第19巻第1号

平成3年3月15日
内

原

容

著

皮膚リーシュマニア症の治療

一Metronidazoleと温熱療法による治療例一一矢後

文子 1−14高知県の大複殖門条虫症一12症例の追加一
一鈴木

了司，今村

京子

15−23

培養熱帯熱マラリア原虫における生殖体形成および受精に対する電子顕微鏡的観察（英文）

・・小野

忠相，中林

敏夫，大西

義博

25一＄1

HIVgp41の構成ペプチドおよびその類似合成ペプチドによるヒト末梢血NK活性の抑制機構（英文）・・清原龍夫，村田建一郎，守屋泰一郎，渡辺正実，

池田 柊一，市丸道人，板倉 英喜

33−38パラグアイ，アスンシオン市における幼児の細菌性下痢症（英文）・・一瀬休生，Cesar

Rose

MongesM．Albomo，Juan

AりMaria

G．Centurion，Dorita

J．Bestard，坂本信，Ricardo

Medina，MorenoA．39−47

臨床的研究

熱帯熱マラリア治療における開始遅延の意義
一海老沢 功，田辺清勝，小原博 49−56

一海老沢 功，田辺清勝，小原博 57−63

マラリア患者の黄疸について

第32回日本熱帯医学会総会講演英文抄録
目

次……………・・…・………・…

65−68

特別講演……・…………・…一………

69−73

一般講演……・………………………

74−124（裏面に続く）日本熱帯医学会

1

日熱医会誌．，第19巻 第1号 1991年，1−14頁

皮膚リーシュマニア症の治療
一Metronidazoleと温熱療法による治療例一
矢後文子

平成2年10月15日受付／平成2年11月15日受理

はじめにJamesand Stephen，1975）があるので，皮疹より原虫を分離培養しながら，Metronidazoleによ日本人の海外滞在地域の拡大と長期化に伴い，る治療を行った。Metronidazoleは，原則として

本来我が国には存在しない疾病の治療を，求めらの疾患の1つである。同症の治療は，皮疹が少数

1日750mgを分3で10日間投薬後，10日間の休薬期間をもって1クールとし，3クール行った。さらにKN株が入浴湯温である42℃で，2時間以上の場合には，一般には局所療法単独または薬剤の継続保温すると，死滅する実験結果（矢後ら，全身投与を併用，皮疹が多数の時や局所療法が適1989；矢後，1990）を応用して，患部を42℃よりさない部位などの場合には，主に薬剤の全身投与

43℃に，合計30時間保温し，治癒した症例を経験が用いられている。治療薬としては，古くからアしたので報告する。

れることがある。皮膚リーシュマニア症もそれら

ンチモン剤が使用されているが，日本では入手しにくく，その副作用も考慮しなければならず，まする報告（Convit
Rahim

and

and Kerdel−Vegas，1965；

Tatar，1966；土田ら，1989）もあり，症

儲膨蜥た皮膚リーシュマニア症に対する効力を，疑問視例：51歳，日本人男性。会社員。
1988年3月15日。

アンチモン剤に代わり得る薬剤が望まれている。

局所凍結療法を2回施行し，アンチモン

今回，サウジアラビアで感染し，アンチモン剤の剤を合計5，700mg筋注したが，完治し筋注と局所凍結療法を受けたにもかかわらず，皮ない皮膚リーシュマニア症と考えられる

疹からリーシュマニア原虫が培養された患者を治

皮疹の治療。

療する機会を得たので，アンチモン剤を使用しな

既往歴：特記事項なし。

い治療法を試みた。まず，本症例より分離培養し現病歴：1987年6月より12月中旬まで，サウジ

たPromastigote型原虫（以下KN株と略記す）

アラビア，リヤドの西北約300kmのブレを，伽∂飾oで原虫の増殖性と運動性を示標に，5

郊外の砂漠地帯に滞在した。滞在中は，刺咬時にイダ市

価のアンチモン剤（Pentostam⑪）と，現在日本で強い痛みを伴う，多数の虫刺を受けた。刺咬され使用されている代表的な抗原虫薬であるた直後に，腹部をピンク色にした白い小さな虫にMetronidazole（Flagy檸）との殺KN株効果を比

気づき，捕らえて圧平すると，血液と思える液体較（矢後ら，1990）し，Metronidazoleが，アンが認められた。刺咬後，掻痒感は2または3週間

チモン剤に変わり得る効力を持つことを確認した。

位は持続したが，1カ月位後には消失した。1987

実際に，Metronidazoleを皮膚リーシュマニア症

年11月初旬，右大腿伸側中央部に掻痒感の強い，患者に投与し，完治した症例報告（Peter，1973；

他の虫刺部位に比べて，出血しやすく治癒しにく東京女子医科大学寄生虫学教室

2
い発赤腫脹に気づいたが，単なる虫刺と思い放置

は直径約1cm（写真1）と小さかったので，同24しておいた。時々，噴火口のように盛り上がった

日直ちに，局所凍結療法が施行された。凍結療法

皮疹の中央の窪んだ部位から浸出液が流出し，下は，直径1．9cmの金属円板を，皮疹に強く押し付着を汚した。12月16日，帰国途中のバーレインのけ，圧縮された炭酸ガスの気化熱を利用して，円ホテルで入浴中，同皮疹が少しも治癒していず，

板を一35。C〜一36。Cに1分位冷却後，一度円板を発赤腫脹部の中央が湿潤なのも気になり，ホテル

離して患部を観察し，再び1分位冷却した。冷却で医師の診察を依頼したが，医師は見つからな時，出血や疹痛はなかった。2月6日，長期問サ

かった。12月19日帰国し，某皮膚科を受診したが，ウジアラビアで治療するのが困難な患者の事情も蚊刺ではないかといわれた。しかし，サウジアラ

考慮され，5価のアンチモン剤（Pentostam亀ビアの人々が，サンドフライの刺咬によって起こ

Wellcome社，England，1mJ中に100mgのる病気を，大変恐れていたことを思い出し，12月

Pentavalentantimonyを含む）を，3ml轡部に筋注した。翌2月7日より，6mlずつ毎日左右の下旬，ブレイダ市の皮膚科医へ，皮疹の所見，すなわち周囲がピンク色の直径約2cmの皮疹で，中轡部に交互に2月15日まで筋注し，合計10回，全央の直径1cm位の部分は硬結を伴う濃い赤色で，投薬量は5，700mgであった。アンチモン剤は，直その中心に1mm位のいつも湿潤な凹部が観察さ接皮疹内には注入しなかった。同剤の筋注は大変れる状態や，皮疹の経過や写真なども送り，翌1988

不快で，投与後30分位すると，注射側の足がしび

年1月初旬，皮膚リーシュマニア症との返信を得れ始め，1時間位続いた。また，注射を開始してた。1月13日某病院皮膚科を受診し，皮膚リーシュ

2日目と3日目には，投与後1時問位から，軽度マニア症とのサウジアラビアの医師の診断を伝えな頭痛，発熱を伴わない悪寒，数度の咳などの感ると，患部は外科的に切除するとのことなので，冒様症状が出現し，また注射期間中は，両上肢と治療は希望しなかった。1月18日，某病院でり一

頭部に掻痒感を生じ，手には赤色の発疹が出た。

シュマニア症についての説明を受け，内蔵型の混医師に症状を訴えたが，頻発する症状とのことで，合感染の可能性も考え，1月23日，治療のためサ

予定通り10回筋注した。食欲は良好であった。1

ウジアラビアヘ再渡航した。1月24日，ブレイダ

月24日に施行した凍結療法部位に接して，2月16

市の国立病院で，視診と触診のみで皮膚リーシュ

日，再び小さな発赤腫脹（5×2mm位）が認めら

マニア症の初期と，再び診断された。発赤腫脹部

れ，視診と触診のみで再発と診断され，前回と同様にして第2回目の凍結療法が行われた。凍結療法部は，冷却した金属円板を皮疹に強く圧迫した

1・一ための白色の陥凹（写真2A）が形成され，ついで水庖（写真2B）になり，潰瘍，壊死を経て，白斑（写真3）になった。凍結療法後も，なお数カ月の経過観察が必要と言われたが，帰国を急ぐ
患者の希望もあり，2月20日帰国した。帰国時，再々発に備え，Pentostamを100m♂持ち帰った。
サウジアラビアの医師からは，完治を期待しても良いと言われたが，帰国後経過観察するも，皮疹が治癒していくようには思えないとのことで，相談を受けた。
Photo．1

Clinical apPearance ofthe lesion on

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patient、初診時現症：1988年3月15日，右大腿伸側の膝関節より体幹寄り約15cmの部位に，直径約3cmの不正円形の脱色白斑（図1，March31，788および写真3参照）が観察された。白斑の周囲には，

3

1
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幅約0．5−1cmの暗褐色の色素沈着が認められた。

易疲労感，疹痛，発熱，紫斑，貧血，リンパ節腫

白斑部内には，直径約5mmの暗赤褐色の硬結を脹，肝脾腫または黄疸などはなく，一般状態は良伴う隆起が2カ所（図1，March31，

好であった。

88と写真3原虫の培養と診断：サウジアラビアでは，視診の皮疹番号1と2；以下図1および写真3と写真
6の皮疹部位を示す番号と凍結療法痕AおよびBと触診のみで，皮膚リーシュマニア症と診断されは共通で，皮番と略記す）観察された。掻痒感，

たが，確定診断をするために，1988年3月31日，

4
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皮番1と2より原虫採取を行った。採取方法は，

the

the

at

検査成績：1988年3月31日，5月19日，7月14

生理的食塩水（以下生食と略記す）約0．2mJを入日および10月6日と4回行った血液一般生化学検れた1mJの注射器に静注針（22G×1％）をつ査値では，A／G比の軽度な低下が認められた以外

け，発赤腫脹部の表皮に平行に浅く注射針を挿入し，静かに回転しながら針先で周囲の組織を破壊

は，異常は認められなかった。A／G比は3月31
日1．3，5月19日と7月14日に1．4（正常範囲

した後，生食の注入吸引を数回繰り返し，原虫をL5−2．2）であったが，10月6日には1．5と正常値含む細胞を採取した。針を挿入した腫脹部は柔らになった。電解質，脂質などは異常なく，肝機能

かく，採取操作は容易であった。しかし，患者は異常もなかった。また貧血は認められず，白血球

約半月後の4月中旬まで，採取部位の鈍重感や違数，白血球分画，赤血球像なども正常で，検尿に和感を訴えた。採取した材料は，斜面に固まらせも異常はなかった。1988年5月19日と同年10月6た6．5mJのNNN培地（Leventhal

and Chea・日に行った腹部エコー検査でも，肝腫や碑腫など

dle，1989）に，生食を5m1加えた中試験管に接種は認められず，検査成績からは，内蔵型の感染はし，25℃で培養した。培養1週目の4月7日，皮番1の採取材料より，多数のリーシュマニア
Promastigote型原虫（KN株写真4）が培養さ示唆されなかった。

治療と経過1）Metronidazoleの経口投与：1988年3月

れたので，本症例を皮膚リーシュマニア症と確定

31日，皮番1より原虫が採取されたが，その後経診断した。

過観察するも，硬結を伴う発赤腫脹部の融合と増

5

鱗灘
豊
懇1

灘

■

一灘

Photo．4Leishmania

NNN

promastigotes

medium（Giemsasseparated from thestain；original

lesionandculturedinmagnification×1，000）．

大（図1のMay19， 88．皮番1，2および6）と新生（同，皮番3，4および5）が認められたの

く浅くなり，また良く動くようになったという。

で，サウジアラビアで投与されたアンチモン剤のし，Metronidazoleは合計7，500mg服用した。服効果は，臨床所見からは認められなかったので，

薬期間が2日間延長したので，休薬日数は8日間

5月30日，12日間かけて1クール目の服薬が終了

副作用も考慮し，以後のアンチモン剤の使用は見と短くなったが，予定どうり6月8日より2クー合わせた。そこで，KN株に殺原虫効果を示した

ル目の投薬を開始した。6月13日頃より，1日1−

Metronidazole（Flagy縛）による治療を5月19

3回の下痢が始まり，口中に金属様の酸味感を覚

日より開始した。同日の投薬直前に，皮疹より原えるようになった。特に，砂糖入りコーヒーを飲

虫採取を行った。採取部位は，3月31日に原虫がんだ直後に，酸っぱい金属様の味を強く感じた。

培養された皮番1と，新たに出現した皮番3（図しかし，他に副作用と思える症状は，認められな

1，May19， 88）である。培養5日目の5月24日，皮番3から多数のPromastigote型原虫が培かった。6月18日に，11日間かけて2クール目が終了し，Metronidazoleの服薬量は1クール目と養されたが，皮番1からは，6月4日まで16日間

合わせ，15，000mgになった。2クール目の休薬観察したが，原虫は検出されなかった。Metroni−

期間に入り5日目の6月23日に，皮疹内の4カ所

dazoleの投薬は，原則として1日750mgを分3（図1のJune23，

88および写真3参照），すなわ

で，10日間内服後，10日間の休薬期間をもって1

ち，5月19日に原虫が培養された皮番3，硬結部

クールとしたが，実際に服薬した1日当たりの薬が増大した皮番5，1988年1月24日施行された第量と分服回数の記録は，表1に示した。服薬開始

1回凍結療法痕のA，および脱色白斑部の皮番11

後5日目の5月23日頃より，患者の観察によれば，

より第3回目の原虫分離を行い，7月14日まで3

手で皮疹を触れると硬結部が服薬前に比べ柔らか週間培養したが，原虫は培養されなかった。口内

6
Table

l

Scheduleoforaladministration

Date

Dose

1

Course1234567891
0㎜
一1
11
21
31
41
51
61
71
81
92
0

Date

2

Dose（mg）Times†

May，19（十）＊500
20

500

21

750

22

1，000

23

500

24

750

25

500
750

27

500

28

500

02
03
04
05
06
07

︻UO

Jme，01

June，080000000000
75

26

29
30
31

Metronidazole

3
Times

500
500
750
750
750
750
750
500
750

Date

Dose

June，28

09
10
11
12
13
14
15
16
17

1，000

18

500

08

500

19

0

09

500

20

0

10

250

21

0

11

22

0

12

23（一）＊

29
30
July，01

02
03
04
05
06
07

2

0

13

0

0
0

24

0

14（一） 0

25

0

15

26

0

16

0

27

0

17

0

0

Subtotal

7，500

7，500

7，500

Total

7，500

15，00022，500＊The markcould not

†The

dose

Refer

to

（十） shows that the parasites were
cultured．

a

day

was

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thethe explanations of Figure

times

l and

cultured

and

from

the

lesion

and

（一）

Times

0
0
07
07
05
5
5
5
5
05
0
75
70
50
50
57

Day

of

shows

that

they

administered．

Photograph3．

の金属様の酸味感は，2クール目の休薬期間中続かった皮番2，5月19日に原虫が採取され6月23

いた。6月19日より27日まで9日間休薬後，6月日には採取されなくなった皮番3，6月23日には28日より3クール目の投薬を開始した。服薬4日

原虫が培養されなく，7月14日痂皮形成が認めら目の7月1日頃より，再び1日2−3回の下痢が生じたため，Metronidazoleは1日500mgで分2にして7日間服用したので，3クール目は13日

れた皮番5と皮番5の痂皮より，第4回目の原虫の分離培養を行ったが，8月4日までの3週間の培養では，原虫は検出されなかった。そして，7間かかった。口内の金属様の酸味感は，再び生じ

月14日の原虫採取操作時には，皮疹に注射針を挿てきた。7月10日，3クール目の服薬が終了し，入または生食を注入する時に，強い抵抗感があり，

Metronidazoleの全服薬量は，22，500mgになっ原虫が採取された3月31日や5月19日の注入操作た。3クール目の休薬4日目の7月14日（図1，時の柔らかさとは，明らかに異なってきた。

July14，88および写真3参照）には，皮疹の大腿すなわち，原虫はMetronidazole投薬開始日で外側の白斑部と色素沈着部の境界が不鮮明になり，ある1988年5月19日には，皮番3より採取された白斑部が縮小し，発毛が認められた。同日，皮疹が，2クール投薬後と3クール投薬後には，試みの4カ所，すなわち3月31日に原虫が培養されなた皮番1，2，3，5および凍結療法痕A，白斑

7部11からは採取されず，Metronidazoleの投薬はとの間の温度は，常に42℃より43℃に保った。保

有効であったと考えられた。

温時間は，継続して1時間より5時間までで，102）局所温熱療法：Metronidazoleの投薬に

月2日まで6日間にわたり12回保温した。最後のより，皮疹より原虫が検出されなくなったが，皮温熱療法後4日目の10月6日に，皮番3と皮番5

疹のすべての部位から原虫採取を行ったわけでは（写真6参照）に発赤腫脹が生じたので，同所よなく，また経過が緩慢なので臨床症状からは，すり5回目の原虫分離を行ったが，原虫は培養されべての原虫が死滅したかどうかを判定することはなかった。さらに，1988年11月26日，89年3月2

困難であった。そこで，生存しているかもしれな

日（写真6）そして1989年7月5日にも，同様ない原虫を殺すために，引き続き局所温熱療法を発赤腫脹が皮番3に生じたので，それぞれ第6回行った。42。Cの恒温水槽水面より，KN株を培養し目，第7回目およぴ第8回目の原虫採取を行ったている試験管のNNN培地の最上部を0．7cm低くが，いずれも原虫は培養されなかった。しかし，沈めて，2時間継続保温すると，原虫は死滅する

実験結果（矢後，白坂，1989；矢後，1990）を治

1988年11月26日の皮番3には，1×2mm大の膿瘍が認められ，89年3月2日の発赤腫脹部は，原療に応用し，皮疹を保温した。保温方法は，表2

虫採取操作時の出血量が多く，欝血があったものに示したように，赤外線または温湯を使用して，と考えられ，1989年7月5日には痂皮形成が認め

合計30時間行った。まず，7月14日の第4回目のられた。これらのことより，繰り返し生じた発赤

原虫採取直後に，赤外綜ランプで皮膚表面の温度

腫脹は，残存原虫に起因するものではなく，細菌を43℃より44。Cに，1時間継続保温した。第2回の感染などによる2次的反応と考えられた。なお，

目の7月16日からは，写真5に示したように，皮経過観察しても，1989年9月8日には発赤腫脹や疹の上にハンカチ大の布を数枚重ねた上に，55℃

硬結は角蜘されなく，9偉10月現在，再発は認め

より60℃位の温湯を入れたビニール袋を載せ，皮られず完治と判定した。

膚と布の間に置いた温度計が，42。Cより43。Cを指すように布の枚数で調節した。温湯の温度が低下するに従って，布を1枚ずつ取り除き，皮膚と布Table2

MethodDate

Ju1．14， 88

infraredTemperature（℃）
lamp

Total

43−44

hot

water＊

42−43

16，

hot

water

42−43

16，

hot

water

42−43

17，

hot

water

42−43

17，

hot

water

42−43

17，

hot

water

42−43

24，

hot

water

42−43

24歩

hot

water

42−43（over43℃on

Sep．25，

hot

water

42−43

Oct．2，

hot

water

42−43

2，

hot

water

42−43

covered by several pieces of

cloth

and

occasion）

warmed with ahrs

ブ⊥ーブ⊥司⊥∩乙23

16，＊Lesion was

Duration（hrs）

123470159470

12345
789012
1⊥6−斗⊥No

Schedule of the班notherapy

vinyl

bag

that

contained

hot water from55℃to60℃which was put on the cloth．As the water grew cool，the cloth
was taken away piece by piece and the temperature between the cloth and the lesion was
kept

constant

at42。Cor43。C．

8{S ;," '; A, ' ･ ･･ ,#,. . ̲jl j * .

' h･ :̲ . ; i‑
Photo 5. Patient treated by thermotherapy, using hot water, at 42'C or 43'C.

Schedule and method of thermotherapy was given in Table 2.

c
*
'*+'

;
'* **

* *+

Z

I

!" ‑ '

vl

' ' '

+ * :

s

Photo. 6 Swelling with redness and desquamation on March 2,'89, at a time when no parasite could be cultured.

9
考

察

人男性に1日750mgを分3で10日問投薬後，10日間の休薬期間を1クールとし，2クールで治療に

皮膚リーシュマニア症治療の第一選択剤は，ア

成功した報告（Peter，1973）や，アフガニスタン

ンチモン剤とされている（輸入熱帯病の薬物治療で感染した24歳の白人男性に，同方法で3クール法に関する研究班，1982；Paul召≠砿，1984；佐々投薬し完治した報告（JamesandStephen，1975）

ら，1986）。本症例は5価のアンチモン剤であるなどがある。本症例でも，Metronidazole投薬開Pentostam⑬を，1988年2月6日より，10日間かけ始日である1988年5月19日には，皮疹から原虫がて合計5，700mg筋注したにもかかわらず，投与培養されたが，2クール投薬後の1988年6月23日

後約1カ月半の3月31日と約3カ月後の5月19日には原虫は培養されず，その後1989年7月5日まに，皮疹より原虫が培養され，同法によるアンチ

で5回にわたり，原虫の採取培養を行ったが，原

モン剤の効果は認められなかった。また，土田ら

虫は培養されなかった。しかし，皮膚リーシュマ（1989）も3価のアンチモン剤であるFua田n⑪

ニア症には，自然治癒があり得るので，Metroni−

を，それぞれ1．5mJ，2．5mJ，3．5mJと3日毎

dazoleによる治療期問と，自然治癒が偶然重なったに，計4回筋注した症例で，注射後の悪心が強いという可能性は否定できない。この点を明らかに上に硬結はむしろ増大し，同剤の効果は認められするには，より多くのMetronidazoleによる治癒なかったと報告している。本患者も，多数の皮膚例が必要であるが，矢後，白坂（1990）のKN株をリーシュマニア症の治療経験を持つサウジアラビ

用いた勉∂伽oの実験結果と合わせ考え，現時点

アの皮膚科医から，経験的にPentostamの投薬ででは，Metronidazoleにより原虫は死滅したもの

は，著しい効果は認められないので，アンチモンと推定している。

剤による治療は推奨しないと言われている。しかMetronidazoleは日本では日常的に，膣トリコ

し，本患者の場合には，帰国を急いで，当時は特モナス症，ランブル鞭毛虫症やアメーバ性赤痢の効薬と信じていたPentostamの投薬を強く望ん治療に用いられている薬剤なので，入手が容易なだために投与されたものである。これらのことか

上，経口投与が可能なので，患者はアンチモン剤

ら，皮膚リーシュマニア症に対するアンチモン剤の投与時のように，注射のために毎日通院したり，の使用については，その副作用を考慮しても，なまたその副作用を考慮して入院する必要はない。

お第一選択剤とすべきかどうか，その効果を再検

Metronidazoleの副作用は，常用量では軽微で，討する余地があるものと思われた。

報告されている主なものには，末梢神経障害があそこで，本患者の治療には，これ以上Pentos−

る。同障害を生じた症例のMetronidazoleの全服

tamを投与しても効果は少ないものと判断し，ア

薬量は，309より3149で，必ずしも服薬量には関

ンチモン剤以外の薬剤を検討した。Metroni−

係していず，その障害も軽度で，服薬を中止すれ

dazole（Flagy1を使用）とアンチモン剤（Pento＄

ば回復した（Bradley召∫認，1977）という。本tamを使用）の薬効の差を比較した矢後ら（1990）

症例の総服薬量は，1日750mgで10日間服薬を3クール行ったので，22、59である（表1）が，の実験，すなわち，小試験管に斜面にしたNNN培

地L3mJに生食1mlを加え，5mg，7mgおよび Metronidazoleの蓄積と副作用を考慮して，原則9mgのMetronidazoleとアンチモン剤をそれぞとして10日間投薬後，10日問の休薬期間を置いた。

れ入れ，KN株を培養すると，原虫の自発的な運動

しかし実際には，表1に示したように，1クール

性は観察した14日後までには，両剤に認められな目には2日間服薬が延びたので休薬日数は8日間，くなった。同様に，1mg，0．5mgおよび0，1mg

2クール目は1日延びたので9日間，3クール目の濃度では，両剤中で原虫は生存し，Metroni−の休薬日数は同様に7日間であった。本症例で観dazoleとアンチモン剤の薬量による差異は，認めら察された副作用と考えられる症状は，1日1−3

れなかった。実際にMetronidazoleを使用して治回の下痢と金属様の酸味感である。酸味感は，療を行った症例では，パナマで感染した24歳の白Metronidazoleが唾液中に分泌されるためと説10

明されている（Ralph，1983）。これらのことから，されたり壊死に陥り，その結果，原虫の生存条件

Metronidazoleは皮膚リーシュマニア症の治療が2次的に悪化し，弱化または死滅するのではなには，アンチモン剤に比して，副作用が軽微で，いかと推察している。一方，温熱療法は，粘膜皮殺KN株効果もあるので，試みる価値がある薬剤

膚リーシュマニア症の治療にも用いられていると考えられた。

（Rodbard，1979）。Berman

andNeva（1981）

局所療法には，外科的切除，放斜線照射，凍結によれば，皮膚リーシュマニア症を生ずるL傭h一

療法および局所への薬物注入などがある。外科的㎜n宛speciesは，37。Cまたはそれ以上の温度で切除は，インドなどでは古くから施行され，小さは，長期間生存できない。また，Ashi召」尻（1980）

な腫瘤では，醜い癩痕も残さずむしろ自然治癒よは，サウジアラビアでの皮膚リーシュマニア症の

りきれいに3週間以内に治癒する（Peter and

発症数は，毎年10月または11月頃より増加しはじ

Killick−Kendrick，1987）という。本症例も，日め，1月または2月に最多数を記録し，4月頃より減少し，7月と8月に最少になるという。この本で外科的切除を勧められたが，既に皮膚リー
シュマニア症には自然治癒もあり得，また全身にような変動は，皮膚が直接触れる外気温の変化と感染が広がる重篤な疾病ではないという知識をは，無関係ではないと考えられる。そこで，温度持っていたので，切除は希望しなかった。外科的負荷による殺KN株効果を，運動性と増殖数を示

切除は，顔面などの不適な部位もあり，また広範

標に実験（矢後，白坂，1989；矢後，1990）し，囲の摘出や部位によっては，機能障害を生ずる場

42。Cで2時間継続保温すれば，原虫が死滅するの合がある。有効で副作用が少ない内科的治療法がを確認し，治療に応用した。患者が不動に近い姿

確立されれば，一般には外科的切除より優先され勢を長時間保たねばならない負担と，低温火傷おるべきであろう。放射線照射は，自然治癒が平均

よび保温時問を中断した場合の殺KN株効果の減

9カ月かかるのに，86例中84例で7または8週間弱などの実験結果（矢後，1990）から，実際にはで治療できたという報告（Peter

Killick−

皮膚温は43。Cを目標に，1日に合計3時間保温

Kendrick，1987）があるが，放射線の人体への影

し，少なくとも1時間は継続し，1時問経過後に響も考慮する必要がある。凍結療法は，患者がサ中断する場合も15分以内とし，3日問以上連続iしウジアラビアの病院で通院中観察していると，痕て保温するのが有効と考えられた。本症例では，跡が残っても差しつかえない部位には，積極的に第1回目は赤外線ランプを利用したが（表2），赤

行っていたという。本症例は，サウジアラビアで外線ランプによる保温では来院しなければならず，

and2回凍結療法を受けているが，1988年2月16日に多忙な患者には負担が大きかった。そこで，治療2回目が終了した1カ月半後の3月31日と，3カ

時期が7月より10月と，外気温の高い時期であっ月後の5月19日に，原虫が皮疹より培養された。

たので，写真5のようにビニール袋に温湯を入れ

矢後，白坂（1989，1990）は，NNN培地で培養中

皮疹を暖めたが，冬期には使い捨て懐炉の使用がのKN株を，7℃に10日間静置して観察したが，原便利と考えている。試みに，市販の使い捨て懐炉

虫は活発な運動性を保持していたので，低温にはの中から，最高温度66。（ち平均温度50℃（都条例

抵抗性を持つと考えている。また，50％にグリセによる測定値），持続時間18時間（日本工業規格にリンを加えた培養液中のKN株を，一80。Cで約1よる測定値）の懐炉を用いて，成人男子の大腿伸カ月間凍結保存した後融解してみると，運動性を側を保温すると，東京の11月下旬で，懐炉と皮膚取り戻し，また継代培養も可能であった。したがっの間に4枚のガーゼを挟んだ時，皮膚とガーゼのて，本症例に施行された一35。C1分間位を2回施

問の温度は約40℃であった。ガーゼの枚数や衣服

行した温度と時間では，原虫は死滅しない可能性を調節すれば，42℃の保温は可能であろう。懐炉が考えられる。凍結療法が本症の治療に有効であによる保温は，低温火傷に注意しさえすれば副作

るならば，低温のために原虫が死滅するのではな

用もなく，仕事をしながらでもできる推薦できる

く，原虫が寄生している組織が，低温により破壊治療法と考えている。本症例では，Metroni一

11dazoleの内服治療を3クール行い，原虫が皮疹より室にも殺虫剤を散布し，下水の蓋の隙間などはふ検出されなくなってから，補助療法として温熱療さぐ。サウジアラビアに滞在中は，皮膚をよく観法を行ったので，温熱療法が殺原虫効果を示した

察し，発赤腫脹や硬結に注意し，帰国時には治療とする直接的な証明にはならない。しかし，原虫薬を持って帰ることなどである。患者は当時，の採取培養を行いながら，温熱療法をMetroni−

Pentostamが特効薬で，日本では入手できず，治dazoleの投薬より先に行う症例数を増やせば1この療経験のある医師はいないと考えていたが，その点はより明らかにできるであろう。

他のことは大変参考になる提言である。実際，

サウジアラビアで，L．ケoρ吻を媒介するサン勘」助o如吻粥の飛行距離は短く，しかも休み休み

ドフライの種は，主に勘励o孟o解欝とS砂 飛行するので，殺虫剤は窓の周囲や扉などにも噴
g召％云o勉吻である（Ashiα磁，1980）。日本では，

霧するのが有効である（Michael召∫磁，1989）。

勘」660渉o吻％sのような体長約5mm（Viqar，

しかし，どんなに用心しても，流行地で感染した

1983）という，患者の言によれば，60wの電球をサンドフライによる刺咬を避けるのは難しい。患近づけてやっと見えるぐらいの，埃のような小さ者は，サウジアラビアの病院で，顔面に罹患したな飛行する虫に刺される経験が乏しかったため，多くの人々，とりわけ鼻部が変色し，鼻下に4cm

最初患者は，掻痒感を原因不明の体調不良による四方位の，一部が黒化した潰瘍形成を生じた2歳ものと思っている。そこで，健康に注意してみた位の女児を見た時には，その児が成人した時のこが，なお同じ症状が続くので，次にはイエダニのとを考え，心が痛んだという。リーシュマニア原発生を考え，衣類などは日光にあて，身のまわり虫の感染のサイクルを断つ努力とともに，有効での清潔に心掛けた。しかし，掻痒感は減少せず，副作用の少ない予防接種の開発と普及が望まれる。

なお注意して観察し，刺咬部位が露出部に多く，皮膚リーシュマニア症患者を治療する場合，内また空気中に浮かんでいる，白い小さな埃にして蔵型の合併の有無には，常に注意する必要がある。

は動きの不自然な浮遊物に気がついた。また刺咬分離される原虫の形態からは，皮膚型と内蔵型のされた直後に，白い腹部をピンク色に染め，フラ鑑別は困難なので，臨床症状や検査成績に加えて，

フラしている小さなハエのような虫を捕らえて，渡航地や渡航経路が参考になる。内蔵型は，中近圧平して血液を認め，初めて吸血する虫による刺東ではイランなどサウジアラビア周辺には，一般咬と判明した。多いときには，1度に3匹も捕ま的に分布しているが，サウジアラビアには少なくえたことがあったという。そして，患者はサウジ（Ashi召∫認，1980），アラビア半島南西部のイエ

アラビアの皮膚科医から，皮膚リーシュマニア症

メンに近い地域にのみ発症例が報告されているの参考文献を入手し，自分の体験と合わせ，同地（Peterに引き続き滞在する同僚のために，以下のこと

滞在していたサウジアラビア，カシーム地区の皮andKillick−Kendrick，1987）。患者がを進言している。勘励o孟o卿欝の飛行距離は，約

膚科医に，1988年1月に送った質問の手紙に対150m（Ashi8∫磁，1980）以内であるので，宿舎のし，本症例が感染したと推定されるブレイタ布近

周囲は150m以上空き地にし，窓には網目の細かい

郊には，内蔵型の発症例はないという返信が得ら

防虫網をはり，暑くても裸で屋外では眠らず，外れた。同市近郊には皮膚型が多く，1975年より出時には，長袖シャツ，長ズボンや靴下などを着

1979年までの5年間に，サウジアラビア各地の病用し，なるべく露出部を少なくする。敷地内では，院より報告された4，352例の皮膚リーシュマニア

イヌ，ネコ，ニワトリ，ウサギなどの動物は飼わ症例の1，853例（42．6％）は，カシーム地区から報ず，ネズミの巣などは片づけ，また外部からもこ告されている（Ashiθ∫磁，1980）。内蔵型の症状

れらの動物が侵入しないように囲いをし，は，持続する発熱，悪寒，紫斑，肝脾腫，貧血，勘励o如吻欝の繁殖のサイクルやヒトとの接触を好中球減少，肝機能異常，IgGの多クローン型増加妨げる。日中，勘勅o孟o卿薦が潜んでいそうな暗によるA／G比の低下などであるが，本患者にはい場所や，洗面所，風呂場，そして出勤時には自A／G比の軽度低下が観察された他には，相当する

12

脹が1カ所だったので，凍結療法が適応され得る検査成績や症状などはなく，また発生年齢も多くは9歳以下の幼児や乳児であることなどを考慮し，などの説明を受け，また皮疹の部位も成人男子の内蔵型の合併はなかったものと判断した。

場合，あまり人目にさらす場所でない大腿であっ皮疹から原虫が培養されなくなった1988年6月23日以後も，同10月6日，1989年3月2日およたことなどもあり，一応の落ち着きを得た。しかび7月14日と皮番3に発赤腫脹が繰り返し生じた。

寄せていない医師には，皮膚リーシュマニア症の

しかし，原虫の採取培養を行っても，原虫は検出

有効で副作用の少ない治療法や，治療薬などにつされなかった。発赤腫脹部には，膿汁，欝血や痂いての情報が得にくい。海外渡航が日常化され，し日本では，特に熱帯病や特殊な皮膚病に関心を皮などが確認されているので，細菌の感染などに日本にはない疾病が，即時に国内にもたらされる

よる二次的な反応と推定しているが，患者は特に昨今，熱帯病についての知識や治療法，特殊な医

皮膚に傷をつけるようなことはしていないという。

薬品の入手方法などについても，日頃から準備しPeterandKillick−Kendr圭ck（1987）によると，ておく必要があると痛感させられた症例であった。

皮疹は傷を受けやすく，リンパ管炎，蜂窩織炎，
丹毒などを起こし易く，また薬剤の注入により，結アレルギーを伴った湿疹性反応を起こすことがあるという。本症例で，繰り返し生じた発赤腫脹も，語

サウジアラビアに滞在し，皮膚リーシュマニア症に感染した51歳の日本人男子に，原則として，感染のほかに何らかのアレルギー性反応を合併しMetronidazoleを1日750mg分3で10日問内服ていた可能性もある。

皮膚リーシュマニア症は，日本では頻発する疾後，10日間の休薬を1クールとし3クール行い，患ではないので，皮膚科医にも診断や治療の経験

その後，皮疹を42℃より43℃に合計30時間保温が少ないと思われる。本患者が，帰国直後に訪ねし，完治した症例を報告した。

た皮膚科医には，蚊刺ではないかといわれ，また会社の同僚からも，小さな皮疹を恐れる患者は，

謝

理解されなかった。幸いサウジアラビアには，人々

辞が大変恐れている皮膚病があることを知っていた御講読賜りました東京女子医科大学寄生虫学教

ので，患者は現地の医師に直接相談し，皮膚リー

室白坂龍曖教授，またサウジアラビアの皮膚リー

シュマニア症と診断された。治療経験の豊かなサ

シュマニア症の治療の現状や，貴重な文献や情報

ウジアラビアの皮膚科医により，皮膚型が内蔵型

を御提供下さいました西山勝治氏，および諸検査に移行することはなく，また滞在していた地域にに御協力戴きました諸氏に深謝致します。

は内蔵型の発症例はなく，皮膚型は生命の危険がなお，本稿の要旨は第58回日本寄生虫学会大会

ない上，特別な治療をしなくとも自然治癒があり（1989年4月）で発表した。

得，1988年1月24日の初診時には，小さな発赤腫

献文1）Ashi，J．，Peter，W．，A1−Gindan，Y、，Abdulaziz，O．，Hawwary，G．H．，θ

Symposiumon

Leishmaniasis：The

collegeof

medicine

andmedical認（1980）：Medical

sciences，KingFaisalUniversity．Kingdam of Saudi Arabia，5−112）Be㎜an，」．D．and Neva，F．A．（1981）：Effect of temperature on multiplication of leishmania

amastigotes within

human

monocyte−derived

macrophages伽∂蜘o，Am．」．Trop．Med．Hyg．，

30，318−3213）Bradley，W．G．，Karlsson，1．J．and3（September）610−611

Rassol，C．G（1977）：Metronidazoleneuropathy，Brit．MedJ．，

134）Convit，J，andKerde1−Vegas，F．（1965）：Disseminated cutaneous leishmaniasis，Arch．Der−

matoL，91，439−447
5）James，K．P．and Stephen，S．S．（1975）：Metronidazole

therapyforcutaneousleishmaniasis，Arch．Denmato1．，111，1343−1344
6）Leventha1，R．and

Cheadle，R．F．（1989）：Medical

Parasitologyl3thed．，140，F．A．Davis

Co．，

Philadelphia7）Michael，K，Dickson，D．D．and

Robert，W．G．（1989）：Parasitic

Diseases，2nd

ed．，182−187，

Spring−Verlag，New York
8）Pau1，CB．，Rodney，CJ．and Eddie，W．C（1984）：Clinicalparasitology，9thed．62−63，Lea＆Febiger，Philadelphia9）Peter，1．L．（1973）：Cutaneousleishmaniasis

treatedwithmetronidazole，J．Amer．Med，Assoc．，

223（12），1378−137810）Peter，
Biology

W．and

Killick−Kendrick，R．（1987）：The Leishmaniases in Biology and Medicine，I

and Epidemiology，63−71．235−262，II Clinical aspects

andContro1，633−635，847−907，

Academic Press，London
11）Rahim，G．E

andTatar，L．H．（1966）：Oriental

12）Ralph，ED．（1983）：Clinicalpharmacokinetics

43−6213）Rodbard，D・（1979）：Treatment

of

sore

in

Iraq，Bull．Endem．Dis．，8，29−54

ofMetronidazole，Clinicalmucocutaneous

Pharmacokinetics，8，leishmaniasis（1etter），New

Eng．J．Med．，

300，1489
14）佐々 学，緒方一喜，石井 明，田中 寛他（1986）：標準医動物学，第1版，23−28，医学書院，
東京15）土田哲也，大原国章，野村克己，小林和代，和田芳武，岡本雅子，山浦 常，松本克彦，石橋康正（1989）：皮膚リーシュマニア症一サウジアラビアでの感染例r臨床皮膚科，43（13），1275−1280
16）矢後文子（1990）：皮膚リーシュマニァ症の治療一Promastigote型原虫による温熱療法の基礎実験
日熱医会誌．，18（2），143−15317）矢後文子，白坂龍畷（1989）：皮膚リーシュマニア症患者より分離された原虫の温度による殺虫効果
寄生虫誌．，38（1），39

18）矢後文子，白坂龍蹟（1990）：皮膚リーシュマニアPromastigote型原虫に対するMetronidazoleとアンチモン剤の薬効の差異，寄生虫誌．，39（増刊号），115

19）輸入熱帯病の薬物治療法に関する研究班一厚生省研究事業一（1982）：輸入熱帯病の薬物治療の手引き，720）Viqar Z．（1983）：Scanning

Maruzen Asia，Tokyo

electronmicroscopy

of

medicalimportant．Arthropods，58−67，

14TREATMENT OF CUTANEOUS LEISHMANIASIS‑REPORT OF A CASE TREATED WITH METRONIDAZOLE AND LOCAL HEAT‑AYAKO YAGO
Received October 15 1990/Accepted Nobember 15 1990

A 51‑year‑old Japanese male who had spent six months in Saudi Arabia was diagnosedby biopsy in his right thigh as having cutaneous leishmaniasis and was successfully treatedwith metronidazole and local heat. The dose of metronidazole used was 250 mg three times
a day, and the drug was given for three ten‑day periods with intervening rest periods of ten
days. Leishmania promastigotes could be cultured from the lesion on the first day that
metronidazole was administered but could not be cultured after two ten ‑ day periods oftreatment with the drug. After medication was given, Iocal heat therapy was applied at
42'C, using hot water in a vinyl bag, for a total of 29 hrs, and heating under an infrared lamp

at 43'C for I hr on one occasion only. Details of heat treatment are given in Table l.

