第22巻第4号平成6年12月

内
原

容

著

長崎県下の1名のC型肝炎（HCV）患者血清中のHCVウイルス遺伝子型別及びE1とE2／NSI糖蛋
白質領域の遺伝子解析（英文）・・鄭薇

埼一……………・………一………・…………・169−177

エクアドル共和国におけるらいの血清疫学的調査（英文）
・・細川

篤，野中

薫雄，Miguel

H．Jurado，

古川

正人，江下

優樹，三森

龍之，片倉

EduardoA．Gomez，和泉

眞蔵，橋口

健

義久……

17H84

マラリア予防薬としてメフロキンを長期投与した際の副作用について・・竹島

茂人…・……………・・…・……………・…・………

185−192

日本における輸血マラリア

ー血小板輸血により感染したと考えられる熱帯熱マラリア1症例を中心に一
・狩野

繁之，鈴木

守・…………・……・……………

193−198

長崎市の南部と北部における犬糸状虫伝搬蚊の個体数と自然感染率の比較（英文）
・小田

力，末永

敏，在津

誠，黒川

憲次，

藤田紘一一郎，小川

保徳，山崎

一郎，飯田

国洋，

三根真理子…………………一一…………・…………

199−206

ソロモン諸島で見いだされたムナケブユ亜属1新種の記載（英文）
・高岡

宏行，鈴木

博………・………・……………・207−210

研究ノート

タンザニアの免疫血清による熱帯熱マラリア原虫の試験管内増殖阻害（英文）一Thomas

B．Nyambo，神原（裏面に続く）廣二…・………・……・211−215

短報
インドネシア在住日本人の寄生虫感染状況（英文）
・山田

誠一，森

有加，藩

悦，赤尾

信明，

月館説子，藤田紘一郎…

217−218

症例報告
エクアドル共和国におけるらいの2例の提示と接触者検診の試み（英文）
一細川

篤，野中

薫雄，JuanJ．Alava

P，

EduardoA．Gometz

L．，HugoM．Jurado

S．，

橋口

義久…一

219−223日本熱帯医学会雑誌発行所

日本熱帯医学会

第22巻第4号

編集者

神

1994年12月15日

印刷

1994年12月15日

発行

原

二

印刷所㈱昭和堂印刷
諌早市長野町1007−2 （⑰854）
奮0957−22−6000

本雑誌の刊行にあたりその費用の一部を文部省科学研究費補助金（研究成果公開促進費）によった。

廣

169

Jpn. J. Trop. Med. Hyg., Vol. 22, No. 4, 1994, pp. 169 177

GENOTYPE IDENTIFICATION OF HEPATITISC VIRUS (HCV) ISOLATED FROM A SlNGLE
JAPANESE CARRIER IN NAGASAKI PREFECTURE
AND GENOME ANALYSIS OF E1 AND E2/NS1
ENVELOPE GLYCOPROTEIN REGIONSWEI‑YUN ZHENG
Received September 20, 1994/Accepted October 30, 1994

Abstract: The nucleotide (nt) sequence of a Hepatitis C virus genome (HCV‑N) which was derived froma single Japanese patient's serum in Nagasaki Prefecture has been determined by multiple clones coverning
22 overlapping regions of the HCV geneme. The sequenced region consisted of 9295 nt, including 248 nt of
5'‑untranslated region (UTR) , a single large open reading frame (ORF) encoding a polyprotein of 3010

amino acids (aa) and a 17 nt of 3'‑UTR. Phylogenetic analysis indicated that HCV‑N belongs to II/1b

genotype of group 1. Two other Nagasaki HCV strains (HCV‑Nl and HCV‑N2) were also sequenced in
the E1 and N‑terminus of the E2/NSI regions. Two hypervariable regions (HVR I and HVR 2) were foundin the N‑terminus of E2/NSI region among 3 Nagasaki strains and 7 other HCV strains with publishedsequences. Two well‑conserved aa sequences were also identified among 10 HCV strains in the E1 and N‑terminus of the E2/NS1 regions. The results will be useful for future underStanding on the pathogenesis,
virological diagnosis and development of vaccine for HCV.

INTRODUCTION

ka et al.. 1992), HCV‑T (Chen et al.. 1992), HCV‑JKl(Honda et al.. 1993) , HC‑C2 (Wang et al.. 1993) , HC‑G9

Hepatitis C virus (HCV) is the major cause of nonA

(Okamoto et al., 1994) and NZLI (Sakamoto et al..

‑nonB (NANB) hepatitis, and chronic infection with1994) . Depending on the HCV sequence similarity, HCV

HCV has been linked to the development of liver cirrho‑

genome can be classified into 6 genotypes: I, II, 111, IV,

sis and hepatocellular carcinoma (Plagemann, 1991).
Many entire and partial HCV sequence informations
published so far have shown that HCV genome consisted

V and VI as reviewed by Sakamoto et al. (1994) , or intoof about 10 Kb single‑stranded positive‑sense RNA with
5'UTR, a single long ORF foll'dtved by 3'UTR. The viralgenome organization re embles that of the flaviviruses
and pestiviruses (Kato et al., 1990; Plagemann, 1991).
Since Choo et al. (1989) cloned the genome of an

RNA virus from the plasma of a chimpanzee inoculatedwith plasma fyom patient with NANB hepatitis anddesignated it as HCV, entire sequences have been report‑
ed for at least 15 HCV strains. There are: HCV‑J (Katoet al., 1990), HCV‑1 (Choo et al.. 1991), HCV‑H (In‑chauspe et al.. 1991), HCV‑BK (Takamizawa et al.,1991), HC‑J6 (Okamoto et al., 1991), HC‑JI (Okamoto
et al., 1992a), HC‑J8 (Okamoto et al., 1992b), HC‑J4/
83, HC‑J4/91 (Okamoto et al., 1992c), HCV‑JT (Tana‑

6 major genotypes: I (a, b, c) , 2 (a, b, c), 3 (a, b), 4, 5 and

6 (Simmonds et al.. 1933b).
Recently, the newest classification has been sum‑
marized by Sakamoto et al. (1994) who classified HCV

genome into 3 major groups with entire publishedsequences. The entire HCV sequence of Japanesestrains have been assigned either to group I (1/1agenotype: HC‑J1; II/1b genotype: HCV‑J, HCV‑BK, HC‑J4/83, HC‑J4/91, HCV‑JT and HCV‑JK1) or group 2(III/2a genotype: HC‑J6; IV/2b genotype: HC‑J8)
according to Sakamoto et al. (1994). In order to knowwhether there is particular HCV in the local area ofNagasakj, Japan, HCV patient serum which came frorn

Nagasaki Prefecture was obtained and the genome
sequence of the HCV‑N in this serum was determinedby multiple overlapping clones.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan

170

In order to see the genetic variability of HCV in the

N gasaki area, HCV genome sequence in 2 more HCV
patient sera in Nagasaki Prefecture (HCV‑N1 and
HCV‑N2) were also analyzed similarly in the E1 and N‑terminus of the E2/NSI envelope glycoprotein regions.
Two hypervariable regions have be found among these 3

Nagasaki HCV strains using the sequence diversity
comparison with published sequence data of 7 HCVstrains.

MATERIALS AND METHODSRNA extraction from HCV patient serum
Serum samples were obtained from NANB patientsin Nagasaki Prefecture which were kindly provided by
the Second Department of Internal Medicine of Nagasa‑
ki University Hospital. These patients were confirmed
to be infected with HCV by anti‑CIOO HCV ELISA Kit(Ortho Diagnostic Systems, Tokyo, Japan) and HCVreverse‑transcription polymerase chain reaction (RT‑PCR) as published by Kurihara (1992). One hundred
microliters of HCV patient serum was mixed with 20 pl

lwaki Co.) was started irnmediately after RT andrepeated 35 times. CDNA product was subjected toagarose gel electrophoresis and visualized by ethidium
bromide staining.

Cloning and sequencing of HCV CDNA product
The amplified HCV CDNA was excised from agar‑
ose gel, phosphorylated with T4 polynucleotide kinase(Nippon Gene Co.) and blunted with T4 DNA polymer‑ase (Takara Co.). The modified CDNA fragment was
ligated into Smal site of pUC19 and transformed into
Escherichia 'coli JM 109 strain. The recombinant pUC19

carrying inserted CDNA fragment was purified withWizardTM Minipreps DNA Purification System (U.S.
A.). The CDNA fragment sequence was determined inboth directions with sense and antisense primers bydideoxy chain termination method using both 35S radio‑isotope‑labeling (DNA Sequencing Kit Version 2.0, U.S.
A.) and fluorescent dye‑labeled DNA sequencing system(373A DNA Sequencer, Applied Biosystems) . To avoid
sequence variabili,ty, 3 colones from each RT‑PCR

product were isolated independently for nucleotide
sequence determination.

of 10% sodium dodecyl sulfate (SDS) and 80 pl ofAnalysis and homology comparison in nucleotide

sterile distilled H.O at room temperature for 5 min.
HCV RNA was extracted with phenol / chloroform andand deduced amino acid sequences were carried out for
HCV‑N and 7 other HCV strains with published entire

precipitated with 3 volumes of ice‑cold absolute ethanol.

sequence, using a computer system with DNASIS Mac
Version 2.2, NEW CD2 system (Hitachi Software Engi‑

After storage at ‑80'C for lhr, HCV RNA was pelleted
in an Eppendolf centrifuge with 15,000xg at 4'C for 30

neering Co., Ltd, 1992).

min. The pellet was washed once in 70% ethanol,
vacuurn dried and dissolved in 100 pl sterile distilled

RESULTSwater. RNA solution was stored at ‑80'C.
Selection and synthesis of oligonucleotide primers

Oligonucleotide primer sequences were selectedbased on the published sequence data (Choo et al., 1991,

Takamizawa et al.. 1991) and gene walking method.
Oligonucleotide primers were synthesized by Applied

The genome characterization of HCV‑N
Multiple cDNA clones were isolated from alto‑
gether 22 overlapping regions which covered almost
entire genome of HCV‑N, except extreme 5' and 3'
terminals (Fig. 1). For each of the overlapping region,

Biosystems Model 392 DNA / RNA Synthesizer andconfirmed for their purity by ion‑exchange gel

3 independent CDNA clones were isolated from the RT‑PCR product and sequenced. The cleavage site of thechromatography (Gen‑pack; Waters) .

polyprotein coded by the ORF of the HCV‑N genome

RT‑PCR

was assigned according to the publications by Okamoto
et al. (1992b) and Honda et al. (1993). Total length of
the sequenced region of HCV‑N consisted of 9295 nt andTen. microliters of RNA solution were added with90 pl of RT‑PCR mixture [100 pmol of each primers, 0.2
mM deoxynucleoside triphosphate, 10 mM Tris (PH 8.
9) , 1.5 mM MgC1,, 80 mM KC1, 0.5 mg of bovine serum
albumin per ml, 0.1% sodium cholate, 0.1% Triton X

3010 aa, respectively (Fig. 2) . The sequenced region canbe divided into 248 nt in the 5'UTR, 9030 nt in a single

ORF and 17 nt in the 3'UTR, respectvely. The ORF was‑lOO, 10 U of reverse transcriptase (Life Science Inc.)

considered to be translated into C (191 aa) , E1 (192 aa) ,and 2U of Tth DNA polyrnerase, a thermostable DNApolymerase (Toyobo Co.)]. The reaction mixture was

E2/NSI (346 aa), NS2 (277 aa), NS3 (609 aa) , NS4 (398aa) and NS5 (997 aa) proteins.
Genotype classification and genome homology compari‑covered by 2 drops of mineral oil and incubated for 10
min at 53'C for RT. PCR amplification (94'C for 60 sec,
53'C for 90 sec and 72'C for 120 sec by thermal cycler;son among HCV‑N and 7 other HCV strains withpublished sequences

171

1 ooo

2000 3000

4000 5000 6000 7000 8000

C E1 E2/NSI NS2

NS3

NS4

gooo

bp

(Kato et al.. 1990), HC‑J6 (Okamoto et al..

1991), HC‑J8 (Okamoto et al.. 1992b), HCV‑JT (Tanaka et al.. 1992) arid HCV‑T (Chenet al.. 1992). Homology comparison between

NS5

HCV‑N and these 7 HCV strains was shownin Table 1.' The most conserved region is the

L ] DD

5 UTR which showed nt homology more than
91.5% among 8 HCV isolates. The sequencehomology of the C protein region was also
highly conserved (nt > 81.1%, aa > 88.5%).
In the putative E1 and E2/NSI regions, the

D D
IEl]

:

HCV‑N showed high homology with HCVli] ll‑BK, HCV‑J, HCV‑JT and HCV‑T (nt > 84.
5%, aa > 85.8%). Whereas HCV‑N showedIii ]Ill i]

low homology with HC‑J6 and HC‑J8 (nt <68.8%, aa < 72.3%). Nt sequence homology

Figure I Physical map of the HCV genome and locations of the
CDNA clones derived from HCV‑N strain. Nucleotides are

between HCV‑N and HCV‑1 in the E1 andE2/NSI regions was 73.7%, 74.5% and aa

numbered from putative 5'end as indicated on the top. The

sequence homology was 76.0%, 81.2%, respec‑

genome organization of HCV is according to Okamoto etal. (1994) , showing coding region from structural proteins:core (O , envevop (EI and E2) to nonstructural proteins(NS1‑NS5) by blocks, untranslated region (UTR) (5' and3') by bars. The order of clone number is based on the

tively. From NS2 to NS5 regions, it wasevident that HCV‑N is closer to HCV‑BK,

HCV‑J, HCV‑JT and HCV‑T and remotefrorn HC‑J6 and HC‑J8, while HCV‑1 seems

to be located intermediate. The total
sequence homology comparison among 8

cloning process.

HCV isolates gave us a conclusion that HCV‑N belongs

Entire sequence informations have been published
for the following 7 HCV strains: HCV‑1 (Choo et al.,to HCV‑BK/HCV‑J/HCV‑JT/HCV‑T genotype. In

1991), HCV‑BK (Takamizawa et al.. 1991), HCV‑J

contrast, HCV‑N is remote from J6 and J8 genotypes,

Table 1

Homology comparison of,nucleotide and deduced amino acid sequence among 8

HCV strains (HCV‑N, HCV‑1, HCV‑BK, HCV‑J, HCV‑JT, HCV‑T, HC‑J6 andHC‑J8). The homology is indicated by %. The nucleotide sequence homology isshown in upper and arnino acid sequence homology is shown in lowbr withparenthesis, respectively.

Region
Total

5'UTR

HCV‑N *&

HCV‑N &

HCV‑N &

HCV‑N &

HCV‑N &

HCV‑N &

HCV‑N &HCV‑1

HCV‑BK

HCV‑ JHC‑ J6

HC‑ J8

HCV‑ JT

HCV‑T

77 . 992 . O91 . 562 . 461 . 791 . 790 . O(84 . 5)(94 . 1)(93 . 7)(70 . 7)(69 . 2)(93 . 9)(92 . 1)' 9'

98 . 494 . o91 . 599 . 299 . 28 . 8

C90 . 796 . o95 . 883 . O81 . 195 . 193 . 9(96 . 3)(97 . 4)(97 . 9)(90 . 1)(88 . 5)(96 . 3)(93 . 7)E1

73 . 789 . o91 . 760 . 156 . 792 . 291 . 1(76 . o)(89 . 6)

(93 . 2)(52 . 9)(51 . 8)(92 . 2)(93 . 8)

E2/NS174 . 5

8 +* 784 . 568 . 866 . 884 . 785 . 3(81 . 2)(88 . 1)(86 . 1)(71 . 1)(72 . 3)(85 . 8)(87 . o)

NS2

71.0

92 . 1(74 . 4)(91 . 7)NS3

78 . 190 . 790 . 469 . 969 . 891 . 189 . 4(89 . O)

(93.4)(91 . 8)(79 . 3)(78 . 8)(94 . 9)(92 . 8)NS478 . 993 . 192 . 766 . 867 . o93 . 589 . 9(88 . 2)(97 . 5)(97 . 2)(74 . 1)(72 . 9)(97 . o)(94 , o)NS578 . 893 . 592 . 759 . 559 . 293 . 191 .8(83 . 7)(96 . 3)(95 . 2)(71 . 8)(71 . 5)(95 . 2)(92 . 5)"'‑*,･ g2 . 7(94 . 9)59 . 858 . 289 . o87 . 8(58 . 7)(56 . 2)(92 . 4)(91 . 3)

172

11a1

蝋㎜oo…一臨

一

601

V

S

O

L

F

T

F

S

P

R

R

AココXコ3CCハOG門，（コロOGgCCコ1恥CGハ鵡ロ

VSYLKGSSGGPLしCPSGHAV
GC鳶一肉
乳G肥門℃ATACCCG

278

GIFRAAVC↑RGVAKAVDF工P

127日

一丁一㍑ひ㎜一κ㎝燃獄㎝鴬㎜
IRTGIRTITTGAPITYSTYC

41A一㏄A（㎝唱σr階1・1で！血X欄㎝

H工NRTAL国CNDSLHTU

1］9賦KCDELAAkLSTしGINAVAY一一一一一
5…一一一…560
561一蝕一一㎜一一一4620㎜⁝

4？6G国SDOKPYCWHYハPRQCGlv
A
一1一℃C㎝
PASDVCGPV騨騨 轡
TTDRLGVPTYT■GEN日TDVL

418YRG【，DVSVIPASGDVVV，VAT

CC配CG轟丁α一℃C（コACGTAC赴CσR一聯GA一㏄

438㎜℃AAC義ATκC㎜CGCA㎜ハA一ハCA瓢ATG納CAGCA

D

A

L

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D

F

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㎜α一CCCハCAT一AC−CAGA㏄黒CH睡LDAACNWTRGERCDLEDR

22i ATAGATCハGハGσ：℃ハGTC⊂GC一ACAハCAGAGTOGCAGATACTGCC㎜
658D R S日L S P L L L S T T E w O I L P C

H−

㎜捧

㎜丁甲

ACCC（一ハハCTT℃ACCATCTTCハ汽GGTTAGGATGTATGTAGGGGGCGTGG八GC

羅

⁝

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GSGP吋LTPRCMVDYPYRL■H
8YPCTVNF↑IFKVRMYVGGVE
AC一臆A蹴AAT㎜伽℃Gハ㏄鵬一CC㎜GGAC脳CGCACCCCATAハCCA湘3丁燃TCムTG漏＝AπGCATU脳：G〔XコロハC−X筑℃ACTA

THP工TKY−MTC岡S轟DLEVVT

1一一㎜一一㎜一…80
8STWVLVGGVLAALAAYCしTT
1GCA㏄㎜℃A一罵皿㎜00一質℃C㏄A㎝5〕4
8GSVVIVGRIILSGRPAVVPD
TCAひCO眠㎝ACA540

TGCTGCTGハTAGCCCAGGCT〔；AGGCTGCCTTAGAGAACCTGGTAG『℃CTCAハTGCGGCGr

1678

rACA

K

＾

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amino

㎜⁝

deduced

O

and

of

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sequence

K

acid

＾

階
SHLPY
㎝㎜
㎜㎜㎜
しGLLQ
轡
㎜c㎝㏄一℃ccA一燃皿α蛇㏄σ㎜
評

01一㎜㎜㎜一皿一㎝A−
1BCGGAVLVGLVPLTLSPYYK』V

⑳五

08

49

㎜⁝

㎜

169

㎜旧

MLLIAQAEAAしENLVVLNAAC㎜CGG羅℃ASVAGAHGVLSFLVFFCAAWY
eeTGGG鷹ATA㎜TA㎜ハ Ψ
PしL
PGAAYALYGV 一
2
一欄一躍鵬㎜c一一㎜
LLしLALPPRAYAMDREMAAS
line

G

㎝

01

GTTCG−ACσrAGσ1「OC八TGGTTGACTATCCGTAC㎜℃TGGCATT

upper

T

VTQTVDFSLDPTFTIETTTv㏄C陥一℃㏄一丁㎜GCAGG㎝AGG《G鵬CAPQDAVSRSQRRGRTGRGRRG
一㎜㏄一一㎝鵬TG一㎜q切IYRFVTPGERPLG翼FDSSVLLLNNTRPPHG国脚FCCTWMNStheM㎜℃AG瓦CGσ鵬AT皿℃Aσr㎜C㏄ハCC口℃ハCCA−A（！Gム（㎜TGFTKTCGGPPCNIGGVGNN
C一一一一一一m㏄㎜㎜㏄㏄一㏄A㏄C㏄㎜A㎝ハCAC㎜爬TG
TL工CPTDCFRkHPEATYTKC

in

4260
133日

1℃AC｛、誠唱ぐ

KA：PLEムIKGGRHL工FCHSK
鳳一一一一一…411

TCRP工DkFPQGWGPITYNNS

1681㏄触e鵬㏄一A一Ae踏CGC㏄σ㎜C＿A㎜AC

shown〕〔誕XでAC【＝【1【コT『AC｛X工「L℃｛X；Gく1A『1で1弔

21㎝一
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58P日PNIεEVALS田TGEVPFYGCGπGCCGCOOCA■mATAAσ『1でGATCハGOGA㎜C〔＝CC∫》］℃A（71』『ACAA（コA八丁AC−

is1』A−1、下1唱州、1A

CHSTDSTTILGICTVLDQAETAGARLVVLA↑ATPPGSVTV

438しACVFYHNKFNSSGCPERしA

respectively．

八ハ

96KドLADGGCSGGAYP11上ぐじレ：
GO巳ACTCAみC「X；AC㎜ACTA㏄AT㎜A『PC臓ACAG「陀（7rGG八㏄AAGCGGAGハ1561「臓一M℃AC脚一一㎜℃CハGハ㏄（㎝α、amino

割て猟『A−1て『κ1

09
8
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T㎜C乱C⊂コ匡賦一A面貞㎜鳳八CAT㎝ハーAACA
4080
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6308

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OB

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492
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35 IO

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61A鴨r惚A
38DVIPVRRGGDG臓GSLLSPkP

8FAGVDGsTVVTGGVOGR
r−rS㎝㎜ eσ一（㎜TCCCAGハGハσ1℃CAG㎝ATAAACACCAACG

deduced］300
IO1日

E：LLGPADSC栂TVFHGAGS民ILVGP賦，GPI
3541C㏄㎜㍑A㎝σ一㏄
C−
10geTOMYTSVDOD』VG■榊ERPGA
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41

and

m一㎝Tハ⊂y麗998SGLPVSAR驚n

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コE＝7コ0σ（三X｝1ロ昭X：ムGT−

π℃AT口

241

α一盛㎝理㎜一㎝一丁㎜㎜燗
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193e938FMKL轟ALTGTYVYDHLTPLQ
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318
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一一一一一一㎜㎜ハC−TGσ一CAα一LR：POAVVDMVAGGH GVLALI

CσE㎜MG鳩CA「鷹＿G0118

IRR龍VDLLVGAAAドCSAMYV
DF

旧C《C搾1℃AC℃ハハAα噛1℃『mlU罵＝｛τATハ（『1、U＝

061轍

Oe08

02

ATGTACG町GG

GY

918L：RACMLVRKVAGG開YVOMA

㎝T

貞ツ2

218D岡工IDSPGCVPCVR且NNLSR
GC㎜㎜AC㎜COC−C−CCACGハ279

1監A一1叉−A｛「Cハ

Q

918

01
86308
0

TAハCCTてコ㎜

C回VALTPTL八ARNPSVPTT
T㎜

1．肖

941㎜一瓢一一A欄GC3000
B98PL撹VLOACISRLP￥FV PAOG
001¶℃A㎜A眠A㎜一CA質ATσ固℃MA皿σr 3060

0387

SS工V1『EAV

ハ｛コしπてり㌧Tてコ聖㎜陛¶

m

1，

W

CVVHPEしISDITkしLVAILG

08

198SG工YHVTNDCS

rκハハ

W

σr一℃（訥一㎜ハσ一℃CAハTTハ

B41㎜甑丁鵬丁一（コ㎜σ1℃鵬職輿一901

工〔X灯Im贈A眠㎜AA1℃⁝

LEDGV飼YA
LLALLSCL

1m1て器1

L

2﹃一

6﹁・

CM一℃

PLVGAPLCGみハRALみ闘Gvnv㎜（xxx㎜A㎜

R

63

87

PRGRROP工PKAR臓P已GRTWA、OPGYP脚PLYGNEGMCWTG■L一一鴬㎜一一㎜℃C
一CT−
LSP聴GS聴PS回GPTDPR医RSR

A

1一一TG㏄A鳳一

08

11
86
13
8
0

一一蝋一…㎜GC【一熈（一一AC㎜
nPODVXFPGGGOIVGGVYLL

601一㎜鵬一㎝㎜㎜瓢㎜燃蹴
118国LG試V工D↑LTCGFADL麗GYI、1瞭㎜1℃κrA

L

8QV回VPPLNVRGG臓DA工ILLT

0101

ハハハα一一一CCCG一漁CC
随ST胴PKPORKTKR国TYR

ハG〔議C㎜℃「

38F

耀㎜㎜一一一蹴㎜躍附1W㎜隅㏄

PRRGPRLCVRAT貰KTSERSQ
C一αコ購

61

201

㎜7踏A〔：CCO〔コ℃

171

TATKO義EAAAPMVESKNRA1・strain．The nucleotide sequence of HCV−N

HCV−N
acid

sequence

of

HCV−N

is

shown

in

the

lower

line，

173

5521 A(::A(7rtllrlTxl,r.AJW,c,w･JvlllmcIAA1,1lrA11,Jvltx a 1 IA･tnc ',v :17vi･ri,N'Aa:, ･t‑

e2el GGalvrAT,Ll ( cslccc(,,oAc:lwv ,Ir v x xx :AL: JVlt ; I u x

l･1S8 E T r w A K R ,( w N F I s c I L, v L A U

2678

5581 TA TWOCA:TCAC!GA: GCATrcACA lrCATCA
1778 L S T L P G N P A I A S L ,, A P T A s I

5640
179C

8341
2698

56d I ACCC rAccClcc:UrrrMc,vtcrlt nRuvlt･(,L:1L :cl

b700
1018

84cn
2718

5701

5760
1838

8461
27]8

S.61 T ATl GCAe
rc38A v G s I G L G x v L v D I L A c y c A

5820
1858

8521
27S8

le58G v A G A L v A F tc v

a s G E v p s T E‑

5800
1879

8S81
2778

187e D L v N L L P A : L s p G A L v v G v v

AceTea,r,LATrrA 5940

rs90

8641
2798

5g41
1898c A G!Gc
A I L R CM:T
R r, v AOG!WCCC
G P G E G A v TGM:cco w M N

6000
l･1B

87O1
2818

6001

6O6o
1938

8761
2838

6120
1958

8821
2859

6180

88Bl
2e78

179GT s p L T T Q N T L L p N : L e G w v A

1818A Q L A P p s A A S A F v G A G I A G A

5981

AT wCA lT!CccCc ACr ;1UI creAeA

l･18R L I A F A S R G N R v S P T H Y v p E
6o61 ccGA rcAGA!cauCCAGCarraCCRTCAClrAccreC! A
1938 s D A A A R v T O I L s s L T I T Q L L

6121
Tr A M
62dl TGCCAACeC : A Ae
1958K !tL H P c s G s w L
OWINEDCST
6181
1978 R eoGA
D v ,,TATecA
D w I c T v L T D F K T w L O s

1 97 e

621O
1998

0941
289a

19･8K L L P O L P G I P p L s C Q R G Y x G
G301 ATCAl l:MCC a!ecccATo! AGCAcAGATCACCceAC
2cn8v w R a D G : ,! L T T C P c G A O I JT G

63aO
2O18

9001
2918

6]6a
2a] 9

9O61
2938

Ga61
TG
2Q38H vAlx
K N G!CA,W:T
S ,1 R I vl GcaIGAOCAP K T C S N AOC
T w tl

642O
205G

9121

6421 TGAACATTCCCCA:rcMcccAT
2058G
F P I N A Y T T G P C T P s p CA PncT
N

61BO
207 8

9181
2978

6.81 A:

2O78y s !t A L w R v A A E E Y v E v T R v G

6540
2098

9241
29g8

6541
AITrcCACT W!1GOCCAI CCAOCTrcCee
2098 D F r, y v T G :, T T D N v ,c C P c o v p

6600
21le

66Ol AGGT Ccc,CCecCelncA
2138R
L R E E v ,t Far
Q v GAGTA
L N O Y L v G
6661P L eACC1,

G660
2138

2118A P E P F T E v D G v R L R R Y A P A c

G720
2158

29S8

n･ rAlx x x I k

r

Rl･to

RLyiGGPLTNSXGONceyRR
ecceCeC CT GC COe!1MTACCaCACAl TAall MGO
CRAS GVLTTSCGNTLTcyLASAACRAAXLQDCT,1LVNGDTa GM:1rcA GGAl CG CCrncGAccar
DLVVICECTGTOE:DAANLRA,,TGACC, T COGeGACCCOCCCCMCCAaMTnco
FTEAIITRysAPPODppOPEV
Aal earMTMCATCA TocATCl
DLELITSCSSNVSVAHDASGCWP AC1:nc CCccAlrOCA
XRVyYLTRDPTTPIARAAWEC TTARHTPVNSWLGNIINYAPT
T I CMGGATeA:TTll A TCCT! :

･9u
8doO
27 1 88d60
27]B

A GAc:rcx:Ac:c･uvf e;crcx7luJU (1‑:G G:Ac:(;

A:T!rc!

