■■■

王

第21巻第2号平成5年6月

内
原

容

著
地卿孟0200κ砒n露のgamontにおける微細構造の観察（英文）・・村田 智昭，井上
嶋田 雅暁，横山
北部タイにおける急性呼吸器感染症の気炎菌（英文）1mmmohistochemical

Demonstration

NervousofLong−termAnti−PGL

Antibody

Specific

System

誠，鹿江

雅光，堀尾

政博，

満，田浦

保穂，中問

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一永武
毅，高橋
淳，田尾
操，広瀬 英彦，
真崎 宏則，渡辺貴和雄，KamUruddin Ahmed．，
力富 直人，松本 慶藏，Prasit Tharavichitkul，Thira Sirisanthana，Ronatrai Ruengverayudh，Tawom Kasomson，Satid Charuroteskulchai，
Sonboon Jiamtaveeviboon，Chintana Uthaisilp，Witaya Swaddiwudhipong，Charmee Chandom l11−115of〃夕60わ46云6吻御妙鵤6in the

Cured

Leprosy・・Masamichi

Patients

Using

Goto，Yasuhikoa〃1妙昭8MinauchiandEiichiSato

117−121

・一……………・……一…一…・…………・……・・………・…・……・………一盛

和世

123−125

研究ノート

ケニア南部におけるツェツェバエ2集団の特性（英文）
臨床報告
輸入四日熱マラリアの1例（英文）一西山 利正，荒木 恒治，天野
福井
博，辻井
正

博之，石坂

重昭，
127−130

会報・記録
日本熱帯医学会幹事会・国際委員会合同会議記録………………一・……一……一・…・・……一 131−132
熱帯医学と医療を語る会ニュースNQ4……・…一……・……一………・・一……一……・……… 133−134
投稿規定…・・……………・・……・…………・…………一・…・……・・…………・………………………

136

一■

107

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 2, 1993, pp. 107 110

FINE STRUCTURE OF GAMONT OFHEPA TOZOON CA NISTOMOAKI MURATA , MAKOTO INOUE2, MASAMITSU KANOE3MASAHIRO HORI04, MASAAKI SHIMADA4, MITSURU YOKOYAMA5
YASUHO TAURAI AND SANENORI NAKAMAl
Received November 30 1992/Accepted February 8 1993

Abstract: Gamont of Hepatozoon canis was observed by light, phase contrast and transmission electron
microscopies. In a thin blood film the gamont was contained within a pale‑blue capsule in the leukocyte. Byphase contrast microscopy, the gamont was seen to be surrounded by a contrasty substance. Transmission

electron microscopy revealed the presence of a parasitophorous vacuolar membrane, two layers in thepellicle of the gamont and a fine fibril‑like structure surrounding the parasitophorous vacuole.

INTRODUCTION
Hepatozoon canis infection is a protozoan parasitic

disease of dogs found worldwide. The various hostanimals have been infected with Hepatozoon spp. and ahuman case of Hepatozoon has been reported in the
Torrid Zone (Craig, 1990). H. canis infection occurs
mainly in India, South Africa, Nigeria, Israel, thePhilippines, Malaysia, North America and Japan (Bent‑
ley, 1905; McCully et al.. 1975; Ogunkoya et al.. 1981;
Elias and Homans, 1988; Carlos et al.. 1971; Rajamanick‑am et al.. 1984/85; Craig et al.. 1978; Murata et al..
1991) .

Little information is available concerning the finestructure of the gamont of H. canis. We therefore
attempted to observe the gamont by light, phase con‑trast and transmission electron microscopies.

MATERIALS AND METHODS
A dog infected with H. canis in Fukuoka Prefecture

was employed in this study. Thin blood films wereprepared as follows. One milliliter of peripheral blood
was mixed with I or 2 units of heparin sodiurn (Green
Cross Co., Japan) . The film was then fixed with meth‑anol for I min and stained with Giemsa stain for 40 min.

An unstained film was observed with a phase contrast
microscope (Nikon Optiphoto, Tokyo) .

Preparation for the transmission electron micros‑
copy (TEM) was made as follows: the heparinized blood
sample was centrifuged at 2,000 rpm for 10 min and the
buffy coat was separated. The specimen was fixed witha mixture of 2.5% glutaraldehyde and 1% osmic acidand suspended in 4% agarose gelatin. The solidified
specimen was dehydrated in a graded series of ethanol
and embedded in Quetol 812 epoxy resin. The ultrathin

sections were stained with uranyl acetate and leadcitrate and examined in JEOL 1200EX electron micro‑
scope at 80 kV.

RESULTSThe blood films stained with Giemsa stain revealed
that the gamont of H. canis had one nucleus dark bluein color in the leukocyte. A capsule with pale‑blue color
was seen to invest the gamont (Fig. 1, arrow) .

By phase contrast microscopy a thick capsule wasclearly observed to surround the gamont (Fig. 2, arrow) .

TEM of the present examination showed that thegamont contained within a parasitophorous vacuole(PV) possessed one nucleus, apical complex, electron‑
dense and spindle‑shaped tnicronemes, electrondense
microbodies and ovoid granules. A thick electronlucid
material was observed to invest the PV in the cytoplasmof the host leukocyte (Fig. 3a, arrow).
At high maguification, two layers were found in the

1 . Departments of Veterinary Surgery, 2 . Veterinary Pathology, 3 . Veterinary Microbiology, Faculty of Agriculture, Yamagu‑chi University, 1677‑1 Yoshida, Yamaguchi 753
4 . Department of Medical Zoology, 5 . Laboratory of Electron Microscopy, School of Medicine, University of Occupational &Environmental Health Japan, 1‑1 Iseigaoka, Yahatanishi‑ku, Kitakyushu 807, Japanl 08

Figure I One nucleus of H. canis gamont can be seen in
the capsule (arrow) in the leukocyte ( x 1.000,
Giemsa stain) .

Figure 2 Phase contrast micrograph showing the cap.
sule (arrow) investing the gamont ( x 400) .
The capsule forms a striking contrast to thegamont .

pellicle of the gamont and a parasitophorous vacuolar

the gamont of an adeleine haemogregarine was reported

membrane (PVM) was seen over them 50 nm in width

to show a double membrane complex that lacked a(Fig. 3b) . These two layers were found to be a thickouter layer and an inner complex of two closely apposed

coated membrane (oufe'r limiting unit membrane) and aplasmalemma (Baker and Lainson, 1967) . The featuremembranes. Subpellicular microtubules coming fromthe apical complex appeared to be seen beneath theseems to be induced by poor fixation or other procedual
differences, as mentioned by Nadlar and Miller (1985) .
Thus, we consider the thick layer and the double mem‑layers. The PV including the gamont was surrounded by
the fine fibril‑like structure in the cytoplasm of theleukoc yte.

brane complex in the gamont pellicle of H, canis toresemble the outer and the inner layers in that of H.
mocassird (Nadler and Miller, 1985) .

