JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

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著
ミトコンドリアゲノムに基づく，フィリピン産肺吸虫の系統学的位置
佐藤 雪太，石上 盛敏，岩下淳，阿部 達也，
湯川 眞嘉，Bayani L. Blas，川島健治郎，吾妻
健 …………………………………………１‐３
ミトコンドリア１
２S rRNA 遺伝子領域から推測した，Oncomelania 属及びTricula 属の系統類縁関係
岡本 宗裕，Chin-Tson Lo，Wilfred U. Tiu，Dongchuan Qui，Pinardi Hadidjaja，Suchart Upatham，杉山広，田口 尚弘，平井 啓久，斉藤 康秀，
波部 重久，川中 正憲，平田 瑞城，吾妻
健 ……………………………………………５‐
１
０

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CONTENTS

容
Japanese Journal of Tropical Medicine and Hygiene Vol. 31 No. 1

原

VOL．
３
１ No．
１

平成１
５年３月

Original article
Sato, Y., Iwagami, M., Iwashita, J., Abe, T., Yukawa, M., Blas, B L., Kawashima, K., and Agatsuma, T.
Phylogenetic status of a lung fluke in the Philippines based on mitochondrial genome ……………１‐３
Okamoto, M., Lo, C., Tiu, W. U., Qui, D., Hadidjaja, P., Upatham, S.,
Sugiyama, H., Taguchi, T., Hirai, H., Saitoh, Y., Habe, S., Kawanaka, M., Hirata, M., and Agatsuma, T.
Phylogenetic relationships of snails of the genera Oncomelania and Tricula
inferred from the mitochondrial 12S rRNA gene ……………………………………………………５‐１０
Proceedings of the 43rd Annual Meeting of Japanese Society of Tropical Medicine………………………………………………………………………………………………………１１‐７２

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は４mm
（決）

１

Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
１,２
０
０３, pp. １‐３

Phylogenetic status of a lung fluke in the Philippines based on
mitochondrial genome
YUKITA SATO１, MORITOSHI IWAGAMI２, JUN IWASHITA３, TATSUYA ABE３, MASAYOSHI YUKAWA１,
BAYANI L. BLAS４, KENJIRO KAWASHIMA５ AND TAKESHI AGATSUMA２＊
Accepted 26, February, 2002
Abstract
Based on a near-complete mitochondrial DNA sequence, the phylogenetic status of a lung fluke collected in
the Philippines was evaluated. The lung fluke from Leyte Island, Philippines, resembles Paragonimus westermani
morphologically and is sometimes regarded as a subspecies, P. westermani filipinus. In this study, all mitochondrial
genes of the Leyte form were sequenced: 12 subunits of mitochondrial enzymes, 2 ribosomal RNA genes and 21
transfer RNAs. The gene order is the same as that of the previously-published P. westermani from Korea. All genes
are transcribed in the same direction. The sequence from Leyte was 88.8% identical to the previously-published sequence (accession No. AF219379). This is further evidence that the Philippine form should be regarded as specifically distinct from the form in East Asia (China, Japan and Korea). This conclusion is strengthened by the observation that the molluscan host of the Philippine form is of a different family from that of the East Asian form.
Key words: mt DNA, complete sequence, Paragonimus westermani, P. filipinus, Philippines

INTRODUCTION
The lung fluke, Paragonimus westermani (Kerbert,
1878) is a well known zoonotic agent found in eastern andsouthern Asia and has been the focus of many taxonomic
and biological studies (e.g. Blair et al., 1999). Several previous studies have suggested that some regional populationsof P. westermani should be regarded as distinct species. For
example, Miyazaki (1981) proposed on morphologicalgrounds that the Philippine form should be recognized as a
distinct subspecies, P. westermani filipinus. Allozyme
analysis and partial mitochondrial DNA sequences demonstrated that Philippine isolates are genetically distant fromstrains found in Taiwan, Japan and North East China (Agatsuma et al, 1988; Blair et al, 1997). Previous analyses ofpartial sequences (e.g. Iwagami et al, 2000) used only shortsequence tracts. Comparisons of complete genome sequences can provide more reliable results and also permit
comparisons of features such as gene order and the structureof non-coding regions. In this study, we present and discuss
the complete sequence of the coding region of the mitochondrial genome for the Philippine lung fluke. In the lightof this, we make recommendations concerning its phyloge１

netic status.

MATERIALS AND METHODS
Adult specimens of a Philippine species of Paragonimus used in this study are those previously reported (Agatsuma et al., 1988). DNA extraction of the specimens is as
described previously (Iwagami et al., 2000). Based on the
14,967bp of mitochondrial DNA sequence from P. westermani (Korean triploid form - database accession No. AF
219379), several PCR primer sets were designed and applied for the present study. Amplified PCR products were
purified by gel-electrophoresis and ethanol-precipitation,
ligated into pGEM-T plasmid vector (Promega) and cloned
inserts sequenced using a dye terminator cycle-sequencing
kit (Applied Biosystems) and an automated sequencer (ABI
310, Applied Biosystems). Multiple sequence alignments
were performed using the programs CLUSTAL V and GENETYXMAC.

RESULTS AND DISCUSSION
In total, 14,199bp were amplified and sequenced. As

Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Japan
Department of Environmental Health Science, Kochi Medical School, Japan
３
Laboratory of Molecular Biology, Akita Prefectural University, Japan
４
Schistosomiasis Research and Training Center, Palo, Leyte, the Philippines
５
School of Health Sciences, Kyusyu University, Fukuoka 812-8582, Japan*Corresponding author: agatsuma@med.kochi-ms.ac.jp, Phone/ Fax: +81-88-880-2535２

２

Fig. 1. Circular map of the mitochondrial genome of a lung
fluke of Paragonimus from Leyte, the Philippines.
There are 12 protein-encoding, 2 ribosomal (12S and
16S subunits) and 21 transfer RNA genes. Transfer
RNA genes are described with amino acid one lettercode. An arrowhead indicates putative direction fortranscription. *VNCR: variable non-coding region.

shown in Fig.1, the putative mitochondrial genes included
12 subunits of mitochondrial enzymes (ATP6, CO1〜3,
Cytb, ND1〜6, ND4L), two ribosomal RNA genes (12SrRNA and 16S rRNA) and 21 transfer RNAs (tRNA). As isthe case in other trematodes, serine and leucine are eachspecified by two different tRNAs. No tRNA corresponding
to glutamic acid (Glu: E) was found. The gene order and direction of transcription was determined by comparison withthe previously reported sequences (Le et al., 2000).
Sequence comparisons between Philippine (2n) andKorean (3n) forms are shown in Table1. The overall difference in DNA sequences of protein-coding genes was
12.40%, in tRNA genes 6.10% and in ribosomal RNAs,
8.03%.
The complete mitochondrial genome of P. westermani
is estimated to be about 21Kbp (Agatsuma et al., 1994). Of
the 14,199 bp we sequenced, 13,196 bp coded for genes and1,003 bp consisted of non-coding regions. The portion remaining to be sequenced is therefore about 6,800 bp andmust constitute the long variable non-coding region(VNCR). Complete sequencing of the VNCR has not beenpossible to date, probably because of the presence of many
repeats. This region corresponds to origin of replication of
mitochondrial genomes in many other organisms, attracting
interest as to why the length of the site in Paragonimus
seems to be much longer than in others.

Table 1. Sequence analysis of the mitochondrial genome of a lung fluke from the Philippines, compared to a triploid
type of Paragonimus westermani from Korea*
Protein-coding
regionNumber ofbases

Number of
substitution

Rate ofdifference (%)

ATP6
CO1
CO2
CO3
Cytb
ND1
ND2
ND3
ND4
ND4L
ND5
ND6

513
1,536
600
645
1,119
891
867
357
1,263
258
1,584
453

62
151
62
70
126
114
131
47
180
18
225
65

12.09
9.83
10.33
10.85
11.26
12.79
15.11
13.17
14.25
6.98
14.20
14.35

Total

10,086

1,251

12.40

16S rRNA
12S rRNA

988
744

76
63

7.69
8.47

Total

1,732

139

8.03

ribosomalRNAtRNA

Number ofbases

Number of
substitution

Rate ofdifference (%)

Ala
Arg
Asn
Asp
Cys
Gln
Gly
His
Ile
Leu (CUN)
Leu (UUN)
Lys
Met
Phe
Pro
Ser (AGN)
Ser (UCN)
Thr
Trp
Tyr
Val
Glu

73
72
71
67
64
62
71
62
62
62
65
65
66
70
66
60
65
66
64
62
63
Not found

0
5
1
6
6
1
4
11
1
0
3
3
0
6
10
5
2
3
4
3
10
Not found

0
4.17
0
7.46
9.23
1.61
5.71
12.70
1.61
1.61
4.62
7.58
0
8.57
13.43
8.33
3.08
4.55
6.25
4.84
15.38
Not found

1,378

84

6.10

Total
*Accession No. AF219379

３

As mentioned above, Miyazaki (1981) regarded thePhilippine lung fluke as a subspecies of Paragonimus
westermani, namely P. westermani filipinus. The justification was that one lobe of one or both testes is detached fromthe remainder and that the molluscan host belongs to a different family from that of P. westermani. Blair (2000) reviewed phylogenetic relationships among species andstrains of the genus Paragonimus and showed P. westermani filipinus to be distinct from populations from Japan,
Taiwan and Malaysia. His study used only 393 bp of themitochondrial CO1 gene. Partial CO1 sequences previously
deposited in the public databases showed 88.7% identity between Paragonimus isolates from the Philippines and Korea,
suggesting that Paragonimus in the Philippines can be regarded as a species distinct from P. westermani from East
Asia (China, Japan and Korea). However this suggestion
was thought to be weak because such short sequence tracts
were used. Our results enabled full comparison of all genesof the mitochondrial genome for the first time and found an
overall identity of 88.8% between Philippines and Korean
worms (Table 1).This result resembles that of the partial sequence analysis discussed above. Despite the lack of clear
molecular criteria to determine species-boundaries within agenus such as Paragonimus, we consider that our results,
and the differences in molluscan host specificity, are goodevidence that the Philippine form is specifically distinct.
The name P. filipinus Miyazaki, 1981 should be applied toit.

ACKNOWLEDGEMENTS
We would like to thank Associate professor D. Blair(James Cook University) and Associate professor Joanna D.
Hare (Kochi Medical School) for reviewing the manuscript.
We also would like to thank Dr. S. Habe (Fukuoka University) for collecting materials in this study. This study was

supported in part by research grants (Nos. 60041052 and
61043047) under the International Scientific Research Program from the Ministry of Education, Science, Sports andCulture of Japan.

REFERENCES
１）Agatsuma, T., Habe, S., Kawashima, K. and Blas, B.L.
(1988): Population genetic studies on isozymes inParagonimus westermani from the Philippines. Jpn. J.
Parasitol., 37, 195-202.
２）Agatsuma, T, Yang, L., Kim, D. and Yonekawa, H. (1994):
Mitochondrial DNA differentiation of Japanese diploidand triploid Paragonimus westermani. J. Helminthol., 68,
7-11.
３）Blair, D., Agatsuma, T., Watanobe, T., Okamoto, M. andIto, A. (1997): Geographical genetic structure within thehuman lung fluke, Paragonimus westermani, detectedfrom DNA sequences. Parasitology, 115, 411-417.
４）Blair, D., Xu, Z. B. and Agatsuma, T. (1999): Paragonimiasis and the genus Paragonimus. Adv. Parasitol., 42, 113222.
５）Blair, D. (2000): Genomes of Paragonimus westermani
and related species: Current state of knowledge. Int. J.
Parasitol., 30, 421-426.
６）Iwagami, M., Ho, L.Y., Su, K., Lai, P.F., Fukushima, M.,
Nakano, M., Blair, D., Kawashima, K. and Agatsuma, T.
(2000): Molecular phylogeographic studies on Paragonimus westermani in Asia. J. Helminthol., 74, 315-322.
７）Le, T. H., Blair, D., Agatsuma, T., Humair, P.-F., Campbell,
N.J.H., Iwagami, M., Littlewood, D.T.J., Peacock, B.,
Johnston, D.A., Bartley, J., Rollinson, D., Herniou, E.A.,
Zarlenga, D.S. and McManus, D.P., (2000): Phylogenies
inferred from mitochondrial gene orders - A cautionary
tale from the parasitic flatworms. Mol. Biol. Evol., 17,1123-1125.
８）Miyazaki, I. (1981): Taxonomical studies of the lung fluke
occurring in the Philippines. Med. Bull. Fukuoka Univ., 8,279-282.

５

Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
１,２
０
０３, pp. ５‐１０Phylogenetic relationships
of snails of the genera Oncomelania and Tricula
inferred from the mitochondrial 12S rRNA gene
MUNEHIRO OKAMOTO１, CHIN-TSON LO２, WILFRED U. TIU３, DONGCHUAN QUI４,PINARDI HADIDJAJA５, SUCHART UPATHAM６, HIROMU SUGIYAMA７,
TAKAHIRO TAGUCHI８, HIROHISA HIRAI９, YASUHIDE SAITOH１０,
SHIGEHISA HABE１１, MASANORI KAWANAKA７, MIZUKI HIRATA１２ AND
TAKESHI AGATSUMA１３＊
Accepted 28, February, 2002
Abstract
The Schistosoma japonicum group and S. sinensium utilize intermediate snail hosts belonging to the genera
Oncomelania and Tricula (Gastropoda: Pomatiopsidae). In the present study, partial sequences of the mitochondrial 12S rRNA gene from 7 subspecies of O. hupensis, two species of Tricula (T. bollingi and T. humida) and O.
minima were examined to infer a phylogeny for these. Nucleotide differences among subspecies of O. hupensis
were less than 6.5% and among species from different genera, 10-12%. The phylogenetic tree obtained in thisstudy indicates that O. hupensis subspecies fell into four distinct clades ; that is, O. h. quadrasi from the Philippines, O. h. lindoensis from Indonesia, O. h. hupensis from Yunnan, China and the remaining 5 subspecies (O. h.
hupensis from other parts of China, O. h. robertsoni from China, O. h. formosana from Taiwan, O. h. chiui from
Taiwan and O. h. nosophora from Japan). The phylogenetic tree also showed that O. minima was placed as sisterto all of the subspecies of O. hupensis. Possible evolutionary relationships among the snail hosts were discussed.
Key Words: Oncomelania, Tricula, mitochondrial DNA, 12S rRNA gene, phylogenetic tree

INTRODUCTION
Species of Schistosoma have been placed in a numberof groups based on, amongst other things, egg morphology
and/or geographical distribution (Rollinson and Southgate,1987). For example, African schistosomes of the S. mansoni
and S. haematobium groups develop in pulmonate snails of
the family Planorbidae, while the oriental species, namely
the S. japonicum group (S. japonicum, S. mekongi, S. malayensis) and S. sinensium, utilize snails belonging to thefamily Pomatiopsidae. Pulmonate snails belong to the sub１class Pulmonata, while pomatiopsids belong to the subclass
Caenogastropoda. These two subclasses diverged a longtime ago (Davis, 1980). Generally, specificity for an intermediate host in the genus Schistosoma is high (Rollinson
and Southgate, 1987). So the phylogeny of these intermediate host snails seems to be very important when the evolution of the schistosome is traced.
Schistosoma japonicum is widely distributed throughout East Asia, China and Japan, and the intermediate host is
Oncomelania hupensis. Since O. hupensis shows geographical variation in morphology, many subspecies have

Department of Laboratory Animal Science, School of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan.
Department of Parasitology, National Yang-Ming University, Taipei, Taiwan, R.O.C.
３
Department of Parasitology, College of Public Health, University of Philippines, Manila, Philippines
４
Department of Schistosomiasis, Sichuan Institute of Parasitic Diseases, Chengdu 610041, China
５
Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia
６
Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand
７
Department of Parasitology, National Institute of Infectious diseases, Tokyo, Japan
８
Department of Anatomy, Faculty of Medicine, Kochi Medical School, Nankoku, Kochi 783-8505, Japan
９
Department of Population Genetics, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan
１
０
Department of Parasitology, Faculty of Veterinary Medicine, Azabu University, Kanagawa, Japan
１
１
Department of Parasitology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan１
２
Department of Parasitology, Faculty of Medicine, Kurume University, Kurume, Japan１
３
Department of Environmental Health Science, Faculty of Medicine, Kochi Medical School, Nankoku, Kochi 783-8505, Japan*Corresponding author: T. Agatsuma; Phone/Fax: +81888802535; E-mail: agatsuma@med.kochi-ms.ac.jp
２

６

been described (Davis et al., 1995). Geographical variation
is so great that some researchers prefer to regard the subspecies as independent species (Woodruff et al., 1988; Nihei etal., 1998). In the present study, phylogenetic relationshipsof the snail hosts of S. japonicum, O. hupensis, and otherrelated species of Oncomelania and Tricula were studied
using the mitochondrial 12S rRNA gene.

MATERIALS AND METHODSSnail samples
Sixteen isolates of O. hupensis, belonging to 7 subspecies, were examined. We also examined O. minima, T. bollingi and T. humida. The geographical origins and locationsof species or subspecies used in this study are shown in Table 1 and Fig. 1.
Preparation of DNA
Genomic DNA from each snail sample was extractedusing Easy-DNA Kit (Invitrogen, USA). DNA extracted bythis kit contained an inhibitor for PCR, and this inhibitor
could not be removed by phenol/chloroform extraction orcommercially available spin column etc. So DNA was purified by 0.5% agarose gel electrophoresis with 0.5 X Trisboric EDTA buffer. After electrophoresis, high molecularweight DNA was cut off with agarose gel and extracted by
QIAEX II Gel Extraction Kit (Qiagen, Germany).
Amplification and sequencing of DNA
Purified genomic DNA was used as a template for amplification of DNA fragments by the polymerase chain reacTable 1. Geographical origins of subspecies or speciesof Oncomelania and Tricula examined.
subspecies or species

Location

Country

Oncomelania hupensis hupensis
O. h. hupensis
O. h. hupensis
O. h. hupensis
O. h. robertsoni
O. h. formosana
O. h. formosana
O. h. formosana
O. h. chiui
O. h. nosophoraO. h. nosophoraO. h. lindoensis
O. h. quadrasi
O. h. quadrasi
O. h. quadrasi
O. h. quadrasi
O. minima
Tricula bollingi
T. humida

Anhui
Hunan
Hubei
YunnanSichuanKaohsiung
Shuili
Yilan
ShimenKofu
KurumeSulawesi
MindoroBohor
Asuncion
Digos
Sado
Fang
Sichuan

China
China
China
China
China
TaiwanTaiwanTaiwanTaiwanJapan
Japan
Indonesia
Philippines
Philippines
Philippines
Philippines
Japan
Thailand
China

Fig. 1. Geographical locations of species or subspecies ofOncomelania and Tricula used in this study.

tion (PCR). Amplification of a part of the mitochondrial 12
S rRNA gene was carried out using universal primers (Kocher et al., 1989). The sequences of the primers were as follows: L1091; 5’-AAA AAG CTT CAA ACT GGG ATTAGA TAC CCC ACT AT-3’ and H1478; 5’-TGA CTG CAG
AGG GTG ACG GGC GGT GTG T-3’. PCR was performed using Ampli Taq DNA Polymerase (Perkin Elmer,
USA) according to the manufacturer’s instructions. After an
initial denaturation step (94°
C for 3 minutes) there were 30
cycles of denaturation at 94°
C for 30 sec, annealing at 50°
Cfor 30 sec and extension at 72°
C for 1min. PCR products
were purified with QIAquick-spin PCR purification Kit(Qiagen). Purified double-stranded PCR products were directly sequenced with the same primers as those of PCRfrom both ends using Dye Terminator Cycle Sequencing FS
Ready Reaction Kit and a Model 377A DNA sequencer(Perkin Elmer).
Phylogenetic analyses
DNA sequence data were aligned using the CLUSTAL
W computer program. The evolutionary distances werecomputed by Kimura’s two-parameter method (Kimura,1980), and the phylogenetic tree was constructed by theneighbor-joining method using the neighbor-joining computer program in the PHYLIP 3.5 phylogeny package(Felsenstein, 1993). The tree was evaluated using the bootstrap test based on 1,000 resampling. A sequence from Littorina littorea was used for the outgroup (Rumbak et al.,1994).

RESULTS AND DISCUSSION
Using this primer pair, fragments of 365 to 368 bp
were amplified in this study. Sequences and partial alignments of 12S rRNA gene are shown in Fig. 2. The maxi-

７

８

Fig. 2. Nucleotide sequence alignment of the 12S ribosomal RNA gene in the mitochondrial
DNA in the three genera, Oncomelania, Tricula and Littorina.

Table 2. Pairwise differences in nucleotide sequences of the 12S rRNA gene among subspecies/species of the genus
Oncomelania and Tricula.
species/subspecies
O.h.hupensis (excluding Yunnann)O.h.hupensis (only Yunnan)**O.h.robertsoni
O.h.formosana
O.h.chiuiO.h.nosophora
O.h.lindoensis
O.h.quadrasi
O.minima
T.bollingi
T.humida

O.h.hup. O.h.h.Yun O.h.rob. O.h.for. O.h.chi. O.h.nos. O.h.lin. O.h.qua. O.minima
0.8*
4.2
0.7
1.1
0.0
1.7
3.6
5.1
9.4
10.0
10.7

4.4
3.7
4.6
4.8
6.4
6.4
9.3
10.2
12.1

1.0
0.8
1.6
3.4
5.3
9.6
10.2
11.0

1.3*
1.3
2.0
3.4
5.0
9.3
10.4
10.7

1.8
3.4
5.0
9.3
10.4
10.7

0.7*
4.2
6.1
9.1
9.9
10.5

0.8*
6.2
8.3
9.8
10.8

0.7*
10.5
11.7
12.5

8.8
9.0

T.bollingi

9.9* : values for intra-subspecies**: The Yunnan strain of O.h.h. was listed separately from the other strains, because it differed in a high degree from any other subspecies of O. hupensis.

９

mum levels of nucleotide variations detected between pairsof species or subspecies for the 12S rRNA gene are shownin Table 2. The values are expressed as pairwise differencesin percentage. Nucleotide differences within the subspeciesof O. hupensis were in general very low. However, in thecase of O. h. hupensis, the intra-nucleotide difference wasvery large, because a Yunnan isolate of O. h. hupensis differed from all of the other isolates. On the other hand, differences among five of the subspecies, O. h. hupensis from
China except for Yunnan, O. h. robertsoni, O. h. formosana,O. h. chiui and O. h. nosophora, were less than 2.0%. Nucleotide differences between these 5 subspecies and O. h.
lindoensis, or O. h. quadrasi or the Yunnan isolate of O. h.
hupensis were larger, being about 3 to 6%. Very large values of nucleotide differences (about 9 to 12%) were obtained between different species.
As shown in Fig. 3, a phylogenetic tree of the snail
hosts was constructed using the neighbor-joining method.
Oncomelania hupensis subspecies are distributed among 4groups. That is, four specimens of O. h. quadrasi from Philippines form a monophyletic clade. Oncomelania h. nosophora, O. h. hupensis, O. h. robertsoni, O. h. chiui and O.
h. formosana form one group. Oncomelania h. lindoensis
and O. h. hupensis from Yunnan make independent clades.
In the meantime, O. minima, T. bollingi and T. humida also
make independent clades, and they are genetically distantfrom each other as well as from all the subspecies of O. h.
hupensis.
The sequence of mitochondrial 12S rRNA gene has
been utilized to infer the phylogeny of various animals (Ko-cher et al., 1989). Rumbak et al. (1994) examined the phylogenetic relationships among 11 species in the genus Littorina, which is a widely distributed marine gastropod, belonging to the same suborder Archaeotaenioglossa as thegenus Oncomelania. In the case of the genus Littorina, nucleotide differences between European species and American species were about 3%. And differences between subgenera in the genus Littorina were about 5% or more. In thepresent study, O. hupensis were distributed among 4 groups,
and nucleotide differences among these 4 groups were 3 to
6%. Although there is no reason to assume that rates of molecular evolution have been the same in Littorina as in thepomatiopsids, comparisons of the percentage differences
between the two studies suggest that the subspecies in O.
hupensis may require re-evaluation as suggested byWoodruff et al. (1988). Despite the wide geographic area
involved, the genetic differences between the subspecies inJapan, Taiwan and China were very small. All of the examined snails in China were collected from the Yangtze basin
except for that of Yunnan Province. In the glacial maxima,
only approximately twenty thousand years ago (Wang andSun, 1994), sea levels were much lower than now, themouth of the Yangtze was considerably closer to Japan andTaiwan, and the Taiwan channel was dry land. This could
explain the high levels of similarity among Japanese, Taiwanese and Yangtse basin samples.
It has been reported that genetic variation among S. japonicum populations in Asia, including China, the Philippines, Japan and Indonesia, is very slight (Bowles et al,1993). However, we have shown that genetic variation
among their intermediate host snails is quite considerable.
Thus, our study supports the idea that S. japonicum has
been recently introduced to many areas where it now occurs,
and has been able to adapt to local strains of Oncomelania,
as suggested in previous papers (Woodruff, 1988; Davis,
1992; Attwood et al., 2002).
Davis (1980) suggested that S. sinensium may constitute a species complex, because of geographical differencesin snail host specificity. Our previous studies showed a large
difference in egg sizes as well as in nucleotide sequence of
CO1 between the two isolates of S. sinensium from China
and Thailand, supporting Davis’s hypothesis (Kawanaka etal., 1998; Agatsuma et al., 2000). The present result of alarge distance value between their intermediate hosts may
suggest coevolution between the hosts and parasites.

ACKNOWLEDGEMENTS
Fig. 3. A phylogenetic tree of the genus Oncomelania andTricula, including the snail intermediate hosts ofSchistosoma japonicum, inferred from 12S rRNA genein the mitochondrial DNA using the NJ method.

We would like to thank Associate Professor David
Blair, James Cook University, and Associate Professor
Joanna D. Hare, Kochi Medical School, for reviewing the１０

manuscript. This study was partly supported by a Grant-inAid for Overseas Scientific Research (T. Agatsuma:
05041098) from the Japanese Ministry of Education (1993).

REFERENCES
１）Agatsuma, T., Iwagami, M., Liu, C.X., Saitoh, Y.,
Kawanaka, M., Upatham, S., Qui, D. and Higuchi, T.
(2001): Molecular phylogenetic position of S. sinensium
in the genus Schistosoma. J. Helminthol., 75, 215-221.
２）Attwood, S.W., Upatham, E.S., Meng, X.H., Qiu, D.C.
and Southgate, V.R. (2002): The phylogeography of
Asian Schistosoma (Trematoda: Schistosomatidae). Parasitology, 125, 99-112.
３）Bowles, J., Hope, M., Tiu, W.U., Liu, X. and McManus,
D.P. (1993): Nuclear and mitochondrial genetic markers
highly conserved Chinese and Philippine Schistosoma japonicum. Acta Trop., 55, 217-229.
４）Davis, G.M. (1980): Snail hosts of Asian Schistosoma infecting man: origin and coevolution. Malacol. Rev.
Suppl., 2, 195-238.
５）Davis, G.M. (1992): Evolution of prosobranch snails
transmitting Asian Schistosoma; coevolution with Schistosoma: a review. Progr. Clinical Parasitol., 3, 145-204.
６）Davis, G.M., Yi, Z., Hua, G.Y. and Spolsky, C. (1995):
Population genetics and systematic status of Oncomelania hupensis (Gastropoda: Pomatiopsidae) throughout
China. Malacologia, 37: 133-156.
７）Felsenstein, J. (1993) PHYLIP. Version3.5.1, Universityof Washington, Seattle.
８）Kawanaka, M., Sugiyama, H., Saito, Y., Habe, S.,
Upatham, E.S., Qui, D., Taguchi, N. and Agatsuma, T.
(1998): Morphometric and molecular genetic comparisons of two geographic strains of Schistosoma sinensium
isolated from Sichuan Province, China and North Thai-land. Proceedings of the 9th International Congress ofParasitology, Chiba, Japan. Monduzzi Internat., Bologna,649-653.
９）Kimura, M. (1980): A simple method for estimating evolutionary rate of base substitutions through comparative
studies of nucleotide sequences. J. Mol. Evol.16, 111-120.
１０）Kocher, T.D., Thomas, W.K., Meyer A., Edwars, S.V.,。。
Paabo, S., Villablanca, F.X. and Wilson, A.C. (1989): Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc.
Natl. Acad. Sci. USA, 86, 6196-6200.
１１）Nihei, N., Kanazawa, T, Blas, B.L., Saitoh, Y., Itagaki, H.,
Pangilinan, R., Matsuda, H. and Yasuraoka, K. (1998):
Soil factors influencing the distribution of Oncomelania
quadrasi, the intermediate host of Schistosoma japonicum, on Bohol Island, Philippines. Ann. Trop. Med.
Parasitol., 92, 699-710.
１２）Rollinson, D. and Southgate, V.R. (1987): The genus
Schistosoma: A taxonomic appraisal. pp1-49. In: Rollinson, D. and Simpson, A.J.G. (eds). The biology of schistosomes from genes to latrines. Academic Press, London,
UK.
１３）Rumbak, E., Reid, D.G. and Thomas, R.H. (1994): Reconstruction of phylogeny of 11 species of Littorina(Gastropoda: Littorinidae) using mitochondrial DNA sequence data. Nautilus Suppl., 2, 91-97.
１４）Wang, P. and Sun, X. (1994): Last glacial maximum in
China: comparison between land and sea. Catena, 23,341-353.
１５）Woodruff, D., Staub, K.C., Upatham, E.S., Viyant, V. and
Yuan, H.C. (1988) Genetic variation in Oncomelania
hupensis: Schistosoma japonicum transmitting snails in
China and the Philippines are distinct species. Malacologia, 29, 347-361.

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Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
１,２
０
０３, pp.１
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７
２

PROCEEDINGS OF THE 43RD ANNUAL MEETING OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
21-22 November 2002, Kochi

President
Yoshihisa Hashiguchi
Professor, Department of Parasitology
Kochi Medical School
CONTENTS
Prize winner’s lecture
JSTM (Japanese Society of Tropical Medicine) Young Investigator Award
The malaria vector in the area surrounded by rice field in Khammaoune Province, Lao PDR withthe discussion of the future ITM strategy in Laos and also Mekong

Kobayashi, J.

President’s lecture
Tropical diseases research and the endemic areas--- A brief review, based on the research/control ofleishmaniasis in South and Central Americas

Hashiguchi, Y.

Special lectures!． The mucosal immune system for the development of new generation vaccine"． Molecular strategies for disease control--- research trials in vector biology--Educational lecture
The great names of history in tropical medicine

Kiyono, H.
Chinzei, Y.

Kobayashi, T.

