Vol.33
No.01
MARCH 2005

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.33 No.01 MARCH 2005

ISSN 1348-8945【CONTENTS】
Original articles
Detection of Giardia cysts in sewage and estimations of giardiasis prevalence among
inhabitants in Hyogo Prefecture, Japan
Oda, T., Kawabata, M. and Uga, S.

1

Epidemiological study on Certrocestus armatus metacercariae in the Chikusa River,
Hyogo Prefecture, Japan
Kimura, D. and Uga, S.

7

Reviews
Review and future perspectives of “development study” in the health sectors
Kobayakawa, T., Nakano, H., Hozumi D., Akashi, H.,
Minamoto, Y., Miyazaki, K. and Ashida, T.

13

Proceedings of the 45th Annual Meeting of Japanese Society of Tropical Medicine

19

Vol.33 No.01 MARCH 2005

Tropical Medicine
and HealthJAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 平島 稲

背は４mm
（決）

Vol.33
No.01
MARCH 2005

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.33 No.01 MARCH 2005

ISSN 1348-8945【CONTENTS】
Original articles
Detection of Giardia cysts in sewage and estimations of giardiasis prevalence among
inhabitants in Hyogo Prefecture, Japan
Oda, T., Kawabata, M. and Uga, S.

1

Epidemiological study on Certrocestus armatus metacercariae in the Chikusa River,
Hyogo Prefecture, Japan
Kimura, D. and Uga, S.

7

Reviews
Review and future perspectives of “development study” in the health sectors
Kobayakawa, T., Nakano, H., Hozumi D., Akashi, H.,
Minamoto, Y., Miyazaki, K. and Ashida, T.

13

Proceedings of the 45th Annual Meeting of Japanese Society of Tropical Medicine

19

Vol.33 No.01 MARCH 2005

Tropical Medicine
and HealthJAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 平島 稲

背は４mm
（決）

Tropical Medicine and Health Vol. 33 No. 1, 2005, pp. 1-5
Copyright! 2005 by The Japanese Society of Tropical Medicine

１

Detection of Giardia cysts in sewage and estimations of giardiasis
prevalence among inhabitants in Hyogo Prefecture, JapanTAKUYA ODA†, MASATO KAWABATA†, and SHOJI UGA‡
Accepted 11, January, 2005

Abstract: We recovered Giardia cysts from sewage and estimated the prevalence of giardiasis among inhabitantsin Hyogo Prefecture, western Japan. Two 200-ml sewage samples were collected once a month from two different
sewage plants (A and B) during a one-year period. After pretreating the samples by filtration and centrifugation in
the presence of alcohol and diethyl ether, an immunomagnetic separation method was applied to isolate Giardia
cysts. The mean number of cysts recovered from plants A and B were 880/L and 2,000/L, respectively. Circadianrhythms in the number of cysts detected on each of two examination days were observed with a peak at 10:00 a.m.,and seasonal variations in cyst numbers were also observed with two peaks in May and December. By using thedata obtained from this survey, we estimated that the prevalence of Giardia infection in habitants in the areas serviced by plants A and B were 0.1-1.1 and 0.1-1.3%, respectively.
Key Words: Giardia intestinalis, sewage, detection method, prevalence

INTRODUCTION
The flagellate protozoan parasite Giardia intestinalis
is widely distributed throughout the world with a highprevalence in developing countries in the tropics and subtropics. G. intestinalis infection is acquired by digestingcysts excreted from infected humans and animals [1]. Approximately 20 to 30% of inhabitants in developing countries in Asia and Africa are infected with this parasite. In developed countries with improved hygiene conditions, meanwhile, Giardia infection also constitutes a public healthconcern [2]. In developed countries, the parasite is broughtback from abroad by infected travelers [3]. Many outbreaksof giardiasis due to ingestion of inadequately treated water
have also occurred in developed countries; five of these occurred in the United States in 1993 and 1994 [4]. Althougha direct smear method is frequently used to detect Giardia
cysts in stool samples, it does not consistently reflect the accurate prevalence of giardiasis in a given survey area. For
instance, 15 to 20% of patients are reportedly diagnosed as
false negative [5], despite the use of appropriate fecal examinations. The need for examination of three consecutive
stool samples has therefore been indicated for accurate diagnosis [6,7].
The reported prevalence of giardiasis in the metropolis
and Tokai regions of Japan ranges from 0.2-0.9% [8,9].
†

However, the study populations consisted of outpatients and/or inpatients who might not have represented the total
population in the area. Furthermore, it is time consuming
and expensive to examine thousands of individuals, and it isdifficult to obtain consent for stool surveys.
Sykora et al. examined 11 sewage plants at different
cities in the USA and detected 642 to 3,375 Giardia cysts
per liter of samples [10], and Jakubowski et al. used thesedata to clarify the relationship between cyst number andprevalence in the respective cities [11]. Although these studies are interesting from the viewpoint of simplicity and/or
economy, they do not discuss the recovery efficiency of
their method or the circadian rhythm of cyst numbers. In
this study, we recovered Giardia cysts from sewage samples using the immunomagnetic separation method at a
fixed time each month and used the results to estimate theprevalence of the intake of Giardia infection among inhabitants in Hyogo Prefecture, Japan.

MATERIALS AND METHODS
Sewage Plants
The present survey was conducted at two different
sewage treatment plants (A and B) in Hyogo Prefecture, Japan, during a one-year period (July 2002 to September
2003). Plant A is located on an 44×10５-" artificial island

International Center for Medical Research and Treatment, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017,
and ‡Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe
654-0142, Japan２

constructed in 1981 in the southern part of the prefecture.
Approximately 15×10３ people live on this island, and plant
A treats only the sewage from this population. There aredogs and cats but no large animals on the island. On theother hand, plant B is located 22 km west of plant A andtreats the sewage of approximately 205×10３ people. Many
livestock animals such as cows, chickens and pigs are bredin this area. The mean daily flow of plants A and B during
the study period was 8×10６ and 66×10６ L, respectively.
Sample Collection and Concentration
Two hundred ml of sewage was collected from eachplant at 10:00 a.m. once a month for one year. Samples
were placed in 250-ml conical tubes into which 2 ml of
10% gelatin solution was added to effectively disperse thesubstances. The tubes were then capped tightly and theircontents mixed thoroughly. In an additional experiment,
samples were collected from plant A every two hours for 24hours to clarify the hourly changes in cyst number. We repeated this experiment twice, on May 28 and August 12,
2003. In addition, sampling was performed for 5 continuousdays in plant A to compare cyst number before (April 14-18,
2003) and after the holiday week (May12-16, 2003).
The sewage sample was filtered through a stainlesssteel sieve with 32µm pores, then centrifuged at 1,000 ×g
for 10 min. The supernatant was aspirated leaving 4 ml ofconcentrate in the tube into which 4 ml each of ethyl alcohol and diethyl ether were added (total 12 ml). The samples
were then mixed and incubated at 60°
C for 5 min. After fur-ther mixing, the contents were centrifuged at 1,000 ×g for
10 min, then the pellet was re-suspended in 15 ml of 0.1％
gelatin solution and centrifuged again under the same conditions. Finally, the pellet was suspended in 10 ml of distilled water (Fig. 1).
Separation Procedure and Counting of Cysts
Giardia cysts suspended in the 10-ml water sample
were purified using an immunomagnetic separation (IMS)
method. In brief, samples were incubated with paramagnetic beads (Dynabeads, Dynal, Oslo, Norway) which were
coated with antibodies raised against Giardia. The resulting
complex of cysts and beads was then magnetically isolated
and washed. The cysts were then dissociated from the beads,
and the beads magnetically removed from the purified sample. The cysts were placed in a well-slide and stained using
a fluorescein-labeled monoclonal antibody (EasyStain,
Biotechfrontiers, North Ryde, Australia) then observed under a fluorescent microscope (BX50, Olympus, Tokyo, Japan). Round or oval fluorescein isothiocyanate (FITC)labeled objects measuring 7-10×12-15 µm with two or
more internal characteristic features (2-4 nuclei, medianbodies, axonemes) were counted as Giardia cysts.

RESULTSThe recovery efficiency of this method was calculated
by spiking one hundred Giardia cysts labeled with red fluorescent dye (Colorseed, Biotechfrontiers, North Ryde, Aus-

Fig. 1 Method for recovery of Giardia cysts from sewage.

３

Fig. 2 Hourly change in number of Giardia cysts in relation
to sampling time. Sewage samples were examined on
May 28 (Day 1) and August 12 (Day 2).

Fig. 3 Monthly change in number of Giardia cysts recoveredfrom each plant. Plant A (●) is located on an artificial
island and Plant B (■) is located in the suburbs.

tralia) into 200-ml sewage samples from plant A; this spiking procedure was repeated three times. The average recovery efficiency was calculated as 40% (37, 42, 40, n=3) andthe number of cysts per liter of sewage sample was corrected according to this recovery efficiency.
Circadian rhythms in cyst numbers in plants A and B
were observed, with a peak at 10:00 a.m. (4,300/L and
7,700/L, respectively) (Fig. 2). The average number of cysts
was 1,700/L and 1,000/L, respectively). The ratio of cyst
number at 10:00 a.m. to the daily average cyst number was
0.22 and 0.24 respectively, and the mean was 0.23.
Monthly investigation samplings were performed at
10:00 a.m.; therefore, cyst numbers were adjusted to theFig. 4 Comparison between number of Giardia cysts before
and after a holiday week. Sewage samples were collected for 2 series of 5 continuous days from April 14□) and from May 12
to April 18 (before holiday week:■
to May 16 (after holiday week:■).

daily average number. The daily average number of cysts ineach examination was equal to the cyst number detected ×
correction factor (0.23; the correction factor is the coefficient used to correct the number of cysts obtained from the
10:00 a.m. sampling period to the daily average).
The seasonal variation in cyst numbers is shown in Fig.
3. The present study revealed that both of the sewage plants
studied were positive for Giardia cysts, with a mean number of 880/L (range: 370- 2,000/L) and 2,000/L (range: 850- 3,900/L) in plants A and B, respectively. Cysts were detected throughout the year in both plants, with the numberof cysts detected in plant B being higher than that in plant A.
Monthly changes in the number of cysts recovered fromplants A and B showed two peaks, in one May and the otherin December. The number of cysts detected after a holiday
week was higher than that before: the average number detected before and after was 1,500/L and 2,500/L, respectively, and the difference was significant (p<0.05 student’s ttest) (Fig. 4).
The estimated number of infected individuals on eachof the examination days was the daily sewage flow × cyst
number of each monthly examination / number of cysts excreted by a patient in one day (1 to 2 ×10８; according toTsuchiya, 1931); the estimated prevalence was the estimated number of infected individuals / population in each

Table 1. Estimation of prevalence of giardiasis on examination days in areas covered by plant A and B
Plant

Quantity of sewage
treated /day (L)*

Number of cysts found(L)**

Population

A

8x10６

370 (min.)880 (ave.)2000 (max.)

15 x 10３B

66x10６850 (min.)2000 (ave.)3900 (max.)

205 x 10３

Estimated
number of patients prevalence (%)
0.1-0.2
15- 300.2-0.5
35- 700.5-1.1
80- 160
280- 560
650-1300
1300-2600

0.1-0.3
0.3-0.6
0.6-1.3

Estimated number of patients=*×**/number of cysts excreted per patient per day (1 to 2 x 10８, according to Tsuchiya, 1931)
Estimated prevalence=estimated number of patients / population in each plant area.

４

plant area.
The prevalence of giardiasis in the areas serviced byplants A and B was estimated using the above results (Table
1). Consequently, the average monthly prevalence was 0.11.1 and 0.1-1.3%, respectively.

DISCUSSION
Giardia is widely distributed throughout the world
with a varying rate of prevalence [2], and it is one of themost common enteric parasites recovered from humans, especially in tropical areas. However, it has become difficult
even in these areas, to obtain consent to sample fecal materials. In the U.S.A., attempts have been made to detect
Giardia cysts in sewage and to estimate giardiasis prevalence [11], but the sucrose floatation and Lugol’s iodine
techniques used are relatively insensitive. For instance, using the sucrose flotation technique, Sykora et al. foundhighly variable results (0.4 to 77.8%) with a mean of 11.0%[10]. With this in mind, we used a modified immunomagnetic bead method [12] to detect Giardia in sewage samples.
As a result, the average recovery efficiency from 200-ml of
spiked sewage samples was 40% and estimations of cyst
concentrations were more precise.
We observed marked seasonal variations in Giardia
cyst recovery, with two peaks in the cold (December) andwetter seasons (May), in this study. These findings wereconsistent with previous results. For example, among Guatemalan children, the incidence of giardiasis was highest
during the cooler and wetter months [13]. Furthermore, in
Saudi Arabia, the highest incidence occurred during September, which is the wettest month of the year [14]. Therefore, cyst numbers in sewage might be related to climaticconditions. Jarroll et al. also suggested that Giardia cysts
find cooler and wetter conditions more favorable for survival [15].
In the present study, the number of cysts detected after
a holiday week exceeded those detected before that week.
This finding observed might be related to the Japanese life
style, that is, many Japanese take vacations from the end of
April to the middle of May and travel to foreign countries,
including developing countries, and this custom is also seenat the end of the year. It is possible therefore those individuals are infected during these overseas trips, especially during trips to endemic areas. The present findings correlated
well with the holiday period in 2003 (April 29 to May 9).
According to the Immigration Bureau, 570,000 Japanese
traveled overseas during May 2003, and of these, approximately 230,000 traveled to developing countries. This suggests that 290 of the inhabitants in the area around plant A
traveled to developing countries.

Kimura et al. examined Japanese travelers who visited
Nepal and India for more than 10 days and who experienced diarrhea when they arrived back in Japan [16]. Theirsurvey revealed that 69 out of 692 (9%) travelers were positive for Giardia cysts. Giardia is the most common enteric
protozoan parasite in Nepal and India [17,18,19,20] and has
remained top of the list for the past decade [17]. Similar imports of Giardia infections into developed countries havebeen reported elsewhere [3]. The reported Giardia cyst
shedding rates in calves ranges from 80 [21] to 100% [22]in British Columbia, Canada. However, since the area serviced by plant A is an isolated artificial island where livestock are not bred, we can rule out the possibility of sewage
contamination by such animals. The hourly variation results
obtained in this study also clearly demonstrated that cyst
concentrations are related to human activity, and the findings strongly suggest that the Giardia cysts were excreted
from inhabitants of this island. This result agrees with theearlier study of Gassmann and Scwartzbrod who suggested
that a peak of Giardia cysts was observed at 10:00 a.m. in
Nancy, France [23].
It is difficult to assess the number of cysts excreted
daily from observations of a single infected individual.
Therefore, we calculated the number of infected individuals
based on the recovery efficiency, monthly survey, and variation in cyst numbers at different sampling times. Tsuchiya
calculated that the number of cysts excreted from an individual/day varied from 0-7 × 10８ with cyst-excreting andnon-excreting days occurring alternatively during the courseof infection [24]. Using this data as a base, we estimated themean number of cysts excreted per day per patient. Accordingly, the mean number of cysts excreted for a period ofduring six weeks after the onset of symptoms was 1 × 10８,
but when calculations were conducted using only the cystexcreting days the value was 2 × 10８.
The infection rate of Giardia in the areas serviced byplants A and B was estimated as 0.1-1.1 and 0.1-1.3%, respectively, and these results were consistent with the results
obtained from fecal examinations in Japan (0.2-0.9%) [8,9].
This study revealed that sewage samples can be used to estimate giardiasis in a particular service area (community) using the IMS method. The method developed for this survey
was easy, cost-effective and time saving, and it allowed for
accurate estimations of the prevalence of giardiasis. We believe it can be used for future surveys of the prevalence ofgiardiasis in other areas.

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５２）Rabbani GH, Islam A. Giardiasis in humans: Populations
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６）Meyer EA, Radulescu S. Giardia and Giardiasis. Adv.
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７）Wolfe MS. Giardiasis. Pediatr. Clin. N. Am. 1979; 26: 295-303.
８）Ichizawa T, Kato H, Mochizuka I, Kurita M, Senoh K,
Suzuki N. Giardiasis in Fujieda and its neighboring
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passage of soft stool in Kochi Prefecture, Japan. Jpn. J.
Trop. Med. Hyg. 1998; 26: 113-116.
１０）Sykora JL, Sorber CA, Jakubowski W, Casson LW,
Gavaghan PD, Shapiro MA, Schott MJ. Distribution ofGiardia cysts in wastewater. Water Sci. Technol. 1991;
24: 187-192.
１１）Jakubowski W, Sykora JL, Sorber CA, Casson LW,
Gavaghan PD. Determining giardiasis prevalence by examination of sewage. Water Sci. Technol. 1991; 24: 173178.
１２）US Environmental Protection Agency. Method 1623:
Cryptosporidium and Giardia in water by Filtration/IMS/
FA. Office of Water, EPA-821-R-01-025 2001.
１３）Farthing MJG, Mata L, Urrutia JJ, Kronmal RA. Natural
history of Giardia infection of infants and children in rural Guatemala and its impact on physical growth. Am. J.
Clin. Nutr. 1986; 43: 395-405.
１４）Kasim AA, Elhelu MA. Giardiasis in Saudi Arabia. Acta
Trop. 1983; 40: 155-158

１５）Jarroll EL, Bingham AK, Meyer EA. Giardia cyst destruction: effectiveness of six small quantity water disinfection
methods. Am. J. Trop. Med. Hyg. 1980; 29: 8-11.
１６）Kimura A, Minekawa Y, Ikeda N, Shimoirisa K, Kusui Y,
Mastumoto Y, Nakabayashi T. Survey of Giardia lamblia
infection in returning travelers with diarrhoea from India
and Nepal at Osaka airport quarantine station. Jpn. J.
Trop. Med. Hyg. 1992; 20: 291-297 (in Japanese).
１７）Rai SK, Bajracharya K, Budhathoki S, Khadka JB, Rai
KK, Shrestha MK, Sharma CM, Nakanishi M, Kubo T,
Shrestha HG. Status of intestinal parasitoses at TU
Teaching Hospital. J.Insti. Med. (Nepal) 1995; 17: 13442.
１８）Rai SK, Hirai K, Abe A, Ishiyama S, Rai G, Ono K, Uga
S. Intestinal parasitoses among school children in a rural
hilly area of Dhading district, Nepal. Npl. Med. Coll. J.
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１９）Ono K, Rai SK, Chikahira M, Fujimoto T, Shibata H,
Wada Y, Tsuji H, Oda Y, Rai G, Shrestha CD, Masuda K,
Shrestha HG, Matsumura T, Hotta H, Kawamura T, Uga
S. Seasonal distribution of enteropathogens detected fromdiarrheal stool and water samples collected in Kathmandu, Nepal. Southeast Asian J. Trop. Med. Public
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２０）Fernandez MC, Verghese S, Bhuvaneswari R, Elizabeth SJ,
Mathew T, Anitha A, Chitra AK. A comparative study ofthe intestinal parasites prevalent among children living inrural and urban settings in and around Chennai. J. Commun. Dis. 2002; 34: 35-39.
２１）Olson ME, Guselle NJ, O’Handley RM, Swift ML, McAllister TA, Jelinsk MD, Morck DW. Giardia and dairy
calves in British Columbia. Can. Vet. J. 1997; 38: 703706.
２２）Ralston BJ, McAllister TA, Olson ME. Prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams. Vet.
Parasitol. 2003; 114: 113-122.
２３）Gassmann L, Schwartzbrod J. Wastewater and Giardia
cysts. Water Sci. Technol. 1991; 24: 183-186.
２４）Tsuchiya H. A study on variabilities in dimensions andnumbers of discharged cysts Giardia lamblia (stiles
1915) from day to day under normal conditions. Am. J.
Hyg. 1931; 13: 544-567.

Tropical Medicine and Health Vol. 33 No. 1, 2005, pp. 7-11
Copyright! 2005 by The Japanese Society of Tropical Medicine

７

Epidemiological study on Centrocestus armatus metacercariae
in the Chikusa River, Hyogo Prefecture, JapanDAISUKE KIMURA and SHOJI UGA
Accepted 11, January, 2005

Abstract: The prevalence of Centrocestus armatus metacercariae in fresh water fishes were examined in Hyogo
Prefecture, western Japan for a period of one year from April 2003 to March 2004. Three species of cyprinoid fish,
namely Zacco temminckii, Zacco platypus, and Pseudogobio esocinus, were found to harbor metacercariae of C.
armatus. The infection rate of metacercariae in Zacco temminckii, Zacco platypus, and Pseudogobio esocinus was
99% (371/377), 100% (8/8), and 100% (2/2), respectively. The mean number of metacercariae recovered was 232
for Z. temminckii, 2,123 for Z. platypus, and 20 for P. esocinus. However, we focused on Z. temminckii in thisstudy because it was found to be the most predominant fish species in the Chikusa River and to have high ametacercariae prevalence as well. In Z. temminckii, metacercariae were distributed in the brain (37%), viscera(35%), muscles (29%) and gills (0%). No metacercariae were recovered from the scales and fins. This is the firstreport of the occurrence of C. armatus metacercariae in the brain region of the second intermediate fish. The prevalence of C. armatus metacercariae was almost constant throughout the year, and no positive correlation was foundbetween the prevalence and seasonal changes. However, a positive relationship was observed between prevalenceof metacercariae and fish length, i.e., the intensity of infection increases with the size of the fish host (p < 0.05).
People in the survey area sometimes eat fish raw, indicating enlightenment as well as the countermeasures to prevent C. armatus infection is needed.
Keywords: Centrocestus armatus, metacercariae, epidemiological study, freshwater fish, Chikusa River, JapanINTRODUCTION
The fluke genus Centrocestus is a group of minute intestinal trematodes belonging to the family heterophyidae.
Centrocestus is widely distributed in Japan, Taiwan, Republic of Korea, and Southeast Asia. Four species of Centrocestus, namely C. armatus [1], C. formosanus[2], C. nycticoracis [3] and C. asadai [4], have been reported to be distributed in Japan. These species normally infect fish-eating
birds. They have also been reported to infect mammals, including man except for C. nycticoracis [5]. Moreover, C.
formosanus var. kurokawai, C. caninus, and C. armatus
have been reported to infect humans in Japan, Thailand, andKorea [6, 7, 8].
The first intermediate host of Centrocestus is a fresh
water snail belonging to genus Semisulcospira [9], the second intermediate host fresh water fish, and birds such as
Nicticorax nycticorax the final host [1]. About 20 species offresh water fish including Zacco platypus, Pseudorasbora

parva, and Carassius carassius are known to serve as second intermediate hosts of C. armatus [1, 10].
Rim et al. [11]reported that metacercariae of C. armatus were found at a rate of 33-100% in five species of fresh
water fish from three rivers in Korea. Furthermore, Sukontason et al. [12] reported the presence of Centrocestus
metacercariae in six species of cyprinoid fish in northern
Thailand. The prevalence of metacercariae in different locations, seasons, and fish species had also been reported.
However, the number of fish examined by Rim et al. waslimited to only 1-15 samples. Moreover, the prevalence offish infection was not described and no precise description
was provided regarding the identification of metacercariae.
In the present study, therefore, the state of Centrocestus metacercariae infection in freshwater fish of the Chikusa
River in Hyogo Prefecture, Japan was investigated from
April 2003 to March 2004. The correlation of seasonalchanges and fish length to metacercariae prevalence wasalso determined.

Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, Kobe 654-0142, Japan
Correspondence: Uga Shoji
E-mail: ugas@ams.kobe-u.ac.jp
Tel & Fax: +81-078-796-4548

８

MATERIALS AND METHODS
Survey area
The study was carried out from April 2003 to March
2004 in the Chikusa River in Hyogo Prefecture, Japan. The
survey area was a midstream section of the river 30 m inwidth and 0.1 to 1.5 m in depth. Various fish species were
captured using a landing net once a month for a period of
one year. The collected fish were kept alive in a cooled-box
with aeration and brought to the laboratory. Taxonomic
identification of the caputured fish species was carried out
according to the fish atlas by Miyaji et al. [13]. In addition,water temperature was measured at the time of each survey.
Examination and observation of metasercariae
The fish brought to the laboratory were kept alive in an
aquarium, with artificial aeration, until the examination of
metacercaria. The body length of each fish sample was alsomeasured and recorded. To obtain the metacercariae fromeach sample, fish was minced and digested using a digestive
solution (0.1% each of pepsin and HCl in distilled water) at
37°
C for1hour. The digested material was filtered through a
325µm mesh to remove large particles and rinsed twicewith saline solution (0.85% NaCl). The sediment was thencentrifuged at 150 x g for 5 minutes. The C. armatus
metacercariae collected were counted under a stereoscopic
microscope.
In other experiments, organs such as the brain, muscles,
gills, viscera, scales and fins were also dissected and digested artificially to determine the distribution of themetacercariae in the fish body. The counting of circumoral

spines for identification of metacercaria was carried out using a light microscope and a scanning electron microscope(T-330A, JEOL, TOKYO). The size of different organs observed for metacercariae was also measured using a micrometer caliper (OLYMPUS, TOKYO) under a light microscope. In addition, the recovered metacercariae were excysted with artificial intestinal juice (0.4% sodium hydrogen carbonate, 1.0% trypsin, 0.85% NaCl) fixed with 10%
neutral buffered formalin and observed under a scanningelectron microscope to elucidate morphological detail.
Statistical analysis
The mean abundance was expressed as mean ± standard deviation (SD). The difference between length of fishand mean abundance was evaluated by one-way factorial
analysis of variance (ANOVA), followed by Fisher’s PLSD
multiple comparison test to identify significant differencesamong the multiple samples. Differences in the prevalenceof infection were tested using χ２ test. A p value less than
0.05 was considered statistically significant.

RESULTS
Throughout course of the survey we caught 511 freshwater fish from Chikusa River. These consisted of 15 different species as shown in Table 1. Eight of the fish species belonged to the cyprinoid family. Table 1 shows the infection
rate of each fish species examined and the mean number ofmetacercariae per fish. Metacercariae were obtained fromonly three cyprinoid fish species, namely Z. temminckii, Z.
platypus, and P. esocinus with a high infection rate (99-

Table 1. Prevalence of metacercariae and mean number of metacercariae recovered in fish
collected from the Chikusa River, Hyogo Prefecture, Western Japan
Species of fish

No. examined

Zacco temminckii*
Rhinogobius sp.
Plecoglossus altivelis
Silurus asotus
Cobitis biwae
Zacco platypus*
Pelteobargus nudiceps
Coreopera kawamebari
Odontbutis obscurus
Pseudogobio esocinus*
Carassius carassius*
Puntungia herzi*
Squalidus gracilis gracilis*
Tribolodon hakonensis*
Cyprinus carpio*

377
50
28
13
12
8
5
5
3
2
2
2
2
1
1

* : Cyprinoid fish** : metacercariae

No. of fish infected
(%)
371
0
0
0
0
8
0
0
0
2
0
0
0
0
0

(99)
(0)
(0)
(0)
(0)
(100)
(0)
(0)
(0)
(100)
(0)
(0)
(0)
(0)
(0)

Mean No. of Mc**per fish
229
0
0
0
0
2,123
0
0
0
20
0
0
0
0
0

９

Fig. 1 Monthly changes in prevalence of metacercariae ofCentrocestus armatus in Zacco temminckii (■) andwater temparature (▲―).

100%). Z. platypus showed the highest mean abundance ofmetacercariae. However, Z. temminckii was the most predominant fish species collected (74%) throughout the sampling period, and it also showed a high infection rate (99%).
Therefore, further examinations were focused on Z. temminckii.
The monthly changes in the prevalence of metacercariae in Z. temminckii are shown in Fig. 1. The metacercariae
infection rate of Z. temminckii was nearly 100% throughout
the year. No positive relationship was observed between
prevalence of metacerariae and seasonal changes and watertemperature.
Throughout the survey, 84,921 metacercariae were recovered from infected 371 Z. temminckii, with a meanabundance of 229 (Table 1). Of the 11,760 metacercariae
found in Z. temminckii, 37% (4,306) were observed in thebrain; 0% (16) in gills; 35% (4,069) in viscera; and 29%(3,369) in muscles (Table 2). No metacercariae were foundin scales and fins. The number of metacercaria was also
computed per gram of fish meat. As a result, the brain
showed the highest number of metacercariae per gram ofmeat (2,057/g), followed by viscera (262/g), muscles (20/g),
and gills (5/g) (Table 2).
The relationship between fish length and the numberof metacercaria per fish is also shown in Fig. 2. The prevaTable 2. The distribution of Centrocestus armatus metacercariae
in various parts of fish
Parts of fish
Brain
Gills
VisceraMuscleScale, Fin

Mean Weight Mean No. of Distribution
No. of Mc/g(g)
Mc
rate (%)
2,057
37
144
0.07
5
0
1
0.22
262
35
136
0.52
20
29
112
5.59
0
0
0
0.47

Thirty Zacco temminckii, 9-10 cm, were examined.

Fig. 2 Relationship of length of Zacco temminckii, mean
■ of Centrocestus
abundance (■) and the prevalence (―)
armatus metacercariae in the Chikusa River, WesternJapan. Data are expressed as mean ± SD (n: 16-65). *:p<0.05.

lence levels were almost the same irrespective of fish length(p > 0.05). Even young fish of four centimeters or less inlength showed an infection rate as high as 90%. The results
also showed that the number of metacercaria per fish was
directly correlated with fish length, i.e., there was an increase in the mean abundance of metacercaria as the fish
length increased (p < 0.05). The number of metacercaria recovered from fish samples with smaller than 5 cm and morethan11cm in length was8and1,069respectively (p=0.017 x
10１７) (Fig. 2).
In addition, the recovered metacercariae were measured under a microscope. The average length was about 200µm and the average width about 100 µm. The metacercariae were mounted on glass slides for detailed observation.
The oral sucker, ventral sucker, and excretory bladder ofmature metacercaria were observed. Light and scanningelectron microscopic observations revealed that the oral
sucker was located at the anterior end, with 44 circumoral
spines arranged in two rows around it (Fig. 3a, 3b). The
ventral sucker was located in the midventral part and the excretory bladder was shown as an “X”-shaped organ locatedposterior to the ventral sucker (Fig. 3a). However, these organs were not observed in immature metacercariae except
for the eyespot that was a remnant of the cercarial stage(Fig. 3c).

DISCUSSION
Seven out of 20 known freshwater fish species wereobserved in this study as second intermediate hosts of Centrocestus armatus [1, 10]. In the present study, metacercariae of C. armatus were detected in only 3 out of the 15
freshwater fish species obtained from Chikusa River,１０Fig. 3 Microscopic photographs of Centrocestus armatus metacercariae.
A. Metacercariae showing the oral
sucker (OS), ventral sucker (VS),and excretory bladder (EB);B. Oral sucker showing the circumoral
spines (CS);C. Immature metacercariae showing
the eyespot (ES).

namely Zacco temminckii, Zacco platypus, and Pseudogobio esocinus. The prevalence of metacercaria in these three
species of freshwater fish was almost 100%. Among the 3
species, Z. platypus showed the highest mean abundance ofmetacercaria. However, since Zacco temminckii was foundto be the most predominant fish species collected from theriver, it can be inferred that this fish played an importantrole as the second intermediate host of Centrocestus armatus in the Chikusa River.
The prevalence and intensity of trematodes infection tosecond intermediate hosts such as fish were said to be associated with seasonal changes [14]. Sukontason et al. [12]mentioned that trematode metacercariae levels were highest
in winter and lowest in the rainy season. Several other studies showed that the emergence of cercariae from the first intermediate host increased as water temperature increased[15, 16]. A study was also conducted on prevalence of cercariae of C. armatus in the Chikusa River by Kimura andUga [17]. They showed that the mean prevalence of cercariae in the snail Semisulcospira libertina was low at 6% (116%) during winter and spring seasons (October-May)
when water temperature was low. In contrast, the period
from June to September when water temperature was higher
showed a very high prevalence. The mean prevalence value
was 39% [17]. The authors conclude that the infection of
cercariae to fish as the second intermediate host had occurred during summer when water temperature was high.
However, the results of the present survey did not show any
seasonal changes in prevalence of metacercariae. Chubb[14] also mentions that changes in the prevalence ofmetacercariae were not very evident at different climatic

seasons of the year. He states that metacercariae could
thrive for a long period of time inside the fish host andtherefore that no drastic changes in metacercariae prevalence were observed at different seasons of the year.
Rim et al. [11] examined the distribution of metacercariae in the different organs of fish using 22 samples of Z.
temminckii and Z. platypus. They reported that the metacercariae were distributed in the gills, muscles, intestinal organs, and scales and fins, and that the intestinal organs
showed the highest mean abundance of metacercariae. In
the present study, the brain, muscles, viscera, gills, andscales and fins of 30 Z. temminckii were examined for presence of metacercariae. The brain region showed the highest
mean abundance (144), followed by the viscera and muscles
with abundance of 136 and 112, respectively. However, thegills showed only a mean of 1, and 0 for scales and fins.
These results were similar to the observation of Rim et al.
[11] except for the presence of metacercariae in the brain
region. In general, metacercariae of any fluke species can befound in the muscles and viscera of the fish body. In thepresent study, however, it is interesting that the infection of
metacercariae of C. armatus was observed in the brain region of the second intermediate host. No related studies
have reported the occurrence of metacercariae of C. armatus in the brain.
Poulin [18] reported the positive correlation of fish
length and mean abundance of metacercariae. Dogiel et al.
[19] also stated that, in fish populations, the intensity of infection by parasites increased with the age and size of the
fish hosts. The same result was observed in the present
study, i.e., the longer the fish length the higher the metacer-

１１cariae infection. At a fish length of 3-5 cm, the mean abundance was 8, but this increased gradually to 1,069 at a fish
length of 11-19 cm (p=0.017 x 10-１７). Since a fish with alarger length has a higher contact area, it is likely that cercariae have more chances for attachment to the fish bodysurface. These cercariae, in turn, could develop into
metacercariae inside the fish body and accumulation could
occur over time. That is why a higher number of metacercariae was observed in older fish with a greater fish length.
It was reported that metacercaria of the four species ofCentrocestus distributed in Japan have a different numberof circumoral spines: C. armatus, C. formosanus, C. nycticoracis, and C. asadai have circumoral spines of 44, 32, 42,
and 38, respectively [10]. Throughout the present survey,
the recovered metacercariae were found to have 44 circumoral spines and were identified as C. armatus.
In conclusion, the prevalence of C. armatus in Z. temminckii was high in the Chikusa River and the fish Z. temminckii is considered to be the most common second host.
The questionnaire survey done in our study area [20] revealed that 52% of the inhabitants have eaten fresh water
fish including Z. temminckii. Moreover, about 10% of inhabitants eat these fish raw, indicating possibility that thepeople living in this area have been infected by C. armatus.
Further enlightenment as well as the countermeasures toprevent the infection is needed.

