Anthrax. Dr.T.V.Rao MD. Dr.T.V.Rao MD 1

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1 Anthrax Dr.T.V.Rao MD Dr.T.V.Rao MD 1

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 member Produce endospores Aerobic 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 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 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. anthracisproduces lethal toxins. Dr.T.V.Rao MD 4

5 Bacillus anthrax Several landmarks 1 st to Observe under Microscope 1 st to communicable disease. 1 st observe the spores ( Robert Koch ) 1 st to prepare for attenuated vaccine. ( Louis Pasteur) Dr.T.V.Rao MD 5

6 20, ,000 cases estimated globally/year Dr.T.V.Rao MD

7 Animal Transmission Most commonly infected by ingestion from contaminated soil or contaminated feed or bone meal Dr.T.V.Rao MD 7

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 B. anthracis Gram-positive, spore-forming, non-motile bacillus Dr.T.V.Rao MD 9

10 Anthrax Bacilli Dr.T.V.Rao MD 10

11 Bacillus anthracis Bacillus anthracis General characteristics Large, Gram positive, non-motile rod Vegetative form and spores Nearly worldwide distribution Over 1,200 strains Dr.T.V.Rao MD 11

12 Bacillus anthracis Gram + rod Facultative anaerobe 1-1.2µm in width x 3-5µm 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

to extremes Survive for decades Taken up by host and

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

Endospore Oxygen required for sporulation 1 spore per cell dehydrated cells Highly resistant to heat, cold, chemical disinfectants, dry periods Protoplast

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

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.

15 Gram Stain Morphology of B. anthracis Broad, gram-positive rod: 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 O 2 Dr.T.V.Rao MD 15

EF and/or LF binds Complex internalized by endocytosis Acidification of

16 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 ionconductive channels Mechanism of Infection Dr.T.V.Rao MD 16

17 Cultural characteristics Aerobe, facultative anaerobe Grows between c 2-3 mm colonies Edge like matted hair Medusa head appearance Blood agar Hemolytic colonies Dr.T.V.Rao MD 17

18 Appearance of Anthrax String of pearl appearance with Pencillin Differentiates Anthrax and Cereus Dr.T.V.Rao MD 18

19 Contain 1 polymyxins 2 Lysozyme 3 Ethylene dioxide 4 Tetra acetic acid Contains EDTA SELECTIVE MEDIUM PLET Dr.T.V.Rao MD 19

20 Biochemical Reactions Gelatin Inverted fir tree appearance Dr.T.V.Rao MD 20

21 Biochemical Reactions Glucose, Maltose and Sucrose fermented with acid but no gas Catalase positive Dr.T.V.Rao MD 21

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

By duckering process (five stages) i.e. lmmersion in 0.

23 By duckering process (five stages) i.e. lmmersion in % soda liquor Immersion in soap liquor; Two immersions in 2% formaldehyde solution; and Rinsing in water Wool and Hair Dr.T.V.Rao MD 23

24 Antibiotics Pencillin Erythrocin Tetracycline Chloramphenicol Occasional strains resistant to penicillin are encountered Dr.T.V.Rao MD 24

as hair, feces, or hides, and is characterized by

25 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 Transmitted Dr.T.V.Rao MD 25

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 Anthrax Cycle Dr.T.V.Rao MD 27

Rely on primate data Minimum infection dose of ~ 1,000-8,000 spores LD 50 of

28 Pathogenesis The infectious dose of B. anthracisin humans by any route is not precisely known. Rely on primate data Minimum infection dose of ~ 1,000-8,000 spores LD 50 of 8,000-10,000 spores for inhalation Virulence depends on 2 factors Capsule 3 toxins Dr.T.V.Rao MD 28

29 Anthrax: Clinical Presentation Cutaneous Inhalational Gastrointestinal Dr.T.V.Rao MD 29

30 Epidemiology of Anthrax in Animal and Human Hosts Dr.T.V.Rao MD 30

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 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 Anthrax: Cutaneous Begins as a papule, progresses through a vesicular stage to a depressed black necrotic ulcer (Escher) Edema, redness, and/or necrosis without ulceration may occur Form most commonly encountered in naturally occurring cases Incubation period: 1 12 days Case-fatality: Without antibiotic treatment 20% With antibiotic treatment 1% Dr.T.V.Rao MD 33

34 Anthrax: Inhalational A brief prodromal resembling a viral-like illness, characterized by myalgia, fatigue, fever, with or without respiratory symptoms, followed by hypoxia and dyspnea, often with radiographic evidence of mediastinal widening. Meningitis in 50% of patients Rhinorrhea (rare) Dr.T.V.Rao MD 34

anthracis virulent Most important role during establishment of disease

35 Glycocalyx 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 Capsule Dr.T.V.Rao MD 35

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 Anthrax Toxin The toxin is a three factions The edema factor, (OF Factor I ) The protective antigen factor ( PA or Factor II ) The lethal factor ( LF or Factor III ) Individually they are not toxic Dr.T.V.Rao MD 37

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 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 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 Clinical Presentation of Anthrax Cutaneous Anthrax 95% human cases are cutaneous infections 1 to 5 days after contact Small, pruritic, non-painful papule at inoculation site Papule develops into hemorrhagic vesicle & ruptures Slow-healing painless ulcer covered with black Escher surrounded by edema Infection may spread to lymphatic's w/ local adenopathy Septicemia may develop 20% mortality in untreated cutaneous anthrax Dr.T.V.Rao MD 41

42 Cutaneous Anthrax CDC, Cutaneous Anthrax Vesicle Development Dr.T.V.Rao MD 42

Right, Eschar of the neck on day 15 of illness, typical of the last stage of the lesion.

