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Potential Impact of Rapid Blood Culture Testing for Gram-Positive Bacteremia in Japan with the Verigene Gram-Positive Blood Culture Test.

1. Introduction

Gram-positive bacteria are the most predominant microorganisms associated

with sepsis in healthcare settings and the most prevalent cause of bacteremia in patients with hematopoietic stem cell transplantation [1, 2]. Enterococcal bacteremia is associated with increased risk of mortality in patients with hematopoietic stem cell transplantation, irrespective of susceptibility to vancomycin [3, 4]. In cardiology units, infective endocarditis, infectious aneurysm, catheter-related bloodstream infections, or surgical site infections after cardiac surgeries are the major infections of which the most common causative microorganisms are Grampositive cocci [5-10]. In both medical units, early detection of Gram-positives and resistant markers is very critical in managing patient care, antibiotic stewardship, and preventing spread of resistant microorganisms.

As early intervention with antimicrobial therapy is associated with improved prognosis with each hour of delay associated with increased mortality, rapid diagnosis is critical [11]. The Verigene Gram-positive blood culture assay (BC-GP) (Nanosphere, Inc., Northbrook, IL) is a sample to result microarray system for identification of common Gram-positive bacteria and major resistance markers directly from positive blood culture. Although a number of studies have previously evaluated BC-GP reporting performance ranging from 92 to 99% agreement with conventional methodology [12-16], one limitation of previous reports has been that many of the studies have been from the United States as well as countries outside of Japan with only one published report of limited scope in Japan [17].

Genetic variation among bacterial lineages circulating in different geographical regions of the world can affect the sensitivity of molecular assays based on oligonucleotide probes to detect organisms or resistance markers. Studies in Hong Kong and Belgium have reported lower BC-GP performance [18,19].

The purpose of this study was to determine the potential impact on patient management and patient outcome of BCGP in specialized hospitalized settings within the Japanese healthcare environment, which faces a number of challenges. An increasingly aging population and the accompanying burden of increasing overall healthcare costs have challenged the healthcare infrastructure. Despite methicillin-resistant Staphylococcus aureus (MRSA) accounting for over 90% of the hospital-acquired infections caused by resistant bacteria in Japan [20], outsourcing of clinical microbiology testing or no weekend coverage is common in many healthcare facilities as part of cost-containment. This paper represents the first comprehensive evaluation of BC-GP in Japan to validate its clinical performance. The simulated blood culture study includes a library of well-characterized healthcare-associated MRSA (HA-MrSA), community-acquired MRSA (CA-MRSA), and vancomycin-resistant enterococci (VRE) strains circulating in Japan.

2. Methods

BC-GP was evaluated at Toranomon Hospital (TH) and Sakakibara Heart Institute (SHI), in accordance with site-specific institutional review board approved study protocols, during June 26, 2012, to March 6, 2013. TH is an 1168-bed general teaching hospital with a 123-bed hematological care unit for hematopoietic stem cell transplantation performing 140 to 160 hematopoietic stem cell transplants per year. The microbiology laboratory at the hospital operates daily during day hours. SHI is a 320-bed teaching hospital specializing in cardiovascular diseases with a caseload of over 1,500 open-heart surgeries per year. The microbiology laboratory in the hospital is operated by an outside commercial reference laboratory. The microbiology laboratory operates during the day shift on weekdays and closed on weekends and holidays.

Blood cultures were performed at SHI using BacT/ ALERT FA bottles and monitoring with BacT/ALERT 3D (bioMerieux, Marcy l'Etoile, France). TH used BACTEC Plus bottles and monitoring with BACTEC 9240 and FX (Becton Dickinson, Franklin Lakes, and NJ). Only one positive blood culture bottle containing Gram-positive cocci or bacilli per patient was included in the study. Two ml of positive blood culture medium was stored at -85C for retesting.

Routine microbiological identification and susceptibility testing of isolates were performed using conventional identification tests such as bile solubility, optochin disk susceptibility, and the MicroScan WalkAway system (Beckman Coulter, Pasadena, CA) at TH and the Vitek 2 system (bioMerieux, Marcy l'Etoile, France) at SHI. Cefoxitin screening for methicillin resistance was performed according to CLSI guidelines [21]. A latex agglutination test for detection of penicillin-binding protein PBP2a was also utilized [22].

