Mycobacteriology

1. Mycobacteriology Leprosy

2. 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 Transmittance and personal safety

3. 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

4. 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. Non-chromogens

5. 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 Photochromogens

6. 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 Scotochromogens

7. 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

8. Rapid Growers : M. chelonae - Arylsulfatase (+); 5% NaCl, iron uptake and Nitrate (-) M. fortuitum - Arylsulfatase (+) ; 5% NaCl, iron uptake and nitrate (+) Pigment Photochromogen Scotochromogen Non-chromogen Tween 80 posistive Tween 80 negative Strong Nitrate Positive Nitrate negative Niacin negative Niacin positive M. scrofulaceum Urease (+) TCH (+)/ PZA (+) TCH (-)/ PZA (-) M. gordonae Urease (-) M. Tuberculosis Nitrate (+) , TCH (+) cat (-) NAP (-) M. kansaii Cat (+) M. marinum M.Avium complex Tellurite and cat (+) M. bovis

9. 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

10. 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) Specimen collection

11. 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 isolation technique: Digestion, Decontamination and Concentration

12. Gram stains poorly due to high lipid content (mycolic acid) in cell wall Fluorescence or Carbolfuschin technique (i.e. Ziel Neelson or Kinyoun stain) Staining method

13. 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 blood Latent or active TB will stimulate interferon-gamma release from leukocytes Measured with ELISA Detection methods

14. 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 Leprosy (hansen’s bacillus)

15. 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 + Clofaziminemultidrug therapy which effectively treats both TB and leprosy (must be used in combination to prevent development of resistant organisms) Vaccines and treatment