Name: Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles
Uploaded: 2024-05-24T13:40:00
Description: Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Studien ble finansiert av det intramurale forskningsstipendet gitt til Dr. Ayush Gupta av AIIMS Bhopal. Vi anerkjenner bidraget fra laboratorieteknikere og fastleger som utførte og leste testene flittig i rutine- og akutttiden.

Studien, finansiert av det intramurale forskningsstipendet gitt til Dr. Ayush Gupta av AIIMS Bhopal, ble godkjent av Institutional Human Ethics Committee (IHEC) ved brev nr: IHEC-LOP/2022/IL072.
MERK: Et prøvevolum på 5 ml ble brukt basert på studier utført av Quesada et al.25 og Munoz-Davila et al.27.
1. Standard inokulumprotokoll (SIP) for bakteriell identifikasjon ved bruk av aMIAST
<ol><li>Br...

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

<ol>
	<li>Rudd, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional,+and+national+sepsis+incidence+and+mortality,+1990&#8211;2017:+analysis+for+the+Global+Burden+of+Disease+Study.">regional, and national sepsis incidence and mortality, 1990&#8211;2017: analysis for the Global Burden of Disease Study.</a> Lancet. 395 (10219), 200-211 (2020).</li><li>Ikuta, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+mortality+associated+with+33+bacterial+pathogens+in+2019:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2019.">Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019.</a> Lancet. 400 (10369), 2221-2248 (2022).</li><li>Liu, V. 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European Committee on Antimicrobial Susceptibility testing. , (2023).</li><li>Clinical and Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+standards+for+antimicrobial+susceptibility+testing.+33rd+ed.">Performance standards for antimicrobial susceptibility testing. 33rd ed.</a> CLSI. Clinical and Laboratory Standards Institute. , (2023).</li><li>European Committee on Antimicrobial Susceptibility testing. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology-EUCAST+rapid+antimicrobial+susceptibility+testing+(RAST)+directly+from+positive+blood+culture+bottles.+Version+1.0.">Methodology-EUCAST rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles. Version 1.0.</a> EUCAST. 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European Committee on Antimicrobial Susceptibility testing. , (2018).</li><li>European Committee on Antimicrobial Susceptibility testing. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zone+diameter+breakpoints+for+rapid+antimicrobial+susceptibility+testing+(RAST)+directly+from+positive+blood+culture+bottles.+Version+6.1.">Zone diameter breakpoints for rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles. Version 6.1.</a> EUCAST. European Committee on Antimicrobial Susceptibility testing. , (2023).</li><li>Soo, Y. T., Waled, S. N. M. B., Ng, S., Peh, Y. H., Chew, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+EUCAST+rapid+antimicrobial+susceptibility+testing+(RAST)+directly+from+blood+culture+bottles.">Evaluation of EUCAST rapid antimicrobial susceptibility testing (RAST) directly from blood culture bottles.</a> Euro J Clin Microbiol Infect Dis. 39 (5), 993-998 (2020).</li><li>Berinson, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCAST+rapid+antimicrobial+susceptibility+testing+(RAST):+Analytical+performance+and+impact+on+patient+management.">EUCAST rapid antimicrobial susceptibility testing (RAST): Analytical performance and impact on patient management.</a> J Antimicrob Chemother. 76 (5), 1332-1338 (2021).</li><li>Strubbe, G., Messiaen, A. S., Vandendriessche, S., Verhasselt, B., Boelens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCAST+rapid+antimicrobial+susceptibility+testing+(RAST)+compared+to+conventional+susceptibility+testing:+implementation+and+potential+added+value+in+a+tertiary+hospital+in+Belgium.">EUCAST rapid antimicrobial susceptibility testing (RAST) compared to conventional susceptibility testing: implementation and potential added value in a tertiary hospital in Belgium.</a> Acta Clinica Belgica: Int J Clin Lab Med. 78 (5), 385-391 (2023).</li><li>Jasuja, J. K., Zimmermann, S., Burckhardt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+EUCAST+rapid+antimicrobial+susceptibility+testing+(RAST)+for+positive+blood+cultures+in+clinical+practice+using+a+total+lab+automation.">Evaluation of EUCAST rapid antimicrobial susceptibility testing (RAST) for positive blood cultures in clinical practice using a total lab automation.</a> Euro J Clin Microbiol Infect Dis. 39 (7), 1305-1313 (2020).</li><li>Martins, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+antimicrobial+susceptibility+of+Enterobacteriaceae+by+disk+diffusion+directly+from+blood+culture+bottles+using+the+EUCAST+RAST+breakpoints.">Rapid antimicrobial susceptibility of Enterobacteriaceae by disk diffusion directly from blood culture bottles using the EUCAST RAST breakpoints.</a> J Glob Antimicrob Resist. 22, 637-642 (2020).