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DetectionofIndirectAgglutination

Praphathip
Adisakdi

Antibodies

toPα解sケon8夕1κs6召n孟on6s奮inEamsobhana，Dom

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Sera

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J pn. J. Tro p. Med. Hyg., Vol.

127, No. 1 1999 pp. 1‑5

DETECTION OF ANTIBODIES TO PARASTRONGYLUS CANTONENSISIN HUMAN SERA BY GELATIN PARTICLE
INDIRECT AGGLUTINATION TEST
PRAPHATHIP EAMSOBHANA , DORN WATTHANAKULPANICH2PAIBULAYA PUNTHUPRAPASA1, ADISAKDI YOOLEK AND SOMKUAN SUVUTTHO
Received October 27, 1998/Accepted December 18, 1998

Abstract: A newly developed agglutination test using gelatin particles as an antigen carrier (GPAT) was
compared with a conventional enzyme‑linked immunosorbent assay (ELISA) for the detection of Parastron‑
gylus cantonensis antibodies in sera from patients. A total of 70 sera was used in the study. Of these, 10 each

wefe from patients with parastrongyliasis, gnathostomiasis, paragonimiasis, cysticercosis, toxocariasis,
filariasis and malaria. The control group consisted of 50 serum samples from normal healthy individuals.
The mean reciprocal titer of the parastrongyliasis patients group was significantly higher than that of thenormal group as well as those of other parasitic infections. The sensitivity nd specificity of the GPAT were100% and 92.4%, respectively. The results of GPAT in detecting P. cantonensis antibodies appeared to beclosely correlated with those obtained with ELISA. The GPAT, however, is more easy, rapid and cheap; it
may also be a test of choice for routine immunodiagnosis of human parastrongyliasis.
Key words: Immunodiagnosis, Parastrongylus ( = Angiostrongylus) cantouensis, gelatin particle indirect
agglutination test (GPAT) , ELISAINTRODUCTIONly, a newly developed agglutination test using gelatin
particle as an antigen carrier has been shown to be a

Human eosinophilic meningitis or meningoence‑

sensitive and specific method for the diagnosis of humanphalitis caused by Parastrongylus (=Angiostrongylus)
cantonensis is endemic throughout Asia and the PacificIslands (Cross, 1987; Kliks and Palumbo, 1992). The

tosomiasis (Yang et al., 1994; Kobayashi, 1995) , Chagas'

most reliable diagnosis of this parasitic disease is basedon the presence of either larvae or juvenile worms in the

cerebrospinal fluid (CSF) from the patients. Suchstrongyloidiasis (Sato and Ryumon, 1990), schis‑disease (Yamashita et al., 1994) and opisthorchiasis(Watthanakulpanich et al.. 1998). GPAT is technicallysimple and can be performed rapidly without specialized
apparatus or facilities. These make it convenient to use

diagnosis nevertheless is rare since worms are seldomfound in the limited volume of CSF taken for analysis.
A variety of immunological tests based on detecting

in the diagnosis of many diseases both in the labora‑

serum antibodies against P. cantonensis has been used to

GPAT could be used to detect serum antibodies of

support the diagnosis. These include intradermal test,
indirect hemagglutination test, immunodiffusion, im‑munoelectrophoresis, complement fixation test, ELISA
and immunoblot test (Tharavanij, 1979; Ko, 1987; Eam‑parastrongyliasis (=angiostrongyliasis) patients. The
results were compared with those of ELISA.

sobhana et al..1997). The enzymatic test system has

tories as well as in the field.

In this study, we attempted to evaluate whether

MATERIALS AND METHODS

become more widely used because of its greater sensitiv‑
ity. The test, nevertheless, requires specific materials,

Antigen preparation
Adult worms of P. cantonensis were obtained from

specialized equipment and expensive reagents. Recent‑the pulmonary vessels of infected Wistar albino rats as

Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
2. Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

Correspondent author: Dr. Praphathip Eamsobhana, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok
10700, Thailand.

E‑mail: sipes@mahidol. ac. th2previously described (Eamsobhana et al., 1997). The
worms were homogenized in a small volume of normal

particles spread out uniformly covering the bottom ofsaline with a glass tissue grinder. The suspension wassonicated and extracted overnight at 4"C in a refrigera‑
tor. Soluble antigens were obtained as supernatant after

was determined as the highest serum dilution giving acentrifugation at 4,000 rpm for 15 min. The protein
content of the extract was determined using a protein
assay kit 11 (Bio‑Rad Labs, USA) .

Enzyme‑linked immunosorbent assay (ELISA)

the well indicated a positive result. The antibody titerpositive agglutination pattern.

To evaluate the results of GPAT, the ELISA wasalso applied for assessment of serum antibodies to P.
cantonensis.

Sera
Serum samples were obtained from five patientsThe ELISA was performed according to the method
described by Voller et al. (1976) with some modifica‑

with parasitologically confirmed parastrongyliasis (3tions. Briefly, wells of microtiter plate (Nunc,

with cerebral parastrongyliasis; 2 with ocular parastron‑

Denmark) were sensitized with 100 pl of P. cantonensis
antigens at a concentration of 5 pg/ml of protein in
carbonic buffer solution, pH 9.6. The wells were succes‑
sively incubated for 2 hr each with 100 pl of blocking
solution (2% skim milk in PBS‑Tween) , serum samples

gyliasis) and five patients with presumptive parastron‑

gyliasis. The latter group was diagnosed as parastron‑
gyliasis based on clinical symptoms and history of expo‑
sure to infection, as well as having high antibody titers

as detected by ELISA.

Sixty heterologous sera were collected from

diluted to 1:100 with PBS containing 1% bovine serum
albumin and 0.05% Tween 20, and peroxidase‑conjugat‑

patients suffering from other parasitic infections. Of
these, 10 sera each were from patients with gnathos‑ed anti‑human immunoglobulins (Dakopatt, Denmark)tomiasis, toxocariasis, filariasis, paragonimiasis,

incubated for 30 min with the substrate (o‑cysticercosis and malaria. A11 these cases were positiveby parasitologic and/or serologic tests for a specificparasite or its products. The normal control group ofsera were obtained from 50 healthy adults who were
negative for any parasitic infection at the time of blood

diluted to 1:1,000 in PBS‑Tween. Finally, the wells werephenylenediamine) solution. The enzymatic reaction
was stopped with 50 pl of 2.5 N sulphuric acid and theoptical density (OD) was measured at 492 nrn with an

ELISA reader (SLT Labinstrument, Australia).
The optimal concentration of the antigens and thecollection. All serum samples were kept at ‑20'C until

optimal dilution for patient's serum and conjugate wereuse.

pre‑determined using a chequerboard titration. For
each test, a negative, a positive and a PBS‑Tween

Gelatin particle indirect agglutination test (GPAT)

controls were included.

The GPAT was performed as previously described(Watthanakulpanich, 1998). Briefly, the pre‑deter‑

A result was considered positive if the OD value
exceeded the mean OD+ 3SD of the values obtained withmined optimal concentration of P. cantonensis antigens

the 50 negative sera.

(50 pg/ml) was conjugated to the artificial gelatin
particles (Fujirebio Inc.. Tokyo, Japan) treated with 5

Statistical analysis

pg/ml tannic acid solution. After conjugation of

Sensitivity and specificity of the tests were deter‑

antigens, the gelatin particles were washed 3 times andfinally made into a 1% suspension in phosphate buffered

mined using the method of Galen (1980). Association
between GPAT and ELISA was evaluated using linear
correlation and regression after the titers of GPAT

saline (PBS), pH 7.0 containing inactivated normal
rabbit serum. These coated gelatin particles were thenready for use.

For estimation of agglutination titer, one drop
containing 25 pl of the antigen‑coated particles suspen‑sion was mixed in the U‑bottomed micro‑wells with anequal volume of test serum in 2‑fold serial dilutions.
The particles were allowed to settle for at least 3 hr atroom temperature and the agglutination patterns in theplates were read according to Campbell et al. (1974);
particles concentrated in the shape of a compact buttonin the center of the well indicated a negative result;were transformed into logarithm (10g2) 'RESULTS
The distribution of GPAT titers in 70 patients and

50 uninfected controls is shown in Figure 1. All 10
patient sera with parastrongyliasis showed positive
agglutination response at serum titer of 1:32 or more
(10g, reciprocal titer ; 5) , whereas negative results were

demonstrated at the lowest serum titer of 1:16 (10g,reciprocal titer 4) in all the normal individuals. The

3

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..*"‑ g'."pFigure I Distribution of GPAT titers (log,) for detectionof serum antibodies against P. cantonensis in 10
patients each with parastrongyliasis (A) , gnath‑

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Figure 2

ostomiasis (B) , toxocariasis (O , filariasis (D) ,

s.*
9""p
Distribution of optical density values in ELISA
for detection of serum antibodies against P.
cantonensis in 10 patients each with parastron‑
gyliasis (A) , gnathostomiasis (B), toxocariasis

paragonimiasis (E) , cysticercosis (F) , and(O , filariasis (D) , paragonimiasis (E) , cysticer‑

malaria (G) and in 50 normal healthy individuals(H). The dotted line indicates cut‑off titer.

cosis (F) , and malaria (G) , and in 50 normal
healthy individuals (H). The dotted line shows
the cut‑off value.

10g2 reciprocal titers in the parastrongyliasis patients

ranged from 5 to 8, with the majority from 6 to 7.

1.G ‑The mean antibody titer (X SD) of normal
healthy individuals was 3.15 0.58. The mean antibody
titer of parastrongyliasis patients group was significant‑ly higher than that of the normal group as well as thoseof other parasitic infections (P<0.01). A cut‑off titerfor the positive antibody response was then establishedat X+3SD of the healthy group which was at log2
reciprocal titer of 4.89. The GPAT was positive for all
the parastrongyliasis patients but negative for the nor‑eeE t4‑c.,'n'r 1.2‑

a
O

e
e
e

b,

o, i.o.

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e
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e

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,,

( .8‑

O
:
Ul .c‑ey = 0.28 x‑0.857' = O.9G8

n= 10

mal controls.

Of the 60 serum samples from other groups ofparasitic infections, 51 (85%) were negative, while 9(15%) were cross‑reactive at the cut‑off titer. The
cross‑reactive serum samples were from patients withgnathostomiasis (5/10), toxocariasis (2/10), filariasis(1/10), and paragonimiasis (1/10). None of the

4.5 b.o 6.0 6.5 7.0 7.5 8.0 8.s
5 i

GPAT tit'r(1'g2'

Figure 3 Correlation between GPAT titers (10g2) and
ELISA values performed on 10 sera from paras‑
trongyliasis patients.

cysticercosis and malaria patients showed cross‑reac‑tion. The sensitivity and specificity of the GPAT were100% and 92.4%, respectively.
The ELISA was carried out using different dilutionsof sera from healthy individuals and from patients withAs shown in Figure 2, all the serum samples fromparastrongyliasis cases (10/10) were positive in theparastrongyliasis. The maximum difference in OD

tomiasis (6/10), toxocariasis (3/10), filariasis (1/10),

values was observed at 1:100 dilution which was used toand paragonimiasis (2/10). Normal parasite‑free indi‑

evaluate all serum samples. The mean OD value (XSD) of the normal group was 0.253i0.107. The meanviduals were all negative. The sensitivity and specificity

ELISA and 48 of 60 sera from patients with otherparasitic infections (80%) were negative. Cross‑reac‑

tions were found with serum samples from gnathos‑plus three standard deviation OD value of the healthygroup sera was then taken as the cut‑off value, OD)>of the ELISA were 100% and 90.2%, respectively.
Figure 3 represents the correlation of GPAT titersand ELISA values on the parastrongyliasis patients. A

0.574 indicated positive results.

significant correlation was found between the two tests,

4

the correlation coefficient was 0.968.

DISCUSSION
The definitive diagnosis of parastrongyliasis is
made when Parastrongylus worms are found in the CSF
of patients. Proven cases of human infection showing

worms are however rare (Punyagupta, 1979) and the
ELISA utilizing either crude or partially purified Paras‑
trongylus antigens is currently used as a reliable method

for immunodiagnosis of the infection (Welch et al.. 1980;

Chen, 1986; Yen and Chen, 1991). Although ELISA isa reliable immunological test for human parastron‑
gyliasis. The indirect agglutination test is technicallysimple to perform, requires no specialized skill, equip‑

ment and facilities, and can be completed within threehours. The advantages of the gelatin particles are theirstability and resistance to mechanical agitation. The
particles are colored and therefore convenient for read‑ing the setting pattern. Moreover, the particles can belyophilized for long term storage after sensitization with

antigen. Therefore, the GPAT can be performed by the
one‑step reaction of the preserved antigen‑particles

sensitive enough for detecting serum antibodies to P.

with a test serum, thus applicable both in less equippedlaboratories as well as in a field survey. Nevertheless,cantonensis, it is expensive, Iaborious and limiting withbecause of the relatively strong cross‑reaction withrespect to the length of time to get results.

other clinically related parasite, Gnathostoma spim er‑
um, the use of more specific antigenic preparation in the

The recently developed inert gelatin particles areactively being employed as an antigen carrier for vari‑
ous diagnostic kits, and GPAT has already been success‑assay will be needed. Experiment on purification of theP. cantonensis specific antigen for future use is under‑

fully applied by a number of investigators for im‑way.

munodiagnosis of various parasitic infections (Sato and
Ryumon, 1990; Yamashita et al.. 1994; Yang et al.. 1994;
Kobayashi et al.. 1995; Wattanakulpanich et al.. 1998).

ACKNOWLEDGMENT

In the present study, we confirmed the findings that

We wish to thank Mr. Ryuichi Fujino and Mr.

GPAT could also be used for immunodiagnosis of

Shuichi Horikawa, Fujirebio Inc., Tokyo, Japan, forhuman parastrongyliasis. The sensitivity of the present

GPAT did not differ from that of the ELISA when the

supplying the gelatin particles. We would like also to
thank Dr. Wanchai Maleewong, Dr. Peera Buranakitjar‑

overall rates of positive reactions among patients' sera

oen, and Mr. Paron Dekumyoy for kindly providing

diagnosed to be parastrongyliasis were compared; inserum samples of parastrongyliasis, gnathostomiasis
and paragonimiasis patients. We are most grateful toProfessor Dr. Yong Hoi Sen, Institute of Biologicalsera of 10 patients with parastrongyliasis, positive anti‑

body response was demonstrated in all of them by the
GPAT and ELISA. When such tests were evaluated forspecificity by testing sera from healthy individuals
presumed to be normal, the result did not show any
false‑positive reactions in each test. A significant

Sciences, University of Malaya, for his critical readingof the manuscript. This work was supported in part by
the Siriraj China Medical Board Grant No. 75‑348‑269
to the senior author.

correlation was observed between GPAT and ELISA.
Sera from patients with other parasitic infections

were also examined by the GPAT and ELISA to deter‑mine the specificity of the tests. Strong positive
responses were observed in a few patients with gnatho‑
stomiasis. Weak cross‑reactive positive reactions also
occurred in sera from a few patients with toxocariasis,
filariasis and paragonimiasis. The responses, however,were lower in the GPAT. The sera from patients withcysticercosis and malaria showed no cross‑reactivity in

REFERENCES
1 ) Campbell, D.H., Garvey, J.S., Cremer, N.E. and Sussdorf,

D. H.(eds.) (1974): Methods in immunology (A Iabora‑
tory text for instruction and research) . pp. 279‑283.
Benjamin, W.A., Inc. Massachusetts
2 ) Chen, S.N. (1986) : Enzyme‑linked immunosorbent assay(ELISA) for the detection of antibodies to Angiostron‑
gylus cantonensis. Trans. Roy. Soc. Trop. Med. Hyg., 80,

both tests. The positive responses in gnathostomiasis,
toxocariasis, filariasis and paragonimiasis patients,
however, seem to correlate with the antigen preparation

398‑405
3 ) Cross, J.H. (1987): Public health importance of Angios‑trongylus cantonensis and its relative. Parasitol. Today,
3, 367‑369

used rather than the assay itself. By using a more

4 ) Eamsobhana, P., Mak, J.W. and Yong, H.S.: (1997):

defined antigen, the cross‑reaction can be expected todecrease (Ko, 1987).

ldentification of Parastrongylus cantonensis specific

The present study indicated that the GPAT can be

antigen for use in immunodiagnosis. Int. Med. Res. J., 1,
1‑555 ) Galen, R.S. (1980): Predictive value and efficiency oflaboratory testing. Pediat. Clin. North Am., 27, 861‑869

6 ) Kliks, M.M. and Palumbo, N.E. (1992): Eosinophilicmeningitis beyond the Pacific Basin: the global disper‑
sal of a peridomestic zoonosis caused by Angiostrongylus
cantonensis, the nematode lungworm of rats. Soc. Sci.
Med., 34, 199‑212
7 ) Ko, R.C. (1987): Application of serological techniques

for the diagnosis of angiostrongyliasis. In: Current
Concepts in Parasitology. R.C. Ko (ed.). pp. 101‑110.
The University of Hong Kong Press
8 ) Kobayashi, J., Sato. Y.. Soares, E.C., Toma, H., Brito, M.

C. and Dacal, A.R.C. (1995): Application of gelatin
particle indirect agglutination test for mass screening ofschistosomiasis in endemic area of Brazil. Jpn. J. Par‑
asitol., 44, 12‑18

NAMRU‑2 Special Publication No. 44, Taipei, Taiwan12) Voller, A., Bartlett, A. and Bidwell, D.E. (1976):
Enzyme immunoassays for parasitic diseases. Trans.
Roy. Soc. Trop. Med. Hyg., 70, 98‑103
13) Watthanakulpanich, D., Waikagul, J., Dekurnyoy. P. andAnantaphruth, M. (1998): Application of the gelatin
particle indirect agglutination test in the serodiagnosisof human opisthorchiosis. Jpn. J. Trop. Med. Hyg., 26,
5‑1014) Welch, J.S., Dobson, C. and Campbell, G.R. (1980):
Immunodiagnosis and seroepidemiology of Angiostron‑
gylus cantonensis zoonoses in man. Trans. Roy. Soc.
Trop. Med. Hyg., 74, 614‑623

15) Yarnashita, T, Watanabe, H., Maldonado, M., Legu‑
izamon, M.A., Watanabe. T., Saito, S., Shozawa, T.,
Sato. Y. and Sendo, F. (1994): Gelatin particle indirect
agglutination test, a means of simple and sensitive ser‑odiagnosis of Chagas' disease. Jpn. J. Trop. Med. Hyg.,

9 ) Punyagupta, S. (1979) : Angiostrongyliasis: clinicalfeatures and human pathology. In: Studies on Angio‑
strongyliasis in Eastern Asia and Australia. J.H. Cross(ed.). pp. 138‑142. NAMRU‑2 Special Publication No.
44, Taipei, Taiwan16) Yang, J., Chuang, C., Nakajima, Y. and Minai, M.
(1994) :Detection of antibodies to Schistosoma

10) Sato. Y. and Ryumon, I. (1990): Gelatin particle indirect

japonicum ova in schistosomiasis patients by gelatin

agglutination test for serodiagnosis of human stron‑
gyloidiasis. Jpn. J. Parasitol., 39, 213‑219

22, 5‑8

agglutination test. Jpn. J. Parasitol., 43, 280‑287

17) Yen, C.M. and Chen, E.R. (1991): Detection of antibody11) Tharavanij, S. (1979): Immunology of angiostron‑

to Angiostrongylus cantonensis in serum and cere‑gyliasis. In; Studies on Angiostrongyliasis in Eastern

brospinal fluid of patients with eosinophilic meningitis.

Asia and Australia. J.H. Cross (ed.). pp. 151‑164.

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J pn. J. Tro p.

Med Hyg Vol 27 No I 1999 pp. 7 ‑12

7SPOROZOITE INVASION OF PLASMODIUM BERGHEI,
RODENT MALARIA PARASITE, TO THE SALIVARY
GLANDS OF THE VECTOR MOSQUITO,ANOPHELES STEPHENSI= AN ELECTRON MICROSCOPIC STUDYKATSUHIKO ANDO1, KEITA KURAISHll, KIMIAKI NISHIKUBol,

TETSU AsAMll, PHILOMENE WAIDHET‑KOUADlol, HIROYUKI MATSUOKA2
AND YASUO CHINZE11
Received November 7, 1998/Accepted January 18, 1999

Abstract: The sporozoite penetration process of a rodent malaria parasite, Plasmodium berghei, into thesalivary glands of the vector mosquito, Anopheles stephensi and sporozoite distribution in the cytoplasm andsecretory cavity in the distal region of salivary glands were observed with a scanning electron microscope
and a transmission electron microscope. In non‑infected mosquitoes, many swellings were observed on theouter surface of the median lobes of salivary glands, whereas many shallow depressions were observed on
the lateral lobes. In infected mosquitoes, sporozoites were concentrated on the distal region of median andlateral lobes of salivary glands and penetration occurred from the anterior end into both lobes. Sporozoites
were about 10 pm long with one end flat and the other round. Small holes through which sporozoites might
have passed were observed on the surface of both median and lateral lobes. A white powder like substance,
which might come from the holes, covered the surface of both lobes. Sporozoites invading the cytoplasm of
the salivary gland cells were surrounded with vacuoles. These sporozoites invaded the secretory cavity and10dged to form bundles.
Key words: Plasmodium berghei, Salivary gland, Sporozoite, Invasion

INTRODUCTION
The salivary glands of mosquitoes play an impor‑tant role in transmission of mosquito‑borne diseases.
Therefore, the structures of mosquito salivary glands
have been studied by numerous researchers and fineinternal structures have also been observed with a trans‑mission electron microscope (TEM) (Wright, 1969;
Janzen and Wright, 1971; Barrow et al., 1975). The

tions (Sterling et al., 1973; Posthuma et al., 1989; Golen‑da et al., 1990; Ponnudurai et al., 1991; Pimenta et al.,1994) . In addition, reports on the penetration process of

sporozoites into salivary glands using scanning electron

microscopy (SEM) have also been published by Sinden(1975) and Meis et al. (1992). However their observa‑
tions did not concentrate on the penetration process of
sporozoites so that number of photos (1 sheet of photos
by Sinden and 3 sheets of photos by Meis) and descrip‑

salivary glands also play an important role in the life
cycle of malarial parasites. Sporozoites released fromtions are not sufficient. Especially, there is no discrip‑

mature oocysts are distributed in the hemolymph ofmosqiutoes and actively penetrate the salivary glandcells. It is not known whether the sporozoites movesporozoite penetration to the lobe is not clear.

actively or passively in the direction of the salivary

lateral lobes of the salivary glands of Anopheles stephen‑glands.

The penetration process of sporozoites into salivary

gland cells and biology of sporozoites have been widely
studied by transmission electron microscopic observa‑

tion of the salivary gland lobe and the direction of
In the present study, we show the penetration proc‑
ess of Plasmodium berghei sporozoites into median andsi by SEM and their distribution in the cytoplasm andsecretory cavity in the distal region of salivary glands

by TEM.

Department of Medical Zoology, School of Medicine, Mie University, Tsu, Mie 514‑0001, Japan
2. Present address : Department of Medical Zoology , Jichi Medical School, Yakushiji 3311‑1, Minamikawachi, Kawachi‑gun,Tochigi 329‑0498, Japan

8

MATERIALS AND METHODS
The parasies, P. berghei ANKA strain clone 2.34,
were kindly supplied by professor R.E. Sinden, Imperial

College, and have been maintained in our laboratory.
They were transmitted to BALB/C mice by A. stephensi
mosquitoes. The blood collected from infected mice(passage‑O mice) was stored at ‑80'C. This blood was

injected intraperitoneally (1xl07 parasites) into
BALB/C mice (passage‑1). When the parasitemia of apassage‑1 mouse reached lO%, a passage‑2 mouse wasvation without damage. Therefore, we dissected thesalivary glands together with the surrounding tisses andmainly observed the distal regions of both the medianand lateral lobes by SEM. In some specimens, we could
observe the intermediate region.

Many swellings corresponding to salivary glandcells were observed on the outer surface of the distal
region of median lobes of both non‑infected and infectedmosquitoes (Fig. 1) . However, these swellings were notso conspicuous in some lobes. Surface morphology oflateral lobes was different from that of median lobes.

prepared by injection of 5 >< 106 parasites from the pas‑

Many shallow depressions were observed on the outersage‑1 mouse fresh blood. A. stephensi females (4‑7

surface of the distal region of the lateral lobes of both
non‑infected and infected mosquitoes (Fig. 1) . No holes

days after emergence) were allowed to feed for I hr ona passage‑2 mouse 3 days after infection (parasitemia,
about 2.0%) at 21'C. Engorged mosquitoes were separ‑
ated, kept for I hr at 21'C to form ookinetes efficiently(Sato et al., 1996) and then maintained at 26'C until
dissected.

Live mosquitoes were fixed by 0.1 ml injection of
2.5% osmium tetroxide in PBS into the thorax with acapillary tube on 16 days post‑feeding. Salivary glands
were carefully dissected 2 hrs after injection and speci‑

mens for SEM were prepared by standard methods andobserved under a JSM‑T200 SEM operated at 10 kv. To
prepare the specimens for TEM, the salivary glands
were dissected from the fresh mosquitoes and prefixedin 1% glutaraldehyde in 0.1 M phosphate buffered saline(PBS) (pH 7.2) for I hr. They were washed three times
in PBS and were post‑fixed with 1% osmium tetroxide
in PBS for 2 hrs. After washing with PBS, they wereexisted on the surface of non‑infected median and lat‑eral lobes. Whereas, on the surface of the middle andlateral lobes infected with malarial parasites, small
holes through which sporozoites might pass were obser‑
ved (Fig. 2). In addition, these lobes were covered witha white powder like substance (Figs. 2, 5) . We supposedthat this powder like substance was saliva which leaked
from the small holes because we couldn't observe it on
the surface of non‑infected lobes. Sinden (1975) obser‑

ved a sponge like matrix on the surface of salivary
glands (10be and area, not mentioned) in A. stephensi
infected with P. yoelii nigeriensis sporozoites but wedidn't observe this structure. The number of sporozoites

concentrated on the surface of lobes was different
among specimens but usually many sporozoites were
present (Figs. 4, 5) except in a few specimens with a few

sporozoites (Fig. 3). No sporozoite was observed on thedehydrated in ethanol and acetone, and embedded insurface of the intermediate region of lateral lobes in this

Quetol 812. Thin sections were stained with 4% uranyl
acetate and lead citrate, and observed under a H‑700HX
Hitachi TEM operated at 100 kv.

study.

RESULTS AND DISCUSSION

The sporozoites observed on the surface of salivary
glands were about 10 pm in length with one end flat andthe other round (Fig. 3). Sato (1998) also observed thesame morphological features in P. berghei sporozoites
which emerged from oocysts. We judged frorn the figure

We dissected 50 pairs of salivary glands from A.

by Knell (1991) that flat and rounds ends were thestephensi (20 non‑infected and 30 infected with malarial

anterior and posterior ends of the sporozoite, respective‑

parasites) . Th salivary glands consisted of paired
glands and each gland consisted of one short median
lobe and two long lateral lobes. The median lobe wasdivided into two, proximal and distal regions, whilely. We observed the round end of the sporozoite outsideof the salivary gland. This fact indicated that thesporzozoites penetrate the salivary gland cells from theanterior end (Figs. 2, 5, 6). During the process oflateral lobes were divided into three, proximal, interme‑

penetration into salivary gland cells, sporozoites attachdiate and distal regions as described by Wright (1969) .

and cross the basal lamina, the plasma membrane of

The whole body was prefixed by injecting osmium

salivary gland cells and then penetrate the cytoplasrn.
Pimenta et al. (1994) observed interaction of P. gal‑linaceum sporozoites with A. aegypti salivary glands bytetroxideto to observe the malarial parasites and thesalivary glands together as they are in the mosquito
body. This made it difficult to separate whole salivary
glands from the other tissues completely for SEM obser‑

TEM and emphasized that the penetration process
appeared to involve the formation of a membrane junc‑

9*,"* *:

1. *='; .

} == ' 'i ';i i:{;! ('; = ';':;ing;;;;; L'

'<

'

̲

' '

' > ‑ s= ) F

:j; ' ' S{' "Figure I Morphology of. the salivary glands of a nan infected mosquito. A. stc'J;hensi. Many swellings wereobserved on the surface of the median lobe (ML) , while shallo depre slons ',vere Observed on thesurface of the lateral lobe (LL) (bar; 50 j em)
Figure 2 Median lobe infected with P. berghei. The surface Of the gland was c0'vered with a white powder
like substance (arrow) (left, bar; 20 ftm) and higher magnification of left micrograph shov rs th rt
sporozoites were penetrating the giand through small hole.s (right, bar; 5 /Im) .
rigure 3 Sporozoites attached to the surface of the median lobe (left, bar; 20 fttm) . Higher. Inagnificationof the sporozoites which were about l=0 ;Im in lengtl‑1 with a flat anterior and round posterior end{r. ight, bar; 2 ;xm) .
AE: anterior end. CP: cytoplasl l, DR: distal regian, DW: duct̲ wa.11, (}L; giand lumen, IR: intennedi?'Ite region. LL: Iateral lr.)lr)i , L: 1 i riddle lobe, P. I+ : posterior end, S: spor :)zoite, SC: sec.retory cavity, V: vac.uole

e,

l O

4

i

s

;

Figure 4 Many sporozoites were concentrated on the surface of median lObes (left' bar; 20 /Im)' Higher
magnification of sporozoites whichvere penetrating the l{)be siml'̲ Itaneously (right' bar; 5 / l] ) 'Figure 5 Lateral lobe infected with P' belgh'ei Was cavered With a white powder like subStance arn )w)(1eft' bar; 20 ;lrn) ' lligber mag̲nification of bott0 1 of the diStal region With many spol azoites
attached' Sorne sporozoites were penetrating tJ̲ e salivary gland (right' bar; 10 'um) 'l*'igure 6

SPorozoites penetrating into the lateral lobes of the salivary gland' anterior encl first with Posterior
end remair'‑ring autside (collection of sporozoites not shown in fig' ures 25, bar; 5 'um) '11""' ; '; :'*' * 'i * " ' "*"
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1 eglon near mtermedlate regron of lateral lobe was obser ed (bar 2 jlm) '12tion between the cell coat of sporozoites and basal

REFERENCES

lamina of the salivary gland.

Sporozoites were surrounded with vacuoles after
penetration into the cytoplasm of salivary gland cells(Fig. 7). Pimenta et al. (1944) observed the same
phenomenon and explained that these vacuoles appear
to be formed by the invagination of the palsma mem‑brane. We observed sporozoite moving just from thecytoplasm to the secretory cavity (Fig,. 7). Spopozoites

in the secretory cavity became lodged to form bundles(Fig. 8) .

A cuticular salivary duct passes through each lobe
and salivary gland cells surround the salivary duct. In
Aedes, the duct continues from the mouth to the end of
the distal region of the lobe (Jansen and Wright, 1971) ,but in Anopheles, it ends in the intermediate zone of the

distal region (Wright, 1969). The duct wall was notobserved at the end of the distal region of lateral lobes(Fig. 8), suggesting that spopozoites in the secretory

cavity move to the lumen of the salivary duct moreeasily at the posterior half of distal region than theanterior half of the distal region. We found a sporozoite

moving toward the probosis in the duct lumen with avery thick wall at the distal region near the intermediate

region of the lateral lobe (Fig. 9). It is doubtful thatsporozoites located around this thick duct could pene‑
trate the duct wall and move toward the probosis.
We observed sporozoites invade from the anterior

1 ) Barrow, P.M., Mclver, S.B. and Wright. K.A. (1975):
Salivary glands of female Culex pipiens: morphologicalchanges associated with maturation and blood‑feeding.
Can. Entomol., 107, 1153‑1160

2 ) Golenda, C.F., Starkweather, W.H. and Wirtz, R.A.
(1990): The distribution of circumsporozoite protein(CS) in Anopheles stephensi mosquitoes infected with
Plasmodium falciparum malaria. J. Histochern. Cyto‑
chem., 38, 475‑481

3 ) Janzen, H.G. and Wright, K.A. (1971): The salivary
glands of Aedes aegy ,ti (L) : an electron microscope
study. Can. J. Zool., 9, 1343‑1345

4 ) Knell, A.J. (1991): Malaria, 1‑93, Oxford University
Press5 ) Meis, J.F.G.M., Wismans, P.G.P., Jap, P.H.K., Lensen, A.

H.W. and Ponnudurai, T. (1992): A scanning electron
microscopic study of the sporogonic development of
Plasmodium falciparum in Anopheles stephensi. Act.
Trop., 50, 227‑236

6 ) Pimenta, P.F., Touray, M. and Miller, L. (1994): The
journey of malaria sporozoites in the mosquito salivary
gland. J. Euk. Microbiol., 41, 608‑624

7 ) Ponnudurai, T., Lensen, A.H.W., van Gemert, G.J.A.,
Bolmer, M.G. and Meuwissen, J.H.E.T. (1991) : Feeding
behaviour and sporozoite ejection by infected Anopheles
stephensi. Trans. R. Soc. Trop. Med. Hyg., 85, 175‑180
8 ) Posthuma, G., Meis, J.F.G.M., Verhave J.P., Gigengack,S., Hollingdale, M.R., Ponnudurai, T. and Geuze H.J.
(1989): Immunogold determination of Plasmdium fal‑end into median and lateral lobes of the salivary glands.

ciparum circumsporozoite protein in Anopheles stephensi

The anterior end of sporozoites also has an apicalsalivary gland cells. Eur. J. Cell Biol., 49, 66‑72

complex (Sterling et al., 1973; Sato, 1998). Therefore,
apical complex apparatus is also considered to play arole in penetration of sporozoites into salivary glands.

ACKNOWLEDGEMENTS
This work was supported in part by the Grant‑in‑
Aid for International Scientific Research to Ando K.
(No. 06045019) from the Ministry of Education, Science

and Culture, Japan. We thank Dr. DeMar Taylor,
Tsukuba University, for critical reading of the manu‑
script.

9 ) Sato, Y., Matsuoka, H., Araki, M., Ando, K. and Chinzei,
Y. (1996): Effect of temperature to Plasmodium berghei
and P. yoelii on mosquito stage in Anopheles stephensi.
Jpn. J. Parasitol., 45, 98‑104

10) Sato, Y. (1998): Development of Plasmodium berghei
ookinetes to sporozoites in the Anopheles stephensi
mosquitoes: An electron microscopic study. Mie Med.
J., 48 (in press)

11) Sinden, R.E. (1975): The sporogonic cycle of Plas‑
modium yoelii nigeriensis: a scanning electron micro‑
scope study. Protistologica, 11, 31‑39
12) Sterling, C.R., Aikawa, M. and Vanderberg, P.J. (1973):

The passage of Plasmodium berghei sporozoites throughthe salivary glands of Anopheles stephensi: An electron
microscope study. J. Parasitol., 59, 593‑605
13) Wright, K.A. (1969): The anatomy of salivary glands ofAnopheles stephensi Liston. Can. J. Zool., 47, 579‑599

Jpn. J. Tro p. Med. Hyg Vol 27 No 1 1999, pp. 13‑17

13

A SEROEPIDEMIOLOGICAL STUDY ON
HEPATITIS B VIRUS, HEPATITIS C VIRUS

AND HUMAN IMMUNODEFICIENCY VIRUS
INFECTION IN NORTHERN THAILAND
PRAPAN JUTAVIJITTUMl, YUPA JIVIRIYAWAT1, AMNAT YOUSUKH ,KAN TORIYAMA', HIDEYO ITAKURA2, MICHITAMI YAN03
AND SHIGEKI HAYASHI+
Received December 3, 1998/Accepted January 26, 1999

Abstract: A total of 1,889 voluntarily donated blood from five provinces in northern Thailand were
investigated for the prevalences of hepatitis B virus (HBV), hepatitis C virus (HCV) and human im‑
munodeficiency virus (HIV) infection. The average positive rates of HBs‑Ag, anti‑HBs, IgM anti‑HBc,
anti‑HCV and anti‑HIV were 8.7, 42.6, 0.5, 1.6 and 1.7%, respectively. The highest positive rates of HBs‑Ag,
anti‑HBs, IgM anti‑HCV and anti‑HIV were found in the age group under 19 years (13.3%) , 40‑49 year oldgroup (47.6%), 20‑29 year old group (0.6%), 30‑39 year old group (2.6%) and 20‑29 year old group (2.9%),

respectively. We found only two cases that were anti‑HIV and HBS‑Ag positive. There are no cases thatwere anti‑HIV and lgM anti‑HBc positive, and that were anti‑HIV and anti‑HCV positive. These results
suggest that in northern Thailand, most of the HBV infections are due to vertical transmission from motherto child, although HBV infection is a major cause of viral hepatitis, HCV infection will become an important

public health problem in the near future and that the prevalence of HIV infection is quite high amongsexually active generations. Therefore, there is a need for more medical attention to these blood‑borne virus

diseases, and we strongly recommend routine blood screening for HCV in every medical institutions innorthern Thailand.

Key words: HBV, HCV, HIV, northern Thailand

INTRODUCTION
Although HBV infection is highly endemic in Asia,
Africa and Middle and South America, having harmful
influences to the population in these countries, the intro‑

the correlation among these viral infections have notbeen well established in northern Thailand. It is the
purpose of this study to determine the prevalences of

HBV, HCV and HIV infections among voluntarily

duction of routine donor blood screening for HBS‑Ag

donated bloods and discuss the transmission routes toformulate prevention strategies of these blood‑borne

has decreased the incidence of HBV infection in many ofvirus infections in northern Thailand.

these countries. In Thailand, HCV infection has become
the major cause of post‑transfusion and sporadic hepati‑tis (Tanprasert et al., 1993). The HIV infected popula‑

MATERIALS AND METHODS

tion is increasing in northern Thailand and it is also well

The total number of 1,889 serum samples were

known that there are high prevalences of HBV and
HCV infections among anti‑HIV positive populations

randomly collected from the voluntary blood donors at(Esteban et al., 1989; Botti et al., 1992; Eysler et al.,Lampang, Lamphun and Phayao Provincial Hospitals innorthern Thailand (Fig. 1) during June to December,
1995 and stored at ‑20'C until examination. Sera were1993; Bryan et al., 1993; Quan et al., 1993; Yousukh etal., 1996). However the seroepidemiological study of
1.
2.
3.
4.

Chiang Mai University Hospital and Chiang Rai,

Department of Pathology, Faculty of Medicine, Chiang Mai University, 110 Intavaroros Rd., Chiang Mai 50200, Thailand
Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852‑8523, Japan
Institute for Clinical Research, Nagasaki Chuo Hospital, 2‑1001‑1 Kubara, Omura 856‑0835, Japan
Department of Gastroenterology, International Medical Center of Japan, 1‑21‑1 Toyama, Shinjuku 162‑8655, Tokyo, Japan14voluntary

MYANMAR

CHlNA

MYANMAR

CHllNGRA］

ゆVlETNAMLAND

CHIANGMご1

CAMBODIA

●

●

from

five

anti−HBs，0．5％forforanti−HCV

and1．7％forof

HBs＿Ag，anti＿HCV

tobe10．9％，2．5％，and2．5％respectively，in

PHAYAOanti−HBs

LAMPANGpositive

provincesAg，42．6％for

LA。s

LAMPHU二donorsaverage

LAO
THAI

blood

Thailand．Theandof46．4％and

rates
IgM

northemHBs−anti−HBc，1．6％

anti−HIV．The
anti＿HIV

IgM

ofare8．7％forhighestpositiveanti−HBc

rates

are

found

Phayaoof12％in

and

Lam−

phun．

THAILAND

Table2summerizes the age distribution of HBs−
Ag，anti−HBs，IgM anti−HBc，anti−HCV and anti−HIV
FigurelMapofnorthempositive

Thailand

rates
testedforthepresence〃onoolonol（theLtd．，North

third

of

HBs−Aggeneration

by

EIA

Chicago，111inois，USA），anti−HBs

E五4（human−subtypes

North

Chicago，111inois，USA）and

ad

and

ay，Abbotttheandusing。4％sz夕郷6

kit，Abbott

titersanti＿HIV

1．6％inby、4％sの

the

old

blood

donors．The

HBs−Ag，anti−HBs，IgM

under19years

Lab．

Lab．Ltd．，

anti−HBs

among

of

were

found

the20−29year

group

to

old，47．6％in
oldand2．9％in

highestpositiveanti−HBc，anti−HCV

be13．3％in

the

group

the40−49year

agegroup，

group，2．6％in

the20−29year

oldthe30−39year

group，respec−tively．

ofThe

prevalence of HBs−Ag，anti−HBs，IgM anti−
and anti−HIV among male donors is

positive samples were calculated andseroprotectionwas
defined as anti−HBs levels≧10mIU／ml（Andre，1989；

HBc，anti−HCV

Yap6渉α1．，1992），IgM anti−HBc by Co郷耀一〃 （γDNA，Abbott Lab．Ltd．，North Chicago，Illinois，USA），

described in Table3．The average positive rates were
9．8％for HBs−Ag，44．0％for anti−HBs，0．4％for IgM

anti−HCV
USA）andanti−HIV

伍y−2（the

third

North
of

the

E五430

（c100−3，HC−34，

anti＿HBc，2．1％for

anti＿HCV

and1．6％for

Lab．Ltd．，North

Chicago，Illinois，

The

rate

HBs−Agby／1660πμC

HC−43，NS5，Abbott

V

generation

EIA

kit，Abbott

Chicago，111inois，USA）．The
results

wasinby、4660渉渉R6607n扉n召n渉伍7−1／

performedusingchi−square

Chiang

Mai

390

Rai

415
380

LampangLamphunofanti−HBc

year

old

group，anti−HCV

test．

and

anti＿HIV

of

found

of52．7％in

O．9％inof3．6％inof2．7％inanti−HIV．

of14．7％wasgroup，anti−HBs

group

HBV，HCV

andHIV

represented

thethe20−29

the30−39year

the20−29year

in

five

provinces

old

old

group．

6（1．5％）

167（40．2％）

3（0．7％）

7（1．7％）

8（1．9％）

150（39．5％）0（0．0％）

7（1．8％）

2（0．6％）

160（46．4％）

4（1．2％）

5（1．5％）

7（2．0％）

9（2．5％）

9（2．5％）

31（1．6％）32（1．7％）

1，889

165（8．7％）804（42．6％）10（0．5％）248

33（13．3％）

20−29

447

30−39

547

40−49

382

≧50

265

QJ4つ0ゴ⊥

Tota1

1，889

6358

≦19

Thailand161（41．3％）

Total

HBs−Agnorthemand1．8％foranti−HIV

166（46．2％）

Numberanti−HCV

3（0．9％）

39（10．9％）Age（years）

in

and

HIV

infections

HBs−Ab92（37．1％）

IgM

by

age

anti−HBc

innorthemThailandanti−HCV

anti−HIV1（0．4％）

0（0．0％）

4（1．6％）

13（2．9％）（8．1％）

156（34．9％）7（L6％）

7（1．6％）（7．9％）

256（46．8％）2（0．4％）

14（2．6％）10（1．8％）（9．2％）

182（47．6％）0（0．0％）

6（1．6％）

5（1．3％）

118（44．5％）0（0．0％）

4（1．5％）

0（0．0％）

804（42．6％）10（0．5％）31（1．6％）32（1．7％）（6．8％）

165（8．7％）

positiveanti−HBs，0．8％

anti−HCV

ant卜HBc

359

HBV，HCVaverage2（0．5％）

IgM

PhayaoofTable4．The

infections

1（0．3％）

Prevalencein

HBs−Ag，39．4％foranti−HBc，0．7％for20（5．1％）27（7．8％）iswere6．5％for

IgM

HBs−Ab37（9．7％）

donorsrates

for

HBs−Ag42（10．1％）

345

Table2ofThe prevalence of HBs−Ag，anti−HBs，IgM anti−
HBc，anti−HCV and anti−HIV positive among female

Number

Provinces
ChiangPrevalenceoldanalysisTable l shows the prevalence of HBs＿Ag，anti−
HBs，IgM anti−HBc，anti−HCV and anti−HIV in1，889
lunder19year

oldgroup，IgM

RESULTS

Table

the

positive

30−39year

Lab．Ltd．，

statisticalhighest15

anti＿HIV．The

highestrates

of

HBs＿Agof8．8％was

and1980inyear

positive

old

old

under

group

l9yearandoldgroup，anti＿HBs

anti−HIV

of3．6％inof48．0％the20−29year

These

group．

Table5demonstrates
anti−HBs

positivetive（90．4％）had

cases

the

the

less

protection

the20−29year

oldpositivein

Thailand．

anti−

prevalenceofand6．5％infemale（p＜0．05）and

level

of

highestlevels≧10mIU／ml

theprevalence

was

found

in

man，1987），Thailand
1ence

region

group

of

HBV

previous

reportrevealed

HBs−Agis7．4％among

to

an

by

the

Table3Prevalencetheintermediate

averagedonatedofinfemale（p＜0．05）．The

blood

HBV，HCVandHBs−Ag

191

28（14．7％）

20−29

336

30−39

366

40−49

≧50

257
147

Total

1，292

≦19

57

20−29

111

30−39

181

40−49

125

≧50

Total

≦19

that

from

that

of

in

the

between9．8％in
also

in

reason

than

males

for

femalehave

and39．4％

thehigherage

positivie

the

the

age

group

over50years．

of

HBs＿Agpositive，a

among

the

older

age

among

male

donorspreva−

HIV

infections

IgM

risk

of

contacting

groupanti−HBc

under19years
and

the

highershows

lowest

rate

the
is

to

the

rate

of

anti＿HBs

is

found

of

regardnorthemtotheanti−HIVanti−HCV

33（9．8％） 118（35．1％）30（8．2％） 193（52．7％）27（10．5％） 122（47．5％）9（2．7％）

1（0．3％）

13（3．6％）7（1．9％）

0（0．0％）

6（2．3％）

3（1．2％）

8（5．6％）

66（46．5％）0（0．0％）

3（2．1％）

0（0．0％）

126（9．8％）569（44．0％）5（0．4％）

27（2．1％）21（1．6％）HIV

infections

among

IgM

HBs−Abfemaledonorsanti−HBc

innorthemThailandanti−HIVanti−HCV

22（38．6％）0（0．0％）

0（0．0％）

2（3．5％）

38（34．2％）4（3．6％）

2（1．8％）

4（3．6％）

13（7．2％）63（34．8％）1（0．6％）

1（0．6％）

3（1．7％）

8（6．4％）

60（48．0％）0（0．0％）

0（0．0％）

2（1．6％）

123

10（8．1％）52（42．3％）0（0．0％）

1（0．8％）

0（0．0％）

597

39（6．5％）235（39．4％）5（0．8％）

4（0．7％）

11（1．8％）5一（8．8％）3（2．7％）

Anti−HBslevelsMale≧10mIU／ml ＜10mIU／ml

by

age

and

sex

in

northemThailand

Total

Female≧10mIU／ml

protective

Thailand2（1．0％）

andinContrary5（1．5％）

HBV，HCV＜10mIU／ml≧10mIU／ml＜10mIU／ml

82（89．1％）

10（10．9％）9（12．9％）21（95．5％）

1（4、5％）

6

36（94．7％）

2（1．8％）

148（94．9％）

8（5．1％）

20−29

112（94．9％）

30−39

175（90．7％）18（9．3％）58（92．1％）

5（7．9％）

233（91．0％）23（9．0％）40−49111（91．0％）11（9．0％）52（86．7％）8（13．3％）163（89．6％）

19（10．4％）9（13．6％）44（84．6％）8（15．4％）101（85．6％）

17（14．4％）53（9．3％）211（89．8％）24（10．2％）

727（90．4％）77（9．6％）

≧50
Tota1

57（86．4％）

516（90．7％）（5．1％）

highest

found

rate

groups．In

in

o1（i

3（0．9％）

70（36．6％）

HBs−Ag61（87．1％）

HBV

unclear．However0（0．0％）

ofmale

prevalence

male

is

higherthe1（0．5％）

Table5Age（years）

male

HBs＿AgHBs−Ab≦19

Numbersuspectedin

HBs＿Agdifference

between44．0％inof

between1970

NumberTable4Prevalenceispositive

littleslightpositive

rate

prevalenceAge（years）

Age（years）

HBs−Agyounger

WHO（Zucker−

infection（2−7．9％）andthat

differsaisof

HBV than females，possibly via sexually transmittedroute in Thailand （Tanprasert窃 α1．，1993）． The

classification

belongs

Thailandanti−HBs

rate

prevalence

There

of

infection

DISCUSSION
the

the

centralseroprotection

northem

Mai（Yousukh6砲1．，1996）．

that

of804

group．

to

Chiangsuggestposi−

it

Accordingin1991infindings

levelsthan10mIU／ml．The（94．9％）of

of8．7％positiveof804anti−HBs

anti−HBs

cases．7270ut

HBs（≧10mIU／ml）and77cases（9．6％）showed
levelsresultThailand（Chainuvati6∫α1．，1990）．

amongthe volmtary donatedblood in l995from five provincial areas of norhtem
Thailand is similar to the previous report of7．9％

in

Our

centralinthe40−49year oldgroup，IgM anti−HBc of3．6％inthe
20−29years old group，anti−HCV of1．8％in the20−29

detected

16

anti‑HBs level more than 10 mlU/ml, the highest preva‑
lence rate was found in the 20‑29 year old group and theAg and anti‑HIV positive and no cases that are lgManti‑ HBV and anti‑HIV positive, and anti‑HCV andrate steadily decreased with increasing age. In addition,
the rate of lgM anti‑HBc positive, which indicates acutegation is necessary to confirm these findings.

anti‑HIV positive in the present study. Further investi‑or recent HBV infection, usually within six months or

The results of our study suggest as follows; 1)

less, is 0.5% among the total donated blood. Thesefindings suggest that the main transmission route of

Although there are some occurences of horizontal infec‑
tion, the transmission route of HBV infection is mainly
due to vertical infection in northern Thailand and it is

HBV infection is a vertical infection during the neonatal

period, however there are some occurrences of theirnportant to prevent HBV infection from HBS‑Ag

horizontal infection of HBV in northern Thailand.
The screening tests for anti‑HCV in blood donors

positive mother to infant, 2) Although HCV infection isless prevalent than HBV infection at present, it will
become an important public health problem in the nearusing the 2nd generation EIA test, revealed 1.6% of anti‑

HCV positive in Bangkok (Nuchprayoon et al., 1993),
1.3% in Khon Kaen, north eastern Thailand (Tomana‑kan, 1994) and 2.4% in Chiang Mai (Hotta et al., 1997).

Our results using the 3rd generation EIA test which
provides an improved sensitivity for the detection ofanti‑HCV seroconversion and the identification of HCV
infected individuals showed 1.6% of anti‑HCV positive
among the donated blood. It has been suggested that
artificial factors such as reusing syringes for somevaccination, which is contaminated with HCV are themain reasons for the higher prevalence in older people inJapan (Yano, 1992). The fact that the prevalence of

future, and it is strongly recommended to introduce
routine screening of anti‑HCV in the donated blood, 3)
The incidence of HIV infection is quite high in northern
Thailand and it will need more medical attention.

ACKNOWLEDGMENTS
We are grateful to the relevant officers in Chiang

Rai, Lampang, Lamphun and Phayao Provincial Hospi‑
tals for their cooperation. This work was supported by
a Grant for International Health Cooperation Research(9C‑4) from the Ministry of Health and Welfare, Japan.

HCV infection is the highest in the 30‑39 year old group,and no positive case is found in the age group under 19
years old, may indicate that the similar factors play arole in the transmission of HCV among the specified age
group in northern Thailand.
Since 1988, Thailand has witnessed an explosive risein the number of HIV‑infected populations (Phanuphak
et al., 1995). In particular, prevalence is the highest in

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J pn. J. Trop. Med. Hyg., Vol. 27, No. 1, 1999, pp. 19‑22

Short communication

EVALUATION OF A RECOMBINANTPROTEIN (RTc24) AND SYNTHETICPEPTIDES IN ANTI‑TRYPANOSOMA CRUZIPOSITIVE SAMPLES FROM BLOODBANK DONORS IN CHAGASIC ENDEMIC

AREAS OF ECUADORANGEL G. GUEVARAl'2, JUAN C. RUlZ C2., RAYMOND L. HOUGHTON4,LISA REYNOLDS4, PAUL SLEATH4, DARIN BENSON4,
ALI OUAISS13 AND RONALD H. GUDERIANl
Received September 14, 1998/Accepted December 14, 1998

Chagas' disease, caused by the hemoflagellate
Trypanosoma cruzi, affects around 17 million people inCentral and South America (WHO, 1997). The mainroute of transmission involves T. cruzi infected
triatomine insect bites, but other mechanisms of trans‑mission such as blood transfusion or blood derived
products have been reported to be responsible for fatal
cases of acute Chagas' disease (Villalba et al., 1992).

Improved vector control by insecticide spraying andbetter management of blood banks in endemic areas are

disease (Burns Jr. et al., 1992).

In Ecuador, although Chagas' disease has been
known to exist since 1927 (Arteaga, 1930), the true
prevalence of the disease is still unknown. In the coun‑try, blood bank screening for Chagas' disease is manda‑
tory but testing in blood banks located in endemic areas

is not regular. Recently, blood donors were found to bepositive for anti‑T. cruzi antibodies in a vector‑freeregion and eve, n in non‑endemic areas of the country(Grijalva et al., 1995, 1997) . Therefore, the screening ofessential to reduce the disease transmission. Transfu‑
sion‑associated transmission is not only a threat in theendemic countries but also in non‑endemic areas due tomigration of T. cruzi‑infected individuals (Kirchoff,1993) and the serological screening of blood banks could
reduce the transfusion‑associated transmission, avoid‑
ing the transportation of contaminated blood with theparasite. Actually, the use of serological screening inblood bank donors is a necessity in Ecuador.

disease control is hindered by the cost of the assays andfrom March to June 1996 (Figure 1). The samples were

the sensitivity and secificity of the antigens used.
Recently, with the development of the DNA technology,
highly specific and sensitive recombinant antigens

randomly collected and no distinction was madebetween volunteers, paid and frequently members.

