第29巻第1号

平成13年3月

内原容著
日本住血吸虫感染マウスにおける宿主テストステロンが宿主一寄生虫相互作用に及ぼす影響（英文）

賀
陳

宏斌，張
仁利，川口
炎，太田 伸生………

仁，吉田
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第41回日本熱帯医学会大会英文抄録

一一一一

・5−85

会報・記録
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・・87
一一・

2001（平成13）年度日本熱帯医学会役員名簿（2001年1月1日現在）
日本熱帯医学会雑誌編集委員名簿……………・…・・……………・…・・一…
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日本医学会だより……………一……………………・・………………

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一89−92

一一一・93・一一・一・・94−95

一一・一・一一96−97

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一100−101lJpn. J. Trop. Med. Hyg., Vol. 29, No. 1, 2000, pp. 1‑4

ROLE OF TESTOSTERONE IN HOST‑PARASITE INTERACTION
DURING MURINE EXPERIMENTAL INFECTION OF
SCHISTOSOMA JAPANICUM
HONBIN HE*'*, RENLI ZHANG1, HITOSHI KAWAGUCHI' , AYAKO YOSHIDAl,

MAKOTO ITOH3, YAN CHEN' AND NOBUO OHTA*
Received February 1 9, 2000/Accepted January 10, 2001

Abstract: We analyzed roles of testosterone on susceptibility to infection during murine experimental schistosomiasis japonica.

Male C57BL/6 mice infected with Schistosoma japonicum showed marked reduction in serum level of testosterone compared with
infection‑free male control mice, suggesting that S. japonicum infection suppressed testosterone level in host animals. Potential rolesof testosterone in the susceptibility to schistosomiasis were tested by preparing mice in which testosterone levels were artificiallymanipulated. At 45 days after infection, we observed that male mice with reduced testosterone level showed significantly higher
worm burden (p<0.05). The area occupied by granulomas in liver was increased in mice of low circulating testosterone level possi‑
bly due to the high worm burden. High level of testosterone also inhibited, to some extent, fecundity of the female parasites. Treat‑ment of infected mice with an inhibitor for testosterone receptor did not alter worm burden, and this suggests that testosterone could
have direct effects on the parasites.

Key Words: Schistosoma japonicum , testosterone, susceptibility, egg granuloma, fecundity

INTRODUCTION

interaction (Wunderlich et al., 1998; Benten et al., 1 992).

In murine experimental Schistosoma mansoni infec‑

It is a rather common observation in epidemiologicaltion, Te was reported to have a resistant function for the in‑studies that prevalence of schistosomiasis in males are

fection (Eloi‑Santos et al ., 1992; Nakazawa et al., 1997).

higher that in females in human populations (Butterworth et

Similarly, hormone balance might modify innate or ac‑

al ., 1984; Rose et al ., 1997; Ministry of Health, China,

quired resistance to schistosome infections in humans, andl 998). The exact reason for such observation is not clear,some biological analysis was attempted for S. mansoni in‑
fection (Hagan et al., 1 998). On the other hand, Iittle

however, biological as well as behavioral factors have been

discussed (Chandiwana and Woolhouse, 1991). Gender‑

information is still available for schistosomiasis japonlca.

dependence in intensity of parasitic infection has been ana‑

We, therefore, extend a further study testing the situation oflyzed, and some of the studies demonstrated that hormonal

Te during murine experimental infection of S. japonicum. In

circumstances affect both incidence and intensity of para‑this study, we measured Te level in infected host mice, andsitic infections (Kamis and lbahim, 1989). Male sex hor‑

examined the effects of Te on worm burden, fecundity, andmones, including testosterone (Te), are known to havepathological lesions due to the parasite eggs. Furthermore,regulatory roles in immune responses of vertebrate animals

we also treated mice with an inhibitor for testosterone re‑(Fulford et al., 1998). Effects of Te in susceptibility to

ceptor during S. japonicum infection to test a possibility

parasitic infections are still controversial, however, accumu‑

that Te has direct effects on the worms.

lated results indicate a critical role(s) of Te in host‑parasite

Department of Medical Zoology, Nagoya City University Medical School, Nagoya, Japan
2

Hunan Institute of Parasitic Diseases, Yueyang, Hunan, China

3

Department of Parasitology, Aichi Medical University, Aichi, Japan

Correspondences to Nobuo Ohta, M.D.
Department of Medical Zoology, Nagoya City University Medical School, 1‑Aza Kawasumi, Mizuhocho,
Mizuhoku, Nagoya 467‑8601 . Japan
Tel. +8 1‑52‑85̲3‑8 1 84, Fax. +81‑52‑842‑0149, e‑mail: nohta@med.nagoya‑cu.ac,jp

2

Evaluation of worm burden and egg count in host mice: All

MATERIALS AND METHODS

mice were sacrificed at 45 days after infection with S.
japonicum, and parasites were recovered from the portal ve‑

Mice and parasites : Six week old male C57BL/6 mice werepurchased from Japan SLC (Hamamatsu, Japan), and werenous system of the infected mice using a conventional per‑infected percutaneously with 25 cercariae of S. japonicum

fusion technique. Adult male and female worms wereof a Chinese isolate kept in Human Institute of Parasitic

counted separately by using a stereomicroscope. Same parts

Diseases, China.

of liver from each group mice were resected, weighed, andTe measurement: Forty five days after the infection, blood

infected with or without S. japonicum were determined by athen digested for 12‑18 hr with 4% KOH for egg enumera‑tion (Cheever et al., 1980). The entire gut of each mouse
was also digested for egg counting. Te levels of the fourradio‑immunoassay (RIA) kit for Te (DPC, Tokyo, Japan)

groups were measured by RIA at a commercial clinical

was taken from the heart, and Te levels in sera of micein line with the manufacture's instruction.

laboratory (SRL, Hachioji, Japan). Because the RIA system

Manipulation of Te level: Male C57BL/6 mice at 6 weeks

in this case was different t rom that of the former description,old (SLC) were devided into four groups of which Te‑levels

Te levels from the two different testing were not compara‑

were artificially manipulated; castration alone as a low‑Tegroup (Group I ), injected with Te after castration as high‑Histopathological examination . The liver was dissected andble (Table I ).

Te mice (Group II ), injected with flutamide (Sigma, St.

immidiately fixed in 10% buffered formalin for morphomet‑

Louis, USA) which is an inhibitor for Te receptor (Walker

ric analysis. Liver sections were embedded in paraffin and

et al., 1 994) (Group lll ), and sham operation as control‑Te

stained with hematoxylin and eosin. We assessed the sizeof granulomas formed around a single egg containing a ma‑

mice (Group IV ). Castration or sham castration was per‑dium pentobarbital anesthesia. Group II mice received 5 mg

ture miracidium by using a video micrometer (VM‑30;
Olympus, Tokyo, Japan). The average of granuloma size

Te propionate (Teikoku Hormone, Tokyo, Japan) daily by

was determined by examining more than ten granulomas ininjecting alternately into the right and left thigh muscles be‑each mice. We also evaluated the mean percentage of granu‑
lomatous area in Imm2 Iiver sections.

formed 3 weeks before experimental infection under so‑

ginning 2 weeks prior to infection and continuing until themice were sacrificed. Mice of Group lll were injected with

Statistical analysis: Results from the different groups offlutamide at the dose of 25 mg/kg subcutaneously in themice were compared one‑to‑one by use of two‑tailed

same time‑schedule as the case of Group II . Mice were in‑fected percutaneously with 25 cercariae of a Hunan isolate

student's t‑test. Regression analyses were performed by
Spearman rank correlation for nonparametric regressionof S. japonicum .

tests and Pearson's correlation coefficient test (Neter, 1985).

A11 results are expressed as mean

SD.

Table 1 Worm recovery and testosterone levels in C57BL/6 mice in infection of S. japonicum

Mouse group
Ex eriment 1

Infection(‑)

Te level (pg/dl )(N=5)
8,413 5,134

270

(‑)

10.0

(‑)

1 55 .6

Total worms
Mature intestinal eggsl

female worm

Group I
(N=9)

Ex eriment 2
Te level (ng/dl )21.1 7.89**

Total worms(Female worms)

14.9

Intestinal egg count24,398(7.7

Mature intestinal eggsl

female worm
Mature intestinal eggsl

I 14* *
!

1 .8

20.4

Group IIGroup HI

(N=6)

(N=9)

5,366 2,147 **72.3

Group IV

(N=8)

79.8

1 89

277

9.8

3.2

(4.3

2.4)

6.5

2. 1

9.7 2.3

I .4* *)

(2.5

I .9)

(4.2

I .2)

I ,499 * *

2,258

536*

2,593

246

3,287 612

1,018

46 1 *

276

1 27

1 25

35.6

188 41.9

32.0

0.0* *

19.2 3.8

20.3

0.0* *

24.3

3 8,606 J: 5,800 **p<0.05, and* *p<0.0 1 , vs.

(N=7)

3.2* *

total intestinal egg (%)

Total liver e countS, japonicum ‑infected

22,614

18,486

Group IV or infection‑free mice.

26, 1 58

10,682

22,7820.0

16,806

3

When we assessed the effect of Te for granuloma for‑RESULTS AND DISCUSSION

mation in liver, there was a more widespread granulomatous

Te levels of four groups are shown in Table I . We ob‑lesion in low‑Te group than in control‑group (Table 2). The

served that there was marked reduction of Te level in the in‑fected mice compared with infection‑free animals (Table 1),mean percentege of the area occupied by granulomas was
significantly different between Group I and Group IV (p<and the difference in those Te levels was statistically signifi‑0.05), although there was no significant difference in meancant in the Student's t‑test (p<0.01). Under such different

diameter of granulomas formed around a single egg of S.
japonicum among any of groups tested here. Total liver

Te levels, numbers of recovered worms also were different

according to Te level. Low‑Te group had increased numberof infected worms than did high‑ or control‑Te groups (p<eggs in Group I were significantly increased in comparison0.01) (Table l). On the other hand, there was no significant

suggest that Te levels have modulatory effects on hepatic le‑with control Group IV mice (p<0.05) (Table 1). These data

defference in worm recovery when flutamide‑treated micesion during murine schistomiasis japonica not through thewere compared with sham castrated ones. Comparison ofenlargement of granuloma size, but through the increase ofworm recovery and Te levels in 29 mice examined showed af ecundity.

significant inverse relationship (r=‑0.374, p<0.05) (Fig. 1).

It is not clear how Te level affects the susceptibility of

The mean numbers of intestinal eggs are shown in Ta‑

murine hosts to S. japonicum infection. One of the possibli‑

ble I . Intestinal eggs in Group I were increased in number

ties is immunological environment being manipulated by Te.

than of high‑ as well as control‑Te groups. Furthermore, theThl cells are supposed to have important roles in protective

percentage of mature eggs per total intestinal eggs inimmunity to S. mansoni in mice (Sher et al., 1 989). In our

flutamide‑treated mice was significantly lower than that inpreliminary study, we did not detect evidence that Te level

control group (p<0.01). And the mean number of mature

was tightly associated with the serum level of cytokines ofintestinal eggs per female worm and the percentage of ma‑ture intestinal eggs in Group I was signficantly increased

Thl and Th2 during murine schistosomiasis japonica (datanot shown). Our finding that flutamide did not change the(p<0.01 ), suggesting the presence of an inhibitory role of Te

disease profile also suggests that Te‑mediated alternation in

for fecundity of female parasites and egg maturation.

the host function can not fully explain the reduced worm
burden observed in the present study, although we tested
function of flutamide at only a single dose. Together with

20

15
t
e)
:$

those, Te seems to have somehow direct inhibitory effects

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o
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o

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5

on the worm survival in host animals. It is needed to study
whether schistosomes express specific receptors for Te pro‑o

duced by host animals.

o

Although previous report testing schistosomiasis man‑oooo o oosoni concluded that egg production per a f emale worm didonot change even in reduced Te‑1evel (Eloi‑Santos et al .,oo

fecundity of female worm of S. japonicum . It might be pos‑

1992), our present observation suggests that Te inhibits
sible that difference in the species of parasites and/or in thestrain of host mice, of difference in conditions for experi‑

o
10

1 OO

1 ,OOO

Serum teststeron

l 0,000(log ng/dl )Figure 1 Correlation between serum testos‑terone levels and worm burdens inmice infected with 25 S. japoni‑cum cercariae. Of 29 mice with S.

japonicum infection examined,
mice with lower testosterone level

showed higher worm burden, andassociation between the two pa‑
rameters was statistically signifi‑

cant (r=‑0.374; p<0.05).

mental infection could provide such difference.

We have to consider about discordance of gender‑
dependent susceptibility to schistosomiasis between miceand humans. In humans, prevalence of schistosome infec‑
tion in males is much higher than in females even when fe‑
males have more frequent water‑contact (Butterworth et al.,1984). It is not clear whether male population has also Te ‑driven higher resistance than females as was observed inmurine infection, and detailed epidemiological analysis
considering Te levels is essential to conclude the role of Te

during human schistosomiasis.
As conclusion, Te level has significant effects on sus‑

ceptibility to S. japonicum in mice. Murine hosts of higher

4

Table 2

Conparisons of granuloma size and density in livers

Mouse group
Group l(N=9)
40.5

Percentage granulomas
Diameters of granuloma (um) 499.6

Group IIGroup nuGroup IV

(N=6)

(N=9)

(N=8)

9.7 *

9.6

8. 1 1 2.9

4.9 1 8.8

8.6

1 3̲ 6.5

3̲ 87.7

97.6 5 12.9

2 10.8 493.7

146.2'p<0.05, vs. Group IV

Te level seem to be resistant against S. japonicum infection,

6) Fulford, A.J.C., Webster, M., Ouma, J.H., Kimani, G. andalthough relatively small number of mice was tested. Re‑

Dunne, D.W. (1998): Puberty and age‑related changes insults in our present study are not inconsistent with other re‑susceptibility to schistosome infection. Parasitol. Today,

ports testing schistosomiasis mansoni, and Te seems to be

14, 23 ‑26

resistant factor for schistosome infection. Based on suchobservation, new therapeutics and prophylaxis could be de‑7) Hagan, P., Ndhlovu, P.D. and Dunne, D.W. (1998): Schis‑tosome immunology: more questions than ans̲wers. Parasi‑veloped for schistosomiasis through a Te‑mediated resis‑
tance. Roles of Te in humans are still not conclusive, and

8) Kamis, A.B. and lblahim, J.B. (1989): Effects of testoster‑further analysis is required for developing a new strategy

one on blood leukocytes in Plasmodium herghei‑infected

for controlling human schistosomiasis japonica.

mice. Parasitol, Res., 75, 61 1‑613

tol. Today, 1 4, 407‑41 2

9) Nakazawa, M., Fantappie, M.R., Freeman, G.L., Eloi‑

AC KNOWLEDGMENTS

Santos, S., Olsen, N.J., Kovacs, W.J., Secor, W.E. and Col‑ley, D.G. (1997): Schistosoma mansoni: Susceptibility dif‑This study was supported in part by a grant for Emerg‑

ferences between male and female mice can be mediateding and Re‑emerging Infectious Diseases, the Ministry of

by testosterone during early infection. Exp. Parasitol., 85,

Health and Welfare, Japan ( 1 2 1 O 1 801 ), a Grant‑in‑Aid for

Scientific Research, the Ministry of Education, Culture and

233‑240
10) Neter, J. (1985): Applied linear statistical models: Regres‑Sports, Japan (12576009), and a grant from the US‑Japansion, analysis of variance, and experimental designs, 2nd

Cooperative Medical Science Program ( 1 999, 2000).

ed., Irwin, Richard D.

ll) Office of Endemic Diseases Control, Ministry of Health

REFERENCESl) Benten, W.P., Wunderlich, F. and Mossmann, H. (1992):

Expert Advisory Committee on Schistosomiasis, Ministryof He,alth, China ( 1 998): A nation‑wide sampling survey onschistosomiasis in 1 995 in China. In Epidemic status of

Testosterone‑induced suppression of self healing Plasmo‑

schistosomiasis in China, Shanghai: Press of Chinese Jour‑

dium chabaudi : an etfect not mediated by androgen recep‑

nal of Schistosomiasis Control, 1‑ 17.

tor? J. Endocrinol., 1̲3̲5, 407‑413

2) Butterworth, A.E., Dalton, P.R., Dunne, D.W., Mugambi,

12) Ross, A.G., Yuesheng, L., Sleigh, A.S., Yi, L., Williarns, G.

M., Wu, W.Z., Xinsong, L., Yongkang, H. and McManus,

M., Ouma, J.H., Richardson, B.A., Siongok, T.K. and Stur‑

D.P. (1997): Epidemiologic features of Schistosoma ja‑

rock, R.F. (1984): Immunity after treatment of humanschistosomiasis mansoni. I . Study design, pretreatment ob‑

ponicum among fishermen and other occupational groups
in the Dongting Lake region (Human Province) of China.

servations and the results of treatment. Trans, Roy. Soc.

Am. J. Trop. Med. Hyg., 57, 302‑308

Trop. Med. Hyg., 78, 108‑123

3) Chandiwana, S.K. and Woolhouse, M.E. (1991): Hetero‑genecities in water contact patterns and the epidemiology

13) Sher, A., James, S.L., Correa‑Oliveria, R., Hieny, S, andPearce, E. (1989): Schistosome vaccines: current progress
and future prospects. Parasitol., 98, s61‑s68

of Schistosoma haematobium. Parasitol., 103, Pt3, 363‑370

14) Wunderlich, F., Mossmann, H.. Helwig, M, and Schillinger,4) Cheever, A.W., Duvall, R.H. and Minker, R.G. (1980):

G. (.1988): Resistance to Plasmodium chahaudi in BIO

Quantitative parasitological finding in rabbits infected with

mice: influence of the H‑2 complex and testosterone. In‑

Japanese and Philippine strains of S. ,japonicum . Am J.
Trop. Med. Hyg., 29, 1‑307‑ 1 3̲ 15

fect. Immum., 56, 2400‑2406

15) Walker, S.E., Besch‑Williford, C.L. and Keisler, D.H.

5) Eloi‑Santos, S., Olsen, N.J., Correa‑Oliveira, R. and Colley,( 1 994): Accelerated deaths from systemic lupus erythema‑

D.G. (1992): Schistosoma manso̲ ni: mortality, pathophysi‑

tosus in NZB X NZW F1 mice treated with the testosterone‑

ology, and susceptibility differences in male and female

blocking drug flutamide. J. Lab. Clin. Med., 1 24, 40 1 ‑407

mice. Exp. Parasitol, 75, 168‑1 75

5Jpn. J. Tro p , Med. Hyg., Vol. 29, No. 1, 2000, pp. 5 85

PROCEEDINGS OF THE 4 1 ST ANNUAL MEETING OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
10‑ 1 1 November 2000, Tokyo

President

Hiroshi Ohtomo
Professor, Department of Tropical Medicine,
Jikei University School of Medicine

CONTENTS
Prize Winner's lecture
JSTM (Japanese Society of Tropical Medicine) Young Investigator Award
A single‑chain antibody fragment specific for the Plasmodium berghei ookinete protein Pbs21 confers
transmission‑blokade in the mosquito midgut

Yoshida, S.

President's lecture
Recent advance of suppressive treatment against falciparum malaria in Japan

Ohtomo, H.

Special lecture
1 Principles and practice of travel medicine: experiences of a major travel clinic in Germany

Nothdurft, H.D.

2 Chemotherapy of malaria in South East Asia
3 A perspective on Thailand's HIV/AIDS epidemic and its prevention and control measures

Khamboonruang, C.

Hien, T.T.

Invited lecture

Clinical experience with curdlan sulfate in Plasmodium falcipalum malaria

Havlik, I. et al .

Symposium: Travel to tropical nations and vaccination
1 Immunization for international travelers in Japanese Red Cross Medical Center
Sonobe, T., Tsuchiya, K t, Komatsu, J. and Oritsu, M.
2 Defference in vaccination schedules and plans of vaccination for travellers

Takayama, N.

3 Problems on the polio vaccine: Is it need to continue polio vaccine?

4 Vaccination at Quarantine station in Japan

Doi, Y. et al.

lwasaki, E. and Ishizuka K.

5 Travel vaccines not marketed in Japan

Hamada, A. et al.

6 The optimal vaccinations for travelers in Japan

Sonobe, T.

Mini symposium
1

Situation of maternal and child health in Cambodia and the MCH Project2

National TB Control Project, Cambodia: struggle in the transition of health policy and aid policy

3

PHC project through human resources development focused on MCH and HIV/AIDS program4

Primary Health Care Project in Cambodian rural villages

5

Promotion of MCH through TBA support in Preykabas District of Takeo Province

6

Present status of infectious disease in Cambodia

Akashi, H. et al .

Onozaki, I.
Kono, S. et al .

Suwa, K.
Tsukamoto, S. et al .

Sato, K.

General presentation
A ‑ I Molecular analysis of alpha‑thalassemia in Nepal: correlation with malaria endemicity

Hamano, S. et al.

2 Molecular analysis of G6PD variants in Southeast Asia
Matsuoka, H. et al .
3 Microsatellite polymorphism in the heme Oxygenase‑1 gene promoter is associated with cerebral malaria

4 Trial to develop novel DNA vaccine using a full‑length‑cDNA Iibrary from Plasmodium berghei ANKAM

et al.

6

Watanabe, J. et al.

Akaki, M. et al.

5

Invasive forms of protozoan parasites have a positive charge at their contact site with host cells

6Bacteria expressing single‑chain immunotoxin inhibit malaria parasite development in mosquitoes Yoshida, S. et al.

7

Expression pattern of mitochondrial Complex H (succinate‑ubiquinone oxidoreductase) in the erythrocytic

stage cells of Plasmodium falciparum

Mi‑ichi, F. et al.

8

Effect of jasplakinollide on the growth and actin cytokeleton of Plasmodium falciparum

9

A malaria patient infected with Plasmodium,falciparum and P. malariae

Mizuno, Y. et al.
Ohnishi, K. et al.

10

Two cases of falciparum malaria with atypical clinical coursell

A case of severe falciparum malaria with prolonged immunohemolytic anemia

12

Chronic auto‑immune thrombocytopenia following recovery from severe falciparum malaria
Kato, Y. and Masuda, G.

13

A case of clinically chloroquine resistant vivax malaria infection

14

A case of falciparum malaria successfully treated with intravenous Artesunate

15

A case recovering completely from severe falcipurum malaria comp]icated with high levels of16

17

A case of severe f alciparum malaria with hyper‑bilirubinemia and nephropathy
The use of the atovaquone/proguanil combination (Malarone'*')18

Analysis of severe and complicated falciparum malaria in Japanese patients caused by delay of diagnosis

and treatment
On the WHO's Guidelines for Severe Malaria, 2000

Ohtomo, H. et al.

19

20

Problems of local transport system for anti‑malaria drugs

Ishiwata, K. et al.

21

Determination of plasma levels of prostaglandins and thromboxane in falciparum malaria patienes in Thailand

22

Detection of Plasmodium vivax from clinical specimens by the use of ICT Malaria Pf/Pv kit

23

An evaluation of ICT Malaria Pf/Pv in imported malaria in Japan

Ohtomo, H. et al .

24

A community‑based malaria control program in Palawan, the Philippines

25

Changes of the spleen rate on Aneityum Island

Kano. S. et al.
Kaneko, A. et al .

26

Active case detection surveys on malaria in recent Laos, with special reference to the comparative efficacy

multiple organ failure

with GC/MS/SIM

of Giemsa staining and Dipstick method on detection of malaria infection

27

29

Tanaka, Y. et al.

Kasai. D. et al.

Yasuoka, C. et al.

Aizawa, M. et al.

Hashizume. K. et al.
Hitani, A. et al.

Kimura, M. et al.

Nakano, Y. et al.

Abe, K. et al .

Kobayashi, J. et al.

Molecular epidemiological evaluation of pyrimethamine/sulfadoxine efficacy in Plasmodium falciparum
patients from Vanuatu archiplago

28

Yoshikawa, K. et al.

Mita, T. et al.

Population genetics and epidemiology: Malaria in Vanuatu
Lum, J.K. et al.
Repetitive dosing of artemisinin and quinine against Plasmodium falciparum in vitro : a simulation of thein vivo pharmacokinetics

Bwijo, A.B. et al.
Nagai, Y. et al.

30

Gene analysis of pfmdr I in mefloquine‑resistant Plasmodium falciparum

31

Mechanisms of parasiticidal activity of tetracyclines on Plasmodium falciparum : a possibility of the plastid

32

Antimalarial activiyty and potent enhancement of the sensitivity of Plasmodium falciparum to chloroquine

as the drug target

by the bisbenzylisoquinoline alkaloid cepharanthin

33Lin, Q.H. et al.
Haruki, K. et al.

The development of new antimalarial drugs‑ in vitro and in vivo antimalarial activity of endoperoxides

Ono, K. et al.
34
35

A potent antimalarial activity of hot‑water extract of Hydrangea macrophylla var. otaksa leaves against

Plasmodium yoelii 17XL in ICR mice Ishih. A.

et al.

Fractionation of antimalarial principle from Hydrangea macrophylla var. otaksa leaves and its activity against

Plasmodium yoelii 1 7XL in ICR mice Takezoe, H.
36 A potent antimalarial activity of hot‑water extracts of plants belonging to the family Saxiragaceae against

Plasmodium yoelii 17XL in ICR mice Sakai, M.
37 An antimalarial activity of hot‑water extract of Dichroa febnfuga leaves or roots against Plasmodium yoelii

et al .

et al.

et al.

B‑l

Three cases of creeping disease due to larval hookworm infection Nakamura‑Uchiyama. F. et al.

2

A case of urinary schistosomiasis with macrohematuria Miki, K. et al.

73

Four imported cystic echinococcosis cases in Japan confirmed serologically4

A severe case of venomous ophathalmia caused by spitting cobra in Central Kalahari

Ito, A. et al.

Nishiyama, T. and Osaki, M.

5Diagnosis of kala‑azar by ELISA using urine samples

Itoh. M. et al .

6

Evaluation of dot‑ELISA for the immunodiagnosis of trematode infections

Araki, K. et al .

A sero‑immunological studies on cysticercosislchikawa, H. et al.

8

Usefulness of Eml 8‑ELISA in monitoring effect of remedies against alveolar echinococcosis: case report

9

Ishikawa, Y. et al.
on long‑term chemotherapy with albendazole
Effect of cytochalasin D on the growth, encystation and multinucleation of Entamoeba invadens Makioka, A. et al.

10

Inhibition of encystation of Entamoeba invadens by antitublin drug oryzalin

Makioka, A. et al.

11

12

Molecular cloning and characterization of peroxiredoxin from Entamoeba moshkovskii
Modifications of staining procedures for the preparations of Entamoeba histolytica

Cheng, X.‑J. et al .

Kumagai, M. et al .

13

Epidemiological study of an epidemic amebiasis in institutions in Japan

Takeuchi, T. et all 4Congenital Toxoplasma infection at placenta resulting in intrauterin growth retardation

15

Intractable recurrent toxoplasmic retinochoroiditis

16

Four cases of cryptosporidiosis

17

Sero‑epidemiological survey of cryptosporidiosis at Sumbawa Island, Indonesia

18

Genetic characterization of Cryptosporidium parvum isolates from 1 4 cattle and 22 patients with diamhea

19Complex II (succinate‑ubiquinone reductase/quinol‑fumarate reductase) of Trypanosoma cruzi mitochondria

20

Characterization of cyanide insensitive oxidase of Trypanosoma brucei brucei expressed in Escherichia coli

21

Expression and properties of dihydroorotate dehydrogenase in Trypanosoma cruzi

22

In BALB/C mice, immune responses and the course of Leishmania major infection were controlled by

Yano, A. et al

Norose. K. et al
Shiota, T. and Arizono, N.
Yanagi, T. et al.

Yagita, K. et al .

Takashima, E. et al.
Kawai, K. et al.
Nara, T. et al.

cytokine expression plasmids delivery with the gene gun

Li, Y. et al.

24

Nashed, B.F. et al.
Th2 cytokines play different roles during the course of infection with Leishmania major
The effects of meglumine antimoniate (Clucantime' ' ') against L. major and L. amazonesis co‑cultured with

25

Leishmaniasis in Equador, with special reference to mucocutaneous forms and man‑biting sand flies,

26

Hashiguchi, Y. et al.
Lutzomyia spp. in the Amazonian endemic areas
Pulmonary infection caused by Rhodococcus equi in HIV‑infected patients: Report of six patients from27

northern Thailand
Influence of HIV infection against community‑acquired pneumonia in adults in Uganda

28

Genetic susceptibility to re‑infection of Schistosoma japonicum in China

29

A population study of the intermediate host of Schistosoma mansoni and the application of remote sensing

30

A questionnaire study to quantify the human behavior at river infested with Schistosoma mansoni Kisu, T. et al .

23

macrophages

Kasem, K.M.A. et al.

Watanabe, H. et al.
Yoshimine, H. et al.
Kikuchi, M. et al.

Mohamed, F.Y. et al .
31

Where do Schistosoma japonicum come from? Agatsuma, T. and lwagami, M.

32

The difference of the echogenic patterns among schistosomiasis japonica and schistosomiasis mekongi?

33

Localization of type IV collagen in granuloma formation and liver fibrosis due to schistosomiasis japonica

Otake, H. et al .

Yoshizaki, M. et al .

36

Influence of ultraviolet rays on infection of Schistosoma mansoni Ohwatari, N, et al.
Usefulness and limitation of COPT in low endemic area of schistosomiasis Ohmae, H. et al.
Urine cytology in an endemic area of schistosomiasis haematobia in Kenya Ohki, T. et al.

C‑1

Hepatic penicilliosis marneffei in Northern Thailand Toriyama, K. et al.

2

A unique drug‑susceptibility pattern of Vibrio cholerae Ol in Laos lwanaga. M. et al.

3

Two infant cases infected with typhoid fever in the Philippines Ozaki, A. et al.

34

35

4
5

Two
cases of melioidosis in Japan Kunishima, H. et al.
Measles serosurveillance study during mass immunization campaign in Malawi: antibody prevalence andserological responses using particle agglutination method Takechi, M. et al.

86

Expression and secretion of mutant cholera toxin and its B subunit in avirulent recombinant Salmonella :7

Evaluation of dengue diagnostic test kits

8

A clinical study on dengue hemorrhagic fever in Metro Manila, Philippines

Construction of cost‑effective oral vaccines against infectious diseases

Shimabukuro, I. et al.

Yamada, K. et al.
Oishi, K. et al.

9

A case of dengue fever complicated with probable acute disseminated encephalomyelitis

lO

Dengue vector situation along urban‑rural ecological gradient11

Molecular and in vitro analyses of dengue‑1 virus strains

12

Risk of West Nile fever and Japanese encephalitis in Japan13

A study on antigenicity of Japanese encephalitis virus isolates in Vientiane, Lao PDR

14

Mucosal immunizations with a Japanese encephalitis virus antigen‑cholera toxin B subunit fusion protein

Yamamoto, Y. et al.
Tsuda, Y. et al.
Ishak. H. et al.

Kamimura, K. et al.
Saito, M. and Fukunaga, T.

induce virus‑neutralizing antibodies in mice: an attempt for the development of edible vaccines against
Japanese encephalitis virus

Tadano, M. et al.

15

What is the role of apoptosis in flavivirus‑induced cytopathic effect?

16

Epidemiological survey on the relation between helminth infection and nasal allergy

17

Epidemiological survey on echinococcosis in Aomori Prefecture, Japan

Kamiya, H. et al.

18

Preliminary report of a survey for intestinal parasitosis at two areas in Nepal

Kanbara, H. et al.

l 9Plevalence of anti‑Toxocara antibodies among children in Jaboatao dos Guarapes, Pernambuco,
northeast Brazil

der Carmen Parquet, M. et al.
Watanabe. N. et al.

Yamasaki, H, et al.

20

Killing ofAcanthocheilonema viteae microfilariae by murine eosinophils

21

Kinetics of myoregulatory factors in the nurse cell formation of Trichinella

22

The prevalence of micro‑albuminuria in diabetic patients attending the Mulago Hospital Diabetic Clinic(MHDC), Kampala, Uganda

Kabole, I. et al.

23

Health care survice for Japanese living in cities of Southeast Asia

Ogawa, Y. et al.

24

Evaluation of GIDEON for imported infections

25

Thirteen‑years experience of the JICA tuberculosis control in Nepal

26

Development of urban dots in Chittagong City of Bangladesh

27

Immunohistochemical identification of phenolyc glycolipid‑1 (PGL‑ I ) in tissues of Buruli ulcer patients

Ishida, K. et al.

Takahashi, Y. et al.

Sakamoto, M. et al.
Osuga, K. et al.

Ishikawa, N.

Mwanatambwe, M. et al.

9Prize Winner's lecture
JSTM (Japanese Society of Tropical Medicine)

Young Investigator Award

A SINGLE‑CHAlN ANTIBODY FRAGMENT SPECIFIC FOR THE PLASMODIUM BERGHEIOOKINETE PROTEIN PBS21 CONFERS TRANSMISSION‑BLOKADE
IN THE MOSQUITO MIDGUTSHIGETO YOSHIDA
Department of Medical Zoology, Jichi Medical School

Pbs21 is a protein expressed on the surface of macro‑

To examine the properties and potential uses of agamete, zygote, ookinete and oocyst stages of the rodent
malaria parasite, Plasmodium berghei, as they develop in/on the mosquito midgut. It has been reported that anti‑Pbs

single‑chain antibody fragment (scFv) for blocking trans‑

21 mAbs effectively block the development of P berghei

mission of malaria parasites to the mosquitoes, we havecloned and sequenced the genes encoding variable regionsof the immunoglobulin heavy and light chains (VH and VL)

ookinetes in vitro and oocysts in Anopheles stephensi mos‑of mAb 13.1. The VH and VL genes were assembled as an

quitoes. The antigen has thus been identif"led as an impor‑

SCFV gene, and expressed in a baculovirus expression sys‑tant target in transmission‑blocking immunity and is amodel for development of transmission‑blocking vaccines.

tem. Following purification of 13.1 scFv, Western blotting

ldeally, transmission‑blocking vaccines should induce hightained the binding specificity of the parent mAb 1 3.1 for

titre, Iong‑lasting transmission‑blocking antibodies after

Pbs21 . Furthermore, 13.1 SCFV bound to the surface of P

single immunization. However, since the vaccine candidate
antigens of Pfs25/28 family are expressed by parasites inberghei ookinete, and blocked oocyst development in mos‑quito midgut by at least 93%, as assessed by oocyst counts

the mosquito stage but not in vertebrate host, boosting of

in mosquitoes. We suggest that the 13.1 SCFV gene could

the immune response following a natural infection has

be useful not only in studying for the mechanism ofnever been expected. Nonetheless the induction oftransmission‑blockade but also in generating, by mosquito

transmission‑blocking antibodies in patiences in combina‑
tion with anti‑malarial drugs could be of considerable im‑production of mosquitoes refractory to malaria.

portance to prevent the spread of the drug‑resistant parasites.

and inhibition ELISA assays confirmed that 1 3.1 SCFV re‑germline transformation, a model system to evaluate the10President's lecture

RECENT ADVANCE OF SUPPRESSIVE TREATMENT AGAINST FALCIPURUM MALARIAIN JAPAN
HIROSHIOHTOMO

Department of Tropical Medicine, Jikei University School of Medicine

The treatment of malaria is chemotherapy that acts
specific for the stage of parasite and results in improvementof symptoms with suppression of fever. The treatment of
falcipalum malaria has been changing by appearance ofFansidar had been distributed from 1980 by the study
group and had been made a main choice for the treament.
Mefloquine has been imported by the study group since
1 991 . The frequency of choice of this drug has been gradu‑

drug‑resistant strains of this parasite and supply of orphan

ally increasing and reached to 77% of falcipalum malaria

drugs from the study group supported by Japanese Ministryof Public Health and Welfare. Although many kinds of anti‑

cases in 1999. Tetracycline or artemisinin and its deriva‑

malarial drug has been developed for the treatment of drug‑

resistant falciparum malaria, only two drugs obtained ap‑

mefloquine. Recently, severe falcipalum malaria has beenfrequently found. 29 to 37% of these patients during 1995

proval for general use from Japanese government, namely

to 1999 were treated with slow intravenous infusion of qui‑quinine powder as a classic anti‑malaria drug and Fansidarnine dihydrochloride or quinine gluconate contributing ef‑

registered in 1987. Under these situations, the study groupfectively for their life saving. Moreover, severe falcipalum

has supported the therapy of imported malaria by some or‑malaria has been also treated with combined administration

phan drugs. During 1970's to early 1980's the period whenwith quinine and artemisinin or its derivatives from 1999, or

imported malaria started to be paid attention in Japan, sin‑

Malarone (atvaquon+proguanil), new drug having been dis‑gle or combined administration of MP tablet, quinine andtributed by the study group from 1998.

chloroquine were frequently used.

tives are used as a drug for combined administration with

11

Special lecture

1 PRINCIPLES AND PRACTICE OF TRAVEL MEDICINE=
EXPERIENCES OF A MAJOR TRAVEL CLINIC IN GERMANY
HANS D. NOTHDURFT, M.D.
Associate Professor for Internal, Tropical and Travel Medicine,

Department of Infectious Diseases and Tropical Medicine,

University of Munich, Germany

What is Travel Medicine?