</li><li>Ekwall-Larson, A., Fr&#246;ding, I., Mert, B., &#197;kerlund, A., &#214;zenci, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+performance+and+potential+clinical+utility+of+EUCAST+rapid+antimicrobial+susceptibility+testing+in+blood+cultures+after+four+hours+of+incubation.">Analytical performance and potential clinical utility of EUCAST rapid antimicrobial susceptibility testing in blood cultures after four hours of incubation.</a> Microbiol Spectr. 11 (2), e500122 (2023).</li><li>Cerrudo, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+EUCAST+rapid+antibiotic+susceptibility+breakpoints+and+screening+cut-off+values+directly+from+blood+cultures+for+the+inference+of+&#946;-lactam+resistance+mechanisms+in+Enterobacterales.">Usefulness of EUCAST rapid antibiotic susceptibility breakpoints and screening cut-off values directly from blood cultures for the inference of &#946;-lactam resistance mechanisms in Enterobacterales.</a> JAC-Antimicrob Resist. 5 (1), 017 (2023).</li><li>Shan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+EUCAST+rapid+antimicrobial+susceptibility+test+for+enterobacterales-containing+blood+cultures+in+China.">Evaluation of the EUCAST rapid antimicrobial susceptibility test for enterobacterales-containing blood cultures in China.</a> J Clin Microbiol. 60 (4), e0255921 (2022).</li><li>Jonasson, E., Matuschek, E., Kahlmeter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EUCAST+rapid+disc+diffusion+method+for+antimicrobial+susceptibility+testing+directly+from+positive+blood+culture+bottles.">The EUCAST rapid disc diffusion method for antimicrobial susceptibility testing directly from positive blood culture bottles.</a> J Antimicrob Chemother. 75 (4), 968-978 (2020).</li><li>Tay&#351;i, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+the+EUCAST+rapid+antimicrobial+susceptibility+test+(RAST)+directly+from+positive+blood+culture+bottles+without+the+advanced+identification+systems.">Implementation of the EUCAST rapid antimicrobial susceptibility test (RAST) directly from positive blood culture bottles without the advanced identification systems.</a> J Antimicrobial Chemotherapy. 77 (4), 1020-1026 (2022).</li><li>Gupta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+study+evaluating+the+effect+of+a+&#8216;Diagnostic+Stewardship+Care-Bundle&#8217;+for+automated+blood+culture+diagnostics.">A prospective study evaluating the effect of a &#8216;Diagnostic Stewardship Care-Bundle&#8217; for automated blood culture diagnostics.</a> J Glob Antimicrob Resist. 34, 119-126 (2023).</li><li>Siddiqui, F., Gupta, A., Purwar, S., Saigal, S., Sharma, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+study+to+reduce+turnaround+time+of+microbiologically+positive+blood+cultures+in+patients+with+sepsis+in+intensive+care+unit.">A prospective study to reduce turnaround time of microbiologically positive blood cultures in patients with sepsis in intensive care unit.</a> Indian J Med Microbiol. 40 (4), 541-546 (2022).</li><li>Gherardi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+the+Vitek-2+Compact+and+Phoenix+systems+for+rapid+identification+and+antibiotic+susceptibility+testing+directly+from+blood+cultures+of+Gram-negative+and+Gram-positive+isolates.">Comparative evaluation of the Vitek-2 Compact and Phoenix systems for rapid identification and antibiotic susceptibility testing directly from blood cultures of Gram-negative and Gram-positive isolates.</a> Diagnostic Microbiol Infect Dis. 72 (1), 20-31 (2012).</li><li>Lemos, T. C., Cogo, L. L., Maestri, A. C., Hadad, M., Nogueira, K. d. a. 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J., Pascual, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+positive+blood+cultures+for+direct+identification+and+susceptibility+testing+with+the+Vitek+2+system.">Use of positive blood cultures for direct identification and susceptibility testing with the Vitek 2 system.</a> J Clin Microbiol. 42 (8), 3734-3738 (2004).</li><li>Munoz-D&#225;vila, M. J., Yag&#252;e, G., Albert, M., Garc&#237;a-Lucas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+Vitek+2+identification+and+susceptibility+testing+of+Gram-negative+rods+directly+and+isolated+from+BacT/ALERT-positive+blood+culture+bottles.">Comparative evaluation of Vitek 2 identification and susceptibility testing of Gram-negative rods directly and isolated from BacT/ALERT-positive blood culture bottles.</a> Euro J Clin Microbiol Infect Dis. 31 (5), 663-669 (2012).</li><li>European Committee on Antimicrobial Susceptibility testing. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control+criteria+for+the+implementation+of+the+RAST+method.+Version+6.0.">Quality control criteria for the implementation of the RAST method. Version 6.0.</a> EUCAST. 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Ved å bruke DIP identifiserte vi RR-GN-ene med betydelig diagnostisk nøyaktighet. Gjennomsnittlig TTI etter positiv flagging av aBC var bare 507 min (~ 8,5 timer). Når det gjøres i forbindelse med EUCAST RAST-metoden for AST-bestemmelse, kan den derfor gi isolatidentifikasjon ved 8 timers AST-lesetid. Denne tilnærmingen har potensial til å implementere EUCAST RAST-metoden og eliminere behovet for massespektrometribasert identifikasjon. Dette er en velsignelse for lavressursmiljøer som ønsker å implementere EUCAS...

