Jpn. J. Infect. Dis., 60, 267-270, 2007

Original Article Dengue Virus Cross-Reactive Hemagglutination Inhibition Antibody Responses in Patients with Primary Dengue Virus Infection Surapee Anantapreecha*, Atchareeya A-nuegoonpipat, Songthum Prakrong, Sumalee Chanama, Areerat Sa-ngasang, Pathom Sawanpanyalert and Ichiro Kurane1 National Institute of Health, Department of Medical Sciences, Ministry of Public Health, , , and 1Department of Virology I, National Institute of Infectious Diseases, Tokyo 162-8640, Japan (Received January 30, 2007. Accepted May 31, 2007)

SUMMARY: Acute and convalescent plasma samples were obtained from 101 confirmed primary dengue cases: 48 cases infected with dengue virus type 1, 10 cases with type 2, 42 cases with type 3 and one case with type 4. The hemagglutination inhibition (HI) titers of individual samples were at levels similar to each of the 4 dengue viruses at both the acute and convalescent stages, irrespective of the dengue virus that infected the patients. The results indicate that HI antibodies to dengue viruses are cross-reactive. When an HI test is used as a diagnostic test for dengue virus infection, the cross-reactive nature needs to be considered when interpreting the results.

PCR), IgM and IgG-capture ELISA. Infecting dengue viruses INTRODUCTION were determined by virus isolation and RT-PCR as previously Dengue viruses belong to the family Flaviviridea, genus reported (5). Antibody-capture ELISA was performed as Flavivirus. There are 4 dengue viruses: dengue virus types 1, previously reported (6). 2, 3 and 4. Dengue virus infection can be asymptomatic, or Collection of blood specimens: Blood specimens were can cause dengue fever (DF) and dengue hemorrhagic fever collected into tubes with EDTA anticoagulant and plasma was (DHF) (1). DF and DHF are a serious health problem in many separated. Plasma specimens were stored in liquid nitrogen tropical and subtropical countries (2). Laboratory tests are until use. All the specimens were transported in liquid nitro- essential for confirmation of dengue virus infection. Various gen to the Arbovirus Laboratory, National Institute of Health types of serological diagnostic methods have been used for (Thai NIH), Department of Medical Sciences, Nonthaburi, confirming dengue virus infection. Among the serological Thailand. tests, the hemagglutination inhibition (HI) test has been used HI test: The HI test was performed as previously reported as the main diagnostic test for a long period of time, although (7); non-specific inhibitors naturally present in the plasma recently the IgM-capture enzyme-linked immunosorbent were extracted twice by mixing 80 μl of plasma with 800 μl assay (ELISA) has been used more widely (3). However, of acetone. Plasma was dried and reconstituted with 800 μl ELISA requires sophisticated equipment which may not be of borate saline buffer, pH 9.0. The antigen was diluted with available in some laboratories. A neutralization test demon- 0.4% bovine serum albumin in borate saline buffer to make 8 strates high levels of virus specificity; however, it takes nearly HA units/50 μl. The treated plasma samples were serially a week to get the results and live viruses need to be used in two-fold diluted from 1:10 to 1:10,240 with 0.4% BABS in a appropriate containment facilities (4). For these reasons, the U-bottom microtiter plate. The positive and negative plasma HI test is still used as the main diagnostic test in many labora- controls were also included in the tests, and they were diluted tories worldwide. In the present study, we precisely analyzed in the same way as the tested plasma samples. Subsequently, the level of dengue virus cross-reactivity of HI antibodies in 25 μl of dengue viral antigen (8 HA units/50 μl) was added primary dengue virus infections. to each well containing 25 μl of diluted plasma and the plates were shaken thoroughly. The plates were incubated at 4°C overnight, and 50 l of 0.33% GRBC in proper VAD MATERIALS AND METHODS μ was added to each well. The plates were incubated at 37°C Patients: Blood samples were obtained from patients with for 1 h. HI titers on the tested plates were read. HI titers were primary dengue virus infection who visited Lampang Hos- demonstrated as a reciprocal of dilutions which inhibited pital in , Maharat Nakhon Ratchasima hemagglutination. Hospital in Nakhon Ratchasima Province, Hospital in Pathum Thani Province, Chareonkrung Pracharak RESULTS AND DISCUSSION Hospital in , Ratchaburi Hospital in Ratchaburi Prov- ince, or Hadyai Hospital in from 1999 to Plasma samples were obtained from a total of 101 con- 2002. Dengue virus infections were confirmed by virus iso- firmed primary dengue cases: 48 cases infected with dengue lation, reverse transcription-polymerase chain reaction (RT- virus type 1, 10 cases with type 2, 42 cases with type 3 and one case with type 4. HI antibody titers were assessed against *Corresponding author: Mailing address: National Institute of antigens of each of the 4 dengue viruses (Tables 1- 4). HI Health, Department of Medical Sciences, Ministry of Public antibody titers were different among patients infected with Health, 88/7 Tivanond Road, Muang, Nonthaburi 11000, Thai- the same serotype. However, titers of individual samples were land. Tel: +66-2-9510000, Fax: +66-2-9511498, E-mail: surapee at levels similar to each of the 4 dengue viruses in both the @dmsc.moph.go.th 1st (acute) and 2nd (convalescent) samples, irrespective of

