CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Amy Carpenter Aquino
April 2021—The interpretive challenges of blood culture identification panels were the focus of an AMP2020 virtual presentation on false-positives and false-negatives and their sources and solutions.
The spotlight was on Proteus, but “it’s not the sole organism we have to worry about,” said Susan Butler-Wu, PhD, D(ABMM), SM(ASCP), director of the clinical microbiology laboratory, LAC+USC Medical Center, Los Angeles, and associate professor of clinical pathology, Keck School of Medicine of USC.
Her co-presenter, speaking on antimicrobial resistance targets, was Richard Davis, PhD, D(ABMM), MLS(ASCP)CM, of Providence Healthcare. (See CAP TODAY, May 2021, for coverage.) Dr. Davis and Dr. Butler-Wu co-wrote a 2020 ASM report titled “Genotypic False Detections from Blood Culture Bottles: Are We Only Seeing the Tip of the Iceberg?”
The Proteus problem prompted their report. “Proteus was being detected by the BCID,” Dr. Butler-Wu said of the BioFire FilmArray panel, “but no Proteus was being isolated from the blood culture bottles.” Such incidents led the FDA to issue a Class II recall.
BD Bactec media were affected in the initial recall in 2018. Subsequent recalls later that year and in 2020 implicated BioMérieux BacT/Alert media.
In a study presented at ASM Microbe 2019, BioFire evaluated the ability of a prototype BCID2 panel with “algorithm and chemistry enhancements to mitigate false-positive results caused by the presence of Proteus and Enterobacteriaceae nucleic acid in sterile blood culture bottles” (Green J, et al. Poster CPHM-967 presented at: ASM Microbe 2019; San Francisco).
Green, et al., wrote that “sterile blood culture media can contain residual nucleic acid from a variety of bacteria likely introduced from raw materials or manufacturing processes.” They acknowledged that the BCID panel was affected by the presence of nucleic acid that triggers Proteus spp. and Enterobacteriaceae detection.
In their study, they tested sterile blood culture media bottles from Becton Dickinson (40 unique media lots of six formulations) and BioMérieux (20 unique media lots of five formulations) for Proteus spp. and Enterobacteriaceae with the BCID and BCID2 panels. Contrived and residual clinical positive blood cultures with Proteus spp. were also tested with both panels for comparison. Testing was performed at BioFire and at five clinical pilot sites.
Blood culture media bottles that were positive for Proteus by BioFire were extracted and amplified using an independent PCR assay to determine the relative concentration of Proteus nucleic acid in the media. Bottles that were negative by BCID were also tested for comparison. The authors found amplification of Proteus DNA even in lots that tested negative by the BCID panel. “So it looked like, at least in the lots they tested, that DNA from Proteus may be omnipresent,” Dr. Butler-Wu said.
“It’s important to note that this is DNA,” she continued. While the bottles are sterile, DNA present in the blood culture media is being detected. “So this false-positive issue appears to be somewhat of a numbers game. Essentially, if you have enough of the DNA present above the limit of detection, then you’re going to get a positive result.”
The authors found that 29 of 67 sterile media (43 percent) tested positive for Proteus with the BCID panel, compared with zero of 67 detections with the BCID2 panel. “These were not detectable with the BCID2 panel because they’ve increased the limit of detection, so it’s not quite as sensitive for the Proteus target,” Dr. Butler-Wu said.
Other findings were as follows:
- Proteus spp. was detected in 16 of 175 (nine percent) of the additional bottles from development studies at BioFire with the BCID panel, compared with zero of 234 detections with the BCID2 panel.
- Enterobacteriaceae was detected in 11 of 175 (six percent) of the additional bottles from development studies at BioFire with the BCID panel, compared with one of 234 (0.4 percent) with BCID2.
- No Enterobacteriaceae detections were observed at pilot sites.
- Proteus nucleic acid was present at levels ranging from 1 × 102 to 1 × 105 GE/mL in sterile media; levels of 1 × 104 to 1 × 105 were linked to the detection of nucleic acid contamination.
- BioFire testing of contrived Proteus positive blood culture samples correctly identified Proteus at 1,000- to 10,000-fold below PBC levels (~1 × 109 CFU/mL).
- BCID2 detected eight Proteus true positive clinical samples confirmed by culture.
Green, et al., concluded that the BCID2 panel was “less vulnerable to false positive detections of Proteus and Enterobacteriaceae caused by nucleic acid contamination observed in specific lots of sterile blood culture media bottles while retaining a high level of sensitivity that is capable of detecting true Proteus PBCs at levels several orders of magnitude below what may be expected in a true clinical sample.”
“The unfortunate reality,” they added, “is that raw materials used to manufacture media are derived from biological sources that have been shown to contain nucleic acid contamination which may continue to confound molecular diagnostics unless materials are screened for and qualified as nucleic acid free in the future.”
Dr. Butler-Wu’s laboratory, which uses Bactec media, went live with the BCID panel at about the time of the emerging false-positive Proteus problem. “So we had to contend with how to handle this,” she said.
Dr. Butler-Wu
Her laboratory’s strategy: “We were very, very conservative.”
“We don’t talk about Proteus in our lab when it comes to BCID,” Dr. Butler-Wu explained. “There are very few instances where we would even report it. We erred on the totally conservative side. The bottom line was if we were seeing a Gram-negative rod and detecting Proteus, we would report it as indeterminate.”
Dr. Butler-Wu pointed out that because of the Enterobacteriaceae call on the BCID panel, “we were seeing that these would be positive for both Enterobacteriaceae and Proteus, but it would turn out to be something else entirely.”
The lab’s conservative strategy worked. By not reporting Proteus, “we saved ourselves a lot of angst with respect to use of this panel,” Dr. Butler-Wu said. “The first day we went live, we had seven false-positive Proteus detections alone. If we had been calling those out on day one of a go-live on a new test, it would have been catastrophic.”
Not reporting Proteus is not a long-term, sustainable solution, she said, “but it’s the one we’ve employed for now.”
In their ASM report, Drs. Butler-Wu and Davis wrote that while a conservative reporting strategy may work for known issues, “it may not be effective when initially encountering a new false positivity issue. Laboratories should therefore always be suspicious for the potential of false positive results any time multiple detections are present and exercise caution when reporting the presence of organisms beyond what is observed by Gram stain.”
They suggested that laboratories mitigate the risk of reporting inaccurate molecular blood culture test results by:
- Ensuring the Gram stain matches the results from the molecular test.
- Confirming that organism morphology matches the molecular test results the next day when growth is visible on solid-growth media.
- Reviewing past cultures from the patient (if present) to ensure consistency.
- Reporting any suspected false-positive results to the manufacturer for investigation.
Although blood culture manufacturing is required to be sterile, DNA contamination can be introduced into the manufacturing process in many ways, she said. “Blood culture media employ a variety of different plant, yeast, or animal extracts, so there is ample opportunity for organisms’ DNA to make it into the product.”
The other solution is for manufacturers of amplification-based panels to increase the lower limit of detection, which is what BioFire did with its BCID2 panel.
The GenMark ePlex BCID panel uses nucleic acid amplification and has Proteus targets, but “there has not been much indication of a problem,” Dr. Butler-Wu said. Still, there are other organisms to worry about.
Several Class II recalls have been issued since 2014 for false-positive microorganism detection associated with certain blood culture media lots when multiplex nucleic acid-based panels are used, she said. The first was “quite worryingly” for Enterococcus and Pseudomonas aeruginosa detection from BacT/Alert standard anaerobic bottles, and a 2019 recall was for false-positive E. coli detections with certain BacT/Alert media.