Download Test Method Validation

Bordetella
Dave Warshauer, Ph.D
Wisconsin State Laboratory of Hygiene
Phone: 608-265-9115
david.warshauer@slh.wisc.edu
January 11, 2012
WISCONSIN STATE
LABORATORY
OF HYGIENE
WISCONSIN
STATE
LABORATORY OF HYGIENE
Bordetella species
•
•
•
•
•
•
•
•
B. pertussis--------------human
B. parapertussis--------human/sheep
B. bronchiseptica------animals/human
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
humidity
• B. pertussis most fastidious of the
Bordetella
– Inhibited by constituents in media
•
•
•
•
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Fatty acids
Metal ions
Sulfides
Peroxides
3
B. Pertussis Gram Stain
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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
• Toxins
– 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
– 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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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
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
– 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
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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
2003----------11,647
2004----------25,827
2005----------25,616
2006----------15,632
2007----------10,454
2008----------13,278
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Number of Specimens Positive for
Whooping Cough (6/1/03 – 10/22/05)
75
Number Positive
60
45
30
15
0
10/22/05
9/24/05
8/27/05
7/23/05
7/2/05
6/4/05
5/7/05
4/9/05
3/12/05
2/12/05
1/15/05
12/18/04
11/20/04
10/23/04
9/25/04
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LABORATORY OF HYGIENE
8/28/04
7/31/04
7/3/04
6/5/04
5/8/04
4/10/04
3/13/04
2/14/04
1/17/04
12/20/03
11/22/03
10/25/03
9/27/03
8/30/03
8/2/03
7/5/03
6/7/03
Bordetella parapertussis
Bordetella pertussis/Bordetella bronchiseptica
Bordetella pertussis
Bordetella pertussis/parapertussis
13
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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
– “Catch up” for 13-18 yr olds
• Issue of “waning immunity”
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Treatment and Prophylaxis
• Macrolides
– Azithromycin
– Erythromycin
– Clarithromycin
• Second choice =
sulfa drugs (SXT)
• Not for infants <2mo
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.
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Specimen Collection
• Nasopharyngeal specimen
– Want ciliated epithelial cells
– Timing critical
• 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
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Transport
• B. pertussis extremely labile
• Direct culture optimal, not feasible
• Transport media
– 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
Polymerase
Chain Reaction
– More rapid than
culture
– More sensitive
than culture
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Laboratory Diagnosis
CULTURE
DFA
PCR
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Culture
• “Gold Standard”
– Essential for public health labs
• 100% specific, but low sensitivity
– (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
• Stainer-Scholte synthetic medium
• Legionella buffered charcoal yeastextract
• Incorporate antibiotics to suppress normal
flora----cephalexin or methicillin
• Incubate 35-36C with high humidity for a
minimum of 7 days
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B. pertussis on Bordet-Gengou
7 days incub. Commercial Medium
“Pearl-like”
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Other Bordetella species
• B. parapertussis
– Colonies within 2-3 days
– On Regan-Lowe appear grey
– On BG have brown pigmentation
• 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
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
illness
– Co-infection with other pathogens occurs
– Culture helps identify other Bordetella
spp.