Department of Parasitology, Tokyo Women's Medical College

日熱医会誌．，第19巻第1号

1991年，15−23頁

15

高知県の大複殖門条虫症

一12症例の追加一鈴木 了司・今村京子

平成2年10月19日受付／平成2年11月22目受理

大複殖門条虫症は日本にのみ見られ，いまだに同一患者からの排出）が存在し，7例が大複殖門

その中間宿主が不明な寄生虫症であるが，高知県

条虫，3例が無鈎条虫，1例が米子裂頭条虫と同では本症例が多く，既に21例に達している（鈴木定された。以下，それらの症例について簡単に記ら，1988）。これら症例は，県内医療機関から同定載する。

を依頼されて見出されたものであるが，県内の医1）県中央部の山間地にある嶺北中央病院に保管

療機関には，未同定，あるいは誤って同定されたしてあった虫体で，患者の住所が大豊町以外は不ままの本条虫の存在が推定される。

明。長さ92．5cm，幅7mmで頭節はない。

そこで，アンケート調査を行ってその存在を確

2）県東部の宮田医院に保管してあった虫体で，かめるとともに，最近の本条虫症について若干の45歳，男性，北川村在住。1973年4月18日に肛門疫学的考察を行った。

より虫体の1／5をぶら下げた状態で受診。割箸で巻取ったもので，長さ43．5cm，幅8mm，頭節を欠方法

く。虫体排出前後の症状は，特に異常ない。ウルメイワシ，マイワシ，アジ，カツオなどの刺身や県医師会の協力を得て，1989年5−7月に，寄生虫症に関連があると思われる462医療機関に対酢のものを毎日のように食べていた。この患者は，し，過去に条虫駆虫とその虫体の有無についての食べない。

上記2種のイワシを区別していた。サバはあまり回答を求めた。虫体が存在する場合には，それら

3）県東部の県立安芸病院の保管虫体で，安芸市を借り受け，または恵与頂き，その虫体の形態を在住の51歳の女性。1987年2月28日に腹痛。夜半調べた。

に便意があり，排便とともに長さ576c偽幅16mm

また，鈴木ら（1988）の報告以降，1989年末まの頭節のない成熟虫体を排出した。魚の嗜好につでに寄生虫学教室に同定の依頼があった大複殖門

いては不明。

条虫の症例をこれらに加えた。

4）症例3と同一病院に保管されていた虫体で，患者に関しては不明。長さは46．3cm，幅5mmの成績

1．アンケート調査による症例
462医療機関のうち，289機関から回答（62．6％）

頭節のない未成熟虫であった。

5）県のほぼ中部に位置する西本医院に保管してあった虫体で，患者は年齢不明の男性，中土佐町在住。1966年11月初旬，長さ615．5cm，幅15mmを得たが，条虫駆虫の経験があると答えたのは15の頭節のない成熟虫体を，排便とともに排出。軽機関であった。このうち，9機関は虫体が既に存

度の貧血以外は特に異常はない。続いて同年12月

在せず，6機関に11例からの12虫体（内2虫体は30日に長さ772．Ocm，幅14mmの頭節のない虫体

高知医科大学寄生虫学教室

16を再び排出した。職業は漁師で，あらゆる海産魚

もって受診。虫体の長さは89．5cm，幅16mmで頭を食べている。

節はない。駆虫はしていないが，その後の再三の6）県西部の県立西南病院に保管中の虫体で，33

検便で陰性。酢のものを含むアジ，サバ，カツオ，

歳の男性，住所不明。1981年5月12日に条虫を排イワシなどの海産魚の生食を好み，イワシの稚魚出したため，Bithionolで駆虫し，同18日に272．1

であるドロメも頻繁に食べている。横川吸虫陽性。

cm，幅15mmの頭節を有する成熟虫を排出した。

2）53歳，男性，土佐市在住。伊藤医院より同定

魚貝類の嗜好は不明。

依頼。1週問前から下痢と腹痛があり，1989年127）症例6の病院に同じく保管中の虫体で，1982月21日に2回と，21日に1回の計3回にわたり虫

年5月末から1日4−5回の水様性の下痢。6月4日夜に下痢便とともに約1mの虫体を排出。続く。後検便は陰性。海産魚を好み，カツオ，マグ

いて5日に長さ不明の虫体を，6日に270cm，幅8mmの頭節のある成熟虫を排出した。標本とし体排出。長さの合計は499cm，幅15mmで頭節を欠ロ，ドロメをよく食べるという。

3）56歳，男性，高知市在住。高知市民病院よりて保管されていたのは，6日の分である。患者は同定依頼。1989年6月15日朝，排便直後に未熟虫

56歳の男性で佐賀町在住。魚貝類の嗜好状況は明体を排出。長さ62cm，幅10mmで頭節はない。患らカ・ではない。

者は健康で，異常は認められない。海産魚の刺身が好きという。

2．同定依頼による症例

4）45歳，男性，高知市在住。高知医大第三内科

鈴木ら（1988）による本症の報告以来，直接により同定依頼。1989年6月28日に排便後，トイレッ

医療機関から同定を依頼された大複殖門条虫症例

トペーパーに付着している虫体を持参して受診。

は5例あった。

長さ4711cm，幅10mmの未熟虫で頭節はない。3

1）68歳，男性，須崎市在住。菅野医院より同定

日前より下痢気味で，受診当日の夜にも虫体の排依頼。1988年12月11日に下痢とともに虫体を排出出があったが，その虫体はない。患者はほとんどしたが，約1mを便槽内に切り捨て，その残りを毎日，カツオ，マグロ，ブリなどの刺身を，また，

ピ

ノ

Ehime

≦10kushima

Prefecture

あ

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芝軸

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檀、

な

昌

や魂，ーノ

ん

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〜／トκチ；lt、．．
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リザノ

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Prefecture

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罫．

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Figure1・廻ノof diplogonoporiasis within

Kochi

PacificOcean

GeographicalPrefecture，

distribution

17

約2週間前に3日ほど連続してドロメを食べてい

海産魚を食べたかは明らかではない。しかし，こる。

の病院では年間，30例近くのアニサキス症患者を5）46歳，男性，春野町在住。1989年7月19日

認めていることから（神崎ら，1987），海産魚類のに，高知市民病院で茸便検査で虫卵が検出され，流通機構の整備，嗜好魚類の変化，内陸部住民の同月21−22日にParomomycin投与。翌23日に長さ284cm，幅13mmの成熟虫を排出。頭節を認め海岸地方への旅行，その他により，今後は海岸部た。

倉旨1生力§弓螢し〉。

ばかりではなく，内陸部にも発生が認められる可これら12例の症例を含め，現在までに県内の本

季節的には不明の3例を除くと，1980年以降，症患者の居住地を地図上に表したものが図1であ1−2月に8例，3−4月に3例，5−6月に7る。ただし，患者の住所不明の1症例は除いた。

例，7−8月に2例，9−10月4例，11−12月に3例となり，1−2月と5−6月に多く，夏期に考察

1．アンケート成績

少ない傾向にある。特に冬期にも患者が見られる
ことは，他県（前嶋ら，1990）と異なる。

本虫寄生による症状は，下痢，腹痛などの消化条虫駆除の経験があったと答えたのは，289機関

器症状があげられているが，今回の場合も，患者中15機関であり，そのうち，虫体が存在したのはは数日前から下痢が続き，その後に自然排出して6機関で，その11例中7例が大複殖門条虫と同定おり，そのほかには症状としては特に異常は認めされ，予測したように新たに本症例を確認するこられない。

とができたが，大複殖門条虫と同定されていたものは全くなく，広節裂頭条虫と同定されていたもの3例，有鈎条虫と同定されていたもの1例，単3．全国的な症例の推移
鈴木ら（1985，1988）は全国の症例を整理したに条虫とされていたもの3例であった。このようが，洩れていた症例（高田ら，1982；Terada8渉な同定結果や，9機関で虫体が既に廃棄されておα」．，1985；板垣ら，1988）や，関東地方を中心にり，検査し得なかったことは，条虫症に対する関各地からの症例（安羅岡ら，1989；赤尾，1989；

心が必ずしも充分でないことを示している。

金子ら，1990；前嶋ら，1990；高尾ら，1990；影井ら，1990）が報告され，また，寺田ら（私信）2．高知県下の本条虫症の未発表分（4例）があり，1989年末までに本症

県下の本症例は今回のアンケート調査による7の合計は162例となった（表1）。87例までは，鈴例，同時期に同定依頼された5例を加えると33例

木ら（1985）の報告と同一のため表から省略しに達した。岩田，安岡（1970）による第1例以た。これらの症例は論文としての発表順であるこ来，1979年までは4例に過ぎなかったが，1980年とが望ましいが，学会のみの報告もあるため，す代には27例（2例は虫体排出日不明で除外）とな

べて，最初に報告された時点の順に並べてある。

り，急激な増加が認められた。更に虫体標本が廃

本報告の12例は1989年に学会発表をしているた

棄された9医療機関では，無鈎条虫としたもの3

め，教室での受け付け順で全国症例の130−141例

例，広節裂頭条虫としたもの1例，不明6例で，に相当する。

この中にも大複殖門条虫が存在していた可能性が

これら症例を虫体の排出時期により分けると，患者は従来指摘されているように，中間宿主が最初に患者が見出された1894年以降1979年までの85年間に85例（排出日不明は10例あるが，著者疑われる海産魚を手に入れやすい海岸に面した市の症例発告時が1979年以前の6例を加えてある）

町村に多いが（図1），海岸から離れた山間地に1であったが，1980年以降の10年間には73例とな例の存在を認めた。この患者は大豊町在住者といり，全国的に症例数が増加している。これを本州

う以外，記録がないため，どこで中間宿主となる

東部太平洋域（福島一静岡），本州西部太平洋域（瀬

ある。

18

Summary of reported cases of diplogonoporiasis in Japan until 1989.

Table 1All cases are re‑numbered according to the date of report.

ReportWorm foundPatient

Case

No. Author

Year Locality

Date

f oundAge Sex

M
46
M
49
M
58
M
65
M
74
M
41
M
55
M
57
M
39
M
63
M
53
M
46
M
36
M
39
M
42
M
50
M
50
M

88 Takada et al.

1981

Osaka

Dec

1980

89 Saiga et al.

1983

1983

Sano
91 Oshima and Amano
92 Oshima and Amano

1984

Kochi
Shizuoka

A pr

Aug

1983

1984

Kanagawa

Apr

1984

Shizuoka
1984 Kochi
1984 Kochi
1984 Kochi
1984 Kochi
1984 Kochi
1984 Kochi
1984 Kochi
1984 Kochi
1984 Miyazaki
1985 Kochi

Jun

1984

90

93 Suzuki et al.
94 Suzuki et al.

95 Suzuki et al.
96 Suzuki et al.

97 Suzuki et al.
98 Suzuki et al.
99 Suzuki et al.
lOO Suzuki et al.
101 Kushima and Imai

l02 Ueta et al.
lO 3

Miyahara and Maejima

1984

May
Jun
Sep

1983

N ov

1983
1984

Feb

1984

Oct

1984
1984

1985

1984

Fukuoka

Dec

1985

1985

Shizuoka
Chiba

106 Kagei

1985

Kanagawa

l07 Terada et al.

1985

l08 Mochizuki et al.

1986

109 Mochizuki et al.

1986

Shizuoka
Shizuoka
Shizuoka
Shizuoka
Shizuoka
Shizuoka
Shizuoka
Shizuoka
Shizuoka
Nagasaki
Kochi
Kochi

llO Mochizuki et al.

1986

111 Mochizuki et al.

1986

ll2 Mochizuki et al.

1986

ll3 Mochizuki et al.

1986

114 Mochizuki et al.

1986

ll5 Mochizuki et al.

1986

116 Inutsuka et al.

1986

ll7 Okamura et al.

1986

Okamura et al.

1986

Yamane et al.
120 Nishiyama et al.

1987

121 Yamamoto et al.

1987

1987

1983

J an

1985

104 Suzuki et al.

1 19

1982

1983

Jun
Jan
Feb

l05 Kanazawa et al.

l 18

Jul

1984

May

1981

A ug

1983

A pr

1985

May

1985

M ay

1985

May
May

1985

63

31

M

M
81
M
50
M
60

43

M

Length

Width

(cm)

( mm )

174 . O

13.0

164 . 44.4

380 . O

18 . O

145 . O

4.0

410 . O

14 : O

341.0 ?

8.0

14 . O

4.0

ll . O

7.0

511 . O

10 . O

130 . O

4.0

97 . O

16 . O889 . 315.0

268 . O

6.0

209 . O

8.1

67 . O

4.5

80 . O

6.5

30 . O

9.0

343 . O

7.3

500 . O

17 . O360 . O

9.0

200 . OlO . O

62 . O

10 . O

280 . OlO . O

50 . O

5.0

180 . O

7.7

60 . O

5.0

1985

47

1985

42

1985

Feb
Feb

1986

N ov

1985

Shimane
Osaka

Feb
Sep

1984

Hyogo
Kanagawa

May
May

1987

64

1987

58

Oct

1986

54

Mar

1987

48

306 . 4

9.0

Sep
Jan

1987

39

393 . 4

10 . O

118 . O

13 . O

Jun
Jun

1985

1986

122 Tanaka et al.

1987

123 Okamura et al.

1988

124 Okamura et al.

1988

125 Okamura et al.

1988

126 Okamura et al.

1988

127 Okamura et al.

1988

Kochi
Kochi
Kochi
Kochi
Kochi

Mar

1988

128 Itagaki et al.

1988

Yamaguchi Jun

1988

129 Yasuraoka et al.

1989lbaraki

Aug

1988

1988

F

45 . O

3.0

43

63 . O

3.0

60

692 . O

9.0

505 . O

2.3

118 . O

4.3

M
M
54
M
32
M
60
M
55
M
F

Ml
F

M
M
43
M
52
M
26
M
57
M

300 . O

5.8

273 . O

13 . O

200 . O

20 . O067 . O

12 . O342 . 5lO . O

45 . O

7.0

248 . O

6.1

490 . O

15 . O

Scolex

+

+

+
+
+

+

19

No. Author

Year Locality

130 Suzuki et al.

1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi
1989 Kochi

131 Suzuki et al.
132 Suzuki et al.

133 Suzuki et al.
134 Suzuki et al.

135 Suzuki et al.
136 Suzuki et al.
137 Suzuki et al.
Suzuki et al.

l 38Worm foundPatient

ReportCase

Date found Age Sex Length Width scolex(cm) (mm)

Dec

1988

J un

1989

M
53
M

89 . 5 ?

68

A pr

1973

45

J un

1989

56

Jun

1989

45

Feb

1987

51

N ov
( Dec

1966

499 . O1989 Kochi
1989 Kochi
1989 Kochi
1989 Fukushima

140 Suzuki et al.
141 Suzuki et al.
142 Kaneko et al.

Jul

1989

May

1981

Jun

1982

Aug
Aug

1989
1988

Se p

1988
1988

15 . O

92 . 57.0

M

43 . 5

8.0

62 . OlO . OM

47 . llO . OF

576 . O

16 . O

46 . 35.0

M615 . 515 . O772 . O

14 . O284 . O

13 . O272 . I ?

15 . O

270 . O ?

0.8

1966
139 Suzuki et al.

16 . O

M
33
M
56
M
46

F

100 . O

8.0

47

M
M
52
M
42
M
41
M
51
M
82
M
57
M
39
M
54
M

600 . O

20 . O50

200 . O

20 . O

49

+
+
+
+

Akao
Akao
145 Akao
146 Akao

1989 Ibaraki
1989 Ibaraki

1989 Tokyo

Oct
Oct

147 Maejima et al.

1990 Tottori

Jun

1978

148 Maejima et al.

1990 Shimane

Mar

1980

149 Maejima et al.

1990 Tottori
1990 Tottori
1990 Tottori

Jul

1984

Apr

1986

Jun

1987

Mar
A pr

1985

156 Takao et al.

1990 Nagasaki
1990 Kagoshima
1990 Yamaguchi
1990 Oita
1990 Saga

1985

157 Kagei et al.

1990 Tokyo

Feb
Dec

1989

Nov

1980

Jun

1982

53

J un

1983

83

F

May

1985

50

M

e

i')J f

ly>.

:,frN

;

v>

D (6.8f**)

143
144

1989 Shizuoka

150 Maejima et al.

151 Maejima et al.
152 Maejima et al.
153 M ejima et al.
154 Maejima et al.
155 Maejima et al.

160 Terada et al.

1990 Chiba
Shizuoka
unpublished Shizuoka

161 Terada et al.

data Shizuoka

162 Terada et al.

Shizuoka

158 Kagei et al.

159 Terada et al.

P i:

S : Cl‑ W f),

D, jL')'1'1ftLd a) 4: 198071 j J

eC

: t r

(

,

i e

‑

* ) t

1970

f eC,

S

<

) IF

]

e E I

,

e

')J

) !".

:‑' !‑ ‑‑ i*

fy5

V>

.

10 . O

64 . O̲̲

13 . O

94 . O

5.5

192 . O

9.0

62

51 . O

15 . O

M
M
81
M
23
M
42
M

5.0
20 . O

Apr

1987

41

1988

58

12 . O

Mar

1989

1989

cj

U

:

1 D

5.5

360 . O

A pr+

100 .O ?
224 . O
20 . O

7.0

57 . O

8.0

36 . O

6.0

M

) i

8.0

135 . O

CDt

De ,

cJ;

.

:

Cv> : , 1984 i', f i 1S 2 !J, f

; 2

JDh
t^

eC196CH:ULl 2 1

6.0

545 . O

6)

D,

> eC

67 . O89 . OeC )t C, 1979#it, 1.40, 1.83. 0.32. 0.20f**'J'I 1 :

‑UJ

1988

15 . O

20 . O

i

P

C )

:

C ! t;

Cv>

C

}

e;:{

I ;e , 1979 El:I : t : .

e 20 !J

20

ゆ

b1
詞！辱

1−
砂

O

3

0
1曳

5！︸﹄

融・gゴ

0．

！

t．

翔

ズ2髪切6
2 鰍る

〃
〃

・

グ

Kochi

Prefecture・。8Figure2

Prefectural

incidence of diplogonoporiasis．

N㎝bersofreportedcases are indicated on the map．

あったが，1980年以降は17例で比較的平均して患であるが，本来の終宿主と考えられるイワシクジ者が見出され，実数としては日本で現在最も多い

ラは北海道，三陸沖でカタクチイワシ，マイワシ，（図2）。

サンマ，サバ，オキアミ類を，コイワシクジラは

人口10万人当たりの発生数については，鈴木ら

日本海でカラヌス，オキアミ，イワシ，イカナゴが既に報告したが，1980年以降1989年までの10年を主要な餌としている（根本，1962：加茂，1969

間の本症例数を，患者数の多い県で改めて計算しより引用）。一方，イワシ，アジ，サバ，カツオ，

直すと（1985年の国勢調査の人口に基づく），長崎

ハマチなどが患者の食生活から指摘され，なかで県0．12，島根県0．25，静岡県0．48，鳥取県0．49，

も，イワシ類が当初から中問宿主として疑われ，

高知県3．10で，高知県は静岡県の6．5倍となり，高平井ら（1976），安羅岡ら（1989）および前嶋ら知県が圧倒的に多い。

（1990）はカタクチイワシの稚魚ではないかとしこのように高知県に本症が多いことは，中間宿ている。

主となる海産魚の回遊状）況と手に入れやすさ，そ高知県の症例からは，今回も魚を特定し得な

れらの魚の生食の多寡や，慣習などが関与していかったが，南西海区水産研究所によると，高知周ると考えられる。

辺で比較的冬期にも漁獲があり，生て喰べることが多く，本州北部には少ないが，もしくは見られ
4．高知県の本症の中間宿主についての検討

本症がIijima

ない海産魚として，ゴマサバ，ウルメイワシ，カandKurimoto（1984）によってタクチイワシ，キビナゴ，ブリ（ハマチ）などが報告されて以来，中間宿主，つまり感染源は不明

考えられる。このうち，キビナゴは高知県西部で

21

主として漁獲され，生で食べられる機会は多く，の高知県の感染源の決定については，今後の研究

愛媛県の4症例中，3例が四国西部の患者ではあるが，図1に示されるように，高知県西部では本に待ちたい。

症の患者は少ないことから疑問視される。

5．米子裂頭条虫症ほか

一方，高知県では，新鮮なイワシの稚魚をヌタ，

今回のアンケート調査により，高知県西部の病もしくは二杯酢で食べるドロメ料理による感染の院から1例の米子裂頭条虫が得られた。本条虫は可能性については，著者（鈴木ら，1985，1988）がYamane6∫磁（1981）および加茂ら（1982）に既に指摘している。この料理は季節により，イワょり，広節裂頭条虫とは異なる種として記載され，シの種類の混合比が異なる。最近，漁獲高が多く，四国からは平井ら（1988）によって愛媛県，およドロメの大半を占めているマイワシは，冬から翌

び高知県から既に見出されているが，今回の症例

年5月位までが最盛期であるが，北方でも漁獲さで，高知県から第2例が得られたことになり，大れ，北海道でも獲れることから，本症の北限を考

複殖門条虫症例と共に，高知県が海産魚を中間宿えると否定的である。ウルメイワシの稚魚は，高主とする寄生虫に感染する機会が多いことを示し

知では秋から翌年初夏にかけて採取され，カタクている。また，無鈎条虫が3例認めたことは，高チイワシは年間採取されるが，6月から秋にかけ

知県に本条虫症が存在することを示す。

て混合の割合が増加する。また，イワシは稚魚をドロメとして食べる以外にも，ウルメイワシ（秋要から冬）の成魚は高知県で生で食べる習慣がある

約が，マイワシ（夏から冬）とカタクチイワシの成高知県では既に21例の本症が報告されているが魚（冬から春）は生で食べない。ゴマサバ（秋か（鈴木，1988），今回12例が追加され，合計33例にら冬）やブリが，幼魚時代に自然感染して感染源達した。また，現在までの我が国の本症の報告をとなる推定，また，平井ら（1976）は自然感染し調べたところ，未発表を含めて162例であり，高知

たカタクチイワシを餌にすることによって，養殖

県は本症が極めて多く，特1に近年になって症例がブリが感染するという推定も否定できないが，餌増加していることを明らかにした。

のイワシは冷凍されている場合が多く，また，サバやプリが遠隔地への輸送が可能なことから，患謝者が海岸部以外にも発生しなければならない。一辞方，イワシの稚魚は生での輸送が困難なこともあアンケート調査に当たり，高知県医師会およびり，高知県での感染源としてはカタクチイワシや各医療機関から多大のご協力を得た。また，水産ウルメイワシの稚魚，なかでも後者が疑われる。

庁南西海区水産研究所古藤力技官，花岡藤雄技

そこで，1988−1989年に高知県沿岸て採取され官には魚類についての御教示を，高知県水産試験たカタクチイワシの稚魚19，868匹，マイワシの稚場山重政則海洋資源科長に，イワシの稚魚の御供

魚17，550匹，ウルメイワシの稚魚6，952匹を圧平与を得た。ここに深謝する。

して解剖顕微鏡下て筋肉，内臓などを検査したが，さらに，鳥取大寄生虫学教室前嶋條士博士に，

plerocercoidを検出し得なかった。しかし，1回

米子裂頭条虫の同定をして頂いたことを付記してに食べるドロメ料理の量は魚の大きさにもよるが，謝意を表する。

約200−300匹であり，この検査数では約220−150
回分にしか相当しないので，これによって寄生の有無を議論するには検査数が不足であり，本条虫

本論文の一部は，第45回日本寄生虫学会西日本
支部大会（1989）で発表した。

22

参考文献
1）赤尾信吉（1989）：最近経験した大複殖門条虫症について，第49回日本寄生虫学会東日本大会プログ
ラム要旨，292）平井和光，酒井雅博，阿波井五郎（1976）：大複殖門条虫について一愛媛県における大複殖門条虫寄生の第4例一，日本農村医誌．，25，599−603
3）平井和光，鳥居本美，鈴木了司，加茂 甫（1988）：四国における米子裂頭条虫，寄生虫誌．，37，13−19

4）板垣国昭，数田行雄，遠藤隆二，山根泰三，上田静雄（1988）：山口県における条虫感染症二例について，山口衛公害センター業績報告集，9，62−66
5）影井 昇，浅野和仁，山浦常雄，松本克彦，白坂龍噴（1990）：関東地区における複殖門条虫感染者の追加と患者の発生状況，第50回日本寄生虫学会東日本支部大会講演要旨，30
6）加茂 甫（1969）：大複殖門条虫に関する研究，寄生虫誌．，18，333−337
7）Kamo，H．，Hatsusika，R．andin

Yamane，Y．（1971）：Diplogonoporiasis

anddiplogonadic

cestodesJapan，Yonago Acta Med．，15，234−246

8）金子隆子，小林正規，竹内 勤，山根洋右，橘 裕司（1989）：福島県いわき市（小名浜地区）周辺に見られた海産魚類由来寄生虫の感染状況；特に胃アニサキス症28例と大複殖門条虫症1例について，第2回東日本寄生虫疾患臨床検討会講演
9）神崎雅樹，溝渕和久，井上和男，沖 勇一，矢野哲也，鈴木了司（1987）：山間部地域におけるアニ
サキス症，第40回高知県医師会医学会講演
10）前嶋條士，矢崎誠一，福本宗嗣，宮原道明（1990）：山陰および九州地方における大複殖門条虫症10
例，寄生虫誌．，39，198−203

11）鈴木了司，岡村宣典，熊沢秀雄，今村京子（1985）：高知県における大複殖門条虫症，寄生虫誌．，
34， 431−439

12）鈴木了司，今村京子，熊沢秀雄，岡村宣典，中川佳子（1988）：高知県の大複殖門条虫症8例の追加，
日熱医会誌．，16，285−291

13）高尾善則，福間利英，井出理恵（1990）：佐賀県における第1例目の大複殖門条虫症，第43回日本寄生虫学会南日本支部大会講演要旨，28
14）高田季久，宇仁茂彦，木俣 勲，井関基弘，塚野賢彦（1982）：大阪における大複殖門条虫症の一例，
寄生虫誌．，31（増），72

15）Terada，M．，Fujiu，Y．andSano，M．（1985）：E丘ectsofsomeneurophamacologicalagentsand
anthelmintics

on

the

motilityofmatureproglottids

of1万弼ogoηoρo捌s

g㎎％薦，Jpn．J．Parasit．，

34，517−51916）安羅岡一男，入江勇治，大前比呂思，西成田

真，小松義成，小泉昭男（1989）：茨城県における大複殖門条虫の第一例，第4回寄生虫疾患臨床検討会講演

23

ADDITIONAL 12 CASES OF DIPLOGONOPORIASISIN KOCHI PREFECTURE
NoRIJI SUZUKI AND KYOKO IMAMURA
Received October 19 19901Accepted November 22 1990

Twenty‑two cases of Diplogonoporus grandis infection have already been reported from
Kochi Prefecture, and in this report, additional 12 cases were described.
In contrast to other regions, Kochi Prefecture aparently exhibits a marked increase in theincidence of cases in recent years.

Since the first description of this tapeworm was reported by lijima and Kurimoto (1984)

in Nagasaki Prefecture, the 162 cases of human infection with the worm were recorded inJapan in the literatures until 1989.

Department of Parasitology, Kochi Medical School Oko cho Nankoku 783 Japan

Jpn J Trop Med Hyg Vol 19 No 1 1991, pp. 25‑31

25

ELECTRON MICROSCOPICAL OBSERVATIONSON GAMETOGENESIS AND FERTILIZATIONIN CULTURED PLASMODIUM FALCIPARUMTADASUKE ONO1, TOSHIO NAKABAYASHI' AND YOSHIHIRO OHNISHI*
Received November 6 1990/Accepted December 14 1990

Abstract: Electron microscopical studies were perfonned on gametogenesis andfertilization of the gametes developed in the prolonged culture of P. falciparum
gametocytes which appeared during the culture of the parasites in the gametocytogenesis‑

induction medium. When the erythrocyte plasma membrane separated from thegametocyte during gametogenesis, a small part of the gametocyte cytoplasm which wasenveloped with the multilayered sheaths of the erythrocyte plasma membrane, wasreleased from the gametocyte into culture medium. We could observe the process ofpenetration of the macrogamete cytoplasm by the microgamete and then the fusion of themembrane surrounding microgamete with the nuclear envelope of inacrogamete. From
the findings of two microgamete nuclei were present in the cytoplasm of a macrogamete
at fertilization, it is assumed that the multiple nuclear‑fusion may occur.

INTRODUCTION
The process of gametogenesis and fertilization of Plasmodium have been examined withelectron microscope (Sinden et al., 1976, 1978; Aikawa et al., 1984) . Sinden et al. (1976)
described the fusion of the plasma membrane of male and female gametes at fertilization in
P. yoelii. Aikawa et al. (1984) observed in P. gallinaceum that the male nucleus appeared totravel through a channel of the endoplasmic reticulum to a region of the female nucleus.
However, these workers did not observe the process of penetration of the macrogamete
cytoplasm by the microgamete and the subsequent nuclear fusion of these gametes. Since
Ono et al. (1986) introduced the gametocytogenesis induction method for P. falciparumstrains which seldom produce gametocytes in the routine culture, it has become easy to study

gametocytogenesis in vitro. Ono and Nakabayashi (1989) found that the gametocytes whichappeared in the gametocytogenesis‑induction medium developed subsequently to the oo‑
kinetes in the prolonged culture medium. In the present experiment, ultrastructural observa‑

tions were carried out on mature gametocytes and the gametes which developed in the
Department of Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita
Osaka 565, Japan
1 Present address; Department of Veterinary Epidemiology, College of Agriculture, Universityof Osaka Prefecture, Mozu‑Umemachi, Sakai, Osaka 591, Japan
2 Present address; Department of Parasitology, School of Medicine, Fujita‑Gakuen Health
Univer sity, Kutsukake‑Cho, Toyoake 470‑11, Japan26

prolonged culture of P. falciparum gametocytes. Studies were focussed upon (1) the releaseof a small part of the gametocytes cytoplasm into culture medium during gametogenesis, (2)the process of penetration of the macrogamete cytoplasm by the microgamete at fertilization,(3) the fusion of the membrane surrounding the microgamete nucleus with the outer mem‑brane of the macrogamete nucleus.

MATERIALS AND METHODSSpecies of Plasmodium: Falciparum‑Vietnam‑Oaknoll (FVO) strain of Plasmodium
falciparum was provided by courtesy of Dr. W.A. Siddiqui, Department of Tropical Medicine,
University of Hawaii in 1983. This strain has been used in the previous reports (Ono et al.

1986; Ono and Nakabayashi, 1989, 1990). Gametocytes are seldom produced in this culture
strain under normal culture conditions. The strain was cultured by the method of Siddiqui(1979) in 100‑ml Erlenmeyer flasks, but Falcon tissue culture dishes were used for the
prolonged cu.Iture of gametocytes which appeared after the gametocytogenesis‑induction.

Induction of gametocytogenesis: Gametocytogenesis‑induction medium (RPMI‑FSC
reactive medium) was used for induction of gametocytogenesis. This medium was preparedby the method described in our previous report (Ono and Nakabayashi, 1989). To put itbriefly, this medium consists of a mixture of 4 ml regular RPMI 1640 medium, 4 ml RPMI‑

FSC medium, and I ml horse serum. RPMI‑FSC medium was prepared by dissolving
powdered RPMI 1640 medium in the mixture of the culture supernatant of the anti‑P.
falciparum antibody producing hybridoma cells and the hybridoma cell lysate. The
hybridoma cells used in the present study were the same cell line as used in the previous
report (Ono and Nakabayashi, 1989) .
Prolonged culture of gametocytes: The method for the prolonged culture of gametocytes
was described previously (Ono and Nakabayashi, 1989) . On day 5 of culture in the regular

RPMI 1640 medium containing 10% horse serurn, the medium was replaced by a mixture of
9 ml RPMI‑FSC reactive medium containing lO% horse serum, 0.5 ml concanavalin A (200pg/ml) , and 0.2 ml caffeine (100 mM/ml) to induce gametocytogenesis. After culturing for

24 hr, the medium was replaced by Waymouth's MB 753/1 medium with para‑aminobenzoic
acid (final concentration, I pg/ml) containing 10% horse serum; thereafter the medium wasrenewed every 24 hr. The parasites, which had been maintained in 100‑ml Erlenmeyer flasks
until the day 3 of culture in Waymouth's medium, were transferred into 60‑mm Falcon tissue
culture dishes for candle jar culture and cultured for 10 days. The temperature was constant‑ly maintained at 37'C through the cultivation. The exfiagellating medium (Carter and Beach,1977) was not used in the present experiment.
Transmission electron microscopic observations: On day 13 of culture in Waymouth's
medium, the parasites were collected by centrifugation at 3,000 rpm for 10 min and thesediment was fixed at 4'C for I hr in 0.01 M phosphate buffer (pH 7.4) containing 2.5%
glutaraldehyde, washed for I hr with 0.01 M phosphate buffer (pH 7.4) containing 0.25 M
sucrose and postfixed at 4'C for I hr with 1.5% osmium tetroxide in isotonic buffer. Afterwashing by centrifugation, the pellet was stained with 1% uranyl acetate solution for I hr.
Then, the samples were dehydrated in absolute ethanol, treated with propylene oxide andembedded in epoxy resin. The sections were double stained with uranyl acetate and leadcitrate.

=={i;i "̲̲'';i i{* "" ; :" :*i^' '" ;"'"> ';'i'=;= =i: { {:="

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i ' ' ' * = '*'* *‑* *" ;}' ii;=* *'**'ii {=+ +*:1::":B:;:::1 i;: ;i::! ;;:;*i**=***'=: ; ; <=!: ::1 = i :: + fi:: :;"' :; :=i !"i'i"'=' ‑' ' "'"i#. . . *̲ ̲.̲

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* * +* +'+ ***:*' :='̲ ;;*" * i '"''=*+" = > '+*'*+ '+* " ' " ' ' ‑‑=+ ' ' " * *'= ' '='=* ‑ ='!('****"* '*; =+* '"";s" ':'

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S'; =' '=.;.'=*=**'‑.; ;'!';.;.',‑=.

*=;':;.=' * ; ‑i: ** ''===:==;=:'= =:' ii= "; ' '; ;{ =;'"= ;' ;'=*..".'.i. {;';"*‑ "';?

' 1ji;* * * '<*' f ;'*.'"**'* ';i{ ****: i== ^+*, j ***.f'* *j = *}iT tl" =',"‑{ ;' ' '{' : ';:i l j;; :' '‑=,{ :, ;'i

"

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'

i; ; =' ' ' *

* '' =i=:=: ;;=;:=;=i= ' '* ^' * ' ' ' ' *'‑ *'+ ;'+^'{= ii =" ' ' ‑:* ":i :'] :+' '*{.
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Figure IA shows same gametocyte during gametogenesis as Fig' IB but a different

section' The extended inner membrane (double arrow heads) of the gametocyte
plasma membrane d. ivides the cytoplasm into two parts of a protruding part and amajor part. The cytoplasm of Protruding part contains ribosomes, a large number ofsmall ve8iclcs' In Fig' IB' remnant of the inner membrane (arrow) and a few
pellicular microtubules (arrow head) are visible clearly beneath the outer membrane(double arrow) in the cytoplasm of a protruding part. epm: : erythrocyte piasma
membrane; pm :::pellicular microtubules' In gametocyte during gametogenesis shownin Fig' IC, the cytoplasm‑like structure (cls) which is enclosed by the disrupted
erythrocyie plasma membrane, contains the similar contents as seen in the protruding

part of gametocyte shown in Figs' IA and B' A small mitochondrion and many
pellicular microtubules (pm) are present in the cytoplasm of the protruding part,
MT :::mitochondriOn' Scale bar equals approx' O'5 pm in Fi s' IA' B' C'

28=･'.,. '' si*:,::.;*'+. :;<'=･;;*

i*::
i*,

;. , .

;*･*:'**':: ":+･,*,,

Figure ID is the same macrogamete as Fig. IE but a different section. A microgamete

is seen between the outer (opm) and the inner (ipm) plasma membrane of a ma‑
crogamete. Pellicular microtubules (pm) , partially dlsrupted inner membrane (arrow
in Fig. ID) , extensive endoplasmic reticulum (ER) and macular desmosomes (arrowhead in Fig. ID) are observed in a macrogamete. The microgamete has a joint
structure (double arrow in Fig. ID) . Fusion of a microgamete plasma membrane(mipm) and the inner membrane Of macrogamete plasma membrane (ipm) can beseen (arrow in Fig. IE) . In the vicinity of this part, two membranes are indistinct inpart (airow head in Fig. IE) . BS*bulbous swelling. In Fig. IF, two condensed

microgamete nuclei (MlN are observed near the nucleus of the macrogamete(MAN). The membrane enclosing a cigar‑shaped nucleus of the microgamete is
fused (arrow in Fig. IF) with the outer one of two disseciated membrane of themacrogamete nucleus. The disrupted inner membrane Of the macrogamete can beseen only in some places (double arrow in Fig, IF) , pm*pellicular microtubules.
Scale bar equals approx. O.5 ;im in Figs. ID and E; O.25 , m in Fig. IF.

29

RESULT
Figures IA and B are the same gametocyte during gametogenesis but in the different
sections. In Fig. IA, a gametocyte is surrounded with the disrupted plasma membrane of anerythrocyte visible as multilayered sheaths. The extended inner membrane of the gametocyte
plasma membrane divides the cytoplasm into two parts of a protruding part and a major part.
Pellicular microtubules are observed below the inner membrane surrounding the cytoplasmof the major part. In Fig. IC, a gametocyte during gametogenesis has a protruding part, as
in Figs. IA and B. The cytoplasm‑like structure which is enclosed by the disrupted eryth‑rocyte plasma membrane, contains many small vesicles. The contents of the structure aresimilar to it of the cytoplasm of the protruding part shown in Figs. IA and B. A Iarge
vacuolated mitochondrion which contains a few tubular crista, is present in the cytoplasm ofa major part. Figures ID and E are the same macrogamete but in the different sections. A
microgamete is seen between the outer and the inner plasma membrane of the macrogamete.

