21/12/2018. Objectives. The Persistence of Pertussis: Infection and Diagnosis. Bordetella. Causes & Symptoms of Pertussis

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1 1 Objectives 2 The Persistence of Pertussis: Infection and Diagnosis Jules Chen R&D Senior Scientist DiaSorin Molecular LLC 1. Understand the causes and symptoms of pertussis 2. Explore the history of pertussis vaccination and waning immunity 3. Discuss laboratory testing options for diagnosis of pertussis and treatment 3 4 Causes & Symptoms of Pertussis MORPHOLOGY/BIOCHEMISTRY Small, aerobic coccobacilli belonging to a group of nutritionally fastidious organisms. Considered an extracellular pathogen but it can also survive within a variety of eukaryotic cell types including macrophages and epithelial cells. MICROSCOPIC APPEARANCE Gram-negative MACROSCOPIC APPEARANCE B. pertussis and B. parapertussisare small, shiny, round and mercury silver in color on Regan-Lowe Agar. With extended incubation, the colonies become whitish-gray. Source: Bailey & Scott's Diagnostic Microbiology Stages of Pertussis Infection 5 Complications of Pertussis Infection 6 Catarrhal Stage 7-10 day incubation period Non-specific symptoms Low-grade fever Sore throat, nasal congestion, malaise etc. Mild, progressive dry cough Paroxysmal Stage Classic whooping cough Post-tussivevomiting Profuse production of mucus Paroxysms occur at night and increase in frequency through weeks 2-3 Organism Burden Convalescent Stage Paroxysms decrease in frequency, duration and severity Mild cough may last up to 6 weeks Syncope temporary loss of consciousness or fainting Sleep disturbance Incontinence Rib fracture Complications among infants: o Pneumonia (22%) o Seizures (2%) o Encephalopathy (<0.5%) Death o Infants especially those who have not received primary vaccination series 1

2 Genus B. pertussis B. parapertussis B. holmesii 7 but only four of them cause respiratory disease in humans B. pertussis B. parapertussis (responsible for up to 20% of pertussislike disease) B. holmesii (infrequent cause of the disease) 8 B. bronchiseptica B. avium B. hinzii B. bronchiseptica (infrequent cause of the disease) 9 pertussis 10 B. pertussis Primary cause of whooping cough (60-70%) with high mortality rate in infants Produces the pertussis toxin (PTx) which may cause a delay in immune response Frequent occurrence of leukocytosis that may resemble leukemia ( 100,000 wbc/mm 3 ) Vaccine-conferred immunity wanes after 7 to 10 years 1. Leber, A.L. Clin Lab Med : B. parapertussis Causes up to 20% whooping cough with milder symptoms 1 Does not express pertussis toxin No vaccine available B. bronchiseptica Can cause respiratory infections in different mammals (e.g. pigs and dogs), rare in humans Older age, induced immunodeficiency, and pre-existing respiratory illnesses are the main risk factors Causes chronic and often asymptomatic respiratory tract infections Is highly resistant to cephalosporins B. holmesii Under-estimated in humans as it is rarely discriminated during diagnosis Mainly a cause of bacteremia and septicemia with some cases of whooping cough Presumed outbreaks in France and Ohio ( ) with 2 fold increased incidence in Spain ( ) Higher response to many common antibiotics Humans are the only hosts of B. pertussis Transmitted by droplets Virulence Factors are responsible for establishing initial infection: Tracheal cytotoxin(tct) Adenylate cyclase toxin Filamentous haemoglutinin(fha) Pertactin Fimbriae Pertussis Toxin (PT) Gram stain of the bacteria pertussis. Credit: CDC entry and multiplication B. pertussis and B. parapertussis enter the trachea and bronchi by inhalation. They attach to the cilia of epithelial cells. Pertussis is superficial infection the organisms remain on mucosal surface and do not invade the tissues. 11 Virulence factors of can be grouped into two major categories: Adhesins and Toxins Name Chemical Nature Site of Action Physiological Effects Pertussis toxin (B. pertussis only) Adenylate cyclase toxin Protein Local Protein Local and systemic Impairs neutrophil chemotaxis, phagocytosis Tracheal cytotoxin Murein Local Increases capillary permeability, hemolysis activity Cytopathologicaldamage to tracheal epithelia Endotoxin Lipopolysaccharide Systemic Fever Fimbriae Protein Local Filamentous hemagglutinin Protein Local Facilitating adherence to respiratory epithelium Adherence and modulation of innate immunity 12 Copyright 2004 Pearson Education, Inc. publishing as Benjamin Cummings Pertactin Protein Local Adherence 2

