量SSN

Japanese

第15巻

Joumalof

TropicalandHygiene昭和62年6月15日

第2号
内

原

Medicine

O304−2146

容著
フィリピンの日本住血吸虫症低流行地におけるCOP反応の疫学的意義（英文）・田中寛，AhedoT・SantosJr・，松田肇，安羅岡一男，Bayani L Blas，中尾稔，荒木 国興，中村哲」 8欝澹，L伽昭痂およびTりφσ os㎜㎜2∫のハムスターとマウス体内での運命一予報一（英文）・橋口 義久，古谷 正人，岡村 宣典

87−96

Di血sionc㎞ber内Lε醜耀π彪出鷹

短97−104

トリメトプリム・サルファメトキサゾールによる三日熱，卵型および熱帯熱マラリア症例の治療成績（英文）・吉田 幸雄，松本 芳嗣，山田稔，塩田 恒三，吉川岡林 加枝，手越 達也，吉川 哲也，小林 祥男，金尚男，龍起 105−116報エクアドル国のシャーガス病流行地住民集団における遅延型皮内反応（英文）・川端 真人，三森 龍之，橋口 義久，Vicenta肌deCoronel117−120

第27回日本熟帯医学会総会講演抄録（1）目
次……
シンポジウム 皿 アジアにおける慈虫病一会

121−123124−125

一般講演（1）…一
英文抄録……

126−137
139−168報
昭和62年度第1回幹事会記録一169−171

投稿規定

日熱医会誌
Japan．J．T．M．H．

日 本熱帯医学会

Japan. J. Trop. Med. Hyg., Vol. 15, No. 2, 1987, pp. 87‑96

87

EPIDEMIOLOGICAL IMPORTANCE OF COP REACTIONSIN AREAS LIGHTLY INFESTED WITHSCHISTOSOMA JAPONIC UM
IN THE PHILIPPlNESHIROSHI TANAKA1, ALFREDO T. SANTOS, JR. 2, HAIIME MATsuoA1

KAZUO YASURAOKA3, BAYANl L. BLAS2, MINORU NAKA04
KUNIOKI ARAK15 AND SATOSHI NAKAMURA1
Received March 25 1987 /Accepted May 15 1987

Abstract : Dwellers in 6 viuages in Talibon and Tlinidad in Bohol, Philippines with anapproxirnate population of 5, 740 were examined for schistosomiasis japonica by stool examinationand circumoval precipitin (COP) test during the period from 1981 to 1984. Eggs were positivein 3.5, o of 4,117 examinees, whereas COP positives were 9.8% in 4.094 examinees. Since
prevalence of egg positives among COP positives increased from 24. 4 and 24.0% to 79. I and48. l%, in 2 villages after repeated stool examinations, respectively, COP reactions by standar‑

dized method seem to indicate the actual infection of individuals. Among 6 viUages, theprevalence did not vary much between 1.4% to 6. 9% by a single stool examination while there

was greater variation between 3. 2% and 22.2% by COP test. Two villages, San Vicente andSto. Tomas, were identified as the area of highest risk. At San Vicente where no snail colony
had previously been found ; a new snail colony was frst identified at Apao Palawan Swamp in July

1984. The disinbution of households of infected persons showing strong COP reactions led tothe suspicion of presence and the eventual discovery of the snail colonies in the area.

INTRODUCTION
Schistosomiasis japonica is a major health problem in the Philippines, having more than
600, OOO of estimated human infections in endemic areas in 1975 (Santos, 1976). Its endemicity

on Bohol Island was frst proven in 1958 by Blas and Dazo (1968). Schistosomiasis was found in
10calized areas with an exposed population of 6,000 in 6 villages with a prevalence of medium
level at that time. Later, the infestation was reported to be as low as 5. 4% in 1980 (Carney etal. ).

l Department of Parasitology, Institute of Medical Science, the University of Tokyo, Minato‑ku 108, Tokyo

2 Schistosomiasis Control Service, Department of Health, Sta Cruz, Manila 2805, the Philippines
3 Departrnent of Medical Biology, Institute of Basic Medical Sciences, University of Tsukuba, Sakura‑mura

305, Ibaraki‑ken

4 Department of Parasitology, Asahikawa Medical College, Asahikawa City 078, Hokkaido
5 Division of Parasitology, Departrnent of Microbiology, Institute of Public Health, Minato‑ku 108, Tokyo
This study was performed as a part of a collaborative project of the Schistosomiasis Control Service,

Departrnent of Health, Philippines and Sasakawa Memorial Health Foundation, Japan for schistosomiasis
control on Bohol Island, the Phiippines

88

The present program was designed and commenced in 1981 to control schitosomiasis almost

completely at these lightly infested areas by means of selective mass‑chemotherapy withpraziquantel supplemented with snail control by mollusciciding. In this program, all dwellers in 6

infested viuages were examined by circumoval precipitin (COP) test and stool examination from1981 to 1984. Results showed that the prevalence in this area was not so low as had beenreported previously. While working for case detection, important roles of COP test wereclarified in the epidemiological surveys.

The present study demonstrates the value of COP test in case detection as an importantparameter and in finding the snail habitat as the hidden source of infection.

MATERIALS AND METHODS
Locality studied : The area studied consisted of 6 Barangays (villages) in 2 Municipalities,

i. e. , San Agustin and San Roque in Talibon and San Vicente, Mabuhay Cabigohan, Sto. Tomas
and Kinan‑oan in Trinidad which are situated on the northeastern part of Bohol Island. Bohol is

ManilaLuaon(
MindoroMasbate

Samar

>
Panay
Leyte

(S

'e pO

rv

C'

;

. ,

Inlested area

!g

BOhol

Mindanao

Davao

Figure 1

Map of the Philippines showing the infested area on Bohol Island.

89

located southwest of Leyie and east of Cebu, Philippines (Figure 1).

The total exposed population in the 6 villages is about 6,000. The area is in a tropicalclimate with much rain the whole year, especially during the rainy season from July to February.

Vfflages are distributed around a cluster of low limestone hius. Rice fields were developed atdepressed narrow belts among hills through which streams and rivers flowed. These water
pathways originated from a small pond or a boggy area often covered with naturally growing
Palawans. The water is supplied from small springs to these wet areas throughout the year
possibly connected with underground water in the hills.

Stool Examination : Stools were examined by the quantitative cellophane thick smear
method (Kato‑Katz). The amount of stool specimens taken quantitatively in a hole of a punched
cardboard for examirration was 54. 5 mg.
COP test : Blood was collected into a heparinized capillary tube by finger prick and closedwith sealing clay at one end. Tubes containing blood were centriftiged late in the afternoon on

the same day and kept standing in a refrigerator. The following morning plasma was separated

by cutting tubes and used for COP test.

COP reactions were performed following the technique by Matsuda et al. (1977) by which

sensitivity was 97.3% using plasma among egg positive persons, and gave no false positive
reaction. Lyophilized eggs were used as antigen and results were read 48 hours after incuba‑tion in a wooden box heated with electric bulbs at 37'C in the day time and not heated at night.
Survey Procedures : Every year, a total of about 1,000 people in I or 2 viffages were
examined. Before sample collection, composition of households was surveyed in the target
population and each person was given an individual number. In surveys up to 1984 before masschemotherapy in each village, people were examined for eggs in stool and COP re ctions withplasma. For mass chemotherapy, praziquantel at a dose of 20 mg/kg was given 3 times in aday .

RESULTSl. Detection of Eggs among COP Positives
Attempt was made to prove actual infections among COP positives by repeated stool
examinations (Tables I and 2). In San Vicente Vmage, the number of COP positives was 103 in

1984. Among then, 86 were examined for Schistosoma eggs in stool by Kato‑Katz method, and21 were found to be positive for eggs during the Ist examination. In the 2nd stool examination,
Table I Increase of egg positives among 86 COP positives after repeated stool examinations by
Kato‑Katz method at San Vicente Village, Trinidad, Bohol, Philippines in 1984

Cumulative
Repetition

No. exam.

No. egg+
+ no.

% +*

21

24. 4%

1

86

21

2

51

43

64

74. 4

3

4

4

68

79. 1

Total

86

68

* Percentage of an initial 86 COP positives.

90

eggs were found in 43 additional patients out of 51 stool samples submitted out of 65 egg
negatives during the Ist examination. In the 3rd examination, 4 cases out of 4 samples
submitted were further found to be egg positive. Of the COP positive group 24.4% werepositive for eggs during the Ist examination and this rose to 79. 1% aiter a 3rd repeat
examination (Table 1).

In the same way, 104 COP positives were examined repeatedly for Schistosoma eggs by
MIFC method at San Roque in 1985, where infestation was lower than in San Vicente Vinage.
In this positive group, 1 1 cases who had been treated with praziquantel before 1985 were
excluded. At each fecal examination, those who were not detected positive for eggs wererequested for further repeated exarninations. By the 5th repeat stool examination, 24. O% of
egg positives among COP positives at the Ist stool examination increased to 48. 1% (Table 2).

Table 2 Increase of egg positives among 104 COP positives after repeated stool examinations

by MIFC method at San Roque Village, Talibon, Bohol, Philippines in 1985

Cumulative
Repetition

No. exam.

No. egg+
+ no.

% +*

l

104

25

25

24. O%

2

49

10

35

33. 7

3

29

7

42

40. 4

4

15

4

6
2

48
50

46. 2

5
Total

l 04

50

48. 1

* Percentage of an initial 104 COP positives.

Since the number of egg positives increased with the repetition of stool examinations andthere were likely non egg‑passers among infected people, COP reactions by this standardized
method were considered to indicate the level of infection which was closer to the actual level
than indicated by a single stool examination.

2. Difference of Infestations among 6 Viuages
In the period from 1981 to 1984, most dweuers in 6 infested villages were examined by bothstool examination and COP test (Table 3). Since the COP test was not conducted at San Roquein 1981, date in 1985 is presented in Table 3. The population within project area based on thehousehold survey was 5,735 out of which 4, 1 17 at a coverage of 71.8% were examined for eggs

and 4,094 or 71.4% were examined by COP test.
In Table 3, results of repeated stool examinations among COP positives at San Vicente andKinan‑oan are presented. If results of the repeated stool examinations are disregarded, percent
positives by a single stool exantination ranged from 1.4% at Kinan‑oan to 6. 9( o at San Vicenteand the difierence among viuages was not so large. The highest prevalence was noticed in San

Vicente fonowed by San Roque and Sto. Tomas.
The results of COP test, however, showed much higher prevalence and a clear difference
among viuages. The highest prevalence was observed in San Vicente fouowed by Sto. Tomas

91

Table 3

Survey results of schistosomiasis japonica by stool examination and COP test in 6
vinages before mass‑chemotherapy on Bohol, Philppines during the period from 1981

to 1984

LocalityYear

TALIBONSan Agustin 1983

San Roque 1981

Po pula‑tion

Stool exarnination

No. exam.

+ no.

cOP test%+

No. exam.

+ no.

%+

1 , 983

1, 137

18

1.6

1,000

32

3. 2

1,314

1, 12457

5. 1

1, 182*

95

8. O

542

461

32

6. 9

464

1 03

22. 2

(465**

72

15. 5)

448
430
495

12

2. 7

52

10. 1

19

4. 4

517
389

83

21.3

7

1.4

542

37

6. 8

(513**

32

6. 2)

4,095

145

3. 5

4, 094402

9. 8(4, I17***

210

5. 1)

TR JNIDAD

San Vicente 1984
M. Cabigohan 1982

668

Sto. Tomas 1982

558

Kinan‑oan 1984

670

Total

5, 735* Performed in 1985.
** Repeated stool examination after COP test.
*** Results including repeated stool examinations.

and M. Cabigohan, where more snail colonies might exist and wider water areas may becontaminated with cercariae.
Results of COP reactions were also valuable in indicafing the lirnited area of highest risk

among infested areas.

3. Detection of the Snail Colony
In San Vicente Viliage, there were no snail colonies hitherto found, although the prevalenceof schistosomiasis was revealed to be 22.2% by COP reactions in 1984.
Considering this high prevalence, presence of snail colonies in this viuage was strongly

suspected. Thus, attempts were made to determine the presence of snail colonies based on theepidemiological survey data.

On a sketch map of this viuage showing distribution of households, egg positives weremarked (Figure 2). Positives were found mostly at the southern 1/4 region of this village. On
the other hand, COP positives scattered more evenly and wid ly over the village with lesser
distribution at northeast region and dense accumulation at west central and southern regions(Figure 3).

When positives with strong COP reactions (type 3 of Yokogawa et al. , 1967 or segmented
precipitation of Yogore et al. 1968) are taken into consideration (Figure 4), positives areconcentrated at the southern region over an area from the fish pond to the upper portion of rice

fields which extend from Apao Swamp to San Vicente Creek. ,Surveys were made to search for swamps at that area and Apao Swamp with naturally
growing Palawans was found. And colonies of intermediate snail host, Oncomelania quadrasi,

92

West

S

!

Ro(pe

/ Tahbon / lNorth
to

･xt

ll

Omaguin Creek

X.

x‑

JL JL

Lsn:

.'

; ..

uth i

Elementary
SehoolVillageHall' APao "e'1

JL JLrSwem"O 'ChapelJL'JL '': JJ!

1

T

Tl

LegendnHouseholdT' O. .' LL JL" T Coconut plantation
X Egg positive

JL
JL

T TT JL JL

n

Pl
l

500m

JL Rice Field

JL

JL

JL
JL

JL
JL
East

JJh JLcreek JL
JL San vicente

JL to
JL T!illidad
JL

Figure 2 Distribution of egg positives at San Vicente Village in July 1984.

were readily discovered there in July 1984 in this viuage. However, no more snail habitats were

found at that time either at upper portions of rice fields or around fish ponds and connecting
canals.

DISCUSSIONSCOP reactions have been widely used for case detection of infections with Schistosoma
mansoni and S. japonicum. There were a few modified procedures of COP technique for S.
japonicum (Yokogawa et al. , 1967, Yogore et al. , 1968, 1979 and Matsuda et al. , 1977). The

highly sensitive method by Y̲okogawa et al. (1967) was effectively used as the screening
diagnostic technique for case finding in an area with a very low prevalence like in Japan.

However, in a highly prevalent area, the sensitive method picks up too many suspectedcases among examinees, including a high ratio of false positives. Consequentiy, nealy all the
population should be examined again by another method, more specifically by stool examination,and consequently the COP test looses its value as the Ist screening test.
In schistosomiasis prevalent area, sensitivity of COP reactions should be properly standar‑
dized (Yogore et al. , 1968, Matsuda et al. , 1977) so that false positives are avoided, even if a few

may be missed from detection. By the technique of COP test with plasma used in this study,

93
X.x
West

San Roque

,c:

,C.,C

ll

,,;'

,s ,,,,

S

,,x.

'$'ix

X'

/ :N.rtbt'/Taubo*i,, Omaguin Creek

,,.

:,.

JL

;

ison

x

l

;dJj,, S

JL

South

,,

X APao,C
x "'C

Elementary ,S ,e

XX

x. x.

,, ･

'$

r Village

,s

X

,e.

Scbool･ Hall1*' swamp
'il'
JL
ll

x

,s,,

Chapel

'O
JJ!JL

T

"c.

JL

, .

n

Pl"

Legend. Household

1

,,.x

JL

,,.

1

̲ '

,( Q..

T

,c

T

n‑

l

JL RiceFleld
T Coconut Pkntation

x‑

T

T

X COP positiveT

vit' c*ek

JL

5(X)m

Figure 3s‑,,.

JL JJLJL
toTrinidadDistribution of all COP positives at San Vicente Village in July 1984.

97. 3% of egg positives showed positive COP reactions and there was no false positive (Matsuda
et al. , 1977).

The COP test is useful in 3 instances at least. Firstly, in epidemiological surveys, thereaction may indicate the actual infection of individual persons, irrespective of evacuation of eggs

in stool. In the 2 villages studied which had low infestation, repeated stool examirration showed

egg positivity rate of 79. I and 48. 1% respectively among COP positives. Yogore et al. (1981)

compared results by ELISA, COP and stool examination (MFCT), and found additional egg
positives after repeated stool examinations only among serologicauy positive individuals.‑ Andthey concluded that repeated stool examinations are necessary as the results by stool examina‑tion underestimate the true infection.

Since some infected people remain negative for eggs due to light infections, very chronic
infections, early stage of infections or shortly after treatment with paraziquantel, it seems that

COP positives detected by this technique in this area are persons highly suspected with
Schistosoma infection and more egg positives will be found by repeated stool examinations or bybiopsy method.
The results by COP provide a good indication to a medical doctor in deciding treatment ofsuspected schistosomiasis even if detection of eggs was negative.

94
,c.x
West

San Roque?<.

';. ,C // North/Talibon. /:X ' omaguincreek

/

' 'C.
X.

JL ;dJj
isondh/' 'fge '. 'C'

Elementary
school

X'

South

Apao

' swamp

; ･･'

B ^

X

' 'O ' chaPel

1

JL

n

' Household

TX

JL X.X

T

.

1

JL

T coconut plantaton

T

LLJJL

T

JL

JL JL san vicente creek JL
JL

JL

T
Figure 4 DiStributiOn Of Strong reactiOnS Of COP (Type 3 Of YOkOgawa et al' ) at San ViCente village inJuly 1984.

The second importance of COP test is to show the infection rate closer to the true rate oftarget populations which is 3. 5% by stool examination and increased to 9.8% by COP test in 6
villages. The small difference in prevalence among vmages by stool examination was magnified,and villages with high prevalence were clearly pointed out by COP test. From the results, 2viuages, San Vicente and Sto. Tomas, were identified as the areas of highest risk, andconsequently the target area of this control program.

The 3rd application of COP reaction in the epidemiological survey was the discovery of newsnail colonies. For this, distribution of strong reactions of COP test was useful. In San
Vicente, no snail colonies were found until 1984, even though schistosomiasis among people had

been found as early as 1958 by Blas and Dazo (1968). From the distribution of strong COP
reactions, suspected foci of infection were delineated in a map, and finauy, the snail colony was

found at Apao Palawan Swamp in July 1984. This discovery led later to that of 3 more snailcolonies around the fish pond and further western region in this viuage.

ACKNOWLEDGMENTS
The present authors wish to show their gratitude to all Philppine members assigned to the95

National Schistosomiasis Control and Research Team at Trinidad, Bohol, especially the present

team leader, Dr. Victoriano Guiritan, and previous leaders. Mr. Rogelio Pangilinan, Miss
Crispina Hinlayagan and Miss Rosalina B. Pacheco.
They would like to dedicate tbis publication to the late Dr. Julian S. Nesefias who has
devoted his whole lif;e to the schistosomiasis control of the Philppines as a staff in the
Schistosomiasis Research and Training Center, Palo, Leyie and as the deputy executive director

in the Schistosomiasis Contorol and Research Service, Ministry of Health, Manila, and who
contributed greatly to this joint program in working on Bohol every year and passed away ofheart attack on August 6, 1985.

REFERENCES
1) Blas, B. L. and Dazo. B. C. (1968) : Schistosomiasis survey and preliminary control work in theprovince of Bohol. J. of Phiippine Med. Assoc. , 44, 80‑87
2) Carney, W. P., Banzon, T., de Veyra, V. and Cross, J. H. (1980) : Intestinal parasites of man innorthern Bohol, Philippines, with emphasis on schistosomiasis. Southeast Asian J. Trop. Med. Pub.
Hlth., 11, 473‑479
3) Matsuda, H., Nosefias, J. S., Tanaka, H. , Santos, A. T. Jr. and Trinidad‑Perez, D. (1977) : Compara‑tive studies on reading criteria of circumoval precipitin reaction of Schistosoma japonicum for field survey

in highly endemic area. Japan. J. Exp. Med. , 47, 369‑375
4) Santos, A. T. Jr. (1976) : Prevalence and distribution of schistosomiasis in the Philippines: A review.

Southeast Asian J. Trop. Med. Pub. Hlth., 7, 133‑136
5) Yogore, M. G. Jr., Lewert, R. M. and Silan, R. B. (1968) : The circumoval precipitin (COP) test in
schistosomiasis japonica. Amer. J. Trop. Med. Hyg. , 17, 65‑71
6) Yogore, M. G. Jr. , Lewert, R. M. and Blas, B. L. (1979) : Schistosomiasis japonica in Barrio San
Antonio, Basey, Samar in the Philippines : 111. The plasma circumoval precipitin test‑procedure and use

in epidemiologic studies. Southeast Asian J. Trop. Med. Pub. Hlth. , 10, 23‑31

7) Yogore, M. G. Jr. , Lewert, R. M. and Blas, B. L. (1981) : Schistosorniasis japonica in Barrio San

Antonio, Basey, Samar in the Philippines. V. The enayme‑linked irnmunosorbent assay (ELISA)
compared with quantitative stool examination and the circumoval precipitin (COP) test. Am. J. Trop.

Med. Hyg., 30, 1252‑1262
8) Yokogawa, M. , Sano. M. and Araki, K. (1967) : Inununosero‑diagnosis of schistosomiasis japonica. 3.
Circumoval precipitation test. Japan. J. Parasitol, 16, 77‑84 (in Japanese with English Sununary)

96

フィリピンの日本住血吸虫症低流行地におけるCOP反応の疫学的意義
田中寛1・ALFREDoT．SANTos，JR．2・松田

安羅岡一男3・BAY姻I荒木L

B猛s2・中尾

国興5・中村肇1

稔4

哲1

フィリピン，ボホール島のタリボンとトリニダッド町内の6村落の約5，740人を対象に，1981年より1984年までに日本住血吸虫の虫卵検査とCOP反応を行った。被験者4，094の内，虫卵陽性率は3、5％であったが，COPでは9。8％であった。2村落におけるCOP陽性者の反復した検便の結果，COP陽性者中の虫卵陽性率は当初の24．4および24．0％から79．1および48．1％へと上昇した。この事から，よく標準化した手法でCOP反応を行えば，住血吸虫の真の寄生率が得られる事が示唆された。6村落
問の虫卵陽性率はL4％から6．9％と差は小さく，COPでは3．2％から22．2％と大きな差を示し，サンビセンテとサントトーマスの2村落が最大の危険地域である事が明瞭に示された。サンビセンテでは

1984年以前には，中間宿主貝の生息地は発見されていなかったが，COP反応強陽性者のいる家族の村
落内の分布調査から，パラワン芋の茂る，アパオ湿地と呼ばれている場所で多数の貝を1984年7月に初めて発見できた。

1東京大学医科学研究所
3筑波大学基礎医学系

2フィリピン保健省住血吸虫対策局
4旭川医科大学
5国立公衆衛生院

Japan. J. Trop. Med. Hyg.. Vol. 15, No. 2, 1987, pp. 97 104

97

THE FATE OF LEISHMANIA BRAZILIENSIS, L.
DONOVANI AND TR YPANOSOMA CR UZI IN

DIFFUSION CHAMBERS IMPLANTEDINTO HAM:STERS AND MICEA PRELIMlNARY STUDYYoSHIHISA HASHIGUCHll, MASATO FURuvA2 AND YoSHISUKE OKAMURA1
Received January 22 1987 IAccepted March 30 1987

Abstract : Leishmania braziliensis and L. donovani were investigated for the transforma‑tion and survival in intraperitoneal (IP), subcutaneous (SO and intrascrotal (IS) difftisionchambers implanted into bamsters and mice. For a comparison, Tmpanosoma cruzi was also
examined by using the same procedure. The 2 Leishmania species revealed an unexpectedly
short survival time, and no transformation was observed in the parasites in chambers implanted

into hamsters or mide. IS chambers seemed to provide a better condition for L. donovani, L.
braziliensis and T, cruzi, as compared with IP and SC chambers in hamsters. In the study, no

IS chambers were examined in mice because of too small size of the scrotum to insert thedifusion chamber. T. cruzi showed a considerably longer period of survival than L. donovani or

L. braziliensis in mice, but not in hamsters. The trypanosome, T. cruzi, transformed from
epirnastigote to trypomastigote and amastigote in IP and SC chambers in mice. These results
seemed to suggest that the factors responsible for the transforrnation and survival of theorganisms might be greatly different between the 2 genera, Leishmania and Tmpanosoma, andalso between the 2 host animals, hamsters and mice.

INTRODUCTION
The parasite of the genus Leishmania is an obligatory intracellular organism as amastigote invertebrate hosts and extracellular one as promastigote in invertebrate hosts. Using such a
protozoan, it seems to be interesting to make analysis in vivo on the interrelation between theparasite in chambers and the host anirnals, by implanting difftision chambers which separate theparasite from a drect physical contact with various host cells by microporous filters. Providingthat the organisms survive, and preferably transform and differentiate in the chambers implanted,

they provide a useful model for investigating both biology of the parasite and development of

irnmunity in the host. Diffusion chambers allow exchange of various difftisible substances
between the parasites in chambers and their hosts. Moreover, if desired, the chamber may beeasily retrieved for examination of the implanted organisms. On the basis of these advantages,

difusion chambers have been used to culture in vivo several species of parasites, and toimmunize host anirnals against protozoans and helrninths.

In Tmpanosoma cruzi, morphogenesis of the parasite and development of the host irnmunity
1

Department of Parasitology, Kochi Medical School, Nankoku City, Kochi 781‑51, Japan

2 Institute for Laboratory Anirnals, Kochi Medical School, Nankoku City, Kochi 781‑51, Japan98

have been studied by using diffirsion chambers implanted intraperitoneally into mice (Logan and

Hanson, 1974). Moreover, in subcutaneous diffusion chambers, T. brucei, T. rhodesiense andT. gambiense multiplied rapidly, persisted for as long as 5 weeks and expressed antigenic
variation (Balion‑Landa et al. , 1985).

In contrast to the genus Tmpanosoma, no such a study has been attempted in the genus
Leishmania. In the present study, therefore, a preliminary trial was undertaken to determine
whether diffusion chamber is suitable for obtaining information on the interrelation between theimplanted parasites and the hosts. As the frst step, the transfonnation and survival of L.
braziliensis and L. donovani were examined in intraperitoneal, subcutaneous and intrascrotalchambers. In the experiment, for a comparison, T. cruzi was also examined by using the sameproce dure .

MATERIALS AND METHODS
Host animals
Adult male BALB /c mice and Syrian hamsters, Mesocricetus auratus, weighing around 30 g

and 130 g, respectively, were used. They were fed with a conunercially prepared diet andwater was provided ad libitum.
Parasites and the in vitro cultivation

The organisms studied were 3 species of protozoans, L. braziliensis. L. donovani and T.
cruzi. The 2 species of Leishmania were kindly supplied by the Department of Parasitology,
Keio University School of Medicine, Tokyo, Japan, and T. cruzi was obtained from a patient with

Chagas' disease in Guayaqul City, Ecuador. The parasites were maintained at 26'C in a serial

in vitro passage, as promastigote in Leishmania and epimastigote in Tmpanosoma, in culture
medium (Aljeboori, 1979) ; a part of in vitro culture was performed by using Pan's medium (Pan,1984). No difference was recognized on the transformation and survival between the parasites

derived from the 2 culture media used. Au the materials used for inoculation of diffusion
chambers were from stationary phase of the culture.
Dtffusion chambers and the implantation or removal
Diffusion chambers were made of a plexglass ring (U‑lOO, diameter : 10 mm, thickness : 2mm) and mimpore filters (pore size : O. 22 pm). The chambers were constracted by cementing
filters to either side of a ring with MF cement (Millipore Filter Co. ). Difftision chambers weresterilized with U. V. for ovemight in the experirnent. These chambers were aseptically ffledwith O. I ml (3 x 105 to I x 106 cells) of the parasite suspension. After loading, the access holeof each chamber was sealed with a plastic plug and then covered with a drop of MF cement andallowed to dry before implantation.
For implantation of a diffusion chamber, the hak of the abdonainal and scrotal regions of theanimals was clipped with an electric clipper, the anirnals anaesthetized with Nembutal, and theclipped regions were washed with , 70% ethanol. An incision about 1.5 cm long was made andthen difftision chamber containing parasites was placed intraperitoneally, subcutaneously andintrascrotally. The incision was closed with sik sutures.

At the removal and examination of these chambers, the animals were anaesthetized andsurgically opened, and the chambers were removed and examined for the parasites at intervals.

99

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The surviral rate, expressed as % recovery (each point recovery no. /initial no. inoculated x 100),of L. braziliensis. L. donovani and T. cruzi in intraperitoneal (IP), subcutaneous (SO and intras‑

crotal (IS) chambers in hamsters. Each point shows mean % of 2 to 4 chambers in which O. I ml/

chamber of promatigotes or epimatigotes (3 x 106 to I x 107/ml) was inoculated. O O, L.
braziliensis in IP chambers ; O e, L. braziliensis in SC ; A A, L. braziliensis in IS ;[1]. L. donovani in IP; I I, L. donovani in SC ; A JL, L. donovani in IS ;O‑‑‑‑O, T. cruzi in IP; e‑‑‑‑e, T. cruzi in SC; l‑‑‑‑1, T. cruzi in IS.

