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Performance assessment of Respiratory Viral ELITe MGB(R) assay for the quantitative detection of influenza A/B and respiratory syncytial viruses

16 May 2020-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: RV ELITe MGB® Panel constitutes a valid and robust system for simultaneous detection and quantification of Flu A/B and RSV.
Abstract: Influenza (Flu) and respiratory syncytial virus (RSV) are responsible for lower respiratory tract infections (LRTIs) associated with significant hospitalization among young children. In the present study, the performances of a triplex PCR assay detecting Flu A/B and RSV were compared with our in-house single-plex assays using 160 stored respiratory specimens previously tested using a panel of laboratory-developed real-time RT-PCR. Of them, 61 were positive for FluA, 41 for FluB, and 58 for RSV. All samples were retrospectively quantified with Respiratory Viral (RV) ELITe MGB(R) Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe InGenius(R) system. Overall, the total percentage agreement observed was 93.4% (57/61) for FluA, 92.7% (38/41) for FluB, and 86.2% (50/58) for RSV. A significant correlation of VL values was observed between the two methods for FluA and RSV ({rho}= 0.91 and 0.84). This finding was supported by the strength of agreement between the two methods, as showed by the linear regression analysis (R2 =0.84 and 0.80). FluB viral load values measured by RV Panel were less significantly correlated ({rho}= 0.77 and R2 = 0.56). The bland-Altman analysis showed how 84.2% (48/57) of FluA and 86.0% of RSV (43/50) samples fell within {+/-}1.0 Log10 variation from our laboratory results, while only 21.1% (8/38) of FluB results fell within this range. The great majority of FluB samples (29/30) outside range had values higher than +1.0 Log10 (median +2.1 Log10 range +1.0 to +3.5 Log10). In conclusion, RV ELITe MGB(R) Panel constitutes a valid and robust system for simultaneous detection and quantification of Flu A/B and RSV.

Summary (1 min read)

MATERIAL AND METHODS

  • A total of 160 respiratory samples, stored at −80 °C in a universal transport medium (UTM™, Copan Italia SpA, Brescia, Italy) and collected from December 2014 through April 2016 at the Molecular Virology Unit of the Fondazione IRCCS Policlinico San Matteo were included in this study.
  • All samples were previously tested using a panel of laboratory-developed (which was not certified by peer review) is the author/funder.
  • All samples were retrospectively quantified with Respiratory Viral ELITe MGB® Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe InGenius® system.

Statistical analysis.

  • All viral RNA load (copies/ml) statistics were performed using log 10 transformed viral load values.
  • Quantitative variables were described as the mean and standard deviation, and/or median.
  • The agreement between the assays was assessed with a Bland-Altman plot (17) and for graphical representation a ± 0.5 Log 10 was considered an acceptable range of variability as also according to other publications (18) .
  • The correlation between (which was not certified by peer review) is the author/funder.
  • The copyright holder for this preprint this version posted May 16, 2020.

RESULTS

  • For FluB samples, no difference in median viral load was observed in high group, while median viral load measured by RV ELITe MGB®.
  • A significant correlation was observed between the LDA and RV ELITe MGB®.
  • The copyright holder for this preprint this version posted May 16, 2020.

DISCUSSION

  • In the field of respiratory infections, a rapid and accurate diagnosis is needed for reducing unnecessary antibiotic usage, preventing transmission, and initiation of specific antiviral therapy (20, 21) .
  • These findings are in keeping with the results of other rapid molecular assays when compared to LDT (22, 23) .
  • Results of the present study encourage the availability of quantitative assays for respiratory virus detection but raise the question that also other respiratory viruses, such as rhinoviruses and parainfluenza viruses, could be included in a quantitative panel due to their increasing frequency of detection also in severe respiratory illness (26, 27) .
  • It will be necessary, a more extended study should be performed, including also negative samples, in order to clarify the clinical impact of this sample-to-result solution within the laboratory workflow.
  • In conclusion, based on the data presented here, the robustness of quantification obtained by the RV ELITe MGB®.

