Anthrax teaching


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Anthrax teaching

  1. 1. Anthrax Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. General Characteristics of Bacillus~ 60 species; Gram-positive or Gram-variable bacilli • Large (0.5 x 1.2 to 2.5 x 10 um) • Most are saprophytic contaminants or normal flora • Bacillus anthracis is most important memberProduce endosporesAerobic or facultatively anaerobic Bacillus spp. are ubiquitous• Soil, water, and airborne dust• Thermophilic (< 75°C) and psychrophilic (>5-8°C)• Can flourish at extremes of acidity & alkalinity (pH 2 to 10) Dr.T.V.Rao MD 2
  3. 3. Anthrax• From the Greek word anthrakos for coal• Caused by spores• Primarily a disease of domesticated & wild animals – Herbivores such as sheep, cows, horses, goats• Natural reservoir is soil – Does not depend on an animal reservoir making it hard to eradicate – Cannot be regularly cultivated from soils where there is an absence of endemic anthrax – Occurs sporadically throughout US – South Dakota, Arkansas, Texas, Louisiana, Mississippi, California recognized endemic areas Dr.T.V.Rao MD 3
  4. 4. A Closer Look at Anthrax• Anthrax is a disease of cattle, goats, and sheep caused by a bacterium, Bacillus anthracis. It is rare for humans to be infected. Most infections that do occur are localized to small cuts in the skin whose edges turn black (hence the name “anthracis”, after anthracite coal). The disease is deadly for humans because B. anthracis produces lethal toxins. Dr.T.V.Rao MD 4
  5. 5. Bacillus anthrax Several landmarks• 1st to Observe under Microscope• 1st to communicable disease.• 1st observe the spores ( Robert Koch )• 1st to prepare for attenuated vaccine. ( Louis Pasteur) Dr.T.V.Rao MD 5
  6. 6. 20,000-100,000 cases estimated globally/year Dr.T.V.Rao MD
  7. 7. Animal Transmission• Most commonly infected by ingestion from contaminated soil or contaminated feed or bone meal Dr.T.V.Rao MD 7
  8. 8. Anthrax • An infectious, Usually fatal disease of warm-blooded animals, especially of cattle and sheep, caused by the bacterium Bacillus anthracis. Dr.T.V.Rao MD 8
  9. 9. B. anthracisGram-positive, spore-forming, non-motile bacillus Dr.T.V.Rao MD 9
  10. 10. Anthrax Bacilli Dr.T.V.Rao MD 10
  11. 11. Bacillus anthracis General characteristics• Bacillus anthracis• Large, Gram positive, non-motile rod• Vegetative form and spores• Nearly worldwide distribution• Over 1,200 strains Dr.T.V.Rao MD 11
  12. 12. Bacillus anthracis• Gram + rod• Facultative anaerobe• 1 - 1.2 m in width x 3 - 5 m in length• Belongs to the B. cereus family – Thiamin growth requirement – Glutamyl-polypeptide capsule – Nonmotile• Forms oval, centrally located endospores Dr.T.V.Rao MD 12
  13. 13. The Spore• Sporulation requires – Poor nutrient conditions – Presence of oxygen• Spores – Very resistant to extremes – Survive for decades – Taken up by host and germinate• Lethal dose 2,500 to 55,000 spores Dr.T.V.Rao MD 13
  14. 14. Endospore• Oxygen required for sporulation• 1 spore per cell• dehydrated cells – Highly resistant to heat, cold, chemical disinfectants, dry periods• Protoplast carries the material for future vegetative cell• Cortex provides heat and radiation resistance• Spore wall provides protection from chemicals & enzymes Dr.T.V.Rao MD 14
  15. 15. Gram Stain Morphology of B. anthracis• Broad, gram-positive rod: 1–1.5 x 3–5 µ• Oval, central to subterminal spores: 1 x 1.5 µ with no significant swelling of cell• Spores usually NOT present in clinical specimens unless exposed to atmospheric O2 Dr.T.V.Rao MD 15
