全自動遺伝子解析装置を用いた

課題番号
：24指207
研究課題名
：全自動遺伝子解析装置を用いた、グラム陰性桿菌菌血症例における迅速菌名同
定・耐性遺伝子同定方法の先進医療適用のための研究
主任研究者名：独立行政法人国立国際医療研究センター
国際感染症センター大曲貴夫
分担研究者名：独立行政法人国立国際医療研究センター
部長
キーワード
：グラム陰性桿菌、遺伝子、耐性遺伝子
研究成果
：
切替照雄
【背景】グラム陰性桿菌菌血症は重篤で死亡率も高い。適切な治療の速やかな開始が必要だが、
現在の検査方法では血液培養提出から菌名同定・感受性試験終了そして最適な治療選択まで 72-96
時間かかってしまう。全自動遺伝子解析装置を用いた迅速菌名同定・耐性遺伝子同定方法を用い
ればこの時間は 24 時間前後に短縮される。本研究ではこの検査技術の国内臨床応用および先進医
療申請のために必要な臨床情報の収集のために施行した。
【研究目的】
本研究は、Nanosphere 社による全自動遺伝子解析装置（Nanosphere Verigine
system）の、グラム陰性桿菌菌血症例における迅速菌名同定・耐性遺伝子同定方法の国内臨床応
用に必要なデータの取得のために行った。
【必要性】グラム陰性桿菌菌血症は重篤で死亡率も高い病態である。この診療において、現在
医療機関の細菌検査室で行われている一般的な検査方法では、血液培養提出から菌名同定・感受
性試験終了まで 72-96 時間程度時間がかかってしまう。これは最適な治療の選択には 72-96 時間
かかることを意味する。グラム陰性桿菌敗血症患者の予後改善のためには、最適な抗菌薬の速や
かな投与が必要不可欠である。よって検体提出から感受性試験結果取得までの時間を如何に短縮
するかが、臨床上極めて重要である。現時点でこれを可能としうるのは遺伝子解析装置を用いた
迅速菌名同定・耐性遺伝子同定法であり、本検査法を用いたグラム陰性桿菌菌血症例における迅
速菌名同定・耐性遺伝子同定および最適治療選択の一連の流れは、先進医療に値するものである。
Nanosphere Inc.（米国）にて開発された VerigeneⓇ System は検体からの核酸抽出からハイブ
リダイゼーションまでを全自動で処理することができる簡易迅速遺伝子検査装置である。DNA マ
イクロアレイを用いているため、１度に多項目を同時に検出することができる。
Verigene System はマイクロ流路処理装置であるプロセッサーとその制御装置であるリーダーか
らなり、測定項目ごとに異なる単回使用のテストカートリッジを使用する。簡易操作、迅速な結
果報告、マルチプレックス測定という点で他の装置と比較して優れており、他の特別な施設を必
要とせずに院内での遺伝子検査の実施を可能とする。本法では血液培養陽性後、2.5 時間で血流
感染症の起因菌（グラム陽性菌）と薬剤耐性遺伝子を検出することができ、従来の血液培養検査
の流れを大幅に改善することが期待されている。
現在、血流感染症用テストカートリッジが開発されており、菌血症の起因菌と薬剤耐性遺伝子
を迅速に検出することができる。本カートリッジには、グラム陽性菌、グラム陰性菌、真菌およ
び薬剤耐性遺伝子が含まれている。これにより、特定の細菌に対し最も適切な抗菌薬治療を 24 時
間以内に開始することが可能となる。現在は、グラム陽性菌用テストカートリッジ（BC-GP）が販
売されており、BC-GP の一部の項目を抜粋して製品化したテストカートリッジ（BC-S）は FDA の
承認を取得している。さらに、グラム陰性菌用テストカートリッジが現在開発中であり、2012 年
初頭に販売予定となっている。欧米では、菌血症起因グラム陽性菌同定用テストカートリッジで
ある BC-GP と従来の生化学的方法を用いたグラム陽性菌の同定結果を比較したところ、BC-GP が
高い感度と特異度を示すことが確認されている 1)-4)。
一方、菌血症の起因菌および薬剤耐性は世界各国でその分布が異なっているため、日本国内で
の臨床性能評価が必要となる。BC-GP に対しては、現在、国内の医療機関 2 施設において従来法
との性能比較研究が実施されている。グラム陰性菌用テストカートリッジは現在開発中の製品で
あり、国内における性能評価研究はおこわなれていないため、本研究が本邦初となる。
【方法】研究では、一般臨床で得られた血液培養陽性検体を用いて、Nanosphere 社による全自
動遺伝子解析装置を用いたグラム陰性桿菌菌血症例における迅速菌名同定・耐性遺伝子同定方法
の従来の検査方法との一致性について検討した。
【結果】
1.
接種されたサンプルの細菌同定(Table3)
血液培養陽性試料 394 検体中 295 検体は、表 2 に列挙されたグラム陰性細菌種のいずれかの単
離物を接種することによって調製した。すべてのサンプルは培養陽性となった。 295 サンプルの
うち、 289 サンプル（ 98.1％）は初回のアッセイで結果を生成した。残りの 6 サンプルでは、
技術的なエラーを意味する「ノーコール」の結果となった。この 6 検体を再試験すると、すべて
の 6 つのサンプルで同定成功し結果が得られた。BC- GN アッセイおよび細菌同定と従来法の方法
の間での菌名における 295 のサンプルの一致率は以下のようであった：