DISCUSSION
The present examination on the gamont of II. canis
seen in the leukocyte showed notable characteristics.
Phase contrast microscopy appeared to indicate that thecontrasty thick material was situated around thegamont. Little information is available on the fineThe gamont of H. canis possessed the nucleus,
apical complex, micronemes, microbodies and ovoid
granules, which appeared to be closely similar to thoseof H. mocassil i, Haemogregarina magna andBabesiosoma stableri (Bashtar et al., 1984; Nadler andMiller, 1985; Paterson et al., 1988; Barta and Desser,
1986; Baker and Lainson, 1967) .

structures of the capsules surrounding the gamonts of H:aeg)pti and H, mocassi,ci (Bashtar et al., 1984; Nadler

ACKNOWLEDGMENTS

and Miller, 1985). According to Nadler and Miller(1985) , the nonstaining capsule seen by light microscopy

The authors gratefully thank Dr. Kazunobu

was considered to correspond to the electron‑lucid
space between the gamont pellicle of H. mocassilci and

Shimoda, D.V.M., Shimoda Animal Clinic for his help tocollect the blood specimens from the dog infected with

the PVM. The ultrastructures of the gamonts of

H. canis.

Haemogregarina magna. Haemogregarina adeleilee andBabesiosoma stableri are well known, but TEM has notshown the existence of a distinct structure around the

REFERENCES

PV (Paterson et al.. 1988; Baker and Lainson, 1967;l ) Baker, J.R. and Lainson, R. (1967) : The fine structure ofBarta and Desser, 1986). Therefore, in this observation,

the gametocytes of an adeleine haemogregarine, J.

the fine fibril‑like structure was found to be situatedProtozool., 14, 233‑238
2 ) Barta, J.R. and Desser, S.S. (1986) : Light and electron

around the PV containing the gamont of H canis;
however, its role in pathogenesis is still unknown.

TEM of the present examination revealed that thepellicle of the gamont of H. canis consisted of the thicklayer and the double membrane complex. The pellicle ofmicroscopic observations on the intraerythrocytic devel‑

opment of Babesiosoma stableri (Apicomplexa,
Dactylosomatidae) in frogs from Algonquin Park,
Ontario, J. Protozool., 33, 359‑368

109

;

f:

i:/ <*i+

Figures 3a and 3b Transmission electron micrographs. (a) : Gamont (G) showing one nucleus(N), micronemes (MN), microbodies, apical complex (AP) and ovoid
granules (O) in the cytoplasm. The thick material (arrow) is seen aroundthe PV containing the gamont ( x 6,000, Bar: I pm) , PV parasitophorous
vacuole (b) : Two layers can be seen in the pellicle of the gamont ( x 100,
OOO, Bar: 50 nm) . The PV is surrounded by a fibril‑like structure. PVM
parasitophorous vacuolar membrane; OL outer layer; IL inner layer; MT
microtubules.

1103 ) Bashtar, A.‑R., Ghaffar, F.A. and Mehlhorn, H. (1984):
Hepatozoon aegD4,ti nov. sp., Z. Parasitenkd., 70, 43‑52

4 ) Bentley, C.A. (1905) : Preliminary note upon a leuko‑
cytozoan of the dog, Br. Med. J., 6, 988
5 ) Carlos, E.T., Atienza, L.M. and Cabiles, C.C. (1971): A

report on the presence of Hepatozoon canis in thePhilippines, Philippine J. Vet. Med., 10, 181‑189

6 ) Craig, T.M., Smallwood, J.E., Knauer. K.W. andMcGrath, J.P. (1978) : Hepatozoon canis infection indogs: Clinical, radiographic, and hematologic findings, J.

A.V.M.A., 173, 967‑972
7 ) Craig, T.M. (1990) : Infectious Diseases of the Dog andCat, 778‑785, W.B. Saunders, Philadelphia8 ) Elias, E. and Homans, P.A. (1988) : Hepatozoon canis
infection in dogs: clinical and haematological findings;
treatment, J. Small Anim. Pract., 29, 55‑62
9 ) McCully, R.M., Basson, P.A., Bigalke, R.D., Vos, V.D.

and Young, E. (1975) : Observations on naturally
acquired hepatozoonosis of wild c lrnivores and dogs in
the Republic of South Africa, Onderstepoort J. Vet. Res.,42, 117‑134

10) Murata, T., Shiramizu, K., Hara, Y., Shimod , K. andNakama, S. (1991): First case of Hepatozoon canis
infection of a dog in Japan, J. Vet. Med. Sci., 53, 1097
‑109911) Nadler, S.A. and Miller, J.H. (1985) : Fine structure of

Hepatozoon mocassini (Apicomplexa, Eucoccidiorida)gamonts and modifications of the infected erythrocyte
plasmalemma, J. Protozool., 32, 275‑279
12) Ogunkoya, A.B., Adeyanju, J.B. and Aliu, Y.O. (1981):
Experiences with the use of imizol in treating canine
blood parasites in Nigeria. J. Small Anim. Pract., 22, 775‑77713) Paterson, W.B., Desser. S.S. and Barta, J.R. (1988) :Ultrastructural features of the apical complex, pellicle,

and membranes investing the gamonts of Haemogrega‑rina m gna (Apicomplexa: Adeleina) , J. Protozool., 35,
73‑8014) Rajamanickam, C., Wiesenhutter, E. and Hamid. J.
(1984/85) : The incidence of canine haematozoa in
Peninsular Malaysia, Vet. Parasit., 17, 151‑157

J pn. J. Tro p. Med.

111

Hyg., Vol. 21 No 2 1993, pp. Ill‑ll5

CAUSATIVE ORGANISMS OF ACUTE RESPIRATORYINFECTIONS IN NORTHERN THAILAND
TSUYOSHI NAGATAKE1, ATSUSHI TAKAHASHI , MISAO TAolHIDEHIKO HIROSE1, HIRONORI MASAKll, KIWAO WATANABE1
KAMURUDDlN AHMEDl, NAOTO RIKITOM11, KEIZO MATSUMOTO1PRASIT THARAVICHITKUL2, THIRA SIRISANTHANA3, RONATRAI RUENGVERAYUDH4,TAWORN KASOMSON4, SATID CHARUROTESKULCHA14, SONBOON JIAMTAVEEVIBOON4,CHlNTANA UTHAISILP4, WITAYA SWADDIWUDHIPONG' AND CHARUNEE CHANuoUN4
Received March 5 1993/Accepted March 30 1993

Abstract: We examined the causative organisms of respiratory infections from 1989 to 1990 at Mae Sot
General Hospital in collaboration with Chiang Mai University, Thailand. We collected sputum from patientwith acute bronchitis and pneumonia to identify the causative organisms by sputum culture andinflammatory sputum cytology. We experienced 72 cases (97 strains of bacteria) of acute broncitis and 17
cases of pneumonia (20 strains of bacteria) in Mae Sot General Hospital. The most frequently identified
pathogens in respiratory infections were Haemophilus influenzae. Streptococcus pneumoniae and Branhamellacatarrhalis. This study shows that Gram‑stained smears and quantitative sputum culture together have asignificant role in enhancing the diagnostic value of expectorated sputum specimens. The causative
organisms between Thailand and Japan were very similar in acute respiratory infections. The mostimportant outcome of this study was the detection of Branhamella catarrhalis in many adult cases with acuterespiratory infection.

