Measure area of induration in mm at 48 hr
 >=15 mm POSITIVE rxn or check for BCG immunization in past
 >=10 mm POSITIVE in immune suppressed or just exposed

3. Cell Mitogen assays for TB screening-IGRAs
(Interferon Gamma Release Assays)
 QuantiFERON-TB Gold (QFT) and T-Spot
 Whole blood tests to screen for cell mediated immunity
to TB complex antigens
 Able to detect both latent TB and active infection
 Specific for TB complex - Useful for screening BCG
immunized population where PPD is falsely positive
 Useful if patient cannot return to have PPD reaction
interpreted
 Tests quantitate the immune response to TB following
the stimulation of the patient’s T cells by TB specific Ags
 Sensitivity is somewhat equal to PPD but better specificity
 What is a “TRUE” positive reaction is the issue! A fluctuating
immune response can ride the positive cut off value.

4. Mycobacteria –Acid Fast Bacilli (AFB)
 Related to the genera Nocardia, Corynebacterium,
and Rhodococcus
 What does the term “Acid Fast” refer to?
 Once stained the rods resist decolorization
with acid alcohol (HCl)
 Very beaded and faded on Gram stain
 Gram stain is NOT a good stain to detect AFB
 Note: Differentiate AFB staining of the mycobacteria from partial
acid fast (PAF) staining used for the Nocardia species.
 AFB acid fast stains use HCl to decolorize
 PAF acid fast stains use H2SO4 to decolorize
 This is a more gentle process and Nocardia will be PAF + but will
stain negative in the “true” AFB stains meant for the mycobacteria.
Gram
AFB

5. Mycobacteria –
General Characteristics
 Aerobic, no spores, slightly curved
or straight rods, rarely branch, vary in length
depending on species
 Survive in the environment for months
 Most species grow on simple substrates
 Mycobacteria include obligate pathogens,
opportunists, and saprophytic species
 High amount of complex mycolic acids and free
lipids in cell wall which give many properties to this
genus including acid fast staining properties

6. Identification of the Mycobacteria
 For decades the identification algorithm started with
the determination of pigment in the light and dark
followed by biochemical reactions
 With expanding taxonomy, biochemical reactions
were not able to separate and identify most of the
newly recognized species so we evolved into High
Performance Liquid Chromatography (HPLC)
methods. HPLC is now obsolete.
 Current methods for identification:
 Genetic probes – RNA/DNA hybridization probes
 MALDI-TOF Mass Spectrometry to analyze proteins
 Sequencing 16 sRNA for genetic sequence information

7. Mycobacteria Taxonomy
 TB complex:
 Mycobacterium tuberculosis
 M. bovis and the Bacillus Calmette-Guerin strain
(BCG)
 M. africanum
 And some very rare species:
 Mycobacterium microti
 Mycobacterium canetti
 Mycobacterium caprae
 Mycobacterium pinnipedii
 Mycobacterium suricattae
 Mycobacterium mungi
 Mycobacteria other than TB – “MOTT”

8. Mycobacteria that cause disease?
 Mycobacterium tuberculosis – the major pathogen of this genus
The others: MOTT
 M. avium-intracullare complex
 M. genavense
 M. haemophilum
 M. kansasii
 M. malmoense
 M. marinum
 M. simiae
 M. szulgai
 M. ulcerans
 M. xenopi
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum

9. Mycobacteria that rarely if ever cause
disease! If so, in immune compromised!
 Mycobacterium gordonae
 M. gastri
 M. celatum
 M. scrofulaceum
 M. terrae complex
 M. smegmatis

10. Algorithm for identification of MOTT
- historically speaking
 Runyon System
 Used to classify those species not in the TB complex (MOTT)
 Based on pigment production when exposed to light & growth rate
 Runyon separates MOTT into four groups using the light
test to promote pigment production
 Photochromogen = Pigment produced with light
 Scotochromogen = Pigment in both light and dark
 Non-photochromogen = No pigment produced
 Rapid Grower = Growth rate (<= 7 days)

11. Light Test – does it produce a yellow pigment
after being exposed to 8 hours of light bulb.
Group I Photochromogen
Turns yellow after light exposure
Group III
Non-photochromogen – No
pigment produced after light exposure
Group II Scotochromogen
Always has yellow pigment