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Figure 3Morphogenetic process of T. cruzi in intraperitoneal (IP) and subcutaneous (SC) chanibers inmice. Each point indicates the rate, expressed as % recovery, of each stage calculated bycounting around 100 protozoans in stained specirnens. O O, epimastigotes in IP ;A, amastigotes in IP; O O, epinrastigotes in SC ;[] [], trypomastigotes in IP; A
JL, amastigotes in SC.
I I, trypomastigotes in SC ; A.
Figure 2

The survival rate, expressed as % recovery, of L. braziliensis. L. donovani and T. cruzi inintraperitoneal (IP) and subcutaneous (SO chambers in mice. Each point shows mean % of 2
to 4 chambers in which O. I ml/chamber of promastigotes or epimastigotes (3 x 106 to I x 107lml)

was inoculated. O O, L. braziliensis in IP chambers ; e o, L. braziliensis in SC ;[] [], L. donovaniinlP; I l, L. donovaniinSC; O‑‑‑‑O, T. cruziinlP; O‑‑‑‑e,T. cruzi in SC.

l02

A sample of the chamber contents was recovered by insertion of a 27 guage of syringe, and thenumber of motile parasites was deterntined by using haemocytometer. An additional sample ofthe chamber contents was recovered, smeared on a clear micros.copic slide, dned, fixed inmethanol, and stained in Giemsa's or Wright's staining solution. The morphological stages
present in the chambers were studied from these stained specimens with the aid of a compound

microscope (1,000 X ). In some cases, a chamber was examined several times at intervals, byrecovering a small quantity of chamber fluid, and then reimplanted into the same host ariunal.

RESULTS
In hamsters, L. braziliensis, L. donovani and T. cruzi showed an unexpectedly shortsmvival in intraperitoneal (IP) and subcutaneous (SO chambers (Figure 1). No marked site
preference of the organisms was observed between IP and SC chambers. All the 3 species ofparasites, however, tended to demonstrate a longer time of survival in intrascrotal (IS) chambers

than in IP and SC ones. In the host animal, no transfomation of the 2 genera, Leishmania andTmpanosoma, was observed in IP, SC and IS chambers, and the majority of the parasites
revealed a small, round and dwarf form with an extremely short flagellum.
In mice, almost all of the leishmanial parasites, L. braziliensis and L. donovani, have died in

IP and SC chambers within I day after implantation (Figure 2). T. cruzi, on the other hand,survived well in both IP and SC chambers. The organisms in SC chambers multiplied during 2
and 3 days after implantation, but thereafter they gradually decteased and reached 34% of theinitial numbers, on day 5. In L. braziliensis and L. donovani, no transformation of the parasites

was observed, while T. cruzi transformed into trypomastigote and amastigote in IP and SC
chambers, as shown in Figure 3. In IP chambers, transformation of T. cruzi from epimastigote
to trypomastigote occurred at 65% on the day I when 15% of the parasites were recovered as
amastigote form. On day 5, around 90% of T. cruzi was recognized as amastigote in both IP
and SC chambers, but a small rate of the parasites still remained epimastigote or trypomastigote.

In the present study no IS chamber was examined in mice, because of too small size of thescrotum to insert the diffusion chamber.

DISCUSSION

The current study was designed to investigate the transformation and survival of L.
braziliensis and L. donovani, together with T. cruzi, in IP, SC and IS chambers in hamsters andmice. In the 2 species of the genus Leishmania, however, an unexpectedly short survival of theparasites was observed especially in IP and SC chambers in the both host anirnals, though theparasites in IS chambers revealed a relatively good result. The parasites, furthermore, could

not perform transformation and most of them were aberrant forms of the promastigote with anextremely short flagellum. The results obtained indicated that the present condition of cham‑bers implanted was not suitable for L. braziliensis and L, donovani. But, it was noticeable thatthe leishrnanial parasites tended to survive for a longer time in IS chambers than in IP and SC

ones in hamsters. The precise reasons for this better survival were not clear, but it might bedue to lower temperature of the scrotum as compared with other body sites, and also due tosome inununological and physiological conditions of the organ. It has been well known that the

intrascrotal temperature in mammals is considerably below the general body temperature

103(Baker, 1986). The intrascrotal region of hamster, therefore, might be a site of choice for the

Leishmania species in such a study.
T, cruzi, on the other hand, survived well performing morphogenesis in IP and SC chambers

in mice, but not in hamsters. As to trasformation of trypanosomes, temperature has been
believed as an important factor by several workers. In T. cruzi, most of the parasites could bestirnulated to transform into trypomastigote, by raising the temperature to 37'C and lowering the

pH in axenic culture or tissue culture (Pan, 1971 ; Trejos et al. , 1963). However, there areother facts that T. cruzi did not transform in the difftlsion chambers either maintained in in vitro

cell culture at 37'C or in several types of in vitro culture media at 26'C or 37'C (Logan and
Hanson, 1974). Thus, the infiuence of temperature might be relatively minor in the transforma‑tion of trypanosomes, as compared with other factors (Logan and Hanson, 1974). The
morphogenetic process of T. cruzi was established when the parasite was maintained in IP
chambers in mice (Logan and Hanson, 1974), as same as the present study. Therefore, thefactors necessary for the transformation of these protozoans should be further studied in future

by using in vitro and in vivo systems.

From the results obtained, it was concluded that the leishmanial parasites, obligatory
intracellular organisms in marnmalian hosts, c9uld not survive for a long time and could not
perform transformation in diffilsion chambers implanted into hamsters or mice. The results alsosuggested that the factors responsible for the transformation and survival of the organisms might

be greatiy different between Leishmania and Tmpanosoma, and also between the 2 host animals,

hamsters and mice.

ACKNOWLEDGEMENTS
We thank the late Professor K. Asami, Department of Parasitology. Keio University Schoolof Medicine, Tokyo, Japan for kind supply of L. braziliensis and L. donovani. Thanks are also
due to Drs. N. Suzuki and H. Osaki of Kochi Medical School, Kochi, Japan for their encourage‑

ment throughout the study.

REFERENCES
1) Aljeboori, T. I. (1979) : A simple diphasic medium lacking whole blood for culturing Leishmania spp,

Trans. Roy. Soc. Trop. Med. Hyg., 73, 1172) Baker, J. R. (1986) : Intrascrotal development of trypanosomes, Parasit. Today, 2, 249
3) Ballon‑Landa, G., Douglas, H. , Colmerauer, M. M. , Goddard, D. and Davis, C. E. (1985) : Growth andantigenic variation of Tmpanosoma brucei. T. rkodesiense and T. gambiense in subcutaneous millipore

chambers, Trans. Roy. Soc. Trop. Med. Hyg. , 79, 24‑28
4) Logan, L. L. and Hanson, W. (1974) : Tmpanosoma cruzi : Morphogenesis in diliusion charnbers in themouse peritoneal cavity and attempted stirnulation of host irnmunity, Exp. Parasitol. , 36, 439‑454
5) Pan, A. A. (1984) : Leishmania mexicana : Serial cultivation of intracellular stages in a cell‑free medium,

Exp. Parasitol. , 58, 72‑80

6) Pan, C. T. (1971) : Cultivation and morphogenesis of Tmpanosoma cruzi in improved liquid media, J.
Protozool. , 18, 556‑560
7) Trejos, A. , Godoy, G. A. , Greenblatt, C. and Cedillos, R. (1963) : Effect of temperature on morpholo‑
gic variation of Schizotmpanum cruzi in tissue culture, Exp. Parasitol. , 13, 211‑218

104

D血1sionchamber内L鵡h耀痂伽z∫」吻廊，L．40 o槻勉およびTりψ伽oso耀on廊のハムスターとマウス体内での運命
一予報一橋口

義久1・古谷正人2・岡村宜典1

宿主動物とL廊伽8物伽卿」伽廊およびL．40η0耀痂との相互関係を伽吻0で解析する一つの試み
として，上記原虫をd血sion chamberに封入，ハムスターおよびマウスの皮下，腹腔，陰嚢（ハムス
ターのみ）へ外科的に移植，継時的にchamberを回収し，まず，それらの世代転換（transformadon）や生存を調べた。また比較のため，Tり卯

030耀o麗ゴについても同様な実験を試みた。その結果，2種のL廊あ㎜吻はハムスターおよびマウスの皮下，腹腔内では，極めて短期間の生存を示したが，ハムス
ターの陰嚢内chamberではより長時問生存した（図1，2）。しかし，いずれの場合にもpromastigoteからamasdgoteへの転換は認められず，原虫は短鞭毛・小形の移行型に止まった。一方，T

捌zゴはハムスターでは生存が短いものの，マウスでは腹腔と皮下の両移植部位において5日以上にわたって
生存し，ep㎞asdgoteからtrypoma甜gote，amas廿goteへの転換も認められた（図3）。以上の結果は，

L8勧伽痂属と丁帥α㎜s㎜属間での世代転換が極めて異なること，また両属原虫の生存期間は，
di血sionc㎞ber内という特殊な条件下においても，宿主動物の種によって異なることが示唆された。

1

高知医科大学寄生虫学教室

2高知医科大学動物実験施設

Japan. J. Trop. Med. Hyg., Vol. 15, No. 2, 1987, pp. 105‑116

105

CLINICAL TRIAL OF TRIMETHOPRIM‑SULFAMETHOXA‑ZOLE IN THE TREATMENT OF VIVAX, OVALE
AND FALCIPARUM MALARIAYUKIO YOSHIDA2, YOSHITSUGU MATSUMOT02, MlNORU YAMADA2TSUNEZO SHIOTA2, HISAO YOSHIKAWA2, KAE OI BAYASH12
TATSUYA TEGOSH12, TETsuvA YoSHIKAWA2AKIO KOBAYASH13 AND RYUKI KIN3
Received January 22 19871Accepted March 30 1987

Abstract : A compound of trirnethoprirn (TMP) and suffamethoxazole (SMZ) was effectivein the treatment of vivax, ovale and falciparum malaria. Two patients with vivax malaria
received 16 mg TMP/80 mg SMZ/kg body weight in 3 divided doses daily for 3 days. A patientwith ovale malaria received 10 mg TMP / 50 mg SMZ/kg body weight in 4 divided doses daily for

4 days. These doses were followed by 15 mg prirnaquine daily for 2 weeks. A patient withfalciparum malaria received 16 mg TMP / 80 mg SMZ /kg body weight in 3 divided doses daily for

3 days. In all patients, radical cures were achieved ; neither relapse nor recrudescence has

occurred. Trirnethoprirn‑sulfamethoxazole succeeded in causing rapid clearance of asexual‑
parasites from peripheral blood of the patients and producing rapid defervescence. No symp‑

tomatic adverse reactions to TMP‑SMZ were experienced.

INTRODUCTION
In Japan, indigenous malaria (falciparum, vivax and malarial malaria) was eradicated by the

1960s. However, the problem of malaria imported from other parts of the world has become
more serious since 1970. During 1972‑1981, 697 imported cases of malaria, including 15
deaths in falciparum malaria, were reported in Japan (Ohtomo and Hioki, 1985). Most of thestandard anti‑malarial drugs including amodiaquine, chloroquine, proguanil, prirnaquine andpydmethamine are not commercially available in Japan. Physicians are anticipating anti‑malarial

drugs which can be easily obtained in Japan.

The effectiveness of trimethoprirn‑sulfamethoxazole (TMP‑SMZ), which is commerciallyavailable in Japan, in the treatment of falciparum and vivax malaria has been reported.
However, no study has been done on the efficacy of TMP‑SMZ in treafing ovale malaria. The
present paper describes satisfactory therapeutic results of this compound in the treaiment ofsymptomatic vivax, ovale and falciparum malaria.

1 Contribution No. 562 from the Department of Medical Zoology, Kyoto Prefectural University of Medicine

2 Departrnent of Medical Zoology, Kyoto Prefectural University of Medicine, Kyoto OO2, Japan

3 Department of Communicable Disease, Kyoto Municipal Hospital, Kyoto 604, Japan

106

METHODS
The cases in the present study include 2 vivax malaria patients (Case I and 2), one ovale

malaria patient (3) and one falciparum malaria patient (4). Au patients were hospitalized andexamined as follows : 1) Thin filrns of peripheral blood were examined for identification of
species and stages of malarial parasite and for parasite counts. The number of parasites / 200

white blood cells were converted into the number of parasites per pl (parasite count) by
calculations based on white blood cell counts taken on the same days. 2) illary temperature
was taken. 3) Routine hemograms were done, recording the number of red blood cells lpl(RBO, the ratio of reticulocyie to whole RBC (Retics), hemoglobin amount (Hb), hematocrit(Ht), the number of platelets /pl (Plts), the number of white blood cells /pl (WBO and difierential

white blood cells count. 4) Liver function tests and other routine biochemistry were performed.

Au patients were treated with an oral TMP‑SMZ compound (BaktarR , Shionogi & Co. ,LTD. , Osaka, Japan). One tablet contained 80 mg TMP and 400 mg SMZ. Cases 1, 2 and 4
were administered 16 mg TMP /80 mg SMZ /kg body weight (Bw) in 3 divided doses daily for 3

days. ･Case 3 was administered 10 mg TMP/50 mg SMZ/kg Bw in 4 divided doses daily for 4
days. The casualties totaled 12x3 tablets in Case I (Bw=OOkg), 8x3'tablets in Case 2(Bw=40 kg), 9x4 tablets in Case 3 (Bw=72 kg) and 14X3 tablets in Case 4 (Bw=70 kg). In
addition, Cases 1, 2 and 3 were administered 15 mg primaquine daily for 14 days fouowing theSera from Cases 2 and 4 were analyzed for the presence of antibodies to erythrocylic stagesof Plasmodium vivax and P. falclparum by an indrect fluorescent antibody technique (IFA).

CASE STUDY
Figures 1‑4 are the courses of treatment showing body temperature, asexual‑parasite

counts and sexual‑parasitemia of the patients.
Case I (Vivax malaria with low parasitemia) (Figure D
Present illness : . On 2 September 1981, a 33‑year‑old Japanese male developed chills andfever and.,visited the Department of Medical Zoology. Kyoto Prefectural University of Medicine

for consultation. Immediate blood examination revealed the presence of P. vivax, and parasite
count was recorded as 675 parasites /pl of blood with 89% trophozoites and 1 1% schizonts. On
3 September he was hospitalized. Tertian‑type high fever attack was found on 4 (39. 7'O and 6(3.9.8'O September and another high parasite count (675lpl) was recorded on 5 September. A
hemogram on 4 September recorded Hb of 14.3 g/dl; Ht 45.8% ; RBC 461 x 104 ; WBC 5,000with 40( o neutrophils, 17% Iymphocyies, 9% monocyies, 33% eosinophils and I % basophils.

On 7 September, Hb was 12.7 g/dl, Ht 40.0%, RBC 40lxl04 and WBC 4,100. A physicalexamination showed a slight hepato‑splenomegaly.
History : The patient suffered his frst attack of malaria in Zaire in March 1975. He had

been there since September 1974 doing anthropological investigations. Until February 1975,

one month prior to the onset of the frst attack, he had continued weekly chloroquine. This
attack was terminated with chloroquine (dose unknown). He returned to Japan in September

1975. During his next visit to Zake from March 1978 to March 1979, the patient sufferedanother malarial attack, which subsided after treatment in a hospital. He made a third visit tol 07

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Figure I Course of temperature, asexual‑parasite count and sexual‑parasitemia in the patient, a33‑year‑old Japanese male with vivax malaria (Case l). The patient was given an oral
compound of trimethopritn‑sulfamethoxazole (ST) at a dosage of 16 mg TMP / 80 mg SMZ/kg
Bw in 3 divided doses daily for 3 days, followed by 15 mg prinraquine daily for 14 days.

Zaire, from August 1979 to February 1980. During this period no further attack was experi‑
enced. On 5 June 1980, 3 months after his return from Zaire to Japan, he developed fever andchius and took chloroquine phosphate ResochinR (dose unknown), without successive primaquine
administration. After that the patient had no suggestion of malaria for 15 months until thepresent episode of nuness.

Treatment : On 9 September 1981, he was started on 12 oral tablets of TMP‑SMZ in 3
divided doses daily (16mg TMP/80 mg SMZ/kg Bw/a day) for 3 days. Beginning 13 Septem‑
ber, 15 mg pritnaquine was administered daily for 14 days.

An examinationi done just prior to this treatment, revealed a parasite count of 82 /pl ofblood with 100% asexual‑parasite. Asexual‑parasite count became O /pl within 24 hours after
the hrst dose was given and remained at O /pl after this titne. Sexual‑parasite count was O /pl 52

hours after the begiming of treatment. Since then no parasites were observed on thin films.

The patient's temperature was normal since 7 September. A hemogram on 1 1 September
recorded Hb of 13.4,g/dl; Ht 38.6% ; RBC 421XI04 ; WBC 5,200 with 47% neutrophls, 30%
lymphocyies, 3% monocyies, 19% eosinophils and 1% basophls. The results of the fonowing
examinations on 21, 24, 28, 29 September were within normal range, except for eosinophilia.
Upon later review of the smears, microfilaria was detected and identified Dipetalonema perstansas previously reported (Yoshida et al. , 1983). Eosinophilia recorded throughout the period
examined might be , due to D. perstans infection. No symptomatic adverse reactions to TMP‑

SMZ were observed. To date, no relapse of malaria has been experienced.
Case 2 (Vivax malaria with high parasitemia) (Figure 2)

Present iUness : On 7 June 1984, a 37‑year‑old Japanese nurse developed chills and fever.

108

ViVax malarla( 37Y, FEMALE )

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Frgure 2 Course of temperature asexual‑parasite count and sexual‑parasitemia in the patient, a37‑year‑old Japanese female with vivax malaria (Case 2). The patient was given an oral
compound of trirnethoprim‑sulfamethoxazole (ST) at a dosage of 16 mg TMP/80 mg SMZ/kg
Bw in 3 divided doses daily for 3 days, followed by 15 mg primaquine daily for 14 days.

On 8 June she made a blood smear and found a malaria parasite. On 9 June she was admitted to
Kyoto Municipipal Hospital and the maralial parasite was identitied as P. vivax. There were 688

parasites/pl on 8 June and 1,075/pl on 9 June ; asexual‑parasites were 97% and 98% of total

parasites respectively. A hemogram on 9 June recorded Hb of 13.6g/dl; Ht 40. 1% ; RBC

427 x l04; WBC 4,300 with 26% neutrophils, 63% Iymphocyies, 10% monocyies and 1%
basophils. Liver function tests and other routine biochemistry were an normal. The highest
parasite count was 25,870 /pl with 93. 9% trophozoites, O. 5% schizonts and 5.6% gametocyteson 12 June. Intermittent tertian paroxysms peaking at the night on 8, 10 and 12 June weremarked, although on 13 and 14 June paroxysms were irregular. The highest temperaturerecorded was 40.2'C on 8 June.
History : The patient had traveled in Thailand, Malaysia, Nepal, India and Australia 4 times

in 10 years ; October 1975 to November 1976, February 1978 to November 1979, February
1980 to July 1980 and October 1981 to September 1983. She took a prophylactic drug against
maralia during the frst trip only. She suffered her frst attack of malaria in India in May 1980.

This attack subsided after takirrg a course of amodiaquine (CamoquinR , dose unknown). In
August 1980, one month after she returned to Japan, a relapse was experienced, which wasterminated with amodiaquine (CamoquinR , dose unknown). This was not followed by prirna‑
quine. In November 1980 she had a normal delivery of a girl. Until 7 June 1984, no replapse
occurred.

Treatnent : From 14 June 1984 in the present therapeutic schedule, she was given 8 oral
tablets of TMP‑SMZ in 3 divided doses daily (16 mg TMP/80 mg SMZ/kg Bw/a day) for 3 days
followed by 15 mg primaquine daily for 14 consecutive days.

109

On 14 June an examination done just prior to this treatment, revealed a parasite count of

12,765/pl with 68% trophozoites, 7.9% schizonts and 24.0% gametocyies. A hemogram on
this day recorded Ht 35.0%, RBC 356 X 104 and WBC 3,000. Asexual‑parasite count became
O /pl within 48 hours after the beginning of treatment. Thereafter the temperature was normal
although a slight fever (37. 7'O on 16 June was experienced. A hemogram on 16 June recorded

Hb of 9.8 g/dl; Ht 31.0%; RBC 312xl04; Retics 1.3%; Plts 66XI03; WBC 6,700 with 57%
neutrophils, 31% Iymphocyies, Il% monocyles and 1% eosinophils. On 19 June the recordings
were Ht 28.0%, RBC 308 X I04 and WBC 4,900. Since then, the Hb and Ht levels gradually
returned to normal over a period of 2 months. The number of red blood cens /pl was 341 X 104

on 26 June, 328 X 104 on 2 July, 397 X I04 on 15 August and 438 x 104 on 19 September.

Hematocrit was 30. 5%, 32.0%, 38.0% and 41.0% respectively. Hemoglobin amount was 10.4
g/dl on 26 June, 12. 7 g/dl on 15 August, and 13.6 g/dl on 19 September. Reticulocyies was
8.0% and Plts was 291Xl03 on 26 June. Liver function tests and other routine biochemistry on

26 June were all normal. No symptomatic adverse reactions to TMP‑SMZ. were observed. To
date, no relapse has been experienced.
Serum was analyzed for antibodies against malarial parasites by the IFA test ; the serum
dilution end‑points were I : 256 against P. vivax and I : 64 against P. faiclparum on 12 June and 2

July. On 15 August these were I : 64 against P. vivax and I : 16 against P. falclparum.
Case 3 (Ovale malaria) (Figure 3)

Present mness : A 29‑year‑old Japanese male developed fever and chius on 29 May and 2
June 1983, 3 months after his return to Japan from Kenya. On 2 June he was admitted to Kyoto
Municipal Hospital when he was temporarily diagnosed as vivax malaria. However, upon later

Ovale malaria( 29Y. MALE )ST im Vllnays
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& ti5 rDATE +++++++++(l983) JUNE
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SEXUAL‑ PARAS I TE

Figure 3 Course of temperature, asexual‑parasite count and sexual‑parasitemia in the patient, a29‑year‑old Japanese male with ovale malaria (Case 3). The patient was given an oral
compound of trimethoprirn‑sulfamethoxazole (ST) at a dosage of 10 mg TMP / 50 mg SMZ/kg
Bw in 4 divided doses daily for 4 days, fouowed by 15 mg prirnaquine daily for 14 days.

110

review of the smears the parasites were identified as P. ovale as previously reported (Matsumoto

et al.,, 1986). There were 200 parasites/pl with 95% trophozoites and 5% gametocyies. A
hemogram on 2 June recorded Hb of 15.4 g/dl; Ht 46. 1% ; Plts 33.1x l03 ; RBC 458 x l04 .

WBC 4,000 with 47% neutrophils, 24% Iymphocyies, 27% monocytes and 2% eosinophils. On
4 J.une the highest parasite count (53,648 /pl with 99.2% trophozoites, 0.4% schizonts and 0.4%

gametocyies) and the highest temperature (40. 7'O was recorded. A hemogram on 6 June
recorded Hb of 13.4 g/dl; Ht 40.1%; Plts 97.0Xl03; RBC 445XI04; WBC 6,400 with 84%
neutrophils, 9% Iymphocyles and 6% monocyies. Intermittent tertain paroxysms peaking at
midnight became marked. Liver function tests and other routine biochemistry which were doneon 3 and 6 June were almost normal except for total bilirubin (1.7 mg/dl on 3 June and 2.3 mg/dl
on 6 June), cholesterol (102 mg/dl on 6 June) and total protein (6.2 g/dl on 3 June and 5.8 gldl

on 6 June). A physical examination on 6 June showed moderate enlargement of the spleen andthe liver.

History : The patient had stayed in Kenya from June 1978 to March 1979, July 1980 to
February 1981, August 1981 to October 1981 and July 1982 to the end of February 1983. Mostof his time was sbent in the Turkana district doing anthropological investigations. During his
frst visit, he irregularly took chloroquine for prophylaxis but he suffered from a malaria‑likeattack and was cured with sulfamonomethoxine. During his following stays in Kenya, heregularly took a prophylactic drug (chloroquine) against malaria. Unul 29 May 1983, no ftirther

malarial attacks had been experienced.

Treatment : On 7 June 1983, he was started on 9 oral tablets of TMP‑SMZ in 4 divided

doses daily (10 mg TMP/50 mg SMZ/kg Bw/a day) for 4 days. Beginnirrg 12 June, 15mg
prirnaquine was given daily for 14 days.

An examination on 7 June, done just prior to this treaiment, revealed a parasite count of
2,993 /pl of blood with 79.5% trophozoites and 20. 5% gametocytes. Asexual‑parasite countbecame O /pl within 36 hours after the frst dose was given and disappeared after this time.
Sexual‑parasite count became O /pl 3. 5 days after the begiuning of treaiment. Since then noparasites were observed on thin films. At midnight on the frst day of treatment the tempera‑ture was 40. 1'C. After that the temperature became normal. Daily hemograms revealed
anemia, with the Hb gradually falling to 10. 6 g/dl on 11 June. A hemogram on the same dayrecorded Ht 33.8%, RBC 345 x 104, Retics 2. 1%, Plts 116xl03 and WBC 4,800. After thatthe hemogram graduauy returned to normal. A hemogram on 27 June recorded Hb of 14. O g /dl; Ht 41.9%; RBC 465XI04; Plts 187xl03; WBC 4,eoO with 54% neutrophils, 26%
lymphocyies, 13% monocyies, 5% eosinophils and l% basophils. Liver function tests and otherroutine biochemistry were all norrnal. No parasites were observed on thin filrns. No symp‑

tomatic adverse reactions to TMP‑SMZ were observed. A hemogram on 29. May 1984, just
one year after the onset of the mness, recorded Hb of 17.2 g/dl; Ht 51.5% ; RBC 521 x 104 ; Plts

175 X 103 ; WBC 6, 700 with 63% neutrophils, 24% Iymphocyies, 8% monocyies, 4% eosinophils

and 1% basophils. To date, no relapse has been experienced.
Case 4 (Falciparum malaria) (Figure 4)

Present inness : A 48‑year‑old Tanzanian male developed fever on 5 August 1984 andvisited the Department of Medical Zoology, Kyoto Prefectural University of Medicine on 9
August for consultation. He was diagnosed as falciparum malaria and immediately hospitalized in

Kyoto Municipal Hospital. Parasite count was 4, 371/pl of blood with 99. 3% trophozoites and111

FalCiparum malaria

ST ( b 3day8 ( 48Y, NALE )

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9 10 Il 12 13 14 15 16 17 18 I9
208 21l 22 2623 2+ + +++ ++ ++ + ++++Figure 4 Course of temperature, asexual‑parasite count and sexual‑parasitemia in the patient, a
48‑year‑old Tanzanian male with falciparum malaria (Case 4). The patient was given an oral

compound of trirnethoprim‑sulfamethoxazole (ST) at a dosage of 16 mg TMP /80 mg SMZ /kg
Bw in 3 divided doses daily for 3 days.

O. 7% gametocyies. The highest parasite count (21,476lpl) was recorded on 11 August and thehighest temperature (39. 7'O was recorded on 10 August. A hemogram on 10 August record'ed
Hb of 14.2 g/dl; Ht 42.9%; RBC 501 X 104; Plts 243XI03; WBC 5,600 with 53% neutrophils,

40% Iymphocyies, 3% monocyies and 5% eosinophils. Liver function tests and other routine
biochemisty which were done on 10 August were alrnost normal except for triglyceride (208 mg/dl), LDH (260 U/1) and CPK (125 U/1).
History : The patient was born and raised in 'Tanzania and did not leave the country until

1959. He stayed in the U.S. A. from 1959 to 1976. After he had returned to Tanzania, hesuffered an attack of malaria (date unknown). He arrived in Japan on 30 April 1984. The nextday, he developed malarial symptoms (fever and headache). From that time until 5 August, heexperienced 7 attacks at two‑week intervals. In early June he took a total dose of 2 gchloroquine sulphate (NivaquineR) over 4 days. The fever disappeared but the headacheremained. On 15 July, he took 400 mg amodiaquine (CamoquinR ) daily for 3 days. After that
he took 400 mg amodiaquine once a week until 5 August.
Treatment : Beginuing 1 1 August 1984, he was started on 14 oral tablets of TMP‑SMZ in

3 dlvided doses daily (16 mg TMP/80 mg SMZ/kg Bw/a day) for 3 days.
An examination on 1 1 August, done just prior to this treatment, revealed a parasite count of

20,440 /pl with 99.9% trophozoites and O. l% gametocyles. Asexual‑parasites disappeared
within 40 hours after the first dose was given, but sexual‑parasites remained for more than 20
days. On the first day of treatment, the temperature was 39. 2'C but after that returned tonormal. A hemogram on 13 August, ' 40 hours after the beginning of treatment, recorded Hb of

13.6 g/dl; Ht 40.2%; RBC 472x 104; Plts 172XI03; WBC 4,900 with 42% neutrophils, 43%
lymphocyies, 6% monocyies and 9% eosinophls. Blood examination was done on 17, 23, 28

112

and 30 August ; 8, 14 and 26 September; and 2 October. Hb ranged irom 14. 7 g/dl on 28
August to 15.8g/dl on 2 October; Ht ranged from 42. 5% on 23 August to 45. 5% on 14
September and 2 October; RBC ranged from 486 X I04 on 23 August to 536 X 104 on 14
September. Liver function tests and other routine biochemistry were alrnost normal except for

CPK (136 U/1). A slight eosinophilia (8‑12%) persisted throughout the examination period.
This was considered to be due to concurrent Necator americanus infection. No symptomatic
adverse reactions to TMP‑SMZ were observed. No recrudescence has been experienced until
April 1985, when the patient returned to Tanzania.
Serum was analyzed for antibodies against malarial parasites by the IFA test ; the serum
dilution end‑points were I : 1,024 on 10 August and I : 256 on 17, 24 and 30 August against P.

falclparum and I : 64 on 17 August and I : 16 on 10, 24 and 30 August against P, vivex.

