D ENGUE BULLETIN
Volume 28 December 2004 D E N G U E
B U L L E T I N V o l u m e
2 8 ,
2 WORLD HEALTH ORGANIZATION 0 0 4 South-East Asia Region Western Pacific Region DDeenngguuee BBuulllleettiinn Volume 28 December 2004
World Health Organization South-East Asia Region Western Pacific Region ISBN 92 9022 256 5 © World Health Organization 2004
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Indexation: Dengue Bulletin is being indexed by BIOSIS and Elsevier’s Bibliographic Databases including, EMBASE, Compendex, Geobase and Scopus Acknowledgements
Editor, Dengue Bulletin, WHO/SEARO, gratefully thanks the following for peer reviewing manuscripts submitted for publication.
In-house Review:
Nand L. Kalra: Reviewed the manuscripts in respect of format check, content, conclusions drawn, including condensation of tabular and illustrative materials for clear, concise and focused presentation and bibliographic references. He was also involved in the final stages of printing of the Bulletin.
Peer Reviewers
1. Dana A. Focks John W. Hock Company P.O. Box 12852 Gainesville, FL 32604 USA E-mail: [email protected]
2. Duane J. Gubler Director Asia-Pacific Institute of Tropical Medicine and Infectious Diseases Leahi Hospital 3675 Kilauea Ave Honolulu, Hawaii 96816 USA E-mail: [email protected]
3. Oon Chong Teik Tropical Medicine & Infectious Diseases Mt Elizabeth Hospital Singapore 228510 E-mail: [email protected]
Dengue Bulletin – Vol 28, 2004 iii 4. Scott Halstead Uniformed Services University of the Health Sciences Bethesda, Maryland USA E-mail: [email protected]
5. Siripen Kalyanarooj WHO Collaborating Centre for Case Management of Dengue/DHF/DSS Queen Sirikit National Institute of Child Health (Children’s Hospital) Bangkok Thailand E-mail: [email protected]
6. Suchitra Nimmannitya WHO Collaborating Centre for Case Management of Dengue/DHF/DSS Queen Sirikit National Institute of Child Health (Children’s Hospital) Bangkok Thailand E-mail: [email protected]
7. Kevin Palmer Regional Office for the Western Pacific P.O. Box No. 2932 12115 Manila Philippines E-mail: [email protected]
8. Michael Nathan World Health Organization Headquarters 20 Avenue Appia 1211 Geneva 27 Switzerland E-mail: [email protected]
The quality and scientific stature of the Dengue Bulletin is largely due to the conscientious efforts of the experts and also due to the positive response of contributors to comments and suggestions.
iv Dengue Bulletin – Vol 28, 2004 From the Editor’s Desk
ver the decades dengue/dengue haemorrhagic fever has emerged as a global public O health problem with countries in Asia and the Pacific sharing more than 70 % of the disease burden. In some of these countries, DHF is gaining hyper-endemicity causing deaths among children. During 2004, Indonesia reported a major dengue outbreak encompassing Central Java, Sumatra and some outer islands. Till the end of July 2004, 69,017 cases of DF/DHF and 770 deaths were registered by Indonesian health authorities. During this epidemic DEN-3 was the predominant serotype.
Sri Lanka also reported a major outbreak with 12,400 cases and 71 deaths as of 23 August 2004. A majority of the cases were reported from five cities: Colombo, Kandy, Gampaha, Kalutara and Kurunegala.
In the South-East Asia Region, Bhutan and Nepal continued to enjoy dengue-free status till 2003 because of their sub-mountainous location. However, during August 2004, Bhutan recorded the first-ever outbreak of DF/DHF in Phuntsholing (population 27,000), a border town with India. During this outbreak a total of 2,544 DF/DHF cases with no deaths were reported. More than 93 % of those affected were persons above 5 years of age. This sent a strong signal to the adjoining DF-free north-eastern part of India and Nepal to take appropriate preventive action.
DengueNet, the WHO Global Surveillance System for management of epidemiological and virological surveillance data for early detection, planning and response, was launched in the South-East Asia and Western Pacific countries during 2004. Each country identified institutions which would participate in the programme.
The current Volume 28 (2004) of the Dengue Bulletin includes contributions from the South-East Asia Region (13), the Western Pacific Region (7), the American Region (5) and the European Region (4).
A supplement, featuring experiences from different countries in social mobilization and communication for dengue prevention and control, is also being issued along with this volume.
We now invite contributions for Volume 29 (2005). The deadline for the receipt of contributions is 30 June 2005. Contributors are requested to follow the instructions carefully while preparing the manuscript. Contributions accompanied by computer diskettes using MS Word for Windows should be sent to the Editor, Dengue Bulletin, WHO/SEARO, Mahatma Gandhi Road, IP Estate, Ring Road, New Delhi-110 002, India, or by e-mail as a file attachment to the Editor at [email protected]. Readers desirous of obtaining copies of the Dengue Bulletin may contact the respective WHO Regional Offices in New Delhi or Manila or the WHO Country Representative in their country of residence.
Dr Chusak Prasittisuk Regional Adviser Vector-borne Disease Control World Health Organization Regional Office for South-East Asia New Delhi, India Contents
1. Annual Changes of Predominant Dengue Virus Serotypes in Six Regional 1 Hospitals in Thailand from 1999 to 2002 Surapee Anantapreecha, Sumalee Chanama, Atchareeya A-nuegoonpipat, Sirirat Naemkhunthot, Areerat Sa-ngasang, Pathom Sawanpanyalert and Ichiro Kurane
2. A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: 7 Demographic and Clinical Features T.U. Brown, K. Babb, M. Nimrod, C.V.F. Carrington, R.A. Salas and M.A. Monteil
3. Ecological Study of Rio de Janeiro City DEN -3 Epidemic, 2001-2002 20 Maria Lucia F. Penna
4. Sero-epidemiological and Virological Investigation of Dengue Infection 28 in Oaxaca, Mexico, during 2000-2001 A. Cisneros-Solano, M.M.B. Moreno-Altamirano, U. Martínez-Soriano, F. Jimenez-Rojas, A. Díaz-Badillo and M.L. Muñoz
5. Spatial and Temporal Dynamics of Dengue Haemorrhagic Fever 35 Epidemics, Nakhon Pathom Province, Thailand, 1997-2001 Wutjanun Muttitanon, Pongpan Kongthong, Chusak Kongkanon, Sutee Yoksan, Narong Nitatpattana, Jean Paul Gonzalez and Philippe Barbazan
6. Sporadic Prevalence of DF/DHF in the Nilgiri and Cardamom Hills of 44 Western Ghats in South India: Is it a Seeding from Sylvatic Dengue Cycle – A Hypothesis Nand Lal Kalra and Chusak Prasittisuk
7. Autoimmunity in Dengue Virus Infection 51 Chiou-Feng Lin, Huan-Yao Lei, Ching-Chuan Liu, Hsiao-Sheng Liu, Trai-Ming Yeh, Shun-Hua Chen and Yee-Shin Lin
8. Inhibition of the NS2B-NS3 Protease – Towards a Causative Therapy for 58 Dengue Virus Diseases Gerd Katzenmeier
Dengue Bulletin – Vol 28, 2004 v Contents
9. Prognostic Factors of Clinical Outcome in Non-Paediatric Patients with 68 Dengue Haemorrhagic Fever/Dengue Shock Syndrome Jaime R. Torres, José M. Torres-Viera, Hipólito García, José R. Silva, Yasmín Baddour, Angel Bajares and Julio Castro M.
10. Dengue Haemorrhagic Fever with Encephalopathy/Fatality at Petchabun 77 Hospital: A three-year Prospective Study (1999-2002) Prasonk Witayathawornwong
11. A New Tool for the Diagnosis and Molecular Surveillance of Dengue 87 Infections in Clinical Samples C. Domingo, G. Palacios, M. Niedrig, M. Cabrerizo, O. Jabado, N. Reyes, W.I. Lipkin and A. Tenorio
12. Clinical and Laboratory Observations Associated with the 2000 Dengue 96 Outbreak in Dhaka, Bangladesh Monira Pervin, Shahina Tabassum, Md. Mobarak Ali, Kazi Zulfiquer Mamun and Md. Nazrul Islam
13. Current Status of Dengue Diagnosis at the Center for Disease Control, 107 Taiwan Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien, Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin and Jyh-Hsiung Huang
14. Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in 118 the Presence of Sodium thiocyanate (NaSCN) Masaru Nawa, Tomohiko Takasaki, Mikako Ito, Ichiro Kurane and Toshitaka Akatsuka
15. Genetic Influences on Dengue Virus Infections 126 J.F.P. Wagenaar, A.T.A. Mairuhu and E.C.M. van Gorp
16. Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in 135 Guangzhou, China, in 2002 Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An
vi Dengue Bulletin – Vol 28, 2004 Contents
17. Induction of Cytotoxic T Lymphocytes by Immunization with Dengue 145 Virus – Derived, Modified Epitope Peptide, Using Dendritic Cells as a Peptide Delivery System Yoshiki Fujii, Hideyuki Masaki, Takanori Tomura, Kiyohiro Irimajiri and Ichiro Kurane
18. Molecular Characterization of Brazilian Dengue Viruses 151 Marize Pereira Miagostovich, Flávia Barreto dos Santos and Rita Maria Ribeiro Nogueira
19. Unusual Emergence of Guate98-like Molecular Subtype of DEN-3 161 during 2003 Dengue Outbreak in Delhi Manoj Kumar, S.T. Pasha, Veena Mittal, D.S. Rawat, Subhash Chandra Arya, Nirmala Agarwal, Depesh Bhattacharya, Shiv Lal and Arvind Rai
20. The Animal Models for Dengue Virus Infection 168 Tao Peng, Junlei Zhang and Jing An
21. Philippine Species of Mesocyclops (Crustacea: Copepoda) as a Biological 174 Control Agent of Aedes aegypti (Linnaeus) Cecilia Mejica Panogadia-Reyes, Estrella Irlandez Cruz and Soledad Lopez Bautista
22. Susceptibility of Aedes aegypti to Insecticides in Viet Nam 179 Vu Duc Huong, Nguyen Thi Bach Ngoc, Do Thi Hien and Nguyen Thi Bich Lien
23. Ovipositioning Behaviour of Aedes aegypti in Different 184 Concentrations of Latex of Calotropis procera: Studies on Refractory Behaviour and its Sustenance across Gonotrophic Cycles Manju Singhi, Vinod Joshi, R.C. Sharma and Keerti Sharma
24. Community-based Assessment of Dengue-related Knowledge 189 among Caregivers Khynn Than Win, Sian Za Nang and Aye Min
25. Students’ Perceptions about Mosquito Larval Control in a Dengue- 196 Endemic Philippine City Jeffrey L. Lennon
Dengue Bulletin – Vol 28, 2004 vii Contents
Short Notes
1. Sero-surveillance in Delhi, India – An Early Warning Signal for 207 Timely Detection of Dengue Outbreaks D. Bhattacharya, Veena Mittal, M. Bhardwaj, Mala Chhabra, R.L. Ichhpujani and Shiv Lal
2. Detection of Dengue Virus in Wild Caught Aedes albopictus (Skuse) 210 around Kozhikode Airport, Malappuram District, Kerala, India B.P. Das, L. Kabilan, S.N. Sharma, S. Lal, K. Regu and V.K. Saxena
3. Entomological Investigations for DF/DHF in Alwar District, Rajasthan, India 213 Kalpana Baruah, Avdhesh Kumar and V.R. Meena
4. Essentiality of Source Reduction in both Key and Amplification Breeding 216 Containers of Aedes aegypti for Control of DF/DHF in Delhi, India B.N. Nagpal, Aruna Srivastava, M.A. Ansari and A.P. Dash
5. Breeding of Dengue Vector Aedes aegypti (Linnaeus) in Rural Thar Desert, 220 North-western Rajasthan, India B.K. Tyagi and J. Hiriyan
Book Reviews
1. A Review of Entomological Sampling Methods and Indicators for 223 Dengue Vectors
2. DengueNet in India 224
3. WHO/WPRO/SEARO Meeting on DengueNet Implementation in South- 226 East Asia and the Western Pacific, Kuala Lumpur, 11-13 December 2003
Instructions to Contributors 232
viii Dengue Bulletin – Vol 28, 2004 Annual Changes of Predominant Dengue Virus Serotypes in Six Regional Hospitals in Thailand from 1999 to 2002
Surapee Anantapreecha*#, Sumalee Chanama*, Atchareeya A-nuegoonpipat*, Sirirat Naemkhunthot*, Areerat Sa-ngasang*, Pathom Sawanpanyalert* and Ichiro Kurane** *National Institute of Health, Department of Medical Sciences, Ministry of Public Health, 88/7, Tivanond Road, Muang, Nonthaburi, 11000, Thailand **Department of Virology I, National Institute of Infectious Diseases Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Abstract A virological study was conducted in six hospitals spread across Thailand from 1999 to 2002. All four dengue serotypes were identified, of which DEN-1 was the most predominant. The predominant dengue serotypes changed every 1-2 years in three of the six hospitals and the predominant serotypes were different in different hospitals. DEN-1 was predominant in two hospitals during three years of the study period (2000-2002). DEN-4 was not isolated, or accounted for only a small percentage of the total isolates, except in one hospital in 2002. Keywords: Dengue virus, dengue virus serotypes, Thailand.
Introduction occurred in a pattern of 2-year cycle, and subsequently in irregular cycles as the The dengue virus consists of four serotypes: disease spread throughout the country. The DEN-1, DEN -2, DEN-3 and DEN-4. The largest outbreak was reported in 1987, with clinical manifestations range from 174,284 reported cases, an incidence rate undifferentiated febrile illness to classic of 325 cases per 100,000 population. It was dengue fever (DF), dengue haemorrhagic followed by another outbreak in 2001, fever (DHF) and to dengue shock syndrome which reported an incidence rate of 224 [1-3] (DSS) . Dengue virus infections are now cases per 100,000 population[2,4,5]. In 2002, important public health problems in many 114,800 dengue cases were reported with tropical and subtropical areas in the world. an incidence rate of 183 cases per 100,000 In Thailand, a dengue outbreak first population. It is important to determine occurred in Bangkok in 1958. It then which of the four serotypes causes the
# E-mail: [email protected]; Tel.: 66 29510000 Ext. 99219, 99220; Fax: 66 25915449
Dengue Bulletin – Vol 28, 2004 1 Dengue Virus Serotypes in Thailand dengue epidemic each year, and whether Figure 1. Location of six provinces where the dominant serotype is the same or the sentinel sites were chosen different in each region in the country. In the present study, we analysed the predominant dengue serotypes at the six regional hospitals located in the four regions of Thailand – north, north-east, central and south regions – in each year from 1999 to 2002.
Materials and methods
Collection of blood specimens A total of 5,160 and 3,619 blood specimens were collected at acute and convalescent stages, respectively, from suspected dengue cases who visited Lampang Hospital in Lampang (north), Maharat Nakhon Ratchasima Hospital in Nakhon Ratchasima 1 = Lampang (north-east), Pathum Thani Hospital in 2 = Nakhon Ratchasima 3 = Pathum Thani Pathum Thani, Chareonkrung Pracharak 4 = Bangkok Hospital in Bangkok, Ratchaburi Hospital in 5 = Ratchaburi and Ratchaburi (central) and Hadyai Hospital in 6 = Songkhla Songkhla (south) from 1999 to 2002 (Figure 1). Blood specimens were drawn into tubes with ethylene diamine tetaacetate (EDTA) anticoagulant and centrifuged. The number of specimens ranged from 1 to 3 per patient. The first samples were collected on the day Virus isolation and determination of hospitalization and the second samples of dengue virus serotypes were collected on the day of discharge. Ten microlitres of acute phase-buffy coat Third samples, if any, were collected samples were inoculated onto monolayer between 10-14 days after the onset of of C6/36 cells in a 24-well plate with symptoms. Plasma and buffy coat were rocking for 90 minutes at room separated at each hospital, kept in liquid temperature[6]. The inocula were discarded nitrogen and then transported to the and replaced by Leibovitz 15 medium (L- Arbovirus Section of the National Institute of 15, Gibco BRL) containing 1% heat- Health, Nonthaburi. Specimens were kept inactivated fetal bovine serum. After 7 days at -70 °C for virus isolation. of incubation at 28 °C, the cultured media were collected, the infected cells were
2 Dengue Bulletin – Vol 28, 2004 Dengue Virus Serotypes in Thailand stained by IFA, and dengue virus serotypes Results and discussion were determined[7,8]. DEN -1 (16007 strain), DEN -2 (16681 strain), DEN-3 (16562 The numbers of samples tested and found strain) and DEN-4 (1032 strain) were used positive for virus isolation are shown in as virus controls in IFA. Table 1.
Table 1. Number of samples used for the analysis
Convalescent Virus isolation Year Hospital Acute sample sample positive
1999 Lampang 128 108 74 Maharat Nakhon Ratchasima 26 18 6 Pathum Thani 0 0 0 Chareonkrug Pracharak 0 0 0 Ratchaburi 35 11 31 Hadyai 1 1 0 2000 Lampang 35 33 15 Maharat Nakhon Ratchasima 22 17 3 Pathum Thani 25 14 4 Chareonkrug Pracharak 15 15 10 Ratchaburi 438 419 238 Hadyai 113 28 22 2001 Lampang 262 238 150 Maharat Nakhon Ratchasima 358 251 176 Pathum Thani 265 170 143 Chareonkrug Pracharak 224 216 99 Ratchaburi 389 363 256 Hadyai 450 231 276 2002 Lampang 339 236 95 Maharat Nakhon Ratchasima 930 600 292 Pathum Thani 154 73 32 Chareonkrung Pracharak 135 128 65 Ratchaburi 490 346 170 Hadyai 326 103 77
All the four dengue serotypes were The predominant dengue virus detected in the six hospitals during this serotypes in each hospital were analysed study. When the total numbers of isolates when more than nine isolates were obtained were analysed, DEN-1 was the predominant in a year. In the Lampang Hospital, the serotype (48%), followed by DEN -2 (27%), predominant serotypes were DEN-2 (44%) DEN-3 (20%) and DEN -4 (5%). in 1999, DEN-1 (86%) in 2000, DEN-1
Dengue Bulletin – Vol 28, 2004 3 Dengue Virus Serotypes in Thailand
(57%) in 2001 and DEN-2 (79%) in 2002. In predominant in four hospitals (Maharat the Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima, Pathum Thani, the predominant serotypes were DEN-1 Chareonkrung Pracharak and Hadyai) and (47%) in 2001 and DEN-1 (57%) in 2002. In two hospitals (Lampang and Ratchaburi), the Pathum Thani Hospital, the respectively. These results suggest that DEN - predominant serotypes were DEN-1 and 1 was the most predominant serotype from DEN-2 (41% each) in 2001 and DEN-1 1999 to 2002 in Thailand; however, the (47%) in 2002. In the Chareonkrung predominant serotypes were different in Pracharak Hospital, the predominant different hospitals. serotypes were DEN-1 (50%) in 2000, DEN - 1 (61%) in 2001 and DEN -1 (65%) in 2002. The results of the present study are In the Ratchaburi Hospital, the predominant generally consistent with those previously serotypes were DEN-1 and DEN-2 (32% reported. The analysis of dengue virus each) in 1999, DEN-3 (52%) in 2000, DEN- isolates at Bangkok Children’s Hospital from 1 (38%) and in 2001 and DEN -2 (74%) in 1973 to 1999 showed changes in dengue [9] 2002. In the Hadyai Hospital, the virus serotypes from year to year . Although predominant serotype was DEN-1 (95%, our study leads to a similar conclusion, the 79% and 44%) in 2000, 2001 and 2002, study was conducted in six regional respectively. hospitals throughout the country and not only in central Bangkok. The predominant serotypes were compared among six hospitals in each of the Each dengue serotype may possess study years (Table 2). In 1999 when data unique characteristics in a dengue epidemic were analysed for two hospitals (Lampang and disease severity. There were and Ratchaburi), DEN-2 was predominant associations between DEN-1, DEN-2 and in one hospital (Lampang), and DEN-1 and DEN-3 and moderately severe dengue DEN-2 were equally predominant in the epidemic years (1984-85, 1989-90, 1997), other hospital (Ratchaburi). In 2000 when and between DEN-3 and severe dengue [9] data were analysed for four hospitals epidemic years (1987 and 1998) . In that (Lampang, Chareonkrung Pracharak, sense, it is important to collect more Ratchaburi and Hadyai), DEN-1 and DEN-3 information on the predominant serotypes were predominant in three hospitals and levels of epidemics. Although the (Lampang, Chareonkrung Pracharak and present study generated information on Hadyai) and one hospital (Ratchaburi), isolates only from the patients who visited respectively. In 2001, DEN-1 was the six hospitals, they are located far away predominant in five hospitals (Lampang, from each other in four regions of Maharat Nakhon Ratchasima, Chareonkrung Thailand: north, north-east, central and Pracharak, Ratchaburi and Hadyai) and south. It is thus assumed that the results of DEN-1 and DEN-2 were equally this study demonstrate, to a certain extent, predominant in one hospital (Pathum Thani). the general features of dengue epidemics In 2002, DEN-1 and DEN -2 were in Thailand.
4 Dengue Bulletin – Vol 28, 2004 Dengue Virus Serotypes in Thailand
Table 2. Proportion of each of the four dengue serotypes determined by virus isolation in the six hospitals
Year Hospital Total DEN-1 (%) DEN-2 (%) DEN-3 (%) DEN-4 (%) 1999 Lampang 74 31 (42) 33 (44) 8 (11) 2 (3) Maharat Nakhon 6 4 (67) 2 (33) 0 (0) 0 (0) Ratchasima Pathum Thani 0 0 (0) 0 (0) 0 (0) 0 (0) Chareonkrug 0 0 (0) 0 (0) 0 (0) 0 (0) Pracharak Ratchaburi 31 10 (32) 10 (32) 8 (26) 3 (10) Hadyai 0 0 (0) 0 (0) 0 (0) 0 (0) 2000 Lampang 15 13 (86) 1 (7) 0 (0) 1 (7) Maharat Nakhon 3 2 (67) 1 (33) 0 (0) 0 (0) Ratchasima Pathum Thani 4 1 (25) 2 (50) 1 (25) 0 (0) Chareonkrug 10 5 (50) 4 (40) 1 (10) 0 (0) Pracharak Ratchaburi 238 99 (42) 9 (4) 125 (52) 5 (2) Hadyai 22 21 (95) 0 (0) 1 (5) 0 (0) 2001 Lampang 150 85 (57) 35 (23) 26 (19) 1 (1) Maharat Nakhon 176 83 (47) 68 (39) 16 (9) 9 (5) Ratchasima Pathum Thani 143 58 (41) 59 (41) 19 (13) 7 (5) Chareonkrug 99 60 (61) 25 (25) 11 (11) 3 (3) Pracharak Ratchaburi 256 98 (38) 43 (17) 96 (37) 19 (7) Hadyai 276 219 (79) 4 (2) 39 (14) 14 (5) 2002 Lampang 95 10 (11) 75 (79) 8 (8) 2 (2) Maharat Nakhon 292 166 (57) 70 (24) 49 (17) 7 (2) Ratchasima Pathum Thani 32 15 (47) 8 (25) 7 (22) 2 (6) Chareonkrug 65 42 (65) 16 (25) 3 (5) 4 (5) Pracharak Ratchaburi 170 21 (12) 126 (74) 12 (7) 11 (7) Hadyai 77 34 (44) 13 (17) 13 (17) 17 (22)
Acknowledgements Dr Vitaya Jiwariyaves and Ms Vanna Pengruangrojanachai of Ratchaburi Hospital, We thank Dr Piyaporn Bowonkiratikachorn Dr Paiboon Vechpanich and Mr Prayuth and Ms Souvapa Pongsathaporn of Kaewmalang of Maharat Nakhon Ratchasima Charoenkrung Pracharak Hospital, Hospital, Dr Wilaiwan Gulgonkarn, Dr Wandhana Sritubtim and Mr Suthichai Dr Aroonrat Suwanarat and Mr Somchai Pongmonjit of Pathum Thani Hospital, Niyomthai of Lampang Hospital and
Dengue Bulletin – Vol 28, 2004 5 Dengue Virus Serotypes in Thailand
Dr Suda Chubuppakarn and Ms Raruay This work was partly supported by grants Jitsakulchaidej of Hadyai Hospital, and other from the Department of Medical Sciences, doctors, nurses and laboratory staff for Ministry of Public Health, Thailand, and the assisting us with the collection of samples. Japan Health Science Foundation.
References
[1] George R and Lum LCS. Clinical spectrum of [7] Henchal EA, Gentry MK, McCrown JM and dengue infection. In Gubler D and Kuno G Brandt WE. Dengue virus – specific and (Eds.) Dengue and dengue hemorrhagic flavivirus group determinants identified with fever. Colorado: US Department of Health monoclonal antibodies by indirect and Human Services, 1997: 89-113. immunofluorescence. Am J Trop Med Hyg, [2] World Health Organization. Monograph on 1982, 31: 830-836. dengue/dengue haemorrhagic fever. WHO, [8] Henchal EA, McCown JM, Seguin MC, Regional Office for South-East Asia, New Gentry MK and Brandt WE. Rapid Delhi 1993. identification of dengue virus isolates by [3] World Health Organization. Dengue using monoclonal antibodies in an indirect haemorrhagic fever. Diagnosis treatment, immunofluorescence assay. Am J Trop Med prevention and control, 2nd edition. Hyg, 1983, 32: 164-169. Geneva: WHO, 1997. [9] Nisalak A, Endy TP, Nimmannitya S, [4] Office of the Permanent Secretary for Public Kalayanarooj S, Thisayakorn U, Scott RM, Health. Annual Epidemiological Surveillance Burke DS, Hoke CH, Innis BL and Vaughn Report 1993. Ministry of Public Health, DW. Serotype-specific dengue virus Thailand. circulation and dengue disease in Bangkok, Thailand from 1973 to 1999. Am J Trop [5] Office of the Permanent Secretary for Public Med Hyg, 2003, 68(2): 191-202. Health. Annual Epidemiological Surveillance Report 2001. Ministry of Public Health, Thailand. [6] Igarashi A. Isolation of Singh’s Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J Gen Virol, 1978, 40: 531-544.
6 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
T.U. Brown*, K. Babb*, M. Nimrod*, C.V.F. Carrington*, R.A. Salas** and M.A. Monteil*#
*Faculty of Medical Science, University of the West Indies, St. Augustine, Trinidad **Caribbean Epidemiology Centre, Port-of-Spain, Trinidad
Abstract
A retrospective analysis of the 1996 DEN-1 epidemic in Trinidad was undertaken to better understand the clinical and demographic expression of dengue infection in the island during one of the larger epidemics in the past 10 years and following the reintroduction of DEN-1 into the island in 1991 after a gap of 14 years. A total of 393 laboratory-confirmed cases were identified. Of these, notes for 157 patients were available for analysis. The epidemic was island-wide, though most cases occurred in the most densely populated county of St. George. There was a slight predominance of females (51.6%) among the cases, and while all age groups were affected, older children and adults comprised the majority. South Asians among the population predominated. Overall, 27 clinical symptoms were reported. The most common were: fever (98.7%), generalized pain (96.2%) and anorexia (63.1%). Rash, arthralgia, retro-orbital pain and haemorrhage (all mentioned in the WHO clinical description for dengue fever) were reported in <50% of cases. Gastrointestinal symptoms were also very common and occurred in over two-thirds of cases at presentation. Bleeding manifestations were reported in 30% of patients and commonly involved the gastrointestinal tract. Features of DHF were noted in only six (4%) patients and there was one fatality. Deficiencies in documented clinical and laboratory monitoring of patients, coupled with a lack of population-specific laboratory reference ranges, may contribute to under- diagnosis of DHF in Trinidad. Keywords: Dengue, DHF, demographic analysis, clinical analysis, Trinidad.
Introduction and potentially fatal form of dengue infection known as dengue haemorrhagic The dengue viruses (DEN), of the fever/dengue shock syndrome (DHF/DSS) is Flaviviridae family, are mosquito-transmitted manifested, primarily through increased viruses that can cause dengue fever (DF). DF vascular permeability and shock. Dengue is an acute febrile illness characterized by exists as four distinct serotypes, DEN-1-4. intense headaches, retro-orbital pain, Infection with one serotype gives life-long myalgia, arthralgia, anorexia and rash. immunity for that virus but not to the others. Additionally, in a minority of cases, a severe Data suggest that secondary infection with a
#E-mail: [email protected]
Dengue Bulletin – Vol 28, 2004 7 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features serotype different from the primary infection case of DHF/DSS[10]. Since then, increasingly enhances the risk of developing larger outbreaks have been documented DHF/DSS[1,2]. The pathological processes throughout the 1980s and the 1990s, that lead to DHF/DSS remain poorly caused by DEN -2 and DEN-4. The first elucidated, but epidemiological studies have cases of DHF/DSS were reported during the highlighted viral and host factors that are 1993 DEN-1 epidemic[11], and this was associated with the development of followed in 1996 by yet another DEN-1 DHF/DSS[3]. outbreak which was characterized by a larger number of reported and confirmed An estimated 100 million people across cases of DHF/DSS[12]. the globe contract dengue annually with about 250,000 persons developing We present here a retrospective analysis DHF/DSS[4]. In the past 20 years, DF and of the 1996 DEN-1 epidemic in Trinidad. DHF/DSS have become significant public This study was done, firstly, to observe the health problems in the Caribbean and in clinical and demographic expression of the Latin America. The discontinuation of the dengue infection in the Trinidad population Pan American Health Organization (PAHO)- during one of the largest epidemics of the initiated Aedes aegypti eradication past 10 years. In particular, we wished to programmes in the 1970s caused a re- assess the ethnic distribution of the disease introduction of the mosquito vector into since there had been previous anecdotal virgin areas and a resurgence of dengue reports of a more severe disease in persons outbreaks throughout the region. of South Asian ancestry. Secondly, the study Furthermore, prior to the 1980s, dengue was done to gain an understanding of the outbreaks in the Americas were caused by clinical and laboratory infrastructure for single serotypes and were geographically dengue in Trinidad and Tobago as part of restricted and largely self-limiting, with no the development of our ongoing dengue- reports of DHF/DSS[5]. The first cases of related clinical research in the island. DHF/DSS documented in this region were identified during the Cuban DEN-2 epidemic in 1981[6] and the pattern of the Methods disease has changed dramatically since A retrospective analysis of the clinical and [4,5] then . Given the strong evidence linking demographic features of patients with the disease severity with secondary laboratory-confirmed dengue from the 1996 [7-9] heterologous infection , increased DEN-1 epidemic in Trinidad, West Indies, hyperendemicity is widely thought to be was conducted. Patients were identified one of the most significant factors from the laboratory database of The contributing to increases in the disease Caribbean Epidemiology Centre in Trinidad [3] severity in the region where all the four (CAREC). The CAREC is a subsidiary of the serotypes are now present. Pan American Health Organization (PAHO) The first dengue outbreak in over 20 and offers infectious disease testing of sera years in the island of Trinidad was reported from patients with suspected dengue in 1977. That outbreak caused by DEN-1 infections. Since 1975, CAREC has resulted in a few DF cases with no reported systematically conducted the isolation and
8 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features typing of dengue viruses in support of viral patients (40%). Information was incomplete surveillance programmes[12]. in 60 (15%) of these cases. The following is an analysis of the clinical and demographic A total of 1,200 samples from data available from the records of the 157 symptomatic suspected dengue cases in dengue-confirmed cases. Trinidad and Tobago were sent to CAREC in 1996 for laboratory confirmation, of which 393 were found positive. We identified Geographical and temporal these confirmed cases of dengue infection distribution of dengue and the medical institutions from which the virus infections samples had originated. The demographic, clinical and laboratory information related One hundred and fifty-four patients’ to each sample was collected from the addresses were available. The estimated respective medical records of each patient. county-specific incidence rates (per 10,000 population) based on these addresses Data were entered into a computerized ranged from 0.60 to 1.87, with the extreme Ò Excel database (Microsoft Excel 2000) and values occurring in the rural counties of analysed according to demographic Nariva/Mayaro and St. Andrews/St. David groupings such as ethnicity, county of respectively (Figure 1). The highest residence, age and gender and by proportion of these 154 confirmed cases frequencies of clinical symptoms. The (46%) occurred in the most densely incidence rates per 10,000 persons were populated county of St. George, in which [13] calculated using the 1990 census data . the capital, Port-of-Spain, is situated. There Proportional data were tested using the Chi was no significant difference in the rates of square test and statistical significance was infection among all the counties (P=0.513; established at P<0.05. All statistical analyses power of test with a=0.05:0.0.301). of the demographic and clinical data were Ò A review of the admission dates to done using SigmaStat Statistical Software Ó health care facilities for 136 dengue- for Windows Version 2.03 (Copyright confirmed patients showed that there was a 1992-1997 SPSS Inc.). Ethical approval for gradual increase in the number of cases this project was obtained from the Ethics from April to June, followed by a large rise Committee of the Faculty of Medical in July (29 cases) to a maximum in the Sciences, University of the West Indies, St. month of August (51 cases). In 1996, the Augustine. rainfall pattern (Figure 2) consisted of a large increase in the rainfall (171.7 mm) between Results April and May, followed by peak precipitation in June (333.1 mm). A The names of the 393 laboratory-confirmed comparison of dengue admissions with the dengue patients were obtained from the monthly precipitation suggests a direct CAREC database for 1996. Of these, 258 relationship between the rainfall and the (66%) could be traced to the source number of cases reported, with a two- institution or the treating doctor. Clinical month lag between the peak rainfall and the records were obtained for a total of 157 peak number of admissions.
Dengue Bulletin – Vol 28, 2004 9 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
Figure 1. Map of Trinidad showing county-specific incidence rates (per 10,000 persons) for dengue infections in 1996 (values in lower half of boxes) based on addresses from 154 confirmed cases. Values in the upper half of boxes show the proportion of the 157 cases found in each county. County divisions are according to the Regional Health Authorities of Trinidad & Tobago
Figure 2. Monthly admissions of 136 laboratory confirmed dengue cases and precipitation levels for the year 1996. Rainfall data obtained from the records of the Trinidad & Tobago Meteorological Office, Piarco, Trinidad
60 350 333.1
300 50 51 287.4 45 250 40 202.6 203.6 207.1 200 174.4 30 150 148.4 20 120.6 Rainfall 1996 (mm) 16 100 No. of confirmed cases 15 10 51.7 9 8 50 28.9 5 2 18.9 30.9 1 2 2 0 2 0 Jul Jan Jun Apr Feb Sep Oct Mar Aug Nov Dec May
Dengue cases Rainfall
10 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
Incidence rates of dengue rate in children (£15 yrs) was not infections in relation to significantly different from that in adults (>15 yrs). An analysis of the estimated gender, age and ethnicity incidence rates by county showed that there Gender and age data were available for all was variation in the age groups with the 157 cases. There was a slight female highest incidence rates for each county. In predominance of 51.6%. three counties, the highest estimated incidence rates occurred in persons 40 years The highest proportion of the cases and over; Caroni (50-59 yrs) (2.9%), Victoria occurred in the 30-34-year age group (40-49 yrs) (2.09%) and St. Andrews/St. (15.9% of the cases), followed by 5-9 and David (40-49 yrs) (3.65%). However, 10-14-year olds (14.0% and 14.6% estimated highest incidence rates were respectively) (Figure 3). The differences observed in younger patients in the counties among the groups were found to be highly of St George (0-9 yrs) (1.89%) and St. significant (P<0.001; power of test with Patrick (10-19 yrs) (2.46%). a=0.05: 0.998). The estimated incidence
Figure 3. Age distribution of 157 confirmed dengue cases
18%
16% 15.9% 14.6% 14% 14.0%
12%
10% 8.9% 8.9% 8.3% 8% 7.0%
6% 5.7% % of confirmed cases 4.5%
4% 3.8% 3.8% 2.5% 2% 1.9%
0% 0-4 5-9 60+ 10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 Age group
Dengue Bulletin – Vol 28, 2004 11 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
Self-reported ethnicity was available for counties. A statistically significant difference 136 patients. Of these, 50% were South was achieved in the county, St. Patrick, Asians, 35% were Africans, 11% were mixed, where the number of South Asian patients and 4% were Chinese and Europeans. As was higher than the African patients seen in Figure 4, a comparison of the (P=0.005; power of test with a=0.05: incidence rates by ethnicity for the whole 0.807), and in the whole group where the sample and by county showed that the incidence rate for South Asians was incidence rates of dengue infection were significantly higher than the mixed the greatest among South Asians for the population (P=0.016; power of test with group as a whole and in three of the four a=0.05: 0.70).
Figure 4. Incidence rates (per 10 000 persons) for 136 confirmed dengue infection patients by ethnicity for whole population and individual counties. County Victoria was excluded since ethnicity was reported in <50% of cases in that county
3 2.44
2.5 2.32
2 1.76 1.58 1.46 1.5 1.42 1.28 1.27 1.11 1.03
1 0.84 0.72 Incidence Rate 0.56 0.5 0.21
0 0 Whole Pop. St. George Caroni St. Patrick St. Andrew/ St. David County
South Asians Africans Mixed
Summary of clinical and gastrointestinal (GIT) complaints (nausea, haemorrhagic manifestations vomiting, diarrhoea and anorexia) were commonly reported (66.2%). Symptoms Overall, 24 clinical symptoms were reported highlighted in black indicate the symptoms in the medical records retrieved. The listed in the WHO clinical description of frequency of these symptoms at dengue fever[14]. Four of these symptoms, presentation is shown in Figure 5. Multiple arthralgia, retro-orbital (R/O) pain, rash and symptoms at the time of admission were haemorrhage, were recorded in <50% of generally reported. In this cohort, cases.
12 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
Figure 5. Frequency distribution of clinical symptoms at presentation in 157 dengue confirmed patients. Symptoms highlighted in black are part of the WHO clinical description of dengue fever
Fever, 156
Anorexia, 99 Pain, 151 Myalgia, 98 Headaches, 95 Vomiting, 90 Dehydration, 56 Upper resp., 52 Arthralgia, 50 R/O pain, 50 Chills, 45 Rash, 42 Diarrhoea, 38 Haemorrhage, 34 Adenopathy, 25 Conjunctivitis, 25 Dizziness, 20 Petechiae, 17 Malaise, 15 Rhinorrhoea, 14 Itching, 11 Hepatomegaly, 7 Short breath, 6 Photophobia, 4
0 10 20 30 40 50 60 70 80 90 100 Percentage of patients
In 34 patients (21.7%), haemorrhagic stool and rectal bleeding occurred in 50% of manifestations (HM) other than petechiae the patients. were reported. The reported haemorrhagic Six cases (approximately 4% of the 157 symptoms and their frequency of cases) could be defined as of DHF based on occurrence are illustrated in Figure 6. More the WHO criteria for the diagnosis of than one form of bleeding was noted in DHF/DSS[14]. Of these, five were female; some patients. In keeping with the high five were South Asians and one was mixed; frequency of gastrointestinal complaints four were children and one case (29-year reported, GIT bleeding including bloody old, South Asian female) died.
Dengue Bulletin – Vol 28, 2004 13 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
Figure 6. Type and frequency of haemorrhagic symptoms among 34 dengue confirmed patients who presented with haemorrhagic manifestations. Symptom highlighted in black is part of the WHO clinical description for DHF
Gastrointestinal bleeding, 29.4%
Gingival bleeding, 29.4% Bloody stool, 17.7%
Urinary tract bleeding, 14.7%
Nasal bleeding, 11.8% Not specified, 5.9%
Heavy menstrual flow, 5.9%
Rectal bleeding, 2.9%
Bloodstained sputum, 2.9%
Discussion densely populated county of St. George. There was a slight predominance of females We reviewed the clinical and demographic and the infection occurred in all age groups. features of 157 dengue-confirmed patients The estimated incidence rates were from a DEN-1 epidemic in Trinidad. This generally higher in patients of South Asian epidemic followed the reintroduction of this ethnicity. Gastrointestinal symptoms were serotype into the island in 1991 after an the most common clinical manifestations at absence of 14 years and the first presentation. Haemorrhagic manifestations, documentation of DHF in the island in 1993. when present, also commonly involved GIT. Approximately 1,200 samples from Six patients met the clinical and laboratory suspected symptomatic patients were sent criteria for DHF and, of these, one died. for laboratory confirmation and, of these, 393 (approx. 33%) proved to be positive. However, the 157 (40%) available records closely reflected the distribution of Clinical and demographic data reported here are from 40% of the confirmed cases patients from public and private hospitals, private doctors and health centres seen for from that outbreak. The outbreak was island-wide with the estimated incidence the 258 names that were traced to their respective sample sources. In the sample of rates being the highest in rural communities, though most cases occurred in the most 258 patients, public hospitals accounted for
14 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
71%, private hospitals 16.7%, and health plastic containers in which water can collect. centres and general practitioners 6.2% each. The main mosquito vector for dengue In the sample of 157 patients for whom viruses in the Caribbean and Latin America medical records were obtained, public is Aedes aegypti, which breeds primarily in hospitals accounted for 67%, private relatively clean, stagnant domestic water hospitals 17.2%, health centres 6.4% and containers. Local research has shown that general practitioners 10%. Over 80% of the important breeding sites for Aedes in patients were seen in hospitals. Trinidad and Tobago include outdoor drums, water tanks, tyres and small discarded The majority of cases (46%) in the bottles and cans[16], which get filled easily sample lived in the most densely populated with stagnant fresh water during the rainy county in Trinidad, St. George’s where season. The lag between the peak rainfall 36.5% of the total population of Trinidad and the number of confirmed dengue cases resides and where the Capital, Port-of-Spain might reflect the period of the mosquito- (POS), and its environs are located[13]. vector population expansion. Dengue, as a mosquito-borne infection, would be expected to spread rapidly in All age groups were affected but over populous areas, so the presence of a large 63% of them were adults (>15 years, the proportion of patients in and around POS is cut off age for paediatric patients in consistent with the experience elsewhere of Trinidad). Since DEN-1 had been dengue infection as an urban reintroduced in Trinidad in 1991, it is phenomenon[15]. Interestingly, our estimates expected that children aged 5 years or less of county-specific dengue incidence (based would constitute a susceptible population. on the sample of 157 patients) yielded rates Infection in this group would be primary in that did not differ significantly between rural which the majority would be asymptomatic. and urban counties in Trinidad. This This may account for children aged 0-4 suggests that despite the variation in years comprising only 8% of the sample. population density, DEN-1 infection was Despite the 1992/93 DEN-1 outbreak in fairly evenly spread across the island. The which over 3,060 cases were reported, island of Trinidad is merely 4,828 km2 and there were still many adults in 1996 who the similarity of the incidence rates across were susceptible to DEN-1. In total, there the island may reflect a combination of a were 3,588 reported cases that year. DEN-1 naïve population and easy However, based on the CAREC experience, accessibility to all parts of the island. only 33% of the clinically suspected dengue cases at that time were actually confirmed The DEN-1 1996 epidemic occurred in by laboratory tests, suggesting thereby that the rainy season with most admissions taking even during that outbreak, many cases with place in July and August, 1-2 months after clinical features consistent with dengue may the peak rainfall for that year in June. in fact not have been of dengue infection. Rainfall is considered to be a risk factor for Clinical symptoms of dengue are known to the development of dengue outbreaks, overlap with many other infections such as especially where there is improper storage measles, hepatitis A, rubella and of water or the presence of receptacles in leptospirosis[17-19]. the domestic environment such as discarded
Dengue Bulletin – Vol 28, 2004 15 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features
The high proportion of cases in the 30- increased incidence in South Asians is 34, 5-9 and 10-14-year age groups suggests interesting and merits further analysis with the possibility of spread between children larger cohorts of dengue-confirmed cases. and parents. In Trinidad and Tobago, 25% The range of symptoms reported in the of the heads of households are 30-39 years 157 Trinidadian dengue-confirmed patients of age with an average of 1.99 children per is similar to those described for patients with household[13]. Trinidadian women often start acute dengue in other parts of the world[18]. their families in late teenage or early Recent-onset fever and generalized body twenties, so parents aged 30-34 years will pains occurred in almost all patients. have children in the age groups 5-9 or 10- Headache, anorexia and myalgia, which 14 years. often occur in the prodrome, were noted in The racial composition of this sample 60% or more of the cases. Gastrointestinal differs from that reported for the general and upper respiratory symptoms were also population, which is composed of South common complaints at presentation. Asians (40.3%), Africans (39.6%), mixed Gastrointestinal symptoms have been (18.4%) and the rest (1.6%)[13]. Thus, there is described as predominant clinical an overrepresentation of South Asians (50%) manifestations in epidemics in which there in our sample with fewer persons of African is an adult-susceptible population[21]. The origin (35%) and of mixed race (11%). Since 1996 epidemic was almost wholly due to hospitalized patients account for 84% of the DEN-1 and the bulk of the study population study population, it may reflect an increased comprised older children and adults. number of South Asians being hospitalized The symptoms of classic dengue as for dengue infection as compared with described by WHO occurred in fewer than other racial groups. Teelucksingh[20] has expected numbers of patients. For example, previously reported a higher incidence of retro-orbital pain and arthralgia were more severe dengue infection and mortality reported in 32% of patients and rash only in South Asian people (colloquially referred noted in 27%. While this may be due to a to as East Indians) in Trinidad. true low prevalence of these symptoms in These data cannot exclude differences the study population, the cultural expression in the environmental factors that might also of symptomatology may also contribute to a increase the exposure of South Asian people falsely low detection of these symptoms. For to more mosquito bites. South Asians have example, in Trinidad, all pains in the head traditionally been the predominant racial region, including orbital pain, are commonly group in agriculture and animal rearing in referred to as “headache”. Similarly, joint Trinidad. In rural communities on the island pains may often be included in “muscle” there is relatively poor piped water supply, pain or be part of a more generalized “body resulting in more water storage, thus pains”. The rash, which is usually transient, providing ideal domestic breeding sites for may be easily missed and even more so in Aedes aegypti[16]. Therefore, farmers in rural dark-skin complexions. areas of Trinidad may be at an increased Haemorrhagic manifestations, including risk of mosquito bites and consequent petechiae, were noted in 51 (32.5%) dengue infection. The tendency towards an patients. This is consistent with other reports
16 Dengue Bulletin – Vol 28, 2004 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features of the prevalence of haemorrhage in dengue the hospitals that were visited. For example, fever[18]. In keeping with the global chest X-rays were performed in fewer than experience, the bleeding manifestations 10% of the patients admitted to private occurred at many sites. Despite the bleeding hospitals but in over 60% of the patients in manifestations in almost a third of the one public hospital. Serial blood tests were patients, the WHO criteria for DHF were performed in approximately 20% of dengue fulfilled only by 6 (4%) patients[14]. Of these, cases in private hospitals but in 40-80% of five were female and one was male, five patients admitted to three public were South Asian and one was of mixed institutions[22]. ethnicity and four were children and two Furthermore, there are no locally were adults. One patient, a 29-year-old developed reference ranges for haematocrit female of South Asian ethnicity died. The by age and sex in use in many laboratories female gender and children were more in Trinidad where the reference ranges prone to the development of DHF/DSS in developed in primarily the Caucasian dengue-endemic areas[3]. While the 1996 populations have been adopted for use. epidemic was predominantly DEN-1, DEN- Moreover, haemoglobinopathies such as 2 was already endemic in the island[12]. thalassaemia and sickle cell traits are not Further, the predominance of persons of uncommon among the Trinidadian South Asian ancestry in this group was also population and these persons tend to have consistent with the observation that certain normal haemtocrit levels below the races were more susceptible to DHF/DSS reference ranges in use. Consequently, such than others. patients presenting with 20% or more The number of cases of DHF/DSS may increase in haemoconcentration may fall have been underestimated. One of the within the adopted reference range, and if essential WHO criteria for establishing the there is no repeat of haematocrit diagnosis of DHF/DSS is the proof of concentration in the convalescent period, increased vascular leakage such as an evidence of haemoconcentration would increase of at least 20% in average have been completely missed and the haematocrit for age and sex; a drop in patient wrongly diagnosed as DF instead of haematocrit of 20% or more following a mild form of DHF[22]. treatment, or clinical evidence of the same In summary, the DEN-1 epidemic in such as pleural effusion, ascites or Trinidad in 1996 was characterized by a hypoproteinaemia[14]. However, since the large number of clinically suspected but plasma leakage can be transient and only unconfirmed cases of dengue infection. evident by laboratory testing in milder forms Laboratory analysis of a third of all reported of DHF cases (e.g. Grade 1 and 2 DHF), it is cases revealed that most of these cases were possible to miss patients with these forms of not of dengue. Furthermore, the variability DHF if clinical monitoring of the in the data available from clinical records, haematocrit levels or tests such as lateral wherever these could be found, resulted in decubitus chest X-rays to detect small less than optimal information retrieval for pleural effusions are not performed. The further analysis. From the available data set, frequency of chest X-rays and serial blood the Trinidadian dengue-infected patients tests in this cohort of patients varied with were mainly older children and adults from
Dengue Bulletin – Vol 28, 2004 17 A Retrospective Study of the 1996 DEN-1 Epidemic in Trinidad: Demographic and Clinical Features rural and urban communities. South Asians cases but features of DHF were noted in were predominant. A wide range of clinical only 4%. Improvement in clinical diagnosis manifestations was recorded, but the most and record-keeping is urgently required to common were gastrointestinal. Bleeding underpin future clinical research in Trinidad. manifestations occurred in over 30% of DF
References
[1] Halstead SB, Chow J and Marchette NJ. [9] Thein S, Aung MM, Shwe TN, Aye M, Zaw Immunologic enhancement of dengue virus A, Aye K, Aye KM and Aaskov J. Risk factors replication. Nature New Biology, 1973, 243: in dengue shock syndrome. American 24-26. Journal of Tropical Medicine and Hygiene, [2] Morens DM, Halstead SB and Marchette NJ. 1997, 56(5): 566-572. Profiles of antibody-dependent enhancement [10] Le Maitre. Dengue fever epidemic in of dengue virus type 2 infections. Microbial Trinidad & Tobago: 1977-1978. Dissertation Pathogenesis, 1987, October, 3(4): 231-237. for the Diploma in Public Health, University [3] Halstead SB. Epidemiology of dengue and of the West Indies, Mona, Jamaica, 1978; dengue haemorrhagic fever. In: Gubler DJ, Chapter 2: 11-18. Kuno G (Eds.), Dengue and dengue haemorrhagic fever. New York: CAB [11] Teelucksingh S, Mangray AS, Barrow S, International, 1997: 23-44. Jankey N, Prabhakar P and Lewis M. [4] Gubler DJ. Dengue and dengue Dengue haemorrhagic fever / dengue shock haemorrhagic fever: Its history and syndrome: an unwelcome arrival in Trinidad. resurgence as a global public health problem. West Indian Medical Journal, 1997, 46(2): In: Gubler DJ, Kuno G (Ed.), Dengue and 38-42. dengue haemorrhagic fever. New York: CAB [12] Campione-Piccardo J, Ruben M, Vaughan H International, 1997: 1-23. and Morris-Glasgow V. Dengue viruses in [5] Gubler DJ. Dengue and dengue the Caribbean. Twenty years of dengue virus haemorrhagic fever. Clinical Microbiology isolates from the Caribbean Epidemiology Review, 1998, 11(3): 480-496. Centre. West Indian Medical Journal, 2003, [6] Pinheiro FP and Corber SJ. Global situation 52(3): 191-198. of dengue and dengue haemorrhagic fever, [13] Central Statistical Office of the Government and its emergence in the Americas. World of Trinidad & Tobago. http://www.cso.gov.tt. Health Statistics Quarterly, 1997, 50(3-4): 161-169. [14] World Health Organization Regional Office for South-East Asia. Prevention and control [7] Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science, of dengue and dengue haemorrhagic fever: 1988, 239 (4839): 476-481. comprehensive guidelines. http://w3.whosea.org/ dengue/searono29/pdff.htm. [8] Kliks SC, Nisalak A, Brandt WE, Wahl L and Burke DS. Antibody-dependent [15] Kuno G. Factors influencing the transmission enhancement of dengue virus growth in of dengue viruses. In: Gubler DJ, Kuno G human monocytes as a risk factor for (Ed.), Dengue and dengue haemorrhagic dengue haemorrhagic fever. American fever. New York: CAB International, 1997, Journal of Tropical Medicine and Hygiene, 61-88. 1989, 40(4): 444-451.
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[16] Focks DA and Chadee DD. Pupal survey: an [20] Teelucksingh S, Lutchman G, Udit A and epidemiologically significant surveillance Pooransingh S Childhood dengue shock method for Aedes aegypti: an example using syndrome in Trinidad. West Indian Medical data from Trinidad. American Journal of Journal, 1999, 48(3): 115-117. Tropical Medicine and Hygiene, 1997, [21] World Health Organization Regional Office 56(2): 159-167. for South-East Asia. Risk factors of [17] Dietz VJ, Nieburg P, Gubler DJ and Gomez I. DEN/DHF epidemics in SEAR countries. Diagnosis of measles by clinical case http://w3.whosea.org/Den1/risk.htm. definition in dengue endemic areas: [22] Nimrod M, Brown TU, Babb K, Carrington implications for measles surveillance and CVF, Salas RA and Monteil MA. An analysis control. Bulletin World Health Organization, of laboratory parameters from confirmed 1992, 70(6): 745-750. cases of dengue haemorrhagic fever in the [18] George R and Lum LCS. Clinical spectrum of 1996 Trinidad epidemic. West Indian dengue infection. In: Gubler DJ, Kuno G Medical Journal, 2003, 52 (Suppl. 1): 17. (Ed.), Dengue and dengue haemorrhagic fever. New York: CAB International, 1997:1- 22. [19] Levett PN, Branch SL and Edwards CN. Detection of dengue infection in patients investigated for leptospirosis in Barbados. American Journal of Tropical Medicine and Hygiene, 2000, 62(1): 112-114.
Dengue Bulletin – Vol 28, 2004 19 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002
Maria Lucia F. Penna#
Escola Nacional de Saude Publica, Fundação Oswaldo Cruz, Rua Leopoldo Bulhões 1480, DENSP, 21041-210 Rio de Janeiro, RJ, Brazil
Abstract Dengue virus serotype 3 was introduced in the Rio de Janeiro metropolitan area in January 2001 which produced a large epidemic during 2001 and 2002. This study looks into the relationship between the urban socioeconomic organization and the dengue attack rate during the epidemic in Rio. This study uses secondary data published in the data website of the city administration, including variables related to sanitation, use of the city area, tax collection, population density, education, income and life expectancy. The model includes as predictors the proportion of households with a well, the proportion of the available area in the city used for commerce and services in general, the proportion of city taxes collected from industries, the mean per capita income and the residential area per inhabitant, all with a statistical significant level of less than 0.05. The model explains 71% of the attack rate variance. The variables included in the model indicated that dengue distribution in the city was related to people’s socioeconomic status and the city organization. Variables that correlate with the movement of people across towns have emerged as the most significant. Keywords: Dengue virus, socioeconomic status, urban organization, Rio de Janeiro.
Introduction introduced in the country in 1990 and 2001, respectively[2]. Once introduced in the Rio After decades of freedom from dengue virus de Janeiro metropolitan area, DEN -3 caused infection, Brazil experienced an outbreak of a large epidemic during 2001 and 2002. In DEN-1 and DEN-4 in areas close to the 2003, DEN-1, DEN-2 and DEN-3 were in borders with Venezuela in 1981-82. circulation in all except the two southern Intensive vector control measures states of the country, with 341,092 cases successfully controlled this outbreak and reported (Figure 1)[3]. checked its spread to other areas in the Despite the use of a variety of control country[1]. In 1986, DEN-1 was introduced in the Rio de Janeiro metropolitan area, strategies, dengue control is a major public health challenge in the world. Demographic which spread to other areas, causing a massive epidemic that later covered the changes occurring in developing countries due to widespread rural-urban migration entire country. DEN -2 and DEN-3 were also
# E-mail: [email protected]; Tel.: 55 21 38829212; Fax: 55 21 38829124
20 Dengue Bulletin – Vol 28, 2004 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002 since the 1960s have resulted in of Aedes aegypti in the 70s is no longer overcrowded cities with multiple deficiencies, applicable to the reality of the social, particularly in housing and basic sanitation. demographic, economic and political The strategy that resulted in the eradication situation in South American countries[4-6].
Figure. Circulation of dengue virus in the Brazilian states, 2003
None DEN-1 and -3 DEN-1 and -2 DEN-1, -2 and -3
Predicting the risk of dengue correctly control measures to be more effective with based on sociocultural factors has been the responsibilities divided between the goal of many authors[7,8], but the dynamics government and communities as per of dengue transmission in urban settings are priorities established on the basis of reliable still poorly understood[9]. The understanding data. of the virus transmission dynamics requires a The present study looked into the theoretical framework that includes relationship between the urban individuals, households and behavioural risk socioeconomic organization and the dengue factors as well as the administrative aspects attack rate during the DEN-3 epidemic in of city management services, use of city Rio de Janeiro city in 2001-2002. It is space and movement of people across the mainly an exploratory study, based on metropolis. This understanding can help available secondary data, that is likely to
Dengue Bulletin – Vol 28, 2004 21 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002 throw some light on how urban settings residence (HOUSE), for industry contribute to dengue transmission. (INDUSTRY), for commerce or service (COMMERCE); the proportion of unused areas (UNUSED); the proportion of the total Methods area of parks and squares (PARKS); the amount of city tax collected per inhabitant Description of study area (TAX); the proportion of city tax of Rio de Janeiro city is located at -22°54'23" commercial origin (COMTAX), industrial south latitude and -43°10'21" west longitude, origin (INDTAX) and service origin (SERVTAX); the mean per capita income at the seashore, with an urban area of 1,255 (INCOME); the life expectancy (LE); the km2, including inland and continental proportion of literacy among inhabitants waters. The municipal area was divided into (LITERACY); and the proportion of children 26 administrative regions (ARs) in 1999 aged 7 to 15 years attending school which were used as spatial units for analysis. The climate is tropical, hot and humid, with (SCHOOL) as independent variables. All proportions were presented as percentages local variations due to altitude differences, and income and taxes as 1,000 reais vegetation and proximity to the sea. The (Brazilian currency). The independent mean annual temperature is 22 °C, with variable was the attack rate for DEN-3 high daily means in summer (30 to 32 °C). epidemic, calculated by the number of Rainfall is 1,200 to 1,800 mm per year, concentrated in summer from December to reported dengue cases during 2001-2002 March. The city has the lowest rate of divided by the population estimate for January 2001, per 100,000 inhabitants. population growth among the Brazilian capital cities, 6.9% between 1991 and 2000. Statistical methods Database A multiple regression model was adjusted to the data using stepwise forward approach, This study used secondary data published in with F to enter = 1 and F to leave = 0.95, the website of the city administration[10], and the author interference to limit the including the proportion of households number of steps in order to prevent a belonging to the sewage collection system (SEWAGE), the proportion of households saturated model. The independent variable belonging to the water supply system was given a log transformation. The software used was Statistica, from Statsoft[11]. (WATER), the proportion of households with a well (WELL), the proportion of households with waste collection by the city authority Results (WASTE), the number of inhabitants per household (INH/HOUSE), the area of Table 1 shows the correlation coefficients residential properties (square metres) per between the independent variable and all inhabitant (M2/INH), the proportion of those variables presented in the model, households in slums (SLUMS), the along with their range. The model included proportion of the total area built for as predictors the proportion of households
22 Dengue Bulletin – Vol 28, 2004 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002 with a well, the proportion of the available partial correlation coefficients for the area used for commerce and services, the variables included in the model, which is a proportion of city taxes collected from measure of the association of each variable industries, the mean per capita income and when the other variables are controlled for, the built household area per inhabitant making it possible to rank their effect. The (Table 2), all with a statistical significant level residues fitted well into a normal less than 0.05. The model explains 71% of distribution and presented no correlation the attack rate variance. Table 3 shows the with the predictors.
Table 1. Descriptive statistics and correlation coefficient with log (attack rate)
Variable Mean Minimum Maximum Correlation
WASTE 1 0.93 1 0.09 SWAGE 1 0.30 1 0.09 WATER 1 0.88 1 -0,30 WELL 0 0.00 0 0,39 M2/INH 24 5.56 56 0.20 SLAM 15 0.00 41 0.04 INH/HOUSE 3 2.36 4 -0.05 LE 72 65.99 78 -0.01 LITERACY 96 90.74 99 -0.38 SCHOOL 90 67.66 113 -0.36 INCOME 670 212.21 2229 -0.13 HOUSE 69 9.89 89 -0.26 COMMERCE 12 3.40 39 0.27 INDUSTRY 7 0.11 21 0.01 UNUSED 981 0.99 19729 0.02 PARKS 2 0.00 25 -0.02 COMTAX 0 0.00 3 -0.20 INDTAX 0 0.00 1 0.30 SERVTAX 99 94.30 100 0.03 TAX 396 2.78 7157 0.19
Dengue Bulletin – Vol 28, 2004 23 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002
Table 2. Regression summary (Standard regression coefficient, regression coefficient, t and P value)
Standard Standard Beta error of B t (20) P level error of B Beta
Intercept 6.360585 0.184526 34.46979 0.000000 WHELL 0.33653 0.145309 7.640343 3.299001 2.31596 0.031294 COMMERCE 0.34087 0.136198 0.024121 0.009638 2.50272 0.021111 INDTAX 0.46826 0.126483 2.084555 0.563072 3.70211 0.001410 INCOME -1.06086 0.273221 -0.001142 0.000294 -3.88279 0.000925 M2/INH 1.12201 0.294558 0.048058 0.012616 3.80912 0.001099
R= 0.84282066; R2=0.71034667; F(5,20)=9.8096; P<0.00007; Standard error of estimate=0.35169
Table 3. Partial and semi-partial correlation
Beta in Partial correlation Semi -partial correlation
WELL 0.33653 0.459859 0.278711 COMMERCE 0.34087 0.488354 0.301187 INDTAX 0.46826 0.637673 0.445527 INCOME -1.06086 -0.655600 -0.467271 M2/INH 1.12201 0.648419 0.458405
Discussion dealt with by the logarithmic transformation of the attack rate[12], as shown by the The exploratory aspect of this study justifies residual analysis. the presentation of a high number of urban and socioeconomic variables in the model. The proportion of households with a These variables present high covariance that well is really a proxy variable of the induces the use of the forward stepwise discontinuity of water supply. The proportion of households that have access method. To avoid a saturated model, the to the city’s water supply network is 95.07% author restrained the number of steps and for the entire city, indicating a high coverage assured that only significant variables were kept in the model by using a relatively high of the city’s water supply network. But the value of F to leave. The rupture of the water supply is not equally effective in all homocedasticy assumption due to the the administrative regions (ARs), with some areas having chronic problems, mainly nature of data (proportions) was successfully discontinuous supply. The presence of a
24 Dengue Bulletin – Vol 28, 2004 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002 well, although small, only in 1.12% of the affecting the presence of potential breeding households (maximum of 11.85% in sites for the vector. This discussion Paqueta AR and 11.06% in Guaratiba AR) is reinforces the relevance of multi-level a proxy variable for the discontinuity analysis in the evaluation of dengue risk problem, for it is a solution that involves factors. financial expenses which is only justified in The inclusion in the model of the the face of an important and lasting problem proportion of the area used for commerce of supply. These results point to the fact that and services and the proportion of city taxes water supply is an important issue in dengue paid by industry shows that urban control, but only in the context of lack of or organization plays an important role in the irregular water supply where the population distribution of dengue. These two variables is forced to resort to storage, which creates are proxy variables for the movement of breeding sites for the vector and not in the people across ARs. The presence of usual context of adequate supply as commerce and general services was suggested by other authors[13].. represented by the proportion of the area The model included mean per capita and the presence of industry as the income as a protective factor meaning that proportion of city taxes, because those are low socioeconomic status of residents of an the variables correlated to the intensity of AR is a risk factor for dengue transmission. A the movement of people. The luxury study in Brazil found no association commerce and services may collect more between the socioeconomic status and taxes than the popular commerce and dengue risk at the individual level[14] and services, but the areas of popular commerce suggested that the previous finding of such and services have a much bigger flow of an association at the aggregate level[15,16] was people. On the other hand, the relative size a fallacy. However, it should be noted that of the industrial area is related to the type of studies that focus on factors at individual industry, as for instance big industrial level are insufficient to address the storage areas, and not to the production or ecological links in the causal chain. the number of workers. Industries that Ecological studies may be, as pointed out by collect more taxes have a larger production Koopman and Languini[17], the only way to and are more likely to have a higher number study risk factors for infectious diseases. The of workers. Other authors[18] indicate that risk of dengue virus infection is not the probability of being reached by a new dependent on the physiological dengue virus is correlated to the intensity of characteristics of individuals, but on the communication among people and the environmental characteristics of the area density of traffic and the road network. It is where the group of individuals live. This important to note that the diffusion of the includes other individuals, the natural epidemic by proximity may be less environment and the way it is transformed important than the diffusion caused by the by humans. The mean per capita income circulation of people in central areas in big has to be interpreted as an environmental cities, which is supported by the present measure concerning the area and not as an finding. This area initially imports infected aggregate measure of individuals, because it individuals from the initial foci of the virus impacts the neighbouring households as where it is newly introduced, resulting in well as the nearby public areas, thus higher local transmission, which, in turn,
Dengue Bulletin – Vol 28, 2004 25 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002 results in exportation of infected individuals people across cities and its significance in to other areas, reinforcing the epidemic all dengue epidemics. over the city. This fact clearly indicates the priority of mosquito control in areas with high levels of population mobility, such as Conclusion the Rio de Janeiro city central area. The current efforts to control dengue The model also included the area of demand a more comprehensive approach, residential property per inhabitant as a risk including health education, community factor for dengue. This variable is closely participation, garbage disposal and proper correlated with the socioeconomic status urban planning, besides chemical and represented by the mean per capita income biological mosquito control measures. In (R=0.864605) that is controlled in the order to improve the efficiency of control model, meaning that, for the same efforts, these activities have to concentrate socioeconomic status, a bigger area of on areas and populations at higher risk, residential property per inhabitant results in which implies early identification of higher bigger dengue risk. This is possibly due to incidence periods and areas and their the existence of empty spaces in properties, characteristics[5]. such as backyards, thus creating greater In Rio de Janeiro, emphasis has been opportunities for the existence of the vector placed on bringing about behavioural breeding sites. This hypothesis has to be changes in the communities to make an investigated for its importance in the impact on the determinants and risk factors selection of priorities for vector control in of dengue through educational interventions. private residences as well as in defining the Health authorities and the press attributed contents of educational interventions. the main responsibility of the problem to The results of the present study allow public behaviour, not clearly distinguishing the establishment of priorities in vector between the responsibilities of the control and educational interventions, government and that of the private citizen[6]. highlighting the importance of the flow of
References
[1] Osanai CH, Travassos-da-Rosa APA, Amaral [3] Secretaria de Vigilância em Saúde. Dengue S, Passos ACD and Tauil PL. Surto de Boletim semana 16; 2004 online dengue em Boa Vista, Roraima. Revista do http://dtr2001.saude.gov.br/svs/epi/dengue/ Instituto de Medicina Tropical de Sao Paulo, boletim/pdfs/Boletim%20semana%2016%20 1983, 1: 53-54. 2004.pdf. [2] Nogueira RM, Miagostovich MP, Schatzmayr [4] Tauil PL. Urbanização e ecologia do dengue. HG, dos Santos FB, de Araujo ES, de Filippis Cad Saúde Pública, 2001, 17(1): 99-102. AM, de Souza RV, Zagne SM, Nicolai C, [5] Pan American Health Organization. A Baran M and Teixeira Filho G. Dengue in blueprint for action for the next generation: the State of Rio de Janeiro, Brazil, 1986- dengue prevention and control. Washington: 1998. Mem Inst Oswaldo Cruz, 1999, 94(3): PAHO, 1999, 1-14. 297-304.