INTRODUCTION
It is well recognized that th.e majority of acute
upper respiratory infections are caused by viral infec‑
tions which are sometimes followed by bacterial infec‑
tions. Bacterial infections cause complications of com‑respiratory infections. It is not known which pathogenicorganisms predominate in Thailand. Therefore, thisstudy was carried out to identify the causative organ‑
isms of respiratory infections in a rural district ofThailand. We examined the causative organisms of

mon cold: acute sinusitis, otitis media, acute bronchitis

respiratory infections from 1989 to 1990 at Mae Sot
General Hospital in collaboration with Chiang Mai

and pneumonia. It is sometimes dangerous for young

University, Thailand. This type of studies are helpful tochildren and aged people to catch cold because of vari‑

understand about the incidence of bacteria causing acuterespiratory infection between the two countries, which

ous complications occur due to secondary bacterial
infections. Respiratory infections cause much mortality
and morbidity (Murray, 1982). This is true both for thehas difference in climate, nutritional status, economyetc.

developed and developing countries. From different
hospitals of developed countries data on bacteria caus‑
ing respiratory infections are regularly published. On

the other hand, such data are not regularly obtained
from developing countries. Moreover the foundation ofchemotherapy of respiratory infections is first of all todetermine pathogens (Matsumoto, 1963). Therefore it
is important to study the bacterial pathogens causing
l

2
3
4

MATERIALS AND METHODS
Location: This study was carried out in Mae Sot
General Hospital in collaboration with Chiang Mai
University (Fig. 1). In Mae Sot, from November to
February is winter, from March to May is summer andfrom June to October is rainy season. This investigation

Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Department of Microbiology, Chiang Mai University, Chiang Mai 50002, Thailand
Faculty of Medicine, Chaing Mai University, Chiang Mai 5002, Thailand
Mae Sot General Hospital, Mae Sot, Tak 63110, Thailand

112

Clinical studies: Our standardized criteria of respi‑

MYANMAR

LAOS

Chiang Mai

ratory infection with causative organisms are as fol‑
lows; (1) Gram stain of sputum smear show plenty ofpolymorphoneuclear leukocytes or macrophages anddominant bacteria located intracellularly and extracel‑lularly, (2) much growth of these bacteria in semi‑quanti‑tative culture or more than 107 CFU/ml in quantitative' Mae sot

culture (Matsumoto et al., 1978) , (3) increase in number

THAILANDof these bacteria coincides with clinical and laboratory
findings. Clinical data corresponding to the specific dataof sputum collection were obtained from patient caserecords at Mae Sot General Hospital.

Ba gkok

CAMBODIARESULTSTime period: The first and third study period werein the winter and the second study period was in therainy season.
Patients: There was a total 392 patients and theirmean age was 43 yrs. A total 249 patients were malewith a mean age of 43.4 yrs and 143 patients were female

ALAYSIA
Figure I The location of Chiang Mai and Mae Sot,
Thailand.

was done at three different time interval, i.e. from
December 1989 to January 1990 (winter) , from July towith a mean age of 42.5 yrs. Details of age, sex andnumber of patients are shown in Table 1.
ldentification of bacterial pathogen: From a total
72 cases of acute bronchitis 97 strains of causative
pathogenic bacteria were isolated. The main pathogenic
bacteria were Haemophilus influenzae (H. influenzae)(44.5%) , Streptococcus pneumoniae (S. pneumoniae) (26.
8%) and Branhamella catarrhalis (B, catarrhalis) (14.
3%). Main causative bacteria, number of cases, strain
isolated in different period of time are shown in Figure

August 1990 (rainy) and from November to December

2.

1990 (winter) .

From a total 17 cases of pneumonia, 20 strains ofpathogenic bacteria were isolated. These bacteria were,Collection of sputum samples: Fresh expectorated
sputum, from patients with respiratory infections sub‑mitted to the Department of Internal Medicine, Mae Sot
General Hospital, were used in this study.

H. influenzae (45%), S. pneumoniae (40%), Klebsiellapneumoniae (K. pneumoniae) (lO%) and B. caiarrhalis(5%). The results are shown in Figure 3.

Sputum smear: Gram staining and Acid fast stain‑

Comparision of causative bacteria of acute bronchi‑

ing were done of sputum smear to observe thetis between Mae Sot General Hospital, Mae Sot,

infiammatory cytology and bacteria. The method forGram staining of sputum specimens was based on
Hucker's modification (Sonnenwirth, 1980) . Ziehl‑Neel‑

Table 1 Age, sex and number of patients attended at the
Department of Internal Medicine, Mae Sot Gen‑

sen's staining was done to recognize acid‑fast bacilli(Cowan and Steel, 1974) .

eral Hospital, Thailand in three different study
periodSputum culture: Sputum was cultured either quanti‑tatively or semi‑quantitatively on TSA agar containing

Periods

1989. 11‑1990. 7‑

1990. 11‑1990. l1990. 8

1990. 12

7% human blood and on chocolate agar. Agar plates

Sex

M

F

M

F

M

F

were incubated at 37'C for 18 hr in ordinary incubator.

No, of patients

79

46

97

44

73

53

Bacteria were identified by standard methods (Cowan
and Steel, 1974) at the Department of Microbiology,
Chiang Mai University, Thailand.

Mean age (y.o.)

