Resistance targets: blood culture ID panel pitfalls

Amy Carpenter Aquino

May 2021—Most of the time, bloodstream infection antimicrobial resistance results achieved with blood culture molecular ID panels will be accurate. When and why they might not be was the focus of an AMP 2020 virtual session.

“I don’t want to lead anyone to believe that these are not good, accurate, and important types of tests,” Richard E. Davis, PhD, D(ABMM), MLS(ASCP)^CM, director of microbiology, Providence Healthcare, Spokane, Wash., said of the panels. “But as laboratorians and people thinking of bringing on tests, we should be aware of what the limitations might be so we can resolve discrepancies and inform clinicians about how exactly these tests operate.”

When it comes to antimicrobial resistance, he said, false-negatives are “far and away the most important. The overall risk is low, but we should be aware that this is not impossible.”

The SENTRY Antimicrobial Surveillance Program data for drug-resistant organisms reveal that rates of methicillin-resistant Staphylococcus aureus (MRSA) isolation from bloodstream infections have stayed relatively stable but vancomycin-resistant Enterococcus (VRE) rates have risen over the years, Dr. Davis said (Diekema DJ, et al. Antimicrob Agents Chemother. 2019;63[7]:e00355–19). “In the Gram-negatives, multi­drug-resistant Enterobacterales from either community- or hospital-acquired sources have increased,” as has the prevalence of extended-spectrum beta-lactamases among E. coli and Klebsiella spp., and carbapenem-resistant Enterobacterales (CRE).

Methicillin-resistant S. aureus is defined by the presence of mecA (or mecC), which encodes a penicillin-binding protein resistant to beta-lactams, so it’s resistant to penicillins and some cephalosporins, Dr. Davis said. “Vancomycin-resistant Enterococcus has vanA or vanB genes that encode a peptidoglycan that has low affinity for vancomycin, so it’s resistant to vancomycin.”

For the Gram-negative determinants, “we’re looking for targets that will confer the extended-spectrum beta-lactamase-type resistance, or the carbapenem-resistant Enterobacterales carbapenemases,” he said. “The ESBL targets are beta-lactamases, like the CTX-M family, and the carbapenemase targets”—bla_KPC, bla_NDM, bla_IMP, bla_VIM, or bla_OXA.

Importantly, not all Gram-negative rod beta-lactamases or other resistance determinants have discrete gene targets, he added.

Dr. Davis categorizes the root causes of false results with rapid diagnostics for bloodstream infections into external (specimen related) and biological (organism- or gene-specific) root causes. (See boxes, next page.) His co-presenter, Susan Butler-Wu, PhD, D(ABMM), SM(ASCP), of LAC+USC Medical Center, addressed false-positive results caused by blood culture bottle contamination (see CAP TODAY, April 2021).

A hypothetical biological root cause of a false-positive result could be, “This organism has a gene that is detected by your PCR, for example, but it can’t encode a functional downstream actual resistance mechanism, so it looks resistant but it’s not,” Dr. Davis said.

For false-negative results, an external root cause could be an assay design that doesn’t detect a known version or variant of a resistance gene. However, a biological root cause could be a novel mutation in the gene target sequence that prevents detection by the PCR—though with functional expression. “These external or biological root causes of false results can be seen in the organism with the most published and reported cases of false antimicrobial resistance detection—MRSA,” Dr. Davis said.

In MRSA, the Staphylococcus genome encodes a number of penicillin-binding proteins, and those contribute to the characteristic thick peptidoglycan layer of Gram-positive organisms. “Those penicillin-binding proteins can be targeted by beta-lactam antibiotics like penicillins, hence the name penicillin-binding protein.”

MRSA develops when a mobile cassette called the staphylococcal cassette chromosome mec (SCCmec) gets inserted into the Staphylococcus aureus genome. “That mecA encodes a PBP2a, a protein that is resistant to being bound by beta-lactam antibiotics.”

When it comes to blood culture panels, some false-negative MRSA results may be due to mecC, which is a rare mecA homologue primarily found in Europe. “It was first identified in livestock and very rarely in bloodstream infections,” Dr. Davis said, adding it can appear sensitive but then become resistant over time or with treatment (Ford BA. J Clin Microbiol. 2017;​56[1]:e01549–17).

While most cases of MRSA are caused by mecA, successful risk mitigation of possible false-negatives comes from improved mec targets for blood culture panels, he said. “Newer panels—like the BCID2, Xpert MRSA/SA, ePlex Gram-positive panels, and BD Max StaphSR—have a mec target that detects both mecA and mecC. So we don’t need to worry about a mecC not being detected if our panel doesn’t specifically target mecC.” Without one of those panels, “there is a chance you might miss a mecC, but it’s still very rare in the United States and outside of Europe,” he said. Screening for phenotypic resistance, via cefoxitin disk or cefoxitin-chromogenic agar, would also be possible if a new panel is not available and mecC is of concern.

A biological root cause resulting in false-negatives, he said, are mutations, insertions, or deletions in the MRSA targets that cause rapid PCR detection tests to fail. In a survey of 252 methicillin-susceptible Staphylococcus aureus blood isolates from the United States and Europe, determined via the Cepheid Xpert MRSA/SA test cleared in 2013, only two isolates (0.8 percent) were phenotypically resistant. Tenover, et al., found that “results for all the isolates were correct” when tested with the updated Xpert MRSA/SA BC assay, which received FDA clearance in 2019 (Tenover FC, et al. J Clin Microbiol. 2019;57[11]:e01195–19).

The authors wrote, “These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare.”

A target-based change to help account for these rare mutations came when Cepheid changed its MRSA-calling rule-based algorithm. The previous algorithm was MRSA = spa and mec and SCCmec. The new algorithm: spa and mec or mec and SCCmec.

“This is a rare event,” Dr. Davis said of false-negatives with biological causes, “but the altered algorithm does help mitigate that potential risk.” He and Dr. Butler-Wu wrote in their 2020 ASM report titled “Genotypic False Detections from Blood Culture Bottles—Are We Only Seeing the Tip of the Iceberg?”: “Surveillance and investigation of discordant genotypic and phenotypic resistance results will be necessary to identify sequence variants not detected by current assays. Manufacturers will hopefully continue to update panels to detect such variants.”

Newer panels with better targets have mitigated much of the risk of false-positive resistance results, Dr. Davis said. In their 2020 report, he and Dr. Butler-Wu wrote that the most common scenario of false-positive resistance detection comes from a mixed blood culture bottle positive for both methicillin-susceptible S. aureus and mecA-containing coagulase-negative Staphylococcus spp. (CoNS), commonly mecA-positive Staphylococcus epidermidis.

“If we look at our Staphylococcus genome,” Dr. Davis said, “if we have a target in our assay that detects mecA and a target that detects a specific Staph aureus gene, such as spa or nuc, both targets are going to be amplified. You would think that indicates MRSA, when in fact it’s two different Staphylococcus species giving positive results for those two different targets.”

Most at risk for false-positive results are the FilmArray BCID—“not the BCID2, which is the most updated version”—the Verigene BC-GP, and the ePlex BCID-GP panels, he said, because they have only mec and S. aureus-specific targets.