第23巻第2号平成7年6月
内
原

容

著

タイ国小児におけるロタウイルス感染症の2年間の追跡調査
一抗原検出と下痢症状一一Jirapom Supawadee，鈴木博，Somboon Suprasert
Prapasri Rangsiyanond，BoonyongPongprot，長谷川斐子
井上

栄，山地

幸雄……・…………・………一…

109−113

ドミニカ共和国における肝炎ウイルスの血清疫学的研究
1．A，B，C型肝炎ウイルス マーカーの保有頻度
・七篠 明久，三舟求眞人，青野 裕士，柴山均
寺尾 英夫，宮田
彬，小澤 秀樹，伊東 盛夫
三角 順一，糸賀敬，Maria Estela Norman
Aracelis Rodriguez German，Mercedes Castro Bello

AbelardoAHidalgoSigaran…一・……一………115−120
1994年ラオス国ビエンチャンで発生したデング熱流行のウイルス学的研究
・・Thongchanh Sisouk，加根村和美，斉藤 美加
Bounlay Phommasack，牧野 芳大，新垣榮馬紹平，Sithat Insisiengmay，福永 利彦…

12H25

インドネシアのスマトラ島で見いだされたホソスネブユ亜属の1新種の記載
・高岡 宏行，D．M．Davies……………・………一… 127−131

長崎市における飼い犬の犬糸状虫の仔虫保有率の27年間の年次的変化に関する研究
一小田
力，末永
敷，在津
誠，森
黒川 憲次，藤田紘一郎，小川 保徳，山崎飯田 國洋，土井 浩一，三根真理子，加藤
短

章夫
一郎
克知

133−137報

モザンビークにおける性感染症の流行
・山田

誠一，藩

悦，森

月館説子，藤田紘一郎…

139−1414﹂任

投稿規定………・…
日本医学会だより・

元

3
17
1

会報・記録

有加，犬尾

109

Jpn. J. Trop. Med. Hyg., Vol. 23, No. 2 (1995) pp. 109‑113

TWO‑YEAR FOLLOW UP FROM BIRTH OF THAICHILDREN FOR ROTAVIRUS INFECTION=
DETECTION OF ROTAVIRUS IN FECESAND DIARRHOEAL SYMPTOM

JIRAPORN SUPAWADEE , HIROSHI SUZUK12, SOMBOON SUPRASERTl,

PRAPASRI RANGSlYANOND , BOONYONG PONGPROT1, AYAKO HASEGAWA3,
SAKAE INOUYE3 and YUKIO YAMAZI+
Received January 26, 1995/Accepted March 6, 1995

Abstract: Twenty‑eight newborns delivered in Chiang Mai, Thailand in October and December, 1988 were
followed up for rotavirus infection for two years after birth. Sixty‑eight percent (19128) of the index
children discharged group A human rotavirus (HRV) antigen and/or viral genomic RNA in their feces
during the two years, and 7.1% (2/28) discharged twice during the period. Seasonal distribution of HRV‑positive fecal specimens was 29% (6121) in January to April, 1989, 9.5% (2/2D in August and September,
1989, 57% (12/21) in December, 1989 to April, 1990, and 4.8% (1/21) in June, 1990.
Eighty‑six per cent (24/28) of the index children developed diarrhoeal symptom and the total numberof the episodes was 67, i.e., 2.39 for one child during the two years. One severe episode, 20% (3115) ofmoderate and 12% (6151) of mild diarrhoeal episodes were correlated to the rotavirus infection.

INTRODUCTION

188 diarrhoeal patients by ELISA in Chiang Mai, Bang‑

kok and Khon‑Kaen (Yamazi et al., 1988) , and madeRotavirus is one of the most important aetiologic
agents of gastroenteritis among infants and children
causing many cases of severe disease and death world‑

some preliminary work on the fecal lgA antibody

wide (Davidson et al., 1975; Kapikian et al., 1980, 1990) .

Hospital based and community based studies in different

newborns delivered at a hospital in Chiang Mai in
October and November, 1988 went followed up fromgeographic areas of Thailand during 1975‑1983 showedbirth for two years for the rotavirus infection. The

that rotavirus was most common cause of diarrhoea
during the cold months (November‑February) (Puth‑

ebjective is to know frequency, time and severity of theavathana et al., 1981, Jayavasu et al., 1982, Louisirirot‑
chanakul et al., 1984, Varavithya et al., 1988). Rotavir‑

first two years of their life.

us was identified in 44% of acute diarrhoeal patients
aged 1‑23 months admitted to Siriraj Hospital, Bangkok

responses to rotavirus infection of the patients in
Chaiang Mai (Supawadee et al., 1988) . In this report, 28

human rotavirus infection of Thai infants during the

MATERIALS AND METHODS(Phijaisanit et al., 1988) , and similar figures were obser‑

Index children and collection of feces

ved also in Japan (Kariyasono et al., 1993) and in

Fourty newborns delivered in the Maharaj Nakorn
Chiang Mai Hospital, Chiang Mai University, ChiangWestern Europe and the United States during the winterseason (Davidson et al., 1975) .

Mai, Thailand in October and November, 1988 wereWe made a seroepidemiological survey of rotavirusin Thailand in 1978 (Maneekarn et al., 1980). In 1984
and 1985, we detected rotavirus antigens in 46% out ofscheduled to be followed up for two years. This timeschedule covered two cycles of dry‑cool and rainy sea‑

1.
2.
3.
4.

sons, so that the distribution of rotaviral infection and

Departments of Microbiology, Family Medicine, and Pediatrics, Chiang Mai University, Chiang Mai, Thailand
Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
Department of Epidemiology, National Institute of Health, Tokyo
Sayama Hakuai Hospital, International Good Will Foundation of Japan, Saitama, Japan110

the response to the infection of Thai newborns in each

Table 1 Rotavirus discharge in f eces a nd diarrhoeal
sym ptomseason were studied. They were selected to cover awide range of environmental and socioeconomical stan‑dards, as far as convenience of the follow‑up work

allowed. This study was approved by the Human
Experimentation Committee, Faculty of Medicine,VolunteerNo.

Diarrhoeal symptom

Rotavirus
in feces

Mild

Moderate

4

Chiang Mai University (N0.9, 1988) and by the World
Health Organization (HQ/89/060317) . Parents of thechildren gave informed, sigued consent. Twelve chil‑

5

9

5

37

16, 19, 42

13

30, 37930

dren did not complete the study because their families
moved away. The data presented refer to the remaining

27

42

36

65

28 children.

30

54
78

85

61

15

A Well Baby Clinic was set up by the Department of
Family Medicine and Pediatrics, Chiang Mai University.
Contact persons with sufficient training visited the14volunteers' homes every week, recorded the clinicalsymptoms, especially gastrointestinal disorders of thechildren, and collected fecal specimens. Extra fecal

11

34

14

21

15

40

18

IZ, 37, 33, 3468

11

19, 6466, 95, 96

38

20

23, 26, 62

samples were collected during diarrhoeal episodes.
Parents were requested to bring their index children

24

40

12,

16

7, 38

every two months to the Well Baby Clinic to be

5

44
61

examined by a pediatrician (P.R.). During the study

35

63

23

66, 72639

67

8, 17, 18

8

69
70

34, 39, 55, 60

59, 6710

2, 34, 42

3

25

70

18
59, Z , 89

period, all participating children received the standard

immunization to diphtheria, pertussis, tetanus, polio,
measles and BCG at the Well Baby Clinic. Fecal speci‑
mens were stored at ‑ 40'C until tested.

772

Detection of rotavirus antigen and determination of

1

74

11, 41, 70

electrophenoty pes

680

23, 71, 80

290

34, 71, 81,

Fecal specimens were tested for group A rotavirus

antigens by a sandwich ELISA and for rotavirus
genomic RNA by polyacrylamide gel electrophoresis(PAGE) at Department of Microbiology, Chiang Mai

Severe

31

59,

,

24

8, 26

79
1

70

Figures in the Table are weeks after birth. Underlines show
weeks within one week of rotavirus discharge in feces.

University, and double checked at N.1.H., Tokyo. The

ELISA was performed according to the WHO standard

1988

The fecal specimens were tested as 10% suspension inphosphate‑buffered saline.
Hyperimmune rabbit
antisera to Wa strain of HRV serotype I was used as
capture antibody, and guinea pig antiserum to Wa strain
and rabbit‑anti guinea pig lgG antibody conjugated withhorseradish peroxidase were used as detection reagents.

1989

1990

6

method with slight modifications (Inouye et al., 1989).

5

8

4

2E

3
2
1

In a preliminary experiment (Yamazi et al., 1988),
rotavirus was not found with electron microscopy in allof the fecal specimens negative for rotavirus antigens byO

this ELISA method. For PAGE of rotavirus RNA, a

Figure 1minor modification of the method of Herring et al.
(Herring et al., 1982) was used. Double stranded viral

RNA was extracted with phenol and chloroform direct‑
ly from 10% fecal suspension. RNA bands were detect‑
ed by silver staining.

ONDJFMAMJJASONDJFMAMJJASO
MonfllMonthly distribution of fecal discharge of group

A rotavirus‑antigen and/or RNA. Fecal samples
collected weekly from newborns delivered in
October and November, 1988 were tested with an
ELISA and PAGE. Closed bar, primary infection.
Open bar, reinfection.

111

Table 2 Correlation between HRV‑discharge and diar‑rhoeal symptom
HRV in feces (%)

Diarrhoeal symptom

Positive

Negative

1 (4 . 8)

O (O)

1

12 (O . 4)

153(14) 6 (29)45 (1 . 6)

Total

Total

asymptomatic primary infection with RNA‑electropher‑
otype S and a symptomatic secondary infection with
RNA‑electropherotype L of the virus. N0.23 had two
asymptomatic infections with RNA‑electropherotype L
and with RNA‑electropherotype S of the virus, respec‑tively.

51

11 (52)

2 , 835 (98)

2 , 846

21 (100)

2 , 892 (100)

2 , 913

Twenty‑eight children were studied for fecal HRV‑dis‑charge and diarrhoeal symptom every week for two years after

birth. Correlation between HRV‑discharge and the symptom
was refered as positive if the virus was found at the same and/or successive week of the diarrhoea.

RESULTS
Rotavirus‑Ag and/or ‑RNA were detected in 19(68%) among the 28 index children from their stool
specimens collected weekly during the two years, andwere found twice in two (7.1%) children in separate
discontinuous weeks. HRV was not found in any fecal
samples from the other nine children (32%) throughout
the two years after birth (Table I ) .

As for the seasonal incidence, six (29%) out of the

HRV‑positive fecal specimens were obtained between
the 1lth and 20th weeks after birth (Jan. to Apr., 1989) ,two (9.5%) were at the 40th and 44th weeks (Aug. andSep., 1989) , 12 (57%) were between the 6lst and 80th
weeks (Dec., 1989 to Apr., 1990) , and one (4.8%) was at

the 90th weeks (June, 1990). From two children who
shed H RV twice, the virus was found at the 19th and64th weeks (Mar., 1989 and Jan., 1990) , and 66th and72nd weeks (Fed. and Mar., 1990) , respectively (Table
1, Fig. 1).

Diarrhoeal symptoms were reported in 24 (86%)

DISCUSSION
According to surveys in Thailand in 1984 and 1985,
children aged 0‑5 years yielded I .5 diarhhoea episoedes
per child per year (Vathanopas et al., 1986, Varavithya
et al., 1988) . These figures are not so different from ourfinding. But Thongkrajai et al. (Thongkrajai et al.,1988) reported that only 13% (44/339) of children infour villages in Khon‑Kaen developed diarrhoea in 16
months. Reason for the discrepancy are not clear.
HRV was detected in 19 (68%) among the 28 index
children from their stool specimens collected weekly
during the two years, and twice in two (7.1%) children
in separate weeks, which indicates that 68% of thenewborn babies of this study group were infected, 7.1%
experienced reinfection but 32% were not infected byrotavirus during the first two years after birth. Sero‑epidemiological data in 1978 showed that 80% of chil‑dren under two years of age had anti‑rotavirus antibody(Maneekarn et al., 1980) .

Seasonal incidence of HRV‑positive fecal speci‑
mens from newborn babies delivered in October andNovember indicated that 29% of the index children
experienced primary infection of rotavirus in the firstdry‑cool season, 9.5% in the first rainy season, 57% in
the second dry‑cool season and 4.8% in the second rainyseason of their life. In tropical areas, rotavirus infec‑

tions are common year round, but increase during
periods of low rainfalls or low humidity and decreaseduring periods of high rainfalls or high humidity, or bothout of the 28 index children and total number of the(Hieber et al., 1978, Black et al., 1980, Suanarto et al.,episodes was 67, i.e., 2.39 for one index child during the1981, Paul et al., 1982). On the contrary, it is wellknown that many infectious diseases including bacterial
gastroenteritis and even influenza prevail in tropicalarea in the rainy season as shown in the Thai Govern‑
ment statistics. The reason why rotavirus prevails in
tropical area in dry‑cool season has been not clearly
explained.
Results of this report suggest that most rotavirus
infections during two years after birth did not cause
severe diarrhoea, as 52% (11/2D of rotavirus infections

two years. Ten (15%) out of the 67 diarrhoeal episodes
were observed at the same or successive weeks of the

fecal HRV‑discharge. Severe intestinal symptome ofdiarrhoea was found only in one case (N0.8) at the weekof the fecal HRV‑discharge, and HRV was detected in

20% (3115) and 12% (6/15) of moderate and mild
diarrhoeal episodes, respectively (Table D . Only 10 out

of 21 HRV‑positive fecal specimens were obtained at
the same or successive weeks of diarrhoeal episodes

Two children (N0.11 and 23) shed HRV twice

which were indicated by the HRV discharge into thefeces were asymptomatic and only one case showedduring the two years after birth. N0.11 experienced ansevere diarrhoea. Asymptomatic infections in full time

(Table I and 2) .

