1991年12月15日

第19巻第4号
内原容著

Immmogenicity
envelopeofslowlyglycoprotein

sedimenting

isolatedfromantigen of

infected

Japanese

culturefluids・・Golam Masud
フィラリア感染好適宿主としてのMongolian

encephalitis virus

MohammadShameem321−330

gerbi1の基礎的検討

一血液学的特性に関する被毛色間の比較一（英文）

一清水

眞澄，七戸

和博，月舘

説子，藤田紘一郎 331−338

Mebendazoleによる糞線虫症治療に関する臨床的研究（英文）一志喜屋孝伸，上地 博之，斎藤

厚，安里

龍二

339−346

AIDS以外の免疫抑制患者におけるニューモシスチスカリニ肺炎の管理（英文）

・・勝又

松田

達哉，河野

茂，古賀

治子，光武耕太郎，東山

宏延，吉富

祐子，

康仁，宮崎義継，

原耕平

347−355

ジャワ島産アシマダラブユ亜属の2新種について（英文）一高岡 宏行，Upik

Kesumawati

Hadi357−370

皮膚リーシュマニア症の治療一Promastigote型原虫に対するアンチモン剤とMetronidazoleの薬効の比較…・矢後文子 371−385
会報・記録

日本熱帯医学会会員への手紙一

387−388

会員消息…一
投稿規定…一……

388

編集部だより389−390391−394一…

395−396日本医学会だより

日本熱帯医学会

日本熱帯医学会雑誌

発行所

第19巻

編集者 板

第4号

平成3年12月15日

印刷

平成3年12月15日

発行

日本熱帯医学会
倉英喜印刷所昭和堂印刷
諌早市長野町1007−2（⑰854）
智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。

Jpn. J. Trop. Med. Hyg., Vol. 19 No. 4 1991, pp. 321 330

321

IMMUNOGENICITY OF SLOWLY SEDIMENTING
ANTIGEN OF JAPANESE ENCEPHALITIS
VIRUS ENVELOPE GLYCOPROTElN ISOLATEDFROM INFECTED CULTURE FLUIDS
GOLAM MASUD MOHAMMAD SHAMEEM
Received July 12 1991/Accepted September 2 1991

Abstract: Slowly sedimenting antigen (SE) and the rapidly sedimenting antigen(RE) , associated with envelope glycoprotein E were prepared from the concentrated
infected culture fluids of Japanese encephalitis (JE) virus. Mice were immunized by eachantigen before and after inactivation of virus infectivity by ultraviolet (UV) irradiation.

The immunogenicity of the antigens determined by the plaque reduction neutralization
test (PRNT), indirect ELISA and hemagglutination‑inhibition (HD test, indicated that
SE was almost equally immunogenic as RE, and that the PRNT epitopes on both antigens
were more immunogenic before UV‑inactivation than after the inactivation.

INTRODUCTION
JE is an acute viral encephalitis and is a serious public health problem in many Asian
countries (Umenai et al., 1985). The etiologic agent, JE virus, belongs to the Flaviviridae
family (Westaway et al., 1985) comprising about 70 viruses (Calisher et al., 1989), of which

many are pathogens for humans and domestic animals (Monath, 1986) . Current JE vaccine
has been developed as formalin‑inactivated and highly purified virion from infected mousebrains and has successfully been used for JE control in Japan, Republic of Korea, and someother countries (Hammon et al.. 1971; Huang, 1982; Oya, 1988; Igarashi, 1988) . Besides
rapidly sedimenting viru antigens associated with complete virion, slowly sedimenting
hemagglutinin or soluble complement‑fixing antigen (SCF) of fiaviviruses have been de‑scribed in the infected mouse brain homogenates or in culture fluids by sucrose gradientsedimentation (Igarashi et al., 1963; Kitaoka and Nishimura, 1965; Smith et al.. 1970;
Shameem et al., 1989). However the immunogenicity of SE has not well been documented.
In order to clarify the immunogenicity of SE and RE, we examined anti‑JE titer of the serafrom mice immunized with SE or RE before and after UV‑inactivation.

MATERIALS AND METHODS
Cells and virus
Aedes albopictus, clone C6/36, cells (Igarashi, 1978) were grown at 28'C and BHK21 cells

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

12‑4 Sakamoto‑machi, Nagasaki 852, Japan322

at 37'C in Roux bottles with 750 cm* volume. The cell growth medium was Eagle's minimum
essential medium supplemented with 0.2 mmol/1 each of 7 nonessential amino acids (Eagle,1959) and 10% heat‑inactivated fetal calf serum (FCS) . The origin of a wild strain JE virus,

JaOArS982, was described by Hori et al. (1986) , and seed virus was prepared in C6/36 cells
at 28'C.

Preparation of slowly sedimenting and rapidly sedimenting antigens of JE virus
The SE and RE were isolated by sucrose gradient sedimentation of concentrated infected
culture fluids as described in our previous communication (Shameem et al., 1989) . In thisexperiment specimens were harvested 36 hr after infection for both cell lines, but incubatedat 28'C for C6/36 and 37'C for BHK21 cells, respectively.

Inactivation of the infective virus by UV‑irradiation
Immunogens (2.5 ml) were spread on a petri dish (60 mm) and exposed to 2 UV bulbs(20 W) at a distance of 42 cm for 5 minutes. After the exposure, infectivity of the virus wasundetectable (Sachiko Matsuo, personal communication) .

Immunization of mice
BALB / c mice were immunized by 4 intraperitoneal injections at I week interval(0.1 ml/mouse/dose) , using either UV irradiated or unirradiated SE and/or RE fractions,which were mixed with an equal volume of Freund's complete adjuvant for the first injection
or incomplete adjuvant for the subsequent injections. One week after the last injection, mice

were individually bled and serum was separated for further test.

Plaque reduction neutralization test (PRNT or N)
This procedure was carried out as described by Hashimoto et al. (1971) with somemodification. Serial two fold serum dilutions beginning from l:lO were made using thediluent of 5% FCS in Eagle's medium and mixed with an equal volume of the seed virus
diluted to yield 100‑200 PFU/0.1 ml. For virus control the working dilution of the virus was

mixed with an equal volume of the diluent. The mixtures were incubated for 90 min at
37'C in humidified atmosphere containing 5% C02. At the end of the incubation, the mixture

was inoculated to the monolayer of BHK21 cells (0.1 ml/welD grown on 24‑well polystyrene
tissue culture plate. Adsorption was carried out at 37'C for 2 hr and the cells were covered
with 1.25% methyl cellulose in the maintenance medium (cell growth medium from which
serum concentration was reduced to 2%). Plates were incubated for 5 days in the same
condition as above and cells were fixed with cold methanol, stained with 0.1% trypan blue in
0.9% NaCl at room temperature for I hr to reveal the plaques. The N was scored as positive
when more than 50% plaque reduction was observed compared with the negative control ofvirus‑diluent mixture. Reciprocal of the highest dilution of the test serum with positiveplaque reduction was considered as N titer.

Sandwich ELISA
The modified procedure of Voller et al. (1976) was described in our previous communica‑tion (Shameem et al., 1989).

323

Indirect micro ELISAThis was carried out according to Voller et al. (1976) using purified JE antigen (100 pglmt). ELISA titer of test specimens was calculated b, comparing the OD with those by
serially diluted standard positive serum as described before (Igarashi et al.. 1981; Morita etal., 1982) . Standard anti‑JE mouse serum was prepared by repeated intraperitoneal inocula‑tion of purified JE virus (Srivastava et al., 1987).

Hemagglutination‑inhibition (HI) test
The procedure was carried out according to Clarke and Casals (1985) using goose red
blood cells in virus adjusting diluent (VAD) at pH 6.6.
Statistical analysis

Student's t test was performed according to the standard procedure.

RESULTS
Mouse immunogenicity of SE and RE before UV‑inactivation
In the initial experiment, mice were immunized with SE or RE fractions of both C6/36
and BHK21 cells without UV‑inactivation. The antibody titer of individual serum from
immunized mice was shown in Table 1. When antibody titers raised by SE and RE werecompared. SE immunogen from C6/36 cells produced less N and HI titers than RE from thesame cells, and these differences were stati tically significant (Table 3) . While, both SE andTable I Immunogenicity of SE and RE before UV‑irradiation, as shown bytiters in log for N. ELISA and HI

Serum
No.

1‑1
l‑2
l‑3
l‑4
2‑1
2‑2
2‑3
2‑4
3‑l
3‑2
4‑1
4‑2
4‑3
4‑4
4‑5

Immunogen

C6/36 SE

C6/36 RE

N
Mean

Titer
1 . 30l . 68l . 60(GMT 48)

O . 293 . 85

4 , 72Mean
4.28i0.40(GMT 19 , 054)

Titer
l . 901 . 904 . 04

1 . 90l . 904 . 492 , 512 . 202.20d:0.25

4 , 16(GMT 158)

4 , 65

1 . 902 . 20

BHK21 RE

Titer

1 . 902.51

BHK21 SE

HI

ELISA

2 . 662 . 51(GMT 457)

2 . 51

4 , 143 . 11

2 . 052 . 20(GMT I12)

4 . 482 . 202 . 08 i O . 16

4 . 062 . 20(GMT 120)

4 , 484 , 494 . 72

4.49:k0.01(GMT 30 , 902)
4 . 22 d: O . 38

(GMT 16 , 595)

O . 302 . 202 . 362 . 51(GMT 229)

2 . 81
2 . 51
2 . 51l . 903 , 893 , 112 . 203 , 962 . 51

Note: GMT =Geometric mean titer2.05 0.31(GMT I12)

2 . 564 . 22l . 902 . 20O . 21

4 . 29:!: O . 24

(GMT 19 , 498)

Mean

O . 22

2.69 0.27(GMT 490)

324

Table 2

Serum
N o.

1‑l
1‑2
1‑3
1‑4
2‑1
2‑2
2‑3
2‑4
3‑l
3‑2
4‑1
4‑2
4‑3
4‑4

Immunogenicity of SE and RE before UV‑irradiation, as shown bytiter ratio of ELISA/N and ELISA/HI

ELISA/N

Immunogen

C6/36 SE

C6/36 REELISA/HI

Titer

Mean

GMT

Titer

2 . 552 . 60 d: o . 40398

1 . 953 . 12

2 . 82

2 , 142 . 142 . 591 . 98

1 . 962.09 0 17

123

2 , 14

BHK21 SE1 . 71

1 . 94

1 . 032.44 :0.22272

2 . 28
1 . 86

4‑5
Table 3o . 41166

1 .38:!:O. 35

24

2. 13i O . 23

134

57

34

2. 22

1 . 142 , 32

2 . 59

BHK21 REl . 65GMT

Mean

2 . 291 . 972.07 0 31

119.1 . 25

2 . 26

1 . 972 . 522 . 21

1 . 99O . 78

1 . 76

1 . 45

1 . 53O .

Statistical analysis on N, ELISA and HI titerand titer ratio difference.s raised by SE and

RE before UV‑irradiation
Iog titer and

Immunogens

titer ratio

SE and RE of C6/36

N
ELISA

P valueo. 02< P <0‑ 05> O ･ 10HIo. 02< P <0‑ 05

ELISA/N
ELISA/HIo. 02< P <0‑ 05

SE and RE of

N

BHK21

ELISA

>0･10

>0.10
o. 02< P <0. 05

HI> O . 10ELISA/N
ELISA/HI>0.10> o . 10

RE from C6/36 cells produced similar ELISA titers. In the case of immunogen from BHK21
cells, both SE and RE produced similar N and HI titers, while ELISA titer produced by RE
was less than the titer by SE and the difference was statistically significant (Table 3). The

antibody titers produced by immunogens from C6/36 and BHK21 cells were almost the same
except that N titer by SE antigen from C6/36 cells was lower than the titer by other antigens(Table 1). Since immunogens of SE and RE from C6/36 cells and SE from BHK21' cells

325

possessed comparable ELISA titers as shown in Table 4, the results may indicate low
immunogenicity of SE from C6/36 cells to induce N antibodies. Lower ELISA titer by RE
from BHK21 cells may be explained by its slightly less ELISA titer (Table 4) . The antibody

titers were compared by their ratios (ELISA/N and ELISA/HD , and the results were shownin Table 2. The SE from C6/36 cells showed higher ELISA/HI ratio than RE from the same
cells with statistically significant difference, while other ratios did not show significant

difference between SE and RE. In this series of experiment a high mortality rate of micearound 40% was observed probably due to encephalitis caused by residual infective virus in
the immunogens (data not shown) . Therefore, it was conceivable that the antibody titers andtheir ratios in Tables I and 2 might reflect the effect of viral antigens produced in mice after

virus replication, besides immunogenicity of inoculated specimens. Therefore, subsequent
experiments were performed by immunogens which had been inactivated by UV‑irradiation.

Mouse immunogenicity of SE and RE after UV‑irradiation
Each immunogens of SE and RE from C6/36 and BHK21 cells were irradiated by UV as
described in the Materials and Methods, and ELISA titers of these immunogens were shownin Table 4. The ELISA titer of SE from both C6/36 and BHK21 cells was reduced around78%, while the titer reduction of RE was less (around 42‑43%) .
The antibody titers of individual mouse serum raised by UV‑inactivated immunogens
were shown in Table 5. Only the HI titer produced by RE from BHK21 cells was higher than
the titer by SE from the same cell, and the difference was statistically significant (Table 7) .

The titer ratio (ELISA/N and ELISA/HD of the individual mouse serum was shown in
Table 6. In this case, only the ELISA/HI produced by SE from C6/36 cells was higher than
the titer by RE from the same cells, and the difference was statistically significant.

Comparison of the immunogenicity before and after UV‑irradiation
The effect of UV‑irradiation on the immunogen was evaluated by comparing theantibody titers (N, ELISA, HD as well as their ratios (ELISA/N, ELISA/HD raised by each
immunogen before and after UV‑irradiation. The statistically significant difference wasobserved only for the reduction in HI titer produced by RE from C6/36 cells and the increasein ELISA/N ratio produced by RE from BHK21 cells (Table 8). Other immunogenicities
were apparently not affected by UV‑irradiation.

Table 4 ELISA titer of immunogens before and after UV‑
irradiation

ELISA titer of immunogen % Reduction

Immunogen before UV after UV in ELISA titer
C6/36

SE

232

50

78 . 45

C6/36

RE

206

116

43 . 69

BHK21 SE
BHK21 RE

229

50

78 . 15186

107

42 . 48

326

Table 5

Immunogenicity of SE and RE after UV irradiation, as shown bytiters in log for N, ELISA and HI

Serum

No Immunogen Titer

1‑l
1‑2
1‑3
l‑4
l‑5
2‑l
2‑2
2‑3
2‑4
3‑1
3‑2
3‑3
3‑4
3‑5
4‑1
4‑2
4‑3
4‑4

C6/36 SE2 . 20l . OO

C6/36 RE

N

ELISA

Mean
l.54:

O. 54

(GMT

35)

Mean

T iterMean

4 . 584.42 0.20(GMT 26,302)

1 . 90l.96 0. 13

4 . 58

1 . 904.11

l . 901 . 904 . 482 . 20l . 604 . 35

1 . 902 . 201 . 98 O. 29

4 . 292 . 20(GMT 95)

4 . 232 . 201 . 604.40 0. 17(GMT 25 118)

4 . 51

1 . 60

BHK21 RE

Titer

1 . OO

1 . 90

BHK21 SEHI

(GMT 69)

4 . 644.51

2 . 512 . 28

1 . 90(GMT 191)

2.51
4 . 43i O . 23

2 . 51(GMT 26,915)

1 . 901 . 304 . 612 . 202 . 204 . 352 . 201 . 904 . 082 . 201 . 90l . 901.90i:O. 24(GMT 79)

O . 292 . 204 . 581.84d:O. 39(GMT 91)

4 . 354 . 50i O . 12

4 . 58(GMT 31,622)

2.20 0.22(GMT 158)

2 . 512 . 663 . Il(GMT 457)

2 . 204 . 442 . 201 . 604 . 61

2 . 81O . 39DISCUSSION
From the results of our immunological studies it appears that the SE of JE virus isimmunogenic in mice to induce N, ELISA and HI antibodies, and the immunogenicity of bothantigens, SE and RE, were almost comparable. Although UV‑irradiation totally inactivatedinfectivity and partially destroyed ELISA antigenicities in both SE and RE, they still retained

their immunogenicities. The different UV‑susceptibility of ELISA antigenicity between SE
and RE from both C6/36 and BHK21 cells may be explained by different target size of theparticle. The RE is the complete virion and possesses larger size and greater number ofrepetitive epitope units than SE, which is smaller size with less number of repetitive units. It

is reasonable to imagine that RE with larger number of repetitive ELISA antigenic epitopes
is more resistant to UV‑irradiation than SE. Kimura‑Kuroda and Yasui (1983) reported thatat least five topographically distinct antigenic determinants including N, HI, and ELISA
epitopes, were present on E protein and the epitopes of HI and N were separated from eachother by monoclonal antibodies.
Recently, Mason et al. (1991) reported that the JE vaccinia virus recombinants possess‑ing JE virus CDNA inserts produced particulate form of antigen containing M (membrane)
and E proteins of JE virus. The antigen migrated in sucrose gradients slower than thecomplete virion, similar to the SE, and induce high level of anti‑JE N antibodies andconferred protection against lethal JE virus infection.

327

Table 6

Serum
No.

1‑l
l‑2
l‑3
1‑4
1‑5
2‑1
2‑2
2‑3
2‑4
3‑l
3‑2
3‑3
3‑4
3‑5
4‑l
4‑2
4‑3
4‑4

Immunogenicity of SE and RE after UV‑irradiation, as shown bytiter ratio of ELISA/N and ELISA/HI

Immunogen

C6/36 SE

C6/36 RE

BHK21 SE

BHK21 RE

Table 7ELISA/HI

ELISA/N
Titer

Mean

GMT

T iter758

2 . 682 . 38 2 . 88 i O . 47
3 . 582 . 683.11

2 . 21

2 . 582 . 282 . 752 . 452 . 09 2 . 45i O . 46281

1 . 782 . 032 . 332 . 61

2 . 31

2 . 982 . 072 . 44 2 . 60i O . 50

398

2 . 132 . 912 . 613 . 31

2 . 41

2 . 152 . 152 . 18l . 882 . 45 2 . 59

O . 32389

1 . 842 . 68l . 472 . 242 . 243 . Ol

1 . 80

Mean
2. 46

288

2. 12 :!: O . 25

131

2 .23 0. 28

170

l. 84

69

O . 31

Statistical analysis on N, ELISA and HI
titer and titer ratio differences raised

by SE and RE after UV=irradiation
log titer and

P value

Immunogens

titer ratio

SE and RE of C6/36

N> O . 10

ELISA

> O . 10HI

> O . lO

ELISA/N
ELISA/HI> O . 10

SE and RE of BHK21

N
ELISA
HI

ELISA/N
ELISA/HI

GMT

0 .21

o. 02< P <0. 05> O . lO
> O . 10

O. 02< P <0. 05> O . 10

>0.05

328

Table 8 Statistical analysis on the difference in antibody titers and titerratios obtained by immunogens before and after UV‑irradiation

Immunogens

P value

N ELISA HIELISA/N ELISA/HISE of C6/36> O . 10>0･10

> O ･ 10> O ･ 10

> O . 10

RE of C6/36

>0.10

>0･10

< O . OO1

> O . 10

SE of BHK21
RE of BHK21> O . 10

> O , lO

> O . 10

> O . 10

> O . 10> O ･ 10> O ･ 10

O . 02 < P < O . 05

>0.lO
>0.10
>0.10

At present, inactivated JE vaccine has been manufactured from infected mouse brain

homogenates, by purifying formaline‑inactivated complete virion using primarily
ultracentrifugation (Takaku et al., 1968) . Till now, Iittle or no attention has been paid forthe recovery of SE during JE vaccine preparation, and most of the SE would have been lostin the supernatant of ultracentrifugation. It would be worthwhile to consider the im‑munogenicity of SE in the crude vaccine preparation before ultracentrifugation in order torecover more immunogens.
Some flaviviruses have been shown to produce slowly sedimenting antigen along with
rapidly sedimenting antigen or complete virion in the mouse brain homogenates or in tissue
culture system (Igarashi et al., 1963; Smith et al., 1970; Shameem et al., 1989). This
information has permitted us to predict that some form of 'incomplete virion' is synthesizedin the early period of replication of JE virus. In the case of dengue type 2 virus, anotherimportant member of flavivirus, Cardiff et al. (1971) argued that most of the incomplete virus
or top component was associated with several unrelated phenomena, viz. defective interfering
particles, adsorbing interfering particles, pleomorphic aggregates of coat structures, ma‑cromolecular capsid precursors and some naturally occurring particles which morphological‑
ly resemble infectious form except that they did not contain no core structure. Alternatively,
SE may represent a protein synthesized during the course of viral replication which might bereleased into the medium, besides complete virion, sharing several biological properties
possessed by envelope glycoprotein E.

