Results of a recent study have shown that the BioFire FilmArray Respiratory Panel (RP) detects one third fewer cases of Bortedella pertussis, the causative agent of whooping cough, when compared to standard PCR-based assays.
However, representatives at BioFire, a BioMérieux subsidiary, told GenomeWeb that this result was not unexpected, and the more specific target in its test has the advantage of reducing the number of false positive results.
Kristen Kanack, BioFire’s VP of regulated products and clinical affairs, and Mari Hoidal,director of marketing, spoke to GenomeWeb about the new study, published online last week in the Journal of Clinical Pathology.
In developing the panel, the firm chose to "provide the most specific assay we could, with the least probability of false-positive results due to contamination," Kanack asserted.
As opposed to testing for an insertion gene called IS481, which can be present in up to 200 copies per bacterium as well as in lower numbers in related Bordetella strains, BioFire chose as its target the promoter for the B. pertussis toxin gene, which is present in one copy per genome.
Hoidal affirmed that the panel has so far met with approval of customers. "The [RP] panel has been a huge success for us and continues to be a major bread-winning product," she added.
The panel runs on the FilmArray platform, which is an automated, multiplexed testing system that uses PCR and melt-curve analysis to detect infectious agents in about an hour. A 2.0 version of the platform was cleared by the US Food and Drug Administration last month.
In general, PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is a concern, according to a 2013 review.
High sensitivity can lead to false positive results, while high specificity can lead to false negative results. Molecular diagnostics makers balance the pros and cons of these outcomes in different ways, depending on the market and health consequences of the test results. With testing for human papillomavirus as a cervical cancer screen, for example, the debate seems to be settling in favor of false positives, despite objections of firms who make more sensitive or more general tests, as previously reported. In non-invasive prenatal testing, however, false positive results, which may have rates as high as 50 percent, could possibly result in termination of healthy pregnancies.
The JCP study of FilmArray RP was conducted by researchers at Children's Healthcare of Atlanta and Emory University, two of whom have served on advisory boards for BioFire and also perform research and development with the FilmArray instrument, according to a competing interests statement in the study.
"The conundrum related to PCR testing for B. pertussis revolves around whether the assay implemented in a clinical laboratory should be highly sensitive (IS481 target) or highly specific (pertussis toxin gene target)," the researchers wrote.
The researchers initially ran a standard PCR-based test from Focus Diagnostics on a set of clinical samples consisting of flocked swabs from patients aged one month to 18 years old.
These samples were originally tested about two years prior to FilmArray testing and then frozen in universal transport medium. The first step was to thaw and retest all the 74 positive samples with the Focus assay. Three samples associated with high Ct values did not repeat as positive results.
In the remaining cohort of 71 positives by Focus PCR, 25 were discrepant for the FilmArray RP. These 25 samples were then sent to the US Centers for Disease Control and Prevention and re-tested using their assay for B. pertussis and B. holmesii, a related bacterium that also happens to carry the IS481 insertion repeat.
The CDC concluded two of the samples did not have B. pertussis, and thus could be called false-positive in the Focus test. Meanwhile, of the 23 samples positive for the IS481 target, 10 also had the toxin gene target, and were considered pertussis-positive. The remaining 13 were considered intermediate, while B. holmesii was not detected in any of the 25 samples.
One of BioFire's considerations in designing the panel was reports of so-called pseudo-epidemics of pertussis, Kanack explained.
A 2010-2011 outbreak in Ohio, for example, later turned out to be a mixed outbreak of B. pertussis and B. holmesii. The IS481 element is also present in B. bronchiseptica strains, she said.
Another concern was contamination of otherwise negative samples. Because the IS481 element is present in such high numbers per cell, even a tiny amount of contamination could register as a positive result.
An apparent outbreak in a rural part of Colorado may have been the result of "environmentally present B. pertussis DNA in clinics from vaccine, clinic standard specimen collection practices, use of liquid transport medium, and lack of clinically relevant PCR cutoffs," according to a subsequent analysis in Pediatrics. And an outbreak of PCR-detected "atypical pertussis" among pre-schoolers in Toronto in 2009 was attributed in part to transient nasopharyngeal B. pertussis carriage.
