私たちは、実験室での技術を実装するために彼らの助けのためにローザンヌ大学病院センターの細菌学研究室の技術者に感謝します。

このプロトコルが開発され、日常的なツールとして実装される前に、当社の機関の研究開発プロセスや倫理的なルールに従う検証されています。 塩化アンモニウム赤血球溶解手順による血液培養細菌ペレットの調製<ol><li>その後の遠心分離のための血液培養陽性の調製。 <ol><li>血液培養キャップを滅菌する。ボトルのキャップ上に70％エタノールを追加し、それを燃やす。 注：層流フードの下に、この手順を実行しないでください。 </li><li>層流フードにボトルを移動します。 20のGシリンジで血液培養の5ミリリットルを収集します。 </li><li>滅菌水（H 2 O）の45ミリリットルを含有する50 mlの遠心チューブに血液培養の5ミリリットルを加え、サンプルを混ぜる。 </li></ol></li><li>溶解遠心分離による血液培養細菌ペレットの調製。 <ol><li>室温で10分間、1,000×gでサンプルを遠心。 </li><li>レモ実験室の真空ポンプで（主にH 2 Oおよび血液細胞を含む）上清VEの。 </li><li>溶液（0.15 M NH 4 Clを、1mMのKHCO 3）を溶解自家製塩化アンモニウム1mlにペレットを再懸濁する。削って、チューブをボルテックスでペレットを分解し、室温で10分間、140×gでサンプルを遠心する。 </li><li>主に赤血球の破片が含まれている上澄みを捨てる。 <ol><li>ペレットは、出血性のままでいる場合、さらに残留赤血球を溶解するために、ペレットを洗浄し、滅菌蒸留H 2 O 2ml中にペレットを再懸濁する。 RTで10分間、140×gでサンプルを遠心し、上清を捨てる。 </li></ol></li><li> H 2 O200μlにペレットを再懸濁</li></ol></li><li>さまざまな選択的および非選択寒天培地上で血液培養のサブカルチャー。 <ol><li> 20のGシリンジで血液培養の1ミリリットルを収集します。 </li><li>血液培養intの1滴を接種血液寒天、チョコレート寒天、マッコンキー寒天とシェドラー寒天上の4つの象限O。 </li><li> 5％のCO 2雰囲気下で37℃で血液寒天、チョコレート寒天プレートをインキュベートし、通常の大気中のマッコンキー寒天および嫌気性条件のシェドラー寒天プレート。 </li><li>異なるメディア上の細菌の増殖に従ってください。 </li><li>細菌の純度を確認してください。 </li></ol></li></ol> MALDI-TOF MSによる2識別<ol><li>血液ペレットからの直接同定。 <ol><li>転送1 MALDIターゲットプレート上の血液ペレットμlの42℃の加熱プラットフォーム上それが乾燥させます。 </li><li> 1μlの70％ギ酸でサンプルをオーバーレイし、42℃の加熱プラットフォーム上それが乾燥させます。 </li><li> MALDIマトリックスを1μl（50％アセトニトリル-2.5％トリフルオロ酢酸中のα-シアノ-4 - ヒドロキシ桂皮酸の飽和溶液）を試料にオーバーレイし、42℃の加熱プラットフォーム上それが乾燥させます。 </li><li> MALに進んでください製造業者によって記載されるようにMALDI-TOF MSシステムとDI-TOF MS分析。 </li><li>識別スコアは、種レベルで無効である場合（スコア&lt;2を持つインスタンスの場合）、血液ペレットサンプルのタンパク質抽出を行う。 </li></ol></li><li>血液培養ペレットからのタンパク質抽出後の同定。 <ol><li> RTで2分間13000×gで、70％エタノール及び遠心1mlでペレットを20μlを混ぜる。 </li><li>上澄みを捨て、ペレットに70％のギ酸を25μlと100％アセトニトリル25μlのを追加。ペレットを再懸濁するために積極的に渦。ボルテックスの前にピペットチップでそれらを分解してタフなペレットを再懸濁。 </li><li>室温で2分間13000×gで遠心分離する。転送1 MALDIターゲットプレート上の上清液（抽出されたタンパク質が含まれている）と、42℃の加熱プラットフォーム上それが乾燥させます。 2.1.2で説明したように進んでください。 </li></ol></li></ol> 3細菌Identificationと自動微生物系による抗生物質感受性試験<ol><li>自動化された微生物のシステムとの細菌同定およびASTのための細菌懸濁液の調製。 <ol><li>自動化された微生物のシステムと互換性の無菌0.45％NaCl溶液3 mlを含有するプラスチックチューブを準備します。 </li><li> 0.6〜0.8のマクファーランド濁度に応じた細菌懸濁液を得るために、0.45％NaCl溶液中に血液培養ペレットの十分な量を加える。濃度計機を用いて濁度を測定します。 注：血液培養細菌ペレットの容積が0.6から0.8のマクファーランド濁度を達成するために、50〜200μlのごとに異なります。 </li></ol></li><li>製造業者によって記載されるように識別のための自動化されたグラム陽性（GP）またはグラム陰性（GN）カードおよびグラム陽性球菌およびグラム陰性菌の抗菌薬感受性試験のための自動化されたASTカードを接種する。 注：IdentificatioMALDI-TOF MSの識別スコア値が&gt; 2であればnはGPまたはGNカードが適用されない。血液寒天培地（GPとGN）とマッコンキー寒天（GN）プレート上の細菌懸濁液を継代培養することにより、細菌懸濁液の純度を確認してください。 </li></ol>ディスク拡散アッセイによる4抗生物質感受性試験 ディスク拡散アッセイは、抗菌薬感受性試験（EUCAST、バージョン3.0、2013年4月、www.eucast.org）に関する欧州委員会によって記述されている。 <ol><li>細菌懸濁液の調製は、ディスク拡散アッセイによる抗菌薬感受性試験を実行する。 <ol><li>無菌の0.9％NaCl溶液3mlを含有するガラス管を準備する。 </li><li> 0.5のマクファーランド濁度に応じた細菌懸濁液を得るために0.9％NaCl溶液中の血液培養ペレットの十分な量を加える。濃度計機を用いて濁度を測定します。 注：血液培養物の容量細菌ペレットを0.6から0.8のマクファーランド濁度を達成するために、50〜200μlに変化します。 </li><li>細菌懸濁液をボルテックスする。 </li></ol></li><li>ミューラーヒントン寒天（MH）またはミューラー·ヒントン寒天培養しにくい有機体寒天（MHF）への細菌懸濁液の接種は（MHが5％脱線維素ウマ血液および20mg、β-ニコチンアミドアデニンジヌクレオチド/ L添加）。 <ol><li>細菌懸濁液に滅菌綿棒を浸し、軽くガラス管の内壁に綿棒を回して余分な水分を取り除く。 </li><li>三方向スワブによって、又は自動プレートローテーターを用いて、MH / MHF寒天プレートの表面全体に均一に細菌懸濁液を広げる。 </li></ol></li><li>接種した寒天プレート上の抗菌ディスクの応用。 <ol><li>手動で乾燥した接種した寒天プレートの表面上または感受性ディスクディスペンサーを使用して、密接にディスクを適用する。 </li><li>適切なテンペでプレートをインキュベート抗菌薬感受性試験EUCASTディスク拡散法（バージョン3.0、2013年4月、www.eucast.org）に記載されているようratureと雰囲気。 </li></ol></li></ol> 5。MRSAの検出のための自動化されたPCRベースの診断テスト<ol><li>マイクロピペットを用いて、20秒の間、PCRベースの診断検査キットと渦を自動化されたMRSAに付属のサンプル用試薬バイアルに血液培養ペレットを50μlを移す。 </li><li> 1ミリリットルのマイクロピペットを使用して、カートリッジの試料ポートに試料を分注する。自動化されたPCRシステムにカートリッジを挿入し、製造業者によって記載されるようにアッセイを開始します。 </li></ol>

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著者は、彼らが競合する金融利害がないことを宣言します。

従来の血液培養陽性診断アプローチと比較して、塩化アンモニウム溶解遠心分離法を用いて、細菌ペレットの迅速な取得が24時間と48時間と24によってASTを報告する時間（ 図1および 16による識別を報告するための時間を短縮3）。 適切な抗生物質治療の迅速な導入が血流感染に罹患している患者の転帰を改善するために極めて重要である。このよ?...