ACKNOWLEDGEMENTS
Dr. GV Paller kindly checked the English manuscript.

REFERENCES
１．Tanabe H (1922) Studien uber die trematoden mit Susswasserfischen als Zwischenwirt. I. Stamnosoma armatumn.g., n.sp. Kyoto Igaku Zasshi 19 (3): 239-252 (in Japanese).
２．Nishigori M (1924) On a new species of fluke, Stamnosoma formosanum and its life history. Taiwan Igakkai
Zasshi 234: 181-238 (in Japanese).
３．Izumi M (1935) A new study of a trematode belonging to
the Genus Stamnosoma and its life cycle. Tokyo Iji Shinji
59 (2948): 2423-2430 (in Japanese).
４．Mishima T (1959) On a new species of the genus Centrocestus with its life history. Tokyo Iji Shinji 76 (5): 291299 (in Japanese).
５．Yamaguti S (1958) Systema Helminthum, Vol. I. Digenetic Trematodes of Vertebrates, pp.705-707 & 872-873.
６．Kurokawa T (1935) On a new trematode of genus Stamnosoma proved from a man. Tokyo Iji Shinji 2915: 293-

298 (in Japanese).
７．Waikagul J (1977) Human infection of Centrocestus caninus in Thailand. Southeast Asian J Trop Med Public
Health 28 (4): 831-5.
８．Hong SJ, Seo BS, Lee SH, Chai JY (1988) A human caseof Centrocestus armatus infection in Korea. Korean J
Parasit 26 (1): 55-60.
９．Takahashi S (1929) A contribution to the life-history of
Stamnosoma armatum Tanabe. Okayama Igakkai Zasshi
4 (8): 1759-1771 (in Japanese).
１
０．Komiya Y (1965) Metacercaria in Japan and adjacent territories. Progress of Med. Parasit. in Japan. Vol. II.
Meguro Parasitological Museum, Tokyo. 135-143.
１
１．Rim HJ, Kim KH, Joo KH, Kim S J, Eom KS, Chung
MS (1996) The infestation states and changing patternsof human infecting metacercariae in freshwater fish in
Kyongsang-do and Kyonggi-do, Korea. Korean J. Parasit
34 (2): 95-105.
１
２．Sukontason K, Piangjai S, Muangyimpong Y, Methanitikorn R, Chaithong U (1999) Prevalence of trematodemetacercariae in cyprinoid fish of Ban Pao district, Chiang Mai Province, northern Thailand. Southeast Asian J
Trop Med Public Health 30 (2): 365-370.
１
３．Miyaji D, Kawanabe H, Mizuno N (1976) Colored illustrations of the freshwater fishes of Japan. Hoikusha Publishing CO., LTD Osaka (in Japanese).
１
４．Chubb JC (1979) Seasonal occurrence of helminthes infreshwater fishes. Part II. Trematoda. In: Lumsden WHR,
Muller R, Baker JR (eds) Advances in parasitology, Vol
17. Academic Press, London, p141-313.
１
５．Taskinen J, Valtonen ET, Makela T (1994) Quantity ofsporocysts and seasonality of two Rhipidocotyle species(Digenea: Bucephalidae) in Anodonta piscinalls (Mollusca: Bivalvia). Int J Parasitol 24: 877-886.
１
６．Lyholt HCK Buchmann K (1996) Diplostomum spathaceum: effects of temperature and light on cercarial
shedding and infection of rainbow trout. Dis Aquat Organ
25: 169-173.
１
７．Kimura D, Uga S (2003) Epidemiological studies on
Centrocestus spp. (Trematoda: Heterophyidae) in
Chikusa River basin, Hyogo Prefecture: infection in the1st intermediate host snail, Semisulcospira libertina. Jpn J
Environ Entomol Zool 14 (2): 97-103 (in Japanese withEnglish summary).
１
８．Poulin R (2000) Variation in the intraspecific relationshipbetween fish length and intensity of parasitic infection:
biological and statistical causes. J Fish Biol 56: 123-137.
１
９．Dogiel VA, Petrushevski GK, Polyyanski YI (1958) Parasitology of Fishes. Oliver and Boyd, London (Translated
by Z Kabata, 1961).
２
０．Kankawa, Y. and S, Uga. (2001) Epidemiological study
on metagonimus spp. in Chigusa River, Hyogo Prefecture,
Japan. Jpn. J. Environ. Entomol. Zool, 12 (3): 147-153.
(in Japanese with English summary).

Tropical Medicine and Health Vol. 33 No. 1, 2005, pp. 13-17
Copyright! 2005 by The Japanese Society of Tropical Medicine

１
３

Review and future perspectives of “development study”
in the health sectors
TAKATOSHI KOBAYAKAWA１, HIROYUKI NAKANO２, DAIRIKU HOZUMI３,
HIDECHIKA AKASHI４, YURIKO MINAMOTO５, KATSURA MIYAZAKI６
and TATSUYA ASHIDA６
Accepted 7, February, 2005

Abstract: The research project titled “Developing Technical Approaches for the Master Plan of the Health Sector
International Cooperation” has been conducted for the duration of three years from 2002 by the support of grantsin-aid for research on international medical cooperation from the Japanese Ministry of Health, Labor and Welfare.
Based upon comprehensive and detailed review on the development studies (DS) in the health sectors so far completed, and throughout extensive and detailed examinations on the various DS related issues at the organized workshops with the parties concerned, the following propositions were made for the orientation and possible improvement of future DS in the health sectors; 1) a master plan in the individual DS to be defined in association with thelevel of strategy with which the study deals, 2) the instruction of surveys and methods to be more crystallized, 3)
appropriate survey methods with reproducibility to be employed, 4) qualitative researches to be complimentarily
exercised with quantitative researches 5) the ownership of DS to be cultivated by adoption of participatory methods.

INTRODUCTION
This article deals with the study results of the research
project titled “Developing Technical Approaches for the
Master Plan of the Health Sector International Cooperation”.
The government project was supported by the Japanese
Ministry of Health, Labor and Welfare which was to be implemented for the duration of three years from 2002. The
purpose of this research project was to determine the orientation of future DS in the health sectors by reviewing thestudies experienced so far and by introducing various study
methods and analytical methods for developing health policies and health planning techniques.
A DS is a form of technical cooperation provided by
the Japanese Official Development Assistance (ODA) to assist in the planning of urgent and high priority development
projects in developing countries through the preparation of
relevant reports [1]. Traditionally, a DS has been conducting
feasibility studies prior to assigned projects, mainly in thefields of social infrastructure including electricity power
supply and road constructions. In recent years, apart from１

these hardware type projects, a DS is also involved in software type projects such as making propositions for the establishment of policies, with which it reflects the diversified
and increasing needs of the developing countries. Since thestart of the DS projects in health sectors, eight studies
which include that of Honduras, have so far been completed(Table 1). DS oriented development assistance aimed at improving the health policy/administrative system is expected
to increase. A DS survey is initiated by the implementationof fundamental surveys. Based on the results of those surveys, a master plan is formed. There are two features withwhich the DS draws the line from other technical cooperation sectors. A DS may propose a policy during the courseof a study but any field surveys, analysis and other related
studies in the report making are actually conducted by aJapanese consultant. However, the know-how in the investigation, analysis, planning and management of a study by
the implementing agency such as the Japan International
Cooperation Company (JICA) or the consultants currently
appear to be not fully competent. Therefore, the primary objective of this research project is to improve future Japanese

Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University
Department of International Cooperation, St. Mary’s Hospital３
Department of Population and International Health, Harvard School of Public Health４
Expert Service Division, Bureau of International Cooperation, International Medical Center of Japan
５
International development consultant
６
Human Development Department, Japan International Cooperation Agency
２

１
４

Table 1. Development study projects with implementation
periods in the health sectors■ The study on the strategies and plans for the upgrading of healthstatus in the Republic of Honduras[January 1995〜October 1996]

Figure 1. Process of formulating a master plan in Malawi
development study
Health situation
analysis

Problem analysis■ The study on strengthening the district health system in the western part of Kenya[August 1997〜November 1998]■ Master plan study on strengthening primary health care services in
the Republic of Malawi

Health problems on supply and demand sides
Identification of risk factors for malnutrition andinfection

Basic strategy
for master plan

Projecting future situation in target period
Consistency with national health development
plan
Essential concept primary health care

[June 1998〜January 2000]■ The development study on reproductive health in the state of Madhya Pradesh, India[November 2000〜March 2002]■ The study on the improvement of health and medical services inthe Lao People’s Democratic Republic

Formulation ofmaster plan

Selection of core health issues
Setting up quantitative targets for the year 2007
Major policy, implementation, monitoring andevaluation
Specification of programmes and projects[March 2001〜November 2002]■ The study on enhancement of district health system for Beni Province Prefecture in the Republic of Bolivia[June 2001〜February 2003]■ The study on the restructuring of health and medical system in the
Republic of Uzbekistan

Selecting ofprioritised
programme

Establishing the order of priority
Project design matrix
Implementing organisation
Cost estimates
Staged implementation plan

Proposal of
specific
programme

Financial planning
Confirmation of validity through general evaluation
Specific implementation plan

[October 2002〜December 2003]■ Master plan study for strengthening health system in the Democratic Socialist Republic of Sri Lanka[March 2002〜December 2003]

ODA projects by means of viewing and subsequently suggesting a future course for the DS in the health sectors. Secondly, the research examines the technical development aswell as the analytical and planning methods related to thehealth sector master plan study.

STUDY METHODS
A comprehensive and detailed review is conducted on
the seven DS projects already completed in Honduras,
Kenya, Malawi, Laos, India, Sri Lanka and Uzbekistan[2,3,4,5,6,8,9]. Extensive analysis and detailed examinations were carried out on the various issues in the healthsectors related to the DS through the workshops organizedby the research group which are limited to the consultants
and the JICA staff. A continuous review was performed on
the operation and the administration in order to assure asmooth and effective implementation of the study. In addition, the review also examined the analytical and planning
methods currently employed for the study in the institutions
both at home and abroad. Besides, as a separate activity, theresearch group performed a follow-up of the DS study in
Laos. It is now also in the process of re-analyzing and renewing the data and information obtained from that ofMalawi in order to search for an appropriate health policy

upon the completion of the study.
For the readers, the implementation procedure of theDS will hereinafter be described as those shown in Figure 1.
The main components of the DS is composed of field surveys, analysis of the survey results and other relevant data,
the formulation of master plans including the selection ofpriority programs/projects, and providing technical cooperation. A detailed procedure on the formulation of a masterplan will be explained by using the example of the DS in
Malawi as is shown in Fig 1.
In developing a master plan, the basic strategies forreaching specific goals need to be drawn up. The strategy
framework includes an outline of policies and programs that
address the roots of the salient health issues. The first cycleof the study demonstrated that a considerable number ofMalawians have relatively favorable access to health facilities, but their health indicators tend to be among the poorest
in the world. Accordingly, the basic strategy for developing
a master plan was directed towards bridging this gap whileat the same time, the future situation of major issues such as
population, economy, and disease conditions were projected
for the respective target years. More importantly, the basic
strategy for the formulation of a master plan was consistent
with the national health development plan and was in accordance with the basic concepts of the local primary health

１
５

care.
In the first cycle of the study, two major issues wereidentified: child malnutrition and maternal health. Then, thestudy came up with a list of specific health problems related
to these two core issues and then it determined the cause
and effect relationships of these problems. The problem tree
exercise aided in proposing interventions to the health problems (a long list) , which were viewed as effective solutions.
Subsequently, each proposed intervention was considered indetail and was integrated into comprehensive projects (ashort list) . The process of the integration and the prioritization of the projects were considered under the basic strategy
or concept as described. Finally, concrete prioritized projects were proposed by taking priority, feasibility, costeffectiveness and efficiency into account.

DEFINITION OF A MASTER PLAN
In this research project, the definition of a master planin the DS was what we described in the beginning of a project. It is a comprehensive and fundamental plan for various
public sectors including the health sectors. The definition ofa master plan may be ambiguous between that of the JICA
and the consultants. Therefore, in reality, the DS achievements may vary in their characteristics, depending on theindividual definitions of the studies. The plural studies appear to define a master plan as the process from a strategy
proposition for the formulation of a plan to the selection ofpriority programs/projects [3,4,6]. Specifically, it generically represents a conceptual master plan based upon a basic study with the existent data, field surveys, priority programs/projects, and action plans. Under such circumstances,the current research project group carried out a discussion
and an analysis on the definition of a master plan prior to
the full research activity.
Goals, strategies, scenarios, resources, programs/projects, and action plans are common components of a masterplan. On the other hand, there could be presumably various
levels of strategies with which a DS deals: the level of national health policy, the level of a priority health program
defined in the national health policy (ex. program for the
improvement of childhood malnutrition) and the level of a
specific implementation activity in certain priority programs(childhood vaccination program) . The mode and the substance of a study may of course vary according to each of
the level concerned. In the present state of affairs, an overall
framework of the master plan should be clearly revealed.
Particularly, it is vital to define the relationship between a
proposed master plan with the priority programs and thecorresponding national health policy. Furthermore, relevance of the master plan ideas with SWAPs (Sector WideApproaches) as well as PRSP (Poverty Reduction Strategy
Paper) should also be considered. Further studies will be
required to define master plans of other sectors.

REVIEW OF A “DEVELOPMENT STUDY” IN GENERAL
A comprehensive review on the DS was performed.
First, the principal issues with respect to the management
and the technical subjects of the DS are described. In termsof management, some recipient countries tend to expect amaterialization of the project following a completion of themaster plan, on the assumption that the DS was a sort offeasibility study prior to a launching of a project. There wasalso indication that the instructions of surveys and methods
used by JICA were extremely comprehensive and detailed
without any given specificities while the process of plan
formulations were complicated. Since the descriptions ofachievements in the context of study were ambiguous andunspecific, a counter-proposal party was necessary in orderto describe the difference of terms to JICA. These processes should be related to the preparatory study. However,since the purpose of the DS was not clearly defined at thetime of the preparatory study, the actual development of thestudy was commenced without a conclusive result of the
preparatory study. Therefore, it is suggested that specification of principles and fields of the study should be clarified
at the time of a preparatory study.
Next, the technical subject on the implementation of
the study was viewed. The field survey implemented in the
DS is the bottom line for the formulation of a master plan.
A field survey is an important opportunity to collect information and data relevant to health sectors in order to understand the health situation of a recipient country. The resultof a field study will be extensively utilized and is itself awider framework than that of a DS. The methods employedby the DS in quantitative researches in the health sectors include household survey, exit interview at health facilities,
growth monitoring, drug and medical supply inventory survey, KAP (knowledge, attitude and practice) survey andhealth manpower assessment [10]. The first issue that one
could point out regarding the quantitative research performed in the DS was the insufficient description of the survey and the analytical methods, which might account forthe difficulty in verifying the relevance and the accuracy ofa study. Similarly, inadequate reference to the planning
methods and processes for the formulation of a master plan
was found. Furthermore, problematic sampling and inappropriate study methods were employed in household surveys.
Recently, qualitative research techniques such as the
Focus Group Discussion, Key Informant Interview and Ob-

１
６

servations have been introduced together with those of thequantitative research into the DS in health sectors. The purpose of a qualitative research is to interpret the phenomenonof a situation in question and why it happened, based on therecognition and interpretation of related parties. More specifically, it is an approach to comprehend the sense of value
and the concept within the social, political and economicalfactors through the behavioral patterns of a party of interest
such as the inhabitants. In the scene of an international cooperation, which is an external intervention, an effective usage of a qualitative research that reflects local needs without
being assertive should be a subject of discussion in the future. When one considers an optimal effect on the practicality of international cooperation in the health sectors, qualitative researches could be complementarily exercised withquantitative researches. For example, the typing of phenomena by a qualitative research could precede a quantitative
research or a qualitative research could also be utilized toreassume the appropriateness of the data collected by aquantitative research [10,11,12,13,14].

TECHNOLOGY TRANSFER
Technology transfer, ownership and follow-up in the
DS are components closely related to each other and will be
described together. In a DS, technology transfer works indifferent manners according to the level of development andmaturity of the counterpart countries. In the countries with ahigh level of problem consciousness as well as ownership,
one could expect their independent operation of the study
even at the level of planning formulation. To the contrary, as
is seen in many African countries with considerably low
level of development, there often can be confusions between a DS and a project finding mission because of thelack of understanding as to what a “development study” is.
For all the DSs conducted in the health sectors so far,with regrets, the ownerships of the studies have not been
well cultivated in Honduras, Kenya and Malawi. However,the participating consultants aptly compensated the ownerships with their expertise in formulating the planning. It
was perhaps from the DS of Laos that the current ownership
was transformed. The Laos study team worked on their task
by implementing strong incentives to the counterparts andadopting participatory methods. Throughout the course of
the study, the Japanese study team kept taking the position
to accord technical supports for the master plan which
would be exercised by the Laos side and consequently thecounterpart actively participated in the implementation of
the study. In the mean time, health forums under the auspices of the Laos Ministry of Public Health were held fivetimes. This was a stakeholder meeting which took placeacross the country with approximately 200 participants that
included the minister, executive officers of the ministry, local health officers, donors and NGOs. Earnest discussions
on programs or strategies were conducted during the courseof the master plan formulation. The final report of the study
was translated into Laos language immediately after thecompletion of the study. This translated version was distributed to the divisions concerned for enlightenment as well as
brainstorming, especially in the regional areas and this undertaking is still underway. The autonomic movement wasenhanced by the efforts of JICA advisor in the ministry.
Thus, the implementation of the DS in Laos permitted any
disputed points on health policy and programs to be on the
grounds for discussion.
In the case of Sri Lanka, the Japanese study team putemphasis on the participation of stakeholders. Specifically,
the counterpart independently organized weekly meetings
and working groups within the structure of the Ministry of
Health in their own realms. Most of the staffs at the positionof directors or higher were involved in elaborating the workof the master plan. In addition, a review panel consisting ofuniversity members, research institute staff and OBs of theministry commented on the draft of the master plan. Regional stakeholder meetings as well as seminars were frequently held to discuss the master plan in progress. Those
undertakings also gave an opportunity to the follow up of
the study.
During the DS of Uzbekistan, the analysis of survey
results and the discussion for the formulation of master plan
were implemented in workshops with specific minor groups
composing of members who were from not only the central
government but also the local governments, internationalorganizations and NGOs et al. The introduction of suchworkshops with diverse members could be a major characteristic of a DS study.
The principle of a Japanese DS implies versatile utilization of a master plan and does not particular assume theexecuting body in the materialization of a proposed program/project. However, a partner country is generally moreinterested in whether or not the consequences of DS could
lead to the implementation of the proposed programs/projects. Therefore, the Japanese side should unveil the definition of the socalled “batting average”. In other words, theyshould clarify the ultimate goal of the Japanese DS; by indicating whether the proposed programs/projects by the Japanese parties or other donors including international organizations would be implemented or whether the DS is only restricted to the master plan study itself. Second thought has
to be given during the processing of a DS and during coordination with the donors in order to find an optimal way.
At the moment, the accomplishment of a DS cannot be１７easily evaluated due to a lack of an established evaluationindex. Therefore, it is necessary to crystallize an index withtwo possible viewpoints: an evaluation of the contract on
the actual implementation of a study and an evaluation of
the extent a master plan can be realized several years after a
DS. The latter should include how a master plan is exploited and materialized. These evaluations should be conducted by not only an internal body, JICA, but also by one
or several other third parties.
Throughout the current research project, improved
quality of DS in the health sectors is definitely recognized,
especially in terms of technology transfer and the development of national ownership. Meanwhile, JICA performed
structural reform and reorganized the former principal assistant team system to five subject departments, namely, human development department, global environment department, rural development department and economic development department. This reorganization allowed a single department to take charge of a particular subject regardless ofthe different modalities for implementation. In addition,
JICA defined “program approach” as an aggregate of activities and policies leading to the comprehensive and coordinated planning and implementation of activities under theownership of the partner government. In the program approach, a variety of modalities, each having different advantages and disadvantages, were made available to meet thedifferent development needs in different countries and sectors. It is expected that projects integrated in the overall coordination framework would contribute more effectively tothe objectives of the overall program. At present, DS is being actively utilized under such programs and some DS
with clear and designated objectives have already been carried out. It is expected that such trend will be happening in
the health sectors soon. With that, it is necessary to prioritize research projects in terms of developing methods ofanalysis for health policies and health planning as well as
developing various study methods based on the program approach.

ACKNOWLEDGEMENT
The authors wish to thank Japan International Cooperation Agency for their assistance with the valuable dataand information. This study was supported by grants-in-aid
for research on international medical cooperation from the
Ministry of Health, Labor and Welfare, Japan.

REFERENCES
１．Development study, Japan’s ODA. Japan International
Cooperation Agency, 1999
２．Final report, the study on the strategies and plans for the
upgrading of health status in the Republic of Honduras,
System Science Consultants, 1996
３．Final report, the study on strengthening the district healthsystem in the western part of Kenya, Pacific Consultants
International, ICNet Limited, 1996
４．Final report, master plan study on strengthening primary
health care services in the Republic of Malawi, St.
Mary’s Hospital, Global Link Management, 1999
５．Final report, the development study on reproductive
health in the state of Madhya Pradesh, India, System Science Consultants Ins, 2002
６．Final report, the study on the improvement of health andmedical services in the Lao People’s Democratic Republic, Pacific Consultants International, 2002
７．Final report, the study on enhancement of district healthsystem for Beni Province Prefecture in the Republic ofBolivia, System Science Consultants Inc, 2003
８．Final report, Master plan study for strengthening healthsystem in the Democratic Socialist Republic of Sri Lanka,
Pacific Consultants International, 2002
９．Final report, the study on the restructuring of health andmedical system in the Republic of Uzbekistan, System
Science Consultants Inc, 2003
１０．Chris J. Jackson & Adrian Furnham. Designing and Analysing Questionnaires and Surveys: A Manual for HealthProfessionals and Administrators. London, Whurr Publishers Ltd. 2000
１
１．Patton, M. Q. (1987) How to Use Qualitative Methods in
Evaluation, London, Sage
１
２．Patton, M. Q. (2002) Qualitative Research & Evaluation
Methods, Sage Publications
１
３．Rice, P. L and Ezyy, D (1999) Qualitative Research
Methods-A Health Focus, Oxford University Press
１
４．Weiss, CH (1998) Evaluation: Methods for Studying Programs and Policies, 2nd ed. , Prentice-Hall

１
９

Jpn. J. Trop. Med. Hyg., Vol. 33, No.1, 2005, pp. 19-82

PROCEEDINGS OF THE 45TH ANNUAL MEETING OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
15-16 October 2004, Tokyo

President
Keizo Yamaguchi, M. D, DMSc
Professor & Chairman,
Dept. of Microbiology & Infectious Disease
TOHO University School of Medicine
CONTENTS
Special lecture
K1
International contribution in the control of tropical diseases through new drug development
Educational lectures
E-1
Role of phosphoinositide 3-kinase in parasite infection
E-2
Why Influenza Kills and will Kill Again
E-3
Current status of dengue vaccine development.
E-4
Bacterial intestinal infection and quorum sensing

Omura, S.

Koyasu, S.
Kawaoka, Y.
Kurane, I.
Kamiya, S.

Prize Winner’s lectures
The JSTM Award of Excellence
K2
Quo Vadis: From Bench To Field
JSTM Young Investigator Award
K3
Drug Susceptibilities and Clinical Manifestations of Mycobacterium tuberculosis in Zambia

Kojima, S.
Mitarai, S.

Symposium 1
S1-0
Symposium 1: Learning Clinical Tropical Medicine in the Topics
Oishi, K.and Nakamura, T.
S1-1
The short term training course in clinical tropical medicine in Philippines
Hasegawa, M.
S1-2
Activities of the student rotation program in San Lazaro Hospital, 2003
Yamashiro, T., Nishizono, A., Kadota, J. and Mifune, K.
S1-3
Clinical clerkship in tropical medicine in Peru.
Iwata, K.
S1-4
The clinical course in tropical medicine at Mahidol University in Thailand
Matsumura, T. et al.
S1-5
Association of increased platelet-associated immunoglobulins and the severityof disease in secondary dengue virus infections
Saito, M., Oishi, K.
S1-6
Development of Malaria Vaccine based on Recombinant SERA
Horii, T.
Symposium 2
S2-1
Role of infectious disease surveillance
S2-2
Risk Management of Infectious Disease at the border.
Role of Quarantine Station in ”Borderless” World
S2-3
Crisis Management for Infectious Diseases in Borderless Era-Role of Central GovernmentS2-4
Infectious disease outbreak and Press reports
S2-5
An outbreak of infectious disease hits civil society hard:
A case study of Japanese expatriate employees and their spouses in Hong Kong

Nakamura, Y. and Yokota, S.

Workshop 1
W1-0
Role of Nursing Pfofession and NGO in Tropical Medicine

Ishikawa, N.and Moriguchi, I.

Taniguchi, K.
Iwasaki, E.
Ushio, M.
Minami, N.

２
０

W1-1
W1-2
W1-3
W1-4

Traditional Medicine in Sri Lanka
Higuchi, M.
Community Health Nursing in Indonesia
Moriguchi, I.
NGO and International cooperation - the African experience
Tokunaga, M.
The Role of Social and Behavioral Research on Tropical Diseases Control : A Case Study of Tuberculosis.
Jintana Ngamvithayapong-Yanai,.

Workshop 2
W2-1
Overview
W2-2-1 Coccidioidomycosis
W2-2-2 A case of chronic dissemiated histoplasmosis emerged during steroid maintenance therapy
for rheumatoid arthritis with secondary amyloidosis and nephrotic syndrome
W2-2-3 Paracoccidioidomycosis
W2-3
Imported mycoses related to zoonoses
W2-4
Causative agents of imported mycoses

Takakura, S.
Igarashi, T.
Sano, A.
Nishimura, K.

Workshop 3
W3-1
Purposes of establishing preventive guidelines against malaria
W3-2
Mosquito Control for Malaria Personal Protection
W3-3
Chemoprophylaxis
W3-4Stand-by treatment
W3-5
Travel advice based on the guidelines for malaria prevention

Kano, S.
Haruki, K.
Okuzawa, E.
Kimura, M.
Hamada, A.

Kamei１, K.
Kawabata, M.

General presentation
1
Role of costimulatory molecules in host resistance to re-infection with Hymenolepis nana oncosphere
Onishi, K. et al.
2
Taeniasis in Bali, Indonesia
Yamasaki, H. et al.
3
The Study Of Ascaris And Ulcerative Colitis Part 2:
Localization Of Mucin 12 On Ascaris Lumbricoides HominisHayashi, I. et al.
4Characterization of the 150-kDa cysteine-rich surface proteins of Entamoeba dispar
Tachibana, H. et al.
5
A survey on preparation against possible resurgence of SARS
Ohara, H.
6
Ecological roles of mongoose in transmission cycle of Japanese encephalitis virus in Okinawa island
Saito, M. et al.
7
Shift of Japanese encephalitis virus propagating in Japan, 1935-2002.
Ma, S. et al.
8Japanese encephalitis in the Philippines
Inoue, S. et al.
9
Immunogenetic analysis of The patients with Dengue Hemorrhagic Fever in Vietnam.
Kikuchi, M. et al.
10
Status of Currently Circulating Dengue Virus Responsible for the Epidemic in Bangladesh in the Year 2002
Islam, M.A. et al.
11
Genetic variation in the 3’non-coding region of dengue virus type 1
Nukui, Y. et al.
12Real-Time Reverse Transcription Loop Mediated Isothermal Amplification (Rt-Lamp) Assayas a Rapid Diagnostic Tool for Emerging Viruses
Parida, M. et al.
13
Characteristic prevalence of HPV genotypes in penile cancer in northern Thailand
Toriyama, K. et al.
14
A new screening method for Salmonella using lemon slices
Midorikawa, Y. et al.
15
Molecular analysis of Haemophilus influenzae isolated from nasopharyngeal aspirate
and cerebrospinal fluid among children in Vietnam
Kaji, C. et al.
16
Buruli and the Ulcers Under the Tropics
Mwanatambwe, M. et al.
17
Epidemiological Study of Leprosy in a local village in Myanmar
Namisato, M. et al.
18
Diagnostic trial for cutaneous mycobacterial diseases by using PCR techniques.
Khan, M.A.K. et al.
19
The influence of circumstances to leprosy skin lesions.
Hosokawa, A. et al.
20
Effect of aphidicolin on Entamoeba excystation and metacystic development
Makioka, A. et al.
21
Molecular cloning and characterization of a protein geranylgeranyltransferase type I from Entamoeba histolytica
Kumagai, M. et al.

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１

22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53

The Curative Efficacy of Metronidazole on Intestinal Amebiasis
Kobayashi, S. et al.
The attachment and entry of Leishmania (Lesimania) major to macrophages:
Observation by scanning electron microscope
Uezato, H. et al.
Development of a sensitive and simple diagnostic method for visceral leishmaniasis using urine samples
Itoh, M. et al.
Establishment of a detection and identification method of Leishmania species
within naturally infected individual sandflies
Kato, H. et al.
Production of recombinant kinesin-related protein of Leishmania donovani
and its application in the urine-based ELISA for the diagnosis of visceral leishmaniasis
Islam, M.Z. et al.
Development and evaluation of a new antibody detection kit (Instant (TM) CHEK-Amoeba ) for amoebiasis
Ayi, I. et al.
The study of antigen for the new tuberculosis diagnosis and application to the serodiagnosis Yoshidome, A. et al.
N-terminus truncated recombinant SARS coronavirus nucleocapsid protein-based ELISA
is more specific for serodiagnosis of SARSYu, F. et al.
Paragonimus ohirai M. in the basin of Maruyama River, Hyogo Prefecture
Yamanishi, H.
A simulation of egg output school children infected with
Schistosoma heamatobium by an immigration-death model
Hamada, Y. et al.
Current epidemic state of Schistosomiasis mekongi in Lao PDR.
Nakamura, S. et al.
Water contact patterns and knowledge of schistosomiasis in north Zanzibar
Ataka, Y. et al.
Daily variation of Schistosoma haematobium egg excretion
Muriu, D.W. et al.
Experimental infections of black flies (Diptera: Simuliidae) with microfilariae
of Onchocerca dewittei japonica, the causative agent of zoonotic onchocerciasis in Japan
Fukuda, M. et al.
Application of urine-based ELISA to monitor the effect of mass-drug administration
for lymphatic filariasis control in a low endemic area
Kimura, E. et al.
A case study of tuberculosis genetic epidemiological study basedon the long-term International Collaborative Field Research in Thailand
Yanai, H. et al.
Study on transmission and infection of soil-transmitted nematode in the suburb of Hanoi,
Vietnam: contamination of vegetables and fingers and nail dirts with parasite eggs.
Fujimaki, Y. et al.
Problems in Dracunculiasis Eradication Activities in Ogou District, Togo
Afetse, Y.D. et al.
A Serosurvey of Human T-cell Lymphotropic Virus type 1 (HTLV-1) in the Dominican RepublicOno, T. et al.
Descriptive epidemiology of imported infectious disease cases from the national surveillance in Japan
Taniguchi, K. et al.
Case series of imported malaria in Okinawa, Japan
Haranaga, S. et al.
Characteristics of Imported Malaria in Japan
Miura, T. et al.
Malaria epidemiology in Lombok ans Sumbawa islands, Indonesia
Kanbara, H. et al.
Malaria control in Lombok and Sumbawa islands, Indonesia
Kanbara, H. et al.
Magnitude of Malaria Problem in Cambodia
Lek, D. et al.
Accumulated infection risks of malaria during five weeks in a living area of an ethnic minority in Vietnam.
Nakazawa, S. et al.
A real-time PCR assay for quantitative detection of pfcrt in Plasmodium falciparumand its application to epidemiology
Katakai, Y. et al.
An investigation of the relationship between release from chloroquine resistance and blood GSH levels
Miyata, Y. et al.
Protection effect on lysis of Malaria infected erythrocytes membrane by bisbenzylisoquinoline alkaloids
Kosuke, H. et al.
Reactivity of blood samples spotted onto filter papers in the WST-8 method for screening of G6PD deficiency
Arai, M. et al.
Structural-effect relationships of mother moieties in drug-resistance releaseing compounds
on the chloroquine resistant mouse malaria
Osa, Y. et al.
Interleukin 4 polymorphism and malaria endemicity in Melanesia.
Dzodzomenyo, M. et al.