Travel Medicine it not Tropical Medicine: Tropical
Medicine is specialised for health risks to the indigenous

Role of the Travel Medicine Clinic
The travel clinic has to address all health needs of theinternational traveler:

population and for infectious diseases. Travel Medicine is‑avoidance of enteric infection, mosquito/insect

specialised for health risks to our population travelling toborne diseases and vaccine preventable illness

developing world.

‑avoidance of blood borne diseases such as hepatitis

Travel Medicine means: development of expertise; new

B and HIVdiscipline; diverse background of practitioners; subject not‑protection from suntaught in medical schools; no formal training. There aredifferent possible backgrounds: Preventive Medicine/Public‑provision and documentation of all necessary vac‑

Health (global impact of travel‑impact of travel on hostcinationscountries), for infectious diseases (tropical medicine), for‑supply or prescription of appropriate medications‑coping with jet lag

gastroenterology (diarrhoea‑hepatitis), for internal medicine/(e.g. antimalarials, antidiarrheals, antibiotics)

family medicine and for surgery (accidents‑infected wounds).

‑instructions for the self treatment

The Practice of Travel Medicine crosses many disci‑plines: For example, with endocrinology/internal Medicine‑provision of referral information (e.g. dialysis in(insulin dosing in patient travelling Rome‑Hongkong), with

India)

obstetricslgyn (pregnancy and travel), with pediatrics (chil‑

Need for Specialisation:

dren/infants and travel), and with internal medicine (chronic‑specific health problems of individual traveller

Travel Medicine is a dynamic discipline

diseases and travel).

‑rapidly changing information

Problems in Travel Medicine‑need for surveillance

Travel Medicine meets some problems: There are diffi‑‑diseases/epidemic/vaccination requirement changes

culties due to the host countries, with climate and infra‑‑need to develop data base on health risks in order tostructure, and with different culture too. For new travel
styles: Iast minute/1ast second booking, adventure travel,‑monitor governmental and regulatory agencies

be able for individual risk assessment

sports activities (climbing, diving). Travel industry is a

(MOH, WHO)

problem too, because of negligence and ignorance. In the‑medical literature/speciality j ournals/computer

medical profession, we can also find as problems ignorance,

based services

contradictory information and excessive restriction of vacci‑‑network of travel medicine specialist and confer‑

nations. Problems are due to the traveller too: Iack of infor‑

ences

mation, refusal of recommendations. As obstacle for pre‑How to establish and run a travel clinic?

ventive therapy, there is resistance of pathogens, costs, ru‑‑Examples from Munich

mours and media reports. For new destinations: Iack of in‑formation and exotic, high risk areas.

12

2 CHEMOTHERAPY OF MALARIA IN SOUTH EAST ASIA
TRAN TINH HIEN, M.D.
Center for Tropical Diseases Ho Chi Minh City, Viet Nam

Malaria is still the most common infectious cause ofvance, particularly in areas where parenteral administrationmortality and morbidity in Viet Nam as it is in other Southis difficult. It is simple and can be done by unskilled per‑East Asian countries. The presence of resistance to avail‑sons. The suppository formulation can provide emergency

able antimalarials and compliance in the target population

treatment of patients at periphery until the patient can be re‑are factors that influence the choice of drugs and regimens.

ferred to a health facility where parenteral treatment is pos‑

The loss of antimalarial drugs to resistance may be one ofsible. It also avoids the risk of parenteral administration

the greatest threats to the control of malaria in the tropic

such as hepatitis or HIV infection.

There are evidences that with its rapid parasite clear‑countries.

Several clinical trials have been conducted to develop

ance, very early treatment of uncomplicated cases with oral

an optimal treatment for drug‑resistant falciparum malaria

or suppository formulation of artemisinin and derivatives,in patients with the disease in different settings. The results

applicable at a primary health care level will help to preventof these trials suggest that a combination of artemisinin (orthe development of complications, consequently reducing

its derivatives) and other antimalarial drugs (mefloquine or

the overall malaria mortality rate.

lumefantril or piperaquine) is the most effective, safe andConcerning P. vivax malaria, chloroquine has been ef‑

practical treatment for acute non‑complicated malaria due

fective in SEA region and its management is less compli‑

to multidrug resistant Plasmodium falciparum . Concerning

doses of artemisinin or analogues are recommended due tocated. However, recently, a study in Thailand has shownthat P vivax was more common in women having their firstbaby and was associated with maternal anemia and a sig‑

their rapid parasite clearance time and other possible anti‑

nificant reduction in birth weight which increased the risk

cytoadherence effects. The rectal administration of qingha‑

of infant mortality and suggest that the use of antimalarial

osu drugs in patients with disease‑related nausea and/or
vomiting would obviously provide a useful therapeutic ad‑

drug against P. vivax in pregnancy may be justified

severe and complicated malaria, parenteral or rectal multi‑

3 A PERSPECTIVE ON THAILAND'S HIV/AIDS EPIDEMIC AND ITS PREVENTION

AND CONTROL MEASURESCHIRASAK KHAMBOONRUANG, M.D ., PHD.
Research Institute for Health Sciences (RIHES), Chiang Mai University

B ackground:

The first case of full‑blown AIDS was reported in
Thailand in August 1 984. Up‑to‑date, the Royal Thai Minis‑

ported in Bangkok on August 1984, HIV‑1 infection has

been spread among homosexual gay men or males who
have sex with males, male commercial sex workers

try of Public‑Health (MoPH) has estimated, as based on(MCSWs) and injection drug users (IDUs). During the frrst

sentinel surveillance, about I .3 million Thais were infectedround of sentinel serosurveillance conducted by the Royal

with Human Immunodeficiency Virus‑1 (HIV‑ 1). Accumu‑

Thai Ministry of Public Health (MoPH) in 1989, an unex‑

lated numbers of AIDS cases and HIV‑symptoms patients inpected high prevalence rate (44%) was detected in direct fe‑

Thailand from 1985‑2000 was reported as 142,027 andmale commercial sex workers (FCSWs) in Chiang Mai

55,635, respectively. Up to June 2000, the infection rate has

been estimated about 329 with a death rate of about 75 per

Province, northern part of Thailand. This finding was re‑ported as a first wave of HIV epidemic of HIV heterosexual

1 O0,000 populations.

transmission in Thailand. Later on, it was disclosed that the

Epidemiology:"epicenter" of HIV infection was located in six upper north‑Few years after the first full‑blown AIDS case was re‑ern provinces. Later, HIV have found their ways to spread13

into their clients, mostly Thai males who have had risk‑

in IDUs and FCSWs was disclosed in 1989, the Thai gov‑

behaviors of having sex with FCSW and came to attend
MoPH's STD clinics after developing STD symptoms andernment has responded decisively and comprehensibly

signs. This risk‑group is called "male STD clinic users" orthrough FCSWs. At the early phase of the epidemic, ap‑"male STD clinic attendees". Subsequently, the infection inproaches to control the transmission was targeted on publicmale STD clinic users has been transmitted to their family,

health problem only. However, in a later phase, a societal is‑launching a control program to reduce a scope of epidemic

wives and/or partners. This leads to an episode of a current

sues related to HIV infection have been included. The con‑"wave" of the epidemic that is a "mother to child" transmis‑

trol program could be discernable in four aspects. Firstly,sion. In parallel to the sentinel serosurveillance among highfor legislative action, announcement to include AIDS in therisk groups which was initiated by MOPH in 1989, a routine

list of notifiable diseases was amended in Communicable

HIV testing of pregnant women attended the MoPH's Ante‑Natal‑Clinics (ANCs), of blood donors from blood banks
across the nation, and male military conscripts have been

Diseases Control Act. Secondly, for administrative action,

included in the survey.

Prevention and Control of AIDS. This Commission is

the National Advisory Committee on AIDS was establishedin 1989. Later, it was called the National Commission fo

Results obtained from the sentinel serosurveillance

chaired by the Prime Minister. The responsibilities of theamong high‑risk groups revealed that MCSWs from three
tourist provinces showed median prevalence of 10%, ranged

Commission are to setting up a policy for national AIDS
control program, to provide a recommendation about annualfrom 9.25% to 12.4% (between 1991‑1999). Infection rates

budget allocation for AIDS control to the government; to coamong IDUs have remained extremely high ‑at 3 1 % to‑ordinate, co‑operate, and to facilitate with organizations/in‑

43% ‑ and are still rising in Bangkok, the Central region,

stitutions dealing with prevention and control of AIDS.

and the south. National HIV prevalence among direct andindirect FCSWs (between 1989‑1999) were 16.7%, (range:
16% to 44.7%), and 6.6% (range: 1.6% to 10.1%), respec‑tively. Prevalence among male STD clinic users (between
1989‑1998) was 8.3% (range: 20/0 to 9%), with an increas‑
ing trend. Results obtained from add.itional HIV testing

Lastly, is to give advice, to provide recommendation for re‑

population showed that a median prevalence among preg‑
nant women was 1.76% (range: 0.8% to 2.3%), and is ele‑
vating. HIV among blood donors was 0.44% and trend is
static. HIV prevalence among male conscripts of 21‑year‑
old was I .6% (range: 0.5% to 4.0%), with a decreasing

across the country. Fourthly, for implementing or practical

HIV health education, HIV counseling, and the promotionof 100% condom use in brothels and sex establishment havetrend.

been considered to be the most effective and successful tool

Molecular Epidemiology :to slow down the epidemic.

In the early phase of the epidemic, HIV‑1 circulating in

infected homosexuals and IDUs was identified as B subtype
or B'subtype. At the early phase of the first "wave" of het‑

erosexual transmission in FCSWs, HIV E subtype was iden‑

searchers in conducting biomedical and behavioral interven‑tion researches submitted for the Commission to review andapproval. Thirdly, for technical action, the MOPH in 1 989

initiated HIV serosurveillance, which has been considered
to be a more efficient effort of monitoring HIV transmission

action, "short‑term" and "medium‑term" action plans tocontrol AIDS have been developed. The extensive outreach

Conclusion:
AIDS arrived in Thailand around 1984, but the national

response was silent. When the infection was explosive
among IDUs and FCSWs, the national response was verytified in majority and B'subtype in minority with a current

vigorous. The national responses that contributed to the suc‑extensive molecular studies, newly isolates of subtype E

cess of the control included national leadership, politicalfrom Thailand is now called "Circulating Recombinantcommitment, strong epidemiological surveillance, exten‑

Form O1 AE" or "CRFOI AE".

sive outreach HIV health education and counseling, and thePrevention and Control:

promotion of 100% condom use in sex establishments. Thisnational response has shown a slow down of the epidemic.

After an explosive epidemic outbreak of HIV infection

14

Invited lecture

CLINICAL EXPERIENCE WITH CURDLAN SULFATE IN PLASMODIUM FALCIPURUM MALARIA
I. HAVLIK1, P. THUMA2, S. LOOAREESUWAN3 AND Y. KANEK04
Department of Experimental & Clinical Pharmacology,
University of Witwatersrand, South Africal

Macha Malaria Research Institute, Choma, Zambia2
Faculty of Tropical Medicine, Mahidol University, Thailand3 andAjinomoto Co., Inc., Tokyo, Japan

Curdlan Sulfate a sulfated I ,3,P‑D‑glucan (CRDS) has

junct medication to artesunate.

been shown in pre‑clinical studies as being effective in in‑

The two arms of treatment in all studies showed simi‑

hibiting P falciparum in vitro , P berghei in vivo and Babe‑lar results for all haematological, biochemical and urine

sia cannis infection in dogs. CRDS has been shown to besynergistic with chloroquine, quinine and artesunate, toanalysis results. In all studies only adverse events recorded

down modulate the immune response in decreasing bothduring CRDS treatment arm was an increase in APTT. This
adverse effect is well documented with CRDS and can be

TNF and NO and direct nonspecific effect on cytoadherence
and rosetting may be predicted as has been described previ‑

easily monitored with the subsequent adjustment of dosing.

ously with other sulfated polysaccharides, e.g. heparin.

on cytoadherence as recorded in phase A where a slight in‑Thus CRDS is a potential candidate as an adjunct medica‑crease in parasitaemia has been observed after CRDS treat‑

There is an indication of the positive effect of CRDS

tion for treatment of severe/cerebral malaria. The clinicalment without subjects presenting with malaria symptoms.

studies have been initiated in four phases to address safety,

In phases B, IIB and C CRDS facilitates clearance of para‑efficacy and potential interaction in vivo of CRDS withsite and fever clearance time (e.g. severity of disease). In

classical antimalarials.

addition, CRDS showed no recrudescence in patients

Phase A ‑randomised, double blind, crossover, placebo

treated with chloroquine in phase B. CRDS seems benefi‑

controlled study (CRDS used alone) in pa‑

cial in cerebral malaria in patients where there is no organ

tients with asymptomatic malaria.

damage present (renal failure and pulmonary oedema).

Phase B ‑randomised, double blind, placebo controlled

In conclusion CRDS was well tolerated in all studies.

study in patients with mild malaria as adjunct

CRDS seems to augment disease process along the line of

medication to chloroquine.

the results obtained from pre‑clinical studies. It seems that

Phase IIB ‑randomised, double blind, placebo con

the group of patients, which will benefit most are sever/trolled study in patients with severe ma‑

cerebral cases with no additional clinical complications
such as renal failure and pulmonary oedema. These compli‑

laria as adjunct medication to artesunate.

Phase C ‑randomised, double blind, placebo controlled
study in patients with cerebral malaria as ad‑

cations are rare in children. This group represents the ma‑
jority of deaths recorded in Africa.

15

Symposium: Travel to tropical nations and vaccinationl IMMUNIZATION FOR INTERNATIONAL TRAVELERS IN
JAPANESE RED CROSS MEDICAL CENTERTOMOYOSHI SONOBEl, K. TSUCHIYAl, JUNKO KOMATSU2 AND MASAE ORITSU2
Department of Pediatric Health Carel and Department of Health Care2,

Japanese Red Cross Medical Center

Our Pediatric Health Care Clinic consists of Well baby

clinic, Psychological counseling and Immunization service.

given simultaneously when needed. Six visitors were only
for consultation without vaccination.

Immunization service is provided to all children including

Adult immunization was also provided at our Healthsick travelers and international children. Proportion of inter‑

Care Clinic. Total number of immunization to international

national travelers was about 2% of all visitors in these serv‑

travelers from January to June 2000 was 351. Destinationices in 1999. Age distribution less than I year of age, I to 9

were as follows; Asia 56%, North America 39%, Europe
2%, South America 1%, Mid East 1%, Africa 1%. Given

years and over 9 years were 66%, 27% and 7%, respectively.

Regarding Area distribution of destination, Asia, North
America, Australia, Europe and Africa was 72%, 24%, I %,1 %, I % and I %, respectively. Among 6,876 immunizations
461 (7%) were given to international travelers: DPT 122(5%), DT 2 (4%), Measles 21 (3%), Rubella 25 (6%),
Mumps 7 (2%), BCG 30 (24%), Polio 52 (22%), HB 140

vaccine was as follows; HB 65, HA 59, Rabies 47, Tetanus

35, DT 35, Measles 30, Rubella 26, Mumps 25. Polio 10,
JE I O, Varicella 4, IG 3, Cholera 2.

The main reason for international travel was job trans‑

fer both in adult and children. They tended to have little

knowledge of immunization. Most children had enough(43%), JE 39 (5%), Varicella 11 (2%), Rabies 7 (100%),

time but most adult travelers did not have enough time be‑

Tetanus 2 (100%), IG 3 (100%). The parenthesis is the pro‑fore their departure.

portion to total number. Immunizations up to 4 kinds were

2 DEFFERENCE IN VACCINATION SCHEDULES AND PLANS
OF VACCINATION FOR TRAVELLERSNAOHIDE TAKAYAMA
Department of Pediatrics, Tokyo Metropolitan Komagome Hospital

The vaccination schedule of Japan is not standard
when seeing in the world. The characteristics of thediseases before their departure. The vaccines that should beJapanese vaccination schedule are as follows; The kinds of

The yellow fever vaccine should be injected when going toat least given are tetanus toxoid and hepatitis A vaccine.

vaccines injected to infants and young children are fewer inthe yellow fever endemic area, even if it is not demandedkind than many other developed countries, a few kinds ofpolyvalent mixed vaccines are adopted, and simultaneousplaned to complete injection of many kinds of vaccines

injections of 2‑3 kinds of vaccines on the same day are notbefore leaving the country by simultaneously injecting 2‑3

generally accepted.

vaccines on the same day.

for entering a country. The vaccination schedule should be

The vaccines necessary for those who are going abroad

The post‑exposure prophylaxis against rabies is added

are divided into 2 categories; namely vaccines demanded
for entering a country and vaccines to preserve one's health.

as a special case of vaccination. To the person who was
bitten by possibly rabid animals in the rabies endemic

The former is only a yellow fever vaccine. Travellers should

regions, the post‑exposure prophylaxis should be given.

complete immunization against vaccine‑preventable16

3 PROBLEMS ON THE POLIO VACCINE: IS IT NEED TO CONTINUE POLIO VACCINE?
YUZURU DOI, HITOSHI HORIE AND SO HASHIZUME
Japan Poliomyelitis Research Institute(Abstract not received on time)

4 VACCINATION AT QUARANTINE STATION IN JAPANEMIKO IWASAKI, KIGEN ISHIZUKA, NOBUYUKI YAGI AND SHUN‑ICHI INAGAKI
Sendai Quarantine Station, Ministry of Health, Labor and Welfare

The first Quarantine Station in Japan was established mune system, are increasing.
Currently, Japanese Quarantine Stations are focusing
about 1 30 years ago. Now the Quarantine Station are set up
at major international ports and airports to prevent patho‑

not only on the prevention of the IHR designated infectious

genic organs from entering country through travelers, im‑diseases like cholera, yellow fever and plaque but also on

ported foods and cargos. Due to the advancement of trans‑

controlling of the infectious disease by vaccination and edu‑

portation and the increased number of travelers, the focus

cation to international travelers before departure to adjustfor "Quarantine" is changing. travelers to various health conditions. Therefore, since 1999
One third of Japanese traveling overseas are in 20's, Japanese Quarantine is giving vaccination not only for chol‑and some of them are backpackers without appropriate pro‑ era, yellow fever and plaque but also polio, diphtheria,
tection and knowledge for preventing infectious disease. In hepatitis A, rabies, Japanese encephalitis, tetanus and mea‑

addition, travelers in 60's and 70's, who have weaker im‑ sles.

5 TRAVEL VACClNES NOT MARKETED IN JAPANATSUO HAMADA, YUTAKA UJITA AND EIICHI OKUZAWA
Japan Overseas Health Administration Center, Labor Welfare Corporation

Recently many Japanese people who travel and stay

are marketed in some developed countries for travelers toabroad have started to take preventative immunization. Re‑

the endemic area. For tickborn encephalitis, injectable inac‑

sults of a survey we conducted on 5,338 Japanese expatri‑
ates staying in developing countries showed that 46.9 ; o oftivated vaccine is available in endemic areas such as East

them were vaccinated against hepatitis A, hepatitis B, teta‑

ter legally in Japan, although yellow fever vaccine can benus, rabies, etc. These vaccines are marketed in Japan. We

legally administered in Quarantine Stations as special case.

also noted that some Japanese expatriates were inoculated

However, Japanese physicians can personally import thesewith vaccines not available in Japan. These include vac‑cines against yellow fever, typhoid fever, meningococcal

vaccines to inoculate Japanese travelers. They must frrst ob‑

meningitis and tickborn encephalitis. In some developed
countries, injectable purified polysaccharide vaccine andInternational Trade and Industry and the Japanese Ministryoral live vaccine are licensed for use against typhoid fever.

physicians more aware of this system. We must also encour‑

These two vaccines are moderately effective and have fewer

age the Japanese government to grant approval to travelside effects. For prevention of meningococcal meningitis,

vaccines not marketed in Japan.

bivalent and quadrivalent purified polysaccharide vaccines

Europe and Russia. These vaccines are difficult to adminis‑

tain temporary permission from the Japanese Ministry ofof Health and Welfare. It is important to make Japanese17

6 THE OPTIMAL VACCINATIONS FOR TRAVELERS IN JAPANTOMOYOSHI SONOBE
Department of Pediatrics, Japanese Red Cross Medical Center

The vaccine givers are required to have the latest

had almost eradicated those diseases. Because USA has anknowledge of travel medicine to give travelers the optimal

excellent vaccination system based on the strict risk control

vaccinations. It became easy to obtain the latest informa‑

policy to save the health and life and property of the people.

tion of endemic diseases by Internet recently. Nowadays,
however, many routine vaccines are also recommended for

Until the consensus of good vaccination system is estab‑travelers other than vaccines for endemic diseases. Thus the

may be used. To promote vaccination for travelers, the vac‑vaccine givers are required to have the sufficient knowledgecine service should be friendly and convenient even forof routine vaccinations not only for adults but also for chil‑busy travelers. For example, the service should be available

lished in Japan, vaccination schedule or method of USA

dren. To make good vaccination schedule, vaccine givers

even at night and on Sunday. Now we are requested to es‑

have to realize that Japan is one of the worst countries totablish our own good vaccination system for the Japaneseprevent vaccine‑effective diseases. To the contrary USA

people including travelers.

18

Mini symposium:l SITUATION OF MATERNAL AND CHILD HEALTH IN CAMBODIA AND THE MCH PROJECTHIDECHIKA AKASHI, SEIKI TATENO AND TAKAKO YAMADAInternational Medical Center of Japan, Bureau of International Cooperation

Health situation in Cambodia: The Maternal Mortality

and training. The new NMCHC was built by Grant Aid

Rate (MMR) in Cambodia was 500, Infant Mortality Ratewas 1 15 (UNIECF 1995). More than 90% of deliveries arefrom Japan opened in 1 997, and several new systems inoccurred at home in rural area. Many births are attended by

the poor, patient registration, independence of nursing divi‑family members or Traditional Birth Attendant (TBA)S.
HIV infection rate is estimated to be around 4%. Bloodsion on management. On clinical, partograph and magne‑transfusion system is weak and there is no ambulance sys‑
tem nationally. Many health facilities, including health cen‑

conference were started. Also trainings of HC midwives
were started, and provincial hospital midwife or doctor

ters (HCs), district hospitals, provincial hospitals and na‑

trainings, hospital management seminar and so on were alsotional hospitals are old, and many medical equipments are

performed. In addition, materials were provided such as

Cambodia were introduced, such as user f ees, exemption forsium treatment were introduced as new techniques, and casebroken. About health personnel, usually TBAS are illiterate,

doppler fetal heart beat detector to district hospitals, TBA

some nurses and midwives cannot measure blood pressure,kits to NGOs, and motorcycles to provincial health depart‑nor read graphs, nor calculate, and some doctors do not un‑ments.

derstand anatomy, physiology and pathology. The seniorstaffs do not know how to transfer their knowledge andtechnique to juniors in practice, and it seems to be difficult
to expect for them to refer the patient to the other health fa‑
cilities.

Results: The number of patients is increasing after the

opening of the new NMCHC, and user fees with exemption
system has high reputation from the Ministry of Health.
More than 1 80 HC midwives were trained and around 20%
of HCs have been covered, and also other donor agencies

Maternal and Child Health (MCH) Project: Japan In‑

sent their midwives in their project areas to the training

ternational Cooperation Agency (JICA) started the MCH

courses in the NMCHC. However, many problems includ‑

project in April, 1995, and its Overall Goal is to reduce theing low government salary and low staff motivation are re‑

MMR in Cambodia, and the Project Purpose is to improve

lating to the national level problems and it is difficult only

National MCH Center (NMCHC) on management, clinicalfor the project to change everything.

2 NATIONAL TB CONTROL PROJECT, CAMBODIA= STRUGGLE IN THE TRANSMISSION
OF HEALTH POLICY AND AID POLICYIKUSHI ONOZAKI
JICA Project Chief Advisor, Chiba Anti‑TB Association[Setting] Situation of TB in Cambodia might be theceeded the capacity of TB services despite the primary suc‑

worst in Asia in prevalence and incidence. Directly Ob‑
served Treatment with Short Course Therapy (DOTS) was

cess of DOTS; HIV/AIDS epidemic may increase burden of
TB even double in near future. While National TB Program

introduced as a national policy in 1994, and it has been suc‑has been enjoying strong vertical structure supported by in‑

cessfully implemented through public hospital network

ternational agencies, Health Sector Reform left TB program

since then. In 1 999, 1 45 TB units in all 23 provinces acrossout of new system: DOTS is not available in newly estab‑lished health centers network. Many poor patients in vil‑the nation provided DOTS, when National TB Control Pro‑
ject by JICA Iaunched.
[Problems] The project extensively reviewed the situ‑

ation and found that the magnitude of the TB problem ex‑lages are dying with TB without visiting hospitals in town.

[Target of the project] The project aims to expand TB serv‑

ices into the community level through new health system.

19[Constrains] Low motivation of the civil servants due

Shift of qualified staff from public sector to private in‑to low salary is one of major constrains. Aid agencies often

cluding NGOS is also an obstacle to sustain a program.

seek outcome in short span, and recipients seek instantbenefit such as cash flow. Those behaviors make us diffi‑

tial. To ensure the best use of resources, we should be flex‑

cult to set a long‑term goal.

ible including reallocation of current budgeting.

LRecommendation] Inter‑agency coordination is essen‑

3 PHC PROJECT THROUGH HUMAN RESOURCES DEVELOPMENT FOCUSED ON
MCH AND HIV/AIDS PROGRAMSATOKO KONO, MARI SATO, TAKASHI SAWADA, YUKO MORIMOTO,
HARUHISA NISHINA AND TORU HONDA
Services for Health in Asian & African Regions

Since 1988, we SHARE have been implementing pri‑mary health program in Cambodia. Especially since 1998
we have PHC project that aims for human resources devel‑
opment through health education focused on MCH and Hrv/
AIDS in Sreu‑sentou Province located north of Phnom Penh.
Activities to achieve such obj ects are as below.

A. Health education and community empowerment targeted
to mothers.

1 ) Health education: To be acquired accurate knowl‑training covered 86 percent of TBA in our target

area. TBA showed active attitude to attend thegroup discussion and role‑playing. We developedand supplied appropriate support information/
education materials and teaching methods. After
training, referral cases from TBA to hospital in‑creased.

2 ) According to increase of HIV/AIDS patients intarget area, the need of training for health staffs isedge about basical health problems by mothers,

also increasing. We held HIV/AIDS training for

health education was performed in 4 villages.
The curriculum includes personal hygiene andhealth staffs in our target area. Under insufficient

sanitation, diarrhea, nutrition, immunization, Io‑

AIDS patients undergo health care in their home

cal environmental hygiene, emergent medication,
the body and health of female, antenatal care,by their families. Health education by health staff

birth spacing, HIV/AIDS, etc.

AIDS patient's families who care patients, anddevelopment of health system to support health2 ) Organization of mother group: We followed up
the villages that finished health education, and fa‑

situation of health system in Cambodia, HIV/to villagers, education of health care skill for HIV/

staffs are needed.

cilitated to organize mother groups. Several
mothers were chosen as leaders of their group.

C. Health education of HIV/AIDS prevention.
1 ) Health education of HIV/AIDS prevention at jun‑

They were strengthened to manage their groupior high school: For junior high school teachers,

having group discussion about health topics thattraining about health education (trainers' training)were interested for them.

was carried. The teachers who attended training

B . Training for health staffs and TBA (traditional birth at‑

tendance).

1 ) TBA training: TBA carries 60 percent of all de‑liveries in Cambodia. We held TBA training to
improve and activate local MCH situation. Our

managed health education to their students.

2 ) Support to Provincial HIV/AIDS Committee:
HIV/AIDS seminar for village mayors, manage‑
ment of HIV/AIDS Day's festival, etc. were car‑
ried.

20

4 PRIMARY HEALTH CARE PROJECT IN CAMBODIAN RURAL VILLAGESKEIKO SUWA
Primary Health Care Project, 24hTV Charity Committee‑Cambodia(Abstract not received on time)

5 PROMOTION OF MCH THROUGH TBA SUPPORT IN PREYKABAS DISTRICT OF
TAKEO PROVINCESATOSHI TSUKAMOTO, MUTSUMI KATO, RYU MORITA, KAZUKO MIYAMOTO,SATOKO YANAGISAWA AND TOMOYUKI IGARI
Japan Overseas Christian Medical Cooperative Service (JOCS)

Since 1997, JOCS has been implementing PHC projectin Preykabas District of Takeo Province covering aboutpurchase/manage TBA:s materials for delivery support such

14,000 people in 1 8 villages with a focus on MCH throughtion. TBA cooperative committee has been established to

TBA support in collaboration with Kampeng Health Center.

purchase/manage materials by themselves. This is function‑

Problems:

ing well. There are good outcomes through these TBA ac‑

as disposal plastic grove, eye medicine and antiseptic solu‑

There is no referral center for MCH nearby the villages

tivities. TBAS have improved their knowledge and skills. A

in case of emergency. Patients have to pay much f or trans‑

good relationship is being developed among TBAS and be‑

portation and consultation for referral. Malnutrition is com‑tween TBA and Kampeng Health Center midwife.

monly seen among pregnant women and children. Tradi‑tional birth attendants (TBAS) in the community lack a

Future perspective and remaining problems:

good knowledge and skills.

Kampeng Health Center, and that of TBA Material Credit to

JOCS project:

TBA themselves. In the next phase of TBA training, JOCS

14 TBAS are identified as key persons for MCH.
JOCS has been doing some activities with TBA such as
TBA meeting, TBA training or TBA material credit. TBA

expects that TBA would be able to do health check for preg‑

meeting is held every month for providing learning oppor‑
tunities for TBAS by sharing their own activities. ThroughManagement of TBA meeting is being handed over tonant women in cooperation with Kampeng Health Center
midwife. How can JOCS support villagers to take an own‑
ership of activities? Cooperation with community develop‑
ment organizations is a way to improve villager's conditions

TBA training. TBAS are expected to improve necessary

in life and health. Another need is an establishment of re‑knowledge and skills so that TBA may be able to find dan‑ferral system and mutual fmance support system for poor

gerous sign of pregnant women, do health education inpeople to borrow money in case of referral.

early stage of pregnancy. Aim of TBA Maternal Credit is to

6 PRESENT STATUS OF INFECTIOUS DISEASE IN CAMBODIA
KIICHI SATO
Kurobe Onsen Hospital

Author was staying in Phnom Penh from Feb/1998 toattached to Ministry of Health was author's office. The op‑

Mar/1999 as one of JICA staff for investigation of infec‑

eration of NMC was consulted by specialist of WHOltious disease in Cambodia. The National Center for Parasi‑

Phnom Penh. During one year, author had been investi‑

tology Entomology and Malaria Control (NMC), which wasgated, what kinds of infectious disease occupied in Cambo‑

21

dia and how to prevent and medication for patients. Just thenus (95) and so on. The number of HIV(+)/AIDS infected

year of 1998 was occupied by outbreak of Dengue fever andpatients was estimated 120,000 (up to 1997) by WHO.

342 children were died. Results of our investigation was as

However the number of parasite positive patients and filar‑

followed. The whole number of reported infectious patients

iasis were unknown. The prevention of malaria and Dengue
fever was continuing by guidance of NMC, regularly. The
purpose of prevention was "Kill the mosquttoes " whichwere about I ,500,000. Among them, respiratory infection,

including pneumonia and severe influenza was largest pa‑
tients (498,781 : occupied 33.3010). Secondary was unknown

mears delivering mosquito net and spraying of Abate (killcaused diarrhoea (297,610). Next was malaria (about

powder for larva). These works were supported by many

200.000) and bacterial/amoebic dysentery ( 196,350) was

kinds of NGO's group. The prevention of parasite and filar‑

the fourth. These four kinds of infectious diseases was oc‑

iasis did not yet perform, especialy for local people. In

cupied 80% of all infection, excluding the parasite, filariasis

Phnom Penh, the staffs of NMC delivered the helminthagueand AIDS. Remained 20% of the infectious diseases were

for infected children, directly. The problem of Tbc will begynecologic infection (puerperal sepsis, etc. 46,524), sex‑

improve very soon, due to the earnest activities of Dr.

ual transmittened disease (45,845), Dengue fever (16,228),

ISHIKAWA, Dr. ONOZAKI and his expert group, supported
by JICA.

tuberculosis (12,838), cholera, measles, meningitis and teta‑

22

General presentation

A‑1 MOLECULAR ANALYSIS OF ALPHA‑THARASSEMIA IN NEPAL=CORRELATION WITH MALARIA ENDEMICITYSHINJIRO HAMANO , SIGERU KOBAYASHI', YASUYOSHI SAKAI*, HIROSHI SHIBATA*,HIROYASU FURUUMl*, TERUKAZU KAWASAKI+ AND YASUYUKI FUKUMAKI*
Department of Parasitology, Graduate School of Medical Sciencel,

Department of Human Geography, Graduate School of Letters, Osaka University2,
Division of Disease Genes, Institute of Genetic Information3 andInstitute of Health Science, Kyushu University

Thalassemia is a prevalent hereditary disorder charac‑terized by impaired synthesis of globin chains. It has beenhigh gene frequency of 63%, while the gene frequency in
the Tamang was only 5%. Four different haplotypes weresuggested that the high frequency of thalassemia might re‑associated with the type of alpha+‑thalassemia in the Danu‑

flect heterozygote advantage due to reduced susceptibility

wars. Nucleotide sequences of the D‑100p region in the mi‑

to malaria. In Nepal, malaria has often occurred in places

tochondrial DNA of the two populations indicated similar

below the altitude of I ,200 m. We carried out a microepide‑nucleotide diversity in each population. The fixation index,

miological study on thalassemia in two neigboring popula‑

FST, representing the degree of genetic differentiation esti‑

tions in Kabhrepalanchok District in Nepal, the Danuwar
and the Tamang. Settlements of the Danuwar are locatedbelow the limit of the malarial zone in Mahadevstan VDC(1,200 m in altitude), whereas those of the Tamang arefound in malaria‑free uplands in Anaikot VDC. Three het‑mated from mitochondrial DNA diversities (FST, 0.0̲5), wassmaller than that obtained from the gene frequencies ofalpha‑thalassemia (FST, 0.55). If we assume neutral mo‑
lecular evolution in the D‑loop region of mitochondrial

DNA, these results suggest that the high frequency oferozygotes for hemoglobin E (HbE) were observed in the
Danuwars. We detected one type (‑alpha 3.71) of alpha+‑

the malarial environment rather than to events such as a bot‑thalassemia that involves a deletion of 3.7 kb, Ieading to atleneck.

alpha+‑thalassemia may be due to biological adaptation toloss of one of two alpha‑globin genes, in the Danuwars, at aA‑2 MOLECULAR ANALYSIS OF G6PD VARIANTS IN SOUTHEAST ASIA
HIROYUKI MATSUOKA', KUNI IWAI*, SHIGETO YOSHIDA*, AKIRA ISHII',
H. FUJI12, FUMIHIKO KAWAMOTO*, SIRO MIWA4 AND A. HIRON04
Department of Medical Zoology, Jichi Medical Schooll,

Department of Clinical Laboratory Medicine, Tokyo Women's Medical University2,
Department of International Health, Nagoya University School of Medicine3

and Okinawa Memorial Institute for Medical Research

We investigated glucose‑6‑phosphate dehydrogenese(G6PD) variants in Southeast Asian countries (Indonesia,

Malaysia, Laos and Myanmer). Blood samples weremainly collected on malaria survey sites in the malaria en‑

demic area. Some samples were collected in hospitals.
Blood of participants was examined for malaria parasites
and G6PD activity with two rapid tests, which were devel‑

5.6%, which was similar rate with previous studies.

After taking informed consent from people of G6PD
deficiency, we extracted genomic DNA from 0.1 ml of pe‑ripheral blood and amplified DNA coding G6PD by PCR.
Each exon of G6PD was analyzed with single strand con‑formation polymorphism (SSCP) method. Some exons

oped by Kawamoto (1992) for malaria and by Hirono

were read by DNA sequencer. Ten variants were detectedamong sixty‑three G6PD deficient subjects. They were(1998) for G6PD. Rate of G6PD deficiency in male was

G6PD Gaohe, G6PD Vanua Lava, G6PD Mahidol, G6PD

23

Coimbra，G6PD Viangchan，G6PD Chatham，G6PD Union，
G6PD Canton and G6PD Kaiping，respectively．A noble

donesia，who

had

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Surabaya．

A−3 MICROSATELHTEPOLYMORPHISMINTHEHEMEOXYGENASE・1
GENEPROMOTERISASSOCIATEDWITHCEREBRALMALAR、IAINMYANMAR
MASArO私KEDAI2，KEN∫IHIRAYAMA1，RAIAWANUBALEE1，0UMAPORNTASANOR3，MIHOKO KIKUCHI1，YUPAPORNWATTANAGOON4，KESARANA−BANGCHANG3，JUNTRA
DepartmentGARBWANG5AND

ofVeterinary

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FacultyofAlliedcaused

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24sures efficient expression of malaria proteins within murine

Results: The five groups were pooled together for statisticalcells.

analyses because they followed the similar course of thedisease. Unexpectedly, the survival rate of the vaccinatedgroups was lower than that of the control group (P=0.053,

Immunization: Plasmids were divided into five groups each

representing 2,000 independent clones. The plasmids were
propagated in E. coli and purified using the Endotoxin‑freeKaplan‑Meyer). There was no significant difference in

QIAGEN Megaprep kit. As a control, the plasmid vector
without inserts were used. For each group, eight female
mice (BALB/C at fifth week) were injected with purified

parasitemia between the two groups. There was no increase

DNA (50 ug/50 u1 saline) initially directly into the spleen,

vaccination had adverse effects on the survival of infectedfollowed by two subsequent intramuscular injections withmice. Fur bristling, shivering, and convulsions were ob‑served which coincided with the time of death. These ob‑

one week interval.

in antibody titer in both groups.

