JP7146765B2 - 改善された迅速な抗菌薬感受性試験のための方法 - Google Patents
改善された迅速な抗菌薬感受性試験のための方法 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/04—Five-membered rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/003—Compounds containing elements of Groups 3 or 13 of the Periodic Table without C-Metal linkages
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Description
本出願は、2016年12月23日に出願された米国仮特許出願第62/438,780号及び2017年4月21日に出願された米国仮特許出願第62/488,454号及び2017年7月20日に出願された米国仮特許出願第62/535,106号の優先権を主張し、これらの開示は参照により本明細書に組み込まれる。
本発明は概して抗菌薬感受性試験に関し、より詳細には臨床試料の迅速な抗菌薬感受性試験に関する。
本発明は、本明細書に記載されている方法が、微生物感染の抗生物質感受性の改善された迅速な決定を提供するという発見に部分的に基づく。本発明はまた、迅速な抗生物質感受性試験(AST)法の有効性及び信頼性が、微生物若しくは抗菌薬又はこれらの組合せの性質及び機能を含むいくつかの要因の変動性に適応することによって大幅に増大し、これによって本発明の多用途のモジュール式でロバストなプラットフォームアッセイシステムを生成するという驚くべき発見に部分的に基づく。
・1種以上の微生物の懸濁液を、複数のチャンバを含むカートリッジに導入するステップであって、複数のチャンバは1種以上の抗菌剤を含む、ステップ、
・初期インキュベーション期間の間、微生物成長を促進する条件下でカートリッジをインキュベートするステップ、
・カートリッジチャンバのサブセットにおいて、微生物成長が閾値を達成したかどうかを決定するために1つ以上のチェックポイントアッセイを実施するステップ、並びに
(a)閾値が達成された場合、1種以上の抗菌薬に対する微生物の感受性を決定するために複数のカートリッジチャンバにおいて複数の異なる成長アッセイを実施し、最小発育阻止濃度(MIC)及び/若しくは定性的感受性結果(QSR)を得るステップ、又は
(b)閾値が達成されていない場合、
(i)閾値が達成され、その後、ステップ(a)を実施するまで、若しくは
(ii)閾値が達成されずに最大18時間が経過し、さらなるアッセイが実施されなくなるまで、
微生物成長を促進する条件下で1回以上のさらなるインキュベーション期間を実施するステップ
を含む。
(a)複数のチャンバへの代謝プローブの添加、
(b)微生物成長を促進する条件下でのアッセイインキュベーション期間、及び
(c)吸光度、蛍光、ルミネセンス、電気化学的シグナル測定のうちの1つ以上を得ること
を含む。
R1は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R2は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R3は独立して、任意選択的に置換されたC6~C10アリール、任意選択的に置換された5~10員ヘテロアリール又はサブ構造Aであり、
サブ構造Aは、
L1は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
L2は独立して、共有結合、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R4は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R5は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールである)
であり、
各々のXは独立して、不在又は一価アニオンである]
を有する。
本明細書に言及されている特許及び科学文献は、当業者に利用可能な知識を確立する。本明細書に引用されている発行された米国特許、許可された出願、公開された米国及び外国出願、参照は、各々が参照により組み込まれるように具体的かつ個々に示されたものと同じ程度まで参照により本明細書に組み込まれる。
