Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections

Background Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients. Methods In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays. Results Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines). SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively. Conclusions This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship), in order for the results to be applied appropriately to the management of patients`infectious processes.

Introduction Bloodstream infections are one of the most serious infectious diseases, with mortality rates of around 30-50% [1]. Factors that make bloodstream infections more difficult to manage and treat include an aging population, chronic diseases, immunosuppression, and most importantly, the increase in antibiotic resistance [2].
Although causative pathogens of bloodstream infections are consistent between studies [3], the empirical treatments used are often inappropriate [4]. This has serious clinical implications for the patient [4,5] since treatment response is directly related to the time that elapses before the appropriate antimicrobial therapy is administered. In some cases, the mortality rate can increase [6] between 6 and 10% for each hour of delay.
The conventional microbiological methods available nowadays for identifying the microorganisms causing bloodstream infections are too slow. Preliminary result based on Gram staining once the blood culture turns positive can be obtained in less than an hour but around 18 to 72 hours may be needed in order to get an accurate result including the antibiotic susceptibility pattern of the pathogen involved. In addition to the risk of administering incorrect treatment, this delay in diagnosis favors the use of broad spectrum antibiotics, implying a high healthcare cost, and the selection of antibiotic-resistant bacteria [1,3,4]. The introduction of new systems of microbiological diagnosis in clinical microbiology laboratories such as matrix-assisted mass spectrometry (MALDI-TOF) [7] or real time PCR [8] have revolutionized microbiological diagnosis producing reliable results in short periods of time. However, these new approaches have significant limitations such as the inability to detect a wide range of antibiotic resistance markers in a same test [9,10], impacting this fact in time to diagnosis.
Sepsis Flow Chip (SFC), (Master Diagnostica, Granada, Spain) is a novel microarray-based diagnostic assay for simultaneous rapid detection of microorganisms causing bloodstream infection and their most important antibiotic resistance markers directly from positive blood cultures in three hours.
The aim of this study was to evaluate for the first time the diagnostic capability of the SFC assay with a collection of microorganisms with a wide variety of genetic resistance determinants and with positive blood cultures samples.

SFC assay
SFC (Master Diagnostica, Granada, Spain) is a DNA microarray based-assay approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD). SFC assay is based on a multiplex PCR amplification using biotinylated primers followed by an automatic reverse hybridization in membrane containing specific probes for detecting the most important pathogens associated with bloodstream infections and the most important genetic resistance determinants in these microorganisms (Table 1).
Positive signals are visualized via a colorimetric immunoenzymatic reaction in a chip membrane (Fig 1) by the HS24 hybridization platform. The HS24 hybridization platform has a built-in camera that captures the image of the chip and then is analized in the platform by the hybrisoft software which identifies the dot pattern that appears on the membrane. Each dot pattern is associated with a microorganism and genetic resistance determinants and the hybrisoft software provide to the user a result. The assay can detect at the species level 12 bacteria and 1 yeast, at genus level 4 bacterial genus and under the generic category "Enterobacteriaceae" all Enterobacteriaceae other than E. coli, K. pneumoniae, M. morganii, Proteus sp. and S. marcescens. SFC assay detects the most important genetic resistance determinants involved in resistance to methicillin and vancomycin in Gram-positive pathogens and determinants related to ß-lactam resistance mechanisms such as ESBLs and carbapenemase production in Gram-negative bacteria ( Table 1). The test is performed directly from a positive blood culture using a minimum volume (10 μl).

Pre-clinical evaluation of the SFC assay
In order to confirm the ability of the system to detect all the microorganisms and their genetic resistance determinants included in the panel, we tested a collection of 109 bacterial strains and 9 fungi strains (S1 Table) in monomicrobial or polymicrobial samples (S2 Table). The microorganisms and its genetic resitance determinants identification were previously characterized phenotypically and then confirmed genotypically. Genotypic identification were carried out by amplification of the 16S rRNA gene by PCR with 16S rRNA universal primers 27F and 1392R followed by DNA sequencing [11], 16s rDNA sequences were assigned using ribosomal database project classifier with the 16S rRNA database (https://rdp.cme.msu.edu). Genotypic characterization of genetic resistance determinants for methicillin resistant Staphylococcus spp. and vancomycin resistant Enterococcus spp. were carried out with Xpert MRSA and Xpert VanA/vanB assay respectively (Cepheid, California, USA). Genotypic characterization of genetic resistance determinants for extended spectrum ß-lactamase (ESBL) and class A and B carbapenemase producers bacterial strains, were carried out by Real Time PCR [12,13]. Class D carbapenemase producers strains characterization were carried out by conventional PCR and DNA sequencing [14].
For pre-clinical evaluation of SFC assay, one single colony of each strain was re-suspended in distilled water at a concentration of 0.5 McFarland (1.5Á108 UFC/ml) and 5 ml were inoculated in a Bactec bottle. Each sample was then manipulated as a positive blood culture following the manufacturer's instruction prior to analysis by SFC assay. Also combinations of some samples were used for the evaluation of SFC in polymicrobial samples (S2 Table).