267 Table 1. HI titers to 4 dengue viruses in plasma samples obtained from patients with dengue-1 virus infection HI titer 1st sample 2nd sample Patient Days after Days after D1 D2 D3 D4 D1 D2 D3 D4 onset onset 1 NI 80 160 80 160 NI 640 1,280 640 2,560 2 NI –1) –– – NI 640 640 640 640 3 NI 40 80 20 40NINDNDNDND 4 NI 20 20 20 40 NI 320 640 640 640 5 NI 40 40 20 20NINDNDNDND 6 NI – – – – NI 160 640 160 320 7 NI 40 40 40 40 NI 160 160 160 320 8 3 40 80 40 160 NI ND ND ND ND 9 3 640 1,280 640 1,280 8 1,280 2,560 1,280 2,560 10 3 10 20 20 40 7 160 640 640 1,280 11 2 – – – – 8 160 160 320 320 12 2 40 40 40 40 9 640 640 640 1,280 13 3 – – – – 520404040 14 4 – – – – 780401020 15 2 – – – – 580404040 16 3 10 20 20 10 740202020 17 3 40 80 80 80 5 160 320 640 320 18 5 10 20 10 10 840201010 19 7 – – – – 12 80408080 20 5 – – – – 920202040 21 4 – – – – 580402020 22 5 10 10 20 10 7 40 80 160 160 23 3 – – – – 8 80 160 160 640 24 4 160 160 320 640 6 40 40 40 160 25 NI – – – – NI 80 320 160 320 26 2 – – – – 6 160 320 640 320 27 1 – – – – 740101010 28 3 – – – – 740201010 29 2 – – – – 680808080 30 3 – – – – 10 40404040 31 4 10 10 10 20 7 80 160 160 320 32 NI – – – – NI 20 10 10 10 33 5 20 40 40 40 1040808080 34 2 10 10 20 10 510102010 35 6 10 10 20 10 720102010 36 5 80 80 40 80 NINDNDNDND 37 4 – – – – 7 160 160 160 320 38 1 – – – – 320101020 39 4 40 40 40 40 7 320 320 640 640 40 4 20 80 40 40 7 160 1,280 640 640 41 3 40 40 40 80 NINDNDNDND 42 6 320 320 320 640 NI ND ND ND ND 43 6 40 160 160 160 NI ND ND ND ND 44 2 – – – – 940404080 45 4 40 40 80 160 7 80 40 320 640 46 NI – – – – NI 10 10 10 40 47 5 40 80 80 160 NI ND ND ND ND 48 NI 20 20 40 40 NI ND ND ND ND Average 33.4 47.9 44.3 57.3 80.0 89.0 89.0 120.4 1): –, The titer was <20. However, when the titer to any of 4 dengue viruses are 20 or greater, <20 was expressed as 10 for calculation. ND, not done; NI, no information. the dengue virus that infected the patients (Tables 1-4). any of the 4 dengue viruses, and the type of infecting dengue Plasma samples were obtained twice from most of the virus can not be determined by the symptoms. This suggests patients: once in the acute phase and once in the convalescent that confirmation of dengue virus infection is often more phase. These plasma samples were analyzed separately. HI important than determination of the infecting dengue virus. antibodies were equally cross-reactive among the 4 dengue Although an HI test is not the best test to discriminate each viruses when using with 1st and 2nd plasma samples. The of the 4 serotypes of dengue viruses because of cross reactiv- results indicate that HI antibodies are dengue virus cross- ity, the extensive cross reactivity suggests that use of anti- reactive at both the acute and convalescent stages in primary gens from any dengue serotype would be sufficient to tell dengue virus infections. DF and DHF are can be caused by whether a person has been exposed to or infected with dengue