12. Runyon Classification System Results
 Group I - Photochromogen – turns yellow when
exposed to light, no color in the dark
 M. kansasii
 M. simiae
 M. szulgai when incubated at 25˚C***
 M. marinum
 Group II - Scotochromogen – yellow pigment in
dark or exposure to light
 M. gordonae
 M. scrofulaceum
 M. szulgai when incubated at 37°C***

13. Runyon Classification cont’d
 Group III – Non-photochromogen – No pigment
produced in the light or dark
 M. avium-intracellulare
 M. haemophilum
 M. xenopi
 Group IV – Rapid growers – grow in 7 days or less
 M. fortuitum group
 M. abscessus
 M. chelonae
 M. mucogenicum
 M. smegmatis

14. Safety in the
AFB Laboratory
 Level 3 biosafety precautions required in AFB laboratories that
process, identify and perform susceptibility testing
 Level 2 Hepa filter approved biosafety cabinet with return air vented
to outside of the laboratory
 Safety cabinets must be certified at least yearly for safe use
 Negative air flow, anteroom, contiguous autoclave
 95 respirator masks or PAPR (powered air purifying respiratory
mask), gloves, disposable gowns must be worn
 Plastic cups with protected lids for centrifugation of specimens
Biosafety Cabinet
PAPR

15. Specimen collection
 Sputum – 3 early morning collection
or 3 specimens at least 8 hours apart
 Bronchial lavage fluid
 Tissues or Lesions
 CSF or sterile body fluids
 Urine – 3 to 5 early morning collection
 Stool – for M. avium-intracellulare complex
 Gastric – children, must neutralize pH (7) of
specimen after collection for AFB to survive
 Blood – for detection of disseminated disease
 Automated systems – AFB blood culture
bottles manufactured for AFB isolation

16. Specimen Processing
Start to Finish!
 5 ml of specimen pipetted into conical tube
 Decontaminate and Liquify specimen for 15 minutes
 Add 5 ml of 4% NaOH (Increases the pH to 9)
 plus N-acetyl-L-Cysteine (breaks up the mucus)
 Fill tube with phosphate buffer to neutralize pH (7.0)
 Centrifuge for 30 minutes – safety cups
 3000 X g to pellet the specimen
 Pour off the supernatant
 Prepare slides from pellet for AFB staining
 Dilute the pellet with small amount of sterile saline
for culture
 Incubate cultures @ 37˚C, 5-10% CO2 for 6–8 wks

17. Why do you Decontaminate Specimens?
 For the slow growing mycobacteria to be cultured
 Must eliminate competing bacteria that grow 24 x faster
 Must also release the AFB from mucus plugs in the sputum
specimens
 Accomplished by exposing the sputum to alkaline/acid solutions
and mucolytic reagents such as 4% NaOH and L-acetyl-L
cysteine
 Mycobacteria are more resistant to killing by acids and
alkaline solutions than most bacteria due to the high
amount of complex lipids in the cell wall – this is used to
rid the specimen of contaminating bacteria and yeast

18. Specimen Decontamination/Digestion
Most often used :
 4% NaOH – for decontamination
 N-acetyl-L-cysteine – liquid faction of mucus
 Expose specimen to solution for 15 minutes
 Used in Special circumstances:
 Oxalic acid for sputum collected from patients with
cystic fibrosis -eliminate mucoid strains of
Pseudomonas aeruginosa
 Oxalic acid should not be used routinely
 Will decrease isolation of AFB in culture
 These solutions kill bacteria and can also kill
AFB if left on specimen > 15 minutes

19. Specimen Centrifugation
 Centrifugation at 3000 X g (fast) with safety cups in
place
 Speed of centrifugation is important - AFB are lipid
laden and they will float if not spun fast enough –
must pellet to bottom of tube so AFB are not poured
off with supernatant
 Helps to determine the sensitivity of the AFB stain
 Pour off supernatant into waste
 Use pellet for stains/culture