26 Dengue Bulletin – Vol 28, 2004 Ecological Study of Rio de Janeiro City DEN-3 Epidemic, 2001-2002
[6] Penna MLP. Dengue control: a challenge for [13] Forattini Oswaldo Paulo and e Brito the public health system in Brazil. Cad Marylene de. Reservatórios domiciliares de Saúde Pública, 2003, 19(1): 305-309. água e controle do Aedes aegypti. Rev Saúde Pública, 2003, 37: 676-677. [7] Hayes JM, Garcia Rivera, Flores Reyna, Suarez Rangel, Rodríguez Mata, Coto [14] Teixeira M, Barreto ML, Costa M, Ferreira Portillo, Baltrons Orellana, Mendoza LD, Vasconcelos PF and Cairncross S. Rodríguez, De Garay, Jubis Estrada, Dynamics of dengue virus circulation: a Hernández Argueta, Biggerstaff BJ and Rigau silent epidemic in a complex urban area. Perez. Risk factors for infection during a Trop Med Int Health, 2002, 7: 757-762. severe dengue outbreak in El Salvador in [15] Vasconcelos PF, de Menezes, Melo LP, 2000. Am J Trop Med Hyg, 2003, 69: 629- Pesso ET, Rodríguez SG, da Rosa, Timbo MJ, 633. Coelho IC and Montenegro F. A large [8] Ashford DA, Savage HM, Hajjeh RA, epidemic of dengue fever with dengue McReady J, Bartholomew DM, Spiegel RA, hemorrhagic cases in Ceará State, Brazil, Vorndam V, Clark GG, Gubler DG. 1994. Rev Inst Med Trop Sao Paulo, 37: Outbreak of dengue fever in Palau, Western 253-255. Pacific: risk factors for infection. Am J Trop [16] Figueiredo LT, Cavalcante SM and Simoes Med Hyg, 2003, 69: 135-140. MC. Dengue serologic survey of [9] C‚mara tecnica e cientifica de schoolchildren in Rio de Janeiro, Brazil, in assessoramento ao controle do dengue. 1986 and 1987. Bull Pan Am Health Organ, Recomendações à Secretaria de Estado de 1990, 24: 217-225. Saude. Secretaria de Estado de Saúde do Rio [17] Longini IM Jr, Koopman JS, Haber M and de Janeiro, Rio de Janeiro, RJ, 2002, 1-11. Cotsonis GA. Statistical inference for [10] Instituto Municipal de Urbanismo Pereira infectious diseases. Risk-specific household Passos, Secretaria Municipal de Urbanismo, and community transmission parameters. Cidade do Rio de Janeiro. Website: Am J Epidemiol, 1988, 128(4): 845-859. http://www.armazemdedados.rio.rj.gov.br/, [18] Muttitanon W, Kongthong P, Kongkanon April 2004. C, Yoksan S, Gonzalez JP and Barbazan P. [11] StatSoft Inc. Electronic Statistics Textbook. Spatial and temporal dynamics of dengue Tulsa, OK: StatSoft. http://www.statsoft.com/ haemorrhagic fever epidemics In: textbook/stathome.html, 2004. Conference Proceedings of Map Asia 2002, GIS Development, 2002, [12] Selvin S. Epidemiologic analysis: a case- http://www.gisdevelopment.net/application/ oriented approach. Oxford University Press, health/planning/healthp0010c.htm. New York, 2001: 321.
Dengue Bulletin – Vol 28, 2004 27 Sero-epidemiological and Virological Investigation of Dengue Infection in Oaxaca, Mexico, during 2000-2001
A. Cisneros-Solano*, M.M.B. Moreno-Altamirano**#, U. Martínez-Soriano***, F. Jimenez-Rojas†, A. Díaz-Badillo‡ and M.L. Muñoz‡
*Escuela Nacional de Medicina y Homeopatía **Escuela Nacional de Ciencias Biológicas-Instituto Poltécnico Nacional ***Universidad Autónoma “Benito Juárez” de Oaxaca †Lab. Est. Salud Pública de Oaxaca ‡Centro de Investigación y Estudios Avanzados-IPN
Abstract A sero-epidemiological-cum-virological investigation was carried out in Oaxaca, Mexico, during 2000- 2001 to assess the incidence of dengue infection and the circulating viruses. A total of 200 serum samples reportedly from dengue patients, based on clinical diagnosis, were collected from Oaxaca´s Central Laboratory of Public Health (in the capital city of the state of Oaxaca). The samples were initially collected from ten regional health centres located across Oaxaca. The sample population for the study included both sexes and age groups with clinical signs compatible with dengue infection. All samples were tested for the presence of dengue virus, mainly by MAC- ELISA and RT-PCR. Ninety-four out of 100 serum samples suspected of dengue were confirmed to be positive. Thirty- two were found positive by MAC-ELISA and 58 were positive by RT-PCR. In addition, the RT-PCR analysis showed that the prevalent serotype in the localities in the study area was DEN-2. However, one isolate of DEN-1 and another of DEN-4 were also detected. The number of infected females was higher than that of infected males and the most affected age group was of people aged under 35 years. The study also highlighted that the sensitivity and specificity of diagnostic tools were crucial for epidemiological studies.
Keywords: Serodiagnosis, MAC-ELISA, RT-PCR, DEN-2, Oaxaca, Mexico.
Introduction by Gubler[3], factors such as demographic and social changes are responsible for the In recent years, dengue fever (DF) / dengue re emergence of dengue. Mexico is haemorrhagic fever (DHF) has emerged as considered an endemic country for dengue major health problem in Mexico. In 1960, and it is reported that major epidemics of the Aedes aegypti mosquito was eradicated DEN-1 occurred on the eastern coast of but it reappeared in 1965[1,2]. As pointed out Mexico during 1979-1980. In 1984-1985,
# E-mail: [email protected], [email protected]; Tel. (5255) 55729-6300 Ext. 62370, Fax (5255) 55396-3503
28 Dengue Bulletin – Vol 28, 2004 Dengue Investigation in Oaxaca, Mexico dengue was diagnosed in 25 of the 32 states Oaxaca is located in the subtropical of Mexico. By then, DEN-1, DEN-2 and region of Mexico at about 1,600 metres DEN-4 were present in the country, and in above sea level (Figure 1A). There is high 1995, DEN-3 was circulating as well. Several demographic pressure and migration to cases of DHF were also confirmed[4]. In different urban zones is common, resulting subsequent years dengue achieved in the establishment of scattered human endemicity in the country. settlements with deficient public services. All these factors contributed to the propagation As per the records of the Mexican of the Aedes aegypti mosquito, resulting in Health Office[5] (Secretaría de Salud, SS), a dengue outbreaks every year in most of higher number of dengue cases were Oaxaca´s communities[6]. recorded in the states of Nuevo León, Tamaulipas, Veracruz and Oaxaca during 1998-2001.
Figure 1. Oaxaca, Mexico (A) United States of México. Oaxaca is located in the west coast (B) Oaxaca is divided by the health authorities in six jurisdictions (I-VI)
Dengue Bulletin – Vol 28, 2004 29 Dengue Investigation in Oaxaca, Mexico
To assess the dengue situation, days of the onset of fever and were epidemiological studies were undertaken to processed for anti-dengue IgM detection make an estimate of the incidence of using IgM capture ELISA (MAC-ELISA) as dengue virus infection and the circulating described by Vorndam et al.[8] Samples from serotypes in some selected endemic areas of healthy donors were obtained at about the Oaxaca during 2000-2001. same time. As a routine practice and with the idea Materials and methods of recording epidemic data, the suspected dengue samples already clinically diagnosed Population study in community health centres were sent to the Central Laboratories in the city of The state of Oaxaca is located on the west Oaxaca (Laboratorio Estatal de Salud coast of Mexico. The Mexican Health Office Pública del estado de Oaxaca, Secretaría de has divided it into six jurisdictions: (I) Salud). In this laboratory, the presence of Central Valleys, (II) Tehuantepec isthmus, dengue virus was confirmed by MAC-ELISA (III) Tuxtepec, (IV) The Coast, (V) The and RT-PCR. Mixteca, and (VI) The Sierra. The presence of dengue virus has been registered in all six Dengue virus isolates jurisdictions (Figure 1B). This study was carried out in ten municipalities distributed Aedes albopictus C6/36 cells were grown in in five jurisdictions in the state of Oaxaca. 48-well tissue culture plates as described by [9] 5 The study population included both sexes Igarashi . Briefly, 2X10 cells were plated in and all age groups[7]. Two population groups 1 ml of minimum essential medium (Gibco- were included in this study: one group BRL, Grand Island, N.Y.) supplemented with consisting of 200 serum specimens from 7% fetal bovine serum (Sigma Chemical Co., patients manifesting signs and symptoms of St. Louis, Mo) and 1% glutamine, vitamins dengue infection, and another group of 50 and nonessential amino acids. After 24 serum samples from healthy controls, all hours of culture, 100 ml of every sera diluted from the same jurisdictions. 1:10 was added to the corresponding well. The mixture was then gently shaken and Sample collection and diagnosis incubated for 60 minutes at room temperature. Cells were then washed with of dengue serum-free medium and cultured at 28 °C Human sera were obtained from 200 with complete medium for at least 10 days. patients presenting clinical manifestations of Cells were harvested for RT-PCR diagnosis. dengue and tested for anti-dengue IgM antibodies. Serum samples were collected RNA extraction by venipuncture, using Vacutainer tubes Total RNA was extracted either from 100 ml (Becton-Dickinson). The clinical samples of serum or from cultured cells by using corresponded with dengue cases reported Trizol LS (GIBCO BRL, Gaithersburg, MD.) during 2000-2001. Dengue-infected according to the manufacturers’ samples were obtained during the first five
30 Dengue Bulletin – Vol 28, 2004 Dengue Investigation in Oaxaca, Mexico recommendations. Ethanol-precipitated RNA reaction mixtures were electrophoresed and was recovered by centrifugation and air-dried. visualised under UV light after ethidium The RNA pellet was re -suspended in 50 ml of bromide staining of the gels. Diethyl-pyrocarbonate (Sigma)-treated water (DEPC water) and used as a template for RT- PCR. Results Diagnosis of the samples by MAC- RT-PCR ELISA Synthetic oligonucleotide primer pairs were Two hundred serum samples initially designed based on published sequence data reported as suspected positive for dengue, for each of the four serotypes of dengue[10,11]. based on clinical reports from the hospital Four fragments of an expected size of 482 where patients were hospitalised, were bp (DEN -1), 392 bp (DEN -4), 290 pb (DEN - submitted for diagnosis based on anti- 3) and 119 bp (DEN-2) were obtained by dengue IgM antibodies detection by MAC- using the SuperScripTM One Step RT-PCR kit ELISA. From these, only 34 samples were in conjunction with PlatinumR Taq positive for IgM antibodiesy. As expected, polymerase (Invitrogen, Life Technologies). the 50 negative-control samples resulted A mixture of 5 ml of RNA, 25 mM of sense negative for anti-dengue IgM antibodies and anti-sense PCR primers, and DEPC (Table 1). water to a total volume of 50 ml was incubated at 85 °C for 5 minutes and then chilled on ice. The tubes-reaction mixture Diagnosis by RT-PCR containing 2X PCR buffer containing 0.4 Once the serum samples were tested for mM of each dNTP, 2.4 mM MgSO4 and anti-dengue IgM antibodies, the results were TM R Super Script RT/platinum Taq Mix, as confirmed by RT-PCR. In this case, only 25 recommended by the manufacturer (In samples from healthy donors were tested. vitrogen TM Life Technologies), was added By this method 58 samples proved to be to the RNA and primers -containing tube. positive for dengue, i.e. 24 more than by The reverse transcription reaction was MAC-ELISA. Interestingly, all samples performed at 50 °C for 30 minutes. positive for MAC-ELISA were also positive Thermocycling began with a hot start at by RT-PCR. Those samples showing 94 °C for 2 minutes followed by 40 cycles positivity for DEN by RT-PCR were further of annealing at 55 °C for 30 seconds, and tested for the four serotypes (DEN-1, -2, -3 extension at 72 °C for one minute and and -4). It was found that the main denaturing at 94 °C for 15 seconds. circulating serotype in Oaxaca during 2000- The PCR conditions for serotype 2001 was DEN-2. Two other serotypes assessment were as follows: 40 cycles of (DEN-1 and DEN -4) were also found (only denaturing at 94 °C for 30 seconds, one case each) (Table 1). annealing at 55 °C for 1 minute, and y extension at 72 °C for 1 minute and, a final Out of 200 samples, originally sent, only 100 samples were found in good condition for evaluation by MAC- extension at 72 °C for 7 minutes. The ELISA or RT-PCR. Other samples deteriorated under transportation/storage conditions.
Dengue Bulletin – Vol 28, 2004 31 Dengue Investigation in Oaxaca, Mexico
Table 1. Positivity for dengue by MAC-ELISA and RT-PCR From one hundred samples tested, from patients with clinical diagnosis of dengue, 36% proved positive by MAC-ELISA and 61% by RT-PCR. DEN-2 was the prevailing circulating serotype
Date of Jurisdiction Locality Number MAC- RT-PCR Serotype Serotype collection of cases ELISA Nov 2000 II Salina Cruz 18 13+/5- 9+/9- DEN-2 DEN-2 Nov 2000 III Tuxtepec 9 2+/7- 5+/4- DEN-2 DEN-2 Nov 2000 II Tehuantepec 10 4+/6- 5+/5- DEN-2 DEN-2 Jul 2000 III Temascal 8 3+/5- 5+/3- DEN-2 DEN-2 Nov 2000 II Juchitan 3 2+/1- 2+/1- DEN-2 DEN-2 Total cases 24+/24- 26+/22- May-Jun IV Huatulco 21 6+/15- 12+/9- DEN-2 DEN-2 2001 Jun-Nov I Oaxaca 7 2+/5- 7+/0- DEN-2 DEN-2 2001 Apr 2001 II Juchitan 2 0+/2- 2+/0- DEN-2 DEN-2 May 2001 II Salina Cruz 1 0+/1- 1+/0- DEN-2 DEN-2 Feb 2001 III Tuxtepec 7 2+/5- 2+/5- DEN-2 DEN-2 Feb 2001 V Tonalá 2 0+/2- 2+/0- DEN-2 DEN-2 Feb 2001 V Huajuapan 6 0+/6- 6+/0- DEN-2 DEN-2 Total cases 10+/36- 32+/14-
Table 2. Distribution of dengue cases Prevalence of infection by by age and sex age and sex Age Male Female An analysis by age and sex revealed a higher (in years) prevalence (61%) of infection in females 1 0 1 than in males (39%) and that the most affected group of people was the under-35- 2-4 0 2 year-olds (Table 2). 5-9 7 10 10-14 5 6 Discussion 15-20 2 3 The Aedes aegypti mosquito’s adaptability to 21-30 0 5 changing environmental conditions has >30 6 6 contributed significantly to the increase in dengue epidemics in the world. Mexico is Total 20 (38%) 33 (62%) considered an endemic country where the
32 Dengue Bulletin – Vol 28, 2004 Dengue Investigation in Oaxaca, Mexico four serotypes of the dengue virus are in and storage in order to send them to the circulation. This study provides some insight reference laboratory for adequate diagnosis. into the dengue epidemic situation in It is worth noting that in several Mexican Oaxaca state, Mexico, in an attempt to states, health authorities are working on contribute to the prevention and control of vector control as well as on facilities for outbreaks of DF/DHF. sample collections to be sent to the Instituto de Referencia Epidemiológica From the 200 serum samples collected (InDRE) in Mexico City for a proper from suspected dengue patients initially diagnosis. It is still, however, a long way for considered for the study, only 100 could be good quality medical care to reach most used. The remaining 100 samples were Mexicans. presumably subjected to non-appropriate storage conditions. From the 100 suspected This report shows that by MAC-ELISA, samples tested, nearly 100% proved to be 36% of the tested samples were found positive for dengue (36% by MAC-ELISA positive for dengue, whereas by RT-PCR up and 61% by RT-PCR). However, the data to 64% of the samples proved to be positive. reported here could be an underestimation Although the sensitivity and specificity considering the several factors that could reported for MAC-ELISA is reported to be influence the laboratory determination good enough for a diagnosis system, it is outcome, such as sample handling and the likely that as a result of inadequate handling diagnosis systems performed at local and storage conditions, some samples hospitals. In some localities of Oaxaca, the reported as negative could in fact be diagnosis for dengue was being positive for dengue when tested by RT-PCR. simultaneously carried out with the No false positive results were found. This diagnosis for rubella and toxoplasma in a raises the question as to how many monoclonal antibodies-based multiplex laboratory assays must be carried out on a assay. In rural communities, however, only suspected dengue sample before reporting it the presence of anti-dengue IgM antibodies as negative. was tested. The use of RT-PCR makes it possible to In this regard, it is possible that some identify the dengue serotype involved; in patients presenting an early secondary this regard this study shows that in Oaxaca, infection in the absence of strong clinical Mexico, the prevalent serotype of dengue manifestations had undetectable levels of virus was DEN -2, although isolated cases of anti-dengue IgM antibodies, since IgG is the DEN-1 and DEN-4 infections were also prevalent Ig isotype at this stage of the found. Some other local reports had also infection. For these cases, it would be mentioned the presence of DEN-3 and necessary to consider some other diagnosis several cases of DHF. techniques such as virus isolation or RT-PCR. Unfortunately, these are difficult to carry out in rural hospitals due to high costs and lack Acknowledgements of suitably trained personnel. We thank Dr F. Javier Sánchez-García for Additional effort is needed to ensure critically reviewing the manuscript. MMBMA appropriate sample collection, handling is an EDI/IPN fellow.
Dengue Bulletin – Vol 28, 2004 33 Dengue Investigation in Oaxaca, Mexico
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[1] Novo S. Breve historia y antología de la [7] Martínez-Soriano U. Tipificación Molecular fiebre amarilla. Salud Pública de México, del virus del dengue en el estado de Oaxaca. 1995, 37: S99-S102. B.Sc. Thesis, 2002, 19-58. [2] Gómez H. Monografía sobre la [8] Vorndam V and Kuno G. Laboratory epidemiología del dengue en México. diagnosis of dengue virus infections. In: Secretaría de Salud, 1992, Vol. 43. Gubler DJ and Kuno G (Eds.), Dengue and dengue hemorrhagic fever. CAB [3] Gubler JD. Epidemic dengue/dengue International, New York, NY, 1997, 313-333. haemorrhagic fever as a public health, social and economic problem in the 21st century. [9] Igarashi A. Mosquito cell cultures and the Trends in Microbiology, 2002, 10: 100-103. study of arthropod-borne togaviruses. Advances in Virus Research, 1985, 30: 21-39. [4] Briseño-García B, Gómez-Dantes H, Argott- Ramírez E, Montesano R, Vázquez-Martínez [10] Lanciotti RS, Calisher CH, Gubler DJ, Chang AL, Ibanez-Bernal S, Madrigal-Ayala G, GJ and Vorndam V. Rapid detection and Ruiz-Matus C, Flisser A and Tapia-Conyer R. typing of dengue viruses from clinical Potential risk for dengue haemorrhagic samples by using reverse transcriptase- fever: the isolation of serotype dengue-3 in polymerase chain reaction. Journal of Mexico. Emerging Infectious Disease, 1996, Clinical Microbiology, 1992, 30: 545-551. 2(2): 133-135. [11] Harris E. A low cost approach to PCR. Ed. [5] Anonymous. Gaceta informativa de la Oxford University Press, USA, 1998, 96-105. Secretaría de Salud, SSA-Oaxaca, 1999. [6] Cisneros AS, Martínez US, Cruz GM, Tovar RG, Moreno MG, Días-Badillo A and Muñoz ML. Dengue fever in the state of Oaxaca, Mexico. The American Society of Tropical Medicine and Hygiene, 50 th Meeting, 2001, Atlanta, Georgia, USA.
34 Dengue Bulletin – Vol 28, 2004 Spatial and Temporal Dynamics of Dengue Haemorrhagic Fever Epidemics, Nakhon Pathom Province, Thailand, 1997-2001
Wutjanun Muttitanon*, Pongpan Kongthong**, Chusak Kongkanon**, Sutee Yoksan***, Narong Nitatpattana***, Jean Paul Gonzalez‡ and Philippe Barbazan†#
*Asian Journal of Geoinformatics; Space Technology Application and Research Program, Asian Institute of Technology. P.O. Box 4, Klong Luang, Pathumthani, Thailand **Department of Geography, Faculty of Education, Ramkhamhaeng University, Bangkok 10110, Thailand ***Center for Vaccine Development (CVD), Institute of Science and Technology for Research and Development, Mahidol University, Nakhon Pathom 73170, Thailand †Research Center for Emerging Viral Diseases (RCEVD) – IRD – Center for Vaccine Development, Institute of Science and Technology for Research and Development, Mahidol University, Nakhon Pathom 73170, Thailand ‡Institut de Recherche pour le Développement (IRD) Ur034, 213 rue La Fayette, 75480, Paris cedex 10, France
Abstract Several environmental factors modulate the distribution of dengue fever (DF), such as climate, density of vector and human populations in urban areas and distribution of herd immunity. In order to identify geographical variables involved in the spread of a DHF process, a Geographic Information System (GIS) has been built to create links between geo-referenced data including medical records and socioeconomic and environmental data. Applied to a retrospective analytical study of DHF epidemics in Nakhon Pathom province 1997( -2001), the GIS allowed a mapping of spatial variations of DHF incidence, the recognition of different temporal incidence patterns and the quantification of the dispersal of outbreaks among defined spatial units. The analysis showed that the diffusion process of these epidemics was of a contagious type as the distance between epidemic areas (sub-districts) was significantly lower than the average distance between every sub-district. This result indicates that these epidemics were likely to be due to the spread of a new or rare virus serotype, from its emergence location in the province to areas with a sufficient density of vectors and a similar limited immune protection against this serotype. Keywords: Dengue haemorrhagic fever, dengue virus, transmission, Geographic Information System, spatial analysis.
# E-mail: [email protected]; Tel./Fax: (66) 2 441 01 89
Dengue Bulletin – Vol 28, 2004 35 Spatial and Temporal Dynamics of DHF Epidemics in Thailand
Introduction dispersion, largely a function of the stochastic movement of incubating/infectious humans Dengue fever (DF) is a viral disease with a and the transport via vehicles of virus-positive worldwide distribution in all tropical areas. females[4]. It is caused by the dengue virus (genus Flavivirus, family Flaviviridae) which presents The understanding of the mechanism of the inter-community spread of DHF during four antigenic forms or serotypes: DEN -1, DEN-2, DEN -3 and DEN-4. In Thailand, epidemic periods is a primary factor likely to lead to an evaluation of the risk of virus Dengue haemorrhagic fever (DHF) a severe [5] form of dengue fever has been endemic transmission and disease dispersal . Moreover, it would provide some guidance since 1958, with a cumulative total of 1,369,542 cases till date[1]. Epidemics occur on the distance from the spatial origin of an epidemic at which preventive control with a periodicity of between two and four years; these epidemics are of significant measures should be applied. concern for the public health authorities. In At a monthly time-scale, the main most of the areas where serotype geographical factors involved in dengue identifications were performed, two or three transmission (urbanization, demography, serotypes were found to be co-circulating[2]. cultural and social characteristics) are stable[6]. A change in the pattern of monthly The dengue virus is an arbovirus (arthropod-borne virus) transmitted by the DHF transmission, such as the emergence of epidemics in an endemic area, should then mosquito Aedes aegypti (L.). Control of the spread of the disease focuses on vector rather be related to factors evolving with time: climate, density of vectors, emergence control strategies based mainly on the elimination of potential breeding sites[3]. A of a new or rare virus serotype, each type of factor inducing a specific pattern of diffusion major attribute of the virus transmission is its [7,8] anthropophilic behaviour, as females mainly of the disease . The emergence of a new serotype in a given population is likely to bite humans and lay eggs in man-made containers near houses (for example, water exhibit particular spatial characteristics. The outbreak would begin where the serotype jars, cans, used tyres). The short flight range of the vector, less than 1 km, contributes to first arrived and then move to places where a low specific herd immunity (towards this the limited spread of the disease by an infected female. Most of the infections by serotype) and a sufficient density of mosquito allow a high level of transmission. dengue viruses are not severe and present asymptomatically, allowing infected patients The spread of a new serotype is then likely to follow the main model of contagious to maintain normal activities. diffusion described for the spread of other Two types of viral spread can be types of moving phenomena[9]. Applied to described: (i) the diffusion of human the diffusion of an infectious disease, it infections as a function of the spatial means that the probability for an area to be distribution of houses and the limited flight reached by a contagious disease will be range of infectious or infected Aedes aegypti inversely correlated to the distance to the females (intra-communal, contagious/ formerly contaminated areas, leading to continuous); and (ii) inter-communal clusters of epidemic areas.
36 Dengue Bulletin – Vol 28, 2004 Spatial and Temporal Dynamics of DHF Epidemics in Thailand
In order to test the validity of this model Department. DHF cases were defined in the frame of dengue dispersal, a study according to WHO criteria[10]. was conducted to describe the spread of significantly higher levels of incidence rate Population and study area (of epidemic significance) among sub- districts. The study, done in a province of Nakhon Pathom province is a part of the Thailand, covering the period 1997-2001, central plain region in Thailand included two DHF epidemics. encompassing the latitude of 13° 38’45.6” N to 14° 10’37.2” N and the longitude of 99° 51’10.8” E to 100° 17’6” E. It covers Materials and methods 2,164 sq km, has a population of 774,276 inhabitants and includes 7 districts and 106 Data collection sub-districts (Figure 1a). The population density ranges from 153 to 623 Data on clinically diagnosed DHF cases inhabitants/sq km. The average surface area were recorded at the Ministry of Public of sub-districts is 20.4 sq. km. The provincial Health, the demographic data were health department reported 14,079 DHF provided by the Administrative Department cases during 1983-2001; two DHF of the Ministry of Interior, and the epidemics occurred in 1997-1998 and geographical maps by the Royal Thai Survey 2000-2001 (Figure 2).
Figure 1. District scale approach: (i) Administrative limits of districts and sub-districts; density of population; main roads; (ii) Average incidence observed before the epidemics (cases/100,000 people), January 1992 – June 1997; (iii) Ratio of the incidence during the first three months of the DHF epidemic compared to the average incidence from January 1992 – June 1997
a) Density of population b) Average Incidence c)Ratio of Incidence 5 0 10 20 km (inhabitants / ha) 01/1992 - 06/1997 07 – 09 1997 vs (cases /100,000) aver. 01/1992 - 06/1997 Main roads > 600 22 - 46 1.6 – 2.8 Districts 350 - 450 8 - 10 1.3 – 1.4 Sub-districts 150 - 350 3 - 7 0.3 – 0.7
Dengue Bulletin – Vol 28, 2004 37 Spatial and Temporal Dynamics of DHF Epidemics in Thailand
Figure 2. Monthly DHF incidence in Nakhon Pathom province, Thailand, from January 1992 to August 2001
300
200 cases
100
0 1-92 7-92 1-93 7-93 1-94 7-94 1-95 7-95 1-96 7-96 1-97 7-97 1-98 7-98 1-99 7-99 1-00 7-00 1-01 7-01 month
Method of analysis In a contagious model for an infectious disease, the spatial entities close to an The study aimed to describe the spatial- infected one were assumed to be more at temporal dynamics at a monthly time-scale risk to become infected than the distant of a DHF epidemic among Nakhon ones. Applied to the diffusion of an Pathom’s 106 sub-districts considered as the epidemic phenomenon, it meant that the spatial units. As a first step, epidemics were distance between the new epidemic sub- defined at the province level as periods of districts and the former ones (observed time (at least two consecutive months) when distance) should be shorter than the average the incidence is higher than the average, (expected) distance between all the sub- plus one standard deviation of the monthly districts. The distance between sub-districts incidence of each month (i.e. January, was defined as the Euclidian distance February, etc.). The average was calculated between their centroids. The expected over the entire 1983-2001 period[11]. distance was the average distance between each ESD and every other sub-district. The During these epidemic months (EMs), observed distance was the average distance epidemic sub-districts (ESDs) were those between each ESD and every other ESD, where the monthly incidence was during the same month (cluster study), or significantly higher than in other sub-districts. from one month to the next (spread study). The threshold for a significantly higher H (null hypothesis) = the average incidence was leveled at the average 0 observed distance (between ESD) was not monthly incidence (per 100,000 different from the average expected distances. inhabitants) plus one standard deviation, observed among every sub-district during H1 = average observed distance that EM. 38 Dengue Bulletin – Vol 28, 2004 Spatial and Temporal Dynamics of DHF Epidemics in Thailand The Z test was used to compare the drowned at 5 km; 10 km; 15 km; 20 km average distances. and out of 20 km. This number is compared to the number of sub-districts centroids The method was applied to the study of distributed in these surfaces to build a two phenomena: (i) the occurrence of relative risk index. clusters of ESD during one month; and (ii) number of ESD in a circle the spread of the epidemic among sub- Relative risk index = districts from one month to the next. A total number of ESD number of sub - districts in a circle cluster is defined here as an aggregation of total number of sub - districts ESD (during one EM) of sufficient size and concentration to be unlikely to have occurred by chance, i.e. if the average Results distance (between these ESD) is shorter than the average distance between all sub- At the district scale, the DHF incidence was districts. The spread of the epidemic is higher in the central-west part of the based on the comparison of observed and province. The epidemic broke out in the expected distances during two consecutive northern district with a medium density of EMs, i.e. the average distance between ESD population (Figures 1b and 1c). At the sub- district scale, the maximum DHF incidence during one epidemic month (EMm) and ESD rate reached 540 cases per 100,000 during the next epidemic month (EMm+1), versus the average distance between ESD inhabitants in July 1997. during EMm and every sub-district during Nineteen EMs were identified in EMm+1. Nakhon Pathom province from January The (discrete) distance, at which an 1997 to August 2001 (Table); the number of epidemic can spread in one month, was EMs in one sub-district ranged from 0 estimated by summing the number of ESD month (in 27 sub-districts) to a maximum of 11 months. centroid during EMm+1 observed inside circles centred on each ESD during EMm and Table. Chronological distribution of epidemic months from January 1997 to August 2001 in Nakhon Pathom province, Thailand Year Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 1997 1998 1999 2000 2001 = Epidemic month Dengue Bulletin – Vol 28, 2004 39 Spatial and Temporal Dynamics of DHF Epidemics in Thailand A total of 49 ESDs were identified significantly lower (P<0.05) than the during the first outbreak (1997-1998) and average expected distance, characterizing 61 during the second outbreak (2000- the occurrence of clusters of ESDs according 2001); 31 sub-districts were epidemic- to the H1 hypothesis. affected during the two outbreaks. The Spread study: The distance observed probability of one sub-district being between ESDs during EM and ESDs during epidemic-affected during the first outbreak m EM , was significantly smaller than the to be epidemic-affected during the second m+1 expected distance (Figure 3), characterizing outbreak as well is not significantly different the contagious spread of DHF among ESDs from a random distribution (P<0.05). according to the H1 hypothesis. Cluster study: During EM, 78.67% of the average observed distances were Figure 3. Study of the spread of DHF epidemic among sub-districts: observed distances are calculated between epidemic-affected sub-districts during one month and the epidemic- affected sub-districts during the next month; expected distances are calculated between all the sub-districts (see text for details) 97-98 epidemic endemic 00-01 epidemic 25 20 15 10 of tambon nb distance (km) 5 0 0 Aug Oct Dec Feb Apr Nov Jan Mar May 97 97 97 98 98 month 00 01 01 01 number of epidemic tambon observed distance expected distance As a consequence, the distribution of Discussion ESDs during EMm+1 in surfaces drowned round ESD during EMm showed a significant The method used for the identification of an [11] (P<0.05) aggregation within the first two epidemic month in the province allowed circles (5 and 10 km). a precise framing of epidemics, defined as periods during which the incidence was 40 Dengue Bulletin – Vol 28, 2004 Spatial and Temporal Dynamics of DHF Epidemics in Thailand significantly higher than the observed distribution of all sub-districts having been average over the complete time series of epidemic-affected at least during one month data (19 years). Meanwhile, this method (67% of the sub-districts) was not could not be used directly at the sub-district significantly different from the spatial scale because of the lack of long-time data distribution of all sub-districts (average series on the incidence at this scale. distances not different, P=0.95). Meanwhile, Moreover, the variance of the DHF the results implied a high degree of spatial incidence in most of the sub-districts was auto-correlation, meaning that neighbouring very high because of the low values of sub-districts shared similar characteristics, incidence often recorded (during the study such as the level of immunity for the period a null monthly incidence was different serotypes (due to a similar reported in 66% of the 5,936 months X sub- epidemiological history) or the density of the districts). Similarly, the village scale (from 3 vector. to 24 villages per sub-district) could not be As shown in Figure 3, the observed used as the spatial unit as the addresses of distances are smaller than the expected patients were often consistent only at the ones, but exhibit similar monthly variations. sub-district scale and many students and This was mainly because of a border effect, pupils did not live in their village. the propagation in sub-districts located in We assumed in this study that sub- neighbouring provinces not being taken into districts could be considered as account. During the months where ESDs homogeneous small areas and that human were located on the periphery of the displacements were sufficient to produce a province, the average distance to other sub- homogenization of the population, allowing districts was larger than during the months consideration of the sub-district as a unit where ESDs were located near the centre of towards DHF transmission. An ‘epidemic’ the province, as several neighbouring sub- pattern can then be identified in any sub- districts located in other provinces district, whatever its density of population: (epidemic or not) were ‘missing’ in the the distribution of ESD was not correlated to calculation. The absolute level of expected the density of population (Pearson’s and observed distances was then directly correlation = -0.24, P = 0.71). Moreover, dependent on the location of the ESD in the we used the incidence rate per 100,000 province. inhabitants to reduce the bias related to the The spread of the epidemic between size of the population. sub-districts followed Hagerstrand’s model The geographical heterogeneity of the that has been used to describe many types environment, e.g. the density of of phenomena, such as the spread of new urbanization or the road network, could also ideas[9] or the waves of innovation which be at the origin of clusters of ESDs. lose their ‘energy’ when the distance from Meanwhile, after the two epidemics, ESDs the source increases[12]. In public health were found to be uniformly distributed over research it has been applied to infectious the entire province, and the spatial influenza[13]. Applied to the DHF epidemic Dengue Bulletin – Vol 28, 2004 41 Spatial and Temporal Dynamics of DHF Epidemics in Thailand in Nakhon Pathom, it means that during the DHF is endemic in Thailand and the epidemic periods the ESDs were the origin different serotypes are largely distributed, as of the emergence of epidemics in at least two or three serotypes are generally neighbouring sub-districts during the next found at the same time in the same area[2,16]. month. The probability of this emergence at Meanwhile, during epidemic periods the m+1 significantly decreased with the relative prevalence of the serotypes varies, distance from the former ESD. This model is as the 2000 epidemic in Bangkok that was of a contagious type and may be opposed to due mainly to the serotypes DEN-1 and a random or homogeneous model. In the DEN-2 (each reaching 42% of total homogeneous models the occurrence of an isolations), whereas the 1994 epidemic was epidemic could be due to a global due mainly to the rise in DEN -4 (36%). But phenomenon, such as an increase in as serology and isolation of viruses are rarely temperature, which should have been performed, the emergence of a DHF observed in any sub-district, leading to a epidemic cannot be forecast by using these random distribution of ESD[14] and an methods. Indirect methods, such as the observed distance not different from the statistical identification of epidemic months, expected distance. are then necessary to identify early the emergence of DHF epidemics. Inside human communities (villages) it has been shown that the spread of DHF The epidemiology of DHF in Thailand is viruses from one house to neighbouring changing[17]. This approach of the houses due to the displacement of infected displacement of epidemics is likely to vectors or hosts follows a pattern similar to contribute to the localization of the origins what we have described between sub- of outbreaks and the delineation of areas at districts[15]. Meanwhile, among communities risk during epidemics, as well as to help separated by several kilometers, the spread public health authorities to focus vector of viruses cannot be due to the active control activities on selected areas. dispersal of mosquitoes or to their transport by car, which is much more rare than the displacement of infected hosts. More than Acknowledgements 80% of infections by dengue virus are The study and the preparation of this paper unapparent or not severe, allowing healthy was supported by the Institut de Recherche carriers to travel. The presence of sufficient pour le Développement (IRD)-Ur034, densities of vectors in destination France, by a fellowship to the Center for communities is also necessary to allow the Vaccine Development, Institute of Science transmission of the virus after it has been and Technology for Research and imported. Development, Mahidol University, Thailand, The contagious distribution and spread and by the Department for Technical and of the two DHF epidemics among the sub- Economic Cooperation, Thailand. We thank districts strongly suggests that they were due Professor Natth Bramapavarati for his to the emergence of a new or rare serotype. constant support. 42 Dengue Bulletin – Vol 28, 2004 Spatial and Temporal Dynamics of DHF Epidemics in Thailand References [1] Annual epidemiological surveillance report, [9] Hagerstrand T. The propagation of innovation 1983-2002. Bangkok: Ministry of Public waves. Lund studies in Geography, Series B, Health, Department of Disease Control, 1952, 4. Bureau of Epidemiology, 2002. [10] World Health Organization. Dengue [2] Vaughn DW, Green S, Kalayanarooj S, Innis haemorrhagic fever: diagnosis, treatment BL, Nimmannitya S, Suntayakorn S, and control, 2 nd edition. Geneva: 1997. Rothman AL, Ennis FA and Nisalak A. Dengue in the early febrile phase: viremia [11] Barbazan P, Yoksan S and Gonzalez JP. and antibody responses. Journal of Dengue Hemorrhagic Fever epidemiology in Infectious Diseases, 1997, 176: 322-330. Thailand: Description and forecast of epidemics. Microbes and Infection, 2002, [3] World Health Organization. Dengue 1(4): 699-705. prevention and control. Executive Board, 109th Session, 2001, EB109/16. [12] Gould PR. Spatial diffusion. Washington, US, Association of American Geographers, 1969. [4] Meade MS, Florin JW and Gesler WM. Medical Geography. London, UK: The [13] Cliff AD, Haggett P and Ord JK. Spatial Guilford Press, 1988. aspects of influenza epidemic. London, Pion Limited. 1986. [5] Cuzick J and Elliott P. Small-area studies: purpose and methods. Chapter 2 of [14] Jackson EK. Climate change and global Geographical & Environmental Epidemiology infectious disease threats. The Medical (Eds. P. Elliott, J. Cuzick, D. English and R. Journal of Australia, 1995, 163(4): 570-573. nd Stern). 2 ed. New York: Oxford University [15] Morrison AC, Getis A, Santiago M, Rigau- Press, 1997. Perez JG and Reiter P. Exploratory space- [6] Kuno G. Review of the factor modulating time analysis of reported dengue cases dengue transmission. Epidemiologic Reviews, during an outbreak in Florida, Puerto Rico, 1991-1992. American Journal of Tropical 1995, 12(2): 321-335. Medicine and Hygiene, 1998, 58(3): 287-298. [7] Focks DA, Daniels E, Haile DG and Keesling JE. A simulation model of the epidemiology [16] Burke DS, Nisalak A, Johnson DE and Scott of urban dengue fever: literature analysis, RM. A prospective study of dengue model development, preliminary validation, infections in Thailand. American Journal of and samples of simulation results. American Tropical Medicine and Hygiene, 1988, Journal of Tropical Medicine and Hygiene, 38(1): 172-180. 1995, 53(5): 489-506. [17] Chareonsook O, Foy HM, Teeraratkul A and [8] Mayer JD. The role of spatial analysis and Silarug N. Changing epidemiology of dengue geographic data in the detection of disease hemorrhagic fever in Thailand. causation. Social Science and Medicine, Epidemiology and Infection, 1999, 122: 1983, 17(16): 1213-1221. 161-166. Dengue Bulletin – Vol 28, 2004 43 Sporadic Prevalence of DF/DHF in the Nilgiri and Cardamom Hills of Western Ghats in South India: Is it a Seeding from Sylvatic Dengue Cycle – A Hypothesis Nand Lal Kalra* and Chusak Prasittisuk**# *A-38, Swasthaya Vihar, Vikas Marg, Delhi – 110 092 **Regional Office for South-East Asia, World Health Organization, New Delhi, India Abstract The Western Ghats of south India, encompassing the Nilgiri and Cardamom hills, are the wettest region of the country. Hills rising upto 3,000 metres receive over 200 cm of rainfall from both the south-west monsoon (June to September) and north-eastern monsoon (October to January). The eastern slopes of the Nilgiri Hills (200-500 metres) are bounded by Coimbatore and Erode districts of Tamil Nadu; whereas the western slopes of the Nilgiri and Cardamom hills are in the state of Kerala. The countryside has rich forests of teak and sandalwood, interspersed by groves of coconut, rubber, pepper, cardamom and banana plantations. Apart from the rich flora, monkeys Macaca( radiata) maintain a strong association in orchards with humans competing for food. The emergence of DF/DHF in this hilly region is a recent occurrence. An epidemiological team from the National Institute of Communicable Diseases (NICD) investigated the first-ever reported outbreak in Coimbatore in 1998. In all, 20 serological positive (IgM) cases were recorded by the city corporation. Five cases came from urban towns and 15 cases from rural areas of the two districts of Coimbatore and Erode. Rural cases were scattered in distantly located villages. Pyramid characterization and clustering of cases was conspicuously absent. No attempt was made to link urban cases to central/peripheral wards, nor the history of movement of patients two weeks prior to the onset of fever was investigated. No increase in fever rate was observed. DF cases did not show any relationship with presence/absence of Aedes breeding. Aedes aegypti detected in urban centres failed to amplify the infection. Kerala state also reported 116 cases in 1997, from Kottayam district. Out of these, 14 cases were confirmed serologically. After a lull of 4 years, 70 probable cases out of 877 were reported from the four districts famous for rubber plantations. Entomological investigations recorded only Aedes albopictus in these areas. Considering the high experimental susceptibility of both species of monkeys, viz. Macaca mulatta and Macaca radiata to yellow fever virus, detection of dengue antigen in field collected Aedes albopictus in Kozhikode (Kerala) and the evidence of transovarian transmission in Aedes albopictus reared from soils of tree holes at Jodhpur – Rajasthan (western India) lend support to the hypothesis that DF in the Western Ghats of South India exists as enzootic monkey – Aedes albopictus – monkey cycle and causes epizootics among rural human population either during periodic amplification of the enzootic cycle or as occupational hazards to the people working in orchards. Keywords: DF/DHF, sporadic cases, Macaca radiata, dengue antigen in Aedes albopictus, transovarial transmission, sylvatic cycle, Nilgiri and Cardamom hills, south India. # E-mail: [email protected] 44 Dengue Bulletin – Vol 28, 2004 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India Introduction Figure. Physical map of Kerala and adjoining areas of Tamil Nadu The Western Ghats of south India encompass two southern states i.e. Tamil Nadu and Kerala (Figure). The crests of the Nilgiri and Cardamom Hills (rising upto 3,000 metres altitude) separate these two states. The eastern slopes of the Nilgiri Hills have a gentle slope and include two important district towns of Tamil Nadu, viz. Erode and Coimbatore, situated at an altitude varying between 200 to 500 metres; each with over one million population. This part of the region receives rains from both the south-west and north-east monsoon. The south-west monsoons become weak, being on the leeward side of the hills, but maximum rains come from the north-east monsoon, the total being <100 cm. The forests are tropical monsoon, which are famous for teak and sandalwood, with patches of arecanut palms[1]. The western side of the Cardamon hills encompasses the state of Kerala. Strong winds of the south-west monsoon lead to the formation of heavy sand dunes in coastal areas and the rain water coming from the steep hills results in formation of shallow lagoons, all along the coast, at places connected to the sea. These lagoons are connected by canals. These backwaters are the characteristics of Kerala state. The banks of these backwaters and sand dunes are dotted with coconut trees[1]. The Region receives heavy rains (>200 Dengue transmission cycles cm) during the south-west monsoon. Hence, Transmission of dengue viruses occurs in two the whole region is very wet and supports cycles, viz. enzootic and epidemic cycles. luxuriant growth. Large tracts of forests have The enzootic cycle is a primitive sylvatic cycle been cleared for raising cash crops, viz. maintained by lower primates (monkeys) and arecanut palms, rubber, banana, pepper canopy dwelling Aedes mosquitoes, as and cardamom plantations[1]. reported from South-Asia[2], Africa[3] and Sri Dengue Bulletin – Vol 28, 2004 45 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India Lanka[4]. Current epidemiological evidence DF in hilly regions of Tamil Nadu suggests that these viruses do not regularly move out of the forests to urban centres but During 1998, a team from the National at times are involved in an epidemic cycle in Institute of Communicable Diseases (NICD), small rural villages or islands[3]. A number of Delhi, investigated the first-ever reported Aedes species may act as reservoirs[2]. outbreak in Coimbatore[8], a town situated at an altitude varying from 300 to 500 The epidemic cycle is confined to large metres on the eastern slopes of the Nilgiri urban centres. The viruses are maintained in Hills. The epidemiological characteristics of the Aedes aegypti – human – Aedes aegypti the outbreak are summed up below: cycles with periodic/cyclic epidemics. Generally all serotypes circulate and give • In all, 20 serologically positive and rise to hyperendemicity. Virus is maintained compatible to DF/DHF cases were either transovarially by the vectors or by reported. Eighty percent (16/20) were continuous low-grade transmission in children below 10 years and two susceptible hosts added to the population. patients aged 16 and 5 died of DHF. DF/DHF in urban cycles is characterized by ‘iceberg’ or ‘pyramid’ phenomenon. At the • Fourteen cases were males. base most of the cases are symptom-less, • Seventeen cases came from Coimbatore followed in increasing rarity, by district and three cases from rural areas undifferentiated fever, DF, DHF or DSS[5]. of adjoining Erode district. Occurrence of multiple cases in a single household or clustering of cases in a locality • Only five cases came from urban is yet another characteristic of this disease[2]. Coimbatore town and the rest (12 cases) were from rural areas of Coimbatore district. DF/DHF in India • Rural cases were scattered in distantly In recent years the first outbreak of DF/DHF located villages. Clustering of cases and was reported from Kolkata (earlier known as pyramid phenomenon was conspicuously Calcutta) in 1963[6]. Since then, more than absent. No attempt was made to link 60 outbreaks have been reported from all urban cases to central or peripheral [7] over the country . Aedes aegypti, invariably wards/zones nor the movement history has been found to be associated with these of patients two weeks prior to the onset epidemics. All the four serotypes, DEN-1, 2, of fever was investigated. 3 and 4, are now circulating in the country. • No relationship could be established between outbreak and increased fever DF/DHF in Nilgiri/Cardamom rate. hilly areas of south India • Eighty-nine percent of blood samples from healthy contact persons from Since 1996-97, there have been reports of urban and rural areas showed dengue sporadic occurrence of DF/DHF cases in virus IgG antibodies. Tamil Nadu and Kerala. 46 Dengue Bulletin – Vol 28, 2004 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India • Entomological investigation recorded DF in Kerala Aedes aegypti in all areas surveyed in urban and rural areas, but failed to Epidemiological data amplify the infection. As per investigations undertaken by the • During the 2003 outbreak in Centre for Research in Medical Entomology Coimbatore town 23 cases of DF were (CRME)[9], the state of Kerala started recorded. Distribution once again reporting DF for the first time in 1997. followed the same pattern, i.e. 5 cases Distribution of DF cases are included in from urban towns and 18 cases from Table 1. rural areas (Source: VBDC, New Delhi) Table 1: Distribution of DF cases in Kerala state during 1997-2001 No. of suspected No. serologically Year Deaths Districts cases positive 1997 116 14 4 Kottayam 1998 67 0 13 Kottayam 1999 1 0 0 Kottayam 2000 0 0 0 Kottayam 2001 877 70 1 4 districts* * Four districts included Kottayam, Idukki, Ernakulam and Thiruvananthapuram– famous for rubber planatations Table 2: Results of AedesY survey of 4 districts of Kerala state during 2001-2003 House index Container index S. Name of Breteau No. No. No. No. Remarks No. district % % index ex. +ve ex. +ve Types of 1. Alappuzha 150 40 26.7 201 63 31.3 42.0 containers and sites of 2. Ernakulam 24 10 41.7 54 41 75.9 170.8 detection of Aedes breeding, viz. 3. Kottayam 70 24 34.2 125 29 23.2 41.4 domestic/peri- domestic; 4. Kozhikode 3,311 895 26.23 17,912 1,077 6.01 31.57 extradomestic not mentioned YPool rearing of larval breeding collected from all localities indicated Aedes albopictus as the major species. Aedes aegypti was encountered in very few places and in scanty numbers (Source: NICD, Delhi, 2004) Dengue Bulletin – Vol 28, 2004 47 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India Entomological data albopictus, when subjected to IFA test, showed the presence of dengue antigen, Entomological investigation initiated by a thereby confirming the transovarial CRME team in 2 localities of Kottayam cycle of the virus[13]. district yielded only Aedes albopictus, and Aedes aegypti was not detected. Sylvan environment of rubber plantations was Hypothesis detected as the unique habitat of the Aedes albopictus[10,11]. Occurrence of DF cases in peripheral and rural areas of Coimbatore and Erode districts Results of yet another entomological in Tamil Nadu and non-amplification of study carried out by the National Institute of infection by Aedes aegypti and sporadic Communicable Diseases (NICD) field station occurrence of DF cases in Kerala in the located at Kozhikode, in DF affected absence of Aedes aegypti points out to districts during 2001-2003, are included in either spillover of enzootic foci of dengue Table 2. during periodic amplification of the sylvatic cycles or occupational hazards in the Antigen detection of dengue virus presence of vertical transmission as During May 2004, a pool of landing evidenced by the Kozhikode and Jodhpur collection of Aedes albopicuts (dessicated), studies. collected from the fringe of forested villages, • Both the Nilgiri and Cardamom hills are 600 metres away from Kozhikode (earlier infested with Macaca radiata, the known as Calicut) International Airport, bonnet monkeys. Enzootic cycle of yielded dengue antigen (processed at CRME, simian malaria caused by Plasmodium Madurai)[12]. cynomolgi and Plasmodium inui, transmitted by Anopheles elegans Vertical transmission by (Anopheles dirus group)[14,15], has been Aedes albopictus detected in the Nilgiri hills. Whereas in A recent study has been carried out at the Kerala, a similar simian foci has been Desert Medicine Research Centre (DMRC), detected at Nilambur district of the Jodhpur (Rajasthan), an institution under the western slope in Macaca radiata, while Indian Council of Medical Research, on in Alappuzha (district in the central possible existence of Aedes albopictus – plains, earlier known as Alleppey) monkey – Aedes albopictus cycle. The monkeys were found negative for lack [16] highlights of the study included that: of Anopheles elegans population . • In a desert ecosystem, both Aedes • Both the monkey species, viz. Macaca aegypti and Aedes albopictus breed in mulatta and Macaca radiata have been tree holes in zoo and monumental found to be highly susceptible to yellow parks, harbouring monkeys, outside the fever virus (flaviviruses) under city limits. experimental conditions[17]. This lends support to the susceptibility of these • Viable eggs retrieved from the soil of monkeys to dengue virus as well. tree holes were reared to adults. Aedes 48 Dengue Bulletin – Vol 28, 2004 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India • Lack of vectorial competence of Aedes • Gubler[20], proposed that at some point albopictus in the amplification of urban in the past, probably with the clearing dengue epidemic has recently been of the forests and development of demonstrated during the investigation human settlements, dengue viruses of the first-ever DF outbreak at moved out of the jungles and into a Phuentsholing, Bhutan in 2004[18]. rural environment where they were, Entomological investigations revealed and still are transmitted to humans by that Aedes aegypti occupied domestic peri-domestic mosquitoes such as Aedes habitats breeding primarily in storage albopictus. containers inside houses, while Aedes In view of the aforesaid, the land use in albopictus bred in tree holes, 55-gallon the Nilgiri hills of Tamil Nadu and in the drums and used tyres in the extra- Cardamom hills in Kerala is under pressure domestic habitats. The overlapping of deforestation to be replaced with cash zone was the peridomestic areas where crops. This has brought monkey populations both species shared breeding in flower much closer to human settlements. vases/trash. A large-scale source Therefore, there is a need for indepth sero- reduction/larvicidal campaign supported epidemiological and entomological studies by deltamethrin fogging in residential with backup of virology support using areas largely eliminated Aedes aegypti molecular tools for genomic sequencing of and the cases came down to single digits viruses obtained from simian and human within a month, while Aedes albopictus sources. Validation of the hypothesis is of still maintained high larval indices. great epidemiological significance as it [19] • Rudnik and Lim , while working in would require radical changes in developing Malaysia, isolated DEN-1, 2 and 3 vector control strategies for Aedes viruses from monkeys and also albopictus-transmitted DF. proposed that the rural dengue vector – Aedes albopictus, may introduce sylvatic virus into the human population. Acknowledgements • Studies in Sri Lanka proved that dengue The author gratefully acknowledges virus causes epizootics among Dr Duane J. Gubler for critically reviewing macaques, rather than being enzootic the manuscript. as observed elsewhere[4]. References [1] L Dudley Stamp. Asia – a regional and [4] de Silva AM, Dittus WP, Amersinghe PH and economic geography. Reprinted Indian Amersinghe FP. Serologic evidence for an edition. New Delhi: B.I. Publications, 1991. epizootic dengue virus infecting toque [2] Gubler DJ. Dengue and dengue macaques (Macaca sinica) at Polonnaruwa, haemorrhagic fever, Clinical Microbiol Sri Lanka. Am J Trop Med Hyg, 1999, 60(2): Review, 1998, 11(3): 480-496. 300-306. [3] Ricco-Hesse R. Molecular evolution and [5] Guzman MG and Kouri G. Dengue: an distribution of dengue viruses type 1 and 2 update. The Lancet Infectious Diseases, in nature. Virology, 1990, 174: 479-493. 2002, 2: 33-42. Dengue Bulletin – Vol 28, 2004 49 A Suspected Sylvatic Cycle in the Nilgiri and Cardamom Hills in South India [6] Ramakrishnan SP, Gelfand HM, Bose PN, [13] Joshi V, Singhi M and Mourya DT. Studies Sehgal PN, Mukherjee RN. The epidemic on determination of possible role of Aedes of acute haemorrhagic fever, Calcutta, albopictus mosquitoes in maintenance of 1963 – epidemiological inquiry. Ind Jour urban cycle of dengue. Desert Medicine Med Res, 1964, 52: 633-650. Research Centre (Indian Council of Medical Research), Jodhpur. Annual Report [7] Yadava RL and Narasimham MVVL. 2002-2003: 58-65. Dengue/dengue haemorrhagic fever and its control in India. WHO/Dengue Newsletter, [14] Ramakrishnan SP and Mohan BN. An 1992, 17: 3-8. enzootic focus of malaria in Macaca radiata radiata, Geoffroy, of Nilgiris Madras state, [8] Singh J, Balakrishnan N, Bhardwaj P, India. Ind J Malariology, 1962, 16: 87-94. Muthadevi P, George EG, Subramani K, Soundararajan K, Appavoo NC, Jain DC, [15] Pattanayak S. A note on natural simian Ichhpujani RL, Bhatia R and Sokhey J. malaria infection in Kerala state, India. Ind Silent spread of DF/DHF to Coimbatore J Malarilogy, 1963, 17: 293-294. and Erode districts in Tamil Nadu, India – [16] Choudhury DS. Investigations on simian 1988. Need for effective surveillance to monitor and control the disease. Epidemiol malaria in India and its potential as a source of zoonosis. Ind J Malariology, 1981, Infect, 2000, 125: 195-200. 18: 28-34. [9] Tyagi BK, Hiriyan J and Tewari SC. [17] Kalra NL and Sharma VP. Yellow fever Investigation on dengue fever in Kerala. threat. Current Science, 1996, 71(12): 948. Centre for Research in Medical Entomology (Indian Council of Medical Research), [18] World Health Organization. WHO mission Madurai – Annual Report 2001-2002: 66-68. to Bhutan – assistance to national [10] Hiriyan J, Tewari SC and Tyagi BK. Aedes authorities for control of dengue outbreak at Phuentsholing, Bhutan (Draft report), 8-9 albopictus breeding in plastic cups and tea- August 2004, WHO/SEARO, New Delhi. vendor spots in Ernakulam city, Kerala state, India. Dengue Bulletin, 2003, 27: 197-198. [19] Rudnik A and Lim TW (Eds.). Dengue fever studies in Malaysia. Bulletin from the [11] Sumodan PK. Potential of rubber Institute for Medical Research, Malaysia, plantations as breeding sources for Aedes 1986, 23: 1-241. albopictus in Kerala, India. Dengue Bulletin, 2003, 27: 197-198. [20] Gubler DJ. Dengue and dengue haemorrhagic fever: its history and [12] Das BP, Kabilan L, Sharma SN, Lal S, Regu resurgence as a global public health K, and Saxena VK. Detection of dengue problem. In: Gubler DJ and Kuno G (Eds.) virus in wild caught Aedes albopictus Dengue and dengue haemorrhagic fever. (Skuse) around Kozhikode airport, Wallingford: CAB International, 1997: 1-23. Malappuram district, Kerala, India. Dengue Bulletin, 2004, 28 (in press). 50 Dengue Bulletin – Vol 28, 2004 Autoimmunity in Dengue Virus Infection Chiou-Feng Lin*, Huan-Yao Lei*, Ching-Chuan Liu**, Hsiao-Sheng Liu*, Trai-Ming Yeh***, Shun-Hua Chen* and Yee-Shin Lin*# *Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan **Department of Paediatrics, National Cheng Kung University Medical College, Tainan, Taiwan ***Department of Medical Technology, National Cheng Kung University Medical College, Tainan, Taiwan Abstract Dengue haemorrhagic fever (DHF) is a complicated disease associated with viral and immune pathogenesis. There is still no effective vaccine to prevent the progression of DHF because of its undefined pathogenic mechanisms. The generation of autoimmunity in dengue virus (DEN) infection has been implicated in dengue pathogenesis. Based on our previous studies showing antibodies (Abs) against DEN nonstructural protein 1 (NS1) cross-reacted with human platelets and endothelial cells, a mechanism of molecular mimicry may contribute to autoantibody (autoAb) production. Here, the generation of autoAbs against human endothelial cells in patients infected with different DEN serotypes is shown. The levels of autoAbs present in different disease stages of DHF and the induction of endothelial cell apoptosis by patient sera were also determined. The results suggest that autoimmune responses are implicated in dengue disease pathogenesis and cause concern in vaccine development. Keywords: Dengue haemorrhagic fever, dengue virus serotype, autoimmunity, autoantibody, endothelial cells. Introduction Presently, the severity of dengue disease is primarily predicted according to the effect Infection with dengue virus (DEN) causes of antibody-dependent enhancement (ADE) dengue fever (DF) – an important in different serotype cross-infections[2,5,6]. In arthropod-borne viral disease in terms of order to effectively control the progression [1,2] morbidity and mortality and may result in of the disease, development of an effective severe dengue haemorrhagic fever and vaccine against DEN infection is needed. dengue shock syndrome (DHF/DSS). There are several vaccine candidates Globally, about 2.5 billion people are at risk undergoing clinical trials[7-10]. Nevertheless, of the infection[3]. A recent dengue outbreak the role that antibodies (Abs) may play in in Indonesia led to a 1.1% case-fatality rate increasing the severity of dengue [4] in 58,301 cases by April 2004 . All four infections[2,7,10] remains a matter of concern DEN serotypes were present in this outbreak. in vaccine development. # E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 51 Autoimmunity in Dengue Virus Infection In addition to the ADE of DEN infection, were 1:25 diluted and incubated with cells at autoantibody (autoAb) production may also 4 °C for 1 hour. After being washed three be involved in dengue diseases[11-15]. We times with PBS, the cells were incubated with demonstrated that the autoAbs generated in 20 ml of fluorescein isothiocyanate (FITC)- DEN infection induced endothelial cell conjugated anti-human IgG or IgM damage[13] and inflammatory activation (in (PharMingen, San Diego, CA) at 4 °C for 1 press). A mechanism of molecular mimicry in hour. The binding activity of Abs to cells was which Abs directed against DEN analysed using flow cytometry (FACScan; BD Biosciences, San Jose, CA) with excitation set nonstructural protein 1 (NS1) is, at least in at 488 nm. part, responsible for the autoimmunity. The relationships of the autoAb levels with Cell death detection dengue serotypes and disease severity are examined in this study. For cell viability determination, cells were stained with eosin Y and counted using light microscopy. Apoptosis-induced DNA strand Materials and methods breaks were analysed by terminal deoxynucleotidyl transferase-mediated Patient sera dUTP† nick-end-labeling (TUNEL) reaction DEN-2 and DEN-3 patient sera were using the ApoAlert DNA Fragmentation Assay collected during the outbreaks in southern Kit (Clontech, Palo Alto, CA). After incubation Taiwan from 1997 to January 1999[16]. DEN- with patient sera for 24 hours, endothelial 6 4 patient sera were obtained from the cells (1´10 ) were fixed and stained Department of Dengue Hemorrhagic Fever, according to the manufacturer’s instructions, Children’s Hospital No. 1, Ho Chi Minh City, and then analysed using flow cytometry. Viet Nam. The disease severity was based on the WHO definition[3]. Normal control sera Statistical analysis from five healthy individuals were used as The statistical difference was analysed using background. unpaired Student’s t-tests in SigmaPlot version 4.0 for Windows (Cytel Software Cell cultures Corporation, Cambridge, MA). Human umbilical cord vein endothelial cells (HUVEC) were cultured in modified M-199 medium as described previously[13]. For Results experiments, 1,000 U/ml trypsin and 0.5 Generation of autoAbs in dengue mM ethylenediaminetetraacetic acid (EDTA) were used to detach cells. patients infected with different serotypes and at different disease Binding activity detection stages After detachment, cells were suspended at Our previous studies demonstrated the 5´105 for flow cytometry. The cells were presence of anti-platelet and anti- washed briefly with phosphate-buffered endothelial cell autoAbs in dengue patient saline (PBS) and fixed with 1% formaldehyde sera[12,13]. The levels of these autoAbs were in PBS at room temperature for 10 minutes, then washed again with PBS. Patient sera † deoxyuridine triphosphate 52 Dengue Bulletin – Vol 28, 2004 Autoimmunity in Dengue Virus Infection higher in DHF/DSS than in DF patient sera. 4 (Table 1). There was no significant The dysfunction of platelets and endothelial difference between different serotype cells caused by the autoAbs was also shown. infections. The levels of autoAbs were The cross-reactivity of patient sera with higher in DHF/DSS than in DF patient sera. endothelial cells was the highest in the acute In addition, the levels of IgM isotype of stage (3-7 days after fever onset) and autoAbs were higher than those of IgG. The subsequently decreased in the convalescent DEN-1 serotype was not tested because we (1-3 weeks after acute phase) and later (8-9 had no DEN-1-infected patient sera. We months) stages. In our previous study, next investigated the endothelial cell cross- patient sera were collected from an reactivity of DHF patient sera at different outbreak of DEN-3 infection. In this study, disease grades. DHF patient sera collected we further examined the autoAb levels from Grades I to IV with DEN -4 infection, produced by patients infected with different according to the WHO definition, were DEN serotypes and the relationship tested. There was no significant difference between the autoAb levels and disease between the four grades of DHF in both severity. The results showed that the levels anti-endothelial cell IgM and IgG (Table 1). of anti-endothelial cell Abs, as determined Due to our limited sample sizes of patient by both the percentages of endothelial cells sera, especially of Grades I and IV, we were reactive with patient sera IgM or IgG and unable to determine whether there was any the mean fluorescence intensity, were correlation of autoAbs with disease severity. similar in patients infected with DEN -2, 3 or Table 1. Anti-endothelial cell IgM/IgG levels in the sera of dengue patients infected with different dengue serotypes and at different disease grades % of endothelial cells reactive Mean fluorescence intensity with patient sera IgM IgG IgM IgG Mean (SD) Mean (SD) Mean (SD) Mean (S D) Normal (n=5) 4.7 (0.5) 2.6 (0.6) 12.8 (1.5) 6.6 (0.8) DEN-2 infection DF (n=5) 38.6 (1.4)*** 14.8 (5.1)* 62.5 (4.1)*** 14.1 (2.1)* DEN-3 infection DF (n=6) 35.1 (3.7)*** 12.7 (2.1)* 65.7 (5.7)*** 15.7 (1.7)* DHF/DSS (n=5) 54.6 (4.4)*** 23.7 (1.9)** 74.9 (4.6)*** 24.6 (3.6)** DEN-4 infection DF (n=5) 35.9 (1.6)*** 15.1 (3.5)* 66.6 (6.3)*** 10.0 (2.3)* DHF (n=36) 50.5 (9.5)*** 24.9 (8.3)** 72.1 (13.9)*** 11.1 (4.4)* Grade I (n=1) 66.9 17.8 83.4 8.8 Grade II (n=26) 51.7 (9.0)*** 25.5 (7.7)* 73.6 (10.4)*** 11.4 (4.3)* Grade III (n=8) 45.4 (8.6)*** 24.7 (10.6)* 64.7 (7.0)*** 13.9 (6.4)* Grade IV (n=1) 42.2 17.9 63.0 11.6 Student’s t-tests: *P<0.05 vs Normal; **P<0.01 vs Normal; ***P<0.001 vs Normal. Dengue Bulletin – Vol 28, 2004 53 Autoimmunity in Dengue Virus Infection Induction of endothelial cell percentages of apoptotic cells from one set of duplicate cultures are shown in the Figure apoptosis by sera of dengue below. The results indicated that cell patients infected with different apoptosis was induced by all patient sera serotypes and the cells underwent a higher percentage of apoptosis when induced by DHF patient Anti-endothelial cell autoAbs caused cell sera than by DF patient sera. Healthy- damage which was characterized by control sera showed only the background apoptosis[13]. The ability of patient sera with level. Cell viability detected using eosin Y different DEN serotype infections to induce staining showed an inverse relationship with endothelial cell apoptosis was tested. the percentages of apoptosis (Table 2). HUVEC were treated with a 1:25 dilution of There was no significant difference in dengue patient or healthy-control sera for endothelial cell apoptosis induced by 24 hours, and cell apoptosis was measured patient sera with different serotype using TUNEL reaction followed by flow infections. cytometric analysis. The histogram and the Figure. Dengue patient sera induced endothelial cell apoptosis Normal DEN-2 DF DEN-3 DF DEN-3 DHF DEN-4 DF DEN-4 DHF 54 Dengue Bulletin – Vol 28, 2004 Autoimmunity in Dengue Virus Infection Table 2. Endothelial cell apoptosis induced by sera of dengue patients infected with different dengue serotypes % of endothelial cell % of cell viability apoptosis Normal (n=5) 94.1 (3.5) 6.9 (2.8) DEN-2 infection DF (n=5) 75.2 (5.4)** 21.9 (5.6)** DEN-3 infection DF (n=6) 81.7 (5.1)** 19.5 (7.1)** DHF/DSS (n=5) 66.6 (10.8)*** 29.7 (9.3)*** DEN-4 infection DF (n=5) 72.0 (9.5)** 22.0 (5.6)** DHF (n=5) 63.3 (15.1)*** 37.2 (5.5)*** Student’s t-tests: **P<0.01 vs Normal; ***P<0.001 vs Normal. Discussion serotypes. The percentages of endothelial cells reactive with DHF/DSS patient sera DHF is a life-threatening disease with poorly were higher than those with DF patient sera. defined pathogenic mechanisms[1,2,5,10]. An However, there was no difference in anti- ADE effect in different serotype cross- endothelial cell Ab levels at different DHF infection is frequent[2,5,6]. Patients may disease grades. The sample size of patient develop severe complications of progressive sera needs to be increased to gain an insight DHF. Vascular leakage and haemorrhagic into the role of anti-endothelial cell Abs in diathesis are the hallmarks in DHF patients. A DHF pathogenesis. These autoAbs exerted number of studies have demonstrated similar effects in the induction of endothelial abnormal immune responses caused by DEN cell apoptosis of patients infected with infection, including cytokine and chemokine different DEN serotypes. production, complement activation and In dengue pathology, various cytokines immune cell activation[10,11,17-20]. In addition, autoimmune responses may be involved in and chemokines including TNF-a, IL-6, IL-8, ‡ DHF pathogenesis[12-15,21]. Dengue patients and RANTES have been detected in patient produced Abs which cross-reacted with sera with DHF/DSS and in DEN-infected [17,18,20] human platelets and endothelial cells[12,13]. endothelial cell culture supernatants . Anti-NS1 produced after DEN infection may, Our recent studies also demonstrate that at least in part, account for the cross- anti-NS1 Abs can stimulate cytokine and reactivity of patient sera with endothelial cells. chemokine production (in press). Therefore, In this study, we further showed that the levels of anti-endothelial cell Abs were similar ‡ regulated upon activation normal T cell expressed and in patients infected with different DEN secreted Dengue Bulletin – Vol 28, 2004 55 Autoimmunity in Dengue Virus Infection both immune activation and apoptosis cross-reactivity of anti-NS1 to host antigens occur in endothelial cells after stimulation and cells and a haemorrhage-like hallmark by autoAbs. in mice. This, taken together with our findings, suggests that a potential pathogenic There are no dengue vaccines available. effect of DEN NS1 vaccine should be taken Yet, several potential vaccines, including into consideration. The possible approaches life-attenuated whole DEN and DNA include gene modifications of DEN NS1 to vaccines, are undergoing clinical trials[7-10]. It truncate or mutate the epitopes that may is hoped that a fusion or a chimera dengue cause the pathogenic effects. vaccine will be developed to provide protection against all serotypes of DEN infection. In addition, DEN NS1 protein Acknowledgement used as a vaccine candidate in mice showed resistance to fatal DEN encephalitis[22]. We thank Dr N.T. Hung from the Passive administration of anti-NS1 Abs also Department of Dengue Haemorrhagic Fever, conferred protection in mice when Children’s Hospital No. 1, Ho Chi Minh City, challenged with lethal doses of DEN[23]. Viet Nam for providing the DEN-4 patient However, these previous studies only sera. The authors acknowledge the editorial monitored the survival rates of mice but did assistance of Bill Franke. This work was not examine the potential histopathological supported by grant NSC92-3112-B006-003 effects. Studies by Falconar[21] showed the from the National Science Council, Taiwan. References [1] Gubler DJ. Dengue and dengue [7] Pang T. Vaccines for the prevention of hemorrhagic fever. Clinical Microbiology neglected diseases – dengue fever. Current Reviews, 1998, 11: 480-496. Opinion in Biotechnology, 2003, 14: 332- 336. [2] Halstead SB. Dengue. Current Opinion in Infectious Diseases, 2002, 15: 471-476. [8] Pugachev KV, Guirakhoo F, Trent DW and Monath TP. Traditional and novel [3] World Health Organization. Dengue approaches to flavivirus vaccines. haemorrhagic fever: diagnosis, treatment, International Journal of Parasitology, 2003, prevention and control. 