Discussion: Our results unexpectedly indicate that DNA

Challenge infection: At one week after the last injection,

servations suggested a possibility that vaccination has a re‑

50,000 eryhrocytes infected with P berghei were injectedverse effect on the cellular immunity resulting in the devel‑

intraperitoneally. Mice were observed daily and tail blood

opment of severe malaria in BALB/C mice which do not de‑

was collected on the every second day for monitoring thevelop cerebral malaria under normal conditions.

parasitemia.

A‑5 INVASIVE FORMS OF PROTOZOAN PARASITES HAVE A POSITIVE CHARGE
AT THEIR CONTACT SITE WITH HOST CELLS

MAYUMI AKAKI*, YAMAJI NAKANO*, EIJI NAGAYASU' , KOUICHI NAGAKURASATORU KAWAI* AND MASAMICHI AIKAWA
Research Institute of Science and Technologyl,

Department of Infectious Diseases2, Tokai University School of Medicine andDepatrment of Medical Zoology, Dokkyo University School of Medicine3

It is known that host membrane is, in general, nega‑

tigotes of T cruzi were positively charged at their flagella.

tively charged. To determine whether surface charges of in‑

These results presented that the specific part of the protozoa

vasive forms of protozoa contribute to invasion or not, we

which makes initial contact with the host cell is positively

examined the surface charges of Plasmodium falciparum,
Toxoplasma gondii, Leishmania amazonensis , Trypano‑soma cruzi and Entamoeba histolytica by atomic force mi‑charged. This indicates that the positive charge at the site ofcontact facilitates binding of the invasive protozoa to nega‑tively charged host cells. This leads to conclude that thecroscopy (AFM) and surface portential spectroscopy.

charge difference between the contact sites of protozoa andWe used an AFM (BioScope AFM, Digital Instruments,
Santa Barbara, CA) equipped with an Extender Electronics
Module (Digital Instruments), which can measure surface

host cell initially brings them together before a ligand‑potential. As a sample, indochina‑1 strain of P falciparum ,positive surface charges. The amebae are highly motile andreceptor interaction occurs.

While trophozoites of E. histolytica did not have any

RH strain of T gondii, LV78 strain (MPRO/BR/73/M1845)

can easily change their form without orientation. Theyof L. amazonensis , Tulahuen strain of T cruzi, and HM1:
IMSS strain, clone 6 of E. histolytica were examined. Dur‑ing examination, they were on indium tin oxide coated glass

phagocytose, not penetrate, host cells. A charge difference

slides (Evers, Osaka, Japan).

sites of other protozoa may also be positively charged, with

As a result, merozoites of P falciparum , tachyzoites ofT gondii were positively charged at their apical ends. Pro‑mastigotes and amastigotes of L. amazonensis , and amas‑

may not be necessary for their interaction with host cells.

This finding strongly suggests that the initial contact
the electric charge possibly playing a major role in host cell
invasion.

25

A‑6 BACTERIA EXPRESSING SINGLE‑CHAIN IMMUNOTOXIN INHIBIT MALARIA PARASITEDEVELOPMENT IN MOSQUITOESSHIGETO YOSHIDA*, DAISUKE IOKA*, HIROYUKI MATSUOKA*,HITOSHI ENDO' AND AKIRA ISHII*
Department of Medical Zoologyl and Department of Biochemistry2, Jichi Medical School

Single‑chain immunotoxins are ideal tools to selec‑

ria, recombinant E. coli harboring the single‑chain immu‑

tively kill infectious agents. In applying this technology tonotoxin gene were introduced into the mosquito midgut byblock transmission of malaria parasites in the mosquito vec‑

membrane feeding. The number of infected mosquitoes andtor, we have constructed a single‑chain immunotoxin com‑posed of a single‑chain antibody fragment (scFv) directed

their oocyst densities were significantly reduced when theto Pbs21 on the surface of Plasmodium berghei ookinetes

berghei‑infected mice. These results indicate not only thatmosquitoes were subsequently allowed to feed on P

linked to a lytic peptide, Shiva‑1. The single‑chain immu‑

a single‑chain immunotoxin with enhanced parasiticidal ac‑

notoxin was expressed in Escherichia coli, and the protein

tivity could form a basis for the development of more effec‑

was purified by a Ni‑NTA column. The single‑chain immu‑

tive malaria therapeutic agents, but also that introduction ofnotoxin was initially shown to exhibit greater killing prop‑

genetically engineered bacteria into anopheline mosquitoes

erties for P berghei ookinetes in vitro compared with the

may offer a practical approach to the regulation of malaria

SCFV Or synthetic Shiva‑ I peptide alone. In an attempt totransmission.

block malaria transmission by genetically engineered bacte‑

A 7 EXPRESSION PATTERN OF MITOCHONDRIAL COMPLEX I(SUCCINATE‑UBIQUINONE OXIDOREDUCTASE) IN THE ERYTHROCYTIC STAGE SELLS OF

PIASMODIUM FALCIPARUM
FUMIKA MI‑ICHI, SATORU TAKEO, EIZO TAKASHIMA AND KIYOSHI KITA
Department of Biomedical Chemistry, Graduate School of Medicine,
The University of Tokyo

Since energy metabolisms of Plasmodium is quite dif‑ferent from that of the mammalian host, the enzymes of en‑

NADH‑fumarate reductase system, of parasitic protozoas
and helminths such as Ascaris suum . Thus, complex IIergy transducing pathway of the parasite are promising tar‑changes its function to adapt various environmental condi‑

get of anti‑malarial drugs. Significant advances have been

tions.

made in our understanding of the importance of Plasmo‑
dium mitochondria, although main energy metabolism in
the erythrocytic stage cells of Plasmodium falciparum wasconsidered to be ATP production by the glycolytic pathway.

However, much more information on the Plasmodium mito‑

Generally, complex 11 is composed of four subunits,

and we have cloned the genes for Plasmodium Fp and lp
subunits which form a catalytic portion of complex II. Anti‑sense DNA for Plasmodium lp showed an inhibitory effecton the growth of cultured P falciparum suggesting thatchondria is required to develop new anti‑malarials.

complex 11 is essential for the survival of Plasmodium inComplex 11 (succinate‑ubiquinone reductase/quinol‑
fumarate reductase) is well known marker enzyme of mito‑

the red blood cell.

chondria in the eukaryotic cell. It is one of the members ofin mRNA and protein level in the life cycle was analyzed.

the TCA cycle enzymes as well as electron transport chain,

From Northern blot analysis, expression of Fp and lp of wasa direct link between major systems for energy metabolism
in aerobic organisms. In addition, complex 11 functions as

highest in schizonts and lowest in ring forms. Immuno blot

quinol‑fumarate reductase in the anaerobic respitatory chain,sion pattern. Intense signal of the lp (33 kDa band) was ob‑

In the present study, expression pattern of complex IIanalysis using antibody against lp showed similar expres‑26

served in schizonts and weak one in ring forms and tropho‑

drial DNA. On the other hand, the expression pattern of an‑zoites. Transcripts of Fp and lp were also highly expressed

other mitochondrial protein dihydroorotate dehydrogenase

at sexual stage gametocytes.

(DHODH) was different from that of complex II. The
mRNA for DHODH was highly expressed in trophozoites

This expression pattern is similar to those of two mito‑

chondrial proteins, subunits of cytochrome c oxidase andand hardly detected in schizonts.

cytochrome b encoded on tandem‑repeated 6 kb mitochon‑

A‑8 EFFECT OF JASPLAKINOLIDE ON THE GROWTH AND ACTIN CYTOKELETON OF
PIASMODIUM FALCIPARUM

YASUTAKA MIZUNO*", ASAO MAKIOKA , SIN‑ICHIRO KAWAZU2,SHIGEYUKI KAN02 AND HIROSHI OHTOMOl
Department of Tropical Medicine, Jikei University School of Medicineland Research Institute, International Medical Center of Japan2

The actin filaments in eukaryotic cells are responsible

served on slide smears of jasplakinolide‑treated cultures, nofor cell motility. To elucidate microfilament functions,actin‑modifying agents have been used extensively.
Jasplakinolide, a naturally occurring cyclic peptide

further growth to ring‑forms occurred. This demonstrates

from the marine sponge. Jaspis sp. is a membrane perme‑cavities in the treated merozoites, but not the apical exten‑

able, actin‑polymerizing and filament‑stabilizing drug. The

nized culture in vitro . Jasplakinolide inhibited the growthsions which were reported previously in tachyzoites ofToxoplasma gondii treated with the drug. Sodium dodecyl
sulfate‑polyacrylamide gel electrophoresis (SDS‑PAGE)
and immunoblot analysis revealed that the jasplakinolideof P. falciparum strain FCR‑3 in a time‑ and concentration‑

treatment led to an increase of F‑actin polypeptide. Thus,

dependent manner, where inhibition was observed after 2

our results indicate that jasplakinolide inhibits the growth ofdays of culture with doses greater than 0.3 uM of drug. The

P falciparum by blocking the invasion of merozoites into

inhibitory effect of jasplakinolide on growth was reversed

red blood cells and, therefore, the drug may become a use‑by removal of the drug. Although development of ring‑

ful tool for studies on the relationship between the actin cy‑

forms to schizonts and liberation of merozoites were ob‑

toskeleton and the process of invasion of the red blood cell.

effect of jasplakinolide on the growth and actin cytoskele‑

ton of Plasmodium ,falciparum was examined in synchro‑the inabiliy of treated merozoites to invade red blood cells.

Electron microscopy showed the formation of cytoplasmic

A‑9 A MALARIA PATIENT INFECTED WITH PIASMODIUM FALCIPARUM
AND P MALARIAE

KENJI OHNISHl, SINSUKE MIYAZAKI AND YASUYUKI KATOH
Department of Infectious Disease,

Tokyo Metropolitan Bokutoh General Hospital

Although malaria is not endemic to Japan, the number

on May 17, 2000, because of a 9‑day of fever and 6 days ofof malaria patients has increased in this country. But cases

diarrhea. He reported he had never had malaria. He hadof malariae malaria, especially in patients coinfecetd with

been in Japan for 10 years, and had traveled in India fromfalciparum malaria, are very rare. We treated a patient coin‑

March 30 to April 28, 2000, then returned to Japan. Labo‑fected with malariae malaria and falciparum malaria, as re‑ratory examination revealed thrombocytopenia (platelet
count: 5.5X 104/mm3) and prothrombin time was 42.8%.
Peripheral blood smears revealed Plasmodium malariae

ported below.
A 36‑year‑old Indian man was admitted to our hospital

27

and P falciparum , and the parasitemia of Plasmodium sp.
was I .O%. He was treated with QuinimaxR in an intrave‑

subtropical areas and this may explain why Japanese physi‑

nously drip May 17 and then oral administration ofcians are unfamiliar with the disease. Although mixed in‑

Malariae malaria has a spotty presence in tropical andMephaquin'*' May 1 8. Neither P malariae nor P falcipa‑

fection is rare, it is important to investigate whether otherrum were identified in his peripheral blood smear sincekinds of malaria coinfect in malaria patients.

May 1 9.

A‑lO TWO CASES OF FALCIPARUM MALARIA WITH ATYPICAL CLINICAL COURSE
KOJI YOSHIKAWA*, TETSUYA HORINO', MASAKI YOSHIDA*, KOHYA SHIBA',TATSUO HOSOYA*, MASAHIRO KUMAGAI' AND HIROSHI OHTOMO'
Department of Internal Medicinel and Department of Tropical Medicine',
Jikei University School of Medicine

The first case is a 42‑year‑old Japanese male who had

traveled on business to Myanmar from March 1 2 to March
30 without malaria chemoprophylaxis.
He had a high fever (39.6 C) on April 8 and was admit‑

ted to our hospital. On admission, he was diagnosed with P
falciparum malaria on blood smear (parasitemia 0.7%) andwas treated with artesunate and mefloquine. Parasites had
disappeared on the next day. But he had a delirium on the

May 7 without malaria chemoprophylaxis.
On May 1 8, he rolled downstairs and was admitted toorthopedics at our hospital, then he was diagnosed withcompound fracture of ankle joint. On admission, body tem‑
perature was 35.8iC and he had a disturbance of conscious‑

ness, oliguria, watery diarrhea and dehydration. Urgent
laboratory data showed thrombocytopenia (Plt 25 X 103/u1 ),renal damage (Cr I .3 mg/dl), hyponatremia (Na 123 mmol/1),

3rd hospital day. His blood examination showed AST

and hyocalcemia (Ca 7.3 mg/dl ). On the next day, P falci‑

2,816 IU/1 , ALT 1,067 IU/1 , T‑Bil 3.6 mg/dl , CK 4,777 IU/

parum malaria were detected in his blood smears (para‑sitemia 4.93%). He was diagnosed severe falciparum ma‑

l , myoglobin I , 100 ng/ml . We think that they were cere‑bral malaria, severe hepatocellular damage and rhabdomy‑
olysis and were caused by the malaria infection. His body
tempareture was normalized after the 10th hospital day. He

laria with algid malaria, cerebral malaria and acute renal

was discharged on the 36th hospital day after a fully recov‑

Parasites had disappeared on the 4th hospital day. He was

failure. He was treated with intravenous quinine infusion,oral artesunate, fluid therapy and hemodialysis (2 times).

ery of all abnormal laboratory data.

discharged on September 4 after a recovery of compound

The second case is a 52‑year‑old Japanese male who
had traveled on business to Zimbabwe from April 27 to

fracture and abnormal laboratory data.

28

A‑11 A CASE OF SEVERE FALCIPARUM MALARIA WITH PROLONGED
IMMUNOHEMOLYTIC ANEMIAYOSHIAKI TANAKA*, AKIFUMI IMAMURA , ATSUSHI AJISAWA* ,MASAYOSHI NEGISHI , GOHTA MASUDA* AND KUMIKO ABE'
Department of Infectious Diseasesl and Department of Laboratory Medicine2,

Tokyo Metropolitan Komagome Hospital(Abstract not received on time)

A‑12 CHRONIC AUT0‑IMMUNE THROMBOCYTOPENIA FOLLOWING RECOVERYFROM SEVERE FALCIPARUM MALARIAYASUYUKI KATO AND GOHTA MASUDA
Department of Infectious Diseases, Tokyo Metropolitan Komagome Hospital

A 49‑year‑old Japanese man made a two‑week visit to

Two weeks later, the platelet count fell to 56,000/mm3.

Gambia. He took no malaria prophylaxis. When he re‑hospital. The temperature was 38.0t, the pulse was 90,

The peripheral‑blood smear showed only thrombocytopenia
and the bone marrow examination was normal. Blood co‑agulation test revealed no abnormalitity for DIC. Ultra‑

and the respirations were 30. The blood pressure was 108/

sonography revealed no splenomegaly. The platelet‑

72 mmHg. On the examination, he was lethargic and jaun‑
diced. His gums were bleeding and liver was palpable at
2 cm below the right costal margins. Thin and thick blood

cells (normal 0.9‑2.5 ng/10' cells). He did not have detect‑films showed Plasmodium falciparum trophozoites; 3% of
the red cells were parasitized. The platelet count was
10,000/mm3 and fibrinogen level 123 mg/dl . The fibrin‑ies to the human immunodeficiency virus was negative.
Antinuclear antibodies and antiphospholipid antibodies
were negative. The mild thrombocytopenia persisted anddegradation products were 40 ug/ml . The diagnosis of se‑

the PAlgG Ievel remained high at 1 8 months after discharge.

turned to Japan, he became febrile and was admitted to ourassociated lgG (PAlgG) Ievel was elevated to 1 1 .96 ng/106
able antiplatelet antibodies in his serum. A test for antibod‑

vere falciparum malaria complicated with disseminated in‑This case was considered a chronic auto‑immune

travascular coagulation (DIC) was made. Quinine (600 mg
every 12 hrs) and minocycline (lOO mg every 12 hours)
were given intravenously followed by orally. Transfusions

thrombocytopenia following recovery of severe falciparum

malaria. Various virus infections including rubella,varicella and infectious mononucleosis, are known to in‑of platelet concentrations and fresh frozen plasma were ad‑duce acute auto‑immune thrombocytopenia. About 10% ofministered to prevent bleeding. On the third day of treat‑these cases develop chronic auto‑immune thrombocy‑

ment, he got afebrile and P. falciparum was not seen in the

topenia. The mechanism of thrombocytopenia in malaria

blood smear. He was discharged on day 28 with a platelet
count of I 04,000/mm*.

remains unknown. But this case suggested that malaria in‑duced auto‑immune thrombocytopenia.

29

A ‑ 13A CASE OF CLINICALLY CHLOROQUINE RESISTANT VIVAX MALARIA INFECTIONDAISUKE KASAl , GOHTA MASUDA*, AKIFUMI IMAMURA*, ATSUSHI AJISAWA ,MASAYOSHI NEGISHI', SHIGEYUKI KANO' AND HIROSHI OHTOMO*
Department of Infectious Diseases, Tokyo Metropolitan Komagome Hospitall ,International Medical Center, Japan2 andDepartment of Tropical Medicine, Jikei University School of Medicine3

A 49‑year‑old Japanese man who had traveled lriyan‑

red blood cell did not decrease. We concluded as chloro‑

jaya, Indnesia since 2 Mar. 2000 till 20 Mar. 2000, hospital‑

quine resistant vivax malaria infection and prescribed Fansi‑

ized with complaint of fever and general malaise. We diag‑dar, so clinical symptom got better. At lriyanjaya, chloro‑

nosed him as vivax malaria infection by smear of peripheral

quine resistant vivax malaria infection is widespreded andblood. Although we prescribed him chloroquine 3 days,

this is the case with it.

high fever still continued and the rate of malaria infected

A ‑ 14A CASE OF FALCIPARUM MALARIA SUCCESSFULLY TREATED WITH
INTRAVENOUS ARTESUNATE

CHIE YASUOKA1'3, AKIRA YASUOKA , YOSHIHIKO YAMAMOTO' , IKUMI GENKA*,TOSHIMITSU HATABU', SHIGERU KOHNO*, SHIN‑ICHI OKAI AND SHIGEYUKI KAN02
AIDS Clinical Centerl , Department of Appropriate Technology,
Development and Tranfer Research Institute2,
International Medical Center of Japan and2nd Department of Internal Medicine, Nagasaki University School of Medicine3

A Nigerian man, who was presumed to be infected in
Nigeria, developed malaria during his visit in Japan. The

in three doses (120 mg given followed by 60 mg 24 hr and48 hr later; total dose was 240 mg). Consequently the para‑patient showed fever with chills, arthralgia and diarrhea.

sites disappeared in 20 hr from his blood but fever still con‑

The blood test revealed thrombocytopenia and anemia.
Ring forms of 0.03% of his RBCS and ICTTMMalariaPf/Pv

tinued in low grade possibly because of cholecystitis. At

test was also positive for Plasmodium falciparum. We pre‑scribed mefloquine to him, but the number of the parasites

the same time of Artesunate treatment, hemoglobinuria
started and anemia worsened partly because of his G6PD
deficiency. All pending problems were improved by thein his peripheral blood did not decrease, and, in fact, theytime he left Japan and those parasited were finally found tocame to increase (maximum 6.66%) 20 hr after the drug

be susceptible for mefloquine by the in vitro susceptibility

treatment. As clinical condition of malaria were liable totest (IC*0<'=100 nmol/ml, IC50=7.86 nmol/ml). This is thechange seriously, intravenous Artesunate (a qinghaosu de‑first reported case of falciparum malaria successfully treatedrivative) was decided to be given additionally to the patient.

with intravenous Artesunate in Japan.

Artesunate was prescribed to him by intravenous injection

30

A ‑ 15A CASE RECOVERING COMPLETELY FROM SEVERE FALCIPURUM MALARIACOMPLICATED WITH HIGH LEVELS OF MULTIPLE ORGAN FAILURE

M. AIZAWA', YASUTOSHI SOMEYA*, MIKITA TANIGUCHl', YASUHIRO ARAKAWA',GEN HASEGAWA*, YASUSHI OHTSUKA*. AKIHIKO HAMAGUCHI', HIDEYUKI KOBAYASHI',KAZUTOSHI TAKIKAWA', TETSUO KIMURA , JUN‑ICHI YOKOYAMA' , TAKEYUKI KITAJIMA*,MASAHIRO KUMAGAI' AND HIROSHI OHTOMO'
Department of Internal Medicine, Daisan Hospitall andDepartment of Tropical Medicine2, Jikei University School of Medicine

A 56‑year‑old male, who had returned from Ginbabue
in Africa, was admitted with consciousness disturbancefell into shock state on the 4th day of hospitalization. He(JCS, 100) after three days history of high fever. Soon after

recovered from shock by using electric defibrillator andfrom acute respiratory distress syndrome by methylpredni‑

his travel history was revealed, he was diagnosed as Plas‑

solone pulse therapy. On the 9th day blood preparation

modium falciparum malaria by the preparation of peripheral

showed no parasitemia and multiple organ failure gradually

blood smear with the parasitemia of 49%. Despite intrave‑

improved. On the 26th day his consiousness was clear, andnous gluconate quinine infusion and artesnate (administra‑tion), his consciousness state, respiratory, renal and hepaticon the 1 17th day he was discharged without any impair‑ments of life quality. We would like to emphasize the im‑function, deteriorated progressively. Although he wastreated with respirator, plasma exchange, hemodialysis,

portance of the loading‑dose IV quinine in the treatment ofcontinuous hemodiafiltration in the intensive care unit, hesevere falciparum malaria.

portance of supportive and general care as well as the im‑

A‑16 A CASE OF SEVERE FALCIPARUM MALARIA WITH HYPER‑BILIRUBINEMIAAND NEPHROPATHY
KIYOE HASHIZUME , TATSURO OSADA*, MICHIHIRO SUZUKI*, MASAHIRO MURAYAMA',JINGORO SHIMADA*, KENJI TAKA14, TAKESHI SAKANE+, MITSUO KAKU5,

MASAHIRO KUMAGAI* AND HIROSHI OHTOM06
Department of Gastroenterology and Hepatologyl, Department of Circulation2,

Department Microbiology3, Department of Immunology and Medicine4,
St. Marianna University School of Medicine,
Division of Molecular Diagnosis, Department of Clinical Medicine,

Tohoku University School of Medicine5 andDepartment of Tropical Medicine, Jikei University School of Medicine6

The patient was a 46‑year‑old male who returned frommalaria. Mefloquine was administrated on the same day

a 10‑day trip to central Africa on January 4, 1 998. The pa‑
tient felt chill and developed fever of 38.6
on the follow‑

rate in red blood cells has increased to 13.7% on the follow‑(the eighth day after the onset), but the protozoan infection

our hospital and admitted on January 9. On peripheraling day. Therefore, quinine hydrochloride was adminis‑
trated, and parasitemia rapidly improved. However, on

blood test, white blood cell and platelet were low: 2,300/ul

January 17, T. Bil and direct bilirubin (D. Bil) were 58.5

and 22,000/u1 , respectively. Blood chemistry revealed in‑

mg/dl and 38.1 mg/dl , respectiely, indicating that jaundicecreases in total bilirubin (T. Bil) (2.7 mg/dl ) and LDH

with predominating D.Bil worsened, and creatinine (Cr)ing day, and visited a physician. The patient was referred to( I ,240 IU/1 ). Splenomegaly was observed on abdominal ul‑

was 6.5 mg/dl , indicating aggravation of nephropathy. In‑

trasonography. On January 12, protozoan were demon‑

trahepatic biliary stasis was suspected for jaundice, andstrated in red blood cells in blood smears (infection rate:

steroid pulse therapy and plasma pheresis were performed.

1 .4%), and the patient was diagnosed as having falciparum

For acute renal failure, hemofiltration was performed. Sub‑

31

performed on February 1 7 revealed bilirubin pigmentation

and Kupffer cells phagocytosing malarial pigments in thesinusoid. We encountered a patient with severe falciparumin hepatocytes, bile thrombus in capillary in blood vessels,malaria accompanied by severe jaundice and nephropathy.

sequently, T. Bil and Cr gradually decreased. Liver biopsy

A ‑ 17

THE USE OF THE ATOVAQUONE/PROGUANIL COMBINATION (MALARONE*)AKIHIRO HITANI '2, MIKIO KIMURA', HARUMI YAMADA*,AIKICHI IWAMOTO

AND HIROSHI OHTOMO

Department of Infectious Disease & Applied Immunology,
Institute of Medical Science, The University of Tokyol,

Narita Airport Quarantine Stationf, Department of Pharmacy, Tokyo University3and Department of Tropical Medicine, Jikei University School of Medicine4

Malarone' ' is a fixed‑dose combination of 250 mg oftients were treated with Malarone' ' ' at the recommended

atovaquone and 100 mg of proguanil hydrochloride. The

dose. The patients with vivax malaria took primaquine at acontinuing spread of drug‑resistant malaria emphasizes thestandard dose after treatment with Malarone( 'need for new antimalarial drugs. Atovaquone is a new

Efficacy: Parasitemia cleared in six patients within sevenbroad‑spectrum antiprotozoal drug with a novel mechanism

tron transport. The combination of atovaquone anddays and no recrudescence developed. Mean PCT (parasite
clearance time) was 72 hr. The cure rate was 100%. One
patient with vivax malaria came back to Madagascar after

proguanil is synergistic in vitro, and clinical studies demon‑

treatment and the long‑term follow‑up was not possible. So

strated enhanced efficacy of the combination compared toeither drug alone for treatment of malaria. Malarone( ' isfar other 2 patients with vivax malaria have had no recur‑

available in many countries for treatment of acute, uncom‑

Adverse events: One patient had a non‑serious adverseof action, i.e., via inhibition of parasite mitochondrial elec‑

plicated f'dlciparum malaria. It is reported that at the recom‑

mended dose (in adults, four tablets once daily for three

rence.
event (urticaria) that necessitated switching of Malarone'*" tomefloquine. One patient had a non‑serious, mild itching.

days), the overall cure rate was >98%. Adverse events (ab‑One case showed slightly elevated levels of ALT and AST

dominal pain, anorexia, nausea, vomiting, diarrhea andcoughing) occurred with similar frequency as in patientsthat continued after the clearance of parasitemia.

treated with other drugs, such as mefloquine, amodiaquine,

tive for treatment of malaria caused by Plasmodium falcipa‑

and chloroquine.

rum , and limited data indicate that this combination is also

We reviewed our experiences of Malarone'*' between

It has been reported that MalaroneR is safe and effec‑

July 1999 and August 2000. This study enrolled 4 patientsactive against the blood stage of P vivax. P ovale, and P
malariae . Therefore, Malarone"" is an important new alter‑with acute uncomplicated falciparum malaria and 3 patients

native for treatment of malaria. However, only limited datawith vivax malaria, 4 of whom were native Africans. All

are available with Japanese patients and further evaluation

patients did not take any antimalarial drugs as prophylaxis.

should be needed.

Initial parasitemia ranged from 100 to 25,000/u1 . All pa‑

32

A‑18 ANALYSIS OF SEVERE AND COMPLICATED FALCIPARUM MALARIA IN
JAPANESE PATIENTS CAUSED BY DELAY OF DIAGNOSIS AND TREATMENTHIROSHI OHTOMO MIKIO KIMURA', MASAHIRO KUMAGAI* AND MASAHI HAYANO'
Department of Tropical Medicine, Jikei University School of Medicinel andDepartment of Infectious Disease & Applied Immunology,
Institute of Medical Science, The University of Toky02

As the incidence of imported malaria increased in Ja‑

l 8% began on the third day or the forth day from the f"rrstpan, fatal of severe Japanese falciparum malaria cases werevisits to doctors. When the treatment had began on the dayreported every year. Analyzing case‑reports, which werecollected by the Research Group for Development of Che‑
motherapeutic Agents against Tropical Parasitic Diseases

or the next day, the rate of severe cases was 10% (5 in 48
cases). On the third day or the forth day, the rate was 39010(5 in 27 cases). On the fifth day or later, the rate reached to(supported by the Japanese Ministry of Health and Welfare)

550/0 (6 in I I cases). The delays of the treatment usuallyand the Research Group for Clinical Evaluation of Orphan

depended on the misdiagnosis. The later the treatment be‑

Drugs against Imported Tropical and Parasitic Diseases(supported by the Human Science Promotion Foundation),gan from the first visits to doctors, the more the rate of se‑vere cases increased. Concerning the days between the on‑

the relationship between the severity of disease and delays

set of symptoms and the beginning of the treatment, theof the treatment were examined. The criteria of severe andsame tendency was shown. When the treatment began oncomplicated malaria in this study were cerebral malaria, se‑the third day or earlier from the onset of symptoms, the ratevere anemia (Hb<5 g/dl ), pulmonary edema, acute respira‑tory distress syndrome, and renal failure (Cre>3.0 mg/dl).

the fifth day, or the sixth day from the onset, the rate wasof severe cases was 4% ( I in 25 cases). On the forth day,Seventy‑four Japanese falciparum malaria case‑reports were19% (5 in 27 cases). On the seventh day or later, the rate

collected from 1991 to 1999. Seventeen cases were severe

reached to 50% (1 1 in 22 cases). In summary, delays of theand complicated, including five fatal cases, and 57 cases

treatment cause severe and complicated malaria. Therefore,
we should diagnose malaria promptly and treat it as soon as

were uncomplicated. Forty‑two percents of patients began
to get anti‑malarial treatment on the day when they visited
to doctors. Twenty‑five percents began on the next day, andpossible.

A‑19 ON THE WHO'S GUIDELINES FOR SEVERE MALARIA, 2000
MIKIO KIMURA', TATSUYA FUJII' AND HIROSHI OHTOMO*
Department of Infectious Disease & Applied Immunology,
Institute of Medical Science, The University of Tokyol,

Self Defence Forces Central Hospital2 andDepartment of Tropical Medicine, Jikei University School of Medicine3

As the clinical experiences of severe malaria are lim‑

loading‑dose IV quinine is stressed because otherwise, theited in our country, guidelines proposed by authoritative

optimal blood concentration could not be attained even un‑

bodies are highly needed. The WHO's guidelines for severe

der hepatic or renal disturbances. The use of artemisininmalaria are one of those and have just been revised 10 years

derivatives especially that of IM artemether is recom‑

after the last edition appeared. We investigated their con‑mended although showing a concern that it could retard thetents caring about the differences compared to the old one.

recovery from comatose state. The oral artesunate may be

Overall, there are no fundamental changes in the new edi‑

useful for patients with high parasitemia that can otherwisetion; however, some treatments are recommended more en‑thusiastically than were before. The importance of thebral malaria, steroids, heparin and aspirin in DIC‑1ike hem‑

develop to severe malaria. Steroids and mannitol for cere‑33

orrhagic complication are not recommended or regarded

sitemia or even in >10% if there are complications such aseven harmful as in the old guidelines. The acute renal in‑

cerebral malaria, acute renal insufficiency and pulmonary

sufficiency is divided into two types, one occurring at theedema/ARDS, although comparative studies which show its

acute stages and the other during convalescence with poorer

efficacy are not available. The treatment of metabolic aci‑

and favorable outcomes, respectively. It is stressed that thedosis with sodium bicarbonate does not seem to be the mostcomplication is often caused by dehydration, necessitating

ideal due to the possible deterioration of tissue acidosis in

fluid infusion before the patients are injected with diuretics

the brain and the induction of sodium overload that can pre‑or dopamines. The indications for hemodialysis include hy‑

cipitate pulmonary complications. Alternatives include
THAM and DCA; however data of their efficacy and safety

perkalemia, metabolic acidosis, fluid overload and the se‑rum creatinine rising rapidly by 2.5‑3 mg/dl/day. As for the

are limited. It is thus revealed that many still remain to betreatment of pulmonary edema/ARDS, the importance of
maintaining CVP between 0‑5 cm H20 is again stressed.

known regarding the appropriate treatment of severe ma‑
laria.

Exchange transfusion should be considered in >30% para‑A ‑ 20

PROBLEMS OF LOCAL TRANSPORT SYSTEM FOR ANTI‑MALARIA DRUGSKENJI ISHIWATA, FUKUMI NAKAMURA‑UCHIYAMA AND YUKIFUMI NAWA
Department of Parasitology, Miyazaki Medical College

Malaria is the most important imported tropical diseasein Japan nowadays. About 100 new cases have been re‑rim and Mephaquin for follow‑up treatment were deliveredby high‑way bus cargo. In both ways, the drugs were deliv‑

corded every year. Nevertheless, some critically importantered within 4 hr. The patient was successfully cured bydrugs to treat malaria or other tropical diseases are nottreatment with these drugs.

available through ordinary drug distribution system in Japan.

Case 2: 30‑year‑old female. She visited Solomon Island on

At the moment, under authorizaton of the Ministry of
Health and Welfare such drugs are provided through thetablets. She felt malaise and developed fever since May 17.

April 29‑May 6, 2000. She did not take any prophylactic

study group, "Orphan drugs for the imported tropical dis‑

She was diagnosed as having falciparum malaria and waseases and parasitic disesases", supported by the Human Sci‑

admitted to the Dept. of Internal Medicine, Kagoshima Uni‑

ence Foundation, Japan. As a member of this study group,
we provided anti‑maralia drugs to two hospitals in Kyushu
to treat severe falciparum malaria patients in Spring 2000.

versity Hospital. She was treated with Plasmotrim followed
by Mephaquin which were delivered by high‑way bus cargo.
The patient was also successfully cured by treatment with

Here we describe our experience of the drug delivery.

these drugs.

Case I : 30‑year‑old female. She and her father visited
Madagascar Island on March 10‑24, 2000. Although theyweek after the onset of the disease. Blood smear examina‑

In the present study, the father of Case I died within a

had taken prophylactic tablets for 3 days after returned totions with suspicion of malaria must be done as early asJapan, both of them developed fever on April 2‑3. Her fa‑

possible when physicians saw patients developed fever

ther's course was rush and, inspite of being diagnosed as

shortly after their return from the endemic areas.

having P falciparum infection on April 6, he died of cere‑

In Kyushu, the orphan drugs for tropical diseases in‑bral malaria with pulmonary edema and renal failure. She

cluding anti‑malaria drugs are deposited in three places; I )also developed signs and symtoms of severe malaria andDept. of Parasitology, Miyazaki Medical College 2) Clinical

was transferred to the ICU, Kumamoto University Hospital.

Division, Inst. Trop. Med., Nagasaki University, and 3) In‑

Because this is our first emergency case to provide anti‑
malaria drugs, we tried to find out the quickest way to de‑fectious Diseases Center, Fukuoka Children's Hospital. Af‑liver drugs. We found that although we could charter a heli‑

copter or an ambulance though the local government, it
would take more than 4 hrs to arrange under permission.
Eventually Qunimax for two i.v. injections were deliveredby our official school car with the driver, and the Plasmot‑

ter our experience of two cases, we found that basicallydrugs can be delivered to any part of Kyushu from one ofthese institutions within 4 hrs via high‑way bus cargo. In
emergency cases, official cars of the institutions can be util‑
ized to shorten the time to deliver drugs.

34

A‑21 DETERMlNATION OF PLASMA LEVELS OF PROSTAGLANDINS AND THROMBOXANEIN FALCIPARUM MALARIA PATIENTS IN THAILAND WITH GC/MS/SIM
YAMAJI NAKANO*, TORU OBATA', EIJI NAGAYASU , SORNCHAI LOOAREESUWAN3,HIROSHI OHTOM0+ AND MASAMICHI AIKAWA*
The Research Institute of Science & Technology, Tokai Universityl,

Institute of DNA Medicine2, Department of Tropical Medicine4,
Jikei University School of Medicine andFaculty of Tropical Medicine, Mahidor University3

We monitored the plasma levels of prostaglandins(PGs) and thromboxane B2 (TXB2) in falciparum malaria
patients in Thailand. We collected samples from 36 normal
adults (NA) and 5 1 falciparum malaria patients before treat‑ment (MP) and 10 falciparum malaria patients who had re‑covered with treatment. The plasma levels of prostanoids

A ‑ 22were measured by using simultaneous analysis of gas
chromatography‑mass‑spectrometry‑selected ion monitor‑
ing method (GC/MS/SIM). TXB2 decreased significantly(p<0.001) and PGF20C, 8‑epi PGF2CC, PGEI and PGE2 weresignificantly increased in MP compared with those in NA,
respectively.