BC-GP testing was performed on positive blood culture showing Gram-positive organisms according to the manufacturer's instructions. Briefly, a well-mixed 350 [micro]l sample of the blood culture media was pipetted into the sample well of the BC-GP nucleic extraction tray, placed onto the Verigene Processor SP for processing and analysis by the Verigene Reader.

An assessment was performed to determine the difference in time between reporting of results using BC-GP and culture-based identification and antimicrobial susceptibility results for 139 positive blood culture broths. For culture-based results, the time required until generation of the final report was the time between the Gram stain reading and entering of final identification and susceptibility results into the laboratory information system. For BC-GP, the time to result was the time between the Gram stain reading and entering of BC-GP results into the laboratory information system.

A challenge set of 208 simulated blood cultures was constructed using type, reference strains, and clinical strains from different geographical regions in Japan to evaluate BCGP. The clinical strains included organisms that presented challenges for commercial identification systems in previous clinical studies at TH and SHI. In addition, a library of well-characterized HA-MRSA, CA-MRSA, and VRE strains from Japan was also tested. Simulated blood culture studies were performed at Miroku Medical Laboratory (Saku City, Nagano Prefecture, Japan). Two hundred and eight strains were adjusted to a turbidity of approximately 100 CFU/ml in sterile saline. Three hundred [micro]l was inoculated into BACTEC Plus Aerobic/F bottles containing 8 to 10 ml of human whole blood (blood type O, Tennessee Blood Services, Memphis, TN) for a final inoculum of 30 CFU/bottle. A BACTEC Plus Anaerobic/F bottle was also inoculated for S. pneumoniae and the S. anginosus group. Each bottle was incubated in the BACTEC system until a positive signal was generated. If BCGP generated negative results, 11-fold dilution of the blood culture medium using sterile distilled water was retested.

Each of the positive blood culture isolates at the two hospital sites was stored in 10% skim milk (Difco) at -85C. Species identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Microflex LT with Biotyper ver. 3.0 software; Bruker Daltonik GmbH, Bremen, Germany) for all strains [23]. If the organism was not identified to the species level by MALDI-TOF MS (score value < 2.0) or identified as Micrococcus, Listeria, Staphylococcus other than S. aureus, Streptococcus other than S. pyogenes, and S. agalactiae, confirmatory testing by PCR-direct sequencing of 16S rDNA or sodA was performed at Juntendo University or Tokyo Women's Medical University [24-26]. Specific-PCR for detecting mecA [27] in all staphylococci and vanA, vanB in all enterococci was performed [28]. The methods used for SCCmec typing of community-acquired or healthcare-associated MRSA used to characterized strains have been previously described [29, 30].

Concordance was determined in comparison to results of the reference methods. There was agreement if BC-GP target detection agreed with the reference method at either the genus or the species level. The ninety-five percent confidence intervals (95% CI) and the paired /-test were determined using GraphPad StatMate (GraphPad Software Inc., San Diego, CA).

3. Results

In this study, the overall identification agreement between BC-GP and the reference method was 327/347 (94%) for prospective blood cultures at two clinical sites and simulated blood cultures. The combined agreement between PCR and BC-GP for mecA detection was 71/73 (97%) for prospective blood cultures and simulated blood cultures.

The combined identification accuracy from both hospital sites was 129/139 (93%). For monomicrobial cultures, the identification accuracy was 121/124 (98%). Agreement between PCR and BC-GP for mecA positivity was 51/53 (96%). Table 1 shows the results from TH. Overall, 96/104 (92%) organisms were correctly identified by BC-GP to the species or genus level including detection of resistance genes. As shown in Table 2, total agreement of BC-GP with the reference method at SHI was 33/35 (94%) organisms.

