Vaccine and
Treatments
KeyIdentificati
on
Isolation technique
Staining method
Specimen Collection
Transmittance and Personal safety
Photochromogens
Scotochromogens
Rapid Growers
Non-chromogen
s
MYCOBACTERIOLOGY
Leprosy
TRANSMITTANCE AND PERSONAL SAFETY Transmitted via:
Airborne droplets (e.g. coughing ) In a confined space (e.g. jail, at home, school, work etc)
General safety: Adequate ventilation; wear masks; early detection and treatment
Lab safety: Use biosafety level 3 cabinets with directional airflow; aerosol free
centrifuges; protective coverings, UV light; periodic TB testing, etc.
Symptoms: Fatigue, weight loss, cough, fever, night sweats. X-ray illustrates cavitary
disease of solitary nodules
NON-CHROMOGENS
M. tuberculosis Rough, buff, about 21 days Key reactions: Niacin and Nitrate Confirmation: Nucleic acid probes or
HPLC (high performance liquid chromatography).
Mainly causes pulmonary illness but can be anywhere.
M. avium-intracelluare (M.A.C) Rough or smooth, slow Can be other Runyoun groups Key reactions: lack of niacin and
nitrate, growth in NAP and TCH. Semi-quant cat and tellurite are positive
Pulmonary, disseminated
M. bovis Generally smooth, buff colonies
with irregular margins seen after 21 days of incubation at 37ºC.
Key reactions: Urease positive, sensitive to TCH
Causes pulmonary illness mainly in livestock but can be transmitted from cattle to humans.
NON-CHROMOGENS
M. xenopi Recovered from water, hot and cold taps Can be scotochromogenic Small, slow growing colonies with dense
centers and filamentous edges Optimum growth at 42 degrees
Celsius Key reactions: Niacin negative, nitrate
negative, Catalase positive, arylsulfatase positive, PZA positive
Slow progressive pulmonary infection
M. haemophilum Rough to smooth buff colonies Optimum growth at 28-32oC Requires heme/hemoglobin for growth Cause lymphadenitis, abscesses, draining
fistulas (but mostly in immunocompromised patients)
M. ulcerans Slow growth, buff smooth and rough
colonies Optimum growth 30-33oC , 6-12
weeks incubation Key reactions: Biochemically inert Bairnsdale ulcer, cutaneous lesions
in tropical regions following soil disturbances.
PHOTOCHROMOGENS
M. kansasii Yellow carotene pigment when exposed to light
Rough colonies with wavy edges and dark centers , slow growth at 37 degrees C
Key reaction: Niacin negative, strongly positive for nitrate and catalase, PZA and Tween 80 positive
Chronic pulmonary disease
M. simiae Smooth, slow growing colonies, 10-21 days at 37
degrees C
Key reactions : Niacin positive (can easily be mistake n for M. tuberculosis), Nitrate negative , catalase positive
Pulmonary disease
M. marinuum Optimum growth at 28oC-32oC Buff colonies seen in the dark, yellow when
exposed to light Slow growing, smooth to rough colonies. Key reactions: Niacin and nitrate negative, catalase negative, PZA positive Cutaneous infections after trauma in water
M. asiaticum Dysgenic and smooth Growth after 15 to 21 days at 37oC Key reactions: Niacin negative, Nitrate negative, Catalase
positive, Tween 80 positive
Rarely causes human infection
SCOTOCHROMOGENS
M. gordonae Smooth yellow-orange colonies , 10-14 days Optimum temperature: 22-37oC Key reactions: Nitrate negative, catalase
positive, Tween 80 positive, urea positive Rarely causes infection Contaminant from tap water (referred to
as “tap-water bacillus”)
M. szulgai Pigment production is temperature dependent Photochromogen at 22oC Smooth and rough colonies Key reactions: slow hydrolysis of Tween 80,
nitrate positive, inability to grow in the presence of 5% NaCl
Pulmonary disease
M. scrofulaceum 4-6 weeks growth at 25-37
degrees Celsius Smooth light yellow to deep
orange colonies with dense centers
Key reactions: Nitrate negative, catalase positive, Tween 80 negative, urea positive
Associated with cervical lymphadenitis in children
RAPID GROWERS
M. fortuitum Growth in 3-5 days of incubation at 37oC Buff rough or smooth colonies Key reactions: Positive 3-day arylsulfatase test,
nitrate positive, NaCl positive, iron positive Skin and soft tissue , “whirlpool footbath”