112

newborns have been reported (Chrystie et al., 1978,

Mahidol University and Ministry of Public Health held

Bishop et al., 1979). Araki et al., who examined fecal

from 1981 to 1986 concluded that there had been no

specimens from 69 diarrhoeal and 46 non‑diarrhoeal
babies aged in average 11.9 months in a Baby Home inTokyo during winter seasons from 1988 to 1991, foundthat 74% (45/6D of HRV‑positive babies had diarr
hoeal symptom but the rest 26% (16/61) were of non‑change of diarrhoeal morbidity, although the mortality
and severity of the disease had satisfactorily reduced(Varavithya et al., 1988) . Rotaviral diarrhoeal disease

was estimated to be responsible for 5,000,000 death

diarrhoea (Araki et al., 1993). Results of our st,udy

annually in less developed countries, and rotaviral ges‑troenteritis is associated with high morbidity in devel‑

could not directly be applied to the entire population of

oped countries (Kapikian et al., 1980). Hence, theChiang Mai, because the number of index children was

situation in Thailand is approaching to these of devel
oped countries. This report, results of two‑year follow‑

small and some inevitable selection of volunteers might
be done, i.e., monthers of good health bahavior would beinterested in and attend this follow‑up study. However,considerably high rate of the inapparent infection can
not be neglected and asymptomatic carrier state could
be a dangerous sourse of the infection.

In our survey, severe diarrhoea was observed inonly one child and rotavirus was responsible for theup after birth of 28 newborns in Chiang Mai might
contribute to the understanding and control of rotaviraldiarrhoea not only in Thailand but also in another partof the world. Typing of isolated viruses, serurn antibody
and secretory fecal lgA responses of the index children
in this study will be described in a further report.

disease, and 20 and 12% of the moderate and mild

REFERENCES

diarrhoeal episodes, respectively, could be correlated to

the rotavirus infection. In other words, correlation
between the severity of diarrhoeal symptom and thefecal HRV‑discharge increased in accordance with theseverity of the symptom. According to Thongkrajai'sreport (Thongkrajai et al., 1988), in children aged one
month to two years rotavirus was responsible for 7/55
(13%) of diarrhoea cases in villages and 791201 (39%)

l ) Araki, K., Chen Z., Tsai, C., Satoh, T., Nishimura, S.,
Tajima, T.. Fujita, Y., Abe, T., Irie, Y., Shinozaki, T.
(1993): Rotavirus infection in a baby home, 1988‑1991,
Clin. Virology, 21, 231‑235 (in Japanese)2 ) Bishop, R.F., Cameron, D.J.S., Veenstra, A.A., Barnes, G.
L. (1979): Diarrhea and rotavirus infection associatedwith differing regions for postnatal care of new bornbabies. J. Clin. Microbiol., 9, 525‑529

of cases at hospital.

3 ) Black, R.E., Merson, M.H., Rahman, A.S.M.M., Yunus,

For rotavirus gatroenteritis outbreak, high secon‑
dary attack rates have been reported (Fonteyne et al.,1978) . Chiba et al., (1986) observed that immunity torepeated infections appears to be serotype dependent,

but Imamura et al. (Imamura et al., 1993) followed
seven cases of rotaviral diarrhoea in 1986‑1992 andfound that one of them was infected twice by type 1
virus. Araki et al. (1993) reported a baby who had an
asymptomatic infection with serotype 4 virus followed
by a symptomatic infection by serotype I in the nextyear.

Prevalence and severity of infectious diarrhoeal
diseases differ in time and areas, i.e., in accordance withthe climate, Ievel of public health and medical care andM., Alim, A.R.M.A., Huq, I.. Yolken, R.H., Curlin, G.T.
(1980): A two year study of bacterial, viral, and para‑

sitic agents associated with diarrhea in rural Bang‑ladesh. J. Inf. Dis., 142, 660‑664

4 ) Chiba, S.. Yokoyama, T., Nakata. S., Morita, Y., Uras‑awa, T.. Taniguchi, K., Urasawa, S., Nakao, T. (1986) :Protective effect of naturally acquired homotypic andheterotypic rotavirus antibodies, Lancet, ii, 417‑421
5 ) Chrystie, I.L.. Totterdell, B.M., Banatvala, J.E. (1978):

Asympotomatic endemic rotavirus infections in the
newborn, Lancet, i, 1176‑1178
6 ) Davidson, G.P., Bishop, R.F., Townley, R.R.W., Holmes,
I.H., Ruck, B.J. (1975): Importance of a new virus inacute sporadic enteritis in children. Lancet, i, 242‑245.
7 ) Fonteyne, J., Zissis, G., Lambert, J.P. (1978): Recurrent

some times with virulence of the pathogen. In Thailand,rotavirus gastroenteritis, Lacet, i, 983
8 ) Herring, A.J., Inglis, N.F., Ojeh, C.K., Snodgrass, D.R.,

National Control of Diarrheal Disease Programme

Menzies, J.D. (1982): Rapid diagnosis of rota virus(CDD) was initiated in 1979 including the use of oral
rehydration therapy (ORT) . Diarrhoeal diseases overthe country ranked as the 2nd leading cause of deathprior to the implementation of the CDD programme, but
in 1985 it was the 5th leading cause of death in infant
under one year (Varavithya et al., 1986) . On the other

hand, the National Diarrhoea Annual Workshops byinfection by direct detection of viral nucleic acid insilver‑stained polyacryamide gel, J. Clin. Microbiol, 16,

73‑4779 ) Hieber, J.P., Shelton, S., Nelson, J.D., Leon, J., Mohs, E.

( 1978) : Comparison of human rotavirus disease in tropi

cal and temperate settings. Am. J. Dis. Child, 132, 853‑
858.

113

10) Imamura, N.. Hanada, N., Shingu, M., Nagai, T. (1993) :Reinfection of rotavirus in children with diarrhoea. J.
Japan, Ass. Inf. Dis., 67, 261‑262 (in Japanese)
1D Inouye, S., Hasegawa, A., Matsuno, S., Katow, S. (1989):(ed.), Virus Diseases in Asia, Mahidol University,
Bangkok, 257‑261.
20) Puthavathana, P., Vanpraper, N., Thakernpol, K., Wasi
C., Thongcharoen, P. (198D: Rotavirus infection in

Changes in antibody avidity after virus infections: Detec‑

Thai people: a preliminary study, J. Med. Assoc. Thai.,tion by an immunosorbent assay in which a mild protein‑denaturing agent is employed, J. Clin. Microbiol., 20,
525‑529
12) Jayavasu, C., Hooniwat, Y., Sagaunwong, S., Jayavasu,

2D Suanarto, Y., Sebodo, T., Ridho, R., Alrasjid, H., Rohde,
J.E., Bugg, H., Barnes, G.L., Bishop, R.F. (1981): Acutediarrhea and rotavirus infection in newborn babies andJ., Chatiyanonda, K. (1982): A Iong term study ofchildren in Yogyakarta, Indonesia, from June, 1978 to

rotavirus infection in Thai infants and children withdiarrheas Southeast Asian J. Trop. Med. Pub. Hlth., 13,29‑32June, 1979, J. Clin. Microbiol., 14, 123‑129
22) Supawadee, J., Inouye. S., Suzuki, H., Yamazi, Y. (1988) :13) Kapikian, A.Z., Wyatt, R.G., Greenberg, H.B., Kalica, A.
R., Kim, H.W., Brandt, C.D.. Rodriguez, W.J., Parrot, R.

H., Chanock. R.M. (1980): Approaches to immunizationof infants and young children against gestroenteritis due
to rotavirus, Rev. Infect. Dis., 2, 459‑469

14) Kapikian, A.Z.. Chanock, R.M. (1990): Rotaviruses. In
Fildes B.M., Knipe D.M. (ed.), Virology, Raven Press,

Ltd., New York, 1353‑1405
15) Kariyasono, H., Shimohara, E., Sugishima. S., Sakuma,

T. (1993): Virological study of pathogens of acutegastroenteritis in children, J. Japan. Ass. Inf. Dis., 67,

261 (in Japanese)
16) Louisirirotchanakul, S., Wasi, C., Satrasook, S., Surak‑

haka, M., Thongcharoen, P. (1984): Rotavirus inpediatric diarrhea at Siriraj Hospital, Bangkok,
Thailand, Southeast Asian J. Tr. Med. Pub. Hlth., 15,
348‑35317) Maneekarn, N., Yamada, Y., Shinozaki, T., Arai, K.,

Yamazi, Y., Watari, E., Suzuki, H. (1980): Seroe‑64, 341‑345.

Rotavirus infection of infants in Thailand: II. Follow‑up
study of fecal antigen and antibody detection, In Thong‑charoen, P., Kurstak, E. (ed.), Virus Diseases in Asia,

Mahidol University, Bangkok, 275‑279
23) Thongkrajai, P., Hahnvajanawong, C., Kowswan, P.,
Sukprasert, S., Thongkrajai, E., Kusalertchariya. S.
(1988) : Rotavitus diarrhea in children and animals ofrural Thailand. In Thongcharoen, P. Kurstak, E (ed.),Virus Diseases in Asia, Mahidol University, Bangkok,
266‑269
24) Varavithya, W., Ramaboot, S. (1986): Diarrhoeal dis‑eases in Thailand, J. Med. Ass. Thai., 69, 46‑49

25) Varavithya, W., Phuapradit, P., Sabcharoen, A.,
Ramaboot, S. (1988) : Profile of diarrheal diseases inchildren in Thailand. In Thongcharoen. P., Kurstak, E.

(ed.), Virus Diseases in Asia, Mahidol University,
Bangkok, 247‑256
26) Vathanopas. K., Indrasukhsri, T., Bunyaratabandhu, P.,

Suthienkul, O., Varavithya, W. (1986): The study ofsocioeconomic, bahavioural and environmental factors
related to diarrhoeal diseases in children under 5 incongested areas of Bangkok Metropolis, J. Med. Ass.

pidemiological survey of rotavirus in Thailand 1978, J.
Nippon. Med. Sch., 47, 570‑571
18) Paul, M.O., Erinle, E.A. (1982): Influence of humidity on
rotavirus prevalence among Nigerian infants and young27) Yamazi, Y., Suzuki, H., Hasegawa. A., Pongprot, B.,

children with gastroenteritis. J. Clin. Microbiol., 15, 212‑

Supawadee, J., Bukkavesa, S., Thongkrajai, P., Inouye,215

S. (1988): Rotavirus infection of infants in Thailand: I.

19) Phijaisanit, P., Santad, S., Vongiraj, A., Wasi, C. (1988):

The etiology and clinical features of acute diarrhoeal
diseases in infants, In Thongcharoen, P., Kurstak, E.

Thai., 69, 156‑162

RNA electropherotype and isolation of virus. In Thong‑charoen, P., Kurstak, E. (ed.), Virus Diseases in Asia,

Mahidol University, Bangkok, 270‑274

115

Jpn. J. Tro p. Med. Hyg ., Vol. 23, No. 2 (1995) pp. 115‑120

SEROEPIDEMIOLOGICAL STUDIES OFHEPATITIS VIRUSESIN THE DOMINICAN REPUBLIC
I. THE PREVALENCE OF MARKERS OF HEPATITIS
A, B AND C VIRUSESAKEHISA SHICHIJO1, KUMATO MIFUNE1, HIROSHI AON02,
HITOSHI SHIBAYAMA3, HIDEO TERA03, AKIRA MIYATA4,HIDEKI OZAWA2, MORIO IT05, JUN‑ICHI MISUM12,
TAKASHI ITOGA3, MARIA ESTELA NoRMAN6, ARACELIS GERMAN RODRIGUEZ6,MERCEDES CASTRO BELL06, ABELARDO A HIDALGO SIGARAN6
Received January 26, 1995/Accepted March 14, 1995

Abstract: A total of 408 subjects (Group D visiting the center of gastroenterological diseases, Aybar
Hospital, Santo Domingo, Dominican Republic and 2,000 apparently healthy individuals (Group ID Iiving in
6 different cities in the Dominican Republic were tested for antibodies against Hepatitis A virus (anti‑
HAV) , Hepatitis B surface antigen (anti‑HBsAg) and Hepatitis C virus (anti‑HCV) . The presence of
HBsAg in their sera was also tested. The study demonstrated that most of the Dominican population are
infected with HAV by the age of 15 years and that H BV infection is still prevalent in the country. The

overall prevalences of HBsAg and of anti‑HBs were 3.2% (Group ID to 4.7% (Group D and 18% (Group
ID , respectively, although HBsAg prevalence rate in young children was very low. In contrast to thepredominant HAV and HBV infections, most of the anti‑HCV‑positive individuals were concentrated in theage groups of higher than 40 (Group ID and 50 (Group D years old and only a few sero‑positive individuals
were observed in the younger population, suggesting that there have been few transmissions of HCV in the
Dominican population in the past 40 to 50 years.

INTRODUCTION

the utmost public health importance for Latin America(Fay et al., 1990). Infection with HBV is known to

Hepatitis A is highly endemic in many tropical andsubtropical areas and occasionally causes large epi‑
demics (Szmuness et al., 1977; Gust & Feinstone, 1989;

cause significant morbidity and mortality resulting from

Hadler, 1991; Yap & Guan, 1993) . However, in these
economically developing countries hepatitis A virus

developing countries, including those in Asia, Africa and(HAV) infection occurs primarily in young children andalmost all have antibodies for HA¥r by the age of 10

hepatitis, cirrhosis of the liver, or hepatocellular car‑cinoma. HBV infection is highly endemic in many
the Western Pacific (Hawkes et al., 1981; Hyams et al.,
1989; Boutin et al., 1990; Bile et al., 1991; Tan et al.,

199D. In such a setting, most persons are infected in(Tsega et al., 1986) . Hepatitis A in adults tends to beearly childhood though both perinatal transmission

more severe than in children. The most effective way of(mother to infant at birth) and horizontal transmission(close contact with other infected persons) . However,reducing HAV infection is to improve standards ofhygiene and sanitation, especially by providing adequate

supplies of clean water and proper disposal of fecal
material.

