ABSTRACT
Eighty Gram-negative bacilli (54 Enterobacteriaceae and 26 nonfermenting Gram-negative bacilli) obtained from multiple institutions in the United States were distributed in a blinded manner to seven testing laboratories to compare their performance of a test for detection of carbapenemase production, the Carba NP test. The Carba NP test was performed by all laboratories, following the Clinical and Laboratory Standards Institute (CLSI) procedure. Site-versus-site comparisons demonstrated a high level of consistency for the Carba NP assay, with just 3/21 site comparisons yielding a difference in sensitivity (P < 0.05). Previously described limitations with blaOXA-48-like carbapenemases and blaOXA carbapenemases associated with Acinetobacter baumannii were noted. Based on these data, we demonstrate that the Carba NP test, when implemented with the standardized CLSI methodology, provides reproducible results across multiple sites for detection of carbapenemases.
KEYWORDS: carbapenemase, antimicrobial resistance, screening, Gram-negative bacilli
INTRODUCTION
Clinical microbiology laboratories around the globe have instituted a range of rapid screening tests for detection of carbapenemase production in the rising tide of carbapenem-nonsusceptible Gram-negative bacilli (GNB). One method, the Carba NP test, offers a rapid and simple colorimetric method for the detection of carbapenemase activity in isolates of GNB. In principle, the test is similar to the classic β-lactamase detection assays described by Escamilla and by Skinner and Wise in the 1970s, in which benzylpenicillin acidimetrically produced a red color after hydrolysis by Haemophilus influenzae β-lactamase (1, 2). With the Carba NP test, hydrolysis of imipenem produces acid and drives the pH indicator, phenol red, from red to yellow.
The Carba NP test has been studied by several investigators (3–14), including evaluations and comparisons to a commercial version (15–20). However, the literature lacks consistency in the Carba NP methodology. The test has evolved several times, including alterations to reagents, and has gone from a microtiter plate-based format performed on bacterial protein extracts to a rapid tube-based test with abbreviated processing. Most studies have been single-laboratory evaluations; there is a lack of multicenter performance assessments utilizing a single, harmonized method.
As part of an effort to standardize the method, the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing performed a multicenter evaluation of the Carba NP test across seven testing sites to evaluate its performance prior to formally recommending the method for use in the 25th Informational Supplement of the Performance Standards for Antimicrobial Susceptibility Testing (M100-S25) (21). Herein, we present a summary of the performance assessment.
RESULTS
The collective sensitivities and specificities for the Carba NP test performed at the seven participating sites ranged from 72.6 to 89.1% and from 88.9 to 100.0%, respectively (Table 1). The results for 11 isolates at 2 sites were invalid (site 3, 3 invalid results, and site 5, 8 invalid results), with invalid results comprising just 2% (11/560) of data points; invalid results were omitted from calculations of sensitivity and specificity. We observed markedly better test sensitivity in the Enterobacteriaceae group than in the non-Enterobacteriaceae group (Tables 2 and 3). The blaOXA carbapenemase genotypes were particularly challenging, as all seven sites reported high rates of false-negative results for blaOXA carbapenemase-harboring isolates, including 4/5 sites reporting false-negative results for blaOXA-48-like–harboring isolates. Sixteen carbapenemase-positive isolates gave false-negative results at any given site; 14/16 of those isolates were falsely negative at all sites. Statistical measurement of collective and organism group site-versus-site sensitivities (see Tables S1 to S3 in the supplemental material) demonstrated high levels of consistency in overall test accuracy for the Carba NP test, with just 3/21 site comparisons yielding a statistically significant difference in sensitivity (P < 0.05) in the collective data analysis (see Table S1).
TABLE 1.
Collective performance characteristics of Carba NP by site
| Site | Sensitivity (%) | Specificity (%) |
|---|---|---|
| 1 | 72.6 | 100.0 |
| 2 | 74.5 | 100.0 |
| 3 | 80.0 | 93.0 |
| 4 | 70.6 | 100.0 |
| 5 | 89.1 | 95.4 |
| 6 | 74.5 | 96.6 |
| 7 | 76.5 | 100.0 |
TABLE 2.
