JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
０巻

第１号
内

Field evaluation of a rapid immunochromatographic test ‘Parachek-F’ in a post-monsoon
Plasmodium falciparum malaria outbreak in villages of South India
SUSANTA KUMAR GHOSH, TIRUCHINAPALLI SUNDARAJ SATHYANARAYAN,
１
３
MALALIKOPPA VEERABHADRAPPA MURUGENDRAPPA and SARALA KUMARI SUBBARAO …………７‐
旋毛虫のミオシンヘビー鎖の抗原性ペプタイド
５‐２１
中田 琢巳，長野
功， ! 志良，高橋 優三 …………………………………………………１

第４
２回日本熱帯医学会大会英文抄録 …………………………………………………………………………２
９‐
８
５

March２００２

日本熱帯医学会表紙!−" ＭＫ 中島 稲Original article
Ishih, A., Sakai, M., Takezono, H., Fujii, K., Sano, M., Asanuma, N., Miyase, T., and Terada, M.
A Potent Antimalarial Activity of Hot-Water Extrects of Plants Belonging to the Family
Saxifragaceae Against Plasmodiumyoeii 17XL in Icr Mice…………………………………………１‐６
Ghosh, S. K., Sathyanarayan, T. S., Murugendrappa, M. V. and Subbarao, S. K.
Field-Evaluation of a Rapid Immunochromatographic Test ‘Parachek-F’ in a Post-Monsoon
Plasmodium Falciparum Malaria Outbreak in Villages of South India
Indigenous diagnostic test kit for P. falciparum ……………………………………………………７‐１３
Nakada, T., Nagano, I., Wu, Z. and Takahashi Y.
An Antigenic Peptide of Myosin Heavy Chain-Like Protein from Trichinella Spiralis ……………１５‐２１
Mitarai, S., Habeenzu, C., Lubasi, D., Kafwabulula, L. M., Kasolo, F. C., Ichiyama, K.,
Terunuma, H., Ito, M., Shishido, H. and Numazaki, Y.
Drug Susceptibilities and Clinical Manifestations of Mycobacterium Tuberculosis in Zambia ……２３‐２８Proceeding of 42st Annual Meetings of Japanese Society of Tropical Medicine………………………………………………………………………………………………………２９‐８５Vol. 30 No. 1

日 本 熱 帯 医 学 会

Hygiene

日熱医会誌

and

会報・記録
日本熱帯医学会会則 ………………………………………………………………………………………８
７
‐
９
０
２
０
０
１（平成１
３）年度日本熱帯医学会役員名簿（２００
２年３月１日現在） ……………………………９１
日本熱帯医学会雑誌編集委員名簿 ………………………………………………………………………９２‐
９３
投稿規定 ……………………………………………………………………………………………………９４‐
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著作権複写に関する注意 …………………………………………………………………………………９６
日本医学会への加盟申請についての公示 ………………………………………………………………９
７
日本医学会だより ……………………………………………………………………………………………９
８

Medicine

ザンビア共和国における結核菌の薬剤感受性と臨床病態
御手洗 聡，Charity Habeenzu, David Lubasi, Lindon M Kafwabulula, Francis C Kasolo,
市山 浩二，照沼裕，伊藤 正彦，宍戸 春美，沼崎 義夫 ……………………………２３‐
２
８

Journal of Tropical

著
ユキノシタ科に属するアジサイ近縁植物の水煮沸抽出液の抗マラリア活性
石井明，酒井美香子，竹添 裕高，藤井香，佐野 真規，浅沼 賀洋，
宮瀬 敏男，寺田
護 ……………………………………………………………………………１‐６

MARCH ２０
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CONTENTS

容
Japanese

原

VOL．
３
０ No．
１

平成１
４年３月

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は４mm

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Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
１,２
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０２, pp.１-６

A POTENT ANTIMALARIAL ACTIVITY OF HOT‐WATER EXTRACTS OF
PLANTS BELONGING TO THE FAMILY SAXIFRAGACEAE AGAINSTPLASMODIUM YOELII 17XL IN ICR MICEAKIRA ISHIH1, MIKAKO SAKAI1, HIROTAKA TAKEZOE1, KAORI FUJII1, MASAKI SANO1,NORIHIRO ASANUMA1, TOSHIO MIYASE2 AND MAMORU TERADA1
Received July 4, 2001/Accepted March 9, 2002
Abstract: Hot-water extracts of 7 plants of the family Saxifragaceae; Hydrangea macrophylla var. Otaksa, H.
macrophylla, H. macrophylla serrata var. acuminata, H. involucrata, H. hirta, H. paniculata, and Cardiandra alternifolia, were screened for antimalarial activity in ICR mice infected with Plasmodium yoelii 17XL. The leaf extract of H. macrophylla var. Otaksa or H. macrophylla had an antimalarial activity against rodent malaria, showing
low parasitemia levels during administration. Following a transient recrudescence of malaria parasites in the
bloodstream of treated mice, no parasites could be detected by a microscopic examination. Mice treated with theleaf extract of H. macrophylla var. Otaksa or H. macrophylla survived during the experiment, though mice in the
non-treated control and other treated groups died with a gradual body weight loss from day 6 to day10 post infection. Furthermore, the antimalarial activity of the hot-water extract of H. macrophylla leaf extract seemed higher
than that of H. macrophylla var. Otaksa leaf extract in respect of the degree of suppression of parasite multiplication and of mouse body weight loss.
Key words: Saxifragaceae plants, Plasmodium yoelii 17XL, antimalarial activity, hot-water extract

INTRODUCTION
Malaria is still one of the major health problems in theworld. The situation worsened with the emergence ofchloroquine-resistant variants of Plasmodium falciparum,and their spread across the globe has stimulated the development of new effective treatments against malaria (Payne,
1987; Winstanley, 2000). In 1972 artemisinin was isolated
from the herb Artemisia annua L. used in traditional Chinese medicine as remedy for chills and fever for more than2000 years (Klayman, 1985). Isolation of artemisinin andthe development of more effective derivatives from thisdrug (Van Agtmael et al ., 1999) have led to further trial for
identifying other medicinal compounds of potential as antimalarial agents from natural products (Iwu et al ., 1986;
Isaka et al ., 1999; Rahman et al ., 1999).
We thus began to investigate experimentally with rodent malaria models antimalarial activity of the plants used
experientially as ingredients of various prescriptions in the1

traditional medical remedies in China and Japan for chills
and fever as antimalarial agents. In a continuation of ourresearch program, the hot-water extract of leaves of Hydrangea macrophylla var. Otaksa showed a high in vivo antimalarial activity against Plasmodium yoelii (Ishih et al .,2001). Furthermore the fraction including the mixture of
febrifugine and isofebrifugine having antimalarial activity
was isolated from leaves of Hydrangea macrophylla var.
Otaksa (Takezoe et al ., 2001). These compounds had already been isolated from Dichroa febrifuga (Koepfli et al .,1947) and from H. umbellata (Abolondi et al ., 1952). Froma practical viewpoint of folk medicine, it is therefore of interest to evaluate the antimalarial activity of plants of thefamily Saxifragaceae which are widely distributed throughout Japan and are not used as antimalarial folk medicines inJapan. This paper deals with an in vivo antimalarial evaluation of the Saxifragaceae plants using ICR mice infectedwith the rodent malaria parasite P. yoelii 17XL to seekplants with large amount of alkaloids, febrifugine andDepartment of Parasitology, Hamamatsu University School of Medicine, Hamamatsu 431-3192
Department of Pharmacognosy, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
Correspondence to: Akira ISHIH, PhD
Department of Parasitology,
Hamamatsu University School of Medicine,
1-20-1 Handayama, Hamamatsu 431-3192, Japan
FAX: 81-53-435-2337
Email: aishih@hama-med.ac.jp2

２

isofebrifugine, or with another antimalarial principles.

MATERIALS AND METHODS
Plant materials
Six species were selected and tested for in vivo antimalarial activity using rodent malaria parasite. The plants
specimens such as Hydrangea macrophylla Thunb. Ser., H.
macrophylla Seringe subsp. serrata Makino var. acuminata
Makino, H. involucrata Sieb., H. hirta Sieb. et Zucc., H.
paniculata Sieb., and Cardiandra alternifolia Sieb. et Zucc.,
were collected in and around the campus of Hamamatsu
University School of Medicine in 1999 or from the Botanical Garden, University of Shizuoka in 1999. H.macrophylla
Seringe var. Otaksa Makino was also used to compare theactivity with other plants.
Hot-water extract
Extracts of the family Saxifragaceae plants:
Five g of air-dried leaves or 20 g of air-dried roots of 6
plants except C. alternifolia, or 5 g of air-dried wholeplants of C. alternifolia put in a herbal bag (Tokiwa Industry) was added with 500 ml of distilled water and boiled.
After removing leaves, roots or whole plants the extract
fluid was finally concentrated to 50 ml . Each hot-water extract (equivalent to 0.1 g/ml extract and 0.4 g/ml extract,
respectively) was stored at 4°
C until use.
Leaf extract of H. macrophylla or H. macrophylla var.
Otaksa :Five, 2.5 and 1.25 g of air-dried leaves of each plant
put in a herbal bag was added with 500 ml of distilledwater and boiled. After removing leaves the extract fluid
was finally concentrated to 50 ml . Each hot-water extract(equivalent to 0.1 g/ml extract, 0.05 g/ml extract and 0.025
g/ml extract, respectively) was stored at 4°
C until use.
In vivo antimalarial activity of hot-water extract
All animal experiments were performed according to
the Guidelines for Animal Experimentation, Hamamatsu
University School of Medicine. Outbred male ICR mice, 8
weeks old, purchased from Japan SLC Inc., were used. For
experiments, the blood stage parasites of Plasmodium yoelii(strain 17XL) from frozen stock were injected into two
mice. The mouse showing 10-15% of parasitemia was bled
under ether anaesthesia to collect P. yoelii 17XL-parasitizedblood. Experimental mice were infected intraperitoneallywith 1x105parasitized erythrocytes. Infected mice treatedwith test samples were monitored for % parasitemia anddays of survival relative to control mice up to day 60 post
infection.
Antimalarial activity of extracts of the family Saxifragaceae
plants:To evaluate antimalarial activity of hot-water extract ofplants, infected ICR mice were divided into three per groupfor activity assay of each hot-water extract, and five mice
were used as non-treated, infected control. Starting on day
3 after injection of parasitized erythrocytes, mice were inoculated through an esophageal tube with a hot-water extract at 75 µl /10 g body weight (b.wt.) in the treated groupand distilled water in the non-treated, infected one, respectively, twice a day for 5 consecutive days.
Comparison of antimalarial activity of the leaf extract of H.
macrophylla with that of H. macrophylla var. Otaksa :Infected mice were divided into four per group for activity assay of hot-water extract (0.1 g/ml extract, 0.05 g/ml
extract or 0.025 g/ml extract), and five mice were used as
non-treated, infected control. Starting on day 3 after injection of parasitized erythrocytes, mice were orally given a
hot-water extract at 75 µl /10 g b.wt. in the treated groupand distilled water in the non-treated, infected one, respectively, twice a day for 5 consecutive days.

RESULTS
Antimalarial activity of the hot-water extract of thefamily Saxifragaceae plants
The results of the investigation of the antimalarial activity of plant extracts in vivo are summarized in Table 1.
Infected mice were orally given each of 13 different extracts
from 7 plants twice a day for 5 consecutive days from day 3
after inoculation of the parasitized erythrocytes. Of 13 extracts, the leaf extract of H. macrophylla var. Otaksa or H.
macrophylla showed an antimalarial activity, although otherextracts showed no effects. Mice in the non-treated control
and the treated groups except one given the extract of H.
macrophylla var. Otaksa or H. macrophylla leaves diedfrom day 5 to day 10 post infection with a gradual bodyweight loss. However, in the groups given H. macrophyllavar. Otaksa extract and H. macrophylla extract, respectively,
the body weight gradually decreased from day 9 and day10,
respectively, but turned to increase in several days, and all
mice survived during the experiment.
Comparison of antimalarial activity of the leaf extract of
H. macrophylla with that of H. macrophylla var. Otaksa
As shown in Fig.1, mice in the non-treated control andgroups given either 0.05 g/ml or 0.025 g/ml extracts diedfrom day 6 to day10 post infection with a gradual bodyweight loss. However, in the group given 0.1 g/ml extractof H. macrophylla var. Otaksa leaves and H. macrophylla
leaves, respectively, the body weight gradually decreased
from day 10 and day 12, respectively, but turned to increasein a few days, and all mice survived during the experiment.
Malaria parasites appeared from day 3 post infection in the
bloodstream of the control and treated groups (Fig. 2). Para-

３sitemia of each mouse in the control and the groups given
either 0.05 g/ml or 0.025 g/ml extract of both plants gradually increased and all of the mice died by day 10. Micetreated with 0.1 g/ml extract of H. macrophylla var. Otaksa
leaves or of H. macrophylla leaves showed low parasitemia
levels during medication. On day 9, however, malaria parasites increased in the bloodstream of the mice treated with
H. macrophylla var. Otaksa leaf extract and each peak parasitemia was more than 50%. Then the parasitemia level decreased and on day 22 parasites in the treated mice could
not be detected by a microscopic examination. On the other
hand, in the bloodstream of mice given H. macrophylla leaf
extract, parasites increased on day 10, and although one
mouse showed 30% of peak parasitemia, other mice showed
less than 10% of parasitemia. Then the parasitemia decreased and on day 22 parasites in the treated mice could
not be detected by a microscopic examination.

DISCUSSION
Our previous study revealed the presence of compounds having an antimalarial activity in H. macrophyllavar. Otaksa leaf extract (Ishih et al ., 2001). Our present results indicate that among 7 plants belonging to the family
Saxifragaceae the hot-water extract of H. macrophylla
leaves or of H. macrophylla var. Otaksa leaves has a potent
antimalarial activity against Plasmodium yoelii 17XL in
ICR mice using approximately 5-fold dosage of clinical one
prescribed in the traditional remedies in China. Hot-water
extract of H. macrophylla leaves seems to have the sametypes of effects on malaria parasites in vivo as the extract of
H. macrophylla var. Otaksa leaves had. Furthermore, theantimalarial activity of the hot-water extract of H. macrophylla leaf extract seemed higher than that of H. macrophylla var. Otaksa leaf extract in respect of the degree ofsuppression of parasite multiplication during medication
and at a recrudescence, and of mouse body weight gain/loss.
This extract exhibited marked suppression of multiplicationof parasites during medication, indicating that the extract

Table Antimalarial screening of plant extracts using Plasmodium yoelii 17XL in ICR mice
Plant species

Ranges of survival days*-

No. mice survived/No. mice used
0/5

Hydrangea macrophylla
Seringe var. Otaksa Makino

Leaves
Roots

3/3
0/3

60
5-7

Hydrangea macrophylla
Thunb. Ser.

Leaves
Roots

3/3
0/3

60
4-5

H. macrophylla
Seringe subsp. serrata Makino
var. acuminata Makino

Leaves
Roots

0/3
0/3

5
4-6

H. involucrata Sieb.

Leaves
Roots

0/3
0/3

5-6
5

H. hirta Sieb. et Zucc.

Leaves
Roots

0/3
0/3

5-7
5-9

H. paniculata Sieb.

Leaves
Roots

0/3
0/3

5-6
5

Whole plants

0/3

5-6

Untreated infected control

Cardiandra alternifolia
Sieb. et Zucc.

Parts tested

5-8Mice were orally given a hot-water extract at 75 µl /10 g body weight in the treated group and distilled water in the untreated, infected one,respectively, twice a day for 5 consecutive days from day 3 post infection with 105 P. yoelii 17XL-parasitized erythrocytes.
*Number of surviving mice was monitored until day 60 post infection.

４

Figure 1 Body weight profile of each mouse in response to treatment with 3 different concentrations of hot-water extracts (at 75 µl /10 g b.wt.) of H. macrophylla var. Otaksa and H. macrophylla. P.yoelii 17XL-infected mice
were orally given leaf-extract, twice a day, for 5 consecutive days from day 3 to day 7 after inoculation intraperitoneally with 105 parasitized erythrocytes. Control group (A) of mice received an equivalent volume ofwater. Each symbol represents an individual mouse. H. macrophylla var. Otaksa leaf extract; 0.1 g/ml (B),0.05 g/ml (C), 0.025 g/ml (D): H. macrophylla leaf extract; 0.1 g/ml (E), 0.05 g/ml (F), 0.025 g/ml (G).

５

Figure 2 Parasitemia profile of each mouse in response to treatment with 3 different concentrations of hot-water extracts (at 75 µl /10 g b.wt.) of H. macrophylla var. Otaksa and H. macrophylla. P.yoelii 17XL-infected mice
were orally given leaf-extract, twice a day, for 5 consecutive days from day 3 to day 7 after inoculation intraperitoneally with 105 parasitized erythrocytes. Control group (A) of mice received an equivalent volume ofwater. Each symbol represents an individual mouse. H. macrophylla var. Otaksa leaf extract; 0.1 g/ml (B),0.05 g/ml (C), 0.025 g/ml (D): H. macrophylla leaf extract; 0.1 g/ml (E), 0.05 g/ml (F), 0.025 g/ml (G).

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has a direct action on parasites. Most of the antimalarials
work against the late trophozoite and schizont stage of themalaria parasite and thus the number of asexual parasites
decreases. Artemisinin derivatives also work against early
trophozoite and ring stages (Van Agtmael et al ., 1999).
Febrifugine and isofebrifugine being active principles
against malaria were isolated from the roots of D. febrifuga
used in Chinese traditional medicine as a treatment for fever
and malaria (Kuehl et al ., 1948; Takaya et al ., 1999), andH. macrophylla var. Otaksa and H. umbellata belong to thefamily Saxifragaceae including D. febrifuga. Thus it is possible that febrifugine and isofebrifugine are the common active alkaloids consisting in the Saxifragaceae palnts and thecontent of principles varies in each plant species. Furthermore, the root extract of H. macrophylla var. Otaksa and H.
macrophylla showed no antimalarial activity, and hence it isof interest to investigate the distribution of effective alkaloids within plants. Since the mechanisms of action of hydrangea compounds are still unknown, the investigation of
the morphologic changes induced in malaria parasites by
the mixture of febrifugine and isofebrifugine will be started.
In the mice treated with the hot-water extract of H.
macrophylla var. Otaksa or H. macrophylla leaves, parasitemia in the bloodstream decreased after a transient recrudescence and malaria parasites could not be detected by microscopic observation, suggesting that the elimination ofmalaria parasites after a recrudescence might be associatedwith host immune mechanisms. It is now generally accepted that cell-mediated immune mechanisms contribute toacquired immunity to blood-stage malaria. Studies are in
progress to investigate the induction of T helper cell subsets
during the course of resolution of primary infection in micetreated with the mixture of febrifugine and isofebrifugine.

REFERENCES
１）Ablondi, F., Gordon, S., Morton, J. II and Williams, J.H.
(1952): An antimalarial alkaloid from hydrangea. II. Isolation. J. Org. Chem., 17, 14-18
２）Ho, M. and Webster, H.K. (1989): Immunology of humanmalaria. A cellular perspective. Parasite Immunol., 11, 105-116３）Isaka, M., Punya, J., Lertwerawat, Y., Tanticharoen, M. and
Thebtaranonth, Y. (1999): Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria. J. Nat. Prod., 62, 329-331
４）Ishih, A., Ikeya, C., Yanoh, M., Takezoe, H., Miyase, T.
and Terada, M. (2001): A potent antimalarial activity ofHydrangea macrophylla var. Otaksa leaf extract against
Plasmodium yoelii 17XL in mice. Parasitol. Int., 50, 33-39
５）Iwu, M.M., Obidoa, O. and Anazodo, M. (1986): Biochemical mechanism of the antimalarial activity of Azadirachta indica leaf extract. Pharmacol. Res. Commun., 18,
81-91６）Klayman, D.L. (1985): Qinghaosu (artemisinin): An antimalarial drug from China. Science, 228, 1049-1055
７）Koepfli, J.B., Mead, J.F. and Brockman, J.A. Jr. (1947): An
alkaloid with high antimalarial activity from Dichroa
febrifuga. J. Am. Chem. Soc., 69,1837
８）Kuehl, F.A.Jr., Spencer, C.F. and Folkers, K. (1948): Alkaloids of Dichroa febrifuga Lour. J. Am. Chem. Soc., 70,
2091-2093
９）Payne, D. (1987): Spread of chloroquine resistance in Plasmodium falciparum. Parasitol. Today, 3, 241-246
１０）Rahman, N.N.N.A., Furuta, T., Kojima, S., Takane, K.,
Mohd, M.A. (1999): Antimalarial activity of extracts of
Malaysian medicinal plants. J. Ethnopharmacol., 64, 249254
１１）Takaya, Y., Tasaka, H., Chiba, T., Uwai, K., Tanitsu, M.,
Kim, H-S., Wataya, Y., Miura, M., Takeshita, M. andOshima, Y. (1999): New type of febrifugine analogues,
bearing a quinolizidine moiety, show potent antimalarial
activity against Plasmodium malaria parasite. J. Med.
Chem., 42, 3163-3166１２）Takezoe, H., Ishih, A., Fujii, K., Sakai, M., Miyase, T. andTerada, M. (2000): Fractionation of antimalarial principle
from Hydrangea macrophylla var. Otaksa leaves and its
activity against Plasmodium yoelii 17XL in ICR mice
[Abstract no A-35]. Jpn. J. Trop. Med. Hyg., 29, 43
１３）Van Agtmael, M.A., Eggelte, T.A. and Van Boxtel, C.J.
(1999): Artemisinin drugs in the treatment of malaria:
from medicinal herb to registered medication. Trends inPharmacol. Sci., 20, 199-205
１４）Winstanley, P.A. (2000): Chemotherapy for falciparum malaria: The armoury, the problems and the prospects. Parasitol. Today, 16, 146-153

７Jpn. J. Trop. Med. Hyg., Vol.３
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FIELD EVALUATION OF A RAPIDIMMUNOCHROMATOGRAPHIC TEST ‘PARACHEK-F’
IN A POST-MONSOON PLASMODIUM FALCIPARUM
MALARIA OUTBREAK IN VILLAGES OF SOUTH INDIA
Indigenous diagnostic test kit for P. falciparum
SUSANTA KUMAR GHOSH1*, TIRUCHINAPALLI SUNDARAJAN SATHYANARAYAN1,
MALALIKOPPA VEERABHADRAPPA MURUGENDRAPPA2 AND SARALA KARUMARI SUBBARAO3
Received October 11, 2001/Accepted March 9, 2002
Abstract: An indigenous rapid immunochromatographic test Parachek-F for diagnosis of Plasmodium falciparum
malaria was evaluated by the field staff in a post-monsoon P. falciparum malaria outbreak in villages of district
Raichur, Karnataka, South India in November 1999. The test functions based on dipstick P. falciparum histidine
rich protein-2 (PfHRP-2) antigen capture assay. Of the 232 uncomplicated clinically diagnosed malaria cases, 158(68.1%) were positive for malaria by microscopy of JSB-stained thick blood smears. Of these, 13 were infectedwith P. vivax, 140 with P. falciparum and 5 had mixed infections of P. vivax and P. falciparum. Malaria patientswere treated with age-specific oral doses of quinine followed by primaquine. Taking microscopy as gold standard,
Parachek-F detected PfHRP-2 antigen in 136 samples (ratio 0.93) and was 93.1% sensitive and 98.8% specific.
Positive predictive value, negative predictive value and efficacy were 99.2%, 89.6% and 95.2% respectively. No
cross reactivity was observed with P. vivax infection. False negative interpretation was associated in 40% (10/25)
lower-grade parasitaemias (parasitaemia <100/µl blood) where sensitivity was only 60%. False positive result was
associated in 1 case (1/74). Cases showing false negative results had taken presumptive treatment with chloroquineprior to the test. Careful microscopical examination on thin smears of such cases demonstrated that the morphologyof the parasites was abnormal and distorted indicating the parasites were affected by chloroquine. The possible roleof chloroquine resulting false negative results is suggested in this communication. Positive correlation between test
bands intensity and parasite density was observed (r=0.137; P<0.05). The test is indigenously developed, rapid,
simple in its application and was found suitable for field condition. Parameters like patients’ conditions, history of
drug intake, morphology of parasites at different developmental stages are to be considered for evaluation of suchtests.
Key words: Presumptive Treatment, PfHRP-2, Immunochromatographic test, Sensitivity, Specificity

INTRODUCTION
In most parts of India malaria is unstable and has reemerged in epidemic forms with high mortality. In the last
decade an increase of about 40% in Plasmodium falciparum
infection has been recorded. Almost all the deaths arecaused by P. falciparum infection and delayed treatment(Sharma, 1999). Malaria case detection and treatment isperformed by the National Anti Malaria Programme
1(NAMP) through active and passive surveillance under primary health care system. A single dose of presumptive
treatment (600 mg chloroquine; adult dose) is administered
to all fever cases at the time of taking of blood samples followed by radical treatment to the malaria positive cases.
The diagnosis is based on microscopical examination of
JSB-stained (Singh and Bhattacharya, 1944) whole bloodby skilled personnel. In all best circumstances, it lags some
important time and thus delays in the delivery of radical

Malaria Research Centre, Field Station (ICMR), Epidemic Diseases Hospital Campus, Old Madras Road, Bangalore - 560 038, India.
Directorate of Health and Family Welfare Services, Ananda Rao Circle, Bangalore - 560 009, India.
3
Malaria Research Centre, 22-Sham Nath Marg, Delhi - 110 054, India.
*
Author for correspondence: Malaria Research Centre, Epidemic Diseases Hospital Campus, Old Madras Road, Bangalore - 560 038.
Email: ghoshmrc@vsnl.net
Telephone: + 91 80 5362115 and + 91 80 5511691. Fax: + 91 80 5299033.
2

８

treatment. The early diagnosis of P. falciparum malaria andinstitution of prompt treatment was emphasized for reducing morbidity and mortality (WHO, 1993). In such situation,
test system developed by the non-microscopic antigen detection of P. falciparum histidine-rich protein-2 (PfHRP-2)
by means of an immunological capture assay has proven to
be very encouraging. The test is rapid, simple and a small
amount of whole blood is required. It does not require any
instrument or electricity, thus found suitable for rural setup
also. The result is very specific, sensitive and can be readwith the naked eyes. Evaluation of several commerciallyavailable test kits e.g., Para SightTM F test (Karbwang et al .,1996; Singh et al ., 1997a) and ICT Malaria! Pf test (Kumar
et al ., 1996; Singh et al ., 1997b; Valecha et al ., 1998;
Ghosh et al ., 2000) and DetermineTM (Singh and Valecha,
2000a) have confirmed the usefulness of these rapid tests.
Recently, an indigenous rapid test kit Parachek-F has beendeveloped (Kumar et al ., 2000). This test kit, like other systems functions similar to the antigen capture assay which
detects the circulating histidine-rich protein-2 (HRP-2) antigen released by P. falciparum (Garcia et al ., 1996). An initiative was taken by NAMP to introduce the rapid diagnostic test in the national programme. In the present study, thiskit was evaluated by field staff with minimal orientation under field condition, considering microscopy as gold standard, in a post-monsoon outbreak of malaria mostly of P.
falciparum in villages of district Raichur, Karnataka, South
India.

MATERIALS AND METHODS
Study area
The Parachek-F test was conducted in two villages of
two Primary Health Centres (PHCs) of district Raichur,south Karnataka during an outbreak of malaria mainly due
to P. falciparum in November 1999. Village Ramdurg(population 2,528) of PHC Gabbur and village Masarkal(population 1,476) of PHC Masarkal were selected for the
study. Generally villages in this area are large, distantly located, semi-town type, having population around 1,500 to
2,500 and well connected by good roads. Each village issurrounded by small rocky hillocks and vast irrigated land.
Open dug wells and rain fed irrigation tanks are the main
source of water. Escape channels from the tanks and seepage water from the hillocks form run-off stream along thevillage side. Malaria vectors Anopheles culicifacies, An. fluviatilis and occasionally An. stephensi were found to breedin such breeding habitats. The area is dry-deciduous withprevailing hot climate. Temperature ranges between 13°
C
and 44°
C. Annual rainfall ranges from 800 to 1,200 mm andprecipitates mainly in the post-monsoon period. Irrigation isrestricted during post-monsoon period. Millets and cotton
are the main cash crops. Rice is cultivated in the catchment
area.
Intensive malaria control activities with fogging ofmalathion (5% technical) and indoor residual spraying withdeltamethrin (2.5% wp) and presumptive treatment withchloroquine to all fever and fever related cases were initiated at the time of investigation. All age and sex groups including pregnant women having recent febrile illness wereenrolled in the study. The test was performed also on thosecases who had received presumptive treatment with chloroquine two to three days before. Such cases wanted immediate results since the reports of blood smear examination
were awaited. Both P. vivax and P. falciparum species were
present and both chloroquine and sulfadoxinepyrimethamine were found resistant in P. falciparum(Ghosh and Sathyanarayan, 2000).
Work plan
The test was conducted under field conditions by thefield staff of Malaria Research Centre, Bangalore. Two scientists, one microscopist and two field health workers wereinvolved in the test evaluation. One scientist was assigned
to enroll names, history of fever, nature of ailments, any intake of anti-malarial drugs from each patient. One worker
was engaged on drawing two sets of blood smears and theother performed the Parachek-F test. The microscopist
stained the blood smears and examined on the spot. At theend, the second scientist correlated the thick smear results
and Parachek-F test. In this way, the test procedure wasblinded at each level. The final readings were rechecked by
the first scientist. Local health staff and village leaders(Gram Pradhans) assisted in carrying out the tests. Verbal
consent of each patient was obtained.
Microscopy of JSB-stained blood smears
Two sets of blood smears (both thick and thin) wereprepared and stained with JSB stain. JSB stain is a combination of two aqueous solutions. The first solution (JSB I)
contains microscopic methylene blue and the second one
aqua yellowish eosin (JSB II). It is a rapid stain. First thethin smears were fixed with methanol and the thick were
dehaemoglobinised by putting simply in distilled water. After completely drying both the smears were dipped into JSB
II solution for 2-3 seconds and subsequently washed thoroughly in buffer water (phosphate buffer; pH 6.8). The
slides were then dipped in JSB I solution for 45 seconds. It
was again washed gently and thoroughly in the buffer. The
stained slides were allowed to dry. One set was examinedon the spot under a compound microscope (Zeiss KF-2,
Germany) at 1,000 x magnification. The second set was

９stained in the laboratory at Bangalore and kept for reference.
A smear was declared negative after counting a minimum of
200 micro fields on thick smears. Thin smears were examined for final identification of the species of parasite. All the
blood smears including discordant results were thoroughly
re-examined by the senior author (SKG). Parasites fromeach positive case were counted against 300-500 whiteblood cells (WBCs). The parasitaemia was calculated assuming 8,000 WBCs/µl blood. P. falciparum cases weretreated with age-specific oral doses of quinine sulphate(adult dose : 600 mg 8 hourly for 7 days) followed by a single dose of 45 mg primaquine. P. vivax cases were treatedwith chloroquine (1,500 mg over 3 days) following by 75
mg primaquine in 5 days. Primaquine was not given to theinfants and pregnant women.
Malaria diagnosis by Parachek-F
The commercial diagnostic kit Parachek-F, indigenously developed by Orchid Biomedical Systems, Verna,
Goa, India was used for rapid diagnosis of P. falciparum
malaria. The kits were supplied to the State Health Department by NAMP for field evaluation. The test system uses
two mice antibodies specific against the immunodominantrepeating sequence from HRP-2 molecules. One antibody is
conjugated to visible colloidal gold and impregnated into
the sample pad while the second one is immobilized in aline on the nitrocellulose strip. The test was performed following strict manufacturer’s instruction (Fig. 1). The kits
sealed in pouches were stored at 4°
C in the PHC andbrought to room temperature before carrying out the tests.
One drop of finger pricked whole blood (〜5µl ) was applied directly on the sample pad just below the arrow markson the test strip. The strip was dipped in 4 drops of clearing
buffer in a clean 12 x 75 mm test tube keeping the arrows
pointing downwards. In the positive samples pink coloured
bands were formed along with control bands within 4-5
minutes. In the negative samples only control bands were
noticed. Presence of control bands validated the tests per-

Test line intensity scale

Negativetest strip

Positivetest strip

Absorbent pad

4.

IntenseControl line
Test line

3.

Easilyreadable2.

Readable

1.

Barelydiscernible

Blood Sample andbuffer application
padFig. 1: Parachek-F test strip.

formed. The final readings were taken after 15 minutes. The
positive test bands were graded (+1 to +4) based on the colour intensity.
Data analysis
To evaluate Parachek-F test, the result of thick smear
examination was taken as gold standard. Variables likenumber of true positives (positive reactors in the assay withpositive microscopy; TP), number of true negatives (negative reactors in the assay with negative microscopy; TN),
number of false positives (positive reactors with negative
microscopy; FP) and number of false negatives (negative reactors with positive microscopy; FN) were measured for
calculating sensitivity, specificity, positive and predictive
values and efficacy of the test (Tjitra et al ., 1999). Sensitivity was thus calculated as TP/(TP + FN), specificity as TN/(TN + FP), positive predictive value (PPV) as TP/(TP + FP)
and negative predictive value (NPV) as TN/(FN + TN). Test
efficacy was defined as (TP + TN)/ total number of cases.
For the purpose of analysis, mixed infection cases having P. vivax and P. falciparum were treated as P. falciparum.
As the subjects having P. falciparum gametocytes only are
considered positive cases in the national programme andhas epidemiological implications, these cases were considered as true positives. All P. vivax cases were treated as
negative.
The correlation between test band intensity and theparasite density was analyzed using Microstat software(Ecosoft. Inc. USA) and significance was determined using
F test considering P <0.05 to be significant.

