1. Mycobacteriology
Just the very basics – meant for board
review or a brief overview of this very
important area of the laboratory.

2. PPD (Purified Protein Derivative)
better known as the TB skin test
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For decades used to determine exposure to TB
Mantoux test – 5 Tuberculin units / intradermal injection
Detects past or current TB exposure and reacts with BCG
immunization
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Not a very sensitive test – up to 25% false negative reactions
Measures delayed hypersensitivity response to TB related antigens
- T cells react to TB related antigens--- lymphokine released ---forms induration at the injection site
Read the reaction at 48 hr - measure induration in mm
 >=15 mm positive or check for BCG immunization in past
 >=10 mm positive in immune suppressed or just exposed
Recent shortages of the PPD (Tubersol) product has led to a
change to using Interferon Gamma Release Assays (IGRA)

3. Cell Mitogen assays for TB screening-IGRAs
(Interferon Gamma Release Assays)
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QuantiFERON-TB Gold (QFT) and T-Spot
Whole blood tests to screen for cell mediated immunity to TB
Able to detect both latent TB and active infection
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NOT meant to replace culture or other diagnostic tests
Is independent of BCG exposure/ specific for TB complex however,
does cross react with a few uncommon Mycobacteria other than TB
 Useful for screening the BCG immunized population where the
PPD cannot be used due to false positive tests
Meant to replace and improve upon the PPD skin test
Whole blood has sensitized T cells --- (normal immune population)
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stimulated by antigen peptides that are specific to TB –(ESAT-6 and
CFP-10) ---secrete cytokine interferon gamma---amount interferon gamma secreted detected is measured using an EIA
assay

4. General Features of Mycobacteria –
aka Acid Fast Bacilli (AFB)
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Closely 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 like species.
 AFB acid fast stains use HCl to decolorize organism
 PAF acid fast stains use H2SO4 to decolorize –
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this is a more gentle process and Nocardia will be PAF + but will be
AFB negative in the “true” AFB stains meant for the mycobacteria.

5. Mycobacteria –
General Characteristics
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Aerobic, no spores, slightly curved
or straight rods, rarely branch, variable in length
depending on the species
Hardy in the environment for months and most grow
on simple substrates
Mycobacteria include obligate pathogens,
opportunists and saprophytic species
High amount of mycolic acids and free lipids in cell
wall which give many properties to this genus
including the AFB staining properties
M. tuberculosis
M. kansasii

6. Identification of the Mycobacteria
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For decades the identification was based on the
production of pigment in the light and dark and
biochemical reactions
With expanding taxonomy, biochemical reactions
are not able to separate and identify some of the
newly recognized species
New methods have evolved for identification:
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HPLC – high-performance liquid chromatography to identify
mycolic acids, good but not the best for definitive speciation
Genetic probes – RNA/DNA hybridization probes
MALDI-TOF Mass Spectrometry to analyze proteins
Sequencing 16 sRNA for genetic sequence identification

7. Mycobacteria Taxonomy
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TB and genetically related organisms that are separated
taxonomically from the other species
TB complex include:
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Mycobacterium tuberculosis
M. bovis
M. africanum
Other vary rare species
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Other Mycobacteria in the TB complex are grouped into “MOTT”
Mycobacteria other than TB
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The Runyon System is used to classify those species not in the TB
complex (MOTT)- divided into four groups:
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Pigment when exposed to light in Light Test
Pigment in both light and dark in the Light Test
No pigment produced in light or dark in the Light Test
Growth rate (<= 7 days) – Rapid grower

8. Light Test – does it produce a yellow pigment
after being exposed to light ?
Group I Photochromogen
Turn yellow after light exposure
Group III
Non photochromogen – never
has pigment
Group II Scotochromogen
Always has yellow pigment
– light or no light exposure

9. Runyon Classification System – Groups
determined by results of the light test
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Group I - Photochromogen – turns yellow when
exposed to light, no color in the dark
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M. kansasii
M. simiae
M. szulgai when incubated at 25˚C
M. marinum
Group II - Scotochromogen – yellow pigment in
dark or exposure to light
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M. gordonae
M. scrofulaceum
M. szulgai when incubated at 37*C

10. Runyon Classification cont’d
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Group III – Non-photochromogen – No pigment
produced in the light or dark
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M. avium-intracellulare
M. haemophilum
Group IV – Rapid growers – grow in 7 days or less
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M. fortuitum group
M. abscessus
M. chelonae
M. mucogenicum
>= 20 species

11. Which ones reported to cause disease?
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Mycobacterium tuberculosis – the major pathogen of this genus
M. avium 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

