Jpn. J. Infect. Dis., 52, 1999

Laboratoryand Epidemiology Communications
The Features of Imported Dengue Fever Cases from 1996 to 1999
Ken‑Ichiro Yamada* , Tomohiko Takasaki , MasaruNawal ,Mikio Nakayama, Yohko T. Arai, Sadao Yabeand Ichiro Kurane
Department of Vl'110logy 1 , National Institute oflnjTectious Diseases,
Toyama ll23‑1, Shinjuku, 7To砂0 162‑8640 and)Department of Microbiology, Saitama Medical School,
Moroyama, Iruma, SaiEatna 350‑0495
Communicated by Hiroshi Yoshikura(Accepted January 1 4, 2000)
Dengue viruses are transmitted by infected mosquitoes,

as previously reported (7,8).

The clinical manifestations of dengue virus infections range

IgM‑capture ELISA wasperformed as previously reported(7,8). Briefly, 96 well microplates were sensitizedwith goatanti‑humanIgM (LL Chain specific)antibody (Zymed IAbora‑

from asymptomatic infection to two forms of illness (3).

tories, Inc., South Sam Francisco, Calif.). One hundred

Dengue fever (DF) is a self‑limited febrile illness. Somemicroliters of serum samples diluted at 1 :101 in phosphatepatients with dengue virus infection develop a severe, life‑

buffered saline (PBS) containing lo啄 calf serum (Wako Puretheatening syndrome called dengue hemorrhagic fever (DHF).

Chemical Industries, Osaka) was added to the wells andincubated for 1 h at room temperature. After washing with
PB S, each of four monovalentand uninfected control antigens

mainly by Aedes aegypti and Aedes albopictus ( I ). There are

four serotypes, namely, dengue virus types l, 2, 3, and 4 (2).

The Wわrld Health Organization categorizes DHF cases into

four grades, from less severe (grade I) to severe (grade 4).

Grades 3 and 4, in which plasma leakage is so profound thatwere added to wells and incubated for 1 h at room tempera‑

shock occurs,arealSo referred to as dengue shock syndrome

ture. Plates were washed and peroxidase‑conjugated human(DSS) (3).
Dengue virus infections are a serious cause of morbidity

anti‑flaviviruS IgG was added and incubated at 1 h at room
temperature. After washing, enzyme substrate, 0‑phenylene‑

and mortalityin many areas of the world: Southeastand SouthAsia, Central and SouthAmerica, the Caribbean,and Africa.
It is estimated that 100 million cases of dengue fever anddiamine 2HCl, and H202 in 0. I M citrare buffer, pH 5.0, wasadded and incubated for 30 min at room temperature. Indexvalue was calculated bythe followlng formula: Index value

250,000 cases of DHF occur annually (4). Thus, DF/DHF is= A492 absorbance with viral antigen/A492 absorbance with

one of the most important infectious diseases in the world.

uninfected control antigen. We definedanIndex value equal

The regions in which dengue virus infectionsare shown to beto or greater than 2.0 as positive.

serious health problems have been expanding,and the number

RT‑PCR was performed as previously reported (7,8)･of DHF cases is increaslng. The World Health Organization

Briefly, RNA was isolated from 0.05 mL of serum specimen

reports that domestic dengue virus infections occur in nearlyusing 0.2 ml of Isogen‑LS (Nippon gene, Tokyo) and O･04 ml

100 countries and reglOnS.

of chloroform. RT and PCR were done in a slngle tube. The

tubes were set in an oil bath‑typethermal programmer (Iwaki,A dengue outbreak occumd in Osaka, Robe, Hiroshima,and Nagasaki from 1942 to 1945, and dengue viruStype ICo. , Tokyo) and subjected to programmed incubation at 53oC

was responsible for the outbreak (5). Dengue virus infectionsfor 10 min for reverse transcription. This was followed byhave never reached epidemic proportions in Japan sincethen,

30‑40 PCR cycles of amplification. PCR products were thenand there are currently no domestic dengue virus infections

subjected to agarose gel electrophoresis. Amplified DNA

in contemporary Japan. However, imported dengue cases havefragments were visualized by ethidium bromide staining･

been reported (6). We have beenperforming serodiagnosis ofTarget size and the pnmer sequences used to amplify eachdengue virus infection upon request from hospitals andserotype of dengue vims were previously reported (7,8)･ HI

clinics. The features of imported dengue cases that wetests adapted for microtiter plates wereperformed using 4

serodiagnosed from 1985 to 1995 at the Department ofhemagglutinin units of dengue‑2 viral antlgen, aS previously

Virology 1 , National Institute of Infectious Diseases, Japandescribed (6). The highest dilution of semm samples which(NIID) were previously reported (6)･

provided positive HI was considered as HI titer･Serum speclmenS Were Obtained from dengue‑suspected

Table 1 shows the number of dengue cases confirmed bycases in clinics and hospitals in Japan from 1996 to 1999,

laboratory tests from 1996 to 1999. The numbers of dengue

and were sent tothe Department of Virology 1, NIID for thecases were 15, 6, 42, and ll in 1996, 1997, 1998, and 1999,

laboratory diagnosis of dengue. In the present manuscript, We

respectively. All of the cases were DF cases;there were noreprt features of these dengue cases. Dengue virus infections

DHF cases during thisperiod of time. Of 74 totalpatients with

were diagnosed by lgM‑capture enzyme‑linked immunosorbent

DF, 53 were maleand 21 were female (1もble 2). The patients'

assay (ELISA), reverse transcriptase‑polymerase chain reac‑tion (RTIPCR), and hemmagglutination inhibition (HI) test,

age distribution is also shown in Table 2. The youngest
patient was 16 years old and the oldest was 62 years old･Approximately half of the cases were between the ages of 21and 30 (inclusive). Each of the three age groups, ll‑20, 31‑*Co汀eSpOnding au血or: Fax: +8ト3‑5285‑ 1 188, E‑mail: kyamada@nih.go.Jp

40, and 4ト50 accounted fわr approximately 15 % of the total

257

Jpn. J. Infect. Dis., 52, 1999
number of cases.

