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Serosurveillance for Japanese encephalitis in children in several districts of West Bengal, India

Aim: To evaluate the prevalence of antibodies to flaviviruses, particularly Japanese encephalitis (JE) in children. Methods: Virological and serological investigations were conducted into JE, along with dengue 2 (Den2) and West Nile viruses, in the
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  Serosurveillance for Japanese encephalitis in children in severaldistricts of West Bengal, India S Chatterjee 1 , D Chattopadhyay 1 , MK Bhattacharya 2 and B Mukherjee 3  ICMR Virus Unit, Kolkata, India; National Institute of Cholera and Enteric Diseases 2  , Kolkata, India; Burdwan Medical College 3  , Burdwan, India Chatterjee S, Chattopadhyay D, Bhattacharya MK, Mukherjee B. Serosurveillance for Japaneseencephalitis in children in several districts of West Bengal, India. Acta Pædiatr 2004; 93: 390–393. Stockholm. ISSN 0803-5253  Aim:  To evaluate the prevalence of antibodies to flaviviruses, particularly Japanese encephalitis(JE) in children.  Methods:  Virological and serological investigations were conducted into JE, alongwith dengue 2 (Den2) and West Nile viruses, in the  Flavivirus  group. The paediatric age group(up to 10 y), in the districts of Burdwan, Bankura, Midnapore and Purulia of West Bengal, India,was assessed for recent activity of these viruses, for 4 consecutive years from 1996 to 1999. Intotal, 2260 sera samples were collected, of which 204 were from acute fever cases; only 72 pairedsera were available. There was a significantly higher incidence of fever cases in children belongingto the schedule caste and schedule tribes. These communities occupy the lowest level in thesociety and are closely associated with pigs.  Results:  No virus could be isolated from the seracollected from fever cases. The results of the serological survey showed the presence of antibodiesto JE virus in only 13.3% of the contact sera, 33.3% of acute sera and 22.1% of convalescent serainvestigated. Only 3.1% of the contact sera had antibodies to Den2 and no antibodies were foundagainst West Nile virus. This indicates that the JE virus is in recent circulation in the population of these four districts, and as the majority of children lack substantial immunity to JE virus, they maybe affected by an epidemic. Conclusion:  Children up to 10 y of age in the four districts of West Bengal are mostly non-immune to JE virus. If an outbreak occurs, the majority of these populations may be affected. Key words:  Children, Japanese encephalitis virus, prevalenceS Chatterjee, ICMR Virus Unit, Infectious Diseases and Beliaghata General Hospital Campus, GB4, 1st Floor, 57 Dr. Suresh Chandra Banerjee Road, Beliaghata, Kolkata 700010, India (Tel.   913323537425, fax.  91 3323537424, e-mail. Encephalitis, especially in children, is very common inIndia, and numerous viral and non-viral agents cancause this disease. The activity of Japanese encephalitis(JE) virus in some of those episodes was firstestablished through serological studies (1). Furtherserological studies (2–3) by Indian workers proved thatJE virus is active in India. Household crowding, lowsocioeconomic status and lack of air conditioningappear to be the risk factors for acquiring JE (4). Sincethen, the incidence of JE has been recorded from all thestates of India. In 1973, an epidemic of JE broke out inWest Bengal, since when it has ravaged differentdistricts in different years. However, the epidemiologi-cal knowledge regarding JE virus activity amongchildren in West Bengal is not very clear.In view of the above facts, the present study wasundertaken to investigate the activity of JE, dengue 2(Den2) and West Nile viruses among children (up to10 y of age) in four districts of West Bengal: Burdwan,Bankura, Midnapore and Purulia. These four districtswere selected because JE cases are still being reportedevery year from these areas and the people of thesedistricts are mainly dependent on cultivation. To raiseeconomic standards, the poorer section of the commu-nity has accepted pig and mini-poultry farming asaccelerated sources of income. These animals areknown