ACKNOWLEDGMENTS
I would like to express my heart felt thanks to Professor Akira lgarashi for his pains‑
taking support, keen interest and critical reading and comments of the manuscript. A partof this work was supported by a Grant‑in‑aid for Scientific Research from the Ministry of
Education, Science and Culture of Japan, No. 61480161 in the year of 1989‑90. The animal
experiment was performed in the Animal Research Center for Infectious Tropical Diseases,
Institute of Tropical Medicine, Nagasaki University. Special thanks are due to Drs. K.
Morita and S. Matsuo and all ,the staff members of this department for their co‑operation
through out the whole period of my.research work.

329

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331

BASIC STUDIES ON THE MONGOLIAN GERBIL AS
A SUSCEPTIBLE HOST TO FILARIAL INFECTION;

COMPARATIVE STUDIES ON HEMATOLOGICALFEATURES BETWEEN THE WILD‑COLOREDGERBIL AND THE COAT COLOR MUTANTS
MASUMI SHIMIZU , KAZUHIRO SHICHINOHE*, SETSUKO TSUKIDATE' AND
KOICHIRO FUJITA'
Received August 16 1991/Accepted October 9 1991

Abstract: Hematological baseline parameters of the Mongolian gerbils were inves‑tigated to be compared between the wild‑colored gerbil (agouti type) and the other coatcolor mutants such as white spotted‑agouti, albino, black and white spotted‑black type.
Erythrocyte counts of the agouti type were higher than those of the coat color mutants.
But, there was no significant difference. A frequent occurrence of polychromasia andbasophilic stippling in circulating erythrocytes was known to be a particular feature of theMongolian gerbil among laboratory animals. The polychromasia and the basophilicstippling were proved to be present in the coat color mutants in the same degree as theagouti type. As to sex dimorphism in erythrocytic values, it was only hemoglobin
concentration that was observed significant sex‑related difference in all coat color gerbils.

In leukocytic values, quantitative sex‑related difference was not seen in this experiment.

A presence of basophils in peripheral blood was observed on all blood films of all coatcolor gerbils. These results confirmed that the coat color mutants of gerbils had the sameunique hematological characteristics as an agouti type.

INTRODUCTION
The Mongolian gerbil (Meriones unguiculatus) has been recognized an useful rodent
having considerable potential for laboratory studies, especially in the field of parasitology, asa susceptible host to filarial infection. The gerbil, ordinary used in laboratories has a wild

coat color called an agouti type. We obtained some new coat color mutants from the gerbil
and reported a comparative study on biological aspects among these mutants (Shimizu et al..
1990) . Some hematological data of agouti gerbils have been reported and it was ascertainedthat the blood of this species manifested some interesting characteristics not generally
observed in other rodents (Mays, 1969; Dillon and Glomski, 1975a; Smith et al.. 1976; Termer
and Glomski, 1978) . But, according to the coat color mutant, only a limited data has been
available that the white spotted‑agouti type of mutants is with a slight anemia (Waring etl2

Department of Laboratory Animal Science, Nippon Medical School, 1‑1‑5 Sendagi, Bunkyo‑
ku, Tokyo 113, Japan
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University,
1‑5‑45 Yushima, Bunkyo‑ku, Tokyo ll3, Japan332

al., 1978) . Hematological baseline parameters of the coat color mutants of gerbils werestudied basically in order to analyze the susceptibility of gerbils to filarial infection. A
comparative study on the blood picture among coat color mutants was carried out in thepresent work and showed some interesting hematological features of the coat color mutantsof the gerbils as well as an agouti type.

MATERIALS AND METHODS
The animals were bred under a conventional condition and housed 5 adults of the samesex in one cage. The room temperature and the humidity were maintained at 24: 2'C and
60 d: 5%, respectively. Ten of 3 to 5‑month‑old gerbils, such as agouti, white spotted‑agouti,

albino, black and white spotted‑black, of both sexes were used in this experiment. Samplesof blood were prepared from the retro‑orbital venous plexu of the ether‑anesthetized gerbils
by means of hepalinized capillary pipettes. All samples were collected between 0900 and lIOO

hours at 1‑month intervals from the animals. Total erythrocyte and leukocyte counts were
performed using hemocytometer under a light microscope. Hematocrit determinations weremade by means of microcentrifugation and hemoglobin concentrations were determined
spectrophotometrically by the cyanmethohemoglobin technique. Dry film blood smears were
stained with May‑Grunwald and Giemsa stain and differential counts were identified at least
100 Ieukocytes per an animal. Blood smears for reticulocyte counts were prepared by using

Brecher's new methylene blue stain. One thousand of erythrocytes were examined and thenumber of cells containing a reticulurn were recorded. The numbers of basophilic stippled
erythrocytes, polychromatophilic cells and cells with Howell‑Jolly bodies were also counted

while examining 1,000 erythrocytes in the smears stained with May‑Grunwald and Giemsa
stain.

Table 1

Total erythrocyte counts and related values in each coat color mutant of Mongolian
gerbils
Erythrocytes Hemoglobin Hematocrit Mean corpuscularMean corpuscularMean corpuscular

Coat color

volume hemoglobin hemoglobinSex( x 10'1/d )(g/dl) (fl) (pg) concentration(o/o)(o/o)

52.0:i:4.6

18 . 6d: 1 .o

33.7 1.0

46.6+1.2

54 . 6d: 5 . O

17,5 1 .6

32.2d:2.0

15 . 7d: O . 6t

47 . 9d: I . 7t

56.3+5.7

18.5d:l .8

14.9d:0.6

45.7+0.859.5d:3.9

19.5 1 .5

32.9 0.7
32.7il.2

8.6+0,915.7 0.6*

47 . 9 i 2 . Ot

56.1 5.8

18 . 5d: 1 .9

32 . 9 I . 6

8.0d:0,5

14.9d:0.7

44.7+1,556.2d:2.4

18 . 9dl 1 .8

33.5 2,2

MaleFemale8.2+0.7
8,0+0.9

15.1 0.4*

18.6 l .6
18.4i2 .4

33 .8d: I .6

14.6d:0.5

45.2+1.1
45.8+2.3

55 . 7d:: 5 . 3

White spotted‑ Male
black
Female8.2+0.7
7,9+0.4

15.6d:1.1'
14.6d:0.8

45.1+1.4

55.4d:5.0
57.Id:3.3

19. 1 l .8

34,6d:1.6

45. 1+1.8

18 . 6:!: l .2

32,5 0.9

Male
Female8.8+0.5
8.6+0.8

16.4d:0.4t

48 . 614.9 0.8

White spotted‑ Male
agouti
Female8.6 1.0*
7,7:!:0,6

Male
FemaleAgoutiAlbinoBlackdifference between male and female* t test, p<0.05
t t test, p<0.01

n = 10, mean

I . Ot57.9 9.0

standard deviation

32 . Oi I . 6

333

Table 2 Incidence of erythrocytes with reticulum, basophilic stippling, polychromasiaand Howell‑Jolly body in each coat color mutant of Mongolian gerbils

Coat color Sex

Reticulocytes Basophilic stippled cel]s Polychromatophilic ce]Is Cells with Howell‑Jolly(/1,000 RBC)(/1,000 RBC)(/1,000 RBC)

bodies (/1,000 RBC)

Male
Female27.4 11.9

8.6+0.4
12.8 2,96.5d:0.3

28.Id: 6.3

l.5+0.7
1.0+1.0

White spotted‑
agouti

Male
Female37 . 910 , 2

6.1+1.8

3.6i0.6

31.2i:13.0

9.0d:3.7

5.9d:2.0

AlbinoMale
Female37.4 7.8

14,0+4.6

6.8d:1.8

34 . 6d: 13 . 1

ll*Od:3.l

5.3d:2.7

Male
Female38.0d: 9.0
30.2d: 5.4

14,4dll,79.0d:1.9

12.5:!:4.0

6.7+2.6

Male
Female34.5d: 7.7
36.0d: 8.3

13.3d:4.5

6.9d:2.8

14.3:!:4,2

6.5+2.6

AgoutiBlack
White spotted‑
black

6.5+2.2

0.6+0.5
O.6+0.5
1.1+1.0
1.5+1.2
1.3+1.2
0.7+0.7

1.3+0.9
1.1+0.7

n= 10, meand:standard deviation

RESULT
The mean values and standard deviations for total erythrocyte counts as well as related
data in the peripheral blood of each color mutant of Mongolian gerbils are presented in Table

1. The average erythrocyte counts for male gerbils were always higher than those forfemales in all color gerbils; the average counts for males were (8.2‑8.8) x 106/pl as comparedto (7.7‑8.6) x 106/pl for females. However, the significant difference between sexes wasproved only in the white‑spotted agouti type of gerbil among all coat color gerbils. The
agouti type of male Was observed to have the highest value of total erythrocyte counts, butthere is no significant difference among all coat color gerbils. No coat color‑related
significant difference in the hemoglobin content was observed, but sex‑related difference wasalso evident in all coat color gerbils. The same tendency was seen in the hematocrit valuesof three coat color gerbils.

Table 2 shows the incidence of erythrocytes with cytologic features in each coat color

mutant of gerbils. The average reticulocyte counts were (27.4‑38.0)/1,000 erythrocytes(RBO for males and (28.1‑36.0)/1,000 RBC for females and sex‑related difference was notseen in this case. That value of reticulocyte was lowest in the agouti type conversely to totalerythrocyte counts. Polychromatophilic and stippled red blood cells were demonstrated
frequently in all blood films in all coat color gerbils (Photos. 1, 2). The average counts ofbasophilic stippled cells were (6.1‑14.4)/1,000 RBC for both males and females and those ofpolychromatophilic cells were (3.6‑9.0) /1,000 RBC. It was observed in all blood films that one

red blood cell had both polychromasia and stippling (Photo. 1). The erythrocytes with
Howell‑Jolly bodies were also observed (0.5‑1.5)/1,000 RBC in all color gerbils (Photos. 2,3) .

Total numbers of cirbulating leukocytes and differentials in each coat color mutant ofMongolian gerbils are shown in Table 3. Total circulating leukocytes were (12.4‑15.6) x 1031
pl for males and (14.2‑16.6) x 103/pl for females. In the circulating leukocyte counts, both

sex‑related and coat color‑related differences were not seen in this study. The lymphocyte
was observed most prevalent in all coat color gerbils of both sexes. Although the number ofbasophils and eosinophils accounted for only small propotion in the peripheral blood, it was

334

.'

** ==* .

," j. , ':='

; .*;',*..""･'.:.;.'** ' ̲*,; + ･ ･ '.

Photo. I Polychromatophilic and basophilic stippled erythrocytes near a lymphocyte
on a blood film of a Mongolian gerbil. May‑Grunwald and Giemsa stain.

S

Photo. 2 An erythrocyte with polychromasia and with a Howell‑Jolly body on a
blood film of a Mongolian gerbil. May‑Grunwald and Giemsa stain.

335

i{ =d

'; { ;' r:*

!

i

:' *'

s

' :*ipmj} ;"Photol 3 A Howell‑Jolly body in an erythrocyte on a blood film of a Mongolian
gerbill May‑Grunwald and Giemsa stainl

Table 3

Total leukocyte counts and differentials in each coat color mutant of Mongolian gerbils
Lymphocytes(%)

Monocytes
(%)

0 6.5
8 6.9

70.6+7.0
72.6 7.0

3.3 1.4
3.1+1.6

7+g.9
6+5.3

70.9 9.2
73.6i5.6

3.9 1.2
1.9+0.9

3+3.6
4,075.5+3,8
74.5+4.2

2.5+1.6
2.0 1.4

0 9.3
0+8.6

67.9+8.8
71.1+8.7

l.5 0.7
2.4+1.3

7+6.9
5+9.6

66.8 6.7
64.8+9.2

2.3+0.8
2.4+1.4

Total leukocytes

Basophils

Eoslnophils

Neutrophils( x 10'1pl)

(%)

(%)

(%)

Agouti Male

13.8+3.6
14.8 3.8

o. 3+0. 5

0.8 0.6

25 .

o. 2

0.

0.3:!I0.5

23 .

White spotted‑ Male

15.6 4.9
15.9 5,8

o. 3

0. 5

1.2:t0.8

23 .

o. 1+0. 3

0.8+0.6

23 .

Albino Male

15.5 3.6
14.8+4.8

o. 4+0. 5

1.3 0.7
l.5 1.5

20 .

FemaleBlack Male
Female12.4+2.6
16.6+5.6

o. 2+0.

0.4+0.5
0.4 0.7

30 .

White spotted‑ Male

15.6 2.9
14.2+4.4

o. l+0. 3

0.1iO.3
0.2+0.4

30 .

Coat color

Sex

Femaleagoutl Female

black Femalen = 10, meano. 2+0. 6

o. l+0. 3

o. 1+0. 3

21. 8

26 .

32 .

standard deviation

confirmed that these cells were present in the all blood films in all color mutants of gerbils.

DISCUSSION
In this paper, we investigated hematological profiles in 4 kinds of the coat color mutantsof gerbils and compared them to the agouti type. There was only one past report on ahamatological characteristics in a coat color mutant of a gerbil (Waring et al., 1978). They
noted that total erythrocyie counts of spotted gerbils were significantly lower than those of

336

an agouti type indicating the presence of a slight anemia. From their description about thelocation of white spots and about breeding data of the mutant, it seemed that their spotted
gerbil was the same mutant as our white spotted‑agouti type. But, their result of erythrocyte

counts was different from ours. The total erythrocyte counts of agouti type were observed
also higher than the other coat color gerbils in our experiment, however, there was no
significant difference and an anemia of white spotted‑agouti gerbils was not observed. The
total erythrocyte counts of agouti type in our experiment were comparable to those of theother investigators (Ruhren, 1965; Mays, 1969; Dillon and Glomski, 1975a; Termer andGlomski, 1978) , but, those of Waring et al. (1978) were about a half level of them.

As their own particular feature of the blood of the Mongolian gerbil, it was known thefrequent occurrence of polychromasia and basophilic stippling in circulating erythrocytes
throughout the life span of agouti gerbils (Ruhren, 1965; Dillon and Glomski, 1975a; Smith etal.. 1976; Termer and Glomski, 1978) . In this study, it was proved that those cells were alsopresent in the blood of the coat color mutants of gerbils as the agouti type. Those cytologic
features were considered a property of immaturity of erythrocytes (Smith et al., 1976) . Highincidence of those cells refiected that immature red cells emerged to the peripheral blood
because of. a brief life span of an erythrocyte (Dillon and Glomski, 1975b) . Although gerbils

have been known to have very wide sensitivity to various parasites compared with the otherlaboratory rodents, the cause of a high sensitivity was not investigated. Since a marked
difference of the gerbil from the other rodents was the continuous presence of stippled andpolychromatophilic cells in the adult gerbils as far as a hematological measurement, the brief
span of blood cells may participate in a wide sensitivity of this animal.
Certain numbers of reticulocytes were also observed. Although there was no statisticaldifference, it was lowest in the agouti gerbil conversely to total erythrocyte counts.

As to sex dimorphism in total erythrocyte counts and related values, there have beensome discussions. Male predominance of hemoglobin concentration, hematocrit, mean
corpuscular volume or mean corpuscular hemoglobin was reported by some investigators(Mays, 1969; Dillon and Glomski, 1975a; Termer and Glomski, 1978) . In this experiment, only
hemoglobin concentration showed statistical sex‑related difference in all coat color gerbils.

The possible existence of quantitative sex‑related difference in the leukocytes has beena feature described for this rodent (Mays, 1969; Dillon and Glomski, 1975a). But, in thisexperiment, sex‑related difference was not observed in total leukocyte counts in all coatcolor gerbils. This finding supported the result of the study by Termer and Glomski (1978) .

The predominant cells in the circulating leukocytes of the gerbils were lymphocytes, acharacteristic common to other laboratory rodents (Wolford, 1986). On the other hand, apresence of basophils in peripheral blood was a specific characteristics of the gerbil and it was

not observed in mice or rats. At the point of existence of both basophils in peripheral bloodand mast cells in connective tissues or mucosal tunics of the gerbil (data not shown), thisanimal can be said more resemble to human being than mice or rats. The gerbil may be usefulfor analysis of the infection kinetics to various parasites, because the animal has both mast

cells in connective tissues and a comparatively larger amount of basophils in peripheral
blood.

Finally, this study confirmed that the coat color mutants had the same uniquehematological characteristics as the wild‑colored gerbil. Further detail hematological andother physiological experiment concerning this animal will help us to know why the gerbil is337

very sensitive to various parasite infections.

REFERENCESl ) Dillon, W.G. and Glomski, C.A. (1975a): The Mongolian gerbil: qualitative and quantitative
aspects of the cellular blood picture, Lab. Anim., 9, 283‑287
2 ) Dillon, W.G. and Glomski, C.A. (1975b) : Erythrocyte survival in the Mongolian gerbil, J. Nucl.

Med., 16, 682‑684

3 ) Mays, A.Jr. (1969): Baseline hematological and blood biochemical parameters of the Mon‑
golian gerbil (Meriones unguiculatus). Lab. Anim. Care, 19, 838‑842
4 ) Ruhren, R. (1965): Normal values for hemoglobin concentration and cellular elements in the
blood of Mongolian gerbils, Lab. Anim. Care, 15, 313‑320
5 ) Shimizu, M., Shichinohe, K., Tsukidate, S. and Fujita, K. (1990): Basic studies on the
Mongolian gerbil as a susceptible host to filarial infection; comparative studies on growth andreproduction among coat color mutants and genetic analysis of coat colors, Japan. J. Trop.
Med. Hyg., 18, 301‑310
6 ) Smith, R.A., Termer, E.A. and Glomski, C.A. (1976): Erythrocyte basophilic stippling in the
Mongolian gerbil, Lab. Anim., 10, 379‑383
7 ) Termer, E.A. and Glomski, C.A. (1978): The cellular blood picture of the Mongolian gerbil
throughout the first year of life; a longitudinal study, Exp. Hemat., 6, 499‑504
8 ) Waring, A.D., Poole, T.W. and Perper, T. (1978): White spotting in the Mongolian gerbil, J.

Heredity, 69, 347‑349
9 ) Wolford, S.T., Schroer, R.A., Gohs, F.X., Gallo, P.P., Brodeck, M., Falk, H.B. and Ruhren, R.

(1986): Reference range data base for serum chemistry and hematology values in laboratory
animals, J. Toxicol. Environm. Health, 18, 161‑188

338

フィラリア感染好適宿主としてのMongolian

gerbilの基礎的検討

一血液学的特性に関する被毛色間の比較一

清水Mongolian

眞澄1・七戸和博1・月舘説子2・藤田紘一郎2

gerbi1（スナネズミ）の被毛色突然変異体（white spotted−agouti，albino，black，

whitespotted−black）の血液学的特性について，野生色（agouti）のものと比較検討した。赤血球数は統計学的な有意差はなかったが，agoutiが最も多かった。他の実験動物には見られないス
ナネズミ独特の性質として，多染性や好塩基斑点を持つ赤血球が多いことが知られているが，それらが被毛色突然変異体の末梢血中にも多数出現することが観察された。赤血球関連の検査値においてすべての被毛色で有意な性差が見られたのは，ヘモグロビン濃度のみであった。白血球数には性差が確認されなかった。白血球中で最も多くの割合を占めたのは，各被毛色ともリンパ球であった。すべての被毛色の各個体の末梢血中に好塩基球の存在が確認された。

以上のように被毛色突然変異体は，agoutiと同様にスナネズミ独自の血液学的性質を持っていることがわかった。

日本医科大学実験動物管理室（〒113東京都文京区千駄木1−1−5）
東京医科歯科大学医学部医動物学教室（〒113東京都文京区湯島1−5−45）

Jpn. J. Trop. Med. H yg ., Vol. 19 No. 4 1991, pp. 339‑346

339

CLINICAL STUDY OF MEBENDAZOLE THERAPYFOR STRONGYLOIDIASIS
KOUSHIN SHIKlYA*, HIROYUKI UECH12, ATSUSHI SAITO* AND
RYUJI AsAT03
Received August 16 1991/Accepted October 9 1991

Abstract: A study was conducted of 225 patients in Okinawa Prefecture, Japan, toevaluate mebendazole (MBZ) therapy for Stron yloides stercoralis. Various schedules
were used with the following results: l) The eradication rates at 12 months after a single

course of MBZ therapy (100 mg of a powder twice a day orally for 28 days) and a
combination therapy (thiabendazole 500 mg powder form three times daily for 5 days
followed by 100 mg of MBZ in powder form twice a day for 9 days, repeated once) were85.0% (17 of 20 patients) and 100% (17 of 17 patients) , respectively. 2) The eradication
rates at 3‑9 months after using MBZ alone in varying dosages were as follows: (a) 100
mg in powder form twice a day for 5 days, repeated at l, 3 and 4 weeks following: 80.0%(24/30) ; (b) 100 mg in powder form twice daily for 5 days repeated at I and 3 weeks
following: 83.3% (5/6); (c) 100 mg in powder form twice a day for 4 days repeated at l,
3 and 4 weeks following: 96.0% (24/25); (d) a 100 mg tablet twice a day for 4 days
repeated at l, 3 and 4 weeks following: 90.6% (29/32). 3) The eradication rate at 24 days
after a 2‑course treatment (a 100 mg tablet twice daily for 4 days repeated I week later)
was 93.8% (15/16). Although the 2‑course treatment was effective, further study seems
necessary as the follow‑up time was too short and the number of patients was too few.