43 Anthrax: Cutaneous Left, Forearm lesion on day 7 vesiculation and ulceration of initial macular or papular anthrax skin lesion. Right, Eschar of the neck on day 15 of illness, 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 Dr.T.V.Rao MD 43

44 Anthrax: Cutaneous Healing after treatment Dr.T.V.Rao MD 44

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.

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 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 Anthrax: Inhalational Mediastinal widening JAMA 1999;281: Dr.T.V.Rao MD 47

48 Mediastinal Widening and Pleural Effusion on Chest X-Ray in Inhalational Anthrax Dr.T.V.Rao MD 48

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 Clinical Presentation of Anthrax Inhalation Anthrax Virtually 100% fatal (pneumonic) Meningitis may complicate cutaneous and inhalation forms of disease Pharyngeal anthrax Fever Pharyngitis Neck swelling Dr.T.V.Rao MD 50

51 Clinical Presentation of Anthrax Gastrointestinal (Ingestion) Anthrax Virtually 100% fatal Abdominal pain Hemorrhagic ascites Paracentesis fluid may reveal gram-positive rods Dr.T.V.Rao MD 51

52 Cutaneous Anthrax: Diagnosis 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 Diagnosis in Humans Isolation of B. anthracis Blood, skin Respiratory secretions Serology ELISA Nasal swabs Screening tool Dr.T.V.Rao MD 53

54 Diagnosis in Humans Anthrax quick ELISA test New test approved by FDA on June 7 th, 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 Clues to diagnosis Aerobic blood culture growth of large, grampositive bacilli provides preliminary identification of Bacillus species Dr.T.V.Rao MD 55

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 Anthrax: Diagnosis Inhalational 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 B. anthracis: Confirmatory Identification Isolate Phage lysis Horse blood (M Fadyean Stain) Capsule Bicarbonate media (M Fadyean stain India ink stain) DFA Capsule antigen Cell wall Dr.T.V.Rao MD 58

59 B. anthracis: Presumptive Identification Clinical specimen (blood, CSF, etc.) Gram stain Capsule production Isolate on SBA Colony morphology Hemolysis Motility Spores Gram stain Malachite green Dr.T.V.Rao MD 59

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 Laboratory Criteria for Identification of B. anthracis Rapid screening assay (PCR- and antigendetection 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 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 Cautions on Treatment Obtain specimens for culture BEFORE initiating antimicrobial therapy. Do NOT use extendedspectrum cephalosporins or trimethoprim/sulfametho xazolebecause anthrax may be resistant to these drugs. Dr.T.V.Rao MD 63

64 Treatment & Prophylaxis Treatment Penicillin is drug of choice Erythromycin, chloramphenicol acceptable alternatives Doxycycline now commonly recognized as prophylactic Vaccine (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 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

Inhibits DNA synthesis Doxycycline 6-deoxy-tetracycline 100 mg tablet every 12h or 100 mg IV every 12h Inhibits

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 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 Immune Protection Against Anthrax Live cellular vaccines "Sterne" type live spore (toxigenic, noncapsulating) Former USSR STI live spore (toxigenic, noncapsulating) "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 AI 3+ ; Freund s; Saponin, Monophosphoryl lipid A; Ribi Dr.T.V.Rao MD 68

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 Vaccine Schedule 3 injections at two-week intervals 3 injections 6 months apart Annual booster Dr.T.V.Rao MD 70

71 Recommended Post exposure Prophylaxis to Prevent Inhalational Anthrax Initial Therapy Duration Adults Ciprofloxacin 60 days (including pregnant 500 mg PO BID women and OR immunocompromised) Doxycycline 100 mg PO BID Children Ciprofloxacin* 60 days 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 at Dr.T.V.Rao MD 71

72 Precautions to Health care Workers Standard contact precautions. Avoid direct contact with wound or wound drainage. Dr.T.V.Rao MD 72

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 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 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 Anthrax Cases, 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

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

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 LD 50 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 Created by Dr.T.V.Rao MD for e learning resources in Developing World doctortvrao@gmail.com Dr.T.V.Rao MD 78

Lecture (14) Amiedi Ph.D.Microbiology

Lecture (14) Amiedi Ph.D.Microbiology AEROBIC BACILLUS Lecture (14) Dr. Baha,H,AL-Amiedi Amiedi Ph.D.Microbiology 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