Symposium
Tropical medicine and emerging/ re-emerging infectious diseases
Ｓ‐!‐
１ Current status of emerging protozoal infectious diseases: cryptosporidiosis, cyclosporiasis, and microsporidiosis
Iseki, M.
Ｓ‐!‐
２ Cryptosporidiosis and cyclosporiasis: the clinical features and occurrence in Japan
Masuda, G.
Ｓ‐!‐
３ Emerging and re-emerging arboviral infectious diseases
Takasaki, T. and Kurane, I.
Ｓ‐!‐
４ Current status of emerging and re-emerging bacterial infectious diseases
Nagatake, T.
Leishmaniasis: from bench to fieldＳ‐"‐
１ Detection of new endemic areas of cutaneous leishmaniasis in Pakistan: a six-year study
Bhutto, A. M. et al.
Ｓ‐"‐
２ Current status of leishmaniasis in Ecuador
Calvopina, M. and Hashiguchi, Y.
Ｓ‐"‐
３ Evolution of the cytochrome b gene of Leishmania and Endotrypanum
Uezato, H. et al.
Ｓ‐"‐
４ Advance in development of anti-leishmanial drugs and research on mechanisms of drug resistance in Leishmania
Katakura, K. et al.
For the fruits of tropical medecine
Ｓ‐#‐
０ For the fruits of tropical medicine
Hamano, S.
Ｓ‐#‐
１ Synthesis of useful antibody molecules using the combinatorial library and the phage display technologies
Yamashiro, T.
Ｓ‐#‐
２ Molecular mechanism of malarial sporozoites infection into the mammalian liver
Ishino, T. et al.
Ｓ‐#‐
３ Involving tropical medicine practicallyAwazawa, T.
Ｓ‐#‐
４ What is the tropical medicine in Japan? --- A question from a young scientist
Kaneko, O.

１２

Hansen’s diseaseＳ‐$‐
１ Adequate treatment and early diagnosis of leprosy
Ｓ‐$‐
２ Around the fundamental leprosy researchＳ‐$‐
３ Present situation of Hansen’s disease in the world
Ｓ‐$‐
４ Present situation of leprosy diseases in Myanmar
Ｓ‐$‐
５ Activities of JICA project for leprosy control and basic health services in Myanmar

Hosokawa, A. et al.
Sasaki, S.
Ishii, N.
Soe, T. A.
Nagao, E.

Tropical medicine in the scope of international contribution
Ｓ‐%‐
１ Role of the JICA Zambia project in AIDS control in Southern Africa
Terunuma, H.
Ｓ‐%‐
２ Tuberculosis control in Nepal and Japanese medical cooperation
Osuga, K.
Ｓ‐%‐
３ Problems of implementing the malaria control project
Kanbara, H.
Ｓ‐%‐
４ Hashimoto initiative and Okinawa infectious disease initiative: Japan’s challenge to global parasite control
Takeuchi, T.
Ｓ‐%‐
５ The progress and the direction of the Asian Center of International Parasite Control Project
Nagai, N. et al.
Ｓ‐%‐
６ Roles and challenges of Japan’s ODA for global health
Kunii, O.
Looking for ideal fieldworks in tropical medicine
Ｓ‐&‐
１ Difficulties encountered in field researches which were used as excusesＳ‐&‐
２ From Voodoo epidemiology to reliable epidemiology
Ｓ‐&‐
３ Filariasis control: how were blood collection and mass drug treatment done?
Ｓ‐&‐
４ Bioethics and biomedical research in developing countries: What should researchers do fromthe viewpoint of bioethics?

Shimada, M.
Yoshimura, T.
Kimura, E.
Kaneko, S.

WorkshopＷ‐!‐
１ Preventive behavior and morbidity of Japanese tourists for Africa
Kikuchi, H. et al.
Ｗ‐!‐
２ Knowledge and preparation regarding the risk of malaria among Japanese international travelers
Kawakami, K. et al.
Ｗ‐!‐
３ Disease trends among Japanese people living in Sortheast Asia
Uchikoshi, A. et al.
Ｗ‐!‐
４ The storage and supply system of orphan drugs for tropical and parasitic diseases
Kimura, M. et al.
Ｗ‐"‐
１ Molecular analysis of α-thalassemia in Nepal: Correlation with malaria endemicity. (")
Hamano, S. et al.
Ｗ‐"‐
２ Study on DNA polymorphism in the DHFR and DHPS genes of Plasmodium falciparum in southern Laos
Imada, Y. et al.
Ｗ‐"‐
３ Molecular analysis of chloroquine resistant related gene in Malawi
Mita, T. et al.
Ｗ‐"‐
４ Generation of transgenic anopheline mosquitoes expressing a gene encoding anti-malaria molecule
Yoshida, S. et al.
Ｗ‐#‐
１ Intranasal inoculation of yeast-produced malaria ookinate surface proteins co-administered with cholera
toxin induces complete transmission-blocking immunity against a rodent and a human malaria parasite
Kishimoto, A.et al.
Ｗ‐#‐
２ High expression of cholera toxin B subunit pentamer in methanophilic yeast Pichia pastoris andits oral immunogenicity in mice
Arakawa, T. et al.
Ｗ‐#‐
３ Expressions of cytokines and monosaccharide transporters in the duodenal mucosa ofgastro-intestinal symptomatic patients in the rural area of Thailand
Lertanekawattana, S. et al.
Ｗ‐#‐
４ Study of Ascaris and ulcerative colitis---Genetic analysis of cross-reactive antigenicity between
Ascaris lumbricoides homeinis antigen and human colon mucosa
Hayashi, I. et al.
Ｗ‐$‐
１ Fusion DNA vaccine against infection with high virulent Toxoplasma
Ishii, K. et al.
Ｗ‐$‐
２ An interleukin-18 expression plasmid administration initiates a protective T helper type 1 immune
response and resolves cutaneous leishmaniasis
Li, Y. et al.
Ｗ‐$‐
３ Protectve role of IgE in mice infected with Plasmodium berghei ANKA
Furuta, T. et al.
Ｗ‐$‐
４ The role of erythrocytes on suppression of immune response inmurine malaria
Tokunaga, M. et al.
Ｗ‐%‐
１ Application of questionnaires for monitoring Schistosoma mansoni infection after mass-treatment
Kisu, T. et al.
Ｗ‐%‐
２ Rapid school health quetionnaire surveys on urinary schistosomiasis in coastal Kenya and Zanzibar Moji, K. et al.

１
３
Ｗ‐"‐
３ Water contact behaviors among schoolchildren in an endemic area of schistosomiasis, Tanzania:
Ｗ‐"‐
４
Ｗ‐#‐
１
Ｗ‐#‐
２
Ｗ‐#‐
３
Ｗ‐#‐
４
Ｗ‐$‐
１
Ｗ‐$‐
２
Ｗ‐$‐
３
Ｗ‐$‐
４

A comparative study of direct observation and 24-hour recall method
Ataka, Y. et al.
School-based and residence-based analyses on infections with S. haematobium and S. mansoni
among children in Moshi, Tanzania
Kaneda, E. et al.
Characterization of the cDNA of the miracidial antigen family of Schistosoma japonicum(Chinese mainland strain)
Yu, C. et al.
Molecular cloning of a novel procyclic form-specific gene (cDNA) from Trypanosoma brucei brucei
Ohshima, S. et al.
Analysis of Leishmania amastigote stage-specific molecules
Sanjoba, C. et al.
The quantification of the genes involved NO production in Leishmania-infected macrophage by real-time PCR
Yamamoto, Y. et al.
Chemotherapeutic approach to African tripanosomiasis: The use of diorganotin and its derivatives
Shiuaibu, N. M., et al.
The induction of TNF-α and iNOS expression by macrophage cells against ONO-4007(a synthetic lipid A derivative), IFN-γ and meglumine antimoniate in vitro study
Khan, M. A. K., et al.
Marine sponges; hopeful sources of new anti-protozoan drugs
Goto, Y. et al.
Combination effects of chloroquine with febrifugine/isofebrifugine mixture against chloroquine-resistantPlasmodium berghei NK65 in ICR mice
Ishii, A. et al.

General presentation
Ｂ‐
０
１ Clinical features of mucocutaneous leishmaniasis in the Amazonian region of Ecuador
Calvopina, M., Hashiguchi, Y.
Ｂ‐
０
２ Malaria epidemiology in Lombok Island, Indonesia
Kanbara, H. et al.
Ｂ‐
０
３ Babesia sp. And other hemoparasites identified in wild rats in Chiang Mai and Mae Hong Son Province, Thailand
Dantrakool, A. et al.
Ｂ‐０
４ Adult black flies attracted to humans and water buffaloes, and natural infections with Onchocerca
larvae in Chiang Mai, in Northern Thailand
Takaoka, H. et al.
Ｂ‐
０
５ Seasonal changes of wild anopheline mosquitoes around Jichi Medical School
Matsuoka, H. et al.
Ｂ‐０
６ A novel GT-mismatch binding protein possibly involved in base excision repair of thermally induced mutation
Yahiro, M. et al.
Ｂ‐０
７ Mitochondrial quinol-fumarate reductase (complex !) from Ascaris suum as a target for chemotherapy
Osanai A. et al.
Ｂ‐
０
８ Chemotactic factors for leukocytes from eggs of Schistosoma mekongi
Owhashi, M. et al.
Ｂ‐０
９ Characterization of recombinant fragments of the 150-kDa lectin (IGL) of Entamoeba histolytica
Tachibana, H. et al.
Ｂ‐
１
０ Involvement of proteasome function in Entamoeba growth and defferentiation
Makioka, A. et al.
Ｂ‐
１
１ Molecular cloning and characterization of farnesyltransferase of Entamoeba histolytica
Makioka, A. et al.
Ｂ‐
１
２ Recent situation of dengue fever and dengue hemorrhagic fever in the Philippins (1995-2001)
Matias, R. R. et al.
Ｂ‐
１
３ A mechanism of thrombocytopenia during the acute phase of dengue virus infection:
Involvement of platelet-associated IgG and anti-dengue virus IgG
Oishi, K. et al.
Ｂ‐
１
４ Evaluation of a hepatitis B vaccination program in Chiang Mai, Thailand
Toriyama, K. et al.
Ｂ‐
１
５ Acute respiratory infections (ARI) among children in Vietnam: Detemination of bacterial
etiology and anitibiotic resistance of pathogens
Rusizoka O. S. et al.
Ｂ‐
１
６ A trial of nosocomial infection control in the Bach Mai Hospital Project for functional enhancement in Vietnam
Ohara, H., Hung, N. V.
Ｂ‐
１
７ Experimental infection into SCID mice with microsporidia isolated from AIDS patient
Juarez S. I. et al.
Ｃ‐
０
１ Analysis of antigen-specific IgG subclasses in serum and urine samples from bancroftian filariasis patients
Qiu, X. -G. et al.
Ｃ‐
０
２ Direct agglutination test (DAT) with urine samples for the diagnosis of visceral leishmaniasis
Islam, M. Z. et al.
Ｃ‐
０
３ Detection of Plasmodium berghei and P. gallinaceum by ICT P. f./ P. V. immunochromatographic test
Arai, M. et al.

１
４
Ｃ‐
０
４ Anti-SERA antibody production as a parameter of anti-malaria immunity in individuals in the Solomon Islands
Ｃ‐
０
５ Serodiagnosis of tuberculosis using MPB64 derived from Mycobacterium bovisＣ‐０
６ A method of blood examinations in overseas mobile clinic--- its clinical application toHato, M. et al.
Yoshidome, A. et al.

the remote areas in Lao PDR

Ishida, T. et al.

Ｃ‐
０
７ Comparison of ultrasonographic images between Schistosoma japonicum and S. mekongi infection

Ohmae, H. et al.
Ｃ‐
０
８ Comparison of ultrasonographic images of liver and spleen by age group and the relationship

Ｃ‐
０
９
Ｃ‐
１
０
Ｃ‐
１
１
Ｃ‐
１
２
Ｃ‐
１
３
Ｃ‐
１
４
Ｃ‐
１
５
Ｃ‐
１
６
Ｃ‐
１
７
Ｃ‐
１
８

between frequency of praziquantel treatment and ultrasonographic images of patients sufferingfrom Schistosoma japonicum and S. mekongi infection in the Philippines and in Cambodia
Chigusa, Y. et al.
The influence of Schistosoma haematobium infection on voiding function
Watanabe, K. et al.
Epidemiological survellance on schistosomiasis in mountainous regions in Sichuan
Chuang, C. -K. et al.
Parasitological survey and related health awareness investigation by questionnaire at a village
in the central region in Ghana
Osada, Y. et al.
Damage in cercariae of Schistosoma mansoni induced by pressure jar in water
Ohwatari, N. et al.
Comparison of intestinal parasitic infections and malnutrition in schoolchildren in urban andrural areas in Hanoi, Vietnam
Fujimaki, Y. et al.
Detection of microorganisms in tap water in Indonesia and Thailand
Uga, S. et al.
Epidemiological analysis of enterohemorrhagic Escherichia coli infection in Indonesia
Ichinose, Y. et al.
The problem on malaria treatment--- About the delay of diagnosis and treatment
Okumura, E. et al.
A severe case of Plasmodium falciparum infection complicated with black water fever
Koyama, K. et al.
Correlation between the recognition by obstetricians about congenital toxoplasmosis and their clinical activities
Naoi, K. et al.

１
５

Prize winner’s lecture
THE MALARIA VECTOR IN THE AREA SURROUNDED BY RICE FIELD IN KHAMMAOUNE PROVINCE.
LAO PDR WITH THE DISCUSSION OF THE FUTURE ITM STRATEGY IN LAOS AND ALSO MEKONG AREA
JUN KOBAYASHI
Bureau of International Cooperation, International Medical Center of Japan

In the 1996, the malaria vector in Laos was not clear.
It was suspected that the same vector in the North Thailand;
Anopheles minimus, An. maculatus and An. dirus, were existed. In Nhommarath District in Khammouane Province,
Lao PDR, a lot of malaria patients were reported in the district hospital located in the center of basin. It was supposedthat patient came from the communities around hospital under the consideration of accessibility to hospital, thus theone community around hospital was selected for malaria
survey. The prevalence of malaria infection in the inhabitant is high as more than 20%, however, only few numberof An. minimus was collected, and An. maculates and An.
dirus was not collected. On the other hand, An. nivipes is
the majority in Anoheles, which was collected by humanbait collection, and the larva of this mosquito was collectedfrom the rice field. Thus, An. nivipes is suspected malaria
vector in this basin surrounded by rice field. At present
time, the distribution of the malaria vector in Lao PDR isestimated according to the results of a lot of surveys. It is
supposed that the existence of An. dirus influence to thehigh endemic of malaria in the southern provinces. Moreover the biting habit of An. dirus in this area is one of thecauses that an impregnated bed net was affected to control
malaria.

The vector control using by impregnated bed net (IBN)
is one component of the recent global malaria control strategy. It is well known that the health education is important
not only to diffuse IBN but also to sustain the project. In the
beginning of 2002, the review of Impregnated Treatment
Materials (ITM) strategy was carried out in Roll Back Malaria Initiative in Africa. The new strategy consists of bothsides between public and private sector in addition to social
marketing. In the Mekong region, the ITM programs werecarried out using by mainly public sector only, because theperipheral health administration system was well established in comparison with African countries. The other reason, that social marketing was not conducted in Mekong
countries, was supposed that the rural areas without suitable
media and market were the serious endemic of malaria.
When the project is being carried out in the wide area fromnow on, however, the only public sector is supposed not tobe able to sustain the project. In fact, ITM project was expanded to whole country in Lao PDR, the re-dipping of
IBN was not carried out in several communities. It is recommended that the joining of private sectors becomes important to sustain the project from now on in Mekong countries.

１
６

President’s lecture
TROPICAL DISEASES RESEARCH AND THE ENDEMIC AREAS -A BRIEF REVIEW, BASEDON THE RESEARCH/CONTROL OF LEISHMANIASIS IN SOUTH AND CENTRAL AMERICASYOSHIHISA HASHIGUCHI
Department of Parasitology, Kochi Medical School, Nankoku, Kochi, Japan

During about 20 years from 1982 to date, we made anintensive investigation on leishmaniasis in Sounth and Central Americas, especially in Ecuador. The research project
was supported at the early phase by JICA, and then supported by the Ministry of Education, Science, Culture andSports, Japan. Such a long term financial support has beenplaying the role of driving force to disclose the bio-medical
features of New World leishmaniasis. In the project, an intensive study has been done, in order to get information on
the disease and its transmission, from multidisciplinary
points of view. The aim of project at the early phase was toconsider a suitable control measure, by disclosing transmission mechanisms of the disease in each endemic area of
Ecuador. By performing country-wide epidemiological surveys, factors relating to the transmission of leishmaniasis,such as the causative agents (6 Leishmania spp.); L.(L.)
mexicana, L.(L.) amazonensis, L.(L.) major-like, L.(V.) braziliensis, L.(V.) panamensis, L.(V.) guyanensis, the vector
sandflies (4 Lutzomyia spp.); Lu. hartmanni, Lu. trapidoi,

Lu. gomezi and Lu. ayacuchensis, and the reservoir hosts (8spp. of mammals); anteaters, sloths (2 spp.), squirrels (2spp.), kinkajous, rats and dogs, have been known at different endemic areas of Ecuador. L.(V.) equatorensis from arborial mammals (sloth and squirrel) was described as a new
species, with no human cases. Andean type of leishmaniasis
was reported for the first time in Ecuador; the disease form
is very similar to Peruvian uta, but the causative agents andvector sandflies are completely different. We tried to searchfor suitable drugs and treatments of the disease, which
would be effective and easily applicable at field conditions;several ointments, lotions and oral drugs such as antimalarials were found to be effective and useful. Furthermore, comparative study was made to have a suitable sampling method applicable for molecular techniques, such as
PCR diagnosis. Besides, in the talk several experienceswere mentioned, demonstrating cases we face to the problems at field surveys, taking blood samples, skin-testing and
etc.

１
７

Special lecture
I THE MUCOSAL IMMUNE SYSTEM FOR THE DEVELOPMENT OF NEW GENERATION VACCINEHIROSHI KIYONO, D.D.S., Ph.D.1,2
1

Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science,
The University of Tokyo
2
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University

The host immune system is equipped with two arms of
immmunity at mucosal and systemic compartments. The
basic foundation of the immune system was establised according to the extensive molecular and cellular characterization of lymphocytes and their associated regulatory molecules in the systemic compartment. To this end, thymus,
bone marrow, spleen and peripheral lymph nodes arethought to be the immunological nest of the systemic immune system. In addition to the systemic immunity, thehost is equipped with the mucosal immune system providing a first line of defense against numerous foreign antigens
which invade the respiratory, intestinal and reproductive
tracts. The mucosal immune system has a difficult task: it
has to protect approximately 400 m2 of mucosal surface
area and it must differentiate between beneficial and unwanted antigens at the site of entry.
For providing a first line of defence at a such large mucosal surface, serectory IgA (S-IgA) is a major antibody.
For the induction of antigen-specific S-IgA immune response at mucosal surfaces, a unique concept of the common mucosal immune system (CMIS) need to be considered. The CMIS consists of inductive sites (e.g., nasopharyngeal associated lymphoreticular tissue [NALT] and gut
associated lymphoreticular tissue [GALT]) and effector sites(e.g., lamina propria and epithelium of the respiratory, intestinal and reproductive tissues). To this end, nasal andoral immunizations have been shown to be effective for the
induction of antigen-specific S-IgA response in the distantmucosal effector compartment. Further, mucosal immunization can induce antigen-specific immune responses in thesystemic compartment. Thus, the estabrishment of two layers of the protective immunity can be accomplished by oral
or nasal vaccine.
NALT is considered to be an important inductive site
for the induction of antigen-specific mucosal and systemic
immunity against nasally-administered vaccine antigen. In

contrast to Peyer’s patch (PP), an example of GALT, cytokine and corresponding receptor regulated molecular machinery for the tissue organogenesis of NALT remains to beelucidated. Based on the result obtained by the histologicaland growth kinetic study of normal and specific gene manipulated mice (e.g., IL-7R-/- and LTα-/-), it was shown that
NALT organogenesis is independently operated from agroup of known cytokine signaling pathway via IL-7R andLTβR for the generation of secondary lymphoid tissue (e.g.,PP).
Our recent investigations have provided new observations that mucosal IgA antibody can be developed in a
CMIS-independent manner. IgA committed B-1 cells expressing IL-5R and IL-15R are responsible for the generation of CMIS-independent IgA, while B-2 cells responding
to IL-5 and IL-6 are the major source for CMIS-dependent
IgA. Taken together, these findings suggested an interestingand new possibility that the mucosal surface in protected by
S-IgA generated from the two distinct sources of mucosal B
cells.
According to these unique features of the mucosal immune system, our current effort is also focused on the development of novel antigen delivery veihicle for the induction of antigen-specific IgA response. Our recent findings
demonstrated that Fusogenic-liposome can effectively deliver antigen to NALT via M cells and epithelial cells. Nasal immunization with Fusogenic-liposome containing vaccie antigen can effectively induce antigen-specific mucosaland systemic immunity. In addition, nasal immunization
with rBCG containg vaccine antigen resulted in the induction of prolonged memory type antibody responses. These
new findings will extend the understanding of unique feature of the mucosal immune system for the development ofmucosal vaccine for the prevention and control of infectious
diseases.

１
８

II MOLECULAR STRATEGIES FOR DISEASE CONTROL---- RESEARCH TRIALS IN VECTOR BIOLOGY ---YASUO CHINZEI
Department of Medical Zoology, School of Medicine Mie University

Vector borne diseases (VBD) are very important intropical diseases. The pathological agents of VBD include
wide range of organisms such as virus, rickettsia, bacteria,
spirochete, protozoa and nematode. Vectors are blood sucking insects such as mosquito, black fly, tsetse fly, sand fly
kissing bug and so on, and also ticks and mites. It is difficult to control or to eliminate VBD because the disease are
depending on very complicated backgrounds and related
deeply to the environments. Vector species and number are
numerous and area specific, and easy to develop insecticide
resistant.
In the last 30 years of 20th century, molecular biology,
molecular genetics and molecular technology have developed very much. Based on these development, some trials
to establish strategies for VBD control by molecular technique have been performed. One of the trials is to produce
a vector which cannot transmit pathogen by genetic transformation. Technology for producing a transgenic mosquito was established first by transforming EGFP gene byusing minos as transposon (Crisanti et al 2000). After sometrials, a transgenic mosquito, Anopheles stephensi, whichcannot transmit malarial parasite Plasmodium berghei wasproduced by transforming a synthetic gene of SM-1 (sali-vary gland / midgut adhesive molecule) (Jacobs-Lorena etal., 2002).
The genome of a malarial vector mosquito Anopheles
gambiae was published very recently (in Nature, October2002). So we can find some more candidate genes to transform a vector mosquito, for instance, malarial resistantgenes against malarial parasite, and can produce more mosquitoes not to transmit malaria. However, before releasingtransgenic mosquitoes we have to develop our technology
to replace the natural population to the transgenic populations of mosquitoes and assess the effects to the natural ecosystem.
In the fruit fly Drosophila, female-specific lethal transgenic insects were developed using a sex specific promoter
to drive the expression of a repressible transcription factor,
which in turn controls the expression of toxic gene product.
This principle is applicable to medically important dipteraninsect mosquitoes. Using this strategies insect population ismore easily controlled than the sterile insect technique.
Some more example trials for developing strategies tocontrol vector borne tropical diseases by tstudies of molecular and genetic biology of vectors were presented in the lecture.

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Educational lecture
THE GREAT NAMES OF HISTORY IN TROPICAL MEDICINETERUYUKI KOBAYASHI
Non-fiction writer

In the present talk, many great names of history inJapanese Tropical Medicine were mentioned. They were
Drs. Yoshio Sawai, Manabu Sasa, Kanzen Teruya, HachiroSato, Yoshito Otsuji and others who worked with tropical
medicine-related health problems/diseases such as, snake(Habu) bite, Tsutsugamushi disease, malaria, filariasis,
schistosomiasis, paragonimiasis and other infectious orparasitic diseases. About 40 years ago, all these diseases
were highly prevalent at country-wide in Japan and they
were great public health problems, because of poor sanitaryconditions and insufficient political atention. For example,
snake (Habu) bite was one of the most serious health problem in subtropical regions of Japan, especially in Okinawa
and Amami-Oshima islands. During 13 years from 1959 to
1971, 3,510 snake bite cases were reported from AmamiOshima island; the average fatality rate was 0.8% in thearea. Dr. Yoshio Sawai, a microbiologist at the Institute ofMedical Science at the University of Tokyo was the person
who hardly worked with the Habu bite. In 1952 he was appointed as Associate Professor and chief of the newlyestablished laboratory of biological products at the Institute.
Antivenin productions including these for Habu venom was
one of the traditional work in the institute. Naturally Dr.
Sawai tried to develop more effective and useful Habu
toxoid. He frequently visited Naze Health Center, AmamiOshima island and observed there many patients sufferedfrom serious and aggressive Habu bites with severe necrosis
at the site of snake bite. He started laboratory investigations
to improve the treatment of Habu bite, together with his coworkers at the Institute. Their purified and lyophilized anti-

venin was favorable to decrease numbers of the severe casesof Habu bites when applied clinically at Amami-Oshima island. However, yearly occurrence of severe clinical cases
were still found in the area, in spite of such a Dr. Sawai’s
untiring efforts. After analyzing many clinical cases ofHabu bites, Dr. Sawai reached to the conclusion that there
might be a limitation in their serum treatment against thesnake bite, because the development of local lesions by thelarge amount of Habu venom was too rapid for the antivenin treatment to prevent. Then, in 1963, Dr. Sawai reported a dihydrothioctic acid mixed with different concentrations of the Habu venom; the preparation inhibited lethal
effect of venom when tested n experimental animals such as
mice, rabbits and guinea pigs. In April, 1965, the first large
scale trial of active immunization using this product wasundertaken in persons living at remote areas with high-riskof Habu bites. In Amami-Oshima and Okinawa islands,
43,446 persons received the Habu venom toxoid during 3
years of active immunization from 1965 to 1967. Statistic
analysis of these trials showed that the venom towoid used
was effective, enough to reduce the rate of necrotic cases by
50%. The improvement of the Habu toxoid developed by
Dr. Sawai’s group has been promoted by the Committeesponsored by the Ministry of Health and Welfare in Japan.
Later, Dr. Sawai expanded his research to Southeast Asia
and India, also contributing greatly to this world-wide life
threatening snake bites; he is 90 years old, living in Ohta
city, Gunma, Japan. Other works by the great names mentioned above were also discussed briefly in the session,
showing many slides.

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Symposium
S−I−1) CURRENT STATUS OF EMERGING PROTOZOAL INFECTIOUS DISEASES:
CRYPTOSPORIDIOSIS, CYCLOSPORIASIS, AND MICROSPORIDIOSIS
MOTOHIRO ISEKI
Deparment of Parasitology, Graduate School of Medical Science, Kanazawa University

Cryptosporidiosis, caused by Cryptosporidium parvum
infection, is recognized as acute, self-limiting, gastroenteritis in immunocompetent humans and persistent and potentially fatal in immunocompromised subjects, particularly
those with AIDS. The number of the infection is estimatedto be 250 millions to 500 millions every year, in both developed and developing countreis. Recently, reports of imported cases among travelers to less developed countries,
domestic infections among both immunocompetent or immunocompromised hosts, and outbreaks associated withpublic water supply are increasing in Japan. Diagnostic
methods for demonstrating the oocyst or antigens in clinicalspecimens are well developed. While, we still have noconsistently effective chemotherapeutic agent for use. Development of effective drugs for treament and of control measures for waterborne outbreaks is urgently needed.
Cyclosporiasis is caused by Cyclospora cayetanensis
infection. The organism is a newly recognized coccidianparasite inhabiting small intestinal epithelial cells andcauses prolonged watery diarrhea, fatigue, and anorexia inhumans was reported at least as early as 1979, it was initially called a coccidian-like body or a cyanobacterium-likebody (CLB). In 1993, the organism was identified as a protozoan of the genus Cyclospora, and the species name of C.
cayetanensis was proposed in 1994. The desease is widelydistributed in tropical and subtropical countries. Since 1996,

more than 10 cases among travelers to developing countries
and a case suspected to be infected by ingesting imported
fresh vegetables have been reported in Japan. Symptomatic
infections
canbetreatedwithtrimethoprimsulfamethoxazole.
Microsporidiosis, caused by infection with several species of the phylum Microspora, is an important opportunistic infectious desease among AIDS patients and appears tohave worldwide distribution. At present, 13 species of microsporidia have been identified as potential pathogens inhumans. The first report of diarrheal syndromes associatedwith microsporidiosis and HIV infection was published in
1985, and the number of case reports increased rapidly after
1990. Chronic diarrheas caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis infection are frequently observed among AIDS patients. While, Enc. hellemand Enc. cuniculi cause keratoconjanctivitis and disseminated infection, Pleistophora spp. and Nosema sp. cause
myositis. Recently, cases of microsporidiosis have been reported in Japan. Microscopic detection of the parasite requires special staining methods and adequate microscopic
techniques. Although two agents, fumagillin and albendazole, have been reported to have activity against microsporidia both in vitro and in vivo, treatment efficacy ispoor except in the case of infections caused by Encephalitozoonidae.

S−I−2) CRYPTOSPORIDIOSIS AND CYCLOSPORIASIS:
THE CLINICAL FEATURES AND OCCURRECE IN JAPANGOHTA MASUDA
President, Tokyo Metropolitan Kiyose Children’s Hospital◆Cryptosporidiosis
Cryptosporidium sp. (mainly C. parvum) causes transient non-bloody diarrhea in normal hosts, while this pathogen produces severe, intractable, and recurrent non-bloody
diarrhea, and in some cases biliary tract and/or respiratory
tract infections in immunodeficient hosts. Gut infections

with this pathogen tend to demostrate various degrees of diarrhea, abdominal cramps, flatulence, general fatigue, nausea and vomiting. Many sporadic diarrheal cases due to thisprotozoa are thought to exist in Japan, however, when suchpatients seek medical care, they are often misdiagnosed tohave culture-negative and antimicobial-refractory diarrhea.

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Large waterborne outbreaks of diarrheal illness due to thispathogen have been reported in Japan involving 461 patients (in Kanagawa-ken, 1994), 8,812 patients (in Saitamaken, 1996), 129 and 170 patients (in Niseko and Toyako
lake side in Hokkaido, respectively, in 2000).
From a prospective study performed at Tokyo Metropolitan Komagome Hospital from 1990 to 2000, Cryptosporidium occysts were positively detected in 18 out of 508
specimens (3.5%) from diarrheal patients who had recently
traveled overseas, in none of 605 specimens (0%) with nohistory of overseas travel, and in 4 out of 140 specimens(2.9%) from HIV infected patients.
◆Cyclosporiasis
Cyclospora cayetanensis causes non-bloody watery diarrhea which clinically resembles cryptosporidial diarrhea
except for the fact that diarrhea with C. cayetanensis tends

to be recurring. The administration of sulfamethoxazole/
trimethoprim is usually effective for this kind of diarrhea.
Cyclosporiasis in immunodeficient patients tends to lead todiarrhea with longer duration than that observed in normal
hosts, and this infection is usually non-fatal even in thesepatients.
A few sporadic cases of Cyclospora enteritis havebeen reported in Japan. A study based on 1,071 stool specimens observed from patients at Tokyo Metropolitan Komagome Hospital from 1996 to 2001 indicated the positive rateof this protozoa among diarrheal illness to be 0.7% (3 out of
410 stool specimens were positive) in patients with a recent
history of overseas travel, none (0/513 specimens) in indigenous diarrheal patients, and 0.7% (1/148 specimens) in
HIV-infected patients.

