Name: rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry
Uploaded: 2014-05-28T13:45:00
Description: Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

<html> 1.. Blood kulturnæringssupper Flag som &quot;positiv&quot; <ol><li> Fjern det signalerede blod kultur flaske fra den løbende overvågning inkubation kabinet og læg det i et biologisk sikkerhedsskab. Bemærk: Flasker kan indeholde farlige mikroorganismer og universelle forholdsregler skal følges. På grund af risikoen for infektiøse aerosoler i prøvetagning, skal alle prøveudtagningsprocedurer udføres i en laminar strømning biosikkerhed klasse II kabinet. </li></ol> 2.. Gram Stain e...

<ol>
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Det er vigtigt, når de anvender MALDI-TOF MS til bloddyrkning bouillon, at stillingen centrifugeringstrin udføres med tilstrækkelig omhu for ikke at remixe de adskilte komponenter. Det er særligt vigtigt at fjerne blod kultur bestanddele og humane cellulære proteiner, herunder hæmoglobin, som kan producere pigge interfererer med MALDI-TOF-spektre. Selvom MS fremstiller anbefale skåret af score på ≥ 2,0 for arter og ≥ 1,7 for slægten identifikation, har andre rapporter foreslået...

Patienter med infektion i blodet (BSI) på grund af bakterier har fortsat høj på hospitalet dødelighed, der spænder fra 6 til 48% 1. Leveringen af passende empirisk antibiotika fremmer overlevelse og i undergruppen af patienter med svær sepsis, hver time forsinkelse på passende behandling korrelerer til nedsat overlevelse 2,3. Derfor et centralt mål for det kliniske laboratorium er hurtigt at opdage, identificere og kommunikere tilstedeværelsen af ​​bakterier i blod kulturer til at informere de kliniske beslutninger. Det er blevet påvist, at den mikrobiologiske laboratorium har den største indflydelse på antimikrobiel behandling på tidspunktet for indberetning af Gram-farvning 4 og for nylig, viste et observationsstudie, at matrix-assisteret laser desorption / ionisering tid søgning massespektrometri (MALDI-TOF MS) udføres direkte på blod kulturnæringssupper indflydelse ordination på over en tredjedel af BSI forårsaget af Gram-negative bakterier 5. 6,7. Teknologien er nu veletableret og er blevet integreret i mange laboratorier til hurtig og præcis identifikation af mikroorganismer isoleret på faste medier 6,8. Den direkte anvendelse af MALDI-TOF MS blod kultur (BC) bouillon, der har signaleret &quot;positive&quot; for mikroorganismer appellerer til både klinikere og laboranter ledere på grund af muligheden for at få en tidligere identifikation af mikroorganismer til en lav pris. Den kliniske anvendelighed af direkte anvendelse af MALDI-TOF MS blod kultur bouillon har været begrænset af den brede vifte af følsomheder observeret sammenlignet med standard fænotypisk kultur baserede metoder til identifikation, med rapporter om vellykkede identifikation af Gram-negative bakterier, der spænder fra 47 til 98,9 % 9-11. Variationen i følsomhed sandsynligvisangår BC bouillon sammensætning, initial bakteriel koncentration, variation i prøveforberedelsesmetoder samt vifte af gram-negative organismer forekommer i undersøgelsesbefolkningsgrupper 9. Sammenlignet med disse andre publicerede protokoller metoden præsenteret her undgår brugen af ​​ethanol, ammoniumchlorid eller yderligere (ikke-matrix) acetonitril. Som et resultat bakteriepelleten vil forblive levedygtig (indtil det punkt, af protein ekstraktion) giver mulighed for potentielle fænotypisk følsomhed prøvningsmetoder, der skal anvendes direkte på disse organismer i bouillon. Derudover har den præsenterede metode vist sig at være billig, pålidelig og hurtig med bakteriel identifikation rådighed inden 25 min af blod kultur Gram farve resultater de, med minimal &#39;hands on&#39; tid 12. Denne metode er en simpel in-house spin-lysis protokol anvender myresyre ekstraktion anvendes direkte til positive blod kulturnæringssupper at identificere gramnegative bacteria med MALDI-TOF MS-teknologi.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

En vigtig rolle i det kliniske mikrobiologiske laboratorium er at give hurtig identifikation af bakterier, der forårsager infektion i blodet. Traditionel identifikation kræver en sub-kultur signalerede blod kultur bouillon med identifikation kun tilgængelig efter kolonier på fast agar har modnet. MALDI-TOF MS er en pålidelig, hurtig metode til identifikation af de fleste klinisk relevante bakterier, når de anvendes til kolonier på faste medier. Anvendelsen af ​​MALDI-TOF MS direkte til blod dyrkningsvæsken er en attraktiv tilgang, da det har potentiale til at fremskynde artsbestemmelse af bakterier og forbedre det kliniske forvaltning. Men et vigtigt problem at overvinde er foranalysen fjernelse af interfererende harpiks, proteiner og hæmoglobin, der er indeholdt i blodet kultur prøver, der, hvis de ikke fjernes, interfererer med MS spektre og kan resultere i utilstrækkelige eller lave forskelsbehandling identifikation scoringer. Desuden er det nødvendigt at koncentrere bacterbl.a. at udvikle spektre af tilstrækkelig kvalitet. Den præsenterede metode beskriver koncentrationen, rensning og udvinding af Gram-negative bakterier giver mulighed for tidlig identifikation af bakterier fra en signaleret blod kultur bouillon.

Den genererede MALDI-TOF MS-spektre er i forhold til den integrerede referencedatabase af spektre. En logaritmisk score er tildelt for tilliden af kampen mellem testen isolat og referencedatabasen isolater med anbefaling af en score ≥ 1.7 kræves for sandsynlig identifikation til slægter niveau (figur 1) og ≥ 2,0 for sandsynlig identifikation arter (figur 2). Rapporter til artsniveau når scoren ≥ 1.7 og de ​​første 5 identifikationer modsvares af Typing software er konsekve...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

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

Anvendelsen af ​​matrix-assisteret laser desorption / ionisering tid søgning (MALDI-TOF) massespektrometri (MS) direkte til blod dyrkningssuppe fremskynder identifikation af bakterier. I denne procedure er en hurtig og pålidelig metode til identifikation af Gram-negative bakterier direkte fra blod dyrkningsvæsken.

Rapid Identifikation af Gram-negative bakterier fra Blood kulturvæsken Brug MALDI-TOF-massespektrometri

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