Acinetobacter spp.、 Citrobacter spp.、Enterobacter spp.、E. coli、K. oxytoca, Proteus
spp.、P. aeruginosa：

K. pneumoniae： 88.2%

S. marcescens：
100%
81.3%
2. 臨床サンプルを用いた細菌同定
2
敗血症患者から得た 99 の血液培養陽性検体のうち、 98 は初回で（ 98.9％）で、BC –GN によ
り菌名が同定された。 99 の血液培養陽性検体から、合計で 107 グラム陰性細菌種を単離した。
99 の血液培養陽性検体を用いた、本法と従来法との菌名一致率は以下である：

Acinetobacter spp., E. coli, K. oxytoca, Proteus spp.、S. marcescens: 100%

Enterobacter spp.88.9%

K. pneumoniae 80%

P. aeruginosa 81.8%
すべての臨床試料のうち、 9 検体は嫌気ボトルで陽性となり、 BC- GN アッセイのすべての結
果は、従来法と一致した。
3.
複数菌陽性の血液培養の検討結果
BC- GN アッセイは 2 又は 3 の臨床分離株を同時接種した血液培養ボトルを用いて検証された。
接種された菌種の以下の組み合わせを試験した：A. baumannii （blaOXA-23) と K. pneumoniae
(blaCTX-M 、blaNDM)、A. baumannii (blaOXA-23) と MRSA、C. freundi・K. pneumoniae (blaKPC) ,
および P. mirabilis、C. freundii (blaKPC)・ E. faecalis・MSSA・E. cloacae (blaIMP)お
よび S. marcescens、 E. faecalis と E. coli (blaCTX-M)、E. coli (blaCTX-M)と P. aeruginosa
(blaVIM)、P. aeruginosa (blaVIM) と MRSE。すべてのグラム陰性細菌分離株は、グラム陽性菌
株を含む他の細菌種の存在にかかわらず BC- GN アッセイによって同定された。 BC- GN アッセイ
は、これらの条件で正常に全ての薬物耐性遺伝子を検出した。 BC- GN のアッセイはまた、グラ
ム陰性病原体であるが、BC -GN アッセイのターゲット間では M. morganii と S. maltophilia を
接種した 2 つの血液培養ボトルで検証された。BC- GN アッセイは、これらの種（データは示さず）
を検出しなかった。
複数の細菌種（2〜3 種）の単離されたサンプルの数は、99 の臨床検体中 7 検体であった。 7
サンプル中 4 サンプルにおいて、 BC- GN アッセイは試料中に含まれる複数の細菌種の菌名を全
て正しく検出した。Enterococcus casseliflavus と E. coli を含む試料において、 BC- GN アッ
セイは、E. coli のみを検出し E. casseliflavus は検出しなかった。残りの 3 つのサンプルのう
ち 1 つでは、 BC- GN アッセイは、複数の細菌種のうち 1 菌種を検出しなかった。
【結論
本研究は血液培養陽性検体内のグラム陽性球菌及び薬剤耐性遺伝子のほとんどが BC- GN アッセイ
により、2 時間の作業時間で識別されることを示した。
3
Subject No.
：24-207
Title
：Evaluation of an automated rapid diagnostic assay for detection of Gram-negative
bacteria and their drug resistance genes in positive blood cultures
Researchers ：
Norio Ohmagari1
Teruo Kirikae2,
1) Disease Control and Prevention Center, 2) Department of Infectious Diseases, Research
Institute, National Center for Global Health and Medicine, Tokyo, Japan
Key word
：Bacterial identification, Blood culture, Drug resistance genes, Gram-negative
bacteria, Sepsis
Abstract
：
We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test®
(BC-GN; Nanosphere, Northbrook, IL, USA), an automated assay for rapid diagnosis of sepsis
caused by 9 Gram-negative bacterial species and for detection of 9 genes associated with
-lactam

