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Mycobacteriology Update 2018



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Mycobacteriology Update 2018

  1. 1. Mycobacteriology Update 2018 Margie Morgan, PhD
  2. 2. Mycobacteria  Acid Fast Bacilli (AFB)  Cell walls contain complex mycolic acids and free lipids, many properties of the mycobacteria due to their presence  Once stained, the rods resist de-colorization with acid alcohol (HCl)  AFB stain vs. Partial Acid Fast (PAF) stain  AFB – HCl used to decolorize – identify Mycobacteria  PAF – H2SO4 used to decolorize – identify Nocardia  Gram stain – Gram positive, weekly stained and appear beaded  Aerobic bacilli, no spores, rarely branch
  3. 3. Identification of the Mycobacteria  For decades, identification algorithm started with the determination of pigment in the light and dark; followed by biochemical reactions, growth rate, and optimum temperature. Obsolete  With expanding taxonomy, biochemical reactions were unable to separate and identify most of the newly recognized species, replaced with High Performance Liquid Chromatography (HPLC) methods. Obsolete  Current methods:  Genetic probes/Amplification  MALDI-TOF Mass Spectrometry to analyze proteins  Sequencing 16 sRNA for precise genetic sequence information
  4. 4. Mycobacteria Taxonomy >170 species  TB complex:  Mycobacterium tuberculosis  M. bovis and the Bacillus Calmette-Guerin strain (BCG – live attenuated strain of M. bovis)  M. africanum  And some very rare species:  Mycobacterium microti  Mycobacterium canetti  Mycobacterium caprae  Mycobacterium pinnipedii  Mycobacterium suricattae  Mycobacterium mungi  Mycobacteria other than TB – “MOTT”
  5. 5. 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
  6. 6. 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
  7. 7. Algorithm for identification of MOTT - historically speaking  Runyon System  Classify 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 that promotes 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)
  8. 8. Light Test – Determine 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
  9. 9. 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***
  10. 10. 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
  11. 11. 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
  12. 12. Specimen collection  Sputum – 3 early morning collection, or 1 early morning, 2 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
  13. 13. 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
  14. 14. Why Decontaminate Specimens?  For the slow growing mycobacteria to be cultured  Must eliminate competing bacteria/yeast that grow 24 x faster – killed by pH change of the 4% NaOH  Must also release the AFB from mucus plugs in the sputum specimens - L-acetyl-cysteine action
  15. 15. Specimen Decontamination/Digestion Most often used :  4% NaOH / N-acetyl-L-cysteine  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
  16. 16. Specimen Centrifugation  Centrifugation at 3000 X g (fast) with safety cups  Speed of centrifugation is important - AFB are lipid laden and they will float if not rapidly centrifuged – must pellet to bottom of tube so they are not decanted with supernatant  Can determine the sensitivity of the AFB stain  Pour off supernatant into waste  Use pellet for stains/culture
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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 TB will form due to cord factor
  22. 22. Direct Detection of TB from Respiratory Specimens by Molecular Amplification  FDA cleared to detect TB complex organisms in smear positive and smear negative sputum specimens:  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  Currently, no FDA cleared assays for MOTT species
  23. 23. PPD (Purified Protein Derivative) Mantoux test/TB skin test  Detects past or current TB exposure  False positive reaction in those with BCG immunization  @25% false negative reactions – usually due to low T cell reactivity or problems with administration  Measures delayed hypersensitivity (T cell response) to TB complex antigens  Measure (mm) area of induration at injection site  >=15mm positive rxn (check for history of BCG)  >=10mm positive in immune suppressed or just exposed
  24. 24. Cell Mitogen assays – Interferon Gamma Release Assays  QuantiFERON-TB-Gold (QFT) and T-spot assays  Whole blood tests to screen for cell mediated immunity to TB specific antigens  Quantitative measurement of gamma interferon due to the CD4 T cell response to TB specific antigens  Specificity for TB is helpful in screening patients immunized with BCG  Detects latent and active infection – does not replace culture  Sensitivity similar to PPD/ improved specificity
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. Pathology of M. tuberculosis Lung with pulmonary TB has apical cavitations with numerous areas of caseous necrosis and massive tissue consolidation
  29. 29. 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
  30. 30. 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.
  31. 31. 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  If patient culture positive after four months/retest/possible resistant strain
  32. 32. 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)
  33. 33. 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
  34. 34. 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
  35. 35. Mycobacterium marinum  Optimum temp for growth 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  Infection associated with trauma occurring in water:  swimming pools (swimming pool granuloma)  cleaning fish tanks  ocean (surfing)
  36. 36. 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.
  37. 37. 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
  38. 38. 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
  39. 39. 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 biochemical reactions  Identify using  GenProbe (AccuProbe) molecular DNA probe  MALDI-TOF  Genetic 16s rRNA Sequencing
  40. 40. 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
  41. 41. M avium-complex in lymph node tissue (Kinyoun stain) Granulomatous inflammation lung tissue (H&E stain) Bowel -Lamina propria expanded from predominately Lymphohistocytic infiltration
  42. 42. 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
  43. 43.  Low virulence, found in nature  Cause: Infections in soft tissue, venous catheter sites and shunts @ 20 species – 5 species 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 Rapid Growers
  44. 44. 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
  45. 45. 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
  46. 46. 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, completely curable with early diagnosis and therapy  Two types of Leprosy  Lapromatous – can lead to severe disfiguring lesions, large numbers of AFB in lesions / +/- co-infection with Strongyloides  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  Found in tropical Africa and Asia
  47. 47. Tuberculoid leprosy Lapromatous leprosy Skin biopsy - AFB seen in nerve fiber AFB in “cigar packets”