Hepatitis B virus (HBV) infection is a disease of

the spread of H BV inection is now preventable by theuse of passive and active immunization with a safe andcost‑effective vaccine. To plan for large‑scale HBVvaccination strategies, it is essential to know the picture

Department of IMicrobiology, 2Public Health and Hygiene, 3lnternal Medicine, 'Biology and *Laboratory Medicine, Oita Medical
University, Hazama‑machi, Oita, 879‑55, Japan and 6the Center of Gastroenterological Diseases, Dr Luis E. Aybar Hospital, Santo

Domingo, Dominican Republic

116

of HBV endemicity and transmission in the region.
It has become feasible to detect the antibody tofrom the serum stocks of the outpatients visiting thecenter of gastroenterological diseases, Santo Domingo,

HCV (anti‑HCV), and there have been many

Dominican Republic, during the period of August to
October, 1993. The sera consisted of almost equal
numbers from 13 different age groups and both sexes.
Age distribution and sex are shown in Table 1. These

epidemiologic studies on the prevalence of HCV infec‑
tion in blood donors and patients with various liverdiseases in the USA, Europe and Japan (Kuhnl et al.,
1989; Tanaka et al., 1992). However, there is no infor‑

sera were tested for the presence of antibodies to hepati‑

mation available in the Dominican Republic. In thetis A virus (anti‑HAV) , HBV surface antigen (HBSAg)

present paper, we report the results of seroe‑

and anti‑HCV.

pidemiological study on HAV, HBV and HCV infection

Group 11 was composed of 2,000 apparently healthy

for the purpose of obtaining information on the current
endemicity of these viruses in the Dominican Republic.
The Dominican Republic is bounded on the north by theindividuals of both sexes and different age groups livingin 6 cities in the Dominican Republic (Fig. 1). These
consisted of 202 subjects from Bani, 192 from La Vega,
211 from Monte Plata, 201 from Samana, 198 from San
Pedro de Macoris and 991 from Santo Domingo, but did

Atlantic Ocean and on the south by the Caribbean sea
and lies within the tradewind belt. The iuhabitants are
partly of Spanish and French descent, but the majority

not include individuals less than 19 years old. Sera wereare of mixed African Negro and European blood.

taken during the period between November 20 and

Voodoo is practiced, but to a lesser extent than inneighboring Haiti. They appear to have no special

December 17, 1993 and stored at ‑ 75'C until the tests

customs affecting the transmission of hepatitis viruses.

were performed.

for the presence of HBSAg, anti‑HBs and anti‑HCV
Methods of serological tests(a) Anti‑HAVMATERIALS AND METHODS

Total (IgG and lgM) anti‑HAV was tested for by
enzyme‑1inked Immunosorbent assay (ELISA) method

Subjects and serum samples
Two groups were evaluated. Group I was composedof 408 serum samples which were picked up at random

using a commercially available test kit (HAVAB, Ab‑bott Lab., North Chicago, Illinois, USA) .
( b ) Hepatitis B serological tests(1) In the case of Group I sera, the presence of
HBsAg was tested for by ELISA method using a com‑

A1,18ntie Oeeen

mercially available test kit (HBSAg, Abbott Lab., North
Chicago, 11linois, USA) .

La vegae

Naiti

Salana(2) In the case of Group 11 sera, HBsAg was tested

for by reversed passive hemagglutination (RPHA)

Uo!Ite Platae(Aucell, Abbott Lab., North Chicago, Illinois, USA) , andby passive hemagglutination (PHA) for anti‑HBsAg

S8nto Do i ngo san PedrQ de 1,8coris

e

Benl

(Sero‑clit‑anti‑HBs, Sankojunyaku, Japan) .

e(c) Anti‑HCVc

C8ribteQn Se8Group I sera were tested for anti‑HCV by an anti‑

HCV second generation enzyme immunoassay (recom‑
binant CIO0‑3, HC‑31, HC‑34, Abbott, Lab., North

Figure I Map of the Dominican Republic and the locations
where the sera were collected.

Table 1

Chicago, 11linois, USA) . In the case of Group 11 sera,
this was performed using the second generation particle‑

Sex and age distribution of the subjects (Group D

tested for anti‑HAV, HBsAg and anti‑HCV
Se x
Male Female Total

Age groups
0‑5

‑10

‑15

‑20

19 13

17 18

16 15

16 14

17 15

16 15

17

32

35

31

30

32

31

32

25

‑30

35

‑40

45

‑50

55

‑60

61‑

15 16

15 16

15 17

15 16

15 15

15 16

31

31

32

31

30

31

15

T ota 1207 201 408

117(

agglutination test (PA test) (FUJI REBIO, INC.,
Japan) . In this case, the titers more than l:32 were

A)

1 OO

taken as sero‑positive.
80

Statistical analysis

A

Statistical analysis was performed using the X2 test80

by Mantel‑Haenszel method. P values less than 0.05

v

were considered significant.

I 40

:'L

zo

RES U LT SoPrevalence of anti‑HA V
Positive rates of anti‑HAV in Group I are shown in

o‑ Il

18

21 2e 31 38

51*

,t

Fig. 2A. Anti‑HAV was detected in 64.7% of those in
the 6 to 10 year old age group. The prevalence then

v

aO

'

increased progressively with increasing ages from 96.8%
in the age group of 11 to 15 years to 100% in the agegroup of 16 to 20 years and essentially 100% positive
rates continued thereafter. It is clear that HAV is veryloe‑widespread in the Dominican Republic. No significant
difference was observed in the prevalence of anti‑HAV
between sexes.

16*

30

A

Prevalence of HBsAg

XFig. 2B shows the prevalence of HBsAg in 13 differ‑v 20

ent age groups in Group I. The overall prevalence of
HBsAg was 4.7%. The rate (6.3%) for HBsAg in maleslO

was significatly higher than that in females (1.5%) (P<0.05) . However, no HBsAg‑positive individuals weredetected in children under 5 years of age or in males
between 6 and 10 years of age. H BsAg‑positive rate
was maximum (13.3% in total, 18.8% in males, 2.4% in

Age g "p.

Figure 2

HBsAg (B) and anti‑HCV (O of Group I sub‑jects. White colurnn and dotted column indicate
males and females, respectively.

females) in the age group from 6 to 20 year old. Table
Table 2Sex

Prevalence by age and sex of anti‑HAV (A) ,Prevalence of HBsAg and anti‑HBs by age and sex in the Dominican healthy
individuals (Group ID

20‑

30‑

40‑

50‑

60‑

70‑

Total

253

206

157

l07

54

33

810

No. of HBSAg‑

lO

lO

9

6

1

3

39

positive(4 . o)(4 . 9)(5 . 7)(5 . 6)(1 . 9)(9 . 1)(4 . 8) a

Age groups
No. oftestedMale

No. of44

38

14(17 . 4)(18.5)

31(19.8)

27anti‑HBs(25 . 2)(25 . 9)(24 . 2)

430

350

208

109

69

24

1190

8

162
(20 . o)positive

No. oftestedFemaleNo. of HBSAg‑

6

9

7

o

1

1

24

positive(4 . o)(2 . 6)(3 . 4)(o)(1 . 5)(4 . 2)(2 . O) b

No. of

50

59

40

23

14

anti‑HBs(11 . 6)

(16 . 9)(19.2)(21 . D

(20 . 3)

positive
Figures in parenthesis indicate per cent.

a : b, P<0.05 by the X2 test with Mantel‑Haenszel method.

6(25 . O)

192
(16 . D

118

Prevalence of anti‑HCV

2 shows the prevalence of HBsAg in Group 11 by age andsex. The overall prevalence of HBsAg was'3.2% (63/2,
OOO). The rate observed for HBsAg was again higher in

As shown in Fig. 2C., the overall prevalence of anti‑

HCV in Group I was 4.7%. A11 young children under 15
years old were sero‑negative. However, there was anmales (4.8%) than in females (2.0%) and this sex
difference was statistically significant (P<0.05) . The

abrupt change in the prevalence in individuals more than
50 years old, the group in which the sero‑positive indi‑prevalence of HBsAg continued to increase graduallyviduals were concentrated. A part (1,401 serum sam‑ples) of Group 11 was alsp tested for anti‑HCV by PA
test (Table 3) . The overall prevalence was 2.6% inwith age and reached 5.7% in males and 3.4% in females
in the 40 year old age group. The HBsAg‑positive rates

once decreased in older age groups (60‑69 years inmales, 50‑69 years in females) but increased again in
individuals more than 70 years old, although the reason
is not clear. This tendency was also observed in Groupmales and 2.2% in females. Although Group 11 wasyounger than Group I, there was again an abrupt
increase in the prevalence in individuals more than 40
years old. The average titer of anti‑HCV was signifi‑

I.

The prevalence of H BsAg in 6 different districts

cantly higher in these individuals than that in individuals

under 39 years of age (Data not shown) .

was compared. The rate ranged from 1.5% in Samana
to 5.6% in Monte Plata. Although the prevalence of
HBsAg in Monte Plata (5.6%) was statistically higher
than that in La Vega (2.0%) or in Samana (1.5%) (P<DISCUSSION

0.05) (Data not shown) , the factors responsible for thisIn the present study, it was demonstrated that mostdifference in the districts were not clarified in theof the Dominican population is infected with HAV bypresent study.

age of 15. This pattern of infection with HAV is consis‑

Prevalence of anti‑HBs
The prevalence of anti‑HBs in different age groups
and sex is shown in Table 2. The prevalence continued

countries where the infection occurs primarily in young
children (Gebreselasse, 1983; Craig et al., 1993). In thetent with the findings observed in other developing

developed world, with improved standards of hygiene
and sanitation, the rate of infection declines and a

to increase gradually with age, reaching 25.9% in males
in the 60‑69 years old group and 25.0% in females in the
70‑79 years old group. However, no significant differ‑significant proportion of the population escapes infec‑
tion in early childhood and the age of primary infection
gradually increases (Hadler, 1991; Yap and Guan, 1993) .
In other words , the rate of infection in early childnood
reflects the sanitary environment in the country. Thus,

ence was observed in the overall prevalence between
males (20.0%) and females (16.1%). When the preva‑
lences of anti‑HBs by age and districts were compared,
the highest incidence for anti‑HBs was found in Monte
Plata population (25.9%) , which was statistically higher

the rate of HAV infection in the Dominican population
would decrease through further efforts to improve thesystem of clean water supply and proper disposal ofthan those in other areas except for that of San Pedro deMacoris (21.2%) . The prevalence rate of anti‑HBs waslowest in Bani (13.4%), followed by La Vega (16.2%)
and Samana (16.9%) . This difference of anti‑HBs rates
by districts appeared to correspond to the H BsAg‑posi‑

fecal material.

The prevalence of 3.2 (Group ID to 4.7 (Group D %of HBsAg carriers in the population and as high as 25%
anti‑HBs seropositive rates, which increase with age,tive rates in those districts.

Table 3Age groups

Prevalence of anti‑HCV by age and sex in the Dominican healthy
individuals (Group ID

20‑

30‑

40‑

50‑

60‑

70‑

Total

166

129

96

78

534

o

7

2

41
3

24

1

1

14

(o . 6)(o)(7 . 6)(2 . 5)(7 . 3)(4 . 2)(2 . 6)Males
No. of tested

No. ofpositive

FemalesNo. of tested

No. ofpositive

316

254

149

79

53

16

867

4

3

5

4

1

2

19

(1 . 3)(1 . 2)(3 . 4)(9 . 8)(1 . 9)Figures in parenthesis indicate per cent.

(12 . 5)

(2 . 2)ll9

indicated that HBV infection still prevails in theseveral other countries (Darwish et al., 1992; Thuring et

Dominican population. However, the HBsAg preva‑
lence rate in young children was very low and began toal., 1993) . Recent studies on the transmission of HCV
have suggested that the maj or source of transmission isincrease only in late childhood. According to the classi‑by blood transfusion and not by perinatal infection

fication by WHO (Zuckerman, 1987) , the above HBsAg(Thaler et al., 1991; Price et al., 1993; Ohto et al., 1994) .

positive rates in the Dominican Republic are classified

The fact that the infectivity of HCV in blood is 10' times

as intermediate prevalence region type (2 to 7%)･ The
rates are higher than those in central Europe and North
America (0.2 to 0.5%) (Zuckerman, 1987) and Japan (O.
8%) (Tanaka et al., 1993) , but apparently lower thanthose in Asian and African countries (Kiire, 1993; WHO,1992). In Latin America, the rates are different fromcountry to country, but appear to be higher in rural
areas with different natives than in urban areas (Van‑derborght et al., 1993; Craig et al., 1993; Kenneth et al.,
1993; Blitz et al., 1994; Pujol et al., 1994).

Although further studies on H BV transmission arenecessary, transmission presumably occurs through per‑cutaneus or permuscosal exposure to infected blood orlower than that of HBV (Brady et al., 1983) would
facilitate HCV‑specific transmission pattern. Thus, it
could be presumed that HCV infection in the Dominican
population might be eliminated in the near future byestablishment of a complete screening system for HCV
infection before blood transfusions.