本明細書に記載されている迅速なAST法は、複数の抗微生物剤を用いて、及び複数の微生物について試験した場合に、臨床検査標準協会(Clinical Laboratory Standards Institute)(CLSI)参照法を使用して得られた結果と一致する正確な結果を提供することができるが、これらの方法は結果を提供するのにCLSI法よりも顕著に短い時間のみを必要とし得る。本明細書に記載されている方法は、標準的な方法と比較して、大幅に低減された時間及び費用にて、適切な治療レジメン、すなわち、特有の抗微生物剤及び特定の投薬量を患者に提供することができる。したがって、本明細書に記載されている方法は、患者の転帰を改善することができ、病院費を下げることができ、抗微生物剤耐性微生物のさらなる進化を低減するのに役立つことができ、したがって、本明細書に記載されている方法は、AST分野における顕著なブレークスルーとなる。
AST法は、ある特定の細菌株におけるMIC又はQSRを決定するために有用であり得るアッセイを実施することができる。1種類のアッセイが、抗微生物剤に対する微生物の感受性を決定する際に、他の種類のアッセイよりも特定の微生物株に対して有効である場合が生じる。本明細書に記載されている方法は、もしあれば、複数の異なるアッセイのうちのどれが、抗微生物剤に対する微生物の感受性を決定するのに適切であり得るかを決定するための手段を提供する。一部の実施形態では、方法は、異なる抗微生物剤-抗生物質の組合せについての異なるアッセイを使用する。
R1は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R2は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R3は独立して、任意選択的に置換されたC6~C10アリール、任意選択的に置換された5~10員ヘテロアリール又はサブ構造Aであり、
サブ構造Aは、
L1は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
L2は独立して、共有結合、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R4は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R5は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールである)
であり、
各々のXは独立して、不在又は一価アニオンである]
を有する。
acid)又はクリプテート配位子との配位錯体を含む表面結合プローブを導入することができる。ある特定の実施形態では、表面結合プローブアッセイは、ユウロピウム、ストロンチウム、テルビウム、サマリウム及びジスプロシウム又はこれらの組合せのような増幅剤を含む。一部の実施形態では、増幅剤は、
チェックポイントアッセイは、微生物成長を確認するために実施され得る。例えば、正確なAST決定を得るために、アッセイはゆっくり成長する細菌株を考慮することができ、したがって本明細書の方法は、十分な微生物成長が起こったかどうかを確認するために初期インキュベーション期間後に行われるチェックポイントアッセイを提供することができる。微生物の成長のような成長は、微生物の数の増殖、長さの増加、体積の増加並びに/又は核酸及び/若しくはタンパク質含有量の増加を含み得る。
上記のように、成長指標は、AST成長アッセイを実施する前に十分な微生物成長を確認するためにチェックポイントアッセイにおいて使用され得る。以下に示されるように、様々な成長指標が利用され得る。
R1は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R2は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R3は独立して、任意選択的に置換されたC6~C10アリール、任意選択的に置換された5~10員ヘテロアリール又はサブ構造Aであり、
サブ構造Aは、
L1は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
L2は独立して、共有結合、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R4は独立して、CN、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールであり、
R5は独立して、任意選択的に置換されたC6~C10アリール又は任意選択的に置換された5~10員ヘテロアリールである)