B520
2750
8580
2778
864o
279B
870o
281S
87GO
2830

r, CAACA:11CAIC,,TarncCCccCCAceT

LWARIIIL'IT'tprSILLAQEO
AGAT acTccATIGAACCAcrTeATC

LGl(ALDCOIYGAcys!EPLD'TWI A:TeA Al raTTaGCGcA
LPQIIERLHCLSAFSLt syS
C AGATCMT
PGEINRVASCLRKLGVPPLR
VWRURARSVRARLLSOGCRAAGTA GACcMGCTcA,nclrAeTCCM

8820
285e
R8Ro
2878
e9do
2898

TAcrrAel, C

9000
29le
eo60
2938

acsCTACTerccc

ATCCKyLFNWAVRTKLXLTP
IPAASRLDLSGWFVAGysGG
AC,
T, T nclrcTAC
DlyHSLSHARPRWP,,LCLLL
TrrcTowi racGc r CCCOTGA
Ls G+G****' R'
LT

gl20
295a
9180
2978
9240
2998
9295
3ou2

Figure 2

respectively. The sequence homology of HCV‑N also

61 2 1 CGCAGC:!COCA COG,,T raGCAareCrCAC:rlCCAI arr,tccGATC

2158 s o L P c E P E P D v A v L T s ,, L T D

6780
217a

showed its closer relationship to HCV‑1 than to HC‑J6

67
217e81p sCCrcCC
H I T A E11
T A MOCO8WF!
K R R L A Rr
G PCCTCCT
P P S

6B40
2198

and HC‑J8 isolates (Table 1). HC‑J8 possessed 23%2198 L A s s s A c
S Q L S A P s L x A T c T T

6841 I CC IGCAC cC

6900
2218

divergence with HC‑J6 and has been clearly identified

e･OI A,rc･A.il5,Lcll'(J'cchoNxx;111, (xr 1'AltlhtlLlt 1lvLt ,l ',rtLn a lrl ･Na:At:A･na:,*q,*o22rs H H D s p D A D L I E A N L l* w R L, L: ,1

2:JB

as a separate genotype from HC‑J6 (Okamoto et al.,

6 961 e AAACATCAOCCOCGIOGAGrcAeA

7020
2258

rMCerACT A,FT]･IllCACrCrrrcc̲ JLCC

223eG c N I T R v E s E N K v v I L D s F D,02 1 co(*rlc Jvx:(x5AoL:Aa JAlr:A(jNxx;Nx:L 7lt･LlrrlLx l;t:,L( x:A(7A,ll'("IL ,Lk:AL:A･r,, s*,

2258 P L R A E E D E R E v s v A E E; I L R R

227B

7 081 AIGCGATQecc,,TA ecC ACMCCC:rCcA1l

714O
2298

2278s R x F P P A b, P I w A R P D y N P P L
7141 T ,TccIGGA; AcccGGAcTA,ro,L･rc(r[ccGcrFA(;;rAcA1[xx71lscccA,ilUc･cAc

2318

?201
C!ccTAA CccCTCCMTAccAccTC CAeAeT
2]18 P A K A P P I P P P R p te R T v v L T E

7260
23]8

7261 CACT 5cAccTCcG lrGT

1320
2358

i]81 AAceerCCG,L cc
AClrcCA CC

2378K c s D v E s y s S b, P P L E G E P G D
74dl cceA:

2398 P D L s D c S w s T v S D E A s E D v v

73Bo
2378

7560
2438
7620
2d58

TcMccc rl Acc

2438s K L P I ,, P L S N
7621 ccAcAAcArrclCeC

40

HCV

7500
2418

CCACAACATI eTn:ATG

7561

(gb]

74la
2]9B

15O1 eclreClrAATgrcarACTCAT GACccoCeccATeATcACccCA OCWL

241B.c c s !1 s y s w T G A ,1 1 T P c A A E E

In order to confirm this conclusion,

phylogenetic trees were constructed base on nucleotide,,oo2298 L E s w K D P D y v P P v v H c c p L P

2338･s T v s s A L A E L A T K T F G s s G s
7321 CecceAeCec rTecTeACCAeAcCl cGAccAC ceAcA
235a s A v D s G T A T A L P D Q T s D D G D

1992b) .

30

Group 1

Group 2sLLR,tHN,1vy
76GO
GMGercACCITrGACACACTecMO

245eA T T s R s A C L R Q K x v T F D R L Q

24?8

7681
2478v LToc!
D N ttTMecAC1:ACCGGGA!CCY R D v L K E tl K A K ACACTrA
S T V

7740
2498

7741 AccCTun Fer

2498 K A K L L s v E E A c X L T P P H s A R

oCCAeAT

7800
2518

7801
ccAMT!Tl Crn , lrC CAocGccolTMccAcA
25la s x F G y G A K D V R N L s s R A v N H

78eo
25]8

7861 TCCOCTC CAC SAGACACCMTTeACAccAccArcA
253e i R s v w x D L L E D T E T P T D T T I

7920
2558

MCc
7921
2558 11IA cMAeAA
x N E v F ccccGcMeccAcx;lc v Q P E K e G R K P Accc
R

7980
2578

7981 TrATO AT!CCCAGA: ATCC 3 Al lvlITACeAI l ;25?8jL I v F P D L G v R V C E K M A L Y D v

8040
2598

8041 T ccAClrTrcCrcAGecceTGAl e ClrATACoGATTCCncl:acTICa:,LAec

8100
2GIB

8rol
2618Q R vCAcCl
E F L v N TCwG
T w K s IQCCerAK K c p M G FAT
s

816O
2638

Hcv‑N HCV‑BKHCV‑J HCV‑JTHCV‑T Hcv‑1 Hc‑J6 Hc‑J8

8161 ATeA GAAl cA

822O
265B

Figure 3 Phylogenetic trees are based on nucleotide diver‑CeT CecCCCCACA

2598 v s T L P Q A v Ft G P s Y G F Q Y s p K

2638 y D T R c F D S T v T E N D I R v E E S
8221 TFrAccAA ccoMGCrMeCAoGccATMACTccarcACAGAcc
2G58 1 y O c c D L A P E A K O A I x s L T E

Figure 2

8280
2678

2o
l 111 bI11 allll2aIV/2b

10

gence (%). Sequence of all 8 HCV strains were
campared and analyzed by the nearest neighbormethod.

174

divergence throughout entire sequence using nearest
neighbor method (Williams and Lance 1977) (Fig. 3).

to know the HCV sequence diversity of the envelope
glycoprotein in local area of Nagasaki, 2 other HCVThis result indicated that 8 HCV strains can be classi‑
fied into 2 groups: the group I contains genotype II/1bcloned and sequenced for the region of nt 623‑1988, aa

Nagasaki strains (HCV‑N1 and HCV‑N2) were also

(HCV‑N, HCV‑BK, HCV‑J, HCV‑JT, HCV‑T) and

126‑580 (nt and aa base on the HCV‑N number) .

genotype I/1a (HCV‑1); while group 2 contains

NSI envelope glycoprotein regions among 3 HCVAlignment comparison of deduced aa sequence among 3
Nagasaki strains and other 7 HCV strains with publi‑
shed sequences were shown in Fig. 4. Two hypervaria‑
ble regions (HVR) were discovered in the N‑terminus

Nagasaki strains and 7 other HCV strains with publi‑of E2/NSI region. HVR I (aa 384‑411) was locatedshed sequences

directly downstream at the beginning of the E2/NS1,
whereas HVR 2 (aa 475‑480) was observed 64 aa down‑
stream from the HVR 1. In the HVR I consisting of 28

genotype 111/2a (HC‑J6) and genotype IV/2b (HC‑J8) .
Sequence variation in the E I and N‑terminus of E2/

Recently, many papers reported that hypervariable
regions existed in the N‑terminus of E2/NSI envelope
glycoprotein region of HCV genome (Hijikata et al..
1991; Weiner et al.. 1991; Honda et al.. 1993). In orderaa, 10 aa residues were well‑conserved among 3 Nagasa‑
ki strains. Whereas only 3 aa
were conserved in this175

HCv‑N LTCGFADLMC YIPLVGAPLG GAARALAHGV RVLEDGVNYA TGNLAGCSFS

E2 / NSl

YIC

Hev‑ lHCV‑N
HCV‑NlBCV‑N2

P

HCV‑ lHev‑BKPP

HCV‑BK

Hcv‑Nl
HCv‑N2P

GND

Hev‑ J

Hev‑ JT

Rev‑Tv

Hc‑ J8

F P

v

I

P

El

Hev‑N
HCv‑NlHCV‑N2
HCv‑ lHCv‑BK
HCv‑ J
HCv‑ JT:V VVVAV

t!C‑J6
HC‑ J8I

H

HCV‑ JTYT

I SS YAHCV ‑ N l

!1CV‑N2
HCV‑ lHCV ‑ BK

HCV‑ J

AG M}!TA M}!T

HCV‑ JT

TD TWQ LQAAVLHV

HCV‑T
HC‑J6

A L

X I TG M

HCV‑N

A MHT

I

HCV‑T

HCV‑ J

H
B
H

225

IFLLALLSCL TTPASAYEVR NVSGIYHVTN DCSNSSIVYE AVDMI I DS peI
A I MHTM
A v MHTPST L
A A LHTHA
A L MHT

v Q
r Q

R
R

HCV‑T
HC‑J6
HC‑J8

P

v
v vv

!1c‑ J6N TWQ LT AVLHLV

HCV‑NI 'DSHCV‑N2GAGS

HCV‑ lHCV ‑ BXSIS TL L

Q

VT I

SI
TI

NV

S

EKVG TIPVS N V VQQ GALEND GT LH IQV N V VKHRGAL

HC‑ J8s

V T DGKL A

A

HCV‑T
HC‑J6

Hev‑Nl
Hcv‑N2M

SF

HCV‑ J
HCV‑ JTHCv ‑ Nj̲S

L

RSL T MI M TA L

325
WGDLCCSVF LVSQLFTFSP RRYETVQDCN CSLYPGHVTG HRMAWDMMM1 1̲
I
LH
I

Hev‑ lG

HCv ‑ BX

HCV‑T
HC‑J6
HC ‑ J8

HHHW T G

I

YLS

I

I

HCV‑ l

HV L

I

Hcv ‑ JI

RCv‑ JT

I

S

HCv‑T
HC‑J6
Hc‑J8

I

G M M M IV QHHWF

V A M IL A MV Q rINFT E

TI

I

Q I

HCV‑ J
HCV‑ JT

HC‑J6
LS

37S

Figure 4

v

R K
K N

MQ
MS S
MQ T
M S SSAMS S

MSA S EA RV
SS GL D RI

ALQ E
TLE E
523

T R

VPTYTWQENE TDVLLLNNTR PPHGNWFGCT WMNSTGFTKT CGGPpCNlcc

A

DL DR

HC ‑ J B

I A

s

D

S

A
A
A

S

N

s

A

S

I

A

F
F

I

Q
Q

v

rv

R

A

HCv‑T
HC‑J6
HC‑J8

K:
KI

P

DQK PYCWHYAPRQ CGIVPASDVC GPVYCFTPSP VVVWTTDRLG
Y
ER
T
s
QM
AV
G
V
AQ PS II GR
PY SCLL
rv
P XP
R
S
KS
G
P P
R
E
T
F
G
P
R
Q
F
G
TEPRDLRs
Q
GP
TEADIQ R
Q
F
G
S
P
VT
MR
G
vs
P p
MR
RT
G
KQ

VL

A

SL TH

SSP

HCV‑ lI F

SL A,iQ

VGNNT

T MA LL MI AA

F AL AH

S

HCV‑N
HCV‑NlHCV‑N2

HCv‑BI<
Hev‑ J
HCV‑ JTRASL TH RAENQ

WS PTAALWS QLLRIPQAW DWAGGHWGV LACLAYYSW GNWAKVLIVM
P
T
IM T A

HCv‑ lLS

K N

SAH QSV F TG PA K

W L H

HCV ‑ BI(nCV ‑ TIQSLV WL Q

RVAS S

PT KI
P

573

HCV‑N
HCV‑NlHCV‑N2SS

HCV‑N
HCV‑Nl
HCv‑N2

HCV ‑ lHCV ‑ BXHCV‑ JTQGL T I MV MS TL L

GLV L

VGV
LA
NRLV M AS

Hev 2

HCV‑N
HCV‑NlHCV‑N2
HCV‑ J

A

TLAQ
SA H

AAK

474
ALNCNDSL}lT GFLAGVFYHN KFNSSGCPER LATCRPIDKF DQGwepITY
T
N
N
N
E QA L AL TY
Q
AL VH
N
s
S LTD
Q
AL TH S
Q
E
H
I AL AH
A
MS}IC‑ J8

evpCVRENNL SRCWVALTPT LAARNPSVPT TTIRRHVDLL VCAAAFCSAM

425

I

IS TVA HSLA TQ
HTV STAHN ARTL GM L
YSS QEAVAG AG TT

A

275

HCV‑N

HVR l

TSVFTSLFS P GASQR LIN TNGSWHINRTVREA T ATGLAKFLVI

LLFAGV

s
s

v

Q
Q
Q
Q
O S
A

Y

G

A

v

A
A

RRA
RRK

580
LI
1;

D

T
H
T

HCV ‑ BK

HCV‑ J
HCV‑ JT

L

HCV‑T
HC‑J6
HC‑J8

G
DF ASMD L
DY s :D L

V
TV

MPF QIAIL
WLL
VF
Alignment of amino acid sequence among 10 HCV strains (HCV‑N, HCV‑Nl, HCV‑N2, HCV‑1, HCVTMILA YAM V EVII IIG A
LTMILA YAA V EL L EIIF

FQ A
A

‑BK, HCV‑J, HCV‑JT, HCV‑T, HC‑J6 and HC‑J8) in the E1 and N‑terminus of E2/NSI regions (aa126‑580). Two hypervariable regions (HVR I and HVR 2) are shown by boxes. Potentral N‑glycosylation sites (N‑X‑S/T) are underlined.

175

Table 2 HCV genotype classification including HCV‑N from this study and 15 entire
HCV strains which have been reported by Sakamoto et al. (1994) .

Genoty peGroup 2

Group 1

Group
I/1a

lc

II/1b

Group 3

III/2a

IV/2b

V/3a

HC‑ J6

HC‑ J8

NZL1

Japan

Japan

HCV‑ JHCV‑BK
HCV‑1
HCV strains

HCV‑H

HC‑ J4/83
HC‑ J4/91

HC‑G9

HC‑ J1

HCV‑ JT

HCV‑T

HCV‑ JK1

HC‑C2

HCV‑N
Japan China Taiwan

Geogra phic

U.S.A.

Area

Japan

IndonesianNew

Zealandregion when a total 10 HCV strains were corrrpared. In

includes HC‑G9 which was derived from Indonesianthe HVR 2 consisting of 6 aa, none of the aa wereconserved among 3 Nagasaki strains. This hypervar‑

strain. While, the genotype I/la of group I includes
HCV‑1 and HCV‑H which were derived frorn' American

iability was also observed when a total 10 HCV strains
were compared simultaneously. In the E1 and N‑termi‑

strains. Although another HC‑JI was Japanese strain, it

nus of the E2/NSI regions, 2 well conserved aa
sequences were observed among 10 HCV isolates: G‑H‑R‑M‑A‑W.TD‑M‑M (aa 315‑323) and W‑F‑G‑C‑T‑W

was supposed to have originated from the US, because
HC‑JI was derived froin a Japanese haemophiliac who
developed hepatitis C after receiving US‑made factor‑M‑N (aa 549‑556). Although 14 N‑glycosylation sites

VIII (Okamoto et al.. 1992a) . In group 2, HC‑J6 and HC‑J8 belong to the 111/2a and the IV/2b genotype, respec‑(N‑X‑S/T) were identified in HCV‑N, one of themtively. Both of them were Japanese strains. In group 3,(NNS at aa No. 475‑477) was unique to this strain andnot present in 2 other Nagasaki strains and 7 other HCV
strains with published sequences. Instead, HCV‑Nl and

NZLI that carne from New Zealand belongs to V/3agenotype (Table 2). Simmonds et al. (1993b) haveclassified 6 major genotypes of HCV from 76 HCVHCV‑N2 possessed another N‑glycosylation site (NFS

isolates, which were almost worldwide collection, using

or NSS at aa No. 250‑252). This glycosylation site isfound also in HCV‑BK, HCV‑J, HCV‑JT, and HCV‑T

phylogenetic analysis of the NS5 region. The 76 HCV
isolates contained entire sequence of HCV‑1, HCV‑H,(Fig. 4). Altogether, only 9 N‑glycosylation sites were

HCV‑J, HCV‑BK, HCV‑T, HCV‑JT, HC‑J6 and HC

conserved in the E1 and N‑terminal of the E2/NS1‑J8 strains and other partial sequence of HCV strains.

regions among 10 HCV strains shown in Fig. 4.

From this classification, only genotype 4, 5 and 6 showed

DISCUSSION

highly restricted geographical distributions, being
apparently confined to Egypt, South Africa and Hong
Kong respectively (Simmonds et al.. 1993b).

Relationship between genotype and geographic area

Two hypervariable regions have already been obser‑of HCV strains including HCV‑N was shown by theirsequence comparison using the classification by Sa‑

ved in the N‑terminus of the E2/NSI region. The

kamoto et al. (1994) (Table 2). The sequence homology
indicated that HCV‑N strain did not possess its local

genotypic character, and similar to HCV‑BK, HCV‑J
and HCV‑JT strain which were isolated from otherareas of Japan. All these strains apparently belong to
the sarne genotype II/1b of group 1. This genotype also

included HC‑J4/83; HC‑J4/91; HCV‑JKI of Japanese
HCV strains, HC‑C2 of Chinese HCV strain and HCV‑T of Taiwan strain. The genotype lc of group 1

number of con erved aa in the HVR I among 3 Nagasa‑
ki strains were higher than among a total 10 strains
including 3 Nagasaki strains and 7 other HCV strains
with published sequences. The numbers of conserved aa(5 aa/28 aa) among HCV strains of genogroup I (HCV

‑N/HCV‑1/HCV‑BK/HCV‑J/HCV‑JT/HCV‑T) werehigher than those (3 aa/28 aa) among 10 HCV strains
which included genogroup 2 (HC‑J6 and HC‑J8). This
result may give us an idea that aa conservation in the

HVR I of the sarne genotype or genogroup of HCV are176

higher than among the different genotype or genogroupof HCV strains. Therefore, the 3 Nagasaki HCV strains
may have been originated from the same ancestor.
In contrast, none of the aa was conserved among 3
Nagasaki strains nor among a total 10 HCV strains in

the HVR 2 which showed higher variability than the
HVR 1. The reason why mutations occur so frequently
in only limited regions such as HVR is not known(Tanaka et al., 1992). The higher degree of divergencein HCV E2/NSI region might reflect the immune selec‑tion and suggest that this region cannot probably be an
ideal target for future vaccine development.
Regarding the N‑glycosylation sites in the E1 and N‑terminus of the E2/NSI region, only 9 sites wereconserved out of the 14 sites seen in HCV‑N. Some of
the glycosylation sites, therefore, would not be essential

for the survival, transmission and maintenance of HCV

in nature. On the other hand, 2 well‑conserved aa

reading and comments of the manuscript.
Special thanks are due to Dr. K. Morita, Dr. S.
Kurihara, Dr. K.Z. Thant and Dr. Y. Tsuda for usefulsuggestion and kindly assistance.

This study was supported by Health Science
Research Grants (Non A‑NonB Hepatitis Research
Grants) from the Ministry of Health and Welfare ofJapan.

REFERENCES
1 ) Bukh, J., Purcell, R.H. and Miller. R.H. (1992):
Sequence analysis of the 5' noncoding region of hepatits
C virus. Proc. Natl. Acad. Sci. U.S.A., 89, 4942‑4946
2 ) Chen, P.J., Lin, M.H., Tai, K.F., Liu, P.C., Lin, C.J. andChen, D.S. (1992): The Taiwanese hepatitis C virus
genome: sequence determination and mapping the 5termini of genomic and antigenomic RNA. Virology,

sequences were found ･in the E1 and N‑terminus of the188, 102‑113
3 ) Choo, Q.L., Kuo, G., Weiner, A.J., Overby. L.R.. Bradley,

E2/NSI regions among the 10 HCV strains compared.
These conserved sequences may be better targets ofD.W. and Houghton, M. (1989): Isolation of a CDNA
clone derived from a blood‑borne non‑A, non‑B viral

vaccine development if they were related with protec‑
tive immunity.

hepatitis genome. Science, 244, 359‑362
4 ) Choo, Q.L.; Richman, K.H., Han. J.H., Berger, K., Lee,
C., Dong, C., Gallegos, C., Coit, D., Medina‑Selby, A.,
Barr, P.J., Weiner, A.J.. Bradley, D.W., Kuo, G. andHoughton, M. (1991): Genetic organization and diver‑

The nt sequence homology of HCV‑N in the 5'UTRshows high conservation comparing with other 7 HCV
isolates (nt > 91.5%) and was used as an ideal target

for PCR amplification to detect HCV RNA (Okamoto
et al.. 1990). But recently, some papers reported thatseveral HCV strains have sequence variation in the 5'
UTR (Lee et al., 1992; Bukh et al., 1992; Simmonds etal., 1993a) . Therefore additional informations would be
required to select optimal primer for PCR diagnosis on

HCV. The functional motifs of the putative encoded
proteins of HCV (Plagemann et al., 1991; Tanaka et al.,1992) have also been found in HCV‑N. There areconsensus sequences of RNA helicase in the NS3 region:

Gly‑Ser‑Gly‑Lys‑Ser‑Thr (aa 1233‑1238) and Gln‑Arg‑Arg‑Gly‑Arg‑Thr‑Gly‑Arg (aa 1486‑1493) , while NS5
region possessed consensus sequence of RNA‑dependent

RNA polymerase: Gly‑Asp‑Asp (aa 2736‑2738). The
sequence heterogeneity in putative structural proteinsand nostructural proteins of HCV provided significant
evidence for genotype classification. The genetic infor‑

mations of HCV‑N genome obtained in this study will
be useful in future understanding on the pathogenesis,
diagnosis and development of vaccine for HCV.

sity of the hepatitis C virus. Proc. Natl. Acad. Sci. U.S.

A., 88, 2451‑2455

5 ) Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M.,
Ohkoshi, S. and Shimotohno, K. (1991): Hypervarible
regions in the Putative Glycoprotein of Hepatitis C
virus. Biochem. Biophys. Res. Commun., 175, 220‑2286 ) Honda, M., Kaneko, S., Unoura, M., Kobayashi, K. and

Murakami. S. (1992): Sequence comparisons for ahepatitis C virus genome RNA isolated from a patientwith liver cirrhosis. Gene, 120, 317‑318

7 ) Honda, M., Kanekd, S., Unoura, M., Kobayashi, K. and
Murakami, S. (1993): 'Sequence analysis of putative
structural regions of hepatitis C virus isolated from 5
Japanese patient with hepatocellular carcinoma. Arch
Virol., 128, 163‑169

8 ) Inchauspe, G., Zebedee, S., Lee, D.H.. Sugitani, M. and

Nasoff, M. (1991): Genomic structure of the humanprototype strain H of hepatitis C virus: comparison with
American and Japanese isolates. Proc. Natl. Acad. Sci.
U.S.A., 88, 10292‑10296

9 ) Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M.,
Ohkoshi. S., Sugimurzi, T. and himotohno, K. (1990):

Molecular cloning of the human hepatitis C virus
genome from Japanese patient with non‑A, non‑B hepa‑
titis. Proc. Natl. Acad. Sci. U.S.A., 87, 9524‑9528

ACKNOWLEDGEMENTS

10) Kurihara, S. (1992): Comparative nucleotide and aminoacid sequences of the envelope protein of five hepatitis C

The author would like to express thanks to Prof. A.
lgarashi for valuable advice and encouragement, carefulviral CDNA clones isolated from HCV infected patients

177

in Nagasaki Prefecture. Trop. Med., 34, 39‑54
11) Lee. C.H., Cheng, C.. Wang, J. and Lumeng, L. (1992):

19) Sakamoto, M., Akahane, Y., Tsuda, F., Tanaka, T.,
Woodfield, D.G. and Okamoto, H. (1994): Entire nu‑

ldentification of hepatitis C viruses with a nonconserved

cleotide sequence and characterization of a hepatitis C
virus of genotype V/3a. J. Gen. Virol., 75, 1761‑1768

sequence of the 5' untranslated region. J. Clin. Mi‑
crobiol., 1602‑1604

12) Okamoto, H., Okada, S., Sugiyama, Y., Tanaka, T.,
Sugai, Y., Akahane, Y., Machida, A., Mishiro, S., Yoshiz‑20) Simmonds, P., McOmish, F., Yap, P.L., Chan, S.‑W.,
Lin, C.K., Dusheiko, G., Saeed, A.A. and Holmes, E.C.
(1993a) : Sequence variability in the 5' non‑coding

awa, H., Miyakawa, Y. and Mayumi, M. (1990): Detec‑tion of hepatitis C virus RNA by a two‑stage polymer‑
ase china reaction with two pairs of primers deduced

region of hepatitis C virus: identification of a new virus

from the 5'‑noncoding region. Japan. J. Exp. Med., 60,
215‑22213) Okamoto, H., Okada, S., Sugiyama. Y., Kurai, K., Iizuka,
H., Machida, A., Miyakawa, Y. and Mayumi, M. (1991):
Nucleotide sequence of the genomic RNA of hepatitis C
virus isolated frorn a human carrier: comparison with

21) Simmonds, P., Holmes, E.C., Cha, T.‑A., Chan, S‑W.,

reported isolates for conserved and divergent regions. J.
Gen. Virol., 72, 2697‑2704

22) Tanata, T., Kato, N., Nakagawa, M., Ootsuyama, Y.,14) Okamoto, H., Kanai, N. and Mishiro, S. (1992a): Full
‑1ength nucleotide sequence of a Japanese hepatitis C
virus isolate (HC‑J1) with high homology to USA iso‑lates. Nucleic Acids Res., 20, 6410
15) Okamoto, H., Kurai, K., Okada, S., Yamamoto. K.. Iizu‑
ka, H., Tanaka, T., Fukuda, S., Tsuda, F. and Mishiro,
S. (1992b): Full‑length sequence of a hepatitis C virus

type and restrictions on sequence diversity. J. Gen.
Virol., 74, 661‑668

McOmish, F., Irvine, B., Beall, E., Yap, P.L., Kolberg, J.

and Urdea. M.S. (1993b): Classification of hepatitis C
virus into six major genotypes and a series of subtypes

by phylogenetic analysis of the NS‑5 region. J. Gen.
Virol., 74, 2391‑2399

Cho, M‑J., Nakazawa, T., Hijikata, M., Ishimura, Y. andShimotohno, K. (1992) : Molecular cloning of hepatitis C
virus genome from a single Japanese carrier: sequencevariation within the same individual and among infectedindividuals. Virus Res., 23, 39‑53
23) Takamizawa. A., Mori, C., Fuke. I., Manabe, S., Mura‑kami, S., Fujita, J., Onishi, E., Andoh, T., Yoshida, I. andgenome having poor homology to reported isolates:

Okayama, H. (1991): Structure and organization of thehepatitis C virus genome isolated from human carriers.

comparative study of four distinct genotypes. Virology,
188, 331‑341

24) Wang, Y., Okamoto, H., Tsuda, F., Nagayama, R., Tao,

16) Okamoto, H., Kojima, M., Okada, S., Yoshizawa, H.,
lizuka, H., Tanaka, T., Muchmore, E.E., Peterson, D.A.,
Ito, Y. and Mishiro, S. (1992c) : Genetic drift of hepatitis

C virus during an 8.2‑year infection in a chimpanzee:
variability and stability. Virology 190, 894‑899

17) Okamoto, H., Kojima, M.. Sakamoto, M., Iizuka, H.,

Hadiwandowo, S., Suwignyo, S., Miyakawa, Y. andMayumi, M. (1994) : The entire nucleotide sequence andclassification of a hepatitis C virus isolate of a novel
genotyoe from an Indonesian patient with chronic liverdisease. J. Gen. Virol., 75, 629‑635

18) Plagernann, P.G.W. (1991): Hepatitis C virus (Brief
Review). Arch Virol., 120, 165‑180

J. Virol., 65, 1105‑1113

Q.M. and Mishiro, S. (1993): Prevalence, genotypes, andan isolate HC‑C2 of hepatitis C virus in Chinese patientswith liver disease. J. Med. Virol., 40, 254‑260
25) Weiner. A.J., Brauer, M.J., Rosenblatt, J., Richman. K.
H., Tung, J., Crawford. K., Bonino, F., Saracco, G., Choo,

Q.L., Houghton, M. and Han, J.H. (1991): Variable andhypervariable domains are found in the regions of HCV
corresponding to the flavivirus envelope and NSI pro‑teins and pestivirus envelope glycoproteins. Virology,
180, 842‑848

26) Williams, W.T. and Lance, G.N. (1977): Statisticalmethods for digital computers: hierarchical clas‑
sificatory methods. 269‑295

179

Jpn. J. Trop. Med. Hyg., Vol. 22, No. 4, 1994, pp. 179‑184

SEROEPIDEMIOLOGICAL SURVEYS
FOR LEPROSY IN ECUADOR
ATSUSHI HOSOKAWAl, SHIGEO NoNAKA1, MIGUEL H. JURAD02,
MASATO FURUYA3, YUKI ESHITA4, TATSUYUKI MIMOR15,
KEN KATAKURA6, EDUARDO A. GOMEZ L.7, SHINZO IZUM18
AND YOSHIHISA HASHIGUCH19
Received July 5, 1994/Accepted Septem.ber 20, 1994

Abstract: Serological exarnination of leprosy in endemic areas of cutaneous leishmaniasis were carried out
using the sera collected during a survey for cutaneous leishmaniasis and several parasitic diseases in
Ecuador. There was no correlation between prevalence rates for leprosy and seropositive rates of theantibodies (anti‑PGL‑1 and LAM‑B antibodies) in the subjects living in several provinces in Ecuador.
Seropositive rates of anti‑PGL‑1 antibodies of the leprosy patients and their families in Los Ranchos,
Department of Manabi, were relatively high (84.6%, 11/13) in comparison with the average seropositive
rates (42.4%, 154/365) of the subjects from other areas of Ecuador. It was suggested that serological surveyof families of leprosy patients might be useful for screening of household contacts in a low endemic areas,such as Department of Manabi, Ecuador.

two diseases cause immunologically similar responses inand M. tuberculosis (Gaylord and Brennan, 1987). The
present paper reports the result of preliminary ser‑ological examinations of leprosy. Furthermore, basedon the results obtained, a brief comment was also madeon the screening method to detect leprosy patients intheir hosts (Bryceson, 1981). Therefore, it may beearly stage of the disease.

