The study was funded by the intramural research grant given to Dr. Ayush Gupta by AIIMS Bhopal. We acknowledge the contribution of laboratory technicians and resident doctors who performed and read the tests diligently during routine and emergency hours.

The study, funded by the intramural research grant given to Dr. Ayush Gupta by AIIMS Bhopal, was approved by the Institutional Human Ethics Committee (IHEC) vide letter no: IHEC- LOP/2022/IL072.
NOTE: A sample volume of 5 ml was used based on studies done by Quesada et al.25 and Munoz-Davila et al.27.
1. Standard inoculum protocol (SIP) for bacterial identification using aMIAST
<ol>
	<li>Wear clean gloves, and inside a Class-IIa biosafety cabinet (BSC), disinfect the septum of +naBC with a swab containing 70% isopropyl alcohol.</li>
	<li>Draw approximately 1 mL of blood-broth mixture using a sterile syringe with a 21 G needle.</li>
	<li>Dispense 1 big drop of the blood-broth mixture from the needle onto the surface of the plated media, namely SBA, CA, and MA. Streak the plates and incubate the culture plates in an incubator at 37 &#176;C &#177; 2 &#176;C under aerobic conditions for 18-24 h.</li>
	<li>After incubation, examine the plates for the appearance of isolated colonies in a BSC and proceed further for identification and AST by aMIAST.</li>
	<li>For each isolate, place an aMIAST polystyrene tube in the aMIAST tube stand and add 3 mL of sterile saline in it using a dispenser attached to the saline bottle.</li>
	<li>Touch three to five morphologically similar colonies with a sterile straight inoculation wire and transfer the bacterial inoculum to the first tube.</li>
	<li>Adjust the turbidity using sterile saline in the inoculated tube between 0.47-0.63 McFarland using a densitometer.</li>
	<li>Place the capillary attachment of the gram-negative aMIAST identification card in the first tube.</li>
	<li>Place the selected cards in an appropriate position on the cassette. The inoculum in the cassette is ready and comes to the aMIAST in filling section. 
	CAUTION: Age of suspension must not exceed 30 min before inoculating the cards.</li>
	<li>Load the cassette into its position in the filler chamber with sample barcode facing inside.</li>
	<li>Close the door and press Fill on the User Interface Screen. Filling is a 70 s cycle. When the cycle is complete the blue indicator light on the system will flash. Placing the cards into the aMIAST system distributes the inoculum within the small chambers of the cards by the machine.</li>
	<li>Remove cassette from filling chamber, close door, and place in loading chamber. Barcodes are automatically scanned and checked against maintain virtual cassette electronic worklist. Straws are automatically sealed, and the cards are automatically loaded into the carousel. Flashing blue arrow on the aMIAST indicates loading is finished.</li>
	<li>Remove cassette waste when finished. See waste disposal procedure in product literature or follow other standard practices. Use remainder of suspension in the tubes to subculture on CLED agar for purity check of the isolates.</li>
	<li>Read the results after the instrument completes the analysis.</li>
</ol>
2. Direct inoculum protocol (DIP) for bacterial identification using aMIAST
<ol>
	<li>Wear sterile gloves, and inside a biosafety cabinet, clean the septum of +naBC with a swab containing 70% isopropyl alcohol.</li>
	<li>Using a sterile syringe with a 21 G needle, take 5 mL of aliquot from the blood-broth mixture of +naBC and transfer it to a serum separator tube (SST) after disinfecting the rubber septum with 70% isopropyl alcohol (Figure 2A).</li>
	<li>Centrifuge this aliquot for 10 min at 160 x g to settle the blood cells in the blood-broth mixture. After the first centrifugation, observe the supernatant in which blood cells will settle down.</li>
	<li>Open the cap of the vial inside a biosafety cabinet, and using a sterile tip and pipette, carefully remove the supernatant and transfer it to a new plain blood collection vial (red top) by removing its top.</li>
	<li>Place the cap on the vial and centrifuge it again for 10 min at 2000 x g. After the second centrifugation, a bacterial pellet will form at the bottom.</li>
	<li>Aspirate the supernatant and discard it using a sterile pipette and tip. The bacterial pellet will remain at the bottom of the tube and will be used to inoculate the aMIAST identification card. 
	NOTE: A serum separator tube was used in the first centrifugation at 160 x g. This was based on studies done by Quesada et al.25 and Munoz-Davila et al.27, where the first centrifugation step was done approximately at 30 x g and 60 x g, respectively. The blood-broth mixture from a +aBC was first centrifuged at low speed in an SST to pellet out the blood cells.</li>
