MagicplexTM Sepsis real-time test for the rapid diagnosis of bloodstream infections in adults

Sepsis is a serious health condition worldwide, affecting more than 30 million people globally each year. In 2017, the World Health Organization (WHO) adopted a resolution on sepsis: “improving the prevention, diagnosis and clinical management of sepsis”, with the aim of improving early diagnosis, management and prevention to save lives. Blood culture (BC) is generally used to diagnose sepsis because of the low quantity of microbes occurring in the blood during such infections. However, approximately 50% of bloodstream infections (BSI) give negative BC, this figure being higher for sepsis, which delays the start of appropriate antimicrobial therapy. This prospective study evaluated a multiplex real-time polymerase chain reaction, the MagicplexTM Sepsis test (MP), for the detection of pathogens from whole blood, comparing it to routine BC. We analysed 809 blood samples from 636 adult patients, with 132/809 (16.3%) of the samples positive for one or more relevant microorganism according to BC and/or MP. The sensitivity and specificity of MP were 29% and 95%, respectively, while the level of agreement between BC and MP was 87%. The rate of contaminated samples was higher for BC (10%) than MP (4.8%) (P < 0.001). Patients with only MP-positive samples were more likely to be on antimicrobial treatment (47%) than those with only BC-positive samples (18%) (P = 0.002). In summary, the MP test reduces the time taken to identify the microbial pathogen, improving the diagnosis of BSI in patients on antibiotic treatment.


Introduction
In 2017, the World Health Organization (WHO) adopted a resolution on sepsis: "improving the prevention, diagnosis and clinical management of sepsis", with the aim of improving early diagnosis, management and prevention to save lives. Blood culture (BC) is generally used to diagnose sepsis because of the low quantity of microbes occurring in the blood during such infections. However, approximately 50% of bloodstream infections (BSI) give negative BC, this figure being higher for sepsis, which delays the start of appropriate antimicrobial therapy and consequently results in worse outcomes and higher mortality rates (1,2). Prompt microbiological diagnosis enables a more appropriate antimicrobial treatment than the empirical combination of broadspectrum antibiotics that have negative effects such as an increased prevalence of resistant pathogens (3).
The microbial diagnosis of sepsis by BC has two main advantages. First, it allows the growth of very small numbers of the microorganism, which is important since the concentration of bacteria in the blood of septic adult patients is usually low (< 10 CFU/mL) (4). Second, this technique allows the isolation of pathogens and, hence, antimicrobial susceptibility testing can be performed. However, BC also has limitations that do not make it an ideal gold standard test, including the long time required for growth detection, the frequent false negative results in patients receiving antimicrobial therapy, and the inability to detect fastidious microorganisms (5).
Molecular tests offer important advantages over BC that could improve the diagnosis of BSI, such as the lower amount of time taken to obtain results by working directly from blood. In addition, the low detection limits of molecular assays might make them more sensitive than BC, enabling the detection of fastidious, non-viable or non-culturable microorganisms, even from patients on antibiotic treatment (5-7). Another advantage of molecular assays is the ability to detect some specific resistance markers, which can provide important information for better treatment. Moreover, the rapid identification of the microorganism can be used to infer antimicrobial susceptibility according to local epidemiology.
The Magicplex TM Sepsis test (MP) (Seegene, Seoul, South Korea) is a multiplex real-time polymerase chain reaction (PCR) that detects more than 90 microorganisms at the genus level (73 Gram-positive bacteria, 12 Gram-negative bacteria and 6 fungi), 27 at the species level and 3 drug-resistant genes (mecA, vanA and vanB) within 6 hours. In this study, we evaluated the ability of the MP test to rapidly detect pathogens causing BSI in adult patients from whole blood compared to conventional BC.  The Seegene Viewer software was used to interpret the results, which were available within 6 hours. All the samples were stored at 2-8°C and processed within 24 h after collection.

Routine microbiological techniques
MP samples positive for CoNS were considered to be true positives if the same microorganism was detected in two sets of BC. Samples that were positive according to MP in the first rt-PCR screening and identified only at the group level (e.g., GNB group A), but negative in the second rt-PCR were considered negative according to MP.
Microorganisms identified by MP only at the genus level, such as Staphylococcus spp.
and Streptococcus spp., were considered to be contaminants if the BC was negative or positive for staphylococci or streptococci that could be identified by MP at the species level.

