Antigenemia Immunoassay Development for the Diagnosis of … · 2019. 5. 5. · • Leptospirosis -...
Transcript of Antigenemia Immunoassay Development for the Diagnosis of … · 2019. 5. 5. · • Leptospirosis -...
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David AuCoin, Ph.D.Associate Professor & Chair
Department of Microbiology and ImmunologyUniversity of Nevada School of Medicine
Lateral flow immunoassay for the diagnosis of melioidosis
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I currently, and have in the past, collaborated with InBiosInternational, Inc. (Seattle WA). My university licenses technologiesto InBios that are produced by my laboratory.
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Disclosure statement
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❖ Biomarker discovery❖ Hybridoma facility❖ mAb analysis ❖ BSL3/ABSL3 ❖ Lateral flow fabrication
❖ University startup❖ On campus❖ STTR/SBIR funding
• Melioidosis - Burkholderia pseudomallei• Anthrax - Bacillus anthracis• Tularemia - Francisella tularensis• Plague - Yersinia pestis• Whooping cough - Bordetella pertussis• Leptospirosis - Leptospira spp.• Lyme disease - Borrelia burgdorferi
• Ebola • Chikungunya virus• Lassa Fever Virus• Hantavirus • Hepatitis B• VEEV
Research areas
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LFI fabrication
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Melioidosis
• Caused by Burkholderia pseudomallei
• Gram-negative, soil dwelling pathogen endemic to SE Asia and northern Australia
• Mortality rates remain high in these regions, patients usually succumb to sepsis
• Resistant to commonly prescribed antibiotics
• Currently there is no vaccine available
• Endemic areas are expanding
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Diagnosis of melioidosis- “gold standard”
5 - 7 days!
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Median count of B. pseudomallei in patient samples
• Blood: 1.1 CFU/mL
• 203/414 – 49% positive
• Urine: 1.5 x 104 CFU/mL
• 56/268 – 21% positive
• Sputum: 1.1 x 105 CFU/mL
• 94/120 – 78% positive
• Pus: 1.1 x 107 CFU/mL
• 23/28 – 82% positive
Am J Trop Med Hyg. 2007 Nov;77(5):812-3.
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CPS – capsular polysaccharide
Monoclonal antibody (mAb)DETECTION
DETECTION
DETECTION
DETECTION
DETECTION
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DTRA funded Pre-submission to the FDA January 2019
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Test line Control line
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CPS
Sample type:
BloodUrineCulture fluidBacterial colony
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• 814 cases since 1999 at LOMWRU
• ~100 cases/year
• mortality 40-50%
• 412 blood cultures tested with AMD LFI
• Compared to latex agglutination and IFA
ຂ"ຂອບໃຈທ(ານ!!+ າຍ+ າຍ*
Evaluation of a rapid diagnostic test for the detection of Burkholderia pseudomallei in the Lao People’s Democratic Republic
Kate L Woods, Latsaniphone Boutthasavong, Caoimhe NicFhogartaigh, Sue J Lee, ViengmonDavong, David P AuCoin, David AB Dance
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• AMD LFI was 99% (99/100) sensitive and 100% (308/308) specific on turbid blood culture bottles
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Evaluation of a rapid diagnostic test for the detection of Burkholderia pseudomallei in the Lao People’s Democratic Republic
• Urine AMD had a positive predictive value of 94% (32/34) for diagnosing melioidosis in this cohort
• AMD sensitivity on stored sera from melioidosis cases during this study, was 13.9% (5/36) when compared to blood culture samples taken on the same day
• In conclusion, the AMD is an excellent tool for rapid diagnosis of melioidosis from turbid blood cultures, and maintains specificity across all sample types
• It is a promising tool for urinary antigen detection, which could revolutionise diagnosis of melioidosis in resource-limited settings
• Further work is required to improve sensitivity on non-blood culture samples
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Time to diagnosis
AMD LFI sensitivity estimation
Latex agglutination Culture
80-90%30% 98-100%Clinical sensitivity
urine
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Detection of CPS in filtered melioidosis patient samples
URINE
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Quantitative antigen-capture ELISA
SERUM
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Intravenous tail injection:
4 μg, 20 μg, and 100 μg of
purified CPS
At 30 min, 2 h, 4 h, 8 h, 12 h, 1 days, 2 days, 4 days and 8 days
Serum Urine Lungs Kidneys Liver Spleen
Determine CPS concentration by antigen-capture ELISA
Organ distributionExcretionModel fitting
Clearance rate
CPS clearance study
Nualnoi et al. PLoS NTD. 2016.