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54
55
56
57
58
59
60
61
62
63

Malaria pressure influenced the frequency of a susceptible allele of TNF-alpha promoter region in Vanuatu
Ubalee, R. et al.
Research of the antimalarial drugs from the natural products in JPMW project
Ishiyama, A. et al.
Antimalarial drug development research against dug-resistant P. falciparum
Kim, H. et al.
The problems medical facilities abroad for Japanese expatriates.
Fukushima, S.
“Research Group on Chemotherapy of Tropical Diseases (abbr.)” during the past and the coming three years.
Kimura, M. et al.
Certification in Tropical Medicine in Germany
Hitani, A. et al.
A preliminary study on the image of tropical medicine and the inclination for medicine
and medical science by the sixth year students of School of Medicine, Chiba University
Naoi, K. et al.
Increasing number of HIV-positive mothers only partly explained the recent declineof birth weight in Republic of Centran Africa
Tokunaga, M. et al.
Physicians’ knowledge, attitude and practice toward sexually transmitted infections
and human immunodeficiency virus infection in Fuzhou city, Fujian, China
Cai, G. et al.
The study of health status for the inhabitants of rural ares on Vientiane province in Lao P.D.R.
Nishiyama, T. et al.

２３Special lectureK 1) INTERNATIONAL CONTRIBUTION IN THE CONTROL OF
TROPICAL DISEASES THROUGH NEW DRUG DEVELOPMENTSATOSHI OMURAThe Kitasato Institute, and Graduate School of Infection Control Sciences, Kitasato University, Tokyo, JapanIn 1987, the Research Center for Liver Diseases of The
Kitasato Institute was designated as a WHO Collaboration
Center for Reference and Research on Viral Hepatitis. The
Kitasato team headed by T. Takahashi has greatly contributed to the control of viral hepatitis in South East Asia
through the large-scale transfer of plasma-derived hepatitis
B virus vaccines, as well as through advances in technicalmethods for vaccine production.
We organized a joint program in 1999 for the screening of antimalarial compounds with the participation ofJapanese Pharmaceutical Companies, The Kitasato Institute,
the Japanese Ministry of Health Labour and Welfare, and
WHO TDR (JPMW). To this joint program, 14 Japanesecompanies and our institute have sent a total of more than
26,000 samples for evaluation of antimalarial activity. Several of the screened candidate compounds have been targeted for further development.
Avermectin was discovered in 1979 and developed

through joint research between The Kitasato Institute andMerck Sharp & Dohme Research Laboratories. Ivermectin,a dihydro derivative of avermectin has shown efficacy forthe treatment of onchocerciasis, which is one of the serious
tropical disease prevalent in tropical Africa and a part of
Latin America.
Administration of mectizan, a formulation of ivermectin for human use, at annual or semi-annual doses of 150
mg/kg is now being used to protect against onchocersciasis.
In 1999, the Mectizan Expert Committee, a joint projectwith Merck & Co., Inc. and WHO, decided to accelerate themectizan treatment program, and in the year 2003, morethan 56 million people in countries with endemic onchocerciasis received the drug. All of the mectizan tablets were donated by Merck & Co., Inc., and The Kitasato Institute, as apartner in the invention of ivermectin, officially agreed to
Merck’s contribution to the onchocerciasis control program.

２
４

Educational lecturesE-1) ROLE OF PHOSPHOINOSITIDE 3-KINASE IN PARASITE INFECTIONSHIGEO KOYASU１
１

Department of Microbiology and Immunology, Keio University School of Medicine ２Japan Science and Technology Agency

We have studied the role of class IA phosphoinositide3-kinase (PI3K) in immune responses using mice lacking the
p85alfa regulatory subunit of class IA PI3K (PI3K deficientmice). We have previously reported that PI3K is important
for the development and functions of B lymphocytes. We
have also found that PI3K is involved in the development ofmast cells as PI3K deficient mice lack gastrointestinal mast
cells but not dermal mast cells. In addition, PI3K deficient
mice have some defects in the induction of Th2 response
and exhibit enhanced Th1response. Accordingly, PI3K deficient mice on a BALB/c background were susceptible to in-

fection by the intestinal nematode, Strongyloides venezuelensis, but were resistant to Leishmania major by eliciting an enhanced Th1response. In vitro studies showed thatnumerous stimuli inducing IL-12 production concomitantly
elicited PI3K activation in dendritic cells (DCs), but both PI
3K deficient DCs and wild type DCs treated with PI3K inhibitor demonstrated increased IL-12 production. The presence of negative feedback mechanisms for regulation of IL12 production during DC activation would contribute toprevention of excessive Th1 polarization causing undesirable immune responses.

E-2) WHY INFLUENZA KILLS AND WILL KILL AGAINYOSHIHIRO KAWAOKA
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science University of Tokyo, Japan

In the past century, there were three influenza pandemics (worldwide outbreaks): the Spanish flu in 1918, theAsian flu in 1957, and the Hong Kong flu in 1968. Amongthese pandemics, the Spanish flu was the most devastating,killing more than 20 million people worldwide. In addition
to these pandemics, yearly influenza epidemics occur, causing increased morbidity and mortality, particularly in vul-

nerable populations like the elderly. Moreover, the economic impact of influenza cannot be disregarded; huge
losses occur as a result of lost work and increased healthcare expenditures during epidemics. In my talk, I will discuss our recent findings that may lead to the development ofantiviral measures to quell the impact of this virus.

E-3) CURRENT STATUS OF DENGUE VACCINE DEVELOPMENT.
ICHIRO KURANE
Department of Virology 1, National Institute of Infectious Diseases

There are 4 dengue viruses: dengue virus types 1, 2, 3,
and 4. These 4 dengue viruses are antigenically related, butdifferent species. Dengue viruses cause dengue fever (DF)and dengue hemorrhagic fever (DHF). DF is a self-limited
febrile illness, while DHF is a fatal illness characterized byhemorrhagic manifestation and plasma leakage. Development of dengue vaccine has been attempted for over 30

years. Several kinds of dengue vaccine candidates are atvarious development stages. The strategy is that 4 monovalent vaccines are developed against each of 4 dengue viruses and a tetravalent vaccine is prepared by mixing the 4
monovalent vaccines.
Two major groups are working on development tetravalent live attenuated dengue vaccine. Vaccines against each２５

of 4 dengue viruses induced expected levels of neutralizing
antibody and very few adverse events were reported. Tetravalent dengue vaccine candidates are at phase II studies.
Yellow fever (YF)-dengue chimera vaccine has also beendeveloped. Yellow fever vaccine 17D strain was used. The
preM and E genes of YF 17D strain were replaced by thoseof each of 4 dengue viruses. Four YF-dengue chimera viruses were mixed and a tetravalent dengue vaccine candidate was prepared. The tetravalent YF-dengue chimera vaccine candidate induced high levels of neutralizing antibodies to 4 dengue viruses. Presence of immunity against yellow fever virus did not inhibit the chimera vaccine from in-ducing dengue virus-specific antibodies. This vaccine iscurrently at phase II study. Dengue-dengue chimera vaccine
using attenuated dengue 2 and attenuated dengue 3 viruses
are also under development and pre-clinical studies havebeen completed.
It is expected that phase Ⅲ study will be done withsome of these candidate dengue vaccines in the near future.
Selection of the countries and areas has been started forphase Ⅲ study. It is, however, still not determined whether
efficacy should be evaluated for prevention of DF or DHF,
or how long vaccinees should be followed to confirm theabsence of increase in DHF.

E-4) BACTERIAL INTESTINAL INFECTION AND QUORUM SENSINGSHIGERU KAMIYA
Department of Infectious Diseases, Kyorin University School of Medicine, Tokyo, Japan

Expression of bacterial genes is regulated depending
on cell density among wide range of bacteria. This regulation system is called as quorum sensing (QS). QS is controlled by autoinducer (AI) produced by bacteria depending
on cell density. As gene expression of bacteria is regulated
by AI, it is possible that QS communicated by AI is considered to be the language of bacteria. Some Gram-positivebacteria produce different types of olidopeptide AI. The oligopeptide AIs are secreted via ATP-binding cassette (ABC)transporter exposed on the surface of bacteria. It has beenreported that more than 25 species of Gram-negative bacteria have QS. AI related with QS in Gram-negative bacteria
is called as AI-1, which is acylated homoserine lactone(HSL). AI-1 is synthesized by LuxI protein. AI-1 freely diffuses through the cell membrane and accumulates at highcell density. At high AI-1 concentration, the Lux R binds totheir cognate AI-1. AI-1/LuxR complex binds to promoterof the target gene and stimulate its transcription. AI-2 isproduced by both Gram-positive and -negative bacteria. As
the function of AI-2 is not limited to the bacteria producing
its own AI-2 and AI-2 communicates QS beyond bacterial

species, AI-2 is called as universal language of bacteria. AI2 has been recently shown to be furanosly borate diester,and is synthesized by LuxS. More than 30 species of bacteria such as Vibrio harveyi, Vibrio fischeri, Escherichia coli,
Salmonella, Shigella, Helicobacter pylori, Bacillus subtilis
and Campylobacter jejuni have been reported to have QS
communicated by AI-2. There have been several reports that
QS communicated by AI-2 is associated with bacterial
pathogenesis. It was reported that decrease in motility andproduction of Vero toxin (Stx2) was detected in luxSinactivated isogenic mutant of enterohaemorrhagic E. coli
(EHEC). Recently, it has been reported that EHEC produces
a novel AI-3, and very interestingly, AI-3 might be also produced by intestinal floral bacteria. It is well known that
EHEC is able to induce apparent infection including hemolytic uremic syndrome (HUS). It is possible that AI-3 produced by intestinal flora assist QS in the infection withsmall number of EHEC. Progress in the research on correlation between QS and pathogenesis in bacterial infection is
expected.

２
６

Prize winner’s lectures
The JSTM Award of ExcellenceK 2) QUO VADIS: FROM BENCH TO FIELDSOMEI KOJIMAAsian Centre of International Parasite Control (ACIPAC), Faculty of Tropical Medicine,
Mahidol University, Bangkok 10400, ThailandTh2 responses are considered characteristic tohelminth infections. Therefore, it is important to clarify thecellular and molecular mechanisms involved in Th2 responses and also the role of the responses in terms of pathogenesis and protection in parasitic infections. We demonstrated IgE-dependent eosinophil cytotoxicity to S. japonicum larvae, and established a monoclonal IgE antibody specific to paramyosin. We detected paramyosin in postacetabular glands as well as in the muscle layers and tegument of young schistosomula, suggesting paramyosin as agood candidate for schistosomiasis vaccine. To better understand the role of Th1/Th2 responses at the molecularlevel in protection against N. brasiliensis and P. berghei infection, we examined IRF-1-/-mice. Unexpectedly, inductionof IFN-γ mRNA occurred in the mutant mice infected with
P. berghei, and the existence of the IL-12-independent
novel pathway distinct from the classical Th1/Th2 differentiation pathway was suggested. Results of a study on a potential role of IL-18 in patients with severe falciparum malaria suggested that IL-18 plays a key role in the disease severity through a pathway of elevating IFN-γ rather than its
IgE inducing activity. In a comparative study in a cerebral
malaria (CM)-susceptible or-resistant strain of mice with P.

berghei ANKA infection, we observed that in the brain,
both strains expressed IP-10 and MCP-1 genes as early as at
24 hr postinfection, and that the expression of thesechemokine genes in KT-5 was induced in vitro upon stimulation with malarial antigen, suggesting that the direct involvement of brain parenchymal cells occurs in response toplasmodial infection, and providing a new aspect to analyse
possible mechanisms of CM. Apart from bench work,
guided by an innermost voice saying “Quo vadis?”, theauthor joined to the JICA-ACIPAC project established under the Global Parasite Control Initiative (Hashimoto Initiative). Many issues came out during the process of the project concerning mainly its management. Fortunately, mostof the issues have been solved by support from the advisoryboard of the project in Japan, in which members of theJapanese Societies of Tropical Medicine and Parasitology
are cordially involved. Thus, just like Pandora’s box, many
parasites and unsolved issues came out even from theauthor’s small experience, but hope still remains there inthat we could overcome disaster caused by infectious diseases eventually, if young scientists would consider unsolved issues to be challenges for themselves.

JSTM Young Investigator Award
K 3) DRUG SUSCEPTIBILITIES AND CLINICAL MANIFESTATIONS OFMYCOBACTERIUM TUBERCULOSIS IN ZAMBIASATOSHI MITARAI
Bacteriology Division, the Mycobacterium Reference Centre,
the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association

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. In Zambia, 73% of the adulttuberculosis 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. The shortages of both
medical resources and knowledge about the disease are im-

２
７pediments to adequate treatment of patients, resulting in an
increase in drug resistant M. tuberculosis. This study was
conducted to obtain data related to the spectrum of drug resistant Mycobacterium tuberculosis. A total of 499 strainsof M. tuberculosis were tested for susceptibilities of isoniazid (INH), rifampicin (RFP), ethambutol (EMB) and streptomycin (SM). Totally 225 patients were also evaluated fortheir 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% and 9.7% to EMB, and3.8% and 3.2% to SM respectively. There were no significant differences between primary and acquired 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 and radiologicalinformation was not useful for the prediction of susceptible
and resistant strains. The HIV serostatus was not associatedwith 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 theefficient implementation of the directly observed treatment
with short course chemotherapy (DOTS) and interrupt thefurther dissemination of resistant M. tuberculosis in thecommunity. The drug resistances must be surveyed continuously to obtain useful clinical perspective and evaluate theeffectiveness of tuberculosis control program.

２
８

Symposium 1
S 1-0) SYMPOSIUM1: LEARNING CLINICAL TROPICAL MEDICINE IN THE TOPICSKAZUNORI OISHI１, TETSUYA NAKAMURA２
１

Department of Medicine, Institute of Tropical Medicine, ２The Institute of Medical Science, The University of Tokyo

Since tropical diseases such as malaria and dengue areobserved only as imported infectious diseases in Japan,
Japanese clinicians have relatively little experience of these
diseases. In this symposium, six speakers made presentations on tropical infectious diseases in the tropics. Four introduced their experiences in a clinical training course, andtwo discussed their clinical research.
Dr. Yamashiro, Oita University, described a short-term
training course for medical students at San Lazaro Hospital,
Metro Manila, Philippines. Dr. Hasegawa also introduced athree-week training course for young medical doctors at thesame hospital. This course was supported by the COE program of Nagasaki University. Dr. Iwata presented in a onemonth clinical training course at Instituto de Medicina
Tropical Alexander Von Humboldt, Lima, Peru. Dr. Matsumura described a six-month diploma course in tropical
medicine and hygiene at Mahidol University in Thailand.

Mariko Saito, a doctoral student of pharmaceutical science, presented data from the Philippines on the mechanismof thrombocytopenia in secondary dengue virus infection.
She also experienced dengue illness as a researcher. Dr.
Horii presented exciting data on the successful developmentof a malaria vaccine called recombinant SERA. He developed this vaccine project and is now evaluating the effects
of SERA vaccine in Apac, Uganda. Scientific achievements
will be of interest to young clinicians who are studying
tropical infectious diseases.
Previous clinical training courses in the tropics were
hampered by a lacking of funds and publicity. The information on training courses presented in this symposium should
be distributed to Japanese clinicians who are interested intropical infectious diseases. Awareness of these courses may
inspire them to enter the field of clinical research in tropical
medicine.

S 1-1) THE SHORT TERM TRAINING COURSE IN CLINICAL TROPICAL MEDICINE IN PHILIPPINES
MAIKO HASEGAWA
Dep. of Vector Ecology & Environment,Inst. of Trop. Med., Nagasaki Uni, Nagasaki, JapanAs the increase in the circulation of goods and people
is making the connection between different regions of theworld tighter, global-scale countermeasures against infectious diseases and enhancement of the disease monitoring
systems in every aspect are required. Physicians in Japanare responsible for the early detection and treatment of diseases, the exchange of information, adequate responses tothe incidence of the diseases, and the improvement of theirown abilities.
The purpose of the short-term training course of tropical medicine in the Philippines is to improve the ability ofclinical physicians involved in tropical infectious diseases
and emerging/reemerging infectious diseases. A one-week
preliminary course was conducted in February, and a threeweek training course will be held in September 2004, in
which 6 physicians, selected from applicants from all overJapan, will participate. The course consists of lectures andclinical training by specialists at San Lazaro Hospital,
which specializes in infectious disease, and at St Luke’s
Medical Center, which is equipped with state-of-the-art
medical facilities, both of which are in Manila. A visit to theWorld Health Organization West Pacific Regional Office(WPRO) is also planned.
Fortunately, I had the opportunity of attending the preliminary course and this year’s course. I consider that thistraining course has three advantageous points. Firstly, trainees can observe the course of tropical diseases of interest.
The experience of observing the course of diseases that are
rare in Japan such as dengue, tetanus, typhoid, and leptospirosis, would certainly strengthen the practical ability of the
trainees. Secondly, problems of the health care systems in
the Philippines makes us realize the importance of global
thinking about the problems of infectious diseases rather
than as domestic problems. For example, the appearance of２
９drug-resistant tubercle bacilli in tuberculosis wards is being
recognized as a problem in the near future. Thirdly, it would
promote exchange of information about the status of, andproblems with, countermeasures against infectious diseases
between staff in WPRO and Japanese institutions. We
learned about the status of avian influenza and the strategyof the WHO in February.

In the present medical system in Japan, it is impossible
to take a leave for 3 weeks to attend the course without theunderstanding of colleagues. It is hoped that the necessity insuch training in tropical infectious diseases will be appreciated by everybody.
In the symposium, I will present the plans of thiscourse held in this September.

S 1-2) ACTIVITIES OF THE STUDENT ROTATION PROGRAM IN SAN LAZARO HOSPITAL, 2003
TETSU YAMASHIRO１, AKIRA NISHIZONO２, JUNICHI KADOTA２, KUMATO MIFUNE３
１

Institute of Scientific Research, Oita University, Oita, Japan２Department of Infectious Diseases,
Faculty of Medicine, Oita University, Oita, Japan３Oita Kouseiren Tsurumi Hospital, Beppu Oita, Japan

Oita University (OU) has a unique educational program in the field of medicine aiming at providing an opportunity for students to learn about infectious diseases not
usually found in Japan. The program has started since year
2003 in collaboration with San Lazaro Hospital (SLH), Manila. Implementation of the program is justified by the Academic Exchange Agreement contracted between OU and
SLH in year 2001. The program has unique characteristics
of: 1) OU gives academic credit for participated students, 2)
OU provides an intensive preparatory program, 3) OU offers a financial support for participants, and 4) SLH staff
prepare program curriculum and do tutoring.Nine medical
students (4th year) participated in the program implemented
in the period of Nov 9 to Nov. 22 year 2003, and 4 OU
staffs (3 lecturers and 1 administrative personnel) accompanied. The students attended Bed-side training in the morning, and lectures in the afternoon. All tutoring and lectures
were made by SLH staffs. In order to facilitate the students
participation in the program, we have provided intensive
preparatory lectures of infectious diseases and of diagnostics before starting of the practice. We have helped the students for preparing a lecture towards SLH staffs regarding

history and a current status of pulmonary tuberculosis in Japan. Eight departments of the Faculty of Medicine OU havebeen involved to support the program. All participants werecovered by travelers insurance and vaccinated for HAV,
Diphtheria, Tetanus, HBV, JEV, Measles, Rubella, Rabies,
and Influenza.The evaluation of activities of the program
was made in both directions. SLH staff gave high rating to
the students in terms of comprehension of lectures and motivation for participating in the program. The students appreciated SLH staff’s teaching and lectures. The students
studied 31 distinctive diseases or syndrome just for 10 days.
Each student prepared 2 case reports for patients with typical symptoms. Adding this, they compiled CD or DVD recording program activities, lecture files, and their own lecture files.The student rotation program is highly effective interms of providing an opportunity for studying infectious
diseases in a short period. In order to improve the program,a sophisticated organization is needed for 1) showing practical and attractive educational contents to the students: and2) offering proper credit for participating in the project tothe staffs of both parties.

S 1-3) CLINICAL CLERKSHIP IN TROPICAL MEDICINE IN PERU.
KENTARO IWATA
Division of Infectious Diseases, Kameda Medical Center

Many facilities provide tropical medicine course.
Some provide microbiological expertise, and the others do
epidemiological skills. Not a lot of institutes, however, havetropical medicine course with actual clinical practice, whichenable us to see real patients and manage them with diagnostic work up and treatment.

３
０

The Gorgas course is one of few these programs,
which is affiliated with The University of Alabama at Birmingham. The course is held at Instituto de Medicina Tropical “Alexander Von Humbolt” (IMT) and Universidad Peruana Cayetano Heredia (UPCH), located in Lima, Peru. The
author was given privilege to spend a month at IMT and
UPCH, learning tropical medicine at bedside. Peru is acountry in South America, shaping a long needle runningfrom north to south. It has variety of geographical charac-

teristics, including coastal area where you can see cholera
patients, or jungle area where you can experience casessuch as malaria or yellow fever. Chagas disease, rabies, andcutaneous leishmaniasis are another examples you can see
in Peru. And of course, many many HIV patients. Clinicalclerkship at outpatient and inpatient settings is invaluable asa clinician. It is highly recommendable course for many
tropical medicine and infectious disease specialists.

S 1-4) THE CLINICAL COURSE IN TROPICAL MEDICINE AT MAHIDOL UNIVERSITY IN THAILAND
TAKESHI MATSUMURA, TETSUYA NAKAMURA, AIKICHI IWAMOTO
Division of Infectional Diseases and Immunology, Institution of Medical Science, Tokyo University

Mahidol University runs a number of internationalcourses. One of them is “the Graduate Diploma in Tropical
Medicine and Hygiene”. An objective of this course is to
produce graduates with knowledge of tropical health problems and diseases. Another is to produce graduates who arecompetent in managing and providing advice about common health problems and diseases of the tropics. The course
runs annually from April to September. The course is taught
in English and is open to participants from all nations. All
the students are required to hold a Medical License andhave clinical experience of more than 1 year. This year’s intake comprises of 32 students from 18 different countries,
including six doctors from Thailand who are working at the
University’s Rajivithi Hospital of Tropical Medicine. Mostof the students are from Asia, especially the neighboring
Mekongi countries.
The DTM&H course consists of 9 compulsory modules and an elective module. The former includes the broad
range of tropical diseases with some emphasis on thosecommon in Thailand, for example malaria, dengue haemorrhagic fever, opisthorchis and gnathostomiasis. Multi-media
lectures are supported by laboratory sessions, involving examination of clinical specimens and demonstrations. There
are also discussion sessions and group work about clinicalcases, literature reviews, and how to address problems indeveloping countries.
There are many opportunities for clinical experience.
In June and July there are two field trips to rural areas. During these trips the students examine stool and blood samples
to assess disease burden and run a small clinic for the local
people. We also collect mosquites, rodents, and ticks tostudy the different vectors of tropical diseases and assess
the control programmes. The Rajivithi hospital has 100
beds and is next door to the faculty. All malaria and othertropical disease cases in the area are referred for treatment.
There is formal bedside teaching from our lecturers; outpatients clinics and the junior doctor introduce us to any patients of interest. We have seen numerous adult cases of severe malaria with cerebral, renal and pulmonary complications.
In the last 2 months of the course students study elective subjects. The elective options include areas such as
Medical Problems in the Tropics, Paediatric Problems in the
Tropics, Nutrition, Vector Control, Immunology, and so on.
The unique features of this course are the wide culture
mix of doctors attending; the access to clinical cases andspecimens; the field trips and close links to brilliant research units.

３１

S 1-5) ASSOCIATION OF INCREASED PLATELET-ASSOCIATED IMMUNOGLOBULINSAND THE SEVERITY OF DISEASE IN SECONDARY DENGUE VIRUS INFECTIONSMARIKO SAITO１, KAZUNORI OISHI１
１

Dept of Internal Medicine, Univ of Nagasaki, Nagasaki, Japan

Dengue virus is a mosquito-borne human viral pathogen that belongs to the genus Flavivirus of the family
Flaviviridae. It has recently become a major public healthconcern particularly in tropical and sub tropical countries
and predominantly in their urban and peri-urban areas. The
Philippines is a tropical country and thousands of cases arereported here every year. Dengue infections remain the major cause of morbidity and mortality in this country. Severe
thrombocytopenia and increased vascular permeability are
two major characteristics of dengue hemorrhagic fever(DHF). The present investigation attempted to elucidate themechanism of their action by observing the relationship between a) platelet count and platelet-associated IgG (PAIgG)
or IgM (PAIgM) and b) disease severity and PAIgG or
PAIgM in 78 patients enrolled in a prospective hospitalbased study. These patients with secondary infection wereadmitted in a hospital in Metro Manila. Forty patients werediagnosed to have dengue fever and thirty-eight had dengue
hemorrhagic fever based on WHO criteria (1997). A decrease in platelet count was observed during the acute phaseof infection, while there was a significant recovery in countduring the convalescent phase. With respect to PAIgG or

PAIgM, a significantly high level of these antibodies wasfound from patients in the acute phase in comparison to thatof the healthy volunteers. There was a significant decreasein either of these antibodies during the convalescent phase.
An inverse correlation between platelet and PAIgG or
PAIgM levels was found in the acute phase. Anti-dengue virus IgG or IgM was eluted from platelets of 10 patients inthe acute phase of secondary infection. Increased levels of
PAIgG or PAIgM were significantly higher in DHF patients
than those of DF. An increased level of PAIgM was associated independently with the development of DHF, representing a possible predictor of DHF with high specificity.
Our present data suggest that platelet-associated immunogloblins involving anti-dengue virus activity play a pivotal
role in the induction of thrombocytopenia and the severityof the disease in secondary dengue virus infections.
Collaborator: Institute of Tropical Medicine, Nagasaki University; Shingo Inoue, Kouichi Morita, St. Lukes Medical
Center; Matias RR, Natividad FF, San Lazaro Hospital;
Alera TP, Dimaano EM

S 1-6) DEVELOPMENT OF MALARIA VACCINE BASED ON RECOMBINANT SERATOSHIHIRO HORII１
１

Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University

Malaria remains as one of the most devastating humaninfectious diseases. The appearance of malaria parasites
with resistance to anti-malarial drugs and mosquito vectors
with resistance to insecticides has made it more difficult toprevent and cure the malaria infection. It is therefore of increasing importance to develop malaria vaccines. For developing an effective malaria vaccine, the antigen moleculeshould be derived from the target antigen in human protective immunity against malaria, however the human malaria
immunity is not well defined to identify the major target antigens. We have worked on malaria vaccine developmentwith a recombinant protein based on N-terminal domain ofP. falciparum serine repeat antigen (SERA). The recombinat

structure was produced in E. coli by the synthetic gene thatis maximized for the protein production. The field researchwith the recombinant SERA molecule revealed that the acquired malaria immunity is largely associated with the sero
positive againt our recombinant SERA. Our findings
strongly suggest that the N-terminal domain of SERA is
one of Achilles heels of malaria parasite. In collaboration
with The Research Foundation of Microbial Diseases ofOsaka University, the recocmbinant SERA protein, SE36,
was produced under GMP conditions. SE36 protein adsorbed to hydoroxyalminum was highly immunogenic in
Chimpanzees. In addition, clinical grade SE36 provided
significant protection in Squirrel monkeys after P. falcipa-

３２

rum challenge infection (Collaborative work with the Department of Molecular Immunology, School of Agricultureand Life Sciences, University of Tokyo).

３３

Symposium 2
S 2-1) ROLE OF INFECTIOUS DISEASE SURVEILLANCEKIYOSU TANIGUCHI
Infectious Disease Surveillance Center, National Institute of Infectious Diseases.

Infectious disease outbreaks continuously occur in theworld, and deforestation and urbanization will increase therisk of emergence of novel pathogens which have lived behind nature for a long time. And current globalization of
travel and trade will facilitate the global spread of disease.
Epidemic of infectious disease in one country might be an
immediate threat to the rest of the world.
Under these circumstances, infectious disease surveillance at global level is quite important for early recognition
and rapid containment of outbreaks of potential international concern. To investigate and follow up outbreak reports, WHO established an innovative mechanism, outbreakverification in early 1997.
Outbreak verification is based on a broad range of information sources, including national institutes of publichealth, WHO offices at regional and national level, theUnited Nations system, non-governmental organizations,
WHO collaborating centers, news medias. After picking up
some events from thousands of rumors, these events are assessed as of potential international importance. Definitionof potential international importance includes (1) Unknown
illness, (2) High mortality or morbidity, (3) international
disease spread or interference with international travel ortrade, (4) International assistance required for containment,and (5) Suspected accidental or deliberate release of bio-

logic agents. Once the event is determined to be publichealth emergency of potential international importance, theprocess of verification by communicating with affected
countries is initiated. Through this verification process,WHO could get more information and discuss about how
international society can support affected country.
But all above activities are voluntary so far. It meansthat if an affected country is reluctant to providing information to the global community, nothing to do. In order to provide global surveillance with legal background, revision ofInternational Health Regulation is now on going.
Of course, good national level surveillance is a base ofthis global surveillance. Each country should detect as earlyas possible for protecting not only people in the country but
also people living in global village. To enhance the surveillance system at national level, many technical approaches
have been made including syndromic surveillance, OTC
surveillance, outbreak surveillance, and so on. But clinician’s awareness of rare infectious diseases or tropical diseases is most important for improving surveillance. Nowadays all physicians in Japan should think about global situation of infectious disease for daily outpatients with proactive measures and preparedness as risk management in thehospital.

S 2-2) RISK MANAGEMENT OF INFECTIOUS DISEASE AT THE BORDER.
ROLE OF QUARANTINE STATION IN “BORDERLESS” WORLDEMIKO IWASAKI
Sendai Quarantine Station

Today, with advancement of transportation, international traffics of people and goods have increased dramatically. This “globalization” has also caused global spreads ofvarious infectious diseases.
Especially, in recent years, there have been many outbreaks of severe and highly infectious zoonosises, and these
diseases spread rapidly in this globalized world.
The SARS outbreak in Asia in 2003 showed us the potential

danger of infectious diseases in today’s world, and it gave
us an opportunity to evaluate our policy on infectious disease control.
In fact, during SARS outbreak, Quarantine Stations at
airports in Japan asked international travelers entering to thecountry to answer a questionnaire and also asked them totake their body temperature. In addition, because it is impossible to identify patients in latency period at the border,

３
４

any health problems after entering the country had to be reported to Quarantine Station and traced.
In addition, because these situations that many of infectious diseases are entering to the county are new to Japanese medical institutions, it is very difficult to manage it forthem. So it is important for Quarantine Stations to support

these institutions to diagnosis and treat infectious diseases.
Furthermore, in this globalized world, every country
recognized that it is also important to prevent any patientswith serious infectious disease from leaving the country. So,
we may need to quarantine anyone leaving the country also.

S 2-3) CRISIS MANAGEMENT FOR INFECTIOUS DISEASES IN BORDERLESS ERA-ROLE OF CENTRAL GOVERNMENTMITSUHIRO USHIO
Director, Infectious Disease Control Division, Health Service Bureau, Ministry of Health, Labour and Welfare

One of the lessons we have learned from the SARS
worldwide outbreak we experienced in 2003 is that there is
no border for infectious diseases, as in the world we live ininternational movements of both people and goods are so
common and rapid with highly developed transportation
systems and prosperous worldwide trade. At the same timeit once again reminded us how severe consequences animal
borne diseases could cause and the need to commit fully tocontrolling through epidemic alert and responses other animal borne diseases such as avian influenza and West Nile
fever which are now increasing in case numbers. We arealso seeing increase of some of the re-emerging diseases
such as tuberculosis which was once thought to be undercontrol. One of the tricky things about infectious diseases is
incubation period; it easily happens that someone is exposed to a virus and get infected in one place and be half aworld away a day later when he becomes symptomatic andspread a disease in the opposite side of the world where hegot the pathogen. We certainly come to recognize the needto improve preparedness for responding to, and mitigating
the public health, economic, and social consequences of any
infectious disease that could possibly pose a serious threat

to global health security.
To cope with such a situation, Japanese central government have been taking necessary measures, including establishing the Law concerning the Prevention of Infectious
Diseases and Medical Care for Patients of Infectious Diseases in 1999. It is based on the old Communicable Diseases Prevention Law which was established more than 100
years ago, but a completely new concepts were introduced
such as surveillance system, laying out proactive plans tocombat infectious diseases outbreak, respect for humanrights and regulation concerning animal borne diseases including introduction of quarantine for animal import. Recent outbreak of SARS in 2003 and US anthrax bioterrorism in 2001 urged us to amend this law in 2003 tostrengthen central government competence to cope withlarge scale outbreak and also to strengthen quarantine system and regulation concerning animal borne diseases and to
improve preparedness for possible use of a biological agent
to cause harm.
Here is a general overview of Japanese national planfor infectious disease control from legislation point of view.

