Post on 05-Feb-2018
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.