Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
DMTC Ltd., Kew, Victoria, Australia.
Microbiol Spectr. 2023 Feb 14;11(1):e0279622. doi: 10.1128/spectrum.02796-22. Epub 2023 Jan 23.
The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/μL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/μL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.
登革热是一种主要由蚊子传播的疾病,对其进行高效、准确的诊断对于临床护理、监测和疫情控制至关重要。鉴定出特定的登革热病毒血清型 1(DENV-1)至 DENV-4 有助于了解登革热疾病的传播动态和传播范围。四种快速、低资源的血清型特异性登革热检测方法使用简单的样品制备试剂,随后进行逆转录等温重组酶聚合酶扩增(RT-RPA)与侧流检测(LFD)技术相结合。结果可直接从临床样本矩阵中在 35 分钟内获得,仅需加热块和移液器进行液体处理。此外,我们证明了快速的样品制备步骤可使 DENV 失活,从而提高实验室安全性。用人血浆和血清对 DENV 进行了加标,检测到的分析灵敏度为 333 至 22,500 中位组织培养感染剂量(TCID)/mL。血液中的分析灵敏度为 94,000 至 333,000 TCID/mL。分析特异性测试证实,每种测试都可以检测到多种血清型特异性株,但不会对其他血清型、密切相关的黄病毒或基孔肯雅病毒的株作出反应。对 80 个人类血清样本的临床测试表明,测试特异性在 94%至 100%之间,DENV-2 测试的灵敏度为 100%,可检测到低至 0.004 PFU/μL,与 PCR 检测的灵敏度相当;其他 DENV 检测可检测到低至 0.03 至 10.9 PFU/μL。总的来说,我们的数据表明,我们的一些快速登革热血清分型检测方法为传统的、劳动密集型的 RT-定量 PCR(RT-qPCR)检测提供了一种潜在的替代方法,后者需要昂贵的热循环仪器、技术专业知识和延长的检测时间。我们的测试在不影响特异性的情况下提供了性能和速度,无论是在人血浆和血清中,还是在资源有限的环境中检测高载量的 DENV ,都有可能成为一种有前途的检测工具。
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