• Necessary for AST and subtyping
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Identification
• B. parapertussis
• B. Pertussis
–
–
–
–
–
–
–
–
Catalase
Oxidase
Urease
Motility
Nitrate
Blood Agar
MacConkey
Fluor. Ab
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+
+
-
–
–
–
–
–
–
–
–
Catalase
Oxidase
Urease
Motility
Nitrate
Blood Agar
MacConkey
Fluor. Ab
+
+
+
V
30
Direct Fluorescent Antibody
• In use since 1960
• Direct detection and ID of isolates
Courtesy of Mike Saubolle, PhD
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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
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
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
• 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
– 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%
167 Pos by BOTH Cult and PCR
5.0%
37.1%
0.8%
6.0%
7.7%
56.9%
27 Pos by Culture ONLY
256 Pos by PCR ONLY
20 culture positive for B. parapertussis
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PCR Crossing Points vs Culture
June 1, 2003- September 10,2004
73.6
68.4
80
% Culture Positive
70
60
46.5
50
40
* No. Specimens
53*
30
21.2
95*
127*
10
20
226*
10
10*
0
13-24.99
25-29.99
30-34.99
35-39.99
40-45
Crossing Point Range
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1200
800
Number Tested
Number of Specimens Tested for Bordetella
by PCR and/or Culture (6/1/03 – 1/29/05)
2000
1600
400
0
1/29/05
1/15/05
1/1/05
12/18/04
12/4/04
11/20/04
11/6/04
10/23/04
10/9/04
9/25/04
9/11/04
8/28/04
8/14/04
7/31/04
7/17/04
7/3/04
6/19/04
6/5/04
5/22/04
5/8/04
4/24/04
4/10/04
3/27/04
3/13/04
2/28/04
2/14/04
1/31/04
1/17/04
1/3/04
12/20/03
12/6/03
11/22/03
11/8/03
10/25/03
10/11/03
9/27/03
9/13/03
8/30/03
8/16/03
8/2/03
7/19/03
7/5/03
6/21/03
6/7/03
Total Tested
38
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Bordetella spp. PCR at WSLH
• Multi-target RT-PCR
– B. pertussis
– B. parapertussis
– B. holmesii
• 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
– B. parapertussis
• RNP
– Inhibition detection
• ptxS1---Not part of multi-plex
– Single copy in B. pertussis and B. parapertussis
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Species Identification
Species
B. pertussis
B. parapertussis
B. pertussis and
B. parapertussis
B. holmesii
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ptxS1
IS481
hIS1001
pIS1001
+
+
+
+
+
-
+
+
-
+
+
-
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Pertussis Serology
• Require acute and convalescent specimens
• Infection in vaccinated individuals
confounds interpretation
• Not included in CSTE case definition
• Can have epidemiologic value
• No standardized assays
• No FDA approved assays
• New CDC developed EIA may provide a
useful serologic tool
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CDC IgG Anti-PT ELISA
• Microwell ELISA format
• Allows for diagnosis of recent
infections with a single specimen
• Useful in later stages of the disease
(>2 weeks from onset
• Post-vaccination antibody levels do not
interfere with diagnosis
• Can be qualitative
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Bordetella holmesii
• First identified in 1995
• Associated with pertussis-like
respiratory disease and pneumonia
– 0.6% of patients
– Role in respiratory disease still unclear
• Bacteremia in asplenic children and
sickle-cell patients
– Endocarditis
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Bordetella holmesii (cont.)
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Bordetella holmesii (cont.)
• Gram negative coccobacillus
• Grows well on SBA in 5% CO2 after
24hr
– Inhibited by cephalexin
• Diffusible brown pigment
– Can be mistaken for alpha hemolysis
• Poor or no growth on Mac
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Bordetella holmesii (cont.)
• Negative for catalase (variable),
oxidase, NO3 reductase, urease,
indole, motility
• Misidentified as Acinetobacter lwoffii
on Vitek 2
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Bordetella bronchiseptica
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Bordetella bronchiseptica (cont.)
• Rarely isolated from respiratory
specimens
• Pertussis-like disease and other
respiratory symptoms
– Infectious bronchitis
• Most frequently in imunocompromised
• Rare cases of bacteremia and
septicemia
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Bordetella bronchiseptica (cont.)
• Has genes for pertussis toxin
– Not expressed
• Positive for catalase, oxidase, nitrate,
urease, motility
• Grows on Mac, SBA
– On SBA, small to medium gray colonies
with beta hemolysis under the colonies
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Bordetella bronchiseptica (cont.)
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Bordetella bronchiseptica (cont.)
• Susceptibility pattern
– Beta-lactamase producer
• R to many penicillins and cephalosporins
• 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
Questions????
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