Bordetella species Bordetella · 2 Parapertussis • Symptoms are similar to pertussis but...

1

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 Hygiene

Phone: 608-265-9115

[email protected]

January 11, 2012

Bordetella species

• B. pertussis--------------human• B. parapertussis--------human/sheep• B. bronchiseptica------animals/human• B holmesii--------------human


B. holmesii human• B. avium-----------------birds• B. hinzii------------------poultry• B. trematum------------animals• B. petrii------------------environmental

2

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

• Fatty acids• Metal ions• Sulfides • Peroxides

3

B. Pertussis Gram Stain


4

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


– 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

5

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


– 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

6

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


• 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.

7

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

8

Transmission

• Respiratory droplets• Highly infectious

– 80-90% of household contacts


9 WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

10

Bordetella pertussis

• 20-40 million cases/year worldwide• 200,000-400,000 deaths

– Primarily among children• Cases/yr in the US

2002 9 771


– 2002----------9,771– 2003----------11,647– 2004----------25,827– 2005----------25,616– 2006----------15,632– 2007----------10,454– 2008----------13,278


12

3

Number of Specimens Positive for Whooping Cough (6/1/03 – 10/22/05)

45

60

75

ber

Po

siti

ve


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


14



16

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


– Recent addition-------Tdap at 11-12 years

– “Catch up” for 13-18 yr olds

• Issue of “waning immunity”

17

Treatment and Prophylaxis

• Macrolides– Azithromycin

– Erythromycin

– Clarithromycin


• Second choice = sulfa drugs (SXT)

• Not for infants <2mo

18

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


• NP aspirates, washes, or swabs• Dacron or rayon swabs

– NO cotton or calcium alginate swaps

• NO throat, sputum, or mouth specimens

19

Nasopharyngeal Specimen


20

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

21

Laboratory Diagnosis

• Culture

• DFA

• Real-time


Polymerase Chain Reaction– More rapid than

culture

– More sensitive than culture

22

Laboratory Diagnosis

CULTURE

DFA


DFA

PCR

23

Culture

• “Gold Standard”– Essential for public health labs

• 100% specific, but low sensitivity(12 60%)


– (12-60%)

• Highest yield– Young children

– Unvaccinated

– Early in cough illness prior to antibiotics

24

5

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 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


26

“Pearl-like”

Other Bordetella species• B. parapertussis

– Colonies within 2-3 days

– On Regan-Lowe appear grey

– On BG have brown pigmentation

• B holmesii


• B. holmesii– Colonies similar to B. pertussis

– Inhibited by cephalexin

• B. bronchiseptica– Colonies within 1 day

– On Regan-Lowe, large, slight brown

27

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

28

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

29

Identification

• B. Pertussis– Catalase +

– Oxidase +

– Urease -

M tilit

• B. parapertussis– Catalase +

– Oxidase -

– Urease +

M tilit


– Motility -

– Nitrate -

– Blood Agar -

– MacConkey -

– Fluor. Ab

– Motility -

– Nitrate -

– Blood Agar +

– MacConkey V

– Fluor. Ab

30

6

Direct Fluorescent Antibody• In use since 1960• Direct detection and ID of isolates


31

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


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

32

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

33

Pertussis PCR (cont.)

• Advantages– Rapid– Extremely sensitive

• <=1 CFU (5ul sample)– Does not require viable organism


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

34

Pertussis PCR (cont.)

• Disadvantages– Stringent requirements to perform PCR– Not presently standardized– More expensive than culture or DFA


More expensive than culture or DFA– PCR inhibitors– Cross reaction with B. holmseii

35

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%

27 Pos by Culture ONLY 0.8% 6.0%

256 Pos by PCR ONLY 7.7% 56.9%

36

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


37

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


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


• Targets– IS481

– plS1001

– hIS1001

– ptxS1

39

Targets• IS481

– B. pertussis (80-200 copies/cell)

• hIS1001– B. holmesii (3-5 copies/cell)

• pIS1001


• 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 + - - +


B. pertussis and B. parapertussis

+ + - +B. holmesii - + + -

41

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

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


• 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


– 0.6% of patients

– Role in respiratory disease still unclear

• Bacteremia in asplenic children and sickle-cell patients– Endocarditis

44

Bordetella holmesii (cont.)


45


• 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

46


• Negative for catalase (variable), oxidase, NO3 reductase, urease, indole, motility


• Misidentified as Acinetobacter lwoffii on Vitek 2

47

Bordetella bronchiseptica


48

9

Bordetella bronchiseptica (cont.)

• Rarely isolated from respiratory specimens

• Pertussis-like disease and other respiratory symptoms


respiratory symptoms– Infectious bronchitis

• Most frequently in imunocompromised• Rare cases of bacteremia and

septicemia

49


• 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

50



51


• 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

52

Thank You


53

Questions????

Bordetella species Bordetella · 2 Parapertussis • Symptoms are similar to pertussis but...

Documents

Transcript of Bordetella species Bordetella · 2 Parapertussis • Symptoms are similar to pertussis but...