resistance. A total of 394 positive blood cultures with Gram-negative bacteria (GNB) were
analyzed using the BC-GN assay. Of the 394 samples, 295 were prepared by inoculating GNB into
blood culture bottles, and the remaining were clinical samples from 99 sepsis patients. Aliquots of
the positive blood cultures were tested by the BC-GN assay. The overall concordance rate between
the BC-GN assay and standard laboratory methods for bacterial identification were as follows:
Acinetobacter spp., Citrobacter spp., Escherichia coli, Klebsiella oxytoca, and Proteus spp., 100%;
Enterobacter spp., 96.3%; Klebsiella pneumoniae, 85.9%; Pseudomonas aeruginosa, 98.4%; and
Serratia marcescens, 85.7%; respectively. The BC-GN assay also detected all
-lactam resistance

genes tested (232 genes), including 42 blaCTX-M, 119 blaIMP, 8 blaKPC, 16 blaNDM, 24
blaOXA-23, 1 blaOXA-24/40, 1 blaOXA-48, 3 blaOXA-58, and 6 blaVIM. The data suggest that the
BC-GN can provide rapid detection of GNB and
-lactam resistance genes in positive blood

cultures and will lead to optimal patient management by earlier detection of major antimicrobial
resistance genes.
Introduction
Sepsis caused by drug-resistant Gram-negative bacteria (GNB) often results in serious clinical
outcomes in patients [1]. Inappropriate initial antibiotic treatment occurs in one third of patients
with severe sepsis due to GNB, which is associated with increased hospital mortality and length of
stay [2]. In contrast, early administration of appropriate antibiotics improves survival of sepsis
patients [3]. Effective antibiotic administration within the first hour of documented hypotension
was related to increased survival of patients with septic shock; however, 50% of septic shock
Researchers には、分担研究者を記載する。
patients did not receive effective antimicrobial treatment within 6 hours of documented
hypotension [4]. To improve diagnosis of sepsis, automated continuous-monitoring blood culture
systems were developed and introduced during the 1990s into clinical microbiological laboratories
[5]. These systems, even with the development of new automated technologies, are recognized as
some of the most important diagnostic tools for sepsis [5]. After a blood culture is positive,
conventional bacteriological procedures still require 2 to 3 days for bacterial isolation,
identification, and antimicrobial susceptibility testing. In addition to the time-consuming
procedures, the emergence and spread of drug-resistant GNB producing various β-lactamases,
including carbapenemase and extended spectrum β-lactamases (ESBLs), has been a serious
problem for treatment of sepsis [6]. Thus, there has been a need for rapid and automated
technology to identify bacterial species as well as detection of drug resistance genes.
Recently, several rapid molecular diagnosis assays for sepsis diagnosis have been introduced and
evaluated; including LightCycler® SeptiFast Test [7], peptide nucleic acid fluorescence in situ
hybridization (PNA-FISH®) [8], and matrix-assisted laser desorption-ionization time of flight
mass spectrometry (MALDI-TOF MS) [9], and a DNA-based microarray platform [Prove-it™
sepsis assay [10] and Verigene Gram-Positive Blood Culture (BC-GP) assay® [11,12]].
The Verigene Gram-Negative Blood Culture (BC-GN) Nucleic Acid Test (Nanosphere Inc.,
Northbrook, IL) is a sample-to-result automated microarray-based, multiplexed assay for species
identification of GNB and detection of their drug resistance genes in positive blood culture bottles.
The Verigene BC-GN assay is designed to directly detect species of GNB from positive blood
culture bottles in continuous-monitoring systems. U.S. Food and Drug Administration does not yet
approved the assay. In this study, we describe the performance of the BC-GN assay using
simulated and clinical samples of positive blood culture bottles.
Materials and Methods
Bacterial strains
A total of 268 stored clinical isolates at the National Center for Global Health and Medicine
(NCGM) were used in the study: 23 Acinetobacter baumannii; 1 Acinetobacter oleivorans; 4
Citrobacter freundii; 14 Enterobacter cloacae; 2 Enterobacter hormaechei; 1 Enterococcus faecalis;
30 Escherichia coli; 6 Klebsiella oxytoca; 43 Klebsiella pneumoniae; 1 methicillin-resistant
Staphylococcus aureus (MRSA); 1 methicillin-sensitive Staphylococcus aureus (MSSA); 1
methicillin-resistant Staphylococcus epidermidis (MRSE); 1 Morganella morganii; 4 Proteus
mirabilis; 4 Proteus vulgaris; 116 Pseudomonas aeruginosa; 15 Serratia marcescens; and 1
Stenotrophomonas maltophilia. Eighteen bacterial strains were donated by Yoshikazu Ishii (Toho
University, Tokyo, Japan), including 2 strains of A. baumannii harboring blaOXA-23; 1 A.
Researchers には、分担研究者を記載する。
baumannii harboring blaOXA-58; 1 Acinetobacter calcoaceticus harboring blaIMP-1; 1
Acinetobacter nosocomialis harboring blaIMP-1; 1 Acinetobacter pittii harboring blaOXA-58; 2 E.
cloacae harboring blaCTX-M-2 and blaCTX-M-9, respectively; 6 E. coli harboring blaCTX-M-2,
blaCTX-M-3, blaCTX-M-9, blaCTX-M-14, blaCTX-M-15, and blaCTX-M-44, respectively; 2 K.
pneumoniae harboring blaCTX-M-2; 1 K. pneumoniae harboring blaCTX-M-9; and 1 P. mirabilis
harboring blaCTX-M-2. Fifteen bacterial strains were obtained from International Health
Management Associates, Inc. (Schaumburg, IL), including 3 strains of A. baumannii harboring
blaOXA-23; 2 A. baumannii harboring blaOXA-58; 1 A. baumannii harboring blaOXA-24/40; 1 A.
baumannii harboring both blaOXA-23 and blaOXA-58; 2 C. freundii harboring blaKPC-2 and
blaKPC-3, respectively; 1 K. pneumoniae harboring blaKPC-2; 1 K. pneumoniae harboring
blaKPC-4; 2 K. pneumoniae harboring blaKPC-11; 1 K. pneumoniae harboring both blaKPC-3 and
blaCTX-M-14; and 1 S. marcescens harboring blaKPC-2.
Identification of bacterial species and PCR
Bacterial isolates were phenotypically identified using the Microscan Walkaway system (Siemens
Healthcare Diagnostics, Tokyo, Japan). Some isolates of K. pneumoniae, P. aeruginosa, and S.
marcescens were analyzed using 16S ribosomal RNA (rRNA) sequencing [13]. Nine genes
associated with
-lactam resistance were detected by PCR using the primers shown in Table 1.