(Taibi et al., 1995) , as well as T. cruzi specific synthetic

used.

peptides, have been useful in the diagnosis of Chagas'

The present study was aimed to determine theprevalence of anti‑T. cruzi antibodies in Ecuadorian
blood banks from two areas endemic for Chagas' dis‑ease. Serum samples from blood donors of the Red
Cross Blood Bank of Guayaquil, province of Guayas(n= 1,423) , and from the Blood Bank of Machala, prov‑
ince of E1 Oro (n= 203) , were obtained during the period

These samples collected were stored at ‑ 20'C until
Total lysate obtained from T. cruzi epimastigotes

Laboratory of Clinical Investigations, Community Services, Hospital Vozandes, HCJB, Casilla 17‑17‑691, Quito‑Ecuador
2. Institute of Molecular Biology, Catholic University Santiago de Guayaquil‑Ecuador
3

CJF INSERM 96‑04 <<Approches Moleculaires et Immunologiques de la Pathogenie des Trypanosomatidae , Centre ORSTOM deMontpellier, France
4 . Corixa Corp, Seattle WA‑U. SA

Corresponding author: Angel Gustavo Guevara PhD. Laboratory of Clinical Investigations, Community Services, Hospital
Vozandes, HCJB, Casilla 17‑17‑691, Quito‑Ecuador
Fax: + +593‑2‑267263/269234 E‑mail: Iabinves@hcjb. org. ec

20t

0.8 pg/ml and 50 pl were coated in microtiter plates, thei coLo"8'A

remaining steps of the ELISA assay was done as de‑scribed for total lysate, the "cut off" value was calcu‑*

lated and values higher than the mean absorbance plus

ll

NUl

l'

Pll ;l/. I ¥ 'I >̲. ‑//'

./'

/

II ‑

tdJ'

:'P' I I 'Ar /'l'l
IN L /
1PERU

8ll‑‑ JI

3 standard deviations [0.187+3 (0.02) =0.247] wereconsidered as positives. The ELISA procedure for the
rTc24 was performed as described in previous reports(Krautz et al., 1995) with slightly modifications, thus,

Immulon I plates (Dynatech Laboratories, USA) were
coated with 100 pl of 0.5 pg/ml of rTc24 antigen andincubated with 0.05 M carbonate‑bicarbonate buffer(pH 9.6) overnight at 4'C. The unbound material was
discarded and plates were blocked with 200 pl of PBS‑

Tween‑BSA solution (PBS pH 7.2, 0.05% Tween 20, 1%
BSA) for 2 h at 37'C. After five washes with PBS pH
Figure I Map showing the Ecuadorian endemic areas for
Chagas' disease ( ///////) and the localities where

samples were collected for the study: Guayaquil(O) and Machala ([])
was used for ELISA as described by Chico et al. (1997)
to test all the sera from blood donors. Briefly, 0.1 pgl
ml of crude antigen per well was coated in ELISA plates
and incubated overnight at 4'C, non‑bound antigen was
discarded and 200 pl of blocking buffer was added andincubated for one hour at room temperaure. The plates

7.4‑0.1% Tween 20 (PBST), 100 pl of human serum
diluted 1:100 in PBST and 0.3% BSA were added andplates incubated for 2 h at 37'C. Then, plates were
washed five times with PBST and 100pl of a 1:5,000
dilution of peroxidase‑ conjugated anti‑human lgG (ychain specific) (SANOFI Diagnostics Pasteur, 92430
Marnes‑1a‑Coquette, France) were added to the wells
and plates incubated for I h at 37'C. After five washes

with PBST and an additional wash with PBS, 100 pl of

10 mg o‑phenyldiamine dihydrochloride (Sigma, St.
Louis, MO 63178 USA) dissolved in 20 ml of citrate

were washed six times with washing buffer and serum

buffer pH 5.0 plus 20 pl of H202 were added to each well

samples, positive and negative controls were diluted 1:50

and incubated 15 min at 37'C. The reaction was stopped
by adding 100 pl of I N HCI to each well. The absorban‑

with washing buffer and 50 pl were added to the corre‑
sponding wells and incubated for 30 min at room temper‑
ature. Then, wells were washed five more times and 50
pl of protein A horseradish peroxidase (Zymed Labora‑
tories) , diluted 1:10,000 in washing buffer was added to

the wells and plates were incubated for 30 min. After
five additional washes with washing buffer, 100 pl of

ABTS [2, 2,‑azino‑di (3‑ethyl‑benzthiazoline sul‑
fonate) I in substrate buffer was added (Zymed) , thereaction was allowed to develop at room temperaturefor 30 min in the dark and stopped by adding 100 pl of
5% SDS and the absorbance of each well was read by an

ELISA plate reader (Titertek Multiscan) at 405 nm.
The "cut off" value was determined by testing 20 sam‑ples from healthy individuals from the endemic areas,values higher than the mean absorbance value plus 3

standard deviations [mean+3SD: 0.139+3 (0.041) =ce was measured against the blank wells at 492 nm bymean of an ELISA reader (Titertek Multiscan) . The"cut off" value was determined as described in the two

previous ELISA tests, values higher than the mean
absorbance value plus 3 standard deviations [mean+
3DS: 0.183+3 (0.025) =0.258] were considered as posi‑tive against rTc24. In all the ELISA tests described,
assays were carried out in duplicate.
Using the total lysate as antigen, 15 (1.1%) of the
1,423 sera collected in Guayaquil were positive; of the

203 samples in Machala 14 (6.9%) were positive(Table 1). These 29 positive samples were tested
against rTc24 and 21 (72.4%) were positive for the
Table 1 Anti‑T. cruzi antibodies against total lysate
antigen in blood donors

0.262] were considered as positive against total lysate.

The positive samples against the total lysate were also

Locality

assayed by ELISA with two additional antigens: a T.

Guayaquil

cruzi specific synthetic peptide 2/D/E (Houghton et al.,Machala1996) and a recombinant T. cruzi protein (rTc24). For
the synthetic peptide, 2/D/E, the antigen was diluted to

Total

Number of samples
testedTotal positive sera(%)
n

1 , 42315(1 . 1)203

14(6 . 9)

1 , 626

29(1 . 8)

21

positive for total lysate antigen with two definedconclussion, we can not define one or another antigen as
the most suitable for diagnosis of T. cruzi infection but

antigens, rTc24 and 2/D/E

the use of highly specific recombinant antigens or syn‑Table 2 Comparison of reactivity of sera from individuals

thetic peptides should be useful to avoid the use of

No. of samples positive for

Total lysate

2/D/E

n (%)

rTc24
n (%)

Guayaquil

15

11

14

Machala

14

10

13

29 (100)

21 (72.4)

27 (93.1)

Locality

Total

* (%)

parasite cultures as required for tests using total lysate

as antigen.

ACKNOWLEDGMENTS
We acknowledge Prof. Y. Hashiguchi from Kochi
Medical School, Kochi, Japan for his kind revision of theantigen. On the other hand, 27 (93.1%) of the samples

manuscript. We also acknowledge Dra. Carmen Romowere found positives against the 2/D/E T. cruzi specificpeptide (Table 2).

de Andrade who helped us with samples collection fromIn Chagas' disease, the present control strategy isdependent upon an efficient vector control program, anddesigned the map. This work was partially supported by

Machala. We thanks Victor Guzm n G. who kindly

FUNDACYT‑PBID 422.

with the development of new tools (fumigantion canis‑
ters, insecticidal paints) the vector‑associated transmis‑

sion of the disease has been interrupted in some of theendemic countries. However, blood transfusion is con‑sidered as a serious danger of T. cruzi infection andscreening of blood units must be compulsory in endemicareas as well as in non‑endemic regions where migra‑
tion of people infected with T. cruzi has been occurred.
Our results support the fact that risk of transfusion‑associated transmission is real especially in areas ofhigh endemicity (e. g. 6.9% in Machala) and it could be
preventable by discarding or treating T. cruzi‑infectedblood. From the results obtained, a regular nationwide
program is required in Ecuador. In the current study, we

studied serum samples from only two blood banks in
two endemic areas but Chagas' disease is described in atleast eight provinces of the country and rural‑urban
migration is important as well.
In relation with the two defined antigens, rTc24 and

REFERENCES
1 ) Arteaga, L. (1930): Inventigaciones sobre la existencia
de la enfermedad de Chagas en la zona del ferrocarril ala Costa. Rev. Univ. Guay., 1, 89‑1012 ) Burns, J.M., Shreffler, W.G., Rosman, D.E., Sleath, P.R.,

March, C.J. and Reed, S.G. (1992): Identification andsynthesis of a major conserved antigenic epitope of
Trypanosoma cruzi. Proc. Natl. Acd. Sci., 89, 1239‑1243
3 ) Chico, M.H., Sandoval, C., Guevara, A.E., Calvopifia, M.

H., Cooper. P.J.. Reed, S.G. and Guderian, R.H. (1997):
Chagas' disease in Ecuador: Evidence for disease trans‑mission in an indigenous population in the Amazon
region. Mem. Inst. Oswaldo Cruz, 92, 317‑320
4 ) Kirchoff, L (1993): American trypanosomiasis (Chagas'
disease) ‑a tropical disease now in the United States. N.

Eng. J. Med., 329, 639‑644
5 ) Grijalva, M.J.. Rowland, E.C., Powell, M.R., McCormick,
T.S. and Escalante, L. (1995): Blood donors in a vector‑

free zone of Ecuador are potentially infected with

2/D/E synthetic peptide tested in this study, the latter isTrypanosoma cruzi. Am. J. Trop. Med. Hyg., 52, 360‑363

seemed to be more sensitive than rTc24. However, sincea negative reaction for rTc24 has been described as a6 ) Grijalva, M.J., Chiriboga, R., Racines, J.R., Escalante. L.

marker of cure of the disease, it is possible to consider

cruzi in the blood supply by the Red Cross blood bank in
Quito, Ecuador. Arn. J. Trop. Med. Hyg., 57, 740‑741
7 ) Houghton, R.L., Benson, D.R., Sheiky, Y.A., Sleath, P.,

that some patients were infected but treated and thepositive responses obtained with the total lysate and the

peptide could be due to the past infection. Unfortunate‑ly, all the positive patients detected in our study arechronic and complete medical records were not avail‑able, the only information about treatment comes frompersonal communications of some of the patients.
Finally, we did not evaluate the negative samples
for total lysate since this antigen could be consideredenough for screening purposes. However, in order toconfirm a Chagas' disease diagnosis at least two positive

tests from a panel of three assays must be required. In

and Rowland, E.C. (1997): Screening for Trypanosoma

Lodes, M., Badaro, R., Krettli. A.U. and Reed. S.G.
(1996): Multiple‑epitope ELISA for the detection ofserum antibodies to Tryt,anosoma cruzi in patients withtreated and untreated Chagas' disease. Transfusion, 36(suppD , 5135

8 ) Krautz, G.M., Galv o, L.M.C., Can ado, J.R.. Guevara‑
Espinoza, A., Ouaissi, A. and Krettli, A.U. (1995): Useof a 24‑kilodalton Trypanos0 na cruzi recombinant pro‑tein to monitor cure of human Chagas' disease. J. Clin.
Microbiol., 33, 2086‑2090

9 ) Taibi. A., Guevara‑Espinoza, A., Schbneck, R.. Ya‑

22hiaoui. B. and Ouaissi, A., (1995): Improved specificity
of Tmpanosoma cruzi identification by polymerase chain

reaction using an oligonculeotide derived from theamino‑terminal sequence of a Tc24 protein. Par‑
asitology, 111, 581‑590
10) Villalba, R., Forn6s, G., Alvarez, M.A., Rom n, J., Rubio,

V., Fern ndez, M., Garcia, J.M., Vifials, M. and Torres,A. (1992): Acute Chagas' disease in a recipient of a bonemarrow transplant in Spain: case report. Clin. Infect.
Dis., 14, 594‑595

11) WHO (1997): TDR Report, Prospects for elimination.
Chagas' Disease, Leprosy, Lymphatic filariasis On‑
chocerciasis. Geneva‑WHO, 23‑35

J pn. J. Trop. Med.

Hyg Vol 27 No 1 1999, pp. 23‑144

23

PROCEEDINGS OF XXXIX ANNUAL MEETINGS OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
25‑27 November 1998, Naha, Okinawa
President

Shigeo Nonaka
Professor Department of Dermatology, Faculty of Medicine,
University of the Ryukyus

CONTENTS
Prize Winner's lecture
JSTM (Japanese Society of Tropical Medicine) Young Investigator Award
Epidemiology of influenza virus infections among children with acute respiratory
infections (ARD in Zambia
Mizuta, K.
President's lecture
Cutaneous diseases in Tropical Medicine especially in Okinawa

Nonaka, S.

Special lecture

Onchocerciasis

Tada, I.

Educational lecture
1 Chronic and intensive solar ultraviolet exposure in tropical areas promotes photoaging

and skin cancer lchihashi, M. et al.

2 Immunomodulatory effects of ultraviolet radiation Horio, T.
Symposium 1: Present and future situation of leishmaniasis research

Introduction Hashiguchi, Y.

1 Classification and diagnosis of Leishmania Mimori, T.

2 Leishmaniasis in Ecuador, with special reference to its Andean form
Hashiguchi, Y. and Gornez L., E.A.
3 Leishrnaniasis in central Eurasia
Matsumoto, Y.
4 Clinical findings of cutaneous leishmaniasis in Ecuador
Hosokawa, A. et al.
5 Molecular biology of drug resistance in Leishmania: gene amplification and P‑glyco‑
proteins
Katakura, K.
6 Expression of heat shock protein correlates with a protective potential against infection

with Leishmania major

Himeno, K.

Symposium 2: Dengue fever and dengue hemorrhagic fever

1 Clinical feature of dengue haemorrhagic fever Tateyama, M.
2 Situation and diagnosis on imported dengue fever in Japan Yamada, K.
3 Breeding habitats of the dengue mosquitoes, Aedue (Stegomyia) spp. in the coastal,
agricultural and mountainous villages in South Sulawesi, Indonesia, 1994‑1996
Miyagi, I. et al.

4 Molecular and in vitro analysis of eight dengue type 2 viruses isolated from patients
exhibiting different disease severities
Mangada, M.N.M. and lgarashi, A.
5 Dengue virus‑specific T Iymphocyte responses and the role of T Iymphocyte in thepathogenesis of dengue hemorrhagic fever
Kurane, I. and Takasaki, T.

24
6Construction and characterization of chimeric Japanese encephalitis/dengue type 4

Tadano, M.

virus

Symposium 3: How can we effectively control infectious diseases in the Tropics?
Session I Immediate reports from current projects in selected areas

1 Parasitic diseases .

2 Leprosy

Shimada, M.
Hatano, K.

3 Malaria‑if we can control the disease or what?

Kano, S.

Session 2 Global strategies for eradication or effective control of infectious diseases

4 Global surveillance for infectious diseases
Nomura, T.
5 Control of infectious diseases control ‑community approaches in developing

6 Global strategy for eradicable infectious diseases Arita, I.
Work shop: For meaningful introduction of advanced technology to studies of malana
epiderniology and control
1 Evaluation of the single‑step screening method for quick detection of glucose‑6‑phos‑lwai, K. et al.
phate dehydrogenase (G‑6‑PD) deficiency in field survey
Kaneko, A. et al.
2 Proguanil polymorphism and efficacy against malaria
3 A novel gene cloning of ookinete surface protein from Plasmodium vivax, and polymor‑
phism in natural parasite isolates
Tsuboi, T.
4 Immunogenetic analysis of the patients with falcipurum malaria in Thailand
Hirayama, K. et al.
5 Antimalaria'l natural products from Dichroa febrzfuga (Joh‑Zan) Kim, H.‑S. et al.
6 Expression of catalytic subunits of mitochondrial complex 11 from Plasmodium falcipar ‑um in Escherichia coli Takeo, S. et al.
Panel presentation
1 Comparative analyses of nonstructural protein NS5 of dengue virus type 1
Ishak, H. et al

2 A study on the approach of the use of insecticide aerosol cans combined with active

surveillance for dengue control Osaka, K. et al.

3 Japanese encephalitis virus recombinant NS5 protein expressed in Escherichia coli

exhibits RNA‑dependent RNA polymerase activity Hasebe, F. et al.
4 Characterization of polioviruses isolated from Pakistani children II

Hasewaga, A. et al
5 The risk of hepatitis virus infection among Japanese staying in developing countries
Hamada, A. et al.
6 Prevalence of human herpesvirus 8 (HHV8) in a young Ugandan population
Mayama, S. et al
7 Comparison of the cytokine induction levels of verotoxin‑producing Escherichia coli

isolated from human, cattle and swine Zang, H‑M. and Yamamoto, T.
8 The isolation frequency of diarrheagenic bacteria in Vientiane, Laoslwanaga, M. et al.

9 Thrombomodulin levels in patients with typhoid fever Ohnishi, K. and Kimura, K.
Wada, A. et al.
10 Identification of the receptor for Aeromonas sobria hemolysin
Namisato, M. et al.
11 The treatment of leprosy; current situation and problems
12 Role of human polymorphonuclear neutrophils in host defense against infection with
Penicillium marneffei
Kudeken, N. et al.
13 Three case report of infection due to Penicillium marneffei with AIDS patients in
Watanabe, H. et al.
northern Thailand

25

14 Study of the salivary gland proteins of malaria vector mosquito, Anopheles stephensiLuo, E. et al.

15 A functional single‑chain Fv directed to a rodent malaria parasite specifically inhibits

the development of oocysts in mosquitoes Yoshida, S. et al.
16 Application to researches and diagnoses of a newly developed malaria parasite detec‑tion method by flow cytometry Saito‑Ito, A. et al.

17 Influence of Schistosoma mansoni infection on susceptibility to Plasmodium chabaudi in

18 Detection of subpatent level of malaria parasites by nested polymerase chain reaction(PCR) and its correlation with microscopy in Myanmar patients Mon, H.M. et al.
19 Chemotherapeutic malaria control trial with diagnosis kit (ICT‑Pf) and single step

G6PD deficiency test in the Solomon islands Nagai, N. et al.
20 Change in malaria vectors and malaria situation in northern Thailand during 30 years

from 1968 Suwonkerd, W. et al.

21 Clinical study on fifteen case of imported malaria Nakamoto, A. et al.
22 Nine cases of imported malaria in Kyushu district during last one year

Ukon, T. et al.

23 Cysteine proteinase gene as a basis for differentiating Entamoeba histlytica andEntamoeba dispar by PCR Rivera, W:L. et al.

24 Bacterial expression of a human monoclonal antibody Fab fragment recognizing an

Entamoeba histlytica surface antigen Tachibana, H. et al.
25 Detection of Entamoeba histlytica ‑specific antigen by sandwich ELISA
Cheng, X‑J. et al.

26 DNA polymerase activity in encysting Entamoeba invadens Makioka, A. et al.
27 Inhibitory effects of aphidicolin on growth and encystation of Entamoeba invadens
Kumagai. M. et al.
28 Seroepidemiological study of entoamoebiasis using micro‑chemiluminescence ELISA in

Nepal Tabuchi, K. et al.

29 Seroepidemiology of cryptosporidiosis Itoh, M. et al.

30 Detection of Cmptosporidium parvum DNA in Apodemus argenteus in residential quar‑ters by PCR Honda, M. et al.

31 Intestinal blockage by carcinoma and Blastocystis hominis infection Horiki, N. et al.
32 Studies on Leishmania species isolated from̲ the great gerbile (Rhombomys opimus) in
Xinjiang Uygur Autonomus region in the People's Republic of China
Sanjoba, C. et al.

33 Leishmaniasis in Turkey

Ozbel, Y. et al.

34 Structure analysis of Try )anosoma cruzi trans‑sialidase gene family: relation to protein

10calization and enzyme activity

Uemura, H. et al.

35 Comparative analysis of CDNA expression profiles between long slender and shortstumpy bloodstream forms of Trypanosoma b. brucei using fluorescent differential
display method
Suzuki, T. et al.
Juares, S. et al.
36 Immunogenetic analysis of Chagas' disease
37 Comparison of the polyrnerase chain reaction with three serological methods for the
diagnosis of Chagas' disease
Juarez, S. et al.
38 Comparative studies on epidemiology of Chagas' disease between Bolivia and northeast
region of Brazil
Miura, S. et al.
39 Study of crossreacting antigenicity between Ascaris lumbricoides antigens and human
Ishida, T. et al.
colonic mucosa
40 Immunodiagnostic application of the recombinant Toxocara canis antigen for humantoxocariasis canis and cati
Yamasaki, H. et al.
41 Strongyloides ratti additive effect of testosterone implantation and carbon injection on

26

the susceptibility of female mice Watanabe, K. et al.
42 Prevalence of Strongyloides stercoralis infection among inhabitants of Yoron Island in
Kagoshima Prefecture and clinical study on symptom in patients with strongyloidiasis
Zaha, O. et al.

43 Seroepidemioloby of an emerging parasitic disease, neurocysticercosis in lrian Jaya,
Indonesia

Ito, A. et al.

44 The resistance of treatment and specific lgG4 antibody titer in HLA‑DRB1*0901
positive patients with strongyloidiasis

Satoh, M. et al.
Asato, R. et al.

45 Strongyloides stercoralis carriers in Okinawa, Japan
46 A case of genital elephantiasis due to possible filaria infection Hamamoto, Y. et al.
47 Detection of circulating Wuchereria bancrofti antigen, filaria specific lgG and lgG4 in
Qiu, X‑G. et al.
chyluria cases in Okinawa
48 Enhancement of IL‑10 production by treatment with rlL‑3 in Trichinella spiralis‑infect‑

Korenaga, M. and Hashiguchi, Y.
ed mice49 Construction of CDNA Iibrary of Trichinella spiralis muscle larvae and the gene
Takahashi, Y. et al.
products
Agatsuma, T. et al.
50 Is the Schistosoma indicum group monophyletic?
51 Sonographic assessment of both urinary tract and hepatic morbidity due to Schistosoma
Katsumata, T. et al.
haematobium
52 Current status of boar.‑meat transmission of paragonimiasis in Japan

Kawanaka, M. et al
53 Morphological variations in next generation of Fasciola gigantica from Zambia
Terasaki, K. et al
54 A case of cystic echinococcosis in a Jordanian patient: treatment with albendazole
Kimura, M. et al.
55 Questionary test and prevalence of intestinal helrnintic infections in Burru, Slawesi,

Indonesia Toma, T. et al.
Japan Toriyama, K. et al.

56 An epidemiological study of malignant skin tumors in western Kenya and Nagasaki,
57 A study of public opinion survey of Japanese residents living in foreign countries

regarding their health Hiroshige, Y. et al.

58 A case of Amblyomma testudinarium bite Hara, N. et al.
59 System of defense frorn ultraviolet rays in wild mammals Ohwatari, N. et al.
60 Suppression of the sweat gland sensitivity to acetylcholine applied inotophoretically intropical Africans cornpared to temperate Japanese Lee, J.B. et al.
61 Installation and maintenance of protective nets against venomous jellyfish on thebeaches of Okinawa Shinjo, M. and Araki, Y.

62 Molecular characterization of the Japanese encephalitis virus representative im‑munotype strain JaGArO1 Mangada, M.N.M. and Tagegami, T.

27

Prize Winner's lecture

JSTM (Japanese Society of Tropical Medicine)
Young Investigator Award

EPIDEMIOLOGY OF INFLUENZA VIRUS INFECTIONSAMONG CHILDREN WITH ACUTE RESPIRATORYINFECTIONS (ARI) IN ZAMBIAKATSUMI MIZUTA
Virus Research Center, Sendai National Hospital

A cornmunity based study was carried out to define
the etiology and epidemiology of viral acute respiratory
infections (ARls) in children. A total of 3,760 throat

and treated as having a malaria infection in Zambia.
However, malaria infections occur mainly in the hot,rainy season (December‑April) in Zambia. Our findings

swab specimens were collected from children with ARI
at three heatlh centers in Lusaka, Zambia between June
1993 and September 1995. Specimens were inoculated
onto the microplate including HEF, HEp‑2, Vero, andindicated that influenza‑like illness with fever and/or

MDCK cell line for virus isolation.
A total of 407 viruses such as influenza virus, enter‑ovirus, adenovirus were isolated (isolation rate 10.8%) .

respiratory symptoms which occur between June andAugust should be regarded as influenza virus infection
rather than malaria infection. This fact also suggested
that a differential diagnosis is difficult but importantand laboratory based investigation is necessary for abetter medical care in developing countries.

In 1993, 54 influenza A/H3N2 were isolated between

Although a worldwide surveillance of influenza

June and November. In 1994, 34 influenza B werevirus infection is carried out by World Health Organiza‑

isolated between May and July. In 1995, one influenza
A/H3N2 was isolated in January and then the same typeof 55 influenza viruses were isolated between June andAugust. These results revealed that influenza virus
infections are one of the most important pathogens of
ARI in children in the cool dry season (June‑August) intion (WHO) , there is few report from sub‑SaharanZambia.

tory for Influenza since 1997.

Most of the patients with fever have been diagnosed

Afica according to the Weekly Epidemiological Recordof WHO. The Virology Laboratory, Lusaka, Zambia,
which have reported a surveillance result of influenza
virus infections in Central African region since 199 , hasbeen functioning as a WHO National Reference Labora‑

29

President's lecture

CUTANEOUS DISEASES IN TROPICAL MEDIClNEESPECIALLY IN OKlNAWA
S HIGEO NONAKA

Department of Dermatology, Faculty of Medicme University of the Ryukyus, Okinawa, 903‑0215, Japan
Abstract: There are tremendous number of diseases with cutaneous manifestations in tropical zone. In thepast, filariasis, malaria, and many other parasitic diseases prevailed in Okinawa. However, malaria andfilariasis have been completely eradicated to date. Okinawa is located in a subtropical zone. Mean
temperature ranges over 20'C from April to Novermber, and high humidity over 80% is one of characteristic
climatological features. In tropical climate, metabolic rate, blood flow rate and physical constitution are
also quite specific. Mobidity statistics reveals a large difference between Okinawa and mainland Japan.
Hypertensive diseases and malignant neoplasms in Okinawa reveal the lowest levels in Ja an. Incidence ofsquamous cell carcinoma of the skin is in a high level in Okinawa. Exposure level of ultraviolet light energy
is measured in four points in Japan by the Meteological Agency. The level of ultraviolet light is the highest

in Okinawa, compared with those in Sapporo, Tsukuba and Kagoshima. Patients with squamous cell
carcinoma on exposed areas showed high percentage in Okinawa. Furthermore, cases of Bowen's disease
and malignant melanoma associated with human papilloma virus infection have been reported in Okinawa.
In addition to these, there is a trend of high incidence in adult T cell lymphoma, Kaposi's sarcoma, andmalignant hemangioendothelioma. Diseases such as multiple piloleiomyorna, pilonidal sinus, and tumors
derived from hair follicle are relatively frequent. Hansen's disease is still encoutered in our outpatient clinic

of Ryukyu University Hospital.

INTRODUCTION
There are tremendous number of diseases withcutaneous changes in tropical zone. Human skin is
affected by environrnental factors such as sun light,

NEOPLASMS (Nonaka et al.. 1996)
The number of total cutaneous neoplasms in Ryu‑
kyu University Hospital from 1983 to 1994 was 720. The
number of actinic keratosis (AK) was 119 cases, that isdust, and high humidity in tropical zone (Nonaka, 1995a,b, c) . Okinawa is located in subtropical zone and its
climate is different from those in mainland Japan. Skin
temperature is higher because of high temperature andhigh humidity. There is a tendency of hyperhidrosis andincidence in female (39 cases in male and 80 cases infemale, that is, male:female=1:2.1). The average ages

hypergrowth of skin flora which are accelerated byof onset were 71.7 years in male and 76.0 years in female.

these conditions. Therefore, superficial infectious dis‑eases on the skin tend to be highly frequented in Okin‑

16.5% of total neoplasms; and squamous cell carcinoma(SCO was 132 cases, that is 18.3% (Takamiyagi et al..

1996). The sexual difference in AK showed higher

The most frequent area of AK was the face (76.5%),

ultraviolet radiations exert a great influence on car‑cinogenesis and immunity to infectious diseases. Cur‑followed by the upper extremities (8.4%), the lip (5.
9%), the ear lobes (5.9%) and the scalp (4.2%). The
number of SCC in male was 64 cases and those in female
was 68 cases. There was no sexual difference in theincidence of SCC. The average age of onset was 68.6
years in male with two cases below 40 years. One caserently, tremendous infectious and parasitic diseases still

was a 27‑year‑old male with old burn scars in his

exist in developing countries of tropical zone. It isuseful to reevaluate the present medical conditions inchildhood, and the other was a 36‑year‑old male withpenile carcinoma. The average age of onset was 76.8
years in female, which means that SCC occurs in higher
age in female than in male. The most frequented areaof SCC was the face (40.2%), followed by the neck (12.

awa compared with those in mainland Japan. Further‑more, enegy level of ultraviolet light is high in Okinawa

compared with those in Japan proper. It is possible that

Okinawa so as to keep the developing countries
improves their medical problems. In this paper, it will

be discussed whether any differences exist or not inincidence of cutaneous neoplasms and infectious dis‑eases between Okinawa and mainland Japan.

9%) , the extremities (12.9%) , the trunk (10.5%) , the lip(9.8%), the genital regions (6.8%) and others. When

30

Table 1 Comparison of distributed areas of squamous cell carcinoma

Area

Ryukyu University

Saitama Medical

Kyushu University

Hos pital9)

Schoollo)

Hospital**)Face

40 . 2%24 . 6%

20 . 2%Scalp

12 . 9%10 . 5%

13.8%

Li p

9 . 8%

8 . 9%

11 . 7%

Genital region

6 . 8%

3 . 6%

2.1%

Extremities

12 . 9%41 . 1%42 . 6%compared with those in Kyushu and Saitama University

sarcoma (KS) are relatively popular in Okinawa. Nine

Hospital, the rate of SCC on the face is larger than thosecases of MHE and 8 cases of KS were reported in
Okinawa from 1983 to 1995 (Bhutto et al.. 1995).

in other two University Hospitals (Table 1). Iri general,

SCC is prevalent in male with outdoor life than infemale (Ikeda and Kiyohara, 1988; Wada and Wada,
1989). However, our data showed that the number offemale patients with AK was twice as much as ' that ofmale in Okinawa (Takamiyagi et al., 1996). On theother hand, there is no sex difference in the incidence ofSCC. This difference between AK and SCC should be
elucidated in Okinawa. Malignant vascular tumors suchas malignant hemangioendotheliorna (MHE) , Kaposi's

Seventeen cases of multiple piloleiomyoma were report‑

ed in Okinawa (Takamiyagi et al.. 1995). There isrelatively a high tendency of this disease because one
hundrod four cases have been reported in Japan thus far.

It is a well known fact that hurnan papilloma virus(HPV) infection has been increasing in the UnitedStates and Europe. Some cutaenous tumors such as SCC
and Bowen's disease are associated with HPV (Sterlingand Kurtz, 1998). Malignant melanoma (Takamiyagi etTable 2 The patients with Hansen's disease observed at Ryukyu University Hospital over the past 16 years (1982‑1997)
type

I

year
1982

1983

BT

BB

7

3

2

5

8

5(1)

2

1

15

4

P.L.

Total
14

2
(14 . 3)

17

2

7

5

2

2

1

2

1990

1

1

1991

1

6 1
1

1

(6 . 2)

1

1

1995

1

1996

1 (1) 2

2

49 (1)

13(1)
6

2 (1)

2

4

3

1

4

3

1

5

2

2

5
1

2

11

12 (2)

36

13 (1)(8 . 1) (36 . 3) (26 . 7) (9 . 6)

( ) : Relapsed cases medicated at other hospitals

42(8 . 1)(8.9)(1 . 5)

1(1)

(1)

1

11

1

(7 . 7) (15 . 4) (7 . 7)
3

1

1997

6

1 (1)

1

1

16

(6 . 2)

1
1

1993

24

3

(4 . 2) (12 . 5)

(15 . 4) (46 . 1) (7 . 7)

1989

11 (1)

2

1(12 . 5) (48 . 3) (31 . 3)
1

1

(9 . 1) (18 . 2)

1

(62 . 5) (16 . 6) (4 . 2)

1988

Total

P.N.

2

(45 . 4) (18 . 2) (9 . 1)

1987

1994

2

LL

(29 . 4) (47 . O) (11 . 8) (11 . 8)

1985

1992

BL

(50 . O) (21 . 4) (14 . 3)

1984

1986

TT

135 (4)(O . 8) (100)31al.. 1998), verruciform xanthoma (Kamiyama et al.,1998) and Bowen's disease (Uezato et al., in press‑a, b)

contributing to tropical medicine to elucidate thesedifferences between the two areas.

associated with HPV were reported in Okinawa.

REFERENCESINFECTIONS AND OTHERS
In 1900, 30,000 cases of Hansen's disease were regis‑
tered in Japan, and 9,458 patients were still registered in1980 (Kon, 1997). Approximately, five hundreds of newpatients were reported in 1955. The reports of newpatients had been dramatically reduced since then, andonly 18 cases including 6 cases in Okinawa, were report‑
ed in 1994. The patients with Hanses's disease observed
at Ryukyu University Hospital over the past 16 years(1982 to 1997) were 135 cases (Table 2) (Hosokawa,
1998). Only one new patient in 1997 was observed. The

number of indeterminate leprosy (D was 11 cases(8.1%); tuberculoid leprosy (TT), 49 cases (36.3%);
borderline tuberculoid leprosy (BT), 36 cases (26.7%) ;middle borderline leprosy (BB), 13 cases (9.6%);
borderline lepromatous leprosy (BL), 11 cases (8.1%);
lepromatous leprosy (LL) , 12 cases (8.9%); purely neu‑
ral type leprosy (PN), 2 cases (1.5%); primary lesion(PL) was one case. Relapsed cases were one case ofTT, one case of BB and 2 cases of LL. Epidemiologicalcharacteristic in Okinawa is that old age group of thepatients are frequent with an increase of high age group.

Low immunity of the aged will influence the onset of
this disease.

Tinea nigra caused by black colored fungi is asuperficial fungal infection generally affecting the skinof palms. The first case of tinea nigra was reported in

Okinawa (Nakama, 1988). Since then, there have been
many case reports in Japan, especially in Kyushu island.

Hyperhumidity and high temperature will be main
exacerbating factors of this disease. Atopic dermatitis
in Okinawa seems to be different compared with that inmainland Japan, that is, the incidence of atopic dermati‑

tis is relatively smaller than those in other areas(Hagiwara and Nonaka, 1995). It is well‑known thatatopic dermatitis is clearly correlated with the role ofclimatological factors: either cold/dry weather leading

to skin dryness, or hot/humid weather leading to hyper‑
hidrosis, itch, and secondary infections as aggravating
factors. To get a clearer view of this disease, further

epidemiologic studies are being awaited.

1 ) Bhutto, A.M., Uehara, K., T,akamiyagi, A., Hagiwara,
K. and Nonaka, S. (1995): Cutaneous malignant heman‑gioendothelioma: clinical and histopathologjcal obser‑
vations of nine patients and a review of the literature. J.

Dermatol., 22, 253‑261

2 ) Hagiwara, K. and Nonaka, S. (1995): A statisticalassessment of atopic dermatitis at Ryukyu University
Hospital from 1988 to 1992. Ryukyu Med. J., 15, 31‑35

3 ) Hosokawa, A. (1998): Tr,eatments of patients with
leprosy at the Ryukyu University Hospital. Jpn. J.
Leprosy, 67, 313‑327 (in Japanese with English abstract)4 ) Ikeda, S. and Kiyohara, A. (1988): Squamous cell car‑cinoma. Epidemiology. 11lustrated Cancer Series, N0.20,
Skin Cancer, Sue, K. et al. (eds.), 39‑43, Medical View
Qo., Tokyo (in Japanese)5 ) Kamiyama, T., Uezato, H., Khaskhely, N.M., Nonaka,
S. and Oshiro. M. (1998) : Detection of human papilloma

virus using an immunohisto‑chemical method andpolymerase chain reaction in verruciform xanthoma: A
case report. Jpn. J. Dermatol., 108, 1283‑1289 (inJapanese with English abstract)6 ) Kon, S. (1997): Epid miology. Diagnosis and Therapyof Leprosy, Ishibashi, Y. et al. (eds.), Medical Science,

Tokyo. 71‑76 (in Japanese)7 ) Nakama, T. (1988): Tinea nigra. Hifuka Mook No. 11,
180‑187, Kanahara Publishing Co., Tokyo (in Japanese)8 ) Nonaka, S. (1995a): Dermatology in Tropical Medicine(1) . Nishinihon J. Dermatol., 57, 66‑75 (in Japanese)9 ) Nonaka, S. (1995b) : Dermatology in Tropical Medicine(2). Nishinihon J. Dermatol., 57, 290‑298 (in Japanese)10) Nonaka, S. (1995c) : Dermatology in Tropical Medicine(3). Nishinihon J. Dermatol., 57, 525‑534 (in Japanese)11) Nonaka, S., Hosokawa, A. and Takamiyagi, A. (1996):
Cutaneous diseases in Okinawa. The Diseases and theirCharacteristics in Okinawa. Nagamori, H. et al. (eds.),

101‑ 111, Kyushu University Press. Fukuoka (inJapanese)
12) Steerling, J.C. and Kurtz, J.B. (1998): Viral Infections.

Rook's Textbook of Dermatology, 1029‑1051. Blackwell
Science, Oxford.
13) Takamiyagi. A., Hagiwara, K., Uehara, K., Miyagi. T.,

Bhutto, A.M., Uesato, H. and Nonaka, S. (1995): A
statistical study of 104 cases of multiple piloleiomyoma
in Japan. Nishinihon J. Dermatol., 57, 76‑79 (in Japanesewith English abstract)
14) Takamiyagi, A., Uehara, K., Nagamine. Y.. Inafuku, K.
and Nonaka, S. (1996): A statistical assessment of solar

CONCLUSION
There are many differences in cutaneous diseases

between Okinawa and mainland Japan. It will bekeratosis and squamous cell carcinoma in Okinawa.
Ryukyu Med. J., 16, 27‑31 (in Japanese with English
abstract)

15) Takamiyagi, A., Asato, T., Nakashima, Y. and Nonaka,

32

S. (1998): Association of hurnan papilloma virus type 16

Nagata, T., Nagamine, Y., Nonaka, S. and Oshiro, M.

with malignant melanoma. Am. J. Dermatopathol., 20,
69‑7316) Uezato, H., Hagiwara, K., Maruno, M., Khaskhely, N.M.

56 in an extragenital Bowen's disease. Acta Der‑

Oshiro, M., Asato, T., Nakashima, Y. and Nonaka, S. (inpress‑a) : Detection of human papilloma virus type 58 ina case of perianal Bowen's disease coexistent with adult
T cell leukemia. J. Dermatol.

17) Uezato. H., Hagiwara, K., Ramji, S., Khaskhely, N.M.

(in press‑b) : Detection of human papilloma virus typematovenereol.
18) Wada, K. and Wada, H. (1989): A statistical study ofsquamous cell carcinomas seen at Kyushu University
Hospital during the past 10 years. Nishinihon J. Der‑
matol., 51, 758‑765 (in Japanese with English abstract)

33

S pecial lecture

ONCHOCERCIASISISAO TADA
Department of Parasitology,

Graduate School of Medicine, Kyushu University

1 . Global view of onchocerciasis

mectin (Mectizan) . Mass treatment by lvermectin1 ) Historical view
The parasite, Onchocerca volvulus, was first detect‑based on the community based system recently acceler‑

ed in a Ghanaian by a German missionary in 1890 and
was nominated Filaria volvulus by Leuckart (1893). No
pathogenicity of the parasite was found yet. In 1915,

ated African Program for Onchocerciasis Control(APOO in Africa, and Onchocerciasis Eradication Pro‑

gram of America (OEPA) in Central and South Amer‑
icas in 1990s.

however, R. Robles reported the new distribution of thisfilarial parasite in Guatemala, Central Arnerica, after
enormous parasitological, clinical and epidemiologicalstudies of coastal erysipela (Erisipela de la costa) . Heclarified that O. volvulus caused ocular lesion and thedenodulization improved the symptoms remarkably.
This is the reason that the Government of Guatemala
launched denodulization campaign in the endemic areas
since 1935. Based on the specimens obtained by Robles
and other information on vector blackfly species,
Brumpt (1919) proposed O. caec 4tiens n.sp., while thisnew species was neglected by various parasitologists.
ldentification of vector insect blackfly by Blacklock in

1926 clarified transmission mechanism and encouraged
vector control trials'in Africa (Garnham and McMahon,1947) in particular the Onchocerciasis Control Program(OCP) in late 1960s.

A filaricide drug Diethylcarbamazine (DEO
showed curative effect, while it caused severe accessory

reactions in the infected people as seen in Mazzotti
reaction. Thus the mass treatment has been considereddifficult before the appearance and registration of lver‑

Table 12 ) Epidemiological view
In 34 countries in the world, 17.7 million people (17.5

millions in Africa, 30 thousands in Arabia Peninsula and140 thousands in America) are estimated to be infected

and 270 thousands are blind. The estimated global
damages by onchocerciasis are 880,000 DALYS. The
infections in Volta River basin covered by OCP since
1974 were greatly reduced from one million people to
only 10 thousands in 1992. No more transmission occur‑
red among children in the OCP area (WHO, 1995).
3 ) Pathology
As is called "River blindness", the ocular lesions
particularly the damage and loss in visual acuity are thecharacteristic features of this disease. Onchocercal

dermatitis and subcutaneous nodules are the otherimportant lesions. The skin disease (pruritus, papules,
excoriations, depigmentation, pachydermia etc.) whichaccompanies strong itching shares half the global
DALYS of onchocerciasis and needs treatments of adultparasites because of its socioeconomic importance. On

Global estimates of the population at risk, infected and blind

RegionPo pulation
at risk ofinfection (millions)

Population

Number blind

inf ected

as a result of

onchocerciasis

Af rica

OCP area:
Original area

17 . 6*

10 , 032

17 6506.0

2 , 230 , OOO

31 700Non‑OCP area

94 . 5

15 , 246 , 800

217 850

African subtotal

118 . 1

17 , 486 , 832

267 200

Arabian peninsula

0.1

30 , OOOoAmericas

4.7

140 , 455

750

122 . 917 , 657 , 287

267 , 950

Extensions

Total* The population given is that which would have been at risk had the OCP not existed(WHO, 1995) .

34

this context, macrofilaricides such as Amocarzine, UMF
078 and etc. are now under clinical trials. The nodules
called onchocercomatas show characteristic distribution
patterns in each continent: No nodules are found on the

successful achievement of OCP, OEPA (started in 1993)
and APOC (started in 1996) were planned and launchedin other endemic areas of the world. Both programs relyon the community directed treatment system based on
the lvermectin and supported by Geographic Informa‑tion System (GIS) for rapid epidemiological mapping.

head in Africa, while half the nodules are on the head inCentral America.

4 ) Control programs
OCP adopted vector control operations by spraying
larvicides such as Temephos (Abate) in the breeding
rivers for Simulium damnosoum complex in the begin‑ning and then added lvermectin delivery system. Basi‑cally 12 thousand km of river were under weekly spray‑
ing of larvicide during rainy season. Rotation of lar‑

2 . Research of human onchocerciasis on humanonchocerciasis by Japanese
No research has ever been performed by Japanese
before a joint study of skin test by us (Tada andFigueroa, 1969). In 1970, I visited a plantation Pana‑jabal, Iocated in Sierra Madre Mountains of Guatemala,guided by Dr. H. Figueroa for the first time. The

vicides and biological larvicide (B. thrungiensis H‑14)

plantation was one of the most highly endemic areas ofwere also adopted later. OCP prevented 9 million

the disease in America. A blind old man has beenchildren born within original OCP area from the risk ofwalking there. The miserable scene shocked me greatly.
Fortunately I was awarded with an opportunity to learn
more about onchocerciasis in Ethiopia being dispatchedonchocercal blindness. Thirty million people areprotected from infection and 100 thousands have been
prevented from going blind. Thus OCP opened up theway for resettlement in fertile areas along the rivers,
previously deserted through fear of disease. OCP activ‑
ity was scheduled to cease in the year 2002. During theby the ODA agency, OTCA (Overseas Technical Coop‑eration Agency, Japan) , next year together with 2
Japanese entomologists and one parasitologist. We
visited several times the endemic areas and obtained

Table 2
Research timeline of human onchocerciasis in Japan
1969

1970
1971
1973
1975

1976
1980
1981
1982
1983

1984
1985
1986
1987
1990s

1997

Result of skin test study on onchocerciasis in Guatemala was reported (1. Tada and H. Figueroa)
I. Tada visited an endemic plantation in Guatemala C.A. to see onchocerciasis

Four Japanese stayed in Ethiopia dispatched by Overseas Technical Cooperation Agency (OTCA) for thetechnical collaboration and study of onchocerciasis (Team leader: T. Ohse)

I. Tada: Brief survey of onchocerciasis in Guatemalan endemic areas dispatched by OTCA. J. Morirequested Japanese Government to support research and control as the Ambassador of Japan
JICA: Dispatch of Basic investigation team to Guatemala (Headed by A. Nakajima)
JICA: Dispatch of Project installation team to Guatemala (Headed by S. Hayashi) to exchange Record of
Discussion (RD)JICA‑Technical cooperation project of onchocerciasis started (Tearn leader: H. Takahashi, Chief of JICA
Steering Committee: S. Hayashi)
JICA: Three‑year extention of research project (Team leader: T. Suzuki)
JICA‑Guatemala: International Congress of Onchocerciasis (Guatemala City)
Min. Education, SC, Japan supported a comparative study of onchocerciasis and its transmission between
South and Central Americas (1) (Headed by I. Tada)
JICA: Collaboration project expired. Among the Japanese experts, Y. Yamagata later joined OCP
Min. Education. SC, Japan: a comparative study of onchocerciasis and its transmission between South andCentral Americas (2) 'JICA: Jos University (Nigeria) project dealt with onchocerciasis (Team leader: H. Takahashi)

I. Tada joined WHO Expert Committee on Onchocerciasis (Geneva) Min. Education, SC, Japan: acomparative study of onchocerciasis and its transmission between South and Central Americas (3)Publishment of 3 volumes of Green books (ed. by I. Tada) dealing with studies supported by Min. Education,

SC, Japan
H. Takaoka: Human infections with zoonotic Onchocerca infections in Japan
I. Tada: Participation to Task Force meetings by TDRIWHO for global onchocerciasis control

35

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36

technical experiences and epidemiological data there. In
1973, I was sent to Guatemala for further investigation

on onchocerciasis by OTCA. I proposed a report of abrief epidemiological survey to the Governments ofGuatemala and Japan asking the survey and control ofthis disease. Mr. J. Mori, the Ambassador of Japan,
requested Japanese government to start a research
project for the control of onchocerciasis through JICA
(Japan International Cooperation Agency) .

1 ) ODA project for the research of onchocerciasis
control

A total of 12 parasitologists and medical entomologists

joined this project. We compared O. volvulus and its

transmission in Guatemala, Ecuador, Venezuela andNigeria using cytogenetic, parasitological, entomologi‑
cal, histochemical nd morpho‑metrical methodologies.
For the comparison of O. volvulus from Central Amer‑ica (Guatemala) and South America (Venezuela), we
adopted the methodologies of Duke et al. (1967) andcompared bilogical similarity of each strain to vector
blackfly by the cross infection. Cytogenetic analysis of

the parasite from Central/South America and Africa
revealed a decisive conclusion on the identity of theThus a technical collaboration project started inparasite (Hirai et al., 1987). Finally we concluded that1975 (Actually it started in early 1976 because of a big
earthquake) and lasted successfully for 8 fiscal years.

Guatemalan Onchocerca was biologically quite similar

The majority of the field works were performed in San

been imported from Africa by the slave trade, too

Vicente Pacaya County. The project yielded various
products in terms of technologies in entomological,
parasitological, ophthalmologic, and epidemiological(Tada, 1983, 1985, 1987),

fields (Suzuki and Mizutani, 1992). In this project a
total of 90 Japanese experts were dispatched and 13
Guatemalan counterparts were awarded with fellowship

to that of South America and thus considered that it had3 ) Aftermath
In 1997, I was invited to two Task Force meetingsof TDR (Filariasis) and in that occasion I could visit a

to visit and learn in Japan. Apart from the scientificallyformer endemic village at Burkina Faso, West Africa.
Although no more transmission has been occurring in

new findings related to onchocerciasis, standardization

this area due to the long lasting and successful controlof various techniques such as immunodiagnosis, in‑operations by OCP, I saw a blind old man walking

seticide spraying, identification of vector blackflies etc.

was intensively promoted. Eventually about 90 scien‑around with stick by himself among actively playing
children. Apparently the villagers have come back totific papers and a set of technical manuals were
produced. Further an international congress of on‑

disappeared. The scene overlapped his counterpart inchocerciasis was convened in 1981 under the auspices of

Guatemala with whom I met 27 years ago. OCP wasGuatemalan government and JICA with the participa‑tion of experts of onchocerciasis control from WHOl
OCP (Tada, 1997).

certainly a successful health program against river‑once abandoned site as the blackflies completely

blindness and hence APOC/OEPA also should reveal
sirnilar good results.

On the other hand, I would like to indicate a serious

2 ) Research project on the origin of Central American
onchocerciasis

During this ODA project, a question has arisen
whether the onchocerciasis in Guatemala was imported
by slave trade from West Africa or was autochtonous incontinental America (Figueroa, 1963). This has been amatter of arguments in Guatemala and Mexico since
when Brumpt proposed O. caecutiens as new species.
Perforation of the skulls of Pre‑Colombian inhabitants

suggested its onchocercal origin and Duke and col‑leagues suggested biological remoteness of O. volvulus
between South American and African parasite strains.
In order to examine this question, my colleagues and I
launched a research project to investigate the origin of
onchocerciasis in Central America being supported by
the grant from the Ministry of Education, Science andCulture, Japan during the period between 1982 and 1987.

fact: After a rush of research on Onchocerca and its
transmission, very few Japanese scientists or, I mightsay, only one, are engaging in the study of Onchocerca
nowadays. Even though we have established the tool
and way of control/elimination of onchocerciasis, isresearch of Onchocerca not necessary any more? Ger‑man scholars belonging to Bernhard‑Nocht Institue ofTropical Medicine are still continuously reporting theirresearch activities based on field laboratory in Liberia.

They range from morphological to molecular biologicalfindings which are very important to understand para‑
sitism and its treatment in general. On this context, I
feel strong necessity to establish and maintain an institu‑

tion in tropical countries collaborating with local staff.

Because Japan lacks adequate number of qualified scien‑tists in infectious and parasitic diseases and tropical
medicine.

37

In the era of emerging and re‑emerging infectious
diseases, field research is very important to promoteresearch of infections and parasitic diseases.

Ufomadu, G.O. (1987): Chromosomes of Onchocerca
volvulus (Spirurida: Onchocercidae) : A comparative
study between Nigeria and Guatemala. J. Helminthol.
61, 43‑46

4 ) Suzuki, T. and Mizutani, K. (1992): Onchocerciasis

REFERENCES
1 ) Duke, B.O.L., Moor, P.J. and DeLeon, J.R. (1967):
Orrchocerca‑Simulium complexes. V. The intake andsubsequent fate of microfilariae of a Guatemalan strainof Onchocerca volvulus in forest and Sudan savanna

forms of West African Simulium damnosum. Ann.
Trop. Med. Parasit., 61, 332‑337

2 ) Figueroa, H. (1963): Historia de la enfermedad deRobles en America y de su descubrimiento en
Guatemala. Editorial Luz., Guatemala.
3 ) Hirai, H., Tada, I., Takahashi, H., Nwoke, B.E.B, andvector control in Guatemala. 43, 273‑286
5 ) Tada, I. and Figueroa. H. (1969): Reacciones cutaneas
al antigeno FPT de Dirofilaria immitis en la oncocer‑
cosis. Rev. Col. Med. Guatemala, 20, 158‑163
6 ) Tada, I. (1983, 1985, 1987): A comparative study on
onchocerciasis between South and Central Americas) .

Shimoda Printing and Co., Kumamoto.
7 ) Tada. I. (1996): Parasitology and international health.
Jpn. J. Parasitol., 45, 465‑473

8 ) W.H.O. (1995): Onchocerciasis and its control. WHO
Technical Series 852, WHO, Geneva.

39

Educational lecture
1

CHRONlC AND INTENSIVE SOLAR ULTRAVIOLET EXPOSURE IN
TROPICAL AREAS PROMOTES PHOTOAGlNG AND SKlN CANCERMASAMITSU ICHIHASHll, MASATO UEDA1, TOHRU NAGANol, KEISHI ARAKll,

SANTOSO CORNAIN2, MUCHTAR HAMZAH2, MpU KANOK02,
HIROYUKI OHN03 AND NOBUO MUNAKATA4
Department of Dermatology, Kobe University School of Medicinel,
Faculty of Medicine, The University of Indonesia',

Department of Preventive Medicine, Nagoya University School of Medicine*and Radiobiology, National Cancer Center Research Institute*
Abstract: DNA damage remaining in the cells several hours after a single exposure to ultraviolet radiation

may play an important role in the sunburn reaction, and chronic repeated exposures may induce genemutations related to carcinogenesis, and immuno‑suppression by UV may be involved in skin cancer
development. Epidemiological studies conducted in people with light skin have shown that sunlight is themost important risk factor for skin cancer, and experimental studies using mice have clarified that ultravio‑
let B (290‑320 nm) radiation is causative of skin cancer. The prevalence and incidence of skin cancer inpeople having darker skin, however, has not yet been fully studied. Therefore, we examined precancerous
lesions and cancer prevalence and incidence in Japan and Indonesia, and analyzed risk and preventive
factors for skin cancer in a joint study between Kobe Universtiy and the University of Indonesia, since 1995.
Pathology‑based cancer registry in Indonesia showed a higher incidence of non‑ melanoma skin cancer(NMSO in males (6.62 per 100,000 population) and females (6.54) than in Japan as reported by IARC. The

ratio of basal cell carcinoma (BCO to squamous cell carcinoma (SCO was about 4:1 for Indonesia andabout 2.04:1 for Japan. Further, a case‑control study suggests that less education, and outdoor working
conditions are risk factors, while diet containing high protein, such as meat and eggs are preventive factors
for skin cancer. Incidences of solar keratosis in Kasai city (35'56'N) and at le‑island (26'42'N) in 1995 were145.6 and 637.0 per 100,000 population, respectively, indicating that the higher ambient solar radiationinduces higher incidence of skin cancer in Japan.

