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An improve dot enzyme immunoassay for serodiagnosis of melioidosis
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14th International Congress on Infectious Diseases (ICID) Abstracts
75.012 An improve dot enzyme immunoassay for serodiagnosis of melioidosis S.A.K. Zainoodin 1,∗ , I. Asma 2 1
Advance Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia 2 Deputy Vice Chancellor, Universiti Sains MAaaysia, Georgetown, Penang, Malaysia Background: Meliodosis in an infectious disease affecting multi-organ system of man and animals caused by a bacterium, Burkholderia pseudomallei. The disease is potentially fatal and early detection is crucial for institution of life saving antimicrobial therapy. Culture isolation remains as a mainstay for definitive laboratory diagnosis of melioidosis. But it is less sensitive, laborious and timeconsuming task. In this report, an improved dot EIA test (patent pending) with user friendly assay protocol for serodiagnosis of melioidosis is described. Methods: Two optimised concentrations of antigens were dotted onto a nitro-cellulose membrane (Microfiltration System, Ca, USA, 0.45uM) divided into 9 mm × 4 mm. The membranes were blocked for 30 minutes in tris-buffered saline (TBS) wiht 5% nonfat skim milk. The membranes were rinsed and cut according to the divided size into test strips and placed in 48 well flat tissue culture plates (Costar 3548, UK.) The test strips were then incubated with patient serum at 1:100 in TBS for 1 hour at room temperature. After incubation, the test strips were washed with TBS for three times and each washing was done for 5 minutes. Subsequently, the diluted alkaline phosphatase conjugated goat anti-human immunoglobulin IgM, IgG and IgA isotypes were added into the respective wells and incubated for 1 hour at room temperature. After incubation the strips were washed three times in TBS as described above. Chromogenic substrate was used in the colour development reaction. Aftwer after 15 minutes the strips washing with distilled water to stop coloue development. The positive and negative results were determined by visual comparison of the dot intensity for each test with the positive cut off control. Results: The sensitivity and the specificity of the assay was 95% and 92%, respectively for the detection of IgG, IgM and IgA antibody isotypes. However, detection of IgG alone showed sensitivity value of 89.2%. The sensitivity value for the detection of IgM alone and IgA alone were 47.7% and 60% respectively.
Dot EIA test results Conclusion: The results showed that combined detection of all three antibody isotypes are required to increase the sensitivity of the assay. In addition, the result from the propective study showed that the test was very useful for early and accurate diagnosis melioidosis in clinical cases. doi:10.1016/j.ijid.2010.02.412 75.013 Diagnostic value of Elisa serological test using synthetic peptides of Mycobacteriun tuberculosis antigens in childhood tuberculosis D. Lopez 1,∗ , F. Giamprieto 2 , J. de Waard 2 , M.A. Patarroyo 3 , C. Fernandez de Larrea 4 , Z. Araujo 5 1
JM de Los Rios Hospital, Caracas, Distrito Capital, Venezuela 2 Instituto de Biomedicina, Caracas, Distrito Capital, Venezuela 3 Fundación Instituto de Inmunología de Colombia, Bogotá, Colombia 4 Servei d’Hematologia. Hospital Clínic de Barcelona, Barcelona, Spain 5 Instituto de Biomedicina, Universidad Central de Venezuela, 4043, D.C., Venezuela Background: One study was conducted to evaluate the efficacy of ELISA serological test for the detection of IgG antibodies against ESAT-6 synthetic peptides and the recombinant ESAT-6 antigen (rESAT-6). Methods: The study was carried out on children with pulmonary tuberculosis and healthy controls (13 cases and 63 controls). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test (epidemiological link with confirmed case), chest radiograph and therapeutic positive test. Relevant body fluids (sputum and gastric fluid) were subjected to biochemical test adenosine deaminase (ADA) and bacteriological tests (Lowëstein-Jensen (LJ) and Ziehl Neelson (ZN)) and PCR. Sera samples were analyzed for antibodies against 5 ESAT-6 synthetic peptides (12033, 12034, 12035, 12036 and 12037) and rESAT-6. The Utility of these methods was evaluated using receiver operating characteristic curve (ROC) analysis.