RESULTSOf the total 232 subjects enrolled in the study (133from Ramdurg and 99 from Masarkal), 128 were males and
104 females. Prevalence of malaria with age wise breakdown given in Table 1 revealed that transmission of malaria
was active at the study area. The youngest one was threemonths female and the eldest one was 80 years male. Microscopical examination on thick smears demonstrated 158(68.1%) were positive for malaria parasites. Of these, 13
were infected with P. vivax, 140 with P. falciparum and 5
had mixed infections of P. vivax and P. falciparum. Of the
145 P. falciparum cases, 107 were with rings only, 32 withrings and gametocytes and 6 with gametocytes only.
The Parachek-F test detected PfHRP-2 antigen in 136(ratio 0.93) P. falciparum patients and was non-reactive to
P. vivax cases (Table 2). Considering P. falciparum rings
only, the test detected 99 of 107 cases (ratio 0.92); while in
the group with gametocytes only 4 out of 6 cases were detected (ratio 0.66). All the 32 cases having concomitant

１０

Table 1

Prevalence of malaria in the affected villages of district Raichur, south Karnataka in November 1999

Patients age Blood smear
Groups (years) examined<17> 1 ‐< 417> 4 ‐< 824> 8 ‐< 1431> 14
Pregnant
Women
Total

Table 2

Positive formalaria
3
12
21
21

1
10
19
17

P. vivax+
P. falciparum
0
1
1
1

Slide Positivity
Rate
42.85
70.58
87.50
67.74

Slide falciparum
Rate
14.28
64.70
83.33
58.06

P. falciparum
Percentage
33.33
91.66
95.23
85.71

P. vivax

P. falciparum

2
1
1
3

147
6

98
3

5
1

91
2

2
0

66.66
50.00

63.26
33.33

94.89
66.66

232

158

13

140

5

68.10

62.50

91.77

Table 3

Performance of Parachek-F assay in 232 clinically diagnosed malaria patients

P. falciparum
P. vivax
P. vivax+P. falciparum
Total positive
Total negative
P. falciparum only
Rings only
Rings + Gametocytes
Gametocytes only
Total positive taking all
P. falciparum cases

Microscopic diagnosis+-

No. Plasmodia positive byThick smear Parachek-F
Ratio
140
13
0
5
5
158
74

136
96

0.86

107

99

0.92

32
6
145

32
4
136

1.00
0.66
0.93

rings and gametocytes showed positive to both microscopy
and PfHRP-2 antigen (ratio 1.00). Thus the sensitivity of
PfHRP-2 assay was 93.1%, the specificity 98.8%, the positive predictive value 99.2%, the negative predictive value
89.6% and the test efficacy was 95.2% respectively. Prior to
the test, 19/135 TP, 10/10 FN, 1/1 FP and 12/86 TN (total
42/232) cases consumed chloroquine as presumptive treatment (Table 3).
Details of parasitaemia in the P. falciparum cases andrelated sensitivity are summarized in Table 4. Parasitaemia
ranged from 17 to 57,120 parasites/µl blood. Compared tomicroscopy, Parachek-F failed to detect infection in 10 of
25 patients (40%) with low parasitaemia (<100 parasites/µl
blood) where 8 were with rings only and 2 with gametocytes only. Thus, the test demonstrated 60% sensitive inlower-grade parasitaemias and 100% in cases having>100
parasites/µl blood. Careful microscopical examination on
thin smears in the lower-grade parasitaemias revealed grossdistortion of the parasite morphology. The rings were verysmall, nugget shaped, chromatin and cytoplasm were compact due to lack of vacuole. The morphology of the gametocytes was also altered having oval and slightly elongatedshapes. The pigments and chromatin were dispersed, dis-

Efficacy of Parachek-F test

Parachek-F +diagnosis -135 (TP)10 (FN)

1 (FP)
86 (TN)

Sensitivity = 135/(135+10) = 0.931; Specificity =86/(86+1) = 0.988; Positive predictive value =135/(135+1) = 0.992; Negative predictive value =86/(86+10) = 0.896; Efficacy = (135+86)/(135+
10+1+86) = 0.952.
All P. falciparum gametocyte cases considered as
positive P. vivax cases considered as negative.
TP - True positive, FP - False positive,
FN - False negative, TN - True negative

Table 4

Sensitivity of Parachek-F test by parasitaemia

Parasitaemia/µl blood

Thick smear
diagnosis

Parachek-F
diagnosis

Sensitivity (%)

17 - 100
101 - 500

25
14

15
14

60
100

501 - 1,000
1,001 - 2,000
2,001 - 5,000

16
53
23

16
53
23

100
100
100

> 5,000

12

12

100

All P. falciparum
cases

145

146

99.2

tributed in the cytoplasm.
Details of correlation between test band intensity andstage wise parasite density is shown in Table 5. Positive correlation (r=+0.137; P <0.05) was noticed in all stages ofparasite except in cases having only gametocytes (r=-0.749;
P >0.05) and band intensity grade 2 (r=-0.306; P >0.05).

DISCUSSION
In the study area malaria was not a serious problem in
the recent past (NAMP, 1998). In the beginning of the study,
there was a confusion of the cause of outbreak even though１１

Table 5

Correlation between parasite density of P. falciparum and colour intensity of test bands
along with different stages of parasite in the test samples.

Parasite Density
Mean ± SD(Range)Band wise
56.60±25.27(17-106)
843.53±1,821.66(0-9,420)
2,379.67±7,384.31(55-41,320)
2,180.59±4,691.93(54-27,290)
6,686.79±2,133.01(220-57,120)
Stage wise
605.0±1,293.54(45-3,245)
3,776.11±9,388.23(17-57,120)
2,461.90±4,693.75(104-27,290)
33,254±8,384.81(17-57,120)

Band
Intensity

Stage of Parasite

r value

F ratio

P value0

R (n=8), G (n=2)+ 0.05550.15

0.0001

1

Neg. (n=1), R (n=18)
G (n=2), RG (n=5)
R (n=25), G (n=1),
RG (n=4)
R (n=21), RG (n=11)+ 0.330

5.56

0.0223- 0.306

3.11

0.0831+ 0.2966.89

0.0109R (n=35), G (n=1),
RG (n=12)+ 0.240

14.56

0.002

(0-4)

G only (n=6)- 0.749

1.30

0.279

(0-4)

R only (n=107)+ 0.81217.28

0.0004(1-4)

RG (n=32)+ 0.159

8.78

0.004

(0-4)

All P. falciparumcases+ 0.13723.17

0.0002

2
3
4

R - rings, G - gametocytes, Neg. - Negative for malaria.

all anti-malarial measures had initiated. The Parachek-F test
clearly identified that the outbreak was due to P. falciparum
malaria showing high sensitivity of 93.1% which is inagreement with similar commercial kits like Para SightTM-F(Beadle et al ., 1994; Karbwang et al ., 1996; Singh et al .,1997a) and ICT-Pf (Kumar et al ., 1996; Singh et al ., 1997b; Valecha et al ., 1998; Ghosh et al ., 2000). Other values
like specificity, positive and negative predictive values andefficacy of the test were comparable. Kumar et al . (2000)
also demonstrated similar results with the same Parachek-F
kit.
As compared to Parachek-F, Para SightTM-F and ICTPf have several washing processes. In this regard, ParachekF is very simple and easy to operate in the field by thehealth workers (Fig. 1). Due to its simplicity, it is costeffective also. Each kit costs approximately U.S.$ 0.64
which is cheaper than other commercial kits viz. Para
SightTM-F and ICT-Pf (between U.S.$ 2.91 and 3.33) but
higher than the suggested price of U.S.$ 0.40 by H. Engers(Singh and Valecha, 2000a). To make accessible, efforts
should be made to produce such kits locally specially in thedeveloping countries where malaria (mainly P. falciparum)
is a real problem. In India, cold chain system has been provided in each PHC under primary health care. Thus, there

will not be any problem to introduce this test system in thenational programme.
There are some limitations in this test for poor interpretation in lower-grade parasitaemias. Low parasitaemia
are most common in many areas where anti-malarial drugs
are easily available and people take self-medication. Suchsituation is very common among travellers who are advised
to cover with chemoprophylaxis (WHO, 1996). Funk et al .
(1999) reported false negative results among travellers up to
72.0% in ICT-Pf and 29.6% cases in Para Sight F whereparasitaemia level was < 0.1%. They did not find these kits
suitable for self-diagnosis. Beadle et al . (1994) observed ina field test in Kenya that sensitivity and specificity markedly decreased in lower-grade parasitaemias. We have observed false negative results in 40% cases having parasitaemia level <100 parasites/µl blood. Karbwang et al . (1996)
observed a lack of correlation between the amount of
PfHRP-2 antigen and small ring form parasitaemia. Better
correlation was observed with the trophozoite load (laterings). Beadle et al . (1994) reported that PfHRP-2 antigen
is produced in trophozoites and early gametocytes. Recently,
PfHRP-2 has been reported to mediate haemozoin formation, which could be inhibited by chloroquine (Sullivan et
al ., 1996). Pandey et al . (1997) have demonstrated that

１２hexapeptide repeats Ala-His-His-Ala-Ala-Asp in PfHRP-2
are heme binding sites in this protein. It could be possiblethat in the early developmental stages of the parasites,
PfHRP-2 molecules are not expressed due to the effect ofchloroquine and demonstrate false negative results. The patients who showed false negative results showed clinicalsymptoms for malaria and had consumed presumptive treatment with chloroquine prior to the test. This was confirmedby the health workers who administered the presumptive
treatment. Further, the morphology of the parasites in thesecases clearly indicated that they were affected by chloroquine as described by Macomber and Sprinz (1967).
Karbwang et al . (1996) observed very low specificity of
50% in Para SightTM F test for detection of multi-drug resistant P. falciparum treatment failure as against 60% in ourstudy cases underwent presumptive treatment. This is possibly due to the drug affected parasitaemia phenomenon.
One false positive case (1/74) detected by Parachek-F
was treated 11 days before the test. This is possible when
persistence of antigenemia in the patient persists even after
the curative treatment (Singh et al ., 1997a, b; Valecha et al .,1998; Kumar et al ., 2000). Recently, Grobusch et al .
(1999) has reported false-positive results in the presence ofrheumatoid factors. False-positive test results could lead toinappropriate treatment of other important causes of fever.
However, this should not be taken as limitation to the test
because timely treatment in many sub-microscopic levelparasitaemias in known malarious areas may prove fruitful.
There are mixed reports on the correlation between thetest band intensity and parasite density. Some reported positive correlation (Kumar et al ., 1996; Ghosh et al ., 2000),
while others negative correlation (Singh et al ., 1997a, Valecha et al ., 1998). Kumar et al . (2000) reported negative
correlation with the same kit as against positive correlationin our study.
Recently, test kits for diagnosis of both P. falciparumand P. vivax are available commercially. One such kit (ICT
P.f./P.v.) was evaluated by Singh et al . (2000b) in Madhya
Pradesh, Central India. The test was 97.5% sensitive and88% specific for P. falciparum. For P. vivax the sensitivity
was only 72% and 99% specific. The authors concerned
about the decline in sensitivity <300 parasites/µl for P. falciparum. The test failed to diagnose P. vivax infection when
the parasitaemia was < 1500 parasites/µl . Tjitra et al .
(1999) also made similar observation.
In conclusion, rapid immunodiagnostic tests are very
useful in the field and in emergencies even having some
limitations, because all test systems do have some selflimiting factors. Proper caution has to be taken for thosesubjects undertaking chemoprophylaxis (especially pregnant women and travellers). The test systems have addi-

tional advantage as the results can be kept as reference for a
fairly long period of time. All the test cards of our study areeasily legible even after more than one and half years of
testing. For evaluation of such test systems, it is very important to consider parameters like patients’ condition, history
like drug intake, parasite morphology, etc. However, thiscannot replace the microscopical diagnosis for routine epidemiological studies where different stages of parasites,
their density etc. are important components, which cannot
be performed by such test systems.

ACKNOWLEDGEMENTS
The authors thank the District Malaria Officer, district
Raichur, the Medical Officers, Primary Health Centres Gabbur and Masarkal and staff of Malaria Research Centre,
Bangalore for assistance in the study.

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１
５Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
１,２
０
０２, pp.１
５-２
１

AN ANTIGENIC PEPTIDE OF MYOSIN HEAVY CHAIN-LIKE
PROTEIN FROM TRICHINELLA SPIRALISTAKUMI NAKADA, ISAO NAGANO, ZHILIANG WU AND YUZO TAKAHASHI
Received October 23, 2001/Accepted February 26, 2002
Abstract: We produced antigenic peptide of Trichinella spiralis newborn larvae (NBL) which seemed to be a part
(approximately one fourth) of myosin heavy chain, and some basic profiles were revealed. The cDNA library wasconstructed from NBL and immunoscreened with an antibody against the parasite. A clone, designated NBL21,
was selected. It contained a cDNA transcript of 1656 bp in length, which encoded 552-amino acids (64868 Da in
the estimated molecular weight). The fusion protein encoded by the clone NBL21 was produced in an Escherichia
coli expression system and affinity purified. NBL21 fusion proteins migrated at 64 kDa and reacted to T. spiralisinfected mouse sera and the antibody against NBL crude antigen. Antisera were developed against NBL21 fusion
proteins, which reacted to a single band migrating at 200 kDa on Western blotting analysis of crude extracts frommuscle larvae, and reacted to hypodermal muscles of T. spiralis on immunohistochemical staining. The antigen
was recognized by the mouse serum obtained from the early phase of infection, but the antigenicity was devoid of
species specificity.
Key words: Myosin heavy chain, Trichinella spiralis, Antigen, Fusion protein

INTRODUCTION

MATERIALS AND METHODSTrichinella spiralis is a parasitic nematode that infects
the skeletal muscle cells of mammals. Many questions remain to be answered about this parasite. Examples include
how they evade host immune attack, and how they can establish parasitism in the host muscle cell. Moreover, thevaccine against this parasite is still far from routine usage.
For progress towards these goals, fusion proteins of
Trichinella have been produced with some success. Vayssier et al . (1999) produced a protein from T. britovi, named
as Tb Hsp70, which is a kind of heat shock protein with amolecular weight of 70 kDa. Sun et al . (1994) produced aprotein Ts39 FP which is a potentially valuable antigen for
vaccine development. Lindh et al . (1998) produced fusion
protein which has properties of a myogenic repressor, andNagano et al . (2001a) produced fusion protein with serine
protease inhibitor activity.
In this study we produced a part of myosin heavy
chain-like protein, and revealed some basic profiles including amino acid sequence and antigenicity. This is the firstreport on antigenic peptide of Trichinella myosin heavy
chain which may afford better understanding of this conservative protein in Trichinella related research.

Parasites
T. spiralis (ISS 413) and Trichinella pseudospiralis(ISS 13) were maintained in mice through oral infection.
Muscle larvae were obtained by a conventional digestion
method (Despomier, 1975). Newborn larvae (NBL) of T.
spiralis were obtained through incubation of five day oldadults in minimum essential medium (MEM) + 10% fetal
calf serum (FCS) + antibiotics (100 µl of streptomycin and
100 unit of penicillin per 100 ml of MEM). The NBL werefrozen and stored until use.
Crude extracts and ES products
Larvae of Trichinella at the muscle stage were isolated
from mice at 2 months post infection using digestion
method. Crude saline extracts of larvae and ES products
were prepared by conventional methods (Wakelin et al .,1994; Wu et al ., 1998).
Preparation of antisera
Polyclonal antibodies against crude extracts of the
NBL were collected from BALB/c mice injected subcutaneously with 30 µg of the crude extracts and Freund’s complete adjuvant followed by two booster injections of 15 µgof the crude extracts mixed with Freund’s incomplete adju-

Department of Parasitology, Gifu University School of Medicine, Tsukasa40, Gifu, 500-8705Japan１
６vant at 2-week intervals.
Infected sera of the parasites and polyclonal antibodies
against ES products were prepared by conventional methods (Nagano et al ., 2001a, b).
cDNA library construction
Construction of a cDNA library from T. spiralis NBL
was performed according to our previous method (Nagano
et al ., 1999). In brief, frozen NBL were homogenized in an
extraction buffer (Quickprep micro mRNA purification kit,
Amersham Pharmacia Biotech, Tokyo Japan), and mRNA
was isolated by affinity chromatography using oligo (dT)cellulose.
Synthesis of cDNA from the resulting mRNA was performed using a Timesaver cDNA synthesis kit (Amersham
Pharmacia Biotech) as described by the manufacturer’s instructions. Resulting cDNAs were ligated into the λZAP II
vector (Stratagene, La Jolla, California USA). The ligateswere packaged in Gigapack Gold III packaging extract(Stratagene).
Immunoscreening
E. coli XL1-Blue MRF’ strain infected with the λZAP
II vector was cultured on the NZY agar plates for 4 hr at 42°
C. And nitrocellulose membrane infiltrated with isopropylβ-D-thiogalactopyranoside (IPTG) was overlaid for 4 hr at
37°
C. After blocking with 5% skim milk in phosphate buffered saline (PBS) overnight at 4°
C, the membrane was reacted with polyclonal antibodies (1/100 diluted) against T.
spiralis NBL. Goat anti-mouse IgG alkaline phosphataseconjugate was then used as the second antibody, and themembrane was incubated in 5-bromo-4-chloro-3-indolyl-ptoluidine salt (BCIP) and nitroblue tetrazolium (NBT) for
development. The positive 66 clones were picked up and rescreened three times.
DNA sequence and homology search
The clone NBL21 was subjected to sequencing inwhich the Thermo Sequenase Cycle Sequencing Kit (Amersham) with bi-directional sequencing protocol and IRD-800
and IRD-700 labeled primers were used. The products weresequenced using an automatic sequencer (Model LIC-4200
L, Aloka, Tokyo Japan). The DNA sequences were assembled and analysed by using DNASIS software (Hitachi Software Engineering, Tokyo Japan). The NCBI BLAST network service at the National Center for Biotechnology Information was used to search for the sequence homology.
Production of fusion protein
The NBL21 clone was subcloned into the expression
vector (pTrc His, Invitrogen, California USA) and trans-

fected to E. coli (DH5α, Toyobo CO. LTD., Tokyo Japan)
according to the manufacture’s instructions. Transformants
were grown in bacterial culture medium at 37°
C. To facilitate protein production, IPTG was added to give a final concentration of 1 mM, and cells were incubated for 3 hr at
37°
C. The induced cells were fractured by ultrasound in 20
mM Tris HCl buffer (pH 8.0). The resultants were precipitated and the pellet was lysed by lysis buffer (20 mM TrisHCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride). The fusion protein was affinity purified using a
HisTrap kit (Amersham Pharmacia Biotech) as described by
the manufacturer.
Production of antibodies against the fusion protein
To produce antibodies against the fusion protein NBL
21, BALB/c mice were injected subcutaneously with 30µgof the fusion protein and Freund’s complete adjuvant. In order to boost the antibodies, after two weeks, the mice were
injected with 15µg of the protein in Freund’s incomplete
adjuvant, and after another one week, 10µg of the protein
was injected into the abdominal cavity. The mice were bled
one week after the final booster.
Immunocytochemistry
Infected muscles of 42-day post infection were fixedwith 4% paraformaldehyde and embedded in OCT compound (Miles Inc., Elkhart, Indiana USA) and frozen.
Cryostat sections (4 µm in thickness) were transferred to albumin coated microscope slides. The sections were immunostained using Histostain SP kit (Zymed Laboratories
Inc., San Francisco, California USA) as described by the
manufacturer’s instructions. Briefly, the sections weretreated with the first antibodies (×100 diluted antibody
against the NBL21 fusion protein or control normal mouseserum) for 30 min at room temperature. After washing, thesections were treated with the biotinylated second antibody(Zymed Laboratories Inc.) for 30 min at room temperature,
washed three times, treated with avidin coupled with peroxdase (Zymed Laboratories Inc.) for 10 min at room temperature, and reacted with diaminobendizone solution(Zymed Laboratories Inc.) for peroxidase staining.
Antigenicity analysis of the fusion protein NBL21
The fusion protein NBL21 was subjected to sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and blotted to nitrocellulose membranes. Membranes were blocked with skim milk and reacted with thefirst antibodies (×100 diluted) including anti NBL antibodies, infected sera, antibodies against excretory and secretory(ES) products of muscle larvae, and control sera for 60 minutes at 37°
C. After washing, the membranes were treated１７with alkaline phosphatase-conjugated anti-mouse IgG for
30 min at 37°
C. The alkaline phosphatase was developed byusing BCIP and NBT.
Specificity analysis of antibody against the fusion protein
NBL21
Reactivity of anti-NBL21 fusion protein antibody waschecked against 4 kinds of antigens including crude antigens and ES products from the two species (T. spiralis andT. pseudospiralis). The antigens were subjected to SDSPAGE and reacted to the anti NBL21 antibodies (×100 diluted) for 60 min at 37°
C. The nitrocellulose membranes
were treated in the same way as described above.
Specificity of fusion protein NBL21
Antigen specificity of the fusion protein was assessedin two ways. The panel of crude antigens of parasites (Dirofilaria immitis, Ascaris lumbricoides, Toxocara canis,
Gnathostoma nipponicum, Sparganum mansoni, Clonorchis
sinensis, Anisakis simplex, Strongyloides stercoralis,Paragonimus westermani, Paragonimus miyazakii, Fasciola
hepatica and Cysticercus cellulosae) was subjected to reaction against anti fusion protein NBL21 antibody by multiple
dot ELISA according to the established method. The othermethod included ELISA where fusion protein NBL21 wasreacted against a panel of human patient sera (trichinosis,
paragonimiasis, dirofilariasis, dihyllobothriasis, anisakiasis,
cysticercosis cellulose hominis, trichuriasis, gnathostomiasis and fasciolasis) according to the established method.
Kinetics of antibody response against the fusion protein
NBL21
Three BALB/c mice were each inoculated orally with
300 infective larvae. Sera from infected mice were collected
before inoculation for a control and at 1, 2, 3, 4, 6, 8 and 10
weeks after inoculation. Antibody titer against fusion protein NBL21 was assessed by means of ELISA according to
the established method.
Nucleotide sequence accession number
The nucleotide sequence reported in this article has
been submitted to the GenBank database and has the accession number AY033438.

RESULTSDNA and amino acid sequences
The cDNA library from NBL gave 66 positive clones,
which comprised of 3 groups of clones. One clone, designated NBL21, from the major group was further subcloned.
The clone NBL21 consisted of 1656 bp nucleotide and en-coded 552 amino acid residues (Fig. 1). A homology study
from NCBI BLAST search revealed significant homology
with myosin heavy chain of some nematodes including
Caenorhabditis elegans (GenBank under accession number
X08067), Onchocerca volvulus (GenBank under accession
number M74066) and Brugia malayi (GenBank under accession number L01625).
Myosin heavy chain of C. elegans had 65% homology
in the DNA sequence and 66% in the deduced amino acidsequence. Myosin heavy chain of O. volvulus had 64% homology in the DNA sequence and 63% in the deduced
amino acid sequence. Myosin heavy chain of B. malayi had
62% homology in the DNA sequence and 64% in the deduced amino acid sequence. In some areas such as aminoacid sequence 260-293 (box in Fig. 1), more significant homology was found; 75-77% homology in the DNA sequence and 88-94% in the deduced amino acid sequence.
Western blotting
The fusion protein NBL21, produced by means of an E.
coli expression system, was migrated at 68 kDa on SDSPAGE and positively immunostained with anti T. spiralis
NBL antibody (arrow in lane 1 of Fig. 2), T. spiralis infected sera (arrow in lane 2 of Fig. 2), T. pseudospiralis infected sera (arrow in lane 3 of Fig. 2), but not with anti ES
product of T. spiralis muscle larvae (lane 4 in Fig. 2), anti
ES product of T. pseudospiralis muscle larvae (lane 5 in Fig.
2) nor with control sera (figure not shown).
An SDS-PAGE analysis revealed that crude antigens
and ES product of muscle larvae were composed of manybands with different molecular weights. Out of many bands,
only one band of 200 kDa (corresponding to the expectedsize) of crude antigens of T. spiralis (lane 2 in Fig. 3) and T.
pseudospiralis (lane 4 in Fig. 3) were positive by immunostaining with the antibody against NBL21 fusion protein. No bands of the ES product of the two species werepositive by the immunostaining (lane 3, 5 in Fig. 3).
Immunolocalization
As shown in Fig. 4 A, strong positive immunostaining
was observed in the internal structures of the muscle larvae.
The staining consisted of a spotty pattern and lining beneaththe cuticle surface, and internal organs were negative by thestaining. The control sera failed to stain any structures (Fig.
4 B).
Antigen specificity
The multiple dot ELISA showed reactivity of the antibody against the fusion protein NBL21 against crude extracts of D. immitis, A. lumbricoides, T. canis, G. nipponicum, S. mansoni, C. sinensis but not against A. simplex, S.

１８

Figure 1 Comparison of a deduced amino acid sequence of the clone NBL21 and its comparable
genes from other nematodes including C. elegans (C. ele; amino acid residues 1314-1865
in the total 1969 residures), O. volvulus (O. vol; amino acid residues 1308-1859 in the total 1957 residues), B. malayi (B. mal; amino acid residues 664-1215 in the total 1313 residues). The asterisks indicate nucleotides, which are identical to those of C. ele, O. vol, B.
mal and NBL21. A box shows high homology region mentioned in the text.

１
９

Figure 2 Western blots analysis of the fusion protein NBL21.
E. coli expressed NBL21 fusion protein was reactedwith anti NBL21 antibody (lane 1), infected sera ofT. spiralis (lane 2), infected sera of T. pseudospiralis(lane 3), anti ES product of T. spiralis antibody(lane 4) and anti ES product of T. pseudospiralis antibody (lane 5). The left side of each lane is molecular marker and the right side is the test sample. The
numbers of the left side is molecular weight. The arrow shows positive band.

Figure 3 Western blot analysis of reactivity of anti NBL21.
The mouse antibody produced against the fusion
protein NBL 21 was reacted with NBL crude antigen (lane 1), T. spiralis crude antigen (lane 2), T.
pseudospiralis crude antigen (lane 4), ES productsof T. spiralis (lane 3) and ES products of T. pseudospiralis (lane 5). The numbers are molecularweights of marker (m).

Figure 4 Immunocytochemical staining of muscle larvae of T. spiralis with antibody against NBL
21 fusion protein (A) and control serum (B). The arrow shows the positive staining.
Figure 5 Dot - ELISA analysis of reactivity of anti NBL21
fusion protein antibody (S) and normal mouse sera
(C) against a panel of crude antigens. 1. P. westermani, 2. P. miyazakii, 3. F. hepatica, 4. C. sinensis,
5. S. mansoni, 6. C. cellulosae, 7. D. immitis, 8. T.
canis, 9. A. lumbricoides, 10. A. simplex, 11. G.
nipponicum, 12. S. stercoralis. Crude antigens fromC. cellulosae, A. simplex and S. stercoralis gave
negative results which may suggest major differencein antigenic epitope of myosin heavy chain fromother parasites.

２０stercoralis, P. westermani, P. miyazakii, F. hepatica and C.
cellulosae. D. immitis and T. canis especially exhibitedstrong reactivity (“S” in Fig. 5). Normal mouse sera (“C” inFig. 5) gave negative or weak positive result.
The fusion protein reacted positive for ELISA against
sera from all patients examined including nematode,cestoide and trematode infection (figures not shown).
Antibody response kinetics
Antibodies against the fusion protein were detectedfrom 4 weeks after the infection and reached a maximum at
8 weeks, and the ELISA titer remained high throughout the
10-week-long experiment (figures not shown).

DISCUSSION
Recent progress in cell biology has enabled us to produce a large amount of proteins with high purity. Taking advantage of this methodology, proteins of a parasite origin
have been mass-produced for a variety of usages including
immunodiagnostic antigen (Chung and Ko, 1999) and vaccine (Sun et al ., 1994; Prema et al ., 1994). Genes encoding
specific antigens of ES products (43, 39 and 53 kDa) havebeen characterized (Sugane and Matsuura, 1990; Zarlenga
and Gamble, 1990; Su et al ., 1991; Vassilatis et al ., 1992).
In this study we obtained the clone NBL21 from T.
spiralis NBL. This clone is likely a part of the myosin
heavy chain from the sequence homology and in situ localization of this protein within the parasite. This study also revealed some basic profiles of the myosin heavy chain including the DNA and amino acid sequence which is discussed with comparable genes of other nematodes such as C.
elegans, B. malayi and O. volvulus. Antigenic specificity
and possible immunodiagnostic usages were also assessed.
Myosin is a fundamental protein shared by all kinds ofcells. The DNA sequence of such a protein should be highly
conservative. This notion is supported by the present study
which showed high homology among other nematodes. In
particular, some areas of the nucleotides are significantly
conserved. Such area may be responsible for its common
function as myosin.
The peptide NBL21 seems to carry immunodominantantigen which is recognized by hosts during the experimental infection. A similar result was reported by Li et al .
(1995) who demonstrated that myosin heavy chain carries
immunodominat filarial antigen. The clone NBL21 had
48% homology with the myosin heavy chain of hosts (humans and mouse), unshared sequence may explain the reasons why the fusion protein NBL21 is highly antigenic
against the hosts.
We have no ready explanation as to how this internal

antigen is exposed to the host immune system. Interestingly,
paramyosin, which is also an internal constituent of parasites, carries immunodominant antigen which may be used
as a protective antigen against schistosome infection (Xu etal ., 1989). Our preliminary experiment, however, showedthat the fusion protein NBL21 does not provoke protective
immunity against challenge infection.
Fusion proteins of parasites can be a candidate for immunodiagnostic antigen as suggested by many authors (Zarlenga and Gamble, 1990; Chandrashekar et al ., 1991; Zarlenga et al ., 1994). The mice infected with Trichinella seem to provoke antibodies at detectable levels against thefusion protein NBL21 from the early phase of the infection,
therefore, the antigen of the fusion protein NBL21 seems to
be Group I antigen (Denkers et al ., 1990) and rapid responding group antigen (Takahashi et al ., 1990). One drawback of this fusion protein is its lack of species specificity.
The antibody against the fusion protein NBL21 reacted
against sera from a variety of parasite crude antigens.
Therefore, this fusion protein can be used for early diagnosis of some parasite infection.

ACKNOWLEDGEMENTS
This study was partially supported by grants from the
Ministry of Education, Science and Culture (12670229).
The authors are thankful to Prof. Y. Nawa, Miyazaki Medical College, who kindly provided a panel of parasite antigens.

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３

Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
１,２
０
０２, pp.２
３-２
８

DRUG SUSCEPTIBILITIES AND CLINICALMANIFESTATIONS OF MYCOBACTERIUM
TUBERCULOSIS IN ZAMBIASATOSHI MITARAI1,a, CHARITY HABEENZU1, DAVID LUBASI1, LINDON M KAFWABULULA1, FRANCIS C KASOLO2,
KOZI ICHIYAMA2, HIROSHI TERUNUMA3, MASAHIKO ITO3, HARUMI SHISHIDO4 AND YOSHIO NUMAZAKI2
Received December 20, 2001/Accepted March 9, 2002

Abstract: The inappropriate use of anti-tuberculosis drugs has resulted in an increase in the drug resistance. This
study was conducted to obtain data related to the spectrum of drug resistant Mycobacterium tuberculosis. A total of
499 strains of M . tuberculosis were tested for susceptibilities of isoniazid (INH), rifampicin (RFP), ethambutol(EMB) and streptomycin (SM). Totally 225 patients were also evaluated for their clinical manifestations. The primary and acquired resistances were observed in 13.9% and 16.1% to INH, 11.1% and 12.9% to RFP, 6.7% and9.7% to EMB, and 3.8% and 3.2% to SM respectively. There were no significant differences between primary andacquired resistances. Multi-drug resistance was observed in 22 strains (4.4%). Clinical manifestation, as measured
by chest roentgenograms, was similar in patients with either drug-susceptible or resistant strains. The clinical andradiological information was not useful for the prediction of susceptible and resistant strains. The HIV serostatus
was not associated with drug susceptibility profiles either. Our study has documented a high incidence of drug resistant M . tuberculosis, with no obvious clinical correlates, which must be considered when implementing a strategy for chemotherapy. As for clinical practices, many defaults during chemotherapy reduced the treatment successes. It is important to assess the efficient implementation of the directly observed treatment with short course
chemotherapy (DOTS) and interrupt the further dissemination of resistant M . tuberculosis in the community. The
drug resistances must be surveyed continuously to obtain useful clinical perspective and evaluate the effectivenessof tuberculosis control program.
Key words: Resistance, Multi-drug, Primary, Acquired, Zambia

INTRODUCTION
The multi-drug resistant Mycobacterium tuberculosis(MDRTB) infection is one of the major problems for tuberculosis control and treatment. In 1993, the emergence oflife-threatening MDRTB in New York City among AIDS
patients was reported (Frieden et al ., 1993). AIDS patientsare susceptible to repeated episodes of new tuberculosis infections. Administration of anti-tuberculosis chemotherapy
results in a high probability of evolution of MDRTB strains1

within the community of HIV infected individuals withweakened immune systems (Gordin et al ., 1996 and Spellman et al ., 1998).
The prevalence of tuberculosis in Sub-Saharan Africancountries has increased in these 10 years. In Zambia, it isapproximately 500 per 100,000 people and 40,417 new patients were recorded in 1996 (WHO report, 1998). In Zambia, 73% of the adult tuberculosis patients are HIV-1 seropositive and 13% of all adult deaths are due to tuberculosis.
With HIV epidemic, tuberculosis is a major cause of death

TB Laboratory in University Teaching Hospital, Private Bag RW1, Lusaka 10101, Zambia
Virology Laboratory in University Teaching Hospital, Private Bag RW1, Lusaka 10101, Zambia
3
Department of Microbiology, Yamanashi Medical College, 1110 Shimogato, Tamaho, Nakakoma, Yamanashi 409-3821, Japan
4
Marumori Hospital, Nagamachi, Taihaku, Sendai 982-0011, Japan
Corresponding Author: Satoshi MITARAI, M.D., Ph.D.
Contact address anda present affiliation:
Clinical Research Division,
The Research Institute of Tuberculosis,
Japan Anti-Tuberculosis Association,
3-1-24 Matsuyama, Kiyose, Tokyo 204-8533, Japan
Tel: 0424-93-3090
Fax: 0424-92-8258
E-mail: mitarai@jata.or.jp
2

２
４(ZAMBART, 1999; Elliott et al .,1996). The shortages ofboth medical resources and knowledge about the disease areimpediments to adequate treatment of patients, resulting in
an increase in drug resistant M . tuberculosis (Bosman,2001).
For effective treatment of tuberculosis, antituberculosis drug susceptibility against the pathogen is essential information. The national anti-tuberculosis programin Zambia has not been operative since 1997. From thattime, the country has not produced the national figures fortuberculosis control. The goal of this study was to obtain
updated information on the drug susceptibility status of M .
tuberculosis infections in Zambia. The readily recognisable
clinical manifestations of tuberculosis infection were also
evaluated for their possible relationship to organism drug
susceptibility.

MATERIALS AND METHODSDefinitions of terms
INH, RFP, EMB, and SM were stipulated as firstline
drugs for treatment of tuberculosis. New case is a patient
who has never had treatment for tuberculosis, or who hastaken anti-tuberculosis drugs for less than 4 weeks. Retreatment case is a patient who was treated previously and requiring retreatment due to relapse, default and treatment
failure. Primary drug resistance is the presence of resistantstrains of M . tuberculosis in a patient with no history ofprior treatment. Acquired drug resistance is the resistancefound in a patient who had previously received at least one
month of anti-tuberculosis treatment, as documented in the
tuberculosis registry, or medical records or by the patient’s
account. Mono resistance is a presence of resistance to single drug. Combined drug resistance is the prevalence ofdrug resistance among all cases of tuberculosis, regardlessof treatment history. Any resistance is the prevalence ofdrug resistance including one designated drug at least. For
example, In the case of INH, it is a total of resistant strains
to INH, INH+RFP, INH+EMB, INH+SM, INH+RFP+EMB,
INH+RFP+SM, INH+EMB+SM, and INH+RFP+EMB+SM.
Clinical specimens
Sputum specimens were collected at the Chest Clinicin the University Teaching Hospital (UTH) in Lusaka, Zambia between March, 1999 and August, 2000. The UTH is
the third referral hospital located in the capital city of Zambia. Patients are referred from the entire Zambia. Suspected
tuberculosis patients who visited the Chest Clinic were requested to submit consecutive three sputum specimens forlaboratory examinations. The specimens are sent to the
UTH laboratory for routine fluorescent smear examination.