44 . 636 . 943 . 446 . 342 . 344 . 4

Total

125

141

126

113
1 OO9.1 1 ‑1 990.1

1 OO0.7‑8

26.30A

24.3010. jn fl uenne

26.3%

H.in fl uenzae
37.99/0

2.79102.7010neumonl ae

B. catarrh

21 .1 olo

K. pneum

1 0.891‑

B. ca tarrhal is

21 .1 okS.pneumonlae 21 .6010

5.201015eases, 20 strains

31 eases, 42 strains
1 99Q.1 1 ‑1 2

9.
3.O910C]

H. in f I uenzae

I

S. pneumoniae
B.catam,alis

K. pneumonl ae

H. in f I uenne
48.40106.10leK. pneumoniae
S.aureus
Ifll

B. catarrhal is
1 2.2910

E:

OthersPolymicrobial Infection.1elo

26 cases, 35 strains
Figure 2 The destribution of pathogenic bacteria isolated from sputum of patients with acute bronchitis inthree different study period.
loo

K. neumonlae
H JnHuenlae

B,cata5%50

H.h,,il crnzae

S. pneumon'ae

45%
B.cafo"hensS. pneums.aureus
K. Pn'umo 'oe

40%1980.1 1‑1990.1 , 1990.74, 1990.1 1‑1217 c8ees, 20 str8lns

Figure 3 The distribution of pathogenic bacteria isolated

from sputum of patients with pneumonia. This
represent the combined results of three different

study period.

othe'slkt ts'8nd Mae sot

d5 cases 97 cases

Figure 4 The distribution of pathogenic bacteria isolated
from sputum of patients with acute bronchitis.

Left column and right column reprensent theresult obtained from lki Island, Nagasaki and
Mae Sot, Thailand respectely.

114

Thailand and lki Public Hospital, Nagasaki, Japan are
shown in Figure 4. The percentage of causative organ‑
isms are similar.

DISCUSSION

Our experience showed that in most of the developing
countries have similar misconception about the recogni‑tion of pathogenic bacteria. This collaboration study

helped the recognition of pathogenic bacteria in Mae
Sot. We also found that Gram‑stained sputum smears

can greatly aid clinicians to make presumptive

In regard to the causative organisms of respiratory
infection some studies of developing countries showedthat the major bacterial pathogens were S, pneumoniae
and H. influenzae (Ghafoor et al., 1990; Mclntosh, 1990;
Vathanophas et al., 1990) .

pneumoniae and B. catarrhalis) of the community

In Mae Sot General Hospital we experienced a total
72 cases of acute bronchitis and 17 cases of pneumonia.

acquired respiratory infections are observed commonlyin the world (Nagatake, 1991; Davies et al., 1990). The

From sputum of acute bronchitis and pneumonia, 97

criteria used in this study would appear to be more
adaptable for routine use in developing countries.

strains and 20 strains of pathogenic bacteria were iso‑lated respectively. The main pathogenic bacteria were
H. influenzae. S. pneumoniae and B. caiarrhalis.
Previously we reported in Japan, B. catarrhalis is

one of the major pathogenic organism in respiratory
infection (Matsumoto et al., 1981; Nagatake, 1985).
Subsequently we reported various important aspect of
this bacteria to elucidate its mechanism of pathogenicity(Ahmed et al., 1990; Rikitomi et al.. 1991; Ahmed et al..

1992). In this study we found in Mae Sot, Thailand, B.
catarrhalis is one of the major pathogen in respiratory
infection. These data show that B. catarrhalis infection
increased simultaneously in different parts of the world(Mcleod et al., 1986; Bartos et al.. 1988). The present

etiological diaguosis, and permit the laboratory toenhance the isolation of pathogenic bacteria by selective

culture methods (Odhiambo et al., 1990). This study
shows that 'big three' pathogens (H. influenzae. S.

Similar type of collaboration studies are very helpful forthe developing countries.

ACKNOWLEDGMENTS
This study was partly supported by the Grant for
Scientific Research for the "Project on the diagnosis andtreatment of acute respiratory tract infection" fromWHO.
REFERENCES
1 ) Ahmed, K., Matsumoto, K., Rikitomi, N., Nagatake T

data also shows that there is a high incidence of B.

Yoshida, T. and Watanabe, K. (1990) : Effect of ampicil‑

catarrhalis in winter. This is also similar to our previous

lin, cefmetazole and minocycline on the adherence ofBranhamella catarrhalis to pharyngeal epithlial cells,
Tohoku J. Exp. Med., 161, 1‑7

observation and we also showed that this increase inci‑
dence of B. catarrhalis infection in winter is due to theincrease attachment of B. catarrhalis to respiratory
epithelium in winter (Mbaki et al.. 1987).

Although H. influenzae is usually regarded as alower respiratory tract pathogen in patients withchronic lung disease. H. influenzae was the main path‑ogen of acute bronchitis and pneumonia in Mae Sot

2 ) Ahmed, K., Rikitomi, N. and Matsumoto, K. (1992):
Fimbriation, hemagglutination and adherence propertiesof fresh clinical isolates of Branhamella catarrhalis.
Microbil. Inununol., 36(10) , 1009‑1017

3 ) Bartos, L.C. and Murphy, T.F. (1988) : Comparison of
the outer mel rbrance proteins of 50 strains of Bran‑hamella catarrhalis. J. Infect. Dis., 158, 761‑765

It is very interesting that the causative organisms of4 ) Cowan, S.T. and Steel, K.J. (1974): Cowan and Steel's
Manual for the Identification of Medical Bacteria, Uni‑
versity Press, Cambridge, pp. 80‑83.
5 ) Davies, B.1. and Maesen, F.P.V. (1990): Treatment ofBranhamella catarrhalis infections, J. Antimicrobi.
Chemother., 25, 1‑7acute respiratory infections between Thailand and6 ) Ghafoor, A., Nomani, N.K., Ishaq, Z., Zaidi, S.Z., Anwar,

Japan were similar. In Mae Sot, doctors had conceptionthat in that area, respiratory infection occured mostly

F., Burney, M.1., Qureshi, A.W. and Ahmad, S.A. (1990) :Diagnosis of acute respiratory tract infections in chil‑by S. pneumoniae. Because in Mae Sot General Hospi‑

dren in Rawalpindi and Islamabad, Pakistan, Rev.

General Hospital. The incidence of H. influenzae pneu‑
monia in adults have been increasing in many countries(Levin et al., 1977; Woodhead et al.. 1987; Maniji et al..
1990) .

tal, instead of rabbit blood human blood is used in agar,
on which it is difficult to recognize H, influnezae. As B.

catarrhalis is a newly recognized pathogen, in Mae Sot
they did not have experience to recognize the bacteria.

