Can Comprehensive Respiratory Pathogen Panels Be Used to Exclude Pertussis Infection? by Colleen Mayhew, MD
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This study examined the ability of comprehensive respiratory pathogen panels (RPAN) to identify cases of pertussis compared to a dedicated pertussis PCR test. The study found that of 102 samples that were positive for pertussis by PCR, the RPAN only identified pertussis in 57% of cases. Additionally, 44% of pertussis PCR-positive samples were negative by RPAN but still showed evidence of coinfection with other respiratory pathogens. The results suggest that solely using an RPAN to evaluate for pertussis infection may miss a significant number of cases.
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Can Comprehensive Respiratory Pathogen Panels Be Used to Exclude Pertussis Infection? by Colleen Mayhew, MD
- 1. Can Comprehensive Respiratory Pathogen Panels Be Used to Exclude Pertussis Infection? Colleen Mayhew, MD Barsan Research Day 2018 Mentor: Allison Cator, MD PhD Co-Investigators: Duane Newton, PhD and James Cranford, PhD Department of Emergency Medicine
- 2. Department of Emergency Disclosures No conflicts of interest to disclose
- 3. Department of Emergency Pertussis Testing Options 1. Dedicated Pertussis PCR 2. Comprehensive Respiratory Pathogen Panel (RPAN) PCR Background
- 4. Department of Emergency Dedicated Pertussis PCR identifies 1/3 more cases than RPAN High rate of coinfection with other pathogens during Pertussis High rate of viral detection in asymptomatic children Background
- 5. Department of Emergency Determine number of cases of Pertussis missed on RPAN Determine rate of coinfection in Pertussis cases missed on RPAN Determine rate of coinfection in all Pertussis PCR positive patients Objectives
- 6. Department of Emergency Microbiology laboratory performed RPAN on all saved Pertussis PCR+ swabs from 3/2015-10/2017 Determined rate of Pertussis identification on RPAN Tabulated presence of additional specific pathogens on RPAN Descriptive statistics were used to analyze demographic and clinical information Methods
- 7. Department of Emergency Results Department of Emergency Medicine
- 8. Department of Emergency Total Pertussis PCR Tests 3/2015-10/2017 3,489 Total Pertussis PCR+ Samples 137 (4%) Samples Available for Testing 102 (74%) Samples Not Saved 35 (26%) Study Cohort
- 9. Department of Emergency Total Pertussis PCR Tests 3/2015-10/2017 3,489 Total Pertussis PCR+ Samples 137 (4%) Samples Available for Testing 102 (74%) Study Cohort
- 10. Department of Emergency Total Pertussis PCR Tests 3/2015-10/2017 3,489 Total Pertussis PCR+ Samples 137 (4%) Samples Available for Testing 102 (74%) Samples Not Saved 35 (26%) Study Cohort
- 11. Department of Emergency Total Pertussis PCR Tests 3/2015-10/2017 3,489 Total Pertussis PCR+ Samples 137 (4%) Samples Available for Testing 102 (74%) Samples Not Saved 35 (26%) Study Cohort
- 12. Department of Emergency Demographics n % Age < 6 months 6 6% 6 - 23 months 9 9% 2-5 years 14 14% 6-11 years 25 25% 12-17 years 32 31% 18-24 years 11 11% > 25 years 5 5% Age Range: 1 month – 73 years Median age: 10 years
- 13. Department of Emergency Demographics n % Testing Location Inpatient 1 1% Ambulatory 91 89% Emergency 10 10% Season Fall 20 20% Winter 21 21% Spring 30 29% Summer 31 30%
- 14. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 15. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 16. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 17. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 18. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 19. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results Adenovirus, Coronavirus, HMPV, Parainfluenza, Rhino/enterovirus, RSV
- 20. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 21. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 22. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 23. Department of Emergency Pertussis PCR+ 102 RPAN Pertussis+ 57 (56%) RPAN Other+ 23 (40%) RPAN Other- 34 (60%) RPAN Pertussis- 45 (44%) RPAN Other+ 20 (44%) RPAN Other- 25 (56%) Results