INTRODUCTION
Leishmaniasis and leprosy are etiologically com‑pletely different diseases, but it has been known that theimportant to know seroepidemiological features of lep‑rosy in endemic areas of cutaneous leishmaniasis. Twotypes of skin tests, Lepromin test for leprosy and Leish‑

mania (Mont negro) skin test for leishmaniasis, weremade on several leprosy patients and thier families.
Using sera which were collected during surveys forcuteneous leishmaniasis and other parasitic diseases
inculuding leprosy, the value of anti‑PGL‑1 (phenolic

MATERIALS AND METHOD
Subjects examined
In this preliminary epidemiological survey of lep‑rosy, an evaluation was made on the serodiagnosis of theological studies of leprosy. PGL‑1 is a major secretory
product of Mycobacterium leprae (Hunter, et al.. 1982).

subjects from the following areas of Ecuador: Los
Ranchos, Portoviejo and Guayabales, Department of
Manabi; Echeandia, Department of Bolivar; Antepara,
Machala, Pinas, Portovelo and Zaruma, Department of
E1 Oro; Pedro Carbo, Guayaquil and Olon, Department
of Guayas; Selva Alegre, Department of Esmeraldas;

LAM‑B is a complex glycolipid found in large amounts

and other areas of Ecuador. A total of 365 subjects (153

(15 mg per g of bacilli) within the cell walls of M. Ieprae

males, 184 females and 28 unknown) , with 3 to 73 years

glycolipid‑D antibodies and anti‑LAM‑B (Lipoar‑abinomannan‑B) antibodies were measured for the ser‑

1. Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903‑01, Japan
2. Centro National de Medicina Tropical, Facultad de Medicina, Universidad de Guayaquil, Guayaquil, Ecuador
3. Institute for Laboratory Animals, Kochi Medical School, Nankoku, Kochi 783, Japan

5.
6.
7.
8.
9.

Department of Parasitology, Kurume University School of Medicine, Kurume, Fukuoka 830, Japan
Department of Parasitic Diseases, Kumamoto University Medical School, Kumamoto 860, Japan
Department of Parasitology, The Jikei University School of Medicine, Tokyo 105, Japan
Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de Guayaquil, Guayaquil, Ecuador
National Institute for Leprosy Research, Higashimurayama, Tokyo 189, Japan
Department of Parasitology, Kochi Medical School, Nankoku, Kochi 783, Japan180

old, were examined for anti‑PGL‑1 antibodies (IgG andin female and 25.8‑year old in total. In the present

the flexor surface of the forearm. The skin test area
was observed for erythema and induration (induration/
erythema in mm) at 48 hours later. Induration size ofmore than 5 mm in diameter at the injection site wassubjects from different areas, the following underlying

considered as a positive reaction.

lgM) and LAM‑B antibodies (IgG and lgM). The mean
age of subjects was 26.2‑year‑old in male, 25.4‑year‑old

diseases were reported: . Departrnent of Bolivar,

toxoplasmosis, 4; Pichincha, cuteneous leishmaniasis, 17,Serological examination
Blood samples were collected by venipuncture. The
sera was separated with a centrifuge at several field
laboratories in Ecuador. The sera were stocked in a

Chagas' disease, 1; Esmeraldas, cuteneous leishmaniasis,

freezer at the temperature of ‑ 20 'C. The value of anti

cutaneous leishmaniasis, 15; EI oro, Chagas' disease, 15

and gnathostomiasis, 1; Guayas, cutaneous leish‑
maniasis, 13, Chagas' disease, 9, gnathostomiasis, 6,‑PGL‑1 antibodies and anti‑LAM‑B antibodies were25.

measured by enzyme‑linked immunosorbent assey

These sera were collected during the surveys for several(ELISA) in a laboratory at the National Institute for
Leprosy Research in Japan (Jzumi et al., 1993). Cut‑off
levels are as follows: PGL‑1‑IgG 0.08 OD (optical den‑

infectious diseases, such as cutaneous leishmaniasis,
gnathostomiasis and Chagas' disease, including leprosy.

sity) units; PGL‑1‑IgM 0.38 OD units; LAM‑B‑IgG 0.25
OD units; LAM‑B‑IgM 0.05 OD units. A criterion for

Sera
Serum samples from 365 subjects were examined.

considering the diagnosis was made as follows: PGL ( + )Skin test
The lepromin tests were performed in eight subjects

suspected for leprosy. An amount (0.1 mD of Mitsuda
lepromin solution was injected intradermally on the
flexor surface of the forearm using the small needle (adisposable needle; size 26G) , and the skin test area wasobserved for erythema and induration (induration/
erythema in mm) at 48 hours later. Erythema size ofmore than 11 mm in diameter at the injection site wasconsidered positive reaction, and reaction with 7 x 10mm was considered an undetermined ( + / ‑ ) Mitsuda

and LAM ( ‑ ) , suspected leprosy; PGL(‑) and LAM( + ) , suspected acid‑fast bacteria infection including
leprosy; PGL‑IgG ( + ) and lgM ( ‑ ) , suspected leprosy(old leprosy and spontaneousry healing subjects) ; PGL‑IgG ( ‑ ) and lgM ( + ) , suspected leprosy.

The diagnosis of leprosy was made on clinical,
bacteriological and immunological grounds, according
to the Rjdley‑Jopling classification (Ridley and Jopling,1996) by the doctors from the Welfare Ministry of
Ecuador.

early reaction.

Leishmania skin tests (Furuya, et al., 1989) weremade in 13 subjects suspected of leprosy (file number G‑1 ‑ G‑13). An amount (0.1 mD of Leishmania promas‑
tigotes antigen solution was injected intradermally on
Table 1

The results obtained are summarized as shown in
Tables I to 6. There was no correlation between preva‑
lence rates for leprosy and seropositive rates of thePrevalence rates of leprosy and anti‑PGL‑1 * antibody positive subjects from different
areas of Ecuador.

Prevalence rate

Department * * ( x 1000)

No.

Total (%)

Male

Female125

3 (20.0%)
3 (18.7%)
61 (30.8%)
10 (55.6%)
12 (80.0%)64 (61.2%)
1 (20.0%)365

154 (42.2%)O . 63‑0 . 91

15

2. El oroO . 63‑0 . 91

16

3. Guayas
4. Pichincha

O . 27‑0 . 38

198

O . 10‑0 . 16

18

ManabiO . 10‑0 . 16

15

6. Esmeraldas

O . 10‑0 . 16

98

7. Other areas

Total

Positive for PGL‑I

examined

1. Bolivar

5.

RESULTS3O

16* * *

32 * * *

5

5

5

6

23

41

O

1

53 (37.9%)

87

(62 .

1%)

* , PGL‑1: phenolic glycolipid‑1; * * , I . Bolivar. Echeandia; 2 . El Oro, five resions; 3 . Guayas,

Pedro Carbo and Guayaquil; 4 . Pichincha, Puerto Quito and Quito; 5 . Manabi, Los Ranchos; 6 .
Esrneraldas, Selva Alegre; * * * , Sex of 13 subjects were unknown.

181

Table 2 Correlation between anti‑PGL‑1 * (IgG) and anti‑PGL‑1 (IgM) antibodies of sub j ects
from differnt areas of Ecuador.

Departments
and cities forvillages

1 . Bolivar

Echeandia
San Francisco
2 . El Oro

PGL (IgG) +PGL (IgM) +O(6 . 7)(o . o)OOONo. and (%) of subjects in each category
PGL (IgG) ‑PGL (IgG) ‑PGL(IgG) +PGL (IgM) ‑PGL (IgM) +PGL (IgM) ‑1

O

Machala

O
O
O
O
O

O

3 . Guayas
Pedro Carbo
Guayaquil
Olon
Others4OO(2 . o)O

8
6
O(4 . 1)

1

4 . Pichincha
Puerto Quito
Quito

2 (11 . 1)

2 (11 . 1)

5 . Manabi
Los Ranchos(26 . 7)

O

Total

*

1

O
O

7 . Other areas(6 . 3)(81 . 2)

16

O
O

13
O
2

O

4

5
5

O

2

2

O

1

49
2

137
115
13

O
6

6

1

(6.6)

6
6
O

(33 . 3)87 (46 . 7)
7

O

O
O

O

(11 . 2)

11

41
41

O

O

1

22 (6.0)25 (6.9)

198
165
15
13

(44 . 5)18
17

(20 . o)

15
13

8
O

O

11

(69 . 2)

4

3
2

1

1
(12 . 3)(24 . 7)

41

1

12
12

15
9
6

O

3

Guale
6 . Esmeraldas
Selva Alegre(80 . o)5

O

Portovie j o7

2 (12 . 5)

Antepara

Zaruma12

1O(o . o)1

Pinas
Portovelo

2 (13 . 3)

Total

1

1

(41 . 8)107 (29.3)

34
34

1

(34 . 7)4211 (57.8)

98
98
5

365

PGL‑1: phenolic glycolipid‑1.

antibodies (anti‑PGL‑1‑ and LAM‑B antibodies) in thesubjects living in several provinces in Ecuador. The

subjects in Ecuador was 69 (19.6%) out of 352.
The results of examinations of leprosy patients andserop,0 itive rates of anti‑PGL‑1 antibodies in Ecuador
was 42.2 %. The seropositive rates for PGL‑1 was higher

their family in Los Ranchos, Department of Manabi
were summarized in Tables 4 to 6. The number ofin female than in male. Correlation between anti‑PGL‑1‑IgG antibodies (PGL‑IgG) and anti‑PGL‑1‑IgM anti‑

subjects examined for serological tests of leprosy was 13

bodies (PGL‑IgM) was summerized in Table 2. The

shown as follows. Only one subject, G‑1, showed un‑

distribution of anti‑LAM‑B antibodies in the subjects

determined ( + / ‑ ) Mitsuda early reaction. Seven otherwith negative anti‑PGl‑1 antibodies in the patients withcutaneous leishmaniasis, gnathostomiasis, Chagas' dis‑

subjects proved to be Mitsuda negative. Leprosy
patients (G‑7, G‑10 and G‑13) except G‑1 showedease and others was summarized in Table 3. In 211 PGL‑1 seronegative persons, 128 (60.7%) were found to be
LAM‑B seropositive; 16 (7.7%), IgG‑positive and lgM‑positive; 16 (7.7%), IgG‑positive and lgM‑negative; 96(46.10%), IgG‑negative and lgM‑positive; and 80 (38.
5%), IgG‑negativ and lgM‑negative. The subjects ofpositive raection to the Leishmania skin test, while 10
other subjects showed negative reaction to the skin test
(Table 4). A subject, G‑7, showed all positive reaction

file number EH57 and EH119 from Esmeraldas and G‑7from Manabi showed positive reaction to all the anti
‑PGL‑1 and anti‑LAM antibodies examined. The num‑
ber of anti‑LAM‑B‑IgG antibody positive nonleprous

ulcerative lesions of feet and neuralgia on the four
extremities. The patient was treated with multi‑drugtherapy (MDT). Other two leprosy patients, G‑10 and(7 males and 6 females) . The results of skin tests wereagainst PGL‑IgG and ‑IgM, and LAM‑IgG‑ and ‑IgM.
He was a borderline lepromatous leprosy patient who
still had active symptoms, such as infiltrated erythema,

G‑13, were positive for PGL‑IgM, and LAM‑IgG and182

Table 3 Distribution of anti‑LAM‑B* antibodies in the subjects, negative for anti‑PGL‑I anti‑
bodies, from differnt areas of Ecuador.

Departments

LAM (IgG) +LAM (IgM) +1 . Bolivar

Echeandia
San Francisco

No. and (%) of subjects in each category
LAM (IgG) +LAM (IgG) ‑LAM (IgG) ‑LAM (IgM) ‑(8 . 3)(8 . 3)oO

2 . El Oro(7 . 7)

AnteparaoMachalao2(15 . 4)O
O

PifiasPortoveloo1

ZarumaoOthers
3 . GuayasoOO(5 . 9)(6 . 6)

LAM (IgM) ‑6 (50.0)

4 (33.4)

3
3

3

7

1

5

4 (30.8)

6 (46.1)

13

O

O

O

1

2

O
3
O
O

3

5

O

4

1

1

65 (47.8)
58

54 (39.7)

Pedro Carbo
Guayaquil
Olon
Others

7

9
8

o

1

4

o

O

2

4 . Pichincha
Puerto Quito

33o(37 . 5)o(o . o)Quito

5 . Manabi
Los Ranchos

8

O
2

O

o

Portovie jooGuayabaleso(25 . o)OOOO(o . o)2

6

3 (37.5)

O

3

O

O

8
8
O

1 (33.3)

2 (66.7)

3

1

2

O

O

9 (26.'5)

34
34

3
3

2

20 (58.8)
20

7 . Others

o

O

O

16 (7.7)

16 (7.7)

96 (46.1)

Total (%)

42
8
O

1

136
115
13

O (0.0)OO(5 . 9)1

12

4

6 . Esmeraldas
Selva Alegre(8 . 8.)Total

LAM (IgM) +1

92 ( 100 . O)

2

80 (38.5)

208* LAM‑B: lipoarabinomannan‑B; * * , PGL‑1: phenolic glycolipid‑1.

‑IgM. A Ieprosy patient, G‑10, with borderline tuber‑‑positive.

culoid leprosy had relatively deep ulcers on both soles.

The type of leprosy in G‑13 was unknown and thesubject had no specific dermatological findings of lep‑rosy. All the leprosy patients were LAM‑IgM‑positive,
and the subjects, G‑1, G‑7 and G‑10 except G‑13, were

COMMENT
Leprosy has a wide r nge distribution in the world.

anti‑PGL‑1‑IgM antibodies in the patients with leprosy

The disease is included among the six most important
infectious diseases which the World Health Organiza‑tion (WHO) planned to stop from being an endemic. In
some regions of Ecuador, the prevalence rates are very

and their families was summarized in Table 5. Twohigh. ' For example, the rate in Department of Los Rios

leprosy patients, G‑1 and G‑7, were positive for PGL‑IgG and ‑IgM. Other two leprosy patients, G‑10 and G

showed 1.17 per 1,000 habitants. Persons living in thestrongly positive for LAM‑IgM, showing more than O.
190 OD units. Correlation between anti‑PGL‑1‑IgG and‑13, were PGL‑IgG‑negative and PGL‑IgM‑positive.
Correlation between anti‑PGL‑1 antibodies and anti
‑LAM‑B antibodies in the patients and their families
was summarized in Table 6. Three patients with lep‑rosy, G‑7, G‑10 and G‑13, were PGL‑IgM‑positive and
LA.M‑B‑IgG‑positive. Indeterminate leprosy patient, G‑1, was negative for only LAM‑IgG. There were no
subjects who were PGL‑IgM‑negative and LAM‑IgG

regions, where the prevalence rate is over 1.0 per 1,000,

would be exposed to serious danger of infection whether

they have or not contact with leprosy patients. The
immediate counter plan for chronic infectious disease
should be considered.
Recently, serodiagnosis of leprosy was considered
as one of the useful methods for early diagnosis (Bu‑chanan, et al., 1983). Although, we could not examine

large numbers of leprosy patients during the current

183

Table 4 Leishmania skin test and Lepromin test in three leprosy families in an endemic area of
cutaneous leishmaniasis, Los Ranchos, Department of Manabi, Ecuador, in 1992.

No. (File no.)Age

1 (G‑ I )12

2 (G‑ 2 )10

3 (G‑ 3 )9

4 (G‑ 4 )

5 (G‑ 5 )11

6 (G‑ 6 )
7 (G‑ 7 )38
41

8 (G‑ 8 )89 (G‑ 9 )15

10 ( G ‑10)31

11 ( G ‑11)612 ( G ‑12)313(G‑13)56

Sex Lepromin

Leishmania

F (+/ )**
F
F
F

M

M
M
M
M
M
M

hypopigmented freckle(‑)

with anesthesia

(‑)
(‑)
(‑)
(‑)
(‑)
(+)

(‑)
(‑)
(‑)
(‑)
(‑)
(‑)

F

Symptoms

skin test

test *

indeterminate
leprosy

none
none
none
none
none
annular and/or infil‑
trated erythemata,nodules with anesthesia

(‑)
(‑)
(+)

(‑)

Type of leprosy

borderline‑

lepromatous

none
none
neuralgia, ulcers with
anesthesi a

(‑)
(‑)
(+)

borderline‑
tuberculoid

none
none
neuralgia, anesthesia

leprosy * * ** Mitsuda early reaction; * * , Mitsuda early reaction is undetermined with 5.7 x 7.5mm ( + / ‑ ) ;leprosy type is unknown.

***

Table 5 Correlation between anti‑PGL‑1 * (IgG) and anti‑PGL‑1(IgM) antibodies in the
leprosy patients and their household contacts in Los Ranchos, Department of
Manabi. Ecuador, in 1992.

PGL (IgG) +PGL (IgM) +PGL (IgG) ‑PGL (IgM) +PGL (IgG) +PGL (IgM) ‑PGL (IgG) ‑PGL (IgM) ‑File

G‑ I , G‑ 5

G‑ 2 , G‑ 3 , G‑ 4none

G‑9 , G‑11

no .

G‑ 7 , G‑12

G‑6 , G‑ 8 , G‑10
2

o

o

7

o

2

Category

G ‑13

No. of leprosy

Total

2

4

* PGL‑1: phenolic glycolipid‑1;

Table 6 Cerrelation between anti‑PGL‑1 * and anti‑LAM‑B * * antibodies in leprosy patientsand their household contacts in Los Ranchos. Department of Manabi, Ecuador, in 1992.

PGL (IgG) +PGL (IgM) ‑LAM (IgM) ‑LAM (IgM) ‑G‑ 1

G‑ 2 , G‑ 3
G‑ 5 , G‑ 6 , G ‑12

G‑9,G 11

PGL (IgM) +LAM (IgG) +PGL (IgM) +LAM (IgG) ‑File

G‑ 4 , G‑ 7 , G‑ 8

no.

G‑10, G‑13

Category

No. of leprosy

3

1

O

o

Total

5

1

5

2

*

PGL‑1: phenol,ic glycolipid‑1;** LAM‑B: lipoarabinomannan‑B;

survey, anti‑PGL‑1 and anti‑LAM‑B antibodies were
examined by using accepted sera from different areas of

Ecuador. The seropositive rates of anti‑PGL‑1 anti‑
bodies in Ecuador was high, showing a rate of 42.2%.
Recently, anti‑LAM‑B antibodies have been measured

to detect the subjects with multibacillary leprosy whose
values of anti‑PGL‑1 antibodies are very low (Izumi etal., 1993). In 215 PGL‑1 seronegative persons, 132 (61.

4%) were found to be LAM‑B seropositive. Some ofthem have the possibility of showing symptoms of lep‑

184

rosy. Anti‑PGL‑ and anti‑LAM‑B antibody positive
subjects would need detailed medical examination torule out the disease by acid‑fast bacteria. A total of 154

subjects (42.2%) were serologicaly suspected of suffer‑

ing from M. Ieprae and 128 subjects (35.1%) were

patients, were strongly positive for LAM‑IgM (data notshown) . As the subjects, G‑5 and G‑12, were positive
for PGL‑IgG and ‑IgM, they should be watched for thedevelopment of the disease symptoms, though no clinicalfindings of leprosy were observed in the present exami‑

suspected of diseases caused by acid‑fast bacteria in‑cluding M. Ieprae. In the examination, tuberculosis,

nation.

tuberculosis cutis and its related diseases such as lupus

Correlation of the skin tests between cutaneous

vulgaris, tuberculosis verrucosa cutis, scrofuloderma,leishmaniasis and leprosy was partly found; three out offour leprosy patients showed positive reaction to Leish‑

erythema induratum Bazin and infectious diseases ofatypical mycobacteria should be taken into considera‑

mania promastigotes antigen. From this result, it would
be speculated that the specific defect of cell‑mediatedtion.

immunity for M. Ieprae might be covered by otherlzumi et al., (1993) reported the distribution of anti

activated cellular immunity.

‑LAM‑B antibodies in non‑leprous sera in Japan andSouth Sulawesi, Indonesia. According to their studies,
the numbers of anti‑LAM‑B‑IgG‑ antibody positive non‑leprous subjects in Japan and Indonesia were 18 (4.9%)
out of 367 and 20 (12.4%) out of 161, respectively. In

comparison, the positive rate of anti‑LAM‑B‑IgG anti‑
bodies in Ecuador was relatively high, showing a rate of

19.6%. From these results, we suspected that there
might be large numbers of inhabitants infected sub‑clinically by the bacillus and some of them have thepossibility of showing symptoms of the disease.
In the present study, no correlation between positive

REFERENCES
1 ) Bryceson, A.D.M. (1981): Clinical, immunological andEpidemiological features of leishmaniasis. Humber, D.
P. ed. Immunological aspects of leprosy, tuberculosis
and leishmaniasis, Addis Ababa, Ethiopia, 32‑38.
2 ) Buchanan, T., Dissanyake, S., Young, D.B., Miller. R.A.,
Acedo, J.R., Harnish, J.P., Khanolkar, S.R. and Estrada‑Parra, S. (1983): Evaluation of significance of anti‑
bodies to phenolicglycolipid of Mucobacterium leprae in
leprosy patients and their contacts, Int. J. Lepr., 51, 658

rates of antibodies (anti‑PGL‑1 and anti‑LAM‑B anti‑3 ) Furuya. M., Mimori, T., Gomez, E.A.L., De Coronel, V.

bodies) and prevalence rates of leprosy was observed inV., Kawabata. M. and Hashigichi, Y. (1989):

each area of Ecuador. In Manabi, Ecuador, the preva‑

Epidemiological survey of leishmaniasis using skin test
and ELISA in Ecuador, Jpn. J. Trop. Med. Hyg., 17, 331‑338

lence rate of leprosy was 0.10‑0.16 per 1,000 inhabitants,

showing a relatively low rate. But the anti‑PGL‑I‑seropositive rates of the patients and their family in

Los Ranchos. Manabi (84.6%) were higher than the
average positive rate (42.2%) of all subject in several4 ) Gaylord, H. and Brenann, P.J. (1987): Leprosy andleprosy bacillus; recent development in characterizationof antigens and immunology of the disease. Ann. Rev.
Microbiol., 41, 645‑675

areas in Ecuador. From the data shown in Table 5, it
was considered that PGL ‑IgG and lgM‑ positive cases5 ) Hunter, S.W., Fujiwara, T. and Brenann, P.J. (1982):

were bacteriologically and immunologically active

glycolipid of Mycobacerium leprae. J. Biol. Chem., 275,
15072‑15078

patients.