INTRODUCTION
In the early 20th century, sunbathing was shown tobe essential to making vitamin D in the skin, which
prevents ricket.

Further, sunlight was believed to protect subjects
from tuberculosis. Those perceived benefits of sunbath‑ing promoted the public to expose their skin to solar
radiation, and a suntan has been regarded as a symbol of
health.

However, epidemiological studies on skin cancer inlight skined people (Urback et al.. 1974) indicated thatsunlight is the most harmful cause of photoaging andcancer development of human skin, based on the find‑ings that: (1) people who live in areas with high ambi‑

ent solar radiation have a higher incidence of skincancer than people who live in areas of lower solar
radiation, (2) people working outside have a higher
incidence of skin cancer than people working indoors,and (3) people who spent their lives in strong sunnyareas during childhood showed a higher incidence ofskin cancer than people who lived in areas with lesssunlight, in groups exposed to the similar level of solar

light after 10 years of age (Kricker and Armstrong,1995) .

Blue eyes, Iight coloured hair and light coloured
skin that burns easily and tans poorly, have been shown
to be primary intrinsic factors for skin cancer.

Experimental studes using hairless mice showedthat ultraviolet light B radiation (waveband ranging
from 290 nm to 320 nm) is a pivotal causal factor of skincancer, possibly due to direct and indirect damage ofDNA, and suppressive effect on immune system.
Molecular studies on skin cancer cells of humans
and other animals indicate that mutations of several
genes relevant to cell proliferation, such as p53 andpatched, may play pivotal roles in UV‑carcinogenesis(Brash et al.. 1991; Hahn et al. 1996).

1. Sunburn may be caused by DNA damage
Xeroderma pigmentosum patients, develop skincancers in childhood, have defective DNA repair of UV‑

induced DNA damage and are easily burned by a small
amount of solar radiation with frequent edema and40bullae formation (Ichihashi et al. 1982). Japanese skin
type I subjects who burn easily and tan poorly have 3‑5

Table 1times more DNA damage [cyclobutane pyrimidine

Factors

dimers and (6‑4) photoproducts] than do skin type II
and 111 subjects who burn and tan moderately, or whoburn rarely and tan heavily, respectively (Ueda et al..

1996). (3) American opossums, who have a photor‑
eactivating enzyme that repairs DNA damage only
when the enzyme is activated by visible or UVA Iight,

required about one fouth of UVB dose necessary to
induce minirnal erythema when the their skin was pre‑Risk factors for skin cancer of Indonesian

Case

Control

OR (95% CI)+ ‑ + ‑

Gender type

15

32

28

64

1 . 07(O . 47‑2 . 44)

Education level

41

6

52

40

5 . 26(1 . 89‑15. 34)

Working outside

20

27

13

79

4 . 50(1 . 84‑11. 17)

Physical protect

24

23

64

28

O . 46(O . 21‑1 . OO)

Smoking

17

30

22

70

1 . 80(O . 78‑4 . 15)+ and ‑ in gender means male and female respectively.
+ and ‑ in education means low and high, respectively.

exposed to UVB and then kept in the dark compared

Hedgehog to Smoothened in the f uture (Fan et al.,with animals exposed immediately to visible light irradi‑1997) .

ation after UVB exposure (Ley, 1985).
These clinical and experimental evidences strongly
suggest that DNA damage remaining in the cells several
hours after UV exposure may play an important role insunburn reaction.

4. Skin cancer and precancer prevalence and incidenceof Japanese who live in Kasai city and le‑island

Studies on skin cancer were initiated in 1992 in
Kasai city (35'56'N) and in 1993 at le‑island (26"42'N).

Four cases of NMSC were found in Kasai city (1992‑
2. The role of p53 in UV carcinogenesis
Sunburn cells are now understood to result from anrole of wild type p53 protein in the prevention of UV
carcinogenesis by increasing apoptosis of UV‑irradiatedcells which had too much DNA damages to be repaired1997) and 11 cases at le‑island (1993‑1997). The num‑
ber of solar keratosis (SK) found every year in each
10cation was more than 10. The total SK patients were
128 and 130 at Kasai city and le‑island, respectively.
The numbers of NMSC in both areas were too small to
calculate the NMSC incidence in Japanese.
The prevalence of SK in Kasai city (Naruse et al..

correctly.

1997) was between 413.4 and 86.8 per 100,000 population,

3. Skin cancer epidemiology in Indonesia
The pathology‑based cancer registry in Indonesia

showed a high incidence of non‑melanoma skin cancer

incidence of SK in Kasai city (Suzuki et al.. 1997) andat le‑island in 1995 were 145.6 and 637.0, respectively(Table 2). SK incidence in le‑island was about 5 times(NMSO in males (6.62 per 100,000 population) andhigher than in Kasai city, indicating that the annual UV

apoptotic process which is controlled at least in part byp53 protein. Ziegler et al. (1994) suggested a possible

and showed decreasing trends from 1992 to 1997. The

females (6.54) , compared with rates in Japan as report‑

dose is an important factor of SK development ofed by IARC (Cornain, 1998). Further, the incidence ofJapanese. Skin type I subjects who burns easily and tan
poorly showed a higher prevalence of SK than skin typebasal cell carcinoma (BCO in Indonesia was about 4times higher than that of squamous cell carcinoma(SCO , whereas the BCC/SCC ratio in Japan was nearly

III subjects who burn rarely and tan heavily. The SK

2: 1, suggesting a role of some unknown environmental
and constitutive factors which may cause such differ‑ences between these two populations. The incidence of
NMSC in Bali in 1996, based on pathology registry, wasTable 2 Incidence of solar keratosis in Kasai city andle‑island (per 100,000 population)nearly 19.7 per 100,000 population, suggesting that solar

Kasai cityradiation may be an important causative factor of skincancer for Indonesians with pigmented skin.
A case‑control study on skin cancer in Indonesia
conducted during 1997 and 1998 suggests that low educa‑tion and outdoor working conditions are risk factors,
while a high protein diet, such as meats and eggs, arepreventive factors of skin cancer (Table 1) .
Molecular epidemiology is expected to clarify therole of some factors altering the signal pathway fromYear examined

Number of people

examinedPatients

Incidenceper 100,000

1993

2

516

16

1994

2 518

12

171 . 2

1995

2 622

10

145 . o

1996

2 706

5

107 . 3

1997

2 562

11

99 . 9223 . 6le‑island

1995

1

014

15

637 . o

1996

1

035

20

625 . 5996

20641 . 31997

Age and sex were standardized to the Japanese population in 1990.

41

prevalence of subjects having more than 5 seborrheic
keratosis was significantly higher than subjects having
less than 5, suggesting that nurnerous seborrheic ker‑

atoses may be a risk for SK and possibly for SCC(Table 3) .

5. Prevention of photoaging and skin malignancy

UV‑induced DNA damage causes UV‑specificmutation, such as C ‑> T and CC ‑ TT alterations in

oncogenes, tumor suppressor genes and cell cycle
regulatory genes, which can result in the development ofcancer cells in the epidermis. Tumor formation in theepidermis can be caused by DNA‑damage produced
directly by UV radiation or indirectly by reactive oxy‑
gen species (Hattori et al.. 1996) , and also by irnmuno‑

ACKNOWLEDGEMENTS
This work was supported in a Grant‑in‑Aid Inter‑national Scientific Research‑Specific Cancer Research‑from the Ministry of Education, Science and Culture ofJapan, in part by the Research Fund for the Effects of

Ultraviolet Ray Influence on Human Health from the
Global Environmental Research, Environmental Agencyof Japan, in part by a grant from Skinryoku‑kai, Kobe
University School of Medicine, Japan, and in part by agrant of Japanese Committee for Sunlight Protection.
The authors appreciate the sponsorship and support of
JSPS and ICMR of Kobe University School of Medicine.
The authors are very grateful to Miss Mari lwao for
editorial assistance.

suppression (Fisher and Kripke, 1977).
Protection of the skin from severe sunburn in child‑

REFERENCES

hood and adolescence reduces the life‑10ng risk ofphotoaging and skin cancer.
Skin cancer prevention education is particularly
important for school children since they spend a large
proportion of their time outdoors.
The Australian, "Slip! Slop! Slap!" guideline, that is;wearing long sleeve clothing, using a sunscreen andwearing a hat, will reduce the risk of skin damage andskin cancer, by protecting skin cells against solar radia‑

tion‑induced DNA damage and immunosuppression.

1 ) Brash, D.E., Rudolph, J.A., Simon. J.A., Lin, A.. McKen‑na, G.J., Baden, H.P., Halperin, A.J., Ponten, J. (1991):

A role for sunlight in skin cancer: UV‑induced p53
mutations in squamous cell carcinoma. Proc. Natl.
Acad. Sci. USA, 88, 10124‑10128

2 ) Cornain, S. (1998): Organization and strategy ofmulticenter study on skin cancer. Programs andAbstracts of Workshop on Multicenter Study on
Etiology and Clinicopathology of Skin Cancer, Jakarta,
Oct.

3 ) Fan, H.E.O.A., Scott, M.P. and Khavari, P.A. (1997):
Induction of basal cell carcinoma features in transgenic

Table 3

Prevalence of solar keratosis (per 100,000 popu‑
lation) in Kasai city and le‑island in relevanceto the number of seborrheic keratosis and smok‑
i ng

human skin ekpressing Sonic Hedgehog. Nature, Medi‑cine, 3, 788‑792

4 ) Fisher, M.S. and Kripke, M.L. (1977) : Systemic altera‑tion induced in mice by ultraviolet light irradiation andits relationship to ultraviolet carcinogenesis. Proc. Natl.

Kasai City

Acad. Sci. USA, 74, 1688‑1692

Prevalence in the year examined
1994

1995

1996

1997

5 )‑ Hahn, H., Wicking, C., Zaphiropoulous, P.G., Gailani, M.

R., Shanley. S., Chidambaram, A., Vorechovsky, I.,(1) Seborrheic keratosis

Holmberg, E., Unden, A.B., Gillies. S., Negus. K., Smyth,I., Pressman, C., Leffell, D.J., Gerrard, B., Goldstein, A.

: 6

496 . O

207 . 1179 . 1143 . 8O‑5

86 . 6

5.4

38 . 240 . 5

ND
ND

ND
ND

142 . 3139 . 7

Wainwright. B. and Bale, A.E. (1996): Mutations of the

163 . 9158 . 4

human homolog of Drosophila patched in the nevoidM., Dean, M.. Toftgard, R., Chenevix Trench, G.,(2) Smoking
yesnole‑island

Prevalence in the year examined
1995

1996

1997

696 . 1407 . 1379 . 186 . 175 . 488 . 2(1) Seborrheic keratosis(2) Smoking

yes ND

no ND

242 . 3264 . 9263 . 9305 . oSex and age were adjusted to the population in 1990.

ND means "not done".

basal cell carcinoma syndrome. Cell, 85, 841‑851
6 ) Hattori, Y., Nishigori, C.. Tanaka, T.. Uchida, K.,
Nikaido, O., Osawa, T., Hiai, H.. Irnamura, S. andToyokuni, S. (1996) : Formation of 8‑hydroxy‑2'‑deoxy‑
guanosine in epidermal cells of hairless mice after
chronic UVB exposure. J. Invest. Dermatol., 107, 733‑
7377 ) Ichihashi, M. and Fujiwara, Y. (1982): Clinical andphotobiological characteristics of Japanese xeroderma
pigmentosum (XP) variant. Br. J. Dermatol., 105, 1‑128 ) Kricker, A. and Armstrong, B.K. (1995): Sun exposure
causes both nonmelanocytic skin cancer and malignant

(42

melanoma. In: Schopka, H. ‑J. (eds.), Environmental

UV Radiation and Health Effects. Bfs‑USH‑171/95,
105‑ 1139 ) Ley, R.D. (1985): Photoreactivation of UV‑iduced
pyrimidine dimers and erythema in the manuspialmonodelphis domestica. Proc. Natl. Acad. Sci. USA, 82,12) Ueda, M., Matsunaga, T., Bito, T., Nikaido, O. andlchihashi, M. (1996): Higher cyclobutane pyrimidine
dimer and (6‑4) photoproduct yields in epidermis ofnormal humans with increased sensitivity to ultraviolet

B radiation. Photodermatol. Photoimmunol.
Photomed., 12, 22‑26

S., Imaizumi. K., Watanabe, S. and lchihashi, M. (1997):

13) Urback, F.. Epstein, J.H. and Forbes, P.D. (1974):
Ultraviolet carcinogenesis: experimental, global andgenetic aspects. In: Sunlight and Man. Pathak, M.1.,

Prevalence of actinic keratosis in Japan. J. Dermatol.

Harber, L.C., Seiji, et al. (eds.), 259‑283. Tokyo Univ.

2409‑2411
10) Naruse, K., Ueda, M., Nagano, T., Suzuki, T.. Harada,

Sci., 15, 183‑187

Press, Tokyo.

11) Suzuki, T., Ueda. M., Naruse, K., Nagano, T., Harada,

14) Ziegler, A., Jonason, A.S., Leffell, J.A., Sharma, H.W.,

S., Imaizumi, K., Watanabe, S. and lchihashi, M. (1997) :Kimmelman, J., Remington, L., Jacks, T. and Brash, D.
E. (1994): Sunburn and p53 in the onset of skin cancer.
Nature, 372, 773‑776

Incidence of actinic keratosis of Japanese in Kasai city,

Hyogo. J. Dermatol., 16, 74‑78

43

2 IMMUNOMODULATORY EFFECTS OF ULTRAVIOLET RADIATIONTAKESHI HORIO
Department of Dermatology, Kansai Medical University, Fumizono 10‑15, Moriguchi, Osaka 570‑8507
Abstract: Sunlight can induce photoallergic reactions both of immediate and delayed types. Endogenousand exogenous chromophores in the skin may be transformed to antigens by light energy. For instance, solar
urticaria is induced by lgE‑mediated allergic reaction, and drug‑induced photoallergic dermatitis is T cell‑

mediated immunologic reaction. On the other hand, ultraviolet radiation may conversely suppress im‑
munologic reactions, affecting a variety of immuno‑competent cells including epidermal Langerhans cell,
keratinocyte, T cell, mast cell and vascular endothelial cell. Exposure to UV Iight inhibits contact sensitiza‑tion to haptens applied not only to the irradiated skin area but also to the non‑irradiated distant skin whenthe exposure dose is relatively high and/or the application skin area is large. In addition, hapten‑specifictolerance develops, which is due to the generation of hapten‑specific suppressor T cells. Similar phenome‑
non can be induced by UVR in delayed type hypersensitivity to microorganisms and tumor immunity. Down
regulation of antigen presentation and production of immunosuppressive cytokines may be involved in these

UV‑induced immunosuppressions.

INTRODUCTION
Recently it is generally accepted that the skin is an

important immune organ which is composed of various
immuno‑competent cells, "skin associated lymphoidtissues (SALT)". These include Langerhans cell, kera‑
tinocyte, T Iymphocyte, mast cell and vascular endothe‑lial cell. We can easily understand that immunologicalproperties of the skin can be modulated by ultraviolet
radiation (UVR) , since the skin is an outermost organ

ate photoallergic reaction is clinically solar urticaria.

The majority of cases with solar urticaria are induced

by endogenous factors (Horio and Minami, 1977). The
responsible allergen may be produced by light energy
from serum factor. In contrast, exogenous photosensit‑

izers have been rarely identified in immediate type(Horio, 1975) . Photoallergic reactions of delayed hyper‑

sensitivity can be induced by either topically orsystemically applied exogenous drugs and chemicals.
The reaction manifests as an eczematous change. The

which is constantly exposed to the sun. Sunlight canmechanism of photoallergic contact dermatitis is closely

affect the immunologic or allergic reactions of the skinsirnilar to that of allergic contact sensitivity. Photoal‑

in two different ways. Allergic reactions may be in‑lergic reaction can be experimentally induced in animals

duced by sunlight in selected subjects on the one hand,using photosensitizing chemicals. The animal model has
clarified that photoallergic contact dermatitis is a T
cell‑ mediated immunologic reaction.

and immunologic reactions may be suppressed by UVR
in all subjects on the other hand. Photoimmunology is afield of research to investigate the effects of light energy

on immune system.
1 . Photoallergic Reactions
When the skin is exposed to the sunlight, various
components in the skin absorb light energy, and some ofthem are transformed into photoproducts, which may
become antigens or haptens. So, we can define thatphotoallergy is an immunologic reaction in which lightenergy may play a certain role in the formation of
antigens. Endogenous nd exogenous substances can betransformed to antigens by sunlight radiation. Theselight‑induced antigens (photoproducts) can develop
both immediate and delayed types of photoallergy.

2. UVR‑Induced Immunosuppression
1 ) Effects on Tumor Immunity
It is well known that UVR induces skin cancers.

The UVR‑induced skin tumors in mice do not growprogressively but are rejected when transplanted innormal syngeneic recipient mice. These tumors canonly grow when transplanted into ,an immuno‑comprom‑ized host, suggesting that the UVR‑induced tumors arehighly antigenic, recognized as foreign tissues, and are

rejected by the immune response of the normal mice.
How then do these highly antigenic tumors grow pro‑

photoallergic reactions; endogenous immediate,
exogenous immediate, endogenous delayed, andgressively escaping the immune response in the primary
UV‑irradiated host? It was demonstrated that exposure
to subcarcinogenic doses of UVR suppressed the genera‑tion of cell‑mediated immune reactions by inducing the
production of antigen‑specific suppressor T cells (Krip‑

exogenous delayed photoallergy (Horio, 1984) . Immedi‑ke, 1984). This indicates that UVR can impair the

Therefore, there may be theoretically at least 4 types of44

Induction of Tolerance

Indu*tio* of Tolerance Induction of CHS

challenge chanengesensitizatio*

challenge¥

sensitizationJf

tow dose sensitizationof
UVB area
on low
UVB
small
on dosesmallofarea

Figure I Local immunosuppression.

Topical application of hapten to UV‑exposed skinarea does not induce contact hyperSensitivity, but
induceS tolerance (left) . Hapten application tonon‑exposed skin can induce hypersensitivity(right) .

fhigh dose of UVB
on large‑ area

Figure 2 Systemic immunosuppression.
When large area of the skin is exposed to high doseof UVB, hapten application even to non‑exposedskin cannot induce contact hypersensitivity.

immunological defense Of hOStS against tumOrs.
dermis, and impairs antigen presenting functions. Appli‑

2 ) Effects on Contact Hypersensitivity

cation of contact allergens to the UV‑irradiated, LC‑

Based on the experimental evidences described

depleted skin results in no induction of hypersensitivity.

above, UVR has been shown to suppress immune

It is possible that UV‑irradiated LC obtains tolerance‑
inducing ability rather than an activating capacity in the

responses to a variety of antigens including sensitizing

chemicals (Toews et al.. 1980) and microorganisms(Howie et al., 1986; Norval et al.. 1994). We have also
asked a question how photo (cherno) therapy exerts theirbeneficial effects on various allergic disorders of theskin (Horio and Okamoto, 1982; Horio and Okamoto,immune system because of impairment of costimulatory
signals between LC and T cells. A group of investiga‑

tors presented experimental evidences that down‑
regulating antigen presenting cells appear in the skinafter UV‑irradiation (Baadsgaard et al., 1988) .

1983) .

The contact hypersensitivity response to antigens
applied topically and the delayed type of hypersen‑

4 ) Effects on Lymphocytes

sitivity response to antigens injected intradermally canlymphocytes in the periphery blood can be altered after

be suppressed not only at the irradiated skin (10cal

UV‑exposure of human body (Morison et al., 1979). In
this report, T Iymphocytes were more sensitive to UVR
than B cells. We indicated that lymphocyte migration
towards chemoattractants was inhibited after in vitro
PUVA treatment (Okamoto et al.. 1985), and that IL‑2

immunosuppression) but also at a site distant frorn theirradiation when exposed to higher dose of UVR (sys‑temic immunosuppression) (Figs. 1, 2). Furthermore,these animals, that obtained UV‑induced immunosup‑
pression, fail to become sensitized to the subsequentsensitization regimen of the same hapten even throughpreviously unexposed skin. These immunosuppressions

It has been shown that subpopulations of

production by T cells in the spleen was suppressed by invivo PUVA treatment in mice (Okamoto et al., 1987).
We also observed that the conversion frorn CD45RA+

may be due to the induction of antigen‑specific suppres‑naive T Iymphocyte to CD45R0+ memory cell by

sor T cells. Therefore, the tolerance can be transfered
by injectirig T cells from UV‑exposed mice into naive

mitogen stimulation was inhibited after UV‑irradiation.

recipients.

5 ) Effects on Mast Cells
The mast cell is an essential immunocompetent cell3 ) Effects on Antigen Presenting Cells

Epidermal Langerhans cell (LO is an antigen

which is involved in lgE‑mediated immediate typehypersensitivity such as urticaria. Skin change ofpresenting cell which plays the first role in the induction

urticaria is induced by chemical mediators from mastof immunologic reactions in the skin when invaded byantigenic agents. A single exposure of UVB (280‑320nm) alone (Toews et al., 1980) or 8‑methoxypsoralencells. We have demonstrated that UVB and PUVA

plus UVA (320‑400 nm) (PUVA) photochemotherapy

1985, 1988). We confirmed clinically the therapeutic
effect of PUVA photochemotherapy on urticaria reac‑(Okamoto and Horio, 1981) depletes LC from the epi‑

irradiation suppressed degranulation and histamine
release of mast cells in vivo and in vitro (Danno et al.,

45

tion (Hashimoto and Horio, 1995).

6 ) Effects on Keratinocytes

The keratinocyte is a major population of the epi‑

dermis constituting approximately 90% of the whole
epidermal cells. Keratinocytes can produce a variety ofcytokines and proinflammatory chemokines. The UVR
enhances the production and release of some of thesesoluble factors such as IL‑1, IL‑10, TNF‑alpha andprostaglandin which can inhibit immunologic reactions(Lugar and Schwarz, 1995). More recently it wasdemonstrated that immunosuppressive neuropeptides,
such as alpha‑MSH and calcitonin gene‑related peptide,are produced by keratinocytes after UV‑exposure(Lugar, 1998).

photo (chemo) therapy.

REFERENCES
1 ) Ba, adsgaard, O., Fox, D.A. and Cooper, K.D. (1998):
Human epidermal cells from ultraviolet‑exposed skinpreferentially activate autoreactive CD4+2H4+ suppres‑sor‑inducer lyrnphocytes and CD8+ suppressor/cytotox‑
ic T Iymphocytes. J. Immunol., 140, 513‑519
2 ) Danno, K., Toda, K. and Horio, T. (1985) : The effect of

8‑methoxy psoralen plus long‑wave ultraviolet (PUVA)
radiation on mast cells; PUVA suppresses degranulationof mouse skin mast cell induced by compound 48/80 orconcanavalin A. J. Invest. DermatQl., 85, 110‑114
3 ) Danno, K., Fujii, K., Tachibana, T., Toda. K. and Horio,
T. (1988): Suppressed histamine release from rat per‑itoneal mast cells by ultraviolet B irradiation; Decreased7 ) Effects on Vascular Endothelial Cells

Associated with immunologic and inflammatory
reactions, various cells infiltrate into the skin fromblood vessels. Prior to the extravasation of inflamma‑

diacylglycerol formation as a possible mechanism. J.
Invest. Dermatol., 90, 806‑809
4 ) Horio, T. (1975) : Chlorpromazine photoallergy; Coexis‑
tence of immediate and delayed types. Arch. Dermatol.,
111, 1469‑1471

tory cells, adhesion molecules must be expressed on
vascular endothelial cells. Highly expressed adhesion
molecules in inflammatory skin are decreased after the
resolution of the disease. We observed that the expres‑sion of E‑selectin and ICAM‑1 on cultured endothelial
cells by TNF‑alpha stimulation was inhibited after UV‑

113, 157‑160
6 ) Horio, T. (1984): Photoallergic reaction; Classificationand pathogenesis. Int. J. Dermatol., 23, 376‑382

exposure (Yamawaki et al.. 1996). Therefore, it ispossible that UVR may have anti‑inflammatory effectultraviolet light on experimental contact sensitization.
J. Invest. Dermatol., 78, 402‑406

at lower dose.

5 ) Horio, T. and Minami, K. (1977) : Solar urticaria;
Photoallergen in a patient's serum. Arch. Dermatol.,7 ) Horio, T. and Okamoto. H. (1982): The mechanisms ofinhibitory effect of 8‑methoxypsoralen and long‑wave8 ) Horio, T. and Okamoto, H. (1983): Immunologic unre‑8 ) Effects on Natural Killer Cells
Natural killer (NK) cell may provide a first line ofdefense against turnor growth and viral infections. The

number and activity of NK cell have been reportedly
decreased after UVB and PUVA irradiation (Schacter
et al.. 1983; Toda et al., 1986).

CONCLUSION
In the past two decades a new field of research,termed photoimmunology, has been developed, since theimmunosuppressive effects of UVR were recognized.
Not only UV‑induced DNA damages but also altera‑tions of immune regulation are important for skin car‑cinogenesis, because the immune system contributes tohost resistance against tumor growth. UV‑induced
immunosuppression can also interfere with the responseto and clearance of some infectious organisms including
bacteria, virus and fungus. From the therapeutic pointof view, immunosuppression, in other words, anti‑aller‑
gic effects may be involved in the beneficial effects ofsponsiveness induced by topical application of hapten to
PUVA‑treated skin in guinea pigs. J. Invest. Dermatol.,
80, 90‑939 ) Howie, S., Norval, M. and Maingay, J. (1986): Exposure
to low dose ultraviolet B Iight suppresses delayed typehypersensitivity to herpes simplex virus in mice. J.
Invest. Dermatol., 86, 125‑128

10) Kripke, M. (1984): Immunologic unresponsiveness in‑
duced by UV radiation. Immunol. Rev., 80, 87‑102
11) Lugar, T.A. and Schwarz, Z.T. (1995): Effects of UV
light on cytokines and neuroendocrine hormones. Krut‑mann, K. and Elrnets, C.A. (eds.) (1995): Photoim‑
munology. pp. 55‑76. Blackwell Science. Oxford.
12) Lugar, T.A. (1998): Immunomodulation by UV Iight;role of neuropeptides. Eur. J. Dermatol., 8, 198‑199
13) Miyauchi. H. and Horio. T. (1995): Detection of action,
inhibition and augmentation spectra in solar urticaria.
Dermatol., 191, 286‑291
14) Morison, W.L., Parrish, J.A. and Blook, K.J. (1979): In

vivo effect of UVB on lymphocyte function. Br. J.
Dermatol., 101, 513‑519
15) Norval, M., E1‑Ghorr, A.. Garssen, J. and van Loveren,
H. (1994): The effect of ultraviolet light irradiation on
viral infections. Br. J. Dermatol., 130, 693‑700

4616) Okamoto, H. and Horio, T. (1981): The effect of 8‑methoxpsoralen and long‑wave ultraviolet light on
Langerhans cell. J. Invest. Dermatol., 77, 345‑346

17) Okamoto, H., Takigawa. M. and Horio, T. (1985):
Alteration of lymphocyte functions by 8‑methoxypsor‑
alen and long‑wave ultraviolet light radiation; Suppres‑sive effect of PUVA on T‑lymphocyte migration invitro. J. Invest. Dermatol., 84, 203‑205

18) Okamoto, H., Horio, T. and Maeda, M. (1987): Altera‑tion of lymphocyte function by 8‑methoxypsoralen and
10ng‑wave ultraviolet light radiation. II. The effect ofin vivo PUVA on IL‑2 production. J. Invest. Dermatol.,
89, 24‑26
19) Schacter, B., Lederman, M.M., Levine, M.J. and Ellner,
J.J. (1983): Ultraviolet radiation inhibits human naturalkiller activity and lymphocyte proliferation. J. Im‑

munol., 130, 2484‑2489

20) Toda, K., Miyachi, Y., Nesumi, N., Konishi, J. and
Imamura, S. (1986): UVB/ PUVA‑induced suppressionof human natural killer activity is reduced by superox‑
ide dismutase and/or Interleukin 2 in vitro. J. Invest.
Dermatol., 86, 519‑522

21) Toews, G.B., Bergstresser, P.R. and Streilein, J.W.
(1980): Epidermal Langerhans cell density determines
whether contact hypersensitivity or unresponsivenessfollows skin painting with DNFB. J. Immunol., 124,
445‑ 453

22) Yamawaki, M., Futamura, S. and Horio, T. (1995):
UVB radiation suppresses TNF‑alpha‑induced expres‑sion of E‑selectin and ICAM‑1 on cultured human umbil‑
ical vein endothelial cells. J. Dermatol. Sci., 13, 11‑17

47

Symposium 1

PRESENT AND FUTURE SITUATION OF LEISHMANIASIS RESEARCHINTRODUCTIONYOSHIHISA HASHIGUCHI
Departrnent of Parasitology, Kochi Medical School, Nankoku‑shi 783‑8505, Kochi, Japan

Abstract: In order to know the global situation of leishmansiasis in the world, the transmission and clinicalforms were briefly discussed, and the prevalence was also reviewed, mainly based on the reports from World
Health Organization (WHO) . Leishmania/HIV co‑infection cases are increasing annually due to different

factors, such as human behavioral, environmental and epidemiological changes, especially in southern
Europe. Spain, Italy, France and Portugal. The co‑infection cases have also been reported from othercountries of different continents, Asia, Africa and Central and South America. In such Leishmania/HIV
co‑infection cases, serologic diagnosis is of little use. To overcome the diagnostic problem in HIV‑infectedpatients, an indirect xenodiagnosis of visceral leishmaniasis using laboratory colonized sandflies wasrecently developed by Spanish workers; the usefulness was shortly discussed in the text as a topic.
Key words: Ieishmaniasis, global situations, epidemiology, Leishmania/HIV co‑infections

Transmission and clinical forms. Leishmaniases

ous secondary infections. The scars and disfigurings

are caused by different species belonging to the genus

caused by leishmaniasis can cause serious social preju‑
dice, and cast out the patient from society (WHO, 1996) .

Leishmania, and the genus divided into 2 subgenera,
Leishmania and Viannia; some 20 species of them arerecourded as causative agents of human leishmaniasis in

Prevalence. The diseases are endemic in 88 coun‑

the world. The parasites are haemoflagellate proto‑tries on the five continents, with a total of 350 million
people at risk, and the diseases afflict at least 12 million

zoans which are exclusively transmitted by the bite of apeople worldwide (WHO, 1996). These numbers aretiny 2 to 3 millirnetre long female sandflies of the genus

however probably underestimates; figures of 2 million

Phlebotomus in the Old World and Lutzomyia in the New

new cases per year, including 1.5 million cases of CL and
500,000 of VL are likely (Desjeux, 1996). Over the last

World. Of the 500 phlebotomine species recorded todate, only about 30 of them have been incriminated as

vectors of human leishmaniasis in the world. The
diseases show a wide range of clinical manifestations
depending on Leishmania species and/or immunologicalor physiological conditions of hosts. They are mainlyclassified into several forms such as visceral (VL) ,ten years, endemic regions of leishmaniasis have been
spreading further afield and there has been a sharp
increase in the number of recorded cases of the disease(Hashiguchi, 1996) . In Tunisia, for example, 1,300 casesof CL were reported in 1983, then the figure had reached6,000 in 1991; in the northern States of Brazil, 2,000 cases

cutaneous (CL), mucocutaneous (MCL) , diffuse

were reported in 1980, and 9,000 in 1990. Furthermore,cutaneous (DCL) , and post‑kala‑azar dermal (PKDL)
leishmaniasis. Among these clinical cases, VL is thein the State of Bihar, India, where 38 out of 42 districts

fever, substantial weight loss, swelling of the spleen andare affected, betwe n 250,000 and 300,000 cases were
estimated in 1992‑five times the official figure (WHO,1993) ; the number of VL cases and the number of deaths

most serious forms characterized by irregular bouts ofliver, and anaemia (Bryceson, 1996). If left untreated,

reported officially from Bihar during 1985‑1992 are

the fatality rate of VL cases can be as high as 100%
(WHO, 1993). CL forms produce self‑healing or non‑healing skin ulcers or nodules leaving permanent scars

shown in Table I (Dhanda et al., 1996). Of all VL caseson the exposed skin surface of the body such as the facein the world, 90% cases occur in Bangladesh, India,
Sudan, and Brazil; 90% of CL occur in Afghanistan,

are usually resistant for the drugs available commer‑

lran, Saudi Arabia, Syria, Brazil and Peru; and 90% of
MCL are reported from Brazil, Bolivia and Peru (WHO,1993) . Thus, Ieishmaniasis endemic regions and clinicalcases are increasing annually without specific measureseffective for the control after the first report fromTurkey in 1756, about 240 years ago. Since 1993, geo‑

cially, and MCL Ieads to partial or total destruction ofgraphic spread of the disease is also found due to the thethe mucous membranes of the nose, mouth and throat

factors related mostly to development, including mas‑cavities and surrounding tissues accompaning with seri‑

sive rural‑urban migration, agro‑industrial projects thatand the upper and lower extremities, conferring a life‑
10ng immunity on the host against the challenge infec‑
tion with the same species or subspecies of the genus

Leishmania. Furthermore, MCL, DCL and PKDL forms

48

Table I Number of kala‑azar cases and number ofpatients is difficult, requiring the use of combination ofdeath reported frorn Bihar during 1985‑1992

several techniques for confirmation of the disease. BoneNo. of deaths

marrow aspirate remains the safest and most sensitiveYear

No. of cases

1985

13 , 030

35

1986
1987
1988
1989
1990

14 , 07946

19 . 267

77

19 , 267

121

30 , 879

475

54 , 650

544

1991

59 , 619

843

1992

75 , 523

1,417

Sourse: Status paper on kala‑azar in Bihar, 1993

being non‑immune urban dwellers into endemic rural
areas, and man‑made environmental changes like dams,
irrigation systems and wells (WHO, 1996).

Leishmauia/HIV co‑infection. Over the past few
years, AIDS and other immunodepressed conditions

techniques, but spleen aspirate and liver biopsy are also

used; in some cases search for the parasites can be donein peripheral blood sarnples (WHO, 1996). Recently,
Molina et al. (1994) developed an indirect xenodiagnosis

of VL in HIV‑infected patients using laboratory colon‑
ized sandflies. Phlebotomus perniciosus females were fed

on a membrane‑feeding apparatus containing 2 ml
blood from each patient, with heparin, EDTA, or sodium
citrate as anticoagulants; ,blood samples were kept at
4'C for 6‑96 hrs until use. Sandfly guts were dissected

and examined for parasites 2‑7 days after membranefeeding (Table 2). Among their diagnostic methods
used, indirect xenodiagnosis was the only method thatproved 100% effective in co‑infected patients, confirm‑
ing it as an alternative in Leishmania/HIV co‑infectedpatients. However, its use should be restricted to those

have increased the risk of Leishmania‑infected people
developing visceral forms, and in certain areas of theworld, especially in southern Europe the risk of co‑infection with HIV is rising due to epidemiologicalchanges; of the first 700 Leishmania/HIV co‑infection

cases where the more usual diagnostic techniques havecases which have been reported to WHO, 673 (96.1%)
were from the regions of southern Europe: Spain (413cases, 59.0%), Italy (130 cases, 18.6%), France (127relapsing rate of co‑infected patients within a period ofcases, 18.1%), Portugal (3 cases, 0.4%). Of these, 627(89.6%) were male, 600 (85.7%) were young adults (20‑40 years old) , and 497 (71.0%) were intravenous drug

users (WHO, 1996). In these Mediterranean basin
Leishmania (Leishmania) infantum is the causative
agent of VL, one of the vectors being Phlebotomus
perniciosus. Approximately 50% of all VL cases inadults of these endemic areas are associated with HIV

infection (Alvar et al., 1992). According to WHO(1996) , in the Americas most Leishmania/HIV co‑infec‑
tion cases are reported from northeastern Brazil, show‑
ing an increase in the risk of overlapping infection ofboth diseases. In eastern Africa, on the other hand, sucha co‑infection has been reported frorn Ethiopia, Kenya,

Malawi and Sudan. In these endemic areas, the risk ofoverlap of leishmaniasis and HIV is increasing due to anumber of factors such as mass migration, civil unrestor war, resettllement programmes, and prostiscuity andprostitution in refugees camps.

Diagnosis and treatment: a topic. Serologic
diagnosis is of little use in Leishmania/HIV co‑infectedpatients, because of abscence of anti‑Leishmania anti‑

failed and there is a strong suspicion of leishmaniasis in
HIV‑infected patiens (Molina et al., 1994). As to treat‑ment for co‑infected patients, pentavalent antimonials
showed a positive response in 83% of cases, with 52%

one month to three years; the main alternative drugs

include pentamidine, amphotericin B and liposomal
amphotericin B (WHO, 1996).
Under such a global situation of leishmaniasis in theworld, the symposium entitled "leishmnaiasis: its pres‑ent and future situation" is intended to disclose theunderlying problems in the identification of causative
Leishmania species, diagnosis and clinical manifesta‑
tions of the disease, epidemiology of the disease in the

Old and New World, parasite resistance for drugs, andthe role of heat shock proteins (HSP) as an escaping
mechanism of the parasite from host defense networks.

REFERENCES
1 ) Alvar, J., Blazquez, J. and Najera, R. (1989): Associa‑

tion of visceral leishmaniasis and human im‑
munodeficiency virus infections. J. Infect. Dis., 160, 560‑

5612 ) Alvar, J., Gutierrez‑Solar, B., Molina, R., Lopez‑Velez,R., Garcia‑Camacho, A., Martinez, P., Laguna. F., Cer‑
cenado, E. and Galmes, A. (1992): Prevalence of Leish‑
mania infection among AIDS patients. Lancet, 339, 1427
3 ) Bryceson, A.D.M. (1996): Leishmaniasis, (ed.), Cook, C.
C., 1213‑1245, Manson's Tropical Diseases (20th ed.) , W.

bodies in 30% of such cases (Alvar et al., 1989).

B. Saunders Comp. Ltd., London, Philadelphia, Tronto,

Therefore, immunologic diagnosis of VL in co‑infected

Sydney and Tokyo.

49

Table2

Xenodiagnosis

ofleishmaniasis

inHIV−co−infected

andnon−infectedpatients：clinicalandparasitological

data

Cases

8

7

9

10

M

M

11

12

M1
6M

6

5

M22南

4

3

2

1
Sex

M

M

M

F

M

M

M

M

Age（years）

36

33

35

25

38

29

22

28

38

27

HIV十

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Hetero

IVDU

IVDU

IVDU

IVDU

IVDU

11

IVC−1

IVD

III

IVC−1

Yes
Yes

Yes
Yes

Yes
No

Yes
Yes

Yes
Yes
3，100

Risk

factors

CDC

HIV

IVDU

IVDU

IVDU

IVDU

IVC−2

1VC−1

IVC−1

IVC−1

PTB，OC

CMV

PCP

Yes

No

EC
Yes

Yes

No

Yes
Yes

group

Associatedinfections

Fever
Splenomegaly

1VC−1

Yes

PTB，EC
Yes

EPTB

OC
Yes

Yes

Yes

Yes
Yes

PCP

2，700

2，400

NA

2，500

4，300

3，600

2，400

1，600

1，900

5，900

3，300

CD4＋（106／」）

40

10

68

56

48

51

144

20

148

ND

ND

440

Haemoglobin（9／1）

8．4

8．3

8．4

9．0

10．1

10

6．5

7．4

Platelets（×109／」）

71

164

NA
NA

7．2

84

75

98

73

179

131

170

13．7
146

12．4
116

十

十

十

十

十

十

十

十

十

十

十

十

Leukocytes（×106／1）

Diagnostic

methodsin（lireCt

GiemsaXe耳OdiagnOStiC

stain

of

Bone−marrow

Blood

十

aspirate

Serology（IFAT）
Bone−marrow
Peripheral
Treatment

of

used

Blood

at4。C（h）

storage

十

十

＜1：40

く1：40

く1：40

十

十

十

十

十

十

十

十

cytomegalovirus

十

and

O

〇

Relapse

Relapse

Relapse

O

O

O

EDTA

HE

HE

HE

HE

HE

HE

HE

10．7

13．8

6

24

6

36

6

24

37．5

14．2

62．7

92．9

48．3

8

transmission；PTB，pulmonary

fluorescent

Leishmaniasis：
inantibody

PublichealthDermatoL，14，417−423

lymphatic

filariasis，re−emer−

gence

and

threatbabesiosis

ofinIndia．

importanceMed．，35，434−435

Leishmaniasis：

its

as

diseases．Int．

an487224696
53．3

tuberculosis；OC，oral

candidiasis；EPTB，extrapulmonary

testing；SC，sodium

050

candidiasis；CMV，
tuberculosis；NA，

citrate；HE，heparin．

7）Molina，H．，Canavate，C．，Cercenado，E．，Laguna，F．，
Lopez−Velez

of

R．and

visceralusing

Alvar，」．（1994）：Indirectxenodiagnosis

Ieishmaniasis

colonized

in10HIV−infectedpatients

P宛18ゐo孟o吻％sヵ召7痂010s％s．AIDS，18，277−2798）WHO（1993）：Theleishmaniasis，1−14，ControlofTropi−

J．Med．Res．，103，46−54

and

十

〇

Clinics6）Hashiguchi，Y．（1996）：

十

HE

of

leishmaniasis

1：1，280〇

done；IFAT，indirectcontroL

1：5，120

HE

K．D．（1996）：Spreadpattem

十

十

＜1：40

〇

5）Dhanda，V．，Das，P．K．，Lai，R．，Srinivasan，R．，Ramaiah，Indian

十

十

十

1：40

HE

user；Hetero，heterosexual4）Desjeux，P．（1996）：

of

十

十

infection；PCP，P彫％窺o邸漉6碗吻pneumonia；EC，oesophageal

available；ND，notaspects

1：320

2

22．8

drug

1：160

SC
6

infections（％）

IVDU，intravenous
not

十

1：80

leishmaniasis（days）

Anticoagulant
Sandfly十＜1：40

monocyteculture

十

十

十
1：160

culture

blood

十
十

importedchanging

cal

Diseases，WHO，Geneva．

9）WHO（1996）：The

leishmaniasis

co−infections，1−4，Fact

Sheet

and五召乞sh耀吻／HIV
No．116，WHO，Geneva．

51

1 CLASSIFICATION AND DIAGNOSIS OF LEI SHMA NIATATSUYUKI MIMORI
Department of Tumor Genetics and Biology, Kumamoto University School of Medicine

Honjo 2‑2‑1, Kumamoto 860‑0811, Japan

Species is a category of living things that ranks
below a genus. A definition of the word "species" isusually made up of related individuals able to produce
fertile offspring in general biology. However, there isno sex in protozoa of the Order Kinetoplastida. Speciesof Leishmania had been mainly classified by the differ‑ence of the clinical manifestations in human, geographi‑
cal distribution and behavior of parasite.
Leishmaniases are widely distributed in tropical andtemperate zones, where they present a considerable
health problem. According to Desjeux (1993) and WHO
report (1998), an estimated 12 million people in 88
countries are affected by these diseases, two million newcases occur each year. The diseases are caused byparasites of the genus Leishmania and reveal visceral,cutaneous and mucocutaneous manifestations depending

to be a member of Mycetozoa or "slime fungi" by
Cunningham (1885). Wright (1903) reported an organ‑
ism in the skin of an Armenian child, and named it
Helcosoma tropicum . Luhe (1906) renamed this organ‑
ism as Leishmania tropica, but he thought it as piro‑
plasms. Rogers (1904) and Nicolle (1908) observed theflagellate stages of the parasites in blood‑agar culture

media and the organisms were classified undertrypanosomatide group. However, the parasites were
thought within the genus Herpetomonas or Leptomonasin trypanosomatide group. Moreover, Wenyon (1926)
suggested that Leishmania was different from Her ‑petomonas and Leptomonas because Leishmania adapted
to vertebrates, whereas Herpetomonas and Leptomonas
could not essentially infect vertebrate hosts.

patient, and the classification of species is important for3 ) New world cutaneous and visceral leishmaniases
Lindenberg (1909) and Carini and Paranhos (1909)
reported the first ca es of autochthonous New world
cutaneous leishmaniasis in southern Brazil, it wasthought that the parasite was Leishmania tropica.

epidemiological and public health surveillance.

Vianna (1911) found the morphological different type ofon the parasite species: Therefore, the identification ofLeishmania species is crucial in choosing the appropri‑
ate treatment s well as predicting the prognosis of theThis paper contains some current topics of theparasite from the patient of disseminated cutaneousclassification and identification of Leishmania parasites

leishmaniasis in Brazil; it was named Leishmania

including molecular biology methods.

brazilienses a name later amended to L. braziliensis by
Matta (1916).
Kirk (1949) had classified Leishmania infecting
human to the following 3 species; Leishmania tropica(Wright, 1903) LUhe, 1906; Leishmania donovani (Laver‑

Isolation of Leishmania parasites
1 ) Old World visceral leishmaniasis
Visceral leishmaniasis was known as Kala‑azar inan and Mesnil, 1903) Ross, 1903; Leishmania braziliensis

India, and as infantile or infectious splenic anaernia in

Vianna, 1911 emend, Matta, 1916. Thereafter, many

the Mediterranean Region in the past. Leishman (1903)
reported intracelluar bodies in the viscera of Kala‑azar
patient of Dum‑Dum in Calcata, India, and recognizedsubspecies were described within species by the differ‑to be them related to the trypanosomes. Donovan

ed lizards Leishmania by the use of the subgeneric name(1903) also discovered the similar bodies from spleen of
the patients from India, and sent its sketch to Ross. The

genus "Leishmania". Moreover Lamson and Shaw

ence of the manifestations in hurnan cases caused byeach strain of the parasites. Saf'janova (1982) separat‑"Saurolelshmania" from mammals Leishmania as sub‑parasite was thought to be a piroplasm and was named(1987) divided subgenus Leishmania to subgenus Leish‑

it Piroplasma donovani by Laveran and Mesnil (1903,) ; itmania and subgenus Viannia by the difference ofwas Ross (1903) who amended the name to Leishmania

promastigote localization in gut of the sandfly. Recent‑donovani in the same year.
2 ) Old world cutaneous leishmaniasis
Descriptions of cutaneous leishmaniasis are foundas Balkh Sore from the first century in Central Asia.

ly, 20 species of Leishmania infecting human are de‑scribed in the WHO report (1990). According thisreport, 12 species; aethiopica, amazonensis, chagasi,
donovani, garnhami, and infantum, killiki, major, mex‑

Parasites were observed in sections of human skinicana, plfanoi, tropica, venezuelensis belong to subgenus

lesions of Delhi Boil in India, the organsm was thought

Leishmania group, and 8 species; braziliensis, guyanensis,

52

panamensis, peruviana, Iainsoni, nalffi, colombiensis ands;iawi belong to subgenus Viannia group.

1989). We have developed highly specific PCR(polymorphism‑specific PCR and arbitrarily primed
PCR) panel to enable the identification of the five major

Classification of Leishmania

Leishmania species which cause cutaneous leish‑
maniases in the New World. PCR diagnosis can be

The classification and identification are done using
the following criteria and methods;
A) Clinical features in human and animal infection

from the leishmanial ulcer lesions (Mimori et al., 1998) .

B ) Morphology by light‑microscopy and electron‑
microscopy
C ) Behavior of the parasite in vector (sandfly) , host

(1aboratory animals) and culture media
D ) Geographical distribution
E ) Host response; Noguchi‑Adler test, excreted factor

serotyping, immunofluorescent test, indirect

applied to formalin‑fixed biopsy and exudate samples

However, a few discrepancies exist between theclassifications based on clinical manifestations andmolecular characterization. Recently, co‑infection with

Leishmania and HIV are increasing in many countries.
The symptoms and signs of these co‑infected patientsare different from classical Leishmania infection(WHO, 1998). Therefore, we must also examine theimmunologic competence of the human host.

haemoagglutination test, cross‑immunity trials inhostF) Biochemistry; Enzyme electrophoretic mobilities,
protein composition, Iipid composition and mono‑clonal antibody reactivitiesG) Molecular biology; Buoyant density of nuclear andkinetoplast DNA, restriction‑enzyme cleavage, fil‑ter hybridization and polymerase chain reaction(PCR) .

ACKNOWLEDGEMENTS
This work received financial supports from theOverseas Scientific Research Program of the Ministryof Education, Science and Culture, Japan, and Japan
International Cooperation Agency. We thank Dr. Ha‑
shiguchi Y., Dr. Furuya M., and Dr. Sharnsuzzaman, S.
M., Kochi Medical Schr, Dr. Nonaka, S., Univ. of Ryu‑

The classification of Leishmania parasites which
infect human had been being based on the clinical fea‑

kyus, Dr. Katakura K., Gunma Univ., Dr. Saya H.,
Kumamoto Univ., Ms. Matsumoto T., Ginkyo College,tures, geographical distributions of the diseases, and thebiological characteristics of the parasites in vectors as

Dr. Gomez E.A., Univ.' Catolica, Ecuador. Mr. Sud R.,

well as laboratory animals until 1980. These criteria

Pratt D., Yale Univ., Dr. Grimaldi Jr. G., Oswaldo Cruz
Inst., Brazil for their co'works.

were not sufficient for the classification of Leishmania
parasite. It is also very difficult to classify Leishmania

Ministerio de Salud Publica, Ecuador, Dr. McMaho‑

species on the basis of morphological features. In 1980,

Leishmania standard reference strains were selected by

REFERENCES

the participants of UNDP/World BankIWHO Special

1 ) Barker, C.D. (1989): DNA diagnosis of human leish‑

Programme Workshop on the Biochemical characteriza‑

maniasis. Parasitol. Today, 3, 177‑184
2 ) Carini, A. and Paranhos, U. (1909): Identification detion of Leishmania, Washington. In the last 2 decades,
the system of classification was supplemented by many
zymodeme (Kreutzer et al., 1987), serodeme (Grimaldi
et al., 1987) and schizodeme (Barker, 1989) analyses.

The methods of using isoenzymes, monoclonal anti‑
bodies and PCR have been widely used for classifying
and identifying field isolates of Leishmania comparing
the reference strains. To explore the genetic basis of
the species and to facilitate the identification of thespecies, a number of methods have been elaborated.
However, the majority of these methodologies are notideally suited for clinical laboratories and field examina‑

tions. We have been performing the diagnosis of leish‑
maniasis in epidemiological survey in Ecuador, SouthAmerica since 1983 (Hashiguchi eds., No. 1, 1987; No. 2,
1990; No. 3, 1992; No. 4, 1994; No. 5, 1997; Mimori et al.,1'Ulcera de Baurti avec le bouton d'Oreint. Bulletin de laSociete de Pathologie Exotique de ses Filiales, 2, 2253 ) Cunningham, D.D. (1885): On the presence of peculiarorganisms in the tissue culture of a specimen of Delhiboil. Scientific Memoirs by Medical Officers of the
Army of India, 1, 21‑31
4 ) Desjeux, P. (1993): Control of Tropical Diseases; The
Leishmaniases. World Health Organization, Geneva, pp.
1‑155 ) Donovan, C. (1903): On the possibility of the occurrenceof trypanosomiasis in India. British Medical Journal, 2,
796 ) Grimaldi Jr., J., David, J.R. and McMahon‑Pratt, D.
(1987): Identification and distribution of New World
Leishmania species characterized by serodeme analysis
using monoclonal antibodies. Am. J. Trop. Med. Hyg.,36, 270‑287

537 ) Hashiguchi, Y. ed. (No. 1, 1987; No. 2, 1990; No. 3, 1992;

humans and wild animals of Ecuador. Am. J. Trop.

No. 4, 1994; No. 5, 1997) Studies on New World leish‑
maniasis and it transmission, with particular reference
to Ecuador. Kyowa Print., Kochi, Japan.
8 ) Kirk, R. (1949): The differentiation and nomenclatureof Leishmania. Parasitology, 39, 263‑273

Med. Hyg., 40, 154‑158
17) Mimori. T., Sasaki. J., Nakata, M., Gomez, E.A., Uezato,
H., Nonaka, S., Hashiguchi. Y., Furuya, M. and Saya, H.
(1998) : Rapid identification of Leishmania species from

9 ) Kreutzer, R.D., Souraty, N. and Semko, M. (1987):
Biochemical identities and differences among Leish‑cific polymerase chain reaction. Gene, 210, 179‑186
18) Nicolle, C. (1908) : Sur trois cas d'infection spl6niquemania species and subspecies. Am. J. Trop. Med. Hyg.,36, 22‑32
10) Lainson, R. and Shaw, J.J. (1987): Evolution, classifica‑tion and geographical distribution. Peters, W., Killick‑Kendrick, R. ed. The leishmaniases in biology andmedicine. Academic press, London, pp. 1‑120
11) Laveran, A. and Mesnil, F. (1903): Sur un protzaire
nouveau (Piroplasma donovani Lav. et Mesn.) . Parasited'une fi vre de l'Inde. Comptes Rendus H bdornadiares
des S ances de 1'Academie des Sciences, 137, 957‑961
12) Leishman. W.B. (1903): On the possibility of the occur‑

rence of trypanosomiasis in India. British Medical
Journal, 1, 1252‑1254

13) Lindenberg, A. (1909): A tllcera de Baurti e seu mi‑
cr6bio. Revista de Medicina de Universidade de S oPaulo, S o Paulo, 12, 116‑120

14) Luhe, M. (1906): Handbuch der Tropenkdrankheiten.
Mense, C. ed., Barth, J.A.; Leipzig, Vol. 3, p. 203

15) Matta. A.A. de (1916):
Sur les leishmanioses
t6gumentaires. Classification g n6rale des leish‑
manioses. Bulletin de la Soci t de Pathologie Exotiqueet de Ses Filiales, 9, 494‑503

16) Mimori. T.. Grimaldi Jr., G., Kreutzer, R.D., Gomez, E.

