Sentinel Level Laboratory Protocols

Transcription

1 Sentinel Level Laboratory Protocols Melissa Bossie, MT (ASCP), CLS (NCA), M. S. Sentinel Laboratory Training 1

2 Sentinel Laboratory Training 2

3 Anthrax Is a zoonotic disease in animals Spores can survive in soil for decades Spores are easily ingested or inhaled by herbivorous animals Human infection results from direct contact with infected animals or animal products Sentinel Laboratory Training 3

4 Contracting Anthrax Three Forms of Anthrax Cutaneous Gastrointestinal Inhalational Sentinel Laboratory Training 4

5 Sentinel Laboratory Training 5

6 Cutaneous Anthrax Spore enters skin Incubation of 2-3 days Papule develops Black eschar forms Anthrax = coal (Greek) Vesicle or eschar should be lifted fluid filled swab aspirate from vesicle Sentinel Laboratory Training 6

7 Gastrointestinal Anthrax Spore is ingested Incubation period of 2-3 days Acute inflammation of intestinal tract Vomiting of blood Severe bloody diarrhea Stool collection - preferably prior to antibiotics Sentinel Laboratory Training 7

8 Inhalational Anthrax Most deadly form Likely form in a bioterrorism event Spore is inhaled Incubation period of 1-6 days Spores are engulfed by macrophages Hemorrhagic mediastinitis Hemorrhagic lymphadenitis Sentinel Laboratory Training 8

9 Inhalational Anthrax Symptoms Fever, cough, malaise, sweat/chills Nausea, vomiting Hemorrhagic mediastinitis/pleural effusion Rapid progression to severe respiratory distress leading to shock/death Specimen Collection Nasal Swab- No clinical value Sputum- early course of disease Blood Culture- later in disease Sentinel Laboratory Training 9

10 Inhalational Anthrax Treatment Early IV antibiotics required Mortality may still exceed 80% Current treatment of choice Ciprofloxacin or Doxycycline Antibiotics for days Long term antibiotics necessary because of spore persistence in lung/lymph node tissue If vaccinated time frame can be reduced Sentinel Laboratory Training 10

11 B. anthracis Meningitis May occur during later stages of disease CSF - lumbar puncture, ventriculostomy Hemorrhagic Meningitis Cardinals Cap Sentinel Laboratory Training 11

12 Sentinel Level Testing B. anthracis Colony Morphology Rough, ground glass colony Irregular edges with comma-like projections Non-hemolytic Sticky, tenacious colony Sentinel Laboratory Training 12

13 24 hr. Growth of B. anthracis on SBA

14 Colony Morphology Large, rough ground glass colony Irregular shaped edges, comma-like projections sticky adheres to the agar Non- Hemolytic Gram Stain Morphology Large gram positive bacilli x 3-5 µm May have spores present Large gram positive rod Hemolysis Bacteria, not a large gram positive rod Not B. anthracis Alpha or Beta hemolysis Not B. anthracis Non-hemolytic Catalase Positive Negative Not B. anthracis Positive Not B. anthracis Motility Negative Cannot Rule Out B. anthracis: Call the OLS TP & BT and refer isolate Sentinel Laboratory Training 14

15 B. anthracis Note Tenacity of Colony Sentinel Laboratory Training 15

16 Anthrax? No, this isolate is hemolytic. Sentinel Laboratory Training 16

17 Sentinel Laboratory Training 17

18 Black Death 14 th Century Sentinel Laboratory Training 18

19 Sentinel Laboratory Training 19

20 Plague Yersinia pestis Three forms of disease Bubonic Septicemic Pneumonic Sentinel Laboratory Training 20

21 Bubonic Plague Most common, naturally occurring, form of the disease Plague bacteria is transported to the lymph node closest to the flea bite site Lymph node becomes inflamed and enlarged = bubo Specimen Collectionaspirate from infected lymph node or bubo Sentinel Laboratory Training 21

22 Septicemic Plague Yersinia pestis infection in the blood Specimen collection- Series of 3 venipuncture specimens taken minutes apart Sentinel Laboratory Training 22