The microtubules remain beneath the pellicular membrane. But, the macrogamete is not
more enclosed in the erythrocyte plasma membrane and the inner membrane of the ma‑
crogamete is disrupted partially. A joint structure is observed in a microgamete. Macular

desmosomes are present between the membrane of a microgamete and the inner membraneof macrogamete plasma membrane. Fusion of these two membranes can be seen in theneighborhood of the bulbous swelling of microgamete. In the vicinity of a fused part, twomembranes are indistinct in a part. Figure IF shows the fusion of the membrane enclosing
the microgamete nucleus with the nuclear membrane of the macrogamete. In Fig. IF, twocondensed microgamete nuclei are observed in the cytoplasm of macrogamete. The ma‑
crogamete is surrounded by two layered plasma membranes, the outer membrane and thedisrupted inner membrane. The latter can be seen only in sbme places. But, a small numberof the pellicular microtubules still remain beneath the plasma membrane. The erythrocyte
plasma membrane is invisible in the neighborhood of the macrogamete.

DISCUSSION

When the erythrocyte plasma membrane separated from the gametocyte during
gametogenesis, a small part of the gametocyte cytoplasm which was enveloped with themultilayered sheaths of the erythrocyte plasma membrane, was released from the gametocyte
into culture medium. This phenomenon is unknown until now. We concerned about whether

the mature gametocytes developed in the midgut of the mosquitoes which engorged a P.
falciparum patient release a part of the cytoplasm as those developed in the in vitro culture

or not. Penetration of the macrogamete by the microgamete is so rapid that the ultrastructu‑

re of the microgamete just after penetration has not been observed until now. In the present

study, however, we could observed a microgamete between the outer and the inner plasma
membrane of the macrogamete. The microgamete is about to penetrate to the cytoplasm of

the macrogamete by fusion of the microgamete plasma membrane and inner membrane ofmacrogamete plasma membrane. Furthermore, we observed the fusion of the membranesurrounding the microgamete nucleus with the outer membrane of the macrogamete nucleus.
A joint structure (double arrow in Fig. ID) in a microgamete has not been observed until
now. This structure is regarded as a convenient structure to detach the nuclear part of30

microgamete which is essential for nuclear fusion.

The multiple fertilization has not been observed in Plasmodium. Nijhout and Carter(1978) found light microscopically in Plasmodium gallinaceum that after the fusion of a
microgamete and a macrogamete, entry of other microgametes into the same nacrogamete
was apparently prevented. But, Aikawa et al. (1984) could not find any evidence for thisprevention by electron microscopic observation of a fertilized macrogamete of P. gal‑linaceum. Electron microscopy in the present study revealed two microgamete nuclei
surrounded by the membrane in the cytoplasm of a fertilized macrogamete. It is interested
in the whether multiple fusion occurs or not.

REFERENCESl ) Aikawa, M., Carter, R., Ito, Y. and Nijhout, M.M. (1984) : New observations on gametogenesis,

fertilization and zygote transformation in Plasmodium gallinaceum. J. Protozool., 31, 403‑413
2 ) Carter, R. and Beach, R.F. (1977): Gametogenesis in culture by gametocytes of Plasmodium
falciparum, Nature, 270, 240‑2413 ) Nijhout M.M. and Carter R. (1978): Gamete development in malaria parasites. Bicarbonate‑
dependent stimulation by pH in vitro. Parasitology, 76, 39‑53
4 ) Ono, T., Nakai, T. and Nakabayashi, T. (1986): Induction of gametocytogenesis in Plas‑
modium falciparum by the culture supernatant of hybridoma cells producing anti‑P. falciparum
antibody, Biken J., 29, 77‑81

5 ) Ono, T. and Nakabayashi, T. (1989): Gametocytogenesis induction in cultured Plasmodium
falciparum and further development of the gametocytes to ookinetes in prolonged culture,
Parasitol. Res., 75, 189‑193

6 ) Ono, T. and Nakabayashi, T. (1990): Gametocytogenesis induction by ammonium compounds
in cultured Plasmodium falciparum. Intemat. J. Parasitol., 20, 615‑618

7 ) Siddiqui, W.A. (1979): Continuous in vitro cultivation of Plasmodium falciparum in humanerythrocytes: Description of a single technique to obtain high yields of parasites. In: Pract.
Tissue Culture Applic. (Edited by Marmorch, K. and Hirumi, H.) , pp. 267‑277 Academic Press
Inc.

8 ) Sinden, R.E., Canning, E.U. and Spain, B. (1976): Gametogenesis and fertilization in Plas‑
modium yoelii nigeriensis: a transmission electron microscope study, Proc. Roy. Soc. London
B, 193, 55‑76

9 ) Sinden, R.E., Canning, E.U., Bray, R.S. and Smalley, M.E. (1978): Gametocyte and gamete
development in Plasmodium falciparum. Proc. Roy. Soc. London B, 201, 375‑399

31

培養熱帯熱マラリア原虫における生殖体形成
および受精に対する電子顕微鏡的観察
小野 忠相1・中林敏夫2・大西義博1

熱帯熱マラリア原虫赤内型の動∂伽o培養に際し，これに抗熱帯熱マラリア原虫抗体産生ハイ
ブリドーマ細胞の上清液を加えて誘発した生殖母体を長期培養すると，生殖体形成，受精，融合
体形成，虫様体形成と発育が進む。この論文では生殖体形成中の生殖母体，雄性生殖母体による雌性生殖体侵入，更に雌雄生殖体による受精を，電子顕微鏡によって観察した。その結果，次の所見が得られた。

1．生殖母体の2枚の細胞質膜の中，内膜が伸展して生殖母体の細胞質の一部が分割される。そして，この部分は生殖母体を包んでいた赤血球膜が生殖母体から剥れる時，赤血球膜に包まれて虫体から培地中に放出された。

2．雄性生殖体は雌性生殖体侵入に際して，まず雌性生殖体細胞質外膜下に入り，その後雄性生殖体膜と雌性生殖体細胞質内膜の融合によって，細胞質に入るように思われた。両膜の間に
maculardesmosomeが見られた。雄性生殖体は，雌性生殖体に入ると核部分がはずれるが，それを容易にすると思われる構造が見られた。

3．雄性生殖体の核を包む膜と，雌性生殖体の核膜の融合，すなわち受精を形態的に初めて観察することが出来た。雌性生殖体細胞質に核が2つ認められ塾．多受精の可能性が示唆された。

大阪大学微生物病研究所原虫学部門
1 現所属；大阪府立大学農学部獣医疫学講座（大阪府堺市百舌鳥梅町）
2 現所属；藤田学園保健衛生大学医学部寄生虫学講座（愛知県豊明市沓掛町）

Jpn. J. Trop. Med. Hyg., Vol. 19 No. I 1991, pp. 33‑38

33

DEFECT OF POST‑TRIGGERlNG NATURAL KILLER
CELL MEDIATED CYTOLYTIC ACTIVITYBY SYNTHETIC HIV ANALOGUE PEPTIDESTATSUO KlYOHARAl, KEN‑ICHlRO MURATAl, TAIICHIRO MORIYAl,
MASAMI WATANABEl, SHUICHI IKEDA2, MICHITO ICHIMARU2
AND HIDEYO ITAKURA1
Received December 1 1990/Accepted January 10 1991

Abstract: Three kinds of peptides termed ABJgl7, ABJ918 and ABJ919 were synthes‑ized and tested their capability of inhibition of Natural Killer (NK) cell mediatedcytotoxicity. ABJ917, which is conserved among various retroviral transmem raneenvelope proteins inhibited NK activities of the peripheral blood lyinphocytes (PBLs) of
both asymptomatic carriers and normal controls. ABJ918 or ABJ919 which correspond to
this conserved region also inhibited NK activities. To know the mechanism of inhibitionof NK cells by these synthetic peptides, conjugate formation assay and triggering assays
were then performed. ABJ917 which inhibited overall NK cytotoxicity did inhibit neither

NK and target tumor cell binding nor NK cell triggering by target K562 tumor cells.
These results show that the inhibition of NK cytotoxic activity by synthetic HIV peptide

is caused in the stage of post‑triggering. Peripheral blood CD4+ cell rate of theasymptomatic carriers of HIV was almost O% when we obtained enough NK activities andthese carriers were still quite healthy. Taken together, th immunosuppression of AIDS
patients is thought to be caused at least partly from some defects of post‑triggering lytic

activity of NK cells by HIV transmembrane peptides.

INTRODUCTION
The hallmark of AIDS is thought to be a selective depletion of CD4 positive helper/
inducer lymphocytes (Fauci, 1988) . But, as far as from our knowledge and experiences,
asymptomatic carriers whose CD4 positive lymphocytes are almost O% in the PBLS are still
healthy with no symptoms untill the onset of opportunistic infections. After disease manifes‑

tation, NK cytolytic activities are. known to be inhibited as other immunological functions(Rook et al., 1983; Wong‑Staal and Gallo, 1985i . 'Therefore the NK cells are thought to play some important roles for initiating disease
manifestation of AIDS. On the other hand, the retroviral transmembrane envelope protein
pl5E and its synthetic analogue peptides are reported to inhibit NK cytotoxicity (Cianciolo
et al., 1985; Harris et al., 1987). And the mechanism 'of this NK suppression was the1

Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 12 ‑ 4Sakamoto‑machi, Nagasaki 852, Japan2

Department of Hematology, Atomic Bomb Research Institute, Naga, saki University School of
Medicine, 12‑4 Sakamoto‑machi, Nagasaki 852, Japan34

inhibition of post‑effector target binding. Here, we tried to analyze further‑i.e. if synthetic

HIV peptide inhibit NK cell triggering or not.

MATERIALS AND METHODS
Effector cells: For the NK cell source, PBLS from HIV asymptomatic carriers or normal
volunteers were prepared after Ficoll Hypaque centrifugation and depletion of plastic dishadherent cells for 30 min at 37'C from heparinized blood.
Target cells: A human myelogenous leukemia cell line K562 was maintained in 5% Fetal

Bovine Serum containing RPM11640 medium and was used as a target tumor cell.
Cytolytic assay: Cytotoxicity assays were performed in a standard 4‑hr 5*Cr‑releaseassay as previously described (Pross'and Baines, 1981) . The following equation was used to

calculate cytotoxicity: % specific lysis=[(cpm test‑cpm medium)/(cpm max ‑cpm
medium) I x 100. The cpm max was determined by counting an aliquot of resuspended target
cells. The cpm medium was determined in wells counting targets only with no effectors
added. Lytic units (LU) were calculated from cytotoxic titration curves; I LU was definedas the number of effector cells required to cause 20% Iysis of 5 x 10' targets.

Binding assay: Effector PBLS were mixed with target K562 cells at a 1:1 ratio as
described previously (Roder and Kiessling, 1987). The cell suspension was centrifuged at
200 X g for 5 min at room temperature, foliowed by incubation on ice for 30 min. The pellet
was gently resuspended and 200 Iymphocytes were counted in triplicate samples.
Triggering assay: To detect the triggering events, phospholipid methylation assay(Hirata et al., 1979) was employed. PBLS (2.5xlO* cells) with or without target K562 cells(5xlO' cells) were incubated in a total volume of 200 pl with 20 pCi of L‑[methyl‑'H]

methionine (87 ci/nmol, I Ci=3.7 x 10‑" MBq, Amersham, Amersham, UK) in 1.5 ml Eppen
dorf tubes. After I hr of incubation at 37'C, the cells were washed once with cold phosphate‑buffered saline (PBS) containing 5 mM L‑methionine (Sigma Chemical Co., St. Louis, MO)
and twice with cold PBS to stop the reaction. The lipids in the cell pellet were extracted
overnight with 750 pl of cold chloroform/methanol (v/v=2/1). After centrifugation at
27,000 X g for 15 min, the chloroform/methanol extract was transferred to borosilicate tubes(Corning Lab. Sci. Comp., Corning, New York) and evaporated to dryness in a Rotavapor RE

120 (Buchi, Toronto, Canada). Dried samples were dissolved in 100 pl of chloroform/
methanol (v/v = 2/1) and aliquots (50 pl) were chromatographed by thin‑layer chromatogra‑phy (TLC) on 20 X 20 cm silica gel G plates (Analtec Inc., Newark, Denver) , using a solvent
system of chloroform/propionic acid/n‑propyl alcohol/distilled water, 2/2/3/1 (v/v) . The

front migrated approximately 15 cm. After drying and staining with iodine (Sigma),
phospholipid spots were scraped and the radioactivity was measured in a liquid scintilation

counter (Beckman LS 7500, Beckman, Irvine. CA).
Synthetic peptides: Three kinds of peptides composed by 17 amino acids were prepared.
ABJ917 is homologous to retroviral transmembrane glycoprotein pl5E (correspond to gp41
for HIV) . ABJ918 and ABJ919 peptides are homologous to ABJ917 but precise structures areminimally changed.

35

Table 1 Amino acid sequence of the synthetic peptides

HIV (gp41)(AB J917)

Leu‑Gln‑Ala‑Arg‑Ile‑Leu‑Ala‑Val‑Glu‑
Arg‑Tyr‑Leu‑Lys‑Asp‑Gln‑Gln‑Leu

CKS17(AB J918)

Leu‑Gln‑Asn‑Arg‑Arg‑Gly‑Leu‑As p‑Leu‑
Leu‑Phe‑Leu‑Lys‑Glu‑Gly‑Gly‑Leu(AB J919)

Leu‑Gln‑Asn‑Arg‑Arg‑Gly‑Leu‑As p‑Leu‑
Arg‑Tyr‑Leu‑Lys‑Asp‑Gln‑Gln‑Leu

Table 2 Inhibition of NK activities of a normal volunteer during incubation
without
NK activities(% inhibition)

1 , 280t+ AB J917 *+ABJ918*+ AB J919 *853

1 , 067

1 , OOO(33 . 4)( 16 . 7)(21 . 9)* Concentration of the synthetic peptides were 5 pM
t Lytic Unit

70
(1333 L. U.)

60

50

l

40

y::f::̲,u

'

30

(1)

20

ABJ918 (211 L. U.):
84.2% InhibitionlOo

5 1 O 20 40
Effector/Target cell Ratio

Figure 1

NK activities of the peripheral blood lymphocytes of an AIDS

asymptomatic carrier before and after adding a synthetic
peptide ABJgl8.
Closed circle shows NK activity (1333 Lytic Unit) against =*Crlabelled target K562 cells before adding ABJ918. Open circle

shows NK activity (211 Lytic Unit) after adding the 918
peptide. 84.2% of NK activity was inhibited by 918 peptide.

36

Target tumor cell
NK celll Binding
Target structure

Recognitionrece ptor

2 Triggering

Triggering
receptor

Triggering molecle

3 Prograrmting forlysis (Lethal hit)

PI CH 3

Acceptor siteo 4 Killer cell
independent,. ‑L‑ ‑ . oo c:)oo olysis

Perforin or NKCF

5 Recycling

Figure 2

Schematic representation of NK cell mediated cytolytic process.
First, NK cell recognizes and binds with target tumor cells. Then, somesaccharides structure (We call it triggering structure) triggers NK cell
metabolic processes (Kiyohara et al.. 1985). Some lytic molecules arethought to be released from NK cells followed by formation of tubular
structure (poly perforin) between NK and target cell and two different

kinds of pores are produced on the target cell membrane. Killer cell
independent lysis is the stage of target cell death accompaning DNA
fragmentation (Apoptotic death) . After target cell death, NK cell detachfrom the target and recycles to another targets.

Table 3 Percent conjugate formationof NK cells and target K562

Table 4 The effect of a synthetic peptide 917

on the phospholipid methylation(triggering) of NK cells with K562

cells

target cells% conjugate formation
without peptide

42,Id:3.0

with 917 peptide

42.8 3.1

Cell mixture

Concentration of the synthetic peptide

917 was 5 pM.

'H‑methyl incorporation into
phospholipid fractions (cpm)

NK aloneO

N K + K562

6 871

NK+K562+917

7 020

RESULTS AND DISCUSSION
Inhibition of NK activities of HIV asymptomatic carriers: As mentioned before, Harris
et al. (1987) showed that NK activity of the normal volunteer was inhibited by the synthetic
peptides. But, if the peptide block NK activity of the AIDS asymptomatic carrier or not has
not been reported yet. So, we decided to see if our synthetic peptides inhibit NK activity of

the carriers. We added 918 peptide (Table D to the NK assay system at the concentration

37

of 5 pg (Fig. 1) . This 17 mer peptide inhibited NK activity of an AIDS asymptomatic carrier

as much as 84%. This is surprisingly high value.
Inhibition of NK activities of a normal volunteer: Next, we tested if these peptides could

inhibit NK activities of normal person instead of asymptomatic carrier. As shown in Table
2, ABJ917 as well as ABJ918 and ABJ919 inhibited normal NK activities. The difference ofthe magnitude of inhibition between normal persons and asymptomatic carriers may suggest
some tendency or difference of sensitivity is present.

The effect of 917 peptide to NK effector target conjugate formation: NK cell mediatedcytotoxicity is known to proceed via several discrete stages including: 1. effector‑target cellrecognition and binding, 2. triggering and activation of the NK effector cells, 3. release andbinding of the lytic factor to acceptor sites on the tumor cell surface, 4. target cell death, and5. effector cell recycling to another target cell (Fig. 2) . To know which stage of these k;lling

process is involved in peptide mediated inhibition phenomenon, we performed Roder's conju‑
gate formation (binding) assay. As shown in Table 3, conjugate formation between NK cells
and target K562 cells was not inhibited at all. Therefore, the synthetic peptide seemed toblock after binding stages.
The effect of 917 peptide to NK effector cell triggering: We defined the triggering stageof NK cell cytotoxic reaction as the stage of some biochemical reactions including membrane
phospholipid methylation coupled to phosphatidyl inositol turnover (Kiyohara et al., 1985) .

So, we examined if the peptide can block phospholipid methylation (i,e. triggering) of NK
cells. As shown in Table 4, 917 peptide did not infiuence NK cell triggering by K562 target
tumor cells. Therefore, 917 peptide block NK cytotoxicity after triggering. Taken together,
the stage of action of the synthetic peptide seemed to be after binding and after triggering.

As the lytic molecules are not determined (there are some evidences that tumor necrosis
factors play some roles in NK cytotoxicity and some people have reported the importance ofthe perforin or esterases in NK cytotoxic reaction) actual site of these synthetic peptide
should be clarified in future.

ACKNOWLEDGEMENT
The authors would like to express thanks to all the members of the Department of
Pathology and Dr. Akiyoshi Utsunomiya, the Department of Bacteriology, Institute ofTropical Medicine, Nagasaki University for their valuable criticism and encouragement.

REFERENCES
1 ) Cianciolo, G.J., Copeland, T.D., Oroszlan, S. and Snyderman, R. (1985): Inhibition oflymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins,
Science, 230, 453‑455

2 ) Fauci, A.S. (1988): The human immunodeficiency virus; infectivity and the mechanism ofpathogenesis, Science, 239, 617‑622
3 ) Harris, D.T., Cianciolo, G.J., Snyderman, R., Argov, S. and Koren, H.S. (1987) :' Inhibition ofhuman Natural Killer cell activity by a synthetic peptide homologous to a conserved region in
the retroviral protein, pl5E, J. Immunol., 138, 889‑894
4 ) Hirata, F.W., Strittmatter, J. and Axelrod, J. (1979) : P‑Adrenergic receptor agonists increase

38

phospholipid

methylation，membrane

fluidityandβ一adrenergic receptor−adenylate cyclase

coupling，Proc．NatL Acad．Sci．USA．，76，368−372
5）Kiyohara，T．，Dennis，J．W．，Boegman，R．J．and Roder，J．C．（1985）：Anexoglycosidasesensitive
triggering site on NK cells

which

is cbupledontransmethylationofmembranephospholipids，J，ImmunoL，135，659−664
6）Pross，H．F．and

parameters

Baines，M．G．（1982）：Studies

affecting7）Roder，J．C．andthecytotoxic

Kiessling，R．（1978）：system．1、Covarianceinnormalandgeneticofhuman

NaturalKillercells．1．乃￠吻o−fmction，lnt．J．Cancer，29，383−390
Target−effector

controlofcytolytic

interaction

and

in

the

Naturaltarget−cel1−binding

Killercell

subpopulations

mouse，Scand．J．ImmunoL，8，135−144

8）Rook，A．H．，Masur，H．，Lane，H．C．，Frederick，W．，Kasahara，T．，Macher，AM．，Djeu，」．Y．，

Manischewitz，J．F．，Jackson，L．，Fauci，A．S．and Quiman，G．V．Jr．（1983）：Interleukin−2
the depressed Natural Killer and Cytomegalovirus−specinc cytotoxic activities ofenhances

lymphocytesfrompatients

with

the

acquired

immunedeficiency

syndrome，J．Clin．Invest．，72，

398−403
9）Wong−Staal，F．and

Gallo，R．C．（1985）：HumanT−lymphotropic

retroviruses，Nature，317，

395−403

HIVgp41の構成ペプチドおよびその類似合成ペプチドによる
ヒト末梢血NK活性の抑制機構
清原龍夫1・村田建一郎1・守家泰一郎1・渡辺 正実1・
池田 柊一2・市丸 道人2・板倉 英音1

AIDS患者末梢血NK活性が抑制されている事が知られているが，このNK活性の抑制機構を解析した。無症候性キャリアのNK活性は抑制を受けておらず，その時のキャリアの末梢血のCD4＋細胞は0％であった。つぎに，HIVの外殼糖蛋白質gp41の一部で，レトロウイルスに保存されている17個のアミノ酸から成るペプチドと，その類似構造をしたペプチドを3種合成した。

これらの合成ペプチドは，正常コントロールのNK活性も，無症候性キャリアのNK活性も共に抑制したが，キャリアに対する抑制が著しかった。正常コントロールNK活性に対する合成ペプ
チドの作用段階は，NK細胞と標的癌細胞の結合にも，標的細胞によるNK細胞のTriggeringにも合成ペプチドが影響を与えないことから，Triggering以後の段階であると考えられた。一般には，AIDSの発症には，HIVがCD4＋細胞に感染し，その機能が損われる事が重要だと考えられており，多くの研究が為されているが，以上の結果より，無症候性キャリアが発症するに至るプロ
セスには，それ以外のNK等の免疫機能の抑制が何らかの役割を果たしていると考えられた。

長崎大学熱帯医学研究所病理学部門
長崎大学医学部原爆後障害研究施設内科

Jpn J Trop Med Hyg Vol 19 No. 1 1991, pp. 39 47

39

AN ETIOLOGICAL STUDY OF･ BACTERIAL
DIARRHEA AMONG INFANTS
IN PARAGUAY IN 1990
YOSHIO ICHINOSEl, CESAR MONGES A.2, MARIA G. CENTURION2,
DORITA MEDlNA2, ROSE M. ALBORN02, JUAN J. BESTARD3,

MAKOTO SAKAMOT04 AND RICARDO MORENO A.2
Received December 5 1990/Accepted January 10 1991

Abstract: An etiological study of bacterial diarrhea in infants under five years of age

in Paraguay from May to July of 1990 was done. Enteropathogenic E. coli (EPEO strainsof known serogroups were isolated from 35.8% of the patients with diarrhea, Salmonella
spp, were isolated from 15.1%, enterotoxigenic E, coli (ETEO strains were isolated from
5.7% , and Shigella and Campylobacter spp. were isolated from 3.8% respectivery. No
enteroinvasive E, coli (EIEO or Vibrio spp. was isolated. Out of 53 diarrheal patients, 34

cases (64.1%) had bacterial diarrheal diseases. The infants under one year of age withdiarrhea were predominant among inpatient and outpatient children under five years ofage and the total number of male diarrheal cases was greater than that of female cases.
Enteropathogenic organisms were isolated from nine out of 12 water samples taken fromthe Paraguay River. The major serotype (018) of the EPEC isolated from the water
samples of the Paraguay River corresponded to the major serotype isolated from patientswith diarrhea. = 'INTRODUCTION

Bacterial diarrheal disease is still one of the main public health problems with highmorbidity and mortality rates, especially in the tropics and subtropics (Merson, 1982).
It is reported that diarrheal diseases accounted for 8.2% of the infant motality rate in

Paraguay in 1989, according to a report by the Ministry of Health, however the etiology ofdiarrhea as well as its prevalence is still unknown. Surveys and studies conducted so far on

diarrhea in Paraguay have been confined mainly to traditional enteropathogens such as
Salmonella and Shigella. The first four cases of diarrhea due to Campylobacter jejuni in
Paraguay were reported by Achucarro et al. (1989) and this was the first report on
1 Department of Bacteriology, Institute Of Tropical Medicine, Nagasaki University, 12‑4

Sakamoto‑machi, Nagasaki 852, Japan
2 Instituto de Investigaciones en Ciencias

de la Salud, Rio de la Plata y Lagerenza C. Correo

2511, Asunci6n, Paraguay
3 Hospital de Clinicas, Catedra de Pediatria, Universidad de Asunci6n, Rio de la Plata y
Lagerenza C. Correo 2511, Asunci6n, Paraguay
4 Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, 12 ‑ 4Sakamoto‑machi, Nagasaki 852, Japan
This study was performed as part of Paraguay/Japan medical coop ration project on Chagas'
disease supported by Japan International Cooperation Agency (JICA) .

40

campylobacter gastroenteritis in Paraguay. However, an etiological investigation including
one concerning gastroenteritis due to E. coli. Campylobacter and Vibrio sp. has not been done.
In this report we tried to clarify the present status of diarrhea, especially the etiologicalagents of bacterial diarrheal diseases among children under five years of age in Paraguay. In
order to treat and control diarrheal diseases, we also investigated the water of the Paraguay
River which is closely associated with the life of the people. In addition, the drinking water

was studied bacteriologically to clarify the degree of enteropathogen contamination resulting

in diarrhea, because diarrheal diseases are recognized to be a fecal‑oral infection, so thatdrinking water and food are considered major sources of infection.

MATERIALS AND METHODS
Rectal swabs (Carrymate media, Ono Pharmaceutical Co. Ltd., Japan) were taken fromchildren under five years of age having diarrhea who visited or were admitted to the
Department of Pediatrics, at the School of Medicine of the Asunci6n University and
LACIMET Hospital from May to July of 1990 and were processed bacteriologically in the
Bacteriology Section of the Instituto de Investigaciones en Ciencias de la Salud. The rectal

swabs were inoculated directly on TCBS, SS, EMB and Skirrow media and incubate at
37'C overnight.

The specimens were, at the same tim , subcultured in alkaline peptone water (pH 8.5)
and selenite broth. After being incubated at 37'C overnight, three loopfuls of bacterial culture

were inoculated on TCBS and SS media. A suspicious colony from each plate was examinedwith the following, media for identification, Kligler iron, SIM, Voges‑Proskauer (VP) , Iysine,

ornithine and Simmons citrate, and was simultaneously checked for the level of citochromeoxidase. Ten colonies of E. coli were inoculated on BTB media to check purity and threecolonies each were checked for setotyping with diagnostic antisera for enteropathogenic E.

coli (EPEO (Denka Seiken, Japan) when E. coli was predominant on the EMB media. The
serotyping for Shigella and Salmonella was done with diagnostic antisera (Denka Seiken,
Japan) .

Skirrow agar medium for Campylobabter containing 5% horse blood, trimethoprim(5 mg/ml) , vancomycin (10 mg/ml) and polymyxin B (2.5 p/ml) was incubated at 37'C for
3 days or 42'C for 2 days in a microaerobic atmosphere containing 5‑10% oxygen generated

by a CampyPak (BBL Microbiology System, Cockeysville. Maryland). The minimum
criteria used for presumptive identification of C. jejunilcoli were: Gram ‑ negative, spiral‑shaped, motile, oxidase positive and growth at 42'C but not at 25'C. The sodium chloride
tolerance test was also employed for further confirmation and classification of Vibrio sp.

Thousand milliliter samples of water were collected in sterile bottles from 12 water
points in the Paraguay River and from nine water points for drinking in two areas in
Asunci6n (Fig. 4).

Each water sample was concentrated to 10 ml with a milipore filter and 3 ml each was
subcultured in double strengthened alkaline peptone water (pH 8.5) , peptone water (pH 7.2)
and selenite broth at 37'C overnight. A portion of the concentrated water sample was also

inoculated on EMB media and incubated at 37'C overnight. The filter paper used forconcentration was placed onto SS media and incubated at 37'C overnight. The bacterial
culture in alkaline peptone water was streaked on TCBS and that in the peptone water and41

selenite broth was both streaked on SS media.
A suspicious colony from each plate was examined with identification media after being
incubated at 37'C overnight. To test the sensitivity against several antibiotics, a single disk

method (Bauer et al.. 1966) was mployed and the results were reported to the pediatricward.
The production of LT and ST was checked by GM1‑ELISA and suckling mouse assay,
respectively, as previouly described (Honda et al.. 1984; Takeda et al.. 1979).

RESULTS
The isolation rate of different organisms and the percentage of all thedetected agents areshown in Fig. 1. From 53 diarrheal patients, 34 cases were recognized to be bacterial
diarrheal diseases. The most dominant bacterial pathogen was enteropathogenic E. coli
( EPEO with 19 isolates ( 35.8% ) which as followed by Salmonella with eight isolates(15.1%). No S. t iphi or S. pqrat phi‑A was isolated. Serotyping of the Salmonella could not

be done thoroughly because there were not enough serotyping kits for diagnosis when they
were isolated. Out of eight isolates identified biochemically as Salmonella, four isolates were

serotyped, two strains were serogroup 04, and one strain each was serogroup 013 and 035.
However, the remaining four isolates were not adequately serotyped because of contamina‑tion during stock. Three strains of enterotoxigenic E. coli (ETEO were, isolated. Heatlabile enterotoxin (LT) was not detected by GM*‑ELISA from all the strains serotyped as
EPEC or ETEC. Two EPEC strains (FA ratio: 0.141, 0.85) and one strain serotyped as ETEC
(FA ratio: 0.096) were revealed to be positive in the suckling mouse assay. Two strains eachof Shigella flexneri 2a ahd Campylobacter jejeenilcoli were isolated. Three mixed infection
cases were found, the included; EPEC O1 and 18; EPEC 086a nd 0157; Salmonella serogroup

20

35 POSITIVE CASES OLrr OF 53 PATIENTS

35.8%

W I TH1

DIARRhEA

(/)

<O1
5

ZO

15. 1%

5 7% 3.8% 3.8%
O

O% O%

EPEC Salmonel ETEC Shjgella canpylQ EIEC VibriOS

Figure l

Pathogenic bacterial agents isolated from diarrheal patients under five

years of age obtained between May and July of 1990.

42

30

I MaleE I Female

2

<o
:
c;

'1

o ....,
i:. . :4;;0

R' 4‑1

A" ' ' IY<

TO :1 ' 'AGE
Figure 2

Age and sex distribution of 34 children with diarrhea.

cl)

!

o
z
1

j

i
i

a

1

SEROTYPE

Figure 3 DistribLtion of Serotyping of enteropathOgenic E' coli iSolated f rom
children with diarrhea'

43

0.4 and EPEC 086a, and the last case was thought to be a S lmonella infection (Fig. 1). No
enteroinvasive E. coli or Vibrio sp. was isolated. A total of 41.5% of the diarrhea cases was,thus, considered to be due to E. coli. The infants under one year of age with diarrhea werepredominant among inpatient and outpatient children under five years of age and the total
number of male diarrheal cases was greater than that of female cases (Fig. 2) .

Eleven kinds of EPEC serotypes were isolated and 018 in the EPEC isolates werepredominant (Fig. 3). Twelve water samples from the Paraguay River were taken at theEcl u a tor'bparaguSOU'TH AMERICA

b

rL

e

7 83 10

1

5e ll

13 1

1

GL) P¥
W:r:u : 'VIL'

6ee 14 5 I
2 1 1 CS

J .! ;

A
PG

,.
11
12
e
1617

89

ASUNCl6No*Figure 4

A map of Asunci6n, Paraguay. The pumbers represent the points
where the water samples (e) and drinking water (1) were obtained.
PG: The Palace of the President of Paraguay, IICS: Instituto deInvestigaciones en Ciencias de la Salud.

44

Table 1 The enteropathogens isolated from water of the Paraguai
River

EPEC (Oi8, 044)

Point

EPEC (018, 026, 0142)
Salmonella (serogroup 013)
Point 6 Salmonella (s6rogroup 021) , EPEC (018, 0146)
Point 7 Salmonella (ND)
Point 8 EIEC (028a,c)
Point
Point

5

Point

9

Point 10

Point 12

ND:

Salmonella (serogroups O1, 3, 19) , Vib rio mimicus

Salmonella (ND)
EPEC (018, 0128)

The isolates were identified biochemically but serotyping of
the isolates was not done.

points shown in Fig. 4. These points were selected as they were near to the areas where
people in relatively low‑socioeconomic conditions live. Enteropathogenic organisms wereisolated from nine points out of the 12 points. Two or three EPECS Were isolated from four
points and the serotype 018 was isolated from all four points. Salmonella was isolated fromfive points.

Each sirain of EIEC and Vibrio mimicus was isolated. Enteropathogenic organisms wereisolated from all the points where people in low‑socioeconomic conditions live. The major
serotype of the EPEC 'isolated from the water sampleis of the Paraguay River corresponded
to the major setotype isolated from the patients with diarrhea (Table 1) .
In the analysis of the drinking water, enterobacteria such as Salmonella. Enterobacter andProteus spp. were isolated from three points out of nine points tested. No enteropathogens

were isolated from the tap water.
In the drug sensitivity test, six or seven kinds of discs containing ampicillin, cephalor‑

idine, cefotaxime, ceftazidime, gentamicin, amikacin, chloramphenicol and trimethoprim
were used. Four strains of EPEC showed resistant against three or four kinds of antibiotics
including ampicillin and cephaloridine and the other pathogenic E. coli strains were relatively
sensitive. One strain of Salmonella was resistant against five kinds of antibiotics including
ampicillin and cephaloridine and the other strains were relatively sensitive to all the antibi‑

otics except for ampicillin and cephaloridine. One strain of Shigella was resistant against
aminoglycosides and chloramphenicol but the other was sensitive to all the antibiotics except

for ampicillin. Two strains of Campylobacter were sensitive against aminoglycosides andtetracycline but resistant to all the other antibiotics.

DISCUSSION
This etiological study of bacterial diarrhea was performed over a short period of time
and the instruments and the materials for making diagnosis were not adequate enough that
we could identify the organisms in detail and thoroughly analyze characteristics such as toxin

productivity. However, additional investigations over an extended period of time will
provide more meaningful results.

45

The followin findings are from the results of this study. Firstly, more than 60% of thediarrheal patients were considered to have bacterial diarrhea, although this, study was
conducted during the dry and cold season. Since the rotavirus is also an important organislTlcausing diarrhea among children during the dry and cold season as previously reported(Koopman et al.. 1984; Br ndt et al.. 1982; Brandt et al.. 1983) , it is necessary to investigate

viral enteropathogens as well as ba6terial enteropathogens. Secondly, the dominant bacter'ial

pathogen that caused diarrhea among inpatients and outpatients was EPEC. It is reported
that EPEC is the leading cause of diarrhea in black South African children and that diarrhea
in white children is largely attributable to rotavirus (Robins‑Browne, 1984) . Of course, it isdif :cult to consider it to be the overall infection rate of EPEC, because the diarrheal patients

who came to the Asunci6n University Hospital arid LActMET Hospital were severe casesand diarrheal patients in low‑socioeconomic areas who do not visit a ho pital unless theircondition becomes severe. Thirdly, the isolation rate of Campylo,bacter jejunilcoli was 3.8%
in this study, which is almost the same as that in other countries '(Chyou et al.. 1988; Tanget al., 1984).

Shimotori et al. '(1986) reported that the overall isolation rate of Campylobacter jejuni

from children with diarrhea in Kenya was 12.6% but in the cold and dry season it Was 6.3% .

It is considered that the infection rate of C. jejunilcoli in Paraguay is within the ranges

reported earlier for developing countries. Forthly, the EPEC serotype of the enteropath‑ogenic organisms isolated from nine out of 'l2 water points in the Paragtiay River correspond‑

ed to that of the diarrheal patients. The outbreak of diarrheal diseases seems to be closely

related to the usage of contaminated water from the Paraguay River. The water level of theParaguay R'iver was elevated because of rainfall during the spring, so a fairly large numberof people who lived on the banks of the river had found refuge on a hill overlooking the river.

The people had to build their houses tempo. rarily in this area. Chlorinated tap water was, ofcourse, supplied to the people of this area. The drinking water was kept in their houses
because the water supply was not connected to individual houses; thus resulting in it becom‑ing contaminated. The, water from the Paraguay River is used for domestic use such as
washing dishes and clothes and bathing. It is, therefore, believed that the establishment of awater supply to this area as well as health education is required. The last point of riote is on
the high mortality rat due' to diarrheal diseases. Firstly, an effective and cheap drug' for

diarrheal patierits should be chosen, with due consideration being

ate drug for

given to the most apbropri‑

ediatric cases. Secondly, particular attention ,should be ,,paid to the care of

hospital patients, and to the improvement of laboratory diagnostic techniques. Thirdly,
effective use of oral rehydration solutibn in rural areas should be promoted because of thehigh mortality rate. Finally, it is important to continue the etiological examination ofdiarrheal diseases.

ACKNOWLEDGEMEN TS
We are grateful to the technicians and physicians who collected the speciinens in theCatedra de Pediatria, Universidad de Asunci6n and Dra. Chizuki Okamoto in LACIMET
Hospital. We also thank Dr. Tatsuyuki Mimori, Dr. Tetsuo Miyashita, Dra. Carmen A. de
Varela, Dra. Myrian Graciela L., Dra. Dolly N. Puerto and Srta. Felipa A. Ruiz for theiradvice and technical assistance and Lic. Yoko Akimoto and Sr. Akira Kokubo for their

46

helpful assistance in this study.

REFERENCES
1 ) Achucarro de Varela, C., Francchi, M.E., Zacur de Jimenez, M., Medina, D., Alborno, R.M.,
Morinigo. M.G., H. de Kaspar (1989) : Campylobacter jejuni.. Primeros Cuatro Casos con
Diarrhea en el Paraguay, 4‑Congreso de Gastroenterologia, Paraguay, 28‑30 de Mayo
2 ) Bauer, A.W., Kirby. W.M.M., Sherris, J.C. and Turck, M. (1966): Antibiotic susceptibility
testing by a standardized single disk method, Am. J. Clin. Path., 45, 493
3 ) Brandt, C.D.. Kim, H.W., Rodrigues, W.J., Arrobio, J.O., Jeffries. B.C., Stallings, E.P., Lewis, C.,

Miles, A.J., Chanock, R.M., Kapikian, A.Z. and Parrott, R.H. (1983): Pediatric viral gas‑
troenteritis during eight years of study, J. Clin. Microbiol., 18, 71‑78
4 ) Brandt, C.D., Kim H.W., Rodrigues, W.J., Arrobio, J.O., Jeffries, B.C. and Parrott, R.H. (1982) :Rotavirus gastroenteritis and weather, J. Clin. Microbiol., 16, 478‑482
5 ) Chyou, S.C.. Leu, Y.J.. Huang, F.Y., Lee. H.C. and Yang, D.1. (1988) : An etiological study of

infectious diarrhea in infants and children in Taipei Area, Acta Paed. Sin., 29, 213‑220
6 ) Honda, T.. Sato, M. and Miwatani, T. (1984): Differential detection of cholera enterotoxin andEscherichia coli heat‑labile enterotoxin by Enzyme‑Linked Imniunosorbent Assays with
antibodies specific to the two toxins. J. Clin. Microbiol., 20, 664‑667

7 ) Koopman, J.S., Turkish, V.J., Monto, A.S., Gouvea, V., Srivastava, S. and Isaacson, R.E.
(1984): Patterns and etiology of diarrhea in three clinical settigs, Am. J. Epi., 119, 114‑123

8 ) Merson, M.H. (1982): The global problem of acute diarrhoeal diseases and the WHO diarr‑
hoeal diseases control programme. In International Symposium on Bacterial Diarrheal Dis‑eases, Osaka, Jap n, 1‑39 ) Robins‑Browne R.M. (1984): Seasonal and racial incidence of infantile gastroenteritis inSouth Africa, Am. J. Epi., 119, 350‑355
10) Shimotori, S., Ehara, M., Watanabe, S., Ichinose, Y., Waiyake, P.G., Kibue, A.M., Sang, F.C.

and Ngugi, J. (1986): Survey on Campylobacter jejuni and enterotoxigenic Escherichia coli in

Kenya, Fukuoka Acta Medica, 77, 584‑590ll) Takeda, Y., Takeda. T., Yano, T., Yamamoto, K. and Miwatani. T. (1979): Purification andpartial characterization of heat‑stable enterotoxin of enterotoxigenic Escherichia coli. Infect.

