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Bκ沈hol467ZαPs甜40窺αll81に対する各種抗菌薬の試験管内抗菌力・細胞内抗菌力および実験的肺炎マウスに対する治療効果の研究（英文）・・普久原 浩，伊志嶺朝彦，普天問光彦，斎藤厚

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Tη卯noso，欄6槻nsJ：パラローザニンが誘出したアキネトプラストクローンに走査電顕に
よってみられるユニークな構造（英文）一Mary Rose Agnes Silva−Tahat，

一ン瀬昭豊，上村春樹，神原廣二………………

9−13

一野田 伸一，Ngethe D．Muhoho，波部 重久
真喜屋 清，青木 克己……………一・・……………

15−20

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・・松田

晋哉

21−24

第36回日本熱帯医学会総会英文抄録
目

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・・35

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会報・記録
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1995年度役員名簿……………・…・……・…一……・・・・99

第24回日本医学会総会1995（名古屋）広報NQ6

・・103

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投稿規定………・………・………・…・……・…………・−105

日本熱帯医学会雑誌編集委員名簿…………………

・・73

1

1

Jpn. J. Trop. Med. Hyg., Vol. 23, No. l, 1995, pp. 1 7

EFFICACY OF ANTIBIOTICS AGAINSTEXTRACELLULAR AND INTRACELLULAR
B URKHOLDERIA PSE UDOMALLEI, A N D

THEIR THERAPEUTIC EFFECTS ON
EXPERIMENTAL PEUMONIA IN MICEHIROSHI FUKUHARA, TOMOHIKO ISHIMlNE,

MITSUHIKO FUTENMA AND ATSUSHI SAITO
Received December 21, 1994/Accepted February 9, 1995

Abstract: We examined the effects of 14 antibiotics on the isolated strains of B. pseudomallei from melioidosis

patients in Thailand. The organisms caused pneumonia and was found to survive in macrophages in mice.
Thirteen strains of B. pseudomallei isolates showed resistance to avariety of antibiotics such as ampicillin,cefazolin, cefotiam, gentamycin, erthromycin, and clindamycin. Piperacillin,'ceftazidime, ceftizoxime, minocy‑cline, ciprofloxacin, Ievofloxacin and ofloxacin were moderately active antibiotics. Imipenem was the mostactive antibiotic. Efficacy of imipenem and levofloxacin were also examined against the organism in themacrophage. Imipenem showed the strong MIC, (MIC=0.125 mg/L) , but a 4 x MIC dose did not inhibit
intracellular growth of the organism in mice peritoneal macrophages. In contrast, a 4 x MIC of levofloxacin
inhibited the intracellular growth although the MIC=1 mg/L. Levofloxacin therapy showed a significantly(P<0.01 or P < 0.05) higher survival rate than imipenem/cilastatin, piperacillin and minocycline against
experimental B. pseudomallei pneumonia in mice. Imipenem/cilastatin and ceftazidirne showed almost thesame effectivity against the. infection, although imipenem/cilastatin required half the dosage of ceftazidime.
Levofloxacin may be the first choice in the treatment of melioidosis caused by B. pseudomallei.
Keywords: Burkholderia pseudomallei; Pseudomonas pseudomallei; Melioidosis; facultative intracellular path‑ogen; Levofloxacin

INTRODUCTION

teristic feature as facultative intracellular bacterium(6) . We have therefore examined the MIC and MBC of
14 antibiotics against isolated L1. pseudomallei as well as

Melioidosis is an infectious disease of man andanimals caused by the Gram‑negative rod species ofthe effects of antibiotics on the intracellular organism inbacterium Burkholderia pseudomallei(formerly

mice macrophage. The therapeutic efficacy of 5 kinds

Pseudonomas pseudomallei) (1) . The disease is endemicin tropical countries which locate between latitude 20'
north and 20' south such as southeast Asiaand northernof antibiotics was also examined by eliciting pneumonia

Australia(2). However, melioidosis has occasionallyoccurred elsewhere probably carried by travellers or
military personnel from such endemic areas (3) . Clinicalwith the isolates in mice. We followed guideline mea‑
sure for biohazard‑control.

MATERIALS AND METHODS

manifestations of melioidosis are septicemia, pneumonia

Bacterial strains

and visceral abscess (4) .

improved by the introduction of ceftazidime to be treat‑

Thirteen strains of 1 . pseudomallei were kindly
provided by Dr. Prasit Tharavichitkul and Dr. Charlie
Phornphutkul, Faculty of Medicine, Chiang Mai Univer‑ment since 1983 (5). Recently, Parksachartvuthi et alsity, Thailand. These strains were kept in sterile skim‑(1990) reported that B. pseudomallei showed is a charac‑

med milk pH 7.4(Difco Inc, Detroit, USA), at ‑80'C,

The mortality rate of severe melioidosis has beenFirst Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑01, Japan2and incubated for 48 hr before use in each experiment.
A11 strains had been recently isolated from sputum or
blood of clinical pneumonia or sepsis cases in Thailand.

well flatbottom tissue culture plate (Nuncon, Denmark)and incubated in the presence of 10mg/L gentamycin for
3 hr in 5% C02/air at 37'C. After incubation, the wells

B. pseudomallei SP‑237 was obtained from sputum cul‑
ture of pneumonia.

medium to remove nonadherent cells. The adherent

Antimicrobial agentsThe following antibiotics were used in this experi‑
ment: ampicillin (Meiji Pharmaceutical Co. Ltd, Japan) ,piperacillin (Toyama Chemical Co. Ltd, Japan) , cef‑
azolin (Fujisawa Pharmaceutical Co. Ltd) , cefotiam(Takeda Chemical Industries, Ltd, Japan) , ceftazidime

were washed five times with sterile warm RPMI
cells were referred to below as the macrophages. B.
pseudomallei SP‑237 (1x 107 cfu) and marophages (1x106) were incubated in RPMI medium for I hr. B.
pseudomallei SP‑237 did not multiply in the medium
after the macrophages were washed five times. New
media with 0.5 mg/L imipenem (4 x MIO , and 4 mglL

(Glaxo Japan Laboratories Co. Ltd, Japan) , ceftizoxime

levofloxacin (4 x MIO were added to wells and incubat‑
ed in 5% C02/air at 37'C. After O, 3, 6, 12, 24 hr, the(Fujisawa Chemistry Co. Ltd, Japan) , imipenem (Banyu
Pharmaceutical Co. Ltd, Japan) , gentamicin (Sionogi &Co. Ltd, Japan) , minocycline (Lederle Japan Ltd, USA) ,ciprofloxacin (Bayer Japan Co. Ltd, Japan) , ofloxacin(Daiichi Pharmaceutical Co. Ltd, Japan) , Ievofloxacin(Daiichi Pharmaceutical Co. Ltd, Japan) , clindmycin

cultured macrophages were harvested in 9 ml of sterile
distilled water, then vortexed for 30 sec to lyse themacrophages cornpletely with out killing the bacteria.
The bacterial suspensions were diluted appropriately,
and 0.Iml was inoculated on nutrient agar to determine
the nurnber of viable . pseudomallei SP‑237 in each

(Upjohn Japan Co. Ltd, Japan) , and erythromycin

well.

(Dainihon Pharmaceutical Co. Ltd, Japan) .

Experimental B. pseudomallei pneumonia in mice model

MIC and MBC determination
The organisms were grown overnight in CSMHB{cation supplemented Mueller‑Hinton Broth (BBL,

Six‑week‑old male Bulb/c mice weighing 18‑20g

were purchased from Shizuoka Laboratory Animal
center (Shizuoka, J Pan) , fed in a specific pathogenfree

Cockeysill, USA) ; Mueller‑Hinton broth 1 Iiter+Mg2+
25mg/L+Ca2+ 50mg/L by incubator in room air at 35'
C}. The inoculum size was determined by diluting anovernight growth of bacteria and comparing the turbid‑
ity with a 0.5 McFarland standard. The bacterial cul‑tures were diluted in 0.9% saline to achieve a final
concentration of I >< 105 CFU/ml. Subsequently thesewere inoculated in U‑bottom microdilution trays along(SPF) environment with a 12 hr light‑dark cycle andused for the experiments. B. pseudomallei SP‑237 wasused throughout this experiment. Mice were anesthet‑ized subcutaneusly with 0.25 ml of 10% pentobarbital
sodium (Dainihon Pharmaceutical Co. Ltd, Japan) andwith control organisms and incubated aerobically for 24
hr at 37"C. The concentrations of antibiotic after inocu‑

Lung stamp specimen from B. pseudomallei SP‑237

lation ranged from 64 to 0.0625mg/L. The MIC wasdetermined as the lowest dilution of antibiotic concen‑
tration that showed no turbidity after overnight incuba‑tion in room air at 35'C. The MBC of. pseudomallei

SP‑237 was defined as the lowest dilution of antibiotic
that completely inhibited growth on nutrient agar (BBL,
Cockeysvill, USA) after a further overnight incubationin room air at 35'C.

infected by the intratracheal route with 3.2 x 103 cfu per
0.05ml.

infected mice
The stamped specimens of the dissected lung wereobtained at 72 hr after infection from mice receiving
levofloxacin therapy or none at all. These specimens

were stained with acridine orange and were observed
through the fluorescence microscope. Levofloxacin
therapy (10mglkg) was initiated at 24 hr after the
inoculation. Levofloxacin was administered orally
twice a day for 60 hr. Lungs of controll mice wereobserved before inoculation.

Intracellular antirnicrobial

ctivity

The mice were injected intraperitoneally with
Roswell Park Memorial Institute(RPMD medium. The
mononuclear cells were adjusted to a concentration of
1 x 106cell/ml, by counting cell in a Neubauer counting
chamber after staining with Tripan blue solution. The
cell suspension (1 mD was placed in each well of a 24

Treatment schedule
Five antibiotics were chosen with relatively highactivity as measured by MIC. The infected mice were
injected subcutaneusly (ceftazidime, imipenem/cilas‑tatin, piperacillin, minocycline, or vehicle), or adminis‑

tered orally (levofloxacin) twice a day for 7 days. Each3dose was 200mg/kg(ceftazicime, piperacillin) , 100mg/

RESULTSkg(imipenem/cilastatin) or 10mg/kg(levofloxacin,
minocycline) starting at 24 hr after infection. Control
mice received 0.Iml of physiological saline(vehicle) .
Each group consisted of ten kinds, n= lO. Mortality wasrecorded daily for 14 days. The statistical significance

MICs and MBCS
The results of drug susceptibility tests with B.
pseudomallei are shown in Table l. Imipenem was theof data was determined by the generalized Wilcoxon

most active antibiotic. Piperacillin, ceftazidime, ceft‑izoxime, minocycline, ofloxacin, Ievofloxacin and cipro‑

test.

floxacin were moderately active antibiotics which had a

MIC90 of ; 4ug/ml. Ampicillin, cefazolin, cefotiam,
Table 1 Minimum inhibitory concentration (MIO of

gentamycin, clindamycin and erythromyc in showed

14 antibiotics against 13 isolates of B.

resistance againstall strains of B. pseudomallei.

pseud omall ei

50%

90%

The MIC and MBC of 13 antibiotics against B.
pseudomallei SP‑237 are shown in Table 2. Imipenem
was most strong bactericidal activity. Piperacillin,

> 64

> 64

ceftazidime, ceftizoxime, and levofloxacin were moder‑ately strong bactericidal activity. Minocycline did not

O . 5‑1

1

1

> 64

> 64
64

> 64
64

1 06.

1 o

Range

Antibiotics

MIC (mg/mL)

PenamsAmpicillin

8‑ > 64

Piperacillin

Cephems
Cef azolin

Cef otiam
Cef tizoxime
Cef tazidime

64‑ > 64
O . 5‑1

1

1

O . 5‑1

1

1

O . 125

O . 125

O . 125

Carbapenem

IPM (4Mlc)a) 104

Imi penem

,
:D
LL

O 10

Quinolones
Of loxacin
Ciprof loxacin
Levof loxacin

2 8
1‑4
l‑4

2

Aminoglycoside
Gentamycin

> 64

4

1

2

1

2

> 64

> 64

1 02

LVFX (4MIC)

1 O1

Tetracyclin

10

Hours
Figure I Intracellular growth of B. pseudomallei in per‑itoneal macrophage prepared from mice. Macro‑

Mynocycline

O . 5‑1

1

1

Lincomycin
Clindamycin

> 64

> 64

> 64

Macrolide
Erythromycin

> 64

> 64

> 64

imipenem was added at I hr after inoculation
with the bacterium. Each point represents then=13mean viable count in cfu S.D. of nutrient agar.

phage monolayers were inoculated, incubatedfor 3 hr and then washed. Either levofloxacin or

Table 2 Comparision of MIC and MBC of 13 antibiotics against B. pseudomallei SP‑237
Antibiotics

MIC MBC (mg/L)

PenamsAmpicillin
Piperacillin

Cef otiam
Cef tizixime
Cef tazidime

Carbapenem
Imipenem

MIC MBC (mg/L)

Quinolones
> 64

0.5

>8
1

Cephems
Cef azolin

Antibiotics
Of loxacin
Levof loxacin

Aminoglycoside
Gentamycin

2

4

1

2

> 64

>8

> 64
> 64

>8
>8

1

2

1

2

Mynocycline

1

8

O . 125

0.5

Lincomycin
Clindamycin

> 64

>8

Macrolide
Erythromycin

> 64

>8

Tetracyclin

4

(a)

(b)

(c)

Figure 2 Fluorescence micrograph of lung stamp specimen from B. pseudomallei infectedmouse by the intratracheal n )ute at / 2 hr after the inoculation(3.2xl03 cfu per 0.05mD.
Intracellular and free organisms vith acridine orange stain were observed (arrow). (a)Normal lung. (b) Lung vithout the treatment. (c) I̲ung̲ vith the treatment; Ievofloxacin(10mglkg) was initiated 24 hr after the inoculation. Treatment was administrated orally
twice a day. Magnification: x 1,000

5

%
1 OO

O Levofloxacln 10mglkg

80

A Mlnocycllne 10m8lkg

F

ca
:)

)
*
:,

D Ceftazidime 200mglkg

*

O 60
CO
L

**

40

**

e Imipenem/cilastatin

1 OOI,8lkg

A Piperacillin 2OOmglkg

l Vehicle

CO20* P<0.05

2 4 6

t f f t tt t t 8t fIot f12ft 14
days
Thera py

day after inoculation
Figure 3 Cumulative survival rate of experimental pneumonia by the intratracheal routewith B. pseudomallei (3̲2 x 103 cfu per 0.05 mD in mice in treatment and vehicle group.
Treatments were given subcutaneusly (ceftazidime, imipenem/cilastatin, piperacillin,
minocyclin, or vehicle) or administrered orally (levofloxacin) twice a day for 7 days.
Control mice received 0.1 ml of physiological saline (vehicle) .

show strong activity with MBC. B. pseudomallei SP‑237

showed resistance as measured by MIC and MBC
against ampicilin, cefazolin, cefotiam, gentamycin, clin‑

damycin and erythromycin.

within 7 days after infection. Incontrast, mice treatedwith levofloxacin showed an increase in survival (80%) .
Levofloxacin therapy showed a significantly (P < 0.01 or
P < 0.05) higher survival rate than minocycline (10%),piperacillin (20%) and imipenem/cilastatin (40%) .

Intracellular antimicrobial activity against B.
pseud omallei

DISCUSSION

The number of bacteria in the macrophages wereshown in Figure 1. The imipenem at 0.5mg/L (4xMIO

Melioidosis was first described in Rangoon in 1912

did not inhibit the intracellular growih of B. pseudomal‑(7) . In recent years, melioidosis has been recognized aslei whereas levofloxacin at 4mg/L (1XMIO remark‑a common cause of illness and death in northeast

ably inhibited.

Thailand (2) . There have been two reports of Japanesemelioidosis patients to date (8, 9). Melioidosis is adisease which is associated with high mortality rate,particularly the pneumonia and sepsis forms (2‑4) .
B. pseudomallei is naturally resistant to many anti‑

Existence of B. pseudomallei SP‑237 in the lung of
infected mice with or without therapy
Intracellular and free organisms were strained inlung stamp specimens from mice infected with B.
pseudomallei SP‑237 without therapy with acridine
orange and observed by fluorescence microscope. No
organisms were observed in the lungs of control miceand one variation of free organis was observed in lungsof the infected mice receiving therapy (Figure 2) .

biotics (10‑12) . The mortality rate of severe melioidosis

was reduced from 70% to 40% by ceftazidime therapy(13) . We expected to find an improved therapy because
the survival rate is still low. We therefore examined theactivity of 14 antibiotics against B. pseudomallei in vivo.

Imipenem showed the highest activity, followed bypiperacillin, ceftazidime, ceftizoxime, minocycline, Ievo‑

Effects of antibiotics against experimental .

floxacin, ciprofloxacin and ofloxacin. The MBC ofpseudomallei pneumonia in mice model

antibiotics were 2‑4 fold less than MIC except in the

The ability of five antibiotics to affect the survival

case of minocycline. Minocycline's MBC may be arate of mice infected with B. pseudomallei is shown in
Figure 3. A11 control mice receiving vehicle alone diedfactor of eight less than its MIC by reason of it being abacteriostatic drug. Livermore et al (1987) reported B.

6

pseudomallei produced beta‑1actamase of the same
weakly inducible but other factor (e.g. poor drug
penetrationb or low target affinity) may contribute to
the beta‑lactams resistance of the organism (14) . B.
pseudomallei is a facultative intracellular growih oforganism such as the genera Salmonella. Shigella and
Listeria (15, 16, 17) . We speculated that B. pseudomallei

survived and multiplied in phagocytes despite of therapy
with effective antibiotic with MIC against the pathogen
in vitro. Beta‑lactams have generally poor penetration
into phagocyies (18) . Gaja et al(1992) found that theratios of intracellular/extracellular drug concentrationof levofloxacin in human neutrophils was 8.83

1.27 (19) .

Imipenem 0.5mglL(4 x MIO did not inhibit the intracel‑lular growih of B. pseudomallei in macrophages, where‑
as levofloxacin 4mglL(4 x MIO could inhibit it. Levo‑
floxacin was significantly more effective than ceft‑azidime, imipenem/cilastatin, piperacillin and minocy‑cline against B. pseudomallei pneumonia in mice. Pene‑
tration of levofloxacin into the lung was presumably
higher than that of ciprofloxacin, as lung concentrations

attained with the related drug ofloxacin after oral
administration was 2‑3 fold higherthan ciprofloxacin(19). Levoxacin, the (S)‑(‑)‑isomer of ofloxacin,
showed generally twice the antibacterial activity ofofloxacin (2D. Ofloxacin given orally was more effec‑
tive than ciprofloxacin against S lmonella typhimurium
infection in mice despite of ciprofloxacin being twice aseffective as ofloxacin in vitro (15) . As S lmonella is afacultative intracellular bacterium, Ievofloxacin may beable to penetrate in to macrophages more effectively
than ciprofloxacin. The ability of B. pseudomallei tosurvive and multiply in macrophages may be supposed
to be the cause of the difficulty of treatment of
melioidosis and the high rate of relapse.

In summary, Ievofloxacin showed high intracellular
activity in vitro and was effective against B. pseudomal‑lei pneumonia in mice in vivo. Levofloxacin may havea useful clinical role in melioidosis caused by B.
pseudomallei in humans.

ACK NOWLEDGMENTS
The authors gratefully thank Mr. I. Nakasone forhis technical assistance and Dr. N. Kusano and Dr. F.
H iga for their encouragement.

REFERENCES
1 ) Yabuuchi, E. and Arakawa, M. (1993): Burkholderia
pseudomallei and melioidosis: Be aware in temperatearea, Microbiol. Immunol., 37, 823‑836
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R. (1984): Melioidosis in far north Queensland: a clini‑
cal and epidemiological review of twenty cases, Am. J.
Trop. Med. Hyg. 33, 467‑473
3 ) Sanford, J.P. (1990): Pseudomonas species (including
melioidosis and glanders) , Principles and practice of
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Wattanagoon, Y., Naigowit, P., Davis, T.M.E., Loonar‑
eesuwan, S. and Pitakwatchara, N. (1989) : Melioidosis:

A major cause of community‑acquired septicemia innortheastern Thailand, J. Infect. Dis., 159, 890‑899
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Septicaemic melioidosis: a review of 50 cases from
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6 ) Pruksachartvuthi, S., Aswapokee, N. and Thankern‑
gpol, K. (1990): Survival of Pseudomonas pseudomallei
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account of the discovery of a hitherto undescribed in‑
fective disease occurring among the population of Ran‑goon. Indian Med. Gaz., 47, 262‑267
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Pseudomonas pseudomallei. Antimicrob. Agents
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12) Dance, D.A.B., Wuthiekanun, V., Chaowagul, W. andWhite, N.J. (1989): The antimicrobial susceptibility of

Pseudomonas pseudomallei. Emergence of resistance invitro and during treatment, J. Antimicrob. Chemother.,24, 295‑309

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Y.K. (1987) : p‑lactamase of Pseudomonas pseudomallei
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M.E. and Mcguire, J.L. (1990): In‑vivo evalution ofofloxacin in Salmonella typhimurium infection in mice,
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9Jpn. J. Trop. Med. Hyg., Vol. 23, No. 1, 1995, pp. 9‑13

TR YPANOSOMA EVANSI= UNIQUE CONCAVITIESON THE SURFACE MEMBRANE OF PARAROSANILINE‑
INDUCED AKINETOPLASTIC CLONES AS REVEALEDBY SCANNlNG ELECTRON MICROSCOPYMARY ROSE AGNES SILVA‑TAHAT*, AKITOYO ICHlNOSE, HARUKI UEMURA
AND HIROJI KANBARA
Received December 20, 1994/Accepted February 28, 1995

Abstract: Pararosaniline‑induced akinetoplastic clones of Tmpanosoma evansi which lack DAPI‑stainable
kDNA network were characterized by scanning electron microscopy. Independent batches of akinetoplastic
parasites from two passages in mice were observed to have similarities with parental kinetoplastic strain
with respect to shape, form, and pleomorphism. The marked difference in surface topography was noted
between the wildtype and the mutant as unique concavities on the cell surface of the latter. These
concavities are variab]e in size, number and extent and may be inheritable. In addition, akinetoplastic cells

were found to undergo active longitudinal binary fission and filopodia formation as reported by others.
These observations suggest that the kDNA‑deficient mutants of T. evansi have maintained their basic
functions of cell division and infectivity and, therefore, the concavities on their surface are not detrimental
to their existence.

Keywords: Scanning electron microscopy, Tmpanosoma evansi, akinetoplastic form, surface concavity

INTRODUCTION
Tmpanosoma evansi is a haemoflagellate whichcauses a wasting disease called surra (also as desren‑
gadera or murrina in local language) in domestic ani‑mals particularly camels, horses and cattle. It has awide geographical distribution ranging from North
Africa, Asia and South America. Although it is mostphologically similar to the wildtype except for themarked difference revealed by transmission electron
microscopy as the replacement of the kDNA network by
an electron‑dense body referred to as the kinetoplast
remnant (Vickerman and Preston, 1976) . In addition,although the fundamental functions of the akinetoplas‑
tic cells are not much different from the kinetoplastids
with respect to viability, infectivity, and‑ability to proli‑closely related to Tmpa, nosoma brucei, it has deviated toferate, we have previously observed that the akineto‑a very simplified life cycle without invertebrate‑related

plastic parasites have a delayed rate of cell division and,stages and, hence, morphology, as an adaptation to atherefore, a slower growih (silva‑Tahat et al., Res., inmechanical mode of transmission by horseflies andpress) .

other biting insects (Hoare, 1967, 1972).

In the present paper, we further characterize para‑rosaniline‑induced akinetoplastic clones of T. evansi by

However, Iike all other members of the order
Kinetoplastida, T. evansi possesses a kinetoplast DNA(kDNA) albeit its reduced size which reflects the lackof respiratory processes in the mitochondrion (Borst
and Hoeijmakers, 1979).. In lieu of this feature, thistrypanosome is susceptible to mutate spontaneously into
forms with altered kDNA, the so‑called dyskinetoplas‑
tic and akinetoplastic forms (Hoare, 1954). Sponta‑
neous mutants account for about 3‑6% of a given popu‑
lation (Inoki et al., 1962). These mutants are mor‑scanning electron microscopy and describe their mor‑
phological features. We show that only the mutants
have unique surface concavities which may be inherita‑
ble and further demonstrate that they undergo active
longitudinal binary fission and filopodia formation in‑
dicating that the concavities are not harmful for theparasites.

Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Japan 852*TO whom all correspondence should be addressed.

10

Figure 1 DAPI fluorescence micrographs. (A) Kinetoplastidwith 2 fluorescent particles, a large nuclear DNA(arrowhead) and a small kDNA (arrow) . (B) An
akinetoplastic form with a single large fluorescentbody, i.e., the nuclear DNA. Bars, I pm.

MATERIALS AND METHODSParasites

Figure 2 Scanning electron micrographs. (A) A representa‑
tive photomicrograph of the kinetoplastid. Notetypical trypanosome surface topography. (B)Akinetoplastic clone bearing concavity at the

A kinetoplastic Tansui strain of Trypanosoma evan‑si isolated from a waterbuffalo was kindly provided by
Dr. K. Fujisaki (National Institute of Animal Health,10wer third of the cell body and a more shallow one
at the anterior region (conjoined arrows) . Short

Tsukuba) and was used in the study. Parasites were
cultured and maintained by passage in 8‑week‑old ICR

arise from the surface of the free flagellum, (O A
number of deep potential concavities (arrow) gath‑mice.
Induction and cloning of akinetoplastic parasites
was accomplished according to Inoki (1960) . Briefly,
the kinetoplastic strain was induced to become akineto

processes of segmented filopodia (arrowhead)ered together at the anterior portion of the akineto‑

plastic body. Bars, I pm.

Electro･n Microscopy

plastic by succe8sive injections of infected mice with 10‑

Infected blood sample was collected by cardiac

20 pglg of pararosaniline. Akinetoplastidy was assess‑ed by 2,4‑diamidino‑6‑phenylindole (DAPD staining ofpuncture. Parasites were isolated and purified by pass‑tail blood samples following the method of Hajduk(1976). Mutants were eventually cloned by single‑cellisolation and propagated in mice. The akinetoplastic
nature of the mutants was further confirmed by Southern hybridization analysis using kDNA probes (silva‑
Tahat et al., in press) .

age through DE52 anion exchange column. Both kineto‑
plastic (passage 5, K5) and established akinetoplastic
clones (passages 3 and 4, AK3 and AK4, respectively) ,which have been maintained in the absence ofthe dye, were suspended and washed in O . 1 6 M phosphate‑saline glucose ( PSG) buffer ( pH 7.4 ) consisting of

0.0113M NaH2P04.2H20, 0.0486M Na2HPO., O.0436M
NaCl, and 0.0555M dextrose prior to fixation in 2%
gluteraldehyde. Bloodstream trypomastigotes were11

Figure 4 Scanning electron micrographs af akinetoplastic
trypanosomes showing filopodia (arrowhead) . (A)Filopodia arise from the flagellum at the anterior
region and from the posterior portion where it is in

Figure 3 Scanning electron micrographs of akinetoplastic
forms during cell division. (A) A trypanosome

which bears a small concavity (arrow) at theposterior end is in the process of longitudinalbinary fission. One of the daughter cells extends afilopodium (arrowhead) from the flage]lum.

close proximity with one of the concavities(arrow) present. The former seems relatively
smooth while the latter appears to be segmented.
(B) Several filopodia Qf various length extending
from the posterior end of the parasite. The longest

appendage appears to be segmented and termi‑

Vesicular structures (asterisk) on the surface ofnates with a vesicular structure (asterisk) . Contin‑

the posterior ceil body may denote primordial
filopodia. Both daughter cells exhibit a slightuations of the filopodia over the cell surface are
8hown adjacent to the concavities. Bars, I pm.

degree of torsion at the anterior regions. (B) Pre‑sumably the final stage of cell division whereby
daughter trypanosomes are still attached at theirposterior broad terminal prior to complete divisionand separation. Note the presence of concavities(arrow) at the fused broad ends of both trypomas‑
tigotes and filopodium (arrowhead) in the same

region of the lower trypomastigote. The upper
trypanosome is very slightly twisted. Bars, I ,!m.

further confirmed by Southern Blot analysis (Silva‑Tahat et al., in press) . The mutants were establishedand cloned in mice, and processed for scanning .as wellas transmission electron microscopy.

In comparison with T, brucei, bloodstream
trypomastigotes of T. evansi exhibited similar shape,

form and pleomorphism as documented by Hoare(1972). While some of them may be long and siender

subsequentiy prepared for scanning and transmission
electron microscopy following standard procedures.

having elongated, flagella, some of them are short and

RESULTS AND DISCUSSION

been observed in both kinetoplastic and akinetoplastic
clones of 7'. evansi. According to Hoare (1972), thestumpy possessing short flagella. These forms have

Pararosaniline caused the deletion of a DAPI‑fluo‑

mean measurements of the stumpy, intermediate, andrescent kDNA in Trypanosoma evansi (Figure l). The

slender forms are 16.819.6 /Im, 19.5‑20.7 pm, and 23.0‑
24.9 /Im, respectively. In the kinetoplastic population,

absence of the kDNA in the akinetoplastic parasites was

12and lined by the cell membrane (Figure 5). These
concavities were apparently absent on the kinetoplastic
body surface (Figure 2).
The functional significance of the concavities foundon the surface of the akinetoplastic cells is not under‑stood. Nevertheless, pararosanil, 1'ne was able to induce

mutation in the kDNA of the kinetoplastid thereby
producing akinetoplastic forms in agreement with theresults of Inoki et al. (1962). Other dyes (e.g., acri‑flavine, ethidium bromide) and antitrypanosomal drugs(e.g., berenil, samorin) have likewise been shown toexert the same effect on other trypanosomes (Hajduk,
1978; Shapiro and Englund, 1990) . Notwithstanding, it is
Figure 5 A transmission electron micrograph of an akineto‑
plastic parasite showing electron‑opaque bodies(arrowhead

within the mitochondrial vesicle(mv) , the hollow space of the concavity (c) , andthe flagellar pocket (fp) . Note that although theconcavity and flagellar pocket appear to be filledwith the same amorphous material, the presence ofthe flagellum (arrow) distinguishes the latter fromthe former. Bar, I ,Im.

also possible that pararosaniline could have produced
the concavities. We cannot rule out the possible mem‑brane destabilizing effects of this dye. Drugs such as
adenine nucleoside trypanocides (e.g., Puromycin, Cor‑dycepin) and diamidines have been shown to interact
with membrane biosynthesis in T'. rhodesiense (Mac"adam and Williamson, 1969, 1972). Further studies on
the mechanism of action of pararosaniline and scanningelectron microscopy of spantaneous mutants may help

the stumpy form mea ured 16.8l9.3 pm in length with amean of 18.3iO.69; the intermediate form was 19.7‑21.6/tm with a mean of 20.8i0.65; and the slender form was
22.9‑26.1 /tm with a mean of 24.1i2.01. On the other
hand, within the akinetoplastic population, the stumpy
form measured 13.8‑19.5 pm with a mean of 17.6 2.94;

clarify this observation.

the intermediate form was 19. .5‑21.8 pem with a mean ofterminal of the parasite (Figure 3) . Longitudinal binary

20.8 0.63; and the slender form was 22.5‑24.7 pm with amean of 23.2iO.99 (representative trypomastigotes areshown in Figure 2) . Although there is some degree ofvariation, these measurements fall within the range Qf
those recorded by Hoare (1972) .
The surface of the streamlined akinetoplastic para‑site body was essentially smooth and relaxed although avery slight degree of torsion was observed in dividing
forms (Figure 3). Of prime interest, however, scanningfission is evident. In addition, attached to the posterior

However, despite the presence of concavities, asignificant number of akinetoplastic trypanosomes wereobserved to be in the process of cell division. Fission
appears to be initiated at the anterior portion bearing

the flagellum and culminated at the posterior broad(proximal to the concavities) and anterior (usuallyfrom the free flagellum) regions of the akinetoplastic
bloodstream trypomastigotes are thread‑1ike structures
known as filopodia (Figure 4). Filopodia were likewise

seen in kinetoplastic parasites (not shown) . The
filopodia are known to possess the variant surface

electron microscopy has illustrated for the first time theglycoproteins, or VSGs, (Vickerman and Luckins, 1969)
and their formation has been implicated as the sheddingof surface antigens (Cherian and Dusanic, 1977) . The

presence of concavities or pockets on the surface ofmore than 90% of akinetoplastic cells in the sample.

terminal vesicular structure at the tip of the filopodium(Figure 4B) appears to indicate that it is in the process

The orientation and extent of twisting of the remaining
akinetoplastic forms did not allow observation of thepresence of concavities. The concavities varied in size,of rounding off to be subsequently detached from theappendage. Similar vesicles were found in transmission

electron micrographs of akinetoplastic forns (notdepth, number and location on the surface membrane

shown) .

(Figures Z‑4) . Most were found at the posterior portion

The concavities were found in (1) establishedof the trypomastigote. An extreme case with respect tomutants collected from two passages in mice, AK3 andsize, depth and number of concavity formati,on was

AK4, which have been grown and maintained in theobserved in some akinetoplastic cells (AK4). Under theabsence of the dye, and (2) both daughter trypomas‑

transmission electron microscope, however, they areseen as hollow spaces filled by an amorphous material

tigotes (Fig. 3B) indicating that they are more likely to
be inheritable features of the akinetoplastic clones. Our13observations of akinetoplastic trypomastigotes undergo‑
ing cell division and filopodia formation clearly imply
that they still perform the fundarnental activities ofmultiplication and infection comparable to the normal
parasites. In conclusion, therefore, pararosaniline‑in‑duced akinetoplastic clones of T. evansi possess harm‑less unique cell membrane concavities.

the fine structure of Tmpanosoma rhodesiense specifi‑
cally associated with drug treatment, Trans. Roy. Soc.
Trop. Med. Hyg., 63:421‑422.
11) Macadam, R.F. and Williamson, J. (1972): Drug effects

on the fine structure of Ttypanosoma rhodesiense:
diamidines, Trans. Roy. Soc. Trop. Med. Hyg., 66 (6) :897‑
904.

12) Shapiro, T.A. and Englund, P.T. (1990): Selective clea‑

vage of kinetoplast DNA minicircles promoted by

ACKNOWLEDGEMEN TS
The authors express appreciation to Dr. Kozo Fu‑
jisaki of the National Institute of Animal Health, Tsu‑kuba City for providing the T. evansi stock and Miss
Miki Kinoshita for technical assistance. This study waspartly supported by research grants from the Ministryof Education, Science and Culture of Japan. M.R.A.S.T.

is a Japanese Government Ministry of Education, Sci‑

ence and Culture (MONBUSHO) scholar.

antitrypanosomal drugs, Proc. Natl. Acad. Sci. U.S.A.,
87:950‑954.

13) Silva‑Tahat, M.R.A., uemura, H. and yanagi, T. 1995.
Pararosaniline‑indeed akinetoplastic Tmpanosoma evan‑si : formation and characterization. J. Protozool. Res.
(In press) .

14) Vickerman, K. and Luckins, A.G. (1969): Localizationof variable antigens in the surface coat of Tmpanosoma
brucei using ferritin‑conjugated antibody, Nature, 224:
1135‑1136.

15) Vickerman. K. and Preston, T.M. (1976): Comparativecell biology of the kinetoplastid flagellates. In: Biologyof the Kinetoplastida. Vol. 1, Lumsden, W.H.R. and

REFERENCES
1 ) Borst, P. and Hoeijmakers, J.H.J. (1979): Extra‑
chromosomal DNA, In: ICN‑UCLA Symposia on Molec‑
ular and Cellular Biology, Vol. 15. Cummings, D.J.,
Borst, P., Dayid, I.B., Weissman, S.M., and Fox, C.F.
(Eds.) , Academic Press, Inc., New York, pp. 515‑531.
2 ) Cherian, P.V. and Dusanic, D.G. (1977) : Tmpanosoma
lewisi: immunoelectron microscopic studies on the sur‑face antigens of bloodstream forms, Exp. Parasitol., 43:
128‑142.

3 ) Hajduk, S.L. (1976) : Demonstration of kinetoplast DNA
in dyskinetoplastic strains of Tmpanosoma equiperdum.
Science, 191:858‑859.

4 ) Hajduk, S.L. (1978): Influence of DNA complexing
compounds on the kinetoplast of trypanosomatids, In:
Progress in Molecular and Subcellular Biology, Vol. 6,
Hahn, F.E. (Ed.), Springer‑Verlag, Germany, pp. 159‑
200.

5 ) Hoare, C.A. (1954) : The loss of the kinetoplast in
trypanosomes with special reference to Ttypanosoma
evansi. J. Protozool., 1:28‑33.

6 ) Hoare, C.A. (1967): Evolutionary trends in mammaliantrypanosomes, Adv. Parasitol., 5:47‑91.

7 ) Hoare, C.A. (1972): The trypanosornes of mammals: azoological monograph. Oxford and Edinburgh: Black‑well Scientific Publications. 749 pp.
8 ) Inoki, S. (1960): Studies on antigenic variation in the
Wellcome strain of Ttypanosoma gambiense. Biken J., 3:
215‑222.

9 ) Inoki, S., Sakamoto, H., Ono. T. and Kubo, R. (1962):

Studies on the Ak forms of Tmpanosoma evansi: II.
Effect of p‑rosaniline on the appearance of Ak forms,
Biken J., 5:127‑131.

10) Macadam, R.F. and Williamson, J. (1969): Lesions in

Evans, D.A. (Eds.). London: Academic Press Inc., Ltd.
pp. 35‑130.