であり、
各々のXは独立して、不在又は一価アニオンである]
を有する。
カートリッジは、微生物の液体懸濁液を保持し、成長を可能にすることができる容器であってもよい。カートリッジの非限定的な例には、培養フラスコ、培養皿、ペトリ皿、バイオアッセイ皿、培養管、試験管、マイクロチューブ、ボトル、マイクロチャンバプレート、マルチチャンバプレート、マイクロタイタープレート、マイクロプレートが含まれ得る。カートリッジは1つのチャンバを含んでもよい。カートリッジは複数のチャンバを含んでもよく、各チャンバは別の空間から物理的に隔離された液体懸濁液を保持することができる空間であり、チャンバの一例はマルチウェル(multiwall)プレートにおけるチャンバである。カートリッジは、1、2、3、4、5、6、7、8、9、10、11、12、24、48、96、192、384、1536個又はこれ以上のチャンバ及びこれらの間の任意の数のチャンバを含んでもよい。カートリッジチャンバの底部は、平坦、円形又はV字形であってもよい。
インキュベーション期間の前にカートリッジを30~45℃に予熱することは、微生物成長を促進するために有利であり得、これにより次に、より速い及び/又はより正確な抗微生物剤感受性試験(AST)決定がもたらされ得る。標準的な空気対流インキュベーターは典型的に、試験パネルを所望の作業温度にするのに30~60分かかるので、予熱は一部の場合に有用であり得る。典型的な所望のインキュベーション時間は8時間未満であり、ほとんどの場合、7時間未満、6時間未満、5時間未満、4時間未満又は3時間未満であるので、予熱は、迅速なASTを実施するための本明細書に記載されている方法と共に使用するのに特に有用であり得る。
溶液混合は、溶液通気を高めることによって、大きな成長溶液体積(例えば、10mL超)における微生物成長速度を促進することが、当業者によって十分に理解される。微量液体希釈法ASTアッセイは一般に、12mm未満の横寸法を有するウェルを含むカートリッジにおいて実施される。12mm未満の横寸法を有するウェル内で適切な溶液混合を達成するために、オービタル振とう振動数は少なくとも毎分500回転(rpm)でなければならない。しかしながら、これらの振動数は、微生物に対する高ひずみ及び高せん断に起因して、12mm未満の横寸法を有するウェル内の微生物成長を阻害する。
感染症は、微生物由来、例えば細菌、真菌細胞、古細菌及び原虫の任意の感染性病原体を含み得る。一部の例では感染性病原体は、細菌、例えば、グラム陽性細菌、グラム陰性細菌及び非定型細菌である。抗微生物剤耐性微生物は、抗微生物剤、すなわち、抗細菌性薬、抗真菌性薬、抗古細菌薬物療法及び抗原虫薬に耐性である微生物であり得る。
、スピリルム・ボルタンス(Spirillum volutans)、スタフィロコッカス属(Staphylococcus)、スタフィロコッカス・アウレウス、スタフィロコッカス・エピデルミデス(Staphylococcus epidermidis)、スタフィロコッカス属種、ステノトロホモナス・マルトフィリア(Stenotrophomonas maltophilia)、ステノトロホモナス属種、ストレプトコッカス属(Streptococcus)、ストレプトコッカス・アガラクチア(Streptococcus agalactiae)、ストレプトコッカス・アビウム(Streptococcus avium)、ストレプトコッカス・ボビス(Streptococcus bovis)、ストレプトコッカス・クリセツス(Streptococcus cricetus)、ストレプトコッカス・フェシウム(Streptococcus faceium)、ストレプトコッカス・フェカリス(Streptococcus faecalis)、ストレプトコッカス・フェルス(Streptococcus ferus)、ストレプトコッカス・ガリナルム(Streptococcus gallinarum)、ストレプトコッカス・ラクチス(Streptococcus lactis)、ストレプトコッカス・ミチオル(Streptococcus mitior)、ストレプトコッカス・ミティス(Streptococcus mitis)、ストレプトコッカス・ミュータンス(Streptococcus mutans)、ストレプトコッカス・オラリス(Streptococcus oralis)、ストレプトコッカス・ニューモニエ(Streptococcus pneumoniae)、ストレプトコッカス・ピオゲネス(Streptococcus pyogenes)、ストレプトコッカス・ラツス(Streptococcus