The sputum specimen yielding a positive smear result wascultured on conventional Lewenstein-Jensen media. The patients with positive smear were enrolled randomly to thestudy under informed consent and subjected to radiologicalexaminations. Some sputum specimens only for drug susceptibility testing were collected from routine laboratory
specimens. Blood specimens for haematological and immunological examination were collected from randomly selected tuberculosis patients.
Smear, culture and drug susceptibility testing
One portion of purulent sputum specimen was stainedwith auramine-phenol method and examined under a fluorescence microscope (200×). The smear positive sputum
specimen was cultured on Lewenstein-Jensen medium containing glycerol (L-J G) or pyruvate (L-J P). The inoculated
tubes were incubated at 37°
C and inspected weekly for up to
8 weeks. The isolates were subjected to Ziehl-Neelsen
staining for acid-fastness, niacin accumulation, pnitrobenzoic acid ( PNBA ) sensitivity, thiophen-2carboxylic acid hydrazide (TCH) and growth at 25 and 42°
C
for identification of M . tuberculosis. Susceptibility testing
to isoniazid (INH), rifampicin (RFP), ethambutol (EMB),
and streptomycin (SM) (Sigma-Aldrich Company Ltd.,
Dorset, UK) was performed using resistant ratio method on
L-J G medium. The resistance ratio is defined as the minimum inhibitory concentration (MIC) of the test strain divided by the MIC of the standard susceptible M . tuberculosis strain in the same set of tests. A resistance ratio of 2 orless defines drug susceptibility, while 4 or more is considered evidence of drug resistance. The concentrations foreach drug were as follows: INH 0.05, 0.1 and 0.2 µg/ml ;RFP 12.5, 25 and 50 µg/ml ; EMB 0.8, 1.6 and 3.2 µg/ml ;SM 10, 20 and 40 µg/ml . Wild standard strains for control
were obtained from the Chest Disease Laboratory (the national reference laboratory in Zambia).
Part of the specimen was also evaluated using the proportion method. The ratio between the number of colonies
on drug-containing medium and the number of colonies on
drug-free medium indicates the proportion of drug resistant
M . tuberculosis present in the population. From these bacterial colony counts, the proportion of resistance can be determined and as a percentage of the total number of viable
colony forming units in the population. Below a certain
fraction, called the “critical proportion”, a strain is classified as susceptible; above that, as resistant. The critical proportion for resistance was taken as 1% in general. The critical drug concentrations were as follows: INH 0.2 µg/ml ;RFP 40 µg/ml ; EMB 2.0 µg/ml ; SM 10 µg/ml .
Clinical data
All patients were queried as to whether they had beenpreviously treated for tuberculosis. Additionally chest roent-２５genograms were evaluated for opacity, cavity, pleurisy andlymphadenopathy. Patients were selected randomly for HIV
evaluation. All patients were enrolled with informed consent and given pre-and post-testing counsel. Blood specimens were obtained for both HIV antibody testing (BIONOR HIV-1&2: BIONOR, Norway) and CD4/CD8 analysis by flow cytometry (Becton-Dickinson, California, USA).
Statistical analysis
Statistical analysis was done using chi-square or
Fisher’s exact test. A p value of less than 0.05 was considered significant.
Ethical consideration
The study protocol has been approved by the ethics
committee of the University of Zambia. Subjects were enrolled after informed consent. Subjects younger than 15
years were excluded from the study.

RESULTS
Drug susceptibility
A total of 499 strains of M . tuberculosis were examined for susceptibility to 4 firstline anti-tuberculosis drugs.
Out of 499 strains, 257 were isolated from sputum specimens of study-enrolled patients. The remaining 242 werefrom sputum specimens for routine diagnosis. Only information about sex and age were available for these 242
specimens. In total, there were specimens from 271 males
and 228 females. The mean age was 31.7±10.3 years old.
A total of 365 (73.1%) patients were under 40 years old.
Sixty strains to INH (12.0%) and 52 to RFP (10.4%) wereresistant respectively. Mono and any resistance to INH wereobserved in 4.6% and 12.0% in combined drug resistanceevaluation. In the same way, mono and any resistance wereobserved in 5.2% and 10.4% to RFP, 2.4% and 7.2% toEMB, and 1.6% and 3.8% to SM respectively. The primary
and acquired resistances were observed in 13.9% andTable 1

16.1% to INH, 11.1% and 12.9% to RFP, 6.7% and 9.7% toEMB, and 3.8% and 3.2% to SM respectively. The multidrug resistance was observed in 22 strains/cases (4.4%).
The primary, acquired and combined resistances are summarised in Table 1. There were no significant differences
between primary and acquired resistances. Twelve strains(2.4%) were resistant to 3 drugs or more.
A total of 297 of the 499 strains were also evaluatedwith the proportion method. There were incidences of combined drug resistance of 11.7% to INH, 9.1% to RFP, 7.5%
to EMB, and 10.4% to SM. There was statistically significant difference between resistant ratio and proportion
method for the analysis of SM susceptibility (p=0.0004).
Clinical data
A total of 509 chest roentgenograms were available for
a comparison of new and retreatment cases. The only difference observed between the two groups was cavity formation(p=0.019). There were no differences in the clinical manifestations when data from 169 drug-susceptible patientswere compared to those from 56 drug resistant patients (Table 2).
Patients were treated with short course chemotherapy
for the standard 8 months period using the combination of
INH, RFP, EMB and pyrazinamide (PZA). Of 509 patients
394 among 433 new cases (91.0%) received this combination therapy while 29 (38.2%) for 76 retreatment cases (Table 3). The WHO standard for retreatment case was 5-drug
combination with INH, RFP, EMB, PZA and SM.
During the same study period, clinical outcomes of tuberculosis patients were monitored for 461 cases including
study population. A total of 225 patients (48.8%) achieved
negative sputum conversion at the time of 8-month while
152 patients (33.0%) defaulted in total. The 8-month mortality of retreatment cases was no higher than of new cases(Table 4).
Fifty-two blood specimens were obtained from ran-

Drug susceptibility patterns of primary, acquired and combined resistance

Number of strains

Primary (%)
208

Acquired (%)
31*

Combined (%)
499

Mono resistance
INH (mono)
RFP (mono)

9 ( 4.3)
11 ( 5.3)

2 ( 6.5)
1 ( 3.2)

23 ( 4.6)
26 ( 5.2)

EMB (mono)
SM (mono)
Resistant to 2 drugs
Resistant to 3 drugs
Resistant to all 4 drugs

5 ( 2.4)
3 ( 1.4)
17 ( 8.2)
4 ( 1.9)0 ( 0.0)

1 ( 3.2)0 ( 0.0)
3 ( 9.7)
1 ( 3.2)0 ( 0.0)

12 ( 2.4)
8 ( 1.6)
29 ( 5.8)8 ( 1.6)
4 ( 0.8)

Any resistanceMulti-drug resistance49 (23.6)
11 ( 5.3)

8 (25.8)
2 ( 6.5)110 (22.0)
22 ( 4.4)*18 of 257 patients had no data about the duration of preceding treatment.

２
６

Table 2

A comparison of clinical manifestation

Category

No. of patients
Mean age±SD(male/female)

Chest roentgenogram findings (%)

New
Retreatment

433 (247/186)
76 ( 42/ 34)

31.7±10.1
31.2± 9.0

FA
74 (17.1)
17 (22.4)

MO
229 (52.9)
42 (55.2)

MI
130 (30.0)
17 (22.4)

Cavity230 (53.1)
52 (68.4)*

LYM
25 (5.8)
1 (1.3)

Pleurisy51 (11.8)
13 (17.1)

Susceptible
Resistant (any)169 ( 89/ 80)
56 ( 37/ 19)31.8±11.0
32.4± 9.7

36 (21.3)
11 (19.6)

88 (52.1)
29 (51.8)

45 (26.6)
16 (28.6)

93 (55.0)
36 (64.3)

11 (6.5)
1 (1.8)22 (13.0)
8 (14.3)

FA = Far advanced: MO = Moderate: MI = Minimal: LYM = Hilar lymphadenopathy* p = 0.019

Table 3Treatment regimen of tuberculosis patients

Initial treatment
INH/RFP/EMB or SM/PZA

Number of patients (%)
New cases Retreatment cases43376394 (91.0)
30 (39.5)

Table 4

Clinical outcomes of tuberculosis patients

Outcome at 8 month
Sputum smear

Number of patients (%)
New cases Retreatment cases44417
251 (56.5)
9 (52.9)

INH/RFP/EMB/PZA/SM
INH/RFP/PZA
INH/RFP/EMB
RFP/EMB/PZA

6 ( 1.4)
12 ( 2.8)
1 ( 0.2)
1 ( 0.2)

42 (55.2)0 ( 0.0)0 ( 0.0)0 ( 0.0)

Positive
Negative
Not done
Default

9 ( 2.0)216 (48.6)
26 ( 5.9)
145 (32.7)

0 ( 0.0)9 (52.9)0 ( 0.0)
7 (41.2)

RFP/PZANo record

2 ( 0.5)
17 ( 3.9)0 ( 0.0)
4 ( 5.3)

Dead
Transferred

42 ( 9.5)
6 ( 1.3)

1 ( 5.9)0 ( 0.0)

domly selected pulmonary tuberculosis patients. The HIVseropositive rate was 69.2% (36/52 patients). Within the 36
HIV-seropositive patients, 34 were available for haematological testing. The mean CD4 lymphocyte number was
375.0±307.9/µl in total. Totally 35.3% (12/34) had lessthan 200/µl CD4 lymphocytes. There was no statistical difference in the drug susceptibility to 4 firstline drugs between HIV positives and negatives.

DISCUSSION
In the present study, the prevalence of anti-tuberculosis
drug resistance in Lusaka, Zambia was high and represents
a major problem for national tuberculosis control. The highincidence of resistant M . tuberculosis in Africa has been
documented in several studies. Weyer and Kleeger (1992)
reported in the 1980’s that the initial resistance in South African adult black patients was 14.2% to INH, 12.1% to SM,
1.8% to RFP and 2.5% to EMB respectively. The incidences,by 1995, in Ivory Coast were 11.3%, 6.9%, 5.3% and 0.3%
respectively (Pablos et al ., 1998). The multi-drug resistance
was reported less than 1% in 1993 in Zambia (Chest Disease Laboratory, 1994), but mono resistance to INH and
RFP, as reported from the Chest Disease Laboratory, was
4.7% and 1.8% by 1994. In the absence of a national tuberculosis control program, the drug resistance incidence has
increased in recent years.
There are several possible explanations for rising drug

resistance. One possible reason is an irregular administration of anti-tuberculosis drugs. It was shown that generalclinicians were following standard combination chemotherapy to treat tuberculosis patients although the Zambian government has not adopted DOTS as a national strategy. However, the treatments were completed in only 56.5% of thepatients with a 32.7% default rate among new cases. Additionally, the mortality during treatment was 9.5%. An unstable drug supply and insufficient awareness in tuberculosis
treatment may be the cause of low treatment completion.
The anti-tuberculosis drugs are provided by the government
without cost for tuberculosis patients in Zambia. However,the supply is neither constant nor sufficient resulting in insufficient treatment at clinics. The shortage of drugs in thehealth institutions discourages patients from coming to thehospital and health clinics. When supplies failed, the purchase of drugs was beyond the financial means of most patients. It is also conceivable that many patients defaulted
from treatment because transportation to the clinic waseither difficult or unavailable.
Another possible reason is relapse or re-infection withresistant strains. HIV prevalence in Zambia is approximately 20% in antenatal women (Handema et al ., 2001).
The epidemic has a potential for the development of M . tuberculosis drug resistance among HIV-infected individuals
as a result of the increased frequency of drug resistance including MDRTB in the population (Bradford et al ., 1996).
However, as indicated in this study, the frequency of drug２７

resistance was not significantly different between HIV positives and negatives. Even for the retreatment cases the frequency of resistance was not higher than new cases. The
data suggest that the resistant strains are spreading within
the community and causing new infection in the population
rather than evolving de novo from each patient.
Multi-drug resistance was observed in 4.4% of total
isolates. Even with this high MDRTB frequency, conversion
to negative sputum at 8 month was achieved in 86.1% (216of 251 evaluated cases) of the new patients with appropriate
treatment. Although the impact of MDRTB on the clinicalsituation at individual level is severe (Goble et al ., 1993;
Mitchison and Nunn, 1986), it may not require any revision
to short course chemotherapy regimen as a national strategy.
The blind use of a 5-drug combination for retreatment cases
may not be efficient, even though that is the recommendation of the WHO (WHO, 1996). The frequencies of drug resistance were not significantly different between primary
and acquired cases. Additionally, most of the patients were
new cases. In this situation, it is not informative to distinguish new from retreatment cases, but rather to follow thepatient, irrespective of drug resistance status, for treatment
efficacy. The suspected resistant patient who shows nonegative conversion of sputum will be transferred to an appropriate referral hospital for further treatment even withsecond and third line drugs.
A clinical indicator of drug-resistant M . tuberculosis
infection would be useful because it is impractical to perform culture and drug susceptibility examinations for eachpatient in Zambia. As mentioned above, the clinical history
was not useful to predict resistance. The chest roentogenogram is quick and feasible examination even in developing countries. However, it was unable to distinguish between drug-susceptible and drug-resistant disease. Also theHIV-serostatus were not informative for the prediction of
drug resistances in the present study. Some techniques forthe detection of resistance using molecular methods havedeveloped in advanced countries (Abe et al ., 2000), butthese may be beyond the resources of developing countries.
Thus, the search for a clinical or physical finding that correlates to drug susceptibility must continue.
In the present study, the resistant ratio method was employed for drug susceptibility testing. The methods used forthe definition of drug resistance did not appear to be critical
although the result figures for SM were different between
resistant ratio and proportion method. The difference observed for SM between resistant ratio and proportion
method was likely due to the critical drug concentrations
employed for examination. The minimal drug concentration
for resistant ratio method was 10 µg/ml and the criticalconcentration for proportion method was also 10 µg/ml .

Taking this concentrations into consideration, if the strains
indicating ratio of 2 were taken as resistant, the frequency
became 9.8% as compared to 10.4% using proportion
method. In this setting, the difference was not significant.
However, it will be important to introduce proportion
method widely in Zambia for data generalisation.
Tuberculosis has risen together with HIV epidemic. As
it is shown in this study, approximately 70% of tuberculosis
patients were HIV-seropositive. However, even in the HIVinfected patients, approximately 65% showed above 200/µl
CD4 lymphocytes. It was almost identical to other reports(Elliott et al ., 1995; Kassim et al ., 1995). Additionally,
only 4 patients (11.8%) had less than 100/ µl CD4 lymphocyte. Good treatment results are expected even in HIVinfected patients with tuberculosis in Zambia despite thedifficulty in curing the severely immunocompromised patient.
The treatment outcome in the present study was far
from satisfactory. It was not a direct objective of this study
to increase treatment success. However, the treatment of tuberculosis was not efficient because of poor cure rate andmany defaults over 30% both in new and retreatment cases.
For reducing treatment failure and default, the DOTS strategy is proven to be effective (WHO report, 2001). As reported in Korea, the prevalence of drug resistance can be reduced with proper treatment (Hong et al ., 1998). Therefore,
it is important to implement DOTS or DOTS like confident
treatment efficiently and monitor drug resistance consecutively. If drug resistant infection is detected, it will be required to employ DOTS-Plus strategy that uses individualised treatment regimens with second-line anti-tuberculosis
drugs.

ACKNOWLEDGEMENT
The present study is an achievement of Infectious Disease Control Project in Zambia (1995-2000) conducted by
Japan International Cooperation Agency.

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F., Hopewell, P.C. and Small, P.M. (1996): The changing
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２８４）Chest Disease Laboratory (1994): Anti-tuberculosis drug
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Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
１,２
０
０２, pp.２
９-８
５

PROCEEDINGS OF THE 42ND ANNUAL MEETING OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
22-24 September 2001, Tokyo

President
Masamichi Aikawa
Professor, Research Institute of Science and Technology
Tokai University
CONTENTS
President’s lecture
The Pathology of malaria
Research Institute of Science and Technology, Tokai University

Aikawa, M.

Key note speech (lecture)
Ⅰ Progress in the global effort to Roll Back Malaria
Ⅱ MIM/TDR task force on malaria research capability strengthening in Africa

Mendis, K.
Zicker, F.

Special lecture! The future of global vaccine research
Takeda, Y.
" Altered immune responses in mice with concomintant Schistosoma mansoni and Plasmodium chabaudi Infection
Troye-Blomberg, M.
Ｓ‐!‐
１ Cholera
Yamasaki, S.
Ｓ‐!‐
２ Pandemic spread of vibrio parahaemolyticus infection: an enteric infection spread from a tropical zone in asia to the world？
Nishibuchi, M.
Ｓ‐!‐
３ Salmonella
Hayashi, H.
Ｓ‐"‐
１ Importance of acute respiratory infections (ARI) in tropical and developing countries
Oishi, K. et al.
Ｓ‐"‐
２ Viral acute respiratory infections in tropical countries−focus on influenza virus infections−Suzuki, H.
Ｓ‐"‐
３ Burden of Haemophilus influenzae type b (Hib) diseases in Bangladesh
Saha, S. K. et al.
Ｓ‐Ⅲ‐
１ Recent developments in HIV/AIDS research
Kitamura, T.
Ｓ‐Ⅲ‐
２ Research and development of recombinant HIV vaccine−Pre‐clinical trial
Honda, M. et al.
Ｓ‐Ⅲ‐
３ Treatment of AIDS
Hatanaka, M.
Ｓ‐Ⅳ‐
１ Trials and prospects of epidemiological survey for babesiosis in Japan and malaria‐endemic areas
Saito‐Ito, A. et al.
Ｓ‐Ⅳ‐
２ Novel strategies for tick control
Fujisaki, K.
Ｓ‐Ⅳ‐
３ Simian malaria parasites as zoonotic pathogens
Kawai, S. et al.
Ｓ‐Ⅳ‐
４ Leishmaniasis as a zoonosis: DNA diagnosis and molecular epidemiology based on PCR
amplification and characteristics of the leishmania miniexon gene
Katakura, K.
Ｓ‐Ⅴ‐
１ Progress of global polio eradication programme
Miyamura, T.
Ｓ‐Ⅴ‐
２ International cooperation in the area of tuberculosis control
Ishikawa, N.
Ｓ‐Ⅴ‐
３ Malaria control program in Lao PDR supported by Japanese grant aid for child health
Kobayashi, J. et al.
Ｓ‐Ⅴ‐
４ Leishmaniasis: its recent topics and research activities in Ecuador
Hashiguchi, Y.
Ｓ‐Ⅴ‐
５ Current activities of regional network on schistosomiasis control in Asia and roles of the Japanese
researchers in the network
Ohta, N.
Ａ‐
１ Changing patterns of antimalarial drug resistance in Plasmodium falciparum mirror the changeof the national malaria treatment policy in Malawi
Bwijo, B. et al .
Ａ‐
２ Analysis of pfmdr1 gene in mefloquine resistant plasmodium falciparum
Begum, K. et al .
Ａ‐
３ Recovery of chloroquine sensitivity and low prevalence of K76T in pfcrt after the change ofmalaria treatment policy in Malawi
Mita, T. et al.

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Drug resistant malaria in Mindanao, the Philippines‐Application of AnaeroPack! malaria culture
system to in vitro drug susceptibility test of plasmodium falciparum in the field
Hatabu, T. et al.
An epidemiological study on malaria in the displaced populations in Viet Nam
Kano, S. and Nakazawa, S.
Recrudescence in vitro after D‐sorbitol treatment over nine days
Nakazawa, S.
Characteristics of cholera toxin‐producing vibrio cholerae strains isolated from seafood in Malaysia
Hsien, C. C. et al.
Flagella filament protein (FliC) induces human β‐defensin-2 (hBD-2) production in host cells
Wada, A. et al.
Enhanced excystation and metacystic development of Entamoeba invadens by cytochalasin D Makioka, A. et al.
Involvement of signaling molecules in the growth and encystation of Entamoeba invadens
Makioka, A. et al.
Bacterial expression of a human monoclonal antibody‐alkaline phosphatase conjugate specific for
Entamoeba histolytica
Tachibana, H. et al.
The regulatory role of nitric oxide (NO) in proliferation of Toxoplasma gondii in mouse brains examinedwith the treatment of L‐NG‐monomethyl‐arginine and QC‐PCR assay method
Mun,Hye‐Seong. et al.
T. gondii infection induces anti‐HSP70 autoantibody in a susceptible C57BL/6 mice by modulating rag genes
Chen, M. et al.
A thought experiment on the link between the risk of congenital toxoplasmosis and the annual infection rates
Naoi, K. and Yano, A.
Phenotype and function of murine peritoneal cavity macrophage derived dendritic cells
Levi, M. et al.
Growth inhibitory effects of triclosan against Babesia spp. parasites
Bork, S. et al.
Geopathological study on malignant tumors in western Kenya between 1979 and 1998
Toriyama, K. et al.
Synergistic effect of two susceptible genetic markers, HLA-B5*0101 and IL-13P-A/A, on thedevelopment of post‐schistosomal liver fibrosis.
Hirayama, K. et al.
A survey on antigenemia of Wuchereria bancrofti and urine antibody titer in rural communities in Nepal
Watanabe, K. et al.
Mathematical model for the transmission of lymphatic filariasis and its applications
Ishii, H. et al.
A trial to detect anti‐B. pahangi IgG4 in urine from children under 5 years old in order toevaluate filariasis control measures
Itoh, M. et al.
Resistance of Blastocystis hominis to metronidazole
Kaneda, Y. et al.
Long term eradication rate of ivermectin therapy for strongyloidiasis on Yoron Island, Kagoshima prefecture
Zaha, O. et al.
Prevalence of oxyuriosis and other helminth at the school of mountaineous region of Nepal:
influence by target selected and blanket treatment
Haruki, K. et al.
A survey on allergy, parasitic infection and nutritious status in school children in Vietnam
Fujimaki, Y. et al.
HIV-1 subtyping in Zanbia
Handema R. et al.
Molecular epidemiology of HIV in Myanmar: emergence of new forms of HIV-1 intersubtype recombinants
Takebe, Y. et al.
Trials of DNA vaccine to Japanese encephalitis virus (JEV): expression of JEV‐envelope protein in
mammalian cells
Feng, Guo‐HE. and Takegami, T.
Evaluation of dengue antigen detection kit
Harada, M. et al.
Biological characteristics of dengue2 viruses isolated from patients with different severities in Ban Hom commune,
rural area of Vientiane, Lao P. D. R.
Saito, M. et al.
Combined detection and genotyping of Chikungunya virus by a specific reverse transcription‐polymerase
chain reaction
Hasebe, F. et al.
A questionnaire survey for dengue control in Blitar municipality, East Java Province,Indonesia:
an evaluation for better communitiy education approach
Siswanto. et al.
Purification of recombinant trypanosome alternative oxidase
Kawai, K. et al.
Dihydroorotate dehydrogenase catalyzing soluble fumarate reductase in Trypanosoma cruzi. Takashima, E. et al.
Comparative analysis of cDNA expression profiles between bloodstream and procyclic forms of
Trypanosoma b. brucei using fluorescent differential disply method
Ohshima, S. et al.
Preliminary analysis of genes expressed during Leishmania paratites infection using cDNA microarray
Uezato, H. et al.

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Defect of Leishmania antigen‐specific interferon‐g production in a patient with diffused cutaneousleishmaniasis in Ecuador
Hamano, S. et al.
Clinical survey of cutaneous leishmaniasis in Ecuador last 10 years (1991〜2000)
Maruno, M. et al.
Flagellin from Burkholderia pseudomallei stimulates pulmonary epithelial cells to induce IL‐8 production
Saito, M. et al.
Clinical and microbiological characteristics of respiratory infections among HIV‐infected patients innorthern Thailand
Asoh, N. et al.
Mycobaterial infection in Thai children infected with human immunodeficiency virus in Thailand Kirikae, T. et al.
Collaboration of medical team and field epidemiologist during the outbreak of Ebola hemorrhagic feverin Uganda
Iwasaki, E.
Detoxifying effects of Achyranthes Japonica extract against Habu venom
Minakami, K. and Jitouzono, T.
The use of travel vaccines by Japanese expatriates in developing countries
Hamada, A. et al.
Survey on patients with imported infectious deseases admitted to the Department of Infectious Deseases
Ohnishi, K. and Kato, Y.
Current status of imported malaria in Japan since April, 1999
Ohtomo, H. et al.
Analysis of malaria surveillance data based on infectious disease prevention law in Japan
Kaku, K. et al.
How could the ISTM’s mailing list Travel Med be utilized？
Kimura, M. et al.
Cerebral metabolic changes in a primate model of severe human malaria as studied by positron emission tomography
Sugiyama, M. et al.
Glucose metabolic change of spleen in malaria‐infected Macaca fuscata: correlation of FDG‐PET resultswith pathological findings
Ikeda, E. et al.
Cerebral ring hemorrhages in squirrel monkeys infected with Plasmodium falciparum
Katakai, Y. et al.
Identification of malarial antigen‐mimicking peptides
Ishikawa, D. et al.
Construction of anti‐P. falciparum/anti‐CD3 bispecific scFv with antiparasiticidal function
Yoshida, S. et al.
Plasmodium vivax transmission‐blocking vaccine: efficacy study on human isolates
Tsuboi, T. et al.
An attempt to identify genes controlling susceptibility to mouse severe malaria
Nagayasu, E. et al.
Biochemical analysis of complex! from plasmodium falciparum mitochondria
Mi‐Ichi, F. et al.
Molecular characterization of Al‐cys peroxiredoxin from the human malaria parasite Plasmodium falciparum
Kawazu, S. et al.
Effect of Minocycline in combination with antimalaria drug on the elimination of Plasmodium
berghei and chloroquine resistant‐P. chabaudi parasites in mice
Hando, K. et al.
Anti‐malarial effects of dipyridamole in vitro and in vivo.
Nakano, Y. et al.
Development of new antimalarial drug‐in vitro and in vivo antimalarial activity of endoperoxides
Ono, K. et al .
A public/private partnership among the Japanese pharmaceutical industry, the government and
WHO/TDR (JPMW) : A research project for discovery of new antimalarial drugs in Japan
Hata, K. et al.
Both mosquito‐derived xanthurenic acid and a host blood‐derived factor regulate gametogenesis of
Plasmodium in the midgut of the mosquito
Arai, M. et al.
Studies on malaria micro‐gemetocyte exflagellation inducing factor in the salivary gland of Anopheles stephensi
Matsuoka, H. et al.

３２

President’s lecture
THE PATHOLOGY OF MALARIAMASAMICHI AIKAWA
Research Institute of Science and Technology, Tokai University

Study of the pathology of malaria has contributed tounderstanding of its pathogenesis. I will summarize mywork of 30 years in the fields of malaria pathology as follows. The main pathologic changes appear to be caused by
the response of the reticuloendothelial system to theparasaite, by the destruction of erythrocytes which together
with their defective production causes anemia and by theobstruction of capillary lumens in the brain and other organs by parasitized erythrocytes (PRBC). Most pathologic
studies of malaria are of P. falciparum which causes severe
malaria. Cerebral malaria is recognized by the blockage ofthe cerebral capillaries by PRBC and associated pathologic
lesions. Based on these findings, the development of malaria vaccines against cerebral malaria is now in progress.
Splenomegaly and hepatomegaly are common findings
among malaria patients. Renal disease also occurs in malaria. In falciparum malaria, mesangiopathic glomeru-

lonephritis causes abnormal urinary sediment including proteinuria and cylinduria. In contrast, with quartan malaria
membrano-proliferative glomerulonephritis leads to a nephrotic syndrome. In pregnancy, falciparum malaria severely
affects the placenta, especially in primiparous women, often
resulting in abortion. Microscopic fields show intervillous
spaces with many PRBC and macrophages. Recently adhesion molecules such as hyaluronic acid and CSA have been
identified. These molecules assist PRBC to cytoadhere totrophoblasts and PRBC sequestration results.
Despite the fact that both the anatomic and microscopic alterations associated with malaria are well recognized, the pathophysiologic changes in malaria infection are
not well characterized. A better understanding of themechanisms of malaria infection may contribute to more efficacious treatment with pharmacological, vaccines and molecular approaches.

３３

Key note speach (lecture)
PROGRESS IN THE GLOBAL EFFORT TO ROLL BACK MALARIAKAMINI MENDIS
Roll Back Malaria Project, World Health Organization, Geneva, Switzerland

The Roll Back Malaria (RBM) partnership to reduce
the global burden of malaria has now been in operation fornearly 3 years. Established on the principles of tackling malaria as a health and development problems and using malaria to strengthen the health systems of malaria endemic
countries, the RBM partnership has increased awareness ofmalaria as a major cause of poverty and underdevelopment,
and inspired action against it at global, regional and country
levels. RBM is a global effort with a major focus on Africa,
which suffers the greatest burden of malaria. The technicalstrategies of RBM are 1) the provision of early and effective
treatment against malaria, 2) prevention against malaria using insecticide-treated nets and other mosquito control
methods, 3) reducing the impact of malaria in pregnancy
and 4) preparedness to tackle malaria epidemics. Technicalissues such as resistance to antimalarial drugs are being
handled at a global and regional level through better surveillance, and arrangements to foster new drug development.
The RBM movement is underpinned by a strong reseachand development (R&D) component-working with partners
at all levels facilitating R&D on implementation and operational research to improve the delivery of existing tools, butalso advocating for longer-term investments for new tools
such as vaccines and new drugs.
Countries in sub-Saharan now have technically soundRBM strategic plans embodying RBM principles. These
strategic plans are technically sound and use evidence based
approaches and are integrated into the national health sector
development plans. RBM is being used by countries to access financial resources through sector-wide approaches andpoverty reduction strategies such as the Heavily Indebted
Poor Country (HIPC) initiatives. Resource short-falls for 12
countries in Africa which are now ready to implement RBM
are in the range of 0.5 billion US Dollars and global efforts
to bridge these gaps are under way. In other regions of theworld, the Mekong countries in Asia, and the Amazon region in South America are working as alliances to roll backmalaria.

MIM/TDR TASK FORCE ON MALARIA RESEARCH CAPABILITY STRENGTHENING IN AFRICAFABIO ZICKER
Research Capability Strengthening, UNDP/World Bank/WHO Special Programme for Research andTraining in Tropical Diseases, Geneva, Switzerland

The medical and social-economic burden of malaria
remains a major public health challenge in Africa. The reduced population access to effective treatment, rapid
spreading of antimalarial drug resistance and vector resistance to insecticides have limited the impact of interventions on malaria morbidity and mortality. The developmentof a sustainable malaria research community in Africa isconsidered a priority to develop and improve public healthinterventions and policies.
The Multilateral Initiative on Malaria (MIM) is an alliance of organizations and individuals working together tomaximize the impact of scientific research against malaria
in Africa (http://mim.nih.gov) It aims to raise international
public awareness of the problem of malaria, promote global

research collaboration and co-ordination to improve malaria
research capacity in Africa. MIM was launched at the conference on “Malaria in Africa: Challenges and Opportunities for Co-operation” held in Dakar (January 1997). Scientists and decision makers from Africa and from several international institutions met with funding organizations andidentified scientific questions to be answered in order to address the problem of malaria in Africa. A multilateral funding mechanism, including Japan, was set up by the UNDP/World Bank/WHO Special Programme for Research andTraining in Tropical Diseases (TDR) to address these research questions and promote research capacity. The MIM/TDR Task Force is supporting a total of 23 multi-country
partnership projects between African scientists (engaged in３４basic and/or applied science) involving 24 African countries
and investigators in developed countries with focus on
strengthening the African research groups to develop effective control tools malaria and improve relevant health policy strategies. The overall aim is to generate new knowledge, develop products and intervention processes relevantto the understanding of the occurrence, distribution, morbidity and control of malaria in Africa.
The Task Force addresses the following priority areas
identified at the Dakar meeting: antimalarial drug policy
and chemotherapy, epidemiology, pathogenesis and immunology, entomology and vector studies, health systems research including social science, and natural products anddrug development. The partnerships has provided opportunity for studying specific aspects of malaria in multiple sites,
establishment of regional networks, and training of PhD and
MSc students.

The projects under way will contribute to the developmentof:・Strategies for mapping, retarding and reversing antimalarial drug resistance・New methodology for improving malaria home management
・Surveillance network on malaria mortality/morbidity tosupport control initiatives
・Network on vector biology, insecticide resistance andvector population genetics
・Identify potential antimalarial compounds from natural
products
・Studies on the relationships between parasite diversity,
immune response, resistance, transmission, infection anddisease patterns
・Better understanding of parasite-vector-host factors involved in severe disease.

３５Special lecture
I THE FUTURE OF GLOBAL VACCINE RESEARCHYOSHIFUMI TAKEDA
Faculty of Human Life Sciences, Jissen Women’s Univewsity

Vaccines are designed to produce long-term protective
immune responses against pathogens and are the most effective medical interventions used to prevent human morbidity and mortality associated with infections diseases.
The eradication of the dreaded viral infection caused byhuman poxvirus is the best success story of how the efficient use of an effective vaccine can eliminate a disease,globally. Dramatic reductions in the incidence of numerous
other childhood diseases, such as poliomyelitis, measles andrubella also show the success of efficient vaccines. To datemost of the diseases where vaccines have been effective are
diseases in which no vectors are involved. A vaccine strategy in diseases in which a vector is involved appears to beconsiderably more complicated and an outstanding exmaple
is malaria. In contrast, enteric diseases like cholera andshigellosis, an effective vaccine should be easier to implement since a vector is not involved in causing the disease
and since there are no known animal reservoirs of thecausative agent. However, the exact type and quality of immunity afforded by natural enteric infections is still not
clearly understood in these enteric infections. Rapid advances in immunology, molecular biology and peptide
biochmistry have fostered the development of new approaches in the development of vaccines including the useof peptide vaccines and genetic vaccines. The future to conquer enteric diseases and other infectious diseases will
largely depend on how successful the host of new entericvaccines will be that are being developed using there new
approaches. The WHO Vaccine Trial Registry documents an
expanding research capacity with an average of 3.9 newstudies per year during 1987-1933 rising to 10.7 per year
during 1994-2000 with nearly 80% of these studies being
conducted in developing countries. This is a good trend forthe future and it is believed that over the next few years effective vaccines will be in place for some of most dreadfulinfectious diseases.

II ALTERED IMMUNE RESPONSES IN MICE WITH CONCOMINTANT SCHISTOSOMA MANSONI AND
PLASMODIUM CHABAUDI INFECTION
M.TROYE-BLOMBERG
Department of Immunology, Wenner Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden

Mixed patasitic infections are common in humanpopulations living in malaria-endemic areas. However, little is known how concurrent infections affect the immunity
and/or pathogenesis to each other. Protection and elimination of blood-stage Plasmodium chabaudi chabaudi As in
resistant mice are characterized by a sequential activation of
CD4+ Th-1 and Th2 cells. The patent egg-laying stage of themurine model of Schistosoma mansoni is associated with astrong Th2 response to Schistosoma as well as unrelated antigens. The present study was performed to examine if S.
mansoni-induced Th2 type of responses would prevent or
inhibit the Th1 response required for the early clearance ofP. chabaudi, thus giving rise to a more severe disease and/or mortality. Our data show that malaria parasitemias weresignificantly higher in mice infected with malaria at thetime point of a patent egg-laying S. mansoni infection. In
vitro stimulation with experiments using spleen cells
showed that TNF-α production was sighnificantly impaired
during the first week of the malaria infection in mice withconcurrent S. mansoni/P. chabaudi infections. In contrast,
the P. chabaudi induced Th1 type as measured by IFN-γ
production, was not affected by the S. mansoni infection.
Taken together our date suggest that schistosoma and malaria infections profoundly affect each other, finding whichmight have implications for the development of future vaccines.