23 Pneumonic Plague 1 o pneumonic plague results from inhalation of plague infected aerosols: Infected person Infected animal Bioterrorism incident 2 o pneumonic plague may result from bubonic plague Sentinel Laboratory Training 23

24 Pneumonic Plague Rarest and most deadly form of the disease Incubation period 2-4 days Rapid progression of disease - fulminant pneumonia, septic shock High mortality without early treatment Specimen collection Hallmark: Hemoptysis = Bloody sputum Bronchial and tracheal aspirates are the specimens of choice Nasal/Throat swab: only useful within 24 hours of exposure Sentinel Laboratory Training 24

25 Pneumonic Plague treatment Almost always fatal if not treated within 24 hours of onset of symptoms Infected patients 10 days Streptomycin Gentamicin Post-exposure prophylaxis 7 days Doxycycline Ciprofloxacin Sentinel Laboratory Training 25

26 Sentinel Level Testing Y. pestis Colony Morphology Pinpoint, gray-white, translucent colonies at 24 hours At 48 hours colonies are non-hemolytic, resemble typical enteric gnb After prolonged incubation, have fried egg appearance Sentinel Laboratory Training 26

27 48 hour growth of Y. pestis Fried egg appearance Sentinel Laboratory Training 27

28 Sentinel Level Testing Y. pestis Gram Stain Morphology Fat gram-negative bacilli bipolar staining 1.0 x 0.5 microns Sentinel Laboratory Training 28

29 Growth in Broth Y. pestis Y. pestis Y. pseudotuberculosis CDC Photo Sentinel Laboratory Training 29

30 Sentinel Level Testing Y. pestis Organism Characteristics: Grows better at room temperature than most enteric gram negative rods May cause problematic identification with automated methods Should be confirmed by classical biochemicals Sentinel Laboratory Training 30

31 Sentinel Level Testing Y. pestis Classical Biochemical Reactions Oxidase: negative Urea: negative Motility: Negative Sugar Fermentation: Only glucose and mannitol are positive Sentinel Laboratory Training 31

32 Sentinel Level Testing Overview Y. pestis Colony Morphology: Lactose negative, Fried egg* Gram Stain Morphology: Gram negative rod, bipolar* Growth in Broth: Flocculent Clumps Oxidase: Negative Urea: Negative Motility: Negative Sugar Fermentation: Only Glucose and Mannitol positive Cannot Rule Out Y. pestis: Call the OLS TP & BT Laboratory *2301 Sentinel Laboratory Training 32

33 Brucellosis Sentinel Laboratory Training 33

34 Brucellosis zoonotic disease Any of four Brucella spp. can cause human infection B. melitensis (from sheep & goats) B. suis (from pigs) B. abortus (from cows) B. canis (dogs rarely causes disease) Sentinel Laboratory Training 34

35 Brucellosis Causes septicemic febrile illness in humans Non-specific symptoms: irregular undulating fevers, headache, profound weakness and fatigue, chills/sweating Focal lesions may occur in bones, joints, genitourinary tract, and other sites Endocarditis common following infection Incubation period is highly variable, ranging from 5 days to 2 months Relapses are common Low mortality rate (<5%), but can be extremely incapacitating Sentinel Laboratory Training 35

36 Specimen collection Brucella spp. Due to the intracellular nature of the organism Blood culture may be positive after 2 nd day of fever Bone marrow may be useful in subacute disease ** Incubate 21 days CSF Synovial fluid Biopsy material from L. nodes, liver, or spleen. Nasal swabs: Only useful in the first 24 hours of exposure. Sentinel Laboratory Training 36

37 Brucellosis treatment Intracellular nature of organism requires combination therapy Doxycycline plus rifampin for 6 weeks Prophylaxis: 3-6 week course following inadvertent lab exposure or exposure via a biological event. Azithromycin shows promising results in most animal models. Sentinel Laboratory Training 37

38 Sentinel Level Testing Brucella spp. Colony Morphology 48 hour growth colonies are small, raised, glistening, white-cream Non-hemolytic May not be visible at 24 hours Sentinel Laboratory Training 38