A.. McMahon‑Pratt, D., Tesh, R.B. and Hashiguchi, Y.
(1989): Identification, using isoenzyrne electrophoresisand monoclonal antibodies, of Leishmania isolated fromformalin‑fixed biopsy samples by polymorphism‑spe‑

infantile

corps de Leishman observ s en Tunisia.

Archives de l'Institut Pasteur, 3, 1‑2619) Rogers, L. (1904): Preliminary note on the development

of Tmpanosoma in cultures of the Cunningham‑Leish‑
man‑Donovan bodies of eachexial fever and kala‑azar.
Lancet ii , 215‑216

20) Ross, R. (1903): (1) Note on the bodies recently de‑scribed by Leishman and Donovan and (2) further noteson Leishrnan's bodies. British Medical Journal, 2, 1261,
1401

21) Saf'janova, V.M. (1982): Classification of the genus
Leishmania Ross. Chapter 11, pp. 95‑101 (in Russian) .
In the Leishmanias. Protozoology, Part 7. p. 220. Acad‑emy of Sciences, USSR All Union Society of Proto‑
zoologists: Leningrad

22) Vianna, G. (1911): S bre uma nova esp cie de Leish‑
mania (Nota preliminar) . Brasil‑M dico, 25, 411
23) Wenyon, C.M. (1926): Protozoology. Vol. 1., Balli re,
Tindall and Cox: London
24) Wright, J.H. (1903): Protozoa in case of tropical ulcer("Delhi sore"). Journal of Medical Research, 10, 472‑
48225) WHO (1990): Control of the leishmaniases. Wrld Hlth
Org Tech Rep Ser No. 793, 1‑158

26) WHO (1998): Leishmania and HIV in gridlock. WHOl

CTD/LEISH/98.9, UNAIDS/98.23

55

2 LEISHMANIASIS IN ECUADOR, WITH SPECIAL REFERENCE TO
ITS ANDEAN FORM
YOSHIHISA HASHIGUCHll AND EDUARDO A. GOMEZ LANDIRES2
Department of Parasitology, Kochi Medical School, Nankoku‑shi 783‑8505, Kochi, Japan* and Department of Tropical Medicine,

Faculty of Medicine, Catholic University. P.O. Box 10833, Guayaquil, Ecuador'

Abstract: In this text, New World leishmaniases were briefly reviewed. In addition, a history of theresearch on Ecuadorian leishmaniasis by the authors' project from 1982 to date was also shortly given. A
total of 7 species of the genus Leishmania as causative agents of the disease were isolated frorn hurnans,sandflies and mammals, and 4 sp cies of Lutzomyia and 8 species of mammals were incriminated as probable
vectors and reservoirs, respectively, in that country. In this paper, a special emphasis was given to Andean
leishmaniasis which was discovered by the authors in 1986 at a small town, Paute, Iocated on the southernpart of Ecaudor, near to the Peruvian borders. The disease form is very similar to Peruvian uta especiallyin clinical features, but the causative agents (Leishmania sp.) and vector sandflies (Lutzomyia sp.) werecompletely different from P̲eruvian ones. Based on the results obtained from our longitudinal surveys on theepidemiology and ecology of the disease in the area, we developed a transmission model of Andean leish‑
maniasis. From the information collected in our studies, we recommended that measures for vector control
should be applied in such an area endemic for the Andean leishmaniasis, during the dry season when thebreeding site of sandflies and the transmission site were limited within and/or around rock crevices andanimal burrows in the open field located at remote area from Paute town.
Key words: Ieishmaniasis, transmission, Andean form, uta, Ecuador

In the New World, Ieishmaniasis is endemic in many
areas of Central and South American countries, and it
constitutes a significant public health problem in eachcountry. Control of the disease in these regions iscornmunication) .

complicated by the variety of factors, such as different

Leishmaniasis in Ecuador. Ecuador is one of thesmall countries in South America through which runs
the equator; Colombia borders to the north and Peruborders to the southeast. The Andes mountain crosses

species of the genus Leishmania, diverse clinical forms
and epidemiological patterns in each endemic area. In

the country from north to south. It divides the country
into three natural regions, the Pacific coastal regions,many regions of the New World, two or more Leish‑

the Andes and the Amazonian regions. Each geographic

mania species are sympatric. The species belonging toregion of the country has specific features relating to thethe subgenus Viannia, are known as the causative
agents of cutaneous (CL) and mucocutaneous (MCL)
leishmaniasis. Among the Viannia group, L. ( V.)transmission factors of leishmaniasis such as terrain,
environment and life style of the inhabitans. In thatcountry, Ieishmaniasis was first reported in 1920 bybraziliensis shows the most wide range of distribution,

Valenzuela (Rodriguez, 1974).

affecting many populations and causing CL and MCL.
In the New World, almost all of the disease forms ofremained one of the least studied of Ecuadorian tropicalleishmaniasis are zoonoses, and man is usually inciden‑

tal host of Leishmania. In the subgenus Leishmania
group, L. (L.) mexicana is mainly reported from theCentral American countries and frorn a pat of thenorthern regions of the South America. The species,
mexicana, amazonesis and pzfanoi in this subgenus areimportant not only as causative agents of CL but also as

those of diffuse type of the disease (DCL) . L. (L.)
chagasi is main or only species which causes visceralleishmaniasis (VL) in the New World with a wide rangeof distribution from Mexico to Brazil; however, thedistribution of L. (L.) infantum which causes VL and/or CL in the Old World is also suspected in Brazil andother South American countries (Momen, H., personal

However, it has

diseases until recently. From 1982 to date, we havestudied almost all of the suspectable endemic foci ofleishmaniasis, in order to disclose the transmission
mechanisms, clinical forms and etc. It was found thatleishmaniasis is widespread in most provinces of
Ecuador, showing a considerable health problem. The
disease occurs in many populations living in rural andmountainous areas on both sides of the Andes, including

those living in the Andean plateau. Of the endemic
zones, 9 provinces are located in the Paciffic coastal
region, 2 are situated in the Andes and 4 in the Am‑
azonean region. As to the causative agents of leish‑
maniasis in Ecuador, 7 species of the genus Leishmania.
L. (L.) mexicana. L. (L.) amazonensis. L. (L.) major‑
like, L. ( V.) braziliensis. L. ( V.) guyanensis. L. ( V.)56panamensis and L. ( V. ) equatorensis, were specifically

identified based on zymodeme, serodeme, schizodeme
and karyodeme analysis. Four species of the genus
Lutzomyia. Lu. trapidoi. Lu. hartmanni. Lu. gomezi andLu. ayacuchensis, and 8 species of mammals; anteaters,
sloths (2), squirrels (2), kinkajous, rats and dogs wereincriminated as possible vectors and reservoirs, respec‑tively, in the endemic areas of Ecuadorian lowland andhighland areas.

and characterized as L. (L.) major‑1ike, by moleculartechniques mentioned above. From the results obtainedin our survey it was suggested that leishmaniases in the

Andean countries would have more complicatedepidemiological and ecological features of the disease indifferent endemic areas than were previously consid‑
ered.

tries. On the Andean plateau and/or highland of

Transmission of Andean leishmaniasis in Ecuador.
In order to disclose a part of transmission mechanism(s) or ecology of Andean leishmaniasis in Ecuador, we
made a longitudinal study on natural infections of Lu.

Ecaudor, we discovered a new type of leishmaniasis

ayacuchensis with L. (L.) mexicana (Fig. 1), in addition(Hashiguchi et al., 1987; Hashiguchi et al., 1991) c, aused

to epidemiological surveys of inhabitants and reservoirs(Gomez and Hashiguchi, 1991; Hashiguchi et al., 1991) .

Andean leishmaniasis in Ecuador and other coun‑by L. (L.) mexicana and L. (L.) major‑like which aretransmitted by sandflies, Lu. ayacuchensis (Takaoka etal., 1990). To date, such a type of leishmaniasis wasfound in three endemic areas of Andean highlands ofEcuador, Paute (2,300 m‑2,500 m above sea leveD,
Alausi (2,300 m‑2,500 m a.s.1.) and Huigra (1,200 m‑
1,500 m a.s.1.). Clinically, the disease forms in the
country were found to be very similar to those in Peru

(Table 1). However, the Leishmania parasites andvector sandfly species from Ecuador are completely
different from Peru and other Andean countries (Table2). In Ecuador, the organisms isolated from humans,
sandflies and dogs were identified as L. (L.) mexicana

Based on these data, we developed a model for Leish‑
mania transmission in the area (Paute) endemic forAndean leishmaniasis of Ecuador (Hashiguchi, 1994).
From the data and the model developed, it was suggest‑ed that transmission cycle of leishmaniasis in the Andes
involved variable overlapping of two sets of the biologi‑
cal entities with a degree of overlap governed by micro‑
or macro‑climatic conditions, depending on the seasonal

climatic conditions in the area. The first set consistedof three categories of habitats ( I ; open field, II; periur‑

ban area and 111; urban area) . Each of these habitats

analysis (Hashiguchi et al., 1991; Katakura et al., 1993) .

was occupied at the same time by humans and domestic
and wild mammals such as dogs and others. The second
set consisted of the relationship between the sandfly

In addition, another species of the genus Leishmania,
was also isolated from humans living in Paute, Ecuador,

vectors and the principal reservoir hosts presumed to be
rats and other rodents. A high intensity of transmission

by zymodeme, serodeme, schizodeme and karyodeme

Table 1 Clinical and parasitological findings of subjects with Andean leishmaniasis in three towns, Paute, Alausi and
Huigra, Ecuador

Locality Age Sex No. of Site oflesions lesions

Paute

6Y
5Y

5M
5M
9M
Alausi

3Y
5Y
7M

8M
Huigra

IY
2Y

9M
2Y

3M
IY
IY

6M

F
F

M
M
M
M
M
M
F
F

M
M
F
F

M

Size oflesionDuration * *Smear or(months)

culture ( +/‑)

identlf ied

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

major‑likemexicana
major‑likemexicana
mexicana

1f ace

15 x 10 mm

14

1

f ace

3x3

7

1

arm

5><5

3

2

f ace

4

1

f ace

3

f ace

5x4, 4x3
2x2
5x5, 3x3, 5x5

1

f ace

1

f ace

1

f ace

2

3

24

5x5
5x5
2x2

3

f ace

lxl, 1><1

4

3

f ace

18

4

f ace

3x5, Ixl, Ixl
5x5, 3x3, 3x3, 3x3

8

1

arm

3x2

2

f ace

2x2, 3x3

1

f ace

1

f ace

1

f ace

5x5
5x3
2x3

*Y=years, M =months; **duration time of the disease as of the examination.
(Modified from Hashiguchi, 1994)

5
4

3

5
9
4

Leishmania sp.

mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana
mexicana

57

Table 2 Leishmania species causing Andean leishmaniasis, and their vector sandf lies and reservoir
hosts, reported from different areas of Andean highland
Areas

Altitudes

Leishmania spp.

Lutzomyia spp.

Mammalian hosts

EcuadorPaute

2 , 300‑2 , 500 m

L. (L.) mexicana
L. (L.) major‑likeLu. ayacuchensis

human, dog and rat

not determined

L. (L.) mexicana
L. (L.) mexicana

Lu. ayacuchensis
Lu. ayacuchensis

human
human
human

Lu. peruensis

human, dog and rat

Alausi2 , 300‑2 , 500 m

Huigra

1 , 200‑1 , 500 m

Peru
Westernslope ofthe Andes

1,300‑2,900 m L. ( V.) peruviana

Lu. verrucarum

Venezuela
Merida800‑1,600m L (L ) garnhami

Lu. youngi

human and o possum

not determinedhuman

Col ombia
Pueblo Rico,

Samniego

1 , 500 m

L. (L.) mexicana(Modified from Hashiguchi, 1994) .

occurred from October to December in open field(habitat I ) , Iargely between the wild mammals living

in rock crevices and the vector sandflies. During the

500

A

y
!i

O

,::

y 4DO

8

a,

O

rainy season from January to February, the sandfly
population increased gradually. The new sandfly gener‑
ations expanded their biting activity towards periurban
area (habitat II), and transmission of Leishmania para‑

B

,1
30
F
a,

QJ

6
s

T,

5

:

q,

SFI 20

4

>

3

o

n,

a,

Q1

sites to humans by sandfly bites intensified in this sea‑son. During March to April there was a high density of
infected sandflies and an increased population of wildrodents living peridomiciliary was also found in theperiurban area (our unpublished data) ; sandfly biting

activity extended into urban area (habitat 11D withmaximum overlap of the domestic and wild hosts andthe vector sandflies. Therefore, many new cases ofhuman leishmaniasis were observed in this season at
habitat 111 (Hashiguchi et al., 1991). During May toJune, the end of the rainy season and the subsequent,!
,Qn,y lO

2

Q,

>

o

l

N FEB ApR MAY JVFg JuL
SPT OCT NOv DECJ '(AR"*. = *=." ***"* ='." "*'C'G SPT

O*=."

Figure I Relationship between sandfly density (A) and natu‑
ral infection (B) with L. (L.) mexicana in an An‑

dean endemic area, Paute, Department of Azuay,
Ecuador. Average density is expressed as thenumber of sandflies captured per hour in eachcollection, and percent of those positives for theparasites, calculated from the rate of sandflies

infected among the 200 flies dissected in eachdecrease in hurnidity caused a rapid decline in the sand‑month. Average temperature and humidity

fly density; sandflies disappeared from the urban areas(IID of Paute town, though they were still observed inthroughout the year ranged from 15'C to 18'C andperiurban areas ( II) and open fields ( I ) . From July toGomez and･>Hashiguchi, 1991) .

from 50% to 80%, respectively (Adapted frorn

September, because of extremely dry conditions inprolonged to May or June lasting about five or six

Andean plateau (10wer terrain) , sandfly density falled

months. On the other hand, it was very short in urban

to a minimum. Nevertheless, rock crevices and animal

areas, Iasting only two months from March to Aprilburrows in the open field allowed the breeding of vector
sandflies even in this dry season. From these results, itwith a high incidence. Thus, changes in the incidence

was suggested that in the open fields Leishmania trans‑mission occurred mainly between wild mammals andsandflies throughout the year, keeping the life cycle ofthe parasite in this habitat ( I ) . In periurban areas ofPaute town, the time of leishmaniasis transmission

and frequency of human leishmaniasis cases in the
Andean endemic regions were considered to be theresult of migrations of sandflies and reservoir hosts
among the three categories ( I , 11 and 11D of the hab‑itats. Based on these ecological analysis of Andean
leishmaniasis in Ecuador, we recommended that in such

58

an endemic area the application of control measures
should be made during the dry season when the tra s‑
mission was extremely limited in open field (habitat I ) ,especially within and/or around rock crevices and ani‑
mal burrows in this area.

transmission, with particular reference to Andean typeof the disease, uta. Jpn. J. Parasitol., 43, 173‑186

3) Hashiguchi, Y., De Coronel, V.V. and Gomez, E.A.L.
(1987): Andean leishrnaniasis in Ecuador. In Studies on

New World leishmaniasis and its transmission, withparticular reference to Ecuador. Hashiguchi, Y. ed.,Kyowa Printing, Kochi, Japan, No. 1, 116‑131

ACKNOWLEDGEMENTS
The authors would like to express their gratitude to

all the following members of the project entitled"Studies on New World leishmaniasis and its transmis‑
sion, with particular reference to Ecuador" supported by

the Japanese Ministry of Science, Culture and Educa‑tion: Drs. S. Nonaka, A. Hosokawa, M. Maruno, A.
Takamiyagi, H. Uezato, M. Furuya, T. Mimori, K.
Katakura, S. Suguri, M. Harada, Y. Matsumoto, R. Sud,
A., M. Carvopifia, A. Guevarra and others.

4 ) Hashiguchi, Y., Gomez, E.A.L., De Coronel, V.V.,
Mimori, T.. Kawabata, M., Furuya, M., Nonaka, S.,
Takaoka, H., Alexander. J.B., Quizhpe, A.M., Grimaldi,
G. Jr., Kreutzer, R.D. and Tesh, R.B. (1991): Andean
leishmaniasis in Ecuador caused by infection with Leish‑

mania mexicana and L. major‑like parasites. Am. J.
Trop. Med. Hyg., 44, 205‑2175 ) Katakura, K.. Matsumoto, Y., Gomez, E.A.L., Furuya,

M. and Hashiguchi, Y. (1993): Molecular karyotype
characterization of Leishmania panamensis. Leishmania
mexicana, and Leishmania major‑1ike parasites: agentsof cutaneous leishmaniasis in Ecuador. Am. J. Trop.
Med. Hyg., 48, 707‑715

REFERENCES

6 ) Rodriguez, J.D.M. (1974): Genero Leishmania. Lec‑
ciones de Parasitologia Humana (5th ed.). Ecuador:

1 ) Gomez, E.A.L. and Hashiguchi, Y. (1991): Monthly

Universidad de Guayaquil, 170‑185
7 ) Takaoka, H., Gomez, E.A.L., Alexander, J.B. and Hashi‑

variation in natural infection of the sandfly Lutzomyia

ayacuchensis with Leishmania mexicana in an endemic
focus in the Ecuadorian Andes. Ann. Trop. Med. Par‑
asitol., 85, 407‑411

2 ) Hashiguchi. Y. (1994): New World leishrnaniasis and its

guchi, Y. (1990): Natural infection with Leishmania
promastigotes in Lutzomyia ayacuchensis (Diptera:
Psychodidae) in an Andean focus of Ecuador. J. Med.
Entomol., 27, 701‑702

59

3 LEISHMANIASIS IN CENTRAL EURASIAYOSHITSUGU MATSUMOTol, TOSHIMITSU HATABUl, CHIZU SANJOBA1,YASUNOBU MATSUMOT02, TETSUHIKO SASAK13, SHlN‑ICHIRO KAWAZU4,YUTAKA NAKA15, KEN KATAKURA6, MAMORU IT07, KOICHI NAGAKURA6
AND MASAMICHI AIKAWA9
Department of Molecular lrnmunology, School of Agricultural and Life Sciences, University of Tokyo ,Laboratory of Global Animal Resource Science, School of Agricultural and Life Sciences, University of Tokyo',
Department of Biological Sciences, School of Sciences, University of Tokyo*,Research Institute, International Medical Center of Japan',

Department of Animal Microbiology and Parasitology, School of Agriculture, Tohoku University ,Department of Parasitology, School of Medicine, Gunma University',
Laboratory of Immunology, Central Institute for Experimental Animals7,
Department of Infectious Diseases, School of Medicine, Tokai University8
and Research Institute of Medical Sciences, Tokai University"

AbstractLirhe ancient Silk Road crossed over boundless deserts, vast pastures, and high mountains andconnected ethnically and culturally diverged areas. It transported not only silk but goods, people, culture
and diseases from one end of the Eurasian landmass to the other. Since that time humans living in the sterilelands had to struggle continuously against natural forces to obtain water. When succeeded, it brought aboutdevelopment of great civilization as well as significant changes in ecology of humans, animals, insects, andpathogenic microorganisms. Such ecological changes sometimes resulted in unexpected endemy of a certain
type of diseases, such as leishmaniasis, a vector born zoonosis. Leishmaniasis, thus, might be one of theman‑made diseases.
Key words: Ieishmaniasis, Leishmania, zoonosis, vector born disease, Central Asia

A vast area of the Central Eurasia has been oc‑
cupied with deserts. Irrigation might bring wealth toresidents and facilitate to socioeconomical development.

However it also trernendously changes ecosystem in theirrigated land, sometimes resulting in new unfavorable
and unexpected endemy of the diseases including leish‑

Republic of China]

Visceral leishmaniasis, kala‑azar, caused by L.
donovani and L. infantum was one of the major para‑
sitic diseases in the vast area of China that lies to thenorth of the Yangtze River. It has been estirnated that

530,000 patients were affected in 1951. As a result ofmaniasis, a vector‑born zoonosis. Leishmaniasis iscaused by parasitic protozoa belonging to the genus

include treatment of patients, control of reservoir hosts,

large scale control efforts against kala‑azar which

Leishmania, and manifested clinically in different forms;

mainly dogs, and residual spraying of insecticides, it iscutaneous, mucocutaneous, and visceral leishmaniasis.
This disease is found in the most parts of the world,

now effectively under control in most of the former
endemic areas. However, kala‑azar has still remained

including the tropical regions of America, Africa and theas an important public health problem in the mountain‑
ous area of Gansu, Sichuan and Shanxi provinces, where
sporadic cases of kala‑azar have still occurred and a
total of 200‑300 cases has been reported annually. In
these areas canine visceral leishmaniasis has still beencommon and the major vector species is P. chinensis.
In recent years, increases of new infections have

Indian subcontinents, Mediterranean, Southwest Asia,
China and Central Asia (Fig. 1).
The parasites arenaturally transmitted by different species of blood‑suck‑ing sandflies in the genus Phlebotomus in the Old World.

Leishmaniasis is mostly zoonotic with canines androdents as major reservoirs. Since 1994 we have perfor‑

med epidemiological researches on leishmaniasis insome areas along the Silk Road that crosses over theCentral Eurasia including Xinjiang Uygur Autonomous
Region of China, the Central Asian countries, and Tur‑

been noticed in the desert area of Xinjiang and thewestern Inner Mongolia (Guan, 1991) . In Xinjiang kala‑azar has been widely and sporadically distributed overthe northwestern fringe of the Tarim Basin, the Turfan‑key (Fig. 2). Topics and recent problems on leish‑

Hami Basin, and the Junggar Basin, mainly affecting

maniasis in those countries are discussed in this review.

children under two years old. The disease is transrnitted

through the vectors P. major wui and P. alexandri.
[Xin jiang Uygur

Autonomous Region of the People's

These two species of sandflies are extensively distribut‑

60

Visceral Leishmaniasis

Cutaneous Leishmaniasis

d

>

.
; *;;

>

i

=
r

Figure 1 Geographical distribution of cutaneous and visceral leishrnaniasis in Central Eurasia.

d
.,
C

P
:t: :: :r' :' .'::;'.'. S;""""

dr

China

l

! K yrg yzstan
91 Uzbekistan: Turkmenistan

I

Azerbaijan
Turke yFigure 2 Location of countries which the authors have performed epidemiological researches
on leishmaniasis since 1994 and have reviewed in this article.

presumably the natural reservoir hosts of the infection.

should be difficult. The only available measure against
the disease at the present is detection and treatment of

Elaborate examination of rodents, hedgehogs, foxes,

the patients.

ed in the field and bite at night. Wild animals aredogs, and bats, however, has so far failed to detect any
Leishmania parasite which causes human visceral leish‑

maniasis. The control of such zoonotic kala‑azar

Recently, cutaneous leishmaniasis has been reported

in Karamy located in Junggar Basin. In this region,
although L. major has not been found, great gerbils,

61

Rhombomys opimus, are infected with L. tl ;ranica and L.
gerbilli vvith high prevalence (more than 30%). It has
been repor, ted that L. turanica causes self‑healing

hrunan skin lesion as proved by human inoculation
experiment (Guan, L. R. et al., 1995) . More recently
DNA analysis of Leishmania parasites from an experi‑mental animal (Lagurus lagurus) which was inoculatedwith biopsy materials from a patient with cutaneousleishrnaniasis in Kararny sugges ted that DNA sequencesof a gene from the isolate was highly homologous to thatof L. i;efa i tum (personal communication from Dr. Li

Ren Guan) . To determine the etiologic species ofLei.thmania for human cutaneous leis hmaniasis in Xin‑
jiang, the further and careful examinations are neces‑

papatasi has been known to transmit the parasites tohumans. Visceral leishmaniasis, in which a major reser‑
ovir host has been considered to be dogs, is also endemic
but sporadic.

Leishmanization, a primitive form of live vaccine

against human cutaneous leishmaniasis used in the
Ottoman Ernpire is still going on in Uzbekistan. Under
the control of the Samarkand ScientificResearch Insti‑
tute of Medical Parasitalogy. Live virulent L. major
promastigotes grown in culture are mixed with promas‑
tigotes lysate, and then inoculated on the arms of humanvolunteers. This produced a local lesion usually lasting
up to a year, after which the person would be immune.
IVlost of countries in this area became independentsary.

in 1991 from the former USSR. Since some confusions
after the independence have still remained on taking

LCentral Asian Countries‑Azerbaijan, Turkmenistan,
Uzbekistan, and Kyrgyzstan]over the public health affairs, the situation of primary
health care is unstable, especially on control of infec‑

Leishmania major Yakimov and Schokhor, 1914

tious diseases̲ Establishment of new control projects

was first described for the parasite from a patient withare in urgent needs.

a skin lesion living in Uzbekistan. Because intensive
researches have been performed by Russian scientists
since that time, the biology and ecology of natural[Turkey]
Cutaneous leishmaniasis has been known for cen‑reservoir hosts and vectors have been studied in detail.
However, this area has still been endemic of the disease

turies in Turkey as Beauty Scar, Oriental Sore, Allepo
Sore or Annual Sore (Fig. 3). It was highly endemic in(Strelkova, 1996). Although reexaminations based onmodern molecular biological techniques are required on
the etiologic agents, four types of leishmaniasis havebeen distinguished mostly based on epidemiological andclinical features; urban type cutaneous leishmaniasis

whole country before 1950. The number of cases de‑creased after 1950 with the beginning of the use of
insectisides for malaria control, and thereafter in‑creased with relaxation of malaria control programs.
Today, because of the development of socioeconomic

caused by L, tropica, rural type cutaneous leishmaniasis

conditions and the improvement of public hygiene, thedisease is effectively under control in most regions
except for southeastern part of Turkey where the dis‑by L, major, kalaazar by L. donova;ei and infantilekala‑azar by L. infantum.
For the rural type cutaneous leishmaniasis, which is

distributed most widely among fcur types of leish‑
maniasis, the maior reservoir is great gerbil, R. opimus.

In fact, distribution of L. major as a pathogenic agentfor zoonotic cutaneous leishmaniasis in this region iscoincident with the range of R. opimus habitat. In R.
opimus population, three species of Leishman:ia. L.
major. L. Iur(Inica, and L. gerbilli are co‑endemic.
Among those three Leishmania species, only L. major isbelieved to be pathogenic for humans. Although theseparasites are easily isolated from Auricula of the great
gerbils, no parasites can be detected in visceral organs.

In this natural host the parasites are seemed to be
maintained without any severe pathological changes.
Thus great gerbils are considered to remain as a sourceof L, major infection for a life. Although L. major has
been isolated from some sandfly species belonging to thegenus Phlebotomus (P. papatasi. P, ardrejevi), only P.

Figure 3 A cutaneous ulcerative lesion of urban typecutaneous leishmaniasis caused by Leishmania
tropica on the face of a patient in Sanliurfa,
Turkey.

62ease is still highly endemic.

The southeastern region of Turkey is a hot and dry

area. The southeastern anatolia project (GAP) is

lated from dogs were also identified as L. infantum.
There is no report on Leishmania promastigotes detect‑
ed in sandflies, however.

Turkey's largest ever integrated project. It was laun‑ched in 1981, covers a region of about 10% of Turkey's

Leishmaniasis is widespread in tropical, subtropical

total area, and will affect at least 6 million people, 10%

and temperate regions, where different combination ofof Turkey's total population. Within this area 22 dums

Leishmania species, vector sandflies, and reservoir ani‑and 19 hydroelectric plants were constructed or are
under construction on the Euphrates, the Tigris andtheir tributaries. The GAP bring about not only themals exists. Humans are considered to be mostly

socioeconomical development but also significant migra‑
tory changes in human, as well as ecology of animals,
insects and parasites. These changes are threatening
public health and leading an increase in the prevalenceof parasitic diseases including leishmaniasis. According

accidental hosts, although in some places, e.g. India, thedisease is anthroponotic. Hence epidemic features ofleishmaniasis varied among areas and therefore control

measures should be different. Careful and detailepidemiological investigations should be needed inadvance to･ establish effective control ineasures against
this disease.

to the reports by the Turkish Ministry of Health, thenumber of leishmaniasis cases in Sanliurfa, which is theregion's main city and has 200,000 population in theurban area, increased from 552 in 1990 to more than 3,
OOO in 1996. Hyperendemicity was observed in certain
quarters of the city around the river crossing. The typeof the disease is urban type cutaneous leishmaniasis, inwhich the etiologic species is L. tropica. In Sanriurfa,

elaborate investigations on reservoir hosts have been
carried out. So far, however, none of the dogs and smallrodents examined were found to be positive serological‑
ly and parasitologically suggesting anthroponotic trans‑mission (Aksoy, 1995). Although P. sergenti is suspect‑
ed to be a vector, the parasites have not yet been foundfrom any species of sandflies. Sporadic cutaneous leish‑

maniasis caused by L. tropica has been also reported
from Ege region and Mediterranean region.
Human visceral leishmaniasis is seen in western andsouthern parts of Turkey along the Aegean and Mediter‑
ranean coasts affecting mainly infants. Although themost cases are seen sporadically, there is an endemic

focus in Manisa district of the Aegean region. The
pathogens has been identified as L. infantum by isoen‑
zyrne and DNA analyses. Although sporadic cases werereported from other regions of Turkey, some of thecases were observed in clusters. Canine visceral leish‑

maniasis is more endemic in Turkey with the higher
prevalence. In a study performed in the Aegean andMediterranean regions of Turkey, 1.6% of 1,150 dogs
were found to be seropositive and in another study, 7%of 478 dogs from 22 villages surrounding Manisa City
were found to be seropositive with reaching a maximum
positivity of 21% in some villages. The parasites iso‑

ACKNOWLEDGEMENT
We thank Dr. M. Ali Ozcel and Dr. Z. Alkan of Ege
Univ., Turkey, Dr. Jun‑Jie Chai and Dr. Xin‑Ping Zuo ofInstitute for Endemic Diseases Research and Control of

Xinjiang Uygur Autonomous Region, China, Dr. Guan
Li‑Ren and Dr. Qu Jing‑Qi, and Dr. Feng Zheng ofInstitute of Parasitic Diseases, Chinese Academy ofPreventive Medicine, Shanghai, China and Dr. Kwang‑
Poo Chang of University of Health Sciences/Chicago
Medical School, Illinois, U.S.A. for valuable discussion

and information. This study was supported in part by
Grant‑in‑Aid for Scientific Research for International
Scientific Research (No. 08041169 and 10041190) from
the Ministry of Education, Science, Culture, and Sports,
Japan.

REFERENCES
1 ) Aksoy, S., Ariturk, S., Armstrong, M.Y.K., Chang, K.P.,
Dortbudak, Z., Gottlieb, M.. Ozcel, M.A., Richards, F.F.
and Western, K. (1995): The GAP project in Southeast‑
ern Turkey: the potential for ernergence of diseases.
Emerg. Infect. Dis., 1, 62‑63

2 ) Guan, L.‑R. (1991): Current status of kala‑azar andvector control in China, Bull. W.H.O., 69, 595‑601

3 ) Guan, L.‑R., Yang, Y.‑Q., Qu, J.‑Q. and Shen, W.‑X.
(1995) : Discovery and study of Leishmania turanica for
the first time in China, Bull. W.H.O. 73, 667‑672
4 ) Strelkova, M.V. (1996): Progress in studies on Central

Asian foci of zoonotic cutaneous leishmaniasis: areview, Folia Parasitologica, 43, 1‑6

63

4 CLlNICAL FlNDlNGS OF CUTANEOUS LEISHMANIASISAND THEIR DIFFERENTIAL DIAGNOSIS IN ECUADOR
ATSUSHI HOSOKAWA1, MOTOYOSHI MARUNO1, ATSUSHI TAKAMIYAG11SHIGEO NoNAKA1, EDUARDO A. GoMEZ. L.2 AND YOSHIHISA HASHIGUCH13
Department of Dermatology. Faculty of Medicine, University of the Ryukus ,Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de Guayaquil'

and Department of Parasitology, Kochi Medical Scho013

Cutaneous leishmaniasis (CL) is a parasitic diseasecaused by various types of Leishmania spp. Man anddomestic animals are infected by sandfly, a vector with
Leishmania parasites.
Clinically, CL is divided into localized, generalized

and diffuse types. CL show various cutaneous manifes‑
tations, such as papules, nodules, ulcers with elevateda relatively large scar. The inflammation of the lesionis also minor. Therefore, the clinical symptoms of thelesions such as reddness and pressure pain, are much
more minor than those of bacterial infection. As the
positive rate of Leishmania parasites of this type is verysmall, diagnosis in the endemic areas of Ecuador has
generally been performed by the history of the presentborders and erythematous plaques. In generalized

illness and cutaneous changes of the patients. Whencutaneous leishmaniasis (GCL) , the eruptions are dis‑

bacterial infection is coexistent at the lesion site, theseminated throughout the entire body surface (Lazo,ulcers tend to become large and the lesions tend to1994). In diffuse cutaneous leishmaniasis (DCL) , which

endure longer. Therefore, disinfection of the lesion isis associated with specific immunodeficiency against
Leishmania, different clinical manifestations such as
nodules, papules and erythematous plaques are observed
throughout the entire body surface, with the exception

very important for treatment. Though various fungi
have been isolated from CL ulcers, their role in theof the scalp, axillary, inguinal, perineal and anal regions.

generally considered that lesions at these sites should be

Cutaneous manifestations in leishmaniasis are very simi‑

treated aggresively at the early stages of the disease. In

lar to those of other infectious and skin diseases.
Therefore, differential diagnosis between CL and otherthe examination for CL in Ecuador, we saw many non‑cutaneous leishmaniasis cases diagnosed as CL anddiseases including leprosy (Jopling, 1984) and deep

treated using meglurnine antimonate, Glucantine. In thismycosis is very important, especially in countries where

symposium, we demonstrated mainly ulcerative skinlesions, observed at the Multin y Casa Hospital inBabahoyo city, Province of Los Rios, Ecuador. Thosethese diseases are relatively common. In Ecuador, CL,
except in its GCL and DLC forms, is clinically divided

into highland type (Andean type) and lowland type.
The highland type of CL, observed in the highland of theAndes Mountains where the temperature and moistureis relatively low, occures as milialy‑to‑pea‑sizedpapules resembling insect bites and furuncles on the face

and four extremities of children. The inflammation of
the lesions is relatively minor compare to that of bacte‑rial infection, although numerous Leishmania parasites
are often detected within the lesions. It has been shown
that, after a sandfly bite, the lesions gradually increas insize for several weeks. Most of the lesions heals sponta‑

neously and form a small scar within half a year. The
10wland type of CL, observed in the lowland with hot

and humid forests, shows the above mentioned

ulceration is still obscure. Because CL Iesions of theauricle and nose are likely to cause deformity, it isulcetative skin lesions required differential diagnosis
between CL and other diseases, as shown in the Table.
Case I (No. I in the Table) , a 44‑year‑old male. The
thumb‑sized ulcers on the legs were diagnosed as beulcus cruris varicosum from the present history and theexistence of typical varix on the lower extremities andatrophie blanch at the center of the ulcer. The indura‑tion was not palpable at the margin of the ulcer. Case
2 (No. 2), a 60‑year‑old female. The ulcers on the legs
were relatively shallow without the elevated border. In
addition, siderosclerosis was observed on the legs. The
lesion was diagnosed to be ulcus cruris varicosum. Case

3 (No. 9), a 48‑year‑old male. The post traumaticcutaneous changes, including ulcer with elevated borderwhere induration is palpable at the margin. After thelesion without elevated border on the leg was considered
to be an ulcer caused by secondary bacterial infection.
Case 4 (No. 10), a 17‑year‑old male. The ulcer on theinfection, the lesions gradually increase in size and formright leg was diagnosed at the local hospital as CL ulcer.

relatively large and deep ulcers. A portion of each

Even though the patient received 19 Iocal injections oflesion heals spontaneously in about one year and leaves

meglumine antimonate, he showed no improvement was

64

Table

Cutaneous changes of non‑cutaneous leishmaniasis cases observed in an endemic area in Ecuador(Multin y Casa Hospital, Babahoyo city, Province of Los Rios, 1994)
Diagnosis performed

No. Age Sex. 44 M
2 . 60 F

3 . 24 M. 63 M
5 . 72 F
6 . 33 F

Cutaneous changes

Site oflesions

Ulcus cruris

ulcer, varix

rt. Iegvaricosum
Ulcus cruris

stasis dermatitis
ulcer, varix

It. Iegvaricosumstasis dermatitis

Diagnonis

Ulcus cruris
varicosum‑in the past(mis‑diagnosed)

Cutaneousleishmaniasis

ulcer, dark brownish
pigmentation, scrach

varicosum

Glucantime(10cal inj, x 5)

erosin, dark brownish
pigmentationleg

Glucantime

legs

Glucantime(i.m. x 14)

Ulcus cruris

Cutaneousulcer, varix

varicosum

leishmaniasis

induration
ulcer
ulcer

1 eg

ulcer,I eg

Leg ulcer. 45 M

Trauma8 . 48 M

Traurna

9 . 17 M

Trauma +Cutaneoussecondaryleishmaniasis

Skin tumor

in the past

legs

mark
Ulcus cruris

Treatment
perf ormed(i.m. x 10)(excision)

dark brownish
pigmentation
ulcer, induration

rt. Ieg

Glucantime(local inj, x 19)

inf ecti on

10. 10 M

11. 53 M
12. 60 M

13. F

Impetigo
contagiosum

crusted lesion,

rt. Ieg

Furuncle?

exudation, erosion
furuncle‑like lesion

cheek

Chromo‑verrucous nodule

dorsal aspectof rt. hand
It. cheeck

Glucantime 2 ml

It. cheeck

Glucantime‑nose(i,m. x 15)

mycosiscontact

Cutaneous

reddish papules,

dermatitis?

leishmaniasis

erythemas

Cutaneous

Mucocutaneous

reddish papules,
ulcer
ulcer, black papules
ulcer, black papules

leishmaniasis

nodule14 20 F15. 40 M
16. 69 M

leishmanisis

B.C.C
B.C.C

(i.m. x 5)

nose
f ace,O peration,oral cavity

Glucantime(i,m. x 30)

rt.; right, It.; Ieft, im.; intramuscular injection, Iocal inj.; Iocal injection, Glucantime; meglumine antimonate, X ; times of

injection of meglumine antimonate. BCC; basal cell carcinoma.

found. This injection caused fibrosis around the lesion
which was palpable as to be induration at the margin.
Case 5 (No. 11), a 10‑year‑old male. Pea‑sized shallow

ulcers without the elevated border appeared on the
10wer extremities in about one week after the child wasbit. Scratch marks were also observed on his lower
extremites. The lesions were diagnosed as impetigo
contagiosum. Case 6 (No. 13), a 60‑year‑old male.

a cauliflower‑like appearance. After various examina‑
tions, the lesion was determined to be a chromomycosis

caused by Fonsecaea pedrosoi (type 4) (Hosokawa, etal., 1997). Case 7 (No. 14), one‑year‑old female. The
reddish papules and erythema were located on the leftcheek. The skin lesion was diagnosed to be CL at a
10cal hospital, and she received five, 5 ml intramuscular

injections of meglumine antimonate for a total of 25 ml.

About one year prior to examination, the patient noticed
a rice grain‑sized scaly papule on the dorsal aspect of

Two weeks after the treatment had ended, no improve‑

his right hand. The scaly papule gradually increased insize to form a superficial nodule. The nodule was non‑The rice‑grain‑sized reddish papules and shallow ulcers

pedunculated, with a nearly flat top, and was raised
about I crn. The surface was rough and irregular withment was visible. Case 8 (No. 14) , a 20‑year‑old female.

were observed on the left cheek. Even though she
received 15 intramuscular injections of meglumine
antimonate, no improvement was observed. Case 9 (No.

6515), a 40‑year‑old male. The shallow ulcer and black
papules were strongly suspected to be basal cell car‑cinoma (BCO . Case 10 (No. 16), a 69‑year‑old male.
The black papules and ulcers on the face were suspected
to be relapsed BCC Iesions. The maxilla was broken,

ensure the accuracy of CL diagsosis, it is very importantand a metastatic lesion was observed at the oral cavity.
At the site of the relapsed lesion, the patient received

tological examinations are not available.

about 30 intramuscular injections of meglumine
antimonate without any improvement. Case 11, a 22‑

to consider the history of the present illness of thepatient and examine the margin of ulcers by palpation;these steps are particularly important in which these

diseases are endemic but parasitological and his‑

REFERENCES

year‑old male. This case was observed in other city andnot presented in the table. Six years prior to admittance1 ) Eduardo, Reyna A., Eduardo, A. Gomez, L., Nonaka, S.,at our clinic, he first noticed discrete papules on his left

cutaneous leishmaniasis: the first report of a par‑asitologically confirmed case in Ecuador. Studies on
New World Leihmaniasis and its Transmission, withknee and right cheek; these began to increase in both
numbers and size, and to spread over the entire body;
papules slovyly evolved into infiltrated plaques andnodules without any ulceration. Over the course ofabout two years, he received different treatments fromdifferent physicians, but without improvement; conse‑
quently, he decided to visit a dermatological dispensary
in Guayaquil. At the dispensary, he was diagnosed with
lepromatous leprosy and medicated for about 5 years.
Based on the results of our examinations, however, hesuffered from DCL (Reyna et al., 1994; Hosokawa et al.,1997). Case 12, a one‑year‑old female. This case wasobserved in the highland area of the Andes and notpresented in the table. In this case, typical lesions of thehighland type of CL, as well as insect bite‑1ike andfuruncle‑like skin changes on the cheek were observed.
Based on the observations of cutaneous changes
described above, special attention should be given tovarious infectious and non‑infectious diseases, including

skin carcinomas, for the differential diagnosis at theexamination of patients with CL. Therefore, in order to

Hosokawa, A. and Hashiguchi, Y. (1994): DiffuseParticular Reference to Ecuador, No. 4, 85‑922 ) Hosokawa, A., Maruno, M., Nonaka, S., Eduardo, A.
Gomez. L. and Hashiguchi, Y. (1997): Clinical compari‑son of cutaneous changes of patients with diffuse
cutaneous leishmaniasis and leprosy in Ecuador. Studies
on New World Leishrnaniasis and its Transmission, with
Particular Reference to Ecuador, No. 5, 137‑146
3 ) Hosokawa, A., Kawasaki, M., Ishizaki, H., Nonaka. S.,

Eduardo A., Gomez, L. and Hashiguchi, Y. (1997): A
case report of chromomycosis from an endemic area for
cutaneous leishmaniasis in Ecuador: differential diagno‑

sis between leishrnaniasis and chromomycosis. Studies
on New World Leshrnaniasis and its Transmission, with
Particular Reference to Ecuador, No. 5, 147‑157
4 ) Jopling, W.H. (1984): Differential diagnosis. Hand
book of leprosy, third ed., 118‑123, William Heineman

Medical Book LTD., London.
5 ) Ramon, F., Lazo, S. and Hashiguchi, Y. (1994): General‑ized cutaneous leishmaniasis: a parasitologically con‑firmed case in Ecuador. Studies on New World Leish‑
maniasis and its Transmission, with Particular Refer‑ence to Ecuador, No. 4, 93‑98

67

5MOLECULAR BIOLOGY OF DRUG RESISTANCE IN LEISHMANIA= GENEAMPLIFICATION AND P‑GLYCOPROTElNSKEN KATAKURA

Department of Parasitology, Gunma University School of Medicine 3‑39‑22 Showa‑machi, Maebashi 375‑8511, Japan
Tel: 027‑220‑8022, Fax: 027‑220‑8025, e‑mail: kenkata @ akagi.sb.gunma‑u.ac.JP

Drug resistance is a major obstacle to treatment
and prevention of many infectious diseases caused by
microorganisms, including parasitic protozoa. A better

understanding of the molecular mechanisms of drug
resistance is important for the design of more rational

drugs and for the discovery of new drug targets ofprotozoan parasites (Borst and Ouellette, 1995; Ullman,
1995). Pentavalent antimonials such as sodium stibog‑

Gene amplification in drug‑resistant Leishmania
In Leishmania, mechanisms of drug resistance havebeen investigated for prornastigotes, usually selected by

stepwise increasing concentrations of various drugs invitro. Gene amplification is frequently observed in the
drug selected promastigote lines. Overproduction of thetarget gene product by gene amplification is also known
as a mechanism of drug resistance in mammalian cells

luconate (pentostam) and meglumine antimoniate(Stark and Wahl, 1984). Amplified DNA sequences(glucantime) have been used for treatment of leish‑
maniasis. However, unresponsiveness to antimonials inleishmaniasis has long been recognized and is now
becoming a gig problem in the endemic areas (Olliaro
and Bryceson, 1993). Although the therapeutic failure
may be explained in part by low immunological compe‑
tence of the host, the emergence of drug resistant
Leishmania has been demonstrated by drug sensitivity

appear as extrachromosomal circular and linear am‑plicons in Leishmania. The amplicons contain genes

tests for clinical isolates in vitro (Grogl et al., 1992;

Faraut‑Gambarelli et al., 1997). Since the mode ofaction of pentavalent antimonials against Leishmania ispoorly understood, Iittle is known about the molecularmechanisms of antimony resistance in field isolates.

encoding enzymes, which are specifically inhibited by

given drugs, and genes encoding membrane proteins
related to mammalian P‑glycoproteins or multidrug
resistance (MDR) proteins (Beverley, 1991; Katakura
and Ohtomo, 1995; Ouellette and Papadopoulou, 1993;
Segovia, 1994) (Table 1).
Selection of wild‑type promastigotes for resistanceto methotrexate (MTX) , an inhibitor of dihydrofolate
reductase (DHFR) , has lead to the first discovery of

DNA amplification in Leishmania. The MTX‑resistant
L. tropica contained two different amplified DNA

Table 1 Gene amplification in drug‑resistant Leishmania

Drugs

Sizes of amplified

inducing gene

DNA in kb

amplif ication(region name)

Genes

Involvement ofgene in drug

Ref erences

resistance"

Circhlar

MTX, CB3717

30 ( R )MTX

36‑87 ( H )PTR1

arsenite

68‑87 ( H )pgpAtunicamycin

30‑63 ( G )

DFMO

20 ?

NAGT

DHFR‑TS

ODC

NDb2), 10)4), 27), 30)20), 28)21), 25)
un publishedpentostam

100

orfSb V

vinblastine

40(V)

yes
yes

6), 15), 18)

daunomycin
tubercidine

30
56

MDR1
MDR1

DFMO

Linear140‑230

orfTOR

ODC
IMPDH

mycophenolic acid

280

MTX

280‑360 ( H )PTR1, pgpA

50

gshl

arseniteUnknown
glucantime*confirmed by transfection

bND, not determined

? (H)

pgpA

yes
yes
yes
yes

ND

16)
5)

yes

7)

ND
ND
ND

34)

17) , unpublished
27)

no

13)

ND

9)

68

sequences, the R and H regions, as circular amplicons(Beverly et al., 1984). The 30 kb amplified R circle

contained the bifunctional DHFR‑TS gene. The R
region amplification was also observed in Leishmania

resistant to CB3717, an inhibitor of thymidylate
synthase (TS) (Garvey and Santi, 1986). In MTX‑
resistant L. tropica, co‑existence of circular (‑36 kb)

appeared at lowet drug concentrations and circularelements were generated at higher drug concentrations(unpublished) . Mycophenolic acid inhibits the purine

metabolism enzyme, inosinic acid dehydrogenase(IMPDH), and the IMPDH genes have been amplified
as linear amplicons in L. donovani lines selected forresistance to mycophenolic acid (Wilson et al., 1991).

reported (Olrno et al., 1995). Recently, it was suggested

Toxic inosine analog such as tubercidin and inosine
dialdehyde induced amplification of 56 kb region ofthat circular amplicons were generated from linear
amplicons in MTX‑resistant L. tarentolae (Grondin etDNA, on which an open reading frame named as the
toxic nucleotide resistance gene ( TOR) was mapped

and linear H DNA (280‑340 kb) amplicons has beenal., 1998) . The H DNA contained two different resistantgenes, pgpA encoding P‑glycoprotein A (Ouellette et al.,1990) and PTRI encoding pteridine reductase(Papadopoulou et al., 1992; Callahan and Beverley,1992). The H region arnplification was also observed inarsenite‑, primaquine‑ and terbinafine‑resistant Leish‑
mania species as circular amplicons in a range of 68 to

170 kb in size (Beverley, 1991; Katakura and Chang,(Detke, 1997) . Finally, circular amplicons were recently

reported in L. tarentolae resistant to sodium stibo‑
gluconate (pentostam) , and a novel open reading frame(orfSbV) coding for 770 amino acids was identified on
the amplicons (Haimeur and Ouellette, 1998).

Recent progress on gene transfection and genedisruption techniques in Leishmania allowed us to verify
the role of amplified genes in the drug resistance. The

1989; Ouellette et al., 1990). Interestingly, amplified

amplified gene was isolated and constructed into a

DNA sequences, which were hybridized to the Leish‑

expression vector. The vectors were transfected, usu‑

mania tarentolae pgpA ( ItpgpA) gene, was observed inally by electroporation, into wild‑type promastigotes toglucantime‑resistant lines of L. ( V.) guyanensis

see if the gene can confer a drug resistance phenotype toobtained through the one‑step but not through stepwise
drug selection (Ferreira‑Pinto et al., 1996). In somearsenite‑resistant L. tarentolae, the gshl gene, which
encodes y‑glutamylcysteine synthetase, was amplified

transfected cells. Most of the genes described above
were confirmed their involvement in drug resistance bytransfection, although the levels of resistance wereas part of a 50 kb linear amplicon or as part of a circular

usually lower than those in drug‑resistant cells estab‑lished by drug selection (Table 1) .

amplicon (Grondin et al., 1997). Amplification of
MDRI genes on extrachromosomal circular arnplicons

P‑glycoprotein gene family and multidrug resistancehas been reported in stepwise selected vinblastine‑resis‑

in Leishmania

tant cell lines of L. enriettii and L. amazonensis (Chowet al., 1993; Gueiros‑Filho et al., 1995). An MDR‑likegene was also amplified in daunomycin‑resistant L.
tropica as extrachromosomal 30 kb circular amplicons(Chiquero et al., 1998) .

Several other drugs also induced amplification of
specific loci. In Leishmania resistant to tunicamycin, anantibiotic that inhibits N‑acetylglucosamine‑1‑phos‑

phate transferase (NAGT), we found that the NAGT
gene was exclusively amplified as extrachromosomal
circular amplicons in a range of 30 to 63 kb in size

Multidrug resistance in mammalian tumor cells is

often mediated by P‑glycoprotein or MDR protein,which contains 12 transmembrane and two ATP binding
domains, a member of the superfamily of ABC (ATP‑binding cassette) transporters (Higgins, 1992). The
first class of leishmanial P‑glycoprotein gene is the
pgpA gene, which is structurally more related to thehuman MRP (MDR‑related) gene than the MDR genes.
The ItpgpA in L. tarentolae and ImpgpA in L. major
were isolated. The ItpgpA gene conferred resistance tooxyanions such as arsenite, trivalent antimony (SbIID(Katakura et al., 1991; Liu and Chang, 1992).

and pentavalent antimony (SbV). The ImpgpA gene,

DL‑alpha‑difluoromethylornithine (DFMO) is an ir‑

however, conferred resistance to arsenite and SbIII, but
not to SbV (Borst and Ouellette, 1995) . Since increased

reversible inhibitor of ornithine decarboxylase (ODO,

Imear (‑230 kb) amplificatron of the ODC gene inlevels of glutathione and trypanothione are importantfor oxyanion resistance in Leishmania, the pgpA protein
may recognize metals conjugated to thiols and transport
the conjugates into intracellular cornpartments, display‑
ing metal resistance by sequestration (Grondin et al.,

DFMO‑resistant L. donovani, in which linear amplicons

1997, L6gar6 et al., 1997).

and DFMO‑resistant Leishmania contained extra‑
chromosomal 140 kb linear DNAs, on which amplified
copies of the ODC gene were located (Hanson et al.,1992). We also found that both circular (‑20 kb?) and69

The second class of leishmanial P‑glycoprotein
gene was isolated as a homologue of the mammalian

contributes to antimony resistance by transporting anti‑

MDRI gene. Human MDRI protein is known as anmony conjugated to a thiol into an unknown subcellular

ATP‑dependent efflux pump that transports various

compartment (L6gar

compounds, including steroids, toxic xenobiotics andendogenous metabolites (Gottesman and Pastan, 1993) .

The Idmdr gene m . onovani is 83% identical to thelemdrl gene in L. enriettii at the amino acid level. Genetransfection experiments demonstrated that both leish‑
manial MDRI genes can confer a low or middle level ofresistance to vinblastine (Henderson et al., 1992; Chowet al., 1993). We have recently isolated L. amazonensis

LaMDRI and LaMDR2 genes by employing the PCR‑
based approach. A genomic library of the L. amazonen‑sis DNA was screened with a 408 bp PCR product withprimers designed for conserved sequences of ATP‑bind‑ing motifs of various P‑glycoprotein genes. Two open

reading frames containing putative two ATP binding
domains and 12 transmembrane domains were deter‑
mined. The LaMDRI gene, encoding 1,341 amino acids,
is 91% identical to the closely related Idmdrl protein,
revealing less conservation in the C‑terminal than in the

N‑terminal transmembrane domains (Katakura et al. inpress). The LaMDRI protein, however, has no potential
N‑glycosylation sites at the extracellular region. Tran‑and arsenite resistance. The pgpA protein probably

et al., 1997; Grondin et al., 1997) .

In addition, a novel gene (orfSbV), showing no signifi‑

cant homology with sequences present in data banks,
was isolated from pentostam‑resistant L. tarentolae,
and was responsible for a low level of resistance topentostam, glucantime, antimony tartrate (SbIID andarsenite (Hairneur and Ouellette, 1998). Further under‑
standing of resistance mechanisms in laboratory strainsis required to elucidate mechanisms of pentavalent
antimony resistance in field strains from patients.

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the aldoketo reductase family confers methotrexate

On the other hand, the LaMDR2 gene, encoding
1,267 amino acids, showed 46% and 29% identity to the

LaMDRI and ItpgpA protein, respectively (unpub‑
lished) . The LaMDR2 gene could not confer resistanceto vinblastine or arsenite, suggesting that the LaMDR2
gene is a new member of leishmanial MDR genes with anovel function.

