Prototype Multi-Agent Lateral Flow Immunoassay (LFI) for the Detection of Lassa
Virus (LASV), Malaria, and Ebola Virus (EBOV) for the Point of Care Detection of
Endemic Infections.
D. Nelson1, S. McCormick2, M. Boisen1,3, L. Branco5, A. Goba4, M. Momoh4, M. Rowland5, M. Heinrich5, D. Abelson6, K. Hastie6, Z. Bornholdt6, M. Fusco6, P. Kulakosky2, R.
Wilson2, E. Saphire6, S.H. Khan4§,J. Schieffelin7, D. Grant4,8, K.R. Pitts1, R.F. Garry3 And The Viral Hemorrhagic Fever Consortium.
1Corgenix,
Inc. Broomfield, Colorado, USA, 2Autoimmune Technologies, LLC, New Orleans, Louisiana, USA , 3Department of Microbiology and Immunology, Tulane University, New Orleans, Louisiana, USA, 4Lassa Fever
Program, Kenema Government Hospital, Kenema, Sierra Leone, 5Zalgen Labs, LLC, Germantown, Maryland, USA, 6Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, California,
USA, 7Sections of Infectious Disease, Departments of Pediatrics and Internal Medicine, School of Medicine, Tulane University, New Orleans, Louisiana, USA, 8Ministry of Health and Sanitation, Freetown, Sierra Leone,
§Deceased
Abstract
Lassa virus (LASV) and Malaria are endemic in regions of Sub-Saharan Africa, the current Ebola virus (EBOV) epidemic establishes this disease in the same region. Patient management becomes difficult considering that all three of these diseases initially present with nonspecific febrile illness. There is a need for a point-of-care test
that can differentially diagnose febrile patients to aid in the appropriate isolation and quarantine measures. Documented cases of malaria positive patients have been sent home from clinics that are later confirmed to have comorbidity with LASV or EBOV creating difficulties for infection control. To assist in this effort Corgenix Inc. and
the VHFC have been working on prototype development of a Multi-Agent Antigen Rapid Test. This dipstick-format lateral flow immunoassay (LFI) incorporates LASV NP, Malaria HRP-II, and EBOV VP40 antigen detection with LASV NP and Malaria HRP-II monoclonal antibodies and EBOV VP40 polyclonal antibodies to detect LASV,
Malaria or EBOV in whole blood or plasma samples. Analytical performance was performed using recombinant antigen to prepare contrived samples. Field trials were conducted on surplus, banked clinical samples at Kenema Government Hospital collected prior to and during the 2014-2015 West African Ebola outbreak. Limit of
Detection (LOD) of the Multi-Agent LFI was 9.4 ng/test for HRP-II, 9.4 ng/test for LASV NP, and 9.4 ng/test for EBOV VP40. Prototype specificity versus n=119 normal serums was 96.6%. In limited field testing, the LFI detected malaria in 11/161 febrile samples and 10/72 asymptomatic controls; Lassa was detected in 12/161 febrile and
0/72 control; Ebola was detected 55/161 febrile and 4/72 controls. This Multi-Agent Antigen Rapid Test prototype exhibited a LOD below the anticipated antigenemia levels during acute illness. Field trails show that this test is capable of point of care triage of febrile cases. This Multi-Agent RDT represents a diagnostic advancement for
point of care detection of high consequence pathogens present in the region of the West Africa EBOV outbreak and can be critical for infection control.
Malaria
A
B
LASV
EBOV
Negative
Malaria
C
Control Line
!
Malaria Test Line
!
Lassa Test Line
!
Ebola Test Line
!
Figure 3: Prototype Multi-Agent RDT Detects Malaria, LASV, and EBOV in Contrived
Samples. LFI strips were challenged with contrived samples: A mix of all 3 recombinant
antigens, Malaria (P.f.) rHRP2, LASV rNP, EBOV rVP40, and bulk normal serum.
Figure 1. Regions of Endemic Febrile Illness and Outbreaks in Africa. (A) Map highlighting endemic
regions of Malaria infection (World Bank) (B) Map highlighting endemic regions of LASV infection (CDC)
(C) Map indicating locations of EBOV infection from 1976-2014 (CDC)
Malaria rHRP2
300ng/test
150ng/test 75ng/test 37.5ng/test 18.8ng/test
LASV
EBOV
Negative
Figure 4: Prototype Multi-Agent RDT Detects Malaria, LASV, and EBOV in
Clinical Samples. LFI strips were challenged with clinical samples: Malaria
(P.f.) serum, LASV serum, EBOV serum, and uninfected whole blood.
