ABSTRACT
Rapid antimicrobial drug administration is crucial for the efficient treatment of sepsis or septic shock, but empirical therapy is limited by the increasing prevalence of multidrug-resistant bacteria. Thus, rapid and reliable antimicrobial susceptibility testing (AST) is needed to start appropriate antimicrobial drug administration as quickly as possible. In the present study, we evaluated the performance of the Reveal rapid AST system. From February to April 2021, 102 positive blood culture bottles (BCBs) from hospitalized patients with bacteremia caused by Gram-negative bacteria were included in the study. All isolates were tested by the Reveal system directly from the positive BCBs in comparison to the DxM MicroScan WalkAway. Essential agreement (EA) and category agreement (CA) were high with 98.5% and 97.1%, respectively. We also determined the susceptibility of 10 highly resistant CDC & FDA AR strains in duplicate. Here, EA was 99.6% and CA 97.9%. The average time to result by Reveal was 5.4 h ± 1.2 h compared to an average of 16 h by DxM MicroScan WalkAway for clinical strains and 3.8 h ± 1.2 h for more resistant CDC & FDA AR strains. Susceptibility determination with the Reveal rapid AST system directly from positive BCBs is for the frequently represented bug-drug combinations a reliable and accurate approach, meeting the European ISO guideline for the performance of AST systems. Moreover, AST directly from blood cultures performed with the Reveal system saves time when compared to the conventional AST, as no subculturing is needed and time to result is very short.
KEYWORDS: Specific Reveal, VITEK Reveal, rapid AST, sepsis, blood culture
INTRODUCTION
Sepsis is a life-threatening organ dysfunction that is caused by a dysregulated host response due to an infection and can lead to shock, multiple organ failure, and death (1, 2). In Germany, e.g., 146,985 explicitly coded sepsis cases and 58,689 (39.9%) associated deaths were reported in 2016 (3). Recently, sepsis has been described as the most common cause of death worldwide, with 48.9 million sepsis cases each year and approximately 11 million deaths associated with sepsis (4). Rapid administration of appropriate antibiotics is crucial for effective treatment of sepsis, and delayed therapy is associated with worse clinical outcome (5–8). The “surviving sepsis campaign” recommends antibiotic treatment within the first hour of recognition of septic shock as well as severe sepsis (9). In septic shock, the initiation of antibiotic therapy within the first hour of documented hypotension increases survival by 79.9%, whereas survival decreases by about 8% every hour of delay over the ensuing 6 h (10). In the case of suspected sepsis, therapy is most often started empirically with broad-spectrum antibiotics even though patients who are infected with multidrug-resistant pathogens are more likely to initially receive an ineffective therapy (11). To provide an adequate antibiotic therapy, identification of bacterial pathogens and antimicrobial susceptibility testing (AST) are required. For rapid identification, the matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) provides accurate identification results with centrifugated pellets from blood cultures for Gram-positive bacteria (88%) and Gram-negative bacteria (96%) (12). However, current standard susceptibility tests are based on the culturing of bacteria. The turnaround time of these diagnostic tools is over 24 h making them inadequate for providing a fast and correct treatment within the first 1–3 h. To overcome this hurdle, new methods and devices for rapid AST direct from positive blood cultures have been developed or are under development. Rapid phenotypical systems for AST directly from blood cultures include the Pheno Test BC (Accelerate diagnostics, Tuscon, AZ, USA), Alfred 60AST (Alifax, Italy), dRAST (QuantaMatrix, Seoul, Republic of Korea), the FASTinov Kit (FASTinov, Portugal), Reveal rapid AST system (Biomerieux, Marcy-l'Étoile, France, formerly Specific diagnostics, San Jose, CA, USA), and rapid disk diffusion according to EUCAST and CLSI guidelines (13, 14).
The aim of the present study was to assess the performance of the Specific Reveal rapid AST System compared to the DxM MicroScan WalkAway used routinely in our laboratory. Specific Reveal rapid AST was recently renamed to VITEK Reveal (Biomerieux, Marcy L'Etoile, France).
MATERIALS AND METHODS
Clinical blood culture samples
The study was performed at Wisplinghoff laboratories (Cologne, Germany) that processed 232,529 blood cultures in 2021 with a positivity rate of 15% (34,801/232,529), of which 38% were Gram-negative bacteria (13,224/34,801).
For the present study, routine positive blood culture bottles (BacT/ALERT FN Plus/BacT/ALERT FA Plus, bioMérieux, Marcy L'Etoile, France) were selected by convenience sampling and screened for Gram-negative bloodstream infections in the time period from 1 February to 31 April 2021. The blood culture bottles (BCBs) were incubated in the BACT/ALERT 3D Microbial Detection Systems (bioMérieux, Marcy L'Etoile, France) until they flagged positive. Time to positivity (TTP) in the blood culture instrument as well as the bottle type (aerobe or anaerobe blood culture bottle) was noted, the bottles were taken out of the BACT/ALERT, and blood samples were Gram-stained throughout the day, 7 days per week. Positive blood cultures were collected to a maximum of 8 strains per day for the study setup. As recommended by the manufacturer (at this time, Specific diagnostics), only blood culture samples that could be processed within 16 h post bottle positivity were included in the study. Blood cultures containing Gram-positive bacteria or fungi were not included in the study. According to the study protocol, the following Gram-negative organisms could be included for performance evaluation of the Reveal rapid AST system: Acinetobacter baumannii, Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa. If a positive blood culture showed Gram-negative bacteremia, cell counting, Reveal testing, and subculturing on agar plates were performed. Cell counts of positive blood cultures were achieved in order to investigate the influence of bacteria growing at different rates as well as the influence of the time from positive notification of BCB to AST (0–16 h). Cell count was measured using the fully automated flow cytometer Sysmex UF-5000 according to the protocol of Mancini et al. and results were noted (15). As described in the protocol of Mancini et al., a 2 × 103 dilution (10 µL of blood culture in 5 mL of 0.9% NaCl) was chosen for quantification of bacteria in blood cultures (15). Antibiotic susceptibility of the bacteria was determined in parallel with the Reveal rapid AST system directly from blood cultures and the DxM MicroScan WalkAway (Beckman Coulter, Sacramento, CA, US), as comparative method/standard of care (further referred to as reference method), from an 18–24 h subculture, the following day. Identification of bacteria to species level was performed using MALDI-TOF MS (MBT Smart, software MBT Compass 4.1, MBT Compass Library revision F version 9 database, Bruker Daltonics, Bremen, Germany) from a 4-h incubated agar culture. If the identification at 4 h resulted in a species that was not noted in the study protocol or the incubated agar showed a polymicrobial growth the samples were excluded from the study. All bacterial isolates included in the study were stored at −80°C in case of later need for retesting.
Bacterial challenge set
In addition to the clinical blood culture samples, 10 Gram-negative challenge strains from the CDC & FDA Antimicrobial Resistance (AR) Isolate Bank with known susceptibilities, including multidrug-resistant strains, were tested from spiked blood cultures with the Reveal rapid AST system. The strains were provided by Specific Diagnostics and are listed in Table S1.