Preparation of positive blood culture samples
Bacterial isolates were suspended in 10-ml Falcon tubes (Becton Dickinson, Tokyo, Japan) in
phosphate-buffered saline, pH 7.4. The suspension was adjusted to McFarland standard 1, and
then diluted to 106 times. A 0.1-ml aliquot was inoculated into 5 ml of human whole blood for blood
transfusion that was scheduled for disposal (Japanese Red Cross, Kanto-Koshinetsu Block Blood
Center, Tokyo, Japan). After mixing of the blood and bacterial inoculum, the sample was injected
into a Bactec plus /F aerobic blood culture bottle (Becton Dickinson). The 0.1-ml mixture was
plated, and the colony-forming unit (CFU) was counted. The average CFU counts of the inoculated
organisms were 23.9 ± 22.6 CFUs per bottle (median: 16, range: 1 – 184). The inoculated blood
culture bottles were incubated in the BACTEC 9050 automated blood culture systems (Becton
Dickinson) until positive. If these positive blood culture bottles could not be run within 12h, they
were refrigerated at 4°C for up to 48h.
Verigene System
The Verigene BC-GN assay detects 9 bacterial species and 9 drug resistance genes (Table 2). The
Verigene System consists of the Verigene Reader and the Processor SP. Following loading of the
Researchers には、分担研究者を記載する。
extraction tray, utility tray and test cartridge into the Verigene Processor SP, 700 μl of positive
blood culture was added to the extraction tray sample well. The Verigene Processor SP extracted
nucleic acids from positive blood culture media. The extracted nucleic acids were automatically
transferred to the test cartridge and hybridized to synthetic specific oligonucleotides attached to
the microarray slide. The nucleic acids bound on the microarray slide were further hybridized to
the second specific oligonucleotides with gold nanoparticles. After 2 hours, the microarray slide
was manually removed and inserted into the Verigene Reader for analysis. The Verigene system
contains internal controls, including negative controls, hybridization controls, and extraction
controls. When any internal control was not valid or the target signals were not adequately higher
than the negative control signals, the Verigene Reader reported “No Call” meaning a technical
error. When the Verigene Reader reported “No Call,” the assay was repeated until it reported
results of the assay other than “No Call.”
Clinical samples
Ninety nine blood culture bottles were obtained from patients with sepsis at the National Center
for Global Health and Medicine (NCGM) with 801 beds and Tokyo Women's Medical University
(TWMU) hospital with 1423 beds as part of routine patient care. Bactec plus /F (Becton Dickinson)
and BacT/Alert (bioMérieux, Tokyo, Japan) blood culture bottles were used in NCGM and TWMU,
respectively. These bottles were incubated in the automated blood culture systems until positive.
After bacterial isolation, bacterial species were identified by the Microscan Walkaway systems
(Siemens Healthcare Diagnostics). The culture-positive samples containing the target species of
the BC-GN assay were tested with the assay. All the storage period of the samples were within 6
days after blood culture positivity and the samples were stored at room temperature until testing.
Only one positive blood culture per patient was included in the study.
Data analysis
Concordance between the BC-GN assay and the conventional microbiological methods was
determined. When discrepant results among the methods were obtained, 16S rRNA of the
inoculated bacteria was sequenced. The sequence similarity was determined using the EzTaxon
server (http://eztaxon-e.ezbiocloud.net/ezt_identify).
Ethical considerations
The study protocol was carefully reviewed and approved by the ethics committee of the National
Center for Global Health and Medicine (No. 1268).
Researchers には、分担研究者を記載する。
Results
Bacterial identification of inoculated samples
Of 394 positive blood culture samples, 295 were prepared by inoculating an isolate of one of the