20. Manual Plating/ Culture Media
 Middlebrook – Synthetic media
 Clear solid agar and liquid media
 Synthetic = chemical ingredients added for optimal growth
 Used for culture and susceptibility testing
 Autoclave for sterility
 Lowenstein-Jensen – Egg based
 Green from addition of malachite green
 Hens egg, glycerol, and potato flour
 Sterilize by inspissation – drying
 Cultures incubated at 37˚C , 5-10% C0₂ for 6-8 weeks

21. Automated Detection of AFB
Bactec MGIT 960, Bacti-Alert and VersaTREK
Liquid Middlebrook 7H9 tubed media for growth
Bactec and Bacti-Alert have same detection method
 As AFB grow in the tubes, the AFB respire CO₂ and the O₂ is decreased.
The lower level of O₂ leads to fluorescence of the tube indicator and indicates
growth in the tube.
Incubation at 37˚C for 6 weeks
Fluoresce
 NAP test – Identification for TB complex
NAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)
Automated test run only on the MGIT 960 instrument
TB complex does not grow in the tube containing NAP
MOTT species grow in NAP
MGIT 960
BACTEC
Bacti-Alert
VersaTrek

22. Acid Fast Staining for Mycobacteria -
only concentrated specimen should be stained
 Carbol Fuchsin based stains
 Carbol Fuchsin is a red colored stain
 Potassium permanganate is the background blue
counterstain
 Two methods:
 Ziehl-Neelsen (ZN) – uses heat to drive stain into lipid
laden AFB
 Kinyoun – uses increased % of phenol to drive stain
into AFB
 Read numerous microscopic fields (5 min)using light
microscope/100x oil objective

23. Fluorochrome based stain
Auramine Rhodamine –
 fluorescent stain, organisms stain fluorescent
yellow with black background,
 Read on 25X or 40X for 2 min, viewing numerous
fields using a fluorescence microscope
 Based on nonspecific fluorochrome that binds to
mycolic acids
Considered more sensitive than ZN or Kinyoun
stains for concentrated specimen patient slide
examination

24. Acid Fast staining of the Mycobacteria
Mycobacterium avium complex
Organisms are routinely shorter than TB
(Kinyoun stain) No cording
M. Tuberculosis - Organisms are
long rods and can appear as if they
are sticking together [cord factor]
In broth
cultures -
ropes of AFB
will form

25. Direct Detection of TB from Respiratory
Specimens by Molecular Amplification
 Two tests FDA cleared to detect TB complex organisms in
smear positive and smear negative sputum specimens:
 Hologic Amplified MTD Test - Transcription Mediated Amplification
 Cepheid Xpert-TB RIF (rtPCR) – detects TB complex and Rifampin
resistance (rpoB gene)
 Amplify a 16S rRNA gene sequence of TB complex
 Sensitivity of assays @ 90% AFB smear (+) specimens and
75% (+) for AFB smear (-) specimens
 Test of diagnosis not cure
 Residual rRNA and DNA can be present up to 6m after a
positive diagnostic test
 Must confirm all specimens with AFB culture and sensitivity

26. Mycobacterium tuberculosis
 Optimal Temp 37˚ C, Grows in 12 –25 days
 Buff colored, dry cauliflower-like colony
 Manual tests for identification – old school
 Niacin accumulation Positive
 Niacin produced from growth of TB on egg containing
medium (LJ medium)
 Nitrate reduction Positive
 Is it really M. tuberculosis or could it be M. bovis?
 Both in TB complex and diseases are similar
 M. bovis = nitrate reduction Negative
 M. bovis does not grow in Thiophene-2-carboxylic hydrazide (T2H),
however TB does grow on this substrate
 Current ID uses Molecular probe, MALDI-TOF, & Sequencing

27. Mycobacterium
tuberculosis
Demonstrates Cord factor –
due to high lipid content in cell
wall, rods stick together and
develop long ropes – unique
to M. tuberculosis
Long AFB – stick together

28. Tuberculosis
 Classic Disease
 Slowly progressive pulmonary infection cough, weight loss, and
low grade fever
 Lesion with calcified foci of infection and an associated lymph node known as
Ghon’s complex
 Dissemination occurs most often in AIDS, elderly, children and with
some medications (Remicade- infliximab)
 Secondary tuberculosis: mostly in adults as reactivation of
previous infection (or reinfection), Granulomatous inflammation
much more florid and widespread. Upper lung lobes are most
affected, and cavitation can occur.
 TB is spread by respiratory droplets
 All patients with a positive concentrated smear require
respiratory isolation precautions
 All patients with high level of suspicion require precautions

