ABSTRACT
Disk diffusion and MIC quality control (QC) ranges were determined for nafithromycin, a new lactone-ketolide, following the completion of a nine-laboratory, Clinical and Laboratory Standards Institute (CLSI) document M23-defined tier 2 study. Five QC strains consistent with the spectrum of activity of nafithromycin were tested: Staphylococcus aureus ATCC 25923 (disk only), S. aureus ATCC 29213 (broth only), Enterococcus faecalis ATCC 29212 (broth only), Streptococcus pneumoniae ATCC 49619 (disk and broth), and Haemophilus influenzae ATCC 49247 (disk and broth). Nafithromycin disk diffusion QC ranges were determined to be 25 to 31 mm for S. aureus ATCC 25923, 25 to 31 mm for S. pneumoniae ATCC 49619, and 16 to 20 mm for H. influenzae ATCC 49247. Nafithromycin MIC QC ranges were determined to be 0.06 to 0.25 μg/ml for S. aureus ATCC 29213, 0.016 to 0.12 μg/ml for E. faecalis ATCC 29212, 0.008 to 0.03 μg/ml for S. pneumoniae ATCC 49619, and 2 to 8 μg/ml for H. influenzae ATCC 49247. All disk diffusion and MIC QC ranges established in this study were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting and were initially reported in the 2017 M100S document. The QC ranges established in this study should be used for determining the in vitro activity of nafithromycin in phase 2 and phase 3 human clinical trials and subsequently for testing patient isolates and isolates in phase 4 surveillance studies.
INTRODUCTION
Nafithromycin, also known as WCK 4873, is a new orally bioavailable lactone-ketolide currently under development by Wockhardt Bio AG (Switzerland) for the treatment of community-acquired bacterial pneumonia (Fig. 1). Nafithromycin has completed phase 1 single- and multiple-ascending-dose studies in Europe and an intrapulmonary pharmacokinetic study in the United States. Nafithromycin is currently in phase 2 clinical studies for the treatment of community-acquired bacterial pneumonia. Nafithromycin was granted qualified infectious disease product (QIDP) status by the U.S. Food and Drug Administration in 2015. In preliminary studies, nafithromycin demonstrated both in vitro and in vivo activity against macrolide-resistant and telithromycin-intermediate and -resistant pneumococci as well as against other pathogens associated with community-acquired pneumonia (1–3).
Chemical structure of nafithromycin.
To monitor the performance of in vitro susceptibility tests, it is essential to establish the limits of variability that are acceptable when testing relevant quality control (QC) strains. Clinical and Laboratory Standards Institute (CLSI) document M23-defined tier 2 studies evaluate the reproducibility of reference antimicrobial susceptibility testing methods within a laboratory, among different laboratories, and among reagent lots (4). QC ranges established by tier 2 studies are reported in applicable CLSI standard documents such as document M100 (5) and are used for determining the in vitro activity of an agent in phase 2 and phase 3 human clinical trials and subsequently for testing patient isolates and isolates in phase 4 surveillance studies. In this report, we describe the results of the multilaboratory CLSI document M23-defined tier 2-qualifying study used to establish expected QC ranges for nafithromycin for applicable ATCC strains tested by disk diffusion and broth dilution. The results of this study were approved at the June 2015 meeting of the CLSI Subcommittee on Antimicrobial Susceptibility Testing and were first reported in 2017 in document M100S (5).
(This study was presented in part at the ASM Microbe 2016 Conference, Boston, MA, 2016 [6].)
RESULTS
Disk diffusion results for nafithromycin for Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247 for the nine participating laboratories are shown in Tables 1↴ to 3. Minor differences in disk diffusion results between the two lots of nafithromycin disks and three lots of agar were observed: 94.9 to 98.9% (by medium lot) and 96.7 to 97.4% (by disk lot) of zone sizes were within the range of 25 to 31 mm for S. aureus ATCC 25923, 98.3 to 99.4% (by medium lot) and 98.5 to 99.3% (by disk lot) of zone sizes were within the range of 25 to 31 mm for S. pneumoniae ATCC 49619, and 98.3 to 100% (by medium lot) and 99.3 to 99.6% (by disk lot) of zone sizes were within the range of 16 to 20 mm for H. influenzae ATCC 49247. The results for telithromycin, the control agent, were within the CLSI control limits for each QC organism tested on each day of testing (data not shown). The following proposed QC ranges, shown in boldface type in each table, were generated: a 7-mm range of 25 to 31 mm for S. aureus ATCC 25923, which included 97.0% of all reported results; a 7-mm range of 25 to 31 mm for S. pneumoniae ATCC 49619, which included 98.7% of all reported results; and a 5-mm range of 16 to 20 mm for H. influenzae ATCC 49247, which included 99.4% of all reported results. Because >95% of all zones reported from at least seven laboratories were within the proposed ranges for S. aureus ATCC 25923, S. pneumoniae ATCC 49619, and H. influenzae ATCC 49247 (4), each of the proposed QC ranges was accepted by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting.
