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Website mycobacteriology 2017 update


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Mycobacteriology Review 2017

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Website mycobacteriology 2017 update

  1. 1. Mycobacteriology Review Update 2017 Margie Morgan, PhD., D(ABMM)
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 29. Pathology of M. tuberculosis Lung with pulmonary TB has apical cavitations with numerous areas of caseous necrosis and massive tissue consolidation
  30. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 48. Tuberculoid leprosy Lapromatous leprosy Skin biopsy - AFB seen in nerve fiber AFB in “cigar packets”