ACKNOWLEDGMEN TS
This work was performed as one of the research
project in the "investigation and clinical treatment ofgastroenterological diseases" in the Dominican Republic

other secretions in children as suggested by Fay et al.,(1990) or still through infected needles, in addition toby the Japan International Cooperation Agency. This
work was supported in part by a grant awarded to oneof the authors, Takashi Itoga, from the Soroptimist

perinatal infection from HBeAg positive mothers toInternational of Japan. Collabolation of Mr. R. FUJlNO

newborns. In Japan, passive immunization and vaccina‑tion have been successfully carried out in newbornsof the FUJI REBIO, INC,. Japan in anti‑HCV tests isgreatly acknowledged.

from HBeAg‑positive mothers to block HBV infection(Yano, 1986) . Thus, HBV vaccination strategies in the
Dominican Republic should be directed with a priority
toward the prevention of perinatal and late childnood
transmission and also aimed at individuals belonging tohigh‑risk groups.
H BsAg prevalence rates were statistically higher
among males than females in both groups of individuals

in the Dominican Republic. This higher prevalence
among males has been observed in other countries(Gebreselassie, 1983; Fonseca et al., 1988). Although
there have been some explanations, such as sex‑specificbehaviour increasing the chance of exposure to thevirus, or some immunological deficiency in males or
10wer titer of HBsAg in female carriers, which in turnresults in a higher detection rate in males (Szmuness,1978) , the underlying mechanism of the sex difference
remains obscure.
HCV infection in the Dominican population showed

REFERENCES
1 ) Bile, K.M., Abdirahman, M., Aden, C., Norder. H.,
Magnius, L., Lindberg, G. and Nilsson, L.H. (199D:
Minor role of hepatitis B virus in the causation ofchronic liver disease in Somalia indicated by a case‑control study, Trans. R. Soc. Trop. Med. Hyg., 85, 104‑1082 ) Blitz, D.L., Monsalve, F., Porto, L., Weir, J., Arteaga,

M., Padron, G., Leon, P. and Echevarria J.M, (1994):
Epidemiology of hepatitis C virus in Western Venezuela:
Lack of specific antibody in Indian communities, J. Med.
Virol. 43, 287‑290
3 ) Boutin, J‑P., Marie, F.F.S., Cartel, J‑L., Cardines, R.,
Girard, M. and Roux, J. (1990): Prevalence of hepatitis

B virus infection in the Austral archipelago, French
Polynesia: identification of transmission patterns forthe formulation of immunization strategies, Trans. R.
Soc. Trop. Med. Hyg., 84, 283‑287
4 ) Bradlly, D.M., Maynard, J.E.. Popper, E.H., Cook. E.H.,

a specific picture. Only a very small proportion ofyounger people are infected with HCV, whereas ser‑

Ebert. J.W.. McCaustland, K.A., Schable, C.A. andFields. H.A. (1983) : Posttransfusion non‑A, non‑B

opositive prevalence in people higher than 40 to 50 years

hepatitis: physicochemical properties of two distinct

has been few transmission of HCV in the Dominican
population in the past 40 to 50 years. This abrupt

agents, J. Infect. Dis., 148, 254‑265
5 ) Craig, P.G., Bryan, J.P.. Miller, R.E., Reyes, L., Hakre,
S., Jaramillo, R. and Krieg, R.E. (1993) : The prevalenceof hepatitis A, B and C infection among different ethnic

increase of prevalence in adults of HCV infection has

groups in Belize, Am. J. Trop. Med. Hyg., 49, 430‑434

old exhibits an abrupt increase, suggesting that there

been reported from Japan (Tanaka et al., 1993) and120

6 ) Darwish, N.M., Abbas, M.O., Abdelfattah, F.M. andDarwish, M.A. (1992): Hepatitis C virus infection in

Favorov, M.O., Fields, H.A. and Liprandi, F. (1994):
Viral hepatitis serological markers among pregnant

blood donors in Egypt, J. Egypt Public Health Assoc. 67,

women in Caracas, Venezuela: implication for perinataltransmission of hepatitis B and C., G. E. N., 48, 25‑28

223‑2367 ) Fay, O.H. and the Latin American Regional Study

20) Szmuness, W., Diestag, J.L., Purcell, R.H., Stevens, C.E.,

Group. (1990): Hepatitis B in Latin America:

Wong, D.C., Ikram, H., Bar‑Shany, S., Beasley, R.P.,
Desmyier, J. and Gaon, J.A. (1977): The prevalence ofantibody to hepatitis A antigen in various part of theepidemiological patterns and eradication strategy, Vac‑cine, vol. 8. Butlerworth & Co. Ltd., sIOO‑sl07
8 ) Fonseca, J.C.F., Simonetti, S.R.R., Schatzmayr, H.G.,

Castejon. M.J.. Cesario. A.L.O. and Simonetti, J.P.
( 1988) : Prevalence of infection with hepatitis B surface

antigen in Amazonas State, Brazil, Trans. R. Soc. Trop.
Med. Hyg., 84, 283‑287
9 ) Gebreselassie, L. (1983): Prevalence of specific markersof viral hepatitis A and B among an Ethiopian popula‑tion, Bull. W.H.O., 61, 991‑996
10) Gust, I.D. and Feinstone, S.M. (1989): Hepatitis A, In:
Progress in liver diseases, vol. 9, Popper, H. & Schaff
ner, F. (editors) . New York: Grune & Stratton, pp 371‑
378
1D Hadler, S.C. (199D: Global impact of hepatitis A virus
infection: changing patterns, In: Hollinger F.B., Lemon
S.M., Margolis H. eds.. Viral hepatitis and liver disease,

Baltimore: Williams & Wilkins, pp 14‑20
12) Hawkes, R.A., Boughton, C.R., Ferguson. V. and Vale,world: a pilot study, Am. J. Epidemiol., 106, 392‑398

2D Szmuness, W. (1978): Hepatocellular carcinoma and thehepatitis B virus: evidence for a causal association,
Prog. Med. Virol., 24, 40‑69
22) Tan, C.C., Guan, R., Yap, I., Tay, H.H. and Kang, J.K.
(199D : Horizontal or vertical transmission of hepatitis

B virus ? A serological survey in family members ofhepatitis B carriers in Singapore, Trans. R. Soc.. Trop.
Med. Hyg., 85, 656‑659

23) Tanaka, E., Kiyosawa, K., Sodeyama, T., Hayata, T.,
Ohike, Y., Nakano, Y., Yoshizawa, K., Furuta, S.,
Watanabe, Y., Watanabe, J. and Nishioka, K. (1992):
Prevalence of antibody to hepatitis C virus in Japanese

schoolchildren: comparison with adult blood donors,
Am. J. Trop. Med. Hyg., 46, 460‑464
24) Tanaka, J., Moriya, H., Sasaki, T., Mito, H., Koyama,T., Noto, H., Yoshizawa, K. (1993): Epidemiology ofT.G. (198D: The seroepidemiology of hepatitis in

hepatitis B infection, Tan‑Kan‑Sui, 27, 495‑500,

Papua New Guinea. II. A Iong‑term study of hepatitis B,
Am. J. Epidemiol., 114, 563‑573( Japanese)

13) Hyams, K.C., Al‑Arabi, M.A., Al‑Tagani. A.A., Mes‑
siter, J.F., Al‑Gaali, A.A. and George, J.F. (1989):
Epidemiology of hepatitis B in the Gezira region of
Sudan, Am. J. Trop. Med. Hyg., 40, 200‑206
14) K6nneth, J.H., Joel, C.G., Richard E.K., Fred, J.D., Philip,

O.M., John, R.T., Ruth, J., Linda, G.R., Maria, H.S. andLlewellyn, J.L. (1993): Initial report of a hepatitis inves‑tigation in rural Belize, Trans. R. Soc. Trop. Med. Hyg.,87, 259‑262

15) Kiire, C.F. (1993): The epidemiology and control ofhepatitis B in Sub‑Saharan Africa, Prog. Med. Virol., 40,

141‑15616) Kuhnl, P., Seidl, S.. Stangel. W., Beyer, J.. Sibrowski. W.

and Flik. J. (1989): Antibodies to hepatitis C virus in
German blood donors, Lancet, 2, 32417) Ohto, H., Terazawa, S., Sasaki, N.. Sasaki, N., Hino, K.,

Ishiwata, C., Kako, M., Ujiie, N.. Endo. C.. Matsui, A.,
Okamoto, H., Mishiro, S. and the vertical transmissionof hepatitis C virus collaborative study group. (1994):

Transmission of hepatitis C virus from mothers to
infants, N. Engl. J. Med., 330, 744‑750

25) Thaler, M.M., Park, C.K., Lander, D.V., Wara, D.W.,
Houghton, M., Wauters, G.V., Sweet, R.L., Han, J.H.
(199D: Vertical transmission of hepatitis C virus,
Lancet, 388, 17‑1826) Thuring, E.G., Joller, J.H.1., Sareth, H., Sokhan, U.,
Reth, C. and Grob, P. (1993): Prevalence of markers ofhepatitis viruses A, B, C and of HIV in healthy individ‑
uals and patients of a Cambodian province, Southeast
Asian J. Trop. Med. Public Health, 24, 239‑249
27) Tsega, E., Mengesha, B., Hansson, B‑G., Lindberg, J.

and Nordenfelt, E. (1986): Hepatitis A, B and Delta
infection in Ethiopia: a serologic survey with demo‑
graphic data, Am. J. Epidermiol., 123, 334‑351
28) Vanderborght, B.O., Reis, A.M., Rouzere, C.D., Silva, R.
S., Yoshida, C.F., Franco, L.G., Maertens, G., Heuverswi
jn, V.H. and Moreira, P.J. (1993): Prevalence of anti‑
hepatitis C virus in the blood donor population of Rio deJaneiro, Vox Sang, 65, 122‑12529) WHO (1992): Control of hepatitis B: the work of WHOm the western pacifrc regron "89‑90", WPR/RC 42/2

WPRO, 169‑17030) Yano, M. (1986): Prevention to HBV maternal‑infant18) Price, A.M.. Brotman, B., Inchauspe, G., Pascual, D.,transmission, Tan‑Kan‑Sui, 13, 591‑197 (Japanese)

Nasoff. M., Hosein, B. and Wang. C.Yi. (1993) : Patterns
and prevalence of hepatitis C virus infection in posttransfusion non‑A, non‑B hepatitis, J. Infect. Dis., 167, 1296‑
1301
19) Pujol, F.H., Rodriguez, I., Martinez, N., Borberg, C.,

3D Yap, I. and Guan, R. ( 1993 ) : Hepatitis A sero‑
epidemiology in Singapore: a changing pattern, Trans.
R. Soc. Trop. Med. Hyg., 87, 22‑2332) Zuckerman, A.J. (1987) : The development of novel
hepatitis B vaccines, Bull. W.H.O., 65, 265‑275

121

Jpn. J. Tro p. Med. Hyg., Vol. 23, No. 2 (1995) pp. 121‑125

VIROLOGICAL STUDY ON DENGUE EPIDEMIC IN
VIENTIANE MUNICIPALITY, LAO PDR, 1994
THONGCHANH SISOUK1, KAZUMI KANEMURA2, MIKA SAIT02, BOUNLAY PHOMMASACK4,YOSHIHIRO MAKlN02,3, SAKAE ARAKAK12, SHAO‑PlNG MA2,SITHAT INSISIENGMAYl, AND TOSHIHIKO FUKUNAGA2,3
Received March 22, 19951Accepted May 10, 1995

Abstract: In May 1994, an epidemic of dengue fever (DF) / dengue hemorrhagic fever (DHF) was reported
in Ban Hom commune in Vientiane municipality. This was after a six‑year period of non‑epidemic DF/
DHF in the municipality. In early June, we collected 81 serum specimel s from children with fever of
unknown origin (FUO) in the commune. Of 81 specimens, 48 were taken from children within 5 days after
the onset of FUO and subjected to dengue (DEN) virus isolation. Four strains of DEN‑1 and 12 strains of
DEN‑2 viruses were isolated from 15 serum specimens (31.3%) ･ A case of dual infection with DEN‑1 andDEN‑2 viruses was detected by enzyme antibody method using type‑specific monoclonal antibodies andreverse transcription polymerase chain reaction (RT‑PCR) . The percentage incidences of the positive virus
isolation from the serum specimens taken on the second and the third day of fever were 41.8% and 21.1%,
respectively, and no virus was isolated from the specimens other than those from these two days. A11 thespecimens were examined by the lgM‑capture ELISA. Thirty‑two specimens (39.5%) were positive for
DEN IgM antibody.

INTRODUCTION
Lao PDR, a land‑10cked country, Iocated in theand virological and serological examinations were car‑ried out on the specimens. This is the first report ofdengue virus isolation in Lao PDR.

Indochina Peninsula, has a total population of about 4.2
milion with an area of 236.8 thousand square kilometers.
In 1985 and 1987, Iarge outbreaks of dengue fever (DF) /dengue hemorrhagic fever (DHF) occurred in the urban

MATERIALS AND METHODS
Study a rea

areas of the Vientiane municipality. In these outbreaks,

The study area, Ban Hom commune, is located1076 and 5232 DF/DHF hospitalized cases were reported
to the Vientiane Municipality Health Service, rcspec‑

south‑west of Hatxayiong district, about 15 km south‑
east of the center of Vientiane municipality and has atively (Bounlu et al., 1992; Fukunaga et al., 1993) . From

total population of 968 residents (Fig. D . It is primarily

1988 to 1993, DF/DHF epidemic did not occur, though

an agricultural commune. In May, the DF/DHF out‑

several sporadic cases were reported in these areas. In

break was first reported in this commune. Severe DF/

1994, an epidemic took place again, expanding to the

DHF cases were sent to the hospitals in Vientianerural areas of the Vientiane municipality, and a total ofmunicipality. The data of hospitalized DF/DHF

1590 hospitalized cases were reported by the middle ofpatients were collected at the Vientiane Municipality
Health Service.

October.
We conducted a survey of the epidemic in a ruralcommune, called Ban Hom, where the DF/DHF epi‑

Serum specimens

demic was the first report in the Vientiane municipality

in 1994. Serum specimens were collected from children

On June Ist, 1994, 81 serum specimens were col‑lected from children 1‑16 years old, with FUO at thewith fever of unknown origin (FUO) in the commune,

Ban Hom Health Center. The serum specimens were

National Institute of Hygiene and Epidemiology, Vientiane, Lao PDR
of Virology
3. Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
4. Vientiane Municipality Health Service, Vientiane, Lao PDR
2. Department

122

stored at ‑70'C in the National Institute of Hygiene and

The infected cells were incubated at 28'C for 7 days.

Epidemiology (NIHE) until transported to the Dept. of
Virology, Faculty of Medicine, University of the Ryu‑kyus.

cells and cultured at 37'C for 3 days. The infected BHK‑

Virus isolation

method using hyperimmune rabbit anti‑DEN serum

Serum specimens taken within 5 days after the
onset of FUO were subjected to virus isolation. The(Ishimine et al., 1987; Okuno et al., 1978) . The DEN
antigen positive cells were further examined for the
serotype by PAP staining with type‑specific monoclonal
antibodies. The serotype was also confirmed by reverse‑transcription polymerase chain reaction (RT‑PCR) .

serum was diluted I : 30 with Eagle's MEM containing
2% fetal calf serum and antibiotics and inoculated onto
C6/36 cells cultured in 25‑cuf flasks (Igarashi, 1978) .

The culture fluid was then inoculated on to BHK‑21
21 cells were screened for the presence of DEN virus
antigens by peroxidase‑anti‑peroxidase (PAP) staining

RP‑ PCR,,

The primers for the detection of DEN virus genome
are described elsewhere (Morita et al., 1991; Morita etal., 1994). Amplification of the fragments of the virus

CHINA

g,
It

,

1

genome was carried out by programmed incubation : 10
min at 53'C for RT once, followed by 35 cycles of PCR,

VIETNAM

consisting of 92'C for I min, 53'C for 1.5 min and 72'C for

2 min. The amplified DAN was run in 2% agarose gel
J,Ce to,,Gutf oflr,

LTonk In

AO S
tlane

Vi

e EO

,e

,p
lddt

NA

KhammouaneSand visualized by ethidium bromide staining.