	<li>Place an aMIAST polystyrene tube in the aMIAST tube stand and add 3 mL of sterile saline in it using a dispenser attached to the saline bottle.</li>
	<li>Using a sterile inoculation loop, take the bacterial pellet from the bottom of the vial and inoculate it in the aMIAST tube</li>
	<li>Repeat steps 1.7-1.14.</li>
</ol>
3. AST by EUCAST RAST protocol4
<ol>
	<li>Keep uninoculated 90 mm circular Mueller-Hinton agar (MHA) plates ready in the biosafety cabinet.</li>
	<li>Wear sterile gloves, and inside a biosafety cabinet, clean the septum of +naBC with a swab containing 70% isopropyl alcohol.</li>
	<li>Using a sterile syringe, aspirate 125 &#181;L &#177; 25 &#181;L of undiluted blood-broth mixture from +naBC and add to each MHA plate in the center.</li>
	<li>Spread the broth gently over the plates using a sterile cotton swab in three directions and apply &#8804; 6 antimicrobial disks on each plate.</li>
	<li>Incubate the plates in an aerobic incubator at 35 &#176;C&#177; 1 &#176;C for 8 h. After completion of incubation, observe the purity of the isolate.</li>
	<li>Read the inhibition zones at 8 h &#177; 5 min. Interpret results using the RAST breakpoint table for short incubation after checking the bacterial identification results in the aMIAST.</li>
	<li>Report AST results only if the isolate is identified as one of the RR-GN and the MHA and CLED plates are growing a single morphotype.</li>
</ol>
4. Quality control
<ol>
	<li>Perform internal quality control for the SIP protocol of aMIAST as per the manufacturer&#39;s instructions using recommended reference strains.</li>
	<li>Perform internal quality control of the RAST method weekly using the recommended reference strain of E. coli as described below.
	<ol>
		<li>Arrange 4 sterile glass tubes in a tube stand. Dispense 3 mL of sterile saline in the first tube and 990 &#181;L of sterile saline in each of the second, third, and fourth tubes.</li>
		<li>Make a 0.5 McFarland suspension of the QC strain from the isolated colonies of an overnight culture on plated media.</li>
		<li>Using a sterile pipette and tip, transfer 10 &#181;L of suspension from the first tube to the second tube.</li>
		<li>After mixing, transfer 10 &#181;L of suspension from the second to the third tube and then subsequently to the last tube.</li>
		<li>From the last tube, take 1 mL of the inoculum using a sterile syringe with a needle and add it to an uninoculated aBC.</li>
		<li>Simultaneously add 5 mL of sterile sheep blood in the aBC using a sterile syringe with a needle and incubate it in the CBCMS till it flags positive due to change in the color of the liquid emulsion sensor at the base of the aBC.</li>
		<li>Repeat steps as explained in steps 1-3 for identification by SIP, DIP and RAST, respectively. The expected results are that the QC strain should be identified correctly by both identification protocols of the aMIAST and the zone diameters in RAST plates should be within the specified range28.</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
<ol>
	<li>Consider microbial identification using SIP as gold standard and if the same identification is obtained by DIP, consider it as concordant.</li>
	<li>If an RR-GN i.e., one of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii complex identified using SIP is discordant in the DIP, consider this as Major error (ME) whereas consider the reverse as very major error (VME) as it has the potential of issuing of a report with wrong identification.</li>
	<li>Calculate the concordance rate for RR-GN as the ratio of the total number of concordant RR-GN to the total number of RR-GN identified by SIP multiplied by 100.</li>
	<li>Calculate ME rate as the ratio of number of +naBCs identified as having a non-RR-GN to the total number of +naBCs with RR-GN identified by SIP multiplied by 100.</li>
	<li>Calculate VME rate as the ratio of number of +naBCs misidentified as having an RR-GN in DIP to the total no. of +naBCs tested by DIP multiplied by 100.</li>
	<li>Calculate the time to isolate identification (TTI) as the time taken to identify the isolate by both protocols from the time of blood culture flagging by the CBCMS.</li>
	<li>Calculate the differences between the DIP and SIP as a reduction in the TTI. Calculate this only for the concordant +naBCs having an RR-GN. Note the respective time points from the clocks of CBCMS and aMIAST.</li>
	<li>Manage data and analyze using a spreadsheet. Use the Mann-Whitney U test for continuous variables, considering a two-sided p-value of &#8804; 0.05 as statistically significant.</li>
</ol>