Sepsis, et viktig globalt helseproblem, er definert som livstruende organdysfunksjon på grunn av en dysregulert vertsrespons på infeksjon. Global Burden of Diseases Study estimerte at det var 48,9 millioner tilfeller av sepsis og 11 millioner sepsisrelaterte dødsfall over hele verden i 2017, som utgjorde nesten 20 % av alle globale dødsfall1. Rundt 2/3 av blodbaneinfeksjoner (BSI) som forårsaker dødelighet skyldes gramnegative bakterielle patogener2. De viktigste årsakene til dødelighet blant gramnegative (GN) er Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa og Acinetobacter baumannii, som står for rundt 40 % av tilfellene blant 33 bakterielle patogener2.
Blodkulturer er fortsatt gullstandarden for diagnostisering av BSI, og rask mikrobiell identifikasjon sammen med resultater fra antimikrobiell følsomhetstesting (AST) er nøkkelen til behandling. Det har blitt anslått at det er en økning på 9 % i odds for dødelighet med hver times forsinkelse i å sette inn passende antimikrobielle midler ved sepsis3. Behandlingstiden (TAT) for mikrobiologisk positive blodkulturrapporter med AST-resultater er rundt 48-72 timer med tilgjengelige mikrobiologiske verktøy i ressursbegrensede omgivelser, selv med automatiserte systemer. Som et resultat av denne undermålige TAT, brukes bredspektrede antimikrobielle midler empirisk, noe som bidrar til det voksende problemet med antimikrobiell resistens (AMR). EUCAST og CLSI erkjenner dette store behovet for å redusere TAT for mikrobiologiske dyrkningsteknikker for sepsis, og beveger seg mot å utføre AST direkte fra positivt flaggede blodkulturflasker (+aBC)4,5.
I 2018 introduserte EUCAST først den raske AST (RAST)-metoden for å bestemme AST ved hjelp av Kirby-Bauer diskdiffusjonsmetode ved korte inkubasjonstider, det vil si 4 timer, 6 timer og 8 timer, direkte fra +aBC 6,7. Metoden er for tiden validert for å bestemme AST for +aBC som inneholder en av de 8 vanligste årsakene til BSI, nemlig E. coli, K. pneumoniae, P. aeruginosa og A. baumannii-kompleks blant gram-negative og Staphylococcus aureus, Enterococcus faecalis, E. faecium og Streptococcus pneumoniae blant gram-positive8. Bruddpunktene for AST-bestemmelse med ulike tidsintervaller er gitt i henhold til mikrobielle arter som er oppført ovenfor. Derfor, før kategorisk tolkning av AST-resultater, er mikrobiell identifikasjon nødvendig. RAST-standarden spesifiserer imidlertid ikke metoden for å muliggjøre mikrobiell identifikasjon innenfor denne tidsrammen.
Flertallet av studiene som evaluerer EUCAST RAST-metoden i deres omgivelser har brukt massespektrometribasert mikrobiell identifikasjon etter kort inkubasjon på belagte medier for å identifisere mikroorganismer 9,10,11,12,13,14,15,16,17 . Massespektrometriinstrumenter er imidlertid ikke allment tilgjengelige, spesielt i lav- og mellominntektsland (LMIC), noe som i stor grad begrenser den potensielle nytten av denne metoden. Få studier har rapportert implementering av denne metoden i sine sentre uten å bruke massespektrometri 18,19,20. Tayşi et al.18 rapporterte en bred kategorisering av GN blant Enterobacterales, Pseudomonas og Acinetobacter spp. basert på gramfargemorfologi og oksidasetest før de tolket AST-resultater. I andre studier fra dette senteret, av Gupta et al.19 og Siddiqui et al.20, ble mikrobiell identifikasjon på artsnivå gjort ved å tilberede en bakteriepellet fra den positivt flaggede blod-buljongblandingen og inokulere den på de konvensjonelle biokjemiske testene. Mens Tayşi et al.18 ikke kommenterte nøyaktigheten av mikrobiell identifikasjon med deres tilnærming, rapporterte Gupta et al.19 at med deres tilnærming i 165/176 (94 %) tilfeller, en RAST-rapporterbar gram-negativ (RR-GN), det vil si en av E. coli, K. pneumoniae, P. aeruginosa og A. baumannii kompleks. Men med sistnevnte tilnærming ble avlesningen av RAST-resultater gjort retrospektivt ved bruk av 8 timers sonediameter bruddpunkter først etter full inkubasjon av konvensjonelle biokjemiske resultater, det vil si 18-24 timer etter inokulering, og gjennomsnittlig tid for rapportering var rundt 2 dager.