Qualtity control strains
Three ATCC strains (E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603 [SHV-18 ESBL-producer]) were used as quality control strains and run once a week for quality control testing.
Antibiotic panels for AST
Commercially available MicroScan Neg MDR MIC 1 panels (Beckman Coulter, Brea, CA, US) were used for susceptibility testing with the Reveal rapid AST system as well as with the reference system (DxM MicroScan WalkAway). The MDR panel contains 35 antibiotics. The following 20 antibiotics (concentration range) were included in the study: amikacin (8–16 mg/L), amoxicillin-clavulanic acid (8/2–32/2 mg/L), ampicillin (4–8 mg/L), aztreonam (1–4 mg/L), cefepime (0.5–8 mg/L), cefotaxime (1–32 mg/L), ceftazidime (1–32 mg/L), ceftazidime-avibactam (2/4-8/4 mg/L), ceftolozane-tazobactam (1/4-4/4 mg/L), cefuroxime (4–8 mg/L), ciprofloxacin (0.06, 0.25–1 mg/L), ertapenem (0.125, 0.5–1 mg/L), gentamicin (2–4 mg/L), imipenem (1–8 mg/L), levofloxacin (0.5–1 mg/L), meropenem (0.125, 1–32 mg/L), piperacillin (8–16 mg/L), piperacillin-tazobactam (8/4–16/4 mg/L), tobramycin (2–4 mg/L), and trimethoprim-sulfamethoxazole (2/38–4/76 mg/L). The antibiotics not listed here were excluded since (i) the European Committee on Antimicrobial Susceptibility Testing (EUCAST) did not publish breakpoints for all species-drug combinations tested in the study, (ii) for some species-drug combinations EUCAST breakpoints were not included in the respective concentration range of the study panel, (iii) at the time of this study some antibiotics were not included in the Reveal rapid AST menu, (iv) ESBL- and AmpC detection was not analyzed. Note, ceftazidime-avibactam, ceftolozane-tazobactam, and amoxicillin-clavulanic acid were not included in the Reveal rapid AST software when the study part with the challenge strains started but were included for the clinical blood culture samples.
Preparation of spiked blood cultures with challenge strains
Spiked blood cultures were used for the study part with the bacterial challenge strains. Frozen isolates were sub-cultured onto blood agar plates. Colonies of a pure overnight culture were diluted into sterile saline to prepare a bacterial suspension according to a McFarland standard 0.5 (approximately 1–2 × 108 CFU/mL). The suspension was further diluted by 106 into sterile saline (102 CFU/mL). One milliliter of the 102 CFU/mL dilution (100 CFUs) was used to inoculate a BacT/ALERT FA Plus aerobe blood culture bottle. As it was only proof of concept no human blood was added. Spiked BCBs were loaded into the BACT/ALERT. Once the bottles turned positive, TTPs were noted, and the bottles were pulled. For testing of the challenge set, AST was performed with the Reveal rapid AST system. All challenge strains were tested twice. For data analysis, reference minimal inhibitory concentrations (MICs) provided by the CDC & FDA were used as comparators. Interpretive categories (S, I, R) were evaluated according to the EUCAST guidelines 2020 (16).
Reveal rapid AST workflow
For Reveal sample preparation, 25 µL of a PBC was directly (without removal of blood cells) diluted 1:1,000 in pluronic water (Beckman Coulter, Sacramento, CA, USA) according to manufacturer’s instructions, and the dilution was used for inoculation of a MicroScan MDR plate using a Renok inoculator (Beckman Coulter, Brea, CA, USA). Subsequently, the plate was covered with the Reveal sensor panel (Specific Diagnostics, San Jose, CA, USA) and sealed automatically in the Reveal plate sealer (Specific Diagnostics, San Jose, CA, USA). The Reveal sensor panel contains an array of seven colorimetric chemical sensors in a nanoporous matrix that are located over each of the 96 wells of the AST plates and respond to metabolic byproducts produced during bacterial growth. Sealed plates were barcode-scanned and loaded in the Reveal rapid AST instrument (Specific Diagnostics, San Jose, CA, USA), where they were incubated at 37°C, constantly agitated to promote the growth of test bacteria and imaged every 10 min by the Reveal system to monitor a change in color of the seven sensors. One well on each AST plate served as growth control (contains growth medium but no antibiotic = growth must occur) and another one as negative control (without media to support bacterial growth = no growth should occur). Results of species identification by MALDI-TOF were entered in the Reveal system if available, anytime during the test. The Reveal software allocated the determined MICs to interpretive categories (S, I, R) according to EUCAST guidelines once the ID of the test bacterium was provided (16). Time to result (TTR) for each antibiotic was noted after each run.
Reference method for AST of clinical blood culture samples
MICs of clinical blood culture isolates of the overnight culture determined by the automated broth microdilution SOC system DxM MicroScan WalkAway served as reference. After Gram-staining, 10 µL each of PBC was spread onto appropriate agar plates (tryptic soy blood agar, MacConkey agar). Plates were incubated 18–24 h at 36 ± 1°C in ambient air. Subsequently, single colonies were diluted in sterile saline (MicroScan prompt inoculation system; Beckman Coulter, Brea, CA, USA), and the further procedure was performed according to the manufacturer’s instructions.
Discrepancies in the interpretive category between the Reveal rapid AST system and the reference system were resolved using MTS gradient strips (MIC Test Strip; Liofilchem S.r.I, Italy) which was performed according to the manufacturer’s instructions. For comparative evaluation, MIC results of the MTS test that were in between of two log2 dilution steps were upscaled to the respective next higher log2 dilution step.
Data analysis
The AST results of the Reveal rapid AST system were compared to the results obtained by the reference method (DxM MicroScan WalkAway) and, if required, by MTS for discrepancy resolution (n = 58/2008). Only the final result (as the result of the MTS strip in the case of a discrepancy) was used throughout the work and for all analyses. Interpretive categories (S, I, R) were determined for all methods according to the EUCAST guidelines (16). Intrinsically resistant bacteria were accounted as resistant according to EUCAST guidance document “Intrinsic Resistance and Unusual Phenotypes” version 3.2 (17).
The total category agreement (CA) was defined as the percentage of isolates classified in the same interpretive category when determined with Reveal rapid AST system and the reference method or MTS. Category discrepancies were classified as follows: (i) very major error (VME) and adjusted VME (aVME) when the test system indicated susceptibility and the reference method or MTS indicated resistance (aVME; only category discrepancies based on more than ±1 log2 dilution steps), (ii) major error (ME) when the test system indicated resistance and the reference method or MTS indicated susceptibility, (iii) minor error (mE) when the test system reported “susceptible increased dosage” (former intermediate) and the reference method or MTS “susceptible” or “resistant”; or the test system reported “susceptible” or “resistant” and the reference method or MTS “susceptible increased dosage”. The essential agreement was defined as the percentage of MIC results that fell within plus or minus one log2 dilution step when compared to the reference method or MTS. Most antibiotics on the test plate showed limited concentration ranges that did not allow for the assessment of evaluable EA (when the MIC result is on-scale for both, the test and the reference method). Thus, on-scale MICs (MICs within the concentration range) as well as off-scale MICs (MICs below or equal to the lowest concentration or above the highest concentration) were considered for EA evaluation. Moreover, if a MIC obtained with the MTS gradient strip or – for spiked samples – a MIC of a respective CDC & FDA AR challenge isolate was below or above the limit of quantification of the MIC test panel (= lowest and highest concentration, respectively, of each drug in the MicroScan Neg MDR MIC 1 panel) and the Reveal MIC was correspondent ≤ or > this was counted as in agreement.