Gram-negative bacterial species listed in Table 2. All the samples became culture-positive. Of the
295 samples, 289 generated a BC-GN result on the first attempt (98.1%) (data not shown). The
remaining 6 generated “No Call ” results meaning technical errors. When retested, all the 6
samples did results (data not shown). The concordance rate of the 295 samples between the
BC-GN assay and the conventional methods for bacterial identification were as follows:
Acinetobacter spp., Citrobacter spp., Enterobacter spp., E. coli, K. oxytoca, Proteus spp., and P.
aeruginosa: 100%; K. pneumoniae: 88.2%; and S. marcescens: 81.3% (left columns in Table 3).
Of 51 samples inoculated with K. pneumoniae isolates (left columns in Table 3), 45 were correctly
detected with the BC-GN assay, but the remaining 6 were not correctly detected. Five of them
were reported as “Not detected”, and the remaining 1 was reported as Enterobacter spp.. The
Microscan Walkaway systems reported these 6 samples as K. pneumoniae. The 16S rRNA
sequences of these samples were more than 99.3% similar to both K. pneumoniae and K. variicola.
Of 16 samples inoculated with S. marcescens (left columns in Table 3), 3 were not detected with
the assay. The Microscan Walkaway systems reported these 3 samples as S. marcescens. The 16S
rRNA sequences of these samples were more than 99.5% similar to both S. marcescens and
Serratia nematodiphila. Their biochemical data on carbohydrates utilization corresponded to those
of S. marcescens [14]. These 3 isolates therefore were diagnosed as S. marcescens.
Bacterial identification of clinical samples
Of 99 blood culture-positive samples obtained from sepsis patients, 98 generated BC-GN results on
the first attempt (98.9 %) (data not shown). From the 99 blood culture-positive samples, a total of
107 Gram-negative bacterial species were isolated (middle columns in Table 3). The concordance
rate of the 99 samples between the BC-GN assay and the conventional methods for bacterial
identification were as follows: Acinetobacter spp., E. coli, K. oxytoca, Proteus spp., and S.
marcescens: 100%; Enterobacter spp.: 88.9%; K. pneumoniae: 80%; and P. aeruginosa: 81.8%
(middle columns in Table 3). Of all the clinical samples, 9 were obtained from anaerobic bottles,
and all the results of the BC-GN assay agreed with those of the conventional methods (data not
shown).
Of the 107 bacterial isolates, 7 isolates (6.5%) were not detected with the BC-GN assay
(middle
columns in Table 3). The Microscan Walkaway systems reported these isolates as Enterobacter
spp. (1 isolate), K. pneumoniae (4 isolates) and P. aeruginosa (2 isolates) (middle columns in Table
3). The sample reported to contain Enterobacter spp. by the Microscan Walkaway systems was
Researchers には、分担研究者を記載する。
reported as “No Call” with the BC-GN assay, and “No Call” result was reported again at the second
test. From the 4 samples reported to contain K. pneumoniae by the Microscan Walkaway systems,
the following bacterial species were isolated; K. pneumoniae in 2 samples: E. coli and K.
pneumoniae from 1: K. pneumoniae and P. aeruginosa from 1. The 16S rRNA sequences of the 4
isolates were more than 99.3% similar to both K. pneumoniae and K. variicola.
From the 2 samples reported to contain P. aeruginosa by the systems, the following bacterial
species were isolated; P. aeruginosa from 1: K. pneumoniae and P. aeruginosa from 1. The 16S
rRNA sequences of the 2 isolates were more than 99.5% similar to P. aeruginosa.
Identification of drug resistance genes
With respect to the BC-GN resistance gene targets, the BC-GN assay reported that, of all the
inoculated 295 samples tested, 184 were positive for one of the 9 drug resistance genes and 18 had
2 genes. These results were completely agreed with those of PCR and/or 16S rRNA sequncing. As
shown on left columns in Table 4, 42 blaCTX-M (2 E. cloacae, 19 E. coli, 2 E. hormaechei, 18 K.
pneumoniae, and 1 P. mirabilis isolates), 119 blaIMP (1 A. calcoaceticus, 1 A. nosocomialis, 1 A.