- 24. Department of Emergency RPAN of All Pertussis PCR+ Patients Pertussis 56 (56%) Rhino/entero 24 (24%) Coronavirus 7 (7%) RSV 6 (6%) Adenovirus 5 (5%) Paraflu 4 (4%) HMPV 2 (2%) Influenza 1 (1%)
- 25. Department of Emergency RPAN only identifies a proportion of pertussis cases Patients with pertussis often have other respiratory pathogens in their nares Co-infection Asymptomatic viral carriage Discussion
- 26. Department of Emergency Risk of incorrectly attributing a patient’s illness to another pathogen Public health impact Communicability of illness Decreased vaccination rates Morbidity and mortality Implications
- 27. Department of Emergency Detailed medical records were not available for half of patients Delayed reanalysis of samples may affect RPAN test results Limitations
- 28. Department of Emergency If concerned about Pertussis infection, use dedicated Pertussis PCR Bottom Line
- 29. Department of Emergency Quality Improvement initiative Survey clinicians on testing practices PDSA affect clinical decision making Cost analysis Prospective study Next Steps
- 30. Department of Emergency Questions? Department of Emergency Medicine
- 31. Department of Emergency Michelle Macy, MD Sarah Tomlinson, MD Acknowledgements
- 32. Department of Emergency Thank You! Department of Emergency Medicine
- 33. Department of Emergency Incidence of Pertussis
- 35. Department of Emergency https://www.cdc.gov/pertussis/clinical/features.html Clinical Course of Pertussis
- 36. Department of Emergency Charge to Patient Cost Pertussis PCR $420 Respiratory Pathogen Panel $703
- 37. Department of Emergency The Assays Department of Emergency Medicine VS Pertussis PCR Respiratory Pathogen Panel
- 38. Department of Emergency Number of cycles needed to detect Pertussis in the RPAN Pertussis- group was higher than in the RPAN Pertussis+ group Mean PCR Cycles RPAN Pertussis+ = 28.0 RPAN Pertussis- = 35.9 ▪Statistically significant p <0.05 Pertussis PCR v RPAN
- 39. Department of Emergency Pertussis PCR+ 10 Repeat Pertussis PCR+ 7 (70%) Repeat Pertussis PCR- 3 (30%) Validation
- 40. Department of Emergency Pertussis PCR+ 10 Repeat Pertussis PCR+ 7 (70%) Repeat Pertussis PCR- 3 (30%) Validation
- 41. Department of Emergency Validation Original Pertussis PCR CT Results Repeat Pertussis PCR CT Results RPAN Pertussis Results 1 26.1 27.4 Pos 2 27.7 25.6 Pos 3 29.3 27.4 Neg 4 29.6 33.3 Pos 5 31.2 32.1 Pos 6 37.6 Neg Neg 7 37.8 Neg Neg 8 38 Neg Neg 9 38.1 33.2 Neg 10 39.5 38.5 Neg
- 42. Department of Emergency Original Pertussis PCR CT Results Repeat Pertussis PCR CT Results RPAN Pertussis Results 1 26.1 27.4 Pos 2 27.7 25.6 Pos 3 29.3 27.4 Neg 4 29.6 33.3 Pos 5 31.2 32.1 Pos 6 37.6 Neg Neg 7 37.8 Neg Neg 8 38 Neg Neg 9 38.1 33.2 Neg 10 39.5 38.5 Neg Validation
- 43. Department of Emergency Original Pertussis PCR CT Results Repeat Pertussis PCR CT Results RPAN Pertussis Results 1 26.1 27.4 Pos 2 27.7 25.6 Pos 3 29.3 27.4 Neg 4 29.6 33.3 Pos 5 31.2 32.1 Pos 6 37.6 Neg Neg 7 37.8 Neg Neg 8 38 Neg Neg 9 38.1 33.2 Neg 10 39.5 38.5 Neg Validation
- 44. Department of Emergency Original Pertussis PCR CT Results Repeat Pertussis PCR CT Results RPAN Pertussis Results 1 26.1 27.4 Pos 2 27.7 25.6 Pos 3 29.3 27.4 Neg 4 29.6 33.3 Pos 5 31.2 32.1 Pos 6 37.6 Neg Neg 7 37.8 Neg Neg 8 38 Neg Neg 9 38.1 33.2 Neg 10 39.5 38.5 Neg Validation
Editor's Notes
- There are two common testing methodologies used to diagnose pertussis infection: Dedicated Pertussis PCR (B pertussis and parapertussis) Comprehensive Respiratory Pathogen Panel (RPAN- multiple viral and bacterial targets including Bordetella pertussis) So… which test should you choose?? -Anecdotally, many clinicians use RPAN testing rather than Pertussis PCR testing to diagnose pertussis But first …. let’s look at the literature…