INTRODUCTION
The infection rate of Strongyloides stercoralis (S. stercoralis) in Okinawa Prefecture,
Japan, was found to be less than 2% of the population when determined by the traditional
methods of direct smear, filter paper culture and formalin‑ether concentration. However,when stool samples were tested by a new technique, the agar plate method, the infection rate
was found to be 8.1% (Arakaki et al.. 1988, 1990; Asato et al.. 1989). We attempted toevaluate the efiicacy of treatment when using mebendazole (MBZ) alone and in combination
with thiabendazole. However, Iiver disfunction developed when MBZ was used alone for a
28 day period and side effects also occurred in the combination therapy (Shikiya et al..
1990a) . We therefore reduced the period of administration of MBZ and repeated the dosage
after intervals, but abnormal liver function was still noted (Shikiya et al,. 1990b, c) . We
finally reduced the dosage to a 4‑day period that was repeated I week later. We are nowlFirst Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207
Uehara, Nishihara, Okinawa 903‑01, Japan

2

Okinawa Prefectural Nago Hospital, 1617‑1 Nago, Nago, Okinawa 905, Japan
Okinawa Prefectural Institute of Public Health, 2085 Ozato, Ozato, Okinawa 901‑12, Japan

3

340

reporting the results of the therapy withthese various schedules. Group. 8. day.s

powdertt

MATERIALS AND METHODS

9

5

t
9

5

2

powderPatients

t

t

t

5

A total of 225 Okinawan patients, 146 3

5

powdermales and 79 females, with strongyloidiasistt5

diagnosed by using the agar‑plate culture

t

5

5

powdermethod were included in this study. 4

t

t

4

Administration Schedules (Figure l) 5

4

t
4

4

powdertt(Table D

4

The patients were divided into 7 6

4

t
4

4

tabletttgroups and different schedules of M BZ or a

4

t

a

combination of MBZ and thiabendazole 7

tabletwere administered as follows:

Group l: 100 mg of MBZ in powder

Mebenda.ole (roo mg x 2/day)form was given twice a day orally for 28: Thiabendazole (500 mg x 3/day)days.

Group 2: thiabendazole powder (500t : chnicat examinationmg) was given three times daily for 5 days

Figure I Administration schedules.

and for the following 9 days 100 mg of MBZ

powder was given twice a day. This combination therapy was administered twice insuccession.

Group 3: MBZ powder was given twice a day for 5 days and was then repeated 1, 3 and4 weeks later.

Group 4: MBZ powder was administered twice daily for 5 days and repeated I and 3
weeks later.

Group 5: MBZ powder was given twice a day for 4 days and repeated 1, 3 and 4 weeks
later.

Group 6: MBZ in tablet form was given twice a day for 4 days and repeated 1, 3 and 4
weeks later.
Sex and age distribution

Table 1
Group

l

2

3

Male 22 (66.7) 14 (53.8) 35 (74.5)
Sex Female ll (33.3) 12 (46.2) 12 (25.5)
Total 33(100 O) 26(100 O) 47(lOO O)
40‑49
50‑59

Age 60‑69(yr) 70‑79
80‑89
No. of patients, (%)

6 (18.2)

6

5

4

7

Total

12

(92 . 3)(62 . 2)24

(53 . 3)ll (68 .8)

146

l

(7.7)

17 (37.8)

21(46 . 7)5 (31.2)

79 (35 . D

13(lOO . O)

45 (100 . O)

45 ( 100 . O)

16 ( 100 , O)

225(100 . O)

28

3 (ll.5)

5 (10.6)

o

(0.0)

3

(6.7)

2

(4 . 4)l(6.3)(18 . 5)20

(64 . 9)(8 .9)

2 (6.D

8 (30.8)

10 (21.3)

2 (15.4)

14(31 . l)lO

(22 . 2)

2

13 (39.4)

11 (42.3)ll (23.4)

7 (13.4)

17 (37.8)

12(26 . 7)7 (43.8)78

11 (33.3)

2 (7.7)13 (27.7)(7.7)

10 (22.2)

17(32 . 8)5 (31.3)

59 (12.9)l (3.0)2 (7.7)8 (17.0)(23 . 1)(2.2)

4

(8 . 9)(6 . 3)3ll

48 (21.3)

20(34 . 7)

(8 . 9)

341

Group 7: MBZ in tablet form was given twrce a day for 4 days and repeated l week
later.

Parasitological Enamination

Fresh fecal samples were examined by using the agar‑plate culture method. The firstpost‑treatment fecal examination was done between 2 to 24 days after the last day oftreatment in all groups. A second post‑treatment fecal examination was done between 3 to
9 months after the last day of treatment in groups I ‑6. In groups I and 2 a third post‑
treatment fecal examination was done 12 months after the last day of treatment.
Clinical Eramination
Clinical examinations were done before, during and after treatment. The examinations
included a clinical history, a complete blood count, serum‑chemistry studies, serologicalstudies and urinalysis. Serum‑chemistry studies included liver function tests as follows: total

bilirubin, gultamic oxaloacetic transaminase (GOT) , gultamic pyruvic transaminase (GPT)and alkaline phosphatase (ALP).

RESULTS
Antiparasitic Effect (Table 2)

Patients who did not complete the course of treatment or who failed to submit post‑
treatment fecal samples were excluded.
The parasitological eradication rates at 12 months after treatment were 85.0% in group
1 and 100% in group 2. The parasitological eradication rates in groups 3 to 6 at 3‑9 months
after treatment were from 80.0 to 96.0%. The eradication rate in group 7 at 24 days after
treatment was 93.8% .
Side Effects (Table 3)

Patients who did not complete the course of treatment were excluded.
Thirteen patients (54.2%) in the thiabendazole‑MBZ group complained of side effects
and therapy was discontinued for two patients because of severe nausea and dizziness which
Table 2 Eradication rates of S. stercoralis according to administration sched‑ules

Grou pdurmg treatment 2̲24 days

after treatment

3‑9 months

12 months

90.3 (28/31)

81.8 (18/22)

77.3 (17/22) 85.0 (17/20)

296.0 (24/25)

lO0.0 (22/22)

100.0 (22/22) 100.0 (17/17)

3

89.1 (41146)

100.0 (46/46)

80.0 (24/30)

484.6 (11/13)

92.3 (12/13)

83.3 (5/6)

597.8 (44/45)

100.0 (45/45)

96.0 (24/25)

693.0 (40143)

97.7 (42143)

90.6 (29/32)

793.8 (15/16)%, (No. of negative/No. of subjects)

342
Incidence of side effects

Table 3

Group
(No. of subjects)

2
(24)

(27)

3
(47)

4
(13)

5

6

7

(45)

(45)

(16)

Heartburn
N ausea

Vomiting
Abdominal pamj BorbDrygnus

7.4 (2)

Diarrhea
Constipation

3.7

20 . 8

(5)

4.2

(1)

4.2

(D

(1)

6.4

(3)

10 . 6(5)

11 . 1

(3)

4.2

(1)

Anorexia

3.7(1)

12 . 5

(3)

4.3

(2)

Headachell . 1(3)

16 . 7

(4)

2.1

(1)

8.3

(2)

2.1 (1)

3.7

(1)

4.2

(1)

(3)

Dizziness or vertigo
Numbness of extremities

6.4

4.4

(2)

2.2

(1)

4.4 (2)2.2(1)

4.4 (2)

6.7

(3)

(2)

2.2

(1)

4.4

6. 3 (1)

6.7

(3)

4.4

(2)

2.2

(1)

4.4

(2)

2.2

(D

2.2 (D

2.1 (1)

Blurring of visionFever

3.7 (D

4.2 (D

General fatigue

7.4 (2)8.3(2)

2.1 (1)4.4 (2)2.2(1)Itching

8.3

(2)

(1)

4.4

(2)

6.6

(3)

Ex anthem a

12 . 5

(3)

4.4

(2)

4.4

(2)

8.3

(2)

8.5

(4)

4.4

(2)

2.1

(D

4.4

(2)

Arthralgia, Lumbago
Urinary frequency
OthersTotal

6. 3 (1)

3.7 (D
7.4 (2)(2)

7.4

40 . 7 ( 11)

2.1

(2)

8.3

54 . 2 (13)34 . O ( 16)0.0 (O) 31.1(14)31 . I (14)12.5 (2)%, (No. of patients)

Incidence of liver disfunction

Table 4

Group
(No. of subjects)

GOT t
GPT t
ALP t

1

2

(28)

(22)

(47)

4

5

6

7

(13)

(45)

(45)

(16)

4.3 (2)7.l (2)

7. 7 (1)9.1 (2)10 . 7

(3)

4.5

(1)

2.1 (D

GOT, GPT t

28 . 6(8)

27 . 3(6)

29 . 8 (14)(3)

48 . 9 (22)51 . I (23)12 . 5(2)

2.2 (1)

GOT, GPT, ALP t

Total

6.3 (1)2.2 (1)23 . 1

GOT, T.bil t

GOT, GPT, T.bil t

6.3

6.7 (3)2.1 (1)T.bil tOOT, GPT, T.bil, ALP t(D

4.4 (2)2.2 (D

6.4 (3)17 . 9

(5)

7.l

(2)

71 . 4 (20)

2.2 (1)

4.5

(1)

4.5

(1)

6.4 (3)50.0(1D

51 . I (24)

2.2 (D
2.2 (D

2.2 (1)30 . 8(4) 57.8(26)68 . 9 (31)25.0 (4)%, (No. of patients)

developed 2 weeks after treatment was begun.
Side effects were observed in 40.7% of group 1, 34.0% of group 3 and O% of group 4.
Incidence of side effects was not significantly different between group 5 (powder) and group

6 (tablet). Side effects were observed in 12.5% of group 7. No special treatment was given

343
for the side effects.

Liver Dibfunction
Patients who did not complete the course of treatment were excluded.
Table 4 shows the incidence of abnormal liver function (elevation of serum total bilirubin

and/or GOT and/or GPT and/or ALP) which occurred during treatment and from 2 to 24
days after treatment.
Liver disfunction was observed in 71.4% of group I and in 50% of group 2 but only one

patient had malaise and jaundice.
In group l, one patient had a total bilirubin of 6.9 mg/dl (normal: 0.3‑0.8 mg/dl) , a GOTof 898 IU/L (normal: 10‑32 IU/L) and a GPT of 1,215 IU/L (normal: 7‑26 IU/L) at I week
after the last dosage; her only symptom was itching. Of the 20 patients in this group who
experienced liver disfunction, 13 patients received a lymphocyte stimulation test and were
negative for MBZ. None of the patients with liver disfunction had allergic reactions stich as
skin rash, fever and eosinophilia.

In group 2, one patient had a GOT of 77 IU/L and a GPT of 174 IU/L at I week after
the last treatment but had no symptoms.
The abnormal liver function of all patients in both groups I and 2 showed a return tonormal in subsequent tests.
Since these liver disfunctions seemed to be dose‑dependent, we reduced the length ofperiods of administration of MBZ and repeated it after intervals. But because 51.1% of thepatients in group 3 displayed a liver disfunction, therapy for the 13 patients in group 4 wasinterrupted before the 4th course.
Incidence of liver disfunction was not significantly different between group 5 (powder)and group 6 (tablet) . Although the GOT after the second course in group 5 (30.3 32.2 IU/
L) was significantly higher than that in group 6 (22.9 12.7 IU/L) , (p<0.05) according to the
Wilcoxan 2‑sample test, there were no significant differences in the GOT of the two groups
after the fourth course (118.0 146.2 vs. 84.1i94.4) and in the GPT after either the second(28.0 63.8 vs. 19.1 16.6) or fourth course (137.0i 162.2 vs. 119.0i 161.1) . The GOT and GPTafter the second course (GOT: 30.3i32.2, 22.9d:12.7; GPT: 28.0i63.8, 19.1il6.6) weresignificantly lower thah after the fourth course (GOT: 118.0d:146.2, 84.Id:94.4; GPT: 137.0:b

162.2, 119.0d:161.1) in both grotips (p<0.05) . The incidence of liver disfunction in group 7

was lower than in the other groups.

DISCUSSION
We decided to use MBZ for a 28 day period for strongyloidiasis because MBZ is effective
against the parasite (Mraval et al.. 1983; Wilson et al., 1983) , because the drug's side effects
are mild and of low frequency (Bekhti et al.. 1977; Keystone et al., 1979; Wilson et al.. 1978) ,and because S. stercoralis has a 17 to 27‑days larval development period in the intestinal
tissue (Grove, 1989; Mraval et al., 1983). However, a high incidence of liver disfunction had

developed in our previous study and because the liver disfunction seemed to be dose‑depen‑
dent (Shikiya et al., 1990a) , we reduced the administration period of MBZ and repeated itafter intervals. Although the incidence of liver disfunction tended to decrease, Iiver disfunc‑tion was still observed in groups 3 and 4 (Shikiya et al.. 1990b) . Since S. stercoralis is located344

mainly in the small intestine, we used powdered MBZ in order to more effectively reach theparasites. Although we considered that a powder would be easily absorbed and therefore
liver disfunction would occur in high incidence, the incidence of liver disfunction and otherside effects, as well as the eradication rate of the parasite were similar for both the powder
and tablet forms of the drug. Since a significant difference was observed in liver disfunction

when examined between the second course and the fourth course in groups 5 and 6 (Shikiya
et al., 1990c) , we finally reduced the period of administration to 4 days (a 10Q mg tablet twice

a day) and repeated it only once a week later.
As high doses of MBZ produces liver disfunction (Berhti et al., 1987; Davis et al., 1986) ,many investigators have tried low‑dose MBZ treatment. Abadi (1985) reported that a single
500 mg dose of MBZ was not effective for a strongyloidiasis patient. Goldsmid (1974) using
100 mg of MBZ twice a day for 3 days, reported a cure rate at 7‑10 days after treatment of
88% (7 of 8 patients) . Musgrave et al., (1979) tried 100 mg of MBZ twice a day for 4 days
and described a cure rate at l0‑20 days after therapy of 67% (14 of 21 patients) .

Although our study showed that one 100 mg MBZ tablet twice a day for 4 days with asecond course of treatment one week later resulted in a 93.8% eradication rate (15 of 16
patients) and in a 25% incidence of liver disfunction (4 of 16' patients) , further study seems

to be necessary as, in most studies to date, the follow‑up time has been too short and thenumber of patients too few.

ACKNOWLEDGMENTS
This study was supported by the 16th Oyama Health Foundation Grant, 1990, Japan andthe Drug Research of Tropical Diseases Grant fror i* the Ministry of Public Welfare, 1990,
Japan.

REFERENCES
1 ) Abadi, K. (1985): Single dose mebendazole therapy for solid‑transmitted nematodes, Am. J.
Trop. Med. Hyg., 34, 129‑133
2 ) Arakaki, T., Hasegawa, H., Asato, R., Ikeshiro, F., Kinjo, F., Saito, A. and lwanaga, M. (1988):

A new method to detect Strongyloides stercoralis from human stool, Japan. J. Trop. Med. Hyg.,
16, 11‑173 ) Arakaki, T., Iwanaga, M., Kinjo, F., Saito, A., Asato, R. and lkesiro, T. (1990): Efncacy ofagar‑plate culture in detection of Strongyloides stercoralis infection, J. Parasitol., 76, 425‑428

4 ) Asato, R., Nakasone, T., Arakaki, T. and lkeshiro, T. (1989)･: Prevalence of S. stercoralis
infection among inhabitants of Okinawa by agar plate method and evaluation of the methods
for detection, Ann. Rep. Okinawa Prefect. Inst. Publ. Hlth., 23, 66‑71 (in Japanese)5 ) Bekhti, A. and Pirotte, J. (1987): Hepatotoxicity of mebendazole. Relationship with serum
concentrations of the drug, Gastroenterol. Clin. Biol., Il, 701‑703
6 ) Bekhti, A., Schaaps, J.P., Capron, M., Dessaint, J.P., Santoro, F. and Capron, A. (1977) :Treatment of hepatic hydatid disease with mebendazole: preliminary results in four cases, Br.
Med. J., 2, 1047‑l051

7 ) Davis, A., Pawlowski, Z.S. and Dixon, H. (1986): Multicentre clinical trials of benzimi‑
dazolecarbmates in human echinococcosis, Bull. W.H.O., 64, 383‑388
8 ) Goldsmid, J.M. (1974): The use of mebendazole as a broad‑spectrum anthelmintic in Rhodesia,

345
S. Afr. Med. J., 48, 2265‑2266
9 ) Grove, D.1. (1989): Strongyloidiasis: a major roundworm infection of man, pp. 212‑214, Taylor& Francis Co., New York
10) Keystone, J.S. and Murdoch, J.K. (1979): Mebendazole, Ann. Intern. Med., 91, 582‑586ll) Mraval, S., Schopp, W. and Bienzle, U. (1983): Treatment of strongyloidiasis with meben‑
dazole, Acta. Tropica., 40, 93‑94
12) Musgrave, I.A., Hawes, R.B., Jameson, J.L., Sloane, R.A. and Quayle, P.A. (1979): Meben‑
dazole: evaluation of a new antihelminthic for trichuriasis, hookworm, and strongyloidiasis,
Med. J. Aust., 1, 403‑405
13) Shikiya, K., Kinjo, N., Ikema, M., Yamashiro, A., Uechi, H., Oyakawa, T., Kinjo, F., Saito, A.,

Nakamura, H., Ohwan, T., Yamashiro, M. and Asato, R. (1990): Comparison of efiicacy on
powder and tablet of mebendazole in the treatment of strongyloidiasis, J. J. A. Inf. D., 65,
681‑686 (in Japanese)
14) Shikiya, K., Kuniyoshi, T., Higashionna, A., Arakaki, T., Oyakawa, T., Kadena, K., Kinjo, F.,

Saito, A. and Asato, R. (1990): Treatment of strongyloidiasis with mebendazole and its
combination with thiabendazole, J. J. A. Inf. D., 64, 1408‑1415 (in Japanese)

15) Shikiya, K., Kuniyoshi, T,, Uechi, H., Oyakawa, T., Kinjo, F., Saito, A., Ikema, M., Nakamura,

H., Yamashiro, M. and Asato, R. (1990): Treatment of strongyloidiasis with mebendazole‑long term eradication and new trials‑, J.J.A. Inf. D., 65, 433‑441 (in Japanese)
16) Wilson, J.F., Davidson, M. and Rausch, L. (1978): A clinical trial of mebendazole in thetreatment of alveolar hydatid disease, Am. Rev. Respir. Dis., 118, 747‑757
1,7) Wilson, K.H. and Kziuffman, C.A. (1983): Persistent Strongyloides stercoralis in a blind loop of

the bowel, successful treatment with mebendazole, Arch. Intern. Med., 143, 357‑358

346

Mebendazoleによる糞線虫症治療に関する臨床的研究
志喜屋孝伸1・上地

博之2・斎藤厚1・安里 龍二3

Mebendazole（MBZ）を用いて225例の糞線虫症患者を治療し，下記の結果を得た。なお，MBZ（100mg）は1回1錠を1日2回経口投与し，［］内は肝機能障害出現頻度を示す。
1．MBZ（粉末）28日間投与の治療1年後の駆虫率：85．0％（20例中17例），［71．4％］

2．Thiabendazole（粉末）を1回1錠，1日3回の5日間投与後，MBZ（粉末）を9日間投与し，それを2コース繰り返す方法の治療1年後の駆虫率：100％（17／17），［50％］3．下記の投与法による3−9カ月後の駆虫率は以下のとおりであった。

1）MBZ（粉末）
2）MBZ（粉末）
3）MBZ（粉末）
4）MBZ（錠剤）

5日間の4コース投与：

80．0％（24／30），［5L5％］

5日間の3コース投与： 83．3％（5／6）、 ［30．8％］

4日間の4コース投与：
4日間の4コース投与：

96．0％（24／25），［57．8％］

90．6％（29／32），［68．9％］

4． MBZ（錠剤） 4日間の2コース投与24日後の駆虫率：93．8％（15／16），［25％］以上のように，MBZ4日問の2コース投与では肝障害の出現頻度が低くなり，駆虫率は高いが症例数が少なく，観察期間が短いのでさらに検討が必要であると思われる。

琉球大学医学部第一内科（〒903−01沖縄県中頭郡西原町上原207）
沖縄県立名護病院（〒905名護市名護1617−1）
沖縄県公害衛生研究所（〒901−12沖縄県島尻郡大里村大里高嶺原2085）

Jpn. J. Trop. Med. Hyg., Vol. 19 No. 4 1991, pp. 347‑355

347

MANAGEMENT OF PNEUMOCYSTIS CARINIIPNEUMONIA IN PATIENTS WITH CONVENTIONALLYCAUSED IMMUNE SUPPRESSIONTATSUYA KATSUMATA, SHIGERU KoHNO, HIRONOBU KoGA,
YUKO YOSHITOMI, HARUKO MATSUDA, KOTARO MITSUTAKE,YASUHITO HIGASHlYAMA, YOSHITSUGU MlYAZAKI AND KOHEI HARA
Received August 27 1991/Accepted October 15 1991

Abstract: Ten non‑AIDS patients with Pneumo(ystis carinii pneumonia were studied.
While the 2 patients with adult T cell leukemia had longer prodromes, the other 8 patients

had acute onset. At presentation a chest radiograph revealed an abnormal bilateral
diffuse shadow in all cases. In 8 patients, diagnostic material was obtained by transbron‑chial lung biopsy and/or bronchoalveolar lavage, and in 2 patients at postmortem. At thetime of diagnosis the serum lactate dehydrogenase value was much higher than prior to
the acute illness, and the AaDO, gradient was highly increased: These appear to be usefulas markers for an initial diagnosis. Other opportunistic organisms were isolated in 5
patients. The concomitant use of pentamidine and cotrimoxazole was relatively welltolerated, but with a high incidence of treatment failure. Corticosteroids appeared to beeffective as an adjunctive therapy.

