Transactions of the Royal Society of Tropical Medicine and Hygiene (2005) 99, 128—141

Tracking the re-emergence of epidemicchikungunya virus in Indonesia

Kanti Larasa, Nono C. Sukria, Ria P. Larasatia, Michael J. Bangsa,Rizal Kosimd, Djauzid, Tony Wandrad, John Mastere, Herman Kosasiha,Sri Hartatia, Charmagne Becketta, Endang R. Sedyaningsihe, H. JamesBeecham IIIa, Andrew L. Corwina,b,c,∗

a US Naval Medical Research Unit No. 2 (US NAMRU-2), Box 3, Unit 8132, Jakarta, Indonesiab

FPO AP 96520-8132, USAc US NAMRU-2, Jl. Percetakan Negara No. 29, Kompleks P2M-PLP/LitBangKes, Jakarta Pusat 10560,Indonesiad Centers for Communicable Diseases — Prevention and Environmental Health (P2M-PLP), IndonesianMinistry of Health, Jl. Percetakan Negara No. 29, Jakarta Pusat 10560, Indonesiae National Institute of Health Research and Development (BaLitBangKes), Indonesian Ministry of Health,Jl. Percetakan Negara No. 29, Jakarta Pusat 10560, Indonesia

Received 5 January 2004; received in revised form 15 March 2004; accepted 16 March 2004Available online 28 October 2004

KEYWORDSChikungunya virus;Epidemic;Indonesia

Summary Twenty-four distinct outbreaks of probable chikungunya (CHIK) etiologywere identified throughout Indonesia from September 2001 to March 2003, aftera near 20-year hiatus of epidemic CHIK activity in the country. Thirteen outbreakreports were based on clinical observations alone, and 11 confirmed by serologi-cal/virological methods. Detailed epidemiological profiles of two investigated out-breaks in Bogor and Bekasi are presented. Human sera were screened using an ELISAfor IgM and IgG anti-CHIK antibodies. Additionally, reverse transcriptase PCR andvirus isolation were attempted for virus identification. The mean age of cases was37 ± 18 years in Bogor and 33 ± 20 years in Bekasi. There was no outstanding case-clustering, although outbreak-affected households were observed to be geographi-cally grouped within villages. The attack rates in Bogor and Bekasi were 2.8/1000and 6.7/1000 inhabitants respectively. Both outbreaks started in the rainy seasonfollowing increased Aedes aegypti and A. albopictus densities.© 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd.All rights reserved.

* Corresponding author. Tel.: +62 21 421 4457 to 4463;fax: +62 21 424 4507.

E-mail addresses: corwinal@namru2.org,corwinal2e@yahoo.com (A.L. Corwin).

1. Introduction

Outbreaks of chikungunya (CHIK) have been re-ported in Africa and many parts of Asia. Epidemic

0035-9203/$ — see front matter © 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.trstmh.2004.03.013

Re-emergence of epidemic chikungunya virus in Indonesia 129

CHIK infections were reported in Bangkok untilthe mid-1970s (Burke et al., 1985), after whichvirus activity virtually disappeared. Sporadic clin-ical CHIK cases were again recognized in Bangkokin 1988 (Lam et al., 2001) with the last reportedCHIK outbreak occurring in Thailand during the 1995rainy season (Thaikruea et al., 1997). Sporadic andepidemic CHIK has also been reported from Cam-bodia (Chastel, 1963), Viet Nam (Dai and Thoas,1967), Laos (Halstead and Udomsakdi, 1966), Myan-mar (Burma) (Khai Ming et al., 1974), Indonesia(Slemons et al., 1984), Malaysia (Lam et al., 2001)and the Philippines (CDC, 1986). The first CHIK out-break in Malaysia was documented in 1998—1999,although CHIK antibody had been found in humansera before then in the absence of clinical dis-ease (Lam et al., 2001). The most recently reportedcases of CHIK in the Philippines were diagnosedfrom three US Peace Corps Volunteers in 1986, end-ing an apparent period of inactivity which began 18years earlier in 1968 (CDC, 1986).

In Indonesia, CHIK antibody was first identifiedduring a serosurvey conducted in 1972, suggestingthat CHIK virus was widely distributed in the ar-eas screened (Kanamitsu et al., 1979). Outbreaksoo‘mddSKaeCYteo1Tbr

avic(a1mmsa

1988), misdiagnosis and underreporting of this dis-ease in dengue-endemic areas is considered com-mon.

The objectives of this report are five-fold: (i) toconfirm epidemic CHIK occurrence associated withfebrile illness and arthralgia, (ii) to identify thecausative etiology, (iii) to determine the ‘measureof impact’ (attack rate) within the community, (iv)to identify possible demographic and/or environ-mental factors contributing to increased risk, and(v) to describe the temporal and spatial movementof epidemic CHIK eastward across the Indonesianarchipelago, from 2001 to 2003.

2. Materials and methods

2.1. Outbreak occurrences

The 24 CHIK outbreak episodes were principallyrecognized from 2001 to 2003, through generaldisease reporting communicated through routinesurveillance, namely weekly reports from commu-nity health centers (PusKesMas) to district andprovincial health authorities, and ultimately, to thenaAcamwpciI(gs(bweb

2

DoB(tdtt

f CHIK have historically been documented through-ut Indonesia. The first outbreak, designated as aknuckle fever’, was inferred from Dutch anecdotalemoirs to have heralded from Batavia (present-ay Jakarta) in 1779 (Carey, 1971). In 1973, a febrileisease outbreak of unknown origin was reported inamarinda and Balikpapan, along the east coast ofalimantan (Indonesian Borneo), presenting signsnd symptoms compatible with CHIK (Kanamitsut al., 1979). Ten years later (1983), a series ofHIK outbreaks occurred in the Special Area ofogyakarta (central Java), with an estimated at-ack rate of 70—90% (Haksohusodo, 1990; Slemonst al., 1984). Additionally, eight other outbreaksf possible CHIK were reported between 1982 and985 throughout Indonesia (Mackenzie et al., 2001).here have been no published accounts of CHIK out-reaks in Indonesia since 1985, until the presenteport.CHIK virus infection is notably characterized by

brupt clinical onset, involving fever and often se-ere arthralgia or arthritis in the extremities. Thiss followed by constitutional symptoms that in-lude maculopapular rash on the trunk and limbsChin, 2000; Jupp and McIntosh, 1988). Symptomsre generally self-limiting and can last from 1 to0 days, although arthralgia or arthritic symptomsay persist for many months afterwards. Becauseild hemorrhagic manifestations have been de-cribed, particularly among cases in Southeast Asiand the Indian subcontinent (Jupp and McIntosh,