In gen.eral, the values of anti‑PGL‑IgM and LAM‑B‑IgG antibodies were used for serological diagnosis ofleprosy because the combination thought to be clinica]ly
useful for the diagnosis in early stage of the disease. As

Structure and antigenicity of the major specific

6 ) Izumi, S., Maeda, M., Van Beers, S., Mdjid, B. and
Kawatsu, K. (1993): Distribution of anti‑LAM‑B anti‑
bodies in leprosy patients and house hold contacts, 66 th
Japanese leprosy Conference, p. 42

for PGL‑IgM and LAM‑IgG. The value of anti‑LAM‑B

7 ) Izumi, S., Maekawa, M., Kawatsu. K. (1993): Distribu‑
tion of Anti‑LAM‑B antibodies in leprosy patients andhouse hold contacts, Report of 26th Joint conference of‑IgM antibody was thought to be unreliable, because of

Tuberculosis and Leprosy (U.S‑Japan Cooperative

shown in Table 6, three leprosy patients were positive

the low of cut off value ( > 0.19 OD unit) . But a strong

positivity of LAM‑IgM might be an indicator for the
diagnosis of leprosy, because three out of four leprosy

Medical Science Program) , 105‑110
8 ) Ridley, D.S. and Jopling, W.H. (1966): Classifacation of
leprosy according to immunity, Int. J. Lepr., 34, 255‑273

185

Jpn．J．Trop．Med．Hyg．，VoL22，No、4，1994，pp．185−192

マラリア予防薬としてメフロキンを長期投与した際の副作用について

竹

島

茂

人

平成6年8月10日受付／平成6年10月30日受理

はじめに
自衛隊はペルシャ湾，カンボジアに引き続きモザンビー
ク共和国に48名の自衛隊員を輸送調整中隊として1993年5月から派遣している。ペルシャ湾に始まる自衛隊の海外への部隊派遣ではいずれも自衛隊医官が同行しておりモザン
ビークにおいても医官2名が同行し隊員の健康管理，及び衛生環境整備を行っている。カンボジア，モザンビーク共
和国ともにマラリアの汚染地域であるため，カンボジア施
設大隊ではマラリア予防薬としてドキシサイクリンを使用していたが，今回，著者等はモザンビーク共和国でメフロキンを使用した。メフロキンの有効性，及びその内服期間を通じて自衛隊員が訴えた体調の変化，及び血液学的変化
でマラリアの副作用と思われる点について述べる。

対を媒介するハマダラカの温床になっている。モザンビーク
共和国は南北に国が延びている為，国連モザンビーク活動（以下ONUMOZ）も北部，中部，南部に分けて活動が行われていた。当第2次モザンビーク派遣輸送調整中隊48名の内38名は首都マプトから約8km離れたマトラに宿営（南部地域の輸送調整業務を担当）し，10名はベイラから約30㎞離れたドントに宿営（中部地域の輸送調整業務を担当）していた（Fig．2）。派遣隊員数は業務を行いうる最少人数であった為，マトラ，ドント共にポルトガル軍の宿営地内に宿営し食事，シャワー等の後方支援はポルトガル軍に依存
していた。

象

今回，対象としたのは第2次モザンビーク派遣輸送調整中隊としてモザンビーク共和国に1993年11月22日から1994
年6月18日の間，派遣されていた48名である。全員男性で平均年齢は32．0歳（SD−5．5）であった。陸・海・空の内訳

は陸上自衛官38名，海上・航空自衛官各5名であった。48
名の既往歴としては1名が約10年前に水腎症の為，片腎摘出術を受けていた。又，日本出国前に全員がB型及びC型
肝炎の検査を受けており，1名がB型肝炎キャリアであった以外は肝炎は認めなかった。

モザンビークの地理的，気候的背景
モザンビーク共和国はアフリカ大陸支部に位置し（Flg．
1）南緯10度から27度，西経30度から41度内に存在する1）。
標高は平均200mと低く，南北に国が延びている為，気候は
北中部は熱帯気候に南部は亜熱帯気候に属する。乾季の時期を除いて高温多湿である。Fig．2に代表的4都市（ナンプラ，テテ，ベイラ，ナマーシャ）の月別平均気温及び降水量を示す2）。季節は11月から4月までの雨期と5月から10月までの乾季に分けられる。標高が低く降水量が多い為，湿地帯が多く，それに加えて高温多湿である為，マラリア
自衛隊札幌病院外科

札幌市豊平区平岸1条12−1

自衛隊札幌病院外科

Mozambique
Figurel The location of Mozambique in Africa．
Mozambique lies between lO。〜27。S and30。

〜41。W．

186

瞥

旧E

Tび

開始した。熱帯熱マラリアの潜伏期間が7日〜30日を考慮して帰国後4週間内服を継続し
た。

J

A

J

4）蚊帳に対するりペラント処置及びその使用指導
蚊帳に対するりペラント処置の効果は数々

O

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Matola（38members）and

第2次モザンビーク派遣輸送調整中隊のマラリアに対する予防衛生

ONUMOZの総司令部には公衆衛生幹部がいたが組織的な防疫活動，駐屯地における衛生指導は行われておらず不定期に殺虫剤が配付されるのみであった。当中隊では以下の4点についてマラリアに対する予防衛生活動を行った。
1）殺虫剤（殺虫スプレー，蚊取り線香，防虫スプレー，
殺虫煉煙剤）の配付と使用勧告。
当初は日本からすべての殺虫剤を追送していたが，殺虫スプレー，殺虫燃煙剤については代用品が現地調達できるようになり追送品は蚊取り線香，防虫スプレーのみとなった。蚊取り線香は各テント毎に配付し毎晩使用するように指導した。防虫スプレーは夜間の外出，旅行時には必ず携行させた。殺虫燥煙剤は2〜3カ月に1回の割合で蚊，その他の病害動物に対して各テント，プレハブ毎炊かせた。
2）駐屯地内の防疫活動
1次隊から駐屯地区の防疫活動は行われており，当初はダイアジノン，スミチオンを野外トイレ，テント周囲及び駐屯地周辺の水たまりに散布していたが，効果が弱い為，1994年2月からペルメトリンに変更し効果を上げた。

3）メフロキンの予防内服
ONUMOZの勧告と同様の250mg／wを出国前1週間から

in

Com−

Dondo（10members）．

マラリア予防薬メフロキンの投与量，投与方法及び副作用調査方法
我々は1）クロロキン耐性熱帯熱マラリアがアフリカ大陸にも多く認められること6），

τomp・

ワラロドロロロのドロロド

閣aputoFigure2The

ペラント処置をされていない蚊帳を使っていた為，2次隊ではペルメトリンによるりペラント処置を施行した。現在リペラント処置を施行された蚊帳が市販されており今後，マラリア流行地域に部隊が派遣される場合にはこれを購入するのが適切であると考えている。

2）WHO，フランス厚生省の推奨するプロト
コール7）8）がONUMOZが推奨するプロトコー
ルと一致する事，からマラリア予防薬として
メフロキン250㎎／wのプロトコールを採用した。メフロキンの内服はモザンビーク着国時，
既に有効血中濃度に達しているように出国前
1週間から始めた。内服開始前には全隊員に服用の必要性，服用方法，副作用等について
パンフレットを配付し，説明した。帰国後は熱帯熱マラリアの潜伏期間7日〜30日を者慮し4週問まで続けた。内服は必ず食後とし多めの水と共に服用するように指導した。メフロキンは現地での休暇期間中も必ず持参させ確実に毎週服用させた。ただし，後で述べる4名についてはモザンビーク入国4カ月で副作用と思われる症状が徐々に増強した為，服用間隔を1週間から2週間に延長している。この4名はメフロキンを計29錠服用した事となるがその他の隊員は設36錠内服した事になる。モザンビークの宿営地内には医務室を設け体調不良時には常時受診できるよう配慮した。又，派遣後1カ月目，3カ月目にアンケートを用いて全隊員の体調をチェックし4カ月目，帰国直前に副作用についてのアンケートを行った。
血液学的検査としては日本出国前，帰国後に血液算球検
査，生化学的血液検査を施行しており又，モザンビーク着
国後3カ月には遠心分離後凍結した血清を日本へ航空機輸送，日本で生化学的血液検査を施行している。
〈結果1一隊員の体調の変化について〉症状としての副作用はすべてアンケートを用いて調査した。それぞれの時期に施行したアンケートの回収率はすべて100％であった。
内服期問を通して重篤な症状は認めなかったが1名に嘔吐（内服し3週間目から1回／月，内服日夜間）が定期的に187認められた。この隊員に対しては消化剤，制吐剤の適宜投与を行うことにより4カ月目には症状は消失し予防内服を250㎎／wで続ける事ができた。モザンビーク到着後4カ月目に施行したアンケートの結果をTable1に示す。全身倦怠感，頭痛の内，各2名は重複している。計16名（33．3％）に副作用があった事となる。いずれも軽度の副作用であっ
たが頭痛2名，全身倦怠感2名が投与間隔の延長を希望した為，この4名に対してのみ以後，帰国後4週間までの投与間隔を1週間から2週間に延長した。残りの14名に対しては症状の強さと本人の希望を考え合わせ従来通りの250
mg／wのままとした。帰国前に再度施行したアンケートでは頭痛，全身倦怠感を訴えていた4名は症状は残っていたがTable1Side

effectswithing

symptoms

werenaire．Two

part

Side

effects

Generalfatigue

of

of

the

the

mefloquin（48persons）Follow−

gottengeneralheadache

Numberofcases

6（12．5％）

Nausea・Vomiting

2（4．2％）

Vertigo1（2．1％）

Diarrhea1（2．1％）

Cell

Llne

Error

Bars：7．1

question−

cases

are

also

group．

8（16．7％）

Headacheby4monthfatigue

Beginning

1W
2
1

of

side

effects

1M

2M
1
2

3M

2
1
1

3
2

軽快していた。帰国後，メフロキン内服を中止して約2カ月半経過した現在，新たな副作用の出現，重篤な副作用の出現は認めていない。

〈結果2一血液検査データの変化〉

日本出国前，モザンビーク到着後3カ月目，そして日本帰国後に生化学的血液検査（3カ月目は遠心分離し日本へ凍結輸送した）を施行している。
血液算球検査については日本出国前と日本帰国後のみの比較になるが，Fig．3に示す通りである。亜熱帯地域での勤務，生活による脱水の影響を受けた為か赤血球は上昇が激しい。これについてはカンボジアにおける派遣でも認められている。白血球も有意に上昇しているがメフロキンとの因果関係については不明である。尚，白血球の上昇に関してはカンボジアでは認められていない。
3カ月目の血液検査では3名（6．3％）の肝機能軽度異常者，6名（12．5％）のガンマGTP異常者，15名（31．3％）のTTT異常者が認められた。帰国後の白血検査では8名（16．7％）の軽度肝機能異常，7名（14．6％）のガンマGTP

異常者，16名（33．3％）のTTT異常者が認められた。

1

ZTT，ビリルビン，LDH，LAP，BUNについては異常を

1

認める者はなかった。肝機能異常を示した隊員と全身倦怠感を訴えた隊員は必ずしも一致していなかった。

1

Chart
Standgrd

Devlation（s）

18

620

17．5

600

Ce

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PRE．WBC

POST．HT

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betweenPre−departureandPost−retum

POST．WBC

188．

Table2

Eight

A一
B一

D一

E
F

G

had

abnormal

liver

shows

the

functionschangeofintest．Thisfunctionsamong

Pre−departure，3monthPost−return

testing

Periods．

No． Liver functionC

memberspost−retum

andに肝機能が上昇した者はA．B．D．Cの4名（8．3％）であった。

Pre−departure3MonthPost−retum

27

19

48

46

47

一
62

｝
16

一

一
35

21

27

61

14

10

一
21

27

14

38

H一

GOT
GPT
GOT
GPT
GOT
GPT
GOT
GPT
GOT
GPT
GOT
GPT
GOT
GPT
GOT
GPT

帰国後の血液検査では肝機能異常を認めた8名の隊員の検査値の推移をTable2に示す。モザンビーク到着後明らか

liver

23

37

36

一
43

』
39

51

『
53

』
18

一
31

一
25

41

41

36

一
26

一
23

一
44

尚，B型肝炎キャリアの隊員はいずれの時点での血液検査でも肝機能には異常は認めなかった。
肝機能異常がメフロキンの副作用である可能性は十分にあるが，脂肪肝，アルコール性肝障害の除外診断を施行していない為，厳密には全てが副作用であるとは言いきれないoメフロキンの予防薬としての有効性について
モザンビークに派遣された自衛隊員ではマラリア患者は1994年8月1日現在，一人も出ていない。しかし，メフロ
キンの予防内服が推奨されているONUMOZ内では多数の

24

一
43

34

19

｝
42

マラリア患者の発生が認められている（Fig．4）。勤務地域
もしくは，居住地域によって罹患率が違ってくる事は当然予想されるし メフロキン耐性マラリアについても考える必要が出てくる。

40

一
40

22

一
35

一
20

25

42

17

19

』
36

自衛隊は48名中38名は南部地域に，10名は中部地域に，
いずれもポルトガル軍と同じ駐屯地内に居住していた。自衛隊が南部，中部業共に一緒に居住していたポルトガル軍のマラリア患者数を見ると（Fig．5）必ずしも自衛隊員にマ一

Jun−93

Ju1−93Aug−93Se−93

Oct−93

Nov−93

D㏄一93

Jan−94Feb−94

1271

32

27

11

30

35

43

3

8

12

Central1228

42
29

7

4

7

19

15

30

43

2
3

North

SOUTH2452
Total

5

18
89

40

4951

7
4

15
13

55

2
1

9

12

40

167
217

Mar−94 Apr−94

May−94

40

40

5

8

55
13

227
272

200
248

300
／

250

］＼．

／
／

＼

North

／

200

、

ノ

へ

＼

＼

ー︑
ρ︐

ρハ

100

Central1228

、

1

150

50

＼

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Malaria

ま止二く

Figure40NUMOZ

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り

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oり

O−ぢO

り

O−望く

Oム考

①−F5﹁

oo

㎝O−晋の

0

1271

、

ノ

SOUTH

2452

Total

4951

68
136

189

ラリア患者が発生していないのは居住地域による違いではないと言える。ポルトガル軍の衛生兵からは，ポルトガル
軍マラリア患者はメフロキンを飲み忘れていた兵士か，副作用の発生を恐れて飲んでいなかった兵士であると情報を得ている。又，ONUMOZ内ではマラリア治療にはほとんどのケースでメフロキンを使用しているがメフロキン耐性マ
ラリアについても1994年8月1日現在報告がない。一以上の事よりメフロキンの予防薬としての効果は今のところモザンビークにおいては非常に高いと考えられる。

考

報道していた。一

当中隊では派遣間そして後も一人のマラリア患者を出すことなく任務を遂行できたが，これはメフロキンの予防内服を含めた予防衛生活動によるものと考えている。なぜな

らばONUMOZでのマラリア発生患者はそのほとんどが予防薬を服用していなかったという情報があり，また駐屯地における防疫活動も著者が知る限りではイタリア軍野戦病院，アルゼンチン軍野戦病院，そして日本隊のみであったからである。

サハラ砂漠以南のアフリカでは85％以上が熱帯熱マラリ
アと従来より言われてきた9）。3つあるONUMOZの野戦病院の1つであるバングラデッシュ野戦病院は熱帯熱マラリ
アがほぼ100％だと言っていた。また，モザンビーク厚生省に我々が直接質問状を提出したところ種類別マラリアの割察

我々が勤務したモザンビーク共和国は亜熱帯地域に位置し標高も低く降水量が多い1）2）ためマラリアを媒介するハマダラカの温床となっていた。又，モザンビークは11月か合を熱帯熱マラリアが95〜99％，4日熱マラリアが1〜5％，卵形マラリアが0．1〜0．5％で3日間マラリアはないと返答を受けた。モザンビークにおいてマラリアの死亡数が多いのは，そのほとんどが致死的となる重症マラリアを引き起こす熱帯熱マラリアであると考えられる。
メフロキンの予防内服はこれまで3カ月を限度として使用するように勧告されていた10）が，今回我々の約8カ月に渡る予防内服では4名の投与間隔延長者はいたものの，いら4月までの雨期と5月から10月までの乾季に分けられていたが我々，第2次モザンビーク派遣輸送調整中隊が派遣された時期は最高気温47度という雨期の真只中であった事も加わり，特にマラリアには注意が必要であった。Fig．4に示すようにONUMOZ内でも2月から4月までは200人／月
以上のマラリア患者発生が認められていた。我々が調査した現地の4つの医療施設でもいずれもマラリアを疾患別患者数のトップにあげていた。又，現地新聞（NOTICIAS5
Apri11994）でもマラリアによる死亡数が第1位であるとJun−93

North

Central77

8
0
0
8

1

South 133
Total 273

Sep−93

Juト93 Aug−93

6
1

63

8

2
0
0
2

Oct−932002

ずれも軽度の副作用を33．3％の隊員に認めるだけで済んだ。

しかし，メフロキン250㎎／wを3カ月以上投与するとそれ

Nov−931
1
1
3

Dec−93300
3

Jan−94

0

1
2
1
4

0
0
0

Feb−945
5
3

13

Mar−94

Ap卜94

1

4
0

2

3

1
4

7

14
12
10

＼

♂

4

2

㌧＼

8 7へ
6

＼

寸①−澄Σ

malaria

蕊∴α＜

寸①−﹂GΣ

Figure5Portugal

寸Oムo﹂

寸①とO﹁

り

①−80

笛①︒︾OZ

㊦①山りO

oo

①−審の

的O−Oコ︵

O︐考﹁

り

φO−ヨ﹁

0

／

63

Central

77

South

133

Total

273

一

＼
＼

North

May−94

2
0
2
4

190

ま．で出ていた軽度の副作用が増強したり，肝機能障害が出

減期が15−33日10）11）である事より薬剤の体内蓄積を考慮し投与間隔を2週間に延長している。アメリカ保健省はメフロキンの体内蓄積を考慮してか，予防内服開始後4週目までは毎週250㎎の内服としその後は250㎎を隔週で内服する﹄した現する可能性がある為，今後長期に渡る内服が必要な場合
は血中濃度をモニタリングし，どのような服用方法が最適であるかを調査する必要があると思われる。投与間隔を延長した4名の隊員については血中濃度分析を行ってはいな
いがメフロキンの最高血中濃度到達時間が2−12時間，半

等18）の報告によればメフロキンを389名に対して12週間，250㎎／wで投与した副作用出現率は延べ12．8％，副作用のために予防内服をメフロキンからドキシサイクリンに変更のは6名1．5％であった。アンケート調査でのSteffen等の報告19）の2L2％，Lobel等20）の47．2％と較べても低率であったがこれは投与期問による差とも考えられる。今回の我々の長期投与による副作用出現率は全体で33．3％でアメリカ平和部隊を対象とした調査（Table3）の39％に較べると若干低かった。

1993年5月にONUMOZが始まってからONUMOZはメ事を推奨している12）。しかし，西アフリカでこの通り内服しフロキンを予防薬として推奨し全部門にこれを配付，またたアメリカ平和協力隊では予防内服を隔週に切り替えた後一ONUMOZの3つの野戦病院の内，アルゼンチン軍，イタリ
ア軍野戦病院の2つはマラリア治療薬の第1選択にメフロにマラリア患者の発生を認めている為，当中隊では250mg／
wを長期に渡り継続した。

キンを採用，残るバングラデッシュ野戦病院についても第2選択にメフロキンを採用していたが1994年8月現在，メ

血液検査では全員の白血球数が有意に上昇していたがHarinasuta等13）の報告では治療量では白血球の減少は認められているものの増加は認められていない。数名に肝機能の上昇を認めた事とあわせて，今後の追加研究が必要と報告がない。ちなみに，モザンビークにおけるクロロキン
耐性マラリアについては30〜50％というデータをモザンビ思われる。

ーク厚生省から得ている。

今回の副作用の内訳を従来から言われてきている副作用
報告3）（Table3）と較べると全身倦怠感が8名（16．7％）と多数であるが他の4項目については従来の報告と一致す

長期にわたりマラリア予防薬としてメフロキンを使用する場合の副作用についての報告は稀であったが，以上より
メフロキンはマラリア予防薬としては，その耐性は認めな
い地域においては副作用も軽く効果の高い薬剤であると思われる。また，従来より3カ月を限度とした予防内服が推奨されていたが耐性獲得を考慮しなければ，注意して使用
すれば6カ月を越える予防内服も可能であると思われた。

る。従来から指摘されている平衡感覚障害14）15）については

1名が眩量を訴えてはいたものの全員，日常生活そして輸送調整業務上も全く支障はなかった。今回は副作用でけいれん等の神経症状の出現16）17）は認めず，メフロキンを中止

フロキン耐性熱帯熱マラリアについてはONUMOZ内でもして他剤へ変更する事もなかった。

カンボジア派遣海上輸送補給部隊として参加した菅沼
Table3

Reports

1．SurveyofonEuropean

Nausea（10．7％）

Vertigo（6．7％）Headache（6．1％）

2．SurveyofGermanoccurrenceof

1／13，000
3．Report4．

staying

in

with

east

mefloquin

Africa：symptomsのため投与間隔を1週間から2週問に延長した隊員が4名いたが投与を中止し薬剤を変更した者はなかった。今回はONUMOZの推奨に近いメフロキンの予防投与を長期に渡（3／201，284）

り行ったが幸いモザンビークではメフロキン耐性マラリアの発生については1994年8月現在報告はない。耐性獲得を考慮しなければメフロキンの長期予防投与は可能と思われ（2cases）

disorder（1case）

Surveyof

第2次モザンビーク派遣輸送調整中隊員48名を対象にマラリアに対するメフロキン250mg／wの予防投与を約8カ月に渡り行った。全身倦怠感16．7％，頭痛12．5％，嘔気・嘔作用を認めた。血液学的検査では全隊員の白血球が有意に上昇しており，また4名の隊員に軽度ではあるが明らかに肝機能の悪化を認めた。投与開始後4カ月後に副作用増強

travelers：

psychicrate

語

吐4．2％，眩量2．1％，下痢2．1％といったいずれも軽度の副C．D．C．：

hospitalization

mentaleffects（prophylactic）

fromconvulsion

side

結

Spanishtravelers：

た。

mental disturbance（vertigo，headache，euphoria，depres−sion，uneasy，insomnia，unstable emotions）（11cases）
eruption

引用文献（3cases）

5．SurveyofAmericanpeace

incidents

of

minor

effectsside

mefloquin

group

39％

chloroquin

group

50％

keeping1）Minister of Education：ATLAS，17，Esselte Map Ser−vice，Maputo Mozambique，1986．
2）Alberto da Barca：MOZAMBIQUE−Perfil Fisico mapas
e graficos，20，Editora Escolar，Mozambique，1992．

force：

quotedfromreference3）

3）小原博：国際協力と健康管理，熱帯医学，25：31−39，

19113）T．Harinasuta

1992．

4）タンザニアのマラリア大流行地域におけるピレスロイドを浸み込ませた蚊帳の試み，ACTA TROPICA，49，3：165−
171，August1991．
5）栗原 毅：ベクター対策における蚊帳の研究，熱帯医学，
24：205−212，

6）New

York

7）WHO：MENS

Times2／12：熱帯医学，24：266−271，1991．

FOR

CHEMOPROPHYLAXISTRAVELLERS，WHO

wkly

REGI−

malariaeta11；Apatients
in

phase

with

ll

clinicaltrial

chloroquin−resistantofmeflo−

falciparum

Thailand．61（2）：299−305，1983．

14）医薬品・治療研究会，正しい治療と薬の情報，5：33−37，
1990．

FRANCAISE

Ministere

リアに関する指針，熱帯医学，25：223−227，1992．
16）J．L．Bem

etall：Mefloquin

Epidem，Rec、，

spontaneousdes95：167−179，1992．

No．51：377−383，1993．

8）REPUBLIQUE

in15）狩野繁之他：カンボジア国内UNTAC職員のためのマラ

1991．

MALARIAquin

Affairesprophylaxis

reports

ofsever

convulsions，Joumalof

TropicalanpsychiatricMedicine

over

view

of

reactions

and

and

Hygien，

epidemoilogigue17）海老沢功二国際旅行医学会議の話題，熱帯医学，24：223−

hebdomadaire，14：103−104，juin1993．
9）WHO；Wkly Epidem．Rec．No．51：377−383，1993．
10）神田錬蔵：多剤耐性マラリアの治療と予防，熱帯医学，25：

18）菅沼利行他：抗マラリア薬「メフロキン」の副作用の検

sociales：

1−13，

Le

point

sur．．．，bulletinofmefloquin

61：169−178，1983．

asantimalarial

dmg．

，

12）Recommendations for the preventation of malaria
among travelers Morbidity and Mortality Weekly
Report March9，1990Vol．39／No．RR−3（U．S，Depart一
卑ent

of

Health討，防衛衛生，41（2）：49−55，1994．
H Machler，R．，Bruppacher，R．et all；Malaria19）Steffen．R

1992．

11）WHO；Development

231，1991．

and

Human

Services，DCD）

chemoprophylaxis among Europian tourrist in tropical
Africa．Bull world Health Organ．68：313−322，1990．
20）Lobe1，H．0．et

term
265

malaria

all；Effectivenessandprophylaxis

mefroquin．」．A．M．A．

：361−364，1991．

withtolerance

of

long−

192

Original Article

SIDE EFFECTS WITH MEFLOQUlN FOR LONG‑TERM

MALARIA PROPHYLAXISSHIGETO TAKESHIMA

Received August 10, 1994/Accepted October 30, 1994

Key words: malaria/mefloquin/side ef f ects/Mozambique/ONUMOZ (Operation des Nations Unies en

Mozambique)

Forty eight members belonged to Japan Self

comparison to pre‑departure. Liver function of four

Defense Force took part in ONUMOZ (Operation desNations Unisis en Mozambique) as the second Move‑cases deteriorated slightly in comparison to predepar‑
ture, three months after arriving, and post‑return. Twoment Control Company from Nov. 1993 to Jun. 1994. I

each members who suffered general fatigue and head‑ache prolonged the period of taking the drug for one
week to two weeks from 4 months after arriving to 4
weeks after returning because the symptoms had wor‑

belonged to the company and stayed in Mozambiqueduring the period. As malaria is rampant in Mozambi‑que, I used mefloquin 250mg/week as prophylaxis follw‑

ing the recommendations of ONUMOZ, W.H.O. and theMinistry of Health in France. In view of the necessary
concentration of the drug and the incubation period of
Plasmodium falciparum, we took the drug from the day

one week before we left Japan to the day four weeks
after returning. The total tablets each members took
was 36 (except for four who dropped out) . We inves‑tigated the symptomatic and laboratory side effects ofthis prophylactic treatment.

The following symptoms were observed as side.
effects of mefloquin: general fatigue 16.7%, headache
12.5%, nausea & vomiting 4.2%, vertigo 2.1%, diarrhea
2.1% (two cases each of general fatigue and headache

sened. But there were no serious side effects during theprophylactic period.

Over 200 members of ONUMOZ contracted malaria
from Feb. 1994 to Apr. 1994, but I had no malaria
outbreak in the Japanese contingent.