rattus)、ストレプトコッカス・サリバリウス(Streptococcus salivarius)、ストレプトコッカス・サングイス(Streptococcus sanguis)、ストレプトコッカス・ソブリナス(Streptococcus sobrinus)、ストレプトコッカス属種、トレポネーマ属(Treponema)、トレポネーマ・デンチコラ(Treponema denticola)、トレポネーマ・パリデュム(Treponema pallidum)、トレポネーマ属種、ウレアプラズマ属種、ビブリオ属(Vibrio)、ビブリオ・コレラエ(Vibrio cholerae)、ビブリオ・コンマ(Vibrio comma)、ビブリオ・パラヘモリチカス(Vibrio parahaemolyticus)、ビブリオ属種、ビブリオ・ブルニフィカス(Vibrio vulnificus)、ビリダンス・ストレプトコッキ(viridans streptococci)、ボルバキア属(Wolbachia)、エルシニア属(Yersinia)、エルシニア・エンテロコリチカ(Yersinia enterocolitica)、エルシニア・ペスチス(Yersinia pestis)、エルシニア・シュードツベルクロシス(Yersinia pseudotuberculosis)及びエルシニア属種が挙げられる。
微生物が細菌である場合、例示的抗微生物剤として、これだけに限らないが、アミカシン、アミノグリコシド、アミノグリコシドアモキシシリン、アミノグリコシド、アモキシシリン、アモキシシリン/クラブラネート、アンピシリン、アンピシリン/スルバクタム、抗毒素、アルスフェナミン、アジスロマイシン、アズロシリン、アズトレオナム、β-ラクタム、バシトラシン、カプレオマイシン、カルバペネム、カルベニシリン、セファクロル、セファドロキシル、セファレキシン、セファロチン、セファロチン、セファマンドール、セファゾリン、セフジニル、セフジトレン、セフェピム、セフィキシム、セフォペラゾン、セフォタキシム、セフォキシチン、セフポドキシム、セフプロジル、セフタロリン、セフタロリンフォサミル、セフタジジム、セフチブテン、セフチゾキシム、セフトビプロール、セフトリアキソン、セフロキシム、セファロスポリン、クロラムフェニコール、クロラムフェニコール(Bs)、シプロフロキサシン、クラリスロマイシン、クリンダマイシン、クロファジミン、クロキサシリン、コリスチン、コトリモキサゾール、サイクロセリン、ダルババンシン、ダプソン、ダプトマイシン、デメクロサイクリン、ジクロキサシリン、ジリスロマイシン、ドリペネム、ドキシサイクリン、エノキサシン、エルタペネム、エリスロマイシン、エタンブトール、エタンブトール(Bs)、エチオナミド、フルクロキサシリン、フルオロキノロン、フルオロキノロン類、ホスホマイシン、フラゾリドン、フシジン酸、ガチフロキサシン、ゲルダナマイシン、ゲミフロキサシン、ゲンタマイシン、グレパフロキサシン、ハービマイシン、イミペネム/シラスタチン、イソニアジド、カナマイシン、レボフロキサシン、リンコマイシン、リネゾリド、ロメフロキサシン、ロラカルベフ、マクロライド、マフェニド、メロペネム、メチシリン、メトロニダゾール、メズロシリン、ミノサイクリン、モキシフロキサシン、ムピロシン、ナフシリン、ナフシリン、ナリジクス酸、ネオオマイシン、ネチルマイシン、ニトロフラントイン(Bs)、ノルフロキサシン、オフロキサシン、オリタバンシン、オキサシリン、オキシテトラサイクリン、パロモマイシン、ペニシリン、ペニシリンG、ペニシリンV、ピペラシリン、ピペラシリン/タゾバクタム、プラテンシマイシン、ポリミキシンB、ポシゾリド(Posizolid)、ピラジナミド、キヌプリスチン/ダルホプリスチン、ラデゾリド(Radezolid)、ラキシバクマブ、リファブチン、リファンピシン、リファンピン、リファペンチン、リファキシミン、ロキシスロマイシン、スルファジアジン銀、スパルフロキサシン、スペクチノマイシン、スペクチノマイシン(Bs)、スピラマイシン、ストレプトグラミン、ストレプトマイシン、スルバクタム、スルファセタミド、スルファジアジン、スルファジメトキシン、スルファメチゾール、スルファメトキサゾール、スルファニリミド、スルファサラジン、スルフイソキサゾール、スルホンアミドクリソイジン、テジゾリド、テイコプラニン、テキソバクチン(Teixobactin)、テラバンシン、テリスロマイシン、テマフロキサシン、テモシリン、テトラサイクリン、チアンフェニコール、チカルシリン、チカルシリン/クラブラネート、チカルシリン/クラブラン酸、チゲサイクリン、チゲサイクリン(Bs)、チニダゾール、TMP/SMX、トブラマイシン、トレゾリド、トリメトプリム(Bs)、トリメトプリム-スルファメトキサゾール、トロレアンドマイシン、トロバフロキサシン、バンコマイシン、及びこれらのジェネリック又はこれらのバリアントが挙げられる。
