Validity of Polymerase Chain Reaction Using Liquid Transport Media During a Pertussis Outbreak

1. Validity of Polymerase Chain Reaction Using Liquid Transport Media During a Pertussis Outbreak Cynthia Schulte, RN, BSN VPD Surveillance Officer Bureau of Immunization New York State Department of Health (NYSDOH)

2. Pertussis Laboratory Testing • Bacterial culture • “Gold standard” • Highly specific • Prone to false negatives because organism is fastidious • Early specimen collection, nasopharyngeal aspirate, proper shipping increase sensitivity • Polymerase chain reaction (PCR) • Rapid results • Highly sensitive • Not highly specific (prone to false positives): may detect other Bordetella spp., DNA in vaccine (clinic contamination) • Not standardized across laboratories • MUST be interpreted in context of signs and symptoms

3. Correct Pertussis Specimen Collection Techniques Nasopharyngeal swab Nasopharyngeal aspiration Images courtesy of CDC. Accessed 03/01/2011 from http://www.cdc.gov/pertussis/clinical/diagnostic-testing/specimen-collection.html.

4. Liquid Solid PCR Transport Media

5. PCR Testing for Pertussis • Not standardized across laboratories • Assay methods, gene targets vary • IS481 as single gene target is especially susceptible to false positives, multiple copies present • Cycle threshold (Ct) values • Large amount of DNA, low Ct value • High Ct values, small amount of DNA • In general, >35-38 equivalent to <1 bacterium (meaning?) • Jefferson County: median Ct 38.9, only 10% Ct<35 • Cutoff values vary by laboratory, can be as high as 50 • Positive, detected, indeterminate, or equivocal

6. PCR and Ct Values — a Fictitious Example

7. Jefferson County • Population ~119,000 • Fort Drum • Young population • 7% aged <5 years • 29% aged <20 years • 40% aged 20–44 years • Total pertussis cases 1992–2009 • 58 (~3 cases/year) Greater than state average

8. Descriptive Epidemiology • 542 PCR+ reports investigated • 103 confirmed cases • 53% male • Median age 5 years (range 5 weeks to 58 years) • Where vaccine status was known (N=93) • 59% were age-appropriately vaccinated • 4% never vaccinated • 85% had minimum 3 DTaP doses required for school entry • 27% of patients ≥11 years had dose of Tdap • 5 case patients hospitalized • 1 mo, 2 mo (2), 4 yr, and 6 yr • No deaths

9. Signs and Symptoms at Time of Testing • Among all PCR+ Patients (N=542) • 77% coughing • 13% catarrhal symptoms only • 10% asymptomatic • Among confirmed cases (N=103) • 100% coughing • 0% catarrhal symptoms only • 0% asymptomatic Testing not indicated

10. Daily Specimen Collections for PCR • >2300 samples tested • 542 positive (28%)

11. Report Count by Date (N=542)

12. Epidemic Curve (N=103)

13. Percent PCR Positive Varied by Pediatric Practice

14. Factors Affecting Outbreak — Testing Issues • Testing of patients with symptoms inconsistent with pertussis • 23% of PCR+ patients did not have cough • High proportion of false positive test results • Sensitivity: ~100% (assumed) • Specificity: 80% • ~90% PCR positive samples had low amounts of DNA detected • Positive predictive value (PPV) of PCR in this outbreak: 19% (103/542) • 4.5% (103/2300) of patients tested were confirmed pertussis cases

15. Factors Influencing Pertussis PCR Test Results • Prevalence of disease among patients tested • Specimen collection technique • Specimens should be collected from the nasopharynx, not the nose or throat • Environmental contamination of clinical specimens • Some pertussis vaccines contain PCR-detectable B. pertussis DNA • Daptacel, Pentacel, Adacel • Liquid transport media for pertussis swabs increases potential for contamination

16. Detection of Pertussis DNA in Vaccines and Clinic Environments • PCR-measurable Bordetella pertussis DNA in • Daptacel • Pentacel • Adacel • Extensive environmental contamination with vaccine DNA; in one study: • 17-87% environmental specimens positive, depending on clinic • Fewer patient samples with Ct > 37 from clinics where above vaccines not used

17. Factors Affecting Outbreak — Co-circulating Pathogens • Bordetella parapertussis (~2%) • Bordetella holmesii (~1%) • Mycoplasma pneumoniae (~13%, 2/16 tested) • 1 hospitalization • Others?

18. Contact Investigation • Recommended antibiotic prophylaxis to 6,900 close contacts of 542 PCR+ patients • Mean: 12 close contacts per report • Range: 0-105 • Nearly 100% self-reported PEP compliance • >6% of entire Jefferson County population received PEP

19. Public Health Actions Taken • Provider education • Health alert • Guidance on specimen collection and selection of patients for testing • Conference call with providers • Additional specimen testing by Wadsworth Center (public health laboratory) • Community vaccination • Vaccine clinics at Jefferson County Public Health Services • >1,200 doses of Tdap • Distribution of vaccine to birthing hospitals

20. Interpretation • Pertussis present, especially early in outbreak • Pertussis never culture-confirmed • “pseudo-outbreak” • Pertussis over diagnosed • Overtesting • False positive PCR results • Possible contamination with vaccine? • Vaccine coverage suboptimal, but might have altered severity of disease

21. Consequences • Unnecessary treatment/PEP of asymptomatic individuals and their close contacts • ~5,500 persons • Healthcare provider offices overwhelmed by testing • County health department overwhelmed by follow-up of patients and their close contacts (especially false PCR+) • 3 full time staff • Unnecessary school and work furloughs

22. CDC Pertussis PCR Testing Recommendations* • PCR complimentary to culture • Culture-confirm outbreak early • Test only patients with clinically compatible disease • Do not test asymptomatic contacts • Test during first 3 weeks of cough • Use proper specimen collection technique and materials • Posterior nasopharyngeal swab or aspirate *Best Practices for Health Care Professionals on the use of Polymerase Chain Reaction (PCR) for Diagnosing Pertussis. 2011.

23. CDC Pertussis PCR Testing Recommendations*, continued • Avoid contamination of specimens with vaccine DNA, particularly if liquid media is used • Prepare and administer vaccines in separate area from specimen collection • Wear gloves for vaccine preparation and administration • Clean clinic surfaces with 10% bleach solution • Glove immediately before specimen collection • Use solid (charcoal) transport media or dry swab • If using liquid media, take care not to handle swab below line • Aspirate kit is closed system, less likely to be contaminated *Best Practices for Health Care Professionals on the use of Polymerase Chain Reaction (PCR) for Diagnosing Pertussis. 2011.

24. CDC Pertussis PCR Testing Recommendations*, continued • Understand limitations of PCR testing • Results variable across laboratories • Interpret results in context of signs and symptoms and available epidemiological information *Best Practices for Health Care Professionals on the use of Polymerase Chain Reaction (PCR) for Diagnosing Pertussis. 2011.

25. Lessons Learned • Rapid provider and community notification can limit the spread of an outbreak • But may also lead to over-testing • PCR is not a perfect test for Bordetella pertussis • To maximize the PPV of PCR, limit testing to patients with cough suggestive of pertussis

26. Thank You • Angie Maxted, MS, DVM Epidemic Intelligence Service Officer Bureau of Communicable Disease Control, NYSDOH EIS Field Assignments Branch, SEPDPO/OSELS, CDC • Elizabeth Rausch-Phung, MD, MPH Medical Director Bureau of Immunization, NYSDOH • Debra Blog, MD, MPH Director Bureau of Immunization, NYSDOH