ONUMOZ provided mefloquin to all members forprophylaxis and two out of three field hospital usedmefloquin for malaria treatment as the first choice, but
no malaria resistant to mefloquin had been reported asof the beginning of August 1994.
We conclude that mefloquin is effective for malaria
prophylaxis in Mozambique, and minor side effects areobserved in only 33.3% of subjects in good health.

overlapped) . Mean WBC was evevated post‑return in

Department of Surgery, Sapporo General Hospital Japan Self Defense Force
Department of Surgery, Sapporo General Hospital Japan Self Defense Force 12‑1 ljo Hiragishi Toyohira Sapporo Hokkaido
Tel. O11‑831‑0161 (EXT. 232)

193

Jpn．」．Trop．Med．Hyg．，Vol．22，No．4，1994，pp。193−198

日本における輸血マラリア
血小板輸血により感染したと考えられる
熱帯熱マラリア1症例を中心に

狩野繁之，鈴木守

平成6年9月2日受付／平成6年10月15日受理

はじめにとして入院した。単純X線写真上第3腰椎に圧迫骨折像が認められ，骨粗懸症と診断された。身長152cm，体重45

1993年の日本人年間海外渡航者数は11，933，620人，また外国人の新規入国者数は3，040，719人であり（法務省入国管kg。現症として右上肢手拳大皮下出血，白血球数の減少，血小板数の減少が認められた。血小板減少症の治療のため，

理局統計），その数は年々増加の傾向を示し，それに伴って日本国内における輸入マラリアの症例数も増加してきている。現在年間罹患者数は報告されるだけでも100名を越え（大友ら，1993），マラリアはもはや珍しい病気ではなくなってきている。しかし，日本においてはマラリアを媒介

4月4日，5日，6日，11日，12日，13日に10単位ずつ合計60単位濃厚血小板輸血を行ったが，改善は認められなするハマダラカの密度が非常に低いため，輸入マラリア（imported malaria）が土着性（indigenous）とならないばかりか（大鶴，1958），マラリア患者からハマダラカを媒介して偶然に感染する導入マラリア（introducedmalaria）の危険性もほとんどない（大友ら，1973）。ところが近年の輸血製剤の需要の高さより，全国における献血者数も1991

年には800万人を越え，いわゆる誘発マラリア（induced
malaria）の範疇にある輸血マラリア（transfusedmalaria）の危険性が論じられるようになってきている。日本における輸血マラリアの報告は酒井（1935）が初めてであり，第二次世界大戦前後に集中しているが，その後散発的に報告され，1985年の矢野ら（1985）が最後である。今回われわ
れは，血小板減少症の治療に濃厚血小板を輸血し，それにより感染したと考えられる熱帯熱マラリアを経験した。わが国における輸血マラリアの文献的考察を加えて報告する。

症

例

患者：70歳，日本人女性。千葉県船橋市在住。海外渡航歴なし。麻薬，覚醒剤等の常用歴なし。
既往歴：1984年6月，A型肝炎で某院入院（20日問）。この時初めて血小板減少症（8万／mm3）が認められた。1988
年4月，鼻出血が止まらず入院（1カ月間）。血小板数4．7
万／mm3，軽度の肝肥大，胆石，右腎嚢胞が認められた。1989
年3月，腰痛を訴えて入院（1カ月間）。白血球数（3400／mm3），血小板数（6．6万／mm3）の減少が認められた。1991
年2月，腰痛のため再度入院（1カ月間）。
現病歴および経過：1991年3月30日，腰痛の悪化を主訴
群馬大学医学部寄生虫学教室〒371群馬県前橋市昭和町3−39−22

かった（Figure1）。4月23日，全身状態が良好であったた
め，一時帰宅して入浴をした。
4月24日，突然の悪寒戦傑，発汗を伴う39。Cの発熱を示した。氷枕するも効果なく，ボルタレンサポTM50mgを投与
し一旦下熱をみた。血液の一般細菌培養は陰性の結果を得た。25日，悪寒を伴う37．6。Cの発熱が再度出現し，翌朝ボ
ルタレンサポTM50mgで下熱した。4月26日，27日，28日，29日と全身倦怠感を訴えるも37。C以下の平熱を維持した。

麹易畳：難轡獲論lo纏諜墜発灘窯
眠傾向を謙あた。

5月1日03：00，嘔気の訴えがあった。06：00，再度嘔気，倦怠感を訴えるも朦朧状態。また，患者は尿閉に陥った。1σ：00，顔色不良，皮膚の黄染が認められた。眼球運動が認められなくなったが，呼鳴には反応した。中脳における出血を疑い，脳CTスキャンを行うが異常を認めなかった．血糖値14mg／dl，インスリン値3．3μU／mlを示
し，低血糖による脳機能低下を疑いブドウ糖を投与するも効果が認められなかった。14：00，呼鳴に反応せず，四肢の硬直を認めた。腱毛反射・対光反射は正常であった。18：00，瞳孔の散大を認めた。20：40，顔色悪く，貧血も高度になり，新鮮血3単位を輸血した。22：15，37．1。Cの発熱。瞳孔は縮小したが，顔面に痙攣が認められ，意識は消失に至った。5月2日01：00，37．9℃の発熱。06：00，38．1。

Cに上昇。顔面，胸部，腹部黄疸著明。意識消失状態。徐々に血圧，呼吸数，脈拍数が低下し，07：50，心停止をきた
し，07：55，永眠した（Figure1）。
血液検査所見：経時的な血液細胞学的検査所見，ギムザ
染色した血液塗抹標本の顕微鏡下に観察したマラリア原虫
寄生赤血球数，間接螢光抗体法（IFAT）による血清中のマラリア抗体価（抗熱帯熱マラリア原虫抗体価：P∫titer，
抗三日熱マラリア原虫抗体価二P∂．titer）をFigure1に，入院時，初回発熱時，死亡直前の主な血液生化学的所見を

194

閣arch

Date

3031

Apr翻
1

2

3

4

PlateletTherapy

5

6

C《）ncentrate（10un詮seach）

鵬、4dηワ1ssめρ

μL匿』｝山』㌧2324252

鴨周レ

閉ay

272829Vbltaren Suppo禰（50r㎎each》

l

l

Whole

12

Blood

（30U耐s）

o

助㌃m鴎↓
（℃》39
38
37
36
35

WBC⊂／mm3》
R8C（x104／mm3》

3000
364

2800
364

3500
37亀

3000
368

17300⑳400
223 233

Hb（9／dl》

12，2

12．2

12．6

12．4

7．2

7，6

Ht（％）

36，1

35．9

36．亀

35．6

22．1

22．9

POt（x104／mm3）

5．8

5．8

4．7

4．1

2，7

2．1

ESR

32

50

60

6

lh｛mm》

髄6．ofρ凱／104脱s

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190

ρ凱t脆●r

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1：256

n凱t醗●r

く1＝亀

1＝16

1：4

FigureTablelClinicalcourse1に，そして5月1日の血液薄層塗抹標本像をPhoto．1に示す。

考察

日本における最初の輸血マラリアの報告は，酒井（1935）
が細菌性赤痢後 ヂストロヒー の1歳6カ月の男児と，百日

咳肺炎の1歳1カ月の男児への輸血療法後に誘発した三日熱マラリアの2例を記載したものである。その後1948年までの輸血マラリアの報告一覧として，貴田，中山（1948）
が諸家の症例29例をまとめている。それらの供血者は，マラリア既往を有するもの11例，マラリア流行地に滞在の事
実のある者5例とあり，明らかに輸血マラリアの危険の高い供血者を含んでいる。また，父，母，兄弟からの供血が多いが，6人の職業供血者からいわゆる売血により供給された血液材料が提供されている。第二次世界大戦終戦前後においては，この職業供血者からのマラリア感染の危険性が強く憂慮されていた（山内，長野，1956）。戦後輸血材料
は主に保存血となり，一回の輸血に複数の供血者の血液が投与されるため，マラリアの感染の危険性も高くなった。
この保存血輸血により誘発されたマラリアの本邦における第1症例が高田（1955）により報告された。保存血中のマ
ラリア原虫の生存期間に関しては，金森（1944）が7日，村瀬ら（1956）が6〜18日，加藤ら（1956）が4〜6℃で14日と記載している。また1960年当時の厚生省基準では4〜6。Cで96時間保存後に輸血材料として使用するとある

of

the

patient．

が，増田（1960）はこの時間以後における血液材料からのマラリアの感染性を実験的に証明している。現在血小板製剤は，分離後常温で保存され72時間以内に使用されることになっており，実際にはもっと早急に半日ほどで輸血に供されることが多い。万一輸血材料にマラリア原虫感染赤血球が含まれていれば，原虫の死滅する可能性は低いと考えられる。実際にわれわれの日常業務で，熱帯熱マラリア患者から採取した血液を12〜24時間室温に置いた後でも，それを培養系に移し原虫を増殖させている。戦後の輸血マラリアの報告例は，1956年までの22例を伊藤ら（1985）が一覧にまとめているが，戦前の報告例を合わせて，日本における輸血マラリアの総数を57例，うち熱帯熱1例，三日熱
41例，卵形1例，不明14例と記載している。その他にわれわれが文献上で調べた限りにおいては，熱帯熱1例（飛田，1952），三日熱11例（神田，1955：日野，1955；阿部，花輪，1956；若林，上原，1956；古寺，1956；宮本，1952；
矢野ら，1985），四日熱1例（花田ら，1966），種不明4例（大鶴，加茂，1954；土屋，1957）の報告がある。即ち，今般報告する症例は日本における輸血マラリアの第75例目，熱帯熱マラリアとしては第3例目，そして初めての死亡例と考えられる。その他に輸血マラリアとは異なるが，誘発
マラリアの症例としては，「覚醒剤常用者の間に特発した不潔な注射器による人工接種例」9例を大鶴，加茂（1953）
が詳細に報告し，森下（1959）が文献上346例（1945〜1958
年）をまとめている。また近年になって天野ら（1976）は針事故によって感染したと最も考えられる熱帯熱マラリア

195

Table

LAP（IU）
ChE（1U）
UN（mg／dJ）
Creatlnin（mg／dJ）

Na（mEq／」）K（mEq／1）α（mEq／」）Ca（mg／d♂）B−Glu（mg／dJ）

T−cho（mg／dJ）
TG（mg／｛萱」）

CRP

2

5

5

γ一GTP（IU）

ハ∪50肖D

ALP（K−U）

0∩U

GOT（IU）
GPT（IU）
LDH（IU）

Apri125、May2

02・
92
・！・
8
26
14
・138
・
55
8・
7
9
・
71
4
5
・7
4
・
00
i
・ 18416963516
4
2
7
1
1
5
731
1
3461
9

T−Bil（mg／dJ）
D−Bil（m露／dJ）

Apr量12

自﹂TTT（U）ZTT（U〉

われわれの今回報告する症例が不幸な転帰をたどったのは，医師をしてマラリアを疑わしめるにはあまりに非定型的な例であったためであると考えられる。現在マラリアの流行のない日本において，麻薬や覚醒剤の常用歴がなく，海外渡航歴もない70歳のいわゆる普通の老女に，その発熱をみてマラリアを第一に疑うことは医師においては困難と言うよりむしろ突飛でさえある。また，マラリアの典型的な症状を示してからの進行も著しく早かった。その理由としてはまず，本患者が従来より重症マラリアに陥った際の一つの指標でさえある血小板減少症に悩まされていたこと

find重ngs

QU
つ0
15要
5
・
2
4
3
・
2
・
3
8
3
2
1
9
0
甲0
曲8
15
75
51
43
！7
92
32
91
・1
33
・／
08
・8
21
41
3
7噛
38曲
231

ALB（g／d1）

Laboratory

047
7
1
7
8
5
6
2
・・
2
・2
・
95
3・
7
53
97
・
・5
3
・2
0
・3
74
7
3
8
2
0
0
6
7
5
1
3
71701318918

Date
TP（9／dJ）lがあろう。死亡時は，出血性症状，FDP値40μg／mJ，plt2．1

万／mm3，PT25，2秒を示し，播種性血管内凝固症候群の基準を充分満たしていた．また，白血球の減少症も認められており，免疫学的にも何らかの不全があったことが疑われ，発熱の程度が感染初期に著しく高く示されなかったのも単なる高齢によるためだけではなかったと考えられる。さらには，慢性肝炎の素因による肝不全への移行の早さが，血清学的データや，・黄疸などの症状より推察される。血溝学的データからはさらに，高度の腎機能障害もうかがわれる（Table1）。死亡直前に至っては，傾眠傾向，眼球運動障
害，意識消失などの脳症状を合併した脳マラリアに陥ったと診断できる。本患者はマラリアによる急速な多臓器不全により死亡したと考えるのが最も妥当であると考えられる。
しかるに，マテリアの治療は本患者には施されなかった。
5月1日に検査室が血液像検査のために作製した薄層塗抹
標本中にマラリア原虫を検出するのが，翌早朝7：55の死
亡時より遅れたためである。原虫種の確定診断のために群馬大学医学部寄生虫学教室に標本が送付され，本患者は熱
帯熱マラリアに感染していたことが判明した（％oto、1）。
さらにわれわれの問い合わせの結果，4月2日，17日，25

4

症例（21歳，看護婦）を報告し，医療従事者におけるマラリアの二次感染の危険性を強く提起している。なおこの看護婦の例は，治療の甲斐なく脳性マラリアを合併し死亡している。

．＿．灘縫難Photo．！Glemsa−st段inedan（iag＆metocyte

blood

smear

of

the

pat圭ent

showingof。P西猷S郷04診寓挽血あ寧）腐グ銘窺．

ring

forms，

196

日の標本が保存されていることがわかり，それらを注意深く検鏡したところ，4月2日，17日の標本には原虫が確認されなかったが，4月25日の標本には赤血球1万個に対して6個という低い感染率の熱帯熱マラリア原虫が検出された（Figure1）。その標本中の原虫は形態的に定型的でなく，また感染率も低いことより，一般病院の検査室におけるスクリーニング作業では診断が極めて困難であり，この標本からの虫体の検出は一般検査技師・医師に要求される能力を越えると考えられた。また，群馬大学医学部寄生虫
学教室において後日行ったIFATによるマラリア抗体検

査により，本患者の5月1日，2日の血清はP∫titerが1：256と高値を示し，血清学的にも熱帯熱マラリアとの診断が下された（Kanoθ≠認，1990）。また4月25日の血清は，P∫titer，P鉱titerともに陰性であった。一般に原虫血症を示してから血清中の抗体価が上昇するまでには数日かかり（Voller，Draper，1982），4月25日は感染のごく初期であったと思料される。以上標本観察とIFATとの所見により，本患者のマラリア感染の経過は，4月4日以来一連にわたって行われた濃厚血小板の輸血によりマラリア原虫が本患者に接種された結果と考えられた。
当該血小板製剤の供給のあった血液センターの協力を得て，カルテに記載された輸血Lot番号の供血者データを調べたところ，供血者は述べ40人（男30人，女10人。日本人
39人，外国人1人。年齢22〜64歳）であることが判明した。
しかしながら個別の感染源調査は行わなかった。血液セン
ターでの献血時には，献血申込書（診療録）に付随して間診表が供血者に手渡され，供血者の現症，既往歴が尋ねられる。肝炎，結核などの感染症，糖尿病，腎臓病などの代
謝疾患，出血傾向に関する血液疾患，さらに心臓疾患の既往に関して聞かれるが，残念なことにマラリアを個別に挙げて尋ねる項目が無い。しかし献血時の医師の問診において，マラリア既往者でその後3年以上経過していない人か

らは採血しないこと，またWHOが指定している流行地からの帰国者については，行き先，生活状態，期間などを詳細に尋ね，感染が疑わしい人からも3年間は採血しないことを日赤の血液センター業務基準としている。マラリア流
行地に旅行する日本人や，流行地から訪日する外国人の数も増えてきている現在，今後医師の問診を強化し，さらに質問表にそれを尋ねる項目を加え，マラリアの二次感染の危険性に備えてゆく必要があるものと考えられる。日赤では輸血用製剤の安全性の確保のため，血液による感染の危
険が高いB型肝炎，C型肝炎，エイズ，成人丁細胞白血病，
梅毒の5つの感染症について検査を実施している。献血申込書には「エイズ検査目的の献血はお断り致します。」と記してあるが，現在においては前述のような金銭目的で供血を行う者こそなくなったものの，熱帯地への渡航帰りに，
いわゆる上記の性行為感染症を調べたい目的で供血を行う者も少なからずあるとされている。その中には々ラリア既

往者，さらには感染ごく初期の患者も含まれてくる可能性
が一般の善意による献血者に比べ高いであろうことが予測され，このような状況にあっては問診票にマラリアに関する質問項目を用意して，供血者全員に対してその質問がなされてしかるべきであろう。さらに供血前1年くらいの間にマラリア流行地への渡航経験があり，なおかつ発熱の既往がある供血者には，血液塗抹標本観察による原虫検出作業を検査に取り入れる必要があろう。また前述のIFATによるマラリアの抗体検査も，マラリアの既往を検査するための確定的な診断法となる（Kano6！磁，1990）。
血小板輸血によって誘発されたマラリアの記載は，Cen−
ter for Disease Contro1（CDC），US Department of
Health，Education and Welfare，Public Health Service（1978）の年次報告に，急性白血病の女性に血小板製剤の投与を行って感染したと考えられる熱帯熱マラリア症例がある。また本邦の伊藤ら（1985）の症例では，濃厚赤血球と同時に血小板製剤の輸血を行っており，その可能性が著者
らによって論じられている。Fajardo，Tallent（1974）は三日熱マラリア患者の血液中の血小板にマラリア原虫が能動的に進入し，そこで生存していると考えられる像を電子顕微鏡で観察しており，血小板自体によってもマラリアの誘発が起こる可能性を強く示唆している（Perkashε！磁，
1984）。また，血小板製剤に混入した原虫感染赤血球によってマラリアが誘発される可能性も否定できない。濃厚血小板製剤への赤血球の混入率は上限2万個／mlとの基準があるが，本患者に投与された血液製剤の供給元である血液センタニにおいては，その数は1000個／ml以下程度との結果を得ている。成分輸血の血小板製剤1単位20mlで，マラ
リア原虫寄生赤血球率が1万個の赤血球に1個程度であるとすれば，一人の供血者からの血液材料中には2個の感染
赤血球が混入する可能性があることになる。よって顕微鏡観察によって原虫血症が検出できるマラリア患者からの血液供給であれば，たとえ血小板製剤であっても混入した赤
血球によってマラリアが誘発されうる。本症例は血小板輸血で誘発されたと考えられるマラリアの日本国内第一例であるが，上記のいずれの機作によって感染が成立したかは断言できない。

大鶴（1958）は，わが国において熱帯熱マラリアの国内感染が終戦直後にあっても非常に少なかった理由について，
日本国内に分布する最も有力なマラリア伝搬者はシナハマ
ダラカであって，主に三日熱マラリア伝搬性が強いこと，
そして熱帯熱マラリアの伝搬に関与するであろうオオツル
ハマダラカの密度は非常に低いことをあげている。本患者は千葉県船橋市に在住していたが，居住地付近は一般住宅地であって，蚊の密度を上げる可能性のある家畜等を飼育する農家も見あたらない。また成田国際空港からの直線距

離も40kmあり，万一マラリア感染蚊が外国から運び込まれたとしても，その飛翔距離内には入っていないと考えられ，本患者が蚊によって媒介されて自然感染を起こしたマ
ラリアであった可能性は無いものと考えられる。しかしながら国内において土着マラリアは消退し，導入マラリアの可能性もほとんど無いとは言え，大鶴（1952）が終戦直後に「何時の日か大陸，南方方面に交を求めることが出来るようになれば，必ずやマラリアの問題は日にそう影のよう197

につきまとうことになるであろう」と予言したとおり，現在においては輸入マラリアがその存在を無視できないほど数を増やしており，それに伴ってもたらされるであろうあ
らゆる危険性に対して，医療従事者においては特に注意を払わなければならない。本報告ではその輸入マラリア患者
から血小板の供血を受けて感染したと考えられ，しかも不幸な転機をとった熱帯熱マラリアの症例を紹介したが，最後に輸血マラリア国内初報告において酒井（1935）が記載
し，現在においても繰り返し強調されるべき文章を引用し，本報告を締めくくる。「輪血時給血者の撰揮に當って，既往症にマラリア の有無を確かめる必要がある。尚給血者の健康状態を吟味する事も大切である。」

10）金森成起（1944）：保存血液中二於ケルマラリア原虫ノ運命
二関スル研究，第3編，保存血液中二於ケル三日熱マラリ
ア原虫ノ感染能力，熊本医学会雑誌，926−930
11）Kano，S．，Waki，S．，Igarashi，1．，Nakazawa，S．，Masuda，

G．andSuzuki，M．（1990）：Retrospectivemalaria

diagno−sis

Japanesebyindirectfluorescentantibody

titrationonpatients，Jpn．J．ParasitoL，39，475−481

12）加藤貞三郎，加藤敏昌，吉富久吉（1956）：保存血に依るマ
ラリア感染，臨床外科，11，1003−100413）神田昭典（1955）：保存血（血液銀行）輸血により発生せる

マラリアの1症例，内科学会誌，内科学会地方会，971
14）増田昭一（1960）：保存血内におけるマラリア原虫の感染性に関する研究，新潟医学会雑誌，74，180−197
15）宮本嘉七郎（1952）：輸血によるマラリヤ感染の1例，日本

外科学会雑誌，53，191

結語

血小板減少症の治療のために濃厚血小板を60単位投与され，それによって熱帯熱マラリアを誘発し，脳マラリアおよび多臓器不全を合併し死亡した70歳の日本人女性の症例を報告した。わが国における輸血マラリアの文献的考察を含め，今後の血液製剤の安全性確保に関しての検討を行っ
た。

謝

辞

本論文作成にあたり，横浜市立大学医学部寄生虫学教室
天野皓昭博士に助言をいただいたことを付記する。
文

献Tallent，C．（1974）：Malarial

ラリア発生状況について，寄生虫学雑誌，42，137
19）大友弘士，小山 力，小早川隆敏，塩之入洋（1973）：三日
熱マラリアの国内感染例，寄生虫学雑誌，22，37
20）大鶴正満（1952）：戦後マラリアの流行学的研究，日医新報，1470，33−37
21）大鶴正満（1958）：わが国において第二次世界大戦による輸入マラリアが土着性とならなかった理由について，医学の動向，22，107−137
22）大鶴正満，加茂 甫（1954）：覚醒剤常用者の間に発生したマラリアについて，日医新報，1555，31−34
23）Perkash，A．，Kelly，N．1．and

1）阿部 換，花輪松夫（1956）：保存血輸血によるマラリア発
生例，治療，38，762−764
2）天野博之，山本利雄，左野 明，高橋泰生，蔵田駿一郎，
市島国雄（1976）：国内二次感染と思われる脳性熱帯熱マラ
リアの一剖検例，日熱医会誌，4，195−205
3）CDC（1978）：Malariasurveillance：Annual Summaryfor
1977，78−8259
4）Fajardo，L．F．and

16）森下 薫（1956）：日本における残留マラリア問題，日医新報，1845，10−1617）村瀬 温，白戸四郎，鈴木 和（1956）：保存血輸血により感染したマラリアの二症例，血液と輸血，3，56−57
18）大友弘士，竹内 勤，増田剛太，赤尾 満，高田季久，荒木恒治，辻 守康，田中 寛（1933）：過去5年間の国内マ

Parasites

within human platelets，JAMA，229，1205−1207
5）古寺秀喜（1956）：保存血輸血により感染したと思われる三日熱マラリア，日医新報，1704，24−25
6）花田十衛，砂部孝和，森下正一郎（1966）：注意すべき輸血
マラリアの経験，日医新報，2208，53−54
7）飛田慎一（1952）：輸血によるマラリヤ感染，日大医学雑誌，27，135−136
8）日野志郎（1955）：保存血輸血により感染した3日熱マラリ
アの1例，内科学会誌，内科学会地方会，971
9）伊藤 章，原野 浩，天野皓昭（1985）：輸血により感染したと思われる卵形マラリア，感染症，15，33−36

ced

parasitizationoffajardo，L．F．（1984）：Enhan−plateletsby

PZ偽

zo4づz6砂z

68堰h切

夕oε1彪Trans．Roy．Soc．Trop．Med．Hyg．，78，451−45524）酒井 潔（1935）：輸血時の注意補遺，児科診療，1，620−623

25）貴田丈夫，中山四郎（1948）：血液注射によるマラリアの1例並びに同症例の文献概説，最新医学，3，498−500
26）高田 蒔（1955）：保存血液の輸血によって感染したマラリ
アの一症例，血液と輸血，2，21−24
27）土屋義貞，板倉 登，山崎栄一（1957）：血液注射によるマラリヤ感染例，児科診療，15，69
28）Voller，A．and Draper，C．C（1982）：Immunodiagnosis
andseroepidemiology

ofmalaria，Br．Med．Bull．，38，173−17729）若林利重，上原武久（1956）：保存血輸血によるマラリア感
染例，日医新報，1695，28−30
30）山内 博，長野武正（1956）：保存血によるマラリア感染
例，臨床外科，11，1001−1002
31）矢野健一，中林敏夫，渡辺知明，藤本輝夫，阪本俊一（1985）：

輸血による三日熱マラリアの1例，日熱医会誌，13，301−306

198

A CASE OF ACCIDENTAL TRANSMISSION OF
PLA SMODI UM FALCIPARUM THROUGH PLATELETTRANSFUSIONSHIGEYUKI KANO AND MAMORU SUZUKI
Received Septernber 2, 1994/Accepted October 15, 1994

[Case] The patient was a Japanese female, 70 yearsof age, Iiving in Funabashi, Chiba, and had never beenplatelet transfusion has been discussed by Fajardo andTallent (1974) , who showed the existence of the parasiteabroad. The woman was suffering from throm‑

in a platelet of a P. v. infected patient by electron

bocyiopenia and received a total of 60 units of plateletconcentrate during the period, 4‑13 April, 1991. On April

microscopy. Nevertheless, since platelet packs may

24, she manifested a fever of 39'C which was success‑fully treated with Voltaren Suppo , a non‑steroidal

by erythrocytes, platelets or both. Still, this report is thecontain some red blood cells, parasites could be carried

oped symptoms of cerebral malaria, Iiver and renal

first case of induced malaria resulting from platelettransfusion, and the 75th case of transfused malaria inJapan since 1935.
As the nurnber of Japanese who went abroad and of

dysfunctions. In the early morning of May 2, she lostforeigners who entered Japan increased, so did theconsciousness and went into cardiac arrest. Giemsa

number of imported malaria cases, which were reported

stained thin blood smears obtained for blood cytologicalto be not less than 100 in 1993. Therefore malaria is nolonger recognized as a very rare disease in Japan, andanti‑inflammatory drug.

Her body temperatureremained normal until May 1, when she suddenly devel‑

examination which were kept at the laboratory of thehospital, Iater revealed that she had contracted Plas ‑modium falciparum malaria. The parasitemia fromexamination of the slides were O% on April 2, O% on
April 17, 0.06% on April 25, 5.5% on May I and 1.9% justbefore her death on May 2. Antibody titers against P.f
antigen were < 1:4 on April 25, 1:256 on May I and May
2. These parasitological and serological results con‑firmed that she accidentally received transmission of P.
f through platelet transfusion.
The possibility of transmission of malaria throughthe risk of transfused malaria from donors who havecome back from malaria endemic places has to be taken
into careful consideration. To date, routine examina‑
tions on blood supplied at Blood Centers are for hepati‑
tis B and C, syphilis, adult T cell leukemia, and AIDS,
but not for malaria. The questionnaire which is used in
obtaining the medical history of donors rarely includes
inquiries on a past history of malaria. Special attention

has to be made for the safety of blood transfusion,
reminding the danger of malaria transmission.

Department of Parasrtology, Gunma University School of Medicine, Maebashi, Gunma, 371 Japan

199

Jpn. J. Trop. Med. Hyg., Vol. 22, No 4 1994, pp. 199‑206

COMPARISON OF POPULATION OF VECTORMOSQUITOES OF DIROFILARIA IMMITIS

AND THEIR NATURAL INFECTION RATESIN SOUTHERN AND NORTHERN PARTS
OF NAGASAKI CITY, JAPANTSUTOMU ODA1, OSAMU SUENAGA2, MAKOTO ZAITSU3
KENJI KUROKAWA', KOICHIRO FUJITA5, YASUNORI OGAWA6ICHIRO YAMAZAK16, KUNIHIRO IIDA6 AND MARIKO MlNE7
Received September 1, 1994/Accepted October 3, 1994

Abstract: Female mosquitoes including Culex pipiens pallens, the main vector of Dirofilaria immitis, andsome secondary vectors, were collected at 4 to 10 sites in Nagasaki City from 1983 to 1989 using light traps,
and the number of the mosquitoes and the natural infection rate with D. immitis were compared between thesouthern and the northern parts of the city. In 1983, Cx. p. pallens was extremely prevalent in the southernpart but not in the northern part. After 1986, the prevalence of Cx. p. pallens became very low in both theparts. The rapid decrease of prevalence of this species may be attributable to the decrease in breeding sites
by improvement of roads and open roadside ditches in parallel with spread of sewage systems. On the basisof the number of infected mosquitoes in Nagasaki City, it was suggested that, Cx. tritaeniorhynchus and Ae.
albopictus are important vectors in addition to Cx. p. pallens, but Ae. togoi does not play a significant rolein the transmission of D. immitis.

INTRODUCTION
We previously reported that the percentage ofhouse dogs having the larvae of Dirofilaria immitis
increased from 1968 to 1983 in the eastern, the westernand the southern parts of Nagasaki City, but this per‑centage decreased during the same period in the north‑ern part of the city (Oda et al., 1993) . We also assumed
that the decrease of the infection rate of house dogs in
the northern part be due to the spread of public sewage
systems contributing to the reduction of breeding sitesof the main vector mosquito, Culex pipiens pallens. To
test the validity of this assumption, we compared thenumber and the natural infection rate of Cx. p. pallens,caught by using light traps, between the southern andthe northern parts of this city during the period from1983 to 1989, and analyzed the data in relation to thedevelopment of public sewage systems. Furthermore,
we attempted to clarify the role of some secondary
vector mosquitoes in the transmission by comparing the
population densities and the natural infection rates.