  16. 16. Mechanism of Infection• Anthrax spores enter body• Germinate & multiple in lymph nodes• PA, EF, LF excreted from bacteria• PA binds to TEM8.• EF and/or LF binds• Complex internalized by endocytosis• Acidification of endosome• LF or EF crosses into cytosol via PA mediated ion- conductive channels Dr.T.V.Rao MD 16
  17. 17. Cultural characteristics• Aerobe, facultative anaerobe• Grows between 12 -45 c• 2-3 mm colonies• Edge like matted hair• Medusa head appearance• Blood agar Hemolytic colonies Dr.T.V.Rao MD 17
  18. 18. Appearance of Anthrax• String of pearl appearance with Pencillin• Differentiates Anthrax and Cereus Dr.T.V.Rao MD 18
  19. 19. SELECTIVE MEDIUM PLET• Contain• 1 polymyxins• 2 Lysozyme• 3 Ethylene dioxide• 4 Tetra acetic acid Contains EDTA Dr.T.V.Rao MD 19
  20. 20. Biochemical Reactions• Gelatin – Inverted fir tree appearance Dr.T.V.Rao MD 20
  21. 21. Biochemical Reactions• Glucose, Maltose and Sucrose fermented with acid but no gas• Catalase positive Dr.T.V.Rao MD 21
  22. 22. Sterilization of environments Floor space/shed/vehicle• Preliminary disinfection using 10% formaldehyde; (1-1.5 It/ sq.m.) or 4%• Gluteraldehydes for at least 2 hours• Cleaning - by washing or scrubbing with hot water• Final disinfection by one of the following disinfectants applied for at least 2 hours.• 10% formaldehyde 4% Gluteraldehydes• 3% hydrogen peroxide or• 1% per acetic acid Dr.T.V.Rao MD 22
  23. 23. Wool and Hair• By duckering process (five stages) i.e.• lmmersion in 0.25-0.3% soda liquor• Immersion in soap liquor;• Two immersions in 2% formaldehyde solution; and• Rinsing in water Dr.T.V.Rao MD 23
  24. 24. Antibiotics• Pencillin• Erythrocin• Tetracycline• Chloramphenicol• Occasional strains resistant to penicillin are encountered Dr.T.V.Rao MD 24
  25. 25. Transmitted• The disease can be transmitted to humans through contact with contaminated animal substances, such as hair, feces, or hides, and is characterized by ulcerative skin lesions Dr.T.V.Rao MD 25
  26. 26. Criteria in Transmission• Skin: direct skin contact with spores; in nature, contact with infected animals or animal products (usually related to occupational exposure)• Respiratory tract: inhalation of aerosolized spores• GI: consumption of undercooked or raw meat products or dairy products from infected animals• NO person-to-person transmission of inhalation or GI anthrax Dr.T.V.Rao MD 26
  27. 27. Anthrax Cycle Dr.T.V.Rao MD 27
  28. 28. Pathogenesis• The infectious dose of B. anthracis in humans by any route is not precisely known. – Rely on primate data – Minimum infection dose of ~ 1,000-8,000 spores – LD50 of 8,000-10,000 spores for inhalation• Virulence depends on 2 factors – Capsule – 3 toxins Dr.T.V.Rao MD 28
  29. 29. Anthrax:Clinical Presentation Cutaneous Inhalational Gastrointestinal Dr.T.V.Rao MD 29
  30. 30. Epidemiologyof Anthrax in Animal andHuman Hosts Dr.T.V.Rao MD 30
  31. 31. Three forms of Anthrax• Cutaneous anthrax – Skin – Most common – Spores enter to skin through small lesions• Inhalation anthrax – Spores are inhaled• Gastrointestinal (GI) anthrax – Spores are ingested – Oral-pharyngeal and abdominal Dr.T.V.Rao MD 31
  32. 32. Cutaneous Anthrax• 95% of all cases globally• Incubation: 2-3 days (up to 12 days)• Spores enter skin through open wound or abrasion• Papule progresses to black Escher• Severe edema• Fever and malaise Dr.T.V.Rao MD 32
