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The performance of RT-PCR compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory.

ABSTRACT: The laboratory diagnosis of dengue has largely relied on serological assays, although many different RT-PCR protocols have been reported. Owing to its limited use, the value of RT-PCR in the clinical laboratory has not been fully evaluated. During the outbreak of severe acute respiratory syndrome (SARS) in Singapore in 2003, RT-PCR to detect dengue viral RNA was used as a rapid diagnostic tool to differentiate dengue from SARS among patients who presented to a hospital designated to manage and quarantine SARS cases. A total of 343 results for RT-PCR and 439 results for serology were analysed and compared with the final discharge diagnosis. Our experience indicates that RT-PCR for dengue can be set up rapidly in a clinical laboratory, with very sensitive and specific results for the diagnosis of dengue, particularly in the first 5 days from onset of symptoms.

SUBMITTER: Barkham TM

PROVIDER: S-EPMC7107224 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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The performance of RT-PCR compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory.

Barkham Timothy M TM Chung Youne Kow YK Tang Kin Fai KF Ooi Eng Eong EE

Transactions of the Royal Society of Tropical Medicine and Hygiene 20051004 2

The laboratory diagnosis of dengue has largely relied on serological assays, although many different RT-PCR protocols have been reported. Owing to its limited use, the value of RT-PCR in the clinical laboratory has not been fully evaluated. During the outbreak of severe acute respiratory syndrome (SARS) in Singapore in 2003, RT-PCR to detect dengue viral RNA was used as a rapid diagnostic tool to differentiate dengue from SARS among patients who presented to a hospital designated to manage and q ...[more]

PMID: 16212996

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Project description:BackgroundEarly detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0.Methodology and principal findingsSpecific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly.Conclusions and significanceThe RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

Dataset Information

The performance of RT-PCR compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory.

Publications

The performance of RT-PCR compared with a rapid serological assay for acute dengue fever in a diagnostic laboratory.

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