3 Adhesins& Toxins 13 Spread 14 Usually spread by coughing or sneezing while in close contact with others. Parents, older siblings or other caregivers can give whooping cough to babies without even knowing they have the disease. Image: Sanofi Pasteur Canada Treatment for pertussis infection 15 Resurgence of Pertussis 16 Antibiotic treatment and prophylaxis play an important role in controlling the spread of pertussis. CDC recommends azithromycin, clarithromycin, and erythromycin. Clinicians can also use Trimethoprimsulfamethoxasole. Eliminates the organism from the respiratory tract. Treat confirmed cases of pertussis and their close contacts. WHO estimates 50 million cases worldwide. 300,000 deaths every year. 4% fatality rate in developing countries. Pertussis by Age Group Pertussis is cyclical and peaks every 3-5 years. 17 Resurgence of Pertussis Changes in Pertussis Reporting by State From 2013 to 2014 *+ Pertussis is a nationally notifiable infectious condition in the U.S. 18 The overall incidence of pertussis has increased since the 1990s. Decrease Increase 18 states saw an increase in pertussis cases reported from 2013 to Infant incidence rate higher than other age groups. * Data for 2014 are provisional and subject to change. + Cases reported through Week 50 in 2013 were compared with cases reports through Week 53 in 2014 There were 17,972 reported pertussis cases in the United States in 2016, including six infant deaths. 3

4 Pertussis -Annual Epidemiological Report 2016 [2014 data] Number of reported pertussis cases per 100,000 population, EU/EEA Vaccination for Pertussis Pertussis vaccines and current recommendations Whole cell pertussis vaccine was introduced in 1940s = wp Acellular pertussis vaccine was introduced in 1996 = DTaP Vaccine recommendations: Infants and children: 5 doses of DTaP (2, 4, and 6 months, months, and 4 through 6 years of age) Adolescents: single dose of Tdap (11 to 12 years of age) Pregnant women: single dose of Tdapduring every pregnancy (27 36 weeks gestation) Adults: every 10 years 21 The pertussis vaccine and disease resurgence What are the theories? Reduced effectiveness of the acellular pertussis vaccine Waning immunity to pertussis over time Both whole-cell and acellular vaccines are effective at reducing disease severity but not transmission, resulting in outbreaks in vaccinated cohorts Lack of cross-protection against B. parapertussisand B. holmesii Unvaccinated populations 22 Working group meeting on pertussis 2013: Possible causes of pertussis resurgence Short-lived adaptive immunity following immunization Suboptimal balance of immune response Need for additional vaccine antigens for optimal protection Insufficient quantity or incorrect balance of antigens Antigen mismatch with circulating strains Lack of vaccine protection against transmission Suboptimal schedule or population coverage Effectiveness of the vaccines in current use Increased awareness, better diagnostic tests, more-complete reporting Burns et al., Pertussis Resurgence: Perspectives From the Working Group Meeting on Pertussis on the Causes, Possible Paths Forward, and Gaps in Our Knowledge. The Journal of Infectious Diseases, 209, Suppl_1, 1 April 2014, S32 S RECENT PUBLICATION The impact of past vaccination coverage and immunity on pertussis resurgence MatthieuDomenechde Cellès, Felicia M. G. Magpantay, Aaron A. King, Pejman Rohani, March 28, Science Translational Medicine Suggests 3 factors that explain the resurgence of whooping cough (based on mathematical analysis): 1. Natural population turnover 2. Incomplete vaccination coverage 3. Slowly waning protection from a highly effective yet imperfect vaccine 24 4