15Jpn. J. Trop. Med. Hyg., Vol. 23, No. 1, 1995, pp. 15‑20

INTERACTION BETWEEN BULINUS GLOBOSUS AND
CLEOPATRA FERRUGINEA AT A TRANSMISSION
SITE OF SCHISTOSOMIASIS IN KWALE, KENYASHINICHI NoDA1, N('.ETHE D MUHOHO SHIGEHISA HABF
KIYOSHI MAKIYA4, and YOSHIKI AOK15
Received December 5, 1994/Accepted February 1O, 1995

Abstract: Snail survey data collected in a srnall village in Kenya from April 1984 to March 1991 were used
to analyze the interaction between Bulipu,is globosus (intermediate host of Schistosoma haematobium) andCleopatra ferruginea (unsusceptible snaiD. An inverse correlation was observed between the two snail
populations. This finding leads to the suggestion that C. fern,lginea has limiting effects on B. globosus
population. The relative penetrative activity of S. haematobium miracidia into the two snail species was also
examined. Miracidia penetrated C. ferruginea as well as B. globosus. Although selective mass‑chemother‑apy has been repeated every 2 years in our study area, the low infection rates in B. globosl,ts were recordedin the year when large numbers of C. ferruginea and small numbers of B. globosus were collected. Therefore,
C. ferruginea seems to diminish the transmission of S. haematobium; C. ferruginea reduc The number of S.
haematobium miracidia which reach to B. globosus.
Keywords: Bulipuis globosus, Cleopatra fc'rruginea, Schistosoma haematobium, snail, biological control,
antagonist, decoy

INTRODUCTION

spots where only two snail species, Bulinus globosus(intermediate host of Schistosoma haematobium) and

The control of snails that serve as intermediate

Cleopatra ferruginea were found. The latter species does

hosts of schistosomes is an effective means of reducing
the transmission of schistosomiasis. However, the use

not serve as an intermediate host for schistosomes orother trematodes of medical or veterinary significanceof chemicals to control snails may have undesirable
effects on the environment. Therefore, we have becomein Kenya (Brown, 1980). We therefore decided tointerested in the use of biological control agents that areboth environmentally less hazardous and cost effective.
Of all biological control strategies, intermolluscan com‑

petition is one of the most attractive mechanisms. The
ideal biological agent would be able to persist in the
habitat of the targe,t snail and induce substantive long‑term depression or eradication of the target snail popu‑

lation. Some potential competitor snails which havereceived considerable attention are Helisoma duryi,
Maris'a cornuarietus and Thiavla granlfera (WHO, 1984).

A research program on urinary schstosomiasis has
been carried out in a small village in the Kwale District,

Kenya since 1981. In our study area, there were someanalyze the data obtained so far to examine whether
intermolluscan competition occurs at natural snail
breeding sites.

As another measure of biological control of schis‑tosomiasis, it has been demonstrated that the infection
rate among intermediate host snails exposed to schis‑tosome miracidia was reduced if unsusceptible snail
species were present. Such snails apparently prevent
miracidia from reaching the intermediate host snails(Chernin, 1968; Upatham and Sturrock, 1973; Laracuente
et al., 1979). To determine if C. ferruginea has thepotential to serve as a miracidial sponge, the present
study also examined the relative penetrative activity ofS. haematobium miracidia into the two snail species.

Department of Medical Zoology, Faculty of Medicine, Kagoshima University. Sakuragaoka, Kagoshima 890, Japan
Centre for Microbiology Research. Kenya Medical Research Institute, P. O. Box 54840 Nairobi. Kenya
3
Department of Parasitology, School of Medicine, Fukuoka University, Jounan‑ku, Fukuoka 814‑01, Japan
Department of Parasitology and Tropical Public Health, University of Occupationa] and Environmental Health, Yahata‑nishiku,
Kitakyushu 807. Japan
5 Department of Parasitology, Institute of Tropical Medicine, Nagasaki University. Sakamoto, Nagasaki 852, Japan
1

2

16

miracidia into B. globosus and C. ferruginea was

MATERIALS AND METHODS
The study area, Mwachinga village, is located in

Kwale District, Coast Province, Kenya. A generaldescription of the study area was given in Noda et al.
(1988). The Kadingo River flows through the village. It
is less than 5 m in width. Some parts of the river dry up

in the dry season, Ieaving small pools. Towards the end

examined in the laboratory. S. haematobium eggs werecollected from school children at Tserezani Primary
School in Kwale District. B. globosus (13x9 mm) werecollected from the Kinango Dam and C. ferruginea (16 x9 mm) were collected from the Kadingo River in Kwale
District. Miracidia were allowed to hatch in dechlori‑
nated tap water. The snails were individually exposedof the dry season, these pools sometimes dry up com‑pletely. Snail surveys were carried out twice eachto 20 miracidia in the wells of a 24‑well tissue culture

month at 7 sites along the river. Villagers had frequentcontact with water at these sites. The sites weresnails were removed from wells, and the remaining
miracidia were counted under a stereoscopic micro‑

identified as 18B, 18C, 19, 21, 22, 23 and 24. Snails were

scope.

plate for 10, 30 and 60 minutes. After exposure, thesampled for 10 minutes by one person using a double‑
layer steel net scoop (4 mm mesh) . The number of each

RESULTS

species of snail collected was determined and recorded.
B. globosus were then put into small petri dishes filledwith 2 ml of dechlorinated tap water. The petri dishes
were kept in a lighted place for more than 2 hours, andthe presence of cercariae of S. haematobium was deter‑
mined under a stereoscopic microscope. Climatic condi‑
tions may be classified into four seasons; a long rainyseason from April to June, a cool dry season from Julyto October, a short rainy season in November, and a hot
dry season from December to March. Each study period
ran from April to March. Snail survey data collectefrom April 1984 to March 1991 were used to analyze theinteraction between the species.
The relative penetrative activity of S. haematobium

Numbers of 1 . globosus and C. ferruginea collected

monthly in each site are shown in Figure 1. Seasonalchanges in total snail population of both species weregenerally associated with the alternating cycle of rainy

and dry seasons. At the end of the long rainy season,snail populations started to increase, reached a peak,and fell during hot dry season. The relative abundanceof populations of B. globosus and C. ferruginea differedby site and year. Therefore, the correlation between
annual numbers of B. globosus and C. ferruginea at eachsite were analyzed. Inverse correlations were observed
between the two snail species. Coefficients of correla‑tion between annual numbers of B. globosus and C.

Table 1. Annual numbers of Bulinus globosus and Cleopatra ferruginea, and coefficient of correlation
between annual numbers of two species at each site
Number of snails
PeriodYear I (April 1984‑March 1985)
Year 2 (April 1985‑March 1986)

Species*
B.g.

Cf
B. g.

C f

Year 3 (April 1986‑March 1987)

B.g.

C.f

Year 4 (April 1987‑March 1988)

B.g.

C f

Year 5 (April 1988‑March 1989)

B.g.

Cf

Year 6 (April 1989‑March 1990)

B.g.

Year 7 (April 1990‑March 1991)

B.g.

Cf
C. f

Coefficient of correlation* B.g. : Bulinus glogosus, C.f : Cleopatra ferruginea

Site

18 B

18 C

19

35
678
79
165
20
735

393
326
606
375
255
450
260
166
801
118
458

458
268
613
134
168
620
260

42
355
442
187
279
89
147
124
‑O . 613

21

22

141

390
561
831
353
422
366
146
409
700
53
706
65
761
68

41

32

629
149
226
106

2 212

59
54
201
129
141
200
137
159
46

812

475

‑o . 650‑o . 629‑o . 257

399
224

34
421

723
48
658
35
475

13

21

104
36
465
127
101
389
106
198
87
104
88

‑o . 237‑o . 627‑o . 523

31

23

24

1

390
480
21
41

825
282
573
295
301
191

17

eoo

Site 1 8B

250

B. globosus
C. ferruginea

200
15* 150

Z

Ioo
;

50
o
1 984

1 985

198e

1 987

1 988

1 989

1 e90

1991

eoo

250

S ite 1 8C

B. globceus
C. ferruglnea

200

b 150
i loo

!..

,.

50
!

o
1 e84

1 985

1 986

1 987

1 e88

1 9CO

1 990 1 991

300
250

Site 1 9

B, globosusC, ferruginea

200
15* 150

loo

50
o
1 984

1 985

1 98e

1 987

1 988

1 98e

1 S90 1 991

300
250

Site 21B, globosus!

/

C. ferruginea

200
15 150

Ioo
so
o
1 984

Figure 1.

1 985

1 986 1 987

1 988 1 990 1 991
1 989

Monthly numbers of ulinus globosus and Cleopatra ferruginea
collected from 7 sites, April 1984 to March 1991.

18
300
250

B. globosus

Site 22C, fetruginea

200
15* 150

Z 100
t

t

50
o
1 984

1 985

1 986

1 987

1 988

1 e89:

1 990 1 991

eoo

250

B, globosus

Site 23

C, ferruglnea

200
15 150

*
100

td

50
o
1 984

1 985

1 e86

1 987

1 988

1 989

1 990 1 991

coo

450

B. globosus

Site 24400

C. ferruginea

850
OOo
15 250

200

ii

z 150
1 oo
';

50

1 9e4 1 985 1 98e

1 987

1 988

1 989

1 990 1 e9 1

Figure 1. Continuation from the f ormer pagehaematobium infected B. globosus collected at 7 sites areyear 4 was comparable to that of year 6. The infection
rate of year 3 was lower than those of year 5 and 7, andthat of year 4 was lower than that of year 6. Years 3
and 4, when the lower infection rates were recorded,shown in Table 2. In our study area, selective mass‑

were those when large numbers of C. ferruginea andchemotherapy with metrifonate was carried out insmall numbers of B. globosus were collected.

ferruginea varied according to collection sites, but were
all negative, ranging from ‑0.237 to ‑0.629 (Table 1).

Annual numbers of C. ferruginea , B. globosus and S.

February 1984, and that with praziquantel in September

The results of the experiments on the relative

1986, 1988 and 1990 (Sato et al., 1988; unpublished date) .

penetration of miracidia into B. globosus and C. fer‑
ruginea are shown in Table 3. Duming the early periodof snail‑miracidia contact, B. globosus was penetrated
by a higher number of miracidia than C. ferruginea (P<0.05, Student's t‑test). Later, however, C. ferruginea
was also penetrated by a high number of miracidia.

Whenever mass‑treatment was given, the overall preva‑
lence in our study area fell rapidly after treatment, butrose again after I year to the pre‑treatment level.
Therefore the level of contamination of river in year 3
was comparable to that of years 5 and 7, and the level in19

Table 2. Annual numbers of Cleopatra ferruginea. Bulinus globosus, and Schis ‑tosoma haematobium infected . globosus collected frorn 7 sites
Number of snails
PeriodCl eo patra

Bu I in us

ferruginea

globosus

Year I (April 1984‑March 1985)
Year 2 (April 1985‑March 1986)
Year 3 (April 1986‑March 1987)*
Year 4 (April 1987‑March 1988)
Year 5 (April 1988‑March 1989)*
Year 6 (April 1989‑March 1990)
Year 7 (April 1990‑March 1991)*

5, 0731 490

2, 837

2 , 892

3, 507

1 , 028

2, 088930

1, 226

3 , 466748

2 , 794835

2 , 258

Inf ected

B. globosus(infection rate)

71 (4.8%)
114 (3.9%)
2 (0.2%)1 (0.1%)111 (3.2%)
61 (2.2%)
25 (1.1%)* The selective mass‑chemotherapy with praziquantel was carried out in September 1986,
1988 and 1990.

Table 3. Number (MeanS.D., N=6) of Schls

tosoma haematobium miracidia remaining
after exposure to Bulinus globosus andCleopatra fermginea (Snails were individu‑
ally exposed to 20 miracidia)

Species

Minutes of exposure
10*30

and achieved levels of relative abundance considerably
higher than noted prior to molluscicide application; M.
tuberculata was slow to recover following molluscicid‑
ing. In our study area, C. ferruginea, also a member of

the Thiaridae, was present and was locally abundant.

An inverse correlation between populations of B.
60

Bulinus globosus 10.1 3.3 7.7i2.4 7.8:t2.9
Cleopatraferruginea 14.7 2.7 11.8i4.7 9.8 3.8* Number of remaining miracidia exposed to two snail
species are significantly different at the 0.05 Ievel (Stu‑dent's t‑test) .

globosus and C. ferruginea was observed in a natural
setting. The data suggest that C. ferruginea has limiting
effects on B. globosus populations. It is likely that C.

ferruginea destroys eggs and young snails of the vector
and competes with vector snails for space and foods.
In addition to the possible role of C. ferruginea as aneffective antagonist to vector snails, the presence of

unsusceptible snails to schistosome may reduce the

DISCUSSION
T. gramfera and T. tuberculata, belong to the gas‑
tropod family Thiaridae, may be effective competitors

infection rate of snails by acting as a miracidial spongeor as a decoy (Chernin, 1968; Upatham and Sturrock,1973; Laracuente et al., 1979). Upatham and Sturrock

with Biomphalaria (reviewed by Pointier and McCul‑(1973) suggest that unsusceptible snails have a signifi‑
cant effect on diminishing transmission, especially whenlough, 1989). The potential of T. granlfera in biologicalcontrol has been investigated in St. Lucia. In four field

chemotherapy has been used to reduce the worm burden
and egg load in the human population. The experiments

trials, B. glabrata was apparently eliminated fromon the relative penetration of miracidia into

marshes and streams 6 to 22 months after the introduc‑tion of T. grantfera (Prentice, 1983). In Martinique, T.
tuberculata was introduced into two groups of water‑
cress beds, and both . globosus and B. straminae were
eliminated from the transmission sites within 3 years ofand C. ferruginea showed that S. haematobium miracidia

the introduction of the competitor (Pointer et al., 1989) .

in year 3 was lower than those in year 5 and 7, and the

Mkoji et al. (1992) reported that Melanoides tuberculata

infection rate in year 4 was lower than that in year 6. As(Thiaridae) co‑exists with Biomphalaria pfelfferi andother pulmonates in Kenyan freshwater habitats, andwas described previously, the level of contamination ofwater at year 3 and 4 was comparable to that of year 5
and 7, and that of year 6 respectively. Our study may
indicate that the predominance of C. ferruginea over B.
globosus reduces the infection rate of snails by reducing
the number of B. globosus, and, in addition, by reducing

possibly acts to regulate pulmonate populations. Aftermolluscicide application, all four snail species (M. tuber‑culata. B. pfelfferi. Lymnaea natalensis and . globosus)

became scarce. Then, . pfelfferi populations recovered. globosus

also penetrated C. ferruginea. In our study area, the low

infection rates in. globosus were recorded in years 3

and 4 when large numbers of C. ferruginea and small
numbers of . globosus were collected; the infection rate

20

the number of miracidia which reach to B. globosus.
C. ferruginea is widely distributed in permanent6 ) Pointier. J. P., Guyard, A. and Mosser, A. (1989) :Biological control of Biomphalaria glabrata and B.

rivers and muddy residual pools in seasonal streams

straminea by the competitor snail Thiara tuberculata in aalong the east coast of Africa (Kenya, Tanzania,
Mozambique and South Africa) where S. haematobium
is endemic (Brown, 1980). C. ferruginea may have apreviously unappreciated effect on diminishing thetransmission site of schistosomiasis in Martinique,
French West Indies. An. Trop. Med. Parasitol., 83, 263‑2697 ) Pointier, J. P. and McCullough, F. (1989): Biologicalcontrol of the snail host of Schistosoma mansoni in thetransmission of S. haematobium in these coastal hab‑

Caribbean area using Thiara spp. Acta Tropica, 46,

itats. C. ferruginea appears to present interesting possi‑

147‑155
8 ) Prentice, M. A. (1983): Displacement of Biomphalaria

bilities as an effective tool for biological control.
Further investigations should be attempted to examine

glabrata by the snail Thiara granlfera in field habitats in

St. Lucia, West Indies. An. Trop. Med. Parasitol., 77,
51‑59

this possibility.

9 ) Sato, K., Shimada, M., Noda. S., Muhoho, N. D.. Katsurnata,

ACKNOWLEDGMENTS
We are grateful to Dr. P. Waiyaki, Director of theT., Sato A. and Aoki Y. (1988) : Efficacy of metrifonate
in a highly endemic area of urinary schistosomiasis in

Kenya. Am. J. Trop. Med. Hyg., 38, 81‑85

Centre for Microbiology Research for administrative
support. We should like to thank Dr. E. S. Brown, the10) Upatham, E. S. and Sturrock R. (1973) : Field investiga‑
tions on the effect of other aquatic animals on the infec‑

British Museum, for identification of snails, and Dr. E. S.

tion of Biomphalaria glabrata by Schistosoma mansoni

Loker for providing helpful suggestions and criticallyreading the manuscript. This study was conductedunder the Kenya‑Japan Communicable Diseases
Research and Control Project, with support from the
Kenya Medical Research Institute and the Japan Inter‑national Cooperation Agency. The publication of thispaper was supported by the Kodama Foundation for
Medical Research. This work is published with permis‑
sion from the director, Kenya Medical Research Insti‑
tute.

REFERENCES
1 ) Brown, D. S. (1980): Freshwater Snails of Africa andtheir Medical Importance. pp. 487. Taylor & Francis,

London2 ) Chernin, E. (1968): Interference with the capacity of
Schistosoma mansoni miracidia to infect the molluscan
host. J. Parasitol., 54, 509‑516

3 ) Laracuente, A., Brown, R. A. and Jobin, W. (1979):
Comparison of four species of snail as potential decoys
to intercept schistosome miracidia. Am. J. Trop. Med.
Hyg., 28, 99‑1054 ) Noda, S., Shimada, M., Sato. K., Ouma, J. H.. Thiongo,

F. W., Muhoho, N. D., Sato, A. and Aoki, Y. (1988):
Effect of mass chemotherapy and piped water on num‑

ber of Schistosoma haematobium and prevalence in
Bulinus globosus in Kwale, Kenya. Am. J.' Trop. Med.
Hyg., 38, 487‑495
5 ) Mkoji, G. M., Mungai, B. N., Koech, D. K., Hofkin. B. V.

and Loker, E. S. (1992): Does the snail Melanoides
tuberculata have a role in biological control of Biom‑phalaria pfelfferi and other medically important African
pulmonates? An. Trop. Med. Parasitol., 86, 201‑204

miracidia. J. Parasitol., 59, 448‑453

11) WHO (1984): Report of an informal consultation on
research on the biological control of snail intermediate

hosts. TDR/VBC‑SCH/SI/84.3.

21

Jpn. J. Trop. Med. Hyg., Vol. 23, No. 1, 1995, pp. 21‑24

A STUDY OF FATIGUE COMPLAINTS AMONG

WORKERS EXPOSED TO HEAT STRESS IN
AN ELECTRONIC COMPANY IN HANOI, VIETNAMSHINYA MATSUDA
Received November 28, 1994/Accepted January 30, 1995

Abstract: The relation between exposure to heat stress in the workplace and the degree of fatigue
complaints was investigated by a questionnaire (composed of 30 iterns) among 39 assembly line workers inan electronic factory in Hanoi, Vietnam. There is a considerable difference in the change of fatigue

complaints before and after work between workers exposed to heat and those not exposed. Among theworkers not exposed to heat stress, the rate of fatigue complaints increased in 8 items, all of which did notshow any statistically significant change during the work. In the case of workers under heat stress, the rateincreased in 25 items, of which nine showed statistically significant increase after working hours. Especially,

the changes were more apparent among the items corresponding to 'drowsiness and dullness'. A special
consideration must be paid to heat stress in tropical countries such as Vietnam, in order to prevent
occupational impairment.

INTRODUCTION

tries like Vietnam, the exhausting working conditions

are very energy consuming both physically and men‑

After the introduction of the new open market

tally. Therefore, a special consideration should be given

policy in 1986, Vietnam has begun to realize a rapid
economic growth. Especially, the permission for foreigncompanies to invest in the country has made it possible
to organize a number of joint ventures between Vietnam

to the working conditions in a tropical environment. In
this report. I present the results of studies focusing on
the relation between the heat stress and complaints offatigue of assembly line workers in an electronic com‑and other countries. It is expected that the living

pany in Hanoi, Vietnam.

conditions of the Vietnamese will be improved graduallyby the stabilization of the national economy. However,this rapid economic development may cause an intensifi‑
cation of labor, especially in the newly developed fac‑
tories, most of which are labor intensive sectors, such as

textile industries and food processing factories. Various"newly adopted" work conditions (i.e., shift work,
assembly line, etc) have been adopted in these work‑
places, where the standards of labor safety and hygiene
are not always respectedl). As a result, various healthproblems at the workplaces have emerged in addition to
the traditional occupational health problems (i.e., silico‑sis, saturism, etc). To evercome these problems, muchattention to occupational safety from the ergonomic
point of view must be paid in the developing countries
like Vietnam. It is well known that most occupational
accidents occur due to the lack of attention of workers
and that a mentally exhausting situation is very apt tocause severe occupat.ional accidents. In tropical coun‑

STUDY AND METHODS
An electronic company in Hanoi, Vietnam wasselected for the study. This factory, which was estab‑lished as a joint venture between Korea and Vietnam,
produces 10 thousand television sets per month on an
assembly line. The average working time is from 8:OO
am to 5:OO pm (there is a two‑hour rest at lunch time) .

The two most important origins of heat in the factory

are the workplaces where the assembled monitor ischecked, and where the soldering machine is located.
Thirty‑nine assembly line workers were investigat‑
ed regarding their working conditions as well as fatigue
complaints before and after work, by using two questi‑

onnaires. All questionnaires were completed by theworkers themselves. The first questionnaire was pre‑pared in order to obtain background information suchas; age, sex, educational level, Iiving conditions (i.e., size

Department of Preventive Medicine and Community Health, University of Occupational and Environmental Health, Japan22of house, nurnber of family members) , monthly income,distance between residence and factory, hazardous con‑ditions at the workplace (i.e., heat, noise, chemicals,in order to monitor the changes in subjective fatigue
symptoms during the working hours.
The basic statistics of the workers are as follows:
Sex distribution: 28 males and 11 females,

radiation, etc). The second questionnaire was thefatigue questionnaire developed by the Japan Associa‑

The average age of workers: 26.8 (SD=8.0, Min=
15, Max=48) ,Number of workers exposed to heat: 28

tion of Industrial Health, which is composed of 30
questions under three categories; the first 10 items are

related to "Drowsmess and dullness", the second 10
items to "Difficulty in concentration", and the last 10
items to "ProJection of physrcal dismtegratron"2). The
questionnaire was administrated before and after work

At first I conducted an analysis stratified by sex,
but as there was no difference between the two, I willshow the result of combined sex analysis in order toTable I Changes in the rate of those who had corresponding fatigue complaints before and after work in an electriccornpany at Hanoi, Vietnam (stratified by the existence of heat stress, n = 39)
Heat ( ‑ ) (n = 1 1)

Complaints

Heat ( + ) (n = 28)

Significance of difference

Before work After work

Signif icance

Before work After work

Significancedue to heat**(%) (%)of change*(%) (%)of change*

Before work After work

I . Drowsiness and dullness

l feel heavy in the head
2 get tired of the whole body
3 Iegs feel heavy

4 give a yawn
5 feel the brain muddled

6 become drowsy
7 feel strained in the eyes

8 become rigid or clumsy in motion
9 feel unsteady in standing
10 want to lie down
Average number of items marked in I

9.1 18.2

p=0.31725.0 67.9

p = O . 005

p = O . 262 p = O . 005

36 . 4 63 . 6

p = O . 109

32 . 1 82 . 1

p = O . 004

0.0 9.1

p=0.801 p=0.205

p=0.317

0.0 17.9

p = O . 043

N.C, p = O . 447

18.2 18.2
18.2 18.2

p = I . OOO

21 . 4 28 . 6

p = O . 361

p = O . 821 p = O . 409

18 . 2 45 . 536 . 4 36 . 4

p=0.109

0.0 21.4

p = O . 028

p = O . 074 p = O . 599

p = O . 139

p = O . 286 p = O . 648

p = I . OOO

35 . 4 53 . 6
32 . 1 64 . 3

p = O . 008

0.0 9.1
0.0 0.0

0.0 14.3

p=O.801 p=0.111

p=0.317

p = O . 068

N.C. p = O . 562

p= I .OOO

9 . I O . O

p=0.317

0.0 0.0

3.6 14.3

p=0.l09

N.C. N.C.
p = O . 490 p = O . 249

1.5 2.2

p=0.054***

1.5 3.6

p = O . oo0$ * *p = O . 066
pi0.949******

p = I . OOO

p = I . OOO

II. Difficulty in concentration

0.0 0.0

p = I . OOO

p=0.0rlO p=0.171

27 . 3 27 . 3

7.1 17.9

p=0 , 109

12 become weary of talking

p= I . OOO

10 . 7 28 . 6

p=0.091

p = O . 197 p = O . 632

13 feel irritable

9.1 9.1
0.0 0.0

p = I . OOO

10.7 17.9

p = O . 180

p = O . 687 p = O . 447

p = I . OO(]o . o (] . o

p = I . OOO

p=0.317

7.1 28.6

p = O . 028

p = O . 312 p = O . 194

p=0.317p=0.317p=0.317

p = O . 482 p = O . 642
p = O . 262 p = O . 051

ll feel difficult in thinking

14 unable to concentrate attention
15 become apt to forget things
16 unable to have interested in things

17 feel uneasy about things

18 apt to make mistakes

18.2 9.1

9.1 9.1
0.0 0.0
9.1 0.0

p = I . OOO
p = I . OOO

p=0.317

3.6 7.1
3.6 7.1

N.C, N.C.

p=0.718 p=0.510

19 unable to straighten up in the posture

27 . 3 27 . 3

p = I . OO(]

25 . O 28 . 6
10 . 7 28 . 6

p = O . O43

p = O . 208 p = O . 632

20 no energy

0.0 9.l
1.0 0.9

p=0.317o . O O . O

p = I . OOO

N.C. p = O . 282

p = o . 676* * *0.8 1 .6

p = (] . OO I * * *Average number of items marked in II

p = o . 202
p=0.696******

III. Projection of physical disintegration

21 have a headache
22 feel stiff in the shoulder

23 suffer low back pai
24 feel oppressed in breathing
25 feel thirsty

26 have a husky voice
27 have a dizziness
28 have a spasm on the eyelids
29 have a tremor in the limbs
30 feel unwell
Average number of items marked in 111

Average number of items marked in Total

27 . 3 36 . 4

p=0.317

9.1 9.1
0.0 9.1
0.0 0.0

p = I . OOO

p=0.317
p = I . OOO

27.3 27.3

p = I . OOO

O . (] O . o

p = I . OOO

9.1 0.0

p=0.317o . O o . op = I . OOO

0.0 0.0
0.0 0.0
0.7 0.8
3.2 3.9

p = I . OOO
p = I . OOO

p=0.341***p=0.195***

25 . O 53 . 6
21 . 4 39 . 3
25 . o 46 . 4

p = O . O25

p = () . 884 p = O . 333

p=0.091

p = O . 346 p = O . 068

p = O . 059

p = O . 077 p = O . 030

3.6 17.9

p = O . 068

p=0.718 p=0.171

17.9 39.3

p = O . 059

p = O . 400 p = O . 356

p = I . OOO

p = O . 718 p = O . 718

7.1 32.1

p=0.018
p=0.317

p = O . 642 p = O . 033

3.6 10.7

p = O . 180

p=0.718 p=0.358
N .C. N .C .

3.6 3.6

0.0 3.6
0.0 0.0
1.1 2.5
3.4 7.8

p = I . OOO

N .C. p = O . 718

p:=0.001***p=0.019
p=0.515******

p = O . OOO

p :1 O . 915* * * p =: O . 032* * **: Wilcoxon test (Before and after) , **: Chi square test or Fisher's exact test (Heat + vs. Heat ‑) , N.C.: Not calculated,***: t‑test

23

increase the statistical stability of the analysis. Thechanges in fatigue complaints were analyzed before andafter work stratified by the existence of heat stress(Wilcoxon test). The differences in the positive rate ofoccupational accidents caused by exhaustive conditionsof a worker due to heat stress, because heat imbalance
may occur even though there is only a slight increase in

fatigue complaints were compared between workers

relationship between heat loss/gain variables and inter‑

under heat stress and those not exposed (Chi square

nal heat production is described by a simple heat bal‑test, Fisher's exact test and t‑test) . The SPSSX statisti‑

ance equation3):

cal package in the computer system of UOEH was
employed in this investigation.

temperature in a tropical country like Vietnam. The

S= (M‑W) :!IC R‑E,
where S=net amount of heat gained or lost by the
body,

RESULTS
Table I presents the results concerning changes insubjective fatigue cornplaints before and after work,
stratified by the existence of heat stress in the work‑
place. There is a considerable difference in the changes

of fatigue complaints between two conditions. Among

M=heat production by metabolism,
W=external work performed,
C = heat transfer by convection,

R=heat transfer by radiation, andE=body heat loss by evaporation.
In a tropical country, because the environmental
temperature is frequently higher than the surface bodythe workers with no heat stress, the rate of workers who

ternperature, radiation and convection can increase thehad corresponding fatigue complaints increased in only
8 items, all of which did not show any statisticallysignificant change between before and after work. In
the case of workers exposed to heat, the rate increased

body temperature. Furthermore, in an extremely warmin 25 items, of which nine showed a statistically signifi‑

completely disturbed. For example, a heat collapse andcant increase after work. Especially, the changes werea heat stroke may easily occur in such a situation.
Luong reported that workers in textile factories inVietnam are usually exposed to temperatures of about

more apparent among the items of 'Drowsiness anddullness' (i.e., feel in the head, get tired of the whole‑moist environment, as found in textile factories locatedin tropical countries, a more troublesome situation
usually arises, where the heat loss by evaporation isbody, Iegs feel heavy, feel the brain muddled, feelstrained in the eyes) . When the average nurnber of

39‑40 'C with 80% humidity in average4). In the present

marked items before and after work in each three major
categories as well as in total was compared, a statisti‑

environment I or 2 'C higher (33‑34 'O than the outdoor

cally significant increase was observed among thehumidity) . Therefore, although we could not detect any
severe cases of heat‑related illnesses, mild forms of
impairment (i.e., irritability, Iassitude, decrease inmorale, and inability to concentrate) due to the pro‑workers under heat stress in all categories (1: p=0.000,
II: p= 0.001, 111: p=0.001, Total: p=0.000; t‑test) , but notamong the workers without the stress. When the num‑
ber of marked items in each category and in total were

cpmpared between the two heat exposure conditions

case, workers under heat stress are working in an
temperature, with 80% humidity (same as the outdoor

longed exposure to the moderately hot climate wereobserved. This situation can be dangerous in work‑

workers exposed to heat with statistical significanceplaces, especially in a place where enough attention tothe occupational safety is not always paid, as is often
observed in developing countries like Vietnam. System‑
atic studies on the effects of exposure to uncomfortably

after work (1: p=0.066, II: p=0.202, 111: p=0.019, Total:

warm environments have clarified that the number of

p=0.032; t=test) .

accidents, sickness and labor turnover increases because

before and after work, respectively, the nuinbers of thetwo conditions before work were almost the same, but

the numbers of marked items were larger among theof their chronic effects on the central nervous system.

DISCUSSION

Inefficiency due to the increase of minor accidents,
errors, and slower work rate is also another important

According to the present results, the existence ofheat stress at the workplace is significantly associatedproblem for developing countries who intend to realize awith the increase of fatigue complaints among theassembly line workers investigated in my study. In

both the Vietnamese workers and their employers do

Vietnam, special attention should be paid to the exis‑
tence of heat stress in factories in order to prevent

rapid economic development. In the current situation,
not seem to pa any special attention to occupationalsafety and hygiene such as heat, noise, and other harm‑
ful substances (i.e., heavy metals, organic solvents and24toxic gas) , although there is a special regulation legislat‑

ed by the government for this purpose5). It is naturalthat a rapid economic development is being given prior‑tional collaborative programs in the field of occupa‑
tional safety and hygiene will be organized in the near

future. Finally, in addition to the epidemiologicality in order to stabilize the society in the current situa‑

research like the present one, physiological studies musttion of Vietnam. In such a situation, it is very difficult

be conducted to establish standards for improving theworking condition in Vietnam.

to give adequate consideration to the working condi‑
tions. According to the results of another question‑
naire6), most of the workers, even though they are
exposed to some occupational hazards have answeredthat they are satisfied with the present working condi‑
tions, because a stable monthly income is assured, even
if there is, a possibility of some occupational healthimpairments in the future. Forty percent of the workers

ACKNOWLEDGEMENTS

have replied that they will endure some occupational

This study was conducted with a financial assis‑
tance from the Nissan Science Foundation. The author
wishes to express his deep gratitude to Mr. N.A. Luong(Director of the National Institute of Labour Protec‑
tion, Vietnam) for his warm‑hearted assistance for therisks to their health in order to earn money to live6). As

present study in Vietnam. The author is also grateful toexperienced in Japan, the ignorance of occupational

Prof. Kahyo (Department of Preventive Medicine andsafety and hygiene will have drastic results in the long

Cornmunity Health, U.O.E.H.) for his helpful criticism.

run. Therefore, special attention must be paid to occu‑

pational safety and hygiene from the early stage ofindustrial development.
A number of studies have been conducted in devel‑
oped countries, in order to determine the TLV (Thresh‑
old Limit Values) of various occupational risks, includ‑ing temperature at the workplace. For example, thedesirable temperatures in the workplace are set as

follows; 30.6 'C (Wet Bulb Globe Thermometer:
WBGT) for light work (235‑405 J/s) , 28.0 'C (WBGT)
for moderate work (up to 235 J/s) 7). However, it is not

REFERENCE
1 ) Thung, D.H., and Trinh, L.V. (1993): Assessment on
pollution of working environment in Vietnam and gen‑eral solution, NILP, Hanoi.
2 ) Industrial Fatigue Research Committee of Japan Associ‑ation of Industrial Health. (1969): Fatigue Scale ofIndustrial Fatigue Research Committee of Japan Associ‑ation of Industrial Health, In: Kyoto Symposium on
Methodology of Fatigue Assessment.
3 ) Hu H. (1988) : Other physical hazards and their effects.

appropriate to apply these standards to developing

In: Occupational Health, recognizing and preventing

countries in the tropics, because they have been devel‑work‑related disease, second ed. (ed. Levy, B.S.), 263‑279, Little, Brown and Company, Boston/Toronto.
4 ) Luong, N.A. (1986) : Some justifications and data forbasis of suggestion to second phase of the project VIE/

oped in western countries, most of which are in thetemperate zone. There is a difference in heat tolerance
and comfortable temperature zone for inhabitants of thetropics and those of other regions. Therefore, different
standards must be established in considering the physi‑
cal and social characteristics of the Vietnamese. How‑ever, it is not easy for the Vietnamese researchers toaccomplish this task because of the shortage of money,

materials and manpower. It is expected that interna‑

86/018. NILP, Hanoi.
5 ) Matsuda, S. (1995) : Ordinance on Labour Protection ofVietnam. The Journal of Science of Labour, 71, 22‑30.
6 ) Matsuda, S. unpublished data.
7 ) Bell, C.R. (1989): Heat and hot work. In: Encyclopediaof Occupational Health̲and safety, Vol. 1, Third ed. (ed:Parmeggiani, L.) , 1018‑1020, ILO, Geneva.

J pn. J. Trop.

25

Med. Hyg., Vol. 23, No. I (1995) pp. 25‑28

PROCEEDlNGS OF XXXVI ANNUAL MEETING OF
JAPANESE SOCIETY OF TROPICAL MEDIClNE
1‑2 December 1 994 , Kagoshima

President
Yoshihito Otsuji(Director General : Kagoshima Prefectural
Comprehensive Health Center)

CONTENTS
Prize Winner's Locture
JSTM (Japanese Society of Tropical Medicine) Young Investigator Award
Antibody frequency distribution curve for risk

S. Kanoassessment of a malaria epidemic
Special Lecture
How to attain the control of tuberculosis in the tropics

N Ishikawa

Overseas exchange by Satsuma Han (in Japanese)

I.

Haraguchi

Establishment of supply system of orphan drug for tropical diseases
and its supporting measure by the ministry of health and welfare

H. Ohtorno
Y. Otsuji

A just simple zeal‑my past 30 years in pursuit of filarial control

Future trend of Japanese contribution to the international

H. Hirumi

collaboration in medical sciences

Symposium :Strategies against tropical infectious diseases

S ‑ I Insect‑borne viral diseases

Y. Takeda et al.
A. Igarashi

N. Yamamoto

S ‑ 2 Acquired immunodeficiency syndrome (AIDS)
S ‑ 3 Chronic liver disease in south Japan:

s‑4
S‑5
s‑6
S‑7
S‑8
S‑9

special reference to chronic liver disease X

T. Arima

Searching for the etiology of HTLV‑1 associated myelopathy (HAM)

M. Osame

Development of bacterial vaccine and its future
H. Watanabe
Eradication of cholera
H. Hayashi
Molecular biological studies on Entamoeba histolytica
Y. Kaneda
S. Kojima
Conception for the development of schistosomiasis vaccine
Modification of strategy needed for tropical infectious disease control
H. Takei

General Presentation:

A ‑ I Studies on pneumonia among the children in Bangladesh Y. Utsunomiya et al .

AA ‑ ‑3 Evaluation
2 Surveyof pertussis in Ghana Y. Kamiya etof murine model infected by Penicillium marneffei N. Kudeken et

al.

al.

A ‑ 4 Efficacy of current clinical diagnosis of acute dilarrhoeal diseases

N. Matsubayashi et al.
in Zambian children
A ‑ 5 Bacteriological investigation on the diarrheal diseases in La Paz
and Sucre, Bolivia in 1993 and 1994
A. Utsunomiya et al .
A‑ 6 Control of children's bacterial diarrhoea in developing countries T. Nakano et al.
A‑ 7 Human rotaviruses isolated in Chaing Mai, Thailand in 1987‑1993

T. Urasawa et al.

A ‑ 8 Heat labile specific antigren of Vibrio cholerae 0139 M. Iwanaga et al .
A ‑ 9 Distribution of genes encoding cholera toxin, zonula occludens toxin,

26

accessory cholera toxin, and eltor hemolysin in Vibrio cholerae of diverse origins

A‑10

A‑11

H. Kurazono et al.
Structure and function of recombinant pig receptor (ST*R) for a Escherihia coli. Heat‑stable enterotoxin (ST*) 111: common motifs in extracellular domainof the membrane associated guanylyl cyclase receptor family including heat‑stableenterotoxin receptor (ST*R)
A. Wada et al.
Epidemiology of hepatitis B virus infection in Zambia, Africa‑The Importance of Horizontal Transmission in childhood and risk factors‑
H. Oshitani et al.

A‑12

Seroepidemiological study on HIV, HBV and HCV Infection

A‑13

Seroepidemiology of hepatitis viruses A, B, C in the Dominican Republic

in Chiang Mai, northern Thailand K. Toriyama et al.
A. Shichijo et al.

A‑14
A‑15

A‑16
A‑17
A‑18

An effective combination therapy with oral fluconazole (FLCZ)
and flucytosine (5‑FO against cryptococcal meningitis (CM)in
AIDS patients in Uganda K. Oishi et al.
Bacterial meningitis among Bangladeshi children attending in a city

hospital K. Ahmed et al.
Activity and therapeutic efficacy of antibiotics against Burkholderia

pseudomallei and its pneumonia in mice H. Fukuhara et al.
Virological study of dengue epidemic in Vientiane, Lao P.D.R. in 1994

T. Sisouk et al.

A‑19

DF/DHF epidemics and control in Vientiane municipality,

Lao P.D.R. in 1994 M. Saito et al.