DETECTION OF PLASMODIUM VIVAX FROM CLINICAL SPECIMENS BY THE
USE OF ICT MAIARIA P. F/P V KITKUMIKO ABE , GOHTA MASUDA', TAKUYA MAEDA3 AND SHIGEYUKI KAN0+
Department of Clinical Laboratoryl, Department of Infectious Diseases2,

Tokyo Metropolitan Komagome Hospital,
Department of Tropical Medicine and Parasitology,
School of Medicine, Keio University3 andResearch Institute, International Medical Center of Japan4

The "gold standard" for both the detection and identifi‑

detecting the malaria specific antigen. Our results in using

cation of the malaria pathogen, Plasmodium sp. is a micro‑

this kits are as follows; (1) in the P vivax positive patients'

scopic examination of Giemsa stained blood specimens, thiswhole blood, 12 out of 19 (63.2%) were positive for P vi‑

parasitic infection is sometimes difficult to diagnose be‑cause the physicians and medical technologists in our coun‑vax. (2) when specimens with very low parasitemia (in‑fected RBC by P vivax < 0.01%) were excluded from thetry are recently not very familiar with these organisms. This

total, the positive results for P. vivax was as high as 80.0%

time, we examined the ICT Malaria P f/Pv kits for identifi‑(12 out of 15). (3) we had difficulties in distinguish it's

cation of Plasmodium vivax in vivax‑malaria patients'

positive band when specimens with low parasitemia. In

whole blood specimens because we previously reported thissummary, this kit is considered to be useful as a supportive

kit has only low positive ratio for diagnosis of vivax‑

tool for the detection and identification P vivax from wholemalaria.

blood specimens, but we must pay attention to its falseposi‑

The ICT malaria P f/P v kit is a kit which has been de‑veloped to identify P falciparum and P vivax by means oftive result in clinical laboratory.

35

A ‑ 23

AN EVALUATION OF ICT MALARIA PF/P V IN IMPORTED MALARIA IN JAPANHIROSHI OHTOMO*, MIKIO KIMURA', MASAHIRO KUMAGAI*,

MASASHI HAYANO AND YASUHARU MATSUMOTO*
Department of Tropical Medicine, Jikei University School of Medicinel

Department of Infectious Disease & Applied Immunology,
Institute of Medical Science, The University of Toky02 andNarita Airport Quarantine Station3

ICT Malaria Pf/Pv (ICT) is a rapid immunochroma‑and 76 cases free from malaria parasites. ICT was sensitive

tographic test for the detection of circulating antibodies of(100%, 35/35) similar to PF test. Both lines were + for 27

Plasmodium falciparum (PD and P vivax (Pv) in whole

Pf cases with relatively high parasitemia, while only linel

blood. Two separate lines have been immobilized across atest strip. Linel is specific for the histidine‑rich protein 2

was + for 8. ICT was sensitive (87%, 26/30) also for Pv.
Both lines were + for the mixed infection, which may re‑antigen (HRP 2) of Pf and line 2 is specific for a common

flect the antigens of 2 species.

antigen to both Pf and Pv. A test diagnosis of Pf and Pv ma‑

One Po case was ICT negative ( ‑ ) and the other I was

laria were respectively made if line I was positive (+) withline 2 +. The common antigen may be expressed also by Po.

or without line 2 and only line 2 was +. We evaluated the

One Pm case was ‑ . Three of 76 cases microscopically freetest for the diagnosis in the imported malaria in Japan, com‑from the parasites were both ICT Iine I and PF test +. They

paring with microscopic detection and ParaSight F (PFhad been treated for Pf malaria or treated by herself withtest). A total of 145 people enrolled in the study were 122

antimalarial for fever, and thus remaining HRP 2 after curepeople returned from malarial country who attended orwould be detected. Except them, ICT was specific for bothwere consulted Jikei University or the University of TokyoPf (lOO%, 106/106) and Pv (99%, I l0/1 1 1, false + in I Po

for about a year from August 1998 to August 1999, and 35
malaria cases (freezed blood was used) who were in hospi‑

ther had history of having antimalarial nor became malariacase). There were 5 cases with PF test + but ICT ‑ who nei‑tal of Jikei University previously. Microscopically, they

afterwards. These PF test + must be false, and ICT probably

were 35 Pf malaria, I mixed infection of Pf and Pv, 30 Pv

has fewer false + than PF test. ICT is thus useful for the di‑malaria, 2 P ovale (Po) malaria, I P malarie (Pm) malaria

agnosis of imported malaria.

A ‑ 24

A COMMUNITY‑BASED MALARIA CONTROL PROGRAM IN PALAWAN,
THE PHILLIPPINESSHTGEYUKI KANO , SHIN ICHIRO KAWAZU PILARITA TONGOL RIVERAELENA ANDINO VILLACORTE', EDITHA CANETE‑MIGUEL*,JOSE ANTONIO SOCRATES+ AND MAMORU SUZUKI'
Research Institute of International Medical Center of Japanl ,Department of Parasitology, College of Public Health,
University of Philippines2,

Alayka Palawan3 and Provincial Health Office,Puerto Princesa, Parawan4, Philippines,

and Department of Parasitology, Gunma University School of Medicine5

Malaria has been considered to be one of the major

In 1 992, we conducted an epidemiologic study of ma‑

health threats to every Palaweno for a long time. For years,

laria in Barangay (village) Mangingisda, Puerto Princesa

different methods and strategies have been used in the con‑City. A unique feature of the study was the active participa‑

trol of malaria in Palawan Island, the Philippines, but theretion of the Barangay Health Committee members in the
promotion and implementation of Community‑Based Ma‑

were problems in sustaining these efforts.

36

lana Control (CBMC) In 1993, a community‑based healthprogram, "Alayka Palawan", became a testimony of this is‑land's commitment to community empowerment.
In 1 996, the Provincial Health Office successfully ob‑

the Governor was launched. The activities included freeblood smears, Iectures, poster exhibits, and displays of
insecticide‑impregnated mosquito nets and larvivorous fishfor biological control.

tained support from Japan International Cooperation

Palawan is the island most severely affected by malaria

Agency for its implementation. Since then, many barangays

in the Philippines. Thus, the CBMC activities so far consti‑

have been mobilized for the control of malaria. The strategy

tute the inception phase for the future malaria eradication in

aimed to increase the awareness of the people and partner

the island. Now the world malaria control is integrated un‑

agencies of their potential role in health care. The Barangay

der the WHO's initiative, "Roll Back Malaria (RBM)".
Soon the implementation phase in Palawan will commence
under the RBM banner in the West Pacific Region. Dedi‑

Health Committee and barangay health workers played an
important role in its activity. They were responsible in pro‑viding appropriate information on the control and preven‑cated effort has to be made through the partnership of peo‑tion of malaria through organized community action.

ple in Palawan Island, WHO and other agencies until they

At last, August 10, 1999 was declared Malaria‑Free
Day and during which the malaria control campaign under

will enjoy the true freedom f'rom malaria.

A‑25 CHANGES OF THE SPLEEN RATE ON ANEITYUM ISLANDAKIRA KANEKO*,3, GEORGE TALEO', TAKATOSHI KOBAYAKAWA* AND

ANDERS BJORKMAN*
Department of International Affairs & Tropical Medicine,Tokyo Women's Medical Universityl,
Malaria Section, Department of Health, Vanuatsu2 andUnit of Infectious Diseases, Karolinska Institute3

Vanuatu consists of 80 inhabited islands in Melanesia,

with hypo‑ to mesoendemic malaria and suitable conditions(Malakula) and one without (Futuna) malaria transmission
have been monitored for comparison. The spleen surveys

for sustained parasite elimination. Weekly mass drug ad‑

were conducted for the children aged two to 1 2 years old.

ministration (MDA) of chloroquine, pyrimethamine/sul‑

AK assessed spleen size in the recumbent or standing posi‑fadoxine and primaquine was conducted on the entire popu‑

tion according to the Hackett's method with calculation oflation of 7 1 8 inhabitants of Aneityum Island for nine weeks

spleen rate (SR) and average enlarged spleen (AES). Thir‑in 1 991 prior to onset of the rainy season. Simultaneously

teen surveys were conducted in Aneityum from January

permethrin‑impregnated bednets were distributed to the en‑l 99 1 to June 1999: two before, two during and nine aftertire population and have been re‑impregnated yearly.

the intervention. Seven surveys were conducted in Malakula

Larvivorous fish were also introduced into several identified

from February 1988 to March 1998, and two surveys werebreeding sites of Anopheles farauti. Periodic blood surveys

conducted in Futuna (February 1992 and July 1997).

for the past nine years have showed complete absence ofPlasmodium falciparum after the MDA and P. vivax from1996 onwards, with the exception of two instances of im‑ported infections (one mixed infection in 1993 and one P
vivax infection in 1999), suggesting that malaria can be

The SRS in Aneityum (48%) and Malakula (36%) with
AES of around I .6 indicated a meso‑endemic situation on

eliminated if the intervention is conducted over the wholearea of transmission and the risk of importation of malaria

both islands before the intervention. In Futuna, the SR wasaround 5% with AES of I .OO, although no parasitaemia was
detected in the two surveys. Since 1996 the SRS in Ane‑ityum (about 4% with AES of I .OO) have been comparable

is controlled within a framework of community consent andto those in Futuna without malaria transmission. In
Malakula, the SRS Oscillated between 30% and 50%. The

participation (Kaneko et al., Lancet, 2000, 356, 1 560‑1564).

observed changes of SRS well correlated with those of para‑

Throughout this period we have also continued tosite rates. Spleen survey is a simple and useful method tomonitor the changes of splenomegaly in children on Ane‑ityum Island. Two additional islands of Vanuatu, one withestimate the geographical differences and the chronologicalchanges of malaria endemicity in given localities with or37
without of control activities. It is necessary to train local

nation.

health workers to standardise the method of spleen exami‑

A‑26 ACTIVE CASE DETECTION SURVEYS ON MALARIA IN RECENT LAOS,WITH SPECIAL REFERENCE TO THE COMPARATIVE EFFICACY OF GIEMSA STAINING
AND DIPSTICK METHOD ON DETECTION OF MALARIA INFECTION
JUN KOBAYASHI*, YUMI IMADA'. HIROSHI TOMA*, TAKESHI ARAKAWA*,KUNI CHINEN*, FABIAN M. MASHAURI', SAKON LAYAMANIVONG3,

BOUAKHAM VANNACHONE', SAMILANE PHOMEPIDA3, TAKAKO TOMA',ICHIRO MIYAGI' AND YOSHIYA SATO*
Department of Parasitology, School of Medicine*,

Department of Medical Zoology, School of Health Science',
University of the Ryukyus andCenter of Malariology, Parasitology and Entomology (CMPE), MOH, Lao PDR3

Lao PDR has been known to be a seriously endemic

The malaria infection was detected in all villages surveyedcountry for malaria in Southeast Asia. The exact prevalence

and the positive rates by Giemsa staining in 1 8 villagesof malaria in Laos, however, is not well known because theranged from 8.0 to 58.29(:o (mean=23.8%). P falciparum

surveillance system to obtain actual prevalence of malaria

malaria occupied 87.8% of malaria species detected and theamong the inhabitants has insufficiently functioned. An ex‑

positive rates of P.f. were 7.5‑57.7% (mean=21 .3%). On thetensive surveys to obtain exact information on malaria
prevalence, therefore, has been desired for future malaria

other hand, the mean positive rates of P.f. by Dipstickmethod in the 20 villages were 22.3%, ranging from 8.5%

control not only in Laos but also in the neighboring coun‑

to 64.5%. The positive rates were generally higher by Dip‑

tries. Since 1 995, the authors have conducted active casestick method than Giemsa staining, but the difference was

detection surveys on malaria among the villagers in a south‑

consistently small. Among the samples, 136 (3.8%) wereern province, Khammouane Province, and the results ob‑

false‑negative in Dipstick method, in which positive results

tained in 1999 were reported in the presentation.

were obtained only in Giemsa staining. On the other hand,
5.4% of the samples gave positive results only in Dipstick

The surveys were conducted in 20 villages where theestimated total population was 5,857. A total of 3,597, ac‑counting for 61.4% of total population, were examined formethod. When the samples positive only by Dipstickmethod were further examined by the PCR method, themalaria infection by microscopical examination using

number of samples in which malaria infection could not be

Giemsa‑stained blood smears and by Dipstick method using

confirmed even by the PCR method were extremely few in

ICT malaria kit (AMRAD Australia) to detect P ,falciparum

the present study. These results indicate that malaria infec‑

antigen in peripheral blood. A part of blood samples col‑tion is seriously prevalent among the inhabitants in southern

lected on filter papers were also examined by the nested‑

Laos and Dipstick method may be a useful tool for rapid di‑

PCR method to evaluate the efficacy of the two methods.

agnosis of malaria infection in active case detection survey.

38

A‑27 MOLECULAR EPIDEMIOLOGICAL EVALUATION OF PYRIMETHAMINE/SULFADOXlNE
EFFICACY IN PIASMODIUM FALCIPARUM PATIENTS FROM THE VANUATU ARCHIPLAGOTOSHIHIRO MITA , AKIRA KANEKO*, GEORGE TALEO', MORRIS KALKOA2,

ANDERS BJORKMAN* AND TAKATOSHI KOBAYAKAWA*
Department of International Affairs & Tropical Medicine,Tokyo Women's Medical Universityl ,Malaria Section, Department of Health, Vanuatu2 andKarolinska Institute, Sweden3

Plasmodium falciparum resistance to cycloguanil andreported in the literature at codon 5 1 (Asn‑His) and an‑

pyrimethamine has been associated with specific point mu‑other at codon 108 (Ser‑Asn). In contrast to dhfr, no iso‑tations in the dihydrofolate reductase (dhfr) and dihydrop‑

late had a mutation in the dhps gene. Although all 40 iso‑teroate synthase (dhps) genes. Vanuatu consists of 80 is‑
lands in Melanesia with seasonal and unstable malaria en‑
demicity. The first‑1ine drug for malaria treatment in chil‑lates had mutations in the dhfr gene at codon I 08 (Ser‑Asn) and at codon 59 (Cys‑Arg), in vivo, 39/40 patients
responded well to single dose of PS. This is in contrast todren aged less than five years old was changed in 1 991 frommany in vitro studies which have demonstrated that isolates

chloroquine to pyrimethamine/sulfadoxine (PS). We havewith mutations in the dhfr gene at codon I 08 (Ser‑Asn)and at codon 59 (Cys‑Arg) are normally associated withstudied in vivo drug susceptibility of 40 P falciparum pa‑
tients to PS on two islands of Vanuatu (Gaua, Santo) andalso investigated the distribution patterns of the dhJ

andmoderate resistance to dhfr‑inhibiting drugs. Thus, our invivo study in Vanuatu clearly demonstrates the clinical effi‑

dhps genotypes in 1 40 P falciparum infections on 4 islands

cacy of PS on P falciparum infections with the putatively(Gaua, Santo, Pentecost and Malakula) by direct sequenc‑
ing. A total of 137 out of 140 P falciparum infections had

resistant dhfr gene. The reason for the discrepancy between

the same mutations at codon 108 (Ser Asn) and at codon
59 (Cys‑Arg) in dhfr The remaining three isolates in
Gaua had the same two mutations, a change not previously

consistent with acquired immunity to host malaria parasite

A ‑ 28in vitro and in vivo studies is as yet unclear, however, it isinfection.

POPULATION GENETICS AND EPIDEMIOLOGY:
MALARIA IN VANUATU

J. KOJI LUM, AKIRA KANEKO AND TAKATOSHI KOBAYAKAWA
Department of International Affairs & Tropical Medicine,Tokyo Women's Medical Universityl

The correlated patterns of mutations within popula‑tions and disease endemicity serve to implicate genes in‑tributions of disease selection and gene flow to patterns ofdiversity. Our analyses indicate that malarial selection has

volved in immunity and resistance to pathogens. In addition,

shaped the frequencies of G6PD deficiency within the ar‑

population founder events and gene flow biased by proxim‑chipelago. In contrast, distributions of CYP2C19 alleles in‑ity or cultural factors can also influence the distributions ofvolved in the metabolism of a wide range of pharmacologi‑

alleles within populations. Here we compare the distribu‑

cally important compounds are correlated with geography,
but not malarial prevalence or neutral autosomal genetic
variation. These data suggest CYP2C19 has been regionallytions of alleles implicated in malarial resistance [glucose‑6‑

phosphate dehydrogenase (G6PD)] and variable drug me‑tabolism Lcytochrome P450 (CYP) 2C19 (CYP2C 19)] withneutral loci [mitochondrial DNA (mtDNA) and autosomal
short tandem repeats (STRs)], geographic proximity, andlinguistic relationships within Vanuatu to evaluate the con‑selected. Our comparisons of functional and neutral genetic

loci provide an unambiguous demonstration of genetic se‑
lection.

39

A ‑ 29REPETITIVE DOSING OF ARTEMISININ AND QUININE AGAINSTPIASMODIUM FALCIPARUM IN VITRO =A SIMULATION OF THE IN VIVO PHARMACOKINETICSBWIJO A. BWIJOl'2, ALIN MOHAMED HASSAN' * NAGAT ABBAAS

ORJAN ERIKSSON4 AND ANDERS BJORKMAN2,5
Department of International Affairs & Tropical Medicine,Tokyo Women's Medical Universityl
Division of Infectious Disease, Department of Immunology, Microbiology,
Pathology & Infectious Diseases, Karolinska Institutet,

Huddinge University Hospital, Huddinge2,

Division of Biophysics & Pharmacokinetics, Department of Pharmacy,
Biomedical Centre, Uppsala University, Uppsala3,

Hospital Pharmacy & Unit of Tropical Pharmacology,
Department of Clinical Laboratory Medicine & Technology,
Huddibge University Hospital, Huddinge4 andDivision of Infectious Disease, Karolinska Institutet,

Karolinska Hospital, Stockholm5, Sweden

Plasmodium falciparum (F32) parasites were exposed

the efficacy was more gradual. On the other hand, dosing

to artemisinin and quinine for 3 and 4 hr respectively, once

once daily appeared to be nearly as effective as twice dailyor twice daily for 3, 5 or 7 days. Between the peaks theparasites were exposed to through concentrations. Continu‑
ous drug exposure was also assessed for comparison. After

posure at peak concentrations of 10 5 M. A concentration of

drug exposure, the cultures were extended for an observa‑

tended to 7 days. There was a strong similarity between es‑

and radical clearance was obtained even after 3 days of ex‑

10‑6 M provided the same effect if the duration was ex‑tion period of up to 30 days to assess the viability of the

timated concentrations of free unbound drug required forparasites remaining after the drug exposure. For artemisinin,

radical clearance in vitro and those empirically required fora critical threshold concentration of 3 X 10 8 M was required

clinical efficacy in vivo. This suggests that the in vitro

for growth inhibition. Dosing twice daily for at least 5 days

model represents an appropriate model for estimating drug

was also critical. Prolonging the duration of drug exposure

efficacy and pharmacodynamics if the in vitro system is

to 7 days further increased the efficacy. For quinine the re‑adapted to simulate the in vivo pharmacokinetics.

sults were quite different. The concentration dependency of

A‑30 GENE ANALYSIS OF PFMDR I IN MEFLOQUINE‑RESISTANT
PIASMODIUM FALCIPARUMYUKIO NAGAI, HYE‑SOOK KIM AND YUSUKE WATAYA
Faculty of Pharmaceutical Sciences, Okayama University

Although mefloquine‑resistance in Plasmodium ,falci‑

tryed to get mefloquine‑resistante parasite. Isolate was ob‑parum is increasing, the molecular mechanism remains un‑tained from mefloquine resistant patient (523a strain). The

clear. Recent reports have shown that the Asn‑86, Ser‑1034,

falciparum is associated with decreased sensitivity of the

523a strain was cultured under mefloquine pressure, after
two years, we succeed to get the parasite which can grow in
1 .5 X 10‑7 M mefloquine concentration (523a R strain). The

anti‑malarials mefloquine.

parasite was cultured under the same condition without

Asn‑1042 and Asp‑1246 alleles of the pfmdr I gene of P

To investigate the association between drug sensitivity

drug as control (523a S strain). Second, using these strains

and sequence polymorphisms in the pfmdr I gene, first we

we measured the half‑maximal inhibitiory concentration

40(IC50) for several anti‑malarials. As a result, in 523a S strain

also have the same sequences.

the IC50 Value of mefloquine is 6.1 X I O‑" M, in 523a R strain,

In conclusion, although the IC50 value of mefloquine isis 6.2 X 10‑8 M. The 523a R strain have about 10 times activ‑

higher in 523a R strain than in 523a S strain, but amino acidity than the 253a S strain. Moreover, 523a R strain in‑sequences are not differrent. 523a R strain, we get in thiscreased resistance to artemisinin and halofantrine and in‑study, is not associated with drug resistance and sequencecreased sensitivity to chloroquine. Third, we analyzed
amino acid sequence by Dye terminator method after par‑polymorphisms in pfmdr I gene. We think that perhaps an‑other mechanism work for drug‑resistance.

ticular domain amplyfied by PCR. Both strains have Tyr‑86,

Ser‑1034, Asn‑1042 and Asp‑1246 of the pfmdr I gene.

Acknowledgements :We wish to thank Dr. Mikio Kimura and Dr. Takahisa Fu‑

There is no change between 523a R and 523a S. The FCR‑3

ruta of University of Tokyo for offering 523a strain to us.

A‑31 MECHANISMS OF PARASITICIDAL ACTIVITY OF TETRACYCLINES ON
PLASMODIUM FALCIPARUM = A POSSIBILITY OF THE PLASMID AS THE DRUG TARGET
Q. H. LIN, KEN KATAKURA AND MAMORU SUZUKI
Department of Parasitology, Gunma University School of Medicine

A plastid‑like organelle, apicoplast, was recently dis‑

three subunits of a eubacterial RNA polymerase genes, suchcovered in the protozoan parasites of the phylum Apicom‑

as rpoB, rpoC I and rpoC2 and these genes are polycistroni‑

plexa, including Plasmodium. The plastids of P falciparum

cally transcribed, the synthesis of the rpoB/C mRNA wascompared to a nuclear‑encoded merozoite surface antigen

carries a 35‑kb circular genome, which encodes large andsmall subunit rRNAS, tRNAS, ribosomal proteins, subunitsof a eubacterial RNA polymerase, a translation elongation(MAS 1) mRNA. As a control, a nuclear‑encoded small
subunit rRNA (ssu rRNA) was also amplified because thefactor and other open reading frames. The fact that the plas‑

ssu rRNA synthesis is unaffected by antibiotics. The rpoB/C

tid contains its own RNAS and prokaryotic proteins associ‑

RT‑PCR products was remarkably decreased by the treat‑

ated with transcription and translation suggests an essential

ment with tetracycline at I OO mg/ml for 1 4 hr, but not forrole of the plastid for malaria parasite survival, although the

8 hr. Minocycline at 60 mg/ml had a similar effect on thefunction of the plastid remains unknown. The prokaryotic

decay of the mRNA. A similar effect was also observed

feature of the plastid also makes this organelle a target forwith the treatment with thiostrepton and ripampin, which

drug development.
We previously reported that tetracycline and minocy‑

appears to be a selective inhibitor of plastid protein synthe‑

sis and an inhibitor of prokaryotic RNA polymerase, re‑cline showed a delayed death activity on P falciparum in vi‑

spectively. However, there is no effect of these antibiotics

tro. In the present study, we examined effects of these anti‑on the levels of nuclear‑encoded MSAI mRNA nor ssu

biotics on the plastid activity of the parasites. We used anrRNA .

assay of plastid‑encoded RNA polymerase mRNA Ievels by
reverse transcriptase‑polymerase chain reaction (RT‑PCR)(McConkey et al., J. Biol. Chem., 272, 1997), because of

inhibit the protein synthesis in the plastid, Ieading the inhi‑

bition of transcriptional activity of the organelle, although

the absence of a direct measurement system for the protein

tetracycline might target mitochondorial protein synthesis.

synthesis in the plastid. Since the plastid DNA contains

These results suggest that tetracycline antibiotics may

41

A ‑32 ANTIMALARIAL ACTIVITY AND POTENT ENHANCEMENT OF THE SENSITIVITY OF

PIASMODIUM FALCIPARUM TO CHLOROQUlN
BY THE BISBENZYLISOQUINOLINE ALKALOID CEPHARANTHINKOSUKE HARUK11, PATRICK G. BRAY2, MINORU ONO* AND STEPHEN A. WARD 'Department of Infectious Diseases, Kyorin University School of Medicinel,

Department of Pharmacology and Therapeutics, University of Liverpool, UK2

and Research Laboratory, Kaken Shoyaku Co. Ltd.3

Cepharanthin is an extract of Stephania cepharantha,

quine against resistant clones by a factor of 15 at a concen‑

widely used in Japan for clinical use. Major components oftration of only 200 nM ( 120 ng/ml ). It is 50 times more po‑

Cepharanthin (CP) are Isotetrandrin (IT), Cepharanthinetent than verapamil, the standard resistance modulator, and(CE), Berbamine (BE), Homoaromoline (HO), Cepharano‑

3 times more potent than the sum of its individual alkaloids.

line (CO) and Cycleanine (CY). We investigated antimalar‑
ial activity of CP and component alkaloids with chloroquine

Combination of component alkaloids acted synergisticallyto sensitize the parasite to chlorquine, possibly explaining

resistant strain K1. And also enhancement of the sensitivity

the enhanced potency of CP. CP differed from verapamil into chloroquine was determined in resistant and sensitivethat it further sensitized clones that are considered to bestrains of Plasmodium falciparum with CP and components.
CP and component alkaloids exhibited moderate antimalar‑fully susceptible, improving the baseline activity of chloro‑

quine. Potent sensitization of parasites to chloroquine in

ial activity, with IC 50 in the range of 1 70 to 3,700 nM (CPvitro coupled with low toxicity suggests that coadministra‑

338, IT170, CE1.020. BE485, HO1,490, CO1,015 and CYtion of CP might extend the clinical utility of chloroquine.

3,700 nM respectively). CP enhanced the activity of chloro‑

A‑33 THE DEVELOPMENT OF NEW ANTIMALARIAL DRUGS‑IN VITRO AND IN VIVO ANTIMALARIAL ACTIVITY OF ENDOPEROXIDESKANAKO ONO*, NAOKI OGURA1, YUKIKO NAGA11, HYE‑SOOK KIM1, KHURSHIDA BEGUMl,
YOSHIAKI HAMADA2, YUJI NONAM12, TAKAHIRO TOKUYASU2, ARAKI MASUYAMA',MASATOMO NOJIMA' AND YUSUKE WATAYA1
Faculty of Pharmaceutical Sciences, Okayama Universityl andFaculty of Engineering, Osaka University2

A number of medicines such as chloroquine and qui‑

2.6X 10‑8 M, in contrast, the 50 ; o inhibitory concentrationnine are available for treatment of malaria, but the rapid de‑of against mouse mammary FM3A cells was 8.0 X I 0‑6 M,

velopment of drug resistance are a serious problem. Medici‑demonstrating that the selective toxicity (>300) was also re‑nal agents based on novel mechanisms of action are, there‑markable. It should be noticed that these compounds arefore, required to overcome emergence of resistance and tosimilar effective of artemisinin (the IC50's against P falcipa‑

control an ever‑increasing number of epidemics caused byrum and FM3A cells were I .8 X 10‑8 M and 1.0 X 10‑5 M, re‑the malaria parasite.

spectively). An in vitro test, morphological changes of ma‑

Now, we have confirmed the potential of I , 2, 4, 5‑

laria parasites during the treatment of N‑89 against P falci‑

tetraoxacycloalkanes as a new class of simple peroxide anti‑

parum were studied. In these results, we have observed amalarial drugs. In the preliminary study, some I , 2, 4, 5‑

condensation of parasite nuclei, shrinked cytosol in tropho‑

tetraoxacycloalkanes have been found to be available forzoites and inhibition of schizont formation by microscopy

chloroquine‑sensitive and ‑resistant P falciparum in vitro.
Especially, I , 2, 6, 7‑tetraoxaspiro [7. 1 1 Jnonadecane (N‑89)

after treatment with 100 times the EC90 Value of N‑89 (2 X
10‑6 M) for 24 hr. The morphological changes in parasites

has a potent antimalarial activity for P falciparum (FCR‑3

induced by N‑89 may be a consequence of several phenom‑

strain) that the IC50 value is 2.5 >< 10‑* M and (KI strain) is

ena including difference in antimalarial activity in vivo.

42
In addition, we investigated the in vivo antimalarial ac‑

treatment of N‑89 for three consecutive days in vivo was as‑

tivities of N‑89 by 4‑day suppressive test. The ED50 value of

sayed. Parasitemia declined have been found within 24 hr

the compound has shown that 12 mg/kg/day (ip), which wasafter drug administration. We used 5 mice for this test, 4

required to cause 50% suppression of P berghei in mice. In

mice have been cured and only I mouse was died. On thethe experiments, the dose of 50 mg/kg/day of N‑89 causes
cure (for 4 mice in 5 tested mice), in which malaria para‑other hand, in treatment with artemisinin, parasitemia de‑clined have been found after 24 hr of drug administration,sites were not observed in circulating blood after 60 days.

but after 72 hr after drug administration, parasitemia be‑

Furthermore, no side effects such as diarrhea, body weight

come increased again. Our studies indicate that N‑89 has aloss and mortality were observed during treatment with N‑

strong and continuous antimalarial activity.

89 at doses by I ,600 mglkg (ip). As a control, mice treated

Our results may be helpful in the design of che‑with artemisinin (50 mg/kg/day, ip) were not cured andmotherapeutic I , 2, 4, 5‑tetraoxacycloalkanes in the world‑

mice died due to P berghei infection. Next, the effect ofwide fight against malaria.

A ‑ 34

A POTENT ANTIMALARIAL ACTIVITY OF HOT‑WATER EXTRACT OF
HYDRANGEA MACROPHYHA VAR. OTAKSA LEAVES AGAINSTPIASMODIUM YOELII 17XLIN ICR MICEAKIRA ISHIH*, HIROTAKA TAKEZOE*, MIKAKO SAKAI*, KAORI FUJII',TOSHIO MIYASE' AND MARORU TERADA'
Department of Parasitology, Hamamatsu University School of Medicinel andDepartment of Pharmacology, School of Phamaceutical Science,
University of Shizuoka2
Malaria is still one of the major health problems in the

yoelii 17XL‑parasitized erythroytes. Starting on day 3 after

world. The emergence and spread of chloroquine‑resistantPlasmodium falciparum has stimulated the development of

injection of parasitized erythrocytes, mice were orally given

new effective treatments against malaria. There is now agroup and distilled water in the untreated, infected one, re‑

new interest in investigation of folk medicine exhibiting aspectively, twice a day for 5 consecutive days. Non‑treateda hot‑water extract at 75 u1 /10 g body weight in the treatedpotent antimalarial activity. We investigated experimentallycontrol mice died with a gradual body weight loss and aan antimalarial activity of Hydrangea macrophylla var.
Otaksa with rodent malaria models. Leaves of this plant
were collected in the campus of Hamamatsu University

rapid increase of parasitemia in the bloodstream from 6 to 7

School of Medicine in August, 1999. Five gram of air‑dried

low parasitemia levels during administration. Following aleaves put in a herbal bag was added with 500 ml of dis‑tilled water and boiled. After removing the leaves the ex‑transient recrudescence of malaria parasites in the blood‑tract fluid was finally concentrated to 50 ml . Hot‑water ex‑
tract was stored at 4iC unitl use. Outbred male ICR mice, 8

a microscopic examination. Leaf extract seems to have 2
different types of effects on rodent malaria parasite, P. yoelii

weeks old, were infected intraperitoneally with I X 10' P

17XL, in vivo.

after infection, but mice given the leaf extract survived dur‑ing the experiment. Mice treated with leaf extract showedstream of the treated mice, no parasites could be detected by43

A‑35 FRACTIONATION OF ANTIMALARIAL PRINCIPLE FROMHYDRANGEA MACROPHYLLA VAR. OTAKSA LEAVES AND ITS ACTIVITY AGAINSTPLASMODIUM YOELII 17XL IN ICR MICEHIROTAKA TAKEZOE*, AKIRA ISHIH*, KAORI FUJII*, MIKAKO SAKAI ,TOSHIO MIYASE2 AND MAMORU TERADA'
Department of Parasitology, Hamamatsu University School of Medicinel andDepartment of Pharmacology, School of Pharmaceutical Science,
University of Shizuoka2

Malaria is still one of the most important tropical dis‑neally with I >< 105 P yoelii 17XL‑parasitized erythrocytes.

eases in the world. One of the reasons is the emergence ofStarting on day 3 after injection of parasitized erythrocytes,

drug resistant parasites. Thus it is important to develop new

mice were orally treated twice a day for 5 consecutive days

antimalarials quickly. In 1972 artemisinin was isolated fromwith the hot‑water extract (5 g leaves/50 ml ) or each frac‑

the herb Artemisia annua L. used in the traditional Chinesetion at two dosages equivalent to 10 g leaves/50 ml and 100 g

medicine. Today artemisinin and its derivatives have be‑leaves/50 ml of the hot‑water extract. Only the less polar al‑come one of the antimalarials for severe malaria. We havealready reported that the hot‑water extract of Hydrangea

kaloid fraction at higher dosage showed the same antimalar‑

ial activity as the hot‑water extract (5 g leaves/50 ml )macrophylla var. Otaksa leaves had a high in vivo antima‑
larial activity against Plasmodium yoelii 17XL in mice. In

The NMR spectra of the less polar alkaloid fraction showed

this study we investigated the antimalarial activity of thethat the signal patterns and chemical shifts of compounds inshowed, though other fractions had no antimalarial activity.

fractions isolated from the hot‑water extract. Four different

the fraction were closely similar to those of febrifugine andfractions were prepared from the hot‑water extract of theisofebrifugine. However, the fraction was 20 times less ac‑

leaves in the usual manner for obtaining alkaloid fractions.

tive than the hot‑water extract.

Male ICR mice, 8 weeks old, were infected intraperito‑

A‑36 A POTENT ANTIMALARIAL ACTIVITY OF HOT‑WATER EXTRACTS OF PLANTS
BELONGING TO THE FAMILY SAXIRAGACEAE AGAINSTPIASMODIUM YOELII 17XL IN ICR MICEMIKAKO SAKAI AKIRA ISHIH HIROTAKA TAKEZOE*, KAORI FIJII*MASAKI SANO', NORIHIRO ASANUMA*, TOSHIO MIYASE' AND MAMORU TERADA*
Department of Parasitology, Hamamatsu University School of Medicinel andDepartment of Pharmacology, School of Pharmaceutical Science,
University of Shizuoka2

We showed that the hot‑water extract of Hydrangea

water and boiled. After removing the materials the extract

macrophylla var. Otaksa leaves had a high in vivo antima‑

fluid was finally concentrated to 50 ml . Hot‑water extract

larial activity against Plasmodium yoelii 17XL. It is there‑

was stored at 4 C until use. Outbred male ICR mice, 8f'ore of interest to evaluate the antimalarial activity of re‑weeks old, were infected intraperitoneally with I X I 05 P

lated plants of the Family Saxifragaceae from a practicalyoelii 17XL‑parasitized erythrocytes. Starting on day 3 af‑

viewpoint of folk medicines. The plants specimens such as

ter injection of parasitized erythrocytes, mice were orally

H. macrophylla, H. macrophylla subsp. serrata var. acumi‑

given a hot‑water extract at 75 u1/10 g body weight in thenata, H. involucrata. H. hirta, H. paniculata, Cardiandra

treated group and distilled water in the untreated, infected

altermfolia and Clerodendron trichotonmum were evalu‑

one, respectively, twice a day for 5 consecutive days. The

ated. Five gram of air‑dried leaves or 20 g of air‑dried roots

extract of H. macrophylla leaves showed an antimalarial ac‑

put in a herbal bag was added with 500 ml of distilledtivity, although other extracts showed no effects. Mice

44

treated with leaf extract of H. macrophylla showed low

macrophylla showed a higher antimalarial activity than thatparasitemia levels during administration. Following a tran‑of H. macrophylla var. Otaksa in respect of suppression ofsient recrudescence of malaria parasites in the bloodstream

malaria parasite increase at a recrudescence and of mouseof the treated mice, no parasites could be detected by a mi‑

body weight loss.

croscopic examination. Furthermore, the leaf extract of H.