Statistical analysis
Mean and median were used as descriptive summary statistics for continuous variables (i.e., TTP, bacterial count) depending on distributional characteristics; categorial variables (bottle type, species, antibiotic drug, error types) were shown as percentages.
Predictive variables such as TTP, bacterial count, bottle type, species, and antibiotic drug as well as dependent variables “occurrence of mE, ME, VME” (outcome parameters) were included as fixed effects along with a random intercept for strain ID in generalized linear mixed models (GLMM) of binomial family. Association of predictive variables with the outcome was tested with a multi-way Analysis of Variance (ANOVA), considering a P-value of <0.05 as statistically significant. The area of technical uncertainty (ATU) was considered no error in this analysis. All calculations were done with R version 4.1.2 (Foundation for Statistical Computing, Vienna, Austria) along with package lme4 (18).
RESULTS
Overall
We found a high overall EA and CA of 98.5% and 97.1%, respectively, between the Reveal and the reference method in the clinical part of the study, as well as for the more resistant CDC & FDA AR bank isolates in the spiked blood cultures (EA 99.6%; CA 97.9%). All quality control strains were within range.
Evaluation of clinical blood culture samples
In detail, 143 routine blood culture samples were randomly collected. Forty-one samples were excluded because (i) 4 were polymicrobial cultures, (ii) 28 of the identified species were not included in the study protocol, (iii) 6 showed no growth on agar plates during the identification time frame, (iv) 1 was not identified by MALDI-TOF, (v) 2 had Reveal System control errors. Thus, a total of 102 blood cultures with 7 different Gram-negative species were included in the study and 2,008 (drugs tested per species see Table S2) strain-drug pairs were tested, of which 10.5% (211/2,008) were tested as resistant by the reference method. EA calculation was not possible for 17 of 2,008 strain-drug pairs because the concentration ranges for ciprofloxacin (0.06, 0.25–1 mg/L), ertapenem (0.125, 0.5–1 mg/L), and meropenem (0.125, 1–32 mg/L) in the MDR panel included skipped concentrations (ciprofloxacin: 0.125 mg/L; ertapenem: 0.25 mg/L; meropenem: 0.25 mg/L, and 0.5 mg/L). The 17 strains showed MIC values that were one dilution step above the highest skipped concentration of ciprofloxacin, ertapenem, or meropenem and were excluded from EA calculation: in the case of ciprofloxacin, 10 strains (1 A. baumannii strain, 3 E. coli strains, 5 K. pneumoniae strains, and 1 P. aeruginosa strain), of ertapenem 2 strains (1 E. coli strain and 1 K. oxytoca strain) and of meropenem 5 strains (3 E. coli strains and 2 P. aeruginosa strains) were excluded. The overall essential agreement for the remaining strains was 98.5% (1,962/1,991) for the Reveal system when compared to the reference method (DxM MicroScan WalkAway) or MTS (in case of discrepancy resolution). In Reveal testing, 6 E. coli strains showed ciprofloxacin MICs in the ATU and were excluded for CA as well as for error calculation. All six strains were tested as susceptible with the reference method. The overall CA was 97.1% (1,944/2,002) with 0.8% (16/2,002) mE, 0.5% (8/1,678) ME, and 16.1% (34/211) VME. Table 1 summarizes all performance parameters in detail.
TABLE 1.
Overall performance of the Reveal rapid AST system when compared to the DxM MicroScan WalkAway (clinical blood cultures)g
| Study details or performance parameter | Number and percentage |
|---|---|
| Study details | No. |
| No. of species collected | 7 |
| No. of antibiotics testeda | 20 |
| No. of strains tested | 102 |
| No. of total strain-drug pairs | 2,008 |
| No. of S strain-drug pairsb | 1,684 |
| No. of I strain-drug pairsb | 113 |
| No. of R strain-drug pairsb | 211 |
| Performance parameter | % (no.) |
| EAc,d,e | 98.5 (1,962/1,991) |
| CAf | 97.1 (1,944/2,002) |
| mEf | 0.8 (16/2,002) |
| MEf | 0.5 (8/1,678) |
| VMEf | 16.1 (34/211) |
The number of antibiotics varies depending on the respective species.
The number of respective strain-drug pairs is the number evaluated by the reference method or MTS (the latter was performed in case of discrepancies between Reveal and the reference method).
Because of the limited concentration ranges for most antibiotics on the test plate, MICs less or equal to the lowest concentration or greater than the highest concentration of a drug were included in the calculation.
Percentage (number) of strains for which the difference in MICs regarding all drugs tested was no more than ±1 log2 dilution when compared to the reference method or MTS.
The concentration ranges of ciprofloxacin, ertapenem, and meropenem included skipped concentrations. If the Reveal system and/or the reference method showed a MIC above the highest skipped concentration, the respective strain was excluded from EA calculation. This was the case for 17 strains.
In Reveal testing 6 E. coli strains showed ciprofloxacin MIC values falling in the area of technical uncertainty (ATU) and were excluded for CA as well as for error calculation. All six strains were tested susceptible with the reference method or MTS.
S, susceptible; I, susceptible increased dosage; R, resistant; EA, essential agreement; CA, category agreement; mE, minor error; ME, major error; VME, very major error.
We also calculated the aVME rate. Thirteen of the 34 VMEs resulted from category discrepancies based on more than a 1 log2 dilution step difference between the Reveal MIC and the MIC determined by the reference method. Thus, the overall aVME rate amounted to 6.2% (13/211).
For the EA, the distribution of all log2 dilution differences between MICs of Reveal rapid AST system and DxM MicroScan WalkAway or Liofilchem MTS test per species is shown in detail in Table S3 and per antimicrobial drug in Table S4.
E. coli (71.6%, 73/102) was the most common pathogen included in our study followed by K. pneumoniae (17.6%, 18/102), K. oxytoca (3.9%, 4/102), E. cloacae (2.9%, 3/102), P. aeruginosa (1.9%, 2/102), C. koseri (0.98%, 1/102), and A. baumanii (0.98%, 1/102). No C. freundii or K. aerogenes—that could be also included according to the study protocol—were observed during the study time period. The average time to result was 5.4 h ± 1.2 h for Reveal and varied slightly between the different species ranging from 4.2 h ± 0.7 h for A. baumannii to 6.0 h ± 1.0 h for P. aeruginosa (see Table 2) compared to DxM MicroScan WalkAway with 16 h time to result after first subculturing.