Infect. Dis., 12 (Suppl. 8) , 907‑914 '7 ) Maniji, H. and Reeve, P. (1990): Haemophilus influenzae
lobar pneumonia in the father of a child with epiglottitis,

Eur. Resp. J., 3, 840‑841

1158 ) Levin D.C., Schwarz M.1., Matthay R.A. et al. (1977):

Bacteraemic Haemophilus influenzae pneumonia in
adults, Am. J. Med., 62, 219‑224

9 ) Matsumoto, K., Uzuka, Y., Nagatake, T., Noguchi, Y.
and Suzuki, H. (1978) : A quantitative sputum culture
method: the signiflcance of the quantity> 107/ml of iso‑lated organisms, Jap. J. Thorac. Dis., 16(2), 77‑89 (inJapanese)10) Matsumoto, K., Nagatake, T. and Watanabe, K. (1981):
Chronic respiratory infections due to Branhamella catar‑
rhalis. Jap. Med. J., 2961, 21‑40 (in Japanese)11) Mbaki, N.. Rikitomi, N., Nagatake, T. and Matsumoto,K. (1987): Correlation between Branhamella catarrhalis
adherence to oropharyngeal cells and seasonal incidenceof lower respiratory tract infections, Tohoku J. Exp.
Med., 153, 111‑12112) Mclntosh, K. (1990) : Creation of a research program todetermine the etiology and epidemiology of acute respi‑

ratory tract infection among children in developing
countries, Rev. Infect. Dis., 12 (Suppl. 8) , 861‑869

13) Murray, P.R. (1982) : Macroscopic and microscopic
evaluation of respiratory specimens, Clin. Lab. Med., 2,

259‑267
14) Nagatake, T. (1985): Clinical significance of respiratory
infection caused by Branhamella catarrhalis with specialreference to p‑lactamase producing strains, Tohoku J.
Exp. Med., 147, 1‑1315) Nagatake, T. (1991): Physiology and pathology oflarynx and airway in the aged. Lower respiratory tract
infection related with bacterial adherence of upper respi‑
ratory tract in the aged, J. Jpn. Bronchoesophagiol. Soc.,
42 (2) , 144‑149 (in Japanese with English abstract)16) Odhiambo, J.A., Oishi, K., Watanabe, K., Nagatake, T.,
Suzuki, H. and MatsuJnoto. K. (1990) : Gram stain andquantitative sputum culture in bacteriological assess‑ment of lower respiratory tract infection. Trop. Med., 32(3) , 115‑12017) Rikitomi, N., Anderson, B., Matsumoto, K., Lindstedt, R.
and Svanborg, C. (1991): Scand. J. Infect. Dis., 23, 559‑56718) Sonnenwirth, A.C. (1980) : Stains and staining proce‑dures. p. 1384. In A.C. Sonnenwirth L. Jarett (ed) .
Gradwohl's Clinical Laboratory Mathods and Diagnosis.
18th Ed. Vol 2. C. V. Mosby Company. St. Louis.

19) Takasugi, M., Matsumoto, K., Ishikawa, H., Ut‑
sunomiya, Y., Kawakami, K. and Yamauchi. S. (1990) :Secondary bacterial infection and changes in pulmonary
function of acute bronchitis, Kagakuryouho no Ryouiki,6, 1300‑1407 (Japanese)20) Vathanophas, K., Sangchai, R., Raktham, S., Pariyanon‑da, A., Thangsuvan, J., Bunyaratabhandu, P., Ath‑ipanyakom, S., Suwanjutha, S., Jayanetva, P., Wasi. C.,

Vorachit, M. and Puthavathana, P. (1990): A commu‑
nity‑based study of acute respiratory tract infection in
Thai Children, Rev. Infect. Dis. 12 (Suppl. 8) , 957‑965

21) Woodhead, M. and MacFarlane, J.T. (1987) : Haemo‑
philus influenzae pneumonia in previously fit adults, Eur.

J. Respir Dis ,, 70, 218‑220

117

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 2, 1993, pp. 117 121

IMMUNOHISTOCHEMICAL DEMONSTRATIONOF MYCOBACTERIUM LEPRAE IN THE NERVOUS
SYSTEM OF LONG‑TERM CURED LEPROSYPATIENTS USlNG A M. LEPRAE SPECIFIC

ANTI‑PGL ANTIBODYMASAMICHI GOTO1,2 YASUHIRO MlNAUCH11 YASUYUKI NoBUHARA3
AND EIICHI SATO'
Abstract: Leprosy (Hansen's disease) has become a curable disease by the advance of chemotherapy,
however, deformities evoked by nerve damages are still a great problem of treatment. We have recently
demonstrated a highly sensitive and specific method to identify the leprosy antigen in skin tissue by
immunohistochemical staining using a Mycobacteriam leprae‑specific antiphenolic lipid‑1 (PGL) monoclonal
antibody. Using this method, we have examined the peripheral nerves of lower extremities, spinal cords andbrain stems of clinically cured (skin slit smear negative more than 10 years) Ieprosy autopsy cases(Lepromatous (L) : n=6, Tuberculoid (T) : n=6) , in which ordinary Fite's acid‑fast staining did not reveal
M. Ieprae. Positive staining was observed as follows: (1) Peripheral nerves: L: 6/6, T: 1/6. (2) Dorsal root
ganglia and posterior spinal roots: L: 6/6. T: 2/6. (3) Anterior roots: L: 0/6, T: 0/6. (4) Spinal cord: L: 6/6,

T: 2/6, observed in posterior horn cytoplasm and anterior horn neurons. (5) Medulla oblongata: L: 6/6, T:
2/6, observed mainly in ambiguus, facial, hypoglossal, cuneate and gracile nuclei. These findings indicatethat M. Ieprae specific antigen remains in the peripheral sensory nerves as well as central sensory and motor
nerves long after the clinical cure, especially in lepromatous patients where definitely abnormal cellularimmunity against M. Ieprae is noted, which suggest the role of motor neurons in the pathogenesis of quiet
nerve paralysis.

every year, and they are usually successfully treated as

INTRODUCTION

outpatients. However, Ieprosy is still a world‑wide

Leprosy is a chronic weakly infectious disease evo‑
ked by acid‑fast bacilli, M. Ieprae. The disease mainly
affects peripheral nerves and skin, but internal organs

active disease affecting more than l0,000,000 peoples inthe developing countries, and common cause of periph‑such as testes, Iiver, spleen and lymph nodes may beeral neuropathy.
Leprosy shows wide variety of clinical and patho‑

involved in severe cases (Jopling, 1988) . Lack of host
cellular immunity against M. Ieprae is responsible for10gical manifestations from tuberculoid to lepromatous
types. In the tuberculoid type, a few dermal macula are

the onset of the disease.

associated with peripheral neuritis.

