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Diagnostic performance of multiplex PCR on pulmonary samples versus nasopharyngeal aspirates in community-acquired severe lower respiratory tract infections.

ABSTRACT: BACKGROUND:PCR-based techniques for the diagnosis of community- acquired severe lower respiratory tract infections are becoming the standard of care. However, their relative ability to identify either atypical bacteria or viruses that cause LRTI from clinical samples from various sources is yet to be determined. OBJECTIVES AND STUDY DESIGN:The aim of our study was to compare the diagnostic yield of nasopharyngeal aspirates with that of pulmonary samples for the etiological diagnosis of severe acute lower respiratory tract infections by multiplex PCR. Patients were adults with community-acquired pneumonia or acute exacerbation of chronic obstructive pulmonary disease. RESULTS:We obtained concordant results for 81 (79%) of the 103 pairs of samples. In 14 of the 22 discordant results, more pathogens were evidenced in the lower respiratory tract samples. CONCLUSIONS:Pulmonary samples had a similar diagnostic sensitivity for virus detection by multiplex PCR as nasopharyngeal aspirates. In contrast, in our study, the diagnostic efficacy of pulmonary samples for Legionella pneumophila over simple aspirates was clearly superior.

SUBMITTER: Robert S

PROVIDER: S-EPMC7185397 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Diagnostic performance of multiplex PCR on pulmonary samples versus nasopharyngeal aspirates in community-acquired severe lower respiratory tract infections.

Robert Sylvie S Lhommet Claire C Le Brun Cécile C Garot Denis D Legras Annick A Mankikian Julie J Goudeau Alain A

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20180829

<h4>Background</h4>PCR-based techniques for the diagnosis of community- acquired severe lower respiratory tract infections are becoming the standard of care. However, their relative ability to identify either atypical bacteria or viruses that cause LRTI from clinical samples from various sources is yet to be determined.<h4>Objectives and study design</h4>The aim of our study was to compare the diagnostic yield of nasopharyngeal aspirates with that of pulmonary samples for the etiological diagnos ...[more]

PMID: 30195090

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Project description:BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R(2)>0.99), signal-to-analyte response (slope 1.0 ± 0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Dataset Information

Diagnostic performance of multiplex PCR on pulmonary samples versus nasopharyngeal aspirates in community-acquired severe lower respiratory tract infections.

Publications

Diagnostic performance of multiplex PCR on pulmonary samples versus nasopharyngeal aspirates in community-acquired severe lower respiratory tract infections.

Similar Datasets

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