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Preventing contamination of PCR-based multiplex assays

11/08/2020 Actualités , Publications 0 118

 

ORIGINAL ARTICLE

Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet

A.Bouam (1,2), J.J. Vincent (3), M. Drancourt (2,3), D. Raoult (2,3) and P.Y. Levy (1,3)

1 POCRAME, Marseille, France

2 IRD, MEPHI, IHU Méditerranée Infection, Aix-Marseille University, Marseille, France

3 IHU Méditerranée Infection, Marseille, France

Keywords

airflow, biosafety cabinet point-of-care,cerebrospinal fluid, false positive, multiplex PCR, polymerase chain reaction, syndromic diagnosis, transcontamination.

Correspondence Amar Bouam, 19-21 Boulevard Jean Moulin,13385 Marseillle Cedex 05, France

.E-mail: mailto:amarbouam@yahoo.fr

2020/0503: received 19 March 2020, revised11 August 2020 and accepted 11 August2020

doi:10.1111/lam.13375

Signicance and Impact of the Study:

PCR-based multiplex assays are frequently used for syndrome-based diagnosis in clinical microbiology laboratories. Commercially available multiplexed real-time PCR assays are closed systems, including DNA extraction, DNA amplication and real-time analysis. However, many studies reported false positives due to in-laboratory pre-analytical contamination of the clinical specimens. Data reported in the present study show for the rst time that the use of a dedicated biosafety cabinet with some preventive measures signicantly reduced the number of false positives. These observations can help clinical microbiology laboratories in the management of the diagnosis of meningitis and other infectious diseases.

 

Abstract:

We retrospectively investigated cases of false-positive diagnoses using the BIOFIRE® FilmArray® meningitis/encephalitis (ME) panel to measure the impact of using a dedicated biosafety cabinet combined with preventive measures to reduce the prevalence of false-positive diagnoses due to pre-analytical in-laboratory contamination. False-positive results were identified by reviewing clinical data, biological parameters and cytology results of cerebrospinal fluid (CSF) samples showing discrepant results between the FilmArray ME panel and routine PCR assays. A total of 327 CSF were analysed over 16 weeks in point-of-care (POC) A and B, over two 8-week periods, periods 1 and 2. The analysis yielded 30 (9 .17%) detection of at least one pathogen including 21/30 (70%) viruses and 9/30 (30%) bacteria. During period 1, POC-A and POC-B manipulated CSF under a non-dedicated hood featuring laminar flow, whereas during period 2, CSFs were manipulated under a dedicated biosafety cabinet without any airflow in POC-A. During period 1, false positives were detected in 3/114 CSF (2.63%) in POC-A and 1/36(2.77%) in POC-B (P = 0.97); during period 2, false positives were detected in0/139 CSF (0%) in POC-A and 1/38 (2.63%) in POC-B (P = 0.23). All false positives were bacterial. The use of a dedicated cabinet without ventilation along with preventive measures during period 2 in POC-A significantly reduced the number of false-positive results (P = 0. 05). Preventive measures described in this study can mitigate false positives when using PCR-based multiplex assays such as the BIOFIRE FilmArray ME Panel for the diagnosis of meningitis and other infectious diseases.

Pour lire la suite de l'article scientifique: Preventing contamination of PCR‐based multiplex assays including the use of a dedicated biosafety cabinet (wiley.com)

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