1. “Update on Pertussis Diagnostics" Maria Lucia Tondella, PhD Pertussis and Diphtheria Laboratory Meningitis and Vaccine Preventable Diseases Branch Division of Bacterial Diseases National Center for Immunization and Respiratory Diseases (proposed)

2. Background Pertussis is an acute respiratory infection caused by Bordetella pertussis Despite high vaccine coverage, pertussis remains a public health problem in the U.S.  25,616 reported cases (2005)  39 (38 children) reported pertussis-related deaths Disease burden is unclear Adolescents and adults may serve as reservoir for transmission to unvaccinated infants who are at the higher risk of severe complications

3. 11-18 yrs >18 yrs <11 yrs DTP 0 10,000 20,000 30,000 1990199520002005 192219301940195019601970198019902000 Year 50,000 0 100,000 150,000 200,000 250,000 300,000 1950-2005, National Notifiable Diseases Surveillance System; 1922-1949, Passive Reports to the Public Health Service. Number of cases Reported Pertussis Cases in the United States, 1922-2005

4. Increased Pertussis Cases Reporting Why the increase? Possible reasons include:  Waning vaccine-induced immunity  Type and quality of vaccines currently used  Lower potency or vaccine failure  Changes in the circulating organism  Improved disease surveillance  Increased availability of laboratory tests  Predictive value for many tests unknown

5. CDC/CSTE Case Definitions Clinical case definition  Cough ≥2 weeks and at least one pertussis symptom: paroxysms, whoop, post-tussive vomiting Confirmed case  Culture positive or  Clinical case and PCR positive or  Clinical case and epi-linked to confirmed case Probable case  Meets the clinical case definition CSTE: Council of State and Territorial Epidemiologists

6. Clinical Diagnosis of pertussis It is complicated by a number of facts:  Previous vaccination or infection  Previously immunized older children and adolescents rarely present a classic “whoop”  Wide spectrum of symptoms  Confusion with other non specific respiratory complaints

7. Laboratory Diagnosis of pertussis It is complicated by:  Stage of disease (catarrhal, paroxysmal, convalescent)  Antimicrobial administration  Vaccination status  Quality/timely collection of clinical specimen  Transport conditions of clinical specimen  Contamination of clinical specimen  Lack of clinically validated and standardized tests

8. Culture Very specific (~100%) Sensitivity varies:  High for young unvaccinated infants with short duration of symptoms  Low (<10%) for adolescents and adults with long duration of cough Slow, minimal period of incubation is 10 days B. pertussis isolation drastically declines after:  2 weeks of cough  Antimicrobial or vaccine administration  Inappropriate collection/transport /growth conditions

9. PCR Included in the CDC/CSTE case definition in 1997 Primary diagnostic test in many laboratories (IS 481 target) Rapid test Potentially more sensitive than culture  Organisms do not need to be viable  May be positive post-antibiotics Disadvantages  Affected by disease phase and antibiotic treatment  No commercial FDA approved tests  No national standardized protocol  Unknown predictive value of results  Potential for false positives (contamination)

10. Potential Problems with IS 481 PCR Assay IS 481 is found  In 50 to >200 copies per B. pertussis cell  In multiple copies in B. holmesii  In one copy in B. bronchiseptica High Threshold Cycle (CT)  High CT indicates very low levels of DNA  Different platforms have different sensitivities  Can be real positives (indicative of a pertussis infection) or  Can be false positives  DNA cross-contamination

11. Serologic Assays Useful for confirming diagnosis  More likely to be positive in adolescents and adults  Typically present late Not part of CDC/CSTE case definition  Exception: MA single point assay with cut-off values (>11yrs age) Preferred assay: IgG anti-pertussis toxin ELISA  Most specific and sensitive Disadvantages  Late (retrospective) diagnosis  Vaccination may confound serology testing  Lack of true acute phase specimens related to nonspecific symptoms in early stage of disease  No universal serologic correlate for protection  No universal serologic marker of disease

12. Positive Pertussis Laboratory Tests Reported to NNDSS* * National Notifiable Diseases Surveillance System Source: Slade, BA. 8th International Symposium: Saga of the Genus Bordetella, 1906-2006. Paris, France. Nov. 7-10, 2006

13. Current status of diagnostic testing for pertussis in the U.S There has been a sharp increase in the number of pertussis cases reported to the NNDSS in 2004/05. Most of the cases were reported by PCR While the number of cases confirmed by culture has been stable, the % of cases confirmed by culture has decreased over time  Cases confirmed by culture: 75% in 1995 to 9% in 2005 PCR testing has rapidly overtaken culture as the primary method for pertussis laboratory diagnosis  Cases confirmed by PCR: <1% in 1995 to 25% in 2005 Serologic testing for confirmation of pertussis is increasing despite the lack of FDA-licensed test  Particularly adults > 20 yrs of age Use of DFA persist in most states due to rapid results Source: Slade, BA. 8th International Symposium: Saga of the Genus Bordetella, 1906-2006. Paris, France. Nov. 7-10, 2006

14. CDC PCR assays two target approach Target sequences  IS 481 – insertion sequence, 50 to >200 copies per cell  Pertussis toxin subunit 1 ( ptxS 1), single copy gene Species IS 481ptxS 1 Bordetella pertussis + (CT <35)+ Bordetella parapertussis -+ Bordetella holmesii + (CT <35)-

15. CDC/FDA anti-PT IgG Serology Assay “User-friendly” kit formulation Ready to use standards (lyophilized) Ready to use controls  At least two levels (49 & 94 EU/ml)  For quantitative result: use standard curve  For qualitative result: compare test samples vs. controls Simple assay  Single dilution of serum sample  Minimal reagent preparation  Microtiter strips  Monoclonal antibody conjugate

16. Clinical Validation Study Estimate the clinical sensitivity, specificity and predictive values of the following diagnostic tests:  CDC/FDA anti-pertussis toxin IgG serologic ELISA  CDC’s combined real-time PCR  IS 481 and ptxS 1  Boston Medical Center’s anti-pertussis toxin secretory IgA Assess clinical usefulness as related to  Age of patient  Stage of disease  Vaccination status  Prior antibiotic therapy

17. Conclusions Clinically validated and standardized tests are lacking No single laboratory test can be considered a gold standard Culture should be performed in combination with PCR Interpretation of PCR results can be challenging In outbreak settings, high CT values of IS 481 real-time PCR should be confirmed by additional testing (other PCR target, culture or serology) Positive PCR results must be interpreted in combination with patients’ symptoms, treatment status and epidemiological factors Serological tests have the potential to contribute to the diagnosis of pertussis when standardized and validated tests are available

18. Acknowledgements Barbara Slade Stacey Martin Pam Cassiday Kai-Hui Wu Kathi Tatti Lucia Pawloski Nancy R. Messonnier Trudy Murphy Margaret Cortese Drew Baughman Gary Sanden Patty Wilkins Bruce Meade Sandra Menzies Vijay Kadwad Amy Poel Kris Bisgard