S 2-4) INFECTIOUS DISEASE OUTBREAK AND PRESS REPORTS
NAOKI MINAMI
News Commentators Division, Japan Broadcasting Corporation, Tokyo, Japan.

(1) The control of SARS and the role of press reports
To prevent the outbreak of SARS, the role of medical
technology and administrative organization is important.
But it is difficult to control it only with them.

When the SARS vaccine and the treatment aren’t established, the citizen must prevent SARS with fundamental
sanitation knowledge , and it is necessary to cooperate withthe public health countermeasures. Part of the press is the３５

receiver of infectious disease information ,and also thesender.
(2) The outbreak of SARS in China and report
The progress of the Chinese SARS outbreak showsthat, if sufficient information hadn’t been provided, the outbreak caused the spread of groundless rumor, and a social
panic followed, when people were sincerely looking for information .
(3) Japanese SARS reports
About Japanese SARS reports, the significant parts
were , the occurrence in the foreign countries, the influenceon the economy and society,the pathogen of this emerging
infectious disease, the route of infection, the diagnostic procedure,and the progress of the research on the prevention
and the treatment, the reports collected strong concern.

Furthermore, the role of the press was to grasp the
problems of the Japanese prevention system of an infectious
disease ,and institute the improvement.
(4) Preparing for the outbreak recurrence.
SARS is the１st infectious disease of the infectious
disease law, and the role of the national government to the
local government was reinforced in Japan.
If it is compared with a moment in 2003, it can be said
that the system of health, medical treatment has been expanded, and you must prepare for it more.
It is important to introduce information to the public
while giving careful consideration to the protection of privacy and from harmful rumor to avoid the social amplification which the public is anxious about infectious disease
outbreak.

S 2-5) AN OUTBREAK OF INFECTIOUS DISEASE HITS CIVIL SOCIETY HARD: A CASE STUDY OF
JAPANESE EXPATRIATE EMPLOYEES AND THEIR SPOUSES IN HONG KONG
YASUHIDE NAKAMURA, SACHIKO YOKOTA
Research Center for Civil Society, Graduate School of Human Sciences, Osaka University, Osaka, Japan

A study by Yokota into “psychosocial support for Japanese Hong Kong residents during the SARS crisis” aimed toinvestigate the feelings and thoughts of Japanese expatriate
employees and their spouses based in Hong Kong at thattime. Field research was conducted from July to August
2003 using semi-structured interviews and a cross-sectionalquestionnaire survey. 116 Japanese businesspeople (response rate: 74%) at10Japanese companies and 70 spouses(response rate: 61%) responded to the questionnaire. The
fact that they could not tell what information was correct (inspite of the large volume of available information) due tothe unknown nature of the disease, stressed 94% of the expatriate employees and 97% of their spouses. 66% of theexpatriate employees and 65% of the spouses responded
that they felt abandoned by Japan. The businesspeople complained about the Government of Japan and their companies
placing first priority on the situation inside Japan. As fortheir sources of information; while they got information
through Japanese media (television or newspapers), most ofthem also relied on the Hong Kong Department of Health’s
web-site and the information pamphlets provided by theirown companies. The Department of Health disclosed infor-

mation in detail on their web-site, while protecting personalprivacy. Companies selected the information that was particularly pertinent for their employees.Crisis management
for infectious diseases should be implemented without making those living in infected areas feel unduly isolated. The
government and the media should consider the humanrights of such people. Psychosocial support offered to themshould be strengthened. For example, warm messages fromoutside the infected area encouraged the people who werefighting against this newly emerging disease or who were
facing the possibility of dying from the disease. People in
infected areas would mostly welcome visits from healthprofessionals during an epidemic. The study revealed thatmany people received sympathy from others within the isolated infected area and that they felt an increased solidarity
with them as a result.We would like to recommend thatgovernments and the mass media disclose detailed information, while respecting personal privacy, and that they report
the facts that are thought to be most reliable at that time.
Remember the lessons learned from the experience of Hong
Kong!

３
６

Workshop 1
W 1-0) ROLE OF NURSING PFOFESSION AND NGO IN TROPICAL MEDICINENOBUKATSU ISHIKAWA１, IKUKO MORIGUCHI２
１

The Research Institute of Tuberculosis Japan Anti-Tuberculosis Association, Tokyo, Japan
２
College of Nursing Art and Science, University of Hyogo, Hyogo, Japan

In this workshop, field based studies on various topics
were reported and discussion was made on the roles ofnursing professions and NGOs in tropical medicine, which
was the first one in the history of Japanese Society of Tropical Medicine (Society hereafter). The speakers were all
nurses in basic education and professionally top-level people with PhD as researchers cum teachers cum field workers.
Their presentations were all impressive and provoking as
all from long-term studies showed more human aspects thandiseases either of the providers at the frontline or clients inthe community, which have not been covered, in the conventional tropical medicine. The expansion of the concept
of conventional tropical medicine to wider holistic one,which should be more action oriented, resulting in the expansion of the topics and members of the Society. Dr.
Higuchi reported the use and perspectives of traditional
medicine from her study in Sri Lanka. Dr. Moriguchi reported the importance of participatory research in healthproblems with the frontline workers in Indonesia. Dr. Jintana showed the role of social and behavioral science research in community care through her study on TB/HIV
care in northern Thailand. Dr. Tokunaga discussed the effective role of NGO at the frontline from her long-term commitment in Africa. This kind of workshop needs to be held
further until a specific program or committee for nursing
and health is institutionalized in the Society.

W 1-1) TRADITIONAL MEDICINE IN SRI LANKA
MACHIKO HIGUCHI
Graduate School of Nursing, University of Shizuoka

Introduction:Traditional medical system in Sri Lanka
was initiated in 3 BC when Ayurvedic medicine was introduced from North India. Currently, Sri Lankans use traditional medicine for the promotion of health and for the prevention and treatment of illnesses even though the modern
health-care system has rapidly increased in Sri Lanka. Sri
Lankans practice different methods of treatment for illnesses, i.e., supplicating with ritual and supernatural powers,
traditional medicine, and modern medicine.The objectivesof this research were to determine the reasons for peoplechoosing between Ayurveda and modern medicine and to
identify the use of home remedies.A total of 286 patientswere interviewed in both Traditional Medical Institutes(TMIs) and Modern Medical Institutes (MMIs) in threecharacteristic areas. The data was collected using individual
survey forms comprising open-ended questions and indepth interviews which were translated from Sinhala to
English. The results show that patients at TMIs had obtained higher education, were from a higher income group,
and were older than those in MMIs. Patients who sufferedfrom pain in the joints, arthritis, or rheumatism sought traditional medication and those with heart problems, hypertension, and diabetes obtained treatment from MMIs. Sixtythree percent of the patients in TMI had previously opted
for treatment from MMIs and 38% of the patients in MMIs
had gone to other MMIs. The patients had changed thetreatment from one MMI to another either because thesymptoms had persisted, the former hospital was distantly
located thus limiting easy access, the former personal physician had recommended the present MMI, extra expenses
were incurred on transportation, or the cost of medicine wastoo high and made it unaffordable. Patients changed from a
TMI to a MMI due to other reasons. They complained that
there was no improvement in the symptoms even after
medication, or the symptoms reappeared, indicating incomplete cure. The patients said that they changed from one
TMI to another because there was no change in the symptoms or that modern medicine could provoke other illnesses,
and being too strong, could cause side-effects.In conclusion,
the reason behind patients choosing each medicine de-３７pended on each research area’s characteristic. Patients in urban areas obtained better accessibility to both TMI and

MMI than those in rural areas.

W 1-2) COMMUNITY HEALTH NURSING IN INDONESIAIKUKO MORIGUCHI
College of Nursing Art & Science,University of Hyogo

The Indonesian government has promoted healthcare
policies based upon primary health care (PHC) since 1978,and has been trying to correct regional differences under its
5th five-year plan since 1989. With a commitment to providing health care services and educating health care workers
in remote areas so that limited resources can be utilized efficiently, the government has striven to increase budgets andreinforce facilities and manpower in areas outside Java.
Healthcare workers are unevenly distributed throughout
Java and in urban areas, and newly graduated physicians
have been dispatched to health centers in remote areas. Dueto a high maternal mortality rate (450 in 1992), trained
community midwives have been dispatched to rural villages
where there are no healthcare facilities. I concluded that
community midwives needed to provide PHC services inorder to correct regional healthcare differences. Beginningin 1992, therefore, I examined the actual conditions community midwives were working under and clarified issues
concerning their activities in South Sulawesi (population:
7.5 million) in cooperation with the province health department. Training and activity methods were also developedwith local community nursing managers to improve thequality of the care provided by community midwives. Oneof the methods involved an investigation into the circumstances of maternal death conducted by community mid-

wives using a verbal autopsy.
In Indonesia, the actual circumstances surrounding
maternal deaths were not known at that time. Community
midwives in South Sulawesi received training in verbal
autopsy to assist investigations into maternal deaths. The
training was held in cooperation with nursing managers ofboth the province and prefectural health departments andafter the training community midwives investigated maternal deaths in the villages where they worked. Investigation
reports and case study reviews followed to clarify directcauses and other factors affecting maternal deaths. The midwives thereby came to recognize the serious problems surrounding maternal deaths and began actively considering
detailed measures to prevent them. This kind of investigation was formerly only conducted by universities and researchers, and results were rarely provided to frontline
health care workers.
Based upon the experience gained in Indonesia, it became obvious that research outcomes should be utilized for
local healthcare in the field of tropical medicine. This includes cooperating with local healthcare workers as well as
researchers; feeding research results back to the workers;
and proposing investigation methods that local workers may
use on an ongoing basis and healthcare activities that suiteach region.

W 1-3) NGO AND INTERNATIONAL COOPERATION - THE AFRICAN EXPERIENCEMIZUKO TOKUNAGA
School of Health Sciences, Nagasaki University, Nagasaki, Japan.

A Japanese NGO “Friend of Africa” has been tackling
infection prevention of HIV andAIDS patients’ support in
the Central Africa Republic since 1992.
The followings are some thoughts through experiencesof NGO activities.
1. I believe it the big mission of NGO to perform educationand technical transfer of the local staff. Because NGO hasabundant field experiences and activities which involve
flexible local residents’ autonomy.
2. It is hoped that mutual cooperation with NGO and ODA
will be performed more powerfully.
3. “Accountability” of NGO has been much discussed.

３
８

NGO should make the standard of crisis as the first priority.

tries, the cooperation with ODA and NGO is an important
subject.

In order to respond to the requests of the developing coun-

W 1-4) THE ROLE OF SOCIAL AND BEHAVIORAL RESEARCH ON TROPICAL DISEASES CONTROL:A CASE STUDY OF TUBERCULOSIS.
JINTANA NGAMVITHAYAPONG-YANAITB/HIV Research Foundation, Chiang Rai, Thailand.

In the traditional model of epidemiological triad (agent,
host and environment), which is a causation model for all
infectious diseases, the social and human behaviors are involved a great deal with host and environment components
and they are determinants of disease occurrence. Tuberculosis (TB) is one of the tropical diseases which is the global
health emergency. TB has been well recognized as a disease
which is closely associated with social and behavioral factors. The paper describes the role of social and behavioral
research in TB transmission, prevention and care. Some key
concepts of sociology and anthropology in health and social

science research methods are discussed. These include attitude and perceptions about TB; gender; stigma; adherence
to TB treatment and qualitative research. By giving the examples from the field research in Chiang Rai, Thailand, theconference’s audiences will recognize how social science
research help the health care workers understand patient’s
attitude and behavior which are associated with TB transmission and prevention. Furthermore, the social science research, which led to intervention and policy for TB control
will be discussed.

３
９Workshop 2
W 2-1) OVERVIEW
KATSUHIKO KAMEIDept of Pathogenic Fungi, Res Ctr for Pathogenic Fungi and Microbial Toxicoses, Chiba University

Mycoses that have been imported into Japan, i.e. coccidioidomycosis, histoplasmosis, paracoccidioidomycosis,
penicilliosis marneffei, and blastomycosis, differ from domestic Japanese mycoses in many aspects: they are highly
contagious, easily disseminated, and able to cause biohazards both in laboratories and in society at large. However,
medical workers in Japan generally have limited knowledge
about these diseases. The numbers of patients of coccidioidomycosis and histoplasmosis have been rapidly increasingin the past decade, with the total numbers having reached
41 and 42, respectively. The increase seen in coccidioidomycosis is particularly dramatic. Most of the coccidioidomycosis patients were infected in the United States, especially in Arizona. As for histoplasmosis, in contrast, as approximately 16% of the patients had no evident history ofvisiting endemic areas, domestic infection was suspected.
Mainly due to continuing increases in international travel
and contact, further increases in both diseases can be expected. A total of 18 patients with paracoccidioidomycosis
have been reported. The number of patients has shown a decreasing trend after a sharp increase in the 90’s during the“Bubble Economy”. Most of the patients were workers mi-

grated from endemic areas such as Brazil, and were on temporary visits to Japan when the disease developed. The patients abruptly increased and then decreased in concert with
the Japanese economy heating up and cooling down. Although only a few patients were reported in the past ten
years, another rise may be on the horizon if the economy
again heats up and many people visit Japan from endemicareas. No cases of penicilliosis marneffei had been reported
until 1998, when the first case was found in an AIDS patient. Then, the second case, also an AIDS patient, followedin 2002. Although the number of patients is still limited, themortality rate of this disease is very high, and its likelihoodof occurrence should not be neglected.No cases of blastomycosis have been reported to date in our country. However,this infection is one of the biggest endemic mycoses in the
United States, and therefore is sure to hit Japan sooner orlater. We have to stay on a high alert.It is clear that these imported mycoses are getting more and more common. Systems for close monitoring of patients, epidemiological studies, and education of medical staffs for their diagnosis andmanagement are urgently required.

W 2-2-1) COCCIDIOIDOMYCOSISMASATERU KAWABATA
Department of Respiratory Medicine, Respiratory Center

53 years old, male was admitted to our hospital complaining of fever, cough and dyspnea on effort after thetravel to Arizona in the United States. He consulted his primary care physician and was diagnosed common cold threedays before the admission. Although he was prescribed
NSAIDS, antitussive and antibiotics, his symptoms had not
improved. The chest X-ray revealed some consolidations in
the right middle lung field and granular shadows bilaterally.
The laboratory data showed increase of white blood cellcounts and rising erythrocyte sedimentation rate. Coccidioidomycosis was suspected for the previous travel history to
Arizona and the clinical features. The thoracoscopic lungbiopsy was performed for the pathological diagnosis. The
specimen was revealed granulomas consist of remarkableinfiltration of eosinophils and giant cell. Some typicalspherules, which were strongly stained by PAS, were foundin the eosinophilic granuloma. We certainly diagnosed coccidiomycosis and started itraconazole 400 mg/day. After thetreatment of three-month regimen of itraconazole, his
symptoms, the laboratory and the X-ray findings were improved. Coccidioidomycosis is caused by Coccidioides immitis, a dimorphic fungus that grows as a mold in the soil.
C. immitis is most commonly found in the deserts of thesouthwestern United States (southern Arizona, central Cali-

４０

fornia, Southern New Mexico, and west Texas) and Central
and South America. Coccidioidomycosis is acquired frominhalation of the spores, arthroconidia. Once in the lungs,
the arthroconidia transform into spherical cells called“spherules”. An acute respiratory infection occurs 7 to 21
days after exposure and typically resolves rapidly. In approximately 40% of infected cases, symptoms present usually as flu-like illness with fever, cough, headaches, rash,and myalgias. Some patients fail to recover and develop
chronic pulmonary infection or widespread disseminated in-

fection affecting meninges, soft tissues, joints, and bone.
Severe pulmonary disease may develop in HIV-infected persons.Patients with self-limited disease or relatively localized
acute pulmonary infections usually do not require antifungal therapy. Antifungal therapy should be given to patients
who have disseminated disease or are under risk of complications due to their underlying immunosuppression andother factors. Amphotericin B and azoles, such as fluconazole, itraconazole, and ketoconazole are used for treatment.

W 2-2-2) A CASE OF CHRONIC DISSEMIATED HISTOPLASMOSIS EMERGED DURING STEROID
MAINTENANCE THERAPY FOR RHEUMATOID ARTHRITIS WITH SECONDARY AMYLOIDOSISAND NEPHROTIC SYNDROMESHUNJI TAKAKURA
Department of Laboratory Medicine, Kyoto University, Kyoto, Japan.

[Case] A78-year-old man with chronic disseminated histoplasmosis emerged during steroid maintenance therapy
without probable travel to endemic area.
[Clinical course] He had received steroid therapy against
rheumatoid arthritis for 10 years. Secondary amyloidosis
with nephrotic syndrome was diagnosed 4 years ago. Daily
dose of steroid was increased temporarily to 60 mg/day but
reduced to maintenance dose of 10 mg/day soon after. Onemonth stay in New York 10 years ago was his only international travel. He has lived in Kyoto and worked as a cargo
worker during World War II and a driver after the war. He
found to have low-grade fever with malaise. A chest and abdominal CT scan revealed enlargement of bilateral adrenal
glands. Pancytopenia and increased acute phase reactant
were found in the blood examination. Intense uptake in thebilateral adrenal glands and numerous spotted uptakes in
the liver was detected by FDG-PET scan.
[Diagnosis] Ultrasound-guided percutaneous adrenal aspirate and bone marrow aspirate yielded Histoplasma capsu-

latumvar capsulatum. Both urinary and serum histoplasma
antigen were positive. We diagnosed him as chronic disseminated histoplasmosis.
[Threapy and course] We started amphotericin B for 0.8 mg/kg/day. Defeverescence and dissapearance of subjective
symptoms were gained after 1 week of therapy. After 3
weeks of therapy, blood cell count and acute inflammatory
markers returned to normal limit. Follow up imaging study
revealed that bilateral adrenal enlargement had continued todecrease in size. Unfortunately, sudden-onset MRSA prosthetic arthritis of his left elbow required urgent surgery andadministration of vancomycin, which lead to rapid deteriolation of renal dysfunction. He expined 3 month after the
diagnosis of histoplasmosis[Conclusion] Histoplasmosis should be suspected even
without obvious internation travel history, and be includedin the differential diagnoses of bilateral adrenal enlargement
and pancytopenia.

W 2-2-3) PARACOCCIDIOIDOMYCOSISTAKESHI IGARASHI
Iwamizawa Rosai Hospital, Iwamizawa, Japan

Paracoccidioidomycosis (PCM) is known as endemic
disease of Central and South America area infected with anadult man. Inhalation of the infective spores is the usual
method of infection. Most patients with the disease are male４
１

agricultural workers in close daily contact with the soil.
Chronic pulmonary paracoccidioidomycosis progresses
very slowly and develops a cough, sputum, loss of weight,
on exertion breathlessness, fever and lung fibrosis progresses when it progresses and develops the respiratory failure .The radiologic patterns of PCM can be micronodular,
infiltrative, or linear. The micronodular are usually less than
3 mm in diameter, sharply defined, and have a uniform density. Cavities are found in approximately one third of thecases and are usually small and multiple, and occur in the
middle field of the lung. The radiologic abnormalities clear
slowly during therapy. Regression may be observed at 3
months or even before that and is usually stable at 6
monthsPCM was reported 17 examples in Japan. We experienced PCM with multiple cavity and fibrosis in lungs lastyear. We will explain in detail about clinical feature of PCM
mainly on this case.

Case: A43year-old Japanese Brazilian came to Japan in2001 years. Since subjective symptoms such as cough, sputum, dyspnea on exertion became severe, he was referred toour hospital because of suspicion of pulmonary tuberculosis
in chest x-ray and CT findings. Through chest x-ray of initial examination showed interstitial shadow in bilaterallungs with nodular, infiltrative or cavitary changes. The mycobacterium tuberculosis was not found. The mycetocyte
with watched budding resembling steerage of the ship
which was characteristic of Paracoccidioides was observedin sputum and transbronchial lung biopsy specimens. We
could culture fungus to show dimorphism of temperaturedependency and diagnosis was established as chronic lungparacoccidioidomycosis. By dosage of ITCZ 200 mg/day,chest x-ray findings and clinical manifestations were improved.

W 2-3) IMPORTED MYCOSES RELATED TO ZOONOSESAYAKO SANO
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan

It is well known that dermatophytes cause zoonotic
fungal infections by the contact with infected or carrier animals. Recently companion animals became very intimate
with their owners’ life. Exotic animals imported without
veterinary check are now common in Japan. Although quarantines for rabies and some viral infections are well established, fungal infections brought by imported animals havebeen ignored. Therefore we have to be on the alert of newly
imported zoonotic mycoses. Actually, a dermatophyte,
Trichophyton mentagrophytes var. erinacei, which has
never been reported in Japan, was isolated from an imported
hedgehog in 2001, although the fungus is not highly pathogenic for humans. Its prevalence in household hedgehogs inJapan was approximately 40%, which suggested that thedermatophyte was already spread whole of the country. As
anticipated, a human case was reported in 2003. This is justa tip of iceberg.
From the aspect of prevalence in Japan, imported mycoses (coccidioidomycosis, histoplasmosis, paracoccidioidomycosis, penicillosis marneffei, and blastomycosis) arenot exceptional. They are probably brought into Japan with
imported animals. Coccidioidomycosis caused by Coccidioides immitis classified as the highest pathogenic fungus is endemic in desert areas of both American continents.

Mammals and reptiles are sensitive to the pathogen.
Histoplasmosis by Histoplasma capsulatum is also reported in humans and many species of animals. Althoughmost of clinical cases were imported, some autochthonous
ones were reported in Japan. Some native cases in both humans and dogs had skin lesions. The clinical manifestations
closely resemble to those of pseudofarcy in horses, which
was endemic in Japan before World War II, and this factsuggested that the humans and dogs were infected as heteroecism. Pseudofarcy is known as a notifiable disease.
Paracoccidioidomycosis by Paracoccidioides brasiliensis is endemic in Latin American countries. The mostimportant natural carrier is nine-banded armadillos. Some
probable cases of dogs were also reported. Penicillosis
marneffei by Penicillium marneffei is endemic in Southeast
Asian countries. Its reservoir is bamboo rats, which becamea fashionable exotic animal a few years ago. Blastomycosis
by Blastomyces dermatitidis is known to be carried by porcupines on their quills. Sometimes dogs in the endemic areas became infected with the fungus by catching porcupines.
Although there is no imported mycosis in animals, except
for autochthonous histoplasmosis, we should pay attentions
to imported or traveling animals.

４２

W 2-4) CAUSATIVE AGENTS OF IMPORTED MYCOSESKAZUKO NISHIMURA
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, JapanThe causative agents of imported mycoses are required
to be carefully handled because they cause diseases even inhealthy subjects. Imported mycoses in Japan are listed up as
follows; coccidioidomycosis caused by Coccidioides immitis, histoplasmosis by Histoplasma capsulatum, paracoccidioidomycosis by Paracoccidioides brasiliensis, blastomycosis by Blastomyces dermatitidis, and penicillosis
marneffei by Penicilium marneffei, which are recognized as
biosafety level 3 pathogens as the same as Bacillus anthracis.
Among those, C. immitis has the highest pathogenicity
with many mortal cases of laboratory infections in U. S. A.
The species produces abundant arthroconidia within one
week, which easily disperse into opened air. It is said that
only 10 arthroconida could cause the disease in humans.
Since the fungus produces a whitish cottony colony withconidia within one week, it is very dangerous to open the
cover or plug of the culture. In cases that patients would
have a possibility to be diagnosed as coccidioicomycosis
based on typical clinical manifestation and on visits to theendemic areas, isolation of the causative agent should bedone under a biosafety level 3 condition and avoid the secondary infection. Common laboratories are permitted only
to do serological diagnosis and/or detection of spherulecells by cytological and histopathological observations.
The causative agents for histoplasmosis and paracoccidioidomycosis are also dangerous and difficult to isolate,because they require special culture conditions and a longtime at least one month to produce characteristic colonies.
The observations of thermally dependent dimorphism need
one additional week.P. marneffei distributed in the Southwest of China and the North of Thailand and Vietnam
should be pointed out to produce very dispersable conidia.
Thus we developed some molecular biological diagnostic methods for clinical materials. The PCR-based detection of species-specific genes from paraffin-embedded tissue samples, biopsy tissue, blood clot, and sputum were attempted to diagnose coccidioidomycosis, histoplasmosis,
paracoccidioidomycosis and penicillosis marneffei. Furthermore, loop-mediated isothermal amplification (LAMP)
method for detecting gp43, species-specific gene, for P.
brasiliensis could confirm the diagnosis within4hours.

４３Workshop 3
W 3-1) PURPOSES OF ESTABLISHING PREVENTIVE GUIDELINES AGAINST MALARIASHIGEYUKI KANO
Department of Appropriate Technology Development and Transfer,
Research Institute, International Medical Center of Japan

Malaria has been successfully eradicated since 1959 in
Japan and we only encounter imported malaria cases now.
The number of Japanese people who were going abroad had
increased up to 16,357,572 in 2001, but decreased to
13,296,330 in 2003 on account of the terrorism in New
York in 2001 and SARS epidemic in 2003, so did the number of imported malaria cases in Japan. In fact, total numberof the patients was around 100-150 annually for the last 10
years, particularly in 2000, the number reached 154, however in 2002, the number decreased to 83.
The significance of the chemoprophylaxis has not been
well recognized in Japan. After the approval of mefloquine
for use by Ministry of Health, Laboour and Welfare, physicians are encouraged to prescribe the drug for international
travelers not only for treatment but also for prophylaxis.

Still, not so many Japanese physicians are happy with thetropical or travel medicine, the guidelines on the preventionof malaria using appropriate drugs have to be established assoon as possible. Mefloquine resistant malaria has been reported widely in Asian malarious areas, and indeed there
are already some reports of its resistance in Japan. We haveto prepare another good chemoprophylactic regimen applying, for instance, atovaquone/proguanil, primaquine, or
doxycycline. Of course, proper anti-mosquito bite behavior
using repellant or mosquito nets are encouraged, in fact,
stand-by emergency treatment should be expected for certain travelers. The need for the medical practices on travel
medicine using the guidelines is envisaged at the outset now
in Japan.

W 3-2) MOSQUITO CONTROL FOR MALARIA PERSONAL PROTECTIONKOSUKE HARUKI
National Defence Medical College, Saitama, Japan

Avoiding mosquito bites are basic protection methodof malaria infection. They are resembles to the diet, exercise
therapy of diabetes and low salt diet for hypertension. Malaria is transmitted by specific mosquitoes, which are belonging to Anopheles spp. The protection methods of mosquito bites are as follows: 1 House and residence protection:
Avoiding to live around water holding place such as marsh,pond, old tires, tins and so on. Living in air-conditioned
house. Living above first floor. 2 Clothes: Wearing long
sleeves and trousers impregnated with insect repellants(DEET) or insecticide (should not use with under ware). 3
Behaviors: Sleep inside the impregnated mosquito net andshould not move around during mosquito active time (usually evening to morning). Using mosquito coils. If it is not
feasible to avoid mosquito bites, although most effective
method to protect malaria infection is avoiding mosquito
bites, most of these cases may be an indication of taking
anti-malarials for prophylaxis and emergency stand bytreatment.

４４

W 3-3) CHEMOPROPHYLAXISEIICHI OKUZAWA
Dept of Research and Information, Japan Overseas Health Administration Center,
Japan Labour Health and Welfare Organization

Recently the number of malaria cases reported in Japan was about 100, and some of them were fatal. Almost of
all victims were defenceless travelers to tropical zone. Mefloquine, the only drug for malaria prevention in Japan, hasbeen available since 2001. However, chemoprophylaxis is
not widely spread among high-risk travelers, and sometimes
used inappropriately. To improve the present situation, Japanese doctors are desired to comprehend the reasonable useof chemoprophylaxis.
1. Legal request for prescription
Mefloquine is under some regulations as well as other prescription drugs.
- Pharmacists, when they sell it, are requested to check doctor’s prescription.
- Doctors, when they prescribe it, are requested to check patient’s physical condition.
- Use of mefloquine for prophylactic purpose is admitted by
MOLH Japan. Only that, medical fees for it are out of coverage by national health insurance.
2. Medical advice to travelers
All travelers to endemic areas should consider the following
measures.
- To be aware of malaria risk
- To avoid mosquito bites- To seek a doctor immediately, if fever develops- To take chemoprophylaxis, if malaria risk is high
3. Indication for chemoprophylaxis
Chemoprophylaxis is strongly recommended if the both of
the following risk present.
- High risk of infection: e.g. Stay in holo-endemic or hyperendemic areas.
- Risk of serious illness: e.g. Absence of proper medical
service at the time of onset.
In the other cases, further assessments are necessary ( relative indication ). Logically, it is supposed to be beneficial incase with high risk of infection.
4. What should be checked and documented by prescribers- Travel schedule to see indication for chemoprophylaxis- Physical condition of the client to exclude contraindication: e.g. drug allergy- Scheduled dates and dose for medication
5. Travel clinic directory
Number of travel clinic in Japan is small yet. This has limited spread of appropriate chmoprophylaxis. A small list of
travel clinics will be attached to the guidelines. It may be
useful for doctors who will not or can not prescribe for preventive use.

W 3-4) STAND-BY TREATMENTMIKIO KIMURA
Infectious Disease Surveillance Center, National Institute of Infectious Diseases

Our future Guidelines for Malaria Prevention may notdeal with stand-by treatment (SBET) in detail. However,
SBET is mentioned in WHO’s recommendations and guidelines of Western countries, and therefore, it will be targeted
during this workshop, aiming at provoking discussions
about its positioning.
SBET is to take antimalarials as an emergency measure when immediate medical attention is not available incase of suspected malaria, because malaria can become severe or even lead to death within a short period. Practically,
prerequisites of SBET include 1) > 7 days after entering amalarious area, 2) fever of > 37.5°
C suggestive of malaria,
and 3) inability to seek medical attention within 24 hours of
disease onset. Furthermore, travelers should make every effort to seek medical attention even after SBET, because thedisease may require other treatments than antimalarial ones,
and even if it is malaria, other antimalarials may have to be
used because of drug resistance. SBET is used either solely
without chemoprophylaxis or as a back-up measure of chemoprophylaxis; however here, we will focus mainly on theformer.
Although in general chemoprophylaxis is recom-

４
５

mended for travel to areas with high risk of malaria, especially sub-Saharan Africa, SBET could be recommended incase of low risk areas, because the risk of drug adverse effects (AEs) due to chemoprophylaxis may overweigh thebenefit of avoiding disease contraction. Furthermore, SBET
could be recommended for travel to high risk areas if chemoprophylaxis is 1) contraindicated, 2) not available, or 3)
not preferred by the traveler.
In selecting chemoprophylaxis and/or SBET, one
needs to understand advantages and disadvantages of bothmeasures as well as other factors including the character of
the traveler. Advantages of chemoprophylaxis include 1)
preventing the disease acquisition (even if it fails, treatment
follows), and 2) less likely to be misused because of thesimple daily or weekly drug intake, and the disadvantages
include1)the possibility of the risk of AEs overweighing thebenefit of avoiding infection, and 2) likely to pose false
sense of security. On the other hand, advantages of SBET
without chemoprophylaxis include 1) the benefit of avoiding death due to malaria overweighing the risk of AEs, and2) insisting the necessity of early treatment with selfresponsibility, and the disadvantages include 1) inability tosuppress the disease acquisition (if it fails, death may follows), and 2) likely to be misused because of the complex
drug intake.
At present, it is not recommended to advise travelers topractice SBET based on the results of the self-use malaria
rapid tests that are available in foreign countries.

W 3-5) TRAVEL ADVICE BASED ON THE GUIDELINES FOR MALARIA PREVENTION
ATSUO HAMADA
Japan Overseas Health Administration Center

The guidelines for malaria prevention were drawn up
in response to the authorization of mefloquine in Japan in2001 by the Minister of Health, Labor and Welfare, which
has made it possible to administer chemoprophylaxis andstand-by treatment here in Japan. The first objective of theguidelines is to clearly cite the conditions under which it isnecessary for chemoprophylaxis and stand-by treatment tobe implemented, so that travelers are able to avoid contracting malaria or possible death. The second objective is toguard against the overuse of mefloquine and to minimize
the incidence of its side effects. These guidelines were created for the average clinician and main antimalarial tablets,
which could be acquired in Japan, were included. Regarding the two types of malaria prevention, only chemoprophylaxis is clearly detailed in the guidelines. As stand-by treatment has quite a few unsolved legal problems, only a briefsummary of it is given in the guidelines.
To begin with, the guidelines emphasize that all travelers staying in or going to regions with a prevalence of malaria should take the necessary personal protection measures
to prevent mosquito bites. The guidelines also mention thetwo conditions under which mefloquine prophylaxis is ad-

ministered, namely absolute indication and relative indication. The essential standard by which absolute indication
can be defined is a situation where travelers face a high possibility of death or contracting serious symptoms due to malaria without taking a chemoprophylaxis regimen. There are
two required conditions for absolute indication. First, if
there is a high prevalence of malaria in the region where thetraveler is staying. Second, if adequate medical facilities are
not present in the place where the travelers are staying.
When both conditions are in existence, absolute indication
occurs and under these circumstances we strongly recommend that mefloquine prophylaxis be taken. Relative indication is defined as the situation where travelers stay in a region where malaria is prevalent but all of the conditions forabsolute indication are not present. In this case, travelers
should take mefloquine only after they have weighed up therisks of infection with the side effects associated with theantimalarial drug.
In this workshop, I hope to discuss the guidelines indetail with the audience and add any relevant contributions
from our discussion.