PLACES AND METHODS
Between 1983 and 1989, we collected mosquitoes
including Cx. p. pallens, which is the main vector mosquito, and three secondary vectors ( Cx. tritaenio‑
rhynchus. Ae. albopictus and Ae. togoi) , using light traps(20 watt black light) , in the southern part of Nagasaki
City where the microfilaria positive rate was high and in

the northern part where the positive rate was low. In
1983, we placed a trap in each of two southern districts*Department of General Education, the School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852, Japan

2Reference Center, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
3Sasebo City Health Center, 1‑10 Yahata‑machi, Sasebo 857, Japan
4Department of Bacteriology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
5Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University, 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo 113, Japan

6Nagasaki City Health Center, 2‑34 Morimachi, Nagasaki 852, Japan
7Scientific Data Center of Atomic Bomb Disaster, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan

200

Table 1 Period and place of mosquito collection in Nagasaki City
collection

Trap, No.

1983

S1 S2

District No. and name
28 27

Jul. 28‑0ct. 21

Fukahori

Place of collection
Building Building

Doinokubi

(13)

1986
Aug. 18‑Sep. 27

Northern part

Southern part

Date and (times) of

Tra p. No.

N1
N2

District No, and name39 34

Nishiurakami

Place of collection
Building Building

Sakamoto

S3 S4

5 25

Tornachi Tomachi

Building Building

N N

3 4

35 35

Takao Takao

Building Building

S5

36

Yamazato

H ouse34 34 34

Sakamoto Sakamoto Sakamoto

House House H ouse

34

Sakamoto

House

(24)

1987
Jul. 1‑Sep. 1
(24)

25

TomachiHouse

N5

S6 S7 S825 25 25

Tomachi Tomachi Tomachi

House House House

N N N

s9

25

Tornachi

House

N9

6 7 8

1988
Jun. 10‑Aug. 11
(27)

1989
Jun. 5‑Aug. 10(20)

S and N show southern and northern parts, respectively.

(Doinokubi and Fukahori) and two northern districts(Nishiurakami and Sakamoto). These four light traps

parts of the city. These light traps were lit three or four

were, as a rule, Iit for operation twice weekly. In 1986,

periods. Fig. I shows the locations where light traps
were placed. The names and lot numbers for Nagasaki

we placed 2 traps in a southern district (Tomachi) and2 in a northern district (Takao), and collected mos‑quitoes three or four times weekly. In 1987 through1989, we placed a trap in 5 houses which had microfilaria‑positive dogs in each of the southern and the northern

N[SH[SONOKI GUN ./'¥‑ '‑'¥ ¥ '/(' '¥.̲ J ¥̲ // / '¥̲ ̲' .

'NI .N2 *N3 Nd /'parts used by Suenaga et al. (1971). The mosquitoes
collected were identified and kept at ‑ 12'C. Under astereomicroscope, female mosquitoes were dissected inphysiological saline and examined for larvae of D.

immitis. The developmental stages of larvae were
decided using the classification of Suenaga (1972) .

I . Population and natural infection rate of the main
vector mosquito, Cx. p. pallens, in the southern andthe northern parts

w5

34 N6
N8
h,9

CHIJIWA BAY
s5

25 s6

‑NACASAK [*BAY s8
/////
.

‑s7

s9

1 . Number of Cx. p. pallens
Table 2 shows the monthly numbers of female Cx. p.
pallens in the southern and the northern parts in 1983. In

two southern districts with considerably high microfilar‑

ia positive rate in dogs where Trap S1 and S2 were27./' 28 sl s2 s3 s4 N

f

N[SHISONOK[ GUN / :.

Figure I A map* showing district No. and place of lighttraps for collection of vector mosquitoes in sourth‑ern and northern parts of Nagasaki City (seeTable I for district No. and trap No.).
*Cited from Suenaga et al., 1971.

City districts were derived from the residence list of the

RESULTS

36 35' .*.' ‑ ¥times weekly. Table I shows the survey sites andoperated, Iarger numbers of mosquitoes were caught inJuly through October, compared with two northern
districts with low positive rate where Trap Nl and N2

were operated. A comparison of the mean numbers
revealed that the number of Cx. p. pallens caught in thesouthern part was about three times larger than that in
the northern part.
Table 3 shows the data obtained in 1986 in Tomachi(a southern district with high microfilaria positive rate)and in Takao (a northern district with a little high201

Table 2(times) of

Jul. (2)Aug. (5)Sep. (4)Oct. (2)Total (13)partin theNo. of females collected in northernNo, of females collected in theindicated trap* in southern partMonth andcollection

Number of Culex pipiens pallens fernales collected by a light trap
in southern and northern parts in Nagasaki City, 1983

S 1*
(Mean)
No.

No. (Mean)

No.

(Mean* * )12( 3.0 )

11

( 5.5 )

67

( 5.15)45 ( 3 46)( 3.5 )

41 8

( 8.2 )
( 2.0 )

( 7.5 )
( 1.5 )

75 12 13

No.
2 4

2 2

( 3.25)( 4.31)

112

N 2*

N 1*

Total

S 2*

5 ( 2.5 )
34 ( 6.8 )4 ( 1.0 )2 ( 1.0 )7

indicated trap*

10(Mean)( 1.0 )
( 0.8 )

No.

No. (Mean * * )5( 2.5 )
( 2.4 )
( 1.75)( 1.5 )

7 ( 1. 75)
16 ( 1.
9 ( 1.
5 ( 1. 25)( 2.08)37 ( 1. 43)

12 3( 0.5 )
( 1.0 )
( 0.77)

Total(Mean)

27

6) 13)* Trap No. is shown in Table l.
**Mean number of females indicates mean per trap per night.

Table 3 Number of Culex pipiens pallens females collected by a light trap
in southern and northern parts in Nagasaki City, 1986

Month and(times) ofcollection

No. of females collected in theindicated trap* in northern partNo. of females collected in theindicated trap* in southern part
S 3*

S 4*

No. (Mean)

No. (Mean)

Aug. (2)Sep. (4)1 ( 0.5 )2 ( 0.5 )o ( 0.0 )o ( 0.0 )Total (6)

3 (05)o ( 0.0 )

N 4*

Total

No. (Mean**) No. (Mean)
1 ( 0.25)
O ( 0.0 )1 ( 0.25)
2 ( 0.25)

No. (Mean)
O ( 0.0 )1 ( 0.25)

No. (Mean**)o ( 0.0 )2 ( 0.25)

1 ( O 17)1 ( 0.17)

2 ( 0.17)

Total

3 ( o 25)

N 3*

* Trap No. is shown in Table 1.
**Mean number of females indicates mean per trap per night.

microfilaria positive rate) . In contrast to the data inin each of three districts (Tomachi in the southern part,

1983, only a few Cx. p. pallens were caught in the both
districts in 1986 (Table 3). Then we placed a light trap

and Sakamoto and Yamazato in the northern part) , andexamined the number of Cx. p. pallens caught during thethree‑year period from 1987 to 1989. As shown in Tablein 5 households keeping dogs positive for microfilariae

Table 4 Number of Culex pipiens pallens fernales collected by a light trap
in southern and northern parts in Nagasaki City, 1987‑1989
No. of females collected in the indicated trap* in southern part

Year and(time) ofcollection

1987 (24)1988 (27)1989 (20)

S 5*

S 6*

S 7*

S 8*

s 9*

Total

No. (Mean)

No. (Mean)

No. (Mean)

No. (Mean)

No. (Mean)o (oo)

5 ( O 21)
3 ( O 11)1 ( 0.05)

o (oo)

2 ( 0.08)

1 ( 0.04)

4 ( O 17)o ( 0.0 )2 ( 0.1 )

No. (Mean**)
11 ( 0.09)
7 ( o 05)
7 ( o 07)

3 ( O 11)1 ( 0.05)

2 (O1)o (oo)
1 ( 0.05)

No, of females collected in the indicated trap* in northern part

Year and(time) ofcollection

1987 (24)1988 (27)1989 (20)

N 5*

N 6*

N 7*

N 8*

N 9*

Total

No. (Mean)

No. (Mean)

No. (Mean)

No. (Mean)

No. (Mean)o (oo)

6 ( o 25)
2 ( 0.07)

12 ( 0.5 )5 ( O 19)
3 ( O 15)

7 ( 0.29)
3 ( O 11)1 ( 0.05)

1 ( 0.04)
o ( 0.0 )No. (Mean**)
26 ( 0.22)
10 ( 0.07)
10 ( O 10)o ( 0.0 )o ( 0.0 )2 (O1)* Trap No. is shown in Table 1.
* *Mean number of females indicates mean per trap per night.

4 (02)

202

Table 5‑1 Anntial changes in rate of population using a sewage system insouthern and northern parts in Nagasaki City, 1983‑1986
Year

District

1983

Part

Population

population

using SS*

(A)

(B)

20 Sako

6 , 934

4 , 80269 . 321 Nita

5, 144

1,410

22 Kitaohura

12 , 548

23 Minamiohura

1986

l 985

RPSS**

Total

Population

population

using SS*

(A)

(B)

6 , 781

5 , 035

74 . 3

27.4

5,022

1 , 671

1 , 379

11.0

12,410

6,999

o

0,0

24 Naminohira

3,531

o

25 Tomachi

15 , 496

26 Kogakura
27 Doinokubi

28 Fukahori

29 Minami

N orthern

RPSS**Total

No. Name

Southem

1984

RPSS*'

Total

Population

population

using SS*

(A)

(B)

6 , 877

5 , 705

83 . o33 . 35 , 052

2 , 552l , 50312 . 1

12 , 185

6 , 848

o

0,0

0,0

3,415

o

o

0,0

15 , 408

5,715

o

0,0

15,034o

0.0

11 , 275

o

0,0

993

o

RPSS**

Total

Population

po pulation

using SS*

(A)

(B)

6,912

5 , 900

85 . 450 . 5

5 , 138

3,218

62 . 6

1 , 751

14.4

11 , 927

2 , 059

17.3

6 , 698o0,0

6 , 598

o

0.0

0.0

3,318

o

0.0

3 , 122

o

0.0

o

0.0

15 , 459

4,719

30 . 5

15 , 490

5 , 19033 . 5

5 , 609o

0,0

6 , 167

4 , 805

77 . 96 , 869

5 , 335

77.7

15,289o

0.0

15 , 382

o0,015,329o

0.0

11 , 452o0,0ll , 241o0.0

lO , 920

o

0.0

0.0

988

o

0.0

986

o

0.0

990

o

0,0

7,515

1,410

18 . 8

7 , 615

1 , 40218.4

7 , 787

1 , 330

17.1

3,4(B/A x 100)

(B/A x 100)

(B/A X 100)

(B/A x 100)

30 Mogi
31 Hayasaka

7 , 504

1 , 490

19.9

5 , 846o0,0

5 , 832o

0.0

5 , 908

o

0.0

5,821

l 99

32 Hiyoshi

l , 615o

0.0

1 , 612o0,0l , 575o0.0

1 , 565

o

0,0

Total

98 , 634

9 , 0819.2

98 , 181

9,619

9.8

98 , 463

20 , 934

21.3

98 , 468

23 , 231

23.6

33 Zenza

6 , 449

4 , 869

75 . 5

6,413

4 , 932

76 . 96 , 236

5 , 035

80 . 7

6, 1945 , 24284 . 634 Sakamoto

10 , 874

6,621

60 . 910 , 848

6,616

61 . Ol O , 724

6 , 767

63 . 1lO , 737

7,271

67.7

35 Takao
36 Yamazato

18 , 620o0,018,984o

0.0

18 , 860o0.0

18,694o

0.0

1 1 , 754

l , 820

15.5

11 , 438

1 , 752

15.3

ll , 531

1 , 799

15.6

11 , 650

l , 748

15.0

37 Nishishiroyama

13 , 352o0,013 , 665o0.0

13 , 982o0.0

14 , 126o

0.0

38 Shiroyama

6 , 153o

0.0

6 , 132o0,06 , OO1o

0.0

6 , 030o0.0

39 Nishiurakami

23 , 790

3 , 063

12.9

19 , 744995

5.0

19 , 649964

4.9

19 , 590

1 , 064

5.4

40 Nameshi

39 , 120

38 , 622

98 . 7

39 , 336

38 , 584

98 . 1

39 , 663

40 , 474l02 . O

39 , 758

40 , 974

l03 . 1

41 Nishikita

12 , 179

404

3.3

12 , 108564

4.7

12,22255 1

4.5

12 , 386551

4.4

42 Nishimachi

17 , 228o

0.0

16,973o

0.0

16,841o

0.0

16,878o0,043 Kawabira

6 , 926o0,0

4 , 478o0,0

4 , 401o

0.0

4 , 392o0,0

166 , 445

55 , 399

33 . 3160 , 11953 , 443

33 . 4160 , 110

55 , 590

34 . 7

160 , 435

56 , 850

35 . 4Total* SS: sewage system$*RPSS: the rate of population utilizing a sewage system.

The sharp decrease in number of Cx. p. pallens inthe southern and the northern parts of Nagasaki City

low in 1983 (9.2%) and 1984 (9.8%). It was doubled in1985 (21.3%) and further rose to 37.0% in 1989. In thenorthern part, sewage systems were already present in1983 and 1984 in 6 of the 11 districts. The RPSS was
about 33.0% in 1983 and 1984, which was much higher
than the rate for the southern part. It is therefore
evident that sewage systems had been better developed

during the 1986‑1989 period seems to be associated within the northern part than in the southern part of this city

the improvement of public sewage systems. Tables 5‑1
population utilizing sewage systems (RPSS) in the totalaround 1983.
Fig. 2 shows the relationship between the RPSS andthe number of Cx. p. pallens caught in individual dis‑population of the southern and the northern parts. The
RPSS values were not so high in Nagasaki City as thosetricts. The values of RPSS used in this figure were
derived from Tables 5‑1 and 5‑2. The RPSS value in4, the number of Cx. p. pallens caught was again verysmall in each district.

2 . Relationship between Cx. p. pallens population anddevelopment of sewage system

and 5‑2 show annual changes in the rate of humanin other cities in Japan, owing to a characteristic topog‑

1983 for the southern part is the average for Fukahori

raphy with a lot of steep slopes, which made difficult toand Doinokubi, and the value for the northern part is theestablish the sewage system. In 1983 and 1984, only 4 ofaverage for Nishiurakami and Sakamoto. Because

the 13 southern districts had sewage systems. After
1985, sewage systems were found in about half of all

Takao, Sakamoto and Yamazato were located close toeach other, the RPSS values in 1984 through 1989 forsouthern districts. The RPSS for the southern part wasthese three districts in the northern part were averaged.

203

Table 5‑2 Annual changes in rate of population using a sewage system
in southern and northern parts in Nagasaki City, 1987‑1989
Year
District

Population

population

using SS*

(A)

(B)

6 , 352

5 , 940

93 . 58.2

5 , 0714,313

85 . 1

2 , 809

24 . 7

11 , 137

2 , 848

25 . 6

6 , 361

o

0.0

6,213

o

0,0

0.0

3 , 036o

0.0

2 , 979o

0.0

6 , 085

40.0

14 , 852

6 , 577

44 . 3

14 , 670

7 , 855

53 . 55,918 550

78 . 88 , 2036,349 1 , 277

77 . 48,471

6 , 82580 . 6

3.4

16 , 480

7.8

16 , 902

1 , 737

10.3

o

0,0

10 , 353o0,010 , 007

o

0,0

978

o

0.0

957

o

0.0

950

o

0,0

7 , 997

1 311

16.4

8 , 083

1 , 30716.2

8 , 239998

12 . 132 . 5Population

po pulation

using SS*

(A)

(B)

6 554

6,217

94 . 976 . 95,216

428

2 , 354

20 , o11 392

6 , 408o0,0

24 Naminohira

3,110

o

25 Tomachi
26 Kogakura

15 , 206
7 , 508

27 Doinokubi

16 , 064

28 Fukahori

10 , 603

29 Minami
30 Mogi
31 Hayasaka

RPSS**

Total

Population

population

using SS*

(A)

(B)

20 Sako

6 , 744

5 , 959

88 . 421 Nita
22 Kitaohura

5 , 330

4 , 097

1 1 , 753

23 Minarniohura

No, Name

RPSS**

Total

RPSS* *

Total

Part

Southern

1989

1988

1987

(B/A x 100)

(B/A x 100)

(B/A X 100)

5 , 803

567

9.8

5,831

1,014

17.4

5 , 6791 844

32 Hiyoshi

l , 550o0,0

1 , 544o0,0

1 , 549

o

0.0

Total

99 , 054

26 84127 . 1

98 , 862

29 , 818

30 . 2

98 , 219

32 , 360

33 , o33 Zenza

6 , 045

5 , 206

86 . 1

5 , 984

5 , 176

86 . 5

5 , 757

5 , 02987 . 434 Sakamoto
35 Takao
36 Yamazato

lO , 679

7 , 260

68 , o10 , 390

7 , 105

68 . 4

10 , 161

6 , 868

67 . 6

18,396o0,018 , 424228

1.2

18 , 054

839

4.6

12 , 005

1 , 760

14 . 7

11 , 715

2 , 727

23 , 3ll , 428

3 , 283

28 . 737 Nishishiroyama

11 , 594o0,0

1 1 , 358o0.0

11 , 020

o0,0

38 Shiroyama
Northern
39 Nishiurakami

5 , 869

O

0.0 5.7

5 , 813

O

0,0 6.6

5 , 775

O

0,0 6.9

19 , 647

l , 116

19 , 466

1,291

19 , 437

1 , 337

40 Nameshi

40 , 188

4 1 , 493

l03 . 230 , 088

40 , 985

102 . 239 , 240

39 , 240

41 Nishikita

12 , 246

583

4,8

12 , 205600

4.9

12 , 008

609

5,l42 Nishimachi

16,826o

0.0

16 , 468o0.0

16 , 374o0.0

43 Kawabira

4 , 340o

0.0

4 , 249o

0.0

4 , 151o0,057 , 835

57,41836 . 4

166 , 160

58 , 1 12

37.2

153 , 405

57 , 205

37.3

Total

lOO . O* SS: sewage system**RPSS: the rate of population utilizing a sewage system.
cutexipie,ts

aL L ens

The number of Cx. p. pallens was expressed by the mean
per trap per night. As seen in Fig. 2, the southern part

females50
4. o

Cl : Southern part
Northern part

:

30
2. o

sharp decrease in Cx. p. pallens. Fig. 2 also indicatesthat the northern part had less Cx. p. pallens and more

10ohad a high density of Cx. p. pallens and inadequate
public sewage systems in 1983, but the sewage systems
became to be spread widely from 1986, resulting in a$

extensive public sewage systems already around 1983,
compared with the southern part. Such sharp decrease$

Sewage Systemof this mosquito in 1986 on seems to be due to a decrease40

in the breeding sites, by improvement of roads and open
roadside ditches in pararell with the spread of sewage

20

system.

lg83 1984 I985 I98G 1987 1989
l 988

Yea'

Figure 2 Annual changes in number of Culex pipiens pallens

females and rate of population using a sewage
system in southern and northern parts in Nagasaki
City, 1983‑1989 (see Tables I to 5‑2).
* Collection was not made.
* * Mean number of females/trap/night.
* * *RPSS shows the rate of population utilizing a sewage systern.

3 . Natural infection rates
Table 6 shows the natural infection rate in Cx. p.
pallens. The percentage of larvae‑having mosquitoes in1983 was zero in the northern part, but it was 0.9% in thesouthern part. Similar results were also obtained in 1986

and 1988. These results suggest,that Cx. p. pallens weremore abundant and the natural infection rate was higher

204

Table 6 Number and percentage of Culex pipiens pallens
females with D. immitis larvae in southern andnorthern parts in Nagasaki City, 1983‑1989
Southern part

Northern partNo. females No. (B) and (%)* No. females No. (B) and (%)*

Yearof females

dissected (A)of females

dissected (A)with larvae

with larvae

1983

112

1**( 0'9)37

O ( 0.0)

1986

3

1 * * (33 . 3)

2o' ( 0.0)

1987

11

o ( 0.0)26

2**( 7.7)

1988

7

1** (14 . 3)10

o ( 0.0)

1989

7

o ( 0.0)10o ( 0.0)

Ae. albopictus and Ae. togoi and their natural infection

rate (the percentage of D. immitis larvae‑carrying
mosquitoes) in the southern and the northern parts ofNagasaki City.
Table 7 shows the annual number of Cx. tritaenio‑
rhynchus for the southern and the northern parts. There
was no significant difference in the annual number ofthis mosquito between the southern 'and the northernparts. The natural infection rate for this species also did
not differ significantly between the two parts of the city.

The number of Ae. albopictus did not differ signifi‑
cantly in any year between the southern and the north‑ern parts (Table 8). The natural infection rate for this* Natural infection rate (B/A x 100)**Ist stage larvae

species tended to be little different between the northern

and the southern parts.
The number and the infection rate of Ae. togoi areshown in Table 9. Any Ae. togoi were not caught in thein the southern part than in the northern part.

II. Population and natural infection rates of threesecondary vector mosquitoes in the southern andthe northern parts

northern part, and the number was very small in thesouthern part. Only one female was found to be infectedin the southern part.

For the 1983‑1989 period, we analyzed the number(the mean per trap per night) of Cx. tritaeniorhynchus.

Table 7 Number of Culex tritaeniorhynchus females collected in light traps and naturalinfection rate in southern and northern parts in Nagasaki City, 1983‑1989
Year and (times)

Southern part
Total No. femalesof collection

1983 (13)1986 ( 6)1987 (24)1988 (27)1989 (20)

Females with(A) collected
and dissected

Mean No.*

115

4 . 43

1** (' 0'9)

19

1 . 59o ( 0.0)

300
593
152

2 . 5014** ( 4.7)
2** ( 0.3)

larvaeNo' (B) (%)***

4 . 391 . 52o ( o o)Northern part
Total No. females(A) collected
and dissected

200
26
150
695
159

Females with

Mean No.*

larvae * *No. (B) (%)***
7 . 691** ( 0'5)

2 . 17o ( 0.0)

1 . 2511** ( 7.3)
6** ( 0.9)

5 . 15o ( o o)1 . 59* Mean number of females/trap/night.
** Ist stage larvae***Natural infection rate (B/AX 100)

Table 8 Number of Aedes albopictus females collected in light traps and natural infection
rate in southern and northern parts in Nagasaki City, 1983‑1989
Year and (times)

Southern part
Total No. femalesof collection

1983 (13)1986 ( 6)1987 (24)1988 (27)1989 (20)(A) collected
and dissected

Mean No.*

Females withlarvaeNorthern part
Total No. females

Mean No.*No' (B) (%)***(A) collected
and dissectedo . 35

1** (11.1)

10o . 391

o . 09o ( o o)4

o . 3425
63
27

O . 21

1** ( 4.0)

o ( 0.0)o ( 0.0)

51
39
16

o . 43o . 47* Mean number of females/trap/night.
** Ist stage larvae***Natural infection rate (B/AX 100)

larvaeNo. (B) (%)***

9o . 27

Females witho ( 0.0)o ( 0.0)o . 291** ( 2.0)
3** ( 7.7)

O . 16

5* * (31 . 3)

205

Table 9 Number of Aedes togoi fernales collected in light traps and natural infection
rate in southern and northern parts in Nagasaki City, 1983‑1989
Year and (times)

Southern part
Total No. femalesof collection(A) collected
and dissected

Mean No.*4
1

2

o . 023o . 022

o . 02

1983 (13)1986 ( 6)1987 (24)1988 (27)1989 (20)Northern part
Total No. females

Females withNo' (B) (%)***(A) collected
and dissected

O . 16

1** (25.0)

oo . 09o ( 0.0)o ( 0.0)o ( 0.0)o ( 0.0)olarvaeFemales with

Mean No.*

larvaeNo. (B) (%)***ooo* Mean number of females/trap/night.
* * Ist stage larvae***Natural infection rate (B/A X 100)

Table 10 Annual changes in mean number of 3 species of vector mosquitoes
and natural infection rate in Nagasaki City, 1983‑1989
Cx. tritaeniorhynchus

Cx. p. pallens

Year
1983
1986
1987
1988
1989

Mean No.*

Natural Inf.**

Infected No.

(A)

(B)

(A X B)

Mean No.*

Natural Inf.* *Infected No.

(A)

(B)

(A X B)Ae. albopictus
Mean No.*

Natural Inf.* *Infected No.

(A)

(B)

(A X B)2 . 87o . 451 . 2915

6 . 06o . 704 . 2420o . 375 . 552 . 0535

O . 2116 . 65

3 . 4965

1 . 88o . ooo . ooooo . 22o . ooo . ooooO . 163 . 85

O . 6160

1 . 886 . oo11 . 2800

o . 323 . ooo . 9600

o . 06

7 . 15o . 4290

4 . 77o . 602 . 8620

o . 383 . 85

1 . 4630

o . 09o . ooo . oooo

1 . 56o . ooo . ooooo . 2215 . 65

3 . 4430* Mean number of females/trap/night.
**Natural Inf. (Natural infection rate in %)III. Comparison of the role in the transmission
among Cx, p. pallens, Cx. tritaeniorhynchus and Ae.
al bopictus

e., construction of large‑scale apartments after about
1982, completion of southern Nagasaki sewage disposal
plants in 1984, establishment of a sewage system for
Tomachi in 1985 (Tables 5‑1 and 5‑2) , and accompany‑

From the number and the natural infection rate offemale mosquitoes of three dominant species collected
to date, the number of infected females was determinedannually as shown in Table 10. Generally speaking, thenumber of Cx. p. pallens in Nagasaki City will further
decrease as the current plan to expand sewage systems

natural infection rate of Cx. tritaeniorhynchus was lower

Suenaga et al. (1973) suggested the principal role ofand the number of the females was much greater thanCx. p. pallens in the transmission of D. immitis in
Nagasaki City, on the grounds that the number wasthat of Cx. p. pallens, but the number of infected females

ing changes in roads and open roadside ditches. The

is put into practice.

was about as same as that in Cx. p. pallens. On the otherlarger and the natural infection rate was higher in Cx. p.

hand, the number of Ae. albopictus, the natural infection

pallens than in Cx. tritaeniorhynchus and Ae. albopictus.

rate and the number of infected females all were similar

In the present study, the infection rate of Cx. tritaenio‑

to that in Cx. p. pallens.

rhynchus was lower but the number was larger than thatof Cx. p. pallens, resulting in similar numbers of infected

DISCUSSION
Before 1980, the number of Cx. p. pallens caught inTomachi (a southern district) was considerably large(Oda et al., 1993). From 1986 on, however, the number
has been almost zero. This change in Tomachi may be
attributable to a decrease in the sites where the mos‑quitoes can breed, following environmental changes, i.

mosquitoes in the both species. This may indicate thatthe relative importance of Cx. tritaeniorhynchus in thetransmission has recently become a little higher in
Nagasaki City as Konishi (1989) reported that thismosquito is important in transmission of D. immitis in
Kobe. Although we can not estimate exactly the popula‑tion size of Ae. albopictus by a light trap because they
are active in daytime, our data showed that the number

206

of infected females of Ae. albopictus was not very differ‑ent from that of Cx. p. pallens. Therefore the ability oftransmission of Ae. albopictus seems to be quite high inthe city. Ae. togoi was caught only in the southern partand its number was very srnall, but the natural infection
rate was high. Keegan (1967) was the first to report D.

immitis‑carrying Ae. togoi. Prior to the present paper,
however, no survey of D. immitis‑carrying Ae. togoi in
Nagasaki City has been published. Since the number of
Ae. togoi caught was very small, this seems to have little

relationship with the transmission of D. immitis, assuggested by Suenaga et al. (1973).

(1967) : Laboratory and field studies of some entomolog‑
ical aspects of canine dirofilariasis problem in Japan.
Jap. J. Saint. Zool. 18, 6‑18.

