In a recent review, estimates of the total burden of Blood Stream Infection (BSI) from population-based studies from North America and Europe were summarized [1]. The BSI incidence ranged between 113 and 220 per 100,000 population-based on reports from eight countries [2]. The serious prognosis for many of these infections and the increasing emergence of antimicrobial resistance making the demands for quick and accurate diagnosis of involved pathogens and their susceptibility to applicable antimicrobial agents exigent [3]. Approximately 25,000 people in Europe and 23,000 people in the United States die every year because of infection caused by antibiotic-resistant bacteria [4].

Within clinical microbiology, much focus is on accurate identification and susceptibility testing, automatization, speed and economy. Introduction of Matrix-assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) made a revolution with respect to establishing accurate, rapid and cheap pathogen identification [5]. Rapidly growing micro organisms often may be convincing identified after 3-6 hour growth on agar plates. A great focus is on fast Antimicrobial Susceptibility Testing (AST) in bloodstream infections in order to help clinicians being able to give the most optimized antimicrobial treatment. QMAC-dRAST^TM (Quanta Matrix Inc., Seoul, Republic of Korea) is a phenotypic AST system based on microfluidic chip technology that enables the observation of changes in a single bacterial cell under antibiotic treatment conditions, with a total turn around time of only 6 h from Gram stain reporting of recognized positive blood cultures [6]. The positive blood culture sample is Gram stained prior to loading the instrument for choosing the right antibiotic panel. On the instrument blood, culture material is automatically mixed with agarose and inoculated into a 96 wells plate with dried antibiotic agents. Each well is followed over time by microscopic detection of changes in bacterial colony formation in the agarose. The performance of this technique has been evaluated in recent publications showing promising categorical (Sensitive, Intermediate or Resistant) agreements (CAs) with standard methods [6-10]. The patented 96 wells plate with dried antibiotics comprises, 19 and 17 antibiotics for the Gram Negatives (GNs) and Gram Positives (GPs), respectively. The size of an instrument is a minor refrigerator (width 59 cm, depth 76.5 cm and 109 cm high, weighting 180 kg), fully automated with random access of up to 12 samples (Quantamatrix.com).

In this study, AST results for 255 recognized positive blood culture bottles from as many patients obtained by QMAC-dRAST^TM were compared to results obtained by the standard procedures performed in the laboratory in order to be able to comment on CAs, error rates, time to result and commenting on laboratory flow.

The QMAC-dRAST^TM set up tested in this study is a newcomer in AST testing of blood cultures detected positive. It is based on single-cell morphological analysis that can determine antimicrobial susceptibility by automatically analyzing and categorizing morphological changes in single bacterial cells under various antimicrobial conditions [12]. Determining factors for optimal antimicrobial treatment are correct bacterial identification, reliable performance and interpretation of AST results and the speed with which these informations can be obtained [13]. Combining the accuracy and speed of MALDI-ToF MS identifications with the accuracy and speed of QMAC-dRAST^TM has proven to holdthe potential of improving the existing examination flow [7-10].

Reports from high-income countries have documented key pathogens to have been and remain being S. aureus, E. coli, Klebsiella species, Ps. aeruginosa, enterococci, streptococci and CoNS [14]. The spectrum of organisms differs for community-acquired and healthcare associated (community and hospital onset) infections. Ps. aeruginosa and staphylococci are clearly associated with healthcare institutions, where as among community-acquired cases there are more typically infections due to Streptococcus pneumoniae and other streptococci, and due to E. coli [1].