３
６

S−I−1) CHOLERASHINJI YAMASAKI
Laboratory of International Prevention of Epidemics, Department of Veterinary Sciences, Graduate School of Agriculture andBiological Sciences, Osaka Prefecture University

Cholera is still a fatal disease in developing countries.
Among 138 serogroups of Vibrio cholerae only O1 serogroup was thought to have potential to cause epidemic andpandemic of cholera. In 1992, however, a non-O1 V. Cholerae was involved in large outbreaks of cholera in thesouthern part of Inida. After preliminary serotyping studies,
this strain was designated as V. cholerae O139 synonym
Bengal. The O139 strain rapidly spread to neibouring countries such as Bangladesh, Nepal, Thailand and China, indicating that this strain had the potential to cause a cholera
pandemic.
Since that time we have initiated several collaborative
studies with National Institute of Cholera and Enteric Diseases, Calcutta on the molecular epidemiology and characterization of Vibrio cholerae strains isolated from diarrheal
patients admitted to ID hospital. Through these studies, wehave demonstrated followings, (1) predominant serofroupof V. cholerae isolated from the patients through the active
surveillance between 1992 to 2000 was O1 serogroup andno O1 serogroup was isolated between January to July in1993 when O139 was predominantly isolated during thisperiod; (2) O1 V. cholerae isolated after O139 epidemic was
not similar to those isolated before O139 epidemics; (3) the
20 kb region responsible for O1 antigen biosynthesis was
replaced by a 36 kb fragment carrying the O139 specificgenes in V. cholerae O139 and the 36 kb gene was most homologous to that in O22 serogroup; (4) the O1 and O139
specific genes were identified and a multiplex PCR, which
can simultaneously amplify the ctxA, O1 specific and O139
specific genes was developed; (5) during the course of surveillance non-O1, non-O139 V. cholerae was also isolated
from patients with severe diarrhea particularly during certain periods and these strain did not carry any known virulence factors normally present in toxigenic V. cholerae O1;(6) antibiotic resistant strains including multidrug resistantand resistance to new quinolones were observed and (7) antibiograms of O1, O139 and non-O1, non-O139 serogroups
differed between each other during any given period.

S−I−2) PANDEMIC SPREAD OF VIBRIO PARAHAEMOLYTICUS INFECTION: AN ENTERIC
INFECTION SPREAD FROM A TROPICAL ZONE IN ASIA TO THE WORLD?
MITSUAKI NISHIBUCHI
Center for Southeast Asian Studies, Kyoto University

High incidence of Vibrio parahaemolyticus infection
has been reported in the tropical and surrounding areas in
Asia since 1996. The research done by our network revealed that it was due to the pandemic spread of V. parahaemolyticus infection. We first investigated the reason fora sharp increase in the number of V. parahaemolyticus infection in Calcutta in February 1996, and found that the increase was due to the infections by a newly emerged V.
parahaemolyticus. The new type of V. parahaemolyticus
was characterized by O3:K6 serovar, possession of the tdh
gene, lack of the trh gene, and unique profile of arbitrarily
primed PCR. The strains belonging to the same type weredetected among those isolated from international travelers
coming from three Southeast Asian countries to Japan.
Subsequently, we conducted a multinational collaborative

study and revealed that infection by the new type of V.
parahaemolyticus occurred very frequently in Bangladesh,
Thailand, Laos, Taiwan, Korea, Japan, and United States.
We developed a new PCR method targeting the bases of the
toxRS operon unique to the new type of V. parahaemolyticus so that it can be detected by this simple PCR method.
Examination of many clinical stains by this PCR (named as
GS-PCR for group-specific PCR) allowed us to find that thestrains belonging to the new type of V. parahaemolyticus
but exhibiting O4:K68 and O1:KUT serobars emerged in
1998. We named the new type of V. parahaemolyticus, including these serovars, a pandemic clone. DNA fingerprinting analyses by pulsed-field gel electrophoresis and ribotyping illustrated clonality of and subtle difference among the
pandemic strains. Next, we were able to isolate an O3: K6

３
７

strain belonging to the pandemic clone from bloody clam in
Hat Yai City in southern Thailand. A total of 317 strains of
V. parahaemolyticus were isolated from diarrhea patientsduring one-year survey in two hospitals in Hat Yai City in
1999. GS-PCR-positive strains accounted for 75.4% of theisolated and 80% of the GS-PCR-positive strains belonged
to O3:K6 serovar. The results demonstrated strong relationship between clinical and environmental O3:K6 strains. In

addition, GS-PCR-positive strains belonging to three newserovars were detected, indicating emergence of the serovariants in this environment. The new serovariants of thepandemic strains were also detected among the international
travelers from Thailand and Viet Nam. We also are examining about 600 clinical strains isolated in Nhatrang, Viet
Nam to further expand our study on the epidemiology of thepandemic strains in Southeast Asian countries.

S−I−3) SALMONELLAHIDEO HAYASHI
Department of Infection Biology. Institute of Basic Medical Sciences, University of Tsukuba

Salmonella causes two major types of infectious diseases, enteric fever and food poisoning. The enteric fever iscommon in tropical countries from where travelers got infected and bring into the developed countries. According to
the census of NIID, Japan, the reported cases of enteric fever in Japan was 148, during the period from April ’99 toNovember ’00. Among the cases, 108 (73%) were imported
from tropical countries and the infected patients were almost men at their age of 20’s. On the contrarily, salmonellar food poisoning which causes limited enteritis in man has
been the most common in developed countries. Salmonella
has been the top ranked food poisoning causative agent for
many years in Japan. S. Typhimurium and S. Enreritidis
have been the common agents but, in 1999, there happeneda diffuse out break by S. Orenienburg that spreadded
through dried squid-snack. We examined the specificity ofthe outbreak strains. The genomic pattern by PFGE was
identical among the outbreak isolates but different fromthose isolated in Thailand and the isolate from the environment. The outbreak isolates had a couple of small plasmids
and this was specific among the isolates from Ibaragi prefecture. The function of the plasmids are under investigation, but the reason why this train caused sudden out breakhas not been clarified.
Genus Salmonella contains more than 2,000 serotypes
and ranges over variety of hosts from lower reptiles tohigher primates. The virulent factors are also very diverese:it has almost all of bacterial virulence factors such as, adhesin, invasin, impedin aggressin, modulin and secretable enzymes. Those natures make it difficult to identify patho-

genic strains to human with a simple and rapid method.
The whole genome analysis was thought to be the most efficient method to solve those complex problems. The nineof the prototype strains have been subjected to the wholegenome analysis but so far none of them is publicized as yet.
The problems seem to lie in the variety or the flexbility of
the genome structure and the complexity of regulatory
mechanism of the gene expression. The five kinds of pathogenicity islands have been characterized but the relation to
the serotypes or the distribution among the species are still
indistinct. To prevent Salmonella infection, it needs a specific detection/identification method for human specificvirulence factors.
We have been challenging to solve these problems bysome different approaches: genetic analysis and identification of human specific virulent genes, detection of the specific gene by rapid PCR, identification of serotype specificgenes by DNA chip and rapid PCR method, development of
ELIZA for detection of enterotoxin in foods and clinicalsamples. With the application of gene subtraction method,
we had been detecting human specific virulent genes andisolated seven of 2kb DNA fragments of which function remain to be identified. The developed methods mentioned
above were briefly introduced.
This study was carried out by the cooperation with, H.
Kurazono, Okayama University, S. Makino, Obihiro Veterinary College, S. Nakayama, NIID Japan, W. Chaicumpa,
Mahidol University, P. Sudarmono, University of Indonesia,
T. Kumao and W. Ba-Thein, University of Tsukuba.

３８

S−II−1) IMPORTANCE OF ACUTE RESPIRATORY INFECTIONS (ARI) IN TROPICAL AND
DEVELOPING COUNTRIESKAZUNORI OISHI, HIROYUKI YOSHIMINE, AND TSUYOSHI NAGATAKE
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University

Acute respiratory infections are common causes ofmorbidity and mortality in tropical and developing countries. A changing pattern of these diseases has, however,been recognized especially in Sub-Saharan Africa, where
there is a high seroprevalence of HIV infection.
A hospital-based prospective study on a total 99 patients with community-acquired pneumonia (CAP) was carried out in Kampala, Uganda between 1996〜1998. We
evaluated the microbiological etiologies, clinical featuresand effectiveness of short-term parenteral ampicillin followed by oral amoxicillin for these patients according toHIV-status. We demonstrated a very high prevalence(74.8%) of HIV-1 infection. No significant difference wasobserved with respect to age, gender, prior antibiotic usage,
symptoms, laboratory data or bacterial etiology between
HIV-1-infected and HIV-uninfected CAP patients. Most of

Streptococcus pneumoniae (19 strains) and Heamophilus
influenzae (8 stains) isolated from HIV-1-infected patientswere penicillin-resistant (94.7%) and b-lactamase producing (75.0%) stains, respectively. We, however, found a highpercentage of good clinical response both in HIV-1-infected(81.3%) and HIV-uninfected (85.7%) among 39 patientswith CAP due to bacterial pathogen. In addition, we documented a clinical feature of fifteen cases of recurrent bacterial pneumonia among 101 HIV-infected persons with CAP
between 2000〜2001. The mean CD4 levels of these patients were found to be 192/ml, and the mean interval between the first and the secound episode was 155 days.
Our present date suggest a clinical importance of bacterial CAP among HIV-infected persons and usefulness of
short-term parenteral ampicillin therapy for these patients in
HIV-endemic, developing countries.

S−II−2) VIRAL ACUTE RESPIRATORY INFECTIONS IN TROPICAL COUNTRIES-FOCUS ON
INFLUENZA VIRUS INFECTIONSSUZUKI HIROSHI
Department of Infectious Disease Control and International Medicine,
Niigata University Graduate School of Medicine and Dental Sciences

Respiratory infections are the most common afflictionsof humans, and mostly are caused by viruses. Children contract on average about half a dozen respiratory illnesses
each year, and adults perhaps two or three. While many
people consider influenza to be a minor disease, this may be
due to be a minor disease, this may be due to confusion in
the diagnosis. Symptoms of influenza resemble those ofother infections and the term is often misapplied to a rangeof minor upper respiratory tract infections as a respiratory
syndrome. More over, episodes of bacterial pneumonia,
and worsening of underlying conditions, may not be recognized as being a consequence of prior influenza virus infections. Laboratory diagnosis is essential to verify the circulation of influenza viruses, and to generate reliable date on
morbidity and mortality, especially in tropical or subtropicalregions.
In most tropical subtropical regions, laboratory-

confirmed influenza can occur throughout the year, withpeaks of increased activity once or twice each year. Wheninfluenza may be not noticed because of a lower peak demand on health services. Furthermore, there were quite
limited epidemilogical date of influenza infections in theseregions due to lack of laboratory personnel and equipment.
Viral isolation is a basic and important technique in thefield of virology despite the development of moderm techniques. One cell line can only culture a limiting numbers ofthese respiratory viruses, and has made it difficult to use thevirus isolation as a routine method for laboratory diagnosis
and epidemiological work of acute respiratory infections(ARI). In order to overcome this difficulty, we used the microplate method for virus isolation, which utilizes five celllines and thus covers a wider range of respiratory viruses.
Application of the method contributes to the routine diagnosis and helps define the epidemiology of viral ARI.

３
９

As influenza has consistently been associated with excess mortality and observed mainly in patients aged 65 orolder, influenza vaccines are strongly recommended incountries for those persons who are at increased risk ofcomplications. Hong Kong and Taiwan have influenza vac-cination program for elderly people, but not other Southeast
Asian countries. In more then 1 peak of influenza occurs
each year, we need to study the appropriate timing of vaccination and selection of the vaccine formation.

S−II−3) BURDEN OF HAEMOPHILUS INFLUENZAE TYPE B (HIB) DISEASES IN BANGLADESH
SAMIR K SAHA1, M. HANIF1, M. RUHULAMIN1, K. MATSUMOTO2, K. OISHI2, T. NAGATAKE2
1

2

Bangladesh Institute of Child Health, Dhaka Shishu (Children) Hospital, Dhaka-1207, Bangladesh.
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

The virtual elimination of Haemophilus influenzae
type b (Hib) disease from industrialized countries by vaccine is a moderm public health triumph. However, the disease is still prevalent in the developing part of the world,
where the vaccine is not available, and causes invasive diseases like pneumonia, meningitis etc. Hib diseases cause
12.9 million deaths per year in under five children and mostof them are in the nonindustrialized countries. In the recent
years, the problem is further aggravated due to the emergence of multidrug resistant Hib strains. Surveillance onburden of Hib diseases is focused on meningitis cases as thediagnosis is straight forward and can yield a very accurate
estimates of over all burden of the disease.
In the study period, from 1994 to 2000, 338 invasive
strains of H. influenzae were isolated from blood and CSF
of pneumonia and meningitis cases respectively. H. influenzae was the most predominant (50%) among the isolates
followed by S. pneumoniae (32%), and 97% of the all H.
influenzae isolates were type b (Hib). A vast majority(91%) of Hib infections were found in first year and only
few (2%) after 2nd year of life.
Over all 35%, 54% and 16% of the strains were resistant to ampicillin, cotrimoxazole and chloramphenicol re-spectively. Most (95%) of the ampicillin resistance was due
to data-lactamase production, as detected by nitrocefin disk,
and the MIC level these strains were>256.0 ug/ml. Cotrimoxazole resistance showed an increasing trend with peak(64%) in 1996 and then declined to the level of 33% in
2000. Resistance to ampicillin remained similar through out
the study period (range 33% to 43%). The most notablefinding was the emergence of resistance to chloramphenicolin 1996 (5%) and its increasing trend in the subsequent
years, which reched to the level of 40% in 2000.
Meningitis caused by Hib resulted death in 34% andsimilar number were survived with sequel. Eighty four percent of the children with Hib diseases were from very poorfamilies who cannot afford to buy the medicines and vaccines for treatment and prevention respectively.
These findings has paramount implications in childhealth as cotrimoxazole is recommended by WHO for
pneumonia, at the community level, and ampicillin andchloramphenicol is the most available and widely used antibiotics in the hospitals of this country. Lastly, the predominance of Hib in causing invasive diseases and emergence ofresistance among them strongly indicates the need of introduction of Hib vaccine in EPI program of Bangladesh.

４０S−III−1) RECENT DEVELOPMENTS IN HIV/AIDS RESEARCHTAKASHI KITAMURA
Director Emeritus, Nattional Institute of Infectious Diseases/Toyama Institute of Health“Panel on AIDS” of US-Japan Medical Science Program (CMSP) has been working with US Panel to combine
the research activities and achievements on HIV/AIDS carried out in both United States and Japan. As the JapanesePanel chair, the speaker has been watching the developments of global HIV/AIDS pandemic and researches tofight against it. Here, as the first speaker of the session, I
would like to present the recent developments in HIV/AIDS
pandemic and researches in various aspects against it.
1) Epidemiology; AIDS was recognized in 1981 by
US-CDC. By its excellent studies based on surveillance it
could identify several key risk factors by March 1983, before the discovery of causing virus by Montagnier in May
1983. By the end of 2000, estimated 36 million people
have been infected with HIV, more than 70% clustering in
sub-Saharan Africa. Number of AIDS orphans, children
lost both parents due to HIV, is increasing rapidly in many
developing countries.
2) Blood safety; Safety of the blood supply has been
ensured by the donor screening based on the detection of
HIV antibody. There is “acute infection phase”, a few
weeks after HIV exposure, where relatively high level ofviremia makes the donor highly infectious despite the“sero-negative” test results. To close such “window period”,
viral nucleic acid testing has been introduced in Japan toclose the “window” by nearly 30%.
3) Vaccine development; Retroviruses have “latent infection period” as a part of regular replication cycle and,during this period, HIV-infected cells can escape the surveillance of immune mechanisms, resulting in the theoretical difficulty of vaccine development. Recently Prof. Kiyono’s group identified “common mucosal immunity”
among various mucosal compartments. Nasal immunization of animals could induce vaginal immunity against recombinant virus with HIV/SIV genes.
4) Chemotherapy; Retroviruses from an exceptionally“chemotherapy-prone” virus group. They have in their replication cycle several specific steps vulnerable to drug interventions. Successful developments of reverse transcriptase
and protease inhibitors substantially improved the prognosis
of HIV/AIDS patients, reducing the progression of HIV disease and death by AIDS. Mother-to-child tansmission are
effectively prevented and occupational exposures can be
managed by chemotherapeutic regimens.
5) Transmission; Transmission efficiency has beenstudied quantitatively by assay of viral RNA genome loads.
Risk of being infected by single episode of heterosexual
contact is estimated to be 0.05-0.15% (infection after 7002000 contacts). Ulcerous or inflammatory lesions in the
genital mucosa enhances the risk of HIV transmission. Genetic deletion of a co-receptor, CCR5, expression make theindividual HIV-resistant.
6) Prevention; UNAIDS put the prevention of general
STDs as the most effective preventive approach against HIV
infection. Human trial of “topical vaginal microbicide”
containing spermicidal contraceptive enhanced the HIVtransmission by nearly 50%, possibly due to inflammatorychanges in mucosa caused by unexpectedly frequent applications.
7) Molecular epidemiolgy; HIV can be identified with
subtypes (clades) based on base sequences of individual
genes. With the time after the introduction of multiple subtypes of HIV in circulation within a region, increasing variety of recombinants were identified. Now the circulating
HIVs are described by CRF (circulating recombinant form)
designation.
8) Drug resistance; Resistances to various antiretroviral drugs are idetified as the mutations in pol genes. Diagnostic tests to identify the infectious viral genomes and possible resistance mutations in the sequence are being developed, in response to the need of effective therpeutic design.

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S−III−2) RESEARCH AND DEVELOPMENT OF RECOMBINANT HIV VACCINE-PRE-CLINICAL TRIAL
HONDA M., NAKASONE T., MATSUO K., IZUMI Y., HAMANO T., OHSU T., TAKIZAWA M.,
SOMEYA K., KAIZU M., HARA T., KAWAHARA M., HORIBATA S., KENEKIYO M., HAMATAKE M.,
AMI Y., SAKAI K., SHINOHARA K., SAKAKI Y., KURATA T., YAMAZAKI S., AND YAMAMOTO N.
National Institute of Infectious Disease

In controlling the HIV infection, preventive HIV vaccine would be of great use, when it was developed efficaciously. Recent considerable progresses have shown that
correlates of protective immunity may due to interaction of
immunological, viral and genetic factors. Furthermore, highlevels of the cellular and humoral immune reponses areprobably responsible for controlling the viral loads that
limit HIV infection and progression of diseases. Mycobacterium bovis bacillus Calmette-Guérin (BCG) and a vaccinia viral strain that trageted in this HIV vaccine development are considered to be the appropriate HIV vaccinevector that satisfy the requirements for HIV vaccine use forhuman, when it would induce high levels of HIV-specificimmunity with maintaining BCG immunity against tuberculosis. The prime-boost vaccine regimen significantly protected all the animals from high levels of palasma viral loadand CD4+ cell loss: a decline in HIV1 set-point levels wasmore than hundred times and CD4+ cell decline was alsosignificantly reduced. This approach induced similar levelsof immunity-specific for HIV-1 Gag in animals consecutively primed with rBCG-HIV/Gag (80 mg orally or 0.1 mg
intradermally) and boosted with rDIs HIV/Gag. Since thevaccine regimen is revealed to be very safe, these studies
suggest a strategy for enhanced induction of T-cell mediatedprotective immunity to HIV-1.

S−III−3） TREATMENT OF AIDSMASAKAZU HATANAKA
Kyoto University, Kyoto and Shionogi Laboratory, Osaka, Japan

AIDS, acquired immunodeficiency syndrome, iscaused by infection of a retrovirus called HIV, human immunodeficiency virus. HIV encodes three enzymes, reverse
transcriptase, protease and integrase, whose inhibition is thetargets of AIDS treatment.
First, nucleoside analogs has been developed as inhibitors of reverse transcriptase followed by non-nucleosides.
As non-nucleoside-inhibitors are less expensive comparedwith nucleoside analogs, developing countries may afford
their use for the treatment of AIDS and prevention of
mother-to-child infection of HIV. Although the onset of resistant strains of HIV towards reverse transcriptase is inevitable by long duration of treatment course, adequate use andchange of drugs may lead long threapeutic effects. Proteaseinhibitors have been developed by mainly molecular design-

ing and are quite effective against resistant strains of reverse
transcriptase inhibitors and presently combination of threedrugs against reverse transcriptase and protease is a major
stream of treatment of AIDS called HAART (highly active
anti-retroviral therapy). At the first time, we have developed an inhibitor of HIV integrase (S-1360), which is undera clinical trial in USA and is expected to be soon available
for the AIDS patients. In developing countries of Africa andAsia, HIV infection is in the midst of its pandemics and isurgent to take action of its prevention and treatment, whichis alerted as emergency by United Nations and WHO. Developed countries including Japan are now taking actions
urgently towards this target in many extensive ways including medical and financial supports.

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S−IV−1) TRIALS AND PROSPECTS OF EPIDEMIOLOGICAL SURVEY FOR BABESIOSISIN JAPAN AND MALARIA-ENDEMIC AREAS
ATUKO SAITO-ITO1, TSUNEZO SHIOTA2, NOBUHIRO TAKADA3, SHUNICHI KUMAGAI4, PRADYA SOMBOON5
Section of Parasitology1 and Division of Clinical Pathology and Immunology4, Kobe Univ.
Graduate School of Medicine, Department of Medical Zoology, Kyoto Prefectural University of Medicine2,
Department of Parasitology, Fukui Medical University3,
Department of Parasitology, Chiang Mai University Faculty Medicine5

Babesiosis is tick-transmitted intraerythrocytic protozoan disease of animals and occasionally occurs in humans.
Most cases of human babesiosis have been caused by Babesia microti, a species of rodents. Cases by B. microti havebeen reported nearly exclusively from the United States. In
1999, we confirmed the first human babesiosis case in Japan. It was demonstrated that the case had been infected by
the blood transfusion from an asymptpmatic donor infectedwith B. microti-like parasites. The sequence of SSUrDNA
of parasites isolated from the patient (Kobe srtain), however,
was distinct from that of B. microti of the United States origin (Gray, GI and AJ strains) nor that of parasites isolated
from wild rats in Otsu where B. microti-like parasites werefirst demonstrated in wild rats in Japan (Otsu strain), although they have the higest homology to one another. Antigenicities of Kobe strain, Otsu srtain and the strains of US
origin are also different from one another.
In these circumstances, we examined antibody-titers
against Kobe strain by IFAT for 995 sera in 2000 and 499
sera in 1981 of outpatients of hospitals in Hyogo under informed consent. Three sera2000 and one in 1981 showed to
be positive at ≧x518 including two positive at ≧x4096.
One serum postive at ≧x518 showed no cross reactivities
against Otsu strain or P. yoelii like the sera of the first patient and the asymptomatic donor. By western blot analysis,
it also detected the antigen of the same size (ca. 40 kD) asthat for the sera of the first patient and the asymptomatic
donor. Thus , it was suggested that at least one outpatient,the donor of the serum, had experienced the actual infection
with Kobe strain. However, three sera that showed cross reactivities against Otsu strain and P. yoelii could be falsepositive. Specificities of IFAT should be reconfirmed.
In the field survey of rats and ticks for Babesia infection in Hyogo in 2000-2001, it was showed by microscopic
observation of smears or PCR that 5 of 9 rats were B.
microti-positive in Rokko area, and all 5 were Otsu type. In
Awaji island, 6 of 22 rats were B. microti-positive, and 4
were Kobe type and 1 was Otsu type (1 was not detemined).
Morever, the probable SSUrDNA of B. microti was amplified from a salivary gland of a tick of Ixodes ovatus collected in Rokko. Its sequence has the highest homology
with those of Kobe, Otsu and US type, but different fromany of them. It was suggested that I. ovatus has some roles
in transmission of B. microti.
In the field survey of rats for Babesia infection in Chiang Mai, Thailand in 2000, Babesia-like parasites wereidentified in 6 of 9 of Bandicota indica. The sequence
analysis of the parasites showed that the parasites are moreclosely related to B. canis and B. divergence than B. microti.
Although futher studies are necessary, the emergence of human babesiosis should be considered in malaria endemic areas.

S−IV−2) NOVEL STRATEGIES FOR TICK CONTROLKOZO FUJISAKINational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine

Ticks are ectoparasites found in almost all parts of theworld. While mosquitoes trasmit most of the important human pathogens such as malaria and yellow fever, ticks surpass all other arthropods in the number of diseases theytransmit to animals and humans. Damage resulting from direct tick feeding activities and the cost of controlling thepathogens they transmit to libestock accounts for the lagest
source of economic losses in the livestock industry. Currently the only widely available practical method to control
ticks is the use of chemical acaricides. This approach is associated with serious limitations such as environmental andfood chain contamination by acaricides. These limitations

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and the growing community awareness regarding the harmful effects of acaricides have stimulated research into alternative tick control methods. Among the several alternative
tick control methods that have been considered, immunological protection of hosts against tick infestation is practical and sustainable. Findings from a series of studies by
an Australian group leading to commercialization of a firstever anti-arthropod vaccine, e. g., TickGARD and Gavac,
provided practical evidence on the use of an immunologicalapproach to control ticks. However the success of this approach is dependent upon identification of key tick molecules that are involved in the mediation of key physiologicalroles for use as candidate vaccine antigens. From this stadpoint, we made here the brief introduction about our recent
achievements on the identification and characterization ofseveral candidate molecules, namely phenoloxidse, serineprotease, peroxiredoxin and troponin, from several tick species.

S−IV−3) SIMIAN MALARIA PARASITES AS ZOONOTIC PATHOGENSSATORU KAWAI1, OHTA NOBUO2, MATSUDA HAJIME1
Deparment of Tropical Medicine & Parasitology, Dokkyo University School of Medicine1,
Department of Mesical Zoology, Nagoya City University Medical School2

The parasites of genus Plasmodium have a wide range
of vertebrate hosts. Although they are generally known tohave strict host specificity, some simian malaria parasites
are infective to human beings, causing zoonotic infectious
diseases. Here we describe simian malaria parasites zoonotic pathogens.
To date, about 25 spices of simian maralia parasites
have been reported. These occur in New World monkeys,
Old World monkeys, gibbons, great apes, and lemurs. Plasmodium of the primate hosts are classified into five groups
according to Giemsa-stained feature of stages found in the
peripheral blood and the periodicity of their erythrocytic
schizogony: falciparum type, vivax type, maraliae type,ovale type and other type. At least six species of simianmalaria parasites (P. brazilianum, P. cynomolgi, P. inui, P.
knowlesi, P. schwetzi and P. simium) are known to infecthumans either naturally or experimentally including accidental laboratory infections.
Generally, the clinical course of infection with simianmalaria parasites in humans is mild with low parasitemia,
and chemotherapy, where necessary, has been effective. Although human malaria infections acquired from non-humanprimates are rare, an accurate diagnosis should be made inany suspected cases. Diagnosis is based on the detection ofparasites in stained thin blood smears but these must be interpreted with great caution because simian malaria parasites are virtually indistinguishable from the erythrocytic
stages of human malaria parasites.
Some epidemiological studies on simian malaria havepointed out the possible role of malaria infected monkeys as
natural reservoirs for infection in humans. P. brazilianum iswidely distributed in several species of monkeys in Central
and South America. This malariae type species is morphologically and molecularly similar to human parasite P. malariae, thus they are virtually identical and should probably
be considered to be a single malaria species. In fact, serological studies in the Amazon Basin have shown the prevalence for anti-P. brazilianum /P. malariae antibodies to behigh both in monleys and in local Indian tribes, suggestingthat this from of malaria is authentic zoonosis in that region.
These findings indicate the possibility that monkeys livingin the rainforest of Amazon area are natural reservoirs forboth simian and human malaria.

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S−IV−4) LEISHIMANIASIS AS A ZOONOSIS: DNA DIAGNOSIS AND MOLECULAR EPIDEMILOGYBASED ON PCR AMPLIFICATION AND CHARACTERISTICS OF
THE LEISHMANIA MINI-EXON GENE
KEN KATAKURA
Department of Parasitology, Gunma University School of Medicine

Leishmaniasis is typical vector-borne and zoonotic disease. The disease is transmitted by the sandfly and morethan 100 vertebrates are known as resevoir hosts. To control leishmaniasis most effectively, an accurate diagnosis ofpatients well as epidemiological studies on reservoir animals and sandfly vectors are required. For DNA diagnosisof leishmaniasis, we have developed a PCR method basedon amplification of the Leishmania mini-exon gene. The
mini-exon gene is tandem repeated as many as 100-200
copies on the parasite genome and the spacer region is defferent in length and in sequence among different Leishmania species. We evaluated the usefulness of this PCR
method in two endemic countries, China and Ecuador.
In Xinjiang-Uigur Autonomous Region in China, there
have been sporadic cases of visceral leishmaniasis. We obtained positive PCR products corresponding to L. donovani
complex in bone marrow or buffy coat samples in 11 out of
13 serologically positive kala-azar patient. However, noneof buffy coat samples from nine patients with antimony
were all PCR-negative. DNA samples from buffy coats of
17 domestic in the endemic area were also PCR-negative.

In addition, we identified parasite isolates from great gerbils (Rhombomys opimus) and sand flies (Rhlebotomus
mongolensis and P. andrejevi) as L. turanica or L. gerbilli
by DNA sequencing of the PCR-amplified mini-exon genes.
In Ecuador, amplification of the mini-exon gene specific to L. braziliensis complex was detected in the skin biopsy sampls from patients with cutaneous leishmaniasis, indicating the usefulness of PCR amplification of the gene fordiagnosis of cutaneous leishmaniasis. Chromosomal locations of the mini-exon gene were different among L. panamensis isolates, futher indicating the DNA karyotype variation among parasites in the endemic region. Furthermore,
DNA sequencing of amplified mini-exon genes of isolates
from a sloth (Choloepus hoffmanni), a squirrel (Sciurus vulgaris) and sand flies (Lutzomyia hartomani) revealed thepresence of Endotrypanum monterogeii and E. schaudinniin Ecuador. Since some of these isolates were reported as L.
equatorensis in the previous paper, it is impottant for thedifferentiation between Leishmania and Endotrypanum for
epidemiological studies on leishmaniasis.

S−V−1) PROGRESS OF GLOBAL POLIO ERADICATION PROGRAMMETATSUO MIYAMURA
Department of Virology II, National Institute of Infectious Diseases

The global polio eradication programme has launchedby world health oraganization in 1988. The main strategy is(i) detecting patients with acute flaccid paralysis (AFP) and(ii) confirming real polio cases both by clinical and virological examination. If the number of AFP patients due
to wild type-polioviruses becomes zero and if the situation
is maintained for a certain period of time (〜3 years), theprogramme is accomplished. In practice, the programme
consists of extensive vaccination and highy effective surveillance. Since the key element is oral attenuated vaccine,
this programme is considered to be, in other words, an experiment to replace wild polioviruses with vaccine strains in
global level.

In the American Region, the programme was accomplished in 1991 and no AFP patients due to wild polioviruses have been reported in spite of extensive vaccinationand surveillance. In our Western Pacific Region, the lastpolio case was found in Cambodia in March 1997, andsince then no genuine polio cases by wild polioviruses havebeen reported. The certification committee declared in October 2000 that transmission of wild polioviruses has completely interrupted and now the region is wild polio-free. In
the European region, the last polio case is considered to be
the one in Turkey in 1998.
The initial target of the WHO programme was 2000.
Although the accomplishments are delayed than expected,

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the programme is now at the very final stage. However atthe very end, the following problems became clear; (i) the
areas where wild polio viruses still exist are the areas withserious internal war and international collaborative efforts
are generally difficult to access (Afghanistan, Somalia, Angola, etc…), (ii) polio-free declaration in the regional level
sometimes induce negative effects for the maintaining thehigh level of vaccination and sustaining good surveillance.

The latter is particularly important because in some areas
where wild polioviruses are no more detected, vaccine derived poliovirus caused poliomyelitis outbreak in low or
non-immunized population. The eradication of poliomyelitis does not automatically mean the abolishment of vaccination. It is the time to consider how we can stop polio vaccination (polio end game).

S−V−2) INTERNATIONAL COOPERATION IN THE AREA OF TUBERCULOSIS CONTROLNOBUKATSU ISHIKAWA
The Research Institure of Tuberculosis, JATA, Tokyo

Global burden of Tuberculosis and DOTS:
Tuberculosis is now a global burden as one third of the
global population has been infected by M.tuberculosis,
causing 8 million new cases and 2 million deaths every year,
WHO has developde a global strategy called DOTS throughthe analysis of the success stories in the world. DOTS consists of the following 5 components; government commitment, microscopic examination for diagnosis to the symptomatics, safe and regular supply of TB drugs, direcr observation of medication with short course regimen, routine monitoring with recording and reporting system. DOTS has yetcovered only half the global population and one third of theestimated number of patients, The international cooperationmust be therefore strengthened to accelerate global expansion of DOTS.
International Cooperation for TB Control by Japan:
Japan has a long history of international cooperation in

the area of TB control since early 1960s both through thegovernment and NGOs. International training courses held
at research Institute of Tuberculosis since 1963 have had aglobal reputation wit more than 1500 participants from 80
contries. New challenges are; donation of drugs which areneeded for DOTS expansion, recruit of the local staff for the
project, or support for the nation-wide expansion of the program. Flexible approaches are needed according to theneeds and effects. Coordination with other and international
agencies such as WHO, or NGOs should be actively made,and global commitment both technical and political need to
be strengthened.
Support System and Base Establishment inside Japan:
The base capacity inside Japan should be further
strengthened, for manpower development for international
works, overseas project support, or collaborative research.

S−V−3) MALARIA CONTROL PROGRAM IN LAO PDR SUPPORTED BY JAPANESE GRANTAID FOR CHILD HEALTH
JUN KOBAYASHI1, 3, SAMLANE PHOMPIDA2, ATARU TUZUKI1, TAKAYAKI SAITO1, and YOSHIYA SATO3
Japan International Cooperation Agency (JICA) Laos Office1,
Center of Malariology, Parasitology and Entomology (CMPE), Ministry of Health, Lao PDR2,
Department of Parasitology, University of the Ryukyus3

In Lao PDR, Malaria control program using Impregnated Bed Net (IBN) stated from 1998 in nation wide according to Roll Back Malaria Initiative. Project in three
central provinces was supported by Japanese Grant Aid forChild Health under the supervision from CMPE, Ministryof Health and JICA expert. Total of 40000 bed nets and Insecticide (Deltamethrin) were donated to CMPE and threeprovincial health offices in the beginning of 1999. Until
end of 2000, it was reported that 81% of bed bet was delivered to the communities. Commune (Sub-district consisted

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６by 5-10 villages) was applied as unit to deliver bed net costeffectively. One village was selected to hold Training ofTrainer (TOT) in each commune to expand to other villages
by district malaria staffs, village leaders or health workers
without support from central level. Total expense to deliver
the bed net using these systems was approximately only US$10000.
Total number of malaria patients and slide positive rateof malaria (SPR) in all public health facilities decreased
markedly in 2000. In the pilot district; Bourapha district inKhammoaune province, it was suspected that IBN program
was well operated according to the analysis the report from
district health office. The report showed 100% coverage tovillages and 3.81 person per IBN as mean coverage in thecommunity. After operation IBN program, KAP study was
carried out to evaluate the program in all communes in pilot

district. High percentage of household where bed net wasn’t set was presented in the remote area. Moreover, lowmalaria knowledge was confirmed among inhabitants wholive in household without bed net. It was suggested thatstrengthening of IEC activities is necessary in especially remote area and poor families. Thus, the new approach to the
inhabitants was applied to sustain the project. To strengthen
the community participation, group discussion and mass diagnosis using rapid diagnosis method were conducted before ordinary health education using video or poster. The
efficacy of these trials will be evaluated in beginning of
2002.
It is expected that improvement of cost recovering system and application of social marketing system support tosustain the project with strengthening IEC activities.