DrscusslON

Trimethoprim‑sulfamethoxazole (TMP‑SMZ) in a fixed‑ratio compound (TMP : SMZ = I : 5),
sometimes designated co‑trimoxazole, is listed as an alternate choice for treatment of infections

due to a variety of microorganisms. One normal strength tablet usually contains 80 mg TMP

and 400 mg SMZ. . This has been marketed under various trade names. The usual recom‑
mended dose for adults is 2 tablets twice daily.
Trirnethoprirn (2, 4‑dlamin0‑5‑[3, 4, 5‑trirnethoxybenzyl] pyrhnidine), one of the pydmidine

derivatives, is a potent inhibitor of the enzyme dihydrofolate reductase (DHFR) in microorga‑
nisms and interferes competitively with the conversion of dihydrofolic (folic) acid to tetrahydro‑

folic (folinic) acid. Trimethoprim has a particularly high affinity for bacterial DHFR but binds

less tightly to the corresponding mammalian enzyme. Sulfamethoxazole (3‑[p‑amino‑phenylsul‑
fonamido]‑5‑methylisoxazole), a semi‑long‑acting sulfonamid, is a structual analog of p‑aminoben‑

zoic acid. Sulfamethoxazole competitively inhibits the utilization of p‑aminobenaoic acid in thesynthesis of dihydr,ofolic acid (folic acid) by microorganisms (Salter, 1982 ; Wormser and Keusch,

1979). In vitro, these agents are shown to be decidedly more active together than either drug
is alone because of sequential blockade of the folate pathway. The efficacy of TMP‑SMZ in
toxoplasmosis (Grossman et al. , 1978), Ieishmaniasis (Kandl, 1973), Pneumosystis pneumonia(Hughes et al. , 1975; Harris et al. , 1980), and malaria has been demonstrated by various
researchers (Salter, 1982).

For treatment of vivax malaria, there are only 2 studies on imported cases in Japan by
Yoshioka and Kosaka (1978) and Yamaguchi et al. (1986), and on resident populations of endemicareas by Minhas and Siddiq (1971) in Pakistan and by Lal (1982) in Punjab, India. Using astandared daily‑dosage of 4 tablets of TMP‑SMZ for 5 days on adult patients, Minhas and Siddiq
demonstrated that fever resolved within 48 hours and parasiternia disappeared in a mean of 1.8

days. Lal compared the responses of parasitemia and fever in resident children with vivax
malaria to standerd doses of chloroquine and to different dosage schedules of TMP‑SMZ. His
study showed that chloroquine produced the most rapid clearance, followed by TMP‑SMZ in highdaily‑dose schedules (8 tablets /day for 2 days or the same dosage followed by 4 tablets /day for 3days), and then TMP‑SMZ in standard dose schedules (4 tablets/day for 3 or 5 days). In Japan,

Yoshioka and Kosaka (1978) used 4 tablets / day of TMP‑SMZ for 7 days, and Yamaguchi et al.
(1986) used 4‑8 tablets / day for 1‑4 days, both with successful elintination of the parasite fromthe blood. In our cases of imported vivax malaria, Case I was given 12 tablets / day (16 mg

113

TMP/80 mg SMZ/kg Bw/day) for 3 days and Case 2 was given 8 tablets/day (16 mg TMP/80
mg SMZ /kg Bw/day) for 3 days, then both were fonowed by 15 mg primaquine daily for 14 days.
This schedule succeeded in producing radical cures. Asexual‑parasites disappeared from theblood of Case I in 24 hours and of Case 2 in 48 hours.
Until this study, no studies of the effects of TMP‑SMZ on ovale malaria had been published.

Nine tablets/day (10 mg TMP/50 mg SMZ/kg Bw/day) for 4 days followed by 15 mg prirnaquine
daily for 14 days succeeded in producing a radical cure. Asexual‑parasitemia disappeared within

36 hours and fever resolved within 24 hours.
The efficacy of co‑trirnoxazole in falciparum malaria has been demonstrated by various
researchers (Benjapongs et al. , 1970 ; Wolfensberger, 1970 ; Fasan, 1971; Yamaguchi et al. ,1986). Benjapongs et al. (1970) gave TMP‑SMZ (4 tablets daily for 2.5‑10 days) to previously‑

untreated acute falciparum malaria and previously‑treated chloroquine‑resistant malaria. Withthe 2.5 day schedule, results were unsatisfactory in both groups. With 4 tablets daily, fever

resolved in an average of 3 days and asexual‑parasites disappeared from blood in 4.3 days.
They concluded that, to get the best therapeutic results, treatment should be continued at least

2 days after the disappearance of parasites from blood ; that is, the total dosage required toachieve a "complete effect" was 30 to 40 tablets. The clinical response to the same dose wassomewhat faster in patients with previously‑treated chloroquine‑resistant falciparum malaria.
Wolfensberger (1970) used 8 tablets daily for 3‑4 days in patients with acute falciparum malaria ;the TMP‑SMZ compared favorably with chloroquine. Fasan (1971) used a single dose of
TMP‑SMZ in falciparurn malaria in semi‑immune Nigerian schoolchildren aged 5‑12 years. The

dose (8 mg TMP/40 mg SMZ/kg Bw) was found to be effective. Fasan also found that half
dosages of the same compound were effective. Yamaguchi et al. (1986) treated 2 imported
cases of falciparum malaria in Japan, one with TMP‑SMZ 4 tablets 3 times a day followed bychloroquine diphosphate 600‑450 mg / day for 4 days, and the other with TMP‑SMZ 3‑8 tablets /day for 4 days. They both recovered completely. Falciparum malaria, Case 4 of our study,

was presumed to be somewhat resistant to amodiaquine ; however, it responded well to
TMP‑SMZ at a dosage of 16 mg TMP/80 mg SMZ/kg Bw/day for 3 days. Asexual‑parasitemia
disappeared within 40 hours and fever resolved within 24 hours.

The only study on malariai malaria has been done by Fasan (1971). However, theirpatients had mixed infections of P. falciparum and P. malariae. He noticed that there was areappearance of quartan parasites 3 days after single doses of TMP‑SMZ(4 mg TMP / 20 mg
SMG/kg Bw). He proposed 2 possible reasons. One is that the individual parasites noted
were gametocyies, which in the case of P. malariae were difficult to differentiate in a thick film.

The other is that TMP‑SMZ at this low dosage was less effective against quartan malaria
parasites.

In the treatment of malaria, TMP‑SMZ has been considered to be a rapidly acting
schizonticide. It has been noted that P. falioparum crescents were detected as late as one to 2

weeks after treatment with TMP‑SMZ, otherwise the response was good. Benjapongs et al.
(1970), concluded that TMP‑SMZ had no effect on gametocyies of P. falciparum and therefore
prirnaquine should be prescribed for malaria patients with TMP‑SMZ to prevent transmission.
In our observation, P. falciparum gametocyies were not cleared from the blood of a patient who

received TMP‑SMZ until 3 weeks folbwing treaiment, otherwise the response was good.
Gametocyles of P. vivax and P. ovale, however, might be cleared from the blood of patients by

TMP‑SMZ, because they disappeared before starting administration of prirnaquine.

114

The combination is generally wen tolerated even with a large dose of long term administra‑tion, as seen in treatment of Pneumorystis infection (20 mg TMP/100 mg SMZ/kg Bw/a day for
14' days) (Hughes et al. , 1975) or prophylaxis against it in children (4 mg TMP / 20 mg SMZ /kg

Bwla day over months) (Harris et al. , 1980) . The most common adverse reactions are mild
gastrointestinal symptoms including nausea, vomiting, 'and less frequently diarrhea, cramps,
gloss.itis, , stomatitis, or jaundlce (Salter, 1982 ; Wormser and Keusch, 1979), Regarding the useof TMP‑SMZ for malaria, Benjapongs et al. (1970) reported slight nausea and vomiting (in 2 of 5

patients) during the frst few days on the 3‑tablets‑daily schedule and noted one case of mildtransient lenkopenia. Lal (1982) reported vomiting was noted in 1･3 of 98 cases, although
vomiting was notably common with chloroquine. In our study, no ,such adverse reaction to

TMP‑SMZ was observed.
A number of hematologic abnonnalities including thrombocyiopenia, Ieukopenia and anemia(macrocytic, megaloblastic, hemolytic or aplastic) have .been described in patients ieceiving
TMP‑SMZ but they are rare (Salter, 1982 ; W, ormser and Keusch, 1979). Patients with known
folic acid or vitamin B12 deficiency are at increased risk from the antifolic effects of the drugs, andpatients with questionably adequate folic‑acid stores, such as pregnant women, the elderly,
patients with malabsorption or mahrutrition, or those with chronic hemolysis (such as sickle‑celldisease), should be carefully observed. , In this study, all patients showed anemia during thetreatment but recovered soon after the treaiment. This might be mainly , due to malarial
infection itself.

In conclusion, our study showed that TMP‑SMZ may be a safe choice to produce radicalcures for malaria caused by any species of malarial parasites, at least vivax, ovale and falciparum

malaria at the dosage described above. Trimethbprim‑sulfamethoxazole should be examinedfurther in the treatrnent of chloquine‑resistant malaria and in those countries in which anti‑
malarial drugs are not easy to obtain.

ACKNOWLED9EMENTS
We are indebted to Professor Mamoru Suzuki of the Departrnent of Parasitology, Gunma
University, School of Medicine for performing the serological tests of Case 2 and 4 in this study.

REFERENCESl) Benjapongs, W. . Sadudi, N. and Noeypatimanond, S. (1970) : Tritnethoprim/sulphamethoxazole com‑bination in the treatment of falciparum malaria, J. Med. Ass. Thailand, 53, 849‑856
2) Fasan, P. O. (1971) : Trimethoprirn plus sulphamethoxazole compared with chloroquine in the treat‑ment and suppression of malaria in African school‑children, Anu. Trop. Med. Parasitol. , 65, 1 17‑121

3) Grossman, P. L. , Krahenbuhl, J. L. and Rernjngtpn, J. S. (1978) : In vivo and in vitro effects oftrirnethoprirn and sulfamethoxaz,ole on Toxoplaswa infection, In : Current chemotherapy, Siegentha‑
ler, W. and Ltithy, R. ed. , 1, 135‑137, American Society for Microbiology, Washington, D. C.

4) Harris, R. E., McCauister, J. A., Alleh,' S. A., Barton, A. S. and Baehner, R. L. (1980) : Preventionof Pneurnocystis pneumonia, A l. J. Dis. Child. ; '13 , 35‑38

5) Hughes, W. T., Feldman, S. and Sanyal, S. K.' (1975) : Treatment of Pneumo(ystis carinii pneumoni‑
tis with trirnethoprirn‑sulfamethoxa:zole, Can. Med. Ass. J. , 1 12, 47‑50

6) Kandil, E. (1973) : ,Treatment of cutaneous leishmaniasis with trirnethoprirn‑sulfamethoxazole com‑bination, Dermatologica, 146, ' 303‑309

1157) Lal, H. (1982) : A comparative trial of oral chloroquine and oral co‑trirnoxazole in vivax malaria, Am.

J. Trop. Med. Hyg., 31, 438‑440
8) Matsumoto, Y. , Matsuda, S. and Yoshida, Y. (1986) : Ultrastructure of erythrocytic stages of Plasmo‑

dium ovale in humans, Am. J. Trop. Med. Hyg. , 35, 689‑696
9) Minhas, A. L. and Siddiq, K. Y. (1971): Trirnethoprirn‑sulphamethoxazole (Septran) in P. falctparumand P. vivax malaria, Medicus (Karachi), 42, 310‑314
10) Ohtomo, H. and Hioki, A. (1985) : Disseminated intravascular coagulation in malaria, Modern Media,

31, 159‑168
11) Salter, A. J. (1982) : Trirnethoprirn‑sulfamethoxazole : An assessment of more than 12 years of use,

Rev. Infect. Dis., 4, 196‑224
12) Wolfensberger. H. R. (1970) : Clinical trials with 'Fansidar', 'Bactrirn', and chloroquine in 300 cases ofacute falciparum malaria, Far East Med. J., 6, 48‑50
13) Wormser, G. P. and Keusch, G. T. (1979) : Trirnethoprirn‑sulfamethoxazole in the United States,
Ann. Intern. Med. , 91, 420‑429
14) Yamaguchi, K., Koga, H., Kohno. S.. Shigeno. Y.. Suzuyama. Y.. Hirota. M.. Saito, A., Hara, K.,
Amagasaki, T. , Iwanaga, S., Okuno, K. , Fujiwara, T. , Murata, I. , Sh zato, K. , Tsutsunti, T. , Goto, J.

and Ishizaki, T. (1986) : Clilaicai evaluation of SMX/TMP on treatrnent of the patients with malaria,

J. Jpn. Ass. Infect. Dis., 60, 1027‑1035 '15) Yoshida, Y., Shiota, T., Yamada, M., Matsumoto, Y., Okabayashi, K., Yoshik wa, H., Takino. T.,
Nakagawa, Y. and Yokota, S. (1983) : Report of an imported case of Dipetalanema perstans in Japan,

Southeast Asian J. Trop. Med. Pub. Hlth. , 14, 341‑344
16) Yoshioka, M. and Kosaka, S. (1978) : A recent case of malaria with reference to its present status,Med. J. Aomori Pref. Central Hospital, 23, 81‑85

116

トリメトプリム・サルファメトキサゾールによる三日熱，卵型
および熱帯熱マラリア症例の治療成績
吉田幸雄1・松本 芳嗣1・山田
岡林

稔1・塩田
加枝1・手越 達也1・吉川

小林

祥男2・金

恒三1・吉川 尚男1

哲也1

龍起2

わが国における輸入マラリア症例は年々，増加の傾向にあるが，現在世界で用いられている抗マラ
リア剤の殆どのものが市販されていない。ところがわが国でも市販されているトリメトプリム（TMP）とサルファメトキサゾール（SMZ）の合剤が三日熱および熱帯熱マラリアに有効なことが知られてきた。われわれも1981年以来経験した4症例に本剤を用いたところ良好な治療効果を得た。まず2例の三日熱マラリアに対して，16mgTMP／80mgSMZ／kg／日を分3し3日間投与し，続いてプリマキン
15mg／日を14日問投与した。1例の卵型マラリアに対しては10mg TMP／50mg SMZ／kg／日を分4し4日間投与し，続いてプリマキン上記量を与えた。さらに1例の熱帯熱マラリアに対しては16mg
TMP／80mgSMZ／kg／日を分3して3日間与え，プリマキンは投与しなかった。いずれの例においても発熱など臨床症状は急速に改善し，治療開始36−52時間後の検血で繁殖体は消失していた。生殖母体は熱帯熱マラリアにおいて治療終了後約10日間検出された後消失したが，他のマラリアでは治療期
間中に消失した。これらはその後，再発あるいは再燃を示しておらず，本治療で根治したものと思われる。今回は比較的大量を短期間投与したが本剤によると思われる副作用は認めなかった。本剤により卵型マラリアの治療に成功したのは本報告が最初である。以上の成績から本剤は，特に他の薬剤の入手困難な国に於て，また他の薬剤に耐性を生じたマラリアについて有用であると考える。

1京都府立医科大学医動物学教室

2京都市立病院伝染病科

Japan. J. Trop. Med. Hyg., Vol. 15, No. 2, 1987, pp. 117‑120

Sh*rt C*117

* i**ti.*

CHAGAS' DISEASE : DELAYED TYPE HYPERSENSITIVITYTO PPD IN RELATION TO SEROREACTIVITY TO

TRYPANOSOMA CRUZI IN ECUADOR

MASATO KAWABATAl, TATSIJYUKI MIMOR12, YOSHIHISA HASHIGUCHIAND VICENTA V. DE CORONEL4
Received December 18 1986/Accepted April 15 1987

Abstract : In a population from an area endemic for Chagas' disease in Ecuador, cell‑
mediated iromune activity was assessed using delayed type hypersensitivity (DTH) reaction to
PPD. Expression of DTH was significantly suppressed in individuals seropositive to Tmpanoso‑
ma cruzi, and the suppression was markedly greater in the age group 10‑39 years old than thoseover 40 years. The results show that cell‑mediated irnmune response is apparently suppressedin patients with Chagas' disease, and tends to be restored in chronic stage. No defference wasnoted in the serum concentration of irnmunoglobulins between patients and controls.

INTRODUCTION
It has been well known that infections with several species of parasites are associated withmodification of immune response to heterologous antigens. Delayed type hypersensitivity(DTH) skin reactions are used as an effective tool to assess cell‑mediated immunity in humans,and have been found to be suppressed in helminthic and protozoal infection (Greenwood et al. ,1973 ; Grove and Frobes, 1979 ; Kawabata et al. , 1983). In patients with Chagas' disease,caused by Tmpanosoma cruzi, the previous reports on DTH to unrelated heterologous antigens
rernain controversial. Expression of DTH reactions in acute chagasic patients was observed tobe less than that in nonchagasic controls (Teixeira et al. , 1978a), whereas no such suppression
was found in the chronic patients (Montufar et al. , 1977 ; Corsini et al. , 1981).

On the other hand, it has been suggested that T. cruzi isolated from various geographic
areas may have distinct properties because clinical manifestations show great regional differ‑ences. In the present study the expression of DTH to purified protein derivatives (PPD) oftuberculin and the quantification of serum concentration of irnmunoglobulins (Igs) was performed

in a population from an endemic area of Chagas' disease in an attempt to assess theirimmunological status in Ecuador.

1 Department of Clinical Pathology, Nihon University School of Medicine, Oyaguchi, Itabashi‑ku Tokyo 173

2 Department of Parasitic Diseases, Kumamoto University Medicai School, Honjo, Kumamoto City,

Kumamoto 8CO
3 Department of Parasitology, Kochi Medical School, Nankoku City, Kochi 781‑51
4 Departamento de Parasitologia, Instituto Nacionai de Higiene y Medichina Tropical, Apartado 3961,
Guayaquil, Ecuador

Correspondence to : Masato Kawabata, M. D.

118

MATERIALS AND METHODS
Subjects : The study was conducted in 2 endemic foci for Chagas' disease, Zaruma andsurrounding areas (Province of El Oro), and Pedro Carbo and surrouding areas (Province of
Guayas). DTH reaction was examined in 255 individuals, while the lg was quantified in 137
individuals. These subjects were examined for their serum antibodies to T. cruzi with acommerciany available indrect hemagglutination (IHA)‑kit (Hema Chagas, Polychaco, S. A. I. C.,

Argentina), and classified into 2 groups according to the seroreactivity. IHA titers of 16 or
greater were interpreted as positive in this study.

DTH reaction : DTH reaction to intradermal injection of O. I ml PPD from Mycobacterium
tuberculosis RT23, equivalent to O. 005 pg of PPD‑s, were measured 48 hours later. Indurationof 5 mm in diameter or more was considered a positive reaction.

Quantification of serum lg : The serum concentration of lgG, IgM and lgA were deter‑
mined by single radial irnmunodiffilsion (SRID) method in agar. The plates were purchased from
Medical Biological Laboratories (M:BL) Co. Ltd. , Japan.

RESULTS AND DISCUSSION

Cell‑mediated immunity was assessed by testing DTH reactions to PPD in individuals
seropositive or seronegative to T. cruzi. Positive DTH were significantiy less frequent inseropositive individuals than in seronegative individuals (Table 1). When the magnitude of thereaction was analyzed in relation to age among the seropositive group, the DTH was weaker inthe 10‑39 year age group (p<0.0D than in the over 40 year age group (p<0.05). The present
results suggest that cell‑medlated immunity was suppressed in seropositive individuals, but was

restored in the older age group, although the difference between 2 groups was not significant.

The normalization trend for cell‑mediated irnmunity shows that the proliferative response oflymphocyies from T. cruzi‑infected mice was suppressed during acute infection, and returned tonormal levels during the chronic stage (Hayes and Kierszenbaum, 1981). Humoral irnmunity
remain suppressed since the antibody response to typhoid vaccine or sheep erythrocyies inpatients with chronic Chagas' disease remains low (Cunninglram et al. , 1980 ; Corsini et al. ,198D.
No significant differences in the serum levels of lgG, IgM and lgA were noted between the

Table I Delayed type hypersensitivity to PPD in a population irom an endemic area of Chagas'

disease in Ecuador

Age group

No of positives') /No. of Examined (%)pb)(years)Sero positives')

Seronegatives

10‑39
40‑

9/34 (26. 5)

64/ 122 (52. 5)< O. O1

22 / 59 (37. 3)

23/40 (57.5)< O. 05a) reactivity to PPDb) p values with chi‑square testc) seroreactivity to T. cruzi by IHA

ll9
Table 2

Serum levels of immunoglobulins in a population from an endemic area of

Chagas' disease in EcuadorlgG

Seropositives')

Seronegatives(n = 96)(n = 41)1555.2 (449.2)b)1470. 9 (433. 5)

lgM

139.9 (67.3)

153.0 (73.3)

lgA

240.7 (87.6)

242:9 (lOO.4)a) seroreactivity to T. cruzi by IHAb)mean (S.D.), mg/dl

seropositive and seronegative groups (Table 2). The results of our study and previous ones
(Lelchuk et al. , 1970 ; Corsini et al. , 1981) indicate a normal level of lg in chagasic patients, but
an elevated level of lg in the acute phase of the infection (Vattuone et al. , 1973 ; Schum 's et al. ,1978). Great care, however, must be taken on the fact that the irnmunological status observedin this study might be restricted to Ecuadorian Chagas' disease caused by different geographicalstrains of parasites.

Our understanding on the irnmunopathological implication of immune suppression and its
restoration on the clinical course of Chagas' disease is lirnited. Since DTH reaction mediated by

T. cruzi‑sensitized T‑lymphocyies has been shown to be involved in the autoimmune destructionof heart cell in vitro (Teixeira et al. , 1978b), restoration of cell‑mediated irnmunity might beassociated with development of chronic heart lesions. In addition, transition from latent tochronic stage, defined by electrocardioigraphic abnormalities, was found in individuals over 40
years old that were seropositive to T. cruzi in Ecuador (Kawabata et al. , 1987). To know the
precise mechanisms responsible for an evolution of chronic lesions in Chagas' disease, further
investigations is needed.

ACKNOWLEDGEMENTS
We wish to express our appreciations to our collaborators in the Instituto Nacional deHigiene y Medicina Tropical, Guayaquil, Hospital de Zaruma, Provincia de El Oro, and Centro de

Salud de Pedro Carbo, Provincia de Guayas, Ecuador.

REFERENCES
1) Corsini, A. C., Vilela, M. S. and Piedrabuena, A E. (1981): Chronic Chagas' disease : Unim, paired

delayed type hypersensitivity reactions in contrast with humoral suppression to typhoid vaccine,
Tropeumed. Parasit. , 32, 82‑86

2) Cumingham, D. S., Grogl, M. and Kuhn, R. E. (1980) : Suppression of antibody responses in humans

infected with Tmpanosoma cruzi. Infect. Immun. , 30, 496‑499
3) Greenwood, B. M. , Whittle, H. C. and Molyneux, D. H. (1973) : Immunosuppression in Gambiantrypanosomiasis, Trans. R. Soc. Trop. Med. Hyg. , 67, 846‑850
4) Grove, D. I. and Frobes, I. J. (1979) : Immunosuppression in bancroftian filariasis, Trans. R. Soc.

Trop. Med. Hyg., 73, 23‑26
5) Hayes, M. M. and Kierszenbaum, F. (1981) : Experirnental Chagas' disease : Kinetics of lymphocyle

responses and irnmunological control of the transition from acute to chronic Tmpanosoma cruzi

120㎞ecUon，Infect・㎞un．，31，

1117−1124

6） Kawabata，M，Izui，S．，Anan，S．，Kondo，S．，Ft血umoto，S．，Zea，G．R andCirculating㎞mune

Guatem曲m7）

complexes

onchocersiasis，

andthe丘possible

InしArcbrelevanceother㎞munologicalparametershlA皿ergy．App1・㎞㎜oL，72，128−133

Kawabata，M．，Uchiyama，T．，M㎞oh，T，Hashiguchi，Y．andelectrocardiographic abnom圃ties withto

Kobayakawa，T．（1983）：

seropositivity

toCoronel，V．V．（1987）：

Tりψ伽oso

Association ofzαon痂血Ecuador， Trans．R．

Soc．Trop．Med．Hyg．，81，7−108）Lelchuk，R，Da㎞assom，A P．，Inglesini，C．L，1Uvarez，M．and Cerisola，J．A．（1970）：Immunoglo−

b血stu磁es血se㎜ofpa廿ents・輌血㎞e蜘偽脚oso血sis（C噸s

出sease），C㎞恥．

㎞uno1．，6，547−555
9）Montufar，0．M．，Musatti，C．C．，Mendes，E

nicChagas

andMendes，N．F．（1977）：Ce皿ular㎞munity

in

chro−

disease，J、Cbn．MicrobioL，5，401−404

10）Sch曲，GA，Sz血m，A，Coarasa，L㎝dV㎞stok，C．（1978）：㎞moglob血concen圃on血treatedh㎜1an acute Chagas｝disease，Am．J．Trop．Med．Hygl，27，473−477

11）Teixeira，八R，Teixeira，G．，Macedo，V．and
nodepression

hl

acute

Chagasdisease，

Prata，八（1978a）：Acq血ed

ce皿一皿ediated㎞mu−

J．C㎞．hlvesL，、62，1132−114112）Teixeira，八R．，Teixeira，G，Macedo，V and Prata，ん（1978b）：Tりφ伽oso魏α ㈱ゴーsensitized T−
lymphocyte mediated51Cr release from hu皿an heart ce皿s in Chagas disease，Am．J．TroP．Med．

Hyg．，27，1097−110713）Vattuone，N．H，Sz肛fman，へandlevelsillhumans with acute or

Cappa，S．M（1973）：Antibody
chronic

response

Tりψ伽os㎜o㎜∫infegtions（Chagas

and㎞m㎜oglobu㎞

disease），J．Trop、Med．

Hy呂，76，45−47

短報エクアドル国のシャーガス病流行地住民集団における遅延型皮内反応

川端真人1・三森龍之2・橋口
VIcENTA

V．DE

義久3

CoRoNEL4

エクアドル国のシャーガス病患者の細胞性免疫機能を検索する目的でPPDに対する遅延型アレル
ギー反応を検討した。シャーガス病流行地の住民255名を対象にT㎝襯抗体をIHA
ga〜Polychacotest（Hemocha−SAIC）で検査すると陽性者は93名，陰性者は162名であった。ツベルクリン反応の陽性率は，IHA陽性者の方が陰性者より有意に低く，特に10−39歳群でその傾向が強かった。この結果からシャーガス病患者では細胞性免疫機能の低下があり，10−39歳群では強い抑制があるが，慢性期になると回復傾向があると推測された。他方，免疫グロブリンをSRIDで測定するとIgG，1喜M，lgAともにIHA陽性者と陰性者の間に差はみられなかった。

