bordetella species bordetella · 2 parapertussis • symptoms are similar to pertussis but...
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BordetellaDave Warshauer, Ph.D
Wi i St t L b t f H i
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Wisconsin State Laboratory of Hygiene
Phone: 608-265-9115
david.warshauer@slh.wisc.edu
January 11, 2012
Bordetella species
• B. pertussis--------------human• B. parapertussis--------human/sheep• B. bronchiseptica------animals/human• B holmesii--------------human
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
B. holmesii human• B. avium-----------------birds• B. hinzii------------------poultry• B. trematum------------animals• B. petrii------------------environmental
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Bordetella• Small gram-negative coccobacillus• Strictly aerobic (except B. petrii)• Oxidize amino acids
– None ferment carbohydrates
• Catalase positive• Optimal growth at 35°C, ambient air with
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
humidity• B. pertussis most fastidious of the
Bordetella– Inhibited by constituents in media
• Fatty acids• Metal ions• Sulfides • Peroxides
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B. Pertussis Gram Stain
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Some virulence factors of B. pertussis• Non-invasive bacterium-does not normally
invade cells• Attachment
– Filamentous hemagglutinin (FHA) and pertactin– Pertussis toxin
• ToxinsTracheal cytotoxin cell death
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Tracheal cytotoxin – cell death– Endotoxin – cytokines, fever– Adenylate cyclase/hemolysin – inhibits lymphocyte
function – Lipooligosaccharide (endotoxin)– Pertussis toxin (PT)* - inhibits immune cells
chemotaxis and acts as an adhesin
* = difference between pertussis and parapertussis
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Whooping Cough
• Bordetella pertussis • Classical whooping cough
– Cold-like illness (Catarrhal phase)• Runny nose, sneezing, low-grade fever, tired
D d ti h f 2 k
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Dry non-productive cough for >2 weeks– “Whooping” cough (Paroxysmal phase)
• Severe cough with vomiting
– Severe disease in infants– 7-10 day incubation (range 5-21)– Epidemic disease every 2-5 years
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2
Parapertussis• Symptoms are similar to pertussis but
typically milder. – cough that occurs in sudden, uncontrollable bursts
• Shorter duration than pertussis (Avg 14 days)
– high-pitched whooping sounds when breathing in after a coughing episode
– posttussive vomiting • Incubation & Transmission Periods similar to
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Incubation & Transmission Periods similar to pertussis
• All age groups can be infected. – Young infants (e.g., <6 months of age) may have
a more severe course• Rarely, death can occur in infants with
underlying health problems or those that are co-infected with B. pertussis.
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Pertussis CSTE Case Definition
• Clinical case definition– Cough > 2 weeks and at least one
symptom: paroxysms, whoop, posttussive vomiting
• Case classification
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Confirmed cases• Culture positive
• Clinical case and PCR positive
• Clinical case and epi-linked to confirmed case
– Probable case• Only meets the clinical case definition
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Transmission
• Respiratory droplets• Highly infectious
– 80-90% of household contacts
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
9 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Bordetella pertussis
• 20-40 million cases/year worldwide• 200,000-400,000 deaths
– Primarily among children• Cases/yr in the US
2002 9 771
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– 2002----------9,771– 2003----------11,647– 2004----------25,827– 2005----------25,616– 2006----------15,632– 2007----------10,454– 2008----------13,278
11 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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3
Number of Specimens Positive for Whooping Cough (6/1/03 – 10/22/05)
45
60
75
ber
Po
siti
ve
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
13
0
15
30
6/7/0
3
7/5/0
3
8/2/0
3
8/30
/03
9/27
/03
10/2
5/0
3
11/2
2/0
3
12/2
0/0
3
1/17
/04
2/14
/04
3/13
/04
4/10
/04
5/8/0
4
6/5/0
4
7/3/0
4
7/31
/04
8/28
/04
9/25
/04
10/2
3/0
4
11/2
0/0
4
12/1
8/0
4
1/15
/05
2/12
/05
3/12
/05
4/9/0
5
5/7/0
5
6/4/0
5
7/2/0
5
7/23
/05
8/27
/05
9/24
/05
10/2
2/0
5
Nu
mb
Bordetella parapertussis Bordetella pertussisBordetella pertussis/Bordetella bronchiseptica Bordetella pertussis/parapertussis
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
15 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Pertussis Vaccine
• Included in childhood vaccines– DTaP at 2,4,6, and 15-18 months, 4-6
years
– Recent addition-------Tdap at 11-12 years
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Recent addition-------Tdap at 11-12 years
– “Catch up” for 13-18 yr olds
• Issue of “waning immunity”
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Treatment and Prophylaxis
• Macrolides– Azithromycin
– Erythromycin
– Clarithromycin
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Second choice = sulfa drugs (SXT)
• Not for infants <2mo
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NOTE: Treatment should be administered during the catarrhal stage to lessen illness. If cough develops, does not affect illness course but may lessen transmission possibility.