2nd edition. Geneva: WHO, 1997. 33: 567-582. [9] Edelman R, Wasserman SS, Bodison SA, [4] World Health Organization. Dengue fever in Putnak RJ, Eckels KH, Tang D, Kanesa- Indonesia-update 4. 11 May 2004. Thasan N, Vaughn DW, Innis BL and Sun W. [5] Halstead SB. Pathogenesis of dengue: Phase I trial of 16 formulations of a challenges to molecular biology. Science, tetravalent live-attenuated dengue vaccine. 1988, 239: 476-481. American Journal of Tropical Medicine and [6] Halstead SB. Neutralization and antibody- Hygiene, 2003, 69: 48-60. dependent enhancement of dengue viruses. [10] Rothman AL. Dengue: defining protective Advance in Virus Research, 2003, 60: 421- versus pathologic immunity. Journal of 467. Clinical Investigation, 2004, 113: 946-951. 56 Dengue Bulletin – Vol 28, 2004 Autoimmunity in Dengue Virus Infection [11] Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH [19] Lin YW, Wang KJ, Lei HY, Lin YS, Yeh TM, and Liu CC. Immunopathogenesis of dengue Liu HS, Liu CC and Chen SH. Virus virus infection. Journal of Biomedical replication and cytokine production in Science, 2001, 8: 377-388. dengue virus-infected human B lymphocytes. Journal of Virology, 2002, 76: 12242-12249. [12] Lin CF, Lei HY, Liu CC, Liu HS, Yeh TM, Wang ST, Yang TI, Sheu FC, Kuo CF and Lin [20] Hung NT, Lei HY, Lan NT, Lin YS, Huang KJ, YS. Generation of IgM anti-platelet Lien LB, Lin CF, Yeh TM, Ha DQ, Huong autoantibody in dengue patients. Journal of VTQ, Chen LC, Huang JH, My LT, Liu CC Medical Virology, 2001, 63: 143-149. and Halstead SB. Dengue hemorrhagic fever in infants: a study on clinical and cytokine [13] Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, profiles. Journal of Infectious Diseases, 2004, Yeh TM, Chen SH and Lin YS. Antibodies 189: 221-232. from dengue patient sera cross-react with endothelial cells and induce damage. [21] Falconar AK. The dengue virus Journal of Medical Virology, 2003, 69: 82- nonstructural-1 protein (NS1) generates 90. antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and [14] Lin YS, Lin CF, Lei HY, Liu HS, Yeh TM, binds to human endothelial cells: potential Chen SH and Liu CC. Antibody-mediated implications in haemorrhagic fever endothelial cell damage via nitric oxide. pathogenesis. Archives of Virology, 1997, Current Pharmaceutical Design, 2004, 10: 142: 897-916. 213-221. [22] Zhang YM, Hayes EP, McCarty TC, Dubois [15] Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM, DR, Summers PL, Eckels KH, Chanock RM Chen SH, Liu CC, Chiu SC and Lin YS. and Lai CJ. Immunization of mice with Endothelial cell apoptosis induced by dengue structural proteins and nonstructural antibodies against dengue virus protein NS1 expressed by baculovirus nonstructural protein 1 via production of recombinant induces resistance to dengue nitric oxide. Journal of Immunology, 2002, virus encephalitis. Journal of Virology, 1988, 169: 657-664. 62: 3027-3031. [16] Lei HY, Huang JH, Huang KJ and Chang C. [23] Henchal EA, Henchal LS and Schlesinger JJ. Status of dengue control programme in Synergistic interactions of anti-NS1 Taiwan - 2001. Dengue Bulletin, 2002, 26: monoclonal antibodies protect passively 14-23. immunized mice from lethal challenge with [17] Rothman AL and Ennis FA. Immuno- dengue 2 virus. Journal of General Virology, pathogenesis of dengue hemorrhagic fever. 1988, 69: 2101-2107. Virology, 1999, 257: 1 -6. [18] Avirutnan P, Malasit P, Seliger B, Bhakdi S and Husmann M. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. Journal of Immunology, 1998, 161: 6338-6346. Dengue Bulletin – Vol 28, 2004 57 Inhibition of the NS2B-NS3 Protease – Towards a Causative Therapy for Dengue Virus Diseases Gerd Katzenmeier# Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand Abstract The high impact of diseases caused by dengue viruses on global health is now reflected in an increased interest in the identification of drug targets and the rationale-based development of antiviral inhibitors which are suitable for a causative treatment of severe forms of dengue virus infections – dengue haemorrhagic fever and dengue shock syndrome. A promising target for the design of specific inhibitors is the dengue virus NS3 serine protease which – in the complex with the small activator protein NS2B – catalyses processing of the viral polyprotein at a number of sites in the nonstructural region. The NS3 protease is an indispensable component of the viral replication machinery and inhibition of this protein offers the prospect of eventually preventing dengue viruses from replication and maturation. After nearly a decade of mainly genetic analysis of flaviviral replication, recent studies have contributed substantial biochemical information on polyprotein processing including the 3-dimensional structure of the dengue virus NS3 protease domain, the mechanism of co-factor-dependent activation and sensitive in vitro assays which are needed for studies on substrate specificity and the development of high-throughput assays for inhibitor screening. This review discusses recent biochemical findings which are relevant to the design of potential inhibitors directed against the dengue virus NS3 protease. Keywords: Dengue virus, NS2B/NS3, polyprotein, protease, inhibitor, treatment. Viral polyprotein processing nucleotides and encodes a single polyprotein precursor of 3,391 amino acid Dengue viruses, members of the Flaviviridae residues[3]. Individual viral proteins are family, possess single-stranded, positive arranged in the order C-prM-E-NS1-NS2A- sense RNA genomes and generate mature NS2B-NS3-NS4A-NS4B-NS5. Proteolytic viral proteins by co- and post-translational cleavages in the N-terminal region of the proteolytic processing of a polyprotein viral polyprotein are mediated by a host precursor catalysed by host cell and virus- signal peptidase and yields three structural encoded proteases [for review see refs. 1, 2 proteins C, prM and E, which constitute the and references herein]. The genomic RNA virion[4]. Before the virion exits the cell, prM of dengue virus serotype 2 contains 10,723 is cleaved by a cellular furin-type # E-mail: [email protected]; Tel.: (662)-800-3624-1237; Fax: (662)-441-9906 58 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease prohormone convertase in the post-Golgi Cleavage sites recognized by the acidic compartment to yield the M protein[5]. NS2B/NS3 protease consist of ‘dibasic’ Cleavages at the NS1/NS2A and residues Lys-Arg, Arg-Arg and Arg-Lys at the NS4A/NS4B junctions are catalysed by a (nonprime) P1 and P2 positions of the signalase bound to the membranes of the cleavage site sequence followed by short ER[6,7]. Proteolytic cleavages in the chain residues such as Gly, Ala and Ser at nonstructural region of the polyprotein are the (prime) P1’ position. The “non- mediated by a heterodimeric complex of canonical” NS2B/NS3 site contains a Gln NS3 with the activator protein NS2B which residue at the P2 position (Figure 1). catalyses in cis (intramolecular) cleavages at the NS2A/NS2B and NS2B/NS3 sites and in The NS3 protease domain trans (intermolecular) cleavages at the NS3/NS4A and NS4B/NS5 polyprotein The existence of a trypsin-like serine junctions[8-10]. Additional cleavages protease domain in the N-terminal region of mediated by the NS3 protease within the C, the flaviviral NS3 proteins was originally NS2A, NS4A and within a conserved C- predicted by sequence comparisons between [14] terminal portion of NS3 itself have been cellular and virus-encoded proteases . The described in the literature[11-13]. NS2B-NS3 endopeptidases of the Flavivirus genus which at present comprises at least 68 known members, are now commonly Figure 1. The 3-dimensional structure of designated as flavivirin (EC 3.4.21.91)[15,16]. the dengue virus NS3 protease The dengue virus 69 kDa NS3 protein is a (shown here is a superimposition of a multifunctional protein with a serine ribbon-presentation of the Ca trace on a protease domain located within the N- space-filling surface model. Residues of terminal 167 amino acid residues[17] and the catalytic triad (His51, Asp75, Ser135) activities of a nucleoside triphosphatase are shown as sticks. The figure was (NTPase) and RNA helicase in the C- [18] generated by Deepview Swiss-Pdb Viewer) terminal moiety . A catalytic triad consisting of residues His51, Asp75 and Ser135 was identified by site-directed mutagenesis experiments and replacement of the catalytic serine by alanine resulted in [19] Ser135 an enzymatically inactive NS3 protein . His51 The NS3 protease is an essential component for maturation of the virus and viable virus was never recovered from infectious cDNA Asp75 clones carrying mutations in the NS3 sequence which abolished protease activity[20]. Interaction of the helicase portion of NS3 with the viral RNA- dependent RNA polymerase NS5 may promote the association of the viral replicase complex to the membranes of the ER[21]. Dengue Bulletin – Vol 28, 2004 59 Inhibition of NS2B/NS3 Protease Figure 2. Schematic of dengue virus polyprotein processing (shown here are sites on the flavivirus polyprotein cleaved by host-encoded proteases and the virus-encoded two-component protease NS2B-NS3) C prM E NS1 NS2ANS2BNS3 NS4ANS4BNS5 NS2B/NS3 protease Signal peptidases Furin protease Unidentified protease The 3-dimensional structure of the N- natural polyprotein substrates. Although the terminal 185 residues of the dengue virus dengue virus NS3 protease exhibits NS2B- NS3 protease domain (NS3pro) was independent activity with model substrates resolved at a resolution of 2.1 Å[22]. The for serine proteases, enzymatic cleavage of overall folding of the protein resembles the dibasic peptides is markedly enhanced with 6-stranded b-barrel conformation typical for the NS2B-NS3 co-complex and the chymotrypsin-like serine proteases. presence of the NS2B activation sequence is Interestingly, the structure of the dengue indispensable for the cleavage of virus NS3 protease is closer to that of the polyprotein substrates in vitro[24]. The initial hepatitis C virus NS3/NS4A co-complex characterization of the co-factor than to the unliganded HCV NS3 protease, requirement for the dengue virus NS3 an observation which is suggestive of major protease had revealed that the minimal structural differences in the co-factor- region necessary for protease activation was dependent activation mechanism of the two located in a 40-residue hydrophilic segment proteases[23]. The substrate binding site of of NS2B[25]. The hydrophobic flanking NS3pro is relatively shallow and contourless regions of the 14 kDa NS2B protein are and specific enzyme-substrate interactions likely to be involved in targeting the were not predicted to extend beyond the protease complex to the membranes of the P2 and P2’ positions of the substrate ER where genome replication occurs. Fusion peptide in the absence of the NS2B co- of the NS2B core sequence to the NS3 factor[22] (Figure 2). protease domain yielded a catalytically active NS2B(H)-NS3p protein, which, upon expression in E. coli and subsequent The NS2B co-factor refolding, displayed autoproteolytic processing at the NS2B/NS3 site conducive The presence of a small activating protein or [24] co-factor is a prerequisite for optimal activity to the formation of a non-covalent adduct . Incorporation of a flexible nonamer linker, of the flaviviral NS3 proteases with their Gly4-Ser-Gly4, between the NS2B core 60 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease segment and the protease domain resulted reaction with the synthetic substrate peptide in a cleavage-resistent protease with GRR-AMC. optimized enzymatic activity against For the HCV NS3-NS4A protease hexapeptide substrates representing native complex, large structural rearrangements polyprotein cleavage junctions[26]. A leading to a catalytic triad, which is recombinant construct representing the full- conformationally optimized for proton length NS2B co-factor linked to the NS3 shuttle during catalysis, were observed as a protease domain was enzymatically active result of co-factor binding[30]. No 3- with peptide substrates derived from the dimensional structure is available for the polyprotein; however, this protein was dengue virus NS2B-NS3 co-complex and completely resistant to proteolytic self- the precise mechanism of co-factor- cleavage[27,28]. dependent activation is not fully elucidated An essential requirement for the correct as yet. In particular, it is an open question as association of the co-factor with the to whether binding of the substrate protease is the presence of hydrophobic contributes to the formation of an ‘induced residues which act as ‘anchor’ for the fit’ conformation as observed with the HCV protease – co-factor interaction. Recently, NS3 protease[31,32]. based on mutagenesis experiments and sequence comparisons of known flaviviral Substrate specificity co-factors, the “Fx3F” - motif was proposed as the common structural element involved So far, only very limited efforts have been [29] in co-factor binding to the protease . The undertaken to analyse the precise substrate “Fx3F” - motif is comprised of two bulky requirements and determinants of cleavage hydrophobic residues separated by three efficiency for the dengue virus NS3 protease. unspecified residues and it was This is surprising in the light of the fact that hypothesized that additional residues development of inhibitors against serine located at the N-terminus of the activation proteases usually starts with optimal peptide sequence would contribute to the stringent substrates derived from the nonprime side specificity of the protease for the wherein the scissile amide bond is replaced polyprotein substrate. A mutagenesis study by an electrophile which reacts with the with the dengue virus NS2B co-factor had catalytic serine residue. revealed that substitutions of the “F” - residues (Leu75 and Ile79 in NS2B of The NS3 protease reacts with small dengue virus type 2) by alanine resulted in model substrates for serine proteases such as preponderant effects on the catalytic activity N-a-benzoyl-L-arginine-p-nitroanilide (BAPA) of the NS3 protease rather than on substrate and activity of the unliganded NS3 protease binding [P. Niyomrattanakit, unpublished towards this substrate is higher than that of data]. A single residue in the N-terminal the NS2B-NS3 co-complex[24], a finding region of NS2B, Trp62, was critical for which suggests that substrate recognition in protease activation and replacement of this the complex requires additional interactions residue yielded a NS3 protease which was extending beyond the P1 side for optimal catalytically inactive in autoproteolysis and activity. A number of fluorogenic tripeptides containing dibasic residues at the P1 and P2 Dengue Bulletin – Vol 28, 2004 61 Inhibition of NS2B/NS3 Protease positions are cleaved by NS2B(H)-NS3pro 10-fold better than the best commercially protease and the best substrate identified in available substrate tested so far, GRR-AMC these experiments was GRR-AMC, which [J. Wikberg, unpublished data]. In the near had a Km value of 180 mM, a kcat value of future, these investigations will likely lead to 0.031 s-1 and a catalytic efficiency expressed the identification of NS3 substrates with -1 -1 [24] as kcat / Km of 172 s M . optimized sequence length and improved binding affinities, which can be applied as Chromogenic p-nitroanilide peptides sensitive probes for enzyme activity in high- representing hexameric sequences of the throughput inhibitor screenings. native polyprotein cleavage junctions were tested in a photometric assay and the most efficiently cleaved substrate derived from Perspectives for inhibitor the NS4B/NS5 site (Ac-TTSTRR-pNA) had a -1 development Km of 346 mM, kcat of 0.095 s and a kcat / Km of 275 s-1 M-1 [26]. Principally, every step of viral Recently, we have shown by a HPLC- morphogenesis, from cell-entry, uncoating, based assay with fluorometric detection that replication and assembly of new virus the NS2B-NS3pro protease incorporating a particles, is a potential target for antiviral full-length NS2B co-factor could cleave N- inhibitors. However, the molecular events in terminally dansylated 12mer peptides the infectious cycle of flaviviruses such as mimicking native polyprotein junctions in dengue virus are characterized only to a the absence of microsomal membranes[28]. very limited extent, making the design of However, this protein was completely specific inhibitors an adventurous task. In inactive in autocleavage and the efficiency contrast, proteases and their inhibitors have of this recombinant protease with the been intensively studied because of their peptide substrate was markedly reduced potential for the development of selective when compared to constructs incorporating antiviral compounds. Although tremendous the 40-residue NS2B core sequence, likely progress in the field is indicated by the due to structural distortions induced by the design and clinical use of antiviral drugs flanking regions of NS2B and inefficient against HIV (AIDS) and hepatitis C virus activation of the protease. proteases, the potential of dengue and related flaviviral proteases for inhibitor Currently, a detailed study on substrate discovery is largely unexploited. In response specificity of the dengue virus NS3 protease to the global problem of the dengue virus is in progress, which uses combinatorial epidemics, considerable efforts are now libraries of internally quenched fluorogenic being devoted to the development of drugs peptides labelled with the aminobenzoyl / which will eventually be suitable for a p-nitrotyrosine reporter pair. For a substrate chemotherapeutic intervention in acute peptide based on the NS3/NS4A cleavage dengue diseases, not only by academic site, Abz-AAGRK?SLTLY(NO2)R-NH2 (? institutions but also by pharmaceutical denotes the cleavage site), a Km value of 141 companies (for example see -1 mM, a kcat of 0.18 s and a kcat / Km of 1262 http://www.nitd.novartis.com). s-1 M-1 was found, which is approximately 62 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease In a first step towards a rational inhibitor suggest the existence of a high-affinity design for the dengue virus NS3 serine binding site in the non-prime region of the protease, inhibition by synthetic peptides enzyme and offer the prospect of mimicking uncleavable transition state developing effective inhibitors against the isosteres of the P6-P2’ residues of the native dengue virus protease by combinatorial polyprotein sites, was demonstrated[26]. The optimization based on the structure(s) of peptides with an a-keto amide in place of native polyprotein cleavage site peptides. the scissile amide bond acted as competitive However, in general, peptide-based inhibitors of the NS3 protease with Ki - inhibitors exhibit poor pharmacokinetic values in the micromolar range. properties and usually the conversion of Replacement of the P1’-P2’ residues by a these structures into less “peptide-like” carboxyl-terminal aldehyde in the compounds (“peptidomimetics”) is required NS3/NS4A-derived peptide (Ac-FAAGRR- to generate drug-like entities. CHO) yielded a competitive inhibitor with a Inhibitor discovery for the dengue virus K of 16 mM. For the dengue virus protease, i NS3 protease is currently limited by the lack the hexapeptides displayed K - values which i of a 3-dimensional structure for the NS2B- were only 2- to 6-fold lower than the K - m NS3 co-complex; however, it can be value for the corresponding substrate, a expected that crystallographic studies and feature which discriminates dengue virus NMR-experiments will provide more insight NS3 from the HCV protease, where product into the structure and catalytic mechanism of inhibitors had binding affinities which were the enzyme in the near future. In addition, one order of magnitude lower than those for powerful computer modelling approaches the substrates[33,34]. exist which may help to obtain information Product inhibition of the HCV NS3 required for a rational drug design even in protease by cleavage-site derived peptides the absence of crystallographic structures. led to the discovery of very potent inhibitors Proteochemometric modelling (PCM) is of this enzyme with IC – values in the 50 currently explored as a tool to analyse the nanomolar range by cyclic optimization of structural and physicochemical properties the inhibitor structures[33,34]. Recently, we which are necessary for the interaction of have shown that peptides representing non- potential inhibitors with the dengue virus prime-side residues of the dengue virus NS3 NS3 protease target structure[35]. Preliminary protease act as competitive inhibitors of the data obtained by this approach suggest that enzyme, whereas prime-side peptides the proteochemometric models are valid appeared to have negligible effects on and useful for the accelerated design of enzyme inhibition at concentrations >1.0 novel inhibitors. This approach does not mM. (S. Chanprapaph, unpublished data). Ki only circumvent the traditional erratic dug - values for hexapeptides derived from all 4 discovery process with its high attrition rates, dengue virus cleavage sites were in the low but also allows to incorporate potential micromolar range and the best inhibitor was resistance of the target and the development based on the NS2A/NS2B site (Ac-RTSKKR- of ‘drug resistance – resistant’ compounds as m CONH2) and gave a Ki of 12 M. In analogy an initial consideration in the design process. to the HCV NS3 protease, these findings The existence of large conformational Dengue Bulletin – Vol 28, 2004 63 Inhibition of NS2B/NS3 Protease ensembles is particularly challenging in the biomedical targets in the dengue virus case of rapidly mutating viral enzymes, polyprotein would even make a therapy where a design against a moveable target feasible, which uses combinations of would require large sets of corresponding different inhibitors and therefore could inhibitors. In the future, these problems are minimize the risk of rapid resistance likely to be addressed by ‘shotgun development. approaches’ to the structure of enzyme- It can also be expected that progress for inhibitor complexes and the identification of inhibitors against the dengue virus protease hot spots of ‘interaction flexibility’ by the will be of large benefit for drug design use of fast, high-resolution methods such as against related human pathogens of the NMR. Detailed accounts on this strategy are flavivirus complex such as yellow fever virus, given in Reference 36. Japanese encephalitis virus and West Nile virus. Conclusions However, in order to bring an effective anti-dengue drug from the ‘bench to the Substantial progress has been made over the past few years in the biochemical bedside’, several questions and limitations need to be addressed. These include the characterization of the dengue virus NS2B- evaluation of prognostic markers for disease NS3 two-component protease. The data, severity, the pathobiochemistry of dengue which are available now, make the dengue haemorrhagic fever and shock syndrome, virus NS3 protease a valid molecular target the problem of selectivity against for the development of antiviral compounds. pharmacologically relevant human proteases A large repertoire of powerful methods for such as furin and the risk of adverse effects. inhibitor development and evaluation exists Potential complications may also arise from which includes state-of-the-art technology in the presence of four related dengue organic synthesis and computer-aided serotypes in the case that their NS3 molecular design. Although there is no proteases show marked differences in their suitable animal model available for dengue inhibition profiles. Intensive efforts and virus diseases, initial screening of potential sustained multidisciplinary research is antiviral compounds would be facilitated by required in the future to cope with the well-established insect and mammalian cell challenging task of a causative treatment for culture systems which are useful to monitor dengue virus diseases. the effects of anti-NS3 inhibitors on the propagation of the virus. Acknowledgements Moreover, alternative drug targets which are present on the dengue virus NS3 We thank Dr J. Wikberg, Department of protein can be exploited for inhibitor Pharmaceutical Biosciences, Uppsala development. These include the binding site University, Sweden, for intensive discussions of the NS2B co-factor to the NS3 protease, and access to data prior to publication. This the NS3 NTPase / helicase portion and the work was supported by grants from the interaction surface of NS3 with the NS5 Thailand Research Fund (TRF). replicase. 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Proc Natl DW, Bazan JF, Fletterick RJ and Rice CM. Acad Sci USA, 1990, 87: 8898-8902. Evidence that the N-terminal domain of [17] Li H, Clum S, You S, Ebner KE and nonstructural protein NS3 from yellow fever Padmanabhan R. The serine protease and virus is a serine protease responsible for site- RNA-stimulated nucleoside triphosphatase specific cleavages in the viral polyprotein. and RNA helicase functional domains of Proc Natl Acad Sci USA, 1990, 87: 8898- dengue virus type 2 NS3 converge within a 8902. region of 20 amino acids. J Virol, 1999, 73: 3108-3116. Dengue Bulletin – Vol 28, 2004 65 Inhibition of NS2B/NS3 Protease [18] Gorbalenya AE, Koonin EV, Donchenko AP protein NS2B: identification of a domain and Blinov VM. Two related superfamilies of required for NS2B-NS3 protease activity. J putative helicases involved in replication, Virol, 1993, 67: 2034-2042. recombination, repair and expression of [26] Leung D, Schroeder K, White H, Fang NX, DNA and RNA genomes. 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[23] Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, [30] Yan Y, Li Y, Munshi S, Sardana V, Cole JL, Markland W, Lepre CA, O’Malley ET, Sardana M, Steinkuehler C, Tomei L, De Harbeson SL, Rice CM, Murcko MA, Caron Francesco R, Kuo LC and Chen Z. Complex PR and Thomson JA. Crystal structure of the of NS3 protease and NS4a peptide of BK hepatitis C virus NS3 protease domain strain hepatitis C virus: a 2.2 A resolution complexed with a synthetic NS4A cofactor structure in a hexagonal crystal form. Protein peptide. Cell, 1996, 87: 343-355. Sci, 1998, 7: 837-847. [24] Yusof R, Clum S, Wetzel M, Krishna Murthy [31] Barbato G, Cicero DO, Cordier F, Narjes F, HM and Padmanabhan R. Purified Gerlach B, Sambucini S, Grzesiek S, Matassa NS2B/NS3 serine protease of dengue virus VG, De Francesco R and Bazzo R. The type 2 exhibits cofactor NS2B dependence solution structure of the N-terminal protease for cleavage of substrates with dibasic amino domain of the hepatitis c virus (HCV) NS3 acids. J Biol Chem, 2000, 275: 9963-9969. protein provides new insights into its activation and catalytic mechanism. EMBO J, [25] Falgout B, Miller RH and Lai CJ. Deletion 2000, 19: 1195-1206. analysis of dengue virus type 4 nonstructural 66 Dengue Bulletin – Vol 28, 2004 Inhibition of NS2B/NS3 Protease [32] Bianchi E, Orru S, Dal Piaz F, Ingenito R, [35] Wikberg JES, Lapinsh M and Prusi P. Casbarra A, Biasiol G, Koch U, Pucci P and Proteochemometrics – a tool for modelling Pessi A. Conformational changes in human the molecular interaction space. In: Mueller hepatitis C virus NS3 protease upon binding G, Kubinyi H (Eds) Chemogenomics. of product-based inhibitors. Biochemistry, Methods and Principles in Medicinal 1999, 38: 13844-13852. Chemistry. Wiley-VCH, Weinheim, 2004, in press. [33] Ingallinella P, Altamura S, Bianchi E, Taliani M, Ingenito R, Cortese R, De Francesco R, [36] Bianchi E and Pessi A. Inhibiting viral Steinkuehler C and Pessi A. Potent peptide proteases: challenges and opportunities. inhibitors of the human hepatitis C virus Biopolymers, 2002, 66: 101-114. NS3 protease are obtained by optimizing the cleavage products. Biochemistry, 1998, 37: 8906-8914. [34] Ingallinella P, Fattori D, Altamura S, Steinkuehler C, Koch U, Cicero D, Bazzo R, Cortese R, Bianchi E and Pessi A. Prime site binding inhibitors of a serine protease: NS3/4A of hepatitis C virus. Biochemistry, 2002, 41: 5483-5492. Dengue Bulletin – Vol 28, 2004 67 Prognostic Factors of Clinical Outcome in Non-Paediatric Patients with Dengue Haemorrhagic Fever/Dengue Shock Syndrome Jaime R. Torres*#, José M. Torres-Viera**, Hipólito García**, José R. Silva**, Yasmín Baddour**, Angel Bajares** and Julio Castro M.* *Tropical Medicine Institute, Infectious Diseases Section, Universidad Central de Venezuela **Clínica Santa Sofía, Caracas, Venezuela Abstract A total of 112 adults with dengue haemorrhagic fever (DHF) admitted at Clínica Santa Sofía, Caracas, Venezuela, were studied during June 1998–June 2001. Capillary leakage (CL) occurred in 28.8% cases, 21.6% experienced bleeding, 9.2% developed pleural effusion (PE) and 9.2% developed acute acalculous cholecystitis (AAC). High correlation was noticed between the length of illness prior to admission (IPA) and length of hospitalization (LH) and levels of Hct, Hb and leukocytes. Significant differences were seen in the length of IPA, LH and level of platelets for patients with or without bleeding (P<0.05) or CL (P<0.005), and in LH for patients with or without PE (P<0.005), or CL (P<0.05). Patients with AAC reached higher leukocyte counts (P<0.05). ANOVA showed an association between IPA and LH, between either of them and levels of Hb, Hct or leukocytes, and between platelets levels and PE, CL or bleeding. The length of IPA and degree of alteration of Hb, Hct, leukocytes and platelets predicted a more severe course in adults with DHF. Keywords: Dengue, dengue haemorrhagic fever, adults, prognostic factors. Introduction been reported in at least 25 countries in the Americas[4]. Venezuela has recorded large Over the last decade, dengue fever has numbers of DHF cases every year, and, in dramatically spread in virtually all Latin 1995, the country reported the largest American and Caribbean countries which outbreak in the region with almost 30,000 are infested with Aedes aegypti. During this dengue cases and 5,000 DHF cases. period, the number of cases reported every Although DEN-1, DEN -2 and DEN -4 had year in these countries jumped from been isolated during this epidemic, DEN-2 250,000 to more than 750,000[1]. was the predominant serotype[5]. Furthermore, recent serological surveys suggest the occurrence of millions of such In contrast with observations made in infections[2]. After its emergence in Cuba in Asian countries, where DHF is almost 1981[3], epidemics or sporadic cases of completely restricted to young children, in dengue haemorrhagic fever (DHF) have the Americas, older age groups are widely involved[6-8]. # E-mail: [email protected]; Fax: + (58-212) 9876590 68 Dengue Bulletin – Vol 28, 2004 Prognostic Factors in Adult Patients with Haemorrhagic Dengue The host’s immune response appears to <105 per mL, and evidence of plasma be a major factor influencing the type and extravasation, such as haemoconcentration severity of disease, as sequential infection (20% increase over base Hct, or 20% with different dengue virus serotypes in the decrease after rehydration), polyserositis, or presence of non-neutralizing antibodies has hypoproteinemia, with or without signs of been strongly incriminated in the circulatory failure, including narrowing of occurrence of DHF/DSS[8-10], and cases of pulse pressure (£ 20 mm Hg), hypotension, DHF/DSS are seldom documented in or shock[15]. The level of severity of the patients with primary infection[11-13]. condition was established according to the Individual factors, such as age, sex, genetic following scale: background and underlying diseases, may (i) Grade I, fever + nonspecific also play a role[14]. constitutional symptoms + positive Since most of the currently available tourniquet test + evidence of clinical and epidemiological data on haemoconcentration and thrombo- DHF/DSS derives from observations on cytopenia; infected children, information is sparse (ii) Grade II, all of the above + regarding prognostic factors of poor spontaneous bleeding, usually evolution among adult patients. In an restricted to the skin ± other sites; attempt to identify potential prognostic factors in this specific setting, a retrospective (iii) Grade III, all of the above + study was carried out among non-paediatric circulatory failure manifested by inpatients with DHF/DSS followed at a rapid and weak pulse, narrowing of single South American private medical pulse pressure (20 mm Hg or less), institution. or hypotension with the presence of cold, clammy skin, and restlessness or agitation, and Materials and methods (iv) Grade IV, all of the above + A total of 112 non-paediatric patients profound shock with undetectable [15] (male/female ratio: 64/48; age range: 15-92 blood pressure and pulse . years; median 36 years) admitted to Clínica Clinical evidence of gross capillary Santa Sofía, Caracas, Venezuela, during a leakage (CL) was defined as the occurrence 36-month period from June 1998 to June of polyserositis, expressed by any of the 2001, who were attended to by the same following: symptomatic pleural effusions, team of physicians, were included in the ascytis, pericardial effusions, gallbladder wall study. The endemic dengue transmission edema and/or acute acalculous cholecystitis. season in the country typically extends from May to October, matching the yearly cycle Acute acalculous cholecystitis (AAC) was of rains. diagnosed according to the following criteria: fever; persistent abdominal pain; All cases fulfilled the diagnostic criteria nausea and vomiting. On physical of DHF/DSS, according to WHO and PAHO examination, occurrence of tenderness or definitions: acute febrile illness with muscle rigidity in the right upper abdominal evidence of bleeding, thrombocytopenia quadrant, epigastrium, or both, and Dengue Bulletin – Vol 28, 2004 69 Prognostic Factors in Adult Patients with Haemorrhagic Dengue Murphy's sign. Additional relevant findings bleeding, and 14 (12.5%) developed pleural were a palpable mass in the region of the effusion on plain chest X-ray films. AAC gall-bladder, jaundice, and mild elevations ensued in 14 (12.5%) cases. No deaths in the serum levels of bilirubin, alkaline occurred (Table 1). phosphatase, and/or transaminases. On ultrasound, demonstration of an enlarged Table 1. Degree of severity of DHF and ³ gall-bladder with thickened wall ( 6 mm) clinical complications in 112 non-paediatric and pericholecystic fluid appearing as a halo, Venezuelan patients or the presence of a diffuse, homogeneous, non-shadowing, medium level echogenicity Severity of within the gall-bladder lumen, were all disease and Number Percentage considered ‘positive’ findings[16]. clinical of cases (%) complication Either specific IgM or IgG seroconversion over a period of 15 days was DHF grade I 71 63.4.9 documented by means of a rapid DHF grade II 23 20.5 commercial qualitative immuno- ® chromatographic test (PanBio Dengue , DHF grade III 18 16.1 Windsor, Australia). or IV Statistical analysis was performed using Bleeding 25 22.3 a StatSoft, Inc. (1995), STATISTICA for Windows, Computer programme manual, Pleural effusion 14 12.5 Tulsa, OK. Either Student’s t-test for the Acalculous 14 12.5 comparison of means from independent cholecystitis samples of unknown variances, Pearson’s correlation analysis, univariate and Pearson’s correlation analysis results multivariate logistic regression analysis was according to clinical complications are performed with CAA, vascular leakage or depicted in Table 2. A significant correlation bleeding as main outcomes, by means of a was found between the levels of Hb and forward stepwise independent variable entry Hct (r=0.762; P<0.0001), and between the in the final model. All calculations were level of platelets and Hb (r=-0.280; two-tailed, and 0.05 significant criteria were P<0.01). Correlation was also observed used. ANOVA analysis of variables was between the number of days of illness prior performed as required. to admission (IPA) and the length of hospitalization (r=-0.233; P<0.05), as well as between the length of hospitalization Results (LH) and the degree of alteration in platelets Out of 112 patients, 71 (63.4%) developed level (r=-0.198; P<0.05). A higher DHF grade I, 23 (20.5%) had DHF grade II, correlation was seen between the length of and 18 (16.1%) had either DHF grade III or hospitalization and the number of days of grade IV. In 37 patients (33%), clinical signs IPA (r=-0.426; P<0.005) for patients with of vascular leakage were evident, 25 dengue grade II or higher, or those with (22.3%) experienced moderate to severe bleeding (r=-0.427, P<0.005). 70 Dengue Bulletin – Vol 28, 2004 Prognostic Factors in Adult Patients with Haemorrhagic Dengue Significant differences were seen in the longer (mean 4.35 days vs. 3.26 days; length of IPA (mean 4.32 days vs. 3.60 days; P<0.05), the length of hospitalization was P<0.05), and the minimum level of platelets longer (mean 4.85 days vs. 3.61 days; (mean 43,000 per mL vs. 65,816 per mL; P<0.001), the serum levels of alkaline P<0.05) for patients with or without clinical phosphatase were higher (mean 183 m/L vs. bleeding, as well as those with or without 91 m/L; P<0.05), and the minimum level of dengue type 2 (mean 4.43 vs. 3.56 days, leukocytes was higher (mean 12,890 per mL and 48,870 vs. 70,800 per mL; P<0.05, vs. 5,026 per mL; P<0.001) in patients respectively). Significant differences were developing vascular leakage. Patients with also found in the length of hospitalization AAC exhibited a significantly longer hospital (mean 5.77 days vs 3.61 days; P<0.005) for stay (4.71 days vs. 3.71 days; P<0.05), and patients with or without pleural effusion, as a higher mean level of peripheral blood well as for those with or without clinical leukocytes (15,986 x mm3 vs. 5,071 x mm3; vascular leakage (mean 4.85 days vs. 3.53 P<0.001). days; P<0.005). The length of IPA was Table 2. Significant differences in disease outcome in 112 non-paediatric Venezuelan patients with dengue haemorrhagic fever according to type of clinical complication Clinical complication Variable Mean value Significance Bleeding Yes Illness prior to admission 4.66 days P<0.05 No 3.70 days Yes Minimum platelet level 43,619 per mL P<0.05 No 61,128 per mL Pleural Yes Length of hospitalization 5.0 days P<0.005 effusion No 3.6 days Signs of Yes Length of hospitalization 4.24 days P<0.05 capillary No 3.53 days leakage Yes Illness prior to admission 4.71 days P<0.001 No 3.56 days Yes Minimum platelet level 45,178 per mL P<0.01 No 68,784 per mL AAC Yes Peripheral blood leukocytes 11,300 per mL P<0.05 No 3,418 per mL Both one-way and multivariate logistic bleeding were illness severity on admission regression analysis revealed that the only according to WHO scale (OR=5.3; variables significantly associated with P<0.001), and length of IPA (OR=1.7; Dengue Bulletin – Vol 28, 2004 71 Prognostic Factors in Adult Patients with Haemorrhagic Dengue P<0.05). Vascular leakage was also Table 4. Logistic regression analysis of associated with illness severity on admission clinical variables associated significantly with in the multivariate regression analysis bleeding in 112 Venezuelan patients with (OR=14.3; P<0.005), as well as with the dengue haemorrhagic fever length of hospitalization (OR=1.79; Univariate Multivariate P=0.001), illness severity on admission Variable (OR=26.5; P=0.001), leukocytes level OR P OR P (OR=1.33; P=0.001), and a prolonged Degree of severity 5.85 0.001 5.3 0.01 aPTTA† (OR=18.9; P=0.001), in the univariate regression analysis. Of note was Length of illness 1.37 0.032 1.7 0.13 prior to admission the finding that whereas patients with bleeding did not remain hospitalized longer, Platelets (nadir) 0.99 0.015 >0.5 those with vascular leakage did (OR=1.79; Platelets <50.000 3.83 0.005 >0.05 P=0.001). Statistically significant results for Pleural effusion 6.3 0.02 >0.05 the one-way and multivariate logistic regression analysis are summarized in Tables OR = odds ratio Blank cells indicate values that should not be included 3, 4 and 5. since they are not significant Table 3. Logistic regression analysis of Table 5. Logistic regression analysis of clinical variables associated significantly clinical variables significantly associated with with capillary leakage in 112 non-paediatric acute achalculous cholecystitis (AAC) in 112 Venezuelan patients with dengue non-paediatric Venezuelan patients with haemorrhagic fever dengue haemorrhagic fever Univariate Multivariate Univariate Multivariate Variable Variable OR P OR P OR P OR P Degree of severity 26.5 0.001 14.3 0.002 Degree of severity 50.5 0.001 >0.05 Blood leucocytes 1.58 0.001 1.34 0.04 Length of 1.79 0.001 >0.05 level hospitalization Abnormal PTT 1.81 0.001 11.3 0.01 Blood leucocytes 1.33 0.001 >0.05 Pleural effusion 9.75 0.001 >0.05 level OR = odds ratio Abnormal PTT 18.9 0.001 >0.05 Blank cells indicate values that should not be included since they are not significant Bleeding 3.51 0.015 >0.05 OR = odds ratio Blank cells indicate values that should not be included Discussion since they are not significant The age distribution for DHF cases in the Americas differs from that observed in South-East Asia[1,3,6,7,8,17,18], where young † Activated partial thromboplastin time 72 Dengue Bulletin – Vol 28, 2004 Prognostic Factors in Adult Patients with Haemorrhagic Dengue children continue to be the age group patients show severe bleeding of the almost exclusively affected. In contrast, an gastrointestinal (GI), or of other sites, age range of 31 to 45 years has been preceding the shock, which may be severe reported for Brazilian patients with enough to cause death[20-21,25]. DHF/DSS[6], while in Puerto Rico, the mean Relevant laboratory findings in DHF age of the patients reported in 1990-91 was cases include thrombocytopenia, haemo- 38 years[7]. Furthermore, during the concentration and hypoproteinemia[15]. A outbreaks in Cuba in 1981 and in drop in platelet count to below 100,000 per Venezuela in 1989, about one third of the mL and an increase of 20% or more in the deaths were among patients older than 14 haematocrit, both resulting from increased years[8,19], and in the 1997 Cuban outbreak, vascular permeability, are consistent findings. all registered deaths were seen among Other signs of plasma leakage include pleural adults[8]. effusion, ascites and hypoproteinemia. The main pathogenic feature of dengue Leukopenia and leukocytosis are common, is an increase in vascular permeability and aminotransferases are usually elevated. leading to loss of plasma from blood vessels, Thrombin, prothrombin and partial which causes haemoconcentration, low thromboplastin times are often prolonged. blood pressure and shock. This may also be Fibrinogen levels decrease and fibrin accompanied by haemostatic abnormalities degradation products may increase. In such as thrombocytopenia, vascular changes patients with DSS, the severity of laboratory and coagulopathy[15]. abnormalities described for DHF tends to be worse. Dilutional hyponatremia and The clinical spectrum of dengue virus hypoproteinemia correlate with disease infection may range from an asymptomatic severity[1] . infection to a severe and rapidly fatal disease[15,20-24]. The most severe end of the In the current series, patients with spectrum of dengue virus infection in longer length of hospitalization exhibited children is represented by dengue shock significantly lower levels of platelets, as well syndrome (DSS)[15]. Adults seem less likely as a higher tendency to severe capillary than children to suffer from DSS. Indeed, in vascular leakage (CVL)‡. Overall, a shorter a retrospective study of 108 adult duration of IPA associated surprisingly with Malaysians with DHF, the overall morbidity a longer length of hospitalization, probably was significant (29.4%) but the case-fatality reflecting the fact that more severely ill rate remained low (2.0%)[25]. patients tended to seek medical attention earlier. Nevertheless, patients with dengue Haemorrhagic manifestations in DHF type II, as well as those with evidence of usually consist of petechiae, ecchymoses, CVL, exhibited significantly more protracted easy bruising and bleeding from IPA. The occurrence of the latter two venipuncture sites. Epistaxis, gum bleeding conditions most likely reflect the delay in and gastrointestinal haemorrhage are less [23-24] common . If improperly treated, shock leads to metabolic acidosis, severe ‡ CVL may induce many other clinical manifestations generalized bleeding and, eventually, [15] and complications besides clinical bleeding, such as death . Unlike children, many adult pleural effusion, ascitis, joint swelling, etc. Dengue Bulletin – Vol 28, 2004 73 Prognostic Factors in Adult Patients with Haemorrhagic Dengue initiating a proper and adequate fluid- Viral, serological and genetic factors replacement treatment, which is the key to may influence virulence. Molecular studies treating DHF in order to compensate for the have identified genetic variation among all 4 loss of plasma from blood vessels due to dengue virus serotypes[31-34]. Of note here is increased vascular permeability[1,15]. the finding that DEN-2 strains associated Of note is the finding that a with grade II DHF or DSS grow to higher considerable percentage of the cases titers in peripheral blood leukocytes than do (12.5%) developed AAC. Recent data DEN-2 strains isolated from mildly ill [35,36] suggest that in children with DHF, a gall- patients . Although characterization of the viral serotypes involved in these patients bladder wall thickening =5 mm on was not performed, DEN -1, 2 and 4 all ultrasonography correlates with a higher risk of hypovolemic shock[26]. However, despite circulated in Venezuela during the period a few scattered reports of AAC complicating when the cases occurred, DEN-2 being the [5] adult DHF patients[27-30], little information most prevalent one . exists in medical literature on the In conclusion, the number of days of pathological and clinical implications of this IPA, the length of hospitalization and the newly recognized condition. It is worth degree of alteration in the level of Hb, Hct, mentioning that while our nine patients with leukocytes and platelets were all predictors AAC exhibited a significantly increased level of a more severe and complicated course in of peripheral blood leukocytes during adult patients with DHF/DSS. The onset of hospitalization, their clinical outcome in leukocytosis must suggest the occurrence of terms of IPA, length of hospitalization or inflammatory complications such as AAC. occurrence of other life-threatening DHF/DSS in the Americas continues to complications, did not differ from that of occur in a significant number of adults, but patients without AAC. Details of the clinical it is not clear whether this relates with the aspects and imaging techniques findings for genetic background of the populations, the this set of patients will be discussed epidemiological events or, else, with other elsewhere. unknown factors. References [1] Istúriz R, Gubler D and Brea del Castillo J. epidemic. Bull Pan Am Health Organ, 1986, Dengue and dengue haemorrhagic fever in 20: 24-30. Latin America and the Caribbean. Infect Dis [4] Gubler DJ and Trent DW. Emergence of Clin North Am, 2000, 14: 121-140. epidemic dengue/dengue haemorrhagic fever [2] Pinheiro FP and Corber SJ. Global situation as a public health problem in the Americas. of dengue and dengue haemorrhagic fever Infect Agents Dis, 1994, 2: 383-385. and its emergence in the Americas. World [5] Salas RA, Tovar D, Barreto A, de Miller E, Health Stat Q, 1997, 50: 161-169. Leitmeyer K and Rico-Hesse R. Serotypes and genotypes of dengue virus circulating in [3] Kouri G, Guzman MG and Bravo J. Venezuela, 1990-1997. Acta Cient Venez, Haemorrhagic dengue in Cuba: history of an 1998, 49 (Suppl 1): 33-37. 74 Dengue Bulletin – Vol 28, 2004 Prognostic Factors in Adult Patients with Haemorrhagic Dengue [6] Zagne SM, Alves VG, Nogueira RM, [16] Nahrwold DL. Acute Cholecystitis. In: Miagostovich MP, Lampe E and Tavares W. Sabiston’s Textbook of Surgery, 15 th ed., Dengue haemorrhagic fever in the state of 1997. W.B. Saunders Company, Editors, Rio de Janeiro, Brazil: a study of 56 1127-1132. confirmed cases. Trans R Soc Trop Med Hyg, [17] Sumarmo, Wulur H, Jahja E, Gubler DJ, 1994, 88: 677-679. Suharyono W and Sorensen K. Clinical [7] Rigau-Perez JG. Clinical manifestations of observations on virologically confirmed fatal dengue haemorrhagic fever in Puerto Rico, dengue infections in Jakarta, Indonesia. Bull 1990-1991. Puerto Rico Association of World Health Organ, 1983, 61: 693-701. Epidemiologists. Rev Panam Salud Publica, [18] Thongcharoen P and Jatanasen S. 1997, 1: 381-388. Monograph on dengue/dengue haemorrhagic [8] Kouri G, Guzman MG and Bravo J. Why fever. New Delhi: WHO Regional dengue haemorrhagic fever in Cuba? 2: An Publications, South-East Asia Series, No. 22, integral analysis. Trans R Soc Trop Med Hyg, 1993, 1-8. 1987, 81: 821-823. [19] Torres JR and Torres-Viera CG. Dengue in [9] Dengue and dengue haemorrhagic fever in Latin America – a unique situation. Dengue the Americas: Guidelines for prevention and Bull, 2002, 26: 62-69. control. Pan American Health Organization, [20] Funahara Y, Sumarmo and Wirawan R. Scientific Publication No. 548, 1994. Features of DIC in dengue haemorrhagic [10] Halstead SB. Pathogenesis of dengue: fever. Bibliotheca haematologica, 1983, 49: challenges to molecular biology. Science, 201-211. 1988, 239: 476-481 [21] Tsai CJ, Kuo CH, Chen PC and Changcheng [11] Gubler DJ. Dengue. In Monath TP (Ed.), The CS. Upper gastrointestinal bleeding in arboviruses: epidemiology and ecology, Vol. dengue fever. Am J Gastroenterol, 1991, 86: II. Boca Raton, CRC Press Inc, 1988: 223- 33-35. 260. [22] Nimmannitya S, Thisyakorn U and [12] Scott RM, Nimmannitya S, Bancroft WH Hemsrichart V. Dengue haemorrhagic fever and Mansuwan P. Shock syndrome in with unusual manifestations. Southeast Asian J primary dengue infections. Am J Trop Med Trop Med Public Health, 1987, 18: 398-403. Hyg, 1976, 25: 866-874. [23] Bhamarapravati N, Tuchinda P and [13] Rosen L. The emperor's new clothes Boonyapaknavik V. Pathology of Thailand revisited, or reflections on the pathogenesis haemorrhagic fever: a study of 100 autopsy of dengue haemorrhagic fever. Am J Trop cases. Ann Trop Med Parasitol, 1967, 61: Med Hyg, 1977, 26: 337-343. 500-510. [14] Bravo JR, Guzman MG and Kouri GP. Why [24] Pinheiro FP. Dengue in the Americas, 1980- dengue haemorrhagic fever in Cuba? 1: 1987. Epidemiol Bull, 1989, 10: 1-8. Individual risk factors for dengue [25] Ibrahim NM and Cheong I. Adult dengue haemorrhagic fever/dengue shock syndrome haemorrhagic fever at Kuala Lumpur (DHF/DSS). Trans R Soc Trop Med Hyg, Hospital: retrospective study of 102 cases. 1987, 81: 816-820. Br J Clin Pract, 1995, 49: 189-191. [15] Anonymous. Dengue haemorrhagic fever: [26] Setiawan MW, Samsi TK, Wulur H, Sugianto diagnosis, treatment, prevention and control, D and Pool TN. Dengue haemorrhagic ed. 2. Geneva, Switzerland, World Health fever: ultrasound as an aid to predict the Organization, 1997. severity of the disease. Pediatr Radio, 1998, 28: 1-4. Dengue Bulletin – Vol 28, 2004 75 Prognostic Factors in Adult Patients with Haemorrhagic Dengue [27] Van Troys H, Gras C, Coton T, Deparis X, [33] Lewis JG, Chang G-J, Lanciotti RS, Kinney Tolou H and Durand JP. Imported dengue RM, Mayer LW and Trent DW. Phylogenetic haemorrhagic fever: aprops of 1 case relationships of dengue-2 viruses: presenting with signs of acute alithiasic correlation with epidemiology. Virology, cholecystitis. Med Trop (Mars), 2000, 60: 1993, 197: 216-224. 278-280. [34] Lanciotti RS, Gubler DJ and Trent DW. [28] Sood A, Midha V, Sood N and Kaushal V. Molecular evolution and phylogeny of Acalculous cholecystitis as an atypical dengue-4 viruses. J Gen Virol, 1997, 78: presentation of dengue fever. Am J 2279-2286. Gastroenterol, 2000, 95: 3316-3317. [35] Morens DM, Marchette NJ, Chu MC and [29] Coton T, Debonne JM, Molinier S, Chaudier Halstead SB. Growth of dengue type 2 virus B, Gras C, Carre D and Raillat A. Alithiasic isolates in human peripheral blood cholecystitis and haemorrhagic dengue. leukocytes correlates with severe and mild Gastroenterol Clin Biol, 1999, 23: 789-790. dengue disease. Am J Trop Med Hyg, 1991, [30] Wu KL, Changchien CS, Kuo CM, Chuah SK, 45: 644-651. Lu SN, Eng HL and Kuo CH. Dengue fever [36] Vaughn DW, Green S, Kalayanarooj S, Innis with acute acalculous cholecystitis. Am J BL, Nimmannitya S, Suntayakorn S, Endy TP. Trop Med Hyg, 2003, 68: 657-660. Dengue viremia titer, antibody response [31] Rico-Hesse R. Molecular evolution and pattern, and virus serotype correlate with distribution of dengue viruses types 1 and 2 disease severity. J Infect Dis, 2000, 181: 2 -9. in nature. Virology, 1990, 174: 479-493. [32] Lanciotti RS, Lewis JG, Gubler DJ and Trent DW. Molecular evolution and epidemiology of dengue-3 viruses. J Gen Virol, 1994, 75: 65-75. 76 Dengue Bulletin – Vol 28, 2004 Dengue Haemorrhagic Fever with Encephalopathy/Fatality at Petchabun Hospital: A three-year Prospective Study (1999-2002) Prasonk Witayathawornwong# Department of Paediatrics, Petchabun Hospital, Petchabun Province, Thailand Abstract During the three-year period from 1 May 1999 to 30 April 2002, there were 1,465 cases of dengue haemorrhagic fever (DHF) admitted to the Department of Paediatrics, Petchabun Hospital. The male to female ratio was 722:743 (1:1.03). Their ages ranged from 80 days to 15 years with a median of 9 years. Thirty-two patients (2.2%) were under one year of age with a median of 8 months, and all except two had primary dengue infection. There were 34 DHF patients with encephalopathy (2.3% of all DHF cases). The male to female ratio was 17:17 (1:1). The median age was 8 years and 11 months (range 10 months to 13 years). Thirty patients (88.2%) were older than 5 years. Thirty and four patients respectively developed encephalopathy in shock and convalescent stages. All 17 fatal cases (1.16% of the total DHF cases, male:female = 8:9) had both prolonged shock and massive gastrointestinal haemorrhage since admission. About 64.7%, 76.4% and 58.8% of the seventeen non-fatal cases (male:female = 9:8) had gastrointestinal haemorrhage, shock state and massive fluid overload since admission respectively. The risk factors for encephalopathy included prolonged shock, severe gastrointestinal haemorrhage, severe hepatic dysfunction and prior fluid overload. Keywords: Dengue haemorrhagic fever, encephalopathy, fatality, gastrointestinal haemorrhage, shock, fluid overload, hepatic dysfunction. Introduction 0.88-5.4%[7-11] first reported in 1976[12]. Although dengue encephalitis existed as The major pathophysiological hallmarks in evident from the direct dengue viral dengue haemorrhagic fever (DHF) are invasion[13-17], the more common conditions leakage and abnormal haemostasis that were encephalopathy secondary to fluid leads to hypovolemic shock and/or extravasation, cerebral oedema, haemorrhage. Generally, vital organs are not hypoperfusion, haemorrhage, hyponatremia, primarily involved in DHF but unusual liver failure and renal failure[2,18-19]. The manifestations, mainly the involvement of treatment of DHF with CNS involvement is the central nervous system (CNS) and severe supportive and symptomatic. Early detection hepatic dysfunction, are increasingly being and proper fluid management of DHF [1-6] detected . The incidence of CNS should be done to prevent any risk factors. involvement in dengue infection was about #E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 77 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) Materials and methods Results There were 1,465 cases of DHF admitted to Of the 1,465 cases of DHF admitted to the the Department of Paediatrics, Petchabun Department of Paediatrics, Petchabun Hospital, Petchabun province, Thailand, Hospital, from 1 May 1999 to 30 April 2002. from 1 May 1999 to 30 April 2002. The The male to female ratio was 722:743 diagnosis methods used followed the WHO (1:1.03). The patients’ age ranged from 80 criteria[20] and about 82% of cases were days to 15 years with the median of 9 years. serologically confirmed using either enzyme- The highest incidence was in the 5-10-year linked immunosorbent assay or age group (57%) and the second highest was haemagglutination inhibition tests. The in the 10-15-year age group (26.2%). Thirty- treatment consisted of general measures two patients (2.2%) were under one year of (closely observed vital signs, general age with the median of 8 months, and all of appearance and serially recorded them except two had primary dengue haematocrit in 24-48 hours after fever, no infection. Eighty-seven per cent of all cases medication except antacid for moderate to were found in the rainy season during May- severe abdominal pain) and fluid therapy October. The patients were from all 11 (minimal amount to normalize vital signs districts in the Petchabun province, 84% and haematocrit: 5% dextrose saline for from the central district alone. There were DHF patients from all villages (178) of all the infants, 5% dextrose Ringer acetate for older subdistricts (17) of the central district. patients, Dextran-40 in normal saline for impending or fluid overload and fresh whole There were 34 DHF patients with blood or packed red cells for significant encephalopathy (2.3%). The male to female bleeding, platelet concentrate and plasma ratio was 17:17 (1:1). The median age was not used). 8 years 11 months (range 10 months to 13 years). Thirty patients (88.2%) were older Patients with encephalopathy (drowsy, than 5 years. Thirty and four patients stuporous, comatose and convulsion) were developed encephalopathy in the shock and closely observed for blood sugar or convalescent stages respectively. There were dextrostix every six hours. Serum 7, 18 and 9 patients with encephalopathy transaminase was done once a day until stage II (drowsy), stage III (stuporous) and recovery. Vitamin K and 10% calcium 1 stage IV (comatose) respectively. Twenty- gluconate were administered for three days. eight patients (82.3%) were referred from Lumbar puncture was performed cautiously district hospitals and 17 patients (50%) had if there was no other risk. a massive fluid overload. Lumbar puncture The statistical analysis included the was performed in 2 non-fatal cases with percentage, mean, standard deviation and normal findings. Their serology indicated range for demographic data and Student’s t secondary dengue response. and chi-square tests for comparing non- About 17 fatal cases (1.6% of total DHF categorized and categorized variables, cases, male:female = 8:9 , median age 8 respectively. years and 4 months, range 10 months to 12 78 Dengue Bulletin – Vol 28, 2004 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) years) had the median duration of 13 hours 9 (52.9%) and 7 (41.1%) cases respectively. for deaths (range 1-26 hours, mean±SD = There was no history of taking 13.4±8.4 hours). Sixteen of the 17 cases acetaminophen more than 60 mg per kg/day. (94.1%) were dead within 24 hours. Aspirin and non-steroidal anti-inflammatory Profound shock and massive gastrointestinal drug (NSAID), Ibuprofen, were taken by each haemorrhage since admission were detected patient for many days during the febrile stage. in all patients. Hepatic failure, comatose Convulsion was detected in 4 cases (Tables 1 stage and massive fluid overload were and 2). detected since admission in 12 (70.6%), Table 1. Clinical manifestations of 17 fatal cases Age Weight L/S DHF Haemorrhage Encephalopathy Fluid Associated Patients Sex TDF/DI Referral* (years) (kg) (cm) grade manifestations signs/onset overload diseases 1NW 1.3 F 81 3/3 3/- 4 GIH Comatose/s + Diarrhea + convulsion 2SS 11.7 F 42 6/7 2/- 4 GIH Comatose/s – - – 3TR 13 M 37 4/3 2/- 4 GIH Stuporous/s + ASA + 4KB 6 F 132 3/3 10/4 4 GIH Comatose/s – Thalasemia + convulsion 5SM 7 M 20 5/6 3/- 4 GIH Stuporous/s – Pneumonia + G6PDdef 6KC 7 F 191 4/4 2/- 4 GIH Comatose/s + + 7UA 2.11 F 21 7/7 3/- 4 GIH Comatose/s + NSAID + convulsion 8WT 10 M 50 5/5 2/- 4 GIH Stuporous/s + + 9KF 6-36.3 M 40 5/6 3/- 4 GIH Stuporous/s – – 10RM 0.10 F 71 4/4 4/- 4 GIH Stuporous/s – + 11JP 10 F 31 4/5 3/- 4 GIH Stuporous/s + ARDS, DIC + 12DP 9.0 M 17 5/6 3/- 4 GIH Comatose/s + + 13BP 9.5 F 231 4/4 5/- 4 GIH Comatose/s – + 14NO 8.4 F 24 6/7 3/- 4 GIH Comatose/s – – 15TW 8.10 M 201 5/6 3/- 4 GIH Drowsy/s – Hypoglycemia + convulsion 16WK 10 M 35 4/4 3/- 4 GIH Comatose/s – + 17PL 12 M 45 5/6 2/- 4 GIH Stuporous/s – – TDF = total duration of fever; DI = duration of illness GIH = gastrointestinal haemorrhage; S = shock stage; kg = kilograms 1,2 = first, second degree protein energy malnutrition * or prior medications especially excessive fluid replacement ARDS = Adult respiratory distress syndrome DIC = Disseminated intravascular coagulation NSAID = Non-steroidal anti inflammatory drug L/S = liver/spleen Dengue Bulletin – Vol 28, 2004 79 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) Table 2. Laboratory investigations of 17 fatal cases Platelets/ AST/ALT PT/PTT* Sodium DHF Duration Patients Hct (max-min) (IU/L) (sec) (mEq/L) serology in hospital 1NW 23,00 (37-) 481/161 20.2/96.6 129 SDI 6 h 2SS 27,000 (50-34) 10,081/2,410 20.9/97.9 135 SDI 24 h 3TR 98,000 (39-21) 1,880/636 29.0/>180 130 SDI 26 h 4KB 35,000 (20-12) 1,346/420 137.0/58.0 130 SDI 7.5 h 5SM 15,000 (28-8) 50/20 26.2/42.0 138 SDI 2 h 6KC 33,000 (47-27) 9,729/4,821 24.0/170 134.9 SDI 21 h 7UA 24,000 (48-37) 10,043/3,734 19.7/171 129.6 SDI 13 h 8WT 17,000 (49-34) 3,537/1,599 20.1/>180 134 SDI 8.5 h 9KF 18,000 (57-47) 600/1,524 13.8/77.7 136 SDI 17.5 h 10RM 22,000 (29-27) 2,582/671 23.1/119.6 128 SDI 6 h 11JP 46,000 (42-6) 3,298/1,274 43.9/472 136.7 SDI 16 h 12DP 12,000 (48-31) 1,452/797 17.1/84.8 137 SDI 24 h 13BP 25,000 (42-34) 10,215/5,015 43.1/172 135 SDI 21 h 14NO 10,675 (35-26) 1,486/631 35.6/>180 138 SDI 1 h 10 m 15TW 2,000 (48-28) 3,940/1,592 35.3/>180 134.0 SDI 12 h 16WK 2,000 (56-32) 234/99 12.6/41.1 136 SDI 20 h 17PL 89,000 (41- ) 17,950/10,446 45.2/160.8 135 SDI 2 h15 m * normal (10-14)/(23-35) sec h = hours, m = minutes SDI = secondary dengue infection There were 17 non-fatal cases district hospitals. Haemodialysis and (male:female = 9:8, median age = 9 years, plasmapheresis were done in 3 renal failure range = 3-12 years). Massive gastrointestinal cases and 2 cases with only supportive haemorrhage, shock and massive fluid treatment. All 17 cases recovered overload were detected since admission in completely without neurological sequelae. 11 (64.7%), 13 (grade 3-9, grade 4-4, The average (mean ± SD) hospitalization 76.4%) and 10 (58.8%) cases respectively. duration and recovery period from There were 5 cases with acute renal failure encephalopathy were 8.3 ± 5.31 days and and one with liver failure since admission. 5.1 ± 3.7 days respectively (Tables 3, 4). Fifteen of the 17 cases were referred from 80 Dengue Bulletin – Vol 28, 2004 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) Table 3. Clinical manifestations of 17 non-fatal encephalopathy cases Fluid Age Weight L/S DHF Hemato Enceph Associated Referral Patients Sex TDI/DI over- (y.m) (kg) (cm) grade manifes signs/onset diseases load 1YB 9 M 25 4/4 3/- 4 GIH Stuporous/s + ARF + 2PS 4.4 F 121 5/3 3/- 4 Petichiae Stuporous/s – Pneumonia – 3ST 10.3 F 29 6/6 3/- 3 GIH Stuporous/s + - + 4SU 6 F 151 7/7 3/- 3 GIH Stuporous/s + Pneumonia, + Tracheitis 5CP 5.9 M 17 7/7 1/- 2 GIH Stuporous/s – - + 6WD 9.9 F 43 5/5 JP 3 GIH Stuporous/s + ARF + 7WB 6.6 M 181 3/3 3/- 3 Petichiae Stuporous/s + - + 8SSr 9 F 41.5 5/5 2/- 3 GIH Stuporous/s – Hypoglycemia, + ARF 9SN 11 F 301 4/7 4/- 2 GIH Stuporous/s – ARF + 10PP 11 M 34 5/5 2/- 4 GIH Stuporous/s + - + 11Sma 9.11 M 28 5/5 5/- 3 GIH Drowsiness/s + - + 12WL 6.11 F 19 5/5 8/6 3 GIH Stuporous/s + Thalasemia, + ARDS, pneumonia 13ThP 7.9 M 22 5/5 4/- 2 GIH Stuporous/s – - + 14SL 5.9 M 251 5/6 5/- 3 Petichiae Stuporous/s + ARF + 15KN 11.11 M 232 5/5 1/- 4 Petichiae Drowsiness/s – - + 16MK 10.4 M 49 6/6 2/- 3 Petichiae Drowsiness/s – - – 17WTh 3 F 13.5 5/7 2/- 2 Petichiae Drowsiness/s + Rhinitis + 1,2 = first, second degree protein energy malnutrition ARF = acute renal failure; ARDS = Adult respiratory distress syndrome GIH = gastrointestinal haemorrhage; JP = just palpable Table 4. Laboratory investigations of 17 non-fatal encephalopathy cases * Patients Platelets/ AST/ALT PT/PTT Sodium DHF Duration ** Hct (max-min) (IU/L) (sec) (mEq/L) serology 1YB 62,000(51-30) 9,420/2,239 23.4/>120 128 SDI 13/7 2PS 17,000(50-26) 2,023/458 14.4/59.9 135 SDI 9/5 3ST 16,000(50-38) 1,098/452 14.7/121.6 130 SDI 4/2 4SU 44,000(46-34) 255/148 16.8/48.5 135 SDI 17/10 5CP 90,000(52-30) 168/110 18.4/64.5 140. 