INTRODUCTION
Pneumocystis carinii pneumonia (PCP) is the most common opportunistic infection which
occurs and is a significant cause of death among patients with acquired immunodeficiency
syndrome (AIDS) . In Japan, the prevalence of AIDS is still low, and PCP is an opportunistic

infection seen mQst frequently in association with certain malignancies or immunosuppres‑sive therapy (Macfarlane and Finch, 1985; Engelberg et al.,' 1984).
Diagnosis of PCP usually requires invasive methods, such as fiber optic bronchoscopy,
although various noninvasive tests, such as chest radiographs and gallium scans, have beenused to diagnose this infection (Oka et al., 1985; Cordonnier et al., 1984) . Recent studies haveshown that the serum LDH value and P (A‑a) 02 gradient might be useful as markers fordiagnosis and prQgnosis of PCP in cases with AIDS (Zaman and White, 1988; Garay andGreene, 1989) .

Several studies have demonstrated that pentamidine has a greater incidence of significant

adverse reactions, and is no more effective in the treatment of PCP, than cotrimoxazole.

Second Department of Internal Medicine, Nagasaki University School of Medicine

Correspondence to: Tatsuya Katumata MD, PhD, Second Department of Internal Medicine,
Nagasaki University School of Medicine, 7‑1 Sakamoto‑machi, Nagasaki 852, J, apan

348

Table 1 Details of patients with pneumocystis pneumonia

Patient Sex (years) disease

Duration of
immun0Su p pressive
therapy

M 63

3 months

Age Underlying
malignant2 . 5 months

1 weekadult T cell

7

F 53

cough, fever

3 dayscough,

3 dayscough, fever,

2 weeks

dyspnea
2 weeks

adult T cell

6 M 32

prodrome and duration

lymphoma2 M 78 malignant
lymphoma3 M 69 leukemia
4 M 47 leukemia
F 73

Clinicalcough, fever,

3 weeks

dyspnealung cancer
renaltransplantation

nephrotic

4 monthslO months

dys pnea

2 dayscough, dyspnea

3 days2 months

asym ptomatic

2 months
7 months
18 months

asymptomatic
dyspnea
asymptomatic

syndrome

8 M 75 dermatomyositis
9 M 51
10 F 44
sarcoidosis

systemic lupus

2 dayserythematodes

Average

58 . 54.9

d: 14 . 6i 5 . 2 months

6.9

7. O days

Pentamidine has therefore been used in patients with PCP who have failed to respond to
cotrimoxazole or who have sustained adverse reactions to this drug combination. PCP cases
from conventionally caused immune suppression are reported to be more fulminant, Iesslikely to relapse, with fewer adverse effects and a greater incidence of treatment failure thanthose from AIDS (Drake et al., 1985; Salamone and Cunha, 1988; Pearson and Hewlett, 1985;
Kluge et al., 1978; Kovacs et al., 1984; Sattler et al., 1988). Therefore in the non‑AIDS

population, the concomitant use of pentamidine and cotrimoxazole has yet to be examined.

We present our experience in fulminant cases of PCP from conventionally caused
immunosuppression admitted to Nagasaki University Hospital during the period 1976‑1989.

PATIENTS AND METHODSThe hospital charts of patients with confirmed PCP between 1976 to 1989 were reviewed.
Only those patients with microbiologically documented disease were included. The following
data was recorded in all cases: age, sex, underlying disease, use of immunosuppressive drugs,
nature and duration of the problem leading to diagnosis, arterial blood gas with calculationof the P (A‑a)02 gradient, Iactate dehydrogenase (LDH) value, chest radiograph findings,

treatment, and outcome. Diaguostic procedures included fiber optic bronchoscopy withbronchoalveolar lavage (BAL) , transbronchial lung biopsy (TBLB) , and postmortem exami‑
nation. Fiber optic bronchoscopy was performed under local anaesthesia with parenteral
sedation. The bronchoscope was wedged into the subsegmental bronchus in the main regionof radiographic abnormality. Bronchoalveolar lavage was performed using at least two349

Table 2 Diagnostic information

Patient

LDH (IU/L) PaO, p (A‑a) 02 Initial chest(torr) (torr)At
1‑2
monthsprior diagnosis
at at
to diagnosis diagnosis
diagnosis

IncreaseradiographDefinitive

diagnosis

by

l

478

896

418 38 5(RA)72 . obilateral
reticular

TBLB

2

587

1 , 132545 44 5 (RA)63 . 5bilateral

BAL

reticular andinfiltrates

3

472

663

191 58 . 2

122(0,31/min)bilateral

reticular andBAL

infiltrates
54 . 4 (RA)1 , 1704

57 . obilateral

reticular andinfiltrates

5

400

734

334

32 . 6 (RA)75 . 6bilateral
reticular

6

322

790

468

59 . 7 (RA)49 . obilateral
reticular

7346

1 , 455

1 , 109

31 . 9 (RA)70 . 1

diff use

BAL
TBLB
BAL
TBLB
BAL
BA L

ground
glass
8

309

l,723 1,414 32 6(RA)82 . 59400

1 , 173773 40 . O (RA)74 . 6bilateral
reticular

BALbilateralpost

reticular andmorteminfiltrates

10

Average353

407
85 . 1

589

236 115(O, 11/min)

1 , 030 610
d:347 :!:391

26 , 3bilateral
reticularpost

mortem69 . 3

23.4* RA: r,oom air
20 ml aliquots of sterile normal saline. Transbronchial lung biopsy was performed under theguidance of fiuoroscopy. The fiuid obtained by lavage was centrifuged at 1,500 rpm for 5 min.
The deposit was spread onto several glass microscope slides and stained to reveal Pneumocys‑
tis carinii cysts using Gomori's methenamine silver stain. All specimens were also stained by

Gram's method. Routine aerobic and anaerobic bacterial cultures were performed on all
samples, as were cultures for mycobacteria and fungi. All patients, except for one patient
diagnosed as PCP at postmortem examination, were treated with a high dosage (8‑16 g/day)of cotrimoxazole given orally together with 4 mg/kg/day of pentamidine isethionate given
either intravenously or intramuscularly. Two of th patients were treated with 600 mg/dayof pentamidine isethionate by inhalation and intravenous corticosterQid adjunctively.

RESULTS
Ten patients met the criteria for inclusion in this study. There were 7 males and‑ 3
females, with ages ranging from 32 to 78. Four patients had an underlying hematologicaldisorder, 2 had renal disease, 2 had collagen disease, I had lung cancer, and I had sarcoidosis.

350

Table 3 Therapy and outcome
Patient

Other opportunisticorganism

1

(‑)

2

(‑)

3

(‑)

4

(‑)

5

Haemo philus
in fluenzae

6
7
8
9

Cand ida
albicans

Therapy
Cotrimoxazole (po)Pentamidine (im)Cotrimoxazole (po)Pentamidine (im)Cotrimoxazole (po)Pentamidine (im)Cotrimoxazole (po)Pentamidine (im)Cotrimoxazole (po)Pentamidine (im)Cotrimoxazole (po)Adversereaction

Intravenous
Corticosteroid

therapy

ventilation

Outcomeof PCP

Mechanical

(‑)

(‑)

(‑)

poor

(‑)

(‑)

(‑)

poor

hypoglycemia

(‑)

(‑)

poor

(‑)

(‑)

(‑)

well

(=)

(‑)

(‑)

poor

thrombopenia

(+)

(+)

well

hy potension

Pentamidine (iv, aer)liver dysfunction

Cand ida
albicans

Cotrimoxazole (po)

(‑)

(+)

(+)

well

(‑)

Cotrimoxazole (po)Pentamidine (iv)
Cotrimoxazole (po)

(‑)

(‑)

(+)

poor

(‑)

(‑)

(‑)

poor

(‑)

(‑)

(‑)

poor

Aspergillus spp.

Pentamidine (iv, aer)

Cyiomegalovirus
10

Can d ida(‑)

albicans

Cyiomegalovirus* aer: aerolized, im: intramusculaly, iv: intravenously, po: per oral

All patients had received immunosuppressive therapy, such as corticosteroids. Eight patients,

all but the two with adult T cell leukemia, had received the therapy for more than 2 months(Table 1) . Three cases were asymptomatic when chest radiography r.evealed an abnormal
shadow, and 5 patients had a rapid progression of cough, dyspnea, and fever, over 2‑3 days.
The patients with adult T cell leukemia had longer prodromes (Table 1) .

The mean P (A‑a)O, gradient at presentation was 69.3i23.4 torr, and the mean LDH
value at presentation was 1,030d:347 IU/L. To assess whether the elevated LDH values werea result of the infection or reflected other aspects of the status of the patients, we reviewed

measurements prior to the acute illness. Previous values, determined 1‑2 months before thediagnosis of PCP, were available in 9 patients, Comparison of these values with those at thetime of diaguosis of PCP showed that all 9 patients had an increased in LDH values of morethan 190 IU/L, with a median increase of 610 391 IU/L (Table 2). At the time of presenta‑

tion, chest radiography revealed a bilateral r ticular shadow in 5 patients, a bilateralreticular shadow and infiltrates in 4, and an
S‑like diffuse ground glass shadow in 1
(Table 2) . Two patients had Pneumocystis carinii cysts demonstrated at postmortem exami‑
nation, but in all other patients diagnostic material was obtained while alive by TBLB and/or BAL (Table 2).
Coexisting pulmonary infections were diagnosed in 5 patients. Candida albicans wasrecognized in 3 patients, Cytomegalovirus in 2, Haemophilus influenzae in 1, and Aspergillus
spp. in I (Table 3). Nine patients received treatment with a high dosage of cotrimoxazole
and pentamidine isethionate. In 3 patients, mechanical ventilation was also used, and in 2 ofthese, intravenous corticosteroid and aerozolized pentamidine were prescribed adjunctively.
Six patients died of progressive pneumonia. Although the other 3 patients recovered from

351

PCP, they died subsequently due to underlying hematological malignancy, heart failure, andmulti organ failure (Table 3).

DISCUSSION
PCP associated with AIDS often presents in an indolent fashion, with symptoms mahifest

for several weeks prior to presentation. Non‑AIDS PCP has a more acute onset, withpulmonary symptoms progressing rapidly to respiratory failure within I week (Macfarlane
and Finch, 1985; Engelberg et al., 1984). In the present study, all patients were PCP cases
from conventionally caused immunosuppression, and 8 patients had a rapid progression ofsymptoms for less than 2‑3 days, with the patients with adult T cell leukemia having longer
prodromes. In one study, patients with hematological disorders had variable prodromes
ranging from 2 days to 4 weeks, which is in accordance with our results (Carter et al., 1988) .

Recent studies have suggested that the serum LDH value and P(A‑a)02 gradient havediagnostic and prognostic implications in patients with PCP associated with AIDS (Garayand Greene, 1989). The serum LDH value is elevated in most AIDS patients when PCP ispresent, and usually increases with the development of the infection' and decreases again withrecovery, although the mechanism for this change is unknown. An association between the
LDH value and survial from PCP was also noted, with survivors having significantly lower
mean serum levels than those who died (El‑sadr and Simberkoff, 1988) . Higher values of
LDH appeared to reflect more extensive interstitial inflamation. A reduction in the diffusing

capacity for carbon monoxide is one of the characteristic abnormalities of pulmonary
function in patients with PCP, which causes alveolar‑capillary block. This abnormality is
attributable to thickening of the alveolar‑capillary membrane by attachment of this parasite
to the epithelial surface (Sankary et al., 1988). The mechanism of the increased P (A‑a)02
gradient has not been fully mentioned. In our study, the LDH value and P (A‑a)02 gradientat presentation were significantly elevated, and there was an increase in the LDH value atpresentation over the previous values. Therefore the elevated LDH values appeared to be a
result of PCP. When the patients with good outcome of PCP were compared with those with

failure, the mean LDH value was 969 287 vs. 1,000i392 IU/L, and the mean P(A‑a)O,
gradient was 62.0i9.48 vs. 74.Id:28.2 Torr. However there was no significant difference
between the two groups. The extremly high degree of the elevation in the LDH value and theincrease in P (A‑a)02 gradient appeared to reflect the severity of the disease, which might
account for the poor outcome in our study group. In the present study, we conclude that thesevalues might be useful as markers for diagnosis of PCP in non‑AIDS patients also.
PCP usually presents on chest radiograph as a diffuse bilateral, progressively coalescing

pneumonia that in its earliest stages often spares the peripheral lung fields. PCP presenting

as a pulmonary nodule or a cavity is rare, and mediastinal lymphadenopathy and pleural
effusion are not believed to occur with this infection (Barrio et al., 1986) . In our study, chest

radiography revealed a typical diffuse bilateral, progressively' coalescing pneumonia, and in
5 patients bilateral diffuse infiltrates were already visible at presentation. This might suggest

an acute onset of the disease in our population and the difiiculty in making an early diagnosis.

It is necessary for the adequate management of PCP to obtain a definitive diagnosis at
an early stage. Non‑invasive methods, such as sputum induction with the use of nebulised
hypertonic saline, are safe, but the sensitivity is relatively low (Yoshida et al., 1978; Pitchenik

352

et al., 1986) . In most studies, transthoracic needle biopsy has not been recommended becauseof its high incidence of complications. Open lung biopsy has been shown to be more reliable,
but is highly invasive, and this method is currently of limited value and has not been usedwidely for the diagnosis of PCP in Japan (Pass et al., 1986; Shorter et al., 1988). Several

studies have demonstrated that fiber optic bronchoscopy with BAL and TBLB is a safe andsensitive method for the initial diagnosis of PCP as well as other opportunistic infections(Oka et al., 1985; Cordonnier et al., 1985) . In the present study, 7 BALS and 3 TBLBS Wereperformed in 8 patients who were diagnosed as having PCP, with no complication. We
suggest that fiber optic bronchoscopy, especially with BAL, is the most safe, sensitive, andrapid method for the initial diaguosis of PCP.
In half of the patients, coexisting pulmonary infections were recognized. In patients 6
and 7, Candida albicans was recognized for the first time in BAL, which was performed after

recovering from PCP. In these cases, fluconazole was given for the treatment of the fungi,
but it was supposed that prophylactic therapy might have prevented this infection. In PCP
cases in the immunocompromised host, great care must also be given to coexisting infections.
Pentamidine isethionate was the first drug demonstrated to have efficacy in the treatmentof PCP. Experience in immunocompromised patients demonstrated about 70% efiicacy, butwith substantial toxicity. Adverse reactions include renal failure, Iiver dysfunction, hypog‑

lycemia, hyperglycemia, pain and swelling at the injection site, hematologic disturbances,hypotension, and pancreatitis (Salamone and Cunha, 1988; Pearson and Hewlett, 1985; Kluge
et al., 1.978; Zuger et al., 1986; Helmick and Green, 1985; Belehu and Naafs, 1982; Stahl‑
Bayliss et al., 1986; Stoner, 1988; Montgomery et al., 1989). Cotrimoxazole became thepreferred therapy, with pentamidine an alternative, in cases of failure and for those withsevere adverse reactions (Kovacs and Masur, 1988; Furio et al., 1988) . It has been suggested

that a combination of cotrimoxazole and pentamidine is no more effective and may be
harmful in the treatment of PCP. PCP in the non‑AIDS population, however, is reported tobe more fulminant, with greater incidence of treatment failure and fewer adverse reactions
than those from AIDS (Drake et al., 1985; Salamone and Cunha, 1988; Pearson and Hewlett,1985; Kluge et al., 1978; Kovacs et al., 1984; Sattler et al., 1988). It therefore seems to be

useful to examine the efficacy and the incidence of adverse reactions in the concomitant use

of pentamidine and cotrimoxazole in the non‑AIDS population. PCP cases with acute onset
tend to progress to hypoxemic respiratory failure requiring mechanical ventilation. The
mortality rate for these patients is significantly high, despite optimal medical management.
Several studies have demonstrated a possible role of intravenous corticosteroids in adjunctive
therapy in cases with acute life‑threatening respiratory failure secondary to PCP. It was also

speculated in some studies that adjunctive corticosteroid therapy might obviate the use of
mechanical ventilation (Gallacher et al., 1989) . The outcome of patients with corticosteroid
therapy and the role of mechanical ventilation have yet to be examined. In the present study,
pentamidine together with cotrimoxazole was prescribed for 9 patients. Although one patient

showed thrombocytopenia and liver dysfunction and one other showed hypotension andhypoglycemia, seven patients sustained mild adverse reactions from the therapy and tolerated
it well. Three of the 9 patients given this tr atment recovered from PCP, which is a relatively

poor outcome. This might be due to the severity of the underlying diseases and failure in
making an ealier diagnosis. The concomitant use of pentamidine and cotrimoxazole in ourstudy seemed no more harmful, but no more effective, than therapy with cotrimoxazole

353

followed by pentamidine. Two patients treated with intravenous corticosteroids and mechan‑ical ventilation have recovered from PCP, and we suggest the possible efiicacy of this regime.
However, we acknowledge the low reliability of this conclusion due to the small population
size and the need for evaluation in a larger patient population.

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tis carinii pneumonia presenting as cavitating and noncavitating solitary pulmonary nodules
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2 ) Belehu, A, and Naafs, B. (1982): Diabetes mellitus associated with pentamidine mesylate,
Lancet, 1463
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Managemant of Pneumocystis carinii pneumonia in the immunocompromised host, New
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tis carinii pneumonia requiring mechanical ventilation, Am. Rev. Respir. Dis,, 137, 1264‑1267

7 ) Engelberg, L.A., Lerner, C.W. and Tapper, M.L. (1984): Clinical features of Pneumocystis
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carinii pneumonia in patients with AIDS and other conditions: Experience in a Philadelphia
University teaching hospital, Pharmacother., 8, 221‑234
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tis carinii pneumonia with corticosteroids in a patient with acquired immunodeficiency syn‑drome, Crit. Care Med., 17, 104‑l05
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Pneumocystis carinii pneumonia, Chest, 95, 769‑772
11) Helmick, C.G. and Green, J.K. (1985): Pentamidine‑associated hypotension and route ofadministration, Annal. Intern. Med., 103, 480
12) Kluge, R.M., Spaulding, D.M. and Spain A.J. (1978): Combination of pentamidine and trimeth‑oprim‑sulfamethoxazole in the therapy of Pneumocystis carinii pneumonia in rats, Antimicrob.
Agents. Chemother., 13, 975‑97813) Kovacs, J.A., Hiemenz, J.W., Macher, A.M., Stover, D., Murray, H.W., Shelhamer, J., Lane, H.
C., Urmacher, C., Honig, C., Longi, D.L., Parker, M.M., Natanson, C., Parillo, J.E., Fauci, A.S.,

Pizzo, P.A. and Mazur, H. (1984): Pneumocystis carinii pneum(5nia: a comparison between

patients with acquired immunodeficiency syndrome and patients with other immunode‑
ficiencies,Ann. Intern. Med., 100, 663‑671

14) Kovacs, J.A. and Masur, H. (1988): Pneumo( stis carinii pneumonia: Therapy and prophylax‑is, J. Infec. Dis., 158, 254‑259

15) Macfarlane, J.T. and Finch, R.G. (1985): Pneumocystis carinii pneumonia, Thorax, 40, 561‑570
16) Montgomery A.B., Debs, R.J., Luce, J.M., Corkery, K.J., Turner, J. and Hopewell P.C. (1989):
Aerosolized pentamidine as second line therapy in patients with AIDS and Pneumocystis carinii

pneumonia, Chest, 95, 747‑750

35417) Oka, M., Noguchi, Y., Matsumoto, Y., Tsurukawa, Y., Kohno, K., Araki, J., Mine, Y., Kanda,
T., Saito, A., Hara, K., Tsukasaki, K. and Amenomori, T. (1985): Five cases of Pneumocystis
carinii pheumonia diagnosed by bronchofiberscope, Japan. J. Thora. Dis., 23, 1052‑1058
18) Pass, H.1., Potter, D., Shelhammer, J., Macher, A., Ognibene, F.P., Longo, D.L., Gelmann, E.,
Masur, H. and Roth, J.A. (1986): Indications for and diagnostic efiicacy of open lung biopsy
in the patients with acquired immunodeficiency syndrome (AIDS), Ann. Thorac. Surg., 41,
307‑31219) Pearson, R.D. and Hewlett, E.L. (1985): Pentamidine for the treatment of Pneumocystis carinii
pneumonia and other protozoal diseases, Annal. Intern. Med., 103, 782‑786
20) Pitchenik, A.E., Ganjei, P., Torres, A., Evans, D.A., Rubin, E. and Baier, H. (1986): Sputum

examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired
immunodeficiency syndrome, Am. Rev. Respir. Dis., 133, 226‑229

2D Salamone, F.R. and Cunha, B.A. (1988): Update on pentamidine for the treatment ofPneumorystis carinii pneumonia, Clin. Pharmacy, 7, 501‑510
22) Sankary, R.M., Turner, J.. Lipavsky, A.. Howes. Jr. E.L. and Murray, J.F. (1988): Alveolar
capillary block in patients with AIDS and Pneumocystis carinii pneumonia, Am. Rev. Respir.
Dis., 137, 443‑449

23) Sattler, F.R., Cowan, R., Nielsen, D.M. and Ruskin, J. (1988) : Trimethoprim‑sulfamethoxazole

compared with pentamidine for treatment of Pneumorystis carinii pneumonia in the acquired
immunodeficiency syndrome, Annal. Intern. Med., 109, 280‑287
24) Shorter, N.A., Ross m, A.J., August, C., Schnaufer, L., Zeigler, M., Templeton, J.M., Bishop, J.

