Accuracy of the Thermo Fisher Scientific (Sensititre™) dry-form broth microdilution MIC product when testing ceftaroline

Diagnostic Microbiology and Infectious Disease 81 (2015) 280–282

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Accuracy of the Thermo Fisher Scientific (Sensititre™) dry-form broth microdilution MIC product when testing ceftaroline☆ Ronald N. Jones a,⁎, Nicole M. Holliday b, Ian A. Critchley c a b c

JMI Laboratories, North Liberty, IA, USA ThermoFisher Scientific, Cleveland, OH, USA Cerexa, Inc, Oakland, CA, USA

a r t i c l e

i n f o

Article history: Received 16 October 2014 Received in revised form 22 December 2014 Accepted 24 December 2014 Available online 30 December 2014 Keywords: Ceftaroline Susceptibility testing Sensititre® Validation

a b s t r a c t Ceftaroline, the active metabolite of the ceftaroline fosamil pro-drug, was the first advanced-spectrum cephalosporin with potent activity against methicillin-resistant Staphylococcus aureus to be approved by the US Food and Drug Administration for acute bacterial skin and skin structure infections. After 4 years of clinical use, few ceftaroline commercial susceptibility testing devices other than agar diffusion methods (disks and stable gradient) are available. Here, we validate a broth microdilution product (Sensititre™; Thermo Fisher Scientific, Cleveland, OH, USA) that achieved 99.2% essential agreement (manual and automated reading) and 95.3–100.0% categorical agreement, with high reproducibility (98.0–100.0%). Sensititre™ MIC values for ceftaroline, however, were slightly skewed toward an elevated value (0.5× log2 dilution step), greatest when testing for streptococci and Enterobacteriaceae. © 2015 Elsevier Inc. All rights reserved.

Ceftaroline is the active metabolite of a new parenteral cephalosporin (ceftaroline fosamil) having potent activity against multidrug-resistant (MDR) Gram-positive cocci including methicillin-resistant Staphylococcus aureus (MRSA) and ceftriaxone-resistant Streptococcus pneumoniae (Flamm et al., 2014; Jones et al., 2013b; Livermore et al., 2014; Moisan et al., 2010; Pfaller et al., 2014). Applying a dosing schedule justified by pharmacokinetic–pharmacodynamic analysis (Van Wart et al., 2014), ceftaroline clinical trials in hospitalized patients were successful against complicated skin and soft tissue infections and community-acquired pneumonia (Frampton, 2013), and those results were confirmed by postapproval effectiveness/safety evaluations (Casapao et al., 2014; Santos et al., 2013; Teflaro™, 2012). New and novel antimicrobials are urgently needed to address infections caused by emerging MDR species (Boucher et al., 2009), and ceftaroline has activity against staphylococci having reduced susceptibility to daptomycin, linezolid, and vancomycin (Sader et al., 2013). To measure ceftaroline activity in clinical microbiology laboratories, quality diagnostic devices are required to recognize its utility as strains of staphylococci have been reported with elevated (N2 μg/mL) ceftaroline MIC values (Mendes et al., 2012). In this presentation, we present a multisite evaluation/validation of a quantitative, dry-form broth microdilution test (Sensititre™; Thermo Fisher, Cleveland, OH, USA) to expand ceftaroline susceptibility tests beyond disk and stable gradient agar diffusion methods (CLSI, 2008) or the use of surrogate marker testing (Jones et al., 2013a). ☆ Note: These data were presented in part at the 2009 ASM meeting, Abstr. C-015. ⁎ Corresponding author. Tel.: +1-319-665-3370; fax: +1-319-665-3371. E-mail address: [email protected] (R.N. Jones). http://dx.doi.org/10.1016/j.diagmicrobio.2014.12.010 0732-8893/© 2015 Elsevier Inc. All rights reserved.

All ceftaroline MIC values were compared over the ≤0.004–64 μg/mL dilution range in the dry-form Sensititre commercial and the frozen-form reference panels (CLSI, 2012; 2014). The MIC endpoints were read manually (CLSI, 2014) and by the Sensititre automated device. Five quality control (QC) organisms were used: S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, S. pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247; all QC results were within published limits (CLSI, 2014) with 67.0% of the MIC values at the midpoint of the QC ranges. Colony counts to monitor the inoculum concentrations across the 3 laboratories and 5 QC strains demonstrated an average density of 3.1 × 10 5 CFU/mL (range, 1.5–5.2 × 10 5 CFU/mL), i.e., acceptable inoculum preparations. Three laboratories participated in this validation using a wellrecognized study design (CLSI, 2008; Jones et al., 2004). Two organism collections (clinical isolates contributed by the sites/challenge strains including resistance phenotypes) were tested as follows: S. aureus (142/27; includes MRSA), coagulase-negative staphylococci (CoNS; 92/19, dominantly methicillin-resistant isolates), enterococci (91/17; includes vancomycin-resistant isolates), β-hemolytic streptococci (βHS; 200/25), viridans group streptococci (VGS; 105/25, includes penicillin-nonsusceptible isolates), S. pneumoniae (152/25; includes penicillin- and ceftriaxone-resistant isolates), Enterobacteriaceae (258/58; includes 32.9% ceftaroline-resistant isolates), nonenteric bacilli (74/11), Acinetobacter spp. (76/6), and H. influenzae (101/50), for a total of 1554 strains. Data were analyzed by comparing the dry-form Sensitire result to the reference MIC for all results and for those where both compared values were within the tested MIC ranges for both methods or “on-scale” (OS). When both values were within +1 doubling dilution step (essential agreement [EA]), this was considered acceptable