液体は、カチオン調整ミューラーヒントンブロス(MHB)のような成長培地を含み得る。この培地は、微生物成長及び安定性を促進するために当業者に周知の添加物を含んでよい。さまざまな抗微生物剤に加えて、異なる検査ウェルは、特定の抗微生物剤に対するAST正確性を改善することが周知である添加物を含む場合がある。例えば、追加的な塩化ナトリウムはオキサリシンを含む検査に加えられてよく、追加的なカルシウムはダプトマイシンを含む検査に加えられてよい。
本明細書に記載される微生物は、生物学的試料由来であってよい。一部の実施形態では生物学的試料は、微生物、例えば細菌及び真菌細胞を含む任意の試料である。生物学的試料は、臨床試料由来であってよい。
対照は、微生物が感受性でない抗微生物剤を含んでよい。例として、アッセイがグラム陽性細菌の感受性を決定するために使用される場合、対照(及び検査インキュベーション)は、グラム陰性細菌を標的化する1種以上の抗微生物剤を含んでよく、アッセイが真核微生物の感受性を決定するために使用される場合、対照(及び検査インキュベーション)は、1種以上の抗細菌性抗微生物剤を含んでよい。
本明細書に記載される方法は、商業的に入手できる装置、特注の装置又はこれらの組合せを使用して自動化された様式で実施され得る。自動化法は、より多くの数のアッセイの実施及びアッセイでの一貫性の増大を可能にする。自動化は、これらの方法の速度及び分解能も増大させることができる。
表面結合アッセイ(表面結合プローブアッセイとも称される)は、シグナル伝達剤を利用する場合がある。シグナル伝達剤は、微生物に結合できる成分(例えば、微生物表面に結合する抗体及び/又はレクチン、微生物表面に非特異的に結合する荷電成分及び/又は機能性成分)並びにシグナルを供給する又はシグナルの産生に寄与する化学成分(例えば、酵素ケミルミノフォア(chemiluminophore)及びランタニドキレート)を典型的には含む。例示的酵素として、西洋ワサビペルオキシダーゼ、アルカリホスファターゼ、アセチルコリンエステラーゼ、グルコースオキシダーゼ、ベータ-D-ガラクトシダーゼ、ベータ-ラクタマーゼ及びこれらの組合せが挙げられる。
本明細書に記載される方法の態様は、所与の微生物に対してどの抗微生物剤が最も有効であるかを判定するための複数の成長アッセイを実施することによって、正確で、低費用な表現型AST結果を述べることができる。本明細書の方法は、処方目的のために所与の有効な抗微生物剤の適切な濃度を提供できる。一部の実施形態では、方法は、所与の微生物によって生じる患者の感染の処置のための推奨の作成を提供する。患者は、本明細書に考察される生物学的試料又は標本の供給源として役立ち得る宿主であってよい。ある特定の態様ではドナーは、脊椎動物であり、任意の動物種(例えば、ヒトのような哺乳動物)を意味することが意図される。ある特定の実施形態では、患者は、これだけに限らないが、ヒト及び非ヒト霊長類、鳥類、は虫類、両生類、ウシ、イヌ、ヤギ(caprine)、キャビティー(cavities)、カラス、エピン(epine)、ウマ、ネコ、ヤギ(hircine)、ウサギ(lapine)、ウサギ(leporine)、オオカミ、ヒツジ、ブタ、ラシーン(racines)、キツネなどが挙げられる任意の動物宿主であり、非限定的に、飼い慣らされた家畜、群れ又は移住動物又は鳥類、外来又は動物学的検体並びにコンパニオン動物、ペット及び獣医師の保護下にある任意の動物が挙げられる。
本明細書に記載される方法は、微生物によって生じた感染を有する患者の処置を提供する。AST決定は、医療専門家又は診断科学者が望ましい行動指針又は処置レジメンに関する患者への推奨を作成できるようにする。一部の実施形態では推奨は、本発明によって提供されて迅速に及びより正確にもたらされる。感染の処置についての推奨は、具体的な抗微生物剤若しくは抗微生物剤の組合せの選択又はそのような抗微生物剤の用量を含む場合がある。一部の実施形態ではそのような推奨は、MIC及び/又はQSR結果に基づいて医師によって提供又は作成される。
並行抗微生物剤感受性アッセイ
本実施例は、並行して(例えば、同じインキュベーション期間を共有して)実施される複数の抗微生物剤感受性アッセイを記載する。