1日本大学医学部臨床病理学教室
2熊本大学医学部寄生虫病学教室
3高知医科大学寄生虫学教室
4エクアドル国立熱帯医学研究所寄生虫部

日本熱帯医学会雑誌 第15巻

第28回

第2号 1987 121−137頁

日本熱帯医学会総会講演抄録（1）

会A本会期：昭和61年11月22日 （土）・23日（日）一場：琉球大学医学部講義室
長：琉球大学医学部教授 大鶴正満

目

田中

次

healthaspects

Westem Pac迅cof廿opical

浅沼靖（国立科学博物館・動物）2

diseases

血Studies

lar

the

3

宏

of tsutsugamusi

Sh虻ai（VVHO）

Epidemiologicalease（scrab（WHO西太平洋地域事務局長）

features oftyphus）in林全勝
4

シンポジウム
1

1

小張

一峰

（琉球大・医・一内科）

神中寛（防衛医大・細菌）

Bactehal（㎞heal

竹田
2 Vkal

3

5

宮崎

千明
concepts

竹内

勤

Study

on廿1e

straills

5ofE，￠如 ￠oθδα

力商如砂あserotyphlg

an（ibiotyp㎞g

Chevof換d肚ect㎞muno−perox−

for

the

Asian

means

恒久

contTolPasmcの薬剤感受性試験への応用

method

及川陽三郎（金沢医大・医動物）

E．AJalal（金沢医大・熱帯医研）

谷荘吉（富士通・川崎病院）

of（㎞hoeal

2

マラリア原虫（PJσs甥04伽魏加㎎勧）感染赤

血球膜からの蛋白質分解酵素の抽出とその二，

dis−

三の性質

Region井上

文英，徳（2，3，5和文抄録なし）（琉球大・医・寄生虫）誠吉（琉球大・医・一生化学）

II アジアにおける蒜虫病
正満門（秋田大・医・微生物）

アのスクリーニング診断およびマラリア原虫

Kidson大鶴

tsutsugamusi

マラリア原虫の迅速・簡易染色法一マラリ（クインスランド医学研究所）

司会

of

（WHO）

Cα卿ρyJoわ㏄伽by Lior s

Meiwen，XuJufen（上海衛生防疫センター）the（新潟薬科大）一般講演（1〜24）

of300

Xie

in

by

σ

1

恵蘭

StTategies

diagnosis

須藤（慶応大・医・寄生虫）

司馬

ease

憲
serolqgic

tropics

and amoebiasis

4

多村
Rapi（i（九州大・医・小児科）
ofC㎞a（Jinan医大・感染症）

i⑫se（IP）

美文（東大・医科研・細菌感染）

CuITent

southemdis−

R勉ε傭毎碑お㎎iロ灘sゴの細菌内増殖機構

diseasediseasesd㎞hea血廿1etsutsugamusi

に関する電子顕微鏡学的研究

熱帯性下痢症（和文抄録なし）

司会

disease血Penhlsu−

Malaysia

Ak辻a

Region中嶋寛（東大・医科研・・寄生虫）

ま日本の蒜虫病に関する医動物学的研究

1

特別講演（和文抄録なし）
Pubhc

121

3

松山

玲子

（琉球大・Rl実験施設）

佐藤良也（琉球大・医・寄生虫）

P伽吻04伽％加怨履感染マウスにおける血液122

inosineのamastigoteに対する作用について

幹細胞の変動について
浅見 鳴子，丸山 治彦，大橋
名和

4

行文

佐竹 弘靖，平岡真，（岡山大・薬・薬品化学）（宮崎医大・寄生虫）

βα伽伽感染に対するトキソプラズマ抗原

Jurieta（TLA）投与マウスにおけるT一リンパ球の動

安治 敏樹，石井Y

K㎞ura，森重

鈴木

直義，関口

信一，佐藤

基佳，

桜井

治久，斉藤 篤志，広瀬恒夫（帯広大・獣医）
尾崎

文雄

松田

京都市におけるβ山田㎞quine一回投与のP，∫gametocyteに対吉田

11

σsホoσs廊ho痂添の感染状

稔，松本

幸雄

芳嗣，手越

（京都府医大・医動物）

広範囲の大腸潰瘍および膿瘍を伴なった重症
中村 敏夫

中村

弘前大・医・寄生虫）（阪大・微研・原虫）

猛，網岡 勝見（中村病院・枚方）

亀井喜世子（JICA北スマトラプロジェクト，

12

鈴木

酵素抗体法による赤痢アメーバ症の診断（予報）

帝京大・医・寄生虫）明山浦（岡山大・医・寄生虫）（東京女子医大・寄生虫）13

東南アジアにおける疫病媒介蚊の羽音等を用いたトラップ法の応用

インドネシア・北スマトラのマラリア対策（2）神田

昆虫学的研究とベクターコントロール
猛，菊地

哲志

一免疫学的検討一正洋

E．A．Jalal（金沢医大・医動物，熱帯医研）

予研・衛生昆虫）
（公害研）

池田

照明

インドネシア共和国北スマトラ海岸地帯にお

佐藤

喜一，堀田

安野 正之

辺に住む人々の間の非常に高いマラリア伝播

8 Tり少415

ヒトスジシマカ，ネッタイシマカのチクング
ニアウイルス感受性に及ぼす吸血の影響につ（聖マリアンナ医大・病害動物）

いて

山西

小西

選ぶか？
P．」．Mhando，柳

秀介，福間

利英，神原

浩

（神戸学院女子短大）

英二，上野山登久（神戸大・医・医動物）

哲雄，

廣二

（長崎大・熱帯医研・原虫）

9

進

英樹

oso 昭伽π】擁80卿伽πs8はjeederce皿を中沢（金沢医大・医動物）（金沢医大・熱帯医研）

ける塩水し好性蚊A肱s観吻加3の棲息地近糸川賢一

14 虫刺創の病理学的研究（第3報）（JICA北スマトラプロジェクト，

7

錬蔵，小川

（聖マリアンナ医大・病害動物）（JICA北スマトラプロジェクト）

高木常，白坂 龍暖

猛（JICA北スマトラプロジェクト）

鈴木

達也，

赤痢アメーバ症の1例

明（JICA北スマトラプロジェクト，

石井

亨

況

インドネシア・北スマトラのマラリア対策（1）

金子

彰，上田

（北大・薬・薬化学）
10

（神戸市）

する効果

6

和久，

明

（岡山大・医・寄生虫）態

5修，綿矢 有佑16

沖縄本島における日本脳炎ウイルスの活動状
況（1985年3月一1986年10月）

加o昭痂

只野

昌之，石嶺

毅，牧野

amas恒goteの伽び伽o㎝ltivation：CarbocycHc

安斎

俊一，福永

利彦

マウスJ774・1細胞を用いるL．

芳大，

123

長谷川英男

（琉球大・医・ウイルス）

下口 和矩，餅田 親子，山口 恵三（琉球大・医・寄生虫）（長崎大・病院・検査部）

17デングウイルスの微量中和試験
石嶺

毅，只野

昌之，福永

草野 展周，内藤 達郎
利彦（長崎大・熱帯医研・病原細菌）（琉球大・医・ウイルス）
奥野

18

良信

河野

茂，斉藤

厚，原

耕平

（阪大・微研・防疫）（長崎大・医・二内科）

デングウイルスのエンベロープ糖蛋白質の解22インドネシア各地の飲料水より検出された

析

Ps6漁伽σs㎜…吻o詔の薬剤感受性と血清

竹上勉（金沢医大・熱帯医研）
関

智代子

堀田

進

型について

（金沢医大・血清学）

奥脇

義行，矢内

（金沢医大・熱帯医研）（女子栄養大・微生物）

19モノクロナール抗体を用いたコレラの迅速診藤田紘一郎，月館 説子

断実用化の研究（長崎大・医・医動物）

柳ヶ瀬康夫，青山和枝，庄司宏（兵庫医大・細菌）
権平

文夫，松田

潤治，平野

杉山

純一，寺田

友次（デンカ生研）

杉山 雅俊

23

勝，

C．P．㎞oa（SanLazaro病院，マニラ）

20バングラデシュで最近分離されたコレラ菌の

澤田

24

清子，須永

寛

（瑞穂短大）

多孔質中空系と銀活性炭を組み合せた減菌水
装置の効力

小原

博，海老沢

功，高柳満喜子（東邦大・医・公衆衛生）

昇，岩永

正明

（琉球大・医・細菌）

21バングラデシュおよび長崎で分離された、4召猶o吻o（順天堂大・医・衛生）

北アフリカ地域における飲料水の水質検査

生物学的諸性状仲宗根寿恵

αs属についての比較検討

一薬剤感受性を中心に一

小松

俊彦，甲原

照子

（予研・腸内ウイルス）

一般講演（25〜68）（次号掲載予定）124

シンポジウムH

アジアにおける慈虫病

r日本の惹虫病に関する医動物学的研究
浅沼靖（国立科学博物館・動物）

フトゲ単独種一満腹回収後，G皿am型Rを分離
一の寄生をうけたハタネズミが同型のRに感染したこ．とを強調しておきたい。本実験でR感染の確

日本における・悲虫病は，近年発生が著しく，かめられたハタネズミは，その後，飼育観察を続1984年度礁は，．患者数971名，発生都道府県数29

け，臓器Rは感染数カ月後には分離不能一R陰性に及んだ。この事実を「新潟，秋田，山形県下に限っ患発隼する」とした過去の蒜虫病に比べるととなること，血清反応（補体結合反応）は1年後今昔の感にだえない。

有毒地点と蒜虫病の発生にも陽性成績を示すことなどが明らかとなった。

各地の蒜虫病媒介者

ところで憲虫病発生

八丈島，白浜（千葉），

富士山麓（静岡）ではタテ，三浦・伊豆両半島南地域の増加は，慈虫病リケッチア（以下Rと略）部，竹田（大分）ではフトゲがすでに媒介者としの大規模な分布拡大によるものではなく，元来存て報告されている。しかし富士山麓では，タテ発在した有毒地点の人における顕性化と考えている

生の全く終息している4月（1982）に患者発生のであってレ、Tamiya（1962）は鼠類のR陽性地点 （従来は9−12月）があり，タテ以外にも媒介種を，すでに46都道府県に見出している。R感染鼠の存在するであろうことが問題となっている。秋は有毒ッッガムシの存在を示すが，後者が全て蒜

田，新潟県では近年春秋の患者が目立つが，浅沼虫病発生に連るとするには問題があり，北海道のらが早くから指摘しているように，同時期に多発如く，札幌，江別などにR感染例，カワムラツツ

するフトゲには常に有毒個体があり，本種を春秋

ガムシ（以下ツツガムシは略），ホッコクにR陽期の蒜虫病媒介者とすることには，最早異論はな

性例があるに拘らず，今なお患者未知の例がある。

いであろう。特に秋田県下の古典的有毒地である

しかし後述のフトゲやタテの分布，特に前者の広十文字地域では，アカによる患者に加えて，フト

いそれを考えると，一般には蒜虫病発生の可能性

ゲ媒介性蒜虫病の発生が確認されている（浅沼ら，を示唆するとしてもよいであろう。

1971）。以上によると，（1）媒介種は慈虫病発生地

新媒介者の証明

少なくともフトゲとタテの2間で異なることがある，（2）複数種が同一地域で媒種が媒介者となりうるとした所以は，浅沼ら介に与りうるということになるが，（2）の例には，（1962）にまとめたが，その後の追加事項として

フトゲとタテの組合せ一伊豆大島の両種におけるは，タテー自然界での人寄生例（富士山麓，長R陽性結果による一が追加される可能性もある。

崎），R陽性例（伊豆大島，富士山麓）。フトゲー
R陽性例（秋田，伊豆大島，富士山麓，長野，岐阜，大分など），R媒介例（秋田，新潟，山形）がある。しかしイカオタマ，ホッコク，ムロトな
どにR陽性例のあることを思うと，新媒介者をフ

4Rjc舵 観α的漉肌gα肌悌8痂の細胞内増殖機構に関する電子顕微鏡学的研究
多村憲（新潟薬科大）培養L細胞系を用いてR．魏∫s㎎ロ耀s（Rt）のトゲとタテの2種に限定するには，まだ問題が残宿主細胞への侵入，細胞内増殖，および細胞からる（浅沼，1983）。

の放出について，電子顕微鏡学的に詳細に検討し，自然感染Rの鼠体内での運命 前述のフトゲのR媒介例（秋田）は，同県下の雄物川河川敷に健康ハッカネズミ116頭を設置した実験によるが，以下の結果を得た。

宿主細胞への吸着と侵入：Rtの宿主細胞への侵入は細胞表面への接着から始まる。この接着は

125

原形質分離を起こしたRtや熱またはUV照射により不活性化したRtでも起こるが，Rtをトリプ
シンで処理すると起こらなくなる。次にRtの接tosisの誘発とPhagosome中への取り込み，着した部分の宿主細胞膜が少し凹み，同時にRtの側面に沿って偽足様の凸起ができる。Phagocy−

中央部にくびれが生じ，2分裂により増殖する。

Phagosomeからの脱出，という過程で進行する。

細胞質内増殖と細胞からの放出：Rtは長軸のtosisの開始である。この場合Rtは必ず形態的にRtの数は経日的に増加するが，感染1日目では内部構造が比較的粗なRtが見られる。しかし旺

intactなものに限られ，原形質分離を起こしたも盛な発育を示す感染3日目頃には電子密度の高い内部構造をもったRtが多数細胞質中に見られ，のや，熱またはUV照射により不活性化したRt
は宿主細胞表面に接着はするが，決してPha− 形も比較的均一な短桿菌状（0．5−0．7×1．0−2．0
gocytosisされない。こうしてRtは宿主細胞の μm）のものが大部分となる。しかし時に，異常に長いRt（0。7×5．0−8．0μm）も認められる。まPhagosome中に取り込まれる。この時2種類のた非常に稀ではあるが，宿主細胞核中で増殖する

Phagosomeが見られる。一つは空胞膜がRtをtiゆtに包み，Rtと空胞膜との間隔が30㎜程度のものである。もう一つはRtと空胞膜の間隔が100㎜以上と巾広いものである。後者の場合，Rtも見られる。感染3日目になると増殖した細胞質内Rtは細胞周辺部に移行し，細胞膜を内側内部のRtは形態が変形しており，Rtがリソソー

enveloped virusが細胞表面からbuddingする形式

ムなどの影響により不活性化されて行く過程のもに似ている。感染4日目になると細胞表面に多数のである。一方前者のdghtphagosome中のRtのbuddingRtが蓄積し，殆ど細胞表面を埋めつは形態的にintactであり，正常な活性を保持してくすほどになる。感染5日目になると細胞はcpeから押し上げるように外に向かう。これは丁度いると思われる。このようなti客htphagosomeにを起こして崩壊し，buddingRtおよび細胞内Rtは空胞膜の一部が断裂しているものが認められる。

は外液中に放出される。このようにRtの増殖サ

その断裂部分からRtは空胞中から脱出し，宿主

イクルは4−5日で，doubHngt㎞eは約9時間で細胞中に放出されるのである。すなわちRtの宿ある。

主細胞への侵入は，細胞表面への接着，Phagocy一

126

一

1

般

マラリヤ原虫の迅速・簡易染色法一マラ
リアのスクリーニング診断およびマラリア原虫の薬剤感受性試験への応用
及川陽三郎
E．A

（金沢医大・医動物）

Jaial（金沢医大・熱帯医研）

谷荘吉（富士通・川崎病院）

マラリア患者の血液中原虫寄生率を求める場合，血液塗抹染色法は標本作製がやや煩雑であるうえ，

講

演

考察：マラリアの診断において血液塗抹染色標本を用いることはマラリアの種類の鑑別には必要であるが，標本作製がやや煩雑であるばかりでな
く，鏡検倍率1，000倍で油浸レンズを用いるため観察に多くの時間と熟練を要する。本法は，標本作成が容易でありまた鏡検も400倍で充分である。
即ち，マラリア流行地においてマラリア患者のス
クリーニングを現場で行う上で極めて有用であると思われる。

鏡検に多くの時間を要する。演者らは，この操作を簡便化する目的で，P伽s㎜4伽翅加響海6∫を用いてマラリア原虫の迅速・簡易染色法を考案し検討した。その結果，標本作製および鏡検に要する時間が大幅に短縮され，寄生率は塗抹染色法による2 マラリヤ原虫（PJα8配o漉郡耽6εrJゐθ ）感

染赤血球膜からの蛋白質分解酵素の抽出とその二，三の性質

井上

文英，徳（琉球大・医・一生化学）結果とほぼ同等であることを第55回寄生虫学会で報告した。今回は，この染色法のマラリアのスク
リーニング診断，およびマラリア原虫のinvitroでの薬剤感受性試験における染色法への応用について検討した。

誠吉

松山

玲子

（琉球大・Rl実験施設）

佐藤良也（琉球大・医・寄生虫）

マラリア原虫（P．加響履）に感染したマウス赤方法および結果：スクリーニング用には，ゲン

血球の形態は不規則に変化しており（Amold8
8L，1969），その赤血球膜ゴーストのSDS−PAGE

チアナ紫原液を0．3％EDTA−2Na・10％メタノー

では，赤血球膜の形態機能を保持しているスペク

ル加生食水で50倍に稀釈して染色液とした。マラ

トリンとバンド5（アクチン）（Mue皿er and Mor−

リア感染マウスの血液をピペットで直径約3mm rison，1981）が減少，または消失していることをになるように指頭上に置き，これをスライドグラ

認めた。一方，赤血球膜の機能維持に関与するエスに触れさせて移し（約0、008mJ），そのうえにンドジニアスなプロティンキナーゼ（Shohetand

毛細管ピペットで本液を1滴たらし（約0．025ml），混合後24×50㎜のカバーグラスをかけGreen喜uist，1978）活性についても正常赤血球の酵素活性に比べて80％近くも減少していることをて鏡検した。赤血球内原虫はほぽ球形で濃紫色に認めた（井上ら，1985）。

染色され，寄生率が0．1％前後でも容易に原虫を他方，マラリア原虫が宿主赤血球内で成長して検出することができた。薬剤感受性試験用にはゲ行く過程で，宿主ヘモグロピン分子をアミノ酸源

ンチアナ紫原液を0．15％EDTA−2Na・10％メタにしていることは知られている（She㎜，1979）

ノール加生食水で400倍に稀釈して用い，培養血が，更に赤血球膜の構成蛋白質をも加水分解して液を本液で5倍に稀釈染色し血球計算盤で寄生率

アミノ酸源として利用していると考えられる。そを求めた。キャンドルジャーでネズミマラリア感

れで，マラリア原虫に感染すると，原虫の蛋白質

染赤血球を塩酸キニーネ添加培養液で2日問培養

分解酵素が赤血球膜の構成蛋白質を加水分解するしたところ，寄生率が対照と比較して有意に減少ことにより赤血球の形態変化を導くと同時に，赤することが観察され，塗抹法による結果と一致し血球膜の機能低下を誘引していくと考えた。

ていた。

そこで私共は，マラリア原虫に由来する蛋白質

127

種で原虫の代謝に必要なアミノペプチターゼを次なった。CFU−Cは感染2〜4日目にやや減少す
るが，6〜10日目には感染前の2倍近くまで上昇の様な方法で抽出した。本酵素活性には合成基質

し，14日目には正常値以下となった。59Fe静注

分解酵素を得る目的で，エキソペプチダーゼの一のL−alanine−p−nitroan皿deかL−1eucine−p−nitroa通一により骨髄，および脾臓での鉄取り込みを調べてMeを用いた。まず，マラリア原虫（P・加㎎履，

みると，4日目より特に脾臓での有意な上昇が見

NK−65株）に感染した，BALB／C，♂，8週齢マられた。

ウスの赤血球膜ゴーストを調製した。本酵素は，これらの結果から，P．加嘔耀感染後早い時期

この膜ゴーストから非イオン性界面活性剤Triton

から血液幹細胞が影響をうけて，感染の進行と共X−100で可溶化（抽出）してセファデックスG−に大きく変動することが示された。

100でゲルろ過を行い，次いでDEAE一セファ
デックスでイオン交換クロマトグラフを行って精4

原（TLA）投与マウスにおけるT一リンパ製した。更に，この精製したアミノペプチダーゼ

球の動態についての物理化学的性質を検討した。

3Bα6θ8εα感染に対するトキソプラズマ抗P伽肌α伽肌わe㎎舵ε感染マウスにおけ

鈴木

直義，関口

信一，佐藤

基佳，

桜井

治久，斉藤

篤志，広瀬

恒夫

（帯広大・獣医）る血液幹細胞の変動について

浅見

鳴子，丸山 治彦，大橋

名和

行文

尾崎

真，

（宮崎医大・寄生虫）

文雄

（神戸市）

TLA乳剤投与マウスにおけるBσ伽彪感染に入破壊を受ける。同時に感染赤血球の処理や原虫対する抵抗性は，TLA非投与マウスに比較して明らかに増強される。そこで，TLA感作によるに対する免疫応答機序により，マクロファージやマウス体内のリンパ球，特にT一リンパ球およびマラリア感染において，宿主赤血球は原虫の侵リンパ球を含めた白血球系細胞にも，大きな変動

T一リンパ球Phenotypesの動態について検討しが見られる。ところが，これらの各種血球系の起た。

源である血液幹細胞のマラリア感染時における動トキソプラズマ破砕溶解抗原（144，000G，2時

態については，殆ど知られていない。そこで今回

問超遠心上清成分）100μgとFIAの乳剤を14日

私達はネズミマラリア（P．加響瓶）感染マウスをおきに2回，筋肉内投与した。初回TLA投与か用いて感染に伴う骨髄幹細胞の変動を調べてみた。

ら28日目にβ．御4肱∫∫感染赤血球1×105個を腹8週齢のC57BL／6マウスに同系マウスより得 腔内接種した。接種前1日目と接種後10日目の胸た感染赤血球105個をLp．接種して感染させ，経腺，脾臓，肝臓内Thy−1（＋）細胞（T一細胞）と時的に大腿骨より骨髄細胞を得た。骨髄細胞中のSIg（＋）細胞（B一細胞）数を計測した。B8わθs毎

多分化能血液幹細胞数を定量する為に，5x105 接種後10日目の胸線萎縮は両群とも著明であった個の細胞を致死量X線照射された同系マウスに静が，TLA感作によって胸線内Thy−1（＋）細胞数

注し，一週問後に脾臓に形成さたコロニー

は，非感作マウスの約2倍以上に増数した。同様（CFU−S）数を実体顕微鏡下で算定した。またに脾臓内Thy−1（＋）細胞数も，βα加s勿接種前1

CFU−Sより一段分化の進んだ血液幹細胞の数を激因子（CSF）存在下で軟寒天培地中で一週問培

日でTLA無処置マウスに比較して3〜6倍に増数し，接種後10日では更に増加した。TLA感作
マウスの肝内Thy・1（＋）細胞は接種前1日で養し，invitroで形成されたコロニー（CFU−C）

0．3×107個と計算されたが，接種後10日目で数を算定した。その結果，感染後2日目より
CFU−Sの上昇がみられ8日目のピーク時には，

3．4×107個と肝内集積が最も著明であった。肝

骨髄細胞1×105個あたりで感染前の約2倍とRESの増殖と活性化および囲管性の単核球の著なった。しかしながら14日目には，正常値以下としい集籏が観察され，これらの単核球は主として，調べる為に，1×105個の骨髄細胞をコロニー刺臓の病理組織学的および免疫組織学的検索では，

128

Ly＋1・2（一），Ly＋閑（＋）のThy−1陽性細胞で対しては，それが消えるまで毎週再検をくり返しあった。

た。Fansidarのみ投与群52例では，DayOでの5

インドネシア・北スマトラのマラリア対策（1）Primaquine一回投与のP．五game・
tocyteに対する効果
金子明（JICA北スマトラプロジェクト，

弘前大・医・寄生虫）

亀井喜世子（JICA北スマトラプロジェクト，

帝京大・医・寄生虫）
石井

鈴木

明

（岡山大・医・寄生虫）

猛GR7L1％がWeek2まで維持され，その後下降して行ったが，Week5でもGR11．5％が残った。
P血1aquineを組み合わせた56例では，DayOでのGR76．7％がday7ですでに30．3％と減少し，Week2で更に減り，Week3の時点でGR7．1％となった。

今回の結果により，Pr㎞aquine45mg一回投与をschizontocideと組み合わせる事が，P．∫game−tocyteの消退を相当程度早めるのに効果があることが明らかになった。

6 インドネシア・北スマトラのマラリア対策（JICA北スマトラプロジェクト）

マラリア流行地における，熱帯熱マラリア患者のchemotherapyでは，schizontocideによるtro−

phozoitesのclearのみならず，媒介蚊への感染形（2）昆虫学的研究とベクターコントロール

鈴木

Nana

S㎞の両村において，1985年8月から12月の問に，passivecasedetecdonを通じて見い出さ

哲志

高木 正洋
（JICA北スマトラプロジェクト，

であるgametocytesの消長に重大な関心が持たれる。当プロジェクトエリア内のKuala Tanl㎜g，

猛，菊地（JICA北スマトラプロジェクト）

予研・衛生昆虫）
安野

正之

（公害研）

研究地域沿岸マラリアの媒介種は，4．甜ルれた熱帯熱マラリア患者の内，Fansidar1，575mg

吻加sで，当地のこの種はexoph丑yのため，DDT

のみ一回投与群131例，Fansidar1，575mg＋Pri一

残留噴霧は効果を期待し得ない。そのため，inte−㎜quine45mgの組み合わせ一回投与群87例につ gratedcontro1による発生源対策を目的に，1978
いて，投与後のgametocyteの消長を追跡調査し年以来基礎研究を続けてきた。また，1986年9月た。尚，両群の全例においてtrophozoitesは，以降，32㎞2の実験地域において，囲operadon

Day7の時点でclearされており，その後re㎝1−を実施中である。

descenseは見られなかった。

対策としては，sourceredu面on／enviro㎜ental

Fansidarのみ投与した群では，DayOとDay2を対応できた25例，DayOとDay7を対応できた

managementを主要な柱とし，発生源の状況に応99例に，gametocyteの有意な変動は認められなを併用する。

かった。

じてグッピーの放飼，殺虫剤（アベート）の散布

海岸の砂丘後背地に散在するラグーンは，これPr㎞aquineを組み合わせた群では，DayOとDay2を対応できた29例にgametocyteの変動は見られなかったが，DayOとDay7を対応できた
72例では，DayOにおいてgametocytepositivity

広く，L魚や殺虫剤による防除がむつかしい。そのrate（GR）73．6％，gametocyte

density（PDI）2．2

drainingについて対策を模索中，s槻面加s幼虫がであったのがDay7ではGR33，3％，PDI1．3と日照の悪い水域から発生しないことから，不用のなった。この減少はwncoxon s

魚池をヤシの葉でおおい，90％の防除効果を得た。

testにより，1％水準を持って有意であった。

更にDay7でg㎜etocyteの残存していた例にを横に連ねるチャンネルを掘削し，水を近くの川に流すことによって，発生を封ずることが出来た。

別の川の河口閉塞によって生じた氾濫原は面積が生物的防除法としては，グッピーをメダンから運び放飼した。池の環境によってその効果は異な

129るが，一度の放飼で4カ月間幼虫の発生をみなかった例もある。殺虫剤については，アベートを

2週間に一度水量の1ppm散布し，良好な防除効果を得た。

8

Tr蹴ραπ080η霧αゐr配cεε9α配ゐεεπ8εはfeed・

er−cellを選ぶか？
P．J．Mhando，柳

中沢

秀介，福間

これらの経験をもとにして近く本格的なopera一

哲雄，

利英，神原

廣二

（長崎大・熱帯医研・原虫）廿onを実施する予定である。実施にあたっては，

CO㎜U⑯の参加による住民の自主的な防除活動

Tりψ伽os㎜伽姻即励∫8sε（Tg）を培養する

を将来の目途とし，実際的なcontrolmanualをつときに，侍養細胞（feeder−ce皿）としてICRマウ

くることを目標にしている。

ス新生仔由来の細胞を用いると，分離して間もな

7

インドネシア共和国北スマトラ海岸地帯における塩水し好性蚊孟η．側π面εeμsの棲息地近辺に住む人々の間の非常に高いマラ
リア伝播

糸川

い脳細胞（NMBC）と筋組織細胞（NMMC）はTgの増殖を助けるが，分離後40日を経過し樹立した

脳細胞，筋組織細胞は侍養細胞としての能力を失う。新しく分離された細胞でも，皮膚細胞や腎細胞は，Tgの増殖を助けない。新しく分離され孝英樹

トリパノソーマは，長期マウスで継代されたトリ（聖マリアンナ医大・病害動物）

インドネシア共和国北スマトラにおいて，JICA

パノソーマより培養するのが困難であると報告されているが，NMBCおよびNMMCはTgの分離による医療協力プロジェクトの活動の一つとして，された時期に関係なく侍養細胞として有効であり，マラリア対策が行われている。海岸地帯に多くの又丁互擁o惚吻郷6や丁猷ゆ耀6θ」にも有効であっマラリア流行巣が存在している事は前回報告した

た。培養トリパノソーマは，107／mJのレベルにが，今回媒介蚊である，A鉱s鰯4σ加sの棲息地

達し，形態上血流型と酷似しており，マウスに感近辺に住む人々の問でマラリア伝播がいかに高い染可能であった。Tgを侍養する細胞を培養皿のかを調べるために，全住民を対象に血液検査を施半面に，他の半面に侍養しない細胞を播き，その行し，malariaendemicityを明らかにした。またこ

上でTgを培養するとTgは前者の側でのみ増殖の地でのA鉱s％rateを調べ，した。増殖速度の速い細胞はトリパノソーマの侍蚊についても検討した。その結果，幼少児では養細胞として適しないという報告もあり，前述の60％と高い原虫陽性率であり，そして年齢と共に各細胞にその増殖をおさえるに足る最少量のX線

陽性率は下がり，中年以降では最低値となり10〜を照射し，侍養細胞として用いてみたが，いずれ20％となった。

の細胞においてもTgの増殖を助けることに関し

吻蜘sのsporozoite

また，寄生虫濃度についても同様な傾向を示し，て変化は見られなかった。Tgが増殖する系では，原虫濃度の高い者は幼少児のみに限られていた。

Tgは侍養細胞の直上あるいは間隙に入って細胞

熱帯熱マラリア原虫生殖母体に関する結果が，と密な接触を保ち，巣状になって増える。以上のもっとも顕著な傾向を示していた。これらの事実ことにより，侍養細胞から増殖因子が出されていは，住民にはかなりな程度でマラリアに対する免ない（あるとしても限極された近傍でのみ有効で疫が成立している事を示唆している。また蚊におある）と考えられ，細胞とTgの問に密な接触をいても，今回の調査によって初めてスポロゾイト