４
６

General presentation
1) ROLE OF COSTIMULATORY MOLECULES IN HOST RESISTANCETO RE-INFECTION WITH HYMENOLEPIS NANA ONCOSPHERE
KOTARO ONISHI１, KAZUHITO ASANO２, KENJI ISHIWATA１, NAOHIRO WATANABE１
１

Dept of Tropical Medicine, Jikei Univ, Tokyo, Japan ２Dept of Physiol(I), Showa Univ, Tokyo, Japan

Host’s intestine is important on the host resistance toorally infectious parasites. However, immune system to intestinal parasites is yet unclear. Mice receiving a single oral
inoculation of Hymenolepis nana (H. nana) eggs acquire astrong protective immunity and a challenge infection witheggs will be completely rejected by inhibiting larval growthin the intestinal villi. This strong resistance to re-infection isestablished on 24 hours after immune infection. This acquired immunity is dependent on CD4 positive T cells. We
focused our studies on in vivo analysis of the role ofcostimulatory molecules in host defence mechanisms
against H. nana re-infection in mice. Primary infection of
H. nana was carried out by giving 1000 eggs on day 0. The
mice were challenged orally with 1000 eggs on day 5 andkilled 4 days later. The protective effect was assessed bycounting the cysticercoids which had developed in the intestinal villi by day 9, 4 days after challenge. Monoclonal antibodies were intraperitoneal injected at 0.5 mg on 2 hours
before and 48 hours after primary infection. These antibodies are neutralizing monoclonal antibodies against mouse
CD80, CD86, ICOS L and CD40 L. Treatment of neither
antibodies on protective immunity failed to suppress acquired resistance to H. nana re-infection. CD28 knock out
mice were carried out as above infection. In this knock out
mice, protective immunity also failed to suppress acquired
resistance. The present results suggest that protective immunity against H. nana oncosphere is establised independent to known costimulatory molecules.

2) TAENIASIS IN BALI, INDONESIAHIROSHI YAMASAKI１, TONI WANDRA２, THOMAS SUROSO２, MINORU NAKAO１,YASUHITO SAKO１, KAZUHIRO NAKAYA３, AKIRA ITO１
Dept of Parasitol, Asahikawa Medical College, Asahikawa, Japan ２CDC, Ministry of Health Indonesia, Jakarta, Indonesia
３
Animal Laboratory for Medical Research, Asahikawa, Japan

１

Indonesia is a multi-ethnic nation which consists ofmore than 17,500 islands and differs in language, religion,
culture, eating-custom and biogeographical characteristic
by the island. Bali Island, located between Java and Lombok Islands, is one of them and a globally famous resort.
The majority of residents is Hindu (93%). From the eatinghabit of Balinese who have eat uncooked pork, has the custom of eating uncooked pork, The island where Balinese
who eat uncooked pork live was endemic area for taeniasis/
cysticercosis due to Taenia solium once. In recent years,
taeniasis due to taeniid cestodes other than T. solium has
been reported. However, whether the causative agents are
Taenia saginata or Taenia asiatica is highly suspicious dueto the morphological similarity between T. saginata and T.
asiatica. In the present study, the taeniid proglottids discharged from the tapeworm carriers in Bali have been accurately identified using multiplex PCR targeting cytochromec oxidase subunit 1 gene (cox1). The survey area was the
Ketewel village in Gianyar district located in the southeast
part of Bali Island. Of 125 inhabitants examined, 32 persons were diagnosed as taeniasis based on the expulsion of
proglottids after treatment with praziquantel. For the molecular identification of the parasites, mitochondrial DNAs
from the parasites were prepared using DNeasy Tissue kit
and multiplex PCR was performed according to the method
established recently１. Consequently, the cox1 fragments of827 bp were amplified from all parasites and were identified that these taeniid cestodes are T. saginata. DNA sequencing of the PCR products also supported these results.
Furthermore, in order to test whether taeniid DNA is detectable in the stool samples, 10 fecal samples from 32 tapeworm carriers were examined. 827 bp-cox1 products weredetected from all stool samples. As mentioned above, mitochondrial DNA analysis of proglottids and stool samples re-４７vealed that human taeniasis in Bali is due to the infection of
T. saginata. Having considered Balinese eating-habits, uncooked beef called lawar is considered to be a possible
source of infection. It seems that change of an eating-habitsof Balinese who are Hindu are reflected. So far, no evidenceof T. asiatica infection has been obtained from Bali, but thefurther investigation is required. １Yamasaki et al. J Clin Microbiol 42: 548-553, 2004.

3) THE STUDY OF ASCARIS AND ULCERATIVE COLITIS PART 2:
LOCALIZATION OF MUCIN 12 ON ASCARIS LUMBRICOIDES HOMINIS ITOKO HAYASHI, SEIJI KANDA, ATSUKO YOSHIDOME, TAKAAKI ISHIDA, TOSHIMASA NISHIYAMA.
Dept. of Public Health, Kansai Med. University

Some researchers reported that the antibody against
Ascaris was detected in patient’s serum of ulcerative colitis(UC). In the 43rd congress (2002), we reported the resultsof analyzing the genes related with cross-reactive antigens
between A. lumbricoides and human colon mucosa by
screening method. In the results, we got 3 clones, transmembrane mucin12, similar to actin (gamma 2 of entericsmooth muscle) and beta-casein-like-protein. In these three,mucin localizes in the digestive organs, especially in the colon, and was reported that the antibody against colonic mucin might contribute to the epitherial cell injury of colonicmucosa through antibody-dependent cell-mediated cytotoxicity (ADCC), and it was detected that this autoantibody(anti-mucin antibody) titers are high in some patients for
UC. A. lumbricoides was likely to harbor in the human intestine, because mucin3 localize richly in human intestine,
and it was guessed to be a suitable environment for A. lumbricoides. In this study, we were investigated by immunostaining method using the human anti-mucin antibody in order to determine the localization of Mucin 12 on A. lumbricoides. In addition, we were investigated that which organs
Mucin 12 localized in human internal organs at the RNAlevels using Northern Blot analysis.
As the results of immuno-staining methods using themomoclonal anti-human Gastric mucin for first antibody
and FITC anti-mouse IgG for secondary antibody, the specific signal was observed in the lateral lines and cluster-likeorgans of A. lumbricoides. Therefore same localizations
were detected by immuno-staining method using the DAB(3,3’-diaminobenzidine). These results were demonstrated
that these signals were specific and not endogenous fluorescent substance in A. lumbricoides. Furthermore in order toconfirm the specificity, immuno-staining was performed using monoclonal anti-human antibody pretreated with purified Mucin12 . As the results, it was observed that these signals were declined clearly. This result was suggested that
Mucin12 had cross-reactive antigenisity to human Mucin in
the lateral line and cluster-like organ of Ascaris. As a resultof Northern Blot, Mucin12 were localized mainly in colon,
rectum, and rarely in jejunum in human internal organs. In
this report, we specified the localization of Mucin12 in Ascaris and human internal organs. We are going to prepareanti-Mucin12 antibody and perform immuno-staining, similarly.

4) CHARACTERIZATION OF THE 150-KDA CYSTEINE-RICH SURFACE PROTEINS OF
ENTAMOEBA DISPARHIROSHI TACHIBANA１, XUN-JIA CHENG１, SEIKI KOBAYASHI２, TSUTOMU TAKEUCHI２
１

Dept of Infectious Diseases, Tokai University School of Medicine, Isehara, Japan
Dept of Tropical Medicine and Parasitology, School of Medicine, Keio University, Tokyo, Japan

２

Entamoeba dispar, morphologically similar to Entamoeba histolytica, is neither pathogenic nor invasive. Comparative analyses of surface proteins of the two species areimportant to determine pathogenic factors and to establish a

specific diagnosis for amebiasis. Recently, we have identified intermediate subunits of galactose- and N-acetyl-Dgalactosamine-inhibitable lectin (Igl), as 150-kDa cysteinerich proteins on the surface of E. histolytica (EhIgl1and４
８

EhIgl2). In the present study, molecular characterization of
the corresponding proteins in E. dispar has been performed.
The open reading frame of two cloned Igl genes from E.
dispar encoded1, 110 (EdIgl1) or 1,106 (EdIgl2) amino acids. The calculated molecular masses and theoretical pI
were 120,886 Da and 5.5 in EdIgl1and 120,347 Da and4.87 in EdIgl2. When the deduced amino acid sequences ofthese Igls were compared, identities were 76.9% between
EdIgl1and EdIgl2, 73.7% between EdIgl1and EhIgl1, and73.0% between EdIgl2 and EhIgl2. To measure quantitatively the expression of the Igls, real-time reverse transcription PCR was performed and the results analyzed by a comparative CT method using actin genes as internal standards.
The expression level was higher in E. histolytica than in E.
dispar, and also higher in Igl1 than in Igl2. These results
suggest that the Igls are essential proteins even in the non-pathogenic ameba. Recombinant Igls were expressed in Escherichia coli and their reactivity with anti-EhIgl antibodies
and patients’ sera was examined. Sera from patients withamebiasis and a polyclonal antibody to the EhIgls reactedwith the EdIgls suggesting the existence of common epitopes between Igls from E. histolytica and E. dispar. On
the other hand, two monoclonal antibodies recognizing
either the conformation or the N-terminus of EhIgl1 did notreact with EdIgls showing the existence of E. histolyticaspecific epitopes. Although we have recently demonstrated
that recombinant EhIgl1 is a useful antigen for the specificand sensitive serodiagnosis of amebiasis, it is important toconsider the antigenic similarity between EhIgls and EdIgls
and their species-specific epitopes for increasing diagnostic
specificity.

5) A SURVEY ON PREPARATION AGAINST POSSIBLE RESURGENCE OF SARSHIROSHI OHARA
Bureau of International Cooperation, International Medical Center of Japan

While Severe Acute Respiratory Syndrome (SARS)
was in epidemic, a large number of nosocomial infections
broke out in hospitals where patients were accommodated.
The epidemic was put under control in July 2003, however
possibility of resurgence still remains. This survey was conducted with the purpose of contribution to the appropriate
response at hospitals in case of resurgence, by analyzing
current situation of preparation against SARS and existing
problems.
Questionnaire survey was performed for hospitals in
the countries where SARS was experienced (41 hospitals in
Vietnam and 95 in China), and those in the country without
experience (5 in Nepal) on awareness among staff, current
situation of nosocomial infection control, ability to accept
SARS cases, etc. These hospitals will possibly accept
SARS cases in case of recurrence. Among them only 1 hospital in Vietnam actually treated SARS cases, and 2 hospitals in Vietnam and 3 in China examined suspected cases.
In Vietnam and China awareness on SARS among hospital staff was significantly higher (66.7% and 27% of thehospitals, respectively, answered “high”) than in Nepal(100% were “low”). Regarding manual, 66.7% of hospitals

in Vietnam and 72.5% in China prepared, while 25% in Nepal. The rate of having nosocomial infection prevention
committee was 85.4% in Vietnam, 95.9% in China and 75%
in Nepal. In Vietnam 95.1% and 97.5% of hospitals wereregarded as having good storage of surgical masks andgloves, respectively. Similar good results were obtained in
China. However, in both countries protection attires such as
N95 masks and goggles were lacking. In Vietnam andChina, 75% and 97.9% of hospitals conducted staff training
for infection control against SARS. In Vietnam and China,
Infection Control Team (ICT) has been established in
51.2% and 71%, respectively, while in no hospital in Nepal.
All the hospitals in Vietnam were requesting Bach Mai Hospital, the hospital that had an experience of SARS containment, for providing technical guidance on SARS control.
In hospitals in the countries that experienced SARS,
growing awareness on SARS, steady preparations against
possible resurgence, and request of staff for further training
were shown. ICT has been established in many hospitals but
had problems in management. On the other hand, in the
country where SARS was not experienced, both awarenessand preparation status were poor.

４９6) ECOLOGICAL ROLES OF MONGOOSE IN TRANSMISSION CYCLE OF
JAPANESE ENCEPHALITIS VIRUS IN OKINAWA ISLAND
MIKA SAITO１, GO OGURA２, TAKU NISHIJIMA２, KATSUHIRO YAMASHITA２, NAOKI MORI１
１

Div of Molecular virology and Oncology, Graduate School of Medicine, Univ of the Ryukyus, Okinawa, Japan２
Lab of Subtropical Zoology, Faculty of Agriculture, Univ of the Ryukyus, Okinawa, Japan

Background
Japanese encephalitis (JE) had been one of the major
public health problems before 1970’s in Japan, including
Okinawa Island. No case has been reported officially in Okinawa Island since 1974, but transmission of JE virus in nature is demonstrated by the high prevalence of JE virus antibody in swine sera, and by the virus isolation from swine
sera and mosquitoes.
Around 20 mongooses (Herpestes javanicus) were imported to Okinawa Island in 1910 in order to control toxic
snake and mice. The number of the mongoose is increasing
and estimated as 30,000 at present and hazardous to theunique wild life.
Objectives
To clarify trole of mongooses in transmission cycle ofJE virus in Okinawa island.
Materials and methods
102 sera were collected from captured sera in all areasof Okinawa Island between 2001 and 2004. Sera from 3
baited mongooses in agricultural area were collected regularly from July to October, 2004. Virus isolation from allsera mentioned above was carried out by inoculation eachsera onto C6/36 cells and Vero cells and incubated for 8
days. Exisitance of infective virus in cultural fluids was
identified by immunostaining method using polyclonal antibodies against JE virus.

Neutralization (N) test were conducted on sera from
captured and baited mongooses by 50% focus reduction
method using Beijing-1(human vaccine strain, genotype 3),
Nakayama (prototype, genotype 3, Oki 431S (Okinawan
isolate from swine in 2002, genotype 1)and Na 54 (Okinawan isolate from swin in 1985, genotype 3).
Results
Virus isolation from captured and baited sera wasfailed.
Prevalence rates of N antibody against JE virus strains
were: 14.7% against Beijing-1, 24.5% against Nakayama,
3% against Oki 431S and 1% against Na 54. N-titers
against JE virus strains were relatively low as less than 125
except one sera with high N-titers between 400 to 3,100.
Temporal change of N antibody titer in sera of one baitedmongoose was observed.
Conclusion
This is the first serological report that mongooses have
JE virus antibodies, and clarified that mongooses are infected by JE virus.
Discussion
We found N-antibodies against JE virus strains inmongooses, however relatively low N-titer might suggests
possibility to be infected by other flaviviruses than JE virus.
Furthermore, the role of mongooses in transmission cycle ofJE virus or other flaviviruses must be clarified.

7) SHIFT OF JAPANESE ENCEPHALITIS VIRUS PROPAGATING IN JAPAN, 1935-2002.
SHAO-PING MA１, KOUICH MORITA２, MASAYUKI TADANO３, MASAO OGAWA４
１

Div of Epidemiology, Dept Infectious Disease, Fac. Med., Oita Univ., Hasama, Oita, Japan
２
Dept Virol, Inst Trop Med, Nagasaki Univ, Nagasaki, Japan
３
Div Mol Virol Oncol, Dept Microbiol, Graduate School of Med, Univ of the Ryukyus ４Oita Pref Inst Health Enviro

We have reported that the dominant genotype of theJapanese strains of Japanese encephalitis virus (JEV)
shifted from genotype 3 to genotype 1 (G1) in the early
1990s. However, the origin of these Japanese G1strains remains unclear. A phylogenetic analysis based on the 196nucleotide (nt) capsid/premembrane region of 77 JE virus

strains isolated during 1935-2002 in Japan revealed the existence of at least six dominant genetic clusters (cluster A-E
and GI). The result of the phylogenetic analysis, which used
the neighbor-joining method, indicated that these clusters of
JEV might have been introduced into Japan from the westof Japan and not from the south via the Ryukyu Islands and５０

Taiwan. The strains that isolated from Japan in the 1980s,
belonged to cluster E, had a 9- to 10-nt deletion in the 5’ter-minal of their 3’ noncoding region.

8) JAPANESE ENCEPHALITIS IN THE PHILIPPINESSHINGO INOUE１,２, MARIA T. ALONZO２, FLORENCIO P. TADENA２, RONALD R. MATIAS２,３,
FILIPINAS F. NATIVIDAD２, and KOUICHI MORITA１
Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan１
Research and Biotechnology Division, St. Luke’s Medical Center, Quezon City, Philippines２
Institute of Biology, University of the Philippines Diliman, Quezon City, Philippines３[Objectives]
Japanese encephalitis virus (JEV) belongs to Genus
Flavivirus, Family Flaviviridae and is the causative agentof Japanese encephalitis, a mosquito-borne disease transmitted by Culex tritaeniorhynchus. In recent years, no serological and virological survey on JEV has been done in thePhilippines. This study aims to determine the seasonality,
age distribution, and geographical distribution of JEV infection in the Philippines.
[Patients and Methods]
One hundred fourteen Serum and 329 cerebrospinal
fluid (CSF) samples extracted in 2002 and 2003 from patients clinically diagnosed as CNS diseases such as viral encephalitis, aseptic meningitis and other acute paralysis were
examined using IgM capture ELISA for JEV. Six hundred
eighty four pig sera were also collected in Tanay, Rizal from
May, 2002 to October, 2003 and then examined their IgM
and IgG against JEV.
[Results and Discussion]
Out of 329 CSF and114serum samples, 38 (11.6%)
and 21 (18.4%) were IgM positive for JEV, respectively.

These results were almost similar with the results of 1999 to
2001. The analysis of 52 laboratory diagnosed JE patients
indicated that among 31 age recorded cases, 25 (80.6%)
were younger than 20 years old. The seasonality of JEV infection was seen from July to November that was coincidedwith rainy season of this area. The geographical distributionof JEV infection was observed in Tarlac province, 150km
north from Metro Manila, had higher incidence.
We also monitored the IgM and IgG levels against JEVamong pigs in Rizal province, 100km south from Metro
Manila, monthly from May 2002 to October 2003. The result of IgG-positive ratio against JEV indicated that thepeak season of JEV infection was from July to September
in 2002 and 2003. It was synchronized with the incidenceof human JEV infection cases.
Although we have not yet isolated the JEV from human, pig and mosquito samples, the results of our serological surveillance on JEV indicated that JEV infection occurred among young age group during hot and rainy seasonfrom July to September.

５１9) IMMUNOGENETIC ANALYSIS OF THE PATIENTS WITH DENGUEHEMORRHAGIC FEVER IN VIETNAM.
MIHOKO KIKUCHI１, VU THI QUE HUONG２, VU THIEN THU NGU２, VO DINH THAM３,
TRAN VAN DAT４, DO QUANG HA２, KOUICHI MORITA５, KENJI HIRAYAMA１
１

Department of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki University, NAGASAKI, Japan
Arbovirus laboratory, Pasteur Institute, Ho Chi-minh City, Vietnam ３Pediatric Hospital No.2, Ho Chi Minh City, Vietnam
４Center for Preventive Medicine, Vinh long Province, Vietnam
５Dept. of Virology, Institute of Tropical Medicine, Nagasaki University

２

Dengue virus infections are serious public health problems in many regions of the world, especially in the South
East Asian countries. Dengue virus infection may lead to
Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF), or
Dengue Shock syndrome (DSS). Two factors are proposed
to be important to produce DHF / DSS. One is viral virulence and the other is host genetic factor. In this study, we
made an experimental design to identify the host gene(s)contributing to the development of DHF/DSS in Vietnamese
by case control study. The patients were clinically diagnosed by WHO criteria (grade I, II or grade III, IV) andtheir peripheral blood samples were collected at the Center
for Preventive Medicine, Vinh long Province (VL), and thePediatric Hospital No.2, Ho Chi Minh City (NDII) in 2002

to 2003. The patients age ranged between 10 months and 15
years. 200 age and sex matched control samples were collected in Vinh long. The number of the patients with mildcases (grade I, II) was 80 from VL, 107 from NDII, and thatof the patients with shock (grade III, IV) was 205 from VL,
66 from NDII. DNA was extracted from each blood sample,
then HLA class I (HLA-A, B) and TNF-α promoter SNPs
typing were performed. There was no significant differencein HLA-A, HLA-B alleles. However, TNFP-D allele wassignificantly increased in severe cases (p=0.0074, Pc < 0.04
OR=2.2895% CI1.23-4.23). The TNFP-D allele might directly contribute to severity of DHF/DSS or might be a genetic marker that has strong linkage disequilibrium withsome responsible gene10) STATUS OF CURRENTLY CIRCULATING DENGUE VIRUS RESPONSIBLE
FOR THE EPIDEMIC IN BANGLADESH IN THE YEAR 2002
MOHAMMED A. ISLAM１, MUZAHED U. AHMED２, NASIMA BEGUM３, NASEEM A. CHOWDHURY３,
AFJAL H. KHAN１, MARIA DEL C. PARQUET１, SOPHIE BIPOLO１, FUTOSHI HASEBE１,YASUO SUZUKI４, KOUICHI MORITA１
１

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Bangladesh
３
Shaheed Suhrawardi Hospital, Shere-e-Bangla Nagar, Dhaka, Bangladesh
４
Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Japan
５
Core Research for Evolutional Science and Technology (CREST), Japan

２【Introduction】Dengue fever and dengue hemorrhagic fever (DF/DHF) has appeared in Bangladesh as an outbreaksince the year 2000. To analyze clinical features of DF/DHF
in Bangladesh and to get detailed information about the currently circulating dengue viruses, we conducted clinicaldata collection, sero-epidemiological surveillance and virus
isolation. This is the first report on molecular biologicalanalyses of dengue viruses isolated in Bangladesh.
【Patients and Methods】A total of 200 serum samples werecollected from the dengue clinically diagnosed patients in

Shaheed Suhrawardi Hospital, Shere-e-Bangla Nagar,
Dhaka, Bangladesh in 2002. All the serum samples were
measured the anti-dengue antibody level by IgM-capture
ELISA and indirect IgG ELISA. For dengue virus isolation,
serum samples were inoculated into mosquito derived C6/
36 cells. Isolated dengue viruses were sero-typed by RTPCR. Based on the envelope gene sequencing data, phylogenetic analysis was done. To clarify the clinical feature of
DF/DHF in Bangladesh, clinical records of 200 dengue patients collected in 2002 were analyzed.

５
２【Results and Discussion】IgM positives and IgG positives
against dengue virus were 59.5% and 65.5%, respectively.
Eight strains of dengue virus were isolated and all of themwere DEN-3. All 8 DEN-3 Bangladeshi strains clustered
within genotype II along with some DEN-3strains from
Myanmar, Thailand and Malaysia and they were closely related to Thailand’s isolates from the1990’s. It suggests that
DEN-3might entered Bangladesh from Thailand during thelast decade. Besides, 8 isolates presented in a distinct subcluster of the genotype, indicating some independent evolu-tion occurred in Bangladesh.Clinical profiles of 200 dengue
patients indicated that most patients showed high frequency
of prodromal symptoms such as fever, headache and vomiting. Also relatively high percentage (70%) of patients
showed hemorrhagic manifestation. Six patients (3%) also
showed plasma leakage (edema and ascities). Peak age distribution of the dengue patients belongs to 21-30 years old
age group. Most of the dengue cases were reported in August and September.

11) GENETIC VARIATION IN THE 3’ NON-CODING REGION OF DENGUE VIRUS TYPE 1
YOKO NUKUI１, SHIGERU TAJIMA１, TOMOHIKO TAKASAKI１,
KAZUHIKO KOIKE２, ICHIRO KURANE１
１

Department of Virology I, National Institute of Infectious diseases, Tokyo, Japan;
２
Department of Infectious diseases, University of Tokyo, Tokyo, JapanWe determined the complete genome sequence of
dengue-1virus, DENV1/hu/Seychelles/NIID41/2004 (NIID
03-41), isolated from a traveler to Seychelles. Sequence
analysis showed that the 10,718-nucleotide-long RNA
genome of NIID03-41 was shorter than that of other dengue-1 (DEN-1) isolates, because there was a 17- nucleotide deletion in the 3’ non-coding region (NCR). A region of approximately 60 nucleotides, which located at the down-

stream of the open reading frame stop codon, showed highsequence variability as compared with other DEN-1 isolates.
Phylogenetic analyses along with the full-length sequencesof 20 DEN-1 virus genomes indicated that the NIID03-41
strain was closely related to those included in the Pacificgroup, not the African group. This is the first report of DEN-1 isolate with a nucleotide deletion in the 3’ NCR.

12) REAL-TIME REVERSE TRANSCRIPTION LOOP MEDIATED ISOTHERMAL AMPLIFICATION(RT-LAMP) ASSAY AS A RAPID DIAGNOSTIC TOOL FOR EMERGING VIRUSESMANMOHAN PARIDA１, HARUMI MINEKAWA２, TSUGUNORI NOTOMI２,
SHINGO INOUE１, FUTOSHI HASEBE１, KOUICHI MORITA１
１

Department of Virology, Institute of Tropical Medicine, Nagasaki University
Eiken Chemical Co. Ltd.,1381-3 Shimoishigami, Ohtawara,Tochigi 324-0036, Japan

２

Loop Mediated Isothermal Amplification (LAMP), a
novel method of gene amplification is emerging as a valuable rapid diagnostic tool for rapid detection of infectious
diseases. We have developed and evaluated one-step single
tube real-time accelerated quantitative reverse transcription
loop mediated isothermal amplification (RT-LAMP) assays
for rapid detection of some of the recently emerged humanviral pathogens viz; West Nile, SARS and Dengue viruses.
The whole procedure is very simple and rapid wherein theamplification can be obtained in 20-30 min by incubating

all the reagents in a single tube with reverse transcriptase
and Bst DNA polymerase at 63°
C. The RT-LAMP assay was
found to be 10 to 100 fold more sensitive as compared toconventional RT-PCR, with a detection limit of 0.01 to 10
PFU of virus. The specificity of the RT-LAMP assay systems for respective viruses was established by crossreaction studies with other closely related viruses. The
evaluation of SARS RT-LAMP assay with 49 suspected and
10 healthy throat wash specimens revealed 100% and 87%
sensitivity and specificity respectively with regard to RT-

５３

PCR. The evaluation of West Nile RT-LAMP assay with 8of spiked and 6 negative human serum samples revealed
100% concordance with RT-PCR in terms of sensitivity andspecificity. The applicability of RT-LAMP assay for clinicaldiagnosis of dengue virus infection was validated by examining limited number of DF and DHF patient serum samples of each serotypes. The RT-LAMP assay could correctly
identified the serotypes with a 100% concordance with one
step serotype specific direct RT-PCR. The field applicationof RT-LAMP assay made easy by employing the SYBRGreen-I dye mediated naked eye visualisation protocol following incubation in a water bath. Thus the RT-LAMP assay reported here has the advantages of rapid amplification,
simple operation and easy detection with potential usefulness for clinical diagnosis and surveillance of viral diseases
in developing countries without requiring any sophisticatedequipments or skilled personnel.

13) CHARACTERISTIC PREVALENCE OF HPV GENOTYPES IN PENILE CANCER INNORTHERN THAILAND
KAN TORIYAMA１, MASACHIKA SENBA１, ATSUSHI KUMATORI２, TOSHIYUKI MORIUCHI２,SHUICHI FUJITA３, AMNAT YOUSUKH４, PRAPAN JUTAVIJITTUM４
１

Dept of Pathology, Inst of Trop Med, Nagasaki Univ, Nagasaki, Japan
Dept of Biochemistry, Inst of Trop Med, Nagasaki Univ, Nagasaki, Japan
３
Division of Oral Pathology and Bone Metabolism, Dept of Developmental and Reconstructive Medicine,
Nagasaki Univ, Nagasaki, Japan ４Dept of Pathology, Faculty of Medicine, Chiang Mai Univ, Chiang Mai, Thailand
２

PURPOSE: Penile cancer(PC) is uncommon in the developed countries such as the US, Japan and European countries. The incidence is higher in some of the African, South
Asian, Southeast Asian and South American countries. The
etiology of PC is not well understood. Lack of neonatal circumsion and phimosis are considered to be the most significant risk factor for PC and also hygienic condition in eachcountry influences the occurrence of PC, however thecausal relationship has never been established. MATERIALS AND METHODS: To clarify the role of human papillomavirus(HPV) in PC, we studied the prevalence andgenotypes of HPV DNA in the paraffin blocks from surgical
specimens of PC and control tissues in northern Thailand,
using Short-Fragment PCR assay. We analyzed 31 cases ofinvasive squamous cell carcinoma (SCC), 1 of verrucous
carcinoma (VC), 1 of epithelial dysplasia (ED) and1of paraffinoma. RESULTS: Overall, the detection rates for HPV
DNA in our study were 81% (25 of 31 cases) for invasive
SCC, 100% (1 of 1 case) for VC, 100% (1 of 1 case) for
dysplasia and 100% (1 of 1 case) for paraffinoma. In all

SCC groups, including VC, 42% (11 of 26 cases) for a solitary infection of HPV-18, 23% (6 of 26 cases) for a solitary
infection of HPV-6, 15% (4 of 26 cases) for double infection of HPV-6 and 18 and 4% (1 of 26 cases) for mixed infection of HPV-6, 18 and 22. 100% (1 of 1 case) of ED andparaffinoma showed solitary infection of HPV-6 and HPV18, respectively. CONCLUSION: The prevalence rate of
HPV DNA in PC in northern Thailand is much higher thanprevious reports from the US and European, South American and African countries. They described that HPV-16
which belongs to high risk types is the most dominant HPV
genotype, HPV-18 which also belongs to high risk types isminor HPV genotype in HPV-positive PCs and HPV-6
which belongs to low risk types are detected mainly in the
benign and premalignant lesions in these countries. However, in our study no HPV-16 is found in HPV-positive PCs
and a premalignat lesion. HPV-18 and HPV-6 are the mostcommon genotypes in HPV positive PCs in northern Thailand.

５４14) A NEW SCREENING METHOD FOR SALMONELLA USING LEMON SLICESYUTAKA MIDORIKAWA１, KAZUE KISHIMOTO１, YUMIKO TAKENAKA１,ATSUKO MAKINO１, SATOSHI NAKAMURA２
１

Suzuka Universitu Medical Science, Suauka, Japan ２Research Institute, International Medical Centre Tokyo, JapanThe salmonella was inoculated in DHL agar and thecircular slice lemon was put in the top, and it was cultivated
at 35C for 18 hours. In 18 hours and circumference of thecircular slice lemon, the black band where 4-5 mm of theferric sulfide was observed. In addition, the part under thecircular slice lemon pinkly changed. In case of Citrobacter
freundii and Proteus mirabillis,there was the dispersion of
the result. This research indicated the method for examining
the Salmonella only in the DHL culture medium.

15) MOLECULAR ANALYSIS OF HAEMOPHILUS INFLUENZAE ISOLATEDFROM NASOPHARYNGEAL ASPIRATE AND CEREBROSPINAL FLUIDAMONG CHILDREN IN VIETNAMCHIHARU KAJI, HIROSHI WATANABE, KIWAO WATANABE,
KAZUNORI OISHI, TSUYOSHI NAGATAKE
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki Uiniversity, JapanThe goal of this study was to investigate the characteristics of Haemophilus influenzae among children less than 5
years of age in Hanoi, Vietnam. Thirty-seven H. influenzae
strains were isolated from the nasopharyngeal swabs (NP)of 37 children with acute lower respiratory tract infections
and 44 H. influenzae strains were isolated from the cerebrospinal fluid (CSF) of 44 children with meningitis. Of the 37
H. influenzae isolates from NP, the serotypes of 30 isolates
were nontypeable, 4 were type b, 2 were type c and one wastype a, whereas those of the 44 isolates from CSF were alltype b. Twenty-six strains (70.3%) from NP and 23 strains(52.3%) from CSF were b-lactamase-producing, and the remaining strains were b-lactamase-negative. PCR analysis to
identify the resistance genes indicated 25 strains from NP
and 21 strains from CSF had the TEM-1-type b-lactamasegene, 11 strains from NP and 22 strains from CSF lacked all
resistant genes, and each strain from NP and CSF had theTEM-1-type b-lactamase gene and the ftsI gene with thesame substitution as the low-b-lactamase-negative
ampicillin-resistant (low-BLNAR) strains. No BLNAR
strain was found. The MICs against 16 antibiotics between
the H. influenzae isolates from NP and CSF were similar,
except for penicillin G and ampicillin. Molecular typing bypulsed-field gel electrophoresis (PFGE) showed that the 37
H. influenzae strains from NP had 22 PFGE patterns without any predominant pattern and 44 H. influenzae strainsfrom CSF had 9 PFGE patterns with predominant patterns a(22 isolates) and b (12 isolates). The PFGE patterns of the 4
H. influenzae type b strains from NP were quite different
from those of 44 H. influenzae type b strains from CSF. Our
results indicates that the BLNAR strains are not prevalentand that 2 predominant types of H. influenzae type b strains
have the potential to spread among children with meningitis
in Hanoi, Vietnam.