ational level Centers for Communicable Diseasesnd Prevention—Environmental Health (CDC-EH).dditionally, outbreak information was communi-ated through anecdotal reporting from districtnd/or provincial sources, and public pronounce-ents in local newspaper accounts. Outbreaksere classified as either laboratory-confirmed orresumed on clinical presentation only. Laboratory-onfirmed outbreaks were those in which spec-mens from patients were positive for CHIK bygM/IgG ELISA and/or reverse transcriptase PCRRT—PCR). Clinically recognized outbreaks wereenerally defined as clusters of persons ill withigns and symptoms compatible with CHIK etiologyfever with symptoms of rash and/or joint pain),ut lacking laboratory test evidence. Specimensere made available for laboratory diagnosticvaluation from 11 of 24 clinically reported out-reaks.

.2. Investigated outbreaks

etailed epidemiological profiles are presentedf two investigated CHIK outbreaks in Bogor andekasi, West Java Province, western IndonesiaFigure 1). The first outbreak investigated, lastingwo months beginning in October 2001, was in Ke-ung Badak and Kebun Pedes kecamatans (subdis-ricts), Tanah Sareal District, on the periphery ofhe city of Bogor which is located approximately

130 K. Laras et al.

Figure 1 Geography of investigated chikungunya outbreaks in Bogor and Bekasi, West Java Province, Indonesia.Outbreak-affected households had ≥1 clinical chikungunya case(s).

56 km south of Jakarta, the capital city of In-donesia. The two subdistricts are situated between106◦43′—106◦51′E and 60◦30′—60◦40′S, at an ele-vation of 200—350m asl. The second outbreak in-vestigated, from February to June 2002, was inKali Jaya village, Cikarang Barat district, BekasiRegency, approximately 45 km east of Jakarta be-tween 106◦48′—107◦27′E and 6◦10′—6◦30′S, at an el-evation of 25m asl.

2.3. Outbreak chronologies

In November 2001, national newspaper accounts re-ferred to a ‘strange’ outbreak of disease near Bogor,reportedly involving approximately 100 cases withsymptoms that included fever, rash, joint pain andloss of ability to walk in some cases. Anecdotal re-ports at the time suggested that the outbreak mighthave been associated with CHIK virus. An investi-gation team was assembled rapidly and deployed

from 30 November to 7 December by the IndonesianMinistry of Health, with institutional representationfrom the Indonesian National Institute of HealthResearch and Development (NIHRD), CDC-EH, andUS Naval Medical Research Unit No. 2 (US NAMRU-2) to support the local district health service’sresponse.

On 6 June 2002, the Bekasi Regency Health Au-thority Office contacted US NAMRU-2 reporting anoutbreak of ‘unknown’ disease in Kali Jaya vil-lage. The outbreak was reported to have begun inFebruary, in one part of the village, and had sub-sequently spread to two other areas. By early ac-counts, an estimated 163 people were believed tobe afflicted. There was considerable confusion inidentifying the cause of the outbreak, as earlier ithad been presumed to be leptospirosis. However,testing on 12 specimens yielded only one reac-tive Leptospira sample by ELISA, whereas sevensera were ELISA-positive for IgM CHIK. An inves-tigation team made up of the same institutions

Re-emergence of epidemic chikungunya virus in Indonesia 131

mentioned in the outbreak response near Bo-gor was dispatched from 17—22 June 2002, insupport of the local district health service’s re-sponse.

2.4. Study subjects

Community-based, case—control investigationswere conducted in both outbreak areas of Bogorand Bekasi. Patients presenting with fever, rashand/or joint pain were identified as suspectedCHIK cases. Healthy family members of cases(those residing in the same household) were alsoscreened to assess possible familial clustering.Controls were randomly selected from healthyindividuals in the affected community, carefullymatched by age and gender. Controls served toexclude possible remnant viral background infec-tions with other outbreak etiologies. One villagewith no recent case reports suggestive of CHIKwas chosen as a control village. For comparison, across-sectional study was carried out in the controlvillage.

2.5. Specimen and data collection

A(aDtrsisUgwsacaui

2

FtttChoi

ing the Dengue Fever Virus IgM Assay (Focus Tech-nologies, Cypress, CA, USA).

2.7. Detection of IgM and IgG antibodiesagainst chikungunya virus

Serum samples were assayed for the presence of theIgM and IgG antibodies against CHIK using an anti-gen capture ELISA (Porter et al., 2004). The sam-ple was considered as positive if the optical density(OD) value exceeded the mean plus three standarddeviations from normal sera.

2.8. Detection of chikungunya viral RNA

Viral RNA was extracted from all sera and mosquitosamples by the Qiagen extraction method (QiagenViral RNA Mini Kit, Valencia, CA, USA), following themanufacturer’s protocols. Viral RNA was detectedusing a nested RT—PCR assay. Nested primer pairswere designed to amplify three structural gene re-gions, Capsid (C), Envelope-2 (E2), and an areaoverlapping the 6K and E1 genes (6K/E1). All nestedprimer sets were also tested against all four dengueseCwt(aC333CCCTAGTCurCfg

2

Scc

fter informed consent, one venous blood sample5—7ml) was obtained from each volunteer casend control subject using a Vacutainer® (Bectonickinson, Franklin Lakes, NJ, USA) without addi-ive. At the same time, day collections of indooresting mosquito populations using a sweep netampling device were made from suspected CHIK-nfected households. Sera and mosquitoes weretored in liquid nitrogen and transferred to theS NAMRU-2 laboratory for further analysis. Demo-raphic information and clinical signs and symptomsere collected from each participant using a study-pecific questionnaire, administered by local healthuthorities. All investigative actions were predi-ated on formal request letters for participationnd assistance through the NIHRD. Informed vol-ntary consent was obtained from all participatingndividuals before entering the study.