S−I−3) EMERGING AND RE-EMERGING ARBOVIRAL INFECTIOUS DISESESTOMOHIKO TAKASAKI and ICHIRO KURANE
Department of Virology 1, National Institute of Infectious Diseases

Arboviruses are one of the major pathogens ofemefging and re-emerging infectious diseases. West Nile
fever (WNV) epidemics since 1999 in the United States anddengue outbreak in Hawaii during 2001-2002 are valuablelessons. Global warming is a favorable condition for in-sects such as mosquitoes and ticks. We need to pay more
attention to emerging and re-emerging arboviral infectious
diseases in Japan as well as in other areas of the world.
Key words: West Nile virus, dengue virus, imported case

S−I−4) CURRENT STATUS OF EMERGING AND RE-EMERGING BACTERIAL INFECTIOUS DISEASESTSUYOSHI NAGATAKE

S−II−1) DETECTION OF NEW ENDEMIC AREAS OF CUTANEOUS LEISHMANIASISIN PAKISTAN: A SIX-YEAR STUDYABDUL MANAN BHUTTO1, RASHID AHMED SOOMRO1, SHIGEO NONAKA2 and YOSHIHISA HASHIGUCHI3
1. Department of Dermatology, Chandka Medical College/Hospital Larkana, Sindh, Pakistan
2. Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
3. Department of Parasitology, Kochi Medical School, Nankoku, Kochi, Japan

Cutaneous Leishmaniasis (CL) is endemic in Pakistan
and is widely spreading. We report some new endemic areasof CL in the country. A total of 1210 cases of CL visited

our department from 1996 to 2001. Among them, 760 were
the residents of Jacobabad, Larkana and Dadu districts of
Sindh province and had never been traveled to the endemic

２２

areas before. These districts have never been reported/recognized as endemic for CL. Others were the residents ofendemic areas of Balochistan province. All the patientswere between 2 and half months and 65 years of age. Three
hundred and ninety two patients were females and 368 weremales. Duration of the disease ranged from 2 months to
one and half year. Most of the patients had single lesion on
the face and/or extremities. Clinically the disease was classified as dry popular type, 407 cases: dry ulcerative type,
335 cases; and wet ulcerative type, 18 cases. No any caseof muco-cutaneous or visceral leishmaniasis was found during this period. Diagnosis was made on clinical presentation; Giemsa stained smear test; histopathological; and polymerase chain reaction (PCR) results. Smear test was positive in 845 cases, while 365 cases were histopathologically

positive. Ultrastructural study was performed using thespecimens of few cases. Leishmania parasites were detected in the dermal tissues as well as macrophages. About
200 cases were studied by PCR, among them 92% werepositive for Leishmania parasite. All the cases were treatedwith the meglumine antimonate 600 mg/day in adults and
15 mg/kg/day in children as intramuscularly for 20 consecutive days.
On the basis of our findings we propose that Jacobabad, Larkana and Dadu districts could be considered
endemic for CL. Wet and dry type lesions indicates thepresence of both the L. (Leishmania) tropica and L. (L.)
major in this tropical region. This will be confirmed by sequencing technique in future.

S−II−2) CURRENT STATUS OF LEISHMANIASIS IN ECUADORMANUEL CALVOPINA and YOSHIHISA HASHIGUCHI
Department of Parasitology, Kochi Medical School, Nankoku-shi, Kochi 783-8505, JapanThe leishmaniasis is endemic throughout Ecuador andthe incidence is increasing annually. Cases had been reported from all geographical regions mainly in populations
living in rural areas. Recent studies found that the disease
is wide spread in most provinces and is considered a healthproblem in the country. National data showed that the rateof cases increased from 4.36 in 1998 to 20.24 in year 2001
per 100000 inhabitants.
The localised cutaneous form (LCL) represents morethan 95.0% of the total cases. The common LCL form isulcer, but non-ulcerated forms such as papulas, plaques andnodules are also seen. Diffuse cutaneous form (DCL) isvery rare. Mucocutaneos form (MCL) had only been reported from the Amazonian lowlands. In an active survey
we found 13 cases of MCL in the region. A confirmed caseof DCL was reported to be produced by L. (L.) mexicana.
Using molecular techniques, seven species of the genus Leishmania has been identified from the Pacific side: L.
(V.) braziliensis, panamensis, guyanensis, equatorensis, L.
(L.) amazonensis, mexicana, and L. (L.) major-like; twofrom the Andes: L. (L.) mexicana and L. (L.) major-like andtwo from the Amazon: L. (V.) braziliensis and L. (L.) lainsoni.

Confirmed vectors of Leishmania in the Pacific sideare Lutzomyia trapidoi, hartmanni and gomezi. More recently Dujardin demonstrate cryptic species in Lu. trapidoi.
In the Andean foci Lu. ayacuchensis has been incriminatedas a vector. In the Amazonian no vector has been found infected, though there are several man-biting species Lu. serrana, Lu. nevesi, etc. Non-human hosts of Leishmania detected to date are sloths, squirrels, anteaters, rats, kinkajousand canines. Dogs are recorded as hosts of Andean L. (L.)
mexicana.
In most endemic areas, leishmaniasis is well known
and patients use several “folk methods” for treating it. The
drug used is Glucantime provided by the government freeof charge. Other drugs such as mefloquine, artesunate, allopurinol, paromomycin and fosfomycin had been tested insmall trials with variable efficacy.
Current control measures rely on chemotherapy to alleviate the disease. No vector control has been implemented.
Extensive vaccination trial has demonstrated that a cocktail
of five killed Leishmania induces significant protection. A
new phase III study (Biobras) with killed promastigotes+
BCG, is in final step and under revision by TDR/WHO.

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S−II−3) EVOLUTION OF THE CYTOCHROME B GENE OF LEISHMANIA AND ENDOTRYPANUM
UEZATO, H1., YAMAMOTO, Y1., RUYO-ACERO, G1., NONAKA, S1., OSHIRO, M2.,
KARIYA, K2., HAHIGUCHI, Y3
1

2

Dpt. Dermato1., Ryukyu Univ Sch. Med.,
Dpt. II Biochem., Ryukyu Univ Sch. Med.,
3
Dpt. Parasitol., Kochi Medical School

Leishmaniasis is caused by parasites belonging to genus Leishmania. Human infections display a spectrum of
manifestations ranging from cutaneous, mucocutaneous togeneralized systemic visceral disease. The genus Leishmania is taxonomically classified into subgenus Leishmania
and subgenus Viannia. The parasites reside in different regions of the digestive organ of sandfly, Phlebotomus (in the
Old World) and Luztomiya (in the New World), and theirclassification is yet to be completed. The cytochrome bgene (Cyt b) is contained in the mitochondrial genome andencodes an enzyme in the respiratory chain of mitochondria.
The Cyt b gene has been used for phylogenic studies foranimals, plants and fungi. Here, we report determination ofcomplete nucleotide sequences of the Cyt b gene in para-sites belonging to the genus Leishmania (14 strains) andthose belonging to the genus Endtrypanum (4 strains). Phylogenic analysis of these parasites, based on the differenceof Cyt b gene sesquense, agreed well with the previous
classification of above three groups. The difference wasconsistent with different geographic distribution of parasites
and with different clinical manisfestations associated withparasites. In addition, we present recent progress in ourgene expression profile analysis of Leishmania-infected
macrophages. We show that expression of the genes encoding MCP (macrophage chemoattractant protein)-5 and
STAT (signal transducer and activator of transcription)-1 are
up-regulated during infection as examined by the quantitative real-time RT-PCR analysis.

S−II−4) ADVANCE IN DEVELOPMENT OF ANTI-LEISHMANIAL DRUGS AND RESEARCHON MECHANISMS OF DRUG RESISTANCE IN LEISHMANIAKEN KATAKURA1, MAMORU SUZUKI1, KOJI YOKOYAMA2, MAKOTO MIYAJI2, KAZUKO NISHIMURA2, OSAMU KANEKO3,
MOTOMI TORII3, HIROSHI FUJISE4, YOSHIHISA HASHIGUCHI5
1

Department of Parasitology, Gunma University School of Medicine,Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University,
3
Department of Parasitology, Ehime University School of Medicine,
4
Laboratory of Pathophysiology, School of Veterinary Medicine, Azabu University,
5
Department of Parasitology, Kochi Medical School
2

Pentavalent antimonial drugs have been used for treatment of leishmaniasis since 1940’s. Antimonial drugs,
however, require to be injected and produce side effects,
thereby oral safety anti-leishmanial drugs have long been
desired. Sterol biosynthesis pathway in Leishmania appears to be very similar to that in yeast. Thus, oral antifungal drugs have also been used for chemotherapy ofleishmaniasis. We found that SS750, a synthesized new triazol anti-fungal compound, was effective on both cutaneousand visceral leishmaniasis in mice. Skin lesions of mice,
which orally received SS750, were smaller than those of untreated control mice infected with Leishmania amazonensis.
Parasite burden in the liver was low in mice orally treatedwith SS750 than that in control mice infected with L. donovani. These results suggest that SS750 may be applicablefor treatment of various types of leishmaniasis, althoughmiltefosine (hexadecylphosphocholine) is now the mosthopeful oral drug for treatment of visceral leishmaniasis.
Studies of drug resistance mechanisms in Leishmania
is necessary for more rational use of drugs and drug combinations to minimize or circumvent resistance, since antimony resistant Leishmania species are found in endemic areas, especially in India. Studies on multidrug resistance inmammalian cancer cells lead the discovery of the ATPbinding cassette (ABC) proteins, which form one of thelargest protein families and its members are present each２４kind of organism from bacteria to human. Among the ABC
proteins, MDR1 (multidrug transporter or P-glycoprotein)
and MRP1 are most involved in drug resistance in human.
MDR1 is localized in the plasma membrane and can extrude a wide range of structurally unrelated hydrophobic
toxic compounds from the cell. MRP1 transports both hydrophobic anticancer agents and glutathione conjugates. In
Leishmania, currently two ABC transporters can mediate
drug resistance in experimental conditions. PGPA, a member of MPR subfamily, appears to transport antimony- andarsenite-thiol conjugates into intracellular vesicles.

Leishmanial MDR1, a homologue of mammalian MDR1,
can confer resistance to a number of anticancer drugs of the
mammalian MDR1 spectrum and appears to be localized in
the multivesicular tubules of lysosomes. We have cloned
and characterized a new member of ABC transporter,
LaMDR2 in L. amazonensis, which can confer resistance to
5-fluorouracil by probably exocytosis. These results suggest that drug resistance mediated by ABC proteins inLeishmania is related to secretory and exocytotic pathwayof the parasites.

S−III−0) FOR THE FRUITS OF TROPICAL MEDICINESINJIRO HAMANO
Department of Parasitology, Fraculty of Medical Sciences, Kyusyu Univ.

The tropics are inevitaby hotbeds of pathogen transmission because of its various natural and socio-economical
conditions, where there are all of the disease transmission
structures including pathogens, vectors and diseases, whichinvestigators have been pursuing. The final goal of “Tropical Medicine” is “To prevent, diagnose and treat diseases be
there, mainly composed of infectious disease”, and “Tropical Medine” is a scientfic field containing all fields concerning it. For the fruits of “Tropical Medicine”, in other words,
in order to contribute to the prevention, the diagnosis andthe treatment of diseases there, what should we do in future?
In this session, young scientists will introduce theirworks in the past and now at first, and then survey a future
concerning the study. Furthermore, for the fruits of “Tropical Medicine”, we would like to discuss about
1. What are deficient in?
2. How can we get the deficient one (knowledge, technique, skill, idea, personal connections, language
ability, funds, a position, fields etc.) ?
3. What kind of strategies we can map out to survive ineach organ while pursuing Tropical Medicine?
, as an individual investigator or as an organization, Japanese Society of Tropical Medicine,.
I hope that this session will encourage young scientists
in making a dream come true.

S−III−1) SYNTHESIS OF USEFUL ANTIBODY MOLECULES USING THE COMBINATORIAL
LIBRALY AND THE PHAGE DISPLAY TECHNOLOGIESTETSU YAMASHIRO
Department of Infectious Diseases, Oita Medical University

DEN infection has been a big concern in a DEN endemic society for decades all over the world. According to
the investigations conducted by the WHO, the worldwide
incidence of dengue fever (DF) is estimated from twentyfive million to fifty million. An incidence of the Dengue
shock syndrome (DSS), an atypical and much more serious
response to the viruses characterized by hemorrhage is con-sidered more than five hundred thousand in the whole world.
Recently, we have constructed a phage library withmaterial of DEN hyper-immune higher primate and fromwhich a panel of Fab specific to DEN virions were isolated.
Biological analysis of those Fabs were performed and described. Those Fabs obtained through a combination of
combinatorial library and phage display technologies

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should be valuable for use in passive prophylaxis or therapy
for humans against dengue infection.
When one attempts a collaborating study with an institute of distinct culture, attention should be paid on the following points. Choose a theme appropriate to demands ofthe society where the study would be conducted. Have theclear objective. Prepare the detailed work plan. Share detailed information among the study team, Make an effort to
reduce mistake-prone steps. Design and perform a pilot
study in order to obtain information needed for refining thestudy. Distribute the results and interpretation obtained
from the study to each person or institute involved. Carefulconsiderration should be paid to the human rights when collecting and handling human samples.
We are aiming at the preparation of human Fabs possessing anti-dengue neutralization activity through the com-binatorial science. Is it possible to construct an Fab libraryusing human material obtained from healthy volunteers who
had been recovered from DEN infection after obtaining fullconsent to participation in a study in DEN endemic area?
The constructed library should contain higher population ofphage clones displaying Fab specifically neutralize DEN
and has an obvious advantage in isolation of DEN neutralizing Fabs over other libraries.
Curretly, a large number of epidemiological surveys aswell as sample collections for determining pathogens or
pathogenesis of microorganisms have been conducted in thefield of tropical medicine, however, it also possess a potential dimension for supplying useful molecules for prophylaxis or therapeutic applications against certain infectious
diseases.

S−III−2) MOLECULAR MECHANISM OF MALARIAL SPOROZOITES INFECTION INTO
THE MAMMALIAN LIVERTOMOKO ISHINO, MASAO YUDA, KAZUHIKO YANO, YASUOCHINZEI
Department of Medical Zoology, School of medicine, Mie University

Even today, malaria is one of the most devastating infectious diseases, and it kills more than two million people
in the world at a year. The emergence of resistant parasites
makes it more difficult to control this disease. Moreover,preventive method, such as vaccine, has never developed
yet.
Malarial sporozoites injected by mosquito into mammalian host rapidly enter circulatory system, then infect hepatocytes, where they develop to thousands of merozoites.
Therefore sporozoite infection of hepatocytes is the obligatory step for malaria transmission to mammalian host.
However its molecular mechanism is unclear yet. Aimed at
elucidation the molecular basis of parasites infection, weapplied a reverse genetial method, as ‘post genome study’.
As it was reproted that infectivity to the liver was acquired after sporozoite invaded the mosquito salivary gland,
we constructed EST database of P. berghei sporozoites col-lected from salivary gland. Then, by gene targeting method,function of some proteins expressed in salivary glands
sporozoite were analyzed. As focused on parasite-host interactions, putative membrane associated or secretory proteins were selected preferentially. Here we report that asalivary gland sporozoite specific protein was identified andit was involeved in parasites’ infectivity of the mammalianliver.
By the way, it was known that administration of radiation attenuated sporozoites induces preventive immunity
against malaria. Thus it was believed that some sporozoite
proteins should be good targets for developing malarial vaccine. Our strategy will faciliate on understanding of themolecular basis of malarial of vertebrate hosts, and maylead to the development of vaccines against malarial transmission.

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S−III−3) INVOLVING TROPICAL MEDICINE PRACTICALLYTOSHIKI AWAZAWA
The Research & Control of Infectious & Prasitic Diseases Project,
Kenya Medical Research Institute/JICA

The presentation focused on two areas.
The first outlined the various approaches in participating international health in the tropical regions. Having
learnt from personal experiences, from working as a staff
member in the health service providing facility such as a
hospital, to a project co-cooridnator in the ministry ofhealth, the disease control programme onto the existing
health infrastructures seems feasible approach. One ofthese infrastructures is the school system. Schools have increasingly been recognized as one of the most convenientand logical springboards from where to promote healthy behaviours and to deliver certain health services. In the context of de-worming campaigns, schools offer one of themost efficient and effective channels to one of the most vulnerable groups.Moreover, the ability of non-medical personnel to deliver the drugs means that the number of children and the speed at which they can be reached is mas-sively accelerated.
The second part of the presentation focused on theparasite control activities at Eastern and Southern Africa
Centre of International Parasite Control (ESACIPAC) of the
Kenya Medical Research Institute. ESACIPAC is one of thethree centres of the Global Prasite Control Initiative, also
referred to as the Hashimoto Initiative. Its mandate is to coordnate capacity building for parasitic diseases control activities in the regional countries, namely, Kenya, Uganda,
Tanzania/Zanzibar, Malawi, Zambia, Botswana and Zimbabwe. Its primary functions include training of personnel
and networking among them. ESACIPAC will involve implementation of school based parasite control activities in
Kenya. The strategy for coodinating effort of all other technical co-operation project funded by JICA (Japan International Co-operation Agency) is also discussed.

S−III−4) WHAT IS THE TROPICAL MEDICINE IN JAPAN? - A QUETION FROM A YOUNG SCIENTIST OSAMU KANEKO
Department of Molecular Parasitology, Ehime University School of Medicine

So-called “Tropial medicine” consists of two researchareas. First area pursuits to study a basic biological aspectof the tropical diseases and develops tools to prevent and/or
treat human beings from diseases by biomedical research.
The other area pursuits to study a public health system todevelop strategies to apply tools produced. These two areas
simultaneously contribute to solve the tropical health problem. Increasing funding in the basic reseach area of the infectious diseases has stimulated the activity of this area andthe number of young researchers has increased in the past
10 years. Furthermore, based on the Hashimoto Initiative,
projects aiming human resource deveopment and information networking to control parasites in Asia and Africa werelaunched with the strong supports by JICA. However, despite of these situations, there is a big gap between the basic
research area and the disease control area. One of the reasons of this gap can be explained by the limited human re-sources of this area, especially in the research area conducted in the tropical field. Improved health situation in Japan has reduced not only infectious diseases, but also reseachers in this area. As mentioned at the beginning, successful control of the diseases requires tools and strategies,
thus our current need is to promote the authentic epidemiology in order to create better strategies to combat tropicaldiseases. Here I propose 3 activities, 1) Promoting more
field studies in the tropical countries to stimulate the academic side of this area; 2) promoting young researchers inthis area and recruit researchers from other areas to increase
the population of this area; and 3) encouraging to attend international meetings of tropical medicine to upgrade thequality of this area. These activities would produce fruitful
outcomes from Japanese Society of Tropical Medicine in
the next 20 years.

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S−IV−1) ADEQUATE TREATMENT AND EARLY DIAGNOSIS OF LEPROSY
ATSUSHI HOSOKAWA, MOHAMMED A.K.KHAN, SHIGEO NONAKA
Department of Dermatology, Faculty of Medicine, University of the Ryukyus

In fact, a large number of patients with leprosy rapidly
decreased by multi-drug-therapy(MDT). But newly detected patients with leprosy has been increased in number inthe endemic areas, especially in India, Brazil and Southeast
Asia etc. Three important measures of leprosy control are
to be discussed, namely chemotherapy, early detection in
the early stage of leprosyand the prejudice for the disease.
1) Chemotherapy; Four leprosy cases with sequelae causedby neuritis which increased after starting MDT and twocases without sequelae were shown. For a patient with borderline group leprosy, who showed severe neuritis and deformity of the fingers after starting of the medication, thechemotherapy was considered to be inadequate. A patientwith an annular erythema on the on left buttock, diagnosed
as to be tuberculoid leprosy, received a modi-fied MDT,
comprising of 450mg of RFP per monthy and 75mg of
DDS for a month, healed completely within a few months.
Two contrastive patients with borderline group leprosy
showed similar infiltrared erythema on the right face werereported. In the course of chemotherapy, the deformity offace became obvious and flacid palsy of the right orbicularis oculi muscle appeared in a borderline tuberculoidmiddle borderline leprosy patient. On the other hand, another patient with borderline tuberculoid leprosy healed
without any sequelae. As shown in the above mentioned

cases, each of them showed different and various responses
to chemotherapy during the course of the treatments.
Therefore the simplified chemotherapy for the simplified
clasiffication considering sensory loss, neuritis and the siteof lesion are need-ed to prevent the sequelae. For example,
n1 indicate the type of leprosy with sensory loss and minor
neuralgie, n2 indicate the type of leprosy with sensory loss
and severe neulargie. h1 indicate the site of lesion at the axillary, inguinal, perineal lesion, buttock and scalp, h2 indicate site of lesion at the hands, feet-leg, face and ear.
2) Early detection; Indeterminate leprosy case healed completely in a few months. Therefore, it is very important forelimination of leprosy to detect leprosy patients in theirearly stage of the disease. For this purpose, paramedical
and non-medical staffs may play a great role.
3) Prejudice; Though the prevalence rate of leprosy in Japan
became below 1.0 per 10.000 approximately 30 years ago,
prejudice for the disease is still remain especially in oldpeople.
Japanese medical personnels can assist to resolve theabove mentioned problems and shortening the period for
elimination or eradication of the dis-ease in the endemic areas.

S−IV−2) AROUND THE FUNDAMENTAL LEPROSY RESEARCH− HOW TO PROCEED THE RESEARCH OF A CLASSICAL INFECTIOUS DISEASE IN GENOME ERA −SHIN SASAKI
Department of Bioregulation, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan

Leprosy is a classical infectious disease caused by Mycobacterium leprae, which was discovered by G. A. Hansen
in 1873. The disease has been documented since antiquity
and still continues to be endemic in some developing countries. M. leprae is an exceptional bacterium because of its
long generation time and no growth in artificial media. Entire sequencing of the bacterial genome revealed numerous
pseudogenes which might be responsible for the very limited metabolic activity of M. leprae. The establishment ofeffective combination chemotherapies in 1970s prove agreat boon to the victim of leprosy. Multiple drug therapy(MDT), which consists of several antibiotics linked to simplified diagnostic regimen, has become increasingly popularin the countries where the disease is endemic. A significant
drop of the disease prevalence has been achieved worldwide,
and the endemicity of leprosy remains in few countries. To
achieve the worlcd without leprosy, we have many questions tobe answered. How the bacteria are disseminated?

２
８

Why intrafamilial infection is likely? Is there any source of
infection other than symptomatic patients? Moreover, no diagnostic marker nor vaccines are available. A compendium

for the current leprosy research is provided and the future
prospects are also discussed.

S−IV−3) PRESENT SITUATION OF HANSEN’S DISEASE IN THE WORLDNORIHISA ISHII
Department of Bioregulation, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan

Lrprosy patients in Japan was reported that about
30,000 in 1900, which decreased to about 16,000 by 1919.
From roughly 1955, due to the improvement of publichealth and appearance of remedies, new patients decreased
repidly. Recently, there are less than 10 new patients per
year. On the other hand, non-Japanese patients (imported
cases) have been on the increases since roughly 1991,
counting about 10 patients per year. It is, therefore, anticipated that there will be no leprosy patients among Japanesein near future. Non-Japanese patients, however, appear
among a large number of employees aged 20s〜30s from
Brazil and the Philippines where leprosy is still endemic.
Global situation owing to multidrug therapy (MDT)
pursued by WHO; more than 10 million leprosy patientsworldwide were cured from 1985 to the end of 2001. The
registered cases decreased to 635,404 by the beginning of
2002 and the prevalence was 1.0 per 10,000 population,
which is a 88% reduction from 1985. After treatment for 6
months in PB cases and for one year in MB cases, they are
regarded as cured and withdrawn from the WHO. The relapse rate is about 0.1% per year. Only few MDT-resistantcases have been found. Physical disability may be seen after peripheral nervous impairment (two to three million

cases), and many have difficulty returning to and attaining
social lives. In spite of the promotion of MDT by WHO, as
many as 760,000 new patients are still registered every year,
and that is presumably due to improved diagnosis and enhanced surveillance for many new cases including ones
overlooked in the past. The countries with many yearly registered new leprosy patients during the year 2001 are; India(617,993), Brazil (41,070), Indonesia (13,286), Bangladesh(10,740), Myanmar (9,684), and Nepal (13,830).
Full control of leprosy has eluded mainly in Angola,
Brazil, India, Madagascar, Mozambique, Myanmar and Nepal. These countries are committed to stepping up leprosy
control activities. Access to information, diagnosis andtreatment with MDT is essential Information campaigns
about leprosy in high risk areas are crucial so that patientsand their families, who were historically ostracized fromtheir communities, are encouraged to come forward and receive treatment. Today, diagnosis and treatment of leprosy
is easy. Essential work is being carried out to integrate leprosy services into existing, general health services. This isespecially important for communities at risk for leprosy,
which are often the poorest of the poor and under-served.

S−IV−4) PRESENT SITUATION OF LEPROSY DISEASE IN MYANMAR
TIN AUNG SOE {M. B; B. S}, Team Leader
Leprosy Control Programme, Myanmar (Leprosy Research Center, NIID, Japan)

The Union of Myanmar is one of the hyper endemic
countries with leprosy disease. Although the leprosy disease burden has significantly reduced to a low level in Myanmar, leprosy elimination activities need to intensify toachieve the elimination goal in time. National Leprosy
Elimination Programme has been already committed toreach the elimination at the national level by reducing theprevalence below one case per 10,000 populations by theend of year 2003. To achieve the elimination of leprosy, theprogramme was fully integrated in Basic Health Services.
It was decided in 1969 to try a gradual handling over of responsibilities of the vertical leprosy control services to thebasic health services personnel. In this presentation I present “the present situation of leprosy disease in Myanmar”

２
９such as disease burden, integration, detection of new cases,
prevalence and detection ratio, routine, activities, community awareness and monitoring and supervision. And I alsopresent “cooperation of Myanmar and Japan (JICA) regarding the leprosy disease”. With the cooperation of other organizations such as WHO, JICA and NGOs, we could accelerate the elimination activities towards the goal and also
other aspects regarding with leprosy disease. There is a pe-

riod very near to elimination of leprosy in Myanmar. We
conduct the acceleration and sustainability of leprosy elimination activities. The application of multi drug therapy(MDT) is progressing rapidly towards the goal of elimination of leprosy as a public health problem. Preparatory activities for post-elimination strategies are also considered
including prevention of disability, disease surveillance, control and prevention tools and etc.

S−IV−5) ACTIVITES OF JICA PROJECT FOR LEPROSY CONTROLAND BASIC HEALTH SERVICIES IN MYANMAR
EIJI NAGAO
National Leprosarium Osima Seisho-en

JICA project team has carried out many kinds of provisions against National Programme for Leprosy Elimination
in Myanmar, from 2000.
We have conducted training courses of midwives,
laboratory technicians, doctors, physiotherapists, nurses,
prosthesis therapists, shoemakers, and persons affected leprosy (PALs).
We recommend (1) that special actions for new casedetection should be continued, until true elimination on
townships and villages level is achieved, (2) that various
kinds of useful training should be continued, and (3) thatvarious kinds of effective enlightenments should be held incommunities, because leprosy is a chronic disease with extermely long incubation term and PALs with disabilities
need many kinds of aids.

S−V−1) ROLE OF THE JICA ZAMBIA PROJECT IN AIDS CONTROL IN SOUTHERN AFRICAHIROSHI TERUNUMA
Department of Microbiology, Faculty of Medicine, University of Yamanashi

The AIDS epidemics is still one of the biggest problems in global health issues. At the end of 2002, over 60
million people were infected with HIV. Forty million are
still living with HIV and 3 million died during the year
2002. More than 90% of these infected people are living indeveloping countries and got infected with HIV throughheterosexual contact. Zambia has a high seroprevalence ofHIV-1. One forth of the adult population in Zambia is infected with HIV. For the past 10 years education has beenemphasized to control the spread of HIV in Zambia. Whileeducation is still very important, it is, however, not enoughto control the HIV epidemic because the number of infected
people is still increasing. Further more over 1 million Zambians are living with HIV.
From 1989 to 2000 JICA Zambia infectious disease
and infectious diseases control programs established the

University Teaching Hospital (UTH) Virology Laboratory
to examine viral diseases such as polio, measles, influenza
and HIV. For HIV, we introduced flow cytometory and
PCR technology at the laboratory in Zambia. In 2001 theJICA HIV/AIDS and TB control project was started inZambia and this project is contributing to the control of the
HIV epidemic. Currently the Virology Laboratory is the
only laboratory which can evaluate quality assurance of
HIV serological testing and monitoring the condition of
HIV-infected patients.
To control HIV/AIDS, this program is closely working
with other working groups such as the national AIDS secretariat, voluntary counseling and testing and care program(VCT), prevention of mother to child transmission program(PMTCT), and treatment and vaccine program. The Virology laboratory is currently conducting training and quality

３０

assurance of HIV serological testing for technologists at
VCT sites, monitoring of drug-resistant HIV-1 and HIV-1
strain surveillance for vaccine development.
I hope through this program many young Zambian andJapanese experts will gain experiences to fight infectious
diseases and will develop better strategies to control HIV/AIDS.

S−V−2) TUBERCULOSIS CONTROL IN NEPAL AND JAPANESE MEDICAL COOPERATIONKATSUNORI OSUGA
Research Institute of Tuberculosis Japan Anti-Tuberculosis Association

In the early 1960s, a Japanese doctor arrived in theKingdom of Nepal. His name was Noboru Iwamaura. As amission doctor, he worked in the mountainous outskirts ofthe Himalayas, visiting villages searching for tuberculosis
patients. Many locals still remember Dr. Iwamura, one of
the nost well known Japanese names in Nepal to date.
With widespread poverty, however, tuberculosis remained
the biggest threat to public health in Nepal.
Following pioneers from the private sector, the Japanese Government started to provide assistance to control tuberculosis in the 1970s. The National Tuberculosis Center
was constructed with the Grant Aid scheme in the 1980s.
Today, this center functions not only as the headquarters of
the National Tuberculosis Program, but also as the regional
TB center for South Asia (SAARC TB Center). Coupledwith the construction of the TB center, a technical cooperation project by JICA was also carried out. Short course
chemotherapy using Rifampicin, which was uncommon indeveloping countries at that time, was also tried as a pilot
study. Technical expertise was provided from the ResearchInstitute of Tuberculosis (RIT), Tokyo. Apart from Japan,
no major donors or intermational agencies provided substantial assistance for TB control in Nepal during this period.

It was in the mid 1990s, when the international arena
began to focus on TB control. WHO Iaunched the newstrategy called “DOTS” and the World Bank supported its
cost-effectiveness. Since then, many new donor partners,
such as Norway, UK, and WHO, joined the effort to combat
TB in Nepal. With multiple partners providing assistance to
the Government of Nepal in different schemes, misunderstanding and friction sometimes occurred. The importanceof coordination among donors and UN agencies was recognized.
Today, nearly 90% of the population in Nepal has access to DOTS, with over 85% treated successfully. Japanese technical coooperation in Nepal now has broader aspects. The current JICA project is focusing on “lunghealth”, targeting childhood ARI and smoking cessation, as
well as TB control in difficult population groups and areas.
Every year, medical doctors and paramedics from Nepal aretrained at RIT; more than 70 Nepalese have been trained in
the past 40 yaers. It is now well recognized that Japanesetechnical cooperation, both through governmental and nongovernmental sectors, has made a significant contribution tohuman resource development and appropriate technology
transfer for TB control in Nepal. Howrver, the approaching
HIV epidemic, apreading poverty, and social instability inrecent years, all pose a major threat to TB control in Nepal.
Long-term collaboration appears necessary to sustain thefight against tuberculosis in Nepal.