29. Pathology of M. tuberculosis
Lung with pulmonary TB has apical cavitations with
numerous areas of caseous necrosis and massive tissue
consolidation

30. TB in HIV/AIDS patients
 Worldwide -TB is the most common opportunistic infection
affecting HIV/AIDS patients
 AIDS patients have higher likelihood to be multi-drug
resistant (resistant to at least INH and Rifampin)
 With progressive decline of cell mediated immunity (low
CD4 count) – greater risk of extra pulmonary dissemination
 Granulomas with/without caseation can occur
Miliary TB

31. M. tuberculosis outside lung
 Scrofula -Unilateral lymphadenitis (cervical lymph node)
most often seen in immune compromised (50%)
 Fine needle aspiration for diagnostic specimen
 M. tuberculosis most common in adults
 M. avium complex and other MOTT (2-10%)
most common in children
 Pott’s disease - TB infection of the spine
 Secondary to an extra-spinal source of infection
 Manifests as a combination of osteomyelitis and arthritis that
usually involves more than 1 vertebra.

32. Susceptibility testing of TB
 Two methods for testing
 (1) Agar dilution (indirect proportion method)
 antibiotics embedded in solid agar and TB inoculated onto media
 (2) Bactec liquid 7H9 medium containing antibiotic solutions
 Tested on automated MGIT 960 system
 Primary TB drug panel / 5 drugs
 Isoniazid Ethambutol
 Pyrazinamide Streptomycin
 Rifampin
 PCR Methods– Hybridization probes used in combination with RT
PCR assay to quantify target DNA is used by public health labs for
rapid determination of MDRTB

33. Mycobacterium bovis
 Produces disease in cattle and other animals
 Spread to humans by raw milk ingestion
 Disease in humans similar to that caused by TB
 Bladder infections in patients treated with BCG [Bacille Calmette-
Guerin] when used as an immune adjuvant to treat bladder cancer
 BCG is an attenuated strain of M. bovis
 It can become “active” and infect the bladder
 M. bovis will be isolated from urine specimens
 Is it M bovis or is it M tb?
M.bovis M. tb
Nitrate Negative Positive
Growth in T2H* No growth Growth
Pyrazinamidase Negative Positive
*(Thiophene-2-carboxylic hydrazide)

34. Mycobacterium ulcerans
 Bairnsdale or Buruli ulcer boil that progresses into a
painless skin ulcer
 Can progress into avascular coagulation necrosis
 African continent and tropical environments
 Optimum growth temp 30˚ C
 All skin lesions should be
cultured at both 30˚ and 37˚C
 Slow growing requiring 3- 4 weeks

35. Mycobacterium kansasii
 Culture 37* C, 10-20 days
 Photochromogen
 Niacin accumulation test = negative
 Nitrate reduction = positive
 Tween 80 positive / tests for presence of lipase enzyme
 68*C catalase positive
 AFB are larger than TB - rectangular and beaded
Shepherd’s crook shape
 Clinical disease mimics pulmonary TB but usually does
not disseminate from lung –
 Predisposition diseased lung (COPD)
 Immune suppressed, pneumoconiosis, alcohol abuse, HIV
 Granulomatous inflammation in lung

36. Mycobacterium marinum
 Optimum temp for is 30˚ C
 Grows poorly or not at all at 37°C
 Grows in 5-14 days
 Photochromogen
 M. marinum found in fresh and salt water
 Associated with trauma occurring in water:
 swimming pools (swimming pool granuloma)
 cleaning fish tanks
 ocean (surfing)

37. Mycobacterium marinum
 Disease: Tender, red or blue/red subcutaneous nodules
develop following trauma
 Biopsy (skin) specimens for culture and histopathology
 Lesions can spread up arm and along lymphatics,
 Clinically appears similar to infections with Sporothrix,
Nocardia and rapid growing Mycobacteria species.