For å redusere TAT av kliniske rapporter ytterligere, foreslår vi en alternativ metodikk for å muliggjøre tidlig identifisering av GN tilstede i +aBC ved bruk av aMIAST. Før introduksjonen av massespektrometribaserte mikrobielle identifikasjonssystemer, ble disse automatiserte identifikasjonssystemene ansett som standarden for omsorg for mikrobiell identifikasjon, der identifikasjon ble muliggjort av kolorimetriske og/eller fluorometriske endringer indusert av testbakterier når de ble inokulert i miniatyriserte biokjemiske tester plassert i en kassett og matchet resultatene med deres isolatdatabase. Gjennomsnittlig tid til identifikasjon i disse systemene er rundt 4 timer til 8 timer, men de er begrenset av det faktum at produsentene anbefaler vekst av mikrober over natten før deres respektive identifikasjonskort kan inokuleres. Dette kravet begrenser i stor grad nytten av dem for å redusere tiden det tar å rapportere.
Få studier har evaluert metoder for direkte å identifisere mikrober fra +aBC ved å bruke disse automatiserte systemene 21,22,23,24,25,26,27. Når det gjelder +aBC som inneholder monomorf GN, viste flertallet av studiene utmerket samsvar mellom direkte inokulering fra bakteriepellet laget av positiv blodbuljongblanding og standard koloniinkubasjon. For gram-positive var imidlertid konkordansratene suboptimale. Siden gjennomsnittlig tid til positivitet av +aBC er mellom 8 timer og 16 timer og identifisering av GN tar ~4 timer til 8 timer i et automatisert mikrobielt identifikasjonssystem, antar vi at ved å bruke direkte inokuleringsprotokoll i den automatiserte mikrobielle identifikasjonen, kan vi fullføre den kliniske rapporteringen av +aBC med GN som har en RR-GN innen 24 timer etter mottak av prøve.
Ramme for studien Denne studien ble utført i det kliniske bakteriologiske laboratoriet til et 950-sengs, akademisk, tertiært omsorgsinstitutt av nasjonal betydning (INI) i Sentral-India fra januar til oktober 2023. Laboratoriet er utstyrt med et kontinuerlig blodkulturovervåkingssystem (CBCMS) og aMIAST. Bakteriologilaboratoriet er i drift døgnet rundt med tilgjengelighet av teknikere og mikrobiologer for behandling og rapportering av eventuelle positivt flaggede blodkulturflasker (+aBC).
Mikrobielle metoder brukt her Arbeidsflyten til studien er vist i figur 1. +aBC-ene som viser monomorfe GN-er (+naBC) ble behandlet ved direkte inokulering av tilsvarende identifikasjonskort for å muliggjøre identifikasjon og AST ved bruk av EUCAST RAST-protokoll. Disse resultatene ble sammenlignet med standard-of-care (SoC)-metoden for +aBCs, det vil si subkulturering på konvensjonelle belagte medier gjennom saueblodagar (SBA), sjokoladeagar (CA) og MacConkey-agar (MA), inkubert aerobt i 16 timer til 24 timer etterfulgt av identifikasjon og AST-kort gitt av aMIAST når isolerte kolonier dukker opp. Blodkulturer som viser grampositive kokker, grampositive basiller, spirende gjærceller og ≥2 forskjellige mikroorganismer på innledende gramfarging eller belagte medier ble ekskludert fra studien.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANTIMICROBIAL DISKS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amikacin disk 30 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD035-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Amoxyclav disk (20/10 &#181;g)</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD063-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Cefotaxime disk 5 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD295E-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Ceftazidime disk 10 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD062A-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Ciprofloxacin disk (5 &#181;g)</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD060-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Co-Trimoxazole disk (23.75/1.25 &#181;g)</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD010-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Gentamicin disk 10 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD016-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Imipenem disk 10 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD073-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Levofloxacin disk 5 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD216-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Meropenem disk 10 &#181;g&#160;</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD727-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Piperacillin-tazobactam disk (30/6 &#181;g)</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD292E-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>Tobramycin disk 10 &#181;g</td>
			<td>Himedia, Mumbai, India</td>
			<td>SD044-1VL</td>
			<td>Antimicrobial susceptibility testing&#160;</td>
		</tr>
		<tr>
			<td>ATCC Escherichia coli 25922</td>