268 Table 2. HI titers to 4 dengue viruses in plasma samples obtained from patients with dengue-2 virus infection HI titer 1st sample 2nd sample Patient Days after Days after D1 D2 D3 D4 D1 D2 D3 D4 onset onset 1 NI – – – – NI 160 320 160 640 2 NI – – – – NI 160 320 160 1,280 3 NI 10 20 20 40 NI 320 640 320 1,280 4 NI 40 40 40 40 NI ND ND ND ND 5 NI 20 20 80 40NI20208040 6 NI – – – – NI 160 320 320 640 7 5 10 40 10 40 NI ND ND ND ND 8 5 40 40 40 40 NI ND ND ND ND 9 3 – – – – 710201010 10 1 40 40 40 80 NI ND ND ND ND Average 22.4 31.7 31.7 44.9 80.0 142.5 113.1 254.0 The footnote and abbreviations are in Table 1.

Table 3. HI titers to 4 dengue viruses in serum samples obtained from patients with dengue-3 virus infection HI titer 1st sample 2nd sample Patient Days after Days after D1 D2 D3 D4 D1 D2 D3 D4 onset onset 1 NI – – – – NI 320 640 320 1,280 2 NI 160 160 160 160 NI 160 160 80 320 3 4 80 160 160 160 7 320 640 640 1,280 4 8 – 40 40 40 11 160 640 320 640 5 3 – – – – 6 10 20 40 40 6 5 10 10 10 20 10 80 320 320 320 7 4 – – – – 8 10 20 40 20 8 2 – – – – 6 20 20 40 40 9 3 – – – – 6 10 20 20 10 10 3 – – – – 7 160 320 320 1,280 11 3 – – – – 780160 320 320 12 NI – – – – NI 160 320 640 640 13 1 10 10 20 10 3 160 320 320 320 14 3 – – – – 7 80 160 160 320 15 1 – – – – 840808080 16 7 – – – – 11 40408080 17 3 – – – – 8 320 320 320 640 18 4 – – – – 910104020 19 4 20 20 2040NINDNDNDND 20 4 80 160 160 640 11 160 320 320 640 21 1 – – – – 310404040 22 3 – – – – 910101020 23 2 – – – – 8 20 20 160 40 24 3 – – – – 510202040 25 4 – – – – 810102020 26 4 – – – – 710104010 27 3 – – – – 8 80 160 160 320 28 2 – – – – 7 80 160 160 160 29 4 20 40 40 40 7 80 160 320 320 30 4 10 40 40 40 7 160 320 320 320 31 1 – – – – 710104010 32 3 10 10 2010 8 10204040 33 1 20 40 80 80 4 80 160 320 320 34 2 10 40 10 10 3 80 1,280 160 160 35 5 10 20 20 40 9 160 320 640 640 36 5 – – – – 710104020 37 4 10 10 2040NINDNDNDND 38 2 – – – – 7 80 160 160 160 39 3 – – – – 720104040 40 3 – – – – 6 160 160 160 640 41 3 40 80 40 160 NI ND ND ND ND 42 1 40 80 160 160 3 160 320 320 320 Average 21.9 36.7 40.0 51.9 48.6 84.4 116.2 129.3 The footnote and abbreviations are in Table 1.