  33. 33. Anthrax: CutaneousBegins as a papule, progresses through avesicular stage to a depressed black necroticulcer (Escher)Edema, redness, and/or necrosis withoutulceration may occurForm most commonly encountered in naturallyoccurring casesIncubation period: 1–12 daysCase-fatality: Without antibiotic treatment—20% With antibiotic treatment—1% Dr.T.V.Rao MD 33
  34. 34. Anthrax: InhalationalA brief prodromal resembling a “viral-like”illness, characterized by myalgia, fatigue,fever, with or without respiratorysymptoms, followed by hypoxia anddyspnea, often with radiographic evidenceof mediastinal widening.Meningitis in 50% of patientsRhinorrhea (rare) Dr.T.V.Rao MD 34
  35. 35. • Glycocalyx Capsule – Sticky, gelatinous polymer external to cell wall• pX02 plasmid• Made up of D-glutamic acid• Non-toxic on its own• Only encapsulated B. anthracis virulent• Most important role during establishment of disease – Protects against phagocytosis & lysis during vegetative state Dr.T.V.Rao MD 35
  36. 36. Virulence Factors• 1 Capsular polypeptide• Anthrax Toxin• Both are coded by separate plasmid• The capsular polypeptide aids virulence by inhibiting phagocytosis, loss of plasmid loss of virulence• How the live attenuated anthrax spore vaccine ( Sterne strain ) Dr.T.V.Rao MD 36
  37. 37. Anthrax ToxinThe toxin is a three factionsThe edema factor, (OF Factor I )The protective antigen factor ( PA orFactor II )The lethal factor ( LF or Factor III )Individually they are not toxic Dr.T.V.Rao MD 37
  38. 38. How Toxicity Manifests• They are not toxic indivually but whole complex produces local edema and generalisaed shock.• PA I which is the fraction which binds to the target cell surface and in turn provides attachment sites for OF or LF facilitating .their entry into the cell Dr.T.V.Rao MD 38
  39. 39. TOXIGENICITY• OF island adenyl cyclase which is activated only inside the target cells leading to the intracellular accumulation of cyclic AMP• Responsible for edema and other biological effects of toxin.• Entry of LF toxin into the target cell causes cell death.• Loss of plasmid which encodes anthrax toxin renders the strain avirulant.• Sterne vaccine strain devoid of Plasmid coding for the capsule polysaccharide. Dr.T.V.Rao MD 39
  40. 40. Pathogenesis• Anthrax spores enter body• Germinate & multiple in lymph nodes• PA, EF, LF excreted from bacteria• PA binds to TEM8.• PA nicked by protease furin – 20-kDa segment off leaving 63-kDa peptide – Heptamer forms• EF and/or LF binds• Complex internalized by endocytosis• Acidification of endosome• LF or EF crosses into cytosol via PA mediated ion-conductive channels• LF cleaves MAPKK 1 & 2• EF stimulates cAMP Dr.T.V.Rao MD 40
  41. 41. Clinical Presentation of Anthrax Cutaneous Anthrax95% human cases are cutaneous infections1 to 5 days after contactSmall, pruritic, non-painful papule at inoculation sitePapule develops into hemorrhagic vesicle & rupturesSlow-healing painless ulcer covered with black Eschersurrounded by edemaInfection may spread to lymphatics w/ local adenopathySepticemia may develop20% mortality in untreated cutaneous anthrax Dr.T.V.Rao MD 41
  42. 42. Cutaneous AnthraxCDC, Cutaneous Anthrax—Vesicle Development Dr.T.V.Rao MD 42