A‑37 AN ANTIMALARIAL ACTIVITY OF HOT‑WATER EXTRACT OF DICHROA FEBRIFUGALEAVES OR ROOTS AGAINST PIASMODIUM YOELII 17XL IN ICR MICEKAORI FUJII', AKIRA ISHIH*, HIROTAKA TAKEZOE*, MIKAKO SAKAI*,NORIHIRO ASANUMA', MASAKI SANO*, TOSHIO MIYASE' AND MAMORU TERADA
Department of Parasitology, Hamamatsu University School of Medicinel andDepartment of Pharmacology, School of Pharmaceutical Science,
University of Shizuoka2

We showed that the hot‑water extract of Hydrangea

parasitized erythrocytes, mice were orally given a hot‑water

macrophylla var. Otaksa leaves had a high in vivo antima‑
larial activity against Plasmodium yoelii 1 7XL though theextract at 75 u1/lO g body weight in the treated group andextract of roots had no activity. Febrifugine and isofebrifug‑

consecutive days. Mice in the non‑treated control and group

ine having antimalarial activity were isolated from the roots

given the old leaf extract (5 g/50 ml) died with a gradualof Dichroa febnfuga. It is therefore of interest to examine

body weight loss and a rapid increase of parasitemia in thethese principles within leaves of D. febnfuga. Old leaves
and roots of D. febnfuga and young leaves were collectedbloodstream. Root extract (5 g/50 ml ) showed an antimalar‑in December, 1 999, and in June, 2000, respectively. Fivegram of air‑dried leaves or air‑dried roots put in a herbal

extract at a dose of 5 g/50 ml and of 2.5 g/50 ml , respec‑

bag was added with 500 ml of distilled water and boiled.
After removing the material the extract fluid was frnallyconcentrated to 50 ml . Hot‑water extract was stored at 4 C

thus the death seemed due to the adverse effects of extract.

until use. Outbred male ICR mice, 8 weeks old, were in‑fected intraperitoneally with I X I O' P yoelii 17XL‑

the seasonal changes of alkaloids' content within leaves andparasitized erythrocytes. Starting on day 3 after injection of

distilled water in the control, respectively, twice a day for 5

ial activity. A11 mice and 50% of mice given the youg leaf
tively, died without multiplication of malaria parasite andBut mice given young leaf extract at a dose of I .25 g/50 ml

survived during the experiment. Present results suggested
also indicated the important ideas f or material collection.

45

B‑l THREE CASES OF CREEPING DISEASE DUE TO LARVAL HOOKWORM INFECTION
FUKUMI NAKAMURA‑UCHIYAMA' , KIYOFUMI EGAWA', KENJI OHNISHI*,TAZUKO YOKOTA4 AND YUKIFUNI NAWA
Department of Parasitology, Miyazaki Medical Collegel,

Department of Dermatology, Kumamoto University School of Medicine2
Depattment of Infectious Disease, Tokyo Metropolitan Bokutoh Hospital3 andDepartment of Dermatology, JR Sapporo Hospital4

Larvae of the dog and cat hookworms, Ancylostoma
caninum and A. brasiliensis are known to cause creeping
disease. Recently three cases of creeping disease due to lar‑val hookworm infection on abroad were referred to ourlaboratory. Two of them were young women (twenties)
traveled Thailand or Malaysia for several weeks. They

reappeared in January 2000. Skin biopsy was performed on
all three cases, but sections of worms morphologically de‑termined Ancylostoma spp. Iarvae were found in the super‑
ficial skin epidermis of only one case. In other cases, justsmall tunnel with eosinophil infiltration in epidermis layer
considered to warm trail was found in the sections.

walked around the bush/seashore on bare feet or lay on the

Although Gnathostoma sp. and Spirurina larvae arebeach. Creeping disease developed shortly after outdoor
episodes. The remainder was an I ‑year‑old Cambodian boy
adopted an American living in Japan. He had a history of
creeping disease just before coming to Japan. The disease

famous causative spices of the creeping disease in Japan,

we should be aware of animal hookworm larvae cause thedisease. Especially the patient developed a creeping disease
with rapid onset after outdoor episodes on abroad.

B‑2 A CASE OF URINARY SCHISTOSOMIASIS WITH MACROHEMATURIAKENTA MIKI, NOBUKI KATO, SHOUICHI ONODERA AND YUKIHIKO OHISHI
Department of Urology, The Jikei University School of Medicine

A 27‑year‑old Japanese man admissioned our hospitalwith dyspnea after six‑month journey to Africa. In thebiginning he was treated by the diagnosis of the eosino‑philla. He had been presented to the Department of Urol‑eral irregular changes on mucous membrane of urinaryogy, because of macrohematuria.

bladder. We biopsied some of those through the cystoscope

Several eggs of schistosomiasis were seen in urinalysis

and many white protuberances were also seen on the mu‑
cous membrane of urinary bladder. We diagnosed this pa‑
tient as a schistosomiasis haematobium by the urinalysis
and his typical clinical progress.

He was treated with insectfuge for two days, andmacrohematuria disappeared three days after this therapy.
After one month of initial therapy, no eggs of schistosomia‑

sis were presented in urinalysis but cystoscopy showed sev‑

and found the eggs of schistosomiasis under the mucous
membrane but no malignant changes were seen histologi‑
cally.

Some of urinary bladder carcinoma related to this dis‑ease were reported in several papers, so we try to follow up
this patient very carefully.

46

B‑3 FOUR IMPORTED CYSTIC ECHINOCOCCOSIS CASES IN JAPANCONFIRMED SEROLOGICALLYAKIRA ITO* , YUJI ISHIKAWA*, YASUHITO SAKO*, MINORU NAKAO*AND KAZUHIRO NAKAYA'
Department of Parasitology* andAnimal Laboratory for Medical Research2, Asahikawa Medical College

From I st April 1999, echinococcosis, either alveolar

to be CE when we carried out immunoblot analysis using(AE) or cystic (CE) is included in the parasitic diseases incrude cyst fluid and purit' led antigen B subunit (8 kDa) of

category 4 of the New Law of Prevention of Infectious Dis‑

Echinococcus granulosus and crude and purified Eml8,

eases in Japan. Two AE cases were reported from Honshu

specific to AE. These all cases recognized the antigen B(the main island) based on or referred to the ongoing serodi‑

subunit without any response to Eml8 and were concluded

agnosis carried out at the Hokkaido Institute of Public
Health. However, one was fascioliasis, the other was CE
but not AE. However, mass‑media reported that the killer
parasite had already invaded into Honshu Island based on

to be CE serologically. After serological analysis, we asked

these misdiagnosed cases. So far, there is no official mes‑

clinicians have any experience to observe such image data

sage that these two were not AE even from the Projectof Health and Welfare, Japan. Under the new law, AE

before. It is pointed out that CE with very unique image
data is not indigenous in Japan but imported cases should
be more common in the future. Curative treatment for thesegroup on the control of AE in Japan sponsored by Ministry

the clinicians to show us the image data. The image datafrom US and CT also showed typical patterns for CE. How‑ever, CE is absolutely uncommon in Japan and almost no

should be confirmed after surgical resection of the lesions.

three zoonotic cestodiases differs critically. Therefore, it is

Asahikawa group has already established differential se‑essential and urgent to establish better resolution for confir‑

rodiagnosis without surgical confirmation for zoonotic
cestodiases including AE, CE and neurocysticercosis,

mation serodiagnosis before surgical treatment. We stresshighly appreciated internationally.

these three diseases from other parasitic or non‑parasitic

Last four years (1997‑2000), we experienced four CE
cases in Japan: one Nepali, one Jordanian, one Chinese androdiagnosis of AE, CE and NCC using more reliable anti‑

one Japanese. These all were serologically easily confirmed

gens and technology without surgery.

that it is easy to obtain critical diagnosis to differentiatediseases and we better shift to introduce a confirmation se‑

B‑4 A SEVERE CASE OF VENOMOUS OPHATHALMIA CAUSED BY
SPITTING COBRA IN CENTRAL KALAHARITOSHIMASA NISHIYAMA* AND M OSAKI'
Department of Public Health, Kansai Medical University1 andHimeji Dokkyo University'

We researched about health status of the Central Kala‑

At the latter, he consulted an ophathalmologist of gen‑hari Bushman (Gwi and Gana Bushmen) in Republic oferal hospital at Gaborone (the capital of Botswana), but he

Botswana. In this research, a first case of venomous of
ophathalmia caused by spitting cobra was experienced.

The pupils were symmetric and their diameters were about

could not recovered. His physical signs were as follows:

Sixty‑five‑year old male, whose job is leather processing,

3 mm and their conjunctivas were without jaundice andlived in New Xade (Ghantsi District, Botswana). He hadanemia. The light reflection of these pupils was not found.

been sputtered at both eyes, in March 1975 and had intense

And other physical signs were almost nothing particular.

eyes pain, eye mucous and palpebral edema, and when he

In conclusion, according to Branch (1998), several
species of venomous snakes are known in southen part ofAfrica. Especially, the venomous snakes, which can sputer

had attacked, he could not wash his eyes because it was a
dry season, and then he lost his eyesight soon.

47

the venom, are Mozambique Spitting Cobra (Naja mossam‑

Kalahari, black color Spitting Cobra is not found but brownbica) and Black‑necked Spitting Cobra (N. nigricollis). But,

Spitting Cobra was sometime seen in this area. Therefore,

in the Central part of Kalahari Desert in Botswana, these

we thought that this case was attacked Mozambique Spit‑cobras have not been investigated, yet. In this research, weting Cobra. Thus this is a first report that the case of visualcollected the materials of the spitting cobra in the Central

disorder by spitting cobra stays in the Central Kalahari.

B‑5 DIAGNOSIS OF KALA‑AZAR BY ELISA USING URINE SAMPLES
MAKOTO ITOH1, MOHAMMAD ZAHIDUL ISLAMl, S.M. SHAMSUZZAMMAN2,
A.K.M. SHAMSUZZAMAN CHOUDHURY3, M.D. AKRAM HOSSAIN3, IFTEKHAR AHMED4,RUSELLA MIRZA4, XU‑GUANG QIU1, NILUFAR YEASMIN*, MASATO FURUYA6,YOSHIHISA HASHIGUCH12 AND EISAKU KIMURA1
Department of Parasitology, Aichi Medical Universityl,

Department of Parasitology, Kochi Medical University',

Department of Microbiology, Mymensing Medical College, Mymensing3,
Department of Microbiology, Rajshahi Medical College4,
Department of Parasitology, Institute of Epidemiology,

Disease Control and Research, Mahakhali, Dhaka5, Bangladesh andInstitute of Laboratory Animals, Kochi Medical University6

For the diagnosis of visceral leishmaniasis (kala‑azar),nese samples (mean+3SD). Anti‑L. donovani lgM and lgA

detection of the causative parasite is the most accurate

titers in urine from kala‑azar patients were very low and themethod. Detecting anti‑Leishmania donovani antibodies inserum samples is an alternative method. In this study, we

sensitivities were 67% and 38%, respectively. A11 urine
samples from healthy people in Bangladesh were negative.

developed a diagnostic method to detect anti‑L. donovani
lgG in urine samples. Anti‑L. donovani lgG, IgA, IgM, andtotal immunoglobulins (total lgs; IgG, IgM and lgA) in se‑

lgG ELISA using serum samples gave 29 positive results(88%) out of 33 kala‑azar patients. The lgG ELISA withurine samples was similar in sensitivity. As collection of

rum and urine samples were measured with ELISA. Among

urine is much easier than serum, this method will be useful48 urine samples from kala‑azar patients, 43 (90%) or 41(85%) samples were judged positive with lgG or total lgs

for epidemiological studies and laboratory diagnosis of pa‑
tients .

ELISA respectively, using cut off points obtained with Japa‑

B‑6 EVALUATION OF DOT‑ELISA FOR THE IMMUNODIAGNOSIS OF TREMATODE INFECTIONSKUNIOKI ARAKI , HIROSHI YAMASAKI', TAKASHI AOKI',RYUICHIRO MAEDA* AND TAMOTSU KANAZAWA+
Department of Microbiology, National Institute of Public Healthl,

Department of Parasitology, Juntendo University School of Medicine2,
School of Veterinary Medicine, Obihiro University3 andDepartment of Parasitology and Tropical Medicine,

University of Occupational and Environmental Health4

In recent years, dot‑ELISA has been used for the im‑munodiagnosis of parasitic diseases. However, the cross‑reactivity caused by the crude antigens used has not been

examined sufficiently. We have already reported that an im‑proved dot‑ELISA is very useful for the immunodiagnosis
of Japanese paragonimiases, amoebiasis and human toxo‑

48

from patients infected with 4 different trematodes, Parago‑ways necessary to decide the endemic Paragonimus species,
because various kinds of Paragonimus exist in China andnimus sp., Fasciola sp., Schistosoma japonicum and S.
mansoni, were examined. One microgram of crude anti‑

tosomiasis japonica and 12 schistosomiasis mansoni. Clear

cariasis. In the present study, a total of 58 serum samples

Southeast Asia. Similar results were obtained for 14 schis‑gens extracted from S. japonicum , S. mansoni, P miyazakii,

spots were recognized against S. japonicum egg, S. japoni‑

P. westermani. Fasciola sp. and Clonorchis sinensis adultcum adult and/or S. mansoni antigens. S. japonicum adultworms were absorbed onto a nitrocellulose membrane. In
addition, S. japonicum egg antigen was also absorbed tocompare the reactivity with S. japonicum adult antigen.
Clear dark purplish spots against P miyazakii and P

antigen is also available for the immunodiagnosis of schis‑tosomiasis japonica, although egg antigen has a higher
specificity than the adult antigen. 8 out of 17 fascioliasis

showed strong cross reactions against P. miyazakii and P

westermani antigens were recognized in all 15 cases of im‑westermani antigens. In these cases, ELISA or other sero‑

ported paragonimiasis. No big difference in the spot inten‑

10gical tests are desirable to obtain a definitive diagnosis.

sity of both Paragonimus antigens was recognized at I : 200

Our results suggest that the dOt‑ELISA is very useful

serum dilution. The cross‑reactions against other trematodefor the immunodiagnosis of imported trematode infections.

antigens were less than those by ELISA. Therefore, dot‑
ELISA is useful for the differential diagnosis between

Further investigations will be needed for the immunodiag‑

paragonimiasis and other respiratory diseases. It is not al‑

well as trematode infections.

nosis of nematode and cestode infections by dot‑ELISA, as

B‑7 A SER0‑IMMUNOLOGICAL STUDIES ON CYSTICERCOSISHIDETAKA ICHIKAWA1, YOICHI IT02, YUKIO HOSAKA3, TOSHIHIKO KOMATSU3,ZHANG YONGSHENG+, XU ZHI‑JIE', DU JUN+,TERUAKI IKEDA ' AND KEIZOU NAGASE6
Department of Medical Zoology, Kanazawa Medical Universityl,
Department of Parasitology, School of Medicine Kitasato University2,

Biomedical Science Association3
Department of Parasitology, Norman Bethune University of Medical Sciences4,
Department of Parasitology, Harbin Medical University5 andDepartment of Medical Zoology, Fujita Health University College6

Immunoserological study of cysticercosis carried out
on sera from patients, healthy chinese, endemic inhabitant

healthy chinese, non‑endemic and endemic area inhabitants,
clealy significant difference were observed.

and non‑endemic inhabitant, using the enzyme‑linked im‑

For the detection of specific antigen by immunoblot

munosorbent assay (ELISA) and enzyme‑linked immu‑

assay, we compared with healthy inhabitant, patient and en‑

notransfer blot assay (EITB) in Jilin Province, China.

For the evaluate of suitable antigen, we prepared the 2materials designated as cyst fluid (CF) and whole cyst(WC) obtained from pig. A total 450 sera, including 1 34

demic inhabitant sera which obserbed positive reaction on

ELISA. Among the sera from patient or endemic inhabitant,some of the bands were recognized on the molecular weight
15.5, 17.5, 18.5, 20, 22, 26.5, 27, 28, 32, 35, 37, 40, 48, 56,

healthy sera, 86 patients sera, 1 15 non‑endemic inhabitants,

62, 82, and 97 kDa of WC and CF antigens. On the other95 endemic inhabitants sera obtained from Changchung inhand, the few of healthy inhabitant sera were very thinly

China. In patients (clinically suspected) sera, 82 (95.4%)

band recognized on molecular weight 48 and 97 kDa of WC

out of 86 were positive. On the other hand, 4 (3.0%) of 134

and CF antigens. We can't find specific antigens which di‑healthy chinese sera showed positive. Sixteen ( 1 3.9%) out

agnosis for cysticercosis until now. But we will tray withof 1 1 5 sera from non‑endemic area were positive. On the

our vest to find the useful ELISA antigen fractions for theother, 33 (34.7%) of 95 sera of endemic area showed posi‑diagnosis of cysticercosis.

tive. In comparison with ELISA value among sera from49

B‑8 USEFULNESS OF EM18 ELISA IN MONITORING EFFECT OF REMEDIESAGAINST ALVEOLAR ECHINOCOCCOSIS =CASE REPORT ON LONG TERM CHEMOTHERAPY WITH ALBENDAZOLEYUJI ISHIKAWA' , YOSHINORI FUJIMOTO', MINORU NAKANO*, YASUHITO SAKO ,KAZUHIRO NAKAYA*, YUTAKA KOHGO' AND AKIRA ITO*
Department of Parasitologyl, 3rd Department of Internal Medicine2 andAnimal Laboratory for Medical Research3,

Asahikawa Medical College

We have reviewed the correlation between clinicalcourses and immunological responses in two patients with

the illness as AE involving the right iliac bone, the liver, andalveolar echinococcosis (AE) involving the liver, the lungs

October 1 8 as radical surgery was thought to be difficult tothe lungs. We started chemotherapy with Albendazole on

and the bone. We operated on both of the patients for the

perform. We felt the effectiveness of the chemotherapy wasbone AE and have administered Albendazole for several
years. Case report: A 39‑year‑old woman from Kitami
City in Hokkaido was admitted to Asahikawa Medical Col‑lege Hospital in August 1989. She had suppurated dis‑confirmed because the intractable discharge stopped 6

charge through a fistula on the right hip with a dull pain.

ment twice against recurrent development of the iliac AE.

Around September 1987, she only had a dull pain but later

However, we have detected no remarkable progression in

she felt a subcutaneous nodular lesion. The lesion was
about the size of her fist without bulge or hyperemia by
March 1988.

the hepatic and the pulmonary AE for the last eleven years.

Percutaneous cutting from a softened part of the lesion

investigation, she was diagnosed as having tuberculous os‑

Movement of Em 1 8‑ELISA value and clinical activity ofAE, e.g. recurrence of iliac focus have almost correlatedwith each other. We will make another case presentation
concerning the correlation between the course of AE andEm 1 8‑ELISA value.
Conclusions: I . Albendazole has been effective forteomyelitis, but anti‑tuberculotics had no effect. The sec‑

both hepatic and pulmonary AE, whereas, the bone fociond curettage performed in November 1988, did not heal
the wound and severe pain brought her to Asahikawa Medi‑have not necessarily been suppressed to full satisfaction. 2.

cal College. We detected not only the specific tissue frag‑of AE that may be difficult to detect using common labora‑ments of AE in a tissue specimen obtained in the previous

tory tests or imaging devices. 3. Em 1 8‑ELISA may be ap‑

surgery but also multiple findings of hepatic AE by ultra‑

plicable not only to detect residual AE after surgery or a re‑sonography (US). Physically she looked well except for thecurrence but also, to help make decisions for treatment or adischarging fistula on the right hip. We finally diagnosed

complete cure.

was perfomed at Kitami Red Cross Hospital in April 1 988.
Persistive pyorrhea followed the treatment, and she then un‑derwent curettage against suppurative osteomyelitis in theright iliac bone three months later. After the total clinicalweeks after the administration of Albendazole. The third
operation was perfomed on the right iliac AE on December
19, 1989. Since that time, she has undergone surgical treat‑

We have made regular examinations through the use of US,

CT, MRI, common laboratory tests and Em 1 8‑ELISA.

Em 1 8‑ELISA is a useful indicator to evaluate the activity

50

B‑9 EFFECT OF CYTOCHALASIN D ON THE GROWTH,
ENCYSTATION AND MULTINUCLEATION OF ENTAMOEBA INVADENSASAO MAKIOKA MASAHIRO KUMAGAI', HIROSHI OHTOMO ,SEIKI KOBAYASHI' AND TSUTOMU TAKEUCHI
Department of Tropical Medicine, Jikei University School of Medicine* andDepartment of Tropical Medicine & Parasitology,
School of Medicine, Keio University'tion of Entamoeba invadens was examined. Cytochalasin

D became multinucleate; more than three nuclei per cellwere observed in 7 1 % of trophozoites grown in the pres‑ence of the drug as opposed to only 5% of those grown in

D blocked the growth of axenic E. invadens strain IP‑ I in a

the absence of the drug. Also, trophozoites grown with cy‑

dose‑dependent manner, which suggests that the drug is ef‑

tochalasin D produced multinucleate cysts following theirfective against this species of Entamoeba as well as against

transfer to encystation medium. Encystation with cytocha‑

E. histolytica strain HM‑1: IMSS as previously demon‑
strated. Encystation of E. invadens as induced in vitro waslasin D was more strongly inhibited among trophozoites
grown in the presence of the drug than among those grown

The effect of cytochalasin D, a specific inhibitor of mi‑

crofilaments, on the growth, encystation, and multinuclea‑

also inhibited by cytochalasin D. This is the first evidence

in the absence of the drug. Also, encystation without cyto‑of the participation of microfilaments in the encystation

chalasin D was less f requently observed among trophozoi‑

process.
Concentrations of cytochalasin D effective for the inhi‑

tes grown in the presence of the drug than among those

bition of encystation were lower than those effective for theof trophozoites induced by cytochalasin D had an inhibitory

inhibition of growth. Trophozoites grown with cytochalasineffect on their encystation.

grown in the absence of the drug. Thus, the multinucleation

B‑10 INHIBITION OF ENCYSTATION OF ENTAMOEBA INVADENS BY
ANTITUBLIN DRUG ORYZALINASAO MAKIOKA MASAHIRO KUMAGAl*, HIROSHI OHTOMO*,SEIKI KOBAYASHl' AND TSUTOMU TAKEUCHI'
Department of Tropical Medicine, Jikei University School of Medicine* andDepartment of Tropical Medicine & Parasitology,
School of Medicine, Keio University2

We have previously demonstrated that the antimicrotu‑

drug, a number of trophozoites survived for at least 3 days.

bule drug oryzalin inhibits the growth of Entamoeba invad‑

In contrast, trophozoites grown in the absence of oryzalin

ens as well as E. histolytica , the former being more resis‑

neither survived nor encysted after their transfer to encysta‑

tant to the drug than the latter. The aim of the present study

tion medium supplemented with the drug, which suggests

was to examine the effect of oryzalin on the encystation ofthat pretreatment of trophozoites with oryzalin contributes

E. invadens using an axenic encystation system in vitro .

to their continued survival as trophozozites, without their

Oryzalin inhibited the encystation of E. invadens strain IP‑ 1

transforming into cysts, in encystation medium. Trophozoi‑

in a dose‑dependent manner. The addition of oryzalin aftertes grown with oryzalin did encyst after their transfer to en‑

the induction of encystation was also inhibitory for encysta‑

cystation medium without the drug.

tion. Trophozoites incubated for I day in encystation me‑

Accumulation of trophozoites in the mitotic phase wasdium with oryzalin did not encyst after removal of the drug.

observed after culture with oryzalin. When cysts prepared

Although trophozoites grown in the presence of 300 uMat day I of encystation, most of which were mononucleate,oryzalin for 2 days did not encyst after their transfer to en‑

were reincubated in the presence of oryzalin for an addi‑
tional 2 days, inhibition of their maturation was observed.

cystation medium containing the same concentration of

51

Thus, oryzalin is a potent mitotic‑phase inhibitor of E. in‑lationship between the cell cycle and encystation of thisvadens and may become a useful tool for studies on the re‑parasite.

B‑11 MOLECULAR CLONING AND CHARACTERIZATION OF PEROXIREDOXIN
FROM ENTAMOEBA MOSHKOVSKIIXUN‑JIA CHENG, YOSHIMASA KANEDA AND HIROSHI TACHIBANA
Department of Infectious Diseases, Tokai University School of Medicine

Recently, the term "peroxiredoxin" has been proposed
for a family of antioxidant enzymes which includes thiore‑

kDa. When the deduced amino acid sequence of E.

doxin peroxidase and alkyl hydroperoxidase. The peroxire‑

histolytica (233 amino acids) via the FastA Program, identi‑

doxin of Entamoeba histolytica seems to be important inprotection from oxidative attack by activated host phago‑ties were 8 1 % on the overlapping 215 amino acids. A dis‑

cytic cells during the ameba's invasion of host tissue and/or

quence of E. moshkovskii peroxiredoxin was shorter thanin protection against its own metabolically produced hydro‑that of the E, histolytica peroxiredoxin.

gen peroxide. The nonpathogenic E. moshkovskii is mor‑
phologically indistinguishable from E. histolytica , but usu‑

The cDNA encoding peroxiredoxin of E. moshkovskii
was subcloned into expression vector pET19b. Recombi‑

moshkovskii peroxiredoxin was compared with that of E.

tinctive difference was that the cysteine‑rich N‑terminus se‑ally is isolated from polluted waters. As a step towards

nant peroxiredoxin was expressed as a fusion protein with aclarifying the biological significance of peroxiredoxin in

histidine tag in Escherichia coli . The recombinant protein

both pathogenic and nonpathogenic Entamoeba , we report
here the molecular cloning and characterization of E.
moshkovskii peroxiredoxin.

was purified by affinity chromatography and then examined

Trophozoites of E. moshkovskii Laredo were axeni‑
cally cultured in BI‑S‑33 medium. A cDNA Iibrary wasconstructed from poly (A) RNA of E. moshkovskii and thenscreened with a 352 bp probe prepared from genomic DNA

removal of H202, in the presence of dithiothreitol, in aconcentration‑dependent manner. Its activity was compara‑by polyclonal antibodies, prepared against the recombinantby PCR amplification using primers pl and p3 for E. histo‑

peroxiredoxin of E. moshkovskii, Iocalized the protein inlytica . The clone containing the longest insert was sub‑the nucleus and cytoplasm of trophozoites. The E.

cloned into a pUC19 vector and then sequenced. The open
reading frame of a cloned CDNA encoded a polypeptide of
217 amino acids with a calculated molecular mass of 24

moshkovskii peroxiredoxin seems to function in protection

B ‑ 12for its ability to remove exogenously added H20, in an in vi‑

tro assay. The peroxiredoxin was capable of catalyzing theble with that of E. histolytica . Immunofluorescent staining

against its own metabolically produced H,02.

MODIFICATIONS OF STAINING PROCEDURES FOR THE PREPARATIONS OF
ENTAMOEBA HISTOLYTICA
MASAHIRO KUMAGAI*, SEIKI KOBAYASHI', TOMOMI OKITA*

AND HIROSHI OHTOMO*
Department of Tropical Medicine, Jikei University School of Medicinel andDepartment of Tropical Medicine & Parasitology,
School of Medicine, Keio University2

Preparation of stained smears of Entamoeba histolytica

has several drawbacks. The iron hematoxylin staining andthe trichrome staining, which have been most widely used,

for example, employ highly toxic mercuric chloride. We
therefore tried to develop simple staining procedures bymodifying Kohn's chlorazol black E staining and the52trichrome staining. Amoebae fixed in suspension withnot be stained with these stains after fixation in the basic so‑either the basic solution of Kohn's staining or Bouin's f"Ixa‑

lution, but were stained after Bouin's fixation. Further thetive were clearly stained with Kohn's stain and thestaining can be combined with a cyst‑concentration tech‑trichrome stain, and can be examined as wet mounts.

nique, which uses the basic solution (instead of formalin)

Permanent preparations were made by processing for
mounting. Amoebae in fecal smears fixed in these fixatives
were also stained with these stains. These procedures wereeasier when the basic solution and trichrome stain wereused. Erythrocyes ingested by trophozoites, however, canB ‑ 13and ether. Consequently, without highly toxic mercuric
chloride, wet mounts and permanent preparations can bemade using permanent stains and preserved cysts can bestained af ter concentration.

EPIDEMIOLOGICAL STUDY OF AN EPIDEMIC AMEBIASISIN INSTITUTIONS IN JAPANTSUTOMU TAKEUCHI', M. SHIMIZU'. SEIKI KOBAYASHll,

TOMOYA SAITOI AND TAKUYA MAEDA*
Department of Tropical Medicine & Parasitology,
School of Medicine, Keio Universityl andDepartment of Parasitology, Kawasaki Medical Scho012(Abstract not received on time)

B‑14 CONGENITAL TOXOPLASMA INFECTION AT PLACENTA RESULTING IN
INTRAUTERIN GROWTH RETARDATIONAKIHIKO YANO*, N. KUBOTA , TOKUZO KASA12, MAMI NAKAYASHIR03,
KAZUMI NOROSEl, HIDEKAZU HATA', MASASHI KOBAYASH11 AND FUMIE AOSAI]
Department of Parasitology, Chiba University, School of Medicinel,

Department of Obstetrics and Gynecology, Chiba Kaihin Hospital' andDepartment of Pediatrics, Chubu Hospital3

and its importance as a public health concern are now rec‑centa were observed, and the infection of T gondii was
confirmed with pathologic and immuno‑pathologic diagno‑
sis together with PCR diagnosis. No evidence of T gondii
infection in the newborn, however, was obtained by sero‑

ognized by many scientists and physicians in European

10gical and chinical diagnosis. Thus, we propose a new

countries, USA, and more recently in Japan. Early diagno‑

classification of congenital toxoplasmosis by adding a piv‑

sis by seroconversion and clinical diagnosis of fetuses fol‑otal concept of "congenital toxoplasmosis at placenta" inlowed by early chemotherapy have been shown to improve

which only IUGR of fetuses occurred without T. gondii‑

the prognosis.

fetal infection in utero. Further validation of the up‑dated

Fetal infections by Toxoplasma gondii can result in
neuological sequelae, congenital malformations, and oculardisorders. The severity of human congenital toxoplasmosis

We report a case of IUGR (intrauterin growth retarda‑

techniques for measuring fetal growth will undoubtedly en‑tion) delivered from the mother infected by T gondii during

hance the ability to diagnose congenital toxoplasmosis pre‑her pregnancy. Malformation and hypofunction of the pla‑

natal ly.

53

B‑15 INTRACTABLE RECURRENT TOXOPLASMIC RETINOCHOROIDITISKAZUMI NOROSE , FUMIE AOSA12, HIDEKAZU HATA*, MASASHI KOBAYASHI*,MASAAKI SHIMADA' AND AKIHIKO YANO*
Department of Parasitology, Chiba University, School of Medicinel andInformation and Reference Center,
Institute of Tropical Medicine, Nagasaki University2

Toxoplasmic retinochoroiditis is the most common

while the patient was still receiving clindamycin. The vitre‑

manifestation of congenital Toxoplasma gondii infection.

ous opacity and retinal edema increased. Although the dose

Many issues remain unsolved regarding the mechanism ofof corticosteroid was increased again, he is not under medi‑

the recurrence and the treatment of ocular toxoplasmosis.

cal control.

Here we report two cases of retinochoroiditis caused byCase 2: This 7‑month‑old boy visited out patient clinic

due to a esotropia. The patient had focal chorioretinal le‑congenital toxoplasmosis.
Case I : This 14‑year‑old boy visited out‑patient clinic

sion with pigmentation at the both macular areas. The latex

due to a decreased and blurred vision in his right eye. He

aggulutination titer was positive (1 : 10,240) for toxoplasmo‑

was born in Senegal. He was apt to have fever and skin ab‑

sis. Computed tomographic scan showed an intracerebral

scesses in the upper part of the body in his infancy andchildhood. He came back to Japan when he was 3 years old.
He was pointed out that his visual acuity was poor when hecalcification. The patient was treated with acetilspiramy‑cine. His father, mother, and elder sister were healthy andhad no abnormal findings in their eyes, although the latex

was 6 years old. Inactive multiple chorioretinal scars sug‑

aggulutination titers were all positive (1:10,240) for

gestive of toxoplasmosis were present in both maculas, in‑toxoplasmosis. He was clinically free of evidence of recur‑

volving the left fovea but not the night. The latex agguluti‑

rent disease throughout the 1 1 years follow up.

nation titer was positive (1:10,240) for toxoplasmosis. In

The disease recurred repeatedly at long or short irregu‑

April 2000, when he was first examined, preretinal hemor‑rhages and retinal edema were present in the right eye. He

larly spaced intervals in spite of the treatment. Medical

was treated with clindamycin and corticosteroid. During

We discussed with regard to current policies in treatment.

treatment of toxoplasmosis may not be effective always.

the tapering off the corticosteroid, active infection recured

B‑16 FOUR CASES OF CRYPTOSPORIDIOSISTSUNEZO SHIOTA AND NAOKI ARIZONO
Department of Medical Zoology, Kyoto Prefectural University of Medicine

The signs and symptoms of cryptosporidiosis causedby Cryptosporidium parvum (Cp) can easily be confusedarrhea usually subsides within a 2 to 3 week period of time.
Therefire, it may be that the timings of natural recovery andwith other intestinal disturbances. In this report we de‑scribed the clinical course of cryptosporidiosis in 4 Japa‑

treatment were the same.

nese with severe diarrhea.

to Kenya was referred to our institute because of high fever

Case 2: A 25‑year‑old Japanese male who had traveled

Case I : A 24‑year‑old Japanese male who had traveledand severe diarrhea. Plasmodium falciparum was detected

to India was referred to our institute because of severe pro‑in his blood smears and also Cp oocysts in his stool speci‑

longed watery diarrhea. Cp oocysts (3.5 X 105/g) were de‑mens. He was treated with pyrimethamine/sulfadoxine andtected in his stool. Stool examinations were negative forquinine and he soon recovered from malaria as well as fromother intestinal protozoa. Bacterial cultures were also nega‑

cryptosporidiosis.

tive for pathogenic agents. He was treated with sul‑

Case 3: A 27‑year‑old Japanese female who had trav‑

famethoxazole/trimethoprim for 6 days and he soon recov‑
ered. In immunocompetent individuals cryptosporidial di‑eled to Malaysia was referred to our institute because of se‑vere prolonged watery diarrhea. Cp oocysts and Blastocys‑

54

tis hominis were detected in her stool specimens. No treat‑ment was done and her symptoms subsided spontaneously

cheal specimens. He was treated with acetylspiramycin
against Cp, ganciclovir against CMV and pentamidine

after 13 days‑prolonged diarrhea.

isethionate against Pc, but diarrhea continued and respira‑Case 4: A 44‑year‑old Japanese male was referred toour institute because of severe 5‑months‑prolonged waterydiarrhea which resulted in a 20 kg body‑weight loss. Labo‑

tory failure appeared. Then he died one month after his
condition was diagnosed. The signs and symptoms of cryp‑
tosporidiosis can be easily mistaken for other intestinal

ratory data showed positive for anti‑HIV antibodies, CD41

disturbances ‑i.e., infections caused by other protozoas, bac‑

CD8=0.009 and 100 CD4T cell count/u1 in his peripheral
blood. Cp oocysts were detected in his stool specimens.

teria, viruses or food poisoning. Physicians must include

CMV showed positive in his large intestinal biopsy speci‑

ining patients with severe diarrhea of unknown origin.

cryptosporidiosis in their differential diagnosis when exam‑mens. Pneumocystis carinii (Pc) were detected in his tra‑B ‑ 17

SERO‑EPIDEMIOLOGICAL SURVEY OF CRYPTOSPORIDIOSIS AT
SUMBAWA ISLAND, INDONESIATETSUO YANAGll, MICHIKO FUKAHOR11 , HISAE YAMAGUCHI] , MACHFUDZ',BARIA L. SUTANT02, YOES P. DACHLAN2 AND HIROJI KANBARAl
Department of Protozoology, Institute of Tropical Medicine,

Nagasaki Universityl andTropical Disease Center, Airlanga University, Indonesia2

We carried out the serological test to detect antibodies

found when the positive rates of different class of immuno‑

against Cryptosporidium parvum in sera of people in threeglobulin were compared between villages. For example, thevillages of Sumbawa Island. Serum samples were collectedfrom villagers selected by random sampling, i.e., 68 frompositive rate of lgG in Labanka, Penyaring and Stowe Blangdesa Labanka IV, 1 10 from desa Penyaring and 107 from

O%, 19.1% and 28.0( 0; that of lgA was 13.20/0, 35.5% anddesa Stowe Berang. Indirect IFA was used for antibody de‑

1 1 .2%. In order to understand the meaning of these differ‑

was 35.3%, 3 1 .8% and 28.0% respectively; that of lgM wastection. Oocysts obtained from SCID mice infected with C.

ences, we carried out the fecal examination in three villages,

parvum were used as antigens. Each serum was diluted

but we could not find out any indicative result, although thewith PBS at 10% and used for the test. IgG. IgM and lgAantibodies were examined separately. The rate of people

transmission mode of Cryptosporidium has been shown to

who had antibodies at least in one class of immunoglobulin

the present report, we discussed plausible reasons for thewas 42.6% in Labanka, 52.3% in Stowe Berang and 57.3%
in Penyaring. The very characteristic differences weredifferences, considering social, geographical, meteorologi‑

be completely different from other intestinal parasites. In

cal and environmental conditions.