Collective performance characteristics of Carba NP for Enterobacteriaceae by site
| Site | Sensitivity (%) | Specificity (%) |
|---|---|---|
| 1 | 77.4 | 100.0 |
| 2 | 80.7 | 100.0 |
| 3 | 90.0 | 85.7 |
| 4 | 77.4 | 100.0 |
| 5 | 90.0 | 93.1 |
| 6 | 83.9 | 95.7 |
| 7 | 83.9 | 100.0 |
TABLE 3.
Collective performance characteristics of Carba NP for non-Enterobacteriaceae by site
| Site | Sensitivity (%) | Specificity (%) |
|---|---|---|
| 1 | 65.0 | 100.0 |
| 2 | 65.0 | 100.0 |
| 3 | 65.0 | 100.0 |
| 4 | 60.0 | 100.0 |
| 5 | 87.5 | 100.0 |
| 6 | 60.0 | 100.0 |
| 7 | 65.0 | 100.0 |
DISCUSSION
We report here the results of the CLSI blinded, multisite trial of a standardized method for the Carba NP assay described in the three most recent CLSI M100S editions, including the 27th edition (21–23). Overall, the performance statistics were consistent with those in prior publications, including issues noted with isolates expressing OXA-48-like carbapenemases and OXA genotypes found in the Acinetobacter baumannii complex (4, 6). In 2015, Bakour et al. described a universal and improved bacterial cell lysis that offered improved performance with OXA genotypes found in the A. baumannii complex (24). These data were not available at the time of our study, and we were not able to evaluate the method improvements proposed by Bakour et al. (24). Despite the necessity for participating laboratories to prepare the bulk of the reagents, there was a high degree of consistency in accuracy across all seven sites. Only three site-versus-site comparisons had statistically significant differences in sensitivity. These results were borderline and detected in the uncorrected comparisons only. Given the technical requirements in preparing reagents, these data suggest that the Carba NP test is highly reproducible when laboratories adhere to a single, standardized method.
In conclusion, the CLSI standardized procedure for Carba NP testing demonstrated good performance using isolates with common carbapenemase genotypes currently found in Enterobacteriaceae in the United States. These data originated from a multisite trial using a well-characterized collection of GNB isolates with a diverse mix of resistance genotypes and phenotypes. Reduced performance of Carba NP is likely in areas that have a predominance of isolates harboring OXA-48-like carbapenemases. Further improvements which alleviate cost and improve reagent stability should be explored, as should a rigorous evaluation of methods and media to improve the performance of the assay with problematic genotypes.
MATERIALS AND METHODS
Gram-negative isolate collection.
Eighty clinical isolates of GNB (54 Enterobacteriaceae and 26 nonfermenting GNB) obtained from multiple centers across the United States were distributed to seven testing laboratories. The species were provided, but the testing laboratories were blinded to the antibiotic resistance phenotypes and genotypes of the isolates. Phenotypic and genotypic characterization of antimicrobial susceptibility was performed on all isolates included in the study (Table 4). The 80 isolates included 51 harboring the most common carbapenemase genes: 4 harbored blaIMP (Klebsiella pneumoniae [n = 2] and Pseudomonas aeruginosa [n = 2]); 10 harbored blaKPC (Klebsiella pneumoniae [n = 2], Klebsiella ozaenae [n = 1], Enterobacter cloacae [n = 4], Escherichia coli [n = 2], and P. aeruginosa [n = 1]), including 3 isolates which coproduced a TEM-1 extended-spectrum β-lactamase and 2 that displayed low carbapenem MICs; 12 harbored blaNDM (E. cloacae [n = 1], K. pneumoniae [n = 3], E. coli [n = 3], Morganella morganii [n = 1], Providencia rettgeri [n = 1], and Acinetobacter baumannii [n = 3]); 5 harbored blaOXA-48-like (K. pneumoniae [n = 3], K. ozaenae [n = 1], and E. aerogenes [n = 1]); 2 harbored blaSME (Serratia marcescens [n = 2]); 8 harbored blaVIM (P. aeruginosa [n = 5] and K. pneumoniae [n = 3]); 1 harbored blaSPM (P. aeruginosa [n = 1]); 2 harbored blaOXA-23 (A. baumannii [n = 2]); 1 harbored blaOXA-24 (A. baumannii [n = 1]); 2 harbored blaOXA-58 (A. baumannii [n = 2]); 1 harbored blaOXA-72 (A. baumannii [n = 1]); 1 harbored both blaNDM and blaOXA-48-like (K. pneumoniae [n = 1]); 1 harbored blaOXA-23 and blaNDM (A. baumannii [n = 1]); and 1 harbored blaOXA-23 and blaOXA-24 (A. baumannii [n = 1]). Carbapenemase-negative isolates (n = 29) were selected to challenge the test with common resistance types, including extended-spectrum β-lactamases, plasmid-mediated AmpCs, OXAs without carbapenemase activity, and carbapenem resistance due to porin modification.