14th International Congress on Infectious Diseases (ICID) Abstracts
75.012 An improve dot enzyme immunoassay for serodiagnosis of melioidosis S.A.K. Zainoodin 1,∗ , I. Asma 2 1
Advance Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia 2 Deputy Vice Chancellor, Universiti Sains MAaaysia, Georgetown, Penang, Malaysia Background: Meliodosis in an infectious disease affecting multi-organ system of man and animals caused by a bacterium, Burkholderia pseudomallei. The disease is potentially fatal and early detection is crucial for institution of life saving antimicrobial therapy. Culture isolation remains as a mainstay for definitive laboratory diagnosis of melioidosis. But it is less sensitive, laborious and timeconsuming task. In this report, an improved dot EIA test (patent pending) with user friendly assay protocol for serodiagnosis of melioidosis is described. Methods: Two optimised concentrations of antigens were dotted onto a nitro-cellulose membrane (Microfiltration System, Ca, USA, 0.45uM) divided into 9 mm × 4 mm. The membranes were blocked for 30 minutes in tris-buffered saline (TBS) wiht 5% nonfat skim milk. The membranes were rinsed and cut according to the divided size into test strips and placed in 48 well flat tissue culture plates (Costar 3548, UK.) The test strips were then incubated with patient serum at 1:100 in TBS for 1 hour at room temperature. After incubation, the test strips were washed with TBS for three times and each washing was done for 5 minutes. Subsequently, the diluted alkaline phosphatase conjugated goat anti-human immunoglobulin IgM, IgG and IgA isotypes were added into the respective wells and incubated for 1 hour at room temperature. After incubation the strips were washed three times in TBS as described above. Chromogenic substrate was used in the colour development reaction. Aftwer after 15 minutes the strips washing with distilled water to stop coloue development. The positive and negative results were determined by visual comparison of the dot intensity for each test with the positive cut off control. Results: The sensitivity and the specificity of the assay was 95% and 92%, respectively for the detection of IgG, IgM and IgA antibody isotypes. However, detection of IgG alone showed sensitivity value of 89.2%. The sensitivity value for the detection of IgM alone and IgA alone were 47.7% and 60% respectively.
Dot EIA test results Conclusion: The results showed that combined detection of all three antibody isotypes are required to increase the sensitivity of the assay. In addition, the result from the propective study showed that the test was very useful for early and accurate diagnosis melioidosis in clinical cases. doi:10.1016/j.ijid.2010.02.412 75.013 Diagnostic value of Elisa serological test using synthetic peptides of Mycobacteriun tuberculosis antigens in childhood tuberculosis D. Lopez 1,∗ , F. Giamprieto 2 , J. de Waard 2 , M.A. Patarroyo 3 , C. Fernandez de Larrea 4 , Z. Araujo 5 1
JM de Los Rios Hospital, Caracas, Distrito Capital, Venezuela 2 Instituto de Biomedicina, Caracas, Distrito Capital, Venezuela 3 Fundación Instituto de Inmunología de Colombia, Bogotá, Colombia 4 Servei d’Hematologia. Hospital Clínic de Barcelona, Barcelona, Spain 5 Instituto de Biomedicina, Universidad Central de Venezuela, 4043, D.C., Venezuela Background: One study was conducted to evaluate the efficacy of ELISA serological test for the detection of IgG antibodies against ESAT-6 synthetic peptides and the recombinant ESAT-6 antigen (rESAT-6). Methods: The study was carried out on children with pulmonary tuberculosis and healthy controls (13 cases and 63 controls). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test (epidemiological link with confirmed case), chest radiograph and therapeutic positive test. Relevant body fluids (sputum and gastric fluid) were subjected to biochemical test adenosine deaminase (ADA) and bacteriological tests (Lowëstein-Jensen (LJ) and Ziehl Neelson (ZN)) and PCR. Sera samples were analyzed for antibodies against 5 ESAT-6 synthetic peptides (12033, 12034, 12035, 12036 and 12037) and rESAT-6. The Utility of these methods was evaluated using receiver operating characteristic curve (ROC) analysis.