TABLE 2.
AST results of the Reveal rapid AST system compared to the DxM MicroScan WalkAway test for 102 clinical Gram-negative strains and all drugs testedg,h
| Species | Reveal average TTR (h) | No. of strains | No. of tested antibiotics per species | No. of AST results of the reference method | % (no.) agreement | % (no.) of errors | Comments | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total | Total used for EA calc. | Total used for CA and error calc. | S | I | R | EAa,b | CA | mE | ME | VME | |||||
| Enterobacterales | |||||||||||||||
| E. coli | 5.4 | 73 | 20 | 1,460 | 1,453c,d,e | 1,454f | 1,229f | 71 | 154 | 98.5 (1,431) | 97.1 (1,412) | 0.3 (5) | 0.4 (5) | 20.8 (32) | mE: CIP (1), PIT (1), CTZ (1), CUR (1), TRS (1); ME: TOB (1), CIP (1), TRS (1), GEN (1), CTV (1); VME: AMC (13), GEN (6), TOB (5), AMI (4), PIT (2), AMP (1), CTZ (1) |
| K. pneumoniae | 5.3 | 18 | 20 | 360 | 355c | 360 | 306 | 21 | 33 | 99.2 (352) | 97.8 (352) | 1.7 (6) | 0 (0) | 6.1 (2) | mE: PIP (3), CUR (2), LEV (1); VME: AMC (1), PIT (1) |
| K. oxytoca | 5.3 | 4 | 20 | 80 | 79d | 80 | 70 | 6 | 4 | 98.7 (78) | 96.3 (77) | 2.5 (2) | 1.4 (1) | 0 (0) | mE: PIP (2); ME: CTT (1) |
| E. cloacae | 5.1 | 3 | 19 | 57 | 57 | 57 | 42 | 1 | 14 | 96.5 (55) | 96.5 (55) | 1.8 (1) | 2.4 (1) | 0 (0) | mE: CEP (1); ME: CTA (1) |
| C. koseri | 4.9 | 1 | 19 | 19 | 19 | 19 | 16 | 1 | 2 | 94.7 (18) | 89.5 (17) | 5.3 (1) | 6.3 (1) | 0 (0) | mE: PIP (1); ME: CTT (1) |
| Non-fermentative GNB | |||||||||||||||
| P. aeruginosa | 6.0 | 2 | 12 | 24 | 21c,e | 24 | 10 | 12 | 2 | 100.0 (21) | 95.8 (23) | 4.2 (1) | 0 (0) | 0 (0) | mE: CEP (1) |
| A. baumannii | 4.2 | 1 | 8 | 8 | 7c | 8 | 5 | 1 | 2 | 100.0 (7) | 100.0 (8) | 0 (0) | 0 (0) | 0 (0) | No errors |
| Overall | 5.4 | 102 | n.e. | 2,008 | 1,991 | 2,002 | 1,678 | 113 | 211 | 98.5 (1,962) | 97.1 (1,944) | 0.8 (16) | 0.5 (8) | 16.1 (34) | mE: PIP (6), CUR (3), CEP (2), CIP (1), PIT (1), CTZ (1), TRS (1), LEV (1); ME: CTT (2), TOB (1), CIP (1), TRS (1), GEN (1), CTV (1), CTA (1); VME: AMC (14), GEN (6), TOB (5), AMI (4), PIT (3), AMP (1), CTZ (1) |
Because of the limited concentration ranges for most antibiotics on the test plate, MICs less or equal to the lowest concentration or greater than the highest concentration of a drug were included in the calculation.
Percentage (number) of strains for which the difference regarding all drugs tested was no more than ±1 log2 dilution.
The concentration range of ciprofloxacin on the AST plate was ≤0.06 mg/L, 0.25–1 mg/L (the MIC of 0.125 mg/L was skipped). If a MIC of the Reveal system and/or reference method was 0.25 mg/L, the respective strain was excluded from EA calculation. This was the case for 1 A. baumannii strain, 3 E. coli strains, 5 K. pneumoniae strains, and 1 P. aeruginosa strain.
The concentration range of ertapenem on the AST plate was ≤0.125 mg/L, 0.5–1 mg/L (the MIC 0.25 mg/L was skipped). If a MIC of the Reveal system and/or reference method was 0.5 mg/L, the respective strain was excluded from EA calculation. This was the case for 1 E. coli strain and 1 K. oxytoca strain.
The concentration range of meropenem on the AST plate was ≤0.125 mg/L, 1–32 mg/L (the MIC values 0.25 mg/L and 0.5 mg/L were skipped). If a MIC of the Reveal system and/or reference method was 1 mg/L, the respective strain was excluded from EA calculation. This was the case for 3 E. coli strains and 2 P. aeruginosa strains.
In Reveal testing 6 E. coli strains showed ciprofloxacin MIC values falling in the area of technical uncertainty (ATU) and were excluded for CA as well as for error calculation. All six strains were tested susceptible with the reference method or MTS.
TTR, time to result; calc., calculation; S, susceptible; I, susceptible increased dosage; R, resistant; EA, essential agreement; CA, category agreement; mE, minor error; ME, major error; VME, very major error; GNB, Gram-negative bacteria; n.e., not evaluable; AMI, amikacin; AMC, amoxicillin-clavulanic acid; AMP, ampicillin; CEP, cefepime; CTA, cefotaxime; CTZ, ceftazidime; CTV, ceftazidime-avibactam; CTT, ceftolozane-tazobactam; CUR, cefuroxime; CIP, ciprofloxacin; GEN, gentamicin; LEV, levofloxacin; PIP, piperacillin; PIT, piperacillin-tazobactam; TOB, tobramycin; TRS, trimethoprime-sulfamethoxazole.
In general, S, I, R categorization based on MIC results that were evaluated by the reference method or MTS (the latter was performed in case of discrepancies between Reveal and the reference method).
Of the overall observed 34 VMEs, 32 VMEs (94.1%) were found in E. coli (see Table 2). VMEs were not equally distributed among the antimicrobial agents tested in this study. Most of the VMEs (41.2%, 14/34) occurred when testing amoxicillin-clavulanic acid (EUCAST concentration) followed by testing the aminoglycosides gentamicin (17.6%, 6/34), tobramycin (14.7%, 5/34), and amikacin (11.8%, 4/34). We investigated whether the number of errors was significantly higher for a particular bug-drug combination than for others, but found no significance (amoxicillin-clavulanic acid_VME_E.coli P = 0.28, gentamicin_VME_E.coli P = 1.0, amikacin_VME_E.coli P = 0.60, tobramycin_VME_E.coli P = 0.33). The distribution of all errors by test drugs is shown in Table 3. The average time to result for the Reveal varied among tested drugs and ranged from 3.6 h ± 0.9 h for ampicillin to 7.3 h ± 1.7 h for piperacillin-tazobactam (see Table 3). The distribution of EA/CA and errors only concerning E. coli are shown in Table S5.