			<td>Microbiologics, Minnesota USA</td>
			<td>0335A</td>
			<td>Recommended Gram negative bacterial strain for quality control in RAST</td>
		</tr>
		<tr>
			<td>BacT-Alert 3D 480</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>412CM8423</td>
			<td>Continuous automated blood culture system</td>
		</tr>
		<tr>
			<td>Biosafety cabinet II Type A2</td>
			<td>Dyna Filters Pvt. Limited, Pune, India</td>
			<td>DFP-2/21-22/149</td>
			<td>For protection against hazardous&#160; and infectious agents and to maintain quality control</td>
		</tr>
		<tr>
			<td>Blood agar base no. 2</td>
			<td>Himedia, Mumbai, India</td>
			<td>M834-500G</td>
			<td>Preparation of blood agar and chocolate agar</td>
		</tr>
		<tr>
			<td>Clinical Centrifuge Model SP-8BL</td>
			<td>Laby Instruments, Ambala, India</td>
			<td>HLL/2021-22/021</td>
			<td>Centrifugation at low and high speed for separation of supernatant</td>
		</tr>
		<tr>
			<td>Dispensette S Analog-adjustable bottle-top dispenser&#160;</td>
			<td>BrandTech, Essex CT, England</td>
			<td>V1200</td>
			<td>Dispensing accurate amount of saline</td>
		</tr>
		<tr>
			<td>MacConkey agar&#160;</td>
			<td>Himedia, Mumbai, India</td>
			<td>M008-500G</td>
			<td>Differential media for Lactose fermenters/ non-fermenters Gram negative bacilli</td>
		</tr>
		<tr>
			<td>Micropipette (100-1000 &#181;L)</td>
			<td>Axiflow Biotech Private Limited, Delhi, India</td>
			<td>NJ478162</td>
			<td>Transferring supernatant after first centrifugation, discarding supernatant after second centrifugation</td>
		</tr>
		<tr>
			<td>Micropipette tips (200-1000 &#181;L)</td>
			<td>&#8206;Tarsons Products Pvt. Ltd., Kolkata, India</td>
			<td>521020</td>
			<td>Transferring supernatant after first centrifugation, discarding supernatant after second centrifugation</td>
		</tr>
		<tr>
			<td>Mueller-Hinton agar&#160;</td>
			<td>Himedia, Mumbai, India</td>
			<td>M173-500G</td>
			<td>Antimicrobial susceptibility testing by Kirby-Bauer method of disk diffusion</td>
		</tr>
		<tr>
			<td>Nichrome loop D-4</td>
			<td>Himedia, Mumbai, India</td>
			<td>LA019</td>
			<td>For streaking onto culture media</td>
		</tr>
		<tr>
			<td>Nichrome straight wire</td>
			<td>Himedia, Mumbai, India</td>
			<td>LA022</td>
			<td>For stab inoculation</td>
		</tr>
		<tr>
			<td>Nulife sterile Gloves</td>
			<td>MRK healthcare Pvt Limited, Mumbai, India</td>
			<td></td>
			<td>For safety precautions</td>
		</tr>
		<tr>
			<td>Plain vial (Vial with red top), Advance BD vacutainer</td>
			<td>Becton-Dickinson, Cockeysville, MD, USA</td>
			<td>367815</td>
			<td>Obtaining pellet after second centrifugation</td>
		</tr>
		<tr>
			<td>Sheep blood</td>
			<td>Labline Trading Co., Hyderabad, India</td>
			<td>70014</td>
			<td>Preparation of blood agar and chocolate agar</td>
		</tr>
		<tr>
			<td>SST II tube, Advance BD vacutainer</td>
			<td>Becton-Dickinson, Cockeysville, MD, USA</td>
			<td>367954</td>
			<td>Supernatant separation in first centrifugation</td>
		</tr>
		<tr>
			<td>Sterile cotton swab (w/Wooden stick)</td>
			<td>Himedia, Mumbai, India</td>
			<td>PW005-1X500NO</td>
			<td>Lawn culture of blood culture broth for antimicrobial susceptibility testing</td>
		</tr>
		<tr>
			<td>Sterile single use hypodermic syringe 5ml/cc</td>
			<td>Nihal Healthcare, Solan, India</td>
			<td>2213805NB2</td>
			<td>Preparing aliquots from +aBC</td>
		</tr>
		<tr>
			<td>VITEK DensiCHEK McFarland reference kit</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>422219</td>
			<td>Densitometer to check the turbidity of suspension</td>
		</tr>
		<tr>
			<td>VITEK saline solution (0.45% NaCl)</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>V1204</td>
			<td>Adjustment of McFarland Standard turbidity</td>
		</tr>
		<tr>
			<td>VITEK tube stand&#160;</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>533306-4 REV</td>
			<td>Stand for proper placement of tubes before ID card inoculation</td>
		</tr>
		<tr>
			<td>VITEK tubes</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td></td>
			<td>Tubes for inoculum preparation</td>
		</tr>
		<tr>
			<td>VITEK-2 Compact 60</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>VKC15144</td>
			<td>Automated identification and AST system</td>
		</tr>
		<tr>
			<td>VITEK-2 GN card</td>
			<td>bioMerieux, Marcy d&#8217; Etoille, France</td>
			<td>21341</td>
			<td>Identification of Gram negative bacilli</td>
		</tr>
	</tbody>
</table>

