TUBEX: High-definition Rapid Diagnostics for Typhoid and Other Diseases

LIM Pak Leong

Chairman, IgGENE

Hong Kong

pllim@iggene.com

NEGATIVE

antibody-coated

microsphere

Slide latex agglutination test

POSITIVE

antigen forms

bridge between

microspheres

clumping

rock

2 min

I.M. Pei

Lourve

Bank of China

magnetic( combinations )

multiple testscoloured( combinations )

small vol

ANTIGENdetection

magneticmagnetic

particleparticle

bacterial ȌȌȌȌ antigen

BLUEBLUE--REDRED

SUPERNATANTSUPERNATANT

TUBEX assay

Ἆ antigen

Indicator blue particlecoated with anti−Ἆ monoclonal

antibody ☺♦

No inhibitor (CONTROL)

magneticmagnetic

particleparticle

In solution

SEDIMENT BY MAGNET

CLEAR (RED) CLEAR (RED)

SUPERNATANTSUPERNATANT

Indicator blue particle

coated with anti−ȌȌȌȌmonoclonal antibody

ȌȌȌȌ antigen

☺♦

ANTIBODYdetection

patient’s anti−ȌȌȌȌ antibody

magneticmagnetic

particleparticle

BLUEBLUE--RED RED

SUPERNATANTSUPERNATANT

More antibody, more blueMore antibody, more blue

INHIBITION; Low noise

IgM>>IgG, no wash

2 min

magnet / COLOR SCALE

Applications

e.g. Enteric fever, Dengue fever … (fever)

typhoid

paratyphoid A, B, C

SALMONELLA

> 2000 serotypes based on O–antigens and H–antigens

ACUTE

infectious diseases

CHRONIC

infectious diseases

Autoimmune diseases

pathogen-specific IgM pathogen-specific IgG Autoantigen-specific IgG

typhoid paratyphoid A

S. Typhi S. Paratyphi A

O9, O12 O2, O12

Bulletin WHO May 2004 82(5)Bulletin WHO May 2004 82(5)

TYPHOIDTYPHOID affects millions in the worldaffects millions in the world

LaboratoryLaboratory diagnosis of typhoiddiagnosis of typhoid

1. Blood / stool culture

* requires many days, specialized lab & staff; 50% detection

2. Serology / immunodiagnosis

1896 Widal test Still widely used, simple, affordable:

Agglutination using whole organisms;

Not sensitive nor specific

1980’s ELISA Sensitive, objective; Long and

cumbersome; Subunit antigens

1990’s “TyphiDot”

Dipsticks

“TUBEX”

Lateral-flow tests

POC tests; Rapid, user-friendly;

Subunit antigens

DiffusionDiffusion

porepore

PORINSPORINS

(34 (34 -- 36K)36K)

CAPSULECAPSULE(Vi, M antigen)(Vi, M antigen)

LIPOPOLYSACCHARIDELIPOPOLYSACCHARIDE

((LPS; OLPS; O--antigen)antigen)

OUTER MEMBRANEOUTER MEMBRANE

PERIPLASMICPERIPLASMIC

SPACESPACE

CYTOPLASMICCYTOPLASMIC

MEMBRANEMEMBRANE

PEPTIDOGLYCANPEPTIDOGLYCAN

75 A75 A

OUTSIDE

CYTOPLASM

FLAGELLUM

(H-antigen)

CYTOPLASM

SALMONELLAcell wall

O‒polysaccharide repeats core sugars lipid A whole LPS

galactose

mannose

rhamnosetyvelose

O–12

O–9

S. Typhi

paratose

O–2

S. Paratyphi A

ELISA

Lateral–flow tests

antianti−−O9 indicatorO9 indicator

magnetic

bead

O12a

O12b

O12cO9

S. Typhi LPS

antianti−−O12b indicatorO12b indicator

magneticbead

O9

S. Typhi LPS

High definition

O9TF

O12TP

4 7 8

Magnetic particlescoated with O9 LPS

Indicator particles coated with anti-:

Typhoid sera

Indicatorparticles

1 2 3 4 5 6

anti-O12TUBEX TP

10 10 10 10 10 10

anti-O9TUBEX TF

Patient

0 0 ND 0 0 2

Paratyphoid A sera

O12 + O9

Culture-confirmed typhoid patientsexamined by various ELISA and TUBEX tests

Yan M. et.al PLOS One Sep 2011

ELISA IgM ELISA IgG TUBEX

SerumDays

fever

S. Typhi LPS

P-LPS M-LPS

S. Typhi LPS

P-LPS M-LPS TF bTF PA bPA TP 12P

Group A1 (early stage)

