Validity of Polymerase Chain Reaction Using Liquid Transport

Media During a Pertussis Outbreak

Cynthia Schulte, RN, BSNVPD Surveillance OfficerBureau of Immunization

New York State Department of Health (NYSDOH)

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

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 swab

Nasopharyngeal aspiration

PCR Transport Media

Liquid Solid

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

PCR and Ct Values — a Fictitious Example

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

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

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

11/1

/201

0

11/8

/201

0

11/1

5/20

10

11/2

2/20

10

11/2

9/20

10

12/6

/201

0

12/1

3/20

10

12/2

0/20

10

12/2

7/20

10

1/3/

2011

1/10

/201

1

1/17

/201

1

1/24

/201

1

1/31

/201

10

20

40

60

80

100

120

140

160

Specimen Collection Date

Sp

eci

men

Cou

nt Press re-

lease

Daily Specimen Collections for PCR

>2300 samples tested

542 positive (28%)

11/1

11/7

11/1

3

11/1

9

11/2

512

/112

/7

12/1

3

12/1

9

12/2

5

12/3

11/

61/

121/

180

10

20

30

40

50

60

70

80

90

Date Reported to County(3-day periods)

Rep

ort

Cou

nt

99% from 1 laboratory

Report Count by Date (N=542)

10/1

10/7

10/1

3

10/1

9

10/2

5

10/3

111

/6

11/1

2

11/1

8

11/2

4

11/3

012

/6

12/1

2

12/1

8

12/2

40

2

4

6

8

10

12

14

16

Cough Onset Date(3-day periods)

Case C

ou

nt

Median 11/11/2010

Epidemic Curve (N=103)

Percent PCR Positive Varied by Pediatric Practice

A B C D0%

10%

20%

30%

40%

50%

60%

Pediatric Practice

Perc

en

t of

Sam

ple

s P

CR

Pos-

itiv

e

Mean of four pediatric practices

Overall mean

Mean of all other

prac-tices

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

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

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

Factors Affecting Outbreak — Co-circulating Pathogens

Bordetella parapertussis (~2%)

Bordetella holmesii (~1%)

Mycoplasma pneumoniae (~13%, 2/16 tested) 1 hospitalization

Others?

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

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

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

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

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.

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.

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.

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

Thank You Angie Maxted, MS, DVM

Epidemic Intelligence Service OfficerBureau of Communicable Disease Control, NYSDOHEIS Field Assignments Branch, SEPDPO/OSELS, CDC

Elizabeth Rausch-Phung, MD, MPHMedical Director Bureau of Immunization, NYSDOH

Debra Blog, MD, MPHDirectorBureau of Immunization, NYSDOH