- While NOT common knowledge… -Jerris et al.. in 2015 compared these two testing methodologies and determined that the dedicated Pertussis PCR assay identifies up to 1/3 more cases of Pertussis than RPAN testing HOWEVER… this particular study was limited to the pediatric population and did NOT look at other pathogens identifiable on RPAN testing In addition to that… -Many studies in the pediatric population have documented a high rate of coinfection during Pertussis infection -Other studies have documented a high rate of viral detection in asymptomatic individuals --Specifically a 2015 viral surveillance study out of Utah found that 44% of the time a virus was detected, patients were asymptomatic Therefore there is a risk of incorrect attribution of symptoms to other pathogens if RPAN is used to assess for pertussis Byrington et al. Community Surveillance of Respiratory Viruses Among Families in Utah Better Identification of Germs Longitudinal Viral Epidemiology Study (BIG-LoVE)
- Therefore, the objectives of my study were:
- Microbiology laboratory performed comprehensive respiratory pathogen panels (RPAN) on all banked pertussis PCR positive swabs 3/2015-10/2017 from inpatient, ambulatory and emergency department settings From there…. The rate of pertussis identification on RPAN was determined The presence of additional specific pathogens was tabulated Descriptive statistics were used to analyze demographic and clinical information
- Approximately 3500 patients were tested for pertussis between 3/2015 and 10/2017
- Of those 137 were positive for pertussis. This is approx. 4% of samples.
- Of the positive Pertussis PCR samples, 102 samples were saved and available for testing
- This was the cohort used in my study
- The ages of patients with pertussis infection ranged from 1 mo to 73 years with a median age of 10 years -We are identifying pertussis in both children and adults --Keep it on your DDx
- At UofM, Pertussis identification occurred predominately in ambulatory clinics. Additionally… It is known that Pertussis infection has no distinct seasonal pattern. As expected, Pertussis was diagnosed in all seasons.
- A total of 102 banked pertussis PCR + samples were tested by RPAN
- The RPAN was negative for pertussis in 44% of cases These are the potential missed pertussis cases if RPAN was used alone for diagnosis
- The RPAN was negative for pertussis in 44% of cases These are the potential missed pertussis cases if RPAN was used alone for diagnosis
- More importantly, The RPAN was negative for pertussis, but positive for other pathogens in 44% of samples This can lead to potential misdiagnosis
- More importantly, The RPAN was negative for pertussis, but positive for other pathogens in 44% of samples This can lead to potential misdiagnosis
- Specifically the pathogens found on RPAN in this group were adenovirus, coronavirus, human metapneumovirus, parainfluenza virus, rhino/enterovirus, RSV
- What about the rate of co-infection in ALL pertussis PCR+ patients?
- What about the rate of co-infection in ALL pertussis PCR+ patients?
- In the following slides we will look at the specific RPAN results for the entire cohort of Dedicated Pertussis PCR+ Patients
- RPAN pertussis+ patients will be represented in yellow RPAN pertussis- patients will be represented in grey
- This heat map displays the RPAN results of ALL Pertussis PCR Positive Patients -Each row represents an individual patient The fist column demonstrates the RPAN results for Pertussis -The yellow shading indicates the 56% of patients that were ALSO Pertussis Positive on RPAN -The grey shading indicates the 44% of patients for which RPAN Pertussis testing was negative, the potential missed cases of Pertussis Looking across each row, you can see the additional pathogens identified in BLACK -The most common additional pathogen identified was rhino/entero virus 24% Interestingly, the distribution of additional pathogens is similar in the RPAN pertussis+ and RPAN pertussis – groups -Statistically speaking, testing positive for other pathogens on RPAN is equally likely in both of these groups. -(2 (1) = 0.2, p = .68) With this information in mind… should RPAN be used to diagnose pertussis??