Table 1 ･ Number of imported dengue cases confirmed at the National

Table 3 shows the countries visited by these dengue patients

Institute of Infectious Diseases from 1996 to1999

before they developed illness. Most of the Japanese dengue
Ye‑

TotetendTTubeersof Deny,eF)fever Dengue he(器F)hagicfever

1 99615(M 14,F 1)*3 526恥男1 997

6(M3,F3)

1 998

42 (M28 , F14)

1 999

ll (M8,F3)

patients were infected withdengue viruses in Southeast andSouth Asia, especially in Thailand, India, the PhilipplneS,
Indonesia, Singapore,and Myanmar. There were patients who
had visited Tahiti, Guatemala, Nigeria, and Cote d'Ivoire in0000

recent years; these patients developed dengue illness after

retumlng tO Japan.

*M and F indicate male and female, respectively･

Althoughthe age distribution of dengue patients ranges
from 16 to 62 years old, approximately 50% of the cases werein the age group of 2 I ‑30. This probably renects the number ofTable 2･ Age distribution of imported dengue cases confirmed at theNationaJ Institute of lnfecdous Diseases from 1996 to1999

travellers who visited dengue epidemic areas. Furthermore, it

is important to notethatthere were patients who visited Oceania,
CentralAmerica,and Africa before developlng dengue illness.

Age Number of DF cases*
1 2 3 4 5 人V 1 1 1 1 l 1

qSqSSFll (15%)Thus, dengue is not to be considered as a differentialdiagnosis

35 (49%)

only in the case of patients who retum from Asian countries.

ll (15%)We believethat these dengue cases account for only a small

10 (14%)part of the total imported cases; the exact number of imported

3 (4%)

cases in Japan remains unknown. The new infectious disease

1 (1%)

control law is effective as of Apri1 1, 1999 in Japan. DM)HF
is one of the diseases which physicians are requested to report*Ages of 3 cases were not known.

bythis law. Thus, it is expected in the near future thatthe
exactannualnumber of dengue cases will be known in Japan.
Nearly 5 million Japaneseannually visit these countries inTable 3･ Countries visited by dengue padents beforethey

developed symptoms
Countrics*

1

tropICal and subtropical areas; nearly 2 millionpeople visit

996

1997

1998

1999Total

2k2 1 1 12194 32 1

Japan from these areas. Therefore, DF/DHF is a slgnificant
infectious disease worthy of more attention fromthe medicalAsia
Thailand21dlmdia

480Phi I ippl neS

11Indonesi a3Si ngapore

1

Myanmar0Malaysia

2boBangl adeshlc0Maldi Yes0ll1Nepal0100Cambodi a0001This work was supported by a grant forthe Research onEmergingand Re‑emerglng Infectious Diseases from Mimistryof Healthand Welfare of Japan,and by a grant for the Program16｡

community in Japan. DF/DHF should be included in the3differential diagnosis when febrile patients have just retumed

81

from tropicaland subtropical reg10nS, including those inAsia,

15012g090

20Oceania, Centraland South America, and Africa. Such adiagnosis should be considered irrespective of sex or age.

2o1lhoWethank the doctors at the clinics and hospitals forproviding us the serum samples for the laboratory diagnosisof dengue.

Oceani aTihi ti010000101 00 0o IJ0 0for the Promotion of Fundamental Studies in HealthSciences

Central Amedca
Guatcmal a

fromthe Organization for PharmaceuticalSafetyand Research(OPSR) of Japan. We are also grateful for grants fromthe
Ministry of Education, Science, Sports and Culture (Grant‑Africa

Nigeria

in Aid for Scientific Research 084571 00, and 09044344), andCote d'Ivoire
Total1564211

forthe Cooperative Research Grant 1999 (1 1‑A‑2) fromtheInstitute of TropICal Medicine, Nagasaki University.

74*When patients visited more than one country, the countries which
patients visited last were counted.

a) One patientalso visited Thailand.

REFERENCESb)One padent also visited Thailand.

1. Monath, T. P. (1994): Dengue:therisk to developed andC) One patientalso visited India.

d)One patient also visited hos and Vietnam.

developlng countries. Proc. Natl. Acad. Sci. USA, 91,e)Two patientsalso visited Cambodia･ One patientalso visited China,

2395‑2400.

2. Henchal, E.A.and mtnak, R. (1990): The dengue viruses.

Vietnamand LAOS･ One patientalsovisited L弧S and Myanmar･o Two patients alsovisited Thai1and･Clin. Microbiol. Rev., 3, 376‑396.
3. World Health Orgamization ( 1997): Dengue haemorrhagic
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A)One patientalsovisited Nepal and India･Health Organization, Geneva.

i) One patientalSo visited Srilanka.

J) One patient also visitedLibcria.
k)Two patients also visited Myanmar･

4. Halstead , S. B. (1988):ねthogenesis of dengue. Challenge

to molecular biology. Science, 239, 476‑481.

5. Hotta, S. (1965): Twenty years of laboratory experience

with dengue virus. p.228‑256. In Saunders, M. andLennette, E. H. (eds.), Medical and Applied Virology.
Green, St I』uis.

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8. Yamada, K., Nawa, M., Takasaki, T., Yatx, S.and Kurane,(1996): Laboratory virological diagnosis of importedI. ( 1 999): IAboratory diagnosis of dengue virus infection

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6･ Yabe, S., Nakayama, M., Yamada, K., K血no, T., Arai,

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