Pertussis Presentation

Pertussis: Old Disease, 

New Dilemmas 

Amy L. Leber, PhD, D(ABMM) 

Nationwide Children’s Hospital 

The Ohio State University College of 

Medicine and Public Health 

• Pathology and 

epidemiology of 

Bordetella infections 

• Current laboratory 

testing for Bordetella 

infections 

Objectives: 

• Methods for improving 

diagnosis and control 

of disease 

Bordetella species 

• Family Alcaligenaceae 

• Small gram-negative coccobacillus 

• Do not utilize carbohydrates, inert 

biochemically 

• Obligate aerobes, optimal growth at 35- 

37 C 

• Respiratory tract of animals 

• 10 species (currently) 

Bordetella species 

• B. pertussis – agent of whooping cough 

• B. parapertussis – pathogen in both humans and 

sheep, milder form of whooping cough 

• B. bronchiseptica – respiratory pathogen in many 

animals, including dogs, pigs, rabbits and cats, but 

also cause pneumonia and bacteremia in humans 

• B. holmesii - blood cultures, young adults with 

underlying disorders, asplenic patients. Isolated 

from sputum and may colonize the respiratory tract 

B. pertussis Epidemiology 

• Humans only 

reservoir 

• Transmission occurs 

via respiratory 

droplets 

• Very Contagious 

• Epidemic cycles 

every 3-5 years 

Bordetella pertussis - Pathogenesis 

• Attachment to host cells 

– Filamentous hemagglutinin 

– Pertactin 

– Fimbriae 

• Virulence factors damage 

host tissue 

– Pertussis toxin 

– Adenylate Cyclase 

– Dermonecrotic toxin 

– Tracheal cytotoxin 

• Does not disseminated 

Amy L. Leber, PhD 

IL Communicable Dis Conference 2012 

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Epidemiology 

• Catarrhal -runny nose, sneezing, low fever, and a mild cough; similar to 

a common cold and gradually become more severe. 

• Paroxysmal - bursts or paroxysms of numerous, rapid coughs. These 

coughing episodes seem to be due to a difficulty in expelling mucus from 

the tracheobronchial tree; end with a long inspiratory effort accompanied 

by the high pitched whoop from which the disease gets its name. 

• Convalescent – gradual recovery but cough may persist for months 

Clinical Case 

• 8 - week old female was seen in the ER 

with 2 week history of paroxysmal 

cough, progressing to her gasping for 

breath and vomiting. 

• Clear chest X-ray, pulse 160, respiration 

70, fever of 38.0 o C. WBC 16,000/mm 3 

with 70% lymphocytes. 

Classic Presentation 

Clinical Case 

15 yo female presents to 

her pediatrician with 

history of coughing fits 

for 4 weeks duration 

interfering with sleep. 

No fever, whoop or 

posttussive vomiting. 

Common Presentation 

Pertussis 

Control of Pertussis 

“A disease of 

adolescents and adults 

that can kill infants.” 

– 90% of deaths from 

pertussis in infants 

• Antimicrobial 

Treatment 

• Infection Control 

Measures 

• Vaccination 

Aust Fam Physician. 2007 Jan-Feb;36(1-2):51-6 



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Pertussis Vaccine 

Childhood Vaccination: 

• 1940s – whole cell 

vaccine introduced (DTP) 

• 1991 – acellular DTaP 

vaccine approved for 4 th 

and 5 th dose 

• 1996 – DTaP approved 

for complete series of 5 

shots. 

http://www.cdc.gov/vaccines/schedules/downloads/child/0-18yrs-pocket-pr.pdf 

Waning Immunity 

Protection following: 

Immunization in Adolescents 

• Natural Infection 10-15 years 

• Vaccination* 

5-10 years? 

*(full childhood series) 

Case Definitions 

Clinical Diagnosis Complications 

• Heterogeneity of disease expression 

• Level of immunity at the time of 

exposure 

• Modification of disease by immunization 

• Mixed infections and infections with other 

Bordetella sp. 

• Low index of suspicion by physician 



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Laboratory Diagnosis 

• Culture – Gold Standard 

• DFA 

• Serology 

• NAAT 

Specimen Collection 

• Nasopharyngeal 

specimen 

– Want ciliated epithelial cells 

– Timing critical 

• NP aspirates, washes, or 

swabs are acceptable 

– Throat swabs are not 

acceptable 

http://www.copanusa.com/downloads/education/ 

Culture 

• Sensitivity 30 – 60% 

• Specificity 100% 

• Factors effecting sensitivity 

– Type and quality of specimen 

– Time specimen obtained in 

the course of illness 

– Appropriate transport 

– Choice of culture media 

– Length of time cultures 

incubate 

Direct Fluorescent Antibody 

• Sensitivity (18-78%) 

• Specificity (56-93%) 

• Advantage 

– More rapid than culture 

• Technically challenging 

• Because of its limitations, CDC does not 

recommend use of DFA testing. 