H. and O'Neill, Jr.J.A. (1988): The usefulness of open‑lung biopsy in the pediatric bonemarrow transplant population, J. Pediat. Surge., 23, 533‑537
25) Stahl‑Bayliss, C.M. and Kalman, C.M. (1986) : Pentamidine‑induced hypoglycemia in patientswith the acquired immunedeficiency syndrome, Clin. Pharmacol. Ther., 39, 271‑275
26) Stoner, C.P. (1988): Aerosol pentamidine for Pneumocystis carinii pneumonia, Drug. Intellig.
Clin. Pharmac., 22, 916‑917
27) Yoshida, Y., Ikai, T., Ogino, K., Takeuchi, S., Yamada, M., Shimada, Y. and Shiota, T. (1978):Studies on Pneumocystis carinii and Pneumorystis carinii pneumonia. V. Diagnosis by cyst
concentration from sputum, Jpn. J. Parasitol., 27, 473‑481

28) Zaman, M.K. and White, D.A. (1988) : Serum lactate dehydrogenase levels and Pneumocystis
carinii pneumonia, Am. Rev. Respir. Dis., 137, 796‑80029) Zuger, A., Wolf, B.Z., EI‑Sadr, W., Simberkoff, M.S. and Rahal, J.J. (1986): Pentamidine‑
associated fetal acute pancreatitis, JAMA, 256, 2383‑2385

355

AIDS以外の免疫抑制患者におけるニューモシスチスカリニ肺炎の管理
勝又 達哉・河野茂・古賀 宏延・吉富 祐子・
松田 治子・光武耕太郎・東山 康仁・
宮崎 義継・原耕平

AIDS以外の免疫抑制状態に，ニューモシスチスカリニ肺炎を発症した，10症例に臨床的検討を加えた。発症様式では，成人丁細胞白血病の2例は前駆症状が長かったが，他の症例では急性の発症を示した。発症時の胸部レントゲンでは，全例において両側びまん性の陰影を認めた。確定
診断は，8例において経気管支的肺生検，または気管支肺胞洗浄によって，2例において剖検によってなされた。診断時には，LDHの値は発症前の値に比べて高度に上昇しており，肺胞気動脈
血酸素分圧較差も高度に開大しており，これらの値が早期診断に有用であることが示唆された。
他の日和見感染の合併は，5例に認められた。治療においては，ペンタミジンとST合剤の併用
は，副作用は少なかったものの，有効率も低かった。コルチコステロイドは，補助療法として有用と考えられた。

長崎大学医学部第二内科学教室（〒852長崎市坂本町7−1）

J pn. J. Trop. Med. Hyg., Vol. 19 No. 4 1991, pp. 357‑370

357

TWO NEW BLACKFLY SPECIES OF SIMULIUM (SIMULIUM)
FROM JAVA, INDONESIA (DIPTERA= SIMULIIDAE)
HIROYUKI TAKAOKA* AND UplK KESUMAWATI HADI'
Received September 6 1991/Accepted October 15 1991

Abstract: Two new blackfiy species, Simulium (Simulium) sigiti sp. nov. and S. (S.)
javaense sp. nov. are described based on the female, male, pupal and larval specimens
collected from Java. Simulium (S.) sigiti is characterized by the resence of a pit‑likecuticular organ at the base of pupal gill, as well as shortened pupal filaments. This is thefirst species of the tuberosum group from Java. The variety of S. (S.) iridescens De
Meijere, 1913 is here elevated to the species status and is given a neiv specific name S. (S.)

javaense sp. nov. This new species is easily distinguished from S. (S.) i.ridescens by the
infiated gill filaments, the absence of tubercles on the cephalic and thoracic integumentsof the pupa, and the russet body coloration of the larva.

INTRODUCTION
The simuliid fauna of the Sunda Islands has not been studied since Edwards (1934)
described 11 new species from Sumatra, Java and Bali, making a total of 19 taxa (including
two subspecies) for this archipelago.

Recently, we made a preliminary survey on the blackfiies in East and West Java andcollected a total of 16 species of Simulium Latreille s. l. including several new species. This
paper treats two new species belonging to the subgenus Simulium Latreille s. str.
The classification follows that of Crosskey (1969). Collecting and rearing methods, aswell as dissection of anatomical parts for description, were mentioned in Takaoka (1983) .
The holotype, allotype and some paratype specimens will be deposited at the Departmentof Parasitology and Pathology, Faculty of Veterinary Medicine, Bogor Agricultural Univer‑sity, and some other paratypes at the Departinent of Entomology, Bogor Museum of Zoology,
Bogor. Indonesia.

DESCRIPTION
Simul ium (Simulium) sigiti sp, nov.

Female. Body length 2.1 mm. Head. Narrower than width of thorax. Frons black, shiny,l2Division of Medical Zoology, Medical College of Oita, Hazama, Oita 879‑55, Japan ,Entomology Laboratory, Department of Parasitology and Pathology, Faculty of Veterinary
Medicine, Bogor Agricultural University, Taman Kencana 3, Bogor 16151, Indonesia

358

and with several dark stout hairs along lateral margins; frontal ratio (width at top of eyes
and that just above antenna to length) 1.3:1:1.3; frons‑head ratio (width of frons at top of eyes

to maximal width of head) 1:4.1. Clypeus black, shiny, probably greyish pruinose and with

scattered dark stout hairs. Antenna composed of 2 +9 segments, dark brown except scape,pedicel and base of Ist fiagellar segment pale. Maxillary palp brownish black, composed of
5 segments with proportional length of 3rd, 4th and 5th segments 1:1:1.7; 3rd segment (Fig. 1)
of moderate size; sensory vesicle medium in size, elliptical, with rugged surface, 0.34 x lengthof 3rd segment, and with small round opening a little distad to the center. Maxillary laciniawith 17 inner and 17 outer teeth. Mandible with ca.30 inner and 12 outer teeth. Cibarium(Fig. 3) with numerous minute tubercles. Thorax. Scutum black, shiny, not patterned andcovered moderately with recumbent, dark brown pubescence, interspe.rsed with long, upstand‑ing dark hairs on prescutellar area. Scutellum black, with long dark hairs. Postscutellum
black, shiny, silvery iridescent when viewed in certain angle of lights, and without hairs.
Pleural membrane bare. Katepisternum longer than depth, and bare. Legs. Foreleg (Fig. 4):
coxa pale yellow; trochanter dark yellow; femur dark yellow basally, gradually becoming
dark medially and black on distal 1/2 or l/3; tibia black with outer median portion largely
pale white and in lights sheeny; basitarsus entirely black, dilated (W:L = 1:4.8) , and with shortdorsal hair crest; rest tarsal segments black. Midleg (Fig. 5) : blackish except basal 1/2 oftrochanter, base of tibia, basal 2/3 or more of basitarsus and basal 1/2 of 2nd tarsomere
whitish yellow; tibia largely sheeny on posterior surface in lights. Hind leg (Fig. 6) : blackishwith trochanter and base of femur dark yellow, and basal 1/3 of posterior surface of tibia,
basal 3/5 of basitarsus and basal 1/2 of 2nd tarsomere whitish yellow; tibia largely sheeny on
posterior surface in lights, and slightly narrower than femur; basitarsus W:L= 1:5, calcipala
short, width to that of basitarsal tip is 1:1.9, W:L ratio 1:1; pedisulcus distinct. All tarsal claws

simple, without subbasal or basal tooth. Femora and tibiae of all legs covered with simple

hairs only. Wing. Length 2.0 mm; costa with spinules and hairs; subcosta haired; basal
section of vein R bare; hairs at base of stem vein dark brown; basal cell absent. Abdomen.
Basal scale black with fringe of dark hairs; 2nd segment dark brown with large, dorsolateral

whitish iridescent spots broadly connected to each other in the middle; tergites 3, 4 and 5
small, dark brown and shining, tergites 6‑9 Iarge, dark brown, shining and with dark hairs.
Genitalia (Figs. 8‑10) . Ventral surface of abdominal segment 7 with large, weakly developed
sternite medially (Fig. 8a) and moderately haired. Sternite 8 (Fig. 8d) well sclerotized, baremedially but with ca. 13 short and long stout hairs laterally on each side; anterior gonapo‑
physes (Fig. 8e) triangular in shape, membraneous, covered with ca. 12 minute setae as wellas numerous microsetae except narrow portion along posterior margin bare; inner borders
slightly curved, narrowly sclerotized, and folded dorsally as shown in Fig. 9. Genital fork
(Fig. 8c) of inverted‑Y form, with well sclerotized stem; arms slender, each with strongly
sclerotized distal ridge and distinct projection directed anterodorsally. Paraproct (Fig. 10)

slightly shorter than wide, with ventral margin concave medially, and covered with ca. 15
short stout hairs ih lateral view; paraproct in inside view somewhat broadly sclerotized along

ventral margin and covered sparsely with microsetae. Cercus (Fig. 10) rounded posteriorly,

ca.0.5x as long as wide, and, covered with numerous short hairs. Spermatheca (Fig. 8b)
nearly globular in shape, well sclerotized with weakly defined reticulate pattern, and withminute internal setae; tube and small adjacent area of spermatheca unsclerotized.

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Figures 1‑18 Adults of Simulium (Simulium) sigiti sp. nov. 1, third segment of female maxillary
palp; 2, third segment of male maxillary palp; 3, cibarium; 4, foreleg of female; 5, midleg of female;6, hind leg of female; 7, hind leg of male; 8, ventral view of female genitalia a, 7th sternite; b,
spermatheca; c, genital fork; d, 8th sternite, e, anterior gonapophysis; f, paraproct; g, cercus; 9,anterior gonapophyses showing dorsally folded inner margin; 10, paraproct and cerctiS in side view;ll, male scutum in dorsal view; 12, male scutum in side view; 13, scale‑like hairs on outer surfaceof male hind femur (scale, 0.02 mm) ; 14, male genitalia in ventral view (coxite and style on rightside, median sclerite and parameres omitted) h, coxite; i, style; j, ventral plate; 15, ventral platein lateral view; 16, ventral plate in end view; 17, style viewed ventromedially showing basal
protuberance with numerous spines; 18, median sclerite.

360

Male. Body length 2.4 mm. Head. Width slightly wider than thorax. Upper eye consistingof large facets in 20 horizontal and vertical rows. Clypeus black, whitish grey pruinose,strongly silvery iridescent when illuminated, and covered sparsely with dark brown hairs.
Antenna composed of 2 + 9 segments, dark brown except scape and pedicel dark yellow andbase of Ist flagellar segment pale yellow; Ist flagellomere somewhat elongated (W:L= l:2.1) .

Maxillary palp composed of 5 segments with proportional length of 3rd, 4th and 5th segments
1:1.1:2.3; 3rd segment (Fig. 2) of normal size with small, globose sensory vesicle which is
0.2 x length of 3rd segment; small rounded opening situated medially. Thorax. Scutum black,with white pruinose pattern composed of an anterior pair of triangular spots with rounded
apex on shoulders extending posteriorly along lateral margins up to base of wing and a largetransverse spot entirely covering prescutellar area which is not contiguous to anterior spots(Figs. 11 and 12) , ‑these pruinose areas with silvery iridescence when illuminated: scutum

uniformly covered with dark brown recumbent pubescences (in lights these pubescences
appear bright coppery) , interspersed with long upright hairs on prescutellar area. Scutellum
black, shiny, white pruinose and with several uptight dark hairs. Postscutellum black, shiny,

white pruinose and without hairs. Pleural membrane and katepisternum as in female. Legs.
Coloration as in female except hind tibia black with base pale whitish yellow, and hindbasitarsus with less pale portion (basal 1/2 or a little more) ; in addition to simple hairs, scale‑like hairs (Fig. 13) are seen densely on outer surface of femora and on both sides of tibiae
of hind leg; fore basitarsus somewhat dilated (W:L ratio 1:4.4) ; hind basitarsus (Fig. 7) much

enlarged (W:L= 1:4.5) , widening toward basal l/2, then nearly parallel sided, and its greatest

width equal to those of hind femur and tibia. Calcipala small (W:L rati0=1:1) and ca. 0.22 x

as wide as basitarsal width. Pedisulcus well marked on 2nd tarsomere. Wing. Length 1.9 mm;other features as in female except subcosta bare. Abdomen. Basal scale blackish with longdark hairs. Terga black, with dark hairs; segrnents 2, 6 and 7 each with a pair of silvery
iridescent areas dorsolaterally, those on segrnent 2 connected broadly to each other in the
middle. Genitalia (Figs. 14‑18) . Coxite in ventral view (Fig. 14h) as long as wide and ca.
0.6 x length of style; style (Fig. 14i) Iength ca. 3 x greatest width at basal 1/4, spatulate,curved inwards, slightly tapering apically and with a subterminal spine; style (Fig. 17) withbasal protuberance produced dorsomedially, bearing numerous spines on its surface. Ventral
plate (Fig. 14j) with base nearly quadrate in shape, having ventrally produced hairy processwith weakly toothed posterolateral margins on proximal 2/3; posterior margin when viewed
ventrally slightly concave; basal arms slightly diverging from each other. Median clerite(Fig. 18) well sclerotized at base, plate‑like and nearly parallel sided. Parameres each withnumerous small hooks.
Pupa. Body length (excluding gill filaments) 2.5 mm. Head. Integument dark golden yellow,
sparsely covered with large, disc‑like tubercles of various sizes (Fig. 20) , Iarger ones
appearing to have more distinct depression in the center than smaller ones; most tubercles
fringed with several microtubercles; head with I facial and 2 frontal pairs of slender, simple

trichomes. Thorax (Fig. 21). Anterior 1/2 of thoracic integument dark golden yellow,
sparsely covered with large disc‑like tubercles (Fig. 2lb) similar to those on the head, though
the larger groups of tubercles much smaller, and central depression of tubercles less distinct,

while posterior 1/2 of thorax pale greyish brown and covered moderately with small, conicalprocesses (Fig. 2lc) ; thorax with 2 dorsal and 2 Iateral pairs of simple trichomes on anterior

361

1/2 as well as I Iateral pair of trichomes on posterior 1/2 (Fig. 21) ; at base of gill, there isa large, pit‑like cuticular organ (Fig. 2la) . Gill (Fig. 22) with 6 very short, slender and darkbrown filaments in pairs, almost sessile, and slightly tapering apically; all filaments subequal

to each other in length (ca. 0.8‑1.0 mm) and thickness, and generally extending close together

forwards, downwards, and then inwards just in front of head ( in some specimens I or 2
filaments, specially of upper and lower pairs, curved upwards or downwards and directedbackwards) ; cuticle of filaments with well‑defined annular furrows, and covered uniformly
with minute tubercles (Fig. 22d) . Abdomen. Tergum 2 on each side with I Iong and 5 short
simple setae, 4 of which are stout and spinous. Terga 3 and 4 each with 4 hooked spines alongposterior margin on each side. Terga 6, 7, 8 and 9 each with a cross row of spine‑combs oneach side, though spine‑combs on segment 6 weakly developed (in some pupae, tergum 6
without spine‑combs) . Tergum 9 Iacking terminal hooks. Sternum 4 with a bifid hook anda minute simple seta close together submedially on each side. Sternum 5 with a pair of bifid
hooks submedially on each side. Sterna 6 and 7 each with a pair of bifid inner and simple
outer hooks widely spaced on each side. Grapnel‑like hooklets absent. Cocoon (Fig. 19) .
Simple, slipper‑shaped, thickly woven, with a thick anterior rim; there is a connecting bandacross the front floor but not forming a definite front wall.

Mature larva. Body length 5.0‑5.2 mm. Body color dark brown (though annular interspacesof abdominal segments 1‑5 narrowly discolored in the alcoholic specimens) . Cephalicapotome (Fig. 23) pale widely on anterior 1/2, and light brown on posterior 1/2, withdarkened areas medially, Iaterally and posteriorly; head spots more or less positive. Antennacomposed of 3 segments and apical sensillum, Ionger than stem of cephalic fan; Iength ratioof segments (from base to tip) 1.5:1.6:1. Cephalic fan with ca. 42 main rays. Mandible (Fig.

26) with a large and a medium mandibular serration but without supernumerary serration.
Hypostomal teeth 9 in number, small with median and corner teeth longer than others; 6
hypostomal bristles lying subparallel to lateral border on each side (Fig. 25) . Postgenal cleft(Fig. 24) nearly parallel‑sided near base, then converging apically, and ca. 2 x length of
postgenal bridge. Thoracic cuticle bare. Abdominal cuticle bare except dorsolateral areas
on last segment moderately covered with short, discolored setae. Rectal gill of 3 Iobes eachwith 2‑3 small secondary lobules ventrally. Anal sclerite X‑shaped, with broadened anteriorarms which are 0.54 x length of posterior ones. Posterior circlet with ca. 78 rows of hooklets

with up to 14 hooklets per row. Ventral papillae absent.

Type specimens. Holotype, slide mounted, reared from pupa collected from Puncak, West

Java, l0.1.1991, H. Takaoka and U.K. Hadi. Allotype c7t, slide mounted, reared from pupa,same data as holotype. Paratypes, I, I c71 reared from pupae, 4 pupal skins and associatedcocoons, and 2 mattire larvae, all slide mounted and same data as holotype.

Distribution. West Java.
Ecological notes. The pupae and larvae were found on slender roots of trees trailing in thewater of a small shaded stream (less than 0.5 m wide; 1,250 m in altitude) running down

through the sloped natural forest near the upper border of open field cultivated for tea
plantation. This species was collected together with S. (S.) iridescens and two Gomphostilbia

362

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Figures 19‑26 Pupa and larva of Simulium (Simulium) sigiti sp. nov. 19, pupa and cocoon attachedto the slender plant root; 20, enlargement of disc‑like tubercles on pupal head (scale, 0.02 mm) ; 21.

lateral view of pupal thorax a, pit‑like organ; b, enlargement of disc‑like tubercles on anterior
half (same scale as in Fig. 20); c, enlargement of conical processes on posterior half (same scale

as in Fig. 20); 22, pupal gill filaments‑ l, enlargement of filament showing annular furrows(scale, 0.02 mm) ; 23, Iarval cephalic apotome; 24, ventral view of head capsule showing postgenal
cleft; 25, Iarval hypostomium; 26, apical tip of larval mandible.

species.

Remarks. This specific name was given in honor of Dr. Singgih H. Sigit, Professor and Dean,

Faculty of Veterinary Medicine. Bogor Agricultural University for his contributions to
medical entomology.
This new species can be assigned to the tuberosum group of Simulium (Simulium)
defined by Rubtsov (1959‑1964) by having the spinous basal protuberance of the style,quadrate ventral plate with ventral projection and toothed posterolateral borders, pruinose

363

scutal pattern in the male, simple claws and unpatterned scutum in the female, and pupal gillwith six filaments.

Although the female, male and larva of this species are very similar to those of mostother known species of the tuberosum group, the pupa of S. (S. ) sigiti sp. nov. is very distinct
in possessing a pit‑like organ at the base of gill (Fig. 2la) , as well as the short, slender gill

filaments which are subequal to each other in length and thickness. The similar pit‑likecuticular structure near the base of pupal gill has been reported in the clathrinum group of
Simulium (Morops) from Australasian region (Smart and Clifford, 1965; Crosskey, 1967).

However, none of the other known species of the tuberosum group has such an interesting
cuticular structure except S. (S.) nignfacies Datta, 1974 described from the male and pupal
specimens collected from Darjeeling, West Bengal, India, which seems to have the similar pit‑
like organ at the base of pupal gill judging from the illustration made by Datta (1974) . The
pupal gill filaments of the latter species are also shortened (1.2 mm long) Iike this species but

the relative thickness of filaments are different, decreasing from above downwards with theouter filament of upper pair being the thickest of all, as in most other species of this group.
In addition, the pupa of S. (S) , nignfacies has spine‑combs on the abdominal segments 8 and9 only.