pittii, 9 E. cloacae, 4 K. pneumoniae, and 103 P. aeruginosa), 8 blaKPC (2 C. freundii, 5 K.
pneumoniae, and 1 S. marcescens), 16 blaNDM (2 A. baumannii, 1 E. hormaechei, 1 E. coli, 11 K.
pneumoniae, and 1 P. aeruginosa), 24 blaOXA-23 (24 A. baumannii), 1 blaOXA-24/40 (1 A.
baumannii), 1 blaOXA-48 (1 K. pneumoniae), 3 blaOXA-58 (2 A. baumannii and 1 A. pittii), and 6
blaVIM (6 P. aeruginosa) were detected by the BC-GN assay.
Of all the 99 clinical samples tested, 12 were positive for one of the 9 drug resistance genes. As
shown on middle columns in Table 4, 11 blaCTX-M (1 E. cloacae, 8 E. coli, and 2 K. pneumoniae),
and 1 blaOXA-58 (1 A. baumannii) were detected by the BC-GN assay.
These results were completely agreed with those of PCR and/or 16S rRNA sequncing.
Data of polymicrobial positive blood cultures
The BC-GN assay was tested on blood culture bottles inoculated simultaneously with 2 or 3
clinical isolates. The following combinations of the inoculated bacteria species were tested: A.
baumannii (harboring blaOXA-23) and K. pneumoniae (blaCTX-M and blaNDM); A. baumannii
(blaOXA-23) and MRSA; C. freundii, K. pneumoniae (blaKPC) and P. mirabilis; C. freundii
(blaKPC), E. faecalis and MSSA; E. cloacae (blaIMP) and S. marcescens; E. faecalis and E. coli
(blaCTX-M); E. coli (blaCTX-M) and P. aeruginosa (blaVIM); P. aeruginosa (blaVIM) and MRSE.
All Gram-negative bacterial isolates were correctly detected by the BC-GN assay regardless of the
presence of other bacterial species, including Gram-positive strains. The BC-GN assay detected all
drug resistance genes correctly under these conditions (data not shown). The BC-GN assay was
also tested on 2 blood culture bottles inoculated with M. morganii and S. maltophilia, respectively,
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which are Gram-negative pathogens but are not among the targets of the BC-GN assay. As
expected, the BC-GN assay did not detect these species (data not shown).
The number of samples from which multiple bacterial species (2 to 3 species) were isolated was 7 of
all the 99 clinical samples. In 4 of the 7 samples, the BC-GN assay detected correctly all of the
multiple bacterial species contained, even in a sample that 3 species contained. In a sample
containing Enterococcus casseliflavus and E. coli, the BC-GN assay detected only E. coli but not E.
casseliflavus, since it detects only Gram-negative bacteria but not Gram-positive ones. In the
remaining 3 samples, the BC-GN assay did not detect one of multiple bacterial species, i,e. the not
detected species were K. pneumoniae in 2 samples and P. aeruginosa in 1 samples, and the
bacterial identification was described above in the ‘Bacterial identification of clinical samples’
section.
Taken together, the results of the present study indicated that most of the GNB organisms and
drug resistance genes were identified with a run-time of 2 hours by the BC-GN assay.
Discussion
There are a number of advantages in using the BC-GN assay to diagnose Gram-negative sepsis.
The BC-GN assay can potentially detect up to at least 3 bacterial species in a single sample,
although we did not do a complete validation. The BC-GN assay, which targeted 9 Gram-negative
bacterial species, will contribute to the diagnosis of Gram-negative sepsis. Of the 10 most
frequently isolated microorganisms in ICU-acquired bloodstream infection in Europe, 7 were
Gram-negative pathogens targeted by the BC-GN assay [15]. Similar results were obtained from
monomicrobial nosocomial bloodstream infections both in ICU and non-ICU wards in the USA [16].
The National Healthcare Safety Network at CDC reported that a significant proportion of central
line-associated bloodstream infections are caused by drug-resistant Gram-negative pathogens in
the USA [17].
We evaluated the performance of the BC-GN assay in detecting various drug resistance genes
using inoculated samples. The assay could detect correctly all of the samples in the study. The
BC-GN assay can also detect multiple genes associated with
-lactam resistance even when they