もたらすことがTgの増殖を推進するのに必要でがこの地の媒介蚊・肋．s吻吻加sから検出され，あると考えられる。

その陽性率は0．6％（2／341）とunstableなvector

である、肋．s㈱吻加sにしては高い結果が得られ，

蚊の方からもマラリア伝播の高いことが証明され
た。

130

9

マウス」774・1細胞を用いるL．doπooα漉amastigoteの加oεかocutivation：Car・

bocyclicinosineのamastigoteに対する作用について佐竹 弘靖，平岡修，綿矢 有佑（岡山大・薬・薬品化学）
Juheta，Y

K㎞ura，森重和久，」774・1細胞内に原虫は殆ど見られなくなった。

これに対し対照群では5日目において約60％のJ774・1細胞に原虫が認められた。また，C−Inoを前投与した場合，感染率を50％に抑えることができた。これらの結果により，C−lnoはL．d伽oびαη∫に対して，そのpromastigoteだけでなくamasti−

goteに対しても殺原虫作用をもつことがわかっ
た。

安治

敏樹，石井

明

（岡山大・医・寄生虫）

松田

彰，上田

亨

10京都市におけるBJα8ocsεεゐo耽加澹の感染状況（北大・薬・薬化学）

目的：寄生原虫の核酸代謝系は，宿主とは異

山田

吉田

稔，松本

幸雄

芳嗣，手越

達也，

（京都府医大・医動物）

なっており，プリンヌクレオチドの新生合成系が、B伽如のs爵㎞f傭Bmmpt，1912は，世界中に欠損し，且つ寄生原虫特有のサルベージ経路を有分布し，長い間，非病原性のヒト腸管内寄生酵母している。我々は，抗寄生原虫剤を開発するにあと考えられてきたが，近年Zierdt一派の一連のたり，このことに着目し，ヌクレオシド・アナロ

研究により本微生物が酵母ではなく原虫の一種とグの抗原虫作用の検索を行った。その結果，考えられるようになった。そして最近，この微生L召勧㎜吻伽砂蜘およびLε勧㎜痂伽o昭擁原

物が下痢の原因となる可能性も指摘されている。

虫（promasdgote）に対し，イノシンアナログであしかしこの微生物の研究は非常に立ち遅れており，るcarbocy∬cinosine（C−Ino）が有効であることを人体寄生の微生物であるにもかかわらず不明な点

見出し報告した。今回我々は，マウスマクロ
ファージ系J774・1細胞を用いて，五．伽ω伽f

月の間，京都市におけるβ．ho痂ηなの感染状況

ama甜goteの伽吻oHke culdva廿on systemを確立を調べた。まず糞便検査を行った1，251人を5つした。さらに，このシステムを用いて，宿主の形のgroupに分けた。Group1（14人）は外国人旅行

態であるamasdgoteに対するC−lnoの効果を検者で，彼らは殆ど消化器障害を訴えており，その討したので報告する。

多くは東南アジア，インド，アフリカを経て来日

方法：マウスマクロファージ系J774・1細胞が多い。今回演者らは，1983年6月から1986年7した者である。Group2（79人）は，消化器障害ををhpopolysaccharide（0．5，μg／mJ，DIFCO，E．ooJ∫

訴えて当大学付属病院を訪れた者であり，Group

0127：B8）を含むRPMl1640培地で1日培養し3（1，000人）は何らかの疾患あるいはその疑いでた。このJ774・1細胞と同数のL．吻o槻ηfpro−当大学の検査室に持ち込まれた検体（入院，外masdgoteを加え，さらに1日培養し，2回洗浄来）である。Group4（122人）は京都市内の2カした後，C−Inoを100％増殖阻害濃度である7．5所の精神薄弱児（者）施設の者で，Group5（36人）

μM加え，C−Ino存在下で5日間培養した。またはそこに勤める職員である。検査はまず糞便のC・hloを前投与し，2時問後にL．吻o％ ∫を加ルゴール染色で検索し，ついで位相差顕微鏡ならえた場合の効果も調べた。L．伽o昭勉を加えてびにギムザ染色により同定した。その結果，

から一定時間ごとに染色（Diff・Quik）し，100個のGroup1では14人中11人（78．6％），Group2では

J774・1細胞内のL．吻

79人中3人（3．8％），Group3では1，000人中12人

o昭初原虫を数え，感染率を求めた。

（1．2％），Group4では122人中21人（17．2％），

結果：C−Ino存在下で培養しているJ774・1細Group5では36人中1人（2．8％）にB．㎞伽お胞内へのム伽o％η∫原虫の感染率はC−Ino投与を見出した。下痢を認めたβ．加痂痂陽性患者

から経日的に減少し，C−Ino投与後5日目での中には，ランブル鞭毛虫，鞭虫など他の寄生虫

131の感染も認められた。またB．加痂痂は便性状が有形ないし軟便でも認められた。B．ho吻廊はわが国においても潜在的にヒトに感染しており，糞便を介して感染が広がるものと思われる。最後

メーバ症を診断されたものである。

12酵素抗体法による赤痢アメーバ症の診断
（予報）

山浦

に協力を頂いた本学・臨床検査部の方々に深謝す

常，白坂

龍暖

（東京女子医大・寄生虫）る。

11

広範囲の大腸潰瘍および潰瘍を伴った重症
赤痢アメーバ症の1例
中林 敏夫

中村

（阪大・微研・原虫）

猛，網岡

勝見
（中村病院・枚方）

患者は48歳，男性，大阪市在住。約20年問，精近年，注目されている酵素抗体法（ELISA）の赤痢アメーバ症への診断の応用性について検討した。抗原にはHJ株を使用し，標識抗体にはペルオキシターゼ標識抗IgG羊血清を，基質は，2，2・azinobis（3−ethylbenzthiazo㎞es面onicadd）を使用した。ELISAの手技は，Matsudaε」砿（1984）に準じ，反応後のプレート（Dynatech，イミュロ

神分裂症のため某病院に入院，加療中であった。

ン1）の吸光度を直読式分光光度計（MIP−32，コ

海外渡航歴はない。1986年2月10日，頭部外傷でロナ）により415㎜の波長で測定した。（1）4。C，中村病院に入院，10日後に治癒，退院した。当時，23。C，37。Cの温度で抗原のプレートヘの感作条件

右腹部の腫瘍の疑が持たれたが，対症治療を与えを検討した所，4。C1日感作で高い吸光度が得らるに止まった。

れた。（2）至適抗原濃度と標識抗体濃度の検討で6月22日，腹部膨満，便秘，発熱を訴え再入院

は，蛋白質として10μg／mが抗原の至適濃度で，した。イレウス病状が強く，腹壁癩痕性ヘルニア

一患者一穴法（血清希釈200倍）を利用するためのを認めた。ロマノスコピーで腸粘膜に異常を認め

標識抗体濃度は6，000倍が適した濃度と判定されなかった。X線像で大腸部にガス貯留が著明で，た。（3）ELISA（一患者一穴法）による赤痢アメー

通過障害が認められた。保存的療法を試みたが効バ症の診断：一般健康人血清（アメーバシスト陰なく，6月27日に開腹手術を施行した。上行結腸性者）200例についてELISAを実施した所全例がに狭窄および壊死があり，盲腸は後膜壁と，右結0．200以下（0．028−0．181）の吸光度を示したの

腸曲部は肝および胆嚢と癒着していた。上行結腸で0．200以上を陽性と判定した。その結果，ア

後面に膿瘍形成が認められた。回腸末端部より横メーバ赤痢患者2例は，0．586，0．610（平均

行結腸中問部までを切除し，腸吻合を行った。術0．598），アメーバ性肝膿瘍患者18例は，0．410〜

後も病状は好転せず，意識レベルも次第に低下しL820（平均1．020），アメーバ性肝膿瘍と潰瘍性た。7月4日，切除結腸組織標本中に多数の赤痢大腸炎の合併患者3例では，0．964〜1．500（平均

アメーバ栄養型を検出した。

L268）の吸光度を示し全例陽性と判定され，赤7月5日よりデヒドロエメチン（40mg，筋注，

痢アメーバシスト陽性者血清32例では2例

1日1回7日問），メトロニダゾール（500mg／日，（6．3％）のみが陽性であった。非アメーバ性肝膿

胃内注入，7日問）投与を開始したが，容態は悪瘍や大腸炎の患者血清6例は全例陰性であった。

化し7月14日死の転帰をとった。切除結腸粘膜はアメーバ症患者血清のELISA抗体価は，400倍が壊死におちいり，黄白色の厚い偽膜で被われてい6例（26．1％），800倍が3例（13．0％），1，600倍た。潰瘍は外膜に達し，穿孔寸前であった。潰瘍が10例（43．5％），3，200倍以上が4例（17．4％）

底部には多数の赤痢アメーバと共に好中球，単球で23例中6例（26．1％）の患者は国内感染であっ等が認められた。腸組織にはリンパ球，形質細胞，た。詳細は現在検討中であるが，ELISAは赤痢

単球等の他に繊維性増殖，血管の新生があり，壊アメーバ症の免疫学的診断に応用しうるものと思

死性肉芽性炎症像を呈した。本症は国内感染例で，われた。

長期間の経過の後に開腹手術後はじめて赤痢ア

132

13

東南アジアにおける疾病媒介蚊の羽音等を用いたトラップ法の応用

神田錬蔵，小川 賢一
（聖マリアンナ医大・病害動物）

ハマダラカの採集では，450Hzから530Hz間の振動数に雌雄共にトラップされている。

以上の実験から，羽音は同一種の雌の発する振動数を中心に，吸血誘引物質を併用することにより，ヌマカは雄が非常に多く誘殺され，イエカ属

疾病媒介蚊駆除法に殺虫剤を用いないで，蚊のでの吸血誘引物質の併用は，コガタアカイエカのもつ物理的生物的性質を逆手に，羽音吸血誘引性

雌を多く誘殺できた。ハマダラカ属は球形のトを利用してトラップする事の考案応用を試みた。

ラップの方が効率が高かった。本法により個体群

これは，蚊のもつ羽音を記録し，これに合わせた動態を推定することも出来，ヌマカ属の駆除に応

振動数の音をスピーカーで発信し，一方これに金用できるものと考えられる。

網ケージに実験動物を入れ，ドライアイスを用い，吸血誘引し，蚊を集める方法である。Mαηsoπ吻，

C％伽および、セ4おには，池庄司が考案した四角のプラスチック薄板を，スピーカーを中央に円筒形に巻き，粘着剤を噴霧し，飛来した蚊をトラップするものである。このスピーカーの下に動物ケージとドライアイス0．5kgを置いた。ハマダ
ラカには円筒に代わり，小動物のハムスターを入れた小型ケージとスピーカーを直径約30㎝の

14 虫刺創の病理学的研究（第3報）
一免疫学的検討一
E．A．Jala1（金沢医大・医動物，熱帯医研）
池田照明（金沢医大・医動物）

佐藤

喜一，堀田

進

（金沢医大・熱帯医研）

我々は，蚊刺咬暴露されたモルモットについて，

ちょうちんに入れ，外側に粘着剤を噴霧した薄い腹腔細胞における肥満細胞脱穎粒反応および

塩化ビニール袋をかぶせ，ちょうちんと袋は予め

homocytotropic

多数の穴を開けて，音と匂いの発散を計った。羽

さらに血清による感作状態のpassive

音には誘殺目的の蚊と同じ振動数のものを用いた。

ついても検討を加えたので報告する。

antibody

responseについて検討し，

transferにこうすることにより，羽音だけで誘殺するよりも，材料および方法：雄HarUey系albinoモルモッ

動物とドライアイスを併用することでより多く蚊

トの背部を週2回ずつ継続的に・セ4ゑs㎎卿あでがトラップされ，ヌマカ類では330Hzから350

蚊暴露し感作した。肥満細胞脱穎粒反応は，腹腔Hzの振動数の羽音に最も多かった。マレーシア

侵出細胞を蚊抗原加培養液で37。C，10分間incu−

セランゴール州，ベルジュンタイ村の亜鉛採掘廃

bateして判定した。Homologous

鉱に溜まった沼地の周囲の草叢において，同一ス

問と8日間の1atentperiodで行い，ELISAは抗原感作した96穴micropleteでperoxidase標識

テーション連続3日毎3回の採集により，初回のPCA反応は24時870頭から3回目には4頭に減少し他の場所でも and一モルモットIgGを用いて行った。蚊抗原は，同様の傾向を示した。尚，間隔は50mを中心に 蚊の頭部・胸部のホモジネートをPBS（pH7．2）
した。一方タイ国アユタヤ省B㎝gPa㎞部落の で抽出することにより得た。
水田地帯で，1986年5月から10月にいたる月毎の結果：蚊暴露開始後2週目に発赤腫脹が出現し消長をこの方法で調べたところ，8月が最高で8週目まで増大したがその後縮小傾向がみられた。

4，590頭をトラップした。この前後の月は明らか

肥満細胞脱穎粒率は蚊暴露開始後3週目より上昇に少なかった。各ステーションを比べ，密度の高し，以後も高率に推移した。感作モルモット血清

い所は他よりも比較的高密度を示した。場所によの24時間PCA反応では，7週目から9週目をり密度の差をみたが，雄蚊はその発生源からの移ピークとする一過性の反応を示した。8日間

動はないようである。C麗1砿師如㎝伽勿耀加sは

PCAでは，8週目に高い抗体価が得られたのみ

吸血誘引による雌蚊のトラップが著明であったが，だった。一方，56。C，30分間の血清の熱処理によヌマカではこれが特に多いとはかぎらなかった。

り，24時間・8日問PCAとも抗体価の大幅な低133

下が認められた。ELISAにより測定したspec面c

IgG抗体は，5週目より抗体値が上昇し8週目にピーグを示したが，その値は，極めて低く，以後抗体値は漸次減少する傾向が認められた。感作モルモットより得たIgE抗体陽性血清の1mJを正常モルモットに静注し，8日目に蚊暴露して，発赤の出現を検討したところ，発赤が出現し，大き16

沖縄本島における日本脳炎ウイルスの活動

状況（1985年3月一1986年10月）
只野

昌之，石嶺

安斎

俊一，福永 利彦

毅，牧野

芳大，

（琉球大・医・ウイルス）長谷川英男（琉球大・医・寄生虫）さも感作モルモットが示した最大平均値に匹敵す

沖縄本島における日本脳炎ウイルス（JEV）のるものだった。56。C，30分問の熱処理により血清

活動状況を知るために，沖縄本島で飼育された豚の感作能の低下が認められた。以上の結果より，のJEV抗体陽性率の調査，およびコガタアカイ

蚊刺咬の結果できる発赤腫脹は，即時性アレル

エ蚊（C．T．）の採集と採集されたC．T．からJEV

ギー反応に起因するものであると結論された。

の分離を行った。

15 ヒトスジシマカ，ネッタイシマカのチクン

グニアウイルス感受性に及ぼす吸血の影響
について
山西

浩

（神戸学院女子短大）

小西英二，上野山登久（神戸大・医・医動物）

1985年3月から1986年10月までの調査期間で80％以上の抗体陽性率が見られた期問は，沖縄本島の北部では，85年6月から12月までの間と86年7月から10月までの間に見られた。中部地区は，

85年7月下旬から10月下旬までの問と86年5月下旬から10月までの間に見られた。南部地区は85年8月中旬から9月下旬までの間と86年8月下旬かネッタイシマカ，2系統のヒトスジシマカおよら9月上旬までの間に見られた。調査期間を通し

びコガタアカイエカに，チクングニアウイルスをての陽性率は北部が50％，中部が47％，南部が経口感染させ，中腸におけるウイルスの消長を調30％で，北部が最も高く，南部が最も低い値を示べた。感染直後，感染1日後には中腸のウイルスした。3地区に共通して陽性率のはげしい変動が量は殆ど同じであった。感染2日後にコガタアカみられた。北部・中部・南部の3地区の抗体保有

イエカではウイルスが全く検出されず，ネッタイ

率の推移を合せてみると，抗体保有率が0％にな

シマカでも一部に少量検出されたにすぎない。ヒ

ることは殆どなかった（調査期問を通して抗体保トスジシマカ兵庫系では，ウイルス量は少なかっ

有率が0％になったのは，86年6月11日の検体だ

た。一方オアフ系では，ウイルス量がやや減少すけであった）。

る程度であった。感染3日後ではオアフ系の中腸2ME感受性抗体は本島全体でみると，85年4で103PFU程度のウイルスが検出され，4日後

月から10月の間と86年5月から10月までの間で検には中腸以外の組織からもウイルスが検出された。

出された。

感染7日後には兵庫系は中腸だけでウイルスがみ

中部地区の3カ所の豚舎で，85年3月から12月

られ，オアフ系では頭部，胸部で106PFU以上までの間で採集されたC．Tの数（1，233匹）に比のウイルスが検出された。14日後にはその傾向がべて，南部地区の一豚舎では86年5月から10月ま顕著になった。このように蚊のウイルス感受性は，での間に多数（10，875匹）のC．T．が採集された。

中腸におけるウイルスの消長に依存していた。

中部地区のプールから1株，南部地区のプールか

ネッタイシマカにおいて，感染前の吸血あるいはら18株のJEVが分離された。南部地区の採集地

再点検によって中腸でのウイルスの増殖への影響

点は，クレソンを栽培する水田に隣接しており，を調べた。今回の実験では，ウイルス増殖に及ぼそれが蚊の発生源と考えられる。中部地区の採集す影響は，はっきり認められなかった。

地点の周囲には発生源が見られなかったので，発生源の有無がC．T．の採集数の差に現われたものと考えられる。沖縄本島では水稲の作付が減少し134ており，「い草」「水芋」「クレソン」等の作付面

ドに比べて，安価であり，多数の検体を少量で迅積も発生源を考える上で重要であると考えられる。

速に処理できるので，DENウイルスの大規模な

沖縄の北部，中部で高い抗体陽性率が長期問続い

血清疫学的研究に広く応用できる。

たこと，2ME感受性抗体が6カ月問にわたって18

見られたことから，沖縄本島では1年間の内かな

デングウイルスのエンベロープ糖蛋白質の解析

り長期間にわたってJEVが活動していることが勉（金沢医大・熱帯医研）

智代子（金沢医大・血清学）

竹上

強く示唆される。

関

蚊の発生源である水田が減少していることと，

堀田

最近の豚の多頭飼育化の傾向とによりJEVの循環が起こりうる豚舎が孤立化しているのではない

進（金沢医大・熱帯医研）

デングウイルス感染成立の過程においてエンベかと思われる。そのために個々の豚舎で独立してロープ（E）糖蛋白質は重要な役割を果たすと考JEVが循環し，豚のJEVに対する抗体陽性率のえられているが，生化学的，免疫学的解析は十分推移が大きく変動したものと推察する。

とは言えない。本研究は，E蛋白質に対する単ク

17

ローン抗体の調整と抗原分析，並びにE蛋白質領

デングウイルスの微量中和試験

石嶺毅，只野 昌之，福永 利彦（琉球大・医・ウイルス）奥野良信（阪大・微研・防疫）域の遺伝子構造の解明をめざし解析を行った。

マウスBalb／cにデングウイルス1型望月株を接種し，追加免疫後4日目に免疫マウス脾細胞を採り，更に脾細胞とSP2／0ミエローマ細胞とを東南アジアのある地域では，フラビウイルスに融合させ，ハイブリドーマを得た。デングウイル

属するデング（DEN）ウイルス，日本脳炎（JE）

ス粒子を抗原に用いたELISA法による2回のスウイルスが共存している。DENウイルス1−4型のサブグループ，JEウイルスの間では赤血球凝集抑制（HI）試験で交差反応がみられ，HI試験

クリーニングの結果，20種以上の単クローン抗体価の調査から，得られた単クローン抗体は少なくでのウイルスの区別は難しい。血清学的に最も特とも3種のグループに分けることができた。即ち，異性の高い方法は中和（N）試験であるが，従来（1）ELISA価，HI価が共に高い，（2）ELISA価はのプラーク法では接種後のincubationを得た。ELISA価および血球凝集阻止（HI）抗体

periodが長高いがHI価は低い，および（3）ELISA価は低い

く，使用する検体血清量も多い。また，多数の検が皿価は高い等の各グループになった。いずれ

体を処理する血清疫学的研究には大量の器材，労のグループに属する抗体も免疫沈降法によってデ力等が要求される。8チャンバースライドとペル

ングウイルスE蛋白質と特異的に反応することをオキシダーゼー抗ペルオキシダーゼ（PAP）染色確認した。他方，エンベロープとの関わりが予想法を併用した迅速なDEN，JEウイルスの中和法

されるE蛋白質のC 末端側を調査する目的で逆が奥野ら（1978）によって開発されたが，8チャ

転写酵素および合成プライマーを利用するヌクレ

ンバースライドが高価で，多数の検体を扱う場合

オチドシークエンシングを行った。E蛋白質のに問題がある。今回，8チャンバースライドの代

C

わりに96穴プレートを用い，DENウイルスの中て配列している疏水性領域であることを明らかに和を試みた。96穴プレートではウイルス希釈とした。又，E蛋白質と隣接する蛋白質との切断部

末端側は，非電荷アミノ酸が20個以上連続しフォーカス数は良く相関し，DEN2，4型では接 位は，黄熱ウイルス等と同じくアラニンーアスパ
種後40時間，DEN1，3型では56時問後に計数し ラギン酸であった。以上の事実からE蛋白質のやすいフォーカスサイズを示した。N試験では8C

チャンバースライド，96穴プレートで得られたN他の領域は外界と接する部分であり，HA部位を抗体価は2倍以内の範囲にあり，良く相関した。

含めいくつかの抗原決定部位が存在することを示

96穴プレートを用いたN法は8チャンバースライ

唆している。

末端領域はエンベープに挿入しており，その

135

19

モノクロナール抗体を用いたコレラの迅速診断実用化の研究

柳ヶ瀬康夫，青山和枝，庄司宏（兵庫医大・細菌）
権平

文夫，松田

潤治，平野

勝，

集陰性となった。

以上本試験は患者検体からのスクリーニングに優れた特異性，検出感度を示し，コレラの迅速診断に有用と考えられる。

20バングラデシュで最近分離されたコレラ菌

杉山 純一，寺田 友次（デンカ生研）の生物学的諸性状

C．P．R面oa（SanLazaro病院，マニラ）

仲宗根

昇，岩永

正明

（琉球大・医・細菌）

従来，コレラ菌の検出同定は，患者検体より分離培養後に生化学的，血清学的同定を行うが，コ1986年1月コレラ患者から分離されたコレラ菌

レラの特性上迅速かつ確実な診断が必要である。

91株の生物学的性状，および6種の抗菌剤に対す

我々は患者検体よりコレラ菌の直接検出を目的とる感受性について調べた。血清型において，79

してホルマリン不活性化菌を免疫原とし，コレラ

株が小川型，10株が稲葉型，2株が彦島型であっ菌および小川型，稲葉型に特異的なマウス腹水型

た。コレラファージIVとポリミキシンBに対すモノクローナル抗体を作製した。この抗体をる感受性により，91株中60株がclassic型，31株

hydroxylaptitegelによるカラムクロマトグラ

がElTor型に分類され，1973年以来姿を消し，

フィーで精製した。精製抗体をラテックス粒子に

1982年再び突如として現れたclassic型コレラ菌

感作して，各クローンの抗体感作ラテックスをス

は，現在もなおバングラデシュにおいて優勢で

ライド凝集反応用試薬として検討を加えた。

あった。EITor菌31株の溶血性で，血液寒天平使用したA感作ラテックスは小川型，稲葉型標

板上でβ一溶血を示したのは21株（67．7％），Feely

準株を特異的速やかに凝集して，01抗原因子A and Pit血mの方法で完全溶血を示したのは8株を認識し，B感作ラテックスは小川型のみに，C（25．8％）であった。ファージタイプで，EITor

感作ラテックスは稲葉型のみに凝集を示し，それ菌はType1，4，5，untypableに分れたが，classic

ぞれ抗原因子B，Cを認識したクローンであると型菌は全株，Muk硝eeのTypeIに属した。El考えられる。

Tor型菌のプロファージ型は，ウボール型14株，そこでフィリピン国立サンラザロ伝染病院におセレベスcured16株，セレベス原型1株であり，いて，このA感作ラテックス試薬を用い下痢患者

ウボール型が多いのが特徴的であった。毒素産生

検体より直接検出とTCBS平板，TSIによる培を培地または培養条件を変えて調べてみると，当養試験を平行して行い，モノクローナル抗体感作教室で考案したElTor型毒素産生用培地のAKl

ラテックスによるコレラの迅速診断実用化の検討培地を用いると，今回分離されたclassic株も高を加えた。

い毒素産生を示し，1973年以前に分離された

1985年，54検体中本試験薬による直接検出およclassic型コレラ菌と相違を示した。6種の抗菌剤

びTCBS培養試験陽性になったものが18例，直に対するMICは，多くの株がクロラムフェニ

接検出陰性で培養試験陽性になったものはなかっ

コールで0．78μg／m1，ストレプトマイシン，アモた。

キシシリンで6・25μg／mJを示し，ミノマイシン，

1986年，検体をアルカリ性ペプトン水に1／100

テトラサイクリン，ナリジクス酸に対しては0．2

接種，2時間増菌培養したものについて検討した。 μ9／m 以下と，6薬剤に対し高度耐性株はみられ
254検体中，①直接検出陽性，培養試験陽性39例。 ず，1980年頃，バングラデシュにおいて問題と②直接検出陰性，培養試験陽性17例，これは定量培養で102〜4／mJ以下で本試薬の検出感度に達しなかった為である。③直接検出陽性，培養試
験陰性3例，これは100。C，10分問加熱処理で凝なったテトラサイクリン耐性コレラ菌は消失して
いた。

136

21バングラデシュおよび長崎で分離された
ノ4εm肌oπα8属についての比較検討

ミクロヨード法にて，そのβ一ラクマターゼ基質

一薬剤感受性を中心に一
下口 和矩，餅田 親子，山口 恵三（長崎大・病院・検査部）

草野

β一ラクタマーゼ産生性の関与が考えられたため，

ABPC，CEXに耐性を示す・4．勿4〆砂h惚を対象に，特異性を検討したところ，本菌はペニシリナーゼ
産生菌であることが示唆された。

22

展周，内藤 達郎

河野

茂，斉藤

厚，原

インドネシア各地の飲料水より検出された
P8e膨面〃⑰侃α8αε剛g加osαの薬剤感受性（長崎大・熱帯医研・病原細菌）

耕平と血清型について（長崎大・医・二内科）

奥脇 義行，矢内 寿恵（女子栄養大・微生物）

目的：今回私達は，バングラデシュの国際腸管感染症センター（ICDDR

藤田紘一郎，月舘 説子

B）より分与された（長崎大・医・医動物）、48釦伽oησs属の薬剤感受性について検討を加え，杉山

長崎にて分離された臨床分解株の成績と比較した

雅俊（順天堂大・医・衛生）

今回は，1985年および1986年に行った，インドので報告する。

対象および方法：供試菌株としては，ICDDR．

ネシア各地の飲料水の細菌学的調査で検出されたBから分与された人，環境由来の105株（A．勿4一Ps餌伽伽αs佛㎎吻sαの化学療法剤感受性テス吻h吻29株，A．

ト，および血清型別の成績を報告する。

砂吻41株，五．3の吻35株）と長崎にて分離された臨床分離株29株（、4．勿伽のhゴー

伽14株，Aoα吻812株，A．so励3株）の計134株を用いた。

薬剤感受性試験は，16薬剤（ABPC，MPC，PIPC，CEZ，CEX，CCL，CTM，CZX，CP，GM，KM，TOB，TC，MINO，ST，NFLX）を用いて，ミクロブロス希釈法（MIC，2000，Dynatech）による12濃度法にて行った。

成績：ICDDR B分与株と長崎臨床分離株を対象とした上記16薬剤の薬剤感受性試験の成績には，著しい相違点は認められず，両者ともABPC，
CEZ，CEX，CCLには耐性を示す株が多く存在し

P5ε％吻㎜σs oε㎎吻sαは，1985年は70検水中から11菌株，1986年は98検水中から19菌株分離された。

これらの菌株について，PCB，PIPC，MZPC，
CER，CET，CFX，CMZ，CPZ，GM，KM，CP，TC，CLおよびNAの14剤の感受性テストを，まずディスク法で行った。その結果，PCB，PIPC，
MZPC，GM，KM，CP，TC，CLおよび第3世代セフェム剤のCPZには良好な感受性が示された。
しかし，第1世代セフェム剤のCER，CET，第2世代セフェム剤のCFX，CMZには耐性であった。
そこで，1986年に分離された19菌株について，たが，その他の薬剤に対してはほぼ全株が感性をディスク法で耐性を示した薬剤のMIC値を求め示した。一方，菌種別に薬剤感受性成績を比較した。その成績は，19菌株の殆どでCER1，000
μg／mJ以上，CET250μg／m 以上，CFX1，000
μg／mJ以上，CMZ500μg／mJ，およびNA1，000てみたところ，、4．勿伽ρh吻，ノ4．oα 吻では殆どの株がABPC，CEX，CEZ，CCLに耐性を示したが，ハ．soδ吻はABPCに対して約50％の株が感μg／mJという極めて高い値を示した。

性を示し，また，CEX，CEZ，CCLに対してはそ

一方，疫学的見地から重要視されるPs舳郷o一

れぞれ約10％，30％，30％の株が感性を示した。

紹s碑㎎物凹の血清型を調べたところ，1985年

次に，菌株毎の薬剤耐駐パターンにづいて比較しの11菌株では6株がE型であった。また1986年のてみると，A妙伽砂履o，A．6α吻6では，ABPC，19菌株では，E型とF型がそれぞれ6株ずつでCEX，CEZなどに多剤耐性を示す株が殆どであっあった。

