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TRANSCRIPT
Overview of diagnostic tools to advance clinical trials
(confirmatory diagnosis, assays for vaccine development)
Devy Emperador – Scientific Officer, Pandemic Preparedness
WHO consultation on RVFV therapeutics and vaccine evaluation - WHO R&D Blueprint Meeting
1st Nov 2019; Geneva, Switzerland
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FIND is a global non-profit driving diagnostic innovation to combat major
diseases affecting the world’s poorest populations
WHO Collaborating Centre for Laboratory Strengthening
& Diagnostic Technology Evaluation
WHO SAGE-IVD member
ISO-certified quality management system for IVD clinical trials
We address market failure by partnering
to develop and deliver diagnostic solutions
to LMICs
ANTIMICROBIAL
RESISTANCE
HEPATITIS C
& HIVMALARIA & FEVER
NEGLECTED
TROPICAL DISEASES
PANDEMIC
PREPAREDNESSTUBERCULOSIS
Geneva
(HQ)
South
Africa
India
Viet Nam
Kenya
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ANTIMICROBIAL RESISTANCE
Without action, 10 million deaths every year
predicted by 2050
MALARIA & FEVER
In Africa alone, over 600 million childhood fevers occur
every year – which may or may not be due to malaria
HEPATITIS C & HIV
71 million people affected by HCV but only
1 in 5 know their status
NEGLECTED TROPICAL DISEASES
Affect more than 1 billion people, yet suffer from
historical lack of attention
PANDEMIC PREPAREDNESS
6 out of 10 WHO “Blueprint” pathogens have significant
unmet diagnostic needs
TUBERCULOSIS
3.6 million cases missed in 2017 and the
majority of infected children go undiagnosed
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In the 2014–16 Ebola
outbreak, a point-of-care
diagnostic could have saved:
70% of
lives lost
80% of
all costs
Diagnostics save lives, and money
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What happens without diagnostics?
Impact of not closing the diagnostic gap
– Delayed identification of outbreaks leading to escalation and spread
– Increased costs of local, national, international response
– Delays in vaccine and therapeutic evaluations
– Limited understanding of epidemiology of disease
Clinical trials for vaccines and therapeutics require well characterized
diagnostics:
– Clear planning for trials based on sound epidemiological data
– Clear enrollment criteria for patients based on definitive diagnosis
– Early identification of at risk populations for tailored clinical trials
Delays in addressing the diagnostic gaps will cause delays in initiation of
clinical trials, increased costs and potentially inappropriate site selection
PANDEMIC PREPAREDNESS
6 out of 10 WHO “Blueprint” pathogens have significant
unmet diagnostic needs
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Diagnostics for Rift Valley Fever
Pathogen ID
– Virus isolation
– RT-PCR
– Antigen detection (ELISA, immunohistochemistry)
Serological tests
– Immunofluorescence assays
– Virus neutralization
– ELISA
Biocontainment needs: High (BSL-3)
Current testing location: Limited to reference laboratories
Source: Hartman A. (2017). Rift Valley Fever. Clinics in laboratory medicine, 37(2), 285–301. doi:10.1016/j.cll.2017.01.004. Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for
emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Paweska JT. (2015). Rift Valley fever. Revue scientifique et technique, 34, 375-389.
Figure: Biomarker dynamics in Sheep Experimentally Infected with Natural RVFV
Note: Viremic period can often be very short, as little as 2 or 3 days.
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Status of commercial assays for Rift Valley Fever
Type Product name Species Company Regulatory Comments
NAT RealStar RVFV RT-PCR Human Altona CE No published independent studies
NAT RVFV Human TIB MolBiol RUO No published independent studies
NAT Rift Valley Virus (RVFV) Real Time RT-PCR
Reagent
Human LifeRiver CE No published independent studies
NAT FTD RVFV Human Fast-Track Diagnostics RUO No published independent studies
NAT Rift Valley Fever Virus RT-PCR kit; EBOV+RVFV
and EBOV+RVFV+YFV PCR kits
Human Genekam Biotech Ag RUO No published independent studies
NAT Rift Valley Fever virus Human PCRMax RUO No published indpendent studies
NAT QPCR Kit, RNA, Rift Valley Fever Virus Human Techne/Cole Palmer RUO No published independent studies
NAT
(in dev)
field-ready RT-PCR, assay for RVFV, "Lab-in-a-
suitcase"
- USDA and GeneReach not known Status unknown
PCR +
Luminex
(in dev)
platform for surveillance, serology, a panel of
diseases, such as RVFV and Blue Tongue
- USDA not known Status unknown
Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever
Diagnostics – FIND Landscape (2017).