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antimonials
Pentavalent antimony (SbV) is likely to be convert‑

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dryl groups of leishmanial components may be a majormechanism of action and/or toxicity, resistance toantimony in Leishmania appears to be multifactorial
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26) Olliaro, P.L. and Bryceson, A.D.M. (1993): Practicalprogress and new drugs for changing patterns of leish‑
maniasis. Parasitol. Today, 9, 323‑328
27) Olmo, A., Arrebola, R., Bernier, V., Gonz lez‑Pacanows‑
ka, D. and Ruiz‑P rez, L. M. (1995): Co‑existence of(1995): Leishmania amazonensis: multidrug resistancein vinblastine‑resistant promastigotes is associated withrhodamine 123 efflux,' DNA amplification, and RNA
overexpression of a Leishmania mdrl gene. Exp. Par‑
asitol., 81, 480‑490

16) Haimeur, A. and Ouellette, M. (1998): Gene amplifica‑tion in Leishmania tarentolae selected for resistance tosodium stibogluconate. Antimicrob. Agents Chemother.,42, 1689‑1694

17) Hanson, S., Beverley, S.M., Wagner, W. and Ullman. B.

(1992): Unstable amplification of the two extra‑
chromosomal elements in a a‑difluoromethylornithine‑
resistant Leishmania donovani. Mol. Cell. Biol., 12,5499‑5507
18) Henderson, D.M.. Sifri, C.D., Rodgers, M., Wirth, D.F.,

Hendrickson, N. and Ullman, B. (1992): Multidrug
resistance in Leishmania donovani is conferred by ampli‑

fication of a gene homologous to the mammalian mdrl
gene. Mol. Cell. Biol., 12, 2855‑2865

19) Higgins, C.F. (1992): ABC transporters: from micro‑organisms to man. Annu. Rev. Cell Biol., 8, 67‑113
20) Katakura, K. and Chang. K.‑P. (1989): H DNA amplifi‑
cation in Leishmania resistant to both arsenite andmethotrexate. Mol. Biochem. Parasitol., 34, 189‑192
21) Katakura, K.. Peng, Y., Pithawalla, R., Detke, S. andChang, K.‑P. (1991): Tunicamycin‑resistant variantsfrom five species of Leishmania contain amplified DNA
in extrachromosomal circles of different sizes with aBiol., 12, 4112‑4122

circular and multiple linear amplicons in methotrexate‑
resistant Leishmania. Nucleic Acids Res., 23, 2856‑2864
28) Ouellette. M., Fase‑Fowler, F. and Borst, P. (1990): The

arnplified H circle of methotrexate‑resistant Leish‑
mania tarentolae contains a novel P‑glycoprotein gene.
EMBO J., 9, 1027‑1033
29) Ouellette, M. and Papadopoulou, B. (1993): Mechanismsof drug resistance in Leishmania. Parasitol. Today, 9,
150‑15330) Papadopoulou, B., Roy, G. and Ouellette, M. (1992): A
novel antifolate resistance gene on the amplified H
circle of Leishmania. EMBO J., 11, 3601‑3608
31) Segovia, M. (1994): Leishmania gene amplification: amechanism of drug resistance. Ann. Trop. Med. Par‑
asitol., 88, 123‑13032) Stark, G.R. and Wahl, G.M. (1984): Gene amplification.
Annu. Rev. Biochem., 53, 447‑491
33) Ullman, B. (1995): Multidrug resistance and P‑glyco‑
proteins in parasitic protozoa. J. Bioenerg. Biomembr.,27, 77‑8334) Wilson, K., Collart, F.R., Huberman, E., Stringer, J.R.

and Ullman, B. (1991): Amplification and molecularcloning of the IMP dehydrogenase gene of Leishmania
donovani. J. Biol. Chem., 266, 1665‑1671

71

6 THE ROLE OF HOST‑DERIVED HOST‑SHOCK PROTElN IN
PROTECTION AGAlNST LEISHMANIA MAJOB INFECTION IN MICEKUNISUKE HIMENO, H. ISHIKAWA AND H. HISAEDA
Department of Parasitology and Immunology, The University of Tokushima School of Medicine
Exposing cells to a variety of stressful conditions
such as elevated temperature, chemical intoxication or

ment in murine toxoplasmosis. The expression of host‑derived HSP65 appears to be crucial in directing theinfection leads to the transcription of a set of genes and,host‑immune systems to achieve protective immunity

subsequently, to the synthesis of a family of polypeptide

against infection with Toxoplasma gondii. Alternatively,

known as heat shock proteins (HSPs) . These proteins
have retained highly conserved animo acid sequences
throughout evolution from prokaryotes to eukaryotes.

highly virulent Toxoplasma may escape from host‑

Because HSPs are found commonly as immunogens

HSP65 expression itself in/on macrophages.
In the present study with murine leishmaniasis, we

within numerous microorganisms, these proteins arebeing studied in detail. However, in infectious states,
HSPs are found both in the host and in microorganisms,
and they are highly homologous, making it difficult todefine their role in infection and immunity. HSPS may
be essential for the adaptation of intracellular parasites
to the harsh/hostile environment of hosts, and for trans‑

immune systern by exerting a suppressive influence on
the ability of y(

T cells to induce HSP65 or by inhibiting

found that HSP65 expressed in/on host macrophages

cellular immune systems. On the other hand, HSPs

also plays an important role in host protection. How‑ever, the expression mechanism was different from thesystem with toxoplasmosis as mentioned above.
As the first step, NKT cells instead of y( T cells
was the primary effecters, which activate Leishmania‑
specific CD4+ T cells by releasing cytokine such as
interferon‑y and others. As the second step, those T
cells induce the expression of HSP65 in/on host macro‑phages and this protein may contribute to host defense

synthesized by host cells as they respond to stress during

repairing the host‑cell darnage with endogenous

certain infections may actually play a role in hostdefense. Thus, HSPs expressed by either the host or

cytotoxic agents like NO and superoxide.

formation of microorganisms into infectious forms. The

HSPs of parasite also function as immunodominant
peptides that can be recognized by host humoral andparasites may have the potential to modulate the host‑parasite interaction.

We already proposed the ouoline of HSP65 involve‑

Finally, CD4+ T cells recognizing host‑derived
HSP65 activate the macrophages further and complete‑ly eliminate the parasites within those macrophages.

73

DENGUE FEVER AND DENGUE HEMORRHAGIC FEVER

Symposium 2

1 FIVE CASES OF DENGUE VIRUS INFECTIONMASAO TATEYAMAl, MASATO TOYAMAl, KEI MIYAG11, ATSUSHI NAKAMOTol, FUTOSHI HIGAl,
MICHIO KOIDE1, ATSUSHI SAITO1, NOBUCHIKA KUSAN02, MASAYUKI TADAN03 AND TOSHIHIKO FUKUNAGA3.
First Department of Internal Medicine. Faculty of Medicine, University of the Ryukyus

Department of Clinical Laboratory, University Hospital of the Ryukyus'

Department of Virology, Faculty of Medicine, University of the Ryukyus'
Abstract: We experienced five cases of dengue virus infection during the period of 1995‑1996. Four weredengue hemorrhagic fever (DHF) and one was dengue fever (DF) . Three of the 4 DHF patients werecategorized as grade I and the other one was graded as grade 2 according to the standards set by WHO.
Dengue type 2 virus was isolated frorn one DHF patient. Fever with other symptoms such as chills, vomiting,headache, general fatigue were presented in all patients. Hemostatic abnormalities were determined in all

DHF patients but was absent in the DF case. All the 4 DHF patients were found to have thrombocytopenia(platelets, 12,000‑100,000). Grade 2 DHF patient had already developed two different type of antibodies
specific for dengue virus, whereas the DF patient had no antibody, thus signifying the severity of grade 2

DHF case.

our conducted cases.

INTRODUCTION
It is estimated that as many as 100 million dengue
virus infections occur annually, principally in tropical

PATIENTS AND METHODS

and sub tropical areas inhabited by the mosquito

Case records of 5 patients, who were admitted

vectors, Aedes aegypti and Aedes albopictus. In Japan wehave no endemic dengue virus infection, so only very few

to the medical ward during the period of 1995 to 1996,
with symptoms sugge ting dengue virus infection were

physicians have the chance to examine dengue virus
infection. With increasing numbers of internationalby the World Health Organization (WHO) is based

travel, physicians should be alert to the possibility ofon the presence of high continuous fever, hemorrhagic

dengue fever when encountering febrile patients return‑ing or coming from endemic countries. This reportmanifestations (including at least a positive tourniquettest) , hepatomegaly, thrombocytopenia (platelet countdescribes the clinical features and laboratory data from< 100,000/mm') and hemoconcentration (hematocrit

Table 1

reviewed. A clinical definition of DHF established

Clinical features of study subjects at entry, tabulated
according to final diagnosis

DHF3 DHF4

Case No

DHF1

DHF2

(Age/Sex)(35y/M)(35y/F)

+

+

+

+

abnormal ities

+

+

+

+

H morrhage
PLT (104/mm3)

+

+

+

+

1.2

1.5

H . 7

5.5

+
+
+
+

+
+

+
+

+

2 , 500

6 , 600

DF(14y/M) (17y/F) (5ly/M)

Clinical details

1 Fever: acutecontinuous

2 . Hemostatic

Tourniquet test
Hemoconcentration
3 . Pain
4 . General fatigue

5. WBC (/mm3)+

7.8+ Unknown Unknown +
+

+

+

+

6 , 100 5 , OOO

DHF, dengue hemorrhagic fever. DF, dengue fever.

1 , 400

74

increased by

20% above baseline value) .

RESULTSOf the 5 subjects, 4 were diagnosed as DHF patientsand one as DF patient.
These of the 4 DHF cases were categorized as grade
1 the other as grade 2 (case 1, in Table 1) on the basisof the standards set by WHO. Dengue type 2 virus was
isolated from one DHF patient. Clinical details ofpatients are given in Table 1. Fever with other syrnp‑
toms such as clills, vomiting, headache, general fatigue

were complained by all patients. Hemostatic abnormal‑
ities were determined in all DHF patients but was notseen in the DF patient. Thrombocytopenia (platelets

ranging from 12,000 to 100,000) was recorded in 4
patients.

DISCUSSION
Host immune responses are important in the path‑ogenesis of DHF, as the risk for DHF is increased atleast 15‑ fold during secondary dengue virus infections.

Further DHFI had already two different type of anti‑
body for specific dengue virus and has been categorized
as grade 2, Therefore, severity may be corelated withthe development of antibody as the DF case had no
antibody.

REFERENCES
1 Halstead, S.B. (1980): Immunological parameters of
togavirus disease syndromes. In: Schlesinger R. W. ed.
The togaviruses. Biology, structure, replication. New

York, Academic Press, 107‑73
2 Halstead. S.B. (1988) : Pathogenesis of dengue: Challen‑
ged to molecular biology. Science, 239, 476‑481

3 World Health Organization (1986): Dengue haemorr‑
hagic fever: diagnosis, treatment and control. Geneva,

WHO, 1‑58

75

2 LABORATORY DIAGNOSIS OF IMPORTED DENGUE CASES
KEN‑ICHIRO YAMADAl, TOMOHIKO TAKASAK11, MASARU NAWA2 AND
ICHIRO KURANEl
Department of Virology 1, National Institute of Infectious Diseases

andDepartment of Microbiology, Saitama Medical School, Saitama2

Abstract: Dengue fever (DF) and dengue hemorrhagic fever (DHF) are major public health ploblems intropical and sub‑tropical countries. Dengue is not endemic in Japan, but there are imported dengue cases.

We have performed laboratory diagnosis of dengue virus infection, using serum samples from suspectedcases. We mainly carry out lgM‑capture enzyme‑1inked immunosorbent assay (IgM capture‑ELISA) andreverse transcriptase polymerase chain reaction (RT‑PCR) , along with immunochromatographic test andhemagglutination inhibition (HD test. Dengue cases can be diagnosed by the combination of serological andmolecular techniques.

Key words: dengue, imported case, RT‑PCR. IgM capture‑ELISA
Dengue virus infections are a major public healthploblem in tropical and sub‑tropical countries. Dengue
is not endernic in Japan today, but it is occasionallysequentially in a single tube. The tubes are set in an oilbath type thermal programmer (Iwaki, Co., Tokyo,

from patients with fever are occasionally brought to ourJapan) and subjeted to programmed incubation: at 53'C
for 10 min for reverse transcription, followed by 30‑40
PCR cycles of amp.1ification. PCR products are thensubjected to agarose gel electrophoresis. Arnplified

laboratory for diagnosis of dengue virus infection. We

DNA fragments are visualized by ethidium bromide

examined serum specimens from 293 suspected cases

staining.

from 1985 to 1998, and approximately half of these cases
were confirmed to be infected with dengue viruses.

Virus isolations are done by inoculating the serum
samples onto C6/36 cells in 24‑well plates (Linbro, Flow

Laboratory diagnostic methods for dengue virus infection

Lab. Limit., Irvine, Scotland) . Vero and monkey fetallung (MFL) cells are used for plaque assays.

imported by travelers who visit tropical areas andbecome infected with dengue virus. Serum samples

We carry out lgM‑capture enzyme‑linked im‑
munosorbent assay (IgM capture‑ELISA) , reverse
transcriptase polymerase chain reaction (RT‑PCR) ,immunochromatographic test and hemagglutination

Detection of dengue viral genome by RT‑PCR
Recent development in molecular biologic tech‑

inhibition (HD assay. We also attempt to isolate vir‑niques, such as PCR, has promised to make rapid diag‑nosis of dengue virus infection. We have used RT‑PCR

uses using the mosquito cell line, C6/36.

for diagnosis of dengue virus infection since 1993. Viral

HI test is conducted according to the method of
Clarke and Casals (1958) using microtiter plates. IgM
capture‑ELISA is used to demonstrate dengue lgM in
the serum samples. Four prototypes of dengue viruses
type I (Hawaii), type 2 (New Guinea O , type 3 (H83)
and type 4 (H241) are propagated in C6/36 cells for 5

genomes are usually detected by PCR in the specimendays at 28'C or 31'C, and the supernatant fluids are usedas the antigen. Presence of dengue virus‑specific lgMand lgG is also exarnined using commercially available
immunochromatographic test (Dengue Rapid Test, Pan‑
Bio Rty Ltd., Brisbane, Australia), according to the
manufacture's instruction.
Reverse transcriptase polymerase chain reaction

collected on days 2‑5 after onset of fever in our study.

Our results are consistant with the report by Morita etal. (1994) that viral genome was detected in the speci‑

mens by PCR with each dengue type‑specific prirners
until days 3‑5 after onset of the disease. Other groupdetected all four serotypes of dengue viruses by RT‑
PCR with the universal primers in clinical specimens
with high sensitivity (Sudiro et al.. 1997). Figure 1
shows the results obtained using serum specimens fromthe patient NK. The patient was infected with dengue

virus in Thailand or Vietnam. Serum specimens werecollected on days 3, 7 and 10 after onset of fever.

(RT‑PCR) is performed as reported (Morita et al.,Dengue virus type 1‑specific band (1ane 1) was obtained1991) with a minor modification. RT and PCR are doneusing day 3 specimen. Dengue‑1 viral genome was not,Please send correspondence to: Ken‑Ichiro Yamada, Ph.D., Department of Virology 1, National Institue of Infectious Diseases,
l‑23‑1 Toyama, Shinjuku‑ku, Tokyo, 162‑8640 Japan
Tel & Fax: 03‑5285‑1188

76

1 2 345 678 9

logic test for diagnosis of dengue virus infection (Bundo(b. p. )‑ 490(DI )**i¥398(D4 )

320(D3)230(D2)and lgarashi, 1985). Serum samples from 89 suspectedcases were examined by serologic tests in our labora‑
tory in 1998, and 44 cases were positive. Dengue virus‑
specific lgM was detected by lgM capture‑ELISA whenserum samples were collected 7 days or later.
It has been reported that lgM responses are cross‑‑reactive for 4 dengue serotypes, and that lglvl capture‑

ELISA cannot be used to determine the infecting virus
serotype (Gubler, 1998). We examined the relationshipbetween the lglvi levels ta each of the 4 serotypes, andPrimer

1 . NK‑3 D1

2.
3. //
!1 D2
D3
4.
//
D4D1
5. D1 (Positive)
6. D2 // D2
7. D3 l! D3
8. D4 ll

D4

9. NegatiVe control Di
Figure I Results of RT‑PCR using serurn specimens fromthe patient NK,

the dengue serotype determined by RT‑‑PCR. We obser‑
ved that the levels of lgM tended to be higher to thehomologous serotype, compared to the levels to theheterologous serotype. Althoug"h lgM capture ELISA isusually used for diagnosis of dengue virus infection
without expecting the determination of serotype, IgM
capture‑ELISA rrLay provide some infonTlation about
the infecting serotype. Further, studies, however, need to

be done concerning serotype specificity of lgM:
responses determined by lgM capture‑ELISA.
A number of connTlercial test kits for anti‑denguelgM and lgG antibodies have become available in thepast few years (Sang et al., 1998b, Kuno, 1998). We
have used a commercial immunochromatographic test
(PanBio; Australia). The results can be obtained in 5

however, detected in day 7 and day 10 specimens. The
results using dengue virus types 2, 3 and 4‑specificminutes, and are generally consistent with thoseprimer pairs were all negative.

investigators (Sang et al., 1998a. Vaughn et al., 1998).

lsolation of dengue viruses from clinical specimens
We usually attempt to isolate dengue viruses fromclinical specimens using C6/36 cells. We put 50 /ll of
human serum on C6/36 cells and culture at 28'C for 7

Combination of RT‑PCR, and lgM captureELISA or

obtained by lgM capture‑ELISA, as reported by otherrapid immunochromatographic test for diagnosis ofdengue virus infection

days. Presence of viruses in th,e culture fluids is checked

There seem to be quite a number of importedby plaque formatl'on on Vero or MFL cells. We also

dengue cases in Japan, although exact number of dengue
patients is not known. Clinical observation is important

examine the culture supernatants by RTPCR. We
sometimes detect virus genome in the culture super‑
natants by PCR when we can not detect dengue virus
genome in the original sera. RT‑PCR is a useful diag‑nostic method according to our experience. However,as described by Gubler (1998), it must be emphasizedthat RT‑PCR should not be used as a substitute forvirus isolation. The availability of virus isolates isimportant fcr characterizing virus strain differences,since the information obtained by isolating viruses iscritical for viral surveillance and pathogenesis studies.

Detection of dengue virusspecific lgM by lgM cap‑
ture‑ELISA and a rapid immunochromatographic testlgM capture

ELISA is the most widely used sero‑

for diagnosis; however, Iaboratory diagnosis is needed toconfirm dengue virus infection. According to aur expe‑
rience, mast Japanese dengue cases are primary dengue

infection in the presence of immunity to Japanese
encephalitis virus. Dengue virus genome can be detected

in serum samples by RTPCR until several days after
the onset of fever. Detection of lgM by lgM capture‑
ELISA or a rapid immunochromatographic test pro‑vides positive results when serum specimens are
obtained on day 7 or later. Thus, we recommend the
combination of RT‑PCR and lgM‑detection for diagno‑
sis. False negative can be avoided by attempting tocheck the presence of both. dengue virus and specific
lgM.

77

ACKNOWLEDGEMENTS
This work was supported by the grant from the
Research on Emerging and Re‑emerging Infectious
Diseases, Ministry of Health and Welfare of Japan, andby the Cooperative Research Grant 1998 (10‑A‑3) of theInstitute of Tropical Medicine, Nagasaki University.

of virus genome from irnported dengue fever and dengue
hemorrhagic fever patients by derect polymerase chain
reaction. J. Med. Virol., 44, 54‑58

7 ) Sang, C.T., Cuzzubbo, A.J. and Devine, P.L. (1998a) :Evaluation of a commercial capture enzyrne‑linked, im‑munosorbent assay for detection of immnoglobulin 'M

and G antibodies produced during dengue infection.
Clin. Diagnostic Lab. Immunol., 5(1) , 7‑108 ) Sang. C.T., Hoon, L.S., Cuzzubbo, A. and Devine, P.

REFERENCES
1 ) Bundo. K. and lgarashi, A. (1985) : Antibodies capture
ELISA for detection of immunoglobulin M antibodies insera from Japanese encephalitis and dengue hemorr‑
hagic fever patients. J. Virol. Methods, 11, 15‑222 ) Clarke, D.H. and Casals, J. (1958): Techniques forhemagglutination and hemagglutination‑inhibition witharthropod‑borne viruses. Am. J. Trop. Med. Hyg., 7,561‑ 577

3 ) Gubler, D.J. (1998): Dengue and dengue hernorrhagic
fever. Clin. Microbiol. Rev., 11, 480‑4964 ) Kuno, G., Cropp. B., Lee, J.W. and Gubler, D.J. (1998):

Evalution of an lgM immunoblot kit for dengue diagno‑
sis. Am. J. Trop. Med. Hyg., 59, 757‑762
5 ) Morita, K., Tanaka, M. and lgarashi, A. (1991): Rapid

identification of dengue virus serotypes by using
polymerase chain reaction. J. Clin. Microbiol., 29, 2107‑
2110
6 ) Morita, K., Maemoto, T., Honda, S., Onishi, K., Murata,M., Tanaka. M. and lgarashi. A. (1994) : Rapid detection(1998b) : Clinical evaluation of a rapid immnochrornato‑
graphic test for the diagnosis of dengue virus infection.
Clin. Diagnostic Lab. Immunol., 5(3) , 407‑409
9 ) Shirtcliffe, P., Cameron, E., Nicholson, K.G. and Wisel‑ka, M.J. (1998) : Don't forget dengue! Clinical features ofdengue fever in returning travellers. J.R. Coll. Physi‑chans Lond., 32(3) 235‑237

10) Sudiro, T.M., Ishiko, H., Green, S., Vaughn, D.W.,
Nisalak.. A., Kalayanarooj, S., Rothman, A.L., Raengsa‑
kulrach, B., Janus, J., Kurane, I. and Ennis, F.A. (1997) :Rapid diagnosis of dengue viremia by reverse trans‑
criptase‑polyrnerase chain reaction using 3'‑noncoding
region universal primers. Am. J. Trop. Med. Hyg., 56(4) , 424‑429

11) Vaughn, D.W., Nisalak. A., Kalayanarooj, S., Solomon,T., Dung, N.M.. Cuzzubbo, A. and Devine, P.L. (1998):
Evaluation of a rapid immunochromatographic test for
diagnosis of dengue virus infection. J. Clin. Microbiol.,36(1) , 234‑238

79

3

BREEDlNG HABITATS OF THE DENGUE MOSQUITOES,A EDES (STEGOMYIA) SPP. IN THE COASTAL, AGRICULTURAL AND

MOUNTAlNOUS VILLAGES IN SOUTH SULAWESI,INDONESIA, 1994‑1996

ICHIRO MIYAGI , HASANUDDlN ISHAK" AND TAKAKO TOMA
Laboratory of Medical Zoology, School of Health Sciences, University of the Ryukyus ,Laboratory of Biodefence Medicine, Faculty of Medicine,

Toyama Medical and Pharmaceutical University' andLaboratory of Environmental Health, Faculty of Public Health,Hansanuddin University, Indonesia3

The breeding habitats of the dengue vector mos‑The house index ranged from 8.3% in Harapan to 82.0%

quito, Aedes aegypti and A. albopictus were studied using

in Pancana, with a mean index of 59.5% for the 6villages. Like the house index, the container andlarval collection method inside and outside houses in 2
coastal (Pancana and Lalolang) , I plain (Lompo Riaja) ,2 hill (Doi Doi and Bette) and I mount (Harapan)
villages of Barru, (total population 150,000 in 52 vil‑lages) South Sulawesi, Indonesia from July 1994 toAugust 1995. And also seasonal appearances of these
species were studied using ovitraps in and around housesin Ujungpandang and Barru, throughout the year fromMarch 1995 to 1996.
A total of 615 houses in the 6 villages was examined

and Ae. aegypti was the dominant species, being abun‑dant indoors especially in the coastal areas. Ae. al‑
bopictus was breeding primarily in outdoor containers in

the hill and mountain areas. Earthen jar was the mostcommon breeding habitat of Ae. aegypti in all villages

surveyed. Drum can was the most common outdoor
breeding habitat of Ae. albopictus in the hill and moun‑tain areas. Larval indices (house, container andBreteau) for the two species were high especially in

Breteau indices ranged from 2.6% and 8.3 in Harapan to53.4% and 174.1 in Pancana, with the mean indices of
29.5% and 113.7 respectively.
The number of Aedes mosquito eggs, particularly in
outdoor ovitraps, increased at the onset of the rainyseason (November) in Barru, but increased mid rainyseason (January) in Ujungpandang. In the dry season(June‑October) , the number of eggs decreased in Barru,

but not in Ujungpandang. Ae. aegypti was dominant inboth indoor and outdoor ovitraps in Barru in the dry
season, while Ae. albopictus was dominant in both indoor

and outdoor ovitraps throughout the year in Ujungpan‑dang.
The high Breteau indices of Ae. aegypti suggeststhat this species may play an important role in thetransmission of dengue hemorrhagic fever in coastal andplain villages. Barru where epidemics of the fever occur
occasionally.

coastal, but apparently low in hill and mount villages.

4 MOLECULAR AND IN VITRO ANALYSIS OF EIGHT DENGUETYPE 2 VIRUSES ISOLATED FROM PATIENTS EXHIBITlNG
DIFFERENT DISEASE SEVERITIESMARLOU NoEL M. MANGADA AND AKIRA IGARASHI
Department of Virology, Institute of Troprcal Medicme Nagasaki Unrversity

Potential genetic determinants of dengue virulence
were studied by sequencing the entire genomes of eight
dengue 2 virus strains isolated from patients exhibiting
different disease severities during an epidemic season innortheastern Thailand in lq. 93. The isolates came fromone dengue shock syndrome (ThNH‑7/93), three den‑gue hemorrhagic fever and four dengue fever patients.
Phylogenetic analysis showed that the isolates belonged

to the Southeast Asian genotype. The 3' noncoding
regions showed distinctive secondary structures with80

one specific structure for the isolate ThNH‑7/93.
Analysis of the predicted polyprotein showed severalamino acid changes scattered mostly in the non‑structrual region. Of 30 positions with amino acidchanges, 7 were unique to the isolate ThNH‑7/93 and 3of those led to radical alterations in amino acid charac‑

ter. Several amino acid changes coincided with previous
studies relating genome sequence and virulence. Mini‑

mal changes in computer predicted protein secondary

structures were observed. Infective particles in the
inoculum for all isolates were approximately equal asmeasure by focus formation on BHK‑21 cells, but thisdid not correlate with the number of plaques formed on
LLC‑MK2 cells. Isolates from patients that. experienced
secondary infections were shown to have significantly
larger plaques than the isolates from primary infection
patients.

81

5

DENGUE VIRUS‑SPECIFIC T LYMPHOCYTE RESPONSES AND
THE ROLE OF T LYMPHOCYTES IN THE PATHOGENESIS OF

DENGUE HEMORRHAGIC FEVER

ICHIRO KURANE AND TOMOHIKO TAKASAKI
Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan

Abstract: Pathogenesis of dengue hemorrhagic fever is one of the main subjects in dengue virus research.
We hypothesize that rapid increase in the levels of cytokines and chemical mediators induces malfunctionof vascular endothelial cells, and leads to plasma leakage and shock. The process may be initiated by
antibody‑dependent enhancement of dengue virus infection, and triggered by serotype‑cross‑reactive T
lym phocytes.

Dengue viruses are the members of the family
Flaviviridae and there are four serotypes, dengue virus

types 1, 2, 3 and 4. The genome of dengue viruses
consists of a single‑stranded RNA nearly 11 Kb in(Kurane et al., 199la) . These clones included serotype‑specific, serotype‑cross‑reactive ahd flavivirus‑cross‑reactive CD4+ CTL clones. Dengue virus‑specific,

length which is plus‑stranded and infectious (Rice et al".,human CD4+ CTL clones established from other donors'
PBMC recognized E, C and NS 1, 2a proteins (Living‑1986). Dengue virus genome codes for three structuralston et al., 1994; Gagnon et al., 1996).

proteins, capsid (O, preM which is a precursor tomembrane (M), and envelope (E) , and seven non‑structural proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b
and NS5 (Henchal and Putnak, 1990).
Dengue virus infections cause dengue fever (DF)and dengue hemorrhagic fever (DHF) . Analysis ofhuman T Iymphocyte responses in dengue virus infection
is important for understanding the mechanisms of recov‑
ery from dengue virus infection and the pathogenesis of
DHF. It is especially important to determine the pro‑

Dengue virus speczfic‑human CD8+CD4‑ T Iymphocytes
Dengue virus‑specific CD,8+ T Iymphocytes also
include dengue serotype‑specific and dengue serotype‑cross‑reactive T Iymphocytes. Dengue virus‑specificCD8+ T Iymphocytes are cytotoxic. CD8+ CTL were
demonstrated in the PBMC of 7 of 8 donors who hadreceived monovalent, Iive‑attenuated experimental vac‑cines of the 4 dengue serotypes (Mathew et al., 1996).

NS3, NS1, 2a and E proteins were recognized by CD8+teins recognized by dengue virus‑specific T

CTL from 6, 5 and 3 donors, respectively. CTL of theselymphocytes. This knowledge would also be useful forthe development of dengue vaccines that induce protec‑
tive immunity, but do not induce immune responses that

7 donors recognized either NS3 or NS1, 2a. CTL fromone donor recognized prM protein. CTL that recognizedNS3 were mainly serotype‑cross‑reactive, while thosewhich recognized NS1, 2a and E proteins were mainly

may lead to immunopathology of DHF.

serotype‑specif ic.

Dengue virus‑speczfic human CD4+CD8+ T Iymphocytes
Dengue virus infection induces dengue virus‑spe‑
cific CD4+ T Iymphocytes in humans. Dengue virus‑
specific CD4+ T Iymphocytes include dengue serotype‑specific, dengue serotype‑cross‑reactive and flavivirus‑

cross‑reactive T Iymphocytes (Kurane et al., 1989;
Green et al., 1993). Dengue virus‑specific CD4+ T
lymphocytes are mainly Th I type and produce IFN‑yand IL‑2. Proteins recognized by dengue virus‑specificCD4+ T Iymphocytes haVe been determined using dengue
virus‑specific T Iymphocyte clones. CD4+ cytotoxic T

lymphocyte (CTL) clones recognized NS3 protein

Epidemiological and immunological studies that suggest

immunopathology of DHF
Following primary infection, Iife‑10ng immunity
develops to protect against repeated infection by thehornologous serotype of dengue viruses; however, pro‑tective immunity to the heterologous serotype is short‑
lived. It is known that antibodies to dengue viruses haveability to augment dengue virus infection of Fcy rece‑ptor‑positive cells such as monocytes (Kurane et al.,199lb) . On the basis of the epidemiological and labora‑
tory studies, it has been hypothesized that antibodies toPlease send correspondence to : Ichiro Kurane, M.D., Department of Virology 1, N. ational Institute of mfectrous Drseases

Toyama, Shinjuku‑ku, Tokyo 162‑8640, Japan
Tel & Fax: 03‑5285‑1169

l ‑23,‑1

82dengue viruses and other serotype‑cross‑reactive
immune responses may contribute to the pathogenesis of

DHF (Halstead, 1988).
We found that the levels of T cell activation
markers, i.e. soluble IL‑2 receptors, soluble CD4 andsoluble CD8 were elevated in the serum samples ofpatients with DHF (Kurane et al., 199lc) . This suggeststhat T Iymphocytes are highly activated in patients with

DHF.

genetic background of the hosts probably also contrib‑
ute to the pathogenesis of DHF.

ACKNOWLEDGMENTS
This work was supported by the grant from theProgram for Promotion of Fundamental Studies in
Health Sciences of the Organization for Drug ADR
relief, R & D Promotion and Product Review of Japan,
by the grants from the Ministry of Education, Science,Sports and Culture (Grant‑in‑Aid for Scientific
Possible immunopathological mechanisms of DHF
In secondary infection with dengue virus of a differ‑ent serotype from that which caused primary infection,
serotype‑cross‑reactive, non‑neutralizing antibodies
increase the number of dengue virus‑infected monocytes
by forming dengue virus‑antibody complexes. Serotype‑cross‑reactive CD4+ T Iymphocytes are activated, andproduce IFN‑y and IL‑2. Increase in the number ofdengue virus‑infected monocytes and augmented
expression of HLA class I and class 11 by IFN‑y facili‑tate the recognition of the epitopes on infected cells bydengue virus‑specific T Iymphocytes, and results in very

Research 08457100, and 09044344) , and by the grant fromthe Research on Emerging and Re‑emerging Infectious
Diseases, Ministry of Health and Welfare of Japan.

REFERENCES
1 ) Gagnon, S.J., Zeng, W.L., Kurane, I. and Ennis, F.A.
(1996) : Identification of two epitopes on the dengue 4
virus capsid protein recognized by a serotype‑specificand a panel of serotype‑cross‑reactive human CD4+
cytotoxic T‑1ymphocyte clones. J. Virol., 90, 141‑147
2 ) Green, S., Kurane, I., Tacket, C.O., Edelrnan, R., Eckels,
K.H., Vaughn, D.W., Hoke, C.H. and Ennis, F.A. (1993):

high levels of T cell activation. Marked T cell activa‑tion results in the production of much higher levels ofDengue virus serotype‑specific human CD4+ T

lymphokines.

live‑attenuated dengue I vaccine: Bulk culture prolifera‑tion, clonal analysis and precursor frequency determina‑

IFN‑y‑activated monocytes may release various
kinds of cytokines upon infection with dengue viruses,

or dengue virus‑infected monocytes may release highlevels of cytokines and chemical mediators as a result oflysis by dengue virus‑specific CD8+ and CD4+ CTL and/or as a result of contact with virus‑specific T
lymphocytes. Furthermore, the production of cytokinesand chemical mediators is induced by other cytokines.
Therefore, once various cytokines are produced, thecornplex network further increases the production ofcytokines and chemical mediators. These cycles could
result in high levels of cytokines and chemical mediators

in a short period of time. Cytokines not only induce theproduction of other cytokines, but also have synergistic
ef f ects.

lymphocyte responses in a recipient of an experimental

tion. J. Virol., 67, 4962‑5966

3 ) Halstead, S.B. (1988): Pathogenesis of dengue. Chal‑lenge to molecular biology. Science, 239, 476‑481
4 ) Henchal, E.A. and Putnak, R. (1990): The dengue vir‑uses. Clin. Microbiol. Rev., 3, 376‑396

5 ) Kurane, I. and Ennis, F.A. (1992): Immunity and im‑
munopathology in dengue virus infections. Seminar.
Immunol., 4, 121‑127
6 ) Kurane, I. and Ennis, F.A. (1994): Cytokines and dengue
virus infection. Seminar. Virol., 5, 443‑448
7 ) Kurane, I., Brinton, M.A., Samson, A.L. and Ennis, F.A.

(199la): Dengue virus‑specific, human CD4+CD8‑
cytotoxic T cell clones: Multiple patterns of virus cross‑reactivity recognized by NS3‑specific T cell clones. J.
Virol., 65, 1823‑1828

8 ) Kurane. I., Mady, B.J. and Ennis. F.A. (199lb):

We hypothesize that rapid increase in the levels of

Antibody‑dependent enhancement of dengue virus infec‑

cytokines and chemical mediators induces malfunction

tion. Rev. Med. Virol., 1, 211‑221
9 ) Kurane, I., Innis, B.1., Nimmannitya, S., Nisalak, A.,of vascular endothelial cells, and leads to plasma leak‑age and shock (Kurane and Ennis, 1992, 1994). These
processes appear to be initiated by antibody‑dependentenhancement of dengue virus infection of monocytes,

and are triggered by serotype‑cross‑reactive T
lymphocytes. It is known, however, that some DHF
patients do not have pre‑existing dengue virus antibody

and develop DHF during primary infection. Therefore,
other factors such as virulence of dengue virus strains or

Meager. A., Janus, J. and Ennis, F.A. (199lc) : Activationof T Iymphocytes in dengue virus infections: High levelsof soluble interleukin 2 receptor, soluble CD4, soluble

CD8, interleukin 2 and interferon gamma in sera ofchildren with dengue. J. Clin. Invest., 88, 1473‑1480

10) Kurane, I., Meager, A. and Ennis, F.A. (1989): Dengue
virus‑specific human T cell clones: serotype‑cross‑reactive proliferation, interferon gamma production andcytotoxic activity. J. Exp. Med., 170, 763‑775

8311) Livingston, P.G., Kurane, I., Lai, C.J., Bray, M. andEnnis, F.A. (1994) : Recognition of envelope protein bydengue virus serotype‑specific hurnan CD4+CD8‑
cytotoxic T cell clones. J. Virol., 68, 3283‑3288

12) Mathew, A., Kurane, I., Rothman, A., Zeng. L.L.,
Brinton. M.A. and Ennis, F.A. (1996) : Dominant recogni‑tion by human CD8+ cytotoxic T Iymphocytes (CTL) of

dengue virus nonstructural proteins NS3 and NS1, 2a,
J. Clin. Invest., 98, 1684‑1692
13) Rice, C.M., Strauss, E.G. and Strauss, J.H. (1986): Struc‑ture of the flavivirus genome. In: Schlesinger, S.,
Schlesinger, M.J. (eds) . The togaviridae and flavivir‑

idae. 279‑326 Plenum Press, New York

85

6

CONSTRUCTION AND CHARACTERIZATION OF CHIMERICJAPANESE ENCEPHALITIS/DENGUE TYPE 4 VIRUS
MASAYUKI TADANO
Department of Vrrology, Faculty of Medicine, University of the Ryukyus

The pre‑membrane (prM) and the envelope (E)clones (# 3 and # 7) frorn genome of chimera, original
cDNA souse and intermediate clone of a chimeric cDNA
were examined. A deletion between nts 76 and 81 in 5'

genes of a full‑length cDNA clone of dengue type 4(DEN4) virus (strain 814669) were replaced with thecorresponding genes of a Japanese encephalitis (JE)
virus (strain JaOH0566). An RNA transcript preparedfrom chimeric CDNA was used to transfect permissive

position 472 (Leu472Arg) of E protein was found inmosquito cells (C6/36). The chimeric virus was

CDNA clones (# 3 and # 7) of chimera and intermediate

recovered at five weeks after transfection, despite only

cDNA clone, but not in original CDNA souse. A nu‑

a few viral‑antigen positive cells were detectedat a

NCR was observed in the all CDNA clones. An aminoacid substitution of leucine for arginine at amino acidPAP staining of infected cells with a panel of mono‑cleotide substitution (G86O and an amino acid substitu‑
tion (Phel33Leu) have occurred during transfection of
RNA and producing progeny virus, but no changes weredetectedin C protein gene of chimera. The cDNA clone

clonal antibodies (MAbs). The prM and E proteins# 3 carried an amino acid change (His689Leu) , but not inderived from JE virus were detected, together with core

the clone # 7. On the other hand, clone # 7 has an aminoacid change (Ala660Thr) , but not in the clone # 3.

week after transfection.
The viral proteins of the chimera were identified by(O protein derived from DEN4 virus. However E
proteins of DEN4 virus and non‑structural proteins

The growth property of the chimera was examined.

(NS1, NS3 and NS5) of JE virus were not detected. The
chimera contained the antigenic epitope (s) that was(were) reactive with a panel of neutralizing Mabs by

The chirnera was restricted in replication in Vero cells
at 37'C, but grew to low titer in same cell line at 32'C
and in C6/36 cells at 28"C. Also, the chimera exhibited

neutralization test.

mouse neurovirulence that significantly reduced

Genomic RNA of the chimera was sequenced

compared with parental JE virus. Significantly, thethrough the structural protein genes (E, prM/M and O

chimera lacked neuroinvasiveness after intraperitoneal

and the 5' non‑cording region (NCR) . Two CDNA

inoculation of Balb/c or C3H/he mice, but produced
antibody by injection of infected cell lysate.

86

Symposium 3 HOW CAN WE CONTROL TROPICAL INFECTIOUS DISEASES IN THE TROPICS?
Session I humediate reports from current project in selected areas

1 ‑PARASITIC DISEASES‑
Masaaki SHIMADA
Information and Reference Center, Institute of Tropical Medicine, Nagasaki University

Since most of parasitic infection do not cause life‑
threatening diseases, they are usually considered lessproblematic than bacterial or viral infections both forindividual health and for public health. The DALY Ioss
due to parasitic diseases other than Malaria in sub‑Saharan Africa is only 1.8% of total loss which is muchsmaller than expected (World Bank, 1995).
When people living in a rural area in Guatemala,Is it so unimportant to control of parasitic diseases?

For the control of parasitic diseases, non‑specificcontrol measures, which sometimes are almost neglect‑
ed in the control of other infectious diseases, have beentaken as a main control measure. Although non‑specificmeasures such as safe water supply, health educationand construction of toilets are very much effective also
on the control of other infectious diseases, the impact iswhere parasitic diseases were prevalent, were asked
what the most important disease is in the area, only

not necessarily taken into account. It must be recog‑

6.2% of the people provided parasitic diseases as mostproblematic ones in their village. This may be one of
the factors which leads to the low participation rate in

DALY Ioss also reduces the DALY Ioss by other infec‑

a control project of parasitic diseases.

nized that the control of parasitic diseases with very low
tious diseases.

The control of infectious diseases by non‑specificmeasure should be revived.

87

2 LEPROSYKENTARO HATANONational Sanatorium Oku‑Komyo‑En, a former worker in Bangladesh sent from Japan Overseas
Christian Medical Cooperative Service

INTRODUCTION

Historically and world wide, Ieprosy is one of the

Is it really true to say that leprosy is the tropicaldiseases which has been recognized with its strong
prejudice and discrimination. Without wiping thisdisease ? Many ambiguities still remain in its etiology,

stigrna away, the early detection or elimination is not

and it is difficult to positively say that this disease haspossible.

the characteristic factors that are only seen in the

Another characteristic which makes the control

tropics. Actually, it is the disease of poverty, rather
than of the tropics. However, nowadays the high preva‑

difficult lies on leprosy inducing M. Ieprae itself: Its

generation time is extremely long. Whereas the genera‑

lence rate of this disease is recognized in the developingtion time of M. tuberculosis is 18 hours, M. Ieprae takes

countries in the tropics.

At the 44th WHO General Conference held in May,

12 days. The long incubation period makes eradicationof the disease difficult. The artificial culture of the M.

1991, a resolution was made to attain the global elimina‑

leprae has been impossible so far, and this is also one oftion of leprosy as a public health problem by the year

the characteristics which makes the research hard.
Since the past, trials have been made to eradicate

2000. In another words, this resolution is meant to
reduce the prevalence rate of leprosy under one per ten
thousands population, and to recognize that this publichealth problem is solved. This was the epoch‑making
event. Since then, much efforts have been made and,statistically the leprosy prevalence rate has drastically

leprosy. Four noticeable turning points in the history ofleprosy control can be described as follows;
1 . Discovery of M. Ieprae. This event precedes that ofM. tuberculosis and many other bacteria.
2 . Discovery of the first effective drug (Promin) in

fallen down. Furthermore, the WHO report of 1998

1943. Discovery of this drug is not much behind instates that "Today, we can be confident that elimination(of leprosy) is within our reach" and "Viewed over all,the leprosy situation in the world gives every reason forsatisfaction". Aside from the minor details, this analysis

tirne from the discovery of the effective drugs of othersounds essentially optimistic.

This paper is going to introduce the statistics of
WHO report, as well as the leprosy condition in Bang‑ladesh, and tries to discuss the possibilities of leprosy
control in the tropics.

Characteristics of Leprosy
The definition of leprosy is as follows; "A chronic
disabling disease of man, mainly attacking the nerves
and skin, caused by the Mycobacterium leprae, mildly
communicable, diagnosable, curably and of good progno‑

infectious diseases.
3 . In 1981, multi drug therapy (MDT) was recorn‑mended by the proposal of WHO's expert committee.
This meeting was actually intended to discuss on DDS
resistant control, but it played a significant role inexpanding the MDT as worldwide standard therapy.
Also, we cannot ignore the simplification of the classi‑

fication which was recommended at the same time. It
made the work possible for the field workers.
4 . The epochal declaration by WHO in 1991, and theworldwide actions which follow, have been very effec‑
tive, holding up the goal of leprosy elimination.

Leprosy in the Worldsis, if recognized early and treated properly".
In leprosy, at its early stage, a patient is not much

First, I want to introduce some statistics from
WHO report. Graph I shows changes in the number ofdisturbed by any particular symptoms, and this is one ofthe diseases which is difficult to detect by the patient
himself. It is especially vulnerable for the poor who arepatients from 1985 to 1997 in 32 endemic countries. It is

more concerned about their daily meal than their own

However, we must pay attention to that there wasa change in definition of leprosy patient who can becounted for statistics. On the PB cases, no change has
been added to the definition. But in the MB cases, thehealth, making this tendency worse. For the early
detection of the disease, the social education is very
important for those people.

a joy to notice that the number of patients drops downfrom four million to less than one million.

88

Prevelence and Detectbn per 100OO

Prevalence and Detectionin 32 endemic countries (Graph I )in top 16 endemic codr,tJies (Gr･ph 2)

L= i=L=‑‑'g
8 745(W'O
40Gon,G 5

35,WX'
8eOCaoo

2WOOO
2O(naao15(ilOaO
1000,C1,

5900OeOj

O

85 88 87 88 89 90 O1 92 93 94 95 96 97

Patient 8tatistic8

The rate of Ghildren, disabled and MB

of CLC (Graph 4)

trend in 32 endemio countries (Graph 3)‑Ple ･fPL

J!:

l

3500

45
40
35
30
25
20

3000
2500
2000
'

‑‑̲̲ i!::Li:::at :"c" =!i"!:: :atateel ‑ Disabl'd 'ate

! ̲MB rate

15
10
5

1 500
1 OOO

500
O

O

J

O
,･,
,O OOee, O'
e oO a'O'CDO'F
O'
COtr)
C' a'
O'

85 86 87 88 89 90 91 92 93 94 95 96 97

Gbdefinition of 1982 states a patient "more than two years

and until bacterial test becomes negative". By thisLepraey new cases of Bangladesh (Greph 5)

definition, many MB cases took more than two years for

1 ZOOD [.

the treatment. Sometimes, it took six years. In the

10ooo

beginning of 1990s, by the instruction of WHO, the termrs limited to "two years". The patient who has complet‑ed this period was no longer counted as a "patient". It

800o

eooo

400Q

is necessary to analyze actually how the number ofpatient decreased by this change of definition.

Graph I also shows change of number of newly

2000

Q

discovered patients. For thirtten years, there is almost

no changes. Much efforts surely have been made during

shown by the rate, but essentially, as there is no change

this period for the early detection, and the fact is that,in the number of newly detected patients, changes are of
the actual nurnber.

locally looking, some area does show distinctive
improvement. However, Iooking around all over theworld, it is difficult to see the distinctive decrease of

new patients.

Three points can be pulled out from these graphs

Graph 2 shows the comparison of number of1 ) MB rate is increasing. Looking locally, MB ratepatients and new patients per ten thousands population

increases as elimination proceeds. It may well said that
this is a good trend. But it is necessary to keep in mind

in the top 16 endemic countries. The rate of MB inthese countries varies frorn 31.7% to 80.6%, and anthat definition of MB was changed by WHO during 13

analysis seems to be needed. Anyhow, there seems to be

years from "over than B.1.2" to "more than B.1.1". Also,

some contradictions which can not be understooddegree 2 by WHO disability table, and the rate of

it is likely that a field worker who experienced recur‑
rent case becomes to feel psychological pressure and,being afraid of making the same mistakes, he tends tosift the diagnosis to more MB side.
2 ) Disability rate of WHO 2 degree seems to be surely
decreasing. The effort for the early detection must be

children during 13 years. In this graph, changes are

effective on this.

between the nurnber of newly discovered patients andprevalence.

Graph 3 shows changes of the MB rate of newly
discovered patients in 32 countries, disabled rate of

89

a NGO Ieprosy project in south‑east of Bangladesh

workers quickly throughout the nation. At first, theyplanned to do the training in 3 months, but because oflack of facilities and manpower for training, the plan
was shortened to one month. After that, each candidate
was alone assigned to the thana hospital. This elimina‑tion project started anyhow with a lot of uncertainties
about workers' knowledge, skill, and motivation. Mean‑time, the NGO side worked together to educate about 20
students at once who were sent from projects all overthe country, and in four months, they were to be readyto go back to each place assigned. They were to workwhere there are five district, first half of the term as thea little longer with their senior workers for more experi‑

only medical doctor in the project and in later half as aences.

3 ) The children's rate does not decrease. In leprosy
where the incubation period is long and the incidence
rate can be hardly known, the children's rate itself canbe the index of incidence rate. It is likely to think thatno decline in the rate implicitly states some serious
situation.

From the field in Bangladesh
Frorn January 1985 to April 1994, I was engaged insenior medical officer given all responsibilities of theThough there was some unevenness over the region,

project, and training a native doctor at the same time.

this was the first control casting‑net on all over Bang‑

From what I experienced there, I would like to talkladesh. Graph 5 shows changes of number of newly

about the leprosy control work.

detected patients in Bangladesh. A drastic increase can

The area where our team covered extended as far
as 28 thousands square kilometers where there lived

be seen just before and after 1994. This is the very effectmore than ten million people. Our leprosy project withten field workers was the only project in the area, andthere was no countermeasure of the government.
Graph 4 indicates number of patients in our project.

As MDT started in 1984, number of patients who werereceiving treatment gradually decreased, but there is no

signs of decrease in the number of newly discovered
patients. Meanwhile we worked energetically to train
volunteers, to share information to medical institutions,and to campaign leprosy education. However, in thisvast area, with the small project as ours, we could not
improve the condition up to where we could satisfy, thatis to see the sign of an interception of the infections. By

the time. when I was to leave the project, we trained new

field workers, and we were able to set up a new field
control system, separated from the leprosy hospital. We
needed this system to respond to the National Leprosyof the National Leprosy Elimination Project whichgovernment and NGO jointly worked, each taking 57%
and 43% of the land in the nation. This action can behighly evaluated. As a result of this effort, for the firsttime in history, prevalence reached a level of one person

per ten thousands population. However, because thisnumber coincides with the number of WHO goal, it wassaid that this plan would be ended successfully. Only
when the prevalence increases more, and after thatincidence decreases, if prevalence again comes down
under the target point, the'n we will be able to consider
that the plan have reached the goal.

Merits and demerits of WHO program
After the resolution at WHO conference in 1991, big
efforts were paid under the leadership of WHO. It hasbrought great advancement. No one denies on thisElimination Project which, at last, started in fall 1993.

matter. At the same tirne, the resolution enabled to

By this time, the number of our field workers wereheighten international interest for leprosy elimination,

about 50. But still we felt inadequate to do the control
work in this large area.

resolution, priority for the leprosy control was raised in

The National Leprosy Elirnination Project started
in 1993 in Bangladesh as the very first National Leprosy

Project. First it was necessary to divide the control

area between government and NGO. As the government
scheme, they decided to send a worker per one thana(small administrative area) hospital. Among 489 thanasin the nation, 57% of area was under government directcontrol. It was an urgent need to educate about 300
workers in a short time and to arrange them in the
country. The government intended to achieve the con‑trol work in 5 years, and they hoped eagerly to send

and to accommodate necessary fund. Also, by thethe developing countries. Also it worked to encourage
government's participation.

It was the WHO's expert committee held in 1981
that prepared WHO's resolution and enabled for it topass. By this proposal, MDT was standardized, and thesimplification of the classification enabled native field

workers to work more effectively. As the result, Ieprosy
control has become world‑wide. These are the big steps

in the work of leprosy control.
However, with the year of 2000 just ahead, there area few things which weigh on my mind for the world wide

90

leprosy control. What I am concerned most is that,ironically, in many areas, Ieprosy elimination statisti‑cally seems to achieve its goal by the year of 2000.

Would a local health worker who is very busily occupied
can make this steady effort for early diagnosis ? Moreefforts will be needed to succeed in the integration of

WHO confronts with many other problems also, andleprosy work, but who will take the responsibility forthey seem to wish to draw the leprosy project as it

this efforts ?

attained its goal and a success.

Moreover in the international society, once theannouncement is made that we are reaching the goal,

I do appreciate the merits of WHO work, but
wonder if the figures reported are the true reflection ofinterest for the control may be lost. If we are to face thethe fact. A WHO report of the Leprosy Elimination

era when acquiring of manpower and financial support

Campaign (LEO is told as follows; in India, during theconcentrated LEC done in relatively short period, two

is difficult, the distribution of free treatment drug may

million thirty thousands people were presented to a
specialist with their suspicious symptoms, and four
hundred twenty three thousands were diagnosed as lep‑rosy. Also, in three towns in Myanmar where there

also become impossible.

In 2lst century, when WHO has finished all the
special efforts that have been made, who on the earth
will be able to take a positive attitude for the leprosy
control in the world, and who will take the leadership ?

were 330 Ieprosy patients and 227 newly detected

These issues are still enveloped in a fog. But somepatients in 1996, LEC was carried out for two months,

system in taking leadership must exist in order to carryout elimination, and eventually true eradication.
The most dreadful thing is that etiology of leprosy

and 311 new cases were found. This is truly the fruit ofmuch efforts, but at the sarne time, this result seems to

imply that as much cases are still left undiagnosed.

Also, my friends at work in the field report manystories. For example, in Niger, an area of two million
people has been left out from the control work, or instill leaves many uncertainties. Moreover, the present
leprosy control rely too much on the effectiveness of
MDT that have been observed, and at some point, there
is an atmosphere that every problem has been solved.

Uttar Pradesh in India, 1,900 cases were found in 1997 in

Is the source of infection only limited to a man ? Isa small project carried only by catholic sisters without a

MDT presently used really all right as it is ? What is thedoctor.

mechanism of recurrent and how do we identify the risk
group ? What is the mechanism of the tissue degenera‑tion ? Is there any means to prevent and alleviate theperipheral nerve damage ? Above all things, is preva‑

Coming to know these incidents, I feel that, with thetarget year of 2000 near at hand, unless information onwhat are left out in the leprosy control are correctly
reported, there will be much problems later.