DEN antibody assays
For the neutralization test, a fifty percent focus
reduction neutralization test in BHK‑21 cells, using
PAP staining method, was employed (Ishimine et al.,
1987; Okuno et al., 1978; Vongxay et al., 1995) . A
neutralizing antibody titer less than I : 20 was defined as

THAILANDnegative.

lgM‑capture enzyme‑linked immunosorbent assay(IgM‑capture ELISA) was essentially the same methodo･s

CAMBODtA

Alf ,e ‑las reported elsewhere (Bundo and lgarashi, 1985; Innis
et al., 1989; Ruechusatsawat et al., 1994). Human lgG

50 7s f OO

was purified from the pooled DEN patient sera by
DEAE‑column chromatography and labeled with horse‑radish peroxidase (Wilson and Nakane, 1978). An
ELISA titer less than I : 100 was defined as negative.

Ph ,btNa ･ aythong

XaRh8r,RESULTSIn the Ban Hom commune, the DF/DHF epidemic

,
,,

R

1

C , ¥dl ver): Chanthabury

R: Sikhottabone
C: Xaysettha): Sisattanak

L JL:iok‑Ban Hom commune

Figure 1 Maps of Lao PDR (upper paneD and Vientiane
municipality (lower paneD. Vientiane municipal‑
ity consists of 8 districts. Ban Hom commune is
10cated in the south‑west of Hatxayfong district.

started in May, reached a peak in June and subsided byAugust, 1994. Fig. 2 shows the numbers of the monthly
hospitalized DF/DHF cases in the commune. During theepidemic, a total of 61 patients were admitted to thehospitals in the central area of Vientiane municipality.
Most of them were children under 15 years old.
Of 81 serum specimens from children with FUO, 48
were considered as acute phase sera (i. e. within 5 days
after the onset of fever) , and subjected to virus isolation.

DEN viruses were isolated from 15 (31.3%) specimens

123
30

q,

2 3 4

.l

(n : 61)

25

20

v

*c 15

,

c;

nj 4ee

Z 10

* 229

5

o‑

,

<

:
ce

s:

h

Montb

sh

ee

Figure 2 Monthly hospitalized DF/DHF cases in Ban Hom

Figure 3 Agarose gel electrophoresis profile of RTPCR

products from B52‑infected culture fluid using
DEN1 primers (1ane 1) , and DEN‑2 primers (1anecommune in 1994.

strains of DEN‑1 and 12 strains of DEN2 viruses wereidentified by PAP staining method using the type‑spe‑2) , from DEN‑1 infected culture fluid using DEN1
primers (1ane 3) and from DEN2 infected culture
fluid using DEN2 primers (1ane 4) . M: DNA size
marker. Expected DNA sizes are indicated on thecific monoclonal antibodies and RT‑PCR method. PAP

right side.

using a mosquito cell line (C6/36) (Table D. Fourstaining of BHK‑21 cells infected with the virus (es)recovered from B52 serum specimen showed positive
reaction with both DEN‑1 and DEN‑2 type‑specificantibodies. The RT‑PCR products from the B52 speci‑
men also showed two bands, identical to those from
DEN‑1 and DEN‑2 prototype viruses (Fig. 3) , indicat‑
ing that the patient, B52, was a case of dual infection

ing antibody to DEN‑3 virus (Table 1).
Fifteen serum specimens, from which DEN viruses
were recovered, were taken on either the second or thethird day of FUO. No virus was recovered from sera

enough symptoms to require admission. It would betaken on other than these 2 days in this study. Amongthese 15 serum specimens, 10 had no neutralizing anti‑
body to DEN virus, 4 had heterologous antibody and one
had homologous antibody to the isolated virus serotype.

noted that the child, B52, had the pre‑existing neutraliz

All the serum specimens were examined for DEN

with DEN‑1 and DEN‑2 viruses. A follow‑up study
revealed that the child, B52, did not develop severe

Table lResults of virus isolation, neutralization test and lgM‑capture ELISA

Serum Days ofVirus

code fever*isolation

B6

2

DEN‑1
DEN=2

2

DEN2

B25
B27
B52
B64

3

DEN‑1 & DEN‑Z

3

DEN2
DEN2

B 73

2

B 76

2

DEN ‑ 2

B80
B96
B98
B99
BIO1
BI02
B110

2

DEN‑2
DEN 2
DEN‑l
DEN 2

2

2
3
3
2

lgM‑ca pture

Neutralization titer to

DEN ‑1

DEN 2

DEN‑3

DEN ‑4

ELISA

56

27
86

DEN ‑2DEN ‑2

24

DEN1

28

*Days after onset of fever, ‑Neutralization titer < 20, or ELISA titer < 100

l 20

124

lgM antibody by lgM‑capture ELISA. Of 81 serum
specimens, 32 (39.5%) were positive for DEN IgM

the Department of Virology, Faculty of Medicine, Uni‑
versity of the Ryukyus.

antibody, indicating confirmed DEN virus infection.

Among 15 serum specimens with positive DEN virus
isolation, only one (6.7%) specimen was positive for thelgM antibody (Table D.

REFERENCES
1 ) Bounlu, K., Tadano, M., Makino, Y., Arakaki, S.,

Kanemura, K. and Fukunaga, T. (1992): A seroe‑

DISCUSSION
In 1994, the outbreak of DF/DHF occurred for the
first time in a rural commune, Ban Hom, Iocated in
Hatxayfonhg district, in Vientiane municipality. By the
middle of October, the number of hospitalized cases withpidemiological study of dengue and Japanese encephali‑
tis virus infections in Vientiane, Lao, PDR, Jpn. J. Trop.

Med. Hyg., 20, 149‑156
2 ) Bundo, K. and lgarashi, A. (1985): Antibody‑capture
ELISA for detection of immunoglobulin M antibodies insera from Japanese encephalitis and dengue hemorr‑
hagic fever patients, J. Virol. Methods., 11, 15‑22

clinical diagnosis of DF/DHF was 431 in the district,including 61 cases in Ban Hom. During the epidemics in3 ) Fukunaga, T., Phommasack, B., Bounlu. K., Saito, M.,
Tadano, M.. Makino, Y., Kanemura, K., Arakaki, S.,

1985 and 1987, most of the hospitalized cases were

Shingo, M., and Insisiengmay. S. ( 1993) :residents of the urban areas of Vientiane municipality.
Thus, DF/DHF seems to be expanding to the rural areasin Vientiane municipality.

Epidemiological situation of dengue infection in Lao
PDR, Trop. Med., 35, 219‑227

The sera taken from the children with FUO werestudied in the outbreak of Ban Hom commune by perfor‑
ming virus isolation and lgG and lgM antibody assays
on the serum specimens collected. Thirty‑two (39.5%)of 81 cases were confirmed as DEN infection by the
DEN‑specific lgM antibody detection, indicating theusefulness of the lgM‑capture ELISA. DEN‑1 and DEN‑2 viruses were isolated from 15 out of 48 serum speci‑
mens, indicating that the main causative viruses were

those two serotypes in the Ban Hom commune. All 15
cases did not develop severe hemorrhagic manifestation
and recovered in several days. Four of the 15 cases wereidentified as having the preexisting antibody to theheterologous DEN serotype by lgG antibody assay. Itshould be noted that the case B52, who had had pre‑existing antibody to DEN‑3, was dually infected with
DEN‑1 and DEN‑2 viruses during the outbreak, but did
not develop a severe form of DEN infection. This kindof serological and epidemiological approach on a larger
scale with both mild and severe cases of DEN infection
would give important information on the pathogenesis

of DHF.

ACKNOWLEDGMENT
We are grateful to the staffs of NIHE, Vientiane
Municipality Health Service, Hatxayiong District Hos‑pital and Ban Hom Health Center for collecting serum

specimens. We also appreciate the residents of Ban
Hom commune for their cooperation in this study. T.
Sisouk is a participant of the training course of theJapan International Cooperation Agency (JICA) , held in4 ) Igarashi, A. (1978): Isolation of a Singh's albopictus cellclone sensitive to dengue and chikungunya viruses, J.
Gen. Virol., 40, 531‑544
5 ) Innis, B. L., Nisalak, A., Nimmannitya, S., Kusalerdchar‑iya, S., CHomgswasdi, V., Suntayakorn S., Puttisri, P.
and Hoke, C. H. (1989) : An enzyme‑1inked immunosor‑
bent assay to characterize dengue infection where den‑gue and Japanese encephalitis co‑circulate, Am. J. Trop.
Med. Hyg., 40, 418‑427
6 ) Ishimine, T.. Tadano, M., Fukunaga, T., and Okuno, Y.
(1987) : An improved micromethod for infectivity assays
and neutralization tests on dengue viruses, Biken J., 30,

139‑1447 ) Morita, K.. Tanaka, M., and lgarashi, A. (1991): Rapid

identification of dengue virus serotypes by using
polymerase chain reaction, J. Clin. Microbiol., 29, 2107‑

2110
8 ) Morita, K., Maemoto, T., Honda, S., Ohishi, K., Murata,M., Tanaka, M. and lgarashi, A. (1994): Rapid detectionof virus genome from imported dengue fever and dengue
hemorrhagic fever patients by direct polymerase chain
reaction, J. Med. Virol., 44, 54‑58
9 ) Okuno, Y., Igarashi, A. and Fukai, K. (1978): Neutrali‑zation tests for dengue and Japanese encephalitis virusesby the focus reduction method using peroxidase anti ‑peroxidase staining, Biken J., 21, 137‑147
10) Ruechusatsawat, K., Morita, K., Tanaka, M., Vongcher‑ee, S., Rojanasuphot, S., Warachit, P., Kana K., Thom‑
gtradol P., Nimnakorn P., Kanungkid, S. and lgarashi,
A. (1994): Daily observation of antibody levels among
dengue patients detected by enzyme‑linked imrnunosor‑
bent assay (ELISA), Jpn. J. Trop, Med. Hyg., 22, 9‑12lD Vongxay, P., Makino, Y., Kanemura, K.. Saito, M. andFukunaga. T. (1995): Seroepidemiological study ofarbovirus infections in Khammouane Province, Lao
PDR, Ryukyu Med. J., 15, 19‑2212) Wilson, M. B. and Nakane, P. (1978): Recent develop‑

125

ments in the periodate method of conjugating horse‑radish peroxidase (HRPO) to antibodies. In Knapp, W.,
Molubar, K. and Wick, G (eds) , Immunofluorescenceand related staining techniques, 215‑224, Elserver/North‑Holland Biomedical Press, Holland

127

Jpn. J. Trop. Med. Hyg., Vol. 23, No. 2 (1995) pp. 127‑131

A NEW BLACK FLY SPECIES OF SIMULIUM( NE VERMANNIA ) FROM SUMATRA, INDONESIA
H. TAKAOKAI and D.M. DAVIES2
Received February 13, 19951Accepted March 6, 1995

Abstract: Simulium (Nevermannia) glatthaari sp. nov. is described based on the pharate female and male,
pupal and mature larval specimens collected from Sumatra, Indonesia. This species is assigned to theruficorne‑group of the subgenus Nevermannia, and is distinguished from S. (N.) aureohirtum Brunetti, theorrly known species of this species‑group in south‑east Asia, chiefly by the number of pupal gill filaments,
and the arrangement of the anterior teeth of the larval hypostomium.

In south‑east Asia, a black fly species Simulium(Nevermannia) aureohirtum Brunetti has long been the
only species belonging to the ruficorne‑group defined by

Crosskey (1969) (e.g. Takaoka and Davies, 1995) . In
the course of taxonomic studies on the Simuliidae of the
Oriental Region, we found another species assignable to

the ruficorne‑group, judged by the genitalia of both
adult sexes, which was collected from North Sumatra
by Dr. R. Glatthaar. Although adult specimens available
are only a few pharate individuals, this species is readily

distinguished from S. (N.) aureohirtum by the numberof the pupal gill filaments and the arrangement of theanterior teeth on the larval hypostomium, and is here
described as the second member of the ruficorne‑group

covered with pubescence. Scutellum with pubescenceand several erect long hairs. Postscutellum bare.
Pleural membrane bare. Katepisternum longer thandeep, bare. Legs. Fore basitarsus slender, cylindrical.
Hind basitarsus parallel‑sided. Calcipala well devel‑
oped (Fig. 3). Claws (Fig. 4) each with a large basal

tooth, 0.35 x length of claw. Color markings not yet
developed. Wing. Costa with 2 parallel rows of shortspines as well as hairs. Subcosta fully haired. Basal
portion of radius fully haired. Genitalia (Fig. 7).
Sternite 8 bare medially, furnished with numerous shortand long hairs on each side. Anterior gonapophysis thin,membranous, rounded, with ca. 10 short setae; innerborders sowewhat separated from each other. Genital

fork with slender stem and wide arms. Paraproct
depressed anterolaterally. Cercus much wider thanin south‑east Asia.

DESCRIPTION
Simulium (Nevermannia) glatthaari Takaoka and Davies,
sp. nov.

10ng. Spermatheca not yet sclerotized.

MALE (dissected out of pupa) . Head. Holoptic; upper
eye consisting of large facets in 22 vertical columns and

FEMALE (dissected out of pupa) . Head. Narrower

22 horizontal rows. Antenna composed of 2 + 9 seg‑than thorax. Frons and clypeus densely covered withpubescence. Frontal ratio 1.6:1.0:1.8. Fronto‑ocularments; Ist flagellomere elongated, ca. 2.0 X as long as

area well developed. Frons‑head,ratio 1.0:4.3. Antenna

sensory vesicle (Fig. 2) small, globular. Thorex. Nearly

composed of 2 + 9 segments. Maxillary palp with 5

as in . Legs. Fore basitarsus slender, cylindrical.
Hind basitarsus narrow, parallel‑sided. Calcipala wellsegments, 3rd segment (Fig. I ) somewhat enlarged, withelongate sensory vesicle, 0.47 x length of 3rd segment.