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S−V−3) PROBLEMS OF IMPLEMENTING THE MALARIA CONTROL PROJECTHIROJI KANBARA
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University

We, the Institute of Tropical Medicine, Nagasaki University have began the JICA partnership project “Malaria
control at Lombok and Sumbawa islande in Indonesia”
since November, 2001. Since activities necessary for the
project included various fields, we, first, formed necessary
sections and appointed a responsible person in each section
as follows. 1) Section for malaria parasite survey and control. The section was devided into two pars ! Regularmonitoring survey and " Case detection and treatment 2)
Section of entomological survey for vector mosquitoes 3)
Section of vector control 4) Section of social survey including education programs for villagers and concerned staff.
Besides, the regular meetings were held four times ayear in which Japanese representatives including me, representatives from Ministry of Health, Jakarta, from Tropical
Disease Center, Airlangga University, from Nusa Tenggara
Barat (NTB) provincial health office and from two district
health offices at West Lombok and Sumbawa participated.
These activities evoked the following problems.
1) Malareia epidemiological conditions were different according to geographycal and environmental conditions.
In Muninting area of West Lombok district two distinct
malaria endemics were found, one in the coastal areas
and the other in the forested and hilly areas.
2) Although we must adopt proper control methods basedon endemic characters in each area, we have only limited number of control methods, and don’t have a fixed
control manual for each character.
3) Malaria control activities are feasible only under systematic coopereation of various organizations, especially the comprehensive cooperation from villagers.
4) Intimate relationship between the central government
and local governments must be established.
In the present project we are acting as a coordinatorand as a financial supporter to establish a model of control
strategy fitting for endemic conditions in each area. For thispurpose we despatched two Japanese young staff for longstay after training them. This typr project will become important for our country to pactise medical cooperation or
support in developing countries. However, the following
problems in our country should be considered for further
development of medical cooperation.
1) Shortage of specialists and practical personnel for control activities.
2) Low evaluation for persons concerning practical works
in the field of preventive medicine including malaria
control.
3) Shortage of educational organs to teach public health indeveloping countries.

S−V−4) HASHIMOTO INITIATIVE AND OKINAWA INFECTIOUS DISEASE INITIATIVE:
JAPAN’S CHALLENGE TO GLOBAL PARASITE CONTROLTSUTOMU TAKEUCHI
Department of Tropical Medicine and Parasitology, School of Medicine, Keio University

In May 1998, at G8 Summit in Birmingham, Mr. R.
Hashimoto, the prime minister of Japan, made a proposalthat the G8 should take a leading role in promoting parasite
control efforts and contributing to the world health. Mr.
Hashimoto’s proposal was endorsed and the resolution tothis effect was incorporated into the official communiquewith the statement that “Control of parasitic diseases including malaria should be implemented”. Since then, Mr.
Hashimoto’s proposal has been called “Hashimoto Initiative”(HI).

As the basic strategy, Hashimoto Initiative raised 4 issues, First, it stressed the necessity of effective international
cooperation for parasite control. Than it raised the active
pursuit of research as the scientific basis for parasite control
and active implementation of the control projects. Finally,
the initiative requested each of G8 countries to reinforce thecapability to deal with parasitic diseases.
Among Japan’s own activities by HI, the most important contribution is the establishment of research and training centers for parasite control in Asia and Africa. As the３２

foothold for this purpose, Mahidol University in Thailand,
and the Kenya Medical Research Institute (KEMRI) in
Kenya, and the Noguchi Memorial Institute of Medical Research (NMIMR) in Ghane were chosen. These centers will
have functions of effective training, technical support, operational research and information/human networking. To
promote such activities, collaboration with international organizations will be accelarated. More importantly, thesouth-to-south cooperation will be strongly promoted.
Among them, the center at Mahidol University became
functional earlier. It is called the Asain Center of International Parasite Control (ACIPAC). Those at KEMRI and
NMIMR are called East and South African Center of Inter-national Parasite Control (ESACIPAC) and West African
Center of International Parasite Control (WACIPAC), respectively, and have already initiated their own activities.
In addition to HI, Japanese Government Iaunched Okinawa Infectious Disease Initiative at the Kyushu-Okinawa
G8 Summit in 2000, where HIV/AIDS, tuberculosis, malaria were listed as the major target of international collaboration. As the chairman country, Japan also proposed parasitic diseases and polio. In this sense, both Hashimoto Initiative and Okinawa Infectious Disease Initiative are expected to be effectively coorodinated in near future, taking aleading role in parasite control at the global level.

S−V−5) THE PROGRESS AND THE DIRECTION OF THE ASIAN CENTRE OF INTERNATIONAL
PARASITE CONTROL PROJECTNOBUHIKO NAGAI, NORIAKI TOMONO, MITSUHIKO IWASHITA and SOMEI KOJIMAAsian Centre of International Parasite Control (ACIPAC)

At the G8 Summit Meetings in 1997 and 1998, former
Prime Minister Ryutaro Hashimoto proposed that the G8
countries should cooperate to support countries affected byparasitic desease. Under the Hashimoto Initiative, Japan International Cooperation Agency (JICA) decided to set up
one centre for parasitic disease control in Asia (Asian Centre of International Parasite Control, ACIPAC) and two in
Africa. ACIPAC was launched as a five-year project inMarch 2000. The partner countries include the Greater
Mekong Sub-region countries of Cambodia, Lao PDR, Myanmar, Thailand, and Vietnam. The overall goal is tostrengthen parasite control programmes in the partner countries through human resource development. JICA’s regional
technical cooperation programme supports ACIPAC activities.
The slogans “Save Wormy Schoolchildren” and “Save
Schoolchildren from Parasites” have been chosen to promote this effort. ACIPAC will advocate parasite control
through school health programmes. ACIPAC will utilizeSouth-South Cooperation through information sharing andhuman resource networking, to help achieve the goal ofparasite control.
In the second fiscal year (2001), the first three-monthtraining programme “School-based Malaria and Soil.transmitted Helminthiases Control for Programme Managers” was conducted from September 17 to December 7.

Twenty-eight trainees who are administrators and technicalofficials from MoH, MoE and provincial Health Offices
participated in this training. Five participants were invitedfrom each of ACIPAC’s partner countreies. There was one
participant each from Japan, the UNICEF Laos office andthe Kenya Medical Research Institute (one of the Hashimoto Initiative African Centres).
After the ACIPAC training, ACIPAC started a smallscale pilot project in each country. Planning for these pilot
projects was done by the students during the ACIPAC International Training. In February and March 2002, as a firststep, baseline surveys of malaria and parasite infection andsocio-behavioral studies were conducted in Laos, Cambodia,
and Vietnam. Myanmar will start its baseline study earlynext fiscal year. At the end of March, ACIPAC held an international workshop, and some of the graduates presented
the results of baseline study.
In Thailand, ACIPAC has two model projects. They
are in Ratchaburi province and Nakorn Si Thammarat province, which are endemic areas for malaria and soiltransmitted helminths, respectively. These model projects
were utilized for field practice in the ACIPAC Intermational
Training Course.
ACIPAC is also planning an information-sharing network for parasitic disease control. ACIPAC has launched
its Web site (http://www.tmacipac.mahidol.ac.th).

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S−V−6) ROLES AND CHALLENGES OF JAPAN’S ODA FOR GLOBAL HEALTHOSAMU KUNII
Economic Cooperation Bureau, Ministry of Foreign Affairs, Japan[Background] Japanese government placed health sector
among priority areas in its Official Development Assistance(ODA), and announced “Global Issues Initiative on HIV/AIDS and Population” in 1994, “International Parasitic Diseases Control (Hashimoto Initiative)” in 1998 Birmingham
Summit and “Okinawa Infectious Diseases Initiative” in2000 Kyushu-Okinawa Summit.
The Okinawa Infectious Diseases Initiative (IDI), reinforcing the Hashimoto Initiative, has six basic strategies: (1)Supporting institutional building of health sector with ownership of the recipient countries, (2) Focusing human resources development, (3) Strengthening partnership withcivil society, donor countries and international organizations, (4) Supporting South-South cooperation, (5) Promoting research activities, (6) Promoting public health approaches at the community level.
[Achievements] IDI pledged US$ 3 billion in five years
and has already spent US$ 1 billion in 1.5 year. It has provided over 50 bilateral projects and has contributed to international organizations such as WHO, UNICEF, UNAIDS,
and to Japanese/International NGOs. In particular Japan established “Policy and Human Resource Development Fund

(PHRD Fund)” in World Bank, “Human Security Fund” as
the trust fund of UN to support their programs, which havebeen made use of over 100 countries. Besides IDI, Japanhas pledged US$ 200 million to the Global Fund to Fight
AIDS, Tuberculosis and Malaria (GFATM), 2nd largest
contribution following the U. S..
[Challenges] More collaboration with stakeholders at theinternational/national/regional level is required to makemost of Japan‘s ODA. Especially it is crucial to make systematic and strategic coordination in (a) human resourcesfrom Japan Overseas Cooperation Volunteers (JOCV),
NGOs to professionals, (b) organizations from JICA/JBIC
to private enterprises, (c) programs from infrastracturebuildings to community development. Japan could contribute to global partnerships to promote cooperation in development assistance.
I hope the Japanese Society of Tropical Medicine
would play more active role in Japan‘s ODA and NGOs’ assistance, providing technical assistance, researches directly
contributing to ODA, and human resource development of
the tropics and Japanese young generation.

S−VI−1) DIFFICULTIES ENCOUNTERED IN FIELD RESEARCHES, WHICH WERE USED AS EXCUSESMASAAKI SHIMADA
Research Center for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University

For the symposium I was given an assignment. What
the chairperson requested me to talk was my personal experience in field works. Therefore the subjects I point out
here are mostly on my personal experience.
1. Selection of target population
As is usual in any field work, target population cannot
always be selected based on scientific evidences. A community is usually selected according to the administrative
boundaries and therefore a political will always influences
the selection. Sample size may not be satisfactory. Sampling methods we apply sometimes do not work well because of the complaint of both selected and unselected people.
2. Members of the population

After the selection, several persons or one in the community are recruited as informants or field workers. It issometimes difficult to choose the best person by the research team. There is always a hierarchy in any community,
which may disturb the best selection.
3. Identification of individuals
It is not easy to identify a person at different times andplaces. Demographic data such as names, sexes, birthdays
sometimes do not help because different names and birthdays could be used at different circumstances. Even pictures may not help especially in case of children. Population movement is another factor that makes the identification difficult.
4. Expectation of the community

３
４What we are expecting in the field research and thatthe community is expecting in the work are sometimes different. Not all of the members of the community are cooperative. The demand of the community, excessive expectations and the existence of uncooperative people may influence the results of the study.
5. Heterogeneity of the population

The target population is not homogeneous like laboratory animals. This heterogeneity is a treasure of field workto obtain interesting results. However it is not easy to collect the data that properly show the heterogeneity of the
population. There are usually limitations in collecting dataon the characteristics of individuals.

S−VI−2) FROM VOODOO EPIDEMIOLOGY TO RELIABLE EPIDEMIOLOGYTAKESUMI YOSHIMURA
Dept. Clinical Epid., Inst. of Industrial Ecological Sciences, Univ. of Occupational and Environmental Health, Japan

An epidemiological study is essential and inevitable in
the field study of tropical medicine, because an epidemiological study shoud only be the method to obtain necessary information for the people live in the target area. But
an epidemiological study is not so appreciated in the societyof tropical medicine because of uncertainty of date. In thispresentation, four points will be discussed in order to establish reliabe epidemiology.
1. Two types of epidemiological studies should be distinguished. One is descriptive type epidemiological study(to describe epidemiological characteristics of target population in a specific area.) The another is hypothesis testing
type epidemiological study (to test hypothesis among human subjects). So called epidemiological study can be referred as one of two types. Study type should be clarified
for further discussion.
2. Goal of the study should be clarified. For descriptive type epidemiological study, representativeness, degreeof data accuracy should be carefully considered.

On the other hand, for the hypothesis testing type epidemiology, comparability between study populations
should be of most importance.
3. Study design should be carefully considered. Study
design should be a balance between informativeness andfeasibility of the study. Which level of information should
we obtain for further action? Feasibility of a study (humansubjects availability, cooperability, human resources, money,time, etc.) is fundamental for operation of the study.
4. Obtained results should be criticized for reasonableinterpretation in terms of three points of view.
1) Is there any biases (Information bias, Selection bias)?
2) Are confounding factors in the study controlled properly?
3) Are statistical test and power problems properly considered?
For reliable epidemiology rather than Voodoo epidemiology, four points stated above should be considered.

S−VI−3) FILARIASIS CONTROL: HOW WERE BLOOD COLLECTION AND MASS DRUG
TREATMENT DONE?
EISAKU KIMURA
Dept. Parasitol., Aichi Medical Univ.

In 1978-81, I worked for the filariasis research andcontrol program in Samoa as a WHO staff member. Bloodcollection by finger prick and venipuncture was repeated
day after day. Under the WHO project, I never thought of

ethical issues in relation to blood sampling, and cannnot remember such things were discussed among WHO staff. As
an international organization, there should never be any unethical affairs with WHO activities. It was an established３
５fact. In Samoa, there is a “matai” system, where decisionby village matais, or chiefs, will overpower individual opinions of villagers. If matais consent blood collection, no villagers can protest. Although it may not be unethical, ourprogram apparently took advantage of the system to carryout researches more easily than in other countries.
In 1973-75, I worked in Ethiopia for smallpox eradication program under WHO. What we did was to vaccinate as
many children as possible. We worked hard in very remotemountainous areas where people often could not expect
even the minimum level of medical care. In 1980, WHOproudly declared the eradication of smallpox, and publishedthe final report. One sentence in it was shocking: Vaccination will no longer be justified because it has serious side
reactions which may be fatal. We vaccinated many children

without discussing any about the danger of the vaccine.
Was it ethically acceptable to vaccinate when we knowsome could suffer from serious reactions and they could
never be attended? Can some people be sacrificed if theaim is for the international benefits and betterment of all
people? We know that benefits are often more for the richthan the poor.
Ethical standards are moral prerequisite for all researchers, but for some civilized people, they have other aspects of avoiding legal complications, and self-satisfaction.
It will be very important for us to participate positively inthe formation of morally sound and practically workablestandards with which field studies in developing countries
can be performed with more confidence of scientific contribution.

S−VI−4) BIOETHICS AND BIOMEDICAL RESEARCH IN DEVELOPING COUNTRIES:WHAT SHOULD RESEARCHERS DO FROM THE VIEWPOINT OF BIOETHICS?
SATOSHI KANEKO
Cancer Information and Epidemiology Division, National Cancer Center Research Institute

The fundamental principle of ethics for medical research on human body had been based on protection of human right, and voluntarily participation to research. Alongwith rapid changes in modern societies, new concepts onhuman right have appeared on the field of biomendical research; that is, right to be let alone and right to control personal information. Furthermore, a process of informed consent to support voluntarily participation has been weighted
in recent biomedical research. These new ethical issues
have been considered to conflict with procedures in biomedical research. To balance the friction between them,several ethical guidelines for biomedical research using human subjects have been published based on the Declarationof Helsinki. Such guidelines recommended strengthening
the review function of ethical review committees (ERCs) tomake an applied biomedical research ethically justifiable.
Although the ethical acceptability of biomedical research indeveloping countries supported or undertaken by developed
countries must have followed the guidelines, it is not welldiscussed until the debates in 1997 on the zidovudine(AZT) clinical trials conducted in developing countries.
These trials were implemented to see whethere zidovudine(AZT) treatment for HIV-infected women prevented perinatal taransmission of HIV. Although these debates resulted
in the revision of the Declaration of Helsinki and an ethicalstandard to clinical trials in developing countries were given,
they did not expand to ethical issues in general biomedical
research or genomic research in developing coutries.
In Japan, the bioethical guideline for epidemiologicalresearch has recently proclaimed by governmental ministries. Since the guideline has just come into effect, there
has been no standard on ERCs and the abilities to review
study protocols are not well evaluated. When it comes tobioethical issues for research in developing countries, thesituations becomes more chaotic. Most institutional ERC
members do not know the situation in developing countries.
Thus researchers must make an effort to inform the particular environments in developing countries to the ERC members. Sometimes, researcheres might have to scientificallyor logically assert reasons to ERC members when the protocol could not exactly follow the bioethical guidelines.
Through such constructive debates, the study protocol
would become more refined, and persuasive. Mutual evolution of ERC members and researchers would not only promote the bioethical standard, but also improve the quality ofthe biomedical studies in developing countries. It is timefor ethics and science to shake hands. The Japanise Societyof Tropical Medicine must take a measure to enhance suchmutual evoluion.

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W−I−1) PREVENTIVE BEHAVIOR AND MORBIDITY OF JAPANESE TOURISTS FOR AFRICAHITOSHI KIKUCHI, AKIHIRO HITANI, YASUHIRO ARAI, TOMOAKI SANO,TSUTOMU YOKOTA AND YOSHIE TANAKA
Narita Airport Quarantine Station

In many advanced countries, travel medicine is developed and taravelers who visit risk areas can access travel
medicine information. But, in Japan, travel medicine is developing and many tourists visit risk areas without or insufficient travel medicine knowledge. To assess the preventive
behavior and health risks of Japanese tourists, a questionnaire study was carried out. Japanese tourists for Africa arefocused. With a cooperaton of a tour company, three hundred tourists of African tours were asked to complete questionnaire. 135 questionnaires ware collected and were analyzed. Male was 58(37%) and the average age was 40.9±
12.4(mean±SD), ranged from 8 to 76. As the infectious
disease information sources, 96(62%) tourists get information from guidebooks, 66(42%) from tour agency, 36(23%)
from internet web, and 30(19%) from friends. TRAVELER’S DIARRHEA: The attack rate of diarrhea was 24%
and food score proposed by Sonneburg was 4.7±2.6.
Compared with the Sonneburg’s data of Kenyan traevlers
that the attack rate was 65.7% and the food score was 5.5±
8.6, the attack rate of Japanese tourists was lower. MARARIA. The knowledge of malaria was asked and 130(80%) knew malaria and 131(84%) intend to prevent formalaria. To prevent malaria, 117(87%) planned to use repellent before travel and 105(78%) actually used, 94(70%)
planned to wear long sleeved and 86(63%) were, 93(69%)
planned to use mosquito coil and 104(77%) used, 26(19%)
planned to use mosquito net and 61(45%) acutually used.
28(20%) planned to take malaria prophylaxis before travel,
13(9.6%) actually took, 12(8.9%) take regularly, 5(3.7%)
stopped after return. These indicate that most of the tourists
know malaria and try to prevent mosquito bite, but theknowledge of malaria prophylaxis is insufficient. VACCINE: Vaccination rate was as follows: yellow fever 108(80%), tetanus 12(8.9%), rabies 6(4.4%), hepatitis A 5(3.7%).
These data support that guidebooks and the travel
agency made a major role for tourists’ prevention. Tourists
have tendency to keep general preventive measures. The
travel agency recommended general precautions actively,
but had difficulty to recommended medical prevention because they could not treat medical problems and travel
medicine facility is very limited in Japan.

W−I−2) KNOWLEDGE AND PREPARATION REGARDING THE RISK OF MALARIA AMONG
JAPANESE INTERNATIONAL TRAVELERSKAWAKAMI KEIKO1, KIMURA MIKIO2, HASHIMOTO MICHIKO3, AOKI KAZUKO3
HAMADA MASARU4, TANIHATA TAKEO1
1

National Institute of Public Health, Tokyo, 2National Institute of Infectious Diseases, Tokyo,
3
Tokyo Quarantine Station, Tokyo, 4Osaka Quarantine Station, Osaka, JapanBACKGROUND : Although Japanese citizens travel increasingly to malaria-endemic areas, little has been described on their knowledge and preparation regarding therisk of malaria. This prompted us to conduct a questionnaire study on these issues targeting this population.
METHODS : During a 2-month period, travelers ≧ 16
years of age vitising Tokyo or Osaka Quarantine Station for
vaccination were asked to answer a self-administered questionnaire. Most of them attended to receive yellow fever
vaccine and therefore, scheduled to visit areas that are, to agreater or lesser extent, malarious. Questions included

knowledge of the transmission route, main symptom andpraimary preventive measure of malaria. They were also
asked whether and how they were informed of the current
malaria situation at their destinations, what they would do
in case of common cold symptoms after return, and whether
they know a medical facility they should visit in case of suspected malaria.
RESULTS : Among a total of 468 individuals visiting thequarantine stations, 284 (61%) responded the questionnaire,with the response rate in Tokyo (questionnaires not delivered individually) and Osaka (delivered individually) being

３
７

40% and 99%, respectively. Of those, 48% cases were subjected to analysis. Those who know the transmission route(mosquito bite), main symptom (fever) and primary preventive measure (avoiding mosquito bite) of malaria accounted
for 85%, 83%, and 69%, respectively. Eighteen percent
stated that the primary preventive measure is vaccination.
Only as few as 40% knew whether their destination is malarious, 19% would visit a hospital specialized for infectious diseases in case of common cold symptoms, and 19%

knew a medical facility they should visit in case of suspected malaria.
Very few travelers (11%) got the information fromtravel agencies on malaria prevalence in their destinations.
CONCLUSION : Japanese international travelers visiting
malarious areas have moderate knowledge of malaria; however, their preparation for the disease is unexpectedly insufficient and needs to be impromved by education.

W−I−3) DISEASE TRENDS AMONG JAPANESE PEOPLE LIVING IN SOUTHEAST ASIA
AKIRA UCHIKOSHI, EIICHI OKUZAWA, NAOKO HONDA, NORIHIKO OOKUBO, ATSUO HAMADA
Labor Welfare Corporation. Japan Overseas Health Administration Center,
Japan Overseas Medical Fund

Purpose and Method: The number of Japanese people living abroad is increasing every year. We conducted researchat two different hospitals in South East Asia (Ram Hospitalin Chiang Mai, Thailand, and the Subang Jaya Medical
Center in Kuala Lumpur, Malaysia) and have investigateddisease trends among adult Japanese patients. We gathereddisease names of adult Japanese (≧16 years old) who visited outpatient clinic in both hospitals from January 2000 to
December 2001, and the data was classified according to
the International Classifications of Deseases (ICD-10).
Results: At Ram Hospital the research covered a total of4315 Japanese patients. The most common were respiratory diseases (739 cases, usually including respiratory tract
infections), which was followed by digestive tract diseases(652 cases, often including dental caries) and infectious diseases (456 cases, many including traveler‘s diarrhea). At
Subang Jaya M. C., the reserch period included 4570 Japanese patients. The most comomon were respiratory diseases (239 cases, mostly respiratory tract infections), which

was followed by ocular diseases (237 cases,showing symptoms of sore and/or swollen eyes) and unclassified clinicalsymptoms (237 cases, often including fever probably due torespiratory tract infections). During times of seasonalchange, respiratory diseases were common in both hospitals
during dry periods. At Ram Hospital, many patients suffered from infectious diseases during the monsoon season.
At Subang Jaya M. C., patients suffered from ocular diseases during the dry seasons.
Conclusion: Most of adult Japanese visited the hospitals
because of acute diseases such as respiratory tract infections,traveler‘s diarrhea, dental caries and sore eyes. However,the number of Japanese who visited the hospitals forchronic diseases (i. e. metabolic disorders or circulatory diseases) was low. During the research period, we offeredhealth consultations to adult Japanese living in the cities,
and many of them suffered from the above-mentioned
chronic diseases. It is necessary to urge them to visit thehospitals for taking medical cares of the diseases.

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８

W−I−4) THE STORAGE AND SUPPLY SYSTEM OF ORPHAN DRUGS FOR TROPICAL
AND PARASITIC DISEASESMIKIO KIMURA1, YUKIFUMI NAWA2, HIROSHI OHTOMO3,RESEARCH GROUP ON CHEMOTHERAPY OF TROPICAL DISEASESInfectious Disease Surveillance Center, National Institute of Infectious Diseases1, Department of Parasitology,
Miyazaki Medical College2, Department of Tropical Medicine, Jikei University School of Medicine3

Despite a significant number of cases with tropical andparasitic diseases, major drugs for those diseases including
several antimalarials are not licensed in our country. The
foundation of our research groups that import, store andsupply those orphan drugs dates back to the year 1980. The
present research group, the Research Group on Chemotherapy of Tropical Diseases, is funded by the Japan Health Sciences Foundation. Recently, the number of institutions wedesignated to store and supply the orphan drugs has been
increased to a total of 23 throughout our country, aimingthat every Japanese physician who needs the drugs can haveaccess without significant delay. We also initiated communications with some European hospitals that are specializingin tropical and parasitic diseases in order to update trends ofthe worldwide drug usage. We also publicized our web
page on which the drugs we introduced, institutions storing
those drugs and other useful information on travel-related
illnesses are described. On our closed listserve our member
physicians are discussing various issues, e.g. the availabilityof some drugs from other member physicians and the diagnostic problems of actual cases.

Until October 2001 when mefloquine was marketed,almost all mefloquine that was used for the treatment of falciparum malaria in our country must have been supplied
from our research group, as chloroquine and primaquine arestill now in the case of vivax malaria. Lives of many patients with severe malaria have been saved by injectable
quinine, and recently with artesunate, supplied from our research group. In addition, we recently experienced fourcases of severe amebic dysentery, three of which were associated with HIV infection, that showed dramatic improvement after receiving injectable metronidazole supplied fromour research group. We also succeeded to introduce threekinds of drugs for African trypanosomiasis from a responsible section of WHO.
Thus, we are intending to grow our reseach group network into a comprehensive one in which we, not only supply the drugs upon request, but also accept and manage consultations regarding various clinical issues of tropical andparasitic diseases including diagnostic and therapeutic matters.

W−II−1) MOLECULAR ANALYSIS OF α-THALASSEMIA IN NEPAL:CORRELATION WITH MALARIA ENDEMICITY (II)SHINJIRO HAMANO1, SHIGERU KOBAYASHI2, AKIRA SUZUKI3, KANJI WATANABE4, TAKU SHIRAKAW3,KAZUNARI ISHII1, HITOSHI TADA1, YOSHIDA AOKI4, KAORU NISHIYAMA3, KUNISUKE HIMENO1
Department of Parasitology, Faculty of Medical Sciences, Kyusyu University1,
Department of. Human Geography, Gradute School of Letters, Osaka University2,
Department of. Health Science, School of Medicine, Kobe University3,
Institute of Tropical Medicine, Nagasaki University, Japan4

Thalassemia is a group of hereditary disease caused by
impaired synthesis of specific globin chain. The disease hasa high frequency in regions endemic for malaria including
the Mediterranean Basin, Africa, Middle East, Southeast
Asia and South China. It has been suggested that the highfrequency of thalassemia might reflect advantage due to reduced susceptibility to malaria. To evaluate this malaria hy-

pothesis, we analyzed thalassemia mutations in four neighboring populations of Nepal, the Tamang, the Newar, theParbate and the Danuwar. The settlements of the Tamang
are lacated in the malaria free upland, while those of the
Danuwar are below the limit of the malaria zone. The
Newar and the Parbate had been livid in the malaria free upland up to 1960s, when malaria eradication program was３
９accomplished, and then transferred to the malaria zone,
where is convenient to cultivation. We detected the 3.7 kb
deletion type of α-thalassemia (-α3.7) with a high gene frequency, 0.54 in the Danuwars, while only 0.05 in the
Tamangs, 0.14 in the Parbates and 0.28 in the Newars,
Haplotype analysis of α-globin gene cluster showed that the
α-thalassemia mutation has four different genetic background in the Danuwars. These results suggest that the highfrequency of α-thalassemia in the Danuwar is due to the adaptation to malarial environment rather than to events suchas a bottleneck.
To examine the relationship between α-thalassemia

and the actual malaria infection, we analyzed the malaria
infection rate by genotype in the malaria zone mentioned
above since 2000. In September 2000, 3 of 12 (25.0%) in
αα/αα, 1 of 35 (2.9%) in -α3.7/αα and 5 of 37 (13.5%) in-α3.7/-α3.7 were positive for plasmodium by PCR. In March
2001, 20 of 63 (31.7%) in αα/αα, 5 of 38 (13.2%) in -α3.7/
αα and 5 of 26 (19.2%) in -α3.7/-α3.7 were positive, and in
February 2002, 21 of 82 (25.6%) in αα/αα, 6 of 39(15.4%) in -α3.7/αα and 2 of 29 (6.9%) in -α3.7/-α3.7 werepositive for plasmodium. These results showed that the malaria infection rate was significantly lower in the population
with α-thalassemia than those without α-thalassemia.

W−II−2) KNOWLEDGE AND PREPARATION REGARDING THE RISK OF MALARIAAMONG JAPANESE INTERNATIONAL TRAVELERSKAWAKAMI, K.

W−II−3) MOLECULAR ANALYSIS OF CHLOROQUINE RESISTANT RELATED GENE IN MALAWITOSHIHIRO MITA1, AKIRA KANEKO1, LUN J KOJIRO1, BWIJO BWIJO1, TAKECHI MIHO2,INNCENT L ZUNGU2, TSUKAHARA TAKAHIRO1, and KOBAYAKAWA TAKATOSHI1
Department of International Affairs and Tropica Medicine, Tokyo Women’s Medical University School of Medicine1,
Community Health Sciences Unit, Ministry of Health and Population2

Although chloroquine resistant Plasmodium falciparum has been spread in Africa since 1970, chloroquine isstill important compound because of its inexpensivenessand safeness. In Malawi, first line drug for treatment of P.
falciparum was officially replaced from chloroquine to sulfadoxine/pyrimethamine in 1993 because of high rate oftreatment failure (80%) and in vitro resistance (48%). To
date, the use of chloroquine has been strictly enforced. We
conducted two drug efficacy surveys of in vitro in 1998 andin vivo in 2000 at Salima district in Malawi and found significant reduction of chloroquine resistant isolates both invitro (3%) and in vivo (9%).
We assayed two genetic mutations implicated in chlroquine resistance (pfmdr1 and pfcrt) from 128 P. falciparum
isolates collected during the 1998 and 2000 resistance studies P. falciparum DNA was extracted from filter paper with
finger prick blood Nested PCR followed by RFLP methods
were performed to determine pfcrt polymorphisms at position 76, 220, 271, 326, 356 and 371, and for pfmdr1 at position 86, 184, 1034, 1042 and 1246.
In pfcrt, mutant type alleles were detected in 2 to 21%

of isolates at position 76, 220, 271, and 371. The prevalence of mutant alleles significantly decreased from 1998 to2000 at position 76 (P=0.007), 220 (P=0.009), 271 (P=0.007) and 371 (P=0.007). Nearly all of mutant alleles
were detected in isolates with mixed infections; i.e. including both wild and mutant. In pfmdr1, high prevalences ofmutant type alleles were detected at position 86 and 184.
No defference was found in these prevalences between 1998
and 2000. In pfcrt, Fst value were significantly high andsimilar level (0.051-0.055) at position 76, 220, 271, and 371
between two populations (P<0.02). Contrary to pfcrt, theFst value at position 86, 184, 1042, and 1246 were not significant in pfmdr1. These results suggested that pfcrt has
four distinct regions (76, 220, 271, and 371) of loci with respect to allele frequency between parasite population in1998 and 2000.
Our results support one possibility that the eliminationof chlroquine pressure has reduced the prevalence of mutant
pfcrt alleles and resulted in a substantial recovery of chloroquine sensitivity. Furthermore, in addition to the K76T
polymorphism chloroquine sensitivity is highly dependent

４０

on other as yet unidentified polymorphisms or loci.