In Japan, Ieprosy became a disease of the past.
Although about 7,000 persons still live in 13 national and3 private leprosaria, most of them are bacteriologicallyinactive (cured) because of effective chemotherapy, yethave physical deformities evoked by leprous neuropath‑y. Recently only about 20 new cases have been reported

E pithelioid

granuloma completely replace the nerve fascicles while
only small number of M. Ieprae are found. In the le‑promatous type, where cellular immunity to M. Ieprae isalmost absent, multiple dermal nodules (lepromas)
appear. These lepromas show numerous M. Ieprae in the
foamy macrophages, but inflammatory reaction is lack‑1 . National Leprosarium Hoshizuka‑Keiaien, 4522 Hoshizuka‑cho, Kanoya‑shi, Kagoshima 893‑21, Japan2 . Department of Pathology, Kagoshima University School of Medicine, 8‑35‑1 Sakuragaoka, Kagoshima 890, Japan
3 . Medical student, Kagoshima University School of Medicine
Presented Xlth International Congress of Neuropathology, Kyoto, September 2‑8, 1990.
This manuscript (special article) is reproduced with author's permission.

118

ing. In the late stage of this type, glove and stocking

PGL. Five micrometer thick sections were stained bytype sensory neuropathy results.

hematoxylin and eosin (HE) and Fite's acid fast stain‑

We could demonstrate the persistence of
mycobacterial antibodies in the skin biopsies of le‑promatous leprosy, even in long‑treated cases whereusual acid‑fast staining was negative. Both polyclonal
antibody against BCG cross‑reacting with many acid‑fast bacilli and monoclonal antibody against M. Ieprae

specific phenolic glycolipid‑1 (PGL) (Hunter et al..
1982) showed positive immunohistochemical staining in

ing.

As primary antibodies, mouse monoclonal anti‑PGL
antibody (ml 1‑21) raised against synthetic natural
trisaccharide (Fujiwara et al.. 1984; courtesy of Fuji‑

Rebio Research Institute, Japan, diluted X 1,000) or
rabbit polyclonal anti‑BCG (Bacillus Calmette‑Gue'rin)antibody (DAKO Japan B124, diluted x 2,000) were used

(60 min at room temperature) . Immunostaining wasparaffin sections of such cases (Goto and lzumi, 1991) .

done as described by Hsu et al. (1981) using biotinylated

We have also summarized our neurological andpathological works of leprosy based on biopsy andautopsy performed in Hoshizuka‑Keiaien. Minauchi

secondary antibodies and ABC complex (Vectastain PK

found persistence of ultrastructurally densely stained
bacterial structure in the myelinated Schwann cells ofclinically inactive cases as well as demyelination and‑4002 for PGL or PK‑4001 for BCG), followed bydiaminobenzidine‑peroxide. Finally, counterstaining
was done by hematoxylin.

RESULTSremyelination. In the longitudinal study of lower
extremity nerves, he also showed sensory nerve̲tropism

Peripheral nerve:

and upward extension of the bacteria and lesions

All cases (6/6) of lepromatous leprosy showed PGLand BCG antigens in most of the examined sural, tibial,
peroneal and sciatic nerves. As there were no essential
differences in the staining sites between PGL and BCG,
the following results will be focused to PGL staining. In(Minauchi et al. in press) . Usually these ultrastructural‑ly proven bacilli did not show positive acid‑fast staining,which led us some problems of specificity.

This study was primarily aimed to prove that thesebacillary structures have the chemical properties of M.

the sciatic nerve, PGL staining was much different

leprae using BCG and PGL immunohistochemistry,

among the nerve fascicles. Positive staining distributed

which is successfully demonstrated here. Moreover, on
the contrary to our expectation, PGL positive staining

sporadically along the axon adjacent to Schwann cell
nuclei as granular or foamy pattern. Old large globi(giant foamy macrophage) were perivascularly recog‑
nized in some lepromatous cases, but these globi were

was also demonstrated in the spinal cord and brain stem.

MATERIALS AND METHODS
Since 1983, autopsy has been permitted in about 21
3 of the deceased individuals in our leprosarium, and

PGL negative. Except for one case where dense
lymphocytic infiltration is noted focally, there were no

systemic examination of nervous system as well asinternal organs has been performed. Among them, six

inflammatory reaction or tissue damages associatedwith PGL antigen. In tuberculoid leprosy, most cases(5/6) showed PGL negative.
In the lumbar dorsal root ganglia and posterior

cases of lepromatous leprosy and six cases of tuber‑spinal roots (Fig. Ia, b) similar positive findings (Le‑culoid leprosy were selected, in which skin slit smears topromatous 6/6, Tuberculoid 2/6) were observed. In theexamine the active leprous skin lesions were negative

ganglia, neuronal soma were also stained. Lumbar
anterior roots of both lepromatous and tuberculoid

for more than at least ten years and leprosy was consid‑

ered to be clinically cured. As controls, three le‑types showed negative PGL staining. Dorsal root gan‑promatous leprosy with positive skin‑slit smear within 5

glia and spinal roots of non‑1eprosy were PGL negative.
There were no diffeneces of staining location, pat‑tern or intensity between six cured lepromatous cases
and three bacteriologically positive cases.

years before death and two non‑leprosy were used.
Sural, tibial, peroneal and sciatic nerves, Iumbar dorsal

root ganglia, anterior and posterior spinal roots at lum‑bar level, spinal cord (lumbar, thoracic, cervicaD and 3‑4 mm step serial coronal sections of brain stem (frompyramidal decussation to superior colliculus) were fixedSpinal cord and brain stem:in formalin and embedded in paraffin. In some cases
peripheral nerves were fixed in glutaraldehyde, but thisstaining than BCG. The spinal cord showed PGL posi‑tive in nerve fibers of dorsal horn and anterior horn

fixation did not suppress the immunohistochemistry ofneuron (Fig. Ic, d). The latter was frequently associat‑

Generally, PGL antibody showed much strongerl 1 9Figure l PGL immunohistochemistry in posterior root (a, b x 380) , spinal anterior horn cell (c, d >< 380) ,gracile nucleus (e, f X 250) , ambiguus nucleus (g, h >< 125) . (i) shows ambiguus nucleus by BCG
immunohistochemistry ( x 125) . Left photographs (a, c, e, g) were taken with red filter for nuclear
information, while right ones (b, d, f, h, i) were taken with blue filter for immunostaining.

120

ed with unusual vacuolation degeneration, but manyneurons preserved their normal shape. Sometimes

from Yamada's study which stresses the acid‑fast sub‑stance located in the pyramidal and other tracts ofbrown immunohistochemical products were difficult totuberculoid leprosy.

Prevention of quiet nerve paralysis, which occurs

differentiate from lipofuscin.

