Mycobacteriology

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Mycobacteriology

  1. 1. Mycobacteriology Just the very basics – meant for board review or a brief overview of this very important area of the laboratory.
  2. 2. PPD (Purified Protein Derivative) better known as the TB skin test    For decades used to determine exposure to TB Mantoux test – 5 Tuberculin units / intradermal injection Detects past or current TB exposure and reacts with BCG immunization     Not a very sensitive test – up to 25% false negative reactions Measures delayed hypersensitivity response to TB related antigens - T cells react to TB related antigens--- lymphokine released ---forms induration at the injection site Read the reaction at 48 hr - measure induration in mm  >=15 mm positive or check for BCG immunization in past  >=10 mm positive in immune suppressed or just exposed Recent shortages of the PPD (Tubersol) product has led to a change to using Interferon Gamma Release Assays (IGRA)
  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 Able to detect both latent TB and active infection     NOT meant to replace culture or other diagnostic tests Is independent of BCG exposure/ specific for TB complex however, does cross react with a few uncommon Mycobacteria other than TB  Useful for screening the BCG immunized population where the PPD cannot be used due to false positive tests Meant to replace and improve upon the PPD skin test Whole blood has sensitized T cells --- (normal immune population)   stimulated by antigen peptides that are specific to TB –(ESAT-6 and CFP-10) ---secrete cytokine interferon gamma---amount interferon gamma secreted detected is measured using an EIA assay
  4. 4. General Features of Mycobacteria – aka Acid Fast Bacilli (AFB)     Closely 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 like species.  AFB acid fast stains use HCl to decolorize organism  PAF acid fast stains use H2SO4 to decolorize –  this is a more gentle process and Nocardia will be PAF + but will be AFB negative in the “true” AFB stains meant for the mycobacteria.
  5. 5. Mycobacteria – General Characteristics     Aerobic, no spores, slightly curved or straight rods, rarely branch, variable in length depending on the species Hardy in the environment for months and most grow on simple substrates Mycobacteria include obligate pathogens, opportunists and saprophytic species High amount of mycolic acids and free lipids in cell wall which give many properties to this genus including the AFB staining properties M. tuberculosis M. kansasii
  6. 6. Identification of the Mycobacteria    For decades the identification was based on the production of pigment in the light and dark and biochemical reactions With expanding taxonomy, biochemical reactions are not able to separate and identify some of the newly recognized species New methods have evolved for identification:     HPLC – high-performance liquid chromatography to identify mycolic acids, good but not the best for definitive speciation Genetic probes – RNA/DNA hybridization probes MALDI-TOF Mass Spectrometry to analyze proteins Sequencing 16 sRNA for genetic sequence identification
  7. 7. Mycobacteria Taxonomy   TB and genetically related organisms that are separated taxonomically from the other species TB complex include:     Mycobacterium tuberculosis M. bovis M. africanum Other vary rare species  Other Mycobacteria in the TB complex are grouped into “MOTT” Mycobacteria other than TB  The Runyon System is used to classify those species not in the TB complex (MOTT)- divided into four groups:     Pigment when exposed to light in Light Test Pigment in both light and dark in the Light Test No pigment produced in light or dark in the Light Test Growth rate (<= 7 days) – Rapid grower
  8. 8. Light Test – does it produce a yellow pigment after being exposed to light ? Group I Photochromogen Turn yellow after light exposure Group III Non photochromogen – never has pigment Group II Scotochromogen Always has yellow pigment – light or no light exposure
  9. 9. Runyon Classification System – Groups determined by results of the light test  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 Group IV – Rapid growers – grow in 7 days or less      M. fortuitum group M. abscessus M. chelonae M. mucogenicum >= 20 species
  11. 11. Which ones reported to cause disease?                Mycobacterium tuberculosis – the major pathogen of this genus M. avium 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