On the other hand, last year an Archives of Pathology study of the impact of the FilmArray RP assay on patient outcomes showed that it "decreases the duration of antibiotic use, the length of inpatient stay, and the time in isolation." Authors of that work include two of the co-authors of the recent JCP evaluation.
BioFire provided materials for the research, and, although two of the researchers were part of the company's scientific advisory boards, Kanack emphasized that the current study was independent. "We collaborate with many people helping to advise us on our panel," she said.
"A lot of thought was put into this, and we stand by the decision for this panel," Kanack said. However, she added, "we are always listening to our customers and new developments in the field, and are open to considering any of our decisions."
Furthermore, BioFire has been aware of the possible disadvantages of a more specific pertussis target for some time, and issued an advisory notice in May of 2013.
"We put it out just for some clarification, although similar information is already included in our packaging," said Kanack.
The CDC guidelines for PCR assays of pertussis note that "IS481 is especially susceptible to falsely-positive results," and points out that "clinical laboratories might report high Ct values as any of the following: positive, detected, indeterminate, or equivocal." In the current study, for example, CDC re-tested samples that were IS481 positive but those negative for the toxin gene were "deemed intermediate."
"Intermediate or indeterminate pretty much mean that there is an element of IS481 that was detected, but it is not confirmed that it is from a pertussis [bacterium]. It could be, but it could not be as well," Kanack said.
The CDC further advises that "interpretation of PCR results, especially those with high Ct values, should be done in conjunction with an evaluation of signs and symptoms and available epidemiological information."
BioFire currently manufactures three FDA-cleared panels. Its respiratory panel was cleared to test for 15 organisms in 2011, when the company was called Idaho Technology, and later granted 510(k) clearance for five additional pathogens. The firm also has FDA-cleared panels for blood culture identification and gastrointestinal infections.
Hoidal said the firm has a lower respiratory panel in development and a meningitis panel that is nearing readiness for FDA submission. The firm's sister company, BioFire Defense, has two Ebola assays on the FilmArray platform which are still available under Emergency Use Authorization.
BioMérieux's $486 million acquisition of BioFire in January 2014 contributed to an annual earnings increase, and BioMérieux expects BioFire to "expand quickly" in the US, led by its FilmArray panels, as previously reported.
However, representatives at BioFire, a BioMérieux subsidiary, told GenomeWeb that this result was not unexpected, and the more specific target in its test has the advantage of reducing the number of false positive results.
Kristen Kanack, BioFire’s VP of regulated products and clinical affairs, and Mari Hoidal,director of marketing, spoke to GenomeWeb about the new study, published online last week in the Journal of Clinical Pathology.
In developing the panel, the firm chose to "provide the most specific assay we could, with the least probability of false-positive results due to contamination," Kanack asserted.
As opposed to testing for an insertion gene called IS481, which can be present in up to 200 copies per bacterium as well as in lower numbers in related Bordetella strains, BioFire chose as its target the promoter for the B. pertussis toxin gene, which is present in one copy per genome.
Hoidal affirmed that the panel has so far met with approval of customers. "The [RP] panel has been a huge success for us and continues to be a major bread-winning product," she added.
The panel runs on the FilmArray platform, which is an automated, multiplexed testing system that uses PCR and melt-curve analysis to detect infectious agents in about an hour. A 2.0 version of the platform was cleared by the US Food and Drug Administration last month.
In general, PCR testing for pertussis is rapid and sensitive, but the lack of standardization and variable specificity is a concern, according to a 2013 review.
High sensitivity can lead to false positive results, while high specificity can lead to false negative results. Molecular diagnostics makers balance the pros and cons of these outcomes in different ways, depending on the market and health consequences of the test results. With testing for human papillomavirus as a cervical cancer screen, for example, the debate seems to be settling in favor of false positives, despite objections of firms who make more sensitive or more general tests, as previously reported. In non-invasive prenatal testing, however, false positive results, which may have rates as high as 50 percent, could possibly result in termination of healthy pregnancies.
The JCP study of FilmArray RP was conducted by researchers at Children's Healthcare of Atlanta and Emory University, two of whom have served on advisory boards for BioFire and also perform research and development with the FilmArray instrument, according to a competing interests statement in the study.
