Clinical performance of ASTA SepsiPrep kit in direct bacterial identification and antimicrobial susceptibility test using MicroIDSys Elite and VITEK‐2 system

Abstract Background Rapid and accurate microbial identification and antimicrobial susceptibility testing (AST) are essential for timely use of appropriate antimicrobial agents for bloodstream infection. To shorten the time for isolating colonies from the positive blood culture, various preparation methods for direct identification using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) system were developed. Here, we evaluated the SepsiPrep kit (ASTA Corp.) for direct identification of microorganisms and AST from positive blood cultures using MicroIDSys Elite MALDI‐TOF MS system (ASTA Corp.) and VITEK‐2 system (bioMérieux). Methods For direct identification, a total of 124 prospective monomicrobial positive blood culture bottles were included. For direct identification, the pellet was prepared by centrifugation and washing twice. For direct AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5. The results from the direct identification and AST using MicroIDSys Elite and VITEK‐2 system were compared to those from the conventional method performed with pure colony subcultured on agar plate. Results Compared to the conventional method using pure colony, correct direct identification rate was 96.5% and 98.5% for 57 gram‐positive isolates and 67 gram‐negative isolates, respectively. For direct AST, among the 55 gram‐positive isolates, the categorical agreement (CA) for staphylococci, streptococci, and enterococci was 96.7%, 98.4%, and 94.1%, respectively. For 66 gram‐negative isolates, the CA for Enterobacterales and non‐fermentative gram‐negative rods was 99.0% and 96.6%, respectively. Conclusions The SepsiPrep kit was easy to use combined with MicroIDSys Elite and VITEK‐2 system and also, the correct identification and AST rate were very high.


| Conventional identification and AST
When automated blood culture system showed positive signal, an aliquot from positive blood cultures was subjected to Gram staining and then subcultured onto the following agar plates: sheep blood agar (Asan Pharmaceutical Co., Ltd.) and MacConkey agar (Asan Pharmaceutical. Co., Ltd.). After overnight incubation, a pure colony from the agar plate was used for MALDI-TOF MS analysis with MicroIDSys system and AST by VITEK-2 system.
For MALDI-TOF MS analysis, colonies were smeared onto the target plate, 1.5 µL of 70% formic acid (ASTA Corp.) and 1.5 µL of a α-cyano-4-hydroxycinnamic acid matrix solution (ASTA Corp.) were added for analysis with the MicroIDSys system. Acquired protein spectrum was compared with the reference library provided by the manufacturer (MicroIDSys CoreDB v1.27), and the score ≥140 was considered acceptable according to the manufacturer's recommendation.
The AST of the isolates was performed VITEK-2 system (AST P601 card for staphylococci, AST-ST01 card for streptococci, AST-P600 card for enterococci, AST-N224 card for Enterobacteriaceae, and AST-N225 cards for non-fermentative gram-negative rods) (bioMérieux). For AST, a McFarland 0.5 standard suspension was prepared using 0.45% saline with the Densichek VITEK colorimeter.

| Direct identification and AST
When a blood culture was flagged positive by the BACTEC FX blood culture system (Becton Dickinson) indicating bacterial growth, and a Gram stain confirmed the presence of the gram-positive or gramnegative bacteria. For cases showing single morphotype by Gram stain, direct bacterial identification was performed using SepsiPrep kit (ASTA Corp.). Briefly, 1 ml of blood culture was transferred to a lysis tube. After vortex for 2 minutes, the tube was centrifuged at 13,000 g for 2 minutes, and supernatant was discarded. Then, the pellet was washed twice with 1 ml of wash solution by vortexing and centrifugation (2 min, 13,000 g). The supernatant was discarded, and the pellet was used for MALDI-TOF MS analysis.
For AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5.
The MALDI-TOF MS analysis and AST are performed in the same way as colony with conventional method. For the AST, the unidentified isolates from direct identification were excluded because VITEK cards were chosen according to the identification obtained by MALDI-TOF. The identification was introduced into the VITEK 2 expert system to allow it to choose the correct interpretive criteria.

| Data analysis
For identification, samples determined to be incorrectly identified were those with invalid identification, and samples with inconsistent results between the direct identification and conventional identification methods. The correct identification rate was calculated as (correctly identified samples/total tested samples) × 100.
Comparison of AST between the direct and conventional method was expressed in terms of categorical agreement (CA), very major error (VME, falsely susceptible), major error (ME, falsely resistant), or minor error (mE, all other errors) according to the CLSI M23Ed5 document. 12

| Antimicrobial susceptibility testing
Among the 124 enrolled blood cultures, 121 which were identified by direct identification were selected for direct AST.
For non-fermentative gram-negative rods, among the 89 bacteria/antimicrobial agent combinations, VME and ME were not found and only three mEs were found with each of the following drugs; ticarcillin/clavulanate, minocycline, and tigecycline ( Table 3).
The bacteria/antimicrobial agent combinations that did not agree with conventional method are listed in Table 4.  was very easy to use because the lysis buffer is freeze-dried and contained in one tube and only two washing steps are needed.
Combining use of MicroIDSys Elite system and VITEK-2 system with blood culture pellet do provide reliable identification and AST results in same day that the blood culture bottles flagged positive.

TA B L E 4
Discrepancies of antimicrobial susceptibility testing results between the direct method and the conventional method

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest relevant to this paper were reported.

AUTH O R CO NTR I B UTI O N S
YJ Park designed the study. YJ Park and IY Yoo analyzed the data and wrote the manuscript. YJ Cha collected the samples. DP Shin, SI Ha, and JH Han participated in experiments. YJ Park supervised the study design and reviewed the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.