たが，、4．50ゐ吻はCEXのみに耐性を示す株が約半数に認められた。これら耐性機序の一つに，

137

23

北アフリカ地域における飲料水の水質検査
澤田 清子，須永寛

小原（瑞穂短大）

博，海老沢 功，高柳満喜子（東邦大・医・公衆衛生）小松

1984年8，月，モンロビア，アビジャン，コナク

俊彦，甲原

照子

（予研・腸内ウイルス）

リ，アルジェ，チュニスおよびトリポリの6都市

において，水道水，レストランや邦人宅の飲料水

水より感染する疾患は，開発途上国では極めて並びに市販の容器入り飲料水について，水質検査

高頻度である。海外に滞在する日本人が増加するを行って次の成績を得た。

に伴い，水の衛生がますます重要な課題となって水道水でも遊離残留塩素が検出されないものがおり，その有効な対策が望まれている。近年，多2／3に達し，殊に大西洋沿岸の3都市とトリポリ

孔質中空系と銀活性炭を組み合わせた滅菌水装置に多かった。また，これらの地域の水道水から大（三菱レイヨン・日本ケミファ）が開発され期待腸菌群が分離された。

が持たれている。その効力を調べるため，特に開モンロビアにおける食堂や，ホテルの飲料水か

発途上国滞在中に水より感染し易いと思われる病らは高率に大腸菌群が検出された。在留邦人家庭

原体を用いてその滅菌性能について実験を行い，の飲料水からも，大腸菌群が検出される例があっ検討した。

た。これらの飲料水は，いずれも濾過器を通した実験菌としては，大腸菌，セラチア，腸チフスものであり，その使用法や管理が適切でないため

菌，コレラ菌，赤痢菌，ネズミチフス菌を用いた。

と考えられた。一方，市販の容器入り飲料水から

108〜109個の生菌を含んだ蒸留水1」を滅菌水装

は，大腸菌群は検出されなかった。

置に通し，得られた液をミリポアフィルターに通飲料水80検体のうち，大腸菌群が陽性のものはした。フィルター上部を遠藤培地に接種して一晩20検体であった。また，分離された21株を菌種別培養し，集落数を測定した。滅菌水装置に菌液をにみると，K，ρ鯉耀ω吻εが1／3で最も多く，E磁直接通す実験の他に，水道水50」または多摩川

oJo㏄卿，C．か6朔鵡κo燭oo8の順であり，E． oJ∫の水151を通す前処置を施した後，1週間放置も2株検出された。種々の薬剤に対する感受性検

して菌液を通す実験も行った。菌液中の菌数は普査（MIC）ではSM，TC，CPに感受性の低いもの

通寒天培地を用い，段階希釈法で測定した。ウイが少数例みられたが，ピペミッド酸にはすべてがルスについては，ロタウイルスを用いて検討した。

高い感受性を示した。

腸菌球の陽性は6検体で，7株が分離された。

6種類の細菌について行った実験の結果，滅菌水装置を通した後の液中には菌はまったく検出さそのうち5株は翫血伽傭吻繊伽sで，翫れなかった。水道水，または多摩川の水を通す前力80σ傭とS止加吻翅が1株ずつであった。なお，処置を施した後でも，液中に菌はまったく検出さ腸球菌陽性の検体はいずれも大腸菌群も陽性でれなかった。ロタウイルスについて行った実験のあった。

水道水の化学的検査では，過マンガン酸カリウ

結果もウイルスは検出されなかった（堀田ら，1984）。

ム消費量が10mg／」以上のものが過半数を占めて主要な腸内細菌およびロタウイルスについていた。総硬度および塩素イオン濃度は，アルジェ

行った実験の結果，病原体は検出されず，良好なおよびトリポリの水道水の殆どが，飲料水の水質性能を有すると思われた。多孔質中空系と銀活性基準値を越えていた。殊にトリポリでは，塩素イ炭を組み合わせることにより，細菌はもちろんウオン濃度が1，000mg／」以上のものもみられ，塩イルスの除去にもかなり効果が期待できる。飲食

辛い水は日常生活に支障を来していた。

物に関する注意や予防接種に滅菌水装置を加える
ことにより，経口感染症の予防に大いに役立つと

24

多孔質中空系と銀活性炭を組み合わせた滅菌水装置の効力思われる。

Japan. J. Trop . Med. Hyg., Vol. 15, No. 2, 1987, pp. 139‑168

139

PROCEEDlNGS OF XXVIII ANNUAL MEETlNG OF
JAPANESE SOCIETY OF TROPICAL MEDIClNE (1)22‑23 Novembe* 1986 Okinawa

CONTE NTS
Specral lecture : Public health aspects of tropical diseases in theWestern Pacific Region
H. Nakajirna

Symposrum : Diarrheal diseases in tropical area
Chaired by K. Kobari and Y. Zimakal Bacterial diarrheal diseases

Y. Takeda

2 Viral diarrhea in the tropics

Y. Miyazaki

3 Current consepts of Entamoeba histolytica and amoebiasis

T. Takeuchi
4 Study on the serotyping and biotyping of 300 strains of CampD'Iobacter by Lior's

methodSirna Huilan et al.

5 Strategies for the control of diarrhoeal disease in the Asian Pacffic Region

C. Kidson

Symposium : Tsutsugamushi disease (scrab typhus) in Asia
Chaired by M. Otsuru and H. Tanaka
1 Medico‑zoological studies of tsutsugamushi disease in Japan, with special reference

to Leptotrombidium pallidum K. Asanuma

2 Studies of tsutsugamushi disease in peninsular Malaysia A. Shirai
3 Epidemiological features of tsutsugamushi disease (scrab typhus) in southern China

Lin Quansheng
4 Electron microscopic studies of intracellular multiplication of Rickettsia tsutsugamushi

A. Tamura
5 Rapid serologic diagnosis of tsutsugamushi disease by means of indrect irnmuno‑

peroxidase (IP) technique T. Suto
General presentation (1‑24)
1 Study of quick and simple staining for malarial parasite ‑ Application for screening ofmalarial parasitemia and detennination of drug sensitivity to malarial parasite

Y. Oikawa et al.

2 Isolation and some properties of proteolytic enzymes from Plasmodium berghei

infected mice erythrocyies F. Inoue et al.

3 Kinetic changes of hemopoietic stem cells in Plasmodium berghei‑infected mice

M. Asarni et al.

4 Changes of T‑lymphocyies after babesia infection in mice ptetreated with toxoplasma

lysate antigen (TLA) N. Suztiki et al.

5 Malaria control in north Sumatra, Indonesia (1) Gametocyiocidal effect of prirna‑

quine A. Kaneko et al.
cotrol T. Suauki et al.

6 Malaria control in north Surnatra, Indonesia (2) Entomological studies and vector

140
7 Extremely high transmission of malaria among those who live near breeding sites ofthe sea‑water mosquito, An. sundaicus, in the coastal area of north Sumatra,Indonesia
H. Itokawa
P. J. Mhando et al.
8 Does Trypanosoma brucei gambiense choose feeder‑cells ?
9 Anti‑parasite activity of carbocyclic inosine against amastigotes of Leishmania dono‑

vani

H. Satake et al.

10 The prevalence of Blastocystis hominis infection in humans in Kyoto city

M. Yamada et al.
ll A serious case of amebiasis accompanied by extensive colic ulcer and abscess
T. Nakabayashi et al.

12 Serologicai diagnosis of amoebiasis by micro‑ELISA H. Yamaura and R. Shirasaka
13 Use of wing sound traps for male mosquitoes of some vector species in fields

T. Kanda and K. Ogawa
14 Pathological study on mosquito‑bitten wound (III) An irnmunological study
E. A. Jalal et al.

15 The influence of blood sucking on the susceptibility of Aedes albopictus and Aedes

aegypti to CHIK virus H. Yamanishi et al.

16 The state of Japanese encephahtis virus activities in Okinawa main island

M. Tadano et al.

17 Micro‑neutralization test of dengue viruses using 96‑well plate and peroxidaseanti‑

peroxidase technique T. Ishimine et al.

18 Analysis of the envelope glycoprotein of dengue virus T. Takegami et al.
19 Monoclonal antibody‑based reversed passive latex agglutination for identification ofVibrio cholerae Ol Y. Yanagase et al.

20 Characterization of Vibrio cholerae O1 recently isolated in Bangladesh

N. Nakasone et al.

21 Comparative study on drug susceptibility of Aeromonas species isolated in Bangla‑

desh and Nagasaki K. Shirnoguchi et al.

22 Drug sensitivity and serotype of Pseudomonas aeruginosa isolated from drinking

water in Indonesia Y. Okuwaki et al.

23 Examination of drinking water in northem African countries

K. Sawada and H. Sunaga
24 Water‑sterilizing capacity of an equipment made of honow multi‑porous fibers andsilber‑activated carbon powders H. Ohara et al.
25‑68 wiu be appeared in the next issue

141

SpesialPUBHCHEALTHASPECTS OFlN

WESTERN

PACIFICHIRosHINAKAJIMATHE

Regional

Malaria

remainsalecturesedousTROPICALDISEASESREGlON

Director，WHOIWPRO，Man∬a，Phihppines

healthproblem innine

countriesoftheWestemPacmc

Region：

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Malaysia，Papua New Guinea，Solomon Islands，Vanuatu and Viet Nam．The major tec㎞cal
problems

areresistImce

resistImce

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oftheoccu∬encediseaseis142

being strengthened, and development of a new vaccine using recombinant DNA technology is
being explored.

Dengue fever / dengue haemorrhagic fever is present in China, Malaysia, the Philppines,
Singapore, Viet Nam and countries of the South Pacific. A lwe attenuated dengue‑2 vaccine has
been prepared and is being tested in Thailand. Research on the preparation of a new dengue‑2
vaccine using recombinant DNA technology is progressing in Australia, Japan and Malaysia.
Hantaan virus, the etiological agent of haemorrhagic fever with renal syndrome, is endemicin China and the Republic of Korea. Serological surveys on rodents collected in Hong Kong,Japan and Singapore have demonstrated the presence of antibodies to Hantaan virus, suggestingthat the virus may be widespread in the Region.

The increasing prevalence of sexuauy transmitted diseases, notably gonorrhoea caused by
penicillinase‑producing strains of Neisseria gonorrhoeae, has been reported in various countries of

the Region, and the possible resurgence of syphilis, due to the use of antibiotics other thanpenicruin in the treatment of peniciurn‑resistant gonorrhoea, has been of concern to many
countries. It is proposed to inihate a surveillance programme on antibiotic resistance.

Cases of acquired immunodeficiency syndrome (AIDS) have been reported in the Region
and it is feared that in due course it might become widespread. Simple diagnostic procedures
are heing developed to facilitate surveillance of the disease.

143

Symposium
Diarrheal diseases in tropical area

1 BACTERIAL DIARRHEAL DISEASES
YOsrnFUMI TAKEDA
Department of Bacterial Infection, The Institute ofMedical Science, The University of Tokyo

Acute diarrheal dlseases are recognized as a major cause of mortality and morbidity inchildren in developing countries in the tropical world. The World Health Organization estimatedthe number of deaths due to acute diarrheal diseases in children under 5 years old in 1980 as

about 4. 6 million in Africa, Asia (excluding China), and Latin America. Several entericpathogens, such as Vibrio cholerae O1, V. cholerae non‑O1, enterotoxigenic Escherichia coli,
enteropathogenic E. coli. Salmonella. Shigella. Campylobacter jejuni and V. parahaemolyticus
have been shown to cause acute diarrheal diseases.
The pathogenesis of these enteric bacteria has been a focus of research. Many toxins
responsible for acute diarrhea have been identified and the physicochemical, immunological andbiological properties of some of these toxins have been exanained. The invasive character ofsome of these bacteria has also been studied and at a molecular level the plasmids encoding
invasiveness have been identified.

Among the many toxins so far reported, cholera enterotoxin is the best understood. It
stimulates membrane‑bound adenyl te cyclase of target cells and increases the intracellular levelof cyclic AMP. Heat‑labile enterotoxin of enterotoxigenic E. coli shows similar physicochemi‑cal, immunological and biological properties to those of cholera enterotoxin. Recently, themolecular structure of heat‑stable enterotoxin of enterotoxigenic E. coli was characterized.
This toxin stimulates membrane bound guanylate cyclase and increases the intracellular level ofcyclic GMP, but not adenylate cyclase. The mechanism by which intracellular increase of cyclic
nucleotides in the target cells causes sigDificant secretion of water is still unknown.

Shiga toxin produced by Shigella dysenteriae has also been well characterized. Although therole of Shiga toxin in Shigella infection is not fully understood, the potent cyiotoxic activity of

Shiga toxin may somehow be related to dysentery caused by the organism.
Recentiy, some E. coli strains that belong to 0157 : H7 were found to produce a toxin
immunologically related to Shiga toxin. Further studies revealed that these strains also produce
a potent cyiotoxin that is irnrnunologically unrelated to Shiga toxin. The roles of these toxins in

E. coli 0157 : H7 infection require further study.

144

2 VIRAL DIARRHEA IN THE TROPICSCHIAKI MrrAZAKI
Department of Pediatrics, Faculty of Medicine, Kyushu University

Acute infectious diarrheal illness is recognized as one of the leading causes of morbidity andmortality in tropical developing areas. Viral agents discovered during the last decade have been

proven to be responsible for a large population of diarrhea with defined etiologic agents.
Especially, from numerous cross‑sectional studies in more than 30 countries (including thetropics), rotavirus has been documented as a major etiologic agent for gastroenteritis in children

under 2 years of age. Fifteen to seventy per cent of the hospitalised cases (especially caseswith severe degree of dehydration) in these studies have been found to be associated withrotavirus. Studies in temperate clirnate areas revealed peak incidence of rotavirus diarrhea inchldren between 6 months and 2 years of age, while a good proportion occurs in infants under 6months of age in developing countries. This discrepancy may due to an earlier exposure tocontaminated environment under poor sanitation in developing areas. Rotavirus diarrhea shows
its peaks of occurence in cooler seasons in temperate climate areas, compare to a year‑throughbut with occasional outbreaks in tropical areas.

From 1982 to 1984, we studied epidemiology of rotavirus infection in Kenya in East Africa,and over 2, OOO diarrheal stools from Kenyan infants and young children were conected at some

hospitals. Under monthly surveillance, an average of 22%, range 8 to 50%, of the stools
showed rotavirus‑positive by ELISA. Age distribution of rotavirus diarrhea was similar to otherreport from tropical countries. By electropherotyping of viral RNAs, rotaviruses assigned tosubgroup 11 were found predorninant in Kenya (sinular to other countries), and the altemation of
predominant strain was observed in the studied area. Monthly detection rate of rotavirus did
not correlate with climatological data.

Although therapy with oral rehydration solution succeeded in decreasing the mortality ofseverely dehydrated diarrhoic children in many developing countries, prophylaxis with vaccine

would have a significant impact. Multiple divergent approaches have been undertaking todevelop live oral vaccine against rotavirus : (1) rotaviruses of animal origin, (2) attenuated humanrotavirus, (3) reassortant hybrid virus, etc. Several strains of anirnal origin including bovine

strain RIT 4237, rhesus strain WWU 18006 and bovine strain WC 3 have been undergoing clinical

trials for their safety, immunogenicity and effectiveness in several countries. In humanrotavirus has two subgroups (1 and ID and at least four serotypes. Human and anirnal
rotaviruses usually share common group antigen. If heterotypic immunity to a given vaccine isfound to be enough for protection against any human rotavirus serotype, these vaccine
candidates could be truely useful. It should be noted that none of these vaccines will likely

protect new rotaviruses lacking cornmon group antigen which have recently caused large
outbreaks of gastroenteritis in China. Anyuvay live vaccine may become a strong weapon
against rotavirus diarrhea of infants and young children in the near future.

145

3 CURRENT CONCEPTS OF ENTAMOEBAHISTOLYTICA AND AMOEBIASISTSUTOMU TAKEUCHI
Department of Parasitology, School of Medicine, Keio University

In the present symposium, recent concepts of Entamoeba histolytica and its infection are
discussed as a model of protozoan diarrheal disease, because numerous significantly novel

knowledges have been obtained on amoeba and amoebiasis.
Amoebiasis is still one of the major health hazards in developing countries. According toWHO, there are 400, OOO, OOO people annualy infected with this protozoan, most of whom areapparently located in the developing nations. On the other hand, the number of cases with thisenteric infection had been dramatically decreasing from 1950 to 1975 in developed countries ;accordingly, it seemed to be of no more significance in their public health. Since 1980,
however, we have had a sudden increment in the number of cases with invasive amoebiasis inJapan. Our later investigation suggests that this is attributable to sexual transmission of thispathogen, at least partially, among biased males. The same pattern of its transmission also has

been confirmed in the United States, the United Kingdom and Canada. Although somedifferences have been found concerning the factors which affect this mode of transmission
between Japan and other developed countries, it is undoubtedly important in relation to AIDS.
New knowledges have also been obtained on the biology of E. histolytica, in particular on its

pathogenesis. It is now evident that cell kiWng and subsequent phagocyiosis are the primarymechanism of its pathogenicity. Current evidences suggest that there are two modes of cellkilling by this protozoan, i. e., one is contact‑dependent and the other contact‑independent cellkilling. The former is considered to be attributed, at least partially, to the action of "pore‑

forming proteins" which deloparized J224 macrophage‑like cells and spleen lymphocyies,
although they were not found on the plasma membrane of amoeba. On the other hand, thecontact‑independent process is made by a protein of molecular weight 35, OOO to 40,000 and a pl

4.5 to 5.0. The activity of this protein is inhibited by N‑acetylgalactosamine, suggesting a
lectin‑like property.

Concerning diagnosis of amoebiasis, serodiagnosis is certainly the primary measure. Cur‑

rently, ELISA, CIE and gel diffusion are widely employed for this purpose. Indirect im‑
munofluorescent antibody technique was found effective only for hepatic amoebiasis. However,the IFA developed in our laboratory was found as effective as gel diffusion and CIE for theserodiagnosis of both intestinal and hepatic amoebiasis. Moreover, of particular interest
concerning this modified IFA was that it qualitatively agreed with gel diffusion at more than 97( owhen tested on asymptomatic cyst passers, suggesting that there are two types of carriers ; one
with and the other without tissue invasion of this parasite.

Chemotherapy of amoebiasis has been prirnarily done by nitroirnidazole derivatives likemetronidazole. However, distinct mutagenicity and carcinogenicity in experimental models, andappearance of metronidazole‑resistant Tritchomonas vaginalis indicate an importance of develop‑

ment of new anti‑amoebic drugs. We have been testing some series of compounds, and foundthat dichlorophene, a halogenated bisphenolic derivative, had potent anti‑amoebic effects. Some146

other compounds of plant origin also have been found effective in treating experirnental amoebic
liver abscess.

4 STUDY ON THE SEROTYPING AND BIOTYPING OF 300 STRAINSOF CAMPYLOBACTER BY LIOR'S METHODSIMA HUILANl, XIE MEIWEN2 AND XU JUFEN2
Consultant in Health Laboratory Technology, WHO Western Pacific Regional Office,
Manilal and Shangllai Hygiene and Antiepidemic Center, Shanghai, China2

Three hundred Campylobacter strains isolated from hurnan (277 strains) and anitnal (23strains) were serotyped by Lior's slide agglutination assay. The typing sera were supplied by

Dr. Lior through waO Geneva.
The result showed that 251 strains (83. 7%) were typable and 49 strains (16.3%) were
untypable. Of the 277 human strains, 233 were typable and of the 23 anirnal strains 18 weretypable. These figures represent 84. 1% and 78. 3%, respectively. The typable strains fromhuman belonged to 31 serogroups and 16 sub‑serogroups (agglutination in paks of sera) of which
the most common serogroups were serogroup 1, 2, 4, 9, 28, 32, 36, 48 and 60. More or lessthese figures are similar to those reported in European countries where, the most common
serogroups are 1, 2 and 4. On the other hand, the very common serogroup 7 in Europe is notfound in Shanghai, China.

According to a new extended biotyping scheme of Lior, 213 (76. 9%) human strains belonged
to Campylobacter jejuni I and II, 64 (23. l%) belonged to Campylobacter coli I and II, whereas allof the 18 anixnal strains (12 strains from swine) belonged to Campylobacter coli I and II. The

result of serotyping and biotyping assay of 300 Campylobacter strains indicated that swine wasone of the important sources of infection of the Campylobacter enteritis in Shanghai, China which

compared with Skirror's report that CampVlobacter coli occupied 5% only among Cam ,lobacter
jejunilcoli group.

A comparison of Lior's and Penner's serotyping method showed Lior's slide agglutination
assay was simple, rapid and easy to perform in the field trail for epidemiological investigation.

The autoagglutination often occurred in slide agglutination when saline suspension was used. It
could successfiilly be eliminated through suspenditrg the bacteria in phosphate buffered saline

containing DNase.

The preparation of the typing sera of Cam ,lobacter wiu be studied further in Shanghai
Hygiene and Antiepidemic Center.

147

5 STRATEGIES FOR THE CONTROL OF DIARRHOEAL DISEASE
IN THE ASIAN PACIFIC REGIONCHEV KIDSON
Queensland Institute of Medical Research, Brisbane, Australia

The patterns of diarrhoeal disease differ greatly from country to country in this region andindeed from one area to another within a given country. These differences relate to acuteversus chronic diarrhoeas, to relative frequencies of particular etiological agents, to the degree ofmorbidity, to the extent of mortahty and to the age group affected.
Given the difiering spectra of geographical characteristics, population size, economic status,

health care service accessibility, social organizatio,n and stage of development of national
programs for the control of diarrhoeal di,seases (CDD), this variability is hardly surprising.
However, the practical implication is that it is essential to have a flexible approach to achieving

the goals of the global CDD program.
In countries such as the People's Republic of China the mortality from acute diarrhoeal
disease is low, whereas in countries such as the Socialist Republic of Vietnam or Bangladesh
there is still significant mortality. The differences relate to both disease patterns and toaccessibility to appropriate health services. Thailand, for example has established an, effective

national CDD program, as has the Philippines, but these countries have had differing successthus far in reducing mortality due to acute diarrhoea.
Morbidity data are not readily available for many countries but morbidity may be high evenwhen mortahty is low, e.g. in China. This type of situation usually reflects adequate treaiment

services but inadequate organization of preventive programs at this time.
In general in this region, as elsewhere in, the world, there is a correlation between the use oforal rehydration therapy and reduced mortality in acute diarrhoea. There is generally a high asso‑
ciation of rotavirus, E. coli and a number of other organisms with acute diarrhoea in children. In‑

deed, the similarity in etiological patterns is remarkable, suggesting that corumon strategies may

be applicable for prevention. However, there are some marked differences from area to area in

the frequency of shigella and amebic dysentery which appear to be related to socioeconomic
and behavioural variables of considerable consequence to the planning of preventive strategies.
A number of important research studies focused on environmental or social interventions arein progress in some countries in the region. Many more are needed to address the key issueof the economic implications of control strategies where the latter may impinge on longstand‑ing agricultural or sanitaion practices. These strategies need to be weighed against other poten‑

tial interventions involving technologies such as immunoprophylaxis using new vaccines.
Although epidemiological information on acute diarrhoea is far from complete in most areas,
data on chronic diarrhoeas and their relationship with nutritional status are far less available. In

this context there are important chauenges for the development of molecular diagnostic tools suit‑able for epidemiological surveiuance to perDait more rational control strategies to be implemen‑ted. These developments need to be combined with socioeconomic analyses to facilitate plan‑ning towards achievable goals in the control of diarrhoeal disease in countries in the region.

148

Tsutsugamusi

1disease（scrab

MEDICO・ZOOLOGICALDISEASE IN

typhus）in

STUDIESJAPAN，W皿H

OF

asia

TSUTSUGAMUSHl

SPECIAL

REFERENCE

TO LEP：7η7ROMBIDπ刀πP既L11）乙曜
KIYosHIDepartmentAnoutbreakofin thefooth皿sofMt．Fujiof廿1edisease血Jap2m．

tsutsug㎜usi
This

type

vectorsAofdiseasehl cer㎞areas

or

the

also廿1ez卿粥カゴ（classicalatMuseum，Tokyo

chigger−bom dckettsiosis amongsuggested

type

existence ofahithertoofoccun』enceof

American soldiers

un㎞o㎜endemicthediseasethe disease）but by otherpresent，五．so協2〃α惚andL．加〃∫伽吻arearea

trImsmitted

species of chiggers

detem血1edasnewL．加〃づ伽吻and L．σ肱吻％sh oundonkl Japan．

occurrence and rickettsial

positivity

rodents，M比η伽szo蛎のε〃∫ill

aclassical

endemic

dj直erence

case

of

the

kl

to・theSciencein1948，estabhshedthe（逓sease），and

monthly

Zoology，National

illcident

not by Lの該o伽o 2δ∫4づ％御σ々

（new

of

AsAMuRA

incidencedisease

of

area

this

in

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cases

in

seasonal

sp血g

as

we皿

asaut㎜肛e幡血ttedbyL．卿伽whereas血ecases血su㎜erm副ybyL．伽醜Asregards

seasonal

to locah廿es；one
Japan，w皿e

occurence

the

type

parts

of

montNy occurence andcohlcided

vdth

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has2peaks

singlepeak

the other type has aor less southem

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is

this

on the

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man

type

occurence

of

cases

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thoughtheinsummerother

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new

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as

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L．々吻sσ娩L．

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L．加〃ゴ4％吻

cases

in

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isolatedfromrodentsasrickettsia丘om

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andthereh1．

｛md五．so吻〃膨are missing or a few in n㎝ber，such

Fuji andsphng

Kanagawa，Shizuoka，Tokyo，Oita

positiveofJapan，dlere

disease，the possible

infestjng

ρσゆσ」8，L．〃3％名oホ06πs商，ムセo伽o吻わあα」αρoextraord㎞ary

asdisease〇αブ砂o 吻，ノV．吻吻吻郷厩and others，and

The

late

positivityof thethese2vector species of new

assumedhl

from late autmm

country，suchdckettsia

hlcidence

z

in

of

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cases

we皿as

a

ofMt．

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have

yet

been

reported

chigger，L，肋ωα卿％名甑149

2 STUDIESOFIN

TSUTSUGAMUSHl

PENINSULARAlqRADISEASEMALAYSIASHIRAI

WHO／WPRO，Man丑a，Ph皿ppines
Early

shovm曲tstudies

initiated by

the

23％

the

padentsofA㎞ost80％of the
Since

there

febr皿e

cases

was

an

jor

one

廿1e

populationthatwere㎜1dertaken

contain血gyear，5years

highesto皿palmscmb

Amy Medical

typhushuman血fectionin centralof（世ferent

and8years，werein廿1e血dividualsofStatespresen廿ng at

Ule

Research

ident面ed

settledvectors

chiggers

conectedwere

Lのホo伽oup

t㎞1％ofthechigger

less

was

rates

typhus．

might

according

to

the

stage

of

detem血e

the

risk

of

Penjnsular
ages．

for

Malaysia

Three

the

vary

much

rates

the

lower

in

the

febr皿ealso

suryey．

examined．

population．Most

A

hl

were

chiggers，rodents，and

patients

prevalence

O．6％血rodents
The

to

setUerpopulations，廿10se

Antibody

prevalence

settledrate

wasjor5years． The㎞pact of ecological changes on出ezわ顔％吻46漉犯s8．

R∫o々爾s勿云s％お麗gπ魏％s肱

md45％fromA

Malaysia had

scnlb

peak

in

the

the

old

older

numberofchiggers

trees．M司orityofo皿pa㎞areas，L．42漉

Asoosんoεηg襯毎伽4記α，a

per

the

sεmadenon−vector，mite．

Infection
rates ofin

hospi回had

host（Rα伽討伽伽勧s）was noted hl the area with2−1／2year

nest−dwe皿ng

Unit

disUゴct

were o丑pa㎞1workers．

indicationo丑pa㎞growth，studies
i皿6ection血areas

Ur匝ted

scrub

and

of

The

were

also

determined

from

the

isolation

therodents，

inthecorrespondingareas

conta直ning

younger

was

noted

in

with

more

maturetrees，with

only

be

direcdy

related

the

an

the

chiggers，11．7％in

were4％in

wor㎞g

infection

h㎜1ans

rates

area

trees．

O．1％血chiggers．

typhusvector

nat皿

al

thek

hosts

succession

areas

or㎞undergro㎜d

population

wit㎞the oil pa㎞1habitat（琵mof theo皿pa㎞scheme．

Bumingthefrom the groundsurface，leaving

only

pocketsAs

thegrasses

under

the

chiggers

and

their

nests．

habitatforthevector

takes

over

as

the

domhlant㎜almamma㎞hosts

wit㎞廿1eforest e㎞nates
covercropisoil palm

mostof infestations
begin

fomled．

The

tochiggers andwit㎞sma皿unbumed

togrow，asuitablehardy1〜．∫∫o㎜πあ螂

habitat． The

vector population

con血uestobu丑dupun旬thet㎞eofh飢ves㎞gandtheanivalofsetUers．Overapedodofa
few years，the

the

thick

setUers，but

betv》eenthegrasses

between the

harborageoil

pa㎞rows．

trees w翌1beparUa皿yel血並natedbyculturalpracticesofforR．伽σ燃s棚be fbmed by the placement ofpa㎞丘onds
As

the

trees

contime

to即ow

and

the

canopycloses，thevectorhabitatisag血面m血shedandthehostsbe瀬ntofomnestsinthehtUep皿esand／ortheo皿pdm
廿ees． The area

chigger

betweenpopu蛤don．

the

tree

does

not

containsuitablegrassestomajntainaTrombicuHd

150

3

EPIDEMlOLOGICA1。FEATURES

OF TSUTSUGAMUSHI DISEASE（SCRUB TYPHUS）IN
LIN
Depa㎞entofTsutsugamushi
inSouth−east

July−Aug．

diseaselncidence

parts

of

Southemtobe greatest

Ule食equency

of血伽sion㎞toUleenzoo盛c

Mortahty

preant㎞icrobial

after a（㎞血hstration
been

demonstrated

other

Ulan

logicalof

Guangzhou，Ch㎞

etiologically

China．In

apPearsin

Diseases，J血an University

Co皿egewas recognizedSouth−west

QuANsHENG

Infectious

Medical

SOUTHERN CHINA

cycle．

Eschars商（廿1ep血cipalin1948and

occur

with

of agesubsequently

seasonal

peakwhich depends

onofcases．

decreases

markedlyAtandleast18species

vector）are

difference hl local isolated rickettsia

is seen血84to98．2％

invaried丘om16％to61．4％which

to harborR廊ゐε∫云s毎云s％云s㎎π吻％sh∫in

L．d㎏あ￠

Guangzhou

between21and40years

therapy years

spec迅c antibiotics．

in

China，casesofnature．

RodentiaA

few

natura皿y血fected．

strahlswhichshouldlnsectivora

havespeciesoflarvaePresenceof

n血or㎞uno−beconsidered

in

mites

vaccinedevelopment．

4

ELECTRON MICROSCOPIC STUDIES OF INTRACELLULAR
MULTIPLICATlON OF RICKET7SL478U7Sひσ朋ひS皿
AKIRA
Department

The

of

TAMuRA

Microbiology，Mgata

Co皿egePhannacymechadsm of penetration of pu面ed Rあ々6鉱吻 s魏s㎎iロ卿％s雇（Gilliam strajn）into

cultured mouse

fibroblasts（L−ce皿s）andhlvesdgatedbyelec廿on血croscopic observadon

血fection．

After10−40π血of

often

seen on the ce狂surface． Theseinmolphology，wh皿erickettsiae

weretogetherwaseithertightlyplasmolysis

taken

up

or

were

were

illto

ln themembranes and the

were

seen

seen．

escape of

only

in

were

obsenred

of

the

hl

the

restrictedainthe cen

the

Large

were

which

phagocytized．

phagocytosisofseemedseveral

loose

often

intactphagosomesberickettsiae

rickettsiae

showeddisintegration

of

signs

of

phago−

ofseveralcase the microorganisms

weredefomed andpenetration

stages inconcluded

that

the

releasedinto

the censpec㎞ensandcontaining
thefrequency of appearanceof10，20and40min

tightcytoplasm by disnlption ofrickettsiae

toAdditiona皿y，enclosed

Pa面al

were

clump

these d逝erentarickettsiae

phagocytized

a

which

into

behlg

whose membranes

phagosomes

observations

enter血itiany

ce皿sphagosomes

occasiona皿y，inrickettsiae

the

the

From the

the

in

fヒom

denatured。

above

the

never

cytoplasm．

be

to

to

were

molphologica皿ytight phagosomes．

growthprocess ofphagosome，and

Rickettsiae

onlyrickettsialof廿1e spec㎞ens prepared at various t㎞es ofce皿s，phagosomesrarely released intosomal

rickettsiae

individua皿yloosely

were

pro伍esheav皿y plasmolyzed ones

rarely observed．
and

the

hlfection，nckettsiae

intactseemedofphagosome

the

a血er

infection，it

of
was

by

phagocytosis

and

are

phagosomal

membrane．

No

other

then

151

mechanisms of penetration were found.
Although the process of penetration of rickettsiae into host cells proceeded within relatively

short period of 10‑40 min after iniection, multiplication of the microorganisms in the cells wereslow and the observations were performed at day‑intervals. At the early stage of infection (24hr of infection), many of the rickettsiae in host cen cyioplasms showed electron‑less dense iuner

structures, but electron‑dense type became dominant at the later stage of infection (72‑120 hr).