A ‑20 Evaluation of filter paper method for the collection and storageof blood and serum specirnens in the assay of anti‑dengue lgM‑ELISA
A. Igarashi et al.

A‑21

A new DNA allele of HLA‑B in Orang Asli population in mainlandof Malaysia

A‑22

Structural analysis of population problem in Vietnam
Mechanism of eosinophil accumulation in cerebrospinal fluidof Angiostrongylus cantonensis infected mice
Clinical study on HTLV‑1 infection and strongyloidiasis
Epidemiology and control of Malaria in Yunnan Province,
People's Republic of China
Malaria epidemiology in Lombok Island, Indonesia;
application of sero‑epidemiological methods
A case of accidental transmission of Plasmodium falciparumthrough platelet transfusion
Limitacion of clinical diagnosis of malaria in PHC program

A ‑23

A‑24
A‑25
A ‑26

A‑27

M. Zaidi et al.

S. Matsuda
K. Yoshimura et al.
O. Zaha et al.

C. Li‑Gang
Y. P. Dachlan et al.
S. Kano et al.

A ‑28
H. Ohmae et al.
A ‑29 DNA diagnosis of malaria in the Socialist Republic of Vietnam H. Miyake et al.
K. Kita et al.
A ‑30 Analysis of mitochondrial DNA from Plasmodium vivax

A‑31

Usefulness of the PCR method with microtiter plate‑hybridizationin the clincal practice of irnported malaria

M. Kimura et al.

A ‑32 The phagocytosis of Plasmodium falciparum and TNF production
by human macrophages
T. Nagao et al.
A ‑33 Human blood lymphocyte responses to Plasmodium falciparum infection
Rzepczyk. C. M et al.

A ‑34 Drug sensitivity of Plasmodium vivax H. Amamo et al.
A ‑35 Age‑specific manifestation of drug resistance of Plasmodium

27

A‑36
A‑37
A‑38
A‑39

falciparum and Plasmodium vivax in malarious islanders in Eastern Melanesia
A. Kaneko et al.
Y.
Midorikawa
et al.
Immunosuppression and malarial parasite
Epidemiological survey of Chagas disease in Macharety village,

A. Uchida

Boqueron pref. Paraguay

S. Miura et al.
Serodiagnosis of congenital Chagas' disease in acute phase
T‑lymphoproliferative response of the patients with Chagas' disease
K. Hirayama et al.
to epimastigote antigen of T. cruzi in Guatemala

K. Yamada et al.
Sequences of E/NSI gene junction from four dengue‑2 virusesof northeastern thailand and their evolutionary relationships with other denge‑2

A ‑40 Studies on the detection of dengue viral genome by RT‑PCR

A‑41
A‑42

virusesClassification of Leishmania parasites, using kDNA finger printing

K. Zin et al.

B‑ 1

T. Mimori et al.
method by arbitrarily primer polymerase chain reaction
A survey of hookworm and Strongyloides infection for adult farmers
living surrounding Surabaya city, and for adult factry workers in Surabaya city,
K. Koga et al.
Indonesia

B‑2

Sero‑epidemiological survey of human toxocariasis in Nepal,

B‑3
B‑4

B‑ 5
B‑ 6
B‑ 7
B‑ 8

Bangladesh and Thailand
An immunological study of cysticercosis in Guatemala
Differential diagnosis of cutaneous leishmaniasis in Ecuador
First case of diffuse cutaneous leishmaniasis in Ecuador
Genetic regulation of cutaneous leishmaniasis in mice infected

K. Kondo et al.
H. Ichikawa et al.
S. Nonaka et al.
A. M. Bhutto et al.

with Leishmania amazonensis
Family examination of leprosy patient in low endemic area for

K. Katakura et al.

A. Hosokawa et al.
the disease in Ecuador
Health education in the control of schistosomiaSis haematobium
in coast province, Kenya: effect on knowledge, attitudes and practices
K. Moji et al.

B‑9Studies on schistosomiasis japonica in Oriental Mindoro, Philippines(1) seroepidemiological survey in school children with blood taken

on filter paper H. Matsuda et al.
area of Kenya K. Takahashi et al.
B‑11 Assay of serum type IV collagen in Schistosoma japonicum infection

B ‑10 Intermediate snail control in Schistosoma haematobium endemic

B ‑12
B ‑13

H. Ohmae et al.
Genetic study of Japanese encephalitis viruses from Malaysia H. Tsuchie et al.
A 27 amino acid coding region on the C‑terminal of Japanese
encephalitis virus E‑protein expressed in recornbinant Escherichia

coli possesses neutralizing epitope(S) S. A. Seif et al.
B ‑14

Isoforms of mRNA for large subunit of cytochrome B558 detectedin CDNA Iibrary of human granulocytes̲ S. Suzuki et al.

B ‑15 Analysis of novel chronic granulomatous disease A. Kumatori et al.

B‑16

Effects of diethylcarbamazine against Wuchereria bancrofti in Fiji:
five rounds of annual single‑dose treatment vs. a course of 28‑dose

tratment spread over 2 years J. U. Mataika et al.
B‑17

Fundamental surveys for the utilization of medicinal plants acainst filariasis

in Kenya (minireview) J. Maki et al.

B ‑18 A new methods for determination of diethylcarbamazine andivermectin in the blood by enzyme‑linked immunosorbent assay Y. Mitsui et al.

B ‑19 Are there sporadically endemic foci of lymphatic filariasis

28

in the Caribbean basin of Central America?

S. Hurtarte et al.

B ‑20 Rates of microfilaria prevalence and clinical filariasis in Chuuk
B ‑21(formerly Truk) state, Federated states of Micronesia
M. Fujiwara et al.
Statistical observations on chyluria patients for the recent twenty

B ‑22years in department of urology, Kagoshima University; inpatientsand outpatients between 1974 and 1993
Y. Mizuma et al.
Clinicopathological characteristics of four cases of pulmonary
dirof ilariasis

B ‑23
B ‑24

H. Hirano et al.

Basic studies on the Mongolian gerbil as a susceptible hostto filarial infection (13) sensitivity to diabetogenic substances
Basic studies on the Mongolian gerbil as a susceptible host to
filarial infection (14) the organ weights of the wild‑colored gerbil

K. Shirota et al.

and the coat color mutants

B ‑25 A new approach for analysis of human sweating
M. Shimazu et al.
B ‑26 Analysis of long‑team heat‑acclimatization in tropical inha,bitants;
comparison of sweat response among Japanese, Thai, and Africans
T. Matsumoto et al.

B ‑27 Vasodilation of the tail skin induced by isoproterenol in chronic

spinal rats K. Tsuchiya et al.

B ‑28 Inorganic ion composition of rain water in Yakushima island T. Andoh et al.
B ‑29 Clinical evaluation of health examinations of Japanese workers
B ‑30

dispatched
to foreign countries M. Fukumura
Bacteriological analysis of stool for 5 years in Kagoshima airport

quarantine station S. Hagio et al.

B ‑31 Asymptomatic infection of Cryptosporidium muris in humans T. Katsumata et al.
B ‑32 Venomous snakebites in Okinawa prefecture in 1993 S. Katsuuren et al.
B ‑33 The neutralization effects of Habu‑antivenom against snake‑venoms
extracted from snakes recently captured in Okinawa Island M. Nozaki et al.
B ‑34 Study on Chinese cobra (Naja atra) toxoid from Guangxi, China
Y. Kawamura et al.

' 'B ‑35 Comparison on the seasonal abundance of Anophel
es mmrmus
in Southern Yunnan province, China and Northern Thailand M. Takagi et al.
B ‑36 Growth curve of mosquito larval population and comparison of

growth rate K. Makiya et al.

B ‑37 In vitro effect of Plant‑extracts, especially those in Guatemala,against ympanosoma cruzi (minireview) J. Maki et al.

B ‑38 Information collecting and access system of medical assessmentin the tropics H. Itakura

B ‑39 Theme of health in North East Thailand M. Umemoto et al.
B ‑40 Environmental health in North East Thailand H. Masuda et al.
B ‑41 Genetic polymorphism of group‑specific component, transferrin andalpha‑1‑antitrypsin in Kagoshima prefecture: the serologicalcharacters of the populations in the Nansei‑shoto K. Ago et al.

29Prize winner's lecture

JSTM (Japanese Society of Tropical Medicine)

Young Investigator Award

ANTIBODY FREQUENCY DISTRIBUTION CURVE FOR RISKASSESSMENT OF A MALARIA EPIDEMIC
SHIGEYUKI KANO
Department of Parasitology, Gunma University School of Medicine

Seroepidemiology is important, in that it provides
more informative period prevalence data which cannot
be obtained through microscopy. In particular seroe‑
pidemiological assessment is valuable when a malaria
control program reaches the more advanced stage andthe endemicity of malaria bocomes very low in thecontrolled areas. On such an occasion, a more suitable
method for epidemiological surveys is required, in orderto detect latent malarial foci in apparently controlledareas. In the present study, follow up epidemiologicalsurveys were conducted in two villages in the Sudan in
1987 and 1989. In 1987, the frequency distribution curveof antibody titers, which was measured by the ABC‑ELISA, revealed a potential danger of future malaria
epidemics in one of the villages, Sennar, showing abimodal pattern with a second peak at titer 1:512. The
subjects who manifested titer of 1:512 were those consid‑ ‑ered to have contracted malaria about the time when thesurvey was done, and they might constitute the probablefocus of succeeding transmission in the community.
Thus the second mode of the curve was regarded as thesignificant indicator of potential risk of malaria epi‑
demics which might occur as a consequence of environ‑mental or man‑made changes. This assumption wasproven to be true after the disastrous flood occurred inreflected in the shape of the frequency distribution curveof antibody titers. Application of this method will be of

considerable value in identification of early or evenpresumptive malaria foci where control measures must
be concentrated.
We also applied this method of malaria assessment
to an indian colony located in the Amazon jungle, wheremalaria was regarded as a minor disease by the inhabit‑
ants. The reported incidence was very low, and in fact,
no parasitemia was detected in the blood smears of agroup of inhabitants obtained through our short visit.

But on the contrary, the ABC‑ELISA revealed a 100%
prevalence of malaria antibodies in the population, andthe frequency distribution curve of the titers showed abimodal pattern suggesting that malaria transmission
was actively taking place in the colony. Therefore,
serological studies which can detect the period preva‑
lence are useful for the assessment of malaria situationin the highly endemic community which is not readily
accessible, or whose populations seem to have acquireda certain degree of immunity depressing parasitemia to
submicroscopic levels.

The wider application of seroepidemiology isenvisioned in defining foci in an endemic area in Pal‑awan, the Philippines, where appropriate control cam‑August 1988, which adversely affected the well im‑
plemented malaria control program in the whole area

paigns are to be conducted. In the community underincluding our study villages. In the study in 1989, in‑mean reciprocal titer during the rainy season as compar‑
ed with that during the dry season. Seasonal changes inthe distribution of high‑ and low‑titer responses, which
is suggestive of the occurrence of recent past malaria

creased malaria prevalence was actually observed by
microscopy. The antibody titers obtained in Sennar
changed into hyperendemic pattern, showing the highest
peak at the highest titer 1:1024. This pattern was thereflection of the very recent past or current infection in
the majority of the people at the time of the survey. On

the other hand in Mobi, the other village under study,

study, there was a significant difference in the geometricepidemics, were also recognized. Using this method ofmalaria assessment, we could also identify the focus
even in a limited area, supporting the observation thatmalaria is not evenly spread over the geographic areas

the frequency distribution curve of the titer, character‑ized by a single low‑titered peak in 1987, changed into abimodal pattern suggestive of a small, recent past out‑

in which it is prevalent, instead, it is highly focal.

break of malaria in the village in 1989. Comparison ofseropositivity and slidepositivity rates did not present
significant difference between the results before andafter the flood. It was thus affirmed in this study thatthe actual prevalence of malaria may be more precisely

frequency distribution curve in malaria seroe‑

Thus, the three studies described above definitely
show the significance of the application of antibody

pidemiology, particularly in generating more precise
and informative malariometric data which is critical in
successful malaria control.

3 O

Special lecture

How to attain the Control of Tuberculosis in the Tropics

NoBUKATSU ISHIKAWA(The Research Institute of Tuberculosis)

Global TB Problem
Tuberculosis (TB) is still one of the major killing
diseases in the world 8 million new cases and 3 million
deaths every year. It can be a priority tropical disease

as more than 70% of those new cases or dead cases
occur in the tropics. Few more points need to bementioned for the problem of TB; 1) It has been neglect‑

New Framework for TB Control Program
WHO has recently issued a policy package for
effective TB program based on the analysis of successcases in TB control. The target should be first to
establish 85% cure rate of the sputum smear positive
patients, and then to develop 70% case‑finding cover‑age. To attain these, a following package program isneeded; i. e. )government commitment, @case finding

ed in the past few decades in most of the developing
countries; 2) Most productive and working age groups

by sputum microscopy with its network and quality

are affected; 3) The epidemiological situation is being

control,

deteriorated in most of the areas partly due to HIV
infection; 4) New move to strengthen the global TB
control program is urgently needed as the situation
would continuously deteriorate if no measures are)adoption of short course chemotherapy,)

secured supply of anti‑TB drugs, Rstandardized
monitoring system for treatment.

However practice is not easy. In macro scale,international financial and technical assistance is need‑taken; 5)Tuberculosis control is one of the most cost‑effective measures according to the recent analysis by

ed and in micro scale, primary health care system andthe World Bank. 'Why does TB not decrease in developing countries?

over trials in pilot areas need to be made to develop asuitable system to each country.

The possible reasons for no reduction or increase of
TB problem in most of the countries in the tropics are;)biomedical characteristics of TB as a chronic disease,

From the author's experience

insufficient socio‑economic development, Rpoor
health infrastructure, )poor TB program, and Rnew
epidemic of HIV infection.
Considering the current epidemic situation of HIV
infection, strengthening of TB control is urgently need‑ed in most of the countries in the tropics under theintensified international cooperation scheme.

community participation need to be promoted. More‑

Sixteen years experiences in Bangladesh by theauthor are demonstrated including participatory action
research for the integration of TB program into Thana(sub‑district) general health services. In the interna‑

tional cooperation, the ultimate goal is not only thetechnology transfer but further system developmentwith full participation of the staff of the country, result‑ing in the empowerment of the people concerned inproblem finding and its solution.

31

OVERSEAS

SpeciallectureEXCHANGE

BY

IzuMI
Faculty

of

law

and

SATSUMA

HAN

HARAGucHI

literature，Kagoshima

島津77万石と俗に言われるが，江戸時代の薩摩藩は，宮崎県の南半分がら琉球王国の与那国島までを含む，ちょうど日本の本州の長さに匹敵する領域だった。このうち琉球王国は，薩摩藩が支配しながらも，国際的には立派な独立王国だった。そこに，薩摩藩の海外とのつながりが出てく
るわけである。

NHKの大河ドラマ「翔ぶが如く」の時代考証をした時に，明治維新の薩摩藩の原動力は何だったのか，その放送だけでは本当の理解は得られないという思いがあって，NHK側に「琉球の風」を茶の間に届けてほしいと願い出た
結果取り上げられた。私は琉球の風が吹いて，翔ぶが如くに明治維新が成就したという考えで，この二つのドラマの時代考証をした。

琉球ルートを通じて，中国の学問，芸術，文化が薩摩に入って来たことは間違いない。長崎は，海外からの商品の窓口だったのに対して，琉球王国と中国とは，国家と国家の交流である。中国福州に琉球館があり，常時100人以上の琉球の人たちがいた。そこに高嶺徳明（中国名・魏士哲）という館付きの通訳官がいた。徳明は，中国人黄会友から手術の秘法を伝授された。徳明が琉球に戻った頃，王国の尚貞王の11歳の孫尚益が，生まれながらの兎唇だった。徳明はその手術をしたのである。1689年のことである。
福州で得た技術で，琉球王の孫に手術を施したということで，薩摩藩が聞き逃がすはずはない。琉球在番奉行付きの医者・伊佐敷道与（1661−1730）が，高嶺徳明から学んで薩摩に帰ったあと，川内川中流域の宮之城佐志領主であった島津家分家の23歳の子息に手術を施したという記録がある。川内市の歴史民俗資料館に，道与の弟子が書いたUniversity

秘伝書が残っているが，この手術は「酸鼻余りに忍びず」と書かれていて，ものすごい手術だったことがわかる。華
岡青洲が，全身麻酔術を施して乳がんの手術をしたのが，その70年後であることを考えると，薩摩の海外とのつながりを示すよい事例だと思うが，全身麻酔がどれほど効いていたのか，麻酔を全くしないで行ったのではなかろうしと，私も確信は持てない。
さかのぼって，関ケ原の戦いで奮戦したのが，加治木に隠居していた島津義弘である。城下町の加治木になぜ医者が多いのか。これは，加治木が海外との貿易港であったことと，戦国時代いつも金瘡手術をしなければならなかったからではなかろうか。島津義弘自身，金瘡手術を心得ていたという記録がある。生涯50回の戦をした人だから，金瘡
手術を心得ていなければ，命永らえなかったと思われる。
義弘の時に，島津氏は徳川に負けた。しかし，薩摩藩はその幕府を倒し，最後の将軍徳川慶喜は，大政を奉還した。
その時点で，五代友厚，寺島宗則らの藩費留学生が海外に赴き，わが国に初めて近代的な学術をもたらした。その流れの中で，明治2年，ウィリス博士が呼ばれ，距児島の外科術がイギリス流として芽生え，鹿児島大学医学部へと発展した。ウィリス博士は，西南戦争直前までいて，治療した人は一万人以上にのぽっている。また門弟の一人が慈恵医大を創設した高木兼寛先生で，先生はやはりイギリス流の医学者である。臨床を常に重視し，地域の課題に応じるというその伝統は，学術的な炎を灯しつづけながら受け継がれ，この日本熱帯医学会も，東南アジア，東アジアにおける本当の意味の国際貢献を果たしていると思う。

32

S pecial lecture

ESTABLISHMENT OF SUPPLY SYSTEM OF ORPHAN DRUG FOR
TROPICAL DISEASES AND ITS SUPPORTING MEASURE BY
THE MINISTRY OF HEALTH AND WELFARE
HIROSHI OHTOMO
Department of Tropical Medicine, Jikei University School of Medicine

There is no doubt that drugs have made a considerable
contribution to the fight against a whole series diseases.

However, one realized that there are some parasitic
infections or tropical diseases of which drug treatment
leaves a lot to be desired or is even to totally inadequate,

because of no available in Japan. In the last 20 years,
patients with so‑called imported tropical diseases wereincreasing gradually in Japan as results of the changesof life style and the internationalized personal inter‑changes. At 1980, the Group for the study of the drug

treatment of imported tropical diseases based on the

research enterprise by the Ministry of Health andWealfare was founded and established an access routeof orphan drugs for parasitic infections and tropicaldiseases for physicians by free distribution at theirrequests: 15 kinds of orpahn drugs were imported andconfirmed qualifications for the drug under investiga7
tions by National Institute of Hygienic Sciences. At
1988, this study group was reorganized into the Groupfor the study of the drug development for tropicaldiseases. Then, over 3,000 patients with parasitic infec‑

tions such as malaria, strongyloidiasis, amebiasis,

echinoccosis and Pneumocystis' " ' werearzwa cpneumoma

treated successfully by orphan drugs supplied from thesestudy group. In addition, supporting measures in Japanfor the development of orphan drugs were operated on
the basis of a pharmapeutical affairs bureau's notifica‑tion at 1985,0n affix materials concerning an applicatin
for permit of production(import) of orphan drug. Under
this mearure, six orphan dugs, thiabendazole, sulfadox‑

ine/pyrimethamine, praziquantel, mebendazole,
pentamidine, albendazolo, had been approved for clini‑
cal use by the Ministry of Health and Welfare.
At April, 1993, a proposed amendment of the Drugs,
Cosmetics and Medical Instruments Act and the Promo‑
tions Foundation Act for Relief and Study of Adverse
Drug Reactions was passed at the 126th session of theDict. Then, with starting research and development

promotion system from October, above two study
groups were re‑reorganized into the Group for the studyof the drug development enterprise. At present, thisstudy group is developing works on supply of orphan
drugs for tropical diseases and clinical investigatitionsof these drugs. This paper briefly covers the achieve‑
ment of orphan drugs supply, and internal and external
circumstance of these drugs.

Special Lecture

A JUST SIMPLE ZEAL‑MY PAST 30 YEARSIN PURSUIT OF FILARIAL CONTROLYOSHIHITO OTSUJI, M.D.
Kagoshima Prefectural Comprehensive Health Center

Filariasis had formerly been prevalent over allities and scrotal hydrocele were found drawn in a

Japan, mainly in Southern Kyushu. In one of ourresearch activities, we tackled control of filariasis.

picture scroll in around 1000. Further, the state offilarial spread in Japan was reported based on varied

Results of our filarial control work are described herein‑

historical materials.

af ter.

1 ) History of World Filariasis:

In around B.C. 1500 the disease was already known in
Egypt in the form of frescoed elephantiasis. In Japan,
on the other hand, elephantiasis of the lower extrem‑2 ) Signs and Symptoms of Filariasis:
We have seen elephantiasis cases of huge penis, scro‑tum, the upper and lower extrernities and the labiumand scrotal hydrocele and chyluria, etc.
3 ) Treatment of Filariasis:

33

Our treatment study was focused on diethylcarbam‑
azine (DEC or Supatonine) regimen (dosage, effec‑

shima 27,450 mf positives were found out of 771,924

tiveness and side effects, etc.), which later was con‑456) of all mf positives (34,350) were found in Kago‑
shima.
6 ) Follow‑up Results of Mass Treatment with DEC:
Follow‑up investigations were made after 10 years on
people treated with DEC in Kiyohara district, Bounot‑

firmed to have long‑lasting effectiveness for remark‑able reduction of microfilaria (mf) count.

About 1,500 people living in district of Kiyohara,
Kagoshima prefecture, were also subjected to ourmass treatment project, which lowered the mf posi‑
tives almost completely to zero level among them.
4 ) Mass Treatment of Filariasis Patients:
Japanese Central Government‑sponsored filariasis

examined during 1962‑1971 period. About 80% (27,su‑cho, Kagoshgima and Kuro island of Okinawa
prefectures, which revealed mf had completely been
eradicated in these places.
7 ) Malayan Filariasis in Cheju Island, Korea:

prevention project was started in 1962. Our concluded
standards for most effective method of filarial control
are as follows:a ) Blood to be taken after 9:OO P.M.
b ) Blood quantity to be taken is 30cmm.
c ) About 100 people are reasonable number per one
medical staff for blood taking.

Study on Malayan Filariasis in Cheju Island, Korea
was performed jointly by universities of Nagasaki,
Kagoshima and Seoul (Korea). Mf positive rate there
was high (20.3%; 353 out of 1,714 examined). Massd ) Total dose of DEC is 70mg/kg/B.W. per person.
e ) Continued and intermittent administration of
DEC to be combined.

by steroid hormone administration. We have come torealize that due consideration is necessary on thef ) Side effects of DEC administration do not require

treatment by DEC was done, with resultant side
effects, especially higher fever onset than in cases ofBancroftian filariasis. The high fever was alleviatedoccasion of international cooperation.

any preventive measures. The subjected people,

8 ) Pharmacodynamic Mechanism of DEC :Microscopic study revealed that DEC affected the

however, should be kept fully‑informed on the side

reproductive cell (sperm, ovum) of Dirofilaria im‑

effects, especially on possible fever onset.

mitis by inhibiting fertilization, which was confirmedg ) Subjected people should be well‑explained about

by us.

filariasis in order to obtain their full cooperation.

Our study has centered mainly on filariasis. In the

Exhaustive public relations are to be promoted in

begining of our study, eradication of filariasis which

this connection.

had been endemic on detached islands and outlying
districts was supposed to be impossible.
Long periods spanning 30 years have passed since thenand I now feel I have accomplished my long‑cherishedpurpose of filarial control. My profound gratitude is

About 15 or more years were required to reach above‑mentioned conclusions. We further have come toknow that on‑the‑spot work in order to confirm therighteousness of our knowledge was needed to over‑come miscellaneous problems.

given to colleagues from Kagoshima University Hos‑pital, Prefectural Government and people who cooper‑5 ) Mass Treatment Intended Mainly to Eradicate
Infection Sources by Administration of DEC:

ated with our filarial eradication project.

The filarial prevention project covered one metropolis

Last but not least, I can never thank late professor
Sato enough for his kind instruction.

and 8 prefectures, where 34,350 mf positives weredetected among 2,039,728 people examined. In Kago‑/

34

Special lecture

FUTURE TREND OF JAPANESE CONTRIBUTION TO
THE INTERNATIONAL COLLABORATION IN MEDICALSCIENCESHIROYUKI HIRUMI
Research Center for Protozoan Molecular Biology Immunology, Obihiro University ofAgriculture and Veterinary Medicine

Japanese contribution to the progress of interna‑
tional medical sciences may be divided into three cate‑experts as well as for the institutions. The ability torecruit the experts under the present system, particular‑gories: (D development of modern technology, (IDly in the medical research/training area, has been hitting

medical aids in the states of international emergency,

the limit in Japan as a whole. A drastic change issuch as a natural disaster, a relief of famines and refu‑

therefor urgently needed in this area. To improve thegees etc., and (IID medical research and training indeveloping nations. Although the Japanese contribu‑
tions to (D and (ID have been progressively improving,a number of aspects in the area of (IID may requireconsiderable improvements to achieve the efficiency ofproblem it is highly recommendable (a) to foundresearch/training centers of internationally high stan‑

dard within existing medical institutions in several
places m Japan and (b) to establish "ODA Posts" withinsuch centers under a novel system which is highly

Japanese aids to developing countries. Firstly, prior tomeritorious to the experts and the centers, aiming at a

the initiation of any aid project in developing nations, it

rapid progress in the promotion of the experts. Thirdly,
the main objective of the medical aid projects should be

is essential to recognize the existence of great differ‑ences among such countries in their culture, Ievels of

industrial and economical development and infras‑
tructures. Therefore it is important to set up aid proi
jects under a flexible aid policy which is highly compat‑
ible to the community of each recipient country. Secon‑
darily the promotion of scientists who would be highly
competitive to conduct medical aid projects in develop‑
ing countries is urgently required. The present system
in dispatching "Japanese Experts" to the aid projects
would be one of the major factors that have been con‑stnctmg the promotron of "Japanese Experts". The
system which recruiting the experts by seconding scien‑tists from universities and/or research organizations

has been accounted for a number of demerits for thedrverted from the present "Technology Transfer" to"International Collaborative Research Projects" andexecute "Problem Solvmg Jomt ProJects" with the col‑laboration of qualified local and Japanese scientists andin the future also with international experts. Objectivesof the projects have to be focused on national problemsof a high priority in the recipient country. Such projects

should be completed within a term, not leaving "a ruin

of ODA" benind. If the improvements suggested above
would be achieved, Japanese aids in this area would
become more efficient and make a greater contribution
to the progress of medical sciences in developing nations
in near future.

35

Sym posium

STRATEGIES AGAINST TROPICAL INFECTIOUS DISEASESYOSHIFUMI TAKEDAI AND ISAO TADA2
Research Center, International Medical Center of Japan*

and Department of Parasitology, Faculty of Medicine, Kyushu University2

In the control of infectious and parasitic diseases,

various conventional treatment measures are becdming
increasingly inadequate with the appearance of drug
resistance in pathogens, insecticide resistance in vectors,

lack of effective vaccines among others. Surveillancesystems are usually badly functioning in many develop‑
ing countries due to devastated economic and social
conditions. Further, emerging new strains ( V. cholerae139) or species (HIV, HTLV, HCV) of virulent micro‑

Vaccine has undoubtedly top priority in the preventionof these diseases and certainly in the past it has conquer‑ed small pox successfully, although various importantviral and bacterial pathogens still remain unchecked.
No vaccines are available for parasitic infections. Indi‑

vidual pathogens have their own mechanism of regulat‑
ing molecular and immunological identities. Intensive
trials on the identification of specific targets in thepathogens will be necessary for breakthroughs.
Recently it has been rapidly recognized that anthro‑organisms are threatening humankind. These humanpathogens are disseminating among the inhabitants
abetted by the behavior and social customs widely

pological approaches to the target communities are verypracticed in tropical and subtropical regions. Tropical

Without understanding knowledge, attitude and practiceimportant and necessary in the control of diseases.

infectious and parasitic diseases are no longer a regional(KAP) of the community, it will not be possible toproblem, but a global concern in the age of frequentinternational movement of refugee, migrant laborers

improve health level effectively. Thus, the combinationand travelers. In this context, we designed this sympo‑

sium to review desirable strategies against diseases
caused by viral, bacterial and parasitic pathogens.

of advanced technologies in biomedical science andactive application of anthropological methodology
should be encouraged in this era.

S‑ 1

INSECT‑BORNE VIRAL DISEASESAKIRA IGARASHI
Department of Virology, Institute of Tropical Medicine,

Nagasaki University

Insect‑borne viruses are mernbers of arboviruses,
many of which are human pathogens. Special attention
has been paid for causative viruses of yellow fever(YF) , Japanese encephalitis (JE) , and dengue, becauseof the clinical severities, number of patients, and epi‑

and limited supply of the current JE vaccine, WHOpostulated to develop the second generation vaccine bymodern biotechnology. Another approach is the local
production of current JE vaccine and integration into
EPI. JE in tropical Asia is an attracting research

demic areas.
YF has been endemo‑epidemic in tropical rain for‑sub j ect .

ests in Africa and South America. Although ideal

the tropics, because of increasing number of patients,

vaccine has been developed, its effective applicationneeds integration into national vaccination program
especially EPI program.
JE has been prevalent in Asia monsoon areas, withlarge epidemics occurring in several developing coun‑

enlarging epidemic areas, and appearance of severe
manifestation of dengue haemorrhagic fever (DHF) .
While dengue vaccine is still under development, andtries in recent years. In order to overcome the high cost

Dengue has been a world‑wide health problem invector control could not provide long‑lasting effect. +Research on the vascular permeability factor and viru‑
lence gene of dengue viruses are challenging subjects.

36

s‑2
ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS)NAOKI YAMAMOTO
Abstract: The etiologic agent of acquired im‑initial HIV infection. This clinical feature of AIDS

munodeficiency syndrome (AIDS) is a retrovirus calledsuggests that some factors other than HIV itself may behuman immunodeficiency virus (HIV) . The CD4 mole‑cule on helper T cells serves as a receptor for HIV

related to the onset of AIDS. On the other hand, therapid replication of HIV seems to directly affect thedevelopment or progress of AIDS. Therefore, anti‑HIV
drug therapy is also essential for AIDS. Most of theinfection. The viral RNA is converted into DNA by its
own reverse transcriptase and incorporated into cellular
DNA, resulting in an establishment of persistent infec‑
tion. In the presence of lymphocyte‑activating factors
or some other factors. HIV is activated in vivo in infect‑
ed individuals, resulting in insufficiency of cell‑mediatedimmunity. Indeed, it takes several years for infectedindividuals to develop clinical symptoms of AIDS after

HIV‑infected persons in Japan are hemophiliacs towhom HIV was transmitted by administration of clot‑
ting factors. What is required now is to protect these
HIV carriers from the development of AIDS as well as
to prevent further spread of HIV.

s‑ 3

CHRONlC LIVER DISEASE IN SOUTH JAPAN=
SPECIAL REFERENCE TO CHRONlC LIVER DISEASE XTERUKATSU ARIMA
Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University
In the early of the 1980 decade, prior to the studies
that lead to the characterization of the HCV genome, we
had the opportunity to treat successfully patients suffer‑

ing from acute and chronic forms of non‑A, non‑B(NANB) hepatitis.

hepatitis, or from patients recovering from acute
NANB hep titis. In fact, recent studies have demon‑
strated high frequency of genetic mutations in the M/E,

which may account for our observations and suggest adifficulty of development of the HCV vaccine.

In 1989, my group cloned CDNA fragment, A‑14

Recently in 1993, 1,300 patients with liver diseases

clone which is specific for NANB hepatitis. A‑14 clone
contains an epitope (AR142) recoguized by the sera fromwere visited our clinic. Liver biopsies were performed

HCV‑infected patients and has 8/11 and l0/11 aminoacid homology to the HCV‑1 and II, respectively. The

Twenty‑three out of 90 patients with chronic active
hepatitis type C were completely responded (CR) to an

for 1,009 patients with liver diseases in the last 5 years.

epitope is localized between amino acid positions 8 to 18

interferon treatment of 3MU, 3 times a week for 24of the core region of the HCV genome.

weeks and antibodies against A‑14 peptides were consid‑
erably reduced in those patients with CR irrespective of
the HCV type found in the patients.

In addition, fifty‑five clones encoding fragments ofthe HCV genome had been isolated by my group. The
RNA and amino acid homologies between these clones
and the prototype HCV, HCV‑1, sequences ranged from
42% to 91%, and from 47% to 94%, respectively. The

In 1993, 43 patients with liver cirrhosis and 52
patients with primary liver cancer had visited our clinic.

Thirteen(30%) and five(lO%) of those cases, respec‑

sequences covered by these clones corresponded to 60%

tively, were of unknown (X) origin. Of these patients, 3of the total HCV genome. However CDNA correspond‑
ing to the M/E and NS2 regions were not obtained byour method. These results suggest that there is veryof which have chronic active hepatitis and 2 who havedecompensated liver cirrhosis, all negative for HBSAg,

low, if any at all, titer of antibodies against M/E andalso negative for HBV and HCV‑PCR in the tissue havebeen studied extensively. All five patients presented

NS2 region in sera from patients with chronic NANB

anti‑HCV antibodies, serum PCR‑HCV negative, and37

corticosteroid treatment, although two of them present‑

results suggest that these patients represent cases ofCAH, Iiver cirrhosis and primary liver cancer due toed plasma cell clustering in the portal area. These

new hepatotropic virus (es) .

chronic active hepatitis which could not be controlled bys‑4
SEARCHING FOR THE ETIOLOGY OF HTLV‑I
ASSOCIATED MYELOPATHY (HAM)
MITSUHIRO OSAME, M.D.
The Third Department of Internal Medicine Faculty of Medicine Kagoshima University

Four hundred and sixty cases were reported for a

The association of spastic paraparesis with human
T‑lymphotropic virus type I (HTLV‑D was demonstrat‑

national prevalence of 0.35/100.000; the geographic dis‑

ed independently in two different areas; Caribian Basinl

tribution of HAM in Japan paralleled that of ATLL. Of

and Japan2 3. A serologic study in Martinique foundthe 460 patients, 26% had a history of blood transfusion,that 59% of patients with tropical spastic paraparesis

and the distribution of the interval from transfusion to(TSP) had antibodies to HTLV‑1.1 Within several

onset of HAM followed a log‑normal distribution.

months another study showed the presence of antibodies
to HTLV‑1 in both serum and cerebrospinal fluid(CSF)
of TSP patients in Jamaica and Colombia. In Japan wealso found an association of HTLV‑1 with spastic par‑aparesis, but because these patients reside in a temper‑

Significantly more transfusion‑associated HAM cases

were reported in HTLV‑1 nonendemic areas(37.6%)
than in endemic areas(20.4%, p= .OO1) and among caseswith no family history of HAM (28%) than in those witha family history(2%, p=.OOD. These results demon‑

ate zone we proposed the term HTLV‑1‑associatedstrate an association between blood transfusion and the

myelopathy(HAM).2‑3 The finding of such patients inJapan suggested HTLV‑1 as a cause of spastic parapar‑

development of HAM and suggest that the transfusionof HTLV‑1 infected blood may load to the developmentof HAM. These data strongly suggest that the blood
supply should be screened for HTLV‑1 before transfu‑

esis because Japan is one of the most endemic areas in

the world for adult T‑cell leukemia‑lymphoma (ATLL)
caused by HTLV‑1 and is free from malnutrition andyaws, which had been considered possible causes of
TSP. Some initial differences noted between TSP and
HAM seem to have originated from the difference inhistorical background which led to the discovery ofthese conditions.' Other investigators have found asimilar association between HTLV‑1 and myelopathy inother areas of the world, and now HAM and HTLV‑I‑associated TSP are thought to be the same disorder.5
A subgroup of cases with HAM has been identified,sion.

The mean age at onset of HAM was 41.2 years(range 5‑75 years) , and gait disturbance was the mostcommon initial complaint. Onset of HAM was gradualin 92% of patients but the disease progressed more
rapidly in older patients than in younger ones. Examina‑tion of cerebrospinal fluid frequently showed abnormal‑

ities and ATL‑like cells were seen in the peripheral
blood smear of 49% of patients. Favorable response tocorticosteroid therapy was reported in 74% of patients. 'which was related to a previous history of blood transfu‑

sion.6 The existence of another subgroup with mother

REFERENCES‑to‑child transmission was also reported.7 The identity(1) Gessain, A. et al., Lancet ii, 407‑411, 1985 (2) Osame,of the viruses of HAM and ATLL was shown throughM. et al., Lancet i, 1031‑1032, 1986 (3) Osame, M. et al.,DNA blotting8 and DNA sequence analysis9 from estab‑

Ann Neurol 21, I17‑122, 1987 (4) Osame, M. et al., Advlished cell line from CSF of HAM patient.Io HLANeurol Sci 31, 727‑745, 1987 (5) Roman, GC. et al.,‑haplotype‑linked high immune responsiveness against
HTLV‑1 has also been reported.ll
From October 1986 through January 1988, we con‑Lancet i, 651, 1988 (6) Osame, M. et al., Lancet ii, 104‑105, 1986 (7) Osame, M. et al., Lancet i, 106, 1987 (8)ducted a nationwide survey of HTLV‑1‑associatedYoshida, M. et al., Lancet i, 1085‑1086, 1987 (9) Tsu‑
jimoto, A. et al., Mol. Biol. Med. 5, 29‑42, 1988 (lO)myelopathy(HAM) in Japan to study its epidemiology,

Hirose, S. et al., Lancet i, 397‑398, 1986 (1D Usuku, K. et

relationship to blood transfusion, and clinical aspects.12

al., Ann Neurol 23, S143‑150, 1988 G2) Osame, M. et al.,38

Ann Neurol 28, 50‑56, 1990 a3) Hematology Reviews 3,

271‑284, 1990

s‑ 5

DEVELOPMENT OF BACTERIAL VACCINE AND ITS FUTURE
HARUO WATANABE
Department of Bactenology, National Institute of health

Several kinds of vaccines are now available in theworld as listed below.
1 ) inactivated whole cell vaccines‑ V. cholerae. B.
pertussis

multivalent vaccine. In addition to the live attenuatedvaccines, polysaccharide‑protein conjugate is also one
candidate as another future bacterial vaccines. In the

below, multivalent vaccines now developing are de‑2 ) Iive attnuatted vaccines‑S. typhi Ty2la, BCG, V.
cho le rae3 ) component vaccine‑DPT, pili4 ) polysaccharide vaccines‑Hib, Vi‑antigen, S.
pneumoniae. N. meningitidis
Inactivated whole cell vaccines and polysaccharide vac‑cines tend to induce humoral immunity but not cellularimmunity, while live attenuated vaccine induces cellularimmunity too. Oral administration of vaccines can
induce mucosal immunity. It is believed that mucosaland cellular immunity is much important than humoral
immunity to prevent bacterial infection in the mucosa.

scribed.