infections
M. chelonae Related to M. abscessus Most commonly isolated rapid grower Rough or smooth buff colonies Growth within 3-5 days of incubation at 37oC Key reactions: Positive 3-day arylsulfatase,
nitrate negative, iron negative, NaCl negative
Disseminated nodular disease in immunocompromised patients
M. smegmatis complex
Buff, rough, wrinkled or coarsely folded growth after 2-4 days Key reactions: negative arylsulfatase, iron positive, nitrate
positive, positive growth in NaCl
Pulmonary, skin, soft tissue and bone infections
M. phlei
3-5 days growth; optimum temperature 30oC Coarsely wrinkled with deep yellow to orange pigment Key reactions: negative arylsulfatase, iron positive, NaCl
negative Rare cause of pulmonary and soft tissue infections
M. thermoresistable
Rare Grows at 52oC
M. abscessus
Subspecies of M. chelonae Chronic lung disease, otitis media following tympanostomy,
disseminated cutaneous infections
Rapid Growers : M. chelonae - Arylsulfatase (+); 5% NaCl, iron uptake and Nitrate (-)
M. fortuitum - Arylsulfatase (+) ; 5% NaCl, iron uptake and nitrate (+)
Photochromogen
Nitrate negative
StrongNitratePositive
M. kansaii Cat (+)
M. marinum
Tween 80
negative
M. scrofulaceu
m
Urease (+)
M. gordonae
Urease (-)
PigmentScotochromog
enNon-
chromogen
Niacin positive
Niacin negative
M. Tuberculo
sisNitrate
(+) , TCH (+) cat (-)
NAP (-)
TCH (+)/PZA (+)
TCH (-)/ PZA (-)
M. Avium complex Tellurite and cat
(+)
M. bovis
Tween 80
posistive
SPECIMEN COLLECTION Sterile wide mouth cup with tightly fitted lid Respiratory:
First morning deep cough sputum, bronchial wash or brushing, BAL or bronchial lavage
Cutanaeous lesions Body Fluids
Pleural, ascitic pericardial, gastric, CSF, urine, joint aspirate, feces, etc.
SPECIMEN COLLECTION Culture media
Egg based media (Lowenstein Jensen)- classical TB medium. Contains malachite green which suppresses the
growth of gram positive bacteria
Agar based media (Middlebrook 7H10) – contains casein hydrolysate (helps isolate isoniazid resistant stains), biotin
and catalase (helps revive damaged bacilli); and albumin (binds toxins)
ISOLATION TECHNIQUE: DIGESTION, DECONTAMINATION AND CONCENTRATION
1. Digestion: N-acetyl-L-cysteine (NALC) disrupts disulfide bonds of mucus,
releasing organism
2.Decontamination 4% sodium hydroxide (NaOH) equally mixed with NALC destroys
bacterial species (i.e. normal flora) other than mycobacterium
Concentration Neutralization: Phosphate buffer of low specific gravity is
added to mixture, and centrifuged at a high rate to concentrate the organism
Albumin dilutes the mixture and heal the damaged cells
STAINING METHOD Gram stains poorly due to high lipid
content (mycolic acid) in cell wall
Fluorescence or Carbolfuschin technique (i.e. Ziel Neelson or Kinyoun stain)
DETECTION METHODS Mantoux screening (tuberculin skin test)
Read 48 hours later Positive skin test due to:
1) exposure to the antigen
2) Recovery from past infection(delayed type (IV) hypersensitivity)
Reading the test: Healthy individuals – Induration (raised hardened area) of 15mm or greater is positive result
Immunocompromised individuals–Induration greater than or equal to 5 mm is a positive result
Quantiferon-TB gold Use whole bloodLatent or active TB will stimulate interferon-gamma release from leukocytesMeasured with ELISA
LEPROSY (HANSEN’S BACILLUS) Spread through direct contact or by inhalation via aerosols
Slow growth (very difficult to culture in routine lab)
Acid fast positive
Two forms: Tuberculoid form – non-contagious hypo-pigmented skin macules with underlying nerve involvement causing loss of feeling
Lepromatous form - highly contagious. Cell mediated immunity absent causing an increase development of the organism
VACCINES AND TREATMENT B.C.G NOT used as a vaccine for TB due to:
Interference with skin test U.S. is a low risk population Varies in its ability to trigger immunity
Dapsone + Rifampin + Clofazimine multidrug therapy which
effectively treats both TB and leprosy
(must be used in combination to prevent development of resistant organisms)
