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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Test Results by Cough Duration St. Croix 2007 19

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

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

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 http://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html

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

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