269 Table 4. HI titers to 4 dengue viruses in serum samples obtained from patients with dengue-4 virus infection HI titer 1st sample 2nd sample Patient Days after Days after D1 D2 D3 D4 D1 D2 D3 D4 onset onset 1480160 160 320 7 80 320 320 640 virus. Preparation of HI antigen and treatment of serum Science Foundation. samples before assay are time-consuming. For these reasons, many laboratories have begun to use other serodiagnostic REFERENCES methods, such as IgM-capture ELISA, in place of the HI 1. World Health Organization (1997): Dengue hemorrhagic fever: diag- test; however, the HI test still has characteristics which are nosis, treatment, prevention and control. 2nd ed. World Health Organi- considered to be advantageous over other methods. As shown zation, Geneva. in the present study, HI antibodies are equally cross-reactive 2. World Health Organization (2002): Dengue and dengue hemorrhagic fever. Fact sheet. 117. among dengue viruses in primary infections. Hence, the HI 3. Clarke, D.H. and Casals, J. (1985): Techniques for hemagglutination test cannot be used to differentiate dengue serotypes in pri- and hemagglutination-inhibition with arthropod borne virus. Am. J. mary dengue infections. Trop. Med. Hyg., 7, 561-573. 4. Endy, T.P., Nisalax, A., Chunsuttiwat, S., et al. (2004): Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia ACKNOWLEDGMENTS and severity of disease in prospective cohort study of DV infection in We would like to thank Dr. Piyaporn Bowonkiratikachorn and Ms. Thailand. J. Infect. Dis., 189, 990-1000. Souvapa Pongsathaporn at Charoenkrung Pracharak Hospital, Dr. Wandhana 5. Anantapreecha, S., Chanama, S., A-nuegoonpipat, A., et al. (2005): Sritubtim and Mr. Suthichai Pongmonjit at Pathum Thani Hospital, Dr. Vitaya Serological and virological features of dengue fever and dengue Jiwariyaves and Ms. Vanna Pengruangrojanachai at Ratchaburi Hospital, haemorrhagic fever in Thailand from 1999 to 2002. Epidemiol. Infect., Dr. Paiboon Vechpanich and Mr. Payuth Kaewmalang at Maharaj Nakhon 133, 503-507. Ratchasima Hospital, Dr. Wilaiwan Gulgonkarn, Dr. Aroonrat Suwanarat 6. Chanama, S., Anantapreecha, S., A-nuegoonpipat, A., et al. (2004): and Mr. Somchai Niyomthai at Lampang Hospital, Dr. Suda Chubuppakarn Analysis of specific IgM responses in secondary dengue virus infec- and Ms. Raruay Jitsakulchaidej at Hadyai Hospital, and other doctors, nurses tion: levels and positive rates in comparison with primary infections. J. and laboratory staff members in these hospitals for assisting us with the Clin. Virol., 31, 185-189. collection of samples. We also thank Dr. Somchai Sangkitporn, the then 7. Sa-ngasang, A. , Wibulwattanakij, S., Chanama, S., et al. (2003): Evalua- acting director of Thai NIH for his support. tion of RT-PCR as a tool for diagnosis of secondary dengue virus infec- This work was partly supported by the grant from Department of Medical tion. Jpn. J. Infect. Dis., 56, 205-209. Sciences, Ministry of Public Health, Thailand and the grant from Japan Health

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