TABLE 4.
Isolate characteristics
| AR Bank IDa | Species | Resistance mechanism(s)b | 2-h Carba NP result by sitec | MIC (μg/ml) of: |
|||
|---|---|---|---|---|---|---|---|
| Ertapenem | Doripenem | Imipenem | Meropenem | ||||
| 32 | E. cloacae | KPC-3, TEM-1B, ACT-16 | +++++++ | >4 | 2 | 4 | 4 |
| 33 | A. baumannii | NDM-1, OXA-94 | +++++++ | NA | >8 | >32 | >8 |
| 34 | K. pneumoniae | IMP-4, TEM-1B, SHV-11 | +++++++ | 2 | 4 | 1 | 2 |
| 35 | A. baumannii | OXA-72, TEM-1D, ADC-25, OXA-66 | −−−−I−− | NA | >8 | >32 | >8 |
| 36 | A. baumannii | OXA-24, OXA-65 | −−−−I−− | NA | >8 | >32 | >8 |
| 37 | A. baumannii | NDM-1, OXA-94 | +++++++ | NA | >8 | >32 | >8 |
| 38 | E. cloacae | NDM-1, OXA-9, TEM-1B, ACT-7, CTX-M-15, OXA-1, OmpC | +++++++ | >4 | >8 | >32 | >8 |
| 39 | K. pneumoniae | OXA-181, CTX-M-15, SHV-26, OmpK35 | −−−−−−− | >4 | 4 | 4 | 4 |
| 40 | K. pneumoniae | VIM-27, CTX-M-15, SHV-11, OXA-1, OmpK35 | +++++++ | >4 | >8 | >32 | >8 |
| 41 | K. pneumoniae | NDM-1, CMY-4, CTX-M-15, SHV-11, OXA-10 | +++++++ | >4 | >8 | >32 | >8 |
| 42 | K. pneumoniae | TEM-1B, CTX-M-15, SHV-1, OXA-10, OXA-1 | −−−−−−− | >4 | 2 | 1 | 2 |
| 43 | K. pneumoniae | SHV-12 | −−I−+−− | >4 | 1 | ≤0.5 | 2 |
| 44 | K. pneumoniae | OXA-9 (internal stop codon), TEM-1A, CTX-M-15, SHV-12, OXA-1, OmK35 | −−−−−−− | >4 | 2 | 1 | 4 |
| 45 | A. baumannii | OXA-23, TEM-1D, OXA-69 | −−−−−−− | NA | >8 | 32 | >8 |
| 46 | K. pneumoniae | VIM-27, CTX-M-15, SHV-11, OXA-1, OmpK35 | +++++++ | >4 | >8 | >32 | >8 |
| 47 | K. pneumoniae | TEM-1A, OmpK35 | −−−−−−− | >4 | 4 | 2 | 4 |
| 48 | E. coli | NDM-1, TEM-1B, CMY-6, CTX-M-15, OXA-2, OmpF | +++++++ | >4 | >8 | 16 | >8 |
| 49 | K. pneumoniae | NDM-1, TEM-1B, CMY-6, CTX-M-15, OXA-1, OmpK35 | +++++++ | >4 | >8 | >32 | >8 |
| 50 | E. cloacae | KPC-4, TEM-1A, ACT-5 | −−+−+−+ | 0.5 | ≤0.12 | 1 | ≤0.12 |
| 51 | K. ozaenae | OXA-181, CTX-M-15, SHV-26, OmpK35 | −−−−I−− | >4 | 4 | 4 | 4 |
| 52 | A. baumannii | OXA-58, OXA-100 | −−−−I−− | NA | 8 | 16 | >8 |
| 53 | E. cloacae | KPC-3, OXA-9, TEM-1A, OmpF | +++++++ | >4 | 4 | 8 | 8 |
| 54 | P. aeruginosa | VIM-4, OXA-50, PAO | +++++++ | NA | >8 | >32 | >8 |
| 55 | E. coli | NDM-1, CMY-6, OXA-1 | +++++++ | >4 | >8 | 8 | >8 |
