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

Autor: Tang HT; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Nörz D; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Grunwald M; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Giersch K; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Pfefferle S; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Fischer N; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Aepfelbacher M; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Rohde H; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany., Lütgehetmann M; University Medical Center Hamburg-Eppendorf, Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany. Electronic address: mluetgeh@uke.de.
Jazyk: angličtina
Zdroj: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology [J Clin Virol] 2024 Aug; Vol. 173, pp. 105693. Date of Electronic Publication: 2024 May 16.
DOI: 10.1016/j.jcv.2024.105693
Abstrakt: 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.
Competing Interests: Declaration of competing interest ML received speaker honoraria and related travel expenses from Roche Diagnostics and Qiagen GmbH. DN received speaker honoraria and related travel expenses from Roche Diagnostics. All other authors declare no conflict of interest.
(Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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