4
Specimen Collection
• Nasopharyngeal specimen– Want ciliated epithelial cells
– Timing critical
• NP aspirates washes or swabs
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• NP aspirates, washes, or swabs• Dacron or rayon swabs
– NO cotton or calcium alginate swaps
• NO throat, sputum, or mouth specimens
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Nasopharyngeal Specimen
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Transport
• B. pertussis extremely labile• Direct culture optimal, not feasible• Transport media
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– If <2hr----0.5-1.0% Casamino Acid Sol’n at room temp
– 2hr – 24hr----Amies with charcoal, room temp
– >24hr----Regan-Lowe or Jones-Kendrick at 4 C
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Laboratory Diagnosis
• Culture
• DFA
• Real-time
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Polymerase Chain Reaction– More rapid than
culture
– More sensitive than culture
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Laboratory Diagnosis
CULTURE
DFA
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
DFA
PCR
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Culture
• “Gold Standard”– Essential for public health labs
• 100% specific, but low sensitivity(12 60%)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– (12-60%)
• Highest yield– Young children
– Unvaccinated
– Early in cough illness prior to antibiotics
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Culture Media• Bordet-Gengou
– Potato infusion, glycerol, sheep/horse blood• Regan-Lowe
– Charcoal agar, 10% horse blood• Jones-Kendrick
– Charcoal agar, yeast extract, starch
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Stainer-Scholte synthetic medium• Legionella buffered charcoal yeast-
extract• Incorporate antibiotics to suppress normal
flora----cephalexin or methicillin• Incubate 35-36C with high humidity for a
minimum of 7 days25
B. pertussis on Bordet-Gengou7 days incub. Commercial Medium
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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“Pearl-like”
Other Bordetella species• B. parapertussis
– Colonies within 2-3 days
– On Regan-Lowe appear grey
– On BG have brown pigmentation
• B holmesii
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• B. holmesii– Colonies similar to B. pertussis
– Inhibited by cephalexin
• B. bronchiseptica– Colonies within 1 day
– On Regan-Lowe, large, slight brown
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Culture Sensitivity• Considered no more than 60% sensitive• Factors effecting sensitivity
– Type and quality of specimen
– Time specimen obtained in the course
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
of illness
– Age of the patient
– Appropriate transport
– Choice of culture media
– Length of time cultures incubate
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Culture for Public Health
• Important if an outbreak is suspected• Isolation of the organism confirms
pertussis– Other agents can cause pertussis-like
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
illness
– Co-infection with other pathogens occurs
– Culture helps identify other Bordetella spp.
• Necessary for AST and subtyping
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Identification
• B. Pertussis– Catalase +
– Oxidase +
– Urease -
M tilit
• B. parapertussis– Catalase +
– Oxidase -
– Urease +
M tilit
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Motility -
– Nitrate -
– Blood Agar -
– MacConkey -
– Fluor. Ab
– Motility -
– Nitrate -
– Blood Agar +
– MacConkey V
– Fluor. Ab
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Direct Fluorescent Antibody• In use since 1960• Direct detection and ID of isolates
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Courtesy of Mike Saubolle, PhD
DFA• Problems
– Poor sensitivity (18-78%)– Requires large numbers of organisms (>104 /ml)– Best when test early in course of illness– Requires skilled and experienced microscopist– Antibiotic therapy can affect binding of DFA
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Antibiotic therapy can affect binding of DFA reagent to cell wall
– Poor specificity (7-44% false positives)• Advantage
– More rapid than culture• No longer recommended
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Pertussis PCR• Optimal diagnostic test
– 70 – 90% Sensitivity
• No prolonged asymptomatic “carrier ”
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
state”– If positive, considered diagnostic
• Recognized by CSTE as official laboratory confirmation of pertussis in addition to culture
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Pertussis PCR (cont.)
• Advantages– Rapid– Extremely sensitive
• <=1 CFU (5ul sample)– Does not require viable organism
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Does not require viable organism• Transport delays and antibiotics do not prevent
laboratory diagnosis– Positive longer than culture– Specific
• Except for detection of B. holmesii if using IS481
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Pertussis PCR (cont.)