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Status of commercial assays for Rift Valley Fever
Type Product name Species Company Regulatory Comments
ELISA IgG ELISA assay ID Screen Rift Valley Fever
Competition (IgM + IgG)
Multi-species ID-vet Veterinary Good performance in 9 labs in 2015 animal
EQA study and animal ring trial
ELISA ID Screen Rift Valley Fever IgM Capture Ruminants ID-vet Veterinary Good performance in 10 labs in 2015 animal
EQA study
ELISA Ingezim FVR DR 13-FVR.K0 (IgM) Ruminants Ingenasa Veterinary No published independent studies identified
ELISA Ingezim FVR Compac 13.FVR.K Ruminants Ingenasa Veterinary Good performance in 1 lab in 2015 animal EQA
study in animals
ELISA Rift Valley Fever recN Ag detection ELISA Ruminants BDSL Veterinary Company no longer exists
IFA RVFV IIFT IgG. Human EUROIMMUN CE No published independent studies
IFA RVFV IIFT IgM Human EUROIMMUN CE No published independent studies
Lateral Flow Ingezim FVR Crom Ruminants Ingenasa Veterinary No published independent studies; does not
discriminate IgG vs. IgM
Dipstick VectorTest RVFV (antigen) Mosquito VectorTest - No published independent studies
ELISA
(in dev)
anti-RVFV IgM Humans ID-vet plan for CE Status unknown
Lateral flow
(in dev)
Device to detect IgM + IgG - USDA not known Status unknown
Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever
Diagnostics – FIND Landscape (2017).
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Status of commercial assays for Rift Valley Fever
Type Product name Species Company Regulatory Comments
ELISA IgG ELISA assay ID Screen Rift Valley Fever
Competition (IgM + IgG)
Multi-species ID-vet Veterinary Good performance in 9 labs in 2015 animal
EQA study and animal ring trial
ELISA ID Screen Rift Valley Fever IgM Capture Ruminants ID-vet Veterinary Good performance in 10 labs in 2015 animal
EQA study
ELISA Ingezim FVR DR 13-FVR.K0 (IgM) Ruminants Ingenasa Veterinary No published independent studies identified
ELISA Ingezim FVR Compac 13.FVR.K Ruminants Ingenasa Veterinary Good performance in 1 lab in 2015 animal EQA
study in animals
ELISA Rift Valley Fever recN Ag detection ELISA Ruminants BDSL Veterinary Company no longer exists
IFA RVFV IIFT IgG. Human EUROIMMUN CE No published independent studies
IFA RVFV IIFT IgM Human EUROIMMUN CE No published independent studies
Lateral Flow Ingezim FVR Crom Ruminants Ingenasa Veterinary No published independent studies; does not
discriminate IgG vs. IgM
Dipstick VectorTest RVFV (antigen) Mosquito VectorTest - No published independent studies
ELISA
(in dev)
anti-RVFV IgM Humans ID-vet plan for CE preliminary studies completed, validation
underway in several academic labs. No
timeline for launch
Lateral flow
(in dev)
Device to detect IgM + IgG - USDA not known Status unknown
Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever
Diagnostics – FIND Landscape (2017).
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Status of lab-developed tests for Rift Valley Fever
Type Publication Description EQA status & Performance
RT-PCR Bird et al. (2007) 2 step real-time assay, 4 – 5 hours. Designed to detect 40
known strains. Designed for high throughput, automation.
8 labs used this protocol in 2012 human EQA study, where it showed
capacity for optimal performance. 2 labs used in 2015 animal EQA with
100% correct.
RT-PCR Drosten et al. (2002) 1 step real time assay. Quantitative. Part of a panel of assays
for 6 VHF pathogens
16 labs used this in 2012 human EQA study, showed capacity for optimal
performance. 3 labs used this in 2015 animal EQA study, 2 labs 100%, 1
lab had unacceptable results.
RT-PCR Drolet et al. (2012) 1 step real-time assay with pathogen inactivation in 1st
step. Study used samples with known status. No independently published
studies of performance.
LAMP Peyrefitte et al. (2008) 1 step endpoint assay. 63 C, 30 minute assay. Qualitative. Study used samples with known status, and artificial samples. No
independently published studies
LAMP Le Roux et al. (2009) 1 step Real time assay, 30 to 45 min assay 1 lab used this protocol in 2012 human EQA, where it showed limitations in
sensitivity.
RPA Eular et al. (2012) 1 step, 42 C, ESEquant tube scanner. < 30 min assay.
TwistDx reagents. Qualitative. Part of panel of 10 biothreat
pathogens.
1 lab used this protocol in 2012 human EQA, with optimal performance.
RT-PCR Weidmann et al. (2008) One step assay, designed against 19 strains 4 labs used this protocol in 2012 human EQA, showed capacity for optimal
performance.
RT-PCR Garcia et al. (2001) 2 step real time Taqman 1 lab used this in 2012 human EQA, did not detect Egypt strain.
RT-PCR Busquets et al. (2010) 1 step real time Taqman 1 lab used this in 2012 human EQA, did not show optimal performance.