The robustness and accuracy of MALDI-ToF MS identifications have been documented for the majority of BSI pathogens irrespective of being the setup from Bruker or from Bio Merieux, though especially non-hemolytic streptococci, including pneumococci, still are challenging with respect to species identification. Directly from recognized positive blood cultures the Sepsityper kit (Bruker Daltonics) has been used also in connection with testing the QMAC-dRAST^TM strategy [7, 8]. In only 18 of 346 (5.2%) incidences of monobacterial infections and in 7 of 38 (18.4%) incidences of polymicrobial infection no identifications were obtained [8], being comparable to results from previous studies [5]. Identifications have to be added to the instrument for finalizing the AST report in order to adjust for expert rules. In our laboratory, we streak positive blood culture material on agar-plates and incubates 3-6 hours making it possible, in by far the most cases, to have identifications and these being ready before AST testing is ready for reporting, which is a prerequisite for interpretation of obtained data by the included expert system based on EUCAST breakpoint recommendations. This is in agreement with the study by Sekercioglu et al. [15], on 1351 positive blood cultures,where a Columbia agar plate with 5% sheep blood was inoculated with one drop from the blood culture broth. After a 5-hour incubation period, a colony from the culture plate was submitted to MALDI-ToF MS. When manufacturer-recommended score values were taken into account, MALDI-ToF MS correctly identified 98.4% of the isolates to the species level with a score of > 2.0, 89.1% with a score between 1.7 and 2.0, and 75.4% with a score of < 1.7.

In the first report from 2014 by Choi et al [12] they compared 189 clinical hospital samples, including extended-spectrum β-lactamase–positive E. coli and K. pneumoniae, imipenem-resistant Ps. aeruginosa, methicillin-resistant S. aureus, and vancomycin-resistant enterococci with BMD testing. A CA of 91.5% with 6.51% mEs 2.56% MEs, and 1.49% VMEs were obtained. Since more studies have been added [7-10]. In Table 5 published studies comparing data obtained by QMAC-dRAST^TM and standard AST methods are given. Four additional studies, including our study have included as well GNs as GPs [6-8], one study focused on GNs [10] and one on staphylococci and enterococci [9]. In the study by Grohs et al. and in our study QMAC-dRAST^TM data were compared mainly to disc diffusion AST; strains with disagreeing results were additionally examined with etest or BMD testing. In the other studies given in (Table 4) BMD ASTs were used for comparison. All in all CAs of 91.1-96.3% have been found, with our study being the one with highest CAs,%. Likewise, differences in VMEs, MEs and mEs were registered. Thus, comparisons have shown comparable results though with some differences in obtained VMEs, MEs and mEs. Especially the VMEs have to be careful evaluated with respect to possible improvements. In our study MIC determinations for most of discrepancies were close to the breaking points, thereby finding that the QMAC-dRAST^TM setup seems very reliable for detecting susceptibilities for by far the most of the compared antimicrobials.

The genera and species QMAC-dRAST^TM has been validated for includes the most frequent isolated GNs and GPs. Genera/species dominating in our study are in agreement with those dominating in the other studies, though strains in our study tends to have a more susceptible pattern. This exemplified in the study by Kim et al. [7] on 119 patients where more GNs were ESBL producing (E. coli and Klebsiella species) and carbapenem resistant (Ps. aeruginosa and Acinetobacter baumannii) as well as the GPs including more vancomycin and ampicillin resistant enterococci and methicillin resistant S. aureus. However, single strains were recognized with these susceptibility traits in our study except that no carbapenemase producing strains were recognized. Most often registered discrepancies, though relatively few, when testing GNs were for susceptibility to piperacillin/tazobactam, ceftazidime, cefotaxime, imipenem and ciprofloxacin. By far the most were within +/- one dilution step and the number of VMEs relatively small. This is close to be in accordance with the study by Kim et al. [7] where however no VMEs were observed for GNs with commonly used antibiotics such as beta-lactam/beta-lactamase inhibitors, broad spectrum cephalosporins and carbapenem. Huh et al. [9] studied 110 detected positive blood cultures with staphylococci and enterococci. AST was performed directly using the QMAC-dRAST^TM and thereafter, colony isolates derived from subculture were used for the QMAC-dRAST^TM, the VITEK-2 system and BMD. The QMAC-dRAST^TM with colony isolates produced more reliable results for staphylococci and enterococci than the direct QMAC-dRAST^TM blood cultures detected positive. On colony isolates, the QMAC-dRAST^TM system performed comparably to BMD and the VITEK-2 system. However, the QMAC-dRAST^TM seems relative robust taking the presumed differences in amount of bacterial cells present into consideration; this in agreement with the setup based on single-cell morphological analysis. When examining GPs, especially discrepancies when examining S. aureus strains for penicillin susceptibility and CoNS (and two S. aureus strains) for inducible clindamycin resistance were noticed. In both, different methods are applied by QMAC-dRAST^TM and our routine for their determination. Regarding penicillin susceptibility, we use a combination of growth zone appearance and clover leaf testing [11], whereas QMAC-dRAST^TM interpretation is based on MICs. Inducible clindamycin resistance we detect as recommended by EUCAST by demonstrating the D phenomenon, which does not seem as applicable when examining in a well as in QMAC-dRAST^TM. Whether small colony variants of S. aureus, potentially responsible for chronicization of an infection [16], will challenge a method using microscopy as the detection method remains to be settled. When examining positive blood cultures, there is an exigent desire/need for the method also being validated to be used for susceptibility testing of other relevant organisms such as Gram positive rods, streptococci (as well pneumococci as hemolytic and non-hemolytic streptococci), Gram negative cocci (especially Neisseria meningitidis), the HACEK group of fastidious GNs, anaerobic bacteria and Candida species. Patient outcome is critically influenced by delayed effective therapy, wherefore fast and accurate pathogen diagnostics, including AST, decisively improves the care of patients [17]. Progress in BSI pathogen diagnostics is based on a bundle approach that includes optimization of the pre-analytical parameters, rapid start of incubation, the use of rapid methods, re-organization (e.g. 24/7, transportation service) and a close involvement of antimicrobial stewardship teams [17]. Time to available antibiotic susceptibility report for blood culture specimens detected positive plays a major role in diminishing the time to being able to institute the most optimal antibiotic treatment. The presented time to report data obtained by QMAC-dRAST^TM in this study, and those mentioned in (Table 4), being of 3-8 hours for blood culture specimens examined, strongly support a further possible improvement in the workflow for handling blood stream infections.

