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 Images courtesy of CDC. Accessed 03/01/2011 from http://www.cdc.gov/pertussis/clinical/diagnostic-testing/specimen-collection.html. Nasopharyngeal swabNasopharyngeal aspiration

4. PCR Transport Media Liquid Solid

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