  43. 43. Anthrax: CutaneousLeft, Forearm lesion on day 7—vesiculation and ulceration of initial macularor papular anthrax skin lesion. Right, Eschar of the neck on day 15 ofillness, typical of the last stage of the lesion. From Binford CH, Connor DH,eds. Pathology of Tropical and Extraordinary Diseases. Vol 1. Washington,DC: AFIP; 1976:119. AFIP negative 71-1290–2. Dr.T.V.Rao MD 43
  44. 44. Anthrax: Cutaneous Healing after treatment Dr.T.V.Rao MD 44
  45. 45. Inhalation Anthrax • The infection begins with the inhalation of the anthrax spore. • Spores need to be less than 5 microns (millionths of a meter) to reach the alveolus. • Macrophages lyse and destroy some of the spores. • Survived spores are transported to lymph nodes. • At least 2,500 spores have to be inhaled to cause an infection.Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of Pathology Dr.T.V.Rao MD 45
  46. 46. Inhalation Anthrax • Disease immediately follows germination. • Spores replicate in the lymph nodes. • The two lungs are separated by a structure called the mediastinum, which contains the heart, trachea, esophagus, and blood vessels. • Bacterial toxins released during replication result in mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of Pathology Dr.T.V.Rao MD 46
  47. 47. Anthrax: Inhalational Mediastinal widening JAMA 1999;281:1735–1745 Dr.T.V.Rao MD 47
  48. 48. Mediastinal Widening and PleuralEffusion on Chest X-Ray in Inhalational Anthrax Dr.T.V.Rao MD 48
  49. 49. Gastrointestinal Anthrax • GI anthrax may follow after the consumption of contaminated, poorly cooked meat. • There are 2 different forms of GI anthrax: 1) Oral-pharyngeal 2) Abdominal • Abdominal anthrax is more common than the oral-pharyngeal form. Dr.T.V.Rao MD 49
  50. 50. Clinical Presentation of Anthrax Inhalation AnthraxVirtually 100% fatal (pneumonic)Meningitis may complicate cutaneousand inhalation forms of diseasePharyngeal anthrax• Fever• Pharyngitis• Neck swelling Dr.T.V.Rao MD 50
  51. 51. Clinical Presentation of AnthraxGastrointestinal (Ingestion) AnthraxVirtually 100% fatalAbdominal painHemorrhagic ascitesParacentesis fluid may revealgram-positive rods Dr.T.V.Rao MD 51
  52. 52. Anthrax: DiagnosisCutaneous Gram stain, polymerase chain reaction (PCR), or culture of vesicular fluid, exudate, or eschar Blood culture if systemic symptoms present Biopsy for immunohistochemistry, especially if person taking antimicrobials Dr.T.V.Rao MD 52
  53. 53. Diagnosis in Humans• Isolation of B. anthracis – Blood, skin – Respiratory secretions• Serology• ELISA• Nasal swabs – Screening tool Dr.T.V.Rao MD 53
  54. 54. Diagnosis in Humans• Anthrax quick ELISA test – New test approved by FDA on June 7th, 2004. – Detects antibodies produced during infection with Bacillus anthracis – Quicker and easier to interpret than previous antibody testing methods • Results in less than ONE hour Dr.T.V.Rao MD 54
  55. 55. Clues to diagnosis• Aerobic blood culture growth of large, gram- positive bacilli provides preliminary identification of Bacillus species Dr.T.V.Rao MD 55
  56. 56. Laboratory Criteria for Identification of B. anthracis• From clinical samples, such as blood, cerebrospinal fluid (CSF), skin lesion (eschar), or oropharyngeal ulcer – Encapsulated gram-positive rods on Gram stain• From growth on sheep blood agar: – Large gram-positive rods – Nonmotile – Nonhemolytic Dr.T.V.Rao MD 56
  57. 57. Anthrax: DiagnosisInhalational Chest X-ray—widened mediastinum, pleural effusions, infiltrates, pulmonary congestion Affected tissue biopsy for immunohistochemistry Any available sterile site fluid for Gram stain, PCR, or culture Pleural fluid cell block for immunohistochemistry Dr.T.V.Rao MD 57