R.N. Jones et al. / Diagnostic Microbiology and Infectious Disease 81 (2015) 280–282

at ≥95.0%. Categorical agreement (CA) was calculated by comparing the categorized MIC results using the US Food and Drug Administration (USA-FDA) ceftaroline product package insert breakpoint criteria (Teflaro™, 2012). Only 5 organism groups (S. aureus, βHS, S. pneumoniae, H. influenzae, and Enterobacteriaceae) could be analyzed for CA, but this included 1036 organisms. One hundred strains were used to determine test reproducibility; each organism tested in triplicate by each of the 3 laboratories. Table 1 presents the direct MIC comparisons of the commercial Sensititre product compared to the reference MIC (CLSI, 2012) for all 1554 organisms, including 1074 OS results. Ten organism groups were analyzed, of which 5 are among those species indicated for ceftaroline therapy by the USA-FDA (Teflaro™, 2012). For all MIC comparisons, 633 (49.0%) and 164 (62.4%) results showed the exact (same) value for the clinical and challenge organism populations, respectively (Table 1). Similarly, the OS comparisons had 41.1–50.3% of MICs having the same ceftaroline value. A slight skewing of Sensititre™ MIC results toward a 2-fold high value was observed for enterococci, βHS, VGS, S. pneumoniae, and Enterobacteriaceae. Overall, the OS comparisons revealed a Sensititre ceftaroline modal MIC that was 1 doubling dilution higher, especially among clinical strains (Table 1), and this trend was more likely to occur among the Gram-positive pathogens (Table 2). When the MIC50 and MIC90 results produced by the Sensititre product were compared to reference (CLSI, 2014) MIC results for all 10 organism groups, an elevated MIC50 was observed for Sensititre with enterococci (4 versus 2 μg/mL), VGS (0.03 versus 0.015 μg/mL), and the Enterobacteriaceae (0.5 versus 0.25 μg/mL). Similarly, ceftaroline MIC90 results by the Sensititre system were higher by 1 doubling dilution for S. aureus

281

Table 2 Summary of Gram-positive (920) and Gram-negative (634) strain MIC results comparing Sensititre™ MIC performance to the reference frozen-form MIC values from 3 laboratories. Organisms (no.tested)/compared MICs

Gram-positive cocci All comparisons (920) OS (684) Gram-negative All comparisons (634) OS (390) All data All comparisons (1554) OS (1074)

Sensititre™ MIC variation log2 dilutions: ≥−2

−1

Exact

+1

≥+2

2 1

37 32

412 291

461a 358a

8 2

2 2

61 53

385 167

185 168a

1 0

4 3

98 85

797 458

646 526a

9 2

EA between methods was 99.2% overall. a Represents a significant skewing toward a higher MIC value and a modal MIC shift.

(1 versus 0.5 μg/mL), βHS (0.03 versus 0.015 μg/mL), VGS (0.25 versus 0.12 μg/mL) S. pneumoniae (0.25 versus 0.12 μg/mL), and H. influenzae (0.03 versus ≤0.015 μg/mL). The EA (results + 1 log2 dilution) was 98.9% (OS = 99.6%) and 99.5% (OS = 99.5%) for the Gram-positive and Gram-negative strains, respectively (99.2% overall). The automated system read results demonstrated a 98.6% EA with 13 of 19 errors being noted among streptococci. Using the contemporary USA-FDA breakpoints for ceftaroline when testing all indicated species (Teflaro™, 2012), the CA was as follows: for S. aureus at 98.2%; for βHS at 99.6%; for S. pneumoniae at 100.0%; for H. influenzae at 100.0%; and for Enterobacteriaceae at 95.6%, i.e., acceptable

Table 1 Comparison of ceftaroline Sensititre™ MIC results to those of the reference broth microdilution method stratified for all tested organisms (1554) and those having OS endpoints. Organisms(no. tested)/collectiona

Sensititre™ MIC variation in log2 dilutions: All comparisons ≥−2

S. aureus Clinical (142) Challenge (27) CoNS Clinical (92) Challenge (19) Enterococcus Clinical (91) Challenge (17) βHS Clinical (200) Challenge (25) VGS Clinical (105) Challenge (25) S. pneumoniae Clinical (152) Challenge (25) Enterobacteriaceae Clinical (258) Challenge (58) Non-enterics Clinical (74) Challenge (11) Acinetobacter spp. Clinical (76) Challenge (6) H. influenzae Clinical (101) Challenge (50) All strains Clinical (1291) Challenge (263) Combined (1554)