SOME ISSUES ON LEPROSY CONTROL AND QUESTIONSFOR THE FUTURE
As stated so far, still there are many problerns left

lence really decreasing in the world ? What is themethod of leprosy control that can be effective todecrease incidence ?

Without answering these many basic questions, andwithout clarifying the etiology of leprosy itself, eradica‑tion of leprosy does not seem possible.

in order to conquer leprosy. As an imminent issue, it issaid that in the year of 2000, Ieprosy control will be
integrated as the object of usual local health system in
the area where the elimination goal is achieved. But as

The great advance has been made in the worldstated before, in the early stage, Ieprosy itself does notleprosy control under the leadership of WHO. At many

give much discomfort for a patient, and hence it does notplaces, the goal of elimination has been achieved.

give a motivation to him to receive treatment voluntary.
Also, it is only after the peripheral nerve damage pro‑

gresses when anyone can diagnose, and at that point, heDespite of above fact, there seems no clear evidence
that prevalence is decreasing in the world.
After the year of 2000, in order to link elimination

would not go and take treatment willingly. Moreover,because of discrimination in a society, he would be
afraid to be diagnosed. For these reasons, there is awith eradication, there are still many hurdles for us tojump over, such as of leadership, integration of leprosy
control to the usual health system, and above all, eluci‑

great possibility that this disease is likely to be neglect‑

dation of etiology which still has much obscurities.

ed if one does not have a positive attitude to detect it.

Many cases have been reported in which, by the local
specialist's efforts, prevalence in that area increased.

SUMMARY91

ACKNOwLEDGMENT
I

the

would

like

presidentofMedicine，also

man

of

giving

I

to

give

the

wish

thankstoJapanesetoextendmy

symposium，Dr．N．Ishikawa
meachancetopresent

theProfessor

S．Nonaka，SocietyofTropicalthankstothe

Chair−

Arita

for

and

Dr．L

situationofleprosy．

93

3 MALARIA‑IF WE CAN CONTROL THE DISEASESHIGEYUKI KANO
Department of Appropriate Technology Development and Transfer,
Research Institute, International Medical Center of Japan,

1‑21‑1 Toyama, Shinjuku, Tokyo, 162‑8655 Japan
Abstract: The 39th Annual Meeting of Japanese Society of Tropical Medicine (President of the meeting
was Professor Shigeo Nonaka, Ryukyu University) held in Okinawa, focused on the control strategy of sometropical infectious diseases, and attempted to give chance of discussion in an important syrnposium orga‑
nized and chaired by Dr. Isao Arita, Chairman of Agency for Cooperation in International Health, Dr.
Nobukatsu Ishikawa, Vice‑Director of the Research Institute of Tuberculosis, and Dr. Akira lgarashi,

Professor of Institute of Tropical Medicine, Nagasaki University. This report summarizes one of thepresentations in the syrposium spoken by the author on world malaria situation, emerging trends of malaria,
as well as the prospect for the global malaria control into the 2lst century.

INTRODUCTION

WORLD EpIDEMIOLOGICAL SITUATION

Malaria has been with man for thousands of years,
however it was only towards the end of the last century
that Dr. Charles Louis Alphonse Laveran discovered themalaria parasite and Sir Ronald Ross demonstrated that
this parasite was transmitted by mosquito bites. Follow‑

cases annually, and 1.5‑2.7 million people die of malaria

ing these Nobel Prize‑awarded discoveries, manyeach year, including approxirnately I million deaths

Now, WHO's estimates of populations at risk
within totalling 100 endemic countries and territories
are 2,300 million in number or 41% of the world popula‑tion. The incidence of malaria is 300‑500 million clinicalresearchers explored and proved how malaria was inter‑among children under 5 years of age (WHO, 1997).

woven into the fabric of life in an extraordinarily varied

and rich way (Litsios, 1996). Then, time had come to

Severe malaria caused by P. falciparum (P. f) is strik‑
ing particularly young children, non‑immune adults, andprepare a definitive statement concerning the principles

women during their first pregnancies.

and practice of malaria eradication by World HealthOrganization (WHO) after the 6th session of the WHO
Expert Committee on Malaria held in Athens, June 1956.
Despite a global effort aimed at its eradication, Iasting
practically from 1955 to 1969 and costing hundreds ofmillions of dollars, malaria was still with the people intropical and sub‑tropical areas. Dr. Marcolino Candau,
Director‑General of WHO at the time, said in January
1969, "The Organization had reached, where the malaria

The problem of the emergence of drug‑resistantmalaria and its worldwide expansion continued to inten‑
sify. Among the countries where falciparum malaria isendemic, only those in Central America and Egypt have
not recorded chloroquine resistant P. f malaria (WHO,1997). Resistance to sulfadoxine/pyrimethamine is
widespread in South‑East Asia and South America.
Mefloquine resistance is also widely reported in South‑
East Asia, and particularly in Thailand (Karbwang anderadication program was concerned, a crucial moment

Wernsdorfer, 1993). Multi‑drug resistance including

in its history. It should be realistic and, while counting

quinine resistance is a matter of great problem. Conse‑

its success, it should not fail to recognize its failures"quently, in certain areas, artemisinin and its derivatives(Litsios, 1996). Now, malaria remains the most impor‑

are coming to be used for first‑line treatment. Resis‑
tance of vivax malaria strains to chloroquine has alsotant of the tropical diseases, and in fact, it is re‑emerg‑

more, controlling malaria is proving to be more andbeen confirmed in some Asian countries (WHO, 1997).
Primaquine resistance of vivax strains in the hepatic

more difficult with increasing levels of drug and insecti‑

stage is another topic which makes it hard to avoid thecide resistance of parasites and mosquitoes. It would be

relapse of vivax malaria. Worsening drug resistancefoolhardy for us to predict when and how malaria will
be conquered.

makes policy decisions on the use of antimalarial drugs

ing to areas where it has been once eradicated. Further‑increasingly difficult.

94

EMERGlNG TRENDS
In fact, an expansion of the geographical areas
susceptible to malaria transmission and a widespreadincrease of potential malaria risk are to be expected as

to include more general social values, such as learing

ability and impact on education. It would be very
difficult now to weigh the cost/effectiveness of themalaria control and its socio‑econornic impact as thesocial structure becomes more and more complicated.

the climate changes. As it is known, malaria incidence
is determined by a variety of factors, particularly the

MALARIA SUMMIT

abundance of anopheline mosquitoes, human behavior,and the presence of malaria parasites. Anthropogenic
climate change directly affects the behavior and geo‑

The worsening malaria problems, as summarized
above, Ied the WHO to convene a Ministerial Confer‑graphical distribution of the malaria mosquitoes and thelife cycle of the parasite, and thus change the incidence

ence on Malaria in Amsterdarn in October, 1992. Healthleaders from 102 countries, as well as representativesof the disease. Indirectly, climate change could also
have an effect on vegetation thus the availability ofbreeding sites. The large‑scale migration and resettle‑
ment of populations from malaria endemic areas into
receptive areas, a movement induced by rural impover‑ishment influenced by climatic change, will play animportant role in the dynamics of the disease.
The re‑emergence of malaria will also be associat‑

from UN agencies and NGOS joined the conference,
which endorsed a Global Malaria Control Strategy

ed with socio‑economic development and expanding‑to provide early diagnosis and prompt treatment;‑to plan and implement selective and sustainablepreventive measures including vector control;‑to detect early, contain or prevent epidemics; and‑to strengthen local capacities in basic and appliedresearch to permit and promote the regular assess‑infrastructure, population growth and urbanization,
natural disaster or civil unrest, and effectiveness ofcontrol measures (Aikawa et al., 1998). Increased riskof malaria due to various environmental changes may
seriously affect human health in the coming century
worldwide.

(GMCS). The goal of the GMCS was to prevent
mortality and to reduce morbidity and social and eco‑nomic loss due to disease through the progressive
improvement and strengthening of local and national
capabilities. The four basic technical elements of thestrategy are:ment of a country's malaria situation, in particular

the ecological, social and economic determinants

THE BURDEN OF MALARIA
Malaria was one of the very first diseases to be
characterized as a "social disease". Sir Ronald Ross in1911 proposed the calculation of the economic loss to thecommunity caused by malaria, based on mortality andof the disease.

The Ministerial Conference was concluded by the
adoption of a World Declaration on the Control ofMalaria. The two major global objectives of the decla‑ration was that, by the year 1997, at least 90% ofcountries affected by malaria implement appropriate(Najera and Hempel, 1996). For example, estimates

malaria control prograrns, and that, by the year 2000,
malaria mortality will have been reduced by at least

focus on days lost from work or output foregone, theloss of wages during sickness plus cost of treatments.

countries. To achieve this goal, it was estimated that

World Bank (1993) measured the global burden of

WHO will require a total of just over 40 million US

disease in units of disability‑adjusted life years(DALYS) , and estimated that 2.6% of the total world
DALYS Was from malaria. Of course, a man cannot be

dollars through the period 1993‑1999 in addition to its

morbidity and "the local values of human life and labor"considered as an exchange value, nevertheless, calcula‑tions are thought to be necessary to justify funds.
Because public health action against malaria often
consisted of the implementation of large scale program
which required the use of considerable public funds, thejustification is needed for their political approval having

either been fully based on economic arguments.
The burden on society was first measured in eco‑nomic terms, such as lost productivity, and later, coming

20% compared to 1995 in at least 75% of affected

regular budget allocation to malaria control (WHO,1993). In fact, WHO allocated extra funds for malaria(10 million US dollars between 1996 and 1997 and thesame amount again from 1997 to 1998) to help countries
develop plans for tackling malaria, training staff andinitiating more effective action.

ROLL BACK MALARIAWorld malaria situation was proposed in discussion
among the leaders of the G8 nations in Birmingham, UK,95

1998, and remained as one of the issues on the agenda aspart of the G8 development focus. The summit discus‑
sions, 3 days after Gro Harlern Brundtland's election,

education and hygiene control, which employed themethod of community participation and community
empowerment. Along with the community, public insti‑

enabled the leaders of the G8 countries to offer strong
support for the new WHO initiative, which is called the"Roll Back Malaria (RBM) " initiative, announced on 13

tutions, parasitologists, and private organizations wereinvolved in a coordinated manner (Aikawa et al., 1998) .

May 1998 by Brundtland herself as new director generalof WHO. There was also a strong political commitment
in affected countries (African Initiative on Malaria),
and now, the research community (Multilateral Initia‑
tive on Malaria) is mobilized and the private sector(Medicines for Malaria Venture) is showing interest.

can play in advancing public health and hygiene, theformer prime misister, Mr. Ryutaro Hashimoto, pointed
out its importance, and stressed the need for interna‑
tional cooperation in this area during the G8 Summit
held in Denver, 1997. Upon the heels of this initiative
was formed a working group on parasite control by the

In recognizing the major role which parasite control

RBM is not another atternpt to eradicate malaria;

Government of Japan. Subsequently, teams of theinstead, the aim will be to halve malaria‑associatedworking group members were dispatched to the devel‑morlality by 2010 and again by 2015. The greatest

oped countries as well as developing countries, in orderemphasis of the initiative is to strengthen health seT‑
vices, so that effective treatment and prevention strat‑egies are accessible to all who need them. It will apply

to accumulate much information on how Japan can playa role in the global parasite control. As a result, aevery existing tool more effectively, building on experi‑
ences of the past‑failures and successes alike (Nabarro
and Tayler, 1998) .

Century" was published and distributed (Aikawa et al.,1998) . This report presented Prime Minister Hashirnoto

RBM was backed up by a financial commitment of
L60 million by the British government to kick‑start.

report entitled "The Global Parasite Control for the 2lst

and the Japanese Government with important ideas tomeet with the preparation for the next Summit held in
Birmingham, 1998.

Now, it is reported that the budget justification of RBM(July 1988‑December 1999) is US$ 7 million for Direct
Country Support, US$ 6 million for Resource Support
Networks, US$ 4.5 million for Global R&D, US$ 1,870
thousand for Core Staff Support, and US$ 500 thousand
for Operational Support (travel, corlsultants, meeting) ,totalling US$ 19.87 million (WHO, 1998). To achieve

Recent Japan's overseas malaria control projects
through Japan International Cooperation Agency are:Solomon Islands Primary Health Care Promotion
Project, Sep. 1991‑Aug. 1996; Laos‑Japan WHO PublicHygiene Promotion Project, Oct. 1992‑Sep. 1997; Mal‑awi Public Hygiene Project, Sep. 1994‑Aug. 1999; andthe above described goal, RBM will not be a "one‑time"
project, and indeed, sustained effort and financing will
be required over the next two decades.

Zimbabwe Infectious Disease Control Project, Jul. 1996‑

JAPAN'S ROLE

We believe that, if Japanese experiences and tech‑niques are employed, and if the G8 and other developed
countries join forces in organizing suitable malaria
control programs according to the specific conditions ofJapan was once a malaria endemic country. Indige‑nous malaria cases were observed even in Hokkaido in
the late 19th centuty. The numbers of malaria cases

Jun. 2001.

endemic countries, more effective control strategies willbe by all means feasible.

gradually decreased; 200 thousand cases in 1903, but 13

thousand in 1945. When the second world war ended,

ACKNOWLEDGMENTS

about 430 thousand soldiers and civilians who returned

to Japan from the war zones suffered from recurrent
malaria. To counter this situation, malaria control
programs were implemented across the country. As aThis work was supported in part by a Grant forInternational Health Cooperation Research (10A‑1)

result, along with subsequent economic development, thelast indigenous malaria case in the main land of Japan

Research on Health Sciences focusing on Drug Innova‑
tion (42204) from The Japan Health Sciences Founda‑

was reported in 1959. Today, only imported malariacase (which exceeds 100 cases per year though) are
reported. In Japan, malaria control measures weretion.

integrated into public health activities, such as healthfrom the Ministry of Health and Welfare, and a

96

REFEREN CES1 ) Aikawa, M., Aoki. Y.. Ishii, A.. Inaba, H., Suzuki, M.,
Takeuchi, T., Tada, I., Tsuji, M., Hara, T. and Morit‑

sugu, Y. (1998): The Global Parasite Control for the
2lst Century‑A Report on Global Parasite Control‑.
9‑14, The Working Group on Global Parasite Control,
Ministry of Health and Welfare, Government of Japan,Tokyo2 ) Karbwang, J. and Wernsdorfer, W.H. (1993): Clinical4 ) Nabarro, D.N. and Tayler, E.M. (1998): The "Roll Back
Malaria" Campaign. Science, 280, 2067‑2068

5 ) Naiera, J.A. and Hempel. J. (1996): The burden ofmalaria. 9‑14, Malaria Unit, Division of Control of
Tropical Diseases, WHO, Geneva
6 ) WHO (1993): WHO's plan of work for malaria control
1993‑1999. Part 1. 1‑17, Division of Control of Tropical

Diseases, WHO, Geneva7 ) WHO (1997) : World malaria situation in 1994. Wkly.
Epidemiol. Rec., 72, 269‑276

Pharmacology of Antirnalarials. 167‑185, Mahidol8 ) WHO (1998): Roll Back Malaria, A global partnership,

University, Bangkok
3 ) Litsios, S. (1996): The tomorrow of malaria. 7‑163,
Pacific Press, Wellington, New Zealand9 ) World Bank (1993): World Development Report 1993,

RBM/Draft/1. 1‑8, WHO. Geneva
25‑29

97

Session 2 Global strategies for eradication or effective control of infectious diseases

4 GROBAL SURVEILLANCE AND NEW LAW REGARDlNG THE PREVENTIONOF INFECTIOUS DISEASES AND MEDICAL TREATMENT GIVEN TO
PATIENTS WITH INFECTIOUS DISEASESTakashi NOMURAInfectious Diseases Division, Health Service Bureau, Ministry of Health and Welfare, Government of Japanmethods have progressed, hygiene standards have im‑

Given the circumstances, we propose the establishmentof a new law in order to promote comprehensive mea‑sures to prevent infectious diseases and improved medi‑
cal care. The new law is to become effective on April

proved, and residents have become more aware of

1, 1999, except for certain provisions.

One hendred years have passed since the Infectious
Diseases Prevention Law was established in 1897. Overthe past century, medical science and treatment

health and hygine issues. The situation has improved
accordingly and incidents such as the death of over
10,000 people from cholera no longer occur. On theother hand, the prevalence of E. coli become social
problem in Japan in 1997, while at the same time a new
infectious diseases called Ebola hemorrhagic fever
which had not been known previously, appeareded out‑

One of the most important factor in new law isdiseases surveillance. If a doctor diagnoses a patient as

having an infectious disease notify the prefectural gov‑
ernor of information specified in Ministry of Health andWelfare. This surveillance system is composed of twoparts. The one is to require the information of all

patients diagnosed by the doctors. The number ofside Japan. It is conceivable that such a disease could be

infectious diseases in this system is about 40. The otherbrought into Japan, given the sheer volume of interna‑
tional exchange today. Also, despite the common belief
that malaria would disappear in the near future it has

is sentinel surveillance including about 30 infectious
diseases. Furthermore, groval surveillance is essential
for infectious diseases control measures. So under the
new law, the national government is required to estab‑lish a system to promote the collection on infectious
diseases, and make efforts to secure global cooperation.

reappeared and has once again become a problematic
infectious disease. By their reappearance in society,infectious diseases are threatening people in a new way.

99

5 CONTROL OF INFECTIOUS DISEASES‑COMMUNITY APPROACHES IN DEVELOPlNG COUNTRIES‑
Takusei UMENAI
Department of Health Policy and Planning, The University of Tokyo

Over the past century, the average sea surface
temperature has increased approximately 0.7'C andINTRODUCTION
Environmental changes such as climate change havea big influence on the emergence and reernergence of
infectious diseases. Recent studies have identified an
upward trend in global temperatures and now estimate
a global mean temperature increase of 1‑2'C by the year

waters. Zooplankton, which feed on algae, can serve as
reservoirs for Vibrio cholerae and other enteric path‑ogens, particularly gram‑negative rods. Large coastal

2050.

blooms may have contributed to the recent multi‑

How to meet the challenge of controling infectious
diseases caused by global environmental changes is themost urgent question to be seriously explored, particu‑
larly taking into consideration of the mobilization andparticipation of people at the grass root level who makeup almost 70% of the total population in the world.

epicentered cholera pandemic in Latin America.

marine growth of algae has been observed responding to

10calized temperature increases in nutrient‑replete

Control
For the control of infectious diseases, both preven‑tive and curative measures are necessary. Preventive
measures include areas such as environmental protec‑
tion, vector control, immunizations education, behavior‑

Epid emio logy

Concerns have been increasing about the relation‑
ship between environmental changes and the spread of
infectious diseases in the world. For example, recent

studies indicated that a global mean temperature
increase of 1‑2'C would enable mosquitoes to extend
their range to new geographical areas, Ieading to in‑creased epidemics of mosquito‑borne diseases such as
malaria and dengue fever.

al changes,
chan es safe water and sanitation. For curative
measures, development of simple and low‑cost diagnosis

methods and effective and low‑cost anti‑microbial
drugs are important.
Development and implementation of even single oneof the above measures is not easy. For example, effec‑

tive policy and measures have not yet been accepted
globally for the control of C02 emissions which has
resulted in a global temperature rise.

dengue, which currently threatens 1.8 billion people,

Another example is a chronic shortage of financial
and human resources for the control of the infectious
diseases, particularly at the grass root level in many
disease endemic countries. To cope with these prob‑lems, several efforts have been taken at the grass root

infects 50 million people and causes 25,000 deaths annu‑

level in developing countries.

It is reported that a temperature rise of 1‑2'C by theyear 2050 could result in an increase in the population atrisk for Malaria, presently estimated at 2‑4 billion
people in the world of 45% to 60%. Similarly, in case ofally, it is indicated that a ternperature rise of 1‑2'C could

result in an increase in the at risk population of several

hundred million, resulting in 20,000‑30,000 more dengue
deaths a year in 2050 (Table 1).
Table I Climate change* and risk of infectious
disease
Disease

1998

2050

Annual deaths

Disease vector control linked withincome generating schemes
1 ) Composite Fish Culture‑Ponds free from aquatic vegetation‑Preventing vector mosquito breeding‑Gain was Rs 5.8 million

2 ) Vector Control Linked with

Malaria
population at risk

Table 2

2.4 billion 3.4‑3.8 billion
2‑3 million 3.4‑5 million

Denguepopulation at risk

1.8 billion Over 2 billion

Annual deaths

25,000 30,000*A temperature rise of 1‑2'C by 2050

Agricultural Development Programme‑Getting rid of noxious aquatic weeds‑Growth alternative aquatic weeds (nitrogen rich and non‑vector mosquito breeding) as a source of green manure
for agricultural purposes‑RS 100 thousand for the community

100

Table 3 Health revenue in one commune in Vietnam
Source

1991

1992

1993

Regular government allocation

o

o

o

User feeso

3 . 405 . 60n/a 17 . 50

21 . 90

Drug revolving fundCommunity regular

1994(in Million VND)

1995 1996(1/2) Total

O 22 . 60
11 . 30

6 . 207 . 4011 . 30n/a 22 . 60

34 . 60 26 . 30 155 . 30
55 . OO

1 . 60 18 . 20

o4 . 805 . 603 . oo3 . 20

Donors

o

o

o

o

o

o

o

Others

o

o

o

o

o

o

o

Total

o 25 . 70

33 . 10contribution

Community Approach
Infectious diseases have been endemic in many
developing countries. In these countries 70%‑80% of

43.80 39 20 218.70
76 . 90themselves.

CONCLUSION

people live in rural areas where they have long sufferedfrorn disease as well as a shortage of resources to cope

By facing unprecedented changes taking place inwith problems caused by disease. Among many efforts

the global climate and the consequent possibility of theso far taken for the control of infectious disease at the

introduction and dissemination of many infectious dis‑eases, efforts to cope with these problems have been

grass root level in these countries, several approaches

have drawn attention. One was the control of Malaria
and Filariasis by people in the rural community ininitiated both globally and locally.

Kerala State in India where vector control was linked to

the grass roots level is quite important and several
approaches have been proven to be workable.

income generation such as fish culture and agricultural

Encouraging people to participate in these efforts at

development (Table 2).

Another example was the development of commu‑
nity health fund project through the introduction of an
essential drug revolving system to improve health ser‑vices including community infectious disease control ata rural community in Vietnam (Table 3).
During the several years of the project, the successof community health fund project encouraged commu‑
nity people to challenge Japanese encephalitis and den‑gue control.
These examples provide an important precedent forpeople living in rural areas of developing countries toinitiate and develop infectious disease control activities

REFERENCES
1 ) Jonathan, A. et al. (1996): Global climate change andemerging infectious diseases, JAMA, 2752 ) Climate change‑Health risk for the 2lst century, The

World Health Report3 ) Panicker, K.N. (1992): Control of brugian filariasis. A
community approach, Misc. Publ. VCRC (19) , Miscella‑
neous Publication of Vector Control Research Centre4 ) RDFS in Vietnam (1997): Building a Model for Suitaina‑

ble Health Care Services, Nippon Foundation

101

6

GLOBAL STRATEGY FOR ERADICABLE
INFECTIOUS DISEASESISAO ARITA
Agency for Cooperation in International Health

Abstract: Initiation of a global eradication programme of selected infectious disease may be oneof the major health activities of international interest in the 2lst century. The article reviewed thecurrent topics in this regard and discussed the conditions with which further eradication may
succeed with reference to the experiences in smallpox and polio eradication programmes.

The 1980's WHO declaration of smallpox eradica‑tion dictated an important lesson, namely "humankind
can eradicate a severe disease by mobilising world
resources and modern technology" (Arita, 1979). The
characteristics of eradicable diseases would be de‑treatment.
These reviews have been reflections of recent andnew interest of world community to promote the worldconsensus, namely if a disease is potentially eradicable,

scribed as ( ) no animal reservoir R no persistent infec‑

programmes.

should we not proceed with implementation of suchtion R availability of effective control measures and )Operational strategy for eradication should meetworld interest for allowing mobilisation of world

with certain conditions. It should last within 10‑15 years

resources.

with globally coordinated efforts in all the continents

The lesson together with eradication efforts of(Arita, 1998) . Global surveillance should be the key that

other diseases was studied by two conferences, first thein Atlanta in Feb. 1998. The latter Conference suggested
diseases‑eradicable as well as diseases of which trans‑

reveals effective method of stopping the transmission
within above mentioned length of time. Otherwise, it
can not be cost‑effective, which is the major advantage
of eradication effort. In principle the execution of
global eradication should be financed by "additional"

mission could be suppressed with effective control to theglobal resources, not obtaining the support from that ofminimum so that the diseases will be no more threat tothe community (Table 1), (Personal communication,

ongoing health programmes. These considerations leadto the necessity for intensive mobilisation of world

Walter Dowdle) . To me, effective control of lymphatic

resources for eradication, should it start.

filariasis is of particular interest as it was prevalent in

The intensified smallpox eradication was launched
in 1967 and succeeded to interrupt the transmission inthree endemic continents, within 10 years, namely in
Latin America in 1971, in Asian subcontinent in 1975 andDahlem Workshop in Berlin, in March 1997 and second‑ly, Conference of Eradication of Communicable Diseases

southern part of Japan there just four decades ago, but
it now disappeared with effective surveillance and drug

Table I CDC Conference, February 1998
Eradication Effective Control
1 acterial Disease+++

Congenital syphilis

Trachoma
Hib
Parasitic Diseases

Dracunculiases
Onchocerciasis

+
+
+
+

Lymphatic filariasis

ChagasMeasles
Rubella
Hepatitis A
Hepatitis B

+
+
+
+

Source: Walter Dowdle's personal comunicationin 1988. The transmission stopped in Americas in 1991
and East Asia in 1997. Global eradication is planned tobe completed in 2000. In both programmes, the mobilisa‑
tions of world resources have been adequate althoughpolio eradication further requires resources since majorfoci still remain in Asian subcontinent as well as inAfrica, South of Sahara.
In smallpox eradication, the continued surveillance

discovered human monkeypox resembling smallpox
infection. Extensive research revealed that the disease
does not frustrate the eradication. In polio eradication,

Viral Diseases

Polio

in Africa in 1977. Global polio eradication was initiatedstudies are underway to elucidate the behaviour ofvaccine virus after the anticipated stoppage of polio+

vaccination. Thus, global eradication of any disease willneed substantial research.
In conclusion, eradication effort will be one of most

l 02

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lacked adequate scientific analysis of feasibility world‑;*'*' "'**i"{"' ""‑ " ''valuable human efforts to eradicate permanently humanmisery, but its initiation must be well prepared andconducted under a strong leadership. The failure af
malaria eradication in 19. 70's was a good lesson as it>

P ? hQ ej

itie m !di[̲*inwide at the inception (Figure 1) .

Toward the 2lst century, it is hoped Japan would

Ci ri tlliC s

contribute significantly to this developrnent.
cir f n tr LnB I J: tc{i n : th8rs'#'; 'REFERENCES'i;' Re‑"'*l s it !tioll ) Arita, I. (1979): Virological evidence for the success of
Individu J:i : !tiO l

I Igure I clobal eradication iS a logical consequence of theevolution of medical technology in the mainstreamof the 2lst century' Nate this is contrasted by therevival in the trend of indiViduahzation on theother extreme end'

Smallpox eradication programme, Nature, 279 (5711),
293‑298
2 ) Arita, I. (1998): Are there better global mechanisms forformulating, implementing and evaluating eradication
programmes?, The Eradication of Infectious Diseases,

Dahlem Workshop Reports. John Wiley & Sons Ltd.

103

SHOP： FOR MEANINGFUL INTRODUCTlON OF ADVANCED TECHNOLOGYSTUDIES OF MALARIA EPIDEMIIOI。OGY AND CONTROL

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survey．

was
field

well

The

withoutanyelectric

equipments
screening

resultofSingle−stepcorrelatedwithformazanイing

condition．

method

104

2 PROGUANlL POLYMORPHISM AND EFFICACY AGAlNST MALARIA
A. KANEKol'6, Y. BERGQVIST2, M. TAKEUCH11, M. KALKOA3, O. KANEK04
T. KOBAYAKAWAl, T. ISHIZAK15 AND A. BJORKMAN6
Department of International Affairs and Tropical Medicine,Tokyo Women's Medical University School of Medicine*,
Dalarna University College, Borlange, and Department of Clinical Chemistry,
Falun Central Hopital, Sweden',

Malaria Section, Department of Health, Vanuatu3,

Department of Medical Zoology, Osaka City Umversity School of Medicme
Department of Pharmacology and Therapeutics,
Graduate School of Clinical Pharmacy, Kumamoto University5 andDepartment of Infectious Diseases, Karolinska Institutet,

Danderyd University Hospital, Sweden6

Variations in the human cytochrome P450 isoen‑

site dhfr mutations. We observed a similarly highzyme CYP2C19 and the parasite dihydrofolate reductase

antimalarial efficacy of proguanil against both infec‑(dhfr) genes, related to poor metabolism of proguanil totions in 62 patients with CYP2C19‑ related poor
metabolizer (PM) genotype as in 33 with extensive

cycloguanil (a strong DHFR inhibitor) and resistance tocycloguanil respectively, have both been assumed to be
associated with poor antimalarial effect by proguanil.

To study this relevance, 95 subjects (median age, 7
years) with uncomplicated Plasmodium falciparum or P.
vivax infections in Vanuatu, where we have recently

found extremely high frequencies of CYP2C19 muta‑
tions, received proguanil treatment for 3 days (adultrelative dose of 300‑500 mg/day) and were followed up
for 28 days. Blood samples were collected on filter

paper for determining human CYP2C19 mutations,blood concentrations of proguanil/cycloguanil and para‑

metabolizer genotype, even though signlficant cycloguanil

blood concentratioles were not detecable in the PMs.
Parasite genotyping revealed two dhfr mutations (resi‑
dues 59 and 108) in all 28 P. falciparum isolates tested,suggesting moderate resistance to cycloguanil. The
results suggest that the parent compound proguanil has
significant intrinsic efficacy against falciparum andvivax malaria independent of the metabolite
cycloguanil.

Proguanil represents an important

antimalarial even in a population with a high frequencyof the CYP2C19‑ related PM genot ipe status.

105

3A NOVEL GENE CLONlNG OF OOKlNETE SURFACE PROTElNFROM PLASMODlUM VIVAX, AND POLYMORPHISM IN NATURAL
PARASITE ISOLATESTAKEFUMI TSUBOI D C KASLOW MAYUMI TACHIBANA1, Y‑M. CAOl, Y. YAKUSHIJlNl,
T. NAGA03, HIROJI KANBARA3 AND MOTOMI TORIll
Department of Parasitology, Ehirne University School of Medicinel,
Laboratory of Parasitic Diseases, National Institute of

A1lergy and Infectious Diseases, National Institute of Health, USA' andDepartment of Protozoology,
Institute of Tropical Medicine, Nagasaki University3

In many malarious regions outside of Africa, devel‑

opment of transmission‑blocking vaccine will require
activity against both Plasmodium falciparum and P.
vivax. Work on P. vivax transmission‑blocking vaccines
has been hampered by the inability to clone the vaccine
candidate genes from this parasite. To search for genes
encoding the ookinete surface proteins from P. vivax, wealigned the gene sequences of the eight known proteinsin P25 subfamily (Pfs25, Pgs25, Pys25, and Pbs25) and in
P21/28 subfamily (Pfs28, Pgs28, Pys21, and Pbs21) andsynthesized degenerate PCR oligonucleotides. We used
genomic DNA, genomic library or splinkerette genomic

library of P. vivax Sall as templates of PCR. To
determine the polymorphism of Pvs25 and Pvs28, weused genomic DNA extracted from P. vivax isolates.
Analysis of the amino acid sequence of Pvs28 revealed asecretory signal sequence, four EGF‑ Iike domains, a(GSGGE/D) * repeat and then a short hydrophobic
region. Pvs28 is the presumed homologue of P21/28
subfamily member because the fourth EGF‑1ike domain
has four rather than six cysteines. Analysis of thededuced amino acid sequence of Pvs25 revealed a simi‑
lar structure to Pvs28. The presence of six rather thanfour cysteines in the fourth EGF‑like domain suggested
that Pvs25 is the homologue of P25 subfamily member.
Comparison of the primary structures of Pvs25, Pvs28,
Pfs25 and Pfs28 obtained from field isolates suggested
that there were some point mutation sites. In conclu‑
sion, we found two novel ookinete surface protein genes,

Pvs28 and Pvs25 from P. vivax that are candidates oftransmission‑blocking vaccine. We also found somepoint mutation sites in Pvs25 and Pvs28 that result in
antigenic polymorphisms.

106

4 IMMUNOGENETIC ANALYSIS OF THE PATIENTS WITH
FALCIPARUM MALARIA IN THAILAN. D
KENJI HIRAYAMAl, O. TASANOR2, MIHOKO KIKUCH13, S. LOOAREESUMAN2,
Y. WATTANAGOON2, K. NA‑BANCHANG2, J. KARBWANG2, AKlNORI KIMURA3,KYOUGO ITOH4, ToZO KANDA5 AND MASAMICHI AIKAWA6
Department of Medical Zoology. Saitama Medical School*
Faculty of Tropical Medicine, Manidol University',
Division of Adult Disease, Medical Research Institute,Tokyo Medical and Dental University',
Department of Immunology, Kuruime University School of Medicine*,
Japan Association for Tropical Medicine5 andResearch Institute of Medical Science, Tokai University5

clinical course of falciparum malaria, polymorphism of

Thirteen cerebral malaria patients were also examined
for their HLA‑B alleles. The frequency of HLA‑B*4601

HLA‑B, DRB1, TNF‑a, promoter region, PECAM

significantly increased in the patients compared with the(CD31) gene were analyzed in the outpatients and hospi‑
tal patients with different clinical severity of malaria.
Two hundred and 20 outpatients with positive bloodsmear of P. falciparum at Mae Sod malaria clinic, Iocat‑

outpatients at Malaria clinic with mild symptoms (Pc<

To investigate the host genetic factors affecting the0.02). TNF‑a 5'‑flanking region which is locatedwithin classIII region of HLA, was revealed to bepolymorphic in ‑238, ‑308, ‑857, ‑863, ‑1031 positions

ed at the border between Thailand and Myanmar, were
examined for their CBC, Hb, Ht, and parasitemia. At

from the initiation codon of TNF‑a. In the patientsthe same time, physical examination including palpebral
anemic change, bulbar icterus, and hepatosplenic palpi‑

857T, ‑863C, ‑1031T that had been detected to be a highthere were at least 5 Iinkage groups and one of the type‑anti‑malarial drugs. We have also collected blood
samples from 14 severe malaria patients at Mae Sod

producer of TNF‑a increased in the cerebral malaria
patients. The same kind of study was the Faculty ofTropical Medicine, Mahidol University. Although Thais
are different ethnic groups from Myanmars, the same
genotype of the TNF‑promoter increased in the cere‑

National Hospital. The nationality of the patients wasbral malaria group.

tation was done by the same physician. After the
diagnosis, all the subjects were immediately treated by

Myanmars (115 from Karen, 105 from other Burmese) .

107

5 ANTIMALARIAL NATURAL PRODUCTS FROMDICHROA FEBRIFUGA (JOH‑ZAN)

HYE SOOK KIM*, YASUHARU SHIBATA*, NAOMI IKEMOTO*, YUSUKE WATAYA ,YOSHIAKI TAKAYA' AND YOSHITERU OSHIMA'
Faculty of Pharmaceutical Sciences, Okayama University*and Faculty of Pharmaceutical Sciences, Tohoku University'

A number of medicines such as chloroquine andas active principles against malaria, and febrifugine wasquinine are vailable for treatment of malaria, but the
rapid developrment of drug resistance is a serious prob‑

this compound was never launched because of theirlem. Medicinal agents based on novel mechanisms ofaction are, therefore, required to overcome emergenceof resistance and to control an ever‑increasing numberof epidemics caused by the malaria parasite. As part ofa multidisciplinary research program on antimalarial
natural products, we are screening plant extracts thatare alleged to have antimalarial activity. The methanol
extract of the roots of Dichroa febrlfuga (Japanese

once submitted to clinical test. However, it is told that

serious adverse reactions, such as vomiting and diar‑rhea. To reexamine their activity and utility, the con‑stituents of the methanol extract of D. febrlfuga havebeen investigated in detail. As the results, powerfulantimalarial compound with higher therapeutical selec‑
tivity than febrifugine was isolated. C‑252, the composi‑

name: Joh‑zan) shows high potency against Plas ‑tion of the roots of D. febrzfuga shows high antimalarial
activity (selective toxicity: 830) between chforoojuirie‑sensitive and resistant strain of P. falciparum in' vitro .

modium falciparum . In China, the roots of D. febrzfuga

This result suggests that C‑252 could fortn the basi's of

have been employed against malaria fevers, and noa new antimalarial agent for the treatment of drug‑

parasite which is resistant to D. febrzfuga has beenreported. Febrifugine and isofebrifugine were isolated

resistant human malaria. ' ' '108

6 EXPRESSION OF CATALYTIC SUBUNITS OF MITOCHONDRIALCOMPLEX II FROM PLASMODIUM FALCIPABUM
IN ESCHERICHIA COLI
S. TAKEol, H. AMlNO1, N. SEKlNE Y SASE E ASO
M. TORI12, T. TsUB012 AND K. KITA1
Department of Biomedical Science, Faculty of Medicine, University of Tokyo*
and Departrnent of Parasitology, Ehime University School of Medicine2

The progress in malaria vaccine development isComplex 11 is generally composed of four subunits, andrapid in recent years, but the schedule for the practical

we have already cloned the genes for two catalytic
subunits (flavoprotein subunit: Fp and iron‑sulfur

use of it is still rather vague. To make matters worse,
chemotherapy of malaria using chloroquine and alterna‑
tive compounds has been followed by the appearance ofresistant parasites as shown in many epidemiologicalsubunit: Ip) from P. falciparum . Northern analysis and

RT‑PCR also demonstrated the expression of two genes
at intraerythrocytic stage parasite.

studies. The major reason for this vicious cycle is that

The physiological function of the enzyme encoded

the modes of action of many antimalarials are poorly

by these senes should be clarified, but the protocol forunderstood. This lack of knowledge would be efficiently
overcome by exploring the biology of Plasmodium in theasexual erythrocytic stage and focusing on preferential

preparing workable quantities of functional mitochon‑dria has not been established yet. So, we planned toreconstruct parasite complex 11 in E. coli. DNA Frag‑targets.

ments that encode putative mature region of each

The morphological difference and the favorable

subunit of Fp/Ip were independently ligated into expres‑effect of low 02 Ievel on the in vitro cultivation of
Plasmodium falciparum suggests that the physiologicalrole of malarial mitochondria, including that in energy
metabolism, is different from human host. The studies
so far did not support the existence of complete TCA"cycle" in Plasmodium , but suggested the functional
parasite electron transport system. For elucidating therole of this organelle, we have focused on complex II(succinate dehydrogenase, SDH) because it is exclusive‑ly mitochondrial enzyme and plays a unique role as adirect link between these two major mitochondrial sys‑
terns. And furthermore, this enzyme functions as fumar‑
ate reductase (FRD) , opposite to SDH, under anaerobic

sion vector, and recombinant protein were successfully

condition in some parasites such as adult Ascaris suum .

expressed. Antisera were raised against each recom‑

binant protein and immunofluorescence assay on
acetone‑fixed P. falciparum and P. yoelii parasitized‑
erythrocytres demonstrated the expression of lp, confir‑

ming the result of Northern analysis. Furthermore, thebinding of prosthetic group flavin adenine dinucleotide(FAD) , which is essential for enzymatical activity ofcorr plex II, to recornbinant Fp was demonstrated using
antibody against FAD. The results encouraged further
effort toward the overall goal of our study, to clarify therole of parasite mitochondria and show targeting point
for organized chemotherapy.

109

Panel presentation

1 COMPARATIVE ANALYSES OF NONSTRUCTURAL PROTElN NS5 OF
DENGUE VIRUS TYPE 1

H. ISHAKl, TSUTOMU TAKEGAM12, K. KAMIMURAI AND H. FUNADAl
Department of Biodefence Medicine, Faculty of Medicine,

Toyama Medical and Pharmaceutical University* andDivision of Tropical Medicine, Medical Research Institute,

Kanazawa Medical University'

Dengue viruses (DEN) cause serious clinical mani‑
festation. However, understanding of virus replication

rate and yields of Mochizuki strain were higher thanthose of A88. Expression of DEN‑1 nonstructural pro‑is still not enough. To clarify the biological activity oftein NS5 was compared between Mochizuki and A88

DEN nonstructural protein, we examined the differencein the expression and antigenicity between two DEN‑1

strains. By western blot analyses, Mochizuki strain NS5
was clearly detected at 3 days post infection, especiallystrains, i.e. Mochizuki and A88 (Fujita et al.. 1997) . The

in Vero cells. On the other hand, A88 NS5 protein was

Mochizuki strain has been passed through mouse brains
and subsequently through mosquito cell cultures for anumber of generations, while the A88 strain isolated
from the blood of DHF patient in Indonesia, has beencultivated in mosquito cell cultures for a number ofgenerations.
Mochizuki and A88 viruses were cultivated in Veroand C6/36 cells. Virus reproduction was confirmed by
RT‑PCR, western blot and virus titration. The growth

not detected, although viral RNAS Were detected by
RT‑PCR using DEN‑1 specific primers. The nucleotide
and amino acid sequence homology are 95% at the NS5
region. The results of western blot indicate that the
antigenicity of Mochizuki NS5 is different from that of
A88 NS5. The properties of virus replication seem to berelated with biological activity of NS5. Further ana‑lyses are necessary.

2 A STUDY ON THE APPROACH OF THE USE OF INSECTICIDEAEROSOL CANS COMBlNED WITH ACTIVE SURVEILIANCE
FOR DENGUE CONTROLKEN OSAKA1, DO QUANG HA2 AND TAKUSEI UMENA13
Infectious Disease Surveillance Center,
National Institute of Infectious Diseases

Arbovirus Unit, Pasteur Institute, Ho Chi Minh City' andDepartment of Health Policy and Planning, The University of Tokyo'

In dengue control, early case detection in sentinel
health facilities, early warning system and prompt coun‑provision of insecticide aerosol cans for neighborhoodsof dengue patients, we had conducted the implementing

termeasures of vector control have been consideredstudy from February to December, 1997 in two com‑munes (Binh Minh and Trang Bom) of southern Viet‑essential. Concerning of early detection, a number ofcommercial kits for anti‑dengue lgM and lgG antibody
detection have become available in the past few years.
On the contrary, there are few progresses in counter‑nam.
The insecticide aerosol cans were provided for theneighborhood of dengue patients in June, July, August,measures for dengue control. WHO recommended theSeptember and October in Binh Minh. The ultra low

use of household aerosol insecticides as the personal

volume fogging of insecticide (usual approach in this
district) were done around the patient's in March, May,
July, August and September in Trang Bom as a control
area. The serum samples of febrile cases collected inprotective measures. To evaluate the feasibility and theefficacy of new approach combining dengue active ser‑ological surveillance among febrile people with the

110numberofcaseTrang Bom．The overall positive rate for dengue IgM
antibody is5．6％（22／396）in Binh Minh and5．7％（43／was48．3％ofpreviousyear（89cases）in

Trang

The

rate

Binh

758）in

Trang

the

cally

numberof

local

healthfacilities

Bom．The

IgM

cases

Minh

positive

wasandAugustamong110febrile
one

month

early

zero

case

was

ybar（56cases）in

and758in

rate

Binh

Trang

and

The

in

Bom

million

and

and

posi−

（which

was

in

July

lower

Trang

positive

in

fever

patients

inWHO

criteria，the

ed

in

Binh

Minh

who

were

total

and

graded

as

third

and

sixteenpatients

were

three

patients

in

forty

forth

3

JAPANESEENCEPHAHTIS

genomic

polyproteinthat

structural

Proteins．

an

been

is

RNA

RNA

in

recombinant

virus

and

analysesNS5B

ofreported，confirming

to393US

of

cost

seven

dollars）．

ultra

Vietnamese

study
an

volumemillionsuggestedand（which

one

was

that

this

the

cost

new

contro1（Acknowledgements：Dainihon

RECOMBINANT

fiveaerosolDonwashmdredequivalentdengueprovidingininsecticide

hadCo．，Ltd．for

in

cost

Vietnamese

dollars）．That

low

Don

advantagecans

apProachku

theactiマity

combined

effectivenessin

Jochugi−cans）．

NS5PROTEIN

virus（JEV）is

still

unknown．

To

and2／30f
asisdengueThe

activ・

HAsEGAwA1，0sAMu
SAKAE
National

Department

Healthofmotifs，wereresin

protein

of

form

and

NS5protein

ing

that

proteinof

JEV

NS5protein．

CHILDREN

purified

completecontainingtheEsohε7づ6h宛601づ

using

metal

recombinant

pro−

AND

SHIN

of

Health2andInstitute

Health，Nagoya

PublicUniversity
resolution

N−terminal

RdRp

FROM

Diseases1，

Publicthe

ISOLATEDInfectious

National

notdeletedactivity，indicat−
for

RdRp

activity

from

the

earth

II

IsoMuRA3the

JEV，thepurifiedrequired

KATo2，SHIGEo

Organization（WHO）madeinisNlsHIo2，YuMIKoofandpossessed

integrity

INouYEI

Institute

expressedcolumn．The

POHOVIRUSESPAKISTANI
AYAKo

of

both

completemutantencephalitis

OF

activityNS5genehistidine−tagged

affinity

virus（HCV）have

CHARACTERIZATION

RdRp

C−terminal

RdRp

the

University

teins had a predicted molecular weight of103kDa（JEV−NS5F）and65kDa （JEV−NS5S）respectively．

Recentlyofassessputative（RdRp）with

Japanese

IGARAsHI

non・

flavivirus

INouE，

Medicine，Nagasaki

large

region．
C

AKIRA

Tropicalapredictedenzymatic
of

AND
of

NS5protein

NS5protein

hepatitis

RdRp

4

of

that

C−terminal

ities．HoweverWorld

thousand

cost

rate

from789／105to381／105．

hundredequivalent

Bom．
dramati−morbidity

one

which

and

into

polymerase

activity

MORITA

structural

NS5suggests

the

The

the

reducedaerosolthe『Our

reducedwasinsecticide

Yu，」．K．TuEI，SHINGo

Virology，Institutetranslated
into

in

l

of

Sequence

proteinmotifsisprocessed

RNA−dependent

RdRp
type

than

VIRUS

HAsEBE，Fu−XuN

KOulcHIDepartment

nonstructuralMinhin1997EXPRESSED IN ESCπERICπ14COLI EXHIBITSRNA−DEPENDENT RNA POLY．MERASE ACTIVITYFuTOSHI

Flavivirus

in

also

to553US

Bom

Mirlh，theofthousandreport−

Trang

was

eightyofdenguehaemorr−

in1997．To compare the patients number of previous
year，the mmber of cases in1997was28．6％of previous

sequence

Bom

provision

spraying

comparedthenumber

Binh

from413／105to118／105，and

in

Minh．

Whenwehagic

morbidity

patients

among137febrile口atients

rate：15．3％）though

Binh

of

Binh

patients

cases

rate：15．5％）and（21positive
tive

peak

positive

Minh（17positive
positivewere396in

Schooltooferadicate

MATsuNo1，

Medicine3
poliomyelitis

by

111

the year 2000 in 1988. Although the number of reported
polio cases has markedly decreased after the introduc‑of type I and 13 strains of type 3 showed differenttion of National Immunization Days (NlDs) in 1994,

RFLP patterns from that of Sabin I and 3, respectively.
A type 2 and a type 3 strains were identical to Sabin

polio has been one of the indigenous infectious diseases

strains. Three strains of type I did not identified by thisin children in Pakistan. We tried to characterise thepolioviruses isolated from poliomyelitis and diarrheal

method. We conducted to determine the nucleotide

patients, 1995‑1996.

sequences of amplified genomic fragments of the wild
poliovirus isolates. Twelve isolates of type I were

Eighty‑one stool specimens collected from patientsclassified into 3 distinct genotypes, and 12 type 3 isolates

were inoculated into La cells (devided from Prof.

were divided into 2 genotypes.
In Pakistan, hundreds of polio cases were reported

Nomoto, Institute of Medical Science, Univ. of Tokyo) .

Isolated polioviruses were identified using antisera topolioviruses and intratypically differentiated by thein 1995 and 1996. Our results show that almost ofdigested with restriction enzymes, Ddel, Hpall or

polioviruses isolated from polio cases were wild types
and one polio case was associated with vaccine strain.
Three or more genotypes of type I and 3 wild poliovir‑

HaeIII. The phylogenetic similarity of these sequencesuses were circulating in this area. For the eradication of

PCR‑RFLP method; amplified PCR products were

was compared using the UPGMA software packages.
Thirty‑three poliovirus strains were isolated fromwild poliovirus infection it is necessary to increase

21 poliomyelitis and 11 diarrheal patients; 18 for type 1,

vaccine coverage in all areas of Pakistan and to identify
infectious agents of poli0‑1ike and acute flaccid paraly‑

1 for type 2, and 14 for type 3. A mixture of type I andsis patients.

2 was isolated from a diarrheal patient. Fifteen strains

5 THE RISK OF HEPATITIS VIRUS INFECTION AMONG
JAPANESE STAYlNG IN DEVELOPlNG COUNTRIESATSUO HAMADAl, E. OKUZAWAl, Y. HIROSHIGE1, Y. KAWABUCHll,
H. NAKAJIMA2 AND T. NISHIKAWAl
Japan Overseas Health Administration Center. Labor Welfare Corporation and
Departrnent of Internal Medicine, Jikei University School of Medicine2

Five cases (6.8%) seroconverted to anti‑HAV and

Hepatitis virus infection is common in Japanesestaying in developing countries. We investigated theone cases (0.9%) seroconverted to anti‑HEV. The

risk of the infection comparing antibodies to the viruses

places of their stay were Asia (4) and Africa (1) in anti‑

in Japanese serum before and after the stay.
Sera of 109 Japanese (Median age: 38.8 y.o., Aver‑age length of stay: 17.2 months) were obtained before
and after the stay in developing countries (Asia: 79,
Middle East: 5, Africa: 8, Latin America: 17). Anti‑bodies to hepatitis A virus (total anti‑HAV) , to B core
antigen (total anti‑HBc) , to C virus (total anti‑HCV)

and to E virus (IgG anti‑HEV) were examined by EIA.
Japanese who had the antibody before the stay or had
vaccinations were excluded from the subjects of eachvirus (No. of subtracted subjects; HAV: 74, HBc: 79,
HCV: 109, HEV: 109).

HAV cases, and Asia in anti‑HEV case. These cases
did not have a history of hepatitis during the stay in the

countries, and the liver functions were normal after thestay. Seroconversion to anti‑HBc or anti‑HCV was notdetected.

Among hepatitis viruses, the risk of HAV and HEV
infections, food and water‑borne infections, are relative‑

ly high compared to HBV and HCV infections which aretransmitted by parenteral exposure. Therefore, preven‑tive measures such as the health education and vaccina‑tion should be provided for Japanese staying in develop‑
ing countries with priority in HAV and HEV infections.