3 SDI 13/10 6WD 36,000(41-30) 13,895/5,200 24.6/77.6 127 SDI 11/7 7WB 4,000(58-34) 2,128/918 15.1/67.8 128 SDI 6/3 8SSr 20,000(48-32) 14,580/5,852 16.4/56.0 134 SDI 10/7 9SN 68,000(49-27) 6,987/3,789 32.5/57.2 138 SDI 7/3 10PP 21,000(42-27) 3,810/1,935 15.3/52.5 129 SDI 4/2 11Sma 34,000(48-33) 2,250/448 14.9/78.9 128 SDI 6/3 12WL 20,000(42-27) 3,436/1,841 15.0/89.3 143 SDI 21/15 13ThP 56,000(42-30) 1,315/549 12.4/61.5 134 SDI 2/2 14SL 45,000(56-33) 4,746/1,470 15.9/1,133 131.6 SDI 2/2 15KN 40,000(49-32) 8,150/4,450 16/72.2 137 SDI 7/4 16MK 3,000(51-35) 2,996/1,482 13.8/64.0 132.8 SDI 5/3 17WTh 60,000(32-30) 275/599 15.4/66.7 128 SDI 4/3 * normal: (10-14 )/(23-35 ) sec ** duration in hospital/consciousness change (days) Dengue Bulletin – Vol 28, 2004 81 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) A comparison between the clinical manifestations and encephalopathy cases manifestations and laboratory investigations are shown in Tables 5, 6 and 7 respectively. of the fatal and non-fatal cases, usual Table 5. Comparison between clinical manifestations and laboratory investigations of fatal and non-fatal cases Clinical manifestations or laboratory Fatal cases Non-fatal cases P-value investigations* (n=17) Shock (grade IV) 17 4 <0.001 Internal bleeding 17 11 0.007 Onset of encephalopathy in shock stage 17 13 0.035 Coma since admission 9 0 <0.001 Platelets (/cubic millimeters)** 29,33.8 (26,662.1) 37,411.7 (24,153.3) 0.362 AST (IU/L) 4,641.4 (5082.7) 4,560.7 (4,545.8) 0.961 ALT (IU/L) 2,108.8 (2,655.1) 1,878.8 (1,838.6) 0.771 PT in sec 25.5 (11.1) 17.3 (5.0) 0.011 PTT in sec 146.1 (98.8) 75.9 (25.5) 0.011 Serum sodium (mEq/L) 133.9 (3.3) 132.8 (4.8) 0.471 * mean (standard deviation, SD) ** minimum value PT=Prothrombin time PTT=Partial thromboplastin time Table 6. Comparison between clinical manifestations of usual manifestations and encephalopathy cases Usual manifestations Encephalopathy Clinical data P-value (n=1431) (n=34) Age <1 yr 31 (2.1%) 1 (2.9%) 0.775 1 – 4 yr 210 (14.6%) 4 (11.7%) 0.661 5 – 10 yr 812 (56.7%) 23 (67.6%) 0.219 10 – 15 yr 378 (26.4%) 6 (17.6%) 0.252 Female 726 (50.7) 17 (50.0%) >0.993 Shock 332 (23.2) 30 (88.2%) <0.001 (grade 3=330, 4=2) (grade 3=9, 4=21) Referal or prior medications 266 (18.6%) 28 (82.3%) <0.001 Internal bleeding 130 (9.1%) 28 (82.3%) <0.001 Malnutrition[21] 716 (50.0%) 12 (35.3%) 0.091 (1°=511, 2°=192, 3°=13) (1°=10, 2°=2) Death – 17 (50%) <0.001 Duration in hospital (days):mean 3.4 (1.38) 83 (5.31)* 0.002 (standard deviation) * only non fatal cases 82 Dengue Bulletin – Vol 28, 2004 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) Table 7. Comparison between laboratory investigations of usual and encephalopathy cases Usual manifestations Encephalopathy Laboratory data* P-value (n=1431) (n=34) AST (IU/L) 257.3 (250.6) 4601.0 (4748.3) <0.001 ALT (IU/L) 119.5 (138.1) 1993.8 (2251.8) <0.001 Platelets (minimum) 70697.9 (2975.7) 33372.8 (2538315) <0.001 PT (10-14 sec) 11.2 (1.1) 21.4 (9.4) <0.001 PTT (23-35 sec) 51.9 (13.4) 111.0 (79.5) <0.001 * mean (standard deviation, SD) Discussion DHF/DSS patients were well-nourished but patients with CNS involvement were more DHF patients from all the 11 districts in the undernourished without significant Petchabun province were included in this difference[24,26]. In this study, patients with study. The minors were referred from 10 usual manifestations and encephalopathy other districts. The adults were from the 178 were underweight[21] by about 50% and villages in all 17 subdistricts of the central 35.3% respectively. Patients with district. This epidemic was similar to the encephalopathy were mainly in stage [22] previous dengue epidemic in 1997 . This III[7,8,18,27] (88.2%) initially developed in data implied that the dengue virus had shock stage (52.9%), more than in febrile or spread nationwide. The percentage of DHF convalescent stage[7,9,28]. Seizure in DHF patients with CNS involvement in this study with encephalopathy had been reported in was 2.3%, the same as in the previous about 18.8-100%[7-9,13,18,24,27,29,30]. In this study[22] as well as in other studies[7-11]. study seizure was detected in 4 out of 34 Female patients were reported to be more patients (11.7%). Two patients (1NW, 7UA), severely affected and accounted for more under 5 years, developed seizure in afebrile fatalities than male patients but without any stage, indicating that the seizure might have significant difference[18,23-25]. In this study, had a specific primary cause[10]. However, the number of both sexes was equal and some children had possible confounding both had usual manifestations and factors such as hyponatremia (3 patients, encephalopathy cases. Young patients, 1NW, 4KB, 7UA), hypoglycemia (15TW), especially those less than 1 year of age, had and liver failure (3 patients, 1NW, 7UA, the tendency to be more severely and fatally 15TW). Other factors could be a history of affected[18,23-25]. Age itself was not a risk previous febrile seizure, co-infection[10] and factor of disease severity in this study. drug ingestion. Although there was one fatal case aged 10 The causes or factors contributing to months (10 RM), 85% of those who died CNS manifestations included the following: were older than 5 years. Most of the direct CNS infection – a rare entity, CNS Dengue Bulletin – Vol 28, 2004 83 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) bleeding[6,18] and severe hepatic dysfunction. occur in severe cases with prolonged shock, Lumbar puncture was done in only two DIC and also hepatorenal syndrome[37]. patients (5CP, 15KN) in the convalescent There were five patients with ARF in this stage with normal findings. Direct CNS study. All of them recovered completely, infection could not be evaluated in this three (6WD, 8SSr, 14SL) with haemodialysis study. There was massive gastrointestinal and plasmapheresis and two (1YB, 9SN) haemorrhage in 28 out of 34 cases (82.3%). with only supportive and symptomatic Acute liver failure could be the direct or therapy. Hyponatremia was a factor in CNS indirect cause of encephalopathy[31,32] and involvement. Serum sodium of an important cause of death in DHF[33]. encephalopathy patients in this study was Twelve of the 17 fatal cases (70.5%) had this slightly low. It was due to excessive severe condition. Severe hepatic hypotonic solution replacement. The risk dysfunction could be due to profound shock, factors of encephalopathy in this study were massive gastrointestinal haemorrhage or profound shock, massive gastrointestinal immune complex mechanism[34]. The liver haemorrhage, excessive fluid overload and pathology in profound shock stage might be severe hepatic dysfunction. Among the fatal centrilobular hepatocellular necrosis[35] or and non-fatal encephalopathy cases, extensive necrosis of hepatocytes usually in laboratory investigations except for a massive or submassive distribution[33]. coagulogram, were not significantly different. Massive gastrointestinal haemorrhage might But fatal cases had more severe shock cause hepatic dysfunction and vice versa. conditions, gastrointestinal haemorrhage Immune complexes, detected in 80% of and onset and depth of encephalopathy DHF patients[36], might be deposited in (Table 5). hepatocytes and then destroyed as in hepatitis B virus infection[34]. Excessive use of hepatotoxic drugs such as acetaminophen, Conclusion an antiemetic drug, might interfere with In conclusion, encephalopathy in DHF was liver function. There was no history of a severe complication with high mortality, excessive use of such drugs in this study. although neurological sequelae in recovered Two fatal cases with liver failure and massive patients were rare. Prevention should be gastrointestinal haemorrhage had taken attempted by early diagnosis and proper aspirin (3TR) and non-steroidal anti- management of fluid therapy. inflammatory drug, Ibuprofen (7UA), for many days during the febrile stage. Both drugs aggravated the gastrointestinal Acknowledgements haemorrhage and aspirin could have The author thanks officials of the Regional induced Reye syndrome[37]. Medical Science Centre, Phitsanuloke, Thalassemia, of which two cases with a Thailand, for DHF serology testing and all large liver and spleen were included in this nursing staff and workers of the Paediatrics study (4KB, 12WL), was a risk factor for Department of Petchabun Hospital for hepatic failure[18]. Acute renal failure (ARF) taking good care of the patients. was a rare complication of DHF but could 84 Dengue Bulletin – Vol 28, 2004 DHF with Encephalopathy/Fatality at Petchabun Hospital, Thailand (1999-2002) References [1] Nogueira RMR, Filippis AMB, Coelho JMO, [11] Attavinijtrakarn P. Hepatic dysfunction in Segueira PC, Schatmayr HG, Paiva FG, dengue haemorrhagic fever in Ramos AMO and Miagostovich MP. Dengue Paholpolpayuhasaena Hospital. 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Pediatrics, 1982, 3: 321. syndrome: a co-relation study with acetaminophen usage. Southeast Asian J Trop Med Public Health, 1990, 21: 694-695. 86 Dengue Bulletin – Vol 28, 2004 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples C. Domingo* #, G. Palacios**, M. Niedrig***, M. Cabrerizo*, O. Jabado**, N. Reyes*, W.I. Lipkin** and A. Tenorio* *Laboratorio de Arbovirus y Enfermedades Víricas Importadas, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera de Pozuelo Km 2 (28220 Majadahonda), Madrid, Spain **Jerome L and Dawn Greene Infectious Disease Laboratory, Columbia University, New York, USA ***Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany Abstract Dengue fever and dengue haemorrhagic fever are amongst the most important challenges in tropical diseases due to their expanding geographical distribution, increasing outbreak frequency, hyperendemicity and evolution of virulence. Here, the use of a RT-nested PCR for both the diagnosis and genetic characterization of dengue infections in clinical samples is described. Keywords: Dengue, dengue haemorrhagic fever, diagnosis, molecular epidemiology, surveillance, glycoprotein E gene, NS1 gene. Introduction infrastructure in most endemic areas are the likely factors contributing to the surge in new Dengue fever (DF), dengue haemorrhagic cases[4] . A major concern is the potential fever (DHF) and dengue shock syndrome spread of dengue fever into the United States (DSS) are considered to be the most of America and Europe due to climate important arthropod-borne viral diseases due warming and the spread of its vector. to the high rates of morbidity and mortality Travellers to areas where dengue is caused by them. Over 2.5 billion people are endemic are a potential source of the spread. at risk of the infection and more than 100 Most infections manifest as a mild febrile countries are home to endemic dengue illness during travel that coincides with the transmission with an increasing incidence of peak of viral shedding and risk for DHF cases[1-3], making dengue an archetypal transmission. Imported dengue virus “emerging” disease. International travel, infections have been reported in several non- urbanization, overpopulation, crowding, endemic countries; and dengue virus poverty and a weak public health infection is one of the most frequent causes #E-mail: [email protected]; Phone: (+34) 918223954, Fax: (+34) 915097966 Dengue Bulletin – Vol 28, 2004 87 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples of febrile illness in tourists and people Materials and methods working in dengue-endemic areas[5,6,7,8]. As early symptoms of DF mimic those of other Virus isolates and clinical samples diseases such as malaria or leptospirosis, Viral RNAs were provided by the National rapid laboratory diagnosis is important for Collection of Pathogenic Viruses (Porton proper patient care. Dengue fever can be Down, Salisbury, UK): serotype 1 dengue diagnosed by virus isolation, genome and virus (DEN-1; strain Hawaii), serotype 2 antigen detection, or serological studies. dengue virus (DEN-2; strain New Guinea C), Samples obtained for the detection of serotype 3 dengue virus (DEN-3; strain H87), dengue virus by cell-culture require proper and serotype 4 dengue virus (DEN-4; strain handling of the sample for viral viability. H241); the RNAs from prototype strains of Serology, even for anti-dengue IgM Japanese encephalitis (JEV), yellow fever antibodies, is feasible only after 5 days (YFV), tick-borne encephalitis (TBEV), following the onset of the symptoms. Thus, Murray Valley encephalitis (MVEV) and St. molecular techniques fulfil an important role Louis encephalitis (SLEV) viruses were used in the diagnosis of dengue infection during its to check the specificity of the dengue virus early stages. assay. Serial dilutions of this genome material The characterization of circulating were prepared to obtain the standards to dengue virus serotypes is important in assess the sensitivity of the assay. surveillance, since the introduction of a new Viremic human sera samples were variant to areas affected by pre-existing obtained from patients with a clinical serotypes constitutes a risk factor for diagnosis of dengue infection (Sera [9] DHF/DSS . By defining intra-serotypic 1794F02; 13VI02; 366VI03; 438VI03). genetic variation, the global distribution and These were travellers who presented at the spread of virus strains can be mapped and Spanish Tropical Medicine Units with [10-13] followed up , and the genetic differences dengue-compatible symptomatology on associated with disease severity can be their return from the Dominican Republic, [10,14-16] identified . Moreover, recent India, Indonesia and Nicaragua, respectively, epidemiological analyses suggest that the and suffered from classical DF as defined by more virulent genotypes are now displacing the WHO criteria[19]. those of lower epidemiological impact[17], [18] resulting in dengue outbreaks . In this Selection and synthesis of context, a methodology for real-time, worldwide surveillance is needed to track oligonucleotide primers dengue strains and help anticipate changes in A RT-nested PCR protocol was developed the epidemiology of the infection. for the detection of the four serotypes of Here we report the amplification and dengue virus in clinical samples. Dengue analysis of a genomic interval spanning the virus primers for amplification and/or E/NS1 junction of the dengue genome for sequencing (Table) were designed based on the detection and typing of all four dengue dengue virus sequences in the public virus serotypes in clinical specimens. This sequence databases, using a computer- assisted analysis (MACAW version 32 sensitive, specific and rapid alternative assay software, 1995, NCBI, Maryland) to requires only a single acute phase serum sample. determine consensus sequences. The Table 88 Dengue Bulletin – Vol 28, 2004 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples shows the sequences and the respective S2176DEN2, S2176DEN3, S2176DEN4, primer positions in the prototype strains of AS2504DEN) and 2.5 U of DNA Taq the four dengue serotypes. To address the Polymerase (Perkin-Elmer). The samples were natural variability of dengue viruses, mixtures subjected to a denaturation step (94 °C, 2 of degenerated primers were used to enable minutes) followed by 40 cycles of hybridization with all known serotypes. denaturation (94 °C, 30 seconds), primer annealing (57 °C, 4 minutes), and primer RT-nested PCR extension (72 °C, 30 seconds) and a further extension step at 72 °C for 5 minutes. Using purified dengue virus RNA as a template, relevant aspects of the RT-PCR and Dengue virus sequence database nested PCR assay (Mg2+ concentration, primers, RT temperature, number of cycles, A dengue sequence database was annealing temperatures) were initially constructed by extracting sequences from optimized to achieve the greatest sensitivity. the NCBI GenBank. Each sequence was A PTC-200 Peltier thermal cycler (MJ identified by name, place, date and Research) was used throughout. 5 µl of viral serotype. Previously described genotypes RNA solution were added to 45 µl of a were taken from the references listed: medium compatible with both the reverse dengue strains genotypes were noted as transcription and PCR amplification steps described by Rico-Hesse for DEN-1, 3 and [17] (QIAGENâ OneStep RT-PCR kit). The RT- 4 and by Twiddy et al. for dengue virus [20] PCR mix contained 1´OneStep RT-PCR type 2 . A manual search was employed buffer, 400 mM of each dNTP, 20 pmol of for all the sequences in GenBank each sense or antisense degenerated primer encompassing the targets of selected primers. (S1871DEN1, 1871DEN2, 1871DEN3, Next, we used BUSSUB, a new tool 1871DEN4, AS2622DEN1, AS2622DEN2, developed at the Bioinformatics Unit of the [21] AS2622DEN3, AS2622DEN4) and an Institute of Health Carlos III . This software optimized combination of Omniscript and simplifies and boosts the process of retrieving Sensiscript reverse transcriptases and HotStar sequences contained between two given Taq DNA polymerase. The RT-PCR reactions flanking regions, improving the final results of were carried out using an initial reverse a search. Genetic characterization was transcription step at 41 °C for 45 minutes performed on a total data set of 113 DEN-1, followed by a denaturation and Hot Star Taq 191 DEN-2, 102 DEN-3 and 153 DEN -4 polymerase activation step (94 °C, 15 sequences. minutes) and 40 cycles of denaturation (94 °C, 30 seconds), primer annealing Sequence analysis of amplified (55 °C, 1 minute), and primer extension products (72 °C, 30 seconds). A final incubation was Original sequence data were first analysed by carried out at 72 °C for 5 minutes. A second the CHROMAS software (version 1.3, amplification reaction (nested PCR) was McCarthy 1996; School of Biomolecular and seeded with 1 µl of the initial amplification Biomedical Science, Faculty of Science and product. The reaction mixture contained 1´ Technology, Griffith University, Brisbane, buffer B (60 mM Tris-HCl pH 8.5, 2 mM Queensland, Australia); forward and reverse MgCl , 15 mM (NH ) SO , 40 pmol of each 2 4 2 4 sequence data of each sample were aligned sense and antisense primer (S2176DEN1, using the programme EDITSEQ (DNASTAR Dengue Bulletin – Vol 28, 2004 89 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples Inc. Software, Madison, Wisconsin, USA). Scores were calculated to test the significance The consensus sequence was compared and of each pair-wise alignment by Monte Carlo aligned to other samples or DNA database simulation on the shuffled sequences. sequences using the programme CLUSTAL X, Statistical analysis was conducted with the version 1.83[22]. Programmes from the MEGA SPSS statistical package (SPSS Software, package[23] were used to produce Chicago, IL). phylogenetic trees using NJ as the method to reconstruct the phylogeny and Kimura-2p as nucleotide substitution calculation method. Results The statistical significance of a particular tree topology was evaluated by bootstrap re- Design of the primers sampling of the sequences 1,000 times. The E/NS1 region of the genome was Published sequences used in the chosen for the development of a RT-nested comparisons were obtained from the PCR. The primers selected specifically GenBank databases. Pair-wise comparisons amplify the four dengue viruses with no of the dengue virus database were done by cross reactivity to other members of the global alignment using the Needleman flavivirus family. A mix of degenerate Wunsch[24] algorithm using the primers representing each serotype was implementation from EMBOSS, the European used to ensure coverage for the highly Molecular Biology Open Software Suite[25]. Z- variable dengue serotypes (Table). Table. Primers used in RT-nested PCR assays and sequencing Primer* SequenceØ Genome position ¶ PCR S1871DEN1 5’-TGGCTGAGACCCARCATGGNAC-3’ 1869 to 1890 RT-PCR S1871DEN2 5’-TAGCAGAAACACARCATGGNAC-3’ 1871 to 1889 S1871DEN3 5’-TCTCCGAAACGCARCATGGNAC-3’ 1863 to 1884 S1871DEN4 5’-TGGCAGAAACACARCAYGGNAC-3’ 1873 to 1894 AS2622DEN1 5’-CAATTCATTTGATATTTGYTTCCAC-3’ 2620 to 2644 RT-PCR AS2622DEN2 5’-CAATTCTGGTGTTATTTGYTTCCAC-3’ 2622 to 2646 AS2622DEN3 5’-CAGTTCATTRGCTATTTGYTTCCAC-3’ 2614 to 2638 AS2622DEN4 5’-TAGCTCGTTGGTTATTTGYTTCCAC-3’ 2624 to 2648 S2176DEN1 5’-ATCCTGGGAGACACYGCNTGGG-3’ 2174 to 2195 Nested S2176DEN2 5’-ATTTTRGGTGACACAGCNTGGG-3’ 2176 to 2197 S2176DEN3 5’-ATCTTGGGAGACACAGCNTGGG-3’ 2168 to 2189 S2176DEN4 5’-ATTCTAGGTGAAACAGCNTGGG-3’ 2178 to 2199 AS2504DEN 5’-TGRAAYTTRTAYTGYTCTGTCC-3’ 2506 to 2527 DEN-1 Nested 2504 to 2525 DEN-2 2496 to 2517 DEN-3 2506 to 2527 DEN-4 *Primers name s beginning with “S” indicate a genome (plus)-sense orientation; names beginning with “AS” indicate a complementary sense orientation. ¶The genome positions are given according to each dengue virus serotype prototype strain (DEN-1; strain Mochizuki, DEN-2; strain Jamaica N-109, DEN-3; strain H87, DEN-4; strain 814669) ØDegenerate positions: N:A/C/g/T, R:A/g, Y:T/C 90 Dengue Bulletin – Vol 28, 2004 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples Dengue virus RT-nested One hundred and sixty-four serum PCR specificity samples from cases of febrile illness associated with travel were tested with the The specificity of the RT-nested PCR was assay. Thirty-seven cases were diagnosed as determined by analysing serial dilutions of of classical dengue fever by the WHO RNA from related flavivirus (JEV, MVEV, criteria[19]. Sixteen of these cases were found SLEV, TBEV, WNV, YFV) and no positive by using our E/NS1 assay. amplification was obtained (data not shown). Convalescent sera were available for 13 of The amplification was successful with these cases; all were later confirmed to be both commercial RNA and serum samples seroconvert. All serum samples found for all dengue virus serotypes as shown positive by RT-nested PCR were collected in (Figure 1), yielding a distinct DNA product the first week after the onset of the of the expected size (328-pb) in agarose gels. symptoms. Figure 1. Amplification products obtained Sequence analysis results through PCR analysis of sera from subjects The phylogenetic trees obtained by the with acute dengue virus infection analysis of the representative strains of the [Arrow indicates 328 bp E/NS1 products. 1% four serotypes and unknown sample agarose gel. MWM: Molecular weight markers; sequences allowed rapid differentiation of 438VI03 (DEN-1); 13VI02 (DEN-2); 1794F02 the corresponding serotype (Figure 2). (DEN-3); 366VI03 (DEN-4)] Pair-wise sequence analysis using Needleman Wunsch global alignment was carried out on the 220bp sequence where a higher amount of sequences were available for comparison. As expected, comparisons between serotypes showed a low sequence MWM 438VI03 13VI02 1794F02 366VI03 similarity and could be easily grouped. An all-against-all sequence comparison was done within each serotype to evaluate the possibility of using sequence similarity to 396 344 classify genotypes. Significant sequence 298 similarity was observed when comparing sequences within the same genotype. This was evaluated by an analysis of variance between groups (ANOVA), comparing the scores of sequence comparisons within genotypes to comparisons between genotypes. Each group was significant to the P<0.001 level. Genotypes with only one member sequence were excluded from the analysis. Dengue Bulletin – Vol 28, 2004 91 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples Figure 2. Phylogenetic tree constructed with the E/NS1 fragment which identifies the four dengue serotypes [Phylogenetic analysis was performed using the Kimura-two parameter model as a model of nucleotide substitution and using the neighbor joining method to reconstruct the phylogenetic tree (MEGA version 2.1 software package)] Den1-Peru9615-00-PERU Den1-FGA89-FRENCHGUYANA1989 Den1-Peru9581-00-PERU Den1-BR90-BRASIL1990 Den1-Br01MR-BRASIL2001 Den1-233-BRASIL1997 Den1-111-BRASIL1997 Den1-409-BRASIL1997 Den1-Peru5198-97 Den1-SingaporeS275-90-SINGAPORE1990 DENGUE SEROTYPE 1 Den1-CAMBODIA-CAMBODIA1998 Den1-GZ80-CHINA1980 Den1-DJIBOUTI1998 Den1-MOCHIZUKI-JAPAN1953 Den1-16007-THAYLAND1964 Den1-A88-INDONESIA1988 Den1-clonePDK27 Den1-WestPac-NAURU1974 Den3-80-2-CHINA1980 Den3-H87-PHILIPPINES1956 DENGUE SEROTYPE 3 Den3-11069 Den2-IQT2913-PERU1996 Den2-539-96-PERU1996 Den2-IQT1797-PERU1995 Den2-131-MEXICO1992 Den2-VEN2-VENEZUELA1987 Den2-44-CHINA1989 Den2-NewGuineaC-NEWGUINEA1944 Den2-43-CHINA1987 Den2-04-CHINA1985 Den2-Mara4-VENEZUELA1990 Den2-N.1409-JAMAICA1983 DENGUE SEROTYPE 2 Den2-CookIslands-AUSTRALIA1997 Den2-16681-THAYLAND1964 Den2-ThNH54-93-THAYLAND1993 Den2-ThNHp36-93-THAYLAND1993 Den2-CO371-THAYLAND1995 Den2-CO166-THAYLAND1966 Den2-CO167-THAYLAND1996 Den2-K0008-THAYLAND1994 Den2-K0010-THAYLAND1994 Den4-TRI94-TRINIDAD1994 Den4-HON91-HONDURAS1991 Den4-MON94A-MONTSERRAT1994 Den4-TRI99-TRINIDAD1999 Den4-BAH98A-BAHAMAS1998 Den4-MON94B-MONTSERRAT1994 Den4-SUR94A-SURINAM1994 Den4-BDS93A-BARBADOS1993 DENGUE SEROTYPE 4 Den4-MEX91-MEXICO1991 Den4-JAM83-JAMAICA1983 Den4-TRI82A-TRINIDAD1982 Den4-814669-DOMINICA1981 Den4-SUR82D-SURINAM1982 Den4-JAMAICA1981 Den4-TRI84A-TRINIDAD1984 YellowFever WestNile JVE 0.1 Sequences that had no known genotype generate a full taxonomy tree, but did fully were classified with respect to the group to differentiate the genotypes (data not shown). which they were most similar. To verify the Even with this simple method, all unknowns utility of this method, a phylogenetic tree were classified correctly into their genotypic was built in parallel with the unknown and group (Figure 3 illustrates one example characterized sequences. Bootstrap values result for each serotype, compared to in the 220bp region were too low to known sequences). 92 Dengue Bulletin – Vol 28, 2004 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples Figure 3. Pair-wise analysis of four dengue strains detected by PCR amplification of 328 bp E/NS1 products from patient sera. Samples are (a) 438VI03, DEN-1 AMERICAN-AFRICAN genotype, (b) 13VI02, DEN-2 INDIAN genotype, (c) 1794F02, DEN-3 COSMOPOLITAN genotype, and (d) 366VI03, INDONESIAN DEN-4 genotype (a)a) Dengue 1 - 438VI03 Nicaragua 2003 (b)d) Dengue 2 - 13VI02 India 2002 1100 1100 1000 1000 900 900 NW Score NW Score 800 800 700 700 COS ASIAN ASIAN AMERICAN ASIAN AFRICAN AMAF ASIAN MAL SP THAI GENOTYPE 2 AMERICAN GENOTYPE 1 Genotype Genotype (c)b) Dengue 3 - 1794F02 Dominican Republic 2002 (d)c) Dengue 4 - 366VI03 Indonesia 2003 1200 1200 1100 1100 1000 1000 NW Score 900 NW Score 900 800 800 700 700 SE Asia/SP THAILAND INDIAN AMERICAS INDONESIA SE ASIA MALAYSIA SUBCONTINENT Genotype Genotype Discussion governments and healthcare workers in planning for potential outbreak situations. The efficient worldwide control of dengue The advent of a simple and accurate virus requires the definition of sources of method for diagnosis and surveillance could epidemic viruses and the precise improve the establishment of these identification of virus genotypes. A key programmes in developing countries objective of DF and DHF surveillance affected by the disease, and in non-endemic programmes is early detection of outbreaks areas where dengue is a travel-acquired to permit the implementation of control infection. measures. DHF outbreaks can be The RT-nested PCR described here anticipated by monitoring the emergence of allows rapid direct diagnosis of acute new genotypes in a region. The need for dengue infection in laboratories without surveillance is warranted, since air travellers BSL-3 (bio-safety level 3) facilities. can quickly move viruses from an endemic area to a receptive area. Dengue virus Pair-wise comparison to classify surveillance should be implemented in sequences has been used for enteroviruses endemic and non-endemic areas to aid and potyvirus[26-28]. Multiple alignment and Dengue Bulletin – Vol 28, 2004 93 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples rigorous phylogenetic methods are preferable Acknowledgements to establish exact lineages of sequence strains and discover recombination events. Pair-wise This investigation received financial support comparisons can substitute if only a high level from the Instituto de Salud Carlos III (ISCIII) of taxonomic classification is desired. Our through research project grants (MPY method allows classification of dengue 1194/02 and C03/04). G. Palacios and WI genotypes using the sequence of the 220bp Lipkin were supported by the Ellison Medical region amplified by the PCR assay. The Foundation and the National Institutes of advantage of pair-wise comparison for Health (AI 51292 and U54 AI57158-Lipkin). classification is its speed, simplicity and C. Domingo was contracted by an agreement availability. The database and classification between the Public Health Division of the scheme provides a repository for sequences, Spanish Ministry of Health (DGSP-MSC) and complementing efforts in tracking dengue the Instituto de Salud Carlos III (ISCIII) for the genotype distribution. A website could be development of the Haemorrhagic Viral deployed wherein clinical laboratories post Fevers Surveillance and Control Programme. their sequences, location and circumstances The authors thank Dr J. Gascón, Dr of isolation. This would allow rapid R. López-Vélez and Dr S. Puente and the centralized analysis detailing the genotype, many scientists who contributed dengue- date and location of the most similar infected patient samples for this work. The sequence isolate in the database. New authors are grateful to Dr J.E. Mejia for genotypes could be rapidly identified by assisting in manuscript preparation. failure to relate them to a described group. References [1] Gubler DJ. Epidemic dengue/dengue [7] Lopez-Velez R, Perez-Casas C, Vorndam AV haemorrhagic fever: a global public health and Rigau J. Dengue in Spanish travelers problem in the 21st century. Dengue Bulletin, returning from the tropics. Eur J Clin 1997, 21. Microbiol Infect Dis, 1996, 15: 823-826. [2] World Health Organization. Dengue and [8] Trofa AF, DeFraites RF, Smoak BL, Kanesa- dengue haemorrhagic fever. Fact sheet, thasan N, King AD, Burrous JM, MacArthy 2002, No. 117. PO, Rossi C and Hoke CH, Jr. Dengue fever in US military personnel in Haiti. JAMA, [3] Clarke T. Dengue virus: break-bone fever. 1997, 277: 1546-1548. Nature, 2002, 416: 672-674. [9] Halstead SB. Pathogenesis of dengue: [4] Lifson AR. Mosquitoes, models, and dengue. challenges to molecular biology. Science, Lancet, 1996, 347: 1201-1202. 1988, 239: 476-481. [5] Jelinek T. Dengue fever in international [10] Lanciotti RS, Lewis JG, Gubler DJ and Trent travelers. Clin Infect Dis, 2000, 31: 144-147. DW. Molecular evolution and epidemiology of dengue-3 viruses. J Gen Virol, 1994, 75 [6] Jelinek T, Dobler G, Holscher M, Loscher T (Pt 1): 65-75. and Nothdurft HD. Prevalence of infection with dengue virus among international [11] Lanciotti RS, Gubler DJ and Trent DW. travelers. Arch Intern Med, 1997, 157: Molecular evolution and phylogeny of 2367-2370. dengue-4 viruses. J Gen Virol, 1997, 78 (Pt 9): 2279-2284. 94 Dengue Bulletin – Vol 28, 2004 A New Tool for the Diagnosis and Molecular Surveillance of Dengue Infections in Clinical Samples [12] Rico-Hesse R. Molecular evolution and [21] Sánchez-Marino JPL-C, G.; Spiteri, I.; distribution of dengue viruses type 1 and 2 Martín-Sánchez, F. BUSSUB: A virtual in nature. Virology, 1990, 174: 479-493. amplicon retrieval software. ISMB/ECCB 2004. Glasgow, 2004. [13] Chungue E, Deubel V, Cassar O, Laille M and Martin PM. Molecular epidemiology of [22] Thompson JD, Gibson TJ, Plewniak F, dengue 3 viruses and genetic relatedness Jeanmougin F and Higgins DG. The among dengue 3 strains isolated from CLUSTAL_X windows interface: flexible patients with mild or severe form of dengue strategies for multiple sequence alignment fever in French Polynesia. J Gen Virol, 1993, aided by quality analysis tools. Nucleic Acids 74 (Pt 12): 2765-2770. Res, 1997, 25: 4876-4882. [14] Leitmeyer KC, Vaughn DW, Watts DM, [23] Kumar S, Tamura K, Jakobsen IB and Nei M. Salas R, Villalobos I, de C, Ramos C and MEGA2: molecular evolutionary genetics Rico-Hesse R. Dengue virus structural analysis software. Bioinformatics, 2001, 17: differences that correlate with pathogenesis. 1244-1245. J Virol, 1999, 73: 4738-4747. [24] Needleman SB and Wunsch CD. A general [15] Pandey BD, Morita K, Hasebe F, Parquet method applicable to the search for MC and Igarashi A. Molecular evolution, similarities in the amino acid sequence of distribution and genetic relationship among two proteins. J Mol Biol, 1970, 48: 443-453. the dengue 2 viruses isolated from different [25] Rice P, Longden I and Bleasby A. EMBOSS: clinical severity. Southeast Asian J Trop Med the European Molecular Biology Open Public Health, 2000, 31: 266-272. Software Suite. Trends Genet, 2000, 16: [16] Rico-Hesse R, Harrison LM, Salas RA, Tovar 276-277. D, Nisalak A, Ramos C, Boshell J, de Mesa MT, Nogueira RM and da Rosa AT. Origins [26] Palacios G, Casas I, Tenorio A and Freire C. Molecular identification of enterovirus by of dengue type 2 viruses associated with analyzing a partial VP1 genomic region with increased pathogenicity in the Americas. different methods. J Clin Microbiol, 2002, Virology, 1997, 230: 244-251. 40: 182-192. [17] Rico-Hesse R. 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Dengue Bulletin – Vol 28, 2004 95 Clinical and Laboratory Observations Associated with the 2000 Dengue Outbreak in Dhaka, Bangladesh Monira Pervin*#, Shahina Tabassum**, Md. Mobarak Ali***, Kazi Zulfiquer Mamun* and Md. Nazrul Islam** *Department of Virology, Dhaka Medical College, Dhaka, Bangladesh **Department of Virology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh ***Shahid Suhrawardi Hospital, Dhaka Bangladesh Abstract A large outbreak of dengue fever (DF)/dengue haemorrhagic fever (DHF) occurred in Dhaka city, Bangladesh, in 2000. The present study was conducted on 105 clinically-suspected cases of DF to confirm the diagnosis, determine the major clinical manifestations and correlate the haemorrhagic manifestations with different dengue serotypes circulating during the outbreak. A total of 97 cases were positive for anti-dengue IgM and were considered as recent dengue infection; 52.6% patients had secondary and 47.4% had primary dengue infection. According to WHO case-definition, 79 cases were classified as DF, 17 as DHF and 1 as DSS. Among the 18 DHF/DSS cases, 14 had secondary and 4 had primary type of antibody response. The mean age of the dengue patients was 29.2±12.9 years and most of them (37.1%) belonged to the 20-29-year age group. All the clinically-suspected patients had fever ranging from 100-104 ºF, but the secondary dengue fever patients had higher (101.6±1.4 ºF) mean body temperature. Common complaints included myalgia (84.5%), headache (82.5%), arthralgia (68.0%), lethargy (80.4%) and retro-orbital pain (49.5%). Rash, especially maculopapular type, was significantly higher in primary infection (P<0.01), while hepatomegaly was higher in secondary infections (P<0.01). Haemorrhagic manifestations were observed both in primary and secondary dengue patients and were mostly associated with serotypes DEN-3. In 22.7% of cases, the platelet count was <1x105/mm3 and was associated more with secondary infection. Haematocrit more than 45% was found in 16.5% patients and a significantly higher association was detected among the secondary dengue fever patients (P=0.02). Although all 4 dengue serotypes were prevalent during the outbreak, DEN-3 was the predominant serotype (70.5%) and was associated with more severe clinical manifestations. Keywords: Primary dengue fever, secondary dengue fever, dengue haemorrhagic fever, serotypes, Bangladesh. # E-mail: [email protected] 96 Dengue Bulletin – Vol 28, 2004 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka Introduction Materials and methods Dengue was first reported in Bangladesh in The study was conducted on clinically 1964 and the outbreak came to be known suspected cases of dengue fever attending as ‘Dacca Fever’[1]. For a long period after the inpatient and outpatient departments of that, dengue cases remained undetected. A Medicine and Paediatrics, Bangabandhu few cases were reported in 1999 before a Sheikh Mujib Medical University (BSMMU) large outbreak occurred in 2000, during Hospital, and patients admitted at the Dhaka Medical College Hospital during June – which 5,551 cases and 93 dengue-related December 2000. Some patients seeking deaths were reported[2,3]. Cases reported diagnostic facilities at the Department of during the outbreaks were mostly diagnosed Virology, BSMMU, were also included in the clinically, except for a few serologically study. diagnosed cases[4]. According to specific inclusion criteria, Several studies have observed that 105 clinically suspected patients with fever sequential or secondary dengue infections (presenting within 5 days of onset with body are more likely to produce a severe form of temperature above 100 °F at the time of the disease. DHF and DSS occur mostly in blood sample collection) and fulfilling the persons with pre-existing dengue antibodies case-definition criteria of dengue fever (DF) acquired actively or passively[5-7]. and dengue haemorrhagic fever (DHF) of WHO[10] were enrolled in the study. The Detection of anti-dengue IgM indicates exclusion criteria defined cases of febrile the diagnosis of recent dengue infection in illness of more than 5 days and/or with both primary and secondary cases. Anti- definite sources of infection, chronic dengue IgM develops earlier than IgG in illnesses like tuberculosis, bronchial asthma, primary infection and is usually detectable congenital heart disease, renal failure, by day 5 of illness and wanes after 1-2 history of bleeding tendency since birth and [8] months . The ratio of IgM and IgG patients who refused to give two blood antibodies determined by ELISA is useful for samples. distinguishing primary and secondary Clinical data were collected through infections[8,9]. In primary infection, the interviewing the patients or their attendants IgM/IgG ratio generally exceeds 1.8 OD [8] and meticulous physical examination of the units in acute or convalescent sera . patients conducted by a doctor. The Detection of an early and excess of IgG tourniquet test was performed in all patients characterizes secondary infection. In the by conventional method[11]. Hepatomegaly present study, dengue patients were and ascites were ascertained by physical classified into primary and secondary cases examination and on reports of on the basis of this concept and it was ultrasonography and X-rays. 5 ml of venous carried out to confirm the clinical diagnosis, blood was collected aseptically from all correlate the clinical manifestations with patients during both the early and laboratory findings and establish the convalescent stages of fever irrespective of association of haemorrhagic manifestations age and sex. This was processed and stored with different dengue virus serotypes appropriately for virus isolation, antibody responsible for the outbreak of 2000. assay, platelet count and haematocrit Dengue Bulletin – Vol 28, 2004 97 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka estimation. Detection of IgM and IgG anti- anti-dengue IgM or IgM and IgG antibodies dengue antibody, isolation of dengue viruses and 38 (36.2%) patients developed only by mosquito inoculation technique and anti-dengue IgG in acute stage by ELISA. serotyping of the isolated viruses were done When these patients were tested in the [12,13] as described previously . Patients were convalescent stage, 21 (20%) were positive classified into DF and DHF or DSS for only IgM and 76 (72.4%) were positive [10] according to WHO, 1997 . for both IgM and IgG antibodies. Eight patients did not develop anti-dengue IgM Statistical analysis and IgG in either of the specimens (Table 1) The numerical data obtained from the study and were also negative on virus isolation. were analysed and the significance of the Thus, a total of 97 (92.4%) cases were difference was estimated by using statistical diagnosed serologically as current dengue methods. The data were expressed in infection, while 8 (9.8%) cases were frequency, percentage, mean and standard considered as non-dengue febrile illness and deviation as applicable. The comparison were excluded from further analysis. Of the between groups was done by Student’s ‘t’ 97 dengue patients, 46 (47.4%) had primary test and ‘Chi square’ and ‘Z’ test as and 51 (52.6%) had secondary infection applicable. All data were analysed by using depending on anti-dengue IgM and IgG the computer-based SPSS programme. antibody level of acute and convalescent Probability less than 0.05 was considered as stage sera (Table 2). Among them, 17 significant. patients developed DHF and one developed DSS. Of the 18 DHF/DSS patients, 14 had Results secondary type of infection and 4 had primary infection. Of the 105 clinically suspected dengue patients, 39 (37.1%) were positive for either Table 1. IgM and IgG dengue antibody positive cases by ELISA Only IgM Only IgG Both IgM and IgM and IgG Time of serum antibody antibody IgG antibody antibody Total collection positive cases positive cases positive cases negative cases n (%) n (%) n (%) n (%) n (%) Acute stage serum 21 (20.0)* 38 (36.2) 18 (17.1)* 28 (26.7) 105 (100) (within 5 days of fever) Convalescence 21 (20.0)** 00 (0.0) 76 (72.4)** 8 (7.6)*** 105 (100) stage serum (within 14-21 days of fever) * Total number of dengue infection cases detected in acute stage serum (21+18) = 39 (37.1%). **Total number of dengue infected cases detected in convalescence serum (21+76) = 97 (92.4%). *** Non-dengue febrile illness cases = 8 (7.6%). Figures in parenthesis indicate percentage 98 Dengue Bulletin – Vol 28, 2004 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka Table 2. Age distribution of dengue patients by primary and secondary dengue infection Primary infection Secondary infection Total Age in years n (%) n (%) n <10 3 (42.9) 4 (57.1) 7 10-19 4 (44.4) 5 (55.6) 9 20-29 20 (55.6) 16 (44.4) 36 30-39 11 (45.8) 13 (54.2) 24 40-49 7 (53.8) 6 (46.2) 13 >50 1 (12.5) 7 (87.5) 8 Total 46 (47.4) 51 (52.6) 97 (100.0) *Mean±SD 27.5±11.0 30.7±14.4 29.2±12.9 P value 0.214 (unpaired student’s ‘t’ test) * Mean of age Figures in parenthesis indicate percentage The involvement of all age groups, Dengue viruses were isolated from 44 especially an adult predominance, was of the 97 dengue patients. The rate of observed. The mean age of the dengue dengue virus isolation was significantly patients was 29.2±12.9 years and most higher (68.2% vs 31.2%) among primary belonged to the 20-29-year age group. The than secondary infection patients (P=0.018). mean age of primary dengue fever patients The isolation rate decreased gradually with was 27.5±11.7 years and that of secondary the increasing duration of fever (see Figure). patients was 30.7±14.4 years. Figure. Isolation rate of dengue virus between primary and secondary dengue patients by day of fever 4/4 9/10 100 5/6 8/11 80 3/5 60 1/3 40 5/18 4/14 4/15 20 Virus isolation rate (%) 1/1 0 1st 2nd 3rd 4th 5th Days of fever Primary Secondary (P=0.018) P value reached from Z test Dengue Bulletin – Vol 28, 2004 99 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka The distribution of clinical manifestations The most common presenting sign was rash, in dengue cases is given in Table 3. The especially maculopapular type, and its mean body temperature of the dengue association was significantly higher with patients was 101.5±1.4 ºF but there was no primary DF cases (P=0.016). Ascitis was significant difference in the mean body observed in 5 (9.8%) cases; all had temperature between primary and secondary secondary DF. Hepatomegaly was present in DF patients. Other common symptoms 13 (13.4%) patients and its association was included myalgia (84.5%), headache (82.5%), significantly higher (P=0.002) in secondary arthralgia (68%), lethargy (80.4%) and retro- DF patients. Abdominal pain (6.2%) and orbital pain (49.5%). Patients with primary lymphadenopathy (4.1%) was less frequent dengue infection presented more commonly among our study patients though abdominal with headache, arthralgia and retro-orbital pain was more common (7.8%) in secondary pain, whereas lethargy was commonly DF patients. associated with secondary dengue infection. Table 3. Distribution of clinical manifestations in dengue patients Types of dengue Secondary Total Clinical characteristics Primary infection P-value infection n=97 n=46 n=51 Mean temperature (°F) 101.4±1.4 101.6±1.4 101.5±1.4 Headache 41 (89.1) 39 (76.5) 80 (82.5) Arthralgia 34 (73.9) 32 (62.7) 66 (68.0) Retro-orbital pain 25 (54.3) 23 (45.1) 48 (49.5) Myalgia 39 (84.8) 43(84.3) 82 (84.5) Lethargy 36 (78.3) 42 (82.4) 78 (80.4) Rash 19 (41.3) 13 (25.5) 32 (32.9) Maculopapular 17 (36.9) 8 (15.7) 25 (25.8) 0.016S Petechial 2 (4.3) 5 (9.8) 7 (7.2) Anorexia, nausea and 16 (34.8) 19 (37.3) 35 (36.1) vomiting Abdominal pain 2 (4.3) 4 (7.8) 6 (6.2) Ascitis 0 (0.0) 5 (9.8) 5 (5.1) Enlarged lymph node 2 (4.3) 2 (3.9) 4 (4.1) Hepatomegally 1 (2.2) 12 (23.5) 13 (13.4) 0.002S Figures in parenthesis indicate percentage P-value reached from chi-square analysis 100 Dengue Bulletin – Vol 28, 2004 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka Haemorrhagic manifestations in primary (12.4%), gum bleeding 12 (12.4%) and and secondary dengue infections are haematemasis/malaena 11 (11.3%) were the indicated in Table 4. The most common other bleeding manifestations. Besides these, sign of bleeding manifestation, i.e. a positive conjunctival bleeding, haematuria and per tourniquet test, was observed in 18 (18.6%) rectal bleeding also occurred in a small patients. Petechiae 15 (15.5%), purpura 12 number of patients. Table 4. Haemorrhagic manifestations among primary and secondary dengue cases Types of dengue Secondary Total Clinical characteristics Primary infection infection n=97 n=46 n=51 Positive tourniquet test 7 (15.2) 11 (21.6) 18 (18.6) Petechiae 8 (17.4) 7 (13.7) 15 (15.5) Purpura 5 (10.9) 7 (13.7) 12(12.4) Epistaxis 1 (2.2) 2 (3.9) 3 (3.1) Haematemesis/melaena 8 (17.4) 3 (5.9) 11 (11.3) Gum bleeding 5 (10.9) 7 (13.7) 12 (12.4) Per vaginal bleeding 2 (4.3) 1 (2.0) 3 (3.1) Conjunctival bleeding 1 (2.2) 1 (1.0) 2 (2.0) Haematuria 1 (2.2) 0 (0.0) 1 (1.0) Per rectal bleeding 0 (0.0) 2 (3.9) 2 (2.0) Figures in parenthesis indicate percentage The haematological features in primary association of >45% haematocrit level was and secondary dengue infections are given detected among secondary DF patients in Table 5. Platelet counts of <1x105/mm3 (P=0.02). was detected in 22 (22.7%) patients and Haemorrhagic manifestations were was more frequently 16 (31.4%) associated mostly associated with DEN-3 and DEN-4 with secondary DF (Table 5). Haematocrit infections; only one patient with DEN-1 value of >45% was observed in 16 (16.5%) infection had per rectal bleeding (Table 6). patients and a significantly higher Dengue Bulletin – Vol 28, 2004 101 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka Table 5. Haematological features in primary and secondary dengue infection Types of dengue infection Secondary Total Haematology Primary infection P-value infection n=97 n=46 n=51 Platelets/cu mm <1´105 6(13.1) 16(31.4) 22(22.7) 0.059 >1´105 40(86.9) 35(68.6) 75(77.3) Haematocrit >45% 3(6.5) 13(25.5) 16(16.5) 0.020S <45% 43(93.5) 38(74.5) 81(83.5) Figures in parenthesis indicate percentage P-value reached from chi-square analysis Table 6. Haemorrhagic manifestations in different serotypes of dengue virus infections Serotypes of dengue viruses Clinical characteristics DEN-1 DEN-2 DEN-3 DEN-4 (n=6) (n=3) (n=31) (n=4) Positive tourniquet test 0 (0.0) 0 (0.0) 6 (19.4) 3 (75.0) Petechiae 0 (0.0) 0 (0.0) 1 (3.2) 1 (25.0) Purpura 0 (0.0) 0 (0.0) 3 (9.7) 1 (25.0) Epistaxis 0 (0.0) 0 (0.0) 1 (3.2) 0 (0.0) Haematemesis/melaena 0 (0.0) 0 (0.0) 4 (12.9) 1 (25.0) Gum bleeding 0 (0.0) 1 (33.3) 4 (12.9) 1 (25.0) Haematuria 0 (0.0) 0 (0.0) 1 (3.2) 1 (25.0) Per rectal bleeding 1 (16.7) 0 (0.0) 0 (0.0) 1 (25.0) Figures in parenthesis indicate percentage Discussion dengue infection. The presence of secondary cases indicated that DF was The secondary dengue infection is usually present in the area and perhaps a low-grade associated with more severe manifestations transmission was continuing during the than the primary infection. In our study, out previous years. Thus, it may be speculated of the 97 dengue cases, 46 (47.4%) had that a considerable proportion of cases, primary and 51 (52.6%) had secondary which were diagnosed as “viral flu” in the 102 Dengue Bulletin – Vol 28, 2004 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka past, may have been due to dengue virus DHF in Cambodian[20] and Thai children[21], infection. On serological test in acute stage, while it was less frequent in patients in our only 39 (37.1%) cases were found to be study. In the present study, statistically positive for dengue antibody. However, significant (P=0.002) hepatomegaly (13.4%) when the serum from the patients was in secondary dengue infection was noted. tested again in the convalescence stage, 97 Hepatomegaly was also a common clinical (92.4%) cases were found to be positive, finding in several other studies[22-24]. Acute indicating that they had recent dengue virus abdominal pain, which is considered as an infection (Table 1). Thus, the detection of a early sign of shock in DF/DHF[10], was significant number of cases could have been present in only 6 (6.2%) patients in the missed if the tests were not done again at present study. This is similar to a study from the convalescence stage. A second test Lucknow, India, where abdominal pain was should therefore be considered at the found in only 5% of cases in an epidemic of convalescence stage to delineate the actual DHF[19]. In some studies, acute abdominal numbers of infection due to dengue. pain was strongly correlated with DHF and [10,24,25] Our study indicates the involvement of DSS . In the present study, ascitis, a all age groups of the population with adult sign of plasma leakage, was present in only predominance. This finding is not consistent 5 (9.8%) secondary infection cases and was with the epidemiological data from other in agreement with the findings of other [25,26] endemic countries in Asia where young studies . [14,15] children were affected . However, adults The positive tourniquet test, which may also be the major victims of DHF as reflects capillary fragility and is used as a reported in different epidemics where guiding test for detecting dengue illness[27], [16,17,18] dengue was endemic . No significant was found in only 18.6% cases in the age or sex difference among patients was present study. There was no statistically observed between primary and secondary significant difference in test positivity infections in the present study. Similar between primary and secondary DF patients observations were also reported from an although it was more frequently positive in [11] outbreak in Fiji . secondary DF. In most studies, comparing Anorexia, nausea, vomiting, abdominal DHF with classic DF, either a higher pain and ascitis were associated more with incidence of tourniquet test positivity in [23,26,28] secondary than primary dengue infections DHF or no difference between the [24,29] (Table 3). In the present study, a statistically two groups , was reported. These significant (P=0.01) association of rash variable findings are probably because the (32.9%) among primary dengue infection pathogenesis of tourniquet positivity and cases was demonstrated. The predominant other bleeding manifestations in dengue type of rash in primary infection was cases are different. Moreover, the defects macular or maculopapular whereas which lead to increase in capillary fragility petechial rash was found frequently in may also predispose to the development of secondary infection. Other studies also shock, thus indicating a better correlation of reported similar association of rash in the test to shock rather than to haemorrhage. DF[11,19]. Petechiae were frequently found in Dengue Bulletin – Vol 28, 2004 103 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka In our study, platelet counts of there is a wide variation in the presence of <1´105/mm3 were observed among 22 various symptoms, which may misguide the patients and were associated more with physician regarding the severity of the secondary than primary infections (Table 4). disease. Therefore, the use of clinical case- Lack of correlation between a moderately definition results in inaccuracies. Thus, in low platelet count and bleeding order to identify the cases accurately, one manifestation in dengue patients has been should take the help of a simple and cheap noted by other investigators[8]. The bleeding diagnostic test that is able to diagnose in dengue is probably due to other factors accurately in the laboratory. In this context with or without the additive effect of ELISA is clearly very promising for the thrombocytopenia. Haemoconcentration, the confirmation of clinical diagnosis and to evidence of plasma leakage and shock, was differentiate between primary and observed in 16 patients with secondary secondary infections. This will guide the infection and the association was statistically clinician to take prompt and meticulous significant (P=0.02). Increased haematocrit clinical management and early hospitalization was found in 95.5% patients with shock in of severe cases. Indeed, it is hoped that contrast to 31.7% cases without shock[21]. accurate laboratory diagnosis will not only reduce the morbidity and mortality but will In the present study, the virus isolation also reduce the economic burden of the rate was significantly higher in patients with patient and the government. primary (68.2%) than secondary (31.2%) infections (P=0.018). Similar results have been reported in a study from Fiji[11]. In Acknowledgements Bantal, Indonesia, the isolation rate was 100% among patients with primary dengue We express our sincere thanks to infection and 57% with secondary dengue Dr Vorndam Vance, CDC Dengue Lab, San infection[30]. However, a study from Juan, Puerto Rico, USA, and Dr Ichiro Thailand reported a high rate of virus Kurane, Arbovirus Research Laboratory, isolation in both primary (98%) as well as NIID, Japan, for their kind support in secondary (93%) infections[31]. The reason providing cell line and anti-dengue for a higher isolation rate in secondary monoclonal antibodies. We are also grateful infection was probably because blood to the patients who participated in the study. samples were collected at an early febrile We would like to thank Mr Tauhid Uddin phase (within 72 hours). In our study, the Ahmed, Ex-PSO, IEDCR, Bangladesh, and isolation rate was also high in both primary Noor-e-Jannat for providing mosquito (100%) and secondary (68%) infections in colony and rearing techniques. We would early febrile period, i.e. on day 1 of fever. also like to thank Dr Bijon Kumar Sil, Ex- Thereafter, the rate of isolation was always Senior Scientific Officer, Bangladesh higher in primary than in secondary dengue Livestock Research Institute, Savar, Dhaka, infection in the subsequent days of fever Bangladesh. We also express our gratitude (Figure). to the Department of Medicine and Paediatrics, BSMMU, and Dhaka Medical In conclusion, the clinical diagnosis is College, Bangladesh, for their support in the not very reliable in dengue infection as study. 104 Dengue Bulletin – Vol 28, 2004 Clinical and Laboratory Observations during the 2000 Dengue Outbreak in Dhaka References [1] Aziz MA, Gorham JR and Gregg MB. “Dacca [9] Kuno G, Gomez I and Gubler DJ. 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Bulletin of Pan American Health Organization, 1984, 18: 213-220. 106 Dengue Bulletin – Vol 28, 2004 Current Status of Dengue Diagnosis at the Center for Disease Control, Taiwan Pei-Yun Shu, Shu-Fen Chang, Yi-Yun Yueh, Ling Chow, Li-Jung Chien, Yu-Chung Kuo, Chien-Lin Su, Tsai-Ling Liao, Ting-Hsiang Lin and Jyh-Hsiung Huang# Center for Research and Diagnostics, Center for Disease Control, Department of Health, 161, Kun-Yang Street, Taipei, Taiwan Abstract A national-level diagnostic laboratory has been set up in Taiwan for routine diagnosis of reported cases of dengue fever (DF)/dengue haemorrhagic fever (DHF), Japanese encephalitis (JE) and yellow fever (YF). The facilities include serological diagnosis, virus isolation by cell culture, molecular diagnosis and molecular tools for epidemiological investigations. To detect and differentiate dengue, JE and YF virus infections, a differential diagnostic system has been developed. For acute-phase sera, virus isolation by cell culture and real-time one-step reverse transcription-polymerase chain reaction (RT-PCR) has been established. For all of the serum samples reported, serological diagnosis of specific antibodies based on envelope and membrane (E/M)-specific capture IgM and IgG enzyme-linked immunosorbent assay (ELISA) are performed. In this report, a case study from Taiwan has been presented with the analysis of 959 serum samples (including some paired sera) collected between day 1-30 of illness from 799 confirmed dengue cases reported in 2002. The results demonstrated that 94.5% of acute-phase serum samples of confirmed dengue cases could be identified as positive or probable with the combined use of real-time one-step RT-PCR and E/M-specific capture IgM and IgG ELISA. Furthermore, a nonstructural protein NS1 serotype-specific indirect IgG ELISA has been developed and used to analyse dengue NS1- specific IgG antibodies. Both E/M-specific capture IgM and IgG ELISA and the NS1 serotype-specific indirect IgG ELISA have been used to detect and differentiate primary and secondary dengue virus infections. In addition, the NS1 serotype-specific indirect IgG ELISA has the potential of replacing the plaque-reduction neutralization test (PRNT) and is being used for a large-scale seroepidemiological study. Keywords: Dengue virus, virus isolation, real-time one-step RT-PCR , E/M-specific capture IgM and IgG ELISA, NS1 serotype-specific indirect IgG ELISA. # E-mail: [email protected]; Tel.: 886-2-26531374, Fax: 886-2-27883992 Dengue Bulletin – Vol 28, 2004 107 Dengue Diagnosis in CDC, Taiwan Introduction haemagglutination inhibition (HI) test and E/M-specific capture IgM and IgG ELISA[8] The dengue viruses cause a broad spectrum are the two most commonly used serological of illness ranging from inapparent infection, techniques for the routine diagnosis of mild undifferentiated fever and classic flavivirus infection. The serodiagnosis of dengue fever to a more severe form, dengue flavivirus is rather complicated due to the haemorrhagic fever/dengue shock syndrome high cross-reactivity of IgG antibodies to (DHF/DSS), resulting in high morbidity and homologous and heterologous viruses. We mortality. The diagnosis of dengue virus have attempted to set up an ELISA system infection based on clinical syndromes is not that can be easily and reliably used to detect reliable, and a confirmation of the infection and differentiate various flavivirus infections. should rely on laboratory diagnosis with the To accomplish this goal, three different detection of the specific virus, viral antigen, forms of ELISA were developed including: genomic sequence and/or antibodies. (i) E/M-specific capture IgM and IgG ELISA; A rapid, simple, sensitive and specific (ii) E/M-specific antigen-coated indirect IgM assay system to detect the virus in the acute- and IgG ELISA; and (iii) NS1 serotype- [9-11] phase serum is essential to improve the specific indirect IgG ELISA . clinical treatment, etiological investigation and disease control of dengue virus Case study of 2002 outbreak of infection. Among the various assays for virus DEN-2 in southern Taiwan detection, virus isolation by cell culture and We present here a case study of the analysis dengue virus antigen detection by ELISA[1,2] of biological material obtained during a suffer from some disadvantages – while the DEN-2 outbreak in 2002 in southern former needs a longer time, the latter has Taiwan, by utilizing the facilities available in low sensitivity. However, recent advances in the national diagnostic laboratory. molecular diagnosis have demonstrated that various RT-PCR protocols can be reliably A major DEN-2 epidemic occurred in used to detect the viral genomic sequence southern Taiwan, affecting Kaohsiung city, with high sensitivity and specificity. More Kaohsiung county and Pingtung county recently, several investigators have reported between October 2001 and December 2002, real-time RT-PCR assays for the detection of with more than 5,000 confirmed cases. dengue virus in acute-phase serum Among these, 227 cases were classified as of samples[3-7]. The real-time RT-PCR assay has DHF with 21 deaths. This outbreak was a many advantages over the conventional RT- repeat of the 1987-1988 DEN-1 epidemic in PCR methods, which include rapidity, many aspects[11,12]. In this report, we present quantitative measurement, lower the results of a total of 959 acute- and contamination rate, higher sensitivity, higher convalescent-phase sera collected from 799 specificity and easy standardization. confirmed dengue patients reported to the Kun-Yang office of the Center for Disease For convalescent sera, detection of Control (CDC), Taiwan, 2002. specific IgM and IgG antibodies based on 108 Dengue Bulletin – Vol 28, 2004 Dengue Diagnosis in CDC, Taiwan Materials and methods performed as previously described[7,9]. The culture supernatants were used as the source of E/M and NS1 antigens for ELISA. Human serum samples The control antigen was prepared by the The serum samples used in this study were same procedure from Vero cells culture collected from the confirmed cases of without viral infection. dengue patients reported to the Arbovirus Laboratory in the Kun-Yang office, CDC, One-Step SYBR Green I Real-Time Department of Health, 2002. A total of 959 RT-PCR acute- and convalescent-phase sera collected from 799 confirmed dengue One-step SYBR Green I real-time RT-PCR patients were analysed. Most of these serum for dengue virus was performed in the TM samples were from the major DEN-2 Mx4000 quantitative PCR system [7] outbreak, together with a few serum (Stratagene) as recently described . Briefly, samples from imported cases contracted a set of flavivirus- (in the NS5 gene region), during travel to the neighbouring South-East dengue- and serotype-specific primer pairs Asian countries. (in the core gene region) was selected and used for analysis. To assure the specificity of Case definitions amplicons produced from SYBR Green I real-time RT-PCR in daily routine screening, A confirmed case of dengue virus infection both flavivirus- and dengue -specific primer was defined as febrile illness associated pairs were used for each of the serum with: (i) the isolation of dengue virus; samples tested. Serum samples found (ii) positive test of real-time one-step RT- positive for initial screening were then PCR; (iii) positive seroconversion or ³ four- tested for serotype by each of the four fold increase in dengue-specific IgM or IgG serotype-specific primer pairs. antibody from appropriately timed paired serum; or (iv) high-titer dengue-specific IgM ELISA and IgG antibody in a single serum specimen where cross-reaction to Japanese E/M-specific capture IgM and encephalitis (JE) had been excluded. Sera IgG ELISA collected during day 1-7 after the onset of symptoms are referred to as acute-phase A modified E/M-specific capture IgM and sera. Early and late convalescent sera refer IgG ELISA was performed to measure the to the specimens collected during day 8-13 dengue-specific IgM and IgG antibodies as and day 14-30, respectively. recently described[13]. Briefly, each microtiter 8 wells strip was coated with Virus isolation by cell culture and 5 µg/ml, 100 µl/well of affinity purified goat virus antigen preparation anti-human IgM (µ-specific) or IgG (g- specific) antibodies, followed by incubation The isolation of dengue virus by cell culture with 1:100 diluted serum, incubation with and virus antigen preparation from culture cocktail contained 1:3 diluted pooled virus supernatants of DEN-1, DEN-2, DEN -3, antigens from culture supernatants of DEN-1, DEN-4 or JE-virus infected Vero cells were DEN-2, DEN-3 or DEN -4 infected Vero cells Dengue Bulletin – Vol 28, 2004 109 Dengue Diagnosis in CDC, Taiwan and 1 µg/ml mAb D56.3, incubation with with goat anti-human IgG conjugated to 1:1,000 diluted alkaline phosphatase- alkaline phosphatase. The enzyme activity conjugated goat anti-mouse IgG (g-specific). was developed and OD was taken 30 The enzyme activity was developed with the minutes later. substrate p-nitrophenyl-phosphate and optical density (OD) was taken 30 minutes Data analysis later. For routine screening, culture supernatant from JE virus-infected Vero cells For E/M-specific capture IgM and IgG ELISA, was used as negative control antigen due to primary dengue virus infection was defined ³ the limited cross-reactivity between dengue- if the IgM:IgG OD ratio was 1.2, or and JE-specific IgM antibodies measured by secondary if the OD ratio was <1.2. For E/M-specific capture IgM ELISA. This was in those sera with positive NS1-specific IgG contrast to the high cross-reactivity of antibody response, NS1 serotyping was dengue- and JE-specific IgG antibodies calculated by the ratio of the highest OD among dengue patients with secondary value and the second highest OD value read infection. from the four dengue serotypes. Positive serotype-specificity is defined if the OD E/M-specific antigen-coated indirect ratio is ³1.2 and negative serotype- IgM and IgG ELISA specificity is defined if the OD ratio is <1.2. Based on NS1 serotype-specific indirect IgG E/M-specific antigen-coated indirect IgM ELISA, primary dengue virus infection was and IgG ELISA were performed as previously defined if: (i) negative NS1-specific IgG described[10]. Two-fold serial dilutions of antibody response was found for sera appropriately timed paired sera diluted from collected between day 1 and 14 of illness, 1:100 to 1:12,800 were analysed to or (ii) positive serotype-specificity for sera determine whether ³ four fold increase of collected ³9 days of illness. Secondary dengue-specific IgM or IgG antibody could dengue virus infection was defined if: be found. This assay has the advantage of (i) positive NS1-specific IgG antibody better sensitivity in the detection of IgM and response was found for sera collected IgG antibody increase than E/M-specific between day 1 and 8 of illness, or capture IgM and IgG ELISA. (ii) positive NS1-specific IgG antibody response and negative serotype-specificity NS1 serotype-specific indirect was found any time after the onset of IgG ELISA infection. NS1 serotype-specific indirect IgG ELISA was performed as previously described[9,13]. Results Briefly, each microtiter 8 wells strip was coated with 5 µg/ml, 100 µl/well of mAbs Dengue surveillance system and D2/8-1, followed by incubation with 1:3 diluted NS1-containing culture supernatants laboratory diagnosis in Taiwan of DEN-1, DEN -2, DEN-3, DEN-4 or JE Taiwan has an integrated programme for viruses-infected Vero cells, incubation of dengue surveillance and control. The serum samples at a 1:50 dilution, incubation dengue prevention and control centre is a 110 Dengue Bulletin – Vol 28, 2004 Dengue Diagnosis in CDC, Taiwan mission-oriented structure jointly sponsored Pingtung county due to the large samples by the Department of Health and the generated by the DEN-2 outbreak. For Environment Protection Administration routine diagnosis, serum samples from the responsible for the planning and execution reported cases were sent to the laboratory of dengue control. To assure the on a daily basis and tested according to the effectiveness of dengue surveillance, three flow chart shown in Figure 1. The periods of report systems are currently associated with time required to complete these tests were the dengue surveillance programme 7 days, 6 hours and 4 hours for virus including: (i) hospital-based passive report isolation, real-time one-step RT-PCR and system; (ii) syndrome report system (under E/M-specific capture IgM and IgG ELISA, the classification of viral haemorrhagic respectively. The results were reported as fever); and (iii) active surveillance system. positive, negative or probable cases. The The Arbovirus Laboratory in Kun-Yang office, probable case was referred to ELISA result Taipei City, CDC, Department of Health, is with only IgM or IgG antibody positive. For responsible for the diagnosis of various negative and probable cases, the flavivirus. In addition, a second dengue convalescent serum samples collected after diagnostic laboratory was set up in the day 14 of the illness were demanded and Fourth Branch, Kaohsiung City, CDC, in July tested for the presence of or increase in IgM 2002 to provide prompt service to and/or IgG antibodies. Kaohsiung city, Kaohsiung county and Figure 1. Flow chart of laboratory diagnosis of dengue virus infection 1-7 days (+) Result Serum Virus isolation Real-Time Probable or 1-30 days RT-PCR (-) Result E/M-specific Current Capture dengue IgM/IgG ELISA (+) Result infection 1:100 dilution >=14 days serum E/M-specific Capture Final Probable or (-) Result IgM/IgG ELISA Result 1:100 dilution Dengue Bulletin – Vol 28, 2004 111 Dengue Diagnosis in CDC, Taiwan Representative results of routine provided a good example of the dynamic diagnosis measured by virus change of dengue virus and specific IgM and IgG antibodies in the acute- and isolation, real-time one-step convalescence-phase sera from patients with RT-PCR, and E/M-specific capture primary or secondary dengue virus infection IgM and IgG ELISA covering all four serotypes. As shown in Figure 1, all of the three assays were Table 1 shows the representative results of performed for acute-phase sera, whereas serum samples analysed by virus isolation, only E/M-specific capture IgM and IgG real-time one-step RT-PCR and E/M-specific ELISA was tested for convalescence-phase capture IgM and IgG ELISA. The results sera. Table 1. Representative results of routine diagnosis of serum samples from reported dengue cases E/M-specific capture IgM and Real-time one -step RT-PCR IgG ELISA Ct value Dengue Dengue Onset Virus OD 405 nm infection serotype days isolation Flavi- Dengue- Serotype- Dengue- JE-IgM Dengue- JE-IgG specific specific specific IgM IgG Primary DEN-1 3 + 28 25 25 0.526 0.237 0.200 0.330 infection 16 2.791 0.951 1.687 1.453 DEN-2 2 + 26 33 29 0.209 0.192 0.182 0.174 15 3.484 0.245 1.165 0.708 DEN-3 3 + 25 31 32 0.478 0.104 0.148 0.228 16 3.074 0.329 2.535 1.704 DEN-4 3 – 26 35 35 0.094 0.082 0.057 0.063 15 2.225 0.148 1.301 0.096 Secondary DEN-1 1 + 27 28 24 0.166 0.151 0.226 0.176 infection 16 1.949 0.469 3.606 1.699 DEN-2 2 + 17 18 20 0.267 0.217 0.222 0.164 18 0.496 0.157 3.482 1.069 DEN-3 1 + 18 23 24 0.175 0.142 0.180 0.216 7 3.631 0.691 3.494 3.474 DEN-4 7 + 20 28 29 1.298 0.105 2.883 0.273 12 1.468 0.108 3.424 0.637 + = positive – = negative Due to the long time needed to isolate reliable and universal RT-PCR protocol that virus using the cell culture method, it has can be used to systemically detect and limited value in rapid diagnosis. The isolated differentiate various flavivirus. To assure the virus, however, is the key material for the specificity of amplicons produced from later studies of molecular epidemiology and SYBR Green I real-time RT-PCR in routine pathogenesis. The real-time one-step SYBR screening, both flavivirus- and dengue- Green I RT-PCR we developed is a simple, specific primer pairs were run (Table 1). 