BC-GP reports the presence of mecA for only S. aureus and S. epidermidis. In this study, of the 102 staphylococcal strains, 72 were either S. aureus or S. epidermidis. Discordant results were due to undetectable mecA S. epidermidis organisms in polymicrobial cultures containing both mecA positive and mecA negative S. epidermidis. Of the 30 Staphylococcus spp., other than S. epidermidis and S. aureus, 21 (70%) were positive for mecA, including 2 S. lugdunensis, which could not be reported as mecA positive by BC-GP.

Table 3 shows the difference in time between the generation of BC-GP results and culture-based final identification and antimicrobial susceptibility results at both hospitals. BCGP results were available at a mean of 28.2 to 51.0 hours before culture-based final identification and susceptibility results at TH. At SHI, BC-GP generated results at a mean of 34.5 to 196.6 hours earlier. In comparing the time to final culture-based identification and susceptibility results at TH and SHI, with the exception of S. aureus, results for S. epidermidis and coagulase-negative staphylococci other than S. epidermidis, enterococci, and streptococci required significantly (P < 0.05) longer time at SHI (83.3, 123.6, 159.1, and 199.1 hours, resp.) compared to TH (40.8, 53.9, 36.1, and 53.5 hours, resp.).

Using simulated Gram-positive blood cultures, BC-GP correctly identified 198/208 (95%) of the organisms (Table 4). Six streptococci (3%) were either incorrectly identified or identified at the genus level only by BC-GP. With respect to the 4 BC-GP false-negative blood culture bottles, 1 S. pyogenes was correctly identified and 1 S. mitis generated a positive Streptococcus genus/S. pneumoniae signal following 11-fold dilution of the blood culture medium. The mecA gene was detected in 20/20 (100%) MRSA organisms representing community-acquired (SCCmec type IIa, IV, and V) and healthcare-associated (SCCmec type I, IIb, III, and nontypeable) strains by BC-GP. BC-GP detected 14/14 (100%) vanA and 20/20 (100%) vanB genes in well-characterized VRE strains from previous studies in Japan.

4. Discussion

The performance of BC-GP observed in our study was similar to previous reports [12-16, 31-33]. As clinical microbiology services are outsourced or not operating during off-shifts during weekdays and closed on weekends and holidays in many hospitals in Japan, rapid diagnostic testing has significant potential to impact patient care by dramatically decreasing the time to organism identification and antimicrobial susceptibility results.

BC-GP detects mecA in all staphylococci based on measurement of signal intensity; however, reporting is restricted to S. aureus and S. epidermidis based on an algorithm in which mecA is only reported when S. aureus or S. epidermidis is detected by BC-GP. Future versions of BC-GP should consider modification of the algorithm to allow reporting of mecA detection for staphylococci other than S. aureus or S. epidermidis as 70% of the 30 non-S. aureus and S. epidermidis strains in our study were methicillin-resistant. Although S. epidermidis is the major coagulase-negative staphylococcal pathogen, other staphylococci such as S. lugdunensis and S. haemolyticus are important pathogens in the healthcare environment [34, 35].

Polymicrobial positive blood cultures generated the majority of the discordance. In contrast, the performance of BC-GP was 121/124 (98%) in monomicrobial clinical blood cultures and 198/208 (95%) in simulated blood cultures. In this study, polymicrobial blood cultures accounted for 14/106 (13%) and 3/33 (9%), respectively, of the positive blood cultures at TH and SH. Combined, polymicrobial cultures represented 17/139 (12%) of the prospective clinical blood cultures which is consistent with previous studies reporting 6 to 20% of all bloodstream infections to be polymicrobial [36-38]. BC-GP correctly identified all of the organisms in 12/17 (70%) of polymicrobial cultures. In previous BCGP studies, the correct identification rates in polymicrobial cultures ranged from 57 to 86% [14-16, 31, 32]. As misleading information can affect clinical diagnosis, resulting in inappropriate selection of antimicrobial agents, there is a need to understand the limitations of BC-GP.