TABLE 3.
| Drug | Reveal average TTR (h) | No. of strains with AST results of the reference method | % (no.) agreement | % (no.) of errors | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total | Total used for EA calc. | Total used for CA and error calc. | S | I | R | EAa,b | CA | mE | ME | VME | ATUc | ||
| Amikacin | 3.9 | 102 | 102 | 102 | 98 | 4 | 99.0 (101) | 96.1 (98) | n.a. | 0 (0) | 100.0 (4) | ||
| Amoxicillin-clavulanic acid (EUCAST) | 4.0 | 99 | 99 | 99 | 72 | 27 | 91.9 (91) | 85.9 (85) | n.a. | 0 (0) | 51.9 (14) | ||
| Ampicillin | 3.6 | 99 | 99 | 99 | 44 | 55 | 99.0 (98) | 99.0 (98) | n.a. | 0 (0) | 1.8 (1) | ||
| Aztreonam | 5.1 | 99 | 99 | 99 | 90 | 1 | 8 | 100.0 (99) | 100.0 (99) | 0 (0) | 0 (0) | 0 (0) | |
| Cefepime | 5.9 | 101 | 101 | 101 | 93 | 3 | 5 | 99.0 (100) | 98.0 (99) | 2.0 (2) | 0 (0) | 0 (0) | |
| Cefotaxime | 5.4 | 99 | 99 | 99 | 92 | 7 | 99.0 (98) | 99.0 (98) | 0 (0) | 1.1 (1) | 0 (0) | ||
| Ceftazidime | 5.3 | 101 | 101 | 101 | 93 | 2 | 6 | 97.0 (98) | 98.0 (99) | 1.0 (1) | 0 (0) | 16.7 (1) | |
| Ceftazidime-avibactam | 6.1 | 101 | 101 | 101 | 101 | 99.0 (100) | 99.0 (100) | n.a. | 1.0 (1) | 0 (0) | |||
| Ceftolozane-tazobactam | 6.4 | 101 | 101 | 101 | 99 | 2 | 98.0 (99) | 98.0 (99) | n.a. | 2.0 (2) | 0 (0) | ||
| Cefuroxime IV | 5.1 | 95 | 95 | 95 | 88 | 7 | 98.9 (94) | 96.8 (92) | 3.2 (3) | 0 (0) | 0 (0) | ||
| Ciprofloxacin | 4.1 | 102 | 92d | 96c | 81c | 3 | 12 | 96.7 (89) | 97.9 (94) | 1.0 (1) | 1.2 (1) | 0 (0) | 5.9 (6) |
| Ertapenem | 6.5 | 99 | 97e | 99 | 98 | 1 | 100.0 (97) | 100.0 (99) | n.a. | 0 (0) | 0 (0) | ||
| Gentamicin | 5.1 | 100 | 100 | 100 | 93 | 7 | 98.0 (98) | 93.0 (93) | n.a. | 1.1 (1) | 85.7 (6) | ||
| Imipenem | 6.5 | 102 | 102 | 102 | 99 | 2 | 1 | 100.0 (102) | 100.0 (102) | 0 (0) | 0 (0) | 0 (0) | |
| Levofloxacin | 4.3 | 102 | 102 | 102 | 89 | 3 | 10 | 100.0 (102) | 99.0 (101) | 1.0 (1) | 0 (0) | 0 (0) | |
| Meropenem | 6.6 | 102 | 97f | 102 | 101 | 1 | 100.0 (97) | 100.0 (102) | 0 (0) | 0 (0) | 0 (0) | ||
| Piperacillin | 5.0 | 101 | 101 | 101 | 62 | 7 | 32 | 100.0 (101) | 94.1 (95) | 5.9 (6) | 0 (0) | 0 (0) | |
| Piperacillin-tazobactam | 7.3 | 101 | 101 | 101 | 92 | 3 | 6 | 97.0 (98) | 96.0 (97) | 1.0 (1) | 0 (0) | 50.0 (3) | |
| Tobramycin | 4.1 | 102 | 102 | 102 | 95 | 7 | 99.0 (101) | 94.1 (96) | n.a. | 1.1 (1) | 71.4 (5) | ||
| Trimethoprim-sulfamethoxazole | 4.0 | 100 | 100 | 100 | 86 | 1 | 13 | 99.0 (99) | 98.0 (98) | 1.0 (1) | 1.2 (1) | 0 (0) | |
Because of the limited concentration ranges for most antibiotics on the test plate MICs less or equal to the lowest concentration or greater than the highest concentration of a drug, were included in the calculation.
Percentage (number) of strains for which the difference regarding all drugs tested was no more than ±1 log2 dilution.
In Reveal testing 6 E. coli strains showed ciprofloxacin MIC values falling in the area of technical uncertainty (ATU) and were excluded for CA as well as for error calculation. All 6 strains were tested susceptible with the reference method or MTS.
The concentration range of ciprofloxacin on the AST plate was ≤0.06 mg/l, 0.25-1 mg/l (the MIC of 0.125 mg/l was skipped). If a MIC of the Reveal system and/or reference method was 0.25 mg/l the respective strain was excluded from EA calculation. This was the case for 1 A. baumannii strain, 3 E. coli strains, 5 K. pneumoniae strains and 1 P. aeruginosa strain.
The concentration range of ertapenem on the AST plate was ≤0.125 mg/l, 0.5-1 mg/l (the MIC 0.25 mg/l was skipped). If a MIC of the Reveal system and/or reference method was 0.5 mg/l the respective strain was excluded from EA calculation. This was the case for 1 E. coli strain and 1 K. oxytoca strain.
The concentration range of meropenem on the AST plate was ≤0.125 mg/L, 1–32 mg/L (the MIC values 0.25 mg/L and 0.5 mg/L were skipped). If a MIC of the Reveal system and/or reference method was 1 mg/L, the respective strain was excluded from EA calculation. This was the case for 3 E. coli strains and 2 P. aeruginosa strains.
TTR, time to result; calc., calculation; S, susceptible; I, susceptible increased dosage; R, resistant; EA, essential agreement; CA, category agreement; mE, minor error; ME, major error; VME, very major error; n.a., not applicable.
In general, S, I, R categorization based on MIC results that were evaluated by the reference method or MTS (the latter was performed in case of discrepancies between Reveal and the reference method).
Figure 1 shows the correlation of the mean TTR and the resistance rate for each antibiotic. For every percent more resistance, TTR decreases by −0.018 h. This is significant with P < 0.0001.
Fig 1.
TTR for Reveal in hours and percentage of resistance (%) stratified by antibiotic. Dots represent the resistance rate/TTR for individual bug-drug combinations.
Evaluation of challenge strains
To analyze the performance of the Reveal rapid AST system with more resistant strains which were not well represented in the clinical part of our study, we performed test-runs on 10 highly resistant strains (including multidrug-resistant strains) from the CDC & FDA AR Isolate Bank in duplicate. Here in total, 244 strain-drug pairs were analyzed, of which 88.5% (216/244) were resistant. EA/CA was as high as 99.6%/97.9% and no VME, 1 ME, (gentamicin), and 4 mE (meropenem and piperacillin-tazobactam) occurred (see Tables 4 and 5).