Immun., 25, 978‑985
12) Tang, R.B.. Hsieh, K.S. and Hwang, B. (1984): Campylobacter jejuni enteritis in children,
Chinese J. Microbiol. Immunol., 17, 226‑236

47

パラグアイ，アスンシオン市における幼児の細菌性下痢症
一瀬

休生1・Cesar

DoritaMongesA．2・Maria G．Centurion2・Medina2・Rose M．Albomo2・Juan J．Bestard3・坂本
信4・Ricardo Moreno A．2

1990年5月から7月までの3カ月間，パラグアイ・アスンシオン市の医療施設において，5歳以下の下痢症例患者を対象に，下痢便の細菌学的検査を行った。53症例から34名（64．1％）に下痢原因菌を検出したが，その結果は病原性大腸菌の19株（35．8％）を筆頭に，サルモネラ8株（15．1％），毒素原性大腸菌3株（5．7％），赤痢菌とカンピロバクターがそれぞれ2株（3，8％）が検出され，このうち混合感染が3例あった。
診断用抗血清による凝集試験で，病原性大腸菌では11種に型別され，018型が最も頻度が高かった。血清学的抗原構造で毒素原性大腸菌に属するものからは，易熱性毒素（LT）は検出できなかったが，乳のみマウスによる耐熱性毒素（ST）の検査で，毒素原性大腸菌から1株が陽性を示し，病原性大腸菌も2株が陽性を示した。
河川水や飲料水の細菌学的検査において，病原性大腸菌，組織侵入性大腸菌，サルモネラ，ビブリオ属などの病原菌の他に，腸内細菌科の細菌も検出された。特にパラグアイ川からは，下痢

便で最優先であったもの≧同型の，Ol8型病原性大腸菌が検出されたこ≧は，この地域住民に水系感染の危険性があることが推察された。

1

またディスク法によって，抗生物質7種類に対する薬剤感受牲試験を行ったが，同一菌種間においても，その感受性パターンは多様性を示した。

パラグアイにおける細菌性下痢症の調査は，従来，サルモネラや赤痢菌などの古典的下痢症については行われてきた。しかしカンピロバクターについては，1989年において，初めて4症例の報告がなされた状況であり，腸管病原性大腸菌やビブリオ属の調査については，まったくなされていない。

パラグアイでの下痢症の疫学的・病因的調査は不十分な状況であるので，この調査を機会に，今後も継続して行っていく必要性を強調したい。

長崎大学熱帯医学研究所病原細菌学部門
国立アスンシオン大学保健科学研究所（IICS）
アスンシオン大学付属病院小児科
長崎大学熱帯医学研究所寄生虫学部門

日熱医会誌．，第19巻

第1号

49

1991年，4g−56頁

臨床的研究

熱帯熱マラリア治療における開始遅延の意義
海老沢

功1・田辺

清勝2・小原博3

平成2年10月24日受付／平成2年12月1日受理

はじめに
現在日本で報告されるマラリア患者は年間約60

マラリアの治療を受けないで死亡した者もいる。

遅延日数（損失）日数：はじめて発熱した日を第1病日として起算し，その後適切な抗マラリア薬

人，実際は約100人，死亡例は1965−1987年の23年を用いて治療を開始するまでに経過した日数を，間の平均が3人である。その他に瀕死の重症例が遅延日数とした（病日一1日に相当する）。遅効性

毎年1−2人ある。これらの重症，および死亡例
はすべて熱帯熱マラリアで治療開始が遅れたもの

あるいは感染熱帯熱マラリア原虫がR3耐性を示して，効果を示さなかった時は有効な治療薬を開である（海老沢ら，1980； Ebisawa窃磁，1980；

始した日までの日数とした。

Ebisawa，1989）。治療開始遅延日数と血液の諸検・査所見を比較して，熱帯熱マラリアの重症化の関結係を述べ，早期診断と治療の重要性を強調したい。

果

1．瀕死重症例の発生要因

患者資料
1966−1989年の24年問に東大医科学研究所内

瀕死重症例はすべて熱帯熱マラリアで，その他の原虫種感染、によるものはなかった。

1）感染地域別発生頻度

科，東邦大学その他の施設で我々が直接，あるい

瀕死の重症例は，アフリカ大陸で感染したマラは間接に関与したマラリア患者432人である。血液リア患者122人中17人（14％），その他の地域で感内マラリア原虫の算出法その他は既に報告した

染したマラリア患者は310人中8人（3％）で，ア（海老沢ら，1990a，b）。

フリカ大陸で感染したものに多く発生している。

使用述語の定義

その理由は，熱帯熱マラリアの頻度がアフリカ大瀕死の重症：意識混濁，昏睡，全身痙攣などの意

陸では70％，その他の地域では30％と，アフリカ

識障害，腎不全，高度の貧血，黄疸，血色素尿，

大陸に熱帯熱マラリアが多し〉ためである。

DIC，肺水腫などを単独あるいは合併して，生命に熱帯熱マラリア原虫に感染した場合には，瀕死

危険な兆候を示したものを瀕死の重症とした。治重症マラリアの発生率は，アフリカで感染した86

療にはクロロキンの筋注，またはキニーネの点滴静注を行い，腎不全合併例では腹膜，あるいは血人からは20％，その他の地域で感染した90人からは9％で，有意な差はなかった。廊ち感染地域に液透析などの積極的治療を必要とした症例を指す。

関係なく，熱帯熱マラリアに感染することが危険

全症例中23人が瀕死の重症に分類された。死亡しである。

た13人はすべて瀕死の重症例であり，その中には

京浜急行川崎診療所
鹿児島大学医学部医動物学教室
埼玉医科大学医動物学教室

50

1

％oo

90

1．Falciparum maIaria

80

2．

A

μ●ヒー

cases

70

60
50

40

〆

30
20

〃
1

Figure1

6
。

2

●

Ol

10

／：

o：moribund、survived●：died…鱒 834567891011121520253035405060
No．of days

Cumulative

percentage

patients

and

days

more．

or

All moribundof timedevelopment

of

lag

moribundand fatal cases arein thetreatment ofconditions afteramalaria

delay

of4

falciparum malaria patients．

2）遅延（損失）日数

内に治療を開始することが必要である。

発熱発作時に悪寒戦藻を伴う三日熱ド卵形およ3）遅延日数に相関する諸種検査成績
予後に最も関係する血液内最大原虫数，腎機能び四日熱マラリアは症状が定型的であり，素人でもマラリアを疑う。これに反して熱帯熱マラリアの指標としてのBUNとクレアチニンの最大値，は・発病初期に悪寒だけを訴えることが多く，そおよび赤血球数と血色素の最低値について，遅延の他の症状が乏しい。

日数との関連を検討した。

そこで，熱帯熱とその他のマラリアを含めた全

1．血液内熱帯熱マラリア原虫数

症例について遅延日数を分析し，その累積百分率a）熱帯熱マラリア原虫の自然増加を図1に示した。これによると，遅延日数は感染

抗マラリア薬を予防内服しない患者：血液検査

原虫の種に関係がない。また発病後5日以内，即で熱帯熱マラリアと診断のついた患者の治療開始

ち6病日以内に治療を開始された者はポ全体の約が遅れたため，マラリア原虫が急激に増殖し患者50％に過ぎない。

は意識不明，腎不全を合併し瀕死の重症になった。

他方，瀕死の重症に陥ったが生存したものを白

血液1μ1当たりの原虫数は，7日問に6，760から丸で，死亡例は黒丸で，それぞれの遅延日数を図1，549，000に230倍増加した（海老沢ら，1978）（表1）。

の下部に記入した。瀕死症例中には，遅延日数4

効力不完全な抗マラリア薬を内服した場合：熱日で治療を開始したが重症であった例もある。死帯熱マラリア原虫にピリメタミン耐性があるナイ

亡例の中には，図に示した日に治療を受けること

ジェリアに滞在中，発病予防にピリメタミンを内なく死亡したものがある。従って熱帯熱マラリア服していた男性が，次第に貧血となり脾腫を発見

患者が重篤な合併症を起こさず，順調な経過をされ，その原因追求のため入院した。その間血液とって治癒するには，少なくとも遅延日数4日以

内には，少量の熱帯熱マラリア原虫があったが発51に1μ1当たり550から10，000に18倍増加しただけ

Table1 Increase of」P，血」6々）αn4郷 inuntreatedである。ピリメタミンで不完全に抑制されていた，

patients

少量の原虫の長期感染によりある程度の免疫がでCase1．

35y，m．

Case2．

38y．m．

Day

ParasiteDay

Parasite

き，熱帯熱マラリア原虫の増殖が抑えられたため

count

of

count

であろう。

（／μ」）

dis．

（／μ」）

b）多数例における観察一治療開始前の最大原虫

5︸3211234
一一一

of
dis．

6，760
91，20056，200371，500
380，200
1，548，800

550

数

2，290

検討した対象は，非マラリア流行地に生まれて830

成人したもので，主として日本人である。治療開1，780

始前10日以内に，何らかの抗マラリア薬を内服し400たものは除外した。

5，000

80人の成績を，遅延日数を横軸に，最大原虫数

6，030の対数を縦軸にとって図2に箱ひげ（box・whis−

10，000ker）図で示した。縦長の矩形は各群50％の症例が，Day l is the day on which the patient
became febrile．The sign−indicates day
before onset of fever． Case 2 had been
admitted for examination of anemia andsplenomegaly of unknown cause、

入る範囲を，上下にのびる直線は最高と最低値の範囲を，箱を横切る線分は平均値を示す。

遅延日数と血液内原虫数の間には1−10日の間
では，正の相関関係が見られた。
y＝4．031十〇．124x，r＝0．359，p＜0．01（n＝80）

熱がなかった。そのうち発熱したので血液標本を

ただし11日以後になると異常に高い値を示すも遡って鏡検したところ，入院時より少量の原虫がのが少数あるが，一般に遅延日数が延びても原虫

血液内にいたことが分った。この例では，9日間数は増加しない傾向が見られた。

⁝

3

the treatmenωs maximum

9 10

㎝

S

y
a

R

1

See

in

n

9
a
1
e
m

lag

malaria

d

■

5 6 7

¶

Time

8

4

2 3

α

＜
P
9
7
5
3
α
＝
r
瓦
4
2
1
α
十
1
3
0
4
＝
y

1
Figure2

1

．

｝

R

コoo﹄
石︸
︸o噌
ミ︾
だコ8︒↑語﹄区も︒〇一噌昌

コ

5 0 5 0 5 0 5 0 5 0
6 6 5 5 4 4 3 3 2 2y←一一一一一一一一一一X

11

21

〜

1

15
parasite comt

in falciparum

patients．

text

for

explanationofbox−whisker figures．The regression line

indicates，yニ4．031十〇．124x，r＝03579，p＜0．OL

52

Table2

Time

lag

in

days∂stheincidenceof the patients withparasite

of

of

over

Time

lOO，000perμJ

Parasite

23，400と69，200である。

count

2．赤血球と血色素a）一搬的傾向

blood

count／μ」

lag in赤血球と血色素の値を遅延日数ごとに箱ひげ図

Total

26

1

59

ズ2ニ3．490，p＞0．05，No

819U94

1⊥22

1⊥11⊥

﹃0︻D6︻﹂8

2〜4
6811
〜〜〜〜

︑⊥QJ579

Total

を作り，さらにそれらの値と遅延日数との間の相関の有無を検討した。図3に示すように，これら検査値と遅延日数ζの相関は遅延日数が1−3日と4」8日の間では，いずれも有意な相関関係（p＜0．01）が証明された。すなわち赤血球については遅延日数1−3日（r＝一〇．480），4−8日

85

significant

difference．

（r＝一〇．408），血色素については1−3日（r＝一〇．524），4−8日（rニー0．444）の値が得られた。いずれも有意な相関係数である。

遅延日数が9日以上の者は，数が少なく検討でc）高い原虫数の出現頻度きなかった。ただし今回の分析で，赤血球と血色

血液1μ1当たりの原虫数が10万を越えるもの

素が最低の98万／μ1と3．2g／dJの値を示した者はの出現頻度を，2日間隔で区切って調べたところ，

遅延日数が11日で，原虫数は1μ」当たり728，500

2病日ですでに10万を越える患者があり，以後そに達して死亡した（海老沢ら，1990a，b）。

の割合は増加の傾向はあるが，統計学的には有意b）遅延日数と高度貧血出現頻度差は見られなかった（表2）。

遅延日数が4日以内と5−11日の群について，

d）遅延日数4日以内と，5−11日の患者の最大赤血球数は1μ1当たり299万以下，血色素は9．9

原虫数g／dl以下の貧血を示すものの割合を比較すると，原虫数の対数を両群について検討し，遅延日数

いずれも5−11日のものに多くでる結果が得られが多い群の原虫数が，有意に多いことが認められた（表3）。また両群の平均値の問に赤血球ではた（p＜0．05）。平均値を実数で示すと，それぞれ約50万，血色素では2．59／dJの差が認められた

r、b．c．

5．0

lag

in

the

2〜

Time

a

17

■I

Figure3

7〜8s

2

9〜0

y
a
d
6 n
9
5
e
m
T
4

1

b
7
4
H1
16
115
11
13
1 2109876543

r．b，c，mi1／μ2

甲treatmentびs minimuni

Hb，g／d2

吉123456791121〜

Time lag8〜in daysr．b．c．counts and Hb

10

〜

17

〜

concentrationin falcipamm malaria patients，Regression lines are l（yニ4．39−0．36x，r＝一〇．480），2（y＝4．89−0．25x，

rニー0・408），3（y＝14・66−1．33x，r＝一〇．524），and4（yニ15．93−0．89x，
r＝一〇．444）．

P

valuesareall＜0．01．

53

Table 3 Time lag in days vs degree of ane‑

2 99 mill p l

3.00 mil 45 (2) 35 ( 7) 80 (9)

Hb

g/d l

8

53 (2) 51 (17) 104 (19)

10 . OHb*
g/d l

12.4 (2.1) 10.0 (2.5) p<0 Ol

Max ‑ Min

18 . 1‑7 . 9 14 . 8‑3 . 2* ( ) Standard deviation in parenthesis.
There was a significant difference in themeans but not in the variances (p>0.05) .

The number in parenthesis indicates mori‑bund but survived or fatal cases.
x2=3.88, p<0.05 for r.b.c. vs time lag
x2=9.800, p<0.01 for Hb vs lag time(

The incidence of moribund cases was not
related to the degree of anemia, but wasmore frequent among the patients with

3.81 (0.69) 3.28 (0.82) p<001

Max ‑ Min 5.53‑2.25 4.76‑0.98

42 (1) 28 ( 5) 70 (6)49 (D 49 (15) 98 (16)

Total

r.b,c. *mil/pl21 (lO) 28 (10)7

4 days 5‑ll days DifferenceTime lag

16 (lO) 24 (10)

r,b,c

9.9

r. b. c. count and Hb concentration
vs time lag in days

4 5 ‑11 Total

Time lag in days

Total

Comparison of the lowest mean

Table 4mia

4 ).

a ) I j5

) !

flonger time lags than with shorter timelags (p<0.01).

j . Ilz >f :L

BUN
mg!de

ec ftLCv>;)h v>.

creatinine
mglcl

200 1 50

1 20
1 oo

4 3

I .5 2
1 .o .9 .8 16

1 2 3 4 5 6 7 9 11 21i i i
8 ID 17

Time lag in days

Figure 4

1 2 3 4 5 6 7 9 11 21S

8 10 17

STime lag in days

Time lag in the treatment vs maximum BUN and creatinine in falcipar‑
um malaria patients.
Note abrupt increase of abnormal values of BUN and creatinine beyond
time lag of 5 days or more.

54

れは，極端に高い値を示すものがあるためである。

例えば遅延日数2日で，他の例は大部分正常範囲が確認された（表5）。

両群の差は，平均値に最も顕著に見られた。

内にあったのに，BUNが92mg／dJ，クレアチニン

BUNの平均値は，両群でそれぞれ16．0と57．1

が16．9mg／dJに達していた症例があった。ここ

mg／dJ，クレアチニンの平均値は1．2と3．4mg／

では平均値はそれに引きずられて上方にずれていdJで，有意差が認められた（p＜0．01，表6）。

る。

一般的傾向としては，BUNとタレアチニンい・考ずれも遅延日数が5日以上になると，平均値と分察散が大きくなっていることがわかる。この傾向は

図1に示したように，熱帯熱マラリア患者が重

原虫数，赤血球数，血色素量いずれの場合よりも症化するのは，遅延日数が5日以上になる時が多顕著な差として認められる。

い。この5日を境として何が起こるかが，今回のb）遅延日数と異常BUN，クレアチニン値の出分析の主要な目的であった。

現頻度

本論文では原虫数，貧血および腎機能を示す

統計学的分析には，2病日で例外的異常値を示

BUNとタレアチニンと遅延日数との相関，およびした例を除いて検討した。この例を除いた者の遅延日数4日以内と5−11日の群の各検査項目のBUNとクレアチニンの平均値±標準偏差は，それ平均値を検討した。・

ぞれ15．3±2．7と1．2±0．1mg／dJであり，平均値

まず注目すべきことは，図2に示した回帰直線に標準偏差の3倍の値を加えても，異常高値例ので遅延日数0，即ち第1病日の原虫数の平均値は，数字に達しない。すなわち，このような異常値を

約10，000と推定されることである。これは初めて

示す例は，統計学的に0．3％以下に過ぎないので除発熱した日を第1病日としたからで，実際にはそ外して計算した。

の数日前から，原虫は血液内で増殖をしていたこ

遅延日数が4日以内と5日一11日の2群に分けて，BUNが2Lmg／dl以上，およびクレアチニン
がL6mg／dJ以上になったものの割合を検討しとを示すものである。いわゆるprepatentの終り

Table6た。その割合は危険率1％以下で後者に多いことTime

lag in daysηs BUN andcreatinine（mg／dJ）

Time

Table51ncidence of abnormal kidney func−tion（BUN and creatinine）びs time
lag in days

BUN，mg／dJ≧21．0 ≦20．O

6

≦4

Time
in

lag

57．1

60．5

Tota1

85

39．7

45．8

No．

Mean

S．D．

31

1・2

0．3

Time

lag

Creat一

Tota1

36

49

85

inine 5〜11 45

3

28

45

Tota1

23

53

76

of

abnonnally

and

high

76

BUN

creatinine

2．5

patient

withcreatinineof16，9mg／dJ

on

of

was

from

illness

BUN

were

3．0

Both

25

and

3．4 4．0

higher in patients with longer time lags
than with shorter time lags（p＜0．01）．A

20

BUN

5．1

31

5〜11

incidence

≦4
Tota1

Tota1

days

The

49

49

≦4

in

5〜11

19

≧1．6 ≦1．5

S．D．＊

16．0

30

Creatinine，mg／dJ

lag

36

Mean

36

5〜11

days

Time

30

No．

≦4

BUN

Tota1

lag

excludedof92
thesignificantly

mg／dJ andsecondtheday

analysiscreatinine was significantly higher inpatients with longer time lags than withas the values were greater than mean十3
S．D．of the patients whose treatment was

shorterstartedtimelags（p＜0．01），

on

the

same dayof illness．

55

から抹梢血液のマラリア原虫は出現するが，一定施しておくべきである。

の数に達しないと発熱しない。prepatentperiod

遅延日数が12日以上のものでは，検査所見に異は潜伏期よりも少なくとも2日以上短いという（Garrham，1988）。この時期には臨床的に倦怠

常に高いものが少なかったのは，重症例はそれまでに死亡してしまってし｝るが，ある程度の免疫が感，頭痛，食欲不振など非特異的症状を訴えるもできて病勢が進展しなかったものであろう。

のが多い。本来はこれら症状の出現時を初発日とすべきであるが，発熱は最も明らかな客観的所見

結であるから，初めて発熱した日を第1病日とした。

語

実際にはこれは4−5病日になっている可能性は

1）全体約420人のマラリア患者中，発病後5日以理解しておくべきである（表1，症例2）。

内に治療を開始された者は，熱帯熱でもその他の諸検査項目は，いずれも遅延日数の増加に伴っマラリアでも約50％である。熱帯熱マラリア患者て悪化する相関関係が認められたが，最も顕著でで瀕死の重症になったものは，遅延日数が4日以あったのはBUNとクレアチニンである。両者とも遅延日数が5−11日のものは4日以内のものに上になったものである。

比べて，顕著な増加ぶりを示している。Marsden（赤血球と血色素の減少），腎機能の低下（BUN

2）発病後11日までの間では原虫数，貧血の程度（1989）は熱帯熱マラリア原虫の標的臓器は脳ととクレアチニンの増加）は遅延日数に平行して悪腎臓で，意識障害と尿量に注意するよう述べてい化している。発病後4日以内と5−11日以内の群るが，的を得た表現である。

では，検討した項目すべてに有意な増加，または小児では，脳性マラリアと貧血が最も普通に見低下が見られた。特に5日を境にした，腎機能のられる致死的合併症であるという（箪01yneux，

急激な悪化が目立つ。

1990）。我々の症例には小児は少なく，貧血が死因と思われた例は26歳の女性1人で，この患者の遅謝

延日数は11日であった。

辞従って熱帯熱マラリア治療上最も注目すべき事

この論文の作成と発表には，一部に公益信託児

項は，臨床的に遅延日数を確定し，意識障害の有玉記念基礎医学研究助成基金の援助を受けたこと

無と尿量に注意する。最小限の項目として原虫数，を感謝する。

BUNとクレアチニン，赤血球と血色素の検査は実

献

文

1）海老沢

功，谷

荘吉，飯田

俊，内 孝（1978）：

マラリア，とくに重症マラリアの治療，日本医事新報，Nα2849，43−48
2）海老沢

功，谷

荘吉，渡辺迫男（1980）：

マラリァτとくに熱帯熱マラリアの予後決定因子について，日本臨床，38，333−335
3）Ebisawa，1．，Muto，T．and

falciparum

Watanabe，M，（1980）：Factors

contributing

tothe prognosis

ofmalaria，Japan．」．Exp．Med．，50，117−122

4）Ebisawa，1．（1989）：Time

limitsinR．，Lobel，H．0．，Haworth，J．and

theinitiationoftreatment

Bradley，nJ．（eds．），Traveloffalciparum

malaria，Steffen，Medicine，158−159，Springer−

Verlag，Berlin，Heidelberg，New York，Paris，Tokyo，Hongkong5）海老沢 功，小原 博，田辺 清勝（1990a）： マラリア患者の血液所見，特に貧血について，日熱
医会誌．，18，239−245
6）海老沢 功，小原 博，田辺誌，103，2051−2055

清勝（1990b）：

マラリア患者の血液と生化学的所見，日本医師会雑567 ) Garnham, P.C.C. (1988): Malaria parasites of man: Iife‑cycles and morphology (excluding
ultrastructure). Malaria‑Principles and Practice of Malariology (ed.) Wernsdorfer, W.H. &McGregor, I., Vol. 1, 61‑96. Churchill Livingstone, Edinburgh, London, Melbourne, New York8 ) Marsden. P.D. (1989): Growing problems of malaria, Brit. Med. J., 299, 1328‑1329
9 ) Molyneux, M.E. (1990): Cerebral malaria in children: clinical implications of cytoadherence,

Am. J. Trop. Med. Hyg., 43. Suppl. 1, 38‑41
Cli*i･*1 st*dy

THE SIGNIFICANCE OF TIME LAG IN THE TREATMENT OF

FALCIPARUM MALARIAISAO EBISAWA*, KIYOKATSU TANABE' AND HIROSHI OHARA3
Received October 24 1990/Accepted December 1 1990

We have previously reported that there exi.sts a time limit for effective treatment of
falciparum malaria. In short, four days after the onset of the illness was found to be the time

limit beyond which severe, Iife‑threatening complications may develop. .
In order to elucidate the level of laboratory variables beyond which the patient's
prognosis is poor, we examined the corfelation between time lag in days vs maximum
patasite count, minimum red blood cell (r.b.c.) count, minimum hemoglobin (Hb) , maximum
blood urea nitrogen (BUN) and creatinine concentration of each patient during the period ofadmission. A Iinear correlation was found between time lag (x) and the maximum parasite
count in log units (y) , y=4.031+0.124x, r=0.3579. All variables among patients whose
treatment was started after 5 to 11 days of time lag (6th to 12th day of illness) weresignificantly worse than the variabfes of patients whose treatment was started after only I to

4 days of lag time. However, the most conspicuous change was observed in the abrupt
increase of BUN and creatinine in the members of the late‑treatment group: the mean BUN
and creatine levels in the early and late treatment groups were 16.0 vs 57.1 and 1.2 bs 3.4
mg/dl ( < 0.01) , respectively. The mean parasite counts of the early and late treatment groups

were 23,400 vs 69,200/pl of blood (p<0.05) , while the mean r. b. c. count and Hb concentra‑tion of the two groups were 3.81 vs 3.28 million per pl and 12.4 vs l0.0 gldl (p<0.01),
respectively.

In addition to the level consciousness of the patient, special attention should be paid tokidney function in the treatment of falciparum malaria patients whose treatment is delayed
beyond the 5th day of illness.

1 Keihin Railway Kawasaki Clinic
2 Department of Medical Zoology, Faculty of Medicine, Kagoshima University
3 Department of Medical Zoology, Saitama Medical College

日熱医会誌．，第19巻第1号

1991年，57−63頁

57

臨床的研究

マラリア患者の黄疸について
海老沢 功1・田辺 清勝2・小原博3

平成2年10月24日受付／平成2年12月1日受理

算には脇本たちのソフトウ手ア（脇本ら，1987）

はじめにを用いて行った。

マラリアでは一定の間隔をおいて溶血が起こり，

黄疸を起こすことが知られている。しかしマラリー

成ア患者の血清ビリルビンを，各感染原虫ごとに比較した研究は少ない。

績1）感染原虫別に見た血清総ビリルビン

重症熱帯熱マラリアは，しばしば激症肝炎，急各感染原虫別に調べた血清総ビリルビンの比較性腎不全を伴った閉塞性黄疸などの死亡診断名がは，一部をすでに報告した（海老沢ら，1990c）。

つけられているので，これらの疾患との鑑別を念

それによると，熱帯熱マラリアでは血清総ビリル

頭において，マラリア患者の血清ビリルビンにつビンの値が0．2〜42．3mg／dJ』と広い範囲にわたっている。血清総ビリルビンの平均値と標準偏いて述べる。

差は，熱帯熱マラリア患者が6．2±8．6mg／dJ

症例と検索方法
症例は前報（海老沢ら，1990a，b）に述べた419（n＝59），三日熱と卵形マラリア患者がL2±0．9
卑mg／dJ（n＝57）で，平均値と分散ともに有意の差がある。

人のマラリア患者のうち，血清ビリルビンを測定

表1にその後の資料を加えて，血清総ビリルビしてあった127人である。患者は複数の病院で検査

ンの値を1，0mg／dJ以下，1．1〜5．0，5．1以上の3されているので，各検査室で行われている成績を群にわけて各原虫ごとに示した。熱帯熱マラリア

そのまま用いた。また各検査項目につき，その患患者には，5．1mg／d1以上の値を示すものが多者が入院中に示した最高値を，その患者の代表値

い。最高値は熱帯熱マラリアで42．3mg／dJ，三日とした。患者の80％は男性で，20−40歳のものが熱マラリアでは6．4mg／dlであった。

80％を占める。

マラリア原虫数の半定量的検査法は，すでに述
べた（海老沢ら，1990a，b）。検査データの統計

症例を熱帯熱と，それ以外のマラリアの2群に分けて検定す
ると有意の差が認められた甲（p＜0．01）。

2）血清総ビリルビン値と予後的処理で原虫数，総ビリルビンとLDHは計測値が意識混濁，昏睡，痙攣などの意識障害，腎不全，

広い範囲にわたるため，対数変換を行って分析し血色素尿，DI（あ肺水腫などを単独あるいは複数合た。データの処理は，群間の検査値の分散と平均併し，生命に危険な兆候を呈したものを，死亡例

値の差，度数分布の差，および2つの変数の相関を含めて瀕死重症と定義した己瀕死重症例は，すは相関係数を求めて，その有意差を検定した。計べて熱帯熱マラリアである。そこで熱帯熱マラリ

京浜急行川崎診療所
鹿児島大学医学部医動物学教室
埼玉医科大学医動物学教室

58
Table

l

Semm

total

bilirubin

and

the

incidenceofsevereillnessinmalariapatients
Total

Infectingbilirubin

Rμ6加n

mg／dJ

解

malaria

P吻砿

≦1．0

19

（0）

28

1．1〜5．0

33

（5）a

24

14（14）b1

66（19）

53

≧5．1

Tota1
The

numberinparenthesisPparasite

Tota1

o∂α」8

P㎜乙

2
4
0
6

1
1
0
2

indicates

moribmd

or

fatal

50
62
15

127
cases．

a：30f5patientsdiedb：70f14patientsdied・
ア患者を血清総ビリルビン値により3群にわけ，うことにする。

瀕死重症例の起こる頻度を見ると，血清ビリルビ

3）原虫数と総ビリルビン

ンが異常値，特に5．1mg／dJ以上になった者に，

ここでは，総ビリルビンが1mg／d！以下の7人

瀕死重症例が多発していることがわかる（表1）。

も加えて検討した。両者とも，最低値と最高値の以下の分析は，すべて熱帯熱マラリアに関して行

開きが大きい。血液1μ沖の原虫数（pc）の対数

y
UnUO

︻コ4つ﹂

●

p．c．

r＝0．470，Pく0．01

20

●

t、b．＝

一〇．763＋0．2了5bg● ●弓秘E．仁﹂君渚一80ト

●

1
0．8

1

R
●

●●
●

09876543

2

一

●

●

log

●

●

●

●

●

●

●

●●

●

●

●

●

2

ニ

3 ● 4●

5 ●

6

民＝し09P．c．ノμ

0．5

●

0．3

●

4．2

●

Figure1M4ximumparasitec・mt（x）asserumt・talbilirubin（y）infalcipa「ummalaria．
RegressionlineRindicates：yニー0．763十〇275x，r＝0、470，p＜0．01，

where

x＝10g

of

parasitecount／μ1，and

yニlog

of

total

bilirubin

mg／dJ．

59

x＝10g pcと総ビリルビン（tb）の対数y＝・logtb

速な溶血によるもので，間接ビリルビンの増加がの相関関係を調べ，有意の相関関係が得られた期待される。総ビリルビンが1mg／dJ以上で，直（図1）。

接ビリルビンが測定してあった34人の総直接，

log

間接ビリルビンおよび直接／総ビリルビン比を図

tb＝一〇、763十〇．275109Pc， r＝0．470，

p＜0．01

2に箱ひげ図で示す。すなわち，中央の縦長の矩

すなわち感染原虫数が多いほど，総ビリルビンの形で検査値の50％が入る区間を，上下に延びる直値も高くなる。例外的に原虫数が1μ1当たり線で最高と最低値を含む範囲を，横に交差する線

250，900の高い値を示したのに，総ビリルビンは分で平均値を示した。データが正規分布するとき0．3mg／d！に止まったものもある。しかし一般的

は平均値は縦長の矩形のほぼ中央にくるので，平には原虫数が多いほど予後は悪いので，総ビリル

均値が上方にずれている．ことは，極端に高い値をビンが高いものほど予後が悪かったこと（表1）

示すものの影響を示しているσと一致する所見である。

総，直接と問接ビリルビンの平均値は，それぞ

4）直接と間接ビリルビン，および直接／総ビリル
ビン比

れ9、4，5．4，3．9mg／dJで，直接／総ビリルビン比の平均値は0．5であった。直接と間接ビリルビンの﹄﹃﹇OO 09876543
d
O
／5
43

マラリアにおける血清ビリルビンの増加は，急平均値と分散には，有意差はなかった。すなわち，

m9

20

08﹃JRU54
．3

2

1．

．2

．1

totaI
d！t ratio

directFigure2Serumtotal，direct

bilimbin

An

in

and

falciparum

upright

quadrangle

found．

Two

lines

maximumandminimum

value．

indirectindirect bilirubinand d／tratio（direct／tota1）ofmalaria．

indicates

above

and

a

below

values．A

range
the

in

which50％ofquadrangle

transverse

line

data

are

indicatetherangesofindicates

the

mean

60

y
0．9
●

≦コ君一蓋でぢ︽ぢΦ﹄看︸OΩ冨匡

●

●

●

0．7
●

0．6

●

●

●

●

0．5

●

0．4

●
●
●

●●
●

0．3

●

●

●●
●

●
・●

0．1

R

●

●

の

02．

●

R：y＝0．397＋0．15610gx，r＝0．4525，P〈0．02

1

2

3

4

5

6

Total
＊Patients

Figure3

●

0．8

78910

20

30

40

50

X

bilirubin，mgld2

with≧1・1mgld20f

total

bilirubin

Correlation between serum total bilirubin（x）andbilirubin（y）in falciparum malaria．

ratio

of direct／total

RegressionlineRindicates：y＝0．397＋0．15610gx，r＝0．4525，p＜0．02．
iu／〜、

間接ビリルビンと平行して，直接ビリルビンも増

20，000加している所見である。

15，0005）総ビリルビンと直接／総ビリルビン比
血清総ビリルビンは，L1から42・3mg／dJの広10，000
iu／〜

8，000

了00

い範囲にわたるので，その対数（x）と直接／総ビ

600
500

6，000

リルビン比（y）の相関関係を調べた。両者の間

400

4，000

に，有意な相関関係が認められた。

300

y＝0．397十〇．15610gx，r＝0．4525，P＜0．02

200

2，000

すなわち，総ビリルビンが増加すると直接／総ビ

150

1，500

清総ビリルビンが30mg／dJに達したのに直接／

総ビリルビン比が0．26の低値を示したものもあった。

6〉その他の検査成績：SGPT，SGOT，γ一GTP

およびLDH
総ビリルビンが1．1mg／dJ以上の高値を示し

1，000

㎜
600
500
400
300

200

20

LDH

SGOT
100

︵U9日

たものについて，肝機能に関係のあるSGPT，0000000 009876543

リルビン比も増加する（図3）。しかし中には，血SGOT，γ一GTPおよびLDHを分析した。各項目

SGPT

γ一GTP

について得られたデータを図2と同様に箱ひげ図によって，図4に示す。平均値はSGPT（70），
SGOT（132），γ一GTP（74），LDH（1，112）でFigure4

SGPT，SGOT，γ一GTP
falciparum

malaria

and

LDH inpatients・

61ある。SGPTよりもSGOTの方が高くなること，
赤血球の崩壊を反映してLDHが最高15，140に達おり，直接／総ビリルビン比の平均値は0．5であっしていることが特徴である。γ一GTPも高い者がンも出ているから，間接型ビリルビンが多少増加

多いが，これは飲酒癖の多いものがあることも考していても，閉塞性黄疸とは容易に区別できよう。

慮する必要があろう。

4）その他の血清酵素反応

た。さらに尿中にはビリルビンとウロビリノーゲ

この他にアルカリフォスファターゼ（Al−P）も熱帯熱マラリア患者ではSGPTだけでなく，土昇した例があるが，問題になるようなことはな

SGOT，γ一GTPの上昇が見られたが，肝炎患者にいので省略する。総ビリルビン値が高くなった患見られるほど高いものではない（岩田ら，1982）。

者では，尿中にもビリルビンが検出され，同時にLDHはマラリアそのものの病態を反映して，極め

ウロビリノーゲンも検出されている。また血清総，て高い値を呈していた。・

直接と間接ビリルビン，SGOT，SGPT，LDHい

高熱と黄疸を伴う，重症熱帯熱マラリア患者をずれもマラリア原虫の消失とともに急速に，多く初めて見る医師にとって，マラリアを念頭におかの場合3−4週問内に正常値にもどった。

ないで診断すると，直接と間接ビリルビンが共に上昇しているので，診断が困難である。しかし3

考察と4の所見を総合すれば，劇症肝炎，あるいは閉塞性肝炎とは，容易に鑑別できよう。

図1と3に示したように，原虫数の増加→（赤血球崩壊の増加）→総ビリルビンの増加→直接／総結

ビリルビン比の増加という一般の図式が明らかに語なった。このことから原虫数が多くなる熱帯熱マ

マラリア患者，特に熱帯熱マラリア患者の血清ラリアが，強い黄疸の出現で問題になることも推

ビリルビンについて検討し，その黄疸が肝炎や閉定できる。

塞性黄疸と異なることを示した。

1）熱帯熱と他のマラリアの差

1）血清総ビリルビンは， 熱帯熱マラリアでは三先ず強調すべきことは，同じマラリアといっても熱帯熱マラリアは他のマラリアに比べ七，血液日熱，および卵形マラリアに比べて，1．1mg／dJ
以上の高値を示すも4）が多い（71％：49％，p＜

内原虫数と貧血や血液生化学的異常所見を呈する0．01）。熱帯熱マラリアでは，42．3mg／dlの最高者が多く（海老沢ら，1990a，b），かつ重症にな値を示したものがある。

りやすいことである。これは血清総ビリルビンの以下の記載は，熱帯熱マラリアの血清ビリルビ

最高値が，熱帯熱マラリアでは42．3mg／dJ，三日ンに関するものである。

熱マラリアでは6．4mg／dlであったことからも

2）総ビリルビンが5．1mg／dJ以上になったも明白である。

のは，重症であった。

2）総ビリルビンと予後

3）血液内原虫数と総ビリルビン値には，正の相

熱帯熱マラリアで，総ビリルビンが5．1mg／dJ

関がある（p＜0．01）。

以上の14人が，すべて瀕死重症と判定された。こ4）総直接，間接ビリルビン，および直接／総ビれは，感染原虫数が多いことを反映するものであ

リルビン比の平均値は，それぞれ9．4，5．4，3．9る。予後判定の上からも，総ビリルビンの上昇はmg／dlと0．5であったる直接と間接ビリルビンの要注意の所見としてよいであろう。

値には，分散と平均値に有意差はない。

3）直接と問接ビリルビンの動向

5）総ビリルビンと直接／総ビリルビン比には，正マラリアの基本的病態は，分裂体による毛細血マ）相関が疹る（p＜0．02）。

管の一時的閉塞と，定期的に起こる溶血であるか

5）肝機能および溶血に関したSGPT，SGOT，ら，間接ビリルビンの増加が推定される。しかし

γ一GTP，

これとほぼ平行して，直接ビリルビンも上昇して

132，74，1，112で特にLDHの最高値が15，140に達およびLDHの平均値は，それぞれ70，62

したことは，溶血の強さをよく示している。

謝以上の所見を総合すると，急性肝炎は勿論，たとえ間接ビリルビンが増加していても，閉塞性黄疸は除外されよう。

辞

この論文の作成と発表に当たっては，一部に公益信託児玉記念基礎医学研究助成基金の援助を受けたことを感謝する。

文
1）海老沢

功，小原

博，田辺

献

清勝（1990a）： マラリア患者の血液と生化学的所見，日本医師会雑誌，103，2051−2055
2）海老沢 功，小原 博，田辺 清勝（1990b）： マラリア患者の血液所見，特に貧血について，日熱
医会誌．，18，23餅245
3）海老沢 功，小原 博，田辺 清勝（1990c）： マラリア患者の血液生化学的所見，日熱医会誌．，18，

247−253
4）岩田晃一郎，田辺清勝，鳴戸 弘，清水純孝，海老沢功（1982）：

マラリア患者の肝障害，肝臓，23，909−9145）脇本和昌，垂水共之，田中版，東京

豊（1987）： パソコン統計解析ハンドブック」1．基礎統計編，共立出

63
Cli*i･*1 *t*dy

SERUM BILIRUBlN IN MALARIA PATIENTSISAO EBISAWA , KlYOKATS J TANABE' AND HIROSHI OHARA3
Received October 24 1990/Accepted December 1 1990

The serum bilirubin of malaria patients was investigated in an effort to show thatjaundice occuring in malaria patients differs 'from hepatocellular (hepatitis) and obstructive

jaundice.
1 . The incidence of abnormally high serum total bilirubin (1.1 mg/dl) was more frequentin cases of falciparum than in vivax and ovale malaria patients put together (71% vs 49% ,p<0.01) . The maximum value of a falciparum malaria patient was 42.3 mg/dl.
The following data were obtained concerning falciparum malaria:
2 . All 14 patients showing 5.1 mg/dl of serum total bilirubin were moribund, and 7 ofthem died.
3 . A positive correlation was noted between the maximum parasite count and serum total
bilirubin (p<0.01) .