2 ) Konishi, E. (1989): Culex tritaeniorhynchus and Aedes
albopictus (Diptera: Culicidae) as natural vectors ofDirofilaria immitis (Spirurida: Filariidae) in Miki City,

Japan. J. Med. Ent. 26(4) : 294‑300.
3 ) Oda, T,, Suenaga, O., Mori, A., Fujita, K., Zaitsu, M.,

Kurokawa, K., Nishioka, T., Itoh, T., and M. Mine
(1993): Studies on epidemiology of Dirofilaria immitis
in house dogs in Nagasaki City, Japan, with considera‑
tions on yearly changes in microfilarial prevalence. Jpn.
J. Trop. Med. Hyg., 21, 231‑237.
4 ) Suenaga. O.. Itoh, T and T. Nishioka (1971): Studies on

the filarial prevalence among house dogs and the mos‑

ACKNOWLEDGMENTS

quito vectors in Nagasaki City, Western Japan. 1. on thenatural infection rate among house dogs in Nagasaki

Professor Y. Wada, Department of Medical Ento‑

City. Trop. Med., 12, 169‑178. (in Japanese with English

mology, Institute of Tropical Medicine, Nagasaki Uni‑summary) .
5 ) Suenaga, O. (1972): Studies on the filarial prevalence
among house dogs and the mosquito vectors in Nagasaki
City, Western Japan. 2. on the susceptibility of fourcommon mosquitoes to the larvae of Dirofilaria immitis
in Nagasaki City. Trop. Med., 14, 32‑40. (in Japanesewith English summary) .
6 ) Suenaga. O., Itoh, T. and T. Nishioka (1973): Studies on
the filarial prevalence among house dogs and the mos‑quito vectors in Western Japan. 5. on the vector mos‑quitoes of Dirofilaria immitis in Nagasaki City. Trop.
Med., 15, 131‑140. (in Japanese with English summary) .

versity kindly read this manuscript and provided
detailed suggestions. The staff in Department of Sew‑
age System in Nagasaki City kindly gave us material

for Sewage System in Nagasaki City. We wish to
express our sincere gratitude to these persons.

REFERENCES CITED
1 ) Keegan, H. L., Betchley, W. W., Haberkorn, T. B.,

Nakasone, A. Y., Sugiyama, H., and Warme, R. T.

207

Jpn. J. Tro p. Med. Hyg., Vol. 22, No. 4, 1994, pp. 207 210

A NEW BLACKFLY SPECIES OF SIMULIUM(MOROPS) FROM SOLOMON ISLANDS, SOUTH
PACIFIC (DIPTERA= SIMULIIDAE)
HIROYUKI TAKAOKAI AND HIROSHI SUZUK12
Received September 3, 1994/Accepted October 5, 1994

Abstract: A new blackfly species, Simulium (Morops) herei sp. nov. is described based on male and pupal
specimens collected from the Solomon Islands, South Pacific. This species is placed in the farciminis‑groupby the pupal gill consisting of I inflated, horn‑like filament and 4 slender filaments. This species is uniquein possessing the parameral hooks in the male genitalia which are absent in all the Papua New Guinean
Morops species. From the known species of the farciminis‑group, this species is easily distinguished by theabsence of the distinct hair crest on the male fore basitarsus, and in the pupa by the plate‑like terminal
hooks, all terga well‑sclerotized, and the normal, wall‑pocket‑shaped cocoon covering whole of the thorax
and abdomen. This is the first record of the farciminis‑group of the subgenus Morops from the Solomon
Islands.

In the Solomon Islands, South Pacific, Stone andMaffi (1971) reported two species of the subgenus
Morops Enderlein (i. e., Simulium (M.) sherwoodi Stone
and Maffi, and S. (M.) sp. nr. avilae Smart and Clifford)

from Guadalcanal Island. In 1992 and 1993 one of us (H.
S.) collected larvae, pupae and adults of blackflies in the

Solomon Islands. One new species belonging to theis 0.22 x length of 3rd segment, and bears small,rounded opening medially. Thorax. Scutum brown uni‑
formly and densely covered with yellow pubescence.
Scutellum with long dark hairs and yellow pubescence.
Postscutellum brown, without hairs. Pleural membranewith ca. 56 yellow pubescence. Katepisternum longer
than deep, with ca. 30 dark hairs on each side. Legs.

subgenus Gomphostilbia Enderlein was already described

Fore basitarsus slightly dilated, ca. 6.0 X as long as(Takaoka, 1994). In this paper we describe one newwide, with usual dorsal hair crest. Hind basitarsus (Fig.

species which is placed in the farciminis‑group of the2) parallel‑sided, much narrower than tibia. Calcipala

subgenus Morops, defined by Crosskey (1967). The(Fig. 2) well developed, ca. 1.4 x as long as wide and ca.

uniqueness of this species within the species‑group, as
well as within the subgenus is indicated.

0.8 x as wide as basitarsus. Wing. Costa with spinules
and hairs; subcosta bare; basal section of vein R fully
haired; hairs at base of stem vein dark brown; basal cell

DESCRIPTION
Simulium (Morops) kerei sp nov
Male. Body length ca. 3.0 mm. Head. Width somewhat
wider than thorax. Upper eye consisting of large facetsin 12 horizontal rows and in 11 vertical columns.
Clypeus black, densely covered with hairs (except
sparsely in the center) . Antenna co.mposed of 2 + 9
segments; Ist flagellomere somewhat elongated, ca. 1.3
X as long as 2nd flagellomere. Maxillary palp com‑posed of 5 segments with proportional length of 3rd, 4th
and 5th segments '1.0: 0.9: 2.1; 3rd segment (Fig. 1) ofnormal size with small, elliptical sensory vesicle which

absent. Abdomen. Basal scale dark with fringe of longyellow hairs. Abdominal segments 2‑9 blackish except
segment 2 pale on basal 1/2; tergite 2 broadly shiny
when illuminated, and tergites 5‑7 each with pair oflarge dorsolateral shiny areas. Genitalia (Figs. 3‑7).
Coxite rectangular in ventral view, ca. 1.5 x as long as

wide. Style short, ca. 3/4 x length of coxite, curved
inwards, tapered apically, with terminal spine. Ventral
plate (Fig. 3) transverse, narrowed posteriorly, withposterior margin nearly straight or undulate or concave(different by angles) in ventral view, moderately setoseon ventral and posterior surface, with short arms
converged; ventral plate much produced ventrally as

1, Division of Medical Zoology, Oita Medical University, Hasama, Oita, Japan 879‑55
2, Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan 852

.',
ltl"
r,t: I tt '/.4

I IIIlli't t','1' 11 t
tl"'blt 1:.1:.::t!Sl;i.} /'ih"}'*t t: : t t :, .:t.'1"t "" ' l$:'il; ･f' ' "'t . '.:;t:; :‑?:

52
1"...'‑:'̲̲‑./,: "]e,･ :i; : ;:1,jf‑ ‑l'i //‑

bj' :E:::/ ･･.･ /:P /1;/ S : 17) i ,,:djla'lll"!s ,1,1'

4/j/j/:] r:f/:;;: I / ';l' :"'f '.

;. ' p̲':‑.'.‑/.:/̲: )･ ,,:,.Xe,1/ "', ̲̲̲.̲ ̲ ,,.‑!''/,;'/7./i,V.1'ell'l,;l
..
llll

6

., .

'LldJrb'I1:IlI

rlt ' tii h' 'I t {It' Ib"I'l:"i'l
' I '

t 1 1

¥'¥:

tttll

3

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7

8

Figs. 1‑9.

Male and pupa of Simulium (Morops) kerei sp. nov. 1, 3rd segment of male maxillary palp
showing sensory vesicle; 2, male hind leg (coxa and trochanter omitted) ; 3, coxite, style (rightside only) and ventral plate in ventral view; 4, ventral plate and median sclerite in lateral view;
5, ventral plate in end view; 6, parameres showing distinct parameral hooks in posterodorsal view;
7, median sclerite; 8, pupal gill (right side only) in lateral view; 9, terminal hooks of pupal abdomenin end view.

seen in Figs. 4 & 5. Parameres (Fig. 6) each with 3
distinct parameral hooks and several undeveloped ones.
Median sclerite (Fig. 7) plate‑like, moderately sclerot‑
ized except distal portion transparent.
Pupa. Body length (excluding gill filaments) ca. 3.0 mm.

Head and thorax. Integument brown, bare except poste‑rior 1/2 moderately covered with small tubercles; anten‑

nal sheath of usual form. Head with I facial pair of

10ng, bifid trichomes and 3 frontal pairs of long, simple
trichomes. Thoracic trichornes 6 pairs (5 anteriorly, 1posterolaterally) , all long and simple except 3 or 4
anterodorsal pairs bifid. Gill (Fig. 8) with I inflated,horn‑1ike filament (0.8‑1.0 mm long) and 4 slender fila‑
ments (1.0‑1.4 mm long) arising at base from dorsal andinterolateral surface of horn‑1ike filament; all filaments

brown except base of horn‑1ike filament transparent
ventrally, and covered uniformly and densely with

209

minute tubercles; horn‑like filament gradually tapered

toward apex, thick‑walled, irregularly with several
annular constrictions; 4 slender filaments very slightly

genus Morops by having hairs on the katepisternum andpleural membrane of the male adult, although female
adult and mature larva are not available.' ThiS species

tapered toward apex, with numerous annular furrows.
Abdomen. All terga brown, sclerotized, with interseg‑

is further placed in the farciminis‑group. df= tlp

mental transparent space; terga I and 2 without tuber‑which consists of I inflated, horn‑like, filam'e̲ nt and 4
slender filaments. However, this speciesliis, ,.,,nique incles; tergum I with long seta on each side; tergum 2 with

6 simple setae on each side, I seta much longer thanothers; terga 3 and 4 each with 4 hooked spines directed
forward along posterior margin on each side; terga 6‑9
each with spine‑combs in transverse row and comb‑like
groups of minute spines on each side; tergum 5 devoidof spine‑comb; tergum 9 with a pair of plate‑liketerminal hooks which are serrated on ourter margins(Fig. 9). Sternum 4 with a simple hook and a few
minute setae on each side; sternum 5 with a pair of bifid

hooks on each side; sterna 6 and 7 each with a pair ofbifid inner and bifid or simple outer hooks; comb‑like
groups of minute spines present medially and laterally
on sternite 4, submedially each on sterna 5‑7 and medi‑
ally on sternite 8; grapnel‑1ike hooklets absent on eachdefined by Crosskey (1967) , by the form̲Qf thubgenus,

pufPal gillpossessing the distinct parameral hooks '*,ip; i"the male
genitalia which are absent in all Morops specie,s:reported
from Papua New Guinea and Australia. '; ,j h,i species

has also a few characters which dart f}om.'=the diag‑noses for the,farciminis‑group: i. e., thab' ence ofdistinct dorsal hair crest on the fore b sitar.*sus, thecocoon of no short form but of normal 'fbtn ;,,eov, eringwhole of the thorax and abdomen, the pupa'l = =e': :g.

wellsclerotized, and terminal hooks not cone=shap d. but
plate‑like with serrate outer margin. Either. pf thesecharacters easily separates S. (M.) kerei from the
known species of this species‑group. The pupal gillitself most closely resembles that of S. (M. ) wantoatense

Smart and Clifford from Papua New Guinea (Smart andside of last segment. Cocoon. Simple, wall‑pocket‑

Clifford, 1965), although in the latter species there areshaped, tightly woven, extending ventrolaterally in some
cocoons but not in others, covering whole of thorax anddistinct tubercles on the surface of horn‑like filament,

abdomen; anterior margin strongly woven and wellbase of the other 2 filaments. Apart from those charac‑ters mentioned above, this species also differs from thelatter species by the narrow male hind basitarsus, andthe absence of saw‑like projections on the pupal anten‑
nal sheaths. The male hind basitarsus is enlarged, andpupal antennal sheaths are provided with saw‑like pro‑defined; floor formed on posterior 1/2.

Female and larva. Unknown.
Type Specimens. Holotype c?1 , dissected out of pupa,
slide mounted together with pupal exuvia, collected at
Noro, New Georgia Island, New Georgia Islands, West‑ern Province, Solomon Islands, 5. IX. 1992, H. Suzuki.
Paratypes I pharate (71 , 3 pupal exuviae, in alcohol,same data as holotype. Holotype and one of paratypes

will be deposited at the Natural History Museum,London, U. K. and other paratypes will be deposited at
Bishop Museum, Honolulu, U. S. A., and at the Carnegie
Museum Natural History. Pittsburgh, U. S. A.

Ecological notes. The pupae were collected from trai‑
ling grass leaves in a small, fast‑flowing stream withwidth of 1‑2 m, running in a natural forest, midpoint
between Munda and Noro, together with S. ( G. ) hiroshii

Takaoka, S. (M.) sherwoodi and S. (M.) sp.

and 2 of 4 slender filaments arise a little distant from thejections in S. (M.) wantoatense and 4 other related
species of the farciminis‑group (the presence of thelatter character has been confirmed in the specimens
held in the Natural History Nuseum, London by H. T.) .

ACKNOWLEDGEMENTS
We wish to thank Dr. A. J. Shelley and Miss Ther‑esa Howard, the Natural History Museum, London, whokindly gave H. T. the opportunity to examine somePapua New Guinean simuliid specirnens needed for thisstudy. One of us (H. S.) is grateful to Dr. N. Kere,

Ministry of Health and Medical Services, Solomon
Islands, for his assistance during the collection trip.

This work was supported by the Grant‑In‑Aid from
the Ministry of Education, Science and Culture of Japan

Etymology. This new species is named after Dr. N.
Kere, Permanent Secretary, Ministry of Health andfor International Scientific Research Programme (No.

Medical Services, Solomon Islands, for his cooperation.

Toyama University of International Studies.

Remarks. Simulium (M.) kerei is assigned to the sub‑03041080), headed by Prof. M. Sasa, president of

210

REFERENCESl ) Crosskey, R. W. (1967) : The classification of Simulium

Latreille (Diptera: Simuliidae) from Australia, New
Gtiinea, and the western Pacific, J. Natur. Hist., 1, 23‑51
2 ) Smart, J. and Clifford, E. A. (1965): Simuliidae (Dipter‑a) of the territory of Papua New Guinea, Pacific Insects,
7, 505‑619

3 ) Stone, A. and Maffi, M. (1971): A new species ofSimulium from Guadalcanal, Solomon Islands (Diptera:
Simuliidae) , J. Med. Entomol,., 3, 299‑300

4 ) Takaoka, H. (1994) : A new blackfly species ofSimulium (Gomphostilbia) from Solomon Islands, South
Pacific (Diptera: Simuliidae) , Jpn. J. Trop. Med. Hyg.,22, 97‑101

211

Jpn. J. Tro p. Med. Hyg., Vol. 22, No. 4, 1994, pp. 211 215

Research Note

GROWTH INHIBITION OF CULTURED
PLASMODIUM FALCIPARUM BY
IMMUNE SERUM FROM TANZANIA.
THOMAS B. NYAMBOI AND HIROJI KANBARA
Received April 22, 1994/Accepted September 15, 1994.

Abstract: The inhibition titers of sera from adult residents in Dar es Salaam, T n ania to intraerythrocytic
growth of Plasmodium falciparum were not correlated to their antibody tit is by' immunofluorescence andenzyme immunoassay, and did not exhibit specificity to the strain withc ftain MSA‑1 gene. Anotherinhibitory factor (s) was found in a low molecular fraction with molecular weight less than 30,000 that was
separated from a high molecular fraction by centrifugation on a meriibrane filter (centriprep‑30, Amicon) .

This factor induced morphological changes of intraerythrocytic parasites and was thought to correspond tocrisis form factor (CFF) by Jensen et al. (1982). The factor was shown to remain in the serum of an adult
Tanzanian who had lived in a non‑malarious area six months prior to the commencement of this study andto be contained in a hyperimmune serum from a Japanese patient even though on a low level,

INTRODUCTION
Various humoral factors in immune sera to inhibit

MATERIALS AND METHODS
Serum samples and parasite strains.

malaria parasite growth were described by Mcgregor

Twenty five serum samples were collected fromand Wilson (1988) . Although intraerythrocytic growth
inhibition has been considered to be associated mostly
with antibodies, Jensen et al. (1982, 1983 & 1984) haveadult malaria patients in Muhimbili Hospital in Dar es

Salaam, Tanzania where malaria is hyperendemic. The
samples were collected in sterile tubes and preserved

endemic areas contained factors independent of anti‑

with 0.05% sodium azide prior to transportation. A
serum sample T was obtained from a Dar es Salaambodies which induced crisis forms of erythrocytic stages

resident, a 39‑year‑old man who had lived in a nonen‑of P. falciparum and which were absent in Indonesian

demic area, Japan for six months prior to this study andimmune sera with high immunofluorescence antibody

another sampe M was from a Japanese malaria patient,a 43‑year‑old man who had contracted the disease while

shown that sera from Sudanese living in malaria(IFA) titer. These results suggested the presence of two

different and ethnogenetically biased humoral mecha‑
nisrn for elimination of asexual blood forms. In thepresent work we examined the inhibitory pattern ofon visit to Mozambique four months before andrepeated recrudescence till the serum collectiQn. Before

use the Tanzanian serum samples were freed of sodium

Tanzanian sera to cultured P. falciparum in relation toazide by Sephadex G‑25 column chromatography, andanti‑malaria antibodies. Furthermore serum samples
were devided into a high (HMF) and a low molecular
fraction (LMF) by centrifugation on a cut off mem‑

were screened for chloroquine (CHQ) according to thebrane filter for Mr 30,000 to specify the growth inhibi‑
tory factors.

modified Hanskins method (Hanskins MM ID as de‑scribed by Mount et al. (1987). Control samples wereobtained from voluntary persons in Japan. Part of theserum samples were diluted 2.5 fold with serum free

RPMI 1640 media, devided into two fractions by
centrifugation at 3,500 rpm for about 55 minutes using
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan*Present address, Department of Biochemistry, Faculty of Medicine, Muhimbili University College of Health Sciences, 65001 Dar
es Salaam, Tanzania

212

centriprep‑30 concentrator (Amicon, USA) at a cut. ‑offhours at room temperature. The plates were read at 400
nm after 30 min incubation with 0.3 ml of substrate (oof Mr 30,000. The filtered low moleculair fra,c, tion(LMF) was kept alone and the remaining high molecu‑
lar fraction (HMF) was adjusted to the original volurne
by adding serum free RPMI 1640 media and kept at 4 C
till further use. Three P. falciparum strains FCR, K1‑Phenylenediamine) at room temperature. The ELISA
titer was expressed as the ratio of OD value of eachsample to that of control.

and Thai 836 were used in the inhibition assays. Briefly

ozoite surface antigen (MSA‑1) (Jongwutiwes et al.,Immunofluorescence assay (IFA).
Dried thin layer blood slides of parasitised cells
were treated with various dilutions of immune sera andincubated in moist chamber at 3TC for one hour. The
slides were then washed 3 times with PBS and incubated1991). P. falciparum parasites were usually cultured inwith 100 times dilute fluorescein conjugated anti‑humangroup O red blood cells adjusted to 5% hematocrit in ahumidified conditions of 5% carbon dioxide, 5% oxygen,lgG (FC fragment) (Organon Teknika Co.) in PBS at
37'C for half an hour. The slides were then 3 times
washed with PBS and mounted with 5% glycerol in PBS
for fluoroscopy. The IFA titer was expressed as the

90% nitrogen and then overlaid media were changed

highest dilution that showed positive.

the strain FCR was used due to its easiness of culture
and the latter two strains were because they had thedistinct genotype of the precursor of the major mer‑

RPMI 1640 medium supplemented with 10% humanserum as described by Tragar and Jensen (1976) atwith fresh ones everyday.
Growth inhibition assays.

Enzyme linked immunosorbent assay (ELISA) .
FCR strain infected red blood cells with 13% par‑asitemia were processed for antigen preparation accord‑

Sorbitol synchronized parasites were prepared as
described by Lambros and Vandergerg (1979) . After anovernight culture the synchronous culture with more

ing to the method by Spencer et al. (1979). Protein
estimate by the method of Lowry in the final super‑
natant which was the source of antigen was 3.2 mg/ml.

than 9,5% trophozoites was adjusted to 0.5 to 2.5%
parasitemia depending on the experiment and to ahematocrit of 5% by freshly washed group O red blood

The antigen was aliquoted and stored at ‑80'C till fur‑cells. Assays containing final volume of 0.2 ml with ten

ther use. Microplates (ELISA Plate, MS‑3496,

times dilute immune serum or LMF or HMF (at final

Sumitomo‑Bakelite) were coated with 0.2 ml antigen

concentration of 10%) were prepared in flat bottomed
96 well culture plates. Where immune serum was used

diluted in 0.06 M carbonate buffer, pH 9.6 at I : 300
overnight at 4'C, washed 3 times with PBS, sealed and(Dainippon Pharmaceuticals) according to the manufac‑
turer's instruction. This solution was also used as adiluent for the following steps. Test serum was diluted

together with the normal serum, the latter was adjusted
to give the filal concentration of 10%. In one experi‑
ment the immune sera were adjusted to final dilutions
from 5 times to 160 times. Culture conditions were as
described earlier and the overlying media were changed
daily for fresh media. Giemsa stained blood films wereat I : 200. 0.2 ml of diluted serum was run in duplicate

examined under the light microscope to ascertain theon the antigen coated microtiter plates, incubated at
37'C for two hours. The plates were washed three times
in PBS‑Tween and incubated with 0.2 ml of dilute (1500) horseradish peroxida e‑conjugated goat lgG frac‑changes. The inhibition rate was expressed as the ratioof the difference of parasitemia between control (O andsample (S) to control; (C‑S/O xIOO.

kept at 4'C till further use. Prior to use in the next step

the microplates were blocked by Block Ace solution

level of parasitemia and possible morphologicaltion to human lgG (Organon Teknika Co.) for two
Table 1. IFA titer and growth inhibition rate after 72h incubation to three P. falciparum strains

by four Tanzanian immune sera.

FCR
Sample No IFA Inhibitron (%) IFA
8Thai 836
K1
Inhibition (%)
Inhibition (%) IFA; 3 , 20057

1, 60079;

3 , 200

58

3, 200

81

5

400

46

400

78

200

74

7

400

41

400

71

400

65

34

;

1 , 60065

3 , 200

79

213

Table 2. CHQ Ievels and serum antibody titers to FCRtion assay of synchronous culture of P. falciparum FCR

strain of immune sera and their inhibition rates
after 72h incubation.

strain (Table 2). Levels of CHQ and its metabolites
above 2.0 pM were considered significant. However,
although serum samples 34, 5, 3 and 4 exhibited highdrug levels close to the 75 pM considered to be themaximum therapeutic serum levels in the treatment ofSample No. CHQ ( /t M) ELISA IFA Inhibrtron (%)
11

O

10

O

8

1.5

2.7
3.0
8.1

34

61 . 5152.3

556 . 37O

362 . 3

4

61 . O

T*

M*

100
800

54
45

3 , 20074

malaria (Staiger et al., 1981) they did not influence the18 . 3; 3 , 200

growth inhibition by the immune serum. We consider

100
400
400

53

final culture volumes because the immune sera were lO
times dilute in the final inhibition assays. This could
also have been augmented by possible CHQ resistance

O

5.6
4.1
5.9
7.5
3.3
4.1

80
61

O

> 18 . 3;

68

1 , 600

65

800
400

65

3 , 200

57

73

A serum from a Dar es Salaam resident living in Japan for
6 months.
b A serum from a Japanese patient with chronic infection for
4 months.

RESULTS AND DISCUSSION

this to be due to the low CHQ concentration levels in theby the parasite strains used.

The analysis of the inhibitory capacity of the LMFS

and HMFS of the serum M, T and 34 on synchronously
growing P. falciparum FCR strain revealed a stronger

inhibition tendency of LMF than HMF except theJapanese serum M (Table 3) , although the ELISA titerby the dilution method (Spencer et al., 1979) for theJapanese serum M was two times higher than the serum
sample 34 which was the highest value 10,240 in Tan‑

Using the preliminary ELISA 9 samples were

zanian samples. Surprisingly the sample T from aselected from 25 for further examination. They had IFA
titers ranging from 100 to more than 3,200 that well

Tanzanian who had lived in Japan for six months still
maintained high inhibitory level of LMF despite his low

corresponded to ELISA titers from 2.7 to 18.3 (Table 2) .

Of these samples four were used in the growth inhibition

antibody titer. This finding may partly explain reported
findings by various groups that P. falciparum infection

assay of P. falciparum strain FCR, K1 and Thai 836

does not cause the severe complication of acquired(Table 1) .

immunodeficiency syndrome, AIDS (Wabwire‑Mangen

The 72 hours inhibition pattern of the immune sera
did not correspond well with their respective antibody
titers (Table I and 2). Although the Thai 836 (MAD 20

et al., 1989. Lucas, 1990 and Simooya et al., 1991).

type) is slightly less susceptible to the inhibition assay

Further analysis of the LMF and HMF inhibition
using the serum T showed that the inhibition was dose‑dependent in either case. Inhibition capacity of thethan the K1 type, the least susceptibility was seen in the

FCR. This we attributed to the fact that FCR strain isbetter adapted to in vitro culture conditions than theother two strains. Eleven serum samples were analysed

80

for a widely used anti‑malaria drug CHQ and its

60

metabolite desethylchloroquine and used in the inhibi‑

,‑g‑‑

a

‑‑h, b

=

o

,

G
J:
Table 3. Growth inhibition after 72h incubation to

P. falciparum FCR strain by LMFS and

E 40

ae

HMFS of different immune sear.

Serum sample

Average inhibition (%)

CONTROL LMF

10
O

HMF

M LMF
HMF
T LMF
HMF
34 LMF
HMF

27
37

20

o

100 20o
dilutlon

57
16

Figure 1 The effect of concentration on the inhibition capac‑

39
33

synchronously growing P. falciparum FCR strain in
48 hours.

ity of LMF (a) and HMF (b) from the serum T oh

214

b

a

Figure 3 Giemsa stained thin blood films of synchronous
culture after 72h culture with a nonimmune serum(a) and with a immune serum T (b). Note thenumber of produced merozoites (‑) .

We have shown here that the LMF with a molecular

15

weight less than 30,000 was a more potent inhibitor than

‑‑ *

a

the HMF which contained the immunogloblins. Infected‑,‑‑ b

red blood cells cultured with the LMF, washed andincubated with fluorescein conjugated anti‑human lgG
could not show malaria antibodies, while with the HMF

10
t

o

we could detect antibodies on the surface of the infectede

red blood cells (results not shown) . We thus report,,tpresence in the Tanzanian immune sera, of an anti

se 5‑malaria factor (s) that is capable of growth inhibitionof intracelluiar parasites and formation of crisis farms,

which support previous findings where it was refered too

20 4c 80 Ioo
60

Time (hotrrs)

Figure 2 Growth of P. falciparum, FCR strain in the pres‑ence of LMF of T serum (b) and in the absence(a) . At time points O, 48, and 96h the predominant

forms were trophozoites while at time points 24
and 76h ring forms predominated.

as crisis form factor (CFF) by Jensen et ai. (1982),
originally by Taliaferro and Taliaferro (1944). We
could not induce crisis forms by the HMF of the immune
serum. This mechanism of malaria defense is possibly aparallel mechanism to antibody defence and our findings
suggest that they do occur together in an individual atvarying degrees of potency. Further studies are neces=sary to define the nature of the factor.

serum fractions was almost absent after 40 times dilu‑

tion (Figure 1). To determine the life stage of theparasite that was affected by the immune sera, thegrowth pattern was monitored everyday. Synchroniza‑tion was observed to start disappearing after 48 hours.
Crisis forms were noted with typical characteristics ofbulky ring appearance, poorly staining IRBC, denaturat‑

ed schizonts and mature schizonts showing remarkably
fewer merozoites (between 4 and 7) as described by

REFERENCESl ) Jensen. J. B.. Boland, M. T. and Akood, M. (1982) :Induction of crisis forms of cultured Plasmodium fal‑ciparum with human immune serum from Sudan. Sci‑ence, 216, 1230‑1233.
2 ) Jensen, J. B., Boland, M. T., Alian, J. S., Carlin, J. M.,

Jensen et al. (1983) instead of Laverania characteristic

Vande Waa. J. A., Divo, A. A. and Akood, M. A. S.
(1983): Association between human serum‑induced
crisls forms in cultured Plasmodium falciparum andof the number of merozoites between 12 and 16 (Figure

clinical immunity to malaria in Sudan. Infect. Immun.

