artus Infl. A/B/H1QS-RGQ Kit CE

Limit of blank (LOB)

The LOB is defined as the highest measurement result that is likely to be observed for a blank sample. For this kit, the cycle threshold (Ct) value in the Test Channel was considered an appropriate LOB. The Ct value of negative samples should remain above a threshold value (45) to generate a result “not detected”. A separate LOB was established for influenza A and influenza B on 61 measurements across two reagent lots. For influenza A-H1, the LOB was established on 67 measurements across 2 regional lots.

Limit of detection (LOD)

The LOD of the artus Infl. A/B/H1 QS-RGQ Kit was established for the3 targeted analyte strains included in the kit, i.e., influenza A/H3N2 (A/Marseille/90454111/2011), influenza B/Yamagata (B/Marseille/74506131/2013), and influenza A/H1N1 (A/Marseille/4590681204/2014 [season 2013-2014]).

Cross-reactivity

The analytical cross-reactivity of the artus Infl. A/B/H1 QS-RGQ Kit was evaluated by testing a panel of40 commonly co-infected pathogens at clinically relevant concentrations. The analytical reactivity of the artus Infl. A/B/H1 QS-RGQ Kit was assessed against 11 influenza A strains and 9 influenza B strains. Results showed no evidence of cross-reactivity and confirmed the reactivity of all 20 influenza strains tested. Carryover and cross-contaminationThese studies were conducted with contrived samples obtained by diluting Influenza A/Marseille/58863121/2012 (H1N1) strain in virocult for positive samples and virocult only for negative samples with Influenza AB and H1 Masters. There was no evidence of carryover or intra-run contamination.

Repeatability and reproducibility

Repeatability and reproducibility of the artus Infl. A/B/H1 QS-RGQ Kit were determined using single site and multi-site studies using2 different viral strains, Infl. B/Yamagata (B/Marseille/74506131/2013) and Infl. A/H1N1 (A/Marseille/4590681204/2014) as circulating in season 2013-2014. Single site studies showed there was no Rotor-Gene Q instrument nor batch effect except for assay A of the Influenza AB Master where a batch effect was observed when the sample was at low concentrations. Multi-site studies confirmed there were no QIAsymphony Sample Preparation (SP) - Assay Setup (AS) modules and site effects.

Interfering substances

The effect of potentially interfering substances was determined in accordance with CLSI guidelines EP07-A2 (1). This included endogenous substances (e.g., mucin,blood) and exogenous substances (e.g., fluticasone, menthol, muciprocin, oseltamivir, oxymetazoline, phenylephrine, saline, tobramycin). The concentrations at which these substances are unlikely to interfere with the assay can be found in the kit handbook.

Performance characteristics

A total of 202 human nasal swab specimens were tested with the artus Infl. A/B/H1 QS-RGQ Kit and the results compared with those obtained previously using laboratory developed tests derived from published method (1) at the University of Marseille, France. RNA was extracted on the QIAsymphony SP using a single batch of QIAsymphony DSP Virus/Pathogen Midi Kit. The RT-qPCR plates were set-up with a single QS-AS module using one reagent lot of the artus Infl. A/B/H1 QS-RGQ Kit. Discrepant results were resolved by testing samples with a third method, the GeneXpert Xpert Xpress Flu/RSV (Cepheid) according to protocols at the University of Marseille, France.

Further information on the performance characteristics of this kit are provided in kit handbook, which can be downloaded from the Resources tab on this webpage.

References

1. World Health Organization (July 2017). WHO information for the molecular detection of influenza viruses. Available at:http://www.who.int/influenza/gisrs_laboratory/WHO_information_for_the_molecular_detection_of_influenza_viruses_20171023_Final.pdf

The artus Infl. A/B/H1 QS-RGQ Kit constitutes a ready‑to‑use assay for the detection and identification of influenza A and B viral RNA and influenza A – H1 Pandemic 2009 strain (H1pdm2009) in nasal swab specimens. The kit contains reagents and enzymes to perform three separate reverse transcription-real time quantitative polymerase chain reaction (RT-qPCR) assays in a single run for the detection and identification of influenza A and B viruses RNA and for the detection of the A - H1pdm2009 virus RNA.

The artus Infl. A/B/H1 QS-RGQ system uses the QIAsymphony Sample Preparation (SP) module and the QIAsymphony Assay Setup (AS) module automated sample preparation and Rotor-Gene Q MDx 5plex HRM instrument for target amplification and detection.

The artus Infl. A/B/H1 QS-RGQ Kit provides all necessary reagents optimized for detection of influenza A and/or influenza B virus RNA and influenza A1-H1 Pandemic 2009 strain (H1pdm2009).

The RT-qPCR template is RNA extracted from human nasal swabs specimens using the QIAsymphony Sample Preparation (SP) module. After RNA extraction, RT-qPCR plates are automatically prepared by the QIAsymphony Assay Setup (AS) module. The RT-qPCR runs are then performed in Rotor-Gene Q MDx 5plex HRM instruments. The kit contains sufficient reagents and enzymes to perform 3 separate RT-qPCR assays in one single run, for the detection and identification of influenza A and B viruses RNA and for the detection of the A - H1pdm2009 virus RNA. Each run is performed with Influenza AB Master and H1 Master together.

To validate and control the full automated workflow, i.e. RNA extraction, plate setup and RT-qPCR reaction, an internal control sequence is automatically added into each sample at the beginning of the RNA extraction. The artus Infl. A/B/H1 QS-RGQ Kit therefore contains2 master mixes.

The Influenza AB Master contains reagents and enzymes for the specific amplification of 2targets:

1. 143bp region of the influenza virus A genome in Green channel, and

2. 94 bp region of the influenza virus B genome in Yellow channel.

The Influenza H1 Master contains reagents and enzymes for the specific amplification of an 80 bp region of influenza virus A – H1 Pandemic 2009 strain (H1pdm2009) genome in Green channel.

The artus Infl. A/B/H1 QS-RGQ Kit contains3 external positive controls (Influenza Control A, Influenza Control B and Influenza H1 Control). Influenza Control A and Influenza Control B are tested within each A/B RT-qPCR run and Influenza H1 Control within each A/H1 RT-qPCR run. These controls verify that each qPCR assay was performed correctly. The validity of each control (Internal and external controls) are automatically assessed by the specific Assay Profile.

The artus Infl. A/B/H1 QS-RGQ Kit is intended for use as an aid in the differential diagnosis of viral upper respiratory tract infection in symptomatic patients when used in conjunction with other clinical and laboratory findings.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other management decisions. The artus Infl. A/B/H1 QS-RGQ Kit is not intended for the detection of influenza C virus RNA. Positive results do not exclude co-infection with other pathogens. The pathogen(s) detected may not be the definite cause of disease. Other laboratory testing and assessment of clinical presentation must be included in the final diagnosis.