An additional concern with polymicrobial cultures is the clinical interpretation of mecA detection and staphylococcal detection. In our 17-polymicrobial cultures, 5 samples yielded 2 or 3 staphylococcal strains with or without mecA. This may lead to unnecessary use of vancomycin or underestimating infection caused by methicillin-resistant staphylococci. Repeating BC-GP on another set of blood cultures may lessen the risk of this problem. For monomicrobial cultures or simulated cultures, all of the discrepancies were observed with streptococci except for 1 S. caprae strain. BCGP misidentification for streptococci included 2 S. mitis identified as S. pneumoniae, no detection of 2 S. pneumoniae, 2 S. anginosus group, and 2 S. pyogenes. Previous reports have also reported similar results for S. mitis, S. oralis, and S. pneumoniae [14-16, 39]. As genetic relatedness among S. mitis, S. oralis, and S. pneumoniae is well known based on >99% homology of 16S rRNA gene sequences [24], BCGP results for S. pneumoniae or Streptococcus with alphahemolysis without anypositive species-specific signals should be carefully interpreted and confirmed by conventional methods such as optochin sensitivity or the bile solubility test. Interestingly, 11-fold dilution ofthe original blood culture medium can lead to detection of the Streptococcus signal and species signal (Table 4). A range of detection depending on the organism by BC-GP has been reported [40].

The major benefit of utilizing BC-GP is the earlier time for reporting identification and resistance determinants from positive blood cultures allowing for earlier selection of appropriate antimicrobial therapy and implementation of infection control measures such as isolation and contact precaution [39,41]. This has the potential of making a significant impact in the Japanese healthcare delivery system. The difference in time between BC-GP results and final culture-based identification and susceptibility results shown in Table 3 at TH is consistent with previous reports [14-16, 18, 32, 41]. On the other hand, earlier results of 80.7 to 196.6 hours for organisms other than S. aureus using BC-GP at SHI reflects the unavailability of clinical microbiology services during weekends and holidays. As clinical microbiology services are outsourced in many hospitals in Japan or limited to one shift during weekdays or not offered during weekends, the potential cost-benefits of retaining blood culture services in hospitals are significant. Furthermore, training of laboratory personnel in the general laboratory to recognize Grampositive cocci and bacilli will allow BC-GP testing over the weekend as well as evenings/nights. BC-GP provides the opportunity for hospitals to retain in-house a very critical laboratory service.

In conclusion, BC-GP provided accurate identification and detection of resistance markers compared with routine culture-based laboratory methods for Gram-positive organisms including CA-MRSA, HA-MRSA, and VRE strains circulating in Japan. Minimizing the time to optimizing antimicrobial therapy using BC-GP may contribute to reduced costs and improved patient care. In 2016, BC-GP received regulatory approval in Japan becoming the first multi-target molecular test for positive blood cultures approved as an in vitro diagnostic device to aid in the diagnosis of bacterial bloodstream infections. Additional studies will be necessary to validate the cost-effectiveness of BC-GP within the context of the Japanese healthcare delivery system.

http://dx.doi.org/10.1155/2017/4896791

Ethical Approval

This study was approved by the TH and SHI internal review boards.

Competing Interests

All the authors declare no competing interests.

Authors' Contributions

Ken Kikuchi designed and carried out the study and drafted the manuscript. Mari Matsuda, Shigekazu Iguchi, Tomonori Mizutani, Kaori Sansaka, Kenta Negishi, Kimie Shimada, Shigeyuki Notake, Hideji Yanagisawa, and Reiko Yabusaki carried out the laboratory works. Keiichi Hiramatsu, Michiru Tega-Ishii, Jun Umemura, Hiroshi Takahashi, Hideki Araoka, and Akiko Yoneyama supervised the data collection and coordinated and participated in designing the study.

Acknowledgments

This study was supported, in part, by a Grant-in-Aid (S0991013) from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), for the Foundation of Strategic Research Projects in Private Universities.