direct microbial identification using an automated microbial identification system to facilitate the eucast rast method without mass spectrometry

Gramnegativ (GN) sepsis er en medisinsk nødsituasjon der behandling i ressursbegrensede omgivelser er avhengig av konvensjonelle mikrobiologiske dyrkningsteknikker som gir resultater på 3-4 dager. I erkjennelse av denne forsinkelsen i behandlingstiden (TAT), har både EUCAST og CLSI utviklet protokoller for å bestemme AST-resultater direkte fra positivt flaggede automatiserte blodkulturflasker (+aBC). EUCAST rapid AST (RAST)-protokollen ble først introdusert i 2018, hvor sonediameterbruddpunkter for fire vanlige etiologiske midler for GN-sepsis, det vil si Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa og Acinetobacter baumannii-kompleks kan rapporteres. Imidlertid er de kliniske laboratoriene som har implementert denne metoden i sin rutinemessige arbeidsflyt avhengige av massespektrometribasert mikrobiell identifikasjon, som ikke er lett tilgjengelig, og utelukker dermed implementering i ressursbegrensede omgivelser. For å omgå det, evaluerte vi en direkte inokulumprotokoll (DIP) ved hjelp av et kommersielt automatisert mikrobiell identifikasjon og antimikrobielt følsomhetstestsystem (aMIAST) for å muliggjøre tidlig mikrobiell identifikasjon innen 8 timer etter positiv flagging av aBC. Vi evaluerte denne protokollen fra januar til oktober 2023 for å identifisere de fire RAST-rapporterbare GN (RR-GN) i den positivt flaggede aBC. De mikrobielle identifikasjonsresultatene i DIP ble sammenlignet med standard inokulumpreparatprotokoll (SIP) i aMIAST. Av 204 +aBC med monomorf GN (+naBC), ble en av de 4 RR-GN identifisert i 105 +naBC ved SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 og A. baumannii-kompleks : 26). Av disse ble 94 % (98/105) korrekt identifisert ved DIP, mens major error og very major error var henholdsvis 6 % (7/105) og 1,7 % (4/240). Når DIP for mikrobiell identifikasjon gjøres ved bruk av EUCAST RAST-metoden, kan foreløpige kliniske rapporter gis innen 24 timer etter mottak av prøven. Denne tilnærmingen har potensial til å redusere TAT betydelig, noe som muliggjør tidlig iverksettelse av passende antimikrobiell behandling.