Maxilla with 10 inner teeth and 13‑15 outer ones.
Mandible with ca. 20 inner teeth and 10 outer ones.
Cibarium lacking denticles. Thorax. Scutum densely

2nd flagellomere. Maxillary palp with 5 segments;

developed. Color markings not yet developed. Wing.
As in. Genitalia (Figs. 5 and 6). Coxite large, much
longer than wide. Style shorter than coxite, broad inlateral view, with apical spine on posterodorsal edge.

Division of Medical Zoology, Oita Medical University, Hazama, Oita 879‑55, Japan
2. Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1, Canada

128

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Figures 1‑7 Adult of Simulium (Nevermannia) glatthaari sp. nov. l, 3rd maxillary palpal segment; 2, d 3rd
maxillary palplal segrnent; 3, distal tip of basitarsus and 2nd tarsal segment ofhind leg; 4,
claw; 5, c 1 genitalia in situ (ventral view) ; 6, Ieft style (1ateral view) ; 7,

genitalia in situ (ventral

vie c; spermatheca and left paraproct and cercus omitted) .

Ventral plate broad, with setose median keel produced
ventrally; arms short, curved inward. Paramere with adistinct parameral hook. Median sclerite simple, rod‑
shaped.

PUPA. Body length (excluding gill filaments) 3.0‑3.8
mm. Head and thorex. Integument dark yellow, moder‑ately covered with small tubercles. Head with 3 pairs of

simple, short trichomes (in some pupae frontal tri‑chomes not detected) . Thoracic trichomes 6 pairs (3anterodorsally, 2 anterolaterally and I posterolaterally) ,all simple. Gill (Figs. 8 and 9) with 8 slender filaments,

2.0‑3.0 mm long, widely diverged, arranged in 3 + 2 + 3 or

2 +3+3 filaments on very short stalks in 4 pupae (Fig.
8) but on relatively long secondary stalks in 3 other

Terga I and 2 yellow, weakly tuberculate; tergum I witha single slender seta on each side; tergum 2 with 4
hooked spines (a little smaller than those on terga 3 and4) and 2 spinous setae on each side (Fig. 13). Terga 3

and 4 each with 4 hooked spines and I small spine oneach side. Terga 7 and 8 each with spine‑combs andcomb‑Iike groups of very minute spines in transverserow. Tergum 5 bare. Tergum 6 occasionally with spine‑
combs consisting of a few spines. Tergum 9 with comb‑
like groups of very minute spines and a pair of stout
terminal hooks (Fig. 17) . Sternum 3 with 4 setae oneach side. Sternum 4 with 2 stout spines and 2 slendersetae on each side; sternum 5 with a pair of bifid hooks
situated close together and I seta on each side; sterna 6

pupae (Fig. 9) , secondary furcation of ventral triplet inand 7 each with a pair of inner bifid and outer simple
hooks widely spaced and I seta on each side. Lateral

1 of 3 pupae located on distal 2/3 thus showing very longsurface of segments 2‑5 each with 3 hook‑like spines on

secondary stalk. All filaments tapered apically, witheach side (Fig. 15) . Cocoon. Wall‑pocket‑shaped,
thinly woven, often extending forward and inward,

numerous weakly defined transverse ridges, covered
with minute tubercles. Abdomen (Figs. 11 and 12).

making narrow anteroventral collar, with no anterodor‑

129

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Figures 8‑18 Pupa of Simulium (Nevermannia) glatthaari sp. nov. except Figs 10, 14, 16 and 18 for S. (N.)
aureohirtum. 8, 9 & lO, gill filaments; Il, dorsal surface of abdomen; 12, ventral surface of abdomen;

13 & 14, spinous setae on tergum 2; 15 & 16, Iateral spines or setae on segments 2‑5; 17 & 18,
terminal hooks. Scale 1.0 mm for Figs. 8,̲ 9 & 10; 0.1 mm for Figs. 11‑18 (in Figs. 11 & 12 scale
applies to enlargements of spines and hooks only) .

130

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Figures 19‑27 Larva of Simulium (Nevermannia) glatthaari sp. nov. except Figs. 20, 23, 25 and 27 for S. (N.)
aureohirtum. 19 & 20, cephalic apotome; 21, ventral surface of head capsule; 22 & 23, antenna; 24& 25, tip of mandible; 26 & 27, hypostomium. Scale 0.2 mm for figs. 19‑21; 0.1 nun for Figs. 22,
23, 26 & 27; 0.05 mm for Figs. 24 & 25.

eral sides, with distinct dark brown head spots (Fig. 19) ;ly; hypostomal setae 7‑9 Iying parallel to lateral mar‑gins. Postgenal cleft (Fig. 2D small, as long as or alittle shorter than postgenal bridge. Thoracic cuticle
bare. Abdominal cuticle bare except dorsal surface ofposterior segments sparsely covered with minute color‑less setae and sides of anal sclerite of last segrnent
covered with numerous colorless setae. Rectal papillaventral surface of head capsule largely darkened.
Antenna (Fig. 22) composed of 3 segments and apicalwith 3 simple lobes. Anal sclerite of usual X‑form, withanterior arms ca. 0.5 x as long as posterior ones; widely

sensillum, as long as or a little shorter than stem oflabral fan; proportional length of 3 segrnents from base
2.0:3.1:1.3. Labral fan with ca. 40 main rays. Mandible(Fig. 24) with enlarged comb‑teeth decreasing in size
from Ist to 3rd tooth; mandibular serrations composedof 2 Iarge teeth somewhat spaced, without supernumer‑

sclerotized at base. Ventral papillae absent although
there is a large bulge produced laterally on each side.
Posterior circlet with ca. 84 rows of up to 22 hooklets

ary serrations. Hypostomium (Fig. 26) with row of 9with pupal skin and cocoon, collected from the foot of
Mt. Sibayak, North Sumatra, 13.IV.1977, by Rolf Glatth‑aar. Paratypes, I pharate c71 , 6 Pupae (3 of these are

sal projection.

MATURE LARVA. Body length 5.0‑6.0 mm. Body
entirely greyish, abdomen gradually widened from Ist to

7th or 8th segment when viewed dorsally. Cephalicapotome brown except narrow pale portion along lat‑

apical teeth; median tooth longer than others which aresubequal in size; Iateral margin markedly serrate apical‑

per row.

TYPE SPECIMENS. Holotype pharate, associated131

thoracic integument with gill filaments only) and 10
mature larvae, same data as holotype. Types will bedeposited in the Natural History Museum, in London, U.
K.

aureohirtum, though rarely simple as reported by Ta‑
kaoka (1976) , and the new species has more rows ofhooklets on the posterior circlet than the latter whichhas 60 to 70 rows (Puri, 1933; Takaoka, 1979). In thespecies were collected from trailing grass leaves in arice‑field drainage brook (width I m, depth 0.3 m andpupa of S. (N.) aureohirtum, spinous setae on the ter‑gum 2 (Fig. 14), those of lateral surface of abdominal
segments 2‑5 (Fig. 16), and terminal hooks on the ter‑gum 9 (Fig. 18) appear to be less prominent than thosevelocity 10 l/sec) , at altitude of 1200 m, at the foot ofof this new species.

Mt. Sibayak, North Sumatra. Collected together withAs in most other known species of the ruficorne‑group, adults of S. (N.) aureohirtum were reported toshow the characteristic color markings of the legs andthe antennae (Puri, 1933). However, such color charac‑

ECOLOGICAL NOTES. Pupae and larvae of this new

this species were mostly pupae and larvae of S. ( Gom ‑phostilbia) sundaicum Edwards and S. (Simulium) ar ‑gyrocinctum de Meijere. One mature larva of S. (N.)
aureohirtum was also gathered.

DISTRIBUTION. Sumatra

ters could not be observed in the immature pharate
specimens of this new species.
It remains to be studied whether the new species is

autogenous, as shown in S. (N.) aureohirtum (Takaoka

REMARKS. This was named after Dr. R. Glatthaarand Noda, 1979) .

who collected the material.
The present new species is easily assigned to the

ACKNOWLEDGEMENT

ruficorne‑group of the subgenus Nevermannia by theunique shape of the genitalia of both sexes, and isdistinguished from the other known species of thisWe thank Dr. R. Glatthaar, Winterthur in Switzer‑
land, who gave us simuliid specimens collected from

species‑group by the eight pupal gill filaments per side
(Figs. 8 & 9) and the arrangement of the anterior teeth

Sumatra. The senior author is grateful to Ms. C. Aoki,
Division of Medical Zoology, Oita Medical University,of the larval hypostomium (Fig. 26). The pupal gillfor her help.

filaments are six per side in S. (N.) aureohirtum, theonly other member of this species‑group in south‑east
Asia, and four in the other known species in the Eth‑iopian, Palaearctic and Australasian Regions (Crosskey,1969). All these known species of the ruficorne‑group
have the anterior teeth of the larval hypostomium ofwhich each corner tooth is prominently higher thanintermediate teeth, as shown in the Fig. 27.

Apart from these characters, this species differsfrom S. (N.) aureohirtum by the coloration of the larval
cephalic apotome (Fig. 19) and the shape and colorationof the larval body. The latter species shows marked
head spots on pale ground color of the cephalic apotome(Fig. 20) and has the pale body color and the abdominal
segments 1‑4 subequal in size. In addition, there is adifference in the relative size of the mandibular comb‑
teeth, of which second tooth is larger than the third inthis new species (Fig. 24) but is subequal to or shorter
than the third in S. (N.) aureohirtum (Fig. 25) . The
larva of S. (N.) glatthaari is also characterized by theshortened third antennal segment compared to the sec‑
ond one (Fig. 22). The rectal papillar lobes are simple

in the new species but usually compound in S. (N.)

REFERENCES
1 ) Crosskey, R.W. (1969): A re‑classification of the
Simuliidae (Diptera) of Africa and its islands, Bull. Br.

Mus. (Nat. Hist.) Entomol. Suppl., 14, 195
2 ) Puri, I.M. (1933): Studies on Indian Simuliidae. Part
VIII. Descriptions of larvae, pupae, males and femalesof S. aureohirtum Brunetti and S. aureum Fries, Ind. J.
Med. Res., 21, 1‑93 ) Takaoka, H. (1976) : Studies on black flies of the
Nansei Islands, Japan (Simuliidae; Diptera) I. On six
species of the subgenus Eusimulium Roubaud, with thedescriptions of Simulium (E.) satumense sp. nov. and S.
(E.) subcostatum koshikiense ssp. nov., Jpn. J. Sanit.
Zool., 27, 163‑180

4 ) Takaoka, H. (1979): The black flies of Taiwan (Dipter‑a: Simuliidae). Pacific Insects, 20, 365‑403

5 ) Takaoka, H. and Davies, D.M. (1995) : The black flies(Diptera: Simuliidae) of Java, Indonesia, Bishop Mus.
Bull. Entomol. (in press)

6 ) Takaoka, H. and Noda, S. (1979): Autogeny of theblackfly Simulium (Eusimulium) aureohirtum (Diptera:
Simuliidae) , J. Med. Entomol., 15, 183‑184

133

Jpn. J: Trop. Med. Hyg., Vol. 23, No 2 (1995) pp. 133‑137

STUDIES ON ANNUAL CHANGES IN MICROFILAR‑
IAL PREVALENCE OF DIROFILARIA IMMITISAMONG HOUSE DOGS OR 27 YEARSIN NAGASAKI CITY, JAPANTSUTOMU ODAl, OSAMU SUENAGA2, MAKOTO ZAITSU3,
AKIO MoR14, KENJI KUROKAWA=, KOICHIRO FUJITA6,YASUNORI OGAWA6, ICHIRO YAMAZAK17, KUNIHIRO IIDA7,

KOUICHI D017, MARIKO MINE8 and KATSUTOMO KAT09
Received February 24, 1995/Accepted April 17, 1995

Abstract: The positive rate of dogs with Dirofilaria immitis microfilariae among house dogs was examinedfor 27 years from 1968 to 1994 in the southern and northern parts of Nagasaki City. Roles of the numberof the main vector, Culex plpiens pallens and of RPSS (rate of population utilizing a sewage system) wereanalyzed statistically in relation to the annual changes in microfilarial prevalence. In the southern andnorthern parts, there was a clear correlation among three parameters of the positive rate in the dogs, thenumber of Cx. p. pallens and RPSS. The present study proved that the decrease in the positive rate of dogs
in both the southern and northern parts is mainly attributable to a decrease in the number of Cx. p. pallens.
which resulted from the decrease of the breeding places of this mosquito following the spread of the publicsewage system and the improvements in the use of roads and open roadside ditches.

INTRODUCTION

among house dogs in southern and northern parts ofNagasaki City over the past 27 years after 1968 to

Oda et al. (1993) previously reported that the posi‑clarify the role of the density of Cx. p. pallens and thetive rate of dogs for microfilariae of Dirofilaria immitis

spread of public sewage systems in reducing Dirofilaria

increased from 1968 to 1983 in the eastern, western andsouthern parts of Nagasaki City, but decreased during
the same period in the northern part. Moreover, Oda etal. (1994a) collected female Culex pipiens pallens, theimmitis infection.