T33a 10 M H H L H M 7 6 1 1 7 3

T35a 9 H H H H H H 5 ND 6 0 7 6

T39a 8 H M L H H H 3 ND 6 0 8 9

T45a 8 H M M H H H 6 ND 8 0 7 7

T53a 11 M M L H H H 2 ND 2 0 6 4

T55a 10 H H H H H H 6 ND 9 0 8 8

T59a 10 H H H L M M 5 ND 3 0 5 5

T67a 8 H M L H H H 5 ND 4 0 4 5

T69a 8 M M M H H H 4 3 7 0 4 6

T73a 8 M L - H H H 3 ND 7 0 6 5

H=high L=lowSensitivity 90.0%

8/10

80.0% 100%10/10

100%M=med –, neg

TUBEX scores: 0 (neg) TUBEX scores: 0 (neg) -- 1010

Culture-confirmed typhoid patientsexamined by various ELISA and TUBEX tests

ELISA IgM ELISA IgG TUBEX

SerumDays of

fever

S. Typhi

LPS

P-LPS M-LPS

S. Typhi

LPS

P-LPS M-LPS TF bTF PA bPA TP 12P

Group B

T31a 8 H H M - - - 4 ND 1 1 4 4

T31b 23 M H M - L - 5 ND 0 0 5 3

T41a 24 H H H - - - 6 ND 0 0 7 6

T57a 8 H M L - M M 3 ND 0 0 5 1

T63a 8 H H M - - L 6 ND 1 0 7 2

T63b 22 M H M - L L 5 ND 0 0 7 4

T65a 10 H H M L - - 3 ND 1 0 4 4

Sensitivity 100%0/7

0%

100%

5/7

71.4%

thymus‒independentresponse

ELISA IgM ELISA IgG TUBEX

SerumDays

fever

S. Typhi

LPS

P-LPS M-LPS

S. Typhi

LPS

P-LPS M-LPS TF bTF PA bPA TP 12P

Group C

T41b 38 - - L - - - 1 ND 0 0 1 0

T43a 9 - - - - - - 2 ND 1 0 0 0

T43b 21 - - - - - - 1 ND 0 0 0 0

T49a 7 - - - - - - 1 ND 1 0 2 0

T49b 23 - - - - - - 2 ND 1 0 3 0

T51a 8 - - - - - - 1 ND 0 0 2 0

T51b 21 - - - - - - 0 ND 0 0 1 0

Sensitivity 0%0/7

0% 14.3%0/7

0%

Culture-confirmed typhoid patients

examined by various ELISA and TUBEX tests

* Non-respondergenetics, immuno-suppression,

antibiotics

Sensitivity ∞∞ Specimen

TUBEX = ELISA*

*

Rahman M. et.alDiagn. Microbiol. Infect. Dis. Jul. 2007

Also with CULTURE

? FALSE / TRUE POSITIVES real signals

Yan M. et.al PLOS One Sep 2011

IgM

ELISA

IgGELISA

S. Typhi

LPS

TUBEX correlates well with IgM ELISA

OD

450 n

m

r2 = 0.07 ; P = 0.0729

10864200

4

2

3

1

TUBEX TF scoreTUBEX TF score

OD

450 n

m

r2 = 0.59 ; P < 0.0001

0

4

2

3

1

1086420

TUBEX TF score

Rahman M. et.al Diagn. Microbiol. Infect. Dis. Jul. 2007 H=high levels

Culture negativeCulture negative, , TUBEX positive, TUBEX positive, febrilefebrile individualsindividuals

Widal test ++ ++ ++ ++ ++ ++

Dengue test –– –– –– –– –– ––

IgM + IgG ELISA HH HH HH HH HH ––

IgM ELISA LL HH LL HH HH ––

IgG ELISA HH HH HH HH HH ––

TUBEX® score 8 8 7 10 7 6

If asymptomaticbut TUBEX-positive:

subclinical infection due to neutralizing antibodies

carrier state

?

?

TUBEX simplifies culture: : Antigen detection

bloodblood

broth

culture

agarculture

biochemicalidentification

antigenicidentification

2 2 -- 4 days4 days

100% detection of S.Typhi and S.Paratyphi A

bacteria by TUBEX TP (18 and 10 isolates, resp.)

Nugraha et al PLOS ONE Nov 2012

Typhoid and paratyphoid A fever still threaten the world.

TUBEX is a simple low-noise high-definition POC test.

Performance of POC tests should be judged by conventional

objective immunoassays, not just culture.

SUMMARY

How sensitive? How specific?

1.

2.

3.