Serology 

• No FDA-approved diagnostic tests 

• Paired sera are desirable for best results 

• Antigens used: PT, FHA, Pertactin 

• No standard criteria 

• Antibodies elicited by infection and 

vaccination, complicating interpretation. 

• CDC – not relied on for case confirmation 

Timing of Serologic Testing 



4

IS Ct 

PCR 

• Majority of Labs using LDA real-time PCR 

• Each laboratory must validate assay 

– PCR +, Culture – 

– Imperfect Gold Standard 

• Conditions and Reagents vary 

• Sample Type and Collection 

Method vary 

PCR in Adolescents/Adults 

Clinical Infectious Diseases 2007; 44: 1216-1219 

Targets for PCR 

• Toxin Gene Promoter (PtxnP) 

– single copy gene 

– specific for B. pertussis 

• Insertion Sequence 481 (IS481) 

– Multicopy gene 

– B. pertussis 50-200 copies/cell 

– B. holmseii 10 copies/cell 

– B. bronchiseptica 

• Insertion Sequence 1001 (IS1001) 

– B. parapertussis 

– 35-50 copies/cell 

PCR at Nationwide CH 

• Testing since 1993, evolution of methods 

• Currently offering Real Time PCR 

– Target: IS 481 for B. pertussis 

IS 1001 for B. parapertussis 

– Extraction: MagNA Pure Compact 

– Amplification: ABI 7500 

– Specimen: 2 NP swabs transported in casamino 

acids 

– Cut-off for positive results Ct ≤ 37 

Real Time PCR 

• Add slide on how to interpret Real time 

PCR 

Crossing 

threshold (Ct) 

40 

35 

30 

25 

20 

15 

PCR Detection of B. pertussis: 

Toxin Promoter vs IS 481 Target 

(Single vs Multi copy) 

Tox Ct -vs- IS Ct 

y = 0.9367x - 5.2046 

R 2 = 0.8317 

10 

10 15 20 25 30 35 40 45 

TOX Ct 



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Setting a Cut-Off value for IS481 PCR 

• High Ct values may represent low 

bacterial load 

– Ct values >35 correlate with 90% outpatients 

We noticed an increase in positivity from Client A. 

• Looked at data from that office only. 

# Tested 

# Pos 

(Ct≤37) 

% Pos 

(Ct≤37) 

# neg w/Ct 

(>37) 

% neg w/Ct 

(>37) 

126 65 51.6% 55 43.7% 

Pediatric Infectious Disease Journal Vol. 13, 

1994 

• Environmental 

monitoring 

performed 

• B pertussis DNA 

detected with PCR 

• Whole cell vaccine 



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Pediatric Infectious Disease Journal Vol. 27, 

2008 

Initial Environmental and Personnel Testing at Site A 

Site A Environmental Samples 

Dr K, Self-Collected 

Dr G, Self-Collected 

Bp IS481 PCR 

Ct 

Neg 

Neg 

Nurse, 1 38.1 

Nurse, 2 39.1 

Nurse, 3 39.3 

Nursing Station: 

Area Near Fridge And Fridge Handle 

(Vaccine And Bp Collection Kit Storage) 

31.7 

Nursing Station - Computers/Phones 34.8 

POC Testing Area 34.8 

Exam Room 2 37.0 




Break Room 36.3 

Lobby, Sick Kids Side 

Uninoculated Collection Kit From Fridge 

Neg 

Neg 

Pediatric Office: Common Practices 

Contamination of Environment with Target 

Vaccine Prep Area 

Ct values 21-25 

Collection Kits 

Pentacel 

• Vial 1 – liquid DTaP–IPV 

• Vial 2 – Lyophilized Hib 

1 2 

Detection of Bordetella pertussis DNA in Acellular Vaccines and in Environmental Samples from 

Pediatric Physician Offices A Leber, et al.. 2010. Interscience Conference in Antimicrobial Agents 

and Chemotherapeutics (ICAAC), Boston, MA. 