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CPS clearance
CPS is cleared rapidly from serum with a short
half-life of 2.9 – 4.4 hours
Nualnoi et al. PLoS NTD. 2016.
* Not detected in major organs
CP
S (μ
g/m
l)
CP
S (μ
g/m
l)
Serum Urine
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Detection of CPS in melioidosis patient urine CPS does not appear to be degraded
Western blot probed with anti-capsule mAb
• Filtered melioidosis patient
urine samples
• No bacteria in sample
• CPS high molecular weight
antigen
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Urine samples - Laos
• Received 42 urine samples from Lao-Oxford-Manhosot Hospital-Wellcome Trust
Research Unit (LOMWRU) courtesy of Dr. David Dance
• Most were melioidosis positive patients - samples blinded
• Samples were 0.2 µm sterile filtered in a biosafety level 3 laboratory
• Verified for sterility using a validated procedure and brought to a biosafety
laboratory 2 for further testing
• LFI read by 3 blinded readers
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LFI results – Laos urine samples
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Quantitative ELISA – CPS concentration
• Melioidosis culture positive patient samples were tested for the presence of CPS
• Quantitative antigen-capture ELISA (analytical sensitivity – LOD is ~7 pg/ml)
Patient Identifier
urine CPS (ng/ml)
Vol (ml)
905* 1247 3
850-1* 535.1 0
919-1* 517.7 0
919-2* 487 3
878-2* 336.3 3
878-1* 178.1 3
861 64.5 0
871 41.7 0
893* 21.5 11
891-1* 14.6 0
891-2* 5.53 0
914-2* 5.11 0
882 3.13 1
884 0.814 1
Patient Identifier
CPS (ng/ml)
Vol (ml)
914-1* 0.522 3
909-1 0.195 2.7
909-2 0.0918 1
885 0.0732 2
No MM 0.062 3
906 0.0589 3
842-2 0.0362 2
904 0.0349 17
859 0.0197 0
842-1 0.017 3
832 0 4
834 0 4
838* 0 18
844 0 3
* Urine culture positive Green: LFI positive Black: LFI negative
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LFI negative urine samples
ELISA borderline
Quantitative CPS ELISA
5X concentrated
ELIS
A s
ign
alEL
ISA
sig
nal
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Serum or urine sample?
• Melioidosis culture positive patient samples were tested for the presence of CPS
Patient Identifier
Urine CPS (ng/ml)
Serum CPS (ng/ml)
905* 1247 210
850-1* 535 0.17
919-1* 5170.28
919-2* 487
878-2* 336 ND
878-1* 178 ND
861 64.5 0.51
871 41.7 ND
893* 21.5 11
891-1* 14.60.077
891-2* 5.53
914-2* 5.11 0.066
882 3.13 0.30
884 0.814 0.66
Patient Identifier
Urine CPS(ng/ml)
Serum CPS (ng/ml)
914-1* 0.52 0.066
909-1 0.20 none
909-2 0.0912 none
885 0.073 ND
No MM 0.062 0.02
906 0.059 none
842-2 0.036 ND
904 0.035 0.017
859 0.020 0.020
842-1 0.017 ND
832 ND ND
834 ND ND
838* ND 0.033
844 ND ND
* Urine culture positive Green: LFI positive Black: LFI negative
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mAb-magnetic particle (MAG) LFI
• Add particles to 1 – 5 ml urine sample
• Incubate for 5 minutes
• Isolate particles with magnet
• Apply magnetic particles to LFI
• Boost in sensitivity vs. low volume/ no
mag enrichment
Magnetic particle200nm