W−II−4) GENERATION OF TRANSGENIC ANOPHELINE MOSQUITOES EXPRESSING
A GENE ENCODING ANTI-MALARIA MOLECULESHIGETO YOSHIDA1, TONY NOLAN2, FLAMINIA CATTERUCCIA2, HIROYUKI MATSUOKA1,CHISATO SEKI1, DAISUKE KONDOH1, AKIRA ISHII1
1

2

Departments of Medical Zoology, Jichi Medical School,
Department of Biological Science, Imperial College of Science, Technology and Medicine

The recent generation of genetically transformed mosquitoes has raised hopes for the production of mosquito
strains that are unable to transmit various parasites and viruses.
We have previously constructed a gene encoding of asingle-chain antibody fragment (scFv) specific for Pbs21
molecule expressed on the ookinete surface. The scFv, designated 13.1 scFv, bound to the surface of P. berghei ookinete, and blocked oocyst development in the mosquito midgut by at least 93%, as assessed by oocyst counts in mosquitoes. To examine whether expression of 13.1 scFv in themosquito midguts could lead to transmission-blockade ofmalaria in the mosquito stage, we generated 5 lines of transgenic Anopheles stephensi mosquitoes that express the 13.1
scFv-Shiva gene under the control of the gut-specific andblood-inducible An. gambiae carboxypeptidase (AgCP)
promoter using Minos germ line transformation. The embryos were microinjected by using glass needles with amixture of Minos-13.1 scFv-Shiva transfer plasmid and Minos transposase plasmid. The Minos transposase mediates
integration into the genome of An. stephensi cells. Hatchedlarvae were analysed on an inverted microscape at a wavelength of 490 nm to detect EGFP expression.
Real-time PCR analysis showed that expression of
13.1 scFv-Shiva mRNA is specifically induced (8-fold) in
the guts of transgenic mosquitoes, with peak expression at〜3h after a blood meal. By 48h after a blood meal, mRNA
abundance retums to a level close to that present before ablood meal. By 48h after a blood meal, mRNA abundance
returns to a level close to that present before a blood meal.
To examine inhibition of oocyst formation in the transgeniclines, wild-type and transgenic mosquitoes were allowed tofeed on the same P. berghei-infected mouse. Oocyst formation in one of transgenic lines were significantly reduced(50%) compared with that in wild-type.

W−III−1) INTRANASAL INOCULATION OF YEAST-PRODUCED MALARIA OOKINETE SURFACEPROTEINS CO-ADMINISTERED WITH CHOLERA TOXIN INDUCES COMPLETE
TRANSMISSION-BLOCKING IMMUNITY AGAINST A RODENT AND A HUMAN MALARIA PARASITEAYANO KISHIMOTO1, TAKAFUMI TSUBOI2, MAYUMI TACHIBANA2, TAKESHI ARAKAWA3,
JETSUMON P. SATTABONGKOT4, NANTAVADEE SUWANABUN4, THANAPOM RUNGRUANG2, HAJIME HISAEDA5,ANTHONY STOWERS6, YASUNOBU MATSUMOTO7, NAOTOSHI TSUJI8, YOSHIYA SATO3,9, and MOTOMI TORII2
Dept. Bio-Production, Sch. Agri., Univ. Ryukyus, 2Dept. Mol. Parasitol., Ehimc Univ. Sch. Med., 3Ctr. Mol. Biosciences,
Univ. Ryukyus, 4Dept. Entomol., Armed Forces Res. Inst. Med. Sci., Thailand, 5Dept, Parasitol. Immunol.,Tokushima Univ., Sch. Med., 6Malaria Vaccine Dev. Unit, Lab. Parasitic Dis., Natl. Inst. Allergy & Infect. Dis., NIH, USA,
7
Dept. Glob. Anim. Sci., Grad. Sch. Agri. & Life Sci., Univ Tokyo, 8Natl. Inst. Anim. Health, Natl. Agri. Res. Org.,
9
Dept. Parasitol., Sch. Med., Univ. Ryukyus
1

Transmission-blocking Vaccines against sexual-stage
malaria parasites have been considered one strategy for malaria control. Recently, several genes encoding ookinete

surface proteins (OSP) have been cloned from Plasmodium
species of rodent, human and avian malaria parasites. To
investigate whether mucosal vaccines against malaria para-４１

site OSPs are feasible strategy for induction of systemictransmission-blocking immunity, the yeast-synthesized P.
yoelii Pys25 protein was administered to DBA/2 micethrough oral or nasal route in combination with cholera
toxin (CT). Intranasal administrations of Pys25 in the presence of CT induced storong systemic humoral immune responses evidenced by high levels of serum IgG1 with low
but detectable levels of IgG2a antibodies, suggesting thatTh-2 type immune responses were predominantly induced
against Pys25 protein. Howeber, in the absence of CT the
intranasal immunization failed to induce any detectable levels of serum antibodies. Oral administrations of Pys25 evenin the presence of CT did not induce antibody responses.
When Anopheles stephensi mosquitoes were allowed tofeed on mice infected with Plasmodium yoelii 17X, which
previously vaccinated intranasally with Pys25/CT, oocyst
formation in mosquito midgut was completely blocked inevery engorged mosquitoes examined. Further, passivc
transfer of immunesera to naı̈ve mice conferred complete
transmission-blocking immunity. Similarly, intranasal immunization of mice with Plasmodium vivax ookinete surface protein, Pys25 induced significant serum IgG with highIgG1/IgG2a ratio. Feeding Anopheles dirus mosquitoes
with mixtures of immunesera and gametocytemic blood derived from vivax volunteer patients in Thailand significantly
reduced both the number of midgut oocyst as well as thepercentage of infected mosquitoes.
The observed
transmission-blocking effect was dependent on immunesera
dilution. These studies demonstrate for the first time thatthe mucosally induced mouse immunesera against malaria
ookinete surface proteins can completely block parasitetransmission to vector mosquitoes, suggesting the possibility of developing non-invasive mucosal vaccines against
malaria.

W−III−2) HIGH EXPRESSION OF CHOLERA TOXIN B SUBUNIT PENTAMER IN METHANOPHILICYEAST PICHIA PASTORIS AND ITS ORAL IMMUNOGENICITY IN MICETAKESHI ARAKAWA1, JIN YAH1, AYANO KISHIMOTO2, YASUNOBU MATSUMOTO3,NAOTOSHI TSUJI4, HHIROMU TOMA5, and YOSHIYA SANO5
1Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus,
2
Deptartment of Bio-production, School of Agriculture, University of the Ryukyus,
3
Laboratory of Global Animal Resource Science, Graduate School of Agricultural Life Sciences, University of Tokyo,
4
Laboratory of parasitic discases, National Institute of Animal Health, National Agricultural Research Organization,
5
Department of Parasitology, School of Medicine, University of the Ryukyus

Cholera toxin B subunit (CTB) has been expressed inbacterial gene expression systems and successfully used forthe induction of mucosal and systemic immune responses tochemically or genetically conjugeted antigens. In the present study we tested the possibility for yeast to express CTB
gene and evaluated the feasibility of recombinant yeastbased oral immunization strategy. We introduced into
Pichia pastoris chromosome 1) the authentic CTB gene
(CTBori, 2) CTB gene deleted with 22-amino acid leader
peptide coding sequence (CTB 1), and 3) CTB gene fusedat the C’ terminus with yeast microsomal retention signal(HDEL)-encoding sequence (CTBHDEL). We found significant levels of CTBori and CTBHDEL pentamers retaining GM1ganglioside affinity were produced and secreted into themedium (30 µg/ml). We detected CTB 1 transcript by RTPCR but the protein was not detected. Yeast-synthesized recombinant CTB was found heat-labile, quickly dissociatedinto monomers when cells were heat-treated at 100°C, but

the protein was almost completely resistant to desiccation.
Next, to evaluate oral immunogenicity of yeast-produced
CTB, yeast or culture supernatant calculated to contain 8-16
µg of pentameric CTB was given to mice by oral gavage
once a week for 6 weeks. Mice fed yeast cells, but not with
culture supernatant, developed CTB-specific serum IgG(predominantly IgG1) and intestinal S-IgA antibodies.
Feeding 10 µg of commercially available CTB protein
mixed with untransformed yeast cells induced lower levelof CTB-specific serum IgG, indicating that recombinant
CTB accumulated within yeast cells has higher oral immunogenicity than naked CTB, probably due to increased resistance to digestive degradation in gastrointestinal tract.
The induced antibody response was maintained at least forsix months when mice were given extra booster feedings ofyeast cells. Taken togegher, these results suggest that yeast
can be used as an efficient vaccine production system aswell as an oral delivery system for vaccination.

４２

W−III−3) EXPRESSIONS OF CYTOKINES AND MONOSACCHARIDE TRANSPORTERS
IN THE DUODENAL MUCOSA OF GASTRO-INTESTINAL SYMPTOMATIC PATIENTSIN THE RURAL AREA OF THAILANDSUJET LERTANEKAWATTANA1, THAVORN WICHATRONG1, KAMRON CHAISAIRI2,RYUICHI UCHIKAWA3, AND NAOKI ARIZONO3
Nongkhai Hospital, Thailand1, Nongkhai Provincial Health Office, Thailand2, Department of Medical Zoology,
Kyoto Prefectural University of Medicine, Japan3

Expression levels of cytokines and GLUT familymonosaccharide transporters in the duodenal mucosa were
examined in patients from Nongkhai, Thailand, who had received gastrofiberscopy because of the gastrointestinal
problems. Duodenal biopsy specimens were collected froma total of 33 patients (24 males and 9 females, 45.0 +/‐13.5
years old): 10 patients showed present or recent intestinal
helminth infections, including strongyloidiasis, taeniasis
and ascariasis (group A), 7 were urea-test positive, indicating Helicobacter pylori infection (group B), and 16 hadneither helminth infection nor urea test positivity (group C).
Total RNA was extracted from the biopsied specimens andsemi-quantitative RT-PCR was performed. Positivities of
IL-13, IL-5 and IFN-γ mRNA expressions in all patientswere 24.2, 60.6 and 100%, respectively, with the highest
IL-13 and IL-5 positivities in group A followed by group C
and B. IL-5 positive rate was also significantly higher
among patients with high peripheral blood eosinophil
counts (>4%) than in patients with low peripheral blood
eosinophil counts. GLUT-1 and GLUT-5 were detectable inall patients. Although GLUT-1 expressions did not differ
between group A, B and C, GLUT-5 expressions were significantly lower in group B than in group C. These results
indicate that helminth and H.pylori infections result in different immunopathological responses in the duodenal mucosa: lower expressions of type 2 cytokines and monosaccharide transporters in H.pylori infection than in helminth
infections.

W−III−4) STUDY OF ASCARIS AND ULCERATIVE COLITIS-GENETIC ANALYSIS OFCROSS-REACTIVE ANTIGENICITY BETWEEN ASCARIS LUMBRICOIDESHOMEINIS ANTIGEN AND HUMAN COLON MUCOSAHAYASHI ITOKO, KANDA SEIJI, YOSHIDOME ATSUKO, ISHIDA TAKAAKI, NISHIYAMA TOSHIMASA
Dept. of Public Health, Kansai Med. University

It is known that the antibody against the Ascaris is
produced in patient’s serum of ulccrative colitis (UC). We
have already reported the cross-reactive antigens between A.
lumbricoides and human colon mucosa are presented. In
this study, to determine the genes related with cross-reactive
antigens between A. lumbricoides and human colon mucosa,
we analysed the screening method at the DNA levels using
the human colon cDNA library (Clontech laboratories Inc.,
CA,USA). λ phage was infected to E. coli. XL-1 Blue,and IPTG induced-protein derived from each plaques weretransferred to the membrane and they were investigated byimmuno-staining method using the anti-A.lumbricoides rabbit serum. We analyzed 50,000 plaques approximately, 3clones were reacted positively as a result of secondary
screening. To determine the sequences of these clones, each
cDNA was analyzed by the DNA sequencer ABI PRISM

310 Genetic analyzer. As the result, one was considered to
be a transmembrane mucin 12 homolog. Other two genes
were similar to actin, gamma 2 of enteric smooth muscle
and beta-casein-like-protein. Mucin exists in the digestiveorgans, especially in the colon and was reported that the antibody against colonic mucin might contribute to the epitherial cell injury of colonic mucosa through antibodydependent cell-mediated cytotoxicity (ADCC) and thisautoantibody levels are high in some patients for UC. Mucin12 gene contains in the coding regions that are homologous to mucin3, mucin4, mucin11 and mucin17 with highfrequency. In these results, A. lumbricoides was likely toharbor in the human intestine, because mucin3 exists richly
in the intestine, and it was guessed to be a suitable environment for A. lumbricoides.

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３

W−IV−1) FUSION DNA VACCINE AGAINST INFECTION WITH HIGH VIRULENT TOXOPLASMAKAZUNARI ISHII1, YOKO NAKANO2, TOHRU SAKAI2, SHINJIRO HAMANO1, AKIHIKO YANO3, KUNISUKE HIMENO1
1

Department of Parasitology, Faculty of Medical Sciences, Kyusyu University, 2Department of immunology & Parasitology,
Tokushima University School of Medicine, 3Department of Infection Mechanisms and Immunity,
The Graduate Course for Medical Doctor for Science, Chiba University School of Medicine

For most obligate intracellular protozoa such as
Toxoplasma gondii (T. gondii) and Trypanosoma cruzi,
available vaccines have not yet been developed, despite thatthose kinds of pathogen are resistant or less effective forchemotherapy in general. Cellular immunity especially CD
8+ T cell response plays essential roles for the protection
against those kinds of pathogens. Effective protective immunity against those infections is, however, hardly inducible by conventional vaccination using proteins/peptides derived from target pathogens. It is a well-established consensus that protein antigens must be ubiquinated and then processed by proteasome within antigen-presenting cells when
MHC class I-restricted CD8+ T cell response is preferentially induced. From these bases, we fused naked DNA of
SAG-1 (major surface antigen of Toxoplasma gondii) withthat encodinig ubiquitin. Thereafter, BALB/c mice were
subcutaneously injected with this fusion DNA using genegun before the infection with a high virulent RH strain of T.
gondii. Strikingly, most mice vaccinated with the fusion
DNA acquired strong protective immunity and controlled
the infection, although DNA vaccine with eigher DNA encoding SAG-1 gene alone or that encoding ubiquitin genealone was not effective. Furthermore, the effect of ubiquitination was confirmed in vitro using a specific inhibitor forproteasome, MG-132. Mice immunized with the fusion
DNA showed a potend cytotoxic activity of CD+ T cells specific for SAG-1 transfected target cells. This kind of vaccine strategy is expected to be usuful for infections withother type intracellular pathogens including viruses andbacteria.

W−IV−2) AN INTERLEUKIN-18 EXPRESSION PLASMID ADMINISTRATION INITIATES A PROTECTIVE
T HELPER TYPE 1 IMMUNE RESPONSE AND RESOLVES CUTANEOUS LEISHMANIASISYONG LI1, YOICHI MAEKAWA2, SHINJIRO HAMANO1, TOHRU SAKAI2, KAZUNARI ISHII1,
MANXIN ZHANG1 and KUNISUKE HIMENO1
1.Department of Parasitology, Graduate School of Medical Sciences, Kyusyu University
2.Department Parasitology and Immunology, Tokushima University, School of Medicine

We investigated the therapeutic effect of an IL-18 expression plasmid administration on susceptible BALB/cmice infected with Leishmania major. BALB/c mice were
subcutaneously challenged with L. major into hind footpad.
Simultaneously, they were weekly treated with 4 µg IL-18
and/or IL-12 expression plasmid DNA with the gene gun.
All of the treated mice showed similarly decreased footpad
swelling and less parasite burden compared with thosetreated with empty vector (control mice). IFN-γ was signigicantly increased at 3 weeks after infection in all treatedmice but not in control mice. Antibody administration demonstrated that CD4+ T cells and IFN-γ played critical rolesin the protection against L. major induced by IL-18 expres-sion plasmid administration. Moreover, TNF-α was also increased in the treated mice. In TNF-α-deficient BALB/cmice, which were also susceptible and rapidlly succumbedto progressive disease after L. major infection, IL-18 expression plasmid administration did not lead to the resistance as in the wild type mice, despite of the high level of L.
major-specific IFN-γ production. This demonstrated thecritical role for TNF-α in L. major infection. These results
suggest that administration of an IL-18 expression plasmidwith gene gun is able to protect susceptible mice against an
intracellular protozoan infection, with shifting of immune
response towards Th1 direction.

４
４

W−IV−3) PROTECTIVE ROLE OF IgE IN MICE INFECTED WITH PLASMODIUM BERGHEI ANKA
TAKAHISA FURUTA1, TAKANE KIKUCHI1, MIKIO KIMURA2 and NAOHIRO WATANABE3
Division of infectious Genetics, Department of Microbiology and Immunology, The Institute of Medical Science,
The University of Tokyo1, Infectious Disease Surveillance Center, National Institute of Infectious Disease2,
Department of Tropical Medicine, Jikei University School of Medicine3

Recently, it is has been reported that IgE is related tothe pathogenesis in severe malaria infection, particularly in
cerebral malaria. IgE levels in the sera of individuals living
in high P. falciparum endemic areas were investigated, approximately 85% of people from these areas had significantly elevated level of total IgE. However, the role andbiological significance of IgE in the protection against P.
falciparum and pathogenesis of malaria are not known. For
these reasons, we decided to examine the possible roles of
IgE responses using IL-5 transgenic and SJA/9 mice, which
are genetically producing high levels of IgE under normal
condition or deficient of IgE production. 1×103 P. berghei
ANKA-infected erythrocytes were injected i.v. into IL-5transgenic and the background mice, and the parasitemia
was measured every day. After the infection of P. berghei,
although the parasitemia was incresed gradually in bothgroups of mice, the parasitemia in IL-5 transgenic mice wasalways lower than that in background mice, and the IL-5transgenic mice showed longer survival. We examined theprotective role of IgE in P. berghei infection using congenitally selective IgE-deficient SJA/9 and the background SJL/
J mice. After P. berghei infection, the parasitcmia in SJA/9

mice was significantly higher than that of SJL/J mice, andthe mortality of SJA/9 mice was also higher than that of SJL/J mice. These results indicated the protective roles of IgE
for P. berghei infection. The production of anti-P. berghei
specific IgE antibody in the P. berghei ANKA-infected
mice was examined by Western blot analysis, the results
showed that anti-P. berghei IgE antibody was also producedin the sera from these mice. Since IgE has both protective
and pathogenic effects in infectious diseases via two major
receptors, the high-affinity receptor FcεRI and low-affinity
FcεRII or CD23, we examined the possibility that those twokind of receptors involved with IgE-mediated resistanceagainst P. berghei infection using WBB6F1-W/WV mice,
which are congenitally deficient mast cells bearing FcεRI
receptor and CD23 Ko mice. After P. berghei infection in
W/Wv and CD23 Ko mice, the parasitemia in W/Wv and CD
23 was always higher than those in the background mice,suggesting that the protective effect of IgE on P. berghei infection was mediated via FcεRI and FcεRII. In conclusion,
IgE and anti-P. berghei ANKA IgE antibody have importantroles for the protection of P. berghei ANKA infection mediated through FcεRI and RII.

W−IV−4) THE ROLE OF ERYTHROCYTES ON SUPPRESSION OF IMMUNE RESPONSE
IN MURINE MALARIAMICHIKO TOKUNAGA, MINAKO OHNO, MAKOTO OWHASHI
Department of Life Science, Faulty of Integrated Arts and Sciences, Tokushima University

It is well known that malaria is associated with hepatosplenomegaly, anemia and myeloid hyperplasia. The increased levels of hemopoiesis and the phagocyte system arethought to be implicated in this pathogenesis. Immune depression during malaria is also a well-recognized phenomenon that likely contributes on the host-parasite relationship.
Previous studies have shown that suppressor T cells ormacrophages are involved this phenomenon. Recently wefound that RBC from Plasmodium-infected mice showed
increased levels of affinity to some FITC-labelled plant
lectins.

In the present study, we examined the possible role of
RBC of Plasmodium-infected mice on the regulation immune response upon Plasmodium infection. Proliferative
response of splenocytes upon stimulation with Con A or allogenic APC increased in the presence of RBC from normal
mice, whereas depressed in the presence of RBC from P.
yoelii-infected mice. Supernatant of RBC lysate showedsimilar effect on the proliferative response with the RBC.
On the other hand, RBC lysate from P. yoelii-infected miceinduced superoxide production from neutrophils. Timecourse study revealed that the stimulatory activity of the４５

RBC lysate coincided with the increase of parasitemia during P. yoelii 17X infection. The stimulatory activity of the
RBC lysate was partially lost after the treatment withheparin-agarose. HPLC analysis equipped with G3000SW
column indicate that the stimulatory factor possesses a mo-lecular weight mass of around 14kd. The relationship between the depression of the immune response by RBC of P.
yoelii-infected mice and superoxide production from neutrophils was discussed.

W−V−1) APPLICATION OF QUESTIONNAIRES FOR MONITORING SCHISTOSOMA MANSONIINFECTION AFTER MASS-TREATMENTTOMOKO KISU1, YUJI ATAKA1, EIKO KANEDA2, KAZUHIKO MOJI1 AND MASAAKI SHIMADA1
Research Center for Tropical Infectious Diseases1, Department of Social Environment2,
Institute of Tropical Medicine, Nagasaki University

The purpose of the study is to determine if adding
some questions about water-contact behavior on signs andsymptoms improve the efficacy of questionnaires for themonitoring of Schistosoma mansoni infection.
<Subjects and methods>
A total number of 1033 schoolchildren aged 7-17
years in four primary schools in Moshi Tanzania were involved in the study. February 2001, they received mass
treatment with praziquantel and mebendazol for the deworming of schistosome and intestinal parasites. Before thetreatment, the children were requested to answer a questionnaire. They were examined for the ova of schistosome andintestinal parasites in stool before the treatment and one
month later. A year later in February 2002, the examinationand questionnaire were carried out again. Only 267 children completed the examination and questionnaire. The
questionnaire consists of the questions on the subjective
symptoms in the previous two weeks and frequency and nature of their water-contact behaviors. The answers of questions recorded as ’yes’, ’no’ or ’I don’t know’. Stool examination was conducted with Kato-Katz procedure. The association between infection and each questionnaire variable
was calculated by using a chi-square test and a logistic binomial regression analysis.
<Results>
1) The prevalence of S. mansoni infection was 67.4% be-fore the treatment and decreased to 6.4% after treatment.
In 2002, one year after the treatment, the prevalence increased to 27.0%. The mean intensities were 29.8 eggs/
gram, 0.3 eggs/gram and 2.5 eggs/gram respectively.
2) Before treatment, constipation (OR=2.91, 95% C.I.;
1.29-6.58, P=0.010) and net fishing (OR=2.30, 95% C.
I.; 1.29-4.11, P=0.005) were significantly associatedwith infection. Sensitivities and specificities were
20.6%, 89.7% and 43.5%, 70.6% respectively. One
year after treatment, only diarrhea (OR=1.91, 95% C.I.;
1.10-3.32, P=0.021) had significant association with infection. The sensitivity and specificity were 61.1%,
54.9% respectively.
3) No water contact behavior had significant association
with infection in this study.
4) When the factor of the previous infection before treatment was introduced to a logistic binomial regression
analysis, both previous infection (OR=1.96, 95% C.I.;
1.04-3.70, P=0.037) and diarrhea (OR=1.88, 95% C.I.;
1.08-3.27, P=0.026) were significantly associated withthe current infection.
In the study area, questions about water-contact behavior added on signs and symptoms did not improve the efficacy of questionnaires for the monitoring of Schistosoma
mansoni infection.

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６

W−V−2) RAPID SCHOOL HEALTH QUESTIONNAIRE SURVEYS ON URINARY SCHISTOSOMIASISIN COASTAL KENYA AND ZANZIBARKAZUHIKO MOJI1, MOHAMED KARAMA2, NG’ETHE MUHOHO2, HABAB CHWAYA34, ALI-FOUM3,SHIMBA KHAMIS3, YUJI ATAKA1, MASAAKI SHIMADA1, AND YOSHIKI AOKI1
Institute of Tropical Medicine, Nagasaki University1, Kenya Medical Research Institute2,
Ministry of Health and Child Welfare, Zanzibar3, now UNICEF Zanzibar4

The WHO’s resolution on schistosomiasis has set thegoal of attaining a minimum target of regular administrationof chemotherapy to at least 75% of all school-age children
at risk of morbidity by 2010. However, systematic data on
the prevalence of infection, the coverage of administrationof chemotherapy and school health education have not beencollected in Sub-Saharan African countries. Therefore, we
conducted rapid school health questionnaire surverys in
Kwale District of Coast Province of Kenya (K), and in thetwo islands of Zanzibar; Unguja (U) and Pemba (P) in 2001.
In total, 264 schools (113 in K, 84 in U, and 67 in P) wereparticipated in the study. Ten boys and ten girls in class 4
were asked to answer questions in these surveys. In
Zanzibar trained health staffs visited schools and conducted
the survey. In Kenya the procedure of survey was explained
to teachers in a district meeting, and teachers themselves
conducted the survey in their schools. The results were collected by mail. Questions asked were knowledge on schis-tosomiasis, presence or absence of self-diagnosed urinary
schistosomiasis, subjective symptoms such as blood in urine,
contact with river water in the previous two weeks, and experiences of urine examination, treatment, and health education. Mean percentages of children answering “yes” toquestions in K, P, and U were as follows (sex combined):
Knowledge on what schistosomiasis is, 73%, 50%, 31%;
Self-diagnosed urinary schistosomiasis, 39%, 21%, 6%;
Blood in urine, 33%, 19%, 6%; Contact with river water inthe previous two weeks, 82%, 57%, 16%; Health education,
34%, 24%, 31%; Urine examination, 26%, 19%, 8%; Treatment, 26%, 35%, 11%. These results indicate the needs ofsysytematic efforts to achieve the WHO’s goal. The governments must annually conduct this kind of rapid monitoring survey on health status of school children, coverage ofschool health servies, and coverage of health education toaccumulate the basic information on school health.

W−V−3) WATER CONTACT BEHAVIORS AMONG SCHOOLCHILDREN IN AN ENDEMIC AREAOF SCHISTOSOMIASIS, TANZANIA: A COMPARATIVE STUDY OF DIRECT OBSERVAITON
AND 24-HOUR RECALL METHODYUJI ATAKA1, EIKO KANEDA2, MASAAKI SHIMADA1, TOMOKO KISU1, KAZUHIKO MOJI1
Research Center for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University1,
Department of Social Environment, Institute of Tropical Medicine, Nagasaki University2

Estimation of exposure to cercarial infested water iscritical for epidemiological and immunological studies in aschistosomiasis endemic area. Questionnaire surveys havebeen used to estimate water exposures, addressing frequency of water contact only, due to its cost-effectivenessand less labor-intensiveness. Direct observation needs morecost and time, but it provides more accurate exposure information. We compared the results of 24-hour recall methodwith direct observation method for clarifying whether 104
schoolchildren living in Lower Moshi area, a Schistosoma
mansoni endemic area, of Tanzania could accurately recall
their water exposures. Individual tracing method was ap-

plied to obtain detailed direct observation data for all watercontact behaviors, and, on the following day, an interview
was carried out to collect corresponding information on allcontact behaviors. Exposure index (EI: %min/day) was calculated by contact duration and intensity of exposure (percent body surface area) using Lund and Browder burn chart.
The most critical water contact behavior for the infection
was swimming/playing in the river and canal among schoolchildren. They used safe water, in most cases, from the tap
or deep well for bathing purpose. We found a significantly
smaller frequency (4.5 vs. 6.4, p<0.001), but longer duration (29.7 vs. 24.6 min, p<0.01), of water contact in the re-４
７call data than directly observed data. For the risk watercontacts, the EIs of water contacts obtained by recall
method were significantly higher than those of direct observation (p<0.001). The EIs of 24-hour recall were significantly correlated with those of direct observation (ρ=0.885,
p<0.01); more strongly among boys (ρ=0.931, p<0.01)
than girls (ρ=0.750, p<0.01). Although the values of the EI

are significantly higher in the recall data and should be corrected in practice, these results show a possibility of replacing direct observation data by detailed recall data. The 24hour recall method is a practical procedure to accurately
quantify water exposure in Lower Moshi area where S.
mansoni has been endemic.

W−V−4) SCHOOL-BASED AND RESIDENCE-BASED ANALYSES ON INFECTIONS WITH
S. HAEMATOBIUM AND S. MANSONI AMONG CHILDREN IN MOSHI, TANZANIAEIKO KANEDA1, TOMOKO KISU2, YUJI ATAKA2, MASAAKI SHIMADA2, AND KAZUHIKO MOJI2
Dept. Soc. Med., Inst. of Trop. Med., Nagasaki Univ.1 Res. Cent. for Trop. Infect. Dis., Nagasaki Univ.2

Schistosomiasis in Africa is not widely and evenly
spread along river lines, but is scattered in clusters in someparticular areas. The areas vary in seasons and years, which
seems to have influence upon the infection with the disease
to human bodies. This paper is to report the characteristics
of distributions for two kinds of schistosomes, S. haematobium and S. mansoni, both existing in the same area, one
from the view of school districts and another from that ofdwelling places.
Subjects for our survey were the children in grades
four and five from Chekereni primary school in Chekerini,and those from Oria primary school in Oria school district,the number of which was 284 in total, the former 199, andthe latter 85. Whether inflection was positive or negative
was determined by examining existence of the eggs in subjects’ feces and urine. Kato-Katz method for S. mansoni,and nuclepore filter paper method for S. haematobium dis-eases were adopted.
Presence of S. mansoni eggs was positive among
50.8% of children from Chekereni primary school whereas
31.2% in Oria primary school. S. haematobium eggs wereobserved among 20% of children in Oria primary school,
while only few children from Chekereni primary school
were infected. No significant difference was observed on
the prevalence of S. mansoni between Chekereni children
coming from Chekereni area and those coming to the sameschool from Oria area (P>0.05). While for that of S. haematobium, a significant difference was observed (P<0.05).
Epidemiological surverys related to the infection withthe parasaites have often been carried out on the basis ofschool districts. The distripution of infection is often biased
by endemic area. We conclude that location of subjects’ inhabitation should also be considered for epidemiologicalsurveys on parasitic diseases.

W−VI−1) CHARACTERIZATION OF THE cDNA OF THE MIRACIDIAL ANTIGEN FAMILY OFSCHISTOSOMA JAPONICUM (CHINESE MAINLAND STRAIN).