.6. Laboratory methods

or detecting active, recent or past CHIK infec-ions, tests were performed using ELISA for de-ecting IgM and IgG antibody, a nested RT—PCRechnique and virus isolation using cell culture.linically-suspected CHIK cases that presented withemorrhagic manifestation that included bleedingf gums and/or epistaxis were additionally exam-ned for IgM antibodies to dengue virus by ELISA us-

erotypes (DEN 1—4), Ross River virus and Japanesencephalitis virus and were found to be specific forHIK. Viral RNA was reverse-transcribed to cDNA,hich was concomitantly amplified in the sameube using the Promega Access RT/PCR Reagent KitMadison, WI, USA), utilizing a cocktail containingll outer primers (C: 5′ATG GAG TTT ATC CCA ACCA 3′, 1-20 and 5′GTT GTT TCG TGG CGC CTG CT′, 240-221; E2: 5′GCA GAC GCA GCG AGG GCC AG′, 1201—1220 and 5′CGT GCT GCA AGG TAG TTC TC′, 1440—1421; 6K/E1: 5′AGT CAA CAG ACC GGG CTAA 3′, 2481—2500 and 5′CGC TCA ATT GCG TAT TTTA 3′, 2740—2721). Inner primer pairs (C: 5′AAC TTTTA CAA TAG GAG GT 3′, 21—40 and 5′CGT TTT GCTTT GCT TCT TA 3′, 220—201; E2: 5′GCT ATT TGTAG AAC GTC AG 3′, 1221—1240 and 5′TAC CGT GCTCG GTC GGG AA 3′, 1420—1401; 6K/E1: 5′GCC CAAGG TAC TGG AGA TG 3′, 2501—2520 and 5′GTG TCGAG AAG CAG TAG GC 3′, 2720—2701) were thensed in separate nested-PCR reactions with sepa-ate reagents (PE Applied Biosystems, Foster City,A, USA) for each gene. The predicted product sizeor the amplicon was 200 nucleotides for all threeenes.

.9. Chikungunya virus isolation

erum was diluted 1:10 in PBS and inoculated ontoonfluent monolayers of C6/36 Aedes albopictusells (ATCC Cell Repository Line 1660) in 24-well

132 K. Laras et al.

culture plates. The plates were then incubated at30 ◦C for 14 days and observed daily for evidenceof cytopathic effects (CPE). At the end of 14 daysor upon recognition of CPE, cells were removedfrom the plates and evaluated for presence of CHIKvirus by an immunofluorescence assay using anti-CHIK (arbovirus group A) hyperimmune mouse as-citic fluid (Pavri, 1973).

2.10. Mosquito collections

All mosquitoes collected from indoor resting siteswere identified to species based on morphological

characters. Mosquitoes were pooled by species andlocality and tested for the presence of CHIK viralRNA using a nested RT—PCR.

3. Results

From September 2001 to March 2003, 24 suspectedoutbreaks of CHIK virus were reported through-out Indonesia (Figure 2). Most outbreaks (83%) oc-curred on the main island of Java, nearly half(46%) occurring in the densely populated provinceof Central Java. CHIK outbreak occurrences were

Figure 2 Temporal and spatial tracking of epidem

ic chikungunya across Indonesia, 2001—2003.

Re-emergence of epidemic chikungunya virus in Indonesia 133

also reported from Aceh (1 episode), North Suma-tra (1), North Sulawesi (1) and Lombok Island (2).These outbreaks occurred in urban (21%), semi-urban (neighborhood and village) (17%) and rural(62%) communities, mirroring roughly the distribu-tion of the human population in Java. The firstinstance of suspected epidemic CHIK occurred inBireun, Aceh province, located in northwestern-most Indonesia in September 2001, progressivelymoving eastward in the country, with transmis-sion last seen in Pasuruan (East Java), Klaten (Cen-tral Java), Tanggerang and Bekasi (West Java), inMarch 2003. Correspondingly, the number of epi-demic CHIK foci increased with time, from just twoin 2001, to eight in 2002 and 14 during the firstthree months of 2003. Seventy-five percent (18/24)of outbreak episodes were reported from Novem-ber 2002 to March 2003 (Table 1). The total numberof reported epidemic-associated cases during thistime period was 5821 (mean 253 patients per out-break event, range 37—1031 cases/event).

Table 1 Reported chikungunya outbreaks in Indonesia, 2001—2003

No. Regency/city Province Month, year Cases Laboratory confirmed

SeOcJaFeMaApNoNoDe

DeJaJaJaJaJaFeFeFeJa

3.1. Laboratory confirmed outbreaks

From the 11 outbreak episodes subjected to lab-oratory follow-up examination, CHIK was incrimi-nated as the etiology in all instances. Overall, evi-dence of recent and acute CHIK infections, based onserology and/or RT—PCR, was demonstrated in 47%(325/696) of serum samples assayed. Confirmationof ten outbreak episodes of CHIK was based on pres-ence of IgM antibody and/or viral RNA, found in 25%to 100% of samples tested. CHIK was implicated inthe Bolaang Mongondow (North Sulawesi) outbreakby presence of IgG antibody alone (Table 1).

3.2. Bogor outbreak

3.2.1. BackgroundHistorical trends from community health cen-ter records dating from 1998 to January 2002

1 Bireun, regency Aceh2 Bogor, citya West Java3 Klaten, regency Central Java4 Bekasi, regencya West Java5 Tegal, regency Central Java6 Purworejo, regency Central Java7 Boyolali, regency Central Java8 Kudus, regency Central Java9 Bolaang Mongondow,

regencyaNorth Sulawesi

10 Bandung, regencya West Java11 Klaten, regency Central Java12 Tegal, regency Central Java13 Jember, regencya East Java14 Cirebon, regency West Java15 Lombok Barat, regency West Nusa Tenggara16 Lombok Tengah, regency West Nusa Tenggara17 Jepara, regency Central Java18 Bandung, regencya West Java19 Yogyakarta, city Yogyakarta

20 Bantul, regencya Yogyakarta Ja21 Pasuruan, regencya East Java Ma22 Tanggerang, citya Banten Ma23 Bekasi, regencya West Java Ma24 Klaten, regencya Central Java Ma

Data from Arbovirus Sub Directorate General, CDC, Indonesian Minavailable.a Laboratory confirmed chikungunya outbreak.b Positive by IgM ELISA and/or reverse transcriptase PCR.c Based on an IgG anti-chikungunya response, because of the delay

No. ofcase sera

Percentagepositive (%)

pt—Nov 2001 420 NC —t—Nov 2001 119 86 63b

n—Oct 2002 127 NC —b—Jun 2002 169 93 56b

rch 2002 78 NC —ril—May 2002 371 NC —v—Dec 2002 73 NC —v—Dec 2002 187 NC —c 2002—Feb 2003 803 222 8c

c 2002—Feb 2003 246 11 100b

n—Feb 2003 185 NC —n—Feb 2003 108 NC —n—Feb 2003 202 141 74b

n—Feb 2003 107 NC —n—Feb 2003 120 NC —b 2003 142 NC —b 2003 37 NC —b 2003 38 23 91b

n—Mar 2003 372 NC —

n—Mar 2003 1031 23 70b

r 2003 654 20 25b

r 2003 47 17 94b

r 2003 NA 50 44b

r 2003 185 10 60b

istry of Health. NC: not laboratory confirmed; NA: data not

in specimen collection relative to acute disease phase.