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mAb-magnetic particle LFI
• 1 ml urine spiked with 40 pg/ml CPS
• Add magnetic particles
• Incubate for 5 minutes
• Isolate particles only with magnet
• Disassociate CPS from mAb – magnet
• Add directly to AMD LFI
• Boost in sensitivity vs. standard volume
Sample volume
100 μl1 ml
40 pg/ml 40 pg/ml
Spiked concentration
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• Suspected melioidosis: diabetics with sepsis or fever; prostate abscess; or deep abscess in lung, liver or spleen
• Should be useful to directly test samples from suspected melioidosis cases so recommended antibiotics can be administered sooner
• AMD LFI sensitivity when testing blood directly is low (20-40%)
• However, higher sensitivity with with urine and pus when testing samples from suspected melioidosis cases
• Following microbiological culture B. pseudomallei may be dismissed as a culture contaminant
• Can be misidentified as Pseudomonas spp. or other organisms by API 20NE and automated bacterial identification systems
• AMD LFI or the latex assay should be used for testing all Gram-negative, oxidase positive bacilli that are isolated from blood culture and that cannot be simply identified as Pseudomonas aeruginosa
Melioidosis diagnostic summary
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AuCoin Laboratoryo Haley Kinneyo Teerapat Nualnoio Kate Pflughoefto Marcie Hollingswortho Michael Dillono Sujata Pandito Dana Reedo Derrick Huao Emily Hannaho Jacob Sorenson
University of Nevadao Paul Bretto Mary Burtnick
o Chad Roy
o Jasmine Ozsurekci
o Bart Currieo Derek Serovicho Mark Mayoo Vanessa Theobald
o Direk Limmathurotsakulo Narisara Chantratitao Gumphol Wongsuvan
o Paul Keimo Jason Sahl
o Frederic Zenhauserno Jian Guo Peng Chen
o Raymond Houghtono Syamal Raychaudhurio Jean Cheno Aarthy Vallur
o David Danceo Kate Woodso Caoimhe NicFhogartaigho Latsaniphone Boutthasavong
Public Health Agency of Canada
o Xianggo Qiu, Ph.D.
o Shihua He, Ph.D.
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Evaluation of a rapid diagnostic test for the detection of Burkholderia pseudomallei in the Lao People’s Democratic Republic
• 241 urine samples
• 15/241 urine samples were B. pseudomallei culture positive
• 13/15 of which were LFI positive
• 21/226 urine culture negative samples were LFI positive
• 19/21 melioidosis confirmed by culture from another site/sample
• suggesting that these were not “false positive” LFI results
• The positive predictive value of LFI on urine for correctly diagnosing melioidosis
in this cohort was therefore 94.1% (32/34; 79.7 – 98.5%) with a disease
prevalence of 35.7% (86/241)
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MM
Urine CPS (ng/mL)
Urine LFISerum CPS
(ng/mL)Serum
LFI
832 0 - 0 -
834 0 - 0 +/-
838 0 - 0.0327 +/-
842-1 0.017 -0 -
842-2 0.0362 -
844 0 - 0 -
850-1 535.1 +0.0171 -
850-2 1.94 +
857 0 - 0 -
859 0.0197 - 0.0198 -
861 64.5 + 0.0514 -
871 41.7 + 0 -
875 0 - 0 +/-
876 0 - 0 -
878-1 178.1 +0 -
878-2 336.3 +