112 Dengue Bulletin – Vol 28, 2004 Dengue Diagnosis in CDC, Taiwan Those serum samples positive for initial Therefore, a positive dengue-specific IgM screening were then tested for serotype by and IgG antibody response can be easily each of the four serotype-specific primer used to detect and differentiate primary and pairs. The analysis of acute-phase serum secondary dengue virus infections. samples demonstrated that the one-step SYBR Green RT-PCR was more sensitive to E/M-specific antigen-coated the virus isolation method and could detect indirect IgM and IgG ELISA two-times more the acute-phase sera with positive dengue-specific IgM and/or IgG for the detection of dengue antibodies[7]. virus infection The E/M-specific capture IgM and IgG Occasionally, there were acute-phase sera ELISA has several advantages in the which tested positive with E/M-specific detection of dengue-specific IgM and IgG capture IgM or IgG antibody response, but antibodies including: (i) high sensitivity; did not show an apparent increase in (ii) high specificity (only for IgM antibody); antibody titers in convalescent sera. Due to (iii) analysis of isotype-specific antibody the higher sensitivity of E/M-specific indirect responses; (iv) easy automation to test large IgM and IgG ELISA (especially for IgG amount of serum samples; and antibody), it can be reliably used to (v) differentiation of primary and secondary determine whether ³ four-fold increase of dengue infections. The results shown in dengue-specific IgM or IgG antibody were Table 1 demonstrated the low cross- presented. Figure 2 shows an example reactivity between dengue - and JE-specific where significant dengue-specific IgG IgM antibody and inverse pattern of IgM:IgG antibody increase in a convalescent serum. OD ratio of primary and secondary infection. Figure 2. E/M-specific antigen-coated indirect IgM and IgG ELISA to detect the increase of dengue-specific IgM and/or IgG antibodies in the pair sera. The serum samples showed a = four-fold increase in dengue-specific IgG antibody titer in the convalescent sera 3.0 2.5 2.0 S9100837A-day 5 IgM 1.5 S9100837B-day 15 IgM S9100837A-day 5 IgG OD 405nm S9100837B-day 15 IgG 1.0 0.5 0.0 50x 100x 200x 400x 800x 1600x 3200x 6400x Patient serum dilution (S9100837) Dengue Bulletin – Vol 28, 2004 113 Dengue Diagnosis in CDC, Taiwan Statistical analysis of results of respectively. The positive rate for E/M- serum samples from confirmed specific capture IgM and/or IgG ELISA was 31.6%, 32.6%, 30%, 39%, 52.6%, 87.5% dengue cases reported in 2002 and 80% for day 1-7 of illness, respectively. Table 2 shows the comprehensive analysis Thus, the combined results of real-time RT- of the results of serum samples from PCR and E/M-specific capture IgM and IgG confirmed dengue cases reported to Kun- ELISA (IgM and/or IgG positive) could detect Yang office in 2002. The results of virus an average 94.5% (89.7% to 97.5%) of isolation, real-time RT-PCR and E/M-specific acute-phase serum samples of confirmed capture IgM and IgG ELISA were analysed dengue cases. The results also showed that separately or in combination from the sera the real-time RT-PCR was more sensitive samples collected on 1-30 day of illness. than virus isolation although very few sera, The positive rate for real-time RT-PCR was which were virus-isolation positive, were 74.7%, 69.5%, 72.3%, 76.6%, 57.7%, missed by real-time RT-PCR. 36.3% and 22.2% for day 1-7 of illness, Table 2. Statistical analysis of results of serum samples from confirmed dengue cases reported in 2002 Days after onset Assays 1 2 3 4 5 6 7 8 9 10 11 12 13 14-30 Total Total serum no. 95 95 130 128 97 80 45 58 27 24 9 16 7 148 959 tested V.I.+ (Virus 53 52 64 58 32 9 2 – – – – – – – 270 isolation) % of V.I.+ 55.8 54.7 49.2 45.3 33.0 11.3 4.4 – – – – – – – – RT-PCR + and 52 50 60 57 31 8 2 – – – – – – – 260 V.I.+ RT-PCR + or 72 68 98 99 57 30 10 – – – – – – – 434 V.I.+ RT-PCR + (Real- 71 66 94 98 56 29 10 – – – – – – – 424 time) % of RT-PCR+ 74.7 69.5 72.3 76.6 57.7 36.3 22.2 – – – – – – – – RT-PCR - (Real - 24 29 36 30 41 51 35 – – – – – – – 246 time) ELISA + (E/M- 20 25 21 27 29 41 27 45 19 23 8 12 7 140 444 specific capture IgM+IgG +) Probable (ELISA 10 6 18 23 22 29 9 9 8 1 1 3 0 8 147 IgM+ or IgG+) % of IgM+ 31.6 32.6 30.0 39.1 52.6 87.5 80.0 93.1 100 100 100 93.8 100 100 – and/or IgG+ RT-PCR -, V.I.-, 3 3 5 3 9 1 3 4 0 0 0 1 0 0 32 ELISA IgM-IgG- 114 Dengue Bulletin – Vol 28, 2004 Dengue Diagnosis in CDC, Taiwan Days after onset Assays 1 2 3 4 5 6 7 8 9 10 11 12 13 14-30 Total RT-PCR + and 3 2 4 5 6 7 2 – – – – – – – 29 IgM+IgG + RT-PCR + and 6 2 4 7 9 13 1 – – – – – – – 42 IgM+IgG - RT-PCR + and 1 3 4 12 5 1 1 – – – – – – – 27 IgM-IgG+ RT-PCR + and 61 59 82 74 36 8 6 – – – – – – – 326 IgM-IgG- RT-PCR - and 17 23 17 22 23 34 25 45 19 23 8 12 7 140 415 IgM+IgG + RT-PCR - and 3 0 8 3 4 14 6 8 7 0 0 3 0 6 62 IgM+IgG - RT-PCR - and 0 1 2 1 4 1 1 1 1 1 1 0 0 2 16 IgM-IgG+ RT-PCR - and 4 5 9 4 10 2 3 4 0 0 0 1 0 0 42 IgM-IgG- RT-PCR + or 88 89 111 120 79 63 35 45 19 23 8 12 7 140 839 ELISA + % (RT-PCR +or 92.6 93.7 85.4 93.8 81.4 78.8 77.8 77.6 70.4 95.8 88.9 75.0 100 94.6 – ELISA +) / Total serum no. tested RT-PCR + or 91 90 121 124 87 78 42 54 27 24 9 15 7 148 917 ELISA IgM+ and/or IgG+ % (RT-PCR +or 95.8 94.7 93.1 96.9 89.7 97.5 93.3 93.1 100 100 100 93.8 100 100 – ELISA IgM+ and/or IgG+) / Total serum no. tested NS1 serotype-specific indirect IgG specific capture IgM and IgG ELISA and NS1 ELISA in the differentiation of JE, serotype-specific indirect IgG ELISA showed good correlation with 95.90% agreement[13]. primary and secondary dengue Most importantly, retrospective sero- virus infections and for the DEN epidemiological studies on serum samples serotyping of primary infection collected from Liuchiu Hsiang, Pingtung county and Tainan city in southern Taiwan, More recently, we have developed a NS1 demonstrated that NS1 serotype-specific serotype-specific indirect IgG ELISA in the indirect IgG ELISA could replace plaque- detection and differentiation of primary and reduction neutralization test (PRNT) for secondary infections. Comparisons of E/M- seroepidemiological study to differentiate JE, Dengue Bulletin – Vol 28, 2004 115 Dengue Diagnosis in CDC, Taiwan primary and secondary dengue virus evaluated the real-time RT-PCR method for infections and for the DEN serotyping of rapid detection of dengue virus in the acute- primary infection[11]. phase sera. In this report, we have presented a detailed analysis of a total of 959 acute- and convalescent-phase sera Discussion collected from 799 confirmed dengue Recent advances in molecular and patients reported to the Kun-Yang office of serological assays have revolutionized the CDC, Taiwan, in 2002. The results laboratory diagnosis of flavivirus demonstrated that 94.5% of acute-phase infection[7,13,14]. Rapid diagnosis of dengue serum samples of confirmed dengue cases virus infection in the acute-phase sera, could be identified as positive or probable which is important for disease control with the combined use of real-time one-step measures and potential treatment, will RT-PCR and E/M-specific capture IgM and require very sensitive and specific assays. IgG ELISA. The results are very encouraging With the maturation of real-time RT-PCR and suggest that these two assays are well- technique, its routine application to clinical suited for routine tests for the early diagnosis and laboratory diagnosis has now become a of dengue virus infection. reality. For serodiagnosis, E/M-specific capture IgM and IgG ELISA has become the new standard assay for the detection and Conclusion differentiation of flavivirus infection. The real-time RT-PCR assay has many The large DEN -2 epidemic in southern advantages over conventional RT-PCR Taiwan, was uncontrolled despite vigorous methods, which include rapidity, quantitative attempts to contain it by the central and measurement, lower contamination rate, local health governments during October higher sensitivity, higher specificity and easy 2001 – December 2002. Although standardization. Therefore, real-time insecticide -resistance was blamed as an quantitative assay might eventually replace important factor for this disaster, other virus isolation and conventional RT-PCR as elements including, political, social, the new gold standard for the rapid diagnosis environmental, community and human of virus infection in the acute-phase serum factors were also responsible for this setback. samples. This epidemic was a strong warning to us and suggested that more effective measures should be sought and applied. There is an Acknowledgements urgent need to improve the surveillance system and laboratory diagnosis which We wish to thank Hsiu-Ling Pan, Yun-Yih would help to identify confirmed cases in Chang and Chih-Heng Chen for their expert the acute-phase sera and respond promptly technical assistance. This work was in part and effectively to control the transmission supported by grants DOH91-DC-2007 and chain. DOH91-DC-2016 from the Center for Disease Control, Department of Health, Along with the progress of the DEN-2 Taiwan. outbreak in 2002, we have developed and 116 Dengue Bulletin – Vol 28, 2004 Dengue Diagnosis in CDC, Taiwan References [1] Alcon S, Talarmin A, Debruyne M, Falconar A, [8] Innis BL, Nisalak A, Nimmannitya S, Deubel V and Flamand M. Enzyme-linked Kusalerdchariya S, Chongswasdi V, immunosorbent assay to dengue virus type 1 Suntayakorn S, Puttisri P and Hoke CH. An nonstructural protein NS1 reveals circulation enzyme-linked immunosorbent assay to of the antigen in the blood during the acute characterize dengue infections where dengue phase of disease in patients experiencing and Japanese encephalitis co-circulate. Am J primary or secondary infections. J Clin Trop Med Hyg, 1989, 40: 418-427. Microbiol, 2002, 40: 376-381. [9] Shu PY, Chen LK, Chang SF, Yueh YY, Chow [2] World Health Organization. Dengue L, Chien LJ, Chin C, Lin TH and Huang JH. haemorrhagic fever: diagnosis, treatment and Dengue NS1-specific antibody responses: control (second edition). Geneva, WHO, 1997. isotype distribution and serotyping in patients with dengue fever and dengue hemorrhagic [3] Callahan JD, Wu SJ, Dion-Schultz A, fever. J Med Virol, 2000, 62: 224-232. Mangold BE, Peruski LF, Watts DM, Porter KR, Murphy GR, Suharyono W, King CC, [10] Shu PY, Chen LK, Chang SF, Yueh YY, Chow Hayes CG and Temenak JJ. Development L, Chien LJ, Chin C, Lin TH and Huang JH. and evaluation of serotype- and group- Antibody to the nonstructural protein NS1 of specific fluorogenic reverse transcriptase Japanese encephalitis virus: potential PCR (TaqMan) assays for dengue virus. J Clin application of mAb-based indirect ELISA to Microbiol, 2001, 39: 4119-4124. differentiate infection from vaccination. Vaccine, 2001, 19: 1753-1763. [4] Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H and Gunther S. [11] Shu PY, Chen LK, Chang SF, Yueh YY, Chow L, Chien LJ, Chin C, Yang HH, Lin TH and Rapid detection and quantitation of RNA of Ebola and Marburg viruses, Lassa virus, Huang JH. Potential application of nonstructural protein NS1 serotype-specific Crimean-Congo hemorrhagic fever virus, Rift valley fever virus, dengue virus and yellow immunoglobulin G enzyme-linked immunosorbent assay in the fever virus by real-time reverse transcription- seroepidemiologic study of dengue virus PCR. 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Dengue Bulletin – Vol 28, 2004 117 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of Sodium thiocyanate (NaSCN) Masaru Nawa*#, Tomohiko Takasaki**, Mikako Ito**, Ichiro Kurane** and Toshitaka Akatsuka* *Department of Microbiology, Saitama Medical School, 38, Moroyama, Saitama 350-0495, Japan **Division of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan Abstract Dengue virus serotype-specific IgM was detected by IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) in the presence of a chaotropic agent, sodium thiocyanate (NaSCN). NaSCN did not affect the reactions between anti-human IgM and patients’ IgM, and between dengue viral antigens and detecting antibody, peroxidase-conjugated flavivirus-specific monoclonal antibody D1-4G2 IgG. Among 18 dengue- confirmed cases, highest IgM responses were detected to infecting serotypes in 14 cases in the presence of 0.5 M of NaSCN. The results indicate that: (i) the protein-denaturing agent, NaSCN, affects antigen- antibody reaction in IgM-ELISA, and enables the differentiation of serotype-specific IgM from cross-reactive IgM; and (ii) IgM responses against the infecting serotypes are higher than those against the other three serotypes in most primary dengue virus infection. In conclusion, the addition of NaSCN to IgM-capture ELISA is useful for highlighting serotype-specific IgM responses in primary dengue virus infections. Keywords: Dengue, IgM-capture ELISA, serotype-specific IgM response, sodium thiocyanate, NaSCN. Introduction tropical and subtropical areas and nearly two million people visit Japan from these areas. Dengue is currently one of the most Therefore, dengue fever (DF) and dengue important arboviral disease in humans. haemorrhagic fever (DHF) has become an Dengue viruses, belonging to the family infectious disease of significance and worthy Flaviviridae, are comprised of four of more attention from the medical antigenically cross-reactive serotypes and are community in Japan. responsible for epidemics in tropical and We have earlier reported the laboratory subtropical countries. Since the dengue outbreaks in Osaka, Kobe, Hiroshima and diagnosis of dengue by reverse transcription polymerase chain reaction (RT-PCR) and Nagasaki from 1942 to 1945, dengue has not [3-5] occurred in an epidemic form in Japan[1]. IgM-ELISA . We demonstrated that IgM- However, imported dengue cases have been ELISA was a reliable diagnostic method and that IgM responses were generally serotype reported[2]. Approximately five million Japanese people annually visit countries in cross-reactive but often highest against # E-mail: [email protected]; Tel.: +81-49-276-1438; Fax: +81-49-295-9107 118 Dengue Bulletin – Vol 28, 2004 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN infecting virus serotype in most Japanese Japan from 2000 to 2002 and sent to the cases[4]. The serotype specificity of IgM Department of Virology 1, National Institute responses in dengue patients has been of Infectious Diseases, Tokyo, Japan. controversial[6-8]. Burke had reported that Prototype dengue viruses were serotype-specific IgM responses propagated in the Aedes albopictus corresponding to the isolated virus type were mosquito cell clone C6/36, and the infected detected in primary dengue virus infection[6]. Gubler had reported that in dengue infection, cell culture supernatants were inactivated by frequent monotypic IgM responses were not incubation with beta-propiolactone at a final correlated with the virus serotype isolated concentration of 0.2% for 30 minutes at [10] from patients[7]. In 1984, Inouye et al.[9] 37 °C as previously reported . Tetravalent demonstrated a new technique for the and four monovalent dengue viral antigens were prepared by the method as previously differentiation of antibody avidity after virus [4,5] infection, i.e. rubella, rota and Japanese reported . encephalitis viruses. They estimated the The IgM capture ELISA was carried out antibody avidity to viral antigen using a low according to the method described concentration of a protein-denaturing agent, previously[3-5]. Anti-human IgM (µ chain guanidine hydrochloride, in the diluent of specific) goat serum and peroxidase- antibody in the ELISA. They concluded that conjugated anti-human IgM were purchased the “stringent immunosorption” technique from Sigma-Aldrich, Inc, USA. The IgG was useful for investigating the antigenic fraction of the flavivirus-specific monoclonal relationship among closely-related viruses. antibody, D1-4G2, was prepared by the In the present study, we examined Protein G affinity chromatography kit serotype-specific IgM responses under (ImmunoPure G IgG Purification kit, PIERCE, stringent conditions in the presence of a USA), and then conjugated with horseradish chaotropic agent, sodium thiocyanate peroxidase by a commercial kit (EZ-Link Plus (NaSCN), in the reaction mixture of dengue Activated Peroxidase kit, PIERCE, USA). viral antigens and patients’ sera. The In order to detect the serotype-specific development of a simple method to reaction between dengue viral antigen and distinguish serotype-specific reaction from dengue virus-specific IgM antibody, patient cross-reaction will be useful not only for serum was treated with the Protein G affinity laboratory diagnosis but also for chromatography kit described above. The seroepidemiological studies. IgG fraction in the serum specimen was removed by adding Protein G beads Materials and methods according to the manufacturer’s instruction. Unless otherwise stated, data were presented Twenty-eight serum specimens from 18 as the mean of independent two-to-three confirmed Japanese dengue cases were used assays. in the study. Serum samples from 22 Japanese subjects with other illnesses, who had never been to areas where dengue was Results and discussion epidemic-prone or endemic, were used as One of the authors has previously reported the control. These sera were obtained for that the addition of NaSCN to the reaction diagnostic purposes in clinics and hospitals in mixture of ELISA highlights serotype-specific Dengue Bulletin – Vol 28, 2004 119 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN reaction between crude dengue viral antigen reactive one. According to these results, we and anti-dengue hyperimmunized mouse decided to use NaSCN in IgM-capture ELISA sera[11]. The antigen-antibody reaction was at the following conditions: (i) NaSCN was affected not only by the concentration of included at a final concentration of 0.5 M in NaSCN but also by the procedures of the 10% normal calf serum-PBS; (ii) peroxidase- NaSCN treatment. A concentration higher conjugated flavivirus- specific monoclonal than 0.7 M inhibited the reaction non- antibody D1-4G2 (D1-4G2) was diluted in specifically, while a concentration lower than 0.5 M NaSCN; and (iii) viral antigens 0.3 M had no effect on the discrimination of captured by patients’ IgM were detected by serotype-specific reaction from the cross- the detection antibody in 0.5 M NaSCN. Figure 1. The effects of NaSCN treatment on antigen-antibody reaction (a) 1.5 Control NaSCN 1 ELISA O.D. 0.5 0 50 60 70 80 90100 200 300 400 Serum dilution (b) 2 Control NaSCN 1.5 1 ELISA O.D. 0.5 0 1 2 3 4 5 6 7 8 Antigen dilution (a) Human serum was serially diluted from 1:50 to 1:400 with 10% CS-PBS containing 0.5 M NaSCN, and IgM was captured on anti-human IgM goat serum-coated wells. IgM was detected with peroxidase-conjugated anti-human IgM goat serum. (b) Serially diluted tetravalent dengue viral antigen was captured on the solid phase sensitized with D1-4G2 IgG, and then detected with peroxidase-conjugated D1-4G2 IgG in the presence of 0.5 M NaSCN. 120 Dengue Bulletin – Vol 28, 2004 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN Figure 1 shows the reactions between with the Protein G beads (UltraLink anti-human IgM and patients’ IgM (a), and Immobilized Protein G Plus, capacity: ~25 dengue viral antigen and the detection mg IgG/ml of the gel, PIERCE). The levels of antibody (b), in the presence of 0.5 M cross-reaction between dengue viral antigens NaSCN in the ELISA. These two reactions and patients’ IgM antibody (a) were were not affected by 0.5 M NaSCN, decreased after the treatment with 0.5 M suggesting that IgM capture and detection of NaSCN in the ELISA (b): The level of reaction dengue viral antigens were not affected in with homologous dengue-1 antigen was less IgM-capture ELISA. affected. The results suggested that the Figure 2 shows anti-dengue IgM titration addition of 0.5 M NaSCN to the reaction curves in the presence or absence of 0.5 M mixture of ELISA may highlight a serotype- NaSCN in the ELISA. The IgG fraction in the specific reaction between crude dengue viral serum specimen was removed by adsorption antigen and anti-dengue IgM antibody. Figure 2. Titration curves of dengue virus specific IgM antibody in the presence or absence of 0.5 M NaSCN in the ELISA (a) 2 v.s.D1 Ag v.s.D2 Ag v.s.D3 Ag v.s.D4 Ag 1.5 1 0.5 0 100 1000 Serum dilution (b) 2 1.5 1 0.5 0 100 1000 Serum dilution A serum specimen obtained from the dengue virus type 1-infected patient was used in the study. The IgG fraction was removed according to the method described in the Materials and Methods section. Protein G-treated serum specimen was serially diluted from 1:20 to 1:2560 in the absence (a) and in presence of 0.5 M NaSCN (b) in 10% CS-PBS, and then reacted on the plates coated with each of 4 dengue viral antigens. Dengue virus-specific IgM antibody was detected with 1:500 diluted peroxidase-conjugated anti-human IgM goat serum. Dengue Bulletin – Vol 28, 2004 121 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN We examined the IgM levels by ELISA all the tested cases, the serotype-specific with the antigen of 4 dengue virus serotypes. IgM levels were the highest against the The table below shows the results of 28 infecting dengue virus serotype than against serum samples from 18 Japanese dengue three other serotypes in the presence of patients. The infecting dengue virus NaSCN. The serotype-specific IgM serotypes were determined by RT-PCR. The responses were more highlighted in the data were presented as the index value presence of NaSCN than in its absence. according to the method described These data suggest that IgM responses to the previously[4]. We defined index values of infected dengue virus serotype determined 2.28 or greater and 26.60 or greater as by RT-PCR in primary dengue infection positive, respectively, in the absence and were the highest. These results agreed with presence of 0.5 M NaSCN in the ELISA. In the report by Burke[6]. Table. Serodiagnosis of dengue by IgM-ELISA with and without 0.5M NaSCN Index values* Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60) D1 D2 D3 D4 D1 D2 D3 D4 1 D3 4 1.20 1.47 2.29 1.42 5.33 3.67 45.67 5.67 9 8.58 7.73 16.40 10.46 110.30 47.67 471.67 273.00 2 D2 7 4.89 52.18 7.96 4.49 128.00 1,246.00 670.00 239.00 14 5.27 37.93 8.87 5.42 68.00 395.00 243.70 179.00 3 D3 6 5.34 3.34 9.60 2.35 50.25 15.00 283.00 39.50 17 6.79 7.07 12.38 4.00 78.00 46.25 413.25 108.50 4 D3 8 5.61 2.84 9.20 2.63 67.25 12.75 305.50 77.00 10 4.77 3.16 9.10 3.45 54.00 14.00 274.20 97.20 5 D2 6 1.13 4.77 1.71 0.81 2.78 9.36 8.00 0.00 13 2.65 12.31 4.49 2.15 10.27 46.27 28.73 14.45 6 D3 6 3.07 1.75 6.55 0.89 11.83 4.00 140.33 2.50 8 4.06 2.40 9.23 1.34 21.00 6.83 198.33 11.83 7 D1 5 2.76 1.46 6.33 1.39 7.86 0.29 20.71 0.00 7 13.51 6.65 11.49 3.67 127.14 72.86 11.43 25.71 8 16.91 9.53 13.28 4.43 143.37 100.00 20.37 42.00 14 24.51 11.49 9.86 3.63 214.50 68.25 34.00 51.13 122 Dengue Bulletin – Vol 28, 2004 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN Index values* Patient RT-PCR Disease day Without NaSCN (cut off = 2.28) With 0.5M NaSCN (cut off = 26.60) D1 D2 D3 D4 D1 D2 D3 D4 8 D1 7 1.73 3.89 1.66 1.45 5.54 6.38 6.00 1.62 11 6.95 10.19 2.27 1.76 26.42 20.25 19.00 10.17 9 D2 5 1.93 3.02 1.28 0.72 1.80 4.70 3.90 0.00 10 D2 ND 0.75 2.98 0.92 0.28 2.13 4.63 3.75 0.00 11 D1 5 1.25 1.28 1.32 0.69 4.00 1.57 10.71 0.00 12 D1 ND 20.35 15.44 15.02 8.00 161.28 36.71 39.90 97.71 13 D1 19 18.37 10.69 10.02 12.75 244.43 22.86 138.14 135.00 14 D1 8 7.55 6.78 6.41 3.37 50.25 11.67 27.92 19.92 15 D4 19 1.12 1.44 1.19 2.41 2.56 3.44 7.22 35.44 16 D1 5 1.36 1.13 1.27 0.27 6.27 1.45 2.45 0.00 17 D1 6 4.74 2.01 5.20 0.98 58.67 3.71 17.58 0.00 18 D1 15 14.18 8.39 8.52 3.58 137.38 33.27 31.00 49.88 D1 to D4, dengue virus type 1 to 4, respectively. ND, not determined. * index values were calculated by the formula A492 with the viral antigen/A492 with uninfected control antigen. The chaotropic agent, NaSCN, is known represent the envelope glycoprotein and NS1, to denature protein structure and inhibit the respectively[11]. Trent et al.[15,16] observed formation of immune complexes as was three antigenic determinants on the envelope exploited for immunoaffinity glycoprotein: (i) flavivirus group- reactive; chromatography[12,13]. There are multiple (ii) complex-specific; and (iii) serotype- factors which affect NaSCN’s ability to specific. Henchal et al.[17] characterized four highlight serotype-specific reaction in antigenic determinants on the envelope comparison with cross-reaction in the ELISA. glycoprotein: (i) flavivirus group-reactive; One of the factors is a characteristic of (ii) dengue complex-specific; (iii) dengue dengue viral antigens, which are prepared subcomplex-specific; and (iv) dengue from the infected mosquito cell culture. It serotype-specific. was reported that a rapidly sedimenting Brandt et al.[14] reported that the DEN-2 haemagglutinin (RHA) was cross-reactive and SCF antigen extracted from infected mouse labile, but the soluble complement-fixing brains was resistant to the treatment with antigen (SCF) was serotype-specific and protein denaturing agents. Falconar and relatively stable against the NaSCN treatment Young[18] reported serotype-specific epitopes in the ELISA[11,14]. The predominant on NS1. These results suggest that polypeptides in RHA and SCF fractions Dengue Bulletin – Vol 28, 2004 123 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN heterologous antigenic determinants on reactive IgM. This procedure may be useful dengue viral antigens contribute to the cross- for determining infecting dengue virus reactivity among the four dengue serotypes, serotypes, especially in primary dengue virus and that the presence of a chaotropic agent infection. in the reaction mixture induces changes of viral antigens and may decrease the cross- reactive antigenicity. Acknowledgement In conclusion, we demonstrated that the This work was supported by a grant for addition of NaSCN to IgM-capture ELISA research on emerging and re-emerging highlighted the detection of serotype-specific infectious diseases from the Ministry of IgM in comparison with serotype cross- Health, Labour and Welfare, Japan. References [1] Hotta S. Twenty years of laboratory [6] Burke DS. 1983. Rapid methods in the experience with dengue virus. In Saunders laboratory diagnosis of dengue virus M and Lennnet EH (Eds.), 1965, Medical infections. In T Pang and R Pathmananathan and Applied Virology. Green, St Louis: 228- (Eds.), Proceedings of the International 256. Conference on Dengue/Dengue Hemorrhagic Fever. University of Malaya, [2] Yamada K, Takasaki T, Nawa M, Nakayama Kuala Lumpur, Malaysia, pp. 72-84. M, Arai TY, Yabe S and Kurane I. The features of imported dengue fever cases [7] Gubler DJ. Dengue and dengue from 1996 to 1999. Jpn J Infect Dis, 1999, hemorrhagic fever. Clin Microbiol Rev, 1998, 52: 257-259. 11: 480-496. [3] Yamada K, Nawa M, Takasaki T, Yabe S and [8] Innis BL. Antibody responses to dengue virus Kurane I. Laboratory diagnosis of dengue infection. In DJ Gubler and G Kuno (Eds.), virus infection by reverse transcriptase Dengue and dengue hemorrhagic fever, polymerase chain reaction (RT-PCR) and Wallingford: CAB International, 1997: 221- IgM-cpature enzyme-linked immunosorbent 243. assay (ELISA). Jpn J Infect Dis, 1999, 52: [9] Inouye S, Hasegawa A, Matsuno S and 150-155. Katow S. Changes in antibody avidity after [4] Nawa M, Yamada K, Takasaki T, Akatsuka T virus infections: detection by an and Kurane I. Serotype-cross-reactive immunosorbent assay in which a mild immunoglobulin M responses in dengue protein-denaturing agent is employed. J Clin virus infections determined by enzyme- Microbiol, 1984, 20: 525-529. linked immunosorbent assay. Clin Diag Labo [10] Nawa M, Takasaki T, Yamada K, Kurane I Immunol, 2000, 7: 774-777. and Akatsuka T. Development of IgM- [5] Nawa M, Takasaki T, Yamada K, Akatsuka T cpature enzyme-linked immunosorbent and Kurane I. Development of dengue IgM- assay for serodiagnosis of dengue using beta- cpature enzyme-linked immunosorbent propiolactone-inactivated dengue viral assay with higher sensitivity using antigens. Dengue Bulletin, 2003, 27: 95-99. monoclonal detection antibody. J Virol Methods, 2001, 92: 65-70. 124 Dengue Bulletin – Vol 28, 2004 Detection of Dengue Virus Serotype-specific IgM by IgM Capture ELISA in the Presence of NaSCN [11] Nawa M. An enzyme-linked immunosorbent [16] Trent DW. Antigenic characterization of assay using a chaotropic agent (sodium flavivirus structural proteins separated by thiocyanate) for serotype specific reaction isoelectoric focusing. J Virol, 1977, 22: 608- between cude dengue viral antigen and anti- 618. dengue mouse antibody. Microbiol [17] Henchal EA, Gentry MK, McCown JM and Immunol, 1992, 36: 721-730. Brandt WE. Dengue virus-specific and [12] Dandlinker WB and de Saussure VA. flavivirus group determinants identified with Antibody purification at neutral pH utilizing monoclonal antibodies by indirect immunospecific adsorbents. Immunochemistry, immunofluorescence. Am J Trop Med Hyg, 1968, 5: 357-365. 1982, 31: 830-836. [13] Avrameas S and Ternyck T. Use of iodide [18] Falconar AKI and Young PR. Production of salts in the isolation of antibodies and the dimer-specific and dengue virus group cross- dissolution of specific immune precipitates. reactive mouse monoclonal antibodies to Biochem J, 1976, 102: 37c-39c. the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol, 1991, 72: [14] Brandt WE, Cardiff RD and Russell PK. 961-965. Dengue virions and antigens in brain and serum of infected mice. J Virol, 1970, 6: 500-506. [15] Trent DW, Harvey CL, Qureshi A and Lestourgeon D. Solid-phase radioimmunoassay for antibodies to flavivirus structural and nonstructural proteins. Infect Immunol, 1976, 13: 1325-1333. Dengue Bulletin – Vol 28, 2004 125 Genetic Influences on Dengue Virus Infections J.F.P. Wagenaar#, A.T.A. Mairuhu and E.C.M. van Gorp Department of Internal Medicine, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands Abstract Dengue virus infections are an important cause of morbidity and mortality in the tropics, with 100 million people infected annually and an estimated 2.5 billion people at risk. Human infections can be asymptomatic or can manifest as the self-limited febrile dengue fever, or the more severe and life- threatening dengue haemorrhagic fever (DHF). There are several possible reasons why some infected individuals might develop a more severe form of the disease than others. Antibody enhancement and viral virulence have been implicated in the pathogenesis of DHF but host genetic factors may also be relevant and predispose some individuals to DHF. This review discusses the possible involvement of a variety of genetic polymorphisms on the course of dengue virus infections. It has been shown that several common genetic polymorphisms can influence the susceptibility to dengue haemorrhagic fever. Gene polymorphisms concerning human leucocyte antigens, antibody receptors, inflammatory mediators and other factors with immunoregulatory effects are described. The study of genetic polymorphisms might provide important insights into the pathogenesis of a more severe disease and could have an impact on the design of future vaccines. Keywords: Dengue haemorrhagic fever, genetic polymorphisms. Introduction headache and muscle and joint pain. Occasionally, dengue virus infections Dengue has become one of the most progress to dengue haemorrhagic fever important arthropod-borne diseases in (DHF) and dengue shock syndrome (DSS). tropical and subtropical regions of the world. These are potentially life-threatening Approximately 100 million cases of dengue illnesses characterized by haemorrhagic infections occur annually, and an estimated manifestations and the loss of plasma from 2.5 to 5 billion people are at risk of dengue the vascular compartment, which may give virus infection[1]. The four serotypes of rise to shock in severe cases. dengue virus (DEN-1, 2, 3 and 4) are Central in the pathogenesis of DHF and transmitted to humans through the bite of an infective female Aedes mosquito and DSS is the loss of endothelial integrity that is believed to be the result of an abnormal may result in dengue fever (DF), an acute viral infection characterized by fever, rash, immune response and a disturbance in # E-mail: [email protected]; Tel.: + 31-(0)20-5124591; Fax: + 31-(0)20-5124783 126 Dengue Bulletin – Vol 28, 2004 Genetic Influences on Dengue Virus Infections immune regulation. Elevated levels of inflammatory response is related to viral several cytokines and chemical mediators, virulence. Several studies have found that which cause capillary leakage and may lead infection with DEN-2 caused more severe to shock, have been found in those suffering disease than other serotypes, suggesting that from DHF and DSS. Replication of dengue the virus phenotype influences the viruses occurs primarily in mononuclear outcome[7,8,11]. In addition, genetic variations phagocytes, which are a major source of within a specific serotype may also account tumor necrosis factor (TNF)-a and other for differences in disease severity, although vasoactive inflammatory mediators. Several reports remain scanty[14]. a studies have demonstrated that TNF- and Hyperendemic transmission of multiple other cytokines that are produced DEN serotypes in a Haitian population and a downstream of TNF- in the inflammatory the apparent absence of DHF and DSS, in cascade, e.g. IL-1b, IL-6 and IL-8, are addition to the observation that black people [2-4] related with disease severity . Other were hospitalized less frequently with DHF inflammatory mediators, like IL-2 and and DSS than the whites during epidemics in interferon (IFN)-g, are released from T Cuba, led to the hypothesis that human lymphocytes that are activated during genetic factors, e.g. gene mutations and gene dengue virus infections. The levels of these polymorphisms, may contribute to variable cytokines are significantly higher in DHF susceptibility[15-17]. Genetic polymorphisms [5,6] and DSS patients than in DF patients . are stable gene variants that usually have There are several possible reasons why minor effects on the regulation or function of some infected individuals might produce a proteins. These subtle chances might very greater inflammatory response than others. well have important consequences for [18] The most favoured hypothesis concerns the susceptibility to the disease . Several studies antibody-dependent enhancement theory. have confirmed that some genetic Several epidemiological studies demonstrate polymorphisms may protect or predispose an that prior infection with a different viral individual to DHF and DSS. Understanding serotype constitutes the largest risk factor for the molecular basis for these differences in DHF[7-11]. In vitro studies demonstrated that susceptibility should provide useful insight in the presence of anti-dengue antibodies at the pathogenesis of DHF and DSS and aid in sub-neutralizing concentrations augment the development of effective therapies and dengue virus infection of Fcg receptor- vaccines. This review attempts to describe positive cells, such as monocytes[12,13]. Based the current knowledge of the role of genetic on these epidemiological and laboratory influences on dengue virus infections. observations, it has been hypothesized that dengue cross-reactive antibodies may increase the number of dengue virus- Human leucocyte antigen infected monocytes during secondary genes infections, and lysis of these dengue virus- The function of the human leukocyte infected monocytes may lead to DHF and antigens (HLAs), encoded by the major DSS. Another possibility why some infected histocompatibility complex (MHC) and individuals might produce a greater whose genes are on chromosome 6, are to Dengue Bulletin – Vol 28, 2004 127 Genetic Influences on Dengue Virus Infections display antigen-proteins to antigen receptors HLA gene regions influence peptide epitope of host T-lymphocytes in order to activate binding[18]. A number of studies have looked cellular host immune responses. HLA genes at the variation in HLA genes and found show great variability and it could well be some of them to be associated with the that specific polymorphisms seen in human severity of dengue virus infections (Table). Table. Effect of HLA and non-HLA alleles on the severity of dengue virus infections Alleles Class Effect Population Reference HLA alleles Class I A1 Susceptibility Cubans 20 A2 Susceptibility Thai 19, 22 A*0203 Protective Thai 22 A*0207 Susceptibility Thai 22 A24 Susceptibility Vietnamese 21 A29 Protective Cubans 20 A33 Protective Vietnamese 21 B blank Susceptibility Cubans/Thai 19, 20 B13 Protective Thai 19 B14 Protective Cubans 20 B44 Protective Thai 22 B46 Susceptibility Thai 22 B51 Susceptibility Thai 22 B52 Protective Thai 22 B62 Protective Thai 22 B76 Protective Thai 22 B77 Protective Thai 22 Class II DRB1*04 Resistance Mexicans 29 Non-HLA alleles Fc gamma-receptor Resistance Vietnamese 30 Vitamin D receptor Resistance Vietnamese 30 HLA class I Polymorphisms in this class I region gene were found to be associated with DHF HLA class I alleles consist of HLA-A, -B, and - disease susceptibility. Chiewslip and Paradoa C; its products have a wide distribution and were the first to report an association are present on the surface of all nucleated between HLA class I and the severity of cells and on platelets. Antigens associated dengue virus infection[19,20]. In two ethnically with HLA class I products will interact with and geographically distinct populations CD8 cells during an immune response. evidence was presented suggesting that HLA- 128 Dengue Bulletin – Vol 28, 2004 Genetic Influences on Dengue Virus Infections A1, HLA-A2 and HLA-B blank increased in haemorrhagic fever[23]. HLA-B52 showed a frequency in DHF patients. A negative strong association with less severe DF. The relationship was found for HLA-B13, HLA- reduced frequency of the HLA-B15 group of B14 and HLA-A29. However, these studies serotypes, including HLA-B62, B76 and B77, had a small sample size and additional in patients with secondary infections, suggests studies with a larger number of patients were that they may be protective against needed. Subsequently, a large case control developing clinical disease in study in 560 study subjects was performed, immunologically primed individuals. By which mainly confirmed the observations contrast, HLA-B46 that also belongs to the made by the two previous studies that HLA HLA-B15 group of serotypes, was increased class I was important[21]. The data in DHF patients with secondary infections. demonstrated that polymorphisms in the Since HLA-B46 is in strong equilibrium with HLA class I region, particularly of the HLA-A HLA-A*0207, it is believed that the effect of gene, were significantly associated with B46 was likely to be an adjunct to that of susceptibility to DHF. Of the 15 alleles A*0207. Finally, HLA-B44 appeared also to studied, two particular alleles were relevant: be protective against the development of patients with HLA-A33 were less likely to severe disease in patients with secondary develop DHF (odds ratio 0.56; 95% dengue virus infections. confidence interval 0.34-0.39), whereas those with HLA-A24 were at an increased HLA class II risk to develop DHF (odds ratio 1.54; 95% confidence interval 1.05-2.25). The HLA-B Class II HLA products consist of HLA-D, -DR, alleles were not associated with DHF disease -DP, and -DQ; they have a more limited susceptibility. distribution on B-cells, macrophages, dendritic cells, Langerhans cells and activated Another case control study, in a Thai T cells. HLA class II alleles have shown to population, also demonstrated that the HLA- play a role in mycobacterial diseases, and A2 locus serotype was associated with their association with hepatitis clearance is disease susceptibility[22]. HLA-A*0203 was also established[24-26]. HLA-DRB1, which is associated with the less severe DF, whereas one of the most polymorphic loci of the HLA HLA-A*0207 was associated with complex in the Mexican population[27,28], was susceptibility to the more severe DHF. studied in Mexican patients suffering from a Interestingly, these associations were only dengue virus infection[29]. Although the found in immunologically primed persons, sample size was relatively small, the but not in immunologically naïve patients investigators found that the frequency of with primary infection. Dengue virus-specific HLA-DRB1*04 was lower in DHF patients. associations were also observed within the Persons homozygous for DRB1*04 were less HLA-B5 group of related alleles, whereby likely to develop DHF than persons who molecularly-determined HLA-B51 alleles were DRB1*04 negative (odds ratio 0.28; were associated with the development of 95% confidence interval 0.12-0.66), DHF after secondary infections. HLA-B51- suggesting a protective effect. The envelope restricted CTL responses to a variety of protein (E) of the virus is responsible for viral viruses have been described, including entry into target cells. The immunological Hantaan virus which also causes a determinants of protein E are probably Dengue Bulletin – Vol 28, 2004 129 Genetic Influences on Dengue Virus Infections processed and presented by HLA class II gene has been associated with antigens. The HLA-DRB1*04 molecule may meningococcal disease and recurrent present these viral antigens to CD4+ respiratory tract infections[33,34]. It chances the lymphocytes leading to an effective immune IgG binding affinity of the receptor with response and therefore protection from DHF. reduced opsonisation of IgG2 antibodies These findings are in contrast to the findings causally associated with the arginine variant. of Loke et al., who studied polymorphisms in Loke et al. found that homozygotes for the the HLA-DRB1 gene but did not find an arginine variant at position 131 of the FcgRIIA [21] association . gene may be less susceptible to DHF[30]. HLA class III Vitamin D receptor (VDR) Genes in the class III region encode a This gene mediates the immuno-regulatory number of proteins, including complement effects of 1.25-dihydroxyvitamin D3, which proteins (C4A, C4B, C2 and Bf), TNF-a and include activating monocytes, stimulating TNF-b[18]. Loke and colleagues studied cellular immune responses and suppressing promotor polymorphisms in the TNF-a gene immunoglobulin production and lymphocyte but did not find an association[21]. No other proliferation[35]. Recently the tt genotype of a studies are reported to have studied HLA single nucleotide polymorphism at position class III polymorphisms. 352 of the VDR gene has been associated with tuberculoid leprosy, enhanced clearance of HBV infection and resistance to Non-HLA host genetic factors pulmonary tuberculosis[36,37]. Expression of The number of studies on polymorphisms VDR may affect susceptibility to DHF since within non-HLA genes remains low. Loke activated B and T lymphocytes express VDR and colleagues investigated the association and 1,25D3 affects monocytes, the main sites [12] between susceptibility to DHF and of dengue virus infection and replication . polymorphic non-HLA alleles: vitamin D The t allele at position 352 of the vitamin D receptor (VDR), Fcg receptor II (FcgRII), receptor (VDR) gene was associated with Interleukin-4 (IL-4), Interleukin-1 repeat resistance to severe dengue, although the alleles (IL-1RA), and mannose-binding lectin exact mechanism needs to be explored. (MBL)[30]. Two of the five genes assessed showed evidence of association with altered Interleukin-4 (IL-4) risk of severe dengue. IL-4, primarily produced by Th2 subset of CD4+ T-cells, regulates B-cell growth, IgG Fcg receptor class switching and suppresses Th1-type [38,39] The Fcg receptor is a widely distributed responses as well . Since this gene affects receptor for all subclasses of IgG and is able both antibody responses and inflammatory to mediate antibody dependent responses during disease, IL-4 promotor enhancement in vitro by binding to virus-IgG polymorphisms were studied in order to find complexes[31,32]. An arginine to histidine a relationship in susceptibility to DHF. substitution at position 131 of the FcgRIIA However, no associations were found in this context[30]. 130 Dengue Bulletin – Vol 28, 2004 Genetic Influences on Dengue Virus Infections Interleukin-1 repeat allele (IL-1RA) infections but also of viral infections in general. The finding of a protective IL-1RA was thought to be a good candidate association with particular HLA or non-HLA- gene as well because IL-1RA is involved in types may encourage the design of future the regulation of IL-1-mediated inflammatory vaccines, whereas polymorphisms associated responses by competitive binding to IL- with the susceptibility to develop a more [40] receptors . But no significant difference severe disease may help to identify certain could be found in the DHF group in addition risk groups in a population. It is therefore of [30] to the controls . great importance to stimulate the study of the interaction of single and multiple Mannose-binding lectin (MBL) polymorphisms in severe dengue virus Several mutations in the MBL gene, which infections. encodes for a protein involved in the The few studies performed thus far have activation of the classical complement demonstrated that host genetic factors can be [41,42] pathway , have been associated with a important in susceptibility to DHF. It is most marked reduction in serum MBL levels and likely that classical HLA class I and class II [43,44] MBL-mediated complement activation . gene products play a crucial role in Polymorphisms in this gene were not proved determining the severity of dengue virus to have any effect on the susceptibility to infections. Two polymorphic non-HLA alleles, DHF. However, this variant allele was the FcgRII receptor and VDR, could also play relatively low in the observed population, an important role in susceptibility to DHF. which limits the statistical power of the Some polymorphic HLA alleles were [30] analysis . observed in several studies, e.g. HLA-A2 in a Thai and Vietnamese population, but differences in susceptibility to DHF were Discussion and future observed[19,21,22]. An explanation for the perspective observed difference may be that a genetic polymorphism is more frequent in a The number of candidate susceptibility and population whereas another is relatively protective genes is expanding rapidly, but infrequent. Overall, such disease associations what is the use of studying these genes in warrant further analysis, but also emphasize relation to DHF? 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American Journal of (CTL) responses to Hantaan virus infection: Tropical Medicine and Hygiene, 2002, 67: identification of virus-specific and cross- 102-106. reactive CD8(+) CTL epitopes on nucleocapsid protein. Journal of Virology, [31] van de Winkel JG and Capel PJ. Human IgG 1999, 73: 5301-5308. Fc receptor heterogeneity: molecular aspects and clinical implications. Immunology Today, [24] Abel L and Dessein AJ. Genetic epidemiology 1993, 14: 215-221. of infectious diseases in humans: design of population-based studies. Emerging [32] Littaua R, Kurane I and Ennis FA. Human Infectious Diseases, 1998, 4: 593-603. IgG Fc receptor II mediates antibody- dependent enhancement of dengue virus [25] McNicoll J. Host genes and infectious infection. Journal of Immunology, 1990, diseases. Emerging Infectious Diseases, 1998, 144: 3183-3186. 4: 423-426. [33] Fijen CA, Bredius RG and Kuijper EJ. [26] Thursz M. MHC and the viral hepatitides. Polymorphism of IgG Fc receptors in The Quarterly Journal of Medicine, 2001, meningococcal disease. Annals of Internal 94: 287-291. Medicine, 1993, 119: 636. Dengue Bulletin – Vol 28, 2004 133 Genetic Influences on Dengue Virus Infections [34] Sanders LA, van de Winkel JG, Rijkers GT, [40] Dinarello CA and Thompson RC. Blocking Voorhorst-Ogink MM, de Haas M, Capel PJ IL-1: interleukin 1 receptor antagonist in and Zegers BJ. Fc gamma receptor IIa vivo and in vitro. Immunology Today, 1991, (CD32) heterogeneity in patients with 12: 404-410. recurrent bacterial respiratory tract [41] Ohta M, Okada M, Yamashina I and infections. The Journal of Infectious Diseases, Kawasaki T. The mechanism of 1994,170: 854-861. carbohydrate-mediated complement [35] MacDonald PN, Dowd DR and Haussler MR. activation by the serum mannan-binding New insight into the structure and functions protein. The Journal of Biological Chemistry, of the vitamin D receptor. Seminars in 1990, 265: 1980-1984. Nephrology, 1994,14: 101-118. [42] Lipscombe RJ, Sumiya M, Summerfield JA [36] Roy S, Frodsham A, Saha B, Hazra SK, and Turner MW. Distinct physicochemical Mascie-Taylor CG and Hill AV. Association characteristics of human mannose binding of vitamin D receptor genotype with leprosy protein expressed by individuals of differing type. The Journal of Infectious Diseases, genotype. Immunology, 1995, 85: 660-667. 1999, 179: 187-191. [43] Madsen HO, Garred P, Kurtzhals JA, Lamm [37] Bellamy R, Ruwende C, Corrah T, McAdam LU, Ryder LP, Thiel S and Svejgaard A. A KP, Thursz M, Whittle HC and Hill AV. new frequent allele is the missing link in the Tuberculosis and chronic hepatitis B virus structural polymorphism of the human infection in Africans and variation in the mannan-binding protein. Immunogenetics, vitamin D receptor gene. The Journal of 1994, 40: 37-44. Infectious Diseases, 1999, 179: 721-724. [44] Super M, Gillies SD, Foley S, Sastry K, [38] Paul WE and Seder RA. Lymphocyte Schweinle JE, Silverman VJ and Ezekowitz responses and cytokines. Cell. 1994, 76: RA. Distinct and overlapping functions of 241-251. allelic forms of human mannose binding protein. Nature Genetics, 1992, 2: 50-55. [39] Swain SL. T-cell subsets. Who does the polarizing? Current Biology, 1995, 5: 849- 851. 134 Dengue Bulletin – Vol 28, 2004 Identification and Phylogenetic Analysis of DEN-1 Virus Isolated in Guangzhou, China, in 2002 Jun-lei Zhang, Rui Jian, Ying-jie Wan, Tao Peng and Jing An# Department of Microbiology, College of Medicine, Third Military Medical University, Chongqing, 400038, People’s Republic of China Abstract Virus was isolated and identified from the serum samples of patients with suspected classical dengue fever (DF) in Guangzhou province, China, in 2002. The serum was incubated with the Aedes albopictus cell line, C6/36, for isolation and 5 of 20 serum samples caused cytopathologic effects on C6/36 cells. A 539-nucleotide (nt) fragment in the NS1 region of the isolated virus genome was amplified using universal primers of 4 serotypes of DEN viruses. By sequencing the primer-extension production and blasting in the GenBank, the isolated strain was the closest to rDEN-1 dalte30 (AY145123), which was an attenuation strain of DEN-1 virus. A 593nt fragment from the Envelope-Nonstructural protein 1 (E/NS1) of these isolates was also sequenced to compare it with published sequences of other DEN-1 viruses. The 240nt from the E/NS1 region of this DEN-1 virus genome was the closest to DEN-1/T14 strain (M32931), which was genotype IV. The phylogenetic analysis of the NS1 (480nt) and E/NS1 (240nt) regions of the gene nucleotide sequences was performed using neighbour-joining methods: 11 strains of DEN-1 virus were divided into three genotypes: I, IV and V, as defined by Rico-Hesse, or Asia, South Pacific and Americas/Africa, as defined by Goncalvez. Based on the sequence information and the definition, the isolated virus (named DEN-1/GZ 2002) strain belonged to DEN-1 and IV or South Pacific genotypes. The suckling mice died on days 10 to 11 after intracerebral inoculation with the isolated virus. TCID50 of the virus was 4.37 log pfu/0.2ml. Small plaques with an unclear edge were seen on Vero cells on days 8 to 9 after infection. Combined with clinical data that thousands of patients only showed DF manifestations, the results suggested that DEN-1/GZ 2002 might be a low-virulence strain. Keywords: Dengue virus, DEN-1/GZ2002, virulence, phylogenetic analysis. Introduction RNA, approximately 11,000 nucleotides long, encoding 10 distinct proteins. The Dengue (DEN) viruses belong to the genus gene order is 5’-C-prM(M)-E-NS1-NS2A- Flavivirus. There are four closely related, but NS2B-NS3-NS4A-NS4B-NS5-3’, expressed antigenically distinct, virus serotypes (DEN-1, as a single polyprotein that is cleaved by DEN-2, DEN -3 and DEN-4). The viral both viral and cellular proteases to form the genome is a positive-sense single-stranded viral polypeptides. The three 5’ proteins are # E-mail: [email protected]; Tel.: +86-23-68752774; Fax: +86-23-65463259 Dengue Bulletin – Vol 28, 2004 135 Identification and Phylogenetic Analysis of Dengue-1 Virus three structural proteins: capsid (C), I) America, Africa and South-East Asia; membrane (M) and envelope (E), and the II) Sri Lanka; III) Japan; IV) South-East Asia, remaining seven are nonstructural (NS) the South Pacific, Australia and Mexico; and proteins[1]. The DEN viruses cause classical V) Taiwan and Thailand[14]. Recently, dengue fever (DF) and dengue Goncalvez set up the phylogeny of 36 DEN - haemorrhagic fever/dengue shock syndrome 1 viruses according full E gene sequences. (DHF/DSS). DF is a non-specific viral febrile Statistical analyses of the validity of illness while DHF/DSS is a severe and fatal branching patterns (bootstrap) also haemorrhagic disease. Up to 100 million suggested five classifications: 1) from Asia; cases of DF occur annually in the world, 2) from Thailand; 3) from sylvatic/Malaysia; with some 2 billion people at risk of the 4) from South Pacific; and 5) from the infection in tropical and subtropical regions Americas/Africa, in which three genotypes of Africa, Asia and the Americas[2,3]. indicated as 1), 4) and 5) were consistent to I), IV) and V) mentioned by Rico-Hesse[15]. The pathogenesis of the DEN virus Aviles-G and collaborators sequenced infection is not clear. Although many factors Capsid-pre Membrane (C/prM) and E/NS1 are proposed to be involved in the DEN regions of 24 recent isolates of DEN-1 from virus epidemiology and occurrence of South America, suggesting that the recent DHF/DSS, the evolution of the DEN viral epidemics in Argentina and Paraguay were virulence and the spread of DEN viruses in due to the re-emergence of a previously different geographical areas are two circulated strain[16]. This indicated that small important features in the epidemiology and gene fragment sequences are available for pathogenesis of the disease. Studies have analysis of viral genetic characterization. suggested that specific viral structures might contribute to the replication capability in Current methods for obtaining DEN the target cells and to their transmission[4]. virus sequences no longer require a viable Co-circulation of novel DEN genotype, virus isolate. In fact, the entire genome another genotype of the same serotype or sequences can be obtained by enzymatic different serotypes of DEN viruses in the amplification of viral RNA template in the same area, might lead to the displacement patient’s blood sample. Unfortunately, there of the native genotype by a new genotype are numerous sequences that contain errors, and thus to the occurrence of DHF/DSS[5]. which have led to serious mistakes in their interpretation. Therefore, it is wise to obtain Several studies have shown that the virus isolates for further genetic phylogenetic trees obtained from small gene characterization[17]. In this study, a viral fragment sequences are congruent with the strain that circulated in Guangzhou, China, trees obtained from an entire gene in 2002 was isolated first, then identified by sequence, although there might be minor amplifying and sequencing 539 nucleotide rearrangements in the terminal branches[6-13]. (nt) in NS1 region of DEN virus genome Using a partial sequence from Envelope- using universal primers. Also, E/NS1 regions Nonstructural protein 1 (E/NS1) junction of the isolated virus were sequenced to region, Rico-Hesse defined five genotypes determine their origin. for DEN-1 viruses isolated worldwide: 136 Dengue Bulletin – Vol 28, 2004 Identification and Phylogenetic Analysis of Dengue-1 Virus Materials and methods primer sequences are: 5'GTG CAC ACA TGG ACA GAA CA 3'(forward) and 5'CTT TCT ATC CAA TAA CCC AT 3'(reverse). Collection of blood samples Their combination generated a 539nt Twenty patients with suspected DEN virus fragment. Briefly, 5 µl of the extracted RNA infection admitted to the Eighth People’s was mixed with 50 pmol (2µl) of reverse Hospital of Guangzhou were enrolled – 12 primer at 70 °C for 5 minutes and cooled males and 8 females aged between 23-57 down on ice. Then 13 µl of RT mix years. The blood samples were collected on containing 4µl of 5X RT XL buffer, 0.5 µl of day 1 to 11 after the onset of fever. The sera 10mM dNTP, 6.0 µl of sterile UHQ water, were separated by centrifugation and stored 0.5µl RNase inhibitor and 20 U of AMV at –70 °C until use. reverse transcriptase XL (TaKaRa, Japan) were added. The mixture was incubated at Viral isolation 42 °C for 1 hour for RT reaction, then heated to 95 °C to inactivate AMV and Aedes albopictus mosquito cell line, C6/36, cooled down on ice. PCR was subsequently was cultured in RPMI 1640( pH6.8~ 7.0) carried out by adding 24 µl of PCR mix containing 10% fetal bovine serum (FBS) containing 2.5 µl of 10X Taq Polymerase and 2 mM glutamine with 5% CO2 at 28 °C. buffer (PROMEGA, USA), 2.5 µl of 25mM 30µ l of the serum sample was diluted with MgCl2, 12.5 pmol each of sense and reverse 1 ml RPMI 1640 containing 2% FBS and primers, 0.5 µl of 10mM dNTP, 2U of Taq was incubated with overnight C6/36 cells for DNA Polymerase (PROMEGA, USA) and 1 hour at 28 °C. After removing the serum, 17.5 µl of sterile UHQ water to the 1st the cells were cultured in RPMI 1640 strand synthesis tube containing 1 µl of ( pH6.8 ~ 7.0 ) containing 2% FBS at cDNA. PCR with denaturation at 94 °C for 28 °C. The cells were observed daily for 30 seconds; annealing at 53 °C for 30 cytopathic effect (CPE)[18]. If CPE was not seconds; and extension at 72 °C for 1 seen initially, the cells were passaged several minute 30 seconds for 30 cycles. The RT- times to confirm whether there was virus in PCR conditions for the amplification of the serum samples or not. nucleotides from positions 2107~2701 coding for the E/NS1 fragment were similar RNA extraction to those used for NS1 fragment, but with an annealing temperature of 58 °C instead of Viral RNA was extracted from the culture 53 °C. supernatants of infected C6/36 with ISOGEN (Nippon Gene, Toyama, Japan) DNA purification and sequencing according to the manufacturer’s instructions. PCR-amplified DNA products were RT-PCR amplification electrophoresed in 1% agarose gels and stained with 1µg/ml ethidium bromide. The Nucleotides from positions 2503~3041 bands of predicted size (539nt for the NS1 coding a fragment of the NS1 gene were fragment and 593nt for E/NS1 fragment) amplified using RT-PCR. The universal were excised from the gel and purified using Dengue Bulletin – Vol 28, 2004 137 Identification and Phylogenetic Analysis of Dengue-1 Virus a Golden Beads Product Purification Kit NS1 region and the E/NS1 junction of our (Songon, CN) according to the isolates were carried out with the neighbor- manufacturer’s instructions. Purified PCR joining method (NJ)[20], calculating bootstrap products were cloned on to pMD18-T confidence intervals of 1,000 replicates. Vector (TaKaRa, Japan) and sent to Co. Character state tree-building algorithms Bioasia, Shanghai, CN, in 15% Glycerol to (PHYLIP package) were also tested. A strict perform sequencing using an ABI Prism 377 consensus bootstrap tree was obtained by Genetic Analyzer. Amplifying primers were using the following programmes: M13-48. (i) SEQBOOT to generate 100 replicas, (ii) DNADIST and NEIGHBOR to acquire Computer analysis the tree of each reiterated data, and (iii) CONSENSE to build a strict consensus The multiple sequence alignment bootstrap tree. Phylogenetic trees were [19] programme Clustalx, version 1.8 , was drawn using TreeView[21]. Published used to obtain an optimal nucleotide sequences used in the analysis are listed in sequence alignment file. Phylogenetic Table 1. analysis of nucleotide sequences from the Table 1. Dengue viral sequences from GenBank used in the phylogenetic analysis Virus Strains Origin Year isolated Accession number DEN-1 FGA/89 French Guiana 1989 AF226687 DEN-1 BR/90 Brazil 1990 AF226685 DEN-1 S275/90 Singapore 1990 M87512 DEN-1 Abidjan Ivory Coast 1999 AF298807 DEN-1 WestPac/74 Nauru 1974 U88535 DEN-1 A88 Indonesia 1988 AB074761 DEN-1 GD05/99 Guangdong, CN 1999 AY376738 DEN-1 GD23/95 Guangdong, CN 1995 AY373427 DEN-1 GZ/80 Guangzhou, CN 1980 AF350498 DEN-1 Mochizuki Japan 1943 AB074706 DEN-1 Djibouti Djibouti 1998 AF298808 DEN-2 TR1751* Trinidad 1954 M32969 * The full-length gene segment sequence of this viral strain is unavailable in GenBank and the NS1 region in the phylogenetic analysis was sequenced by us 138 Dengue Bulletin – Vol 28, 2004 Identification and Phylogenetic Analysis of Dengue-1 Virus The resulting unrooted trees were out- Figure 1. Cultured C6/36 cells: grouped to the sequence of DEN-2 (TR1751), (a) C6/36 cells without DEN-1 infection; which was kindly provided by Dr Oya A (b) Cell-cell fusion and syncytia on C6/36 (National Institute of Infectious Diseases, cells was seen on day 6 after infection Japan) and isolated from a DF patient[22,23]. with DEN-1/GZ2002 The RT-PCR amplification of the NS1 region of this virus strain using universal primers and sequencing of amplified products was done as described above. Biological character detection The virus isolated from the serum samples was propagated in C6/36 cells (MOI = 1) and the titer was determined by the plaque assay using Vero cells monolayer culture Figure 1 (a) under methylcellulose overlay medium. TCID50 of the virus was also measured by the plaque assay and calculated using Reed- Muench method[24]. Eighteen suckling mice were intracerebrally (ic) inoculated with 104 pfu/mouse of the virus for observing clinical signs and their survival time. Results General observation Figure 1 (b) After two passages, 5 of the 20 serum Figure 2. Plaque formation on Vero cells specimens caused CPE on C6/36 cells. was detected on day 8 after infection Infected C6/36 cells became syncytia and A. Blank control; B. Vero cells were cell-cell fusion. Some cells showed necrosis infected with DEN-2/Tr1751; C and D. Vero and got detached from the plate at a late cells were infected with DEN-1/GZ2002 stage of the infection (Figure 1). The virus was propagated in C6/36 cells and the highest titer was 5×105pfu/ml at day 6 post- infection (pi). Using the Reed-Muench method, TCID50 of the virus was 4.37 log pfu/0.2ml. Plaque formation on Vero cells was detected on day 8 or 9 after infection and they were small and unclear (Figure 2). After ic inoculation with isolated virus, all of the suckling mice showed kyphoscoliosis and paralysis of hind legs and died on days 10 to 11 after infection. Dengue Bulletin – Vol 28, 2004 139 Identification and Phylogenetic Analysis of Dengue-1 Virus Comparison of nucleotide which was an attenuation strain of DEN-1 sequences virus. According to the full-length gene sequences of rDEN-1 dalte30 (AY145123), As shown in Figure 3, a 539nt fragment we designed the primers to amplify the from the NS1 region (positions 2503 to nucleotides of a fragment from the E/NS1 3041) was amplified using the universal region (positions 2107 to 2701). The 240nt primer by RT-PCR and the amplified from the E/NS1 region (position 2309 to products were sequenced to determine the 2548) of this DEN-1 virus genome is the relationships among the 5 isolates and their closest to DEN-1/T14 strain from Australia origin. Comparisons of the NS1 region of isolated in 1981 (M32931), which was the 5 isolates virus strains showed little genotype IV, their divergence was 2%. divergence, so we chose one as the Based on sequence information and representative strain to compare with the definition by Rico-Hesse, our isolates sequences published in GenBank. The belonged to DEN-1 and genotype IV, nucleotide sequence of this strain was the named DEN -1/GZ2002. closest to rDEN-1 dalte30 (AY145123), Figure 3. Agarose gel analysis of the cDNA products from RT-PCR of RNA samples isolated from the supernatant of the infected C6/36 cells (a) After amplification with the universal primer, a band of 539nt for the NS1 fragment was seen. Lanes 2-6: PCR amplified DNA products from different samples respectively; lane 7: blank control; lanes 1 and 8: 1Kb DNA Ladder marker (GeneRulerTM ) (b) After amplification with a primer for 593nt fragment of E/NS1 region, an expected band about 593nt was seen (lane 1), Lane 2: 1Kb DNA Ladder marker (GeneRulerTM ) Figure 3 (a) Figure 3 (b) 140 Dengue Bulletin – Vol 28, 2004 Identification and Phylogenetic Analysis of Dengue-1 Virus Phylogenetic analysis branches (Figure 4). From the NJ trees in our study, 11 strains of the DEN-1 virus were We compared 480nt from the NS1 region divided into three genotypes coinciding with (position 2516 to 2995) and 240nt from the I, IV and V as defined by Rico-Hesse or Asia, E/NS1 region (position 2309 to 2548) of this South Pacific and Americas/Africa, as DEN-1 virus genome with sequences of defined by Goncalvez. Our isolated DEN - other published DEN -1 viruses. The 1/GZ2002 strain belonged to genotype IV or phylogenetic analysis of the NS1 (480nt) South Pacific genotype. In addition, it was and E/NS1 (240nt) regions of the gene found that an arrangement of the S275/90 nucleotide sequences was performed using strain showed a major difference between NJ methods respectively. Two NJ trees were the two trees, which supports the previous set up and they showed a similar result, hypothesis that the S275/90 strain is a except for a little difference in terminal recombined strain[25]. Figure 4. Phylogenetic relationship of DEN-1/GZ2002 to previously characterized DEN-1 viruses. Two phylogenetic trees were set up using 240nt from the E/NS1 region, position 2309 to 2548 (a) and using 480nt from the NS1 region, position 2516 to 2995 (b) of these DEN-1 virus genomes. The resulting unrooted trees were out-grouped to the sequence of DEN-2 (TR1751) Discussion vector and the environment. DEN virus evolution is also determined by many It is evident that the DEN virus complex interactions. Some genetic changes epidemiology is determined by many factors, that occur during the natural transmission including those in the host, the virus, the cycles of the DEN virus might affect its Dengue Bulletin – Vol 28, 2004 141 Identification and Phylogenetic Analysis of Dengue-1 Virus virulence and cause the disease. Therefore, in DEN virus evolution and the NS1 understanding DEN virus variation is sequences were a highly conserved region. especially important to clarify the In our study, after sequencing E/NS1 (240nt) pathogenesis of DEN virus infection. and the NS1 (480nt) gene region, we Although current methods of obtaining DEN analysed the phylogenetic relationship of 11 virus sequences no longer require viable DEN-1 virus representatives of three virus isolates, virus isolates are necessary for genotypes coincidence with I, IV, and V as further studies of the biological and genetic referred to by Rico-Hesse (1990), or Asia, characteristics[17]. In this study, we isolated South Pacific and Americas/Africa, as DEN-1 virus that circulated in Guangzhou in defined by Goncalvez. This indicated that 2002 and analysed its possible origin and the isolated virus belonged to genotype partial biological features. IV/South Pacific. In an In vitro experiment, the virus Tolou completed the genome sequence caused typical CPE on C6/36 cells such as analysis to demonstrate the likelihood of syncytia and cell-cell fusion, which was recombination between different strains of continuously shown when the virus DEN-1 virus. The region of 1295~2592nt passaged several times. Their titer was considered to be a hot spot for the maintained a level about 5× 105 pfu/ml recombination[25]. We sequenced one during passages. On Vero cells, a small and region outside (2516~2995nt) and one unclear plaque, a biological marker of region inside (2309~2548nt) the possible attenuation, was seen. The suckling mice recombination site. No recombination event survived for 11 days after ic inoculation. occurred in our strains, at least in the Interestingly, the sequences of 539nt from regions analysed. However, no virus isolates the NS1 were very close to rDEN-1 dalte30 meeting the stringent criteria for (AY145123), which was an attenuation recombination have yet been described and strain of DEN-1 virus. Combined with it remains to be determined whether and at clinical data where thousands of patients what frequency DEN viruses undergo only showed DF clinical signs, our results recombination in nature[17]. indicated that the isolated virus might be a The evolution of DEN viruses has had a low-virulence strain. major impact on their virulence for humans As is known, a comparison of full-length and on the epidemiology of the DEN virus gene segment sequences is impractical, around the world. It is necessary that a more especially when looking at a large number complete and systematic survey of DEN-1 of samples. Recently, several studies have samples be undertaken before a link shown that the phylogenetic trees obtained between specific genotypes and the from small gene fragment sequences are virulence of these viruses can be established. highly congruent with those obtained from an entire gene sequence, except for a minor rearrangement in the terminal branches, Acknowledgment suggesting thereby that small gene fragment This work was partially supported by grants sequences were effective for the study of nos. 30170848 and 30300303 from the [6-13] viral genetic characteristics . The E/NS1 National Science Foundation of China junction sequences were a useful indicator (NSFC). 