to be the favoured sources of   Flavivirus infection. Very hot summers and high humiditycharacterize the climate of these four districts through-out the year. The cold weather starts from mid-November and lasts until the end of February. Theperiod from March to May represents summer. Themonsoon is from June to September, although thundershowers occur frequently in April and May. About 80%of the annual precipitation falls during June toSeptember. The stagnant water in the paddy fieldsaffords a suitable place for mosquitoes to breed, andthese insects act as a vector for JE virus. Domesticanimals and cattle form the reservoir for JE virus. Themajority of people in these four districts live under thesame roof as their animals, which brings animals intoclose association with humans, and thus the community ©  2004 Taylor & Francis. ISSN 0803-5253      DOI 10.1080/08035250310007448  becomes exposed to JE infection through mosquitoes.Overall, antibodies to JE and dengue are present in14.5% and 2.77% of children living in these areas,respectively, but no antibodies to West Nile virus havebeen detected. Materials and Methods Patients admitted to the paediatric and general medicineward of Burdwan Medical College, Burdwan, BankuraSammilani Medical College, Bankura, and two districthospitals of Midnapore and Purulia were studied. Fever,confusion, stupor, coma, convulsion and neck stiffnesswas considered as major clinical features in the presentstudy. Neurological deficits were considered but werenot found among the cases in the study. The cases wereclinically diagnosed as encephalitis, and the possibilityof bacterial and prokaryotic aetiology was excludedthrough investigations at the local hospitals.In total, 2260 blood samples, of which 204 were fromfever cases, admitted to these medical colleges andhospitals were collected during the 4 y of the study.Samples were collected by venous puncture from cases,as well as from contacts from family members and fromthe localities where the patients resided. A total of 72convalescent blood samples (paired sera) was alsocollected from clinical encephalitis cases. In all of thecases, samples were only collected from children (agedup to 10 y). Contact samples were collected frombrothers, sisters and other close relatives of the casesof the same age group, as well as from their classmatesand playmates. Sera were separated from the collectedblood samples and transported on wet ice to the virusunit, Calcutta, where they were stored at   80 ° C untiltesting.All sera were tested within 3–4 mo of reaching thevirus unit. From the 204 acute samples, attempts at virusisolation were made in infant Swiss mice. The undilutedserum was inoculated intracerebrally in 1 litre in babymice (2–3 d old). The mice were observed for 15 d. Thebrains of the sick mice were harvested and inoculatedinto nutrient broth to exclude any bacterial infections.Separate tests were then done for the presence of JE,dengue and West Nile antigens on microscope slides,stained by the appropriate indirect fluorescent antibodytechnique (5) or by haemagglutination tests using thestandard method (6). The acute-phase sera were alsoinoculated into Vero cell cultures with two blindpassages and observed for 15 d for cytopathic effects. Results All 2260 samples collected for this study wereevaluated. Among them, 204 sera were collected fromacute fever cases (up to 4 d from the onset of fever) and72 sera were collected during the convalescent stage.The rest of the 1984 sera were collected fromneighbours and contacts. Attempts were made to isolatethe virus from the 204 acute samples, but no virus couldbe isolated from any of them, either by mouseinoculation or through tissue culture. The haemagglu-tination inhibition (HI) test (6) was used to detectantibodies to JE, Den2 and West Nile viruses.In interpreting serological results, the standardcriteria (2) were followed. Samples were consideredpositive if they had a fourfold rise in titre in the pairedsera. The results are shown in Tables 1 and 2. Theresults of 72 convalescent and 1984 contact samplesfrom the four districts are shown in Table 1. InBurdwan, only 10 out of 28 (35.7%) paired sera hadthe fourfold rise in JE