RT-PCR Mwaengo et al. (2012) 2 step real time assay 1 lab used this in 2012 human EQA, did not show optimal performance.
RT-PCR Sall et al. (2002) Nested PCR assay, qualitative 6 labs used this in 2012 human EQA with mixed performance, some issue
with false positives.
RT-PCR Sanchez-Seco (2003) Nested PCR assay, qualitative 1 lab used this in 2012 human EQA, did not show optimal performance.
RT-PCR Wilson et al. (2014) Real time RT-PCR multiplex, detects 3 segments (different
dyes) allowing DIVA. Includes armored RNA ampli. control
Study used samples with known status. Low sensitivity reported for
quadriplex assay. No independent published studies
RT- PCR Liu et al. (2016) Taqman assay card for 26 pathogens, for outbreak
investigation or surveillance
Publication reports performance on > 1000 samples from various sources,
but small N for many rare pathogens
Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever
Diagnostics – FIND Landscape (2017).
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Status of lab-developed tests for Rift Valley Fever
Type Publication Description EQA status & Performance
ELISA Paweska et al. (2003) Separate IgG sandwich and IgM capture assays for ruminants,
using irradiated whole virus as the antigen
IgG and IgM assays compared in van Vuren et al. (2010), against
samples with known status: good performance, IgM assay detected
seroconversion earlier (day 4).
ELISA Paweska et al. (2005) IgG sandwich and IgM capture assays for humans made using
irradiated whole virus as antigen, validated on human samples
Compared in van Vuren et al. (2010), against samples with known
status. Some problems with high viremia
ELISA Paweska et al. (2007) IgG assay for humans, made using recombinant N protein.
Validated in human samples
No independent EQA studies identified. Excellent performance in ~
3000 samples. Comparator was VNA
ELISA van Vuren et al. (2007) Separate IgG, IgM indirect ELISAs for humans, ruminants, uses
recombinant N protein.
Study used samples with known status. No independent EQA studies
identified.
ELISA Fafetine et al. (2007) Separate IgM and IgG assays for ruminants based on recombinant
N protein, validated in ruminants
Study used samples with known status. IgG assay compared in van
Vuren et al. (2010), less sensitive than IgG LDT 1 in detecting early
response in sheep
ELISA van Vuren et al. (2009) Sandwich ELISA for ruminants, humans. Uses polyclonal capture
sera generated using recombinant antigen. Does not dis-tinguish
IgM vs. IgG. Validated preincubation of samples at 56 C 1 hour
(biosafety).
Sensitivity poor compared to VNA. No independent EQA studies
identified
ELISA Jackel et al. (2013) Assay for ruminants uses recombinant Gn protein expressed in E.
coli, and detects IgG in sheep and goats
No independent EQA studies identified. Excellent performance in ~
2000 samples. Comparator was VNA
ELISA (DIVA) McElroy et al. (2009) 2 parallel ELISAs distinguish natural infections from vaccinations
(recombinant N and NSs proteins). Validated for goat, human.
Does not distinguish IgM vs IgG.
No independent EQA studies identified. Study used samples with
known infection status
ELISA Williams et al. (2011) IgM capture ELISA for ruminants, using rabbit anti-sheep IgM on
plate, and recombinant N protein conjugated to HRP for detection.
Validated on sheep
Some challenges in 2013 animal ring trial. Comparator was ID-vet
competition ELISA
ELISA Ellis et al. (2014) Non-species-specific assay, uses recombinant N protein expressed
in E. coli to coat plate, and N-protein-HRP conjugate for detection.
Detects both IgM and IgG. Validated on sheep and cattle.
No independent EQA studies identified. Comparator was ID-vet
ELISAs
VNA Winchger Schreur (2017) Avirulent RVFV which expresses eGFP, takes 48 hours and is
more sensitive than classic VNT. Not species specific.
No independent EQA studies identified. Comparator was classical VNA
Source: Mansfield KL et al. (2015). Rift Valley fever virus: A review of diagnosis and vaccination, and implications for emergence in Europe. Vaccine, 33(42), 5520-5531. https://doi.org/10.1016/j.vaccine.2015.08.020. Rift Valley Fever
Diagnostics – FIND Landscape (2017).
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Recommendations for future planning
Diagnostic gaps for RVFV
– Validated, commercial serology assays for human specimens
– Diagnostics for lower-level laboratories
– Understanding of correlate(s) of protection
Lab capacity must be assessed and strengthened prior to beginning vaccine clinical trials and
epidemiological studies
Laboratory tests must be evaluated prior to starting clinical trials
– If appropriate tests are not available sufficient time must be allowed for R&D
– Access to well-characterized samples and reference standards can accelerate test evaluation
Procurement, import and export are additional considerations that can lead to unexpected delays in starting a
study
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Swiss Development and Coop eration;
State Secretariat for Education, Research and Innov ation
Netherlands EnterpriseAg ency
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partners who make the work of
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