Sepsis, an important global health problem, is defined as life-threatening organ dysfunction due to a dysregulated host response to infection. The Global Burden of Diseases Study estimated that there were 48.9 million cases of sepsis and 11 million sepsis-related deaths worldwide in 2017, which accounted for almost 20% of all global deaths1. Around 2/3rd of bloodstream infections (BSI) causing mortality are due to gram-negative bacterial pathogens2. The leading causes of mortality amongst gram-negatives (GN) are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii,&#160;which account for around 40% of cases amongst 33 bacterial pathogens2.
Blood cultures remain the gold standard for diagnosing BSI, and rapid microbial identification along with antimicrobial susceptibility testing (AST) results is the key to management. It has been estimated that there is a 9% increase in odds of mortality with each-hour delay in instituting appropriate antimicrobials in sepsis3. The turnaround time (TAT) of microbiologically positive blood culture reports with AST results is around 48-72 h with the available microbiological tools in resource-limited settings, even with automated systems. As a result of this subpar TAT, broad-spectrum antimicrobials are used empirically, contributing to the burgeoning problem of antimicrobial resistance (AMR). Recognizing this dire need to reduce TAT for microbiological culture techniques for sepsis, EUCAST and CLSI are moving towards performing AST directly from positively flagged blood culture bottles (+aBC)4,5.
In 2018, EUCAST first introduced the rapid AST (RAST) method for determining AST by Kirby-Bauer disk diffusion method at short incubation times, i.e., 4 h, 6 h and 8 h, directly from +aBC6,7. The method is presently validated for determining AST for +aBCs containing one of the 8 most common causes of BSI namely E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii complex&#160;amongst gram-negatives and Staphylococcus aureus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae amongst gram-positives8.The breakpoints for AST determination at various time intervals are provided as per microbial species listed above. Hence, before categorical interpretation of AST results, microbial identification is necessary. However, the RAST standard does not specify the method to enable microbial identification within this time frame.
The majority of studies evaluating the EUCAST RAST method in their setting have used mass spectrometry-based microbial identification after short incubation on plated media to identify micro-organisms9,10,11,12,13,14,15,16,17. However, mass spectrometry instruments are not widely available, especially in low to middle-income countries (LMICs), which greatly limits the potential usefulness of this method. Few studies have reported implementation of this method in their centers without using mass spectrometry18,19,20. Tay&#351;i et al.18 reported a broad categorization of GN amongst Enterobacterales, Pseudomonas, and Acinetobacter spp. based on gram stain morphology and oxidase test before interpreting AST results. In other studies from this center, by Gupta et al.19 and Siddiqui et al.20, species-level microbial identification was done by preparing a bacterial pellet from the positively flagged blood-broth mixture and inoculating it on the conventional biochemical tests. While Tay&#351;i et al.18 did not comment upon the accuracy of microbial identification with their approach, Gupta et al.19 reported that with their approach in 165/176 (94%) cases, a RAST reportable gram-negative (RR-GN), i.e., either of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii complex. However, with the latter approach, the reading of RAST results was done retrospectively using 8 h zone diameter breakpoints only after the full incubation of conventional biochemical results i.e., 18-24 h post-inoculation, and the average time for reporting was around 2 days.
To reduce the TAT of clinical reports further, we propose an alternative methodology to enable early identification of GN present in +aBCs using aMIAST. Before the introduction of mass spectrometry-based microbial identification systems, these automated identification systems were considered the standard of care for microbial identification, where identification was enabled by colorimetric and/or fluorometric changes induced by test bacteria when inoculated in miniaturized biochemical tests harbored in a cassette and matching the results with their isolate database. The average time to identification in these systems is around 4 h to 8 h however, they are limited by the fact that the manufacturers recommend overnight growth of microbes before their respective identification cards can be inoculated. This requirement greatly limits their usefulness in reducing the time to report.
Few studies have evaluated methods to directly identify microbes from +aBCs using these automated systems21,22,23,24,25,26,27. In the case of +aBCs containing monomorphic GN, the majority of studies showed excellent concordance between direct inoculation from bacterial pellet made from positive blood-broth mixture and standard colony incubation. However, in the case of gram-positives, the concordance rates were suboptimal. As the average time to positivity of +aBCs is between 8 h and 16 h and identification of GN takes ~4 h to 8 h in an automated microbial identification system, we hypothesize that by employing direct inoculation protocol in the automated microbial identification, we can complete the clinical reporting of +aBCs with GN having a RR-GN within 24 h of sample receiving.
Setting for the study 
The present study was conducted in the clinical bacteriology laboratory of a 950-bed, academic, tertiary care institute of national importance (INI) in Central India from January to October 2023. The laboratory is equipped with a continuous blood culture monitoring system (CBCMS) and aMIAST. The bacteriology laboratory is functional round-the-clock with the availability of technicians and microbiologists for processing and reporting any positively flagged blood culture bottle (+aBCs).
Microbial methods used here 
The workflow of the study is shown in Figure 1. The +aBCs showing monomorphic GNs (+naBC) were processed by direct inoculation of corresponding identification cards to enable identification and AST using EUCAST RAST protocol. These results were compared with the standard-of-care (SoC) method for +aBCs i.e., subculturing on conventional plated media through sheep blood agar (SBA), chocolate agar (CA), and MacConkey agar (MA), incubated aerobically for 16 h to 24 h followed by identification and AST cards given by aMIAST when isolated colonies appear. Blood cultures showing gram-positive cocci, gram-positive bacilli, budding yeast cells, and &#8805;2 different micro-organisms on initial gram staining or plated media were excluded from the study.