Multivalent vaccines
1 ) Iive attenuated vaccines(S. typhi Ty2la, BCG) ex‑
pressing foreign antigens (tetanus toxin, HIV, Pas ‑modium etc.) or small peptides corresponding to foreign
B‑ or T‑cell epitopes.
2 ) polysaccharide‑protein conjugate
Surface polysacharide
capsular polysaccharide: H. influenzae type b,
S. pneumococci. N. meningitidis etc.
carrier proteins:So, one direction to develop future bacterial vaccines is

toxoids: DPT, LT, CT, VT etc.
viruses: F protein of measles, protective

to use live attenuated vaccines as a carrier for

antigens of varicella, rubella etc.

s‑ 6

ERADICATION OF CHOLERAHIDEO HAYASHI
Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba

Although more than one hundred years have passed
since Koch identified Vibrio cholerae as a causative

isolated from environment sources do not have virulence
genes like ctx, ace, zot etc., the organism proliferate only

agent of cholera in 1882, the eradication of the disease orin human intestine and the root of infection is no othereven the control of the organism have not been wellestablished as yet. The major problem preventing us
from the eradication may be as follows: 1. where andway but just fecal‑oral mean by eating raw fish or

how V. cholerae survive or persist in natural environ‑ment? 2. how the organism aquire flexibility for muta‑tion to survive under environmental stresses? and 3. why
the vaccines so far evaluated by field trials failed toshelter or reservoir in our environment. We have tocontinue endle s effort to identify the reservoir byapplying advanced technology with deep insigt into theshow high efficacy? The followings are some ideal

The organism is provided with a magnificient capa‑
bility to adapt to environmental stresses for their own

strategies for the eradication of the disease.

The organism is unable to survive for a long period
or to proliferate in the natural environment, strains

vegetables like seashell, algea, seaweeds, or oyster, allof these evidence indicate that there should be a specificecology of the vibrios.

survival. Such adaptational responce is initiated byrecognizing the stress and then the signal is transduced

39

to a certain effector gene through the signal transduc‑tion system. The gene expressions are regulated by

to depress the production of vilurence factors or torepress the adaptational changes so as to keep thesome upper ranking genes via sequential reactions, fororganism susceptible to certain drugs or to im‑

instance, the production of the vilulence factors like ctx.

munological defencing factors of human. We need tofocus one of the research projects on this matter toobtain more detailed information and to create moreace, zot, and tcp are enhanced by tOXT which is controled

by tOXR and ToxS as well as by the other signals fromthe environmental stresses. Physical, chemical or evenbiological stresses clearly induce many kinds of stressproteins in the organism, i.e. heat shock proteins, theroles of which have not been well characterized. These
proteins should involve in the adaptational responcesuch as acquiring drug resistance or serotype conver‑sion. The organism has been mutated from the calssicaltype to Eltor type through the past pandemics, althoughthose were all O1 type, and recently, 0139 caused pan‑effective strategy for the eradication.

Many cholera vaccines have been invented in US,
Sweenden and/or Australia by applying gene technology
and have being tested in field trials, but so far up to
today, the efficacy has not been satisfactory. It is truethat the convalescent patient acquirs vibriocidal humor‑

demic in the south west Asia of which population
seemed to have been immunized by O1 type. In theal antibodies lgA, IgG and lgM which last for 3 mQnths
to one year. The invented vaccine were all effective assame level as the natural aquired immune. The involve‑
ment of memory T cell has been unclear, that is, molecu‑
lar cascade reaction from antigen recognition and pre‑endernic area, most of the isolates seemed to gain drug

sentation through secretion of antibody or some ant

resistance, i.e. tetracycline resistance. It is evident that‑vibtio agents that prevent the organism from adhesion,
proliferation, and/or production of the toxins should be

the organism gains a de nove property, most probably
via mutation or transformation from the other organism
under stressed condition and that the stress induced

proteins including signal transduction components
should play key role in the gaining process. If thesequential procedures are made clear at molecular level,

clearly characterized.

The problems mentioned above should be inves‑tigated extensively at a well organized institution by
efficiently organized scientists group supported by thenational foudation.

then we can nail down which component is the key one

s‑7

MOLECULAR BIOLOGICAL STUDIES ON ENTAMOEBAHIS TOL Y TICA

YOSHIMASA KANEDA
Department of Infectious Diseases, Tokai University School of Medicine

It is estimated that 500 million people in the world

nizes the 30,000 Mr antigen, also can distinguish between

are infected with Entamoeba histolytica, of whom 40

pathogenic and non‑pathogenic strains. However,million have developed disabling colitis or extraintes‑
tinal abscesses, which will result in at least 40,000
deaths. However, more than 90% of infected individuals

zymodeme patterns and reactivities of monoclonal anti‑
bodies are phenotypic properties. Indeed, it has beenreported that zymodeme conversion from nonpath‑usually are asymptomatic. This is probably due toogenic to pathogenic (or the reverse) can occur within adifferences in the pathogenicity of amoebic strains.

cloned culture of some strains of E. histolytica during the

Although it is difficult to morphologically discriminate

process of axenization under appropriate growth condi‑
tions. Therefore, molecular biological studies on the

between pathogenic and non‑pathogenic strains, it has
been reported that biochemical and immunological dif‑ferences exist. Sargeaunt and coworkers (1978, 1988)

demonstrated that amoebic organisms isolated from

pathogenic properties of the amoeba are needed. The
DNA sequence coding the 30,000 Mr antigen was detect‑
ed not only in pathogenic isolates but in nonpathogenic

patients with dysentery show isoenzyrnatic patterns thatisolates as well. However, in comparing both DNA

differ from those isolated from asymptomatic carriers.
We reported that a monoclonal antibody, which recog‑

sequences, minor differences were observed. In addi‑tion, we designed oligonucleotide primers on the basis of

40

the nucleotide sequence encoding the 30,000 Mr antigen,and have shown that the polymerase chain reaction
technique using these primers is useful for determining
pathogenicity. Numerous studies on the genetic analysis

of pathogenic and nonpathogenic strains suggest that E.
histolytica is a complex of two species, one pathogenic,

the other nonpathogenic.

.s‑ 8

CONCEPTION FOR THE DEVELOPMENT OFSCHISTOSOMIASIS VACCINESOMEI KOJIMA
Department of Parasitology, Institute of Medical Science, The University of Tokyo

Schistosomiasis is widely distributed in developing
countries and affects some two hundred million people,
despite the fact that extensive efforts to control theblock the invasion of schistosomes or reduce the fecun‑dity of the parasite in order to avoid severe damages of

disease have been continuously made in many endemic

the hosts to deposited eggs. On the other hand, it is also

areas. Since the eradication of snail intermediate hosts

necessary to clarify the targets of the effectors, such as

is absolutely difficult and reinfection easily occurs
among people living in the endemic areas even after asuccessful chemotherapy, the development of vaccines
against schistosomiasis is urgently required. For thisthat which molecule or which stage of schistosomes is

purpose, it is important to clarify effector mechanisms,

on one hand, by which final hosts including humans canvarious tissues which result from immune responses ofcritical in stimulating the effectors. In this symposium,

recent observations including those of our own labora‑
tory will be summarized and the important steps whichshould be taken into consideration in the development ofschistosomiasis vaccine will be discussed.

S‑9
MODIFICATION OF STRATEGY NEEDED FOR TROPICALINFECTIOUS DISEASE CONTROLHIDEO TAKEI
Faculty of Letters, Chiba University

Medical science has already accummulated suffi‑

The Programs of reeducating medical personnel,

cient knowledge and has developed efficient technology
for tropical infectious disease control. And many Pro‑
grams of tropical disease control have been launchedwithout achieving planned and expected results. A caselike the extinction of smallpox is rare. The task ofand reorganizing them for new, or more extended, tasks
seem to have achieved fairly good results. A case of
Tuberculosis Control Program presented here might beclassified as this type.

On the contrary, however, when some Program

efficient tropical disease control still remains as a har‑entered directly into communities, with the intention todest one to accomplish in most of the developing coun‑

failed? Was technology employed inefficient? Financial

educate ordinary community members to change their
daily habits, it met with unexpected obstacles, evenwhen Health Education seems to have been successful.
This was the case of Schistosomiasis Control Programsupport was poor? Or medical personnel involved, ofin East Africa.

those countries, Iacked knowledge for the task or failed
to learn it? Judging from reports presented here at thisca es? From the scientific point of view, i.e. seen as anannual meeting, these conditions do not seem respon‑sible for poor performance of the Programs.

applied practice based on the biomedical model of dis‑ease, both Programs are reasonable enough without any

tries.

What was wrong with those Programs which had

What makes the difference between these two

41

doubt. A crucial difference between the two is thetarget groups, in the former case a group of medical
personnel and in the latter ordinary community mem‑bers. This fact suggests that the crucial factors in thesecases are social, cultural and historical in nature.

tion of diseases, which determines their priority forseeking medical assistance, and their survival strategy
is based on it. When the target disease of a Program,
more precisely, its signs and symptoms are culturally
considered as benign ones, people will not change theirAmong various sociocultural factors, what should

behavior easily. Accepting what the Program recom‑

be taken into account, I argue, is the survival strategy

mends, sparing time for participating in its activities
means, for them, putting asides for a while their daily

and its underlying cosmology of the target groups. In
the case of medical personnel, receiving further educa‑
tion, participating earnestly to acquire new skills,
practicing newly acquired knowledge and skills, allthese might fit into their survival strategy, because
these can improve their competence as a salaried per‑son. For the providers of the Program, medical person‑
nel have another advantage. They are already educatedto be able to share biornedical view point. It is notdifficult to persuade them cooperate efficiently to fulfill

their assigned task.

In the case of ordinary people, conditions are com‑pletely different. They do not share such view point, norwould be interested to learn and change their behaviour
when there is not any other benefit to encourage theirmotivation. Moreover, they have their proper classifica‑

activities, which would be of certain significance in theirsurvival. They would never accept any change recom‑
mended by the Program personnel before analysing its
merits and demerits. If the Program can not show any
tangible merits according to their way of thinking, it is
not strange that they consider it useless to listen to theadvices of outsiders, who can survive without worrying
about foods for today.
What is needed in formulating "New Strategy" fortropical infectious disease control is to understand peo‑
ple's survivan strategy, including the disease classifica‑tion underlying it. Then, frorn the beginning of planninga Program, its planners and coordinators would be able
to take such understanding into account for achieving
better performance.

42

General Presentation

A‑ 1

STUDIES ON PNEUMONlA AMONG THE CHILDREN IN BANGLADESH
YOSHIAKI UTSUNOMIYAl, KAMRUDDlN AHMEDl, KIWAO WATANABE1, MISAO TAOl,
GLENDA CAROL, MARTlNEZ FRANCO1, NAOTO RIKITOMll, TSUYOSHI NAGATAKEl, KEIZO MATSUMOTol,SAMIR K. SAHA2 AND RUHUL AMlN2
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japanl

and Dhaka Shishu Hospital, Sher‑e‑Bangla Nagar, Dhaka, Bangladesh2

In Bangladesh pneumonia causes much morbidity
and mortality among the children. The pathogen caus‑
ing bacterial pneumonia among Bangladeshi children is
not exactly known. We started this project in Dhaka
Shishu Hospital, Dhaka, Bangladesh to know the exact

pathogen of pneumonia by culturing pharyngeal andnasal swab. From May 1993 to October 1993 a total 157
cases were included in this study. There were 102 (65%)

male and 55 (35%) female patients. The age ranged
from 21 days to 11 years with the mean age of 14.4
months. Most of the patients carne from the urban slum
area. According to the nutritional status there were 117(75%) of normal body weight, 14 (9%) of under weight,

14 (9%) of Kwashiorkor and 11 (7%) of Marasmus

pneumonia and 27/156 (17.3%) cases of lobar pneumo‑nia. Penicillins and gentamycin were administered inmost of the patients. From the pharyngeal culture

isolated bacteria were H. influenzae (16%), S.
pneurnoniae (14%), E. coli (11%), B. catarrhalis (8%),
P. aeruginosa (8%) , S. aureus (6%) , E. cloacae (2%) , K.

ozanae (2%) and 23% isolated bacteria were mixed
flora. From the nasopharyngeal culture isolated bacte‑
ria were B. catarrhalis (27%), H. influenzae (19%), S.

aureus (16%), S. pneumoniae (6%), P. aeruginosa(6%), K. pneumoniae (6%), E. coli (3%), K. ozanae(3%), E. cloacae (1%) and 12% isolated bacteria weremixed flora. The isolated H. influenzae 76% were non‑typable, 18% were type‑b and 6% type e.

cases. There were 129/156 (82.7%) cases of broncho‑

A‑ 2

SURVEY OF PERTUSSIS IN GNANAYASUHIKO KAMIYA PATIENCE AKPEDONU H E K LONGMATEY2, F.K. NKRUMAH2KIYOSU TANIGUCH12,3, HIROKI HOR13 AND HITOSHI KAMIYA"
Department of Pediatrics, Shiga University of Medical Science*,

Noguchi Memorial Institute for Medical Research', Department of Padiatrics,

Mie University School of Medicine' and National Mie Hospital'
A total of 56 clinical pertussis cases were identified

Cyclodextrin Solid(CS) medium and/or Bordet‑Gengou

in a sentinel survey at five polyclinics in Accra, Ghana(BG) medium.
The isolation rate was 16.7% on each medium,for one year between August, 1992 and 1993 by using thecase definition as 14 days of cough plus paroxysms ofthough CS medium allowed srnaller number of cells of

cough, whooping or vomiting. This active survey

B. pertussis to grow in experimental test.

identified higer number of pertussis cases than passive

In the nine positive cases, seven were under one
year old and eight had not been three times or moreimmunized with DTP vaccines.
These findings show that pertussis vaccine was
effective for the prevention of illness and should besurveillance data in Ministry of Health. By age, 51
cases were under five years old, including 13 under one
year.

Of all 56 cases whose nasopharygeal swabs werecollected and stored in Amies transport medium, ninecases yielded positive culture of Bordetella pertussis on

administered fully as early as EPI schedule.

43

A‑ 3

EVALUATION OF MURINE MODEL INFECTED BY
PENICILLIUM MARNEFFEINoRIFUMI KUDEKENl, NoBUCHIKA KUSANO , FUTOSHI HIGA , KAZUYOSHI KAWAKAMI*,
ATSUSHI SAITO*, PRASIT THARVICHITKUL2 AND THIRA SIRISANTHANA3First Department of Internal Medicine, School of Medicine, University of the Ryukyus ,Department of Bacteriology', Department of Internal Medicine*and School of Medicine, Chiang Mai University, Thailand

with the P. marneffei is the common opportunistic infec‑

tion of anti‑CD4 monoclonal antibodies (GK1.5).
After two weeks, the survival rate of mice with I x
108 cfu/mouse was O%, I >< 107 cfu/mouse was 50% and
3 x 106 cfu/mouse was 100%. On the histopathologicalexamination, the lungs of infected mice showed infiltra‑tion in patients with the human immunodeficiency virus

tion of mononuclear cells and the destruction of alveolar(HIV) in northern Thailand. We made murine model

structures at two weeks. After inoculation of I x 108
cfu/mouse, the viable P. marneffei were isolated fromthe lungs, spleen and liver on 3,7 and 10 days. The
mortality of the mice depleted of CD4 Iymphocytes islower than that of control mice (p<0.05). The numbers

Penicillium marneffei, a dimorphic fungus that is
endemic in southeast Asia, causes deep‑seated infection
in humans and rodents. Next to pulmonary tuberculosis
and cryptococcal meningitis, disseminated infection

infected by P. marneffei and evaluated the effects of anti

‑ CD4 antibodies on this model.

Specific pathogen‑free BALB/C mice (male,weight; 20‑25 g) were infected with intratracheal inocu‑
lation of the conidia of P. marneffei within the range of3 x 106 to I x 108 cfu/mouse. The strain was Penicillium

marneffei isolated from blood of patients with HIV. For

the counts of viable P. marneffei of the organ, thehomogenates prepared from the lungs, spleen and liverin diluted water with 0.1% Tweeen80 were cultured on

Sabouraud dextrose agar.

To deplete CD4+lymphocytes, mice were received intraperitoneal injec‑of viable P. marneffei in the lungs and spleen of the mice

depleted of CD4+ Iymphocytes were significantly in‑creased (p<0.001) than that of the control mice at 13
days.

These results suggested that CD4+ Iymphocytes
play the role in the destruction of alveolar structures by

the infiltration of mononuclear cells and the removal of

P. marneffei on this model.

A‑ 4

EFFICACY OF CURRENT CLlNICAL DIAGNOSIS OF ACUTEDIARRHOEAL DISEASES IN ZAMBIAN CHILDRENNoBUYUKI MATSUBAYASHll, MASAHIRO WATANABE1, G.J. BHAT2,HITOSHI KAMIYAI AND MlNORU SAKURAll
Department of Paediatrics, Mie University School of Medicine*and Department of Paediatrics, University Teaching Hospital, Lusaka, Zambia'

INTRODUCTION: In most developing countries,

clinical diagnosis was reviewed and evaluated.

acute diarrhoeal diseases are diagnosed as dysentery

METHOD: From May 1992 to May 1993, 690 ofand non dysentery, according to macroscopic stool

3624 children with acute diarrhoeal diseases admitted to

examination due to limited laboratory facilities. In thisDiarrhoea Training Unit (DTU) of University Teaching
Hospital, Lusaka, Zambia were recruited.

sense, dysentery might include diarrhoea caused byother type of pathogens. World Health Organization
recommends adequate rehydration for both dysentery
and non dysentery, and antibiotics only in exceptional
cases such as bloody diarrhoea. In this study current

The inclusion criteria for this study were; 1. chil‑dren less than six years. 2. History of ADD as definedby WHO (passage of two or more loose/watery stool inpast 24 hours lasting less than 14 days) 3. No history of44

priot antibiotic therapy.

Medical evaluation includes history by way of ques‑tionnaire noting clinical features of diarrhoea episode;physical examination noting degree of dehydration andconcurrent diseases; Iaboratory work out including stool
macro/microscopy, stool culture, polyvalent anti sera
test for EPEC, ELISA kit test and electron microscopy
for viruses.

found sensitive to EPEC had significantly lower CFR(O%) while those on other antibiotics (21:9%) and not
on any antibiotics (14.3%) had higher CFR respectively.

DISCUSSION: The study r veals that EPEC was
one of major causative pathogens of acute diarrhoeal
diseases in Zambian children and has the highest CFR.
Antibiotics treatment of acute diarrhoea with EPEC
infection has not been established yet. However, treat‑

RESULT: Among Dysentery cases, Shigellament with nalidixic acid of the acute diarrhoeal diseases

Dysenteriae was the first causative pathogen (23.2%)
whilst EPEC was second causative pathogen (13.2%).
On the other hand, Rotavirus was the major pathogen

had significantly lower CFR in this study. We thus
propose that not only dysentery cases but Non Dysen‑(28.2%) in Non Dysentery cases while EPEC was thesecond causative pathogen (15.8%). In terms of casefatality rate (CFR), EPEC had the highest percentage(15.8%) , while both Shigellae and Rotavirus was about

9%. The group (EPEC infected cases both in Non

tery cases as well with EPEC infection should be treatedwith appropriate sensitive antibiotics. Diagnostic proce‑dure for easy detection of EPEC infection is necessary inthe clinical evaluation of acute diarrhoeal diseases forprompt institution of appropriate treatment in order toreduce mortality.

Dysentery and Dysentery) on nalidixic acid which was

A‑ 5

BACTERIOLOGICAL INVESTIGATION ON THE DIARRHEALDISEASES IN LA PAZ AND SUCRE, BOLIVIA IN 1993 AND 1994
AKIYOSHI UTSUNOMIYAl'+, DANIEL ELto‑CALVO1, ARMANDO ALBERT BENITES REYEsl,
ERNESTO SANZETENEA CASTRol, ENRIQUE RODRicUEzl, CAROLlNA TORRES2,JENNY I. ZAMORA DE CORZ03, JUAN LUIS URiAl, ARNOLD HOFMAN‑BANG SOLETol,
AKEMI KA15 AND KAZUMICHI TAMURA6
Instituto de Gastroenterologia Boliviano‑Japon6s La Paz*, Sucre'. Cochabamba',
Bolivia; Department of Bacteriology, Institute of Tropical Medicine,

Nagasaki University, Japan'; Department of Microbiology, Tokyo Metropolitan
Laboratory of Public Health, Japan= and Department of Bacteriology,
National Institute of Health, Japan'

The aim of this study is to clarify the etiology andecology of bacterial diarrheal diseases in La Paz (alti‑

tude 3,800 m with a thin atmosphere, chilly, Iow humid‑

In the age group 5 years and under (0‑2 years plus
3‑5 years) , the isolation rate was 29.8%, and EPEC,
Shigella. ETEC were also predominant. While in the ageity) and in Surce (altitude 2,800 m) , to contribute to thegroup 17 years and older, the isolation rate was 30.2%,

health of inhabitants.

and the predominant enteropathogens were EPEC, V.
cholerae. Salmonella, ETEC and Shigella. In the age

Twelve hundred and thirty‑four diarrheal rectal
swabs were collected from outpatients and inpatientsduring the period between July 1993 and July 1994.

The numbers of isolated enteropathogens were as
follows: 139 isolated strains (11.3%) for enteropath‑ogenic Escherichia coli (EPEO , 80 (6.5%) for Shigella,

55 (4.4%) for enterotoxigenic E. coli (ETEO , 39
(3.2%) for Salmonella, 29 (2.4%) for Vibrio cholerae, 27(2.2%) for enteroinvasive E. coli (EIEO and I (0.1%)
Verocytotoxin‑producing or enterohemorrhagic E. coli
(VTEC or EHEO , respectively.

group between 6 and 16 years, the isolation rates wererelativery low in each pathogen. Among EPEC isolates,

044, 055, Olll were predominant, and 06, 0169, 0153

for ETEC, 028ac was most common for EIEC. Among
10 strains of E. coli 0157, only one strain produced
Verocytotoxin (VT 2). Regarding the serovar of Shigel‑la. S. flexneri 2a, 3a, and lb were prodominant. In thecase of Salmonella, S. Enteritidis was the most predomi‑
nant, followed by S. Typhi, S. Poona and S. Paratyphi B.
Out of 29 cholera cases, 25 strains belonged to serovar

45

Ogawa and remaining 4 were serovar Inaba.
The drug sensitivity tests were performed by discand minimal inhibitory concentrations (MICs) methods

with ABPC, or SXT, and rarely with GM, KM andusing aminobenzil‑penicilin (ABPO, sulfamethox‑
azole/trimetoprim (SXT) , trimetoprim (TM) , nalidixic

for the treatment of bacterial diarrheal diseases, andresistant strains will increase and persist in the nearacid (NA), gentamicin (GM), kanamycin (KM),

future. While, NA showed high and KM, GM showedtetracycline (TO , chloramphenicol (CP) , erythromycin(EM), cefazolin (CEZ), ciprofloxacin (CIP), amikacin

relatively high susceptible results in the sensitivity test.

(AMK) and amoxicilin (AMPO. The resistant EPEC,
ETEC, and Shigella strains against ABPC, SXT weremost prominent.
In Bolivia, diarrheal patients are treated mainly

others drugs. From these results, it is possible to saythat ABPC and SXT are ineffective and invalid drugs

Therefore, it is very important to consider changing theantibiotics for the treatment of diarrheal diseases. NA,

KM or GM are recomended for the available chemother‑apeutants.

A‑ 6

CONTROL OF CHILDREN'S BACTERIAL DIARRHOEAIN DEVELOPlNG COUNTRIESTAKASHI NAKANol, HITOSHI KAMIYA2, TAKESHI HONDA3, HIROKI HORI I ,MASAHIRO WATANABEI AND NOBUYUKI MATSUBAYASHI
Mie University School of Medicine*, Mie National Hospital'
and Research Institute for Microbial Disease, Osaka University'

Children's bacterial diarrhoea is one of the major
health problems especially in developing countries. We
conducted the survey of etiological agents and comparedwith clinical conditions in diarrhoeal children of African

region. Moreover, we tried to clarify the source andmode of bacterial enteric infection. The main purposeof our study is to establish the effective control mea‑sures for children's infectious diarrhoea in developing
countries.

significant, the incidence of Sh. dysenteriae was similarly

higher in the diarrhoeal group. Shigella species werealso isolated to some extent from non‑diarrhoeal chil‑dren. The isolation rates of the other bacterial enter‑
opathogen were not significantly higher in the diarr‑
hoeal group. In formulating effective and practicable
control measures, including antibiotic therapy, attention

should be paid not only to children with diarrhoea but
also the non‑diarrhoeal ones.

(Japan International Cooperation Agency) projects in
African region. They are Noguchi Memorial InstituteThe hospital‑based studies in Ghana (September‑Novernber, 1992), Zambia (May 1992‑May 1993) andKenya (1993‑) showed the most frequent isolates offor Medical Research (Ghana), Zambia University

EPEC followed b'y Shigella. Salmonella andTeaching Hospital (Zambia) and Kenya Medical

Campylobacter. The severity of diarrhoeal illness didn't
correlate with isolation rates of bacteria.

We collaborated with three institutes of JICA

Research Institute (Kenya). Our study was also suppor‑
ted by the Grant for International Health Cooperation

The bacteriological investigations for water source

Research from the ministry of health and welfare,and home‑kept water of diarrhoeal children were per‑Japan.

formed. The piped water and rain water were not

The community‑based study in Ghana (June 1987

Campylobacter. EPEC and ETEC. The isolation rate ofcontaminated, but the water from well and river wascontaminated with bacteria. Several kinds of bacteria
were isolated from home‑kept water. Health education
programmes such as 'handling of clean water' are also

Sh. flexneri was significantly higher in diarrhoeal chil‑needed for the prevention of diarrhoeal diseases.

‑May 1988) showed that Shigella species were the mostfrequently isolated bacteria agents, followed bydren. Although the difference was not statistically

46

A‑ 7

HUMAN ROTAVIRUSES ISOLATED IN CHAlNG MAI, THAILAND
IN 1987‑1993

TOMOKO URASAWA1, KOKI TANIGUCHll, NOBUMICHI KOBAYASHI , KEIJI MISEl,SHOZO URASAWA*, AYAKO HASEGAWA2 AND YUKIO YAMAZ13
Department of Hygiene and Epidemiology, School of Medicine, Sapporo Medical University*,
National Institute of Health, Japan' and Nippon Medical School'

Stool specimens were collected from pediatricpatients with diarrhoea who were admitted to threehospitals in Chaing Mai (93% specimens were from
McCormick hospital) during 6 consecutive years 1987
‑1993. Four hundred fifty‑seven stool specimens shownto contain group A rotaviruses by the demonstration ofviral antigen by ELISA and / or of viral RNA in PAGE

Although almost all strains examined showed theusual correlation between subgroup, G serotype andRNA electropherotype found in human rotaviruses (i.e.
subgroup I‑G serotype 2‑short RNA pattern, subgroup
II‑G serotype 1,3 or 4‑long RNA pattern, or subgroup
I+11‑G serotype 3‑long RNA pattern) , seven strains
having unusual characters were isolated. Strain Mc35

were subjected to this study. Ninety percent of thespecimens were from those less than 24 months of age.
Determination of subgroup and G (or VP7) serotype

had subgroup I‑G serotype 10‑long RNA electropher‑antigens of group A rotavirus was carried out by ELISA
with subgroup (1 and ID ‑ and G serotype(1‑4) ‑specific

9‑long RNA electropherotype, and in RNA‑RNA

monoclonal antibodies or PCR with pairs of serotype

human rotaviruses. Mc930 and 1129 strains had G‑specific oligonucleotide primers.

serotype 6 antigen that was usually found in bovine

Of 398 strains (87.1%) that could be subgrouped, 46.

8% were subgroup I, 37.0% were subgroup II, and 3.5%
were subgroup I+11. Of 374 strains (81.8%) serotyped,
169 (37.0%) were G serotype 1, 179 (39.2%) G serotype
2, 8 G serotype 3, and 11 G serotype 4. Our 6 years'

otype, a property hitherto found in bovine rotaviruses.
Strain Mc323, 345, 697 and 955 had subgroup I‑serotype

hybridization they were more related to porcine than torotaviruses.

These results suggest interspecies transmission andgenomic reassortment of animal rotaviruses in nature.
This study was supported in part by a Grant fromserotypes changed by year with dominant serotypes

Diarrheal Disease Control Program, WHO (Principal
investigator: Y. Yamazi) and was carried out withcollaboration from B. Pongprot, J. Supawadee and S.

appearing in the order of G serotypes I +2, G serotype 1

Suprasert, Chaing Mai University.

survey showed that the relative frequency of individualand G serotype 2.

A‑8
HEAT LABILE SPECIFIC ANTIGREN OF VIBRIO CHOLERAE 0139
MASAAKI IWANAGA, MASAO TANABE, NoBORU NAKASONE, SATOSHI NAKAMURA,YASUKO HONMA, KAZUTOSHI NAKASHIMA, TOMOKO OwAN, TSUYOSHI SUNABE
AND M. JOHN ALBERT*
Department of Bacteriology, Faculty of Medicine, University of the Ryukyus.
International Centre for Diarrheal Disease Research, Bangladesh*

It has been said that new cholera vibrio, Bengal, is some important differences in the antigenic substances
almost the same with traditional cholera vibrios except between new and traditional cholera vibrios, as far as
nonagglutinability of the Bengal strains with O1 antiser‑ the sugar chain of LPS is not the protective antigen.
um. However, there is a fact that post infection protec‑

We have established a monoclonal antibody

tive immunity of the traditional cholera is not cros‑sreactive to the new cholera. Therefore, there must be(MAb72) which clearly distinct Bengal strains from non‑0139 Vibrio cholerae. The antigenic substance for

47

MAb72wascharacterizedsoantigenislocatedsurfaceBengalstrains，2）the

at

cell

far

antigenisheating（100C，1min），3）thewashing

but

the

not

cells，and

the

as
as

umn

inactivatedbyantigenextractedby6）thereeasilyisisreactivereactive

after

the

treatment

antigendoes

notpassthroughextracted

of

agglutinogenantigenextractcut）ultrafiltration

follows；1）the

the

with

to

is

protein

MAb72

SDS，4）themembrane，5）inchromatography
elutedisafiltrationsephacrylbeforeblue

outcommonOmpS，7）the

gel

using

epitopeantigendextran（2，000kDa），

in

is

col・

S−1000，the

the

outer

resistanttomembranechrolofor−m．

PM100（100kDa

A−9
DISTRIBUTIONZONUl。AOCCLUDENS

National

andof

CHOLERAPAL1・2，PRAsANTA

KARASAwA1，TATsuYA

Department

ENCODING

yIBR10CHOLER」4E

KuRAzoNo1，AMIT

TADAHIRo

of

GENES

TOXIN，ACCESSORY
IN

HIsAO

OF

ResearchofCholera

and

AND

of

Institute，Intemational

thosebelongingtothenon−0139）serogroups

non−01and

were

and

O139andACE

non−0139（non−01：

regionwithbatteryed

examinedaof

YosHIFuMI

Medicalabsence

OIofpresence

of

the

HLY

environment

and

food

HLY

the

importancedisease
ity

of

these

factors

in

the

OI

clinicalcholera．Among

the

genes

of

the

strains

intactandalsostrains

of

OI

had

possessedwithnaturalHLY

deletionsthestrainsandpossessed

gene．Thereof

O139serogroup．Amongtheregion

genes

rare，

genetic

the

CT，ZOT

and／

and

OI

serogroup

examined，theelement

HLY

gene

to

the

of

core

dominant・

isolatesandnon−01：non−0139

O139strains

locatedappears

the

core

the

the

genes

tion

of

environmentalofγ．oho伽鷹．AII

gene．Althoughofclinicalisolates，major−

pathogenesis

comprisingtheunderstandcombination

comprising

the

the

to

genes

OI

the

and

detected．The

with

tion

strainsalsovirulence

the

study

wild

were

the

Japan3

but

this

among

of

among

DNA probes specific for cholera toxin（CT），zonula
occludens toxin（ZOT），accessory cholera toxin（ACE）
and eltor hemolysin（HLY）to determine the distribu−
genes

NAIR1・2，

TAKEDA1・3

University1，Center

genes

among

of

HEMOLYSIN

Diseases，Calcutta，lndia2

or

serogroups

ELTOR

ORIGINS

Medicine，Kyoto

Enteric

Alargecollectionof1，154strainsof四6万06hol6鰯召including

TOXIN，AND

DIVERSE

diverseorig三ns

TOXIN，

K．BAG1・2，G．BALAKRIsH

MIHARAI

Microbiology，Facultylnstitute

OF

CHOLERA

in

the

be

a

examinedcore

naturalof玩6ho伽π8as

regioninandattenua・

compared

to

all

the

virulence−associated

gene

was

the

most

conservedofbiotypes

in玩oho伽88independent

serogroups．

A−10
STRUCTURE（STaR）FORA

AND

FUNCTlONE8chεrJεhOF

RECOMBINANT

αcoJ広HEAT−STABI。E

PIG

RECEPTORENTEROTOXIN（STa）lll：

COMMON MOTIFS IN EXTRACELLULAR DOMAIN OF THE MEM［BRANE−ASSOCIATED GUANYI。YI。CYCI。ASE RECEPTOR FAMILY INCLUDING
HEAT−STABLE ENTEROTOXIN RECEPTOR（STaR）
AKIHIRODepartment

Membrane−associated
ptors

are

a

familyofofguanylyl

proteins

WADA

AND

Bacteriology，Institutecyclase（GC）rece・

which

regulate

intracel−

1ular cyclic GMP content in animal tissues．Several
kinds of GC receptor have been reported：NPR−A（GCofTosHIYAHIRAYAMA

TropicalMedicine，Nagasaki

University一A），NPR−B（GC−B），NPR−C，STaR（GC−C），retGC，andor SPGC）．The structural features shar・

sapGC（APGC
ed

by

these

novel

hormone−bindingGC

receptors

includeandomain，asingletransmembrane

extracellular

48

domain, and an intracellular signaling domain com‑prised of a kinase homology domain and a GC catalytic
domain. The sequences of the extracellular domains are

weakly homologous (17‑43 % amino acid identity),
whereas the intracellular domains are 36‑78 % identical.

found including a GP motif (CX20‑25GPXCXY and
CX20‑25GPXXXCXY) and a DXXGD motif. To define
the fundtional role of the DXXGD motif, each aminoacid in the DNCGD sequence of pig STaR containing an
Ha‑epitope (Ha‑STaR) was mutated to an Ala residue.

We compared the amino acid sequences of 13 GC

Three Ha‑STaR mutants which were substituted atreceptors. For the homology search an algorithyn for
multiple sequence alignment was used. Six amino acidresidues are highly conserved in the extracellulardomain of all 13 membrane‑associated guanylyl cyclase

conserved Asp347, Gly350, or Asp351 residue in the
DXXGD motif did not retain STa‑binding and GC‑activ‑

receptors [C72, G98, P99, Clol, D347, and D351 in pig heatcritical for conformational stability and/or activation of‑stable enterotoxin receptor (STaR)]. By considering

the GC catalytic domain.

ities. Accordingly, the conserved amino acids of Asp,
Gly, and Asp in the DXXGD motif of GC receptors arecommon amino acids present, unique sequences were

A‑11

EPIDEMIOLOGY OF HEPATITIS B VIRUS INFECTION IN ZAMBIA, AFRICA‑THE IMPORTANCE OF HORIZONTAL TRANSMISSION
IN CHILDHOOD AND RISK FACTORS‑HITOSHI OSHITANll, HRIOSHI SUZUKll, FRANCIS C. KASOL02, KATSUMI MIZUTA2MWILA MpABALWAN12, NAKANDU P. LU02 AND YOSHIO NUMAZAKll
Virus Research Center, Sendai National Hospital, Sendai*

and Virology Laboratory, Universi,ty Teaching Hospital, Lusaka, Zambia'

It is known that s,ub‑Saharan Africa is a highendemic area of hepatitis B virus (HBV) infection.
However precise mechanism of HBV transmission is‑rPHA, HBCAb‑PHA and HBSAb‑ELISA.

still to be defined, and control of HBV by vaccination
has not been introduced in most of countries in Africa.

[Results] I . An average of 6.5 % of pregnant women(137/2,098) was positive for HBSAg, and HBeAg waspresent in 16.1 % (22/137) of HBsAg positive sera.
2 . Prevalence of HBsAg and antibody were signifi‑

An epidemiological study of HBV was carried out incantly higher in rural areas than in Lusaka urban area.

Zambia as a part of Infectious Diseases Protect, a tech‑3 . Both HBsAg and antibody prevalence increased byage in children of urban community.
4 . History of injection and blood transfusion were

nical cooperation of Japanese Government, supported by
Japan International Cooperation Agency (JICA) .

[Material and Methods] I . Normal pregnant women:a total 2,098 pregnant women attending to three (3)urban health centers in Lusaka and four (4) district

significant risk factors for HBV infection in children of

hospitals in other provinces.

medium to high endemic area for HBV.

2 . Children in urban community: a total 598 children

2 . Horizontal transmission in childhood might be rriore

aged 6 months to 15 years in urban community of
Lusaka who were recruited by house to house holdsurvey.

important transmission route than vertical transmission.
3 . Medical practices such as injection and blood trans‑
fusion were significantly correlated with prevalence ofurban community.

[Discussion] I . It was shown that Zambia is a

3 . Samples: serum was collected from pregnant

HBV markers.

women, and capillary blood was collected on filter paper
from children.

control measure of HBV including prevention of risk

4 . Laboratory Methods: HBsAg‑rPHA, HBeAg

f actors.