142 Dengue Bulletin – Vol 28, 2004 Identification and Phylogenetic Analysis of Dengue-1 Virus References [1] Trent DW, Manske CL, Fox GE, Chu MC, [9] Morzunov SP, Feldmann H, Spiropoulou CF, Kliks SC and Monath TP. 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TREEVIEW: An application to General Virology, 2001, 82: 1283-1290. display phylogenetic trees on personal computers. Computer Applications in the Biosciences: CABIOS, 1996, 12: 357-358. 144 Dengue Bulletin – Vol 28, 2004 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus – Derived, Modified Epitope Peptide, Using Dendritic Cells as a Peptide Delivery System Yoshiki Fujii*, Hideyuki Masaki**#, Takanori Tomura**, Kiyohiro Irimajiri* and Ichiro Kurane*** *Department of Pharmacotherapy, Kinki University School of Pharmaceutical Sciences, Higashi-Osaka, Osaka, Japan ** First Department of Biochemistry, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan ***Department of Virology 1, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan Abstract A single 9-amino acid peptide of the defined murine cytotoxic T lymphocyte (CTL) epitope (named peptide-1), which corresponds to the amino acid residues 298-306 (GYISTRVEM) of NS3 of dengue virus serotypes DEN-2 and 4, was examined for induction of specific CTLs. Immunization of BALB/c mice subcutaneously with the peptide-1 emulsified with complete Freund adjuvant (CFA) did not induce specific CTLs. The peptide-2 (GYISTRVEL), in which the residue (M) at 9th position of the peptide-1 was substituted for L, was prepared. The peptide-2 possessed the complete H-2Kd-binding motif. Intravenous immunization with 5x10 5 dendritic cells (DCs) pulsed with the peptide-2-induced specific CTLs. Furthermore, subcutaneous immunization with the peptide-2 emulsified with CFA-induced CTLs which lysed peptide-1-pulsed target cells as well as peptide-2-pulsed ones. These results indicate that immunization with dengue virus-derived CTL epitope peptide induces specific CTLs, and that DC can be used as a vehicle for the modified epitope peptide. Keywords: Dengue virus, cytotoxic T lymphocyte, dendritic cell, epitope, peptide, binding motif. Introduction have CTL epitope expressed in host cells by infection with live viruses, or by Major histocompatibility complex (MHC) administration of an expression plasmid class I – restricted, CD8+ cytotoxic T vector (i.e. DNA vaccine) in which the lymphocytes (CTLs) are known to play an epitope gene is incorporated. The other is essential role in the recovery from viral immunization with a defined CTL epitope infection by lysing virus-infected cells[1]. peptide. The former strategy is more There are two major strategies to induce physiological; however, the preparation of CTL-mediated protective immunity. One is to immunogen is often difficult and there is a # E-mail: [email protected]; Tel.: +81-72-366-0221, Ext.3152; Fax: +81-72-366-0206 Dengue Bulletin – Vol 28, 2004 145 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus potential risk that the immunogen may be the Animal Facility of Kinki University School pathogenic. Immunization with a CTL of Medicine under conventional conditions. epitope peptide is relatively easy; however, Mice were used at the age of 6 to 12 weeks. the epitope varies depending on T cell receptor repertoires and MHC class I Cells haplotypes. Furthermore, the peptide Murine mastcytoma line, P815 (H-2d), was administrated into the body may be used as target cells in CTL assays. The cells degraded or washed away soon. Thus, the were maintained in RPMI 1640 medium peptide is usually less immunogenic for CTL (Sigma, St. Louis, MO) with 5x10-5M 2- induction, and an appropriate delivery mercaptoethanol (2-ME), 100U penicillin, system is necessary for induction of CTLs[2,3]. 100mg/ml streptomycin, 10mM HEPES, and 10% heat-inactivated fetal calf serum Dendritic cells (DCs), which are potent (Complete medium) at 37 °C in 5% CO . antigen-presenting cells, are postulated to be 2 one of the peptide delivery systems for inducing CTLs[2,4]. It has been reported that Peptides intravenous immunization with DCs pulsed The peptide-1 (GYISTRVEM), which with virus-derived peptides, or tumour- corresponds to the amino acid residues 298- [5-7] derived peptides, elicits specific CTLs . 306 of NS3 of dengue virus types 2 and 4, Dengue viruses cause dengue fever and and the peptide-2 (GYISTRVEL) were dengue haemorrahgic fever/dengue shock synthesized with 9-fluorenylmethoxycarbonyl syndrome (DHF/DSS). Vaccine development chemistry by Sigma Genosis, Japan. The against dengue virus infection has not been purity was determined to be 95.0% for the accomplished yet. It is important to analyse peptide-1 and 96.2% for the peptide-2 by CTL responses elicited by a single epitope in reverse phase HPLC. order to understand the role of CTLs in dengue virus infection. In the present study, Induction of dendritic cells we employed a single 9-amino acid (a.a.) BALB/c mouse bone marrow cells (9x105) peptide, in which C-terminal residue was were cultured in 1ml of AIM-V medium replaced to provide the complete H-2Kd- (Invitrogen, Carlsbad, CA) supplemented with binding motif. This peptide was a derivative 20ng/ml mouse GM-CSF (R&D Systems, of the defined H-2Kd-restricted 9-a.a. CTL Minneapolis, MN) in 24-well plate at 37 °C epitope peptide that corresponds to the in 5% CO . On days 4 and 6, 50 to 75% residues 298-306 of NS3 of dengue virus 2 volume of the culture medium was changed types 2 and 4[8]. We examined whether with the fresh one supplemented with the intravenous immunization with bone same amount of GM-CSF. On day 7, cells marrow-derived DCs pulsed with this peptide were harvested, subjected to flow cytometry elicited specific CTL response. analysis and used as DCs for immunization. Materials and methods Flow cytometry analysis Bone marrow-derived cells (4x105) were Mice incubated with 1mg of FITC-conjugated anti- d d Female BALB/cAJcl mice were purchased mouse I-A /I-E antibody (BD PharMingen, from Clea, Japan, and were maintained in San Diego, CA), PE-conjugated anti-mouse 146 Dengue Bulletin – Vol 28, 2004 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus CD86 antibody (BD PharMingen), and and 10 U recombinant mouse IL-2 was biotinylated anti-mouse CD11c (BD added. On day 7, the cells were harvested PharMingen) in 100ml of phosphate buffered and used as CTL (cytotoxic T lymphocyte) saline (PBS) containing 0.02% NaN3 effector cells. Mice were also immunized by (PBS/NaN3) at 4 °C for 20 minutes. Same subcutaneous injection with 1 n mole of the amounts of FITC-conjugated rat IgG2a,k (BD peptide emulsified with complete Freund PharMingen), PE-conjugated rat IgG2a,k (BD adjuvant (CFA) into two-foot pads. In this PharMingen) and biotinylated hamster IgG immunization protocol, draining lymph node group 1, l (BD PharMingen) were used as (popliteal lymph nodes) cells were used as isotype controls. The cells were washed three the effector cells after stimulation in vitro with times with PBS/NaN3 at 4 °C, and then the peptide as described above. When mice incubated with 0.1mg of streptoavidin- were immunized with the peptide-1, the cells conjugated Cy-ChromeTM (BD PharMingen) were stimulated in vitro with peptide-1- pulsed spleen cells. in 100ml of PBS/NaN3 at 4 °C for 20 minutes. The cells were washed three times, fixed with 1ml of PBS containing 1% paraformaldehyde, Cytotoxicity assays and analysed by a FACS Calibur (Becton P815 cells (1x106) were pulsed with the Dickinson, San Jose, CA) and CELL QuestTM m version 3.3 software. peptide at a concentration of 10 M in complete medium at 37 °C for 3 hours. The cells were labelled with 100 mCi of Immunization and CTL induction 51 Na2 CrO4 (NEN Life Science Products, Bone marrow cells (10x106) stimulated with Boston, MA) for one hour, then washed three GM-CSF for 7 days were incubated in the times and suspended in complete medium. presence of 10mM peptide-2 in 1ml of AIM-V Peptide-pulsed, 51Cr-labelled cells were at 37 °C for 2 hours. The cells were washed seeded in 96-well V-bottom plate at 1.5x103 two times with RPMI-1640. Peptide-2-pulsed cells in 100ml of complete medium per well. cells (2x106) were injected intravenously into Effector cells were added to the plate to BALB/c mice. Four weeks later, the spleens make various effector/target ratios (E/T ratios) were collected, minced into single cell in a total volume of 0.2ml per well, and the suspension, erythrocyte-lysed, and treated plate was incubated at 37 °C in 5% CO2 for with anti-CD4 antibody (BD PharMingen) at four hours. The supernatant fluids were 7 the rate of 1mg/1x10 cells and 10% baby harvested with a Supernatant Collecting rabbit complement (Cederlane, Hornby, Ont, System (Skatoron, Lier, Norway), and 51Cr Canada) to deplete CD4-positive cells. Five content was measured by a gamma counter million cells were then co-cultured with the (Aloka model ARC-300). Maximum 51Cr same number of peptide-2-pulsed, 33Gy X- release was determined by adding 0.1% ray-irradiated syngeneic spleen cells in 2ml of Triton X, and spontaneous 51Cr release was EHAA medium (Sigma) supplemented with determined with the wells that contained 100mg/ml nucleic acid precursors, 2mM L- target cells and medium only. Assays were -5 glutamine, 5x10 M 2-ME, 100U penicillin, performed in triplicate, and the mean value 100mg/ml streptomycin, 10mM HEPES, and was used to calculate percent-specific lysis 10% fetal calf serum in 24-well plate at with the following formula: % specific lysis = 37 °C in 5% CO2. On day 4, half volume of 100 x [(release with effector cells – the medium was replaced with fresh one, spontaneous release) / (maximum release – Dengue Bulletin – Vol 28, 2004 147 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus spontaneous release)]. Spontaneous release were cultured with GM-CSF for seven days, did not exceed 28.9% of the maximum by flow cytometry three colour analysis, and release. evaluated the purity of DC. As shown in Figure 1a, 25.07% of the bone marrow cells strongly expressed both I-Ad/I-Ed and CD86. Results and discussion The percentage of CD11c-positive cells in these double positive cells was 95.81% It is known that mature murine DCs strongly (Figures 1b and 1c). In contrast, freshly express major histocompatibility complex isolated bone marrow cells did not express (MHC) class II antigen, co-stimulatory these surface molecules (data not shown). molecules CD80 and CD86, and CD11c, the These results suggest that bone marrow cells a chain of p150/95 b2-integrin[9,10]. We were differentiated into DCs during the examined the expression of MHC class II culture with GM-CSF for seven days and that antigen I-Ad/I-Ed, CD86, and CD11c on DCs accounted for one-fourth of the entire BALB/c mouse bone marrow cells, which population. Figure 1. Expression of the dendritic cell markers on bone marrow cells after culture in the presence of GM-CSF for 7 days (a) (b) (c) R1 CD86 CD11c CD11c I-Ad/I-Ed I-Ad/I-Ed CD86 [BALB/c mouse bone marrow cells were cultured for 7 days in AIM-V medium with 20 ng/ml of murine GM-CSF. The cells were stained with anti – I-Ad/I-Ed – FITC, anti-CD86 – PE, anti-CD11c – biotin, and streptoavidin – Cy-Chrome TM. (a); The cells in the region (R1) strongly expressed I-Ad/I-Ed and CD86, and accounted for 25.07% of the entire cultured bone marrow cells. (b) & (c): The cells in the region (R1) were examined for the expression of CD11c. 95.81% expressed CD11c.] We first attempted to induce specific induce specific CTLs might partly be due CTLs by two foot pad immunizations with to the low binding affinity to H-2Kd MHC the peptide-1 (GYISTRVEM) emulsified class I molecule, because the peptide-1 with CFA. Specific CTL activity was not possesses only one anchor residue (Y) for detected in draining lymph node cells. binding to H-2K d molecule. We, therefore, (Table, Experiment no. 1). We speculated prepared the peptide-2 (GYISTRVEL) in that the inability of the peptide-1 to which the last residue M of the peptide-1 148 Dengue Bulletin – Vol 28, 2004 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus was substituted for L in order to provide were DCs and it was reported that the complete H-2Kd-binding motif[11]. intravenous immunization with 1x105 to 5x105 purified DCs pulsed with peptides It was reported that immunization induced antiviral immunity[5]. Spleen cells with virus epitope peptide–pulsed DCs from the mice immunized with peptide-2- efficiently induced virus -specific CD8+ pulsed DCs lysed peptide-2-pulsed P815 CTLs and protective immunity[5,6]. We cells in a dose dependent fashion after attempted to induce specific CTLs by stimulation in vitro with X-ray-irradiated, immunization with peptide-2-pulsed DCs. peptide-2-pulsed syngeneic spleen cells in We intravenously injected 2x106 bone the presence of recombinant IL-2 for marrow cells, which were stimulated with seven days (Table, Experiment no. 2). This GM-CSF for seven days and pulsed with result demonstrates that intravenous the peptide-2, into BALB/c mice. One- immunization with peptide-2-pulsed DCs fourth of the cultured bone marrow cells induced peptide-2-specific CTLs. Table. Induction of specific CTLs by immunization with peptide-2-pulsed dendritic cells and the peptide-2 emulsified with complete Freund adjuvant Experi- % Specific lysis* ment Immunization E/T ratio Peptide-1- Peptide-2- Non-pulsed no. pulsed pulsed 1. Peptide-1/CFA 20 5.4 7.9 Not done 100 22.9 21.9 Not done 2. Peptide-2/CFA 10 6.5 Not done 36.8 20 8.4 Not done 55.2 40 9.9 Not done 65.4 Peptide-2-pulsed DC 10 20.9 Not done 24.5 20 18.7 Not done 35.8 40 24.0 Not done 44.2 3. Peptide-2/CFA 5 12.7 23.0 23.9 10 20.8 30.4 34.2 20 28.7 46.3 48.3 * 51Cr–labeled P815 mastcytoma (H-2d) pulsed with the peptide (10µM, 3hours) were used as target cells. Specific CTL activity was observed in pulsed ones. (Table, Experiment nos. 2 and the draining lymph node cells after 3). These results suggest that the peptide-2 immunization with peptide-2/CFA. which has the complete binding motif to H- Interestingly, CTLs induced by peptide- 2kd molecule can induce specific CTLs 2/CFA demonstrated lower levels of non- when used with CFA, and that peptide-2- specific cytotoxic activity to P815 cells than specific CTLs also lyse original peptide-1- those induced by peptide -2-pulsed DCs. pulsed target cells. Induction of higher levels Peptide-2/CFA-induced CTLs lysed peptide- of non-specific cytotoxicity by immunization 1-pulsed target cells as well as peptide-2- with DCs may be due to the high antigen Dengue Bulletin – Vol 28, 2004 149 Induction of Cytotoxic T Lymphocytes by Immunization with Dengue Virus presentation ability of DCs to prime various activiity. The data, however, only suggest repertoires of T cells. The other possibility is how efficiently measurable specific CTLs the difference in the source of lymphocytes. can be induced in vitro. It is plausible that We observed that spleen-derived intravenous immunization with peptide- lymphocytes tended to show higher levels of pulsed DCs induces higher levels of specific non-specific lysis than lymph node -derived CTLs in vivo and protective immunity cells (data not shown). It seems that the against viral infections. Moreover, CFA is not draining lymph node cells from mice accepted for human use. Thus, immunized with the peptide and CFA are a immunization strategy using peptide-pulsed better source of CTLs than the spleen cells DCs is still worth investigating for induction from those immunized with peptide-pulsed of CTL-mediated anti-dengue virus DCs because of low non-specific cytotoxic immunity. References [1] Gotch F, Gallimore A and McMichael A. syngeneic irradiated HIV-1 envelope derived Cytotoxic T cells – protection from disease peptide-pulsed dendritic cells. Int Immunol, progression – protection from infection. 1993, 5: 849-857. Immunol Lett, 1996, 51: 125-128. [7] Porgador A, Snyder D and Gilboa E. [2] Toes RE, van der Voort EI, Schoenberger SP, Induction of antitumor immunity using bone Drijfhout JW, van Bloois L, Storm G, Kast marrow-generated dendritic cells. J Immunol, WM, Offringa R and Melief CJ. 1996, 156: 2918-2926. Enhancement of tumor outgrowth through [8] Spaulding AC, Kurane I, Ennis FA and CTL tolerization after peptide vaccination is Rothman AL. Analysis of murine CD8+ T-cell avoided by peptide presentation on clones specific for the dengue virus NS3 dendritic cells. J Immunol, 1998, 160: 4449- protein: flavivirus cross-reactivity and 4456. influence of infecting serotype. J Virol, 1999, [3] BenMohamed L, Belkaid Y, Loing E, Brahimi 73: 398-403. K, Gras-Mass H and Druihe P. Systemic [9] Maraskovsky E, Brasel K, Teepe M, Roux ER, immune responses induced by mucosal Lyman SD, Shortman K and McKenna HJ. administration of lipopeptides without Dramatic increase in the numbers of adjuvant. Eur J Immunol, 2002, 32: 2274- functionally mature dendritic cells in Flt3 2281. ligand-treated mice: multiple dendritic cell [4] Steinman RM. The dendritic cell system and subpopulations identified. J Exp Med, 1996, its role in immunogenicity. Annu Rev 184: 1953-1962. Immunol, 1991, 9: 271-296. [10] Son YI, Egawa S, Tatsumi T, Redlinger RE Jr, [5] Ludewig B, Ehl S, Karrer U, Odermatt B, Kalinski P and Kanto T. A novel bulk-culture Hengartner H and Zinkernagel RM. method for generating mature dendritic cells Dendritic cells efficiently induce protective from mouse bone marrow cells. J Immunol antiviral immunity. J Virol, 1998, 72: 3812- Methods, 2002, 262: 145-157. 3818. [11] Romero P, Corradin G, Luescher IF and [6] Takahashi H, Nakagawa Y, Yokomuro K and Maryanski. H-2Kd-restricted antigenic Berzofsky JA. Induction of CD8+ cytotoxic T peptides share a simple binding motif. J Exp lymphocytes by immunization with Med, 1991, 174: 603-612. 150 Dengue Bulletin – Vol 28, 2004 Molecular Characterization of Brazilian Dengue Viruses Marize Pereira Miagostovich#, Flávia Barreto dos Santos and Rita Maria Ribeiro Nogueira Laboratory of Flavivirus, Department of Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Fiocruz Avenida Brasil 4365 Manguinhos, Rio de Janeiro 21040-360, RJ, Brazil Abstract Many countries in Central and South America as well as Brazil have been characterized by a rise in dengue endemicity. Since 1986, dengue infection has gained endemicity in these countries and more than 3 million dengue cases have been reported along with the emergence also of the severe forms of the disease. Once intratypic variations among dengue virus (DEN) serotypes have been associated with the disease severity, the molecular characterization of DEN becomes an indispensable tool for the laboratories performing virological surveillance programmes. In countries endemic for DEN, as in Brazil, the monitoring of DEN activity should be an ongoing programme to detect the eventual introduction of new serotypes/genotypes to curb the impact of the circulating strains. Here, the molecular epidemiological studies performed on Brazilian DEN strains are presented in order to contribute to a better understanding of the dengue epidemiology in the country. Keywords: Dengue viruses, molecular epidemiology, genotypes, Brazil. Introduction frame (ORF) of over 10,000 nucleotides that encodes a polyprotein precursor of about The dengue (DEN) virus belongs to the 3,400 amino acid residues which is co- and Flaviviridae family, genus Flavivirus, and it has post-translationally processed by the host cell four distinct antigenic serotypes (1 to 4) that and virus-specific protease to yield structural cause a spectrum of diseases ranging from and nonstructural proteins. The coding asymptomatic, mild, undifferentiated fever protein region starts with the sequence for and classic dengue fever (DF) to more severe the core (C), precursor of membrane (prM/M) forms known as dengue haemorrhagic fever and the envelope (E) structural proteins, [1,2] (DHF) and dengue shock syndrome (DSS) . followed by a series of seven nonstructural The DEN virus is a spherical particle of (NS) proteins ordered as follows: NS1, NS2A, approximately 500 Å in diameter, lipid NS2B, NS3, NS4A, NS4B and NS5. The ORF enveloped that includes one segment of a is flanked by two untranslated regions (5’ and single-stranded positive sense RNA with 3‘ UTR) and has a type I cap at its 5’ end ~11,000 nucleotides in length. The genomic (m7GpppAmp) and appears to lack a 3’ - RNA contains a single long open reading terminal poly A tract[3,4]. # E-mail: [email protected]; Fax: 55-21-2598-4373 Dengue Bulletin – Vol 28, 2004 151 Molecular Characterization of Brazilian Dengue Viruses Electron micrographs show that the characterization of DEN strains isolated in virion is characterized by an electron dense Brazil during the last 18 years since dengue core that consists of an isometric became endemic and surveillance nucleocapsid, made up of a single C (100 programmes were implemented in the amino acids) protein, surrounded by a country. double lipid layer, whereas both E (495 amino acids) and M (75 amino acids) proteins are associated[5]. E-glycoprotein is DEN-1 the major surface protein and as showed by The first molecular analysis for DEN Brazilian crystallography, the flat elongated dimmer strains characterization was performed by an [6] extends parallel to the viral membrane . The analysis on genome fragments from DEN-1 E protein is associated with a number of by using restriction endonuclease (RE) biological activities, being the most important enzymes. In this study, restriction fragment antigen with regard to virus biology and heterogeneity by Hae III digestion of cDNA [7] immunity . products was used to map the distribution of The intratypic variation of DEN was DEN-1 topotype found in the American demonstrated by the fingerprinting method region. The strains isolated in the state of Rio that determined genetic variants within each de Janeiro from human serum specimens [23,24] serotype and employed the term topotype to from 1986 to 1994 were grouped in the define variants representing samples from the American (Caribbean) topotype, recognized same geographical region[8]. From 1990 as the only one circulating in the onwards, the molecular analysis by the partial Americas[8,25]. The percentage of the similarity sequencing of the DEN genome gathered the observed among DEN-1 Brazilian strains topotypes in genomic groups (genotypes) and ranged from 60% to 94%, showing the became an important tool to determine their evolution of those samples since its genetic variation and to identify risk factors introduction in the state in 1986. associated with the transmission of particular By the sequencing of 240-nucleotides [9] strains . (nts) spanning the E/NS1 junction (111 nts The E gene has been the most from the 3’ end of E gene and 129 nts from commonly surveyed gene in dengue the 5’ end of NS1 gene), the DEN-1 Brazilian molecular epidemiology[10-15], although genes strains were classified as belonging to that encode nonstructural proteins and non- genotype I which comprises of strains from [9] coding regions have also been used in the the Americas, Africa and South-East Asia . [16-22] phylogeny studies . The DEN-1 Brazilian strains were also Once intratypic variations among analysed by using one-step amplification with different serotypes were associated with the four primers that target regions spanning disease severity, technologies for the polymorphic endonuclease restriction molecular characterization of DEN became specific sites (RSS-PCR) and all of them were an indispensable tool for laboratories grouped into subtype C, which corresponds performing virological surveillance to the largest genotypic group of DEN-1 [9,26] programmes. This paper presents the most described as genotype 1 . The RSS-PCR relevant results of the molecular has become an alternative tool routinely used, 152 Dengue Bulletin – Vol 28, 2004 Molecular Characterization of Brazilian Dengue Viruses which has allowed the characterization of DEN-2 from Brazil, Colombia, Mexico and DEN strains for molecular epidemiological Venezuela have a common progenitor with studies performed in endemic countries, those from South-East Asia[36]. providing rapid identification of viruses In Brazil, the first molecular currently circulating[27-30]. characterization of DEN-2, introduced in the Another study performed with the DEN- country in 1990[37,38], was performed by RE 1 Brazilian strains compared the complete analysis and showed a similarity of 80% with sequences of three strains isolated in 1990, the 1981 Jamaica isolate, suggesting the 1997 and 2001. The genome analysis of spread of those viruses from the Caribbean those strains revealed a remarkable region to South America[25]. conservation of the structural proteins and 27 The geographical origin of DEN-2 amino acids substitutions in the nonstructural Brazilian strains was also established by the genes, and 12 of them in the NS4B-NS5 and direct sequencing of cDNA fragments nine specific to strains BR/97 and BR/01. amplified by the polymerase chain reaction Those findings also suggested that of a fragment encoding amino acids 29 to 94 recombinant events might have occurred, in the E gene. Considering a divergence of since some amino acids substitutions were 6% between the nucleotide sequences as a previously identified in DEN-1 strains cut-off for genotype classification, it was sequenced so far[31]. The evidence that the demonstrated that the Brazilian strains genetic diversity of DEN might be generated belonged to the South-East Asian/American by recombination among those viruses has genotype. The comparison of the three DEN- been described[32-34]. 2 strains isolated in Rio de Janeiro, two of them obtained from classic dengue cases and DEN-2 one from a fatal case, did not identify the markers for virulence in the region studied[10]. The DEN-2 fingerprinting analysis of the American strains showed that this serotype The analysis of DEN-2 samples isolated exists in the American continent as two in the states of Rio de Janeiro, Ceará, Bahia, topotypes representing strains from the and Alagoas between 1990 and 1995 was Caribbean region (Puerto Rico) and from the performed by the partial sequencing of nts Americas, India and South Pacific[8]. By the 1685 and 2504 encompassing the E gene and demonstrated the spread of this serotype sequencing of the E/NS1 junction those [39] topotypes could be related to genotype I from Rio de Janeiro to other states . (Native American) and to genotype III (South- All the characteristics observed in the East Asian/American), respectively. This latter Brazilian DEN-2 genotype were confirmed by genotype was introduced into the Americas the full-length analysis of the nucleotide and in 1981, and was responsible for the first amino acids sequence (GenBank access # DHF/DSS epidemic that occurred in the AF489932)[40]. The Asian-specific non- continent and spread throughout the region conserved amino acid differences, previously over the next two decades[35,36]. The direction described by Leitmeyr et al.[41] as well as of the transmission from South-East Asia to additional differences specific to the Brazilian the Americas was demonstrated as well, since strain were found in E, NS3, and NS5 Dengue Bulletin – Vol 28, 2004 153 Molecular Characterization of Brazilian Dengue Viruses genes[40]. Changes in the E protein could strains presented the same pattern, affect the immunogenicity or cell presenting consistent and reproducible entry/tropism, whereas changes in NS3 amplicons of 582bp and 100bp and, (helicase/protease) and NS5 (RNA-dependent occasionally, extra amplicons of 676 bp or RNA polymerase) could affect replication 150 bp[29]. The DEN-2 Brazilian RSS-PCR efficiency. In addition, differences in the pattern was consistent; however, it did not predicted secondary structure of the 5’ and match any of the RSS-PCR patterns 3’ untranslated regions were found between previously described by Harris et al.[46] Once the South-East Asian/American and native the method was developed with DEN-2 American genotypes; in these regions, the isolates obtained from 1964-1986, the Brazilian isolate was identical to the South- ongoing evolution of those viruses over the East Asian/American strains in sequence and last 15 years could explain the genetic consequently in the predicted secondary diversity observed. Despite those structures[40]. These similarities with the observations, the sequence of the E/NS1 South-East Asian/American genotype were gene junction (GenBank Access # AF529064 also reported recently for the Martinique to AF529078) showed that the Brazilian 703/98 strain after a complete analysis of the DENV-2 strains still belonged to the South- genome[42]. East Asian/American genotype[29]. In Brazil, this DEN-2 genotype was responsible for some clinical features, mainly DEN-3 related to the severity of the disease. In regions where DEN-2 accounted for primary Different from the DEN-3 genotype IV infections, as in the states of Bahia and (topotype Caribbean) responsible for Espirito Santo[43], the most common clinical epidemics in the ‘60s and ‘70s, the DEN-3 feature consisted of classic fever, with re-introduced in the American continent after frequent exanthema, pruritus and a few an absence of 17 years belonged to genotype severe cases. However, in other states where III (topotype Sri Lanka), represented by DEN-2 circulated after extensive epidemics strains from Sri Lanka and India, which are caused by DEN-1, as in Rio de Janeiro, Ceará, associated with DHF/DSS cases in those Pernambuco and Rio Grande do Norte, an countries[13,47]. increase in the number of severe cases was By the time of DEN-3 isolation in observed. The first DHF/DSS case was Brazil[48], RSS-PCR was extremely valuable reported in Rio de Janeiro after the which once allowed the rapid introduction of DEN-2 in 1990, and it was characterization of the first strain as subtype accompanied by an increasing number of C, confirming the introduction and direction hospital admissions resulting from DEN-2 of transmission of those viruses from Central [44,45] secondary infections . to South America[28,46]. By using RSS-PCR we were able to After the RSS-PCR analysis, the nucleic analyse geographically and temporally acid sequencing from positions 278 to 2550 distinct Brazilian DEN-2 strains encompassing of DEN genome was performed (Access ten years (1990 to 2000). The analysis of the number AY038605) and the parsimony RSS-PCR products showed that all Brazilian analysis generated a phylogram assigning a 154 Dengue Bulletin – Vol 28, 2004 Molecular Characterization of Brazilian Dengue Viruses Brazilian DEN-3 strain to genotype III, The severity of the disease and the reconfirming those data[13,28]. The similarity occurrence of deaths resulting from primary rate of a Brazilian DEN-3 strain to others infections during the DEN-3 epidemic in the represented by the same subtype III ranged state of Rio de Janeiro in 2002 could be from 96% to 98% and 98% to 99% for explained partially by the virulence of this nucleic acid and deduced amino acid particular genotype[28,54]. Fatal cases, resulting sequences, respectively. The comparison of from primary dengue infections, were the Brazilian DEN-3 with strains isolated in previously described[55] before the DEN-3 Guatemala showed a total of 14 nucleic acid genotype III introduction in Brazil. However, substitutions, with one of them resulting in an the highest number of DHF/DSS cases that amino acid change from histidine to occurred in the state were due to secondary arginine[28]. infection by the South-East Asian/American DEN-2 genotype[45]. Those findings As a result of several DEN-3 epidemics in corroborated the previous observations that Latin American countries, a large number of some DEN strains can be more virulent than DEN-3 genome sequences have been others, representing an important risk factor recently deposited in the GenBank[49-51]. A for DHF/DSS and that antibody-dependent phylogenetic study compromising DEN-3 enhancement (ADE) itself does not explain all strains isolated in Sri Lanka, East Africa and cases of severe disease[51,56-58]. Recently, it Latin America confirmed the establishment of was suggested that the more virulent the new DEN-3 genotype[51]. According to genotypes were now replacing those that had the author, there are two separate lineages a lower epidemiological impact throughout formed within genotype III: Group A the world[59]. consisting of isolates from 1981 to 1989 in Sri Lanka and Group B which was expanded in three distinct clades including isolates from Conclusion 1989 to 1998 in Sri Lanka, strains isolated in East Africa from 1985 to 1993 and isolates In the last few decades, dengue has spread as from 1994 in Latin America. The a pandemic in the American continent, phylogenetic analysis suggested that genotype starting in the Caribbean islands and III was introduced from the Indian expanding to North, Central and South [60] subcontinent into East Africa in the 1980s America . In this context, the dengue and from Africa into Latin America in 1994, epidemiological profile in Brazil has changed showing a single genotype introduction in the from a non-endemic to a hyperendemic one. continent and its subsequent diversification[52]. Since the 1980s when the first DEN strains In the same year, Peyrefitte et al.[53] showed a were isolated, more than 3 million dengue high similarity between the DEN-3 cases and nearly 2,090 DHF/DSS cases have Martinique and the Brazilian strains. been reported in the country Furthermore, the complete genome (www.funasa.gov.br)[61,62]. characterization of the Brazilian DEN-3 The endemicity of dengue in 25 out of sequence (AY679147) strain confirmed an the 27 federative units, the remarkable insertion of 11 nts in the 5´ non-coding virulence of the DEN-2 and DEN-3 region of the genome as previously described genotypes and the risk of the introduction of for the Martinique strain[53]. Dengue Bulletin – Vol 28, 2004 155 Molecular Characterization of Brazilian Dengue Viruses DEN-4 in the country highlight the alarming disease, not because of the virulence- dengue epidemiological picture in Brazil. In inherited properties but because antibodies this scenario, use of rapid methods for DEN from a primary infection may enhance identification and molecular characterization infection with one genotype while are indispensable tools in the virological neutralizing infection with a distinct one[52,65]. surveillance laboratories, mainly due to an Given the limited options available for obvious need to characterize dengue dengue control, active surveillance genotypes before a major outbreak programmes with continuous monitoring of occurs[26,46,63]. The partial sequencing of DEN dengue infection in communities is still one strains genome has also been used routinely of the strategies available to detect the for DEN molecular epidemiological studies, introduction of new serotypes/genotypes, and, and it recently characterized the co-infecting consequently, to prevent the occurrence of genotypes of DEN-1 and DEN-2 in a patient epidemics, thus minimizing the impact of the presenting classic dengue fever in São circulating strains. Paulo[64]. The knowledge of the virus genotype circulating in a particular region has also Acknowledgements implications for the potential introduction of To Conselho Nacional de Desenvolvimento vaccines, allowing the evaluation of the Científico e Tecnológico (CNPq), Fundação genomic relations between the viruses used de Amparo a Pesquisa do Estado do Rio de in vaccine development and the circulating Janeiro (FAPERJ), Fundação Oswaldo Cruz strains. The ability of pre-existing dengue (FIOCRUZ), PAPES III – FIOCRUZ. Thanks antibodies to neutralize better certain DEN are also due to the staff of the Laboratory of variants than others has been demonstrated. Flavivirus for their technical assistance. Some strains may produce a more severe References [1] Westaway EG, Brinton MA, Gaidamovich SY, [5] Kuhn RJ, Zhang W, Rossmann MG, Pletnev Horzinek MC, Igarashi A, Kaariainen L, Lvov SV, Corver J, Lenches E, Jones CT, DK, Porterfield JE, Russell PK and Trent D W. Mukhopadhyay S, Chipman PR, Strauss EG, Flaviviridae. Intervirol, 1985, 24: 183-192. Baker TS and Strauss JH. Structure of [2] World Health Organization. Dengue dengue virus: implications for flavivirus haemorrhagic fever: diagnosis, treatment organization, maturation, and fusion. Cell, and control. Geneva: WHO, 1997. 2002, 108: 717-725. [3] Chambers TJ, Hahn CS, Galler R and Rice [6] Rey FA, Heinz FX, Mandl C, Kunz C and CM. Flavivirus genome organization, Harrison C. 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Bull World Health Organ, 1980, 926. 58: 1-21. Dengue Bulletin – Vol 28, 2004 159 Molecular Characterization of Brazilian Dengue Viruses [58] Watts DM, Porter KR, Putvatana P, Vazquez [62] Nogueira RMR, Miagostovich MP and B, Calampa C, Hayes CG and Halstead SB. Schatzmayr HG. Dengue virus in Brazil. Failure of secondary infection with American Dengue Bulletin, 2002, 26: 77-83. genotype dengue 2 to cause dengue [63] Lanciotti RS, Calisher CH, Gubler DJ and haemorrhagic fever. Lancet, 1999, 354: Vorndam V. Rapid detection and typing of 1431-1434. dengue viruses from clinical samples by using [59] Rico-Hesse R. Microevolution and virulence reverse transcriptase-polymerase chain of dengue viruses. Adv Virus Res, 2003, 59: reaction. J Clin Microbiol, 1992, 30: 545-551. 315-341. [64] Santos CLS, Bastos MAA, Sallum MAM and [60] Gubler DJ. Epidemic dengue/dengue Rocco IM. Molecular characterization of hemorrhagic fever as a public health, social dengue viruses type 1 and 2 isolated from and economic problem in the 21st century. concurrent human infection. Rev Inst Med Trends Microbiol, 2002, 10: 100-103. Trop São Paulo, 2003, 45(1): 11-16. [61] Da Silva Júnior JB, Siqueira Júnior JB, Coelho [65] Kochel TJ, Watts DM, Halstead SB, Hayes GE, Vilarinhos PTR and Pimenta Júnior FG. CG, Espinoza A and Felices V. Effect of Dengue in Brazil: current situation and dengue-1 antibodies on American dengue-2 prevention and control activities. Epidem viral infection and dengue haemorrhagic Bulletin, 2002, 23(1): 1-7. fever. Lancet, 2002, 360: 310-312. 160 Dengue Bulletin – Vol 28, 2004 Unusual Emergence of Guate98-like Molecular Subtype of DEN-3 during 2003 Dengue Outbreak in Delhi Manoj Kumar*#, S.T. Pasha*, Veena Mittal*, D.S. Rawat*, Subhash Chandra Arya**, Nirmala Agarwal**, Depesh Bhattacharya*, Shiv Lal* and Arvind Rai* *National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi – 110 054, India **Sant Parmanand Hospital, 18 Shamnath Marg, Delhi – 110 054, India Abstract With a view to identifying the molecular subtype of the circulating dengue virus responsible for a major outbreak of dengue fever (DF) / dengue haemorrhagic fever (DHF) in and around Delhi during the post- monsoon period in 2003, 32 serum samples were collected from clinically suspected cases. These were subjected to reverse transcription/polymerase chain reaction (RT/PCR) for amplification of 511 bp C- PreM gene region of the dengue virus. Seven specimens, yielding a satisfactory quantum of viral RNA, were subsequently processed for automated nucleotide sequencing. Five of the seven analysed isolates showed close DNA sequence homology with Guate96-98 strains of DEN-3 virus, whereas two turned out to be genotype IV of DEN-2. Earlier, DEN-2 (genotype IV) had been identified as the etiological agent during a major DF/DHF outbreak in Delhi in 1996 and also in 2000. Though DEN-2 continues to prevail, DEN-3, having a close sequence homology with Guate96-98 strains, seems to have entered India for the first time in late 2003, resulting in a major DF/DHF outbreak. How the Guate96-98 strain of DEN-3 entered India remains to be linked epidemiologically. Keywords: Dengue outbreak, molecular typing, CpreM gene, DEN-3, Delhi. Introduction alone, a number of outbreaks of dengue virus infection were recorded in 1967, 1970, Dengue fever/dengue haemorrhagic fever 1982, 1988 and 1990[9-13]. Again in 1996, a (DF/DHF) is caused by one or more of the major DHF outbreak, resulting in 10,252 four antigenically-related dengue virus cases with 432 deaths, occurred in and serotypes DEN-1 to DEN-4. It is widespread around Delhi. DEN -2 genotype IV was the in tropical and subtropical countries in the predominant etiological agent[14,15]. world and is a serious cause of morbidity Delhi and its adjoining areas were again and mortality, threatening about one third of the total human population[1-3]. Many struck by a major outbreak of DF/DHF between September and December 2003. outbreaks and epidemics of DF/DHF have The present study was undertaken to unveil been reported in different parts of India the predominant molecular subtype of the during the past four decades[4-8]. In Delhi dengue virus involved in this outbreak. # E-mail: [email protected]; Tel./Fax: 91-11-23912960 Dengue Bulletin – Vol 28, 2004 161 Emergence of DEN-3 in Delhi in 2003 Materials and methods Virus RNA isolation Clinical specimens Thirty-two serum samples were subjected to dengue viral RNA isolation. 140µl sera A total of 32 serum samples from clinically sample was processed for RNA isolation using suspected acute cases of DF/DHF were QIAamp viral RNA Kit (QIAGEN, Germany) collected from different hospitals in Delhi using standard kit protocol. Finally, viral RNA between September and December 2003. was eluted in 30µl nuclease-free water. Most of the serum samples were collected within the first five days of the clinical onset. Reverse transcription/polymerase Samples were transported to the laboratory chain reaction (RT/PCR) within six hours of collection and stored at – RT-PCR was carried out using previously 70 °C until processed. Thirty-two sera are reported D1 and D2 primers that were M13 considered adequate to establish the tailed for the convenience of nucleotide predominant molecular subtype of dengue sequencing. This primer set is capable of virus. amplifying all the four types of dengue viruses (DEN-1 to DEN-4). Primer D1 (with M13F tail) (5’-TGTAAAACGACGGCCAGTTCAATATGCTGAAACGCGCGAGAAACCG-3’) (M13 forward primer sequence underlined) Primer D2 (with M13R tail) (5’-CAGGAAACAGCTATGACCTTGCACCAACAGTCAATGTCTTCAGGTTC-3’) (M13 reverse primer sequence underlined) Complementary DNA (cDNA) synthesis followed by 40 cycles of 95 °C for 30 and gene amplification of 511bp CpreM seconds, 55 °C for 30 seconds, 72 °C for 1 gene region of the dengue virus was minute and final extension for 72 °C for 10 performed using one step GeneAmp RNA minutes and hold at 4 °C. Appropriate Gold RT PCR Kit (Applied Biosystems, USA) positive and negative controls were used in with D1 and D2 primers for detection and RT/PCR. PCR products were typing of all the four types of dengue virus[16]. electrophoresed on 1.5% agarose gel along Briefly, 50ml reaction mix containing final with 100bp DNA ladder marker (MBI concentration of 1X of 5X RT buffer, 1.5mM Fermentas, USA) and were visualized on gel MgCl2, 200mM dNTPs, 5mM dithiotheratol documentation system (Biometra, Germany). and 10pmol of D1 and D2 primers, 10U RNAase inhibitor, 15U of MuLV MultiScribe Gene sequencing and phylogenetic reverse transcriptase, 2.5U of Amplitaq Gold analysis DNA polymerase and 5 µl of extracted viral RNA. RT was performed at 42 °C for 20 PCR amplicons were purified using minutes on GeneAmp 9700 PCR System. Centricon-100 columns (Millipore, USA) Then pre-hold at 95 °C for 10 minutes and subjected to automated dideoxy chain 162 Dengue Bulletin – Vol 28, 2004 Emergence of DEN-3 in Delhi in 2003 termination nucleotide cycle-sequencing from 18,000 to 250,000. Most of the using commercial ABI PRISMTM Big Dye samples collected within five days from the Terminator Cycle Sequencing Kit with onset of the fever were selected for RT-PCR Amplitaq DNA Polymerase FS, following the testing. manufacturer’s protocol and run on ABI Out of the 32 serum samples subjected PRISMTM 310 Genetic Analyser (Applied to RT-PCR, only seven yielded amplification Biosystems, USA). Nucleotide sequences of 511bp C-PreM gene region of dengue were edited and aligned using Sequence virus. Lane 1-14 in the Figure included Navigator Software. Subsequently, blast clinical samples, 100bp DNA ladder marker search (http://www.ncbi.nlm.nih.gov/blast) in lane M and negative control in lane Neg. and phylogenetic analysis using DNA Star Automated nucleotide sequencing of these software, were done to reveal the dengue seven RT/PCR products revealed two groups virus molecular subtype. of sequences, the first group had five almost identical sequences, while the second group Result and discussion had two similar sequences. All samples were subjected to blast search, which revealed During the study, a majority of the subject that five isolates had a very close sequence patients had clinical symptoms of DF and homology (=98%) with Guate98 AB038478 only sporadic cases presented symptoms of strain of dengue type 3 (DEN -3), while two DHF/DSS {DF: 24 (75%), DHF: 7 (21.9%) turned out to be DEN-2 and showed =99% and DSS: 1 (3.1%)}. The samples belonged homology with Delhi96 AF047394 strain to all age groups ranging from 5 to 50 years. Delhi 2000 strains (Table). These Cpre M The male-female ratio was 18:14. The mean gene sequences were submitted in the Gene platelet count was 77,120, which ranged Bank wide accession AY 706094-AY706099. Figure. PCR products visualization on 1.5% agarose gel Dengue Bulletin – Vol 28, 2004 163 Emergence of DEN-3 in Delhi in 2003 Table. Clinical and molecular analysis data of seven samples, yielding RT-PCR positivity for 511bp CpreM gene of dengue virus Assigned S. Sample Serum Clinical Platelet RT-PCR genotype Age/Sex No. ID* collection state count 511bp on blast search 1 04DEL03 22.09.03 DF 30/F 89,000 +ve DEN-3 2 06DEL03 24.09.03 DF 20/M 79,000 +ve DEN-2 3 10DEL03 07.10.03 DHF 15/M 39,000 +ve DEN-3 4 11DEL03 08.10.03 DF 14/F NA +ve DEN-3 5 16DEL03 01.10.03 DF 34/F 18,000 +ve DEN-2 6 19DEL03 04.10.03 DHF 40/M 38,000 +ve DEN-3 7 26DEL03 13.10.03 DHF 16/M 36,000 +ve DEN-3 Out of 32 samples subjected to RT-PCR for 511bp Cpre M gene of dengue virus, only 7 turned positive, while 25 did not show any amplification. All five DEN-3 strains had =98% nucleotide sequence homology with Guate98 strains. Both DEN-2 strains had =99% nucleotide sequence homology with already circulating Delhi96 and Delhi2000 strains. NA: Information not available Several studies have shown that dengue although cases of DEN-1 were also virus infection has been endemic in detected[19-21]. different parts of India, as documented for During the present study, the majority over four decades[17,18], and almost all the of the patients had clinical symptoms of DF four known serotypes of dengue virus (DEN - and only sporadic cases presented with 1 to DEN-4) have been reported. The symptoms of DHF or DSS. The samples metropolitan city of Delhi witnessed several referred to our laboratory for molecular outbreaks of DF/DHF in 1967, 1970, 1982, characterization were accompanied by IgM 1988,1996 and 2000. DEN -1 and DEN-3 serology results. When cross-checked, we viruses were associated with the 1970 found that five RT/PCR-positive samples epidemic, DEN-1 and DEN -2 with the 1988 were IgM-negative, while two RT/PCR- epidemic, while genotype IV of DEN-2 was positive samples were dengue IgM-positive. responsible for the major DHF outbreak in Previous studies had also shown that most, 1996[15]. Our previous findings revealed but not all, RT/PCR-positive samples had genotype IV of DEN-2 as the predominant negative IgM serology. The reason for this is type circulating from 1996 onwards, based attributed to the fact that the virus had not on RT-PCR and C-PreM gene sequencing, been recovered from most of the DF/DHF patients beyond the 5th day of the onset of 164 Dengue Bulletin – Vol 28, 2004 Emergence of DEN-3 in Delhi in 2003 the symptoms, while detectable levels of Americas had also been reported[25]. The dengue-specific IgM antibodies appear after first epidemic of DEN -3 in Jamaica and the 4th or the 5th day. Our findings re- Puerto Rico was witnessed in 1963, which established that the ideal time for the was followed by another epidemic of DEN - collection of samples for molecular test was 3 in Colombia and Puerto Rico in the mid- between days 1-5. 1970s, and in the Pacific islands in early 1980s[26]. In 1994, a new strain of DEN-3 The nucleotide sequence alignment and was introduced in the Americas, causing a blast search of the seven RT/PCR-positive major epidemic of DF/DHF in Nicaragua samples in the present study, when and an outbreak of DF in Panama[27]. But compared with those of earlier ones, this DEN-3 was genetically different from revealed that only ~29% (2/7) belonged to the DEN-3 strains which circulated in the the already prevalent genotype IV of DEN- Americas earlier. Interestingly, in 1994, this 2; whereas the majority ~71% (5/7) showed DEN-3 genotype was reported to have a close genomic homology (=98%) with close identity with those strains which GUATE98 AB038478 strain of dengue type caused DHF epidemic in some of the South- 3 (DEN-3) which caused a widespread East Asian countries around the same dengue outbreak in Guatemala in the late period[28]. This DEN -3 strain subsequently Nineties[22]. A similar strain is also reported spread from Asia to Central America and to have been reintroduced in Marlinique Mexico in 1995 and caused major (French West Indies)[23] and in Rio de epidemics. A classic example of the Janerio, Brazil (unpublished data vide Gene replacement of one type by the other is Bank Accession No. AY679147). The first evident from the fact that, in 1971, DEN-2 reported evidence of DEN-3 in Delhi was in was introduced into the Pacific areas 1970, based on serotyping, but no genomic followed by a new strain of DEN-1 in 1975 data of the 1970 strain of DEN -3 is available. and DEN-4 by 1979, and in early 1980s by It is difficult to ascertain, after a long gap of yet another new strain of DEN -3[29]. These 33 years, whether the old 1970 strain of reports support our current findings that, DEN-3 had re-emerged during the current although DEN -2 (genotype IV) has been outbreak; or Guate98 DEN -3 strain predominant in northern India over the past (prevalent in South American countries) had few years, Guate96-98-like DEN-3 strain been introduced for the first time in India. dominated during the major outbreak of DF The changing epidemiology of different in Delhi in 2003. subtypes of dengue virus and their co- existence and/or replacement of one type by the other is well documented[24]. During Acknowledgements the present outbreak, we found DEN-3 as We thank Ms Priyanka, Ms Kamini Singh the predominant type, but it did not seem and Ms Seema George for their technical to completely replace the previously assistance. The secretarial assistance of circulating DEN -2. Prior to 1977, co- Mr A.K. Manchanda, Ms Kiran Bhatt and existence of DEN-2 and DEN -3 in the Ms Sarita Kumar is acknowledged. Dengue Bulletin – Vol 28, 2004 165 Emergence of DEN-3 in Delhi in 2003 References [1] Gubler DJ and Clark GG. Dengue/dengue disease. Epidemiol Infect, 2000, 125(1): haemorrhagic fever and emergence of a 195-200. global health problem. Emerg Infect Dis, [9] Balaya S, Paul SD, D’Lima LV and Parvi KM. 1995, 1(2): 55-57. Investigations on an outbreak of dengue in [2] Monath TP. Dengue: the risk to developed Delhi in 1967. Indian J Med Res, 1969, 57: and developing countries. Proc Natl Acad 767-774. Sci, USA, 1994, 91: 2395-2400. 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Indian J Med Res, 1969, 57(8): 1392-1401. [13] Srivastava VK, Suri S, Bhasin A, Srivastava L [6] Chaturvedi UC, Kapoor AK. Mathur A, and Bhardwaj M. An epidemic of dengue Chandra D, Khan AM and Mehrotra RM. A haemorrhagic fever and dengue shock clinical and epidemiological study of an syndrome in Delhi: a clinical study. Ann epidemic of febrile illness with Trop Paediatr, 1990, 10: 329-334. haemorrhagic manifestations, which [14] Broor S, Dar L, Sengupta S, Chakraborty M, occurred at Kanpur, India in 1968. Bull Wali JP, Biswas A, Kabra SK, Jain Y and Seth World Health Organ, 1970, 43(2): 281-287. P. Recent dengue epidemic in Delhi, India. [7] Padbidri VS, Dandawate CN, Goverdhan In: Saluzzo JF and Dodet B (Eds.): Factors in MK, Bhat UK, Rodrigues FM, D’Lima LV, the emergence of arbovirus diseases. Kaul HN, Guru PY, Sharma R and Gupta NP. Amsterdam: Elsevier, 1996: 123-128. An investigation of the aetiology of the 1971 [15] Singh UB, Maitra A, Broor S, Rai A, Pasha ST outbreak of febrile illness in Jaipur city, India. and Seth P. Partial nucleotide sequencing Indian J Med Res, 1973, 61(12): 1737-1743. and molecular evolution of epidemic [8] Singh J. Balakrishnan N, Bhardwaj M, causing dengue 2 strains. J Infect Dis, 1999, Amuthadvi P, George EG, Subramani K, 180: 959-965. Soundararajan K, Appavoo NC, Jain DC, [16] Lanciotti RS, Calisher CH, Gubler DJ, Chang Ichhpujani RL, Bhatia R and Sokhey J. Silent G and Vorndam V. Rapid detection and spread of dengue and dengue haemorrhagic typing of dengue viruses from clinical fever to Coimbatore and Erode districts in samples by using reverse transcriptase – Tamil Nadu, India. Need for effective polymerase chain reaction. J Clin Microbiol, surveillance to monitor and control the 1992, 30: 545-551. 166 Dengue Bulletin – Vol 28, 2004 Emergence of DEN-3 in Delhi in 2003 [17] Kalra NL, Kaul SM and Rastogi RM. [23] Peyrfitte CN, Paris PC, Perennec M, Bessaud Prevalence of Aedes aegypti and Aedes M, Martial J, Kenane, Durand JP and Tolou albopictus vectors of dengue and dengue HJ. Genetic characterization of newly haemorrhagic fever in North, North-East and reintroduced dengue virus type 3 in Central India. Dengue Bulletin, 1997, 21: Martinique (French West Indies). J Clin 84-92. Microbiol. 2003, 41(11): 5195-5198. [18] Mathew T, Nayar M, Gupta JP, Suri NK, [24] Gubler DJ. Epidemic dengue/dengue Bhola SR, Ghosh TK, Suri JC, Talwar V and haemorrhagic fever: A global public health Pattanayak S. Serological investigations on problem in the 21st century. Dengue Bulletin, arbovirus activity in and around Delhi, a 1997, 21: 1-15. five-year study. Indian J Med Res, 1979, 69: [25] Gubler DJ and Trent DW. Emergence of 557-566. epidemic dengue/dengue haemorrhagic [19] Kumar M, Rai A, Pasha ST and Datta KK. fever as a public health problem in the Molecular detection and typing of dengue Americas. Infect Agents Dis, 1994, 2: 383- viruses in clinical specimen from dengue 393. outbreak in Delhi in 2000. The Proceedings [26] Gubler DJ and Casta-Valez A. A programme of the International Conference of Indian for prevention and control of epidemic Public Health Congress, New Delhi, 14-16 dengue and dengue haemorrhagic fever in April 2001. Puerto Rico and the US Virgin Islands. Bull [20] Singh UB and Pradeep Seth. Use of PAHO, 1991, 25: 237-247. nucleotide sequencing of the genomic [27] Centers for Disease Control. Imported cDNA fragments of the capsid/pre dengue-United States, 1993-1994. MMWR, membrane junction region for molecular 1995, 44: 353-356. epidemiology of dengue type 2 viruses. Southeast Asian J Trop Med Public Health, [28] Lanciotti RS, Lewis JG, Gubler DJ, and Trent 2001, 32(2): 326-335. DW. Molecular evolution and epidemiology at dengue-3 viruses. J Gen Virol, 1994, 75: [21] Das PK, Parida MM, Saxena P, Kumar M, 65-75. Rai A, Pasha ST and Jana AM. Emergence and continued circulation of dengue-2 [29] Gubler DJ. Dengue and dengue (genotype IV) virus strains in northern India. haemorrhagic fever. Its history and Journal of Medical Virology, 2004, 74(2): resurgence as a global public health problem. 314-322. In DJ Gubler and G Kuno (Eds.), Dengue and dengue haemorrhagic fever, CAB [22] Usuku S, Castillo L, Sugimoto C, Noguchi Y, International, Wallingford, Oxon OX108DE, Yogo Y and Kobayashi N. Phylogenetic UK, 1997: 1-23. analysis of dengue-3 viruses prevalent in Guatemala during 1996-1998. Arch Virol, 2001, 146(7): 1381-1390. Dengue Bulletin – Vol 28, 2004 167 The Animal Models for Dengue Virus Infection Tao Peng, Junlei Zhang and Jing An# Department of Microbiology, College of Medicine, Third Military Medical University, Chongqing, 400038, People’s Republic of China Abstract Currently, the mechanisms involved in the pathogenesis of DHF/DSS remain poorly understood and there is no effective vaccine available to prevent infection with DEN virus. The lack of a reliable small animal model that mimics dengue disease is a major obstacle. In this paper, the development of small animal models such as mice for dengue virus infections is reviewed. Keywords: Dengue virus, animal model, mice. Introduction However, the mechanisms involved in the pathogenesis of DHF/DSS remain poorly Dengue (DEN) viruses are mosquito-borne understood and there is no effective vaccine RNA viruses, which belong to the genus available to prevent infection with any of Flavivirus (family Flaviviridae), and are the four serotypes of DEN virus. A major grouped into four antigenically distinct types technical barrier is the absence of a suitable (DEN-1, DEN-2, DEN-3, and DEN-4). Every animal model that mimics DEN disease, year, they infect millions of people and can including DHF/DSS. So far, there are only cause a mild-to-debilitating febrile illness three known hosts for DEN virus infections: (classical dengue fever, DF) or life- mosquitoes, humans and lower primates[2]. threatening syndrome (dengue Although these lower primates infected with haemorrhagic fever/dengue shock syndrome, wild type DEN viruses develop viremia, they DHF/DSS). In recent years, the geographical generally manifest only very mild or no range of dengue in tropical and subtropical clinical signs of disease[3]. Since the regions of the world has extended and appearance of the severe combined DHF/DSS is occurring in new areas and with immunodeficiency (SCID) mice in 1983[4], increased incidence[1]. Cardinal signs of efforts have been made to develop new DHF/DSS include haemorrhage, abrupt small animal models that may be useful for onset of vascular leakage and shock, the development of a future DEN virus accompanied by severe thrombocytopenia vaccine and for studying the pathogenesis of and massive complement activation. DEN virus infections. # E-mail: [email protected]; Tel.: +86-23-68752774; Fax: +86-23-65463259 168 Dengue Bulletin – Vol 28, 2004 The Animal Models for Dengue Virus Infection Animal models based on virus titers were detected in the peripheral blood and the brain tissues, indicating that SCID mice DEN virus had replicated in the infected The SCID mice, which do not produce K562-SCID mice. Other serotypes of DEN functional T and B cells and lack detectable viruses were also used to infect the K562- immunoglobulin (Ig), can support DEN - SCID mice, and the mortality rates of the susceptible human cell lines xenografts, and infected mice varied with different challenge this system has been employed to study strains, suggesting that this animal system DEN virus infection in vivo. SCID mice might potentially be utilized to define the reconstituted with human peripheral blood virulence of various human DEN isolates lymphocytes (hu-PBL-SCID) have been used and to characterize the molecular for studies on the pathogenesis of infection determinants for such viral virulence. K562- with the human immunodeficiency virus SCID mice were also challenged with DEN - (HIV)[5,6] and for research on treatment of 2 virus and received antibody administration HIV infection[7]. at the same time or one day earlier, and the results revealed that these mice exhibited a The hu-PBL-SCID mice were firstly reduction in mortality and a delay of evaluated as an animal model for DEN viral paralysis onset after DEN virus infection. [8] infection in 1995 . SCID mice were These results indicated that an in vitro injected intraperitoneally (ip) with hu-PBL neutralizing antibody also defended K562- for reconstitution and successful SCID mice against DEN-2 virus infection. engraftment was demonstrated by the presence of a high serum level of human Target cells and organs for DEN virus IgG. Hu-PBL-SCID mice were ip-infected replication in humans remain unclear. with DEN-1 virus. Unfortunately, only 5 of Unusual clinical manifestations, mostly 19 hu-PBL-SCID mice showed sensitivity to cerebral and hepatic symptoms, have DEN-1 virus. become more common in patients with DEN virus infection in recent years[10,11]. The It was suggested that the main reason involvement of liver cells in the for the low DEN infection rate was a scanty pathogenesis of DEN virus infection has number of appropriate human target cells in been indicated by abnormal liver function, the reconstituted mice. Thus, investigators pathological findings and detection of viral searched for more DEN-susceptible human antigen in hepatocytes and Kupffer’s cells at cell lines to improve the infection rate of the biopsies[12]. It was reported that DEN virus SCID mouse model. One promising could replicate in a human heptocarcinoma candidate was K562 cell, an cell line, HepG2, and infectious particles erythroleukemia cell line. SCID mice were were released into the culture medium[13,14]. engrafted ip with K562 cells (K562-SCID Therefore, HepG2 cells were transplanted [9] mice) . After intratumor injection into the into SCID mice to develop an animal model peritoneal tumor masses of DEN-2 virus, for studying the pathogenesis of DEN virus K562-SCID mice showed neurological signs infection[15]. The replication of HepG2 cells of paralysis and died at approximately 2 in host mice was confirmed by an increase weeks post-infection (pi). In addition to of serum human albumin and propagation being detected in the tumour masses, high of HepG2 cells in the liver. At 7-8 weeks Dengue Bulletin – Vol 28, 2004 169 The Animal Models for Dengue Virus Infection after transplantation, HepG2-grafted SCID such as arching of the back, ruffling of the mice were ip-infected with DEN-2 virus. A fur and slowing of activity appeared at end high titer of the virus was detected in the of day 4 pi. The presence of DEN-2 virus in liver and serum but not in the brain in the the blood was confirmed on day 2 pi by early stage of the infection. When the mice reverse transcriptase-polymerase chain showed paralysis, the highest titer of virus reaction (RT-PCR). The development of the was detected in the serum and brain. DEN - experimental DEN -2 virus infection in 2 antigens were also found in HepG2 cells mouse model was accompanied by the virus of the liver in the early stage and some reproduction in all animals. Within 5 and 6 neurons of the brain in the late stage. Upon days pi, all mice showed severe sickness clinical examination, thrombocytopenia, with anorexia and weight loss ending in prolonged partial thromboplastin time, limb paralysis and 100% mortality rate was increased haematocrit, blood urea nitrogen noted at 7 days pi. The most impressive and tumour necrosis factor a (TNF a) were changes were seen with TNF-a, which seen in the paralyzed mice. Moreover, mild abruptly and steeply increased 24 h before haemorrhages in the liver and tarry stool in death. Serum levels of interleukin (IL)-1ß, IL- the small intestine were observed in some 6, IL-10, IL-1 receptor antagonist and mice. soluble TNF receptor I continuously increased during the time of infection. All of above animal models based on Treating animals with anti-TNF-a serum SCID mice with transplanted DEN - reduced the mortality rate down to 40%. susceptible human cells mimic some of the This model supports the view that the aspects of human disease, which may be activation of innate immune response is at helpful for studying DEN virus infection, least partially responsible for mortality in especially in the areas of viral pathogenesis, DEN-2 virus infection, and in line with this virus-host interaction and vaccine concept, anti-TNF treatment significantly development against DEN infections. reduces the mortality rates. Therefore, However, it is generally agreed that inbred 4-week-old BALB/c mice are useful DHF/DSS is an immune-mediated disease, models to research the immune activation and since SCID mice are unable to produce of host in DEN-2 virus infection. the innate immune response, this may impose some limitations on the use of these animal models to extrapolate the situation in Gene knockout mouse human DHF/DSS. (AG129) model There is evidence that alpha and beta BALB/c mouse model interferons (IFN-a/ß) and gamma IFN (IFN-?) Inbred four-week-old BALB/c mice were might be involved in human DEN virus found sensitive (haplotype H-2d) to the infection[17,18]. In addition, exogenously challenge with dengue virus type 2 (strain administered IFN appears to protect mice P23085)[16]. Mice were ip-infected with a from DEN virus challenge[19]. This dose of 5 LD50 of the mouse-adapted DEN - information suggested that mice defective in 2 virus, and the first clinical manifestations their IFN response might provide a suitable 170 Dengue Bulletin – Vol 28, 2004 The Animal Models for Dengue Virus Infection model for DEN virus infection. paraplegia after virus infection, they Intraperitoneally administered mouse- recovered after one month. However, there adapted DEN -2 virus was uniformly lethal in was transient thrombocytopenia at 10-13 AG129 mice[20], which lack IFN-a/ß and days pi. When the mice were re-infected IFN-? receptor genes, regardless of age. The with the same DEN-2 virus two months later, mice showed neurological abnormalities, thrombocytopenia was manifested again at including hind-leg paralysis and blindness at 10 days after infection. Anti-platelet 7 days pi, and died at 12 days after infection. antibody was also generated after injection. The immunized mice were protected from And there was strain variation in DEN-2 virus challenge, and the survival time virus infection; the A/J strain was more increased following passive transfer of anti- sensitive than BALB/c or B6 mice. These DEN polyclonal antibody. To determine results show that this DEN-2 virus-infected which aspect of the IFN response was mouse system accompanied by thrombo- critical in protecting these mice from DEN cytopenia and anti-platelet antibody may be virus infection, animals individually deficient a suitable model to study the pathogenicity, in either IFN-a/ß (A129) or IFN-? (GKO) especially immune activation in DEN virus functions as well as BALB/c controls were infection. On the other hand, A/J mice had subjected to a similar DEN virus challenge. to be inoculated with a large quantity of None of these mice exhibited any overt DEN-2 virus; a dose of less than 1×108 pfu symptoms of illness, indicating that for DEN per mouse was not effective in causing virus infection, IFN-a, -ß, and -? paraplegia. Furthermore, viremia was low abnormalities in combination were and transient in A/J mice compared with necessary for the mouse-adapted virus to be that in SCID or IFN-deficient AG129 mice. lethal when the ip challenge route was used. DEN-2 virus could not be isolated from the These results demonstrated that AG129 blood of infected mice; it could only be mice were a promising small animal model detected by sensitive RT-PCR in A/J mice. for DEN virus vaccine trials. Conclusion A/J mouse model Although the above-mentioned new small DEN virus infection causes DF and animal models, which mimic some of the DHF/DSS. No animal model is available that aspects of human DEN virus disease, may mimics this clinical manifestation. The facilitate not only the study of DEN immunocompetent mouse (A/J strain) was pathogenesis but also the evaluation of anti- reported as a mouse model for DEN virus DEN virus as well as vaccine development, infection that resembles the thrombo- there are still drawbacks in each model, cytopenia manifestation[21]. Intravenous especially in mimicing DHF/DSS. Presently, injection of DEN-2 virus into A/J mice the molecular mechanisms underlying the induced paraplegia at 2-3 weeks, while the pathogenesis of DHF/DSS remain unknown, mock-infected controls were normal. such as the receptors of DEN virus which Viremia detected by RT-PCR was found are still not clear. Even though the use of transiently at two days but at no other time transgenic animals has been proposed in the after infection. Although A/J mice developed quest for an animal model, it is apparent Dengue Bulletin – Vol 28, 2004 171 The Animal Models for Dengue Virus Infection that one needs to know more about the Acknowledgment mechanisms involved in the pathogenesis of DHF/DSS at the molecular level before one This work was partially supported by grants can construct a transgenic animal to serve as nos. 30170848 and 30300303 from the a model for use in research on the National Science Foundation of China pathogenesis, vaccine development and (NSFC). therapy for DHF/DSS. References [1] Rico-Hesse R, Harrison LM, Salas RA, Tovar combined immunodeficient (SCID) mouse D, Nisalak A, Ramos C, Boshell J, de Mesa as an animal model for dengue viral MT, Nogueira RM and da Rosa AT. Origins infection. The American Journal of Tropical of dengue type 2 viruses associated with Medicine and Hygiene, 1995, 52: 468-476. increased pathogenicity in the Americas. [9] Lin YL, Liao CL, Chen LK, Yeh CT, Liu CI, Virology, 1997, 230: 244-251. Ma SH, Huang YY, Huang YL, Kao CL and [2] Gubler DJ. Dengue viruses. In R.G. Webster King CC. Study of dengue virus infection in and A. Granoff (Eds.), Encyclopedia of SCID mice engrafted with human K562 cells. virology. Academic Press, Inc., San Diego, Journal of Virology, 1998, 72: 9729-9737. California, 1994: 324-331. [10] Rajajee S and Mukundan D. Neurological [3] Schlesinger RW. Dengue virus. New York, manifestations in dengue hemorrhagic fever. Spriger-Verlag, 1977: 72-73. Indian Pediatrics, 1994, 31: 688-690. [4] Bosma GC, RP Custer and MJ Bosma. A [11] Thisyakorn U and Thisyakorn C. Dengue severe combined immunodeficiency infection with unusual manifestations. mutation in the mouse. Nature, 1983, 301: Journal of the Medical Association of 527-530. Thailand, 1994, 77: 410-413. [5] Mosier DE. Immunodeficient mice [12] Kuo CH, Tai DI, Chien CSC, Lan CK, Chiou xenografted with human lymphoid cells: SS and Liaw YF. Liver biochemical tests and new models for in vivo studies of human dengue fever. The American Journal of immunobiology and infectious diseases. Tropical Medicine and Hygiene, 1992, 47: Journal of Clinical Immunology, 1990, 10: 265-270. 