Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses

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@article{6eae1ee6eeb24497983a649536843a8c,
title = "Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses",
abstract = "BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %.DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.",
keywords = "Humans, Multiplex Polymerase Chain Reaction/methods, Respiratory Tract Infections/virology, Sensitivity and Specificity, High-Throughput Screening Assays/methods, Viruses/isolation & purification, Virus Diseases/diagnosis, Automation, Laboratory/methods, SARS-CoV-2/genetics, COVID-19/diagnosis, Molecular Diagnostic Techniques/methods",
author = "Tang, {Hui Ting} and Dominik N{\"o}rz and Moritz Grunwald and Katja Giersch and Susanne Pfefferle and Nicole Fischer and Martin Aepfelbacher and Holger Rohde and Marc L{\"u}tgehetmann",
note = "Copyright {\textcopyright} 2024 The Author(s). Published by Elsevier B.V. All rights reserved.",
year = "2024",
month = aug,
doi = "10.1016/j.jcv.2024.105693",
language = "English",
volume = "173",
pages = "105693",
journal = "J CLIN VIROL",
issn = "1386-6532",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses

AU - Tang, Hui Ting

AU - Nörz, Dominik

AU - Grunwald, Moritz

AU - Giersch, Katja

AU - Pfefferle, Susanne

AU - Fischer, Nicole

AU - Aepfelbacher, Martin

AU - Rohde, Holger

AU - Lütgehetmann, Marc

N1 - Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.

PY - 2024/8

Y1 - 2024/8

N2 - BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %.DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.

AB - BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %.DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.

KW - Humans

KW - Multiplex Polymerase Chain Reaction/methods

KW - Respiratory Tract Infections/virology

KW - Sensitivity and Specificity

KW - High-Throughput Screening Assays/methods

KW - Viruses/isolation & purification

KW - Virus Diseases/diagnosis

KW - Automation, Laboratory/methods

KW - SARS-CoV-2/genetics

KW - COVID-19/diagnosis

KW - Molecular Diagnostic Techniques/methods

U2 - 10.1016/j.jcv.2024.105693

DO - 10.1016/j.jcv.2024.105693

M3 - SCORING: Journal article

C2 - 38820916

VL - 173

SP - 105693

JO - J CLIN VIROL

JF - J CLIN VIROL

SN - 1386-6532

ER -