Molecular test to detect Dengue, Zika & Chikungunya and run on the QuRapID LV platform. Dr David Edge BioGene Dr Emily Adams LSTM

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1 Molecular test to detect Dengue, Zika & Chikungunya and run on the QuRapID LV platform. Dr David Edge BioGene Dr Emily Adams LSTM 03/05/17

2 Technology was initially developed for the rapid detection of sepsis causing bacteria. Adapted in /2016 for the detection of Ebola- the QuRapID technology demonstrator for the EbolaCheck project.

3 Add 25ul of whole blood <50 mins Add 450ul of extraction buffer by fixed volume pipette. Close reaction vessel cap and place into instrument for extraction step-vessel is subjected to 4 cycles of -18 to 4 0 C followed by 5 seconds at 70 0 C Simplified results displayed indicating which pathogen is detected 30 min Q-PCR step 5 min Reverse Transcription step

4 Extraction is performed in tube by rapid cyclical freezing and thawing of the sample in the actual RT-PCR mastermix. Freezing the sample introduces mechanical damage from ice formation. Thawing the frozen sample generates osmotic shock as water flows rapidly back into the cell. Direct processing of blood, no lab access required-simply add fingerprick of blood into PCR reaction vessel.

5 Technical limitations to blood in QPCR. 1. Inhibition of amplification 2. Inhibition of fluorescence signals Identical RT-QPCRs at 0% and 6% whole human blood using standards reagents and instrumentation-signal/amplification suppressed over 95% Spectrum resulting from SYBR green QPCR with (right) and without (left) human blood at 10%-95% inhibition of fluorescence

6 Optics for QPCR detection in the presence of up to 20% whole blood High powered laser spectrophotometry-makes possible triplex detection of fluorophores in the presence of up to 20% blood. Real-time RT-QPCR in the presence of 12% whole human blood- 1 spectrum captured for each of the 45 PCR cycles.

7 Mastermix-optimised for consistent amplification at up to 12% whole blood added directly into the PCR. Has been tested for a range of matrices including swabs, sputum. Mastermix reliably amplifies at up to 12% whole blood Optimised for 8% blood

8 Detection of Ebola Zaire direct from whole human blood (in publication) Three replicates of armoured RNA surrogate containing EBV GP/NP inserts at molecules per reaction and at 8% whole blood.

9 Figure 1. Figure 2. Figure 1. Showing A) efficiency of lysis for an enveloped virus, expressed as number of cycles against Ct value. B) Performance of the dedicated one step RT-QPCR mastermix in the presence of varying percentages of whole blood-the Ebolacheck assay was optimised for 8% whole blood. C) Detection of 66 viral genome equivalents 2016 contained in 5ul of whole blood (8% of final reaction volume). Figure 2. Showing standard curves generated for live Ebola virus detected in the 62.5µl closed tube Ebolacheck assay. A) The 1976 Ebola strain spiked into reactions at 3 serial dilutions and plotted as plaque forming units of the original viral culture against detection Ct B) a plot of genome equivalents per reaction against Ct for two Ebola strains and an internal control armoured RNA. The graphs demonstrate the linearity of the detection methodology.

10 Summary: A rapid technology-multiplexed detection of viruses from whole blood~50 minutes. Working on assays for Zika, Chikungunya, Dengue serotyping, Ebola, Lassa, Malaria and others. In-field point of care diagnostics. No requirement for laboratory facilities, cold chain or expert users Automated analysis and random access. Reduced costs per test by removing time and effort of RNA extraction. Ability to be adapted easily to alternate matrices and targets. Extraction method is completely linear with a LLOD of <4,000 genome equivalents/ml blood. The only current molecular approach that can detect multiple RNA viruses from a single whole blood sample.

11 6 samples at 5.35x10 7 /ml Zika Chik NTC Assays for 1. Ebola, Lassa, Marburg, Rift valley fever, Crimean Congo. Den1 Den3 2. Dengue 1-4 serotyping, Zika, Chikungunya.

12 Wider ideas Assay will render virus non-infectious. Human RNA markers are also in the blood sample. Works on a range of matrices and targets. Thanks DFID/Save the Children/Wellcome trust-ebolacheck DOH QuRapID LV Innovate UK for funding the background IP PHE- Dr Kevin Richards, Dr Miles Carroll Dr Sterghios Moschos LSTM

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14 In LSTM and BioGene were awarded a MRC Zika Emergency fund project. Design of direct from blood molecular tests for Zika, Dengue (typed) and Chikungunya in the Americas. Prospective evaluations in Brazil and Guatemala Response to other viral/bacterial/parasitological infections with design of new probes as required.

15 Probe design and field collection and testing Visual OMP used for probe design and optimisation, able to visualise secondary structure of target region Testing on live viral culture in LSTM s Category 3 laboratories, including Dengue serotypes, Zika and Chikungunya (to come) Prospective collection of patient samples in Guatemala and Brazil field testing commencing in July 2017 on whole blood. Comparison with reference standard molecular tests in referral laboratory Sample collection to be created and happy to discuss sample types and access

16 Co-infection of DENV and CHIKV present in 32% of positive samples () CHIKV DENV 46

17 Sensitivity required? Low viraemia in acute Zika infection

18 What would speed up the process of product development, validation and distribution Development and supply of cheap enzyme in order that large volume reactions can be used. Funding larger field collections in Guatemala for storage, testing and distribution

19 Thomas Edwards Prof Luis Cuevas Chris Williams Kavit Shah Adam Tyler Nelson Nazareth Leticia del Carmen Castillo Signor - Guatemala Eunis Donis Ricardo Gurgel - Sergipe