Statistical analysis
Statistical analyses were performed using the SPSS software (SPSS, Chicago, IL, USA).
Differences were considered significant when P < 0.05.
A total of 140 pathogens were detected among 132 positive samples (  Additional clinical information, including underlying disease and the antibiotic or antifungal therapy administered on the day of sampling, was recorded for the patients with 36 MP-positive and BC-negative or contaminated results (Table 3)

Pseudomonas aeruginosa
Acute gastroenteritis and community-acquired UTI --

Staphylococcus aureus
UTI in a patient with permanent bladder catheter -Amoxicillin/clavulanic acid

Enterococcus faecalis
Catheter-related blood stream infection in a patient transplanted due to acute myeloid leukemia BC positive for E. faecalis 6 days before sampling.
Daptomycin ICU: intensive care unit; BC, blood culture; MP, MagicPlex assay a Only antimicrobial/antifungal therapy potentially effective against pathogen detected and initiated before sampling

Discussion
Several molecular assays have been recently tested for the direct molecular identification of pathogens in blood samples (8)(9)(10)(11)(12)(13). However, few studies have focused on commercially available PCR-based tests other than the SeptiFast test for the detection of BSI (14,15). In the present study, we evaluated the Magicplex TM Sepsis test (MP), comparing it with conventional BC. We observed that MP showed a sensitivity and specificity of 29.2% and 95%, respectively. Only a few studies have evaluated the sensitivity and specificity of MP (10,(16)(17)(18)(19). However, if they had included all the MP and BC results, the sensitivity of MP would have been 38%, PPV 17% and specificity 65%. Taking into account all these results, we conclude that MP can be useful for the rapid detection of pathogens causing sepsis.
The low sensitivity of MP is probably due to the low bacterial concentration in whole blood and the low sample volume of 1 ml used. There are several strategies that can be applied to improve bacterial recovery, such as removing or significantly reducing the amount of human DNA present in the whole blood sample. This step is included in the MP and SepsiTest assays. Another possibility is to introduce an additional incubation step prior to extraction (20). Although this approach can increase microbial concentrations, it lengthens the whole process and compromises the main advantage of the molecular assays, which is the rapid procurement of results. Another strategy is to increase the initial volume of the blood sample (> 1 ml) (21), which has been reported to give promising results and greatly improve the detection rates of PCRbased assays (10) or increase the amount of bacterial DNA, as shown by Trung et al. (22).
We observed 36 samples that were positive according to MP and negative according to BC. This could have been due to the fact that almost half of these samples were obtained from patients on antibiotic treatment, which could explain the negative BC results. The presence of cell-free DNA or non-viable microorganisms or even contaminating DNA could explain the other 19 discordant results (23). Importantly, MP detected 5 out of the 14 Candida spp. and 11 out of the 13 S. aureus isolates in our study. Early detection of these two pathogens is essential due to the high mortality rates and risk of haematogenous complications associated with these microbes. In addition, Candida requires a longer time for growth in BC than other pathogens, delaying the initiation of treatment by several days. Candida spp. was the third most frequently occurring pathogen detected in our study. As expected, most of the positive samples were obtained from ICU patients. We detected a small number of resistance genes. Although the three mecA genes detected by MP were confirmed by routine susceptibility testing, 4 methicillin-resistant CoNS were not identified by MP.
Furthermore, the only vanB-positive E. faecalis detected by MP was not isolated by BC.
Therefore, the accuracy of MP in detecting resistance genes should be further evaluated.
Despite the many advantages of molecular assays, these tests have several important limitations. First, these methods require specialised equipment and technical experience and are usually expensive. The MP test also has several manual steps that make it laborious and increase the risk of possible contamination. Moreover, its low sensitivity makes its implementation as a routine test difficult in clinical microbiology laboratories. Finally, the sensitivity and specificity of molecular assays vary according to the test, the extraction method, the algorithm used to evaluate the results, the comparative method and the study population. Data published to date support the use of PCR-based tests for specific groups of patients, such as critically ill patients in ICU, those with suspected candidaemia or patients receiving broad-spectrum antibiotics (24,25). There is no consensus about the interpretation of BC-negative and PCRpositive results (low pathogen concentrations, non-viable bacteria and DNAemia, etc.).
Further studies are needed to evaluate the role of new molecular assays in the routine microbiological diagnosis of BSI.
In conclusion, sepsis is a time-dependent disease that requires early diagnosis and prompt appropriate treatment to improve prognosis. The MP assay provides results within 6 hours, thereby significantly reducing the amount of time for diagnosis and seems to be especially useful in patients on antimicrobial treatment. Nevertheless, the assay has to be optimised, mainly for greater automation and to facilitate its introduction into routine laboratory workflow. Furthermore, the microbial detection limits of the molecular assay need to be improved, probably by new extraction protocols and greater sample volumes to improve the positive and negative predictive power so that it can be a useful tool in clinical practice, especially regarding its impact on antibiotic use.
Financial support: This study was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, and co-financed by the European Regional Development Fund (ERDF) "A Way to Achieve Europe" and the Spanish Network for