55

B‑18 GENETIC CHARACTERIZATION OF CRYPTOSPORIDIUM PARVUM ISOLATESFROM 14 CATTLE AND 22 PATIENTS WITH DIARRHEAKENJI YAGITA SINJI IZUMIYAMA YUJI TACHIBANA*, GOHTA MASUDA+,
MOTOHIRO IZEKI*, KOJI FURUYA', TOSHIO KUROKI',ICHIRO FURUKAWA' AND TAKURO ENDO*
National Institute of Infectious Diseasesl

lkeda Schience C0.2, Tokai University School of Medicine3,

Tokyo Metropolitan Komagome Hospital4, Osaka City University Medical Scho015,
Hokkaido Institute of Public Health6 andKanagawa Institute of Public Health7

Polymerase Chain Reaction combined with Rsa I Re‑

tients linked to the first waterborne outbreak in Japan instriction Fragment Length Polymorphism (PCR‑RFLP)

September 1994 were of genotype 2, and those linked to theanalysis of a threonine‑rich open reading frame gene fromsecond waterborne outbreak in June 1996, were of genotype

Cryptosporidium parvum was applied to 36 specimens col‑1 . Among isolates from sporadic cases, 2 were of genotype

lected from patients with cryptosporidiosis associated with

2, and 10 were of genotype I . Specimens from 3 patients

two waterborne outbreaks, sporadic human cases and live‑
stock (neonatal calves) from other areas. The sizes of thewith sporadic cryptosporidiosis who were all immunologi‑

fragment amplified with the primer pair (Sense: 5'‑fragment amplified was 521 bp spanning 2 Rsa I sites, and

CTCTTAATCCATCATTACAAC‑3 ' ;Anti‑sense : 5 ' ‑was digested into three fragments (55, 190 and 276 bp).

AGCAGCAAGATATGATACCG‑3') for C. parvum anthro‑

One specimen from a patient with a sporadic case yielded

ponotic genotype I is 521 bp, and for C. parvum zoonotic
genotype 2 is 5 1 8 bp. The genotype I fragment contains 2

both genotype I and the new genotype. The latter two

Rsa I restriction sites, and is digested into three DNA frag‑that there is a strong preference for one host. These find‑ments (55, 62 and 404 bp). The genotype 2 fragment con‑ings strengthen the concept of C. parvum genetically clus‑

cally healthy adults yielded a new genotype. The size of the

genotypes failed to visibly infect nude mice, demonstrating

tains 3 Rsa I sites, and is digested into four DNA fragments

tering into subgroups that correlate with infectivity, trans‑(55, 62, 128 and 273 bp). Fourteen isolates from neonatal

mission of infection to humans, and with the resulting clini‑

calves were exclusively of genotype 2. Isolates from pa‑

cal manifestations.

B‑19 COMPLEX 11 (SUCCINATE‑UBIQUINONE REDUCTASE/QUINOL‑FUMARATE
REDUCTASE) OF TRYPANOSOMA CRUZI MITOCHONDRIAEIZO TAKASHIMA1, SHIGERU MJNEK12, HIROKO HIRAWAKE*, HIROKO MIYADERA*,JUNKO SHIMADA*, TAKESHI NARA*, TAKASHI AOK13 AND KIYOSHI KITA'
Depertment of Biochemical Chemistry, Graduate School of Medicine,
The University of Tokyol ,Department of Applied Biological Schience, Science University of Toky02 andDepartment of Parasitology, Juntendo University, School of Medicine3

In the present study, protocol to isolate active mito‑

brane where it plays a role in mammalian type mitochon‑chondria from epimastigote of T cruzi was established anddria as a component of the TCA cycle and electron transport

succinate‑ubiquinone reductase (SQR) was partially puri‑chain. In addition, some organisms living in anaerobic con‑fied. Furthermore, a CDNA for Fp subunit, which contains
binding sites of flavin and substrates of T cruzi SQR was

dition use complex 11 as quinol‑fumarate reductase (QFR),

cloned and characterized.

plex 11 in the T cruzi mitochondria remains unknown, al‑though several reports suggested the presence of fumarate

Complex 11 is located in the inner mitochondrial mem‑

which catalyzes reverse reaction of SQR. The role of com‑

56

reductase (FRD) which is a part of QFR activity in the mi‑

2,035 bp has a 35‑bp spliced leader sequence and the ORF

tochondria.

encodes 609 amino acids. Amino acid sequences around
FAD binding and substrate binding regions were highly

Mitochondria were isolated from cultured T cruzi epi‑
mastigote by using glass‑beads treatment and following dif‑conserved. Southern blotting analysis showed that this gene

ferential centrifugations. Then complex 11 was solubilized

from the mitochondria with sucrose monolaurate and sepa‑

is single copy in the T cruzi genome. Northern blotting
analysis revealed that T cruzi Fp mRNA contains long 3'‑

rated by DEAE‑cellulofine in the presence of the detergent.

noncording region. Amino acid sequence of T cruzi Fp

SDH activity was eluted from the column as a single peak

showed higher homology to that of eukaryote rather thanthat of the bacterial QFR. Amino acid composition of Fp
peptide of purified preparation determined by MS analysis
was correlated well with that deduced from the CDNA se‑with FRD activity. Immunoblotting showed a 70 kDa band
was recognized with the anti‑bovine Fp antiserum suggest‑
ing that this protein band is Fp subunit of T cruzi.

Then, a CDNA for Fp subunit of the enzyme has beenquence.

cloned by homology proving and RACE. The CDNA with

B‑20 CHARACTERIZATION OF CYANIDE INSENSITIVE OXIDASE OFTRYPANOSOMA BRUCEI BRUCEI EXPRESSED IN ESCHERICHIA COLIKEISUKE KAWAI*, KOICHI NIHEI , YOSHIHISA FUKAI* , YOSHISADA YABU',NOBUKO MINAGAWA*, AKIO YOSHIMOTO*, KAZUO NAGAI+ AND KIYOSHI KITA*
Department of Biochemical Chemistry, Graduate School of Medicine,
The University of Tokyol,
Department of Medical Zoology, Nagoya City University School of Medicine2,
Department of Biochemistry, Niigata College of Pharmacy3 andDepartment of Bioengineering, Tokyo Institute of Technology4

African trypanosome is a parasite, which causes Afri‑against trypanosomes. It is also reported that TAO is highly

can sleeping sickness of human and nagana disease of cat‑homologous to alternative oxidase of plant mitochondria,

tle. Because, at present, any drug against trypanosomes has

but biochemical analysis has not been carried out due to thestrong side effect and is not effective for chronic patients, adifficulty of purification. Therefore, we have been trying to

new effective drug is awaited. We found ascofuranone iso‑lated from Ascochyta visiae inhibits specifically mitochon‑

TAO. Our final goal is clinical development of ascofura‑drial respiration of blood stream form of Trypanosoma

none .

overexpress a recombinant TAO in E. coli to characterize

brucei brucei at very low concentration. In addition, we

We constructed FN102/pTAO by transformation offound that the parasite in the blood disappeared quickly by

CDNA for TAO into heme‑deficient E. coli . In this strain,

the treatment with ascofuranone in combination with glyc‑

because of the deficiency of heme, cytochrome bo complex

and cytochrome bd complex of E. coli don't show quinol

erol .

Experimental evidences showed that a target of ascofu‑

oxidase activity, and we can analyze solely quinol oxidaseranone is cyanide insensitive terminal oxidase localizeded

activity of rTAO. Then, we established the large scale cul‑in mitochondrion of blood stream form of T b. brucei. This

ture system on which about 80% of membrane protein is

enzyme is referred to as TAO (trypanosome alternative oxi‑rTAO. Kinetic analysis of rTAO in this membrane indicateddase) and, unlike cytochrome c oxidase of mammalian mi‑
tochondria, functions as quinol oxidase. Because mammals

that ascofuranone inhibits quinol oxidase activity of TAO
competitively with quinol, suggesting that aromatic struc‑

don't have TAO that is essential for re‑oxidation of reducing

ture of ascofuranone is recognized by quinol binding site ofequivalent generated during glycolysis in the glycosome,
TAO is expected as a candidate of target for chemotherapy

the enzyme.

57

B‑21 EXPRESSION AND PROPERTIES OF DIHYDROOROTATE DEHYDROGENASE
IN TRYPANSOMA CRUZITAKESHI NARA, MASAO ODAKA AND TAKASHI AOKI
Department of Parasitology, Juntendo University School of Medicine

Dihydroorotate dehydrogenase (DHOD, E.C.1.3.3.1)

expressed in Escherichia coli and the enzymatic properties

is the fourth enzyme of the denovo pyrimidine biosynthetic

were analyzed. The recombinant DHOD tagged with hexa‑

pathway and thought to be one of possible targets for cancer

histidine at the N‑terminal was recovered in the soluble

and infectious diseases. We have previously reported apyrimidine‑biosynthetic gene cluster that contains fivegenes encoding all six enzymes, including DHOD, of thepyrimidine biosynthetic pathway in Trypanosoma cruzi .

fraction and highly purified by using the affinity column forPhylogenetic relationships among DHOD sequences were

marate as an electron acceptor and inhibited by a final prod‑shown to be related to their localization and catalytic prop‑

uct, orotate. The kinetic values for dihydroorotate and fu‑

erties in the cell. The T cruzi DHOD is classified to the cy‑

marate were 0.01 mM, 0.11 mM, respectively. These re‑the tagged sequence. The T cruzi DHOD displayed thespecific absorbance at 368 nm and 455 nm, characteristic toa flavoprotein. The DHOD activity was dependent to fu‑

tosolic group utilizing fumarate as an electron acceptor,

sults indicate that the T cruzi DHOD provides the distinct

whereas the animal DHODS are of membrane‑bound utiliz‑

character from that of mammalians, suggesting the possible

ing oxygen via respiratory chain. In order to characterizetarget of chemotherapy.

the T cruzi DHOD, the recombinant T cruzi DHOD was

B‑22 IN BALB/C MICE, IMMUNE RESPONSES AND THE COURSE OF LEISHMANIA MAJOR
INFECTION WERE CONTROLLED BY CYTOKINE EXPRESSION PLASMIDS
DELIVERY WITH THE GENE GUNYANG LI, HIROYUKI ISHIKAWA, TOHRU SAKAI AND KUNISUKE HIMENO
Department of Parasitology and Immunology,
School of Medicine, The University of Tokushima

DNA vaccination has been shown to elicit a long‑

treated with IL‑ 12 gene developed slighter footpad swelling

lasting immune response in a variety experimental models.

and parasite burden than did control mice. On day 42

The concept of DNA immunization has been applied re‑postinfection, mice treated with IL‑12 gene were capable ofcently to gene therapy using vector constructs that encode

generating more IFN‑Y as well as L. major‑specific lgG2atiters than did control mice, while that of IL‑4 and lgG1

cytokine gene. The particle‑mediated method for gene de‑livery with a gene gun utilizes a shock wave to accelerate

was decreased. In contrast, treatment with IL‑4 plasmid

DNA‑coated gold particles into target cells or tissues. ThisDNA exacerbated the disease. The results demonstrate thatgene delivery method is effective in various somatic tissues

gene gun‑based IL‑ 12 plasmid DNA treatment can skew thein vitro and in vivo . In this report, we investigated the ef‑

fect of cytokine gene treatment to Thl/Th2 response and theimmune response in a Thl direction in L. major infected
BALB/C mice, while IL‑4 DNA treatment may promote Th2

course of Leishmania major infection. BALB/C mice were

response. Further studies are in progress to determine theinfected with L. major subcutaneously into hind footpad.

mechanisms of gene gun‑based cytokine plasmid DNA

Simultaneously, they were begun to be treated with 4 ug oftreatment.

IL‑ 12 or IL‑4 plasmid DNA by gene gun weekly. Mice

58

B‑23 TH2 CYTOKINES PLAY DIFFERENT ROLES DURING THE COURSE OF INFECTION
WITH LEISHMA NIA MAJOR
BAHER F. NASHED, YOICHI MAEKAWA, TIANQIAN ZHANG AND KUNISUKE HIMENO
Department of Parasitology and Immunology,
School of Medicine, The University of Tokushima

Leishmania spp. are dimorphic obligate intracellularfect and so forth. Incomplete Freund's adjuvant (IFA)

parasites. Flagellated promastigotes replicate and differen‑which is one of the oil adjuvants was used to investigate Th‑

tiate in the gut of the sandfly vector and are transmitted to adifferentiation in experimental cutaneous leishmaniasis andvertebrate when the sandfly takes a blood meal. Survival of

to study the effector roles of the different Th cytokines (IL‑the parasite within a vertebrate host is dependent on the
successful entry into a macrophage and transformation into

4, IL‑5, IL‑13, IL‑10, IL‑9, and IFN‑Y) in this immune de‑the amastigote form. They cause a wide range of infectious

tigen (SLA) in IFA (IFA/SLA) can make the resistantprocesses from asymptomatic to cutaneous, mucosal or vis‑
ceral manifestations, depending on the species of the para‑mouse strain (DBA/2) susceptible to L. major infection
with shifting the immune response from Thl to Th2. This

site and the type of immune response induced. The immune

could be attributed to the modulation of the cytokine patternviation. Twice immunization using soluble leishmanial an‑

responses and clinical outcome in leishmanial infections arewith marked Th2 cytokine up‑regulation by this immuniza‑

dependent in part on the patterns of cytokines produced.

tion scheme. IL‑4 and IL‑13 are considered to be the mostExperimental cutaneous leishmaniasis is a useful model

important susceptibility factors in mounting the disease‑in studying the mechanism regulating immune responses
between Thl and Th2. Mice susceptible to Leishmania ma‑

promoting Th2 response in mice susceptible to L. major in‑
fection. The production of these cytokines is sustained in

jor infection such as BALB/C are associated with the induc‑susceptible mice until the chronic phase of the infection.

tion of the disease‑promoting Th2 response, while the resis‑

While the levels of these two cytokines decrease markedly

tant mice such as DBA/2 develop the protective Thl re‑in the resistant strains of mice. However, it is difficult tosponse. Genetically determined resistance and susceptibil‑
ity are clearly related to the development of polarized CD4'

explain the mechanism that establishes Th2 response with
only these two Th2 cytokines, because we could not detect

Thl and Th2 responses, respectively. Cytokines are thesignificant difference in the production of IL‑4 and IL‑ 1 3

most important stimulus in influencing the development offunctionally polarized CD4' Th effector responses.

early after the infection between the healer and the non‑Adjuvants are known to enhance the immune re‑pear to have a significant role in determining the course ofsponses through the combined effect of several factors: pro‑healer groups of mice. On the contrary, IL‑5 and IL‑ 10 ap‑
the disease early after the infection.

10nged release of antigen, migration of cells, mitogenic ef‑

B ‑ 24

THE EFFECTS OF MEGLUMINE ANTIMONIATE (GLUCANTIMER) AGAINST
L. MAJOR AND L. AMAZONESIS CO‑CULTURED WITH MACROPHAGESKHAN MOHAMMED ABUL KASEM*, MOTOYOSHI MARUNO*, NOOR MOHAMMED KHASKHELY',SHIGEO NONAKA*, YOSHIHISA HASHIGUCHl' AND EDURADO A. GOMEZ LANDIRES3
Department of Dermatology, Faculty of Medicine, University of the Ryukyusl,

Department of Parasitology, Kochi Medical Scho012 andDepartment of Tropical Medicine, Catholic University Guayaquil,

Guayaquil, Ecuador3

Meglumine antimoniate (MA) has been widely used

However, no report has been found about how MA affects

for leishmaniasis. Anti‑1eishmanial effect of MA has beenstudied on the point antipromastigote activity till date.

the parasite‑intake of macrophage. In this study, we exam‑
ined the parasite activity co‑cultured with macrophage un‑

59

der several concentrations of MA.

Leishmania promastigotes co‑cultured with macrophages

Promastigotes of Leishmania major and L. amazonesis,
were treated at the concentration ranged from O%, 0.0001%

the reduction in number of promastigotes. The observation(0.085 ug/ml), 0.01% (8.5 ug/ml) and 1% (850 ug/ml) of
MA. Simultaneously, macrophage (J774) co‑culture wasincubated with the both strains of Leishmania parasites andand treated with O% to I % concentrations of MA, revealedof Giemsa stain, disclosed that macrophages treated withMA, contained many promastigotes outside and a few num‑
ber of amastigotes inside but macrophages treated without

also exposed to drug as same dosages. The number of para‑MA, showed the opposite results. The promastigotes ofsites was counted by hemocytometer after 48 hr of incuba‑tion, stained by neutral red and Giemsa staining. Electron‑

Leishmania parasites treated with O% to I % concentrations

microscopic study was also performed to investigate the ac‑them in neutral red stain. Electron‑microscopic study re‑tivity of MA against amastigotes, formed inside of macro‑vealed that L. major infected macrophages treated with MA

phages. For which, infection of macrophages with L. majorcontained less amastigotes as compared to cells treatedpromastigotes was done prior to administration of MA atwithout MA.
Our data suggests that MA seems to have a weakconcentration of 20 mg/ml .

of MA, showed no significant difference of staining within

The number of promastigotes of L. major and L. ama‑

leishmaniacidal effects. As an anti‑leishmanial effects, MA

zonesis treated without MA, was proliferated but the reduc‑inhibit the proliferation of promastigotes only but also maytion in number of promastigotes was observed treated withinterfere the promastigotes entry into the macrophages.

1 % to 0.0001% concentrations of MA. Both strains of

B‑25 LEISHMANIASIS IN EQUADOR, WITH SPECIAL REFERENCE TO
MUCOCUTANEOUS FORMS AND MAN‑BITING SAND FLIES, LUTZOMYIA SPP.
IN THE AMAZONIAN ENDEMIC AREAS
YOSHIHISA HASHIGUCHI' , TATSUYUKI MIMOR12, EDUARDO A. GOMEZ L.3,
MANUEL CARVOPINA H.+, ANGEL GUEVARRA E.5, RODORIGO ARMIJOS4 AND

SHIGEO NONAKA6
Department of Parasitology, Kochi Medical Schooll,

Department of Tumor Genetics and Biology,

Kumamoto University School of Medicine2,
Department of Tropical Medicine3 andInstituto de Tropical Molecular Biology5, Catholic University, Equador,

Instituto de Immunology, Quito Central University4 andDepartment of Dermatology, Faculty of Medicine, University of the Ryukyus5

To disclose the epidemiological features of leishmania‑

and reservoirs, 4 species of the genus Lutzomyia , Lu. ta‑

sis in Ecuador, intensive field research and laboratory works

rapidoi, Lu. hartmann, Lu. gomezi and Lu. ayacuchensis,

have been done from 1 982 to date. During the period, al‑

and 8 species of mammals, anteaters, 2 spp. of sloths, 2 spp.

most all of the suspectable areas endemic for the disease inof squirrels, kinkajous, rats and dogs, were incriminated atthat country were surveyed, by performing investigations

different endemic areas of Ecuador, respectively. The datafor patients, vector sand flies, and reservoir animals. Nine

mentioned above, however, were mainly obtained from theprovinces of Ecuador are located in the Pacific regions; 2, inendemic areas of lowlands and Andean slopes of Pacific re‑the mid‑Andes; and 4, in the Amazonian regions. To date, 7

gions, and Andean plateau of the country, but not from thespecies of the genus Leishmania , L. (L.) mexicana , L. (L.)

Amazonian regions. In the present paper, we mentioned theamazonensis, L. (L.) major‑like, L. (V) braziliensis , L. (V)data from the Amazonian endemic areas, Loreto, Napo, by

guyanensis, L. (L.) panamensis , and L. (V) equatorensis(the last species may be belonging to the genus Endotrypa‑

performing epidemiological surveys including the examina‑num), were identified based on their zymodeme, serodeme,
schizodeme and karyodeme analysis. Regarding to vectors

flies. The results obtained revealed that there were consid‑

tions of patients and the collections and dissections of sanderable numbers of patients with mucocutaneous leishmania‑

60

lection using protected human baits, 43 in total belonging tocollected at the Amazonian leishmaniasis‑endemic areas,
only the last species, Lu. gomezi was already incriminated

7 species of the genus Lutzomyia were collected, and dis‑

as a probable vector of the disease at the Pacific lowlands

sected, but none of them were positive for Leishmania pro‑and Andean slopes of Ecuador. Further studies are required

mastigotes; the man‑biting flies were Lu. tortura , Lu. carre‑

in order to disclose the epidemiological and vector entomo‑

rai thula, Lu. flaviscutellata, Lu. olmeca bicolor, Lu. geni‑

logical features of the disease in the Amazonian regions.

sis in addition to those with cutaneous one. In the fly col‑culata , Lu. yuilli yuilli, and Lu. gomezi. Among these flies

B ‑26 PULMONARY INFECTION CAUSED BY RHODOCOCCUS EQUIIN HIV‑INFECTED PATIENTS: REPORT OF SIX PATIENTS FROM NORTHERN THAILAND
HIROSHI WATANABE*, SHINOBU KOBAYASHI', KIWAO WATANABE*, KAZUNORI OISHI',WEERAYUT KOSITSAKULCHAI', TIPPAYA SANCHAI', KHEMRASSAMEE KUNSUIKMENGRA12,
SUMPUN KAHINTAPONG', BANYONG KHANTAWA*, PRASIT THARAVICHITKUL*,
THIRA SIRISANTHANA' AND TSUYOSHI NAGATAKE
Department of Internal Medicine, Institute of Tropical Medicine,
Nagasaki Universityl ,Nakornping Hospital2 and Faculty of Medicine, Chiang Mai University3

We report six human immunodeficiency virus (HIV)The mean CD4 Iymphocyte count in 5 of tested patients

infected patients (5 males and one female, average age, 32.2

was 8/mm* (CD41CD8 rati0=0.05). Eight isolates of R. equiyears) with pulmonary infection (four with pneumonia andpatients who presented with fever and productive coughin 6 patients were sensitive to imipenem, minocycline,
erythromycin, clarithromycin vancomycin and teicoplanin(minimum inhibitory concentration, MICs I ug/ml ), butwere admitted to Nakornping Hospital in northern Thailand.

resistant to most P‑lactam antibiotics. Four isolates were

Chest roentgenograms showed pulmonary infiltration
and/or cavitary lesions. Their conditions were poor because

sensitive (MICs I ug/ml) and four resistant (MICs 64
ug/ml ) to rifampicin. Three patients were treated withof severe anemia, and transfusion was necessary in 5 of 6erythromycin+rifampicin while the other two were treatedpatients. The etiologic microorganisms identified in sputum
smears were gram‑positive and acid‑fast coccobacilli before

with anti‑tuberculous drugs and penicillinG+gentamicin.
However, three patients subsequently died because of respi‑

culture results were available. Only one of the six cases

ratory failure due to pulmonary infection caused by R, equi.

two with lung abscess) caused by Rhodococcus equi. These

was a mixed‑infection by R. equi and Salmonella enteritidis.

B ‑ 27

INFLUENCE OF HIV INFECTION ON COMMUNITY‑ACQUIRED PNEUMONlA
IN ADULTS IN UGANDAHIROYUKI YOSHIMINE*, KAZUNORI OISHI*, MASAYUKI ISHIDA*, KIWAO WATANABE*,TSUYOSHI NAGATAKE*, HAWA NALWOGA', PHILIPOMBASII' AND ROY MUGERWA'
Department of Internal Medicine, Institute of Tropical Medicine,
Nagasaki University I and

Department of Internal Medicine, Makerere University2

Hospital‑based prospective study on 48 patients with

community‑acquired pneumonia (CAP) has been carriedout in Kampala, Uganda from September 1999 to June

2000. We evaluated the bacterial etiologies employing
quantitative sputum culture and Toluidin blue‑O (TBO)
staing, clinical features, and prevalence of rerurrent pneu‑

61

monia. We found high prevalence (81.2%) of HIV‑1 infec‑
tion among enrolled patients. Mean CD4 positive lympho‑

thirty‑three ( 15%) patients infected with HIV‑ I had recur‑

cyte counts of patietns infected and uninfected with HIV‑ 1

these patients decreased from 1 32.0/u1 at the first episoderent pneumonia. Mean CD4 positive lymphocyte counts of

was 191.4/u1 and 509.5/u1 , respectively. Major bacterial

to 61 .O/u1 at the second episode. The mean period between

pathogen were Streptococcus pneumoniae (11 strains),

the first and the second episode was 8 1 .6 days. All of fiveBranhamella catarrhalis (9 cases), and Haemophilus injlu‑

cases had different bacterial pathogens in each case. Recur‑

enzae (8 strains). One patient infected with HIV‑ I had arent bacterial infection may progress the decline of cellular

typical clinical features of Pneumocystis carinii pneumonia.

immunity in each individual .

He was successfully treated with co‑trimoxazole. Five of

B ‑ 28

GENETIC SUSCEPTIBILITY TO RE‑INFECTION OF
SCHISTOSOMA JAPONICUM IN CHINAMIHOKO KIKUCHI*, LIU JIANXIANG', HE NA2, JIANGHONG YAN1,ZHAO QlNGHA13, YUANG HONG‑CHANG2, LIN TANG‑TANG2, SHOJI ZHANG3 AND
KENJI HIRAYAMA*
Department of Medical Zoology, Saitama Medical Schooll,
Department of Epidemiology, School of Public Health,Shanghai Medical University2 andJiangxi Provincial Institute of Parasitic Disease, Nan‑chang3, PRC Chma

Previous field studies showed that resistance to re‑infection of S. mansoni or S. haematobium is positively as‑

sons could get a infection regardless of resistance. There‑

fore we excluded them from our study. Thirty six persons

the balance between the levels of effective anti‑adult worrn

were re‑infected and 50 were not infected. There was noage difference between them. The serum levels of immuno‑lgE and of blocking lgG4 antibodies. In 1995, we haveglobulines were positively correlated to intensity of infec‑

done a re‑infection study of S. japonicum in Zhuxi Villagetion, but that looked no relation to resistant to infection. No

near Poyang Lake, Jiangxi Province, where schistosomiasis

HLA‑DRB I allele was significantly associated to resistant

had been highly endemic (around 40%). In March, the rateor susceptible group. However, DRB I * 1 101 and DRB I *sociated with age, and that the resistance is determined byand intensity of infection was quantified by modified Kato‑

1501, that were associated with protection and susceptibil‑

Katz methods, then all subjects (n=100) received praziquan‑

ity to Shistosomal fibrosis (Hirayama et al ., 1999), Iooked

tel treatment. After the confirmation of 100% cure rate, theprotective and susceptible to re‑infection in Zhuxi, respec‑

water contact investigation by the questionnaire method wastively. Taken together, T cell immunity that is controlled bycarried out during the transmission season. Then, they were

host immune‑response genes (HLA‑ClasS II) seemed to be

re‑examined for their fecal eggs in December. The subjects

more important than B cell in the protective immunity to S.

whose water contact index were more than 20, were 94010

japonicum infection.

re‑infected and we suspected that those highly exposed per‑62

B‑29 A POPULATION STUDY OF THE INTERMEDIATE HOST OF SCHISTOSOMA MANSONIAND THE APPLICATION OF REMOTE SENSING

FATMA Y. MOHAMEDl, KIYOSHI SHIRATORI', E. S. MASSAWE3, R.J. SHAY03 ,TOMOKO KISU4 AND MASAAKI SHIMADA+
Department of Epidemiology, Ministry of Health, Tanzanial ,Kilimanj aro Agricultural Training Center Proj ect JICA2,

Kilimanjaro Aglicultural Training Center Tanzania3 andInformation and Reference Center of Tropical Medicine,
Institute of Tropical Medicine, Nagasaki University4

To determine the risk of infection, the density of therigation canals of paddy fields. The density of the snail wasintermediate host of Schistosoma mansoni was studied for10‑ 100 times higher in the river than in the irrigation canals

two years at transmission sites in a village at Lower Moshi,

of paddy fields. The snails appear to change in number de‑Tanzania. The specific objectives are to identify the specificpending on the stagnation and vegetation of the water, butseason, condition and site with high risk of infection.
Snails were collected with I O‑time‑scooping, or 5‑minute‑

not on the water temperature. The possibility of the appli‑

picking where scooping was not applicable. Biomphalaria

infection by analyzing the correlation between the popula‑

pfelfferi was found to be widely distributed in a river and ir‑tion of the snail and vegetation will be discussed.

cation of remotely sensed variables to determine the risk of

B‑30 A QUESTIONNAIRE STUDY TO QUANTIFY THE HUMAN BEHAVIOR AT
RIVER INFESTED WITH SCHISTOSOMA MANSONITOMOKO KISU*, SUSUMU TANIMURA2, E.S. MASSAWE*, R.J. SHAY03,
KIYOSHI SHIRATOR14, FATMA Y. MOHAMED' AND MASAAKI SHIMADA*
Information and Reference Center of Tropical Medicinel,
Departrnent of Social Environment2, Institute of Tropical Medicine, Nagasaki University,

Kilimanjaro Aglicultural Training Center Tanzania3,

Kilimanjaro Aglicultural Training Center Project JICA4 andDepartment to Epidemiology, Ministry of Health, Tanzania5

Quantification of human behavior is essential to deter‑

vidually. Intestinal helminths were also examined quantita‑mine the risk of Schistosoma infection. However the quan‑tively and school attendance was taken into account as well.

tification of the behavior is not easy to estimate and it is dif‑

Kato‑Katz technique was applied for the stool examination.

ficult to relate the behavior to the infection individually.

Children were asked if they went to river or not, what for

Three hundred eighty three primary school children aged 7‑

and so on, for 10 successive weekdays. The prevalence of S.

14 were studied for their infection with Schistosoma man‑mansoni and Ascaris lumbricoides, hookworm, Trichurissoni by a stool examination, for their presence at school andtrichiura were 49%, 13%, 16%, 25%. The intensities of in‑for their water contact behavior by questionnaire in January

fection, EPG (geometric mean of egg count per gram), wereand February, 2000, at Moshi, Tanzania. The specific ob‑

10.2, I .6, I .O, and 2.0, respectively. The possibility if quan‑jectives were to determine if it is feasible to quantify the de‑tification of water contact activity through questionnaire

gree of individual water contact by questionnaire in a shortand the relationship between the intensity of infection andperiod and to determine if there is a correlation between thethe degree of water contact will be discussed.

degree of water contact and the intensity of infection indi‑

63

B‑31 WHERE DO SCHISTOSOMA JAPONICUM COME FROM?
TAKESHI AGATSUMA AND MORITOSHI IWAGAMI
Department of Environmental Health Science, Kochi Medical School

In his hypothesis on coevolution of Asian schistosomes

speculated that an ancestral species of the S. indicum groupand snails, Davis (1980,1992) implies that the ancestors ofof Asian origin may have been brought to Africa, colonized

the S. indicum species group and its associated pulmonate

and diversified in pulmonate snails in Africa. Afterwards,

snail hosts (Indoplanorbis and Lymnaea) were African andhuman schistosomes in Africa may have speciated from thearrived in Asia via the Indian plate. However, our recent

chromosome analysis using the C‑banding pattern sug‑diversified schistosomes as suggested by Despres et al .
(1992). S. hippopotami placed in a basal position of the Af‑

gested that the S. japonicum group is the original type, andrican species could be an ancestral species for the African

the African species represents the derived type (Hirai et al .,species, as have been suggested (Despres et al ., 1995).

2000). Further, Snyder and Loker (2000) also argued that,

Therefore, Iarge mammals such as hippopotamus or ele‑on the base of their LSUrDNA data, Schistosoma appears tohave originated in Asia, colonized and diversified in pul‑

phants could be a suitable host for a common ancestral spe‑monate snails in Africa, and then recolonized Asia. The
present study revealed that members of the S. indicumgroup have a close affmity with the African human schis‑tosomes and supported a hypothesis of the Asian origin of

they have longer life span and bigger capacity for harbor‑

the genus Schistosoma . However, we believe that the S. in‑to the genus Bivitellobilharzia discovered in Sri Lanka sug‑dicum group may have originated in Asia, but not recolo‑
nized Asia. Because nucleotide differences in both ITS2
and COI regions between the S. indicum group and the Af‑
rican group are still considerably large; this should havecies of the African group and the S. indicum group, since
ring a number of parasites that enable them to maintain andcarry the parasites to wide areas in a long span. Very re‑cently, molecular data of elephant schistosomes belonging
gested that the elephant schistosomes were placed in a morebasal position among Asian species of Schistosoma than S.

incognitum (Rajapakse et al ., 2000). Existence of theseschistosomes in Asia including Orientobilharzia which wasman. Therefore, it would be difficult to explain the above

placed among species of Schistosoma (Snyder and Loker,
2000) would support the hypothesis of Asian origin of

hypothesis of "recolonization by early humans activity" as

Schistosoma .

happened long before domestication of wild animals bysuggested by Barker and Blair (1996). Instead, it could be

B ‑32 THE DIFFERENCE OF THE ECHOGENIC PATTERNS AMONGSCHISTOSOMIASIS JAPONICA AND SCHISTOSOMIASIS MEKONGI?
HIDEHIRO OTAKE*' 2, YUICHI CHIGUSA', HIROSHI OHMAE*, SINUON MUTH4,DUONG SOCHEAT+, JUN MATSUMOT02, MASASHI KIRINOK12 AND HAJIME MATSUDA'
Faculty of Nursing, Kure Universityl,

Department of Tropical Medicine and Parasitology,

Dokkyo University School of Medicine2,
Department of Tropical Medicine and Parasitology,
Institute of Basic Medical Sciences, School of Medicine,

University of Tsukuba3 andNational Malaria Centre, Cambodia4Voge et al . in 1978. Those two schistosome species weretold to be quite resembled each other at the appearance ofmediate hosts, Oncomelania spp. and Y‑Neotricula aperta
were quite different each other compare with the morpho‑
logical similarities of the parasites. Even though no new

their eggs. The habitats and morphology of the snail inter‑patient was reported with Schistosoma japonicum since

Since Schistosoma mekongi was first described by641978 when the ultrasonographic investigation was intro‑

Cambodia in order to observe the echogenic pattern of the

duced for schistosomiasis cases in Japan. We were able to

network with schistosomiasis mekongi for the first time.
Totally around 400 adult patients who seriously affectedreport the echogenic pattern of the network on 1 1 young pa‑

tients, 9 to 14 years old, among elementary school children

Appearance of the echogenic pattern of the network with S.

with S, mekongi were examined for the abdominal ultra‑
sonography. However, none with the echogenic pattern ofthe network was found through our investigation. Thesejaponicum has been confirmed with not only for adult cases

data suggest the echogenic pattern of the network appeared

but also for younger group. Our investigation was con‑ducted in 1999 and 2000 at several highly endemic loca‑

though our investigations could not be performed undertions inside the two provinces, Stung Treng and Kratie, in

ideal circumstance.

at the highly endemic areas in Mindanao Island, Philippines.

on only S. japonicum among schistosome species even

B‑33 LOCALIZATION OF TYPE IV COLLAGEN IN GRANULOMA FORMATION AND
LIVER FIBROSIS DUE TO SCHISTOSOMIASIS JAPONICA
MAYUMI YOSHIZAKI, MASAMI NAKAMAE AND HIROSHI OHMAE
Department of Tropical Medicine and Parasitology,
Institute of Basic Medical Sciences, School of Medicine,

University of Tsukuba

Type IV collagen is a main component of basement

until eleven weeks after the infection. By immunopa‑

membrance in the liver parenchyma. A significant increase

thological method, type IV collagen was detected along thein the levels of serum was reported among Schistosoma ja‑

wall of portal veins in the liver from five weeks to elevenponicum infected patients with periportal fibrosis.

weeks after the infection. But it was not clearly detected in

Localization of types I, 111, and IV collagen concomi‑tant with biochemical serum analysis were performed inmurine schistosomiasis japonica. Each mouse was infected
percutaneously with 30 cercarie. From five to eleven weeks

granulloma formation around ovas of schistosomiasis.

Serum levels of type I collagen and procollagen‑III‑
peptide did not show high elevation in the early stage of theafter the infection, seven mice were sacrificed every two

infection. As the progress of periportal fibrosis due toschistosomiasis japonica, they became to show high levels.

weeks. Before sacrifice, serum levels of types collagen I

And they were detected in granulloma formation due toand IV, and procollagen‑III‑peptide were examined. Patho‑

schistosomiasis in the liver.

10gical changes of the liver were examined by Hematoxylin

Type IV collagen is detected along the wall of portal

and eosin and Masson trichrome stainings. And localiza‑tion of types I, Ill and IV collagens were detected by im‑vein in the liver from early stage of the infection. The se‑munopathological methods.

changes due to schistosomiasis than the other types of colla‑

Five weeks after the infection, serum level of type IV

collagen showed significant elevation and kept high levels

rum level of type IV collagen is more sensitive to periportal
gen such as types I and 111.

65

B ‑ 34

INFLUENCE OF ULTRAVIOLET RAYS ON INFECTION OF SCHISTOSOMA MANSONINOBU OHWATARI*, YASUNORI FUJIMAKI', ATSUSHI KUMATORl*,

KAN TORIYAMA+ AND EIKO KANEDA*
Department of Environmental Physiologyl , Department of Parasitology2,

Department of Biochemistry3, Department of Pathology4 andDepartment of Thermal Adaptation5,
Institute of Tropical Medicine, Nagasaki University

There are reports that ultraviolet rays exposure changes

immunology ability. So we examined that the changes of
immunology ability induced by artificial ultraviolet (UV‑B)

500 cercariae with abdominal skin cut hair of all mice underanaesthesia.