  58. 58. B. anthracis: Confirmatory Identification Isolate Capsule DFAPhage lysis Capsule antigen Horse Bicarbonate Cell wall blood media (M’Fadyean (M’Fadyean stain Stain) India ink stain) Dr.T.V.Rao MD 58
  59. 59. B. anthracis: Presumptive Identification Clinical specimen (blood, CSF, etc.) Gram stain Isolate on SBACapsule production Colony morphology Hemolysis Motility Spores Gram stain Malachite green Dr.T.V.Rao MD 59
  60. 60. Laboratory Criteria for Identification of B. anthracis• From clinical samples, such as blood, cerebrospinal fluid (CSF), skin lesion (eschar), or oropharyngeal ulcer – Encapsulated gram-positive rods on Gram stain• From growth on sheep blood agar: – Large gram-positive rods – Nonmotile – Nonhemolytic Dr.T.V.Rao MD 60
  61. 61. Laboratory Criteria for Identification of B. anthracis• Rapid screening assay (PCR- and antigen- detection based) for use on cultures and directly on clinical specimens• Confirmatory criteria for identification of B. anthracis – Capsule production – Lysis by gamma-phage – Direct fluorescent antibody assay (DFA) Dr.T.V.Rao MD 61
  62. 62. PCR Assay• Detection time: - PCR only takes several hours ex) Rapid-cycle RT-PCR can be finished within 1-2 hours• Can start early treatment of Anthrax• There are many different types of PCR assays for the detection of Anthrax such as multiplex PCR, enter bacterial repetitive intragenic consensus-PCR (ERIC-PCR), and long-range repetitive element polymorphism-PCR.• Rapid diagnostic methods provide answers in minutes or hours instead of days. Dr.T.V.Rao MD 62
  63. 63. Cautions on Treatment• Obtain specimens for culture BEFORE initiating antimicrobial therapy.• Do NOT use extended- spectrum cephalosporins or trimethoprim/sulfametho xazole because anthrax may be resistant to these drugs. Dr.T.V.Rao MD 63
  64. 64. Treatment & ProphylaxisTreatment• Penicillin is drug of choice• Erythromycin, chloramphenicol acceptable alternatives• Doxycycline now commonly recognized as prophylacticVaccine (controversial) Laboratory workers Employees of mills handling goat hair Active duty military members Potentially entire populace of U.S. for herd immunity Dr.T.V.Rao MD 64
  65. 65. Treatment• Penicillin – Has been the drug of choice – Some strains resistant to penicillin and doxycycline• Ciprofloxacin – Chosen as treatment of choice in 2001 – No strains known to be resistant• Doxycycline may be preferable Dr.T.V.Rao MD 65
  66. 66. Treatment• Before 2001, 1st line of treatment was penicillin G – Stopped for fear of genetically engineered resistant strains• 60 day course of antibiotics• Ciprofloxacin – fluoroquinolone – 500 mg tablet every 12h or 400 mg IV every 12h – Inhibits DNA synthesis• Doxycycline – 6-deoxy-tetracycline – 100 mg tablet every 12h or 100 mg IV every 12h – Inhibits protein synthesis• For inhalational, need another antimicrobial agent – clindamycin – rifampin hrax.html – chloramphenico Dr.T.V.Rao MD 66
  67. 67. Trends on Vaccine• BioThrax/Anthrax vaccine absorbed – Made by Bioport – Route of exposure not important• Administered subcutaneously – .5mL at 0, 2, and 4 weeks, and at 6, 12, & 18 months, & booster doses at 1 yr intervals• PA from attenuated, nonencapsulated Sterne strain absorbed onto aluminum hydroxide – Contains no dead or live bacteria in the preparation – Antibodies to PA prevent binding to the target cell & confer protection from anthrax.• 95% of vaccinated Rhesus monkeys survived lethal doses of inhaled anthrax• A December 22, 2003 ruling temporarily halted the Department of Defense’s anthrax vaccination program – Lifting of that injunction on January 7, 2004 Dr.T.V.Rao MD 67
