Introduction. Abstract

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1 Comparison between Haemagglutination Inhibition (HI) Test and IgM and IgG-capture ELISA in Determination of Primary and Secondary Dengue Virus Infections Atchareeya A-nuegoonpipat a, Songthum Prakong a, Areerat Sa-ngasang a, Sumalee Chanama a, Pathom Sawanpanyalert a, Ichiro Kurane b and Surapee Anantapreecha a a National Institute of Health, Department of Medical Sciences, Ministry of Public Health, 88/7 Tivanond Road, Mueang, Nonthaburi 11000, Thailand b Department of Virology 1, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo , Japan Abstract The results of HI test and antibody-capture ELISA in laboratory diagnosis of primary and secondary dengue virus infections were compared. Of the 1123 cases serologically confirmed to be dengue, 42 were classified as primary infection and 947 were classified as secondary infection by both HI and ELISA tests, an agreement of 88%. Of the 173 cases determined to be primary infection by HI, 131 revealed discordant results by ELISA. Three of the 950 cases that were determined to be secondary by HI were determined to be primary infection by ELISA. The results indicated that there was discrepancy between HI test and ELISA in determination of primary or secondary infections in some of the confirmed dengue cases, and suggest that a third test such as neutralization test should be included for those showing discordant results. Keywords: Haemagglutination inhibition (HI) assay, antibody-capture ELISA, primary dengue virus infection, secondary dengue virus infection. Introduction Dengue viruses belong to the family Flaviviridae, genus Flavivirus. There are four dengue serotypes, viz. DENV-1, 2, 3 and 4. The patients infected with dengue viruses can be asymtomatic, or can develop dengue fever (DF) or dengue haemorrhagic fever (DHF). [1,2] DF and DHF are a serious health problem in many tropical and subtropical countries. Clinical observation is important for the diagnosis of DF and DHF; however, laboratorial tests are essential for confirming dengue virus infection. Various types of serological tests are used for confirmation. Haemagglutination inhibition (HI) test and antibody capture enzyme-linked immunosorbent assay (ELISA) are widely used as the main serological tests in many laboratories, [3-7] although they have crossreactive reactivity with other flaviviruses. In the present study, we compared HI and ELISA tests in the laboratory diagnosis of primary and secondary dengue virus infections, according to the currently used diagnostic criteria. atcharee@dmsc.moph.go.th; Ext.99220; Fax: Dengue Bulletin Volume 30,

2 Materials and methods Eleven hundred and twenty-three dengue patients were analysed in the study. These patients were hospitalized in six provincial hospitals in Thailand: Charoenkrung Pracharak Hospital at Bangkok, Pathum Thani Hospital, Ratchaburi Hospital, Maharaj Nakhon Ratchasima Hospital at Nakhon Ratchasima province, Lampang Hospital and Hadyai Hospital at Songkhla province, during Blood specimens were obtained from patients for diagnostic purpose. The number of blood specimens ranged from one to three per patient. The first sample was collected on the day of hospitalization and the second sample on the day of discharge. The third sample was occasionally collected after the discharge day. Plasma and buffy coat were separated at the hospitals, kept in liquid nitrogen and then transported to the Arbovirus Section, National Institute of Health, Thailand. Specimens were kept at 70 C for virus isolation and at 20 C for haemagglutination inhibition (HI) test and IgM and IgG-capture ELISA. HI test was performed as previously reported with four haemagglutination activity (HA) units of DENV-1, 2, 3 and 4 antigens except that the assay was modified to a microtiter format. [8] The plasma samples were treated by 12.5% kaolin to remove non-specific inhibitor and by packed goose red blood cell to remove naturally agglutinin. Antigens were produced by sucrose acetone extraction from the brains of suckling mice infected with the following prototype virus strains: DENV-1 Hawaii, DENV-2 New Guinea C, DENV-3 H- 87, and DENV-4 H-241. All the specimens were also tested for dengue virus-specific IgM and IgG by antibody-capture ELISA as described by Innis et al. with a minor modification. [5] The cut-off points for IgM and IgG were 40 and 80 units, respectively. [9] Primary and secondary infections were determined by HI test according to the WHO criteria [1] and by ELISA test according to the criteria by WHO and Innis et al. with a minor modification. [1,5] Virus isolation was accomplished by inoculating buffy coat separated from 238 acute-phase specimens on C6/36 cell culture. Indirect immunofluorescence assays were performed with type-specific monoclonal antibodies to confirm isolation and to determine serotype. [9-11] Detection of dengue virus RNA and serotype identification were also performed in 584 acute phase plasma by the method reported by Yenchitsomanus et al. [12] However, all the data are not shown. Results and discussion Of the 1123 dengue-confirmed cases, 173 and 950 cases were determined to be primary and secondary infection, respectively, according to the HI criteria. Plasma samples from these cases were also tested by IgM and IgG-capture ELISA. As a result, out of the 173 cases determined to be primary by HI test, 42 and 131 cases were determined to be primary and secondary, respectively. Of the 950 cases which were determined to be secondary by HI test, 3 and 947 cases were determined to be primary and secondary, respectively, by ELISA (Table 1). The ratios between primary and secondary infections determined by HI and ELISA were significantly different (P<0.001). Representative data of plasma samples, which demonstrated compatible and incompatible results, is shown in Table 2. Table 1: Comparison of the results between HI and antibody-capture ELISA tests 142 Dengue Bulletin Volume 30, 2006

