Comparison of Bordetella species identification among differing rt-PCR assays in the United States

ABSTRACT In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012–2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC’s assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC’s assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.


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Reviewer #1 (Comments for the Author): Commercial Bordetella assays are typically unable to differentiate between B. pertussis/B.holmesii (IS481) and B. parapertussis/B.bronchiseptica (pIS1001).To examine the distribution of Bordetella species in clinical specimens, a total of 3,984 positive Bordetella specimens collected over 7 years (2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019) were retested at the CDC using species-specific targets that allow differentiation among the various Bordetella species.The CDC assay confirmed 85% of results produced by two large commercial laboratories.Although a rather straightforward study, it included a large sample set and confirms the specificity of common laboratory tests/methods that use IS481 and pIS1001 for detecting Bordetella species.Also, the small amount of B.
holmesii (1.4% of samples) detected indicates that the prevalence of B. holmesii is low in the US and detection of IS481 by laboratory tests is primarily associated with B. pertussis infection.
Major comments: IS481 has been reported in the literature to also be found in Bordetella bronchiseptica.However, only one potential B. bronchiseptica was found in this dataset for IS481 positive specimens.This should be mentioned in the discussion along with possible explanations for the discrepancy.
Reviewer #2 (Comments for the Author): Cole et al. performed a molecular study evaluating the performance of 3 different molecular assays for detection of Bordetella (and subsequent species).The team exhaustively tested >3900 samples that were previously positive for Bordetella and showed that overall agreement in Bordetella species detection and identification was 85%.Most discrepant results were reported as indeterminates by the CDC assay and only a small portion of B. holmesii was identified from previously tested samples in the commercial labs.The Ct values among the assays were also evaluated which showed variable concordances.Overall, their findings suggest that commercial labs produce accurate Bordetella results with minor differences.
Major comments: 1. Are there any studies (new data, or previously published) regarding the limit of detection of these three assays, especially the assay at the CDC.Since most of the discrepant results tend to be indeterminates, I wonder if this is a sensitivity/limit of detection issue, rather than false-positive from one assay?If the lower limit of detection is not the same among the assays, it is very hard to judge these weak positives.As mentioned in line 268-270, all those factors can affect assay performance and it is a concern of this paper that those factors are mainly at play.2.Similar to the above comments, line 226, the Ct difference between two assays is ~3 which is typically around 1 log, a rather big difference (10X nucleic acid).Similarly, the mean is also on the end of the spectrum so once again, the performance/limit of detection of these assays are important.This help answers what is the purpose of comparing the Ct values (figure 1)? 3. Figure 1.Please elaborate clearly in the text the purpose of figure 1 and the studies.Please describe the findings in each panel (from A-F).What are the implications?The r ranges from 0.63-0.82.However, if the performance of these assays are not comparable then comparing Ct values don't offer much value.4. While the results were written clearly in the text, as one tries to look at the numbers in the tables and read the text, it gets really confusing.Most of the numbers (%s) written in the Results text body cannot be found on the tables.Please add either an overall or total column to depict those exact numbers.It is hard for the reader to compare the table and text together given how the paper is written now.(For example line 203, it says B. holmesii was detected in 1.5% of specimens, but there is not even a 1.5% anywhere in table 2).Please ensure the values you included in the Results body can be easily found in all of tables 1-3. 5. Please include either Supplemental table 1 into the main body or include most of the interpretative criteria (such as Ct cut-offs) in the text.This information is useful to understand the Ct values and also, potential performance differences.
Minor comments 1. Figure 1  Thank you for your review and comments on our manuscript.We have addressed the comments and responded to the questions from the reviewers in the revised version of the manuscript attached in this submission.We have included both a marked-up and clean version of the manuscript for your reference.We have also included the responses to the reviewers' comments and edits to the manuscripts below.The responses are italicized while the edits are italicized and underlined.
Please do not hesitate to contact us if you require any more information from us.Thank you for your consideration and for an opportunity to publish our work with Microbiology Spectrum.