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Patient Results vs. Vaccines Used 

on Site 

Environmental Contamination 







How to Mitigate Environmental Contamination 

• Test only symptomatic 

patients 

• Separate sample collection 

from vaccine administration 

• Wear gloves 

• Consider different sample 

type or transport (dry 

transport for NP swabs)* 

• Clean* 

Practice A –Effect of Cleaning on IS481 DNA in 

Environment 10% Bleach following by Isopropyl Alcohol 

Location of Wipe Testing 

Pre-clean 

Postclean 

Fridge with vaccine and counter 33.7 39.2 

Exam Room 6, exam table 37.6 neg 

Exam Room 6, door handle/light 

switch 

33.8 38.9 

Exam Room 6 sind area /scales 36.6 neg 

Break Room 36.2 neg 

http://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html 

Leber et al., Unpublished data. 

Increasing Incidence 

Increasing Incidence in Adolescents 

• Decreased vaccination due to concerns 

• Waning adolescent and adult immunity 

• Lower protection from acellular vaccines 

• Increased awareness by physicians 

• More sensitive laboratory testing 

• Strain Variability 

• Other Bordetella Species 



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Increasing Incidence in Adolescents 

Booster Vaccine 

• 2005 –Tdap 

approved for 

Adolescent Booster 

• 2006 – Adult Booster 

FIGURE 3. Incidence of confirmed and probable pertussis among 

persons aged ≤19 years, by patient age and vaccines received* — 

National Notifiable Diseases Surveillance System, United States, 

January 1–June 14, 2012 

Other Bordetella Species? 

Bordetella parapertussis 

• 1-5% of pertussis patients 

• Same virulence factors except 

Pertussis toxin 

• Little cross-immunity between B. 

pertussis and parapertussis 

MMWR: July 20, 2012 / 61(28);517-522 

Immunization is not protective for B. 

parapertussis. 

Not a reportable disease 

Other Bordetella species? 

Outbreak in Central Ohio 2010-2011 

Anatomy of an Outbreak 

Role of B. holmesii 

• B pertussis outbreak was confirmed 

– IS481 positive 

– BptxPr positive 

• B. holmesii was co-circulating 

– IS481 positive 

– IS1001-like positive 

• Isolated in culture 

• Serology (CDC) on limited subset showed: 

– Antibody to PT in B. pertussis infected 

– No antibody in B holmesii infected 

Unpublished data 



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Impact of Target Copy Number on Ability of 

PCR to Confirm IS481 Positives 

Interpretation of PCR Results: 

PCR Target 

(Copy #) 

IS481 

(Bpert 200; Bholm 10) 

Toxin Promoter 


IS-1001 like 


Unpublished data 

Organism and Mean Cts of 

IS481 Positives 

B pertussis 

B holmesii 

26.3 28.8 

34.5 Neg 

Neg 29.7 

IS481 

Target 

PtxPr 

IS1001 

like 

Interpretation 

+ + − B. pertussis 

+ − + B holmesii 

+ − − ??? 

Targeting IS481 vs Pertussis Toxin Promoter 

IS481 +/ Toxin promoter − / IS1001like − 

• True positive for B. pertussis but: 

– Unable to confirm due to relative insensitivity of PCR for 

single copy toxin promoter vs repetitive IS481 target 

• False positive for B. pertussis due to: 

– Contamination of PCR reactions with low level B 

pertussis DNA from environmental sources 

• vaccine DNA in office practice 

• amplicon DNA in lab 

• genomic DNA in either setting 

Laboratory Diagnosis of Pertussis 

Unanswered Questions: 

• Change the cutoff for IS481 PCR 

– Eliminate some of the “false” positives? 

• Routinely do Bptxp and Bholmseii PCR on all 

IS481 positives. 

– Report out B holmesii? 

• Change to a specific, single copy target 

• Incorporate serology into testing algorithms 

• Resurrect culture 

The Pertussis Dilemma: 

• “…remains endemic 

in the United States 

despite routine 

childhood vaccination 

for more than half a 

century and high 

coverage levels in 

children for more than 

a decade.” 

http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5503a1.htm 

Conclusions 

• Pertussis continues to be an endemic disease 

affecting infants and adolescents/adults. 

– Immunization of those with waning immunity is 

important 

• Laboratory diagnosis has improved 

– PCR being the new (but flawed) “gold standard”. 

– A well standardized, widely available serology is 

needed 

• Increasing incidence of disease is multifactorial. 

– Efficacy of vaccine for long term immunity 

– Role of B. holmesii and other species 

– Environmental contamination 



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Thank-you 

Questions? 

amy.leber@nationwidechildrens.org 



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Pertussis Presentation

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