3 Table 2: Representation of data of plasma samples, which showed compatible and incompatible results between HI and ELISA tests *U: Unidentified Dengue Bulletin Volume 30,

4 In the present study, HI test and antibodycapture ELISA were compared in determination of primary and secondary dengue virus infections. Of a total of 1123 cases, 134 cases demonstrated disagreement between HI test and ELISA (Table 1). Fourfold or greater changes in HI titer between paired specimens and the titer equal to or less than 1:1280 in the convalescent sample are considered to be the criteria for primary infection. [1] HI test does not discriminate subtypes of immunoglobulin, while antibody-capture ELISA discriminates IgM and IgG. [1,5] It is of interest that the ratio of average IgM to IgG ELISA unit in plasma samples from dengue cases confirmed to be primary by HI test was lower than 1.78 on disease day 7 or later (data not shown). It is possible that the IgM to IgG ratio of 1.78 may not pick up all the primary infections. HI test has been the standard test for the confirmation and classification of dengue infections. [13] In 1997, WHO added antibodycapture ELISA test to serological confirmation. [1] The results in the present study demonstrated the incompatibility between HI test and antibody-capture ELISA, especially among those who were determined to be primary infection by HI test. Plaque reduction neutralization test (PRNT) has also been used, because it generally shows a monotypic reaction to the infecting virus in primary dengue virus infection. [14] Chuan-Liang Kao et al. also verified that the PRNT serotyping generally exhibits a monotypic reaction to the primary infecting virus. [15] We, therefore, suggest that the third test such as PRNT be applied to the cases demonstrating incompatibility between the HI test and ELISA. Acknowledgements We would like to thank Dr Piyaporn Bowonkiratikachorn and Ms Souvapa Pongsathaporn at Charoenkrung Pracharak Hospital, Dr Wandhana Sritubtim and Mr Suthichai Pongmonjit at Pathum Thani Hospital, Dr Vitaya Jiwariyaves and Ms Vanna Pengruangrojanachai at Ratchaburi Hospital, Dr Paiboon Vechpanich and Mr Payuth Kaewmalang at Maharaj Nakhon Ratsrima Hospital, Dr Wilaiwan Gulgonkarn, Dr Aroonrat Suwanarat and Mr Somchai Niyomthai at Lampang Hospital and Dr Suda Chubuppakarn and Ms Raruay Jitsakulchaidej at Hadyai Hospital, and other doctors, nurses and laboratory staff in these hospitals for assisting us with the collection of samples. This work was partly supported by the grant from the Department of Medical Sciences, Ministry of Public Health, Thailand, and the grant from the Japan Health Science Foundation. References [1] World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. Geneva; p [2] George R, Lum LCS. Clinical spectrum of dengue infection. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever, Colorado: US Department of Health and Human Services; p [3] Bundo K, Igarashi A. Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients. J Virol Methods 1985 May;11(1): [4] Lam SK, Devi S, Pang T. Detection of specific IgM in dengue infection. Southeast Asian J Trop Med Public Health 1987 Dec;18(4): Dengue Bulletin Volume 30, 2006

5 [5] Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S, Puttisri P, Hoke CH. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg Apr;40(4): [6] Yamada K, Takasaki T, Nawa M, Kurane I. Laboratory diagnosis of imported dengue cases. Jpn J Trop Med Hyg 1998;27:75-7. [7] Nawa M, Yamada KI, Takasaki T, Akatsuka T, Kurane I. Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 2000 Sep;7(5): [8] Clarke DH, Casals J. Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Am J Trop Med Hyg 1958 Sep;7(5): [9] Chanama S, Anantapreecha S, A- nuegoonpipat A, Sa-gnasang A, Kurane I, Sawanpanyalert P. Analysis of specific IgM responses in secondary dengue virus infections: levels and positive rates in comparison with primary infections. J Clin Virol 2004 Nov;31(3): [11] Henchal EA, McCown JM, Seguin MC, Gentry MK, Brandt WE. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg 1983 Jan;32(1): [12] Yenchitsomanus PT, Sricharoen P, Jaruthasana I, Pattanakitsakul SN, Nitayaphan S, Mongkolsapaya J, Malasit P. Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR). Southeast Asian J Trop Med Public Health 1996 Jun;27(2): [13] World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. Geneva; p [14] Innis BL. Antibody responses to dengue virus infection. In: Gubler DJ, Kuno G, editors. Dengue and dengue hemorrhagic fever, Colorado: US Department of Health and Human Services; p [15] Kao CL, King CC, Chao DY, Wu HL, Chang GJ. Laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health. J Microbiol Immunol Infect 2005 Feb;38(1):5-16. [10] Henchal EA, Gentry MK, McCown JM, Brandt WE. Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg 1982 Jul;31(4): Dengue Bulletin Volume 30,