入院患者における血流感染および敗血症は、罹患率および死亡率の主な原因である。したがって、感染血流に関連した死亡率は、入院患者の37％に約14％で観察され、集中治療室に35％の患者1-3を増加させることができる。感染性病原体の迅速な同定は、最適な抗菌処理を案内すると、抗菌薬治療4,5の成功の結果を高めるために極めて重要である。陽性の血液培養からのグラム染色の迅速な分析がすでに抗菌療法6,7が、感染性因子の正確な同定の適応に重要な影響を持っている患者への最適な適応抗生物質治療を提供するために必要である。例えば、異なる抗生物質による治療レジメンは、グラム染色によって区別す​​ることが困難であり、腸球菌および連鎖球菌による菌血症以下に実装されなければならない。同様に、種での識別がLEVエルは、8ラクタムβに増加した抵抗性を付与する染色体がAmpC遺伝子をコードするグラム陰性腸内細菌を検出する必要がある。 血液培養陽性の場合、従来の診断アプローチは、生化学検査、異なる選択培地上での成長および自動化された微生物同定システムを含むさまざまなアプローチを有する追加のインキュベーションの前に識別の数時間を必要とする別の寒天プレート上の感染性病原体を、継代培養することである。従来の診断アプローチの結果までの時間は約1〜3日間である。 微生物の迅速な同定のためのマトリックス支援レーザー脱離/イオン化飛行時間型質量分析（MALDI-TOF）技術の出現は、迅速に陽性の血液から直接、また、寒天プレート上で増殖させたコロニーから微生物しかしを識別するための新しいツールを提供している培養物（ 図1）9-12。のTh血液培養から感染性病原体を識別するために、MALDI-TOFのEの使用は、数分の代わりに伝統的な方法が必要とする時間と曜日に、結果までの時間を削減しました。 Croxatto ら 13で説明したように、MALDI-TOF同定の効率は、微生物の純度と量など、さまざまなパラメータに依存しています。これらの2つの基準が簡単に寒天プレート上で増殖させた個別のコロニーから得られたが、MALDI-TOF識別を妨害する可能性が複数の細胞およびタンパク質の成分を含んでいてそのような血液培養などの複雑なサンプルからの細菌の濃縮および精製のための分析前処理が必要である。 血液培養から各種微生物&#39;単離方法は、サポニンまたは細菌抽出9,14、血清分離方法10のための他の中性洗剤法を含む多くの研究が、溶解遠心分離方法12において使用されている</sアップ&gt;などsepsityperキットとして市販ソリューションを提供しています。当社の細菌学診断研究所は、MALDI-TOFによる細菌や酵母の迅速同定および自動識別システム（ 図2）15 を許可し 、塩化アンモニウム赤血球溶解に基づく簡単な血液培養細菌ペレット製剤を開発しました。この血液培養ペレット製剤はまた、グラム染色、メチシリン耐性黄色ブドウ球菌 （MRSA）の迅速な検出、およびと抗生物質感受性試験用のPOCT-PCRのような自動化されたPCRベースの診断テストのような他の直接下流のアプリケーションのためのサンプルを提供自動化されたASTシステムおよび/ ​​または寒天プレート上のディスク拡散アッセイ（ 図3）による。 Prod&#39;hom らが説明したように、本 ​​研究では、血液培養細菌ペレットを製造するための異なるステップを説明します。15（ 図4）。また、解除しますMALDI-TOF 15による同定、識別（ID）と、 腸内細菌やブドウ球菌および自動化されたPCR-のための自動化されたシステム16と抗生物質感受性試験（AST）：血液培養ペレット上で実行できる主なアプリケーションの3のためのプロトコルをスクライブMRSA 17の検出のためのベースの診断テスト。