  12. 12. Mycobacteria that rarely if ever cause disease! If so, in immune compromised!       M. gordonae M. gastri M. celatum M. scrofulaceum M. terrae complex M. smegmatis
  13. 13. Specimen collection         Sputum – 3 specimens - early morning or at least 8 hours apart Bronchial lavage fluid Tissues or Wounds CSF or sterile body fluids Urine – 3 to 5 early morning collections Stool – M. avium complex only Gastric – for children, must neutralize the pH of specimen for AFB to survive Blood – disseminated disease  Automated systems – AFB blood culture bottles manufactured for AFB isolation
  14. 14. Specimen Processing in the AFB Laboratory   Level 3 Safety 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 Must have negative air flow in laboratory, anteroom for dressing and washing hands 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
  15. 15. Specimen Processing Start to Finish!   5 ml of specimen put in the conical tube Decontaminate and Liquify for 15 minutes     Fill tube with phosphate buffer to neutralize pH Centrifuge for 30 minutes      Add 5 ml of 4% NaOH (Increases the pH to 9) plus N-acetyl-L-Cysteine (breaks up the mucus) 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
  16. 16. How and why do you perform Specimen Decontamination?   Mycobacteria are more resistant to killing by acids and alkaline solutions than most bacteria due to the high amount of lipid in the cell wall – this is used to rid the specimen of contaminating bacteria and yeast For the slow growing mycobacteria to be cultured / must eliminate competing bacteria that grow 24 x faster and release the AFB from mucus plugs in the sputum specimens - this is accomplished by exposing the sputum to alkaline/acid solutions and mucolytic reagents such as 4% NaOH and L-acetyl-L cysteine
  17. 17. 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 can be used for cystic fibrosis sputum specimens to eliminate the resistant (mucoid) Pseudomonas strains   Oxalic acid should not be used routinely, it is too harsh and will decrease isolation of AFB These solutions kill bacteria and can also kill AFB if left on specimen > 15 minutes
  18. 18. Specimen Centrifugation      Centrifugation at 3000 X g (fast) 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 into the waste This determines the sensitivity of the AFB stain so proper centrifugation speed is important Pour off supernatant Use pellet for testing
  19. 19. Plating -Selection of Plate and Tubed Culture Media  Middlebrook – Synthetic media  Clear agar and liquid media  Synthetic = chemical ingredients added for optimal growth  Used for culture and susceptibility testing  Can Autoclave for sterility  Lowenstein-Jensen – Egg based  Green media due to malachite green)  Hens egg, glycerol, and potato flour  Sterilize by inspissation – drying  Cultures on solid media incubated at 37˚C , 5-10% C0₂ for 8 weeks
  20. 20. Automated Detection of AFB  Automated systems:  BACTEC MGIT 960 and BACTI-ALERT Instruments  Use Liquid Middlebrook 7H9 tubed media for growth  Both systems have same detection method BactiAlert System    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 BACTEC MGIT 960 NAP test – Identification for TB NAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone) An automated test on the BACTEC 960 for TB identification MGIT 960 TB does not grow in the NAP containing tube BACTEC Other Mycobacteria species grow in NAP
  21. 21. Acid Fast Staining for Mycobacteria Carbol Fuchsin based stains Stain the AFB Red CF is the red colored stain/potassium permanganate is the background counterstain (blue)  Ziehl-Neelsen (ZN) – uses heat to drive stain into AFB  Kinyoun – uses phenol to drive stain into AFB  Read for 5 min on oil objective  Fluorochrome based stain  Auramine Rhodamine – fluorescent stain, organisms stain fluorescent gold, read on 25X or 40X for 2 min on a fluorescence microscope  Generally considered to more sensitive than ZN or Kinyoun
  22. 22. Acid Fast staining of the Mycobacteria Mycobacterium avium complex Organisms are routinely shorter than TB M. Tuberculosis - Organisms are long and can appear as if they are sticking together [cord factor]