Sincerely, Matthew Cole
Responses to reviewers o Major comments ▪ IS481 has been reported in the literature to also be found in Bordetella bronchiseptica.However, only one potential B. bronchiseptica was found in this dataset for IS481 positive specimens.This should be mentioned in the discussion along with possible explanations for the discrepancy.o Cole et al. performed a molecular study evaluating the performance of 3 different molecular assays for detection of Bordetella (and subsequent species).The team exhaustively tested >3900 samples that were previously positive for Bordetella and showed that overall agreement in Bordetella species detection and identification was 85%.Most discrepant results were reported as indeterminates by the CDC assay and only a small portion of B. holmesii was identified from previously tested samples in the commercial labs.The Ct values among the assays were also evaluated which showed variable concordances.Overall, their findings suggest that commercial labs produce accurate Bordetella results with minor differences.o Major comments: ▪ Are there any studies (new data, or previously published) regarding the limit of detection of these three assays, especially the assay at the CDC.Since most of the discrepant results tend to be indeterminates, I wonder if this is a sensitivity/limit of detection issue, rather than false-positive from one assay?If the lower limit of detection is not the same among the assays, it is very hard to judge these weak positives.As mentioned in line 268-270, all those factors can affect assay performance and it is a concern of this paper that those factors are mainly at play.
• Agreed, this is a limitation of the study, especially since we retested specimens.We did not perform a direct comparison of assays to determine relative lower limits of detection.While we do not know the lower limit of detection for the commercial assays, ours are very similar to the original publication, Tatti et al., 2011(doi: 10.1128/JCM.00601-11),ranging from <1 genome copy for IS481 to 1 genome copy for ptxS1 per PCR reaction.Despite that limitation, the highly correlated Ct values between assays suggests highly similar results.The CDC assay cut-off for IS481 at Ct < 35 and indeterminate range of 35 -<40 is important for our surveillance work and may not reflect the interpretation cut-offs for other assays.• Lines 304-306: To determine lower limit of detection among PCR assays, a side by side comparison would be needed, rather than retesting frozen specimens, as was done here.▪ Similar to the above comments, line 226, the Ct difference between two assays is ~3 which is typically around 1 log, a rather big difference (10X nucleic acid).
Similarly, the mean is also on the end of the spectrum so once again, the performance/limit of detection of these assays are important.This help answers what is the purpose of comparing the Ct values (figure 1)?
• Recognizing that our Ct values in the upper range differed from the commercial assays, we felt it was important to see how well Ct values correlated across all samples, as shown in figure 1.Since we were retesting frozen specimens, some DNA degradation is expected, and any loss in nucleic acid would be more apparent in samples with low signal (high Ct value).▪ Figure 1.Please elaborate clearly in the text the purpose of figure 1 and the studies.Please describe the findings in each panel (from A-F).What are the implications?The r ranges from 0.63-0.82.However, if the performance of these assays are not comparable then comparing Ct values don't offer much value.
• Thank you for the comment; we have added the following text to the discussion in lines 269-273: o We compared C T values using r c to better describe the differences of Bordetella PCR testing across the performing laboratories (Figure 2).Our study data for both commercial laboratories combined showed good correlation in IS481 and pIS1001 values.Lower correlation observed with the combined commercial laboratory pIS1001 target (Figure 2F) compared to the IS481 target Figure 2C) can likely be explained by lower sample size of submitted BppBb specimens.▪ While the results were written clearly in the text, as one tries to look at the numbers in the tables and read the text, it gets really confusing.Most of the numbers (%s) written in the Results text body cannot be found on the tables.Please add either an overall or total column to depict those exact numbers.It is hard for the reader to compare the table and text together given how the paper is written now.(For example line 203, it says B. holmesii was detected in 1.5% of specimens, but there is not even a 1.5% anywhere in table 2).Please ensure the values you included in the Results body can be easily found in all of tables 1-3.Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.Data Availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact ASM production staff immediately with the expected release date.
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Sincerely, Siu-Kei Chow Editor Microbiology Spectrum should come before Figure 2 in the text.2. Are these not original, unpublished data?If so, "Data source: CDC Pertussis and Diphtheria Laboratory" should not be referenced in your figures/tables.3. Line 186.Awkward wording.Perhaps change to "Commercial laboratory testing detected BpBh in 92%..." Dr. Siu-Kei Chow, PhD Editor Microbiology Spectrum May 24, 2024 Dear Dr. Chow and reviewers: To examine the distribution of Bordetella species in clinical specimens, a total of 3,984 positive Bordetella specimens collected over 7 years(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)were retested at the CDC using species-specific targets that allow differentiation among the various Bordetella species.The CDC assay confirmed 85% of results produced by two large commercial laboratories.Although a rather straightforward study, it included a large sample set and confirms the specificity of common laboratory tests/methods that use IS481 and pIS1001 for detecting Bordetella species.Also, the small amount of B. holmesii (1.4% of samples) detected indicates that the prevalence of B. holmesii is low in the US and detection of IS481 by laboratory tests is primarily associated with B. pertussis infection.

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We thank the reviewer for your comment.While true that IS481 is also found in B. bronchiseptica, B. bronchiseptica primarily causes respiratory tract infections in domesticated animals and rarely causes zoonotic infection in humans.Our sample consisted only of clinical specimens taken from patients with suspected pertussis and did not include any animal specimens.We have made the following changes to address this

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Thank you for the comment; we have made the following changes to better display our results o Added Table 2 to show assays interpretations for the commercial laboratories and CDC to better show counts presented in Results text o Included two new summary lines in newly named Table 3 labeled "Any B. holmesii detected" and "Total matching" to properly show the counts and percents presented in the Results section.▪ Please include either Supplemental table 1 into the main body or include most of the interpretative criteria (such as Ct cut-offs) in the text.This information is useful to understand the Ct values and also, potential performance differences.• Thank you for the comment; the interpretive criteria and Ct cutoffs are mentioned at various points through the Materials and Methods.Please see lines 104-114 and 152-166 for the inclusion of the criteria into the main body.o Minor comments ▪ Figure 1 should come before Figure 2 in the text.• Thank you for your comment; the order of these figures has been updated.▪ Are these not original, unpublished data?If so, "Data source: CDC Pertussis and Diphtheria Laboratory" should not be referenced in your figures/tables.• Thank you for your comment; that text has been removed from the figure legends.▪ Line 186.Awkward wording.Perhaps change to "Commercial laboratory testing detected BpBh in 92%..." • Thank you for your comment; we have updated line 189: "Commercial laboratory testing detected BpBh in 92%..."