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Application of an Improved Method for the Recombinant K39 Enzyme-Linked Immunosorbent Assay To Detect Visceral Leishmaniasis Disease and Infection in Bangladesh

Application of an Improved Method for the Recombinant K39 Enzyme-Linked Immunosorbent Assay To Detect Visceral Leishmaniasis Disease and Infection in Bangladesh K. M. Kurkjian 1 , L. E. Vaz 1 , † , R. Haque 2 , C. Cetre-Sossah 1 , ‡ , S. Akhter 2 , S. Roy 2 , F. Steurer 1 , J. Amann 1 , § , M. Ali 2 , R. Chowdhury 2 , Y. Wagatsuma 2 , ¶ , J. Williamson 1 , S. Crawford 1 , R. F. Breiman 2 , ∥ , J. H. Maguire 1 , # , C. Bern 1 , and W. E. Secor 1 , * 1 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Division of Parasitic Diseases, Branch of Parasitic Diseases, Public Health Service, Department of Health and Human Services, Atlanta, Georgia 2 Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh ABSTRACT Several serology-based immunoassays are used to diagnose visceral leishmaniasis (VL), a chronic protozoan parasitic disease caused by the Leishmania donovani complex. These tests are primarily designed to diagnose the most severe clinical form of VL, known as kala-azar. However, leishmanial infection is frequently asymptomatic and may manifest only as a positive serologic response or positive leishmanin skin test. We modified a previously described enzyme-linked immunosorbent assay (ELISA) that detects patient antibodies reactive with the recombinant Leishmania protein K39 (rK39) to confirm suspected kala-azar and to detect asymptomatic infection in a community study in Bangladesh. With the inclusion of a standard curve on each ELISA plate, the rK39 ELISA was more repeatable (kappa coefficient of agreement = 0.970) and more reliable compared to the original method (kappa = 0.587, P < 0.001). The cutoff point for a positive antibody response was chosen based on the 99th percentile of the ELISA distribution for the negative-control sera. However, we found that sera from all patients with active kala-azar yielded values more than twice the magnitude of this cutoff. Using receiver-operator characteristic curves, we determined a second cutoff value predictive of kala-azar. Using these criteria, the sensitivity and specificity of the modified ELISA for kala-azar were 97.0% and 98.9%, respectively, for sera from our study population. We hypothesize that individuals with antibody levels greater than the 99th percentile of the negative controls but less than the cutoff point for kala-azar have asymptomatic leishmanial infections. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical and Vaccine Immunology American Society For Microbiology

Application of an Improved Method for the Recombinant K39 Enzyme-Linked Immunosorbent Assay To Detect Visceral Leishmaniasis Disease and Infection in Bangladesh

Abstract

Application of an Improved Method for the Recombinant K39 Enzyme-Linked Immunosorbent Assay To Detect Visceral Leishmaniasis Disease and Infection in Bangladesh K. M. Kurkjian 1 , L. E. Vaz 1 , † , R. Haque 2 , C. Cetre-Sossah 1 , ‡ , S. Akhter 2 , S. Roy 2 , F. Steurer 1 , J. Amann 1 , § , M. Ali 2 , R. Chowdhury 2 , Y. Wagatsuma 2 , ¶ , J. Williamson 1 , S. Crawford 1 , R. F. Breiman 2 , ∥ , J. H. Maguire 1 , # , C. Bern 1 , and W. E. Secor 1 , * 1 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Division of Parasitic Diseases, Branch of Parasitic Diseases, Public Health Service, Department of Health and Human Services, Atlanta, Georgia 2 Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh ABSTRACT Several serology-based immunoassays are used to diagnose visceral leishmaniasis (VL), a chronic protozoan parasitic disease caused by the Leishmania donovani complex. These tests are primarily designed to diagnose the most severe clinical form of VL, known as kala-azar. However, leishmanial infection is frequently asymptomatic and may manifest only as a positive serologic response or positive leishmanin skin test. We modified a previously described enzyme-linked immunosorbent assay (ELISA) that detects patient antibodies reactive with the recombinant Leishmania protein K39 (rK39) to confirm suspected kala-azar and to detect asymptomatic infection in a community study in Bangladesh. With the inclusion of a standard curve on each ELISA plate, the rK39 ELISA was more repeatable (kappa coefficient of agreement = 0.970) and more reliable compared to the original method (kappa = 0.587, P < 0.001). The cutoff point for a positive antibody response was chosen based on the 99th percentile of the ELISA distribution for the negative-control sera. However, we found that sera from all patients with active kala-azar yielded values more than twice the magnitude of this cutoff. Using receiver-operator characteristic curves, we determined a second cutoff value predictive of kala-azar. Using these criteria, the sensitivity and specificity of the modified ELISA for kala-azar were 97.0% and 98.9%, respectively, for sera from our study population. We hypothesize that individuals with antibody levels greater than the 99th percentile of the negative controls but less than the cutoff point for kala-azar have asymptomatic leishmanial infections.
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