were present in the sample simultaneously. These properties will be very useful in clinical
situations, because multidrug-resistant bacteria and polymicrobial infections are important
causes of failure in the treatment of sepsis. However, various 
-lactam resistance genes, including
carbapenemases and ESBLs, have been reported [6]. Drug resistance genes continue to evolve,
with new variants emerging in populations exposed to these agents. It is necessary to carefully
monitor the spread of drug resistance genes and the BC-GN assay must be continuously updated
to detect newly reported genes associated with
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-lactams.

A number of new diagnostic methods have been applied for rapid detection of pathogens in positive
blood cultures. A real-time PCR-based assay (LightCycler SeptiFast Test) can detect
Gram-negative and Gram-positive bacteria and fungi in a single assay, although it cannot detect
drug resistance genes [7]. PNA-FISH can detect 3 Gram-negative pathogens, including E. coli, K.
pneumoniae, and P. aeruginosa, but can not detect their drug resistance genes [8]. MALDI-TOF
MS can detect bacterial isolates in blood culture, but cannot detect 2 or more bacterial species in
polymicrobial samples or drug resistance gene products [18]. A DNA-based microarray platform,
Probe-it sepsis assay, can detect various Gram-negative and Gram-positive pathogens in blood
cultures. Nevertheless, it could not detect drug resistance genes other than mecA [10].
The BC-GN assay misidentified a total of 16 isolates from the same number of samples (Table 3).
Of them, 10 were reported as K. pneumoniae by the Microscan Walkaway systems. The 16S rRNA
sequence revealed that the 10 misidentified isolates shared over 99% similarity to both K.
pneumoniae and K. variicola. Other group reported that when identified using routine methods, K.
variicola might be included within K. pneumoniae [19]. It will be necessary to clarify the clinical
importance of these isolates.
A limitation of this study is that we could not fully evaluate the detection performance of the
BC-GN assay to detect some drug resistance genes, such as blaOXA-24/40 and blaOXA-48. We also
admit that we could not fully evaluate clinical effectiveness of the BC-GN assay, because we
examined concordance rates between the BC-GN assay and the conventional methods using
inoculated samples and clinical ones. We plan to do prospective study to determine the clinical
impact of the assay including selection of antibiotics and length of stay.
In conclusion, the Verigene system BC-GN assay effectively detected Gram-negative bacterial
isolates and their drug resistance genes in positive blood cultures in a rapid and accurate manner,
and it was remarkably sensitive, specific, and faster than conventional methods. This new
microarray platform can be easily introduced into microbiological laboratories in various clinical
settings. It will provide better sepsis management, such as potential time savings over routine
laboratory methods, administration of appropriate antibiotics, and early de-escalation.
Acknowledgements
This work was supported by the grant of National Center for Global Health and Medicine (24-207).
We thank Japanese Red Cross Blood Center for providing human whole blood. We also thank
Yoshikazu Ishii (Toho university, Tokyo, Japan) and International Health Management
Associates, Inc. for providing bacterial strains.
Transparency and Declaration