Author Spotlight: Accelerating Diagnostic Accuracy with Direct Identification of Gram-Negatives from Blood Culture Bottles

Direkte mikrobiell identifikasjon ved hjelp av et automatisert mikrobielt identifikasjonssystem for å lette EUCAST RAST-metoden uten massespektrometri

This research aims to evaluate the diagnostic accuracy of Gram-negatives directly from positively flag blood culture bottles by automated microbial identification system. The purpose is to utilize it as a tool for early clinical reporting by EUCAST rapid antimicrobial susceptibility testing method. EUCAST RAST method was recently introduced in 2018 to enable reporting of anti-microbial susceptibility testing results within four to eight hours of positive flagging of automated blood culture bottles.It involves performing everywhere disk diffusion method directly from positively flagged bottle and reading results at either four, six, or eight hours. For clinical reporting by EUCAST RAST method, the microbial identification is a prerequisite for determining the interpretative category of AST results. The major challenge in implementing this method is early identification of the microbes within eight hours, especially in resource poor settings, which lacks mass spectrometry.We found that the direct inoculum protocol was around 94%accurate in identifying a RAST reportable Gram-negative, which includes Escherichia coli, Kiebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex. This approach of identifying Gram-negatives directly was around four times faster than the standard approach of identification after overnight incubation. We will now focus on assessing the impact of this approach of clinical reporting in Gram-negative sepsis on patient outcomes in terms of length of hospitalization and mortality rate.Furthermore, it can also be used as a tool to reduce anti-microbial usage to facilitate anti-microbial stewardship.

Generelle resultater I løpet av studieperioden ble 240 +naBC identifisert av aMIAST ved bruk av både DIP og SIP. Av disse ble 15 % (36/240) +naBC funnet å være polymikrobielle etter inkubasjon over natten på det belagte mediet. Av de 204 +naBC-ene var andelen RR-GN identifisert av SIP 51,5 % (105/204). Blant dem var 47,6 % (50/105) E. coli, 19 % (20/105) K. pneumoniae, 8,6 % (9/105) P. aeruginosa og 24,8 % (26/105) A. baumannii-kompleks . En detaljert beskrivel...

EUCAST har utviklet en protokoll for direkte antimikrobiell følsomhetstesting (AST) for automatiserte blodkulturer. Imidlertid kan dens avhengighet av massespektrometribasert mikrobiell identifikasjon unngås ved å bruke en direkte inokulumforberedelsesprotokoll i et automatisert mikrobielt identifikasjonssystem. Denne tilnærmingen kan gi AST-rapporter innen 24 timer etter prøveinnsamling.

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques ...

Denne forskningen tar sikte på å evaluere den diagnostiske nøyaktigheten til gramnegativer direkte fra positivt flaggede blodkulturflasker ved hjelp av automatisert mikrobielt identifikasjonssystem. Hensikten er å bruke den som et verktøy for tidlig klinisk rapportering ved hjelp av EUCASTs metode for rask antimikrobiell følsomhetstesting. EUCAST RAST-metoden ble nylig introdusert i 2018 for å muliggjøre rapportering av antimikrobielle følsomhetstestresultater innen fire til åtte timer etter positiv flagging av automatiserte blodkulturflasker.Det innebærer å utføre diskdiffusjonsmetode overalt direkte fra positivt flagget flaske og lese resultater etter enten fire, seks eller åtte timer. For klinisk rapportering etter EUCAST RAST-metoden er mikrobiell identifikasjon en forutsetning for å bestemme tolkningskategorien for AST-resultater. Den største utfordringen med å implementere denne metoden er tidlig identifisering av mikrobene innen åtte timer, spesielt i ressursfattige omgivelser, som mangler massespektrometri.Vi fant at den direkte inokulumprotokollen var rundt 94 % nøyaktig når det gjaldt å identifisere en RAST-rapporterbar Gram-negativ, som inkluderer Escherichia coli, Kiebsiella pneumoniae, Pseudomonas aeruginosa og Acinetobacter baumannii-kompleks. Denne tilnærmingen med å identifisere gramnegative direkte var rundt fire ganger raskere enn standardtilnærmingen for identifikasjon etter inkubasjon over natten. Vi vil nå fokusere på å vurdere effekten av denne tilnærmingen til klinisk rapportering ved gramnegativ sepsis på pasientutfall når det gjelder lengde på sykehusinnleggelse og dødelighet.Videre kan den også brukes som et verktøy for å redusere antimikrobiell bruk for å lette antimikrobiell forvaltning.

Research

JoVE Journal

Medicine

Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

Standard Inoculum Protocol (SIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

Standard inokulumprotokoll (SIP) for bakteriell identifikasjon ved bruk av et automatisert mikrobiell identifikasjon og antimikrobielt følsomhetstestsystem (aMIAST)

Direct Inoculum Protocol (DIP) for Bacterial Identification Using an Automated Microbial Identification and Antimicrobial Susceptibility Testing System (aMIAST)

Direct Inoculum Protocol (DIP) for bakteriell identifikasjon ved hjelp av et automatisert mikrobielt identifikasjons- og antimikrobielt følsomhetstestsystem (aMIAST)