4 . The mean values of total, direct and indirect bilirubin and the ratio of direct / total(d/t) bilirubin were 9.4, 5.4, 3.9 mg/dl and 0.5, respectively. There were no significant
differences in the mean and variance of direct and indirect bilirubin.
5 . A positive correlation was found between serum total bilirubin and d/t ratio (p<0.02) .

6 . The mean values of SGOT, SGPT, y‑GTaP and LDH were 70, 132, 74 and 1,110, respec‑tively. The maximum value of LDH was 15,140. The above data may prove to be helpful indifferentiating jaundice in malaria patients from hepatocellular and obstructive jaundice.

2
3

Keihin Railway Kawasaki Clinic
Department of Medical Zoology, Faculty of Medicine, Kagoshima University
Department of Medical Zoology, Saitama Medical College

Jpn. J. Trop. Med. Hyg., Vol. 19, No. 1, pp. 65‑124

65

PROCEEDlNGS OF XXXII ANNUAL MEETINGS OF
JAPANESE SOCIETY OF TROPICAL MEDIClNE
1‑2 N*+. b.* 1990, Y*k*h* *President

Tomoo Oshima(Professor: Department of Parasitology, Yokohama City University
School of Medicine)

CONTENTS
Special lecture:

1 The international health aids of Japan: its past and future T. Shimao
2 The fundamental nature of Chagas' disease: a view provided by immvne

responses and idiotypes Daniel G. Colley

General presentation:

1 Ways of the Japanese Society of Tropical Medicine and the Japan
Association for International HealthT. Matsumura et al.
2 Medical problems of the Japanese staying at the oversea development
countries

K. Fuj ita et al.

3 Present status of the medical check of the Japanese staying at theoversea development countries
A. Sudo et al.
4 Malaria studies in Bangladesh
T. Kurihara
5 Assessment of epidemics by frequency distribution curve of malaria
antibody titers

S. Kano et al.

6 Epidemiology of malaria through questionnaire conducted on JICA
experts working in the tropical and temperate area T. Shibuya et al.
7 A follow up study of malaria indirect fluorescent antibody positivity
rates of Japanese stayed in the endemic countries M. Suzuki et al.
8 Investigation of imported malaria cases in 1988‑1989 H. Ohtomo et al.
9 The localization of neuraminidase on stages of Tr)panosoma cruzi
T. Aji et al.

10 Chemotaxis of elongated bloodstr eam forms of Tr)panosoma vivax (IL1392) against pollen grains of Fuchsia hybrida and Saintpaulia ionan‑tha H. Hirumi and K. Hirumi

11 Leishmaniasis and its vector sandfly in Paraguay Y. Hashiguchi et al.
12 Therapeutic effect of Sulfamoyldapsone (SDDS) in mice infected with

Leishmania donovani K. Morishige et al.

13 Epidemiological studies on viral hepatitis among long‑term sojourners
in the developing countries and evaluation of preventive measures
H. Ohara et al.
14 Infection rate of intestinal parasites in Japanese inhabiting tropicaloversea countries ‑study of past 10 years‑ S. Tsukidate et al.

66

15 Bacteriological and chemical study of drinking water in the tropical
M. Sugiyama et al.
countries ‑study of past 7 years‑
16 Establishment of specific T cell lines which could respond to soluble egg

T. Amano et al.
antigens of Schistosoma mansoni
17 Regulation of the T cell response to Schistosoma japonicum egg antigen
N. Ohta et at.
by concomitant cellular and humoral mechanisms

18 Cross‑reactive Ids on rabbit anti‑CEA antibodies stimulate anti‑Idlymphocytes responses by cells from CBA/J mice infected with Schis‑
M. Nakazawa et al.
tosoma mansoni
19 Immunogenicity of ultraviolet‑attenuated Schistosoma mansoni cercar‑
H. Kamiya et al.
iae
20 A community based study on measles epidemics I. Factors affecting
S. Isomura and D.S. Akram
spread of measles vaccine
21 Study on the mechanism of thermal acclimatization in the pika (whistle

M. Kosaka et al.
rabbit)22 Continued low microfilaria prevalence in American Samoa in the past
E. Kimura et al.
17 years
23 Present condition of Chagas' disease in Northeast Brazil: electrocardio‑
S. Miura et al.
graphic abnormalities and serodiagnosis
24 Reevaluation of the insecticide residual splaying as a control measure
K. Ogata et al.
of malaria in Guatemala

25 Malaria control programme in Dar es Salaam and Tanga, Tanzania:
activities and results on the first year (1988/89)

T. Ikemoto et al.

26 Geopatholbgical study on malignant tumors in Western Kenya, East
AfricaK. Toriyama et al.

27 Enteropathogenic E: coli (EPEO and enterotoxigenic E. coli (ETEO
isolated in Northeast Thailand and their resistance to antibiotics

P. Gasaluck et al
28 The relation between protein and toxin production in V. cholerae
A. Mazuji et al.

29 Causative organisms o. f respiratory infections ‑community aquired

infection in Northern Thailand H. Hirose et al.
30 The possibility to acquire Strongyloides stercoralis infection among
pupils in the endemic aieas of strongyloidiasis in Okinawa, Japan
R. Asato et al.

31 A comparative study on an improvement of the agar plate method
regarding safety (sealing) , in Northern Thailand S. Kasuya et al.
32 How sensitive is the agar plate method for Strongyloides stercoralis?

K. Koga et al.
33 Four additional cases of gnathostomiasis doloresi and applicability of

ELISA diagnosis H. Maruyama et al.

34 An integrated application of an insectgrowth regulator, pyriproxyfen
and sound trap system for the control of the vector of Japanese enceph‑

alitis in an open paddy field in Thailand V. Kerdpibule et al.
35 Comparisons of genetic maps of two Aedes mosquito species ‑with

67

special emphasis on filarial susceptibility loci‑ T. Tadano
36 Non‑Mendelian population of a Triatoma infestans strain, a main

vector insect of Chagas' disease H. Hirai and I. Tada
37 Phylogenetic analysis of three species of Paragonimus, human inciden‑
tal parasites in Thailand and Japan I. Isozymes analysis

C. Komalamisra and T. Kanda
38 Two‑dimentional analysis of a putative eggshell precursor protein of

the female Schistosoma japonicum M. Kawanaka and H. Sugiyama
39 Fasciola cystein protease: species specificity and availability as an

antigen for enzyme‑linked immunosorbent assay (ELISA)
H. Yamasaki et al.
40 Avidin binding components in parasites
M. Itoh and S. Sato
41 Effect of chemotherapy with piquitong (Praziquantel) on murine schis‑tosomiasis mansoni and changes of serum antibody levels Y. Ito et al.

42 Detection and quantitative assay of Mamusi snake venom andantivenom by ELISA‑with special reference to in vivo effect of theantivenom
43 Sea snake bites in Okinawa

A. Sakai et'al.

Y. Araki and Y. Tomihara

44 Ixodid ticks and incidence of Borrelia burgdorferi in Nagano Prefecture,

Japan (preliminary report)
K. Uchikawa et al.
45 Recurrent outbreaks of tsutsugamushi disease and fiuctuation of vector
population densities
K. Uchikawa

46 Epidemiological study on schistosomiasis mansoni in rural areas
around Recife, Brazil
M. Tanabe et al.
47 Characteristics of ultrasonographic findings and liver dysfunction of
Schistosoma japonicum infection in Leyte, Philippines

H. Ohmae et al.
48 Ultrasonographic and pathophysiological changes after ‑treatment with
Praziquantel in Schistosoma japonicum infection in Leyte, Philippines
H. Ohmae et al.
49 Basic studies on Mongolian gerbils as a susceptible host to filarial

infection (5) serum lipid K. Shichinohe et al.

50 Basic studies on Mongolian gerbils as a susceptible h6st to filarial

infection (6) biochemical assay M. Shimizu et al.
51 Basic studies on Mongolian gerbils as a susceptible host to filarial

infection (7) serum protein K. Shichinohe et al.
52 Comparative studies on sensitivity to Brugia pahangi among coat color

mutants of Mongolian gerbile K. Fujita et al.
53 Falciparum malaria with bone marrow abnormalities resembling malig‑nant histiocytosis A. Hamada et al.

54 Use of Mefloquine in treatment of imported falciparum and ･vivax

malaria H. Amano et al.

55 Incidences of DIC complication in Japanese patients with malaria

K. Tanabe
56 The significance of time lag in the treatment of falciparum malaria

68

I. Ebisawa

57 DNA diagnosis of falciparum malaria Y. Wataya et al.
58 Serologic evidence of t0: ocariasis in Northeast Brazil

K. Nagakura et al.
59 Sero‑epidemiological survey of human toxocariasis in tropical zone
K. Kondo et al.
60 Serologic typing of clinical cases with amebiasis by gel diffusion

precipitin test E. Okuzawa et al.

61 The distribution Naegleria sp. and Acanthamoeba sp. in Kanto area of

62 Prevalence of Cryptospororidium infection among house rats. Rattus
rattus and R. norvegicus and the experimental infection in R. rattus

H. Yamaura et al.
63 The function of dengue virus specific proteins in virus reproduction:
Biological activities of NS5

E. Zulkarnain et al.

64 Mechanism of enhancement of arbovirus multiplication in mosquitoes
ingesting microfilariae of Dirofilaria immitis by histopathology andE. M. Zytoon et al.
electron microscopy
65 Development of microfilariae of Dirofilaria spp. studied by i,v vitro useof hemolymph of Aedes albopictus mosquitoes

E. M. Zytoon et al.

69Special lecture

1 THE INTERNATIONAL HEALTH AIDS OF JAPAN= ITS PAST AND FUTURETADAO SHIMAO
Regular Trustee of Anti‑TB Association,
Honorable Director of TB Institute

Present world health services seem to be classified into 3 patterns. The first one is seenin the advanced countries in where infectious diseases are seldom and people are sufferingfrom malignant tumors and adult diseases and their population patterns are shifted to aged
people. In this group the geriatric diseases are the main target of health service of the society.

The second one is seen in NIES or oil‑producing countries in where the diseases patterns are
just behind of the advanced countries and there are little serious problems in their healthservices. The third types are recognized among the most of the developing countries where
many infectious and parasitic diseases are prevailing and infant mortalities are very high andthe feasibilities of medical and sanitary services are very low due to the financial difiiculties.

These situations need rapid improvement. The populations proportions of people living inthese three types against those of whole world are 19% in the first group, 46% in the second
group and 35% in the third one. On the other hand the global spread of HIV infection andenvironmental hazards on human health are the common threat throughout the world. The
most urgent problem to be solved is the unbalanced medical service in the world. The
difficulties of improving the level of health services in the greater majority of the developing

countries are derived from the delays of their economical development. In some developing
countries their economical crisis resulted in big slow downs of health services. In thosecountries, populations are increasing rapidly and there are no hope of ljmproving their healthservice by elevating people's living standards. Effective provisions of health and medical
services in these countries are very difficult due to their scanty health budgets and poor
educational and social services. The front health service systems are not be well organized

and the developments of PHC are very slow. The systems of controlling diseases andmanaging health services are very poorly established. What is more, there are serious
problems of the migration waves of farmers from their home villages into big urban area
after natural disasters, severe drought and civil wars which make neiv slums in every big
cities. HIV is prevailing in and out of Africa, As a natural course of event in those areas

TB are much more furious ,than any past time.
The Japanese oversea health aids started in 1960. In those days it was just like big scale

traveling clinics, however, it changed the styles as the medical aids projects in 1970. Aftet
the terminations of reparations of 11 world war in the latter half of the nineteen‑seventies, thefree medical aids started and increased yearly. In the nineteen‑eighties, combination of free

medical aids and technical cooperation became the major parts of oversea health aids ofJapan.
Today Japan is the. second biggest economical major power, and is responsible forsupporting the developments of developing countries in cooperation with other advanced

70

countries. Amount of the Japanese ODA is now biggest in the world, however, the order ofthe rate of ODA against GNP among advanced countries has been as low as the 13th. Thismeans the effort of Japanese GQvernment to h^elp retarded countries is not enough comparing
with those of other advanced countries. From humanistic view point the medical service
should be very important in ODA. The level of Japanese medicine is fairly high and theJapanese pharmaceutical industries and medical instrument industries are also in high level
and they have enough abilities to support developing countries.
Japan possesses 'also enough knowledges and experiences in succeeding in control ofparasitic and infectious diseases in the past and is able to offer them to Qversea medical stuffs

in developing countries by training them in Japan.

Dr. H. Nakashima has become the first Secretary General of WHO from Asia and theworld is expecting Japan to play his big roll in the international health service.
But Japan has many disadvantage as follows in playing his rolls in international services.

We have little experiences in international cooperations in health service. We are suffering
the shortage of stuffs and specialists in this field. We don't have enough knowledges and ideas
in international health service. We don't have the exact system of criteria on the evaluationof our foreign medical aids. The Japanese are in general very weak in foreign language.
Very few Japanese are well versed in foreign languages besides English. Medical instrument
and drugs produced in Japan are made only for Japanese use. Very little informations areavailable about WHO and JICA in Japan and people don't know how they can work ininternational fields. The communication of the Government and NGO is very poor.
Japanese oversea medical aids have been originally established only by the requests ofthe partners and as a rule Japanese officials have had no original active intention. This
attitude resulted in tremendous wastes of medical instruments and facilities of aids projects.

Medical aids from J.apan in the future must be taken into account of the intornational
unbalance of health service and also social unbalances within the countries.
Medically developing countries must be classified in 3 classes A. B. C and their geographi,‑cal circumstance, cultural and linguistic situation should be cautiously studied. Before
starting aids to some country, they must examine and evaluate whether the past aids to thatcountry from Japan had been effective or not. Offer the most suitable medical aids projectfrom Japan and if it seems to be more suitable to ask the aids from WHO do not hesitate toask it to WHO. Regarding the maximum term of medical aid, in some project the limited 5
years is too short and much longer term should be considered case by case.

71

2 THE FUNDAMENTAL NATURE OF C IAGAS' DISEASE:
A VIEW PROVIDED BY IMMUNE RESPONSES AND IDIOTYPESDANIEL G. COLLEY
Research Career Scientist, Veterans Affairs Medical Center,and the Professor of Microbiology and Immunology, and of Medicine,
Vanderbilt University School of Medicine, Nashville, Tennessee,
U. S. A.

The etiologic agent of Chagas' disease is the protozoan Tr)panosoma cruzi, which infects

10‑15 million people in endemic areas throughout Latin America, and is naturally transmitted

by insect vectors of the family Reduviidae. Infection can also occur by congenital passage,orally, by laboratory accident, and in organ transplants and blood transfusions. About 15,000
T. cruzi infections a year occur in Brazil by contaminated blood transfusions, and 5‑51% of
the blood units recently tested in Bolivia were serologically positive. Transfusion cases in the

United States have been traced to immigrant Latin American blood donors. Recent immigra‑
tions of people of Japanese heritage from Brazil to Japan, might be something Japaneseparasitologists and tropical medicine workers need to consider in the future.
There are 3 Iife‑cycle forms of T. cruzi. Epimastigotes multiply in the midgut of theinsect vector, then differentiate in the hind‑gut into infectious trypomastigotes, which are
excreted with the feces or urine after the insect takes a blood meal. They enter the body by

mucous membranes or abraded skin, enter mammalian host cells, escape from thephagolysosomal vacuole, and differentiate into amastigotes. Intracellular amastigotes
multiply in host cells, redifferentiate to trypomastigotes, to be released upon rupture of thecel I .

Acute Chagas' disease can be a mild or severe illness of 1‑2 months or asymptomatic.
Morbidity can be localized (chagoma at the site of infection) , and/or systemic (fever, edema,

lymphadenopathy, enlargement of the heart, electrocardiographic changes) . Acute disease isusually accompanied by general immunosuppression, blood and tissue parasitemia, and canbe fatal. Usually, symptoms and parasitemia decrease in a few months, followed by lifelong
chronic infection with little morbidity, in which parasites are difficult to demonstrate.
Serologically‑positive, chronic asymptomatic patients are termed indeterminate (1) . Morbi‑

dity‑associated lesions develop in 20‑30% of chronic patients, after a variable number ofyears (usually 10‑20). Severe disease often inyolves myocardiopathy (Cardiac disease; C) ,ranging from minor electrocardiographic changes to sudden death by heart failure. Severe"digestive" megasyndromes can also develop.

Chagasic myocardial lesions are widely distributed lymphocytic/histiocytic
inflammatory infiltrates, often with multinucleated giant cells and plasma cells. Cardiac

muscle and neuron destruction occurs, followed by progressive fibrotic replacement. The

cellular participants in these lesions are only now being phenotypically defined.
Asymptomatic I‑patients who die of other, unrelated causes have identical (but less intense)

lesions. Longitudinal studies in endemic areas estimate the risk of I‑patients developing
overt cardiopathy in the third decade of life is 2‑5% per year.
The immunopathogenesis and immunoregulation of these cardiac lesions is of primary

72

interest in my laboratory and to our collaborators. This presentation will focus on the
immunologic profiles which occur during the chronic stages of the infection, and the findingthat both responses against T. cruzi antigens, and their immunoregulation, and responses
agaipst idiotypes associated with these anti‑ T. cruzi responses correlate with the ptesence ofthe different clinical forms of the infection.

The pathogenesis of chronic Chagas' disease is still in question. This is primarily because
whether the patient has the l‑, C‑, or digestive‑form, it is difiicult to detect organisms, either

as blood stage trypomastigotes or tissue‑dwelling amastigotes. Because of this it is popular

to consider that T. cruzi may induce an autoimmune state (anti‑heart muscle; anti‑nerve)that then,perpetuates itself in the presence or absence of organisms. Circumstantial evidence

for this theory is based on numerous findings in chagasic patients of lymphocytes andantibodies that respond to, or react with T. cruzi antigens and self antigens. Yet, as with any

proposed autoimmune condition, it is difiicult to prove a cause‑and‑effect relationship.

Chagasic patients' peripheral blood mononuclear cells (PBMO respond to T. cruzi
epimastigote antigenic extracts (EPD. If these responses are subdivided into levels of
resp,onsiveness and analyzed, almost all of the low responders are C‑patients. The responsesof these patients are also augmented the most by removal of adherent macrophage suppressor
lls, and their responses are partly augmented by indomethacin. Chronic I‑patients. are

medium or high responders, and their PBMC exhibit little or no adherent cell‑mediatedimmunoregulation. Comparisons of patients' Ab responses by Western blotting against
separated EPI and their cell‑mediated responses by T cell‑Western blotting have also shown
some differences between C‑ and I‑patients. PBMC from I‑patients responded more often tohigh molecular weight components (100‑150 kD) , while both I‑and C‑patients responded well
to moieties between 28‑32 and 48‑57 kD. The Ab reactivity profiles were similar to those of
PBMC. All chagasic patients' sera had Abs against a T. cruzi GP57/51 antigen, and the levelof all chronic patients' PBMC resp6nses to crude EPI correlated strongly with their responses
to highly purified GP57/51.

Patients with chronic Chagas' disease have peripheral blood anti‑idiotype (anti‑Id) T
cells that respond to anti‑EPI antibodies immunoafflnity‑purified from the sera of patients.

Some patients' PBMC anti‑Id responses to anti‑EPI Ids from C‑patients (Id‑O are inhibitedby chloroquine (Group l) , but some other patients' PBMC anti‑Id responses to Id‑C are notinhibited by chloroquine, anti‑HLA Class 11 antigens, or sodiurn azide (Group 2). Almost
70% of the patients in Group I are asymptomatic, and 100% of those in Group 2 have severe
disease. This is a striking clinical correlation with a given type of anti‑Id responsiveness,.

The direct (non‑processed; non‑MHC‑presented) stimulation of anti‑Id T cells from C‑
patients by Ids expressed on anti‑EPI antibodies from C ‑ patients could provide an im ‑munopathogenic basis for disease. Anti‑Id specific rabbit sera can detect Id differences in theanti‑EPI Abs from pooled or individual C vs. I‑cases. Competitive ELISA assays andWestern blot analyses of the Ids confirms that the Ids on I‑patients' anti‑EPI Abs areassociated with the primary structure of lg heavy and light chains, while the Ids on C ‑patients' anti‑EPI Abs are defined by intact, non‑denatured Ab molecules.
The chronic, endemic nature of Chagas' disease provides optimum opportunities forstrong maternal/Kneonatal Id interactions that could influence subsequent immune response
and immunoregulatory abilities of children born of infected mothers. Such interactions do
occur naturally, because the cord blood mononuclear cells from chagasic mothers' children

73

respondtoldsonanti−EPIAbs．Ahypothesisbasedonidiotype−inducedpathologyand
immunoregulationwill

account

aspects

for

many

be

described亡hatof thecould，throughimmunology

andpathology

matema1／neonatalof

Chagasinfluences，disease．

74

General presentation

1 WAYS OF THE JAPANESE SOCIETY OF TROPICAL MEDICINEAND THE JAPAN ASSOCIATION FOR INTERNATIONAL HEALTH
TAKEO MATSUMURA, SHOJI UGA, ATSUKO ITO

AND TOSHIKO SAWAYAMA
Department of Medical Zoology, Kobe Univershty School of Medicine

The Japanese Society of Tropical Medicine was founded in 1959 and the First Annual
Meeting was held in the autumn of that year under the presidency of Prof. Kaoru Morishita
now dec ased, of Osaka University. Since then all areas of tropical medicine, including
parasitology, bacteriology, medical entomology, infectious diseases, public health, nutrition
and acclimatization have been studied on the basis of factors such as climate, biology, culture(socioeconomic) and genetics. The results of the studies have contributed not only to sciencebut also to the welfare of human beings. The achievements of these societies have improved
the health of resid nts of the tropics, and also promoted international cooperation.

The very begining of tropical medicine as a separate field of medicine was probably thefoundation of the Liverpool School of Tropical Diseases in 1898. Soon after, the name of theschool was changed to the Liverpool School of Tropical Medicine. The first prospectus of theschaol, issued in 1900, gave as its objectives:(1) The training of workers in the special subject of tropical medicine,(2) The promotion of research into tropical diseases,(3) The organization of measures for prevention of diseases in the tropics,(4) The clinical care of patients suffering from tropical diseases (This objective of theschool was omitted accidently in the first prospectus but added I ter) .
These objectives have pointed out the direction of tropical medicine since then.
In Japan, international cooperation in the field of health was much encouraged by "Japan
Salon for International Health" held in June, 1983, by medical doctors and persons in related
fields. Various fields are part of the promotion of international health. It is necessary toenhance health of all persons everywhere; this concept was the starting point for the fouda‑tion of the Japan Association for International Health in 1986. The First Annual Meeting washeld in March 1986 under the guidance of Prof. Emeritus Naoichi Zuyama of Tokyo
University. The members of this association are medical practitioners, including medical
doctors, other workers in medical fields, and students. All want to contribute to the resolutionof the problems of disease and to the establishment of health and welfare, through interna‑
tional cooperation in collaboration with the inhabitants of developing countries, in particular.

These two groups plan close cooperation in the future, both having 'as their goal thehealth of persons all oyer the world.

75

2 MEDICAL PROBLEMS OF THE

JAPANESEDEVELOPMENTSTAYINGATTHE

OVERSEACOUNTRIES

KolcHIRo FuJITA1，MAsATosHI SuGIYAMA1，SETsuKo TsuKIDATE1，
AKIRA SuDo2，HIRosHI OHARA3，KAzuHIRo SHlcHINoHE4，

YosHIYuKl OKuwAKIラ，YAsuKo
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PRESENT STATUS OF THE，MEDICAL CHECK OF THE JAPANESESTAYING AT THE OVERSEA DEV皿、OPMENT COUNTRIESAKIRASETsuKo

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76

persons there.

We discuss here the results obtained from medical check and analysis the infection routesof viral hepatitis of type A as well as type B.

4 MALARIA STUDIES IN BANGLADESH
TAKESHI KURIHARA
Department of Medical Zoology, Teikyo Unrversrty School of Medicme

Malaria continues. to be a major public health problem in Bangladesh, according to the

Directorate of Health Services of the Bangladesh Government. Annual incidence of malaria
cases reported in the the country for the past 10 years has remained more or less static with

only slight annual fluctuations. The maximum number recorded was 45,902 in 1981, which
was 0.42 per 1,000 population. The country has been divided into three strata in terms ofmalaria transmission potential and activitie developed accordingly. Stratum‑1 is firstpriority areas which have a total 10 million population, most of them in the eastern part ofthe country. The proportion of Pl. falciparum infection showed an increasing trend andconstituted approximately 67% of all cases in 1989. The majority of these were from theChittagong District and it was suspected there were cases scattered in other districts which
had been brought irL from the hilly areas. Some 34 species of anopheline mosquitoes havebeen recorded in the country, four of which, namely An. dirus. An. philippinensis. An.
minimus and An. sundaicus are considered important vectors. An.' dirus from hilly andforest areas, in particular, seems to be playing a very important role.

5 ASSESSMENT OF EPIDEMICS BY FREQUENCY DISTRIBUTIONCURVE OF MALARIA ANTIBODY TITERSSHIGEYUKI KANO1, S. H. EL SAF12, F. EL D. M. OMER2
P. T. RIVERA3, A. A. EL GADDAL2 AND MAMORU SUZUKll
De artment of Parasitology, Gunma University School of Medicine*,

Blue Nile Health Project, Wad Medani. Sudan2 and Department of
Parasitology, University of Philippines, College of Public

Health. Manila, the Philippines3

When a malaria control campaign comes to an advanced stage, microscopic observationof blood smears may miss lower parasitemias than microscopic threshold. On such anoccasion, a suitable method for seroepidemiology is required in order to detect latent malarial

foci in the controlled areas. A follow up surveys were conducted at two villages, Sennar andMobi, in the Sudan Gezira in December 1987 and January 1989. In 1987, 80 school children
were examined in Sennar and 5 children with parasitemia were detected, while, in Mobi, noneout of 46 manifested parasitemia. The statistical analysis showed no significant difference of

77

endemicity between the two villages. Antibody positivity rates of Sennar and Mobi were
52.5% and 34.8% respectively. Again significant statistical difference was not noted between

the villages. However notable difference of endemicity derived from the type of frequency 'distribution curves of malaria antibody titers was recognized: A bimodal distribution of titers

was obtained with sera collected in Sennar, and antibQdy pattern characterized by a single
low‑titerd peak resembling an exp6tential curve was obtained with those collected in Mobi.

The former type of curve suggests that the population sampled lived in an endemic area
where transmission had recently occurred, and the latter shows that malaria was absent ortransmission had been interrupted in the area. Thus a potential higher risk of malaria
epidemics in Sennar' than in Mobi was estimated in December 1 87. In August 1988, a heavy
rainfall caused a flood of the Nile in the Gezira afea and a malaria pidemic did occur in
Sennar triggered by the flood. In January 1989 in Sennar, 34 out of 72 exarhinees manifested

parasitemia, positivity rate of antibody was 75.0% , and the second mode of the frequency
distribution curve became higher than that obtained before the flood. While in Mobi, 7 outof 90 manifested parasitemia and antibody positivity rate was 29.0%, which was the samelevel of endemicity as obtained in 1987. However the antibody pattern chan ed into bimodal
one, which suggested a small outbreak of malaria caused by the fiood. It was affirmed in thissurvey that t, he endemicity of malaria can be precisely refiected in the shape of the frequency

distribution curve ' of antibody titers. And, with this method, potential risk of malaria
outbreak in Senhar was foreseen in'l987 before the fiood that triggered 'the epidemic in 1988.

6 EPIDEMIOLOGY OF MALARIA THROUGH QUESTIONNARECONDUCTED ON JICA EXPERTS WORKING‑ IN

THE TROPICAL AND TEMPEBATE AREATOSHIRO SHIBUYA, KISEKO KAMEI, MICHIO MIYAZAKI,TAKAYA IKEMOTO AND RYUICHIRO MAEDA
Department of Parasitology, Teikyo University School of Medicine

A questionnaire was conducted on JICA experts living in various places on the situation,
countermeasu're and problems of malaria in their area in 1963. The objectives of the survey

was to collect informations on1 ) Iocal situations of malarial transmission2 ) distribution of drug resistant malaria
3 ) personal history of catching rrralaria

4 ) basis of diaguosis
5 ) actual conditions on prophylactic measure, treatment and means to obtain antimalarial

drugs, and6') health problems they are facing in general
Samples were selected ramdomly from 1,11Q expert . 211 questionnaires were sent and
136 replies were collected. Results are largely as follows:1 ) Malaria is endemic in most countries of their duty in Asia, Africa and Central‑SouthAmerica. And it is comfirmed that experts in Africa are exposed to higher risk of78

infection than those in other regions from the view point of daily behavioral range.
2 ) There was considerable difference in the informations on the situation of drug resistantmalaria between those obtained by the present study, and those reported by WHO, whichplight be due to unsatisfactory understanding of respondents regarding to the presence ofresistant malaria parasite and its effect on the health problem in the countries in warm
climates.

3 ) Incidence of malaria among Japanese experts was highest in Africa showing 22%, thatin both Africa and Cental‑South America was approximately 6%.
4 ) Diagnosis was mainly based on the clinical signs and symptoms, the confirmed cases byparasitological examination was only 27%.
5 ) Generally speaking, the experts possess proper knowledge about prophylaxis of malria.
Distribution of chloroquine resistant malaria is expanding recently. Moreover, multidrug

resistance, resistance against newly developed mefloquine have been reported, causing some
confusions in the countermeasure by the authorities.
It is suggested from the results that proper measure should be taken regarding the use of

new drugs and health education as well as clinical diagnosis and treatment for Japaneseworking in malaria endemic areas most of whom is not immune against the disease.

UP STUDY OF MALARIA INDIRECT FLUORESCENT
7A FOLLOW
ANTIBODY POSITIVITY RATES OF JAPANESE STAYEDIN THE ENDEMIC COUNTRIESMAMORU SUZUKI*, RUMIKO KOSUGll and HIROSHI NARUT02
Department of Parasitology, Gunma University School of Medicine'and Japan Overseas Cooperation Volunteers, Japan International

Cooperation Agency'

Since 1982, we have conducted malaria serological survey with indirect fluorescentantibody test on Japanese who had stayed in malarious areas for 2‑3 years and returned
home. Five hundred and eighty three females and 2,127 males of 1‑71 years old with peak age

at 25 were tested. The endemic countries they stayed are 11 African 13 Asian and 6 Latin

American countries. The returner from Africa made the majority of the examinees. In
Africa, it was noted that positivity rate in 1987‑1990 increased comparing to that tested in

1982‑4. In some countries, as high as 40% positivity rate was recorded in recent years. In

Asia, stable positivity rate was noted in Papua N w Guinea and in Solomon islands.
Significant positivity rates were recorded in the Philippines and Malaysia in some years,
however, positive cases turned to negligible rates or to null in the following years. Japaneseare non‑immune to malaria, and likely to take prophylactic drugs. This follow‑up study will

work as a parameter for malaria incidence and the prevalence of drug resistance malaria,
particularly in Africa where malaria monitoring systems are still insufiicient in general.

79

8 INVESTIGATION OF IMPORTED MALARIA CASES IN 1988‑1989
HIROSHI OHTOMO*, SUEHISA TAKADA2, TsUNEJI ARAK13
TSUTOMU TAKEUCHI', MORlYASU TsUJ15, GOTA MASUDA6

ANb MiTSURU AKA07
Department of Parasitology, Gifu University School of Medicine*,

Department of Medical Zoology, Osaka City University School of
Medicine', Department of Parasitology, Nara Medical College',
Department of Parasitology, School of Medicine Keio University',

Department of Parasitology, University of Hiroshima School of

Medicine=, Tokyo Me, tropolitan Komagome Hospital'and 0Saka City Momoyama Hospital'
Since 1960, no indigenous malaria has been detected in Japan. In our country, however,increase in imported malaria is recently drawing attention again as the international travelis getting more active. The present report is to introduce the current status of imported
malaria in Japan, which is based on the results of the investigation on the incidence ofmalaria, carried out by the authors for 2 years in 1988 and 1989 inclusive.
The total number of malaria cases detected for 2 years was 185 (83 in 1988 and 102 in1989). All were imported. The patients were made up 132 Japanese (71.4%) and 53
foreigners (28.6%). The malaria cases consisted of '149 males (80.5%) and 36, females(19.5% ) . As a whole, the majority of the patients were males in the 20‑40 year age group.
Of the 185 cases, 56.8% were caused by Plasmodium vivax, 35.1% by P. falciparum, 2.2% by

P. malariae, 1.6% by P. ovale and mixed infection res ectively. The species was notdetermined in the remaining cases. In the majority of patients (64.1%) were acquired in theSoutheast Asia and We, stern Pacific areas, in 30.9% the origin wa Africa area. Malaria cases

grouped accordihg to the period of time between the date of arrival in Japan and the onsetof illness was analysed. Falciparum malaria started within I month after arrival in Japan in
all cases, while only 52.0% of the vivax malaria cases began to show its clinical symptoms
in the same period of time and 28.0% toOk as long as 6 months to over a year untill the onsetof the symptom. These results suggest that surveillance concerning malaria onset is required,in general, for at least I month after arrival for falciparum malaria, and 6 months to one year

for vivax malaria. Regarding the prognosis of patients, relapse concerned in 8.9% of thevivax malaria, but no death was reported.
Malaria is endemic to a wide region of the torrid and subtropical zones and it is thoughtthat as many as two hundred million people are infected with malaria and that one million
die of the disease every year. Recentry, this disease has become a serious problem of public
h alth due to increase in imported cases in Western countries and also Japan,

80

9 THE LOCALIZATION OF NEURAMINIDASE ON STAGES OFTR YPANOSOMA CRUZITOSHIKI AJ11, MIERCIO E. A. PEREIRA2 and MASAMICHI. AIKAWA3
Department of Parasitology, Okayama University School of Medicinel,
Department of Geographical Medicihe and Infectious Diseases,
Tufts University Medical School, Boston, U. S. A.2 and Institute of

Pathology, Case Western Reserve University, Cleveland, U. S. A.3

A growing body of evidence indicates that neuraminidase is important in mediatinginteractions between Tmpanosoma cruzi and host cells. This parasite has three develop‑
mental forms which differ in their expression of neuraminidase activity. he vitro studies havedemonstrated that living trypomastigotes have the highest enzymatic activity. Neur‑aminidase was shown to be heterogeneously distributed among strains of T. cruzi with thehighcst level of activity being associated with the least virulent strains. Previous work using

indirect immunofluorescence and immunoprecipitation assays suggested that neuraminidase

is located on the surface membrane of trypomastigotes. In this study, we used im‑
munoelectr6n microscopy to directly demonstrate the distribution and localization of neur‑aminidase in various stages of T. cruzi.

Cultures of the Sylvio X‑10/4 strain of T. cruzi were used in all experiments. Im‑

munoelectron microscopy using TCN‑2, a monoclonal antibody specific for the neur‑aminidase, was performed to determine the precise localization of the parasite enzyme. In
agreement with previous observations, TCN‑2 reacted with tissue culture trypomastigotes,
but not with epimastigotes, amastigotes or intracellular forms in intermediate stages ofdevelo pment.
Neuraminidase was localized on the surface of tissue culture trypomastigotes, in particu‑
lar in the fiagellar pocket region. Neuraminidase was also detected in the Golgi apparatusof the trypomastigotes, suggesting that the enzyme is modified post‑translationally. In
agreement with this suggestion, digestion of neuraminidase with N‑glycanase, an enzymethat releases N‑linked oligosaccharides, decreased the molecular weight of the polypeptides

that make up neuraminidase: Neuraminidase was also found in intra‑ and extracellulartrypomastigotes.
Immunoprecipitation by TCN‑2 of metabolically labeled parasites revealed that neur‑aminidase of intracellular trypomastigotes is molecularly indistinguishable from the enzyme
of extracellular parasites.

81

10 CHEMOTAXIS OF ELONGATED BLOODSTREAM FORMS
OF TRYPANOSOMA VIVAX (IL 1392) AGAINST POLLENGRAINS OF FUCHSIA HYBRIDA AND SAINTPAULIA IONANTHAHIROYUKI HIRMI AND KAZUKO HIRUMI
Cell Biology Laboratory, International Laboratory for Research on

Animal Diseases (ILRAD), P.O. Box 30709, Nairobi, Kenya

Bloodstream forms (BSFs) of a West African stock of Tr)panosoma vivax (one of threemajor pathogens of African trypanosomiasis) are infective for rodents and transform from
actively dividing normal forms (N‑BSFS) at the rising parasitemia to non‑dividing elongatedforms (E‑BSFS) at the terminal stage of infection.
The present study revealed that these two forms showed different responses to pollen
grains of Fuchsia hybrida as well as of Saintpaulia ionantha when the grains were mixed with

the infected mouse blood. Although the N‑BSFs showed no response to the graips, the E‑
BSFS Showed rapid swarming around the grains within 30 seconds. The result demonstrated,
for the first time, the existence of chemotaxis in the BSFS Of a salivarian trypanosome against

a natural substance.
This reaction was observed by placing 3‑5 drops of tail blood of the infected mouse bloodover an area where the grains were smeared on a microscope slide and immediately examin‑
ing by phase‑contrast microscopy. The test slides could be stored at least for two months
indicating that the substance on the grains which attracted the E‑BSFS, is highly stable atroom temperature. Subsequent examinations of pollen grains of Hemerocallis fulva and two
species of Lilium revealed the absence of the attractant on the pollens of these plants.
The flndings suggest that (1) the chemotaxis of E‑BSFs is highly stage specific, (2) theattractant is present on the pollens of specific plants, such as Fuchsia and Saintpaulia, and (3)this system may be useful for studying the mechanism underlying the stage transformation of
T. vivax, particularly from the dividing stage to the non‑dividing stage. Identification of thechemical nature of the attractant is presently underway.

