Evaluation of Three Immunoglobulin M Antibody Capture Enzyme-linked Immunosorbent Assays for Diagnosis of Japanese Encephalitis Julie A. Jacobson,* Susan L. Hills, Jennifer L. Winkler, Mammen Mammen, Butsaya Thaisomboonsuk, Anthony A. Marfin, and Robert V. Gibbons Japanese Encephalitis Project, PATH, Seattle, Washington; Department of Virology, United States Army Medical Component -Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Division of Global Migration and Quarantine, Centers for Disease Control and Prevention, Seattle, Washington Abstract. Japanese encephalitis (JE) virus is a major cause of neurologic infection in Asia, but surveillance has been limited. Three JE immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay kits have recently been developed. The aim of this study was to evaluate their sensitivity, specificity, and usability using 360 acute-phase serum samples containing JE, dengue, or neither IgM antibody. The kits, manufactured by Panbio Limited, Inbios International, Inc., and XCyton Diagnostics Ltd, had high sensitivities of 89.3%, 99.2%, and 96.7%, respectively. The specificities were 99.2%, 56.1%, and 65.3%, respectively. When dengue IgM-positive samples were excluded, the kits had specificities of 98.4%, 96.1%, and 96.1%, respectively. The Panbio kit includes both JE and dengue antigens and appears to have an advantage in settings where dengue virus co-circulates, although further assessments in clinical settings are needed. This information is helpful in considering options for strengthening the laboratory component of JE surveillance. INTRODUCTION Japanese encephalitis (JE) virus is the leading cause of viral neurologic disease and disability in Asia. 1,2 Disease manifes- tations primarily occur in children, leading to either death or long-term neurologic disability in ≈ 70% of those with clinical illness. 3–6 In many countries known to be at risk for JE trans- mission, the burden of disease is not known. The clinical pre- sentation of JE cannot be accurately differentiated from other etiologies of meningoencephalitis; therefore, laboratory diag- nostic confirmation is required for definitive diagnosis. Because affordable JE diagnostics are generally not avail- able, cases of encephalitis and other severe neurologic ill- nesses that are truly due to JE virus may not be recognized or reported to public health officials. Limited awareness of dis- ease burden means less chance to control this vaccine- preventable disease. To address this situation, the World Health Organization (WHO) has planned initiatives to sup- port and build laboratory capacity to strengthen JE surveil- lance. 7 Identification of one or more reliable, simple, and available diagnostic tests would facilitate this effort. Although neutralizing antibody assays of paired serum samples are considered the gold standard for JE diagnostics, the capacity to perform such assays is rarely present in many affected nations. Instead, the simpler immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC ELISA) for cerebrospinal fluid (CSF) and serum has become the practical standard for the diagnosis of JE. 2,8–10 “In-house” JE ELISA diagnostics are available at many re- search facilities; however, the performance of in-house tests in less sophisticated field laboratories has generally proven unsatisfactory (Nisalak A, unpublished data). The ELISA format for confirmatory diagnosis of JE infec- tion in Asia is attractive for several reasons. In the context of the goal of measles elimination, WHO has established a Measles Laboratory Network to strengthen laboratory capac- ity for measles testing. 11 Because ELISAs are also widely used for measles diagnosis, the equipment and trained per- sonnel to perform ELISAs already exist in many places and JE testing can be easily integrated. The time required to com- plete testing is relatively short, particularly with these newly developed commercial kits (which is a particular advantage over some in-house kits). Although not commercially available at the time of this study, three standardized JE MAC ELISA diagnostic kits had been developed with the intent for commercial sale: the Japa- nese Encephalitis–Dengue IgM Combo ELISA Test manu- factured by Panbio Limited, the Japanese Encephalitis IgM ELISA manufactured by InBios International, Inc., and the JEV CheX kit manufactured by XCyton Diagnostics Ltd. 12–15 All three tests use a cell culture-derived recombinant particu- late JE antigen; the Panbio test also uses recombinant dengue 1–4 antigens. The Department of Virology, United States Army Medical Component–Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), a regional reference labora- tory for Asia designated as a WHO Collaborating Center, has an in-house JE MAC ELISA. This assay has been widely used across the region for many years and was shown to be very accurate when compared with JE hemagglutination inhibition assay results during the test’s development. 16 The goal of this study was to evaluate the sensitivity, specificity, and usability of the three diagnostic kits against this regionally recognized JE MAC ELISA using specimens containing either JE IgM, dengue IgM, or neither. The inclusion of dengue IgM positive specimens was considered necessary because dengue virus co- circulates in many regions of Asia where JE virus is found, and the potential for cross-reactivity between flaviviruses is well recognized. 3,4 MATERIALS AND METHODS Samples. Serum samples without personal identifiers were selected from the AFRIMS archived samples. The panel of 360 acute-phase samples consisted of 121 JE IgM-positive/ * Address correspondence to Julie A. Jacobson, Japanese Encepha- litis Project, PATH, 1455 NW Leary Way, Seattle, Washington 98107. E-mail: jjacobs@path.org Am. J. Trop. Med. Hyg., 77(1), 2007, pp. 164–168 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene 164