S−V−4) LEISHMANIASIS: ITS RECENT TOPICS AND RESEARCH ACTVITIES IN ECUADORYOSHIHISA HASHIGUCHI
Department of Parasitology, Kochi Medical School, Nankoku, Kochi Japan

Leishmaniasis is one of the eight target diseases of the
Improved Control of TDR diseases (WHO, 2000). The diseases are endemic in 88 countries on the five continents,
with a total of 35 million people at risk, and the diseases afflict at least 12 million people worldwide; the numbers are
however probably underestimated; figures of 2 million newcases per year, including 1.5 million cases of cutaneousleishmaniasis (CL) and 500,000 of visceral leishmanisis(VL) are likely (Desjeux, 2001). In this symposium, thefollowing points are mentioned as recent topics in leishmaniasis research. 1) Epidemiological changes due to
Leishmania/HIV co-infection. Such cases are increasing
yearly and the co-infection is prevalent in 33 countries, including non-endemic areas, such as Germany, Switzerland,
United Kingdom, Belgium, Denmark and etc. In the coinfection areas, transmission cycles are also changing fromzoonotic endemics to anthroponotic epidemics; person toperson direct trasnsmissions by syringe in drug users arefound, especially in south-western Eurpe. 2) Vaccine trials.
Recent vaccine trials performed for CL in Iran and for VL
in Sudan, and for CL in Brazil, Ecuador and Colombia werebriefly reviewed, including immunotherapy done in Venezuela. 3) Chemotherapy for leishmaniasis. Available therapy for the disease was reviewed and special emphasis wasgiven to the trials with miltefosine as oral drug for VL usedespecially in India. Besides, leishmaniasis research project
carried out during about 18 years from 1982 to 2001 was
retrospectively evaluated, mainly emphasizing on the following points: 1) characterization and determination ofLeishmania spp., vector sandflies, and reservoirs at each endemic areas of Ecuador; 2) therapy trials using paromomycin ointment and Glucantime lotions as local treatment andMephaquin and artesunate as oral treatment; 3) detection of
epidemiological characteristics of each area and etc.; 4) development of material collections in the field condition formolecular diagnostic tools.

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S−V−5) CURRENT ACTIVITIES OF REGIONAL NETWORK ON SCHISTOSOMIASIS CONTROLIN ASIA AND ROLES OF
THE JAPANESE RESEARCHERS IN THE NETWORKNOBUO OHTA
Department of Medical Zoology, Nagoya City University Medical School, Nagoya, Japan.

Asian schistosomiasis, schistosomiasis japonica, is endemic in east-Asian countries especially in China and thePhilippines. In comparison with other human schistosomiasis, the disease control is difficult because of the presence ofreservoir hosts and also environmental factors. Under the
support of WHO, a regional network for controlling Asianschistosomiasis (RNAS) was established as international
cooperation activities including the two endemic countries
as well as other countries such as Japan and Australia, inwhich research on schistosomiasis has been accumulated.
Endemic situation is different between China and the Philippines, therefore, control strategies are different between
the two countries. In the previous joint RNAS meetings,
methods for epidemiological evaluation, developing new diagnostic tools, and vaccine development were the common
interest. Japanese researchers are requested to join the cooperative activities in such fields.
Endemic situation of schistosomiasis japonica in both
two countries at a turning point, because WHO gave relatively intense supports for the disease control in the past
decades. Situation has been much improved, however, support by WHO was already over by the end of 2000. In

China, the number of endemic Prefectures and the numberof advanced cases showed significant reduction, and betterorganized countermeasures are needed. For example, selected chemotherapy is the choice in many places in China,
however, such program management is now not easy because of financial problem. Reduced endemicity means difficult step for disease eradication and needs much morebudget and human resources. The same situation is also in
the Philippines. Along with social development, endemicity
is deeply affected by some artificial conditions. The threegauge dome in China is the case, Epidemiological suevey
in Human, Hubei, Sichuan and Jiangxi Provinces are underway.
Cost-effective countermeasure is the most important
subject this region. Technical and scientific assistance fromJapan will be good contribution. In line with such situation,
Japanese researchers are joining partnership for vaccine development, and for developing new epidemilogical evaluation. RNAS activity has just started, and should be expanded though the partnership in east-Asian countries. Thisis, in turn, a good opportunity for Japanese reseachers, since
schistosomiasis is still attractive field of research.

４８A−1) CHANGING PATTERNS OF ANTIMALARIAL DRUG RESISTANCEIN PLASMODIUM FALCIPARUM MIRROR THE CHANGE
OF THE NATIONAL MALARIA TREATMENT POLICY IN MALAWI
B. BWIJO1, A. KANEKO1, T. TSUKAHARA1, M. TAKECHI1, 2, I. L. ZUNGU3 Y. MORIYAMA4,J. K. LUM1 AND T. KOBAYAKAWA1.
Department of International Affairs and Tropical Medicine, Tokyo women’s Medical University, Tokyo, Japan1,
Yachiyo Hospital Hiroshima, Japan2,
Community Health Sciences Unit, Ministry of Health and Population, Lilongwe, Malawi3,
Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand4.

In Malawi chloroquine was replaced by sulfadoxine/
pyrimethamine (SP) in 1993 because of increasing chloroquine failures of Plasmodium falciparum cases. Seven
years after the change in malaria treatment policy, we evaluated the sensitivity of P . falciparum to different antimalarial drugs both in vivo and in vitro in asymptomatic children
and adults in Salima District, Malawi.
A total of 504 individuals were examined for parasitaemias. Of them, 78% were infected with malaria. Monoinfections of P . falciparum and P . malariae accounted for
97% and 1% of the positives respectively, while 2% had
mixed infections. Children <6 years were mostly infected(93%) and had high parasite rate and spleen rate.
Of 173 subjects selected for in vivo trials, 61 weretreated with chloroquine, 46 with SP, and 66 with Malarone(atovaquone/proguanil) and were followed for 28 days. The
in vivo sensitivity response of the three drugs tested was not
significantly different (78-88%, p>0.05), however, Malarone group showed a significantly higher proportion of RIII/
RII resistance than chloroquine and SP groups (9% vs. 2%,

p<0.02).
The in vitro sensitivity of 104 P . falciparum isolates,
to chloroquine, SP, pyrimethamine, amodiaquine, quinine,and mefloquine was also assessed using WHO test kits.
Ninety eight percent of the parasites failed to mature at thelowest concentration of chloroquine (0.2µmol /l BMM)
precluding EC50 and EC90 estimates. Although 44% of theisolates showed schizont maturation at the highest concentration of pyrimethamine, 83% were inhibited at the cut-off
concentration of SP combination.
Our study in Malawi shows the recovery of chloroquine sensitivity by comparing both in vivo and in vitro
tests of the same P . falciparum isolates. The combinationof sulfadoxine and pyrimethamine was effective in the presence of pyrimethamine-resistance isolates, indicating theadvantage of drug combinations as opposed to single drug
therapy in clinical practice. Thus, if chloroquine is removed
for a sufficient period of time, it might be effectively used
once again in combination with other drugs to treat malaria
in Sub-Saharan Africa.

A−2) ANALYSIS OF PFMDR1 GENE IN MEFLOQUINE RESISTANT PLASMODIUM FALCIPARUM
KHURSHIDA BEGUM1, HYE-SOOK KIM1, YOHEI OKUDA1, YUSUKE WATAYA1MIKIO KIMURA2 AND TAKAHISA HURUTA3
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan1,
National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan2
and The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan3

Objective: Drug resistance is a major problem for the treatment of malaria. Therefore new antimalarials are necessary
to combat resistant parasites. Mefloquine is an importantdrug, but resistant parasite are also appeared and now increasing. Our objective was to analyze the mechanism of
mefloquine-resistant of Plasmodium falciparum parasite todevelop new drugs.

Methods: A mefloquine-resistant strain Plasmodium falciparum 523a (isolated from mefloquine-resistant patient)
was continuously cultured under progressively increased
mefloquine pressure. After two years, we got resistant parasite (designated as 523a R strain) and now can able to growat 2.5×10-7 M mefloquine concentration. The original
strain was cultured without drug under the same condition

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９

considered as control (523a S strain). The sensitivity toother antimalarials against these strains (523a S and 523a
R) have also been performed. The amino acid residues at
86, 1034, 1042 and 1246 positions of pfmdr1 gene in chromosome 5 of P . falciparum (523a S and 523a R) were analyzed using Dye Terminator method followed by particulardomain amplified by PCR and compared with previously
reported amino acid residues which thought to involved mefloquine resistance. The size of chromosome 5 has beenmeasured. We performed cloning from resistant strain anddetermined the sensitivity to other antimalarials against
clone/24.
Results and Discussion: It is found that, there is no difference in amino acid sequence between mefloquine sensitive(523a S) and mefloquine resistant (523a R) strain, although
resistant strain showed about 39 times higher IC50 value
than sensitive strain. Moreover, 523a R strain showed decreased sensitivity to artemisinin and halofantrine and in-creased sensitivity to chloroquine. The overexpression of
pfmdr1 mRNA has been observed and it is about 7.2 times
higher than sensitive strain. Our Pulsed Field Gel Electrophoresis result revealed chromosome 5 contained a mixed
population of sensitive and resistant parasites. To get moreresistant parasites we performed cloning. Clone/24, thehighest resistant clone showed similar results as 523a R
strain to other antimalarials, increased sensitivity to chloroquine and decreased sensitivity to artemisininin and halofantrine.
Our results may explain amino acid sequence should
not related to drug resistance and sequence polymorphismof pfmdr1 gene. As overexpression of gene pfmdr1 foundabout 7.2 times higher than sensitive strain, it is likely thatoverexpression of pfmdr1 gene may be associated in mefloquine resistance mechanism. Now we are measuring thesize of chromosome 5, overexpression of mRNA and gene
analysis of pfmdr1 against clone/24.

A−3) RECOVERY OF CHLOROQUINE SENSITIVITY AND LOW PREVALENCE
OF K76T IN PFCRT AFTER THE CHANGE OF MALARIA TREATMENT POLICY IN MALAWITOSHIHIRO MITA1, AKIRA KANEKO1, BWIJO BWIJO1, TAKAHIRO TSUKAHARA1, YOSHINARI MORIYAMA1,MIHO TAKECHI2, J. KOJI LUM1, INNOCENT L. ZUNGU2, AND TAKATOSHI KOBAYAKAWA1
1

Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan
2
Community Health Sciences Unit, Ministry of Health and Population, Lilongwe, Malawi

In 1993 Malawi officially withdrew chloroquine as thefirst-line drug for the treatment of uncomplicated cases ofmalaria due to an increasingly high rate (47%) of resistance.
During field surveys conducted in 1998 and 2000 we investigated in vitro chloroquine resistance (CQR) and later assayed two genetic polymorphisms (K76T in pfcrt and N86
Y in pfmdr1) suggested to contribute to CQR in obtained
Plasmodium falciparum isolates. Among 96 isolates exam-

ined, only 9% showed CQR while 7% and 48% carried the
K76T and N86Y mutations respectively. In contrast, theprevalence of K76T in other African countries with ongoing
chloroquine use was 41-83%. Thus, our findings suggestthat the reduced drug pressure accompanying the change innational drug policy in Malawi has resulted in a significantly lower prevalence of the K76T mutation and consequently a substantial recovery of chloroquine sensitivity.

５０A−4) DRUG RESISTANT MALARIA IN MINDANAO, THE PHILIPPINES-APPLICATIONOF ANAEROPACK! MALARIA CULTURE SYSTEM TO IN VITRODRUG SUSCEPTIBILITY TEST OF PLASMODIUM FALCIPARUM IN THE FIELD.
TOSHIMITSU HATABU1, JUN SUZUKI2, REUBEN F. VALENZUELA3, ELENA A. VILLACORTE3,
PILARITA T. RIVERA3, SHIN-ICHIRO KAWAZU1, and SHIGEYUKI KANO1
Research Institute International Medical Center of Japan1,
The Tokyo Metropolitan Research Laboratory2,
and Department of Parasitology, College of Public Health, University of the Philippines Manila3

Since chloroquine-resisant Plasmodium falciparum
was reported in 1960’s, drug-resistance or decreased susceptibility for drugs of malaria parasites have expanded
their distribution. Therefore, it is important to carry out the
drug susceptibility in the field to obtain the epidemiologicalinformation against the drug-resistant malaria parasites indetail. However, it is different to isolate the fresh parasiteor to examine the drug susceptibility for antimalarial drugs
in the field because of the limited experimental conditions
needed for the cultivation of the P . falciparum in vitro. In
this report, drug susceptibility of P . falciparum isolates wasdetermined using AnaeroPack! malaria culture system withportable thermostat in the field of Mindanao Island, thePhilippines. Blood samples were obtained from 4 symptomatic Filipino patients with P . falciparum who admitted to
the Davao regional hospital, in Tagum City. The aim of thestudy was clearly explained to all the patients and informed
consent was obtained from them. Erythrocytes were resuspended in RPMI 1640 medium supplemented with 10% human serum. The culture plates including the blood samples
were incubated at 37°
C with portable thermostat. The 4 isolates were examined for the chloroquine-susceptibility byadding the drug to the cultured samples at serially diluted
concentrations. After 36 hr incubation, 2 isolates grew fromring form to schizonts. Then, a 100% inhibitory concentration (IC100) of one isolate was observed to be 1.28 µM andthat of the other isolate was 0.64 µM. Two other Isolates of
4 grew to trophozoite as late as after 48 hours’ incubation
and were also thought of as isolates of a certain degree ofresistance. AnaeroPark! malaria culture system with portable thermostat was proved to be powerful and useful to
conduct the drug susceptibility test in the field condition.

A−5) AN EPIDEMIOLOGICAL STUDY ON MALARIA IN THE DISPLACEDPOPULATIONS IN VIET NAM
SHIGEYUKI KANO1, SHUSUKE NAKAZAWA2
Research Institute, International Medical Center of Japan1,
and Institute of Tropical Medicine, Nagasaki University2

Malaria incidence is determined by a variety of factors,
particularly the abundance of anopheline mosquitoes, thepresence of malaria parasites, and human behavior. Reemergence of malaria is thus often associated with socioeconomic development and expanding infrastructure, population growth and urbanization. We report an example ofmalaria epidemics in the displaced populations in Viet Nam.
Study area was a newly colonized village called “Ben
Tre-A New Economic Area” in Binh Phuoc province, which
was located about a 100km to the north of Ho Chi Minh
City and quite near to Cambodia border. People had moved
from the area called “Ben Tre” near the mouse of Mekong
river and settled there. Seventy families including 203 peo-

ple were residing in the area and just started to cultivate theland growing rubber trees, nuts, pepper and rice for theirsupport. Access to the village was not easy by walking but
only feasible by a small motor bike. Electricity had notbeen supplied in the area.
We visited the place on the 13 June, 2001, with a microscope, and made thick blood films from 99 villagers whocame to see us for free blood smearing for malaria. Fivecases (12 y. o. male, 15 y. o. male, 42 y. o. female, 44 y. o.
female, 65 y. o. female) showed positive slides out of the 99,
which was considered to be very high in number as could
be obtained in a single day survey. They said they were
sleeping in the mosquito nets, but that no spraying had been

５１

done in the area. They were taking no counter measuresagainst mosquito bites when they were working in rubber
woods in carly morning. The main vector in the area wasreported to be Anopheles minimus.
The large-scale migration and resettlement of populations from malaria endemic areas into receptive areas were

big problems in many parts of the world, however, on thecontrary, a movement induced by rural impoverishment influenced by the over population from a non-endemic area tohighly endemic areas will also affect the dynamics of thedisease badly.

A−6) RECRUDESCENCE IN VITRO AFTER D-SORBITOL TREATMENT OVER NINE DAYSSHUSUKE NAKAZAWA
Institute of tropical Medicine, Nagasaki University

Drug treatment of falciparum malaria is occasionallyfollowed by a recrudescence. Recrudescences have beenthought to occur due to drug resistant prasites or due to alow plasma concentration of drug. There have been several
reports of discrepancy between in vivo and in vitro resistanttests. Recrudescent parasites obtained from patients weresensitive in the in vitro test. Those reports imply parasites
survive drug treatment in some manner other than drug resistance.
Inactive parasites may escape from the effect of drug.
More than 1.2×109 erythrocytes infected with cloned parasites were exposed to D-sorbitol treatment 12 hourly fornine days, then maintained in standard medium. D-sorbitol
treatment destroys trophozoite and schizont infected erythrocytes and reduces parasite numbers to less than 1/500 per

one cycle (48 hours). Parasites reappeared from 4 to 6
weeks after the treatment and multiplied. Erythrocytes infected with recrudescent parasites were as vulnerable to Dsorbitol treatment as those were before recrudescence experiments.
As D-sorbitol treatment causes 500-fold reduction inparasite numbers per cycle, parasite numbers are presumed
to be decreased to less than 1/5004.5 (=1.4×10-12) for ninedays. This reduction is far beyond the initial parasite number. Ring-form infected erythrocytes are resistant to thetreatment. The results suggest that small percentage of parasites may remain at ring-form stage, so that they survive
the treatment. And after treatment they are activated andlead to an observable infection.

A−7) CHARACTERISTICS OF CHOLERA TOXIN-PRODUCING VIBRIO CHOLERAE STRAINSISOLATED FROM SEAFOOD IN MALAYSIACHIEN-HSIEN CHEN1, TOSHIO SHIMADA2, NASRELDIN E. HUSSEIN3,
SON RADU3, AND MITSUAKI NISHIBUCHI4
Graduate School of Medicine, Kyoto University1,
School of Health Science, Tokyo Medical and Dental University2,
Department of Biotechnology, Faculty of Food Science and Biotechnology, University Putra Malaysia3,
Center for Southeast Asian Studies, Kyoto University4International spread of enteric infections is becoming avery important issue since the movement of humans andfoods across the international borders is increasing year by
year. It is known that cholera is endemic in the Bengal area,
but cholera outbreaks have been reported in the surrounding
areas. We have been studying whether the causative agentof cholera is widely distributed in the environment of suchareas surrounding the Bengal area. Vibrio cholerae O139
emerged in the Bengal area in 1992 and caused a big outbreak. We previously isolated cholera toxin-producing
strains of O139 serotype from the seawater in Malaysia. We
therefore examined the seafoods retailed in this country inthis study. We purchased and examined the fresh seafoods
at 10 sampling sites between 1998 and 1999. As the result,

５
２

20 strains of V . cholerae carrying the ctx gene were isolated from shrimps, squids, crabs, and shellfish. They belonged to serotypes O1 Ogawa (one strain), O139 (14strains), and R (rough, but their rfb genes being of the O1
type, 5 strains). All the ctx-positive strains produced cholera toxin in vitro and possessed the tcpA gene of El Tor
type. The ctx-positive strains of O1 and O139 serotypes
had TLC (toxin-linked cryptic) element, but those of R serotype did not. The R-serotype strains were similar to theclinical O1 Ogawa strain isolated in Kelantan in 1999 because they were negative for the TLC element and shared

the same antibiogram. We previously found similar clinicalstrains in southern Thailand up until March 1998. We compared the environmental strains by various DNA fingerprinting techniques along with the clinical strains isolated inIndia, Bangladesh, and Malaysia. The pulsed-field gel electrophoresis method gave the highest resolution and the results indicated that environmental O139 strains may havebeen introduced from India in 1993 and persisted in Malaysian environment since then and that environmental strainsof O1 and R serotypes may have served as the source ofcholera in Malaysia.

A−8) FLAGELLA FILAMENT PROTEIN (FliC) INDUCES HUMAN β-DEFENSIN-2 (hBD-2)
PRODUCTION IN HOST CELLSAKIHIRO WADA, KEN-ICHI OGUSHI, AND TOSHIYA HIRAYAMA
Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University.

Antimicrobial peptides such as human β-defensins are
crucial for host defense at mucosal surfaces. Expression of
defensin genes is regulated in response to infection and inflammation. To evaluate the role of defensin in S. enteritidis infection, bacterial factors responsible for induction ofhuman β-defensin-2 (hBD-2) mRNA expression in Caco-2
human carcinoma cells were determined. Salmonella enteritidis supernatants of these strains induced hBD-2 mRNA
expression in Caco-2 human carcinoma cells. The supernatant factor was heat-stable and proteinase-sensitive. Using
luciferase as a reporter gene for a 〜2.1 kb hBD-2 promoter,the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. After purification by anion exchange
and gel-filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with theamino-terminal sequence, AQVINTNSLSLLTQNNLNK,
which is identical to that of the flagella filament structuralprotein (FliC) of S. enteritidis. Consistent with this finding,the 53-kDa protein reacted with anti-FliC antibody, which
prevented its induction of hBD-2 mRNA in Caco-2 cells. In
agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody andthe lysate with recombinant FliC but not control lysate has
hBD-2-inducing activity in culture supernatant.
FliC did not enhance luciferase activity in Caco-2 cells
transfected with control luciferse-reporter plasmid (pGL3),
or hBD-2 reporter plasmids without NF-κB site (pGL3-197
and pGL3-938mt), consistent with involvement of NF-κB
in the induction of hBD-2 by FliC. In gel retardation analyses, FliC increased binding of NF-κB (p65 homo dimer) to
hBD-2 gene promoter sequences. These data show that S.
enteritidis FliC induces hBD-2 expression in Caco-2 cells
via NF-κB activation, and thus plays an important role inupregulation of the innate immune response.

５３A−9) ENHANCED EXCYSTATION AND METACYSTIC DEVELOPMENT OF ENTAMOEBAINVADENS BY CYTOCHALASIN DASAO MAKIOKA1, MASAHIRO KUMAGAI1, HIROSHI OHTOMO1, SEIKI KOBAYASHI2 AND TSUTOMU TAKEUCHI2
Department of Tropical Medicine, Jikei University School of Medicine1,
Department of Tropical Medicine and Parasitology, Keio University School of Medicine2

Excystation and metacystic development are the process by which Entamoeba initiates infection. Although theprocess was described previously, little is known about themechanism involved. The process of excystation and metacystic development includes movement of the protoplasm ofcysts, separation it from the cyst wall, escape of the entire
protoplasmic contents through a minute perforation in thecyst wall, and finally production of 8 trophic amoebae.
There is, however, no study on the relation of the actin filaments to the excystation and metacystic development of Entamoeba. Therefore, we examined the effect of three actinmodifying drugs, cytochalasin D, latrunculin A, and jasplakinolide, on the excystation and metacystic developmentof E. invadens by transfering the cysts to growth medium
with the drugs. Cytochalasin D unexpectedly increased thenumber of metacystic amoebae of E. invadens during incu-

bation. Metacystic development, which was determined by
the number of nuclei of metacystic amoebae, was faster inthe culture with cytochalasin D than without the drug.
These results suggest that cytochalasin D enhances the excystation and metacystic development. In contrast, latrunculin A and jasplakinolide inhibited these process. No excystation occurred in encystation medium even in the presence of cytochalasin D, suggesting that growth medium isessential for excystation. Excystation was further enhanced
when the cysts were incubated with cytochalasin D before
culture in growth medium with the drug. The enhancing effect of cytochalasin D on the excystation and metacystic development was abrogated by jasplakinolide. Thus the results indicate that cytochalasin D, unlike latrunculin A andjasplakinolide, caused enhancement of the excystation andmetacystic development of this parasite.

A−10) INVOLVEMENT OF SIGNALING MOLECULES IN THE GROWTH AND ENCYSTATIONOF ENTAMOEBA INVADENSASAO MAKIOKA1, MASAHIRO KUMAGAI1, HIROSHI OHTOMO1,SEIKI KOBAYASHI2 AND TSUTOMU TAKEUCHI2
Department of Tropical Madicine, Jikei University School of Medicine1,
Department of Tropical Medicine and Parasitology, Keio University School of Medicine2

Signaling molecules are responsible for numerous cellular responces including cell proliferation and defferentiation. There are, however, no studies on signaling for thegrowth and encystation of Entamoeba. Therefore, we determined whether signaling through protein kinase C (PKC),
mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (Pl 3-kinase) is involved in the growthand encystation of E. invadens by using several inhibitorsof these enzymes. Among four inhibitors (staurosporine,
chelerythrine hydrochloride, calphostin C, and D-erythrosphingosine) of PKC used, the former two inhibitied thegrowth and encystation of E. invadens. As staurosporine isa potent but not selective inhibitor of PKC, chelerythrine
was used for further experiments. Addition of chelerythrine
after induction of encystation was also effective for inhibi-tion of encystation. The chelerythrine effect on trophozoites in encystation medium was irreversible, whereas it was
reversible in growth medium. All MAPK and MAPKK inhibitors including SB203580, apigenin,forskolin, olomoucine, prostaglandin E2, hypericin, and PD98059 had no effects on the growth and encystation of this parasite. Wortmannin, a specific inhibitor of Pl 3-kinase, also inhibitedthe encystation of E. invadens. Addition of wortmannin after induction of encystation reduced the number of cysts
formed. Like chelerythrine, the wortmannin effect on trophozoites in encystation medium was irreversible. These
results are the first evidence for participation of PKC and Pl
3-kinase in the growth and encystation of E. invadens, contributing to further understanding of Entamoeba growth andencystation and their inhibition.

５４A−11) BACTERIAL EXPRESSION OF A HUMAN MONOCLONAL ANTIBODY-ALKALINE PHOSPHATASECONJUGATE SPECIFIC FOR ENTAMOEBA HISTOLYTICAHIROSHI TACHIBANA1, YUTA NAKATA1, XUN-JIA CHENG1,YOSHIMASA KANEDA1 AND MASATAKA TAKEKOSHI2
Departments of Infectious Diseases1 and Molecular Life sciences2, Tokai University School of Medicine

We previously produced human monoclonal antibody
Fab fragments specific for Entamoeba histolytica in Escherichia coli. For application of these Fab fragments fordiagnostic purposes, an experssion vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E .
coli was designed and constructed. E .coli PhoA gene was
fused to the 3’ terminus of the gene coding the heavy chain
Fd region. As human antibody genes, the kappa and Ed
genes from one of the previously prepared antibody clones,
CP-33, were used. The expressed fusion protein was purified by affinity chromatography using histidine-tag binding
resin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein revealed two maior bands
with apparent molecular masses of 25 and 75 kDa. To con-firm whether these bands represent the kappa chain and thefusion protein of Fd and PhoA, Western immunoblot analysis was performed. As expected, the 25-kDa band was recognized by an anti-human kappa chain goat antibody, andthe 75-kDa band was detected by an anti-PhoA rabbit antibody. When the fusion protein of CP33 and PhoA was incubated with formalin fixed trophozoites of E . histolytica
and developed with substrate, the trophozoites were specifically stained. These results demonstrate that the bacterial
expression of a human monoclonal antibody-PhoA conjugate specific for E . histolytica is possible and that the antibody can be used to detect E . histolytica antigen without
the use of chemically conjugated secondary antibodies.

A−12) THE REGULATORY ROLE OF NITRIC OXIDE (NO) IN PROLIFERATIONOF TOXOPLASMA GONDII IN MOUSE BRAINS EXAMINEDWITH THE TREATMENT OF L-NG-MONOMETHYL- ARGININE AND QC-PCR ASSAY METHOD
HYE-SEONG MUN, KAZUMI NOROSE, FUMIE AOSAI, MEI CHEN,HIDEKAZU HATA AND AKIHIKO YANO
Department of Infection and Host Dcfense, Gradualc School of Medicine, Chiba University

IFN-γ-activated macrophages have been demonstrated
as important effector cells T . gondii infection via NOmediated killing mechanism. Previous studies demonstrated that the trealment with L-NG-monomethyl arginine(L-NMMA) of mice increased T . gondii numbers analyzed
by using direct microscopy and immunofluorescence assay,
although these estimation methods of T . gondii in tissues
seem unreliable because T . gondii is an intracellular protozoan. In the present study, we examined the regulatory roleof NO in proliferation of T . gondii by the use of our own
established the quantitative competitive-polymerase chain
reaction (QC-PCR) targeting SAG1 gene of T . gondii
BALB/c and C57BL/6 mice were intraperitoneally (i.
p. ) injected with PBS (as a control) or L-NMMA (175mg/kg), and the mice were i. p. infected with 200 or 400 cystsof T . gondii. All mice treated with L-NMMA died within
13 days post-infection. In contrast, 33% of the control

BALB/c and C57BL/6 mice treated with PBS and infectedby 200 or 400 cysts of T . gondii died within 10 to 11 days
post-infection and the remainder survived. These data
showed that the treatment of L-NMMA reduced the host resistance of both susceptible C57BL/6 and resistant BALB/cmice to tigh dose infection of T . gondii. In an attempt tocharacterizc whether injection of L-NMMA results in enhancement of proliferation of T . gondii in brains of infected mice, the number of T . gondii in the brains wasmeasured using QC-PCR targeting SAG1 gene 8 weeks after T . gondii infection. The number of T . gondii in thebrains of L-NMMA-treated BALB/c and C57BL/6 mice
was significantly higher than that in control mice. These
data indicate that the treatment of L-NMMA increased thenumber of T . gondii in brains of BALB/c and C57BL/6
mice. After infection with T . gondii, peritoneal macrophages from PBS-treated BALB/c and C57BL/6 mice pro-５
５

duced high levels of NO. Peritoneal macrophages from LNMMA-treated BALB/c and C57BL/6 mice, however, produced low levels of NO. Thus, L-NMMA blocked NO synthesis of peritoneal macrophages of T . gondii-infected mice.

Activated macrophages of T . gondii-infected micedown-regulated proliferation of T . gondii in the brain anddissemination of T . gondii to the brains of infected mice
via NO-dependent effector mechanisms.

A−13) T. GONDII INFECTION INDUCES ANTI-HSP70 AUTOANTIBODY IN A SUSCEPTIBLE
C57BL/6 MICE BY MODULATING RAG GENES
MEI CHEN, FUMIE AOSAI, KAZUMI NOROSE, HYE-SEONG MUN AND AKIHIKO YANO
Department of Infection and Host Defense, Graduate School of medicine, Chiba University

Introduction: Toxoplasma gondii (T . gondii) an obligate
intracellular protozoan parasite, is an important cause ofmorbidity and mortality, especially in congenital toxoplasmosis and immunocompromised hosts. We have reported
anti-T. g. HSP70 antibody as well as anti-mHSP70 autoantibody produced in dominantly T . gondii-infected C57BL/6(B6; a susceptible strain) mice. The mechanism of autoantibody formation in T . gondii-infected mice is not well defined.
The lymphoid-specific genes, recombinationactivating genes (RAGs) catalyze V(D)J recombination. The
purpose of this study was to investigate the mechanisms of
autoimmunity induced by T . gondii-infection.
Material and Methods: BALB/c and B6 mice were perorally infected with 5 cysts of Fukaya strain. Control uninfected and infected BALB/c and B6 mice were sacrificed on
day 0, 3 and 7 after T . gondii-infection. Spleen, MLN, BM
and PECs were collected for the determination of the expression of RAG gene. The T . gondii numbers in the tissues of uninfected and T . gondii-infected BALB/c and B6
mice were analyzed by quantitative competitive-PCR.
Results: The level of RAG1 mRNA expression significantly
decreased in the spleen and MLN of B6 mice on day 3 andrecovered and reached a plateau 1 wk after T . gondii infec-

tion. Before T . gondii infection, the expression of RAG1
was not observed in PECs of B6 mice. One week after T .
gondii infection, RAG1 and RAG2 expression was inducedin PECs of T . gondii-infected B6 mice. The level of RAG1
expression in BM of B6 mice gradually increased after T .
gondii infection. These data indicated that the level of
RAG1 expression was transiently down-regulated in the
spleen and MLN 3 days after the infection, then a higher
level of RAG1 expression was observed in the spleen, MLN,
BM and PECs of B6 mice 7 days after T . gondii infection.
Significant changes in the RAG1 expression in the spleen,
MLN, BM and PECs of BALB/c mice was not shown after
T . gondii infection. On the other hand, the numbers of T .
gondii in the spleen and MLN of BALB/c and B6 micereached a peak 7 days after the infection and then decreased.
No T . gondii was detectable in BM and PECs in BALB/cmice throughout the course of infection.
Conclusion: Out data indicated that the expression of RAG
was transiently down-regulated in the spleen and MLN but
not in BM and PECs of T . gondii-infected a susceptible B6
mice and the levels of RAG expression correlated with the T.
gondii numbers existent in the tissues.

A−14) A THOUGHT EXPERIMENT ON THE LINK BETWEEN THE RISK OF CONGENITALTOXOPLASMOSIS AND THE ANNUAL INFECTION RATESKOJI NAOI AND AKIHIKO YANO
Department of Infection and Host Defense, Chiba University Graduate School of Medicine

Toxoplasmosis, an infectious disease induced by
Toxoplasma gondii (T . gondii) an obligate intracellular
protozoan, is commonly benigh or asymptomatic when acquired by healthy persons. However, it may give harmful

effects to fetus when pregnant women contract a primary
infection congenital toxoplasmosis (CT).
The risk of CT reflects the total number of women
who acquire a primary infection in their pregnancy. Hence,５
６

it also correlates with that of women who acquire a primary
infection in the gestational period: the age equal to or older
than 15 years and younger than 45 years. In the present
study, we made a theoretical analysis with a simple mathematical model on the total number of women in the gestational period who get a primary T . gondii infection,
through which we found out the link between the risk of CT
and the annual infection rates of T . gondii. Considering thedifferences in behavioral patterns, different annual infection
rates, the rate before the gestational period (r1) and that after the period (r2), were applied to out model.
Our present study indicates, in the case that annual infection rate is constant (r1 = r2), 3.67% of annual infection
rate maximizes the risk of CT, while the rates vary if it ispresumed that r1 is different from r2. Adding to it, changes
in r1 would make a shift of the risk curve, while those in r2
would move the location on the same risk curve.
Based on our study, the risk of CT in Japan currently
stands st a relatively lower level. However, the study implies that a possible epidemic of T . gondii infection could
raise the risk steeply. From the standpoint of risk management, public intervention programs are required.
Our study also implies that public interventions for CT
could generate different outcomes, depending on whether
they target women before the gestational period or after theperiod. And a successful program in certain area could induce a harmful effect in another. Tailor-made medicine isrequired in public health policy as well as clinical medicine.

A−15) PHENOTYPE AND FUNCTION OF MURINE PERITONEAL CAVITY
MACROPHAGE DERIVED DENDRITIC CELLSMAKALA LEVI, NISHIKAWA YOSHIFUMI, KAMADA TAKENORI. XUAN XUENAN,
FUJISAKI KOZO, SUZUKI NAOYOSHI AND NAGASAWA HIDEYUKI
Natl Res Ctr for Protozoan Dis, Obihiro Univ of Agri Vet Med

We describe a two-step culture system using peritoneal
macrophages from P30 (SAG-1) transgenic mice as asource of dendritic cell (DC) progenitors. The proliferative
capacity of the progenitors was smplified in the first step of
the culture (day 0-7) using a combination of early cytokines: interleukin 4 (IL-4); and granulocyte-macrophagecolony-stimulating factor (GM-CSF). The second step of
the culture started at day 7 with the removal of early growth
factors to allow differentiation and final maturation of DC

during 2 days of culture with interferon gamma (IFN-γ)
plus Toxoplasma lysate antigen (TLA) as maturing agents.
Here, we show that the resulting DC population exhibits
typical dendritic cell morphology, expresses MHC class II
and co-stimulatory molecules CD80 at higher levels, consistently synthesize IL-12, efficiently stimulate T cell responses in vitro using two way mixed lymphocyte reaction(MLR) and are able to present soluble antigen to CD3+
spleen T cells.

A−16) GROWTH INHIBITORY EFFECTS OF TRICLOSAN AGAINST BABESIA SPP. PARASITESSABINE BORK, NAOAKI YOKOYAMA, HIROSHI SUZUKI, HIDEYUKI NAGASAWA, KOZO FUJISAKI,CHIHIRO SUGIMOTO AND IKUO IGARASHINational Research Center for Protozoan Diseases, Obihiro Univ. of Agri. and vet. med.