4 . There is a urgent need to establish cost effective

49

A−12
SEROEPIDEMIOLOGICAL STUDY ON HIV，HBV AND HCVlNFECTlONS IN CHIANG MAI，NORTHERN THAILAND
KAN

TORIYAMA1，AMNATYoUsuKH2AND

of

Pathology，InstituteofTropicalofof

Medicine，Chiang

Department
alld

We

performedaDepartment

Pathology，Faculty

seroepidemiological

study

on

the

HIDEYoITAKURAl

Medicine，Nagasaki

Teknika

Mai

Universityl

University，Thailand2

B．V．）．The

HepatitisB

surface

Antigen（HBs

Mai，Thailand．The

sera

from

−Ag）was tested by Reverse Hemagglutination（ThaiRed Cross）．HBs−Ag were detected in200ut of276HIV
seropositive blood donors（7．2％）and680ut of844HIV
seronegative blood donors（8．1％），respectively． HCV

1，120blood

donors（male：952，female：168）were

testedfor

antibodies

anti−HIV

antibody

generationanti−HIV

EIA）．The

prevalenceofhepatitis

B（HBV）and

hepatitis

C

virus

（HCV）infections among the human immunodeficiency
virus（HIV） antibody seropositive and seronegative
blood

using

donorsill

indirect

Particle

Chiangbyusing

EIA（Abbott3rd

positive

sera

were

confirmed

by

ELISA（Anti−HIV，1／2Behring）andGel

Aggulutination（GPA−ant卜HIV，

Kyowa

Hakko）．The anti−HCV antibody was assayed in alI
sera by2nd generation EIA kit（Hepatostika C，Organon

wereseropositive
antibody

These

blood

seronegative

results

population

tion

detectedinof276HIV

antibody

donors（6．2％）and60ut

of844HIVbloodsuggestbelongs

tonorthem

in170ut

donors（0．7％），respectively．

thatanti−HIV

high

risk

a

antibody

group

of

positive

HCV

infec−

Thailand．

A−13
SEROEPIDEMIOLOGY
lN

OF
THE

HEPATITISDOMINICAN

VIRUSES

A，B，C

REPUBLIC

A．SHlcHIJol，H．AoNo，H．SHIBAYAMA，H．TERAo，A．YIYATA，
H．OzAwA，M．ITou，J．MlsuRl，K．MIFuNE AND T．IToGA
DepartmentofMicrobiology，Oita

First，408sera（GroupI）selected

at

the

sera

Department

Clinical

toryofcollected

theDominican

atCenterRepublic

the

of

of

randomGastroenterology，Aybar

were

examined

for

the

from

Labora・antibody，and

hepatitis

secondly，2，000healthy

C

sera

positive

rate

was

less

age

groupsSoutheast

Asia

of

anti−HAV

hashygienic

andsanitary

toobserve

tions

the

future．（2）The

among

in

Group

I

the

Africa．

improvementofoverall

was4．7％．The

rate

the

many

prevalence

was17．

difference

wasobservedinclassificationby

intermediate

antigenpositive

WHO，theabove

prevalencewaspositive

rate

regionhigher

rate

was

in

this

type（HBs

than

that

in

examinedbyusing the2nd generation recombinant ELISA kit．The
overall prevalence of anti−HCV was4．7％，indicating
the

rate

in

Americas

HBsAg

those

females（16．1％）．Accord・

rate，2−7％）and

antibody

thatsignificantlyIanti−HBs

was

those

are

in

Group

of

the

Japan．（3）HCV

Group

higherprevalencecountry

thecondi・
oftosignificant

index

studieshygienic

wasin

Becausebeconditions，futhernecessary

overallamong

females（2．0％）like

tロg

of

of

（P＜0．05）．The

in

males（20．0％）and

those

to

HBsAg

significantly

that

that

also

no

prevalence

shown

of

was

7％but

countries

andprevalence

rate

betweenII）

resultbeen

females（1．5％）（P〈virus（HCV）

years．This

tooverall

was3．2％．The

in（Groupthan70％in

identical

0．05）．The

males（4．8％）than

to10years and reached97％in a age groupold and100％in age groups of more than20
almostmales（6．3％）than

prevalen6eof6monthsof15years

is

in

II

collected at various6regions of the country were
examined for HBV antibody and HBsAg． （1）HAV
antibody

higherUniversity1

Hospital，ofhepatitisAvirus（HAV）antibody，hepatitisBvirus（HBV）antigen（HBsAg）and

Medical（1．0％）．This

betweenthemarkedlyandhigher−HCV

wasgroupspositive

found
of

when

compared

Europe（0．2−1．4％）and
rate

did

males（4．9％）and

in

age

is

a

the

only

in

up

to40，but

small

not

differ

in

with
Japan

significantly

females（4．5％）．Anti
numbershowedofabruptindividuals
increasesin50

age groups of above 50 : 10, 20, and 22% at age groups

The present study is a joint one with A. Hidalgo, M.

of 51‑55, 56‑60 and above 61, respectively. In the present

B. Castro, L.V. Sora, A. German and E.N. Maria

study, however, the reason why higher positive rates(Centef of Gastroenterology, Aybar

were observed in higer age groups could not be elucidat‑

Dominican Republic)

Hospital,ed.

A ‑1 5

AN EFFECTIVE COMBINATION THERAPY WITH ORAL
FLUCONAZOLE (FLCZ) AND FLUCYTOSlNE (5‑FO AGAINSTCRYPTOCOCCAL MENINGITIS (CM) IN AIDS PATIENTSIN UGANDAKAZUNORI OISHI SATOSHI MITARAI HIROSHI YAMASHITAl, KIWAO WATANABEl,
TSUYOSHI NAGATAKEl, ROY MUGERWA2 AND KEIZO MATSUMOTO1
Dept of Int Med, Inst of Trop Med, Nagasaki Univ and Dept of Med, Makerere Univ, Uganda2

We evaluated a clinical efficacy of anti‑fungal

chemotherapy with FLCZ and 5‑FC for CM in AIDS
patients in Uganda. This study was designed to com‑pare the clinical efficacy of FLCZ and 5‑FC for first 2

weeks with that of FLCZ alone. In this study, we
employed a dose of 200 mg for FLCZ, because theclinical efficacy of FLCZ at 200mg was comparable tothat of FLCZ at a 400mg in the previous study. 57
patients were enrolled for this study. These patients
receiving each regimen showed the similar mean age (34y.o.), male sex (35‑48%), and incidence of oral can‑didiasis (60‑79%). No significant difference was shownin the CD4 Ievels (73‑81/mm3) or CD4/8 ratios (0.41)
before treatment between two groups.

early death within 2 weeks and significantly increased
the survival rate (61%; P <0.05). No serious ddverse
events of these antifungal drugs was obaserved in theboth group. We also compared CD4 Ievels of patients ineach treatment group between before and after primary
therapy. We found a similar decrease of CD4 Ievels inboth treatment group, but a statistical significance wasdemonstrated only in the group receiving the combina‑
tion therapy. CD4 Ievels in peripheral blood appears to
decline with the progression of HIV infection despite aneffective anti‑fungal chemotherapy.
In this paper, we demonstrated an efficacy of combi‑
nation therapy with FLCZ at a dose of 200mg and 5‑FC

We could evaluate survival rate of 46 patients at thefor AIDS patients with CM in Uganda. This combina‑
tion therapy may prevent the early death of theseend of primary therapy for 2 months. Although thepatients, and also provide a higher survival rate at thegroup receiving FLCZ alone showed a survival rate ofend of treatment and prophylaxis for 6 months.

35%, the group receiving FLCZ+5‑FC prevented theA ‑1 6BACTERIAL MENlNGITIS AMONG BANGLADESHI CHILDREN
ATTENDlNG IN A CITY HOSPITALKAMRUDDlN AHMED*, YOSHIAKI UTSUNOMIYA1, SAMIR K. SAHA2, KIWAO WATANAGE ,RUHUL AMlN2, GLENDA CAROL MARTINEZ FRANColTSUYOSHI NAGATAKEI AND KEIZO MATSUMOTol
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japanand Dhaka Shishu Hospital, Sher*e‑Bangla Nagar, Dhaka, Bangladesh'

Bacterial meningitis causes much morbidity andmortality among the children of Bangladesh. The real

incidence and prevalence of meningitis is still unknown.

Since 1993 we started to study bacterial meningitis

51

among children attending Dhaka Shishu Hospital,

isolated. Among 7 cases same species of bacteria were

Dhaka, Bangladesh, a hospital only for children. Until
now 103 cases with suspected bacterial meningitis has
been studied. The male and female cases were 60 and 43
respectively. The age ranged from 6 days to 12 years
old children. In this study cerebrospinal fluid (CSF) ,blood and swab from the nasopharynx were cultured at
the same time. There was bacterial growth in 61 speci‑
mens of CSF. The isolated bacteria were H. influenzaein 34 cases, S. pneumoniae in 13 cases, N. meningitis in
8 cases, Gram negative bacilli in 3 cases, E. coli in 1
case, V. metschnikovii in I case and F. meningosepticum

also isolated from the blood. Only in one case H.
influenzae was isolated from nasopharynx, CSF andin I case. In all cases of meningitis single pathogen was

blood culture. All strains of H. influenzae were typable.

85.4% were of type b and 14.6% were non‑b‑type of H.
influenzae. We suggest the introduction of vaccine
against H. influenzae might decrease the incidence ofmeningitis among the children of Bangladesh. The highrisk group in meningitis cases were (i) patients withlower socioeconomic status (ii) meningitis by H. in‑fluenzae and (iii) patients treated with penicillin plus

chloramphenicol. Further studies are progressing to
identify the serotypes of S. pneumoniae.

A‑17

ACTIVITY AND THERAPEUTIC EFFICACY OF ANTIBIOTICSAGAINST BURKHOLDERIA PSEUDOMALLEI AND ITS
PNEUMONIA IN MICEHIROSHI FUKUHARAl, FUTOSHI HIGAl, ATSUSHI SAITOl, NoBUTIKA KUSAN02,

PARASIT THRAVICHITKUL3 AND CHARLIE PHORNPHUTKUL3First Department of Internal Medicine, University of Ryukyus*, Central clinical Labotatory,

University of the Ryukyus Hospital2 and Chiang Mai University, Thailand'

Melioidosis is an important infectious disease witha high morbidity and mortality rate in tropical areas.

Levofloxacin(10mglkg) was significantly more
effective than ceftazidime (100mglkg) , imipenem/cilas‑

Recently, melioidosis has been carried elsewhere byworkers or travellers from an endemic area.
We confirmed Burkholderia pseudomallei (formerly
Pseudomonas pseudomallei) to be the causative path‑tatin(50mglkg) , piperacillin(100mglkg) and minocy‑cline (lOmglkg) against B. pseudomallei pneumonia inmice. Imipenem/cilastatin and ceftazidime were almost

ogen of melioidosis in vitro and in vivo. 13 strains of B.

dosage for imipenem/cilastatin was half that of ceft‑azidime. Acridine orange stained preparations of lungsof surviving mice not administered any antimicrobial

pseudomallei isolated from Thailand showed resistanceto a variety of drugs such as ampicillin, cefazolin,
cefotiam, gentamycin, erthromycin, and clindamycin.
Imipenem showed the highest antibacterial activity as

equally effective against the infection but the required

agent were observed for intracellular viability of multi‑

plied bacteria through fluorescence microscopy 72 hr

measured by its MIC, but a I x MBC(4xMIO dose ofafter infection.

imipenem did not inhibit intracellular growth of macro‑phages. In contrast a I x MBC(2 x MIO of levofloxacin
inhibited intracellular growth of macrophages despite of

Levofloxacin may have a useful clinical role intreating melioidosis caused by B. pseudomallei to be
facultative intracellular growth of organism in humanlesser MIC than imipenem.

such as the genera Salmonella. Shigella and Listeria.

52

A ‑1 8

VIROLOGICAL STUDY OF DENGUE EPIDEMIC
IN VIENTIANE, LAO P.D.R. IN 1994

THONGCHANH SISOUKl, KAZUMI KANEMURA2, MIKA SAITOl,2,4, BOUNLAY PHOMMASACK3,YOSHIHIRO MAKlN02, SAKAE ARAKAK12, TOSHIHIKO FUKUNAGA2 AND SITHAT INSISIENGMAYl
National Institute of Hygiene and Epidemiology, Lao P.D.R.*,

Department of Virology, Faculty of Medicine, University of the Ryukyus',

Vientiane Municipality Health Service, Lao P.D.R.= and JICA Lao PHC ProJect

In 1994, dengue (DEN) epidemic occurred after a

Sixteen dengue virus strains were isolated from 15

silent interval of 7 years in Vientiane Municipality. By
17th of October, a total number of 1590 dengue fever andserum specimens. The positive rate of virus isolation
was 41.8% on the second day and 21.1% on the third dayof illness. Double infection with DEN‑1 and DEN‑2 wasfound in one case with type‑specific monoclonal anti‑

dengue hemorrhagic fever (DF/DHF) cases were re‑ported. The outbreak was first reported in May fromBan Hom commune, in a rural district, about 15 km
southeast from the center of Vientiane. On Ist of June,a survey was conducted at this commune. . A total of 107
serum specimens were collected from the children under
16 years‑old who had fever within 5 days were tested forbodies and further confirmed by RT‑PCR. All serum
specimens were tested for DEN‑specific lgM by lgM‑
capture ELISA. The lgM antibody could be detected on
the second day and the positive rate increased thereafterand reached 60% on the fifth day of illness.

virus isolation using cultured mosquito cells (C6/36).

A ‑1 9DF/DHF EPIDEMICS AND CONTROL IN VIENTIANE
MUNICIPALITY, LAO P.D.R. IN 1994

MIKA SAITOl,5,6, BOUNLAY PHOMMASACK2, KHAMPE PHONGSAVATH3, BOUNLEUA PHILAVONG',
DUANGDAO SUKHALUNG", TRAYKHOUANE PHOUTAVONG5, SITHAT INSISIENGMAY5, S. SALAMANY7,YOSHIHIRO MAKlNol, TOSHIHIKO FUKUNAGAI 'AND KIYOSHI SUZUK16
Department of Virology, Faculty of Medicine, University of the Ryukyus , Vientiane Municipality
Health Service2, Sethathirath Hospital", Mahosot Hospital', National Institute of Hygiene andEpidemiology*, JICA Lao PHC project' and Ministry of Public Health Lao P.D.R.'

Dengue fever and dengue hemorrhagic fever (DF/where the ACU had not conducted its activity. The

DHF) is one of the most important public health prob‑

morbidity rates of the rural districts were higher thanthose of the urban districts. No epidemic was reported

lems in Lao P.D.R. In Vientiane Municipality, thecapital of Lao P.D.R., Aedes Control Unit (ACU) has
been organized and conducted dengue control activitiesin 4 urban districts which include larval survey, healtheducation and insecticide spray, to reduce the larvae

density. In the beginning of 1994, Dengue Control
Network was set up to strengthen surveillance system,
laboratory capability and health education, and to makean effective response to the situation. In 1994, epidemicof DF/DHF occurred in Vientiane Municipality after aninterval of 7 years, and a total of 1590 cases with 5
deaths were reported by 17, October, 1994. The out‑
break occurred in 4 urban districts and 2 rural districts

in another 2 adjacent rural districts. At Ban Homcommune (population is 968), which belongs the rural
district, 17 and 23 cases were reported in May and June,

respectively. The larvae density was high (Breteau
Index was 188) at the end of May, 1994. On receiving
information of dengue outbreak through Dengue Control
Network, health education, Iarval survey, Iarviciding
and mass fogging of insecticide were carried out imme‑
diately. In August, the Breteau Index and the reported
cases in this commune decreased to 7.9 and O, respective‑

ly. From these observation, it is recommended that thedengue control should be more strengthened in

53

Vientiane Municipality. (We are grateful to Dr. Senk‑

ham Boudala for his assistance)

A ‑20

EVALUATION OF FILTER PAPER METHOD FOR THE COLLECTION
AND STORAGE OF BLOOD AND SERUM SPECIMENS IN THE ASSAY
OF ANTI‑DENGUE IGM‑ELISAAKIRA IGARASH11, KOUICHI MORITAl, SUNTHAREE ROJANASUPHOT2, SUDARAT RUANGTRAKIT2,
SUMLEE DUANCHANDA2, AREERAT SRIJUGGRAVALOVONG2, SOMYOS KANUNGKIT3, PICHAI THONGTRADOL3
AND PISIT NIMNAKORN3
Department of Virology. Institute of Tropical Medicine, Nagasaki University ,Virus Research Institute, National Institute of Health, Thailand'and Nakorn Phanom Provincial Hospital, Thailand‑

Dengue virus infections have been prevalent in themens, in parallel after storage period of 1, 3, 4, and 5

tropics worldwide, and is counted among the leading
cause of hospitalization and death among children insoutheast Asia. Laboratory diagnosis on dengue has
most frequently been carried out by serology, usingclassical hemagglutination‑inhibition (HI) tests.
Recently, IgM‑ELISA was introduced into dengue ser‑months. The results on (1) aliquoted and frozen speci‑

ology to improve diagnostic efficiency. Because of theconvenience, most of the specimens for dengue serology
have most been collected, stored and shipped as filter
paper dried blood. Although the suitability of filter
paper method was assessed for the HI test, it has notbeen critically evaluated for the lgM‑ELISA.
During June to July 1993, 135 hospitalized dengue

patients were bled for their acute and convalescentmens showed high reproducibility, both in terms of
diagnostic results and ELISA titer. The lgM‑ELISA on
filteraper dried specimens, on the other hand, showeddecreasing numbers of primary dengue cases according
to the storage period, while the number of secondary
dengue cases did not change so much. The lgM‑ELISA
titer of filter paper dried specimen also showed adecreasing trend as the storage period was prolonged.

The decrease was more pronounced when serum wasdried rather than the whole blood, resulting in thedecreased titer for secondary cases also.

The results showed that when the filter paper
method was used for sample collection, storage andspecimens. A part of̲ the specimens was directly driedon filter paper (Advantec No. 1) for storage at room

shipment, whole blood instead of serum should be usedtemperature for 1, 3, 4, and 5 months. Serum wasmonthseparated from each of the remaining specimens and abe false negative results for primary dengue cases.
Differential stability of anti‑dengue lgM ELISA anti‑
body titers in primary and secondary dengue cases in‑dicated some qualitative difference between the lgM
antibodies produced in the primary and secondary den‑portion of it was dried on the filter paper and stored asabove. The remaining serum specimens were aliquoted
and stored at ‑20'C.
lgM‑capture ELISA was carried out according to
the AFRIMS method on (1) aliquoted serum, (2) filter
paper dried blood and (3) filter paper dried serum speci‑

and the lgM‑ELISA should be carried out within one
om the sample collection. Otherwise there will

gue infections.

54

A‑21

A NEW DNA ALLELE OF HLA‑B IN ORANG ASLI POPULATIONIN MAINLAND OF MALAYSIA
MOHD. ZAIDI ABU SAMAHl, KENJI HIRAYAMA2, NASURUDDlN HJ. ABDULLAHl,
ONG KIAN JOOl, LOKMAN HAKIM3, MAK JOON WAH3, KATUSI TOKUNAGA4,
LlN LING4, HIROSI TANAKA3, SOMEI KOJIMA5, MIHOKO KIKUCH12 AND EITARO HOR12
Institute of Medical Research, Malaysia*, Department of Medical Zoology,

Saitama Medical School', JICA‑IMRTropical Disease Project3, Japan Red
Cross Blood Center', Medical Research Institute, University of Toky0

To analyze the genetic factors influencing the sus‑
ceptibility or resistance to Malaria in orang asli popula‑

Serologically, it was typed for B62 with Bw4.

tion, we have typed for their HLA‑B using DNA typing(sequence‑specific oligonucleotide probes: SSO method)sequence that makes pockets fitting to antigenic pe‑

and conventional serology. I14 blood samples from
Bentong Village and 58 frorn Gombak hospital werebeen reported to be resistant type against malaria in
Gambia, p9 pocket of B77 was completely similar to thatobtained to analyze.

of B53. Therefore, it was suggested that the same

Using SSOP method here revealed amino acidptides. When compared with B53 sequence that has

We found one specific type that has a unique

infectious agent functioned as an important evolutionalsequence of Exon 2 of HLA‑B containing at least one
recombination between Bw75.1 and Bw4. The antigenpressure on HLA genes. Determining the whole
sequence of this type is under investigation.

frequency of this type was estimated to be around 40%.

A ‑22

STRUCTURAL ANALYSIS OF POPULATION PROBLEM IN VIETNAM
SHlNYA MATSUDA
Department of Preventive Medicine and Community Health,
University of Occupational and Environmental Health, Japan

Structure of population problem in Vietnam wasanalyzed based on a fertility model developed by theCommittee on Population and Demography. In thisinvestigation, I employed the correlation analysis andthe regression analysis in order to clarify factors as‑sociated with fertility, based on the following data: 1989

census data, 1989 statistical yearbook, results of questi‑

onnaire distributed in the communities. According to

It is predicted that the fertility level in Vietnam will

decrease steadily because of the change of the reproduc‑tive behavior accompanied with a rapid economic devel‑

opment and the urbanization. However, this process
will result in a number of social pathology, i.e., street
children, abandoned aged, increase of the crime, etc. In

order to realize a sound economic development, they
have to implement some integrated program, especiallythe results, the most important factors associated withthe fertility level in Vietnam are the literacy rate ofa special consideration is necessary for the education

women in reproductive age, the proportion married

more, the development of a social supporting system foramong the female in reproductive age, and the infant
and early childhood mortality rate of the region. In

the elderly must be prepared from now in order to cope

addition to these factors, the availability of health facil‑

ity also showed a statistically significant correlation

decrease of the fertility. It is expected to implement
some international cooperative programs between Viet‑with the fertility level.

nam and Japan.

and the employment of women and minority. Further‑with a coming aging society resulted in the rapid

55

A‑23

MECHANISM OF EOSINOPHIL ACCUMULATION IN CEREBROSPINALFLUID OF ANGIOSTRONG YL US CANTONENSIS INFECTED MICEKENTARO YOSHIMURA, HIROKO SUGAYA AND KAZUTO ISHIDA
Department of Parasitology, Akita University School of Medicine

nude mice fail to induce CSF eosinophilia (Sugaya &significant CSF eosinophilia. Moreover, A. cantonensis
infected nude mice were iv transferred with eosinophils
which had been labeled in vitro with a Zynaxis' PHK‑26
fluorescent staining kit. This experiment showed that
almost all of eosinophils accumulated in the recipient

Yoshimura, 1988). In an attempt to determine the

CSF were labeled ones, suggesting that eosinophils

In Angiostrongylus cantonensis infection, cere‑
brospinal fluid (CSF) eosinophilia is provoked in
nonpermissive hosts (mice, guinea pigs and humans) but
not in permissive rat host, and furthermore, infectedmechanisrn (s) of CSF eosinophilia in nonpermissive

accumulated in CSF were transferred eosinophils them‑hosts, therefore, A. cantonensis infected nude mice (AFselves, but not eosinophils newly produced by the recipi‑‑nu/nu) were iv transferred with CSF eosinophils har‑vested from donor AF‑nu/ + mice previously infectedwith the same parasite, and then examined for CSF

ent mice.

eosinophilia. This study revealed that CSF eosinophilia

could be provoked in nude mice infected with A.
cantonensis for 17‑20 days but not in uninfected normal

nude mice, both of which were transferred with CSF
eosinophils (eosinophil purity = 90%) harvested from

An additional experiment was conducted to deter‑mine whether eosinophil chemotactic factor(s) (ECF)
derived from young adult worms (YA) are involved ineosinophil accumulation in the mouse CSF; two kinds ofmonoclonal antibodies against ECF‑YA (Ishida & Yo‑shimura, 1992) were mixed and then, a total of 8 mg of
the mixed antibody, dividing into four injections, wasinfected donor nu/ + mice, suggesting that the presenceof A. cantonensis worms in the brain was essential forthe induction of CSF eosinophilia. In order to determine

given to recipient nude mice before and after eosinophil

whether the source of eosinophils to be transferred

important finding in the current study was that A.

affect the results of eosinophil accumulation in recipientcantonensis infected nude mice iv transferred with CSF
eosinophils yielded significantly lower worm recovery
than those transferred with medium alone, suggestingthat newly transferred eosinophils directly migrated to

CSF, intraperitoneal eosinophils (eosinophil purity =77%) from donor nu/+ mice previously infected with
Mesocestoides corti were iv transferred to nude mice
infected with A. cantonensis for 17 days. This study
demonstrated that the nude mice transferred even withtransfer. This antibody treatment, however, failed toblock CSF eosinophilia in the recipient mice. The mostCSF and played an important role in killing intracranial

worms.

the intraperitoneal eosinophils were capable of inducing

A ‑24

CLINICAL STUDY ON HTLV‑1 INFECTION
AND STRONGYLOIDIASISOSAMU ZAHAl, NAGISA KlNJOl, TAKESHI UEHARA , AKIHIKO KOUCHll
JUNICHI OsfflROl, KOUSHlN SIKIYA1, FUKUNORl KlNJO1'2
AND ATSUSI SAITOU1'2
Ist Department of Internal Medicine* and Research Center of Comprehensive Medicine',

Faculty of Medicine, University of The Ryukyus

Strongyloides stercoralis infection and anti‑HTLV There were 2 cases with hyperinfection syndrome,‑1 antibody have been examined in patients in hospital‑ one disseminated strongyloidiasis with meningitis andized since July 1988 to August 1994. 115 asymptomatic cases. Strongyloides stercoralis

56

infections were 12.3% in male and 5.9% in female.
Anti HTLV‑1 positive rate were 17.9% in male and23.9% in female. Strongyloides stercoralis infections
were 22.5% in male and 11.1% in female who were caseswith HTLV‑1 infection, which were statistically higher
than cases without HTLV‑1 infection.

Two hundred ninty‑five patients with malignant
neoplastic leision, respiratory or digestive organs were

investigated in this study, which revealed the patientswith Strongyloides stercoralis infection has been highly

complicated by malignancy than patients without
Strong yloides stercoralis infection.

A ‑25

EPIDEMIOLOGY AND CONTROL OF MALARIA IN YUNNAN
PROVlNCE, PEOPLE'S REPUBLIC OF CHlNA
CHE LI‑GANG, DIRECTORThe Research Institute of Malaria Prevention

and Treatment of Yunnan Province

Several disasterous malaria epidemics in the Yun‑nan Province were recorded in the past. The railway
construction scheme started in 1901 caused a epidemic

1102 in 1954, while, Qnly 6 death was reported in 1965.
This success was interrupted by a political disorder, andprevailed in the region with 200,000 recorded deaths. In

overcoming the trouble, in 1993, API of total Province
was lowered to 4.01. However, dead cases in the sarne
year were 15. The present control efforts are concen‑
trated along the borderline zone where the chloroquine

1919, another malaria epidemic occurred in Simao
where only 1000 people out of 70,000 total population
could escape from the infection. In the epidemics occur‑
red in 1933, 30,000 casualities were resulted. A system‑

atic malaria control programme was set out in 1956 andhad been continued successfully. In 1954, annual para‑site incidence(APD was 234(/10,000); the rate de‑creased sharply to 11.08 in 1965. Recorded death was

in 1973, API peaked at 56.18 with 271 deaths. After

resistant falciparum malaria makes serious problem.
Also, population movement causes a difficulty in thecontrol operation. Research activity so far, studies on

the risks of global warming on malaria epidemics arebeing worked with Japanese experts.

A‑26

MALARIA EPIDEMIOLOGY IN LOMBOK ISLAND, INDONESIA;APPLICATION OF SERO‑EPIDEMIOLOGICAL METHODSYOES P. DACHLANl, SOEDARTO1, SRI HIDAJATI B.S.1, BARIAH IDEHANl, KUSMARTISNAWATll,
ANI SAFRIAHl, MACHFUDzl, SUBAGYO YOTOPRANOTO1, SRI SUBEKTll, EDDY SOEWANDOJO1,PARWATI S.B.1 ET AL., MARTON02, BAMBANG WIRATMAD12, KUNTOR02, AGUS SUTANT03,
UAENAL ARIFlN3 ET AL., SUBAKTlNlNSIH4, TATONG HARIYANT04, K. TANABE5, I. MIYAG15,

S. UGA5, F. KAWAMOT05 AND H. KANBARA5
Airlangga University, Surabaya*, Department of Health, Province of East Jawa',

Department of Health, Province of Nusa Tenggara Barat", University of Braijaya' and Adviser5

Based on the preliminary epidemiological survey of

peptides were introduced and evaluated for their appli‑malaria in the Lombok island, Indonesia in 1991, weselected three subvillages and conducted the regularcability. As reported previously antibodies to CS

malaria survey at intervals of two months from August,to. determine the epidemic season. Two subvillages

1992 to June, 1993.
In the survey the sero
‑epidemiological methods using CS and RESA repeat

showed the similar epidemic pattern, namely two peaksof seropositive rate on August and April which wererepeats were short‑lived, and consequently were useful

57

supposed to be caused by Anopheles spicies breeding inbrackish water along the seaside such as lagoons, rivers
and fish ponds. On the other hand the other subvillage
showed two separate epidemic patterns, one was similar
to that in the former two subvillages but the other was

distinct from it. This distinct epidemic was found on ahilly area 3‑4 km apart from the sea and showed two
lower peaks of seropositive rate on October and June.
Causative Anopheles species remain to be determined.

A‑27

A CASE OF ACCIDENTAL TRANSMISSION OF PLASMODIUMFALCIPARUM THROUGH PLATELET TRANSFUSIONSHIGEYUKI KANO AND MAMORU SUZUKI
Department of Parasitology, Gunma University School of Medicine

The patient was a Japanese female, 70 years of age,25, 1:256 on May I and May 2. These parasitological andresident of Funabashi, Chiba, and had never been
abroad. The woman suffered from thrombocytopenia

serological results confirmed that she accidentallyand received a total of 60 units of platelet concentratesion.

during the period, 4‑13 April, 1991. On April 24, she
manifested a fever of 39"C which was successfully treat‑
ed with Voltaren SuppOTM, a non‑steroidal anti‑inflam‑

The possibility of transmission of malaria throughplatelet transfusion has been discussed by Fajardo andTallent (1974) , who reported the existence of the para‑matory drug. Her body temperature remained normalsite in a platelet of a P. v. infected patient by electron

until May 1, when she suddenly developed symptoms of
cerebral malaria, Iiver and renal dysfunctions. In the

microscopy. Nevertheless, platelet packs may containsome red blood cells and parasites may be present inearly morning of May 2, she lost consciousness and went

erythrocytes, platelets or both. The questionnaire which

into cardiac arrest. Giemsa stained thin blood smears
obtained for blood cytological examination which wereis used in obtaining the medical history of donors rarely

kept at the laboratory of the hospital, Iater revealed thatreceived transmission of P.f through platelet transfu‑

includes inquiries on a past history of malaria. Special
attention should be made for the safety of blood transfu‑

O% on April 2, O% on April 17, 0.06% on April 25, 5.5%

sion, taking into consideration of the danger of malariatransmission. This report is the first case of induced
malaria resulting from platelet transfusion, and the 75thon May I and 1.9% just before her death on May 2.

case of transfused malaria in Japan since 1935.

she had contracted Plasmodium falciparum malaria.
The parasitemia from examination of the slides were

Antibody titers against P.f antigen were < 1:4 on April

A‑28

LIMITACION OF CLINICAL DIAGNOSIS OF MALARIAIN PHC PROGRAMHIROSHI OHMAE , YURI J. UCHIDA', YUTAKA MIZUSHIMA', HIROMI KATO',TOSHIAKI TANAKA+, ARBlNO BOBOGARE=, SAI KIRIMAOMA AND AKIRA ISHn'
Bureau of International Cooperation, International Medical Center of Japan*,
Department of Medical Zoology, Jichi Medical Scho012
Department of Internal Medicine, Toyama Medical and Pharmaceutical University',
Department of Internal Medicine, Nara Medical College",
Medical Reserach and Training Institute, Honiara, Solomon Islands5
and Department of Parasitology, National Institute of Health'

Malaria is one of the most sufferable infections in

the world. About 550 million people are at risk f rom

58

malaria and over one million people are estimated to diemonths. After the repeticious treatrnent based on thefrom malaria every year. To prevent death fromresults of clinical diagnosis in the PHC activity, sensitiv‑

malaria, early clinical diagnosis and prompt treatment

ity of clinical diagnosis was reduced in 2 villages. Afterare recommended, and incorporated in the primary
health care (PHO systems. However, prompt treat‑

12 months, the sensitivity of clinical diagnosis was
reduced from 36.7% to 18.9% in the A village and was
reduced from 37.5% to 15.4% in the B village. However,the PRS did not change and kept holoendemic levels.

ments based on the results of early clinical diagnosis are

not always enough to reduce the endemicity of malaria.
We investigated the accuracy of clinical diagnosis
of malaria in 2 malaria endemic villages (A and B) in
northeastern Guadalcanal in the Solornon Islands. In
January 1993, the parasite rate(PR) was 73.1% and thesensitivity ,of clinical diagnosis was 36.7% in the Avillage. In the B village, the PR was 58.5% and sensitiv‑
ity of clinical diagnosis was 37.5%. The residents ineach village had been followed microscopically every 6

Only by clinical diagnosis, many patients of malaria

could not be diagnosed as malaria. Sensitivity of clini‑
cal diagnosis of malaria was reduced in PHC activity inholoendemic area. Many aymptomatic malaria parasite
carriers, especially asymptomatic gametocute carriers
could not be treated by the dignosis based on febrileclinical syptoms.

A ‑29

DNA DIAGNOSIS OF MALARIA IN THE SOCIALISTREPUBLIC OF VIETNAM

HIROFUMI MIYAKE1, FUMIHIKO KAWAMOT02, MASATUGU KIMURA3,OSAMU KANEK04 AND YUSUKE WATAYA
Faculty of Pharmaceutical Sciences, Okayama University*,

Department of Medical Zoology, Nagoya University School of Medicine',
Department of Biophysics, Osaka City University School of Medicine'and Department of Medical Zoology, Osaka City University School of Medicine*

We have developed a colorimetric assay "microtiterplate‑hybridization" (MPH) to detect amplified nucleic
acids of malaria parasites on a microplate well. Thismethod has high sensitivity and specifity more than
microscopic examinatjon. And this assay system allows
us to detect and identify the four species of humanmalaria parasites. A pair of oligonucleotide primers
were designed for the amplification of the conserved
region of the gene coding for the 18S small subunit
Species‑specific oligonucleotide
ribosomal RNA.

crotiter plate‑hybridization and microscopic examina‑tion using the blood samples. The microscopic examina‑tion was prepared two set of thin blood smear. One set
was stained with acridine orange (AO) and the other set

was stained with Giemsa. They were examined by the
local microscopists. The 110 blood samples among the
154 samples were compared with MPH and AO, and 53

samples were compared with MPH and Giemsa. The
difference between the results of MPH and AO is about

18%, and MPH and Giemsa is about 19%. Among 15

probes were also designed and immobilized on mi‑

samples judged as P.f/P.v. mixed infection by MPH,

crotiter wells. Target sequence of malaria parasites incarried out the epiderniological trial in the Socialist
Republic of Vietnam, from July to August, 1994. Blood
samples (10 pl each) were obtained from 154 donors by

only 4 samples was judged as P.f/P.v. mixed infection
by microscopy. The case of mixed infection, judgementof species in microscopic examination was very diffi‑
cult. Therefore, this MPH diagnostic method is indicat‑
ed with effective tool on epiderniological research. On
AO, we think that because of insufficient time to checksmear, the mistakes happened.
It was well known that the malaria parasite wasalmost P.f and P.v., scarcely P.m.. in Vietnam. But we
had found that 2 samples from within 29 samples in Phu

finger puncture. We have detected by two ways, mi‑Rieng was P.m. Therefore, we think that P.m. may

human blood was amplified by the polymerase chain
reaction (PCR) and the PCR‑amplified product wascaptured by the species‑specific probe on the microplate
well. The biotin‑streptavidin system was used to detect

the captured materials. Positive samples gave yellow

color by the chromogenic reaction. We have been

59

exist high rate depend on place.

be a new malarial species which has different target

We found a sample that was positive by micros‑
copy, but negative by MPH. We think the sample might

region of 18SrRNA gene from other human malaria
species.

A‑30

ANALYSIS OF MITOCHONDRIAL DNA FROM PLASMODIUM VIVAXKIYOSHI KITAl, AKATSUKI KOKAZEl, NATSUKO UEl, SOMEI KOJIMAl,
PATRICIA LIM2 AND JOHN MAK2
Department of Parasitology, The Institute of Medical Science, The University of Tokyo*
and Biotechnology Centre, Institute for Medical Research, Malaysia'

Over the past decade, more evidence has accumulat‑
ed to suggest an active role of the intraerythrocytic
Plasmodium mitochondrion. In addition, recent effort todefine the mitochondrial DNA of malaria parasites haverevealed unique feature of the genome. It is a circularly

permuted tandem repeat of 6 kb that encodes fragment‑

ed both large and small subunit ribosomal RNAs.
Mitochondrial genomes of Plasmodium have received
much interest as they encode some components of therespiratory chain, including cytochrome c oxidasesubunit I and 111 (COI and 11D and cytochrome b (CYb)
as well as rRNAS, and the respiratory chain has beensuggested to be a target for antimalarial compounds.
However, sequence data for the 6 kb element of humanmalaria is available only from Plasmodium falciparum,
although those from murine and avian parasites havebeen reported. Due to the limited amount of starting
material, isolation of pure mitochondrial DNA and its
sequence analysis are difficult in the case of otherhurnan malaria, such as P. vivax. P. ovale and P. malar‑iae. In this study, primers specific for P. falciparum

COIII gene were used in polymerase chain reaction(PCR) to amplify a part of P. vivax COIII gene.

Val‑14 to Ser‑138) , were synthesized. Total DNAS of P.

vivax were prepared from two patient, one Malaysianand one Malaysian student from India. PCR productswith expected size were cloned into TA vector andsequenced. Alignment of the nucleotides of P. vivax
COIII with published sequence of P. falciparum COIII
showed nucleotide homology of 88%, while amino acidsequence comparison gave a homology of 91%. It isinteresting to note that sequence data of COIII for both
Malaysian and Indian isolates of P. vivax were identical,

although relatively higher frequency of mutation in the

mitochondrial DNA has been suggested. In contrast to
the great similarity between two Plasmodium species,

similarity between P. vivax COIII and human COIII(28%) was much lower than that between yeast COIII
and human COIII (40%) . Deletions were observed in thetransmembrane helix 11 and two hydrophilic segments
for both P. vivax and P. falciparum. These deletions arefound only in the Plasmodium enzymes, suggesting thattertiary structure of COIII and interaction between thesubunits in Plasmodium cytochrome c oxidase may bedifferent from those of other organisms. The unique

Sequence data of the partial P. vivax COIII gene is thenfeature of the respiratory component found in this study
may make this enzyme as a target for specific diagnosis

compared with those of P. falciparum and other organ‑

and antimalarial compounds. This study was performed

isms.

as part of the Institute for Medical Research and JapanP. falciparum specific primers, which amplify 373
nucleotides coding the partial peptide of COIII (fromInternational Cooperation Agency Research Project on
Tropical Diseases.