５５16) BURULI AND THE ULCERS UNDER THE TROPICSMILANGA MWANATAMBWE１, KAZUHIRO SHICHINOHE２, MASUMI SHIMIZU-SUGANUMA２,
NORIHISA ISHII３, SEIKAN SUZUKI４, GORO ASANO５
１

Department of Pathology, Kinshasa University, DRC ２Department of Laboratory Animal Science, Nippon Medical School,
Tokyo, Japan ３Leprosy Research Institute, National Institute of Infectious Diseases, Tokyo, Japan
４
Medical Wellfare College, Tokyo, Japan ５Hakujikai Memorial Hospital, Tokyo, Japan

Buruli ulcer, caused by Mycobacterium ulcerans is
considered the third most prevalent mycobacteriosis after
Tuberculosis and Leprosy. A largely neglected disease, it
represents a serious treat to public health in poor and remote rural areas of endemic countries. Emerging since theeighties as an important cause of human suffering, the disease has been reported or suspected in more than 30 countries in the world. Although Africa remains the most affected region, Asia bears as well some endemic regions like
Australia, India and Malaysia whether Buruli ulcer is an
emerging or re-emerging remains subject of controversy.
But, that Buruli ulcer is a neglected disease fits perfectly in
the WHO definition of Neglected diseases. Although Buruli
ulcer can prove extensively damaging to the skin, its awareness is very limited in the medical community as well as in

the general public. This results in its under-recognition andthus its under-reporting. Its under-diagnosis or most frightening, its misdiagnosis can lead to life threatening consequences. We report here a series of 96 specimens of skin biopsy, obtained from the Agroyesum Catholic Mission Hospital in the Ashanti district of Ghana. Thirty % of the specimen in the series proved to be misdiagnosed lesions, otherthan Buruli ulcer. Among these, 1/3 were malignant lesions
ranging from fibrosarcoma to malignant melanoma. The remaining were inflammatory tumorus conditions. The aim of
the present study is to emphasize that Buruli ulcer can leadto confusion with the wide spectrum of tropical both parasitosis and infectious conditions that can mimic its different
clinical presentation.

17) EPIDEMIOLOGICAL STUDY OF LEPROSY IN A LOCAL VILLAGE IN MYANMARMASAKO NAMISATO１，２, YOSHIKO KASHIWABARA３, MASANORI MATSUOKA３
TSUYOSHI FUJIWARA４, HIDEOKI OGAWA５, KHIN NWE OO６, KYAW KYAW７
１

Deputy Director General in National Kuryu-Rakusenen Sanatorium, ２Nirvana (NGO)３
Leprosy Research Center in National Institute of Infectious Diseases
４
Institute for Natural Science, Nara University
５
Department of Dermatology in Juntendo University School of Medicine
６
Immunology Research Division, Department of medical Research
７
Central Special Skin Clinic in Yangon General Hospital (CSSC)

In many developing countries where life threatening
diseases such as malaria, tuberculosis have been getting big
attention of government and international organizations,
there are also other diseases which have less prevalence andless severity. They are called neglected diseases and areusually not acutely fetal. However, the QOL of sufferers areoften greatly impaired leading to the vicious cycle of poverty. Leprosy typifies a neglected disease.
After introduction of Multidrug therapy in 1982,
global prevalence of leprosy has greatly decreased. However, yearly number of new cases has not decreased satisfactory. Although early detection and treatment is in the main-stream of leprosy control, another intervention is desired
based on the recent epidemiological studies.
We have been supporting leprosy control activity in Myanmar where leprosy is highly endemic.
Materials & Methods
1 From 2000, prevalence of Mycobacterium leprae (ML) infection has been studied in K-village (population: 306),
except in 2002 and 2003, study was difficult because of therestricted situation. ML-infection was identified by ML specific anti-PGL-I antibody titer, measured by ELISA.
2 Drug resistance-related mutation was examined in leprosy
patients visited CSSC. Cardinal drugs for chemotherapy;

５
６

DDS, RFP, and OFLX were examined. This time, preliminary study can be shown.
Results
1 From the prevalence of PGL-I-positivity, about 40% ofvillagers were once infected with ML until 20-30 year-old.
During study period, 3 villagers developed leprosy.
Case1was found in 2001 to have MB (multi-bacillary) leprosy. She presented high level antibody in 2000 and 2001,
and then it decreased in 2004. Case 2 was diagnosed in
2002 to have PB (pauci-bacillary) leprosy. She presented
high titer in 2000 but low in 2001 and 2004. Case 3 wasfound in 2003 to have PB leprosy. His antibody was negative during all study-period. (The shift of antibody is shownin the graph.)

2 Drug resistance-related mutations were found in 2 out of
29 cases examined. From the past history, they were considered to be primary resistant cases.
Conclusion
1 High prevalence of ML-infection was shown in K-village.
We may predict some possible cases of developing MB leprosy by measuring PGL-I antibody. However, PB candidates cannot be predicted with this method. Chemoprophylaxis targeting the villagers having high antibody titer might
be one way to prevent new MB cases, but cannot cover the
PB cases.
2 It’s a big threat that primary drug resistance was detectedin endemic situation. More vigorous study is required inthis field.

18) DIAGNOSTIC TRIAL FOR CUTANEOUS MYCOBACTERIAL DISEASESBY USING PCR TECHNIQUES.
MOHAMMED ABUL KASEM KHAN１, HAJIME ARAKAKI１, HIROSHI UEZATO１,BENIKO KINZYOU１, KAZUHIRO INAFUKU１, MIDORI SINNHAMA１, SAYURI ONAGA２,ISAMU NAKASONE２, NOBUHISA YAMANE２, and SHIGEO NONAKA１
１

Department of Department, Division of Organ-related Medicine, School of Medicine,
Faculty of Medicine, University of the Ryukyus, Okinawa, Japan２
Department of Clinical Laboratories, Faculty of Medicine, University of the Ryukyus, Okinawa, JapanFor the diagnosis of Mycobacterium tuberculosis (TB)
infection, identification of bacteria by Ziehl-Neelsen stain
and culture methods are very useful. Eventually, sometimes
it is very difficult to detect bacteria especially in skin smaples. Now-a days, DNA-DNA hybridization and amplification of Mycobacterium tuberculosis DNA have been considered as a new method for the diagnosis. This new method
requires cultured samples. When culture of bacteria fails,
then PCR analysis become the more important useful
method, which requires a small quantity of DNA takenfrom the tissue samples. In this study, we reported a total of
five cutaneous mycobacterial patients, in which four cases

were scrofuloderma and another was warty type. All cases
were confirmed as positive reactivity to Ziel-Neelsen stain
and culture. The confirmation can be made on positive culture only in our cases, but we performed PCR analysis anddirect-sequence analysis, by using DNA product of the skintissues instead of cultured sample for species specificity.
PCR analysis is the easy and simple method as compared toother diagnostic tools. We confirmed the all fives cases were
infected with mycobacterium tuberculosis. From these observations, we considered that PCR analysis might be usefulfor the diagnosis for cutaneous tuberculosis.

５７19) THE INFLUENCE OF CIRCUMSTANCES TO LEPROSY SKIN LESIONS.
ATSUSHI HOSOKAWA１, CHOMAR KAUNG MYINT２，３, SHIGEO NONAKA１
１

Department of Dermatology, Ryukyu University School of Medicine, Okinawa, Japan,
Preventive & Social Medicine Department, Institute of Dental Medicine, Union of Myamar,
３
Department of Environmental Sociology, Ryukyu University School of Medicine, Okinawa, Japan
２

Today, most of countries endemic for leprosy are situated in the tropical zone. Therefore, in consideration of thehistopathology and epidemiology of leprosy, the evaluationof its temperature and UV etc. are considered to be important.
The purpose of our study is to find out the correlation
between conditions of circumstances and skin lesions ofleprosy patients who were observed at the Ryukyu University Hospital.
Material and method; From 1982 to 2004, 147 new patients and seven relapsed patients with leprosy visited at out
outpatient clinic of dermatology. We recorded the sites andsymptoms of skin lesions and the date of outbreak of thedisease written in their charts.
Result: 26 cases showed the eruption on the face
(17.7%). In 32 cases (21.6%), the first site of lesion wasface and/or neck, and in 16 cases (10.8%, 16/147) lesions
were localized at face and/or neck. Number of patients who
noticed the disease was largest in June. In addition, relatively many cases showed the eruption at the knee joint areaand elbow where skin temperature was slightly low, and nolesion was observed at the scalp, axillary, inguinal, perineal
and annal regions.
Summary; Clinically, we could observed the correlation between the cutaneous changes of leprosy and UVB, temperature and skin temperature.

20) EFFECT OF APHIDICOLIN ON ENTAMOEBA EXCYSTATION
AND METACYSTIC DEVELOPMENTASAO MAKIOKA１, MASAHIRO KUMAGAI１,SEIKI KOBAYASHI２, TSUTOMU TAKEUCHI２
１

Dept of Tropical Medicine, Jikei Univ School of Medicine, Tokyo, Japan.
Dept of Tropical Medicine & Parasitology, Keio Univ School of Medicine, Tokyo, Japan.

２

The effect of aphidicolin, a specific inhibitor of thereplicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined.
The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin werealso examined by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblotting. Excystation, which
was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited byaphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development,
when determined by the number of nuclei in amoeba, wasalso inhibited by aphidicolin, because the percentage of 4nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug.
The addition of aphidicolin to cultures at day1of incubation

reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect ofaphidicolin on excystation and metacystic development was
reversed by removal of the drug. Pretreatment of cysts withaphidicolin before transfer to a growth medium containing
the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 inthe cultures with aphidicolin, reacted strongly with rabbit
anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, whichwere predominant at day 3 in cultures without aphidicolin,no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.

５８21) MOLECULAR CLONING AND CHARACTERIZATION OF A PROTEINGERANYLGERANYLTRANSFERASE TYPE I FROM ENTAMOEBA HISTOLYTICA
MASAHIRO KUMAGAI１, ASAO MAKIOKA１,
TSUTOMU TAKEUCHI２, TOMOYOSHI NOZAKI３
１

Dept of Tropical Medicine, Jikei Univ School of Medicine, Tokyo, Japan.
Dept of Tropical Medicine & Parasitology, Keio Univ School of Medicine, Tokyo, Japan.
３
Dept of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.

２

Ras superfamily small G proteins act as a molecularswitch of signal transduction involved in cell proliferationand differentiation, cytoskeletal reorganization, and vesicular transport. Protein prenylation, a class of posttranslational
lipid modifications, is necessary for these proteins to associate membrane structures and function. Protein prenylation
includes protein farnesylation and protein geranylgeranylation, which are catalyzed by protein farnesyltransferase(FT), protein geranylgeranyltransferases type I (GGT-I) andtype II. It has been revealed from the study of mammals andyeasts, that these enzymes are consisted of α and β subunits,
and that FT and GGT-I have a common α subunit. The inhibitors for protein prenyltransferases inhibit the carcinogenesis caused by mutations of Ras proteins, and also inhibit the proliferation of protozoan parasites such as Trypanosoma. We have been studying on the protein prenyltransferases of Entamoeba histolytica (Eh), as a control
molecule of cell proliferation and differentiation, and as apossible target of chemotherapy. We previously cloned andsequenced EhFT and characterized the recombinant pro-teins. Here we reported on EhGGT-I.
The β subunit of EhGGT-I had 337 amino acids, andshowed 22 to 28% identities to those of human, yeasts andArabidopsis thaliana. The recombinant EhGGT-I, expressed by Escherichia coli, was obtained as a complex of
38 kD and 35 kD proteins. The rabbit antiserum raised
against the recombinant EhGGT-I reacted with EhGGT-I,
but did not reacted with rat GGT-I. In contrast to rat GGT-I,
which geranylgeranylates human H-Ras-CVLS, but does
not geranylgeranylate the mutant H-Ras-CVLL, EhGGT-Iβ
geranylgeranylated both substrates. In mammals, Ras proteins are farnesylated by FT, and Rac proteins are geranylgeranylated by GGT-I. EhGGT-I, however, geranylgeranylated Ras proteins such as EhRas 1-CIMF and EhRas 2CELL as well as Rac proteins including EhRacA-CLLF
and EhRacG-CSLF. EhGGT-I was much more resistant toinhibitors for mammalian GGT-I than rat GGT-I.
In conclusion, EhGGT-I was significantly different
from mammalian GGT-I, and thus would become a possible
chemotherapeutic target of amoebiasis.

22) THE CURATIVE EFFICACY OF METRONIDAZOLE ON INTESTINAL AMEBIASISSEIKI KOBAYASHI１, EIKO IMAI１, TAKUYA MAEDA１, TSUTOMU TAKEUCHI１
１

Dept of Tropical Medicine and Parasitology, School of Medicine, Keio Univ, Tokyo, Japan

The prevalence of amebiasis of these three years tends
to increase in Japan. And over five hundred cases of mainly
symptomatic amebiasis have been reported per year. The
majority of these reported cases were intestinal amebiasis.
Metronidazole (Mz) is usually single used as a medicine fortreating amebiasis in Japan. Mz has significant therapeutic
effect especially on invasive amebiasis because of its immediate absorption from intestine. And Mz is also used fortreatment of noninvasive intestinal amebiasis by means ofincreasing its dosage and dosing period. However, recently
measurable failure cases of Mz have been reported. We havealso experience of Mz failure in treatment of mass infectionof Entamoeba histolytica (prevalence of cyst positive cases/
80 persons; 30%) in a rehabilitation facilities for persons
with intellectual disabilities, despite of repeat prescriptionsof Mz, maximum 8 times for 2 years. Coprophilia was estimated one of the reason of reinfection of cysts of E. histolytica and the reason of failure of Mz treatment. Finally, themass infection of E. histolytica was eliminated by combination chemotherapy of Mz and Diloxanide flooate(Dlx) as
lumen drug. By the result of Mz treatment in mouse (C3H/
HeJ) intestinal amebiasis model, Mz (12.5mg/kgx7days)
administered via intraperitoneal was more effective than via
orally administered Mz with same dose. This drug kinetics

５
９

may indicate that amebae are colonized and proliferated byadhesion with or by invasion in intestinal mucosa. These results showed some difficulties of monotherapy of Mz. And,

if unlicensed Dlx is unobtainable, some combinations ofprobiotics for improvement of intestinal bacterial flora andconstipation with Mz may be effective.

23) THE ATTACHMENT AND ENTRY OF LEISHMANIA (LESIMANIA) MAJOR TO MACROPHAGES:OBSERVATION BY SCANNING ELECTRON MICROSCOPEHIROSHI UEZATO１, HIROTOMO KATO２, BHUTTO ABDUL MANAN３,KEN KATAKURA４, SHIGEO NONAKA１, YOSHIHISA HASHIGUCHI５
１

Dept of Dermatology, Univ of the Ryukyus, Okinawa, Japan
Dept of Veterinary Hygiene, Yamaguchi Univ, Ymaguchi, Japan
３
Dept of Dermatology, Chandka Medical College/Hospital, Larkana, Pakistan
４
Lab. of Parasitology, Graduate School of Veterinary Medicine, Hokkaido Univ, Hokkaido, Japan
５
Dept of Parasitology, Kochi Medical School, Kochi Univ, Kochi, Japan
２

Leishmaniasis starts with the injections of the
Leishmania protozoan promastigates into the skin at thetime of blood suctions by a female sandfly. The infection
with leishmania is established when the Leishmania protozoans start their own intracellular multiplication after having been phagocytized by the host macrophages. In the earliest stage of the infection of leishmaniasis, therefore, theattachment of the promastigates to the macrophages is es-

sential. We incubated in vitro a mixed culture of macrophages (JM774-1A) and Leishmania (Leishmania) majorfor 6 hours and observed the form of the attachment between the two by a scanning electron microscope. We foundthat the attachment between the two occurred at the site of
the parasite body in addition to the previously reported sites
such as the flagellar tip, flagellar base, and aflagellar tip(posterior pole).

24) DEVELOPMENT OF A SENSITIVE AND SIMPLE DIAGNOSTIC METHOD FOR VISCERALLEISHMANIASIS USING URINE SAMPLES
MAKOTO ITOH１, MOHAMMAD ZAHIDUL ISLAM１, HIDEKAZU TAKAGI１,YOSHIHISA HASHIGUCHI２, EISAKU KIMURA１
１

Dept of Parasitology, Aichi Medical Univ School of Medicine, Aichi, Japan
２
Dept of Parasitology, Faculty of Medicine, Kochi Univ, Kochi, Japan

We previously reported a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the diagnosis ofvisceral leishmaniasis (VL) using urine samples that can beeasily and safely collected.
Antibodies specific to Leishmania antigens were detected by the ELISA in non-concentrated urine samples
from VL patients. However, the urine-based ELISA needs aplate reader to measure optical density. On the other hand, adirect agglutination test (DAT), which uses Leishmanis
donovani promastigote as antigen (DAT antigen), does not
require sophisticated equipment and has been used widely.
In this study, we report a new DAT that has enough sensitiv-

ity to detect antibodies in urine samples.
A96-well microtiter plate was coated with anti-human
IgG. Urine samples were applied to each well and incubatedovernight. After washing the plate, DAT antigen was applied to each well. Agglutination was observed after overnight incubation.
With this new DAT, 55 out of 56 VL patients (98%)
were diagnosed as positive and all endemic normals (n=59)
and Japanese controls (n=24) were negative. Adaptation ofthis sensitive, specific and simple method to a mass survey
of VL is now in progress.

６０25) ESTABLISHMENT OF A DETECTION AND IDENTIFICATION METHOD OFLEISHMANIA SPECIES WITHIN NATURALLY INFECTED INDIVIDUAL SANDFLIESHIROTOMO KATO１, HIROSHI UEZATO２, KEN KATAKURA３, MANUEL CALVOPINA４,JORGE D. MARCO４, PAOLA A. BARROSO４, TATSUYUKI MIMORI５, MASATAKA KORENAGA４,SHIGEO NONAKA２, YOSHIHISA HASHIGUCHI４
１

Dpt. of Veterinary Hygiene, Yamaguchi Univ. Yamaguchi, Japan
２
Dept. of Dermatology, Univ. of the Ryukyus, Okinawa, Japan
３
Lab. of Parasitology, Graduate School of Veterinary Medicine, Hokkaido Univ., Hokkaido, Japan
４
Dept. of Parasitology, Kochi Medical School, Kochi Univ., Kochi, Japan
５
Dept. of Tumor Genetics and Biology, Graduate School of Medical Science, Kumamoto Univ., Kumamoto, JapanThe surveillance of prevalent Leishmania and sandfly
species in endemic areas is important for prediction of therisk and expansion of leishmaniasis. Usually, the infectionof sandlies with Leishmania promastigotes has been examined by dissection of each sandfly under a microscope.
However, for this purpose, sandflies should be fresh andhighly skilled technique is required for dissection of tiny
sandflies. In addition, this process takes a relatively longtime and is not suitable for examination of a large numberof sandflies. In the present study, we attempted to establish
a PCR-based detection and identification method of
Leishmania species within naturally infected sandflies.
First, we tested the specificity and sensitivity of our PCR
method with primers specific for Leishmania minicircle kinetoplast DNA using genomic DNA equivalent to 10, 1 or
0.1 parasites from five reference strains of Leishmania species distributed in Ecuador as templetes. As the result, weamplified minicircle kinetoplast DNA from all the Leishmania species examined and detected 0.1-1 parasites in these
species. In addition, we successfully detected Leishmania

minicircle DNA by PCR from ethanol-fixed sandflies infected with Leishmania promastigote under microscopy.
We next applied this method to ethanol-fixed field-captured
sandfly samples. After extraction of DNAs from individual
sandfly samples, PCR amplification was performed as described above. We detected the minicircle DNA in 6 out of
183 (3.3%) sandflies with this method, while 5 out of 143(3.5%) were positive for Leishmania promastigotes in thesame areas under microscopic examination. The species
were determined Leishmania (Leishmania) mexicana by
nucleotide sequencing of the Cytochrome b gene. Additionally, all the Leishmania-positive sandflies were identified as Lutzomyia ayacuchensis by the restriction enzyme
digestion of the PCR-amplified 18S rRNA gene fragments.
Since this combined method is relatively easy and process alarge number of samples, it will be a powerful tool for rapid
identification of prevalent sandfly and Leishmania species
as well as monitoring the infection rate in sandfly populations in endemic areas.

26) PRODUCTION OF RECOMBINANT KINESIN-RELATED PROTEIN OF
LEISHMANIA DONOVANI AND ITS APPLICATION IN THE URINE-BASEDELISA FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS
MOHAMMAD ZAHIDUL ISLAM１, HIDEKAZU TAKAGI１, MAKOTO ITOH１,YOSHIHISA HASHIGUCHI２, EISAKU KIMURA１
１

Dept of Parasitology, Aichi Medical Univ School of Medicine, Aichi, Japan
２
Dept of Parasitology, Faculty of Medicine, Kochi Univ, Kochi, JapanFor the serodiagnosis of visceral leishmaniasis (VL), a
recombinant antigen, rK39 (a part of L. chagasi kinesin related protein), has shown very good sensitivity and specificity. Here we report the production of recombinant kinesinrelated protein with a molecular weight of 42-kDa (rKRP

42) of L. donovani, a homologue to rK39, and the value of
this antigen in urine-based ELISA for the diagnosis of VL.
To obtain the gene coding the rK39 homologue, PCR
amplification was performed from L. donovani genomic
DNA. Then the amplified PCR product was subcloned in６１

pBluescript KS (-) and sequenced. The PCR product was
1071 bp, which was just one repeat (39 amino acid) larger
than the rK39 antigen. Amino acid sequence showed 89.3%
identity and 98.7% homology to the rK39 antigen. The PCR
product was cloned in pTYB12 expression vector. The
rKRP42 was purified with Ni-NTA column, then with Chitin column, and used in the urine-based ELISA for the diagnosis of VL.
The ELISA showed 94.3% sensitivity (66 positives

among 70VL samples) and 99.6% specificity (1 positive
among 240 non VL samples). The specificity was determined with urine samples of 59 endemic healthy controls,
80 Japanese controls, 58 malaria, 13 tuberculosis, 23 cutaneous leishmaniasis, and 7 other diseases. This assay system showed similar sensitivity and specificity to our previously reported urine-based ELISA with acetone-treated L.
donovani promastigotes antigen. The use of this rKRP42
will ensure the stable supply of antigen.

27) DEVELOPMENT AND EVALUATION OF A NEW ANTIBODY DETECTION
KIT (INSTANT(TM)CHEK-AMOEBA ) FOR AMOEBIASISIRENE AYI１, NOBUAKI AKAO１, KUNIOKI ARAKI１, EIJI HAYASHI１,
KOICHIRO FUJITA１, SEIKI KOBAYASHI２, TSUTOMU TAKEUCHI２, JYUN-ICHI WATANABE３,MIHOKO IMADA４, ALBERT CHU５
１

Section of Environmental Parasitology, Graduate School of Tokyo Medical and Dental University, Tokyo, Japan２
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo, Japan
３
The Institute of Medical Science, University of Tokyo, Tokyo, Japan
４
Sam Ratulangi University, Madado, Indnesia５EY-Lab. Inc., Hong Kong, China

After the enforcement of the Infectious Diseases Control Law in April 1999 (partially amended in November
2003), amoebiasis was included in Category V of the law
and all physicians were to take note that it was no longer aquarantinable disease. Since then, the chances of medical
examination and treatment of amoebiasis in out-patientsclinics have increased, and antibody tests of the disease arein higher demand nowadays. Diagnostic tests based on existing methods like plate ELISA, gel precipitation test andimmunoblot assay require a couple of days to obtain laboratory results. In addition, commercially available test kits foramoebiasis often show false negative results. We hereby
present a report on a rapid test kit Instant (TM) CHEKAmoeba for the detection of anti-Entamoeba histolytica antibodies from patients’ serum. The kit does not need any
special apparatus and the result of a test could be decided
within one minute.
The test is easy to perform. Twenty microliters of test
serum diluted fourfold (1:3) is added to a nitrocellulose
membrane unto which E. histolytica HM-1 strain antigen
has been adsorbed and allowed to be naturally absorbed(approx. 15 sec). A solution of Protein A conjugated colloidal gold particles is immediately added and allowed to beabsorbed (approx. 15 sec). Then, 3 drops of washing buffer
A and 3 drops of washing buffer B (included in kit) areadded in that order to wash off excess reagent (30 sec). In
the case of antibody positive reaction a red/pink spot appears in the center of the device at the point of washing withbuffer A. It is that simple to conduct the test.
This new kit was used in testing dot-ELISA-tested serum samples from 44 hepatic and 64 intestinal amoebiasis
patients and a 100% agreement rate was obtained. Sera
from patients with non-amoebic protozoa infections, ulcerative colitis and normal individuals, consisting of both males
and females, were used in investigating the kit and no positive results were observed in all the cases. We believe thatso far the Instant (TM) CHEK-Amoeba is the only antibody
detection system that has the peculiarity of a matching sensitivity to the already established dot-ELISA and besides it
is very handy and the results can be decided in the shortest
possible time.

６２28) THE STUDY OF ANTIGEN FOR THE NEW TUBERCULOSIS DIAGNOSISAND APPLICATION TO THE SERODIAGNOSISATSUKO YOSHIDOME１, SEIJI KANDA１, ITOKO HAYASHI１, TOSHIMASA NISHIYAMA.１
１

Dept. of Public Health, Kansai Med. University,

In order to identify the active tuberculosis, it is necessary to search the antigen that has the high specificity andgood sensitivity for the diagnosis. In the field of developing
countries, to establish the screening method for the active
tuberculosis patients is very important. At first, we established the BCG cDNA library. Briefly, RNA was extracted
from the Mycobacterium bovis (BCG Tokyo strain), andcDNA was synthesized using the random primer and theadapter with restriction enzyme was attached to both end of
the fragments, and ligated into the expression vector. As aresult of immuno-screening using the tuberculosis patients’sera, 15 positive clones were identified. The sequences ofthese clones were examined and searched their homologies
by GenBank. One of their clones was identified as CONSERVED HYPOTHETICAL PROTEIN of M. bovis (ORFof this gene is 288 bp and the molecular weight is about 12kDa). This region was highly preserved between M. bovis
and M. tuberculosis. This result suggested that this antigen
could be applied to the diagnosis of the human tuberculosis

and was named BCG1. We confirmed the expression of recombinant BCG1 as a fusion protein (40 kDa) with GST by
SDS-PAGE. Therefore, the reactivity of this antigen against
the patient’s serum was examined by Western Blotting, andspecific signal was detected by patient’s serum but not by
healthy subjects. These results were suggested that this antigen BCG1 was reacted with the patient of tuberculosis specifically. Furthermore, we examined the specificity of thisantigen by Dot-blot analysis. The dilutions of the antigen
were prepared by each 4 fold from 156 mg/ml. The reactionof patient’s serum was disappeared at 4,096 fold dilution
and healthy subject disappeared at 64 fold dilution. The difference of the reactivity was 64 fold between patient andhealthy subject. These results were suggested that BCG1
antigen was useful for diagnosis of active tuberculosis patients in view of the peculiar sensitivity with patient’s serum.
We are going to examine the reactivity of BCG1 antigen
against each condition of patients.

29) N-TERMINUS TRUNCATED RECOMBINANT SARS CORONAVIRUS NUCLEOCAPSIDPROTEIN-BASED ELISA IS MORE SPECIFIC FOR SERODIAGNOSIS OF SARSFUXUN YU１, SHINGO INOUE１, MAI QUYNH LE２, HONG THI CAM THAI１,
FUTOSHI HASEBE１, KOUICHI MORITA１
１

Department of virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
２
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia.
The disease has affected 30 countries in 5 continents, withmore than 8400 cases and more than 910 deaths. Since therecognition of the SARS coronavirus (SARS-CoV) as theagent causing SARS in April 2003, enormous international
efforts to develop laboratory tests for SARS have occurred.
Despite sensitive methods for detecting virus genetic material, detection of virus in the early stages of illness may still
be difficult, and the final assessment of whether an individual has been infected may depend on detection of antibody
to virus.To develop a safe and reliable diagnosis method for
SARS, we cloned the whole range and the N-terminal 121
amino acids truncated SARS-CoV nucleocapsid protein (Nprotein and N-１２１protein) gene into plasmid and expressed
them in E. coli. Serum samples collected from 175 healthy
volunteers in Vietnam before the SARS outbreak occurred
were used to for the assessment of our newly developed indirect IgG ELISA using recombinant N protein and N-１２１
protein as assay antigens. While ELISA using N protein
showed relatively high nonspecific reaction which was 38/
175 (21.7%) and the titer ranging 1:100-1:3200, howeverN-１２１ protein could drastically reduce it up to 11/175 (6.3%)
and the titer ranging 1:100-1:200. To assess the sensitivityof IgG ELISA using N-１２１ protein, serially collected serum
samples from 37 SARS probable (including 15 cases confirmed with RT-PCR) cases were examined. Thirty six patients (97.3%) showed IgG seroconversion and the titer was

６３

ranging 1:600-1:204, 800. The timing of IgG seroconversion against N-１２１ protein after onset of illness were22.2% in the first week, 69.4% in the second week andreached to 100% in the third week.Our data indicate that
our newly developed SARS-CoV N-１２１ protein-based IgG

ELISA is a safe, specific and sensitive test for diagnosing
SARS-CoV infection. It could serve as a simple, sensitive,
and specific test for clinical diagnosis and large-scale seroepidemiological studies.

30) PARAGONIMUS OHIRAI M. IN THE BASIN OF MARUYAMA RIVER, HYOGO PREFECTURE
HIROSHI YAMANISHI
Kobe Gakuin Women’s College, Kobe, JapanIn Maruyama River in the north Hyogo Prefect., theepidemiological study of Paragonimus ohira is carried out
by Yoshida (1955) and Miyamoto (1961) 50 years ago. The
investigation was carried out in the holm of Kikuyasima in
Maruyama River. Seasarma dehaani which was second intermediate host of P.ohira in the Kikuyasima lived in great
numbers. In April 2002, the infection rate of metasercaria(MC) of P.ohira in the S.dehaani was 84.9 per cent, 68.8
per cent in May, 61.5-67.4 per cent in July, 78.0-95.6 percent in August, 92.0-93.3 per cent in September and 100 per
cent in November. The average infection rate was high with
78.6 per cent. It tended to be same in 2003 and the average
infection rate was 87.0 per cent. In this holm, Assiminea japonica and A.parasitologica were distributed. Cercariae of
P. ohira could not be detected. In comparison with the reports 50 years ago, the distribution of the S.dehaani in
Maruyama River had been limited and the infection rate of
MC were high 50 years ago similarly in Kikuyasima.

31) A SIMULATION OF EGG OUTPUT SCHOOL CHILDREN INFECTED WITHSCHISTOSOMA HEAMATOBIUM BY AN IMMIGRATION-DEATH MODELYOSHIKI HAMADA, MASAAKI SHIMADA, KAZUHIKO MOJI
Research Center for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Monthly Schistosoma haematobium egg counts in 10
ml of urine after treatment fluctuated remarkably within asubject of schoolchildren in an African community. Thiskind of fluctuation has been thought as a technical limitation of the urine examination.It has been assumed 1) thatthe number of pairs of adult worms of S. h increases relatively constantly after the treatment and it does not fluctuatemuch, 2) that the egg production per pair of worms is relatively unchanged (although crowding induces reduction ofegg production per female worm), and 3) that the observed
fluctuation of egg output, therefore, is caused both by uncertainty of egg excretion to urine and by volume change ofurine.
Daily fluctuation of egg counts for consecutive days,
however, was smaller than monthly fluctuation in this community. This suggests that the large monthly fluctuation is
not due to the technical limitation.There is a possibility oflarger fluctuation of number of adult worm pairs in a humanbody than it has been thought.To simulate and verify this, amathematical model was applied. We built an immigrationdeath model that assumed that the observed egg output in
urine reflected the real number of egg production. We created two parameters in a stochastic process: Mortality (orlife expectancy) of adult worms, and frequencies of infection. The number of cercariae penetrating at one time of infection was adjusted by the frequency to have the same acquisition per unit of time.Egg outputs of four patterns, combination of mortality (high or low), and frequency (constantand less constant), were calculated for 1000 days. The
simulation model with high mortality of adult worms andless frequent infection matched most with the observed pattern of egg counts. The mean life expectancy is as short as
six months in this model. Because this study is based on asimplified assumption, further studies are needed to verify
the plausibility of this model/hypothesis.