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Ken Kikuchi, (1,2,3) Mari Matsuda, (3) Shigekazu Iguchi, (1,3) Tomonori Mizutani, (3) Keiichi Hiramatsu, (3) Michiru Tega-Ishii, (2) Kaori Sansaka, (2) Kenta Negishi, (2) Kimie Shimada, (2) Jun Umemura, (2) Shigeyuki Notake, (4) Hideji Yanagisawa, (4) Hiroshi Takahashi, (5) Reiko Yabusaki, (6) Hideki Araoka, (6) and Akiko Yoneyama (6)

(1) Department of Infectious Diseases, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

(2) Sakakibara Heart Institute, 1-16-1 Asahimachi, Fuchu, Tokyo 183-0003, Japan

(3) Department of Infection Control Science, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

(4) Miroku Medical Laboratory Inc., 659-2 Innai, Saku, Nagano 384-2201, Japan

(5) East-West Diagnostics/Theranostics, San Francisco, CA 94105, USA

(6) Department of Infectious Diseases, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan

Correspondence should be addressed to Ken Kikuchi; kikuchi.ken@twmu.ac.jp

Received 18 October 2016; Revised 27 November 2016; Accepted 18 December 2016; Published 20 February 2017

Academic Editor: Jose Ramon Blanco
Table 1: Performance of the BC-GP assay at Toranomon Hospital.

                                         BC-GP (total)

Organism                    Number of    Number (%)
                            organisms    of isolates

                                         Correctly        Not
                                         identified     detected

Staphylococcus                  78         73 (94)        4(5)
  S. aureus                     16         16(100)
    Methicillin-sensitive       9          9 (100)
    Methicillin-resistant       7          7 (100)
  S. epidermidis                37         34 (92)        2(5)
    Methicillin--               2           1(50)      1 (50) (a)
    sensitive
    Methicillin-resistant       35         33 (94)      l(3) (b)
  S. lugdunensis                1          1 (100)
  Other CNS                     24         22 (92)        2(8)
    S. caprae                   9           8(89)        1(11)
    S. hominis                  8          8 (100)
    S. haemolyticus             4           3(75)      1(25) (d)
    S. capitis                  1          1 (100)
    S. schleiferi               1          1 (100)
    S. simulans                 1          1 (100)
Streptococcus                   9           7(78)        1(11)
  S. agalactiae                 1          1 (100)
  S. anginosus group            1          1 (100)
    S. constellatus             1        1 (100) (e)
  Other streptococci            7           5(72)        1(14)
    S. mitis                    2           1(50)
    S. inf antis                2           1(50)      1 (50) (g)
    S. tigurinus                2          2 (100)
    S. oralis                   1          1 (100)
Enterococcus                    17         16 (94)        1(6)
  E. faecalis                   2           1(50)      1 (50) (h)
    Vancomycin-sensitive        2           1(50)        1(50)
  E. faecium                    15        15 (100)
    Vancomycin-sensitive        15        15 (100)
Total                          104         96 (92)        6(6)
Other nontarget Gram-           15
positives
  Bacillus                      3
    B. subtilis                 2
    B. cereus                   1
  Corynebacterium               12
    C. striatum                 9
    C. jeikeium                 3

Total isolates                 119

                                       BC-GP (monomicrobial cultures)

Organism                                   Number of    Number (%)
                                           organisms    of isolates

                            Incorrectly                 Correctly
                             identified                 identified

Staphylococcus                  1(1)           71         70(99)
  S. aureus                                    16        16 (100)
    Methicillin-sensitive                      9          9 (100)
    Methicillin-resistant                      7          7(100)
  S. epidermidis                1(3)           34        34 (100)
    Methicillin--                              1          1 (100)
    sensitive
    Methicillin-resistant    1 (3) (c)         33        33 (100)
  S. lugdunensis                               1          1 (100)
  Other CNS                                    20         19(95)
    S. caprae                                  7           6(67)
    S. hominis                                 7          7 (100)
    S. haemolyticus                            3          3 (100)
    S. capitis                                 1          1 (100)
    S. schleiferi                              1          1 (100)
    S. simulans                                1          1 (100)
Streptococcus                  1(11)           6           5(83)
  S. agalactiae                                1          1 (100)
  S. anginosus group
    S. constellatus
  Other streptococci           1(14)           5           4(80)
    S. mitis                 1 (50) (f)        2           1(50)
    S. inf antis                               1          1 (100)
    S. tigurinus                               1          1 (100)
    S. oralis                                  1          1 (100)
Enterococcus                                   16        16 (100)
  E. faecalis                                  1          1 (100)
    Vancomycin-sensitive                       1          1 (100)
  E. faecium                                   15        15 (100)
    Vancomycin-sensitive                       15        15 (100)
Total                           2(2)           93         91(98)
Other nontarget Gram-                          10
positives
  Bacillus                                     2
    B. subtilis                                1
    B. cereus                                  1
  Corynebacterium                              8
    C. striatum                                5
    C. jeikeium                                3