TABLE 4.
Overall performance of the Reveal rapid AST system with blood cultures spiked with CDC & FDA AR bank isolates compared to CDC & FDA AR reference datai
| Study details or performance parameter | Number and percentage | ||
|---|---|---|---|
| Study details | No. in 1st test | No. in 2nd test | No. in 1st and 2nd test |
| No. of species tested | 6 | 6 | 6 |
| No. of antibiotics testeda | 14 | 14 | 14 |
| No. of strains tested | 10 | 10 | 20 |
| No. of total strain-drug pairsb | 122 | 122 | 244 |
| No. of S strain-drug pairsc | 11 | 11 | 22 |
| No. of I/ATU strain-drug pairsc,d | 3 | 3 | 6 |
| No. of R strain-drug pairsc | 108 | 108 | 216 |
The number of antibiotics varies depending on the respective species.
Of 123 strain-drug pairs, one MIC result for piperacillin-tazobactam and K. aerogenes was missing by the Reveal system, resulting in 122 evaluable strains-drug pairs.
The number of respective strain-drug pairs is the number based on CDC & FDA AR reference data. EUCAST clinical breakpoints were used for categorization.
CDC & FDA AR reference MIC of one K. aerogenes strain for ciprofloxacin was falling in the area of technical uncertainty (ATU) and was excluded for CA as well as for error calculation.
Because of the limited concentration ranges for most antibiotics on the test plate, MICs less or equal to the lowest concentration or greater than the highest concentration of a drug were included in the calculation.
Percentage (number) of strains for which the difference regarding all drugs tested was no more than ±1 log2 dilution.
The concentration ranges of ciprofloxacin, ertapenem, and meropenem included skipped concentrations. If the Reveal system and/or CDC & FDA AR data showed a MIC above the highest skipped concentration, the respective strain was excluded from EA calculation. This was the case for one strain each in test one and two.
For meropenem and CDC & FDA AR MIC data, eight strains in each test had MIC values of >8 mg/L or >16 mg/L. On AST plates used in the study, the highest concentration is 32 mg/L for meropenem, resulting in MIC values of >32 mg/L if a strain grows at 32 mg/L. Thus, the eight strains were excluded from EA calculation.
S, susceptible; I, susceptible increased dosage; R, resistant; EA, essential agreement; CA, category agreement; mE, minor error; ME, major error; VME, very major error.
TABLE 5.
AST results of the Reveal rapid AST system with blood cultures spiked with CDC & FDA AR bank isolates compared to CDC & FDA AR reference datag
| Drug | Reveal average TTR (h) | No. of strains with CDC & FDA AR reference AST data | % Agreement (no.) | % (no.) of errors | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Total | Total used for EA calc. | Total used for CA and error calc. | S | I | R | EAa,b | CA | mE | ME | VME | ATUc | ||
| 1st test | 3.8 | ||||||||||||
| Amikacin | 3.4 | 10 | 10 | 10 | 4 | 6 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Aztreonam | 4.9 | 7 | 7 | 7 | 7 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | |||
| Cefepime | 3.8 | 8 | 8 | 8 | 8 | 100.0 (8) | 100.0 (8) | 0 (0) | 0 (0) | 0 (0) | |||
| Cefotaxime | 3.4 | 7 | 7 | 7 | 7 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | |||
| Ceftazidime | 3.6 | 8 | 8 | 8 | 8 | 100.0 (8) | 100.0 (8) | 0 (0) | 0 (0) | 0 (0) | |||
| Ciprofloxacin | 3.2 | 10 | 9d | 9c | 9 | 100.0 (9) | 100.0 (9) | 0 (0) | 0 (0) | 0 (0) | 10.0 (1) | ||
| Ertapenem | 3.6 | 7 | 7 | 7 | 1 | 6 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | ||
| Gentamicin | 3.3 | 9 | 9 | 9 | 1 | 8 | 88.9 (8) | 88.9 (8) | 0 (0) | 100.0 (1) | 0 (0) | ||
| Imipenem | 4.3 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Levofloxacin | 3.6 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Meropenem | 5.4 | 10 | 2e | 10 | 1 | 1 | 8 | 100.0 (2) | 90.0 (9) | 10.0 (1) | 0 (0) | 0 (0) | |
| Piperacillin-tazobactam | 5.3 | 7f | 7 | 7 | 1 | 1 | 5 | 100.0 (7) | 85.7 (6) | 14.3 (1) | 0 (0) | 0 (0) | |
| Tobramycin | 3.2 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Trimethoprim-sulfamethoxazole | 3.2 | 9 | 9 | 9 | 9 | 100.0 (9) | 100.0 (9) | 0 (0) | 0 (0) | 0 (0) | |||
| 2nd test | 3.9 | ||||||||||||
| Amikacin | 3.5 | 10 | 10 | 10 | 4 | 6 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Aztreonam | 4.9 | 7 | 7 | 7 | 7 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | |||
| Cefepime | 3.9 | 8 | 8 | 8 | 8 | 100.0 (8) | 100.0 (8) | 0 (0) | 0 (0) | 0 (0) | |||
| Cefotaxime | 3.4 | 7 | 7 | 7 | 7 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | |||
| Ceftazidime | 3.6 | 8 | 8 | 8 | 8 | 100.0 (8) | 100.0 (8) | 0 (0) | 0 (0) | 0 (0) | |||
| Ciprofloxacin | 3.3 | 10 | 9d | 9c | 9 | 100.0 (9) | 100.0 (9) | 0 (0) | 0 (0) | 0 (0) | 10.0 (1) | ||
| Ertapenem | 3.6 | 7 | 7 | 7 | 1 | 6 | 100.0 (7) | 100.0 (7) | 0 (0) | 0 (0) | 0 (0) | ||
| Gentamicin | 3.3 | 9 | 9 | 9 | 1 | 8 | 100.0 (9) | 100.0 (9) | 0 (0) | 0 (0) | 0 (0) | ||
| Imipenem | 4.6 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Levofloxacin | 3.6 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Meropenem | 5.6 | 10 | 2e | 10 | 1 | 1 | 8 | 100.0 (2) | 90.0 (9) | 10.0 (1) | 0 (0) | 0 (0) | |
| Piperacillin-tazobactam | 5.3 | 7f | 7 | 7 | 1 | 1 | 5 | 100.0 (7) | 85.7 (6) | 14.3 (1) | 0 (0) | 0 (0) | |
| Tobramycin | 3.1 | 10 | 10 | 10 | 1 | 9 | 100.0 (10) | 100.0 (10) | 0 (0) | 0 (0) | 0 (0) | ||
| Trimethoprim-sulfamethoxazole | 3.2 | 9 | 9 | 9 | 9 | 100.0 (9) | 100.0 (9) | 0 (0) | 0 (0) | 0 (0) | |||
Because of the limited concentration ranges for most antibiotics on the test plate, MICs less or equal to the lowest concentration or greater than the highest concentration of a drug were included in the calculation.
Percentage (number) of strains for which the difference regarding all drugs tested was no more than ±1 log2 dilution.