The antimicrobial biocide triclosan ｜5-chloro-2 (2.4dichlorophenoxy) phenol｜ has been reported recently toinhibit the growth of Plasmodium falciparum in vitro and P.
berghei in mice (Nature Medicine, 7: 167-173, 2001). In
the present study, we have evaluated the growth inhibitory
effect of triclosan against the hemoprotozoan, ticktransmitted parasites, Babesia egui B . caballi and B . bovis

The growth inhibitory tests were carried out in vitro following the method of lgarashi et at. (Expt. Parasitol., 90: 290293, 1998). Different concentrations of triclosan wereadded to cultures containing an initial 1% parasitemia ofBabesia parasites in 24-well plates and incubated at 37°
C.
The medium was changed daily and the percent parasitemia
was monitored on Giemsa-stained blood smears for five

５
７

days. Parasite growth of B . egui and B . caballi was inhibited at drug concentrations of 20-200 µg/ml , while growth
inhibition of B . bovis was noted at 100-500 µg/ml . Initial
findings in in vitro studies are encouraging and suggest theuse of triclosan as a potential drug against equine and bovine babesiosis. To further determine the efficacy of triclosan in vivo, we have started studies on its effect on Babesia rodhaini-and B . microti-infection in mice.

A−17) GEOPATHOLOGICAL STUDY ON MALIGNANT TUMORS IN WESTERNKENYA BETWEEN 1979 AND 1998
KAN TORIYAMA1, JANE WASIKE-SIMINYU2, MASACHIKA ISEKI1 AND MASACHIKA SENBA1
1

Department of pathology, Institute of Tropical Medicine, Nagasaki University
2
Histology Section, Rift Valley Provincial General Hospital, Nakuru, Kenya

An ethnogeographic variation in the frequency of different types of tumors is mainly attributed to the interplayof varied causative factors such as exposure to ultraviolet
light, chemical carcinogens, oncogenic viruses, genetic factors, and cultural practices among various populations. This
study analyzed histopathologic specimens in western Kenyafor 20 years during 1979 to 1998 with a focus on the following objectives; to examine ethnogeographic distribution
and to relate the tumors to putative environmental causative
factors. The most common malignant tumor was uterine
cervix cancer, which is widely distributed in this area, fol-

lowed by malignant lymphoma, including Burkitt’s lymphoma, which is more prevalent among the Luo around theLake Victoria, and squamous cell carcinoma of the skinamong the Kalenjin in Tropical Highlands. In addition, several malignant tumors such as Burkitt’s lymphoma, Kaposi’s sarcoma, penile cancer, squamous cell carcinoma of
the urinary bladder showed characteristic ethnogeographic
distributions. These findings suggest that environmental
factors, including oncogenic viruses, and cultural practices
seem to play a main role in the development of malignant
tumors in western Kenya.

A−18) SYNERGISTIC EFFECT OF TWO SUSCEPTIBLE GENETIC MARKERS,
HLA-B5*0101 AND IL-13P-A/A,
ON THE DEVELOPMENT OF POST-SCHISTOSOMAL LIVER FIBROSISKENJI HIRAYAMA1, MIHOKO KIKUCHI1, HONGGEN CHEN2, RATAWAN UBALEE1,
TONG YIN2, XIAONAN GU2, JIANXIANG LIU3, SHAOJI ZHANG2, AND HONGCHANG YUAN3
Department of Molecular immunogenetics, Institute of Tropical Medicine, Nagasaki University1,
Jiangxi Provincial Institute of Parasitic Disease2,
Department of Epidemiology, School of public Health, Shanghai Medical University3.

In 1995, over 100,000 patients have been estimated to
be infected with Schistosoma japonicum in Jiangxi province. One of the most serious complications is postschistosomal liver disease that develops within several years
after the infection. Schistosomal egg antigen specific CD4+
T cells play a major role in the formation of granuloma
through Th2 type cytokine production in experimental
schistosomiasis mansoni. To identify the host genetic factors affecting the prognosis after infection, we have examined DNA polymorphism of 192 unrelated past patients
who were living in the endemic area named Yushan, Jiangxi,

including 36 individuals with no fibrotic change in the liver(Grade O) and 156 patients with typical fibrotic change(Grade I through Grade III), diagnosed by ultrasonography (Cairo, 1991). After extensive analysis of HLA-class IIregion and polymorphic immune response related genes(TNF-α promoter, IL-4R, IL-4 promoter, IL-4, IL-13 promoter, IL-13, region), the HLA-DRB1*1101 and HLADRB5*0101, IL-13 promoter A / A genotypes were revealed
to be associated with protection and susceptibility to fibrosis. Moreover, HLA-DRB5*0101 and IL-13P-A / A both ofwhich were susceptible markers to fibrosis were synergisti-

５８cally elevated the odd’s ratio. This effect was considered tobe mediated though IL-13 production up-regulation in theantigen specific CD4+T cells stimulated by HLA-DRB5*
0101.

A−19) A SURVEY ON ANTIGENEMIA OF WUCHERERIA BANCROFTI AND URINE ANTIBODY TITER
IN RURAL COMMUNITIES IN NEPAL
KANJI WATANABE1, MAKOTO ITOH2, SHINJIRO HAMANO3, SHIGERU KOBAYASHI4,SASHI SHARMA5, GOPAL P, ACHARYA5, YOSHIKI AOKI1
1

Department of Phrasitology, Institute of Tropical Medicine, Nagasaki University,2
Department of Phrasitology, Aichi Medical University,
3
Department of Microbiology and Immunology Faculty of Medical Sciences, Kyushu University,
4
Department of Human Geography, Graduate School of Letters, Osaka University, Japan
5
Institute of Medicine, Tribuvan University, Nepal

In order to determine the prevalence of the bancroftian
filariasis in rural communities in Nepal, serum and urine
samples from inhabitants in two rural villages (Judigaun,
Kotyang) were examined. Serum and urine samples of 244
inhabitants in Judigaun, which locates 30 km from Kathmandu, were collected in 2000, 2001. Stored serum andurine samples collected from inhabitants in Kotyang in October 1998 at before survey were also used. Serum samples
were examined for W. bancrofti antigens by using Og4C3
assay. Immunoglobulin G4 against filarial antigen in theurine samples were measured by using filarial antigen

coated 96-well micro titer plates and peroxidase conjugatedsecond antibodies to human IgG4. Among 238 serum samples and 244 urine samples from Judigaun people, 60(25.2%) serum samples and 124 (50.8%) urine samples
were positive with regards to W. bancrofti antigenemia andfilarial IgG4 titer respectively. Among 117 serum samples
and 364 urine samples from Kotyang people, 18 (15.4%)
serum samples and 90 (24.7%) urine samples were also
positive with antigenemia and IgG4 titer respectively. These
results show that rural area are also endemic of W. bancrofti
and more detailed study will be expected.

A−20) MATHEMATICAL MODEL FOR THE TRANSMISSION OF LYMPHATIC FILARIASISAND ITS APPLICATIONSHIROYUKI ISHII1, HIROFUMI ISHIKAWA2, AND YUKIO OHGA1
Department of Environmental Synthesis and Analysis,
Graduate School of Natural Science and Technology, Okayama University1,
Department of Environmental and Mathematical Sciences,
Faculty of Environmental Science and Technology, Okayama University2

Our study aims at evaluating the effect of vector control on the prevalence of Iymphatic filariasis caused byWuchereria bancrofti using computer simulations based on
the stochastic transmission model, and especially it is focused on the continuous effect for a post control period.
In Pondicherry, South India, Vector Control ResearchCentre had carried out the integrated vector control program
against filariasis and malaria for five years (1981-1985),
and reported a substantial decrease in both vector population and the transmission index for W . bancrofti during theperiod of vector control. We improve the transmission
model proposed by A. P. Plaisier et al . (1998) to be fitted
for a prevalent situation in Pondicherry on the basis of precontrol epidemiological data there. The population dynamics of mosquito vectors has been simulated by the carrying
capacity model of the larval mosquitoes environment.
Our simulations show that in the vector control area
the rate of microfilaremia in human population is decreasing slowly while the mean number of L3-larvae carried bymosquito population is decreasing in an early period of a５９control project. For a post control period, they also showthat the prevalence revives in a several years and is beyond
the initial prevalence level because of a decline of immunity
level if an effect of vector control looses thoroughly, but theprevalence stays at low level for a long time if a continuous
effect remains at least 10%. We conclude that in a post control period keeping of a little continuous effect contributes
to control or elimination of lymphatic filariasis.

A−21) A TRIAL TO DETECT ANTI-B. PAHANGI IGG4 IN URINE FROM CHILDREN UNDER 5 YEARSOLD IN ORDER TO EVALUATE FILARIASIS CONTROL MEASURES
MAKOTO ITOH1, MIRANI V. WEERASOORIYA2, NIPUL K. GNAWARDENA3, YASUNORI FUJIMAKI3,
XU-GUANG QIU1, EISAKU KIMURA1
Department of Parasitology, Aichi Medical University School of Medicine1,
Filariasis Research Unit, University of Ruhuna, Sri Lanka2,
Department of Parasitology, Institute of Tropical Medicine, Nagasaki University3

The global program to eliminate lymphatic filariasis
has been launched. For the program, it is essential to findendemic areas and evaluate the effect of control measures.
Surveillance of infectious status of young children will be
useful for the evaluation, since successful control prevents
new infections and children born after the control measuresare expected to harbor no filarial parasites.
We developed a sensitive and specific ELISA for bancroftian filariasis using urine samples. In this study, we applied the ELISA method to evaluate filarial infections
among young children in a bancroftian filariasis endemic
area.
Subjects and Methods
Urine samples were collected in Matara District, Sri
Lanka from 206 children under 5 years old including 38 infants. Samples from their mothers and / or fathers (301 samples) were also collected. Urine collection bags (Atom Pediatric Urine Collector) were used for small children and in-fants. Anti-Brugia pahangi IgG4 antibodies in urine samples were measured by ELISA.
Results and Discussion
Urine samples from small children could be collectedeasily and safely. Filaria antigen specific IgG4 antibody
were positive in 10.5% (4/38) of infants (≦1 year old),
7.5% (3/40) of the age group 1-2 years, 2.3% (1/43) of theage 2-3 years, 22.5% (12/47) of the age 3-4 years and26.3% (10/38) of the age 4-5 years. A remarkable increasein the positive rate was observed at age 3 years old andabove, indicating that the filarial infections occur very earlyin life in this area. The positives in infants have antibodies
transferred from their mothers.
These results show that the measurement of filarial antigen specific IgG4 antibodies in urine samples from children is easy and useful for evaluation of the parasite control
program.

A−22) RESISTANCE OF BLASTOCYSTIS HOMINIS TO METRONIDAZOLEYOSHIMASA KANEDA1, NORIYUKI HORIKI2, XUN-JIA CHENG1, HIROSHI TACHIBANA1
Department of Infectious Diseases, Tokai University School of Medicine1;
Division of Internal Medicine, National Mie Chuo Hospital2

Blastocystis hominis has been found on numerous occasions in the human intestinal tract. Although most casesof B . hominis infection are symptom-free, symptomatic infections have been reported, and the pathogenicity of thisunicellular organism remains controversial. When a B .
hominis infection is diagnosed, chemotherapy is usuallyprescribed. At present, the drug of first choice is metronidazole. We used metronidazole to treat 15 infected individuals
whether or not they were symptomatic. Of the 15 infectedindividuals, 11 proved refractory to treatment even thoughlarge doses of metronidazole were administered. Because
none of our surveyed individuals were immuno or otherwise

６０

deficient, any GI symptoms were believed to have been due
to the infection with B . hominis. It is informative that all
symptoms disappeared after treatment. To elucidate thecausative factors of the chemotherapeutic failure, we monitored the in vitro effects of metronidazole on B . hominis
isolates obtained from the refratory individuals both before
and after treatment. Metronidazole was effective in inhibiting growth of control isolates obtained from non-treatedcases and the reference strain (Nand II), although it took 4hours to manifest efficacy. In contrast, the refractory B .
hominis isolates showed a slightly increased resistance invitro to metronidazole. On the basis of the present results, it
seems that treatment failure may have resulted from insufficient exposure to metronidazole and that Blastocystis may
acquire a degree of resistance after initial exposure.

A−23) LONG TERM ERADICATION RATE OF IVERMECTIN THERAPY FOR STRONGYLOIDIASISON YORON ISLAND, KAGOSHIMA PREFECTURE
OSAMU ZAHA, KAZUTO KISHIMOTO, HIDENAO OMIJA, NOBUHUMI UCHIMA, TETSUO HIRATA, NAGISA KINJO,AKIRA HOKAMA, HIROSIHI SAKUGAWA, FUKUNORI KINJO AND ATSUSHI SAITOFirst Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus

In our present study, we investigated the epidemic of
Strongyloides stercoralis on Yoron Island, Kagoshima Prefecture. Furthermore we assessed the long term eradication
rate of ivermectin therapy for chronic strongyloidiasis andadverse effects of the drug. Yoron Island is situated in thesouthernmost area of Kagoshima Prefecture. Of the 611
persons examined by using agar plate culture method, 64(10.5%) were found Strongyloides-positive. By age, thepositive rate was highest in 60s (16.0%), following by 70s(13.8%), 80s (12.5%), 50s (6.9%) and 40s (6.5%), Of those
who were found Strongyloides-positive, 32 persons (17males and 15 females) desired to have treatment. HTLV-1
antibody positive rate was 25.0% (8 / 32), of which males
were 23.5% and females 26.7%. To each patient, approximately 100 µg/kg of ivermectin was administered before
breakfast. The same treatment was repeated after 2 weeks.
During the period of administration of ivermectin, Liverdysfunction was observed in one patient. However, it was ofmild with only slight increases in ALT, and no particulartreatment was required. As subjective adverse effect, a feeling of vertigo was noted in one patient after administrationof 2 courses. In this case, too, no particular treatment wasnecessary. In the 32 patients who received treatment, theeradication rates in 2 weeks and 4 weeks after first treatment were 100% (32/32 patients) respectively. In thefollow-up investigation conducted one and four year later, 2cases of recurrence (30/32, 26/28) were found respectively.
Both of the 2 patients were HTLV-1 antibody positive.
Ivermectin is being used widely all over the world as adrug for Onchocerca volvulus, and it shows high anthelmintic activity against S. stercoralis too. Ivermectin showed excellent anthelmintic effects with mild toxicity. We therefore
consider ivermectin is the most useful agent for the treatment of chronic strongyloidiasis at present.

A−24) PREVALENCE OF OXYURIOSIS AND OTHER HELMINTH AT THE SCHOOLOF MOUNTAINEOUS REGION OF NEPAL:
INFLUENCE BY TARGET SELECTED AND BLANKET TREATMENT
KOSUKE HARUKI1, 2, TAKASHI FUJINO1, MORIYASU TSUJI1, JEEVAN BAHADUR SHERCHAND2
Department of Infectious Diseases, Kyorin University School of Medicine1,
Infectious and Tropical Disease Research Centre / Nepal2

Helminth is one of major cause of economical loss indeveloping world. JAITI (Japan Agriculture In-service
Training Institute) Basiphant School was established in

1993 at mountainous region located about 80 km southwestof capital city Kathmandu in Nepal. The school children
were treated by mebendazole twice a year since 1993. We６
１have started school health project in 1998. Two hundred
forty school children aged six to twenty first were the target.
The prevalence of helminth was 60% at initial time in 1998
and considered that the result was contributed to blanket
treatment. We examined the stool and selected positive
cases and treated them by albendazole but not any medication to negative cases. One years interval showed highprevalence of helminth over 80%. The result suggested highre-infection rate and necessity of blanket treatment. We
have started blanket treatment by albendazole twice a year
then after two years later in 2001 the prevalence was decreased to 58%. But the prevalence of Enterobius infection
using scotch tape method was 43% in 1999 and 52% in
2001. This data indicated several speculation to re-consider
the strategy of the project as follows: 1. Target selected
treatment is not an adequate method to control helminth infection in the school. 2. Re-infection rate is supposed to behigh and even by six months blanket treatment may not be
expect to control the prevalence less than 50%. 3. Healtheducation programme involving families of children has notbeen performed yet. The infection cycles in each family andcommunity may contribute high re-infection rate at theschool. 4. Increased prevalence of Enterobius infection may
due to improved technique by children to collect the specimen. 5. Treatment performed twice in a year by albendazole
does not decrease the prevalence of Enterobius infection.
This may due to high infectivity of Enterobius ova and poor
hygienic condition particular clothes and bed linens. 6. Improving hygienic condition by health education may only beable to decrease the prevalence of helminth infection lessthan 50% combined with blanket treatment in the school.
We are planning to involve families of children then community to health education programme in near future.

A−25) A SURVEY ON ALLERGY, PARASITIC INFECTION AND NUTRITIOUS STATUS
IN SCHOOL CHILDREN IN VIETNAMYASUNORI FUJIMAKI1, DAO TO QUYEN2, YUKI SATO3, NGUYEN THI DANG4, HA HUY KHOI2,
DO THI KIM LIEN2, YOSHIKI AOKI1, SHIGERU YAMAMOTO3, FUSAO OTA5
1

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

A survey on allergy, parasitic infection and nutritious
status in school children in Hanoi, Vietnam was carried out
to know relationships among allergy, parasitic infection,and nutritional status. Eighty eight children with an averageage of 10.6 years old were examined. For intestinal parasitic
infection their feces samples were examined using KatoKatz technique. To survey their allergic diseases or symptoms all the children were done by use of a questionnaire.
Daily food intake was assessed by the ‘24 hour recall’
method. Their weight and height were measured. Anthropometric indices were analyzed using the standard US National Center for-Health Statistics charts and tables. Body
mass index (BMI) was also calculated. Blood samples werecollected and analyzed for serum IgE, total protein, albumin
and lipid in plasma.
Results: 1) Prevalence of parasitic infection (83.0%)
was very high. Trichuris trichura was a most dominantparasite (80.7%). 2) Only 9 children had a history of allergy. Six out of them were infected with parasitic infection

and no relationship between the presence of allergy andparasitic infection was observed. 3) The level of total IgE
in serum was significantly higher in children with parasite(474.4 IU/ml ) than that in those without parasite. 4) Nutritional status of chidren examined was poor regardless ofparasitic infection. Thirteen and sixteen children were considered to have malnutrition by weight-for-age and heightfor-age analysis respectively. 5) The mean serum level of
total protein, total cholesterol, triglyceride etc were allwithin the normal range. 6) For nutritional intake, the meanenergy was 1413.9Kcal, showing about 70% of recommended dietary allowance in Vietnam. No difference in nutritional intake such as protein, lipid and carbohydrate wasobserved between the children with and without allergy andparasitic infection.
Conclusions: In Vietnam the prevalence of intestinal
parasitic infection was high but the number of children witha history of allergy was few. Nutritional status of children
was very poor regardless of parasitic infection. No relation-

６
２

ships between allergy and parasitic infection, and between

allergy and nutritional intake were observed.

A−26) HIV-1 SUBTYPING IN ZAMBIARAY HANDEMA1, HIROSHI TERUNUMA1, FRANCIS KASOLO2 HIROTAKE KASAI1, XUEWEN DENG1,
ATSUYA YAMASHITA1, NAOKI YAMAMOTO3, MITSUO HONDA4, MASAHIKO ITO1
1

Department of Microbiology, Yamanashi Medical University, Yamanashi,
2
Virology Laboratory, UTH, Lusaka,
3
Department of Microbiology and Molecular virology, Tokyo Medical and Dental University, Tokyo,
4
AIDS Research Center, NIID, Tokyo

Introduction: Sub-Saharan Africa has the highest HIV
prevalence and AIDS burden. The ultimate goal of controlling the HIV-1 pandemic is to develop safe and efficacy
vaccines. However, the relatively high level of genetic divergence between the group M clades has led to the hypothesis that multiple vaccines against HIV-1 may have to
be made against the different subtypes of the virus. Sequence information on most of the nine subtypes is currently limited, suggesting that more information will beneeded if subtype specific vaccines are to be produced. We
are currently carrying out a nation-wide molecular epidemiological study that seeks to access the distribution of HIV-1
subtypes in Zambia. Objective: To document the HIV-1
subtypes circulating in Lusaka, Luapula and Northern provinces in Zambia. Methods: Two hundred and thirty samples
collected from antenatal women in seven clinics in Lusaka
province were initially screened for HIV using antibody assays. Twenty-five and eight HIV-1 seropositive whole blood
samples were collected from provincial headquarters ofNorthern and Luapula provinces respectively. Molecularepidemiology of HIV-1 subtypes was investigated by direct
sequencing of PCR products of gag p17 and env gp120 C2V3-C3 fragments and phylogenetic analysis was done. Results: Sixty-three samples from Lusaka province were HIV-

1 positive. The number of HIV-1 gag p17 and env gp120
sequences obtained per province were 56 for Lusaka, 20 for
Northern, and 8 for Luapula. Thus a total of 84 sequencesfrom 92 (91%) HIV-1 positive samples were obtained. HIV1 gag/env subtype C per province were 51 (91%) for
Lusaka, 20 (100%) for Northern, and, 8 (100%) for Luapula.
One (1.8%) HIV-1 gag/env subtype A-like, one (1.8%)
HIV-1 gag/env subtype G-like, and one (1.8%) A/C and one(1%) D/C recombinants were detected from Lusaka samples.
A high conservation of the CTL peptide epitopes was observed among HIV-1 gag and env subtype C-like viruses.
conclusions: HIV-1 subtype C is predominant in Lusaka,
Luapula and Northern provinces. Two new HIV-1 gag/env
subtype A-like and subtype G-like have been identified forthe first time in Zambia in this study suggesting that more
subtypes could be in existence. Two suspected recombinants, A/C and D/C, have also been detected in this study.
We are now doing full length genome analysis to determine
the nature of recombination of these samples. Some of themore conserved CTL epitopes among different HIV-1 subtypes should be considered in vaccine constructs in order tohave cross clade immune responses. There is need for acontinued work of HIV subtyping to enhance the efforts being made towards the development of HIV-1 vaccines.

６３A−27) MORECULAR EPIDEMIOLOGY OF HIV IN MYANMAR: EMERGENCE OF NEW FORMS
OF HIV-1 INTERSUBTYPE RECOMBINANTS
Y. TAKEBE*1, K. MOTOMURA1, S. KUSAGAWA1, H. H. LWIN2, AND M. ZAW21

2AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan;
AIDS Prevention and Control Programme, Department of Health, Yangon, Myanmar.

Background: UNAIDS estimated that 530 thousands people has been infected in Myanmar (as of the end of 1999)
since the HIV-1 was introduced in 1989. Highest HIV
prevalence is observed among injecting drug users (IDUs)(63%) and female commercial sex workers (fCSWs) (38%).
In order to study the molecular epidemiology of HIV-1 in
Myanmar, we investigated the molecular nature of circulating HIV strains and the interrelationship of the epidemic
with those in surrounding countries.
Methods: The nucleotide sequences of various segments in
HIV-1 genomes were determined and subtyped for 84 specimens from Mandalay, Central Myanmar. The near fulllength sequences of plausible recombinant HIV-1 isolates
were determined after PCR-cloning and were subjected tothe phylogenetic and recombination breakpoints analyses.
Results: Phylogenetic analyses based on gag (p17) and env(C2/V3) sequences detected 13 subtype C (16%), in addition to the previously identified subtype B’ (Thai-B cluster
within subtype B) (19/84, 23%) and CRF01_AE (37/84,

44%) that are likely to be originated from neighboring Thailand. The majority of subtype C in Myanmar belongs to theIndia-China cluster of subtype C. Notably, the remaining 15
specimens (18%) showed discordance between gag and env
subtypes, including 4 gag B’/env C (B’/C), 3 C/B’, C/E and
B’/E, and 2 E/B’. These discordant specimens were foundmost frequently among IDUs (10/34, 29%). The analyses
based on near full-length HIV-1 sequences revealed theunique chimeric structures between co-circulating subtypesof B’, C and E. in four isolates.
Conclusions: The data suggest that multiple subtypes of B’,
C and CRF01_AE are co-circulating in Central Myanmar,
leading to the evolution of new forms of HIV-1 intersubtype
recombinants. The multiple occurrences of the recombinants with unique genome structures suggested on-going recombination between these three circulating subtypes inCentral Myanmar. Our finding would provide information
critical for future vaccine strategies aimed at this particulararea in Asia.

A−28) TRIALS OF DNA VACCINE TO JAPANESE ENCEPHALITIS VIRUS (JEV): EXPRESSIONOF JEV-ENVELOPE PROTEIN IN MAMMALIAN CELLSGUO-HE FENG, TSUTOMU TAKEGAMI
Division of Tropical Medicine, Medical Research Institute, Kanazawa Medical University

Japanese encephalitis virus (JE) is a serious mosquitoborne viral disease of major public health important in Asia.
Recently it has been reported that DNA vaccine is effective
to protect the virus infection including JE virus. Expression
level of transfected plasmid appears to be related with theefficacy of DNA vaccine. Here we constructed two kinds ofexpression vectors, pJE and pJME for JE envelope (E) protein and compared the expression level in several mammalian cells. Both expression vectors containing FLAG tag
were transfected with liposome (DMRIE-C reagents,
GIBCO) into cultured mammalian cell lines, i.e. HepG2
and KN73 derived from human liver, COS-1 and Vero derived from monkey, and BHK cells. To detect expression
proteins, Western blot using anti-FLAG or anti-E sera was

performed. In the transfected cells, 72kDa (prM+E) and 53
kDa(E) protein were expressed under the control of CMV
promoter. The expression of E protein in the transfection
with pJME was usually higher than that of pJE. It is likelythat prME protein is cleaved to E protein in the cells transfected with plasmid. In addition, expression level was dependent on the amounts of DNA transfected, and different
among the cell lines. Protein expressions in HepG2, KN73,
COS1 were relatively higher than those in Vero and BHK
cells. The expression efficacy of E and other proteins may
be influenced by the host factors including peptidase. It is
important to check the features of protein expression for thedevelopment of an effective DNA vaccine.

６４A−29) EVALUATION OF DENGUE ANTIGEN DETECTION KIT
MAKOTO HARADA1, KEN-ICHIRO YAMADA2, TOMOHIKO TAKASAKI2, ICHIRO KURANE2
Yokohama Quarantine Station1, National Institute of Infectious Diseases2

Dengue fever (DF) and dengue hemorrhagic fever(DHF) have been a serious health problem in most of thetropical and subtropical countries in the world. Dengue virus is not endemic in Japan, however, there are imported
cases. According to the Japanese new infectious disease
control law, which became effective on 1st April 1999, DF/
DHF is one of the infectious diseases to be reported by physicians.
We perform reverse transcriptase polymerase chain reaction (RT-PCR) as one of the laboratory diagnostic methods for virus infection, especially with sera obtained duaring a febrile period. Dengue antigen detection Kit (Globio)
has recently been on the market. We examined this test kit
for sensitivity and specificity in comparison with RT-PCR.
77 serum specimens were collected from denguesuspected febrile patients and sixty serum specimens (asnegative controls) were collected from exanthema subitum
patients who had never been to dengue epidemic area. They
were tested by RT-PCR, IgM capture-ELISA and Dengue
Antigen Detection Kit (Globio).

77 serum specimens collected from febrile patientswere divided into three groups by the results of RT-PCR
and IgM capture-ELISA. Twenty-two samples in 1st groupwere RT-PCR (+) and IgM capture-ELISA (-). Twenty-fivesamples in 2ndgroup were RT-PCR (-) and IgM captureELISA(+). Thirty samples in 3rd group were RT-PCR (-) and
IgM capture-ELISA(-). The results obtained by the antigen
detection kit were as follows. !Five (23%) of the 22 samples in first group were determined to be negative. "Eight(32%) of the 25 samples in second group were determined
to be positive. #Six (20%) of the 30 samples in third groupwere determined to be positive. More over, 5 (8%) of 60
sera from exanthema subitum patients were determined to
be positive.
These results demonstrated that this kit has nonspecific reaction and the sensitivity is not as high as RTPCR. However, if high specificity and sensitivity are established by some modification, this technique may be usefulfor rapid diagnosis, in certain facilities.

A−30) BIOLOGICAL CHARACTERISTICS OF DENGUE 2 VIRUSES ISOLATEDFROM PATIENTS WITH DIFFERENT SEVERITIESIN BAN HOM COMMUNE, RURAL AREA OF VIENTIANE, LAO P. D. R.
MIKA SAITO, MASAYUKI TADANO AND TOSHIHIKO FUKUNAGA
Department of Virology, Faculty of Medicine, University of the Ryukyus

Dengue outbreak occurred in a rural commune (BanHom, 4 km2）Vientiane, Lao PDR in 1994. Five strains ofdengue virus type 2 (DEN2) were isolated from patientswith different clinical severities in this area within 15 days.
In order to identify possible viral factors in the pathogenesisof dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), biological characteristics of these 5 strains
were compared.
Each strain passaged 3 times in the mosquito cell line C6/
36 was tested for mouse-neurovirulence, plaque size and virus production in cultured cells, fresh peripheral bloodmononuclear cells (PBMC), and dendritic cells. Basic characteristics of the 5 isolates are ; (name: severities, primaryor secondary infection, sampling date) 1) UOFP: Fever of

unknown origin, primary, June 1, 2) UOFS: Fever of unknown origin, secondary, June 1, 3) DF: dengue fever, secondary, June 15, 4)DHF: DHF grade I, secondary, June 15,5) DSS: DHF grade III, secondary, June 15. Prototype DEN
2 (New Guinea C strain) was used as a control.
Mouse neurovirulence was determined by intracerebral
inoculation in 3-day-old ICR mice with each strain at 104focus forming units (ffu). Death rates at 3 weeks after inoculation of DSS, UOFP were 0%, DHF 17%, UOFS 26%, DF
38%, NGC 100%. Plaque size in C6/36 cells were; UOFP,
DHF(7.56-7.95 mm), UOFS, DF, DSS (5.13-5.30 mm).
Plaque formation in LLC-MK2 cells was not observed forany of the 5 isolates but for NGC (1.52 mm). The production of virus particles in culture fluid was determined in ffu/

６
５

ml using PAP staining method. C6/36 cells could provide
efficient replication of all isolates ranging 3-9x 107 ffu/ml
and NGC, 6x108 ffu/ml . LLC-MK2 cell line showed significantly lower production for every isolate (2x103-3x104
ffu/ml ) than C6 / 36. The production of virus particles were
not detected in infected culture fluid of PBMC for any of
the 5 isolates, while infection of all isolates and NGC in
PBMC was confirmed by indirect immunofluorescenece re-action. In immature dendritic cells -like cells generated
from PBMC by addin IL-4 and GM-CSF to culture fluid, all
5 isolates could produce virus particles efficiently ranging
1.2-1.8x 105 ffu/ml and have peaks of growth curves at 34
hours after infection.
All biological characteristics studied here did not correlate with the clinical severity of the patient from whom
the virus was isolated.

A−31) COMBINED DETECTION AND GENOTYPING OF CHIKUNGUNYA VIRUS BY A SPECIFIC
REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION
F. HASEBE1*, M. C. PARQUET1, B. D. PANDEY1, M. SINNIAH2, AND K. MORITA1
1

2

Department of Virology, Institute of Tropical Medicine, Nagasaki University
Division of Virology, Institute for Medical Research, Ministry of Health Malaysia

A reverse transcription-polymerase chain reaction (RTPCR) was developed for the detection of chikungunya virus(CHIK) infection. Based on the non-structural protein 1(nsP1) and glycoprotein E1 (E1) genes of CHIK, twoprimer sets were designed. Total RNA were extracted fromthe cell culture fluid of Aedes albopictus C6/36 cells inoculated with the S27 prototype virus, isolated in Tanzania in
1953, and the Malaysian strains (MALh0198, MALh0298
and MALh0398), isolated in Malaysia in 1998. For bothsets of RNA samples, the expected 354 and 294 base pair(bp) cDNA fragments were effectively amplified from thensP1 and E1 genes, respectively. The sensitivity of the RTPCR was approximately 5 plaque-forming units (PFU) for
the nsP1 primer set and 50 PFU for the E1 primer set whenapplied to the S27 strain. RT-PCR products from both S27
and Malaysian strains were sequenced and compared. For
the 314 bp-long sequences of the nsP1 gene, the nucleotides
were 96.8% to 97.1% homologous and the amino acid sequences deduced for the S27 and Malaysian strains wereidentical. For the 257 bp-long sequences of the E1 gene, theS27 prototype and the Malaysian strains showed a 96.5%
and 97.6% homology for the nucleotide and the amino acidsequences, respectively. Phylogenetic analysis was conducted for the Malaysian strain and other virus strains isolated from different regions in the world endemic for CHIK,
using partial E1 gene sequence data. The Malaysian strains
isolated during the epidemics of 1998 fell into a cluster
along with other members of the Asian genotype.

A−32) A QUESTIONNAIRE SURVEY FOR DENGUE CONTROL IN BLITAR MUNICIPALITY,
EAST JAVA PROVINCE, INDONESIA:AN EVALUATION FOR BETTER COMMUNITY EDUCATION APPROACHSISWANTO1, 2, MASAAKI SHIMADA2, RUYONGA JOSEPH2, 3, TOMOKO KISU2, PAIMAN SUPARMANTO1
Hlth Serv Res Ctr, Natl Inst Hlth Res Dev, MOH, Indonesia1, Res Ctr Trop Inf Dis, Inst Trop Med, Nagasaki Univ2,
District Hlth Serv, Hoima District Local Gov, Uganda3

The vaccine against Dengue virus infection has notbeen produced so far. The only method to prevent dengue
fever (DF) and dengue hemorrhagic fever (DHF) is the control of vector mosquitoes. In line with these facts, the Ministry of Health of Republic Indonesia has been carrying out
an anti-DHF-campaign of mosquito control both via healthcenters and mass media. In order to assess the effectivenessof the campaign, a questionnaire survey was conducted in acommunity. The objectives of the study were (i) to explore
the knowledge, attitude and practices of the people about
DHF control and to identify the relating factors, and (ii) toassess the performance of DHF anti-DHF-campaign. The

６６

survey was conducted in Blitar municipality, East Java
Province, Indonesia. By using multistage random sampling,
198 households from 4 villages (kalurahan) were selected.
The head of a household or the other member, such as
wives and children, of the household was interviewed byusing semi-open questionnaire (guided interview). Analysis
was made using SPSS Release 10 (SPSS Inc.). The items in
the questionnaire were sex, marital status, position in theirfamily, age, education level, occupation, questions about thesource and the mean of dengue information and questions
to determine their knowledge, attitude and practice for thecontrol of mosquitoes.
Out of 198 respondents, the heads of the household
were 103 consisting of 87 males and 14 females, wives

were 74 and children were 21. The mean age of the respondents was 44.0 with a range of 13-77. Fourteen did not
graduate from any school, 83 graduated from elementary
school, 41 from junior high school, 54 from high school
and 6 graduated from college or university. Twenty four
were farmers, 37 were merchants, 22 were civil servants, 22
was working at private company, 33 were unskilled workers
and 60 were unclassified.
Although most of the respondents (184/198) were
aware of dengue fever (DF) and dengue hemorrhagic fever(DHF), a lot of heterogeneity was observed on their knowledge, attitude and practice. The factors that associate withthe heterogeneity will be demonstrated and discussed.

６７B−1) PURIFICATION OF RECOMBINANT TRYPANOSOME ALTERNATIVE OXIDASEKEISUKE KAWAI1, COHICHI NIHEI1, YOSHIHISA FUKAI1, YOSHISADA YABU2,NOBUKO MINAGAWA3, KAZUO NAGAI4, KIYOSHI KITA
1

Department of Biochemical chemistry, Graduate School of Medicine, the University of Tokyo,
2
Department of Medical Zoology, Nagoya City University, Medical School,3
Department of Biochemistry, Niigata College of Pharmacy,
4
Department of Bioengineering, Tokyo Institute of Technology.