In the step sections of brain stem of lepromatous

insiduously without acute neuritic episodes, is an impor‑leprosy, motor neurons such as (1) ambiguus nucleus, (2)facial nerve nucleus, (3) hypoglossal nucleus, and sensory

tant problem for the successful treatment of leprosy(WHO Expert Committee on Leprosy, 1988). Thick‑system such as (4) gracile nucleus and tract (Fig. Ie, f) ,ened nerve trunks are usually observed in this condition,(5) cuneate nucleus and tract, (6) vestibular nucleus, (7)but there are also some cases of quiet nerve paralysis

spinal trigeminal nucleus and tract, (8) solitary nucleus

without swollen nerves. The results obtained in ourpresent study may add a new viewpoint for the further

were PGL positive. Among them, (1) to (5) were almost
constantly positive, whereas the other were variablefrom case to case. In the positive nuclei, most strongly

understanding of pathogenesis of the quiet nerve paraly‑sis.

stained part is neuronal soma with granular or foamy
patterns, but perineural zone also showed small foamy
patterns. In two cases, micro‑granulomas were detect‑
ed in close association to PGL positivity (Fig. Ig‑i) . In

tuberculoid leprosy, 2/6 cases were PGL positive, but

two non‑leprosy control were PGL negative. There
were no diffenences between six cured lepromatous and

ACKNKOWLEGDEMENTS
We thank excellent technical assistance of Mr. M.
Minaminakamichi, Ms. M. Shirai, Ms. A. Karizono andencouragement by Dr. M. Suzuki (former director of
Hoshizuka‑Keiaien) .

three bacteriologically positive cases.

REFERENCESDISCUSSION

1 ) Fujiwara, T., Hunter, S.W., Cho, S.N., Aspinall, G.O. andIn the peripheral nerves, the PGL antigen (M. Ieprae

Brennan, P.J. (1984): Chemical synthesis and serology

and its degeneration product) was preserved even longafter the clinical cure. Solid M. Ieprae was stained as
granular pattern, while non‑solid (dead) M. Ieprae as
foamy pattern in the skin biopsy study. In the nerves
both patterns were observed, which suggest that thesebacilli still survive, although these bacilli are usuallyof disaccharides and trisaccharides of phenolicunstained by conventional Fite's staining. We do notknow whether this fact is merely a technical problem or
essential problem of M. Ieprae infection.

By the investigation of autopsy cases in Hoshizuka‑Keiaien, we have found secondary degeneration of
spinal dorsal column due to sensory neuropathy, which
is severe in lepromatous leprosy, and degeneration of
optic system in blind cases (Goto and Minachi, 1985).

Yamada reported the presence of acid‑fast sub‑stance in the brain stem of leprosy using periodic acid‑pretreated acid‑fast staining (Yamada, 1984), but wecould not get similar results except for non‑specificstaining. Thus, until recently we have believed that M.

leprae invade up to dorsal root ganglia and do not
penetrate the central nervous system.
Mitsuda described acid‑fast bacilli in the soma of
facial nerve nucleus, ambiguus nucleus and spinal ante‑rior horn nucleus (Mitsuda, 1952). Our present study

confirm his work from the immunohistochemical stand‑point. On the contrary, our results were mostly different

glycolipid antigens from the leprosy bacillus and prepa‑

ration of a disaccharide protein conjugate for ser‑odiagnosis of leprosy, Infect. Immun., 43, 245‑252

2 ) Goto. M. and Minauchi, Y. (1985) : Central nervous
system lesions observed in leprosy autopsy, Nippon Rai
Gakkai Zasshi, 54, 1443 ) Goto, M. and lzumi, S. (1991): Light‑and electronrni‑
croscopic immunohistochemistry using anti‑PGL‑1 anti‑
body specific for Mycobacterium leprae. Int. J. Lepr., 59,
195.

4 ) Hsu, S., Raine, L. and Fanger, H. (1981): Use of avidin‑

biotin‑peroxidase complex (ABO in immuno‑perox‑
idase techniques: a comparison between ABC and un‑labelled antibody (PAP) procedures, J. Histochem.
Cytochem., 29, 577‑580

5 ) Hunter, S.W., Fujuwara, T., Brennan, P.J. (1982):
Structure and antigenicity of the major specificglycolipid antigens of Mycobacterium leprae. J. Biol.
Chem., 257, 15072‑15078
6 ) Jopling, W.H. (1988) : Handbook of Leprosy, 4th ed. .

Oxford, Heinemman.
7 ) Minauchi, Y., Goto, M. and Osame, M.: Leprous Neuri‑tis, In Roman GC. ed. Boca Rapon (Florida) , CRC Press,
in press8 ) Mitsuda, K. (1952): Atlas of leprosy, Papers of Leprosy

Vol.VI. Kyoto, Nankodo
9 ) WHO Expert Committee on Leprosy. (1988) : Technical
Report Series 768. Geneva, WHO, 19‑20

12110）Yamada，N．（1984）：Histopathologicalcentralnervoussystemin−rosalinin

positivematerials

relationtissueinjury，Nippon

67−80

to

investigation

leprosy．1．Distributionin

leprosyRai

withGakkaiofofparaspecialZasshi，53，

123

Jpn. J. Tro p. Med. Hyg., Vol. 21, No. 2, 1993, pp. 123 125

Research Note

MORPHOMETRIC CHARACTERISTICS OFGLOSSINA PALLIDIPES POPULATIONSON THE SOUTH KENYA COAST
KAZUYO ICHIMORI
Received January 18 1993/Accepted February 25 1993

Abstract: In this study both spatial and seasonal variations in the size of tsetse fiies from the south Kenya

coast were investigated. The work was done at two sites; in Rongomwagandi (in Shimba Hills National
Researve) and in Muhaka Forest. Wing length was used as an index of fiy size. In a cross‑sectional study
the size of fiies from two study sites was compared. Significant differences were found between the size offlies from Rongomwagandi and Muhaka Forest. In a longitudial study the size of flies from two study sites

was compared over a six month period. The population of flies at Rongomwagandi showed larger seasonalvariation in size than those in Muhaka. Additionally, all flies collected were aged using the wing fraymethod. More older flies were observed in dry season than in rainy season.

worthogs and buffalos. Muhaka Forest outside the

INTRODUCTION

Shimba Hills National Researve (4'20'S, 39'32'E, alt. 35

Etten (1982) demonstrated the existence of diver‑sity in behaviour between tsetse populations. For them) is a region of forest relicts in rural areas and had
both domestic and wild animals including cattle, goats,
warthogs, bushpigs and bushbabies (Fig. l) .

effective control of tsetse flies and trypanosomiasis, it isBlue and white biconical traps (Challier et al., 1977)

also important to understand the variability of otherwere used for sampling tsetses. Flies were collectedusing 2‑5 traps in each place. Traps were operated forcharacteristics between different tsetse populations.