PLACES AND METHODS

main vector of Dirofilaria immitis. from 1983 to 1989,

The survey was conducted in four districts: a south‑and found that in 1983 Cx. p. pallens was extremely

ern district (Tomachi) and three northern districts(Sakamoto, Takao and Yamazato) . From 1983 to 1994,
blood examination was conducted in April or May fromprevalent in the southern part but not in the northernpart, and after 1986, the prevalence of this mosquito
became low in both parts, in parallel with the spread of
sewage system. In this study, we analyzed statisticallythe annual changes of Dirofilaria immitis infection rates

about 400 registered dogs in these four districts at thetime of vaccination against rabies. One drop (about
20mm3) of blood was collected from the ear lobe of

1 Department of General Education, The School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852,

Japan
2 Reference Center, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
3 Sasebo City Health Center, 1‑10 Yahata‑machi, Sasebo 857, Japan
4 Department of Medical Zoology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
5 Department of Bacteriology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
6 Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University, 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo
113, Japan
7 Nagasaki City Health Center, 2‑34 Morimachi, Nagasaki 852, Japan
8 Scientific Data Center of Atomic Bomb Disaster, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
9 Department of Physical Therapy, The School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852,

Japan

134

individual dogs. Blood samples were subjected to Giem‑

sa staining and examined for microfilariae under astereomicroscope.
Mosquitoes were collected using a light trap (16watt black light) from 1972 to 1989 in Tomachi andfrom 1967 to 1994 in Sakamoto. Each trap was usuallyoperated once or twice a week in the May‑October

2 . Incidence of new infection among the dogs
The incidence of new infection in each year from1984 was calculated as in Table 2 by dividing the numberof dogs which turned from negative to positive formicrofilariae by the number of dogs examined among
those which had been negative in the previous year. In
each district, this incidence decreased from year to year

period. The details of the sites and methods of mosquito
collection were previously reported (Oda et al., 1993;
1994a) . The districts in four parts (eastern, western,(Table 2). Fig. I shows the relationship between theincidence of new infection and the percentage of dogs
having microfilariae for the southern part (Tomachi)

southern and northern) of N agasaki City were derived

and the northern part (data in Sakamoto, Takao andYamazato were combined) of Nagasaki City. In bothfrom the residential list used by Suenaga et al. (1971).

parts of the city, there was a significant positive correla‑tion between the positive rate of dogs and the incidence

RESULTSof new infection (P<0.0D . Thus the percentage of dogs

1 . Annual changes in microfilarial prevalence of

having microfilariae decreased clearly from year to

Dirofilaria immitis among house dogs
Table I shows the annual changes of the positive
rate (percentage of dogs having microfilariae) in eachof the four districts during the 1968‑1994 period. The
annual changes in positive rate were examined by theyear owing to the reduction of new infection in both thelinear trend test of Armitage (1955) . The positive rateof dogs generally decreased in all districts, althoughsome fluctuations were noted. This decrease during the27 years was siguificant (P<0.0D in all districts.

Table 1southern and northern parts of the city.

3 . Annual changes in positive rates of dogs, the num‑
ber of Cx. p. pallens and the rate of public sewage

systemFig. 2 shows annual changes in the positive rate ofdogs, the number of Cx. p. pallens females caught andthe rate of human population utilizing a public sewage
system (RPSS) . As described previously (Oda et al.,Annual changes in microfilarial prevalence of Dirofilaria immids among house dogs in southern andnorthern parts in Nagasaki City
Northern part

Southern part

District nameDistrict nameYear

Tomachi

Sakamoto

Takao

Yamazato

Total

No. dogs No. (%) of No. dogs No. (%) of No. dogs No. (%) of No. dogs No. (%) of No. dogs No. (%) of
exarnined positive dogs examined positive dogs examined positive dogs examined positive dogs examined positive dogs

64 30 (46 . 9)

123 61 (49 . 6)

229 106 (46 . 3)

33 15 (45 . 5)

43 14 (32 . 6)

41 13 (31 . 7)

117 42(35.9)

149 57 (38 . 3)

32 5 (15 . 6)

134 34 (25 . 4)

32 6(18.8)

198 45 (22 . 7)

137 41(29 9)

63 17 (27 . O)

161 41 (25 . 5)

46 7 (15 . 2)

270 65(24 D

55 8 (14 . 6)

181 38 (21 . O)

1968

100 34(34 O)

42 15 (35 . 7)

1977

120 42 (35 . O)

1983
1984
1985

107 44 (41 . D

52 16 (30 . 8)

74 14 (18 . 9)1986

129 41 (31 . 8)76 23 (30 . 3)

57 9 (15 . 8)

30 6 (20 . o)

163 38 (23 . 3)

212 31 (14 . 6)

1987

127 36 (28 . 3)

73 13(17.8)

103 13 (12 . 6)

36 5 ( 13 . 9)

1988

137 36 (26 . 3)

70 24 (34 . 3)

177 30(16 9)

39 5 (12 . 8)

286 59 (20 . 6)

1989

155 41 (26 . 5)

46 14 (30 . 4)

148 26 (17 . 6)

57 7 (12 . 3)

251 47(18.7)

1990

125 24(19.2)

74 10 (13 . 5)

164 24 (14 . 6)

59 3( 5.D

297 37 (12 . 5)

1991

51 7 (13 . 7)

86 8( 9.3)

166 21 (12 . 7)

313 33 (10 . 5)

1992

145 26(17.9)

83 10 (12 . O)

154 17(11 O)

1993

74 11 (14 . 9)

1994

137 20(14 6)

32 o( 0.0)
56 3( 5.4)

149 16 (10 . 7)

61 4( 6.6)
56 3( 5.4)
30 1( 3.3)
54 2( 3.7)

78 7( 9.0)

293 30 (10 . 2)

140 8( 5.7)
259 21( 8.D

135

Number and percentage of dogs infected newly with Dirofilaria immitis microfilariae

Table 2

Northern part

Southem part

District nameDistrict name

Takao

Sakamoto

Tomachi

Year

Total

Yamazato

No. dogs' No. (%) of No. dogs* No. (%) of No. dogs' No. (%) of No. dogs* No. (%) of No. dogs$ No. (%) of
examined dogs newly examined dogs newly examined dogs newly examined dogs newly examined dogs newly
infected.

inf ected.

infected.

inf ected.

inf ected.

1984

46 8 ( 17 . 4)

20 4 (20 . o)

40 8 (20 . o)1986

36 6 (16 . 7)

1987

1989

49 4( 8.2)
58 4( 6.9)
63 4( 6.3)

26 3(11.5)
25 2( 8.0)
32 3( 9.4)

1990

66 4( 6.D

57 4( 7.0)25 o( 0.0)24 l( 4.2)
28 o( 0.0)
61 5( 8.2)
79 7( 8.9)70 1( 1.4)

12 1( 8.3)
19 1( 5.3)
12 O( 0.0)
15 O( 0.0)
20 o( 0.0)
28 1( 3.6)30 o( 0.0)

89 9 (10 . D

1985

1991

14 1( 7.1)
21 O( 0.0)
70 1( 1.4)
79 1( 1.3)

87 1( 1.D
89 1( 1.D

. 29 .o( 0.0)

1988

1992

1993
1994

32 1( 3.D
25 3 (12 . O)

20 1( 5.0)
40 1( 2.5)
44 1( 2.3)
33 o( 0.0)36 o( 0.0)

75 2( 2.7)
64 1( 1.6)

31 1( 3.2)
28 o( 0.0)30 o( 0.0)

70 4( 5.7)
61 3( 4.9)
75 3( 4.0)
113 6( 5.3)
132 11( 8.3)

120 2( 1.7)
156 2( 1.3)
164 3( 1.8)
136 2( 1.5)
130 1( 0.8)

*No. dogs examined : Number of dogs examined among those which had been negative in the previous year.
Southorn part
Positive rate

ge50
ey ‑ 1.789x + 8.008; r ‑ 0.85S (p(0.01)
y O y ‑ 1.096x + 14.914; r * 0.862 (p(0.01)

SO

*30
20

'

10

*

O

8 16
122

O ̲･‑

40

SdO
･

Cx. b. PalLens

84
:4
8

o
e
o>

IJ

GOO .･‑" '<{) ‑ eNORTH

30'e e‑" O‑‑‑ SOUTH

20:JJ‑,J‑

10

*, tj
,'

oO

68

o I o I s 20 2sxs

New infection rate (%)
Figure I Relationship bctween positive rate of dogs and new
infection rate in southern and northern parts in

Nagasaki City.
1994a) , the numbers of Cx. p. pallens females in southernand northern parts were obtained at Tomachi and
Sakamoto, respectively. The RPSS for Tomachi wasregarded as the RPSS for the southern part of Nagasaki
City, and the RPSS's for the northern part was calcu‑

lated by averaging the RPSS for Sakamoto, Takao andYamazato. In the southern part, the positive rate ofdogs remained unchanged between 1968 and 1983 but it
decreased gradually thereafter, the number of Cx. p.
pallens was considerably high until about 1980 but it was

‑50
i !

S'o

ii

! }i {1

20

e
O

RPss

60
dO

O

u'

o

O
80

j'f

'

' i ,,,F

eo929470 72 74 76 78 80 82 84 86
Northern part
Positive rate

*･ 30

.

20

'‑ ro

o

16

Ct. b. paltens

s t2

*5 88
: 4
o
80

RF

')e60

1d
20‑
a'

ji

6870727476788082848688909294

Figure 2 Annual changes in positive rate of dogs, mean
number of Culex pipiens pallens per night and rateof population utilizing a sewage system (RPSS) .
* Data not available

136
e y = 14. 110x + 18.972: r = 0.782 (p<0.01)

y Oy

O y = 3. 588x ‑ 33. 957; r = 0.522 (p>0.05)

Oy

so
50 y

e
,,,

o

,g

o:‑

..1"I"'6'30

̲o' co

10

Od,

30

O
:‑

20

.p

O ‑‑‑ SOUTH

8' ‑Q: ̲o.1o2

Log No. fema I es

RPSS : Rate of population utilizing a sewage
system.

quite low after that year, and the RPSS increased sharp‑ly after about 1985. These patterns of aunual changes in

e x ' ‑O. 022z + o. 77; r = ‑O. 531 (p<0.01)

the three parameters for the northern part were approx‑
imately equal to those observed in the southern part,
excepting that the percentage of dogs having microfilar‑

O x = ‑O. Q33z + o. 23: r = ‑o. 825 (p<0. ol)x

2.0

iae began to decrease earlier in the northern part

between two of the three parameters (Fig. 3‑5) . In thesouthern part, the correlation between the positive rateof dogs and the number of Cx. p. pallens was not strong,probably because the number of available data in pair
was not sufficient. However, it is noteworthy that thepercentage of positive dogs in this part had a significant

correlation with the RPSS (P<0.0D as in Fig. 4. A

Relationship between positive rates of dogs and
RPSS in southern and northern parts in Nagasaki
City.

f emales.

calculated a correlation coefficient and a regression linez

75

RPSS (%)

Figure 4

Figure 3 Relationship between positive rates ofdogs and number of Culex pipiens pallens(around 1977) than in the southern part, and that the
number of Cx. p. pallens caught in the northern partbegan to decrease in about 1977. These features of thenorthern part probably reflect the fact that public sew‑
age systems began to spread in about 1970.
To analyze the relationships among the positive
rate of dogs, the number of Cx. p. pallens and RPSS, we

O ‑‑‑ SOUTH

25 1 oosoX1e ‑ NORTHe ecli o o ‑. ,g̲..

o.2

r= ‑o. 8868 (p<0. ol)***‑. ooO‑O. 24lz + 37. 391 ;

10

,

eoeoe
e

cl'

o
eL

e

t

20

O NORTH

r= ‑O. eo99 (p<0. ol )

*,, e

c5

̲J o

33. 730:

o

40
' '
5'

o

40
*f*

‑O. 53lz

e
S

1 .O
co

o
,g
5

1*

e
e

o

!:,

Jo

e
e

‑1 .O

e NORTHO ‑‑‑‑ SOUTH

o
o

‑2.0

o

10 20 30 40 SO 60
RPSS( 96 )Figure 5

Relationship between number of Culex pipiens
pallens females and RPSS.
RPSS : Rate of population utilizing a sewage
system.

mosquito following the spread of public sewage systems.

significant correlation between the number of Cx. p.

pallens and the RPSS was also noted (P<0.0D, as

DISCUSSION

shown in Fig. 5.
In the northern part, a clear correlation was noted

The present study revealed correlations among thein any combination of two among three parameters of

positive rate of dogs, the number of female Cx. p. pallens

the positive rate of dogs, the number of Cx. p. pallenscaught and the RPSS, and that the correlation was

and RPSS (Fig. 3‑5). From these results, it was con‑particularly strong between the positive rate of dogs andcluded that the decrease in the positive rate of dogs inboth the southern and northern parts is attributable to a

the RPSS. In view of these findings, the decrease in thepercentage of positive dogs observed apparently in bothparts of this city after 1983 may be explained as follows.

decrease in number of Cx. p. pallens, which was an
outcome of a decrease of the breeding places of thisIn ptiblic sewage systems, the sewage including all house

137

sewage but excluding rain water is directly sent to thesewage processing plant, Under this sewage system,
clean rain water containing little organic substance inopen road side ditches does not constitute significant
oviposition sites of mosquitoes. As a result, the sewage
system reduced the number of breeding places of Cx. p.
pallens. The number of breeding places of this mosquito

in 1989 and 1993, the percentage of dog owners who used
the preventive drugs was not high (Oda et al., 1994b) .
Therefore, these drugs do not seem to contribute greatly
to reducing Dirofllaria immitis infection.

ACKNOWLEDGMENTS

was further reduced by the improvement of roads andProfessor Y. Wada, Department of Medical Ento‑

open roadside ditches, in addition to the development ofmology, Institute of Tropical Medicine, Nagasaki Uni‑

public sewage systems. Accordingly the number ofversity kindly read this manuscript and provided

female Cx. p. pallens decreased, and the transmission ofdetailed suggestions. Dr. T. Fukuda, President, andmembers of the Society of Veterinarians in Nagasaki

Dirofilaria immitis to dogs by mosquitoes was reduced.
Contrarily, the positive rate of dogs increased great‑ly during the 1968‑1977 period, in Koga, Toishi, Fukuda

and Hayasaka (the four newly populated residential
areas at margins of Nagasaki City) (Suenaga et al.,1980). This is explained by the fact that no publicsewage systems were available in any of these four
districts at that period. The lack of sewage systems
probably resulted in the formation of many breeding
places of Cx. p. pallens, Ieading to the increase in thepercentage of positive dogs by a large number of vector

mosquitoes.
If the number of Cx. p. pallens in both the southernand northern parts remains unchanged or decreases
from the current level on, the positive rate of dogs willfurther decrease. The current number of Cx. p. pallens
may have already approached critical vector density, as
pointed out by Wada et al. (1984) , but this needs to befurther studied. As pointed out in a previous paper (Odaet al., 1994a) , it is also likely that secondary vectors(Culex tritaeniorhynchus and Aedes albopictus) will playa relatively more important role in the future, following

a decrease in the number of Cx. p. pallens.

The number of stray dogs may be another factor
influencing the transmission. The number, as given by

the number of arrested dogs (unpublished data) de‑creased in about 1972 and thereafter remained littlechanged in Nagasaki City. From this, stray dogs are notconsidered to be an important factor for the decrease of
positive rate.

According to our questionnaire survey in 1989 and
1993, the number of households which keep dogs indoors
has been increasing (Oda et al., 1994b) . It is not clear
whether this change contributed greatly to the reductionin positive rate of dogs.