- NO!! As we saw … RPAN only identifies a proportion of pertussis infections -In this cohort, RPAN does no better than a coin flip in detecting pertussis Furthermore… the presence of other pathogens cannot be used to exclude pertussis -There was no statistically significant difference in the rate of additional pathogen detection between RPAN pertussis + and RPAN pertussis - groups Those ADDITIONAL pathogens may represent co-infection OR asymptomatic viral carriage -But, it is impossible to differentiate between the two
- Therefore, if RPAN is used for pertussis diagnosis rather than dedicated pertussis PCR, there is a risk of incorrectly attributing a patient’s illness to another pathogen This has serious public health implications given communicability of illness, decreased vaccination rates decreased heard immunity, & morbidity and mortality especially in infants
- There were a few limitations to my study… Detailed medical records were not available for half of the patients. -Their ambulatory clinic’s records are not on MiChart. -Therefore we were unable to correlate patient’s test results with signs/symptoms/laboratory work up/disposition Delayed re-analysis of samples may affect RPAN test results -However, DNA should NOT significantly degrade over time -Validation testing through repeat dedicated Pertussis PCR testing is being completed -Among 10% of samples already tested, there does not appear to be any statistically significant degradation -Validation testing of remaining samples currently underway
- Therefore… If concerned about Pertussis infection, use dedicated Pertussis PCR Only use RPAN if identification of other specific targets will change your clinical management -Such as mycoplasma or flu -All others have no specific treatment
- So… the next steps include a QI initiative to ensure clinicians are ordering the right test for their specific clinical question The first step is to survey clinicians on their current testing practices From there… begin a PDSA cycle to work to influence testing practices -Education -Clinical support tools in the EMR Additionally -Cost analysis -Prospective study
- I would also like to thank Dr. Macy and Dr. Tomlinson for their help with this project
- Pertussis incidence over time CDC - National Notifiable Diseases Surveillance System
- Pertussis incidence by age group
- Pertussis PCR has optimal sensitivity during the first 3 weeks of paroxysmal cough onset when bacterial DNA is still present in the nasopharynx. After the fourth week of cough, the amount of bacterial DNA rapidly diminishes, which increases the risk of obtaining falsely-negative results.
- Cost charged to “out of pocket” patients
- Reason is rooted in basic science Pertussis PCR assay detects an insertion sequence present in many copies (IS 481) -More sensitive -However, also found in Bordetella Holmesii genome RPAN assay for pertussis detects a single gene (Pertussis toxin) -More specific -Problem = Cases of pertussis could me missed
- Ct = Cycle threshold = Number of PCR cycles needed to detect a pathogen Lower numbers = more concentrated sample Higher numbers = more dilute sample Above 40 = negative test In this study, the number of cycles needed to detect Pertussis in the RPAN Pertussis – group was higher than in the RPAN Pertussis + group This was statistically significant In other words, the samples with high Cycle Numbers on original Pertussis PCR testing (more dilute samples) are more likely to test negative for pertussis on RPAN (likely due to sampling error and inherent decreased sensitivity of RPAN compared to Pertussis PCR) The samples with low Cycle Numbers (high concentration of pathogen) on original Pertussis PCR testing are more likely to test positive for pertussis on RPAN
- Repeat Pertussis PCR was preformed on 10% of samples for validation purposes Pertussis was confirmed in 7/10 of samples, but ….
- Repeat dedicated PCR was negative in 3/10 of cases Possibilities include: Specimen degradation Sampling error / low level of target False positives
- This is a chart is organized by Ct results
- Degradation less likely given original and repeat Ct are similar -Among 10% of samples there is NO statistically significant difference between original and repeat Ct values using a paired t-test. -The mean scores were virtually identical, M = 33.5 vs. M = 33.7, t (9) = -0.31, p = 0.76. -Complete validation testing is currently underway
- The samples that were negative for pertussis on repeat testing all had higher original Ct’s This indicates a lower concentration of Pertussis DNA Therefore, this is likely related to sampling error
- Samples with lower Ct = higher concentration of pertussis were more likely to have Positive RPANs (higher specificity) Unable to determine false positives from this data set because no specific testing for the B homesii was completed