６４32) CURRENT EPIDEMIC STATE OF SCHISTOSOMIASIS MEKONGI IN LAO PDR.
SATOSHI NAKAMURA１, HAJIME MATSUDA２, MASASHI KIRINOKI２,
SHIGEHISA HABE３, VIROJ KITIKOON４, TORU WATANABE５, NAOKO NIHEI６,
SOURAXAY PHROMMALA７, BOUNGNONG BOUPHA７, NAO BOUTTA８.
１

Research Institute, International Medical Centre of Japan, Shinjuku, Tokyo, Japan
Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine, Tochigi, Japan
３
Animal Science Centre and Department of Parasitology, School of Medicine, Fukuoka University, Fukuoka, Japan
４
Department of Social and Environmental Medicine, School of Tropical Medicine, Mahidol University, Bangkok, Thailand
５
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan
６
Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan
７
National Institute of Public Health, Ministry of Health, Vientiane, Lao PDR.
８
Cabinet, Ministry of Health, Vientiane, Lao PDR.
２

Abstract
A preliminary investigation on Schistosoma mekongi
infection in human, animal and snail host was performed at
Khong district, Champasack province in Lao PDR in May
2004. The results of stool examination conducted at 7 villages that contained 2 newly surveyed ones, revealed in between 20% to 50% of high endemic prevalence. Children in
10-15 years of the age group were the most prevalent at
60% was confirmed. Adult developed stage patients werealso confirmed in the area. No egg was detected at 19 samples of fresh pig stools collected among the villages. On thesnail vector collections, both alpha- and gamma- race Neotricula aperta snails were also detected at the shallow rocky
streams of the Mekong basin.
Key words: Schistosoma mekongi, Neotricula aperta, infection, Khong, Champasak, Lao PDR.

33) WATER CONTACT PATTERNS AND KNOWLEDGE OF SCHISTOSOMIASIS IN NORTH ZANZIBARYUJI ATAKA１, KAZUHIKO MOJI１, ALI F. MGENI２, ALIPO N. KHAMIS２, MASAAKI SHIMADA１
１

Research Center for Tropical Infectious Diseases, Inst. Trop. Med., Nagasaki Univ., Nagasaki, Japan
２
Ministry of Health and Social Welfare, Zanzibar

Water contact patterns have been one of the foci in theschistosomiasis epidemiological studies. The villagers living in the schistosomiasis endemic area contact cercarial infested water frequently for various domestic purposes intheir daily life. These contacts are, however, difficult to
avoid due mainly to the unavailability of alternatives, suchas safe water supply. Furthermore, the villagers especiallychildren and young adolescent wittingly contact risky water
for bathing/playing purposes. Although villagers’ knowledge and perceptions of schistosomiasis is critical information for planning control program, they were ignored inmany occasions. As a result, little is known about the relationships between exposure to risky water and knowledge
and perceptions of this disease. In this study, we conducted
researches to elucidate the interrelationships between watercontact behaviors and knowledge and perceptions of schistosomiasis among villagers (N=648) living in Bandamaji,

an urinay schistosomiasis endemic area in the northZanzibar. For water contact behaviors, direct observation
was performed using time-saving spot check method with
six visits/day from 6:00 till 18:00 for all water contact sites(N=41), and knowledge and perceptions on schistosomiasis
were collected by interview survey with systematic questionnaire. The urine examination was also conducted foregg counts. High mean score (8.6/10.0: SD=1.8) for theknowledge of schistosomiasis etiology among villagers excluding small children (N=537) implied that the villagers
understood considerably about the risk of infection in theriver/pond water. The questionnaire on risky water contacts
also clearly indicated that the villagers contacted risk water
frequently in spite of their awareness of infection risk. As
the result of logistic regression analyses, “washing utensils/
tools/bicycle” (p<0.01) and “fishing” (p<0.001) were thesignificant factors related to infection for males and “wash-６５

ing clothes” (p<0.01) for females, in addition to “agegroup” factor. The direct observation clarified the frequencyof risky water contacts, 6% (104:male=81, female=23) of
1791 water contacts (male=304, female=1487) during14
consecutive days, and the risky water contacts were observed more among males (27%: 81/304) than females (2%:23/1487). These results corresponded with the percentage
and intensity of egg positives, and with villagers’ answers to

the questions on water contacts. In conclusion, control programs which simply recommend avoidance of contacts withrisky water contribute little to reducing infections, andtherefore the reasons of risky water contacts should be carefully considered; for instance, a program with supply ofsoaps which are soluble and will lather easily even in hardwater of the wells in this area.

34) DAILY VARIATION OF SCHISTOSOMA HAEMATOBIUM EGG EXCRETIONDAVID WARUNGE MURIU１, YOSHIKI HAMADA１, KANJI WATANABE１, YOSHIKI AOKI１, KAZUHIKO MOJI１,YAWO DOTSE AFETSE１, MOHAMED KARAMA２, N’GETHE MUHOHO２, and MASAAKI SHIMADA１
１

Institute of Tropical Medicine, Nagasaki University and ２Kenya Medical Research InstituteEggs of Schistosoma haematobium were collected by
two methods for 62 schoolboys for 3 days of a week in aurinary schistosomiasis endemic community of coastal
Kenya. Spontaneous urine after 10:30 am was collected and
10 ml of the urine was filtered for collecting eggs. Thismethod is referred as the 10 ml spontaneous urine method(eggs/10 ml). The second urine, one hour after the previous
spontaneous urine was collected and all the volume of theurine was filtered for collecting eggs. This method is referred as the one-hour urine method (eggs/hour). Prevalenceof egg-positive after three urine examinations was 95.2%

for egg/10 ml and 93.5% for egg/hour. Three-day mean oflog converted eggs/10 ml and eggs/hour of each boys correlated very well (R=0.866), although the value of egg/hour
was twice larger than that of egg/10 ml (mean of log (1+eggs/hour) was 2.33 with standard deviation (SD) of 1.04,
and mean of log (1+eggs/10 ml) was 2.01 with SD of 0.92.
paired t-test, p<0.001). Mean of coefficients of variance(CV) of log converted egg/hour was 23.5% and smaller thanthat of egg/10ml was 32.4% (p<0.05). The value of the CVs
measured in a week seemed smaller than that of three repeated measures over three months.

35) EXPERIMENTAL INFECTIONS OF BLACK FLIES (DIPTERA: SIMULIIDAE)
WITH MICROFILARIAE OF ONCHOCERCA DEWITTEI JAPONICA,
THE CAUSATIVE AGENT OF ZOONOTIC ONCHOCERCIASIS IN JAPANMASAKO FUKUDA１, HIROKO SATO２, HIROYUKI TAKAOKA２
１

Institute of Scientific Research & Department of Infectious Disease Control, Oita University, Oita, Japan.
２
Department of Infectious Disease Control, Faculty of Medicine, Oita University, Oita, Japan.

Zoonotic onchocerciasis is very rare in humans. Only
11 cases including five cases in Oita, Japan, have been reported. The causative agent in Japanese cases was all Onchocerca dewittei japonica Uni, Bain et Takaoka, from wildboars. To determine the vector (s) of this parasite, microfilariae of O. dewittei japonica were injected intrathoracically into 40 newly-emerged females of five Simulium spp.
from Oita. The flies were maintained with sucrose solution
at 25°
Ｃ. After10days post-infection, the third-stage larvae
were found in 2 of 14 S. aokii (Takahasi) dissected, 4 of 6 S.

arakawae Matsumura, 2 of 4 S. bidentatum (Shiraki), 1 of
1 S. japonicum Matsumura and 3 of 4 S. rufibasis Brunetti.
The infective third-stage larvae were 750-1,000 µm long by
18-20 µm wide. It has been proved for the first time thatthese five black-fly species are able to support the larval development of O. dewittei japonica. This finding may suggest a possibility that black flies play a role in the transmission of zoonotic onchocerciasis due to O. dewittei japonica
in Japan.

６６36) APPLICATION OF URINE-BASED ELISA TO MONITOR THE EFFECT
OF MASS-DRUG ADMINISTRATION FOR LYMPHATIC FILARIASIS CONTROLIN A LOW ENDEMIC AREAEISAKU KIMURA１, MAKOTO ITOH１, MIRANI V WEERASOORIYA２, YASUNORI FUJIMAKI３,
MOHAMMAD Z ISLAM１, TAKENORI AOYAGI１, YOSHINORI ISOGAI４
１

Dept of Parasitology, Aichi Medical Univ School of Medicine, Aichi, Japan
Dept of Parasitology, Faculty of Medicine, Univ of Ruhuna, Galle, Sri Lanka
３
Dept of Parasitology, Inst of Trop Med, Nagasaki Univ, Nagasaki, Japan
４
Faculty of Social and Information Scinence, Nihon Fukushi Univ, Handa, Japan２

The 50th World Health Assembly made a resolution toeliminate lymphatic filariasis as a public health problem
from the world by 2020. The basic strategy for the elimination is mass treatment of all the people in endemic areas
with a drug combination of diethylcarbamazine and albendazole. In Sri Lanka, the mass drug administration (MDA)
campaign was launched in 2002. For the evaluation of
MDAs, many countries, including Sri Lanka, employ a traditional night blood survey to detect microfilaria (mf) carriers, which is one of the most difficult field activities. An
ICT card test that detects filarial antigen with day blood in
10 minutes is very effective but not popular due to high cost.
Blood sampling that people (especially young children) dislike is another barrier, which is getting higher as the number
and seriousness of filarial infections are going down. Considering these problems and importance of children as asentinel group, we have developed a new ELISA that uses
urine as a sample for serum. The ELISA (urine ELISA) detects filaria-specific urinary IgG4, and its high sensitivity
and specificity have been shown in various studies. As afirst trial to study the usefulness of urine ELISA as a monitoring tool of nationwide MDAs, it was applied in Wuchereria bancrofti-endemic areas in Deniyaya, a mountainous tea
plantation district in southern Sri Lanka. Urine samples
were collected in the daytime from 699 schoolchildren aged
6 to 16 years in 2002 and 2003, before and about 1 year after the first MDA. The MDA reduced the IgG4 positive ratefrom 7.9% to 3.3%. Thirty-eight (69%) of 55 positive children in 2002 became negative after treatment. On the other
hand, 6 of 644 negative children turned positive in 2003.
The results indicate that IgG4 antibodies among children reduce relatively quickly by treatment, implying that the antibody detection is as good as antigen detection in terms ofnegative conversion. Urine ELISA is a very sensitive
method to detect early infection. Sample collection is noninvasive, and therefore most suitable for pediatric subjects.
The ELISA needs well-equipped facilities, however. It isnecessary to improve the method to be more simple and lesscostly so that it can be applicable in filariasis endemic areas.

37) A CASE STUDY OF TUBERCULOSIS GENETIC EPIDEMIOLOGICAL STUDY BASED ON
THE LONG-TERM INTERNATIONAL COLLABORATIVE FIELD RESEARCH IN THAILANDHIDEKI YANAI, SURAKAMETH MAHASIRIMONGKOL,PATHOM SAWANPANYALERT, NORIO YAMADA, NAOTO KEICHOTB/HIV Research, Depatment of Research, The Research Insitute of Tuberculosis, Tokyo, JAPANThai NIH, Ministry of Public Health, Nonthaburi, Thailand
TB/HIV Research Foundation, Chiang Rai, Thailand
The Research Institute, International Medical Center of Japan

Background and Objective: Since we do not have an exact
animal model for tuberculosis (TB), we need to study thehuman tuberculosis in the field. Genetic epidemiological research may contribute the breakthrough finding. We haveconducted a study of the human genetic susceptibility to

Mycobacterium tuberculosis based on the long-term collaboration with Thai Ministry of Public Health and otheragencies (since 1993) in Northern Thailand.
Methods: The project set up an active computerized
population-based surveillance on TB and HIV/AIDS since６７

1987 in collaboration with the Chiang Rai provincial healthoffice. Chiang Rai province is the northernmost remote
province In addition to the database; the project started thecollection of the culture isolates of M.tb since 1996. We had
begun the study on the genetic susceptibility on tuberculosis
in 2002 after a receiving the approval of the ethic committee of Thai Ministry of Public Health. Since our study is offamilial-based in nature, we began collecting complete or
incomplete TRIO (TB patient and their parents or substitutes) for TDT (transmission /disequilibrium test), ASP: affected sib-pair for Sib-Pair Analysis, and ARP: affected
relative pair for alternative methods to detect the linkage.
Results: We identified 17,216 who were registered in 19872001 in the surveillance. Among them, we detected 8,586
pulmonary AFB-smear-positive TB cases and we found 144
families to that were affected by TB and thus met our enrollment criteria (ASP 25, ARP 49, Trio 64, Incomplete Trio

6). We confirmed 811 TB patients who did not meet the criteria, 73 who were in the prison, 260 who had moved toother provinces, and 3,756 who had already died. 1,725 TB
cases could not be located. We began a prospective study
since 2002 January and we have identified 20 ASPs, 34
ARPs, 97 Trios, and 67 In-complete Trio by the end of 2003.
We identified one family with 10 TB patients (among them
5 were sibling). We had made the continuous dialogues withThai counterparts and made the Material Transfer Agreement in August 2003.
Discussion: We were able to set up the field research site,which is possible to conduct the sensitive researches likehuman genetics based on the mutual trusts. Thai counterpart
emphasized the equal partnership based on the long-term
collaboration and in particular requested training and education for young Thai scientists. In addition, issue of the intellectual property need to be clear.

38) STUDY ON TRANSMISSION AND INFECTION OF SOIL-TRANSMITTED NEMATODE
IN THE SUBURB OF HANOI, VIETNAM: CONTAMINATION OF VEGETABLES
AND FINGERS AND NAIL DIRTS WITH PARASITE EGGS.
YASUNORI FUJIMAKI１, NGUYEN THI VIET HOA２, SHINICHI NODA３, SHOJI UGA４,LE KHANH THUAN２, LE DINH CONG２, YOSHIKI AOKI１, KAZUHIKO MOJI１
１

Dept of Parasitology, Institute of Tropical Medicine, Nagasaki University, Japan
National Institute of Marariology, Parasitology and Entomology, Hanoi, Vietnam
３
Research Center for the Pacific Islands, Kagoshima, Japan
４
Dept of Medical Technology, Kobe University, School of Medicine

２

To establish more efficient and effective control strategy against soil-transmitted helminth (STH) infections,mode of transmission of STH infections in endemic area
should be elucidated. We have been carrying out the project
to know how or to what extent the environment is contaminated with STH eggs in a village at the suburb of Hanoi, Vietnam. We report here parasitic contamination level of vegetables, fingers and nail dirts in the study site. The vegetables(nine leafy type, four root type and two fruit type) were examined. Most of them are eaten raw by villagers. Samplesof finger-washing fluid and nails were obtained from school
children and inhabitants in 33 households. Parastie eggs
were separated from these samples by soaking in 0.5%
Tween 20 and recovered by floatation method using saturated NaNO３. Examinations of these samples were done
once in both rainy and dry seasons. Six and seven species ofparasite eggs were recovered from sample of vegetables andfingers/nails, respectively. The recovery rate of the eggs
from these samples was not different by season. Out of a to-tal number of 317 vegetables examined throughout the year,
25.9% was positive for any of the eggs. Leafy type of vegetables was most highly contaminated (30.5%, 73/239). Outof the six species recovered, Ascaris and Trichuris eggs
were detected in 20.5% and 7.9% of the samples, respectively. Mean number of eggs recovered from egg-positive
vegetables was 2.7-7.0 for Ascaris, and 2.0-2.2 for Trichuris in rainy and dry seasons, respectively. For examinationof fingers and nail dirts, the total number of 508 people,
506 finger-washing and 326 nail dirts samples was examined throughout the year. Out of them, 8.5% of people,
7.5% of finger washing and 3.1% of nail dirts were positive
for any of the eggs. Enterobius vermicularis eggs weremost highly recovered (3.5%), followed by Ascaris (1.0%)
and Trichuris (0.6%). The number of E. vermicularis eggs
recovered from egg-positive samples was 1-37. Those of
Ascaris and Trichuris eggs were only 1 in all positive samples. In conclusion, leafy type of vegetables is considered to
be one of important sources of STH infections in our study６
８

area. Fingers and nail also are one of possible sources but

the possibility might be lower.

39) PROBLEMS IN DRACUNCULIASIS ERADICATION ACTIVITIES IN OGOU DISTRICT, TOGOYAWO DOTSE AFETSE, MASAAKI SHIMADA, DAVID WARUNGE MURIU,CAI GUO XI, AND KAZUHIKO MOJI
Institute of Tropical Medicine, Nagasaki University

Togo is one of the 14 remaining dracunculiasisendemic countries of the world. In 2000, 75,223 cases ofdracunculiasis were reported from these 14 African countries, and Togo reported 811 indigenous cases from 147 villages, the six largest numbers after Sudan, Nigeria, Ghana,Burkina Faso, and Niger. About a half of the cases (386)were reported from Ogou district. In the present study weobtained monthly reports on dracunculiasis eradication activities in Ogou district for 22 months in 2002 and 2003.
During this period, 51867 households were visited, and 380
dracunculiasis cases were found, of which 327 (86%) were
contained cases. Contained cases mean 1) the infected per-son was detected within 24 hours of the emergence ofguinea worm through the skin, 2) actions (i.e. occlusive
bandages, counselling, and care of the patient until theworm is pulled out) were taken to prevent the person fromcontaminating sources of drinking water, and 3) these two
conditions were confirmed by a supervisor within 7 days ofoccurrence. The eradication programme distributed morethan 30,000 clothes for filtering drinking water. Although
the number of dracunculiasis cases is decreasing, integration of this vertical eradication programme with the primary
health care system of communities is necessary for successful eradication of the disease.

40) A SEROSURVEY OF HUMAN T-CELL LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1)
IN THE DOMINICAN REPUBLICTETSURO ONO１, YOSHIHIRO ISHIMATSU１, TAVERAS DELFIS１,DISLA MILDRE１, GARCIA BARBARA１, YOSHIHIRO MAKINO２
１

Centro de Educacion Medica de Amistad Dominico-Japonesa, Santo Domingo, .Dominican Republic２
Department of Infectious Diseases, Faculty of Medicine, Oita University, Oita, Japan.

HTLV-I is known as a causative virus of ATL, HAM/
TSP, HAB, HAAP and Uveritis and distributed in the world
wide. The people infected with HTLV-I are also widely distributed in the Caribbean region. However, the investigation
concerning the HTLV-I in the Dominican Republic (R.D.),
located in the Caribbean region, has not done up to the present time and the situation of the HTLV-I infection remains
uncertain. Then, we investigated to grasp the situation of infection by using the serum from a total of 3,075 people,
1,069 from men and 2,006 from women, at the 6 cities andthe capital in the R.D. during the period from 2001 to 2002.
The measurement of the HTLV-I antibody was carried out

by the indirect fluorescent antibody method using the MT-2cell an antigen. An HTLV-I antibody titer of 40 or over wasconsidered positive. A total of 55 (1.82%), 41 women(2.09%) and 14 men (1.33%), revealed antibody positive tothe HTLV-I. The antibody positive rates among the cities
were 0.78%-2.88%. A low tendency was seen in the southern part of cities compared with northern part of cities. Although the antibody positive rate of the women appeared
higher than men, but a no significant difference was seen. A
big difference (0%-5.97%) was observed among the different age groups.

６９41) DESCRIPTIVE EPIDEMIOLOGY OF IMPORTED INFECTIOUS DISEASE CASESFROM THE NATIONAL SURVEILLANCE IN JAPANKIYOSU TANIGUCHI１, MIKA SHIGEMATSU１, NOBUHIKO OKABE
Infectious Disease Surveillance Center, Nationnal Institute of Infectious Diseases

Some diseases listed on the national surveillance in Japan have high reporting rates of imported cases. For example, among six diseases, cholera, bacterial dysentery, typhus,paratyphus, dengue fever, and malaria, 62 to 100 percentsof cases were imported from the year 2000 through 2003.
Many have no effective vaccine available and personal protection through behaviour change is the only preventive
measure. Consequently, the information on health issues indestination as well as disease incidence among Japanese became more important for travel planning.
The data of imported cases was analysed in order to illustrate epidemiological characteristics of the diseases. In
total, 2786 cases were reported as acquired abroad. Peaks ofcases by diagnosed date were observed during holiday seasons and the period shortly after, March to May and August
to September in most of the six diseases. Besides this seasonality, some clusters attributing outbreaks and epidemic
in destination country observed.
All other diseases except Malaria, major travel destinations prior to the onset were Thailand, Indonesia, India, Vietnam and China which counted as endemic area of tropicaldiseases. Malaria, on the other hand, most of the cases reported visits to countries in African continent. Young adult
was the most affected age group (20 to 39 years), although

the outbound travellers were evenly distributed by approximately 20% among four age groups of adults. Speculatingfrom the months of visits and age distribution of reports,
persons suffered from bacterial dysentery and cholera
seemed to be travelled for holiday. However, those sufferedfrom dengue fever could speculate as composed from people travelling for holiday and for business in same way.
This descriptive epidemiology of cases reported
through mandatory reporting system well supports the results from other studies. Those from India and Nepal reported about ill-prepared Japanese travellers, unvaccinated,
no preparation for repellent, and without knowledge of endemic infectious diseases in advance. Especially among
young adults, preference of adventurous and more interactive travel style resulting in increased opportunity of exposure to the pathogenic agents. At the same time, travel as
daily life attitude of senior traveller also leading to thechance of infection.
Despite several recent health threats in internationalsociety, such as sever acute respiratory syndrome and H5N1
avian influenza, the number of Japanese visiting abroaddemonstrates upward trend. Therefore, we have obligation
to provide, accurate and ready to use information from surveillance to travellers in need in timely manner.

42) CASE SERIES OF IMPORTED MALARIA IN OKINAWA, JAPANSHUSAKU HARANAGA１, MASATO TOUYAMA１, TAKASHI SHINZATO１,FUTOSHI HIGA１, MASAO TATEYAMA１, ATSUSHI SAITO１
１

Division of Infectious Diseases, Department of Internal Medicine,
Graduate School and Faculty of Medicine, University of the Ryukyus

The aim of this study was to clarify the characteristics
of cases of imported malaria.We reviewed the charts of all
27 patients (18 males and 9 females) who discharged with adiagnosis of imported malaria from the University Hospital
Ryukyu and the related hospitals in Okinawa, between 1989
and 2004. Malaria was diagnosed by identification of malaria parasites using Giemsa staining method of thin andthick blood smears, and also using new method of antigen
detection kit and of PCR for these 5 years.The mean age ofpatients was 31 years old (range: 5 to 64), and 16 patientsof them were Japanese who traveled foreign countries. All
patients were given diagnosis of malaria within 24 hours after their access to hospitals. The majority of malaria species
was Plasmodium vivax (13 patients), followed by Plasmodium falciparum (12) and Plasmodium malaria (1). Mixed
infection, identified using PCR method in addition to Giemsa staining, was found in two patients with P. falciparumand P. ovale, and with P. vivax and P. malaria. Appropriate

７０

treatment with antimalarial drugs was started immediately
after making accurate diagnosis. There were no patientswith adverse effects of the drugs. Recurrence of farciparum
malaria occurred in two patients, and relapse of vivax malaria was suspected in one patients. Chemoprophylaxis failure was found in 10 patients because they did not take drugs
according to recommended prescription. Twelve patients

experienced malaria infections in several times, but no one
prepared for prevention of re-infection in correct way.Microscopy, rapid antigen test, or PCR diagnosis where possible, should be carried out in order to confirm malaria infection because these rapid diagnostic tests for malaria may be
useful. Education on correct prevention and chemoprophylaxis for malaria should be much given to travelers.

43) CHARACTERISTICS OF IMPORTED MALARIA IN JAPANTOSHIYUKI MIURA１, MIKIO KIMURA２, TOMOHIKO KOIBUCHI１,
HITOMI NAKAMURA１, TOKIOMI ENDO３, TAKASHI ODAWARA１,TETSUYA NAKAMURA３, AIKICHI IWAMOTO１
１

Division of Infectious Diseases, Advanced Clinical Research Center, Instituteof Medical Science, University of Tokyo, Tokyo, Japan ２Infectious Diseases Surveilance Center,
National Institute of Infectious Diseases, Tokyo, Japan ３Deapartment of Infectious Diseases
and Applied Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Whilst exchange of human resources between Japanand developing countries increases, imported malaria is getting more important for public health in Japan. We analyzed
153 of new malaria cases presented to our hospital from1992 to 2001 for further understanding of current situationof malaria in Japan. The total number of malaria cases in Japan has been constantly over 100, and our cases has accounted for 14.9% of all cases in Japan. The predominantmalaria species has been Plasmodium falciparum throughthe decade (51.9%). The most frequent contraction area was
Africa (54.2%), followed by Asia (20.9%) and Oceania(19.6%). For the treatment of falciparum malaria, mefloquine was the most commonly used antimalarial drug
(82.5%), and atovaquone/proguanil has been in use since1999. The analysis of 101 Japanese cases revealed clinicalissues that had to be kept in mind to manage malaria patients in Japan. Firstly, high relapse rate of vivax malaria after completion of primaquine treatment was observed incases imported from Oceania. Secondly, although the number of chemoprophylaxis failure due to inadequate regimen
decreased, substantial number of travelers without chemoprophylaxis still suffered from malaria. Finally, median duration from onset of illness to first visit to clinics was 3.0
days, which was unacceptably long for better prognosis.
These results indicate that development of travel medicine
to provide current information to health care providers, andeducation campaign to enterprises, travel companies and individual travelers, are warranted to reduce morbidity andmortality due to imported malaria

７１44) MALARIA EPIDEMIOLOGY IN LOMBOK ANS SUMBAWA ISLANDS, INDONESIAHIROJI KANBARA１, KAZUMI YOSHINAGA１, MASAHIRO TAKAGI１,YOSHIHIDE MAEKAWA１, I.K. GERUDUG２, AAN SURYANATHA３,ISKANDAR YAM４, YOES P. DACHLAN５
１

Institute of Tropical Medicine, Nagasaki Univ, Nagasaki, Japan
Nusa Tenggara Barat Provincial Health Office, Mataram, Indonesia
３
Meninting Health Center, West Lombok, Indonesia
４
Utan Rhee Health Center, Sumbawa Besar, Indonesia
５
Tropical Disease Center, Airlangga Univ, Surabaya, Indonesia

２

Introduction; This work was done as the cooperative project
between Indonesia and Japan under the JICA partnershipprogram.
Purpose; Malaria epidemiological conditions are clarified todecide the most suitable control measures at the project areas.
Project areas; The subdistrict Batulayar under Meninting
health center in Lombok island and the subdistrict Utan under Utan Rhee health center in Sumbawa island were chosen.
Methods; Spleen examination was applied to schoolchildren in the first and second grade of all the elementary
schools. Regular blood examinations and entomologicalsurveys were done at subvillages selected based on theabove results. Case detection and treatment was practiced
by newly organized teams in health centers.
Results and discussion; In the subdistrict Batulayar two different types of malaria endemics were found, one was at
hilly forested areas and another was at coastal areas. The

former was found to be more prevalent and to be maintained by a vector mosquito species, Anopheles balabacensis which breeds in stagnant water along rivers through valleys and small water pools formed around streams or
springs in the forest. The transmission season was obscure,because seasonal difference in transmission seemed to bevaried owing to small environmental differences between
subvillages. The latter was maintained by a vector mosquito
species, An. sundaicus which breeds in brackish water
mainly of lagoons formed at exits of rivers to the sea in thedry season and was relatively suppressed by long control
activities executed by the local governments. In the subdistrict Utan malaria endemics were found only along the coast.
The transmission vector was determined as An. subpictus
that breeds in brackish water mainly of fish ponds developed in the mangrove forest after cutting trees. No malaria
endemic was found on hilly areas. These results indicatedthat efforts of malaria control should be focused on forest
malaria endemic.

45) MALARIA CONTROL IN LOMBOK AND SUMBAWA ISLANDS, INDONESIAHIROJI KANBARA１, KAZUMI YOSHINAGA１, MASAHIRO TAKAGI１,YOSHIHIDE MAEKAWA１, I.K. GERUDUG２, AAN SURYANATHA３,ISKANDAR YAN４, YOES P. DACHLAN５
１

Institute of Tropical Medicine, Nagasaki Univ, Nagasaki, Japan
Nusa Tenggara Barat Provincial Health Office, Mataram, Indonesia
３
Meninting Health Center, West Lombok, Indnesia
４
Utan Rhee Health Center, Sumbawa Besar, Indonesia
５
Tropical Disease Center, Airlangga Univ, Surabaya, Indonesia

２

Introduction; This work was done as the cooperative project
between Indonesia and Japan under JICA partnership program.
Purpose; Malaria control activities are carried out based on
epidemiological data. Subsequently the most suitable ma-

laria control methods are proposed.
Project areas; The subdistrict Batulayar under Muninting
health center in Lombok island and the subdistrict Utan under Utan Rhee health center were chosen.
Methods; 1) Reduction of human sources, The case detec-

７２tion and treatment teams visit subvillages according to theirschedule which are informed to villagers through influential
persons in advance, find out malaria patients from clinicallysuspected patients using ICT malaria diagnostic kits andtreat them. 2) Distribution of insecticide treated bed-nets
and insecticide indoor residual spray are executed to cut thetransmission of malaria parasites between vectors and humans. 3) Trials for larval control are done. 4) Appearance ofdrug-resistant Plasmodium falciparum is examined.
Results and Discussion; 1) The activity of case detection
and treatment was shown to be universally effective for malaria control, specially under the meso-endemic condition.
2) Anti-adult-mosquito methods were effective against

coastal malaria but not forest malaria. Besides, these methods were costly and laborious. Bed-nets were more acceptable to villagers than the indoor spray. 3) Larbal control
methods are limited in practical use. 4) Chloroquine resistant parasites were very prevalent in both project areas. SPresistant parasites also seemed to be common considering
the follow-up study in Sumbawa. From these results we
proposed that the most effective and economical control
method is the continuous activity of case detection andtreatment. The activity is also useful to educate villagers
and to obtain trust from villagers. Bed-nets should be used
as a supplemental measure based on entomological studies
and budget size.

46) MAGNITUDE OF MALARIA PROBLEM IN CAMBODIA
DYSOLEY LEK, AKIRA KANEKO, HIDEAKI ETO,AMANE SUZUKI, TAKATOSHI KOBAYAKAWA
Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University, Tokyo, Japan.

Malaria continues to be a major public health problem
in Cambodia. It is the main obstacles to the country’s development. About 1.6 million people out of 12.3 million total
population are at risk of malaria, who are living in villages
located within or with the distance of 1 km from the forest.
These people can therefore be classified into the following
4 categories; (I) people reside in forested villages, (II) people reside in the villages at 200 m distance from forest, (III)people reside in the villages between 200 to 500 m distances from the forest and (IV) people reside in the villages
between 500 to 1,000 m distances from the forest. Abouthalf a million people live in the high transmission forest areas defined as the category (I) with intense malaria transmission; malaria is most severe in isolated villages surrounded by forests. Prevalence rates range from about 15%
to 40% in villages near or in forested areas to 0-3% in theplains and rice field areas. Factors that contribute to malaria
risk include predominance of falciparum malaria, poor
health infrastructure especially in the high transmission areas, continuing shortages of appropriate diagnostics anddrugs, poor communication and transportation systems, re-sulting in treatment delays, presence of specific high risk
groups including military personnel, forest and mining
workers, in forest villages, children under the age of five
years are the primary risk group, predominance of unqualified private medical practitioners and pharmacists who still
do not follow national treatment guidelines, abundance of
fake drugs on the market and spreading drug resistance inparasites in different pockets of the country. To reduce malaria disease burdens in the high transmission areas, early
diagnosis and treatment approach should be improved inpublic health sector systems, and also private sector throughsocial marketing strategy, improve coverage of preventive
measure using impregnated bed nets and social marketingof hammock net with insecticide K-O TAB, increase awareness and care-taking practices on malaria prevention andproper health seeking behavior , improve the behavior
change communication in promoting malaria safer behavior
in target population and strengthen the institutional capacityof the national malaria control program at central, provincial, operational district and commune levels.

７３47) ACCUMULATED INFECTION RISKS OF MALARIA DURING FIVE WEEKSIN A LIVING AREA OF AN ETHNIC MINORITY IN VIETNAM.
SHUSUKE NAKAZAWA１, LE DUC DAO２, NGUYEN VAN TUAN２,NGUYEN DUC GIANG２, TRUNG VAN HANH２, TOSHIHIKO SUNAHARA１,HARUKI UEMURA１, NOBUYUKI NISHIKIORI１, TARO YAMAMOTO１, KAZUHIKO MOJI１.
１

Institute of Tropical Medicine, Nagasaki University,
National Institute of Malariology, Parasitology and Entomology.

２

Eighty-three of ethnic minority and twenty-five Kinh
school children were diagnosed on malaria by microscopy.
The children with slide positive were treated with artesunate
and observed for the following five weeks. Thirty children
out of 83 were slide positive at the first examination. Fiftynine children were slide positive at any point during the period. The remaining 24 out of 83 ethnic minority and 25
Kinh children were slide negative throughout the period.
Cumulative positive rate was compared between thetwo ethnic minority children with slide positive and negative at the first examination. The former was not positive
one week after treatment but small number of them began toturn positive again from the second week after treatment.

Finally, 50% of them were positive during the period. The
latter became positive constantly from week 1 and the increment curve of cumulative positive rate from week 1 to 4
was parallel to that of the former group from week 2 to 4.
Body temperature was not significantly different between Kinh school children and minority school children
and even between minority school children with positive
and negative slide.
The results suggested that the minority school children
should be asymptomatic carriers and the effect of treatment
to such people in an endemic area should not be maintained
for a long time.