Clinical evaluation of the SFC assay
Further, a total of 202 positive blood cultures obtained consecutively from unique patients with bacteremia were included in the evaluation of the SFC assay. Blood cultures from patients with bacteremia were collected in Bactec plus/F aerobic and anaerobic culture bottles (Becton Dickinson, New Jersey, USA) and incubated on BD BACTECTM 9240 (Becton Dickinson, New Jersey, USA). When the BD BACTEC system detected a positive sample, the blood culture bottle was removed from the system and analyzed in parallel by our standard microbiological methods and by SFC assay. Only one blood culture bottle per patient was analyzed by SFC regardless of whether it was an aerobic or anaerobic bottle. An aliquot of 500 μl of each blood culture sample included in the study was preserved at -20˚C for future determinations in case discrepant results were obtained with the different methodologies and reproducibility assays.
According to our standard microbiological diagnostic protocols, positive blood cultures were processed for Gram staining and subcultured onto different culture media. The microorganisms were identified using MALDI-TOF (Burker Daltonics, Bremen, Germany) and this was complemented by biochemical identification and susceptibility testing by WalkAway (Beckman, San Francisco, USA) following the EUCAST guidelines [15] for analysis of antibiotic susceptibility. Confirmatory susceptibility studies were performed by E-test and by molecular methods. Methicillin resistant Staphylococci were confirmed by Xpert MRSA. Bacterial strains suspected of being carriers of ESBL or carbapenemases were confirmed by molecular methods previously described [12].
Positive blood cultures were processed in parallel by SFC assay. A volume of 10 μl of positive blood culture was diluted 1/10 with distilled water to a final volume of 100 μl and a volume of 4 μl of this dilution was used to perform the assay. Amplification reactions were carried out in a thermal cycler (Veriti Thermal Cycler, Applied Biosystems, California, USA) following manufacturer's instructions, reverse hybridization and analysis of the results were automatically conducted with the hybriSpot HS24 platform (Master Diagnostica, Granada, Spain). The time necessary to obtain a result from a positive blood culture with SFC assay is about three hours and the platform can process up to 24 samples simultaneously.
Once the study was completed, 20 samples randomly selected from the preserved blood culture collection were reanalyzed in order to study the reproducibility of the results obtained by SFC assay.
For the evaluation of the SFC assay in positive blood cultures, sensitivity and specificity with 95% confidence intervals and concordance between conventional methods and SFC assay were calculated using SPSS v 17.0. The samples used for this study were surplus of clinical diagnosis and the data were analyzed anonymously, according to the law of biomedical research; it was not necessary to obtain informed consent. This study was approved by the local ethical committee of the Hospital General Universitario of Alicante (CEIC PI2015/39).

Pre-clinical evaluation results
When SFC assay was tested using a collection of bacteria and fungi in monomicrobial samples, including multiresistant bacterial strains and representing all the species and genetic resistance determinants included in the test, it was seen to identify all the bacteria and C. albicans strains. Bacterial strains and other Candida spp. as C. parapsilosis, C. tropicalis, C. krusei and C. glabrata not included in the SFC detection panel were not detected as was expected.
In polymicrobial samples the identification of all bacterial strains tested was not as good as in monomicrobial samples, the assay showed some limitations in samples with more than 3 of Enterobacteriaceae members. Regarding to the detection of genetic resistance determinants in polymicrobial samples, the assay showed no limitations in samples up to three microorganisms. The results of the tests performed with SFC on polymicrobial samples are shown on S2 table.