Total isolates                                103

                            BC-GP (monomicrobial cultures)
Organism

                              Not      Incorrectly
                            detected    identified

Staphylococcus                1(2)
  S. aureus
    Methicillin-sensitive
    Methicillin-resistant
  S. epidermidis
    Methicillin--
    sensitive
    Methicillin-resistant
  S. lugdunensis
  Other CNS                   1(6)
    S. caprae                1(33)
    S. hominis
    S. haemolyticus
    S. capitis
    S. schleiferi
    S. simulans
Streptococcus                             1(27)
  S. agalactiae
  S. anginosus group
    S. constellatus
  Other streptococci
    S. mitis                            1 (50) (f)
    S. inf antis
    S. tigurinus
    S. oralis
Enterococcus
  E. faecalis
    Vancomycin-sensitive
  E. faecium
    Vancomycin-sensitive
Total                         1(1)         1(1)
Other nontarget Gram-
positives
  Bacillus
    B. subtilis
    B. cereus
  Corynebacterium
    C. striatum
    C. jeikeium

Total isolates

(a) Polymicrobial culture of methicillin-sensitive and methicillin-
resistant 5. epidermidis.

(b) Polymicrobial culture with E.faecium.

(c) Correctly identified as Staphylococcus, but not as 5.
epidermidis, S. aureus, or 5. lugdunensis.

(d) Polymicrobial culture with 5. tigurinus.

(e) "S. anginosus group" identified by the BC-GP assay is defined as
"correctly identified" for each species.

(f) Identified as 5. pneumoniae.

(g) Polymicrobial culture with methicillin-resistant 5. epidermidis.

(h) Polymicrobial culture with Escherichia coli.

Table 2: Performance of the BC-GP assay at Sakakibara Heart
Institute.

                                 BC-GP (total)

Organism                     Number of    Number (%)
                             organisms    of isolates

                                           Correctly         Not
                                           identified      detected

Staphylococcus                   24         24 (100)
  S. aureus                      7          7 (100)
    Methicillin- sensitive       6          6 (100)
    Methicillin-resistant        1          1 (100)
  S. epidermidis                 12         12 (100)
    Methicillin- sensitive       2           2(100)
    Methicillin-resistant        10         10 (100)
  S. lugdunensis                 1          1 (100)
  Other CNS                      4           4(100)
    S. hominis                   2          2 (100)
    S. haemolyticus              1          1 (100)
    S. capitis                   1          1 (100)
Streptococcus                    8          8 (100)
  S. pyogenes                    1            0(0)
  S. agalactiae                  1          1 (100)
  S. anginosus group             2          2 (100)
    S. anginosus                 2         2 (100f) (b)
  Other streptococci             4           4(100)
    S. oralis                    2          2 (100)
    S. sanguinis                 1          1 (100)
    S. parasanguinis             1          1 (100)
Enterococcus                     2           1(50)          1(50)
  E. faecalis                    1            0(0)       1 (100) (c)
    Vancomycin-sensitive         1            0(0)       l(100) (c)
  E. faecium                     1          1 (100)
    Vancomycin-sensitive         1          1 (100)
Listeria spp.                    1          1 (100)

Total                            35         33 (94)          1(3)

Other nontarget                  1
Gram-positives

Corynebacterium striatum         1

Total isolates                   36

                                      BC-GP (monomicrobial cultures)

Organism                                    Number of    Number (%)
                                            organisms    of isolates

                             Incorrectly                  Correctly
                              identified                  identified

