3 The diagnostic tests

3 The diagnostic tests

The interventions

The LightCycler SeptiFast Test MGRADE

3.1 The LightCycler SeptiFast Test MGRADE (Roche Diagnostics) is a CE‑marked, in vitro, diagnostic, real‑time polymerase chain reaction (PCR) test that simultaneously detects and identifies DNA from 25 bacterial and fungal pathogens. The test needs 1.5 ml of ethylenediaminetetraacetic acid (EDTA)‑treated whole blood.

3.2 The LightCycler SeptiFast Test MGRADE involves 3 distinct processes: specimen preparation by mechanical lysis and purification of DNA, real‑time PCR amplification of target DNA in 3 parallel reactions (gram‑positive bacteria, gram‑negative bacteria and fungi), and detection using fluorescence‑labelled probes specific to the target DNA. The test takes a minimum of 6 hours, depending on laboratory workflow.

3.3 The SeptiFast Identification Software set v2.0 analyses the samples and generates a report, which contains all the relevant laboratory data and details of the identified species. The software also includes a crossing point cut‑off rule, which is intended to reduce the positive rate for coagulase‑negative Staphylococci and Streptococcus species based on the assumption that they are contaminants and not causal agents when the crossing point value is less than 20.

3.4 If Staphylococcus aureus is identified in a sample, an aliquot of the SeptiFast Test MGRADE eluate can be further tested for the MecA gene using the LightCycler SeptiFast MecA Test MGRADE. The test can determine the likely methicillin resistance of Staphylococcus aureus through PCR, using the LightCycler 2.0 instrument.

3.5 The test has an analytical sensitivity of 100 colony‑forming units/ml for coagulase‑negative Staphylococci, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumonia and Streptococcus mitis. The minimum analytical sensitivity for all other pathogens detected by the LightCycler SeptiFast test MGRADE is 30 colony‑forming units/ml.


3.6 SepsiTest (Molzym Molecular Diagnostics) is a CE‑marked PCR in vitro test for detecting bacterial and fungal DNA in 1 ml of k‑EDTA- or citrate‑treated whole blood. The test is able to identify species from more than 200 genera of bacteria and 65 genera of fungi.

3.7 The SepsiTest involves 3 distinct processes: extracting and purifying microbial DNA using centrifugation, universal PCR and sequencing. The PCR result, which is available after 4 hours, indicates whether bacteria or fungi are present in the sample. Amplicons from positive samples are then sequenced to confirm the PCR result and to determine which bacteria or fungi species are present. If readable sequences are available from sequence analysis, bacteria and fungi can be identified using the SepsiTest‑BLAST online tool. Sequencing results may be available in 3 to 4 hours depending on the analyser used.

3.8 The analytical sensitivity of SepsiTest ranges from 10 to 80 colony‑forming units/ml, depending on the target species.


3.9 The IRIDICA BAC BSI assay (Abbott Laboratories) is a CE‑marked, in vitro, diagnostic test for detecting and identifying DNA from bacteria and candida in 5 ml of whole blood treated with EDTA. The test can also detect the MecA (Staphylococcus‑specific methicillin resistance), vanA and vanB (Enterococcus‑specific vancomycin resistance), and KPC (gram‑negative associated carbapenem resistance) genes, which are associated with antibiotic resistance.

3.10 The test is designed for use with the IRIDICA system, which combines broad‑range PCR with electrospray ionisation time‑of‑flight mass spectrometry to amplify and detect pathogens. The estimated time to result is at least 5 hours and 55 minutes.

3.11 The IRIDICA analysis computer consists of a proprietary database and software, which identifies the organism present in the sample by comparing the sequence of the sample with a library of known sequences.

3.12 The BAC BSI assay is able to identify more than 780 bacteria and candida. The mean limit of detection for the assay is 39 colony‑forming units/ml, with a range of 0.25 to 128 colony‑forming units/ml depending on the target species.

The comparators

3.13 Two comparators are included, blood culture alone and blood culture with MALDI‑TOF mass spectrometry:

  • Blood culture alone refers to the incubation of whole blood followed by the identification of pathogens by traditional microbiology techniques.

  • Blood culture with MALDI‑TOF mass spectrometry refers to the incubation of whole blood followed by the identification of pathogens using MALDI‑TOF mass spectrometry.

  • National Institute for Health and Care Excellence (NICE)