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H. Takahashi is a consultant to Nanosphere. The remaining authors have no reported potential
conflicts of interest.
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Researchers には、分担研究者を記載する。
課題番号
：24指207
研究課題名
：全自動遺伝子解析装置を用いた、グラム
陰性桿菌菌血症例における迅速菌名同定・耐性遺伝子同
定方法の先進医療適用のための研究
主任研究者：
独立行政法人国立国際医療研究センター
国際感染症センター
大曲
分担研究者：
独立行政法人国立国際医療研究センター
部長
切替
照雄
キーワード：グラム陰性桿菌、遺伝子、耐性遺伝子
貴夫
全自動遺伝子解析装置を用いた、
菌血症例における迅速菌名・耐性遺伝子同定
48－72時間が必要
敗血症疑い
患者
血液
培養
分離菌
菌種同定・薬
剤感受性試験
(病院検査室)
菌種同定・薬
剤耐性因子
迅速検出
2.5時間に短縮
検査法の感度・特異度を検討する
最適治療
開始
課題番号
：24指207
研究課題名
：全自動遺伝子解析装置を用いた、グラム陰性桿菌菌血症例における迅速菌名同
定・耐性遺伝子同定方法の先進医療適用のための研究
分担研究者名：独立行政法人国立国際医療研究センター
キーワード
：グラム陰性桿菌、遺伝子、耐性遺伝子
研究成果
：
【研究目的】
部長
切替照雄
Nanosphere 社による全自動遺伝子解析装置（Nanosphere Verigine system）の、
国内で分離されたグラム陰性桿菌株を用いて装置の評価をする。そのうえで臨床研究で従来法と
比較し異なった結果が出た場合の原因を細菌学的に解析し明らかにする。
【方法】研究では、一般臨床で得られた血液培養陽性検体を用いて、Nanosphere 社による全自
動遺伝子解析装置を用いたグラム陰性桿菌菌血症例における迅速菌名同定・耐性遺伝子同定方法
の従来の検査方法との一致性について検討した。
【結果】
1.
接種された検体の細菌同定(Table3)
K. pneumoniae 分離株を接種した血液培養陽性試料 51 検体のうち 45 検体が正しく BC- GN アッ
セイで菌名同定されたが、残りの 6 検体では正しく同定されなかった。そのうち 5 検体では、「検
出せず」、残りの 1 検体では、Enterobacter 属として報告された。従来法である Microscan
Walkaway system では、これらの 6 つの検体を K. pneumoniae と同定し報告した。これらの検体
の 16S rRN 核酸配列は、K. pneumoniae および K variicola の両方に 99.3％を越える一致性を示
していた。
S. marcescens を接種した 16 検体のうち、 3 検体では菌名が同定されなかった。Microscan
Walkaway system はこれらの 3 検体を S. marcescens として報告した。これらの検体の 16S rRNA
配列は、
S. marcescens および S. nematodiphila 両方に 99.5％以上を越える一致性を示していた。
炭水化物の利用率関する生化学的性状は、S. marcescens のもの[ 14 ]と同一であった。これら 3
分離株は、したがって、S. marcescens と判定された。
2.
臨床検体を用いた細菌同定
敗血症患者から得た 99 の血液培養陽性検体のうち、 98 検体は初回で（ 98.9％）で BC –GN
により菌名が同定された。 99 の血液培養陽性検体から、合計で 107 グラム陰性細菌種を単離し
た。107 細菌分離株のうち、 7 株（ 6.5％）は、BC -GN アッセイで検出されなかった。Microscan
Walkaway system は、これらを Enterobacter spp. (1 株), K. pneumoniae (4 株)そして P.
aeruginosa (2 株)と同定した。Microscan Walkaway system で、Enterobacter spp. と同定され
た菌は BC -GN アッセイで No Call となり、2 回目の検査でも同じく No Call との結果であった。
Microscan Walkaway system によって K. pneumoniae と同定された 4 株から、K. pneumoniae
株、E. coli 1 株、K. pneumonia
2
1 株、および P. aeruginosa 1 株を単離した。これら 4 分
離株の 16S rRNA 配列は、肺炎桿菌および K variicola 両方に 99.3％を越える同一性を示してい
た。BC-GN システムによって緑膿菌を含むと報告されら 2 検体からは、緑膿菌 1 株と、肺炎桿菌
および緑膿菌分離した。 2 分離株の 16S rRNA 配列は緑膿菌に対して 99.5％以上一致していた。
3. 抗菌薬耐性遺伝子の同定
BC- GN 耐性遺伝子に対して、 BC- GN アッセイは、試験した全ての 295 検体中、 184 検体で 9
つの薬剤耐性遺伝子のいずれかに陽性であり、 18 検体は 2 つの遺伝子を検出したことを報告し
た。これらの結果は、PCR および/または 16S の rRNA sequncing のそれと完全に一致した。表 4
左のコラムに示されているように、 42 の bla CTX -M （E. cloacae
2 株, E. coli
19 株,
E.
hormaechei 2 株, K. pneumonia 18 株, and P. mirabilis isolates 1 株）、 119 の blaIMP （ A.
calcoaceticus 1 株, A. nosocomialis 1 株, A. pittii 1 株, E. cloacae 9 株, K. pneumonia
4 株, P. aeruginosa
103 株）、8 の blaKPC（C. freundii 2 株, K. pneumonia 5 株, S. marcescens
1 株) 、 16 の blaNDM （A. baumannii 2 株, E. hormaeche 1 株, E. coli 1 株, K. pneumonia 11
株,、P. aeruginosa 1 株）、 24 の blaOXA - 23 （A. baumannii ２4 株）、 1 つの blaOXA-24/40
（ A. baumannii 株 1）、１つの blaOXA - 48 （K. pneumonia 1 株）、 3 つの blaOXA - 58
（A. baumannii 2 株と A. pittii
1 株）、および 6 つの blaVIM （ P. aeruginosa 6 株）が
BC -GN アッセイにより検出された。
試験した全ての 99 の臨床検体のうち、 12 株は、 9 の薬剤耐性遺伝子のいずれかについて陽性
であった。表 4 の中欄に示されるように、 11 の bla CTX -M（E. cloacae
1 株, E. coli 8 株
そして K. pneumonia 2 株）、および 1 つの blaOXA -58 （A. baumannii 株）が BC- GN アッセ
イによって検出された。これらの結果は、完全に PCR および/または 16S の rRNA sequncing のそ
れと合致した。
【結論】
本研究は血液培養陽性検体内のグラム陽性球菌及び薬剤耐性遺伝子のほとんどが BC- GN アッセ
イにより、2 時間の作業時間で識別されることを示した。
2

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