11 LEISHMANIASIS AND ITS VECTOR SANDFLY IN PARAGUAYYOSHIHISA HASHIGUCHll, MASATO FUkUYA2 and MASATO KAWABATA3
Department of Parasitology* and Institute for Laboratory Animals',

Kochi Medical School and Department of Clinical Pathology,
School of Medicine Nihon University'

Cutaneous and mucocutaneous leishmaniasis are endemic in Paraguay; the disease occurs
in a wide range of the country. It is, therefore, a considerable problem of public health,especially in the north‑eastern area, where agricultural human population is very high,because of the suitable (fertile) terrain for cultivation. Little information, however, has been

available on epidemiological features of the disease, such as infection rates of inhabitants,

82

sandfiies and wild and domestic mammals with Leishmania in each endemic area of Par‑aguay.
Historical notes: Many clinical cases were reported in the country since Migone (1913)
described a case of visceral leishmaniasis (it is considered, at present, to be an imported bne) .

Recently, Yamasaki et al. (1988, 1989) briefly mentioned current status of the disease andschizodeme characterization of several Leishmania isolates in the country. As to the causa‑
tive agent of leishmaniasis in Paraguay, only one species, L. braziliensis, has been recorded
hitherto by Grimaldi et al. (1989) who examined Leishmania isolates from patients living inthe south‑eastern area of the country.
Object of the present survey: For a better understanding of the epidemiological featuresof leishmaniasis in Paraguay, a preliminary survey on natural infections of humans, wild anddomestic mammals and vector sandflies was made.
Materials and methods: Examinations were performed in four endemic areas, Limoy,
Departamento de Alto Parana, Brazilero‑cue and Cantera‑voca, Departamento de Caaguazu
and Tavai, Departamento de Caazapa. Physical (dermatologicaD examinations of inhabit‑
ants were made; skin test (Montenegro reaction) was also employed by using antigens
prepared from L. panamensis promastigotes (Furuya et al., 1989)
Results: In the examination of 149 inhabitants (63 males and 86 females) in Limoy, Alto

Parana, 66 (44.3%) were positive for dermal lesions, while 74 (49.7%), for intradermal
reactions. These positivity rates had a tendency to be higher in males than in females; therates increased with age in both sexes. Frequency distribution of dermal lesions in skin test‑

positive persons was 55%, Iower extremities; 24%, upper extremities; 11%, trunk; and lO%,
face. From these results, it was estimated that sandfiies in the area preferably fed on lower
parts of the body surface. Two of the 66 (3.0%) subjects with dermal lesions revealed severe

mucocutaneous manifestations, Iosing nasal septum completely. Similar epidemiological
data were also obtained in inhabit nts of Tava‑i, Departamento de Caazapa. In the present
survey of sandflies, 5 species of the genus Lutzomyia, intermedia, whitmanni, shannoni.
migonei and jischeri, were collected at different endemic areas. A total of 615 sandflie weredissected, of which only one (Lu. whitmanni) from Brazilero‑cue was positive for leish‑
manial promastigotes, showing a very low rate of infection. At present, however, this species
is estimated to be a potential vector of leishmaniasis in the country. To date, 12 species of

Lutzomyia have been reported in Paraguay; the sandfly fauna, therefore, seems to be very
poor (Alexander, pers. comm.). Although liver and spleen punctures of rats and wild deers
were performed for in vitro culture, all were negative for the parasite.

83

12

THERAPEUTIC EFFECT OF SULFAMOYLDAPSONE (SDDS)
IN MICE INFECTED WITH LEISHMANIA D ONO VANIKAZUHISA MORISHIGEl, TOSHIKI AJI , AKIRA ISHH*,

YUSUKE WATAYA2 AND TATSUJI YASUDA3
Department of Parasitology, Okayama University Medical School*
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences' and Institute of Environmental Medicine'

Okayama University

BALB/C mice infected with Leishmania donovani were treated with sulfamoyldapsone(SDDS) for appraising therapeutic effect. The mice were infected with I x 10' L. donovani
promastigotes 2S‑15M strain via tail vein. SDDS was administered five times by intramus‑
cular (i. m.) or intraperitoneal (i. p.) injection on alternate day after infection of L. donovani.

SDDS administered orally .was done once a day for thirteen days after infection. In thesecond week after infection, impression smear of the liver was prepared to determine theparasite load which was expressed as Leishman Donovan unit (LDU) by 1,000 hepatic cell
nuclei. In the mice infected with L. donovani, the group administered SDDS 100 mg/kg orally
showed 69% inhibitive effect as compared with the group administered saline. This was
confirmed by a repeated experiment. On the other hand, mice injected with Pentostam, apositive control drug, showed 79% inhibition. The groups administered SDDS 20 mglkg
orally, SDDS 20 mglkg i. m. and SDDS 10 mg/kg i. p. showed 47%, 41% and 35% inhibitiveeffect respectively. The groups administered metronidazol 50 mglkg orally and mebendazol
50 mg/kg orally showed 54% and 43% inhibitive effect respectively. SDDS 100 mglkg orally
can be useful for treatment of L. donovani in future because that had high inhibitive effectas well as Pentostam.

13 EPIDEMIOLOGICAL STUDIES ON VIRAL HEPATITISAMONG LONG‑TERM SOJOURNERS IN THE DEVELOPING
COUNTRIES AND EVALUATION OF PREVENTIVE MEASURESHIROSHI OHARA , ISAO EBISAWA', EITARO HORll, KOICHIRO FUJITA3

AND HIROSHI NARUT04
Department of Medical Zoology, Saitama Medical School*
Keihin Railway Kawasaki Clinic', Department of Medical Zoology,
Faculty of Medicine, Tokyo Medical and Dental Universityi

and Japan International Cooperation Agency'

It is known that acute viral hepatitis is very common among sojourners in developing
countries. In order to conduct effective health control epidemiological studies were made on
viral hepatitis which occurred among Japanese staying in developing countries, and evalua‑tions were carried out on preventive measures against it.

84

The subjects of present study were a group o'f Japanese people staying in developing
countries for two years. Mid year population of the group was 1,732 in 1988. Period of thepresent study is ten years from 1979 to 1988. The study was conducted based on the reports
from ofiices in each country, survey trips and serological studies on the subjects.
In 1979 frequency of hepatitis A (HA) was very high showing 79% of total hepatitides.
However after starting of inoculation of human immune serum globulin (ISG) the frequency
of HA declined remarkably. Marked statistical significance was recognized in the efiicacy of
ISG.

Among 35 cases of hepatitis B (HB) (34 males and I female) , 2 derived from HBeantigen carrier while the remaining 34 were regarded as infected during their stay indeveloping countries. No cases of HB were recognized among those who got injection of HB
vaccine and statistical significance was recognized in the efiicacy of HB vaccine. The rateof people whose HB marker terned positive during their stay is showing a tendency of
increase (4.2% in 1987) . The frequency of infection with HB virus is especially high in Asianand African countries where the carrier rates of native people were also high.

Ten cases of non‑A non‑B hepatitis was recognized. Among them 7 were infected in
Asia, I in Africa and I in Middle America. The route of infection of the most of the cases

were regarded as oral one.
When Japanese people stay in developing countries, the danger of contracting with acuteviral hepatitis is high. Especially a,mong younger generation, carelessness easily leads tocontraction with HA. As preventive measures, inoculation of ISG is effective as well as
paying attention to foods and drinks. The chances of infection with HB virus is also high.
As routes of infection, sexual intercourse in considered to be important as well as medical

practices. The efiicacy of HB vaccine to those who stays in developing countries was
confirmed.

14 INFECTION RATE OF INTESTINAL PARASITESIN JAPANESE INHABITING TROPICAL OVERSEACOUNTRIES ‑STUDY OF PAST 10 YEARS‑SETSUKO TSUKIDATE , KOICHIRO FUJITAl, MASATOSHI SUGlYAMAl
AKIRA SUuo2, KAZUHIRO SHICHINOHE3, HIROSHI OHARA', NoBUAKI AKA05

KAORU KoND05, YASUKO OGASAWARA6 AND TAKEO ASAKURA6
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical
and Dental University*, National Medical Center', Department ofLaboratory Animal Science, Nippon Medical School', Department ofMedical Zoology, Saitama Medical School', Department of
Parasitology, Kanazawa University School of Medicine*and Japan Association for Tropical Medicine'

Many Japanese inhabiting tropical oversea countries are suffering from intestinal prot‑ozoa and helminths diseases. We carried out to check intestinal parasites of Japanese in 8

countries of Asia and Oceania, 9 countries of Middle East and East Africa and 12 countries

85of South America.
High positive infection rates were obtained from Japanese in these areas, such as 16.7%

in Panama, 12.8% in Pakistan and 9.1% in Indonesia at the h vestigation in 1989. People in
Asia and Oceania area were infected with highest of 5%, those in South America were of
4.5% and those in Middle East of 3.4%.
People in Asia, Oceania were apt to infect with helminth, on the other hand, those in
Middle East were inclined to infect with protozoa.

15 BACTERIOLOGICAL AND CHEMICAL STUDY OF DRINKINGWATER IN THE TROPICAL COUNTRIES‑STUDY OF PAST 7 YEARS‑MASATOSHI SUGlYAMA , SETSUKO TSUKIDATE*, KOICHIRO FUJITA ,YOSHIYUKI OKUWAK12, KAZUHIRO SHICHINOHE3, HIROSHI OHARA*,YASUKO OGASAWARA* AND TAKEO ASAKURA5
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical
and Dental University*, Department of Microbiology, Kagawa
Nutrition College', Department of Laboratory Animal Science, Nippon
Medical School', Department of Medical Zoology, Saitama Medical
School' and Japan Association for Tropical Medicine=

Not a few Japanese living or traveling in tropical areas suffer from diarrhea and otherintestinal infections, one of the major causes of which is considered contaminated drinking

water.
Examination of drinking water used in houses of Japanese inhabiting in 8 countries ofAsia and Oceania, in 9 countries of Middle East and East Africa and in 10 countries in SouthAmerica were conducted as a past of the health management of Japanese in tropicaldevelopmental countries.
Results were obtained as follows I ) Most of tap water specimens collected contained
no free available chlorine 2 ) Tap water specimens were obtain found contaminated withcoliform bacilli and sometimes with E. coli. 3 ) In some countries of North Africa and Near

East tap water was very high in hardness.

86

16 ESTABLISHMENT OF SPECIFIC T CELL LINES WHICH
COULD RESPOND TO SOLUBLE EGG ANTIGENS OFSCHISTOSOMA MA NSONITERUAKI AMANol, TOMOO OSHIMAI AND D.G. COLLEY2
Department of Parasitology. Yokohama City University School of

Medicine* and Department of Immunology, Vanderbilt University
School of Medicine, Nashville'

In schistosomiasis, the granuloma formation around the deposited eggs in portal vein ismost important pathological phenomenon. It is also well known that cellular and/or humoral

immune responses are correlated to regulate this disease. In schistosomiasis mansoni,
cellular immune mechanisms are suspected to be more important than humoral immune
system to modulate and regulate this disease. As T cell clones can be established to respondsoluble egg antigens of S. mansoni (SEA), we will be able to understand more well themechanisms of granuloma formation and the functions of T cells.
In this study, we tried to establish T cell lines or clones from CBA/J mice infected with

S. mansoni. Spleens were aseptically removed from the 6 weeks infected CBA/J mice, whichwere infected with 30 cercariae of S. mansoni (Puerto Rican strain) with the tail exposure
method. Homogenized spleen cells were cultured in 96 fiat bottom wells with RPMI 1640,
including 10% FCS, 3% Penicillin‑Streptomycin (Gibco) and 5 pg/ml SEA. At I week later,

culture supernatant was taken out and new complete medium, including 20% MLA‑144
culture supernatant as IL‑2, was added into each wells. As the cells in wells were multiplying,

the supernataht were･ removed and new complete medium including SEA was put into eachwell with 5 x 105 irradiated feeder cells. After this, the supernatant was changed to newcomplete medium, including IL‑2, or SEA with feeder cells as antigen presenting cells oncea week. On the way, the multiplying cells were checked to respond to SEA antigen or IL‑2
with 3H‑TdR up take test. In many cases, those T cell lines had lost to respond to SEA, but

could respond only to IL‑2. Only one T cell line could respond to SEA (10,865 c.p.m.: background 193 c.p.m.) . As these cells were cultured with normal spleen cells in 24 wells including

SEA‑coated beads, these cells aggregated around beads at 5 days of cultivation. However,as these T cells were cultured with irradiated feeder cells, granuloma formation was notfound around beads. The normal spleen cells could not aggregate around beads, as only thosecells were cultured with beads. From this result, we may suspect that this T cell line be able

to work. as helper cells to make granuloma around eggs.

87

17 REGULATION OF THE T CELL RESPONSE TO SCHISTOSOMAJAPONICUM EGG ANTIGEN BY CONCOMITANT CELLULAR

AND HUMORAL MECHANISMSNOBUO OHTAl, AKIRA ISHIll and YUKIO HOSAKA2,
Department of Parasitology, Okayama University School of Medicine*and Department of Parasitology, National Institute of Health'Regulation system of Schistosoma japonicum egg antigen‑specific T cell response wasanalyzed in humans. Individuals with previous infection of S. japonicum failed to show
vigorous response of helper/inducer T cell response (CD4+) to a particular fraction (18 kD>

of soluble egg antigen (SEA) , while strong response was observed for other SEA fractions.

The diminished T cell response was restored when peripheral blood lymphocytes werepretreated with anti‑HLA‑DQ monoclonal antibody (mAb), or with anti‑CD8 mAb. Thissuggested the existence of HLA‑DQ‑controlled, a SEA fraction (18 kD) ‑specific suppressor
On the other hand, serum‑mediated regulation of SEA‑specific T cell response was also
tested. When we added autologous serum to SEA‑specific human T cell lines (DC3+, 4+, 8‑ ) ,we observed suppression of T cell proliferation to the SEA fraction of 18 kD. This suppres

sive activity was detected in the lgG2 subclass. Serum‑mediated suppression was dose‑
dependent and antigen‑specific. T cell response to the SEA fraction was modulated in thepresence of 100 pglml autologous as well as allogeneic infected lgG2'
Considering our finding that the CD8+ suppressor T cells had no effect on established T
cell lines, the suppressor T cells and infected lgG2 had distinct regulatory mechanisms. Those
results suggest that T cell response to a particular component (s) of SEA is strictly regulated

through both cellular and humoral mechanisms in human chronic schistosomiasis japonica.

18 CROSS‑REACTIVE IDS ON RABBIT ANTI‑SEA ANTIBODIESSTIMULATE ANTI‑ID LYMPHOCYTES RESPONSES BY CELLSFROM CBA/J MICE INFECTED WITH SCHISTOSOMA MANSONIMASATOSHI NAKAZAWA1, TERUAKI AMANol, TOMOO OSHIMAAND D. G. COLLEY2
Department of Parasitology, Yokohama City University School of

Medicine* and Department of Microbiology and Immunology,
Vanderbilt University School of Medicine, Nashville'

Many recent studies have investigated the possible roles of idiotypic/anti‑idiotypicinteractions in schistosomiasis mansoni and japonica. In those works, syngenic idiotypic
antibodies were used in human and experimental animals. In this study, we described asystem in which spleen cells from S. mansoni‑infected mice proliferated in vitro upon

exposure to blastogenic anti‑SEA antibodies from rabbits immunized with soluble eggs

88

antigen of S. mansoni (SEA), but those of normal mice did not.
Male CBA/J mice (obtained from Charles River Japan, Inc.) were infected with tailexposure method of 30‑45 cercaria of Puerto Rican strain of S. mansoni.
Polyclonal anti‑SEA antibodies were prepared from individual serum of rabbits immun‑

ized with SEA by afflnity chromatography on a SEA‑Sepharose column. Normal rabbits lgG(NRlgG) was prepared from the pooled sera of healthy rabbits by the method of ammonium
sulfated precipitation.

Polyclonal purified anti‑SEA antibodies were separated in 10% SDS‑polyacryl‑amide
gel electrophoresis and stained with Coomassie blue stain. The visible bands of purified anti‑

SEA antibodies were those that correspond to immunogloblin heavy and light chains and no
SEA components. The individual purified anti‑SEA antibodies had showed different lgGbands by Western‑immunoblotting methdd.
Lymph node and spleen cells (5xl05 cells/welD were cultured for 3 days in 96 well
microtiter plates in 200 pl RPMI 1640, containing lO% heat‑inactivated fetal calf serum, 3%
penicillin‑streptomycin (Gibco) and either 4 pg/ml SEA or 40 pglml anti‑SEA antibodies.
Cells were plused for the final 8 hr of culture with 0.5 pCi 3H‑TdR, harvested on glass fiber
filters and incorporated radioactivity was counted in liquid scintillation counter.
Until 6 weeks after infection, spleen cells did not respond to purified anti‑SEA antibqdies

from immunized rabbits. At 8 weeks after infection, spleen cells responded storongly to anti‑
SEA antibodies. However, the individual urified anti‑SEA antibodies had induced different

responses from 8 weeks infected mice. The specificity of the responses was demonstrated by
the inability of cells of all‑stage infected mice to heterogenic NRlgG. Furthermore, spleen
and lymph node cells from individual uninfected mice did not respond to anti‑SEA antibodies
nor to NRlgG. Similary, cells from uninfected mice did not respond to SEA, while cells from

6 to 12 weeks infected mice responded strongly.
These data demonstrated that T Iymphocytes from S. mansoni‑infected CBA/J mice canbe stimulated to proliferate by exposure to heterogenic antibodies with specificity for schis‑tosomal antigens.

19 IMMUNOGENICITY OF ULTRAVIOLET‑ATTENUATEDSCHISTOSOMA MANSONI CERCARIAE
HARUO KAMIYA, HIROSHI SATO, TOSHIHIRO OzAKI,

MASAHIRO KUMAGAI AND TAKASHI INABA
Department of Parasrtology, Hirosaki University School of Medicine

It has been well demonstrated that highly gamma irradiated cercariae of Schistosoma
mansoni elicit the high level of immunity against the challenge infection (Smithers andDoenhoff, 1982; Dean, 1983; Kamiya, 1986) . Even though the nature of this immunity has been

mainly analyzed in mouse model, the newer vaccinated laboratory animal model may provide
the new facets of immunity to elucidate its mechanisms. And also, the nature of stimulus has
a profound influence on immune expression in laboratoy animals.
In these aspects, we investigated the immunogenicity of ultravidlet (UV)‑attenuated89

cercariae of S. mansoni. Cercariae of S. mansoni were irradiated at 254 nni with UV Iamp.

The parasites exposed by 15 or 18 mJlcm2 of irradiation were not recovered by portal
perfusion at 21 days post infection. This fact was confirmed according to chase of theirradiated parasite migration by means of tissue mincing method and also indirect immuno‑
histochemical staining.
Moreover, Hartley guinea pigs, ICR mice, male and female Mongolian gerbils vaccinatedwith cercariae attenuated by 18 mJ/cm2 UV‑irradiation showed 44, 51, 13 and 22% immunity
against ,challenge infection, respectively. This revealed that different kinds of vaccinatedanimals exhibit different level of resistance .against the challenge infection.

To investigate the mechanisms of protective immunity induced by UV‑attenuatedcercaria vaccination, we examined, the peripheral eosinophil kinetics of vaccinated guinea
pigs.

Vaccinated guinea pigs did not show any peripheral eosinophilia until the end bf experi‑

ment at 12 weeks post vaccination, although peripheral eosinophil number in guinea pigs
infected with 500 normal cercariae significantly increased after 8 weeks post infection.
However, when vaccinated guinea pigs were challenged with normal cercariae at 5 weeks ofvaccination, peripheral eosinophila was evoked at 3 weeks of post‑challenge.
We also mentioned the significant role of epidermal Langerhans cells and lymphocyte
proliferation in lymph node of guinea pigs vaccinated with UV‑attenuated cercariae.
Therefore, vaccine model using UV‑attenuated cercariae will provide precious informa‑tions on the mechanisms of protective immunity.

20 A COMMUNITY BASED S UDY ON MEASLES EPIDEMICS
I. FACTORS AFFECTING SPREAD OF MEASLES VACCINESHIN ISOMURAI AND D. S. AKRAM2
Aichi Prefectural Institute of Public Health*and Department of Pediatrics, Civil Hospital, Karachi2

Previous studies conducted by Takasu and Ahemed (1987) disclosed two remarkablecharacteristics of SSPE in Karachi; (1) High incidence of the disease in the area, and (2)relatively older contraction of preceding measles infec'tion in many of the cases. In relation

to these findings, we conducted a preliminary investigation on clinical epidemiology ofmeasles in children in the city. In order to clarify the incidence and clinical pictures ofmeasles, mothers and grandmothers were interviewed asking for their concepts on clinicalpictures of the disease and history of measles attack in their children. Interviewers were also

asked questions on factors affecting acceptance of immunizations, e. g. information resources

and motivation to attend immunizations, key person to take children to clinic, and reasons ofabsence of immunizations.
During a period from 16th to 24th November 1988, and 28th October to 24th November
1989, 126 mothers, grandmothers and fathers who accompanied their dhildren to Civil

Hospital Karachi (Hospital group) and 50 families in Neelam Colony in Karachi (Commu‑
nity group) were interviewed.

90(1) Clinical manifestations of measles, "khas.era" in Urdo usually developed high fever for

more than seven days with generalized exanthema and severe cough. Once the diseaseattacked a child, he/she had got long lasting immunity. These findings may indicate that"khasera" has an entity very similar to measles and it has not been confused with other viral

exanthematous diseases such as rubella and enterovirus infections. As to complications,many of the interviewers talked of many kind of serious complications of measles. Impor‑tance of immunizations are well recognized to the interviewers. (2) The interviewers had atotal of 814 children. All but 5 had po repeated episode of measles. This finding again
supports the idea that "khasera" gives long lasting immunity, same as measles. Age of thecontraction of measles distributed widely from infancy to 12 years old but most of them wereattacked before 6 years of age, mainly between 9 and 18 months. Relationship between
measles vaccination and measles attack was not clearly defined. However, there were many
vaccin̲ e failures according to their immunization records. A survey on seroconversion ofchildren after the measles immunization has now been in progress. (3) There was no relation

between the age, occupational and educational backgrounds and the answers of the inter‑viewers. There was no difference between the two groups, Hospital and Community. (4) Mostof the mothers felt responsible to take their children to immunization but in some combined
families with a large number in a household the decision makers to take children to immuni‑
zation havd still been grandparents. (5,) Clinical staff and local health educators and mass

media such as TV have been well utilized for information resources of immunization. (6)Distribution of medical facilities was one of the most urgent factors to spread the immuniza‑tion.

21 STUDY ON THE MECHANISM OF THERMAL ACCLIMATIZATION
IN THE PIKA (WHISTLE RABBIT)
MITSUO KOSAKA, JIA‑MlNG LEE, TAKAAKI MATSUMOTO,
GUO‑JIE YANG, KOICHI NAKAMURA, KATSUHIKO TSUCHIYA,MASAKI YAMAUCHI AND JUAN AMADOR
Department of Environmental Physiology, Institute of Tropical

Medicine, Nagasaki University

The aim of this study is to identify the inductions of HSPs (heat shock proteins) and
CSPs (cold shock proteins) in weak heat‑ and strong cold‑tolerant pika in order to clarify
the mechanism of its character of weak heat‑tolerance and to examine the physiologicalcorrelation between thermo‑tolerance and HSPs or CSPs. (1) HSP 70 in rats and HSP 68 in
rabbits were induced by the heat‑shock. In pika rabbits, the induction of HSP by the heat‑shock was not detected by 10% SDS‑PAGE not only in liver tissue but also in those tissues
such as kidney, adrenal gland, spleen, brain and skeletal muscle. ‑ According to previous
research, tissues of liver, kidney, adrenal gland and spleen have relatively large ability of
inducing HSP 70 than other tissues in hyperthermia of rat. In further study, Western blotting

method with anti‑HSP 70 antibody was performed. HSP 70 was detected in liver tissue ofall rats under heat shock, but only one showed positive in all pikas. These results suggest that91

the difficulty of induction of HSPs somewhat relate to the characteristic of weak heat ‑tolerance in pika rabbits. (2) Induction of HSP 68 by chronic heat load instead of heat‑shock

was observed on 10% SDS‑PAGE. In the experiment of Western blotting method with anti‑
HSP 70 antibody, the 68 kD protein responded to anti‑HSP 70 antibody but the spot ofstaining showed smaller M. W. size. This discrepancy may be explained as a gradation ofproteins including 68 kD protein. (3) Contrarily in cold‑shock experiments, induction of CSP

was not identified in rat, rabbit or pika. But one result was found that 32 kD protein in
cytosol fraction vanished in one rat after cold‑shock. Four kinds of plasma proteins '(55, 27,
25, 20 kD) vanished in chipmunk, a hybernator, during and after the period of hybernation has

been reported in previous paper. Therefore, the present finding showed the similarity to theprevious report of chipmunk and suggests that not only hybernation but also short term of
cold‑shock may lead to the similar result. Furthermore, physiological significance of HSP
and CSP was discussed from the viewpoint of thermal acclimation.

22 CONTINUED LOW MICROFILARIA PREVALENCE IN
AMERICAN SAMOA IN THE PAST 17 YEARSEISAKU KIMURA, YOSHIKAZU AIRA AND KIYOTOSHI KANEKO
Department of irarasit logy, Aichi Medical University

American Samoa which consists of 4 islands with the total population of 35,000 is the US
territory in the South Pacific. The climate is hot and wet through the year. Most inhabitants

are ethnic Samoan and there is a considerable amount of population movement between the
territory and the neighboring independent country of Western Samoa. Filariasis in this area
is caused by diurnally subperiodic type of Wuchereria bancrofti which is transmitted by two
main vectors, Aedes polynesiensis and Ae. samoanus.
The yearly change of microfilaria (mf) prevalence rates was studied in the years from1974 to 1990 (up to July) , in order to know if filariasis has been increasing or decreasing after

two island‑wide mass treatments with diethylcarbamazine (DEO in 1963 and 1965, which
successfully reduced the prevalence rate from 20.4% in 1962 to 0.36% in 1967. Subjects
included in th‑‑‑e ,study were food handlers, applicants for government jobs, visa applicants and.

drivers of commercial vehicles, who were legally requested to have a physical checkup
including a mf test.

As a whole, the mf prevalence was found to have been kept at a low level (0.8‑2.6%) in
the past 17 years. The rates were maintained particularly low in 1982‑1987. This period
coincided with the DEC mass drug administrations (MDAS) carried out in Western Samoa in

1982, 1983 and 1986. The continued low prevalence in American Samoa seemed to have
reduced clinical filariasis significantly in the past 8 years. Possible factors to keep the low

prevalence in American Samoa are;1 . improvement of living conditions,2 . reduced mosquito breeding sites, and3 . the MDAS in Western Samoa.

92

23 PRESENT CONDITION OF CHAGAS' DISEASE INNORTHEAST BRAZIL: ELECTROCARDIOGRAPHICABNORMALITIES AND SERODIAGNOSISSACHIO MlURA1, HIROSHI TACHIBANA2, SEIKI TATEN03,
J. F. SILVA3, P. JUNGMANN3, M. M. MEDEIROS3,WILSON Jr. OLIVEIRA4 AND C. B. MoURA'

Department of Parasitology, School of Medicine, Keio University*,

Department of Parasitology, Tokai University School of Medicine',

Laborat6rio de Imunopatologia Prof. Keizo Asami Universidade FederaldPernambuc03 ̲and Hospital Oswaldo Cruz Universidade

Federal de Pernambuco'

Antibodies against T. cruzi of out patients at Hospital Oswald Cruz in Recife, weredetected by IFA and ELISA. Examinations were performed on men and women aged O to 85
years with an average age of 47.5 years. The patients were divided into 3 groups. A group,
54 cases (38.8% ) who had already been diagnosed as Chagas' disease. B group, 55 cases (39.

6%) who were diagnosed some heart diseases. Group C, there were 30 cases (21.6%) who
were going to the ambulatory butits and clinical diagnosis remained unclear.
Of 54 patients of Group A, complete ECG records were available for 47 as follows: 24(51.5%) with CRBBB, 12 (25.5%) LAH, 9 (19.1%p) PVC, 7 (14.9%) AV‑block, 8 (17%) LPH,
4 (8.5% ) RBBB, I (2.1%) LBBB, including plural findings, wheres 7 of these had normal ECG
findings with positive IFA and ELISA titers for anti‑T. cruzi antibodies in these cases. B

group, complete ECG records were available for 20. Three of ten were serological positive
for T. cruzi infection, the first one whose chief symptom was dyspnea showed AV‑block, thesecond one with pericarditis showed LAH. The third, 51‑years‑old house wife who lived in

Nazar6 da Mata where about 130 km northeast from Recife had been bitten by a Triatoma,
vector of T. cruzi. Her ECq showed PVC though she had no symptom. Iri group C, both IFA
and ELISA gave a positive result in 13 (43.3%) out of 30. Complete ECG records wereavailable for 7 of 30, as follows: 3 CRBBB, 2 PVC, 2 AV‑block, I RBBB. Our serodiagnosis
gave a positive result for T. cruzi antibodies in 85 (61.2%) with an average of 47.8 years.
Especially in this inspection, it is of interest that 3 (2%) out of 85 had been infected bytransfusion. Moreover there were two probable congenital cases. Concerning of them anewborn baby, X‑ray diagnosis gave a cardiomegaly and both IFA and ELISA were positive.
The other one is 22‑years‑old man whose mother had a diagnosis of Chagas' disease. He wasborn in Recife and has never been lived in endemic area of Chagas' disease, but both IFA and

ELISA were positive. Most of the patients who were serologically positive were fromsurrounding zone of Recife (Nazar6 da Mata, Limoeiro, etc.). Follow‑up on congenital ortransfusion infections of T. cruzi should be paid more attention in this city.

93

24 REEVALUATION OF THE INSECTICIDE RESIDUAL SPRAYING
AS A CONTROL MEASURE OF MALARIA IN GUATEMALAKAZUKI OGATAl'2, TOKUKO UMlN03, TAKAHARU IKEDA4
AND RouoLFO ZEISSIG B.5
Department of Parasitology, Yokohama City University School of
Medicine*, Japan Enviroumental Sanitation Centef2,
Department of Medical Zoology, Teikyo University School of Medicine3,
Tsukuba University' and Divisi6n de Malaria,
Ministerio de Salud Publipa, Guatemala5

In order to reevaluate the effdctiveness and to improve the measure of residual spraying,

the village scale experiment was carried out in La Avellana, Guatemala, during the period
from March 1988 to October 1990, on Anopheles albimanus of main vector of malaria in thiscountry. As the results of preliminary observations in 1988, it was revealed that the vector
bit man evenly indoors and outdoors and got away outdoors shortly after sucking blood andthe resting mosquitoes were found out predominantly on roof of shed. From the results ofinsecticide studies, propoxur was the choice selection by reasons of the high insecticidal
activity especially on short time contact, the high residual effect and sirong vapor action.
In 1989, propoxur was treated 3 cycles by the improved method in the village, covering

about 2,400 houses and the area of about 200 km2. In 1990, deltamethrin, which is usedcurrently ori a nationwide s ale, was tested. As the results, the vector density did not
remarkably decreased, in spite of that the residual activity on the surface of the houses lasted

for long time and the mosquitoes rested on the insecticide residues without notable avoidance.

However, the reduction of the parous rate and the remarkable change of the endophagus
behavior were observed. After the application of the insecticide, an endophagus rate fell

down from about 50% to 30‑40%. By contraries, the number of cases by an active casedetection decfeased obviously, namely 1.55% and 1.43% of positive rates before and shortly
after the launching of the spraying fell down to O, O and 0.27% after 13, 15 and 17 months
respectively. But the decrease of number of cases by passive case detection was not remark‑able. As a conclusion, it seemed that the decrease of malaria cas s might occur presumably
although the ?ector density did not always fall. It is likely presumed th t the impact of theinsecticiding was not so strong due to the change of an endophagus behavior. According toanother resul.ts, which investigated a relation between an insecticide spraying and the inci‑
dence of malaria in considerable villages in the southern region, the positive correlation wasapproved to exist between both ones.
The residual spraying of insecticide seemed to be effective to some extent to control
malaria in Guatemala. But, it was assumed that the effectiveness decreases considerablycompared with one in the past, due to the change of the behavior rincipally.

94

25 MALARIA CONTROL PROGRAMME IN DAR ES SALAAM AND
TANGA, TANZANIA: ACTIVITIES AND RESULTS
ON THE FIRST YEAR (1988/89)
TAKAYA IKEMOTO1, KIYOSHI MAKIYA', J. H. MARIJANI3

AND HIROSHI TANAKA4
Department of Parasitology, Teikyo University School of Medicine*,

Department of Medical Zoology, University of Occupational andEnvironmental Health', Ministry of Health, The United Republicof Tanzania' and Institute of Public Health, Chiba Prefecture'

Dar es Salaam and Tanga are port towns situated on the east coastal Tanzania, where
the temperature range is 24‑28'C, the humidity high comparatively in all seasons. Malaria isendemic in the both towns with high degree of the transmission caused by the principal
vectors of Anopheles gambiae and An. funestus. Although the transmission may not beperennial, varying to some extent with the season, no season is consistently free from malaria

resulting in a considerable degree of immune response in all age groups of inhabitants,
particularly in the adults.

The activities of the urban malaria control programme in Dar es Salaam and T.anga havepractically started in 1988 through integrated vector mosquito control measures. Namely,
larviciding and environmental management are applied in the central area of the cities, andresidual house spraying with fenitrothion in the urban‑rural fringe areas. The larviciding
area in Dar es Salaam is 200 km2, and the residual spraying covers 50,obO houses twice a year
on the plan.

The parasite rate is surveyed among 2,000 and 800 apparently healthy schoolchildren
aged 6‑8, once a month and once two month in Dar es Salaam and Tanga, respectively. In
addition to the blood examination, data of clinical suspected malaria cases are collected
regularly from 48 dispensaries in Dar es Salaam, and from 9 in Tanga for epidemiologicalevaluation.

Entomological evaluation is based on systematic field sampling of malaria vector popula‑tion from geographical representative locations within the insecticiding operation area andnon operation area. For this purpose 60 catching sites in 12 areas of Dar es Salaam and 12
catching sites in 4 areas of Tanga were selected.
Before the control operation in 1988, the parasite rate among schoolchildren proved to be
34% (October, ranged 5.5‑73.5% among 6 schools surveyed) in Dar es Salaam, and 42% (May‑

June, 25.6‑64.2, 4 schools) in Tanga. Plasmodium falciparum was found in more than 99% of
the positive films. Other malaria species were found as a mixed infection with P. falciparum.
P. malariae being dominant comparatively in Dar es Salaam and Tanga, but P. ovale or P.
vivax was not detected in each area, respectively.
The population densities of the vector mosquitoes decreased after the residual housespraying for 1‑2 months at least. The parasite rate among children was also on the decreasein the operation areas, reducing the rate by at least 70% for a period of 6 months following
completion of spraying, thus achieving the first year objective.

95

26 GEOPATHOLOGICAL STUDY ON MALIGNANT TUMORSIN WESTERN KENYA, EAST AFRICA
KAN TORIYAMA, HIDEAKI ETo, SATORU KOMURO, TAI‑ICHIRO MORIYAAND HIDEYO ITAKURA
Department of Pathology, Institute of Tropical Medicine,

Nagasaki University

We performed the geopathological study on malignant tumors (MT) in western Kenya,
East Africa. The western region of Kenya stands almost exactly astride the equator. It
accounts for almost one thirds of the whole country in area and about one half in population.

Natural environments show various features, such as, hot and dry desert areas, cool andmoist tropical highlands and hot and dry tropical savannahs. Also various ethnic groups
inhabit in this area. Western Kenya is composed of three provinces; Western, Nyanza andRift Valley Province. , ̲
The results were as follows: 1) Out of 30,452 surgical pathological specimens at
provincial and district hospitals in western Kenya during 10 year‑period between 1979 and1988, 5,386 cases were histologically diagnosed as MTs. 2) The most common MT in western
Kenya was cervical cancer (CC, 1,104 cases, 20.5%) , followed by malignant lymphoma (ML,
including leukemia, 794, 14.7%) , skin cancer (SC, 777, 14.4%), malignant soft tissue tumor(MST, 223, 4.1%), esophageal cancer (EC, 221, 3.9%)', breast cancer (BC, 189, 3.5%) and

Kaposi's sarcoma (KS, 150, 2.8%). 3) The most common MT in male was ML (including
leukemia, 477, 20.8%) , followed by SC (399, 17.4%), EC (170, 7.4%), KS (128, 5.6%) , MST(128, 5.6%), prostatic cancer (97, 4.2%) and hepatocellular carcinoma (78, 8.4%). 4) The

most common MT in female,was CC (1,104, 37.1%), followed by SC(376, 12.7%). ML(including leukemia, 277, 9.3%) , BC (164, 5.5%), ovarian cancer (99, 3.3%) and MST (95,
3.2%). 4) CC, ML and SC were equally distributed in western Kenya ethnogeographically. 5)
On the other hands, KS, Burkitt's lymphoma (BL), fibrosarcoma (FS) , penile cancer (POand EC showed specific ethnogeographical distributions in western Kenya. These findings
suggest that some etiological factors, such as, natural environments, Iife styles and probably

in some part, genetic factors might have some .relations with onco enicities of KS, BL, FS,

PC and EC.

96

27 ENTEROPATHOGENIC E. COLI (EPEC) AND ENTEROTOXIGENIC
E. COLI (ETEO ISOLATED IN NORTHEAST THAILAND AND
THEIR RESISTANCE TO ANTIBIOTICSPlYAWAN GASALUCK YUTAKA MlnoRIKAWAl, MASAYUKI IMAll
AND HITOSHI YoS'iuMURA2
Department of Public Health* and Department of Laboratory Medicine',
Mie University School of Medicine

Escherichia coli samples from Northeast Thailand were examined the pathogenicity. To

determine the enterotoxigenic E. coli (ETEO , which is one of the most predominantdiarrhoeal pathogen in tropical developing countries, twenty three specimens from stool ofboth asymptomatic (9 samples) and diarrhoeal patients (14 samples) from children under 5

years old were collected from Srinagarin Hospital, Khon‑kaen University in Thailand.
Those diarrhoeal samples have no information of another pathogen such as Vibrio. Shigellaor Salmonella. The heat‑1abile (LT) enterotoxin of E. coli was detected by both Biken test
(Modified Elek method) and latex agglutination method (Denka Seiken) . Heat‑stable (ST)enterotoxin'of E. coli was detected by suckling mice method. Enteropathogenic Escherichia
coli (EPEO were detected with antisera (Denka Seiken) , then antibiotic susceptibility ofthese E. coli samples were also tested (BBL Kirby‑Bauer method) because increasing ofantibiotic resistance bacteria also becoming problem in tropical developing countries such as

Thailand.
Though none of LT enterotoxigenic E. coli was detected by both Biken test and latex
agglutination method, a ST enterotoxigenic strain was detected from a diarhoea sample.

This samples have reconfirmed by ELISA (Denka Seiken method). Three samples fromasymptomatic and four samples from diarrhoea were ETEC serotype. Both asymptomaticand diarrhoea samples included EPEC and ETEC showed antibiotics resistance to such as
streptomycin, ampicillin, tetracycline, chloramphenicol, kanamycin or sulfathoxazole‑
trimethoprim.
Though. the purpose of this study was to detect enterotoxigenicity of E. cdli, healthycarriers of EPEC and resistance of antibiotics of E. coli were also pointed out.