It is known that the preventive drugs of diethylcal‑bamazine and ivermectin which has recently been devel‑
oped have the excellent effects against Dirofilaria im ‑mitis. However, according to our questionnaire survey

City really cooperated with our research. The staff in
Department of Sewage System in Nagasaki City gave us
information for sewage system. We wish to express our
sincere gratitude to these persons.

REFERENCES
1 ) Armitage. P. (1955): Test for linear trends in propor‑
tions and frequencies, Biometrics 11, 375‑386
2 ) Oda, T., Suenaga, O., Mori, A., Fujita, K., Zaitsu, M.,

Kurokawa, K., Nishioka, T., Itoh, T., and M. Mine
(1993): Studies on epidemiology of Dirofilaria immitis
in house dogs in Nagasaki City, Japan with considera‑
tions on yearly changes in microfilarial prevalence, Jpn.

J. Trop. Med. Hyg., 21, 231‑237
3 ) Oda, T., Suenaga, O., Fujita, K., Zaitsu, M.. Kurokawa,

K., Ogawa, Y., Yamazaki, I., Iida, K., and M. Mine
(1994a): Comparision of population of vector mos‑quitoes of Diofilaria immitis and their natural infection

rates in southern and northern parts of Nagasaki City,
Japan, Jpn. J. Trop. Med. Hyg., 22, 196‑202
4 ) Oda, T., Suenaga, O., and M. Mine (1994b) : A question‑
naire survey of living environments of house dogs andmeasures taken by dog owners to prevent zoonotic
parasite, Dirofilaria immitis infection in Nagasaki City,
Japan, Bull. Sch. Allied. Med. Sci., Nagasaki Univ., 8,

9‑165 ) Suenaga, O., Itoh, T., and T. Nishioka (197D: Studies
on the filarial prevalence among house dogs and themosquito vectors in Nagasaki City, Western Japan. 1.
On the microfilarial prevalence among house dogs in
Nagasaki City, Trop. Med., 12, 169‑178 (in Japanesewith English summary)6 ) Suenaga, O., Nishioka, T., and T. Itoh (1980): On thetransition of canine heartworm prevalence among dogs
in Nagasaki City, Western Japan. 1. The filarial preva‑
lence among dogs in the city, Trop. Med., 22, 35‑45. (inJapanese with English summary)7 ) Wada, Y., Tsuda, Y., and O. Suenaga (1984) : Transmis‑
sion dynamics of Dirofilaria immitis in a southwesternpart of Japan, Trop. Med., 31, 35‑47

139

Jpn. J. Tro p. Med. Hyg., Vol. 23, No. 2 (1995) pp. 139‑141

Short communication

HIGH PREVALENCE OF SEXUALLY TRANSMITTEDDISEASES IN AN AREA IN MOZAMBIQUE
SEIICHI YAMADA, YUE PAN, YUKA MORI,
GEN INUO, SETSUKO TSUKIDATE and KOICHIRO FUJITA
Received March 14, 1995/ Accepted April 7, 1995

Each year 10 milliori Japanese travelers andtreatment for the infection. The true prevalence ofworkers visit various foreign countries, including Asia
and Africa. Sexually transmitted diseases (STD) s are

STDS can be derived only from data on unselected

now more common in many developing countries than in
the developed nations (De Schamphelerie et al., 1990;
Mosha et al., 1993; Nkowane, 199D. Evidence indicatesthat the classic STDS facilitate the transmission ofhuman immunodeficiency virus (HIV) infection (Pepin
et al., 1989). Particularly compelling is the need to

Our objective was to investigate the incidence of
STDS in people in rural areas of in Mozambique and to
identify factors involved in the dissemination of theQuelimane in Mozambique in 1991 and 1994. Blood

prevent fetal death, prematurity, and neonatal complica‑

samples were taken from 88 people, 59 males (average

tions by STDs, especially in developing countries

age; 32.01:!: 11.65, range; 12‑60) and 29 females (average

populations.

inf ections.

We conducted our investigation in an area around(Schulz et al., 1987).

age; 22.6ld:8.25, range; 11‑42) in 1991, and from 38

Whereas many studies have been conducted on theprevalence of STDS in urban populations of developing

males (average age; 29.03d:8.03, range; 18‑52) in 1994.

countries, data are sparse on their prevalence in rural

All subjects were socially active and believed them‑selves to be healthy. Laboratory tests of serum wereareas, there is little reliable information on the risk of

conducted by a commercial laboratory, BML co. in

STDS especially in the developing countries. This isbecause most studies on the prevalence of STDS havebeen conducted in selected populations, such as in‑patients or outpatients being administered medical

Tokyo. The methods used were reversed passive
hemagglutination (RPHA) for hepatitis B surface
antigen and passive hemagglutination (PHA) for hepati‑
tis B surface antibodies, Glass plate tests, agglutinin,

Table la Subjects testing positive for STDS in 1991

STS
Male
FemaleTPHA FTA‑ABS*
4/5
0/1

8
4

8
4

Table lb

STS TPHA
Male 16

17t HIVt H BsAg
Syphilis
6
3

3
3

1

1

H BsAb

Sub j ects

15
7

59
29

Chlamydia

Sub j ects

22

38

Subjects testing positive for STDS in 1994

FTA‑ABS*

Syphilis'

3/7

14HIVt H BsAg H BsAb
l

3

12

* : Ratio of positive tests to number of people testedt : Serologically diagnosed syphilis:
Either findings:
1 ) STS (+) and TPHA (+)

2 ) STS (+) and TPHA (‑) and FTA‑ABS (+)
t : Afterward there was no remnant serum that was positive for HIV‑2 using ORTHO HIVCHEK

System 3 andLabblot‑2.

Department of Medical Zoology, Tokyo Medical and Dental University School of Medicine. 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo 113,
Japan.

140

Table 2a Number of serologically proven STDS
among subjects tested in 1991: HBV,
syphilis and HIV

of STDS in 1994 than in 1991. This difference may result
from the number of tested cases 30 out of 38 in 1994 andNone Single Double Triple Subjects

ll out of 88 in 1991, respectively, since public healthservices who reduced in the rural villages.

Male
36
Female 1 7

19

3

l

11

1

O

59
29

Table 2b Number of serologically proven STDS
among subjects tested in 1994: HBV,

Treponema pallidum, HIV and
Chlamydia trachomatis

None Single Double Triple Subjects

Maleemployed test methods. We found a higher prevalence

7

12

13

6

38

We could get little informations on STD symptoms
from interviews with the subjects. Thus, the near‑total
absence of reported symptoms in these cases precludes ascreening program for STDS based on symptoms alone.
Vuylsteke et al (Vuylsteke et al.,1993) have report‑
ed a high prevalence of Haemophylus ducreyi antibodies

in the Mozambican population. We did hot have theproper assay system to test for this classic v, enereal
disease.

As mixed infections are common, syphilis andtreponema pallidum hemagglutination (TPHA) andfluorescent treponema antibody absorption (FTA‑
ABS) for syphilis, enzyme‑linked immunosorbent assay(ELISA) (HTLV‑3EIA, Dainabbott) and western blot
(Labblot‑1, Fuji‑Rebio) for HIV‑1, and fluorescentantibody for lgG against Chlamydia trachomatis.
As shown in Table la, in 1991, we found no signifi‑
cant difference between the male and female subjects inincidence of STDs. Nine of the 88 subjects (10.2%)
were serologically diagnosed with syphilis; two (onemale and one female) were HIV carriers; and 28 were
infected with hepatitis B virus (HBV) . As shown in
Table 2a, 35 subjects (39.7%) had at least one of the
STDS we tested for.

In 1994, 14 of 38 subjects were serologicallydiagnosed with syphilis; one was an HIV carrier; 15 were

infected with HBV; and 22 were infected with tra‑chomatis: (Table lb) . A total of 31 (81.6%) either had

STDS or a history of STDS and half of the 38 people
tested in 1994 had a history of two or three different
STDS (Table 2b) .
These findings suggest a high prevalence of STDS in

the general population of Quelimane. According to
Lijestrand et al (Lijestrand et al., 1990) the prevalenceof active syphilis in pregnant women in six of thecountry's 10 provinces ranged from 1.6% (Muiane, com‑chlamydia screening for every STD patient should beinstituted. Mozambique in the 1990's appears to befacing problems associated with syphilis and other
STDS Similar in severity and magnitude to those facedby the western world in the early 1900's (De Schrvyer
and Meheus, 1990). We also need to be concerned aboutother non‑tested STDs.
The low prevalence of HIV infection contrasts withthe prevalence of the other STDs. Only two males andone female (2.4%) were confirmed as HIV‑positive.
Because of the civil war, Mozambique has been relative‑
ly isolated from neighbouring countries such as Malawi,

Tanzania, and Zambia, all of which have a highprevalece of HIV infection. However, the high preva‑
lences of STDS that we found suggests that circum‑
stances in Mozambique are favourable for an explosive
HIV epidemic, especially now that the civil war has
ended and the roads are again open.
In conclusion, because of the very high rates of STD
in Mozambique and the great potential for the spread of

HIV infection, STD control and management must
assume top priority. Case management at the primary
health care level provides an oppotunity to educate
people about their high‑risk behaviour. Although theacquired immune deficiency syndrome, human papil‑
lomavirus infection, and other newly identified STDs,

munity‑based survey) to 9.8% (Maputo, suburban

are attracting much attention in Japan, we must remainviligant for the serious sequelae of syphilis and gonor‑

antenatal care unit) . Possible explanations for the highroea, especially in areas where these classic STDS

prevalence of STDS we found include the collapse ofremain uncontrolled.

health services during the civil war and changing due tothe customs in rural areas.

The incidence of chlamydial infection in Quelimane

was higher than the reported global incidence (DeSchryver and Meheus, 1990). We consider this result tobe due in part to the difference in the test methods

REFERENCESl ) De Schamphelerie, I., Van De Velden, L., and Van Dijck,
E. (1990): Malaies sexuellement transmissibles dans lapopulation feminine a Pikine, Senegal, Ann. Soc, belge.

141Med. Trop., 70, 227‑235

2 ) Lijestrand, J., Bergstrom, S., Nieuwenhuis, F. andHederstedt, B. (1985): Syphilis in pregnant women in
Mozambique, Genitourin. Med., 61, 355‑358

HIV infection in Africa: a review of data published in
1990, AIDS, 5(suppl 1) s7‑sl56 ) Pepin, J., Plurnmer, F., Brunham, R.C., Piot, P., Camer‑
on, D.W. and Ronald, A.R. (1989): The interaction of

HIV and other sexually transmitted diseases: an3 ) Lindstrand, A., Bergstrom, A., Bugalho, A., Zanconato,G., Helgesson, A. and Hederstedt, B. (1993) : Prevalenceof syphilis infection in Mozambican women with second

oppotunity for intervention, AIDS, 3, 3‑97 ) De Schryver A. and Meheus, A. (1990): Epidemiology oftrimester miscarriage and women attending antenatal

sexually transmitted diseases global picture, Bull. World

care in second trimester, Genitourin. Med., 69, 431‑433

4 ) Mosha, F., Nicoll, A., Barongo, L., Borgdorff, M.,

Health Organiz., 68, 639‑654
8 ) Schulz, K., Cates, W. and O'Masra, P.R. (1987) : Preg‑

Newell, J., Senkoro, K., Grosskurth, H., Changalucha, J.,

nancy loss, infant death, and suffrering: Iegacy of syphi‑

Klokke, A., Kellewo, J., Velema, J., Muller, A.S.,
Rugemalila, J. and Hayes, R. (1993) : A population‑based study of syphilis and sexually transmitted dis‑lis and gonorrea in Africa, Genitourin. Med., 62, 320‑3259 ) Vuylsteke, B., Bastos, R., Barreto, J., Crucitti, T., Fol‑gosa, E., Mondlane, J., Dusauchoir, T., Piot, P. and Laga,ease syndromes in North‑Western Tanzania. 1. Per‑

M. (1993): High prevalence of sexually transmitted

valence and incidense, Genitourin. Med., 69, 415‑420

disease in a rural area in Mozarnbique, Genitourin. Med.,

5 ) Nkowane, B.M. (1991): Prevalence and incidense of69, 427‑430

JUNE1995

VOL．23NO．2

CONTENTS
Original article
Supawadee，」．，Suzuki，H．，Suprasert，S．，Rangsiyanond，PりPongprot，B．，Hasegawa，A．，Inouye，S．and Yamazi，Y．
Two−Year

FollowDetection

of

up

from

Rotavims

Birth

in

of

Feces

Thai

and

Childrenfor

Diarrhoeal

Rotavims

Infection：
・・109−113

Symptom…一Shichijo，A．，Mifune，K．，Aono，H．，Shibayama，H．，Terao，H．，Miyata，A．，

Ozawa，H．，Ito，M．，Misumi，J．，Itoga，T．，Norman，M，E，German，A，R．，Bello，M．C．and Sigaran，A．A．H．
Seroepidemiological Studies of Hepatitis Viruses in the Dominican Republic
I．The

PrevalenceofMarkersofHepatitis

A，B

and

C

・・115−120

Viruses…

Sisouk，T．，Kanemura，K．，Saito，M．，Phommasack，B．，Makino，Y．，

Arakaki，S．，Ma，S．，Insisiengmay，S．and Fukmaga，T．
Virological Study on Dengue Epidemic in Vientiane Mmicipality，Lao PDR，1994・
Takaoka，H．and Davies，D．M．
A New Black Fly Species of S加π1伽窺（1▽ω6捌α朋勿）from Sumatra，Indonesia・

一121−125

・・127−131

Oda，T．，Suenaga，0．，Zaitsu，M．，Mori，A，，Kurokawa，K，Fujita，K，Ogawa，Y．，Yamazaki，L，Iida，K，Doi，K，Mine，M．andStudies

on

among

House

Short

AnnualDogs

Changesinfor27years

Kato，K

Microfilarial

PrevalenceinCity，Japan……

Nagasaki

Dづz碗Zα吻加吻お一133−137

communication

Yamada，S．，Pan，Y．，Mori，Y．，Inuo，G．，Tsukidate，S．and

High

of

Prevalenceof

Sexually

Transmitted

Diseasesin

Fujita，K

an

Area

in

Mozambique・

一139−141