"The conundrum related to PCR testing for B. pertussis revolves around whether the assay implemented in a clinical laboratory should be highly sensitive (IS481 target) or highly specific (pertussis toxin gene target)," the researchers wrote.
The researchers initially ran a standard PCR-based test from Focus Diagnostics on a set of clinical samples consisting of flocked swabs from patients aged one month to 18 years old.
These samples were originally tested about two years prior to FilmArray testing and then frozen in universal transport medium. The first step was to thaw and retest all the 74 positive samples with the Focus assay. Three samples associated with high Ct values did not repeat as positive results.
In the remaining cohort of 71 positives by Focus PCR, 25 were discrepant for the FilmArray RP. These 25 samples were then sent to the US Centers for Disease Control and Prevention and re-tested using their assay for B. pertussis and B. holmesii, a related bacterium that also happens to carry the IS481 insertion repeat.
The CDC concluded two of the samples did not have B. pertussis, and thus could be called false-positive in the Focus test. Meanwhile, of the 23 samples positive for the IS481 target, 10 also had the toxin gene target, and were considered pertussis-positive. The remaining 13 were considered intermediate, while B. holmesii was not detected in any of the 25 samples.
One of BioFire's considerations in designing the panel was reports of so-called pseudo-epidemics of pertussis, Kanack explained.
A 2010-2011 outbreak in Ohio, for example, later turned out to be a mixed outbreak of B. pertussis and B. holmesii. The IS481 element is also present in B. bronchiseptica strains, she said.
Another concern was contamination of otherwise negative samples. Because the IS481 element is present in such high numbers per cell, even a tiny amount of contamination could register as a positive result.
An apparent outbreak in a rural part of Colorado may have been the result of "environmentally present B. pertussis DNA in clinics from vaccine, clinic standard specimen collection practices, use of liquid transport medium, and lack of clinically relevant PCR cutoffs," according to a subsequent analysis in Pediatrics. And an outbreak of PCR-detected "atypical pertussis" among pre-schoolers in Toronto in 2009 was attributed in part to transient nasopharyngeal B. pertussis carriage.
On the other hand, last year an Archives of Pathology study of the impact of the FilmArray RP assay on patient outcomes showed that it "decreases the duration of antibiotic use, the length of inpatient stay, and the time in isolation." Authors of that work include two of the co-authors of the recent JCP evaluation.
BioFire provided materials for the research, and, although two of the researchers were part of the company's scientific advisory boards, Kanack emphasized that the current study was independent. "We collaborate with many people helping to advise us on our panel," she said.
"A lot of thought was put into this, and we stand by the decision for this panel," Kanack said. However, she added, "we are always listening to our customers and new developments in the field, and are open to considering any of our decisions."
Furthermore, BioFire has been aware of the possible disadvantages of a more specific pertussis target for some time, and issued an advisory notice in May of 2013.
"We put it out just for some clarification, although similar information is already included in our packaging," said Kanack.
The CDC guidelines for PCR assays of pertussis note that "IS481 is especially susceptible to falsely-positive results," and points out that "clinical laboratories might report high Ct values as any of the following: positive, detected, indeterminate, or equivocal." In the current study, for example, CDC re-tested samples that were IS481 positive but those negative for the toxin gene were "deemed intermediate."
"Intermediate or indeterminate pretty much mean that there is an element of IS481 that was detected, but it is not confirmed that it is from a pertussis [bacterium]. It could be, but it could not be as well," Kanack said.
The CDC further advises that "interpretation of PCR results, especially those with high Ct values, should be done in conjunction with an evaluation of signs and symptoms and available epidemiological information."
BioFire currently manufactures three FDA-cleared panels. Its respiratory panel was cleared to test for 15 organisms in 2011, when the company was called Idaho Technology, and later granted 510(k) clearance for five additional pathogens. The firm also has FDA-cleared panels for blood culture identification and gastrointestinal infections.
Hoidal said the firm has a lower respiratory panel in development and a meningitis panel that is nearing readiness for FDA submission. The firm's sister company, BioFire Defense, has two Ebola assays on the FilmArray platform which are still available under Emergency Use Authorization.
BioMérieux's $486 million acquisition of BioFire in January 2014 contributed to an annual earnings increase, and BioMérieux expects BioFire to "expand quickly" in the US, led by its FilmArray panels, as previously reported.