38. Mycobacterium szulgai
 Grows at 37 ˚C in 12 - 25 days
 Scotochromogen at 37˚C / Photochromogen at 25˚C
 Only AFB that has a different light test result based on
temperature of incubation
 Rare cause of pulmonary
disease in adults
Symptoms similar to TB 25˚ C - Photochromogen
37˚ C - Scotochromogen

39. Mycobacterium xenopi
 Optimum temp 42˚C,
 Capable of growing in hot water systems
 Grows in 14 - 28 days in culture
 Scotochromogen
 Egg nest type colony on agar plate
 Rare cause of pulmonary disease
 Clinically disease is like TB
 Occurs in patients with preexisting lung disease and
HIV/AIDS

40. M. avium-intracellulare complex
 M avium & M intracellulare most common but complex
includes eight species of environmental and animal related AFB
 Biochemically and genetically very difficult to distinguish
 Growth at 37 ˚C / 7 – 21 days
 Non-photochromogen
 Smooth colony
 Inert in biochemicals
 Identify using
 GenProbe (AccuProbe) molecular DNA probe
 MALDI-TOF
 Genetic 16s rRNA Sequencing

41. M. avium-intracellulare complex
 Opportunistic infection
High organism load in intestine, liver, spleen and bone marrow
Can be recovered from blood cultures
Involvement of GI tract causes diarrhea
 Positive AFB stool smears
 Tissue Biopsy with inflammation
AFB small and not beaded
 Do not have cord factor
Pathology - Necrotizing rather than
granulomatous inflammation

42. M avium-complex in lymph node tissue
(Kinyoun stain)
Granulomatous inflammation
lung tissue (H&E stain)
Bowel -Lamina propria expanded from
predominately Lymphohistocytic infiltration

43. M. avium complex clinical correlation
 HIV/AIDS
 Disseminated infection in end stage AIDS disease
 Nonspecific low grade fever, weakness, weight loss,
picture of fever of unknown origin
 Abdominal pain and/or diarrhea with malabsorption
 Normal host
 In adults mostly pulmonary disease, much like TB
 marked % of cases – elderly adults with lung
damage (COPD)
 M. chimaera
 Contaminated Heater–cooler units using tap water identified as a
source of surgical site infections. Units caused an airborne
transmission of M. chimaera over an open surgical field

44. Rapid growing Mycobacteria species
 Low virulence, found in nature
 Cause: Infections in soft tissue, venous catheter sites
and shunts @ 20 species – 5 spp. most common
 M. fortuitum – skin and surgical wound infections
 M. chelonae- skin infections in immune suppressed
 M. abscessus - chronic lung infection and skin infection in
immune suppressed
 M. smegmatis
 M. mucogenicum
 Grows in <=7 days = rapid grower
 Biochemical reactions:
 All species are Arylsulfatase test positive
 M. fortuitum: Nitrate Positive and Iron Uptake positive
 M. chelonae and M. abscessus: Nitrate Negative
 MALDI-TOF and 16 S RNA Sequencing for identification

45. Miscellaneous
 M. gordonae –
 Rare! Cause of Infection
 Scotochromogen
 Major laboratory water contaminant in cultures
because it is commonly contaminating water
systems
 Use sterile/distilled water in AFB culture
processing to prevent contamination

46. Miscellaneous pathogenic species
 Mycobacterium haemophilum
 Requires addition of hemoglobin or hemin to
culture media for growth
 Will not grow on LJ or in automated systems without the
addition of hemin supplements
 Disease: Painful subcutaneous nodules and
ulcers, primarily in AIDS patients or immune
suppressed
 Lymphadenitis in children

47. Mycobacterium leprae
 Leprosy – Hansen’s disease
 Begins with anesthetic skin lesions,
peripheral neuropathy with nerve thickening,
numbness in earlobes or nose, loss of eye brows
 Two types of Leprosy
 Lapromatous – severe disfiguring lesions, large numbers of
AFB in lesions / associated with co-infection with Strongyloides
stercoralis
 Tuberculoid -less severe/fewer lesions, lower numbers of AFB
in lesions
 Will not grow on laboratory AFB media
 PCR on tissue for definitive diagnosis
 Armadillo is the natural reservoir

48. Tuberculoid leprosy
Lapromatous leprosy
Skin biopsy - AFB seen in nerve fiber
AFB in “cigar packets”