antibody titre. Out of 509 contactsamples, only 94 (18.5%) were positive for JE antibodyand 12 (2.4%) were positive for dengue. In Bankura,antibodies to JE and Den2 were observed only in 15.7%and 3.6% of the 521 contact samples, respectively,whereas 47.3% of the paired sera had the fourfold rise inJE antibody titre. Among the 544 contact sera inMidnapore, antibodies to JE and Den2 were observedonly in 10.3% and 5.3%, respectively, and 27.8% of thepaired sera had the rise in antibodies to JE only. InPurulia, the prevalence of HI antibody to JE was only7.6% among the contact samples, which was muchlower than in Burdwan, Bankura and Midnapore.However, in the case of paired sera (although thesample size was too small) only 1 out of 7 (14.3%)samples showed the fourfold rise in titre, whichconfirmed JE virus infection. Only 2 out of 410(0.5%) contact sera were positive for Den2 antibody. Table 1.  Results of serological investigations of   Flavivirus  infections in four districts of West Bengal. DistrictPaired patient sera a Sera from contact neighbours n  JE Den2  n  JE Den2Burdwan 28 10 (35.7) 0 509 94 (18.5) 12 (2.4)Bankura 19 9 (47.3) 0 521 82 (15.7) 19 (3.6)Midnapore 18 5 (27.8) 0 544 56 (10.3) 29 (5.3)Purulia 7 1 (14.3) 0 410 31 (7.6) 2 (0.5)Total material 72 24 (33.3) 0 1984 263 (13.3) 62 (3.1)Data are shown as  n  (%). a West Nile antibody activity not found in any sera.  The results of the HI test of the sera collected fromthe acute fever cases are presented in Table 2. In total,204 samples of sera were collected from patients withacute illness. The overall antibody prevalence to JE wasobserved in only 22.1% of the cases. In Burdwan, only16 out of 65 (24.6%) acute sera were positive for JEantibodies. In Bankura, antibodies to JE were observedonly in 14 sera out of 57 acute samples (24.6%). InMidnapore and Purulia, the prevalence of antibodies toJE was 16.6% and 20%, respectively. In none of thesamples collected from cases of acute illness wereantibodies to Den2 or West Nile observed. Discussion This study was exclusively aimed at a search for Flavivirus  infection, particularly JE, in the sera of children from the districts of Burdwan, Bankura,Midnapore and Purulia, West Bengal. Although at-tempts at virus isolation were made by inoculating theacute samples in 1–2-d-old Swiss albino mice andthrough tissue culture, no virus was isolated from any of the sera tested. Japanese workers have reported thatvirus isolation is rare from cases after 7 d of onset (7). Incontrast, antibodies against the two  Flavivirus  antigensJE and Den2 could be demonstrated. The prevalence of the disease depends on the prevalence of vectormosquitoes and other factors, which were not investi-gated in the present study. Antigens to three members of the  Flavivirus  family (JE, Den2 and West Nile) wereused in this study to detect the respective antibodies inthese sera, but no antibodies to West Nile were detectedin the acute, convalescent or contact sera.The results presented in Table 2 show that the overallprevalence of antibodies to JE in the acute sera was22.1%, while in the paired sera it was 33.3% and in thesera from the contacts it was 13.3%. Antibodies to Den2were present in 3.1% of the contacts and neighbours(Table 2). No samples were positive for West Nileantibodies and it is very difficult to explain the absenceof this antibody. The sera samples positive for dengueantibody were solely from the contacts and neighboursand were present with JE antibody, which provesdengue infection at some undetermined time.From the results it is apparent that the possibleaetiological agent of the present illness was the JE virus.Inapparent infection of a large number of children playsa great role in the containment of future epidemics.Moreover, many of the ecological conditions in thesedistricts do not differ substantially from those incountries where the disease has been prevalent (8).In the district of Burdwan, the involvement of mosquitoes, birds (9) and domestic animals (10), whichplay a zoonotic role in the maintenance cycle of the JEvirus, and seroepidemiological studies on JE after someepidemics (11–12), are well documented for the activityof JE virus.Similar observations