134 K. Laras et al.

show this outbreak episode to be the first in-stance of epidemic CHIK transmission in the af-fected area. Historical data on signs and symp-toms compatible with CHIK found no evidenceof prior outbreak occurrence in the previousthree years. Monthly community health centerrecords dating back to January 1998 presentedsigns and symptoms compatible with CHIK sug-gesting that it had first appeared in August2001. Analysis of trend, with respect to feverof unknown origin (FUO) also shows a gradualincrease of CHIK disease beginning in August2001, with peak occurrence coinciding with out-break recognition between August—November 2001(Figure 3).

3.2.2. Clinical epidemiologyClinically recognized CHIK cases (119) wereidentified during the outbreak period from thecommunity health centers. No fatalities relatedto CHIK occurred in these two outbreak-affectedvillages. The mean age of recognized cases was36.5 ± 18 years (range 3—87 years). The propor-tional distribution of cases in the age groups ≥ 40years, 30—39 years, 20—29 years, 10—19 years and<10 years was 44, 19, 18, 14 and 5% respectively,and the age-specific attack rates (AR) in the af-fected villages were 4.6 cases per 1000 persons,3.7/1000, 2.7/1000, 1.6/1000 and 0.5/1000 respec-tively. The male-to-female case ratio was 1:2. Themost notable signs and symptoms among 108 cases

Figure 3 Undifferentiated fever cases from chikungunya ouand Kali Jaya) and control villages (Kayu Manis and Harja Mekbased on monthly reports of government health clinics, two y

tbreak-affected villages (Kebun Pedes & Kedung Badakar) in Bogor and Bekasi, West Java Province, Indonesia,ears preceding the outbreak.

Re-emergence of epidemic chikungunya virus in Indonesia 135

Figure 4 Frequency of signs and symptoms of clinically diagnosed chikungunya cases from outbreaks in Bogor andBekasi, West Java Province, Indonesia.

interviewed were arthralgia (81%), fever (78%) andmalaise (75%) (Figure 4). Duration of illness (mor-bidity) averaged 6.7 ± 5.05 days, with a range of1—30 days.

3.2.3. Epidemic curveThe period of epidemic CHIK transmission (cumu-lative 16 weeks) began at week 35 (August 2001)and ended at week 50 (December 2001). There wastemporal uniformity in the two affected villages(Figure 5). CHIK introduced itself slowly until week40 (October 2001), when the first of three dramaticrises in cases were recorded. The epidemic curvereflected periods of intermittent or oscillating pe-riods of activity, with repeated outbreak peaks oc-curring during weeks 44 and 48 (November 2001).At the close of the outbreak (week 50), only fivesuspected CHIK cases were recorded.

3.2.4. Case distributionClinically recognized CHIK cases were distributedin only two (Kedung Badak and Kebon Pedes) of the11 villages comprising Tanah Sareal district. Out of119 clinically defined cases, 69 were from KebunPnbe1ioath

and symptoms of CHIK. There was, however, ge-ographical case clustering of affected householdsobserved in both Kebon Pedes and Kedung Badakvillages.

Figure 5 Epidemic curves of clinical chikungunya casesin Bogor and Bekasi, West Java Province, Indonesia, basedon active case detection involving house-to-house visitsby community health center staff.

edes village, where the outbreak was first recog-ized, and 50 from Kedung Badak village. The ARsased on community health center case records andxtrapolated from census data, were 3.4 cases per000 persons in Kebun Pedes, and 2.3/1000 personsn Kedung Badak. CHIK cases were identified in 55%f 94 households surveyed in the outbreak-affectedrea. There was little evidence of familial case clus-ering in that 73% of 52 CHIK affected-householdsad just one family member with compatible signs

136 K. Laras et al.

Table 2 Laboratory results from investigated chikungunya outbreaks in Bogor and Bekasi, West Java Province,Indonesia

Village Percentage of positive sera

IgM ELISA (%) PCR (%) Culture IgG ELISA (%)

Outbreak-affected villageBogorKebon PedesCases (n = 50) 62 12 All negative 82Controls (n = 18) 11 6 All negative 67

Kedung BadakCases (n = 36) 40 17 14 47Controls (n = 27) 22 15 All negative 37Family member of cases (n = 25) 48 All negative 4 52

BekasiKali JayaCases (n = 93) 43 6 1 73Controls (n = 124) 10 0 Not done 40Family member of cases (n = 21) 19 All negative Not done 71

Non outbreak-affected vllageKayu Manis (Bogor) (n = 99) All negative 9 All negative 13Harja Mekar (Bekasi) (n = 55) All negative All negative Not done 6

3.2.5. Laboratory resultsFrom 86 case sera, 63% showed laboratory evidenceof recent CHIK infection by IgM ELISA (45) and/orRT—PCR (13) and/or viral isolation (5). Eleven of45 control sera obtained from healthy individualsin the outbreak-affected villages, had evidence ofrecent CHIK infection: eight by IgM ELISA and fiveby RT—PCR. CHIK IgG was detected in 67% (58/86)of case sera and 49% (22/45) of the control sera. In52% of sera obtained from 25 healthy family mem-bers from affected households (≥1 case), anti-CHIKIgM by ELISA was detected (12) or virus was iso-lated (1). Thirteen of 25 (52%) healthy family mem-bers also had anti-CHIK IgG indicating past expo-sure. Twelve had both IgM and IgG antibodies. Inthe control village of Kayu Manis, 9 and 13 (n = 99)specimens were positive by RT—PCR and IgG ELISA,respectively (Table 2).