  23. 23. Direct Detection of TB from specimens by Amplification    Gen-Probe (Hologics) TMA and Cepheid Xpert-TB RIF FDA cleared Can only test respiratory specimens per FDA Detect TB complex in AFB smear positive and negative respiratory specimens        The Xpert TB/RIF test can also detect Rifampin-resistance associated mutations of the rpoB gene Respiratory specimens must first be decontaminated and concentrated prior to testing for Gen-Probe. The Cepheid Xpert method can test non-concentrated sputum. Amplifies a 16S rRNA gene sequence of TB Sensitivity @ 90% AFB smear (+), 75% AFB smear (-) Test of diagnosis not cure Residual rRNA can be present up to 6m after diagnosis Still must perform culture and susceptibility testing
  24. 24. Mycobacterium tuberculosis    Optimal Temp 37˚ C, Grows in 12 –25 days Buff colored, dry cauliflower-like colony Manual tests for identification  Niacin Positive - accumulation of niacin, a product produced form growth on this egg containing medium (LJ medium)- must be performed on culture growing on LJ medium  Nitrate reduction – Reducing nitrate to nitrite = Positive  Confirmation of TB vs M Bovis    M bovis = nitrate negative M. bovis does not grow in Thiophene-2-carboxylic hydrazide (T2H) Molecular identification:  Gen-Probe AccuProbe DNA/RNA hybridization identifies TB complex organisms with excellent accuracy
  25. 25. Mycobacterium tuberculosis Demonstrates Cord factor – due to high lipid content in TB organisms they stick together and can develop long ropes – unique to TB
  26. 26. Tuberculosis  Classic presentation      Slowly progressive pulmonary infection Coughing, weight loss, low grade fever Biopsy of lung most often caseating granulomas Organisms seen in a concentrated AFB stain indicates that the patient is capable of infecting others Never report a non-concentrated sputum AFB stain   high % of false negative stain results TB is spread by respiratory droplets  All patients require respiratory isolation precautions
  27. 27. TB in HIV/AIDS patients    Worldwide -TB is the most common opportunistic infection affecting HIV (+) Pulmonary TB most usual presentation With progressive decline of cell mediated immunity (low CD4 count) – greater risk of extrapulmonary dissemination  Granulomas with/without caseation
  28. 28. TB can be a cause of Scrofula    Unilateral lymphadenitis Nodes drain to skin surface Two most common causes:   M. tuberculosis M. avium-complex
  29. 29. Susceptibility testing of TB  Two methods   (1) Agar dilution -antibiotics embedded in solid agar (2) Bactec liquid 7H9 medium with antibiotic solutions   Primary drug panel tested for TB consists of 5 drugs     Tested on the automated MGIT 960 system Isoniazid Pyrazinamide Rifampin Streptomycin Ethambutol Molecular Beacon testing – hybridization probes used with RT PCR assay to quantify target DNA. Used for rapid determination of MDRTB – detect INH and RIF resistance
  30. 30. Susceptibility of TB   Most TB isolates are susceptible to the 5 primary TB drugs Populations more likely to be resistant to one or more drugs include    HIV/AIDS Immigrant populations MDRTB refers to the multi-drug resistant strains of TB   At least resistant to INH and RIF – the two most common drugs used to treat TB Can be resistant to additional first line drugs
  31. 31. M. bovis    M. bovis produces disease in cattle and other animals  Spread to humans by milk ingestion  Most common disease symptoms like that of TB M. bovis can cause bladder infections in patients treated with BCG [Bacille Calmette-Guerin] used as an immune adjuvant to treat bladder cancer  BCG is an attenuated strain of M. bovis  It can become “active” and cause infection in the bladder Is it TB – or is it M Bovis?  M bovis = nitrate negative, M TB = nitrate positive  M. bovis does not grow in Thiophene-2-carboxylic hydrazide   TB grows in this compound M. bovis does not produce Pyrazinamidase enzyme  TB produces PYRZ enzyme
  32. 32. Mycobacterium ulcerans   Disease: Painless boil turning into ulcer known as the Bairnsdale or Buruli ulcer Can progress into avascular coagulation necrosis Found primarily African continent Laboratory identification  Optimum temp 30˚ C  Slow growing  Niacin and nitrate = Negative All skin lesions should be cultured at both 30˚ and 37˚
  33. 33. Mycobacterium ulcerans    Infection begins as boil and develops a painless ulcer known as Bairnsdale or Buruli ulcer  can progress into avascular coagulation necrosis  Found primarily in the African continent Laboratory identification Optimum temp 30˚ C  All skin lesions should be cultured at both 30˚ and 37˚C  Slow growing 3- 4 weeks Negative – niacin and nitrate