On the number of cercariae invaded, it in skin group

exposure influences to infection of Schistosoma mansoni .

was significantly many cercariae compared with it in con‑After ultraviolet rays exposed to DDY mice, the valueof CD41CD8 in the mice decreased gradually for one week
and was minimum value which was significant differencecompared with non‑exposure mice at one week after. After‑

trol group and lung group (p<0.01), but it was no significant

wards, it recovered to the normal value for 2 weeks.

difference between control group and lung group. There
were not significant dit ferences among three groups on theegg of S. mansoni.

On the number of adult worm recovered, it in lungThe 24 DDY mice separated to three groups of control,group was significantly many cercariae compared with it inskin and lung. Control group had not without UV‑B irradia‑

control group and skin group (p<0.01). UV‑B exposure wastion, and skin and lung groups were irradiated at 400 J/m2

encouraged cercariae invasion into skin, but it was not dueof UV‑B two times for one week interval. Skin group was
infected by cercariae of S. mansoni at one week after theto skin damage by UV‑B in skin group results. On the other

UV‑B irradiation, and lung group was infected by it at one

hand, Iung group data is suggest that immunology ability
about one week after infection, when many cercariae gath‑day after the UV‑B irradiation. Challenge infections were

ered in lung was important to inhibit cercariae growing.

B‑35 USEFULNESS AND LIMITATION OF COPT IN LOW ENDEMIC AREAOF SCHISTOSOMIASISHIROSHI OHMAEl, HAJIME MATSUDA2. YUJI IRIEl, I. BALUNGA1, L. PORTILL03,
R.B. PACHEC04, V.S. GUIRITAN4, B.L. BLAS5,KAZUO YASURAOKAI AND HIROSHI TANAKA6
Department of Parasitology, Institute of Basic Medical Sciences,

University of Tsukuba, School of Medicine*,

Department of Tropical Medicine & Parasitology,

Dokkyo University School of Medicine2,
Schistosomiasis Training and Research Center3,

Schistosomiasis Control Team of Boh014,
Schistosomiasis Control Servise5, Philippines andDepartment of Parasitology, The Institute of Medical Science,

The University of Toky06

In high or low schistosomiasis endemic area, useful‑and a low endemic area, Bohol Island, Philippines.

ness of immunological tests is different. We compared theThere are about 270 schistosomiasis endemic villages

changes of prevalence of schistosomiasis detected by Kato‑in Leyte Island and the area of snail colonies is very wide.

Katz's stool examination and positive rate of circum oval

So in Leyte, selective or non‑selective mass treatment has

precipitin test (COPT) in a high endemic area, Leyte Island

been a main measure for control of schistosomiasis and66

snail control measures have not been so active. In Leyte,

heavy endemic village, infected snails had been found until

the prevalence of schistosomiasis decreased from 8% in1986 to 4% in 1996, however, COPT positive rate kept 30‑

1992. In this village, the prevalence of schistosomiasis de‑

40% .

creased from 8.4% in 1982 to 2.6% in 1988, however
COPT positive rate showed 21.3% and 14.1%. And six

In Bohol Island, there are eight schistosomiasis en‑

years after the disappearance of infected snails, COPT posi‑

demic villages and the area of snail colonies is limited.
Each village has 700‑2,000 inhabitants and total population

tive rate became O% among primary school children in

is about I 0,000. Through yearly selective mass treatment

1996.
By a transmission of schistosomiasis from the snail tosis japonica had decreased from 4.2% in 1982 to 0.1(; o inhuman, COPT shows positive reaction and it continues for
two years after the treatment with praziquantel. COPT is atogether with snail control, the prevalence of schistosomia‑
1996. In three villages, infected snails rapidly disappeared,

good indicator to evaluate frequency of transmissions inand no snails have been found since 1990. In these villages,

low endemic area of schistosomiasis. And in the final stage

the prevalence of schistosomiasis had decreased from 3.8%of control measures COPT is also useful to evaluate elimi‑

in 1982 to 0.80/0 in 1988. And COPT positive rate had also

nation of schistosomiasis.

decreased from 10.1% in 1982 to 2.2% in 1988. In the

B ‑36 URINE CYTOLOGY IN AN ENDEMIC AREA OFSCHISTOSOMIASIS HAEMATOBIA IN KENYATOMOHARU OHKI', HIDEHIKO YAMAUCHll, HIROSHI MATSUYAMA*,
MASANOBU ANAM12, MASACHIKA ISEK13, N.D. MUHOH04 AND YOSHIKI AOK11
Department of Parasitologyl , Department of Pathology3,
Institute of Tropical Medicine, Nagasaki University,

Department of Pathology, Nagasaki Univetsity Hospital2 andKenya Medical Research Institute4

Urinary schistosomiasis has been linked to bladdertained from I ,516 villagers in an endemic area and f'rom

cancer since the turn of the 20th century. However, the in‑

294 villagers in non‑endemic area. The cytological reports

cidence in the community is still unknown due to a scarcity

were divided into 3 class V, I class IV, 6 class 111, 22 class IIof a population‑based studies. Although reported sensitivity

and I ,484 class I. Those of non‑endemic area were all classfor urine cytology detection of' bladder cancer is approxi‑

I. The patient age for the 4 cytologically detected bladdermately 40%, urine cytology has been an integral part ofcarcinomas ranged between 38 and 70 years. One of thepatients was male. Squamous metaplasia, RBC, WBC andbladder cancer screening. And this is technically feasible,

relatively inexpensive and readily acceptable by the com‑schistosome eggs were encountered in 2, 2, 2 and I of themunity. We conducted a urine cytologic survey on the in‑patients. Cytologically diagnosed tumors included 2

habitants of an endemic and non‑endemic area of schisto‑

squamous and 2 transitional cell carcinomas. A11 villagers

somiasis haematobia in Kenya. The target populations werevillagers who were all 20 years and over. Urinary cytology

with class 111 had squamous metaplasia. Our result is com‑parable to that from screening bladder cancer in the high‑was performed using a slight modification of the standard

risk group of a rural Egyptian population infected with S.

techniques. The smear reports were classified according tohaematobium (El‑Bolkainy et al ., 1982).

Papanicolaou (1946). Single voided specimens were ob‑

67

C‑l HEPATIC PENICILLIOSIS MARNEFFEI IN NORTHERN THAILAND
KAN TORIYAMA*, AMNAT YOUSUKH2, MASACHIKA ISEKI* AND HIDEYO ITAKURA*
Department of Pathology,
Institute of Tropical Medicine, Nagasaki Universityl andDepartment of Pathology, Faculty of Medicine, Chiang May University, Thailand'

Penicillium marneffei, which is the only dimorphic
member of the genus Penicillium , was first isolated fromwith multiple granulomas, which contain a few number ofthe liver of a bamboo rat (Rhizomis sinensis) in Vietnam inerate cellular reaction, 3) 14 cases of Mixed type (M),which showed intermediate features between D and G types.

1956. P. marneffei shows globose‑to‑oval shaped yeast‑like

intra‑ and extracellular P marneffei and with mild to mod‑cell, which measures 2‑3 X 2‑7 um with obvious septa. P

Clinically, the highest elevation of AST Ievel was found in

marneffei infection is one of the major opportunistic infec‑

tions among the HIV infected individuals in Southeast Asia,

D (mean level; 252), followed by M (mean level; 245) andG (mean level; I 08). The highest AL‑P Ievel was observed

particullary in Northern Thailand. We performed a clinico‑in M (mean level; 535), followed by G (mean level; 473)

pathological study of hepatic penicilliosis marneffei in Chi‑and D (mean level; 389). These findings suggest that D in‑

ang Mai, Northern Thailand, examining 30 cases of AIDS

fections cause the hepatocyte injury, the bile ducts arepatients with disseminated P marneffei infection. Hepatic

mainly damaged by G infections and M infections show thelesions of the liver biopsy specimens were histologicallyboth of hepatocyte and bile duct injuries and the degrees ofclassified to three types as follows; 1) 10 cases of Diffuse

immunosuppression of the patients might influence the his‑

type (D) with diffuse infiltration of histiocytes, which con‑

tological and clinical differences of hepatic penicilliosis

tain a numerous number of P marneffei and without signifi‑

marneff ei .

cant cellular reaction, 2) Six cases of Granuloma type (G)C‑2 A UNIQUE DRUG‑SUSCEPTIBILITY PATTERN OF VIBRIO CHOLERAE O1 IN LAOSMASAAKI IWANAGA*, CLAUDIA TOMA*, NAOMI HIGA*,SITHAT INSISIENGMAY' AND BOUNNANH PHANTOUAMATH'
Department of Bacteriology, Faculty of Medicine, University of the Ryukyus'

and Center for Laboratory and Epidemiology, Ministry of Health, Lao PDR2

Cholera epidemic due to polymyxin B sensitive E1 Tor
vibrios appeared in southern part of People's Democratic

trimethoprim were about 256 times higher than thoseagainst the previous isolates, they were mostly 640 ug/ml

Republic of Lao since 1998.

(trimethoprim 32 ug/ml , sulfomethoxazol 608 ug/ml) or

During the past 7 years (1993‑1999) in Laos, we haveinvestigated the changes of drug susceptibilities of Vibrio

more. Eleven percent of the isolates (11/99) were as sensi‑

cholerae O1. Until 1996, the susceptibilities were almost
as expected and cholera disappeared in 1 997. When cholera

ug/ml ). Polymyxin B sensitive strains were isolated fromoutbreak resurfaced in 1998, the susceptibilities of isolated

tral Lao) but not from Northern districts. In 1 999, the sus‑

V cholerae O I against tetracycline, sulfomethoxazoltrime‑

ceptibility pattern was almost the same with that in 1998

tive to polymyxin B as the classic cholera vibrios (MIC<2
Southern part of the country and the City of Vientiane (cen‑thoprim, chrolamphenicol and polymyxin B were quite dif‑

except polymyxin B to which 58% of the isolates (21/36)

ferent from the previously isolated organisms. Minimum

became sensitive.

Inhibitory Concentrations (MICs) of tetracycline and chro‑

In 2000, the stool samples were not collected from thelamphenicol against the isolates in 1998 were 3.13 or 6.25

ug/ml which were about 16 times higher than the MICs

epidemic area in the South but only 3 strains because of
traffic disturbance. The 3 strains were sensitive to poly‑against the previous isolates. The MICs of sulfomethoxazol‑

myxin B, whereas the all strains from the North were resis‑

68tant. The susceptibility pattern to the other drugs was al‑

most the same with those in 1999.

E1 Tor hemolysin which was neutralized by the correspond‑
ing antiserum. Polymyxin B susceptibility test is no longer

Genetic analysis using PFGE suggested that the clones

the tool for biotype classification of V cholerae Ol. Eco‑of the isolates in 1998 and 1999 were closely related re‑logical and epidemiological consideration should be madegardless of the drug‑susceptibilities, but different from thefor the polymyxin B sensitive El Tor vibrios.

isolates in 1995. Polymyxin B sensitive isolates produced

C‑3 TWO INFANT CASES INFECTED WITH TYPHOID FEVER IN THE PHILIPPINESAKIRA OZAKI*, EMI TAKAGll, KEISUKE TOKIEDA*, HIROKO YAMAKAWA*,MINORU UMEHARA*, JUN ISHIHARA*, MITSUO SAKAMOTO' AND HIROKO SAGARA2
Department of Pediatrics[ and Department of Infectious Disease2,

Yokohama Municipal Citizen's Hospital

If an overseas trip returnee (especially returned fromingestion reduced and the patient was admitted to this de‑developing countries) have had an onset of fever, it is neces‑

partment on August I O, 2000. At the time of admission,sary to perform diagnosis and clinical examinations, taking

slight distention of the abdomen was found. The feces cul‑

imported infections into consideration. This time we reportture of the day before admission was Salmonella 07 group
positive. Blood culture of the time of admission was Sal‑

our experiences of two infant cases with typhoid fever who
had infected in the Philippines.

monella Typhi positive. Abdominal US revealed hypertro‑

Case I : Male, infant aged one year. The patientphy of gallbladder wall. We administered two antimicrobes,

stayed in the Philippines, the homeland of the patient's
mother, from January 1999 to late in April 1999. Since

cefotaxim (CTX) by IV and NFLX orally. The fever

April 28, 1999, the patient became febrile and also had anof the therapy to the complete alleviation of fever. Afteraround 39 C persisted and it took twelve days f rom the start

onset of diarrea. Then the parient visited this department ondischarge the patient had no fever and the systemic condi‑

April 30, 1999. The administration of a medicine for intes‑

tion was well, but the feces culture for confirming eradica‑

tinal disorders improved diarrea, but the fever persisted andtion of Salmonella Typhi bacilli was positive and fever
manifested again. Then the patient readmitted to this de‑partment because of recurrence. We administered amoxillin

the patient admitted to this department on May 1 2, 1999.
At the admission, the patient had the distention of abdomen.

Clinical examination data at admission were WBC 8,900/ul
Hb 8.9 g/dl , GOT 354 IU/1 , GPT 284 IU/1 and CRP 2.5 mg/dl . Abdominal ultrasonography (US) revealed hypertrophyof enteric wall. Blood culture at admission was Salmonella(AMPC) perorally. Two days after, the fever was alleviated.

No recurrence was found thereafter.

Discussion: The improvement of sanitary conditions has
made the infection with typhoid fever in Japan rare. The

Typhi positive. We started IV of ABPC. Later it was foundthat the strain of Salmonella Typhi in the patient was ABPC

number of report of typhoid fever in infant in Japan is also

resistive. Therefore, we changed ABPC IV to peroral ad‑

cially to South East Asia and developing countries) typhoidministration of norfloxacin (NFLX). Two days after, the fe‑fever must be considered in case of differential diagnosis.

ver was alleviated. NFLX was administrated perorally for

Regarding int'antile typhoid fever, general condition is not

two weeks in total. No recurrence was found thereafter.

Case 2: Male, inf ant aged eight months. On July 29,

severely worsened and it often tends to be diagnosed ascommon cold. Feces and blood culture test are important

rare. However, if a patient have ever been abroad (espe‑

2000, the patient returned from the Philippines where thefor the differential diagnosis of typhoid fever. As antimi‑

patient was grown up from birth. Since August I , 2000, fe‑crobial therapy for typhoid fever, new quinolon type antimi‑

ver of around 38 C manifested. Since August 5, 2000, thepatient had an onset of diarrea, then fever of 38t to 40

crobial drugs are mainly used recently because of increaseof resistive Salmonella Typhi strains for chloramphenicol

persisted. Then on August 7, 2000, the patient visited this(CP), ABPC, and ST mixture and consequentry, recurrence

department. The clinical examination were, WBC 9,380/ul
and CRP 3.8 mg/dl . Then we doubted bacterial enteritis
and we administrated fosfomycin (FOM). However, waterof disease. In these two cases, NFLX was effective for thecase I , but recurrence was obserbed in the case 2 in spite of

the administration of NFLX. Infants are in disadvantageous

69
situations regarding the treatment of typhoid fever, for ex‑

system for enabling the administration of TFLX and LVFX

ample, tosufloxacin (TFLX) and levofloxacin (LVFX) thatto infants with typhoid fever in case the benefits of treat‑

are frequently used in adults with typhoid fever has no indi‑

ment exceed the risk of administration of TFLX and LVFX.

cation to infants. Therefore we believe that to improve the

C‑4 TWO CASES OF MELOIDOSIS IN JAPANHIROYUKI KUNISHIMA' , TOSHIAKI SUZUK12, REIKO SEKI*,TOSHIO NAKAMURA' AND MITSUO KAKU*
Department of Respiratory Internal Medicineland Department of General Internal Medic,ine2, St. Marianna University School of Medicine andDepartment of Molecular Diagnostics, Tohoku University Graduate Medicine3

Melioidosis is amphixenosis caused by Burkholderia
pseudomallei. Its patients have been commonly observedin the Southeast Asia region between latitude 20' N and

Medical history: The patient was diagnosed with diabetes

20' S. This bacterium can infect from injured skin percuta‑

three years ago, but had received no treatment.

ese, immigrated to Japan 10 years ago, has never traveled
abroad since.

neously or through the respiratory tract, and forrn an ab‑

Case history: On Oct. I , 1999, the patient visited a hospital

scess on any organ of the body. There are few effective

for abdominal pain on the left side. A conspicuously highmedications for it. Both case fatality rate and recurrence

level of blood sugar was observed and he was admitted torate of this infectious disease are high in Southeast Asia.

the hospital for the treatment of diabetes. An abscess on theReported in this study are two cases of melioidosis ob‑

spleen was suspected after abdomen CT. On Oct. 8, the pa‑

served at our hospital.

tient was transferred to our hospital for complete examina‑(Case 1) A 69‑year‑old male patient. Chief complaints: fe‑tion and further treatment.

ver, breathing difficulty. Medical history: The patient was

Developments: We treated the patient conservatively by ad‑diagnosed with diabetes in 1 979 and has been on a dietary

ministrating cefazolin 2.0 g/day, but with little improvement.

regimen since then.

On Oct. 15, drainage of splenic abscess was performed. Af‑

Case histry: On Jan. 30, 1998, the patient traveled to Myan‑ter identifying B. pseudomallei at the same site, we diag‑mar, and several days later, started showing symptoms ofcommon cold. At'ter the fever exceeded 38 , the patient

penem I .O g/day and minocycline 200 mg/day were admin‑

returned on Feb. 3. Two days later, he visited a nearby hos‑istered. After that, the patient made a rapid recovery.

pital and was administered ofloxacin and ceftazidime, but
did not recover. On Feb. 1 8, he visited our hospital. Upondeclining tendency in pharmaceutical sensitivity of this bac‑observing water retention in the upper left of the chest on

terium to antibacterial drugs. In the two cases we studied,

nosed it as melioidosis. For antibacterial medication, mero‑(Discussion) In recent years, researchers have reported aplain films, and a honeycomb pattern on the right lower

sensitivity was low on such drugs as the P‑lactam antibacte‑limb, the patient was admitted to the hospital.

rial. Since the bacteria in this study is known to proliferate

Developments: By the pleural effusion centesis, the patientin a cell, we found it highly possible that the patient in Casewas diagnosed with empyema. A drainage of the thoracic
cavity was performed while administering panipenemlbe‑

It then developed further due to a suppression of the im‑

2 was inadvertently infected with the bacterium in Vietnam.

tamiprom I .O g/day, and clindamycin I .2 g/day. This wasfollowed by an incision to discharge pus for the honeycomb

mune system, which had been caused by diabetes. Al‑

pattern. After identifying B. pseudomallei at both sites, wepan, it is detectable by a general bacteriological examina‑

though this kind of afferent infectious disease is rare in Ja‑

diagnosed it as melioidosis. After that, the patient made ation. As travelers to Southeast Asia are on the rise, it willrapid recovery.

be necessary to exercise adequate caution to detect this in‑(Case 2) A 33‑year‑old male patient. Chief complaints:

fectious disease.

Abdominal pain on the left side. Personal history: Vietnam‑

70

C−5 MEASLES SEROSURVEILLANCESTUDYDURINGMASSIMMUMZATIONCAMPAIGNINMALAWl：ANTIBODYPREVALENCEANDSEROLOGICALRESPONSES USINGPARTICLEAGGLUTINATIONMETHODMIHOTAKECHIl，MIKIKOISAACH

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71

Immunizations through gastrointestinal tract might

efficiently secreted to the culture media confirmed by GMl‑provide an effective way of stimulating both local and sys‑

ELISA method. The mCT molecule may function as a mu‑

temic immune responses. However, oral vaccines, the mostconvenient way of vaccine antigen delivery method, mustbe formulated in such a way that vaccine antigens are pro‑cosal adjuvant for co‑locally produced and/or secreted for‑tected from protein degradations occur in the gastrointesti‑

We are also interested to find whether such secreted mCT
provide mucosal adjuvantisity for co‑expressed pathogen‑derived antigens. In addition, we are producing Salmonella

nal tract. Vaccine antigens produced and delivered byavirulent recombinant Salmonella may be provided such

eign protein antigens derived from other pathogenic micro‑organisms such as malaria and Japanese encephalitis virus.

protection as they pass through the stomach and intestine.

secreting fusion proteins between mCT and microbial anti‑

In addition, purification processes are not required for anti‑

gens for effective induction of systemic immune responses

gens produced in recombinant Salmonella. Salmonella‑against fused antigens. Avirulent recombinant Salmonelladelivered vaccine antigens are efficiently presented to cells

may provide cost‑effective vaccine antigen production andof mucosal immune systems such as Peyer's patches for in‑delivery systems for induction of local and systemic im‑duction of both humoral and cell‑mediated immune re‑mune responses against many life‑threatening infectious
agents to which new type of low‑cost vaccines are need forsponses.

We have expressed nontoxic mutant of cholera toxin(mCT) in avirulent Salmonella and found that the mCT was

the improvement of health of humans and domestic animals.

C‑7 EVALUATION OF DENGUE DIAGNOSTIC TEST KITS
KEN‑ICHIRO YAMADA1, TOMOHTKO TAKASAKI*, MASARU NAWA2 AND ICHIRO KURANE*
Department of Virology I , National Institute of Infectious Diseasesl andDepartment of Microbiology, Saitama Medical Scho012

Dengue virus infections are a major public healthquito cell clone C6/36. Three commercial diagnostic assays,

problem in tropical and sub‑tropical countries of the world,a lgM‑capture ELISA (MRL, California, USA), an im‑

and dengue is considered as one of the most important re‑

munochromatographic card assay and a strip assay (PanBio,

emerging infectious diseases. There are currently no do‑

Brisbane, Australia) were evaluated for detection of lgM tomestic dengue virus infections in Japan; however, imported

dengue viruses. The specificity of the in‑house ELISA withdengue cases have been reported. Detection of specific lgM

a flavivirus group specific monoclonal antibody, D1‑4G2‑4‑

by lgM‑capture enzyme‑1inked inununosorbent assay (ELISA)

1 5 was higher than that with patients' IgG, and lower levels

and of dengue virus genome by reverse transcriptase polym‑of specific lgM was detected in the serum samples. These

erase chain reaction (RT‑PCR) have been widely used as

results suggest that the modified dengue lgM‑ELISA withlaboratory diagnosis. Commercially available kits for the

monoclonal antibody has multiple advantages over thedetection of anti‑dengue virus lgM have recently been de‑original in‑house ELISA. We compared the in‑houseveloped. These standarized assays have greatly enhanced

ELISA with the commercial ELISA kits. Fifty‑seven serum
samples from confirmed and suspected dengue cases were
tested by the in‑house and the commercial ELISAs. The re‑sults were consistent between the in‑house ELISA and thecommercial one, except for one sample. The results by the

our ability to effectively and efficiently diagnose dengue vi‑

rus infections. In the present study, we evaluated the den‑gue diagnostic test kits in comparison with in‑house diag‑nostic assays. Four prototype dengue strains (type I , Ha‑waii; type 2. New Guinea C; type 3, H87; and type 4, H
241) were used as the antigens for in‑house lgM‑capture
ELISA. Viruses were grown in the Aedes albopictus mos‑new commercial kits (DEN‑25, new type and DEN‑25S,
strip type) were different from those by the in‑house ELISA

and the commercial ELISA kit.

72

C‑8 A CLINICAL STUDY ON DENGUE HEMORRHAGIC FEVER IN
METRO MANILA, PHILIPPINESKAZUNORI OISHl , CELIA CARLOS', DOLLY CINCO', SHINGO INOUE2,
RONALD MATIAS', FUTOSHI HASEBE*, KOUICHI MORITA*, FILIPlNAS NATIVIDAD',AKIRA IGARASHI* AND TSUYOSHI NAGATAKE*
Department of Internal Medicinel, Department of Virology2,
Institute of Tropical Medicine, Nagasaki University and

St. Lukes Medical Center, Quezon City, the Philippines3

Dengue hemorrhagic fever (DHF) is characterized byacute fever associated with hemorrhagic diathesis and a ten‑patient with DHF grade IV died due to prolonged thrombo‑
cytopenia and generalized hemorrhage. The platelet counts

dency to develop shock, which is distinct from dengue fever.

in DHF were significantly lower than those in DF (42,000

However, the mechanisms responsible for bleeding in DHF
remain unclear. To elucidate the mechanisms of hemor‑brinogen levels in DHF were significantly lower than thoserhagic diathesis in DHF, we evaluated the clinical featuresin DF. No significant difference was found in prothrombin

and hematologic data including platelet counts, fibrinogentime between DF and DHF. Disseminated intravascular co‑in DF vs. 109,000 /u1 in DHF; p value <0.001). The fi‑levels, bleeding time, prothrombin time, fibrin degradation

agulation (DIC) was found only in 1 1 . I % of patients withproducts (EDP) and anti‑platelet antibody in sera among 65

DHF and in 5.2"/, of patients with DF by DIC scorespatients with virologically confirmed dengue infection.

(symptoms and laboratory data described above). Platelet
binding antibody was not detected in sera from either pa‑

From our preliminary data, we diagnosed dengue fever(DF) (n=38) and DHF (n=27) by the WHO criteria. The

tients with DF and DHF. Our data indicate that DHF ismean age is 10.8 and 8.6 years old in DF and DHF, respec‑

closely associated with severe thrombocytopenia and is nottively. Bleeding manifestations other than subcutaneous
hemorrhage was observed in 48% of patients with DHF. A

bocytopenia in DHF requires further investigation.

frequently associated with DIC. The mechanisms of throm‑

C‑9 A CASE OF DENGUE FEVER COMPLICATED WITH
PROBABLE ACUTE DISSEMINATED ENCEPHALOMYELITIS
Y. YAMAMOTOAKIHIRO HITANI MIKIO KIMURA M MAEDA', K. WASHIZAK13,
TETSUYA NAKAMURA* AND AIKICHI IWAMOTO *Departmenrt of Infectious Diseases & Applied Immunology,
Institute of Medical Science, The University of Tokyol,

Higashi Hospital Kitazato University2 and Yokohama Rosai Hospital3

Dengue fever is mosquito‑borne viral infection andgue fever because of positive lgM antibody to dengue virus.

primarily a disease of older children and adults in south‑east

On the 9th day after onset, he experienced neurological

Asia and western Pacific countries. The dengue virus is a

symptoms including confusion, bilateral visual disturbance,

Flavivirus and Aedes aegypti is a major vector. Recently,

sensory disturbance and mild paraplegia of lower extremi‑

several factors including demographic changes and in‑ties, which was gradually worsened to complete palsy be‑creased chances of air travel contribute to increased epi‑

low Th7 Ievel by the 1 1 day after onset. He was transferred

demic/endemic dengue activity. Forty percent of world

to our hospital on Feb. 25, 2000.

population is currently under constant threat of this infec‑

MRI revealed multiple spots of T2WI high lesions inspinal cord of Th 7 ‑ 1 1 without abnormal fmdings in brain.

tion.

We report here a dengue fever case of a 58‑year‑old
Japanese male who lived in Brazil. He had fever, anorexia,
and rash on Jan. 1 5, 2000, and was made diagnosis of den‑

CSF study showed slightly increased cell number and pro‑tein concentration. From his clinical course and MRI, we
suspected his neurological symptoms as acute disseminated73

encephalomyelitis (ADEM). Thus, 3 cycles of methylpred‑
nisolone (1,000 mg/day) for three days were administrated

nating disease of central nervous system. It usually occurs

intravenously, and bilateral sensory disturbance and para‑

acutely and exhibits monophasic course about I or 2 weeks
after viral infection and vaccination. There have been some

plegia were improved step by step after each cycle. After 3

reports about the involvement of central nervous system inmonths from the onset, his spinal MRI did not show any ab‑

dengue fever and dengue haemorrhagic fever, such as multi‑

normal findings, and he could walk with help of a stick, al‑ple neuropathies, mononeuropathy, and meningoencephali‑tis. However, we could not find reports of ADEM caused
by dengue fever. Thus, this seems to be the first case of
ADEM associated with dengue fever.

though mild bilateral visual disturbance, dysuria, and dy‑schezia remained unchanged.

ADEM is an immune‑mediated inflammatory demyeli‑

C‑lO DENGUE VECTOR SITUATION ALONG URBAN‑RURAL ECOLOGICAL GRADIENTYOSHIO TSUDA*, WANNAPA SUWONKERD' AND MASAHIRO TAKAGI*
Department of Medical Entomology,
Institute of Tropical Medicine, Nagasaki UniversitylandOff"rce of Vector‑Borne Disease Control 2,

Ministry of Public Health, Thailand2

Larval survey of dengue vectors were conducted from
July 1996 to September 1997 in Chiangmai Province, Thai‑area> urban area. House survey of breeding containers

land. Three study villages at urban (Sri Phoom), semi‑urban (Mae Rim) and rural area (Mae Taeng) were selectedtion of breeding containers among the study areas. The sea‑

for the survey to clarify the spatial distribution of Aedes ae‑

February‑November 1997 did not dif'fer significantly among

gypti and Ae. albopictus along urban‑rural ecological gradi‑

the study areas. However, there were significant area differ‑showed significant differences in the number and composi‑
sonal pattern of rainfall recorded at the study areas froment. The results of ovi‑trap survey showed clear differences

ences in the average distance between houses, average tree

in larval density among the study areas. The average den‑height, and percentage of area with vegetation cover. Thesesity of Ae. aegypti larvae was the highest at urban area andresults suggested that urbanization is one of important fac‑

intermediate at the rural area. Although we expected thetors influencing density and distribution of dengue vectors.

lowest density of Ae. aegypti at rural area, it was observed

However, there might be additional ecological factors to ex‑

at semi‑urban area in this study. The results for Ae. al‑plain area differences in dengue vector situation.

bopictus were opposite to Ae. aegypti, semi‑urban> rural

C Il MOLECULARAND IN VITRO ANALYSES OF DENGUE‑1 VIRUS STRAINSHASANUDDIN ISHAK*, TSUTOMU TAKEGAMI' AND KIYOSHI KAMIMURA*
Department of Biodefence Medicine, Faculty of Medicine,

Toyama Medical and Pharmaceutical Universityl andDivision of Tropical Medicine, Medical Research Institute,

Kanazawa Medical University2

The genomic RNA of two dengue‑ I virus strains wassequenced and compared with other published strains toin 1 943) in Vero cells were significantly higher and their in‑

identify regions of dengue genome that might contribute tofectivity decreased slightly slower than those of A88 strain(isolated in 1988) after freezing at ‑ 80t. Analysis of de‑the viral growth characteristics in vitro . The growth rate

duced amino acid sequences indicated charge differences,and yields of the mouse‑adapted Mochizuki strain (isolated

mostly observed at the envelope (E) protein of Mochizuki

74

strain. The other charge differences were observed at cap‑

glycosylation site. Radical amino acid differences for A88

sid (C), pre‑membrane (prM), non‑structural NS1, NS4b,
and NS5 proteins. A unique amino acid, 1le‑69 for Mochi‑

strain were observed at the cleavage site of C/prM junction,

E, NS3, NS4a, and NS5 proteins. These differences might

zuki at E protein resulted in the loss of an Asn‑67‑1inked

be related to their different growth characteristic in vitro .

C‑12 RISK OF WEST NILE FEVER AND JAPANESE ENCEPHALITIS IN JAPANKIYOSHI KAMIMURA*, AKIRA IGARASHI', TSUTOMU TAKEGAM13, ISHAK HASANUDDlN ,YOSHIKAZU SHIRAI' , YOSHIHISA KUSUI+ AND TOSHIAKI TAKASU*
Department of Biodefence Medicine, Faculty of Medicine,

Toyama Medical and Pharmaceutical Universityl,
Department of Virology, Institute of Tropical Medicine, Nagasaki University2,

Division of Tropical Medicine, Medical Research Institute,

Kanazawa Medical University3 ,Kansai Airport Quarantine Station4 andDepartment of Neurology, Nihon University, School of Medicine5

A survey of viruses in Culex tritaeniorhynchus of Ka‑

adults and 1 52,000 young birds per year, and they are notrachi and environs, Pakistan, was conducted from 1985 to
1993. A total of 208,375 Cx. tritaeniorhynchus were sub‑mitted to virus isolation. The Japanese encephalitis virus

inspected for viral infection. Living mosquitoes are also

(JEV) was not detected but 4 serotypes of West Nile virus

from Africa, Middle East or other parts of the world. The(WNV) were isolated from I ,865 Cx. tritaeniorhynchus ofmosquito vectors for WNV or JEV, such as Cx. tritaenio‑

found in the hold of airplanes, with a possibility of intro‑

duction of infected mosquitoes in Japan by flights coming

20 pools collected on July 6‑8, 1986 in Karachi. In therhynchus. Cx. pipiens pallens, Cx. pipiens molestus, Aedes

cerebrospinal fluid collected from 24 patients with acute en‑

albopictus, Ae. japonicus and Ae. vexans are commonly
found in Japan. Moreover, in August 1998, the JEV wascephalitis in Karachi during February‑August 1 992, thegenome of WNV and JEV were detected in 8 and I samples,
respectively. The results suggested that WNV but not JEVisolated, in high proportion, from Cx. tritaeniorhynchus
collected in Uchinada, Ishikawa Prefecture. Besides this,is present commonly in Karachi, with occasional incidences

an increase in antibody titer against JEV was detected inof JEV infection that is transported by migratory birds com‑

sera of pigs during August and September. All these facts

ing from Siberia in October. Similarly, the migratory birds

suggest a high risk of occurrence of West Nile fever andcome from India or Middle East to Japan in May‑June,
when the mosquitoes also become more active. The num‑

Japanese encephalitis in Japan in the future, and a continu‑

ber of birds imported to Japan is estimated to be 420,000

animals are of significant importance.

C ‑ 13ous surveillance with isolation of virus from birds and wild

A STUDY ON ANTIGENICITY OF JAPANESE ENCEPHALITIS VIRUS ISOLATESIN VIENTIANE, LAO PDR
MIKA SAITO AND TOSHIHIKO FUKUNAGA
Department of Virology, Faculty of Medicine, University of the Ryukyus

Epidemics of Japanese encephalitis (JE) have not been

(LaVS 56 and LaVS 145) from slaughtered swine sera inrepored in Lao PDR, the inland country of Indo‑china pen‑

Vientiane belong to Genotype II, distributing from northern

insula surrounded by JE epidemic countries such as Thai‑
land and Vietnam. We reported that two JE virus isolates

Thailand to Cambodia, and that the sera of two JE patientsof Vientiane did not neutralize both of Vientiane isolates,

75

but neutralized Chinese strain (Beijing‑1) significantly. Inof antiserum for Beijing‑ I . And the neutralization patterns

this study antisera against 2 Vientiane strains (LaVS 56, andof the Vientiane patients' sera was compared with those of

LaVS 1 45), Thai Chiang Mai strain (P19, Genotype II) and

Chiang Mai (North Thailand) and Okinawa (Japan). Chi‑

2 standard strains, Nakayama (Japan, Genotype I), andBeijing‑1 (China, Genotype I) were made in BALB/C miceang Mai patients' sera neutralized JE viruses in the order of

Nakayama, Beijing‑1> P19 > LaVS56, LaVS145: this pat‑and used to analyze antigenicity by cross‑neutralization test.

tern was apparently different from that of Vientiane patients:

Anti‑Nakayama and anti‑Beijing sera neutralized signifi‑
cantly their homologous strains but not P19, LaVS56 nor

Okinawan patients' sera significantly neutralized Nakayama

LaVS 145. Neutralizing reactivity of antisera for P19, LaVS

These results suggest (1) The two JE virus Vientianestrains (LaVS56, LaVS145, genotype II) are antigenically

56, and LaVS145 was similar to each other: the three anti‑

and Beijing but not P19, LaVS56 nor LaVS 1 45.

sera significantly neutralized Nakayama, Beijing‑1, LaVS
56, LaVS145, and P19 strains. The results indicate that
LaVS56 and LaVS 145 are the same antigenicity as P19, but

infected with JE virus that is antigenically the same as

distinct from Nakayama and Beijing strains. Neutralization

Beijing‑1 strain. (3) There are JE viruses with at least two

pattern of 2 JE patients' sera of Vientiane was similar to that

distinct antigenicities in Vientiane.

the same as P19 which belong to antigenicity of ThCMAr
6793. (2) Two JE patients in Vientiane seemed to have been

C‑14 MUCOSAL IMMUNIZATIONS WITH A JAPANESE ENCEPHALITIS VIRUS
ANTIGEN‑CHOLERA TOXIN B SUBUNIT FUSION PROTEIN INDUCE VIRUS‑NEUTRALIZINGANTIBODIES IN MICE= AN ATTEMPT FOR THE DEVELOPMENT OF EDIBLE VACCINESAGAINST JAPANESE ENCEPHALITIS VIRUS
MASAYUKI TADANO*, TAKESHI ARAKAWA2, S‑P. MA , KAZUMI KANEMURA1,
HIROSHI TOMA', AYANO KISHIMOT03, Y. MATSUMOT0+, SIGETOSHI SAT03,
ICHIRO KURANE5, YOSHIYA SATO' AND TOSHIHIKO FUKUNAGA*
Department of Virologyl , Department of Parasitology2,

Faculty of Medicine and Department of Bioproduction, School of Agriculture3,
University of the Ryukyus,

Laboratory of Global Animal Resource Science,
Department of Global Agricultural Sciences, Graduate School of Agriculturaland Life Sciences, The University of Toky04 andDepartment ofVirology 1, National Institute of Infectious Diseases5

Recent advancement of plant genetic engineering tech‑mucosal immune systems exhibited by cholera toxin B

nology has made many genetically modified (GM) plants

subunit (CTB) for more efficient induction of mucosal andcommercially available. Our group is currently construct‑systemic immune responses. In this study, we expressed afusion gene between Japanese encephalitis virus (JEV) E
glycoprotein and CTB genes in Agrobacteria. We also con‑ducted immunization experiments for evaluation of the fu‑

ing several trans enic plants expressing vaccine antigen

genes derived from virus and parasitic microorganisms.
These GM plants may be used in future for induction of
mucosal and systemic protective immune responses against
infectious agents. The advantages of the GM plant‑basedsion protein as mucosal vaccines.

vaccines are their ease of large‑scale production and cost‑Experimental methods and Results: A fusion gene (E gly‑

effectiveness in comparison with conventional fermentation‑

coprotein/CTB) has been constructed and inserted down‑
stream of the cauliflower mosaic virus 35S promoter in aplant expression vector pB1121. The constructed plasmid
DNA was introduced into Agrobacterium tumefaciens LBA

based recombinant protein production systems. In addition,
vaccines produced in edible plants may be used as oral vac‑cines for the induction of protective immune responses.
However, protein components without specific affinity for

4404, a bacterial vector used for plant transformation. The

mucosal tissues are generally weak in their immunogenicity.

transformed bacteria was found to produce the fusion pro‑

Therefore, we took advantage of specific affinity fortein retaining specific affinity for G *‑‑ ganglioside, a recep‑

76

tor of cholera toxin. Several groups of mice were immu‑

oral immunizations with the JEV vaccine did not develop

nized with crude extracts of the fusion protein in nasal, oral

detectable levels of virus‑specific antibodies even in theand intra‑peritoneal routs. Immunization was conduced

presence of cholera toxin. Antisera containing anti‑fusion

once a week for a total of 4 immunizations. A week after

protein or anti‑JEV vaccine antibodies were analyzed forthe last immunization, antibodies were analyzed. In con‑trast with mice immunized intra‑nasally and orally, mice

their virus neutralization ability in vitro . Antisera devel‑

immunized intra‑peritoneally produced strong anti‑bacterial

nasal immunization strongly neutralized the virus in vitro .

antibodies without anti‑fusion protein antibodies. Bothgroups of mice immunized in nasal and oral routs devel‑
oped specific antibodies against the fusion protein. We

Anti‑fusion protein antibodies also exhibited weaker but

have also immunized mice with commercially available

cially available JEV vaccine are mucosally immunogenic

JEV vaccine in nasal and oral routs to determine the mu‑

for induction of virus‑neutralization antibodies.

cosal immunogenicity of the vaccine. Mice immunizedThis study was supported in part by research grants
provided by the Ministry of Health, Labor and Welfare andintra‑nasally developed anti‑virus antibodies whose immu‑

nogenicity was significantly enhanced when the vaccine
was mixed with a mucosal adjuvant cholera toxin. However,oped in mice immunized with the JEV vaccine by intra‑

significant virus‑neutralizing capability. This study indi‑cated that E glycoproteinICTB fusion protein and commer‑

Bio‑oriented Technology Research Advancement Institution(BRAIN) .