Laboratory findings identified 63% of ‘symp-tomatic’ cases and 11% of ‘asymptomatic’ controlsas confirmed CHIK infections. Symptomatic CHIK-positive cases comprised 50, 88, 64, 61, and 60%in the age groups <10, 10—19, 20—29, 30—39, and≥40 years, respectively. Age-specific proportionsas to asymptomatic representation among CHIK-

RT—PCR, and 32.2 ± 16.6 by virus isolation. No sig-nificant difference (P = 0.199, analysis of variance[ANOVA]) was seen when these figures were com-pared with mean age of (i) laboratory negative,clinically recognized cases (40.7 ± 21.6 years); (ii)laboratory positive, village-affected controls (34.3± 17.4 years); and (iii) laboratory negative, village-affected controls (40.9 ± 17 years) (Table 3). Therewas little agreement between laboratory resultsinvolving 86 case specimens examined of which54 were found positive from the two outbreak-affected villages. No specimen was ‘positive’ by allthree diagnostic methodologies, whereas 32 werefound ‘negative’ by all three tests.

3.2.6. Vector ecologyDaytime indoor resting collections for adultmosquitoes captured 120 mosquitoes of which 60%were identified as Culex quinquefasciatus Say, 39%A. aegypti (L.), and 1% A. albopictus Skuse.

3.2.7. Climatic influencesThere was negligible variability in average monthly24-hour maximum—minimum ambient tempera-tures from January 2000 to December 2001( ◦iTca

positive cases was 0.6, 17, 38, and 15%, in the agegroups <10, 10—19, 20—29, 30—39 and≥40 years re-spectively. The mean age of laboratory confirmed,clinically recognized CHIK cases was 37.8 ± 18.3years: 38.5 ± 18.9 by IgM ELISA, 33.2 ± 15.5 by

24—26.2 C), however, the seasonal fluctuationsn rainfall were pronounced (79—491mm/month).here was an increase in rainfall from August 2001orresponding to the leading edge of the outbreaknd subsequent outbreak months of September to

Re-emergence of epidemic chikungunya virus in Indonesia 137

Table3

Agedistribution

oflaboratory

confi

rmed

chikungunyainfectionin

BogorandBekasi,WestJava

Province,Indonesia

Age(years)

Laboratory

confi

rmed

a

Bogor

Bekasi

Cases

Family

mem

bers

Controlsb

Cases

Family

mem

bers

Controlsb

1—9

2/4(50%

)6/10

(60%

)0/0(0%)

1/1(100%)

2/3(67%

)0/0(0%)

10—19

7/8(87.5%

)4/7(57%

)3/5(60%

)2/7(14%

)1/7(14%

)0/12

(0%)

20—29

9/14

(64%

)1/3(33%

)1/6(17%

)13/20(65%

)1/4(25%

)5/33

(15%

)30—39

11/18(61%

)0/1(0%)

3/8(37.5%

)7/20

(35%

)0/1(0%)

1/29

(3%)

≥40

24/42(59.5%

)2/4(50%

)4/26

(15%

)16/43(37%

)0/6(0%)

6/50

(12%

)Mean

±SD

years(range)

37.8

±18.3

(3—87)

14.9

±13.2

(3—42)

34.3

±17.4

(12—

60)

34.5

±14.2

(1—70)

12.8

±8.7(5—23)

32.8

±9.4(22—

46)

aPositive

byIgMELISAand/or

reversetranscriptasePC

Rand/or

virusisolation.

bFrom

outbreak-affectedvilla

ges.

December 2001: 142 cumulative mm versus a cu-mulative mean of 320 ± 91.8mm (ranging from 224to 445mm/month). The monthly rainfall (mm) andtemperature (◦C) data were obtained from BogorMeteorology and Geophysics office.

3.3. Bekasi outbreak

3.3.1. BackgroundHistorical trends over a four-year period (January1998 to July 2002) from Kali Jaya community healthcenter data showed no evidence of sporadic or epi-demic CHIK, based on review of reported clinicalsigns and symptoms before the outbreak period.When data extraction from clinical records was re-stricted to FUO cases, possible CHIK was detected inSeptember 2001, four months before the outbreak(Figure 3).

3.3.2. Clinical epidemiologyNo deaths were attributed to CHIK infection amongthe 169 outbreak-related cases in Kali Jaya. Themean age of clinically recognized cases was 33.3± 19.9 years (range 2—85 years). The highest pro-portion (37%) of CHIK was in the age group ≥40y3tt(msoyycgwf

3Ti(t7f

3Adilol

ears, followed by the age groups 20—29 (28%),0—39 (16%), 10—19 (12%), and <10 (7%) years. Ex-rapolation from Bekasi Regency population statis-ics (census data 2001), translated to the villageKali Jaya) level, provided for an overall esti-ated AR of 6.7 cases per 1000 persons, with age-pecific ARs of 11.4 cases/1000 persons (≥40 yearsf age), 8/1000 (30—39 years), 13/1000 (20—29ears), 4.0/1000 (10—19 years), and 1.5/1000 (<10ears). The male:female case ratio was 1:1.6. Prin-ipal clinical signs and symptoms included arthral-ia (89%), malaise (83%), and fever (76%) (Figure 4),hile the duration of illness (morbidity) rangedrom 1 to 60 days (mean 7.9 ± 8.1 days).

.3.3. Epidemic curvehe outbreak period lasted 23 weeks, startingn week 5 (January 2002) and ending week 27July 2002). The epidemic curve (Figure 5) showedhree dramatic increases in cases beginning week(February), week 18 (May) and week 24 (June),ollowed by an abrupt decline.

.3.4. Case distributionll suspected and confirmed outbreak cases wereetected from only one village, Kali Jaya, locatedn Cikarang Barat district, which comprises 11 vil-ages. The outbreak cases were restricted to onlyne of seven Rukun Wargas (RW, areas within a vil-age normally consisting of between 300 and 750

138 K. Laras et al.

households each), and further distributed withinonly two of three Rukun Tetanggas (RT, smallerunit areas within an RW). Seventy-nine percentof suspected CHIK cases were residents of RT 1,and 21% from RT 2. Cases were identified in 51%(91/180) of households surveyed, 24% of whichhad more than two family members with symp-toms compatible with CHIK. There was some ev-idence of familial case clustering because 47% of91 households had more than two cases per familyunit.