28 THE RELATION BETWEEN PROTEIN AND TOXIN PRODUCTION
IN VIBRIO CHOLERAEAKIKO MAZUJ11, YUTAKA MIDORIKAWAI , PlYAWAN GASALUCK*,MASAYUKI IMAll, YOSHITO IWADE2 AND AKlRA SUGlYAMA2 .
Department of Public Health, Mie University School of Medicine*and Institute of Public Health, Mie Prefecture'

Relation between cholera toxin (CT) and protein production was tested for develop the
better detection of CT. Five V. cholerae samples were inoculated to the syncase broth. These97

samples were incubated by both shaking and static condition under 30'C. Cholera toxin was
detected by RPLA (Denka Seiken) method. Cell protein of V. ckolerae was measured by the
Lowry's method. Protein which produced in the supernatant was determined by means of Bio‑
Rad method. The shaking incubation was superior to the static incubation for both total
protein production and CT production by V. cholerae. Though CT det ction became positive
after 15 hrs passed and protein production was rapidly under the shaking incubation, it took
48 hrs by means of static incubation to get both CT positive result goes and enough protein
production. But as far as the result of amount of the protein produced in supernatant after
48 hrs incubation, V. cholerae strains showed higher protein production by the static incuba‑tion method than the shaking incubation.
This study showed that amount of protein produced by V. cholerea reflected the CT
detection and also showed that by 48 hrs incubation without shaking, the amount of protein
production increased and also detection of CT became possible.

29 CAUSATIVE ORGANISMS OF RESPIRATOBY INFECTIONS‑COMMUNITY AQUIRED INFECTION‑ IN NORTHERN THAILAND
HIDEHIKO HIROSEl, TAKESHI NAGATAKEl, NAOTO RIKITOMI*,
ATSUSHI TAKAHASHll, KAZUNORI OISHI*, MASAKAZU TAKASUG11,YOSHIAKI UTSUNOMlYA1, KIWAO WATANABE*, KEIZO MATSUMOTO1,THIRA SIRISANTHANA2, PRASIT THARAVICHITKUL2, TAWORN KASOMSON3
AND RONNATRAI RUENSVIRAYUDH3
Department of Internal Medicine, Institute of Tropical Medicine,

Nagasaki University*, The Faculty of Medicine, Chiang Mai University'and Mae Sod General Hospital'

Bacterial respiratory infections are one of the important disease in tropical area. It is

not known which pathogenic organisms are predominate in Thailand. Therefore, this study
was carried out to identify the causative agents.

Purpose : 1) To identify the pathogenic organisms responsible for bacterial respiratory
infections. 2) To identify suitable antibiotic available in Thytiland after discussing withphysicians of Mae Sot General Hospital. 3) Compared the result of Thailand with those in

Nagasaki.
Material and Methods : Out patient and in‑patient of Mae Sot General Hospital during
1989. Il.‑1990. 1. and 1990. 7.‑1990. 8. To determine the causative bacteria, sputum werecollected from patients. Gram‑stain and acid fast stain were done to observe theinfiammatory cytology and bacteria. Sputufn culture was also done. Identification of isolated

bacteria was done at the Laboratory of Chiang Mai University.
Results : ' Rate of causative organisms in Mae Sot area. 1989. Il.‑1990. 1. (n=48): H.
influenzae 41.7%, S. pneumoniae 27.1%, B. catarrhalis 20.8%, S. aureus 4.2%, K. pnemoniae
4.2%, E. cloaca 2.1%, 1990. 7.‑1990. 8. (n=24): H. inf. 41.7%, S. pne. 37.5%, B. cata. 4.2%,
K. p,ne. 16.7%,

Discussion : 1) We found that H. inf., S. pne. and B. cat. were main causative agents of

98

respiratory infections in Mae Sot area. The occurence of respiratory infections by B. cat.

decreased in summer and increased in winter. These results are similar to that of in
Nagasaki. 2) By MICgo , we found that TC is unsuitable for H. inf. S. pne. and B. cat. as
first choice of antibiotic, with the cooperation of pysicians from Mae Sot General Hospital,
we must determine the flrst choice of antibiotic more suitable than TC.

30 THE POSSIBILITY TO AQUlRE STRONGYLOIDES STERCORALISINFECTION AMONG PUPILS IN THE ENDEMIC AREAS OF
STRONGYLOIDIASIS IN OKINAWA, JAPANRYUJI ASATO*, TAMIO NAKASONE , CHOKEI YOSHIDA*, TAMIKI ARAKAKITSUYOSHI IKESHIR03 AND MASAAKI SHIMADA'
Okinawa Prefectural Institute of Public Health*, Department ofBacteriology, School of Medicine, University of the Ryukyus',

Center for Preventive Medicine Okinawa'
and Department of Medical Zoology, Universrty of Occupatronaland Environmental Health‑In Okinawa Prefecture, Japan, high prevalence of Strongyloides stercoralis infection has

been demonstrated among adult inhabitants by agar‑plate culture of feces. However, its
prevalence among young people and the possibility to acquire new infections have not beenadequately known. An epidemiological survey with questionnaires and fecal examinations
was made to evaluate the possibility to acquire S. stercoralis infection among pupils in Miwa

and Takamine areas, Itoman City, where the positive rate of S. stercoralis infection among

adult inhabitants was proved to be 20.5% and 15.8%, respectively. In Miwa and Takamine
areas, 96.1% and 81.5% , respectively, of the adult inhabitants with Strongyloides infection are

farmers or engaged in farming partly, 53.3%.o and 33.3% , respectively, used human feces as
manure before 1972, the year of the retrocession of Okinawa to Japan, and 33.3% and 14.8%,
respectively, continued to fertilize w.ith human feces after 1972. Moreover, 6 farmers still
apply their stools on the fields, and I inhabitant offers his family's excreta to a farmer as

manure. Among the primary school and junior high school pupils in Miwa area, 23.5% and41.5%, respectively, participate in farming, and 2.6% and 10.1%, respectively, are often
barefooted at farming. In Takamine area, 19.7% and 27.9% of the primary and junior highschool pupils, respectively, also participate in farming, and 2.2% and 1.9%, respectively, areoften barefooted at farming. The usage of human feces as manure and the participation of
barefooted pupils in farming at the present time may cause infections with S. stercoralis
among the pupils. Actually, S. stercoralis was detected in each one among 782 primary schooland 240 junior high school pupils of Miwa area by the agar‑plate culture of the feces,although Strongyloides infection was not detected among 455 pupils of Takamine area. An
educational campaign on hygiene for the inhabitants may be necessary to prevent Stron‑
gyloides infection among the pupils.

99

A COMPARATIVE STUDY ON AN IMPROVEMENT OF
THE AGAR PLATE METHOD REGARDING SAFETY (SEALING)
IN NORTHERN THAILAND
31SHIRO KASUYA , KAORI KOGAl, CHIRASAK KHAMBOONRUANG2,
MIKI M‑OR13, KEIKO TOMITA3 AND HIROSHI OHTOMO1
Department of Parasitology* and Students', Gifu University

School of Medicine and D.epartment of Parasitology, Faculty of

Medicine, Chiang Mai University, Thailand'

The agar plate method, a superior method for the detection of Strongyloides stercoralis

from stool, has been improved regarding safety standards in this study. Agar dishes aresealed with adhesive tape to prevent filariform larvae from crawling out of the dishes. Outof 54 specimens collected from children in Chiang Mai (Thailand) , the coincidence rate
(positive number in both methods+negative number in both methods divided by the total
number) between the sealing method and the customary method is 94.4%. Sealing depresses

neither the detection rate nor the development of worms. This improvement not only
improves safety, but it also helps to eliminate such cumbersome problems as offensive smellof the cultured stool and break out of maggots, as pointed out in previous studies.

32 HOW SENSITIVE IS THE AGAR PLATE METHOD FOR
STRONG YLOIDES STERCORALIS?

KAORI KOGA, SHIRO KASUYA AND HIROSHI OHTOMO
Department of Parasitology, Gifu University School of Medicine

We have been reported that the agar plate method is much more reliable to detect
Strongyloides than other traditional stool examination methods.
This method is, in short, when a fecal sample is placed at the center of an agar plate,worms crawl out onto the agar media during incubation (27'C, 2 days) , and then characteris‑tic tracks become detectable.
The difficulty in detecting Strongyloides primarily depends on a small number of larvaein the feces, because of a small number of eggs produced by one female parasite, and in casefew worms parasitizes. And appearance of larvae into the feces may be often intermittent.
We have studied how sensitive the agar plate method was when few worms were in thesamples. We made several kinds of feces mixture with various larvae density, and tried themethod.
The feces from a patient was diluted serially with water at first, and then mixed withfeces from healthy persons. Five kinds of mixture with 4 x 103, 4 x 102, 40, 4, 0.4 Iarvae/2 g,

respectively (We used 2 g mixture per one agar plate.) were made: We placed 2 g mixture
to each agar plate, and incubated at 27'C for 2, 5, 8 and 11 days respectively (5 plates each) .
On the second day, all plates were positisve in the groups of 4 x 103 to 4 Iarvae/plate, and100

3/5 were positive in the group of 0.4 Iarvae/plate. O, n the 5th, 8th and llth day, similar results

were obtained.
The conclusion is that we can detect only one alive Strongyloides larva using the agar
plate method. This method was very sensitive to Strongyloides.

33 FOUR ADDITIONAL CASES OF GNATHOSTOMIASIS DOLORESIAND APPLICABILITY OF ELISA DIAGNOSISHARUHIKO MARUYAMAl, YUKIFUMI NAWA1, JUN ICHI IMAIAND KATSUMI OGATA2
Department of Parasitology' and Dermatology',

Miyazaki Medical College

Genus Gnathostoma is naturally a parasite of wild animals. Although this genus includes
more than 10 species, only one species, namely G. spinigerum, had long been thought to cause

human gnathostomiasis. Recently, however, confirmed human gnathostomiasis cases causedby G. hispidum. G. nipponicum, or G. doloresi were reported. Thus, all Gnathostoma spp.
seem to cause the larva migrans in humans. Since human gnathostomiasis is caused byingesting the third stage larvae in fishes, amphibians or reptiles, this disease is one of theimportant food‑born parasitic diseases. In Miyazaki Prefecture, Kyushu, Japan, we havealready reported a total of 10 (three confirmed and seven suspected) cases of gnathostomiasis

doloresi during 1985‑88. Although the additional case was not found･ in 1989, four cases
including one 'confirmed case newly appe red in 1990, indicating that G. doloresi infection isstill endemic in this area.

The new confirmed case was a 55‑years old house‑wife having past history of having
eaten raw slices of the flesh of freshwater fishes which were caught by her husband. Since
6 April 1990, she had been suffering with right‑lQwer abdominal pain and had an appendec‑
tomy on the 12th April in the local hospital. On 13 April, a rapidly extending creeping
eruption with pain and itch developed just above the site of appendectomy. She admitted to

the Department of Dermatology because gnathostomiasis was strongly suspected. At thetime of admission, Iaboratory examination data related to parasitic diseases were as follows;
WBC 9,500, Eo 13.9%, IgE 50.5 IU/L, CRP (+) , immediate type skintest against G. doloresi
antigen ( ‑ ) . Since linear vesicle formation was noticed at the top of the creeping eruption,

this part was biopsied. When the biopsied specimen was carefully examined under adi'ssecting microscope, a parasite‑like creature was moving inside the vesicle. An intact
living worm of approximately 3 mm length was dissected out, and, this worm was identified
as the juvenile adult of G. doloresi because it had 7 Iines of hooklets on the head‑bulb; the 2nd

and 7th rows were incomplete.
Although her husband also noticed a mobile indulation with mild pain and itch on his
right lumbar region on 20 May 1990, the indulation was too large to be extirpated. His
laboratory data was; WBC 4,800, Eo 25.5%, IgE 10,833 U/L, CRP (‑) , immediate type skintest for G. doloresi ( + ) . About one month later, the indulation spontaneously disappeared.
Sinde the skin test of this couple gave an opposite result, we carried out specific antibody

lOl

detection by lgG‑ELISA for these and other gnathostomiasis cases to test the applicability
for the diagnosis. The results show that 3 out of 4 confirmed cases were positive, whereas 2
out of 5 suspected cases were positive by the ELISA. Our results were markedly different

from those previously reported high positivity of ELISA diagnosis for gnathostomiasis
spinigerum in Thailand or in Guatemala. This discrepancy seems to be due to, at least inpart, the intensity and the duration of infecti6n. Further study is required to clarify this point.

34 AN INTEGRATED APPLICATION OF AN INSECTGROWTHREGULATOR, PYRIPROXYEN AND SOUND TRAP SYSTEM FOR
THE CONTROL OF THE VECTOR OF JAPANESE ENCEPHALITISIN AN OPEN PADDY FIELD IN THAILAND
VANIDA KERDPIBULE*, Tozo KANDA2, T. DEESINl
AND S. LEEMINGSAWATl
Faculty of Tropical Medicine, Mahidol University, Thailand*and Department of Medical Zoology, St. Marianna University
School of Medicine'

Realization of integrated application of sound trap system and an insectgrowth regula‑tor, pyriproxyfen (IGR) was succes fully proven at an open paddy field in Sai Noi District,

Nontaburi, Thailand. A blocked site with 2,900 m2 was treated with pyriproxyfen (IGR) by
the concentration of 5 ppb of granule in 5% of chemical. The depth of the water was ke'pt
about 5 cm and the length of the rice plant was 25 to 30 cm during the experiment. The
mosquitoes collected were mono‑specific Culex tritaeniorhynchus. The mosquito population

at the site dominantly responded to 350 and 1,000 Hz in the comparison among soundfrequency responses. After application of the chemical during 6 days consequent overnight
trapping (18:OO pm to 6:OO am) was performed by emitting these two frequencies froin 16
traps. Then the trappings were performed to monitor the population of the vector on 23rdand 33rd day after the application of the IGR by using 10 traps. The number of the vector
trapped decreased from 2,347.7 on the commencement day to 971.9 Ist day. A slight increas‑
ing was proven to 1,990.3 on the 2nd. This level of the trapping numbers was kept till 33 days,

though some decreasing were proven on 15th and 23rd.
These results mint that the integrated control method had enough ability to decrease thenumber of the vector at the study site, however, the increasing of the number on the 2nd day

suggested the migration of the vector from neighboring area. The continuation of theovernight‑trapping for five nights strikingly proved significant decreasing of the populatioriof the vector before the effect of IGR.

The investigation of the parity of the vectors collected by sweeping also proved
significant decreasing due to the function of IGR, being nulliparous rate of 6.0% on the 15th

from 46.0% on 6th, whereas trapped number of the vectors was not much changed. Thisphenomena can be suggested because of migration of parous vectors from neighboring area
not treated. Thus in open field the utility of combination of these two control method were102

proven to contrlbute effectively for interruption of the transmission of Japanese encephalitis

by extreme reduction of risky infected vector and to keep lower vector density in suchendemic open field.

35 COMPARISONS OF GENETIC MAPS OF TWO AEDES MOSQUITOSPECIES ‑WITH SPECIAL EMPHASIS ON FILARIALSUSCEPTIBILITY LOCI‑TAKEO TADANO
Department of Medical Zoology, St. Marianna University
School of Medicine

Genetic maps of two mosquito species, Aedes (Stegomyia) albopictus. Ae. (Finlaya) togoi,

have been constructed in our laboratory. These maps can be utilized for genetic analysis offilarial susceptibilities of Brugia malayi. B. pahangi, or Dirofilaria immitis.

Comparisons of genetic maps of these two species and Ae. (Stegomyia) aeg pti suggestthat chromosomal translocations or inversions have occurred among the three linkage groups(2N=6) of the three Aedes species during the evolutionary time. But there is a good
indication that the region surrounding the sex‑determining locus (M/m) is similar in thegene order to each other in the three species and so this region has been conserved during theevolution.

In the genetic map of Ae. aeg)pti (Munstermann and Craig, 1987), Iinkage group I (sexchromosome)' contains the following segment; f(‑f ')‑(2‑3 map units)‑Me (malic

enzyme) ‑ ( 5 units) ‑ M / m (sex) ‑ ( 3 units) ‑ Aap ( acid phosphatase). The above‑
mentioned f '‑f 4 are the locus ( or loci) for susceptibilities to four filarial species, B.
malayi, Waltonella flexicauda, D. repens, and D. immitis, respectively. Although any filarial

susceptibility locus has not been located in the genetic map of either Ae. togoi or Ae.
albopictus, Iinkage group I of both species includes a gene order very similar to the above
segment in Ae. aeg pti: i. e. Me‑(ca. 5 units) ‑M/m‑(3‑5 units) ‑Aap in Ae. togoi; also,
Me‑(5 units) ‑M/m‑in Ae. albopictus. Such tracing of gene loci in linkage group I gives
an assumption that the filarial susceptibility locus may be close to Me in both Ae. togoi andAe. albopictus, although cross experiments remain to be done to verify this assumption.

36 NON‑MENDELIAN POPULATION OF A TRIATOMA INFESTANS
STRAIN, A MAIN VECTOR INSECT OF CHAGAS' DISEASE
HIROHISA HIRAI AND ISAO TADA
De artment of Parasitic Diseases, Kumamoto University
School of Medicine

The cytogenetic informations have often contributed to the control programmes for103

discriminating insect vectors of endemic diseases. Chromosome observation of triatominebugs, medically important vectors of American trypanosomiasis (Chagas' disease) , wasattempted. We demonstrated by C‑banding technique that a Brazilian strain ofTriatoma
infestans (2n = 22) included at least three cytotypes in terms of constitutive heterochromatin

polymorphism. The first type designated as cytotype A has six chromosomes (chromosomes1‑3) with enlarged C‑heterochromatin blocks at both terminals. Likewise, cytotypes B andC possess seven and eight such chromosomes, respectively. The chromosomes characterized
by the C‑block were numbered by the order of size from large to small. Chromosomes I and2 had prominent C‑bands at the both terminal regions one side of which is larger than theother. Chromosomes 3 and 4 were characterized by a unilateral heterochromatin block. The
homology of these chromosome pairs was confirmed based on pairing patterns at metaphase
I of males. A total of 85 bugs examined, included 39 cytotypes A, 26 B and 20 C, respectively:

Cytotype B seems to be the hybrid between cytotypes A and C, because chromosome 4 ofcytotype B is heteromorphic for terminal C‑band ( ‑ / + ) , whereas it is homomorphic ( ‑ / ‑ )in cytotype A and ( + / + ) in cytotype C. The frequency of cytotype B was significantly low

from the expected value (A, 31.8; B, 40.4; and C, 12.9) by Hardy‑Weinberg expectation test,

suggesting non‑Mendelian population in mating. We propose an assumption that there aresome reproductive barriers between cytotypes A and C, and thus the viability of their F1(cytotype B) is reduced significantly. Although we need further genetic experiments among
the various cytotypes, the terminal C‑bands in chromosomes 1‑3 and heteromorphism inchromosome 4 mentioned above would potential markers for discriminating vector individ‑uals.

37 PHYLOGENETIC ANALYSIS OF THREE SPECIES OF PARAGONIMUS,
HUMAN INCIDENTAL PARASITES IN THAILAND AND JAPAN1. ISOZYMES ANALYSISCHALIT KOMALAMISRA AND TOzo KANDA
Department of Medical Zoology, St. Marianna University

School of Medicine

According to an occurrence of human incidental parasites, Paragonimus miyazakii, one of

the harmful zoonotic agents causing Paragonimjasis among man and animals were reported
in many cases in Japan as well as in Thailand due to Paragonimus heterotremus. These
parasites whose natural definitive hosts as animals were considered to be related species
because their pathogenicity and clinical manifestation in human were very much similar suchas high frequency of plueural effusion, high eosinophilia of the blood, pleural exudate andspontaneous pneumothorax.
Another species from Thailand, P. siamensis was also considered to relate with P.
miyazakii and P. heterotremus as well. Epidemiological studies found that there was no reportof P. siamensis infect man in this country inspite of the crab intermediate host of this parasite,

the inhabitant were fond of eating raw crab.
Although, the life cycle and morphological characters among 3 species of Paragonimus

l04

were disctiminated on each other. An attempt to determine the genetic relationships among
all of them was performed. However, cytological qualification were very difficult to see
especially euchromatin and heterochromatin.
Thus, emphasis on the genetic differentiation among 3 relate d species P. miya akii fromJapan, P. heterotremus and P. siamensis from Thailand were assessed by electrophoretic
analysis of allozymes. Twenty one loci of sixteen enzymes examined; Acid phosphatase,
Adenylate kinase, Aldehyde oxidase, Esterase, General protien, Glucose phosphateisomerase,

Glucose 6 phosphate dehydrogenase, Alpha glycerophosphate dehydrogenase, Hydro‑
xybutylate dehydrogenase, Hexokinase, Malic dehydrogenase, Malic enzymes, Octanal
dehydrogenase, Phosphoglucomutase, 6 phosphogluconate dehydrogenase and Tetrazolium
exidase were analysed by using polyacrylamide vertical slab gel electrophoresis and esti‑
mated the genetic drifts between the 'phenotypic frequencies of respective pair combinations

among the 3 species of Paragonimus. The genetic distance between P. miyazakii and P.
heterotremus, between P. miyazahii and P. siamensis and between P. heterotremus and P.
siamensis were 1.0036, 1.2942 and 1.4297 respectively. Comparing these values with standard
genetic distance described by Ayala and Kiger (1980) , these suggested that the 3 Paragonimus
could be classified into an independent species from each other, and also indicated that P.
miyazakii was genetically closest to P. heterotremus than P. siamensis.

However, P. ohirai is another one of interesting species in Japan. Epidemiology, Iife
history and pathogenicity of this parasite are similar to P. siamensis from Thailand.
Therefore, the phylogenic relationships between these 2 parasites will be studied further.

38 TWO‑DIMENTIONAL ANALYSIS OF A PUTATIVE EGGSHELL
PRECURSOR PROTEIN OF, THE FEMALE SCHISTOSOMA JAPONICUM
MASANORI KAWANAKA AND HIROMU SUGIYAMA
Department of Parasitology, National Institute of Health

Production of eggs in schistosomes is dependent upon the integrated functioning of two
different cell types‑ the ovum and the vitelline cells. The parasite egg is formed fromapproximately 30 vitelline cells and one fertilized ovum which become encapsulated by
eggshell within the ootype. It has been suggested that the formation of the eggshell in the
ootype occurs via the rapid enzyme‑dependent cross‑linking of precursor proteins rovided
by the vitelline cells. This may occur by the oxidation of tyrosine residues of the precursor

protein but has never been adequately investigated. In adult worms of Schistosoma
japonicum, we have obtained experimental results that a major female‑specific protein withapparent molecular weight of 34 kD, as determined by SDS‑PAGE, was suggested to be a
precursor of the eggshell proteins. It has proved difficult to detect maior female‑specificproteins by direct staining of SDS gels. This 34 kD protein was mainly identified byradiolabelling of parasites in vitro with [14C] tyrosine and fiuorography of SDS gels (Kawana‑ka, 1990).

On the other hand, it is known that eggshell hydrolysates of S. japonicum are very richin glycine and poor in methionine (Byram and Senft, 1979) . In in vitro experiments, we havel05

observed in autoradiograms that the synthesized protein with [14C I glycine in vitelline
cells was utilized in formation of the shells of newly formed eggs. As may be expected, a[14C]glycine‑labelled female‑specific protein was demonstrated at 34 kD in the parasite
protein on fiuorography of SDS gels. A single band in the one‑dimentional gel, however, isusually composed of several polypeptide species having different isoelectric points. In orderto determine whether the identical polypeptide species were being detected in the region 34
kD of the female protein, we performed two‑dimentional gel electrophoresis of [1*C]tyrosi‑n ‑, [*･C]glycme and [35S]methionme labelled parasite protems
In [14C]tyrosine‑1abelled proteins, at least five spots with apparent molecular weight of

34 kD region were exclusively observed in the female and these pl values were 6.0, 5.8, 5.5,
4.8 and 4.6. By labelling the female protein with [1*C] glycine followed by separation by two‑
dimentional electrophoresis, three spots with pl values 6.0, 5.8 and 5.5 were identified and they

were thoroughly corresponded to [14C] tyrosine‑labelled spots. Any female‑specific polype‑ptide was not detected at 34 kD region by labelling the parasites with [35S]methionine. The
results were suggested that a putative eggshell precursor protein of S. japonicum (34 kD) was
resolved into three polypeptide species with different pl values.

39 FASCIOLA CYSTEIN PROTEASE= SPECIES SPECIFICITY
AND AVAILABILITY AS AN ANTIGEN FOR ENZYME‑LINKEDIMMUNOSORBENT ASSAY (ELISA)HIROSHI YAMASAKI, TAKASHI AOKI AND HIROSHI OYA
Department of Parasitology, Junt ndo University School of Medicine

Preyious work in our laboratory indicated the presence of a cysteine protease in theintestinal epithelial cells of Fasciola adult worms. We, believe that the enzyme is excreted
into the intestinal lumen as a digestive enzyme and induces the lgG antibody production in
the host animal. In this study, we have examined the species specificity and availability ofthe enzyme as an antigen for ELISA in the diagnosis of human fascioliasis.
The Fasciola protease fraction, partially purified by amonium sulfate precipitation and
Sephadex G‑75 column chromatography, contains a major protein ivith a molecular weightof 27 kD that was identified as the enzyme by specific monoclonal antibodies in an immuno‑,
blot analysis. For comparison, a partially purified Schistosoma mansoni cysteine proteasecontaining a major protein with a molecular weight of 31 kD was also used. In our ELISA
system, we used the following conditions: (1) the concentration of the enzyme antigen used tosensitize the microtiter‑plate wells was 5 pg/ml; (2) the dilution with PBS of the sera
examined was 1:1,000; (3) peroxidase‑labeled anti‑human lgG conjugate (1:1,000) and (4) the
substrate ABTS for color development were used; (5) absorbance at 405 nm was measured.
When the Fasciola cysteine protease was used as the antigen for ELISA, the sera frompatients with fascioliasis (N = 13) gave the highest absorbance of 1.603i0.394 (mean SD) .
For the active fascioliasis sera, no false negative case was observed. Hol ever, the heter‑
ologous reactions between the Fasciola enzyme with the schistsomiasis japonica sera
(Philippines, N = 13) and with the schistosomiasis mansoni sera (Brazil, N = lO) yielded the106lower absorbance of 0.51ld:0.278 and 0.573 0.168, respectively, statistically significant
comparing with the value of fascioliasis sera at the p < 0.001 Ievel. Similarly, the absorbancevalues for the sera from patients with paragonimiasis (N=3) , angiostrongylosis (N= 1),
gnathostomiasis (N = 1) , sparganosis (N = 1) , and amebiasis (N = 1) were 0.425i0.024, 0.307,
0.278, 0.179, and 0.309, respectively, and close to the values for the normal human sera (0.228:
0.095) .

When the S. mansoni cysteine protease and the patient sera (homologous combinations)were examined in ELISA, an absorbance of 1.248 0.573 was obtained. Cross‑reactions withthe schistosomiasis iaponica sera were observed, but the average absorbance values (0.792)
were lower than that of schist0 0miasis mansoni sera (1.248) . No cross‑reaction between the

Schistosoma protease and the fascioliasis sera was observed.
The results obtained in this study indicate that the Fasciola and Schistosoma cysteine
proteases, even at their partially purified state, are highly species‑specific as the sensitive

ELISA antigens for the immunodiaguosis of human parasitic diseases. Therefore, it ispredicted that Fasciola protease antigen will serve as a valuable ELISA antigen in
epidemiological surveys of human fascioliasis, including the heterotopic parasitism andimmature liver‑fluke's migration.

40 AVIDIN BlNDlNG COMPONENTS IN PARASITES
MAKOTO ITOH AND SHIGEFUSA SATO
Department of Medical Zoology, Nagoya City University Medical School

Strong and specific afiinity of avidin for biotin has been widely utilized for enzyme
immunoassay. Biotin is also known as vitamin H and certain growth factors.
We recently found that some parasites contain avidin binding comporients (ABO in their

extracts. In this study, we first investigated ABC in extract of various parasites. The
extracts were dotted on a membrane and ABC were detected by peroxidase conjugated avidin‑
D. Then, ABC positive extracts were applied to SDS‑PAGE, electrically transferred to amembrane and ABC patterns were observed on the membrane by treatment with peroxidase

conjugated avidin‑D and a substrate. Parasites used are as follows. Trematode: Par‑agonimus miyazakii. P. westermani. P. ohirai. Fasciola sp., Clonorchis sinensis and Schistosoma

japonicum ; Nematode: Gnathostoma doloresi. Toxocara canis. Dirofilaria immitis. Trichuris
vulpis and Angiostrongylus cantonensis; Cestode: Diphyllobothrium latum. Spirometra erinacei.

Hymenolepis diminuta. H. nana. Taenia saginata and Dipylidium caninum.
Among the extracts studied, those from T. canis. G. doloresi. D. Iatum. S. erinacei and all

the trematode except S. japonicum contained much ABC. A Iittle amount of ABC weredetected in extracts of S. japonicum and A. cantonensis and no ABC was detected in those ofother parasites with the method used. ABC patterns of the extracts studied differed eachother. Even those of close related Paragonimus species, P. miyazakii. P. westermani and P.
ohirai, could be discriminated by their ABC patterns. No individual variation was observedmong flukes of the same species. These observations suggest that ABC pattern can be oneof the markers for identification of parasites.

107

41 EFFECT OF CHEMOTHERAPY WITH PIQUITONG (PRAZIQUANTEL)ON MURINE SCHISTOSOMIASIS MANSONI AND CHANGESOF SERUM ANTIBODY LEVELSYOUICHI ITOl, FUSAKO OSADA1, JUN DU'
AND NoRIAKI KAJIHARA3
Department of Parasitology, Kitasato University School of Medicine*,

Department of Parasitology, Norman Bethune University of Medical
Science' and Yamanashi Prefectural Institute for Public Health'

In mice experimentally infected with Schistosoma mansoni, piquitong ( Paraziquantel )that synthesized in Shanghai, China, administered orally at the levels of 750 mglkg (Group‑1) , 150 mglkg (Group‑2) or medium alone (Group‑3) for two consecutive days of two months
after infection. Mice were autopsied and examined one, two, four, eight, 12, 16 and 20 weeks
after treatment.
The results of the piquitong treatment with doses of 750 mglkg for two consecutive days
showed that none of worms recovered from the mice within least 7 days after treatment.
The mature schistosome eggs in the liver and intestine disappeared and the number of thecalcifled eggs became to increase in the mice of Group‑1 from eight weeks after treatment.
High levels of lgG and lgM antibody titers against adult and egg antigens by ELISA wereobserved in sera of all mice groups until 20 weeks after treatment. On the other hand lower
titer of lgA antibody against egg antigen was observed from eight weeks after treatment in
the group of cured mice.

42 DETECTION AND QUANTITATIVE ASSAY OF MAMUSHISNAKE VENOM AND ANTIVENOM BY ELISA ‑WITH SPECIAL
REFERENCE TO IN VIVO EFFECT OF THE ANTIVENOM
ATSUSHI SAKAI, MITSUKO HATSUSE AND YoSH.IO SAWAIThe Japan Snake InstituteRadioimmunoassay (RIA) and enzyme‑linked immunosorbent assay (ELISA) methods
are used for detection of snake venom in patients to identify the biting snakes and assessmentof antivenom potency. There are few report of the venom movement in patients and animals
and the in vivo effect of antivenom. It is suggested that the venom is absorbed at least
partialy via the lymphatics and some is excreted in the urine from the result that the highlevels of the venom were detected from lymph node, kidneys and‑ blood in the snake bite
victim by Australian elapid snake. And it is reported that Crotalus venom showed' strong
localization to renal tubules and blood v ssel walls in r,ats injected intraperitoneally.

We investigated the potency of the antivenom by monitoring the Mamushi venom in theserum and injection site of rats using ELISA. The venoin concentration in the serum of rats

injected intramusculary reached a maximum at 3‑6 hrs after the injecton, while in the

108

injection site it decreased rapidly during 6 hrs. However, the venom could be detected in theserum and injection site even at 48 hrs after injection.

The venom levels in rats injected with the antivenom intravenously or intramuscularly
30 min after the venom injection were measured. Only 50% of the venom was detected at 30
min after i. m. injection of the antivenom, whereas no venom was detected after i. v. injectionof the antivenom. The venom in the injection site also decreased by the antivenom injection.
However, the venom 'was detected in the injection site during 24 hrs after i. m. injection of theantivenom, whereas no venom was detected at 6 hrs after i. v. injection of the antivenom.
Further investigation was carried out for monitoring the antivenom concentration in thesera of rats and rabbits following i. v. and i. m. injection of the antivenom. The antivenom

concentration in the serum of rats injected intravenously with 50 units of the antivenom
showed 6 units/m'l ･at I hr, while it reached a maximum (1 unit/ml) in the serum of rats
injected intramuscularly 6 hrs after injection, and it needed two days to reach a maximum(1.1 unit/ml) in rabbit injected intramuscularly with the antivenom (500 units) .

These results showed that the patients in snake bites must be administrated with theantivenom by i. v. injection.

43 SEA SNAKE BITES IN OKINAWA
YASUTETSU ARAKI AND YASUHIRO TOMIHARA
Department of Habu, Okinawa Prefectural̲ Institute of Public Health

We have a very limited knowledge about the realities of sea snake bites in Japan. The
only record we have is a report made by Mishima (1963) on a death case caused by Hydrophis
cyanocinctus in Kagoshima Prefecture. Yet the report was not detailed enough to clarify thenature of the bites. In January 1989, an injury case by H. cya,iocinctus occurred in Naha,
Okinawa. The victim narrowly survived the actident after one month's hospitalization,

though he received no treatinent by sea snake antivenin. ,We conducted on‑the‑spot surveys of the sea snake bites during 18 months fromFeb,ruary 1989 through July 1990. Of the 7 wounded persons, 6 (85.7%) died. The elapsed
time between inju, ries to consequent deaths were 2 to 5 hrs for 4 persons, and 15 to 20 hrs for

2 persons. Our surveys include a death case by Laticauda semlfasciata which we presume isthe first record ever reported in the world. The following circumstances are the main reasons

why we have been unable to clarify the sea snake bites in Okinawa.
1: The Prefectural Government has been without an adequate information system due to theabsense of sea snake antivenin that is supposed to deal with the sea snake bites.
2: When bitten, the victims fall into a critical condition so quickly that they usually die or

become fatal before hospitalized. This fact has been preventing doctors from obtaining fullknowledge about the sea snake bites.

l09

44 IXODID TICKS AND INCIDENCE OF BORRELIA BURDORFERIIN NAGANO PREFECTURE, JAPAN (PRELIMINARY REPORT)KIMITO UCHIKAWA , KOICHI MURAMATSU2, KENJI MIYAMOTO

AND MlNORU NAKA03
Department of Parasitology, Shinshu University School of Medicine*,

Nagano Research Institute of Health Pollution' and Department of

Parasitology, Asahikawa Medical College'

Lyme disease described as a separate entity 'in 1977 in USA has also been reported
sporadically from Hokkaido, Ka oshima and Nagano Prefectures since 1987. It is of prior
importance to have a thorough in estigation of the epidemiology of this tick‑born disease inJapan. Since the disease is a typical zoonosis, we have various means to realize incidencesof spirochetal infection in given areas. We think, however, it is most efflcient to demonstratespirochetae in unfed ticks. So we collected ticks by fiagging the vesitation at 20 sites locatedmostly. in the national or prefectural parks in Nagano Prefecture in May and June, 1990.
lxodes persulcatus and I. ovatus were most abundant in grassy and low‑shrub habitats on andalong lanes and tracks in forests. Borrelia burgdorferi, the etiologic agent of Lyme disease,

was detected with IFA adopting the monoclonal antibody H5332 and/or isolated from theabove 2 ticks from 18 sites, indicating a wide dissemination of the pathogen throughout thewhole prefectufe. The infection rate was unexpectedly high and more than 20% on anaverage in the adults of bothpecies. Similar prevalence levels may be recorded in adults ofcommon ixodid ticks in some of the 8 prefectures that neighbor Nagano Prefecture.

45 RECURRENT OUTBREAKS OF TSUTSUGAMUSHI DISEASEAND FLUCTUATION OF VECTOR POPULATION DENSITIESKIMITO UcHIKAWA
Department of Parasitology, Shinshu University Schoo,1 of Medicine

The recurrent outbreaks of tsutsugamushi disease were noticed in the middle of the
1970's. The causes of the rapid increase of patients have been discussed in conection with thechanges in usage of both antibiotics and pesticides, remarkable improvements in diagnostic
techniques and some other artificial factors from the beginning of the 1980's, ignoring possible

chages in vector population densities. We intended to supplement the shortage in theconsideration of causality with ecological studies of the main vectors, Leptotrombidium
pallidum and L. scutellare. L. pallidum was densely populated on grasslands newly formedon abandoned farms and on larger paddy banks that have recently been made' during the
paddy zone reconstruction program. Environme,ntal chages brought about by the abandon‑
ment Qf farms in depopulated areas and the construction of many larger banks in paddy zones
are thought to have provided suitable habitats for L. p llidum and are probably responsible
for the recurrent outbreaks of tsutsugamushi disease transmitted by this chigger. L. scutella‑

110

re was shown to be populated at well‑drained sites on the eastern slope of Mt. Fuji, Shizuoka

Prefecture, and the specific habitat for this vector was the sparse Miscanthus grassland
developed on the sunny granular soilbed. However, we can not evaluate these findings inrelation to the recent outbreaks of the disease transmitted by L. scutellare yet.

46 EPIDEMIOLOGICAL STtJDY ON SCHISTOSOMIASIS MANSONIIN RURAL AREAS AROUND RECIFE, BRAZILMASANOBU TANABE1' 2, JOSE FELIPE GoNCALvES3, SEIKI TATEN02,
KATSVYA MARUYAMA4, ISAO OKAZAKI , TETSUICHI SHIBATA6,SEIICHI INAYAMA6 AND TSUTOMU TAKEUCHll
Department of Parasitology*, Department of Preventive Medicine
and Public Health' and Pharmaceutical Institute', School of Medicine

Keio University, Laborat6rio de Imunopatologia Prof. Keizo Asami
Universidade Federal de Pernambuco', Centro de Pesquisas Aggeu

Magalhaes' and Department of Internal Medicine, Kurihama Hospital'

Parasitological, clinical, ultrasonographic and clinico‑biochemical studies have been

carried out on 485 inhabitants in Sao Lourenco, 25 km northwest of Recife, and on 619
inhabitants in Cabo, 50 km southeast of Recife, Pernambuco, Brazil. Stool examination
demonstrated a high prevalence of intestinal parasitic infections. Moreover, the prevalenceof Schistosoma mansoni in the inhabitants of Sao Lourenco and Cab

were 82.1 and 9.8%,

respectively.

Ultrasonographic examination of the subjects of Sao Lourenco demonstrated a frequentabnormarity such as hepatic peri‑portal fibrosis, splenomegaly and visualization of splenicvein. There was a positive correlation between the degree of splenomegaly and thesehepatosplenic abnormarities and betwe n the degree of hepatic peri‑portal fibrosis andsplenic vein, whereas there seems little correlation between the degree of these hepatosplenic

abnormarities and the intensity of infection as monitored with EPG.