４８

W−VI−2) MOLECULAR CLONING OF A NOVEL PROCYCLIC FORM-SPECIFIC GENE (CDNA)
FROM TRYPANOSOMA BRUCEI BRUCEISHIGERU OHSHIMA, TAKASHI SUZUKI, YOSHISADA YABU, MARIKO HATOH and NOBUO OHTA
Dept. Mol. Parasitol., Nagoya City Univ. Grad. Sch. Med. Sci.

Trypanosoma brucei brucei protozoa spreads between
the mammal host and the tsetse fly vector in its lifecycle. In
order to control Trypanosoma, we analyzed geneexpression profiles of blood stream and procyclic (insect)
forms of T. b. brucei using the method of fluorescence diffierential display. About 12000 cDNAs were surveyed, anda total of 39 differentially regulated cDNA bands were identified. One markedly differential expressed cDAN, which
was up-regulated in procyclic from, was selected and fulllength of which was obtained by 5’RACE method. The result showed that the gene is 3304bp encoding an ORF of
813 amino acids. Genbank database search revealed that
this gene has 38% identity with Trypanosoma cruzi (T.
cruzi) Cyclin 5 gene. T. cruzi Cyclin 5 was known to bebound to TzCRK1 (T. cruzi Cdc2p-related kinase 1) which

is one of Cdks (cyclin dependent kinase). The function of T.
cruzi Cyclin5 is still uncertain but presumed to play an important role(s) in controlling the cell. It is thus suggested
that the novel procyclic form-specific gene (cDNA) from T.
b. brucei also plays an important role in the process of thecell cycle similar to T. cruzi Cyclin5.
The result of RT-PCR demonstrated that the gene was
not expressed in long slender blood stream form, while it
was expressed in short stumpy (pre-adapted) form, which
can change to procyclic form when the blood is sucked bytsetse fly. Therefore, it seems likely that this gene has animportant function in lifecycle change of T. b. brucei fromblood stream to procyclic forms. In order to clarify thefunction of the gene, RNAi-mediated gene knock down
analysis is under way.

W−VI−3) ANALYSIS OF LEISHMANIA AMASTIGOTE STAGE-SPECIFIC MOLECULESCHIZU SANJOBA1, TETSUHIKO SASAKI2, YASUYUKI GOTO1, TAKASHI ONODERA1 and YOSHITSUGU MATSUMOTO1
Dept. of Mol. Immunology, School of Agriculture and Life sciences, Univ. of Tokyo1,
Dept. of Biological Sciences, Univ. of Tokyo2

A Leishmania protozoan differentiates from the promastigote to the amastiogote during its digenetic life cycle
between invertebrate and vertbrate hosts respectively. The
promastigote can be easily cultivated in vitro. This has resulted in Leishmania promastigotes being investigated in awide range of basic biological sciences and immunology onleishmaniasis. In contrast, in vitro cultivation of the amastigote stage is still a challenge. As a result, relatively little
is known about amastigote stage-specific molecules. The
proliferation of amastigotes in macrophages results in cell
rupture and tissue damage, giving rise to the various symptoms associated with leishmaniasis. Thus, detailed investigation of amastigotes in teams of protein levels is crucial tounderstand the pathogenesis of leishmaniasis. Moreover,these studies will help to understand the survial mechanisms of amastigotes during infection within the mammalian host. The identification of amastigote stage-specificproteins was addressed by proteomic approaches using thetechnique of two-dimensional electrophoresis. The protein

expression patterns of Leishmania major amastigotes purified from cutaneous lesions in experimentally infected Rag2
knock out mice (BALB/c) and cultured promastigotes werecompared. The majority of protein spots occurred between
28 kDa and 94 kDa; at least 32 spots from amastigotes and
43 spots from promastigotes were clearly visualized on a
Coomassie Brilliant Blue -stained gel. although 11 protein
spots were found to be expressed by both amastigotes andpromastigotes, and the remaining 21 protein spots were specific to amastigotes and 32 spots were promastigote specific.
These results demonstrated that L. major expresses stagespecific proteins. Sequentially, to detect amastigote stagespecific antigens, proteins from promastigotes and amastigotes were analyzed by western blotting using the polyclonal antibodies from a rabbit immunized with homogenatesof purified L. major amastigotes. Three specific spots ofamastigotes were detected. Determination of amino acidsequences of these amastigote stage-specific antigens is
now on going. These studies will contribute to the develop-

４
９ment of imunological diagnosis or treatment, or effective
vaccine against leishmaniasis with the new concept thatamastigote stage-specific antigents should be targets forcontrol measures.

W−VI−4) THE QUANTIFICATION OF THE GENES INVOLVED NO PRODUCTION
IN LEISHMANIA-INFECTED MACROPHAGE BY REAL-TIME PCR
YAMAMOTO, Y.

W−VII−1) CHEMOTHERAPEUTIC APPROACH TO AFRICAN TRYPANOSOMIASIS:
THE USE OF DIORGANOTIN AND ITS DERIVATIVESNASIR MOHAMMED SHUAIBU1,2, HIROJI KANBARA1, TETSUO YANAGI1, AKITOYO ICHINOSE4, A. D. AMEH2,
J. J. BONIRE3 and JONATHAN ANDREW NOK2
Department of protozoology, Institute of Tropical Medicine, Nagasaki University1,
Biochemistry Department2,
Chemistry Department3,
Ahmadu Bello University, Zaria, Nigeria, Central Laboratory, Institute of Tropical Medicine, Nagasaki University4

African trypanosomiasis is an endemic disease in nature with recurrent outbreaks, affecting both human and his
domestic livestock; with no vaccine at near sight. The disease is fatal if untreated and chemotherapy remains themost powerful strategy to date, albeit narrow therapeutic index. Despite the tremendous progresses made in the identification and characterisation of biochemical and molecularproperties of trypanosomes, potential drug target to produce
an “ideal trypanocide” proves elusive. Current drugs thatare available are old, scarce, less effective, toxic and encounter parasite resistance. Therefore there is an urgent
need for new drugs for the treatment of this disease. The
development of in vitro drug screening assays has improved
the search for compounds against pathogenic trypanosomes.
The proliferation of trypanosomes is rapid and has been likened to some types of cancer cells and all the currently useddrugs against human African trypanosomiasis (HAT) haveanti-cancer effect or vice versa. DFMO was first developed
as anti-cancer drug, but now has therapeutic activity against
Gambian sleeping sickness, even in the late CNS-involved
stage. Organometallics have also been reported to haveboth anti-cancer and anti-trypanosomal effects. To date

only organoarsenic melarsoprol that cures all forms of HAT
albeit undesirable and often fatal side effects. African Trypanosome are partially auxotrophic for lipids and are salvaged from the host environment for the synthesis of conventional membrance and GPI anchor.
In an attempt to explore the chemotherapeutic potentialof organotin and to broaden the search for much needed trypanocide an approach was made against three species oftrypanosoma, using organometallic dibutyltin dichloride
and its fatty acid derivatives as antitrypanosomal agents invitro. The compounds mediated the killing of the parasites
with minimum inhibitory concentration (MIC) values ranging from 76.0-597.0 ng/ml, 53.2-597.0 ng/ml and 106.4600.0 ng/ml for Trypanosoma brucei brucei, T. b. gambiense and T. b. rhodesiense, respectively. Our observed
selectivity indices of these compounds against murinederived fibroblasts are not as high as the currently used trypanocides. These observations suggest the chemotherapeutic potentials of organotins and the approach could be usefulin either developing new lead compounds or improve theuse of the current trypanocide in use.

５０W−VII−2) THE INDUCTION OF TNF-α AND iNOS EXPRESSION BY MACROPHAGE CELLS AGAINSTONO-4007 (A SYNTHETIC LIPID A DERIVATIVE), IFN-γ AND MEGLUMINE ANTIMONIATEIN VITRO STUDY
MOHAMMED ABUL KASEM KHAN1, MOTOYOSHI MARUNO1, NOOR MOHAMMAD KHASKHELY1, HIROSHI UEZATO1,EDUARDO A. GOMEZ LANDIRES2, YOSHIHISA HASHIGUCHI3 and SHIGEO NONAKA1
1) Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa,
2) Department of Tropical Medicine, Catholic University OF Guayaquil, Guayaquil, Equador,
3) Department of Parasitology, Kochi Medical School, Kochi, Japan

Introduction: In leishmaniasis, TNF-α induces a lytic
mechanism in protecting against Leishmania parasites
through stimulating inducible nitric oxide synthase (iNOS),
which catalyzes to nitric oxide (NO) by activated macrophage cells. The purpose of this study to clarify the antileishmanial action of ONO-4007 (a LPS derivative) or
IFN-γ or meglumine antimoniate as a single or alternately
in combination of ONO-4007 plus IFN-γ, induced by TNFα and iNOS expression.
Material and Methods: 1) Promastigtes of L. major(MHOM/SU/73/5ASKH) and murine macrophage cell line,
J774 were cultured in RPMI and DMEM medium respectively. 2) ELISA for TNF -α: Macrophages were cultured
and infected with L. major promastigotes for 24 hrs. The
drugs were administered as a single regime: ONO-4007(0.01, 0.10 and 1.0 mg/ml), IFN-γ (1, 10 and 100U/ml),
meglumine antimoniate (0.085, 8.5 and 850 µg/ml) and also
in combination regime: ONO-4007+IFN-γ for 24 hrs. TNFα in the supernatant was assessed at 6-24 hrs incubation by
ELISA. 3) Immuno-histochemical study: Macrophages were
cultured and incubated for 24 hrs with ONO-4007, IFN-γ,
and also in combination of ONO-4007+IFN-γ. After 6, 12
and 24 hrs of incubation with these drugs, fixation of thecells were done with 100% ethanol alcohol and immunostaining performed by using primary antibody against
iNOS (Wako, Japan).
Results and Conclusion: ONO-4007 alone and in combination of ONO-4007 plus IFN-γ, induced the significant
higher level of TNF-α following 6-24 hrs of incubation at
three different concentrations as compared to control. TNFα production by infected-Mφ cells in response to IFN-γ andmeglumine antimoniate as a single regime did not induce
the significant higher level at three different concentrations
as compared to contorol. Immunohistochemical observations revealed that the positive reactivity for iNOS expression were observed at 6 hrs of incubation and the intensityof the staining pattern gradually increased following 12 hrs
and 24 hrs of incubation, in response to ONO-4007 andIFN-γ as a single and also in combination regime of ONO4007+IFN-γ. From these observations, we suggest that
ONO-4007 either alone or in combination of IFN-γ mightact as an antileishmanial agent by inducing TNF-α andiNOS production by macrophage cells in vitro study.
Meglumine antimoniate exerts its antileishmanial action
through different mechanism but not involving thecytokine-immune pathway.

W−VII−3) MARINE SPONGES; HOPEFUL SOURCES OF NEW ANTI-PROTOZOAN DRUGSYASUYUKI GOTO1, YOICHI NAKAO2, CHIZU SANJOBA1, YUKO KATAKAI1, SUKMAWATI BASUKI1,
SHIN-ICHIRO KAWAZU3, TAKASHI ONODERA1 and YOSHITSUGU MATSUMOTO1
Department of Molecular Immunology, University of Tokyo1,
Department of Aquatic Natural Product Chemistry, University of Tokyo2,
Research Institute of International Medical Center of Japan3

In recent marine sponge has received much attention asa source of unique chemicals, which affect eukaryotic cells.
Those chemicals include mycalolide B and jasplakinolide,which shows anti-tumor activities and effects on the structure of actin filaments, respectively. In this study we exam-

ined the anti-protozoan activities of extracts from various
marine sponges. The lipophilic and hydrophilic layers frommarine sponges were examined on the point of inhibition
activities against Plasmodium falciparum and Leishmania
amazonensis. Inhibition activities against P. falciparum５
１erythrocytic stages in vitro were examined by direct counting on Giemsa stained smears. For anti-leishmanial activities, L. amazonensis LV78 promastigotes transfected withenhanced green fluorescent protein (EGFP) gene were used.
The fluorescence intensity of transfected promastigotes wasin proportion to the number of promastigotes, indicatingthat this method could be the high throughput screening
method. Among extracts from 93 marine sponges tested
against P. falciparum, 8 lipophilic and 4 hydrophilic layers

showed > 50% inhibition at the concentration of 1 µg/ml.
For anti-leishmanial activites 5 lipophilic layers among
those from 61 marine sponges showed > 50% inhibition at
the concentration of µg/ml. Further fractionation of theselipophilic layers by HPLC, the 50% inhibition concentration (IC50) values of 4 fractions against P. falciparum and 4
fractions against L. amazonensis were < 100 ng/ml. These
results suggest that marine sponges are hopeful sources of
new anti-protozoan drugs.

W−VII−4) COMBINATION EFFECTS OF CHLOROQUINE WITH FEBRIFUGINE / ISOFEBRIFUGINEMIXTURE AGAINST CHLOROQUINE-RESISTANT PLASMODIUM BERGHEI NK65 IN ICR MICEAKIRA ISHIH1, TOHRU SUZUKI1, MACHIKO WATANABE1, TOSHIO MIYASE2, MAMORU TERADA1
Department of Parasitology, Hamamatsu University School of Medicine1,
Department of Pharmacognosy, School of Pharmaceutical Science, University of Shizuoka2

Of strategies on controlling malaria which is one of the
major health problems in the world, chemotherapy is theprimary defense against malaria. Under world-wide emergence of chloroquine-or multiple drug-resistant variants of
Plasmodium falciparum, new approaches to treatment ofmalaria are urgently required as well as a development of
new effective drugs. Recently, several reports have been
published in combination therapy. In this study, we reportour results on combination of chloroquine (CQ) with febrifugine/ isofebrifugine mixture (FI) in ICR-P. berghei NK65
model. The alkaloid fraction was prepared from the driedleaves of H. macrophylla var. Otaksa using a porous polymer gel (Mitsubishi Diaion HP-20) column and a cation exchanger (Amberlyst 15) column. Outbred male ICR mice,
7 weeks old, were infected intraperitoneally with1x105 P.
berghei NK65-parasitized erythrocytes. 1) From day 4 after injection of parasitized erythrocytes, mice were treatedorally with a single dose of 40 mg/kg of CQ base, a twoday dosage of 20 or 40 mg/kg of CQ base or a three-day

dosage of 20 mg/kg of CQ base in the treated group. Distilled water was given in the untreated, infected mice. Micein the untreated control showed a progressively increasing
parasitemia leading to death. A treatment of CQ alone
showed little effect against P. berghei NK65 infection, andall mice died up to day 17 with an increasing parasitemia.
2) A four-day dosage of 1 mg/kg, twice a day, of FI alone
showed a little effect, but all mice died up to day 27. All
mice treated with FI showed low parasitemia levels during adrug administration and following a few days, but then malaria parasites increased in the bloodstream of the treatedmice until death. On the other hand, mice treated with CQ
plus FI survived during the experiment. Malaria parasites
in the mice given CQ plus FI decreased on day 6 and thencould not be detected by a microscopic examination duringobservation period. Studies are in progress to clarify themechanisms of combination effects of CQ plus FI against
the parasite.

５２B−1) CLINICAL FEATURES OF MUCOCUATANEOUS LEISHMANIASIS IN THE
AMAZONIAN REGION OF DCUADORMANUEL CALVOPINA1,2 and YOSHIHISA HASHIGUCHI2
1

Unidad de Inmunologia y Medicina Tropical, Universidad Central, Ecuador
2
Dept.Parasitology Kochi Medical School, JapanPURPOSE: To determine in an active survey the presence of mucocutaneous leishmaniasis (MCL) in persons living at north east of the Amazonian region of Ecuador.
INTRODUCTION: MCL is the most serious and destructive clinical form of American cutaneous leishmaniasis.
Figures of skin lesions facial deformities have been represented on pre-Inca pottery from Ecuador, Peru and Colombia. Although MCL was reported since 1924 and is believed to be endemic in the Amazonian region, relatively little active research has been done. Data reported in Ecuadorian medical journals during 1920 to 1987 showed 260
cases, of these 18 (6.9%) were MCL.
STUDY AREA, PATIENTS & METHODS; The study
was carried out in the provinces of Sucumbios and Orellana. Individuals suspected to have MCL were clinicallyand parasitologically examined using Giemsa-stained
smears, culture of aspirated, PCR and histopathologic studyof skin biopsy. Sandfly collections were done by protected
human bait. Itraconazole was the drug used to treat all patients.
RESULTS: 13 patients were found positive. The age
ranged from 22 to 67 years. All patients have got lesions in

nasal mucosa, septum and turbinate, 5 in upper lip and 4 inpharynx.
Lesions in early stages encountered were
erithema, bleeding and crusts, nasal infiltration, nodules andgranulomas in the septum. Large evolution determined septal perforation, swelling of upper lip, protuberant nose anddisphonia. 12 of 13 had evidence of previous scars. 7 antrophophilic Lutzomyia could be identified.
DISCUSSION & CONCLUSIONS The progressive
inflammatory destruction seen in the more advanced cases
resulted in septal perforation (7 cases), and invasion of thelarynx causing dysphonia (4 cases). 7 (53.8%) were classified as having severe MCL. Severe disease might follow along period of evolultion and/or be attributed to the virulence of L.(V.) braziliensis. None of the subjects had evidence of active CL. These findings are in agreement withprevious observations but contrasts with those reported
from Colombia. These differences may reflect the parasite
species and ecological location. This active search favors
the description of the lesions in early and late stages. This
report emphasises the importance of an active search in theendemic communities for early diagnosis and treatment toprevent irreversible disfigurement.

B−2) MALARIA EPIDEMIOLOGY IN LOMBOK ISLAND, INDONESIAHIROJI KANBARA1, MASAHIRO TAKAGI1, YOSHIO TSUDA2, KAZUMI YOSHINAGA1, YOSHIHIDE MAEKAWA1,
YOES P. DACHLAN3, I.K. GERUDUG4, H.L. SEKARNINGRAT5
Institute of Tropical Medicine, Nagasaki University1, National Institute of Infectious Diseases2, Tropical Disease Center,
Airlangga University3, Nusa Tenggara Barat Provincial Health Office4, West Lombok District Health Office5

We had carried out the malaria epidemiological survey
at Sokotong in Lombok island and at Stowe Brang, Penyaring and Labanka IV in Sumbawa island from 1991 to 2000.
The result revealed that malaria endemics in surveyed areas
were restricted to coastal areas where vector mosquitoes,
Anopheles subpictus bred. Therefore, we had the preconception that malaria endemic in Lombok island should be
restricted to coastal areas before starting the JICA partnership projekt “Malaria control in Lombok and Sumbawa island”. The Muninting Health Center covering area was se-lected as the area for malaria contorol in Lombok. Immediately after the preliminary survey that examined the spleensize of schoolchildren at grade 1 and 2 of all the primaryschools in the area, we noticed that malaria endemic in thisarea was distributed not only in the coastal area but also in
the forested and hilly area. Thereafter, we have been conducting parasitological surveys and entomological surveys.
Parasitological surveys were done by the regular monitoring
survey at four subvillages four times a year and by casefinding and treatment that was done by three teams on the５３

occation of circulating visit to each subvillage, and revealed
that most of malaria cases appeared in forested and hilly areas and a few cases in coastal subvillages located at thenorthern part of the project area. In forested, hilly areas
malaria casea were found constantly through the year.
Since in coastal areas malaria usually shows a seasonal epidemic, there still remains a possibility of a sudden occurrence of malaria epidemic although only a few cases were
found at the moment. On the map of two subvillages forthe regular survey, malaria cases were plotted. No biased
distrbution was not demonstrated. The entomological teamdemonstrated that the main vector in the forested area wasAnopheles balabacensis and that in the coastal was An. sundaicus. The parasitological team examined cerebral malaria
cases at the Mataram hospital and found that most of thecases were from forested and hilly areas. This indicatedthat malaria information which we had obtained previously
was biased by the old preconception. Although we began
the first trial of distribution of impregnated mosquito nets
and residual indoor spraying in selected subvillages, we
suppose that these trials would be effective in the coast but
not in the forest judging from the results of the entomological survey.

B−3) BABESIA SP. AND OTHER HEMOPARASITES IDENTIFIED IN WILD RATS
IN CHIANG MAI AND MAE HONG SON PROVINCE, THAILANDANCHALEE DANTRAKOOL1, ATSUKO KAWAI1, PRADYA SOMBOON2, ATSUKO SAITO-ITO1
Section of Parasitology, Graduate School of Medicine, Kobe University1, Department of Parasitology,
Faculty of Medicine, Chiang Mai University, Thailand2

Wild rodents are thought to be the most important reservoir hosts of zoonotic infectious diseases especially whena tick is involved as a vector. Human babesiosis is a typicalcase of emerging tick-transmitted parasitic diseases induced
by wild rodent reservoirs. Thus, human babesiosis, which
is mostly caused by rodent babesias, Babesia microti, has
occurred mainly in the United States, and a few sporadic
cases have recently emerged in the other countries including
Japan. In order to infer the possibility of the emergence or
latent existence of human babesiosis in the malaria endemicarea, we started in 2000 to investigate the prevalence ofBabesia infection in wild rodents in Chiang Mai and Mae
Hong Son Province, Thailand and partly reported elsewhere.
Here, we report the results of the consecutive investigation
and analysis regarding Babesia infection as well as the detection of other hemoparasites in wild rats in the area.
During 2000 to 2002, the epizootiologic surveys forwild rats were carried out at several sites in Chiang Mai and
Mae Hong Son Province, Thailand. In total 47 wild rats, including 30 Bandicota indica and 17 Rattus exulans, werecaught. The blood smears showed that 17 of 30 B. indica
were parasitized by Babesia sp. (parasitemia 0.03-11.02%)
but not in any of 17 R. exulans. All of Babesia-positive B.
indica were captured in Chiang Mai but not in Mae Hong
Son Province. Twelve of 17 Babesia-positive B. indica

were infested with ticks of Haemaphysalis spp.. No otherkinds of ticks were identified on any of captured wild rats.
All of the ss-rDNA sequences of 6 Babesia isolates fromdifferent rats were completely identical without regard to
the site and the year for capture of rats. The phylogenetic
analysis based on the sequence of ss-rDNA showed that theThai Babesia sp. is most closely related with canine babesias, B. canis and B. gibsoni, but not so with B. microti.
Two dogs kept near the site where the prevalence of Babesia infection in wild rats was high were found negative withBabesia parasites by PCR as well as blood smears. Regarding other hemoparasites, two Babesia-positive B. indica
showed the simultaneous infection of Hepatozoon-likeparasites (gamonts) in leukocytes. Hepatozoon is also
known to be transmitted by ticks. The ss-rDNA sequence
analysis supported that the parasite is most closely relatedwith Hepatozoon sp.. On the other hand, Trypanosoma sp.
was identified only in R. exulans (3/17), but not any of B.
indica. Although human babesial infection has not been reported in Thailand, the risk of zoonotic babesial infection
cannot be denied at present. Indeed, many ticks bites are
noticed for human as well as for domestic dogs in the area.
Serologic survey using the Thai babesial infection is holdamong human population in the area.

５４B−4) ADULT BLACK FLIES ATTRACTED TO HUMANS AND WATER BUFFALOES, AND
NATURAL INFECTIONS WITH ONCHOCERCA LARVAE IN CHIANG MAI, IN NORTHERN THAILAND
HIROYUKI TAKAOKA1, CHHARU AOKI1, MASAKO FUKUKA1, WEJ CHOOCHOTE2 AND ODILE BAIN3
Department of Infections Diseases, Oita Medical University1, Department of Parasitology, Chiang Mai University2,
Prasitologie comparée et Modèles expérimentaux, associé à 1’INSERM (U567), et Ecole paratique des Hautes Etudes,
Muséum National d’Histoire Naturelle3

Several Simulium species were investigated as to theirbiting habits and natural infections with filarial larvae at
Ban Pan Fan, Chiang Mai Province, in northern Thailand.
Female adult flies landing on or flighting around a humanand a water buffalo were collected during the daytime from06.00 to 19.00 hours on 22 June 2001. As a result, 217 S.
nodosum, 86 S. asakoae and 2 S. nigrogilvum were obtained from a human attractant, and 416 S. nodosum, 25 S.
nakhonense, 16 S. asakoae, 4 S. fenestratum and 2 S.
nigrogilvum, from a water buffalo. The blood-feeding was
confirmed only for S. nodosum and S. nigrogilvum on humans, and for S. nodosum and S. nakhonense on water buf-

falos. Dissections of these simuliids showed that S. nodosum was naturally infected with developing filarial larvae.
Two types of microfilariae were distinguished but only one
type of infective larvae. These larvae resembled Onchocerca suzukii, a parasite from a wild Japanese bovid, suggesting that an unknown Onchocerca species from ruminants was transmitted in Thailand. Infection rates with allstages of larvae and third-stage larvae were 2.3% (14/608)
and 1.0% (6/608), respectively. This is the first report ofnatural infections of black flies with Onchocerca larvae inSoutheast Asia, and the involved black-fly species is anthropo and zoophilic.

B−5) SEASONAL CHANGES OF WILD ANOPHELINE MOSQUITOES AROUND JICHI MEDICAL SCHOOLHIROYUKI MATSUOKA, MEIJI ARAI, SHIGETO YOSHIDA, AKIRA ISHII
Department of Medical Zoology, Jichi Medical School

Abstract
We have collected wild mosquitoes around Jichi Medical School, Tochigi Prefecture, for two years (2001-2002).
A light trap was hanged overnightly in a cattle hut 2 km far
from Jichi Medical School, and the number of mosquitoes
collected was counted next day. Mosquito species collected were Anopheles sinensis, Culex tritaeniorhynchus,
Culex pipiens pallens etc... An. sinensis, which can transmit vivax malaria, was collected from June to September, inparticular, middle July to beginning of August. On the peakday, more than 1800 of An. sinensis female mosquitoes

were collected. Almost all An. sinensis had fed blood. Cx.
tritaeniorhynchus, which can transmit Japanese encephalitis,
made a peak, more than 1600 per night, in August. Three
weeks interval was observed between peaks of An. sinensis
and Cx. tritaeniorhynchus. Both mosquito species might
feed on cattle and oviposit in paddy fields several hundred
meters far from the cattle hut. In winter, no adult mosquitoes were collected from the cattle hut. We should noticethat these vector mosquitoes still live together with us in Japan. We will follow these mosquitoes’ number as the information for the future.

５５B−6) A NOVEL GT-MISMATCH BINDING PROTEIN POSSIBLY INVOLVED IN
BASE EXCISION REPAIR OF THERMALLY INDUCED MATAION
MAKI YAHIRO1, YOSHITO FUJII1, SHUJI. YONEI2, and MICHIO NAKAMURA1
1

Department of Host-Defense Biochemistry, Institute of Tropical Medicine,
Nagasaki Universtity, and 2Laboratory of Radiation Biology, Graduate school of Science, Kyoto University

Watson-Crick-type base pairs are extremely strict ingenomic DNA because of high specificity of polymerase
activities and error-correcting machineries. Even nonreplicating DNA is exposed to error-prone stresses such as reactive oxygen species, radiation and high temperature thatmodify bases or cut backbone DNA strands. Among mismatches found in DNA, a G/T mispairing is most common
because cytosine is frequently deaminated after an enzymatic methylation. We report here a serendipitously discovered novel G-to-T-mismatch binding protein (nGTBP).
This protein bound to a CYBB promoter-originated 23-mer heteroduplex with a G/T mismatch at bp-177.
None of heteroduplexes with the rest 15 possible base pairs
at bp-177 bound to nGTBP. Similar heteroduplexes with G/
T or T/G at different bp positions failed to recruit nGTBP.
DNA heterodupolex-nGTBP comlex was not disturbed by

an MSH6 consensus oligmer nor anti-MSH6 antibody on anelectrophoresis mobility shift assay. In additon, the nGTBP
was detected in MSH6 deficient cell lines. The protein
minimally required a middle 14-mer heteroduplex of theoriginal 23-mer and TRTGNB as the core sequence with the
G mispairing with T. Neither oxidized T [thymineglycol]
nor oxidized G [8-oxoguanine] at bp-177 of 14-mer homoduplex made the duplex recruit the nGTBP. Deaminated
A [hypoxanthine] and deaminated C [uracil], however,made the duplex bind to the nGTBP. These results suggestthat the nGTBP primarily recognizes deaminated“matched” pairs. Deaminations occur by spontaneous hydrolysis that is increased by high temperature. Accordingly
this nGTBP may be involved in the repair of thermally injured deoxynucleotides.

B−7) MITOCHONDRIAL QUINOL-FUMARATE REDUCTASE (COMPLEX Ⅱ) FROMASCARIS SUUM AS A TARGET FOR CHEMOTHERAPY
ARIHIRO OSANAI1, NORIKO SHINJYO1, HIROKO MIYADERA1, TING WANG1, HISAKO AMINO1,SHIGEHARU HARADA2, AND KIYOSHI KITA1
Department of Biomedical Chemistry, School of Medicine, The University of Tokyo1,
Department of Protein Structural Biology, School of Pharmaceutical Sciences, The University of Tokyo2

Ascaris suum, a parasitic nematode, lives in host small
intestine, where oxygen tension is limited and develops a
unique anaerobic energy generation system. In this systemcalled NADH fumarate reductase system, mitochondrialcomplex Ⅱ catalyzes fumarate reduction and functions as aterminal oxidase. The difference between A. suum andmammalian complex Ⅱ that functions as succinate dehydrogenase gives us an idea that complex Ⅱ must be a promising target for chemotherapy. In this study, we purified A.
suum complex Ⅱ to almost homogeneity and performed
enzymatical studies and crystallization.
First, we established a new method of mitochondria
preparation from A. suum muscle and we could prepare 500
mg protein of mitochondria from 500 g (about 400 nematodes) muscle. Second, we performed 2 kinds of anion-

exchange chromatography and could purify about 7.5 mg ofcomplex Ⅱ from 1000 mg protein of mitochondria.
In enzymatical analylsis, we showed that A. suum
complex Ⅱ had higher affinity to fumarate than bovine
complex Ⅱ and that A. suum complex Ⅱ could function as
fumarate reductase by this high affinity to fumarate. Some
inhibitors were tested and we showed that TTFA and carboxin, the inhibitors of bovine complex Ⅱ, did not inhibit A.
suum complex Ⅱ and this suggests that the structure of inhibition site must be different between A. suum and bovine
complex Ⅱ. So flutolanil can be a promising lead compound of anthelmintics.
In trials of crystallization, we could obtain the crystal
with the size of 30 micrometer reproducibly. Stabilizationof A. suum complex Ⅱ activity by additon of 1 mM malo-

５６nate and screening of wide range of detergents led to the
crystal growth of A. suum complex Ⅱ. Now the X-ray diffraction study is undergoing.

This study shows that complex Ⅱ is a promising target
for chemotherapy and is a first step for development of new
anti-parasitic drugs.