3.3.5. Laboratory resultsOverall, 46% of 93 case sera examined had evidenceof recent CHIK infection, compared with only 10% of124 asymptomatic control sera from the outbreak-affected village, 19% of 21 sera from healthy fam-ily members of cases, and none of the 55 sera fromthe control village. Forty-three percent of case serawere positive by IgM ELISA, 6% by RT—PCR, and 1%by virus isolation. Detection of IgG anti-CHIK among(i) cases, (ii) village-affected controls, (iii) healthyfamily members of cases, and (iv) matched controlsfrom the non-outbreak village, was 73, 40, 71, and6% respectively (Table 2). Laboratory findings re-

3.3.6. Vector ecologyDaytime indoor resting collections for adultmosquitoes captured 224 mosquitoes in Kali Jayafrom which 91% were identified as C. quinquefas-ciatus Say, 6% as A. albopictus and 3% as A. aegypti.Residual insecticide fogging just prior to the inves-tigation, however, likely nullified any meaningfuldata collection. All attempts to detect evidence ofCHIK virus by RT—PCR failed.

3.3.7. Climatic influencesThere was little variability in the average 24-hourmaximum—minimum ambient temperatures fromJanuary 2001 to August 2002, ranging from 26.2 to29.6 ◦C. Rainfall increased dramatically during theperiod leading up to the beginning (January andFebruary 2002) of the outbreak. Cumulative rainfallfor January (1094mm) and February (837mm) 2002greatly exceeded the mean cumulative monthlyrainfall (177mm, range 1—565) for pre-outbreakmonths of January to December 2001. The heavyrains resulted in significant flooding in Bekasi Re-gency. There was a precipitous drop in recordedrainfall of only 132mm in March 2002 and it re-mained below 150mm/month from April to July fol-lff

4

Tds(SlIpi1ocmspnioc1

pc

vealed confirmed infection in 46% ‘symptomatic’and 10% ‘asymptomatic’ controls from Kali Jaya.Age-specific proportions of confirmed infection rel-ative to clinical symptomatic group status were100, 29, 65, 35, and 37% in the <10, 10—19, 20—29,30—39, and≥40 year age groups respectively. In thecontrol groups, no asymptomatic infections werefound among the <20 year age group, whereas 15%of those 20—29 years, 3% of those 30—39 years, and12% of those ≥40 years of age were considered tobe asymptomatic infections.

There were significant differences (P = 0.03,ANOVA) between mean ages in laboratory diag-nosed CHIK cases based on confirmatory method:33.5 ± 13.5 years by IgM ELISA; 33.8 ± 13.1 yearsby RT—PCR; and 70 years by virus isolation. Themean age of laboratory confirmed CHIK cases (34.5± 14.2 years), regardless of the testing method-ology used, did not vary significantly (P = 0.2,ANOVA) by mean age from: (i) laboratory negative,clinically defined cases (40.6 ± 16.9 years), (ii)laboratory positive, village-affected asymptomaticcontrols (32.8 ± 9.4 years), and (iii) laboratorynegative, village-affected controls (36.7 ± 15.1years) (Table 3). There was poor test agreement(specificity and sensitivity) between the three as-say methods; only one serum (out of 43 confirmedcases) was positive by all three applied laboratorymethods. In contrast, 50 case sera from 93 exam-ined were found ‘negative’ by the three diagnosticassays.

owed by a rapid decline of cases. The monthly rain-all (mm) and temperature (◦C) data were obtainedrom Bekasi Meteorology and Geophysics office.

. Discussion

he re-emergence of epidemic CHIK across In-onesia after almost 20 years of quiescence isimilar to that previously experienced in IndiaPavri, 1973), Myanmar (Khai Ming et al., 1974),ri Lanka (Vasinjak-Hirjan et al., 1949) and Thai-and (Burke et al., 1985; Thaikruea et al., 1997).n Myanmar, a large outbreak of CHIK was re-orted in 1984, 12 years after the last reportednstance of epidemic occurrence (Thaung et al.,975; Thein et al., 1992). This apparent silencef virus and disease activity is an epidemiologi-al feature that distinguishes epidemic CHIK fromost other vector-borne viral diseases that shareimilar vectors and transmission dynamics, princi-ally dengue viruses. It has generally been recog-ized that re-emergent and epidemic CHIK occursn cycles of seven to eight years, although intervalsf two and three decades have been reported inountries like Uganda (Lanciotti et al., 1998; Pavri,986).Some of the outlying (outside of Java island) pur-

orted CHIK outbreak episodes lacking in laboratoryonfirmation may have in fact been attributed to

Re-emergence of epidemic chikungunya virus in Indonesia 139

dengue virus, commonly associated with epidemicsof febrile disease throughout Indonesia (Corwin etal., 2001; Sukri et al., 2003). Nevertheless, it iscritical in viewing the relatively few instances inwhich laboratory confirmation was possible againstthe background of archipelago-wide epidemic oc-currence.

Most (62%) of the outbreak episodes during therecent Indonesian epidemic occurred in denselypopulated rural localities on Java, although mostcases were recognized from urban and semi-urbanareas. Epidemic CHIK has been predominantly anurban feature in Asia, whereas in Africa, outbreakshave been characterized as being smaller in scaleand affecting principally rural populations (Juppand McIntosh, 1988). In Indonesia, as in many otherdengue-endemic countries in Southeast Asia, spo-radic occurrences and outbreaks of CHIK are likelyto be underreported as the disease is not commonlyincluded in clinical diagnostic algorithms used inthe region. A coincident rise in FUOs months be-fore the suspicion that CHIK was involved in theoutbreaks in Bogor and Bekasi attests to the gen-eral unfamiliarity of the disease for most healthcare professionals, while negligible FUO cases wererficplae

dohiitb(flwh

aM1ldbTidi

by ELISA IgM and/or RT—PCR. Anti-dengue IgM wasidentified from only one case, in the absence ofIgM anti-CHIK. Hemorrhagic manifestations associ-ated with recent CHIK infection has been reportedfrom Thailand, previously, where 8% of hemorrhagicfever cases seen in Bangkok were attributed to CHIK(Burke et al., 1985). A hospital-based study in Myan-mar revealed similar rates of hemorrhage, such asepistaxis (9%), petechiae (8%), and melena (1%), as-sociated with sporadic clinical CHIK (Thein et al.,1992).