have also been made inBankura (13–14). However, clear epidemiological dataregarding JE virus activity among children in Burdwan,Bankura, Midnapore and Purulia districts are notavailable.The difference in the titre of acute- and convalescent-phase sera was highly significant. As it was observedonly against JE virus, it could be concluded that thepresent illness was probably due to the silent andendemic activity of JE virus in that area. Although thepercentage positivity of the HI antibody to JE virus washighest in Burdwan and Bankura (where JE virusactivity has already been established), JE virus activitywas also observed in children in Midnapore and Puruliadistricts. Of the large sample studied, only a negligiblenumber of children had the JE antibody, while the resthad no antibodies to JE virus.In summary, this study shows that the JE virus is incirculation in these districts of West Bengal, and werean epidemic or outbreak to occur in these districts,where the majority of the population is non-immune, itmay result in massive mortality. A disaster due to JEvirus infection among children could be prevented bymass vaccination, eradication of vector mosquitoes andsanitary improvements.  Acknowledgements.—  The authors express their sincere gratitude toall the staff members of ICMR Virus Unit Calcutta, particularly ShriPK Roy, Sri MK Sengupta, and Sri A Mitra, for their help and theirassistance in carrying out the laboratory investigations. References 1. Smith Burn KC, Kerr JA, Gatne PB. Neutralizing antibodiesagainst certain viruses in the sera of residents of India. J Immunol1954; 72: 248–572. Carry DE, Myers RM, Pavri KM. JE studies in Vellore, SouthIndia. Part II. Antibody response to patients. Indian J Med Res1968; 56: 1319–293. Banerjee K, Desai PK. Survey of Arbovirus antibodies in SouthIndia. Indian J Med Res 1973; 61: 344–514. Aaskov JG, Phillips DA, Wiemers MA. Possible clinicalinfection with Edge Hill virus. Trans R Soc Trop Med Hyg1993; 87: 452–55. Naim RC. Fluorescent protein training. Baltimore, MD: Wil-liams and Wilkins 19646. Clarke DH, Casals J. Techniques for haemagglutination andhaemagglutination inhibition with arthropod borne viruses. Am JTrop Med Hyg 1958; 7: 561–77 Table 2.  Results of serological investigations of the acute seracollected. DistrictNo. of acutesera testedNo. of positive cases (%)JE Den2Burdwan 65 16 (24.6) 0Bankura 57 14 (24.6) 0Midnapore 42 7 (16.6) 0Purulia 40 8 (20.0) 0Total 204 45 (22.0) 0 392  S Chatterjee et al.  ACTA PÆDIATR 93 (2004)  7. Kanda Inoue Y. An attenuated mutant of Japanese Encephalitisvirus. Bull Wld Hlth Org 1964; 30: 181–58. Chakravarty SK, Sarkar JK, Chakravarty MS, Mukherjee MK,Mukherjee K, Das BC, Hati AK. The first epidemic of Japaneseencephalitis studied in India—virological studies. Indian J MedRes 1975; 63: 77–829. Bhattacharya S, Chakrabarty SK, Chakrabarty AS, Ghosh KK,Palit A, Mukherjee KK, et al. Density of   Culex visunui  andappearance of JE antibody in sentinel chicks and wild birds inrelation to Japanese encephalitis cases. Trop & GeographicalMed 1986; 38: 46–5010. Nandi KK, Mukherjee KK, Chakrabarty SK, Chakrabarty MS.Activity of Japanese encephalitis among certain domesticanimals in West Bengal. Indian J Med Res 1982; 76: 499–50311. Ghosh SN, Rodrigues FM, Seth GP, Tongaonkar SS, Padbini VS,Gupta NP. Investigations on the outbreak of Japanese encepha-litis in Burdwan district, West Bengal. Part II. Serological surveyof human population. Indian J Med Res 1975; 63: 1472–712. Mukhopadhyay DB, Mukherjee B, Bagchi SB, Chakrabarty M,Mukherjee MK. An epidemiological investigation of Japaneseencephalitis outbreak in Burdwan district of West Bengal during1987–1988. Indian J Publ Health 1990; 34: 107–1613. Banerjee K, Sengupta SN, Dandawate CN, Tongaonkar SS,Gupta NP. Virological and serological investigations of anepidemic of encephalitis, which occurred at Bankura district,West Bengal. Indian J Med Res 1976; 64: 121–3014. Chatterjee AK, Banerjee K. Epidemiological studies on theencephalitis epidemic in Bankura. Indian J Med Res 1975; 63:1164–79Received March 3, 2003; revisions received Sept. 30, 2003; acceptedOct. 6, 2003
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