Simulium (S.) puliense Takaoka, 1979 from Taiwan seems to be close to this new species
by having the somewhat shortened pupal gill filaments (ca. 1.5 mm long) as well as thesimilar arrangement of spine‑combs on the pupal abdomen. However, unlike this new
species, pupal filaments of the Taiwanese species are different in relative thickness, i.e., outerfilament of upper pair is a little thicker than other five filaments which are subequal in
thickness to each other.
The pupa of S. (S.) aenelfacies which was described from Sabah (Edwards, 1933) andlater assigned to the same species group by Takaoka (1983) is yet unknown. The comparisonof female genitalia (Takaoka, 1983) shows that there are differences in the number of hairson the eighth sternite and anterior gonapophyses, and in the shape of paraproct.

Simulium (Simulium) javaense sp. nov.
Simulium iridescens De Meijere 1913 var. ?: Edwards, 1934, Arch. Hydrobiol., Suppl., 13, 114(male, pupa and larva) .

Female. Body length 2.4 mm. Head. Narrower than width of thorax. Frons black, shiny,
thinly greyish pruinose and with several dark stout hairs along lateral margins; frontal ratio

1.2:1:1.1; frons‑head ratio 1:3.8. Clypeus black, shiny, greyish pruinose and with scattereddark stout hairs; silvery iridescent when illuminated. Antenna composed . of 2 + 9 segments,

dark brown except scape, pedicel and base of Ist fiagellar segment pale. Maxillary palp
brownish black, composed of 5 segments with proportional length of 3rd, 4th and 5th seg‑ments 1:1.1:1.8; 3rd segment (Fig. 27) not enlarged; sensory vesicle small, elliptical, withrugged surface, 0.22 x length of 3rd segment, and with small round opening. Maxillary
lacinia with 14 inner and 14 outer teeth. Mandible with ca. 26 inner and 13 outer teeth.
Cibarium (Fig. 29) with numerous minute tubercle. Thorax. Scutum black, shiny, thinly grey

pruinose, not patterned and covered moderately with recumbent, dark brown pubescence,
interspersed with long, upstanding dark hairs on prescutellar area. Scutellum black, with364

long dark hairs. Postscutellum black, shiny, silvery iridescent when viewed in lights, andwithout hairs. Pleural membrane bare. Katepisternum longer than depth, and bare. Legs.
Foreleg (Fig. 30): coxa pale yellow; trochanter dark yellow with distal l/3 brown; femur
brown basally, and much darkened distally; tibia black with outer median portion largely
pale white and in lights sheeny; basitarsus entirely black, dilated (W:L = 1:3.7) , and with thickdorsal hair crest; rest tarsal segrnents black. Midleg: blackish except basal 4/5 or more ofbasitarsus and basal 1/2 of 2nd tarsomere whitish yellow; tibia largely sheeny on posterior
surface in lights. Hind leg (Fig. 31) : blackish with trochanter and base of femur dark yellow,
and base of tibia, basal 2/3 of basitarsus and basal l/2 of 2nd tarsomere whitish yellow; tibia

largely sheeny on posterior surface in lights, and slightly narrower than femur; basitarsus
W:L=1:5.2, calcipala short, width to that of basitarsal tip is 1:2, W:L ratio 1:0.7; pedisulcus
distinct. All tarsal claws each with small, subbasal tooth (Fig. 33) . Femora and tibiae of alllegs covered densely or moderately with scale‑like hairs (Fig. 32) as well as simple hairs onouter surface. Wing. Length 2.0 mm; costa with spinules and hairs; subcosta bare; basal
section of vein R bare; hairs at base of stem vein dark brown; basal cell absent. Abdomen.
Basal scale black with fringe of dark hairs; 2nd segment dark brown with large, dorsolateral
whitish iridescent spots broadly connected to each other in the middle; tergites 3 and 4 small,dark brown and shining, tergites 5‑9 Iarge, dark brown, shining and with dark hairs.
Genitalia (Figs. 34‑37). Ventral surface of abdominal segment 7 widely expanded posterior‑ly, covering sternite 8 almost entirely, and moderately haired on anterior 1/2 but bare on
posterior 1/2; plate‑like sternite undeveloped. Sternite 8 well sclerotized, bare medially andlaterally but with 14 or 15 Iong stout hairs submedially on each side; anterior gonapophyses

triangular in shape, rounded on posterointernal tip, membraneous, covered with a few minutesetae as well as numerous microsetae except narrow portion along posterior margin bare;inner borders nearly straight and narrowly sclerotized. Genital fork of inverted‑Y form, withwell sclerotized stem; arms slender, each with strongly sclerotized distal ridge and distinct

projection directed anterodorsally (well discernible when viewd laterally‑Fig. 35) . Para‑proct narrowed ventroposteriorly in lateral view (Fig. 37) , covered uniformly with numerous
short hairs on outer and ventral surfaces and also narrowly on inner surface (Fig. 36) ; whenviewed from inside, paraproct (Fig. 36) widely transparent with several minute setae
medially and not forming wide, darkly sclerotized plate, although narrowly sclerotized along
ventral margin near base. Cercus (Fig. 37) rounded posteriorly, ca. 0.5x as long as wide, andcovered with numerous short hairs. Spermatheca nearly globular in shape, well sclerotizedwith weakly defined reticulate pattern, and with minute internal setae; tube and small
adjacent area of spermatheca unsclerotized.

Male. Body length 2.5 mm. Head. Width as wide as thorax. Upper eye consisting of large
facets in ca. 18 horizontal and vertical rows. Clypeus black, whitish grey pruinose, st.rongly

iridescent when illuminated,nd covered sparsely with dark brown hairs. Antenna composedof 2 + 9 segments, entirely dark brown with base of Ist flagellar segment pale; Ist flagellomere

somewhat elongated (W:L=1:1.7). Maxillary palp composed of 5 segments with propor‑
tional length of 3rd, 4th and 5th segments 1:1.1:2.1; 3rd segment (Fig. 28) of normal size withsmall, elliptical sensory vesicle which is 0.3x length of 3rd segrnent. Thorax. Scutum black,with white pruinose pattern composed of an anterior pair of triangular spots, a large trans‑verse spot on prescutellar area which is contiguous to anterior spots by a narrow band along

365

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lateral margins (Fig. 38)‑these pruinose areas with silvery iridescence when illuminated;scutum uniformly covered with dark brown recumbent pubescences ( in lights these pubescen‑
ces appear bright coppery) , interspersed with long upright hairs on prescutellar area.

Scutellum, postscutellum, pleural membrane and katepisternum as in female. Legs. Only
following observations were possible due to incomplete condition of legs in dissected speci‑
mens: coloration probably as in female; scale‑like hairs on outer surface of femora and tibiae
also as in female; fore basitarsus somewhat dilated (W:L ratio 1:4.5) ; hind basitarsus enlar‑

ged, probably as wide as or wider than hind tibia, with distinct calcipala; pedisulcus wellmarked on 2nd tarsomere. Wing. Length not measured; other features as in female.
Abdomen. Basal scale blackish with long dark hairs. Terga black, with dark hairs; segments
2, 4, 5, 6, 7 each with a pair of silvery iridescent areas dorsolaterally, those on segment 2
connected broadly to each other in the middle. Genitalia (Figs. 39‑42) . Coxite in ventral view
1.5 x as long as wide and ca. 0.77x length of style; style (Fig. 40) Iength 2.2x greatest width

at basal 1/4, spatulate, curved inwards, tapering apically and with a subterminal spine.
Ventral plate: in ventral view nearly quadrate in shape with short basal arms diverging fromeach other; in lateral view sharply increasing its thickness from anterior to posterior margins

thus appearing triangular and with thumb‑Iike projection directed ventrally in the center
which is setose on anterior surface but bare on its apex (Fig. 41); in end view trapezoid,slightly narrowed apically, with apical margin concave and irregularly dentate medially andalmost smooth and bare (a few scattered minute setae in one specimen) on posterior surface(Fig. 42) . Median sclerite (Fig. 39b) of shoehorn shape, thin, and slightly narrowed apically.

Parameres (Fig. 39a) each with numerous small hooks.
Pupa. Body length (excluding gill filaments) 2.2‑2.5 mm. Head and thorax. Integument dark
yellow, almost bare except posterior 1/2 of thoracic integument covered moderately withsmall tubercles; head with I facial and 2 frontal pairs of simple trichomes, and thorax (Fig.
43) with 5 pairs of simple trichomes dorsally, all subequal in length. Gill (Figs. 43 and 44)
with 6 greyish, short, infiated filaments in pairs, shortly stalked, and tapering apically; allfilaments subequal to each other in length and thickness; I of 2 filaments of upper and middle

pairs directed inwardly and downwardly just in front of head, and I of upper paired‑filaments

directed upwardly and abruptly bent forwardly; cuticle of filaments becoming somewhat
rugged towards apex and with rough annulations but not forming well‑defined reticulate
patterns, and covered uniformly with minute tubercles. Abdomen. Tergum 2 on each sidewith I Iong and 5 short simple setae, 3 of the latter stouter than other 2 setae. Terga 3 and4 each with 4 hooked spines along posterior margin on each side. Terga 7 and 8 each witha cross row of 3‑5 spine‑combs on each side. Tergum 9 with comb‑like groups of minutespines but devoid of spine‑combs like those on segments 7 and 8, and also lacking terminal
hooks. Sternum 5 with a pair of bifid hooks close together submedially on each side. Sterna
6 and 7 each with a pair of bifid or simple hooks widely spaced on each side. Grapnel‑likehooklets absent. Cocoon (Figs. 45 and 46) . Simple, slipper‑shaped, thickly woven, with athick anterior rim; there is a connecting band across the front but not forming a definite front
wall.

Mature larva. Body length 4.5‑5.2 mm. Body color greyish with broad russet band dorsallyon each abdominal segrnent, those bands often discolored medially to varying extents.

367

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Figures 43‑51 Pupa and larva of Simulium (Simulium) javaense sp. nov. 43, dorsal view of head andanterior half of thorax of the pupa showing inflated gill filaments and thoracic trichomes; 44, pupal
gill filaments in lateral view; 45, cocoon in dorsal view; 46, cocoon in lateral view; 47, Iarval
cephalic apotome; 48, ventral view of larval head‑capsule showing postgenal cleft; 49, Iarval
hypostomium; 50, apical tip of larval mandible; 51, ventral view of larval rectal gill showing small
finger‑like secondary lobules.

Cephalic apotome (Fig. 47) pale yellowish on anterior 1/2, also pale or light brown on
posterior 1/2, and much darkened on peripheral areas except near anterior border; head spots
more or less positive except submedial cross spots near posterior border turned to negative.

Antenna composed of 3 segments and apical sensillum, Ionger than stem of cephalic fan;length ratio of segments (from base to tip) 1.9:2.1:1. Cephalic fan with ca. 40 main rays.
Mandible (Fig. 50) with a large and a medium mandibular serration but without supernumer‑
ary serration. Hypostomal teeth 9 in number, small with median and corner teeth longer thanothers; 7 or 8 hypostomal bristles diverging posteriorly from lateral border on each side (Fig.
49). Postgenal cleft (Fig. 48) deltoid in shape, widening posteriorly, ca. 2.5x length of

postgenal bridge. Thoracic cuticle bare. Abdominal cuticle bare except dorsolateral areas
on last segrnent moderately covered with short, discolored setae. Rectal gill (Fig. 51) of 3

lobes each with 1‑3 small secondary lobules ventrally. Anal sclerite X‑shaped, withbroadened anterior arms which are 0.52 x length of posterior ones. Posterior circlet with ca.

368

90 rows of hooklets with up to 16 hooklets per row. Ventral papillae absent.

Type specimens. Holotype, slide mounted, reared from pupa collected near Pujon, East

Java, 19. XU: . 1990, H. Takaoka. Allotype c ' , slide mounted, dissected from pupa, same data

as holotype. Paratypes, 2reared from pupa, I c71 dissected from pupa, 3 pupal skins andassociated cocoons, and 5 mature larvae; all slide mounted except I

and 3 mature larvae

in alcohol, same data as holotype.
Ecological notes. The pupae and larvae were found attached to slender plant roots and sticks
in a small fast‑fiowing stream (width ca. 0.3 m) covered densely with shrubs on the hilly slope

just above the mountainous road climbing from Kediri to Batu. The stream crosses this road
before joining the main river which runs parallel to the road. This species was collected
together with S. (S.) iridescens and S. ( Gomphostilbia) spp.
Distribution. East Java.

Remarks. Edwards (1934) reported this species as a variety of Simulium (Simulium)
iridescens de Meijere, 1913 based on the male, pupal and larval specimens taken from East
Java. The male of this species is very similar to that of S. (S.) iridescens in many features

including the genitalia, as pointed out by Edwards (1934). There seems to be a slightdifference in the size of the triangular pruinose scutal spots on the shoulders which are in S.

(S.) iridescens much larger, Ieaving smaller median and submedian areas black. In thefemale, it is also very difiicult to separate this from the latter, although the eighth sternite ofS. (S.) iridescens seems to bear a little fewer stout hairs.
On the other hand, this new species is readily distinguished from S. (S. ) iridescens by the

inflated gills on the pupal stage. Further, there is a difference in the cephalic integument
which is smooth in this species but is covered moderately with small tubercles in the latter
species. The larva of this species is also very similar to that of S. (S. ) iridescens but differsfrom the latter by the presence of dorsal russet bands on abdomen.
The male of S. (S. ) javaense shows similarities to S. (S. ) nebulicola Edwards, 1934
described from a male taken at Tosari, East Java, but differs from the latter in the shape ofthe genitalia, as well as coloration of legs. The pupa of the latter species recently collectedby us has the six slender gill filaments similar to that of S. (S.) iridescens.

This new species appears to be closely related to the species of the melanopus group,
reported from the Philippines, North Borneo and North Sulawesi (Takaoka, 1983; Takaoka
and Roberts, 1988) in having the similar male scutal pattern, the unpatterned female scutum,
the female claws with a subbasal tooth, six‑filamented pupal gills and the larval postgenal

cleft deep but not reaching the posterior border of hypostomium. However, the shape of theventral plate easily separates the male of this species from those of the melanopus group, andthe female lacks a ventrointernal sclerotized plate on the paraproct which is found in all themember species of this group. The slipper‑shaped cocoon also separates this species from themelanopus group, the cocoons of which are all shoe‑shaped.

369

ACKNOWLEDGEMENTS
We thank Dr. Singgih H. Sigit, Professor of Department of Parasitology and Pathology
and Dean of Faculty of Veterinary Medicine, Bogor Agricultural University for his generos‑
ities and kindness in providing laboratory space and facilities and arranging collecting trips

during this investigation. Our appreciation also goes to all the staffs of Entomology Labora‑

tory, Department of Parasitology and Pathology, Bogor Agricultural University for their help

in various ways. The senior author would like to thank Dr. D.M. Davies, Professor Emeritus,

McMaster University, Ontario, and Dr. H. Takahashi, Tokyo for their encouragement andsuggestion.

The research was supported by a grant‑in‑aid from Ministry of Education, Science andCulture, Japan to H.T.

REFERENCES
1) Crosskey, R.W. (1967): The classification of Simulium Latreille (Diptera: Simuliidae) from
Australia, New Guinea and the Western Pacific, J. Natur. Hist., l, 23‑51
2) Crosskey. R.W. (1969): A re‑classification of Simuliidae (Diptera) of Africa and its islands,
Bull. Br. Mus. Natur. Hist. (Entomol.). Suppl., 14, 195
3) Datta, M. (1974): Some black flies (Diptera: Simuliidae) of the subgenus Simulium Latreille(s. str.) from the Darjeeling area (India). Oriental Insects, 8, 15‑27

4) De Meijere, J.C.H. (1913): Simuliidae In: Studien uber Sudostasiatische Dipteren VII. Tijd,
Ent., 56, 328‑333

5) Edwards, F.W. (1933) : Diptera Nematocera from Mount Kinabalu, J. Fed. Malay St. Mus., 17,
223‑296
6) Edwards, F.W. (1934) : Deutsche Limnologische Sunda‑Expedition. The Simuliidae (Diptera)of Java and Sumatra, Arch. Hydrobiol., Suppl., 13, 92‑138
7) Rubtsov. I.A. (1959‑1964): Simuliidae (Melusinidae) . In: Lindner, E.. Fliegen palaearkt. Reg.
3, 1‑689

8) Smart, J. and Clifford. E.A. (1965): Simuliidae (Diptera) of the teritory of Papua and New
Guinea, Pacific Insects, 7, 505‑619

9) Takaoka, H. (1979) : The blackfiies of Taiwan (Diptera: Simuliidae) : Pacific Insects, 20, 365‑
403
10) Takaoka, H. (1983): The blackflies (Diptera: Simuliidae) of the Philippines, pp. 212, Japan
Society for the Promotion of Science, Tokyo
11) Takaoka, H. and Roberts, D.M. (1988): Notes on blackflies (Diptera: Simuliidae) from
Sulawesi, Indonesia, Japan. J. Trop. Med. Hyg., 16, 191‑219

370

ジャワ島産アシマダラブユ亜属の2新種について高岡宏行1・Upik

Kesumawati

Hadi2

1990年12月から1991年1月にかけて，ジャワ島において吸血性昆虫ブユの採集調査を行い，得られた標本を検討した結果，数種の新種が含まれていることが分かった。本論文では，ブユ属アシマダラブユ亜属に属する2新種の記載を行った。1種は，伽加m躍吻グループに属し，蝋の呼吸管基部近くの外皮に小孔を有し，呼吸管糸が6本とも極めて短いなど，他の近似種に見られない特徴を有する。本グループに属するブユ種は，これまでにインドネシアからは報告がなく，本種が初めての記録である。他の1種は，これまでS吻％伽吻師48so8郷の変種とされていたブユであるが，蝋の呼吸管糸が袋状に大きくなっているなど，幾つかの形態的な違いがあることから，S．
師吻so飢sから独立させ，新種名を与えて記載を行った。一

大分医科大学医動物学教室（〒879−56大分県大分郡挟間町医大ケ丘1−1）
インドネシア国ボゴール農科大学獣医学部寄生虫・病理学部門昆虫学研究室

日熱医会誌．，第19巻 第4号

1991年，371−385頁

371

皮膚リーシュマニア症の治療
一Promastigote型原虫に対するアンチモン剤とMetronidazoleの薬効の比較一
矢後 文子

平成3年8月7日受付／平成3年10月1日受理

緒る薬効を，初∂伽oで原虫の運動性と増殖状態を言

示標に，比較検討した。

リーシュマニア症の第r選択剤は，重金属であるアンチモン剤とされている（熱帯病治療薬の開材料および方法

発研究班，1990；Pau1ε∫α」．，1984；WHO，1990）。

かつて重金属は，医薬品として頻用されていたが，

1）L廊h㎜％勿原虫：

安全で有効な薬品が，開発されるようになったの

原虫は，サウジアラビアのブレ イダ市に1987年

で，治療薬としての価値は激減した。しかし，現6月より同年12月まて滞在し，皮膚リーシュマニ

在でも使用されている，数少ない重金属にアンチア症に感染した51歳の日本人男性の右大腿伸側のモン剤がある。同剤は，化学的にも生物学的にも

皮疹より，1988年3月31日分離し（KN株），NNN

ヒ素剤に類似し，重篤な副作用を伴う物質（グッ

培地（Leventhal

トマン，ギルマン，1975）なので，皮膚リーシュて，使用した。

andCheadle，1989）で継代しマニア症患者への投薬を，躊躇する医師も多い。

内蔵リーシュマニア症と異なり，生命を脅かさない皮膚リーシュマニア症の治療に，時にはショッ

2）薬効試験：アンチモン剤は，5価のPentostam⑪（Well−

ク症状などの副作用を伴う治療薬は適当ではなく，come社，England，1m珍中に100㎎のより安全で日本でも入手し易い薬剤が望まれていpentavalent

る。この度，サウジアラビアで皮膚リーシュマニす）を，metronidazoleは，Flagy1⑪（塩野義製

ア症に罹患し，5価のアンチモン剤を，10日間に薬株式会社，基礎実験用原末，以下M剤と略記す）

わたり合計5，700㎎筋注後，約3カ月経過してを使用した。試験管（内径1cmX高さ10cm）内にも，皮疹から原虫が検出された患者を治療する機

1．3謡のNNN培地を斜面に固め，0．1m尼の会を得た。本例以外にも，アンチモン剤の皮膚リー

promastigote型原虫を含む生理的食塩水（以下シュマニア症に対する有効性に，疑問が持たれる

原虫生食と略記す）を入れ，さらにP剤またはM

症例（Convit

剤を溶解した生理的食塩水（以下薬剤生食と略記

andKerdel−Vegas，1965；Rahim

antimonyを含む。以下P剤と略記

andTatar，1966；土田ら，1989）がある。そこで，

す）を0．9謡加え，2rCで静置培養した。各群は，

現在日本で使用されている代表的な抗原虫薬であ2本の試験管を使用した。薬剤の投入は，原虫生るmetronidazoleで，皮膚リーシュマニア症が食と薬剤生食を同日に加えた初日投薬法と，原虫治癒したという報告（Peter，1973；James