60

A‑31

USEFULNESS OF THE PCR METHOD WITH MICROTITER PLATE‑HYBRIDIZATION IN THE CLlNICAL PRACTICE OF IMPORTED MALARIAMIKIO KIMURA1 HIROFUMI MIYAKE2 AND YUSUKE WATAYA2
Institute of Medical Science, University of Tokyo*

and Department of Pharmaceutical Sciences, Okayama University'

The novel and convenient PCR assay with mi‑site densities. So far, all of 24 cases of falciparum

crotiter plate‑hybridization has been developed mainly

malaria and 25 of 26 cases of vivax malaria wereby Wataya and Yamane. This method targets the 18S

diagnosed species‑specifically by this PCR assay. Also
all of 7 cases of ovale malaria and 2 cases of malariaerRNA genes of malaria parasites that are specific foreach species following amplification with the biotinylat‑

malaria were PCR‑positive. By determining the blooded primer set common to all four parasite species. Ten
pl of blood was first hemolyzed with saponin and thesamples taken in convalescent stages of some patients,
the method was revealed to detect as few as 10 para‑sites/pl blood. In addition, there are 4 smear‑negative,
PCR‑positive results in patients who had already taken
anti‑malarial medicine, suggesting these cases being in

DNA was dissolved with proteinase K treatment.
Biotinylated PCR product is hybridized with specificprobes that are coated onto microtiter plate wells andsubsequently visualized after reaction with alkaline
phosphatase‑conjugated streptavidin and the substrate

taken from non‑malarial patients showed negative PCR

p‑nitrophenyl phosphate. This PCR method wasresults for each parasite species.

already applied to field studies in the Solomon Islands

Thus, this method was shown to be extremely usefulfor clinical practice of malaria, e.g., diagnosis andspecies identification in low parasitemia, detection of
mixed parasite infection, evaluation of anti‑malarial
treatment and also for the retrospective diagnosis ofand Vietnam mainly by Wataya and others, and wasfound to be useful for the species‑specific identificationof malaria parasites especially in low parasitemia.

In this study this method was applied to imported

the convalescent stage of malaria. Thirty samples

malaria from various regions of the world. In somepatients, serial blood samples were assayed and themalaria after having taken anti‑malarial medicine.

results were compared with clinical findings and para‑valuable tool for field studies.

These data also provide evidence that the method is a

A‑32

THE PHAGOCYTOSIS OF PLASMODIUM FALCIPARUM
AND TNF PRODUCTION BY HUMAN MACROPHAGES
TAKAYOSHI NAGAO, HARUKI UEMURA, TETSUO YANAGI AND HIROJI KANBARA
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University

It's known well that Plasmodium falciparum (P.f.)
induce TNF production of host's peripheral monocytes

and tissue macrophages. Now it is regarded as themechanism of TNF induction that the soluble substancereleased from malarial infected RBC at the period of
rupture stimulate the monocytes in the circulation
directly. But the phagocytosis of malarial parasites byreticuloendothelial macrophages in spleen and liver arelarge amount, so these stimulation are also speculated

as important factor inducing TNF from tissue macro‑phages. So at this time we carried out the incubation ofhuman peripheral monocytes with RBC infected with
cultured P.f. and measured TNF induced into the super‑
natant of coculture and observed phagocytosis of para‑sites. And the infected RBC was separated into thesoluble and insoluble fraction and TNF inducing abilityof each fraction was compared. Next we derived tissue

macrophages from human peripheral monocytes andmeasured the phagocytosis of parasites and the amountof TNF secretion and compared them with that of fresh
isolated monocytes.
Methods: Cultured P.f. was synchronized to ring61

form with 5% sorbitol and grew it to mature troph‑ozoites and schizonts by going on the culture after
synchronization. The monocytes isolated from humanperipheral blood were stimulated with each infected
RBC of different life stage obtained as above process,and we assayed TNF production and phagocytosis.
Next the infected RBC was lysed by freeze‑and‑thaw
method and soluble fraction was separated from insol‑uble fraction by high speed centrifugation and mem‑brane filter. The human monocytes were stimulatedwith each fraction and TNF production was compared.
And next human peripheral monocytes were incubatedtrophozoites and schizonts with new ring form caused
monocytes to produce high value of TNF and high rateof phagocytosis of malarial pigments. From the resultof experiment which monocytes were stirnulated withsoluble and insoluble fraction separated from infected
RBC, it became clear that both fraction induced TNF
and insoluble fraction, especially malarial pigments,

were phagocytosed very easily by the monocytes.
However, both soluble and insoluble fraction induced
very little amount of TNF production from activatedmacrophages derived from peripheral monocytes,
phagocytosis of insoluble fraction (malarial pigments)for 6 days then they differentiated into tissue macro‑phages. And similarly they were stimulated with infect‑
ed RBC, and soluble, insoluble fraction from infected

was accelerated conversely.

RBC and compared with fresh monocytes isolated frombesides soluble substance reported at past. And theacceleration of phagocytosis and decline of TNF pro‑

peripheral blood about TNF production andConclusion: The factor which induce TNF produc‑tion of human monocytes exists in the insoluble fraction

phagocytosis.

duction of derived macrophages suggested that

Results: When human peripheral blood monocytes
were stimulated with infected RBC, the stage of latephagocytosis of parasites is not a direct stimulation to

induce TNF production.

A‑33

HUMAN BLOOD LYMPHOCYTE RESPONSES TO
PLASMODIUM FALCIPARUM INFECTION*RZEPCZYK. C.M. *ANDERSON K, *STAMATIOUS TOWNSEND E +ALLOWORTH A+MCCORMACK J, +WHITBY M.
*Queensland Institute of Medical Research and *Dept of Medicine,
University of Queensland. Brisbane, Australia

Using flow cytometry, the response of humanperipheral blood lymphocytes (PBL) to Plasmodium

there was a well defined population of y(falciparum infection was studied in patients who present‑

CD45RO. This proportional representation of y6 T cells

ed at hospitals in Brisbane with acute P. falciparum
infection. Some patients were followed longitudinallyfor up to six months after infection. We were particu‑
larly interested in the y6 T cell response to malaria

subsets appeared comparable to that seen in malaria

infection, although aP T cells, and natural killer (NK)control subjects where only a proportion of y(cells were also chartered.

showed HLA‑DR expression. The high levels of HLA‑DR expression on y( T cells were found to be

Peripheral blood lymphocyte profiles of malaria
patients were variable. While y6 T cells were elevatedin some patients, they were not in others. These differ‑ences did not correlate readily with the patients' previ‑

ous malaria history. The mean percentage of y( T cells
in the blood of 17 patients during the acute stage of
infection was 6.1 :!: 4.7 SD (range 1‑17), whereas thevalues obtained for 17 malaria naive control subjects
was 3.8 2.7 SD (range 1‑9). In patients the largest

fraction of y(

T cells were CD4‑ and CD8‑, although

T cells expres‑sing CD8. The majority of y( T cells expressed

naive control subjects. During the acute stage of infec‑

tion, the majority of y6 T cells expressed HLA‑ DR
antigens. This in marked contrast to the finding in
T cells

maintained in some patients for at least two months
after treatment. In some patients NK cells were ele‑
vated above the normal range with a maximum percent‑age of 35% being observed in one patient. There wasalso activation of afi T cells during infection. The
relative contribution of the different sub‑populations tomalarial immunity and/or pathology still needs to be
determined.

62

A ‑34

DRUG SENSITIVITY OF PLASMODIUM VIVAXHIROYUKI AMANO , SAORI HIGASH12, TOSHIMASA NISHIYAMA2,SHIGEAKI ISHIZAKA2 AND TSUNEJI ARAK12
Department of Overseas Medical Services, Tenri Hospital*and Department of Parasitology, Nara Medical University2

Drug‑resistant strains of Plasmodium falciparum,
are well known around the world, although chloroquine
remains drug of choice for Plasmodium vivax treatment
even now. However, some problems have arisen recent‑ly, such as a chloroquine‑resistant strain, and low sensi‑

tivity of sulfadoxine/pyrimethamine (SP) and prima‑quine in treating P. vivax.

This report presents our experience with the low
sensitivity of P. vivax to SP and the drug sensitivity of
P. vivax in literture is discussed.

A 21‑year‑old Japanese male student developed afever (42'O for 6 days after coming back from thewas 0.294%. Treatment with chloroquine (total 1,500mg
base for 72 hours) was started on the second hospitalday, followed radical therapy with primaquine (30mgx
14ds) , and parasitemia completely disappeared. Clear‑ance of fever and parasitemia required 36 hours and 72
hours, respectively. There were no side effects, andneither relapse nor recrudescence has occurred sincethen.

Our previous study showed that the cure rate ofsulfamonomethoxine/pyrimethamine against P. vivax
in 'laria is 100% and SP showed the same rate. Gener‑
ally, although SP is effective, it is considered less effec‑

Thailand‑Myanmar border in April, 1993. Although

tive than chloroquine against P. vivax and according totreatment with SP in one hospital eliminated his malar‑ial symptoms, fever recurred twice within 14 days after

failed in 40% ,of cases. Additionally, this and a previous

treatment and he visited our hospital 2 days after thesecond recrudescence. On admission, the patient had
clear consciousness and no abnormal signs except he‑patomegaly (2 cm). Important laboratory findings
were; hemoglobin 11.0g/dl, platelet 28.3 x 104lpl, CRP 6.

9mg/dl, FDP<10pg/ml, creatinine 0.9mg/dl, glucose
118mg/dl, total cholesterol 125mg/dl ahd LDH 654lU.
Abdominal ultrasonicated echogrophy showed mild he‑patosplenomegaly. Thin blood smear revealed troph‑ozoites and gametocytes of P. vivax and parasitemia

a Thai report, SP 2 tablet‑therapy against P. vivax
case of P. vivax malaria that we treated with SP 3tabsx
5days revealed that there seems to be a low SP‑sensitive

strain of P. vivax. Although our case was cured bychloroquine, cases of chloroquine‑resistant P. vivax
malaria have been reported in Papua New Guinea andIndonesia since 1989. Low primaquine‑sensitive strainsof P. vivax have been known in Southeast Asia andLatin America since the 1950's. Therefore, it is neces‑sary to be more prudent in the treatment of P. vivax
malaria.

A‑35

AGE‑SPECIFIC MANIFESTATION OF DRUG RESISTANCEOF PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX
IN MALARIOUS ISLANDERS IN EASTERN MELANESIAAKIRA KANEKol, GEORGE K. TALE02 AND HARUO KAMIYAl
Department of Parasitology, Hirosaki University School of Medicine*and Malaria Section, Department of Health, Vanuatuf
Vanuatu is located at the southeast perimeter of themalarious band extending from Southeast Asia to east‑

site rate (PR) 13.5%: 679 Plasmodium falciparum (Pf),847 P. vivax (Pv) , 15 P. malariae (Pm), and 18 mixed

ern Melanesia. From 1988 to 1991 we conducted mass

infections. 165 of Pf cases had gametocytes. We

blood surveys in 33 areas of 11 islands in Vanuatu andexamined a total of 11,590 islanders with overall para‑

analysed age‑specific PRs. PR sharply increased from O

to 1‑2 years for both Pf and Pv. For Pv, PR was the

63highest (15.9%) in subjects aged 3‑4 years, and thenalso significantly higher than that in the latter (21.2%) :dramatically decreased by age. For Pf, PR stayed in the

p<0.01. In vivo response of Pf to Fansidar (pyrimeth‑range of 5.6‑8.6% between age I and 19 years. As aamine 75mg + sulfadoxine 1,500mg on Day O as adultdose: PYR/SDX) was observed in 172 cases with no R3,
5 R2 and 4 R1. During the follow‑up to these Pf cases,
Pv parasitaemia appeared in 24 cases after PYR/SDX

result, a reversal of the Pf/Pv ratio was found at 10‑11

years. Pf gametocytes were detected in all age groups
but its PR was higher in subjects aged 1‑14 years than inthose more than 15 years.
For selected positive cases of the above surveys, we
carried out drug resistance tests using the modified

WHO methods. In vitro response of Pf to chloroquine
was observed in 55 cases with 9 resistance (R) and 17
borderline cases. That to mefloquine was observed in 25
cases with no R and that to quinine in 36 with one R. In
vivo response of Pf to chloroquine (600mg base on Day
O, I & 2 as adult dose: CHL) was observed in 153 caseswith 2 R3, 5 R2 and 36 R1. The appearance rate of R2/

administration, but in only I case after CHL. The
appearance rate of Pv after PYR/SDX was the highest(33.3%) in subjects aged 0‑4 years and 0.0% in thosemore than 15 years. In vivo response of Pv to CHL wasobserved in 102 cases with only one R2. That to PYR/
SDX was observed in 58 cases with 2 R3, 6 R2 and 3 R1
without significant differences between age‑groups.
For chemotherapy of residents in malaria‑endemicareas, it is important to consider not only the drug

R3 in subjects aged 0‑4 years (15.2%) was significantly
higher than that in more than 5 years (1.7%): p<0.01.

resistance in the parasites but also the disease preva‑
lence by age in the hosts. It seems that the formation of
premunitinon by age suppresses the clinical manifesta‑

The appearance rate of Rl in the former (51.9%) wastion of drug resistance.

A‑36

IMMUNOSUPRESSION AND MALARIAL PARASITE
MIDORIKAWA YUTAKAI AND QUAZI MANJURUL HAQUE2
Suzuka University Medical Sciences & Technology*
and Departrnent Public Health, School of Medicine, Mie University'

Relation between Deoxspergualin (DSG) an im‑
munosupressive agent and malarial parasite was stud‑

ied. Several chemicals incruding DSG, which are

infection and DSG treatment were counted and mea‑
sured. The number of leucocyte was increased bymalarial infection and decresed by DSG treatment.

seemed to be the chandidate of malareal treatment,

Same as the result of leucocyte number, erythrocyte,were tested both in vitro and in vivo. Inhibitive effect ofhemogulobin concentration and hematocrit % weredecreased by DSG treatment. These fact shows thatDSG on malarial parasite in vitro was not good effect asin vivo, comparing other chemicals. So the mechanismof inhibition of DSG on malarial parasite was studied invivo with using mice infected by Plasmodium berghei.
Hematological results were collected in this study. The

number of leucocyte and erythrocyte, amount ofhemogulobin concentration and hematocrit % during

DSG inhibit cell segmentation of blood cells in mouseand especially, when immature erythrocyte production
was inhibited by DSG, malarial parasite was also inhib‑
ited the infection because malarial parasite has thetendency of infecting the immature erythrocytes rather
than the matured cells.

64

A‑37

EPIDEMIOLOGICAL SURVEY OF Cl :AGAS DISEASE IN

MACHARETY VILLEGE, BOQUERON PREF. PARAGUAYAKIHIKO UcHIDAl, J. MARTlNEZ2, YASUSHI KAWAKAMll AND MASAO SAN02
Dept. Environmental Biology, Azabu Univ.* and IICS, Asuncion Univ.2

A total of 101 (2‑78 years old) inhabitans of Ma‑
charety in Boqueron Prefecture, Paraguay, 47 men and

54 women, were examined immunologically and par‑asitologically along the field survey conducted. Resultsof immunological (ELISA) test revealed that sera from15 men and 18 women showed positive results at adilution of 1:80, while 10 of them replied that they werediagnosed as Chagas disease before. Although 11 per‑only five of them were ELISA positive, the remaining

three were ELISA negative. ECG abnormalities were
found in just 9.0% and were rather lower when compar‑
ed with other studies. The abnormalities found were as
follows: Sinusal bradichardy with left‑deviation (lessthan 55 beats per minute) , Sinusal bradichardy, Leftdeviation, Complete blocking of right branch with for‑ward‑left hemiblocking.

sons (5 men and 6 women) replied that they had

As for triatomine in houses, 84 (93.3 %) of 90 people

Romana sign, but only 4 of them showed positive resultsby ELISA test. A unilateral bipalpebral edema of the 7
persons was likely due to by other diseases.
Serological studies were performed among 101 peo‑ple, from which 55 were subjected to ECG studies. Eightof them had been found to have ECG abnormalities, but

replayed "yes" and 72 (88.0 %) said that they had been
stuck by the bugs in bed during the night. In every housesurveyed, 2‑26 (on average 11.6) bugs were found andindentified as Triatoma infestans the major vector of T.
cruzi.

A‑38

SERODIAGNOSIS OF CONGENITAL CHAGAS' DISEASE
IN ACUTE PHASESACHIO MIURA1, TUNEARI SEKIGUCHll, TOSHIAKI KAMIYA2,KAORU MATAYOS13 AND TUTOMU TAKEUCH11
Department of Tropical Medicine and Parasitology, School of Medicine, Keio University ,Departrnent of infectious disease, Christian University Bolivia'

and Hospital Colonia Okinawa N' I, Internal Medicine'

Chagas' disease, coursed by the parasitic protozoan
Trypanosoma cruzi ( T. cruzi), is a sever problem for
public health, that's affects several million of people in

25 sera of newborn were collected from umblicalcord at birth and trypanosoma was detected by Strout
method and the mothers corresponding with them wereCentral and South America. The prevalence of ser‑showed positive for lgG by ELISA. In this time we

opositivity for Chagas' disease has declined in parallelwith vector control activities in this area. Generallytransmission route are three: (1) Vectorial, (2) Transfu‑
sion, (3) Vertical transmission. Today's great problem isstudied for newborn cases by immunofluorescence anti‑
body tests (IFA) , ELISA and Immunoblotting. Then 24
out of 25 congenitally newborn had detected lgG anti‑
bodies to T. cruzi but for lgM positive were showed incongenital infection of T, cruzi coursed by verticaltransmission in productive age group. Recent report ofthe congenital Chagas' disease has been 2‑5% in urban

only 6 cases. Also, immunoblotting exhibited 13 positiveand 23‑81% in endemic area in Bolivia. Congenital
Chagas' disease of newborn have been poor aspects fordiagnosis and need more studies for standardization ofserodiagnosis in acute phase of them.

and 12 negative out of 25 cases. Only one case showed
all results of serodiagnosis were negative althoughparasitemia had been positive. Comparing these resultswith chronic Chagas' disease studied in Recife (BraziD
showed different pattern, that is immunological exami‑
nations of chronic Chagas' disease in Recife had good

65

agreement with various serodiagnosis.
In conclusion from these studies, we suggest that
IFA is more stable methods than others. A method forearly detection of infected newborns is essential fortreatment.
Further studies are contemplating the problem inthese aspects.

A‑39

T‑LYMPHOPROLIFERATIVE, RESPONSE OF THE PATIENTSWITH CHAGAS' DISEASE TO EPIMASTIGOTE
ANTIGEN OF T. CRUZI IN GUATEMALAKENJI HIRAYAMA*, EDMUNDO VELASQUEZ2, MARTIN GIL'. TAMARA OBlsp03,
JULIO ARGUETA+. DIANA FREIRE5, MARGARITA PAZ'. TETSUO YANAGI'. KAZIJKI OGATA+,EITARO HORI AND ISAO TADA7
Department of Medical Zoology, Saitama Medical School , San Carlos University',
Roosevelt hospital', JICA‑Guatemala research project of tropical diseases',Institute of atomic energy*, Nagasaki University Institute of Tropical Medicine"and Kyushu University Faculty of Medicine'

Proliferative response of peripheral mononuclear
cells of the patients with Chagas' disease against
epimastigote antigen of T. cruzi was investigated using
10 outpatients of Roosevelt hospital with heart disease

the major antigens of epimastigote of T. cruzi by T celllevel and as well as B cell level, we prepared partiallypurified fractions (32 fractions) after passing throughion‑exchange chromatography (DEAE‑memsep,

from endemic area in Guatemala and using 20 individ‑

Milipore, USA) by using sodium chloride gradient (0‑0.

uals who had been diagnosed as trypanosomiasis byserology in Santa Maria lxhuatan where JICA project

pattern of responsiveness to each fractions except frac‑

had been going on since 1990.

There observed a good correlation between serology

4N). Two seropositive individuals showed differenttion 29 that stimulated both. The interesting finding
was that when we examine their antibody reactivity to(Indirect Hemagglutination Test; IHA, Seroim‑

those fractions, both did not show any reactivity against

munodiagnostic, Co, USA) and T Iymphoproliferative

fraction 29. Thus it was suggested that fraction 29

response to epimastigote antigen except the fact thateven seronegative individuals sometimes responded tothe antigen moderately. Those sero negative responders
were significantly responded compared with Japanese
non‑exposed population. For further identification ofpreferentially stimulated T cells rather than B cells in

those patients. Further study should be needed using
more patients with different clinical forms of Chagas'
disease.

A ‑40

STUDIES ON THE DETECTION OF DENGUEVIRAL GENOME BY RT‑PCR
KEN‑ICHIRO YAMADA, YUKI ESHITAl, MITSUHIRO NUMATA,

SADAO YABE AND TADAHIKO KITANO
National Institute of Health,

Kurume University. School of Medicine‑PCR was performed as previously described (Morita,
The present study was carried out to detect D1, D2,
D3 and D4 viral genome from the cultured fluid of et al., J. Clin. Microbiol. 29:2107‑2110, 1991). C6/36 cell
infected mosquito cells, patients' sera and the prepara‑line was grown at 28 C in MEM supplemented with 10 %tion of mosquito extract by rapid RT‑PCR method. RT fetal bovine serum and 0.2 mM of non‑essential amino

66

acids and TRA‑284‑SF cell line was grown in a serum‑free medium that consisted of an equal volumes offrom the preparation which were prepared the mixtureof an equal volume of virus solution and uninfectedLeibovitz (L‑15) and tryptose phosphate broth. All fourmosquitb extract. The inhibitor effect could be removed

serotypes of dengue viral genomes could be detectedfrom the cultured fluid of infected two mosquito celllines. It was detected that the cell‑associated viralgenome was 24 hr and the extra‑cellular viral genome

by heating (100 C, 5 min.) or treatment of 30 ug/ml
trypsin solution. Obvious DNA band was observed on
the preparations which were treated with heating (100C, 5 min.) in case of the infected mosquito samples.
These results suggest that the inhibitors containing
mosquito extract were some heat‑labile proteins. All

was 30 hr after infection in TRA‑284‑SF cells, respec‑tively. D1 viral genome was detected from the sera ofpatients 3 days after the onset of disease. Although theamplified DNA band was detected from the virus solu‑
tions, the amplified DNA product could not be detectedfour serotypes of dengue viruses could be plaque assayedwith TRA‑284‑SF cell cultures using the overlay mediaof MEM contained I % methylcellulose.

A ‑4 1SEQUENCES OF E/NSI GENE JUNCTION FROM FOURDENGUE‑2 VIRUSES OF NORTHEASTERN THAILAND AND THEIREVOLUTIONARY RELATIONSHIPS WITH OTHER DENGUE‑2 VIRUSESKYAW ZlN THANT, KOUICHI MORITA AND AKIRA IGARASHI
Department of Virology, Institute of Tropical Medicine, Nagasaki University

We determined the 240 nucleotide sequences of the
E/NSI gene junction of four dengue‑2 uiruses by theprimer extension dideoxy chain termination method,
These viruses were isolated from the dengue patientswith different clinical severities in Nakhon Phanom,caused silent mutations. All 56 isolates studied wereNortheastern Thailand in 1993. The results wererelated to prototype New Guinea C strain.
We also observed the variation in nucleotide andcompared with the 52 published dengue‑2 sequences ofthe same gene region. Sequence divergence of four new

isolates varied from 4.17% to 5.42% compared withdengue‑2 prototype New Guinea C strain whereas it
varied from 5.42% to 6.67%, and 6.67% to 7.09% whenclassified into five genotypic groups by constructing thedendrogram. The results indicated that four new iso‑lates from Northeastern Thailand belong to genotype IIof dengue virus serotype 2, and were most closelyamino acid sequences among clusters of isolates(Thailand‑1980, Malaysia‑1989 and Thailand‑1993)
which were obtained from the dengue patients withstrain, respectively. Most of the nucleotide substitutions

different clinical severities. Significance of thesegenetic differences have been discussed to draw thepossible correlation between genetic variability andwere found at the 3rd position of the codons which only

virulence factor.

compared with Jamaica 1409 strain and PR159/S1

A ‑42

CLASSIFICATION OF LEISHMANIA PARASITES,
USlNG KDNA FINGER PRINTlNG METHOD BY

ARBITRARILY PRIMER POLYMERASE CHAlN REACTION
TATSUYUKI MIMOR11, JIICHIROU SASAKll, MOTOMI NAKATA2 AND HIDEYUKI SAYAl
Departrnent of Oncology, Kumamoto University School of Medicine, 2‑2‑1 Honjo, Kumamoto 860, Japanand Biomedical laboratory, Sumitomo Electric Industries2
Leishmaniases are widely distributed in Central andSouth America and Europe, Africa and Asia except

South‑East Asia, where they present a considerable
public health problem.
The identification of mor‑

67

phologically similar parasites responsible for different

DNA (kDNA) net‑works were collected by centrifuga‑

clinical features of the disease is very important. Identi‑

tion (16,000 rpm for 90 min at 4 'O , extracted by phenol

fication techniques, isoenzyme electrophoresis, mono‑clonal antibodies, DNA hybridization, molecular kar‑chloroform and precipitated by ethanol. These kDNA
were sued for templates of AP‑PCR. The quontity (10‑500 ng) of templates were checked using two Leish‑
mania kDNAS. The six primers were tested. Amplifica‑

yotypes, restriction fragment length polymorphism,
polymerase chain reaction have been performed in diag‑procedure and/or difficulty for judgment. Recently,

tion was carried out in a thermal cycler (Perkin‑Elmer)
for initial 5‑10 cycles of low stringency (1 min at 94 'C,
l min at 37 or 42 'C, I min at 72 'O and 25‑30 cycles ofhigh stringency (1 min at 94 'C, I min at 60 'C, I min at

nosis and epidemiological survey of leishmaniasis.
These techniques, however, have some defects like as anecessity of a large quality of materials, complicated

PCR finger printing method by arbitraily primed

72 'O. At the end of the PCR cycles all tubes werepolymerase chain raection (AP‑PCR) was developed on

incubated for 7 min at 72 'C to allow the amplification

the analysis human DNA polymorphisms. Moreover,process to go to completion. The PCR product wassome preliminary studied of classification by thisanalyzed by 6 % polyacrylamide gel electrophoresis andmethod were reported on bacteria and parasite. In thisstudy, we have performed the identification of Leish‑

silver staining.

mania parasites in South America using AP‑PCR.
We used in this study WHO reference and standerd
Leishmaia strains and Leishmania isolates from wild
mammals and humans in Ecuador, South America werealso examined for utility value of this method. These
isolates have been checked previously, using isoenzyme
electrophoresis and monoclonal antibodies. Leishmania

The kDNAS fragment bands were shown more clear
on polyacrylamide gels under the following AP‑PCR
amplification condition; 10 cycles of low stringency (1min at 94 'C, I min at 37 "C, I min 72 'O and 30 cyclesof high stringency (1 min at 94 'C, I min at 60 'C, I min

at 72 'O . All experiments were performed on thisamplyfy condition. Six primers were tested using differ‑promastigotes were cultured in Schneider's medium,ent specices, and primer‑ LS3 was more useful one for
identification between Leishmania complex. From these

harvested parasites were resuspended in SE buffer (0.15

results, the AP‑PCR may be valuable approach for

M NaCl, 0.1 M EDTA, pH 8.0) Iysed with sarkosyl anddigested with proteinase k at 60 'C. The kinetoplast

identifying and distinguishing Leishmania parasites.

B‑ 1

A SURVEY OF HOOKWORM AND STRONGYLOIDES INFECTION
FOR ADULT FARMERS LIVING SURROUNDlNG SURABAYA
CITY, AND FOR ADULT FACTORY WORKERS IN SURABAYA
CITY, INDONESIAKAORI KOGA‑KITAl,2, DANIEL HUSEA3, SRI SUBEKT13, IG.N. GDE RANUH3SHIRO KASUYA2 AND YUZO TAKAHASH12
Department of Public Health, Faculty of Medicine, Kagoshima University*,
Department of Parasitology, Gifu University School of Medicine2
and Tropical Disease Research Center (TDRO , Airlangga University*

Recently, the agar plate method has been shown tobe successful in detecting Strongyloides stercoralis infecal materials, while traditional methods have beenreported to be unreliable. We have been reporting thatthe agar plate method is very efficient, based on resultsof our previous studies in Thailand and Cambodia, notthe agar plate method in the prevalent areas. We havebeen studying about improvement of procedure as an
appropriate technology for developing countries, so thatthe usage of the agar plate method would be globally
advantageous in the diaguosis of strongyloidiasis andonly for its reliability but also for its field applicability

hookworm infections that are broadly prevalent in tropi‑
cal area in the world. We have been reporting that sincein developing countries. We have also been reporting

prevalence rates of Strongyloides in our previous study

that hookworm infection is also detected successfully byin Thailand and Carnbodia were much higher than

68

previous surveys performed without the agar plate
method, it is suggested that global prevalence of Stron ‑gyloides infection is much higher than the present esti‑

mation.
In this study, we made a survey of hookworm andStrongyloides infections for adult farmers living sur‑rounding Surabaya City, and for adult factory workers

in Surabaya City, Indonesia, using the agar plate
method.
Hookworm infections were detected in 108 (31%) of
344 fecal samples from adult (over 20 years old) vil‑lagers living surrounding (within 40 km) the city, and inquite high among farmers living in the villages in thisarea. On the other hand, these infection rates were low
among residents of the city, and there was a significant
difference between farmers and factory workers.

Samples from diarrhea cases of small children(under 5 years old) were also examined. However nohookworm or Strongyloides infections were detected in
75 samples from a hospital. These parasites did notseem to be playing a significant role in agents of thediarrhea in small children in this area.

Prevalence rates of Strongyloides were again muchhigher than previous surveys performed ‑without the17(4%) of 417 samples from adult factory workers.

agar plate method also in this area, among adult

Strongyloides infections were detected in 69 (20%), andin 9 (2%) , respectively. From these results, it was shown

farmers. These results supported the suggestion that
global prevalence of Strongyloides infection is muchthat Strongyloides and hookworm infection rates werehigher than the present estimation.

B‑ 2

SERO‑EPIDEMIOLOGICAL SURVEY OF HUMAN TOXOCARIASISIN NEPAL, BANGLADESH AND THAILAND
K. KONDOl, N. AKAOl, T. OHYAMA T OKAZAWA Y TSUDA
K. YAN04, M.R. AMIN5 AND W. CHOOCHOTE6
Dept. of Parasitology, School of Medicine, Kanazawa Universityl,

Dept. of Public Health, School of Medicine, Kanazawa University2,

Faculty of Pharmaceutical Sciences, Kanazawa University3,
Inoue Hospital', Dept. of Micro‑biology, Faculty of Medicine, Dhaka University'and Dept. of Parasitology, Faculty of Medicine, Chaing Mai University6

The authors have been engaged in sero ‑Chaing Mai, Thailand (2.50%). The mean antibody

epidemiological survey of human toxocariasis in tropi‑
cal zone, especially, Southest Asia. We have got 344

against TcnLES is the highest in Dhaka, Bangladesh (1.
49i0.34) among other for investigated areas including
those in Ishikawa, Japan (controD.
These for areas have hot and humid climate and aresera in Nepal (98 sera in Dharan and 246 sera in
Nepalganj) , 37 sera in Dhaka, Bangladesh and 80 sera in

Chaing Mai, Thailand. We conducted the serologicaltest by ELISA against ES antigen of Toxocara canis
larvae (TcnLES).
The rate of the antibody titer against TcnLES

epidemic in soil transmitted parasitic diseases.

antigen is Nepalganj, Nepal (8.94%) and the lowest in

higher than in control area.

Although there is no clear on the antibody titer against

TcnLES between control and four areas, it is obviousthat the antibody titer of these four areas are relatively

69

B‑ 3

AN IMMUNOLOGICAL STUDY OF CYSTICERCOSIS IN GUATEMALAHIDETAKA ICHIKAWA1, NoBUAKI AKA02, LETICIA CASTILL03, EDMUNDO VELASQUES4,
YOUICHI IT05, KAZUKI OGATA3, ISAO TADA6 AND KAORU KOND07
Department of Medical Zoology, Faculty of Medicine, Kanazawa Medical University*,
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University2,
JICA project', San Carlos University', Department of Parasitology, Faculty of Medicine, Kitazato University=,
Department of Parasitology, Faculty of Medicine, Kyushu University'and Department of Parasitology, Faculty of Medicine, Kanazawa University'

We examined the antigenicity and its nature ofCysticercus cellulosae, a major health problem in
Guatemala, which causes serious lesions in central ner‑vous system and skeletal muscles. Antigenic composi‑tions of the cyst fluid (F) , scolex (S), membrane (M),and whole cyst (O of the cysticercus from pig obtainedin "E" area of Guatemala were compared by using SDS‑PAGE with those from F obtained in China aiming at

the Chinese were 100, 60 and 48 kDa judging from thequantiative aspect of protein concentration. ELISA
assay was performed by using F as the antigen on 78
randomly selecsed sera from the inhabitants of Santa
Maria lxhuat n. Seven (8.97%) in the samples showed
positive and the average antibody titer was 1.18d:0.14,
Among 43 sera from suspected cysticercosis cases, stor‑

ed at ‑80'C in the Division of Malaria, Ministry of

the selection of F‑antigen suitable for serodiagnosis.

Health, ten (23.26%) samples revealed positive and the

All these four antigens were composed of more thantiter was 1.26:!:0.29. There was no significant difference

twenty bands and no differences were seen in the protein

components among them in spite of minor differences in

between two groups in their average titers. An im‑
munoblot assay of serum sample out of 4 from thisprotein concentrations. The main components ofvillage with higher titers recognized 48, 42, 39 and 23

Guatemalan F were of 60 and/or 54 kDa, while those ofkDa bands in F.

B‑ 4

DIFFERENTIAL DIAGNOSIS OF CUTANEOUS
LEISHMANIASIS IN ECUADORSHIGEO NoNAKAl, ATSUSHI HOSOKAWA1, ABDUL MANAN BHUTTOI AND YOSHIHISA HASHIGUCH12
De, partment of Dermatology, Research Center of Comprehensive Medicine, Faculty of Medicine,

University of the Ryukyus* and Department of Parasitology, Kochi Medical School

Cutaneous leishmaniasis is a protozoal disease in‑fested by sand flies and widespread in tropical zone of
the world. Clinically, there are three types, that is,visceral, mucocutaneous and cutaneous type of leish‑
maniasis. We have investigated the skin changes of
cutaneous leishmaniasis in Ecuador since 1988. Most
popular cutaneous change was ulcer, but non‑ulcered
cutaneous changes such as papules, plaques and nodules
were also seen frequently. The symptom we have to beclearly demarcated ulcers are formed on the tibial
region. Seven patients had been diagnosed as cutaneousleishmaniasis and treated previously with meglumine
antimonate. However, the ulcer of these patients still

continued at the time of first examination. Par‑
asitological examination did not show any positive reac‑tion for leishmaniasis. The most important point ofdifferential diagnosis in the leg ulcer was the presence ofithcing sensation in the surrounding area of the ulcers.

most careful for differential diagnosis of this disease
was an ulcer formation. Ulcus cruris, a leg ulcer, is one

There was high incidence of leg ulcer in old age group.
Generally, no indurations exist around the ulcer, where‑of the diseases which we should be careful for differenti‑

as clearly detectable induration were in the patientswith leg ulcer treated with topical injection of meg‑lumine antimonate. In younger age group, impetigo andation. The patients with the leg ulcer often work for
agricultural purposes in the farm of endemic areas. The

70

traumatic injury were also considered as a cause of

mucocutaneous leishmaniasis because of the invasive

ulcer, but no induration existed around these ulcers. In

lesion of the nasal mucosa. The differential diagnosis ofendemic areas, we saw one patient with chromomycosis,
and a patient with basal cell carcinoma. The patientcutaneous leishmaniasis should be performed carefully

with basal cell carcinoma had been diagnosed asasitological and histopathologycal examinations.

not only by clincal examination, but also by par‑B‑ 5

FIRST CASE OF DIFFUSE CUTANEOUS LEISHMANIASISIN ECUADORABDUL MANAN BHUTTO1, SHIGEO NoNAKAI AND YOSHIHISA HASHIGUCH12
Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa*
and Department of Parasitology, Kochi Medical School'

Diffuse Cutaneous Leishmaniasis (DCL) is a spe‑cific type of leishmaniasis in which the lesions dissemi‑nates all over the body. We report a case of DCL having

the nodules and plaques on all over the body since 5
years. Recently, he developed the verruca vulgaris
lesions on the hand and foot. The parasites were iso‑lated and characterized as the Leishmania (Leishmania)
mexicana. Skin test with leishmania antigen was nega‑tive, but tuberculin, candidine and trichophytone
antigens showed positive reactions. Histologically,
macrophages were heavily parasitized and vacuolated,
and infiltrated in all over the dermis. By immunohisto‑
chemical stain, T cells were located in the sections.

Electron microscopically, HPV particles were found in
the verruca lesions. Morphology of the parasites weresimilar with that of normal leishmania parasites, andthey were phagocytized by the macrophages and few
parasites were seen under degeneration. Association ofvirus infection and negative delayed reaction with leish‑

mania antigen represents the immunological depressionof the patient. The unresponsiveness of T cells to theleishmania antigen may be due to the host defect (s)which are unknown. The high load of parasites in thesections may be due to the T cell defect which ultimate‑ly results in the spread of disease.