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

13. Specimen collection
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Sputum – 3 specimens - early morning
or at least 8 hours apart
Bronchial lavage fluid
Tissues or Wounds
CSF or sterile body fluids
Urine – 3 to 5 early morning collections
Stool – M. avium complex only
Gastric – for children, must neutralize
the pH of specimen for AFB to survive
Blood – disseminated disease
 Automated systems – AFB blood culture
bottles manufactured for AFB isolation

14. Specimen Processing
in the AFB Laboratory
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Level 3 Safety 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
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Safety cabinets must be certified at least yearly for safe use
Must have negative air flow in laboratory, anteroom for dressing and
washing hands
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

15. Specimen Processing
Start to Finish!
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5 ml of specimen put in the conical tube
Decontaminate and Liquify for 15 minutes
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Fill tube with phosphate buffer to neutralize pH
Centrifuge for 30 minutes
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Add 5 ml of 4% NaOH (Increases the pH to 9)
plus N-acetyl-L-Cysteine (breaks up the mucus)
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

16. How and why do you perform Specimen
Decontamination?
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Mycobacteria are more resistant to killing by acids and
alkaline solutions than most bacteria due to the high
amount of lipid in the cell wall – this is used to rid the
specimen of contaminating bacteria and yeast
For the slow growing mycobacteria to be cultured / must
eliminate competing bacteria that grow 24 x faster and
release the AFB from mucus plugs in the sputum
specimens - this is accomplished by exposing the
sputum to alkaline/acid solutions and mucolytic reagents
such as 4% NaOH and L-acetyl-L cysteine

17. Specimen Decontamination/Digestion
Most often used :
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4% NaOH – for decontamination
N-acetyl-L-cysteine – liquid faction of mucus
Expose specimen to solution for 15 minutes
Used in Special circumstances:
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Oxalic acid can be used for cystic fibrosis sputum specimens to
eliminate the resistant (mucoid) Pseudomonas strains
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Oxalic acid should not be used routinely, it is too
harsh and will decrease isolation of AFB
These solutions kill bacteria and can also kill AFB if left on specimen
> 15 minutes

18. Specimen Centrifugation
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Centrifugation at 3000 X g (fast)
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 into the waste
This determines the sensitivity of the AFB stain so
proper centrifugation speed is important
Pour off supernatant
Use pellet for testing

19. Plating -Selection of Plate and Tubed
Culture Media
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Middlebrook – Synthetic media
 Clear agar and liquid media
 Synthetic = chemical ingredients added for optimal growth
 Used for culture and susceptibility testing
 Can Autoclave for sterility
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Lowenstein-Jensen – Egg based
 Green media due to malachite green)
 Hens egg, glycerol, and potato flour
 Sterilize by inspissation – drying
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Cultures on solid media incubated at 37˚C , 5-10% C0₂ for 8 weeks

20. Automated Detection of AFB
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Automated systems:
 BACTEC MGIT 960 and BACTI-ALERT Instruments
 Use Liquid Middlebrook 7H9 tubed media for growth
 Both systems have same detection method
BactiAlert System
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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
BACTEC MGIT 960 NAP test – Identification for TB
NAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)
An automated test on the BACTEC 960 for TB identification MGIT 960
TB does not grow in the NAP containing tube
BACTEC
Other Mycobacteria species grow in NAP

21. Acid Fast Staining for Mycobacteria
Carbol Fuchsin based stains
Stain the AFB Red
CF is the red colored stain/potassium permanganate is the
background counterstain (blue)
 Ziehl-Neelsen (ZN) – uses heat to drive stain into AFB
 Kinyoun – uses phenol to drive stain into AFB
 Read for 5 min on oil objective
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Fluorochrome based stain
 Auramine Rhodamine – fluorescent stain, organisms stain
fluorescent gold, read on 25X or 40X for 2 min on a fluorescence
microscope
 Generally considered to more sensitive than ZN or Kinyoun

22. Acid Fast staining of the Mycobacteria
Mycobacterium avium complex
Organisms are routinely shorter than TB
M. Tuberculosis - Organisms are
long and can appear as if they are
sticking together [cord factor]

23. Direct Detection of TB from specimens
by Amplification
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Gen-Probe (Hologics) TMA and Cepheid Xpert-TB RIF FDA cleared
Can only test respiratory specimens per FDA
Detect TB complex in AFB smear positive and negative respiratory
specimens
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The Xpert TB/RIF test can also detect Rifampin-resistance
associated mutations of the rpoB gene
Respiratory specimens must first be decontaminated and concentrated
prior to testing for Gen-Probe. The Cepheid Xpert method can test
non-concentrated sputum.
Amplifies a 16S rRNA gene sequence of TB
Sensitivity @ 90% AFB smear (+), 75% AFB smear (-)
Test of diagnosis not cure
Residual rRNA can be present up to 6m after diagnosis
Still must perform culture and susceptibility testing