生食のみで3日間培養し，4日目に生虫を確認しandStephen，1975）があるので，アンチモン剤とmetronidazoleのpromastigote型原虫に対すた後，薬剤生食を投入した4日後投薬法の2方法で行った。薬剤生食を投入後，少なくも14日間，

東京女子医科大学寄生虫学教室（〒162東京都新宿区河田町8−1）

372

原虫の運動性と増殖状態を観察した。そして，14の基準で記録した。

日目（または15日目）に，鞭毛または虫体部に自十十十

発的な動きが認められる原虫（以下運動性原虫と1視野の運動性原虫の最大数が10未満

1視野の運動性原虫の最大数が10以上略記す）が観察されなくなった場合には，培地の100未満

液体部を0．1m￡採り，0．9謡の薬剤を含まない生

1視野の運動性原虫の最大数が100以上十十十食を入れた新培地に継代し，さらに少なくも14日

5視野中，運動性原虫が検出されない間（通算28日間）培養（以下再培養と略記す）観なお，観察14日目までに，運動性原虫が認めら察した。なお，P剤投薬群をP群，M剤投薬群をれなくなり，再培養しても運動性原虫が観察されM群，初日投薬法を用いた実験群を初日投薬群，なかった時には，死滅と推定した（矢後，1990）。

4日後投薬法を用いた実験群を4日後投薬群と略
記した。

成

績

1）原虫の運動性を阻止するアンチモン剤と3）原虫の運動性と増殖状態の記録：
試験管内の原虫生食を充分撹拝後，スライドにMetronidazoleの薬量の比較：

1滴載せ，400倍率の顕微鏡下で5視野観察し，得a）初日投薬群における阻止量られた1視野当たりの運動性原虫の最大数を，次原虫の運動性を阻止するP剤とM剤の薬量を比Table1The

effects

andmultiplication

mg†

0

Drug

none

pentavalentofantimony andpromastigotes

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373

較するために，各試験管に原虫生食0．1配と，P

原虫の自発的な運動性は阻止できないが，運動性剤またはM剤を1㎎，3㎎，5㎎，7㎎および9を投薬14日目までに阻止するには3㎎以上，さら㎎をそれぞれ0．9謡の生食に溶解した薬剤生食をに2日目以後14日目まで阻止するには，5㎎以上

同日に加え，原虫の運動性と増殖状態を14目間観必要であった。しかし，P剤とM剤による差はな

察し，表1を得た。

かった。

b）4日後投薬群における阻止量1㎎の薬剤を投入した群（以下1㎎群と略記す）

では，表1の試験管番号3および4（以下試番3−

初日投薬群で，原虫の自発的な動きが，観察期4と略記す）のP群と試番5−6のM群で観察され間中，一度も認められなかった5㎎，7㎎そしてるように，原虫は薬剤生食投入後14日目まで生存9㎎群（表1の試番11−16）では，培地や原虫に原し，対照の0㎎群（試番1−2）との差は，認め因がある可能性を否定できない。また，実際にサられなかった。3㎎群（試番7−10）でも，P剤をンドフライに刺咬された場合，掻痒感や疹痛で虫投入した試番7には，運動性原虫は観察期問中検刺に気づいても，治療は原虫が増殖し，特有の皮出されなかったが，M剤を投入した試番9でも，疹が形成されてから，開始される場合が多い。そ運動性原虫は3日目に観察されたのみで，また試

こで，より実状に添うモデル実験として，原虫生番10でも観察9日目以後には運動性原虫は検出さ

食0．1配のみを3日間培養し，4日目に原虫が生れず，P群との著しい差はなかった。5㎎，7㎎

存増殖しているのを確認してから薬剤生食を投入

そして9㎎群でも，P群（試番11，13および15），

し，その後は初日投薬群と同様に観察を行い，表M群（試番12，14および16）ともに，運動性原虫2を得た。

P剤およびM剤を1㎎投入した試番3−6では，は，観察2日目以後認められなかった。

すなわち，P剤およびM剤ともに，1㎎群では，Table2The

effects

andmultiplication

mg†

0

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374

14日目（培養18日目）まで生存した。5㎎，7㎎（試番5−6），1．25量（試番7−8）および2．50量

そして9㎎群（試番11−22）のP剤中では，投薬1（試番9−10）でも，P剤同様，原虫は14日間生存

日目より3日目以後には，運動性原虫は観察されした。しかし，M剤の3．75量の1．875㎎群の試番なかったが，M剤でも，5㎎群の試番13で投薬9日目以後，試番14では3日目以後，7㎎群の試番

11では，3日目より14日目まで，さらに再培養し

17では5日目以後，試番18では7日目以後，9㎎
群の試番21では5日目以後，試番22では7日目以

4日後投薬群（表3−b）でも，初日投薬群と同様にP剤のLOO量（試番15−16），M剤のLOO量（試後，それぞれ14日目まで，運動性原虫は観察され番17−18），L25量（試番19−20），2．50量（試番21−なくなった。

22）では，原虫は14日間生存した。しかし，M剤

すなわち，1㎎群では，原虫はP剤またはM剤

た28日目まで，運動性原虫は検出されなかった。

の3．75量の1．875㎎群の試番23では，11日目｛培中で，14日目まで生存したが，5㎎，7㎎および養開始（以下培養と略記す）15日目／以後，試番9㎎群では，両剤中で投薬14日目までには，運動24では10日目（培養14日目）以後より，再培養し性が認められなくなった。しかし，運動性を停止た21日目（培養25日目）までは，運動性原虫は，させるのには，P剤よりM剤の方が日数を要した。

検出されなくなった。

2）治療に使用される薬量の比によるアンチモン

は，P剤の1．00量およびM剤の1．00量，1．25量おすなわち，P剤の0．5㎎を1．00量とした場合に剤とMetronidazoleの原虫の運動性への阻止

よび2．50量では，初日投薬法でも4日後投薬法で効果の比較：

も，原虫の生存は阻止できなかったが，3．75量のP剤とM剤とでは，治療に使用する薬量が異な

1．875㎎では，両法で運動性原虫の出現を一時的り，一般には1日に成人で，P剤は600㎎を1回筋注し，M剤は750㎎を，分3で経口投与する。
しかし，M剤はアメーバ性赤痢や，ランブル鞭毛に阻止し得た。

b）P剤1㎎を1．00量とした場合
P剤のLOO量を前実験（表3）の倍量の1㎎と虫症でも，時には1日に通常の約3倍量の2，000

し，M剤をP剤と同量の1㎎，1．25量のL25㎎より2，400㎎（Martinez−Palomo，1986；㎎，2．5量の2．5㎎そして3．75量の3．75㎎使用しWHO，1990）を投薬することがある。そこで，Pて，同様の実験を行い表4を得た。

剤とM剤を，同量で比較するのは実際的ではない

初日投薬群（表4−a）では，P剤（1．00量）のので，P剤の600㎎を基準にし，1量と表記すると，M剤の通常の使用量である1日750㎎（1錠

試番3で，原虫は再培養した28日目まで生存した。

250㎎を3錠）はL25量，1，500㎎（6錠）は2．50

後，2．50量の試番9では2日目以後，試番10では量，そして2，250㎎（9錠）は3．75量になる。伽

4日目以後，さらに3．75量の試番11および12では∂伽oのモデル実験でも，P剤の1量を0．5㎎，1

観察1日目より，それぞれ再培養した28日目まで，㎎，1．5㎎そして2㎎とした時，それぞれのP剤

運動性原虫は検出されなかった。

しかし，M剤の1．25量の試番8では，5日目以の薬量に相当するM剤の1量，1．25量，2．50量そ

4日後投薬群（表4−b）でも，P剤（1．00量）

して3．75量使用し，初日投薬法と4日後投薬法の試番15−16では，14日目まで原虫は生存したが，で，原虫の運動性と増殖状態を観察した。

M剤の1．25量（試番19−20）と，2．50量（試番21−22）a）P剤0．5㎎を1．00量とした場合
P剤の0．5㎎をLOO量とし，M剤を同量の0．5では，14日目までには運動性が消失した。なかでも，試番22では，再培養した21日目（培養25日目）㎎，L25量の0．625㎎，2．50量の1．25㎎そしてまで，運動性原虫が観察されなかった。そして，3．75量のL875㎎溶解した薬剤生食を，原虫生食

3．75量の試番24では，再培養した28日目（培養32に加えて培養し，表3を得た。

日目）まで，運動性原虫が観察されなかった。

初日投薬群（表3−a）のP剤の0．5㎎（試番3−4）では，原虫は14日間生存した。M剤の1．00量

すなわち，P剤のLOO量を1㎎とした場合，初日投薬法でも4日後投薬法でも，必ずしも原虫の375
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none

1（5）

十十
十
十十
十十

1．875

3．75

十十
十十十十・十十十

十
十十 十十
十十 十
十十
十十 十十 十十
十十 十
十
十十 十
十
十
十
十

9
●

●

十
十
十

●

十

8

5

4

●

1．25

●

mg
Day

十
十十
十十

0．625

●

Drug

No．

O．5mg

2︻﹂

4th−day

at

50

・

3

1⊥0乙

28

1．00

2

●

M＊

P＊
0．5

十十十十十十・antimony

unit

十十十・十・十十十十・十十十

（b）

21，

pentavalent

0

10
11
12
13
14

when

︻﹂0

1
2
3
4
5
6
7
8
9

＊十 十 十 十十十十 十十十・十十十・十・十十十十・十十十

Day O

00

1

No．＊

multiplication of promastigotes of1，1．25，

cultures＊

none

mg＊
Ratio

and

metronidazole

0ブ⊥

Dmg

of

十

十

十十

十

十

●

●

●

●

＊Refer to the explanations in Table1．
†Refer to the explanations in Table2．

●

376
Table4The

effectsonthemovement

2．50and3．75units

of

and

multiplication

metronidazole

whenof promastigotes of1，1．25，

pentavalent

antimony

at

l

mg

regarded as l unit（a）

Initial

day

︻﹂5
7ワ5

●

十十十・十十

55 十十十・十十

ワ

十十
十

十

十十

7

22

23

十

●

13（17）

14q8）

十21（25）

・

28（32）

・

十

十

十

十十

●

●

●

●

to the explanations in Table1．
†Refer to the explanation in Table2．

十
●

24

十

十十十一十

十

QJ3 十十21

十十十 十
十十十・十十・

十

十

十

0

20

12（16）

十

5︻り
20乙

19

十十十・十十・十十十十一・十十十十十・十十・十十一十十

十十十十・十十・十十十十十十十55 十■＋＋．＋＋．十＋＋十＋．

M

26乙

18

1⊥1⊥

17

000 十十十・十十・十十十十十

16

1⊥−⊥

15

000 十十十・十十・十十十十十

P

1⊥1⊥

十

10q4）
11q5）

14

十

9α3）

00 十十十・十十・十十十十十

7qD
8q2）

13

十十
十
十十十・十十・十十十十十

6（10）

十十

none

Ratio

5（9〉

・

●

cultures†

mg

O†（4）

﹁

4th−day

十

一

・

十

一 一 一

28

十
十
十
十

■

●

・

1（5）
2（6）
3（7）
4（8）

十
一

十十十
・十十十十

21

十十
十
十十
十十

十十十
・十十十十

十十
十十
十十
十十

Drug

Day

十十 十
十
十十 十十 十十十十 十十 十十十十 十十 十十十 十十 十十

十

十

12

●

10
11
12
13
14

No

十
十

11
一

十
十
十
十

十十
十十
十十
十

十
十

10

QJ3

一

十十
十十
十十
十十

一
十

9

十十・

十十十 十

十
十

十

0

十
十

5︻0

十
十
十十 十

22

1
2
3
4
5
6
7
8
9

︻﹂5
0乙2

・

1⊥1⊥

・

十十十
・十十十十

（b）

O

・＊

M＊

000

No．＊

﹁⊥1⊥

Ratio

1⊥1⊥

00

mg
Day

P＊

none

000

Drug

cultures＊

十十

十十

十十 一

一

十十

十．

十H一 十モ十十377

運動性は阻止できないが，M剤の2．50量またはしたそれぞれ28日目まで，運動性原虫は観察され3．75量では，初日投薬法では，投薬後少なくも4なかった。さらに，2．50量（試番9−10），3．75量

日目から再培養した28日目まで，4日後投薬法で（試番11−12）では，1日目または2日目以後よは，投薬後14日目までには，原虫の運動性を停止

り，再培養した28日目まで，運動性原虫は観察ささせ得た。

れなかった。

c）P剤1．5㎎を1．00量とした場合

4日後投薬群（表6−b）でも，P剤の1．00量（試P剤の1．00量を，表4の実験よりさらに0．5㎎

番15−16）では，投薬1日目より再培養した28日目

増量し，L5㎎とし，M剤をP剤と同量のL5㎎，

まで，運動性原虫は検出されなかった。しかし，1．25量の1．875㎎，2．50量の3．75㎎・3．75量の，M剤でも1．00量（試番17−18）より3．75量（試番23−

5．625㎎使用し，同様な実験を行い，表5を得24）まで，少なくも投薬11日目以後より，再培養

た。

した28日目まで，運動性原虫は観察されなかった。

初日投薬群（表5−a）では，P剤の1．00量を投すなわち，1．00量を2㎎とした場合，P剤の薬した試番3で，培養6日目から15日目まで，ま1．00量中では，両投薬法で1日目以後，運動性原た試番4では5日目から8日目，および再培養で，運動性原虫が観察されたが，M剤の2．50量（試番

虫は観察されないが，M剤でも初日投薬群の2．50
量および3．75量，4日後投薬群の1．06量，1．25

9−10）および3．75量（試番11−12）では，試番9量，2．50量および3．75量中では，再培養しても運の観察2日目に，運動性原虫が観察されたのみで，動性原虫は観察されなかった。

その後再培養しても，運動性原虫は観察されなかった。

4日後投薬群（表5−b）でも，P剤の1．00量（試番15−16）では，14日目まで運動性原虫が観察され

考察

皮膚リーシュマニア症の治療には，一般にたが，M剤では，L25量（試番19−20）で13日目とpentavalent

14日目には，運動性原虫が観察されなくなり，さmetronidazole（M剤）により治癒したと考えら

らに2．50量（試番21−22）および3．75量（試番23−

れる症例報告がある。Peter（1973）は，パナマで24）では，投薬3日目（試番24）より10日目試番

感染した24歳の白人男性に，M剤を1日750㎎分

21）以後には，再培養しても，運動性原虫は検出3で，10日間投薬後，10日間の休薬期間を置いてされなかった。

1クールとし，2クールで治癒した例を，James

すなわち，初日投薬群でも，4日後投薬群でも，

and

antimony（P剤）が使用されるが，Stephen（1975）は，アフガニスタンで感染P剤の1．00量（1．5㎎）では，原虫の運動性を阻した24歳の白人男性に，同方法で3クール投与し，止できないが，M剤の2．50量（3．75㎎）および完治した例を報告している。また，サウジアラビ

3．75量（5．625㎎）では，原虫の運動性は，14日

アで感染し，局所凍結療法を2回施行し，さらに目までには消失し，再培養しても，運動性原虫はP剤を合計5，700㎎，10日間にわたって筋注した

観察されなかった。

が，治癒しなかった51歳の日本人男性に，M剤をd）P剤2㎎を1．00量とした場合同方法で3クール投与し治癒した自験例（矢後，P剤の1．00量を，表5の実験より，さらに0．5㎎

1991）などがある。ところが，M剤を無効または増量し2㎎とし，同様な実験を行い，表6を得

必ずしも有効とは認められないという報告もある。

た。

例えば，Gri伍thsandSodeify（1976）は，南イ

初日投薬群（表6−a）では，P剤のLOO量（2ランの24例に，投薬量や投薬方法の詳細は不明で㎎）の試番3−4では，観察1日目より，再培養しあるが，2重盲検法でM剤による治療を行い，何た28日目まで，運動性原虫は認められなかった。

らかの効果が認められたのは6例のみで，10例はしかし，M剤でも，LOO量の試番6では7日目以

無効，6例は来院しなかったので結果は不明，2

後，1．25量の試番7では5日目以後より，再培養

例は12カ月より21カ月の間に完治したが，自然治

378
Table5The

effects

onthemovement

1．50，2．50and3．75unit
mg
（a）

is

regarded

Initia1−day

ワ 民U

8

9

︻﹂0

32

0

7

1．25

十

十・十十十十十・十・十十・十十十・十十十十十・十・十十・十

10

5．625

3．75
12

11

●

十
●

●

●

●

●

●

●

●

●

●

●

●

●

十
一

十

十十
一

一
十
十

●

●

十

十十

●

●

十十
十

十
十

十

十

●

●

in

Table1．

十十十十→一 十十

十十

十

十十十

十

十十 十

●

●

十

十

●

●

■

●

●

●

十十
十十

explanations

●

十十

十十十

the

・

3．75
23『 24

22

十十

十

5

十

十

一﹂UO

十十

5．625

●

o

一十・

十十・

十十・

・十十

●

28（32）

21

十十十十

十十・十・十十十十十十十十十十十十＋20

十十十十十十・十十・十十一十十十十

19

ワ

1．25

18

十

QUn乙

1．875

0

●

M

︻00

十

●

十十・十・十十一十十十

十

粁＋

●

十十十一十十

十

⊥

●

⊥

0

十十

●

21（25）

to

6

17

15qg）

＊Refer

︻﹂0

1⊥1⊥

︻﹂0

●

●＋七

14（1④

1．875

5

16

十十 十十 十十十十 十十 十十

・十十

11㈲
12q6）
13q7）

15

十十十十十十9α3）
10α心

十十・十・十十十十十十

7αD
8⑰

14

十

6（10）

1⊥1⊥

13

（4）†

M＊

P

00

Ratio

1（5）
2（6）
3（7）
4（8）
5（9）

l．5

cultures†

none

No．

pentavalent antimony of●

十十
一●﹃料＋＋＋●一・一一・︑一4th−day

when

十
十

mg
O

4

…
・

Drug

Day

十

3

・・一・一一十十十・十・十十・十

22
29

十

10
11
12
13
14
15

（b）

P＊

2

＊ ＋
＋＋＋＋
・・十・十十十十十・十・十十・十

O
1
2
3
4
5
6
7
8
9

metronidazoleof1．00，

unit

1⊥1⊥

00 ・・十・十十十十十・十・十十・十

1

No．＊

of

of promastigotes

cultures串

none

mg＊
Ratio

Day

l

50
nU

Drug

as

and multiplication

●

●

十十 十十
十
十十
†Refertotheexplanation

inTable2．

379
Table6 The

effects on the movement

125，2．50and3．75units
mg

isregardedasandmultiplicationof metronidazole

of

when

promastigotes

pentavalent

of1、00，antimonyof2l unit（a）Initiai−daycultures＊

5

3

︻﹂ワ

ワ

・十

12

■

●

十十

十

十十

十十

十十

十

十十

十
●

十十 十十
十十 十十
十十 十十

●

十
十十

十
十十

4th−day cultures†

5

0

﹃0弓

7QJ

0に﹂

十十十

24

十十

十十

十十

十十

十

十十

十十

十

十

十十 十

●

●

●

●

●

十

十

十十

十

十

十

十

十

●

十十

十十
十

●

十十
十
●

●

●

●

十十
十

十十

●

＊Refer to the explanations in Table1．
†Refer to the explanation in Table2．

十十

十十

十・

十十十十

十十

●

23

十十

十﹁十

斗⊥ΩU

励の3
2
22

14⑲

十

13（1の

52

12（16）

十

11G5）

22

十

十十

．●

21

一

十十十十十

10（1勢

20

19

一

十
■

5

gq3）

●

十十

18

︻﹂0乙

8q2）

十十︑十

6⑩
7qD

十

5（9）

十十

17

り乙 ⊥

4（8）

十

16

十十十十十

3（7）

15

M

十十十十

2（6）

十十・十・十十十十十・十・十十

1（5）

十

O（4）†

14

nO
UO

13

2ブ⊥

Ratio

2︷⊥

00

mg

No．

P

none

000

Drug

Day

11

●

●

十十

10

十十

（b）

9

・十

十十

0

十十

0︻﹂

●

︻﹂0乙

十

●

8

・十

十

7

十十十

十

5

十

十

5りム

・

12
8
2

10
11
12
13
14

・＊

十

6

十十

1
2
3
4
5
6
7
8
9

5

4

十十十・十

O

3

十十

Day

2

M＊
21⊥

1

2i⊥

00

mg＊
Ratio
No．＊

P＊

000

none

000
21

Drug

十

380

癒の可能性もあると報告している。Walton召地！．

がもつSH基の方が，ヒトのSH基よりP剤の作（1974）は，パナマ地峡で感染した14歳から52歳の用を受けやすい，（3〉ヒトより原虫の代謝経路の方6例に，M剤の投与を試みたが，6例中5例は無効であったと報告し，有効であった1例には，1部が阻害された場合には，生体全体に及ぼす影響