5 RECENT PUBLICATION continued Domenech de Cellès et al., RECENT PUBLICATION continued Domenech de Cellès et al., Phenomenon of the honeymoon period Study Conclusions & Perspectives Long-lived immunity derived from natural infections ending (elderly) Very low disease incidence following start of vaccination program in late 1940s Overall reduction in transmission Reduced the risk of natural infection during childhood to vaccinated and also to the general population (including those who avoided vaccineor for whom the vaccine was not as effective) Rise in the number of susceptible adults sets the stage for the pertussis resurgence, especially among adults The return of pertussis signals the end of the honeymoon period Current pertussis vaccines provide lifelong protection to 55 percent of people and protect 90 percent of people for more than a decade Patterns of pertussis incidence previously attributed to rapid vaccine waning are actually consistent with higher contact rates once children enter school Primary school children and teenagers are the "core transmission group" mainly responsible for spreading the disease. Efforts aimed at curtailing transmission in the population at large, and especially in vulnerable infants, are more likely to succeed if targeted at school children, rather than adults. 27 Testing Recommendations 28 There are two approaches to diagnosis: direct and indirect. Diagnosis of Pertussis Both the WHO and CDC provide recommendations on diagnostic testing based on timing of symptoms and patient presentation. J. Cherry, B. Seaton. Patterns of parapertussis Respiratory Illnesses: ClinInfect Dis. (2012) 54(4): Figure is adapted from WHO Culture 29 Serology 30 60% sensitive with highest recovery in infants and specimens collected within first 2-weeks of cough Useful to detect mutations and emerging antimicrobial resistance CDC Initiative to recover more B. pertussis isolates Media: Regan-Lowe or Bordet- Gengoumedia aerobically for 7 days at C Source: Not often used; requires acute and convalescent specimens Detecting specific anti-pt antibodies Cannot be used as a diagnosis during the year following vaccination Limited diagnostic value for treatment or prophylaxis decisions 5

6 Nucleic Acid Amplification Tests (NAATs) 31 Diagnosis of Infections 32 IS481: B. pertussis ( copies) o Present in B. holmesii(10-50 copies) o Present in 3% B. bronchiseptica cluster I strains IS1001: B. parapertussis(22 copies) o Present in 29% B. bronchiseptica cluster I strains his1001: B. holmesii(3-5 copies) IS1002: B. pertussis (<10 copies), B. parapertussis (<10 copies), B. bronchiseptica(1 copy) Pertussis Toxin (ptx): B. pertussis (1 copy) reca: B. holmseii(1 copy) Insertion sequence elements are the primary target for NAAT assays Optimal Timing for Diagnostic Testing (weeks) Method Specimen Test Window Turn-Around-Time Performance / Application Culture Nasopharyngeal Swab (NPS) 0-2 weeks 7-14 days Molecular Nasopharyngeal Swab (NPS) 0-4 weeks A few hours Serology Serum 2-8 weeks A few hours Centers for Disease Control and Prevention, Low sensitivity Confirm outbreak Timely results with improved sensitivity over culture Not for early diagnosis Vaccine causes positive results Challenges Using the Molecular Approach 1 Multicopy genes help to support diagnostic sensitivity Detect at <1 CFU, may detect colonization or carrier state Risk specificity due to presence of IS481 in B. pertussis and B. holmesii Mitigation: o Periodic surveillance of B. holmesii in population o Addition of second target for increased specificity Multicopy Genes IS481 IS1001 Interpretation POS NEG B. pertussis/holmesii NEG POS B. parapertussis 33 Challenges Using the Molecular Approach 2 Addition of pertussis toxin and recaincrease specificity for B. pertussis and B. holmesii Risks sensitivity at low organism burden Multicopy Genes Toxins IS481 IS1001 ptxp reca Interpretation POS NEG POS - B. pertussis NEG POS NEG NEG B. parapertussis POS NEG NEG POS B. holmesii POS NEG NEG NEG B. pertussis/holmesii 34 Pseudo-Outbreaks Some pertussis vaccines found to contain PCR-detectable B. pertussis DNA (2012) Environmental sampling identified B. pertussis DNA from the vaccines in clinic environment. Accidental transfer of the DNA from environmental surfaces to a clinical specimen can result in specimen contamination and falsepositive results. Simplexa Direct CDC -Best Practices for Health Care Professionals on the use of Polymerase Chain Reaction (PCR) for Diagnosing Pertussis Mandal et al Pediatrics Feb;129(2):e