OS comparisons

−1

Exact

≥+2

≥−2

−1

Exact

0 0

2 0

75 15

65 12

0 0

0 0

2 0

74 15

65 12

0 0

0 0

8 0

62 9

21 10

1 0

0 0

5 0

39 4

17 9

0 0

0 0

2 0

56 15

33 2

0 0

0 0

2 0

23 14

33b 2

0 0

0 0

1 0

72 11

127b 14b

0 0

0 0

1 0

43 1

74b 11b

0 0

1 0

5 3

35 14

63b 7

1 1

1 0

5 2

27 11

62b 7

1 1

1 0

10 6

32 16

104b 3

5 0

0 0

9 6

24 16

63b 3

0 0

0 2

28 11

11 27

119b 18

0 0

0 2

25 9

76 19

115b 18

0 0

0 0

5 0

44 10

25 1

0 0

0 0

5 0

35 9

24 1

0 0

0 0

12 2

59 3

5 1

0 0

0 0

12 2

23 0

4 1

0 0

0 0

3 0

87 44

10 6

1 0

0 0

0 0

5 0

3 2

0 0

2 2 4

76 22 98

633 164 797

572 74 646

8 1 9

1 2 3

66 19 85

369 89 458

+1

Comparisons listed as (−) and (+) have MIC values lower and higher than the reference method result, respectively. a Clinical = recent clinical isolates; challenge = challenge isolates having key resistance phenotypes from stock culture collections. b Significant skewing of MIC mode toward a higher result.

+1

460b 66 526b

≥+2

1 1 2

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performance. CA was high (95.6–100.0%) and error types remained low for false susceptibility (0.0–0.4%, very major error), false resistance (0.0%, major error), and minor type (0.0–4.4%, highest for Enterobacteriaceae). The reproducibility phase of the experiment (100 strains) exhibited results +1 log2 dilution within and between laboratories for 96.0 (streptococci, H. influenzae) to 100.0% of results. Identical MIC modes were noted for 60.0–100.0% of organisms in the evaluation, again lowest for the streptococci. Ceftaroline, the only β-lactam marketed in the United States with proven activity against MRSA (Frampton, 2013), has been increasingly applied in clinical practice since 2010 including for MDR Gram-positive pathogen infections (Casapao et al., 2014; Flamm et al., 2014; Sader et al., 2013; Santos et al., 2013). To direct ceftaroline treatments and provide postregulatory approval resistance surveillance (Mendes et al., 2012), clinical microbiology laboratories require quality, validated susceptibility testing devices (CLSI, 2008; Jones et al., 2004). To this end, we presented a multilaboratory validation study (Tables 1 and 2) of Sensititre™ demonstrating highly acceptable EA (99.2%), CA (95.3–100%), and reproducibility (96.0–100.0%). Finally, this dry-form broth microdilution product (Sensititre™) generally produces a slightly higher ceftaroline MIC (0.5 × log2 dilution step) result when compared to the reference CLSI (2012) method, regardless of manual or automated reading of endpoints. Because ceftaroline-nonsusceptible S. aureus and streptococcal clinical isolates are so unusual (Flamm et al., 2014; Jones et al., 2013b; Mendes et al., 2012; Pfaller et al., 2014), such organisms should be confirmed by repeated testing or referral to a reference laboratory (CLSI, 2012; 2014; Flamm et al., 2014). Acknowledgments We wish to express our appreciation to the JMI Laboratories (P. Rhomberg, J.M. Streit; North Liberty, IA), Kaiser Permanente (L. Sykes; Cleveland, OH), Forest/Cerexa (D. Biek, J. Ge, K. Krause, G. Williams; Oakland, CA), and Thermo Fisher Scientific (C.M. Bastilli, C.C. Knapp, S.B. Killian; Cleveland, OH) staff members for scientific and technical assistance in performing this study. This investigation was supported by a grant from Forest/Cerexa, and JMI Laboratories also received compensation fees for services in relation to preparing the manuscript, which was funded in part by this sponsor. JMI Laboratories has additionally received research and educational funding in 2012–2014 from – Achaogen, Actelion, Affinium, American Proficiency Institute, AmpliPhi Bio, Anacor, Astellas, AstraZeneca, Basilea, BioVersys, Cardeas, Cempra, Cubist, Daiichi, Dipexium, Durata, Fedora, Furiex, Genentech, GlaxoSmithKline, Janssen, Johnson & Johnson, Medpace, Meiji Seika Kaisha, Melinta, Merck, Methylgene, Nabriva, Nanosphere, Novartis, Pfizer, Polyphor, Roche, Seachaid, Shionogi, Synthes, The Medicines Co. (Rempex), Theravance, ThermoFisher, Venatorx, Vertex, Waterloo, and some other corporations. Some JMI

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