African trypanosoma is a parasite, which causes African sleeping sickness of human and nagano disease of cattle.
Because at present drugs against trypanosomes have strong
side effect and are not effective for chronic patients, a neweffective drug is awaited now. We found that ascofuranone
isolated from Ascochyta visiae inhibits specifically mitochondrial respiration of blood stream form of Trypanosoma
brucei brucei at very low concentration. In addition, we
found that injection of ascofuranone into infected micemade blood stream form of T.brucei brucei die out.
It is said that the target of ascofuranone is cyanide resistant terminal oxidase located at mitochondrion of bloodstream form of T.b.brucei. This enzyme is referred to as
TAO (trypanosome alternative oxidase), and functions
ubiquinol oxidase unlike cytochrome c oxidase of mitochondrion of mammal. Because mammals don’t have TAO
that is essential for reoxidation of reducing equivalent generated during glycolysis, TAO is expected as a candidate oftarget for chemotherapy against trypanosomes. It is also reported that TAO is highly homologous to alternative oxidase of mitochondria of plants, but biochemical analysis has
not been carried out due to difficulty of purification. Therefore, we made overexpression of recombinant TAO (rTAO)
in E.coli for the purpose of characterization of TAO, andturning ascofuranone to clinical use.
We established the condition on which about 80 percent of membrane fraction from large-scale culture of E.coli
was rTAO. And, examination of solubilizing rTAO withvarious detergents revealed that digitonin is the most suitable detergent to solubilize rTAO. Digitonin-extaractedrTAO was purified by nickel column chromatography andenzyme assay of purified rTAO indicated that ascofuranone
inhibited ubiquinol oxidase activity of rTAO competitively
with ubiquinol, suggesting that aromatic part of ascofuranone is recognized by ubiquinol binding site.

B−2) DIHYDROOROTATE DEHYDROGENASE CATALYZING SOLUBLE FUMARATE REDUCTASE
IN TRYPANOSOMA CRUZIEIZO TAKASHIMA1, DANIEL KEN INAOKA1, TAKESHI NARA2, MASAO ODAKA2, TAKASHI AOKI2,
KOZI INAKA3, AND KIYOSHI KITA1
Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo1.
Department of Parasitology, school of Medicine, Juntendo University2.
Maruwa food industry Co.,Ltd..3

Succinate production resulting in reduction of fumarate is one of the essential processes controlling redox
homeostasis for many organisms living under anaerobic
conditions. However, Trypanosoma cruzi, a parasitic protozoa causing Chargas’disease, excretes a considerable
amount of succinate even they uses TCA cycle and aerobic
respiratory chain. Thus, it has been believed that unknown
metabolic pathways are involved in the succinate production of the parasite. Present study shows the first evidence
to characterize soluble fumarate reductase (FRD) of T.cruzi

at the molecular level, which has been considered to be a
key enzyme to understand the unknown pathways for the
succinate production.
We presented here that T.cruzi soluble methylviologen(MV) -FRD consists at least 2 kinds of protein. One of the
FRDs is attributable to dihydroorotate dehydrogenase(DHOD), catalyzing oxidation of dihydroorotate (DHO) toform orotate, a 4th enzyme of the pyrimidine de novo biosynthesis pathway. This indicates that not only T.cruzi
DHOD functions as a part of the pyrimidine de novo bio-

６８synthesis pathway but also the enzyme may play an important role in fumarate/succinate metabolism. Using recombinant T.cruzi DHOD that was expressed in E.coli, it wasdemonstrated that the DHOD is biochemically belonging tofamily 1A enzymes that is soluble, homodimeric protein

containing FMN. T.cruzi has unusual FRDs system in addition of aerobic respiratory chain. Further characterization
FRDs of T.cruzi would provide us the complete view of theparasitic metabolism.

B−3) COMPARATIVE ANALYSIS OF cDNA EXPRESSION PROFILES BETWEEN BLOODSTREAM
AND PROCYCLIC FORMS OF TRYPANOSOMA B.BRUCEI USINGFLUORESCENT DIFFERENTIAL DISPLY METHODOHSHIMA S., SUZUKI T., YABU Y., HATOH M., OHTA N.
Dept. Medical Zool., Nagoya City Univ. Med. School

There are 200,000-300,000 Africa narcolepsy patients
who are newly infected by Africa Trypanosoma every year.
In addition, many domestic animals die from Trypanosoma
infection. The Trypanosoma protozoa spread between the
mammal host and the tsetse fly vector in its lifecycle. In order to control Trypanosoma protozoa in the future, we analyzed geneexpression profiles between blood stream andprocyclic (insect) forms of Trypanosoma brucei brucei using the method of fluorescence differential display.
Briefly, total RNA of long slender, short stumpy andprocyclic forms were reverse-transcribed. PCR was performed with combinations of 10-mer arbitrary primer andrhodamine CT16A, G, and C primers. After electrophoresis,
differentially expressed cDNA bands were detected withfluorescent image analyzer.
As a result, about 10,000 cDNA (120 primer combinations) were surveyed, and a total of 39 differentially regulated cDNA bands were identified. One markedly differential expressed cDNA, which was upregulated in procyclic
form, was selected and sequenced. No high similarity was
found for this cDNA fragment, with Genbank databasesearch. Thus, 5’RACE is now under way, for cloning of the
full-length cDNA.

B−4) PRELIMINARY ANALYSIS OF GENES EXPRESSED DURING LEISHMANIAPARATITES INFECTION USING cDNA MICROARRAYHIROSHI UEZATO1, KIMIKO TAKEI1, 2, NOOR MOHAMMAD KHASKHELY1, MOTOYOSHI MARUNO1,SIGEO NONAKA1, MASATO UMIKAWA2, KEN-ICHI KARIYA2, MICHEAL COLLIER3, JAMES BRISTOW3,EDUARDO A. GOMEZ L4, MASATO FURUYA5, AND YOSHIHISA HASHIGUCHI6
Department of Dermatology and 2Department of Biochemistry II, Ryukyu University School of Medicine,3
Department of Pediatrics, University of California, San Francisco, USA,
4
Department Tropical Medicine, University of Guayaquil, Ecuador,
5
Institute of Laboratory Animal, Kochi Medical School, 6Department of Parasitology, Kochi Medical School
1

Purpose; The Leishmania is the causitive agent of leishmaniasis. It multiplies as the extracellular promastigotes andlives and replicates intracellularly in the mammalian macrophages. Macrophages play an important role agansit invasive microorganisms and viruses. It is very interesting why
Leishmania parasite inhabits in the phagolysomes. On theother hand, cDNA microarray allows the simultanous parallel expression analysis of many genes. We report analysis

data of gene expression during Leishmania parasites infection using cDNA microarray.
Materials and method: Total RNA were extracted frommacrophage derived Balb/c mouse (J774-A1) and Leishmania Leishmania major (MHOM/SU/73/5ASKH) infected
macrophages for 48 hrs culture. Total RNA derived
Leishmania-infected macrophages labeled Cy5 (red) andcontrol RNA labeled Cy3 (green). Two probe are combied,６９then hybridized at 64°
C to microarray slide. Raw scanning
image were processed using microarray image analysis software.
Results and discussion: Comparison between non-infected
macrophages and Leishmania-infected macrophages verified the involvement of many genes. About one hundred

genes were found to be up-regukated in Leishmaniainfected macrophage. Leishmania afected gene expressionof diverse range of cellular pathway and function, including
cell adhesion, cytokine, farnesylation, metabolism, RNA
handling, signaling, transcripition, transport factor and EST.

B−5) DEFECT OF LEISHMANIA ANTIGEN-SPECIFIC INTERFERON-G PRODUCTION IN A PATIENTWITH DIFFUSED CUTANEOUS LEISHMANIASIS IN ECUADOR
HAMANO S.1, MIMORI T.2, E. A. GOMEZ L.3, M. CALVOPINA H.4, MARUNO M.5, HIMENO K.1,
NONAKA S.5, HASHIGUCHI Y.6
1

Dept. Parasitol., Facul. Med. Sci., Kyushu Univ., 2Dept. Tumor Gen. Biol., Kumamoto Univ. Sch. Med.,
3
Dept. Trop. Med., Catholic Univ., 4Inst. Immunol., Quito Central Univ.,
5
Dept. Dermatol., Facul. Med., Univ. Ryukyus, 6Dept. Parasitol., Kochi Med. Sch.

Cutaneous leishmaniasis is clinically divided into classical localized and diffused types. Clinical manifestationsof diffused cutaneous leishmaniasis (DCL) are many nodules and papules on the whole body, but no ulcer. Those of
localized cutaneous leishmaniasis (LCL) are one or several
nodules, papules and ulcers, of which margin elevates likebank. DCL was actually not as rare as it was considered before. Many cases were recognized in the New world, especially in Brazil and Venezuela. DCL Patients shows humoral immunity against leishmania antigens, but not cellular immunity and delayed type hypersensitivity. To clarify
leishmanian antigen-specific immune response and pathological characteristic of a patient with diffused cutaneousleishmaniasis.
The study was performed in Ecuador. A DCL patientcaused by L. (Leishmania) mexicana, 6 patients with active
LCL, 6 patients healed from LCL, 6 healthy doners who
have been lived in urban area in Ecuador and were naïve to
Leishmania infection were examined for leishmanianantigens-specific immune response. PBMCs prepared fromabove people were cultured with or without leishmanian antigen, PPD and ConA for 72 hours, and culture supernatants
were collected and transferred to Japan. The concentrationsof IFN-g and IL-4 in the supernatants were measured by
ELISA.
PBMCs prepared from patients with active LCL andthose healed from LCL produced enough amount of IFN-g
in response to leishmanian antigen, PPD and ConA. Although PBMCs prepared from a patient with DCL also produced enough amount of IFN-g in response to PPD and
ConA, but not in response to leishmanian antigen. Six
healthy doners who have been lived in urban area in Ecuador also produced enough amount of IFN-g in response to
PPD and ConA, but not to leishmanian antigen. IL-4 was
not detected in any culture supernatants. Immune system ofa DCL patient was shown to be in anergy or deviation to Th
2 against leishmanian antigen.

７０B−6) CLINICAL SURVEY OF CUTANEOUS LEISHMANIASIS IN ECUADORLAST 10 YEARS (1991〜2000)
MOTOYOSHI MARUNO1, ATSUSHI HOSOKAWA1, HIROSHI UEZATO1, SHIGEO NONAKA1,GOMEZ E. A. L.2 YOSHIHISA HASHIGUCHI3.
Dept. of Dermatology, University of the Ryukyus1,
Dept. of Tropical Medicine, University of Catholic Saniago de Guayaquil, Ecuador2,
Dept. of Parasitology. Kochi medical School3.

We analyzed clinical and epidemiological data on cutaneous changes of leishmaniasis recorded from 1991 to 2000
in Ecuador. The study areas were included Provinces ofManabi, Los Rios, Bolivas, Pichincha, Azuay, and Esmeraldas located at the Pacific coast and the slope of Andean
mountains. A total of 570 cutaneous leishmaniasis patients,
324 males and 246 females, living in Ecuador were examined for this study. Each patient was thoroughly examinedclinically and parasitologically. The mean age of the patients was 19.82 years (±0.89 s.e.) in males, 17.45 years(±1.00s.e.) in females and 18.80 years (±0.67s.e.) in total.
Patients less than 20 year-old occupied more than half ofall the patients examined. The total mean onset of cutaneous lesions was 6.17 months (±0.41s.e.); 5.58 months (±0.39s.e.) in males, 6.96 months (±0.56s.e.) in females. 32
cases (6.5%) in total showed the duration period of lesions
more than 13 months. The popular types of lesions in-cluded ulcer, nodule, erythematous plaque and papule. The
most popular was ulcer formation. The patients with ulcer
occupied more than 50% of the total. More than half of thelesions were located on the face and the extremities. Almost half of the lesions were solitary. The cases with morethan ten multiple lesions were seen in a total of 16 patients.
Ultrastructural observations showed poor parasitophorous
vacuole (PV) formation in the skin specimens in this study.
It is needed to examine whethet PV formation should be related to the duration of the diseases. Cutaneous leishmaniasis in Ecuador may differ from the other areas because of
the defference of the parasite spesies, various behaviours ofthe vectors and the different life style of the inhabitants.
Therefore, medical doctors assigned to the treatments for
cutaneous leishmaniasis in Ecuador have not only medical
knowledge but also entomological, ecological, environmental and anthropological knowledge.

B−7) FLAGELLIN FROM BURKHOLDERIA PSEUDOMALLEI STIMULATES PULMONARY EPITHELIALCELLS TO INDUCE IL-8 PRODUCTION
M. SAITO 1, BENJAMAS INTRAOKA 1, K. OISHI 1, A. WADA 2, T. HIRAYAMA 2, T. NAGATAKE 1.
Dept of Int Med and Bacteriology, Inst of Trop Med, Nagasaki Univ.

The causative agent of melioidosis, Burkholderia pseudomallei (Bp) is one of the most important causes of mortality of septicemia and acute fulminant pneumonia in
Northeastern part of Thailand. Increased plasma levels of
IL-8 is associated with a high mortality in cases of septicemia, and is considered to be a better predictor of mortality.
In this study, we examined the activity of IL-8 induction bypulmonary epithelial cell line (A549 cells) in response to
the culture supernatant of B.p (CSBP), and determinedwhich components were responsible for IL-8 induction by
these cells.
We examined the IL-8 inducing capacity of CSBP ofseven clinical isolates, and found potent activities of IL-8
induction in response to CSBP from these strains. A time-and concentration-dependent IL-8 production by A549 cells
in response to CSBP (561 strain) was observed. Treatment
with proteases and heating of CSBP significantly decreased
IL-8 inducing activity. A gel filtration on chromatographyof the CSBP on a Superose 12 column demonstrated one
separate fractions showing IL-8 inducing activity. These
were estimated to have molecular masses higher than 450
kDa.
Bacterial flagellin has been reported to stimulate NFkb activation through toll-like receptor 5, which subsequently induces IL-8 production and other cytokines. We,
therefore, doveloped an immunoblot assay for condensed
filtrate of CSBP (MW cut off > 100 kDa) employing antiflagellin of B.p antibody. Anti-B.p flagellin recognaized７
１

the band MW of 43 kDa which corresponding to the known
MW of B.p flagellin. These data suggest that one of major
IL-8 inducing factors in CSBP strain is flagellin of B.p.

Flagellin of B.p may contribute to dense neutrophil accumulation in the airway of patients with pulmonary mellioidosis.

B−8) CLINICAL AND MICROBIOLOGICAL CHARACTERISTICS OF RESPIRATORY INFECTIONSAMONG HIV-INFECTED PATIENTS IN NORTHERN THAILAND
NORICHIKA ASOH1, HIROSI WATANABE1, SINOBU KOBAYASI1, KIWAO WATANABE1, KAZUNORI OISHI1,WEERAYUT KOSITSAKULCHAI2, TIPPAYA SANCHAI2, KHEMRASSAMEE KUNSUIKMENGRAI2,
SUMPUM KAHINTAPONG2, BANYONG KHANTAWA3, PRASIT THARAVICHITKUL3,
THIRA SIRISANTHANA3 AND TSUYOSI NAGATAKE1
1

Dept.of Internal Med., Inst. of Tropical Med., Nagasaki Univ., Japan,
2
Nakornping Hosp., Thailand,
3
Dept. of Microbiol., Faculty of Med., Chaing Mai Univ., Thailand

Background: Prevalence of HIV infection is increasing in
Thailand. However, clinical and microbiological characteristics of respiratory infections among such patients is notclear at present.
Methods: We prospectively analyzed the types and characteristics of respiratory infections in 160 HIV-infected patients (161 episodes, 112 males and 48 females, mean age
33.3 years) who were admitted to Nakornping Hospital innorthern Thailand.
Result: The mean peripheral blood CD4 lymphocyte count(CD4/CD8) in 105 patients was 60.0/mm3 (0.1). The mostcommon diagnoses included pneumonia in 82, pneumocystis carinii pneumonia-like interstitial pneumonia in 33, pulmonary mycobacteriosis in 28, pulmonary nocardiosis in 8

and lung abscess in 8. The most common organisms detectde in the sputum included Haemophilus influenzae (34),
Pseudomonas aeruginosa (9), Streptococcus pneumoniae(9), Staphylococcus aureus (8), Penicillium marneffei (7)and Rhodococcus equi (6). Twenty-three cases (14.3%)
had mixed infections. Severe fungal infection caused by
Penicillium marneffei in 15, septicemia in 11, lifethreatening meningitis in 4 (cryptococcal in 3 and tuberculous in 1) and were noted, resulting in 20 fatalities (12.4%).
Conclusions: Several types of organisms and respiratory infections exist in HIV-positive patients in northern Thailand.
In such cases, early diagnosis and treatment are important
for a satisfactory clinical outcome.

B−9) MYCOBATERIAL INFECTION IN THAI CHILDREN INFECTED WITH HUMANIMMUNODEFICIENCY VIRUS IN THAILANDTERUO KIRIKAE2, NARIS WARANAWAT1, VIPA TREERATWEERAPHONG1, ANURAK MUNSRICHOOM1,AKIO NAKAMURA2, YASUHITO TOKUMOTO2, FUMIKO KIRIAE2, NAMIKO MORI2,
MITSUO HONDA2 AND TAWEE CHOTPITAYASUNONDH1
Queen Sirikit National Institute of Child Health, Bangkok, Thailand1,
Department of Infectious Diseases and Tropical Medicine, Research Institute, International Medical Center of Japan, Tokyo, Jpan2,
Vaccine Research and Development Group, AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan3

More than ten per cent of chidren, who are born tomothers infected with human immunodeficiency virus-1(HIV), are still infected with HIV in Thailand. Half ofthese HIV-infected children contract serious respiratory infections, including tuberculosis (TB) and other common

bacterial infections. In the present study, we established amycobacteriology laboratory in a children hospital in Thailand, and carried out the following TB diagnosis examination for specimens from 105 chilren suspected with tuberculosis infection, including 14 HIV-infected children; acid-７
２

fast staining, OGAWA eggbased culture, the MIGT culture
system (Becton Dickinson) which monitor levels of oxgenby fluorescence, PCR diagnosis (Apmricore, Roch Scientific System) for M.tuberculosis, as well as M.avium and M.
intracellulare, and a strain-specific DNA-hybridazationbased diagnosis kit, DDH Mycobacteria (Kyokutoh). In 91
HIV-negative children, 7 specimens form 7 children among81 specimens was positive for M.tuberculosis. No nontuberculous mycobacterium was not detected in these specimens. In 16 HIV-infected children, among 16 specimens, 4

specimens from 2 children was positive for M.tuberculosis,
7 specimens from 5 children was positive for nontuberculous mycobacteria, such as M.gordonae, M.Szulgai, M.triviale, M.simia, M.chelonae, M.scrofulaceum and M.intracellulare. Three specimens from 2 patients was positive forboth M.tuberculosis and nontuberculous mycobacterium.
These results indicates that HIV-infected children were infected with M.tuberculosis as well as nontuberculous mycobacteria.

B−10) COLLABORATION OF MEDICAL TEAM AND FIELDEPIDEMIOLOGIST DURING
THE OUTBREAK OF EBOLA HEMORRHAGIC FEVER IN UGANDAEMIKO IWASAKI
Sendai Quarantine Station, Ministry of Health, Labor and Welfare

On October 16,2000,WHO confirmed that the outbreak of febrile disease in Gulu district of Uganda wascaused by Ebola hemorrhagic fever. WHO immediately
sent personnel and started necessary measures in collaboration with Ugandan Ministry of Health. At the same time,
the WHO requested international support, and the UnitedStates, France, Italy and Doctors Without Frontiers dispatched the personnel. Japan provided the financial support
in the initial phase. In addition, Japanese government decided to dispatch medical specialists and sent 2 doctors on
October 30.
The WHO established teams for the following 5 activities:
1. Field Epidemiology 2. Patient Treatment 3. Diagnostic
Laboratory 4. Public Education 5. Management and Support
In this region, the smallest social unit is a nucleus family, and these blood relatives gather and form a colony. The
chracteristic of this type of society is that groups of cosely
related are scattered in a community. In this society, infectious disease rapidly propagates in the community throughfamily and colony. Therefore, early identification and isola-tion of infected is established became primary activities.
The mobile team identified potential infection from contact
early by follow-up of infected, and they were also in chargeof education for prophylaxis of the disease and discovering
undetected cases through regional patrol.
In the previous outbreaks of Ebola viral hemorrhagic
fever, hospital infection and consequent reintroduction tocommunity became a problem. By practicing the barrier
nursing technique, the medical team prevent hospital infection.
With the collaboration of the mobile team and the patient treatment team, the outbreak of Ebola viral hemorrhagic fever in Gulu district of Uganda ended in February
2001.
In the outbreak such as Ebola hemorrhagic fever, thesocial structure affects transmission because of the “infection by contact”. Therefore, in controlling these diseases,
epidemiological considerations including social structure ofcommunity, habit and behavior have to be taken into account, and the collaboration between patient treatment andfield epidemiology is essential.

７３B−11) DETOXIFYING EFFECTS OF ACHYRANTHES JAPONICA EXTRACT AGAINST HABU VENOMKOREBUMI MINAKAMI1 AND TYOSEI JITOUZONO2
Kagoshima University School of Health Sciences1 and Jitouzono Clinic2

Achyranthes japonica (Inokozuchi) has been believed
as the herb to cure a venomous snake’s bite in Kagoshima,
Japan. We tried to examine biological effects of Inokozuchi
for confirming this ancient tradition.
Leaves from fresh Inokozuchi were cut into pieces andground into pastes. Pastes were filtered and filtrated liquid
was centrifuged at 5,000 rpm for 20 minutes and 5°
C. Supernatant was freeze-dried. Freeze-dried Inokozuchi powder was dissolved in 1/60 M PBS and added Trimeresurus(Habu) venom (Sigma). These were mixtured and left for
one hour in a room temperature. Five doses of Habu venom-Inokozuchi mixture were injected to mice and rabbits andcalculated anti-lethal (ED50) and anti-hemorrhagic reactions of Inokozuchi extract. Five doses of Habu venomboiled (one minute) Inokozuchi mixture were also injected
to rabbits and calculated anti-hemorrhagic reactions of
Inokozuchi extract. Tail pinch methtod was used to evaluate an analgesic level in mice administered Habu venom or
Habu venom-Inokozuchi mixture.

Survival rates showed that median lethal dose in themice administered Habu venom was 4.6 mg/kg of test Habu
venom (LD50) and that median lethal dose in the mice administered Habu venom-Inokozuchi mixture was 10.5 mg/
kg of test Habu venom (ED50). The ratio of LD50 to ED50
suggests that Inokozuchi reduced lethal toxicity of Habu
venom by half. Fifty micro grams of Inokozuchi decreased
hemorrhagic reaction due to Habu venom by 72.3%, although Habu venom-boiled Inokozuchi mixture could notdecreased hemorrhagic reaction due to Habu venom. Inokozuchi suppressed tail pinch response due to Habu venom
by 60%. Local necrosis could not be suppressed by Inokozuchi.
Therefore, Inokozuchi was proved to be antivenom of
Habu venom and that involved effective substances were
heat-unstable as a protein. Anti-hemorrhagic reactions of
Inokozuchi is presumed to be catabolism of toxic proteins
owing to enzymes of Inokozuchi.

B−12) THE USE OF TRAVEL VACCINES BY JAPANESE EXPATRIATESIN DEVELOPING COUNTRIESATSUO HAMADA, YUKA UJITA AND EIICHI OKUZAWA
Japan Overseas Health Administration Center

We conducted a survey to clarify the level of use of
travel vaccines by Japanese expatriates in developing countries. In 1999, we distributed a questionnaire to Japanese
adults (more than 3000) living in Asia (East, South East andSouth), the Middle East, Africa and Latin America. As a result, 2895 questionnaires were recovered and the data wereanalyzed.
The analysis showed that 1398 Japanese (48.3%) were
inoculated with some type of travel vaccines before departure. Compared with a similar survey conducted in 1994
and 1998, the ratio of inoculations (42.7% and 45.6%, respectively) was significantly higher (p < 0.05). Japanese in
Africa showed the highest percentage (90.0%), followed bySouth Asia (67.1%), Latin America (48.6%), South East
Asia (43.6%), the Middle East (37.0%) and East Asia
(34.5%). More than 20% in all areas were inoculated with

hepatitis A and B vaccines and tetanus toxoid. The ratios
were regionally high for rabies vaccine in Africa (44.1%)
and South Asia (39.2%), yellow fever vaccine in Africa(84.7%), and Japanese encephalitis vaccine in South Asia
(26.1%). In each area, the ratios of meningitis, typhoid fever and cholera vaccines were low. In tropical Africa, theratios of hepatitis A and B vaccines were around 20% in
1994, which increased to above 40% in 1999. However, theratio of cholera vaccine decreased from 40.7% in 1994 to
9.4% in 1999.
Recently, many Japanese expatriates have begun to use
travel vaccines bifore departure. However, more than half
still do not use vaccines at all. Therefore, it is vitally important to make Japanese expatriates more aware of the importance and effectiveness of travel vaccines.

７４B−13) SURVEY ON PATIENTS WITH IMPORTED INFECTIOUS DISEASES ADMITTED
TO THE DEPARTMENT OF INFECTIOUS DISEASESKENJI OHNISHI AND YASUYUKI KATO
Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital

Two hundred ninety two patients were admitted to the
Department of Infectious Diseases, Tokyo Metropolitan
Bokutoh General Hospital from January 1,2000 to December 31,2000, and 30 of them were infected with some infectious diseases in foreign countries. Fifteen were Japanesemen, 10 were Japanese women, and other 5 patients were
foreign people. The presumptive place of contraction was
India in 6 patients, Thailand in 4 patients, Philippines in 4
patients, Indonesia in 3 patients, Tanzania in 2 patients,
China in 1 patient, Bangladesh in 1 patient, New Caledonia
in 1 patient, Australia in 1 patient, Kenya in 1 patient, Nigeria in 1 patient, Uganda in 1 patient, South Africa in 1 patient, Mali or Senegal in 1 patient, Thailand or Nepal or In-

dia in 1 patient, and unknown in 1 patient. The number ofpatients of infectious enteritis was 13 (3 were bacillary dysentery, 2 were Campylobacter enteritis, 2 were enteropathogenic E. coli enteritis, 1 was paratyphoid fever, 1 was
Aeromonas enterits, and 4 were enteritis due to unknown
organism), malaria were 6 (3 were falciparum malaria, 1
was ovale malaria, 1 was falciparum malaria with ovale malaria, and 1 was falciparum malaria with malariae malaria),
dengue fever was 3, AIDS was 3, pneumonia was 2, pyeronephritis was 1, bronchitis was 1, and entero viral meningitis was 1. Twenty nine patients became to be well anddischarged, but 1 Ugandan man of AIDS was died.

B−14) CURRENT STATUS OF IMPORTED MALARIA IN JAPAN SINCE APRIL, 1999
HIROSHI OHTOMO1, MIKIO KIMURA2, SHIGEYUKI KANO3, TSUTOMU TAKEUCHI4,
MASASHI HAYANO1, MASAHIRO KUMAGAI1, NAOHIRO WATANABE1
Departoment of Tropical Medicine, Jikei University School of Medicine1,
National Institute of Infectious Diseases2, Research Institute, International Medical Center of Japan3,
Department of Tropical Medicine and Parasitology, Keio University School of Medicene4

Epidemiologic and therapeutic aspects of imported
malaria in Japan have been examined since 1980 by sending
questionnaires every year to more than 1,700 major hospitals.The new “Infectious Disease Prevention Law” cameinto force on April 1,1999. Imported malaria cases fromthat time till December 31,2000 were analyzed. 227 cases
were reported, including 110 vivax malaria cases, 91 falciparum malaria cases, 11 ovale malaria cases, 2 quartan malaria cases, 5 mixed infections cases, and 8 cases due to unknown species. The rate of relapse of vivax malaria is
12.4% and the rate of recrudescence of falciparum malariais 2.3%. Two fatal cases with falciparum malaria (2.3%)
were reported. 179 cases (78.9%) were Japanese, and 48
cases were foreigners. 175 cases (77.1%) were male, and52 cases were female. Concerning of possible place of contraction with vivax malaria, 28 cases (25.5%) were originated from Indonesia, 17 cases (15.5%) from India, 15
cases (13.6%) from Papua New Guinea. With falciparum
malaria, 14 cases (15.9%) were originated from Tanzania or

Kenya, 13 cases (14.8%) from Nigeria, 8 cases (9.1%) from
Ghana. The purposes of travel for Japanese malaria cases
were sightseeing (34.9%), working for NGOs (12.0%),
working as Japan Oversea Cooperation Volunteers (7.8%),
reporting (7.8%), and academic investigations (6.0%).
Concerning the drugs chosen for falciaprum malaria, mefloquine alone occupied 38.6% and combination therapies
occupied 45.5%. Concerning the drugs chosen for erythrocytic stages of Plasmodium vivax, chloroquine alone, quinine alone, Fansidar alone, and combination therapies occupied 67.6%, 12.0%, 8.3% and 6.5%, respectively. For prevention of relapse of vivax malaria, primaquine was used in
81.5% vivax malaria cases. These works were done by the
Research Group for Development of Chemothrapeutic
Agents against Tropical Parasitic Diseases (supported by
the Japanese Ministry of Health and Welfare) and the Research Group for Clinical Evaluation of Orphan Drugs
against Imported Tropical and Parasitic Diseases (supportedby the Human Science Promotion Foundation).

７５B−15) ANALYSIS OF MALARIA SURVEILLANCE DATA BASED ON INFECTIOUS DISEASEPREVENTION LAW IN JAPAN
KOKI KAKU1, HIROSHI TAKAHASHI2, TAKAAKI OHYAMA2, KEN OSAKA2,MIKIO KIMURA2, NOBUHIKO OKABE2
Field Epidemiology Training Program Japan, National Institute of Infectious Diseases1,
Infectious Disease Surveillance Center, National Institute of Infectious Diseases2

Background: Although indigenous malaria was eliminatedin Japan in 1962, over 100 imported malaria cases are reported among Japanese travelers to and foreign visitorsfrom endemic areas each year. According to the research of
the orphan drugs for tropical diseases, diagnosis and treatment are often delayed, and 110-130 malaria cases were reported each year and a total of 14 deaths were reported from1990-1999.
Methods: To identify the current problems and the publichealth importance of malaria in Japan and to describe therisk factors for malaria infection and fatal outcome, we analyzed data from the national malaria surveillance system for
April 1999 to March 2001. To estimate the number of Japanese travelers and foreign visitors, we reviewed the annual
statistical report of legal migrants.
Results: During the study period, 288 malaria cases werereported, 198 in Japanese and 90 in foreigners. Falciparum
malaria (n=116) accouted for 60.3% of the cases from SubSaharan Africa and 24.1% of the cases from South-East
Asia. Six patients (5 Japanese, 1 foreigner) with fatal cases
were diagnosed a mean of 11.8 days after onset of symptoms (range, 5-26) and 110 non-fatal cases a mean of 6.7

days (range, 0-90) (p=0.32). Additional deaths that may
have occurred after reporting are not included in the data,
because the system does not request such follow-up information.
In 2000, 7 million (39.4%) out of 17.8 million Japanese travelers went abroad to malaria endemic countries/territories. Among them, 36 malaria cases were reported for
Sub-Saharan Africa (174.0 cases/100,000 travelers) and 41
cases (1.5/100,000) for South-East Asia (relative risk,
114.5; p < 0.001).
Conclusions/Public Health Impact: Each year, over 100
imported malaria cases are reported among Japanese travelers. Japanese travelers, especially those visiting countries
with a high risk for falciparum malaria, need to be educatedabout malaria prevention and the importance of early treatment. Japanese physicians should ask patients about theirtravel history and consider malaria in febrile patients returning from malaria endemic countries to avoid delay in diagnosis. The surveillance system should be improved to include the prognosis of cases to enable calculation of thecase fatality rate and better assess risk factors for fatal outcomes.

B−16) HOW COULD THE ISTM’S MAILING LIST TRAVELMED BE UTILIZED?
MIKIO KIMURA1, NOBUHIKO OKABE1, YUKA UJITA2 AND DAVID O. FREEDMAN3
Infectious Disease Surveillance Center, National Institute of Infectious Diseases1,
Japan Overseas Health Administration Center2,
Travelers Health Clinic, University of Alabama at Birmingham3

With the ultimate goal of improving travelers’ health,the International Society of Travel Medicine (ISTM) utilizes multiple electronic communication modalities. In order to disseminate and share information, and to improvemutual understandings these means include 1) a web page,2) mass e-mail distribution through which important alerts
and advisories are unilaterally delivered to all 2000 ISTM
members, 3) TravelMed, a mailing list on which 450 voluntarily subscribed ISTM members from >30 countries arefree to propose and interactively discuss travel medicine issues of their own choosing, and 4) GeoSentinel, a surveillance network of 25 sites worldwide reporting on travelrelated illnesses and their geographic origins.
When we analyzed TravelMed postings from May toAugust 2001, the total number of issues discussed was 106,
composed of 81 infectious disease topics and 25 noninfectious diseases topics related to the health of travelers.
Among those infectious disease issues about half were on

７
６

vaccination and one fourth on malaria. The number of responses to each issue varied from zero to !21, with a peakat 2 responses.
Responses from host-country experts to inquiries about
the disease situation in that location or on the availability ofa given vaccine or on antimalarial in that country are extremely useful to subscribers. For some discussion topics,
contradictory responses are posted. Fortunately in thesecases, the respondents often present basic ideas, Personal
experiences or the literature from which their opinions arederived. However, more active participation by leading experts in travel medicine should be further encouraged.
Overall, TravelMed could be a very useful means of discus-

sion and is expected to facilitate improvement of travel
medicine practices.
We (M. K. & Y. U. ) have initiated the provision ofJapanese summaries of TravelMed postings. At the moment,these summaries are disseminated on a mailing list operated
by the Japan Overseas Health Administration Center inwhich 140 participants responsible for travelers’ health aretaking part. By recruiting additional coordinators, the entire
contents of TravelMed could be summarized with less burden on each individual. Moreover, we intend to provide
these summaries on an independent web page in the near
future in order to facilitate the easy availability of the TravelMed archives.

B−17) CEREBRAL METABOLIC CHANGES IN A PRIMATE MODEL OF SEVERE HUMAN
MALARIA AS STUDIED BY POSITRON EMISSION TOMOGRAPHYSUGIYAMA M.1, IKEDA E.1, KAWAI S.5, HIGUCHI T.2, ZHANG H.2, M. NASIM KHAN2, ICHIKAWA A.2,
INOUE T.2, YAMAGUCHI H.3, KATAKURA K.4, ENDO K2, SUZUKI M.4
1

Gunma Univ. Sch. Med., 2Dept. Nucl. Med., 3Dept. Parasitol., 4Sch. Health Sci.,
5
Dokkyo. Univ. Sch. Med. Dept. Trop. Med. Parasitol.

Plasmodium falciparum infection can cause a severe
neurological syndrome termed Cerebral Malaria (CM). CM
is seen mainly in children and nonimmune tourists in tropical countries where infection with Plasmodium falciparumis endemic. The neuropathological hallmark of CM had
been the sequestration of erythrocytes containing parasites
in cerebral capillaries and venules. However, detailed
mechanisms of altered consciousness during CM remains
controversial. Although there have been several studies on
hemodynamic changes in CM, direct measurements on the
cerebral metabolic rate of glucose (CMRglc) have not yet
been reported at all. In this study, local-CMRglu of Japanese monkeys (Macaca fuscata) infected with Plasmodium
coatneyi, a primate model of severe human malaria withcerebral involvement, were studied by using high-resolution
positron emission tomography (PET) using 2-[18F] fluoro2-deoxy-D-glucose ([18F] FDG) for the first time. FDG-

PET data for two cases suggest that CMRglc in frontal andtemporal cortex was significantly reduced, while those ofbasal ganglia do not change. CMRglu reduction in cerebral
cortex seem to be heterogeneous in severly infected monkey’s brain. Reduction of full brain metabolism agree wellwith the “luxually perfusion” as indicated by previously reported hemodynamic changes in human CM. Neuropathological study of the same monkeys as studies by FDGPET showed that preferential sequestration of parasitized
red blood cells appeared in the cerebral microvasculature.
However, petechial hemorrhage, focal endothelial cell damage or necrosis were not observed at all in the HE stained
brain tissues. This implies that the reduction of CMRglc
does not caused by damages in brain parenchyma. We
think that the CMRglc reduction in CM may have a protective role to local hypoxia secondary to mechanical blockageof vessels by sequestration.