7 38 Fast & Flexible Testing Simplexa Direct Kit (Cat. #: MOL2750) Intended use: on the LIAISON MDX instrument for the qualitative detection and differentiation of pertussisand parapertussis from nasopharyngeal swabs from patients with signs and symptoms of infection of the respiratory tract Direct Amplification Disc Multi-use disc, 8-wells Targets IS481 for B. pertussis IS1001 for B. parapertussis Direct Amplification Disc Multi-use disc, 8-wells Fast & Flexible Testing Simplexa Direct Kit (Cat. #: MOL2750) Sample: 50 µl of nasopharyngeal swabs (NPS) in transport medium Run as few as 1 and up to 8 samples at a time Time-to-result: about 60 min Moderate complexity Complete traceability easily track all needed information including sample information, product lot numbers, and QC data Specimen Handling and Storage Simplexa Direct Kit Simplexa (Cat.#: MOL2750) Direct Kit (Cat. MOL2750) Nasopharyngeal swab (NPS) samples stored in RemelM4, RemelM4RT, RemelM5, RemelM6, UTM, BD UVT and Liquid Amies(ESwab) transport media Specimens should be transported on ice and stored at 2 to 8 C for up to 7 days post collection. If there is a greater than 7 days delay before testing the specimen, store the specimen at -70 C Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing Simple and Easy Workflow Simplexa Direct Kit (Cat. #: MOL2750) Ready to use Single use reagents for no waste (24 reactions) No mixing, pouring or rehydrating reagents Simple and Easy workflow No extraction needed, direct sample input Direct Amplification Disc Multi-use disc, 8-wells 41 DAD Technology DAD with Sample and Reagent placed onto LIAISON MDX Instrument uses centrifugal force to meter correct amount of sample (Underside of the Disc) Laser opens Sample valve 42 Centrifugal force moves sample through channel to amplification chamber Fluid checks are performed to ensure sample addition and prevents false negatives if a sample is accidentally not loaded Liquid sensor to ensure adequate sample volume metered No precise pipetting required Hands on time:< 1 minute / sample Sample metering capabilities ensures correct sample inputs, no precision pipetting Direct amplification of sample no DNA/RNA extraction required 7

8 DAD Technology High heat and centrifugation lyse the cells Nucleic acid is released Laser opens reagent valve and centrifugation moves reagent to amplification chamber Thermal cycling initiates PCR process Direct amplification of sample no DNA/RNA extraction required Simplexa Direct Assay Interpretation Interpretation IS481 (FAM) IS1001 (CFR610) IC (Q670) pertussis Detected Not Detected NA* parapertussis Not Detected Detected NA* pertussis & parapertussis Detected Detected NA* Negative Not Detected Not Detected Detected Invalid Not Detected Not Detected Not Detected * NA = not applicable, valid result can be Detected or Not Detected. 45 Clinical Agreement Samples were prospectively collected from 6geographically diverse sites between , from patients with signs and symptoms of infections. 1,482 B. pertussis and 1,289 B. parapertussis results were evaluated by Simplexa and a reference method. 56 samples were contrived for B. parapertussis at various concentrations across the clinical range of the assay (2-50 X LoD) and randomized among 56 negatives for a total of 112 additional samples. Simplexa was compared to either a FDA cleared NAAT or a composite reference method consisting of two well-characterized real-time PCR assays followed by confirmation of positive PCR amplification products with bi-directional sequencing. 46 Composite Reference Method Algorithm SEQ1 SEQ2 Interpretation + + /- + + / Fresh Clinical Agreement Prospective Prevalence of Fresh Enrolled Simplexa Direct Specimens 47 Frozen Clinical Agreement Prospective Prevalence of Frozen Enrolled Simplexa Direct Specimens 48 Prevalence pertussis Overall 10.3% (47/457) parapertussis 1.1% (2/176) Prevalence pertussis Overall 7.2% (82/1134) parapertussis 1.5% (17/1134) Prospective Prevalence of Fresh Enrolled Simplexa Direct Specimens by Site Site pertussis % (6/60) 2 0.0% (0/0) 3 3.7% (4/108) % (17/71) % (17/167) 7 5.9% (3/51) parapertussis 1.3% (1/75) 0.0% (0/8) 0.0% (0/37) 0.0% (0/18) 3.8% (1/26) 0.0% (0/12) Prospective Prevalence of Frozen Enrolled Simplexa Direct Specimens by Site Site pertussis 1 9.4% (20/213) 2 5.9% (3/51) 3 7.9% (29/369) 5 3.3% (4/122) 6 6.9% (26/379) parapertussis 2.8% (6/213) 2.0% (1/51) 1.4% (5/369) 0.8% (1/122) 1.1% (4/379) 8