Clinical evaluation results
Further, a total of 202 blood cultures containing 225 organisms were included in the study. One hundred and eighty eight isolates were in monomicrobial blood cultures and 37 in polymicrobial blood cultures.  (1/17). Two blood cultures harbouring S. parasanguinis and S. viridans were not detected by SFC assay. In identifying Streptococcus spp. the overall concordance between our standard microbiological diagnostic protocols and SFC assay was 88% (15/17) and reached 100% (9/9) for S. pneumoniae.
Enterococcus spp. at the genus level were also detected in 14 blood cultures by SFC assay and by MALDI-TOF and WalkAway, thus, showing a concordance between methodologies of 100% (14/14). These isolates were distributed among the species E. faecalis (13/14) and E. faecium (1/14).
Only one blood culture with L. monocytogenes was identified by SFC assay showing 100% concordance with our standard microbiological diagnostic protocols.
SFC assay only failed to identify K. oxytoca (2/5) and E. cloacae (2/6) in polymicrobial blood cultures, whereas there was 100% concordance between the two methodologies for these microorganisms in monomicrobial samples.
Only 4 positive blood cultures seen to be harbouring Campylobacter jejuni, Bacteroides fragilis, Alcaligenes xylosoxidans and Pseudomonas putida by MALDI-TOF and WalkAway and were negative by SFC assay since these microorganisms were not included in the assay.
Regarding the detection of yeasts in positive blood cultures, 83% of agreement was found between MALDI-TOF and SFC. All positive blood culture samples with C. albicans (5/5) identified by MALDI-TOF were detected by SFC assay and only one sample with C. parapsilosis was not detected, as expected according to the technical indications of SFC assay.
In sum, 95% of the isolates included in this study were identified by SFC assay. The details are shown in Table 2.
When antibiotic resistance markers were analyzed for gram-positive microorganisms, SFC assay detected 52 methicillin resistant Staphylococci (mecA positive), as confirmed by oxacillin E-test and molecular methods as Xpert MRSA, showing 100% concordance between the two methodologies. These methicillin resistant Staphylococci were distributed between S. epidermidis (31/52), S. hominis (16/52), S. capitis (2/52), S. aureus (2/52) and S. haemolyticus (1/52). Vancomycin resistance markers such as vanA or vanB could not be evaluated in positive blood culture since there was a lack of positive blood cultures with vancomycin resistant Enterococcus spp. In the study of antibiotic resistance in Gram-negative isolates our standard microbiological diagnosis methods as cefotaxime E-test combined with inhibitors of class A, B or D ß-lactamases identified the presence of 11 cefotaxime resistant isolates, which 9 were positive for ESBL production (Table 3) as were confirmed by molecular approach [12]. SFC assay detected 9 samples with genetic resistance determinants which could explain the 81% of cefotaxime resistant isolates. The main resistance marker associated with cefotaxime resistance identified by SFC assay was blaCTX, (identified in 4 blood cultures, 1 harbouring E. coli and 3 harbouring K. pneumonia), followed by blaSHV identified in 2 blood cultures harbouring E. coli. Only 2 blood cultures harbouring E. coli and identified as cefotaxime resistant isolates compatible with blaAmpC overproduction by our standard microbiological diagnostic protocols and as were confirmed by molecular approach [13] were not detected by SFC assay since this antibiotic resistance determinant is not included in the SFC assay panel.
Regarding evaluation of carbapenemase detection in blood culture by SFC assay, there were only 4 blood cultures harbouring Pseudomonas aeruginosa carbapenem resistant isolates determined by imipenem/meroperem E-test and none were positive for carbapenemase production, as confirmed by molecular approach [13,14] (Table 3). Only 3 blood cultures harbouring A. baumannii were positive for the blaOXA-51 resistance marker on SFC assay, although these isolates were not resistant to carbapenems as revealed by imipenem E-test.
Full concordance with previous results were found when 20 randomly selected blood cultures were reanalyzed by SFC (data not shown).