  34. 34. Mycobacterium kansasii         Cultured at 37* C in 10-20 days Photochromogen – turns yellow after light exposure Niacin test = negative Nitrate reduction = positive Tween 80 + tests for lipase enzyme 68*C catalase + Acid fast stain: cells are long, rectangular and beaded, larger than TB/ Shepherd’s crook Clinical disease mimics pulmonary TB but does not disseminate – predisposition to diseased lung
  35. 35. Mycobacterium marinum  Optimum temp for culture is 30˚ C   Photochromogen   Growth in 5-14 days pigment is produced after light exposure M. marinum in both fresh and salt water   Swimming pools, fish tanks, water cooling towers Disease known as “Swimming pool granuloma”
  36. 36. M. marinum Disease   Tender, red or blue/red subcutaneous nodules after trauma to skin Lesions can continue to extend up arm and spread along lymphatics,  Clinically appears like Sporotrichosis
  37. 37. Mycobacterium szulgai   Grows at 37 ˚C in 12 - 25 days Scotochromogen at 37˚C and Photochromogen at 25˚ C       Unique characteristic of this species The only AFB that has a different light test based on temperature Niacin negative Nitrate positive Unusual cause of disease Rare Lung infections  25˚ C - Photochromogen Symptoms similar to TB 37˚ C Scotochromogen
  38. 38. Mycobacterium xenopi      Optimum temp is 42˚C so it is capable of growing in hot water supplies Grows in 14 - 28 days in culture Scotochromogen Egg nest colony on Middlebrook agar  Observe features under microscope Rare cause of pulmonary infections  clinically like TB  mostly in patients with preexisting lung disease  Can be seen in HIV/AIDS patients
  39. 39. M. avium-intracellulare complex M avium and M intracellulare Biochemically and genetically difficult to distinguish the species Opportunistic infection in HIV/AIDS  High organism load can be seen in AFB stain in intestine, liver and spleen  Can be seen in bone marrow  Organisms variable in size but mostly short      Smaller than TB Do not have cord factor Positive blood cultures are common Involvement of GI tract can cause diarrhea  Positive AFB smears in stool Pathology - Necrotizing rather than granulomatous inflammation
  40. 40. M avium-complex in tissue – Kinyoun AFB stain M. avium complex Kinyoun AFB stain variable in size No cording
  41. 41. M. avium-intracellulare  Laboratory –      Growth at 37 ˚C / 7 – 21 days Non-photochromogen Smooth colony Inert in biochemicals Identify using    GenProbe (AccuProbe) molecular identification MALDI-TOF Genetic 16s rRNA Sequencing
  42. 42. M. avium intracellulare clinical correlation  HIV/AIDS     Disseminated in end stage disease Nonspecific low grade fever, weakness, weight loss, FUO Abdominal pain and/or diarrhea with malabsorption Normal host   Pulmonary disease, much like TB, marked % of cases - older women with history of smoking
  43. 43. Rapid growing Mycobacteria  Laboratory    Growth at 37˚ C in <=7 days Most positive in arylsulfatase test Many new species but most common:     Nitrate reduction test    M fortuitum group – variety of infections, skin and surgical wound infection M. chelonae- skin infections in immune suppressed M. abscessus - lung infection Positive Negative M. fortuitum M. chelonae, M. abscessus Iron Uptake – M. fortuitum positive
  44. 44. Miscellaneous  M. gordonae –      Rare! Cause of Infection Major laboratory water contaminant in cultures ”tap water bacillus” Use sterile water in culture workup to prevent contamination Scotochromogen M. paratuberculosis –   Association with Crohn’s disease Inconclusive evidence for causation
  45. 45. Miscellaneous  M. haemophilum    Requires hemoglobin or hemin for growthin culture Will not grow on LJ or in automated system without the addition of hemin supplements Painful subcutaneous nodules and ulcers, primarily in AIDS patients or immune suppressed
  46. 46. M. leprae       Leprosy – also known as Hansen’s disease Leprosy begins with anesthetic skin lesions and peripheral neuropathy with nerve thickening Presenting presentation - numbness in earlobes or nose Lapromatous leprosy - disfiguring lesions, large numbers of AFB in lesions / co infection with Strongyloides common Tuberculoid leprosy -less severe/fewer lesions, lower numbers of AFB in lesions Will not grow on artificial media   Armadillo is the natural reservoir PCR on tissue for definitive diagnosis
  47. 47. Lapromatous leprosy Cigar packets of AFB Tuberculoid leprosy

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