CDC & FDA AR reference MIC of one K. aerogenes strain for ciprofloxacin was falling in the area of technical uncertainty (ATU) and was excluded for CA as well as for error calculation.
The concentration range of ciprofloxacin on the AST plate was ≤0.06 mg/L, 0.25–1 mg/L (the MIC of 0.125 mg/L was skipped). If a MIC of the Reveal system and/or CDC & FDA AR bank MIC was 0.25 mg/L, the respective strain was excluded from EA calculation. This was the case for 1 K. aerogenes strain.
For meropenem and CDC & FDA AR MIC data, eight strains had MIC values of >8 mg/L or >16 mg/L. On AST plates used in the study, the highest concentration was 32 mg/L for meropenem, resulting in MIC values of >32 mg/L if a strain grows at 32 mg/L. Thus, the eight strains were excluded from EA calculation.
Of eight strain-drug pairs, one MIC result for piperacillin-tazobactam and K. aerogenes was missing by the Reveal system, resulting in seven evaluable results.
TTR, time to result; calc., calculation; S, susceptible; I, susceptible increased dosage; R, resistant; EA, essential agreement; CA, category agreement; mE, minor error; ME, major error; VME; very major error.
The time to result in the highly resistant CDC & FDA AR strains was lower than in the more susceptible clinical strains (5.4 h ± 1.2 h) with 3.9 h ± 1.2 h (P < 0.0001).
Analysis of the factors that may have an influence on the occurrence of errors
The experimental setup and read-out allowed multiple variables, namely culture bottle type (aerobe vs anaerobe), the bacterial count in the blood from the positive blood culture bottle, the time to positivity (TTP, time until a blood culture was flagged positive), the species, and the tested antibiotic drug to have a potential impact on the occurrence of mE, ME, and VME.
In total of the 102 blood cultures included in the study, 53 were aerobe and 49 anaerobe bottles resulting in 1,030 drug-strain combinations in aerobe and 978 strain-drug combinations in anaerobe BCB. The type of blood culture bottle had no influence on the occurrence of category errors. No error occurred in 96.8% (997/1,030) of drug-strain combination from aerobe bottles and in 97.4% (953/978) from anaerobe bottles (P = 0.386). VME/ME/mE occurred in 1.75% (18/1,030)/0.48% (5/1,030)/0.97% (10/1,030) aerobe bottles and in 1.64% (16/978)/0.31% (3/978)/0.61% (6/978) anaerobe bottles (P = 0.846/0.525//0.368). We also performed this analysis on bug-drug level but again, and we found no significant influence of the bottle type on any of the errors (data not shown).
Bacterial count (CFU/mL) in the BCB at the time of testing (Sysmex) had no influence on the overall failure rate (BCBs with no failure 2,038 ± 1,593 CFU/mL (n = 1,950) vs BCBs with errors had bacterial counts of 2,233 ± 1,651 CFU/mL (n = 58) (P = 0.357).
BCBs with VME/ME/mE had a bacterial count of 2,342 ± 1,784 CFU/mL (n = 34)/2,027 ± 931 CFU/mL (n = 8)/2,104 ± 1,701 CFU/mL (n = 16), while BCBs with no errors had bacterial counts of 2,038 ± 1,591 CFU/mL (n = 1,974)/2,043 ± 1,597 CFU/mL (n = 2,000)/2,043 ± 1,594 CFU/mL (n = 1,992) (P = 0.270/0.978/0.879).
The time to positivity of the BCB (time in the BACT/ALERT before flagging positive) had no influence on the overall error occurrence rate (BCB with no error 13.0 h ± 9.0 h (n = 1,950) vs BCBs with errors 15.0 h ± 12 h (P = 0.073). BCBs with VME/ME/mE had a TTP of 15.0 h ± 11.0 h (n = 34)/11.0 h ± 3.0 h (n = 8)/18.0 h ± 16.0 h (n = 16) while BCBs with no errors had TTPs of 13.0 h ± 9.0 h (n = 1,974)/ 13.0 h ± 9.0 h (n = 2,000)/13.16 h ± 8.9 h (n = 1,992) (P = 0.194/0.465/0.045).
These results show longer TTP for samples with minor errors compared to the samples with no minor errors, indicating that TTP could have an impact on the occurrence of minor errors.
Analysis of the variables in a multivariate setting using GLMMs revealed no influence of TTP, BCB type, bacterial count, antibiotic drug, or species on the occurrence of categorical errors (Tables S6 and S7 ).
DISCUSSION
To our knowledge, this is the first study with the Reveal rapid AST (VITEK Reveal) system analyzing resistance testing of Gram-negative strains including Enterobacterales, directly from positive routine blood cultures by using a test panel that includes EUCAST breakpoints for respective drugs and interpretation according to EUCAST guidelines.
We found a very high overall EA and CA of 98.5% and 97.1%, respectively, between the Reveal rapid AST system and the reference method in the clinical part of the study as well as 99.6% and 97.9%, respectively, in the study part with bacterial challenge strains. Tibbetts et al. published similar performance characteristics for the Reveal system when using panels and breakpoints according to the Clinical and Laboratory Standards Institute (CLSI) (19). However, compared to the study of Tibbetts et al., we found a higher percentage of VME with 16.1% (n = 34/211, MicroScan AST plates) versus 1.3% (n = 3/232, Vitek2 AST cards) and 1.2% (n = 4/313, Sensititre AST plates), respectively, in Tibbetts study (19). In general, in both studies (our study and Tibbetts et al.), the number of resistant clinical isolates was low (<14%) (19). Low number of resistant strains may lead to a higher occurrence of VMEs if guidelines for MIC evaluation without an intermediate category are used, e.g., EUCAST, as it was in the present study.
In our study, the highest number of VMEs per drug occurred with amoxicillin-clavulanic acid (n = 14). It should be considered that EUCAST has no intermediate category for amoxicillin-clavulanic acid, and there were a large number of strains in the study that had MICs around the EUCAST breakpoint (susceptible ≤8 mg/L, resistant >8 mg/L) (16). Thus, measurement inaccuracies of only 1 log2 dilution step can lead to a VME. If only VMEs were considered which resulted from category discrepancies based on more than a 1 log2 dilution step difference between the Reveal MIC and the MIC determined by the reference method, the rate drops from 51.9% (14 VMEs/27 MICs determined as resistant to amoxicillin/clavulanic acid by the reference method) to 18.5% (= aVME: 5 VMEs/27 MICs determined as resistant to amoxicillin/clavulanic acid by the reference method). The aVME rate seems to be more realistic if drugs with S/R instead of S/I/R breakpoints are analyzed. However, the problem in susceptibility testing with amoxicillin-clavulanic acid, under consideration of EUCAST breakpoints, was also highlighted by Soares et al. in a study with 286 E. coli strains (including 159 amoxicillin-clavulanic acid resistant strains). Here, amoxicillin-clavulanic acid MICs around the EUCAST breakpoints also resulted in high VME percentages ranging from 10.5% for the Phoenix System (Becton Dickinson) to 27.3% for the epsilon test (“Etest method”, strips from AES) (20).