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1
Research Articles - JCM
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Performance assessment of Respiratory Viral ELITe MGB® assay for the quantitative
3
detection of influenza A/B and respiratory syncytial viruses.
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Antonio Piralla,
a*
Federica Giardina,
a*
Alice Fratini,
a
Davide Sapia,
a
Francesca Rovida,
a
Fausto
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Baldanti
a,b
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Molecular Virology Unit, Microbiology and Virology Department, Fondazione I.R.C.C.S.
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Policlinico San Matteo, Pavia, Italy
a
; Department of Clinical-Surgical, Diagnostic and Pediatric
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Sciences, Università Degli Studi di Pavia, Pavia, Italy
b
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Running title: Multiplex assays for the quantification of FluA/B and RSV.
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*Antonio Piralla and Federica Giardina contributed equally to this article.
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Address correspondence to Antonio Piralla, a.piralla@smatteo.pv.it
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Text word: 1735
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Abstract: 253 (max 250)
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097303doi: bioRxiv preprint

2
Abstract
22
Influenza (Flu) and respiratory syncytial virus (RSV) are responsible for lower respiratory tract
23
infections (LRTIs) associated with significant hospitalization among young children. In the present
24
study, the performances of a triplex PCR assay detecting Flu A/B and RSV were compared with our
25
in-house single-plex assays using 160 stored respiratory specimens previously tested using a panel
26
of laboratory-developed real-time RT-PCR. Of them, 61 were positive for FluA, 41 for FluB, and
27
58 for RSV. All samples were retrospectively quantified with Respiratory Viral (RV) ELITe
28
MGB® Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe
29
InGenius® system. Overall, the total percentage agreement observed was 93.4% (57/61) for FluA,
30
92.7% (38/41) for FluB, and 86.2% (50/58) for RSV. A significant correlation of VL values was
31
observed between the two methods for FluA and RSV (ρ= 0.91 and 0.84). This finding was
32
supported by the strength of agreement between the two methods, as showed by the linear
33
regression analysis (R2 =0.84 and 0.80). FluB viral load values measured by RV Panel were less
34
significantly correlated (ρ= 0.77 and R2 =0.56). The bland-Altman analysis showed how 84.2%
35
(48/57) of FluA and 86.0% of RSV (43/50) samples fell within ±1.0 Log10 variation from our
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laboratory results, while only 21.1% (8/38) of FluB results fell within this range. The great majority
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of FluB samples (29/30) outside range had values higher than +1.0 Log10 (median +2.1 Log10
38
range +1.0 to +3.5 Log10). In conclusion, RV ELITe MGB® Panel constitutes a valid and robust
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system for simultaneous detection and quantification of Flu A/B and RSV.
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Keywords: Multiplex PCR, Real-time, respiratory viruses, InGenius, Quantitative results.
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097303doi: bioRxiv preprint

3
Influenza viruses type A and B (Flu A/B) and respiratory syncytial virus (RSV) are responsible for
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lower respiratory tract infections (LRTIs) associated with significant hospitalization among young
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children, elderly and immunocompromised patients (1-5). The incidence, morbidity, and mortality
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of Flu as compared to RSV varies from season to season (6). A rapid diagnosis allowing an
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appropriate decision regarding treatment and/or improved cohorting and isolation strategies to
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prevent transmission is a major concern on respiratory virus infections (7-9). In fact, in the last
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decade, the introduction of nucleic acid amplification tests (NAATs) have shortened turnaround
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time (TAT) and increased sensitivity for respiratory viruses (10). Furthermore, the multiplex RT-
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PCR approach is a validated strategy to detect a large number of respiratory viruses (8).
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Quantitative NAATs have been useful in terms of monitoring the reduction of viral load and thus
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the clinical efficacy of specific therapy (11,12). Different viral load levels have been associated
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with a higher risk of complications and severe disease in adults and children (13-15). In addition,
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the determination of viral load for different viruses in co-infections could be useful to distinguish
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which virus is the real pathogen and which the bystander (16). All these issues have to be
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interpreted in the context of available clinical and diagnostic information in order to improve
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clinical management. However, the use of quantitative NAATs in the diagnosis of respiratory
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viruses has largely been debated.
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In the present study, the performances of a triplex-PCR assay detecting and quantifying Flu A/B
61
and RSV were compared with our laboratory developed single-plex assays using positive stored
62
clinical specimens.
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MATERIAL AND METHODS
65
Study samples. A total of 160 respiratory samples, stored at −80 °C in a universal transport
66
medium (UTM™, Copan Italia SpA, Brescia, Italy) and collected from December 2014 through
67
April 2016 at the Molecular Virology Unit of the Fondazione IRCCS Policlinico San Matteo were
68
included in this study. All samples were previously tested using a panel of laboratory-developed
69
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097303doi: bioRxiv preprint