39 72 hour growth of B. abortus on SBA Sentinel Laboratory Training 39

40 Sentinel Level Testing Brucella spp. Gram stain Morphology Very tiny: x 0.6 microns Faintly staining Gram-negative coccobacilli Fine sand Sentinel Laboratory Training 40

41 Sentinel Level Testing Brucella spp. Automated identification: Most likely will not identify this organism Classical biochemicals: Oxidase positive Urea positive.sometimes rapid** Nitrate - positive Motility negative Does not need X or V factor Sentinel Laboratory Training 41

42 Sentinel Level Testing Overview Brucellosis Colony Morphology: Small, raised, glistening whitecream colonies after 48 hours Gram Stain Morphology: Very tiny, faintly staining, gram- negative coccobacilli Oxidase positive Urea positive, sometimes rapid Nitrate - positive Motility negative X & V not required for growth Cannot Rule Out Brucella spp.: Call the OLS TP & BT Laboratory x 2301 Sentinel Laboratory Training 42

43 Sentinel Laboratory Training 43

44 Tularemia Francisella tularensis A disease of wild animals Especially rabbits and rodents. No human to human transmission Man naturally infected by: Skin exposures (e.g. hunters skinning rabbits) Bites from infected ticks and flies Eating undercooked infected meat Rarely by inhalation Sentinel Laboratory Training 44

45 Tularemia Approximately 200 cases per year in U.S. Sentinel Laboratory Training 45

46 Tularemia Symptoms Ulceroglandular Nonhealing ulcers on extremities, lymphadenopathy Oculoglandular Painful, purulent conjunctivitis, lymphadenopathy Glandular Like ulceroglandular form, but no ulcer Sentinel Laboratory Training 46

47 Tularemia Symptoms Oropharyngeal Sore throat which is painful beyond its appearance & does not respond to penicillin Pneumonic Dry, nonproductive cough, dyspnea, fever Gastrointestinal Can range from mild, unexplained persistent diarrhea to bowel ulceration and death Typhoidal (Systemic) Bacteremia, delerium, prostration, and shock Sentinel Laboratory Training 47

48 Tularemia Symptoms Ulceroglandular CDC Photo Sentinel Laboratory Training 48

49 Tularemia Treatment Preferred antibiotics for infected patients Streptomycin Gentamicin Post-exposure prophylaxis Doxycycline Ciprofloxacin Sentinel Laboratory Training 49

50 Specimen collection Francisella tularensis Pulmonary Sputum, bronchial or tracheal washing Glandular / Ulceroglandular form Lymph node aspirate or biopsy Ulcer aspirate, swab, or scraping Septicemia Blood culture Sentinel Laboratory Training 50

51 Sentinel Level Testing F. tularensis Colony morphology Usually too small to see at 24 hours Chocolate Agar: 48 hours; colonies will be shiny, gray and flat, with smooth, entire edges. Cysteine Heart Agar (or BCYE): 48 hours; colonies will be greenish-white with a buttery consistency Sentinel Laboratory Training 51

52 Francisella tularensis Chocolate at 48 hrs. Sentinel Laboratory Training 52

53 Sentinel Level Testing F. tularensis Gram Stain Morphology Extremely tiny: x 0.2 microns Poorly staining Pleomorphic, gram-negative coccobacillus Sentinel Laboratory Training 53

54 Sentinel Level Testing F. tularensis Automated identification: Most likely will not identify this organism Classical biochemicals: Oxidase negative Motility negative Urea negative Nitrate - negative Sentinel Laboratory Training 54

55 Sentinel Level Testing Overview F. tularensis If tularemia is highly suspected; and/or Tiny, poorly staining gram-negative coccobacilli; and Slowly growing organism with characteristic colony morphology; and Routine automated methods fail to identify this organism; and (if performed) Oxidase, urease, nitrate, and motility are all negative; Cannot Rule Out F. tularensis: Call the OLS TP & BT Laboratory and refer isolate x 2301 Sentinel Laboratory Training 55

56 Botulinum Toxin Sentinel Laboratory Training 56

57 Botulism Clostridium botulinum Spore forming, anaerobic organism commonly found in soil Widely distributed in nature May cause food borne botulism, wound botulism, and infant botulism Sentinel Laboratory Training 57

