BACKGROUND

The control of nosocomial infection like MRSA and the management of operational costs are two important drivers in hospitals. Considering these objectives, ELITe InGenius™, an innovative sample-to-result solution featuring a universal extraction with 12 independently controlled Real-Time PCR modules, was developed.Conventional Real-Time PCR workflow involves complex equipment, highly qualified staff and extensive hands-on-time. On the contrary, ELITe InGenius combines on the same platform industry-leading technologies for nucleic acid extraction and RT-PCR and reduces the hands-on-time to few minutes.

ELITe InGenius is a fully automated and flexible cassette-based sample-to-result solution enabling laboratories to perfom multiple and independent Real-Time PCR and run custom panels with mixed parameters according to their specific requirements. • The universal extraction protocol for viral and bacterial DNA/RNA is performed in a ready-to-

use, prefilled unitary cartridge and based on Magtration® technology & sonication producing high nucleic acid yield even with small sample volume in a short turn-around-time. The remaining extracted nucleic acid is collected in an elution tube for additional testing or archiving.

• The Real-Time PCR amplification is performed with the Real-Time PCR ELITe MGB® assays in a dedicated PCR vessel with autocapping function. Each of the 12 unitary RT-PCR modules is independently controlled and consists of 6-optical channels. Several types of PCR tests can be run in parallel from one extracted sample regardless the thermal profile or the chemistries used (Figure 1).

• All the operations from sample preparation to result analysis are automated and guided by an intuitive software.

MRSA/SA ELITe MGB® is a triplex assay which detects and differentiates MRSA and SA using DNA purified from nasal swabs. The unique design targeting exclusively conserved sequences for Staphylococcus aureus (LDH1) and methicilin resistance detection including, mecA and mecC, was initially CE-IVD validated with conventional PCR instrument.

MATERIALS AND METHODS

MRSA Nucleic Acid Extraction ValidationSimulated nasal matrix specimen were used, consisting of one negative and two positive: one MRSA and MSSA negative containing S. epidermidis, one moderate MRSA strain, one moderate MSSA positive.Swabs were prepared by absorbing 100 µL of the corresponding sample from the tube. Bacteria were recovered from the swabs by vortexing them in 0.5 mL of TSB for 10 sec. Then 10 µL of a 1:10 dilution of MRSA/SA Internal Control was added to 200 µL of each swab sample and samples were extracted by using ELITe InGenius instrument and ELITe InGenius™ SP 200 a prefilled, ready-to-use extraction cartridge. Elution volume was setup to 50 µL.

Clinical StudyTen certified clinical samples (4 negative and 6 positive) from Hospices Civils de Lyon were processed as a preliminary validation test.Results obtained with ELITe InGenius system using MRSA/SA ELITe MGB (ref: M800346, ELITechGroup Molecular Diagnostics) were compared to the CE-IVD validated protocol of MRSA/SA ELITe MGB assay with the EasyMAG™ extraction (BioMerieux) and an ABI 7500 Fast Dx PCR instrument (Life Technologies).

Reproducibility StudyA range of dilutions of MRSA genomic DNA was tested. Four levels corresponding to 2,500, 500, 100 and 20 copies respectively were tested in triplicate with three different instruments at three different sites to assess the reproducibility of ELITe InGenius RT-PCR modules.

PURPOSE

The aim of this study was to verify the analytical and the clinical performance of the new sample-to-result ELITe InGenius system for MRSA/SA screening in comparison to conventional CE-IVD PCR workflow.

CONCLUSIONS

In comparison to conventional PCR workflow, ELITe InGenius™ improves the ease-of-use, reduces the time-to-result per sample, limits the risk of contamination and saves operator time while ensuring highest clinical performance for MRSA/SA testing in hospital settings. This feasibility study will be completed with clinical studies for the CE-IVD marking.

Innovative Sample-to-Result Solution to Improve the Workflow of MRSA/SA Testing

Extraction ValidationEquivalent results to the reference method were obtained by applying sample sonication.

Clinical TestingAll clinical samples were correctly identified: 4 MSSA, 2 MRSA, 4 negative.

Reproducibility StudyBoth targets: mecA (channel 1) and LDH1 (channel 3) were accurately determined regardless the concentration of the samples. The reproducibility (%CV) of the amplification between the modules was 0.23%, 0.20%, 0.66% and 1.59% for the following concentrations of genomic DNA: 2,500, 500, 100 and 20 copies respectively.

Sample Result mecACt FAM (Ch 1)

SACt AP559 (Ch 3)

ICCt AP642 (Ch 5)

Control valid 26.63 29.02 Undet

Neg negative Undet Undet 29.06

Neg negative Undet Undet 30.04

Neg negative Undet Undet 30.67

Neg negative Undet Undet 30.62

MSSA positive MSSA Undet 28.82 30.01

MSSA positive MSSA Undet 27.57 30.37

MSSA positive MSSA Undet 28.49 31.10

MSSA positive MSSA Undet 28.45 30.11

MRSA positive MRSA 28.44 28.14 30.43

MRSA positive MRSA 22.99 23.00 Undet

SampleMean Ct LDH1

ΔCt

Mean Ct mecA

ΔCt

Mean Ct IC

ΔCtELITe

InGeniusEasyMAG

ABIELITe

InGeniusEasyMAG

ABIELITe

InGeniusEasyMAG

ABIMRSA 31.44 31.99 -0.55 31.79 32.75 -0.96 28.33 30.29 -1.96

MSSA 32.15 32.90 -0.75 n.a. 38.71 n.a 30.20 30.17 0.03

S. epidermis Undet n.a. n.a. Undet n.a n.a 30.56 n.a n.a

Extraction cartridge:1 to 8 wells: 1:lysis, 2: PK; 3: carrier, 4: magnetic beads, 5: binding buffer, 5-6: wash buffers 1 & 2, 8: water

PCR Cassette: Universal PCR vessel

Sample loading

Sample preparation

RT PCR Amplification

Samples & Reagents Dispensing

• Pre-programmed assays to be run for each sample• Multiple PCR from one sample capability• Random access tests programming & sample batch processing

• Scanned sample tubes: mixed matrices• ELITe InGenius SP 200• ELITe InGenius PCR Cassette• ELITe MGB® RT-PCR reagents : mixed parameters

• Walk away• Flexible mode : extraction and/or amplification & detection• Melt-curve capability

• Approve results• Print report• Upload data to LIS

SELECT REVIEWSTARTLOAD

WORKFLOW

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RESULTS

Healthcare Infection Society 2014 Conference

Figure 1 : ELITE InGenius working area

S. Patanè, Y. Belousov, G. Stefanuto, W. Mahoney, F. Gorreta and A. EstampesELITechGroup Molecular Diagnostics

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