C‑15 WHAT IS THE ROLE OF APOPTOSIS IN FLAVIVIRUS‑INDUCED CYTOPATHIC EFFECT?
MARIA DEL CARMEN PARQUET*, FUTOSHI HASEBE*, ATSUSHI KUMATORI',
KOUICHI MORITA* AND AKIRA IGARASHI
Department of Virologyl and Department of Biochemistry2,
Institute of Tropical Medicine, Nagasaki University

Introduction: Japanese encephalitis (JE), St. Louis en‑

cephalitis (SLE), Murrary Valley encephalitis (MVE) andWest Nile fever (WNF) viruses, are members of the family
Flaviviridae . These viruses are members of the JE anti‑

tometry and chromatin condensation by Hoechst staining.
Results: Flow cytometry analysis revealed that at 48 hr

pi 68% and 69% of cells were infected by WNFV andgenic complex and associated to encephalitis. Flaviviruses

MVEV, respectively. The percentage of cells infected by
JEV and SLEV, however, was above 90%. Seventy two hpi

produce cytopathic effect in a variety of continuous cell cul‑

a rise in cell mortality up to 30% was observed for cells in‑tures. In recent years, a growing number of viruses havefected with JEV, MVEV and SLEV; and 76% for WNFV

been shown to induce cell death by a mechanism called
apoptosis. The purpose of this study was to investigate

infected K562 cells. Propidium iodide staining of the nu‑

whether the observed cytopathic effect due to flavivirus in‑

fection in a human mononuclear cell line (K562) could besubgenomic DNA content (43% for JEV, 44% for MVEV,
49% for SLEV, 50% for WNFV) as compared to mock in‑

related to apoptosis.

fected cells (2%). Similar percentages of K562 cells show‑

Materials and Methods: C6/36 cell line was used forviral growth and BHK‑21 cell line was used for virus titra‑tion by focus formation assay. An input m,o.i. of I FFU/

cleus revealed that approximately 50% of K562 cells had

ing chromatin condensation by Hoechst staining was ob‑
served for JEV, MVEV and SLEV, although for WNFV thisvalue was above 80%.

cell was used and mock infected cells were used as negative

Discussion: In this study we demonstrated that the cy‑

control. Cells were harvested at various times post infec‑
tion (pi) and infected cells were identified by flow cytome‑

topathic effect observed in JEV, SLEV, WNEV and MVEV
infection of the human mononuclear K562 cell line pro‑

try using FITC Iabeled anti‑flavivirus group specific anti‑

gresses with cell shrinkage, chromatin condensation and abody. Cell survival was determined by trypan blue exclu‑

rise in the percentage of cells showing subgenomic DNA

sion test. Apoptotic cells were identified by DNA content
analysis (hypotonic fluorochrome solution: PI 50 ug/ml incontent. All these, characteristics of cell death mediated by

0.1% sodium citrate plus 0.1% Triton X‑100) by flow cy‑

apoptosis.

77

C‑16 EPIDEMIOLOGICAL SURVEY ON THE RELATION BETWEEN HELMINTH INFECTION
AND NASAL ALLERGY
NAOHIRO WATANABE*, TORU IMAI', TOMOHIKO ENDO AND YUKIFUMI NAWA
Department of Tropical Medicine & Parasitologyl andDepartment of Otorhinolaryngology2, Jikei Umversrty School of Medicme andDepartment of Parasitology, Miyazaki Medical College3

The increase of allergic patients has arisen an issue oflation of I ,283. 26% of residents of this area were infected

the effect of helminth infection in allergy. Our animal ex‑

minth infection suppresses onset of allergic diseases. How‑

with A. suum . The infection was determined by anti‑A.
suum lgG ELISA. 273 peoples received clinical examina‑tion by otorhinolaryngologists. Anti‑cedar lgE was de‑tected in 55% of A. suum‑infected population and 27% of
uninfected population. Cedar pollinosis was diagnosed in
32% of infected and 1 5% of uninfected population. Anti‑
house dust lgE was detected in 74% of infected and 34% of

periments demonstarted that helminth infection induced po‑

tentiated lgE production and the hyper lgE resulted inblockade of lgE receptors on mast cells. The former is aug‑
menting factor and the latter is suppressing factor for al‑

lergy. Many epidemiological studies suggested that hel‑
ever, these studies were performed mainly in developing

uninfected population. Allergic rhinitis by house dust wascountries where bacterial and viral infections were endemic.

diagnosed in 53% of infected and 21 ; o of uninfected popu‑

These infections also interfere allergy.

lation. In both cases positive rate of lgE antibodies and theTherefore, rigid epidemiological study under clean andprevalence of nasal allergy in infected population were

defined circumstances is necessary. Our survey is con‑

twice higher than those in uninfected population. These re‑ducted to evaluate the prevalence of nasal allergy in humans

sults indicate that A. suum infection augments the preva‑

infected with Ascaris suum in Kyushu, Japan. The survey

lence of nasal allergy through potentiated lgE antibody pro‑

was carried out in southern part of Kyushu with total popu‑

duction.

C ‑ 17EPIDEMIOLOGICAL SURVEY ON ECHINOCOCCOSISIN AOMORI PREFECTURE, JAPANHARUO KAMIYA, YASUSHI IHAMA, HIROSHI SATO AND TAKASHI INABA
Department of Parasitology, Hirosaki University School of Medicine

Since alveolar echinococcosis has been seriously en‑

mus spp. and Urotrichus talpoides in Aomori Prefecture

demic in Hokhaido, it is suggested that the disease will be

were investigated, since 1990, no animal infected was de‑spread to Aomori Prefecture near at Hokkaido, most North‑tected.

ern part of Honshu, mainland Japan. In Aomori Prefecture,
therefore, the surveillance system is required to detect in its

2 . Survey on E. multilocularis infection in domestic ani‑malsearliest stage of prevalence for echinococcosis. We carried

A. Pigs: In Aomori Prefecture, nearly 800,000 pigs areout epidemiological survey for Echinococcus multilocularis

going to be killed in a year at the prefectural meat inspec‑

infection among wild animals and also domestic animals.

tion center. During the inspection of 1998, 3 pigs were

1 . Survey on E. multilocular infection is in wild animals

found infected with E. multilocularis . Those pigs were

A. Definitive host: Although from 1 997 to 1999, foxes(Vulpes vulpes japonica), raccoon dogs (Nyctereutes pro‑not originated in Hokkaido and confirmed to be bred in

cyonoid es viverrinus ), martens (Martes melampus

tion source such as infected foxes or porcine feed con‑melampus) and weasels (Mustela sibirica itatsi and M.

taminated with E, multilocularis eggs were somewhere

Aomori Prefecture. This issue indicated that the infec‑

nivalis namiyei) were investigated, no animals werearound the pigpen. It should be stressed that the life cy‑

found infected.

cle of E. multilocularis possibly has been established al‑

B. Intermediate host: Since Microtus montebelli, Apode‑

ready in Aomori Prefecture. Therefore, epidemiological78
survey of the disease in mainland Japan is strongly neces‑

fected with the parasites, the surveillance on dogs andsary for prevention of the spread of E. multilocularis in‑cats must be intensively carried out for the public healthfection among wild animals.

in future .

B. Dogs and Cats: Although no animal was found in‑

C‑18 PRELIMINARY REPORT OF A SURVEY FOR INTESTINAL PARASITOSIS AT
TWO AREAS IN NEPALHIROJI KANBARA*, TETSUO YANAGI*, HANAKO TAKAHASHI', SANDRA JUAREZ ,AI MURAKAMI*, JEEVAN BAHADUR SHERCHAND* AND BASU DEV PANDY+
Department of Protozoology,
Institute of Tropical Medicine, Nagasaki Universityl,

Department of Biodefence Medicine, Faculty of Medicine,
Toyama Medical and Pharmaceutical University2,
Department of Microbiology, Parasitology and Public Health Research Laboratory,
Tropical Diseases Research Center, Teaching Hospital, Tribhuvan University3 andInfectious Disease Hospital4, Kathmanzu, Nepal

We carried out a survey of intestinal parasitosis at a

blia , 14% and 6 : o with Entamoeba histolytica , 5% and 6%

wet season, July and August, 2000 in Nepal to clarify actualwith E. coli, 2% and l( o with lodamoeba butschlii, 29; o andinfectious patterns, especially, of intestinal parasitosis

l% with Cyclospora cayetanensis, and 1% and l% withcaused by Protozoa such as amebiasis, cryptosporidiosis
and cyclosporiasis. We chose two areas for the investiga‑

Blastocystis hominis, respectively. All parasites, even As‑

tion to compare and examine the different environmental

tive correlation between parasites and an age group did notfactors related to infectious patterns. The survey was done

exist in both areas. Cryptosporidium was never detected in

caris , were found out in every age bracket, and any distinc‑

at a slum area along Tripreswor Street in Kathmandu and aany stool samples from both areas even by a floating

rural agricultural village, Khokana, Iocated a few kilometers

method to concentrate oocysts with sucrose. The antibodies

far from the capital. The populations of the slum and theto C. parvum oocysts were examined with immunofluores‑

village were about one thousand and five thousand, respec‑

cence assay, and then the positive rates of lgM, IgG, andtively. We collected and examined 225 stool and serumlgA were O%, 4% and 21% in the slum, and 1%, 0.5% andsamples in the former, and 386 in the latter. The infectious

6% in the village, respectively. Up to now we did not con‑rates in the slum and the village were 32% and 44% withclude anything about the survey study from the last data. In

Ascaridae, 20% and 1 1% with Trichuridae, 3% and 4%
with hookworm, I % and 2% with Hymenolepididae , O%
and 0.2% with Taeniidae, 6% and 13% with Giardia lam‑the near future we are going to accumulate data more tocomplete it.

79

C‑19 PLEVALENCE OF ANTI‑TOXOCARA ANTIBODIES AMONG CHILDREN IN
JABOATAO DOS GUARAPES, PERNAMBUCO, NORTHEAST BRAZILHIROSHI YAMASAKI*, RAOEUL COELT02, LUIZ B. CARVALHO JR.2,
KUNIOKI ARAK13, EMILIA P. PEREZ4, TSUTOMU TAKEUCHI* AND TAKASHI AOKl*
Department of Parasitology, Juntendo University School of Medicinel,

Departamento de Bioqufmica, Laborat6rio de Immunopatologfa Keizo Asami‑LIKA,
Universidade Federal de Pernambuco‑UFPE2
Department of Microbiology, National Institute of Public Health3,

Departamento de Parasitologfa, Centro de Pesquisas Aggeu Magalh es‑FIOCRUZ4
and Department of Tropical Medicine and Parasitology,
School of Medicine, Keio University5

In Brazil, epidemiological surveys on human toxo‑

et al. (2000) except that tetramethylbentzidine was used as

cariasis have been conducted in Sao Paulo, and Pernambuco,

a substrate. Anti‑Toxocara antibodies were detected in 28

northeast Brazil, and high prevalence rates (40%) have beenout of 215 samples when the recombinant T canis antigen
was used. Among these positive cases, 20 were also in‑fected with Ascaris, Trichuris , hookworm and so on. No

repored (Virginia et al ., 1991 ; Moreira‑Silva et al ., 1998).

However, the high prevalence rates were considered to be
due to the low specificity of the excretory‑secretory (ES)antigen used in the surveys. Recently, the authors devel‑parasite eggs were detected in two cases. The data on para‑site infection in the remaining 5 cases were not available. In

oped a recombinant Toxocara canis larval antigen with ahigh specificity for the immunodiagnosis of human toxo‑

order to clarify the specificity of the recombinant antigen

cariasis. In April and May, 2000, the authors had an oppor‑

rologically positive for Ascaris suum adult antigen were se‑tunity to examine the prevalence of anti‑Toxocara antibod‑

lected. In tropical and subtropical areas of the world, as‑ies using the recombinant T canis antigen in Jaboat o dos

cariasis, trichuriasis and ancylostomiasis are the most com‑

Guararapes, near Recife, Pernambuco, northeast Brazil. In

mon parasitic diseases, and crossreactions reduce the reli‑against other parasitic infections, 1 6 children who were se‑the present survey, a tota] of 2 1 5 serum samples from chil‑ability of the results when conventional antigens are used.

dren ages from I to 17 years old were examined for the de‑However, no serum sample from ascariasis crossreacted

tection of anti‑Toxocara antibodies by ELISA. Among 21 5
children, parasite eggs, including the most common species

with the recombinant T canis antigen. From these results,

Ascaris, Trichuris , hookworms, and Schistosoma mansoni,
which are endemic to this area, were detected in 163 cases

is highly specific and provides more reliable diagnostic re‑(82%) and none were found in 35 cases (18010).

ELISA was performed by the procedure of Yamasaki

C ‑ 20it has been confirmed that the recombinant T canis antigen
sults in epidemiological surveys of human toxocariasis.

This study was supported in part by Japan Interna‑
tional Cooperation Agency (JICA).

KILLING OF ACANTHOCHEILONEMA VITEAE MICROFILARIAE
BY MURINE EOSINOPHILISKAZUTO ISHIDA*, HIROKO SUGAYA*, KIMIYUKI TSUCHIYA' AND KENTARO YOSHIMURA*
Department of Parasitology, Akita University School of Medicinel andExperimental Animal Center, Miyazaki Medical College2

which is possibly associated with the killing of Acantho‑of previously infected Millardia meltada whereas murine
eosinophils from the peritoneal cavity of IL‑5 transgenic
mice which had been treated with CdS0+. Microfilarial mo‑cheilonema viteae microfilariae in vitro and in vivo .

tility was assessed visually and its viability by MTT reduc‑This paper attempts to elucidate whether murine
eosinophils are capable of producing nitric oxide (NO)A. viteae microfilariae were harvested from the bloodtion method (Comley et al. , 1989). Production of NO by

80

eosinophils was assayed by measuring the accumulation ofphils were more effectively activated when both IFNY andnitrite during culture using Griess reaction (Ding et al. ,LPS were simultaneously present in the culture.

1988).

To determine the possible association of eosinophils

When A. viteae microfilariae were co‑cultured with
IFNY‑ or LPS‑activated‑eosinophils, they showed more re‑mice and their counterpart normal C3H/HeN mice, were in‑duced viability, in terms of morbidity and survival rate, thantravenously injected with microfilariae and their peripheral

those co‑cultured with non‑activated eosinophils. IFNY‑

microfilarial levels monitored. Interestingly, IL‑5 trans‑

activated eosinophils produced a significantly higher
amount of nitrite in the culture supernatants than non‑filarial density, with its almost complete disappearance by

activated eosinophils. The addition of aminoguanidine, in‑day 14 postinjection, whereas, in C3H/HeN mice, microfi‑

hibitor of NO synthesis, to the assay system caused a recov‑

larial density remained very high up to this time.

with microfilaremia in vivo , eosinophilic IL‑5 transgenic

genic mice provoked a rapid reduction in peripheral micro‑

ery of microfilarial viability and a reduced nitrite produc‑

These data suggest that eosinophils are involved in thetion by eosinophils. The degrees of NO production and mi‑
crofilaricidal activity by activated eosinophils are correlated

disappearance of A. viteae microfilariae from peripheral
blood, and also that NO derived from eosinophils could be awith the numbers of eosinophils used. Moreover, eosino‑

candidate effector molecule for killing microfilariae.

C‑21 KINETICS OF MYOREGULATORY FACTORS IN THE NURSE CELL FORMATIONO F TRICHINELLAYUZO TAKAHASHI, ATSUSHI MATSUO, TAKUMI NAKADA,ZHILIANG WU AND ISAO NAGANO
Department of Parasitology, Gifu University School of Medicine

It is well known that establishment of parasitism ofanalysis of such regulatory factors after infection with T

Trichinella results in transformation of muscle cells lead‑spiralis and T pseudospiralis . MyoD and myogenin, origi‑

ing to the nurse cell formation. This transformation can be

confirmed in both capsule forming species (T spiralis) and

nated from satellite cells, were expressed from the earlyphase of cystogenesis in T spiralis infection, during which

non‑capsule forming species (T pseudospiralis). The basic

time satellite cells were most activated. The expression re‑question resides in why terminally differentiated cells suchturned to the normal level after 1 8 days from the inf ection

as muscle cells can differentiate to another phenotype thatwhen the cyst was complete, and no satellite cells were acti‑

has never been anticipated before the infection and probably

vated. In T pseudospiralis infection, they were also ex‑

never encoded in host's DNA.
In this study we developed a detection system for myo‑pressed from the early phase of cystogenesis, but continu‑

genic regulatory factors such as MyoD, myogenin and
MRF4. Adapting the method we performed a longitudinal

infection, satellite cells were continuously activated. MRF4,

ously expressed at least up to 43 days post infection. In thisoriginating from muscle cells, was always highly expressed.

81

C ‑ 22

THE PREVALENCE OF MICRO‑ALBUMINURIA IN DIABETIC PATIENTSATTENDING THE MULAGO HOSPITAL DIABETIC CLlNlC (MHDC),KAMPALA, UGANDAIBRAHIM KABOLE, A.P. NAMBUYA, E. KIGONYA AND M.A. OTIM
Department of Medicine, Faculty of Medicine, Makerere University,

Kampala, Uganda
Background: One of the most serious complications of dia‑tended the MHDC during the months of January and Febru‑

betes mellitus is diabetic nephropathy. If detected early,

ary 1999 and fulfilled the study criteria. One hundred andhowever, this complication can be arrested or even reversed

twenty seven patients selected from the MHDC were en‑with appropriate intervention. Micro‑albuminuria is usuallyrolled in the study. These patients were randomly selected

an early marker of this complication and also a signal forfrom among the patients attending the MHDC during theintervention. Unfortunately, routine monitoring of micro‑

study period. Sixty‑four of these were Type I diabetics andalbuminuria is not done in the Mulago Hospital Diabetic
Clinic (MHDC), and therefore the prevalence of micro‑
albuminuria among patients attending MHDC is not known.

to 79 years, and they were not known to have any overt

The rationale of this study was to determine the prevalence

Results: The prevalence of micro‑albuminuria was 69%.

sixty‑three were Type 11 diabetics. Their age range was 20complications of severe renal disease.

of micro‑albuminuria among patients attending MHDC, toOverall, the level of glycaemic control was poor among theprovide some insight into the magnitude of incipient neph‑study patients, with 53% of the patients having haemoglo‑
binAlc, of 1 1 % and above, representing very poor control.

ropathy, which is a stage of transition to full blown diabetic

nephropathy.
Study Objectives: The objectives of this study were to de‑Ninety four percent of the patients with micro‑albuminuria

termine the prevalence of micro‑albuminuria among dia‑patients had mild to moderate hypertension. The majority

also had raised haemoglobinA*c̲ Seventy‑five (60%) of the

betic patients attending the MHDC, to find out some of the(68%) of the patients with hypertension also had micro‑

factors associated with micro‑albuminuria, and to estimate

albuminuria.

the level of glycaemic control among the study patients.

Conclusion: Micro‑albuminuria was a common fmding

Design: This was a cross‑sectional descriptive study in‑volving diabetic patients attending the MHDC.

among the study patients. This may suggest that incipientnephropathy is common among diabetic patients attending

Setting: The study was carried out in the MHDC Iocated in

the MHDC. The implication of this finding is that if appro‑

the medical outpatient department of New Mulago Hospital,

priate measures are not put in place, these patients are likely

Kampala.

to develop overt diabetic nephropathy.

Subjects: The study subjects were diabetic patients who at‑

C‑23 HEALTH CARE SURVICE FOR JAPANESE LIVlNG IN CITIESOF SOUTHEAST ASIA
YASUTAKA OGAWA* , RIE YOSHIDA' , CHIKAKO ARAKAWA* AND HIROSHI OHTOMO'
Divlsion of Hazard Assessment, National Institute of Industrial Healthl andDepartment of Tropical Medicine, Jikei University School of Medicine2

Purpose: We planed a survey aiming to understand theand sixty six families responded from 4 cities, 44 (H), 32

needs of medical health service for Japanese staying in Ha(P), 3 1 (V) and 59 (Y), respectively.

Noi (H), Phnom Penh (P), Vientiane (V), and Yangon (Y).

Methods: Questionnaire consists of questions asking family

Subjects: Questionnaires were distributed to transient Japa‑

status, about satisfaction of climate and housing, the num‑

nese residents or permanent Japanese residents throughber of servants, who cook meals, about drinking water,

each regional association of Japanese Society. One hundred

about satisfaction of child life, about their offered medical

82

services from employee, about local medical services, andabout their desired medical services.

Results: The percentage of the multiple member households

compared with other three places. Percentage of people
who desired the medical services from Japan at 4 cities
were 65.9% (H), 53.1% (P), 77.4% (V) and 71.1% (Y), re‑was highest at Ha Noi (86.3%) and that of the single mem‑

spectively. The levels were high at all 4 cities and there

ber household was highest at Yangon (55.5%). The percent‑age of people who were unsatisfied with hygienic level ofwere no big differences between them. Desired services
were obstetric, gynecologic, pediatric and dental cares,

their houses at 4 cities were 29.5% (H), 28.10/0 (P), 45.1%

which are thought to have less priority to employers.

(V) and 62.7% (Y), respectively. Yangon was highest com‑pared with other three places. About 970/O of Japanese ofConclusion: In conclusion, we surveyed by questionnaire toknow the needs of medical services for Japanese living ineither city bought drinking water or boiled their water be‑

Ha Noi, Phnom Penh, Vientiane and Yangon. The results

fore drinking. The percentage of people who were unsatis‑suggested that the level of unsatisfaction depended on spe‑

fied with medical services offered from there employee at 4

cific socio‑economical and political status of each country,

cities were 36.3% (H), 15.3% (P), 31,8% (V) and 43.1%

such are economical spurt in Vietnam, politically unstable‑(Y), respectively. It should be noticed that politically unsta‑

ness in Cambodia, economical retardation in Laos, and eco‑ble Cambodia was better compared with other three places.
The percentage of people who were unsatisfied with local

nomically stagnated because of politically closed to outer

medical services at 4 cities were 38.1% (H), 53.1% (P),
58.0% (V) and 66.1% (Y), respectively. Although the level

more medical services from Japan, especially the areasof unsatisfaction was high at all cities, Ha Noi was lowest

and dentistry.

world in Myanmar. Japanese living in either country wants
where employers neglect; those are gynecology, pediatrics,

C‑24 EVALUATION OF GIDEON FOR IMPORTED INFECTIONSMITSUO SAKAMOTO*, MIKIO KIMURAI AND HIROKO SAGARA'
Department of Infectious Disease, Yokohama Municipa] Citizens Hospitall

andDepartment of Infectious Disease & Applied Immunology,
Institute of Medical Science, The University of Tokyo

Object: Imported infections are increasing with the number

Citizens Hospital and the Department of Infectious Diseasesof overseas travelers increases. Quick and accurate diagno‑& Applied Immunology, Tokyo University were eligible forsis of these diseases imported infections is crucial. How‑

medical institutions in Japan. Therefore, aggravation ofthis study. Imported infections were malaria (n=29), ty‑
phoid or paratyphoid fever (n=23), dengue fever (n=16),
bacillary dysentery (n=12), hepatitis A (n=8), Mediterra‑these diseases due to delay of correct diagnosis and not only

nean spotted fever (n=2), Lyme disease (n=1), and Lassa fe‑

the death of infected patients but also the risk of secondlyver (n= I ).

infection to those around the infected patients are possible.

Results: Of all cases, the accuracy of diagnosis with correct

Under such circumstance, development of aid system that

diagnosis ranked any order was 90.2%. Correct diagnosis

can diagnose imported infections correctly is needed seri‑

was ranked f"rrst and second to fifth in 5 1 . I o/c and 29.3%, re‑ously. GIDEON is a comprehensive software developed by
C.Y. Informatics Ltd. for information gathering, diagnosis,

spectively. The accuracy of diagnosis was highest for ty‑
phoid or paratyphoid fever (100%), Mediterranean spotted

and treatment of imported infections. The diagnosis mod‑fever (100 fO: only two cases), dengue fever (93.8%), ma‑ule in GIDEON is a computer program that can diagnose

laria (93.1%), and bacillary dysentery (91.7%). As forpatients based on the overseas travel history, the symptoms,

Lassa fever (the sole case in Japan), the diagnostic result ofand the findings of clinical findings of a patients. We stud‑

GIDEON was deniable due to the inflammation of salivary

ever, the awareness toward imported infections is poor in

ied the diagnostic accuracy of GIDEON retrospectively,

gland but excluding this inflammation from data inputs, thebased on its diagnostic results on the real cases.

diagnostic result was Lassa fever with 100% of possibility.

Materials and Methods: Ninety‑two admission cases who

Discussion: The problem of GIDEON was that it diagnosed

have been confirmed diagnosis as imported infections at thethe correct diagnosis as "not deniable but rare" or as deni‑

Department of Infectious Diseases Yokohama Municipal

able" in some cases. GIDEON diagnosed the cases with

''

83

malana as "deniable" if the cases had sore throat and rela‑rare. However, many of these incidents diagnosed "rare"tive bradycardia. GIDEON also diagnosed the cases withby GIDEON were not rare in reality.

typhoid or paratypoid fever as "deniable" if the cases had

GIDEON was useful in diagnosing imported infections

hematuria. Decrease of platelets, Iiver dysfunction, infec‑

to some extent. However it has various problems. Relying,, bytion in India, Thailand and Viet Nam were judged''rare

diagnosis only upon GIDEON would be very dangerous,

GIDEON for typhoid or paratyphoid fever. In the caseswith hepatitis A and bacillary dysentery, GIDEON diag‑but it was worthy enough in diagnosing patients from broad

nosed that severe illness and necessity of admission were

cates into consideration.

C ‑ 25point of view, with taking much information GIDEON indi‑

THIRTEEN‑YEARS EXPERIENCE OF THE JICA TUBERCULOSIS CONTROL IN NEPAL
KATSUNORI OSUGA*, NORIO YAMADA*, MIWA NAKAGAWA', NOBUKATSU ISHIKAWA' ,TORU MORI*, MASAKAZU AOKI' AND TADAO SHIMAO*
Research Institute of Tuberculosisl , Japan Anti‑Tuberculosis Association2

and Japanese Foundation for AIDS Prevention3

In Nepal tuberculosis has been a major public healthproblem among adult population, with the Annual Risk of
TB Infection (ARTI) calculated as 2.2%. Dr. Noboru lwa‑
mura, who came to Nepal in early 1960s, was one of thepioneers in this field. To tackle the problem at the national

level, technical cooperation by JICA started in 1987. First,

completion rates were highest in the health facility‑DOT.
Therefore the strict DOT based at health facilities was cho‑sen as the method for introduction of DOTS in Nepal in
1996. Four pilot areas were selected (National DOTS Dem‑
onstration Area) in the plain to introduce strict healthfacility‑based DOTS. All the pilot areas were able to

the National Tuberculosis Centre (NTC) was constructed inachieve the high cure rate of over 85%. Encouraged by the

Kathmandu Valley to establish the headquarters for the Na‑

initial result, the NTP gradually expanded DOTS coveragein the plain. High cure rate was continuously observed intional Tuberculosis Programme (NTP). Suitable strategies
to combat TB with were then explored in the project pilot
areas. After the official adoption of the Directly Observed

the expansion areas. While the DOTS strategy was success‑fully introduced in those areas, where the access to health

Treatment, Short Course (DOTS) strategy by the NTP incare facilities is relatively good, geographically difficult ar‑

1995, the project's strategies also shifted to focus on DOTS.

level to strengthen the management of the NTP. To develop

eas such as hilly and mountainous areas remained uncov‑
ered by DOTS. People living in those areas would have towalk for many hours to receive DOT at the nearest healthhuman resources, training was provided at the Research In‑

f acility.

stitute of Tuberculosis (RIT) for program managers andlaboratory technologists. The RIT also provided long andused in some areas, the JICA project utilized the Female

At the same time the project provided assistance at central

short‑term consultants on TB control to Nepal.

Necessary supplies and equipment, such as TB drugs,
microscopes, X‑ray machines, vehicles, etc. were also pro‑

While hospitalization during the intensive phase isCommunity Health Volunteers (FCHV) as treatment super‑visors in those areas. The FCHV are unpaid volunteers,
working in the community for the benefit of the community.

vided for the smooth operation of the project. The goals
and the targets of the project were set in accordance with

By spending time and money for transportation on theirown, the FCHV collects medicine once a week to give DOT

the WHO strategy; 70% case detection rate and 85% cureat the patients' residence in their own villages. Perhaps

rate of the new smear positive pulmonary tuberculosis cases.

there is no single solution to the problem of DOTS in geo‑

Overall case detection rate reached approximately 60%.
Analysis was made to compare ways of supervision of TB
patients' drug taking. When the health faci]ity‑DOT wascompared with the family‑DOT and non‑DOT, the cure andgraphically difficult areas. Suitable methods should be se‑lected in each community, to improve the coverage of
DOTS in the country.

84

C ‑ 26

DEVELOPMENT OF URBAN DOTS IN CHITTAGONG CITY OF BANGLADESHNOBUKATSU ISHIKAWA
Research Institute of Tuberculosis, Japan Anti‑Tuberculosis Association

Tuberculosis is a major public health problem in Bang‑nearest clinic, microscopic centers are increased in number,ladesh and effective program called DOTS has recently

recording/reporting system is standardized, training is given

been successfully implemented in rural areas. However, TB

to all concerned staff, and so on. As a research component,

control system has not yet been established in most of thethe progress has been periodically monitored, and evalu‑

big cities as in many other developing countries. The Chit‑

ation workshops have been held every 6 months throughtagong City is the second largest city in Bangladesh, withparticipatory process. After the operation since 1996, pa‑

the population of 3.3 million. 300/0 of the population istients diagnosed at the TB clinic were referred back to thethought to be a slum area. The City has a Chest Clinic/TB

nearest city clinic to have a TB drug under DOT. NGO

Clinic, 32 urban health centers and NGO clinics. Estimatedclinics were also involved for DOT. The TB services have

TB patients are 3,000 per year. Before the project, there
was no systematic DOTS in the city and majority of the pa‑

been more decentralized. As the outcome, the number of
sputum positive patients increased up to above 500; thetients used only TB clinic. TB program used to be under

number of the patients registered at urban city centers is in‑

the central government program. But due to the decentrali‑creasing while that in TB clinic is decreasing. And theirzation and health sector reform, Iocal government has be‑treatment success rate has been improving above 70%.

come fully responsible for TB program. Before new system

Achievements are: overall decentralization is going on;

was started, TB clinic diagnosed nearly 600 patients, ofwhom only 17̲5 were sputum AFB positive and their treat‑

treatment success is improving; case finding increased, but

ment completion was only 32 ; c. Thus the service quality
was very poor. Our institute started an operational researchcommitment by city corporation has increased, but not suf‑

to demonstrate a model TB control system DOTS for urban
area in Bangladesh with central government (DOH), Chit‑and DOH; to form city TB control committee under city
mayor and to nominate TB program manager; to renew thedevelopment plan; to hold periodic review meetings; andtagong City, BRAC and WHO. In the beginning, all com‑ponents of DOTS was introduced by the initiative of thecentral governmnet; namely, drugs are supplied by the cen‑about 500 is still low against 3,000 estimated; politicalficient. For further development, we recommend to the city

external supports both technical, moral and financial are
still needed from central government, WHO and elsewhere.

tral government; patients are given drugs under DOT at the

C‑27 IMMUNOHISTOCHEMICAL IDENTIFICATION OF PHENOLYC
GLYCOLIPID‑1 (PGL‑1) IN TISSUES OF BURULI ULCER PATIENTS
M. MWANATAMBWEl, M.YAJIMA2, K. SUZUK12, S. ETUAFEL3, K. HIBIYA1,

KAZUHIRO SHICHINOHE4, MASUMI SHIMIZU‑SUGANUMA4. K ASIEDU5,
N.YAMADAI AND G. ASANOl
Department of Pathologyl and Laboratory Animal Sciences4,

Nippon Medical School,
National Leprosy Research Institute2, St. Martin Hospital3 andGrobal Tuberculosis Program, WH05

Buruli ulcer is a skin necrotizing disease caused bybacterial infection are highly suspected. Phenolyc glycolip‑

Mycobacterium ulcerans , which is closed to M. Ieprae andids play crucial roles in the survival of some mycobacteria

M. tuberculosis . Although a toxin of the polykritide family

in phagocytic cells of infected patients. We sought to inves‑

has been incriminated in the pathogenesis of the disease,tigate the immunoreactivity of monoclonal antibodies to
PGL‑ I , which was once believed to be specific to M.

phases of defect in the inflammatory reaction to the myco‑

85

leprae, in tissues of patients infected with M. ulcerans.

formly detected as strongly positive, in a confluent pattern,

Thirty specimens of surgically obtained skin flaps from pa‑

mimicking clusters of AFB, regardless of the clinical form

tients clinically diagnosed as Buruli ulcer, originating fromof the disease. The antigen was detected around extracellu‑

the Ashanti County of Ghana, were classified as follows:

lar and intracellular AFB (Foamy macrophages, adipocytes)

Plaqes 3 cases (10%), nodules 10 cases (33%), ulcerated

distributed in necrotic dermal tissues, fragmented collagennodules I case (3%), deep ulcer bed 7 cases (23%), healing

bundles, fatty lobules, septae and occasionally in wall ofulcer 9 cases (30%). Eighty‑three percent of submitted
specimen were histopathologically compatible with infec‑

role of PGL‑ I , in phases of Buruli ulcer where the striking

tion by M. ulcerans . Acid fast bacilli (AFB), were demon‑

blood vessels. These findings suggest at first, the possible

strated by Fite‑Faraco and Harada stains in I plaque, 5 nod‑absence of reactive inflammatory response has been ob‑
served. Secondly, they are another indication of the nonules, 4 deep ulcer beds, 2 healing ulcers and O ulcerated

specificity of this antigen to a specific member of the Genus

nodules. In all AFB positive specimens, PGL‑ I was uni‑

Mycobacterium .

VOl．29NO、1

MARCH−2001

CONTENTS
Originala血cle
He，H．，Zhang，R，Kawaguchi，H．，Ybshida，A．，Itoh，M．，Chen，Y

andOhta，N．

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