1. Overview of Pertussis Diagnostics
Kathleen M. Tatti, Ph.D.
Pertussis and Diphtheria Laboratory
Meningitis and Vaccine Preventable Diseases Branch
National Center for Immunization & Respiratory Diseases
Division of Bacterial Diseases

2. Diagnostic Testing is Important to Public Health
Laboratory
Confirmation
Surveillance
Public Health
Laboratories
Prevention
and Control
Measures
Epidemiology

3. Diagnostic Needs Clinical vs. Public Health
Clinical setting
Optimizes sensitivity
Rapid turnover
Public health setting
Optimizes specificity
Confirmation of etiology
Prevention and control measures

4. Laboratory Diagnosis of pertussis is Complicated by
Stage of disease (catarrhal, paroxysmal, convalescent)
Antimicrobial administration
Vaccination status
Quality/timely collection of clinical specimen (s)
Transport conditions
Contamination of clinical specimen
Lack of clinically validated/ standardized tests

5. Pertussis Diagnostics
Culture
Real-Time PCR (R-PCR)
Serology
Culture
Serology
PCR

6. Specimen Collection
Specimen type will impact ability to isolate bacterium
Nasopharyngeal (NP) aspirates yield similar or higher rates of recovery than NP swabs (rayon, nylon, or polyester)
Throat and anterior nasal swabs yield unacceptably low rates of recovery

7. Culture “Gold Standard” 100% specific, but low sensitivity
Essential for public health labs
Feasible for clinical labs
100% specific, but low sensitivity
Most sensitive within first two weeks after cough onset
Highest yield
Young patients
Unvaccinated patients
Patients early in cough illness prior to antimicrobials
Incubation time 4-10 days
Gram stain of B. pertussis

8. Public Health Impact of Pertussis Culture
Particularly important if an outbreak is suspected
Isolation of the bacterium confirms pertussis
Other respiratory pathogens often cause similar clinical symptoms
Co-infection with other pathogens does occur
Colony morphology helps to identify other species of Bordetella
Necessary for antimicrobial susceptibility testing
Nasopharyngeal flora
B. pertussis

9. PCR Included in the CDC/CSTE case definition in 1997
Primary diagnostic test in many laboratories (IS481)
Rapid test
Potentially more sensitive than culture
Organisms do not need to be viable
May be positive post-antibiotics
Affected by disease phase
No commercial FDA approved tests
No national standardized protocol
Potential for false positives

10. R-PCR Assay-IS481 Present in three Bordetella spp.
50 to >200 copies in B. pertussis
8 to 10 copies in B. holmesii
0 to 7 copies in B. bronchiseptica
High Ct value could indicate
Positive test for B. pertussis
False positive
Positive for
B. holmesii
B. bronchiseptica
Real-Time PCR Amplification Plot

11. Multi-target R-PCR Allows for Speciation
Species
ptxS1
Multiplex
IS481
hIS1001
pIS1001
B. pertussis + -
B. parapertussis
B. pertussis and
B. holmesii

12. CDC R-PCR Pertussis Outbreak Algorithm
(Ct<35)
(35≤Ct<40)
IS481-
(Ct≥40)
ptxS1+
(Ct<40)
B. pertussis
B. parapertussis1
ptxS1-
B. holmesii2
Indeterminate
Negative
1 Confirmed by pIS1001 target
2 Confirmed by hIS1001 target

13. Falsely-positive PCR Results during Outbreak Investigations
Hospital: NH 2006
Community: CO 2009
Community: OH 2010

14. Falsely-positive PCR Results
Use of IS481 as a single target assay
High Ct values interpreted as positive results
B. holmesii misdiagnosed as B. pertussis
Contamination of clinical specimens during collection
B. pertussis DNA present in some vaccines
Confirmed by environmental sampling of clinics

15. Pertussis Serology Originally designed for vaccine evaluation
Routinely used for diagnosis in other countries
Included as part of Massachusetts’ case definition
Useful for confirming diagnosis, especially during outbreaks where culture is not performed
Can be used later in disease

16. CDC/FDA IgG Anti-PT ELISA Kit
If present, specific IgG antibodies in the serum will bind to the PT adsorbed to the wells
The concentration of the PT IgG antibodies is directly proportional to the intensity of the color

17. Public Health Use of Pertussis Serology
Outbreak
Sera
Positive
Indeterminate
Hospital 2006 39 1 5
School 2007
169 49 10
Community 2009 15

18. Complementary Diagnostics St. Croix Outbreak 2007
Proper transport medium allowed
Excellent recovery of B. pertussis
Direct plating not possible locally
Culture was performed at CDC
Overnight shipment not available 18

19. Test Results by Cough Duration
St. Croix 2007 19

20. Optimal Timing for Diagnostic Testing (weeks)
Cough Onset 2 4 6 8 10 12
Culture
PCR
Serology

21. Conclusions No single laboratory test can stand alone for diagnosis
Labs should maintain culture capabilities
Adoption of multi-target R-PCR methods will allow
Confirmation and speciation among Bordetella spp.
Serology is a useful method for diagnosing pertussis
Especially in adults
Later stages of the disease
Diagnosis is an important part of surveillance

22. CDC Approach to Improve Pertussis PCR Diagnostic Testing Practices
Best practices guidance of pertussis PCR
Developed in response to
Recent pseudo-outbreaks
Falsely-positive PCR results
Target clinicians to optimize the use of PCR
Avoiding contamination of clinical specimens with B. pertussis DNA
Interpreting PCR results properly

23. Acknowledgements Pertussis and Diphtheria Lab CDC/MVPDB Epi Team
M. Lucia Tondella
Pam Cassiday
Lucia Pawloski
Kathryn O'Connell
Kansas Sparks
Monte Martin
Michelle Bonkosky
Amber Schmidtke
Aditi Kapasi
CDC/MVPDB Epi Team
Nancy Messonnier
Tom Clark
Stacey Martin
Sema Mandal
Amanda Faulkner
Stanley Wei
Manisha Patel

24. THANK YOU! National Center for Immunization & Respiratory Diseases
Division of Bacterial Diseases , Meningitis and Vaccine Preventable Diseases Branch