112

6

PREVALENCE

OF
A

SATosHI

HUMAN

YOUNG

DepartmentofofDermatology，Iwate

Medical

University1，

TropicalMedicine，Liverpool

SchoolTropical

DepartmentofMedicalThe

Microbiology

University

NsambyaWe

studiedsthesarcoma

POPULATION

MAYAMA1，TosHIHIDE AKAsAKA1，Luls CuEvAs2，DAvID
THoMAs ScHuLTz3AND Plous OKoNG4Division

KaposiHERPESVIRUS8（HHV8）INUGANDANseroprevalenceandassociatedherpesvirustransmission

and

of

of

Genitourinary

Medicine2，

Medicine，Liverpool3and

Hospital，Uganda4duringof（KSHv／

early50％）before

childhoodandreachedtheofpuberty．KSHV／HHV8wasage

HHV8），among215Ugandan children，adolescents andyoung adults．We measured antibodies to a latent

holdsize，previous

nuclearantigen（LANA）andencod−

girlfriendsoredorf65．

KSHV／HHV80ccured

among

Ugandanchildren

by

a

lytic

Infection

with

7

cycle

protein

COMPARISON
OF

OF

THE

Research

the

hagic

and

hemolytic

colitispredominant

Verotoxinsarethought

developmentof

hemorrhagicofsystemic

to

be

VTEC

erythrocytes（hemolysis）．In
theactionsofing

cytotoxin，investigatedstrains

syndrome

and

with

have

this

the

the

from

AND

TATSuO
andIntemational

VTEC

A

additionof160human

of

boy／

of

KSHV

AND

Soy

transformationoncontainsanpore−form・

1y＋），aclinicalofhuman，swine

and

EntHlyincattle，fromthe

601歪HB101is

Human

EntHlyaously（Bettelheim，1995）．

Bacteria

described

wereprevi−

grown

on

CaCl2

and

derivedplasmidafrom

F60（0128，EntH−

human

VTEC

human

isolated

designed

pF60：Tn1．This

plasmid
from

5〆一

was

determinant

were

infection．E

isolate（EntHly一）．

fromperipheral

blood．

Bacteria（VTEC，EntHly＋HB101，HB101strains）from
ovemight

FBS

cultureswere

inoculatedbrought

intolog−phasegrowthfor5hr，supematants

were

harvestedandmediumcultureand

intofiltered．Approximately105monocytes
the

αwere

supematants

assayed

in

ELISA

VTEC

culturestrains

cattle（65％）were

isolated

varied

RPMI1640／10％

wereincubatedtimes．IL−1βandsupematantsbysterile

with

the

TNF
use

kit．

（1）Most
and

for

cell

plasmid−encoded．Inaswith10mM

gene．EntHly＋HBlOI

isolateHemolysin

performed

supplement

nonpathogenic

Japan in 1996were investigated for production of
EntHly． Twenty−three cattle and ll swine VTEC
strains were obtained from our culture collection．
were

base

with

of

assaysJapan

EntHlymonocytes

withisolatedof

TCTCGCCTGATAGTGTTTGGTA−3〆werebyfocusedLEVELS一GGTGCAGCAGAAAAAGTTGTAG−3

lyse

to

pattem．

SWINE

Agar

amplifyability

horizontal

Medicine，Centerto

study，westrainstransfusions，number

stasus．Transmissiontofrequencies

VTEC

blood

maritala

supply，house−

and5％defibrinated sheep blood．PCR for detection of
was performed for some VTEC strains．Primers

5

infections．

total

Tryptic

both

the

not

qualityfollows

water

（40−

YAMAMOTO

Medical

EntHlyoccurrence

thelevelsthe

INDUCTION

Tropicalhumans．

and compared the初∂伽o cytokine responses inducedby both EntHly＋and EntHly−strains．We fomd that amarked increase in IL−1β production induced by
EntHly−producing strains，and suggested that EntHly
may play an important role in pathogenic mechanisms
of

E

of

adult

ES（】πER1（辺1400Ll

Diseasehemorr−intheenterohemolysin（EntHly），a

isolatedofHowever，instrains

ZHANG

Infectious

of

with

HUMAN，CATTLE

Esoh6万6h毎ool1

cause

associatedcolitiscomplications．

Verotoxins，mostVTECuremicofInstituteVero（Shiga−1ike）toxin−producingare

CYTOKIN

FROM

Hul−MIN
Department（VTEC）strains

associated

VEROTOXIN−PRODUCING

lSOI。ATEDSMITH2，

from

swine

isolated

EntHlycontrast，all

were

fromhuman（98％）

positive，the

EntHlyofthe

EntHlywas

VTECstrains

negative．

（2）An

113

increased IL‑1p and TNFcl! Ievels were determinedfrom monocytes stimulated by EntHly+ VTEC culture

In conclusion, IL‑1p, the cytokine which enhances
the cytotoxic potency of Verotoxin toward hurnan en‑

supernatants. (3) EntHly+ recombinant strain (EntHly+
HBIO1) remarkably induced the IL‑1p productions. In
contrast, HBIOI strain did not.

strains. EntHly may contribute to the development of
VTEC infections.

dothelial cells, was induced by EntHly‑producing

8 THE ISOLATION FREQUENCY OF DIARRHEAGENlC BACTERIAIN VIENTIANE, LAOSMASAAKI IWANAGA1, NoBORU NAKASONEl, NAOMI HIGA1,TETSU YAMASHIRol AND SITHAT INSISIENGMAY2
Department of Bacteriology, Faculty of Medicine, University of the Ryukyus
and National Institute of Hygiene and Epidemiology, Vientiane,

People's Democratic Republic of Laos'

The etiological agents of diarrhea in Vientiane,
Laos, were studied in 1996 and 1997. A total of 880
diarrheal cases visiting medical facilities were examined
for Shigella. Salmonella, Escherichia coli. Vibrio, Aer ‑645) , 11% (97/880) , respectively.

dmonella. Cwa ylobacterand rotavirus were of relatively low frequency with O.
6% (5/880), 4.4% (39/880), and 6.1% (9/148), respec‑tively. Aeromonas and V. cholerae were not found.
Shigella strains isolated comprized 82 S. flexneri. 60 S.
sonnei. 4 S. boydii, and I S. dysenteriae (not type 1).

omonas. Campylobacter and rotavirus. Shigella. enter‑otoxigenic E. coli and enteropathogenic E. coli were
found to be main causative organisms. The isolation

Almost all Shigella isolates were highly resistant tofrequency of Shigella. enterotoxigenic E. coli and enter‑tetracycline, about 50% to ampicillin.

opathogenic E. coli was 16.8% (148/880), 17.2% (111/9 THROMBOMODULlN LEVELS IN PATIENTS WITH TYPHOID FEVER
KENJI OHNISHI AND KYOKO KIMURA
Department of Infectious Dineases,

Tokyo Metropolitan Bokutoh General Hospital

Serum thrombomodulin (TM) and creatinine levels
were determined in 7 male Japanese febrile patientswith typhoid fever and in 6 male Japanese healthy
controls. The serum TM values of the patients andcontrols were 5.04dll.69 FU/ml and 2.93i0.74 FU/ml,
respectively, and a significant difference was identified

between them (P<0.025). The serum creatinine levelsof the patients and controls were 0.93 0.16 mg/dl and
0.77 0.10 mg/dl, respectively, and the fact that no

significant difference was identified between them may

indicate that delayed clearance from kidney was notpredominant as explanations for the elevated serum
levels of TM. Although the numbers of patients andcontrols were small and more studies are waited, we
found elevated serum TM Ievels during the course of a
febrile S. serovar Typhi infection and serum TM Ievels
may reflect the disease acitivity of typhoid fever.

114

10 IDENTIFICATION OF THE RECEPTOR FOR

AEROMONAS SOBRIA HEMOLYSlN

AKIHIRO WADA1, AlplNG WANG2, SHIGERU KoHN02, TOMOHIKO NoMURA3,YOSHIO FUJI14, KEINOSUKE OKAMOT03 AND TOSHIYA HIRAYAMAl
Department of Bacterrology, Institute of Tropical Medicine,

Nagasaki University*,

Second Department of Internal Medicine,
Nagasaki University School of Medicine',
Department of Biochemistry3 and Institute of Pharmacology*,

Faculty of Pharmaceutical Sciences, Tokushima Bunri University

The motile Aeromonas species, particularly A.
ogens associated with acute gastroenteritis in both
adults and children. Aerolysin secreted by the human

There have been few studies on the toxins produced
by A. sobria, and the biological properties of thesetoxins still remain unclear. A. sobria hemolysin ishighly similar (68% at the amino acid leveD to aer‑pathogen A. hydrophila is one of the important virulence

olysin. These toxins may have the same functional

factor and is well characterized. Aerolysin is secreted
as a dimeric inactive precursor which can be activatedhemolysin is responsible for the diarrhea of A. sobria.

hydrophila and A. sobria, have been recognized as path‑by proteolytic clevage of a C‑terminal peptide. Bothaerolysin and its protoxin interact with the target cells

activities. Recently, Fujii et al. demonstrated that

because isogenic mutant strain lacking the hemolysin
gene did 'not cause diarrhea. Accordingly, it is veryby binding to specific receptors. To date, all identified

important to clarify the receptor molecule for A. sobria

receptors for aerolysin were found to be GPI anchored.
However, different receptors were found in different
cell types and a given cell type was found to have morethan one receptor. For example, aerolysin was shown tobind to Thy‑1 on T‑1ymphocytes, to 47 kDa receptor on

hemolysin on the intestinal cells to understand the signal

pathways that leads to diarrhea at the molecular level.

Using immunoprecipitation method with hemolysin and

rat erythrocytes, and to 80 kDa receptor on baby ham‑its antibody, here we report that a cell surface glyco‑
protein (p66) with a molecular mass of 66 kDa can bindto hemolysin in intestine 407 cells, the human intestinalster kidney cells.

cell line.

11 THE TREATMENT OF LEPROSY; CURRENT SITUATION

AND PROBLEMSMASAKO NAMISATO1, K. HIKITA2, M. MAEDAI AND H. OGAWA3'National Hospital Tama‑Zenshoen ,Bureau of International Medical Center of Japan' andDepartment of Dermatology, Juntendo University School of Medicine*

Leprosy had been proved to be a really curable
disease after WHO Study Group on Chemotherapy of
Leprosy recommended MDT (multidrug therapy) as thetion of MDT for MB cases should be shortened to 12

most effective treatment in 1982. In 1992, the Member
States of WHO declared their intention to eliminate
leprosy as a public health problem by the year 2000.
Elimination was defined as the reduction of prevalence
to less than one case per 10,000 population. At thefor the cases of single patch. Although these proposals

beginning of 1998, there were still 32 countries where theprevalence was beyond the global target. In 1997, WHOExpert Committee on Leprosy proposed that the dura‑

months (from 24 months) and single dose of ROM(rifampicin+0floxacin+minocycline) can be indicatedwere approved at the 15th International Leprosy Con‑
gress in September 1998, the duration of treatment ofleprosy has been controversial subject.

Through the comparison of the situation of leprosy
in 2 countries that we studied, one is Morocco as therepresentative of low endemic country and the other is
Myanmar as a well known endemic country, some of the115

curr,ent global problems for leprosy control became

tem is one of the ideals but this regimen might have notclear. We also studied some of our cases retrospectively

been afforded without great support of NGO. In Myan‑mar, there seems to be considerable amount of backlog

to consider about the appropriate duration of chemo‑
thera py.

(hidden) cases that are the suspicious sources of infec‑

In Morocco, great reduction of prevalence of lep‑rosy has been done during these 15 years, having appro‑tion.

priate regimen and enough support of NGO. In Myan‑

Since the MDT of 24 doses cannot be considered to
be enough for LL cases from our experiences, we aremar, although the prevalence decreased rapidly after theconcerning about possible relapse cases that may

implementation of MDT, there was no decrease of newcases. From our experiences, LL case with high Bacte‑emerge more often after the shortening of MB regimen

rial Index showed late reversal reactions even after 10

Leprosy, as a super chronic disease, needs long
follow up with careful observation. Only after enoughduration of follow‑up, we can judge the efficiency ofmonths from the completion of MDT.
For the success of Leprosy Control, enough durationof treatment and follow‑up is necessary along withis introduced.

current regimen.

effective chemotherapy. In this aspect, Moroccan sys‑

12 ROLE OF HUMAN POLYMORPHONUCLEAR NEUTROPHILSIN HOST DEFENCE AGAlNST INFECTION
WITH PENICILLIUM MA RNEFFEI
N. KUDEKEN, K. KAWAKAMI AND ATSUSHI SAITOFirst Department of Internal Medicine, Faculty of Medicine,

University of the Ryukyus

Penicillium marneffei is an important opportunistic

fungal pathogen. Host defense mechanisms against P.
marneffei are not fully understood. We investigated thefungicidal activity of human polymorphonuclear leuko‑
cytes (PMN) against P. marneffei. The yeast cells were
cocultured in vitro with peripheral blood‑derived PMN
for 24 hr. Microscopic examination was also performed
to examine the germination of yeast cells and theirkin (IL) ‑8, interferon (IFN)‑y and tumor necrosis
factor (TNF) ‑a on their activity. Among these cyto‑kines, GM‑CSF, G‑CSF and IFN‑y induced PMN fun‑gicidal activity, but other ones did not or showed amarginal effect. In contrast, all these cytokines enhan‑

ced the inhibitory activity of PMN against the mor‑
phological changes of fungus. These antifungal activ‑
ities were most strongly induced by treatment with GM‑

transformation to hyphal from during culture. Un‑

CSF. The combined use of any of the above cytokines

stimulated PMN inhibited the growth of fungus only at
phological change even at a lower E/T ratio. In further

failed to synergistically enhance antifungal PMN activ‑
ity. Our results demonstrated that cytokine‑activated
PMN exert a significant antifungal activity by suppress‑

experiments, we examined the effects of various PMN‑
activating cytokines including granulocyte‑macrophagecolony stimulating factor (GM‑CSF) , G‑CSF, interleu‑

results suggest that PMN may contribute to host resis‑
tance to infection against this fungal pathogen.

a higher effector/target (E/T) ratio and the mor‑

ing the growth and germination of P. marneffei. Our

116

13 THREE CASE REPORT OF INFECTION DUE TO PENICILLIUM
MARNEFFEI WITH AIDS PATIENTS IN NORTHERN THAILAND
HIROSHI WATANABE*, SHlNOBU KOBAYASHI , KIWAO WATANABE , KAZUNORI OISH11,
NORIHUMI AsoU2, KEIZO MATSUMOT02, TAKESHI YAMARYOU3, TlpAYA SANCYAI+,KHEMRASSAMEE KUNSUIKMENGRAI+, SUMPUN KAHlNTAPONG+,
PRASIT THARAVICHITKUL5, THIRA SIRISANTHANA6 AND TSUYOSHI NAGATAKE1
Department of Internal Medicine, Institute of Tropical Medicine,

Nagasaki University ,Aino Kinen Hospital', Iki Public Hospital', Nakorn Ping Hospital* andDepartment of Microbiology, Faculty of Medicine= andDepartment of Medicine', Chiang Mai University

We had a co‑operative study about "Treatment of
Acute Respiratory Infection in Thailand" from April,
1994 to March, 1997. And we are studying about "Treat‑ment of Respiratory Infections with AIDS in Thailand".
Disseminated infection with the fungus Penicillium

marneffei is one of the most common opportunistic
infections in AIDS patients in northern Thailand. We

He was treated with penicillin G and gentamicin, but hedied 2 days after admission. P. marneffei was detectedfrom sputum in the day of his death. Second case was
24 year‑old male with similar symptom. He was treatedwith antibiotics and amphotericin B, and his clinicalcourse was good. P. marneffei was detected from blood

report 3 cases of infection due to P. marneffei with AIDS

and skin on admission. Third case was 31 year‑old
female with similar symptom. She was treated with 'patients who were adrnitted to Nakorn Ping Hospital in
Chaing Mai in order to treat community acquired pneu‑
monia and review the literature. First case was 33 year‑

antibiotics and antifungal therapy, and her clinicalcourse was good. P. marneffei was detected from bloodon admission.

old male who complained of fever, cough and sputum.

14 STUDY ON THE SALIVARY GLAND PROTElNS OF MALARIAVECTOR MOSQUITO, ANOPHELES STEPHENSIEN JIE LUO, HIROYUKI MATSUOKA, SHIGETO YOSHIDA, KUNI IWAI,

MEIJI ARA11 AND AKIRA ISHII
Department of Medical Zoology, Jichi Medical School

During blood feeding, Anopheles stephensi extreme‑

into the midgut. Moreover, on feeding of An. stephensi

ly reduces the volume of the salivary glands. To

whose molecular size were 33, 35, 45 and 70 kDa, deplet‑

infected with Plasmodium berghei, in prediuresis we
found sporozoites which might come from the salivary
gland of the mosquito and furthermore we detected thesalivary protein, which molecular size were 90 kDa,
from the midgut content of the mosquitoes after fullblood meal. These results indicate that mosquitoes

ed after the blood feeding. Proteins in the salivary

secret their saliva into the host and then take their own

glands were consumed when mosquitoes were probing

saliva into the midgut together with blood.

observe the saliva consumption, we collected and dis‑sected the salivary glands of An. stephensi before feed‑
ing, during probing, and after fully blood feeding. By the

SDS‑PAGE and Western blotting, several proteins,

on mouse, and even when mosquitoes were taking blood

117

15 A FUNCTIONAL SlNGLE‑CHAlN FV DIRECTED TO A R,ODENTMALARIA PARASITE SPECIFICALLY INHIBITSTHE DEVELOPMENT OF OOCYSTS IN MOSQUITOESSHIGETO YOSHIDA , HIROYUKI MATSUOKA1, E. LUO1, KUNl IWA11,
MEIJI ARA11, R.E. SlNDEN' AND AKIRA ISHII
Department of Medical Zoology, Jichi Medical School* andInfection and Immunity Section, Department of Biology,
Imperial College of Science, Technology and Medicine, London, UK.

Pbs21 is a membrane protein identified on the

blood from the mice. After 14 days, the mosquito

macrogamete, zygote, ookinete and oocyst stages of the

midguts were dissected and the number of oocysts werecounted.
Large amounts of the 13.1 SCFV Were produced in
insect cells infected with the recombinant baculovirus.

rodent malaria parasite Plasmodium berghei. A mono‑clonal antibody (mAb), called 13.1, directed to Pbs21
has been reported to inhibit the development of oocysts
in Anopheles stephensi mosquitoes.

Following purification on a Ni affinity column, a single

1) To clone and express a single‑chain antibody
fragment (scFv) from 13.1 hybridoma cells and analyze
its functional activities in vitro. 2) To determine
whether the molecularly engineered SCFV can block theprotein was eluted from the column, as determined bymalaria transrnission in vivo (mouse model) .
The V regions of heavy and light chains were cloned

oocyst development in the mosquito midguts wasand linked by a sequence encoding for (Gly+Ser,) andSDS‑PAGE. Immunoblotting and inhibition ELISA
revealed the 13.1 SCFV exhibit similar binding properties
as the parent 13.1 mAb. Finally, significant inhibition ofachieved with inoculation of 13.1 scFv.
Our data show that the 13.1 SCFV produced by the in

expressed by a recombinant baculovirus. Following

vitro baculovirus expression system fully retains the

purification, the SCFV Was analyzed the binding activity

antigen binding properties and the functional properties

by immunoblotting, inhibition ELISA and IFAT.
Furthermore, the SCFV Was injected into miceof the parent 13.1 mAb. In addition, this functionallyintravenously and mosquitoes were allowed to feedrefractory mosquito against malaria parasites.

16

active SCFV may be used as a model for generating a

APPLICATION TO RESEARCHES AND DIAGNOSES OF

A NEWLY DEVELOPED MALARIA PARASITE DETECTIONMETHOD BY FLOW CYTOMETRYATSUKO SAIT0‑ITol, YASUMASA AKA13, S. HE1, MIKIO KIMURA4KAZUYUKI TANABE5, MASATO KAWABATA2 AND TAKEO MATSUMURAl
Department of Medical Zoology

and International Center for

Medical Research2, Kobe University School of Medicine,Toa Medical Electronics Co. LTD. Research Division3

Department of Infectious Diseases and Applied Immunology,
Institute of Medical Science, The University of Toky0+ andLaboratory of Biology, Osaka Institute of Technology=

Lack of effective vaccine and wide spread of drug

plenty of blood samples for diagnosis purposing malaria

resistance has still made malaria a serious public healthcontrol in the field. The conventional microscopic
examination as golden standard to detect and countconcern. Basic studies aiming at development of vac‑cines and drugs will be more energetically pursued andin these studies in vitro and in vivo it will be required toanalyse a lot of sample at the same time, detecting andcounting parasites. It is also necessary to examine amalaria parasites is a time consuming and rather subjec‑

tive method depending on the technical ability of exam‑
iners. In this situation, we have developed a simple,
sensitive and objective flow cytometric method to detect

118

and count parasites freed by a newly designed lysing
solution and stained with acridine orange in cultured or

falciparum or P. vivax infected) patient blood samples
were also analysed by this method. Although parasites

patient blood samples. The parasite number analysed

could be detected in all of the patient samples examined,by this method correlated closely with parasitemia overthe sensitivity in clinical blood samples was lower than

0.005% in cultured samples. Stage‑specific parasites
could be analysed differentially from one another. The
effects of antimalarial drugs on parasite growth were
examined by three methods (microscopic examinationof thin smears stained by Giemsa, [H3] uptake study,

that in cultured samples. We have attempted the deviceof the preparation of samples and the improvement of

this method. We have also tried to apply flow

and this flow cytometric method) and very similar dose‑

cytometry with imaging divice, which may be enable toresponse curves were obtained. Seven malaria (P.

differentiate four species of Plasmodium in this method.

lysing solution to differentiate parasites from platelets
to get satisfactory sensitivity for clinical application of

17 INFLUENCE OF SCHISTOSOMA MANNONI INFECTION ON
SUSCEPTIBILITY TO PLASMODIUM CHABAUDI IN MICEAYAKO YOSHIDA , HARUHIKO MARUYAMA , TERUAKI AMAN02,
KAZUYUKI TANABE3 AND NOBUO OHTA1
Department of Medical Zoology, Nagoya City University Medical School*
Department of Parasitology, School of Medicine Yokohama City University'and Laboratory of Biology, Osaka Institute of Technology*

In mice and humans infected with Schistosoma

susceptible A/J mice, S. mansoni infection resulted inmansoni, the polarization of helper T cell responses arehave demonstrated that S. mansoni infection changed

significantly decreased mortality, and parasitemias
were reduced to subpatient levels 22 days after P.
chabaudi infection. In vitro IL‑10 and IFN‑y produc‑

not only immune responses but also the course of infec‑

tion for P. chabaudi antigen in spleen cells were signifi‑tion for other pathogens. Considering the fact thatcantly increased by S. mansoni infection, however, S.

induced to a parasite specific antigen. Several studies

people in S. mansoni endemic areas often have multiple

mansoni infection did not affect the level of NO

parasitic infections simultaneously, S. mansoni infection

might affect the onset and development of other para‑

metabolite, NO . In addition, serum and spleen cells
from S. mansoni infected mice did not transfer thesite diseases. In the present study, we infected mice withresistance against P. chabaudi.

S. mansoni, and challenged it with Plasmodium chabaudi
thereaf ter.

Our findings suggest that S. mansoni infection

Mice were infected intraperitoneally (i.p.) with 106

provided susceptible mice with the resistance against P.
chabaudi and influenced the cytokine production induced

P. chabaudi AS‑parasitized erythrocytes (PRBO.

by P. chabaudi. Further analysis about the mechanism

Eight weeks prior to the P. chabaudi infection, somemice were infected with 25 cercariae of S. mansoni. In

and effector of this resistance is in progress.

119

18

DETECTlON OF SUBPATENT LEVEL，OF MALARIA PARASITES BYNESTED POLYMERASE CHAIN REACTION（PCR）ANDITS CORRELATION W皿H MICROSCOPY IN MYANMAR PATIENTS
HLA

MYAT

MON1，HARuKI

UEMuRA1，MARLAR
AND

Department

of

HIRoJI

Protozoology，InstituteNagasaki

Clinical

THAN2，MAsATsuGu

Malaria

Research

KIMuRA3

KANBARAlof

TropicalMedicine，

University1，

Unit，Defence

Services

GeneralHospital，Yangon，Myanmar2andLaboratoryofBiophysics，Osaka

City

an

ogy

altemative

approachfordetection

study，weappliedamplification

of

used

of

malariaanestedsmallaNA）gene，tocompare

its

among

Myanmardetectsubpatent

level

PCR

methodofpatients

male

admittedtoServices

GeneralHospitalrandomly

selected

for

aged

Malariacollection

entmalaria

endemic

initialMicroscopy

and

PCR

methods

of

P乙

18

negativeofand

z7z6窺，

Pヵ1吻α7％郷and
4

negative

blood

smear

was

of96

tive

for

more

than5days．This

showedthatnestedusefulindiffer−Myanmarandofsubpatent

for

level

parasitaemia

ofcases，however，we

DNA

even

within4days

will

be

discussed．

CONTROL

TRIAL

Chloroquine
mass

of

treatmenthasinepidemiological

been

field

data

and

CenterofIslandfor

Medicine

Medical

Medicalformalariastudy．Howevertheanalysesmathematicalmodel

analyses that primaquine，which can effectively killmalaria gametocytes，should be involved to control
malaria prevalence． However it is well known thathemolysis might occur whenprimaquine administeredto
patients who lack glucose6−phosphate dehydrogenase（G6PD）．

Therefore

G6PD

deficiency

testshouldMedical
Kobe

of

the

blood

smear

didnotPCR

presence

nega−

detectthetreatment．Individ−

be

W皿H

CenterofJapan1，

Research，

University2，

Training

andZoology，Jichi

usedfurthernega−

IsHII4Intemationalcommonly

the

AKIRA

Cooperation，Internationalofofparasite

KAwABATA2，A．BoBoGARE3，

Intemational

Department

in

some

cases

MALARIAJ．LEAFAsIA3AND

Facultydetection

the

where

NAGAI1，MAsATo

Solomondetectsensitive

cases．IntheP

P伽砿mixed

in

is

of

cases，respectively．

could

DIAGNOSIS KIT（ICF−PF）AND SINGLE STEP G6PD
DEFICIENCY TEST IN THE SOLOMON ISLANDS

of

cases

cases，we

19CHEMOTHERAPEUTIC

Bureau2

parasitaemia

ual

NoBuHIKO

andthesamplestorevealed74and82
1

after

parasiteinandz乖zl6彦）

DNA

of

areas

zo4づ％

Among

tive

from

zs

to

Unit，Defence
Yangon，Myanmar werecame

relatedroutine

total

blood

symptoms

andofbetween18and48years

in

the

infection

Research

May1998．Patients

of

that

patientshave

吻砿，3and8casestheRNA（SSUrR−

parasitaemia．A

Jamary1997and
parts

to

malariaontostartedcases

present

based

Schoo13

malaria．

methodol−theribosomal

sensitivity

MyanmarClinicalsensitive

infection。In

subunitmicroscopy
theasMedicalthey

Microscopy has been an essential method for the
diagnosis of malaria．Polymerase chain reaction（PCR）
is

University

Research3andMedicalSchool4

incorporated before primaquine treatment．
In this study we tried to diagnose falciparum
malaria with commercial kit（ICT−Pf）and newly deve1−
oped

singlemass

treatment

step

screening

with

G6PD

primaquine

deficiency

as

well

as

test

beforechloroquineand Fansidar．We conducted the survey in malaria
endemic area in Solomon Islands in January1998．The
study

subjectsage

was15．2years

were39210cal

inhabitants

old．Among

and

their

them293weremeanexamined

120

by both ICT‑Pf kit and microscopic examinations with

Giemsa staining. Compared with AO test, the sensitivity

Acridine Orange (AO) and Giemsa staining. The

and specificity of ICT‑Pf kit were 0.85 and the 0.84,
respectively. Anyone can easily diagnose Pf in a few
minutes by ICT‑Pf kit without any training. ICT‑Pf kit
is suitable for Pf diagnosis especially in the field.
Furthermore we could detect the deficiency within 40

remaining 99 subjects were diagnosed only by micro‑
scopic examinations. Primaquine was not given in thecase of G6PD deficiency.
ICT‑Pf showed that 83 (28.3%) of 293 subjects werepositive for Plasmodium falciparum (Pf) . G6PD defi‑
ciency test showed that 29 (7.4%) of 392 subjects were
G6PD deficient. The sensitivity and specificity of ICT‑
Pf kit were 0.85 and 0.82, respectively compared with20

min by the newly developed G6PD deficiency test.
Therefore we could rapidly treat malaria patients on theexamination site with chloroquine and primaquine after
the combination of these two rapid diagnosis methods.

CHANGE IN MALARIA VECTORS AND MALARIA SITUATIONIN NORTHERN THAILAND DURING 30 YEARS FROM 1968

WANNAPA SUWONKERD1, YOSHIO TSUDA2, SOMSAK PRAJAKWONGI AND MASAHIRO TAKAG12
Office of Vector Bone Diseases Control 2, Ministry of Health, Thai Govermentl andDepartment of Medical Entomology, Institute of Tropical Medicine,

Nagasaki University'

To evaluate a long‑term change in ma'laria and its
vector situation, and to avoid missing and scatteringvaluable information in malaria epidemiological surveys

and studies carried out by Office of Vector Borne
Diseases Control 2 (previous Malaria Center 2) withcollaborators in the past time, reformatting, computing
and analysis of all vailable data of malaria concernedin northern Thailand have started. Firstly all readable
stocked data at provincial, district and county malaria
offices were photocopied, examined qualities, and decid‑
ed the format for the original electric database, which

was composed of 5 items (general back ground, vector

control measurement, environmental conditions,malaria incidence and vector abundance) of 34 parame‑
ters. It was found that a total of 4,061 surveys had been

completed for 30 years since 1968 in 13 provinces withmore than 400 surveys in 7 provinces. Problems foranalysis are: that about 20% of the data lacks key
parameters such as the number of human baits and soon, that data before 1968 are too weak to analyze, andthat the surveys had mainly been focused to problem
areas, period, and recent 10 years. Nevertheless, arough analysis based on 436 data sets of Maehongson

Province, where malaria has been being serious,
revealed clear temporal changes in vector and malaria
situation suggesting recent natural and social environ‑mental changes in the province. We believe the comple‑
tion of this database and analysis is helpful to yield
more reasonable and harmonious malaria control strat‑
egy under unstable environment with radical developing.

21 CLlNICAL STUDY ON FIFTEEN CASE OF IMPORTED MALARIA
ATSUSHI NAKAMOTO, TAKASHI SHlNZATO, KAZUMASA TOYODA, MASAO TATEYAMA,
K. KOIDE, KAZUYOSHI KAWAKAMI, NoBUCHIKA KUSANO AND ATSUSHI SAITOFirst Department of Internal Medicine, Faculty of Medicine,

University of the Ryukyus

Ten imported malaria patients admitted to our
hospital and 5 to other hospital in Okinawa within lastten years were studied. These cases comprised 8(53.3%) Plasmodium vivax (Pv), 6 (40.0%) P. falcipar‑
um (Pf) and one unidentified case. Eighty percent ofthese were in their 20s and 30s. Ten (66.7%) wereJapanese and 5 (33.3%) were non‑Japanese. Four of 8
Pv cases were contracted in Southeast Asia, 2 were in
India and 2 were in Africa. Chroroquine or fansidar wasused in the acute phase treatment of most Pv cases, and121then primaquine was used for radical cure. One Pv malecase contracted in Indonesia was relatively resistant toconventional radical therapy with primaquine (15 mg
daily for 14 days) . He received mefloquine and radicallycontracted in Philippine developed severe cerebral
malaria with renal failure and DIC. He was treated withintravenous qunine, mefroquine and fansidar, and need‑ed hemodialysis and plasma exchange. In these manage‑

cured by 2 cycles of primaquine 15 mg daily for 14 days.

ment, he had a remarkable irnprovement within aThree of 5 Pf cases were contracted in Africa and 2
were in Southeast Asia. Mefroquine or fansidar wasused in most cases of Pf. In one case of chloroquinemonth.

resistant Pf, fansidar w, as effective. One Pf male case22

A11 15 patients had a good improvement finally inthis study, but drug resistance is becoming a problem
also in imported case of malaria.

NlNE CASES OF IMPORTED MALARIA IN KYUSHU;
CLlNICAL ANALYSIS OF DURING LAST ONE YEARTOMOO UKON, KAZUSHI MOTOMURA, KAZUNORI OISHI, TOMOKO ONIZUKA,HIROSHI WATANABE, HIRONORI MASAKI, SHlNOBU KOBAYASHI AND TSUYOSHI NAGATAKE
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University

We experienced nine cases of imported malaria

Because sulfadoxine pyrimethamine was not effective

during last one year in the Kyushu district and contribut‑

enough for one tertian case and one falciparum case, theed to treatment of these cases. Of these, seven wereconsulted for drug supply, and two were our cases.
These cases involved six tertian, two falciparumand one ovale malaria. Among consulted cases, sul‑
fadoxine pyrimethamine was used for four cases, doxy‑cycline for one, doxycycline and sulfadoxine pyrimeth‑amine for one case, doxycycline and quinine for one
case. Both of our cases were treated with mefloquine.

treatment was altered to quinine and minocycline,
mefloquine, respectively. We supplied primaquine to six
cases of tertian and ovale as well as mefloquine to one

falciparum case. Orphan antimalarial drugs were notchosen for initial treatment for all consulted cases.
There seemes to be an urgent need for immediate supplyof these antimalarial drugs.

23 CYSTElN PROTElNASE GENE AS A BASIS FOR

DIFFERENTIATlNG ENTAMOEBA HISTLYTICA AND
ENTAMOEBA DISPAR BY PCR
WlNDELL L. RIVERAl, RONALD R. MATIAS2, GLORIA L. ENRIQUEZ3AND HIROJI KANBARA1
Department of Protozoologyl and Department of Molecular Epidemiology2,
Institute of Tropical Medicine, Nagasaki Unive. rsityl andDepartment of Molecular Epidemiology, College of Science,
University of the Philippines'Th

enteric protozoan parasite, Entamoeba his‑

E. histolytica.

tolytica is the causative agent of human amebiasis. It

A WHO‑Pan American Health Organization‑

has long been known that although about 500 million

United Nations Educational, Scientific, and Cultural
Organization Expert Panel recently recommended thedevelopment of irnproved methods, for the specificpeople each year have amebiasis, only about 10% experi‑ence symptomatic disease. After much research andargument, it is now generally accepted that the previous
classification E. histolytica, is now separated into two

diagnosis of E. histolytica infection. To this end, we are

species: noninvasive E. dispar and potentially invasive

E. histolytica from E. dispar based on the gene coding forable to design new sets of primers that uniquely identify

122

E. histolytica cysteine proteinase, an implicated viru‑
lence factor in the pathogenesis of amebiasis and plays

CACTTCA‑3') and WRG‑4 (5'‑TTTCTAACTATC‑a key role in tissue invasion and disruption of hostdef enses.

reference strains but not in E. dispar. A11 DNA extracts
from E. histolytica and E. dispar cysts collected from the

GTTGTTGGAGTCTCTAGC‑3') primer pair, a 570 bp

Philippines are presently being tested against the newsets of primers. At the same time, the sequence of theUsing WRG‑1 (5'‑CAGTTGATTG‑GAGAAGTATTATGAA‑3') and WRG‑2 (5'‑CAT‑CAACATTCTT‑3') primer pair for all E. histolytica

nucleotide was amplified in both E. histolytica and E.

amplified PCR product is being determined for the

dispar. On the other hand, a 755 bp nucleotide wasconstruction of a possible probe in the identification of

amplified using WRG‑3 (5'‑TTCTAAAAACAATAAA‑

pathogenic Entamoeba.

24 BACTERIAL EXPRESSION OF A HUMAN MONOCLONAL ANTIBODYFAB FRAGMENT RECOGNIZlNG AN ENTAMOEBA HISTLYTICASURFACE ANTIGENHIROSHI TACHIBANA1, X.‑J. CHENGl, KATSUOMI WATANABE1, YOSHIMASA KANEDAl,
MASATAKA TAKEKOSH12 AND SEIJI IHARA2
Department of Infectious Diseases

andDepartment of Molecular Life Science', Tokai University School of Medicine

Despite the medical importance of Entamoeba his ‑tolytica, an effective vaccine to prevent amebiasis has
not yet been developed. However, it has been reported

that passive immunization with human anti‑amebic
antibodies obtained from patients with amebic liver

introduced into E. coli. Bacterial colonies were transfer‑

red to nitrocellulose membranes and then screened forthe production of antibodies by incubation with E.
histolytica antigens followed by horseradish peroxidase‑
conjugated antibodies of the patient. One positive clone

antibodies may be useful in attempts to reduce mortality

was selected for further analysis. The monoclonal Fabfragment reacted with 10 reference strains of E, his ‑tolytica, as shown by an indirect fluorescence antibody

from amebiasis by passive immunization. We havetest using fixed trophozoites. In contrast, the antibody

prepared recombinant human monoclonal antibody Fabfailed to react with other enteric protozoan parasites.

fragments in Escherichia coli.

Incubation of intact trophozoites with the antibody
fragment localized the epitope on the cell surface.
Western immunoblot analysis showed that the molecu‑

abscess exerted a protective effect against amebic liverabscess formation in a mouse model. Therefore, such

Lymphocytes were separated from the peripheral
blood of a patient with an amebic liver abscess. Poly(A) RNA was isolated from the lymphocytes and thenlar mass of the E. histolytica antigen recognized by thegenes coding for the light chain and Fd region of theheavy chain were amplified by a reverse transcriptase‑monoclonal antibody Fab fragment was 260 kDa underpolymerase chain reaction. Amplified DNA fragments
were ligated with a plasrnid vector pFabl‑His2 andinhibited the adherence of E. histolytica to human eryth‑nonreducing conditions. The Fab fragment significantly
rocytes.

25 DETECTION OF ENTAMOEBA HIS TL YTICA ‑SPECIFIC ANTIGEN BY
SANDWICH ELISA
X.‑J. CHENG, HIROSHI TACHIBANA AND YOSHIMASA KANEDA
Department of Infectious Diseases, Tokai University School of Medicine

Entamoeba histolytica and Entamoeba dispar are guishing between E. histolytica and E. dispar is impor‑mor phologically inseparable, but only E. histolytica is tant for clinical and epidemiological reasons. We have
responsible for invasive amebiasis. Therefore, distin‑prepared several antibodies specific for E. histolytica

123

and/or E. dispar. We tried to detect E. histolytica
antigen (s) by sandwich enzyme‑1inked immunosorbent
assay (ELISA) using an E. histolytica‑specific mono‑clonal antibody. The wells of ELISA plates were coatedwith rabbit polyclonal antibody to E. histolytica. Vari‑ous concentrations of E. histolytica antigen were added,
incubated at 23'C for I hr, then washed with phosphatebuffered saline (PBS) containing Tween 20. The plates
were then treated with horseradish peroxidase‑labelled

was suspended in PBS, 25 trophozoites per well weredetected by the system. Even if the antigen was mixed
with feces, 50 trophozoites per well could be detected.
On the other hand, when E. dispar. Giardia intestinalis.

and Blastocystis hominis were used as antigen at aconcentration of 1,000 per well, positive reactions did

not occur. The sandwich ELISA system using themonoclonal antibody seems to be useful for the detec‑tion of E. histolytica.

monoclonal antibody and substrate. When the antigen

26 DNA POLYMERASE ACTIVITY IN ENCYSTlNG ENTAMOEBA INVA DENSASAO MAKIOKA*, MASAHIRO KUMAGAI*, HIROSHI OHTOMO*,SEIKI KOBAYASHI' AND TSUTOMU TAKEUCHI'
Department of Tropical Medicine, Jikei University School of Medicine

andDepartment of Tropical Medicine and Parasitology,
Keio University School of Medicine'

There is no axenic encystation system forpolymerases, aphidicolin and N‑ethylmaleimide inhib‑

Entamoeba histolytica. In this respect E. invadens, aited the activity, but 2', 3'‑dideoxythymidine‑5'‑triphos‑parasitic protozoan of reptiles, is a useful model for E.

phate did not. Thus sensitivity of the E. invadens
activity to salt and inhibitors of mammalian DNA

histolytica, because it resembles the human pathogen inmorphology and life cycle and encysts in vitro. Using

polymerases was basically the same as that of E. his ‑this model system, we examined the level of DNA

tolytica in our previous results. The level of DNApolymerase activity of E. ipwadens during encystationinduced in vitro. We first characterized DNA polymer‑

polymerase activity of cysts decreased as encystation
proceeded when corripared with that of trophozoites.

ase activity of trophozoites of E. invadens comparing it

Thus the results indicate that encystation is ac‑
companied by a Ted ced, Ievel of DNA polymerasewith that of E. histolytica and found that the activity of

E. invadens was lower than that of E. histolytica at pH
2, 4 and 6, and was higher at pH 8 and 10. The activityof E. invadens was completely inhibited by high concen‑

activity so that this correlates to the previous reportthat two nuclear divisions during cyst maturation occur

without DNA synthesis.

trations of K+. Among inhibitors of mammalian DNA

27 INHIBITORY EFFECT OF APHIDICOLlN ON GROWTH

AND ENCYSTATION OF ENTAMOEBA INVADENSMASAHIRO KUMAGAll, ASAO MAKIOKAl, HIROSHI OHTOMol,SEIKI KOBAYASH12 AND TSUTOMU TAKEUCH12

Department of Tropical Medicine, Jikei University School of Medicinei and

Department of Tropical Medicine and Parasitology,
Keio University School of Medicine'
Aphidicolin, a, mycotoxin, is a specific inhibitor of

inhibited DNA polymerase activity and growth ofeukaryotic nuclear replicative DNA polymerases andEntamoeba histolytica trophozoites. Because no axenic
culture medium which induces encystation is available
for E. histolytica, E. invadens, a parasitic amoeba oftreatment with this drug accumulates the cells at theG1/S border. We previously reported that aphidicolin

124

reptiles, which can encyst in axenic condition, is a usefulmodel for encystation of E. histolytica. The present
study was conducted to determine whether encystation

as well as growth of E. invadens is affected byaphidicolin. Trophozoites of IP‑1 strain of E. invadens
were axenically cultured in BI‑S‑33 medium and encys‑

was intensely inhibited when trophozoites were cultured
with aphidicolin before being transferred to encystation
medium containing the drug compared with that without
the pretreatment. The inhibitory effect of aphidicolinon growth and encystation was reversible because thesewere restored when the drug was removed by replacing

tation was induced by glucose depletion and reduction ofwith drug‑free medium. These results indicate theosmotic pressure using 47% LG medium. The growth of
E. invadens was inhibited by aphidicolin in a dose‑
dependent manner. Encystation was also inhibited byaphidicolin in a dose‑dependent manner. Encystation

reversible inhibitory effect of aphidicolin on growth and28encystation of E. invadens, and suggest the importanceof DNA synthesis before the onset of encystation.

SEROEPIDEMIOLOGICAL STUDY OF ENTOAMOEBIASIS USlNG

MICRO‑CHEMILUMlNESCENCE ELISA IN NEPAL

KOICHIRO TABUCH11, TOSHIMASA NISHIYAMA1, TAKAAKI ISHIDAl, HIROSHI TAKEUCHll,
S. KANDAl, IPPEI MOHR12, SUSUMU SAKATA3, SATORU SHIMIZU4, TAKASHI KISH15
KAZUKO HIRA16, YOSHIMI OHN07, JEEVAN B. SCHERCHAND8, A.B. JOSH19

AND TSUTOMU TAKEUCHllO
Department of Parasitology, Department of Hygiene', andSecond Department of Physiology3, Nara Medical University,
Tenri University4, Seisen Junior College5. Osaka City University',

Mukogawa Women's University7, Institute of Medicine, Tribhuvan University ,Institute of Medicine, Kathmandu, Nepal" andDepartment of Tropical Medicine and Parasitology,
Keio University School of Medicine "Entamoebiasis histolytica is a worldwide distribut‑

teau. The examination of Nepalese sera results as

ed disease, especially in tropical and subtropical regions.

follows: The average of chemilurninescence value of the

However, few sero‑epidemiologic researches of thistest sera were 4,121 1,848 counts per second (cps).
Data over the converged average 2 S.D. (3,578 2,317

disease has been reported in Nepal, so far. In this study,

we measured the specific antibody of Entamoeba his ‑tolytica in the sera of healthy Nepalese by micro‑cps) were regarded as positive. As a result, 25/220 test

cherniluminescence enzyme‑1inked immunosorbent

negative. Classified with regions, 21/116 test sera (18.

sera (11.4%) were positive, and 195/220 (88.6%) wereassay (micro‑chemiluminescence ELISA) , and analyzed1%) were positive in Kathmandu, 41104 (3.8%) were

the sero‑positive rate.

positive in Dzong. In sex, 14/103 test sera (13.6%) werepositive in male, 11/115 (9.6%) were positive in female.

Test sera were collected from two hundred andtwenty Nepalese, aged sixteen through eighty‑four inthe capit l, Kathmandu, and in a village of central
highland, Dzong in March, 1997. The method of micro‑
chemiluminescence ELISA was conducted as follows: 3

Comparing to colorimetric ELISA, which is usually used
for clinical test for amoebiasis, micro‑chemiluminescen‑

pg/ml of the antigen of Entamoeba histolytica wasmethod for overseas seroepidemiological research, inwhich large number of test sera should be collected.

placed in a microtitre plate. After washing, 1:1,000

ce ELISA needs less arnount of test serum. Therefore

this micro‑chemiluminescence ELISA is suitable

ed. The plate was then washed, alkaline phosphate

Since the people whose antibody indicated positive, wereasymptomatic, their physical condition of entamoebiasis

labelled anti‑human lgG, diluted 1:50,000, was added,
and the plate was incubated. After further washing,seems to have had focuses on the gut without apparent
symptom, or have already cured althought they had had

Lumi‑phos 530TM was added. The luminescence was

focuses before.
The positive rate in an urban area was much higher

diluted test serum was added and the plate was incubat‑

determined when the enzymic reaction reached a pla‑

125

than that in a rural area. Moreover, the positive rate ofdifference in the classifications are to be studied.

male was higher than that of female. The causes of such

29 SEROEPIDEMIOLOGY OF CRYPTOSPORIDIOSISMAKOTO ITOH1, X‑G. QlUl, MOTOHIRO ISEK12, TATSUYA KATSUMATA3,ISAO KIMATA2, HANESANA VISANOUI AND EISAKU KIMURAl
Department of Parasrtology, Aichi Medical University ,Department of Medical Zoology,
Osaka City University School of Medicine' andDepartment of Parasitology, Institute of Tropical Medicine,
Nagasaki University*

Cryptosporidium parvum is one of the causative

were ELISA positive and the rate was double of theagents for diarrhea and distribute worldwide. In Japan
until an outbreak in 1994. Although cases of the infec‑

oocyst positive rate (4.2%) . Oocysts were detected only
in younger age groups, under 10 years old. On the otherhands, ELISA positive cases were lower in a age grouption in abroad are increased, it is suspected that some ofunder I year old (5%) compared with older age groups

the cases have not been properly diagnosed, since its
symptom, diarrhea, usually last within 1‑2 weeks natu‑

positive rate (5.8%) exceeded its ELISA positive rate

little attention had been paid to the protozoan infection(11‑15%) . In the age group under I year old, the oocyst

rally. In this study, we used a quantitative ELISA(5.0%). Anti‑C. parvum lgG Ievels were higher inmethod to measure antibodies to the parasite antigens
and compared the results with those obtained by a stool
examination.

younger age groups. It was suggested that older age

Serum samples from Japanese students, Japanesecriptosporidiosis patients, and Indonesian patients withdiarrhea admitted to a hospital in Surabaya. IgG anti‑

bodies to C. parvum were quantitatively measured bygroups had acquired not complete but partial resistance
to C. parvum infection and this may account for theiroocyst negative and low anti‑C. parvum lgG Ievels of
the ELISA positives.

Anti‑C. parvum lgG Ievels measured in Japanesepatients after the onset of diarrhea shows that the levels

ELISA.

were positive in a limited period, during 2 to 6 weeks

The cut off point of this ELISA system was calcu‑
lated as mean +3SD of 139 Japanese students' anti‑C.
parvum lgG. Only one student's sample was judged as
positive. Out of the 516 Indonesian samples, 45 (8.7%)

post the onset of diarrhea. This may explain that
ELISA and oocyst double positives are rare and ELISA
positive rate was double of the oocyst positive rate.

30 DETECTION OF CRYPTOSPORORIDIUM PAR VUM DNA IN
APODEMUS ARGENTEUS IN RESIDENTIAL QUARTERS BY PCRMIKIKO HONDAl, FLOR DE MARIA LEON FLOREsl, SANDRA JUAREzl' 2

TETSUO YANAG11 AND HIROJI KANBARAl
Department of Protozoology, Institute of Tropical Medicine andDepartment of Molecular Biology of Diseases,
School of Pharmaceutical Sciences2, Nagasaki University

Waterborne outbreaks of cryptosporidiosis havebeen relevant to contamination by animal and humanexcretions containing Cmptosporidium oocysts. It has
been already reported that just neonatal calves excrete

the oocysts, but not adult cattle because they acquire

immunity to the parasites. However the transmission
route of the parasites to calves is still unknown.

We surmised that small animals living near cattle

126

sheds, such as wild mice, were the carriers/reservoirs ofto the oocyst wall and also C. parvum DNA was detect‑

C. parvum which supplied the oocysts to big animals,

ed in the intestinal tissue by a nested PCR (Ochiai et al..

e.g., calves, whose excreta contained a lot of oocysts and1998) .

they might contaminate environmental water source.
Forty three wild mice (32 mice of Apodemus spe‑
cious. 10 mice of A. argenteus and a mouse of Eoth‑

Cmptosporidium in intestinal organs was more useful to

We found that the reproductive stages ofprepare DNA templates for a PCR‑based test than theoocysts in feces because it was tricky to break oocyst

enomys smithii) were captured with traps in the suburbsof Nagasaki City, Jan. 1998. The intestinal contents offive mice (12%) out of them were positive to a specificwall to extract DNA from them, and the infection

FITC Iabelled monoclonal antibody (Wako 297‑54301)

daily milieu near a city.

31

source of Cryptosporidium unexpectedly exsists in a

INTESTlNAL BLOCKAGE BY CARClNOMA AND
BLASTOCYSTIS HOMINIS INFECTION

N. HORIKll, YOSHIMASA KANEDA3, M. MARUYAMA1, Y. FUJITA1, C. TANAKA2,
S. MlNAT02, X. CHENG3 AND HIROSHI TACHIBANA3
Department of Internal Medicine

and Clinical Diagnostic Laboratory2,

St. Luke's International Hospital andDepartment of Infectious Diseases, Tokai University School of Medicine3
Blastocystis hominis is frequently identified in stool

specimens submitted to clinical microbiology labora‑
tories. However, its pathogenic potential remains poor‑
ly understood and controversial. Recently, we detected

appears the B. hominis infection was coincidental andnot related to the neoplastic growth. We suggest thatthe intestinal obstruction and concomitant stool reten‑tion, plus hemorrhage from the cancerous lesions, may

heavy infections of B. hominis in 4 individuals withintestinal obstruction due to cancerous growth. After

have permitted the more abundant growth of B.

resection of the occluding masses, the infections sponta‑

ship between intestinal obstruction and a concomitantneously resolved, without specific chemotherapy. It

B. hominis infection.

hominis. This is the first report of a possible relation‑

32 STUDIES ON LEISHMANIA SPECIES ISOLATED FROM THE GREATGERBIL (RHOMBOMYS OPIMUS) IN XlNJIANG UYGUR AUTONOMUS
REGION IN THE PEOPLE'S REPUBLIC OF CHlNA
CHIZU SANJOBAl, T. HATABUl, YUKO NAKAMURAl, TAKESHI MIZUSHIMAl,
KEN KATAKURA2, SIN‑ICHIRO KAWAZU3, MAMORU ITOH4, YUTAKA NAKA15,
KOUICHI NAGAKURA5, MASAMICHI AIKAWA6 AND YOSHITSUGU MATSUMOTol
Departrnent of Molecular Immunology, Faculty of Agriculture University of Tokyo*,

Department of Parasitology, Gunma University School of Medicine2,

Research Institute of Community Health and Medicine,International Medical Center of Japan3,
Department of Immunology. Central Institute for Experimental Animals+,

Department of Animal Microbiology and Parasitology, Tohoku University5,
Department of Infectious Diseases6 and Research Institute of Medical Science',

School of Medicine, Tokai University

The great gerbil, Rhombomys opimus, is a well‑
documented reservoir of Leishmania major in the cen‑tral Eurasia, the causative agent of Old World zoonotic

cutaneous leishmaniasis, and is also known to be the127natural host of L. gerbilli and L. turanica. To under‑
stand the transmission cycles of Leishmania infection
among the great gerbils, an epidemiological research

was done in 1997 at Karamay, Xinjiang Uygur
Autonomus Region in the People's Republic of China

where cutaneous leishmaniasis has recently beenendemic. As a part of the research, 3 isolates of Leish‑

mania parasites (KMA2, 4, and 7) were established incultures from the auricula of gerbils and five isolates
were established from 3 species CP. mongolensis (KMP3
and 4), P. andrejevi (KMP2), Seregentomyia arpaklesis(KMPI and 6)] of sandflies. To identify the species ofLeishmania isolates, the sequences of a region of themini‑exon gene which is unique in trypanosomatid prot‑

turanica (MRHO/CN/92/Qitai) and L. major(MHOM/Israel/83/LT252), KMP3 and KMA4 wereidentified as L. gerbilli, and KMP2, KMA2 and KMA7
were identified as L. turanica. L. major has not beenfound in this study. To demostrate the pathogenicity ofthese species, histopathological examinations were per‑formed on HE stained tissue sections of great gerbils
from which the isolates were established. The large
number of amastigotes were observed in the auricula ofthe infected gerbils, but no significant pathologicalchanges were seen. No amastigotes were observed inspleen and liver. These results indicate that the coexis‑
tence of L. gerbilli and L. turanica in a population of thegreat gerbils in Karamay and L. gerbilli and L. turanica

ozoa were determind. Based on the sequence compari‑

are transmitted by P. mongolensis and P. andrejevisons with L. gerbilli (MRHO/CN/60/GERBILLD, L.

respectively to the great gerbils.

33 LEISHMANIASIS IN TURKEY
YUDSUF OZBEL , CHIZU SANJOBA2 AND YOSHITSUGU MATSUMOTO1
Department of Parasitology, Medical Faculty Ege University andDepartment of Molecular lrnmunology,
Faculty of Agriculture University of Tokyo'
Leishmaniasis, both visceral and cutaneous is still aendemic and sporadic regions has not been carried out

health problem in Turkey. Visceral leishmaniasis (VL)
is consistent with Mediterranean type of VL with a fatal

yet. The parasites isolated from dogs were also

debilitating disease which is most encountered in infants

between 0‑15 years old. Human VL is mainly observed

Cutaneous leishmaniasis is seen sporadically inmost regions except southeastern part of Turkey wherein western and southern regions, rarely other regions, ofthe disease is still higly endemic. Up to date, a total of

Turkey along Ege and Mediterranean coasts as endemicor sporadic. Strains isolated from human cases werereported. Since there is no report on Leishmania

identified Leishmania infantum using different technics.

promastigotes detected in sandflies, some species canCanine VL (CVL) is also endemic and showing a

only be considered as suspect vector of leishmaniasis inhigher prevalance than human VL. Reservoir studies in

identified as L. infantum.

17 different Phlebotomus species and subspecies wereTurkey.

34 STRUCTURE ANALYSIS OF TRYPANOSOMA CRUZITRANS‑SIALIDASE GENE FAMILY=RELATION TO PROTElN LOCALIZATION AND ENZYME ACTIVITYHARUKI UEMURA, S. CHIN, MIE KATO AND HIROJI KANBARA
Department of Protozoology, Institute of Tropical Medicine,

Nagasaki University

Trans‑sialidase of Trypanosoma cruzi is a uniquemucin‑1ike acceptor molecules on the parasite surface.

enzyme which catalize the transfer reaction of terminal

The sialylated molecules and the enzyme itself aresialic acid from host derived glycoconjugates to theinvolved in host‑parasite interactions and initial stage

128

of trypomastigote invasion into host cells. Trans‑

ized at the different chromosomes and may be regulated

type is tandem repeats of 12 amino acid unit and theseare followed by hydrophobic amino acid sequence whichis replaced by GPI anchor structure. No these repeats
and no GPI exist in trans‑sialidase of epimastigote.
These are consistent with the analysis of the epimas‑
tigote type protein. One of the important information
remained to be obtained is the localization of trans‑

independently. Comparative analysis of the obtained

sialidase in epimastigote and the genes which give

genes revealed that catalytic domain of these two trans‑sialidase molecules shear more than 80% of similarities,

enzyme activity. Here we show that there are at least
four types of C‑terminal amino acid sequences in trans‑sialidase genes expressed in epirnastigote and we haveobtained enzyme activity from only one of these types,

sialidase is highly expressed in trypomastigote stage
and the genes for these proteins are arranged in clustersof tandem array. The other type of trans‑sialidase isdetectable at the epimastigote stage of the parasite.
The trans‑sialidase genes of these two stages are local‑

however amino‑ and carboxyl‑terminal regions have nosimilarities. Most remarkable differences are t their
C‑terminals. The C‑terminal region of trypomastigote

by expressing in E. coli.