Staphylococcus                                  22         22 (100)
  S. aureus                                     7          7 (100)
    Methicillin- sensitive                      6           6(100)
    Methicillin-resistant                       1          1 (100)
  S. epidermidis                                11         11 (100)
    Methicillin- sensitive                      2          2 (100)
    Methicillin-resistant                       9          9 (100)
  S. lugdunensis                                1          1 (100)
  Other CNS                                     3          3 (100)
    S. hominis                                  2          2 (100)
    S. haemolyticus
    S. capitis                                  1          1 (100)
Streptococcus                                   8           7(87)
  S. pyogenes                  l(100)a          1            0(0)
  S. agalactiae                                 1          1 (100)
  S. anginosus group                            2          2 (100)
    S. anginosus                                2        2 (100) (c)
  Other streptococci                            4          4 (100)
    S. oralis                                   2          2 (100)
    S. sanguinis                                1          1 (100)
    S. parasanguinis                            1          1 (100)
Enterococcus
  E. faecalis
    Vancomycin-sensitive
  E. faecium
    Vancomycin-sensitive
Listeria spp.                                   1          1 (100)

Total                            1(3)           31         30 (97)

Other nontarget                                 1
Gram-positives

Corynebacterium striatum                        1

Total isolates                                  32

                               BC-GP (monomicrobial cultures)

Organism

                                 Not        Incorrectly
                               detected      identified

Staphylococcus
  S. aureus
    Methicillin- sensitive
    Methicillin-resistant
  S. epidermidis
    Methicillin- sensitive
    Methicillin-resistant
  S. lugdunensis
  Other CNS
    S. hominis
    S. haemolyticus
    S. capitis
Streptococcus                                 103 (a)
  S. pyogenes                               1 (100) (b)
  S. agalactiae
  S. anginosus group
    S. anginosus
  Other streptococci
    S. oralis
    S. sanguinis
    S. parasanguinis
Enterococcus
  E. faecalis
    Vancomycin-sensitive
  E. faecium
    Vancomycin-sensitive
Listeria spp.

Total                                           1(3)

Other nontarget
Gram-positives

Corynebacterium striatum

Total isolates

(a) Identified to the genus level, but not to the species level.

(b) "S. anginosus group" identified by the BC-GP assay is defined as
"correctly identified" for each species.

(c)Polymicrobial culture with 5. epidermidis.

Table 3: Difference in time to final identification and antimicrobial
susceptibility report.

                      Difference in time to result (a)

Organism              Sakakibara Heart Institute

                          Mean (h)         Range

S. aureus             34.5 (P < 0.05)    21.5-46.8
S. epidermidis        80.7 (P < 0.05)    23.9-160.9
Coagulase-negative    121.1 (P < 0.05)   25.9-217.4
  staphylococci
Enterococcus spp.     156.6 (P < 0.05)   95.9-217.4
Streptococcus spp.    196.6 (P < 0.05)   42.3-502.6

                      Difference in time to result (a)

Organism              Toranomon Hospital

                          Mean (h)          Range

S. aureus             28.2 (P < 0.05)     19.6-47.0
S. epidermidis        38.3 (P < 0.05)     21.6-72.5
Coagulase-negative    51.4 (P < 0.05)     21.4-72.2
  staphylococci
Enterococcus spp.     33.6 (P < 0.05)     23.4-47.7
Streptococcus spp.    51.0 (P < 0.05)     23.4-69.2

(a) The difference in time between BC-GP result and final culture-
based identification and susceptibility results.

Table 4: Detection of Gram-positive bacteria and resistance genes in
simulated blood cultures by BC-GP.

Organism                               Total
                                     no. of
                                     strains    correctly
                                                identified

