1. Vaccine and Treatments Key Identification Isolation technique Staining method Specimen Collection Transmittanc e and Personal safety Photochromo gens Scotochromo gens Rapid Growers Non- chromogens 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

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.

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.

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

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

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

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

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.

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)

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

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)

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

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

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 + Clofazimine multidrug therapy which effectively treats both TB and leprosy (must be used in combination to prevent development of resistant organisms)