Studies have reported on the importance of initial antibiotic coverage, coverage after guidance by Gram stain of recognized positive blood culture smears and improvement of coverage when guided by MALDI-ToF MS identifications [18]. Vlek et al. [18] reported on 253 episodes of blood stream infections that MALDI-ToF MS directly performed on positive blood culture broths reduced the time until definitive identification of bacterial species by 28.8 hours and increased the proportion of patients on appropriate antimicrobial therapy within 24 hours by 11.3% to 82%. In the study by Kim et al. [8] MALDI-ToF MS identification and QMAC-dRAST^TM testing were applied on 359 BSI episodes, where involved pathogens were divided into pathogens having a susceptible or resistant pattern. When reporting based on MALDI-TOF MS results on susceptible/resistant pathogens, optimal targeted treatment was found respectively in 79%/63%, unnecessary broad-spectrum treatment in 16%/5%, suboptimal treatment in 4%/1%, and appropriate antibiotic treatment 100%/68%. Adding QMAC-dRAST^TM results to the decision making raised the percentage of optimal antibiotic treatments to 98.2%. The global emergence of antibiotic resistant microorganisms makes it desirable being guided as quickly as possible in relation to antibiotic susceptibility [3]. In that respect phenotypic methods as the QMAC-dRAST^TM seems desirable as they illustrate all resistance mechanisms expressed in a micro organism.

From a laboratory flow and technician aspect, the procedure about loading the instrument was very easy and intuitive. The QMAC-dRAST^TM is a newcomer on the market and when the data transport from the instrument to a LIMS system will be done automatically the combination of easy loading, the automatized running procedure, easy testing termination, automatic data transport (in addition to automatic alarming when ready for reporting) seems to add important benefits to the current testing availabilities. The saved time from blood culture taken to availably of testing results being present will be able to improve antibiotic treatment of patients.

The genera and species QMAC-dRAST has been validated for includes the most relevant GNs and GPs in relation to positive blood culture specimens, though the desire for also being able to test other blood stream infection relevant pathogens, including streptococci, are exigent. The high CA for as well GNs as GPs are promising. The QMAC-dRAST is a newcomer on the market and when the data transport from the instrument to a LIMS system will be done automatically the combination of easy loading, the automatized running procedure, easy testing termination, automatic data transport (in addition to automatic alarming when ready for reporting) seems to add important benefits to the current testing availabilities. The presented time to report data obtained by QMAC-dRAST in this study being of 3-8 hours for blood culture specimens examined strongly support a further possible improvement in the workflow for handling blood stream infections.