Amoxicillin-clavulanic acid was not tested by Tibbetts et al. as the CLSI panel includes ampicillin-sulbactam instead (19). In contrast to EUCAST, the CLSI interpose an intermediate category for beta-lactame/beta-lactamase inhibitors like amoxicillin-clavulanic acid (susceptible ≤ 8/4 mg/L; intermediate = 16/8 mg/L; resistant ≥ 32/16 mg/L), which results in minor and not in very major errors (21). Accordingly, Tibbetts et al. found no VME but a high number of mE of ampicillin-sulbactam when testing the Reveal rapid AST system (n = 29/33.7% in comparison to Sensititre; n = 16/18.4% to Vitek 2), which may reflect the general problem with testing aminopenicillin combination substances such as beta-lactam/beta-lactamase inhibitor combinations in strains with a MIC around the breakpoints discussed in several other studies (19, 20, 22, 23).
In both studies, low CA while high EA for amoxicillin-clavulanic acid (this study 85.9% CA vs. 91.9% EA) and ampicillin-sulbactam (Tibbetts et al. Senistitre 66.3% CA vs 98.8% EA; Vitek 2 81.6% CA vs 90.8% EA) was observed (19). In our case, all 14 strains showing a VME for amoxicillin-clavulanic acid had Reveal MICs of ≤8 mg/L categorized by EUCAST as susceptible and MicroScan WalkAway MICs of 16 (n = 9), 32 (n = 4), and 64 (n = 1) categorized as resistant.
In our opinion, this arises the demand for the implementation of an ATU for amoxicillin-clavulanic acid MICs—like EUAST has already implemented for disc diffusion of this antibiotic drug (16). The European standard for the evaluation of MIC test systems EN ISO 20776-2:2007 already has addressed this problem and defines test systems with a high EA of >97% as having acceptable accuracy even if the VME rate is >3% (24).
The second most common VME rate in our study was found for aminoglycosides [gentamicin (n = 6/34), tobramycin (n = 5/34), and amikacin (n = 4/34)]. Other studies like Cayci et al. have shown with various testing systems, including standard automated testing systems like the Vitek 2, the unreliability of aminoglycoside testing in Gram-negative bacteria and occurrence of VME (22, 23, 25, 26). However, in our study, the high VME rates may have occurred for the same reasons as discussed above for amoxicillin-clavulanic acid (low rate of resistant strains for these antibiotics and strains with MICs around the respective EUCAST S/R breakpoint). If only VMEs were considered which resulted from category discrepancies based on more than a 1 log2 dilution step difference between the Reveal MIC and the MIC determined by the reference method discussed above for amoxicillin/clavulanic acid, the VME rate drops for amikacin from 100% to 25%, for gentamicin from 85.7% to 14.3%, and for tobramycin from 71.4% to 14.3%.
A study from Couchot et al. using Reveal rapid AST system with EUCAST 2021 breakpoints to the evaluation susceptibility testing for Pseudomonas aeruginosa from blood cultures found VME and ME mostly in piperacillin/tazobactam, ceftazidime, ceftolozane/tazobactam, fluoroquinolones, and amikacin. Since we only included two P. aeruginosa strains in our study, we have no significant result for this species, but looking at the entirety of our strains, the problematic in testing beta-lactam/beta-lactamase inhibitor combinations and aminoglycosides seems to be similar to our study. Also, a high count of the ME strains in piperacillin/tazobactame were with MIC around the breakpoints similar to the amoxicillin/clavulanate errors in our study (27).
In the present study, we observed that there is a correlation between resistance rate and TTR. If strains were highly resistant to an antibiotic drug, they had a shorter TTR when tested with the Reveal rapid AST system, e.g., ampicillin (3.6 h), compared to strains that were susceptible to a drug such as meropenem (6.6 h). In line with this, the average time to result for all antibiotics in the clinical data set was significantly higher compared to that of the study part with highly resistant CDC & FDA AR strains. In our opinion, using the Reveal system for AST has a particular advantage at this point, as patients with resistant strains benefit especially from rapid diagnostics since empirical therapy is often ineffective here (13).
Nevertheless, it is also important to consider that the reveal procedure may not definitively rule out the possibility of polymicrobial infections. Therefore, we respectfully suggest an 18–24-h subculture, potentially on different agar plates, as a final control measure.
There were some limitations to this study. First, only a few Gram-negative species were included in the study because strains were collected by a routine manner. VMEs with clinical study strains occurred only while testing E. coli and K. pneumoniae which is probably the result of a bias caused by the lower number of other species. Thus, significant conclusions may be allowed only for those two species. Phenotypic AmpC and ESBL tests were not measured in this study because the Reveal system did not support the analysis at the time of the study. Since most clinical isolates in our study were highly susceptible to most antibiotics without significant numbers of ESBL, AmpC, or carbapenemase production, we used the challenge strains with highly resistant strains. Challenge strains were incubated in spiked blood culture bottles without human blood and thus the results are theoretically not comparable, ergo only used for proof of concept. The discrepancy analysis was not carried out according to the ISO 20776-2 (2021-12) standards but was performed using a completely different method (MTS gradient strip). Important antibiotics for multidrug-resistant strains like ceftolozane-tazobactam and ceftazidime-avibactam were included in the study but still from the 35 gram-negative antimicrobials in the test panel only 20 could be analyzed at this time. Due to panel concentrations, some antibiotics like aztreonam for Pseudomonas could not be included in the study. Also, one of the most used antibiotics in German hospitals ampicillin-sulbactam was not part of the test panel (https://avs.rki.de/Content/ReferenceData/AIReport.aspx). Because most antibiotics on the test plate showed limited concentration ranges, we considered on-scale MICs (MICs within the concentration range) as well as off-scale MICs (MICs below or equal the lowest concentration or above the highest concentration) for EA evaluation which may lead to overestimation of EA. To close these gaps further studies—also with Gram-positive isolates that were not part of our study—are necessary.
In conclusion, the Reveal rapid AST system is a reliable and accurate approach for the frequently represented bug-drug combinations, meeting the European ISO guideline for the performance of AST systems. The Reveal system provides MICs that could be interpreted by EUCAST or CLSI guidelines. Moreover, AST directly from blood cultures performed with the Reveal system saves time when compared to the conventional AST (MicroScan WalkAway) used in the present study, especially in resistant bacterial strains.
ACKNOWLEDGMENTS
We thank the laboratory staff of Wisplinghoff Laboratories for general assistance.
Consumables and testing system were provided by Reveal.
Contributor Information
Nathalie Jazmati, Email: n.jazmati@wisplinghoff.de.
Erin McElvania, NorthShore University HealthSystem Department of Pathology and Laboratory Medicine, Evanston, Illinois, USA.
ETHICS APPROVAL
The study was excluded from review by the responsible ethics committee due to the chosen study design.
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/jcm.00692-24.
Tables S1 to S7.
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
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Associated Data
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Supplementary Materials
Tables S1 to S7.