B‑ 6

GENETIC REGULATION OF CUTANEOUS LEISHMANIASIS IN
MICE INFECTED WITH LEI SHMA NIA A MA ZONENSI S
KEN KATAKURA, NAOHIRO WATANABE AND HIROSHI OHTOMO
Department of Tropical Medicine, Jikei University School of Medicine

Outcome and subsequent development of the skinexhibited a susceptible phenotype with progressive lar‑lesion of cutaneous leishmaniasis is varied with Leish‑

ger lesions throughout the 120 day infection period,

mania species and genetic background of the host.

whereas SJL/J mice showed really a resistantLittle information, however, is available in terms ofgenetic regulation of American cutaneous leishmaniasis.
Using mouse models, we analyzed genetic regulation of
cutaneous leishmaniasis caused by the infection withphenotype with no apparent lesions. The Fl (BALB/
c x SJL/J) mice exhibited an intermediate lesion

Leishmania amazonensis. Promastigotes of L. am ‑azonensis were inoculated into the shaven rumps ofmice, and host's susceptibility was determined withseveral inbred mouse strains by measurement of theskin lesion diameters. BALB/C and C57BL/6 mice

growth phenotype. In the F1 X BALB/C backcrossprogeny, 11 and 8 mice showed susceptible and interme‑

diate phenotypes, respectively. In the F1 x SJL/J
progeny, 34 mice exhibited an intermediate phenotype
and 38 animals showed a resistant phenotype. Further‑more, Iinkage analysis was carried out with respect to
the Lsh (Bcg) and Scl2, which are mapped on chromo‑

71

some I as a natural resistant gene for L. donovani

crosatellite markers in the vicinity of Lsh and Scl2 Ioci.

infection and on chromosorne 4 as a control gene for L.
mexicana infection, respectively. For this purpose, we

As a result, mouse susceptibility to L. amazonensis

used microsatellite repeats [ (CA)n] as chromosome
markers, which are demonstrated to be polymorphic
among different mouse strains and can be detected byresults suggest that murine cutaneous leishmaniasis
caused by L amazonensis infection is regulated by apolymerase chain reactions. Fifty to seventy‑two back‑leishmaniasis by other Leishmania species.

infection was linked to neither the Lsh nor Scl2. These

single gene different from genes reported for regulating

cross mice (FI x SJL/J) were analyzed with mi‑

B‑ 7

FAMILY EXAMINATION OF LEPROSY PATIENT IN LOW ENDEMIC AREAFOR THE DISEASE IN ECUADOR

ATSUSHI HOSOKAWAl, SHIGEO NoNAKAl, JUAN J. ALAVA P2, EDUARDO A. GOMEZ L.3
HUGO M. JURADO S.4 AND YOHIHISA HASHIGUCH15
Department of Dermatology, Research Center of Comprehensive Medicine, Faculty of Medicine,
University of the Ryukyus, Ninhihara, Okinawa 903‑01, Japanl,
Universidat Tecnica de Portoviejo, Portviejo, Manabi, Ecuador',

Departamento de Medicina Tropical, Facultad de Medicina,
Universidad Catolica Santiago de Guayaquil. Guayaquil, Ecuador3,
Centro Nacidal de Medicina Tropical, Guayaquil, Ecuador4
and Department of Parasitology, Kochi Medical School, Nankoku, Kochi 783, Japan
Four cases of leprosy patients were seen in an aerea

waist, back, the extensor aspect of the legs and the feet.

endemic for cutaneous leishmaniasis, Los Ranchos, theDepartment of Manabi. Two cases of them (borderline
lepromatous leprosy and indeterminate leprosy) in asingle family were reported. Other two leprosy patientsHair loss was not observed at any lesion of the bodysurface. Clinical examination; The Lepromin test waswere examined in the current examination. Futherpositive. Histological findings; Rete peg disappeared.

more, the existence of other four leprosy patients in the

Clear subepidermal clear zone was observed. Though

village was determined. From the result, it was suggest‑ed that family examination of leprosy patient might berelatively large number of epithelioid cells and lymo‑

usefull for detection of leprosy in a low endemic areas
for the disease, such as Department of Manabi. A nine

banded armadillo that were kept by the family had no
acid‑fast bacilli in the liver.

Case l: a 41‑year‑old man. Present histoly: In 1977,

negative. Value of anti phenolic glycolipid (PGL) ‑1 andantilipoarabinomannan (LAM‑B) antibodies were all

hocytes infiltrated in the dermis, epitheliod cell
granuloma was not observed. Bacterial index showed
3+; SFG index 5 (1‑2‑D . Nasal scraping was negative.
Case 2: The patient was a 12‑year‑old female. A
daughter of case 1. Present history. When we examined
the family of case I patient, hypopigmented fleckle witherythema appeared on the abdomen and a doctor

anesthesis on the extensor aspect of the left thigh wasdiagnosed him as borderline lepromatous leprosy innoticed. Hair loss was not observed. The type of

1982. Present condition; Infiltrated erythemata on theface, pea sized reddish nodules on the earlobe, eryth‑emata on the trunk were observed. Deformity of nose,
hands, feet and deep ulcer on the soles were observed.
The anesthesia and dry skin were observed on almost all

leprosy was indeterminate group. Clinical examination;
Lepromin test was undeterminable. Value of PGL‑1 andover the body excep for the axillary, the epigastric, thelower abdornen, the inguinal, the perineal and the anal

region. The ichthyosis‑like change was seen on the

LAM‑B antibodies were positive except anti‑PGL‑IgM.
Histological findings; small number of lymphocytes infil‑

trate around the capillaries and appendages. There was
no epithelioid cell granuloma in the dermis. No acis
bacilli was observed by acid fast staining.

72

B‑ 8

HEALTH EDUCATION IN THE CONTROL OF SCHISTOSOMIASISHAEMATOBIUM IN COAST PROVlNCE, KENYA= EFFECT ON
KNOWLEDGE, ATTITUDES AND PRACTICESKAZUHIKO MOJ11, YOSHIKI AOK12, TOSHIKI AWAZAWA2, MASAAKI SHIMADANGETHE MUHOH04 AND MELANlE KATSIV04
Department of Public Health, School of Medicine, Nagasaki University*,
Department of Parasitology, Institute of Tropical Medicine, Nagasaki University',

Department of Medical Zoology, School of Medicine, University ofEnvironmental and Occupational Health*and Kenya Medical Research Institute'The health education is expected to complement thecontrol measures of schistosomiasis. It helps people
understand that their own behaviour is a key factor in

1993, 85% of villagers acquired the knowledge that thedisease is caused by the infection of schistosome. There

reduce prevalence of disease. The present study deals
with the effect of the health education on knowledge,

was a remarkable decrease in the number of the vil‑lagers who did not know the sign and symptom of thedisease (from 36% t ) 6%).
The changes in attitudes and practices were also

attitudes and practices of people in an endemic focus ofsignificant. The number of villagers who used the safe

schistosomiasis haematobium in Kenya. In 1991, before
the start of health education programme, 190 male orfemale heads of the households, 96% of the total house‑holds of the study area, were interviewed. Among thern
138 heads were interviewed again in 1993, after thecompletion of education for I year. The educational
materials used in our health education activities werewater for bathing increased from 88 to 96%. The

spreading the disease and the changes in their practices

arnount of tap water used in a household increased from
9.9 to 20.9 Iitter/day/adult. Number of villagers withlatrin in their houses increased frorn 11 to 24%. How‑ever, both the number of villagers who took the urine

the manual of health educafion in schistosomiasis publii

examination and the percentage of the villagers who
took the drug did not increase. The prevalence ofdisease in our study area remained relatively high,shed by WHO and our own version of video tepes.
Health education improved people's perception andevery 2 years.

although the mass‑chemotherapy has been repeatedknowledge of schistosomiasis. In 1991, 62% of villagers

The present study revealed that health education

recognized schistosomiasis as the extreamly serious

cultivated the knowledge of schistosomiasis in the vil‑lagers, but did not modify their attitudes and practices
so as to reduce the risk of infection in the study area.

disease. In 1993, the perception of schistosomiasis as aserious disease increased to 85%. Only 58% of people
reported accurately the cause of the disease in 1991. In

73

B−9

STUDIES ON SCHISTOSOMIASIS JAPONICA
IN ORIENTAL MINDORO，PHIHPPINES（1）SEROEPIDEMiIOLOGICAL SURVEY IN SCHOOLCHILDREN WITH BLOOD TAKENON
HAJIME

MATsuDA1，YuIcHI

KAzuo

of

MedicalInstituteAnwithenzyme−linked

a

crude

antigen

メ勿on伽吻eggs

vey
in

for
the

was

immunosorbent

from

used

in

a

schistosomiasisjaponica
ofVictoria

inAugust1994．A

taken

tive

from

each

schoolprick

andsampling

testing
night

this
in

controlsera

densityof

total

0r30．4％of

gradesschoolsandofsurvey

the

lst

to6th

to

dry

on

Rosi

Type

I）．Before

containing1％BSA

more（barrios）of

7％，respectively．The

Tween

than

O．3wasof1，308children

Victoria

them

and

were

was

each

Pola

found

this

The

was

in

barriosandofresults

Malabo，San

located

near

the

Lake

of

highest

risk．

This

is

due

to

high

vectortheir

breedingsites．

thepresent

detectedtransmission

加sJ

preferastudy，significantly

with

ELISA
that

tions，thussuggestingforshould

done

be

the

O．g％α

examined，and398
togirls．TheDuonganplate．An

antibodies

Pola

with

areas

test

In

in

greatlythesincepositive

were

were
barrios

were

Tagbakin

than

these

were

elementary

Victoria

variedthepositive
optical

at

rate

boys

known

positive．

in

that

and

localities

Pakyasinprobablybeandprevalence

higher

get1：

to

eight

indicated

Narciso，Urdaneta
Naujanover−differenttheNarciso，Urdaneta，Duongan，

positive

toconsideredthequantita−

dissolved

at4。C

pool

from

Malabo，San

in

75．4％，47．1％，40．7％，33．7％，24．1％，22．7％，13．6％and10．

schools

of

in

of

II，Bethel，Malayas

was36．0％．

specimen

includedschools

blood

amomt

rates

Central

Mindoro，

paper（Toyowere

Tsukuba2

S．力ρo箆吻吻．Positive

sur−

allowedserumofHealth，Philippines3

of

blood

negativeofMedicine1，

small

filterPBS

Sciences，University

KIRINoKI1，

L．BLAS3

of

Pola，Oriental

child

was

SchoolService，Department

Soh∫s≠oso吻σin9elementary

dried

200dilutions．AA

andfingerMedicalControl

seroepidemiological

town

was

Basic

BAYANI

University

assay（ELISA）

lyophilized

Philippines
throughof

YoKol1，MAsAsHI

E．BuGAwAN3AND

Zoology，Dokkyo

Schistosomiasis

PAPER

CHIGusA1，HAJIMEYAsuRAOKA2，LYDIA

Department
andF皿，TER

for

ELISA

than

with

repeated

potential

inmarshyarea

morepositives

stool

stool

for

examina−examination

positives．

B−10

INTERMEDIATE SNAIL CONTROL IN S㎝ISTOSO崩
E4E％4TOB1乙曜ENDEMIC AREA OF KENYA
KENlcHI
Hokkaido

InstituteofPublicTAKAHAsHI1，SHINIcHI NoDA2，SHIGEHlsA HABE3，
NGETHE D．MUHOHo4AND YosHIKI AoKI5

Health1，Department

Department

of

Kenya
and

Departmentofof

MedicalParasitology，Faculty

Medical

in

Kwale

In

Mwachinga

in

upper

out
River

dries

District，Kenya．

Village，the

part
up

in

snail

of

the

Kadingo

the

hot

dry

controlseason，with

Medicine，Kagoshima

Institute4

Tropicalformation

Medicine，Nagasaki

of

small

University5

pools．Towards

the

end

of

the

dry

season，these pools may dry up completely．However，snail hosts can survive under the vegetation which
maintainan

Snails

in

the

pools

were

subsequent

river

bed

was

dried

by

carried

University2，

University3，

KadingowasRiver．The

ofof

Medicine，Fukuoka

Research

Parasitology，InstituteAttempts were made to control snail population（B％1ぎ纏s gloδos祝s）in Mwachinga and Mtsangatamu
Villages

Zoology，Facultyofappropriate

temperatureandeliminatedbyclearing

humidity、

scooping，andvegetation

duringthe

74

period between October 1992 and October 1993. Snailsurveys were carried out twice each month at 7 sites.
Annual number (June‑May) of collected snail before
snail control was 666‑3,855. Only 5 snail were collectedduring the period between June 1993 and May 1994.
In Mtsangatamu village, the modification of river
stream was carried out on January and February 1994 in
the part of the Mtsangatamu River of which flow isperennial. The snail habitats were reduced by clearingof water plants and concentrating the stream water in anarrow channel. Snail survey were carried out every
three months at 25 sites. The annual number of col‑lected snails in 1992 and 1993 was 507 and 430, respec‑tively. No snail hosts were collected by snail survey
after modification.

B‑1 1

ASSAY OF SERUM TYPE IV COLLAGEN IN SCHIS TOSOMAJAPONICUM INFECTIONHIROSHI OHMAEl, YASUSHI MATSUZAK12, TAKESHI NARA3,
YUJI IRIE4, BAYANl L. BLAS5, GERUNDIO P. PORTILL06,

ORLAND S. SY6 AND KAZUO YASURAOKA4
Bureau of International Cooperation, International Medical Center of Japanl,
Institute of Clinical Medicine, Tsukuba University',

Institute of Medical Sciences, Tokyo University3,
Institute of Basic Medicine'

Schistosomiasis Control and Research Service, Manila
and Schistosomiasis Hospital, Leyte, The Philippines6

We previously reported ultrasonographic (US) andserologic abnormalities in patients with Schistosoma

were performed on 52 patients at Schistosomiasis Hospi‑
tal in Leyte, Philippines in 1990 and 1994. A significant

japonicum in 1990. The US Iiver irnages were classified
into 4 patterns, according to the development of peripor‑detected in the patients diagnosed as periportal fibrosis

tal fibrosis. Among various serum indicators of liverby US. High levels of type IV collagen were also founddamage, the serum levels of total bile acid (TBA)

in sorne of the patients dignosed as no or mild periportal

correlated with the development of liver fibrosis in S.
japonicum infection.

fibrosis by US. The serum type IV collagen level wasincrease in the levels of serum type IV collagen was

more sensitive to detect the periportal fibrosis than theType IV collagen is a main component of basement

serum levels of TBA and Procollagen‑III ‑Peptide. A

membrance. In the liver, type IV collagen is present insignificant decrease in the serum type IV collagen level

the basement membrance in the parenchyma area. It

was also found after praziquantel treatment. The serum
type IV collagen level was proved to be one of the mosthas been reported that the serum type IV collagen levels
reflect the liver fibrosis and the validity of IFN treat‑ment in the patients with HCV.

US examinations concomitant with biochemical
serum analysis including analysis of type IV collagensensitive tool to monitor the severity of hepatic fibrosis

caused by S. japonicum infection, as well as the improve‑

ment resulting from praziquantel treatment.

70r

B‑12

GENETIC STUDY OF JAPANESE ENCEPHALITIS
VIRUSES FROM MALAYSIAHIDEAKI TSUCHIEl, KAZUMASA ODA2, INDRA VYTHILlNGAM3,

RAVlNDRAN THAYAN3, BALASUBRAMANIAM VIJAYAMALAR3, MANGALAM SINNIAH3,
MOHAMMAD MOFAZZAL HOSSAlNl, TAKASHI KURIMURAI AND AKIRA IGARASHI'
Research Institute for Microbial Diseases, Osaka University ,Japan International Cooperation Agency',
Institute for Medical Research3

Malaysia and Institute of Tropical Medicine, Nagasaki University'

Ten Japanese encephalitis (JE) virus strains weresuch as Japan, China, and Taiwan. WTP/70/22 strain

isolated from Culex mosquitoes caught in Selangor,

isolated from Malaysia in 1970 and JE‑827 strain iso‑lated from Sarawak in 1968, belong to another distinct
genotypic group. Our Malaysian JE virus strains, theMalaysia in 1992. In order to analyze the 10 JE virus
strains isolated from Malaysia in 1992, we examined a
240 nucleotide sequence from the pre‑M gene region of

WTP/70/22 strain, and the JE‑827 strain might havein Asia. No difference in the nucleotide sequences wasobserved among our 10 JE virus isolates from Malaysia.
Our JE virus strains belong to the largest genotypic

evolved from a common ancestor and have circulated inmosquitoes in Malaysia. Alternatively, new genotype of
JE virus including our Malaysian isolates might havebeen extending in Malaysia recently via introduction of
the virus by migrating birds or by international trans‑

group that includes strains isolated in temperate regions

portation systems such as jet airplane.

the strains, and studied their genetic relationships to theother JE virus isolates from a variety of geographic area

B‑13

A 27 AMlNO ACID CODlNG REGION ON THE C‑TERMINAL OF JAPANESEENCEPHALITIS VIRUS E‑PROTEIN EXPRESSED IN RECOMBlNANTESCHERICHIA COLI POSSESSES NEUTRALIZlNG EPITOPE(S)SAID ALI SEIF, KOUICHI MORITA, SACHIKO MATSUO,FUTOSHI HASEBE AND AKIRA IGARASHI
Department of Virology, Institute of Tropical Medicine, Nagasaki University,
1‑12‑4, Nagasaki, 852, Japan

In order to localize denaturation‑resistant neutral‑izing epitope (s) in the C‑terminal 180 amino acids ofmunogenicity by western blotting and mouse immuniza‑tion respectively. Among the four recombinant fusion

Japanese encephalitis virus (JEV) E‑protein, four

proteins, the highest neutralization antibody titre wasobtained by the one expressed by the recombinant clone

recombinant clones encoding different or overlapping
nucleotide sequences were constructed by PCR from arecombinant plasmid pS22. The amplified fragments

pRIT2T‑B3, which carried the coding sequence ofamino acid number 373‑399 of JE virus E protein. The

were cloned into PCR 1000 vector, and then transferred

results indicate that this short region of 27 amino acids

into Escherichia coli expression vector pRIT2T. The
inserted genes were expressed as fusion proteins withprotein‑A and examined for their antigenicity and im‑

sequence near the C‑terminal of JEV E protein posses‑
ses neutralizing epitope (s) and is hereby earmarked asa promising candidate for a synthetic peptide vaccine.

76

B‑14

ISOFORMS OF RNA FOR LARGE SUBUNlT OF CYTOCHROME B558
DETECTED IN ･DNA LIBRARY OF HUMAN GRANULOCYTESSHOICHI SUZUKI, ATSUSHI KUMATORI AND MICI IO NAKAMURAThe Department of Biochemistry, Institute of Tropical Medicine Nagasaki

Reactive oxygens play important roles in defence
against invading bacteria and in the regulations ofvarious biological responses in hosts. Most of these
oxygens are generated by NADPH oxidase on the sur‑face of phagocytes upon stimulation. Membrane‑boundcytochrome b558 is an essential constituent of NADPH

screened by plaque hybridization under low stringency.

oxidase for the generation of superoxide anion. It is a

Clone S7, S15 and S16 contained identical and non‑iden‑

heterodimer of a 91‑kD glycoprotein (large subunit)
and a 22‑kD polypeptide (small subunit). As the large
subunit is expressed only in myelogeneous cells at thetical sequences to large subunit CDNA. The clone S7
had first adenine of TATA box in putative promoter

late stage of differentiation, it should have a key role inmany 'responses associated with reactive oxygen
species. We therefore tried to isolate isoforms ofmRNA for the large subunit that may have different
types of information for regulatory domains.

For getting homologous clones to mRNA of thelarge subunit, CDNA Iibrary of human granulocytes was

Seven clones were obtained and sequenced on a Phar‑macia automatic sequencer.
Clone S1, S13 and S18 had identical sequences fromexon 6 to exon 12, exon 7 to exon 13 and exon 12 to exon

13, respectively of the cDNA for the large subunit.

region. This result therefore indicates an alternative
initiation site for mRNA for the large subunit. Clone S3

has mosaic sequences, 5' portion of which seems to
encoding a novel peptide containing more than 132
amino acids, and 3' portion of which encodes 40 aminoacids identical to the C‑terminal of the large subunit.

This clone suggests the presence of an isoform ofmRNA for the large subunit.

B‑15

ANALYSIS OF NOVEL CHRONlC GRANULOMATOUS DISEASE
ATSUSHI KUMATORll, SHOICHI SUZUK11, FUTOSHI KURIBAYASH11,MICHIO NAKAMURA* AND YOSHIRO TSUJI
The Department of Biochemistry, Institute of Tropical Medicine Nagasaki University*

and The Department of Pediatrics, Nagasaki University School of Medicine'Patients with chronic granulomatous disease

patient's leukocytes using biochemical and molecular(CGD) suffer from severe bacterial and fungal infec‑
tions beginning in early childhood. Their phagocytes
cannot kill microbes efficiently due to deficiency of

biological technique.

superoxide‑generating NADPH oxidase activity. The
NADPH oxidase is a multi‑component enzyme consist‑ing of a large (gp91‑phox) and a small subunit (p22‑phox) of cytochrome b558, p47 and p67, and genetic
defects in any one of the components lead to CGD.
Defects in gp91‑phox are most common in CGD because
its gene lies on X‑ chromosome. We found a novel X‑linked CGD patient of 61 year‑old man who was notphils was measured by chemilumi‑nescence after stimu‑

MethodSuperoxide generative activity in peripheral neutro‑

lating them with phorbol myristate acetate (PMA) .
Expression of each component for NADPH oxidase wasanalyzed by western blot analysis of neutrophil extracts

using specific antibodies raised against them. Amountof gp91‑phox and p22‑phox mRNA was determined by
Northern blot analysis using total RNA extracted fromdiagnosed until the patient had first severe infections at

peripheral mononuclear leukocytes. Protein coding
region in gp91‑phox mRNA was amplified into three

the age of 43 years. To clarify the cause of the CGD, weoverlapped fragments by RT‑PCR method. The am‑

analyzed genetic defects of NADPH oxidase in theplified DNA fragments were cloned into pBluescript and77

sequenced by the dideoxynucleotide chain termination
method.
Result and Discussion
Patient's neutrophils did not generated superoxide
in response to PMA. The patient showed undetectableamount of cytochrome b558 subunits on a western blot
analysis of neutrophil extracts. The patient's neutro‑
phils express cytosolic components of NADPH oxidase(p47 and p67) normally. On a Northern blot the patientmononuclear leukocytes, but the amount of p22‑phoxmRNA was normal. We did not found any abnormal‑
ities in coding region of gp91‑phox mRNA of the patient,

nevertheless, on other cases decreases in its mRNA
accompanying with abnormality in the sequence. These
results indicate that major cause of the CGD is due todecrease in transcriptional rate of the gp91‑phox genederived from genetic defects in regulatory regions gov‑
erning transcription of the gene.

showed apparently low levels of gp91‑phox mRNA in

B‑16

EFFECTS OF DIETHYLCARBAMAZlNE AGAINST WUCHERERIABANCROFTI IN FIJL FIVE ROUNDS OF ANNUAL
SINGLE‑DOSE TREATMENT VS. A COURSE OF 28‑DOSE

TREATMENT SPREAD OVER 2 YEARS
J.U. MATAIKAI AND E. KIMURA2

Wellcome Virus Laboratory, Suva, Fijil and Aichi Medical University2
Parasitological and clinical effects of annual single

ment, the reduction was quick and remarkable, but a‑dose treatments with diethylcarbamazine citrate

clear increase in the mf rate and density was observed 2(DEO given at 6 mg/kg in 5 consecutive years (a total

years after the completion of the treatment. It is pos‑sible a few additional annual doses would surpass theefficacy of the multi‑dose scheme. The 5 annual treat‑ments were effective in reducing filarial fever, thoughdose of 30 mglkg) and a 28‑dose DEC treatment at 5
mg/kg spread over 2 years (a total of 140 mglkg) werecompared using about 15,000 people in 82 villages in Fiji,

where diurnally subperiodic Wuchereria bancrofti trans‑mitted mainly by Aedes polynesiensis and Aedes pseudo ‑the 28‑dose treatment showed better clinical effects. In

scutellaris is endemic. Assesed at 5 years after theyears, both mf rate and density increased. After servingfor 3 years as a control group, the people were treatedcommencement of treatment, the former scheme
reduced microfilaria (mf) rate from 6.5% to 0.9% (an86% reduction) and mf density (geometric mean of mf

control villages, where no treatment was given for 3

with 2 rounds of annual single‑dose treatment with

5 (a 76% reduction). The 28‑dose scheme reduced mf
rate from 11.6% to 0.9% (a 92% reduction) and mf

DEC. The study indicates that 5 rounds of annual single‑dose treatment, with the total dosage of only about afifth of the extensive 28‑dose treatment, resulted incomparable effects to the latter.

density from 29 to 5 (an 83% reduction) . In the annual
treatments, the reduction in the mf rate and density was

UNDP/World BankIWHO Special Programme forgradual but steadily progressive; in the 28‑dose treat‑

Research and Training in Tropical Diseases.

count/60 pl of blood among the mf positives) from 21 to

The senior author received support from the

78

B‑17

FUNDAMENTAL SURVEYS FOR THE UTILIZATIONOF MEDICINAL PLANTS ACAINST FILARIASIS IN

KENYA (MlNIREVIEW)

MAKL J.*, ITO, Y. , KOFI‑TSEKPO, W.M.2, AKAL K.'
FUJIMAKI, N.', MrrUL Y.+ AND AoKL Y.'
Dept. Parasitology, Kitasato University School of Medicine*,

Kenya Medical Research Institute',

JICA Project team leader=and Dept. Parasitology, Kitasato University School of Medicine'

Obstinate parasitic diseases like filariasis haveannoyed Kenyan inhabitants, especially those in rural

against filariae in India where filariases are rampant.
The bark of Indian medicinal plant, Azadirachta indica

areas. If drugs with high efficacy and low prices

is said to be useful for the treatment of filariasis there

without severe side effects were readily available, the(Comley, 1990, Trop. Med. Parasit. 41:1‑9).
In East Africa, three medicinal plants, Cyphostemma
nierense. Psychotria tanganyikensis and Xeroderris stuhl‑

present situation would be improved. It appears thatplant‑derived drugs would meet this requirement. From
this viewpoint, the present authors have started prelimi‑
nary surveys and preparation for the possible utilizationof medicinal plants against filariasis in Kenya. This
communication is a brief review of the survey of anendemic area of the infection with Wuchereria bancrofti.

information on herbal therapy of filariases, and medici‑
nal plants harvested.

The researchers of Kenya Medical Institute(KEMRD and Japan International Cooperation Agency(JICA) visited the southeast region of Kenya for themannii are thought to be useful for the treatment ofelephantiasis(Kokwaro, 1993, Medicinal Plants of East
Africa, 2nd edition, 401, Kenya Literature Bureau) . In
kenya, the leaves of A. indica have gained popularity
among inhabitants hoping treatment and prophylaxis ofmalaria. The possible efficacy of the leaf extract
against filariae cannot be denied to the best of thepresent author's knowledge.

For the start of the new work, Kofi‑Tsekpo &Maki(1994. Research Proposal submitted to KEMRI, 3

epidemiological survey of W. bancrofti infection and the‑4) selected about 20 species of plants to be tested in thecollection of medicinal plants. In Kwale district there
have been found three villages Lutsangai, Gandini andefficacy against filariae. Medicinal plants, A. indica,
Clausena anisata, Edithcolia sp., Maytenus sp., Taber ‑naemontana stapfiana and Toddalia asiatica collected inDzivani where the inhabitants are suffering from theinfectious disease caused by W. bancroftic (Gachihi et al.,

1994, Jpn. J. Trop. Med. Hyg. 22:41). Mosquitoes
sprayed down in houses and collected were dissectedunder a stereoscopic microscope and observed under aTaita Hills and Taveta District were brought back to
the laboratory in KEMRI for the preparation of crude

drugs(Maki & Kofi‑Tsekpo, 1994, JICA Progress
Report) .

light microscope for the possible detection of the 3rd

The extracts will be subjected to in vitro and in vivo

‑stage larvae. The vectors were demonstrated to be
three kinds of mosquitoes, one species of Culex and two

assays. Jirds infected with Brugia pahangi are readily
used for in vivo tests. In vitro maintenance of adultspecies of Anopheles (Mwandawiro, personal communi‑

filariae has been studied by a number of workers (see thecation) .

review by Maki(1991, Kitasato Arch. Exp. Med., 64:179

Published papers on medicinal plants with possible
efficacy against filariases from the viewpoints of eth‑nobotanical and experimental studies have been col‑lected by the present authors(Maki & Kofi‑Tsekpo,

1994, JICA Progress Report). A number of plant
species, for instance Andrographis paniculata (Dutta &Sukul, 1982, J. Helminth., 56:81‑84) and Zingiber of‑
ficinale(Dutta & Sukul, 1987, J. Helminth., 61:268‑270)

have been examined in the efficacy of the extract‑182)). The Nl medium or a 1:1 mixture of NCTC135
and IMDM is thought to be one of the best media for thein vitro maintenance of adult . pahangi under the gas
phase of 5% CO, in nitrogen or air. The worms will bemaintained in vitro so that the effect of the extracts on

their movement and microfilarial production might beclosely observed.

79

B−18

A

NEW

METHODSANDFOR

DETERMINATION

IVERMECTIN

IN

THE

OF

BLOOD

lMMUNOSORBENT
YOsHINoRI
Department

A

newsimple

and

tion

of

were

successfullyofMITsuI1，YAsuNoRI

Parasitology，Institutesensitive

methods

diethylcarbamazine（DEC）andfor

ENZYME−LINKEDASSAYFuJIMAKIIofDIETHY1、CARBAMAZINE

BY
AND

TropicalYosHIKI

conjugate．Response

determina−

tive

ivermectin（IVM）

AoKIl

Medicine，Nagasaki

curve

was

University1

for

DEC

in

betweenland1000ng／mL

the

For

assay

conjugatewasexpressly（ELISA）．DEC and IVM were structurallymodified andconjugated to bovine serum albumin for use as im−

ELISA

by

simultaneously−biotinwith

anti−IVM

mmogen．An

antiserum

IgG

then

bound

elicited

micecompetitiveinFor−Lysinesolid

developed．The

enzyme−linked

rabbitsofconjugate

expressly

phaseconducted

antigen．A

by

on

for

DEC

and

IVM

were

DEC

in

the

competitive

simultaneously

for

use

indirectincubating

was

and

DEC

as

the

ELISA

was

with

anti

The

of

gas
An

the

for

competi・

fluid，IVM−biotinA

competitive

incubating

antibody

determination

peroxidase

ourchromatography

withimmunoglobulinG

in

prepared．
overIVM−biotinofimmeasurable

theanti−mouse

IVM

range

sensitivity−DEC antibody over DEC−ploy−L−Lysine solid phaseand then determining the bound mice immunoglobulin

sheep

of

resulting

IVM

goat

and

anti

detectedbyinsystem

IVM

this

IVM

rabbitAvidinwas

O．1−10ng／ml．

fluid，DEC−poly−L

prepareddetermination

peroxidase．Response

respectively．

determination
was

are

immunosrobent

specific

andmethods

themethods

methods

and

are

high

as

those

methods

is

that

HPLC．

advantageof

ourrequireonlyofconcentration

drug

as

lOμl

of

serum
in

the

for

accurate

blood．

B−19
ARE

THERE

F皿，ARIASISSERGIoSPORADICAH。YIN

THE

HuRTARTE1，IsAo

SHINJIRo

Department

of

FOCl

OF

LYMPHATICCENTRALAMERICA？

A．1，SALvADoR

HERRERA3，

OF

OcHoA

MATsuo4，NoBoRu

MAsuDA4AND

KEIToKu

Division，Ministryof

Health，Guatemala1，

Parasitology，FacultyofMedicine，Kyushu

National
alld

BASIN

TADA2，J．ONoFRE

HAMAMO2，RYU
Malaria

ENDEMICCARIBBEAN

KyushuHospita1，Puerto

University

Association

BABA4

University2，

Barrios3

of

TropicalLymphatic filariasis have been found distributing
widely in Caribbean basin such as Puerto Rico andDominica．The disease was considered to have been

Guatemala．Due

transported

Centralrevealedrepor−

resident of Puerto Barrios（53year old male）from
Livingston and the other（36y．o、female）living inMariscos near Izabal lake．A night blood survey on
randomly selected56inpatients from the vicinity of
Puerto Barrios and admitted to National Hospitalfrom

Africaby

America，however，lymphatic

ted

only

from

Costa

Puerto

Limon

area

−15％oftheresidents

theQuepos．No

otherCentralAmericansomeusobserved

area

of

of

urban

trade．In

were

ever

Rica．Butts（1947）investigated

of

this

were

two

Puertoin1946andforfound1

microfilariae

ofCieneguita，Jamaica，Bataan

findings

countries

country

positive％％6h6名召吻δ伽6名φガin
and

slave

filariasis

onlymphatic

except

cases

of

Costa

leg

Barrios，afilariasis

inRica．In1993，

elephantiasis

in

the

Caribbean

port

of

nary

surveyAninterview

revealed

twotoMedicine4

was

this
done

observation
atPuertorecord，aBarrios

in

study

at

variousinstitutions

cases

of

typicalleg

negative．In

the

blood

prelimi−July，1994．

andstreets

elephantiasis，one

examination，approxi−

mately30cmm of blood from the fingertip was micro−
scopically examined．The present study could not findpositive

microfilaremic

persons，whilethepresence

of

80

1eg

elephantiasis

country

will

from

eastemsuggestcoastalsporadicregions

distribution

of

of

the

filariasis

lymphaticin

Caribbean

Guatemala，Central

side

and

Lake

Izabalbasin

inAmerica．

B−20
RATES OF MICROF皿，ARIA PREVALENCE AND CLINICALFILARIASIS IN CHUUK（FORMERLY TRUK）STATE，FEDERATED
MAMoRuFuJIwARA1，KloSI

Department
and
In

we

Department

order

to

determine

carried

out

blood

加％6名φぎinfection．Since

systematic

the

and

state・wideofof

Healthpresent

OF

MICRONESIAANIoL2AND

Parasitology，Aichi

State

examinedoffilariasis，

surveyson肱6h6泥7勿average rates of hydrocele，elephantiasis and endema
were3．4％，0．4％and O．9％respectively．There was athereportbysurvey

has

been

those

locatedPipkin（1953），nopositive

maderiasisfor40years

was2．6％．The

on9islands

averagemf

theislands

mf

rates

the

capital，Moen，showed

low

On

the

other

villages

from

the

capital，showed

rates．High

villages

mf

rates

on3islands．The

significantly03）．Thehigherhighestthanmf

hand，themoderate

rate

that

of

rates

of

age≧15years

forbetween

rates

of

the

the1930s，filariasis

ofclinlcal

fila・

was

not

an

no

of

obtained

in3

rate

autochthonous

might

be

case

explainedofpartlyreporteddiseaseHowever，in1951−52，mf rates in Truk District was12−27％（Pipkin，1953）．This remarkable increase of mf

highEsaki（1938）even

important

was

to

and

rates

of

locatedarea

thevillages（r＝0．622，p＜0．02）．

thisfilariasis

by

a

in

that

there

Micronesia．

large−scale

immigra・observed

males

that

the

mf

studied466adult

moen

and

neighboring

vementofmales（3．2％）wasage≧20years（62−10．3％）．We

males

Micronesia2

females（1．7％）（p二〇．

of

were

mf

of

tion of Japanese，many of whom were from Okinawa
where四．6朋o名碗ガwas highly endemic．After Pipkin s
study，Andoαα乙reported the prevalence rate of7．9％
in1982．Thus，our mf prevalence rate（2．6％）indicatesof7−10％weremf

correlation

and

In

States

in

rate

on

near
far

was

villagesthan1％．

relativelyofmicrofilaria（mf）．The

examinedlessmf

population）in14villages

for

KIMURAl

Universityl

State，Federated

inChuuk．Atotalof2193people（about5．1％ofthe
total

EIsAKuMedicalServices，Chuukrateclinical

STATESchronic

amongclinicalsigns．The

rate

life

has

been

decrease

at

least

islands．Urbanization

andimpro−

conditions

on

the

might

be

the

main

in

reason．

B−21

STATISTICAL OBSERVATlONS ON CHYLURIA PATIENTS FOR
THE RECENT TWENTY YEARS IN DEPARTMENT OF

UROLOGY，KAGOSHIMABETWEEN1974AND1993

MIzuMA，ITARu

MIYAwAKI

Urology，Facultyof

YosHIHIRo
Department
In

this

chyluria

report，wepatients

1974and1993inforpresent

theofstatisticalrecentDepartment

ofUNIVERSITY；INPATIENTSOUTPATIENTS

twentyobservations

years

of

patients

betweenIn

Urology，Kagoshima

University．

Comparative
of

aging

and

follows：average

numberofdata

decrease

of1974and1993showsintendency

numberofchyluria

age，55．3years

oldand68．6years

patients，34and11，percentageofpatientsasold，chyluria

AND

YosHITADA

Medicine，Kagoshima
for

all

out

AND

OHI

University
patients，2．0％and

o皿department，Nagata

investigatedthelymphatic

O．5％．

and

Okamotoetofchyluria

patients

flow

aL

using lymphography for determination of．the etiology ofthe disease in1967．They revealed prominently dilated，

kinkedandflow

contrastofobstruction

complicatedoflymphatic

vessels

media

into

the

renal

thoracic

duct

was

not

with

back

calyces．Organic

recognized．It

has

81

been suggested that these pathological status of lym‑
phatic channels result in stasis of lymph flow, with theIt was declared that filarial infestation was
eradicated by 1975 in Kagoshima prefecture. But

subsequent development of lymphatic pressure. Thischyluria is still one of the important urological diseases

may product fistularisation of lymphatic vessels into theurinary tract.

chyluria is a chronic manifestation of filariasis.

here in the southern part of Kyushu Island, because

B‑22

CLINICOPATHOLOGICAL CHARACTERISTICS OF FOUR CASES
OF PULMONARY DIROFILARIASISHIROSHI HIRANol, TERUMASA SASHIKATAI and TAKEO MATSUMURA2
Department of Pathology, Hyogo Medical Center for Adults*
and Department of Medical Zoology, Kobe University School of Medicine'

Pulmonary dirofilariasis, which is caused by Dir‑
ofilaria immitis, the dog heartworm, is a rare disease.

However, it is being diagnosed more frequently eachyear, and the differential diagnosis of this disease andmalignancies is of increasing importance. To the best of

Gieson stain, Masson trichrome stain, silver stain, andhematoxylin‑eosin stain were used. The silver stain
was the most useful for showing recognizable worm
structure: a smooth cuticle, well‑developed musculature,
an internal coelom, and intestinal and genital tracts.
The patients seemed to have been infected several years

our knowledge, only about 60 cases have been reported
in Japan. Four cases of pulmonary dirofilariasis werediagnosed at the Hyogo Medical Center for Adults in the
period 1984 to 1993. Chest x‑ray films taken during
health check‑ups showed a single spherical nodule 10 to
30 mm in diameter in each patient. All lesions were in

earlier, because some larvae had degenerated and were
not recognizable except for their smooth cuticle andmusculature. We undertook immunohistological investi‑
gations of the operative specimens, which did not reactwith alpha‑smooth muscle actin, but did react with thethe peripheral portion of the inferior lobe of the right
lung. All patients lacked clinical symptoms and report‑

serum of a rabbit hyperimmunized with D. immitis.
According to the literature, cross‑sections of manyed no contact with dogs. In three cases, results ofhematological examinations were within normal limits,dirofilariasis lesions show necrosis, endoarteritis,

and in the remaining case, mild eosinophilia was found.
Biochemical tests of blood gave normal results. Tests
with an antibody to the causative agent were not done.

or more of these findings. The parasites were associat‑

ed with thrombus formation within the pulmonary

Cytological and bronchoscopic examinations of thethe worms. Pulmonary infarctions are usually wedge

lungs gave no abnormal findings. Therefore, the dir‑ofilariasis could not be readily differentiated frommalignant disease without surgical resection of thelesion. Pulmonary dirofilariasis is not life‑threatening,

so resection should be avoided, when possible. Never‑theless, all four patients underwent surgery (lobectomy

eosinophilia, and fibrosis. Each of our patients had one

artery. Coagulation necrosis of this artery surrounded‑shaped on chest x‑ray films because the arteries embol‑

ized by a thrombus supply a wedge‑shaped area. Risher
et al. have suggested that pulmonary dirofilariasis
shows coin‑shaped lesions on x‑ray films because theworms release a toxin that causes arteritis followed by
inf arction.

in three cases, and resection of the lesion in one case) .