24. Mycobacterium tuberculosis
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Optimal Temp 37˚ C, Grows in 12 –25 days
Buff colored, dry cauliflower-like colony
Manual tests for identification
 Niacin Positive - accumulation of niacin, a product produced
form growth on this egg containing medium (LJ medium)- must
be performed on culture growing on LJ medium
 Nitrate reduction – Reducing nitrate to nitrite = Positive
 Confirmation of TB vs M Bovis
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M bovis = nitrate negative
M. bovis does not grow in Thiophene-2-carboxylic hydrazide (T2H)
Molecular identification:
 Gen-Probe AccuProbe DNA/RNA hybridization identifies TB
complex organisms with excellent accuracy

25. Mycobacterium tuberculosis
Demonstrates Cord factor –
due to high lipid content in TB
organisms they stick together
and can develop long ropes –
unique to TB

26. Tuberculosis
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Classic presentation
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Slowly progressive pulmonary infection
Coughing, weight loss, low grade fever
Biopsy of lung most often caseating granulomas
Organisms seen in a concentrated AFB stain indicates
that the patient is capable of infecting others
Never report a non-concentrated sputum AFB stain
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high % of false negative stain results
TB is spread by respiratory droplets
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All patients require respiratory isolation precautions

27. TB in HIV/AIDS patients
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Worldwide -TB is the most common
opportunistic infection affecting HIV (+)
Pulmonary TB most usual presentation
With progressive decline of cell mediated
immunity (low CD4 count) – greater risk of
extrapulmonary dissemination
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Granulomas with/without caseation

28. TB can be a cause
of Scrofula
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Unilateral lymphadenitis
Nodes drain to skin surface
Two most common causes:
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M. tuberculosis
M. avium-complex

29. Susceptibility testing of TB
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Two methods
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(1) Agar dilution -antibiotics embedded in solid agar
(2) Bactec liquid 7H9 medium with antibiotic solutions
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Primary drug panel tested for TB consists of 5 drugs
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Tested on the automated MGIT 960 system
Isoniazid
Pyrazinamide
Rifampin
Streptomycin
Ethambutol
Molecular Beacon testing – hybridization probes used
with RT PCR assay to quantify target DNA. Used for
rapid determination of MDRTB – detect INH and RIF
resistance

30. Susceptibility of TB
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Most TB isolates are susceptible to the 5 primary TB
drugs
Populations more likely to be resistant to one or
more drugs include
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HIV/AIDS
Immigrant populations
MDRTB refers to the multi-drug resistant strains of
TB
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At least resistant to INH and RIF – the two most common
drugs used to treat TB
Can be resistant to additional first line drugs

31. M. bovis
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M. bovis produces disease in cattle and other animals
 Spread to humans by milk ingestion
 Most common disease symptoms like that of TB
M. bovis can cause bladder infections in patients treated with BCG
[Bacille Calmette-Guerin] used as an immune adjuvant to treat
bladder cancer
 BCG is an attenuated strain of M. bovis
 It can become “active” and cause infection in the bladder
Is it TB – or is it M Bovis?
 M bovis = nitrate negative, M TB = nitrate positive
 M. bovis does not grow in Thiophene-2-carboxylic hydrazide
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TB grows in this compound
M. bovis does not produce Pyrazinamidase enzyme
 TB produces PYRZ enzyme

32. Mycobacterium ulcerans
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Disease: Painless boil turning into ulcer known as the
Bairnsdale or Buruli ulcer
Can progress into avascular coagulation necrosis
Found primarily African continent
Laboratory identification
 Optimum temp 30˚ C
 Slow growing
 Niacin and nitrate = Negative
All skin lesions should be cultured
at both 30˚ and 37˚

33. Mycobacterium ulcerans
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Infection begins as boil and develops a painless ulcer
known as Bairnsdale or Buruli ulcer
 can progress into avascular coagulation necrosis
 Found primarily in the African continent Laboratory
identification
Optimum temp 30˚ C
 All skin lesions should be
cultured at both 30˚ and 37˚C
 Slow growing 3- 4 weeks
Negative – niacin and nitrate