48) A REAL-TIME PCR ASSAY FOR QUANTITATIVE DETECTION OF PFCRT IN
PLASMODIUM FALCIPARUM AND ITS APPLICATION TO EPIDEMIOLOGYYUKO KATAKAI, KANAKO KOMAKI-YASUDA,
SHIN-ICHIRO KAWAZU, SHIGEYUKI KANO
Dept Appropriate Technology Development and Transfer,
Research Inst, International Medical Center of Japan, Tokyo, Japan

It has been reported that mutations in the transporter
gene pfcrt of Plasmodium falciparum had strong correlation to its chloroquine (CQ) resistant phenotype. Individual
malaria patients often harbor a mixture of genetically distinct parasites, i.e. parasite population with wildtype alleleof pfcrt (susceptible to CQ) and that with the mutant allele(resistant to CQ). Treatment of the patients with CQ probably selects parasite population, which has the mutant allele.
However, currently used typing methods for pfcrt are unable to quantify the proportions of the wildtype and mutant
alleles in one patient. In this study, we adapted a real-time
PCR with Taqman (R) probes (TaqMan (R) PCR) for differentiation of the pfcrt alleles in P. falciparum-infected blood
samples.
The mutation in pfcrt, which has the most significant
association with CQ resistance, is substitution of threonine

for lysine at codon 76 (K-76T). The TaqMan (R) PCR wasthus used pfcrt-specific primers and the probes each specific to wildtype (K76) and mutant (T76) alleles. PlasmidDNAs with sequences from 76K and 76T alleles were prepared as standards. In vitro cultures of two laboratory
strains, FCR-3 (K76 allele) and K-1 (T76 allele), weremixed in various proportions. DNA samples were extracted
from them, and subjected to the TaqMan (R) PCR. Proportions of K76 and T76 alleles estimated by the PCR wereconsistent with those expected.
Thirteen blood samples collected from malaria patientsin Palawan island of Philippines were subjected to the
TaqMan (R) PCR. K76 alleles were detected in 11 samples,of which 6 had T76 alleles simultaneously. There was no
sample, which had T76 allele alone. Two samples contained
only small amount of DNA and any alleles were detected.

７４Ratio of K76 and T76 alleles varied from 8.7:91.3 to 44.5:
55.5. This result may reflect moderate prevalence of CQ resistant malaria in the island.
Although decisions for antimalarial drug policy will

never be entirely based on molecular survey alone, suchdata can offer important information. TaqMan(R) PCR may
be useful method for estimating the prevalence of CQ resistant P. falciparum in malaria-endemic region.

49) AN INVESTIGATION OF THE RELATIONSHIP BETWEEN RELEASEFROM CHLOROQUINE RESISTANCE AND BLOOD GSH LEVELSYOSHIYUKI MIYATA１, SEIKI KOBAYASHI２, YUMIKO OSA３, KOUICHI TAKINO３,
YOKO SATO３, YUKA HIKIMA３, MASAKAZU SAKAGUCHI１, HIROAKI TAKAYANAGI４,TSUTOMU TAKEUCHI２, HIROSHI NAGASE３
１

POLA Chemical Industries, Inc.,Yokohama, Japan ２Dept of Tropical Medicine and Parasitology,
School of Medicine, Keio Univ, Tokyo, Japan ３School of Pharmaceutical Sciences,
Kitasato Univ, Tokyo, Japan ４Faculty of Engineering, Seikei Unv, Tokyo, Japan

It is a serious problem for the role-back malaria protocol that chroloquine-resistant malaria spreads to all over theworld. And also it is expcted to conquer the chloroquineresistance in malaria therapie. To this aim, there is one important strategie, release from the chloroquine-resistance bydrugs, in order to make malaria protzoan sensitive to chloroquine.It is said that the resistance of malaria to chloroquine is related to ABC (ATP binding casett) transporters.
We have revealed that it could release this resistance to inhibit ABC transporter by Dibenzosuberyl piperazine analogs. Dibenzosuberyl piperazine analogs have little circula-tory effect and also little central nervous effect. Dibenzosuberyl piperazine analogs would have specific inhibition
effect on the ABC transporters ,which are related to thechloroquin-resistance of malaria. To assign those ABCtransporters, we investigated about the influence to GSH
levels in blood by Dibenzosuberyl piperazine analog (D67).
It is said that GSH levels in resistant malaria are higher thansensitive ones, and it is suggested that SG-X pump, one of
ABC transporters,whose substrates are glutathioneconjugates, would be related with the mechanism of thechloroquine resistance.

50) PROTECTION EFFECT ON LYSIS OF MALARIA INFECTED ERYTHROCYTES MEMBRANE
BY BISBENZYLISOQUINOLINE ALKALOIDS
KOSUKE HARUKI１, GIANCARLO BIAGINI２, MINORU ONO３,
MASAHIKO SEGAWA１, TOMOMI KATAGIRI１, YUTAKA SAKURAI１,
PATRICK G BRAY２, STEVEN A WARD２
１

Department of Hygiene, National Defence Medical College, Tokorozawa, Saitama, Japan
Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine
３
University of Tokyo, Tokyo, Japan２

We are investigating the reversal effect by bisbenzylisoquinoline alkaloids on chloroquine resistant Plasmodium
falciparum so far. The alkaloids have membrane stabilizing
activity and also inhibit P-glycoprotein on multi drug resistant cancer cells. We are considering that the mode of actionof reversal by alkaloids is different from P-glycoprotein inhibition. The reasons are as follows: 1 Chemosensitizer
such as verapamil, which inhibits P-glycoprotein at the or-der of micro M, but the alkaloids inhibit at the order of nM.
2 The alkaloid reverse even chloroquine sensitive strain
whilst verapamil does not. Here, we investigated the membrane stabilizing effect due to the alkaloids using haemolysis technique with sorbitol (osmotic) and KSCN (nonosmotic). We examined the strain TM6 at late trophozoite
stage with 5% sorbitol and KSCN by photometer at 540 nm
observing haemoglobin release. Either with sorbitol and７５

KSCN, haemolysis was inhibited by 200 nM and 50 micro
M of the alkaloids. As a cosequence of these results are suggesting that membrane stability effect of the alkaloid may

play partial role of reversal effect of chloroquine resistantmalaria parasite. Further investigationwill be required toelucidate this phenomenon.

51) REACTIVITY OF BLOOD SAMPLES SPOTTED ONTO FILTER PAPERS
IN THE WST-8 METHOD FOR SCREENING OF G6PD DEFICIENCYMEIJI ARAI１, KAZUKO KOSUGE１, BERNARD A. OKECH２, HIROYUKI MATSUOKA１
１

Division of Medical Zoology, Dept of Infection and Immunity, Jichi Medical School, Tochigi, JAPANCentre for Biotechnology, Research and Development, Kenya Medical Research Institute, Nairobi, Kenya

２

Deficiency of glucose-6-phosphate dehydrogenase (G6PD), the most common enzymopathy, causes acute haemolytic anaemia usually triggered by oxidative drugs or foods.
It is therefore very important to assay G6PD activity, particularly in malaria-endemic areas where primaquine, atypical oxidative drug, is widely used for treatment. Recently, a simple and rapid screening method for G6PD deficiency which utilises a water-soluble tetrazolium salt, WST8, was established. The WST-8 method has been demonstrated to be suitable for field conditions, particularly for on-site malaria surveys. Although the original WST-8 method
uses whole blood as a sample, it would be worth using
blood samples spotted onto filter paper, because processing
many samples at the same time in the laboratory will enable
to make more precise evaluation of individual G6PD activity than on-site conditions. We tested the reactivity of blood

samples spotted onto four types of filter paper, 31ETCHR(31ET), 3MM Chr (3MM), and P81filters from Whatman,
and Advantec No.2 (AD2) filter. The reactivity with fresh
samples was highest in31ET, followed by AD2 and 3MM at
similar levels, and lowest in P81. In order to examine thestability of the blood spotted onto each filter, the bloodspotted filters were kept at 4 degrees centigrade or room
temperature (25-28 degrees centigrade) for 1-56 days, andexamined for their reactivity by the WST-8 method. Within
14 days after blood-spotting, all four types of spotted-blood
samples stored at room temperature showed significant reduction (31-45% of the fresh sample) in reactivity, whilesamples kept at 4 degrees centigrade retained their reactivity (70-92% of the fresh sample). Our results indicate that
Whatman 31ET-CHR filter is the most suitable for the WST-8 method with using spotted-blood samples.

52) STRUCTURAL-EFFECT RELATIONSHIPS OF MOTHER MOIETIES IN DRUG-RESISTANCERELEASEING COMPOUNDS ON THE CHLOROQUINE RESISTANT MOUSE MALARIAOSA Y１, KOBAYASHI S２, MIYATA Y３, TAKINO K１, SATO Y１, HIKIMA Y１,
SAKAGUCHI M３, TAKAYANAGI H４, TAKEUCHI T２, NAGASE H１
Kitasato University, Tokyo, Japan, ２Keio University, Tokyo, Japan, ３Pola Chemical Industries, Inc., Yokohama, Japan.
１
School of Pharmaceutical Sciences, Kitasato Unv, Tokyo, Japan
２
Dept of Tropical Medicine and Parasitology, School of Medicine, Keio Univ, Tokyo, Japan
３
POLA Chemical Industries, Inc., Yokohama, Japan, ４Faculty of Engineering, Seikei Unv, Tokyo, Japan１

The spread of malaria and resistance to antimalarial
are becoming a serious problem. Since the study on the
drugs reducing the drug-resistance of malaria would be useful to overcoming the problem of the multidrug-resistant(MDR) Plasmodium (P.) falciparum, we designed and synthesized new compounds that have both reduction activityof chloroquine (CQ)-resistance and antimalarial activity.

The compounds were compoused of piperazine (P), aromatic moiety of dibenzosuberanyl, diphenylacethyl anddipenylmetyl (A) and aliphatic moiety (B). A-P structure reduced CQ resistance, but it action was weak than the compounds of A-P-B, A-P-A and P-B structure had not reduction effects. In conclusion, the structure having A-P-B isnecessary to both reduction effects.

７６53) INTERLEUKIN 4 POLYMORPHISM AND MALARIA ENDEMICITY IN MELANESIA.
MAWULI DZODZOMENYO１, MIHOKO KIKUCHI２, RATAWAN UBALEE２,
HIKOTA OSAWA１, TAKAHIRO TSUKAHARA１, MARITA TROY BLOMBERG３,
TAKATOSHI KOBAYAKAWA１, KENJI HIRAYAMA２, AKIRA KANEKO１
１

Department of International Affairs and Tropical Medicine, Tokyo Women’s Medical University, Tokyo, Japan.
Depertment of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
３
Malaria Research Laboratory, Karolinska University Hospital, Stockholm, Sweden.

２

Previous studies in sub-Saharan Africa have suggested
the protective role of the Th-2 cytokine IL-4 in malarial disease and associations between the IL4-590T mutation andthe elevated levels of Plasmodium falciparum-specific IgE.
The frequency of the IL4-590T allele varies among geographical populations; eg. 0.14 in Japanese and 0.71 in the
UK. We conducted a population-based study to investigate
the distribution of the IL4-590T allele and its relation tomalaria infection and disease in East Sepik of Papua New
Guinea (hyper-endemic, n=80) and on three islands in the
Vanuatu archipelago: Malakula (meso-endemic, n=272),
Aneityum (hypo-endemic, n=476) and Futuna (nonendemic, n=138). DNA was extracted from blood spottedon filter paper collected during mass blood surveys and 200-bp fragment of the 5’-flanking region from positions-1111
to-1310 of the IL4 gene was amplified using PCR. Pyrosequencing using specific designed oligonucleotide sequences
was used for genotyping IL4-590 position. IL4-590T allele
frequencies were 0.39, 0.40, 0.48 and 0.42 in East Sepik,
Malakula, Aneityum and Futuna respectively. The observed
allele frequencies of the IL4-590T are rather consistentamong the surveyed Melanesian populations but different
from those reported in far Eastern Asian and European
populations. We could not see any association between IL4590T allele frequency and malaria endemicity.

54) MALARIA PRESSURE INFLUENCED THE FREQUENCY OF A SUSCEPTIBLE ALLELE
OF TNF-ALPHA PROMOTER REGION IN VANUATURATAWAN UBALEE１, TAKAHIRO TSUKAHARA２, MIHOKO KIKUCHI１,
KOJI LUM３, MAWULI DZODZOMENYO２, AKIRA KANEKO２, KENJI HIRAYAMA１
１

Dept. of Molecular Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Department of International Health and Tropical Medicine, Tokyo Womens Medical College, Tokyou, Japan３
Department of Anthropology, Binghamton University, Binghamton, New York, USA

２

Tumor necrosis factor- alpha (TNF- alpha) is one of
the key cytokines that influence the pathology of microbial
infections. The genetic susceptibility to severe form of falciparum malaria is associated with TNF- alpha promoter gene
polymorphism (TNFP alleles). In our previous study, we
identified a TNFP-allele characterized by a T to C transitionat position -857 (TNFP-D allele) as a strong susceptibility
marker for cerebral malaria in Myanmar. To investigate
whether malaria selection pressure on this susceptibility
marker has influenced its prevalence, the frequency of the
TNFP alleles was estimated in six islands in Vanuatu, Mela-

nesia within the South Pacific. Within the Vanuatu archipelago there is an overall decreasing North to South cline ofmalaria endemicity. Of the four alleles of the TNFP genedetected, the susceptible TNFP-D allele frequency decrease
corresponded to the malaria endemicity increase; TNFP-D
varied from 0.55 in the lowest malarial endemicity population to 0.26 in the highest malarial endemicity population (r= −0.855, p=0.03). This is the first report indicating thatmalaria selection pressure has selected an allele related toimmunity.

７７55) RESEARCH OF THE ANTIMALARIAL DRUGS FROM
THE NATURAL PRODUCTS IN JPMW PROJECTAKI ISHIYAMA１, KAZUHIKO OTOGURO１, HIDEAKI UI２, MIYUKI KOBAYASHI１,
HITOMI SEKIGUCHI１, YOKO TAKAHASHI３, ROKURO MASUMA３, SHINYU NUNOME３,HARUKI YAMADA３, SATOSHI OMURA１
１

Research center of Tropical Diseases, The kitasato Institute, Tokyo, Japan
２
School of Pharmaceutical Science, Kitasato University, Tokyo, Japan３
kitasato Institute for Life Science, Kitasato University, Tokyo, JapanThe JPMW project was established in October 1999
through a public/private partnership (PPP) among the Japanese Pharmaceutical Companies, Ministry of Health, Labour and Welfare, Japan (MHLW), and WHO/TDR, in order to discover new type of antimalarial drugs in Japan by
the Japanese efforts. Our center (KSC) is carrying out theevaluation of antimalarial activity for the compounds fromthe chemical libraries of 14 Japanese companies and thenatural products of our institute, both in vitro and in vivo,using the multi-drug resistant strain of Plasmodium falciparum and the rodent malaria model, respectively. In thispresentation, we report the antimalarial compounds fromthe natural products in this project. By 15 October 2004,
KSC has assayed 16,446 natural products etc as in vitro
screening. As a result, we selected the candidate materials
as 23 natural products that had selective and potent antimalarial activities, which must be examined further isolationand purification of active compounds from those materials.
In the course of the research from the microbial metabolites,
we found that the five known antibiotics (X-206, K-41,
polyketomycin, borrelidin and leucinostatin A) produced byactinomycete strains K99-0413, KP-4050, K99-5147, OM0060 and a fungal strain FKI-0266, respectively, had potent
and selective antimalarial activities in vitro. Further, wefound that takaokamycin (identified with hormaomycin) etc,
own compounds in the antibiotic library of our institute, had
potent activity in vitro. In the course of the research fromthe plant resources, we found that two known biflavonoids
as sikokianins B and C were isolated from the root of Wikstroemia indica, had moderate antimaralial activities in vitro. Furthermore, the in vivo antimalarial activities of X206, K-41 and borrelidin exhibited more potent activities
than those of the existed antimalarial drugs as artemether,artesunate and chloroquine. Especially, K-41 and borrelidin
has the possibility of the lead compounds as the new type of
the antimalarial drugs, further detail studies of K-41 andborrelidin are in progress. There are the first reports to show
antimalarial activities of the above compounds. Now, we are
ongoing to measure the antimalarial activities of other natural compounds and synthetic compounds derived from thenatural products, and also ongoing to isolate active substances from the other natural products.

56) ANTIMALARIAL DRUG DEVELOPMENT RESEARCHAGAINST DUG-RESISTANT P. FALCIPARUM
HYE-SOOK KIM１, MASATOMO NOJIMA２, ARAKI MASUYAMA２,YUSUKE WATAYA１, ALY NAGWA１
１

Department of drug informatics, Faculty of Pharmaceutical Sciences, Okayama University
２
Osaka University, suita, Japan

Emerging and re-emerging infectious diseases have recently been considered to be worldwide problems, and malarial infection, which is a re-emerging infectious disease,has been attracted attention. Three hundred million or more
people are infected with malaria and 1.5-2.7 million people
die every year, and malarial infection is a major cause ofdeath in developing countries (WHO report). Malaria para-site (Plasmodium falciparum, which contributed most of
the death) has been acquiring resistance to various antimalarial drugs. For malaria control, the development of a novel
drug, that overcomes the drug-resistance malaria parasite, ismost important. We are screening about 5,500 samples containing
naturalproducts ,organic
compounds ,microorganism-derived products, combinatorial compounds

７８

and marine products thatare supposed to have antimalarial
activity. As the results, several compounds with high selective antimalarial activity were obtained using the in vitro

and in vivo assay against P. falciparum in vitro and P. berghei in vivo. In this conference, the current status of the development of novel antimalarial drugs is introduced.

57) THE PROBLEMS OF MEDICAL FACILITIES ABROAD FOR JAPANESE EXPATRIATES
FUKUSHIMA SHINJI
Japan Overseas Health Administration Center, Yokohama, Japan

Introduction
Our objective for carrying out this survey was to shedsome light on the standard of consultaion at the local medical facilities and the problems Japanese expatriates and theirfamilies had experienced related to the consultation.
Method and Materials
This survey was carried out between November 2002
and October 2003 and 393 subjects took part. (One family
was treated as one person). The subjects were Japanese expatriates and their families, who had received a health check-up at the Japanese Overseas Health Administration Center
after returning to Japan from abroad. This included both
permanent and temporary returnees. We conducted interviews with the subjects for this study, which dealt with thefollowing issues:
1) whether they had received a consultation at a local treatment facility2) if they had experienced any problems during the consultation① medical service(language difficulties, communication
problems, the consultation system itself, the payment
system of medical costs)② quality of medical treatment(the standard of the staff at
the medical facilities, the hygiene of the medical facilities
and equipment)③ medical costs④ drugResults
The average age of the subjects who took part in our
survey was 42.9 years of age. Their average length of stayin a foreign country was 35 months. Out of the 393 expatriates, 125 had lived in developed countries and 268 had lived
in developing countries. (The developed countries included
North America, Western Europe, Australia, New Zealand).
In total, 306 subjects (77.9%) had received a medical consultataion in the foreign country in which they had lived. Of
these, 99 subjects had lived in developed countries and 207
in developing countries. Of the 306 subjects, who had received consultations abroad, 189 of them (61.8%) complained about problems associated with the consultation (s).
67.7% of the complaints came from subjects who had lived
in developed countries, 58.9% from those who had lived indeveloping countries. The majority of complaints in developed countries were related to the medical service andmedical costs ranked second. Whereas most of the grievances in developing countries were connected with the quality of the medical treatment and this was followed by the
medical service.
Discussion
The number of expatriates who received consultations
in local medical facilities was relatively large and based on
the results of this survey it appears that many of them experienced problems or difficulties related to the consultation.

７９58) “RESEARCH GROUP ON CHEMOTHERAPY OF TROPICAL DISEASES (ABBR.)”
DURING THE PAST AND THE COMING THREE YEARS.
MIKIO KIMURA１, TETSUYA NAKAMURA２, HIROSHI OHTOMO３, YUKIFUMI NAWA４
１

Infectious Disease Surveillance Center, National Institute of Infectious Diseases,
Department of Infectious Diseases and Applied Immunology, Institute of Medical Science, University of Tokyo,
３
Department of Tropical Medicine, Jikei University School of Medicine, ４Miyazaki University

２

The Research Group on Chemotherapy of Tropical
Diseases (abbr.) was launched in 1980 as one of research
projects of the then Ministry of Health and Welfare, andsince then, it has been contributing greatly in the treatmentof tropical and parasitic diseases using medicines that are
not licensed in our country. During the fiscal years Heisei
13-15, the Research Group was funded by a research grantfor “Researches on Health Sciences Focusing on Drug Innovation” from the Japan Health Sciences Foundation, andcontributed to licensing mefloquine as a therapeutic andprophylactic antimalarial, and ivermectin as an antistrongyloidiasis medicine. Since the fiscal year Heisei 16,
again funded by the Japan Health Sciences Foundation, weare expected to conduct activities, reconsidering the researchers and the medicine stockpiling institutions.
During the past three years, we created our web site
aiming at facilitating access to those medicines. Furthermore, we created a listserv aiming at efficient use of the
medicines, and promoting the consultation activities about
diagnosis and treatment of tropical and parasitic diseases.
We exchanged information with specialized European hospitals and succeeded to introduce three kinds of antiAfrican trypanosomiasis (sleeping sickness) medicines
through contacts with the European surveillance network

for travelers’ diseases, TropNetEurop. We treated patientswith scabies, malaria, amebiasis and strongyloidiasis, andused ivermectin, diloxanide furoate, chloroquine, primaquine, in descending order. Injectable metronidazole wasused in severe amebiasis cases that could not intake oral
medicines, and showed excellent efficacy. We are always
trying to analyze our data on the medicine use for feedback
to the improvement of therapy. According to our data,atovaquone/proguanil combination showed almost equal efficacy as compared with mefloquine in the treatment of falciparum malaria, and it showed a better safety profile due tothe absence of neuropsychiatric adverse effects (AEs) thatwere occasionally seen in mefloquine. When patients withscabies were treated with ivermectin, the medicine showed
excellent efficacy, and the occasionally observed AEs were
tolerated.
In the fiscal year Heisei 16, under the renewed Research Group, we introduced nitazoxanide for cryptosporidiosis, and will start a therapeutic study targeting thisprotozoal disease in immunosuppressed individuals, e.g.,
HIV infected ones. We will also promote consultation activities on our listserv with the “telemedicine” approaches,
especially by attaching images such as microscopic photographs of malaria parasites and various CT/MRI images.

59) CERTIFICATION IN TROPICAL MEDICINE IN GERMANYAKIHIRO HITANI１, MIKIO KIMURA２
１

Health Care Center, Health Check, Medical Institute Zenjinkai, Yokohama, Japan
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan

２

There are two certifications related to tropical medicine in Germany: (1) certification of tropical medicine approved by the German Medical Association; (2) certificationof travel medicine approved by DTG (German Society ofTropical Medicine and International Health). Physicians,
who have previously obtained a board certification in specialties such as internal medicine, pediatrics, surgery, andgynecology, are eligible to apply for the postgraduate resi-

dency training that are approved by the German Medical
Association. The certification requires at least one year ofresidency in Germany, over one year of clinical experience
with patient care in tropical countries, and taking a course
to obtain the Diploma of Tropical Medicine (DTM) degree
or its equivalent in Europe. During the given period of residency, applicants must diagnose over 100 cases of tropicaldiseases microscopically by themselves, and experience８０over 300 travel health consultations including vaccinationand malaria prophylaxis. Tropical diseases designated forthe residency program consist of 16 major ones. Applications are examined by the accreditation council of the German Medical Association. Certification of travel medicine
requires a qualification of physician. This training program
consists of lectures of 32 hours for a 4 day period, and amultiple choice examination. Lectures cover topics such as
the basic knowledge of travel medicine, infectious diseases
that are essential for travel medicine practice, vaccinology,
malaria prophylaxis, aviation medicine, and management
issues of diving. After successful completion of the course,
certification is awarded by DTG. Recertification is needed
every three years by participating in a training session for

one day. At present, approximately 500 physicians have obtained the certification of tropical medicine, and are permitted to advertise their designation. The lists of physicians
certified as a travel health practitioner by DTG are distributed widely among travelers, travel agencies, and pharmacies. As the acquisition of those certifications could lead toan increasing number of patients/clients, many physicians
are willing to participate in the training programs. In Germany, specialists of tropical medicine and travel medicine
work in closer cooperation with travel related industries
such as travel agencies and airline companies. This networking has a significant influence on the surveillance, prevention, and treatment of diseases.

60) A PRELIMINARY STUDY ON THE IMAGE OF TROPICAL MEDICINE AND
THE INCLINATION FOR MEDICINE AND MEDICAL SCIENCE
BY THE SIXTH YEAR STUDENTS OF SCHOOL OF MEDICINE, CHIBA UNIVERSITY
KOJI NAOI１, NATSUKO KAMEYAMA２
１

Dept of Infection and Host Defense, Grad Sch Med, Chiba Univ, Chiba, Japan. ２Sch Med, Chiba Univ, Chiba, Japan.

In this research, the image on tropical medicine by
medical students and their inclination for medicine andmedical science were investigated. 108 sixth year students(male: 61, female: 47) of School of Medicine, Chiba University, were encouraged to participate in the study, of
which 70 students (male: 41, female: 29) replied to thequestionnaire in June 2004. Besides their attributes (age,sex. etc.), their interest in and image on tropical medicine(ex. narrow-wide), and their inclination for medicine andmedical science (ex. domestic-international) were evaluatedwith a scale of1to5. Adding to basic statistical analysis oneach factor, the association between interest in tropical
medicine and 18 factors (12 for image, 6 for inclination)
was analyzed with a multiple regression model.
The average of interest in tropical medicine was 2.88,
indicating that medical students are relatively less interested
in it. The general image on tropical medicine was relatively
wide, warm, relatively soft, relatively noisy, relatively
bright, relatively prospective, international, developing nations, scarce budget, relatively clinical, relatively new tech-

nology and relatively specialist. The inclination of students
was clinical, relatively domestic, relatively specialist, relatively acute-disease-treatment and relatively developed nations. No statistical difference by sex was observed in almost all factors.
Multiple regression model indicated the inclination fordeveloping nations, the image of generalist, bright and hardon tropical medicine as the factors to raise up the interest
with statistical significance. The inclination for generalist
and the image of warm, basic science, plentiful budget,
calm and new technology was also implied as the factors
without statistical significance.
This preliminary study suggests that relatively lower
interest on tropical medicine by medical students is backed
by their own inclination for developed nations, domestic
and clinical medicine as specialist. It is impressive that theimage of tropical medicine as basic science of new technology backed by plentiful budget with the mind of generalist
would increase the interest of medical students.

８１61) INCREASING NUMBER OF HIV-POSITIVE MOTHERS ONLY PARTLY EXPLAINEDTHE RECENT DECLINE OF BIRTH WEIGHT IN REPUBLIC OF CENTRAN AFRICAMIZUKO TOKUNAGA, KAZUHIKO MOJI, KAZUYO OOISHI,YUUKO NAKAO, MAKIKO YAMASAKI
School of Health Sciences, Nagasaki University, Nagasaki, Japan.

Birth weight of 6,904 singleton babies in Boy-Rabe
Maternity Clinic of Bangui of Republic of Central Africa
between 1998 and 2003 was analysed for their recent decline. Mean birth weight was 2,867 gram and its standard
error (SEM) was 6 gram. Low birth weight (LBW) babies
were 17.0%. Mean age of mother was 23.2 years old andmean parity was 3.0. Multiple regression analysis revealed
that birth weight was independently related with sex of babies, age of mothers, parity of birth, and year of birth (all, p&LT; 0.001). Mean birth weight was the heaviest in 1998
(2934gram with SEM of 15 gram) and the lowest in 2002
(2827gram with SEM of 16 gram). Mean birth weight ofthe same clinic between 1988 and 1990 was reported 2963
gram with SEM of 6 gram, heavier than the value in 1998
(p&LT; 0.001). Decline of birth weight between 1998 and2003 was observed most obviously among mothers of 30

years old with their third parity or more, and among thoseof 20 years old with their second parity. Between 2001 and
2003, 3995 pregnant women were tested for HIV in thisclinic and 539 of them (13.5%) were HIV-positive. There
were 101 babies (56 boys and 45 girls) born to confirmed
HIV-positive mothers among the 6904 mothers, and theirmean birth weight was 2763 gram with SEM of 52 gram,being significantly lighter than the mean birth weight of theother mothers between 2001 and 2003 (2841 gram with
SEM of 9 gram, n=3352). The proportion of low birthweight among babies born to confirmed HIV-positive mothers was 23.8%. The increasing number of HIV-positive
mothers, however, only partly explained the recent declineof birth weight. Major causes of decline of birth weightmust be further studied.

62) PHYSICIANS’ KNOWLEDGE, ATTITUDE AND PRACTICE TOWARDSEXUALLY TRANSMITTED INFECTIONS AND HUMAN IMMUNODEFICIENCYVIRUS INFECTION IN FUZHOU CITY, FUJIAN, CHINAGUOXI CAI１, EIKO KANEDA１, TARO YAMAMOTO１, MASAAKI SHIMADA１,
KAZUHIKO MOJI１, XIAONAN WU２, JIHUI GUAN３, ZHENG LIN２, JIE CHEN２, BAOJUN PAN２
１

Research Center for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
２
Public Health School of Fujian Medical University, Fuzhou, China
３
Fujian Centre for Health Promotion and Education, Fuzhou, China

A cross-sectional questionnaire survey concerning human immunodeficiency virus infection (HIV) and sexuallytransmitted infections (STIs) was carried out in Fuzhou city,
capital of Fujian province, China, and 461 physicians working in 9 general hospitals, 8 health centres and 86 private
clinics participated. The proportion of physicians graduatedfrom medical college or over was 90% in general hospitals,
35% in health centres, and 28% in private clinics. Accordingly, the proportion of respondents who stated HIV-relativeknowledge items correctly over 60% was 86%, 73% and
52% respectively. Gonorrhoea, condyroma acuminate,syphilis, Chlamydia urethritis and genitalia herpes were themost treated STIs by them. HIV treatment was rare except

large hospitals. About two-thirds of the respondents believed that they had risk of HIV occupational exposure, and
59% of them are not willing to take care of HIV/AIDS patients. Over 90% of respondents requested additional education about HIV, particularly information about the prevention of HIV occupational exposure (75%), despite morethan a half of them had HIV re-education in the past 3 years.
This study revealed the gap between knowledge and attitudes among health care workers about HIV infection, especially in the bottom of the healthcare system. Such attitudes
and knowledge deficiencies have the potential to impact
negatively on the quality of care, as well as on patientpractitioner safety. More effective training on HIV knowl-

８
２edge and attitude is necessary for clinicians in China.

63) THE STUDY OF HEALTH STATUS FOR THE INHABITANTS
OF RURAL ARES ON VIENTIANE PROVINCE IN LAO P.D.R.
NISHIYAMA T.１, HAYASHI E.２, KANDA S.１, ISHIDA T.１LEE K.３,
HANDOU K.４, SISONTONE B.５, AMANO H.１
Dept. of Public Health, Kansai Medical University, ２Tokyo Medical and Dental University,
３
Institute of Medical science, Tokyo University, ４Natonal Institute of Infection Diseases,
５
Dept. of Curative Medicine, Ministry of Health, Lao P.D.R.

１

The investigation of mobile clinic at rural areas in developing countries has several difficulties, for example touse electric power, to carry heavy medical instruments andso on. In this study we tried to make up an integrated mobile clinic for the rural areas in developing countries. And
we found the results of general physiological data, serum
biochemistry data by filter paper method, and parasitological data by stool examination (Kato method, Formalin ether
method) and blood rapid tests (OptiMAL etc.). The subjects were the 387 inhabitants (uplander; male: 203, female:184) at Phone Muang village on Hinheup district in Vientiane province, Lao P.D.R. In the results of biochemistry:
ALT (22.7±7.1 IU/l, n=221), AST (29.2±10.9 IU/l, n=221),γ-GTP (38.8±32.6 IU/l, n=221), BUN (11.8±4.4 mg/
dl, n=221), Creatinin (0.56±0.23 mg/dl, n=221), Total chelesterol (146.1±0.23 mg/dl, n=220), Triglyceride (121.3±
30.1 mg/dl, n=220). In the data of blood examination: Hb(13.1±1.7 g/dl, n=221). In stool examinations, several
parasite eggs were found, and results as follows: ascasiasis(21.8%), trichuriasis (31.2%), Hook worm diseases (20.5%)
etc. All inhabitants treated with mebendazole (adults: 500
mg, single dose; children: 250 mg, single dose). And the results of blood test of parasitic diseases were malaria (5.0%)
and amebiasis(3.6%).
Clinical diagnosis in the field were respiratory diseases(21.0%)、muscle & bone diseases (13.0%) and digestive diseases (10.5%). In conclusion, this mobile clinic was performed high medical levels as well as urban areas in Lao P.
D.R. And the health ststus of this village inhabitants wererelatively better conditions than other rural villages in lao P.
D.R. Because this village are located near the main road
(Rute 13) and peoples have a jobs as woodcutter and farmer
and income were relatively higher than other rural villages.