4
assays (LDA) real-time RT-PCR as previously described (9). Of them, 61 were positive for Flu A,
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41 for Flu B and 58 RSV. Samples were categorized by viral load as high (>10
6
RNA copies/ml),
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medium (10
4
-10
5
RNA copies/ml) and low (10
2
-10
3
RNA copies/ml). All samples were
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retrospectively quantified with Respiratory Viral ELITe MGB® Panel (ELITechGroup Molecular
73
Diagnostics, Puteaux, France) processed using ELITe InGenius® system.
74
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Respiratory Viral ELITe MGB
®
Panel. The archived respiratory samples were processed
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according to the manufacturer’s protocol on InGenius, a completely automated cassette based
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sample-to-results solution combining a universal extraction and independently controlled Real-time
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PCR thermal cycler (ELITechGroup Molecular Diagnostics, Puteaux, France). Briefly, 200 ul of
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respiratory were carefully transferred into a dedicated tube and loaded on the InGenius instrument
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for testing. Finally, the InGenius instrument was supplied with extraction/amplification Internal
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Control (IC), the RV ELITe MGB amplification Master mix, and extraction and amplification
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cassette consumables provided by the manufacturer (ELITechGroup Molecular Diagnostics,
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Puteaux, France). Results interpretation was performed according to the instruction manual of the
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RV ELITe MGB
®
assay. Quantitative results expressed as log
10
RNA copies/ml were measured
85
comparing the cycle threshold (Ct) values obtained and interpolated with a standard curve (serial
86
dilutions of DNA plasmid) for FluA, FluB and RSV.
87
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Statistical analysis. All viral RNA load (copies/ml) statistics were performed using log
10
89
transformed viral load values. Quantitative variables were described as the mean and standard
90
deviation, and/or median. Correlations between two quantitative variables were measured by the
91
Spearman correlation test. The agreement between the assays was assessed with a Bland-Altman
92
plot (17) and for graphical representation a ± 0.5 Log
10
was considered an acceptable range of
93
variability as also according to other publications (18). Descriptive statistics and linear regression
94
lines were performed using Graph Pad Prism software (version 5.00.288). The correlation between
95
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097303doi: bioRxiv preprint

5
the quantitative results was computed as the concordance correlation coefficient (CCC) of the
96
measurements, according to Lin (19) using MedCalc® software (Version 9.4.2.0).
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RESULTS
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A total of 160 respiratory samples with viral load ranging from 120 to 54574920 RNA
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copies/ml for FluA, from 180 to 31370040 RNA copies/ml for FluB and from 100 to 94513860
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RNA copies/ml were analysed. Overall, the total percentage agreement observed was 93.4% (57/61)
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for FluA, 92.7% (38/41) for FluB and 86.2% (50/58) for RSV (Table 1). In detail, all FluA- (4/61)
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and FluB-positive (3/41) samples not detected by RV ELITe MGB® Panel belonged to low viral
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load group (10
2
-10
3
RNA copies/ml), with viral load ranging from 270 to 900 RNA/copies ml for
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FluA and from 225 to 900 RNA/copies ml for FluB. Among 8 (13.8%) RSV-positive samples
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resulted negative by RV ELITe MGB® Panel, 1 (1.7%) had medium viral load (25650 RNA/copies
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ml) and 7 (12.1%) had low viral load ranging from 180 to 810 RNA/copies ml.
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Positive samples were stratified based on viral load into three groups named high, medium
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and low (Figure 1). Viral loads were comparable in samples included in the high (p=0.11) and
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medium (p=0.84) group for FluA as well as for RSV (p=0.07 and p=0.74) (Fig. 1A and 1C).
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Conversely, a significantly difference of viral load was observed for FluA and RSV in low viral
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load group (Fig. 1A and 1C; p<0.001). For FluB samples, no difference in median viral load was
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observed in high group, while median viral load measured by RV ELITe MGB® Panel was greater
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in the medium and low groups (Fig. 2A, p<0.001).
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A significant correlation was observed between the LDA and RV ELITe MGB® Panel for
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FluA and RSV assays (= 0.91 and 0.84) also supported by the strength of agreement observed by
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the linear regression analysis (R
2
=0.84 and 0.80) (Fig. 2A and 2C). In addition, the two assays
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showed good concordance, with a CCC of 0.86 (95% CI, 0.79 to 0.91) and 0.81 (95% CI, 0.70 to
119
0.88) for FluA and RSV assays, respectively (Table 2). FluB viral load values measured by RV
120
ELITe MGB® Panel were less significant correlated to those quantified by LDA (= 0.77 and R
2
121
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted May 16, 2020. ; https://doi.org/10.1101/2020.05.14.097303doi: bioRxiv preprint

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Frequently Asked Questions (1)
Q1. What are the contributions in "Performance assessment of respiratory viral elite mgb® assay for the quantitative detection of influenza a/b and respiratory syncytial viruses" ?

253 ( max 250 ) 20 21 ( which was not certified by peer review ) is the author/funder.