Binary fission of rickettsiae by forming a constriction at the middle of long axis was often
observed at the middle stage of infection (72‑96 hr) in the cyioplasm or at the periphery of thehost cens. Although many rickettsiae were 1.2‑2.5pm in length and 0.5‑0.8pm in width,abnormally long rickettsiae, 10‑15pm in length, were sometimes seen. Addltionally, intranuc‑lear proliferations of this rickettsia were observed, but the incidence was very low. In thespecimens prepared after 72 hr of infection, release of the microorganisms were seen at the hostcell surface by lifting up cell membrane from inside to outside, Iike as the budding profile ofenveloped viruses. The budding rickettsiae increased in number with the lapse of time. In athin section of the later stage of infection (96‑120 hr), detachment of the outer and iuner leafletsof the host cen nuclear membrane and formation of vacuoles were observed. Then the censwere round up and, in some ceus, the cyloplasmic membrane disappeared and rickettsiae inside
the cells were released into the culture fluid.

5 RAPID SEROLOGIC DIAGNOSIS OF TSUTSUGAMUSHI DISEASE BY
MEANS OF INDIRECT IMMUNO‑PEROXIDASE (IP) TECHNIQUETSUNEHISA Suro
Department of Microbiology, Akita University School of Medicine

Tsutsugamushi disease had been recognized as a serious disease in Japan for more than one
handred years. However, over the years the number of cases has been steadily declining with

only a few cases being reported duritrg the years of 1965 to 1975. The disease seemed to havebeen nearly elirninated.

Since 1976, however, the reported cases of the disease began to increase in many parts ofthe country, even where the disease had never been identified previously. The disease appears

to be transmitted by Leptotrombidium pallidum in the early summer and autumn months and by
Leptotrombidium scutellare during the winter months.
This significant increase in the number of reported cases of Tsutsugamushi disease in Japan

has occured concurrently with the wide spread use of beta‑lactam antibiotics which are noteffective in treating Tsutsugamushi disease. The use of these antibiotics led to the occurenceof several fatal cases of Tsutsugamushi disease which were confirmed by a specific serologicalexarnination following death.

These unfavorable occurence have served as a stimulus to develop a rapid and accurate
serologic diagnostic procedure employing the indirect immuno‑peroxidase (IP) technique. This
technique is performed on a glass slide using minute dots of antigen prepared by concentrating
particles of rickettsia growa in L‑cell cultures.

An accurate diagnosis can be made within a few hours after receiving the serum specimens

152

by observing the titers of the specific lgG and lgM antibodies in the serum of the patient who issuspected of having the disease or has a fever of unknown origin.
Using this procedure we have detected a total of 465 cases of Tsutsugamushi disease, irom

19 prefectures, out of 1,368 cases of suspected disease or in cases with a fever of unknown
origin or in patients with exanthema of unknown cause during the 6 year period from May 1980

through June 1986.
In the 7 patients that expired the drect cause of death was disserninated intravascular
coagulation (DIO syndrome. = They were all confirmed seropositive for Tsutsugamushi diseaseby means of our IP method within a day of arrival of the serum specimen which was collected at a
variable periods before death. The most patients responded rapidly afteir the correct diagnosis

was made and adequate treaiment with tetracycline or chloramphenicol was initiated.
This IP procedure is proposed as the most efficient method for rapid and specific diagnosis

of Tsutsugamushi disease at present time in Japan, where beta‑lactam antibiotic therapy iscommonly used as irLitial treaiment of fevers associated with infectious diseses.

153

General presentation

1

STUDY

OF

QUICK

AND SIMPLE STAINING＿APPLICATION

FOR

AND

SCREENING OF

DETERMINATlONTO

OIKAwA1，ELYusRAR

DepartmentofMedicalTropical
HealthWe

previously

reported

at

about

new method

for

more

s㎞ple

quick thanparasitemia

andcount． The

countby thsmethodtheappHcation

of

dmg

sensitivity

toof

SHoKlcHI

Medical

University2and

Kawasaki

Hospita13ofJapaneseparasite（PstainedtheSocietyofParasitology

αs吻04伽郷わ8㎎加づ）．

This

methodblood創m

method in

betweenmethodwasmorescreeningofmalarial

parasitemia

screening，Gentian

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the

blood
fbr

mm

the

TANl3

University1，Institutecon elation

methodthethIm

the case

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O．9．

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ths
and

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report，we
the

was

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this

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the Giemza s

PARASITE

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amual

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Medicine，Kanazawa

the55th

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staj1頸ng

OF

MALARIAL

YosABuRoofFOR

by10％methanol

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placediton the sHde glass（about O．008mJ）． A

stajning

solutionwas

droppedontheblood，

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staineddark

i㎡ectedred

Gentianviolet

and

blood

ce皿s

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theparasitemia

sensitivity

for qu㎞e

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seconds㎞狂ar withofdiscovered

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andobserved五ke

were

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was

kl

the

method．

withblood mmsta面ngbystaining

it takes some hours forwith㎞mersion

oiL

sample scaming

were

easy，because

cosidered

to

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more

usefUltreatmentinthemalaria

reduc廿on of

for

was

In

new

the

of

this

blood創m

microscopicaly

sensitivity

blood

decreasemala梱spedes，but

drug

the

was

about

test

of

O．1％，malaria

drugsensitivity，

by10％methanol

andO．15％EDTA−2Nad丑uted5t㎞es

this

st血血g

by

soludontheparasitemia

wasobserved

clearlyonmethod，andtheparasitemia

count

was

disc血面nationofmethod．

methodma㎞1g

sca皿廿ng

necessary

for

the

preparationand scaming the s㎝ple

wasscreeningofendemic

area

insteadof theparasite，the

is

the

staining method，the preparation maldng and thetheparasiten植a．

For

sta㎞g

for

ma㎞al

parasitemia

with Bロrker−丁血k hemocytometeL At the study of thecandle jar

the result from the

s

tllis

was d皿uted400t㎞es

countedGiemzabyvitro−cultured

dihydrochlohde，thecultureConsideration：a ba皿． Wheneas皿yenoughmalarial

new method

at×400・

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the

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154

2

1SOLATlON

AND SOMEENZYM［ESFROM

PROPERTIES

1NFECTEDFuM田IDEThe

Fhlst

Depa】由ment of

of

An

was

pu面ed

ToKu1，REIKo

YosHIYAoffrom

Expe丘ments

Medicine，University

mousechromatography

by

gel

mtration．

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p一血住o圃deas
but

not

by

essential

modmedbyenzyme has
wasx−100andan apparent

monitored

the

s㎞ple

methodwith

hlfected

involving

on

with

selective

Sephadex

molecular weight of

conveniently

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A−25．This

Fac描ty2andofanon−ionic detergent Thton

MATsuYAMA2

SATo3

parasites（PZαs郷04伽卿δ8怨力砿NK−65strain）bySephadex

PROTEOLYTICERYTHROCYTESBiochemistory1，RI

Parasitology3，Schoolaminopeptidase

MICE

INouE1，SEIKIcHI
AND

OF

PL4S財01）1乙曜BERσ朋1

malaria

extraction

G−100and

about100，000asL−alanine−p−nitroan直1ide

or

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measured

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sulfhydryl

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p−chloromercuribenzoic

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which

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and
is

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rapidly

unaffectedbyaminopeptidasemo（medby

Hg2＋andmonoiodoacetic

has

an

slowlyacid，orN−

methy㎞ale㎞ide．

3

KINETICCHANGES OF HEMOPOIETIC

STEM

PL4S跡01）1㎝4、BERσ丑E1−INFECTEDMEIKo AsAMI，HARuHIKo

CELLS

IN

MICE

MARuYAMA，MAKoTo

OwHAsHIAND YuKIFuMI NAwA
Department

DrasticofParasitology，Miyazaki

hematological changes aredestruc廿on

of

red

ce皿s

and

observed during

acdva廿onof㎞mmeMedicalmalaria

as

wen

as

Co皿egeparasite

infectionmononuclear

becausephagocyteofthesystem．

舳ough齢esebloodce皿s肛eon蜘ted丘omco㎜onprecursor，te㎜edhemopoieUcstem
ce皿s，notmuch

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infection． In theexan血edhl

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The number
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in

vitro

The number

decreasedasthechangesstudy，therefore，㎞etic

hemopoietic

soft−agar

and

for

of

stem

cels duhng malaria

hemopoietic

stem

ce皿s

were

colonyassay，whichαm

assay，whichcanbone㎜ow was measured

detect pluゴpotential

detectcommitted

stem

by2d迅erentcels（CFU−S），

stem

ce皿s（CFU−C）towards

macrophages．

ofofchangesstem ceHs in the

CFU−S started to血creaseon

day4，reachedto subnoun訓level by day14．The㎞e廿cthatofhemopoietic

2δ8㎎物8歪一樋ected血ce．

systems：in vivo spleen colony

granulocyte

samepresent

zo4づ％

of

has

CFU−S

with

In addition to in vivo

andshghUykl

delayed

vitro

c㎞ge

of

a

peak

on

days6−8，Imd

CFU−C

was

essent洲y

in

ma皿ow

then

the

onset．

colonyassays，5gFe

uptakebone

andspleen

was

155
also

examined．

spleenofIron

hlfected

uptake

mice

in

was

the

seven

bone

ma∬ow

t㎞es higherofinfectedt㎞that

in

mice

the

was

twice

respective

and

organ

of

that

in

the

ur山㎡ected

mice．

These results indicate

thathemopoietic

stem

ce皿s

are

acdvated

during

an

acute

stage

ofP．

わε㎎8h初㎞ection．

4

CHANGESOF T・1。YMPHOCYTES AFTER BABESIA INFECTlONlN MICE PRETREATED W皿H TOXOPLASMALYSATE

NAoYosHl

SuzuKI1，SHINlcHI

ANTIGEN（TLA）

SE田GucHI1，MoToYosHI

ATsusHl SAITo1，TsuNEo
SchoolWiththe changesofVete血aIy

oflymphocytes

inthe

and

histopathologica皿y血miceπ，伽初5which

was

fatal

to

the

whole

in

rodents．
thymus㎡ce

were6．5×107andO．9x107ce皿sly．

The totalofnumber

AND

FuMIo

l

City2

thymus，spleen，Hverandbloodwere

exan血ed
with

day

day

Kobe

treatedThe nu恥berl

SAKu㎜1，OsAKl2

Medicine，Ob丑血o Universityl andbioche血callywas3．7×107ce皿s

HIRosEI

SATo1，HARuHlsA

with

of

beforeTLA before血oculation

Thy−1positive（十）ce皿s

inoculation（b．L）wh皿e

in

both

Bα加s毎

untreated

those

in

mice

TLA−treatedb．L

and10days

after

inoculation（a．L），respective−Thy−1（十）ce皿s inthe spleen

was3．9×107and11．7×1071day

b．i．

and10days乱L，respectivelyinuntreatedconαolsand12．9×107and15．6×107，respec廿velyin
TLA−treated
10daysmice．

a．i．wascells inA

remarkablea㎞ost10jold

the liver hl

asincrease ofcompared

TLA treated miceThy−1（十）ceHs

withthat of

was

otmd

l

in

the且verday b．i．． The

aromd

pedvascularof

TLA−treatedincrease

ducts10days

mice

ofThy−1（十）a．i．，however，

show血g fbw Thy−1（十）cells around the ducts in untreated mice． These Thy−1（十）cens
aromd pehvascular ducts were m血y Lyt1 2positive and Lyt2・3nega ve ce皿s．Increase ofThy−1（＋）ce皿shl

the且verand spleen㎝dowing to T−cels released f士om thedecreasethymus

and

of

those

in

aocked

to

the

the

thymusareorgans throughpresumably

the

blood

ckculadon．

5

MALARIA CONTROL IN NORTH SUMATRA，INDONESIA（1）GAMETOCYTOCIDAL EFFECT OF PRIMAQUINEAKIRA KANEKo1・2，KlsEKo
North

Sumatra

HealthKAMEI1・3，AKIRA IsHll1・4AND TAKEsHI

Promotion

Project，JICA1，Departmentof

SuzuKllParasitology，H辻osaki University School of Medicine2，Department of Parasitology，TeikyoUniversity

SchoolParasitology，Okayama

ofMedicine3andUniversity

SchoolDepartmentofof

Medicine4

Conside血g比echemo血erapyag血stl組cip㎜m蜘adents血itsende血c肛ea，itis㎞portant

not

or皿y

to

clear

trophozoitsasc㎞icalcure，but

also

to

clear

gametocytes

which

are

156

infective to vector mosquitoes. During passive case detection at Kuala Tanjung and Nana Siam,

Sep. ‑Nov. 1985, 131 falciparurn patients were treated by Tx : Fansidar 1,575 mg lx, 87 weretreated by Tx: Fansidar 1,575 mg+Prirnaquine 45 mg lx.
After drug administration, these falciparum patients were followed up to observe thegametocyle fluctuation. During this study, trophozoites were cleared by day 7 in all of thesefalciparum cases without recrudescense.
In the group treated only by single Fansidar, 25 cases were exantined on day O and day 2,

and 99 on day O and day 7. In both subgroups, no significant change was observed ingametocyie load between day O and day 2 or day 7.
In the group treated by the combination of Fansidar and Prirnaquine, 29 were examined on
day O and day 2, in which no significant change of gametocyie load was observed. But 72 cases,

examined on day O and day 7, showed 73. 6% gametocyie positivity rate (GR), 2.2 gametocyie

density (PDD on day O; and 33.3% GR, 1.3 PDI on day 7. The difference was significant by
Wilcoxon's test, (p<0.01).

The gametocyie‑positive cases on day 7 were tried to be followed up weekly till gameto‑
cyies disappeared. In 52 cases treated only by Fansidar, 71.1% GR on day O continued till
week 2, after that it declined but stiu as high as 11.5% in week 5. In 56 cases treated by
Fansidar+pritnaquine, 76. 7% GR on day O had already decuned to 30.3% on day 7, after that
GR declined futher to 7. 1% in week 3.
This study showed the treatment of single Prirnaquine 45 mg together with schizontocide
was effective in promoting the gametocyie clearance of falciparum malaria patients.

6

MALARIA CONTROL IN NORTH SUMATRA, INDONESIA (2)ENTOMOLOGICAL STUDIES AND VECTOR CONTROLTAKESHI SUZUKll, TETSUSHI KIKUCH11, MASAHIRO TAKAG11'2
AND MASAYUKI YASUNOl,3
North Sumatra Health Promotion Project, JICA1, Department of Medical
Entomology, National Institute of Health2 and National
Ins titute for Environmental Studies3

The vector of the coastal malaria in our study area is An. sundaicus, which can not be
controlled by DDT residual spraying, because of its exophilic nature. Based on this, studies
have been concentrated on integrated control measures of the vector at its larval stage. A trial

operation was started in a 32 km2 area in September 1986.

The lagoons scattered behind a beach sand bank were infested heavily with the larvae.
They were controlled effectively by draining of the water through a chamel leading to a nearby
river. Taking advantage of the fact that its breeding is linaited only in sunny ponds, a control

trial was done with success by shading of the ponds with coconut leaves. Biological control wasdone by releasing guppies, P. reticulata, into the ponds or ditches which were unable to avail

source reduction / environmental management measures. Temephos was also applied in thebreeding sites with the dose of I ppm, which was effective for 2 weeks.

Based on the information obtained through such trial operation, a large‑scale control

157
operation

is

now

under

planl血g，a㎞ing

at

the

eventualbu皿d−upofvector

controlmeasures

to

AMONG

THOSE

be

u血zedby曲gers血emselves血血t肛eunderco㎜血ty圃cipadon．

7

EXTREMELY HIGH TRANSMISSION
WHO

LIVE

NEAR

BREEDINGOF

MALARIA

S皿ES

OF

THE

SEA−WATER

M［OSQUITO，AIV．S乙WP盟CσS，IN THE COASTALAREA OFNORTHHIDEKI

IToKAwA

Department of Medical Zoology，SaintA mass−blood
as

one

of

examination

activities

detemゴne

the

on

was

the

endemicity

mdertaken

project

among

personshighOn

hand，the20％）．Also，highparasite

6ndings

means

inUniversity，School

the

who

groups（0−9years）．

otherMariamacoastalfor the promotion

vector，ノ肋．s朔血ゴo％s． Verythe

SUMATRA，INDONESIA

d6nsity

of

Hved

of

area

of

Notth

health．

The

a㎞for

near breeding

casesthat the existe血ce of

were

rates

fo㎜d

ill

adultsonly in

Sumatra，Indonesia，

sites

parasite rates（around60％）were
parasite

Medicille

the

the

of

surveyobtainedtoin young

showedrelatively

young

age

high−level㎞munity against malana

wasthe responsibleagelow（10−groups．Theseamong

the examined

persons・
Sahvary創and dissections were perfomed on34L4肱s％π4αあ％ε，which had been co皿ected
from

the

same

area，and2positive

cases werethat Aη，s％η吻づ伽sb】血1gsmosquito−densities

oL4鉱3％％血あ粥（more

8

about

high

obtained（0．6％）．

trans㎡ssionsofmalaria in

This

the

sporozoite

rate

means

area，where

very

high

study

than30／man／hour）wereusua皿yobtained．

DOES皿yP盟OSO岨」BRUCE1σ躍BIEハ石8E
CHOOSE FEEDER・CELLS？

PETER

J．MHANDo，TETsuo

YANAGl，SHusuKE NAKAzAwA，TosHIHIDE FuKuMA AND
Department of Protozoology，Institute

In
and

the

cultivation ofmuscle

fbr

the

Imd

musclederived

could

of

the

derived

ce皿s

lost

seeded

on

the

one

half

inoculation

of

Tg．

were

reportedthat

supportingthek

rapid即owing

ceUs

growthtrypanosomes

The

and

Tropical

KANBARA

Medicine，Nagasaki

Tりψαηso吻αわ概ε歪g8励伽s召，newlycels

cu1偵va血onof

HIRoJlsupport

new
area

no

were

morphologica皿yincreased
cels

not

change was noticed hl
identical

the

half

only

thek
to

contraryaculturethearea

contjnuous

exposedtoprepared newbom

organisms．

Supportingofonsupportweretheabihty．

the othercould

non−supporting

of

Tg．On

suppo面ngandparasites

but

gro幅h

isolate of

their

ce皿s

the

University

They

were

estabhshed
ce皿s

and

dish

of

growthnewbombrahl

ava狂able

mouse

brain

non−supportingcelsrespectively

suppo血ng
of

mouse

also

prior

ce皿s．

to

It

the

was

trypanosomes． Thus，

theminimum

X−inradiation

suppor血g

ab皿ityfor Tg．

The cultured

retainedi㎡ectivity

bloodstream

foms

and

to

suppress

for

158

mice. Growing Tg showed nest‑like clusters in intercellular space. This suggested thenecessity of close contact of Tg with cells rather than soluble growth factor from them.

9 ANTI‑PARASITE ACTIVITY OF CARBOCYCLIC INOSINE AGAINSTAMASTIGOTES OF LEISHMANIA DONOVANIHIROYASU SATAKEl, OSAMU HIRAOKA1, YUSUKE WATAYAl, JULIETA Y. KIMURA2
KAZUHISA MORISHIGE2, TOSHIKI AJ12, AKIRA ISHn2

AKIRA MATSUDA3 AND TOHRU UEDA3
Faculty of Phamraceutical Sciences, Okayama Universityl, Department of
Parasitology, Okayama University Medical Scho012 and Facultyof Pharmaceutical Sciences, Hokkaido University3

A promising approach for the development of chemotherapeutic agents possessing selective
toxicity is the one based on certain unique features in structure or function of the parasite thatdiffer from those of the host cells. Leishmania are incapable of synthesizing purines de novo andare thus dependent on the host for the source of preformed purines. This qualitative differencein the enzymes of purine salvage and interconversion pathways between host and parasite
suggests a rational approach towards the design of agents selectivity toxic in the parasite. We

reported that carbocyclic inosine is a potent inhibitor of promastigotes of Leishmania but
significantly less toxic to mammalian cells.

In the present experirnents, J774 ･ 1, a mouse macrophage line, was cultured in RPMI 1640
medium containing ipopolysaccharide. The adherent cells were exposed to promastigotes of L.

donovani for I day, and treated with the drug. On the other hand, the macrophages werepretreated with the drug for 2 hours, and were exposed to promastigotes of L. donovani. Drugactivity is then assayed by counfing the number of infected host cells / 100 host cells. The
results indicate that carbocyclic inosine is active against amastigotes of L. donovani in vitro.

10 THE PREVALENCE OF BLASTOCYSTIS HOMINIS INFECTION
IN HUMANS IN KYOTO CITYMINORU YAMADA, YOSHITSUGU MATSUMOTO, TATSUYA TEGOSHIAND YuKIO YoSHIDA
Departrnent of Medical Zoology, Kyoto Prefectural University of Medicine

For several decades, Blastocystis hominis Brumpt, 1912 has been considered to be aharmless yeast in the human intestine. However, Zierdt and his coueagues emphasized that itwas not a yeast but a protozoa, though its taxonomic position was not established. In later
studies B. hominis was receiving increased attention as a cause of diarrhea. The present study
demonstrates the prevalence of B. hominis infection in Kyoto City, Japan, between June 1983and July 1986. Twelve hundred and fifty‑one persons who submitted stool specimens for159

parasite examiuation were divided into 5 groups. Group I consisted of 14 foreign tourists.
Most of them had visited South East Asia, India and Africa just before coming to Japan, and moreor less complained of digestive trouble. Group 2 consisted of 79 patients who were admitted to

Kyoto Prefectural University Hospital with digestive trouble. Group 3 consisted of 304 inpa‑
tients and 696 outpatients in this hospital without digestive complaint. Group 4 consisted of 122

mentally deficient persons in 2 private care facilities, and Group 5 were 36 staff members of

those facilities. The stools were hrst examined for B. hominis by iodine‑stained drect smear

method, then confirmed by phase contrast microscopy and Giemsa stain. The results are as
follows : In Group 1, 11 of 14 tourists (78.6:v) were positive for B. honainis. In Group 2, 3 of

79 patients (3. 8%) had B. hominis. In Group 3, 12 of I , OOO inpatients and outpatients (1.2%)

had B. hominis. In Group 4, 21 of 122 mentally deficient persons (17.2%), and in Group 5, oneof 36 staff members (2.8%), were positive for B. hominis. Of the 48 persons positive for B.
hominis in total, 30 had other parasites, for example Giardia lamblia. Entamoeba coli. Enteromo‑
nas hominis. Iodamoeba buetschlii, Ascaris lumbricoides. Trichuris trichiura. Necator americanus,

Clonorchis sinensis, Metagonimus yokogawai, Schistosoma mansoni, and Echinostoma spp. . It
was noticed that B. hominis was found not only in loose and soft stool but also in formed stool.

In conclusion, careful examination may reveal B. hominis stiu existing among Japanese people,

especially in mentally deficient persons isolated in the closed facility. Furthermore, it isinteresting to note that foreign visitors are strongly contanainated with this parasite.

11 A SERIOUS CASE OF AMEBIASIS ACCOMPANIED BY
EXTENSIVE COLIC ULCER AND ABSCESSTOSHIO NAKABAYASHll, TAKESHI NAKAMURA2 AND KATSUMI AMIOKA2
Department of Protozoology, Research Institute for Microbial Diseases, Osaka
Universityl, and Nakamura Hospital, Hirakata, Osaka2

The patient was a 48 years old male resident of Osaka and had been hospitalized withschizophrenia for alrnost 20 years. He had no experience of oversea travel. When he wasadnaitted with head trauma in Nakamura Hospital on February lO, 1986, a right abdominal tumor

was noted.
On June 22 of the same year, he was admitted again with ileus symptoms accompanied byabdominal distension and cicatrical hemia on the abdominal wall. Constipation and remittant
fever were noticed. Romanoscopy revealed neither redness nor ulcer on the intestinal mucosa.
By X‑ray exantination, gas retention with passage disturbance was observed in the colon. The

laparotomy was carried out on June 27. The ascending colon was stenotic with necrotic
portions and retroperitoneal abscess was found along the colon. The cecum and the rightflexture adnered tightly to the retroperitoneal wall and the liver, respectively. The intestine

between the cecum and the middle part of the transverse colon were resected and anastomosed.
After the operation, the patient did not tum better and the level of consciousness became

reduced. On July 4, many trophozoites of Entamoeba histolytica were detected in the sectionsof the resected colon. Next day, antiamebic chemotherapy was started with dehydroemetine(40 mg, i. m. , once a day for 7 days) and metronidazole (500 mg per day, injected through1 60

stomach tube for 7 days). However, the therapy showed no effect but the patient died on July
14, 17 days after the operation.

The mucosa of the resected colon was necrotic and covered with a thick yelbwish
pseudomembrane. The ulcer reached the adventiba with some foci of perforation. Numbersof E. histolytica all of which were trophozoites, phagocytizing erythrocyies, were found in theulcerative foci and even in the deep layer of colon. The case was undoubtedly of an internal
infection of amebiasis of which the accurate diagnosis was just made by tissue examination of theresected colon after the operation.