We thank Dr. E. Konishi for the hyperimmunized

The pathological evaluation of the resected specimens
made possible a definitive diagnosis. The elastica van

rabbit serum and Dr. S. Uga for his preparation of D.
immitis microfilariae embedded in BJ‑4.

82

B‑23

BASIC STUDIES ON THE MOI GOLIAN GEkBIL AS A SUSCEPTIBLEHOST TO FILARIAL INFETION (13) SENSITIVITY TO

DIABETOGENlC SUBSTANCESKEIJIRO SHIROTAl, KATSUYA HAGA2, KOICHIRO FUJITA2,MASUMI SHIMIZU2,3 AND KAZUHIRO SHICHINOHE2,3
Tobu Co‑medical Technical College*,
Department of Medical zoology, Faculty of Medicine, Tokyo Medical and Dental University'and Department of Laboratory Animal Science, Nippon Medical School'

We reported that encephalomyocarditis virus M
variant, which induced diabetes in mice, did not cause

hyperglycemia in the Mongolian gerbil (Meriones un‑

hyperglycemia, polyposia and polyphagia in the gerbils
since 3 days after injections. Degrees of hyperglycemia

guiculatus) although it arose some lesion in the pancreas

were dose‑dependent. The animals injected with morethan 150 mglkg of alloxan or 200 mglkg of strept‑of the animal. We examined sensitivity of the gerbil toozotocin became weakened and some of them died.

diabetogenic substances using alloxan and strept‑Those data suggested that the optimum dose of alloxan
and streptozotocin were 125 and 150 mglkg, respectively

ozotocin in the present study.

Fifty to 200 mglkg of alloxan or streptozotocin
were intravenously injected into three to 4 month‑oldgerbils. Bodyweight, food and water intake and blood
glucose level were measured during I month and his‑
tological observation were performed.

to induce experimental diabetes in gerbils. Since
pyknosis and degranulation of B cells of pancreas wereobserved in histological examination, Iack of insulin
induced by lysis of B cells might caused crisis of diabetic

symptoms in gerbils.

More than 100 mglkg of both chemicals induced

B‑24BASIC STUDIES ON THE MONGOLIAN GERBIL AS A SUSCEPTIBLE HOST TO
FILARIAL INFETION (14) THE ORGAN WEIGHTS OF THE WILD ‑COLOREDGERBIL AND THE COAT COLOR MUTANTS

MASUMI SHIMIZU*'3, KAZUHIRO SHICHINOHEl'3, KAZUYOSHI KAT02

KATSUYA HAGA3 AND KOICHIRO FUJITA3
Department of Laboratory Animal Science* and Department of Emergency and Critical Care Medicine, Chibahokuso Hospital'

Nippon Medical School
Department of Medical zoology, Faculty of Medicine, Tokyo Medical and Dental University'

We performed an anatomical study of Monogolian
gerbils including the coat color mutants to establish
baseline data of this animal and to know whether theiranatomical feature relate to their high and wide suscep‑tibility to parasites.

Fifty each of male and female gerbils having agouti,
albino, black or other kind of coat color were used inthis study. Those animals were 3 to 12 months old andthey had been used for controls of other experiment or

reproduction. The organ weights of the heart, Iung,liver, spleen, pancreas, kidneys, adrenals and thymus
were measured after each animal was bled under ether‑anesthetization. Each organ/body weight ratios was
also calculated.

Those data of the coat color mutants were compa‑
rable to those of the wild‑colored gerbils. But thethymus of Mongolian gerbils was remarkably different
from that of mice or rats. The weight of this organ
generally reduced by age in mice or rats, but weight lossof the thymus was very little in the Mongolian gerbil
and mature one still had a certain size of the thymus. It
seemed to be a peculiar character of this animal and it
may related to its susceptibility to various parasites.

We also immunohistologically examined a surface

83

antigen of the thymocytes of the gerbil iricluding thecoat color mutants using anti‑Thy‑1.1 and anti‑Thy‑1.

2 monoclonal antibodies. Thy‑1 antigen has been

to Thy‑1.1, whereas the response to Thy‑1.2 was com‑pletely negative. The coat color mutants of the gerbil
showed the same response. The results indicated thatit consists of 2 types in mice, i.e. Thy‑1.1 and Thy‑1.2.

the Thy‑1.1 antigen existed in the Mongolian gerbil anddistribution of Thy‑1 antigen did not have the coat color

Each strain of mice has either Thy‑1.1 or Thy‑1.2.

variations in the gerbil.

known as a cell marker for T‑1ymphocyte in mice andCortical thyrnocytes of the gerbil were strongly positive

B‑25

A NEW APPROACH FOR ANALYSIS OF HUMAN SWEATINGMUNENORI SHIMAZU1, TAKAAKI MATSUMOTol, MITSUO KOSAKAl
NoBU OHWATARll, KATSUHIKO TSUCHIYA1, YOSHITO UEYAMA2,3,*
KOJI URAN02,3, YUKO KATAKA12,3 AND MUNEO SAIT03
Department of Environmental Physiology, Institute of Tropical Medicine, Nagasaki University ,Kanagawa Academy of Science and Technology=, Central Institute for Experimental Animals'
and Department of Patholgy, School of Medicine, Tokai University'

mouses' dorsal skin was investigated by intra‑cutaneous

pilocarpine > adrenaline > no chernical. VIP was also
tried, however, none of response of sweating was in‑
duced. Histological investigation of the transplanted

administration of chemical stimulants. Experiments

human skin was also performed in HE stain. In thewere carried out in an environmental chamber (Ta:26'C,

histological specimen, eccrine sweat glands were foundin rather good shape. Blood vessel and a bit of degener‑
ate marks of nerve fibers existed around eccrine glands.
It was confirmed that eccrine sweat glands in the trans‑

Inducing of eccrine sweating of human skin trans‑

planted into several strains of immuno‑deficiency

rh:60%) without anesthesia. Using Minor's method,sweat spots were visualized and recorded with a video

macroscope (NIHON KHODEN:VMS‑1300). For in‑ducing sweat, 0.0lml of different kinds of chemical
stimulants (adrenaline, pilocarpine, atropine andpilocarpine) were administered intra‑cutaneously. One
chemical was used in a day to induce sweating. In atypical case, sweat volume induced by each chemical
was in the following order; pilocarpine > atropine +planted human skin were considerably well kept its
original structures and maintained the moderate func‑tion of sweat secretion. From this finding, it was sup‑posed that eccrine sweating of transplanted human skin

was dominantly cholinergic and slightly adrenergic
under the condition of the lacking of innervation.

B‑26

ANALYSIS OF LONG‑TERM HEAT‑ACCLIMATIZATION IN
TROPICAL INHABITANTS ‑COMPARISON OF SWEAT RESPONSE
AMONG JAPANESE, THAI, AND AFRICANS‑TAKAAKI MATSUMOTO1, MASAKI YAMAUCH12, MITSUO KOSAKA2,DAVID SAM2, NOBU OHWATAR12, ZONG‑WEI LU02
YU CA02, AKIHIRO TAIMURA2, EIKO KANEDA2
AND JEONG‑BEOM LEE2
Department of Thermal Adaptation

and Department of Environrnental

Physiology=, Institute of Tropical Medicine, Nagasaki University

In order to clarify the mechanisms of long‑term accli‑
matization to hot environment, sweat response and heat

tolerance in tropical inhabitants were examined. Ten
Thai, 6 Africans (3 Tanzanian, I Kenyan, I Ghanian and84

1 Senegalese) and 11 Japanese were the subjects in thisstudy. In a chamber at 26.6'C and 33% rh, sweat was
induced by leg immersion into a hot bath at 43'C for 30

min. Oral and skin temperatures were measured withthermistors and local sweat rates on the chest andabdomen were measured with capacitance hygrorneter‑ventilated capsule method. Two of 6 Africans did not
sweat during 30 min heat load, therefore, heat load wasapplied for another 10 min. Sweat onset time in
Africans (21.60d:8.22 min, meand:SD) and in Thai (16.
60 5.95 min) were significantly longer than that in

and abdomen were 10.16i5.00 mglcm2 and 6.8ld:4.16
mglcm2 in Japanese, 1.39d:0.97 mg/cm2 and 2.37 1.29
mg/cm2 in Thai and 2.71 1.63 mglcm2 and 1.68dl0.88
mg/cm2 in Africans, respectively. There was no signifi‑

cant difference in the initial oral temperature before

heat load among Japanese, Thai and Africans. Oraltemperature in Africans as well as in Thai was kept
lower compared with that of Japanese during heat load.
There was no significant difference in threshold oral
temperature for sweat onset among Japanese, Thai andAfricans. From these results, it is concluded that though

Japanese (9.04d:2.27 min) . Local sweat volume in

the amount of sweat is smaller in tropical inhabitants,

Africans as well as in Thai was significantly smaller
than that in Japanese. Local sweat volume on the chest

heat loss ability is better compared to that in Japanese.

B‑27

VASODILATION OF THE TAIL SKlN INDUCED BY ISOPROTERENOLIN CHRONIC SPINAL RATS

KATSUHIKO TSUCHIYA AND MITSUO KOSAKA
Department of Environmental Physiology, Institute of Tropical Medicine,

Nagasaki University

Effect of isoproterenol (adrenaline p‑agonist) on

after 60 min of the injection. The index of blood flow inthe tail artery was studied in cervical spinal rats. The

the tail, dTtail (dTtail= Ttail‑Ta) was 3.7:!:0.6'C before

cervical cords of male Wistar rats were transected at
the level of C8/Thl under pentobarbital anesthesia.
Spinal rats were reared in the room of 30'C. After the
spinalization, body weight decreased progressively.
Heart rate (HR) was calculated by the R‑R interval of

the injection. By the injection of Isop, dTtail initiallydecreased and thereafter gradually increased to be overthe value before the injection. Mean dTtail was 1.9 0.

ECG. Rectal (Tre) , tail skin (Ttail) and air (Ta)temperatures were detected by thermistor probes under
the unrestrained condition. Isoproterenol hydrochloride

8'C after 10 nin, and 4.7 0.7'C after 60 min of theinjection. These changes in dTtail were statisticallysignificant (p<0.05) compared to the value before theinjection. In this study, the injection of Isop induced
both vasoconstriction and vasodilation in the tail of the(Isop, Proternol‑L, Nikken Kagaku, Tokyo) 0.Img/kg

chronic spinal rats. Fregly(1983) suggested that the tailwas injected intraperitoneally to rats after 2‑3 weeks ofvasodilation by Isop was not direct effect on vasculature

the spinalization. Under resting condition, mean HR

but reflex vasodilation due to an increase in metabolic(MiSE, N=5) was 290 17 beats/min. By the injec‑

rate. But the tail vasodilation by Isop in the spinal ratstion, HR increased by about 50% and high level of HR
was sustained for 30 minutes(min) and declined toward

can not be explained by reflex vasodilation via thethe initial level. Before the injection of Isop to conscious

hy pothalamus.
References: I ) Fregly MJ (1983) Pharmacology 27:150

rats, mean Tre was 37.8d:0.4'C. By the injection of Isop,‑159.

Tre increased gradually. Mean Tre was 38.4 0.9'C

85

B‑28

INORGANIC ION COMPOSITION OF RAIN WATER INYAKUSHIMA ISLANDTETSUO ANDOHl, KAZUYUKI NAKAMATA2 AND SUMlNORI AKIBAl
Faculty of Medicine, Department of Public Health, Kagoshima University*

and Yakushima Public Health Centre, Kagoshima Prefecture'

The samples of rain water on Jan. 13, Feb. 23 andurban distric, however, the nitrate‑ion ones of them ofMarch 7 in 1994 were collected at Anbou district and theformer were lower than those of latter. From thethree points of Nagata district in Yakushima Island.
The measuring of inorganic ions of rain water gave theknowledge of a main characteristic of hydrogen‑ion

results, the sulfate‑ions of rain water of Yakushima

concentrations (pH values) of it. The mean of pH

Island may partly originate from China, and the exhaustgas of automobiles on the island may little contributethe nitrate‑ions of rain water. The chlorine‑ions of rainvalues on March was significantly lower than that on
January and February, and that at Nagata district was
lower than that at Anbou. Twice out of twelve collec‑

water of the island may originate mainly from thetions of rain water indicated the lower pH values below

Nagata district, as same as those of Sakuragaoka from
Mt. Sakurajima. The hydrogen‑ and inorganic ions ofrain water of Yakushima Island should be continuallymonitored.

5.0, namely acid rain. The chlorine‑ and sulfate‑ion

concemtrations of the samples of Yakushima Island
were higher than those of Sakuragaoka‑Kagoshima, ordiffusion of sea water, but partly from volcanic gas of

Mt. Shin‑dake in Kuchinoerabu Island, 15km from

B‑29

CLINIBAL EVALUATION OF HEALTH EXAMlNATIONS
OF JAPANESE WORKERS DISPATCHED TO FOREIGNCOUNTRIESMOTOYUKI FUKUMURA, ATSURO HAMADA, MAKIYO MACHIDAAND SATORU KOJIMAJapan Overseas Health Administration Center

In order to evaluate health of Japanese workers

their missions regarding their life styles including diet,dispatched to foreign countries, health exam. were per‑amount of sleep, and physical exam.

formed on 52 Japanese workers before and after their

missions. The study involved 42 men and 10 women

Results of the tests taken after the workers' return
were compared with results prior to their departure.

(mean age:35 1) dispatched to foreign countries for an

The lifestyles(diet, sleep, and physical exercise) of

average of one year. Their missions were in both

the workers changed significantly during their stays
abroad. The changes were more marked in the workers

developing countries (39 subjects) and developed coun‑tries (13 subjects) . The developing countries included
Indonesia(11 subjects) , China (8), Singapore (4) etc.
The developed countries were the U.S.A. (9), Germany(2) and England (2).

dispatched to developed countries than those dispatched
to developing countries.
No significant difference were found in laboratorydata‑including liver function and serum lipid, blood

We perfomed the health exam. immediately before

pressure, and body weight‑before and after their stays.

and after the workers' stays. The exam. were: standard
hematoserological tests, urinalysis, ChestXP, electrocar‑

Although health problems were not detected in the

dio‑gram(ECG) , blood pressure measurement, gastro‑
intestinal XP, and abdominal ECHO.
Participants were also interviewed before and after

workers whose lifestyles changed significantly, it may
nevertheless be necessary to educate the workers in how
to maintain healthy lifestyle in order to prevent healthproblems caused by changes in environrnent and life‑

86
style.

B‑30

BACTERIOLOGICAL ANALYSIS OF STOOL FOR
5 YEARS IN KAGOSHIMA AIRPORT QUARANTINESTATION
SATORU HAGIO AND BISHO SAKUMOTO
Kagoshirna Quarantine Station

An attempt to evaluate the local airport quarantine

system was done in Kagoshima International Airport.
Kagoshima is one of the entrances of south‑east Asian

countries, where various infectious diseases arefound. In the same period, 3 cholera, 9 dysentery and 1
paratyphoid fever patients entered the local infectious

disease wards in Kagoshima prefecture. In Kagoshima

checked by quarantine officer for 5 years (1989‑1993) .

Airport as a small local international airport, quaran‑tine system is not functioning well. Even in local air‑port, health consultation and adequate privacy‑oriented
information service of tropical medicine for the trav‑

Bacteriological examination for their stool was done.

elers are necessary. Amendment of Quarantine law and

14 positive results were obtained (V. para‑

Prevention of infectious disease law is recommended forthe more effective control of imported infectious dis‑

endemic. According to the Quarantine law, 51 travelers

with diarrheal symptoms by their declaration were

haemolyticus=5, Enteroinvasive E. coli=2, P. shigel‑
loides=6, and Salmonella=1). No V. cholerae O1 wasease.

B ‑3 1ASYMPTOMATIC INFECTION OF CRYPTOSPORIDIUM
MURIS IN HUMANSTATSUYA KATSUMATA2, DANIEL HOSEA , EDDY BAGUS WASITOl,
PITONO SOEPARTol, IGN GDE RANUHI SHlNJI YOSHIKAWA2, MITSUO KAKU2,SHIGERU KOHN02 AND KOHEI HARA2
Tropical Disease Research Center, Medical School, Airlangga University

and Second Department of Internal Medicine, Nagasaki University School of Medicine2

In mammals, Cryptosporidium parvum andCmptosporidium muris are accepted as distinct and valid

species. The substantial differences in oocyst size aswell as location allow the two species of Cryptospor‑

idium infectious for mammalian hosts to be distin‑
guished. While C. parvum has been recognized as acause of diarrhea in humans, infection by the parasite,
C. muris has never been reported in humans.

Of 6328 subjects examined for Cryptosporidium in
Surabaya, Indonesia, two were excreting oocysts muchlarger than those of C. parvum. and these were mor‑
phologically identified as oocysts of C. muris. The
patients were a 4‑year‑old and a 6‑year‑old healthy
female children. More than 104 oocysts per lg of feces
were found by Sheather's sugar flotation combined withphase‑contrast microscopy and modified Kinyouin acid‑fast stain. Daily examination revealed that oocyst
excretion continued for the next 5 and 6 days respective‑

ly without abdominal symptoms. In our study group, theprevalence of C. muris infection(0.03%) was lower thanthat of C. parvum infection(1.4%) . Both cases infectedwith C. muris were found in rainy season when C.
parvum infection was more prevalent also.
The present cases showed that C. muris can infecthumans. Natural infection with C. muris has beenreported only in rodents and ruminants, while C. muris
can be experimentally transmitted to mice, rat, guinea
pigs, rabbits, dogs, and cats. Although human C. muris
infection seems to be rare and asymptomatic, the infec‑
tion might cause some positive symptoms in immunosup‑

87pressed subjects. Further studies on the source of trans‑

epidemiology of C. muris infection in humans are need‑mission, Iocation of the parasite, pathogenicity, and

ed.

B‑32

VENOMOUS SNAKEBITES IN OKINAWA PREFECTURE IN 1993
SEIKI KATSUREN and MASATOSHI NoZAKI
Habu Study Section, Okinawa Prefectural Institute of Health and Environment.
During the year 1993, 159 venomous snakebites werereported in Okinawa Prefecture. 102 bites were caused
by Habu (Trimeresurus jlavoviridis), 40 cases by Saki‑shimahabu ( Trimeresurus elegans) and 17 cases byHimehabu ( Ovophis okinavensis). There were no fatal‑
ities reported.

Venemous snakebite usually takes place in earlysummer and autoumn. In 1993, it was reported that
there were 22 cases in October, 16 cases in Novemberand 10 cases in May.
In the case of Habu bites 32 incidents (32%) took

OO. Many of the daytime Habubite cases occurred insugarcane fields while farmers were working. In otherwords, farming activity brought individuals closer toHabu. On the other hand, many of the incidents occur‑
ring between 18:OO and 06:OO took place within house

hold properties. In other word, Habu invaded humancommunities.
Due to the advancement of medical techniques, well‑provided medical institutes and well‑developed trans‑
portation enabled the victims to reach the medical carein a short time. Case of death are rarely reported andplace in agricultural fields, followed by 20 incidents

the number of cases with severe parmanent damage(20%) in domestic dwellings, 14 (14%) indoors, 11

have decreased.

(11%) on grassy lands, 10 (10%) on roads and 3 (3%) in

Because of serum disease caused by the use ofantivenom, for many victims of Sakishimahabu andmountains and forests. In the case of Sakishimahabu
incidents 17 cases (43%) occurred in the fields the mostHimehabu which are less poisonous than Habu,

commbn location. 6 cases (35%) of Himehabubites, themajority, took place in around domestic dwellings.
Although the Habu is nocturnal, many incidents
happened during the daytime rather than evenings. In
1993, many Habu bites took place around noon and 20:

antivenom has not been used. Even Habu bite cases,
antivenom was used for 75% of the victims. In recent
years, if the victims suffer only slight symptoms,
antivenom is not used so often as it used to be.

B‑33

THE NEUTRALIZATION EFFECTS OF HABU‑ANTIVENOMAGAINST SNAKE‑VENOMS EXTRACTED FROM SNAKES
RECENTLY CAPTURED IN OKlNAWA ISLANDMASATOSHI NOZAKI, TAKAO KAMURA AND SEIKI KATSUREN
Habu Study Section, Okinawa Prefectural Institute of Health and Environment

Several kinds of venomous snakes, not indigenous
to Okinawa, have been imported to Okinawa for various

land and road sides in the farming areas. The incidents
are causing a considerable worry to the nearby commu‑

purposes such as production of alcholic beverages

nities.

containing the whole corpses of snakes, snake powder
production, mongoose and snake fight shows for the
tourist indurtry and display purposes.

Some of these snakes which had escaped fromamusement sites were recently captured on agriculturalThough it is very difficult to estimate whether thesesnakes reproduce in Okinawa or not, neutralization testsof venoms and habu‑antivenom were carried out against
the eventuality of attacks.

Hemorrhagic activity was assayed by intracutaneous

88

injection into rabbits and intramuscular injection intomice. Lethal toxicity was tested by intravenous injec‑tion into mice.

The results were as follows:
1 . LD50 of snakes recently captured in Okinawa island
was as follow: Habu ( T. flavoviridis) venom: 35.0 (31.

8‑38.5)pg, Sakishima habu (T. elegans) v.: 48.0 (45.

1.80 (1.53‑2.07)pg, Hybrid v.: 0.51 (0.41‑0.6Dpg,
and Taiwan‑habu v.: 0.56 (0.46‑0.67)pg. Thai‑cobra
v. didn't show hemorrhagic activity.
2 . In intravenous injection into mice, 0.1 ml of habu

antivenom neutralized lethal activity of 162pg (4.
6LD50) of habu v. 99pg (2.lLD50) of Sakishima‑habu
v. 49pg (2.3LD50) of Hybrid v. and 120pg (4.0LD50) of

9‑50.2)pg, hybrid of Habu and Sakishima‑habu v.:

Taiwan‑habu v., but habu antivenom could not neu‑

21.0 (19.1‑23.Dpg, Taiwan‑habu (T. mucros‑

tralize Thai‑cobra v. In intramuscular injections into

quamatus) v.: 30.0 (26.8‑33.6)pg, Thai‑cobra (N.

mice, 0.1rnl of habu antivenom neutralized hemorr‑
hagic activity of 30pg of Habu v., 30pg of Sakishmahabu v., lOpg of Hybrid v. and 30pg of Taiwan‑habu v

kaouthia) v.: 8.7 (7.9‑9.5)pg.

MHD (Minimum Hemorrhagic Dose) of each snake
were Habu v.: 0.56 (0.46‑0.67)pg, Sakishima‑habu v.:

B‑34

STUDY ON CHlNESE COBRA (NAJA ATRA)TOXOID FROM GUANGXI, CHlNA
Y. KAWAMURAl, A. SAKA11, Y. SAWAll

AND N.‑P. WAN2
Japan Snake Institute, Yabuzuka‑honmachi, Nitta‑gun, Gunma 379‑23, Japanl
and Guangxi Medical University. Nanning, Guangxi, China2

Detoxification of ethanol fractionated Chinese
cobra (Naja atra) venom of formalin, and the im‑venom. Thus, it was calculated that Iml of themunogenicity by the toxoid have been investigated. The
venom was supplied by the Guangxi Medical University,

venom. From the protection test, immunized guinea

antivenorn neutralized 40 or 50 pg (4‑5 mlds) of thepigs could survive against intramuscular injection of 0.3

Nanning, China. Immunogenicity of the toxoid was

or 0.6 mg (1 or 2 mlds) of the venorn. Further, 6 out of

tested by injecting the toxoid subcutaneously into guinea

8 guinea pigs survived against the venom of 1.2 or 2.4 mg

pigs. 0.4 or 0.5 ml of antivenom taken from 15 immun‑
ized guinea pigs neutralized 20 pg (2 mlds) of cobra(4 or 8 mlds) .

B‑35

COMPARISON ON THE SEASONAL ABUNDANCEOF ANOPHELES MINIMUS IN SOUTHERN YUNNAN
PROVlNCE, CHlNA AND NORTHERN THAILAND
MASAHIRO TAKAG11, LI‑GANG CHE2, YOSHIO TSUDAl

AND WANNAPA SUWONKWERD3
Department of Medical Entomology, Institute of Tropical Medicine, Nagasaki University*,

The Research Institute of Malaria Prevention and Treatment of Yunnan Province'
and Malaria Center Region 2, Ministry of Public Health, Thai Government'

Seasonal abundance of Anopheles minimus, one of thethe adults baited by 2 human or I baffalo and larval

important malaria vectors on foothills and forest fringes

collection by dipping in slow running streams and theirin southeast Asia was investigated in southern Yunnan
Province and in northern Thailand. Night collection forsurrounding areas were conducted monthly. In Yunnan
Province, adults peaked in September, decreased after‑

89

ward, and could not be collected in November. The
second peak of adults was encountered in April. In
northern Thailand, An. minimus was much more abun‑dant than in Yunnan Province, and showed a large peakduring the first half of the dry season (November‑Janu‑ary). Differences on the seasonal pattern in abundanceof the adults may be due to difference in the averagetemperature between the two study areas. Cool condi‑tion during the dry season in Yunnan Province mayhinder rapid increase of An. minimus, even if conditionsof streams such as the water current were stable. This
speculation was agreed with results obtained by larval
surveys again.

B‑36

GROWTH CURVE OF MOSQUITO LARVAL POPULATIONAND COMPARISON OF GROWTH RATEKIYOSHI MAKIYA AND MARIKO NISHIYAMA
Department of Parasitology and Tropical Public Health,

University of Occupational and Environmental Health

It is important to know whether mosquito larval

Comparison was made among these 3 models to esti‑

population is young or old in order to decide effective

mate growth rate of larval populations in the field. As

control measure against mosquito‑borne diseases. And

a result, Bertalanffy curve, Lt=Li[1‑exp(‑k(t‑it is theoretically possible to estimate growth rate oflarval populations of mosquito in the field by following
successive change of a single age group (cohort) in ageof Aedes albopictus and Tripteroides bambusa. In thisequation, Li is final stage of larval population to be(instar) distribution.

reached and k is growth rate to specify respective

However, it is often difficult to follow a single
cohort because age distribution of larval populations iscurves.

overlapped among different cohorts in the field. Cassie'

and 12 T. bambusa larval populations surveyed with
oviposition traps in a small hill with dense trees.

s method was introduced to analyze such a polymodal
age distribution to follow successive growth of a singleto) ) J , was proven to fit best to mosquito larval growth

Bertalanffy curves were fitted to 18 A. albopictus

larval cohort. Average larval age and standard devia‑
tion of the cohort were calculated by simulation pro‑

Correlation coefficients showing the curve fitness weresignificantly high for all the larval populations. Aver‑age growth rate (k) was estimated to be 0.104/day forgram of Cassie's analysis in case of polymodal age
distribution. Growth curve of larval population was

T. bambusa and 0.224/day for A. albopictus, meaningthat A. albopictus grew about 2 times as fast as T.

made by connecting these average larval age serially.

bambusa larval populations. It became possible to know
and to compare growth rate of mosquito larval popula‑Among several models for organism growth, 3
growth curves, i.e. D Logistic, 2) Gompertz and 3)
Bertalanffy models were often used for this purpose.

tions quantitatively by using this growth curve.

90

B‑37

IN VITRO EFFECT OF PLANT‑EXTRACTS,ESPECIALLY THOSE IN GUATEMALA,

AGAlNST YRYPANOSOMA CRUZI (MlNlREVIEW)
J. MAKll, Y. ITOl, I. TADA2, K. OGATA3,

N. GARCIA4 AND A. CACERES4
Kitasato University School of Medicine , Faculty of Medicine, Kyushu University',

Cooperative JICA Project of Research by Guatemala/Japan for Control of Tropical Diseases'

and School of Chemistry and Pharmacy, University of San Carlos, Guatemala

Since Nussenzweig et al. (1953, Rev. Paulista. Med.
42: 57‑58) demonstrated the in vitro efficacy of gentian

Bixa orellana. Neurolaena lobata. Tagetes lucida andviolet on Trypanosoma cruzi, the plant‑origin drug has
been used for the in vitro treatment of the protozoal
parasites contained in human blood to be transfused.

Guatemala. The drugs to be tested were extracted from

For the decades, many other medicinal plant‑derived

against T. cruzi in vitro. This communication is a

1 ml epimastigote suspension in LIT culture medium(Tulahuen strain, 3 x 106/ml) and 0.1 ml extract‑LIT
medium at the final concentration of 250mg/ml was

minireview of such in vitro studies with special refer‑agitated in small holes of a plate and incubated at 28'C

ence to those in Guatemala.
About 20 articles have described the lethal efficacy
of medicinal plant extracts against epimastigotes andtrypomastigotes of T. cruzi in vitro all over the world

for 48 hours. The negative control was free from any
extract. The in vitro efficacy of the crude drugs was(Maki & Caceres, 1994, JICA Report, pp.14). In the

incubation, 10 pl were aliquoted from each well and put

report, the articles, and names of plants and compounds
were presented in a table. To the best of the authors'

on a glass (Neubauer chamber) . The number of para‑sites was counted under a microscope ( x 400). The

knowledge, no articles except preliminary reports by
Maki & Caceres(1994, ibid.) and Caceres et al.(1995,
Jpn. J. Parasit. 44, in press) have been published on theefficacy of gentian violet was confirmed. The compari‑son of the counted number of parasites in experimentalgroups with that in the negative control suggested thesubject in Central American countries, though the coun‑

possibility that the extracts of the four plants were moredrugs have been tested experimentally for their efficacy

Smilax regelii were collected in Mazatenango,
the plants with ethanol and prepared as described in thereport(Maki & Caceres, 1993, ibid.). The mixture of O.

compared with that of a reference drug, purified gentian
violet at the final concentration of 250pg/ml. After thetries like Guatemala are rich in medicinal plantor less effective against T. cruzi epimastigotes under the

resources(Maki & Caceres, 1993, JICA report, pp.28;

present condition. Among others, the extract from N.

Caceres et al., 1993, Enfermedades tropicales enlobata was shown to be fairly effective in killing T. cruzi

Guatemala. Informe Anual 2, 140‑143) .
One of the newest studies on in vitro efficacy ofin vitro at the significant reduction rate of 60%. The
dose‑effect relationship in vitro and the possible sup‑Guatemalan plant extracts against T. cruzi(Maki &Caceres, 1994, ibid.) is described in somewhat morepressive effect of the extract on the proliferation of thedetail as follows. The four kinds of medicinal plants,

protozoa in mice are now being studied.

91

B‑38

INFORMATION COLLECTlNG AND ACCESS SYSTEMOF MEDICAL ASSESSMENT IN THE TROPICS
HIDEYO ITAKURA
Department of Pathology, Institute of Tropical Medicine, Nagasaki University

We as health professionals are constantly asking:1) how can we keep up‑to‑date with current informa‑tion and trends in the tropics? 2) how can we diagnose
and treat exotic diseases in the tropics? 3) how can we
instantly get access relevant information in the tropics?
4 ) what therapy should we use in the tropics? 5) where

the same tirne, easy access to such information is also
important. The plan for establishment of such system inthe tropical medicine is under investigation by special‑ists in various academic fields including epidemiologists,

microbiologists, parasitologists, geopathologists, publichealth specialists, and information technologists. The

can we go for expert advice in the tropics? Informationcollecting and access system is a computer‑based net‑plan should include: 1) preparation of network, 2)

work and a comprehensive software tool that can help
you. It is important to collect information in medical
fields in the tropics to perform medical activities withnew information and data, and 4) maintenance of theestablished network. The proposed system should beworldwide and useful to every specialist in medical

modernized medicine in appropriate methods and ways.

f ields.

accumulation of products of software, 3) stockpiling of

The information from tropical areas should be fresh. At

B‑39

THEME OF HEALTH IN NORTH EAST THAILANDMICHIYO UMEMOTol, JUNKO TATEMATSU , KUNIYASU SUZUKll,
KENJI YAMAGAl, TOMOKO KUNll , HIROMI SUZUK11
AND YOSHlNARI MORIYAMA2
Suzuka University of Medical Sciences & Technology
and Teikyo University, Faculty of Medicine'

Though, Thailand is one of tropical developing
country, recentry the economic growth is remarkable.
Life style of rural area was also changed. In North east

Thailand, change of health situation and problem of
medical subject were studied. Price of groceries were
10% that of Japan except egg which price was same as

study was carried out was famous season of epidemic
diarrhoea. Outbreak of diarrhea due to bacterial infec‑
tion was not seen, and geriatric diseases seemed predom‑
inant rather than. A Iot of females 20 y.ears to 40 years

old who diagonized was overweighted girl. The reason

why hypper‑tension patients were not many was

Japan. Relatively, daily necessaries such as books, pens,

because the costom of little intake of salt. Twelve

papers, etc. were more expensive than groceries. Eating
habit was same as 10 years before as far as their eating

infant were tested for finding the carrier of pathogenic
bacterria. Non‑OI Vibrio cholerae as isolated from oneof them. Numbers of delivery times were 2 times, andthis fact suggest that effect of birth control by Thaigovernment had been popular in rural village of North
East Thailand.

sticky rice as the staple food. But the divercification offood was also seen in the dish compared with situationof 10 years before, because spread of using money
became popular in the village. Though, the season this

92

B‑40

ENVIRONMENTAL HEALTH IN NORTH EAST THAILAND
HIROYUKI MASUDAl, KAZUYA HATA , KEN OSUMll,
RIKl KUWABARAl, EIKO YAMANAMll, MAKIKO MURAMATSU1,
HARUMI ITO AND YOSHlNARI MORIYAMASuzuka University of Medical Sciences & Technology
and Teikyo University, Faculty of Medicine'

Construction of toilets and preparing big jar for
stocking rain water of drinking purpose for dry season
were major sanitary project in Thailand. Frequency ofdrinking under ground water became less than before.
On the other hand, because the cutting forest, the cli‑mate changed to dry condition and salt soil from under‑
ground increase the concentration of NaCl%. Environ‑mental research of a rural village in Northeast Thailand

had done in March 1994 dry season for this study.
Concentration of NaCl in water both drinking anddomestic, bacterial contamination of water by coli‑
forms, fishes and domestic animals by salmonella werestudied in this research. Though, ten years before,
people in the village used rain water in rainy season andtime when last reaseach had done in this village, most of

hausing equipped a large jar and they used rain water as

drinking water even in dry season in this research.
Majority of hause in the village had refrigerator and,drinking water was usually colled. Most of drinking
water was contamined by coliforms, because chlorina‑
tion had not done. Vibrio alginolyticus was isolated
from fresh water fishes in pond of this village whichshowed a little NaCl concentration. Non‑OI V. cholerae
and V. fulvialis were isolated from a duck in the village.

As mentioned above, especially system of drinking
water shows, circumstance of Northeast Thailand im‑proved recentry, and still have problems of Vibrio con‑tamination due to high NaCl concentration of water.

shallow well in dry season for drinking purpose at the

B‑41

GENETIC POLYMORPHISM OF GROUP‑SPECIFIC COMPONENT,
TRANSFERRIN AND ALPHA‑1‑ANTITRYPSIN IN KAGOSHIMAPREFECTURE: THE SEROLOGICAL CHARACTERS
OF THE POPULATIONS IN THE NANSEI‑SHOTO
KAZU1'OSHI AGO AND OSAO TSUGANEZAWA
Departn'e t of Legal Medici**, Faculty of Medici e, Kag'shim* U**iversity

Distribution of group‑specific component(GO,revealed. Six different types of rare variants weretransferrin(TF) and alpha‑1‑antitrypsin(PD variants

observed, and the distributions of the variants were notsignificantly different among the four populations.

were investigated to clarify characters on serologicalgenetics of Mainland, Tanegashima, Yakushima andAmami Oshima populations in Kagoshima Prefecture.

Tanegashima, Yakushima and Amami Oshima are[TF types] The frequencies of common alleles(TF*C1,

TF*C2) in Mainland, Tanegashima, Yakushima andAmami Oshima were within range of the values ofislands in the Nansei‑Shoto. Typing was performed byisoelectric focusing with acrylamide gels followed byimmunoprint for GC and protein staining for TF and PI.

which the allele frequencies had been obtained at 12
districts in Japan. A new variant with a band which[GC types] Geographical cline of GC*1F, GC*1S andved in the populations of Amami Oshima and Yaku‑

GC*2 frequencies in Japan was examined by use of datashima. This variant was tentatively designated Dama.

estimated in this study and 23 data which had been

The TF*Dama frequencies were 0.0055 in Amarni

obtained at 17 districts. Existence of geographical cline

Oshima and 0.0010 in Yakushima. From these findings,

that the GC*2 frequency gradually increased and GC* IS

the Dama is likely to be characteristic of the population

frequency decreased from Aomori to Okinawa wasin Amami Oshima.

more cathodically located than the Dchi band was obser‑

93[PI types] The frequencies of common allele (PI*M1,

PI*M2 and PI*M3) in Mainland, Tanegashima, Yaku‑shirna and Amarni Oshima were within range of thevalues of which the allele frequencies had been obtained

at 10 districts in Japan. Interesting two rare variants

which were tentatively designated Eama and Pyal were
found in this study. The Eama was restrictively obser‑
ved in the population of Amami Oshirna only (PI'Eama

frequency: 0.0063) and the Pyal in the population ofYakushima only(PI*Pyal frequency: 0.0059). The
Eama was identified with I by the comparison test forreference serum. The I is extremely rare in the otherJapanese populations so far investigated. From thesefindings, the Eama and the Pyal are likely to be charac‑

teristic of the populations in Amami Oshima and Yaku‑shima respectively.

MARCH1995

VOL．23NO．1

CONTENTS
Original article
Fukuhara，H．，Ishimine，T．，Futenma，M．and Saito，A。
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