チェックポイントアッセイは、十分な微生物成長を確認するために使用され得る。
微生物を標識し、定量するためにユウロピウムクリプテート分子を使用する表面結合増幅アッセイは、図8に実証のとおりAST結果を決定するために利用され得る。E.コリ及びdS.アウレウス(左パネル)又はクレブシエラ・ニューモニエ(右パネル)をMES緩衝液、pH6中、le5~le9の範囲の濃度で96ウェルマイクロプレートに接種した。細菌を含む各ウェルに、及び対応する対照ウェルに、ユウロピウムクリプテート-ジアミン(Cisbio)を66ng/ウェルで加え、次に5%溶液のグルタルアルデヒドをユウロピウムクリプテートを含むウェルに加えた。反応溶液を、ウェル内の細菌の外面を選択したレポーターを用いて標識化することを促進するために30分間、インキュベートした。次に検査プレートを、細菌をプレートの底に沈殿させる一方で、すべての未会合レポーターを上清に残すようにThermo Scientific Heraeus Multifuge X3を、2500rpmの速度で2.5分間使用して遠心分離した。次にプレートを、洗浄緩衝液の添加の前に上清及び未反応レポーターを除去するためにBioTek Multiflo Xプレート洗浄機を使用して吸引した。この洗浄手順を、すべての未反応レポーターを完全に除去するためにもう1度繰り返した。ユウロピウムクリプテート-ジアミンを含むウェルを読み取り緩衝液中に再構成し、BioTek HIプレートリーダー上で時間分解蛍光を使用して読み取った。
図9A及び9Bは、十分な微生物成長を決定する成長閾値が達成された後だけに、代謝プローブをマイクロプレート上の追加的なウェルに加える場合に、代謝プローブがAST結果を決定するために利用され得ることを示している。これは、それらの欠点を伴わずに成長指標の有利点を可能にする:シグナルは、生きた微生物から主に生じるが、成長阻止及び毒性作用は、初期インキュベーション期間から除かれる。カチオン調整ミューラーヒントンブロスを含む96ウェルプレートを使用して、細菌を、分光光度計を使用して確認されたマクファーランド値0.5に達するようにコロニーを生理食塩水に希釈することによって調製した。これを1:20で生理食塩水中に希釈し、10μlの接種菌液を各ウェルに加えた。指標レザズリンを接種の時点又は3時間45分間後のいずれかで特定のウェルに加えた。接種したプレートを35℃、150rpmで振とうしながら4時間45分間インキュベートした。このインキュベーション後、蛍光(Ex560/Em590)をレザズリンを含むウェルから測定した。図9A及び図9Bにおけるデータは、陽性成長対照ウェルにおいて測定された蛍光と非接種ウェルにおいて測定された蛍光との比として示されている。接種ウェルにおいてと非接種ウェルとの蛍光シグナルの比は、レザズリンが初期細菌インキュベーション後に加えられた場合にさらに大きかった。
インキュベーションに先行するカートリッジの予熱
本実施例は、赤外線放射加熱を利用するカートリッジ予熱を記載する。赤外線予熱器についての実験設定は、VJ Electronix(VJ IR-1C)からの既製の加熱装置及び96ウェルマイクロプレートを保持するための特注の固定器具(fixturing)からなった。熱データ回収をNational Instruments CompactDAQ Chassis、National Instruments Resistance Temperature Device(RTD)analog module(NI 9216)及び8個までの密封RTD(Omega、HSRTD-3-100-A-40-E)によって実施した。
インキュベーションの際のカートリッジの揺動
2つの代表的な微生物、P.アエルギノーサ及びS.アウレウスの希釈物を、2つの標準的384ウェルマイクロプレートに導入し、一方のマイクロプレートを、回数150rpm及び半径25mmでオービタル振とうする(すなわち、揺動する)ようにしたインキュベーターに置いた。他方のマイクロプレートをインキュベーターに置き、静置した。3時間後、微生物成長を600nmでの光学密度測定によって決定した。図13は、3つの条件下でインキュベートした代表的な微生物の増強された成長比を示している。成長比は、インキュベーションの際に静置された384ウェルマイクロプレートについて600nmでの光学密度測定を、同様に接種され、150rpm、半径25mmで振とうしながらインキュベートした384ウェルマイクロプレートと比較することによって決定された微生物成長である。図14は、同様の成長増強が96ウェルマイクロプレートにおいて達成されたことを示している。