3) . Inhibition was observed at all stages as characterised

41, 1302‑1311.
3 ) Jensen, J. B., Hoffman, S. L., Boland, M. T., Akood, M.

by low parasitemia and a negative gradient in the
immune serum, implicating intracellular growth impair‑ment (Figure 2).

A. S., Laughlin, L. W., Kurniawan, L. and Marwoto, I"･1.
A. (1984) : Comparison of immunity to malaria i,n Sudan

215

and Indonesia: Crisis form versus merozoite‑invasion

immunodeficiency syndrome (AIDS) in Zambia. Anal.

inhibition. Proc. Natl. Acad. Sci. USA, 81, 922‑925.
4 ) Jongwutiwes, S., Tanabe, K., Nakazawa, S., Uemura, H.

lO) Spencer, H. C., Collins, W. E., Chin, W. and Skinner, J. C.

and Kambara, H. (1991): Coexistence of GP195 alleles
Plasmodium falciparum in a small endemic area. Am. J.
Trop. Med. Hyg. 44, 299‑305.
5 ) Lambros, C. and Vanderberg, J. P. (1979): Synchroniza‑tion of Plasmodium falciparum erythrocytic stages inculture. J. Parasitol. 65, 418‑420.

6 ) Lucas, S. B. (1991): Missing infections in AIDS. Trans.
R. Soc. Trop. Med. Hyg. 84 supp. 134‑38.
7 ) Mcgregor, I. A. and Wilson, R. J. M. (1988): SpecificTrop. Med. Parasitol. 85, 269‑270.

(1979): The enzyme‑linked immunosorbent assay(ELISA) for malaria; I. The use of in vitro‑cultured
Plasmodium falciparum as antigen. Am. J. Trop. Med.
Hyg. 28, 927‑932.
11) Staiger, M. A., Nguyen‑Dinh, P. and Churchill, F. C. II(1981) : Sensitive high perfomance liquid chromatogra‑
phic analysis of chloroquine in body fluids. Application

to studies of drug resistance in P. falciparum. J.
Chromatography, 225, 139‑149.

immunity: aquired in man. In, Malaria, vol 1, Werns‑

12) Taliferro, W. H. and Taliaferro, L G. (1944): The effectdorfer, W. H. & Mc Gregor, I. (editors) . London:of immunity on the asexual reproduction of Plasmodium

Churchill Livingstone, pp. 559‑619.
8 ) Mount, D. D., Patchen, S. B., Williams, S. B. and Chur‑

13) Tragar, W. and Jensen, J. B. (1976) : Human malaria

chill, F. C. (1987) : Colorimetric and thin layer
chromatographic methods for field assay of Chloroquine
and its metabolites in urine. Bulletin WHO, 65 (5) , 615

brasilianum. J. Inf. Dis. 75, 1‑32.

parasite in continuous culture. Science, 193, 673‑675.
14) Wabwire‑Mangen, F., Shiff, C. J., Vlahov, D., Kline, R.,9 ) Simooya, O. O., Mwendapole, R. M. and Sikateyo, B. M.

Serwada, D., Sewankambo, N. K., Mugerwa, R. D. andQuin, T. C. (1989) : Immunological offects of HIV‑1
infection on the humoral responce to malaria in an(1991). Severe falciparum malaria and the acquired

African population. Am. J. Trop. Med. Hyg. 41, 504‑511.

‑623.

217

Jpn. J. Trop. Med. Hyg., Vol. 22, No. 4, 1994, pp. 217 218

SURVEY OF INTESTINAL PARASITE
INFECTIONS IN JAPANESE
LIVING IN INDONESIASEIICHI YAMADA ), YUKA MOR11), YUE PAN1)NOBUAKI AKA02), SETSUKO TSUKIDATE*), AND KOICHIRO FUJITAl)
Received August 22, 1994/Accepted October 15, 1994

More than ten million Japanese travelers andfive of helminths were identified in stool samples of 256

workers visit various foreign countries including tropics

individuals (Table 1) . The rate of infectioin was highestwith Blastocystis hominis, 35 of the 256 (13.7%). Giardia

each year. Intestinal parasitic infections are one of
public health problems in developing countries today(Minakami et aD. Unfortunately there are few reliable
information on the risk of parasitic infection in develop‑

lamblia. Endolimax nana, and Trichuris trichiura wereobserved in 4 (one Japanese and 3 native Indonesians) of

256 (1.6%) stool samples. Ascaris lumblicoides.

ing countries. It is because most of the studies on

Iodomoeba buetschlii and hookworm were detected inprevalence of parasitic infection in developing countries

stools of 5 native Indonesians only. Enterobius ver ‑were done in selected individuals, such as those in thehospital or others administered medical treatment for
the infection. The true prevalence of parasitic infection

micularis was found in 2 of the 76 Japaneses so tested. A

should be derived from data on unselected populations(Chacin‑Bonilla et al 1992) .

community‑based study of Blastocystis and other intesti‑

nal parasites in the Asaro Valley of Papua, New Guinea,

showed an extraordinarity high prevalence (from 0.4%
to 58.4%) and variety of protozoan infections. The age‑specific prevalence of Blastocystis infection resembled

Our objective was to investigate the incidence ofintestinal infection caused by protozoa and other para‑sites in Japanese living in Indonesia, and to identify

that of Entamoeba coli and Endolimax nana, indicating
similarity in their mode of transmission (Ashford andfactors that may play a role in the dissemination of the

Atkinson 1992). Zierdt et al (1967) proposed that

inf ection.

Blastocystis hominis is not a yeast, as had been thoughtThis investigation was conducted in 9 cities ofIndonesia in 1993: [Djakarta, Barongang, Semarang,

for many years, but rather a protozoan, and suggested

Surabaja, Probalinggo, Ujung Pandang, Merak,
Menado, and Luwuk]. Samples of stool and blood

Table I Intestinal parasitic infections in Indonesia

collected from 231 Japanese and 25 natives of Indonesia.

Blastorystis hominis

Each stool specimen was fixed in 10% formalin andcentrifuged at 2500 rpm for 3 minutes. Aliquots ofEndolimax nanaslurries were microscopically examined by the formalin

Trichuris trichiura

Giardia lamblia

Japanese

Indonesians

25/231
2/231
3/231
1/231
0/231

10/25
3/25
1/25
3/25
3/25
n. t.* *‑ether method. The remaining aliquots were mixed

Hookwormwith a drop of Lugoul's solution (5% iodine, 10% KI in
100ml of distilled water) . and examined for the pres‑Enterobius venniculuris *ence of protozoa and helminths. A cellophane tape

Heterrophyes heterophyes

2/76
1/231
1/231

Asaris lumbricoides
Iodomoeba buetschelii

0/231
0/231

method was used in collecting specimens to be examined
for Enterobius vermiculuris in 76 Japanese residents.
We assessed the prevalence of protozoa and otherparasites in stool of Japanese residents of Indonesia andamong native population. Five species of protozoa andEntamoeba coli

0/25
0/25
1/25
1/25

positive for parasites / tested residents* : by cellophane tape method* * : not tested

Department of Medical Zoology, Medical and Dental University School of Medicine, 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo 113, Japan
Department of Parasitology,Kanazawa University School of Medicine, 13‑1 Takara‑machi, Kanazawa 920, Japan

218

this organism might be pathogenic. Two patients hold‑ing Blastocystis hominis were sometimes suffering from

rate, the parasitic infection may be blocked if Japanesediarrhea, and the other residents did not show any

These results suggest that there is a risk for visitors

symptoms. We cannot consider that Blastocystis

apart from Indonesian society and rural environments.
to infect intestinal parasites in Indonesia.

hominis may be pathogenic. Both Sheehan et al (1986)

and Pikula (1987) reported a positive association
between B. hominis and E. histolytica. E. histolytica wa

not found in Japanese and Indonesian residents.
Serological evidence of parasitic infection was diffi‑

cult to evaluate, as we could not differenciate an active

infection from that of a prior infection by Entamoeba
histolytica (Ximenez et al 1993). While stool samples
were negative for E. histolytica in all 256 stool samples,

14 subjects were positive for anti‑E. histolytica anti‑
bodies in blood by using ELISA. It must also be careful
to infect E. hystolytica in Indonesia as it is consideredthat at least there were past histories infected with E.
histolytica. Five native indonesians had Blastocyctis

hominis and antibodies tb E. histolyticla. Japanese
residents, who kept Blastocystis hominis, had not anti‑
bodies to E. histolytica. (data not shown)
In this study, the incidence of parasitic infection in

the Japanese residents with less than one year stay was

O%. As the parasitic infection rate of Japanese withmore than one year stay was more than 10%, there may
be a risk for Japanese to infect parasites in Indonesia.

Meanwhile residents of three Japanese communities[Luwuk, Merak, and Menado] showed a zero infection

REFEREN CES
Ashford, R. W. and Atkinson, E. A. (1992) : Epidemiology ofBlastocystis homiilis infection in Papua New Guinea: age‑prevalence and associations with other parasites, Ann.
Trop. Med. Parasitol., 86, 129‑136
Chacin‑Bonilla, L., Bonilla, E., Parra, A. M., Estevez, L. M.,

Morales, L. M. and Minakami, K., Anteson, R. K. andAppawu, M. A. (1992) : Effects of nutritional defficiency

on parasitic infection of the infant in greater Accra,
Ghana, Jpn. J. Trop. Med. Hyg., 20, 143‑147
Pikula, Z. P. (1987): Blastocysds hominis and human disease,
J. Clin. Microbiol., 25, 1581

Sheehan, D. J., Raucher, B. G. and McKitrick, J. C. (1986):
Association of Blastocystis hominis with signs and symp‑
toms of human disease, J. Clin. Microbiol., 24, 548‑550
Ximenez, G., Leyva, O., Moran, P., Ramos, F., Melendro, E.
I., Ramiro, M., Martinez, M. C., Munoz, O., Kretschmer,
R. and Ar llano, J. (1993): Entamoeba histolytica: anti‑
body response to recent and past invasive events, Ann.
Trop. Med. Parasitol., 87, 31‑39
Zierdt, C. H., Rude, W. S. and Bull. B. S. (1967): Protozoan
characteristics of Blastorystis hominis, Am. J. Clinical
Parasitol., 48, 495‑501

Jpn. J.

219

Tro p. Med. H yg ., Vol. 22, No. 4, 1994, pp. 219‑223

Case report

CASE REPORT OF LEPROSY AND A TRIAL OFSCREENlNGS FOR THE FAMILY MEMBERS
IN ECUADOR
ATSUSHI HOSOKAWA, SHIGEO NoNAKA, JUAN J. ALAVA P,
EDUARDO A. GOMEZ L., HUGO M. JURADO SAND YOSHIHISA HASHIGUCHI
Received September 20, 1994/ Accepted October 25, 1994

Abstract: Four cases of patients with leprosy were seen in an area endemic for cutaneous leishmaniasis, Los

Ranchos, Departrnent of Manabi, Ecuador. Two cases of them (borderline lepromatous leprosy andindeterminate one) in a single family and result of screenings for the family members were reported. It wassuggested that family examination of leprosy patient might be useful for early detection of leprosy in a low
endemic areas for leprosy, such as Department of Manabi. A nine banded armadillo kept by the family was
examined, but no acid‑fast bacilli was observed in the liver materials.

leprosy patients were also examined. The current paper

INTRODUCTION

deals with the two cases in a single family in detail and

Leprosy and leishmaniasis are etiologically com‑pletely different diseases but it has been known that thetwo diseases have similar cutaneous manifestations(Jopling, 1984; Butto et al., 1994). Therefore, bothpreliminary screenings for the family members of lep‑rosy. Furthermore, based on the result obtained a briefcomment was also made on the screening methods todetect leprosy patients in early stage of the disease.

diseases should be differentiated in the endemic areas.
During a survey for cutaneous leishmaniasis in Ecuador,***
No‑1 3No.6
N0.7 .

N0‑1 O

N0,9. 1. 5. 4. 3 2

Noll.12.

*Imale lep'osy patient, **ete*ale leprosy pat,ent[] male. t Jfemale

Figure 1 The pedigree of leproSy patientS m Los Ranchos Department of Manabi Ecuador

2.
3.
4.
5.

Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903‑01, Japan
Universidad Tecnica de Portoviejo, Portoviejo, Manabi, Ecuador.
Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de Guayaquil, Guayaquil, Ecuador
Centro Nacinal de Medicina Tropical. Facultad de Ciencias Medicas, Universidad de Guayaquil, Guayaquil, Ecuador
Department of Parasitology, Kochi Medical School, Nankoku, Kochi 783, Japan220

not palpated (Fig. 3). Annular and infiltrated eryth‑

CASE 1The patient was a 41‑year‑old‑male (N0‑7) .

emata were scattered on the trunk. Anesthesia was

Family history. His grand father, an uncle, an aunt,

a nephew, a cousin, and a daughter were reported as

observed on all these eruptions. The body surface wasdry except for the axillary, the epigastric, the lower

suffering from leprosy by interview to the famiiy (Fig.

abdomen, the inguinal, the perineal and the anal r, egion.

l) .

Present history. About 15 years ago, erythemata
with anesthesia appeared on the abdomen. The eruption

The ichthyosis‑1ike change was seen on the region of thewaist, the back, the extensor aspect of the legs and thefeet (Fig, 4). Although the deep ulcers on the soles had

gradually increased in number on the face, the trunk andthe sharply demarcated bank, it did not have the indura‑

the four extremities. At the same time, there was arising in body temperature, and neuralgia appeared inboth exteremities. In 1982, a doctor from the Welfare

tion at the margin of the ulcer that was observed in
cutaneous leishmaniasis. The patient had anesthesia at
the uicers and could walk without pain. The big toes
were mutilated and remaining tces were shortened (Fig.
5). Bilate‑ral apehands and clawhands were seen and

Minl"stry diagnosed him as leprosy.

Treatment. A 100mg per day of DDS (4, 4'
MDT) wasfissures caused by trauma on the right paim weremarked (Fig. 6). There was no loss af hair at any

started according to MDT for multi‑bacillary leprosy

region of the body. Hypertrophy of the ulnar nerves

recommended by the World Health Organization

were palpated at the elbows.

(WHO) (Noodeen, 1991). His prescription was asfollows; DDS (lOOmg/day) and B663 (50mg/day) ,Laboratory examination.

‑diaminadiphenylsulfone) was regularly prescribed for

two years. In 1990, multidrug therapy

every day; B‑663 (300mg/day) and Rifampicin (600mg/l ) The lepromin test: negative.
2 ) Value of antiphenolic glycolipid (PGL)‑ I andday) , once a month,Present illness. Infiltrated erythemata on the face,result were PGL‑ I (IgG) O.089 OD unit, P(*.L I (IgM)

destruction of nasal septum and deformity of the nose

l.693 OD unit, LAMB (IgG) O.545 OD unit and LAM‑B

were observed (Fig. 2) . Pea sized reddish noduies on the(IgM) O.281 OD unit.
3 ) Examination of sensory function: Anesthesia

earlobes were soft and the induration af the lesions were

Figure 2anti‑lipoarabinomannan (LAM) B antibody: Positive

Deformity of the nose Figure 3 Pea sized nodule with sen‑ Figure 4 The ichthyosis‑1ike change

because of destruction of sory loss on the earlobe ofon the extensor aspect of 1. eg

nasal septum and infiltrated Case l,

and foot. Deformity of toes

erythema with sensory loss
on the face (‑) of Case 1

are also obseved.

(No, 7: 41‑year‑old male).

221

i = '

=+･+･ ･+'+*

Figure 5 Deep ulcer on'the sole and shartened and mutilat‑
ed toes cn the foot af Case 1.

Figure 6 Apehand and claw‑hand of both hands of Case

was observed on all over the body surface except for the

CASE 2

scalp, the axillary, the epigastric, the inguinal, the per‑ineal and the anal region.

4 ) Histological findings of the specimen takenfrom the infiltrated erythema on the cheek: Rete peg

disappeared. Clear subepidermal zone was observed(Fig. 7). Dermis was edematous. Though relativeiy
large number of epithelioid cells and lymphocytes in=filtrated in the dermis, epithelioid cell granuloma was

not observed. By acid‑fast staining (The Fite‑Feracol.

The patient was a 12‑year‑old female (No‑1). A
daughter of Case I (Fig. 1).

Present history. When we examined the family of
the Case I patient, hypopignented fleckle on the exten‑
sor aspect of the left thigh was noticed. The patient had
been living in the house at the vill ge of Los Ranchos,
Depatment of Manabi since her birth.
Present illness. Palm sized hypopigmented freckle
with anesthesia on the extensor aspect of the left thighstaining Method) , acid fast bacilli were observed (Fig.

(Fig. 9) . Hair loss was not observed at any region of the8). Biopsy index: 3+, SFG index, 5; SFG value, 12‑1.

body surface. Hypertrophy of peripheral nerve was not

By skin slit smear of the left earlobe, acid fast bacilli

pal pated.

stained by Ziehl‑Neelsen's staining were found̲ Bacte‑rial index showed 3 + ; SFG index, 4; and SFG value, 1‑2‑2. Nasal scraping was negative. The tyPe of leprosy
was borderline lepromatous.

Laboratory examination;1 ) Mitsuda eary reaction (48 hrs) : 7.5mm x
7.5mm/7.5mmx 7.5mm, undeterminable (+/‑) .
2 ) Value of anti‑PGL‑ I and LAM‑B antibodies:
Positive results were PGL‑ I (IgG) 0.ll7 OD unit, PGL‑ I (1gM) O.804 OD unit and LAM‑B (IgM) O.191 OD･ 2‑

Figure 7 Case l, clear subepidermai zone is presented( ) , and typical epithclioid cell granuloma is
not presented (HE, x 100).

Figure 8 Case 1, Mycobacterium leprae (‑) observed indermis (Fite‑Feraco staining Method, x 1,000).

222

mycobacterial disease (infectious in some cases) , pri‑marily affecting the peripheral nervous system andsecandarily involving skin and certain ather tissues(Jopling, 1984) . Leprosy has a wide range distribution

in the world. Leprosy is included among the six mostimportant infectious diseases, as well as leishmaniasis,

which the WHO planned to stop from being an endemic,
and is still a public health problem in developing coun‑tries including Ecuador. In Ecuador, 110 patients with
leprosy were newly deagnosed in 1991; the prevalence
rate of leprosy in Ecuador was relatively iow (O.25 perl,OOO habitants in total) in comparison with that ofendemic areas such as South east Asia and Africa, 0.1‑4.
9 and 0.1‑6.9 per 1,000 hab. respectively (MCDOugall etaL, 1989). It was relatively low (0.10‑0.16 per 1,000hab.) in Department of Manabi, from where the present
cases were reported. As to future group examinations
for leprosy, in a low endemic area, such as Departmentof Manabi, the screening of leprosy family would be
Figure 9 Palm sized hypopigmented freckle with
anesthesia on the extensor aspect of thigh ofCase 2 (‑).
unit. Negative resut was LAM‑B (IgG) O.060 OD unit.
3 ) Examinatibn of sensory function: Anesthesia
was observed only at the lesion of the left thigh.

effective and useful in consideration of a program forthe early detection. The deformities of Case I could be
prevented by early detection and adequate treatment.
Case 2 was fortunately detected in early stafge of thedisease by a doctor during the present examination.

In Okinawa. Japan, for example, graup leprosy
examinations had been done in alrnost all regions of theprefecture during about 20 years by specialists who were

no epithelioid cell granuloma in the dermis. No acid fast

appointed by the prefectural goverer (Saikawa, 1989).
The examination was called as a general medical check‑up and not as a mass examination for leprosy, because
in Okinawa, many of the inhabitants have been preju‑

bacilli was observed in the skin tissue section stained by

diced against leprosy patients as well as the inhabitants

acid‑fast staining (Ziehl‑Neels.en and the Fite‑Feraco

in Ecuador. Members of the group examinations were

staining Methods). The patient was diagnosed as in‑determinate leprosy according to the histological andconsisted of a leprologist, public health nurse from the4 ) Histological findings of the specimen takenfrom the lesian; small number of lymphocytes infiltratearound the capillaries and the appendages. There waspublic health center of each district, manager of leprosy

clinical criteria of Ridley and Jopling (1966).

and tuberculosis from the prefectural office, a dermatol‑

Besides the two cases mentioned above, other two
leprosy patients (one with borderline tuberculaid andother with unknown type) were examined in the current

ogist, an internist and a clinical technologist etc. In thegroup examination, therefore, various examinations
were done to find skin diseases, infectious diseases

study. In addition to these four cases, the existence ofinncluding leprosy and tuberculosis, circulatory diseases

other four patients with leprosy in this village wasdetermined. Besed on these examinations and inter‑such as hypertension, diabetes mellitus and other inter‑view, the figure of the pedigree was depicted, including

nal disorders. The data from this medical check‑up
were reported to each subject by the public healthrelationships among leprosy patients in the endemic

center. If some pathological result was observed, thearea for cutaneous leishmaniasis of Los Ranchos,
Department of Manabi (Fig. 1). A nine banded arma‑

subjects were examined in detail at the nearest hospitalor public health center. Leprosy patients detected in the

dillo kept by the family was examined, but no acid‑fastbacilli was observed in the liver materials.

general medical check‑up were treated by the le‑Comments;tion.

Leprosy (Hansen's disease) is a chronic

prologists. Their family members were also exam,ined
in detail and some of them received preventive medica‑
Recently, serodiagnosis of leprosy was considered

/

/

223

as one of the useful methods for early diagnosis

REFERENCES(Bchanan, et al., 1983). The serodiagnosis have also1991). During the current family examination, ser‑

1 ) Abe, M., Ozawa, T., Minagawa, F. and Yoshimoto, Y.
(199D: Immunoepidemiological studies on subclinicalological examination for 13 subjects was also carriedout. Three leprosy patients were positive for P L‑ I
(IgM) and LAM‑B (IgG) , and three out of four leprosy
patients were strongly positive for LAM‑B (IgM) (data2 ) Buchanan, T., Dissanyake, S., Young, D. B., Miller, R.
A., Acedo, J. R., Harish, J. P., Khanolka, S. R. andEstrada‑Parra, S. (1983) : Evaluation of significance ofantibodies to phenolic glycolipid of Mycobacterium le‑been carried out by leprologists in Okinawa (Abe et al.,infection in leprosy, Jap. J. Leprosy, 60, 72‑84

not shown) . Based on the results, it was suggested that
serological examination of families of leprosy might beprae in leprosy patients and their contacts, Int. J. Lepr.,

useful for screening of house hold contacts in a low
endemic area for leprosy.

3 ) Butto, A. M., Nonaka, S. and Hashiguchi, Y. (1994):
First cace of difftiSe curtaneous leishmaniasis in

These systems of group leprosy examination in
Okinawa might be a good guide for a future program for51, 658‑659

Ecuador, Jpn. J. Trop. Med. Hyg., 22 (suppulement) , 973 ) McDougall, A. C. and Yawalkar, S. J. (1989) : Distribu‑

the early detection in Ecuador. In consideration of
economical and man‑power resources, some modifica‑tion of leprosy, Leposy, second de., 11, CIBA‑EIGY
LTD, Basel4 ) Jopling, W. H. (1984) : Difinition, Epidemiology andtions will be needed to implement this system of group

World Distribution, Handbook of leprosy, third ed., 1,

leprosy examination into Ecuador.

William Heineman Medical Book LTD, London

Cutaneous manifestations in leprosy sometimes5 ) Jopling, W. H. (1984): Differential diagnosis. Hand‑

showed a similarity to those of cutaneous leishmaniasis.

book of leprosy, third ed., 121, William Heineman
Medical Book LTD, London

Early nodular lesions of cutaneous leishmaniasis are
similar to those of lepromatuous leprosy (LL) and thetype of cutaneous leishmaniasis most likely to be con‑fused with LL is the disseminated anergic form. In postkala azar dermal leishmaniasis, hypopigrnented flekle

appear on trunk and limbs, which should be distin‑
guished from those observed in indeterminated leprosy(Jopling, 1984). In the present preliminary examination
for leprosy, anestesia at the lesions was the most impor‑tant symptom for the diagnosis.

The patients (Cases I and 2) kept a nine banded
armadillo for their food. In the smear specimens of theliver, no acid‑fast bacilli and Leishmania amastigotes
were observed. Wild armadillo with leprosy were foundin U. S. (Smith et al., 1978; Marchiondo et al., 1980).

Although no such a report was described in Ecuador, theexamination should be continued to ascertain whether
leprosy was a zoonosis or not, and whether the nine
banded armadillos were natural resorvoirs of the path‑ogen in Ecuador.

6 ) Kosaka, K., Yoneda, K., Makiko, M., Mori, T. and Itoh,T. (1978) : Nude mouse for reseach in leprosy, Int. Lepr.
Congr. Proc. XI , 37‑43
7 ) Marchiondo, A. A., Smith, J. H. and File, S. K. (1980):
Naturally occuring leprosy‑like disease of wild armadil‑los, ultrastructure of lepromatous lesions, J. Reticuloen‑
dothel. Soc., 27, 311‑325

8 ) Noordeen, S. K. (199D: Global review of MDT Imple‑
mentation, Wld. Hlth. Org. techn. rep. ser., 1169 ) Ridley, D. S. and Jopling, W. H. (1966) : Classification of
Leprosy According to Immunity, Int. J. Lepr., 34, 255‑273

10) Saikawa, K. (1989) : Epidemiology of leprosy. Jap. J.
Leprosy, 58, 143‑156
11) Sheperd, C. C. (1965): Stability of M. Ieprae and temper‑
ature optimum for growih, Int. J. Lepr., 33, 541‑547

12) Smith, J. H., File, S. K., Nagy, B. A., Folse, D. S.,
Buckner, J. A., Webb, L. J. and Beverding, A. M. (1978) :Leprosy‑1ike disease of wild armadillo in French
Acadiana, Luisiana. J. Reticuloendothel. Soc., 24, 705‑719

Research note
Thomas B. Nyambo and Kanbara, H.
Growth Inhibition of Cultured Plasmodium falciparum by Immune Serumfrom Tanzania.

･211‑215

Short communication
Yamada, S., Mori, Y., Yue Pan, Akao, N., Tsukidate, S. and Fujita, K.
Survey of Intestinal Parasite Infections in Japanese Living in Indonesia･217‑218

Case rerort
Hosokawa, A., Nonaka, S., Juan J. Alaba P, Eduardo A. Gomez L.,
Hugo M. Jurado S. and Hashiguchi, Y.
Case Report of Leprosy and a Trial of Screenings for the Family

Members in Ecuador･219‑223¥

VOL．22NO．4

回ECE

BER1994

CONTENTS
Original article
Wei−Yun Zheng
Genotype Identification
from

of

a

SingleEI

andof Hepatitis C Virus（HCV）Isolated
Carrier in Nagasaki Prefecture and Genome Analysis

Japanese

E2／NSI

Envelope

Glycoprotein

Hosokawa，A．，Nonaka，S．，MiguelMimori，T．，Katakura，K．，Eduardo

Seroepidemiological

Regions

・・169

177

H．Jurado，Furuya，M．，Eshita，Y．，
A．Gometz L．，Izumi，S．and Hashiguchi，Y、

Surveysfor

LeprosyinMefloquinforLong−Term

・・17H84

Ecuador……

Takeshima，S．
Side

Effects

Kano，S．andA

Case

throughofwith

Malaria

Prophylaxis（inJapanese）一・・18H92

Suzuki，M．
Accidental

PlateletTransmission

Transfusion（inofPJ4s規04」麗彫力lo加7％窺Japanese）………

一193−198

Oda，T．，Suenaga，0．，Zaitsu，M．，Kurokawa，K．，Fujita，K．，

Ogawa，Y．，Yamazaki，1．，Iida，K．and

Comparison

of

and

Their

Naturalof

Nagasaki

New

South

BlackflyMine，M．

of

VectorMosquitoesof

Infection

Rates

in

and

City，Japan

Takaoka，H．andA

Population

SouthemjD伽瞬肱吻ガ窺窺痂sNorthemParts
一199−206・一Suzuki，H．
SpeciesofS加κ1勉窺（〃ozρρs）from

SolomonIslands，

Pacific（Diptera：Simuliidae）…一（Contents

continued

・・207−210

onreverseof

thiscover）