７７B−18) GLUCOSE METABOLIC CHANGE OF SPLEEN IN MALARIA-INFECTED MACACA FUSCATA:CORRELATION OF FDG-PET RESULTS WITH PATHOLOGICAL FINDINGSEIJI IKEDA1, MUNEHIRO SUGIYAMA1, SATORU KAWAI2, TETSUYA HIGUCHI3, HONG ZHANG3,
M. NASIM KHAN3, AKIHIRO ICHIKAWA3, TOMIO INOUE3, HARUYASU YAMAGUCHI4, KEN KATAKURA1,
KEIGO ENDO3 AND MAMORU SUZUKI1
Department of Parasitology, Gunma University School of Medicine1,
Department of Tropical Medicine and parasitology, Dokkyo University School of Medicine2,
Department of Nuclear Medicine, Gunma University School of Medicine3,
Gunma University School of Health Science4

Severe falciparum malaria shows a wide variation in its
clinical presentation, such as hypoglycemia, cerebral malaria, renal failure, and pulmonary edema, in addition to theclassical features of spikes of fever, hemolytic anemia, andhepatosplenomegaly. These serious clinical symptoms may
precipitate various organs into alterations in glucose metabolism. Glucose metabolic changes in severe malaria,
however, have not yet been investigated.
In this study, we applied whole body high-resolution
positron emission tomography (PET) using 2-[18F] fluoro2-deoxy-D-glucose ([18F] FDG]) in order to detect the effect of malarial infection in glucose metabolism, using
Japanese monkey (Macaca fuscata) infected with Plasmodeum coatneyi, aprimate model of severe human malaria.
The PET scans were performed for three infected and threeuninfected animals. The standard uptake value (SUV) , a

semiquantitative index of tissue uptake of FDG, was produced for each case. We also examined pathologicalchanges of tissues of various organs.
The SUV of the spleen significantly increased after infection, up to twice to six times as much as that before infection. However, those of the liver, heart, and lung remained unchanged. Pathological examinations of thesplenic tissues revealed marked expansion of white pulps,
strong expression of MHC class II molecules on B cells anddendritic cells in lymphoid follicles, and enlargement ofmacrophages which had phagocytosed malarial pigments.
These pathological findings suggest the activation of the inflammatory cells in the spleen. The activation of these cells
probably caused the significant rise in splenic glucose metabolism.

B−19) CEREBRAL RING HEMORRHAGES IN SQUIRREL MONKEYS INFECTED
WITH PLASMODIUM FALCIPARUM
YUKO KATAKAI1, BASUKI SUKMAWATI1, SYOSAKU HATTORI2,
TAKASHI ONODERA1, YOSHITSUGU MATSUMOTO1
Department of Molecular Immunology, University of Tokyo1,
Amami Laboratory of Injurious Animals, Institute of Medical Science, University of Tokyo2

The severity of neurological symptoms in malaria patients might be correlated with cerebral pathologic featuresin the brain. Ring hemorrhages are frequently found in thebrains of patients with falciparum malaria. We report here
the squirrel monkeys infected with plasmodium falciparum
Indochina-I/CDC revealed cerebral ring hemorrhages similar to that observed in human malaria cases.
Bolivian squirrel monkeys (Saimiri sciureus boliviensis) were used in this study. A splenectomised monkey was
inoculated intravenously with cryopreserved parasitized red
blood cells (PRBC) infected with P. falciparum Indochina-I/CDC as a donor of fresh PRBC. On day 16, the blood wascollected and followed by removing plasma, white blood
cells and PRBC containing mature stage parasites. The
fresh erythrocytes include with 109 PRBC were inoculated
to spleen-intact monkeys. Peripheral blood was collected
and the parasite counts on Giemsa stained thin blood films
were monitored every day. Clinical signs were also observed daily. Neurological signs were assessed in accordance with “Blantyre coma scale” presented by world
health organization. Between 7 and 11 days after inoculation, autopsy or necropsy was done. Histopathological ex-

７８

amination was performed on their cerebrum and cerebellum.
PRBC were found on day 1 followed by abrupt development of parasite counts. Clinical signs included anorexia,
inactivity, convulsions and lethargy occurred about 1 week
after the inoculation. In several monkeys, coma scale
reached at < 3 as the state of unrousable coma. In grossfindings, petechiaes were seen on the cut surface of whitematter in the cerebrum and the cerebellum. In microscopic
findings, ball, ellipse or ring shaped hemorrhages were seenin the cerebral and cerebellal parenchyma. These hemorrhages were usually situated around the occluded vessels in
the white matter of the cerebral tissue just below the cortex.
In the cerebellum, hemorrhages were generally adjacent to
the granular layer within the white matter. They were 200〜500 µm size in the major axis. In ring hemorrhages, aperivascular necrotic zone separated the exravasated erythrocytes from blood vessels.
In summarry, bolivian squirrel monkeys inoculatedwith P. falciparum Indochina-I/CDC infected fresh erythrocytes caused cerebral disorder and hemorrhagic lesions
which is typical of human cerebral malaria. Due to difficulty of obtaining fresh tissues from patients, mechanism
for the occurrence and participation in the neurologicalsymptoms remain obscure. Understanding of the mechanisms would greatly benefit from the existence of a model
for cerebral malaria in an experimental host closely related
to man. Bolivian squirrel monkey might be a good model
for studying ring hemorrhages associated with human falciparum malaria.

B−20) IDENTIFICATION OF MALARIAl ANTIGEN-MIMICKING PEPTIDESDAI ISHIKAWA1, EIJI NAGAYASU2, YAMAJI NAKANO2, MAYUMI AKAKI2, SORNCHAI LOOAREESUWAN3,MASAMICHI AIKAWA2, AND TAKAO TAKI1
1

Molecular Medical Science Institute, Otsuka Pharmaceutical Co. Ltd.,
2
Total Science Technology Institute, Tokai University,
3
Faculty of Tropical Medicine, Mahidol University

Malaria antigen-mimicking peptides were selected bybiopanning using a pentadecamer random peptide displayed
phage library and anti-malarial anti-sera from 5 plasmodium falciparum-infected patients. Seventy-five positive
phage clones were selected by ELISA assay after 3rd roundbiopanning. From the sequence analysis, 8 positive phageclones were classified as patient IgG-binding peptides and
34 clones were classified as IgM-binding peptides. There
were several consensus alignments in IgG-and IgM-binding
peptides. Each consensus alignment-bearing phage clone
showed the same or similar reactivity pattern in their classes

when binding affinity of the 42 individual phage clone to 20
patient sera and 10 control sera were examined by ELISA,
suggesting the consensus alignments are critical for binding
to the antibodies. When mixed 4 represent peptides wereused for ELISA, the mixed peptides reacted with 40% and
10% of the patient sera from the uncomplicated malaria andthose from sever and complicated malaria, respectively.
Whether the relationships between symptoms and the reactivity are presence or whether the antibodies against these
peptides inhibit the malarial infection are under studying.

７９B−21) CONSTRUCTION OF ANTI-P. FALCIPARUM /ANTI-CD3 BISPECIFIC scFv
WITH ANTIPARASITICIDAL FUNCTIONYOSHIDA S., KOBAYASHI T., ARAI R., MATSUOKA H., ISHII A.
Dept. Med. Parasitol., Jichi Med. Sch.

A novel bispecific single-chain antibody fragmernt(biscFv) has been constructed to address the possibility of a
new malaria therapeutic drug development. The biscFv
consists of two different single-chain antibody fragments
linked by a flexible peptide linker (Gly4 - Ser) 3. One specificity of the two Fv fragments is directed against MSP-1
molecule on the surface of Plasmosium falciparum merozoites and the other is directed against the CD3 antigen ofhuman T cells; the former reacts with any strains of P. falciparum but doesn’t have the growth inhibition activity. The
construct was expressed in insect cells using recombinantbaculovirus system. The antigen-binding properties of the
biscFv are indistinguishable from those of the correspond-

ing univalent single-chain antibody fragments. In the presence of human peripheral blood mononuclear cells (PBMC),
the biscFv specifically induced IFN-γ and TNF-α in P. falciparum in vitro culture. These data suggest that the bridge
formation between malaria parasites and T cells induces cytokine production at high level near the parasites, presumably resulting in activating NK cells and macrophages andthen killing the parasites in vivo. Thus, the biscFv possesses highly selective malaria targeting properties and activates T cells simultaneously. Cell-mediated immune response induced by the biscFv could be great advantage forthe passive immunotherapy against malaria infection.

B−22) PLASMODIUM VIVAX TRANSMISSION-BLOCKING VACCINE:
EFFICACY STUDY ON HUMAN ISOLATESTAKAFUMI TSUBOI1, JETSUMON SATTABONGKOT2, HAJIME HISAEDA3,
MAYUMI TACHIBANA1, MOTOMI TORII1
Department of Molecular Parasitology, Ehime University School of Medicine, Japan1,
Department of Entomology, Armed Forces Research Institute of Medical Sciences, Thailand2,
Department of Parasitology and Immunology, University of Tokusima, School of Medicine, Japan3

Antibodies against Plasmodium vivax ookinete surface
proteins expressed in yeast (yPvs25 and yPvs28) were produced in mice. Theses sera elicit potent transmissionblocking activity to P. vivax Sall strain. To test the efficacyof this vaccine candidate in the natural parasite populations,antisera against yeast expressed recombinant proteins, yPvs
25 and yPvs28, were produced in mice and rabbits. The efficacies of the antisera were tested with human isolates atmalaria clinics in northwestern Thailand. Blood was collected from patients who came to the clinics and whose
blood smears was positive for P. vivax. Antibodies fromeither mice or rabbits were diluted with normal human serum and added to patients’ red blood cells. After mixing theinfected blood was fed to Anopheles dirus using membranefeeding method. Mouse serum immunized with PfMSP1-

19expressed in yeast or pre-immunized rabbit serum wasused as control for mosquito feeding. Mosquito infection
was observed on day 7 post blood feeding. Ten to twenty
mosquitoes were dissected for midguts and examined foroocyst development. Number of oocyst was recorded andcompared among different treatments. For most human isolates, sera from mice immunized using alum as adjuvant
showed complete inhibition of oocyst development. Sera
from rabbits immunized with yPvs25 or yPvs28 + alum wasless inhibitory than the mouse sera. Sera from rabbits immunized with yPvs25 or yPvs28 + Freund’s adjuvant wasmore inhibitory, but still less than the mouse sera. The inhibitory activity correlated with the antibody titer measured
by ELISA on recombinant protein.

８０B−23) AN ATTEMPT TO IDENTIFY GENES CONTROLLING SUSCEPTIBILITY
TO MOUSE SEVERE MALARIAEIJI NAGAYASU1, MAYUMI AKAKI1, YAMAJI NAKANO1, SATOSHI MAKINO2, GEN TAMIYA2,
KOICHI NAGAKURA3, MASAMICHI AIKAWA1
Researcn Institute of Science and Technology, Tokai University1,
Division of Molecular Life Science, Tokai University School of Medicine2,
Division of Infectious Diseases, Tokai University School of Medicine3

Plasmodium berghei ANKA (PbA) infection in the C
57BL/6 (B6) strain of mice leads to the development of experimental severe malaria (ESM) and kills almost all of theanimals 6-12 days after infection. In contrast, the DBA/2(D2) strain of mice shows strong resistance to ECM, a majority of animals survive from ECM and dies 20 to 30 days
after infection by other malaria-related complications, suchas severe anemia and hyperparasitemia. So far, little is
known about the genetic basis that accounts for the different
susceptibility to ECM. In order to localize such genes on
the mouse linkage map, we carried out a genome wide
screening using an F2 intercross population of susceptible(B6) and resistant (D2) strains.
Experimental infections were achieved by intraperito-

neal inoculation of 1 x 105 parasitized red blood cells. F2
animals were phenotyped either as ESM susceptible or as
ESM resistant based on survival until day 14 after infection.
Prior to the infection, genomic DNA were preparedfrom mice. A total of 93 informative microsatellite locialong all autosomes and X-chromosome were genotyped.
Marker-Trait associations on each marker were assessed bychi-square tests.
We identified a genetic region on mid-chromosome 18
that showed statistically significant linkage to ESM susceptibility. We will continue to make effort to identify a true
ESM susceptibility gene on this region by both positionaland functional approach.

B−24) BIOCHEMICAL ANALYSIS OF COMPLEXII FROM PLASMODIUM FALCIPARUM MITOCHONDRIAMI-ICHI FUMIKA1, TAKASHIMA EIZO1, TAKEO SATORU1, KOBAYASHI TAMAKI1,
TAKAMIYA SHINZABURO2, KITA KIYOSHI1
1

Grad. Sch. Med., Univ. Tokyo, 2Dept. Parasitol., Juntendo Univ.

Since the energy metabolism of Plasmodium falciparum is quite different from that of mammalian host, the enzymes of energy transducing pathway of the parasite arepromising targets of anti-malaria drugs. Energy requirement during the erythrocyte stage is provide by metabolizing glucose by anaerobic gycolysis. A functional TCA cycle is absent and ATP synthase has not been characterized inthis stage mitochondria because α-ketoglutarate dehydrogenase doesn’t function.
However, recent studies have shown the presence offunctional respiratory chain in Plasmodium mitochondria,
and the new anti-malarial drug, atovaquone, has beenshown to inhibit the electron transfer of the bc1 complex(complex III) and to collapse the mitochondrial membranepotential. The presence of an NADH-ubiquinone oxidoreductase (complex I) has been suggested by ability of NADH
-linked substrates, such as glutamate, to stimulate ADP

phosphorylation in trophozoites treated with digitonin. Evidently, mitochondrial electron transport system is essential
for the survival of plasmodium in the host, although ATP is
supplied mainly by glycolysis.
Complex II is one of the respiratory components and is
the oxidization-reduction enzyme, which catalyzes conversion of succinate and fumarate. Complex II plays a key rolein the energy metabolism of parasites such as Ascaris suum.
The mitochondrial respiratory chain of A. suum changes
dramatically during the life cycle. Complex II from the mitochondria of adult A. suum exhibits high quinol-fumarate
reductase (QFR) activity and plays a key role in anaerobic
metabolism as a terminal oxidase in the NADH-fumarate
oxidoreductase system. In contrast, complex II from freeliving third stage 1arvae (L3) shows much lower FRD activity, and functions as a succinate dehydrogenase (SDH) during aerobic respiration. If it considers that malaria parasite

８
１

growths under low oxygen, also in P. falciparum, it is likelythat complex II functions as QFR and plays an importantrole in an energy metabolism system. Saraveratum et al .
have purified complex II of P. falciparum, and we havecloned the genes of enzyme for the catalytic subunits,
SDHA for flavoprotein (Fp) and SDHB for the iron-sulfur
protein (Ip).
In the present study, using N2 cavitation, we established a protocol to prepare the active mitochondria fromplasmodium falciparum.
This mitochondrial fraction
showed SDH and SQR (Succinate-ubiquinone oxidoreductase) activity of complex II. The specific activity of SDH
and SQR (5.64 nmol /min/mg and 4.74 nmol /min/mg, respectively, at 25°C) was higher than that of the homogenate

from the saponin-treated P. falciparum (approx. 1 nmol /min/mg, at 37°C) reported by Suraveratum et al .. The malarial enzyme activity was inhibited by competitive inhibitor
malonate, but a known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone (1mM), had no inhibitory effect on the malarial SDH activity. The chalcone
showing antimalarial activity against the in vitro growth of
P. falciparum had no inhibitory effect on the malarial SDH
activity, although rat mitochondria was inhibited by Licochalcone A which inhibits the activity of fumarate reductase(FRD) of Leishmania major promastigote. These results
suggest property of malarial complex II is different fromthat of the host mammalian enzyme. Thus. It could be an
excellent target for antiprotozoal drugs.

B−25) MOLECULAR CHARACTERIZATION OF A1-CYS PEROXIREDOXIN FROM THE HUMAN
MALARIA PARASITE PLASMODIUM FALCIPARUM
SHIN-ICHIRO KAWAZU1, KANAKO KOMAKI1, SATORU KAWAI2, NOZOMU IKENOUE3,HIROYUKI ISHIKAWA1 AND SHIGEYUKI KANO1
Research Institute, International Medical Center of Japan1,
Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine2,
Gunma University School of Health Sciences3

The human malaria parasite Plasmodium falciparum is
exposed to reactive oxygen species that are generated fromits endogenous metabolism (e. g. haemoglobin digestion)and during external attack by the host immune system. In
addition, several anti-malarial drugs (e. g. chloroquine andartemisinin) can burden the parasite with substantial oxidative stress as a part of their anti-malarial effects. Peroxiredoxins are recently described family of antioxidants that are
ubiquitous in living cells for metabolizing hydrogen peroxide and hydroxy1 radicals. We report here the molecularcloning and characterization of a 1-Cys peroxiredoxin fromP. falciparum. Approximately 250 bp of the 5’ region ofperoxiredoxin cDNA was obtained from the P. falciparum
expressed sequence tags (ESTs). The missing 3’ region of

the cDNA was amplified from the trophozoite mRNA by
rapid amplification of cDNA ends (RACE) technique. The
full-length cDNA of the parasite’s peroxiredoxin (PfPrx-2)
encoded a 220 amino acid polypeptide with a predicted molecular mass of 25.2 kDa. The presence of the PVCT consensus sequence flanking the conserved Cys47 confirmedthat the PfPrx-2 was the 1-Cysperoxiredoxin. This is thefirst report of 1-Cys peroxiredoxin in protozoan parasites.
The antioxidant activity of eliminating hydrogen peroxideby the recombinant PfPrx-2 protein was demonstrated using
the ferrithiocyanate system. An elevated expression of the
PfPrx-2 protein seen in cytoplasm of the trophozoites, thestage with active metabolism, suggests an association of theperoxiredoxin with the parasite’s intracellular redox control.

８２B−26) EFFECT OF MINOCYCLINE IN COMBINATION WITH ANTIMALARIA DRUG
ON THE ELIMINATION OF PLASMODIUM BERGHEIAND CHLOROQUINE RESISTANT-P. CHABAUDI PARASITES IN MICEKIYOMI HANDO1. 2, QUING HUA LIN1, KEN KATAKURA1 AND MAMORU SUZUKI1
Department of Parasitology, Gunma University School of Medicine1,
Department of environmental health and laboratory sciences, Gunma University Graduate School of Health Sciences2

The emergence and spread of drug resistant plasmodium falciparum is a major problem for chemotherapy ofmalaria. Development of potent anitimalarial is important,but it is also necessary to investigate the possible use of antimicrobial agents with wide spectrum effect, which havealready been accepted for clinical use in patients with various infectious diseases. A group of tetracyclines has beenused for the treatment of drug-resistant falciparum malaria.
Tetracylines were usually combined with qunine since these
antibiotics require a longer time for an appearance of the inhibitory effect than other antimalarial drugs.
We previously reported that minocycoline (MC)showed more strong inhibitory activity against in vitro
growth of drug-resistant P. falciparum parasites than conventional tetracycline and doxycycline. In the present study,
we examined whether MC is useful for treatment of murine
malaria when it is combined with artemether (AM) or chloroquien (CQ). Random bred ICR mice were intraperitoneally inoculated with 1×105 erythrocytes infected with P.
berghei NK65 strain and the parasitemia was followed.

Mice treated with MC alone at a does of up to 50 mg/kg for
7 days or AM alone at a dose of up to 2.5 mg/kg for 4 days
were all dead. However, when mice were treated with MC(50 mg/kg) for 7 days in combination with AM (2.5 mg/kg)
for 4 days, all mice were cured. In the next experiment,
mice were intraperitoneally inoculated with 1×106 erythrocytes infected with P. chabaudi 3CQR strain, with showed
CQ-resistant phenotype. Mice received CQ at a dose of 5
mg/kg for 4 days exhibited a similar level of parasitemia
compared with that observed in untreated control mice, although an appearance of parasitemia was delayed in thetreated mice. However, when mice were treated with CQ(5 mg/kg) for 4 days in combination with MC (50 mg/kg)
for 7 day, parasites in the peripheral blood was completely
eliminated. These findings suggest that minocycline should
be further studied in clinical cases as an excellent candidate
for chemotherapy of chloroquine-resistant malaria in combination combined with other antimalarials such as artemether and even with chloroquine.

B−27) ANTI-MALARIAL EFFECTS OF DIPYRIDAMOLE in vitro and in vivo
YAMAJI NAKANO1, EIJI NAGAYASU1, MAYUMI AKAKI1, SHIN-ICHIRO KAWAZU2,SHIGEYUKI KANO2, MASAYOSHI ITOH3, MASAMICHI AKIKAWA1
Research Institute of Science and Technology, Tokai Univ1, Research Institute, International Medical Center of Japan2,
The Foundation of Research Institute for Production Development3

We examined anti-malarial effects of dipyridamole(DIP), a coronary vasodilator, in vitro and in vivo. Plasmodium falciparum Indochina 1 (IC-1), a chloroquine (CQ)sensitive strain, and K-1, a multi-drug-resistant strain weregrown in the presence of DIP or CQ. At 72hr culture time,
DIP reduced 50% of parasitemias in IC-1 and K-1 culturesat concentrations of about 50 and 70 µM, respectively. In
both cultures of IC-1 and K-1, CQ reduced the parasitemiasin the presence of 10 or 50 µM DIP more effectively than in
the absence of DIP. Parasitemias in IC-1 and K-1 cultures
incubated with 500 µM DIP treated red blood cells (RBCs)

were lower than those in cultures with DIP-non treated
RBCs, suggesting that parasitemias decreased as the resultof DIP binding to the RBC membrane. We examined effects of DIP on survival rates of C57BL/6N mice infectedwith Plasmodium berghei ANKA. Among ten infected
mice, eight mice died at 10th day after infection, whereas
two mice died among the infected mice administrated with
DIP (1 mg/day/mouse) for three days via the tail vein.
These results suggested that DIP had anti-malarial effects
both in vitro and in vivo.

８３B−28) DEVELOPMENT OF NEW ANTIMALARIAL DRUG-IN VITRO AND IN VIVO
ANTIMALARIAL ACTIVITY OF ENDOPEROXIDESKANAKO ONO1, NAOKI OGURA1, HYE-SOOK KIM1, KHURSHIDA BEGUM1, YOHEI OKUDA1,
KEN-ICHI SUZUKI1, KOHEI YOSHITANI1, YOSHIAKI HAMADA2, YUJI NONAMI2, TAKAHIRO TOKUYASU2,ARAKI MASUYAMA2, MASATOMO NOJIMA2 AND YUSUKE WATAYA1
Faculty of Pharmaceutical Sciences, Okayama University1, Faculty of Engineerings, Osaka University2

A number of medicines such as chloroquine and quinine are available for treatment of malaria, but the rapid developments of drug resistance are serious problems. Medicinal agents based on novel mechanisms of action are,therefore, required to overcome emergence of resistanceand to control an ever-increasing number of epidemics
caused by the malaria parasite.
We have confirmed the potential of 1, 2, 4, 5tetraoxacycloalkanes as a new class of simple peroxide antimalarial drugs. In the preliminary study, some 1, 2, 4, 5teraoxacycloalkanes have been found to be available forchloroquine-sensitive and -resistant P. falciparum in vitro.
Especially, 1, 2, 6, 7-tetraoxaspiro [7.11] nonadecane (N89) has a potent antimalarial activity for P. falciparum that
the IC50 value for FCR-3 strain is 2.5x 10-8 M, that for K1
strain is 2.6x 10-8 M and the selective toxicity (>300) is also
remarkable. In addition, we investigated the in vivo antimalarial activity of N-89 by 4-day suppressive test. The ED50
value of N-89 has shown 12 mg/kg/day (ip), which was required to cause 50% suppression of P. berghei in mice. In
the experiments, the dose of 50 mg/kg/day of five of N-89
treated mice were all cured, in which malaria parasites were
not observed in circulating blood streams after 60 days.

Furthermore, no side effects such as diarrhea, body weightloss and mortality were observed during treatment with1,600 mg/kg of N-89 (ip). As a control, mice treated withartemisinin (50 mg/kg/day, ip) were not cured and micedied due to P. berghei infection.
We evaluated the effect of oral treatments of N-89 (160mg/kg) for three consecutive days beginning on the day of
1% parasitemia. Parasitemia declined have been found after
24 hours of drug administration, all mice were cured withno parasite reincrease or toxicity. Conversely, on similar
treatment with artemisinin, malaria parasites were still observed in their blood streams, parasitemia become increased
again. Our studies indicate that N-89 has a strong and continuous antimalarial activity.
To determine the mechanism of action, morphologicalchanges of malaria parasites during the treatment of N-89
against P. falciparum were studied. After treatment with
100 times the EC50 value of N-89, we have observed a condensation of parasite nuclei, shrinked cytosol, inhibition ofearly and middle trophozoite parasite and little hemozoin
formation.
Our results suggest that endoperoxide, N-89, is a new
antimalarial candidate against drug resistant malaria.

B−29) A PUBLIC/PRIVATE PARTNERSHIP AMONG THE JAPANESE PHARMACEUTICAL INDUSTRY,
THE GOVERNMENT AND WHO/TDR (JPMW) :A RESEARCH PROJECT FOR DISCOVERY OF NEW ANTIMALARIAL DRUGS IN JAPANKAZUHIKO HATA1, KAZUHIKO OTOGURO2, HARUKI YAMADA3 AND SATOSHI ŌMURA3
JPMW Coordination Center1, Research. Center for Tropical Diseases, Kitasato Institute2,
Kitasato Institute for Life Sciences, Kitasato University3

The JPMW project was established in October 1999
through a “public/private parthership” (PPP) among theJapanese Pharmaceutical Companies, Ministry of Health,Labour and Welfare, Japan (MHLW), and WHO/TDR, inorder to discover new type of antimalarial drugs in Japan by
the Japanese efforts. This project is expected to run for five
years. Research Center for Tropical Diseases, The Kitasato

Institute, is identified as the screening center for the project
under WHO contract as the Kitasato Screening Center(KSC). KSC is carrying out the evaluation of antimalarial
activity for the compounds from the chemical libraries of 14
Japanese companies, along with some of its own natural
products, both in vitro and in vivo, using the multi-drug resistant strain of Plasmodium falciparum and the rodent ma-

８４laria model, respectively. In many ways the JPMW project
was nurtured in the context of such international initiatives
as the Roll Back Malaria Initiative that was proposed by
WHO in 1998 and the Hashimoto Initiative that was proposed by the Government of Japan in the same year.
The financial support to the KSC screening operations
is granted from MHLW through WHO/TDR technical service agreements (Principal investigator: Ōmura, S. ) An entity called JPMW Coordination Center (JCC) is playing a
key interface role as “the hub” in this project. By 31 May
2001, KSC has assayed 9,212 samples as in vitro screening.
The results have been registered into WHO/TDR database,and individual companies were notified. Furthermore, KSC
has assayed 32 compounds as in vivo screening by 31 May
2001. Out of these the KSC results showed 3 compounds

that had an activity score 3 in vivo.
In this presentation, we report the background, systems
and current status of this JPMW project. There is not manysuccessful cases of public/private partnerships in the world.
The fact that this project has been strategically supportedby the International Affairs Division of MHLW and blessedby the Japan Pharmaceutical Manufacturers Association(JPMA) would mean that JPMW was recognized as auniquely Japanese contribution to a major international
technical cooperation scheme. Further, we think that it ispossible for the development of the new anitimalarial drugs
in Japan, going beyond the screening project. The Medicines for Malaria Venture (MMV) is taking an active note of
the JPMW outcome.

B−30) BOTH MOSQUITO-DERIVED XANTHURENIC ACID AND A HOST BLOOD-DERIVED FACTORREGULATE GAMETOGENESIS OF PLASMODIUM IN THE MIDGUT OF THE MOSQUITOMEIJI ARAI1, 2, OLIVER BILLKER2, 3, HOWARD R. MORRIS4, MARIA PANICO4, MELAINE DELCROIX4,DARREN DIXON5, STEVEN V. LEY5 AND ROBERT E. SINDEN2
Department of Medical Zoology, Jichi Medical School1,
Department of Biology and Department of Biochemistry4, Imperial College of Science, Technology and Medicine, London, UK,
Abteilung Molekulare Biologie, Max-Planck-Institut fur Infektionsbiologie, Berlin, Germany3,
Department of Chemistry, University of Cambridge, Cambridge, UK5
2

Gametogenesis of Plasmodium in vitro can be inducedby the combined stimulus of a 5°
C fall in temperature andthe presence of xanthurenic acid (XA). In vitro experiments showed that P. gallinaceum (EC50 = 80nM) is much
more sensitive to XA than P. berghei (9 µM), P. yoelii (8µM), and P. falciparum (2 µM). However, in the mosquito
vector we do not know whether the temperature shift and
XA are the only gametocyte activating factors (GAF), nor
do we know with certainty the true source (s) of XA in themosquito blood meal. Previous studies indicate that XA is
the only source of GAF in the mosquito. By defining, andthen contrasting, the ability of an XA-deficient mutant ofAedes aegypti, with the wild type mosquito to support exflagellation and ookinete formation in vivo, we determined

the roles of parasite-, mosquito- and host blood-derived
GAF in the regulation of gametogenesis of P. gallinaceum.
Removal of both host and vector sources of GAF totally inhibited both exflagellation and ookinete production, whilst
the lack of either single source resulted in only a partial reduction of exflagellation and ookinete formation in themosquito gut. Both sources can be effectively replaced/substituted by synthetic XA. This suggests 1) both mosquitoand vertebrate-derived factors act as GAF in the mosquito
gut in vivo; 2) the parasite itself is unable to produce any
significant GAF activity. Studies are underway to determine whether vertebrate-derived GAF is XA. This data
may form the basis of further studies of the development of
new methods of interrupting malarial transmission.

８５B−31) STUDIES ON MALARIA MICRO-GAMETOCYTE EXFLAGELLATION INDUCING FACTOR
IN THE SALIVARY GLAND OF ANOPHELES STEPHENSIHIROYUKI MATSUOKA, MAKOTO HIRAI, WANG JICHUN, SHIGETO YOSHIDA, AKIRA ISHII
Department of Medical Zoology, Jichi Medical School

During blood feeding, the salivary glands of Anopheles
stephensi extremely reduces protein contents. We investigated midgut contents and prediuresis fluid of An. stephensi.
In the midgut contents after full blood meal, we foundsome proteins reacted with rabbit anti-salivary gland serum,
which did not react with the midgut contents of mosquitoes
before feeding. This suggests that these proteins come fromthe salivary glands. After feeding we also found sporozoites in prediuresis fluid of An. stephensi, which had been infected with plasmodium berghei. These sporozoites may
come from the salivary glands, because the density ofsporozoites in prediuresis fluid correlated with the numberof sporozoites in the salivary glands. These results indicatethat An. stephensi mosquitoes draw back their own saliva
into their midguts when they feed blood (Med Entomol
Zool 51: 13-20, 2000).
We next found gamete activation factor (GAF), which in-

duced exflagellation of Plasmodium microgametes, in thesalivary glands of An. stephensi. The exflagellation was induced in a concentration-dependent manner in the supernatant of salivary gland’s crude homogenate. Total amount of
GAF in the salivary gland was higher than that in the midgut and the head. GAF in the salivary glands was heat stable and low molecule (<3,000 molecular weight). Analysisof the supernatant by capillary electrophoresis and UV absorbance profile showed that the salivary glands contained
xanthurenic acid, which was previously identified as GAF
in the head of An. stephensi. The exflagellation inducing
activity in the salivary gland declined immediately after ablood meal, implying that GAF was in the saliva, and wasdelivered into the midgut together with the blood and induced exflagellation in the midgut (Biochem Biophys Res
Commun 287: 859-864, 2001).

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
０巻

第１号
内

Field evaluation of a rapid immunochromatographic test ‘Parachek-F’ in a post-monsoon
Plasmodium falciparum malaria outbreak in villages of South India
SUSANTA KUMAR GHOSH, TIRUCHINAPALLI SUNDARAJ SATHYANARAYAN,
１
３
MALALIKOPPA VEERABHADRAPPA MURUGENDRAPPA and SARALA KUMARI SUBBARAO …………７‐
旋毛虫のミオシンヘビー鎖の抗原性ペプタイド
５‐２１
中田 琢巳，長野
功， ! 志良，高橋 優三 …………………………………………………１

第４
２回日本熱帯医学会大会英文抄録 …………………………………………………………………………２
９‐
８
５

March２００２

日本熱帯医学会表紙!−" ＭＫ 中島 稲Original article
Ishih, A., Sakai, M., Takezono, H., Fujii, K., Sano, M., Asanuma, N., Miyase, T., and Terada, M.
A Potent Antimalarial Activity of Hot-Water Extrects of Plants Belonging to the Family
Saxifragaceae Against Plasmodiumyoeii 17XL in Icr Mice…………………………………………１‐６
Ghosh, S. K., Sathyanarayan, T. S., Murugendrappa, M. V. and Subbarao, S. K.
Field-Evaluation of a Rapid Immunochromatographic Test ‘Parachek-F’ in a Post-Monsoon
Plasmodium Falciparum Malaria Outbreak in Villages of South India
Indigenous diagnostic test kit for P. falciparum ……………………………………………………７‐１３
Nakada, T., Nagano, I., Wu, Z. and Takahashi Y.
An Antigenic Peptide of Myosin Heavy Chain-Like Protein from Trichinella Spiralis ……………１５‐２１
Mitarai, S., Habeenzu, C., Lubasi, D., Kafwabulula, L. M., Kasolo, F. C., Ichiyama, K.,
Terunuma, H., Ito, M., Shishido, H. and Numazaki, Y.
Drug Susceptibilities and Clinical Manifestations of Mycobacterium Tuberculosis in Zambia ……２３‐２８Proceeding of 42st Annual Meetings of Japanese Society of Tropical Medicine………………………………………………………………………………………………………２９‐８５Vol. 30 No. 1

日 本 熱 帯 医 学 会

Hygiene

日熱医会誌

and

会報・記録
日本熱帯医学会会則 ………………………………………………………………………………………８
７
‐
９
０
２
０
０
１（平成１
３）年度日本熱帯医学会役員名簿（２００
２年３月１日現在） ……………………………９１
日本熱帯医学会雑誌編集委員名簿 ………………………………………………………………………９２‐
９３
投稿規定 ……………………………………………………………………………………………………９４‐
９５
著作権複写に関する注意 …………………………………………………………………………………９６
日本医学会への加盟申請についての公示 ………………………………………………………………９
７
日本医学会だより ……………………………………………………………………………………………９
８

Medicine

ザンビア共和国における結核菌の薬剤感受性と臨床病態
御手洗 聡，Charity Habeenzu, David Lubasi, Lindon M Kafwabulula, Francis C Kasolo,
市山 浩二，照沼裕，伊藤 正彦，宍戸 春美，沼崎 義夫 ……………………………２３‐
２
８

Journal of Tropical

著
ユキノシタ科に属するアジサイ近縁植物の水煮沸抽出液の抗マラリア活性
石井明，酒井美香子，竹添 裕高，藤井香，佐野 真規，浅沼 賀洋，
宮瀬 敏男，寺田
護 ……………………………………………………………………………１‐６

MARCH ２０
０
２

CONTENTS

容
Japanese

原

VOL．
３
０ No．
１

平成１
４年３月

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は４mm