微生物生存率決定のためのテトラゾリウム類似物の使用
本実施例は、テトラゾリウムベースの分子が微生物生存率の決定において代謝プローブ及び成長指標として使用され得ることを示している。これらの分子は、(1)同じインキュベーション期間を共有して表面結合アッセイと共に実行される代謝プローブアッセイにおいて、及び/又は(2)十分な微生物成長を決定する成長閾値が達成された後だけに、代謝プローブをマイクロプレート上の追加的なウェルに加える場合に、AST結果を決定するために利用され得る。
MIC検証のための二重アッセイの実施
本実施例は、各試料について2つの異なるアッセイ方法を使用するASTベースMICアッセイが任意の単一のアッセイを使用するよりもより良い確証をもたらし得ることを示す。パーセント補正スコア(percent correct score)を代謝アッセイ又は表面結合アッセイについて、各種の30を超える株のアルゴリズム的に呼び出されたデータ(algorithmically called data)に基づいて作成した。このスコア化システムでは、基本的な一致は、2つのアッセイについてのMICが1回の2倍希釈までで互いに異なっている場合に達成したと見なした。図29は、2つの種、A、K.ニューモニエ及びB、S.アウレウスについての代謝アッセイ又は表面結合アッセイに対するパーセント補正スコアを示している。図に示すとおり、2つのアッセイ間はかなり一致しているが、パーセント補正スコアは使用された抗生物質に基づいてアッセイ内で異なっていた。例えば図29Aでは、ゲンタマイシン(GEN)についての表面結合アッセイは、K.ニューモニエについての代謝アッセイよりも、アルゴリズム的に呼び出されたデータとより良い一致を示した。95%対83%。このような場合では表面結合アッセイは、微生物、K.ニューモニエへの抗微生物剤ゲンタマイシンのMICについてさらに決定的、明白かつ説得力がある結果をもたらした。一方、抗微生物剤セフトリアキソン(CRO)は、代謝アッセイがアルゴリズム的に呼び出されたMICデータとの100%一致を達成して、代謝アッセイ及び表面結合アッセイの両方に高い程度の正確性を示した。
Claims (12)
- 微生物の抗菌薬感受性を決定する方法であって、
1種以上の微生物の懸濁液を、複数のチャンバを含むカートリッジに導入するステップであって、複数のチャンバは1種以上の抗菌剤を含む、ステップ、
初期インキュベーション期間の間、微生物成長を促進する条件下でカートリッジをインキュベートするステップ、
カートリッジチャンバの第1のサブセットにおいて、微生物成長が閾値を達成したかどうかを決定するために、成長指標を使用する1つ以上のチェックポイントアッセイを実施するステップ、並びに
(a)閾値が達成された場合、1種以上の抗菌薬に対する微生物の感受性を決定するためにカートリッジチャンバの別の第2のサブセットにおける代謝プローブアッセイ及びカートリッジチャンバの別の第3のサブセットにおける表面結合プローブアッセイを含む成長アッセイのセットを実施し、前記代謝プローブアッセイ及び表面結合プローブアッセイに基づき、最小発育阻止濃度(MIC)及び/若しくは定性的感受性結果(QSR)を得るステップ、又は
(b)閾値が達成されていない場合、
(i)閾値が達成されるまで微生物成長を促進する条件下で1回以上のさらなるインキュベーション期間を実施し、その後、ステップ(a)を実施するステップ、若しくは
(ii)閾値が達成されずに最大18時間が経過するまで微生物成長を促進する条件下で1回以上のさらなるインキュベーション期間を実施し、及び、さらなるアッセイが実施されないステップ
を含む、方法。 - 前記1つ以上のチェックポイントアッセイが、代謝プローブアッセイ、表面結合プローブアッセイ、化学プローブアッセイ、生化学プローブアッセイ、酵素生化学プローブアッセイ、ATPアッセイ、核酸プローブアッセイ、二本鎖核酸プローブアッセイ、光学密度アッセイ、視覚アッセイ及びpH分子プローブアッセイからなる群から選択される、請求項1に記載の方法。
- 代謝プローブが、7-ヒドロキシ-10-オキシドフェノキサジン-10-イウム-3-オン(レザズリン)を含む、請求項1又は請求項2に記載の方法。
- 表面結合プローブが、ランタニドとジエチレントリアミン四酢酸又はクリプテート配位子との配位錯体を含み、及び好ましくは、表面結合プローブが、少なくとも1つのユウロピウム、ストロンチウム、テルビウム、サマリウム又はジスプロシウムを含む、請求項1~3のいずれかに記載の方法。
