Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

Kayla G Barnes,Daniel J Park,Cameron Myhrvold,Testimony Olumade,Matt Boisen,Donald S Grant,Gustavo Palacios,Brian M Sullivan ,Luis M Branco ,Molly Kemball,Brett Beitzel,John Demby Sandi,Abdulwasiu Bolaji Tiamiyu ,Mambu Momoh,Catherine A Freije,Fehintola V Ajogbasile ,Jessica N Uwanibe ,Amber Carter,Zahra Parker ,Anna E Lachenauer,Bonnie Dighero-Kemp,Andrés Colubri ,Ikponmwosa Odia,Adam Nitido,Katherine J Siddle,Aaron E Lin,Kayvon Modjarrad,Chloe K Boehm,Michael Iroezindu,Lisa E Hensley,Stephen F Schaffner,Mihret F Amare,Robin Gross,Robert F Garry,Samar B Mehta,Hayden C Metsky,Christian Happi ,Sameed M Siddiqui ,Pardis C Sabeti

doi:10.1038/s41467-020-17994-9

Abstract

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.

Highlights

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases
The most widely used diagnostic for LF viral detection, a reverse-transcriptase quantitative PCR (RT-qPCR) developed against Josiah strains derived from Sierra Leone, has had false-positive and false-negative results when tested against recent clade II samples from Nigeria[11,12]
We developed a SHERLOCK Ebola virus (EBOV) assay to target the L gene of the EBOV Zaire strain, which accounts for the majority of known clinical cases of EBOV infections, including the two largest and most recent EVD epidemics[17,18]

Summary

Introduction

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Ebola virus (EBOV) and Lassa virus (LASV) pose immediate, severe threats to human life and public health, as demonstrated by ongoing outbreaks of EBOV disease (EVD) in the Democratic Republic of the Congo (DRC) and Lassa fever (LF) in Nigeria Despite their high morbidity and mortality, EVD and LF are difficult to diagnose, because early symptoms, including fever, vomiting, and aches, are often indistinguishable from those of more common tropical diseases[1,2,3]. Our recent work has shown the high sensitivity of SHERLOCK and HUDSON in detecting Zika virus and dengue virus directly from bodily fluids[14], allowing for a fully point-of-care diagnostic Utilizing this system, we develop a diagnostic test for EBOV and LASV that can be deployed in any setting, requires minimal processing of infectious materials, and accurately reports test results in a userfriendly format

Methods

Results

Conclusion