Staphylococcus                         54        54 (100)
  S. aureus                            35        35 (100)
    Methicillin-sensitive, mecA-       15        15 (100)
    Methicillin-resistant, mecA+       20        20 (100)
      Health-care associated           10        10 (100)
      Community acquired               10        10 (100)
  S. epidermidis                        1        1 (100)
    Methicillin-sensitive, mecA-        1        1 (100)
  S. lugdunensis                        8        8 (100)
  Other CNS                            10        10 (100)
    S. hominis                          2        2 (100)
    S. haemolyticus                     2        2 (100)
    S. saprophyticus                    2        2 (100)
    S. capitis                          1        1 (100)
    S. cohnii                           1        1 (100)
    S. warneri                          1        1 (100)
    S. pseudintermedius                 1        1 (100)
  Streptococcus                        87        77 (88)
  S. pyogenes                           9         8(89)
  S. agalactiae                         8        8 (100)
  S. dysgalactiae                       8        8 (100)
  S. anginosus group (b)               10         8 (80)
    S. anginosus                        3         2(66)
    S. constellatus                     4         3(75)
    S. intermedius                      3        3 (100)
  S. pneumoniae                        22        20 (91)
  Other streptococci                   30        25 (86)
    S. mitis                           12         9(75)
    S. oralis                           4        4 (100)
    S. infantis                         2        2 (100)
    S. mutans                           2         1(50)
    S. sobrinus                         1         0 (0)
    S. sanguinis                        1        1 (100)
    S. parasanguinis                    1        1 (100)
    S. peroris                          1        1 (100)
    S. australis                        1        1 (100)
    S. tigurinus                        1        1 (100)
    S. cristatus                        1        1 (100)
    S. gordonii                         1        1 (100)
    S. gallolyticus                     1        1 (100)
    S. lutetiensis                      1        1 (100)
Enterococcus                           57        57 (100)
  E. faecalis                          32        32 (100)
    Vancomycin-sensitive               18        18 (100)
    Vancomycin-resistant, vanA+         4        4 (100)
    Vancomycin-resistant, vanB+        10        10 (100)
  E. faecium                           25        25 (100)
    Vancomycin-sensitive                5        5 (100)
    Vancomycin-resistant, vanA+        10        10 (100)
    Vancomycin-resistant, vanB+        10        10 (100)

Listeria spp.                           5        5 (100)

Micrococcus spp.                        5        5 (100)

Total                                  208       198(95)

Organism                               No. (%) of isolates

                                       not       incorrectly
                                     detected     identified

Staphylococcus
  S. aureus
    Methicillin-sensitive, mecA-
    Methicillin-resistant, mecA+
      Health-care associated
      Community acquired
  S. epidermidis
    Methicillin-sensitive, mecA-
  S. lugdunensis
  Other CNS
    S. hominis
    S. haemolyticus
    S. saprophyticus
    S. capitis
    S. cohnii
    S. warneri
    S. pseudintermedius
  Streptococcus                        4(5)          6(7)
  S. pyogenes                       1 (11) (a)
  S. agalactiae
  S. dysgalactiae
  S. anginosus group (b)                            2 (20)
    S. anginosus                                  1 (34) (c)
    S. constellatus                               1(25) (c)
    S. intermedius
  S. pneumoniae                                    2(9) (d)
  Other streptococci                  3(10)         2 (7)
    S. mitis                          1 (8)       2 (17) (e)
    S. oralis
    S. infantis
    S. mutans                         1(50)
    S. sobrinus                      1 (100)
    S. sanguinis                                          S. parasanguinis
    S. peroris
    S. australis
    S. tigurinus
    S. cristatus
    S. gordonii
    S. gallolyticus
    S. lutetiensis
Enterococcus
  E. faecalis
    Vancomycin-sensitive
    Vancomycin-resistant, vanA+
    Vancomycin-resistant, vanB+
  E. faecium
    Vancomycin-sensitive
    Vancomycin-resistant, vanA+
    Vancomycin-resistant, vanB+

Listeria spp.

Micrococcus spp.

Total                                  4(2)         6 (3)

(a) Not detected initially, but positive using 11-fold diluted blood
culture sample.

(b) Streptococcus sp. belonging to the S. anginosusgroup is
identified as S. anginosus group by BC-GP.

(c) Positive signal for Streptococcus, but no signal for S. anginosus
group.

(d) Positive signal for Streptococcus, but no signal for S.
pneumoniae.

(e) Misidentified as S. pneumoniae.
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Title Annotation:Research Article
Author:Kikuchi, Ken; Matsuda, Mari; Iguchi, Shigekazu; Mizutani, Tomonori; Hiramatsu, Keiichi; Tega-Ishii,
Publication:Canadian Journal of Infectious Diseases and Medical Microbiology
Article Type:Report
Geographic Code:9JAPA
Date:Jan 1, 2017
Words:5956
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