- 異なる成長アッセイが、(a)逐次的、又は(b)同時に実施される、請求項1~5のいずれかに記載の方法。
- 1つ以上のチェックポイントアッセイチャンバが、抗菌薬を含まない、請求項1~6のいずれかに記載の方法。
- 閾値が、前記微生物に応じた値を含む、請求項1~7のいずれかに記載の方法。
- カートリッジが、96個、384個又は1536個のチャンバを含む、請求項1~8のいずれかに記載の方法。
- 1種以上の微生物が、臨床試料に由来し、及び、好ましくは、前記臨床試料が、エシェリキア属種(Escherichia spp.)、エンテロコッカス属種(Enterococcus spp.)、スタフィロコッカス属種(Staphylococcus spp.)、クレブシエラ属種(Klebsiella spp.)、アシネトバクター属種(Acinetobacter spp.)、シュードモナス属種(Pseudomonas spp.)、エンテロバクター属種(Enterobacter spp.)、ストレプトコッカス属種(Streptococcus spp.)、プロテウス属種(Proteus spp.)、アエロコッカス属種(Aerococcus spp.)、アクチノマイセス属種(Actinomyces spp.)、バシラス属種(Bacillus spp.)、バルトネラ属種(Bartonella spp.)、ボルデテラ属種(Bordetella spp.)、ブルセラ属種(Brucella spp.)、カンピロバクター属種(Campylobacter spp.)、クラミジア属種(Chlamydia spp.)、クラミドフィラ属種(Chlamydophila spp.)、クロストリジウム属種(Clostridium spp.)、コリネバクテリウム属種(Corynebacterium spp.)、エールリヒア属種(Ehrlichia spp.)、フランシセラ属種(Francisella spp.)、ガードネレラ属種(Gardenerella spp.)、ヘモフィルス属種(Haemophilius spp.)、ヘリコバクター属種(Helicobacter spp.)、ラクトバシラス属種(Lactobacillus spp.)、レジオネラ属種(Legionella spp.)、レプトスピラ属種(Leptospira spp.)、リステリア属種(Listeria spp.)、マイコバクテリウム属種(Mycobacterium spp.)、マイコプラズマ属種(Mycoplasma spp.)、ナイセリア属種(Neisseria spp.)、ノカルディア属種(Nocardia spp.)、パスツレラ属種(Pasteurella spp.)、リケッチア属種(Rickettsia spp.)、サルモネラ属種(Salmonella spp.)、シゲラ属種(Shigella spp.)、ステノトロホモナス属種(Stenotrophomonas spp.)、トレポネーマ属種(Treponema spp.)、ウレアプラズマ属種(Ureaplasma spp.)、ビブリオ属種(Vibrio spp.)、エルシニア属種(Yersinia spp.)、カンジダ属種(Candida spp.)、イサタケンキア属種(Issatchenkia spp.)、ブラストミセス属種(Blastomyces spp.)、コクシジオイデス属種(Coccidioides spp.)、アスペルギルス属種(Aspergillus spp.)、クリプトコッカス属種(Cryptococcus spp.)、ヒストプラズマ属種(Histoplasma spp.)、ニューモシスチス属種(Pneumocystis spp.)、スタキボトリス属種(Stachybotrys spp.)、スポロスリックス(Sporothrix)、エキセロハイラム(Exserohilum)、クラドスポリウム(Cladosporium)、リングワーム(ringworm)、ムコールミセテス(mucormycetes)及びこれらの組合せからなる群から選択される微生物を含む、請求項1~9のいずれか一項に記載の方法。
- ステップが、抗菌薬感受性試験のための自動プラットフォームにおいて実施される、請求項1~10のいずれかに記載の方法。
- カートリッジがマイクロプレートであり、複数のチャンバがウェルであり、並びに、1つ以上のチェックポイントアッセイのために使用されるマイクロプレートのウェルが前記1つ以上の抗菌剤を含まず、及び、複数の成長アッセイのために使用されない、請求項1に記載の方法。
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