Lassa rNP
9.4ng/test
4.7ng/test
2.3ng/test
300ng/test
75ng/test
150ng/test
37.5ng/test
Ebola rVP40
18.8ng/test
9.4ng/test
4.7ng/test
300ng/test
2.3ng/test
150ng/test
75ng/test
37.5ng/test 18.8ng/test
9.4ng/test
4.7ng/test
2.3ng/test
Figure 5: Limit of Detection (LOD) Visual Read-outs for Prototype Multi-Agent RDT. The Multi-Agent RDT detects Malaria, LASV, and EBOV Recombinant Antigens with an LOD of18.8, 9.4, and 2.3ng/test, respectively.
LFI strips were challenged with a serial dilution of either Malaria rHRP2 antigen, LASV rNP antigen, or EBOV rVP40 antigen from 2.3-300ng/test.
B
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
Ebola VP40 Antigen LOD Curve
1.00
C
0.90
Mean Test:Control Reflectance Ratio
Figure 2. Schematic of a Lateral Flow Immunoassay (adapted from http://www.cytodiagnostics.com/
images/lateral_flow_assay.jpg). LFIs are designed to provide analyte detection capability outside of a typical
laboratory environment. (A) A sample of serum, whole blood, or saliva is added to the sample pad. (B) The sample
migrates into the conjugate pad by capillary action, where analyte in the sample contacts colloidal gold-labeled
antibody. The labeled antibody-analyte complex then migrates along the nitrocellulose membrane to the wicking
pad. (C) As the labeled antibody-analyte complex passes the test line, a second antibody present in the test line
can capture and concentrate the labeled antibody-analyte complex. Excess labeled antibody flows on to the control
line, where a third antibody present in the control line that recognizes the labeled antibody isotype can capture and
concentrate the labeled antibody. A pink color at the test line indicates the presence of analyte in the sample, and
pink color at the control line confirms that the test is valid.
1.00
Mean Test:Control Reflectance Ratio
A
Lassa NP Antigen LOD Curve
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
50
100
150
200
250
300
Lassa Test Line
Ebola Test Line
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.00
0
50
100
Concentration of Antigen (ng/test)
Malaria Test Line
0.90
0.10
0.00
0
1.00
Mean Test:Control Reflectance Ratio
Malaria HRP2 Antigen LOD Curve
150
200
250
300
0
50
100
Antigen Concentration (ng/test)
Malaria Test Line
Lassa Test Line
Ebola Test Line
150
200
250
300
Antigen Concentration (ng/test)
Malaria Test Line
Lassa Test Line
Ebola Test Line
Figure 6: Limit of Detection (LOD) for the Prototype Multi-Agent Antigen RDT. LFI strips were challenged with a serial dilution of either (A) Malaria (P.f.) rHRP2 antigen, (B) LASV rNP antigen, or (C) EBOV
rVP40 antigen from 2.3-300ng/test. The Multi-Agent RDT detects all three antigens in a dose-dependent manner. LOD for the Multi-Agent RDT is as follows: Malaria HRP2 is detected down to 18.8ng/test (N=5), for
Lassa NP detection down to 2.3ng/test (N=5), and Ebola VP40 detection down to 9.4ng/test (N=5). Test:Control Reflectance Ratio measured by Qiagen ESEQuant LFR.
Conclusions: We have developed and characterized a prototype Multi-Agent LFI RDT capable of detecting LASV, Malaria, and EBOV proteins in sample matrix in a specific, concentration-dependent
manner, with a LOD below the anticipated antigenemia levels present during acute illness. Importantly, this concentration dependency was discernible without the aid of any instrumentation, suggesting a
path forward for the optimization of a rapid point-of-care test that can be used in austere environments. This latest Ebola outbreak demonstrates that point-of-care testing, such as the prototypes discussed
here, is essential to distinguish between Ebola, Lassa, and Malaria during disease outbreaks and in endemic regions. This Multi-Agent RDT represents a diagnostic advancement in the detection of high
consequence pathogens present in the region of the West Africa EBOV outbreak.
w w w. v h f c . o r g
T h i s w o r k i s p a r t o f a n o n g o i n g e f f o r t b y t h e Vi r a l H e m o r r h a g i c F e v e r C o n s o r t i u m w h o s e m e m b e r s
c o n s i s t o f Tu l a n e U n i v e r s i t y, C o r g e n i x M e d i c a l C o r p . , Vy b i o n I n c . , A u t o i m m u n e Te c h n o l o g i e s I n c . ,
S c r i p p s R e s e a r c h I n s t i t u t e , H a r v a r d U n i v e r s i t y, B r o a d I n s t i t u t e , U n i v e r s i t y o f Te x a s M e d i c a l B r a n c h .