日1，500㎎を分3で，10日間投薬後，10日間休薬

は，原虫の方に強い，（4）ヒトの方が原虫より代謝し，再び20日間投薬した。このように，M剤が無速度が速いので，P剤を速やかに他の無害な物質

効または必ずしも有効とは認められないという報に変え得るなどである。また，きわめて広い抗原告がある。症例数も少なく，投与法も一定してい虫作用や，抗菌性のスペクトルを持つM剤の作用ない上に，本症には自然治癒があり得るので，臨機序の詳細も，明らかではない。M剤は，生理的床例からM剤の有効性の有無に結論を出すのは難なpHではイオン化せず，容易に嫌気性菌や細胞しい。そこで，原虫（KN株，promastigote型）．

内に取り込まれ，最終的にはDNAの分裂や合成に対する効果を，P剤とM剤を用いて，勉び伽oを阻害し，殺菌作用や細胞障害を生ずると考えらで，運動性と増殖状態を示標に実験した。まず，れている（AmericanSocietyofHospital

P剤とM剤を同量使用した初日投薬群（表1）でmacists，1990）。P剤が，M剤より早期に原虫の運が単純なので，SH基が障害を受け，代謝経路の一Phar−は，薬量による両剤の効果に著しい差は認められ動性を停止するのは，P剤が原虫のSH基を阻害ないが，4日後投薬群（表2）では，M剤の方がすると考えれば，核酸の合成を阻害すると考えら

P剤より，原虫の運動性を停止するのに日数を要れるM剤より，その効果は速やかに発現すると推した。例えば，5㎎のP群（表2の試番11−12）で定される。また，初日投薬法と4日後投薬法によは，薬剤投入後1日目より，原虫の運動性が観察る差を，同量のM剤で比較すると，例えば，表1

されなくなったが，M群の試番13では，運動性原の初日投薬群（試番9，10，12，14および16）と，虫が消失したのは9日目以後であった。7㎎でも，

表2の4日後投薬群（試番9，10，13，14，17，

P群の試番15−16に比較すると，M群の試番17−18

18，21および22）では，4日後投薬群の方が，原では，より長い日数原虫の動きが観察され，9㎎

虫の運動性を阻止するのに，より日数を要していでも，同様であった。他にも例えば，表6−bのPる。この現象も，4日後投薬群では，原虫の分裂群の2㎎の試番15−16では，投薬翌日より28日目ま増殖，すなわち核酸の合成が，初日投薬群よりあで運動性は停止したが，M群の試番17−18では，投る程度進行してから，M剤が投入されるので，薬薬9日目または11日目以後，運動性が停止した。

剤の作用を受ける時期が遅延する原虫も生じ，まこのように，M剤の方がP剤より，原虫の運動性

た，原虫数も増加しているので，1原虫に対するを停止するのに日数を要している。一般に，P剤

薬量も少なくなり，運動性を停止するまでに，日の生体に対する薬理作用は，SH基と容易に反応数を要すると推定している。同様の現象は，表4，することによると考えられている（American

表5および表6の，それぞれのa表の試番5−12

Societyと，b表の試番17−24にも観察される。

of

Hospital

Pharmacists，1990）。しかし，リーシュマニア症に対する作用機序は，不明

薬物の効果を判定するのには，動物の半数を殺である（グッドマン，ギルマン，1988）。酵素など

す50％致死量（LD5。値）や，目的とする薬物効果のSH基を阻害すると推定されるが確証はなく，を動物の半数に発現させる50％有効量（median

また直接的な殺原虫作用も，組織との接触によりeffectivedose：ED5。値）などが用いられる。成績活性化されることもないと考えられている。宿主の項で得られた結果からは，分裂増殖中の原虫を，であるヒトにも，SH基を持つ酵素などは存在す

100％生存させる薬量の算出は困難なので，LD5。

るが，P剤がヒトより原虫に強い障害を与える理値に代わる値を次のように定義し，L値と名づけ由として，グッドマン，ギルマン（1975）は，次た。すなわち，投薬14日目（または15日目）またのような可能性を考察している。（1）P剤がヒトのは再培養で，試みた2試験管中に，ともに運動性細胞より原虫に，より速やかに浸透する，（2〉原虫

原虫が認められる薬量の最高値を生存最高薬量，

381

また再培養した28日目までに，2試験管中にともみ分布した場合で，最低濃度は1謡の生食と1．3に運動性原虫が観察されない薬量の最低値を死滅

醜のNNN培地の合計2．3m尼に均等に分布した

最低薬量とし，（生存最高薬量＋死滅最低薬量）÷

場合である。原虫を死滅させ得るM剤の最低濃度2を求め，L値とした。まず，P剤の初日投薬群を，L表より求めると，4日後投薬群で，P剤とのL値を算出すると，生存最高薬量は表5−aの試同じ2㎎である。そこで，2㎎が培地の合計2．3m尼

番3−4の1．5㎎で，死滅最低薬量は，表6−aのに均等に分布した場合，すなわち2㎎／2．3謡≒

試番3−4の2㎎である。そこで，L値は（L5＋0．87㎎／m尼が，原虫を死滅させ得るM剤の最低濃2）÷2＝1．75㎎になる（L表参照）。同様に，P度になる。同値は，他の原虫，例えばM剤が初剤の4日後投薬群の生存最高薬量は，表5−bの試η伽oで，0伽4毎彪励伽のほとんどの種に対し

番15−16のL5㎎で，死滅最低薬量は，表6−bのて，発育を阻止する濃度の0．0008−0．032㎎／m￡

試番15−16の2㎎，したがって，L値は初日投薬や，Eη如吻08加群と同じL75㎎となり，投薬法による差はなかっ

昭gJ翅傭の0．003㎎／m尼以下（American

た。同様に，M剤の初日投薬群の生存最高薬量は，

表5−aの試番7−8の1．875㎎で，死滅最低薬量

etyofHospitalPharmacists，1990）に比較すると，高濃度である。青河ら（1971）によれば，健L表

康婦人5例に，M剤250㎎を，1回経口投与した

薬
剤

投 薬

法

生 存 死 滅L
最高薬量最低薬量

配sずo砂∫廊αやT7露ho吻onαs

Soci−時の血中濃度は，2時間値が最大で，比色法で0．02

値㎎／謡である。実験で得られた，最も低濃度である

L5
P 初日投薬群
4日後投薬群
1．5

2．0

1．75

0．87㎎／溜は，青河らのヒトで得られた血中濃度

2．0

1．75

の約44倍になり，初∂伽oでα伽伽彪励枷の

初日投薬群

1．88

2．5

2．19

発育を阻止する有効濃度の約27倍である。Mat・

2．5

2．0

2．25

tockandPeters（1975）は，勉∂伽oで，cellline

M 4日後投薬群中のL．吻ε蛎08％のamastigote型原虫が，アンは，表4−aの試番9−10の2．5㎎，したがって，チモン剤のsodium

L値は（L875＋2．5）÷2≒2．19㎎になる。同値を生ずるには，5−10㎎／m尼という高濃度が必要でstibogluconateにより変性

は，P剤の初日投薬群のL値のL75㎎より，約あると報告し，実験で使用した種と実際の感染例

0．44㎎高値である。同様に，M剤の4日後投薬群の種の差を考察している。Beveridge（1963）の生存最高薬量は，表4−bの試番21−22の2．5㎎

は，ヒトやハムスターのリーシュマニア症に対しで，死滅最低薬量は表6−bの試番17−18の2㎎て有効な5価のアンチモン剤が，promastigote

で，生存最高薬量の方が，死滅最低薬量より高値

型原虫には効きにくく，伽び伽oでは生体内よりで，前3者とは異なるが，型通り計算すると（2．5＋

高濃度が必要であると報告している。そして，原2）÷2＝2．25血gで，P剤の4日後投薬群のL75㎎より高値である。これらのことから，M剤のL

虫に5価のアンチモン剤が接触する前に，生体内値は，P剤のL値より高値で，すなわち，P剤と察している。このように，初碗zoと初吻o，同じ効果を生ずるには，P剤より多量の投薬が必または実験条件により，有効濃度に差が生ずる原要と推定される。

因には，原虫の種の差やamastigote型とでは，代謝などにより3価になる必要性なども考実験に使用した薬量と，ヒトの治療量を比較しpromastigote型の差，発育や分裂過程の差や，生てみると，1試験管に投入したP剤またはM剤の体内では，薬剤が代謝され他の物質に変化したり，

薬量は，0．5㎎（表3）より9㎎（表1および2）

他の物質との相乗作用などもあり，特にM剤は，である。薬剤が，培地のウサギ血液や寒天などにクロラムフェニコールなどにも認められるように吸着されず，また変性などもなく，均等に溶解分

pHに依存し，低酸や無酸ではバイオアベイラビ

布したと仮定すると，薬剤の最高濃度は，薬剤がリティの低下（Ogata切α」、，1985）が生ずるの

原虫生食0．1謡と薬剤生食0．9m￡の合計1m疋にので，経口投与による胃酸との接触が必要であり，

382

また体内では光による分解などもないなどの，多ルマン，1988）。通常，P剤は成人1日1回60伽g

くの条件の差によるのであろうと推察している。

（または10㎎／㎏）を，6日間から10日間筋注し，

5価のアンチモン剤が，一般的には内蔵型に有10日間休薬する（PetersandKillick−Kendrick，

効と考えられているのに，皮膚型には著しい効果

1987；Reynolds，1989）。ところが，近年，特に内が認められないとする報告（ConvitandKerdel−

蔵リーシュマニア症には，休薬期間は不要で，よVegas，1965；Rahim

Tartar，1966；土田ら，り大量をより長期に，例えば1日850㎎を，少な

1989；矢後，1991）があるのは，アンチモン剤のくも20日間投与する治療法（WHO，1984）があand

体内での分布の差にも，原因があると考えられる。

る。しかし，P剤の副作用には，関節痛，頭痛，

5価のアンチモン剤は，肝臓や脾臓により高濃度

腹痛，顔面浮腫，嘔吐を伴う激しい咳，肝炎，徐に分布するので，皮膚に寄生する，例えばL．

脈，呼吸困難，ショック症状などが知られている

70加oαより，肝臓や脾臓に寄生するLので，皮膚リーシュマニア症のように，感染部位

40ηo槻勉の方に高濃度で作用し，結果的に内蔵が皮膚に限局し，生命を脅かさない疾患の治療に，型に有効になる可能性がある。一方，M剤は全身

重篤な副作用を持つ薬剤を，大量に長期問投薬すに比較的均等に分布し，組織内の濃度は，多くのるのは控えたい。一方，M剤はランブル鞭毛虫症

場合血清と同じである（Ralph，1983）。したがっやトリコモナス症などの治療には，1日750㎎をて，P剤とM剤を同量投与しても，肝臓や脾臓に分3で，5日間より10日問投薬するが，重症の嫌高濃度に分布するP剤の皮膚の濃度より，体内に気性細菌感染症では，1日49を3週問使用する均等に分布し，蓄積作用もあるM剤（Ralph，1983）

ことがある（American Society of Hospitalの皮膚の濃度のほうが高くなり，M剤は皮膚リー

Pharmacists，1990）。このように，M剤は，P剤

シュマニア症に，より有効なのであろうと推察しより大量を長期問経口投与でき，その副作用も末ている。

梢神経障害の他は軽微で，同障害も服薬を中止す

本実験は閉鎖系なので，薬剤の投与は1回のみると治癒し（Bradley召∫α1．，1977），原虫に対すで実験を行ったが，M剤の服薬を繰り返すと，ヒる薬効も，伽痂mの実験結果からは，P剤と著トの血中や組織内の濃度は増強する。Ralph（1983）によれば，M剤500㎎を1回経口投与ましい差がないので，現時点では試みる治療剤の1つと考えられる。

たは静注した時の平均血中濃度は，約0．01㎎／m￠

であるが，500㎎を8時間毎に静注すると，血中結濃度は0．015−0．025㎎／m尼になる。自験例（矢後，1991）では，血中濃度は測定してないが，臨床症

論

皮膚リーシュマニアpromastigote型原虫に状から蓄積作用と考えられる所見が得られた。す対するアンチモン剤（pentavalent

antimony，

なわち，M剤を1日750㎎，分3で10日間投薬後，Pentostam⑬使用，P剤）とmetronidazole

10日間の休薬期間を1クールとして，3クール投（FIagyl⑪使用，M剤）の薬効の差を，」薬すると，1クール目では特別な副作用は認めら

で原虫の運動性と増殖状態を示標に比較した。試れなかったが，2クール目では，服薬開始5日目
より1日1−3回の下痢が始まり，口中に金属様の験管内に斜面につくった1．3配のNNN培地に，酸味感を生じた。3タール目では，下痢の始まり

薬剤を溶解した0．9m尼の生理的食塩水（薬剤生食）

伽o

原虫を含む0．1m尼の生理的食塩水（原虫生食）と，も服薬開始4日目と早くなり，回数も1日2−3回を加え，少なくも14日間観察した。薬剤の投入は，と増加し，服薬終了後も継続し，口中の金属様の薬剤生食と原虫生食を同日に加えた初日投薬法と，酸味感も，服薬13日目に少し軽減したにすぎな

原虫生食のみで3日間培養し，4日目に薬剤生食かった。一方，P剤には蓄積作用はなく，腎からを加えた4日後投薬法の2方法で行い，次の結論の排泄は速やかで，筋注後約6時問以内に，投与を得た。

した80％以上が尿中に出現する（グッドマン，ギ（1）原虫生食に，P剤またはM剤を，それぞれ

383

1㎎，3㎎，5㎎，7㎎および9㎎溶解した薬剤

4日後投薬法の2．50量および3．75量中では，P剤生食を加えると，1㎎中では，原虫は両剤の両投では原虫は生存できても，M剤中では14日後まで

薬法で，14日間生存した。5㎎，7㎎および9㎎には運動性は消失し，再培養しても運動性原虫は，中では，両剤の両投薬法で，14日後までには，自観察されなかった。

発的な運動性を示す原虫（運動性原虫）は，観察
されなくなった。

し謝（2〉P剤の0．5㎎，1㎎，L5㎎および2㎎を辞

量，2．50量および3．75量になるように原虫生食に御指導御講読賜りました，東京女子医科大学寄生虫学教室白坂龍暖教授，Pentostam⑬の入手に加えると，P剤の1㎎を1．00量とした時の，M剤御便宜を計って下さいました西山勝治氏に深謝致それぞれ1．00量とし，M剤をP剤の1．00量，1．25の初日投薬法の2．50量，3．75量，およびP剤の1．5します。尚，本稿の内容は，第59回日本寄生虫学㎎を1．00量とした時の，M剤の初日投薬法および会大会（1990年4月）において発表した。

文献1）青河寛次，山路邦彦，松下光延（1971）：Metronodazoleによる丁万oho解o紹s槻g初σ傭感染の化学療法

補遺，産婦の世界，23（2），183−186

2）American Societyof

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8）グットマン，ギルマン（1988）：薬理書（下）薬物治療の基礎と臨床，第7版，1316−1318，廣川書店，東京
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therapy forcutaneousleishmaniasis，Arch

Dermatol．，111，1343−134410）Leventhal，R．and

Cheadle，R．E（1989）：Medical Parasitolosy，3thed．，p．140，F．A．Davis

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Philadelphia11）Martinez−Palomo，A．（1986）：Humanparasitic

diseases2；Amebiasis，pp．189−212，Elsevier，

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Ann．Trop．Med．Parasitol．，69（3），359−371

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14）Ogata，H．，Aoyagi，N．，Kaniwa，N．，Ejima，E．，Takagishi，Y．，Ogura，T．，Tomita，K．，Inoue，S．

andZaizen，M．（1985）：Bioavailability ofmetronidazole from sugarcoated tablets inhumans．

1Effectofgastric acidity andcorrelation

with ％擁如dissolution rate，Int．Pharm．，23，277−288

15）Pau1，C．B．，Rodney，C．J．and Eddie，W．C．（1984）：Clinicalparasitology，62−63，Lea＆Febiger，

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Philadelphia16）Peter，1．L．（1973）：Cutaneous1378−1379
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Clinicalleishmaniasis treatedwith metronidazole，JAMA，223（12），Killick−Kendrick，R．（1987）ゴThe Leishmaniases in Biology and Medicine．II

AspectandContro1，pp．855−893，Academic18）Rahim，G．F．and Tatar，L．H．（1966）：Oriental

19）Ralph，E，D．（1983）：Clinical

Press，London

sore in Iraq，BulL End．Dis．，8，29−54

pharmacokinetics

20）Reynolds，」．E．F．（1989）：Martindale，Theof metronidazole，Clin．Pharmacokinet，8，43−62

extra

pharmacopoeia，29th

Pharmaceutical Press，London
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ed，pp．677−679，The常，松本克彦，石橋康正（1989）：皮膚リーシュマニア症一サウジアラビアでの感染例一，臨床皮膚科，一43（13），1275−128022）Walton，C．B．C．，Paulson，J．E．，Arjona，M．A．，Peterson，C．A．（1974）：American cutaneous leish−

maniasis，Inemciencyofmetronidazole23）WHO（1984）：，Tech．Rep．Ser．701，The
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385

TREATMENT FOR CUTANEOUS LEISHMANIASIS‑A COMPARISON BETWEEN THE EFFECTS OF METRONIDAZOLEAND PENTAVALENT ANTIMONY ON THE MOVEMENT

AND MULTIPLICATION ･OF, LEISHMANIA PROMASTIGOTES‑AYAKO YAGO
Received August 7 1991/Accepted October I 1991

The effects of metronidazole and of pentavalent antimony (P) on the movement andmultiplication of Leishmania promastigotes were compared. Leishmania was isolated fromthe skin of a Japanese man who had spent time in Saudi Arabia. The promastigotes were
cultured at 25'C in test tubes that contained 1.3 ml of NNN medium and 1.0 ml of 0.85w/v%

NaCl. Metronidazole or P was added to cultures by one of two protocols. Either promas‑
tigotes were cultured with the drug of choice from the initial day nd these cultures werecalled initial‑day cultures. Alternatively, promastigotes were cultured for 3 days initiallywithout drugs and then a drug was included from the 4th day. These cultures were called 4th‑

day cultures. The movement and multiplication of promastigotes were observed for at least
14 days after initial exposure to either drug.

(1) Promastigotes were alive in the tubes that contained I mg of metronidazole or P until
the 14th day of exposure to either drug in both initial‑day cultures and 4th‑day cultures, but

their movements were not observed in the tubes that contained 5 mg, 7 mg or 9 mg of
metronidazole or P after a 14‑day exposure to either drug in both cultures.
(2) In a clinical setting, P is injected as 600 mg (defind here as I arbitrary unit) per adult

per day and metronidazole is given at a dose of 750 mg (1.25 units) , 1,500 mg (2.50 units) or

22,500 mg (3.75 units) . P at 0.5 mg, I mg, 1.5 mg and 2 mg was regarded in separate tests asequivalent to I unit and metronidazole at 1.00, 1.25, 2.50 and 3.75 times each dose of P wasadded to promastigotes. As shown in tube No. 3 in Table 4 and tube No. 3 and No. 4 in Table
5, the parasites survived in the presence of I mg or 1.5 mg (1 unit) of P, but their movements
were not observed in the presence of 2.5 mg or 3.75 mg (2.5 units) and 3.75 or 5.625 mg (3.
75 units) of metronidazole in initial‑day cultures. In 4th‑day cultures, as shown in Table 5,

the parasites also survived with 1.5 mg (1 unit) of P, but their movements were not observedwith 3.75 mg (2.5 units) and 5.625 mg (3.75 units) of metronidazole. In these conditions, theeffects of metronidazole were superior to the effects of pentavalent antimony.

Department of Parasitology, Tokyo Women's Medical College, 8‑1 Kawada‑cho, Shinjuku‑ku,
Tokyo 162, Japan

ISSN 030 2 t 46

JAPANESE JOURNAL OF
TROPICAL MEDIClNE AND HYGIENE
Vol. 19 No. 4

December 1991

CONTENTS
Original article

Shameem, G.M.M.
Immunogenicity of Slowly Sedimenting Antigen of Japanese Encephalitis Virus
Envelope Glycoprotein Isolated from Infected Culture Fluids """321‑330

Shimizu, M., Shichinohe, K., Tsukidate, S. and Fujita, K.

Basic Studies on the Mongolian Gerbil as a Susceptible Host to Filarial

Infection; Comparative Studies on Hematological Features between theWild‑colored Gerbil and the Coat Color Mutants

331‑338

Shikiya, K., Uechi, H., Saito, A. and Asato, R.

Clinical Study of Mebendazole Therapy for Strongyloidiasis """"

339‑346

Katsumata, T., Kohno, S., Koga, H., Yoshitomi, Y., Matsuda, H., Mitsutake, K.,

Higashiyama, Y., Miyazaki, Y. ahd Hara, K.
Management of Pneumocystis carinii Pneumonia in Patients with Conventionally

Caused Immune Suppression 'Takaoka, H. and Hadi, U.K.
Two New Blackfly Species of Simulium (Simulium) from Java, Indonesia(Diptera: Simuliidae)

347‑355

357‑370

Yago, A.
Treatment for Cutaneous Leishmaniasis ‑a Comparison between the Effects ofMetronidazole and Pentavalent Antimony on the Movement and Multiplicationof Leishmania Promastigotes‑ (in Japanese) ""Jpn. J. Trop.
Med. H yg.

JAPANESE SOCIETY OF TROPICAL MEDIClNE

371‑385