58 Clostridium botulinum Neurotoxin (BoNT) produced by Clostridium botulinum One of the most toxic compounds known to man -1 gm could kill 1 million people Botulinum toxin type A is 100,000 times more toxic than Sarin gas Toxin Sentinel Laboratory Training 58

59 Botulinum Toxin Symptoms Pupil dilation, double vision, drooping of eyelids Dry mouth, slurred speech Symmetrical descending flaccid paralysis May culminate abruptly in respiratory failure Patient usually alert and orientated Afebrile (intoxication not infection) Incubation period hours, dependent on amount of toxin Mortality rate 5-10% Sentinel Laboratory Training 59

60 Botulinum Toxin Treatment Equine botulinum antitoxin Screen for hypersensitivity to serum Give before onset of symptoms or as soon as possible thereafter Minimizes nerve damage and severity of disease Will not reverse existent paralysis Availability and delay of antitoxin is limiting factor Sentinel Laboratory Training 60

61 Botulism Specimen Collection Serum: 15-20ml - obtain ASAP after onset of symptoms, before antitoxin given Tissue: store and transport anaerobically Stool: g Enema: 20cc from sterile water return Food: leave in original containers if possible Sentinel Laboratory Training 61

62 Sentinel Level Testing C. botulinum toxin There are no Level A tests for botulism toxin The diagnosis of botulism is made clinically, i.e., based on the patient s case history and physical findings Health care providers suspecting botulism should contact the, Threat- Preparedness & Bioterrorism Response Laboratory Suspected C. botulinum toxin: Call the OLS TP & BT Laboratory and refer isolate or specimen immediately x 2301! Sentinel Laboratory Training 62

63 Sentinel Laboratory Training 63

64 Ricin Toxin Sentinel Laboratory Training 64

65 Ricin Is a toxin extracted from a castor bean from the plant, Ricinus communis Ricin does not partition into castor oil because it is watersoluble Ricin toxin contains two parts, Ricin and RCA (hemagglutinin) Ricin contains an A chain, ribosome inactivating protein, and a B chain, cell binder 1,000 X less toxic than Botulinum toxin Sentinel Laboratory Training 65

66 Ricin Poisoning Gastrointestinal and Inhalational Castor bean is eaten or toxin inhaled Symptoms occur within hours Respiratory distress within 8 hours after exposure 1 mg can kill an adult 1 castor bean can kill a child Sentinel Laboratory Training 66

67 Ricin Poisoning Gastrointestinal Symptoms Abdominal pain Vomiting Diarrhea, sometimes bloody Severe dehydration Decrease in urine Decrease in blood pressure If death has not occurred in 3-5 days, the victim usually recovers. Sentinel Laboratory Training 67

68 Inhalation of Ricin Toxin Symptoms Cough Respiratory distress Pulmonary edema Respiratory failure Multisystem organ failure Weakness Influenza-like symptoms Fever Myalgia Arthralgia Sentinel Laboratory Training 68

69 Ricin Toxicity Treatment There is NO TREATMENT Get the ricin off or out of the body as quickly as possible Supportive medical care to minimize the effects of the poisoning Help victims breathe Give them intravenous fluids Give them medications to treat conditions such as seizure and low blood pressure Flush their stomachs with activated charcoal (if the ricin has been very recently ingested) Wash out their eyes with water if their eyes are irritated. Sentinel Laboratory Training 69

70 Specimen collection Ricin Toxin Store at room temperature : Whole blood-5-7 ml (lavender top with EDTA); Whole blood-5-7 ml (grey top with with NaF or KF); and Whole blood-10 ml serum (red top-do not use SST/gel or separate serum) Freeze as soon as possible: hour sputum Urine-25 ml in sterile container Tissue biopsy Sentinel Laboratory Training 70

71 Sentinel Level Testing Ricin Toxin Collect Specimens Call the OLS TP & BT Laboratory and refer immediately x 2301! Sentinel Laboratory Training 71

72 THE END Sentinel Laboratory Training 72