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
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			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
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			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
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			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
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		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
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			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

血流感染および敗血症は、罹患率および死亡率の主な原因である。菌血症を患っている患者の成功した結果は、最適な抗生物質治療を導くために、感染因子の迅速な同定に依存する。陽性の血液培養からのグラム染色の解析が急速に行われ、すでに大幅に抗生物質投与計画に影響を与えることができます。しかし、感染因子の正確な同定は、依然として最適な標的治療を確立するために必要とされる。ここでは、ディスク拡散アッセイまたは自動ASTシステムによるMALDI-TOF MSによる同定、抗生物質感受性試験（AST）などのいくつかの本質的なダウンストリームアプリケーションのための試料とすることができる陽性の血液培養から簡単かつ迅速に細菌ペレット製剤を提示自動化されたPCRベースの診断試験による。血液培養、細菌のペレットに直接適用これらの異なる識別およびASTシステムの性能は非常にSiである通常、寒天プレート上で増殖させた単離されたコロニーから得られた性能にmilar。従来のアプローチと比較して、細菌ペレットの迅速な取得が有意に識別およびASTの両方を報告する時間が短縮される。したがって、血液培養陽性以下、MALDI-TOFによって同定はそれぞれ8〜18時間内、自動ASTシステムやディスク拡散アッセイによりASTの結果に対し、1時間未満以内に報告することができる。同様に、迅速なPCRに基づくアッセイの結果は、菌血症の報告後2時間未満で臨床医に伝えることができる。まとめると、これらの結果は、血液培養細菌ペレットの迅速な調製が同定およびASTのターンアラウンド時間に、従って、血流感染に罹患している患者の成功した結果に大きな影響を与えることを示している。

Prod&#39;hom ら 15による研究において、78人の患者から122血液培養陽性の塩化アンモニウム溶解遠心分離によって得られた細菌ペレットを、MALDI-TOF MSにより分析した。 122血液培養陽性のうち、95（77.9％）が正しく属レベルでの種レベル一つ（0.8％）で同定された。残りの26（21.3％）の血液培養ペレットMALDI-TOFによって信頼性の識別を与えなかった。このうち、21が13連鎖球菌と5コ?...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

陽性の血液培養からの急速な細菌ペレット製剤は、MALDI-TOFによる識別、グラム染色、抗生物質感受性試験およびPCRに基づく試験のような用途のための試料とすることができる。結果は、急速に血流感染症に罹患している患者の転帰を改善するために臨床医に伝えることができる。

迅速な細菌同定および抗生物質感受性試験のための血液培養ペレットの調製

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K ビュー.  University Hospital Center and University of Lausanne. 陽性の血液培養からの急速な細菌ペレット製剤は、MALDI-TOFによる識別、グラム染色、抗生物質感受性試験およびPCRに基づく試験のような用途のための試料とすることができる。結果は、急速に血流感染症に罹患している患者の転帰を改善するために臨床医に伝えることができる。 

ビデオ: Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

監視形成との抗真菌薬感受性試験のための96ウェルマイクロタイタープレートベースの方法カンジダアルビカンスバイオフィルム

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

免疫・感染学

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

タンパク質調製物、動物の予防接種と中和の検出：遺伝子組換えRBDベースのSARSのワクチンのためのプロトコル

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

の識別のための1.5時間の手順腸球菌</em直接血液培養から>種

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

血液培養から細菌性病原体の検出および抗菌剤耐性テストのための一日のワークフロースキーム

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

ダルババンシンの参照ブロス微量希釈法<em&gt;インビトロ</em好気的に成長する細菌の&gt;感受性試験

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

血液処理のための迅速な方法は、ヒト血液中の血漿ペプチドレベルの収量を増やすには

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...