TUBEX: High-definition Rapid Diagnostics for Typhoid and Other Diseases. LIM Pak Leong Chairman, IgGENE Hong Kong
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1 TUBEX: High-definition Rapid Diagnostics for Typhoid and Other Diseases LIM Pak Leong Chairman, IgGENE Hong Kong
2 Slide latex agglutination test rock 2 min NEGATIVE POSITIVE antibody-coated microsphere antigen forms bridge between microspheres clumping
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5 I.M. Pei Lourve Bank of China
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7 small vol coloured ( combinations ) multiple tests magnetic ( combinations )
8 TUBEX assay No inhibitor (CONTROL) CLEAR (RED) SUPERNATANT Indicator blue particle coated with withanti Ἆ anti Ȍmonoclonal antibody monoclonal antibody BLUE-RED SUPERNATANT ANTIBODY detection More antibody, more blue BLUE-RED SUPERNATANT ANTIGEN detection magnetic particle ἎȌ antigen patient s anti Ȍ antibody magnetic particle bacterial Ȍ antigen magnetic particle In solution SEDIMENT BY MAGNET INHIBITION; Low noise IgM>>IgG, no wash
9 magnet / COLOR SCALE 2 min
10 Applications ACUTE infectious diseases CHRONIC infectious diseases Autoimmune diseases pathogen-specific IgM pathogen-specific IgG Autoantigen-specific IgG e.g. Enteric fever, Dengue fever (fever) typhoid paratyphoid A, B, C SALMONELLA > 2000 serotypes based on O antigens and H antigens typhoid paratyphoid A S. Typhi S. Paratyphi A O9, O12 O2, O12
11 TYPHOID affects millions in the world Bulletin WHO May (5)
12 Laboratory diagnosis 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
13 SALMONELLA cell wall FLAGELLUM (H-antigen) Diffusion pore OUTSIDE PORINS (34-36K) CAPSULE (Vi, M antigen) LIPOPOLYSACCHARIDE (LPS; O-antigen) O OUTER MEMBRANE PEPTIDOGLYCAN PERIPLASMIC SPACE CYTOPLASMIC MEMBRANE 75 A CYTOPLASM CYTOPLASM
14 O polysaccharide repeats core sugars lipid A whole LPS ELISA Lateral flow tests O 12 S. Typhi galactose O 9 tyvelose mannose S. Paratyphi A O 2 rhamnose paratose
15 anti O9 indicator anti O12b indicator O12b O9 O12a O12c O9 S. Typhi LPS magnetic bead S. Typhi LPS magnetic bead High definition
16 Typhoid sera Magnetic particles coated with O9 LPS Indicator particles coated with anti-: O9 TF O12 TP O12 + O9 Paratyphoid A sera Indicator particles Patient anti-o12 TUBEX TP anti-o9 TUBEX TF 0 0 ND 0 0 2
17 Culture-confirmed typhoid patients examined by various ELISA and TUBEX tests ELISA IgM ELISA IgG TUBEX Serum Days 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) TUBEX scores: 0 (neg) - 10 T33a 10 M H H L H M T35a 9 H H H H H H 5 ND T39a 8 H M L H H H 3 ND T45a 8 H M M H H H 6 ND T53a 11 M M L H H H 2 ND T55a 10 H H H H H H 6 ND T59a 10 H H H L M M 5 ND T67a 8 H M L H H H 5 ND T69a 8 M M M H H H T73a 8 M L - H H H 3 ND H=high L=low M=med, neg Sensitivity 90.0% 8/ % 100% 10/10 100% Yan M. et.al PLOS One Sep 2011
18 Culture-confirmed typhoid patients examined by various ELISA and TUBEX tests ELISA IgM ELISA IgG TUBEX Serum Days 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 ND T31b 23 M H M - L - 5 ND T41a 24 H H H ND T57a 8 H M L - M M 3 ND T63a 8 H H M - - L 6 ND T63b 22 M H M - L L 5 ND T65a 10 H H M L ND /7 100 Sensitivity 100% 0% % 5/7 71.4% thymus independent response
19 Culture-confirmed typhoid patients examined by various ELISA and TUBEX tests ELISA IgM ELISA IgG TUBEX Serum Days fever S. Typhi LPS P-LPS M-LPS S. Typhi LPS P-LPS M-LPS TF btf PA bpa TP 12P Group C T41b L ND T43a ND T43b ND T49a ND T49b ND T51a ND T51b ND Sensitivity 0% 0/7 0% 14.3% 0/7 0% * Non-responder genetics, immuno-suppression, antibiotics * * Sensitivity Specimen TUBEX = ELISA
20 TUBEX correlates well with IgM ELISA 4 r 2 = 0.59 ; P < IgM ELISA S. Typhi LPS IgG ELISA OD 450 nm OD 450 nm TUBEX TF score r 2 = 0.07 ; P = Also with CULTURE Rahman M. et.al Diagn. Microbiol. Infect. Dis. Jul. 2007? FALSE / TRUE POSITIVES real signals TUBEX TF score 10 Yan M. et.al PLOS One Sep 2011
21 If asymptomatic but TUBEX-positive:? subclinical infection due to neutralizing antibodies Culture negative, TUBEX positive, febrile individuals? carrier state Widal test Dengue test IgM + IgG ELISA H H H H H IgM ELISA L H L H H IgG ELISA H H H H H TUBEX score Rahman M. et.al Diagn. Microbiol. Infect. Dis. Jul H=high levels
22 TUBEX simplifies culture: Antigen detection blood 2-4 days biochemical identification antigenic identification broth culture agar culture 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
23 SUMMARY 1. Typhoid and paratyphoid A fever still threaten the world. 2. TUBEX is a simple low-noise high-definition POC test. 3. How sensitive? How specific? Performance of POC tests should be judged by conventional objective immunoassays, not just culture.
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