34. Mycobacterium kansasii
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Cultured at 37* C in 10-20 days
Photochromogen – turns yellow after light exposure
Niacin test = negative
Nitrate reduction = positive
Tween 80 + tests for lipase enzyme
68*C catalase +
Acid fast stain: cells are long, rectangular and beaded,
larger than TB/ Shepherd’s crook
Clinical disease mimics pulmonary TB but does not
disseminate – predisposition to diseased lung

35. Mycobacterium marinum
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Optimum temp for culture is 30˚ C
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Photochromogen
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Growth in 5-14 days
pigment is produced after light exposure
M. marinum in both fresh and salt water
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Swimming pools, fish tanks, water cooling towers
Disease known as “Swimming pool granuloma”

36. M. marinum Disease
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Tender, red or blue/red subcutaneous
nodules after trauma to skin
Lesions can continue to extend up arm and
spread along lymphatics,
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Clinically appears like Sporotrichosis

37. Mycobacterium szulgai
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Grows at 37 ˚C in 12 - 25 days
Scotochromogen at 37˚C and Photochromogen at 25˚ C
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Unique characteristic of this species
The only AFB that has a different light test based on temperature
Niacin negative
Nitrate positive
Unusual cause of disease
Rare Lung infections
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25˚ C - Photochromogen
Symptoms similar to TB
37˚ C Scotochromogen

38. Mycobacterium xenopi
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Optimum temp is 42˚C so it is capable of growing in hot
water supplies
Grows in 14 - 28 days in culture
Scotochromogen
Egg nest colony on Middlebrook agar
 Observe features under microscope
Rare cause of pulmonary infections
 clinically like TB
 mostly in patients with preexisting lung disease
 Can be seen in HIV/AIDS patients

39. M. avium-intracellulare complex
M avium and M intracellulare
Biochemically and genetically
difficult to distinguish the species
Opportunistic infection in HIV/AIDS
 High organism load can be seen in AFB stain in intestine, liver
and spleen
 Can be seen in bone marrow
 Organisms variable in size but mostly short
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Smaller than TB
Do not have cord factor
Positive blood cultures are common
Involvement of GI tract can cause diarrhea
 Positive AFB smears in stool
Pathology - Necrotizing rather than granulomatous inflammation

40. M avium-complex in tissue –
Kinyoun AFB stain
M. avium complex
Kinyoun AFB stain
variable in size
No cording

41. M. avium-intracellulare
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Laboratory –
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Growth at 37 ˚C / 7 – 21 days
Non-photochromogen
Smooth colony
Inert in biochemicals
Identify using
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GenProbe (AccuProbe) molecular identification
MALDI-TOF
Genetic 16s rRNA Sequencing

42. M. avium intracellulare clinical
correlation
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HIV/AIDS
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Disseminated in end stage disease
Nonspecific low grade fever, weakness, weight
loss, FUO
Abdominal pain and/or diarrhea with
malabsorption
Normal host
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Pulmonary disease, much like TB,
marked % of cases - older women with history of
smoking

43. Rapid growing Mycobacteria
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Laboratory
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Growth at 37˚ C in <=7 days
Most positive in arylsulfatase test
Many new species but most common:
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Nitrate reduction test
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M fortuitum group – variety of infections, skin and surgical
wound infection
M. chelonae- skin infections in immune suppressed
M. abscessus - lung infection
Positive
Negative
M. fortuitum
M. chelonae, M. abscessus
Iron Uptake – M. fortuitum positive

44. Miscellaneous
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M. gordonae –
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Rare! Cause of Infection
Major laboratory water contaminant in cultures
”tap water bacillus”
Use sterile water in culture workup to prevent
contamination
Scotochromogen
M. paratuberculosis –
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Association with Crohn’s disease
Inconclusive evidence for causation

45. Miscellaneous
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M. haemophilum
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Requires hemoglobin or hemin for growthin
culture
Will not grow on LJ or in automated system
without the addition of hemin supplements
Painful subcutaneous nodules and ulcers,
primarily in AIDS patients or immune suppressed

46. M. leprae
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Leprosy – also known as Hansen’s disease
Leprosy begins with anesthetic skin lesions and
peripheral neuropathy with nerve thickening
Presenting presentation - numbness in earlobes or
nose
Lapromatous leprosy - disfiguring lesions, large
numbers of AFB in lesions / co infection with
Strongyloides common
Tuberculoid leprosy -less severe/fewer lesions, lower
numbers of AFB in lesions
Will not grow on artificial media
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Armadillo is the natural reservoir
PCR on tissue for definitive diagnosis

47. Lapromatous leprosy
Cigar packets of AFB
Tuberculoid leprosy