| 56 | A. baumannii | OXA-23, OXA-66 | −−−−I−− | NA | >8 | >32 | >8 |
| 57 | M. morganii | NDM-1, CTX-M-15, OXA-1 | +++++++ | 2 | 8 | 8 | 4 |
| 58 | E. coli | TEM-52B | −−−−−−− | 1 | 0.25 | ≤0.5 | 0.25 |
| 59 | Proteus mirabilis | TEM-1B | −−−−−−− | 0.5 | 4 | 8 | 1 |
| 60 | E. cloacae | ACT-7 | −−+−−−− | 0.25 | ≤0.12 | ≤0.5 | ≤0.12 |
| 61 | E. coli | KPC-3, OXA−9, TEM-1A | +++++++ | >4 | 4 | 4 | 4 |
| 62 | Enterobacter aerogenes | −−−−−−− | 1 | ≤0.12 | ≤0.5 | ≤0.12 | |
| 63 | A. baumannii | OXA-23, OXA−24, OXA-65 | −−−−+−− | NA | >8 | >32 | >8 |
| 64 | P. aeruginosa | SPM-1, OXA-50, PAO, OXA-56 | +++++−+ | NA | >8 | >32 | >8 |
| 65 | E. cloacae | ACT-15 | −−−−I+− | 1 | ≤0.12 | ≤0.5 | ≤0.12 |
| 66 | K. pneumoniae | OXA-232, OXA-9, TEM-1A, CTX-M-15, OXA-1, OmpK35 | −−I−−−− | >4 | >8 | 4 | >8 |
| 67 | E. coli | TEM-1B | −−−−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 |
| 68 | K. pneumoniae | NDM-1, OXA-232, OXA-9, TEM-1A, CTX-M-15, SHV-11, OXA-1 | +++++++ | >4 | >8 | >32 | >8 |
| 69 | E. coli | NDM-1, TEM-1B, CMY-6 | +++++++ | >4 | 8 | 8 | 8 |
| 70 | A. baumannii | OXA-58, OXA-100 | −−−−−−− | NA | 8 | 32 | >8 |
| 71 | Klebsiella oxytoca | OXY-2-8, OmpK36 | −−−−+−− | >4 | 2 | 1 | 8 |
| 72 | E. cloacae | TEM-1B | −−−−I−− | 1 | ≤0.12 | ≤0.5 | ≤0.12 |
| 73 | E. cloacae | −−−−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 | |
| 74 | E. aerogenes | OXA-48 | −−++++− | 2 | 2 | 4 | 2 |
| 75 | K. pneumoniae | OXA-232, CTX-M-15, SHV-1, OXA-1, OmpK35 | −−−−−−− | >4 | >8 | 8 | >8 |
| 76 | K. pneumoniae | VIM-1, SHV-30 | +++++++ | 0.5 | 4 | 4 | 1 |
| 77 | E. coli | −−I−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 | |
| 78 | A. baumannii | ADC-25, SHV-5, OXA-71 | −−−−−−− | NA | >8 | >32 | >8 |
| 79 | K. pneumoniae | TEM-1B, CTX-M-14, SHV-11, DHA-1 | −−−−−−− | >4 | 8 | 16 | 8 |
| 80 | K. pneumoniae | IMP-4, TEM-1B, OKP-B-2, OXA-1, SFO-1 | −++++++ | >4 | 8 | 4 | 4 |
| 81 | E. coli | TEM-1B, CMY-2 | −−−−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 |
| 82 | P. rettgeri | NDM-1 | +++−+++ | >4 | >8 | 32 | 8 |
| 83 | A. baumannii | NDM-1, OXA-23, PER-7, OXA-69 | +++−+++ | NA | >8 | >32 | >8 |
| 84 | E. coli | TEM-1B | −−−−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 |
| 85 | E. coli | CMY-2, OmpF | −−−−−−− | 2 | ≤0.12 | ≤0.5 | 1 |