9 B. pertussis Clinical Agreement 49 B. parapertussis Clinical Agreement 50 Prospectively Collected Concordant Fresh Samples FDA cleared Detected Not Detected Total Simplexa NAAT Detected Not Detected Total %PPA 100.0%(36/36) 95% CI: 90.4% to 100.0% %NPA 97.9%(326/333) 95% CI: 95.7% to 99.0% Prospectively Collected Concordant Fresh Samples Sequencing Detected Not Detected Total Simplexa Detected Not Detected Total %PPA 100.0%(2/2) 95% CI: 34.2% to 100.0% %NPA 100.0%(174/174) 95% CI: 97.8% to 100.0% Prospectively Collected Concordant Frozen Samples Sequencing Detected Not Detected Total Simplexa Detected Not Detected Total %PPA 91.9%(68/74) 95% CI: 83.4% to 96.2% %NPA 98.7%(1026/1039) 95% CI: 97.9% to 99.3% Prospectively Collected Concordant Frozen Samples Sequencing Detected Not Detected Total Simplexa Detected Not Detected Total %PPA 100.0%(13/13) 95% CI: 77.2% to 100.0% %NPA 99.6%(1096/1100) 95% CI: 99.1% to 100.0% B. parapertussis Clinical Agreement 51 Reproducibility 52 Contrived Concordant Frozen Samples Sequencing Detected Not Detected Total Simplexa Detected Not Detected Total %PPA 100.0%(56/56) 95% CI: 93.6% to 100.0% %NPA 100.0%(56/56) 95% CI: 93.6% to 100.0% 56 samples were contrived for B. parapertussis at various concentrations across the clinical range of the assay (2-50 X LoD)and randomizedamong56 negatives foratotalof 112 additional samples Three investigative sites assessed the device's inter-site, interday and inter/intra-assay reproducibility. Sample Panel Member Total % 95% CI pertussis A639 LP (FAM) 100.0% (90/90) 95.9% to 100.0% pertussis A639 MP (FAM) 100.0% (90/90) 95.9% to 100.0% parapertussis A747 LP (CFR 610) 100.0% (90/90) 95.9% to 100.0% parapertussis A747 MP(CFR 610) 100.0% (90/90) 95.9% to 100.0% Native negative naso-pharyngeal swab (UTM) 100.0% (90/90) 95.9% to 100.0% Positive Control(FAM) 100.0% (90/90) 95.9% to 100.0% Positive Control(CFR 610) 100.0% (90/90) 95.9% to 100.0% Total Agreement low positive (LP) approximately 1-2 X LoD medium positive sample (MP) approximately 3-4 X LoD 100.0% (540/540) 99.3% to 100.0% High total percent agreement across three different sites performed across 5 different days, demonstrating robustness of the assay. Interfering Substances pertussis parapertussis (IS481) (IS1001) Potentially Interfering Substance Active Ingredient Concentration % Detection % Detection (# Detected/# Tested) (# Detected/ #Tested) Albuterol sulfate Albuterol sulfate 10 mg/ml 100% (3/3) 0% (0/3) Ampicillin powder Ampicillin 10 mg/ml 100% (3/3) 0% (0/3) Azithromycin powder Azithromycin 10 mg/ml 100% (3/3) 0% (0/3) Beclomethasone dipropionate Beclomethasone dipropionate 10 mg/ml 100% (3/3) 0% (0/3) Blood NA 10% v/v 100% (3/3) 0% (0/3) Chloraseptic sore throat spray Phenol 10% v/v 100% (3/3) 0% (0/3) Ciprofloxacin Ciprofloxacin 1.25 mg/ml 100% (3/3) 0% (0/3) Erythromycin Erythromycin 10 mg/ml 100% (3/3) 0% (0/3) Flonase Nasal Spray Fluticasone propionatecorticosteroid 10% v/v 100% (3/3) 0% (0/3) Mucin Mucin 10 mg/ml 100% (3/3) 0% (0/3) Mupirocin Mupirocin 10 mg/ml 100% (3/3) 0% (0/3) 2.5 mg/ml 100% (3/3) N/A Rifampicin Rifampicin 5 mg/ml N/A 100% (8/8) Robitussin DM Robitussin DM 10% v/v 100% (3/3) 0% (0/3) Saline Nasal spray-sodium chloride Sodium chloride 10% v/v 100% (3/3) 0% (0/3) Sudafed PE Phenylephrine 10 mg/ml 100% (3/3) 0% (0/3) Zicam 12 hrs spray OxymetazolineHCl 10% v/v 100% (3/3) 0% (0/3) 53 Limit of Detection (LoD) The Limit of Detection (LoD) was determined for the Simplexa Direct assay using quantified stocks of 2 strains of pertussis(a639 & BAA-589) and parapertussis (A747 & E595) serially diluted into native negative nasopharyngeal swab in Universal Transport Media (UTM). LoDis the lowest concentration that could be detected as positive > 95% of the time. species pertussis parapertussis strain LoDConcentration (CFU/mL) A BAA A E

10 Summary Pertussis or whooping cough is a highly contagious disease of the respiratory system caused by pertussis and parapertussis. There are different opinions on the root cause of the resurgence of pertussis including the new theory of the end of the honeymoon period. Simplexa Direct detects and differentiates B. pertussis and B. parapertussisdirectly from NP swabs without extraction in about an hour