  68. 68. Immune Protection Against Anthrax• Live cellular vaccines – "Sterne" type live spore (toxigenic, noncapsulating) – Former USSR STI live spore (toxigenic, non- capsulating) – "Pasteur" type (mixed culture, reduced virulence)• Sterile, acellular vaccines – US "anthrax vaccine adsorbed" (AVA)—not licensed for use in civilian populations – UK "anthrax vaccine precipitated" (AVP)• Recombinant PA research vaccines – AI3+; Freund’s; Saponin, Monophosphoryl lipid A; Ribi Dr.T.V.Rao MD 68
  69. 69. Vaccination• Cell-free filtrate• Licensed in 1970• At risk – Wool mill workers – Veterinarians – Lab workers – Livestock handlers – Military personnel Dr.T.V.Rao MD 69
  70. 70. Vaccine Schedule• 3 injections at two-week intervals• 3 injections 6 months apart• Annual booster Dr.T.V.Rao MD 70
  71. 71. Recommended Post exposure Prophylaxis to Prevent Inhalational Anthrax Initial Therapy DurationAdults Ciprofloxacin 60 days(including pregnant 500 mg PO BID women and ORimmunocompromised) Doxycycline 100 mg PO BIDChildren Ciprofloxacin* 60 days 10–15 mg/kg PO Q 12 hrs. Change to OR amoxicillin Doxycycline: if susceptible >8 yrs. and >45 kg: 100 mg PO BID >8 yrs. and <45 kg: 2.2 mg/kg PO BID <8 yrs.: 2.2 mg/kg PO BID *Ciprofloxacin not to exceed 1 gram daily in children Patient information sheets MD Dr.T.V.Rao at 71
  72. 72. Precautions to Health care Workers• Standard contact precautions. Avoid direct contact with wound or wound drainage. Dr.T.V.Rao MD 72
  73. 73. Anthrax Has Been Used As a Bioweapon• Because it is deadly, noncontagious, and dispersed by spores, anthrax has always been considered a good candidate for a bioweapon (table 3). Late in 2001, this possibility became a reality. Letters containing anthrax spores were sent to several news reporters and two United States Senators. Five people died of inhalational anthrax as a result of exposure to these spores. Dr.T.V.Rao MD 73
  74. 74. Weaponization & Bacillus Anthracis:Why is this Agent Considered to be the Department of Defense’s Number-One/Two Biological Threat? A sample of anthrax bacteria at the National School of Biological Sciences, Mexico City Dr.T.V.Rao MD 74
  75. 75. Analysis of the 2001 US Anthrax Attacks Above anthrax-containing envelopes Above anthrax-containing envelopes postmarked September 18th, 2001 postmarked October 9, 2001 *Also believed to be three or more other envelopes that were never found Dr.T.V.Rao MD 75
  76. 76. Anthrax Cases, 2001• 22 cases – 11 cutaneous – 11 inhalational• 5 deaths (all inhalational) – Index case in Florida – 2 postal workers in Maryland – Hospital supply worker in NYC – Elderly farm woman in Connecticut Dr.T.V.Rao MD 76
  77. 77. Analysis of the 2001 US Anthrax Attacks• Anthrax in Envelopes – Concentration of about 1 trillion spores per gram – 2 grams anthrax per envelope – Each letter contained ~200 million times average LD50 – All anthrax was unmilled, contained a certain type of silica to reduce electrostatic charges and was of the Ames strain – all characteristic of US weapons-grade anthrax Dr.T.V.Rao MD 77
  78. 78. Created by Dr.T.V.Rao MD for “ e“ learning resources in Developing World • Email Dr.T.V.Rao MD 78