• Disadvantages– Stringent requirements to perform PCR– Not presently standardized– More expensive than culture or DFA
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
More expensive than culture or DFA– PCR inhibitors– Cross reaction with B. holmseii
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WSLH Pertussis Data July 2003-July 2004 %Pos of Total % of Positives
3330 Tested by Culture and PCR
450 Pos by Cult and/or PCR 13.5% 100%
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
167 Pos by BOTH Cult and PCR 5.0% 37.1%
27 Pos by Culture ONLY 0.8% 6.0%
256 Pos by PCR ONLY 7.7% 56.9%
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20 culture positive for B. parapertussis
7
73.668.4
46.5
40
50
60
70
80
e P
os
itiv
e
PCR Crossing Points vs Culture
* No Specimens
June 1, 2003- September 10,2004
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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21.2
10
0
10
20
30
% C
ult
ure
13-24.99 25-29.99 30-34.99 35-39.99 40-45
Crossing Point Range
53* 95*127*
226*10*
* No. Specimens
Number of Specimens Tested for Bordetella by PCR and/or Culture (6/1/03 – 1/29/05)
1200
1600
2000
r T
est
ed
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
38
0
400
800
6/7/0
36
/21/03
7/5/0
37
/19/03
8/2/0
38
/16/03
8/30
/039
/13/03
9/27
/031
0/11/03
10/2
5/031
1/8/03
11/2
2/031
2/6/03
12/2
0/031
/3/04
1/17
/041
/31/04
2/14
/042
/28/04
3/13
/043
/27/04
4/10
/044
/24/04
5/8/0
45
/22/04
6/5/0
46
/19/04
7/3/0
47
/17/04
7/31
/048
/14/04
8/28
/049
/11/04
9/25
/041
0/9/04
10/2
3/041
1/6/04
11/2
0/041
2/4/04
12/1
8/041
/1/05
1/15
/051
/29/05
Nu
mb
e
Total Tested
Bordetella spp. PCR at WSLH
• Multi-target RT-PCR – B. pertussis
– B. parapertussis
– B. holmesii
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Targets– IS481
– plS1001
– hIS1001
– ptxS1
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Targets• IS481
– B. pertussis (80-200 copies/cell)
• hIS1001– B. holmesii (3-5 copies/cell)
• pIS1001
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• pIS1001– B. parapertussis
• RNP– Inhibition detection
• ptxS1---Not part of multi-plex– Single copy in B. pertussis and B. parapertussis
40
Species Identification
Species ptxS1 IS481 hIS1001 pIS1001B. pertussis + + - -B. parapertussis + - - +
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
B. pertussis and B. parapertussis
+ + - +B. holmesii - + + -
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Pertussis Serology
• Require acute and convalescent specimens• Infection in vaccinated individuals
confounds interpretation • Not included in CSTE case definition
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Can have epidemiologic value• No standardized assays• No FDA approved assays• New CDC developed EIA may provide a
useful serologic tool
42
8
CDC IgG Anti-PT ELISA
• Microwell ELISA format• Allows for diagnosis of recent
infections with a single specimenU f l i l t t f th di
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Useful in later stages of the disease (>2 weeks from onset
• Post-vaccination antibody levels do not interfere with diagnosis
• Can be qualitative
43
Bordetella holmesii
• First identified in 1995• Associated with pertussis-like
respiratory disease and pneumonia0 6% f ti t
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– 0.6% of patients
– Role in respiratory disease still unclear
• Bacteremia in asplenic children and sickle-cell patients– Endocarditis
44
Bordetella holmesii (cont.)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Bordetella holmesii (cont.)
• Gram negative coccobacillus• Grows well on SBA in 5% CO2 after
24hr
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
– Inhibited by cephalexin
• Diffusible brown pigment– Can be mistaken for alpha hemolysis
• Poor or no growth on Mac
46
Bordetella holmesii (cont.)
• Negative for catalase (variable), oxidase, NO3 reductase, urease, indole, motility
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Misidentified as Acinetobacter lwoffii on Vitek 2
47
Bordetella bronchiseptica
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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9
Bordetella bronchiseptica (cont.)
• Rarely isolated from respiratory specimens
• Pertussis-like disease and other respiratory symptoms
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
respiratory symptoms– Infectious bronchitis
• Most frequently in imunocompromised• Rare cases of bacteremia and
septicemia
49
Bordetella bronchiseptica (cont.)
• Has genes for pertussis toxin– Not expressed
• Positive for catalase, oxidase, nitrate,
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
, , ,urease, motility
• Grows on Mac, SBA– On SBA, small to medium gray colonies
with beta hemolysis under the colonies
50
Bordetella bronchiseptica (cont.)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
51
Bordetella bronchiseptica (cont.)
• Susceptibility pattern– Beta-lactamase producer
• R to many penicillins and cephalosporins
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
• Anti-pseudomonal penicillins usually sensitive
– Mostly resistant to SXT
– Most S to amoxicillin-clavulanic acid, tetracycline, gentamicin, and quinolones
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Thank You
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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Questions????
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