The mosquito vectors associated with the twooutbreaks remain speculative, in so far as field col-lections were restricted to species discriminationonly. In Bekasi, extensive insecticide applications(fogging) against adult mosquitoes immediately fol-lowing outbreak recognition complicated proper as-sessment. The relative contribution of A. aegyptiand A. albopictus is not known, although it is pre-sumed that A. aegypti was the primary vector re-sponsible for transmission in all outbreaks in In-donesia. Although the most abundant indoor restingmosquito was C. quinquefasciatus, this ubiquitousspecies is refractory to infection with CHIK virus andthus is not considered a vector (Jupp and McIntosh,1

dwtolrhsbstseibrisirmraglac

o

ecorded from control villages. Epidemic curvesrom the two investigated outbreaks revealed sim-lar trends and progression; a gradually increasingase load early on, followed by three distinctiveeaks in disease incidence. CHIK outbreaks in Thai-and (Thaikruea et al., 1997) and Malaysia (Lam etl., 2001) exhibited the same pattern, reflected inpidemic curves described in west Java.Age-specific attack rates associated with epi-

emic CHIK increased with age in both investigatedutbreaks. Similar demographic disease profilesave been recognized in other outbreaks, includ-ng that described during the Malaysian epidemicn 1998. The two-fold greater female represen-ation among cases seen in both Indonesian out-reaks was comparable to that reported in MalaysiaLam et al., 2001). This outbreak feature may re-ect a greater occupational exposure associatedith women’s daytime activities in and around theome.Hemorrhagic presentation has occasionally been

ssociated with acute CHIK infections (Jupp andcIntosh, 1988; Nimmannitya and Mansuwan,966). In general, hemorrhagic presentation re-ated to CHIK in outbreak settings has not beenocumented from the region, including recent out-reaks in Malaysia and Thailand (Lam et al., 2001;haikruea et al., 1997). In both outbreaks describedn Java, 15 of 179 (8.3%) suspected CHIK cases wereescribed as having hemorrhagic presentation dur-ng illness; 73% (11/15) of which were CHIK-positive

988).The CHIK outbreaks in Bogor and Bekasi began

uring the start of rainy seasons, during which thereas a rapid transition from low to high precipita-ion. Additionally, the first outbreak peak in casesccurred five (Bogor) and four (Bekasi) weeks fol-owing the start of the 2001 and 2002 rainy seasonsespectively. The association with climatic eventsas also been described in other CHIK outbreakettings (Jupp and McIntosh, 1988). In urban out-reaks where A. aegypti is the vector, possiblyupplemented by A. albopictus in Asian countries,he seasonal distribution of CHIK transmission hashown a relationship with rainfall patterns in sev-ral countries, generally increasing in response toncreased precipitation. At least two separate out-reak episodes occurred at the beginning of theainy season (June—August 1995) in Thailand, withncreases in case recognition a month into the weteason (Thaikruea et al., 1997). Exceptions existn other areas of South and Southeast Asia whereainfall is not markedly seasonal and cases of CHIKay occur throughout the year (Halstead, 1966). Aeasonable assertion would be that the increasedbundance of rainwater-filled containers providesreater success rates for mosquito oviposition andarval development, resulting in higher densities ofdult vectors (Jupp and McIntosh, 1988), thus in-reasing risk of virus transmission.A distinguishable, albeit non-linear trend was

bserved in the eastward progression of epidemic

140 K. Laras et al.

CHIK occurrence across the Indonesian archipelago,resulting in 24 recognized outbreak episodes overa 19-month period in 2001—2002. It is likely thatmany more outbreaks occurred in the country thatwere not reported. The long duration between epi-demic disease occurrences in Indonesia likely con-tributed to the general underreporting and poorrecognition of CHIK during sporadic episodes andlong intervals of inter-epidemic activity. The ap-parent temporal distribution of virus has beenattributed to the agency of human movementfrom different localities and islands, a very com-mon activity in Indonesia. This extensive seriesof outbreaks beginning in Sumatra and extend-ing to Lombok and Sulawesi were considered allthe more remarkable given that there has beena notable absence of recognized CHIK diseasereported clinically in Indonesia for nearly twodecades. To our knowledge, CHIK did not spreadto islands further east in the country, remain-ing confined to the Oriental zoogeographical re-gion (Mackenzie et al., 1994). The ferocity withwhich this epidemic presented itself after yearsof apparent inactivity caught health authoritiesacross the archipelago nation off-guard. Countries

tance. Primers used for the RT—PCR were designedby James McArdle. These investigations could nothave been conducted without the full support ofUmar F. Achmadi and Soemarjati Arjoso from theCenters for Communicable Diseases — Preventionand Environmental Health (P2M-PLP) and the Na-tional Institute of Health Research and Develop-ment (LitBangKes), Indonesian Ministry of Health,Jakarta, respectively.This work received financialsupport from the US Department of Defense andthe Global Emerging Infections System (GEIS).

References

Burke, D.S., Nisalak, A., Nimmannitya, S., 1985. Disappearanceof Chikungunya virus from Bangkok. Trans. R. Soc. Trop. Med.Hyg. 79, 419—420.

Carey, D.E., 1971. Chikungunya and dengue: a case of mistakenidentity? J. Hist. Med. Allied Sci. 26, 243—262.

CDC, 1986. Chikungunya fever among U.S. Peace Corps Volun-teers — Republic of the Philippines. MMWR Morb. Mortal.Wkly. Rep. 35, 573—574.

Chastel, C., 1963. Infections humaines au Cambodge par le viruschikungunya ou un agent etroitement apparente. III. Epi-demiologie. Bull. Soc. Pathol. Exot. Filiales. 1, 65—82.

C

C

D

H

H

H

J

K

K

L

neighboring Indonesia, yet to witness a resurgenceor an emergence of epidemic CHIK, would findit useful to learn from Indonesia’s recent experi-ence.

5. Disclaimer

The opinions and assertions contained herein are ofthe authors’ own choosing and are not to be con-strued as official or as reflecting the views of theUS Navy or the Department of Defense.

Conflicts of interest statementThe authors have no conflicts of interest concerningthe work reported in this paper.