While six enzymatic activities in the sera (GOT, GPT, 7‑GTP, MAO. OCT and
ALPase) , which reflect a liver function, were determined on 334 sera of the subjects in Sao

Lourenco, only ALPase activity significantly increased in the sera of the subjects infectedwith S. mansoni.
The concentration of three serum markers of liver fibrosis [P(IIDP‑NP, Iaminin and

IRpPH] were determined by radioimmunoassay or enzymeimmunoassay. There was no
significant increase in P (IID P‑NP concentration in the sera of S. mansoni‑infected subjects,

whereas a significant elevation was observed in the laminin and IRpPH concentrations in thesera of egg‑positive subjects. Moreover, we also demonstrated a significant elevation ofhydroxyproline concentration in the urine of egg‑positive subjects.

These findings probably suggest cross‑sectional examinations using ultrasonography,and quantitative analysis of serum ALPase activity, serum marker of liver fibrosis (laminin

and IRpPH) and urinary hydroxyproline concentration provide a more accurate informa‑tions on morbidity and pathologic state of the subjects infected with S. mansoni.

111

47 CHARACTERISTICS OF ULTRASONOGRAPHIC FINDINGS AND LIVER
DYSFUNCTION OF SCHISTOSOMA JAPONICUM INFECTION
IN LEYTE, PHILIPPlNESHIROSHI OHMAE1, MASATAKA HAYASH12, BAYANI L BLAS
YUJI IRIEI AND KAZUO YASURAOKAl
Institute of Basic Medical Sciences, University of Tsukuba*,

Kofu City Hospital' and Schistosomiasis Control andResearch Service, DOH, Manila, the Philippines'

Ultrasonographic (US) examination was performed on 45 newly infected cases and 45
reinfected cases with schistosomiasis japonica, in Schistosomiasis Hospital, Palo, Leyte, thePhilippines. US Iiver images were classified into 4 patterns (type O: normal pattern, type 1:

linear pattern, type 2: nodule pattern, type 3: network or fishscale pattern) . Correlation
between US patterns and the liver functions was studied. In the chronic cases in Japan, casesof type 3 were common, while those of types I and 2 were rare. In Leyte, however, patientsof type 3 were less than those of types O and 1. Types O and I were commonly found by US
in newly infected cases, and type 3 in long‑t rm infected cases. Big splenomegaly withcollateral vessels was also found in long‑term infected cases. There was no correlation
between US findings and spleen index. No correlation was found between US findings and thelevels of aspartate aminotransferase, alanine aminotrahsferase, y‑glutamyltransferase andlactate dehydrogenaze. The level of alkaline phosphatase in all groups of the infectedpatients showed significant elevation over the healthy controls. Significant elevation of thelevel of total bile acid was also observed in the patients wlth types 2, 3 and liver cirrhotic
pattern.

48 ULTRASONOGRAPHIC AND PATHOPHYSIOLOGICALCHANGES AFTER TREATMENT WITH PRAZIQUANTELIN SCHISTOSOMA JAPONICUM INFECTION IN LEYTE, PHILIPPINESHIROSHI OHMAE1, MASATAKA HAYASH12, BAYANI L. BLA 3,
YUJI IRIE AND KAZUO YASURAOKAl
Institute of Basic Medical Sciences. University of Tsukuba ,Kofu City Hospital' and Schistosomiasis Control and ResearchService, DOH, Manila, the Philippines'

Ultrasonographic (US) examination was performed on 32 newly infected cases and 20
reinfected cases of schistosomiasis japonica, every 3 or 6 months in Schistosomiasis Hospital,

Palo, Leyte. Philippines. An improvement of the thickening echogenecity of the wall of portal
vein and echogenic bands in the liver lobes was found by US examination 6 rrionths after PZQ
treatment. Typical network pattern or fishscale pattern did not change after the treatment.
Splenomegaly decreased in 42 patients without collateral vessels, but not in 10 patients with

112

collateral vessels. There was no significant difference in results of liver function tests suchas the 'levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,choline‑esterase and lactate dehydrogenase between before and after PZQ treatment.
Significant decrease of the level of peripheral total bile acid (TBA) was observed in patientswith and without periportal fibrosis after PZQ treatment. In cases with network pattern, thelevel of procollagen‑lll‑peptide tended to decline. US examination was useful for evaluating

the portal hypertention and for comparing the changes in the US images of liver fibrosis
before and after PZQ treatment. As a parameter for evaluating the liver dysfunction andfibrosis of hepatosplenic schistosomiasis japonica, TBA appeared to be more reliable than theother liver function tests.

49 BASIC STUDIES ON MONGOLIAN GERBILS AS A SUSCEPTIBLE HOST
TO FILARIAL INFECTION (5) SERUM LIPID
KAZUHIRO SHICHINOHE1, MASIJMI SHIMIZU1, TETSUO IEDOKORO',SETSUKO TSUKIDATE2 AND KOICHIRO FUJITA2
Department of Experimental Animal Science, Nippon Medical School*
and Department of Medical Zoology, Faculty of Medicine,Tokyo Medical and Dental University'

The Mongolian gerbil is used as laboratory animal in the fields of virology, cerebral
neurology, endocrinology and parasitology. Especially, in the parasitology, the Mongolian
gerbil is very useful as the susceptible host to filarial infection. But, there is almost no reportabout biological characters in this animal. In order to know biological characteristics of thegerbils including the coat color mutants, we made comparative study on biochemical assay
among coat color mutants of gerbils and Wister rats. In this examinatioh, we studied on theserum lipid components in agouti (wild) gerbil compared with those in the coat color mutants
such as white spotted agouti, albino, black and white spotted black type maintained in ourlaboratory. . Five adult gerbils of each sex or each coat color which are 14 to 15 weeks old,

totalling 50 gerbils were used. Gerbils and rats were kept in conventional condition at
24i2'C room temperature and 65
5% relative humidity. Blood samples were obtained fromtheir hearts under anesthetization with ether. Sera were separated and stored at ‑ 20'C until

examination. Serum lipid components were analyzed by Hitachi 736 autoanalyzer.
Total cholesterol level of each coat color gerbil was within the range of 67‑171 mg/dl.
Other lipid component values were 14‑39 mg/dl in free‑cholesterol, 50‑135 mg/dl in ester‑choresterol, 40‑219 mg/dl in triglyceride, 91‑190 mg/dl in phospholipid, 0.27‑0.88 mEq/1 in
NEFA and 65‑314 mg/dl in p‑Iipoprotein, respectively. There were not significant difference

on the serum lipid component levels among coat color mutants or sexes of the gerbils. All
lipid component levels were higher than rats.

113

50 BASIC STUDIES ON MONGOLIAN GERBILS AS A SUSCEPTIBLE HOST
TO FILARIAL INFECTION (6) BIOCHEMICAL ASSAY
MASUMI SHIMIZU*, KAZUHIRO SHICHINOHE*, TETSUO IEDOKORO'SETSUKO TSUKIDATE' AND KOICHIRO FUJITA'
Department of Experimental Animal Science, Nippon Medical School*
and Department of Medical Zoology, Faculty of Medicine,Tokyo Medical and Dental University'

Mogolian gerbil has been used very widely as a experimental animal. Especially in theparasitology, this animal has been very useful as a susceptible host of experimental filariasis:

But there are few reports on basic characteristics of Mongolian gerbil and it is recognized tobe necessary in recent years. In this experiment, we made comparative study on biochemical
assay among coat color mutants of Mongolian gerbil and Wistar rats.

Animals were kept under conventional condition at 24 d: 2 'C room temper ture and65 5% relative humidity. Five adult animals of each sex or each coat color were used in thisstudy. Blood samples were obtained from the retro‑orbital venous plexus of the anesthetizedanimal with ether by heparinized capillary pipettes. Sera were separated and were kept
under ‑ 20'C until assay. Biochemical assay was made by using Hitachi 736 autoanalyzer.
GOT, GPT, LDH, ALP and CPK Ievels of gerbils were very higher than rafs and they
were most highest levels in the rod'ents according to the past reports. Ch‑E Ievels of gerbils

had no sex difference and that is different from rats. AMY, UA, BUN, CRE data showedalmost same level as rats. Na, K and Cl levels of gerbils were higher than rats and otherrodents. There were no significant difference between sexes or coat color mutants of gerbils.

51 BASIC STUDIES ON MONGOLIAN GERBILS AS A SUSCEPTIBLE HOST
TO FILARIAL INFECTION (7) SERUM PROTEIN
KAZUHIRO SHICHINOHE MASUMI SHIMIZU , TETSUO IEDOKORO'SETSUKO TSUKIDATE2 AND KOICHIRO FUJITA'
Department of Experimental Animal Science, Nippon Medical School*
and Department of Medical Zoology, Faculty of Medicine,Tokyo Medical and Dlental University'

In order to know biological characteristics of the gerbils including the coat color
mutants, in this experiment, we studied on' the serum protein components of agouti (wild)
gerbil compared with those of the coat color mutants which are white spotted agouti, albino,
black and white spotted black type maintained in our laboratory.
Five animals of each sex or each coat color which are 14 to 15 weeks old, totalling 50
gerbils were used. Blood samples were obtained from their hearts under anesthetization withethey. Sera were separated and stored at ‑ 20'C until examinations. Total protein was
analyzed by Hitachi 736 autoanalyzer and the serum protein fractions were examined by the

114electrophoretic method.
Average total protein value of each coat color gerbil was within the range of 5.2‑6.8 gl
dl. A/G ratio of each coat color gerbil were around 1.5. The typical pattern of serum protein
fractions of gerbils had 5 peaks. Those were 57.8‑61.2% in albumin, 1.9‑2.7% in al globulin,
8.8‑ll.2% in a2‑globulin, 4.4‑9.2% in p‑globulin and 18.1‑23.6% in y‑globulin, respectively.

There was not significant difference on the serum protein components among coat color
mutants of the gerbil.

Our examination appeared that A/G ratios of the conventional Mongolian gerbils includ‑ing coat color mutants are almost same value as that of human, 1.5.

52 COMPARATIVE STUDIES ON SENSITIVITY TO BRUGIA PAHANGIAMONG COAT COLOR MUTANTS OF MONGOLIAN GERBILSKOICHIRO FUJITA , SETSUKO TSUKIDATE1, TETSUO IEDOKORO*,

MASUMI SHIMIZU2 AND KAZUHIRO SHICHINOHE'
Department of Medical Zoology, Faculty of Medicine,Tokyo Medical and Dental University* and Department of Experimental
Animal Science, Nippon Medical School'

In this oxperiment, we made comparative study on sensitivity to Br gia pahangi among
coat color mutants of Mongoloan gerbil which widely used as a susceptible host to filarial
infection.

Coat color mutants of gerbils such as agouti, white spotted agouti, albino, black andwhite spotted black type were kept under conventional condition and 8‑9 weeks old, male andfemale animals were used. Infective larvae (L3 ) of Brugia pahangi were obtained from Aedesaeg)pti mosquitoes infected 14 days previously by blood meals on infected gerbils. Qerbils
were infected by subcutaneous injection of 100 L3 at inguinal region. Blood samples weretaken from the retro‑orbital venous plexus every week and percentage of eosinophils andmicrofilaria counts per 30 pl blood were determined. After observation of 25 weeks, animals
were bled and their organ weights were measured.
As mf appearance rates in each coat color of male gerbils were 100% except black typeand female gerbils also showed 67‑100% positive rate, it appeared that all coat color mutantsof Mongolian gerbils also have sensitivity to B. pahangi.
In each coat color of male gerbils, microfilariae began to appear at 8‑9th week after
infection and attained to peak level of 100‑300 counts at 15‑20th week. In only albino andblack type gerbils, mf counts continued to rise until 25th week. In female gerbils,
microfilariae also began to appear at 9‑12th week and attained to peak level at 15th week.
But mf counl:ts were very lower than male gerbils and hig,hest count in female gerbils were
only 65 per 30 pl blood.

Eosinophil responses were quite different betweeen males and females or between mf
positive gerbils and mf negative them. Namely, mf positive male gerbils showed two distinct

phases of eosinophil responses, one in the prepatent phase at 3rd week and another in thepatent phase at 9‑lOth week, in contrast to only former response in mf negative gerbils. In

115

mf positive female gerbils, eosinophil responses did not show clear phase. On the other hand,eosinophil responses showed some difference among coat color mutants. Mf positive female
gerbils showed almost low response but only albino female were observed 11.5% at 7th week
and continued to show 3.5‑5.5% until 18th week.
In ofgan weight examination after observation, weight of spleen showed remarkabledifference between control group and infection group.

53 FALCIPARUM MALARIA WITH BONE MARROW ABNORMALITIESRESEMBLING MALIGNANT HISTIOCYTOSISATSUO HAMADA1, NAOHIRO WATANABE*, HIROSHI TANAKA2
AND AKIO KOBAYASH11
Department of Parasitology* and 2nd Department of Internal
Medicine', Jikei University School of Medicine

We recently examined a patient with Plasmodium falciparum malaria, whose bonemarrow transiently showed a similar finding of malignant histiocytosis.
A 24 year old Japanese businessman was admitted to our hospital with 4 days history ofhigh fever and chill. He had been in Madras, India for one month just before this admission

but had never been in malaria endemic area before this trip. On admission, high fever,

jaundice and hepatosplenomegaly were observed and laboratory examination showedmarked anemia and thrombocytopenia. The patient was diagnosed as Plasmodium falcipar‑
um malaria by blood smear (parasite density: 140,000 ring form/mm3) , and he was treatedwith quinine hydrochloride (1.5 g/day for 7 day) . After 3 days, fever disappeared and no ringform was detected on the blood smear. A bone marrow aspiration was performed on
admission and revealed a normally cellular marrow with increased histiocytes (8.5%) . The
majority of the increased histiocytes showed intense hemophagocytosis and atypical his‑tiocytes were also apparent. These bone marrow findings were consistent with malignant
histiocytosis. Two months after the malaria treatment, the bone marrow was reexamined.
The number of histiocytes was normal and neither hemophagocytic nor atypical histiocytes
were observed.
Our present report is of the first malarial case to show malignant histiocytosis‑like
syndrome. Epidemiological studies have shown that the occurence of malignant histiocytosis
was significantly higher in tropical area than in temperate zone. It has been hypothesized that

this high incidence might be related to repeated infections of tropical infectious organisms,
particularly malaria. Our case apperars to support a role for malaria infection in the etiology
of malignant histiocytosis.

116

54 USE OF MEFLOQ,UINE IN TREATMENT OF IMPORTED FALCIPARUM
A,ND VIVAX MALARIAHIROYUKI AMANO , TETSUO SAKA12, TAKEHIKO OKUN03
TOSHIMASA NISHIYAMA', YUZO TAKAHASHI' AND TSUNEJI ARAKI'
Department of Oversea Medical Services*, Department of Internal Medicine2 andDepartment of Pediatrics', Tenri Hspital andDepartment of Parasitology, Nara Medical University'

Mefloquine (a 4‑quinolinemethanol derivative, structual analogue of quinine) is usuallyused in combination with sulfadoxine/pyrimethamine, however, antimalarial treatment withmefloquine alone has not been performed because of unavailable in Japan. This report deals

with our clinical experience of mefloquine alone therapy on two cases with imported
Plasmodium falciparum and P. vivax malaria.
The first case, a 2‑year‑old Japanese girl, had the febrile attack on her way home fromlake‑side area of Tanganyika, Zaire in March, 1990. Although treatment with chloroquinein Kenya e, Iiminated her malarial symptoms, 7 days after discharge she had fever in Japanand visited our hospital 5 days after the recrudescence. On admission, her temperature was
38.2'C by axilla. Anemia and hepatomegaly were found. The important laboratory findings
were; hemoglobin 8.1 g/dl, platelet 11.2 x 10'1pl, CRP 4.5 mg/dl, FDP < 10 pg/ml, creatinine

0.3 mg/dl, glucose 100 mg/dl, total cholesterol 133 mg/dl and LDH 2,268 IU. The thin bloodsmear revealed ring forms of Plasmodium falciparum and parasitemia was 11,925lpl. The
treatment with mefioquine (375 mg x 2 days) was started on the first hospital day andparasitemia completely disappear. The clearance of fever and parasitemia was 12 hrs and 60
hrs, respectively. She had been displeased, irritable and insomnia since her admission, andthese symptoms were continued 3 days after administration of mefloquine. And addition toloss of appetite, she complained of transient nausea and abdominal pain after chemotherapy.
The second case was a 26‑year‑old Japanese male, had the febrile attack on his way homefrom Papua New Guinea in June, 1990. He was seen in our hospital 7 days after onset of thedisease. On admission, the patient had clear consciousness although complained of severe
fatigue. The important laboratory findings were; hemoglobin 14.3 g/dl, platelet 5.7 x 10'1pl,

CRP 7.4 mg/dl, FDP< 10 pglml, creatinine 1.0 mg/dl, glucose 121 mg/dl, total cholesterol

99 mg/dl and LDH 673 IU. Abdominal ultrasonicated echography showed splenomegaly.
The thin blood smear revealed trophozoites and gametocytes of Plasmodium vivax andparasitemia was 6,266lpl. The treatment with mefloquine (total 1,500 mg for 24 hrs) wasstarted on the second hospital day and parasitemia completely disappear. The clearance offever and parasitemia was 36 hrs and 60 hrs, respectively. No side effects was found.
Mefioquine has nearly same antimalarial actions as quinine, being effective against both
Plasmodium falciparum and vivax. Its half time in vivo (T=14 ds) is so long that the single

dose treatment is recommended. In this study we followed the drug information given by thecompany. Its side effects including dizziness, vomiting, diarrhea, bradicardia and neuropsy‑
chiatric changes are usually mild, transient and tolerable, which was reconfirmed by thepresent cases.

117

55 INCIDENCES OF DIC COMPLICATION IN JAPANESE PATIENTS WITH

MALARIA

KlYOKATSU TANABE
Department of Medical Zoology, Faculty of Medicine,Kagoshima University

Forty eight patients with falciparum malaria (14) and vivax malaria (34) were evaluat‑
ed retrospectively as to whether DIC (disseminated intravascular coagulation) had been
complicated or not. For this purpose, four kinds of laboratory data in coagulation test, thatis, serum fibrin‑degradation products (FDP) , platelets, fibrinogen, and prothrombin time
were estimated according to the diagnostic criteria for DIC complication which was reported
at 1988 by the Study Group. for DIC complication which had been organized by the Ministryof Health and Welfare in Japan. Serum concentration of FDP was elevated in 8 cases (57%)

of falciparum malaria and in 3 cases (9%) of vivax malaria. Thrombocytopenia was seenin 12 cases (88% ) of falciparum malaria and in 30 cases (86% ) of vivax malaria. Prothrom‑

bin time elongated in 4 cases (8%) and plasma concentration of fibrinogen decreased in 3
cases (17% ) . Among the coagulation tests, abnormality grades in FDP concentration had
closest association with DIC in malaria cases, therefore FDP test is regarded as dne of themost important and indispensable test in malignant malaria for checking the DIC complica‑tion.

According to incidences of DIC complication among these 48 cases, only 4 patients met
the criteria for the diagnosis (the score must be over 7 points) . All patients with DIC
complication were infected with falciparum malaria and three of them developed to cerebral

disturbance (cerebral malaria). One case of vivax malaria was suspected of the DIC
complication (6 points) .

As to therapy for DIC, heparin‑sulfate was administered as an anti‑coagulant therapy in

3 out of 4 cases with the DIC complication. The result of the treatment indicated that 3
patients, all of them had manifested cerebral disorders, were cured without complication ofrenal disturbance, whreas one patient escaped the anti‑coagulant therapy developed renal
insufiiciency (serum concentrations of BUN and creatinine elevated at the peak values of 92
and 17 mg/dl, respectively) . These clinical data suggest that the anti‑cdagulant therapy may

be necessary for treating patients with DIC complication even when the DIC is caused bymalarial infection.

118

56 THE SIGNIFICANCE OF TIME LAG IN THE TREATMENT OF
FALCIPARUM MALARIAISAO EBISAWA
Keihin Railway Kawasaki Clinic

We have previously reported that there exists a time limit for effective treatment of
falciparum malaria. In short, 4 days after the onset of the illness was found to be the time
lim'it beyond which severe, Iife‑threatening complications may develop. In order to elucidate
the level of laboratory variables beyond which the patient's prognosis is poor, we examinedthe correlation between time lag in days vs maximum parasite count, minimum red blood cell(r.b.c.) count, minimum hemoglobin (Hb) , maximum blood urea nitrogen (BUN) andcreatinine concentration of each patient during the period of admission. A Iinear correlation
was found between time lag (x) and the maximum parasite count in log units (y) , y = 4.031 +0.124x, r=0.3579. All variables among patients whose treatment was started after 5 to ll
days of time lag (6th to 12th day of illness) were significantly worse than the variables ofpatients whose treatment was started after only I to 4 days of lag time. However, the mostconspicuous change was observed in the abrupt increase of BUN and creatinine in themembers of the latetreatment group: the mean BUN and creatinine levels in the early and latetreatment groups were 16.0 vs 57.1 and 1.2 vs 3.4 mg/dl (p<0.01), respectively. The meanparasite counts of the early and late treatment groups were 23,400 vs 69,200/pl of blood (p<0.05) , while the mean r. b. c. count and Hb concentration of the two groups were 3.81 vs 3.
28 million per pl and 12.4 vs 10.0 gldl (p<0.01) , respectively.
In addition to the level consciousness of the patient, special attention should be paid tokidney function in the treatment of falciparum malaria patients whose treatment is delayed
beyond the 5th day of illness.

57 DNA DIAGNOSIS OF FALCIPURUM MALARIAYUSUKE WATAYAl, FUMIE KUBOCHll, YUKO OHYAl, SATORU NAKAGAM12TATSURO KAWASOE2, AKIO YAMANE2 AND AKIRA ISHll3
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences, Okayama University*, Central Research Laboratories,
Wakunaga Pharmaceutical Co., Ltd.' and Department of Parasitology,

Okayama University Medical Scho013

Techniques utilizing DNA hybridization probes for the rapid detection of Plasmodium

falciparum had been reported. However, these methods had many problems about thesensitivity and the stability of these probes. We have developed two types of plasmid‑born

DNA probes: One represents the junction DNA sequence (410 bp) of the DHFR (dihy‑
drofolate reductase) ‑TS (thymidylate synthase) gene. The other repres nts the DHFR DNA(790 bp) . When these probes were used, the target sequence was amplified by PCR

119(polymerase chain reaction) to increase the sensitivity. We used the Universal Probe System{Yamane et al. (1988) : Nucleic Acid Res., Symposium series,' 19, 93‑95}. This system consistsof two probes; a primary probe prepared from a chimeric phage‑plasmid vector (pUCfl)
containing a sequence complementary to a target, and a biotinLlabeled secondary probe
complementary to a portion of the primary probe, which is detect d by the BCIP/NBT
method. This system has a high sensitivity to the target DNA because the secondary probe
is labeled with many biotin molecules.
We showed that the junction DNA was more sensitive than the DHFR DNA as target todetect of P. falciparum and the limit of detection was 10' parasites in 100 pl human blood with

DHFR‑TS junction probe.

58 SEROLOGIC EVIDENCE OF TOXOCARIASISIN NORTHEAST BRAZILKOUICHI NAGAKURA1' 2, YOSHIMASA KANEDAI AND SEIKI TATEN02
Department of Parasitology, Tokai University School of Medicine*and Laboratorio de Imunopatologia Prof. Keizo Asami (LIKA),
Universidade Federal de Pernambuco, Brazil'

Tropical eosinophilia syndrome (TES) is found most commonly in areas where filariasis

occurs, notably South‑East Asia and the east coasts of Africa and Brazil. The salientfeatures of TES are asthma‑like symptoms associated with hypereosinophilia. However, thesame symptoms can be caused not only by infection with filarial parasites, but also bybacteria, fungi and other helminth parasites such as Ascaris. Ancylostoma. Strongyloides,
Schistosoma and Toxocara spp. (Manson's Trop. Med.). Although Toxocara is known tocontribute to TES, few cases have been reported from tropical regions (Lynch, 1988). In
Brazil, T. canis infection in dogs is relatively common; the rate was 59.8% in Sao Pauloduring 1980‑1985 (Cortes, 1988) . Moreover, in humans, two cases of infant toxocariasis(visceral larva migrans) were reported in Sao Paulo, in 1984 (Kawakami, 1984) . In order todetermine the incidence and status of toxocariasis in northeast Brazil, where TES is very
common, 54 sera from children with a history of asthma and hepatomegaly (eosinophilia:> 740 cells/pl) were screened serologically.

The enzyme‑1inked immunosorbent assay detected 21 positive sera (40%). Many ofthese cases ( 15 of 21) were positive for Dilofilaria immitis as well. Six of the Toxocara‑

positive were detected, in some children, were symptomatic. Their sera were further
examined by the Toxocara species‑specific Ouchterlony's diffusion‑in‑gel test (Nagakura,1990) and one Toxocara canis and one T. cati infection were identified. This is the first reportof serologically defined toxocariasis in Brazil, and the status of the disease in the tropics was

discussed.

120

59 SERO‑EPIDEMIOLOGICAL SURVEY OF HUMAN TOXOCARIASISIN TROPICAL ZONEKAORU KoNDOl, NoBUAKI AKAOl, TAKAAKI OHYAMAl
MOTOHITO SAN02, KOUICHI NAGAKURA3, ENG‑RIN CHEN4
P. RADOMYOS5, B. D. SATTE6 AND M. A. HAFEEZ7
Department of Parasitology, School of Medicine, Kanazawa Universityl

Department of Parasitology, Hamamatsu University School of Medicine2
Department of Parasitology, School of Medicine. Tokai University3

Department of Parasitology, Kaoshiung Medical College',

Bangkok School of Tropical Medicine, Mahidol University, Thailand=
Department of Zoonoses. Haffkin Institute, Bombay, India6
and Department of Pathology. Jabalpur Medical College, India7

Sero‑epidemiological survey of toxocariasis was carried out on 241 individuals (8 areasin 5 countries) Iived in tropical zone. The serum samples were collected from 41 in Brazil(Recife) , 49 in Taiwan (Taichung and Kaoshiung) , 57 in Thailand (Bangkok), 21 in In‑
donesia (Napu in Sulawesi Island) , and 73 in India (Darbhanga, New Delhi and Bombay) . In
this survey, we performed ELISA using Tbxocara canis larval ES (TcnLES) as antigen, andcalculated the positive rates and mean antibody titers against TcnLES antigen.
The inhabitants in Napu showed the highest positive rate (14.2%) and mean antibody
titer (1.52 0.35) among the eight areas. The people who possessed positive antibody titers
for TcnLES had also high antibody titers for Schistosoma japonicum and Dirofilaria immitis
antigens. Napu is known to the endemic area of schistosomiasis and filariasis. Therefore, we
considered that mixed infection or brevious sensitization had occurred in this area. In Recife,

the positive rate and mean antibody titer were 7.32% and 1.49i0.44, respectively, those
figures were secondarily among 8 areas. Although the place was endemic area of filariasis,
we could not detected the antibodies against another four helminth antigens indluding
dirofilaria worm extracts.

There were no significant differences among the positive rate for TcnLES antigen in

Taiwan (4.08%), Thailand (1.75%) and India (1.37%) as compared with those in Japan(1.37%). However, the mean antibody titers for TcnLES antigen in 4 countries were
distributed 1.13 0.39 to 1.23 i 0.43, which were significantly higher than that in Japan. These

results indicated that geographic and ecological factors were essential for understanding of
etiology of toxocariasis.

121

60 SEROLOGIC TYPING OF CLINICAL CASES WITH AMEBIASISBY GEL DIFFUSION PREECIPITIN TESTEIICHI OKIJZAWAl, SEVERA R. N. MOTTA1, TSUTOMU TAKEUCHll

AND GOUTA MASUDA2
Department of Parasitology, School of Medicine, Keio University*and Department of Infectious Diseases, Tokyo Komagome
Metropolitan Hospital2

Although gel diffusion precipitin test (GDP) is a standard serodiagnostic method forinvasive amebiasis, its detailed characterization has not been attempted. In this study, we
analyzed the precipitin bands pattern using the fractionated antigens. The re ult fromapproximately 300 sera suggests that the patterns change reflecting clinical course.
Through this analysis, five groups of precipitation bands were recognized and designatedas P, Q, PAI (greek), R, S bands respectively, and each sera could be classified into 4
serotypes based on the GDP patterns as follows.
1 ) Acute serotype, defined by presence of Q or S in the absence of typical P nor R,
contained approximately 50 cases. Clinical feature of these cases was variable; fromasymptomatic cases to liver abscess. However, in symptomatic cases, virtually all of thesecases were fresh. It is interesting that cyst passing cases with liver abscess were involved

into this type. With some pair sera, this serotype seems to develop from seronegative casesand change to chronic serotype in I month.
2 ) Chronic serotype, defined by presence of predominant R bands to P bands, contained
about 70 cases. Clinical feature of this type is chronic olitis. Eighty percent of chronic
colitis cases showed this serotype. Liver abscess cases with chronic colitis seem to be
exceptional. This type seems to develop from the acute serotype approximately 2 month
later from onset (bloody stooD .
3 ) Advanced type, defined by, presence of strong P bands, contained 25 cases. Clinicalfeature of this type is liver abscess with chronic colitis, which have past history of bloodystool, present colitis and no cyst passing in stool. This serotype seems to develop fromchronic serotype in the case of liver abscess, and change to chronic serotype about 2 monthafter metastatic sign (fever over 38'O . '4 ) Negative or weak pattern: cases difficult to identify.

This serologic procedure is useful for not only clinical follow‑up, but for patho‑
physiological study of amebiasis.

It would be of a great value to distinguish acute and chronic amebiasis, because theformer has a variety of clinical symptoms and sometimes very severe status. On the other
hand the latter shows characteristic serological response. It is likely that the amebiasis is atremendously heterogeneous syndrome clinically judging from serologic aspects.

122

61 THE DISTRIBUTION OF NAEGLERIA SP. AND ACANTHAMOEBA SP.
IN KANTO AREA OF JAPANSHlNKICHI AKAO
Department of Parasitology, National Defense Medical College

The isolation and culture of free living parasitic amoeba has been reported elsewhere.
For the present study, author surveyed pond and river waters in Kanto area of Japan. Author
isolated 6 strains of Naegleria and 3 strains of Acanthamoeba. The one strain of Naegleria

proved pathogenic to mice.
The isolated strain of Naegleria was observed by electron microscopy. In these experi‑ments, Iamellar structures were found in the cytoplasm of Amoeba. Author reported before,
these lantellar structures concerned with the virulence of Naegleria sp. Further pathogenic
studies of these organelles are necessary to clarify their functions.

62 P. REVALENCE OF CRYPTOSPORIDIUM INFECTION AMONG
HOUSE RATS, RA TTUS RATTUS AND R. NORVEGICUS AND
THE EXPERIMENTAL INFECTION IN R. RA TTUSHISASHI YAMAURAl, RYUKOH SHIRASAKA1, HIROKO ASAH11

TSUTOMU KOYAMA2 AND MITSUGU MOTOKI
Department of Parasitology, Tokyo Women's Medical College*,
Department of Parasitology, National Institute of Health'and Apex Sangyo Co. Ltd."An epidemiological survey for Cr)ptosporidium infection was carried out on house rats.
Rattus rattus (roof rats) and R. norvegicus (brown rats) , captured in the Tokyo Metropolitan
District. Of.a total of 231 house rats, consistiong of 175 roof rats. 48 brown rats, and 8 species‑

indeterminate rats. 32 (13.9%) were found to be positive for Cryptosporidium oocysts in theirfeces. The incidence of the infection was 17.7% for roof rats and 2.1% for brown rat,
respectively. In roof rat groups classified by body weight, the incidence was consistently high(13.8% ‑25.0%). There was no significant difference between the incidence of infection andthe sexes. The size of the oocy ts of the isolates from roof rats measurd (3.7i0.22) x (4.8

0.3) pm. Roof rats experimentally inoculated with C,)ptosporidium oocysts from naturally
infected rats began to shed oocysts on days 2 to 3 post inoculation (PD. The number ofoocysts in feces peaked on days 5 to 8 PI, declined rapidly, and thereafter markedly small
numbers of oocysts were detected intermittently until day 60 PI. None' of the roof rats
showed any apparent clinical symptoms such as diarrhea in experimental infection.

123

63 THE FUNCTION OF DENGUE VIRUS SPECIFIC PROTEINS IN VIRUS
REPRODUCTION= BIOLOGICAL ACTIVITIES OF NS5EDWARD ZULKARNAINl, NoBUYA FUJITA2, SUSUMU HOTTA
AND TSUTOMU TAKEGAMI*
Virus Laboratory, Division of Tropical Medicine,

Medical Research Institute, Kanazawa Medical University*and Kyoto Prefectural Institute of Hygiene and Environmental Science'

Dengue virus (DV) infection makes serious problem in tropical society. However, themechanism of DV replication and functions of nonstructural proteins in viral life cycle areunknown. It is speculated from amino acid sequence analysis that NS5 may be viral RNA
polymerase.
Td clarify the function of NS5, we used a monospecific antiserum against Japanese

encephalitis virus (JEV) NS5. Antiserum against JEV‑NS5 (anti‑NS5) detected DV‑1
specific NS5 by Western blot (WB) and immunofluorescence antibody (IFA) analyses. The
results indicated that DV‑1 NS5 was cross‑reactive with JEV‑NS5 and localized at theperinuclear and membrane fractions in the infected cells. On the other hand, activity of invitro RNA synthesis of DV‑1 infected cell extracts was much more lower that of JEV infected
ones and this might be related with the big difference in viral growth. Taken together withthe data using anti‑NS5, ability of DV‑NS5 seems to be different from JEV NS5.
Next we compared virus growth of 3 kinds of newly isolated DVs from Indonesia (Fujita
et al., 1898). In the early st ge of virus reproduction, i. e. until 2 days after virus infection,

3 strains grew more rapidly than that of Mochizuki strains. However, anti‑NS5 could detect
NS5 from only Mochizuki‑infected cells. This result suggests that the immunogenicity of
NS5 from newly isolated strains differs from that of Mochizuki. This immunogenic discrep‑

ancy seems to be related with biological activities of NS5. The comparison of amino acidsequence indicated that NS5 contains a variable region at the upstream of GDD sequence.
This might be virus strain specific and has some roles in the function of NS5.

64 MECHANISM OF ENHANCEMENT OF ARBOVIRUS MULTIPLICATION IN
MOSQUITOES INGESTING MICROFILARIAE OF DIROFILARIA IMMITIS BYCISTOPATHOLOGY AND ELECTRON MICROSCOPYEIMAN M. ZYTOONl, TAKEO MATSUMURA* AND HIROSHI IT02
Department of Medical Zoology* and Department of Pathology',
Kobe University School of Medicine

Aedes albopictus mosquitoes of the Miki strain did, not support the multiplication of
chikungunya virus after oral infection. We investigated whether simultaneous ingestion of
the virus and microfilariae of Dirofilaria spp. would encburage viral infection and dissemina‑tion throughout these mosquitoes. We allowed the mosquitoes to ingest difibrinated sheep

124

blood mixed with 3 x 108 PFU of virus per milliliter and 15 microfilariae per mosquito by an
artificial feeding technique and compared the infection rate in the midgut and salivary glandwith those of control mosquitoes ingesting the virus without microfilariae. The mosquitoes
ingesting both agents were infected at a high rate. The virus titer of separate homogenatesof mosquitoes' bodies and legs was significantly higher in these mosquitoes than in thecontrols. In four repeated experiments, the viral infection rates were high r as the dose ofvirus increased provided that microfilariae were also ingested. Microfilariae seem to enable

the virus to enter the hemocoel through the holes they punctured in the midgut. We studied
this histopathologically.

Fourteen days after infection, the salivary gland of the mosquitoes was examined under
an electron microscope. Some viral particles were found scattered in the salivary gland.
Probably when this'･virus multiplies in the hemocoel and salivary gland of a mosquito, it canbe more effectively transmitted to human beings when they are bitten.

65 DEVELOPMENT OF MICROFILARIAE OF DIROFILARIA SPP.
STUDIED BY IN VITRO USE OFHEMOLYMPH OF AEDES ALBOPICTUS MOSQUITOESEIMAN M. ZYTOON, YOSHIYUKI MATSUKAWA AND TAKEO MATSUMURA
Department of Medical Zoology, Kobe University School of Medicine

Earl (1959) first attempted to cultivate microfilariae of Dirofilaria immitis in vitro, andWeinstein (1963) observed the development of microfilariae up to larvae the late first stage(sausage form) with various supplements to their media. There are few reports of develop‑
ment in vitro beyond this stage. Here, we studied whether microfilariae of Dirofilaria spp.
could develop in organ culture and how the culture method could be simplified. Up to now,the development of microfilariae has been studied in a variety of tissue culture media throughthe alternating gas phase of the cultured cells. Microfilariae were obtained aseptically fromthe blood of dogs/a dog with microfilarimic blood (55,000‑60,000 microfilariae/ml). The
density of the microfilariae was adjusted to 2 x 102 per milliliter of the medium to be tested.

The media used were NCTC 109, Leibovitze L‑15, RPMI 1640, Tr 17, and MEM. We added
cells of the Aedes albopictus cell line C6/36, 40% heat‑inactivated fetal calf serum, 0.1%

sodium casinate, and 0.5% A. albopictus hemolymph. The media were tested with andwithout supplements. The supplements supported larval development to the late first stage
and, at a lower percentage, to the second stage, in NCTC 109 and Leibovitze L‑15 media.

Control media without supplements supported survival for only a few days, and themicrofilariae did not develop.

We thank Drs. Katsuhiko Ando, Jun Mitsuhashi, Hiroshi Yamanishi, and Norimi
Yamamoto for their collaboration throughout this study.

Proceedings of XXXII Annual Meetings of Japanese Society of Tropical Medicine

Contents """""""""""'1"""i'i"'i"""""""""I""""""""""""""'i"""""""""
Special lecture"""""""""""""""""""""""""""'i'i""""""""'1"""""""""""

65‑68
69‑73

General presentation """"""""""'tl ""I"'i'tl'i""""" " """I""""""""""""I"""I'

74‑124ISSN 0304‑2ld6

JAPANESE JOURNAL OF
TROPICAL MEDIClNE AND HYGIENE
March 1991

Vol. 19 No. 1

CONTENTS
Original article

Yago, A.
Treatment of Cutaneous Leishmaniasis‑Report of a Case Tr ated with Metronidazole and Local Heat‑(in Japanese) """"""""'
Suzuki, N. and Imamura, K.

1 ‑14

Additional 12 cases of Diplogonoporiasis in Kochi Prefecture(in Japanese) """"""""""'

15‑23

Ono, T., Nakabayashi, T. and Ohnishi, Y.

Electron Microscopical Observations on Gametogenesis and Fertilizationin Cultured Plasmodium falciparum """"""'

25‑31

Kiyohara, T., Murata K., Moriya, T., Watanabe, M, Ikeda, S.,lchimaru, M. and Itakura, H.
Defect of Post‑triggering Natural Killer Cell Mediated Cytolytic Activity

by Synthetic HIV Analogue Peptides ""'33‑38lchinose, Y., Monges A.C., Centurion, M.G., Medina, D.,

Albomo, R.M., Bestard, J.J., Sakamoto, M. and Moreno A.R.
An Etiological Study of Bacterial Diarrhea Among Infants in Paraguay

39‑47

in 1990 ････････････'･････････‑

Clinical study

Ebisawa, I., Tanabe, K. and Ohara, H.

The Significance of Time Lag in the Treatment of Falciparurn Malaria(in Japanese) """""""""'

49‑56

Ebisawa, I., Tanabe, K. and Ohara, H.

Serum Bilirubin in Malaria Patients (in Japanese) """""""""(Contents continued on reverse of this cover)Jpn. J. Trop.

Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDIClNE

57‑63