Medium lot, disk lot, and interlaboratory comparisons for nafithromycin disk diffusion testing results for S. aureus ATCC 25923a
Medium lot, disk lot, and interlaboratory comparisons for nafithromycin disk diffusion testing results for S. pneumoniae ATCC 49619a
Medium lot, disk lot, and interlaboratory comparisons for nafithromycin disk diffusion testing results for H. influenzae ATCC 49247a
MIC results for nafithromycin for S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, S. pneumoniae ATCC 49619, and H. influenzae ATCC 49247 for the nine participating laboratories are shown in Tables 4↴↴ to 7. No differences in MIC results among the three broth manufacturers were observed. The results for telithromycin, the control agent, were within the CLSI control limits for each QC organism tested on each day of testing (data not shown). The following proposed QC ranges were generated: a three-doubling-dilution range of 0.06 to 0.25 μg/ml for S. aureus ATCC 29213, which included 100% of all reported results; an initial three-doubling-dilution range of 0.016 to 0.06 μg/ml for E. faecalis ATCC 29212, which included 98.5% of all reported results and was expanded to include 0.12 μg/ml (0.016 to 0.12 μg/ml) due to the significant shoulder at 0.06 μg/ml; a three-doubling-dilution range of 0.008 to 0.03 μg/ml for S. pneumoniae ATCC 49619, which included 100% of all reported results; and a three-doubling-dilution range of 2 to 8 μg/ml for H. influenzae ATCC 49247, which included 100% of all reported results. Because >95% of all MICs reported from at least seven laboratories were within the proposed ranges for S. aureus ATCC 29213, E. faecalis ATCC 29212, S. pneumoniae ATCC 49619, and H. influenzae ATCC 49247 (4), each of the proposed QC ranges was accepted by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting. The proposed QC ranges for all five ATCC strains tested for disk diffusion and broth dilution are summarized in Table 8.
Medium lot and interlaboratory comparisons for nafithromycin MIC testing results for S. aureus ATCC 29213a
Medium lot and interlaboratory comparisons for nafithromycin MIC testing results for E. faecalis ATCC 29212a
Medium lot and interlaboratory comparisons for nafithromycin MIC testing results for S. pneumoniae ATCC 49619a
Medium lot and interlaboratory comparisons for nafithromycin MIC testing results for H. influenzae ATCC 49247a
Quality control ranges for nafithromycin disk diffusion and broth microdilution testing of five ATCC strainsa
DISCUSSION
Establishing QC ranges for antimicrobial susceptibility testing is critical for ensuring accurate and reproducible reporting of results (4). This is achieved, in part, by the testing of carefully selected QC strains with known susceptibilities to the antimicrobial agents tested (4). The goals of a QC program are to monitor the precision (reproducibility) and accuracy of susceptibility testing procedures, the performance of reagents used in tests, and the performance of persons who carry out the tests and report the results (4).
The current study design met CLSI document M23 requirements for tier 2 QC studies to establish disk diffusion and MIC ranges for a new antimicrobial agent: 7 laboratories/independent sites, 3 medium lots (different manufacturers), 2 disk lots, and 10 replicates of each QC strain per laboratory. More than 95% of all MIC values reported from the nine participating laboratories were within the QC ranges shown in Table 8. QC results for nafithromycin were highly reproducible in this document M23-defined tier 2 study. The results from this multilaboratory QC study were intended to provide initial ranges for routine susceptibility testing using disk diffusion and broth microdilution methods (7, 8) as nafithromycin progresses through phase 2 and phase 3 human clinical trials. All QC ranges for nafithromycin were presented to the CLSI Subcommittee on Antimicrobial Susceptibility Testing in June 2015 and were approved for future publication in document M100 (5).
MATERIALS AND METHODS
Nine laboratories participated in this CLSI document M23-defined tier 2 study (4). They were Laboratory Specialists, Inc., Westlake, OH (L. Koeth); the Clinical Microbiology Institute, Wilsonville, OR (M. Traczewski); the UCLA Medical Center, Los Angeles, CA (J. Hindler); Thermo Fisher Scientific, Oakwood Village, OH (C. Knapp); Microbial Research, Inc., Fort Collins, CO (D. Bade); the University of Washington Medical Center, Seattle, WA (S. Rivera); the University of Rochester Medical Center, Rochester, NY (D. Hardy); Loyola University Medical Center, Maywood, IL (P. Schreckenberger); and International Health Management Associates, Inc. (IHMA), Schaumburg, IL (M. A. Hackel). Each of the participating laboratories is an experienced microbiology facility, and each laboratory followed CLSI procedures for disk diffusion (7) and broth microdilution (8) testing exactly as written. Supplies, including frozen broth microdilution panels, agar plates, and disks, were distributed by IHMA to the other eight sites.
Five ATCC QC strains consistent with the spectrum of activity of nafithromycin were tested: Staphylococcus aureus ATCC 25923 (disk only), S. aureus ATCC 29213 (broth only), Enterococcus faecalis ATCC 29212 (broth only), Streptococcus pneumoniae ATCC 49619 (disk and broth), and Haemophilus influenzae ATCC 49247 (disk and broth). Telithromycin was tested in parallel with nafithromycin as a control agent of the same class (4).