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

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H., Campa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+antimicrobial+susceptibility+testing+of+Gram-positive+cocci+in+blood+cultures+by+direct+inoculation+into+the+BD+Phoenix+system.">Rapid identification and antimicrobial susceptibility testing of Gram-positive cocci in blood cultures by direct inoculation into the BD Phoenix system.</a> Clin Microbiol Infect. 16 (7), 986-991 (2010).</li><li>Barreales, A., Lara, M., Hern&#225;ndez, I., D&#237;ez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+susceptibility+testing+of+Gram-positive+cocci+in+BacT/ALERT+blood+cultures+by+direct+inoculation+into+the+Vitek+2+system.">Rapid identification and susceptibility testing of Gram-positive cocci in BacT/ALERT blood cultures by direct inoculation into the Vitek 2 system.</a> Rev Esp Quimioter. 24 (3), 131-135 (2011).</li></ol>

The authors have nothing to disclose.

Using DIP, we successfully identified the RR-GNs with considerable diagnostic accuracy. The mean TTI after positive flagging of aBC was only 507 min (~ 8.5 h). Thus, when done in conjunction with the EUCAST RAST method for AST determination, it can give isolate identification at 8 h AST reading time. This approach has the potential to implement the EUCAST RAST method obviating the need for mass spectrometry-based identification. This is a boon for the low-resource settings who wish to implement the EUCAST RAST method in ...

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.

<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

Gram-negative (GN) sepsis is a medical emergency where management in resource-limited settings relies on conventional microbiological culture techniques providing results in 3-4 days. Recognizing this delay in turnaround time (TAT), both EUCAST and CLSI have developed protocols for determining AST results directly from positively flagged automated blood culture bottles (+aBCs). EUCAST rapid AST (RAST) protocol was first introduced in 2018, where zone diameter breakpoints for four common etiological agents of GN sepsis, i.e., Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex can be reported. However, those clinical laboratories that have implemented this method in their routine workflow rely on mass spectrometry-based microbial identification, which is not easily available, thus precluding its implementation in resource-limited settings. To circumvent it, we evaluated a direct inoculum protocol (DIP) using a commercial automated microbial identification and antimicrobial susceptibility testing system (aMIAST) to enable early microbial identification within 8 h of positive flagging of aBC. We evaluated this protocol from January to October 2023 to identify the four RAST reportable GN (RR-GN) in the positively flagged aBC. The microbial identification results in DIP were compared with the standard inoculum preparation protocol (SIP) in aMIAST. Of 204 +aBCs with monomorphic GN (+naBC), one of the 4 RR-GN was identified in 105 +naBCs by SIP (E. coli: 50, K. pneumoniae: 20, P. aeruginosa: 9 and A. baumannii complex: 26). Of these, 94% (98/105) were correctly identified by DIP whereas major error and very major error rates were 6% (7/105) and 1.7% (4/240), respectively. When DIP for microbial identification is done using the EUCAST RAST method, provisional clinical reports can be provided within 24 h of receiving the sample. This approach has the potential to significantly reduce the TAT, enabling early institution of appropriate antimicrobial therapy.

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

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

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.

General outcomes 
During the study period, 240 +naBCs underwent identification by aMIAST using both DIP and SIP. Of these, 15% (36/240) +naBCs were found to be polymicrobial after overnight incubation on the plated media. Of the 204 +naBCs, the proportion of RR-GN identified by SIP was 51.5% (105/204). Amongst them, 47.6% (50/105) were E. coli, 19% (20/105) K. pneumoniae, 8.6% (9/105) P. aeruginosa and 24.8% (26/105) A. baumannii complex. A detailed description of...

EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

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

direct-microbial-identification-using-an-automated-microbial

Research

JoVE Journal

Medicine

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

To begin, swab the septum of an automated blood culture bottle containing monomorphic gram-negative organisms, with 70%isopropyl alcohol, inside a Class II A biosafety cabinet. Then, draw one milliliter of blood broth mixture into a sterile syringe equipped with a 21-gauge needle. Dispense a big drop of the mixture onto the surface of a plated media.After streaking the plates, incubate the cultures at 37 degrees Celsius for 18 to 24 hours. Examine the plates for isolated colonies inside the biosafety cabinet. Next, dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle.Now pick up three to five morphologically similar colonies with a sterile straight inoculation wire. Transfer the inoculum into the first tube. Use saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63 McFarland.Then, place a capillary attachment to the gram-negative aMIAST identification card in the first tube. Position the selected cards on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.Close the door and press Fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The standard inoculum protocol identified 105 out of 204 tested isolates as RAST reportable gram-negatives, with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex being the primary organisms identified.

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

To begin, swab the septum of an automated blood culture bottle, having monomorphic gram-negative organisms with 70%isopropyl alcohol inside a class 2A biosafety cabinet. Draw five milliliters of blood broth mixture into a sterile syringe equipped with a 21 gauge needle. Next, disinfect the rubber septum of a rubber separator tube.Transfer the blood broth mixture into the tube. Centrifuge the sample at 160 G for 10 minutes. Now, carefully pipette out the supernatant inside a biosafety cabinet.Transfer the supernatant to a new plain blood collection vial. Place the capped vial in a centrifuge at 2000 G for 10 minutes. Now, aspirate the supernatant with a sterile pipette.Then dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle. Using a sterile inoculation loop, pick the bacterial pellet from the bottom of the vial and inoculate it into the aMIAST tube. Use sterile saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63.Then place a capillary attachment of the gram-negative aMIAST identification card in the first tube. Position the selected cards appropriately on the cassette. Now, load the cassette into its position in the filler chamber with the sample barcode facing inward.Close the door and press fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now, transfer the cassette from the filling chamber into the loading chamber.Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The results of seven positive blood culture bottles, identified using the direct inoculum protocol, were discordant until the species level.Of the 105 RAST reportable gram negatives identified by the standard inoculum protocol, 98 were correctly identified by the direct inoculum protocol. Among 99 non-RAST reportable gram negatives, a RAST reportable gram negative was identified in four positive blood culture bottles.

Antimicrobial Susceptibility Test (AST) Using the EUCAST Rapid Antimicrobial Sensitivity Testing (RAST) 

To begin, place two uninoculated 90 millimeter Mueller-Hinton agar plates in a biosafety cabinet. With gloves on, clean the septum of an automated blood culture bottle containing monomorphic gram-negative organisms with 70%isopropyl alcohol. Use a sterile syringe to aspirate 100 to 150 microliters of undiluted blood broth mixture from the blood culture bottle.Add the sample to the center of a Mueller-Hinton agar plate. Now, use a sterile cotton swab to gently spread the broth over the plates in three directions. Then, apply about six or fewer antimicrobial discs on each plate.Transfer the plates to an aerobic incubator at 35 degrees Celsius for eight hours. When inoculation is complete, observe the purity of the isolate. Read the inhibition zones within eight hours, give or take five minutes.Use the RAST breakpoint table for short incubation to interpret the results after checking the bacterial identification results in the AMI-AST.

Quality Control Testing for Rapid Antimicrobial Sensitivity Testing (RAST)

For the weekly quality control testing of the RAST method, place four sterile glass tubes on a tube stand. Dispense sterile saline into the tubes. Next, transfer the QC strain from the isolated colonies of an overnight culture into the first tube to create a 0.5 McFarland suspension.With a sterile pipette and tip, transfer 10 microliters of the suspension from the first tube to the second tube. Once mixed, transfer 10 microliters of the suspension from the second to the third tube, and then to the last tube. Next, take one milliliter of the inoculum from the last tube with a sterile syringe.Transfer the inoculum into an automated blood culture bottle after swabbing the septum with 70%isopropyl alcohol. Simultaneously with a separate sterile needle, add five milliliters of sterile sheep blood into the same blood culture bottle. Incubate it in a continuous blood culture monitoring system until it flags positive.

453 Views.  All India Institute of Medical Sciences, Bhopal.  EUCAST has developed a direct antimicrobial susceptibility testing (AST) protocol for automated blood cultures. However, its dependence on mass spectrometry-based microbial identification can be obviated by using a direct inoculum preparation protocol in an automated microbial identification system. This approach can provide AST reports within 24 h of sample collection.

Accelerated Antimicrobial Susceptibility Testing in Blood Culture Bottles

Summary