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)

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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

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Reported Pertussis Cases in the United States,
1922-2005
30,000
gt18 yrs
20,000
11-18 yrs
10,000
lt11 yrs
0
Number of cases
1990
1995
2000
2005
DTP
1922
1930
1940
1950
1960
1970
1980
1990
2000
Year
1950-2005, National Notifiable Diseases
Surveillance System1922-1949, Passive Reports
to the Public Health Service.
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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

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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

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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

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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

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Culture

• Very specific (100)
• Sensitivity varies
• High for young unvaccinated infants with short
  duration of symptoms
• Low (lt10) 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

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PCR

• Included in the CDC/CSTE case definition in 1997
• Primary diagnostic test in many laboratories
  (IS481 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)

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Potential Problems with IS481 PCR Assay

• IS481 is found
• In 50 to gt200 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

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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 (gt11yrs 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

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Positive Pertussis LaboratoryTests 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

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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 lt1 in 1995 to 25 in
  2005
• Serologic testing for confirmation of pertussis
  is increasing despite the lack of FDA-licensed
  test
• Particularly adults gt 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

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CDC PCR assaystwo target approach

• Target sequences
• IS481 insertion sequence, 50 to gt200 copies per
  cell
• Pertussis toxin subunit 1 (ptxS1), single copy
  gene

Species IS481 ptxS 1
Bordetella pertussis (CT lt35)
Bordetella parapertussis -
Bordetella holmesii (CT lt35) -
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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

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Clinical Validation Study

• Estimate the clinical sensitivity, specificity
  and predictive values of the following diagnostic
  tests
• CDC/FDA anti-pertussis toxin IgG serologic ELISA
• CDCs combined real-time PCR
• IS481 and ptxS1
• Boston Medical Centers anti-pertussis toxin
  secretory IgA
• Assess clinical usefulness as related to
• Age of patient
• Stage of disease
• Vaccination status
• Prior antibiotic therapy

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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

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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