185-191. [13] Marianneau P, Megret F, Olivier R, Morens [6] Mosier DE. Viral pathogenesis in hu-PBL- DM and Deubel V. Dengue 1 virus binding SCID mice. Seminars in Immunology, 1996, to human hepatoma HepG2 and simian 8: 255-262. Vero cell surface differs. Journal of General Virology, 1996, 77: 2547-2554. [7] McCune JM, Namikawa R, Shih CC, Rabin L and Kaneshima H. Suppression of HIV [14] Marianneau P, Cardona A, Edelman L, infection in AZT-treated SCID-hu mice. Deubel V and Despres P. Dengue virus Science, 1990, 247: 564-566. replication in human hepatoma cells activates NF-kB which in turn reduces [8] Wu S-JL, Hayes CG, Dubois DR, apoptotic cell death. Journal of Virology, Windheuser MG, Kang Y-H, Watts DM and 1997, 71: 3244-3249. Sieckmann DG. Evaluation of the severe 172 Dengue Bulletin – Vol 28, 2004 The Animal Models for Dengue Virus Infection [15] An J, Kimura-Kuroda J, Hirabayashi Y and [19] Cole GA and Wisseman CL Jr. Pathogenesis Yasui K. Development of a novel mouse of type 1 dengue virus infection in suckling, model for dengue virus infection. Virology, weanling and adult mice. 1. The relation of 1999, 263: 70-77. virus replication to interferon and antibody [16] Atrasheuskaya A, Petzelbauer P, Fredeking formation. American Journal of TM and Ignatyev G. Anti-TNF antibody Epidemiology, 1969, 89: 669-680. treatment reduces mortality in experimental [20] Johnson AJ and Roehrig JT. New mouse dengue virus infection. FEMS Immunology model for dengue virus vaccine testing. and Medical Microbiology, 2003, 35: 33-42. Journal of Virology, 1999, 73: 783-786. [17] Kurane I, Innis BL, Nimmannitya S, Nisalak [21] Huang K-J, Li S-YJ, Chen S-C, Liu H-S, Lin A, Meager A, Janus J and Ennis FA. YS, Yeh T-M, Liu C-C and Lei H-Y. Activation of T lymphocytes in dengue virus Manifestation of thrombocytopenia in infections. High levels of soluble interleukin dengue-2-virus-infected mice. Journal of 2 receptor, soluble CD4, soluble CD8, General Virology, 2000, 81: 2177–2182. interleukin 2, and interferon-gamma in the sera of children with dengue. Journal of Clinical Investigation, 1991, 88: 1473-1480. [18] Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A and Ennis FA. High levels of interferon alpha in the sera of children with dengue virus infection. The American Journal of Tropical Medicine and Hygiene, 1993, 48: 222-229. Dengue Bulletin – Vol 28, 2004 173 Philippine Species of Mesocyclops (Crustacea: Copepoda) as a Biological Control Agent of Aedes aegypti (Linnaeus) Cecilia Mejica Panogadia-Reyes*#, Estrella Irlandez Cruz** and Soledad Lopez Bautista*** *Department of Biology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines **Research Institute for Tropical Medicine, Alabang, Muntinlupa, MM, Philippines ***Department of Medical Technology, Emilio Aguinaldo College, Ermita, Manila, MM, Philippines Abstract The predatory capacity of two local populations of Mesocyclops aspericornis (Daday) and Mesocyclops ogunnus species were evaluated, for the first time in the Philippines, as a biological control agent for Aedes aegypti (L) mosquitoes. Under laboratory conditions, Mesocyclops attacked the mosquito first instar larvae by the tail, side and head. The mean of first instar larvae consumed by M. aspericornis and M. ogunnus were 23.96 and 15.00, respectively. An analysis of the variance showed that there was a highly significant difference between the mean number of first instar mosquito larvae consumed by M. aspericornis and by M. ogunnus, which indicated that the former is a more efficient predator of dengue mosquito larvae. The results of the small-scale field trials showed that the mean number of surviving larvae in experimental drums was 63.10 and in control drums was 202.95. The Student t-test of means indicated that there was a significant difference between the mean number of surviving larvae in the drums with and without M. aspericornis. The findings indicated that M. aspericornis females were good biological control agents, for they destroyed/consumed about two-thirds of the wild dengue mosquito larvae population. Keywords: Mesocyclops aspericornis, Mesocyclops ogunnus, biological control agent, Aedes aegypti, Aedes albopictus, Philippines. Introduction aquatic animals of their own size including small fishes and mosquito larvae. Some Copepods feed on paramecium (ciliates adults are able to tear pieces out of the protozoans) while naupli feed on body of their victims with their strong Chilomonas spp. The adult planktonic mandibles. In the Philippines, around 14 copepods utilize diatoms as the principal species of Cyclops have been recorded, of food, while predacious adult copepods which Mesocyclops aspericornis and feed on protozoa, rotifers and several M. ogunnus are more common[1,2]. # E-mail: [email protected] 174 Dengue Bulletin – Vol 28, 2004 Mesocyclops as a Biological Control Agent of Aedes aegypti In Brazil, a study[3] reported that under Laboratory trials laboratory conditions, four different strains of M. aspericornis showed the potential as In a 600 ml beaker, 500 ml filtered tap biological control agents of Aedes aegypti water with pH 7 was poured. Then 50 larvae. In Viet Nam, under laboratory Aedes aegypti L1 obtained from laboratory conditions, M. aspericornis consumed a culture and a female M. aspericornis were mean 23.75 L1 and killed a mean 13.43 added at the same time. The same L1, or a total of 37.18 L1 within 24 hours. procedure was done for M. ogunnus. The M. ogunnus, on the other hand, consumed control group did not receive Mesocyclops. a mean 8.48 L1 and killed a mean 7.54 L1 The experiment was replicated six times or a total of 16.02 L1 within 24 hours[4]. In and was observed every day for five Australia, six species of Mesocyclops were consecutive days, with daily replacements evaluated as biological control agents of of new L1. The daily number of L1 Aedes aegypti[5]. Of these, M. aspericornis destroyed or consumed by M. aspericornis was found to be the most effective and M. ogunnus and those that died in the predator. This study attempted to evaluate, control group were determined. for the first time, the potential of the local Copepods’ feeding behaviour was population of two species of Mesocyclops observed. as biological control agents of Aedes aegypti (L) under laboratory and field Field trials conditions in the Philippines. Permission to conduct the study in Estero de Tanque located at P Nieto Street, Methodology Barangay 674, Zone 73, Paco, Manila, was secured from the community health Mesocyclops culture officials and Barangay chairperson. Members of the households were Mesocyclops aspericornis and M. ogunnus informed regarding the procedures to be were raised in laboratory following the undertaken and the possible benefit they techniques adopted by Marten and could derive from it. Consent from the Thomson[6]. To ensure the establishment of caretakers of the sample households was a single species culture, a female with egg also secured. sacs was captured and placed in a petri A preliminary study was conducted for dish for examination under a dissecting 15 days. Twenty houses were chosen as microscope before it was transferred to a study sites. Two drums per household or a wide-mouthed beaker containing 100 ml total of 40 drums were emptied and filled of mixed culture of Paramecium caudatum almost to the brim with tap water, pH 7.2. and Chilomonas sp. (food of Mesocyclops). A litre of water as sample from each drum Populations of Chilomonas sp. and container was collected in sterile bottles P. caudatum in the culture bottle were and brought to the laboratory to exclude maintained using sterile wheat seeds. the presence of fungi, bacteria and Sample specimens from copepods culture indigenous copepods. Mosquito eggs that were sent to Maria Holynska, Museum hatched into larvae in drums were and Institute of Zoology, Warsaw, Poland, monitored and collected daily for for identification. Dengue Bulletin – Vol 28, 2004 175 Mesocyclops as a Biological Control Agent of Aedes aegypti recording and identification. Water pH, Results and discussion water temperature and ambient temperature were taken daily. Drums Laboratory trials were checked daily for the presence of mosquito eggs and larvae with the aid of a The results of the predatory capacity of magnifying glass. Drums with mosquito M. aspericornis and M. ogunnus as larvae were marked and female copepods evaluated under laboratory conditions and were introduced in experimental drums analysis of variance are presented in using the ratio one Mesocyclops Tables 1 and 2. The mean L1 consumed aspericornis per 50 Aedes aegypti first by M. aspericornis was 23.96 while that of instar larvae (L1). No copepods were M. ogunnus was 15.00. Control means introduced in the 20 control drums. were 0.63 L1 and 0.60 L1, respectively. Observation was made daily for 15 The findings showed that there was a consecutive days. Surviving larvae were highly significant difference between the collected from all drums and brought to mean number of Ae. aegypti L1 consumed the laboratory for recording and species by M. aspericornis and that of M. ogunnus identification. and the control group. Table 1. Predatory capacity of female Mesocyclops aspericornis vs Aedes aegypti first instar larvae in 500 ml filtered water under laboratory conditions Statistical Treatment Mean F value Tabular f significance Experimental group 23.96 338.83 7.12 Highly significant Control group 0.63 Table 2. Predatory capacity of female Mesocyclops ogunnus vs Aedes aegypti first instar larvae in 500 ml filtered water under laboratory conditions Statistical Treatment Mean F value Tabular f significance Experimental group 15.00 319.18 7.12 Highly significant Control group 0.60 To determine if there was a difference difference between the mean number of between the mean number of L1 L1 consumed by M. aspericornis and by consumed by M. aspericornis and M. ogunnus. The study showed that M. ogunnus, the analysis of variance was M. aspericornis was a more efficient carried out (Table 3). The findings showed predator of Aedes aegypti larvae. that there was a highly significant 176 Dengue Bulletin – Vol 28, 2004 Mesocyclops as a Biological Control Agent of Aedes aegypti Table 3. Comparison of the predatory capacity of M. aspericornis and M. ogunnus under laboratory conditions Statistical Treatment Mean F value Tabular f significance M. aspericornis 23.96 36.11 7.12 Highly significant M. ogunnus 15.00 Table 4. Predatory capacity of Mesocyclops aspericornis vs Aedes larvae in small-scale field trials using drum containers Statistical Treatments # Replicates Mean + SD P value significance Experimental group 20 63.10 + 59.43 0.0002 Significant Control group 20 202.95 + 140.43 Field trials laboratory and field conditions. These copepods could be effectively used for the The results of the field trials are presented in control of Aedes breeding in non-removable Table 4. containers, viz. drums and used tyres. The mean L1 in experimental drums was 63.10 and 202.95 L1 in control drums. To determine the difference between the mean Acknowledgements number of surviving larvae in the drums with The World Health Organization and the M. aspericornis and in the drums without M. Yaman Lahi Foundation, Inc. – Emilio aspericornis, a Student t-test was used. The Aguinaldo College provided financial findings indicated that there was a significant support for this project. We acknowledge difference between the mean number of the kind assistance of Dr Kevin Palmer of surviving larvae in the drums with and the WHO Western Pacific Region; Dr Jose without Mesocyclops. This significant Paulo E. Campos of Yaman Lahi Foundation, difference suggests that M. aspericornis is a Inc. – Emilio Aguinaldo College (YLFI-EAC), good biological control agent, for it Philippines; Dr Remigio M. Olveda of the consumed about two-thirds of the wild, Department of Health – Research Institute dengue mosquito larvae population. for Tropical Medicine (DOH-RITM), Philippines; Dr Maria Holynska of Museum and Institute of Zoology – Polish Academy Conclusion of Science (MIZ-PAS), Poland; Dr Vu Sinh The results of the study showed that Nam of the National Institute of Hygiene Mesocyclops aspericornis was an efficient and Epidemiology (NIHE), Viet Nam; and predator of Aedes aegypti larvae both in Dr Brian H. Kay of the Queensland Institute of Medical Research (QIMR), Australia. Dengue Bulletin – Vol 28, 2004 177 Mesocyclops as a Biological Control Agent of Aedes aegypti References [1] Mamaril AC and Fernando CH. Freshwater [4] Nam VS, Tien TV, Huan TQ, Yen NT, Kay B, zooplankton of the Philippines (Rotifera, Marchand R, Marten G, Holynska M and Cladocera, and Copepoda). Natural and Reid J. Mesocyclops of Vietnam Part I - Applied Science Bulletin, 1978, 30(4): 109- laboratory evaluation as biological agent for 221. control of Aedes aegypti. Dengue Bulletin, 1999, 23: 89-93. [2] Tuyor JB and Baay MO. Contribution to the knowledge of freshwater copepods of the [5] Brown MD, Kay BH and Hendrikz JK. Philippines. Asia Life Sciences, 2001, 10(1): Evaluation of Australian Mesocyclops 35-43. (Cyclopoida: Cyclopidae) for mosquito control. J Med Entomol, 1991, 28(5): 618- [3] Kay BH, Cabral CP, Sleigh AC, Brown MD, 623. Ribeiro ZM and Vasconcelos AW. Laboratory evaluation of Brazilian [6] Marten G and Thompson G. Copepod Mesocyclops (Copepoda: Cyclopidae) for production and application for control mosquito control. J Med Entomol, 1992, procedure for New Orleans Mosquito Board, 29(4): 599-602. City of New Orleans, USA, 1997. 178 Dengue Bulletin – Vol 28, 2004 Susceptibility of Aedes aegypti to Insecticides in Viet Nam Vu Duc Huong#, Nguyen Thi Bach Ngoc, Do Thi Hien and Nguyen Thi Bich Lien Department of Entomology, National Institute of Malariology, Parasitology and Entomology, B.C. 10 200 Tu Liem, Hanoi, Viet Nam Abstract During 2000-2002, studies on the susceptibility of Aedes aegypti to insecticides were conducted at 22 places in 11 provinces and cities in four different regions of Viet Nam. Aedes aegypti was found susceptible to malathion, but resistant to DDT in almost all the study sites. It continues to be susceptible to the pyrethroid group of insecticides (permethrin, lambda-cyhalothrin, deltamethrin and alpha- cypermethrin) in many places in the North and Centre regions, but is resistant to these insecticides in many places in the South and Central Highlands in Viet Nam. However, the species was found highly and widely resistant to etofenprox. Keywords: Aedes aegypti, pyrethroids, insecticides, Viet Nam. Introduction Both malaria and DHF were endemic in many mountainous, forested and coastal Insecticidal measures, especially in the plain areas of Viet Nam where house spray outbreak-risk areas, are the most important and bednet treatment were applied for for the control of Aedes aegypti, the main years. DDT was widely used before 1990 vector of DHF. Many insecticides of the and later lambda-cyhalothrin, permethrin group organochlorine (DDT), organo- and deltamethrin[5] were introduced. In phosphorous (fenthion, malathion and 1999, Aedes aegypti was found resistant to temephos) and pyrethroid (permethrin, DDT and some insecticides of the deltamethrin, lambda-cyhalothrin, etc.) have pyrethroid group in many places in Nam Bo been used for the malaria control programme (the South), Central Highlands, but not yet [6] and for Aedes aegypti. Aedes aegypti has to malathion . This study provides more data on the susceptibility of Aedes aegypti to been resistant to DDT since the early 1960s insecticides in different regions of Viet Nam. and cross-resistant to many insecticides of the pyrethroid and temephos groups in many countries, but is not yet resistant to Materials and methods malathion[1-3]. When this species is resistant to the insecticides of the pyrethroid group, Time and study regions the organophosphorous ones could take their During 2000-2002, studies were conducted [4] place . at 22 places (located in 11 provinces and # E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 179 Susceptibility of Aedes aegypti to Insecticides in Viet Nam cities) – six places in the North, six in the The susceptibility to the insecticides was Centre, six in the South and four in the evaluated on the following criteria: Central Highlands. • Mortality 98-100%: Susceptible to insecticide. Methods • Mortality 80-97%: Possibility of The WHO standard bioassay tests (1998)[7] resistance to insecticide. were followed and the papers treated with • Mortality <80%: Resistant to insecticide. DDT 4%, the control paper with OC (organochlorine control), malathion 5% and the control paper with OP (organophosphate Results control) and 5 insecticides of the pyrethroid group (permethrin 0.75%, lambda- North cyhalothrin 0.05%, deltamethrin 0.05%, Of the six study places, Aedes aegypti was alpha-cypermethrin 30mg/m2 and etofenprox found resistant to DDT at five places and 0.5%) with the same control paper PY possibly resistant to DDT at one place; it (pyrethroid control). was susceptible to malathion at five places The tests were done at a temperature of and possibly resistant to this insecticide at 25 °C ± 2 °C and humidity of 75-85%. one place. It was susceptible to all the tested The unfed, F1 mosquitoes, one or two days insecticides of the pyrethroid group such as old – at least 150 mosquitoes for each permethrin, lambda-cyhalothrin, deltamethrin insecticide, 100 for the test and 50 for the and alpha-cypermethrin in at five places and control, were used. Twenty-five mosquitoes the possibility of resistance to these four were put in each test tube and the per cent insecticides existed at one place. Aedes mortality count was done 24 hours after the aegypti was resistant to etofenprox at four exposure. The mosquitoes in the resting places and possibly resistant to etofenprox at tubes were then fed with glucose 10% in two places (Table 1). soaked cotton. Table 1. Results of suceptibility tests on Aedes aegypti to insecticides in the North and Centre, Viet Nam % Mortality Lambda - Alpha- S. No. Places Permethrin Deltamethrin Etofenprox DDT Malathion cyhalothrin cypermethrin 0.75% 0.05% 0.05% 4% 5% 0.05% 30mg/m2 1. Thi Cau (Co) 100 100 100 100 68 57 100 Bac Ninh (T) Bac Ninh (P) 2. Phu Lang (Co) 100 100 100 100 92 94 100 Que Vo (D) Bac Ninh (P) 3. Cat Ba (S) 100 100 100 100 42 14 100 Cat Hai (D) Hai Phong (C) 180 Dengue Bulletin – Vol 28, 2004 Susceptibility of Aedes aegypti to Insecticides in Viet Nam % Mortality Lambda - Alpha- S. No. Places Permethrin Deltamethrin Etofenprox DDT Malathion cyhalothrin cypermethrin 0.75% 0.05% 0.05% 4% 5% 0.05% 30mg/m2 4. Niem Nghia (Co) 91 92 94 95 92 21 100 Le Chan (D) Hai Phong (C) 5. Ly Thai To (Co) 99 96 (200) 100 100 18 21 97.33 Hoan Kiem (D) (150) Ha Noi (C) 6. Thinh Liet (Co) 100 99 98 98 37 60 98 Thanh Tri (D) Ha Noi (C) 7. Thach Phu (Co) 100 100 100 100 100 63 100 HaTinh (T) Ha Tinh (P) 8. Duc Tho (S) 100 100 100 100 93 37.39 100 Duc Tho (D) (115) Ha Tinh (P) 9. Song Cau (S) 100 100 100 98 51 4 100 Song Cau (D) Phu Yen (P) 10. No. 6 (Co) 100 100 100 91 76 2 100 Tuy Hoa (T) Phu Yen (P) 11. Dong Luong (Co) 94 96 97 95 16 2 100 Dong Ha (T) Quang Tri (P) 12. Trieu Do (Co) 100 100 100 100 86 11 100 Trieu Phong (D) Quang Tri (P) No. 1 - 6 in the North No. 7 - 12 in the Centre Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City Figures in parenthesis indicate the number of mosquitoes tested Central possibly resistant and susceptible to etofenprox at two and one places, Aedes aegypti was found resistant to DDT respectively (Table 1). but was susceptible to malathion in all six places. It was susceptible to four insecticides South of the pyrethroid group such as permethrin, lambda-cyhalothrin, deltamethrin and Aedes aegypti was resistant to DDT at all six alpha-cypermethrin in five places and places; but susceptible to malathion at four possibly resistant to them in one place, and places and possibility of resistance to resistant to etofenprox in three places, and malathion at two places; it was resistant to Dengue Bulletin – Vol 28, 2004 181 Susceptibility of Aedes aegypti to Insecticides in Viet Nam permethrin and lambda-cyhalothrin at four resistance to alpha-cypermethrin at two places and the possibility of resistance to places and possibility of resistance to alpha- permethrin and lambda-cyhalothrin at twor cypermethrin at four places as well as places. It also showed resistance to resistance to etofenprox at all six places deltamethrin at one place and possibility of (Table 2). resistance to deltamethrin at five places; Table 2. Results of susceptibility tests on Aedes aegypti to insecticides in the South and Central Highlands, Viet Nam % Mortality Lambda - Alpha- No. Places Permethrin Deltamethrin Etofenprox DDT Malathion cyhalothrin cypermethrin 0.75% 0.05% 0.05% 4% 5% 0.05% 30mg/m2 1. No. 6 (Co) 62 67 90 82 4 2 81 Ben Tre (T) Ben Tre (D) 2. BinhThuan (Co) 94 90 97 92 53 20 100 Binh Dai (D) Ben Tre (P) 3. Binh Khanh (Co) 18 24 84 75 1 0 99 Can Gio (D) Ho Chi Minh (C) 4. Binh Trung Tay (Co) 96 93 97 96 20 11 95 No. 2 (D) Ho Chi Minh (C) 5. An Loc (S) 79 56 84 96 1 0.8 90 Binh Long (D) (125) Binh Phuoc (P) 6. Tan Xuan (Co) 8 28 19.33 25 7 2 99 Dong Xoai (T) (150) Binh Phuoc (P) 7. Plei Can (S) 36 32 51 28 1 1 100 Ngoc Hoi (D) Kon Tum (P) 8. Quyet Thang (Co) 57 61 66 47 1 1 100 Kon Tum (T) Kon Tum (P) 9. Buon Trap (Co) 5 11 41 40 0 6 97 Krong Ana (D) Dak Lak (P) 10. Thang Loi (Co) 24 36 63 34 23 0 98 Buon Me Thuot (C) Dak Lak (P) No. 1 - 6 in the South No. 7 - 10 in the Central highlands Co: Commune; S: Small town; D: District; T: Town; P: Province; C: City Figures in parenthesis indicate the number of mosquitoes tested 182 Dengue Bulletin – Vol 28, 2004 Susceptibility of Aedes aegypti to Insecticides in Viet Nam Central Highlands were not completely comparable with the observations made by Reiter and Gubler Aedes aegypti was resistant to DDT, (1997)[3], and Vu Duc Huong and Nguyen permethrin, lambda-cyhalothrin, deltamethrin, Thi Bach Ngoc (1999)[6]. Aedes aegypti was alpha-cypermethrin and etofenprox at all possibly resistant to malathion in some four places in this region and susceptible to places. This discrepancy in different regions malathion at three places and showed was possibly due to the longer and more possibility of resistance to malathion at one extended use of the insecticides of the place (Table 2). pyrethroid group in malaria and dengue haemorrhagic fever control programmes and in agriculture in the Southern and Central Discussion Highlands. It is therefore suggested that the Aedes aegypti was susceptible to malathion susceptibility tests should be conducted on and resistant to DDT at almost all study all insecticides before use. Moreover, the places in Viet Nam. It was still susceptible to cross-resistance of Aedes aegypti to the four insecticides of the pyrethroid group insecticides belonging to the pyrethroid (permethrin, lambda-cyhalothrin, deltamethrin group should also be checked. Aedes and alpha-cypermethrin) in many places in aegypti was highly and widely resistant to North and Centre Viet Nam, but resistant to etofenprox and further studies should be these insecticides in many places in the conducted in this context. South and Central Highlands. These results References [1] Yap HH, Cheng NL, Foo AES and Lee CY. [5] Phan VT. Malaria epidemiology and malaria Dengue vector control: present status and control in Vietnam. Medical Publishing future prospects. Kaoshiung J Med Sci, House, Hanoi, 1996: 218-241 (in 1994,10: 102-108. Vietnamese). [2] World Health Organization. Dengue [6] Huong VD and Bach Ngoc NT. haemorrhagic fever: diagnosis, treatment, Susceptibility of Aedes aegypti in south prevention and control. 2nd edn. Geneva: Vietnam. Dengue Bulletin, 1999, 23: 85-88. WHO, 1997: 48-59. [7] World Health Organization. Report of the [3] Reiter P and Gubler DG. Surveillance and WHO informal consultation. Test control of urban dengue vectors. Dengue procedures for insecticide resistance and dengue haemorrhagic fever. Colorado: monitoring in malaria vector, bio-efficacy CAB International, 1997: 425-454. and persistence of insecticides on treated surfaces. Geneva: WHO, 1998. Document [4] Kantachuvesiri A. Dengue haemorrhagic WHO/CDC/CPC/MAL/98.12: 1-43. fever in Thai society. Southeast Asian J Trop Med Public Heath, 2002, 33(1): 56-67. Dengue Bulletin – Vol 28, 2004 183 Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera: Studies on Refractory Behaviour and its Sustenance across Gonotrophic Cycles Manju Singhi, Vinod Joshi#, R.C. Sharma and Keerti Sharma Desert Medicine Research Centre (Indian Council of Medical Research), New Pali Road, Jodhpur 342 005, India Abstract Dengue fever associated with dengue haemorrhagic fever is gaining endemicity in India. Due to lack of any chemotherapy against this arboviral infection, the control of the disease depends largely on preventive measures against Aedes mosquito vectors. A wild shrub, Calotropis procera, commonly growing in the desert areas of Rajasthan has shown a remarkable effect as a larvicide against Aedes aegypti. However, different water concentrations of this biocide have also brought forward very important observations on the ovipositioning behaviour of Aedes aegypti. At 0.7% concentration of latex, the ovipositiong was avoided by the gravid female mosquitoes and this behaviour continued till three gonotrophic cycles. However, at lower concentrations (0.2% and 0.1%) of the larvicidal latex, the refractory behaviour of ovipositioning could not be retained up to the third gonotrophic cycle. The concentration of latex such as 0.7% and 0.2% were observed as ovicidal also and this effect continued across all the gonotrophic cycles. The behavioural observations reported in the present study may serve as significant information on choosing bio-larvicides for vector control against dengue. Keywords: Dengue, Aedes aegypti, Calotropis procera, ovipositioning, refractory behaviour, gonotrophic cycle. [2-4] Introduction have been reported . It has also been established that dengue virus undergoes Dengue fever and dengue haemorrhagic transovarial transmission across generations fever (DF/DHF) is gaining endemicity in of Aedes aegypti under natural as well as many states in India[1]. In the absence of experimental conditions[5,6]. Latex of chemotherapy and vaccines, vector control, Calotropis procera, a milky weed plant largely based on larval control, is the only growing all across the desert areas in the option available. In Rajasthan, a number of state has shown promising larvicidal epidemics of dengue associated with DHF properties in a series of laboratory # E-mail: [email protected] 184 Dengue Bulletin – Vol 28, 2004 Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera experiments carried out against Aedes The experiments were conducted at 25- aegypti (unpublished data, Desert Medicine 30 °C (room) temperature and at relative Research Centre). In an attempt to assess humidity of about 60-70%. The eggs of each the value of this agent as an oviposition generation were counted under a dissecting attractant for use in ovitraps, it was microscope in the control as well as discovered that the agent showed experimental sets. All the eggs were oviposition refractoriness instead. The immersed in plain water to observe whether present communication incorporates the exposure to larvicide had any ovicidal effect findings of these studies. on them. Materials and methods Results and discussion Six experimental sets were designed for the Table 1 shows relative observations on study. Cages of 30 cm3 size, with wooden different concentrations of latex of frame and iron mesh with muslin cloth on Calotropis procera on the ovipositioning one side, were used as units of present behaviour of Aedes aegypti. In experimental experiments. In each cage, 16 gravid cage A, while in the control set, 65 eggs females of Aedes aegypti were released. The were laid, in the corresponding lethal concentration of latex in water, which concentration of 0.7% of the same cage, no showed the highest (0.7%), moderate (0.2%) eggs were laid. In this cage all the and no mortality (0.1%) effect in the mosquitoes showed persistent refractiveness larvicidal efficacy experiments, were of ovipositioning across all gonotrophic prepared and put in beakers in the cages A, cycles from G1-G3. In cage B where 0.2% B, C, D and E while cage F was kept without concentration was offered along with latex solution and contained only water. A control, refractive behaviour up to two beaker containing plain water was also cycles only was observed while in the third placed in each of the six cages to serve as cycle (G3), 127 eggs were laid. Similar control. While in cage D all the observations were made in cage C. It was experimental concentrations were placed interesting to note that in cage D where all along with the control, in cage E all the the experimental concentrations were concentrations were placed without choice offered to mosquitoes along with choice of of a control. In the sixth cage (cage F) two control, even in G1 cycle no refractiveness beakers with plain water were placed was shown. In experiment E where choice without any experimental lethal of control (plain water) was not offered, concentration. The eggs laid by female maximum preference for ovipositioning was Aedes aegypti were counted after 48 hours shown in lowest concentration (0.1%). The in each of the experimental cages. Second different preference among different and third blood meals were provided to concentrations continued across all the facilitate G2 and G3 (gonotrophic cycles) to gonotrophic cycle (Table 1). mosquitoes. Dengue Bulletin – Vol 28, 2004 185 Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera Table 1. Ovipositing preference of Aedes aegypti in different larvicidal concentrations of latex of Calotropis procera Latex concentration (%) in experimental cages Cage A B C D E F Eggs laid C 0.7 C 0.2 C 0.1 C 0.1 0.2 0.7 0.1 0.2 0.7 C C within 48 hrs G1 65 0 156 0 180 0 109 18 18 6 101 51 11 102 110 G2 93 0 156 1 149 0 100 0 0 0 33 10 7 96 83 G3 275 0 230 127 275 52 270 98 31 0 215 150 20 233 197 C: Control G1-G3: Gonotropic cycles Table 2. Effect of latex on the percentage viability of eggs of Aedes aegypti Experimental cages Cage A B C D E F Latex (%) C 0.7 C 0.2 C 0.1 C 0.1 0.2 0.7 0.1 0.2 0.7 C C Eggs 65 0 156 0 180 0 109 18 18 6 101 51 11 102 110 immersed Eggs 55 0 78 0 126 0 78 0 0 0 6 2 0 96 103 hatched % eggs 84.6 0.0 50 0.0 70 0.0 73 0.0 0.0 0.0 5.9 4.0 0.0 93.1 93.6 hatched C: Control Table 2 shows the results of the Dengue fever associated with DHF has experiment carried out to study the viability become a problem of public health of eggs that were exposed to different importance. Available evidence shows that larvicidal concentrations and to control the virus undergoes vertical transmission groups. The eggs laid in experiments A, B, C across generations of mosquitoes[6]. Larval and D showed no hatching, while in control, therefore, is the most effective experimental cage E where no control had approach to restrict the vector and virus been kept, the eggs laid up to a sustenance in nature. The wildly grown concentration 0.2% showed some viability plant, Calotropis procera, has shown very and in cage F where only controls were kept encouraging results in its different lethal the laid eggs showed 93% viability. concentrations. However, if this plant 186 Dengue Bulletin – Vol 28, 2004 Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera species is to be used as a material of choice, domestic mosquito fauna in such premises its other aspects such as the ovipositioning will lay the eggs but they will lose their behaviour of gravid females towards the viability to hatch into larvae (Table 2). larvicide can also become known. The role of Calotropis procera as a The significance of the reported larvicide has been reported by other workers observations is that the refractiveness from India[7]. However, contrary results have developed by the species is sustained across been reported by some workers[8] where two gonotrophic cycles when one larvicidal insect growth regulators actually enhanced concentration was offered with a ovipositioning. corresponding control (Table 1). However, The observations reported by us not only when all the concentrations viz. 0.1 %, present the results of the ovipositioning 0.2 % and 0.7% were of fered with one behaviour induced by a bio-larvicide, but control, such avoidance was not shown. also add the information on relative Similarly, in the experimental set E where all preference in ovipositioning and its concentrations of latex were made available sustenance across gonotrophic cycles when without any control, the maximum egg different larvicidal concentrations were laying was preferred in 0.1 % of latex. The offered. data suggest fine chemo-sensation in Aedes aegypti where the ovipositioning female could distinguish to choose the least Acknowledgements larvicidal concentration for its egg laying. The authors gratefully acknowledge the The observation showed that if in the guidance and inspiration received from various types of domestic containers where Mr N.L. Kalra, Member, Scientific Advisory water is stored a non-lethal concentration of Committee of the Desert Medicine Research latex (0.1%) is used, the refractiveness of Centre, Jodhpur, India. ovipositioning will not be there and the References [1] Dengue alert in South-East Asia Region. outbreak of febrile illness in Jaipur city, India. New Delhi: World Health Organization, Indian Journal of Medical Research, 1973, Regional Office for South-East Asia, 2004 61(12): 1737-1743. (http://w3.whosea.org/index.htm, accessed [4] Chouhan GS, Rodrigues FM, Shaikh BH, 25 August 2004). Ilkal MA, Khangaro SS, Mathur KN, Joshi KR [2] Ghosh SN and Sheikh BH. Investigations on and Vaidhye NK. Clinical and virological the outbreak of dengue fever in Ajmer city, study of dengue fever outbreak in Jalore city, Rajasthan. Part II: Results of serological tests. Rajasthan, 1985, Indian Journal of Medical Indian Journal of Medical Research, 1974, Research, 1990, 91: 414-418. 62: 523-533. [5] Joshi V, Singhi M and Chaudhary RC. [3] Padbidri VS, Dandawate CN, Goverdhan Transovarial transmission of dengue 3 virus MK, Bhat UK, Rodrigues FM, D'Lima LV, by Aedes aegypti. Transaction of Royal Kaul HN, Guru PY, Sharma R and Gupta NP. Society of Tropical Medicine and Hygiene, An investigation of the etiology of the 1971 1996, 90: 643-644. Dengue Bulletin – Vol 28, 2004 187 Ovipositioning Behaviour of Aedes aegypti in Different Concentrations of Latex of Calotropis procera [6] Joshi V, Mourya D and Sharma RC. [7] Girdhar G, Deval K, Mittal PK and Persistence of vertical transmission of Vasudevan P. Mosquito control by dengue-3 virus through vertical transmission Calotropis latex. Pesticides, 1984, 26-29. passage in successive generations of Aedes [8] Moore CG. Insecticide avoidance by aegypti mosquitoes. American Journal of ovipositioning Aedes aegypti. Mosquito Tropical Medicine and Hygiene, 2002, News, 1977, 37: 291-293. 67(2): 158-161. 188 Dengue Bulletin – Vol 28, 2004 Community-based Assessment of Dengue-related Knowledge among Caregivers Khynn Than Win*#, Sian Za Nang** and Aye Min*** *Health Systems Research Division, Department of Medical Research (Lower Myanmar), Myanmar **Health Education Bureau, Department of Health Planning, Myanmar ***Vector Borne Disease Control Programme, Department of Health, Myanmar Abstract The study was conducted in Thaketa township in Myanmar involving 405 respondents aged 18 years and above. It was aimed: (i) to explore the extent of dengue-related knowledge among caregivers; (ii) to identify the exposure of community members to the existing IEC materials; and (iii) to find out the factors related to high knowledge scores. The findings were triangulated by results from personal interviews, focus group discussions and observational checklist. The difference of mean scores among males and females was not statistically significant. Knowledge scores of the caregivers were not statistically different whether there was a primary DHF case at home or not. Almost 60% of the interviewees had received information on DHF by watching television and they observed that television was the most effective medium. Females with more than six years of schooling, persons who had access to pamphlets/posters, television, newspapers and journals got higher scores than the unexposed group. Less than 15% were not exposed to any of the IEC materials. Aedes aegypti larvae were found in 67% of water storage tanks and 15.9% of flower vases when using observational checklist. Focus group discussions were held for drafting IEC materials. Community members were more interested in the mode of DHF transmission to children rather than in the elimination of the Aedes mosquitoes. A low practice score was observed in those with high knowledge level, which means that high knowledge does not necessarily lead to high practice. Less than half of the respondents had seen posters and pamphlets. IEC materials need to be improved so that they present the message most effectively and they should be extensively distributed in the community. Keywords: Dengue, caregivers, community, IEC material, knowledge score, Myanmar. Introduction termed DHF as one of the diseases under national surveillance. DHF is also listed as Dengue haemorrhagic fever (DHF) is the 17th priority disease in Myanmar. One of endemo-epidemic in 12 out of 14 states and the strategies devised in NHP for the divisions in Myanmar and is transmitted by prevention and control of DHF is Aedes aegypti. Most of the reported cases production of guidelines for basic health staff are under 15 years of age[1]. The Myanmar (BHS) as part of the information, education National Health Plan (NHP) (1996-2001) and communication (IEC) programme. # E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 189 Assessment of Dengue-related KAP among Caregivers IEC initiatives are based on the concepts materials; and (iii) to find out the factors of prevention and primary health care. They responsible for high knowledge scores. create awareness, increase knowledge, change attitudes and motivate people to adopt new ideas[2]. Methods and materials Communication participation appears This community-based cross-sectional study to be one of the most promising innovative was based on multistage sampling to identify means to prevent and control DHF. Simple 405 caregivers in Thaketa township, Yangon. elimination of vector-breeding water This area was one of the dengue endemic collections or “source reduction” is the regions in the Yangon division and the case- possible answer to the problem. Community fatality rate (CFR) was 1.65% in 2002. Both activities are identified mainly as reduction quantitative and qualitative data collection of non-essential water containers, protection methods, including observation checklist, of water containers from larvae breeding, were used in the study. The study sample larviciding and release of larvivorous fish. included household members aged 18 years Community participation needs to be and above. The households with children sustained by dissemination of health were selected randomly. In these messages through various channels[3]. households, we chose one subject from each household, regardless of sex. The Existing IEC materials in Myanmar respondent was a relative of the child included health talks routinely carried out in (mother/father/grandfather/grandmother/ schools and in the community. Health brother/sister/uncle/aunt). Mothers were the messages were distributed through radio, key persons to be interviewed after taking television, newspapers and journals before their consent. The questionnaire was pilot- and during the epidemic season. Pamphlets tested for clarity and validity; all questions were developed locally in states and were reviewed by epidemiologists, public divisions. However, it is necessary to find health experts, health educators and by out the most appropriate IEC materials and investigators experienced in conducting means that would be relevant for various community-based surveys in DHF. In order communities. to ensure the accuracy and completeness of This study attempted to improve the data, our surveyors were trained before and existing IEC materials on DHF control based after pre-testing. on the knowledge and practice of child-care Ten sessions of Focus Group providers in the Thaketa township of the Discussions (FGDs) were performed among Yangon division in Myanmar. DHF control basic health staff (BHS), general practitioners, will be more effective in the future by Maternal and Child Welfare Association strengthening community participation on (MCWA) members, other volunteers case information and source reduction. including Ward Law and Order Restoration This research aimed to: (i) explore the Council members, voluntary fire brigade members, etc., for recommendation of extent of dengue-related knowledge among existing IEC activities (including television, caregivers; (ii) identify the exposure of radio, newspapers, local journals and community members to the existing IEC pamphlets). In every FGD, the moderator 190 Dengue Bulletin – Vol 28, 2004 Assessment of Dengue-related KAP among Caregivers explained thoroughly the purpose of Table 1. Dengue-related knowledge conducting the discussions. The Township responses of caregivers Health Centre, Ward Law and Order Restoration Council offices and homes were Responses Frequency Percent chosen for holding FGDs. DHF is common in 254 44.4 children 3-8 years of age Caregivers were those who took care of children at home, or supervised at the DHF is transmitted by 331 81.7 the mosquito health centre or clinic, or advised parents on home care of a child with fever. Mosquito species of 204 61.6 DHF vector is Aedes Data checking, cleaning and validation were performed using Epi-info 6.0, and data Biting time of mosquitoes 266 80.4 is at daytime analysis was conducted using SPSS 10.0. P<0.05 was used as the definition of Aedes breed in statistical significance. The study period • clear water 135 33.3 lasted one year starting in June 2002. • polluted water 166 41.0 The Aedes mosquito 253 62.5 breeds inside the house 144 35.6 Results • flower vases 96 23.7 • ant traps About respondents The Aedes mosquito 292 72.1 The sex ratio of the respondents was 7:1 for breeds outside the house females and males. The mean age was • water containers 178 61.0 35.9±10.3. The majority of the respondents • old tyres, broken pots, 75 25.7 were aged 18-35 years, literate and and coconut shells dependants. About 8.6% of them had • blocked gutters 9 2.2 ³ received 12 years’ school education. DHF transmission is 348 85.9 Nearly 40% lived in their own wooden highest in the rainy houses. Most of the households (77.8%) had season 1 to 2 children under 15 years of age. Only DHF may be fatal 382 94.3 34 (8.4%) of the households that participated in the interview had a child There is a vaccine for 243 60.0 prevention of DHF with history of DHF. A previously infected 293 72.3 The difference of mean score among child may get DHF again. males and females was not statistically significant (P=0.271). If the child is febrile, 262 64.7 DHF should be observed Dengue-related knowledge responses The total knowledge scores were categorized as low (0-19) and high (20-39). The responses of caregivers are included in The percentage of the high score group was Table 1. more than that of the low score group Dengue Bulletin – Vol 28, 2004 191 Assessment of Dengue-related KAP among Caregivers (68.6% vs 31.4%). Signs and symptoms of Existing exposure to IEC material in DHF were fever (57.3%), vomiting (51.6%), the community purpura (36.3%), drowsiness (28.1%), cold extremities (17%), etc. Table 2 contains the responses of the communities to IEC materials. Knowledge score of respondents with and without history Table 2. Exposure to IEC materials in the of DHF case in their homes community Knowledge scores with and without past Community members exposed (n) Percent to IEC materials history of primary DHF cases at home are given in the Figure. • had seen pamphlets 197 48.6 • listened to radio 137 33.8 • watched on television 246 60.7 Figure. Past history of primary DHF cases • read in newspapers/journals 133 32.8 at home and knowledge scores • Percent exposed to any type 352 87.0 Low High of IEC 300 • Percent not exposed to any 53 13.0 258 IEC 250 • Percent exposed to all types 87 21.5 of IEC 200 Are the facts easily recognized? 150 • Yes 317 78.3 113 • No 23 5.7 100 Knowledge scores The percentage of people watching 50 14 20 television was the highest as compared to other types of exposure. Nearly half of the 0 respondents had seen the pamphlets about Present Absent DHF (48.6%). Exposure to radio talks and Past history of primary DHF cases at home information in newspapers and journals was very low. The extent of respondents The figure illustrates that the knowledge exposed to any type of IEC was 87%. The scores of the caregivers were not statistically facts in those IEC materials were concise and easily recognized (78.3%) (Table 2). different according to the presence of primary DHF case at home (P=0.137). Almost 60% of the interviewees felt that television was the most effective medium for Prevention of mosquito bites dissemination of knowledge on DHF in the community. To prevent and protect children from mosquito bites, the following measures were The logistic regression model of taken: use of mosquito nets (47.9%), use of knowledge score by the respondents’ repellants (47.2%), wearing of long sleeves characteristics and exposure to health (12.6%), others (8.6%), and none (5.2%). education media is given in Table 3. 192 Dengue Bulletin – Vol 28, 2004 Assessment of Dengue-related KAP Among Caregivers Table 3. Logistic regression model of knowledge scores, by respondents’ characteristics and exposure to health education materials Odds ratio Variables (n) Percent (95% confidence interval) Respondent's characteristics Sex • Male 54 13.3 1 • Female 351 86.7 0.379 (0.198-0.727)** Years of formal schooling • 0 to 5 (r) 133 32.8 1 • 6 to 20 272 67.2 0.588 (0.364-0.950)* Mean years of schooling 7.6±3.6 Exposure to health education media Pamphlets/posters • Not seen 197 48.6 1 • Seen 208 51.4 0.478 (0.291-0.784)** Television • Not watched (r) 159 39.3 1 • Watched 246 60.7 0.353 (0.218-0.571)*** Newspapers/journals • Not read (r) 272 67.2 1 • Read 133 32.8 0.443 (0.245-0.803)** * Significant at P<0.05; ** P<0.01; *** P<0.001; (r) = Reference category An analysis of the findings suggested Cross-check by observation that females with over six years of schooling checklist were significantly related to total knowledge scores. Survey respondents with 6-20 years The researchers used an observation of schooling were more likely to obtain high checklist to support the findings. Nearly scores than those with 0-5 years of 55% of the households had two to three water containers. Although 46.5% of the schooling. Respondents who were exposed water containers had lids, only 19% were to health education media such as covered tightly. Larvae were found in 67% pamphlets/posters, television, newspapers of the water storage tanks and 15.9% of the and journals obtained higher scores than the flower vases at the time of interviews. Gutter unexposed group. The respondents who blockage was observed in 3.2% of cases. watched television were more likely to score Old tyres, coconut shells and tins were higher than those who did not (Table 3). found in the compounds and larvae existed in half of these solid wastes. Dengue Bulletin – Vol 28, 2004 193 Assessment of Dengue-related KAP among Caregivers Approaches considered for vector breeding places. The subjects knew improvement of IEC activities little of blocked gutters as possible breeding sites. Nearly 80% thought that DHF could Many recommendations were extracted be prevented by immunization. A majority from FGDs for the improvement of existing of the interviewees used measures to IEC materials. It was suggested that messages prevent children from being bitten by should be short and clear. Pamphlets should mosquitoes. A very small percentage did not be widely distributed among the community, use any measure. Some points that require especially in schools. The same messages emphasis for community awareness include: could be published in newspapers and • Aedes is the main vector for DHF. journals once a week before and during the rainy season. Health magazines and • Aedes only breeds in clear water, not in magazines on astrology were the most polluted water. preferred media. They also pointed out the • The biting time of Aedes is daytime. most suitable times for telecast and broadcast of health messages. Regular clearing of • Blocked gutters should be mentioned as gutters should be mentioned in the health possible breeding sites for Aedes in message as well as in the book, “Facts for existing IEC materials. Life”. The fact that “DHF may attack again a • There is no vaccine available for the previously-infected child” was important to prevention of DHF. remind mothers of the danger. Cartoons and art competitions and exhibitions on DHF The chance to get high scores was would be the most effective media for better in females with high education. schoolchildren and mothers. Caregivers with low educational levels should be targeted for health education. The level of knowledge scores was not related to Discussion the history of a previously infected child at The mean scores of male and female home. Knowledge scores may be changed respondents were not significantly different through exposure to various media. Also, in the community. Community members sufficient numbers of IEC materials should knew a lot (more than 80%) about be distributed in the community. Only a transmission of DHF by mosquito bite, the small percentage of the interviewees were biting habit of Aedes and the resultant not exposed to any type of existing IEC. Ways fatality. Nevertheless, only 61.6% correctly and means should be found to improve identified the main vector of DHF. Nearly people’s exposure by further discussions with 20% answered that Aedes usually bit at community members and health workers nighttime. Some people were still confused using participatory approaches. that polluted water was also a breeding Pamphlets/posters, television, newspapers place. Most of the interviewees responded and journals are still popular media for the that the common breeding sites of Aedes public. Television is the most effective were water containers and flower vases. Not medium and various types of programmes, more than 30% identified ant traps, broken such as songs, comedies, short movies with pots, tins, old tyres and coconut shells as the famous actors, actresses, etc., apart from 194 Dengue Bulletin – Vol 28, 2004 Assessment of Dengue-related KAP among Caregivers discussions and health talks can be telecast. with volunteers and local NGOs should also However, radio is still a valuable tool in the be strengthened. health education process because of its easy availability ad popular use in semi-urban and rural areas. The effectiveness of various Acknowledgement existing IEC materials should be reviewed for We express our gratitude to the Township further improvement. Medical Officer and basic health staff in Many water containers were not Thaketa township for their support. We are covered tightly to prevent larval breeding. grateful to volunteers, NGO members and Social mobilization for sustainability of the study area residents for their active larvae control activities should be participation in the study. implemented. Coordination and cooperation References [1] Tha NO. A study on the larval control [3] Yoon SY. Community participation in the operations for DHF prevention in control and prevention of DF/DHF: Is it Sanchaung township, Yangon, Myanmar, possible? Dengue Newsletter, 1987, (13): 7- 2000-2001 (Unpublished report). 14. [2] World Health Organization. Information, education and communication. Lessons from the past: perspectives for the future. Department of Reproductive Health and Research, World Health Organization, Geneva, 2001. Dengue Bulletin – Vol 28, 2004 195 Students’ Perceptions about Mosquito Larval Control in a Dengue-Endemic Philippine City Jeffrey L. Lennon# Foundation University, School of Education, Dumaguete City, Negros Oriental, The Philippines Abstract A study was carried out among university students to find out their perceptions about mosquito larval control in a dengue-endemic Philippine city. This formative research was conducted to obtain information for formulating future school-related dengue control strategies. The study was carried out in- class through a semi-structured, open-ended question format. The study yielded information on students’ perceptions about the most important measures for mosquito larval control and perceived reasons why people did not implement them. The study also explored the opinions of the students by gender. The study yielded students’ knowledge on the types of mosquito larval habitats. Little was expressed about specific indoor mosquito larval control nor about the frequency of conducting source-reduction activities. Perceived barriers to constructing mosquito larval control centred on themes such as apathy, laziness and lack of time. Further studies are necessary to follow-up on these themes in depth. Keywords: Dengue, formative research, mosquito larval control, students, open-ended questionnaire, Philippines. Introduction Consequently, it has become endemic in Dumaguete city, Philippines[12]. The control of Aedes aegypti mosquito Aedes control is largely based on source larvae is essential for the control of dengue fever (DF) and dengue haemorrhagic fever reduction. Therefore, knowledge of the (DHF)[1]. The need to know the perceptions types of mosquito breeding sites is a prerequisite for health personnel, of key informants is necessary in order to schoolteachers and children and the better address the dengue-related control community at large for the control of issues in a specific area or community[2-5]. dengue. Various types of containers have Schools are potential mosquito breeding sites[6-8]. Also, primary, secondary and been identified as potential mosquito tertiary school-age students are principal breeding sites. These include plastic and targets of the Aedes mosquitoes[9-11]. metal containers, animal-feeding dishes, tyres, flower vases, coconut shells and water Dengue has become a steadily increasing storage drums[3,4,13,14]. health problem in the Philippines[9]. # E-mail: [email protected] 196 Dengue Bulletin – Vol 28, 2004 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City The knowledge about the types of College of Education, Dumaguete city, breeding containers alone is not enough to Philippines. There were 36 female and 7 achieve mosquito control. Attitudes and male subjects. All subjects were Filipinos. beliefs impact a person’s knowledge about The participants were students in a college- mosquito control. For example, the belief based “personal and community hygiene” that dengue is not a fatal or serious problem course. This study took place prior to any impairs a person from carrying out adequate course coverage or discussions about mosquito control practices. Some people dengue fever or any of the mosquito-borne believe that mosquitoes within the home diseases. The participation was voluntary and outside are different. So it is believed and no student in the class declined it. The that mosquitoes inside the house do not results and data were confidentially held. All [15] carry disease . results were tabulated and reconciled so Gender-related responsibilities for that the responses would be anonymous. control of certain mosquito breeding containers might also exist. There may be Procedure different responsibilities for one gender Two open-ended semi-structured questions inside the residence compared to the were administered to the subjects on outside surroundings. Or, there may be a January 16, 2003. The students’ instruction distribution of clean-up activities depending upon ownership of specific items such as was the facilitation of the questionnaire. tyres[15]. The two questions were as follows: One study in Mexico indicates that (i) What is the best way to control informants devalued the use of screens in mosquito larvae? dengue prevention and believed that the (ii) Why don’t some people use the screens were not effective in keeping out measure you suggest for question mosquitoes[2]. No. 1? Since dengue is already endemic in this study city in the Philippines, it was The questions were given verbally. The presupposed that there would already be a students were also told that they were high level of awareness about dengue in allowed to give more than one response per general. So, this study sought to explore the question. The students gave written more specific topic related to dengue responses to the questions. The responses control, through source reduction. were classified by gender. There were no Therefore, this objective of the study was to predetermined categories. Categories of explore the opinions of Philippine university responses were created from post-survey education students about the mosquito results. As categories emerged, sub- larvae control in a dengue endemic city. categories were created according to their relationship to the broader response Materials and methods categories. The time allotted for the whole process of issuing instructions to the Subjects students and completion of response-writing The subjects consisted of 43 university was approximately 15 minutes. major students at Foundation University, Dengue Bulletin – Vol 28, 2004 197 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City Results activities, activities not specified as inside or outside, activities related to specific types of On the responses to Question 1, the containers, use of insecticide, unrelated principal categories that emerged activities, and not familiar with mosquito concerning mosquito larvae control activities larvae (Table 1). were as follows: outside activities, inside Table 1. Responses to Question 1 Category Male Female Total I. Outside activities A. General outside activities A.1. Clean surroundings or yard 5 22 27 A.2. Clean surroundings daily 1 1 2 B. Specific water-containing items outside B.1. Turn over coconuts 1 0 1 B.2. Dispose of water in coconut shells 0 1 1 B.3. Put tyres in a safe place 0 2 2 B.4. Dispose of tyres 1 0 1 B.5. Dispose of stagnant water in drums 0 1 1 C. Other environmental activities C.1. Clean canals 1 5 6 C.2. Burn grass 0 1 1 C.3. Put trash cans in a safe place (to avoid rain-water entry) 0 1 1 C.4. Throw garbage properly 0 5 5 D. Activities involving other participants D.1. Clean-up drive participation 0 1 1 II. Inside activities A. Clean house (No specific activities mentioned) 0 4 4 III. Activities not specified as inside or outside A. Non-specific cleaning A.1. Clean things with stagnant water 1 0 1 A.2. Clean or eliminate stagnant water 1 5 6 A.3. Empty all stagnant water 0 2 2 A.4. Avoid or prevent stagnant water 0 4 4 A.5. Dispose of anything where mosquito eggs are laid 0 1 1 A.6. Kill mosquito larvae in stagnant water (not specified) 0 2 2 A.7. Clean things where mosquito larvae are laid 0 2 2 (non-specific item) 198 Dengue Bulletin – Vol 28, 2004 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City Category Male Female Total B. Activities related to non-specific types of containers B.1. Prevent water from entering open containers 0 1 1 B.2. Cover any water container 0 8 8 B.3. Clean water containers 0 1 1 B.4. Dry water containers 0 1 1 B.5. Empty containers 0 1 1 C. Clothes-hanging arrangement 0 1 1 IV. Activities related to specific types of containers A. Avoid open cans 0 1 1 B. Cover water containers such as gallon containers 0 1 1 C. Cover water barrels and tanks 0 2 2 D. Put open cans in a safe place 0 3 3 E. Dispose of cans 1 0 1 F. Cover jars 0 1 1 G. Cover basins 0 1 1 H. Clean bottles 0 1 1 I. Replace flower vase water (no time mentioned) 1 0 1 J. Replace flower vase water daily 1 1 2 K. Replace flower vase water weekly 0 1 1 L. Cover garbage 0 2 2 M. Put garbage in a can (No mention of placing over) 0 1 1 V. Other control-related activities A. Spraying insecticide 0 4 4 VI. Unrelated activity A. Remove an injured person 0 1 1 VII. Not familiar with mosquito larvae 0 1 1 Total 14 94 108 n 7 36 43 No. of students with multiple responses 5 29 34 Dengue Bulletin – Vol 28, 2004 199 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City Table 2. Responses to Question 2 Category Male Female Total I. Knowledge -related categories A. Lack of education 1 1 2 B. Lack of knowledge 0 5 5 C. Lack of consciousness of surroundings or environment 0 3 3 D. Lack of consciousness of health 0 3 3 E. Specific responses related to knowledge – – – E.1. Do not know that the suggested method of larval control 1 1 2 is effective E.2. Do not know the cause and effect of the mosquito to 0 1 1 dengue E.3. They think that they know everything about mosquito 1 0 1 control already E.4. Not aware of the consequences 0 2 2 E.5. Do not know that the mosquito is dangerous 0 2 2 II. Attitude-related categories A. Don’t care – apathy 1 9 10 B. Don’t care because they have enough money to pay 0 1 1 hospital bill C. Lazy 3 7 10 D. Busy 2 6 8 E. Since people are busy, they only use mosquito sprays to 1 0 1 kill adult mosquitoes instead of mosquito larvae F.1. No time 0 6 6 F.2. Insufficient time or little time 0 1 1 G. No money especially to pay for insecticide spray 0 1 1 H. Tired 1 1 2 III. Practice-related categories A. Lack of cooperation with others 0 2 2 B. They just use insecticide spray instead of source 0 1 1 restriction environment control IV. Invalid or unrelated responses A. Misunderstood the question (Answered in the affirmative) 1 8 9 B. Either answered Question 1 incorrectly or unfamiliar 0 2 2 with the term “mosquito larvae” V. No response 1 0 1 Total 13 63 76 n 7 36 43 Students with multiple responses 4 20 24 200 Dengue Bulletin – Vol 28, 2004 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City For the responses to Question 2, the Female subjects responded to all but principal categories that emerged one response, “activities not specified as concerning reasons why people do not inside or outside” (Table 1, Category III). undertake mosquito larvae control were as Perhaps, the male respondents were not follows: knowledge-related categories, carrying out these activities. The male attitude-related categories, practice-related response emphasis on general response categories, invalid or unrelated responses items Table 1 A, “General outside activities” and no response (Table 2). may suggest their lack of specificity in the details necessary to conduct proper dengue- related mosquito control. The largest Discussion response in Table 1, Category III, was on the In response to Question 1, both female and section “cover any water container” male students rated “clean surroundings” (Category III B.2). This practice is consistent with recommendations throughout the (Table 1, Category I A.2) with the largest literature[4,6,7,13,14]. However, these responses number of responses. This reflects the need in Table 1, Category III B.2, are less than for emphasis on specific mosquito control one-third the number of the responses given efforts while developing training and health in Table 1, Category I A.1. education strategies[5,14]. The training of students in The higher rating of outside activities environmental surveys and in the compared to inside activities reflects the environmental action plan known as “4 perceived importance of outside clean-up, o’clock habit”, has previously been and conversely, the relative lack of successful[6]. It is suggested that this be importance of inside clean-up and control projected on a larger scale to include other of inside mosquito breeding sites. This may members of the community. follow the perception as recorded in literature that “house mosquitoes” do not The responses in “activities related to transmit dengue[2,5]. Perhaps students specific types of containers” Table 1, believe that the Aedes mosquitoes do not Category IV, were scattered across the item live inside houses or other living quarters. sub-categories by the respondents. Many of these response items could be used as No male subject gave any response inside containers. Not only is it necessary to related to indoor mosquito control measures cover, empty, and clean containers, but also (Table 1, Category II). Besides the to indicate the frequency of the activity in perception that the indoors lack importance order to conduct adequate mosquito control. in mosquito control, the males may have Only the responses in Table 1, Category IV J, deemed the indoors as the domain of and Category IV K, both dealing with females. The role of women as homemakers flower-water replacement, and Category I needs to be considered in vector control A.2 “clean surroundings daily” mentioned programmes. This was reviewed in a the frequency and timing of activities. This [15] number of international settings . amounted to four responses out of a total of 108 responses in Table 1. Perhaps, Dengue Bulletin – Vol 28, 2004 201 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City programmes need to stress health education mosquito larvae. However, only a minority on the mosquito life-cycle and include gave specific details either in types of emphasis on the time interval to conduct control measures or in the timing of control successive mosquito control clean-ups. The activities. lack of responses on the frequency of For Question 2, the greatest number of environmental clean-up is in agreement responses among the knowledge-related with previous literature on the incomplete category was the general category, “Lack of understanding about the mosquito life-cycle, knowledge”, Table 2, Category I B. There and thus, the need to include this topic in was no concentration of specific knowledge- future health education programmes[2,5]. related responses. There were nearly twice Only four female respondents and no as many attitude -related responses as male respondent in Table 1, Category V A, knowledge-related responses. Attitude indicated that insecticide spraying was the responses were greater than knowledge most important mosquito control measure. responses for both male and female subjects The overwhelming majority of the (Table 2). Since dengue is endemic in respondents discussed general or specific Dumaguete City[12], and various health environmental clean-up activities. Source education programmes have continued for reduction of mosquito breeding sites years, it is reasonable to agree that there is a without the use of adult insecticides has high general awareness about dengue. been stressed in dengue-related health However, knowledge alone is generally not education programmes in Honduras[3], sufficient to change attitudes and Mexico[4], and the Philippines[6,13,16]. Since behaviour[17]. Health education to increase larvicides are not currently being used or only knowledge without addressing health promoted in this Philippine study site, the behaviours has also been ineffective in the respondents most likely were referring to the dengue control experience[5]. use of adult insecticides. In contrast, dengue The greatest numbers of responses were control programmes in Puerto Rico utilized in the attitude-related categories of “Don’t both source reduction of mosquito breeding care – apathy”, Table 2, Category II A, and sites and insecticide use[14]. However, for “Lazy”, Table 2, Category II C. The developing countries, regular adult aerosol responses of apathy or laziness could have insecticides were deemed to be too been a result of a weak belief in the expensive for regular home use, and thus effectiveness of the proposed measures from not recommended for routine control[13]. Table 1 to control mosquitoes and dengue. Only one respondent as seen in Table 1, The health belief model may help to explain Category VI, gave a response to an activity these responses. Two key components of unrelated to mosquito control. Also, there the health belief model are “perceived was only one respondent as seen in Table 1, benefits” and “perceived barriers.” A lower Category VII, who responded to the perception of benefits coupled with category, “Not familiar with mosquito elevated barriers may result in a lower larvae”. These responses validated that the possibility for change[18]. Likewise, people respondents in general had awareness about having low self-efficacy (a construct of the 202 Dengue Bulletin – Vol 28, 2004 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City Social Cognitive Theory and also the Health the collective efficacy for the desired Belief Model), or, in other words, behavioural change activities. High levels of confidence in doing something[18,19] could perceived collective efficacy may increase have resulted in their lack of interest to carry the likelihood of a group carrying out out the suggested mosquito control tas ks. desired behavioural change activities[21]. Therefore, promoting strategies to increase The large number of responses found in collective efficacy may enhance the Table 2, Categories II D, II E, II F1 and II F2, likelihood that community mosquito control related to the factors of insufficient time to activities will be carried out and sustained. perform clean-up tasks is also suggested in the literature[15]. While people may know Asking the questions orally, rather than about various individual mosquito breeding in a written form, had some limitations. This sites source reduction tasks, they may lack may have contributed to the nine the self-confidence necessary to perform a respondents who misunderstood Question 2. regular, comprehensive environmental They answered Question 2 by explaining clean-up task. Thus, skill development on their reason of choice for the best mosquito how to conduct the steps of an larval control, rather than explain why environmental action plan for dengue people were not using the best mosquito control should be emphasized in order to larval control method. See these responses increase self-efficacy and mosquito control in Table 2, Category IV A. behaviours. This promotion of skill This form of questioning was also development may, in turn, increase limited in the lack of in-depth follow-up. personal efficiency to perform source The procedure in this study did not allow for reduction tasks and thus, decrease the follow-up of such responses as the reason perception of time as a limitation to perform for “apathy” as a response to Question 2. mosquito control activities. Unlike other studies that used interviews[2- There were only two responses to Table 5,14] or focus groups[4,5], the facilitator of the 2, Category III A, “Lack of cooperation with questions did not interact with the others”. This low number may have been respondents , nor probe for follow-up due to a high value placed on the important responses. relational imperative or supportive Filipino The method and procedure also had its norm of “bayanihan” or cooperation[20]. Yet, strengths. In spite of utilizing open-ended there were still responses to perceived lack questions, the procedure was very efficient of cooperation as a cause for lower rate of in time and in organization. Unlike a mosquito larval control activities. Addressing previous study where interviews lasted for this norm through various communications an hour per respondent, and at home[5], and other health promotion means may respondents in this study were able to help in increasing mosquito larvae control complete the questions in a matter of activity. minutes, and in one sitting. Increasing the self-efficacy of individuals All questions were completed at the in the community may help in increasing same time, reducing potential biases that Dengue Bulletin – Vol 28, 2004 203 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City could result from interaction with others in • Efforts are needed to create awareness the outside environment. Also, having the regarding frequency of mosquito students complete the questions individually, activities. without interaction with fellow students and • There should be an in-depth reduced biased or blended responses. exploration of the reasons behind the The procedure also demonstrated the perceived causes of non-participation in strength of producing a large variety of mosquito larval control. Especially, the response categories, multiple responses and reasons behind “apathy” and “laziness” overall total responses in a very short period as perceived causes for non- of time. This was evident for both sexes and participation in mosquito larval control in response to both questions. should be further explored. • Activities such as an environmental Summary and control action plan and creation of a mosquito control “checklist” and recommendations implementation for houses and schools • The study is suggestive that there was an as a means to increase self-efficacy understanding by most students of the should be promoted. term “larvae”, and its general • The determinants of collective efficacy relationship to mosquito and dengue in mosquito larval control and dengue control. This was exemplified by the control should be explored. low number of incorrect responses to Teachers play an important role in content unrelated to mosquito larval facilitating of health promotion in dengue control as indicated in Table 1, endemic areas. Students and teachers Categories VI and VII, and Table 2, should be properly oriented to carry out Categories III B, IV B and V. personal, school and community mosquito • There were no male responses for and dengue control measures. Antecedent indoor control measures to Question 1. to this is an understanding of students’ Also, there were almost no specific perceptions about mosquito-related dengue mosquito control measures mentioned control. The in-class semi-structured by the male respondents. Further question method is one tool to carry out this studies should explore the possible type of formative research. gender relationship to mosquito control practices. Acknowledgement • The majority of male and female respondents did not mention indoor The author is associated with the mosquito larval control. Therefore, International Technical Assistance Group future mosquito control programmes (ITAG), Seattle, WA, USA. ITAG’s support is should stress the importance of indoor gratefully acknowledged. Also, thanks to mosquito larval control measures. Chona F. Lennon and Fernando N. Florendo for their assistance. 