879 0 - 0 -
881 0 - 0 -
882 3.13 + 0.301 +
883 0 - 0.0237 -
884 0.814 + 0.662 +/-
MM
Urine CPS (ng/mL)
Urine LFISerum CPS
(ng/mL)Serum LFI
885 0.0732 + 0 -
889 0 - N/A N/A
890 0 - 0.0184 -
891-1 14.6 +0.0768 +/-
891-2 5.53 +
893 21.5 + 0 -
900 0 - N/A N/A
901 0 - 0.021 +/-
903 0 - 0 -
904 0.0349 - 0.01744 -
905 1247 + 210 +
906 0.0589 - N/A N/A
909-1 0.195 -N/A N/A
909-2 0.0918 -
912 0 - 0 -
914-1 0.522 +0.0661 -
914-2 5.11 +
916 0 - 0 -
919-1 517.7 +0.028 -
919-2 487 +
No MM 0.062 - 0.0293 -
Blue: positive in urine (ELISA and LFI) not serum - 3 blinded readersGreen: positive in both urine and serum (ELISA and LFI)- 3 blinded readersPink: positive in urine (ELISA and LFI) but not necessarily in serum
CPS Quantitation/ Analysis in Laos Human Patient urine and serum samples
cc
**
**
**
*
*
c
***
c*c
c
**
**
c: urine concentrated*: urine neat
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MM
Bp culture
positive LFI result LOMWRU LFI urine culure
Urine CPS
(ng/mL)Urine LFI
Serum CPS
(ng/mL)Serum LFI
832 HC,TS - Neg NG 0 - 0 -
834 HC - Neg NG 0 - 0 +/-
838 UR - Neg BPs 0 - 0.0327 +/-
842-1 UR,TS - Pos NG 0.017 -
842-2 UR,TS - Neg NG 0.0362 -
844 HC - Neg NG 0 - 0 -
850-1 HC + Pos BPs 535.1 +
850-2 HC + ? ? 1.94 +
857 HC, P - Neg NG 0 - 0 -
859 HC - Neg NG 0.0197 - 0.0198 -
861 HC, TS,P + Pos NG 64.5 + 0.0514 -
871 HC, TS + Pos NG 41.7 + 0 -
875 P - Neg NG 0 - 0 +/-
876 TS,P - Pos NG 0 - 0 -
878-1 HC, UR + Pos BPs 178.1 +
878-2 HC, UR + Pos BPs 336.3 +
879 HC, TS, P - Neg NG 0 - 0 -
881 HC, TS - Neg NG 0 - 0 -
882 TS,P + Pos NG 3.13 + 0.301 +
883 P - Neg NG 0 - 0.0237 -
884 HC, TS, UR + Pos NG 0.814 + 0.662 +/-
0 -
0.0171 -
0 -
Blue: positive in urine (ELISA and LFI) not serum - 3 blinded readersGreen: positive in both urine and serum (ELISA and LFI)- 3 blinded readersPink: positive in urine (ELISA and LFI) but not necessarily in serum
HC = HaemocultureTS = Throat swabP = PusUR = UrineSP = Sputum
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MM
Bp culture
positive LFI result LOMWRU LFI urine culure
Urine CPS
(ng/mL)Urine LFI
Serum CPS
(ng/mL)Serum LFI
885 P,SP,TS + Pos NG 0.0732 + 0 -
889 HC, P, UR - Neg NG 0 - N/A N/A
890 HC, TS,P - Neg NG 0 - 0.0184 -
891-1 UR + Pos BPs 14.6 +
891-2 UR + Pos BPs 5.53 +
893 HC, UR + Pos BPs 21.5 + 0 -
900 P,SP,TS - Neg NG 0 - N/A N/A
901 P - Neg NG 0 - 0.021 +/-
903 P - Neg NG 0 - 0 -
904 HC,TS - Neg NG 0.0349 - 0.01744 -
905 HC + Pos BPs 1247 + 210 +
906 P,SP,TS - Neg NG 0.0589 - N/A N/A
909-1 HC,TS - Pos NG 0.195 -
909-2 HC,TS - Neg NG 0.0918 -
912 TS - Neg NG 0 - 0 -
914-1 HC, TS,P + Pos BPS 0.522 +
914-2 HC, TS,P + Pos BPS 5.11 +
916 P - Neg NG 0 - 0 -
919-1 HC,P, UR + ND BPs 517.7 +
919-2 HC, P, UR + Pos BPs 487 +
No MM N/A - Pos NG 0.062 - 0.0293 -
0.0768
N/A N/A
0.0661 -
0.028 -
+/-
Blue: positive in urine (ELISA and LFI) not serum - 3 blinded readersGreen: positive in both urine and serum (ELISA and LFI)- 3 blinded readersPink: positive in urine (ELISA and LFI) but not necessarily in serum
HC = HaemocultureTS = Throat swabP = PusUR = UrineSP = Sputum