B−8) CHEMOTACTIC FACTORS FOR LEUKOCYTES FROM EGGS OF SCHISTOSOMA MEKONGI
MAKOTO OWHASHI1, JUN MATSUMOTO2, MASASHI KIRINOKI M2, VIROJ KITKOON3, HAJIME MASTUDA2
1
2

Department of Life Science, Faulty of Integrated Arts and Sciences, Tokushima University,
Dokkyo University, School of Medicine, 3Mahidol University, Faculty of Tropical Medicine

We previously showed that soluble extract of eggs
(SEA) of Schistosoma-japonicum possesses intense chemotactic activities for eosinophils or neutrophils, and pointed
out a contribution of the granulomatous lesions around deposited eggs in the liver. Schistosoma mekongi is one of the
newly recognized species of Schistosoma that is closely related to Schistosoma japonicum. However, little information is available about the pathogenesis of S. mekongi infection.
In the present study, we examined leukocyte chemotactic activity of SEA of S.. mekongi using multi-well microchemotaxis chambers equipped with Millipore membranefilters with a pore size of 3 µm. SEA of S. japonicum wasused as a positive control. S. mekongi SEA showed lower
eosinophil chemotactic activity than S. japonicum SEA. In

contrast, S. mekongi SEA showed more intense neutrophil
chemotactic activity than S. japonicum SEA. Western blot
analysis revealed that S. mecongi SEA generally showed asimilar antigenecity with S. japonicum SEA, whereas S.
mecongi SEA showed lower affinity to an antibody against
the high-molecular weight ECF purified from S. japonicum
SEA. Toluene-extractable fraction of S. mekongi SEA
showed comparable chemotactic activities with that of S. japonicum SEA, whereas toluene-nonextractable fraction of S.
mekongi SEA showed lower chemotactic activity than thatof S. japonicum SEA did. These results indicate that a defect of glycosilation of the high molecular weight components of SEA resulted in the lower grade of eosinophil chemotactic activity of S. mekongi SEA than S. japonicum
SEA.

B−9) CHARACTERIZATION OF RECOMBINANT FRAGMENTS OF THE 150-KDA LECTIN (IGL)
OF ENTAMOEBA HISTOLYTICAHIROSHI TACHIBANA1, XUN-JIA CHENG1,2 AND YOSHIMASA KANEDA1
1

Department of Infectious Diseases, Tokai University School of Medicine, Japan,
Department of Medical Microbiology, Medical Center, Fudan University, China

2

Carbohydrate-protein interactions initiate the contactdependent cytotoxicity for which Entamoeba histolytica
was named. It is well known that the 260-kDa galactose(gal)- and N -acetyl-D-galactosamine (GalNAc)-inhibitablesurface lectin of the ameba mediates adherence to humancolonic mucins, colonic epithelial cells, neutrophils, anderythrocytes. Recently, we identified another Gal/GalNAc
lectin, a 150-kDa surface antigen recognized by monoclonal
antibodies (mAbs). The mAbs inhibited amebic adherenceto erythrocytes and Chinese hamster ovary (CHO) cells,
erythrophagocytosis, and cytotoxicity to CHO cells. The
150-kDa lectin, named as intermediate subunit (Igl), is acysteine-rich protein consisting of 1101 amino acids andcontaining multiple CXXC motifs in amino acid sequences.
In the present study, various lengths of recombinant fragments of Igl were prepared in Escherichia coli and the reactivity of the mAbs to the fragments was examined for epitope localization. Two of four adherence-inhibitory mAbs
reacted with the recombinant protein of full length but withnone of the partial fragments, demonstrating that conformation of the protein is important. The epitopes of the twoother inhibitory mAbs were localized in the parts of aminoacids 603-753 and amino acids 989-1088. These parts may
have the carbohydrate recognition sites and could be vac-

５７cine candidates.

B−10) INVOLVEMENT OF PROTEASOME FUNCTION IN ENTAMOEBA GROWTH
AND DIFFERENTIATIONASAO MAKIOKA1, MASAHIRO KUMAGAI1, HIROSHI OHTOMO1,SEIKI KOBAYASHI2 AND TSUTOMU TAKEUCHI2
Department of Tropical Medicine, Jikei University School of Medicine1, Department of Tropical Medicine and Parasitology,
Keio University School of Medicine2

Protein degradation by ubiquitin-proteasome pathway
plays an essential role for a wide variety of cellular processes in eukaryotic cells. Studies with selective proteasome
inhibitors such as lactacystin have demonstrated possible
contribution of protein degradation by this pathway in cellular processes. Therefore, we examined whether proteasome inhibitors affect growth and encystation as well as excystation of Entamoeba. Three proteasome inhibitors, lactacystin, clasto-lactacystin β-lactone, and MG-132, wereused for Entamoeba histolytica and E.invadens. All thesedrugs inhibited E. histolytica growth in a concentrationdependent manner, while lactanystin was most potent for
the inhibition and MG-132 showed the inhibitory effectonly at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited by these inhibitors and was moresensitive to the drugs than the growth, where the β-lactone
was most potent for inhibition of encystation. The inhibitory effect of lactacystin and the β-lactone on encystation
was slightly or little abrogated by the removal of the drug,
respectively. Multinucleation occurred in E. histolytica trophzoites treated with these drugs, being most marked bylactacystin. Electron microscopy revealed that treatment of
E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules
in the cytoplasm. Lactacystin, β-lactone and MG-132 hadno or little effect on the excystation and metacystic development of E. invadens. These results show that proteasomefunction plays an important role for Entamoeba growth andencystation, but has no obvious effect on the excystationand metacystic development.

B−11) MOLECULAR CLONING AND CHARACTERIZATION OF FARNESYLTRANSFERASEOF ENTAMOEBA HISTOLYTICAASAO MAKIOKA1, MASAHIRO KUMAGAI1, TSUTOMU TAKEUCHI2, AND TOMOYOSHI NOZAKI3,4
Department of Tropical Medicine, Jikei University School of Medicine1, Department of Tropical Medicine and Parasitology,
Keio University School of Medicine2, Department of Parasitology, National Institute of Infectious Diseases3,
PRESTO, Japan Science and Technology Corporation4

Protein farnesylation, which is catalysed by farnesyltransferase (FT), is one of the post-translational lipid modification of proteins such as low molecular weight GTPase
Ras, and thereby enables Ras to attach membranes and interact with other proteins. To elucidate significance of protein farnesylation in Entamoeba histolytica and to evaluate
its FT as a target for chemotherapy, we have started biochemical characterization of FT of this parasite. We obtained cDNA encoding α-and β-subunits of FT (FT α andβ) of E. histolytica by PCR amplification using primers de-signed on the fragments of corresponding genes found in
the TIGR E. histolytica genome database. The genes of E.
histolytica FTα and FTβ encoded 298 and 375 amino acids,
respectively, and showed 24-36% positional identity withthose of human, Arabidopsis thaliana, Saccharomyces cereviside, and Trypanosoma brucei. The coding regions andthe ribosome binding sites between them were cloned intandem in pQE31, and expressed in E.coli. The 38 kDa and
43 kDa proteins corresponding FTα and FTβ, respectively,
were identified by SDS-PAGE. The recombinant E.histo-

５
８

lytica FT showed a FT activity against wild type human recombinant Ras (-CVLS) but not against mutant human Ras(-CVLL). We then prepared 4 recombinant E.histolytica
Ras homologues (-CIMF, -CELL, -CSVM, -CVVA) to examine their substrate activities. Among them, only Ras (-CVVA) showed the substrate activity against the recombinant E. histolyca FT, suggesting a difference in substratespecificity between human and E. histolytica FT. These results suggest that E. histolytica FT may become a possible
target for chemotherapy.

B−12) RECENT SITUATION OF DENGUE FEVER AND DENGUE HEMORRHAGIC FEVER
IN THE PHILIPPINES (1995−2001)
RONALD R.MATIAS1,3, SHINGO INOUE1,4, MARIA T. D. CINCO1, CORAZON C. BUERANO1,3, KAZUNORI OISHI5,
KOUICHI MORITA4, FUTOSHI HASEBE4, JHOE A. R. ALFON1, DEU J. M. CRUZ1, MARY ANN. PANCHO1,
MA LUISA G. DAROY1, EFREN M. DIMAANO6, CELIA C. CARLOS2, AKIRA IGARASHI4, FILIPINAS F. NATIVIDAD1
1

Research & Biotechnology Division, 2Department of Pediatrics, St. Luke’s Medical Center, Philippines, 3Institute of Biology,
University of the Philippines, Diliman, Philippines・, 4Department of Virology, 5Department of Internal Medicene,
Institute of Tropical Medicine, Nagasaki University, 6Pavilion 1, San Lazaro Hospital, Philippines[Introduction] Dengue fever (DF) and dengue hemorrhagic
fever (DHF) cases were reported approximately 13,000
every year and its mortality rate was 1% among children
and adolescent in the Philippines. The Research andBiotechnology Division, St. Luke’s Medical Center and theInstitute of Tropical Medicine, Nagasaki University havedone collaborative study on dengue for 7 years (1995-2001)
in Metro Manila. To know the impact of DF and DHF in
the Philippines, we conducted virological, serological andclinical surveillance using blood samples and clinical records of dengue suspected / clinically diagnosed patients.
[Materials and Methods] Using 3,564 blood samples, virus isolation using C6/36 mosquito cells and RT-PCR were
performed as viroloical examinations. Hemagglutination
inhibition test (HI test), IgM-capture ELISA and IgG indi-

rect ELISA were done as serological examinations.
[Results and Discussion] The peak season of dengue virus
infection was from July to November. The dengue outbreakoccurred every three years that were in 1995, 1998 and2001. Dengue confirmed cases were 53% of the examinedcases and approximately 80% of them were dengue secondary infection examined by HI test and IgG indirect ELISA.
The major serotypes of isolates were DEN2 and 3 in 1995
but it gradually changed to DEN 2 and 1 by 2001. The phylogenetic analysis of DNE2 envelope region indicated theDEN2 virus local strains circulate within the Philippines.
The severity of clinical symptoms based on WHO criteria
could classify dengue as DHF grade 2 (38%), DF (31%)
and Grade 3 (10%) among 863 dengue laboratory confirmed cases.

B−13) A MECHANISM OF THROMBOCYTOPENIA DURING THE ACUTE PHASE OF DENGUE VIRUS
INFECTION: INVOLVEMENT OF PLATELET-ASSOCIATED IGG AND ANTI-DENGUE VIRUS IGG
OISHI K1, INOUE S2, HASEBE F2, KUMATORI A3, MORITA K2, NAGATAKE T1, ALFON JAR4, CRUZ DJM4, CINCO MTD4,
MATIAS RR4, ALERA TP5, DIMAANO EM5, NATIVIDAD FF4
Dept of Int Med1, Virology2 and Biochemistry3, Inst of Trop Med, Nagasaki Univ. Res & Biotech. Div.,
St. Luke’s Med Center4, Pavilion1, San Lazaro Hosp.5, Philippines

Although the public health impact of dengue is rapidly
increasing, the mechanism of thrombocytopenia in this disease remains unknown. To elucidate this mechanism, therelationship between platelet-associated IgG (PAIgG) andplatelet count in 83 patients with secondary dengue virus

infection was investigated in a prospective-hospital-based
study. A significant increase in PAIgG levels as well as asignificant decrease in platelet count were found in all casesof secondary infection, compared with 57 healthy volunteers. Only two of the 83 patients with secondary dengue

５
９virus infection were positive for anti-platelet IgG in plasma.
The PAIgG levels in 53 patients with a platelet count ofless than 100x10３/µL were significantly higher than those in
30 patiets with platelet counts greater than 100x10３µL or
those from healthy volunteers. A significant inverse correlation between the two parameters was found. The low
baseline platelet counts during the acute phase in 12 patients significantly increased during the convalescent phase,

while the increased PAIgG levels of the acute phase in thesepatients significantly decreased during the convalescentphase. Involvement of anti-dengue virus IgG was also
shown in platelets from 8 patients.
These findings suggest a pivotal role of PAIgG formation, involving anti-dengue virus IgG, in the inducement oftransient thrombocytopenia during the acute phase of secondary dengue virus infection.

B−14) EVALUATION OF A HEPATITIS B VACCINATION IN CHIANG MAI, THAILAND
KAN TORIYAMA1, MASACHIKA SENBA1, PRAPAN JUTAVIJITTUM2, AND AMNAT YOUSUKH2
Department of Pathology, Institute of Tropical Medicine, Nagasaki University1,
Department of Pathology, Faculty of Medicine, Chiang Mai University2

In 1992, the Government of Thailand started a nationwide vaccination program for mother-to-infant transmissionof hepatitis B virus (HBV), following a pilot program in
Chiang Mai and Chon Buri. We report the long time efficacy of this vaccination program in Chiang Mai. Ninety
three percent of the urban and 60% of rural children had received adequate vaccination in the 4 to 6 year-old age group.
The prevalence of HBsAg and anti-HBs positivity was 0.9
and 42.8% in the children who were adequately vaccinated,compared to 7.2 and 24.6% in the children who were not orinadequately vaccinated, respectively. The prevalence of
HBsAg and anti-HBs positivity of the adequately vaccinated children was 1.1 and 33.8% in urban areas and 0.5
and 28.2% in rural areas, respectively. These findings indicated that the program has a certain level of efficacy, however it can be hardly said to be sufficient, comparing withthe result of Japan. Several factors such as the commercial
source of the vaccines, improper transportation and storage
systems and insufficient methods of the administration
might reduce the effectiveness of vaccination.

B−15) ACUTE RESPIRATORY INFECTIONS (ARI) AMONG CHILDREN IN VIETNAM:
DETERMINATION OF BACTERIAL ETIOLOGY AND ANTIBIOTIC RESISTANCE OF PATHOGENSRUSIZOKA OS1, WATANABE K1, CHEN M1, NGUYET NT3, THI NT3, DUNG NT4, ANH NTC2, HUONG PLT2,
ANH DD2, Oishi K1, Nagatake T1.
Dept of Intern Med1, Inst of Trop Med, Nagasaki Univ. National Inst of Hyg and Epidemiol2, National Institute of Pediatrics3,
Bach Mai Hospital4, Hanoi, Vietnam

Acute respiratory infections (ARI) are common causesof morbidity and mortality in tropical and developing countries. This study was designed to determine whether aquantitative culture method employing bronchial secretion
obtained by nasopharyngeal swab is useful for determination of causative bacterial pathogens of ARI among children,and to determine the antibiotic susceptibility of major bacterial pathogens.
We enrolled 102 patients with ARI and 43 control patients without ARI. A chest radiograph was taken and bronchial secretion was obtained from each patient by naso-

pharygeal swab, and the quantitative culture of bronchialsecretion was done.
The isolation rate of bacterial pathogens was 51.0%(25/49) in patients with bacterial pneumonia (BP), 30.2%(16/53) in patients with acute bronchitis (AB) and 18.6% (8/43) in control (CT) patients, respectively. The isolation
rate in BP, but not in AB, was significantly higher than thatin CT. Major pathogens isolated were S.pneumoniae (n=12) and H.influenzae (n=11). Major bacterial pathogens of
102 patients of ARI were S.pn (45.5%) and H.inf (36.3%),
respectively. We determined the MICs of them and also ex-６０amined drug resistant genes of these strains by PCR. A
very high rate of penicillin-resistant S.pn (85%) and βlactamase producing H.inf (70%). Most of PRSP strains
had penicillin-binding protein (PBP) mutations in all of
pbpla, pbp 2x and pbp2b, and were belong to serotype 19F
or 23F.

Our data suggest a quantitative culture method using
bronchial secretion is a useful tool for the bacterial diagnosis of BP in pediatric patients, and demonstrate a high rateof β-lactam antibiotic resistance in major bacterial pathogens of pediatric patients of ARI in Vietnem.

B−16) A TRIAL OF NOSOCOMIAL INFECTION CONTROL IN THE BACH MAI HOSPITAL PROJECTFOR FUNTIONAL ENHANCEMENT IN VIETNAMHIROSHI OHARA1, NGUYEN VIET HUNG2
Bureau of International Cooperation1, International Medical Center,
Department of Nosocomial Infection Control, Bach Mai Hospital2

A technical cooperation project has been implemented
in Bach Mai Hospital in Vietnam since January 2000 withthe purpose of upgrading the quality of medical care service
in the hospital and disseminating the benefit of better medical care to wide areas. In this project, nosocomial infection
control was integrated in the program under the recognition
that effective control is indispensable to improve the qualityof medical care. This is the first trial as a hospital project indeveloping countries. In this study, the process of the introduction of nosocomial infection control was summarizedaiming to realize better control in the developing countries.
The activities of nosocomial infection control in the
project are as follows:
1. Setting up of Nosocomial Infection Control Department
and allocation of staff
2. Supply of essential equipment and training of the staff
3. Inspection of current situation of nosocomial infection
control in some hospitals
4. Making out a manual, teaching materials, pamphlets,
posters, etc.
5. Organizing training courses and evaluation

6. Carrying out a fact-finding study for 1,266 in-patients in
Bach Mai Hospital
After the start of the project consciousness of nosocomial infection control among hospital staff is increasing
and control system is being created. The average score of
the tests conducted after the training courses showed the increase of 29.4 compared with pre-tests, and the trainees hadhigh satisfaction. The fact-finding study revealed infection
rate with 5.77% (73/1,266). As causative agents, Pseudomonas aeruginosa, Acinetobacter baumanii, Escherichia coli,
Enterococcus, Klebsiella pneumoniae revealed high frequency. 19 out of 73 cases were regarded as infections associated with surgical procedures.
Nosocomial infection rate in developing countries ishigh and effective control method has been increasingly requested. It should be stressed the importance of basic sanitary procedures such as hand washing. Enlightenment using manuals, teaching materials, and conducting training
courses are also effective. In addition, appropriate use ofantibiotics based on the results of monitoring causative
agents is desired.

６１B−17) EXPERIMENTAL INFECTION INTO SCID MICE WITH MICROSPORIDIA ISOLATEDFROM AN AIDS PATIENT
Juarez SI1, Ichinose A2, Iwasaki T3, Jongwutiwes S4, Yanagi T1, Kanbara H1
1

Dept. Protozool., Inst. Trop. Med. Nagasaki Univ.,
2
Central Lab., Inst. Trop. Med. Nagasaki Univ.,
3
Dept. Pathology., Inst. Trop. Med. Nagasaki Univ.,
4
Dept. Parasitol., Fac. Med., Chulanlongkorn Univ. Bangkok, Thailand

We found out a case of keratitis was caused by a species of microsporidium which was first isolated from thecase. We successfully established an in vitro culture technique for the microsporidium, and we gathered from themorphology by transmission electron microscopy (TEM)that the species was Trachipleistophora anthropophthera in
all likelihood.
The specimen of corneal biopsy was obtained from a
42 years old HIV-infected patient at King Chulalongkon
Memorial Hospital, in Thailand. The man suffered fromchronic keratitis in his left eye for more than 6 months. Several corneal scrapes stained for bacterial, fungal and viral
agents were all negative. The corneal specimen was inoculated into a 25 cm2 culture flask with fibroblasts derived
from newborn mouse brain, and maintained with MEM culture medium supplemented with 5% fetal bovine serum and
5% newborn calf serum at 26C, 29C or 37C. The microorganism could multiply at the same proliferation speed at thethree different temperatures. Many an oval-shaped spore,
which was compatible with microsporidium, was detectedunder a light microscope after the smear of foci in fibroblasts was stained with chromotrope 2R or Calcofluor
White M2R. The cell cultures were periodically scraped,
fixed in glutaraldehyde, and examined with transmission

and scanning electron microscope. Bisporous and multisporous vacuole-like sporophorous vesicles were observed
simultaneously in foci of the microsporidia under TEM.
They proved the species that was identified as T. anthropophthera, not as T. hominis.
To investigate the course of the infection, the spores
from cultures were inoculated into SCID mice intraperitoneally (ip) or intramuscularly (ip). The mice were sacrificed to look for which tissue and organs were infected withthe microsporodia. We initiated the tissue cultures of the
SCID mice to isolate spores from each cultivation. We examined the stump preparation and pathological sections,and detected DNA of the microsporidium by PCR in eachorgan. In the im mice, the infection site was limited in theoriginal part. In the ip mice, the spores became spread into
the general body except the lung, probably through lymphatic circulation. These findings clearly indicate that microsporidia can be infectious into almost organs of a SCID
mouse. Immuno-histological assay will be helpful to confirm accurately further which tissue and organs are infected.
Since intraperitoneal and intramuscular inoculation is artificial, we will infect by different transmission routes (ocular,
intranasal and oral inoculation) into SCID mice in the near
future.

６２C−01) ANALYSIS OF ANTIGEN-SPECIFIC IGG SUBCLASSES IN SERUM AND URINE SAMPLES FROMBANCROFTIAN FILARIASIS PATIENTSXU-GUAN QIU１, MAKOTO ITOH１, MIRANI V. WEERASOORIYA２, MOHAMMAD ZAHIDUL ISLAM１,
AND EISAKU KIMURA１
Dept. Parasitol., Aichi Medical Univ.１, Dept. Parasitol., Univ. of Ruhuna, Sri Lanka２

A sensitive and specific urine ELISA, which detects filarial antigen-specific antibodies, is suitable for field studies.
However, basic information on the immunoglobulins andtotal protein in urine and their correlation to those in serum
has not been well studied. In this study, antigen-specific
IgG subclasses are compared using paired serum and urine
samples from bancroftian filariasis patients.
Seventy-six paired serum and urine samples from theantigen positives were used in this study. They were collected from residents in Matara, Sri Lanka, an endemic areaof bancroftian filariasis. Blood sample, 100 µl, was taken
on a filter paper strip. Collected urine samples were addedwith sodium azide. Bancroftian filaria-derived antigen was
detected in serum samples eluted from the filter paper using
an antigen detection kit (Trop-Ag W. bancrofti). AntiBrugia pahangi female adult worm specific IgG subclasses
were measured by ELISA. Serially diluted standard serum
samples were applied to each ELISA plate to make a standard curve for each subclass. Each IgG subclass was shownwith unit calculated from the standard curve. Urine samples were used directly and serum samples were diluted

5000 times for IgG1 and IgG4, and 100 times for IgG2 andIgG3. Protein levels in urine samples were measured using
Protein Assay (Bio Rad).
Both IgG1 and IgG4 to B. pahangi antigens were detected in serum and urine samples: their levels in serumand urine samples were correlated well (r=0.67, P<0.0001
and r=0.73, P<0.0001, respectively). Strong correlation between the IgG1 and IgG4 levels both in serum and urine
samples were also observed (r=0.71, P<0.0001 and r=0.70,
P<0.0001, respectively). Both IgG2 and IgG3 subclasses tothe antigens in urine samples were low and some of themwere not detectable. Very few part (0.1〜0.001%) of serum
IgG1 and IgG4 were found in urine samples. Serum/urine
ratio of IgG1 and IgG4 correlated well (r=0.73, P<0.0001).
Although 5 out of 75 urine samples showed moderately
high protein levels (30〜55mg/dl) and their anti-B. pahangi
IgG4 levels were high, the remaining 70 urine samples werenormal (<30mg/dl). These observations suggest that presence of anti-B. pahangi IgG4 in urine, which is utilized for
diagnosis, is not due to leak caused by abnormality of kidney function.

C−02) DIRECT AGGLUTINATION TEST (DAT) WITH URINE SAMPLES FOR THE DIAGNOSIS OF
VISCERAL LEISHMANIASIS
MOHAMMAD ZAHIDUL ISLAM１, MAKOTO ITOH１, RUSELLA MIRZA２,３, IFTIKHAR AHMED２,XU-GUANG QlU１, AND EISAKU KIMURA１
Department of Parasitology, Aichi Medical University School of Medicine１, Department of Microbiology, Rajshahi medical College,
Bangladesh２, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University３

Due to high sensitivity, specificity and ease to perform,
direct agglutination test (DAT) has been widely accepted forthe diagnosis of visceral leishmaniasis (VL) especially inthe field. However this method requires blood, which hinders the sample collection in the field particularly with pediatric subjects. In this study we applied urine, as a sample
for DAT instead of blood.
We prepared trypsin treated, formalin fixed, andCoomassie brilliant blue stained Leishmania donovani promastigotes as DAT antigen. The quality of the antigen was

standardized with the commercially available antigen produced by Koninklijk Instituut voor de Tropen, Amsterdam.
Fifty microliter of urine sample was applied without concentration to a well of a V-shaped 96-well microtiter plate,
to which 50 µl DAT antigen was added. Then the plate
was incubated overnight at room temperature. The results
were judged with standard positive and negative samples.
The sensitivity of the assay was 66.7% (38 positives/
57 VL samples). The specificity of the assay, determinedwith 137 non-VL samples, including 59 non-endemic

６３

healthy controls, 58 malaria, 13 tuberculosis and 7 otherdiseases, was 94.1% (129 negatives/137 non-VL samples).
We previously reported a urine-based ELISA for the
diagnosis of VL, which showed 95.0% (57 positives/60 VL
samples) sensitivity and 95.3% (204 negatives/214 non-VL
samples) specificity. One parasitologically confirmed sample became negative with urine-based ELISA, where as, inurine-based DAT that sample became positive. As the sensitivity of the urine-based DAT is still inferior, we are trying
to improve it. Urine-based DAT has several advantages
over conventional DAT. (1) Urine collection is non-invasive.
(2) The assay system can process more number of samples
in one plate, which will reduce the amount of antigen, thecost, time and labour/test.

C−03) DETECTION OF PLASMODIUM BERGHEI AND P. GALLINACEUM BY ICT P.F./P.V.
IMMUNOCHROMATOGRAPHIC TESTMEIJI ARAI, HIROYUKI MATSUOKA AND AKIRA ISHII
Department of Medical Zoology, Jichi Medical School

ICT P.f./P.v. is an immunochromatographic dipstick
test which detects histidine-rich protein, HRP-2 of Plasmodium falciparum. ICT P.f./P.v. can also detect P. vivax andother non-P. falciparum parasites with a pan-specific antibody. Before employing ICT P.f./P.v. for routine screening,extensive evaluation of its usefulness must be made. It isvery important to study at which extent of parasitaemia a
newly-infected patient shows a positive result by ICT P.f./P.
v., and how its reactivity are affected during clinical course.
Several groups have already reported such information.
However, under field conditions, difficulty in confirming‘newly-infected’ among the malaria-positive subjects, persistent post-treatment antigenaemia, cross reactivity withrheumatoid factor, and poor skills of local microscopists,should be taken into account. Recently, we found the panmalarial antibody of ICT P.f./P.v. recognises non-humanparasites, P. berghei and P. gallinaceum. In this report P.
berghei-irfected mice and P. gallinaceum-infected chickens
which had been infected by either merozoite injection or
mosquito bite, were tested for microscopic examination and
ICT P.f./P.v. test. Lowest numbers of parasite in the blood
containing P. berghei or P. gallinaceum detected by ICT P.f.
/P.v. were 8,400 and 10,000 parasites, respectively. Reportedly the pan-specific antibody shows good detection sensitivity on blood samples containing parasites more than 500/
µl (7,500 parasites in 15 µl), which is close to our result on
P. berghei. Thus the mouse model seems to be useful forthe study of detection sensitivity on onset parasitaemia after
mosquito bite. Further study on persistent post-treatment
antigenaemia is underway. P. berghei-mouse system may
provide a good model for evaluation of ICT P.f./P.v. andother rapid diagnostic tests for malaria.

C−04) ANTI-SERA ANTIBODY PRODUCTION AS A PARAMETER OF ANTI-MALARIA IMMUNITY IN
INDIVIDUALS IN THE SOLOMON ISLANDSMARIKO HATO１, JUN FU１, TOSHIHIRO HORII２, KAZUYUKI TANABE３, HIROSHI OHMAE４, AKIRA ISHII５,
MASATO KAWABATA６, JUDSON L. LEAFASIA７ AND NOBUO OHTA１
１

Dept. Med. Zool., Nagoya City Univ. Med. Sch., ２Dept. Mol. Protozol, Inst. Microbial Dis, Osaka Univ., ３Div. Biol., Osaka Inst.
Technol., ４Dept. Mad. Biol., Tsukuba Univ. Sch. Med, ５Dept. Med. Zool., Jichi Med. School, ６Inst. Centr. Med. Res., Kobe Univ.
Sch. Med, ７Solomon Islands Med. Train. Res. Inst, Solomon Islands

Serine-rich antigen (SERA) of Plasmodium falciparumis one of the most promising vaccine candidates against
erythrocytic stage of the parasite in humans. Based on the
recent in vitro observations showing effective SERAmediated protective immunity, the present study was con-ducted to uncover the SERA-mediated protective immunity
in individuals in endemic areas of falciparum malaria
through the sero-epidemiological approach. People in
Guadalcanal, where transmission of falciparum malaria ismeso or hyper endemic, were divided into three categories;

６
４

slide(-)/symptom(-), slide(+)/symptom(-), and slide(+)/
symptom(+). Number of each group was 70, 19 and 26, respectively. The N-terminal portion of SERA (SE36’) andthe N-terminal 6 blocks of MSP1 (Ml/6) were used for
ELISA. We observed that total IgG production specific forboth SE36’ and M1/6 was significantly higher in slide(-)/
symptom(-) group than in slide(+)/symptom(-) group (p<0.01). The difference was much more apparent in SERAspecific IgG levels than that in M1/6-specific IgG. When
we compare IgG３ levels in the two groups, no symptomatic
individuals of 15 yr or more showed detectable level of SE

36’-specific IgG３. On the other hand, we did not observe
any difference in polio virus-specific antibodies between tbe
two groups. Out of 70 slide(-)/symptom(-) group, 14 were
negative for SERA-specific IgG, while all of the group
showed positive IgG to M1/6. Strong association was observed for degree of parasitemia and SE36’-specific IgG.
No individuals with parasite density of 100 and more had
detectable level of SE36’-specific IgG. Together with those,
we concluded that SERA is a promising vaccine candidadefor human falciparum malaria in our seroepidemiologicalstudy in the Solomon Islands.

C−05) SERODIAGNOSIS OF TUBERCULOSIS USING MPB64 DERIVED FROM MYCOBACTERIUM BOVISATSUKO YOSHIDOME１, SEIJI KANDA１, ITOKO HAYASHI１, MASAO UEMOTO２,
KOICHI TOMODA３,TOSHIMASA NISHIYAMA１
１

Dept. Public Health, Kansai Med. University, ３Tsuda Hospital, ３Saiseikai Suita Hospital

It is known that the number of patients for tuberculosis
is gradually increased in the worldwide, including in Japan.
Especially in the developing countries, it’s important to
identify the patients for active tuberculosis. In the previous
study, the MPB64 antigen was efficient by the patch test forthe diagnosis of active tuberculosis. In addition, there wasanother report for MPB64 in which PCR analysis was useful to detect the diagnosis of tuberculous pleuritis and meningitis. In this study, we try to find MPB64 has the highspecificity and sensitivity for the diagnosis of the active tuberculosis. mpb64 cDNA was kindly donated from Dr.
Matsuo (Nagasaki University) and cloned into the expression vector by the GATEWAY technology system (invitrogen Inc.,). The recombinant protein MPB64 was expressed
as a fusion protein with His-tag for the C-terminus and theM. W. was guessed about 30 kDa. Western blot analysis
was done using a patient’s sera for active tuberculosis andhealthy subjects as a control. The result was shown that a
specific band for 30 kDa was detected with the patient but
not for the control. Therefore this band was also detectedby the immunoreactive with anti-His Tag antibody. So thiswas MPB64-specific reactivity for the patient’s serum.
Then MPB64 protein was affinity-purified using His-tag
and the IgG titers in the sera were measured by ELISA
method. The mean value of patients was higher than that ofhealthy subject. And, there is a tendency to decrease theIgG titers time-dependently. It is considered that IgG titers
specific for MPB64 were decreased by the effect of thetreatment progress in sera. These results suggested that
MPB64 might to be a valid antigen for the patient of active
tuberculosis and there is a possibility to use the screening
method of the patient for the active tuberculosis in the fieldof developing country.