For disk diffusion studies, two lots of 15-μg nafithromycin disks (lot 351202 and lot 351203) from one manufacturer were available at the time of testing (MAST, Merseyside, UK); disks were prepared by using nafithromycin powder (batch 12; 95.3% assay purity) provided by Wockhardt Bio AG. Telithromycin disks (15 μg) were prepared by BD/BBL (Sparks, MD) using telithromycin powder (CAS 191114-48-4) purchased from Toku-E (Bellingham, WA). Mueller-Hinton agar (MHA), MHA with sheep blood, and Haemophilus test medium (HTM) agar were purchased from three manufacturers for disk diffusion testing: Hardy Diagnostics (Santa Maria, CA), Remel (Lenexa, KS), and BD/BBL (4). The agar lot numbers were H11-15064 (Hardy), 652358 (Remel), and 5019658 (BD/BBL) for MHA; H21-15057 (Hardy), 642981 (Remel), and 5048528 (BD/BBL) for MHA with sheep blood; and H07-15064 (Hardy), 657915 (Remel), and 5026978 (BD/BBL) for HTM agar. Each ATCC strain was tested and the results were read according to genus/species-specific CLSI standards for disk diffusion testing (7, 9).
The two lots of nafithromycin disks and the telithromycin disks were tested against the three QC strains for disk diffusion (S. aureus ATCC 25923, S. pneumoniae ATCC 49619, and H. influenzae ATCC 49247) on the appropriate agar type provided by the three manufacturers, for 10 replicates each at every testing site. Nafithromycin results per QC strain were accepted only when the QC strain results were within the CLSI-approved range for telithromycin (9). Testing generated 60 nafithromycin results (zone diameter values in millimeters) per site and a total of 540 zone diameters for each QC stain tested at all nine sites. Telithromycin was tested with one lot of disks on three different medium lots for 10 replicates, yielding 30 results per site. No more than four inocula were tested on any given day. All inoculum densities were confirmed by colony counts of the initial inoculum on drug-free agar medium (7, 9).
Reference frozen-form broth microdilution panels were prepared by IHMA according to CLSI guidelines (8, 9) and shipped frozen to the other eight participant sites. Nafithromycin stock solutions were prepared by using a one-half volume of water (solvent), dissolved by the dropwise addition of glacial acetic acid (acetic acid not to exceed 2.5 μl/ml), and diluted to its final concentration by using water (5). Cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB with 3% laked (lysed) horse blood, and HTM broth were each purchased from three manufacturers. CAMHB broth was purchased from Oxoid (Basingstoke, Hampshire, England) (lot 1583507), Fluka (St. Louis, MO) (lot BCBG3391V), and BD/BBL (lot 2277394). HTM broth was purchased from Hardy Diagnostics (lot 105069), Remel (lot 633652), and Teknova (Hollister, CA) (lot h580002c1501). Laked (lysed) horse blood was purchased from Hemostat Blood Products (Dixon, CA) (lot 230595-5). Participating laboratories were instructed to read endpoints in accordance with the guidelines in CLSI document M07-A10 (8). Each laboratory was instructed to prepare and test 10 separately adjusted inocula of each of the four following QC strains: S. aureus ATCC 29213, E. faecalis ATCC 29212, S. pneumoniae ATCC 49619, and H. influenzae ATCC 49247. Each inoculum was tested on supplied panels containing medium from each of the three medium manufacturers. No more than four inocula were tested on any given day. Panels included nafithromycin and telithromycin as a control. All inoculum densities were confirmed by colony counts of the initial inoculum on drug-free agar medium (8, 9). Nafithromycin results per QC strain were included in the data set only when the telithromycin QC was within its range. Testing generated 30 MIC (micrograms per milliliter) results per site, for a total of 270 MIC results for each QC strain tested. Telithromycin was tested on one lot of medium, generating 10 MIC results per site. Proposed QC ranges for nafithromycin for all ATCC strains for both disk diffusion and broth dilution testing were generated by using document M23 criteria (CLSI RangeFinder tool [http://clsi.org/standards/micro/rangefinder/ ]) (4) and the method of Gavan et al. (10), which uses the overall median ± half the median range of the individual laboratory measurements as determining parameters.
ACKNOWLEDGMENTS
M.A.H. and D.F.S. are employees of IHMA. J.A.K. is a consultant for IHMA and an employee of the University of Manitoba and Diagnostic Services Manitoba. None of the IHMA authors or J.A.K. has a personal financial interest in the sponsor of this paper (Wockhardt Research Center).
All authors provided analysis input and have read and approved the final manuscript.
FOOTNOTES
- Received 22 June 2017.
- Returned for modification 13 July 2017.
- Accepted 24 July 2017.
- Accepted manuscript posted online 26 July 2017.
- Copyright © 2017 American Society for Microbiology.