35 COMPARATIVE ANALYSIS OF CDNA EXPRESSION PROFILESBETWEEN LONG SLENDER AND SHORT STUMPY BLOODSTREAMFORMS OF TRYPANOSOMA B. BRUCEI USlNG FLUORESCENTDIFFERENTIAL DISPLAY METHODTAKASHI SUZUKll' 2, YOSHISADA YABU*, ICHIRO KANAZAWA' AND NOBUO OHTAl
Department of Medical Zoology, Faculty of Medicine, Nagoya City University

and CREST Japan Science and Technology'

African trypanosomes are member of the order
Kinetoplastida, causing human sleeping sickness andnagana disease in cattle. During the ascending par‑asitemia, Iong slender, actively dividing forms predomi‑
nate in the host blood and tissue fluids. At the peak ofparasitemic wave, Iong slender forms differentiate into
non‑dividing, short stumpy forms, which have a limited
life span (24‑36 hr) in the host blood. This mechanismof slender to stumpy differentiation is still unclear.

Therefore in order to clarify the mechanism, we
compared the CDNA expression profiles between long
slender and short stumpy forms of pleomorpic clone of
Ttypanosoma b. brucei (GUT at 3.1) using Differential
Display (DD) techniques.
Long slender forms were harvested from in vitro
cultured bloodstream forms. Short stumpy forms of thesame clone were harvested from 5‑6 days after infectionof Balb/c mice and isolated from blood by DEAE‑cellu‑

10se chromatography. Total RNAS of bo, th forms wereisolated and subjected to the DD analysis. Totally,
about 8,000 CDNA bands were compared. As a result, 16
CDNAS Showed increased expression in short stumpy
forms compared to long slender forms, while 33 cDNAs

showed decreased expression. One cDNA which was
expressed in long slender forms, not in short stumpy
forms, was randomly selected, a full‑length of which

was cloned with 5' RACE and subjected to sequence
analysis. BLAST homology search revealed that the
CDNA (gene) is a novel variant surface glycoprotein(VSG) gene. However, the relation of the VSG‑expres‑sion and the difference of the nature of those forms isstill unclear.

129

36 IMMUNOGENETIC ANALYSIS OF CHAGAS' DISEASESANDRA JUAREsl, KAYANO AIDA2, MIHOKO KIKUCH12, KENJI HIRAYAMA2,TETSUO YANAG11, MARIA PAULA DE LEON3, SATOSHI KANEK04, OSCAR AYAU4,JULIO ARGUETA4, VIVIAN MATTA3, TOSHIO SONE2, KYOGO IT05 AND ISAO TADA6
Departrnent of Protozoology, Institute of Tropical Medicine,

Nagasaki University ,Department of Medical Zoology, Saitama Medical Scho012
Department of Histocytology, School of Pharmacy, San Carlos University3,
Japan International Cooperation Agency*,
Department of Immunology, Faculty of Medicine, Kurume University* andDepartment of Parasitology, Faculty of Medicine, Kyushu University6

Chagas' disease is caused by a protozoan parasite
Trypanosoma cruzi. Whether the infected individuals

MICA‑A5 was increased in the seropositives but thedifference was not significant after correction (x2 = 7.62,

are able, to eliminate the organism or develop chronic
Chagas' disease with cardiopathy is unpredictable. In
the present study, in order to reveal the host genetic

Pc=0.223, OR=2.04, 95%CI 1.01‑4.13) .
The increased risk for the individuals who had bothfactors that influence susceptibility to Chagas' disease,higher than the sum of the increased risk for HLA‑B35

we typed 44 seropositive Chagas' patients and 138 ser‑onegative healthy controls in Guatemala for their HLA‑(ORA*‑1 = 2.15) , indicating that the effects of HLA‑B35. DRB1. MICA (MHC class I chain‑related gene A)
and TNFa promotor alleles. The frequency of HLA‑

fore, we suggest that the susceptibility to infection with

B35 was significantly increased in the seropositives

T. cruzi is determined by the interaction of two genes,(x2=17.95, Pc<0.0004, OR=4.69, 95%CI 2.22‑9.92).

HLA‑B35 and MICA‑A5.

HLA‑B35 and MICA‑A5 (ORB35/A5 1=16.55) was much
(ORB35‑1=5.41) and MICA‑A5 positive individuals
and MICA‑A5 on susceptiblity were synergistic. There‑

37 COMPARISON OF THE POLYMERASE CHAlN REACTION WITH
THREE SEROLOGICAL METHODS FOR THE DIAGNOSIS OFCHAGAS' DISEASESANDRA JUAREZ1' 2, TETSUO YANAG11 AND HIROJI KANBARAl
Department of Protozoology Institute of Tropical Medicine

andDepartment of Molecular Biology Diseases,
School of Pharmaceutical Sciences', Nagasaki University

PCR‑based diagnostic test for Chagas' disease wasnation test (RLAT) , immuno‑double diffusion test

after the collection with just EDTA not with guanidine‑
EDTA (Avila et al.. 1991).
To establish the PCR protocol for Chagasic diagno‑

cornpared with three serological methods; rapid aggluti‑(IDDT) and ELISA. We tested 143 blood samples

sis we compared two different DNA extraction

collected more than two years ago at a hospital in an
active transmission area, Guatemala. Forty eight out of

methods: phenol‑chloroform or IsoQuick kit (ORCA

143 were seropositive by the three methods. Twenty
four samples out of 48 seropositive cases showed the
PCR products of Trypanosoma cruzi. Five hemocultures(21%) out of 24 cases with positiveness by PCR became
positive. The results by PCR did not highly correspondwith those of serological tests. One of the reasons issurmised that the blood samples were too old to get
successful PCR products because the samples were kept

BP2 (Silber et al.. 1997) specific primers to nDNA or
121/122 (Gomes et al.. 1998), specific primers to kDNA.
The PCR product obtained from phenol‑chloroform
extraction revealed smears instead of a target productResearch, Inc.) , and two different sets of primers: BP1/

on an electrophoresis, on the other hand IsoQuick
showed apparently a distinctly positive band in a clear

background.
The primers BP1/BP2 cannot distinguish T. cruzi

130(692 bp) from T. rangeli (615 bp) each other since therespective PCR products is almost similar size. In thecase of the primers 121/122, the target size of T. cruzition with IsoQuick kit and the specific primers to kDNA

(121/122) for Chagasic PCR diagnosis. These primers
are useful for PCR diagnosis in the cases of a single(330 bp) is closed to that of T. rangeli (approx. 350 bp)

infection with T. cruzi or T. rangeli, but in the cases ofso that the both parasites can be neither identified fromthe main targets. However the size of artifact productsby hairpinning is respectively different between them;the multiple infection probably the PCR by the primers
makes a wrong diagnosis. The PCR‑based test is a kindof complemental diagnostic examination, that is, thethat of T. cruzi (approx. 650 bp) is smaller than that ofnegativeness by PCR diagnosis means always non‑T. rangeli (>700 bp). T. cruzi and T. rangeli parasites
are distinguishable by just one set of primers (121/122) ,but only in the case of single infection, not multi‑infec‑

chagasic. The results of not only PCR‑based tests but
also serological tests and others should be consideredtion.

epidemiological studies.

for the decision of Chagas' disease and for

We can recommend a combination of DNA extrac‑

38 COMPARATIVE STUDIES ON EPIDEMIOLOGY OF CHAGAS' DISEASE
BETWEEN BOLIVIA AND NORTHEAST REGION OF BRAZILSACHIO MlURA1, H. HORl02, J. ADAUT03, T. KAMIYA4, N. IIBOSH15

MASANOBU TANABEl, SEIKI KOBAYASH11, T. NOZAKll

SHlNJIRO HAMAN06 AND TSUTOMU TAKEUCH11
Department of Tropical Medicine and Parasitology,
School of Medicine, Keio Universityl,
Department of Parasitology and Tropical Medicine,
University of Occupational and Environrnental Health'
Epidemiologia Escola Nacional de Satide Ptiblica, Rio de Janeiro, Brazil*

Christian University' and Centro Nacional de Enfermedades Tropicales*,Santa Cruz, Bolivia andDepartment of Parasitology, Faculty of Medicine, Kyushu University'

In the present studies we compared several aspectsof triatomine bugs and seroepidemiologic features on
Trypanosoma cruzi ( T. cruzi) infection in Santa Cruz‑
Bolivia and the northeast region of Brazil. The sero‑prevalence on T. cruzi infection were evaluated by HA,
IFA and ELISA. The anti‑T. cruzi antibodies in Abapo
and El‑Torno were positive rate at 84.3% and 61.1%
respectively. In addition, we found 31 cases of suspected

congenital Chagas' disease arnong 1,200 newborn babies,
based on the parasiternia positive of T. cruzi by theStrout method. It has been confirmed that congenital
infection of T. cruzi by the xenodiagnosis. In contrast
to Santa Cruz, acute or congenital Chagas' disease havenot been recently reported in the northeast region ofBrazil. We could captured many Triatoma infestans, theprimary vector of T. cruzi have greatly reduced houses

infestations in Abapo and El‑Torno, T. cruzi positive

rate of them were 78% and 83% respectively, but innortheast region of Brazil, scarcely captured T.
brasiliensis and T. pseudomaculata and 8.6% of themshowed positive of T. cruzi. Analysis of the relationshipbetween the reactors and triatomine bugs, interviewed
for inhabitants when collected blood showed that mostof them were free of such knowledge. These observa‑
tions suggest there is a distinct difference in the preva‑

lence of T. cruzi infection between Santa Cruz‑Bolivia
and Northeast region of Brazil. The vector control will

constitute the main factor in the control of Human
Chagas' disease in Santa Cruz of Bolivia. Other factors

have poor condition of economical, sociological andeducational for health care to inhabitants in endemic
area.

131

39

STUDY OF CROSSREACTlNG ANTIGENICITY BETWEENA SCA BIS LUMBRICOIDES ANTIGENS AND HUMAN COLONlC MUCOSATAKAAKI ISHIDAl, TOSHIMASA NISHIYAMA1, HIROSHI TAKEUCHll, SEIJI KANDAl,
TSUNEJI ARAKll, ICHIRO HIRATA2 AND KEN‑ICHI KATSU'
Department of Parasitology, Nara Medical University* andSecond Department of Internal Medicine, Osaka Medical College'

The present study was conducted to determine thement with horseradish peroxidase‑labeled goat anti‑

crossreacting antigenicity between Ascaris lumbricoides

rabbit lgG.

antigen (AS Ag.) and human colonic mucosa (Cm Ag.).
A. Iumbricoides were homogenized in phosphate‑buffer‑
ed saline (PBS) to prepare As Ag. and rabbits were
immunized with As Ag. in order to produce anti‑A.
lumbricoides serum (anti‑As abs) , anti‑As abs was

As a result, crypts of colonic mucosa, especiallybasal membrane part, were stained using immunofluor‑
escence technique. Moreover, we detected that mainly

absorbed in E. coli to eliminate anti‑E. coli serum.

chemiluminescence Western blotting analysis, andSeven normal human colonic mucosa were obtained
from biopsy specimens at colonoscopy and those wereisoelectric point of both proteins were pl 5.8.

respectively homogenized in PBS and Cm Ag. were

antigenicity between As Ag. and Cm Ag. and molecularweights of these cross‑reacting antigens were almost

pre pared.

molecular weights 41 kDa and 38 kDa proteins werecross‑reacting antigens between As Ag. and Cm Ag. by

We demonstrated that the presence of common

Immunohistological staining of embedded colonicmucosal section was performed by immunofluorescenceequal to antigen detected by colitis colon associated lgGtechnique using anti‑As abs.
Sodium dodecyl sulfate‑polyacrylamide gel electro‑

ulcerative colitis by DAS.

phoresis (SDS‑PAGE) and two‑dimensional gel electro‑

reacting antigens and antigen detected by CCA‑IgG. In
conclusion, it is thought that these cross‑reacting
antigens might be closely related with the pathogenesis

phoresis were performed against Cm Ag. andchemiluminescence Western blotting analysis of Cm Ag.
using anti‑As abs were performed followed by treat‑(CCA‑IgG) which was found in the colonic mucosa of

There was a resemblance between these cross‑of ulcerative colitis.

40 IMMUNODIAGNOSTIC APPLICATION OF THE RECOMBlNANTTOXOCARA CANIS ANTIGEN FOR HUMAN TOXOCARIASISCANlS AND CATIHIROSHI YAMASAKll, KUNIOKI ARAK12, Z. NGAH3, J.W. LIM4, J.W. MAK4,

T. RADZAN3, Y. WATANABE5, KIYOSHI KITA6 AND TAKASHI AOK11
Department of Parasitology, Juntendo University School of Medicine*,
Division of Microbiology, National Institute of Public Health'Institute for Medical Research3 and Universiti Putra, Malaysia, Selangor+,

Department of Biochemistry, Dalhousie University, Canada= andDepartment of Biomedical Chemistry,
The University of Tokyo, Graduate School of Medicine6

The diagnosis of human toxocariasis canis caused

by infection with the second stage larvae (L2) of
Toxocara canis depends mainly on the detection of anti‑

T. canis antibodies by ELISA using T. canis L2 ex‑cretory‑secretory antigens (TES). However, the TES
commonly used cross‑reacts with sera from patients
infected with various helminths, and is laborious toobtain in large amounts from cultured T. canis L2' So,
we developed a recombinant antigen to substitute for
TES in the immunodiagnosis of human toxocariasis. A
clone (clone 2) derived from a T. canis L2 cDNA Iibrary

was selected for protein expression. IPTG‑inducible
pET‑32b ( +) plasmid was used as an expression vector.

Recombinant T. canis protein (38 kDa) produced in132

Escherichia coli was purified using TALON*" Metalof 114 samples from patients infected with 13 different

Affinity Resin, and its specificity as an antigen was
evaluated by lgG‑ELISA using 118 serum samples fromhelminths. In the case of TES used for comparison,cross‑reactions were seen in 49 cases out of the 114

patients infected with 15 different helminths. The

serum samples and involved 10 different helminth infec‑

recombinant antigen reacted intensely with all 4 serum

tions, indicating that the specificity of the recombinantsamples from suspected toxocariasis patients at an

antigen is much higher than the TES widely used. The
recombinant T. canis antigen developed can be recom‑
mended for the immunodiagnosis of human toxocariasis
and may provide more reliable diagnostic results thanthe currently available TES.

antigen concentration of 125 ng/ml and serum dilutionof 1:200, yielding no false negative reactions. Cross‑reactivities were observed in only one case each ofgnathostomiasis, paragonimiasis, and spirometriasis out

41

STRONGYLOIDES RATTI ADDITIVE EFFECT OF TESTOSTERONEIMPLANTATION AND CARBON INJECTION ON
THE SUSCEPTIBILITY OF FEMALE MICEK WATANABE SHlNJIRO HAMANO, K. NODA, MASATAKA KOGA
AND ISAO TADA
Department of Parasitology, Faculty of Medicine, Kyushu University

There is sex difference in susceptibility to parasites.

treated with testosterone and carbon was significantly

In Strongyloides ratti‑mice model, it was known thathigher than that treated with testosterone alone orcarbon alone. Furthermore, it seemed to be the sum ofworm number in each treatment group. Serum testos‑terone level was elevated in testosterone and carbon
treated group as compared with that of testosterone
treated group. To adjust serum testosterone level
equally between these groups, the amount of implanted

male mice were highly susceptible to S. ratti than female

mice and testosterone implantation could render female

mice susceptible similar to native male mice. Thiseffect was observed during migrating phase of S. ratti.
Recently it was reported that testosterone could affectvarious cell's function, including macrophages. We tried
to examine whether the testosterone could affect suscep‑tibility through macrophages. Testosterone was im‑planted in female mice in advance and macrophages
were blocked by carbon injection. After carbon injec‑
tion, 2,000 S. ratti L3 were infected subcutaneously. The

effect of each treatment was assayed by counting worm
number arrived to the head of infected mice. Testoster‑
one treatment alone or carbon treatment alone effec‑
tively increased the worm number as previously report‑

ed by others. The worm number observed in mice

testosterone was adjusted before S. ratti L3 infection.
Testosterone (adjusted dose) and carbon treatment also

increased the worm number equally to the sum of eachtreatment alone. This experiment revealed that testos‑terone and carbon treatment had an additive effect on
susceptibility in female mice. It might be suggested that

testosterone could increase the susceptibility of female

mice to S. ratti independently of macrophages because

testosterone could affect the worm recovery after
macrophage blockade.

133

42 PREVALENCE OF STRONGYLOIDES STERCORALIS INFECTION
AMONG INHABITANTS OF YORON ISLAND IN KAGOSHIMAPREFECTURE AND CLlNICAL STUDY ON SYMPTOM INPATIENTS WITH STRONGYLOIDIASISO. ZAHAl, H. HIRATA1, S. MIYAG11, A. HOKAMAl, N. KlNJOUl, H. SAKUGAWAl,
F. KINJOUl, ATSUSHI SAITO1, H. NAKAMURA2 AND N. YAMANE2First Department of Internal Medicine, Faculty of Medicine

University of the Ryukyus* andDepartment of Clinical Laboratory, University Hospital of the Ryukyus2

Strongyloides stercoralis which is soil‑dwelling
nematode is prevalent in the tropics and subtropics soil.

In humans, infestation with this nematode most com‑monly involves the upper small intestine. Clinical signs

and symptoms are minimal digestive syndrome. How‑ever, in the presence of immunosuppression, invasivestrongyloidiasis may develop into a life‑threatening
disease.

We examined 611 cases inhabitants of Yoron Islandin Kagosgima Prefecture. The patients with S. stercor‑

alis was 64 cases. The positive rate was higher insixties.

The most common complaints of 64 patients with S.
stercoralis were borborygmus (45.6%), abdominal pain
(26.3%) and diarrhea (26.3%).
We treated 32 patients with ivermectine and symp‑
toms improved after treatment described below: The
cure rate was 100% after 4 weeks after. Sixteen of thepatients (88.9%) with digestive syndrome improved.
Seven of the 9 patients (77.8%) with itching of anus,
four of the 5 patients (80.0%) with discomfort of phary‑nx and seven of the 12 patients (58.3%) improved.

43 SEROEPIDEMIOLOGY OF AN EMERGlNG PARASITIC DISEASENEUROCYSTICERCOSIS IN IRIAN JAYA, INDONESIAAKIRA ITO1, TONY WANDRA2, RIZAL SUBAHAR5, GlNDO SIMANJUNTAK2,
THOMAS SUROS02 AND SRI S. MARGON03
Department of Parasitology, Asahikawa Medical College*,
Communicable Disease Control and Environmental Health,
Ministry of Health, Indonesia' andDepartrnent of Parasitology, Faculty of Medicine, University of Indonesia*

Based on our previous report on taeniasis/cysticer‑
cosis in Indonesia as an emerging disease (Simanjuntak
et al. 1997, Parasitology Today, 13, 321‑323), we havedone seroepidemiological study of cysticercosis in lrian294) has revealed that 12 (67%), 20 (20%), O (O%) and13 (12 from endemic and I from non‑endemic areas)(17%) cases were serologically confirmed cysticercosis,
respectively. It has been known that three districts in

Jaya, Indonesia. For this study, we have examined alrian Jaya Province have already been contaminatedtotal of 138 serum samples including 18 from suspected
neurocysticercosis (due to anamnesis of epileptic sei‑with taeniosis/cysticercosis (Wandra et al., submitted
for publication) . We have found one case of cysticer‑

zures) , 31 from suspected cysticercosis (due to subcuta‑
neous or muscle nodule(s) ) , 12 from taeniosis (due tocosis from non‑endemic area from another district.

fecal examination or anamnesis of expulsion of prog‑
lottids) and 77 frorn people at risk (47 from endemicarea, and 30 from non‑endemic area). Highly sensitive

10gically confirmed cysticercosis cases of people at risk
have epilepsy, subcutaneous nodule (s) , indeed. There‑

and specific serodiagnosis by both immunoblot andJaya. We are interested in doing serological study of the

ELISA (Ito et al., 1998, Am. J. Trop. Med. Hyg., 59, 291‑

local people in Papua New Guinea.

Follow‑up study has revealed that most of the 13 sero‑

fore, we suppose that cysticercosis is spreading in lrian

134

44 THE RESISTANCE OF TREATMENT AND SPECIFIC IGG4ANTIBODY TITER IN HLA‑DRB1*0901 POSITIVE PATIENTSWITH STRONGYLOIDIASISMASAO SATOHl, HIROSHI TOMA2, YOSHIYA SAT02, MASAHIRO TAKARA3,
YOSHIYUKI SHIROMA*, SUSUMU KIYUNA4,MIHOKO KIKUCH11 AND KENJI HIRAYAMA1
Department of Medical Zoology, Saitama Medical Schooll
Department of Parasitology, Faculty of Medicine,
University of the Ryukyus'
Gushikawa Clinic* and lzumizaki Hospital"
Strongyloidiasis is difficult to be completely treat‑group (P<0.05) . IgG4 antibody titers were higher in theed. To find host factors involved in response to treat‑ment, patients were analyzed specific antibody titersand HLA‑DRl 1 alleles. Higher titers of lgG4 antibody
were observed in the non‑cured group than in the cured

DRB1*0901 positive group than in the negative group(Pc<0.05) . These results suggest that DRB1*0901 is apossible genetic marker for resistance to treatment thatis associated with elevation of lgG4 antibody titer.

45 STRONGYLOIDES S TER CORA LIS CARRIERS IN OKlNAWA, JAPANRYUJI AsATOl, TAMIKI ARAKAK12, TSUYOSHI IKESHIR03, NAOMASA OsHIROl,
ATSUSHI OHNO1, JUN KUDAKAl, KIYOMASA ITOKAZUl,

MASAHARU NAKAMURAI AND MASAAKI SHIMADA*
Okinawa Prefectural Institute of Health and Environment ,Shirakawa Clinic', Okinawa Preventive Medicine Cooperation3 andInformation and Reference Center,
Institute of Tropical Medicine, Nagasaki University'
A surprisingly high prevalence rate of Strongyloides

Rheumatoid factor, platelet, cholinesterase, LDH andstercolaris infection in Okinawa has been reported

gamma‑GTP were compared between S. stercolaris(Asato et al.. 1992). The impact of infection, however,

although the infection is generally considered oppor‑

carriers and non‑carriers. The differences were foundonly in the number of WBC, eosinophile and lymphocyte.
They were significantly higher in carriers than in non‑tunistic.

carriers.

1. It is likely that the transmission of S. stercolaris does

3. A relationship between HTLV‑1 infection and dis‑seminated strongyloidiasis has been reported. Amongon the health status of patients has not been well studied

not exist because the age‑prevalence has been decreas‑

ing constantly both in Itoman from 1983‑1995 and in
Osato from 1989‑1995.
2. Using the health check data obtained in 1989‑1992
from a health center, the results of chest X‑ray, serum
cholesterol, neutral fat, HDL, GI tract examination,
urine protein, urine blood, BUN, cleatinin, uric acid,albumin, alkaline phosphatase, GOT, GPT, ZTT, serum
bililbin, serum protein, blood sugar, haemoglobin,heamatocrit, RBC, WBC and its analysis, ESR,

the S. stercolaris carriers, total lgE Ievel was compared

between HTLV‑1 carriers and HTLV‑1 non‑carriers.
The lgE Ievel was higher in males than in females, andtended to be higher in HTLV‑1 non‑carriers both inmales and females.
4. In the record of the cause of death from 1990‑1994 in
Okinawa S. stercolaris infection was reported, as a causeof death, only in 3 cases among 21,608 deaths.

135

46 A CASE OF GENITAL ELEPHANTIASIS DUE TO POSSIBLE
FILARIA INFECTIONYOSHIAKI HAMAMOTO, HIROAKI TAKAHARA, KOICHIRO YAMAMOTO

AND MASAHIKO MUTO
Department of Dermatology, Yamaguchi University School of Medicine
In Japan, elephantiasis is a very rare disease char‑acterized by the aspect of lesional skin which is similar

observed. Histopathologic examinations revealed thatacanthosis with hyperkeratosis, fibrosis of the dermis

to that of the elephant. The latest case of filaria‑in‑and dilatation of lymphatic vessels. Lymphography

duced elephantiasis was reported in 1982 by Shirahama
et al. in Japan. A 62‑year‑old man, born in Okinawa,
had a history of fever with shivering from age of 25 tofrom the left third toe‑finger revealed that obstructionof thoracic duct and reflux of contrast medium on
kidney and scrotum. Repeated examinations for mi‑

30 years at a rate of one time a week followed bycrofilaria in the midnight urine were negative. Based on

swelling of the genital region. Before the onset of theclinicopathological and epidemiological studies, thedisease he had been in Bolivia where filariasis prevailed.

When he visited our hospital, the skin of his penis anddiagnosis of genital elephantiasis was established. The
lesional skin was resected and free skin graft was done
onto the penis shaft. Skin defect on scrotum was sutur‑
ed. The postoperative course was uneventful, except fora small ulcer formation on the scroturn at the operation

scrotum was rough, verrucous, thickened and coloreddark brownish. Milky discharge from the scrotum waswound with bacterial infection, which had been im‑proved in a few weeks.

Ten years before visiting our hospital, surgical treat‑ment was performed elsewhere. However, a few months
after the operation the same eruption was recurred.

47 DETECTION OF CIRCULATlNG WUCHERERIA BANCROFTIANTIGEN, FILARIA SPECIFIC IGG AND IGG4 IN

CHYLURIA CASES IN OKlNAWA
XU‑GUANG QlUl, MAKOTO ITOHl, YUZO KOYAMA2, YOSHIHIDE OGAWA2,MIRANl V. WEERASOORIYA3, YASUNORI FUJIMAKI* AND EISAKU KIMURA1
Department of Parasitology, Aichi Medical University ,Department of Urology, Faculty of Medicine, University of the Ryukyus',

Department of Parasitology, Faculty of Medicine, University of Ruhuna" andDepartment of Parasitology, Institute of Tropical Medicine,

Nagasaki University'

The diagnosis of filarial chyluria is made simply

gyloides ratti were used to absorb antibodies cross‑based on patients' history of living in the past endemic

reactive with these antigens.

areas of filariasis. In this study, we studied (1) if we canAbsorption with combined antigens of D. immitis
and Anisakis reduced cross‑reactive antibodies, andclearly separated mf positive Sri Lankans, chyluria
patients and normal controls. If the average antibody

obtain immunological evidence to support the filarial
origin of chyluria, and (2) if there is any immunologicalmethod useful to make a diagnosis of filarial chyluria.

Serum samples from 16 chyluria patients in Okin‑awa were tested. Sera from 14 healthy Japanese were
negative controls, and 7 sera from Sri Lankan carriersof microfilariae (mf) were positive controls. Circulat‑

ing W. bancrofti antigen was assayed by ELISA using
Og4C3 monoclonal antibody. Filaria‑specific antibodies
were detected using Burgia pahangi antigens. Antigens

of Dirofilaria immitis. Anisakis simplex and Stron‑level of the healthy control + 3SD is regarded as a cut‑
off point, 7 of 14 chyluria patients could be diagnosed asfilarial. Antibodies of lgG4 subclass, which cross‑react‑
ed very little with other parasites antigens and associat‑

ed with active filarial infection, were detected in one
chyluria petient. In conclusion, for the diagnosis offilarial chyluria, (1) measurement of B. pahangi‑
reactive lgG4 or (2) measurement of B. pahangi‑

136

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137

50 IS THE SCHISTOSOMA INDI C UM GROUP MONOPHYLETIC?
TAKESHI AGATSUMA1, PlNARDI HADIDJAJA2 AND STEPHEN AMBU3
Department of Bioresource Science,
Obihiro University of Agriculture and Veterinary Medicine
Departrnent of Parasitology, University of Indonesia' andDepartment of Tropical Ecology, Institute for Medical Research3

Four Schistosoma species, S. indicum. S. spindale. S.

worms. Adult worms for each species were grouped to
extract DNA. Amplified regions are the 2nd internal

incognitum and S. nasale found in India and Southeast
Asia develop in snails of the family Lymnaeidae, andtentatively referred as to the S. indicum group, having
terminal ‑or subterminal‑ spined eggs (Rollinson andtranscribed spacer (ITS2) and the cytochrome c oxidasesubunit 1, CO1. Primers, PCR conditions, and computor

Southgate, 1987). However, S. incognitum does not

1997; Blair et al.. 1997). The Neighbour Joining method

analyses were described elsewhere (Agatsuma et al..

possess a true terminal spine to its egg and its intermedi‑

was performed to construct phylogenetic trees for theate host belongs to the genus different from that of theother three species. In the present report, phylogenetic

two DNA regions. Both of the trees showed that S.

relations between S. spindale and S. incognitum andand S. spindale was included into an African cluster, or

their relations with other Schistosoma groups were inves‑specifically the S. haematobium group. Affinity between
S. spilrdale and S. haematobium has been also obtained

tigated using PCR‑amplified nucleotide sequences. For
S. spindale, naturally infected snail species of Indo‑planorbis were collected in Malaysia and maintained in
the laboratory of IMR. For S. incognitum. Rattus rattus

were trapped in Jakarta, Indonesia to obtain adult

incognitum was not clustered with any species groups,

by the S18 rRNA gene tree analysis using the Fitch‑
Margoliash least squares method (Johnston et al.. 1993) .

The present result clearly indicates that the S. indicumgroup is not monophyletic but paraphyletic.

51 SONOGRAPHIC ASSESSMENT OF BOTH URINARY TRACT
AND HEPATIC MORBIDITY DUE TO SCHISTOSOMA HAEMATOBIUMTATSUYA KATSUMATA1, ANTHONY M. KASOM02, TOSHIKI AWAZAWA1,
NGETHE D. MUHOH02 AND YOSHIKI AOKI*
Department of Parasitology, Institute of Tropical Medicine,

Nagasaki University and
Kenya Medical Research Institute'

Since present aim of the global control ofschitosomiasis is to reduce morbidity rather than toreduce transmission or even eradicate the disease,ultrasonography has widely been used in a number of

The investigations were conducted in the Tserezani
village, Coast province, Kenya. In February 1998, 225
and 222 school children were examined for liver andurinary tract respectively by ultrasonography. Urinary

epidemiological studies in schitosomiasis endemic areas.

bladder morbidity was graded by the classiaication

The method has proved to be feasible and useful to
examine the morbidity. Pathology due to Schistosoma
haematobium is examined exclusively for the urinary

systern proposed by Medhat, A. et al. (1997). The bodyheight‑dependent reference values pub. Iished in Senegal

tracts so far. However, recently periportal fibrosis ofliver has also been described in S. haematobium infection

ing the periportal fibrosis. Urine passed by each subject

by using ultrasonography. We commenced thethe nucleopore filtration method.
Pathological lesions of urinary bladder were obser‑

ultrasonographic studies to examine the extent of liver
pathology, in addition to urinary tract morbidity in anarea where schistosomiasis haernatobia is highly
endemic and no intervention was introduced so far.

(Y:azdanpanah, Y. et al.. 1997) were applied for evaluat‑

at midday was microscopically examined for eggs by

ved in 160 (72%) subjects. Arnong these, 75 (34%)
subjects had ultrasonographic score of grade 111, while
only 6 (2.7%) subjects had kidney congestion. Peripor‑138

tal fibrosis were shown in 54 (24%) subjects. The
comparative analysis using logistic regression modelshows that both urinary bladder morbidity and peripor‑

In conclusion, a relatively high morbidity due to S.
haematobium infection in an area where no intervention

has been done was demonstrated.

tal fibrosis are associated with presence of eggs in urine.

52 CURRENT STATUS OF BOAR‑MEAT TRANSMISSION
OF PARAGONIMIASIS IN JAPANMASANORI KAWANAKA, HIROMU SUGIYAMA AND KEIKO KATO
Department of Parasitology, National Institute of Infectious Diseases

Human paragonimiasis has been reported in mostparts of Japan. In the early 1950s, a total number ofpatients with the lung flukes in Japan were estirnatedfrom three to five hundred thousand. The inhabitants ofendemic areas had a custom of eating crabs, Eriocheirgonimiasis in our laboratory. Among them 23 cases
were from Kyushu and the half (11) of them wereassociated with eating the flesh of wild boar.

japonicus or Geothelphusa dehuani, the second intermedi‑

2 ) The incidence of Paragonimus spp. in boars wasassessed. 100‑200 g of muscle taken frorn each boar
was examined for the larvae. One of 67 wild boars

ate hosts of the lung flukes. Human infection mainly

from Kyushu was found to be infected with theoccurred an accidental transfer of the encysted larvae tolarvae. In addition, a total of 106 serum samples
from different regions were checked specific anti‑
body by ELISA using P. westermani antigen. Out of
59 collected from Kyushu, 44 (75%) were reacted

the mouth through the handling of crabs when preparing
them for food. In parallel with the recent socio‑eco‑nomic changes, the prevalence has dramatically de‑creased with the health education, the use of effective
drugs and changing people's eating habits. On the other
hand, sporadic cases of paragonimiasis due to anotherpositively. Out of 39 collected from the middle part

infection mode, boar‑meat transmission, have been

was found from domestic boars.
3 ) Boar hunters have to be considered at risk forfound in the southern part of Kyushu Island since themiddle of 1970s. It has been demonstrated that wildboars in the endemic area harbored larval forms of P.
westermani in their musdles, and they can easily maturein the definitive host. Many outbreaks in recent years
have been associated with eating the flesh of boar as
wild game in Kyushu.
1 ) During the last three years, a total of 32 casesof Honshu Island (Kinki area), 16 (41%) werepositive. In contrast, no positive case (8 examined)boar‑meat transmission of paragonimiasis. We
made inquiries the hunters about an eating customof the flesh of boars. Four groups of boar‑huntersfrom different regions in Kyushu replied this questi‑

onnaire. More than sixty percent (44 examined) ofhunters have experienced in eating the flesh of boarin the raw (sashimi) .

were diagnosed by serological tests as para‑

53

MORPHOLOGICAL VARIATIONS IN NEXT GENERATION OF
FA SCIOLA GIGANTICA FROM ZAMBIAKUNlO TERASAK11, T. ITAGAK12, TOSHIYUKI SHIBAHARA3 AND YASUTAKA NODA4
St. Mary's Junior College*,

Department of Parasitology, Faculty of Agriculture, Iwate University2,

Laboratory Research Center, Faculty of Medicine, Tottori University" andInstitute of Animal Experiment, Kurume University School of Medicine+

Purpose: Research individual genetic variations in themorphology in F. gigantica.

Materials and Methods

1. Collect F. gigantica from
Lusaka, Zambia. 2. Obtain eggs from a F. gigantica.

139

3. Cultivate at 30'C for about 2 weeks. 4. InfectLymnaea ollula with the hatched miracidia. 5. Keep
L. ollula at 25‑30'C for 30‑40 days. 6. Store themetacercaria in vinyl bag at a low temperature. 7.
Administration of the metacercaria to 6 wild goatsfrom Madara isld., Saga, orally. 8. Obtain adultflukes from biliary ducts 129‑130 days after infec‑
tion. 9. Measurement of body length and breadth.

coefficient of correlation; ‑0.509

Consideration: Results may be considered as follows:
1 ) Variation of body length and body breadth in next
generation from a F. gigantica have wide range, butrange is smaller than the individual variation mea‑
sured by other researchers.

2 ) There is a negative correlation between bodylength and body breadth, and the method of prepar‑
ing specimens yield a difference between both val‑

Results: body lengthXbody breadth=32‑43 x 11‑15(average 36.4 i3.0 X 12.7

1.2)

ues.

54 A CASE OF CYSTIC ECHlNOCOCCOSIS IN A JORDANIAN PATIENT:TREATMENT WITH ALBENDAZOLEMIKIO KIMURA1, AIKICHI IWAMOTO , YOJI NISHIMURA2, MAKI HASHIMOT03,

TAMI EGAWA' AND AKIRA IT05
Department of Infectious Diseases and Applied Immunology*,

Department of Surgery and Transplantation' and Nursing',
Institute of Medical Science, University of Tokyo,

Tokyo International Centre, Japan International Cooperation Agency+ and

Department of Parasitology, Asahikawa Medical College*
A case of cystic echinococcosis (CE) identified in a

Jordanian male is presented. He has come to Japan asa JICA participant and was subjected to abdominal
ultrasonography because of microscopic hematuria,
which incidentally revealed an intrahepatic cystic lesionof 6‑7 cm in diameter with several daughter cysts in its

The results of successive imaging procedures clarified
the amelioration of the CE after the start of chemother‑apy, as revealed by the disappearance of the daughter
cysts and the accumulation of echo‑dense materials.
After three cycles of albendazole he has left Japan as
had been scheduled previously.

inside. Substantially the same cystic lesion was

In Jordan the positive rate for antibodies against E.

revealed by CT scan, which was surrounded by a capsulegranulosus were reported to be 2.4% and 5.8% among

with some calcification. Serological tests with im‑

the general population and those attending outpatientmunoblot using antigen extracts from both Echinococcus
granulosus and E. multilocularis revealed a positive bandclinics, respectively. Another study ddne in those areas

for antigen B from E. granulosus but none for Em 18
from E. multilocularis, a pattern that is compatible with

CE. Because of his special situation as a JICA partici‑
pant, surgical treatments were not adopted and he wasput on the albendazole treatment which consisted of 28days of drug administration and 14 days of cessation.

showed that 13% of sheep and cattle carry the larval
worms, and 14% of dogs carry the adult worms. Physi‑
cian should be alert to the possibility of CE when en‑
countering intrahepatic cystic lesions in individuals
from endemic countries. Application of excellent sero‑
logical methods is recommended to confirm the diagno‑
sis.

140

55 QUESTIONARY TEST AND PREVALENCE OF INTESTlNAL
HELMlNTIC INFECTIONS IN BARRU, SULAWESI, INDONESIA
TAKAKO TOMA1, ICHIRO MIYAG11, HIDEO HASEGAWA2, KIYOSHI KAMIMURA3
YUKO TOKUYAMA1, M. SELOMO', D. DAHLAN4, I. MIJID', I. HASANUDDIN',

R. NGATIMlN4 AND MOTOYOSHI MOG15
Laboratory of Medical Zoology, School of Health Sciences,
University of the Ryukyus*,

Oita Medical School', Toyama Medical and Pharmaceutical Universityi,
Faculty of Public Health. Hansanuddin University, Indonesia' andSaga Medical School=

A questionnaire with parasitological study was
carried out on the inhabitants of 4 villages of Barru
District, Sulawesi Is., Indonesia from 1994 to 1995. A(farm) , seaside or riverside.

A total of 654 fecal samples were examined bymodified Kato‑Katz thick smear method. Fivequestionnaire was made on life style and sanitary condi‑
tions. In 482 houses of 4 villages, interview for items ofnematode species, Ascaris lumbricoides. Trichuris tri‑

questionnaire sheets was made to owner, housekeeper

unidentified Rhabditoids of free‑living nature weredetected. Cestode, Hymenolepis nana, infection wasproven. A11 of hookworm examined by modified Har‑
ada‑Mori culture technique were N. americanus.

and children of same family by us with staffs of Barru

Health Center. In Pancana and Lalolang, 37.7% and
50% of men in the inhabitants surveyed were fishermen,

chiura. Necator americanus. Strongyloides stercoralis andwhile in Lompo Riaja and Pattappa, 38.6% and 65.5%
were farmers. The highest proportion of official
workers was 33.7% in Lompo Riaja. Educational level
was low, and 88.4% of Pancana, 90.4% of Lalolang,

The prevalence of Ascaris and Trichuris infections
was significantly lower in persons who had graduatedsenior high school or university. The prevalence of

62.1% of Lompo Riaja and 91.2% of Pattappa hadelementary or under elementary school education.

persons with latrine or without it, while in the preva‑
lence of Ascaris and Trichuris, there was significant
difference. In persons without own latrine, 62.1% of thepersons had Ascaris and 44.9% had Trichuris, and mostof them use seaside for evacuation. The difference inWhile in Lornpo Riaja, 30.8% of the inhabitants had
graduated senior high school or university. The percent‑age of own latrine was 30.3% in Pancana, 13.2% in
Lalolang, 31.9% in Pattapa and 60% in Lompo Riaja

hookworm infection was not different between the.

Ascaris and Trichuris hookworm infections was notwhich is significantly highest rate. The 5.4% to 11.4% ofshown between the persons with helminthic medicine or

the latrines overflowed in rainy season. The people

without it.

without latrine usually defecated mainly in rice field

56 AN EPIDEMIOLOGICAL STUDY OF MALlNGNANT SKlN TUMORSIN WESTERN KENYA AND NAGASAKI, JAPANKAN TORIYAMA, MASACHIKA ISEKI AND HIDEYO ITAKURA
Department of Pathology, Institute of Tropical Medicine,

Nagasaki University

The skin provides protection against ultraviolet
light and mechanical, chemical and thermal insults andacts as a barrier to invasion by micro‑organisms, but, on

Japan. The cases of the atomic bomb survivors in
Nagasaki were excluded. The most common malignant
skin tumor in western Kenya was squamous cell car‑the other hand, the skin is directly damaged by these

cinorna (SCO , followed by malignant melanoma (MM) ,harmful factors. In this report, we discuss the results ofbasal cell carcinoma (BCO, skin appendage tumor(AT) and Bowen's disease (BD) . In Nagasaki, BCC
was the most common tumor, followed by SCC, BD,

a comparative epidemiological study on malignant skintumors in western Kenya, East Africa and Nagasaki,

141MM, extra‑mammary Paget's disease (PD) and AT.
BCC and BD were rare skin tumors and no case of PD
was found in western Kenya. Although the most com‑mon primary site of SCC in western Kenya was thered in the lower extremities'foot in western Kenya and
45% of the cases in Nagasaki. The higher prevalencesof SCC in the penis were found among the ethnic groups,

who have no custom of male circumcision in western

10wer extremities'foot, followed by the penis and scalp'

Kenya. These findings suggest that the inhabitants inneck, the face was the most common site, followed byities'foot in Nagasaki. Most SCCs developed fromwestern Kenya have a higher resistance against ultravio‑
let light than the peoples in Nagasaki and natural andsocial environments and ecological situations, including

antecedent lesions such as burns, scars and chronic
ulcers in western Kenya. Over 80% of MN cases occur‑

specific customs, influence the occurrence of thesemalignant skin tumors.

the shoulder ' upper extremities 'hand and lower extrem‑

57 A STUDY OF PUBLIC OPlNION SURVEY OF JAPANESE RESIDENTS
LIVlNG IN FOREIGN COUNTRIES REGARDlNG THEIR HEALTH
YUKA HIROSHIGE, EIICHI OKUZAWA, MIZUE HONDA, ATSUO HAMADAAND TETSUO NISHIKAWA
Japan Overseas Health Administration Center

Health problems on Japanese living abroad weresurveyed by questionnaires in order to provide the better

difference of the frequency of abnormal findings at
health checkup before leaving Japan was not statisti‑

health education.
Questionnaires regarding their life and health had

cally significant between developing countries and devel‑

been distributed to Japanese residents through theJapanese societies from January 1995 to November 1997.

health checkup, and caution for foods and water, which
are the preventive measures for health problems, wereTotal 953 answers in 19 countries were returned andmentioned more frequently in those from developing

analyzed.

countries than those from developed countries.

Infectious diseases were the most common illness
which Japanese were afraid of in developing countries,
while "adult diseases" were less common In contrast

A variety of self‑defensive measures have been
performed to prevent infectious diseases by Japanese
residents especially in developing countries. However,"adult diseases" and mental stress were still serious
health problems without any effective countermeasuresin both developed and developing countries. Therefore,
preventive measures for such problems are required.

"adult diseases" were more common in developed
counries than infectious diseases. Mental stress was
mentioned in 30‑40% of the whole subjects, and practi‑
cal troubles were described in 60‑70% of them. The

oped countries. Immunization before leaving, annual

58 A CASE OF AMBL YOMMA TES TUDINA RI UM BITENoRIAKI HARAl, SHICHU HoUl, TATSUYA MIYAZAK11, AKIO YAMAKAGE1,
SOJI YAMAZAKI , SATORU KAWAI', YUICHI CHIGUSA' AND HAJIME MATSUDA'
Department of Dermatology and Department of Medical Zoology',
Dokkyo University School of Medicine
A 73 year‑old man, who lived in Tochigi prefecture,presented with a bean‑sized brownish tick on his rightshoulder. He has been to the mountain in Shizuoka 19
days before, where he seemed to have been attacked.
The lesion was excised with the surrounding skin. The
parasite was identified as an adult female tick named

Amblyomma testudinarium.
A. testudirlarium widely distributes over the SouthAsia, Southeast Asia and the southern and western partof Japan. The patient with tick bite of A. testudinariumis, however, very rarely found in Tochigi which is locat‑

ed in the middle‑northern part of Japan.

142

59 SYSTEM OF DEFENSE FROM ULTRAVIOLET RAYS IN WILD MAMMALSNOBU OHWATARI, EIKO KANEDA, JEONG‑B. LEEI AND MITSUO KOSAKA
Department of Environmental Physiology,
Institute of Tropical Medicine, Nagasaki University

Wild mammals possess systems of life protections
from the ultraviolet rays (VU) . We make clear theologically.

defensive ability and relationship between it and envi‑

significant low, and the damage of skin tissue in ratsronmental conditions. The hair of some species of wild
mammals has a two color structure. It is black near the
body, and is body color the end. The black part of thehair (black layer) may be effective in absorbing UV andcompared with other animals was hard. Because black

infrared radiation. Therefore we investigated theeffects of black layer on skin protection to UV. The
study used Yellow rats ( Citellus dauricus, N = 9) , Squir‑

1 ) Transmittance of UV through the black layer islayer absorbed UV efficiently. This result suggests thatthe black layer has an effective role to protect the skintissue from UV.
2 ) The hair length depends on environmental condi‑
tions in particular temperature, and transmittance of
UV through the black layer is low in longer hair. So

rels (Eutamias sibiricus. N = 12), Jirds (Meriones un‑
guiculatus, N = 15) and Pikas (Ochotona curzoniae, N =efficiency of defense from UV is difference among17) with black layers, and Wistar rats (N=12) fortions.

controls.

3 ) Yellow rats live in where ambient temperaturechanges widely in a day. The hair length of them isshort, but epidermis is thick compared with other

Experiment I: Transmitted UV were measured
through hair and skin tissue, black layer and skin tissue,

and skin tissue. Experiment II: For pretreatment, thespecies depended on their living environmental condi‑

back hair of animals was cut in three ways; all hair wascut, and part of the body color hair was cut, and no hairspecies. Long hair induce increase core temperature, so
thick epidermis protect skin tissue from UV.
These wild mammals possess the protecting ability

was cut. The animals were irradiated by UV‑B at 2.013
mW/cm2 for two days, and the cell degeneration of skinwith black layer of hair and/or thick epidermis toprotect skin from UV, and environmental conditions

and subcutaneous tissues were then analyzed path‑influence the protecting ability.

60 SUPPRESSION OF THE SWEAT GLAND SENSITIVITY TO
ACETYLCHOLlNE APPLIED IONTOPHORETICALLY IN

TROPICAL AFRICANS COMPARED TO TEMPERATE JAPANESEJEONG‑B. LEE1, 2, TAKAO MATSUMOT03, TIMOTHY OTHMANl, JAMES NICHOLAS2

AND MITSUO KOSAKA1
Department of Environmental Physiology,
Institute of Tropical Medicine, Nagasaki Universityl,

Department of Cell Physiology, School of Medicine,

Nagasaki University2 andSecond Department of Physiology, Aichi Medical University*

Tropical inhabitants possess the ability of heat‑
tolerance through permanent residence in the tropics.
Previously, we have shown that tropical African andThai subjects regulate the core temperature with lessarnount of sweat against heat compared to temperate
Japanese subjects and that suppression of sweating in
tropical subjects was attributed to suppression in both

central and peripheral sudomotor mechanisms. To

elucidate the peripheral mechanisms of the suppressed
thermal sweating in tropical natives, sweating responses
to acetylcholine (ACh) , a primary transrnitter of thesudomotor innervation, were cornpared between
Japanese and Africans. All experiments were carriedout in a climatic chamber (24 0.5'C, relative humidity
40 3%) at 2‑5 p.m. between October 1997 and June 1998

in Nagasaki, Japan. Upon arrival into the climatic

143

chamber, the subject wore light indoor clothing and sat
on a chair for 60 min before the experiment. Quantita‑

output per single gland was 20.4% Iarger (P<0.001) in
the Japanese compared to the Africans. The Japanese

tive sudomotor axon reflex test, QSART (LOW et al.,showed the a 0.17'C higher oral temperature and a 0.30'C

1983; Lee et al., 1997), was performed to quantitatively

higher forearm skin temperature compared to theevaluate glandular ACh‑sensitivity. ACh was ionto‑
phoretically administered on the forearm. Directly
activated and axon reflex‑mediated sweat responses
were evaluated by quantitative sudomotor axon reflex
test. The sweat onset‑time was 0.72 min shorter (P<0.01) and the sweat volumes were 72%‑110% higher(P<0.01) in the Japanese than the Africans. Iodine‑
impregnated paper method revealed that sweat glanddensity was 50.6% higher (P<0.001) and sweat gland

Africans (P<0.05, respectively) at rest under a ther‑moneutral condition. ACh iontophoresis did not produce
any influences on oral temperature, but increased the
10cal skin temperature in both the Japanese and theAfricans. These results indicate that suppressed ther‑mal sweating in Africans is, at least in part, attributed

to the suppressed glandular sensitivity to ACh throughboth recruitment of sweat glands and sweat output pereach gland.

61 INSTALLATION AND MAlNTENANCE OF PROTECTIVE NETS AGAlNSTVENOMOUS JELLYFISH ON THE BEACHES OF OKlNAWA
MASAKI SHINJOI AND YASUTETSU ARAKI'
Department of Preventive Medicine, Ryukyu Universityl andOkinawa Prefectural Chuo Health Center'

Venomous marine animals inhabiting the coastal

in Okinawa, and are known to cause sting accidents toareas of Okinawa islands are known to be hazardous oreven fatal to swimmers or divers particularly in thesummer season when marine recreational activities areat their peak. Most of the incidents are caused byswimmers on the beaches.
The net was first installed in the Sunset‑beach(Chatan town) in 1989, but accidents continued eachyear. After making several constructional improve‑

Coelentrata, particularly Chiropsalmus quadrigatus, as

ments to the net e.g., (1) adjusting the gap between theseen in the two fatal cases since last year. This species

net and water surface, (2) narrowing the gap betweenof jellyfish inhabit coastal areas ranging from the Ryu‑

the bottom of the net and seabed, (3) strengthening the

kyu Archpelago to Southeast Asian countries. To avoid
such marine animal‑related fatal accidents, several

both ends of the net, the number of annual incidents
drastically decreased in comparison with numbers ofbeaches in Okinawa have installed protective nets

accidents reported in previous years (5 reported cases inagainst the jellyfish invasion. The following report is1993) .

based on our recent surveys regarding the current status
of installation and maintenance of protective nets in thealmost completely eliminated the inside‑the‑net acci‑island of Okinawa.

dents. However, the following concerns have been(a) Time and location: through August to September

raised over how to increase the efficiency of protection:

Introduction of such improvements to the nets

1998, at 8 beaches equipped with the nets (including1) the seaweed sticking to the mesh, 2) the size of thethe main island of Okinawa and neighboring

mesh, 3) the height of nets, 4) the fixed part of net's bothislands) .

ends, 5) the gap between the net and bottom of the net
and the water surface and the seabed, 6) the rift of themesh, 7) the way of monitoring and recording, 8) coun‑(b) Method of survey: (1) interviewing with those whoare in charge of the management of the protective
nets, and (2) observation of the nets by diving.

In the northeastern coast of Australia, the beachmanagers have installed nets against Chironex fleckerithat belong to the Box‑jellyfish and the nets are foundtermeasures in typhoon seasons.
At some beaches, it was found that the omission ofcleaning nets caused extensive seaweed growth. A
small tidal current, as is often seen at an artificial beach,to be useful for the prevention of jellyfish invasion. C.

is known to offer a favorable habitat for this species ofquadrigatus is the same species as C. fleckeri, can beobserved during a period from June through September

jellyf ish.

144

62

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MARCH1999

VOL．27NO．1

CONTENTS

Original article
Eamsobhana，P．，Watthanakulpanich，D．，Punthuprapasa，P．，Yoolek，A．and
DetectionofAntibodies

Particle

Indirectto勘昭sケon8yl％s

Agglutination

o観孟on6πsゑs

in

Human

Sera

by

Suvuttho，S．
Gelatin

Test……………一・一………一…・…一……・……・…………

・1−5

Ando，K，Kuraishi，K，Nishikubo，K．，Asami，T．，Waighet−Kouadio，P．，Matsuoka，H．
and Chinzei，Y．
Sporozoite Invasion of PZαs窺o伽吻加忽h切，沢odent Malaria Parasite，to the Salivary
Glands of the Vector Mosquito，ノ464εs s妙h6nsJ an Electron Microscopic Study

・7−12

Jutavijitt㎜，P．，Jiviriyawat，Y．，Yousukh，A．，Toriyama，K．，Itakura，H．，Yano，M．andHaya面，S．

A

Seroepidemiological Study on Hepatitis B Virus，Hepatitis C Virus andHuman Immunodeficiency Virus Infections in Northem Thailand・…∵・……・……・…………13−17

Shortcommunication

Guevara，A。G．，RuizC．，」．C．，Houghton，R．L．，Reynolds，L．，Sleath，P．，Benson，D．，

Ouaissi，A．and Guderian，R．H．
Evaluation of a Recombinant Protein（rTc24）and Synthetic Peptides inAnti−丁肋碗oso吻αon観Positive
Chagasic

Proceedings

Endemic

Areas

of

XXXIX

AnnualMeetingofContents・・一一・・・・・…

Prize
President

Special

wimer
s

s

lecturelectureEducational

Samples

from

Blood

Bank

Donorsin

Ecuador…・…・………一…・…・…一……………・……一…

ofJapanese

Society

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