fastidious gram negative rods respiratory ii clinical laboratory science program carol larson msed,...
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![Page 1: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/1.jpg)
Fastidious Gram Negative RodsRespiratory II
Clinical Laboratory Science Program
Carol Larson MSEd, MT(ASCP)
Please click audio iconto hear Carol’s narration
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• Fastidious– Complex / extensive nutrient requirements
• Faint staining Gram Negative Rods– Safranin counterstain for >2 minutes– Substitute carbolfuschin for safranin
• Serological testing useful
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General Information
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• Causes Pertussis / Whooping cough
• Spread by airborne droplets
• Virulence factors– Attachment to ciliated epithelium of the
upper respiratory tract– Exotoxin – tracheal cytotoxin– Exotoxin - Pertussis toxin– Cell wall endotoxin
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Bordetella pertussis
Clinical Significance
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• Nasopharyngeal swab or aspirate
• Plate at bedside– Regan-Lowe media– Bordet-Gengou media– Methicillin or cephalexin added to media
to inhibit normal flora
• Perform DNA probe testing on aspirate
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Bordetella pertussis
Specimen Collection
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• 35ºC, ambient air for at least 7 days
• Colony morphology– No growth on
BAP & MAC– Bordet-Gengou– Regan-Lowe
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Bordetella pertussis
Growth Characteristics
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• Gram stain– Small, faintly staining gram-negative
coccobacilli
• Oxidase +
• Nitrate –
• Urea –
• Nonmotile
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Bordetella pertussis
Identification
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• DNA probes for direct detection in:– Specimen– Culture confirmation
• Direct fluorescent antibody test
• Agglutination methods
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Bordetella pertussis
Serological Testing
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• Erythromycin is drug of choice
• Vaccination
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Bordetella pertussis
Treatment & Prevention
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What media is used to culture Bordetella pertussis?
Regan-Lowe media and Bordet-Gengou media. Bordetella pertussis will not grow on BAP or MAC. Methicillin or cephalexin can be added to the media to inhibit normal flora.
![Page 10: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/10.jpg)
• Causes acute respiratory infection
• Grows on BAP and sometimes on MAC
• Bordet-Gengou media
• Oxidase negative
• Urease positive (24 hours)
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Bordetella parapertussis
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• Immunocompromised patients
• Grows on BAP and MAC
• Bordet-Gengou media
• Oxidase positive
• Urease positive (3-4 hours)
• Motile
• Nitrate positive
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Bordetella bronchiseptica
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How can you differentiate between Bordetella pertussis and B. parapertussis?
Colony morphology on Bordet-Gengou media, growth on BAP/MAC, oxidase, and urease.
![Page 13: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/13.jpg)
Capnocytophaga species
Clinical Significance• Normal oropharyngeal flora
• Associated with:– Periodontal disease– Sepsis
• Granulocytopenia• Malignancies
– Endocarditis– Animal bites
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Capnocytophaga species
Specimen Collection• Respiratory specimens
• Capnophilic
• Facultative anaerobe
• Set up BAP, CHOC
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• BAP colony morphology– Slight yellow, nonhemolytic, spreading over
agar surface (at 48 hrs)– Center of colony moist, mottled
• No growth on MAC
• Only grows in presence of CO2 (microaerophilic and anaerobic)
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Capnocytophaga species
Growth Characteristics
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Capnocytophaga species
Identification• Fusiform, filamentous gram-negative
rod
• Oxidase –
• Catalase –
• Motility = gliding, no flagella so ittwitches
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What atmosphere does Capnocytophaga require to grow?
Higher concentrations of CO2 (capnophilic) – microaerophilic or anaerobic conditions.
