1. Investigation of Suspected False-Positive PCR Results for Bordetella Pertussis
Rachel Wiseman1, MPH; David G. Bastis2, MPH; Jill Campbell3, RN, MPH; Valerie Wheelock4, MD
March 30, 2011
1.Texas Department of State Health Services, 2. Williamson County and Cities Health District 3. Austin/Travis County Health Department 4. Barton Creek Pediatrics

2. Reported Cases of Pertussis in Texas 1980-2010*
2009: 3,358 pertussis cases
2003: PCR routine in Texas
* Provisional as of March 23, 2011

3. Background Inconsistent clinical presentation
Increase in PCR+ B. pertussis results
Noted by private clinicians
These PCR+ patients had either:
Inconsistent clinical presentation
Recent (within 6 mos) pertussis diagnosis/treatment
In two distinct regions of Texas
All clinicians used same lab (Lab A)

4. PCR Background Low disease burden Contaminant
No FDA approved PCR test for pertussis
Every lab validates its own cutoff values for PCR testing
PCR test uses # of cycles to establish presence of DNA
Lower cycle times, more DNA
Higher cycle times, less DNA
Low disease burden
Contaminant

5. Media Background
Lab A uses liquid transport media for B. pertussis PCR testing
Liquid, not swab, tested
Texas DSHS Lab uses no media for transport for pertussis PCR tests
Swab tested

6. Previous Studies
Some pertussis vaccines contain pertussis DNA detectable by PCR
Pertussis DNA has been detected in clinical environments by PCR
Liquid media may detect environmental contaminant DNA more readily than no/solid media

7. Environmental Sampling Methods
Health department staff visited one clinic site
Swabbed patient rooms, nurses’ station and vaccine handling areas
Door knobs
Counters
Refrigerator surfaces
Samples tested by DSHS Lab using PCR

8. Environmental Sampling Results
17 samples collected
1 (5.9%) positive for B. pertussis DNA
Exam room door handle

9. PCR Comparison Methods
Phase 1
Two clinics collected dual nasopharyngeal swabs on patients presenting with pertussis-like symptoms
Parallel testing was performed by Lab A and DSHS Lab
Phase 2
The labs traded 10 patient samples for re-testing

10. PCR Comparison Results
Lab A
DSHS Lab
B. pertussis not detected
28 (43.8%)
63 (98.4%)
B. pertussis detected
16 (25.0%)
Equivocal
19 (29.7%)
B. Parapertussis detected
1 (1.6%)
Total 64
54.7% discordant

11. <40 (35-40 repeat, release)
PCR Cutoff Values
Lab A
Cycle Times
DSHS Lab Cycle Times
Positive
<40 (35-40 repeat, release)
<35
Equivocal/
Indeterminate
40-45
35-39
Negative
>45
>39

12. Traded Samples Lab A DSHS Lab A Tests DSHS’s Sample DSHS Tests
Lab A’s Sample
Patient 1 + -
- (>39 CT)
Patient 2
Patient 3
Ind (35 CT)
Patient 4 Eq
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
They can validate ours—no DNA in our samples. WE can’t validate theirs, indicates low levels of DNA AND issues with cutoff values.

13. Conclusions
B. pertussis DNA can be identified in the clinic environment
Discrepancies between Lab A and DSHS Lab results
Differences in cutoff values
Differences in results
Lab A not able to replicate results using swabs without liquid media

14. Discussion
Environmental contamination of clinical swabs is more likely to affect outcome of testing when liquid media is used for transport/testing
Cutoff values may need to be adjusted
Equivocal results difficult for clinicians to interpret
High cutoffs may be picking up low level (environmental) DNA

15. Health Department Response
Texas distributed information to health departments and providers
Outlined study results
Recommended:
Separate vaccine handling areas
Decontaminate clinics
Use new gloves for NP swabs
Do not handle swabs below “break” point
CDC issued Best Practices for PCR for Pertussis

16. Implications of False + Pertussis
Missed school, work days
Unnecessary antibiotic treatment for cases AND contacts
Increased work for health departments and schools
Can create “outbreak,” worry
Clinical picture of pertussis distorted
True burden of pertussis becomes unknown

17. Questions? Rachel Wiseman, MPH Rachel.Wiseman@dshs.state.tx.us
(512) ext. 2632