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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 2/3^rd 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, 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 +aBC^6,7. 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 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-organisms^{9,10,11,12,13,14,15,16,17}. 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 spectrometry^18,19,20. Tayşi 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 Tayşi 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 systems^{21,22,23,24,25,26,27}. 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 ≥2 different micro-organisms on initial gram staining or plated media were excluded from the study.

Protokół

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

1. Wear clean gloves, and inside a Class-IIa biosafety cabinet (BSC), disinfect the septum of +naBC with a swab containing 70% isopropyl alcohol.
2. Draw approximately 1 mL of blood-broth mixture using a sterile syringe with a 21 G needle.
3. 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 °C ± 2 °C under aerobic conditions for 18-24 h.
4. After incubation, examine the plates for the appearance of isolated colonies in a BSC and proceed further for identification and AST by aMIAST.
5. 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.
6. Touch three to five morphologically similar colonies with a sterile straight inoculation wire and transfer the bacterial inoculum to the first tube.
7. Adjust the turbidity using sterile saline in the inoculated tube between 0.47-0.63 McFarland using a densitometer.
8. Place the capillary attachment of the gram-negative aMIAST identification card in the first tube.
9. 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.
10. Load the cassette into its position in the filler chamber with sample barcode facing inside.
11. 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.
12. 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.
13. 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.
14. Read the results after the instrument completes the analysis.

2. Direct inoculum protocol (DIP) for bacterial identification using aMIAST

1. Wear sterile gloves, and inside a biosafety cabinet, clean the septum of +naBC with a swab containing 70% isopropyl alcohol.
2. 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).
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Using a sterile inoculation loop, take the bacterial pellet from the bottom of the vial and inoculate it in the aMIAST tube
9. Repeat steps 1.7-1.14.

3. AST by EUCAST RAST protocol⁴

1. Keep uninoculated 90 mm circular Mueller-Hinton agar (MHA) plates ready in the biosafety cabinet.
2. Wear sterile gloves, and inside a biosafety cabinet, clean the septum of +naBC with a swab containing 70% isopropyl alcohol.
3. Using a sterile syringe, aspirate 125 µL ± 25 µL of undiluted blood-broth mixture from +naBC and add to each MHA plate in the center.
4. Spread the broth gently over the plates using a sterile cotton swab in three directions and apply ≤ 6 antimicrobial disks on each plate.
5. Incubate the plates in an aerobic incubator at 35 °C± 1 °C for 8 h. After completion of incubation, observe the purity of the isolate.
6. Read the inhibition zones at 8 h ± 5 min. Interpret results using the RAST breakpoint table for short incubation after checking the bacterial identification results in the aMIAST.
7. 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.

4. Quality control

1. Perform internal quality control for the SIP protocol of aMIAST as per the manufacturer's instructions using recommended reference strains.
2. Perform internal quality control of the RAST method weekly using the recommended reference strain of E. coli as described below.
   1. Arrange 4 sterile glass tubes in a tube stand. Dispense 3 mL of sterile saline in the first tube and 990 µL of sterile saline in each of the second, third, and fourth tubes.
   2. Make a 0.5 McFarland suspension of the QC strain from the isolated colonies of an overnight culture on plated media.
   3. Using a sterile pipette and tip, transfer 10 µL of suspension from the first tube to the second tube.
   4. After mixing, transfer 10 µL of suspension from the second to the third tube and then subsequently to the last tube.
   5. From the last tube, take 1 mL of the inoculum using a sterile syringe with a needle and add it to an uninoculated aBC.
   6. 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.
   7. 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.

5. Statistical analysis

1. Consider microbial identification using SIP as gold standard and if the same identification is obtained by DIP, consider it as concordant.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Manage data and analyze using a spreadsheet. Use the Mann-Whitney U test for continuous variables, considering a two-sided p-value of ≤ 0.05 as statistically significant.

Wyniki

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...

Dyskusje

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 ...

Materiały

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