Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation.

Zhan Qiu Mao,Shingo Inoue,Jean Claude Balingit,Futoshi Hasebe,Le Khanh Hang Nguyen,Thi Thanh Ngan Nguyen,Mizuki Fukuta,Noboru Minakawa,Co Thach Nguyen,Kouichi Morita,Thi Quynh Mai Le,Meng Ling Moi,Thi Thu Thuy Nguyen

doi:10.3390/pathogens10121558

Abstract

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Highlights

IntroductionThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that causes severe respiratory symptoms, has recently caused a major global healthcare concern
To evaluate the real-time RT-qPCR method on heat-inactivated samples as a potential alternative diagnostic method, primer-probe sets targeting SARS-CoV-2 envelope (E) and nucleocapsid (N) genes were tested through chemical inactivation and heat inactivation methods using virus-infected cell cultures and nasopharyngeal swab samples from a COVID-19 patient after symptom onset
The results indicated that heat inactivation and direct real-time RT-qPCR of spiked samples reduced the levels of detectable SARS-CoV-2 RNA in samples with two viruses (DENV-2 and SARS-CoV-2)

Summary

Introduction

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that causes severe respiratory symptoms, has recently caused a major global healthcare concern. In severe and critical COVID-19 cases, mortalities are largely driven by severe respiratory failure related to interstitial pneumonia in both lungs and acute respiratory distress syndrome [3]. Initial clinical features may be similar in both dengue and COVID-19, along with shared laboratory parameters such as thrombocytopenia and leucopenia [5]. The similarity in clinical manifestations is of significant public health concern, especially in countries where dengue is endemic, as it remains a challenge to clinically differentiate dengue from COVID-19 at initial presentation. As dengue and COVID-19 require different clinical management, this may pose a serious public health threat that may lead to adverse consequences, in dengue-endemic countries

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