| 86 | E. coli | TEM-1B, CTX-M-14 | −−+−−−− | 2 | ≤0.12 | ≤0.5 | ≤0.12 |
| 87 | K. pneumoniae | SHV-12, OmpK35 | −−−−−−− | 0.25 | ≤0.12 | ≤0.5 | ≤0.12 |
| 88 | A. baumannii | NDM-1, OXA-64 | +++++++ | NA | >8 | >32 | >8 |
| 89 | E. coli | CMY-2 | −−+−−−− | ≤0.125 | ≤0.12 | ≤0.5 | ≤0.12 |
| 90 | P. aeruginosa | KPC-5, OXA-50, PAO | +++++++ | NA | >8 | >32 | >8 |
| 91 | S. marcescens | SME-3 | +++++++ | >4 | >8 | >32 | >8 |
| 92 | P. aeruginosa | IMP-14, OXA-50, VEB-1, PAO, OXA-10 | +++++++ | NA | >8 | >32 | >8 |
| 93 | E. cloacae | KPC-6, TEM-1B, ACT-16, OXA-1 | +++++++ | 4 | 1 | 4 | 1 |
| 94 | P. aeruginosa | OXA-50, PAO | −−−−−−− | NA | >8 | >32 | >8 |
| 95 | P. aeruginosa | OXA-50, PAO | −−−−−−− | NA | 4 | 32 | >8 |
| 96 | K. ozaenae | KPC-3, OXA-9, TEM-1A, SHV-1 | +++++++ | >4 | >8 | >32 | >8 |
| 97 | K. pneumoniae | KPC-3, OXA-9, TEM-1A, SHV-11 | +++++++ | >4 | >8 | >32 | >8 |
| 98 | K. pneumoniae | KPC-2, OXA−9, TEM-1A, OmpK35 | +++++++ | >4 | 8 | 16 | >8 |
| 99 | S. marcescens | SME-3 | +++++++ | >4 | >8 | >32 | >8 |
| 100 | P. aeruginosa | VIM-2, OXA-50, PAO | +++++++ | NA | >8 | >32 | >8 |
| 101 | A. baumannii | OXA-24, OXA-65 | −−−−I−− | NA | >8 | >32 | >8 |
| 102 | A. baumannii | ADC-25, OXA-66 | −−−−−−− | NA | 4 | 2 | 4 |
| 103 | P. aeruginosa | IMP-1, OXA-50, PAO | +++++++ | NA | >8 | >32 | >8 |
| 104 | E. coli | KPC-4, TEM-1A | +++−+++ | 1 | 0.25 | 2 | 0.5 |
| 105 | P. aeruginosa | OXA-50, PAO, OXA-2 | −−−−−−− | NA | >8 | 2 | >8 |
| 106 | K. pneumoniae | NDM-1, OXA-9, TEM-1A, CTX-M-15, OXA-1 | +++++++ | >4 | >8 | >32 | >8 |
| 107 | K. pneumoniae | OXA-9, TEM-1A, SHV-83, CTX-M-2, OXA-10, OmpK35, OmpK36 | −−−−−−− | >4 | >8 | 8 | >8 |
| 108 | P. aeruginosa | VIM-2, OXA-50, PAO, OXA-4 | +++++++ | NA | >8 | >32 | >8 |
| 109 | K. pneumoniae | TEM-1B, CTX-M-15, SHV-11, OXA-1, OmpK36 | −−−−−−− | >4 | 8 | 2 | >8 |
| 110 | P. aeruginosa | VIM-2, OXA-50, PAO | +++++++ | NA | >8 | >32 | >8 |
| 111 | P. aeruginosa | VIM-2, OXA-50, PAO, OXA-4 | +++++++ | NA | >8 | >32 | >8 |
Antimicrobial Resistance Bank, Centers for Disease Control and Prevention. ID, identification number.
Carbapenemase genes are in boldface.
+, positive; −, negative; I, indeterminate.
Carba NP test procedure.