６５C−06) A METHOD OF BLOOD EXAMINATIONS IN OVERSEAS MOBILE CLINIC―ITS CLINICAL APPLICATION TO THE REMOTE AREAS IN LAO PDR―TAKAAKI ISHIDA１, TOSHIMASA NISHIYAMA１, HIROYUKI AMANO１, YASUKO TAMADA１, BOUTHONG SISOUNTHORN２,
AMPHAYVANH PANHANOUVONG２, MITSUE UEDA３,４, CHIEKO MIYAKE３
Department of Public Health, Kansai Medical University１
Curative Department, Ministry of Health, Lao PDR２
Aichi Medical Foundation of Diagnostic Technology３
Department of Public Health, Aichi Medical University４

In this study, we carried out overseas mobile clinic in
Lao PDR from April to May (for 21 days) in 2002, and examined the serum biochemical tests and blood examinations
using newly developed filter paper plasma skimming film
which is easy to separate without centrifuge.
The sera were collected from the inhabitants of
DakEuy village (168 subjects) at SeKong Province and
NonTao village (68 subjects) at OudomXay Province. In
this time, we were able to examine by the skimming filter
for GOT (AST), GPT (ALT), γ-GTP, BUN, Creatinine, Total cholesterol, HDL-cholesterol, Triglyceride, Amylase and
Uric acid by the filter paper for Hemoglobin, HBs Ag and
HCV Ab by the rapid blood sugar analyzer for Blood Sugar.
The results of these examinations were GOT 35.5±
16.7 IU/l, GPT 21.3±5.7 IU/l, γ-GTP 35.5±l4.1 IU/l,
BUN 11.5±3.4 mg/dl, Cr. 0.4±0.1 mg/dl, T―CHO 135.0±21.2 mg/dl, HDL-CHO 31.7±6.4 mg/dl, TG 107.9±
45.6 mg/dl, AMY 113.4±82.0 U/l, UA 3.3±0.9 mg/dl, Hb

16.1±1.8 g/dl, BS 88.6±l3.6 mg/dl, in DakEuy village. In
NonTao village, GOT 20.1±12.1, GPT 21.3±5.7, γ-GTP
33.9±15.4, UA 5.4±1.0, Hb 13.3±2.1, BS 94.8±l8.3. In
the 4 cases of liver disfunction, we have a chance to measure HBs Ag and HCV Ab. Two cases were positive for
HCV Ab, but no case was positive for HBs Ag.
There was no person who showed higher than upper
range of normal limit of Total cholesterol, Uric acid and renal function. And, the patient of diabetes mellitus was notdiagnosed in these inhabitants. In the DakEuy village, hemoglobin values of the inhabitants were higher, because it
might be influence that this village located in the highlandover 1,000m altitude.
Therefore, it was concluded that these methods(plasma skimming film and filter paper), which were developed as screening test in Japan, were suitable for the mobile
clinic of rural areas in the developing country.

C−07) COMPARISON OF ULTRASONOGRAPHIC IMAGES BETWEEN SCHISTOSOMAJAPONICUM AND S. MEKONGI INFECTIONSHIROSHI OHMAE1, YUICHI CHIGUSA2, HIDEHIRO OTAKE2, ORLAMDO S. SY3, EUINCE ILAGAN4,MUTH SUNUON5, DUONG SOCHET5, HAJIME MATSUDA2
1Institute of Basic Medical Sciences, University of Tsukuba 2Department of Tropical Medicine & Parasitology, School of Medicine.
Dokkyo 3Schistosomiasis Research & Control Hospital, Palo, Leyte, Philippines 4Schistosomiasis Control Team of Miudro,
Department of Health, Philippines 5National Malaria Center, Ministry of Health, Cambodia

In order to estimate liver fibrosis due to Schistosoma
japonicum and S. mekongi infections, ultrasonographic(US) examination were used in the schistosomiasis endemicareas of Philippines and Cambodia. Typical liver damage
due to schistosomiasis japonica can be detected by US, as“network pattern” or “fish scale pattern”. Advanced bridging fibrosis reveals these patterns, and periportal fibrosis
shows other changes such as thickening of portal vein wall.
Severe thickening of portal vein wall and collateral vessels

are frequently found in elder cases with network pattern.
But in young cases, we find no relationship between network pattern and periportal fibrosis detected as thickeningof portal vein wall.
In S. mansoni infections, based on the degree of periportal fibrosis, liver fibrotic changes are classified into somepatterns and these patterns do not include network pattern.
In S. mekongi infections, typical network pattern has not befound. And except for the pattern like irregular fatty liver,６６

most of the patterns of liver fibrosis are similar to those ofschistosomiasis mansoni.
US examination is broadly used in developing countries as one of the appropriate techniques, because this technique is easy and cheap. US has been used for morbidity

studies of schistosomiasis. In S. mansoni infections, formorbidity studies, we have a standard criteria of US diagnosis. In S. japonicum and S. mekongi, infections, standard
criteria of US diagnosis is expected.

C−08) COMPARISON OF ULTRASONOGRAPHIC IMAGES OF LIVER AND SPLEEN BY AGE GROUP AND
THE RELATIONSHIP BETWEEN FREQUENCY OF PRAZIQUANTEL TREATMENT ANDULTRASONOGRAPHIC IMAGES OF PATIENTS SUFFERlNG FROM SCHISTOSOMA JAPONICUM AND
S. MEKONGI INFECTION IN THE PHILIPPINES AND IN CAMBODIA
YUICHI CHIGUSA１, HIROSHI OHMAE２, EUNICE J. ILAGAN３, NINA G. BARZAGA４, MUTH SINUON５,CHEAM SAEM６, DUONG SOCHEAT５, MASATO KAWABATA７ AND HAJIME MATSUDA１
Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine１, Basic Medical Sciences, University ofTsukuba２, Schistosomiasis Control Team, Department of Health, Philippines３, University of the Philippines４, National Malaria Center５,
Provincial Health Department, Kratie６, Ministry of Health, Cambodia, International Center for Medical Research, Kobe University７

We conducted studies in Oriental Mindoro, Philippines
for schistosomiasis japonica (Sj) during July and August
2002 and in Kratie, Cambodia for schistosomiasis mekongi(Smek) during May and June 2002. The patients were examined by ultrasonography (US) and questioned concerning the frequency of praziquantel (PZQ) treatment since
1995, in which the drug were given at a dosage of 40mg/kg
body weight in Cambodia and 40-50mg/kg body weight in
the Philippines. The number of patients examined were 234(170 males: 64 females) in the Philippines and 382 (214males: 168 females) in Cambodia.
The patients were classified into two groups, i.e. 19
years of age or younger and those at least 20 years old. The
frequency of PZQ treatment since 1995 was classified as A)
0 to 2 times, B) 3 to 5 times and C) more than 6 times.
Spleen size was estimated by both US and palpation andwas classified as A) within normal limits (WNL), B) lessthan 15 cm from the costal arch (less than 15) and C) over
15 cm from the costal arch (over 15). Periportal thickeningof portal vein and collateral circulation of the splenic vein

were also observed and recorded by US.
Percentages of patients with splenomegaly in groupsA) WNL, B) less than 15 and C) over 15 were 96.0%, 4.0%
and 0% in the Philippines and 80.2%, 19.8% and 0% in
Cambodia, respectively among the younger patients, and91.8%, 8.2% and 0% in the Philippines and 35.3%, 57.4%
and 7.4% in Cambodia, respectively among the older patients. Periportal thickening of the portal vein was observedin 54.0% (Sj) and 50.5% (Smek) of younger patients and53.3% (Sj) and 66.3% (Smek) of those over 20 years of age.
Collateral circulation of the splenic vein were observed by
US in 0% (Sj) and 4.2% (Smek), respectively in patients of
the younger age group but in 2.2% (Sj) and 30.5% (Smek),
respectively of the older age group.
As shown in the results, frequent PZQ treatment reduced the incidence of periportal thickening of the portal
vein but not those of hard enlarged spleen and collateral circulation of the splenic vein. These observations indicatethat early diagnosis and PZQ treatment of schistosomiasis
japonica and mekongi is important.

６７C−09) THE INFLUENCE OF SCHISTOSOMA HAEMATOBIUM INFECTION ON VOIDING FUNCTION
KANJI WATANABE１, MITSURU NOGUCHI２, KOICHIRO NOMATA２, NGETHE D MUHOHO.３ AND YOSHIKI AOKI１
１

Department of Parasitology, Institute of Tropical Medicine, ２Department of Urology. School of Medicine,
Nagasaki University, ３Kenya Medical Research. Institute, Kenya

To estimate the influence of the Schistosoma haematobium infection on the voiding function quantitatively, urinary flow velocity and residual urine volume after urinationin primary school boys were measured using portableuroflow meter (RMS-100, Tyouryou-systems, Japan) andultrasound machine (Bladder Scan BVI3000, Alcare, US.).
This study was done in two areas in Kenya during February, 2002. One area was Kiambu district near Nairobi,
the capital of Kenya. Schistosoma haematobium infeciton
was not endemic in this area. The other study area was
Kwale district, Coast Province, which lay in the east of the
Kenya. The S. haematobium infection was endemic in thisarea. Study subjects were mainly primary school boys because the uroflow meter was designed to measure the malevoiding function only. Before examination, 500 ml of juice
was given to each students. When they felt bladder full,they started to pass the urine into the uroflow meter. The
uroflow meter measured the maximum flow velocity, aver-

age flow velocity, total voiding time, total voiding volume.
After urination, the students were examined about residual
urine volumes. The S. haematobium eggs number in 10 mlof urine was also examined using Nucleo-pore filer.
The eggs count revealed that the boys in Kiambu were
not infected with S. haematobium but 50% of the boys in
Kwale were infected. The uroflow meter analysis showed
no difference in the proportion of the maximun uroflow velocity to the total voiding volume between boys in Kiambu
and in Kwale. Furthermore the residual urine volumes werealso not different between the boys. The same kind of examination to the adults showed that the proportion of the
maximum uroflow velocity to the total voiding volume in
Kwale adult males was lower than that in Kiambu adultmales. From these results, we concluded that S. haematobium infection did not cause the voiding dysfunction in thechildren but it caused the dysfunction in adults.

C−10) EPIDEMIOLOGICAL SURVEILLANCE ON SCHISTOSOMIASISIN MOUNTAINOUS REGIONS IN SICHUAN
CHENG-KUO CHUANG１, NORIAKI KAJIHARA２, HONG-ZHI YIN３, LIAN-GUO ZHAO３, KAI-YOU SHA３,MASARU MINAI２ AND YASUO NAKAJIMA１
Department of Parasitology and Immunology, Faculty of Medicine, University of Yamanashi１, Yamanashi Prefectural Institutefor Public Health２ and Anti-schistosomiasis Station of Xichang City, Sichuan, China３

The area infested and the population infected with
Schistosoma japonicum in China, reduced after the control
program. The infested area and the infection rate, however,appear to have increased in some highlands in Sichuan andYunnan. The primary objective was to study the epidemiology and the factors impeding the control in the areas.
Surveyed were five villages near Qionghai Lake,
Xichang City, Sichuan Province, in which the fecal examination of villagers in 2000 had revealed the positive rate for
S. japonicum ova of over 30%, of around 10% or of 3-5%:
Two sections from each village were investigated.
Results: Cercaria-positive Oncomelania snails were
found in a section of Xinmin Village (7.7%); that of Zhanglin Village (0.9%) and that of Gucheng Village (0.7%).

The trapped and dissected field mice were free of schistosomes.
In Xinmin Village where the egg-positive rate of over
30% had been found in the previous examination, the fecal
samples from the villagers one year after the treatment revealed the positive rate of 33.9% in Sections 3 and that of
65.6% in Section 7 for miracidia.
Discussion: The villagers cultivate vegetables using
human excreta as manure and working bare-footed. They
wash in irrigation ditches. The snail inhabits in the irrigation canal near the hamlet and at the bottom and side wallsof the irrigation ditches in the farms. More positive snails
were found near the houses. The dominant species of fieldmice is Apodemus chevreri, which seems to supply ova

６８

poorly in spite of its frequent contact with water. Although
the infected human and domestic animals were treated, theinfection rate has not reduced in a village. The sanitary life-

style has to be changed and the household water has to be
independent of the farming water to solve the problem.

C−11) PARASITOLOGICAL SURVEY AND RELATED HEALTH AWARENESS INVESTIGATIONBY QUESTIONNAIRE AT A VILLAGE IN THE CENTRAL REGION IN GHANAYOSHIO OSADA１,２, KWABENA M. BOSOMPEM２, SOMEI KOJIMA３, NOBUO OHTA４
Department of Parasitology and Tropical Public Health, University of Occupational and Environmental Health１,
Noguchi Memorial Institute for Medical Research/JICA infectious Diseases Project２,
The Asian Centre for International Parasite Control３,
Nagoya-city University, Graduate school of Medical Sciences, Department of Molecular Parasitology４

Background: In Ghana, as well as in other sub-Saharancountries, many parasitic infections affect people. In suchsituation, irrigation projects have been introduced to increase agricultural productivity and income. It has been
also pointed out that irrigation often results in expansion ofmosquito-borne and/or water-borne parasitic infections.
Therefore, continuous efforts to keep health of residents
living around the area are important. We conducted healtheducation on endemic parasitic diseases in a village (Okyereko, located in the coastal area in Ghana) in which a small-scale irrigated agriculture promotion project (SSIAPP) by
JICA was under implementation. Here we show the resultsof a baseline survey of endemic parasitic diseases andhealth awareness investigation in the village. Methods:
Urine and stools have been collected from residents in thevillage for parasitological assay. Eggs in the stools weredetected by Kato-Katz method and eggs in the urine weredetected by Nucleopore membrane filtration. Questionnaires on awareness of parasitic diseases present in the village were administered to residents aged more than or equal
to 10 years old. Results and Discussion: Schistosoma haematobium infection was the most prevalent disease among
those we examined. Approximately 50% of the residents

aged 10 to 29 years old were infected. Second highly
prevalent helminth infection was hookworm infection,
which affects 23% of the residents. Schistosoma mansoni,
Trichuris trichiura and Ascaris lumbricoides infections
were also observed. Additionally, in a previous study, lymphatic filariasis (Wuchereria bancrofti infection) was shown
to be endemic in this village (Dzodzomenyo, et al ., 1999).
Thus, as for helminthiasis, we can conclude that urinary
schistosomiasis, hookworm infection and lymphatic filariasis are highly endemic in this village. Analysis of awareness and behavior of residents on such parasitic diseasesrelated symptoms revealed that they had a tendency to seek
medicines from chemists or peddlers rather than to see doctors. Only approximately 20% and 10% of the persons who
had disease-related symptoms tend to see doctors, in thecase of malaria and urinary schistosomiasis, respectively.
Analysis of their knowledge on transmission route of parasitic diseases showed that they had a relatively correctknowledge of transmission of malaria, urinary schistosomiasis and intestinal infections. In contrast, they did not know
the relationship of mosquito bite and lymphatic filariasis.
This fact may be due to the long time progression of thechronic symptoms of the disease.

６９C−12) DAMAGE IN CERCARIAE OF SCHISTOSOMA MANSONI INDUCED BY PRESSURE JAR IN WATERNOBU. OHWATARI１, YASUNORI. FUJIMAKI２, ATSUSHI. KUMATORI３, KAN. TORIYAMA４, EIKO. KANEDA５
Department of Environmental physiology１, Parasitology２, Biochemistry３, Pathology４, Social environment５,
Institute of Tropical Medicine, Nagasaki University

We experimented about influence of the water pressure
vibration for a cercariae of Schistosoma manson. Cercaria
comes out of a shell of intermediator into water by light,
and cercaria has a form peculiar which connection department of head and tail is narrow. Therefore, mechanical energy of the water pressure vibration may be influenced tocercaria with strong damage.
In the first, we were examined some following load tocercaria of each group with 50 cercaria in water. The ratiosof tail loss were 10.0% by magnetic rotation field for 30
minute, 13.9% by static water pressure for 30 minute,
27.0% by magnetic rotation field and low speed stream for
30 minute, 87.2% high-speed stream for 10 minute, respectively. Then control group was 0% at ratio of tail loss.
In the next, cercaria were exposed mechanical energy
by 46 kHz of ultrasonic wave generator was load for 1 minute, 2 minutes, 3 minutes and 5 minutes in water. The connection department of head and tail was amputated by themechanical energy, and the ratio of tail loss reached 100%for less than 4 minutes as a result of regression line in statistical analysis.
Furthermore, we were examined the ultrasonic wave
load to infected shell of intermediate host for one minute.
Body of the shell took shelter in the depths of conch during
ultrasonic wave exposure. Cercaria without tail was 99.2%
in which cercaria flowed out of the shell after ultrasonicwave exposure only one minute.
In this study, it was suggested that following. The tailof cercaria which appeared from a shell evolved for a purpose to arrive at final host in a short time. But the smallpart between head and tail of cercariae was weak against
high-speed stream or water pressure vibration induced byultrasonic wave generator. Interference pattern reflected ofultrasonic wave at inside wall of a shell was very effective
for amputation of cercariae, in which configuration of theconch that the depths narrow progressively. Therefore, it ismost effective to expose shell to pressure jar in water for
amputation of cercariae tail.

C−13) COMPARISON OF INTESTINAL PARASITIC INFECTIONS AND MALNUTRITIONIN SCHOOLCHILDREN IN URBAN AND RURAL AREAS IN HANOI, VIETNAMYASUNORI FUJIMAKI１, DAO TO QUYEN２, YUKI SATO３, NGUYEN THI DANG４, HA HUY KHOI２, DO THI KIM LIEN２,
YOSHIKI AOKI１, SHIGERU YAMAMOTO３, FUSAO OTA５
１

Department of Parasitology, Institute of Tropical Modicine, Nagasaki University, ２National Institute of Nutrition, Hanoi, Vietnam,３
Department of Applied Nutrition, ５Department of Food Microbiology, School of Nutrition, The University of Tokushima,
４
National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam

Surveys on parasitic infections and nutritious status inschoolchildren were conducted in urban(U) and rural(R) areas of Hanoi, Vietnam. Data of both parameters obtained
from 75 and 104 children in U and R respectively were analysed and compared for the prevalence and intensity ofparasitic infections and nutritious status between the two areas. The school children were examined for parasites intheir fecal samples, their daily food intake, weight andheight, and anthropometric indices following the Kato-Katz
technique, ‘24 hour recall’ method and the standard US National Center for Health Statistics charts and tables, respectively. Results: 1) Overall prevalence of parasitic infec-

tions was significantly higher in R (96.2%) than that in U
(83.0%). In both areas Trichuris trichiura was the mostdominant parasite. 2) The geometric mean of EPG (eggcounts per gram feces) of Ascaris and Trichuris was significantly higher in R than that in U. 3) Weight and height
were significantly greater in U than those in R. 4) Weightfor-age analysis showed that 13 (18.1%) children in U and
59 (56.7%) in R were malnutritioned. 5) Height-for-age
analysis showed that 16 (21.3%) children in U and 51(49.0%) in R were malnutritioned. 6) A stratified analysis
by Mantel-Haenszel chi-square test showed no relationshipbetween the prevalence of infections, malnutrition and the７０sites examined. 7) No significant differences in total calorie
intake was observed between the normal and malnutritionalgroups. Conclusions: Schoolchildren in the rural area in
Hanoi, Vietnam had a higher prevalence and intensity ofparasitic infections, and a higher rate of malnutrition thanthose in the urban area. There was no significant difference

between nutrition status and the prevalence of parasitic infections with the same calorie intake in normal and malnutritional groups. (*Supported partly by Grants-in-Aid (No.
11691211) from the Ministry. Educ. Sci. Sport. & Cult, Japan).

C−14) DETECTION OF MICROORGANISMS IN TAP WATER IN INDONESIA AND THAILAND
SHOJI UGA１, TAKUYA ODA２, KENJI KIMURA３, DAISUKE KIMURA１,
KOESDARTO４, MARGONO５, KUNAUKU６ AND NUALTA６
Department of Medical Technology, Faculty of Health Science, Kobe University School of Medicine１,
Water Quality Laboratory of Kobe City Waterworks Bureau２, Water Quality Center, Maezawa Industry Inc.３,
Airlangga University, Indonesia４, University of Indonesia５, Prince of Songkla University, Thailand６

Water samples were directly collected from house faucets at each collection site and filtered through membranes.
The membranes were brought to Japan and examined for
protozoan parasites by immunomagnetic separation. Coliform and Escherichia coli were examined at each sample
collection site using commercially available kits. A total
115 water samples were examined and 37 (32%) were revealed positive for four types of microorganisms. Thesemicroorganisms were two kinds of bacteria (coliform andEscherichia coli) and two species of protozoa (Giardia intestinalis and Cryptosporidium parvum). Of those detected,
coliform was the most common and was found in all threeareas with a mean detection rate of 30%. G. intestinalis
and C. parvum were found at the rates of 9% and 1%, respectively. Bacterial contamination was closely related tothe concentration of residual chlorine but not protozoan
contamination. The water samples that were positive forany of the four microorganisms showed a tendency to havelower residual chlorine concentrations and higher turbidities
compared with negative samples. It is important to supply
safe water in order to maintain people’s health because mostof the people surveyed ordinarily drank tap water without
treating it. Continued efforts are needed to maintain andimprove drinking water quality.

C−15) EPIDEMIOLOGICAL ANALYSIS OF ENTEROHEMORRHAGICESCHERICHIA COLI INFECTION IN INDONESIAYOSHIO ICHINOSE１, TAKAO TSUJI３, EIJI KAWASE３, TEIZO TSUKAMOTO４, YASUHIKO WATAYA５,
TAKUJI KUMAGAI６, MASAAKI SHIMADA２, YOES P. D.７, EDDY B. WASITO７ AND MASAAKI IWANAGA８
Department of Bacteriologyl１ and Research Center for Tropical Infectious Diseases２, Institute of Tropical Medicine,
Nagasaki University, Department of Microbiology３, School of Medicine Fujita Health University, Osaka Prefecture Institute of
Public Health４, Wataya Clinic５, Kumagai Clinic６, TDRC, Airlangga University７ and Department of Bacteriology,
School of Medicine, Ryukyus８

Enterohemorrhagic Escherichia coli (EHEC), which
can occasionally cause big outbreaks and sporadic cases indeveloped countries, is rarely isolated as causative agents ofdiarrheal diseases in developing countries. In this study, we
perform an etiological investigation on diarrheal cases and acarrier rate of enteropathogens among non-diarrheal cases
from two medical facilities in Surabaya, focusing on EHEC,

and the carrier rate of EHEC among domestic animals suchas cattles, pigs and goats. In addition, serological analysis
on the serum IgG level of anti-VT1 and anti-O157 LPS antibodies among Indonesian and Japanese pediatric cases
was performed to clarify real situation of EHEC infection inIndonesia.
It was revealed that six strains of Vibrio cholerae O1,

７１

six strains of Shigella, three stains of Salmonella, one strainof enteroinvasive Escherichia coli, 19 strains of enteropathogenic Escherichia coli and only one strain of EHEC
strain (O8:H4) were isolated from diarrheal and nondiarrheal patients. Thirteen strains of EHEC were isolated
but no EHEC O157 was isolated from cattle stool samples.
Three strains of EHEC O157 were isolated from goats. No
EHEC was isolated from pigs. The production pattern ofverotoxin was completely identical to that of toxin genes
detected by PCR. The fingerprinting pattern of genomic

DNA by PFGE was almost identical to those of strains isolated from sporadic cases in Nagasaki prefecture.
The serum levels of anti-VT1 and anti-O157 antibody
determined by ELISA among pediatric cases in Indonesia
increase by age since neonatal period, while those in Japan
seem to decrease once after birth until they begin to increase at four to five years of age. These findings suggestthat previous exposure to EHEC or VT1, or VT1 related antigens could evoke and sustain high levels of serum antibody against VT1 and O157 LPS.

C−16) THE PROBLEM ON MALARIA TREATMENT―ABOUT THE DELAY OF DIAGNOSIS AND TREATMENT―ETSUSHI OKUMURA１, TAKASHI NISHIMORI２, KEISUKE HASHIMOTO３, KENICHI KATSU４, TSUNEJI ARAKI５
Department of Medical Technology, Kochi Gakuen Junior College１
Department of Clinical Technology, Kubokawa Hospital２
Department of Internal Medicine, Osaka Police Hospital３
Department of ２nd Internal Medicine, Osaka Medical College４
Department of Internal Medicine, Shinsei Hospital５

In our experience to treatment for five cases of malaria
patients, we had great deal of serious situation. That waslateness to the diagnosis and treatnent.
Case 1: A middle school boy of 15 years old causedby ovale-malaria; His diagnosis was two days behind theadmission and treatment was five days lingered because ofdifficulties to obtain anti-malaria medicine.
Case 2: A trading company employee of 44 years oldcaused by falciparm-malaria; He made a long stay in Lagos
in Nigeria from 1973 to 1975 and had arisen a complicationof cerebral symptoms, but Chloroquine and Primaquine
were very good efficacious to him nevertheless.
Case 3: A woman of 40 yeas old university lecturer inEnglish caused by vivax-malaria; She olso started to apply
the medicine after six days of the diagnosis. The reason was
one and the same as case 1.
Case 4: A Vietnamese seaman of 33 years old causedby vivax-malaria; He had not been comfirmed his recovery
perfectly. Because his cargo boat had left Osaka port toPusan in Korea just after the diagnosis on this evening.

Case 5: A male patient, 41 years old American, whoteaches English at the Berlitz school in Osaka; He had recovered before becoming encephalopathy, although he wasa case of Chloroquine-resistant falciparum-malaria. He hadtaken instant effect on Quinine-injection in spite of renal
disorder.
Fortunately, we had not experienced miserable cases.
To countermeasure the delay of diagnosis
1. Precise sampling and exact observation (Giemsa stain;
Buffer solution, not pH6.4 but pH7.2〜7.4).
2. Enlightenment of Polymerase chain reaction technique,
Fluorescent antibody technique etc..
To countermeasure the delay of treatment
1. Vid. Today’s Therapy. Vol. 44. 2002 (Edit. Sachio Takasu
M.D. Igaku-shoin Co. Ltd). In this textbook, the name ofOrganization in Japan which keep anti-malaria medicine
is printed with telephone number.
2. Qinghaus; ask Guilin No2 Pharmaceutical Factory
Guangxi, China Republic.

７２C−17) A SEVERE CASE OF PLASMODIUM FALCIPARUM INFECTION COMPLICATEDWITH BLACK WATER FEVER.
KOYAMA, K.

C−18) CORRELATION BETWEEN THE RECOGNITION BY OBSTETRICIANS ABOUT CONGENITALTOXOPLASMOSIS AND THEIR CLINICAL ACTIVITIESKOJI NAOI１, OSAMU HAYAKAWA２, YOSHIO SAKANO２ AND AKIHIKO YANO１
Department of Infection and Host Defense, Chiba University Graduate School of Medicine１
Obihiro-Tokachi Association of Obstetricians and Gynecologists２

In the present study, correlation between the recognition held by obstetricians about congenital toxoplasmosis(CT) and their clinical activities was investigated with aquestionnaire for 15 subjects, or obstetricians in TokachiObihiro area, Hokkaido, Japan. Besides their attributes, obstetricians were asked about their clinical activities on CT,
such as serum antibody tests and explanation for pregnant
women, as well as their recognition about the risk of CT.
According to the present study, obstetricians who are negative for the explanation are relatively older with longer
clinical experience. Obstetricians who have experienced
some cases of CT are more positive for the explanation thanthose who have not. For serum antibody test to T. gondii,
on the other hand, no correlation is observed with statisticalsignificance. On the recognition about the risk of CT, selfemployed obstetricians have lower estimates on the risk ofinfection for pregnant women (RIW) as well as that for fetus (RIF) than employed obstetricians. Obstetricians who
have not experienced any cases of CT also hold lower esti-mates on RIW and RIF than those who have.
Obstetricians for medical clinics indicate their lower
estimates on the damage to fetus (DF) than those for private
general hospitals. It is also indicated, the older and with thelonger clinical experience, the lower estimates on RlW, RIF
and DF. The number of cases of CT, experienced by obstetricians, positively correlates with their estimates on RIF.
The present study implies that relatively older selfemployed obstetricians for medical clinic, with longer clinical experience but few cases of CT, are typically negative
for the explanation, backed by their lower estimates on therisk of CT. The results of the present study, we believe, reflect a tragedy that CT has been paid less attention here in
Japan. As we showed before, the lower risk estimates on
CT are not in line with recent epidemiological evidence.
Comprehensive epidemiological research is required to inspect their recognition from the view point of EBM (Evidence Based Medicine).

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
１巻

第１号
内

著
ミトコンドリアゲノムに基づく，フィリピン産肺吸虫の系統学的位置
佐藤 雪太，石上 盛敏，岩下淳，阿部 達也，
湯川 眞嘉，Bayani L. Blas，川島健治郎，吾妻
健 …………………………………………１‐３
ミトコンドリア１
２S rRNA 遺伝子領域から推測した，Oncomelania 属及びTricula 属の系統類縁関係
岡本 宗裕，Chin-Tson Lo，Wilfred U. Tiu，Dongchuan Qui，Pinardi Hadidjaja，Suchart Upatham，杉山広，田口 尚弘，平井 啓久，斉藤 康秀，
波部 重久，川中 正憲，平田 瑞城，吾妻
健 ……………………………………………５‐
１
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第４
３回日本熱帯医学会大会英文抄録 …………………………………………………………………………１
１
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会報・記録
日本熱帯医学会会則 ………………………………………………………………………………………７
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３年度（平成１
５年度）日本熱帯医学会役員名簿（２００
３年３月１日現在） ………………………７
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日本熱帯医学会雑誌編集委員名簿 ………………………………………………………………………７８‐
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投稿規定 ……………………………………………………………………………………………………８０‐
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著作権複写に関する注意 …………………………………………………………………………………８２

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 稲 中島

March２
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日熱医会誌

MARCH ２０
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CONTENTS

容
Japanese Journal of Tropical Medicine and Hygiene Vol. 31 No. 1

原

VOL．
３
１ No．
１

平成１
５年３月

Original article
Sato, Y., Iwagami, M., Iwashita, J., Abe, T., Yukawa, M., Blas, B L., Kawashima, K., and Agatsuma, T.
Phylogenetic status of a lung fluke in the Philippines based on mitochondrial genome ……………１‐３
Okamoto, M., Lo, C., Tiu, W. U., Qui, D., Hadidjaja, P., Upatham, S.,
Sugiyama, H., Taguchi, T., Hirai, H., Saitoh, Y., Habe, S., Kawanaka, M., Hirata, M., and Agatsuma, T.
Phylogenetic relationships of snails of the genera Oncomelania and Tricula
inferred from the mitochondrial 12S rRNA gene ……………………………………………………５‐１０
Proceedings of the 43rd Annual Meeting of Japanese Society of Tropical Medicine………………………………………………………………………………………………………１１‐７２

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は４mm（決）