Acknowledgments

The authors gratefully acknowledge the coopera-tion from the respective health authority officesin Bogor and Bekasi, West Java and all govern-ment community health centers and other insti-tutions for assisting in the investigations. Specialthanks to Nursyarofah, Ratna P. Kusumaningrum,Yayu Fathiyah, Rohani Simanjuntak, Erlin Listyan-ingsih, and Chairin Ma’roef and other laboratorytechnicians in the US NAMRU-2 Virology Programfor specimen/data processing and laboratory assis-

hin, J. (Ed.), 2000. Control of communicable diseases manual,seventeenth edition. APHA, Washington, pp. 37—39.

orwin, A.L., Larasati, R.P., Bangs, M.J., Wuryadi, S., Arjoso,S., Sukri, N., Listyaningsih, E., Hartati, S., Namursa, R., An-war, Z., Chandra, S., Loho, B., Ahmad, H., Campbell, J.R.,Porter, K.R., 2001. Epidemic dengue transmission in south-ern Sumatra, Indonesia. Trans. R. Soc. Trop. Med. Hyg. 95,257—265.

ai, V.Q., Thoas, N.T.K., 1967. Enquete sur les anticorps anti-chikungunya chez des enfants Vietnamiens de Saigon. Bull.Soc. Pathol. Exot. Filiales. 4, 353—359.

aksohusodo, S., 1990. Chikungunya viral disease outbreak inYogyakarta, Indonesia. Presented at the Tropical Disease Re-search Report Symposium, National of Health Research andDevelopment, Ministry of Health of Republic Indonesia andNAMRU-2, Jakarta, Indonesia, 13—14 July 1990.

alstead, S.B., 1966. Mosquito-borne haemorrhagic fevers ofSouth and South-east Asia. Bull. World Health Organ. 35,3—15.

alstead, S.B., Udomsakdi, S., 1966. Vertebrate hosts of Chikun-gunya virus. Bull. World Health Organ. 35, 89.

upp, P.G., McIntosh, B.M., 1988. Chikungunya virus disease. In:Monath, T.P. (Ed.), The Arboviruses: Epidemiology and Ecol-ogy, II. CRC Press, Boca Raton, FL, pp. 38—152.

anamitsu, M., Taniguchi, K., Urasawa, S., Ogata, T., Wada,Y., Saroso, S.J., 1979. Geographic distribution of arbovirusantibodies in indigenous human populations in the Indo-Australian archipelago. Am. J. Trop. Med. Hyg. 28, 351—363.

hai Ming, C., Thain, S., Thaung, U., Tin, U., Myint, K.S., Swe,T., Halstead, S.B., Diwan, A.R., 1974. Clinical and laboratorystudies of haemorrhagic fever in Burma, 1970—1972. Bull.World Health Organ. 51, 227—235.

am, S.K., Chua, K.B., Hooi, P.S., Rahimah, M.A., Kumari,S., Tharmaratnam, M., Chuah, S.K., Smith, D.W., Sampson,I.A., 2001. Chikungunya infection — an emerging disease inMalaysia. Southeast Asian J. Trop. Med. Public Health 32,447—451.

Re-emergence of epidemic chikungunya virus in Indonesia 141

Lanciotti, R.S., Ludwig, M.L., Rwaguma, E.B., Lutwama, J.J.,Kram, T.M., Karabatsos, N., Cropp, B.C., Miller, B.R., 1998.Emergence of epidemic O’nyong-nyong fever in Uganda af-ter a 35-year absence: genetic characterization of the virus.Virology 252, 258—268.

Mackenzie, J.S., Lindsay, M.D., Coelen, R.J., Broom, A.K., Hall,R.A., Smith, D.W., 1994. Arboviruses causing human diseasein the Australasian zoogeographical region. Arch. Virol. 136,447—467.

Mackenzie, J.S., Chua, K.B., Daniels, P.W., Eaton, B.T., Field,H.E., Hall, R.A., Halpin, K., Johansen, C.A., Kirkland, P.D.,Lam, S.K., McMinn, P., Nisbet, D.J., Paru, R., Pyke, A.T.,Ritchie, S.A., Siba, P., Smith, D.W., Smith, G.A., van denHurk, A.F., Wang, L.F., William, D.T., 2001. Emerging vitaldiseases of Southeast Asia and the Western Pacific. Emerg.Infect. Dis. 7 (Suppl.), 497—504.

Nimmannitya, S., Mansuwan, P., 1966. Comparative clinical andlaboratory findings of confirmed dengue and chikungunya in-fections. Bull. World Health Organ. 35, 42—43.

Pavri, K.M., 1973. Editorial: epidemic of fever with severe jointpains: chikungunya virus. Maharashtra Medical Journal 20,219—220.

Pavri, K., 1986. Disappearance of chikungunya virus from Indiaand South East Asia. Trans. R. Soc. Trop. Med. Hyg. 80, 491.

Porter, K., Juffrie, M., Tan, R., Istary, J., Suharyono, W., Su-taryo, Widjaja, S., Ma’roef, C., Listiyaningsih, E., Kosasih,H., Hueston, L., McArdle, J.L., 2004. A serological study ofchikungunya virus transmission in Yogyakarta, Indonesia: ev-idence for the first outbreak since 1982. Southeast Asian J.Trop. Med. Public Health 35, 408—415.

Slemons, R.D., Haksohusodo, S., Suharyono, W., Harundriyo, H.,Laughlin, L.W., Cross, J., 1984. Chikungunya viral diseasein Jogyakarta, Indonesia. In: Presented at the 33rd AnnualMeeting of the American Society of Tropical Medicine andHygiene, Baltimore, MD, USA, 2—6 December 1984.

Sukri, N.C., Laras, K., Wandra, T., Didi, S., Larasati, R.P.,Rachdyatmaka, J.R., Osok, S., Tjia, P., Saragih, J.M., Hartati,S., Listyaningsih, E., Porter, K.P., Beckett, C.G., Prawira, I.S.,Punjabi, N., Suparmanto, S.A., Beecham, H.J., Bangs, M.J.,Corwin, A.L., 2003. Transmission of epidemic dengue hem-orrhagic fever in easternmost Indonesia. Am. J. Trop. Med.Hyg. 68, 529—535.

Thaikruea, L., Charearnsook, O., Reanphumkarnkit, S., Dis-somboon, P., Phonjan, R., Ratchbud, S., Kounsang, Y., Bu-ranapiyawong, D., 1997. Chikungunya in Thailand: a re-emerging disease? Southeast Asian J. Trop. Med. PublicHealth 28, 359—364.

Thaung, U., Ming, C.K., Swe, T., Thein, S., 1975. Epidemio-logical features of dengue and chikungunya infections inBurma. Southeast Asian J. Trop. Med. Public Health 6, 276—283.

Thein, S., Linn, M.L., Aaskov, J., Aung, M.M., Aye, M., Zaw,A., Myint, A., 1992. Development of a simple indirectenzyme-linked immunosorbent assay for the detection of im-munoglobulin M antibody in serum from patients following anoutbreak of Chikungunya virus infection in Yangon, Myanmar.Trans. R. Soc. Trop. Med. Hyg. 86, 438—442.

Vasinjak-Hirjan, J., Hermon, Y., Vitarana, T., 1949. Arbovirusinfections in Ceylon. Bull. World Health Organ. 41, 243—249.