A standard procedure based on the work of Vasoo et al. (3) was distributed to the participating laboratories. Solution A (0.05% phenol red and 0.1 mM ZnSO4 [prepared from stock; Sigma-Aldrich, St. Louis, MO], final concentration in deionized water, adjusted to pH 7.8 with 0.1 N NaOH) was prepared fresh with and without 6 mg/ml imipenem (United States Pharmacopeial Convention, Rockville, MD) prior to testing. Two microcentrifuge tubes were prepared per isolate, adding 100 μl of SoluLyse buffer (Thermo Fisher Scientific, Pittsburgh, PA). For each isolate, a 1-μl loopful of culture was emulsified into each tube and the suspension vortexed for 5 s. KPC-positive K. pneumoniae (ATCC BAA-1705), KPC-negative K. pneumoniae (ATCC BAA-1706), and blank control tubes were included for each batch of samples tested. One hundred microliters of solution A without imipenem was added to one of the tubes in the isolate tube set, and 100 μl of solution A with 6 mg/ml imipenem was added to the second tube. The tubes were vortexed and incubated for 2 h at 35 ± 2°C. The tubes were examined for color at time intervals of 10, 30, 60, and 120 min over the course of incubation. Colors in the range of orange to yellow were considered positive, while the range of red to red-orange was considered negative. A color interpretation aid was provided as part of the standardized procedure. The tube with solution A without imipenem was used as a negative control. A positive color in this tube was considered invalid; tests with invalid results were repeated once.
Whole-genome sequencing and resistance genotyping.
For whole-genome sequencing (WGS), DNA was extracted with the Maxwell 16-cell low elution volume (LEV) DNA purification kit (Promega, Madison, WI). Genomic DNA with an absorbance ratio of 1.8 to 2.0 was fragmented to ∼800 bp by Covaris (Woburn, MA) ultrasonic fragmentation. Libraries were prepared using the NuGen Ovation ultralow DR multiplex system 1-96 kit (San Carlos, CA), multiplexed, and sequenced with 2 × 250 bp chemistry on the Illumina MiSeq, version 2.0 (San Diego, CA). Sequencing reads were filtered for quality (Q20 and higher), and reads shorter than 50 bp were discarded from the data set using SolexaQA version 3.1 (25). Reads were assembled with SPAdes 3.1.0 using four k-mer sizes (k = 41, 79, 85, and 97) (26). Afterwards, trimmed reads were mapped back to each assembled genome, using Burroughs-Wheeler Aligner (BWA) for contig error correction (27). Acquired antimicrobial resistance (AR) genes were detected using c-SSTAR, a command line version of SSTAR (https://github.com/chrisgulvik/c-SSTAR), and the ResFinder database (28, 29). AR genes located on the genome assemblies with 100% coverage and >99% sequence identity to genes listed in the ResFinder repository, as well as outer membrane porin genes that had 100% coverage and >80% sequence similarity, were reported.
Statistical analysis.
Sensitivities and specificities were calculated for individual sites using the genotype-based result as the gold standard. Comparisons of sensitivities and specificities between sites were performed using McNemar's test. P values of less than 0.05 were considered statistically significant. Both unadjusted and false discovery rate-adjusted P values were calculated to adjust for multiple comparisons (see Tables S1 to S3 in the supplemental material). Traditional approaches were used for controlling error rates in the presence of multiple comparisons, including strong and weak control of familywise error rates, using techniques such as the Bonferroni correction (30). The false discovery rate approach has been shown to be more powerful than methods like the Bonferroni correction that control false-positive rates (31). Thus, we report both unadjusted and false discovery rate-corrected P values. Analysis was performed using SAS version 9.4 (SAS Inc., Cary, NC).
Accession number(s).
Raw sequencing reads and MIC data were deposited under BioProject accession number PRJNA292904 (BioSamples SAMN04014873 to SAMN04014952).
Supplementary Material
ACKNOWLEDGMENTS
R.P. reports grants from BioFire, Check-Points, Curetis, 3M, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company. R.P. is a consultant to Curetis, Roche, Qvella, and Diaxonhit. In addition, R.P. has a patent on Bordetella pertussis/parapertussis PCR with royalties paid by TIB, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an antibiofilm substance issued. R.P. serves on an Actelion data-monitoring board. R.P. receives travel reimbursement and an editor's stipend from ASM and IDSA and honoraria from the USMLE, Up-to-Date, and the Infectious Diseases Board Review Course.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00244-17.
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