12

SEROLOGICAL DIAGNOSIS OF AMOEBIASIS BY MICRO‑ELISAHISASHI YAMAURA AND RYUKOH SHIRASAI
Department of Parasitology, Tokyo Women's Medical College

In the present study, the reliability of the technique of enzyme‑linked iuununosorbent assay(micro‑ELISA) for the detection of amoebiasis was exarnined. For the assay, peroxidase‑
conjugated anti‑human lgG (Miles‑Yeda) and ABTS, 2,2'‑azino‑di‑(3‑ethylbenzthiazoline sulfonicacid) were used as the conjugate and substrate, respectively. Serum samples from 23 patientsof amoebiasis and 200 healthy residens (amoeba cyst negative) were diluted at I : 200 andexamined.
The results of maximum absorbance among normal control sera was O. 181 (0.028‑0. 181),
and 0.410 (0.410‑1.820) at the minirnal absorbance among amoebiasis. Hence, the absorbanceof O. 2 was considered to be the highest value of negatives above which a case would be positive
for amoebiasis or highly suspective.

Sera from 32 cyst passers examined, only 2 or 6. 3% were positive for the reaction.

The results indicated that micro‑ELISA technique with ABTS developed in the present
study is applicable to the survey of amoebiasis.

13

USE OF WING SOUND TRAPS FOR MALE MOSQUITOESOF SOME VECTOR SPECIES IN FIELDS
Tozo KANDA AND KENICHI OGAWA
Department of Medical Zoology, St. Mariarma University

Vector control of malaria is crucially important than any other method, such as drug
administration, irnmunization of communities to protect from malaria transmission. It is similarly

important in a certain endemic area of subperiodic malayan filariasis, such as Taiping, Malaysia,

where the achivement of vector control became indispensable. Because existence of animalreservoirs of this filaria, one soure of infection stiu r mains its endemicity despite DEC(diethylcarbamadine) administration to human parasite carrier.

Meanwhle, in the vector control the limitation of the use of insecticides is requiring to161

promote the developments of control methods other than insecticides, such as physical, biologicaland biochemical control methods. Among various biological characters by species, taxon or
population some differences of specific characters iufiuence or relate to their mating behaviour or

activity in their genetic divergence in nature. Thus the finding of difference in wing‑beatfrequencies among sibling or very closely related populations, one of sexual isolation mechanism

in genetic divergence has been proved and utilized to collect or trap mosquitoes.
Due to diffculty to find or promote swarming of some vector species of malaria and filariasis

in nature, wing sound trapping with some kinds of biting attractants and / or stimulants was
performed around their breeding places at biting time after sunset. The experiment was carriedout by using 3 kinds of attiactants and / or stimulants using alone or in combinations. These

three were wing sound trap, an anirnal in screened cage, and dry ice. When the sound having afrequency close to that of wing‑beat of the same taxa of species was used. The most effective
trap was a combination of a fitted frequency of wing sound with anirnal dry ice. For the specties

of genus Mansonia the sound of 330 Hz to 350 Hz combined with above two attractants wereable to coilect 870 males and 839 males within one hour from 15 minutes after sunset. For
Aedes albopictus the sound of 480 Hz with anirnal and dry ice was most effective, whereas thenumbers of males captured varied greatly according to the time of trapping. The trap used for
Aedes and Mansonia was a plastic cylinder having a speaker set in the middle of tube and adnesiveplastic sheets covering inner surface at both ends of the tube, however, for Anopheles a plastic
lantern covered with a polyvinyl bag having the glue spread on its surface was more efficient totrap mosquito than the former type of trap.

The reduction of the number of male mosquitoes from the population of Mansonia wasachieved more remarkably by the repetition of trapping for several times at the breeding area
than setting traps in the biting area by females, such as houses which were far from breeding

places. The age of male Mansonia was determined by checking mesothoratic furcum. The
analysis of the results of trapping and of age composition gave the information of the size anddynamics of population and daily production of both sexes of Mansonia. The trapping method
mentioned earier was proved to be useful in the ecoiogical investigations as well as for thedevelopment of control method without use of insecticides, and future integrated control

methods.
The trapping method was also applied to investigations of population dynanaics of Genus

Mansonia and Culex tritaeniorhynchus at a rice field, Ban Pa Inn, Ayutaya, Thailand. The
trapping surveys have been performed for once a month from May 1986 up to date. Highly
prevalent months of both mosquitoes were from June to September, especially August was thehighest in the number of trapped males of Genus Mansonia, whereas their females trapped were
not many. In this plaiu swanpy area there were many breeding focuses where male mansonias
trapped were constantly more than others.
The trapping of C. tritaeniorhynchus proved 2 peaks in its numbers, one was in May andanother was in August, though their trapped numbers were not significantly high. The use ofbiting attractants, alaimal in cage and dry ice trapps collected more in the number of females of C.

tritaeniorhynehus. The trapping system of wing‑sound and biting attractants was effective bothfor physio‑biological control of Mansonia and for survey of population dynamics of Mansonia.
Culex tritaeniorhynchus and Anopheles.

162

14PATHOLOGICAL

S皿JDY

ON

MOSQUITO・BITTENWOUND（IIl）

AN IMMUNOLOGICAL STUDY
ELYusRAR
Departement

ALYAsAR
of

JALAL1・2，TERuAKI

MedicalAND

SusuMuZoologyl

and

㎞zawa

Guineapigs

were

sensitizeddegranulation response
were

investigated．

In

IKEDA1，KHcHI

HoTTA2

Institute

Medical

SATo2

for

TropicalMedicine2，

University

by biting to A召4εsα卿あ2t㎞es

and homocytotropic

antibody

ad（htion，theability

of

semm

to

of

mast

ceUs

from

per

responsedu血gpassivelytransfer

week

the

Mast

ce皿s

sensitization

thesensitivity

pehod
was

also

血vesdgated．
The

degranulation

sign而cantly

peritoneal

increased ffom the3rd week，anddegranuladon

remain24−hourshowedPCA

high㎜皿the12th
peak

pigs

a hi菖h response

relatively

low

only

on

measured bysensitizedbymosquito

bitingpercentage ofweek Hom㏄ytotropic andbodies measured byresponse on weeks7to9，wh丑e

PCA showed

level asguineathen even though fluctuated，theweek8．

Serum

ELISA，but

homocytotropic

IgG

againstantibodies

mosquito

spec皿c IgG levelby8−dayantigenhlcreasedshowedshghtly d皿ing

weeks5to8．

Nomd蜘eapigs面ected血廿avenously舳1mlof8−dayPCAposidvese㎜showed
hypersensitive

The sames㎞1reaction

se㎜previously

on

the

heatedcha皿enge

with

the

jor30mhlutes at56。Cmosquito

showedreacdon．G血eapigs輌ected瓢thnonn田se㎜showedno

bites8days

less

after

wheal

i呵ection．

d㎞eterofs㎞

s㎞reacdon．

Theaboveres皿tsweresu㎜血ed曲t：1）At血es曲㎞e輌舳eappe肛mceof
thes㎞1reactions，thepercentage of

mast

ce皿s

degranulation

increased．

2）Themosquito

bites

induced the production of hom㏄ytotropic antib（〕dies，mainlyof IgE

class．

3）Se㎜fromsensittzed

results

provedthatreaction

an㎞alscanpassively

bites

is

the

e五citedbyTHE

INFLUENCE OF BLOOD

15

mosquitotransfbr

sensitivity．

an㎞mediatea皿ergic

These

SUCKING ON THE SUSCEPTIBILITY

TO

Kobega㎞in

Women

s

and

vims

amountpresent

infeeding． A

after

School

low

of

of

and

A8．

other

MedicalZoology，Medicine2o肱s，Aε．α卿あandnon−susceptible

susceptible

TAKu UENoYAMA2

Department

appropdate days ofhead−thorax，midgut

C鬼孟螂αθ初o名勿犯oh％s，adays after

University

mosquitoes of Cズ．孟γ吻ε勉oz勿／4．召．αのの∫o伽s were dissectedIn

Co皿egelA五7θyp77

VIRUS

KoNlsHI2AND

Kobe

FemaleCHIKYAMANIsHI1，EUI
Juniors㎞

reac廿on．

OF、4EDES ALBOPICTOS AND、4E1）ES

HIRosHlthetheinfection

abdominal

control，thez勧）あwere

v㎞s

Oahu

and Hyogo

with

CHIK

parts

of

was

observed

present

h112％of

v㎞s

the
in

strains ofto quant脅themosquito
themidgutmidgut

body．
untn3

spec㎞ens．

163

The other part of abdomen and the head‑thorax did not show detectable iufectivity after the Ist
day of infection. The Hyogo strain of Ae. albopictus, moderate in susceptibility, was observed in60( o of midgut specimens. The Oahu strain, highly susceptible, indicated that the virus waspresent at higher titers and at higher proportions. All midgut specimens were positive for
infectivity. The susceptibility of midgut was especially important for virus multiplication. In
Ae. ae pti, effect of feeding and infection on virus multiplication was examiued.

16 THE STATE OF JAPANESE ENCEPHALITIS VIRUS
ACTIVITIES IN OKINAWA MAIN ISLAND
MASAYUKI TADANO1, TsuvosHI ISHIMINEl, YoSHIHIRO MAKINofTOSHIKAZU ANZAll, TOSHIHIKO FUKUNAGA1

AND HIDEO HASEGAWA2
Department of Virologyl and Department of Parasitology2,
School of Medicine, University of the Ryukyus

In order to investigate the prevalence of Japanese encephalitis virus (JEV) in Okinawa mainisland, 3 methods were conducted during a period of March 1985 through October 1986 : (1)detection of JEV antibody in swine sera collected at two‑week intervals at the slaughter house(2) collection of vector mosquitoes (Culex tritaeniorhyncus : CT) by light trap (3) isolation of JEVfrom field caught mosquitoes (CT). Serum antibody positive rate over 80% was observed
durirrg periods June‑December 1985 and July‑Octoher 1986 in the northern area, July‑October1985 and May‑October 1986 in the central area and August‑September 1985 and August‑
September 1986 in the southem area. Fluctuations of antibody positive rate were observed inall 3 areas. Overall positive rates were 509: o in the northern area, 47% in the central area and

30% in the southem area. In most cases, overall positive rate was not decreased to O( othroughout the year (except only once at June 1 1, 1986). Anti‑JEV IgM class antibody was
detected during a period Aptil through October 1985 and May through October 1986. Total
numbers of CT collected were 1,233 in the central area (March‑December 1985) and 10,875 in
the southem area (May‑October 1986). One strain (from the central̲area) and 18 strains (fromthe southem area) of JEV were isolated from the pools of these mosquitoes. Due to the recent
drastic decrease of rice field in Okinawa main island, the breeding sites of CT decreased, which

resulted in the decrease of vector mosquitoe population. Pigiarms are becoming larger scaleand locate remote from the mosquito breeding sites. So, JEV may circulate within narrow area.
Thus resulted in the fluctuation of antibody positive rate. In the southern area, the site wheremosquitoes were couected was closely located to the watercress field where clean water wasalways fiurng in. These fields (other than rice field) will become important CT bleeding sites in

Okinawa main island.

164

17 MICRO・NEUTRAHZATlONTEST

OF DENGUE

VIRUSESUSING96・WELL PLATE AND PEROXIDASE．
ANTl−PEROXIDASE TECHNIQUE
TsuYosHI IsHIMINE1，MAsAYuKI TADANo1，TosmHIKo FuKuNAGAl
AND
Department

of

YosHINoBu

Virology，School of

Depa血nent

forofMedicine，University

Preventive

Microbial

OKuNo2ofthe

Ryukyusl

andMedicine，Research InstituteDiseases，Osaka

University2

hl South−east Asia，there are areas where Japanese encephalitis v血1s（JEV）and dengue
血uses（DEN1−4），both belohg to Flaviv面dae，are concomitantly prevailing． In these areas，hemag創utination

inhibition test for

serodiagnosis

betweenv㎞ses．

case，neutraHzation test（NT）can be usef皿． However，

these

conventional
pedod．

handled．
methodplaqueSo，it is

ln

fom血g

methodpractical

for

not

Recently，rapid

has

been

this

NT

developed．

Here，weconelationwasThe

appropdate

hlcubation

This

has

歴．
be

methodhandled

at

an

18

study

more sample

st皿hasadvantage

ANALYSIS

DEN2andmorelots ofthe

have

were class而ed into

in肌ISA but not

望oup could

㎞d，we

in

HI

Medic㎞elanddetem豆nedthebesamplesittonumber

of

and

costs

DEN1−4．

High

focus

fomled．

and

DEN3was56

shortsamplessuitablecanforpedod．

GLYCOPROTEIN

Department

toan㎞portantplay

fmctionSusuMu HoTTAlof

Serology2，

University

of

E

role

hl

dengueprotein，westrain．Fromv㎞s

prepared

the

data

i皿6ection，Hke
monoclonal

of

an

anti−

enzyme一㎞ked

he㎜gglutination圃bition（HI）assay，those monoclonal

at least3groups，i．e．（1）highand（3）high

precipitate E

thebe handled ill

SEKI2AND

Medical㎞munosorbent assay（ELISA）andand

ENVELOPE

toDENGUE VIRUS

bodies against dengue virus type1，Mochizuki
antibodies

toKanazawa

biologicalmanyapphedespecia皿yfor

Haviv㎞ses． To elucidate

hand㎞gand

DEN4was40hr，while DENl
shde

have

peroxidase−anti−peroxidase

that（1）more

Instituteseems

longer incubation
spec㎞ens

is

TAKEGAMI1，CHIYoKo

Envelope（E）glycoproteinofin

TsuToMu

Tropicallotcross−reactivity

methodTHEOFsolutiontheeconomical．Micro−NT

samples

OF

in

of

and takes
asHde andproblem

over8−chambert㎞e（2）it isvolumeusillg96−we皿plateofbecausestudy，where

dilution of v並us

penod

where

appncable

usi㎎8−chambermicro−NT

between thethe same

seroepidemiologicalmethoddeveloped

nots roepidemiologicalHowever，it

expensive．

observed

needs

is

titre

proteinnucleotide

of

in

Hl

denguesequence

prote㎞，ushlg reverse tエanscrip惚se and

but

vims

not

in

Iabeledcontaining

synthedc

pr㎞er．

titre

in

ELISA

and

ELISA．MonoclonalHI，（2）highantibodies

with35S−methior血1e．

COOH−temlinus
An血o

acid

On

titre

in

the

each

other

of（1engue v㎞ls−E

sequence

deduced

at

its

165

COOH−tem血us血dicateshydrophobicity．

protehl

into

may

be

detemゴnants

embedded

virus

These

data

suggest廿1at COOH−tem血al region of E

envelope and

other

part

of

E

proteinhas

severalantigenic

conta』㎞ng hemag幻utklation site．

19

MONOCLONALLATEXANTIBODY・BASEDAGGLUTINATIONREVERSEDFORPASSIVE

IDENTIFICATlON

OF四BR10CπOLERAE O1
YAsuo

YANAGAsE1，KAzuE AoYAMA1，Ko SHoJI1，FuMlo GoNDAIRA2，
MATsuDA2，MAsARu HIRANo2，JuNIcHI SuGrYAMA2，

JuNJI

YuuJI
DepartmentofBacteriology，Hyougo

Biological

Science2and

of

We
of

successfuHy

V二6ho

prepared

8名πθandhave

TERADA2AND

HealthCATHERINE

Co皿egeRepubhcSanof Medicine1，DenkaofL旧zarotheaPhihppines

InstituteofMinistry

Hospital，M㎜丑a3

monoclonal andbodies

developed

P．RA画oA3

against soma髄c andgen丘acdons a，b

latex agglu廿na廿on

for

latex

agglutination test．A sman portion was d丘ecUy

thepresence

onto

sensitivity
choleramedium to detect

the latex

reagents

wassumcient

Phjlippinesof肌o海oJε勉召．

fordetection

The

were

tested byresult showedthatof玖oho伽鷹01in stools

fヒom

patients．

It

latex

TCBS

of

the

and c

I乙o海oJ召名ロ8types．

suspected

streakedinofmonoclonal

antibody−sensitizedcholeraidentificadon

Stool spec㎞ens from308patients

the

of

test

was

concluded

that

the

spec而city

agglutination

test

were

antibody−sensitizedlatex

agglut血ation

higherandsensitivityt㎞thosetestofmethodofthepolyvalent
for

the

monoclonalantiseradiagnosis

andofantibody−sensitized

that

acute

the

monoclonalcholeraisvery

useful．

20

CHARACTERIZATlON

OF四BR10CHOLERン4E「01RECEN皿。YlSOLATEDNoBoRuDepartmentofBacteriology，School

Intemational

Nillety−one
ex肛n血edforthe91isolates

Centreforthekbiologicalindicated
detemjned

Tor

out

lwANAGAIofAND

behaviourthat600f

bycholeraeof31revealedthemclassical

and9ElRoGER

Medicine，University

D㎞hoeal Diseaseof

EEcKLEs2
theRyukyusl andResearch，Dhaka，Bangladesh2

in

Bangladesh

andsensidvity

to6an仙microbial

belongto theclassical biotype

phage

IV

and

beta−haemolysis

complete haemolysis in broth（Feeley㎝d
101naba（oneBANGLADESH

strains of y必短o oんoJ8α紹01，isolated

biotype，asstrains

N㎜soNE1，MAsAAKI

IN

polymyxin

on blood

PittmansinJanuary1986，wereagents．

Biotyp血g

and31to theBsensitivity

test．

agar

plate，but

only8strains

method）．

SerotypingTor），and2H聾【oj㎞a（one classica1，and

one

El

Twenty−one

of

Tor
El

revealed

indicated790gawa，
El Tor）．

Phage

166
typingbelongedtoMuke巧eeclass岨ed血to4groups：one strahl eachshowedinland5，19hl

group． Prophage

CelebesthataH

typ血g oftype，andproductivity
resistantclassical

oneElvibrios

type

Tor

vibrios

resulted in14strains

stra血ofodginalCelebes．

s

type1．ElTor

vibrios

type4，and10were

of

Ubol

were

untypable

type，16stra血s of cured

In classical vibhos，there

was a

high

of cholerae toxin as compared to those isolated before1973． There was no

strainagainst chloramphenicol，streptomycin，tetracychne，minocychne，amoxic皿n andnah（㎞c acid．

21

COMPARATIVE STUDY ON DRUG SUSCEPTIBILITY OF AERO盟0ハ乙4S
SPECIES ISOLATED IN BANGLADESH AND NAGASAKl
KAzuNoRI

SHIMoGucHI1，CHIKAKo MocHIDA1，KEIzo YAMAGucHI1，NoBucHIKA

KusANo2，TATsuRo

NAITo2，SIGERu

KoHEI
Departmentofof

Laboratoryofof

KoHNo3，ATsusI

SAITo3，K．M．S．Azlz4

Medidne，Nagasaki

Bacteriology，InstituteDepartment

HARA3AND

University

SchoolTropical Medicine，Nagasaki

Intemal Medicine，Nagasaki

Medicine3and ICDDR，B

of

Medicine1，Department

University2，The2nd

University

Schoolof

Hospita1，Bangladesh4

Drugsuscepdb皿tytestwaspe面medondle134s廿血s ofA脚吻o郷spesiesto the16
Imt㎞icrobial agents includingABPC，MPC，PIPC，CEZ，CEX，CCL，CTM，CZX，CP，GM，KM，
TOB，TC，M工NO，ST and NFLX。M㎞ahnhibitory concentration（MICs）were detem血1ed bythemicrobrothd皿ution

The134strajlls

lntemational
isolatedtheCenterMost ofprovidedbothan廿n並crobial

Dianhea

hospitahzed

was

as

soδ幅σwere

MIC2000（Dynatech）．

Diseaseinconsistedofthe105strahls㎞〔皿yResearch，Bangladesh（ICDDR．B）and

Nagasald University
thatresistant

to ABPC，CEZ，CEX

fヒom

ICDDR

consisted

The29strains仕om

such

using

ICDDR．B was a㎞ost s㎞nar tostrainsso痂勉strains．Most

dmgs

by

￠onσs species

Hospital．

of

the

and

The

straills

CCL，but

drug

providedby

the29strains

susceptibilityofisolatedfromNagasaki．

susceptible

to

the

other

agents．

The105strai皿s

strains．

A8m

fbr

from patients

strains

tec㎞que

of

of

the

to

of29」4．勿4猶のゐ吻，4L4．oω伽and35、4．soわ吻

Nagasaki consisted
strains

ABPC，CEZ，CEX

susceptible

B

of14A．勿伽砂h吻，12／4．oの初and3／4．

of！1．勿4脚h吻and．4．σσ吻8showed

Imd

CCL． On

the

ABPC

and10−30％of

otherresistance

hand，about50％of

the＄trains

were

the

susceptibletomultiples廿ainsofA．

to

CEZ，CEX

and

CCL．
As

for

the

andbiotic resist｛mtand／4．68〃毎θwereA

soδ吻were

resistant

ABPC

to

production

resistantβ一lactamasepattemtoanconcurrently，although

CEX

only．

was

considered

as

one

both ABPC and CEX． Then，the

勿4名砂h吻was

examinedtospecies，nearly

CEX

勿4猶のh露αto勉ソ4名ρρhづ」σapPeared

ofeachandsubstrate

of

the

dmg

profilesthestrains

the40％ofresist2mce

ofA．勿40ゆh吻

thestrahlsmechanismsofβ一lact肛nase

produced

of

of

A．

by／4．

by

the micro−iodometric assay． Theβ一lactamase produced by／4．

bepenici皿㎞se．

167

22

DRUG

SENSITIVITY ANDSEROTYPE、4、ERUO卿0＆41SOLTEDWATER
YosHIYuKl

IN

OKuwAKI1，HlsAE

OF

FROM

lNDONESIAYANAI1，KolcHIRo

SETsuKo TsuKIDATE2AND MAsATosHl
Department of Microbiology，Kagawa

Zoology，Nagasaki

University

Hygiene，Juntendo

The

present

磁郷ゆstudy「reports

osαisolated

ffom

the

Nutrition

Schoolof

University

results

d血dngofwater in

PSEUDO盟0醐SDRINKING

SuGIYAMA3

Co皿ege1，Department

Medicine2and

Schooldmg

FuJITA2，ofof

Department

MedicalofMedicine3

sensitivity

andserotype

of

Psε％吻

zoησs

Indonesia in1985and1986．

The

number of isolated strahs were ll in1985and19in1986．
Sensitivity testing to chemotherapeutic agents such as PCB，PIPC，MZPC，CER，CET，

CFX，CMZ，CPZ，GM，KM，CP，TC，CL

A㎞ost

an

strains were

t1血d generation

generation
MIC

cephemsensitive

dmg）．

to

were

method．ResistImce

concetrations

Serotypes

of

aH

were

strajns

fo皿1d

23EXAMINATIONCPZ（thethe血噛stthesecondtoandchemicalresidualchlo血ewascephemdrugs（CFX，CMZ）．

which seemed

to

CER，CET，CFX

and

CMZ

ll

strains in1985．

and

E

F

type6among

be resistant by

type6among19strains

DRlNKlNG

were

found

disk

agar

at

very

WATER lN

In1986，

respectively．

NORTHERNCOUNTRIESANDHIRosHISuNAGA

J皿ior Co皿ege，Nagoya

examinations were

Momovia，Abidjan，Conahy，1Mger，Tur岨s andFree

generation

shown

or1，000μg／mJ．

KIYoKo SAwADA

Bactehologicalmethod．

to

AFRICAN

Mizuhod血1sionandE typeOFagarsensitivity，however，was

were class迅ed

in

disk

any

strains

exceeding250μg／mJ

isolated

however，serotypes

ofby

PCB，PIPC，MZPC，GM，CP，TC，CL，NA

Scarcelydmgs（CER，CET）and

examinedvalues were measured in those strains

diffusion
上亘gh

cephemand NAdetected only

carried

out

of

a

thkd

d】血11dng

wat6r

conected

tap

water

in

Tripoh．
kl

less

than

of

total

samples

examined．Co邸o㎜bac皿wereiso蛤ted丘om⑳waterinthe3cidesontheAdandccoastand
Tripoh whereresidualchlorine

was

less

often

fomd．

D血dng

water

samplescollected

atrestaurantsinMonroviawerejound仕equendycontaminatedbycoMombac皿．Drin㎞gwaterinprivateresidences

which

had been passedsugges血9

Probablehlc舳aUonof血adequate

waters

the

were

from

on

marketsafe

throughthe61ter was often contaminated，

use of廿1e fUter． On Ule odler hand，t丑ble

contan血ation

of

the

bac皿1．

Of21str血sofcoMombac皿iisolated，K．勿ε％魏o 伽wasmost丘equendyseen，fo皿owedby

E磁oJo㏄σε，C．加朔鵡K．o甥ooαand also2strains of E．oo砿 MIC tests revealed that

168

there were found 2 resistant strains to SM and TC, respectively, and one to CP, though all
strains were susceptible to pipemid acid.
Seven strains of enterococci were also isolated including 5 strains of St. faecalis liquefaciens

and a strain of St. faecalis and St. faecium, respectively.

Total hardness and chloride ion concentration of tap water in Alger and Tripoli were in
general higher than those in other cities. Especially in Tripoli almost all specimens exceeded

300 mg/1 in hardness and 200 mgll in chloride ion concentration. In some specimens the latter
was over 1,000 mg/1 and salty tap water was the obstacle in making tea in daily life.

24 WATER‑STERILIZING CAPACITY OF AN EQUIPMENT
MADE OF HOLLOW MULTI‑POROUS FIBERS AND
SILBER‑ACTIVATED CARBON POWDERSHIROSHI OHARAl, ISAO EBISAWA1, MAKIKO TAKAYANAGll

TOSHIHIKO KOMATSU2 AND TERUKO KOHARA2
Department of Public Health, Toho University School of Medicinel andDepartment of Enterovirology, National Institute of Health2

The incidence of water borne iufections is extremely high in the developing countries. In
the face of the increasing number of the Japanese sojourning in such countries, the supply of theclear water is becoming more and more important ; and any effective measures which assure thesupply of clear water is desired. Recently a new type of water sterilizer made of hollow
multi‑porous fibers and silber‑activated carbon powders was developed. In order to examine
the efficacy of the equipment, the water sterilizing capacity was tested using various pathogens

which were prevalent in the developing tropical countries.
Escherichia coli. Serratia marcescens. Salmonella typhi. Vibrio cholerae. Shigella saunei and

Salmonella typhimurium were used as the experirnental bacteria. Sterilized water containing
l08‑l09 of bacteria per one liter was filtrated with the sterilizer. The filtrate was passed
through a millipore filter and the filter was cultivated on the Endo's medium. Besides drect

filtration of the bacteria contained water, 2 other experiments were performed after pre‑treatment of the sterilizer ; i. e. a large amount of unclear river water or the tube water was
passed through the filter.

As a result of 3 knds of experirnents carried on 6 kinds of bacteria, no viable bacteria was

detected in the water obtained after filtration with the sterilizer. An experirnent was also
carried out using Rota virus, and no virus was detected after filtration.

The combination of hollow multi‑porous fibers and silberactivated carbon powders wasconsidered to be effective to eliminate bacteria and virus as welk The equipment, in combina‑

tion with vaccination and precaution against taking raw foods, seems to be much effective inprevention against oral infections in the developing countries.

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol. 15 No. 2

June, 1987

CONTENTS
Original article
Tanaka, H., Santos, A. T. Jr., Matsuda, H., Yasuraoka, K., Blas, B. L., Nakao, M.,
Araki, K. and Nakamura, S.
Epidemiologicad Importance of COP Reactions in Areas Lightly Infested with
Schistosoma japonicum in the Philppines . . . . . . . . . . . . . . . . .

87‑96

Hashiguchi, Y. , Furuya, M. and Okamura, Y.
The Fate of Leishmania brazilieusis. L. donovani and Tmpanosoma cruzi in
Difftsion Chambers Implanted into Hamsters and Mice‑a preliminary study‑

97‑104

Yoshida, Y. , Matsumoto, Y. , Yamada, M. , Shiota, T. , Yoshikawa, T. , Okabayashi, K.,
Tegoshi, T. , Yoshikawa, T. , Kobayashi, A. and Kim, R.
Clinical Trial of Trirnethoprirn‑Sulfamethoxazol in the Treatment of Vivax, Ovale
and Falciparum Malaria ................

105‑116

Short comlnunication
Kawabata, M., Mimori, T. , Hashiguchi, Y. and Coronel, V. V.
Chagas' Disease : Delayed Type Hypersensitivity to PPD in Relation to
Seroreactivity to Tmpanosowa cruzi in Ecuador . . . . . . . .

117‑120

Proceedings of XXVIII Annual Moeting of Japanese Society of Tropical
Medicine (1)Contents

139‑140

Special lecture . . . . . . . . . . .

141‑142

Symposium Diarrheal diseases in tropical area

143‑147

Tsutsugamushi dlsease (scarab typhus) in Asia
General presentation (1)148‑152
153‑168

Publrshed byJAPANESE SOCIETY OF TROPICAL MEDIClNE
c/o Institute of Tropical Medicine, Nagasaki University

12‑4 Sakamoto‑machi, Nagasaki 852, Japan