Antigenemia Immunoassay Development for the Diagnosis of … · 2019. 5. 5. · • Leptospirosis - Leptospira spp. • Lyme disease - Borrelia burgdorferi • Ebola • Chikungunya

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

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

❖ 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

LFI fabrication

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

DTRA funded Pre-submission to the FDA January 2019

Test line Control line

CPS

Sample type:

BloodUrineCulture fluidBacterial colony

• 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

• AMD LFI was 99% (99/100) sensitive and 100% (308/308) specific on turbid blood culture bottles


• 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

Time to diagnosis

AMD LFI sensitivity estimation

Latex agglutination Culture

80-90%30% 98-100%Clinical sensitivity

urine

Detection of CPS in filtered melioidosis patient samples

URINE

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Quantitative antigen-capture ELISA

SERUM

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.

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

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

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

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

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

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

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

• 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

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.


• 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)

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

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 -


HC = HaemocultureTS = Throat swabP = PusUR = UrineSP = Sputum

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 -

+/-


HC = HaemocultureTS = Throat swabP = PusUR = UrineSP = Sputum

Documents

Antigenemia Immunoassay Development for the Diagnosis of … · 2019. 5. 5. · • Leptospirosis - Leptospira spp. • Lyme disease - Borrelia burgdorferi • Ebola • Chikungunya