Discussion
SFC assay was seen to be a robust, fast and easy to use tool, which can be easily implemented in clinical microbiology laboratories since it includes the most common of microorganisms normally detected in these infections [16][17][18] and allows the resistance mechanisms of greatest clinical importance in their treatment to be characterized. When SFC works with the hybri-Spot HS24 platform, the assay is fully automated and requires a minimal sample manipulation time, which means that it may be used with positive blood cultures obtained the same workday. In this way the results are made available and may be acted upon within the first few hours of evolution of the patient's infectious condition.
Although SFC takes a little longer to produce results than does mass spectrometry, it provides information about the presence of genetic resistance determinants in clinical material. Because antibiotic resistance is a heterogeneous process involving many factors; for example, according to our results in carbapenem resistance P. aeruginosa the absence of a carbapenemase does not imply that it is sensitive to carbapenems since other mechanisms as efflux pumps and permeability to antibiotic are involved. The lack of concordance between antibiotic susceptibility and genetic resistance determinants it is one of the main limitations of diagnosis systems based on detection of genetic resistance determinants like SFC assay. By the other hand the detection of genetic resistance determinants directly on clinical samples in early stages of the infection is very useful when it comes to optimizing empirical treatment as quickly as possible or commencing the process of antibiotic de-escalation and will thus most likely improve the clinical management of patients [19][20][21]. In addition, rapid diagnosis [22,23] will make it possible to reduce the use of certain broad spectrum antibiotics and help to control the serious problem of antibiotic resistance [24] since it has been reported that hospitals are excellent compartments for the selection of resistant bacteria due to overuse and misuse of antimicrobial agents [6].
Compared to other similar systems [9] that are also currently used for diagnosis of positive blood cultures such as FilmArray BCID [21,25] or Verigene BC-GP and BC-GN [26,27], the concordance with classical methods based on culture dependent and antibiotic susceptibility testing is similar [28,29] but the main strength of SFC assay is that it provides a wider panel for identification of bacteria and genetic resistance determinants for Gram-negative organisms including the majority of carbapenemases of clinical interest described to date (blaSME, blaKPC, blaNMC/IMI, blaGES, blaIMP, blaGIM, blaVIM, blaSPM, blaSIM, blaNDM, blaOXA-23, blaOXA-24, blaOXA-48, blaOXA-51, blaOXA-58) in a single test [30,31]. This is of the utmost importance bearing in mind the extensive propagation in certain geographical areas of Gram negative bacteria with this resistance mechanism, in particular Klebsiella pneumoniae [16].
The use of this type of diagnostic method is revolutionizing the management of these patients, reducing the time they have to wait before receiving the appropriate antibiotic therapy [9,20]. However, its clinical utility is even greater if the data are managed by a multidisciplinary group (stewardship) allowing the results to be applied in an optimal manner [5,32,33]. Antimicrobial stewardship together with this type of diagnostic system also implies significant financial savings for the healthcare system since hospital and intensive care unit length of stay is reduced together with total hospital costs and even mortality [22,32].
The increase in antibiotic resistance in our hospitals [34] is an extremely serious public health problem [2,24]. Due to its complexity, it needs to be approached by multidisciplinary groups in which clinical microbiology [19] should provide fast diagnostic methods [35][36][37] capable of detecting the most prevalent multi-resistant microorganisms [38][39][40], especially those that are carbapenemase carriers, which in addition to causing infections that are difficult to treat may be associated with outbreaks in a hospital setting [41,42]. The system we evaluated will most likely prove to be a useful tool in the management of patients with bacteraemia and with the most common fungemia (C. albicans) and also help to control antibiotic resistance [5,43]. Although SFC detects fungal infections caused by C. albicans, it does not have the capacity to detect fungemia caused by other Candida spp. as has been shown in pre-clinical and clinical evaluation of the assay. It provides useful information for the treatment of patients just a few hours after the blood culture becomes positive [22], which is much quicker than in the case of classical microbiology, and so will help to improve the use of antibiotics. However antimicrobial susceptibility testing cannot yet be replaced by molecular systems based on identification of genetic resistance determinants, as SFC assay due to antibiotic resistance is a heterogeneous process involving many factors.
One of the main limitations of our validation study has been the lack of clinical samples with VRE and carbapenemase producer bacteria. Regarding with these major public health issue, where early report of antibiotic resistance would be very helpful for patient management by clinicians; further interventional studies with patients not only with VRE infections but also with SARM or carbapenemase producer bacteria should be done with SFC assay in combination with multidisciplinary group (stewardship) in order to see the SFC assay impact over different patients populations for example in ICU or non-ICU patients.
From our knowledge this is the first study of validation of SFC assay, SFC showed concordance with our standard microbiological diagnostic protocols of 96.2% in monomicrobial and 89.1% in polymicrobial blood cultures and an overall concordance of 92.6%. Although the results seem promising and better than to other similar diagnosis platforms, further studies with increased sample size and patient population must be done in order to evaluate other outstandings aspects that have remained in this work as detection of VRE and carbapenemase producers bacteria in blood culture.
Supporting information S1 Table. Collection of microorganisms representing all the species and genetic resistance determinants included in the SFC assay and results obtained by SFC in pre-clinical evaluation assay.