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Francisella tularensis
Clinical Significance• Causes Tularemia – an acute febrile,
HIGHLY INFECTIOUS disease
• Acquire by:– Direct contact with infected animals
(rabbits)– Bite from an insect– Inhalation of infectious aerosols
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Francisella tularensis
Specimen Collection• Inflammatory material from infected site
• Wear gloves and use biosafety hood
• Do not aerosolize or allow contact with skin or mucous membranes
• CDC: Biosafety Level 2 pathogen
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Francisella tularensis
Specimen Processing• Requires cysteine / cystine for growth
• Glucose-cystine blood (Francis’) agar
• Grows on– Chocolate– BCYE– Modified TM
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Francisella tularensis
Growth Characteristics• Strict aerobic
• 35°C with 5-10% CO2 for 7 days
• Colony morphology– BAP & MAC = No growth– CHOC = Small, gray
alpha-hemolytic colonyat 2-5 days
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Francisella tularensis
Identification• Pale staining gram negative coccobacilli
• Oxidase –
• Catalase – to weak +
• Glucose fermenter
• Nonmotile
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Francisella tularensis
Serological Testing• Most cases diagnosed serologically
• DFA tests may be performed on specimen
• ELISA and agglutination tests
• Four-fold rise in titer is diagnostic
• Single titer of >=1:160 is presumptive
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• Streptomycin is drug of choice
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Francisella tularensis
Treatment & Prevention
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What substance is required in culture media in order to grow Francisella?
Cysteine / Cystine
![Page 26: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/26.jpg)
What is the best method for determining if a patient has Tularemia and Why?
Serological testing is best. To actually culture the organism in the laboratory has a high risk for laboratory personnel becoming infected.
![Page 27: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/27.jpg)
• Legionnaires’ disease
• Pontiac Fever
• Transmission: inhalation of the organism in aerosols
• Legionella pneumophila serogroup 1
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Legionella species
Clinical Significance
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• BAL, BW, lung biopsy, pleural fluid
• Avoid aerosolization
• Transport ambient temperature
• Requires cysteine and iron salts for growth
• Buffered Charcoal Yeast Extract agar– Selective media: BCYE + antibiotics
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Legionella species
Specimen Collection
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• Aerobic
• 35°C in 5-10% CO2 for 10 days
• Colony morphology– BAP & MAC = no growth– CHOC = grows slowly– BCYE = convex, grayish
white, glistening with an entire edge at 2-4 days
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Legionella species
Growth Characteristics
![Page 30: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/30.jpg)
• Thin, faintly staining short to filamentous GNR
• Oxidase wk +
• Catalase wk +
• Non-”F”
• Non-”O”
• Motile:polar flagella
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Legionella species
Identification
![Page 31: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/31.jpg)
• Specimen screen & Isolate ID– DFA stain and DNA probe
• IFA test of choice (serum)– Four-fold rise in titer to at least 1:128
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Legionella species
Serological Testing
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• Susceptibility testing not routinely performed
• Drug of choice: Erythromycin alone or with Rifampin
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Legionella species
Treatment and Prevention
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What substance is required in culture media in order to grow Legionella?
Cysteine and iron salts.
![Page 34: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/34.jpg)
What populations are most prone to Legionella infections?
Men, cigarette smokers, people with underlying disease, immunocompromised/immunosuppressed patients, people who drink alcohol and nosocomial infections.
![Page 35: Fastidious Gram Negative Rods Respiratory II Clinical Laboratory Science Program Carol Larson MSEd, MT(ASCP) Please click audio icon to hear Carol’s narration](https://reader035.vdocument.in/reader035/viewer/2022081603/5697bf761a28abf838c81008/html5/thumbnails/35.jpg)
• Looked at several organisms
• Clinical significance
• Specimen collection, transport & processing
• Growth characteristics & identification
• Serological testing
• Treatment and prevention
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Fastidious GNR
Summary
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Who am I?
Reagin-Lowe media Gram Stain
Bordetella pertussis
Causes Whooping Cough
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Who am I?
BCYE agar Gram Stain
Legionella species
Causes Pontiac Fever