204 Dengue Bulletin – Vol 28, 2004 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City References [1] Gubler DJ. Aedes aegypti and Aedes aegypti- [9] Custodio GGV. (Ed.). National objectives for borne disease control in 1990s: top down or health, Philippines, 1999-2004: Kalusugan bottom up. American Journal of Tropical Para sa Masa. HSRA Monograph Series Medicine and Hygiene, 1989, 40: 571-578. (Series No. 1), 1999 (On-line Serial). [2] Kendall C, Hudelson P, Leontsini E, Winch P, Retrieved on 1 November 2000, from Lloyd L and Cruz F. Urbanization, dengue http/www.doh.gov.ph/noh/default.shtm. and the health transition: anthropological [10] Gubler DJ. Dengue and dengue contributions to international health. haemorahagic fever. Clinical Microbiology Medical Anthropology Quarterly, 1991, 5: Reviews, 1998, 11: 480-496. 257-268. [11] Pinheiro FP and Corber SJ. Global situation [3] Leontsini E, Gil E, Kendall C and Clark GG. of dengue and dengue haemorrhagic fever Effect of a community-based Aedes aegypti and its emergence in the Americas. World control programme on mosquito larval Health Statistics Quarterly, 1995, 50: 161- production sites in El Progreso, Honduras. 169. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1993, 17: 267-271. [12] Dengue cases by Barangay, Dumaguete City, Philippines, City Health Office, 2003. [4] Lloyd LS, Winch P, Ortega-Canto J and Kendall C. The design of a community- [13] Advocacy and Health Promotion Service. based health education intervention for the Best way to prevent dengue haemorrhagic control of Aedes aegypti. American Journal fever. Cebu City, Philippines, Department of of Tropical Medicine and Hygiene, 1994, Health. 50: 401-411. [14] Winch PJ, Leontsini E, Rigau-Perez JG, Clark [5] Winch P, Lloyd L, Godas MD and Kendall C. GG and Gubler DJ. Community-based Beliefs about the prevention of dengue and dengue prevention programmes in Puerto other febrile illnesses in Merida, Mexico. Rico: impact on knowledge, behavior and Journal of Tropical Medicine and Hygiene, residential mosquito infestation. American 1991, 94: 377-387. Journal of Tropical Medicine and Hygiene, [6] Lennon JL. Knowledge of dengue 2002, 67: 363-370. haemorrhagic fever by Filipino University [15] Winch PJ, Lloyd LS, Hoemeke L and students. Dengue Bulletin, 1996, 20: 82-86. Leontsini E. Vector control at the household [7] Swaddiwudhipong W, Chaovakiratipong C, level: An analysis of its impact on women. Nguntra P, Koonchote S, Khumklan P and Acta Tropica, 1994, 56: 327-339. Lerdlukanavonge P. Effect of health [16] Lennon JL and Gonzales MJCP. The education on community participation in utilization of college of education students in control of dengue hemorrhagic fever in an a dengue health education campaign. urban area of Thailand. Southeast Asian Philippine Journal of Education, 1995, 73: Journal of Tropical Medicine and Public 393, 421, 425-426. Health, 1992, 20: 200-206. [8] Strickman D and Kittayapong P. Dengue [17] Green LW and Kreuter MW. Health and its vectors in Thailand: introduction to promotion planning: an educational and the study and seasonal distribution of Aedes environmental approach, Second Edition. larvae. American Journal of Tropical Mountain View, CA, USA: Mayfield Publishing, 1991: 155. Medicine and Hygiene, 2002, 67: 247-259. Dengue Bulletin – Vol 28, 2004 205 Students’ Perceptions about Mosquito Larval Control in a Dengue Endemic Philippine City [18] Strecher VJ and Rosenstock IM. The health [20] Andres JD and Ilada-Andres PB. belief model. In: K Glanz, FM Frances and Understanding the Filipino. Quezon City, BK Rimer (Eds). Health behavior and health Philippines: New Day Pub, 1987: 55-56. education: Theory, research and practice, [21] Bandura, A. Exercise of human agency Second Edition, San Francisco, CA, USA: through collective efficacy. Current Jossey-Bass Pub, 1997: 41-59. Directions in Psychological Research, 2000, [19] Bandura A. Self-efficacy: the exercise of 9: 75-78. control. New York: WM Freeman Pub. 1997. 206 Dengue Bulletin – Vol 28, 2004 Short Note 1 Sero-surveillance in Delhi, India – An Early Warning Signal for Timely Detection of Dengue Outbreaks D. Bhattacharya*, Veena Mittal* #, M. Bhardwaj*, Mala Chhabra*, R.L. Ichhpujani** and Shiv Lal* *National Institute of Communicable Diseases, 22-Sham Nath Marg, Delhi – 110 054, India **Directorate General of Health Services, Nirman Bhavan, New Delhi – 110 011, India In India the first major outbreak of dengue These samples were tested for dengue by fever (DF) accompanied with dengue haemagglutination inhibition (HI) test[3] or haemorrhagic fever (DHF) was reported in IgM Capture ELISA Test[4]. A titre of Kolkata (Calcutta) in 1963[1]. More than 60 ³ 1:1280 in HI test in acute phase serum is outbreaks have been reported since 1956 to considered a presumptive diagnosis of a date[2]. Of these two major outbreaks of current dengue infection[5]. Samples positive DF/DHF occurred in 1996 and 2003 in for IgM antibodies against dengue virus Delhi and its adjoining states. Surveillance is indicate recent infection with dengue virus. the most cost-effective approach for The results of the sentinel sero- prevention and control of dengue. A strong surveillance from 1996 to 2003 are surveillance system will help in detecting summarized in the Table. The analysis of early warning signals of an outbreak, data over the period of eight years shows that instituting timely and appropriate control dengue strikes Delhi every year. The measures, assessing the impact of positivity ranges from approximately 13%- intervention measures and early containment 33%, except in 1996 and 2003 when dengue of the outbreak. Considering the above facts, fever occurred in epidemic proportions along the arbovirus laboratory at the National with DHF. The positivity in these two years Institute of Communicable Diseases, Delhi, was 53.4% and 57.8% respectively. has started sero-surveillance and monitoring of dengue fever in Delhi since 1996 as an The month-wise distribution of samples ongoing activity. It was intended to develop tested from 1996-2003 shows that the an early warning signal for timely detection of positivity for dengue starts appearing in the an impending outbreak and institution of month of August and reaches a peak in preventive and control measures in high-risk October and continues till mid-November areas. and then a decline starts and the last cases are reported upto 2nd week of December. Sera samples of clinically suspected The data for 2003 also shows a similar trend cases of DF and/or DHF are received from (Figure). various hospitals of Delhi round the year. # E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 207 Sero-surveillance in Delhi – An Early Warning Signal for Timely Detection of Dengue Outbreaks 0 0 0 0 0 5 0 16 14 433 450 +ve 1,438 2,356 (57.8%) 7 5 8 2003 13 10 11 11 43 35 701 877 2,351 4,072 Tested 0 0 0 0 0 0 0 0 1 12 18 11 42 +ve 2.8%) (1 6 2002 18 19 19 34 12 23 17 31 55 60 34 328 Tested 0 0 0 0 0 0 1 6 9 44 94 129 283 +ve (33.2%) 2001 19 21 11 10 13 22 22 43 35 127 304 225 852 Tested 0 0 0 0 0 0 0 1 2003 in Delhi 13 74 13 – 112 213 +ve (30.6%) 8 9 8 9 2000 11 26 18 32 56 77 96 188 254 6 Tested 0 0 0 0 1 0 0 0 5 17 35 35 93 +ve (19.2%) 5 7 1999 11 12 11 12 11 27 71 40 145 130 482 Tested 1 0 0 0 0 0 1 0 5 38 21 erology during the years 1996 140 206 s +ve (23.7%) 1998 21 11 24 28 67 27 31 48 70 47 201 293 868 Dengue Tested 0 0 0 1 0 0 0 3 1 65 26 159 255 Table: +ve (33.3%) 5 6 4 8 1997 39 33 15 20 81 15 169 369 764 Tested 0 0 0 0 0 0 0 0 3 73 74 343 493 +ve (53.4%) 1 4 3 0 3 0 0 3 1996 11 159 643 104 931 Tested Year/ JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC Total Month 208 Dengue Bulletin – Vol 28, 2004 Sero-surveillance in Delhi – An Early Warning Signal for Timely Detection of Dengue Outbreaks Figure. Month-wise samples tested/positive for dengue antibodies in Delhi during the year 2003 2,500 2,000 1,500 1,000 500 0 July May June April March August January October February November December September Tested Positive The studies for the estimation of the to dengue virus can act as an early warning House Index (HI) of mosquitoes also shows signal for an impending outbreak. The that the house index for Aedes aegypti, the above observations show that serological vector of dengue fever, starts building-up surveillance throughout the year, especially during the rainy season, i.e. from July and during the outbreak-prone period, can play reaches a peak in August-September[6]. an important role in the detection of early cases. A regular monitoring of suspected dengue cases by detection of IgM antibodies References [1] Ramakrishnan SP, Gelfand HM, Bose PN, [4] Lam SK, Devi S and Pang T. Detection of Sehgal PN and Mukherjee RN. The specific IgM dengue infection. Southeast epidemic of acute haemorrhagic fever, Asian J Trop Med Public Health, 1987, 18: Calcutta. Ind J Med Res, 1964, 52: 633-650. 532-538. [2] Epidemic preparedness – dengue fever, [5] Gubler DJ. Serological diagnosis of DHF and DSS. CD Alert, Monthly dengue/dengue haemorrhagic fever. Dengue Newsletter, National Institute of Bulletin, 1996, 20: 20-23. Communicable Diseases, Delhi, June 2001, [6] Katyal R, Singh K and Kumar K. Seasonal Volume 5 (16). variation in Aedes aegypti population in [3] Clark DH and Casals J. Techniques for Delhi, India, Dengue Bulletin, 1996, 20: 78- haemagglutination and haemagglutination 81. inhibition with arthropod borne viruses. Am J Trop Med, 1958, 7: 561-573 Dengue Bulletin – Vol 28, 2004 209 Short Note 2 Detection of Dengue Virus in Wild Caught Aedes albopictus (Skuse) around Kozhikode Airport, Malappuram District, Kerala, India B.P. Das*, L. Kabilan**, S.N. Sharma*, S. Lal*, K. Regu*** and V.K. Saxena* *National Institute of Communicable Diseases, 22 Sham Nath Marg, Delhi – 110 054, India **Centre for Research in Medical Entomology, 4 Sarojini Street, Chinna Chokkikulam, Madurai – 625 002, India ***National Institute of Communicable Diseases, Kozhikode Branch, Kerala, India Introduction in and around the airport area. The survey was carried out in May 2004. In India, outbreaks of dengue fever (DF)/dengue haemorrhagic fever (DHF) have been reported in various parts of the Materials and methods country during the past four decades[1]. The Kozhikode airport is situated at 11° .15' Aedes aegypti is the only vector that has so N latitude and 75° .49' E longitude, in a hilly far been implicated in dengue area of Malappuram district, Kerala. It transmission[1,2], even though Aedes became functional as an international albopictus is known to be present in some airport in 1988. A larval survey was carried of the peri-urban and rural areas[2]. Recently, out in various types of water-holding a survey was carried out in the Kozhikode containers to detect the breeding of Aedes (earlier known as Calicut) airport area of (Stegomyia) mosquitoes, both inside the Malappuram district, Kerala. During 2002 airport premises and its periphery up to and 2003 (up to July), 75 and 150 clinical about 600 metres. The larvae were dengue fever cases, respectively, were identified as per the method described reported from the district[3]. Earlier, reports earlier[6,7]. Adults of Aedes (Stegomyia) of Aedes survey in Kerala had shown the mosquitoes were collected while landing on presence of Aedes albopictus in rubber human baits by aspirator tube in a forested plantation areas[4] and in plastic cups[5]. residential area about 600 metres away This communication presents the results from the airport. of the detection of dengue virus from the The wild Aedes albopictus females wild and dry preserved, adult females of caught from outside the Kozhikode airport, Aedes albopictus and their breeding indices the adults reared from larval collections 210 Dengue Bulletin – Vol 28, 2004 Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport from inside the Kozhikode airport and the positive for Aedes albopictus and two for city, and the adults of Aedes aegypti reared Aedes vittatus (container index 15.1% and from larval collection from the 0.7% respectively). The most preferred Thiruvananthapuram international airport containers for Aedes albopictus breeding were separated sex-wise, pooled by species were discarded tyres, coconut shells and (about 15 adults per pool) and transported plastic containers. The average landing rate to the Centre for Research in Medical of Aedes albopictus on humans was 20 Entomology (CRME), Madurai, Tamil Nadu, females/human bait/hour. in a dry state, for detection of dengue virus. The methodology followed was similar to Dengue virus detection in that used for the detection of JE virus and Aedes albopictus based on the protocol developed and standardized by CRME[8]. However, the Of the three pools of Aedes albopictus antibody (D3-5C9-1) was diluted at 1:5000 tested for dengue virus infection following as being followed by CRME. antigen-capture enzyme immunoassay (EIA), one pool was found positive for dengue virus (OD-0.32), thereby indicating dengue Results and discussion viral activity in this mosquito species. The mosquitoes in the positive mosquito pool Aedes survey were collected as landing collection around the Kozhikode airport on 28 May 2004, and The survey for larval infestation in 52 transported as dry specimens to the CRME houses/premises around the airport area laboratory and processed on 8 June 2004 revealed 16 premises as positive for Aedes (Table). Earlier dengue virus was also albopictus breeding (house index 30.7%). A isolated from Aedes albopictus collected in total of 272 wet containers searched for a village in Vellore district of Tamil Nadu[9]. Aedes breeding revealed 41 containers as Table. Aedes mosquito pools tested for dengue virus infection by ELISA No. of pools tested/No. of adults/No. of Collected on Mosquito pools positive Locality (Processed species on) Wild caught adults Reared adults (landing) (immature) Kozhikode airport 27/05/04 – 1/15/0 (08/06/04) Residential area 28/05/04 1/10/1* – Aedes around the airport (08/06/04) albopictus City area 28/05/04 – 1/20/0 (2 kms from (08/06/04) airport) * Positive pool Dengue Bulletin – Vol 28, 2004 211 Detection of Dengue Virus in Wild Caught Ae. albopictus (Skuse) around Kozhikode Airport The present study confirms that antigen- Centre for Research in Medical Entomology, capture enzyme immunoassay is a useful Madurai, Tamil Nadu, is gratefully surveillance tool for monitoring dengue virus acknowledged. The authors are thankful to infection in mosquitoes. Mrs V. Thenmozhi and Mr S. Venkatesan, CRME, for providing laboratory assistance. Acknowledgements The provision of laboratory facilities and the permission for the study by the Director, References [1] Yadava RL and Narasimham MVVL. [6] Das BP. A simple modified method for Dengue/dengue hemorrhagic fever and its mounting mosquito larvae. J Commun Dis, control in India. Dengue Newsletter, 1992, 1986, 18: 63-64. 17: 3-8. [7] Das BP and Kaul SM. Pictorial key to the [2] Das BP, Sharma SK and Datta KK. common Indian species of Aedes Prevalence of Aedes aegypti at the (Stegomyia) mosquitoes. J Commun Dis, International port and airport, Kolkata (West 1998, 30: 123-127. Bengal), India. Dengue Bulletin, 2000, 24: [8] Tewari SC, Thenmozhi V, Rajendran R, 124-26. Appavoo NC and Gajanana A. Detection of [3] Anonymous. Annual Report of the Centre Japanese encephalitis virus antigen in for Research in Medical Entomology, Indian desiccated mosquitoes: an improved Council of Medical Research, 2002-2003: surveillance system. Trans Roy Soc Trop 82. Med Hyg, 1999, 93: 525-26. [4] Sumodan PK. Potential of rubber plantation [9] Tewari SC, Thenmozhi V, Katholi CR, as breeding source for Aedes albopictus in Manavalan R, Munirathinam A and Kerala, India. Dengue Bulletin, 2003, 27: Gajanana A. Dengue vector prevalence and 197-98. virus infection in a rural area in south India. [5] Hiriyan J, Tewari SC and Tyagi BK. Aedes Trop Med Int Health, 2004, 9(4): 499-507. albopictus (Skuse) breeding in plastic cups around tea-vendor spots in Ernakulam city, Kerala state, India. Dengue Bulletin, 2003, 27: 195-96. 212 Dengue Bulletin – Vol 28, 2004 Short Note 3 Entomological Investigations for DF/DHF in Alwar District, Rajasthan, India Kalpana Baruah#, Avdhesh Kumar and V.R. Meena National Institute of Communicable Diseases, 22 Shamnath Marg, Delhi-110 054 Introduction is usually of a short duration (July-August), the average rainfall being 61.16 cm. The During 2001, small outbreaks of DF/DHF highest temperature during June goes up were reported in many districts in to 47 °C whereas the lowest may go down Rajasthan, including the capital city of to freezing point. Jaipur and the industrial city of Alwar. There was a total of 1,820 laboratory- Alwar town has a number of industrial confirmed cases (based on serology using units. Migration of labour thus poses an kits for IgG and IgM antibodies) with 30 increased threat for malaria and other deaths (CFR: 1.65%)[1]. The present vector-borne diseases, including DF/DHF. communication deals with the The city has irregular piped water supply entomological investigations carried out by resulting in water storage practices for the National Institute of Communicable household purposes. In rural areas no such Diseases, Delhi, during the outbreak piped water supply system exists; therefore, period in a few urban and rural areas of water from wells, bore-wells and natural Alwar district that were affected. streams is used for household purposes with minimal storage practices. Study area Larval survey Alwar district is situated in the north- eastern part of Rajasthan between 27°4¢ An Aedes survey, as per WHO guidelines[2], and 28°4¢ north latitude and 76°7¢ and was carried out in four localities out of 10 77°13¢ longitude. The central part of the in urban areas and in five localities under district is occupied by the Aravali hills. The four primary health centres (PHCs) in rural population of the district is 29,90,862 areas, all reporting fever cases. The results (2001 census), of which 85% is of the Aedes survey are given in Table 1. predominantly rural. The monsoon season # E-mail: [email protected] Dengue Bulletin – Vol 28, 2004 213 Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India Table 1. Aedes aegypti larval indices in the urban and rural areas, Alwar, Rajasthan Name of Total Houses House Containers Containers Container Breteau the locality houses found Index searched positive Index Index (or villages) searched positive Urban areas Sonwa 30 10 33.3 62 17 27.4 56.7 Karaulikund 30 9 30.0 57 13 22.8 43.3 Arya Nagar 20 4 20.0 47 4 8.5 20.0 Kalakuan 25 9 36.0 111 15 13.5 60.0 Rural areas Indok 25 0 – 47 0 – – Madhogarh 20 0 – 32 0 – – Malakhera 25 0 – 34 0 – – (Kalachara) Bhartahari 25* 5 20.0 20 5 25.0 20.0 Tiraha Kushalgarh 10* 1 10.0 15 1 6.7 10.0 * Shops and nearby houses just outside the villages Results and discussion Container Index 6.7% to 25.0% and Breteau Index from 10.0 to 20.0 only. The shops used earthen pots for storing drinking water Larval surveys wherein co-breeding of Aedes aegypti and In urban areas, the House, Container and Aedes albopictus was detected. The Breteau indices ranged from 20.0% to dwelling houses along the shops were also 36.0%, 8.5% to 27.4% and 20.0 to 60.0, found positive, as Aedes breeding was respectively. Mixed breeding of Aedes detected in cement containers used for aegypti and Anopheles stephensi was also providing drinking water to cattle. detected in cement tanks in some areas. The area-wise infestation by containers In comparison, the House Index was nil is given in Table 2. In the urban areas, out in rural residential areas as no mosquito of the total of 49 positive containers, breeding could be detected; however, 67.35% were domestic water-storing shops in the marketplaces near the villages containers (like cement tanks, clay pots and and their adjacent houses were found to be overhead tanks), followed by 30.61% positive. The House/Premise Index in these evaporation coolers and the remaining localities ranged from 10.0% to 20.0%, 2.04% trash. 214 Dengue Bulletin – Vol 28, 2004 Entomological Investigations for DF/DHF in Alwar District of Rajasthan, India Table 2. Area-wise infestation of Aedes aegypti in the urban areas, Alwar district, Rajasthan Type of container Evaporation Cement Overhead Clay pots Others Area coolers tanks tanks % % % % % S S S S S +ve +ve +ve +ve +ve Sonwa 27 18.5 16 56.3 12 16.7 7 0 – – Karaulikund 19 21.1 21 38.1 17 5.9 – – – – Arya Nagar 24 8.3 9 11.1 14 7.1 – – – – Kalakuan 37 10.8 27 25.9 41 4.9 4 25.0 2 50.0 Total 107 14.0 73 34.2 84 8.3 11 9.1 2 50.0 S=Searched Adult surveys (Rajasthan), where people from the periphery picked up the infection during Landing collections were also undertaken in their day visit to the city area where Aedes the same urban and rural areas where larval aegypti breeding indices were very high. surveys were carried out. In urban areas the adult density of Aedes aegypti ranged from 2.0 to 7.0 per man-hour. The houses with Acknowledgments poor ventilation and light yielded higher numbers. The authors are grateful to the Director, National Institute of Communicable In rural areas both Aedes aegypti and Diseases, Delhi, for providing the Aedes albopictus were detected in shopping opportunity and necessary facilities to areas only. The cases reported from these undertake this investigation. The rural areas could be attributed to population cooperation extended by the staff of the movement from rural to urban areas (city) Chief Medical Officer’s office, Alwar district, during daytime for earning their livelihood and the technical support of the staff of the or shopping purposes. A similar observation NICD, Alwar branch, is gratefully [3] was made earlier by Kalra et al. in Ajmer acknowledged. References [1] Anonymous. Report of State Health [3] Kalra NL, Ghosh TK, Pattanayak S and Authority, Jaipur, 2001 (unpublished). Wattal BL. Epidemiological and [2] World Health Organization. Prevention and entomological study of an outbreak of control of dengue and dengue haemorrhagic dengue fever at Ajmer, Rajasthan (1969). J fever – comprehensive guidelines, 1999, Com Dis, 1976, 8(4): 261-279. WHO Regional Publication, SEARO 29. Dengue Bulletin – Vol 28, 2004 215 Short Note 4 Essentiality of Source Reduction in both Key and Amplification Breeding Containers of Aedes aegypti for Control of DF/DHF in Delhi, India B.N. Nagpal, Aruna Srivastava#, M.A. Ansari and A.P. Dash Malaria Research Centre, 20 Madhuban, Delhi - 110 092 Delhi, the Capital of India, reported the city Municipal Corporation of Delhi (MCD). first-ever outbreak of dengue fever in The MCD organized a campaign aimed at 1967[1]. Since then the city has experienced source reduction, which was backed by cyclic epidemics every 2-3 years. During vehicle-mounted thermal fogging for the 1996, a large epidemic swept the city when control of the outbreak. However, the 10,252 cases were hospitalized and 423 reduction in the DF incidence was not deaths were recorded[2]. The disease has found to be commensurate with the inputs now become endemic and the yearly made into the campaign. At the request of incidence has varied between 160 and 300 the MCD, a team of experts from the cases of DF/DHF, with a couple of deaths[3]. Malaria Research Centre undertook a Aedes aegypti has been invariably found to comprehensive survey in five affected be associated with these outbreaks in Delhi. localities, viz. Dayanand Colony (Lajpat Overhead tanks (OHTs) and ground level Nagar Phase IV), Lajpat Nagar (Phases I and tanks (GLTs) as key containers, and II), Kotla Mubarakpur, Dayalpur Extension evaporation coolers have repeatedly been and Harsh Vihar – Tulsi Niketan, to identify reported as seasonal amplification sites[4,5]. both the key containers (OHTs and GLTs) The key containers maintain mosquito and the amplification containers breeding throughout the year, whereas (evaporation coolers, earthen pots, flower coolers amplify the Aedes population from vases, household ornamental fountains, etc.) May to November, and thereafter they go during November 2003 as per WHO dry[6]. techniques. The results of the study are included in this communication. During 2003, DF/DHF resurged in some localities in Delhi and a total of 2,604 The results of the larval survey of Aedes DF/DHF cases and 33 deaths were aegypti in five dengue-affected localities in registered up to 9 November 2003 by the Delhi as mentioned above are given in the # E-mail: [email protected], [email protected] 216 Dengue Bulletin – Vol 28, 2004 Source Reduction for DF/DHF Control in India Table. The results revealed that among the also interact with householders and key containers, out of 243 OHTs and 20 impress upon them to remove small GLTs, 64 OHTs (26.34%) and 8 GLTs (40%) water collections indoors/outdoors, were found positive for breeding of Aedes specially water evaporation coolers. aegypti. On the other hand, out of the 533 During the survey it was found that evaporation coolers, which are identified as fogging generally lacked a ‘pre-fogging the major amplification-breeding site, public information campaign’ requiring the checked, none was found positive for public to keep the house doors/windows breeding of Aedes aegypti. Ornamental open to permit the entry of fog. Health fountains inside the drawing rooms and workers, while interacting with mud-pots containing water for birds were householders, invariably talked about the supporting the heavy breeding (>1,000 removal of breeding from coolers and tanks larvae in each site) of Aedes aegypti. The but did not point out other sites of breeding. maximum positivity of OHTs was found in The crosschecking teams of the MCD also Kotla Mubarakpur (39.39%), followed by laid emphasis on inspection of evaporation Phases I and II of Lajpat Nagar (30%) and coolers as focal points and did not verify Dayanand Colony (20.69%). Two OHTs OHTs/GLTs and trash materials, as they located on the roofs of the market area were found it a time-consuming affair. In urban found supporting the breeding of Aedes areas, for security reasons or houses being albopictus. In a children’s home for boys locked, access by health workers into the belonging to the Social Welfare Department houses was also a constraint. of the Delhi Government, five out of 12 OHTs were found positive for Aedes aegypti In view of the aforesaid, it can be while two were found dry. It would thus concluded that: (i) source reduction should appear that during the MCD campaign, cover both the key containers as well as the source reduction efforts had concentrated amplification breeding sites, viz. evaporation on evaporation coolers, and key containers coolers and other trash articles breeding the and other trash materials breeding the vector species; (ii) the fogging operation species remained intact in domestic habitats. should be preceded by a pre-fogging information campaign in order to seek full cooperation of communities to derive IEC Activities of MCD optimal benefits from fogging; (iii) the IEC The IEC activities of the MCD generally campaign based on KAP studies should be covered the following aspects: prepared with particular emphasis on community participation; (iv) special efforts (1) Creating awareness through media and should be focused on behavioural change in spreading vocal messages through the accordance with the guidelines laid down use of loudspeakers in DF-affected by WHO[7]; and (v) health staff of the MCD localities, distribution of pamphlets, should be trained in entomological etc.; surveys/techniques related to Aedes (2) MCD workers who visit houses for breeding, prevention and its control. physical verification of breeding sites Dengue Bulletin – Vol 28, 2004 217 Source Reduction for DF/DHF Control in India – – % 100 100 100 100 1 2 2 5 – – +ve 1 2 2 5 – – Examined Other domestic containers 0 0 0 0 0 0 % 0 0 0 0 0 0 Amplification containers +ve 69 45 147 138 134 533 affected localities of Delhi during November 2003 - Evaporation coolers Examined – – – % 38.46 42.86 40.00 5 3 8 – – – +ve (GLTs) 7 13 20 – – – Ground level tanks Examined 0.0 % 5.88 20.69 30.00 39.39 26.34 Key containers 0 2 12 24 26 64 +ve 5 58 80 66 34 243 des aegypti in key and amplifier containers dengue Overhead tanks (OHTs) Examined – Breeding of Ae Locality Table. : NIL Dayanand Colony (Lajpat Nagar Phase IV) Lajpat Nagar (Phases I and II) Kotla Mubarakpur Dayalpur Extension Harsh Vihar Tulsi Niketan Total * Ornamental fountain and trash – 218 Dengue Bulletin – Vol 28, 2004 Source Reduction for DF/DHF Control in India Acknowledgement former Deputy Director, National Vector- Borne Diseases Control Programme, for his The authors are thankful to Dr K.N. Tiwari, valuable suggestions and guidance. We Municipal Health Officer-cum-Director, acknowledge the efforts of the field staff for Health Services, Municipal Corporation of assisting in carrying out the survey. Thanks Delhi, for providing assistance in the are also due to Mr Sanjeev Gupta for his conduct of the study, and to Mr N.L. Kalra, help in various ways. References [1] Balaya S, Paul SD, D'lima LV and Pavri KM. [5] Ansari MA and Razdan RK. Seasonal Investigations of an outbreak of dengue in prevalence of Aedes aegypti in five Delhi in 1967. Indian Journal of Medical localities of Delhi, India. Dengue Bulletin, Research, 1969, 57: 767-774. 1998, 22: 28-32. [2] Sharma S, Sharma SK, Mohan A, Wadhwa [6] Katyal R, Gill KS and Kumar K. Seasonal J and Dar L. Clinical profile of dengue variation in Aedes aegypti population in haemorrhagic fever in adults during the Delhi, India. Dengue Bulletin, 1996, 20: 1996 outbreak in Delhi, India. Dengue 78-81. Bulletin, 1998, 22: 20-27. [7] Parks W and Lloyd L: Planning social [3] Katyal R, Kumar K, Gill KS and Sharma RS. mobilization and communication for Impact of intervention measures on dengue fever prevention and control. A DF/DHF cases and Aedes aegypti indices step-by-step guide. WHO, Geneva, 2004. in Delhi, India: an update 2001. Dengue Bulletin, 2003, 27: 163-167. [4] Krishnamurthy BS, Kalra NL, Joshi GC and Singh NN. Reconaissance survey of Aedes mosquito in Delhi, Bull Ind Soc Mal Com Dis, 1965, 2: 56-67. Dengue Bulletin – Vol 28, 2004 219 Short Note 5 Breeding of Dengue Vector Aedes aegypti (Linnaeus) in Rural Thar Desert, North-western Rajasthan, India B.K. Tyagi# and J. Hiriyan Centre for Research in Medical Entomology, Indian Council of Medical Research, 4 Sarojini Street, Chinna Chokkikulam, Madurai 625 002, India Aedes aegypti, the vector of dengue fever, is considered worthwhile to highlight the widely present in India[1,2], including the association of Aedes aegypti and tankas in Thar desert in north-western Rajasthan. sustaining the vector population under Jalore town in the Thar desert experienced extreme xeric conditions. the first-ever epidemic of dengue fever in A total of 33 villages in the three 1985. Entomological studies carried out in districts of Jodhpur, Jaisalmer and Sri Jalore during 1990 and subsequently in Ganganagar (now incorporated partly in the 1996 observed extensive breeding of Aedes newly created Hanumangarh district) were aegypti[3,4]. Recently, dengue fever again surveyed for the presence of tankas (Table struck the Thar, this time in Jodhpur district, 1). Compared to those of Jodhpur and warranting a serious review of the vector Jaisalmer, most villages in Sri Ganganagar ecology in the region, particularly in view of are highly irrigated and adequately supplied the prodigious breeding of Aedes aegypti in with the conduit-water system. Four major discarded household and community-based types of water storing facilities were underground water reservoirs called tankas, identified which supported the breeding of of which thousands are formed in different Aedes aegypti. About 13.6% of the tankas, forms in the Thar[3]. These tankas, which which constituted 77.1% of the four water originally attracted only the malaria vector bodies, were found to be breeding Aedes Anopheles stephensi as long as the water aegypti. It is noteworthy that the typical remained potable, started breeding Aedes century-old traditional earthen-type tanka aegypti only after being discarded by local was almost invariably present in most populations in the wake of the recent villages of Jodhpur (88.8%) and Jaisalmer availability of conduit-based water supply (85.7%) but less so in Sri Ganganagar (17%) under Indira Gandhi Nahar Pariyojana where canal-based water storage sources (IGNP canal project). It is, therefore, # E-mail: [email protected] 220 Dengue Bulletin – Vol 28, 2004 Aedes aegypti Breeding in ‘Tankas’ in the Thar Desert had greatly reduced the existence of the while the fourth instar larvae (7.8%) was tankas. As many as 11 (24.4%) out of a total present the least, followed by pupae (2.5%). of 45 community tankas existing This situation clearly indicated a sustained peripherally in Kanasar village were breeding of Aedes aegypti in the tankas. abandoned for want of proper water storage Cement tanks, invariably constructed in facility and rendered uncared for by the close vicinity of bore-wells, for cattle villagers. All such tankas, wherein the water drinking bred Aedes aegypti as soon as the turned turbid in course of time including water there turned turbid due to prolonged vegetation growth, Aedes aegypti was use by cattle, sometimes with Anopheles invariably found to breed, replacing in the subpictus. None of the beris, another type of process its earlier and original occupant, the earthen reservoir in the desert environment, malaria vector Anopheles stephensi. The first supported the breeding of Aedes aegypti. instar larvae abounded the most (52.3%), Table 1. Distribution of different kinds of tankas and beris in various villages in three districts currently under varying degrees of irrigation and/or conduit water supply from canals No. of villages with different types of ‘tankas’ Cement Typical intra- Metallic Central Peripheral Beris No. of tanks District domestic mobile community village tankas villages earthen tankas tankas tankas assembly – + ++ – + ++ – + ++ – + ++ – + ++ – + ++ Sri 17 9 5 3 4 13 0 0 17 0 17 0 0 17 0 0 2 12 3 Ganganagar Jodhpur 9 1 5 3 0 8 1 0 9 0 0 8 1 8 1 0 0 7 2 Jaisalmer 7 1 6 0 0 7 0 0 7 0 0 6 1 6 1 0 0 6 1 Total 33 11 16 6 4 28 1 0 33 0 17 14 2 31 2 0 2 25 6 – = Absence of breeding habitat + = Presence of breeding habitat, with potable water breeding Anopheles stephensi ++ = Presence of abandoned breeding habitat positive for Aedes aegypti breeding Conclusion the tankas in the rural areas of the Thar desert is considered to be a rather recent The dengue vector, Aedes aegypti, has so far phenomenon, possibly due to the easy been collected from the Thar desert only accessibility of newly provided conduit- from townships and/or desert fringe areas in water supply to villages, which has led to the vicinity of urban environment, breeding the abandoning of the traditional water mostly in household pitchers and cement reservoirs. tanks[2,4,5]. The breeding of Aedes aegypti in Dengue Bulletin – Vol 28, 2004 221 Aedes aegypti Breeding in ‘Tankas’ in the Thar Desert References [1] Kalra NL, Wattal BL and Raghvan NGS. [4] Joshi V, Mathur ML, Dixit AK and Singhi M. Distribution pattern of Aedes aegypti in India Entomological studies in a dengue endemic and some ecological considerations. Bull Ind area Jalore, Rajasthan. Indian J Med Res, Soc Mal Comm Dis, 1968, 5: 307-334. 1996, 104: 161-165. [2] Kalra NL, Kaul SM and Rastogi RM. [5] Tyagi BK. A note on the breeding of vector Prevalence of Aedes aegypti and Aedes mosquitoes in cement tanks and pit latrines. albopictus vectors of DF/DHF in north, J Appl Zool Res, 1994, 5: 149-151. north-east and central India. Dengue Bulletin, 1997, 21: 84-92. [3] Chouhan GS, Rodrigues FM, Sheikh BH, Ilkal MA, Khangaro SS, Mathur KN, Joshi KR and Vaidhye NK. Clinical and virological study of dengue fever outbreak in Jalore city, Rajasthan, 1985. Indian J Med Res, 1990, 91: 414-418. 222 Dengue Bulletin – Vol 28, 2004 Book Review 1 A Review of Entomological Sampling Methods and Indicators for Dengue Vectors Dana A. Focks Infectious Disease Analysis, Gainsville, Florida, USA TDR / IDE / Den / 03.1 This review was developed in response to a abundance, temperature, and sero- recommendation of the WHO Informal conversion rates in the human population. Consultation on Strengthening The document reviews and critiques Implementation of the Global Strategy for current methods, focusing especially on Dengue Fever/ Dengue Haemorrhagic Fever sampling methods that provide information Prevention and Control, held in October of on (1) the risk of transmission as a function 1999, urging “the refinement of existing of vector abundance, and (2) the relative or entomological indicators and/or the absolute importance of the various types of development of new indicators that better containers in the environment. This second reflect transmission potential.” The aspect is essential when considering a Consultation “recommended that such suppression strategy designed to minimize indicators should provide clear, meaningful costs or to improve sustainability by information for communities as well as for targeting only a subset of the breeding programme managers and policy-makers.” containers for control or elimination – Whereas the traditional Stegomyia indices specifically those container types that are (the House, Container, and Breteau indices, responsible for the majority of adult and various related derivations) are of some production. In reviewing current and operational value for measuring the generally-used sampling methods, each is entomological impact of larval control discussed with respect to transmission risk interventions against the mosquito vectors of assessment and evaluated in terms of being dengue virus, they are not proxies for adult useful for either “research or special studies” vector abundance. Neither are they useful or as a practical operational tool providing for assessing transmission risk because they useful information for planning and do not take into consideration the management of vector control programmes. epidemiologically important variables, including adult vector and human Dengue Bulletin – Vol 28, 2004 223 Book Review 2 DengueNet in India Weekly Epidemiological Record, 2004, 79(21): 201-203 Epidemic dengue fever (DF) and dengue implementation in South-East Asia and the haemorrhagic fever (DHF) have emerged as Western Pacific was held in Kuala Lumpur a global public health problem in recent on 11-13 December 20032. The objective decades. In fact, the problem has become of the meeting was to expand the pilot hyperendemic in many urban, periurban project to these two regions, building upon and rural areas, with frequent epidemics. the lessons learned from the pilot project in The South-East Asia Region is one of the the Americas. Based on the regions at highest risk of DF/DHF, recommendations of this meeting, two accounting for 52% of the global risk. country workshops were organized in India Dengue outbreaks now occur in India, as in in March 2004, supported by the other high-burden countries in the Region, WHO/CSR and USAID project to strengthen such as Indonesia, Myanmar and Thailand. surveillance in India. The first took place in New Delhi on 11-12 March 2004 with the Strengthening epidemiological and northern states and the second in Bangalore laboratory surveillance of dengue and on 16-17 March 2004, with the southern dengue haemorrhagic fever including, the states. WHO collaborating centres attended implementation of DengueNet 1, is one of both meetings. The proceedings and the priorities of the global and regional recommendations from the New Delhi strategies for dengue prevention and control. meeting were discussed at the Bangalore DengueNet is WHO’s global data meeting to ensure that the consensus management system created on the Internet recommendations addressed national issues, to collect and analyse standardized needs and priorities. Experts from health epidemiological and laboratory surveillance service departments of all states and the data with the objective to improve capacity Delhi City Corporation, the National for effective national and international Institute for Communicable Diseases (NICD), planning for the prevention and control of the National Institute of Virology (NIV) in epidemic dengue and DHF. Pune, the WHO Department of Following the pilot use of DengueNet in Communicable Disease Surveillance and the Americas, a joint WHO Response, WHO Representative for India HQ/SEARO/WPRO meeting on DengueNet and SEARO participated. 1 1 See No. 36, 2002, pp. 300-304. 2 See No. 6, 2004, pp. 57-62. 224 Dengue Bulletin – Vol 28, 2004 DengueNet in India The main objective of the workshops significantly improved its surveillance system, was to strengthen disease surveillance and including strengthening of laboratory response to vector-borne diseases using services. Uttaranchal, although lacking a DengueNet as an entry point. Work focused good network of laboratories, was included specifically on assessing current surveillance because of its close proximity to Delhi. practices (including the use of case It was also decided to designate two definitions, reporting formats and WHO laboratory collaborating centres mechanisms for flow of information), (WHO CC) for northern and southern states laboratory facilities and tests for DHF, on to ensure practical use of these facilities. identifying and strengthening regional Accordingly, NIV-Pune will continue as collaborative laboratories and on establishing WHO CC for the south, and it is a framework for participation in DengueNet. recommended that WHO designate NICD Experiences both from India and from to serve as WHO CC for the northern states the region on surveillance and control were (following a request from NICD). The two discussed. The need for an integrated centres would be responsible for training, approach to surveillance of vector-borne quality control, use of standard procedures diseases and application of lessons and and networking at national and international experiences from malaria and other vector- level. borne diseases was identified. The Based on the recommendations of the consensus was to implement DengueNet in workshops, a follow-up meeting has been accordance with the Integrated Disease planned to develop an activity plan for 2004 Surveillance Programme (IDSP) that is focusing on laboratory strengthening, starting in India. This would require capacity training, disease and vector surveillance, building for disease surveillance and networking, information sharing and response at national, state and district levels, reporting to DengueNet. NICD will take the through training of health workers and lead role in this follow-up activity, which is programme managers and strengthening of scheduled for the end of May 2004, laboratory services. working closely with all major stakeholders 3. Given the vastness of the country, the heterogeneity of the disease burden and the organizational network, the group recommended piloting the participation of selected states in global DengueNet through 3 Major stakeholders include the Indian Council for focal points at state and national level. Medical Research, IDSP Cell, Delhi City Corporation, NICD – WHO CC for Training and Epidemiology, States in which implementation would be WHO CC for Rabies Epidemiology, WHO Regional piloted included Delhi, Karnataka, Reference Laboratory for Polio Surveillance, National Maharashtra, Tamil Nadu and Uttaranchal. Reference Laboratory for SARS and Avian Influenza, Maharashtra and Tamil Nadu have a well state nodal officers of the pilot states and states that developed disease surveillance system and a were not represented in the earlier meetings, national - level institutes such as the Central Bureau of Health network of public health laboratories at both Intelligence, Vector Control and Research Centre – district and state level, with strong linkages WHO CC for Filariasis and Vector Control, National to national-level laboratories. Delhi, Vector Borne Disease Control Programme and NIV – following the recent dengue outbreak, has WHO CC for Arboviral Disease Diagnosis and Research and National Influenza Surveillance Centre. Dengue Bulletin – Vol 28, 2004 225 Book Review 3 WHO/WPRO/SEARO Meeting on DengueNet Implementation in South-East Asia and the Western Pacific, Kuala Lumpur, 11-13 December 2003 Weekly Epidemiological Record, 2004, 79(6): 57-62 Dengue/DHF – Global public Rationale for DengueNet1 health problem DengueNet, WHO’s global surveillance system for dengue fever and DHF, has been Epidemic dengue fever and dengue created as a web-based central data haemorrhagic fever (DHF) have emerged as a management system to collect and analyse global public health problem in recent standardized epidemiological and virological decades, with the development of data in a timely manner and to present hyperendemicity in urban and peri-urban epidemiological trends as soon as new data centres of many tropical and subtropical are entered. Strengthening epidemiological countries. Asia-Pacific countries have more and virological surveillance of dengue and than 70 % of the disease burden; in several DHF, including implementation of of them, DHF has become a leading cause of DengueNet, for early detection, planning and hospitalization and death among children. response is one of the four main priorities of Latin America and the Caribbean appear to WHO’s global prevention and control be following the same DHF epidemic trend, strategy, adopted in resolution WHA55.17 in with the disease affecting all ages and case- May 2002. DengueNet, when fully fatality rates as high as 10-15 % in areas with implemented, will facilitate WHO’s global limited health service infrastructure. The outbreak and response activities and support African and Eastern Mediterranean regions the GOARN2. are much less affected. Air travel is also Epidemiological and laboratory-based facilitating the rapid global movement of surveillance is required to monitor and guide dengue viruses and increasing the risk of dengue/DHF prevention and control DHF epidemics through the introduction of programmes whether these are based on new serotypes. Globally, 2.5 billion people vector control or possible future vaccination live in areas where dengue viruses can be with a safe, effective and affordable vaccine. transmitted: an estimated 50 million dengue Recent and encouraging research infections occur each year, with 500,000 developments have made it likely that a cases of DHF and at least 22,000 deaths, dengue vaccine will become available. As a mainly among children. Although dengue is a consequence, the public health community notifiable disease in many endemic countries, only a small proportion of cases are reported 1 See No. 36, 2002, pp. 300-304. to WHO. 2 Global Outbreak Alert and Response Network. 226 Dengue Bulletin – Vol 28, 2004 DengueNet Implementation in South-East Asia and the Western Pacific needs to define the burden of dengue for representing 20 island countries in the society, so that adequate cost-benefit Americas, and after changes to the analyses can be presented to government supporting computer hardware, software, leaders before they decide to use the vaccine. and routines, a second meeting was Standardized global dengue surveillance data, convened jointly with WHO/WPRO/SEARO one of the principal results expected from the and the WHO collaborating centre for establishment of DengueNet, have become dengue/DHF at the University of Malaya in critical. Kuala Lumpur, 11-13 December 2003. The objective was to expand the pilot to South- East Asia and the Western Pacific, building on Phased implementation of the lessons learned from the pilot conducted DengueNet in the Americas. About 70 participants included national epidemiologists, laboratory First meeting in San Juan, specialists, and clinicians from 19 Asia-Pacific Puerto Rico, July 2002 countries, three countries in the Americas, six WHO collaborating centres, and WHO HQ, The first DengueNet meeting was held jointly regional and country staff4. with WHO/PAHO and the WHO collaborating centre for dengue/DHF at the The plenary presentations and Centers for Disease Control and Prevention discussions focused on: (1) the challenges (CDC Dengue branch; San Juan, Puerto Rico). and need for standardized global Its objective was to describe and demonstrate epidemiological and laboratory surveillance DengueNet to prospective users and to of dengue and DHF; (2) the activities of the launch a pilot, building on the existing Pediatric Dengue Vaccine Initiative (PDVI); reporting systems and network of dengue (3) the national surveillance and reporting laboratories in the Americas. Epidemiologists systems in the participating countries in and virologists from eight countries in the South-East Asia and the Western Pacific; Americas, three countries in Asia and five (4) the activities of the participating WHO WHO collaborating centres provided collaborating centres; (5) the WHO global recommendations for the administrative and technical procedures involved in making DengueNet operational. 4 Participants included: § South-East Asian and Western Pacific regions: Second meeting in Kuala Lumpur, national programmes from Bangladesh, Malaysia, December 20033 Cambodia, China, Fiji, French Polynesia, India, Indonesia, Lao People’s Democratic Republic, After pilot use of DengueNet by four Maldives, Malaysia, Myanmar, Nepal, Philippines, Member States and one network Sri Lanka, Singapore, Thailand, Viet Nam; the bi- regional Mekong Basin Disease Surveillance Network; WHO collaborating centres and 3 research institutes in Australia, India, Japan, This meeting was organized by the WHO Department Malaysia, Thailand. of Communicable Disease Surveillance and Response, Global Alert and Response, jointly with the WHO § Americas: DengueNet pilot country Brazil; WHO Regional Offices for South-East Asia and the Western collaborating centres in Cuba and USA; interim Pacific, and the WHO Collaborating Centre for director of the Dengue Pediatric Vaccine Initiative dengue/DHF at the University of Malaya in Kuala (PDVI). Lumpur, Malaysia, with technical and financial support § WHO: HQ; regional offices (PAHO, SEARO, from the US Centers for Disease Prevention and Control. WPRO); country offices (India, Malaysia). Dengue Bulletin – Vol 28, 2004 227 DengueNet Implementation in South-East Asia and the Western Pacific strategy and regional programmes; • to provide early warning of potential (6) WHO’s global outbreak and response outbreaks of dengue disease or of the activities and GOARN; (7) the DengueNet introduction of dengue viruses into pilot and lessons learned; (8) presentation of epidemiologically silent areas, for the DengueNet and a “hands on” session with purpose of implementing timely control the “new” prototype web site in Global Atlas. measures and notifying decision-makers Two working groups were convened. The in institutions whose occupations or first reviewed and defined the epidemiological livelihood may be affected; data and reporting requirements for • to strengthen and standardize DengueNet, modifications needed to the epidemiological surveillance of DF and present format, identification of countries DHF; for expanding the DengueNet pilot to Asia- Pacific regions, and roles and responsibilities • to promote the use of standardized of national and international partners. A clinical case definitions and reporting subgroup also reviewed and defined the criteria for dengue illnesses, permitting objectives of DengueNet. The second comparisons between countries and working group reviewed the existing over time; laboratory capacity in South-East Asian and • to strengthen the network of Western Pacific countries in relation to collaborating centres and national DengueNet, identifying the current needs laboratories for serotype determination (and gaps) for laboratory standards, quality and strain characterization; control, and dengue serological diagnosis and virus isolation, as well as for reporting • to promote improvement in the quality and information exchange. The group made of laboratory data reported at national recommendations that focused on and international levels; strengthening regional dengue laboratory • to provide a standardized database for diagnosis capacity, so that laboratories epidemiological research and analysis; participating in DengueNet are able to report data of the highest quality possible • to provide data useful for estimating the within their working environment. burden of disease (including the social and economic burden) on a national, regional, or global scale; Meeting outcomes • to support the improvement in national and international alert and response Objectives of DengueNet capacity for dengue/DHF outbreaks; The participants agreed that the overall and objective of DengueNet is to improve • to promote the free and timely exchange capacity for effective national and of epidemiological information between international planning for the prevention affected countries, their neighbours, and and control of dengue and that the specific other stakeholders in order to facilitate objectives for implementing this global and promote dengue control activities surveillance system are: within the region. 228 Dengue Bulletin – Vol 28, 2004 DengueNet Implementation in South-East Asia and the Western Pacific Recommendations of the • In collaboration with WHO country and laboratory working group regional offices, WHO collaborating centres should provide reference To strengthen the regional dengue reagents to national laboratories – laboratory diagnosis capacity, the standard inactivated antigens, participants of this group made the monoclonal antibodies, standard sera, following recommendations for national cell lines for virus isolation, and laboratories, WHO collaborating centres, prototype dengue virus strains. WHO, and government health authorities. Laboratory training Quality control • WHO should organize regional training • Quality control/proficiency testing courses on laboratory diagnosis of should be undertaken by the national dengue and other flaviviruses. laboratory/WHO collaborating centre • WHO should develop a laboratory for other laboratories in the country concerned. manual for dengue diagnosis. • WHO HQ should establish a global • A reference centre should be established at the WHO Collaborating technical advisory group, including representatives from collaborating Centre for Tropical Viral Diseases, Nagasaki, Japan, to undertake centres, to meet annually to advance laboratory training, capacity building, coordination of quality assurance/ control for other WHO collaborating reagents, quality issues, and DengueNet. centers and designated national laboratories in the two Regions. Reporting and information exchange • The Nagasaki reference centre should With regard to collection of laboratory data coordinate, organize, and distribute a and information transfer, the group WHO panel of reference sera for identified a strong need for government validation of tests/kits/rapid tests by health authorities to develop a reporting WHO, national laboratories, and WHO system to collect, centralize, and collaborating centres. disseminate these data, identify key laboratories to participate in this system, and Reference services designate a focal point for DengueNet. The group recommended that WHO • Countries that do not have facilities for virus isolation should send appropriate support national health ministries to assess current laboratory status in Asia-Pacific samples to a WHO collaborating centre of their choice after consultation with countries and to plan mechanisms to strengthen laboratories for DengueNet. A that centre. draft DengueNet laboratory assessment tool • WHO should recommend capacity- is available for review and use. building for virus isolation to the ministries of health of countries that The group expressed appreciation of the efforts made to develop DengueNet and lack facilities. Dengue Bulletin – Vol 28, 2004 229 DengueNet Implementation in South-East Asia and the Western Pacific recommended that WHO work with - Countries should provide annual partners to develop strategies for raising epidemiological data by sex and crucial resources. age groups. Recommendations of the Rate calculations epidemiology working group - Both incidence and mortality rates The group reviewed currently available data should be expressed per 100 000 and reporting practices in the Asia-Pacific mid-year population; countries countries in relation to DengueNet. The should provide updates to discussion was organized around the DengueNet in the event of principal epidemiological variables of time, significant change. place and personal characteristics, plus - The system should not show information about the virus. The group incidence and mortality rates for made recommendations on the countries that report data only from modifications to be made to the present sentinel sites. format of the DengueNet prototype in Global Atlas, on strengthening Virus serotype data – all available epidemiological surveillance, and on a framework for implementation of the - Data should be entered (when DengueNet in Asia-Pacific regions with provided by the laboratory) as the emphasis on the quality of available data cumulative number of isolations of and the active participation of national each serotype in the country from 1 programmes. January. Data collection General recommendations Epidemiological data For countries - To accommodate currently available Countries should promote implementation case classification reporting practices, of the WHOrecommended surveillance countries should provide three standards for dengue. (Participants were categories – DF cases, DHF/DSS provided with copies of these standards.) (dengue shock syndrome) cases, total dengue cases (DF/DHF/DSS). For DengueNet These data should be provided monthly, at state/department level A country information page should be by large countries and at island provided on the DengueNet web site for all level by island nations. country-specific information, definitions, and methods used (e.g. sentinel site - Countries should provide data, information, reporting by time of onset or monthly when available, on notification, case classification other than “dengue deaths” (probable or according to the WHO definition, etc.). confirmed). 230 Dengue Bulletin – Vol 28, 2004 DengueNet Implementation in South-East Asia and the Western Pacific WHO-recommended surveillance Country participation standards should be made available on the DengueNet web site. A major outcome of the meeting was that representatives of all participating countries showed interest in collaborating with Roles and responsibilities of DengueNet and agreed to present the meeting’s recommendations to their health the partners in this network ministries. Country participation will require Countries will collect, validate, and provide a letter of request from WHO to the epidemiological and laboratory data. They ministry of health; ministry authorization for will designate the participating centres and participation and designation of a national focal points, and WHO country offices will DengueNet focal point; and, for some facilitate the process. WHO collaborating countries, an external budget. centres will provide laboratory support, The DengueNet pilot will be expanded proficiency panels, and training to national to countries in American, South-East Asian, laboratories. WHO regional offices will and Western Pacific regions in 2004 after implement the country support activities, modifications to the system have been and WHO HQ will maintain and moderate made in Global Atlas. The lessons learned the DengueNet web site. WHO regional from the pilot will be used to develop a offices and HQ will seek financial support consensus framework for DengueNet for dengue surveillance activities. implementation for standardized global surveillance of dengue and DHF. Dengue Bulletin – Vol 28, 2004 231 Instructions for Contributors The Dengue Bulletin welcomes all original research papers, short notes, review articles, letters to the Editor and book reviews which have a direct or indirect bearing on dengue fever/dengue haemorrhagic fever prevention and control, including case management. Papers should not contain any political statement or reference. Manuscripts should be typewritten in English in double space on one side of white A4 size paper, with a margin of at least one inch on either side of the text and should not exceed 15 pages. The title should be as short as possible. The name of the author(s) should appear after the title, followed by the name of institution and complete address. E-mail address of the corresponding author should also be included. References to published works should be listed on a separate page at the end of the paper. References to periodicals should include the following elements: name and initials of author(s); title of paper or book in its original language; complete name of the journal, publishing house, or institution concerned; volume and issue number, relevant pages and date of publication, and place of publication (city and country). References should appear in the text in the same numerical order (Arabic numbers in parenthesis) as at the end of the article. For example: (1) Nimmannitaya S. Clinical spectrum and management of dengue haemorrhagic fever. The Proceedings of the International Conference on Dengue Haemorrhagic Fever, Kuala Lumpur, September 1-3, 1983:16-26. (2) Gubler DJ. Dengue and dengue haemorrhagic fever: Its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G (ed.), Dengue and dengue haemorrhagic fever. CAB International, New York, NY, 1997, 1-22. (3) Nguyen Trong Lan, Nguyen Thanh Hung, Do Quang Ha, Bui Thi Mai Phuong, Le Bich Lien, Luong Anh Tuan, Vu Thi Que Huong, Lu Thi Minh Hieu, Tieu Ngoc Tran, Le Thi Cam and Nguyen Anh Tuan. Treatment of dengue haemorrhagic fever at Children's Hospital N.1, Ho Chi Minh City, 1991-1996. Dengue Bulletin, 1997, 22: 150-161. Figures and tables (Arabic numerals), with appropriate captions and titles, should be included on separate pages, numbered consecutively, and included at the end of the text with instructions as to where they belong. Abbreviations should be avoided or explained. Graphs or figures should be clearly drawn and properly labeled, preferably using MS Excel, and all data clearly identified. 232 Dengue Bulletin – Vol 28, 2004 Instructions for Contributors Articles should include a self-explanatory abstract at the beginning of the paper of not more than 300 words explaining the need/gap in knowledge and stating very briefly the area and period of study. The outcome of the research should be complete, concise and focused conveying the conclusions in totality. Appropriate keywords and a running title should also be provided. Articles submitted for publication should be accompanied by a statement that they have not already been published, and, if accepted for publication in the Bulletin, will not be submitted for publication elsewhere without the agreement of' WHO, and that the right of republication in any form is reserved by the WHO Regional Offices for South-East Asia (SEARO) and the Western Pacific (WPRO). One hard copy, original and clear figures/tables and a computer diskette indicating the name of the software, of the manuscript should be submitted to: The Editor Dengue Bulletin WHO Regional Office for South-East Asia Mahatma Gandhi Road New Delhi 110002 India Telephone: 91-11-23370804 Fax: 91-11-23379507, 23370972 E-mail: [email protected] Manuscripts received for publication are subjected to in-house review by a professional expert and are peer-reviewed by experts in the respective disciplines. Papers are accepted on the understanding that they are subject to editorial revision, including, where necessary, condensation of the text and omission of tabular and illustrative material. Original copies of articles will not be returned. The principal author will receive 10 reprints of his article published in the Bulletin. A PDF file can be supplied on request. Dengue Bulletin – Vol 28, 2004 233