In this study, comparison was made of the sizes ofGlossina pallidipes in Rongomwagandi (Shimba Hills
National Researve) and Muhaka Forest in Kenya south

24 hr at sampling stations.

coast.

mounted on a glass slide. The length of the middle partof the fourth longitudinal wing vein was then measured

The right wing was removed from each fly and

Furthermore, a longitudinal study of tsetse mor‑phometrics in spatially separated populations was done.

(Jackson, 1946). The wing fray category was also

Since the larva of tsetse fly develops in uterus, variation

recorded (Minter, 1982) .

in size of adult fly reflects, in part, the favourability ofthe environment for the parent females, and may be

RESULTS AND DISCUSSION

related to the adult mortality rate and vectorial capac‑

There was a significant difference in the size of flies

ity (Dransfield et al.. 1989).

from Rongomwagandi and Muhaka Forest. It is known
that the size of flies is influenced by humidity, tempera‑

MATERIALS AND METHODS

ture and food availability (Jackson, 1952; Dransfield etal., 1989). In Shimba Hill National Researve, wild
animals were abundant and especially, Rongomwagandi,

Flies were collected from 2 sites on south Kenya

coast. Rongomwagandi in the Shimba Hills National
Researve (4'5'S, 39'25'E, alt. 180 m) consists of amoraine of forest and savannah. Only wild animals

a bush area, could be a habitat of animals. On the other
hand, in Muhaka Forest, both wild and domestic animals
were present, though there were significantly less gamewere present in this area including bushbucks, bushpigs,

International Centre of Insect Physiology and Ecology (ICIPE) P.O.Box 30772, Nairobi, Kenya,

Present Address: WHO, P.O.Box 77. Apia, Western Samoa

124

Rongomwegend

L' j l̲̲ I I j

I

l

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= Meilmbon]

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d :t : 1': lr:E1(::lr::lr'/‑ ̲̲ i.*,,>, =. =..*..

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1̲.

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YLfl'IflTlfli r
lrlflflr'r'rtrlr

lr r:fIfLIdrIflflclr'lr‑
IfE1;̲1Hr

Figure 1 Map of the South Kenya Coast showing Rongomwagandi and Muhaka Forest.

animals there than in Shimba Hills National

1.95

Researve.

1.9

Seasonal changes in the mean size of G.
pallidipes, as indicated by wing vein length, weremonitored in June (rain season) , September (dryseason) and December (short‑rain season) , 1989
in two study sites (Fig. 2). Both male and female
fiies from Shimba Hills were significantly larger

::

eo

1.8

c:
e)
s::

l‑

1‑

1 . 85

‑‑+

J‑

T+'.

1.T5

a5

>

bo

1.7

' ' ' ̲ . , ̲̲ ̲T'‑ ‑ ‑̲ ̲ ̲ L‑ T'i'.̲

‑ *r =

in June and December. However, in September

1 . 65"‑ 'flies from Muhaka were larger. Size of tsetses in

1 .6

Shimba Hills was varied, whereas in Muhaka size
was rather stable.

It seems that there was not much difference
in environmental conditions such as temperatureand humidity between these two places, although
the micro‑climatic condition might be different.

l] RMG/FEMALE + MHK/FEMALE <RMG/MALE A MHK/MALE

Figure 2 Changes in mean wing vein length (In the dry season the game animals in Shimba Hills
migrate to the other side of the Researve where f ood

was available, but at Muhaka Forest both wild anddomestic animals stayed all the year round.
Since size may be related to the mortality rate ofstandard
error) of Glossina pallidipes in Rongomwagandi(RMG) and Muhaka Forest (MHK) .

seasons.
If we assume that wing fray age category of tsetse

is related to the number of blood meals they have taken,
then populations containing large' numbers of older flies

flies (Dransfield et al., 1989) these results suggest thatwill have greater potential for disease transmission.
When planning the timing of tsetse control programs it

the transmission dynamics of trypanosomes in the two

is important to take into account the age structure of flysites may be different.

populations.

Wing fray age structures of fly populations from thetwo sites changed seasonally (Fig. 3). About 50% of
populations were old flies ( > category 2) at both Ron‑ACKNOWLEDGEMENTS

gomwagandi and Muhaka Forest in June. In September
older flies predominated in both populations (73.4%
were old flies at Rongomwagandi and 61.9% at Muhaka
Forest) . Younger flies appeared in December. ThisI wish to express my sincere thanks to Prof̲, T.R.
Odhiambo, the Director of the ICIPE, Dr. L.H. Otieno,

data suggests that fly survival was greater or breeding

for their help. The Japan Society for the Promotion of
Science is gratefully acknowledged.

rates were lower in September than in the other two

Programme Leader. Tsetse Research Programme,
ICIPE for their support, and to the field staff of ICIPE

125

RONGOMWAGANDI

50
(:,

:)
C
)
Lu )F

uJ
DhL

o

1

2 3

4

5 6

1 2 3 4 5

6

1 2 3

4

5

6

2 3 4 56MUHAKA FOREST

50:=:=1 ::1h(O::;i:i j;;

:qCCJ

Lu
Cl)

k
h

Lu

Cl)

:'

･Ill

O

1 2 3 4 5 6 1 2 3 4 5
wING FRAY wING FR4Y

JUNE SEPTEMBER

6

1

WING FR41YDECEMBER

Figure 3 Age structure by wing fray categories of the populations of Glossina pallidipes in June,
September and December (dark bar=female, Iight bar=male) .

REFEREN CES
1 ) Challier, A., Eyraud, M., Lafaye, A. and Laveissiere, C.

(1977): Amelioration du rendement du piege biconiquepour glossines (Diptera, Glossinidae) par l'emploi duncone inferieur bleu, Cah. ORSTOM Ser. Entomol. Med.
Parasitol., 15, 283‑286
2 ) Dransfield, R.D., Brightwell, R., Kiilu, J., Chudhury, M.F.

and Adabie, D.A. (1989): Size and mortality rate of
Glossina pallidipes in the semi‑arid zone of south west‑
ern Kenya, Med. Vet. Entomol., 3, 83‑95
3 ) Etten, J. van (1982): Comparative studies on the fat
reserves, feeding and metabolic strategies of flies from
two allopatric populations of Glossina pallidipes Austen
in Kenya, Acta Tropica, 39, 157‑169
4 ) Jackson, C.H.N. (1946): An artificially isolated genera‑tion of tsetse flies (diptera) , Bull. Ent. Res., 37, 291‑299

5 ) Jackson, C.H.N. (1952): Seasonal variations in themean size of tsetse flies, Bull. Ent. Res., 43, 703‑706
6 ) Minter. D.M. (1982): Tsetse fiies. In: Tropical Diseases.

(ed. Manton‑Bar, P.E.C.) 791‑801, London

127

J pn. J. Trop. Med. Hyg., Vol. 21, No. 2, 1993, pp. 127‑130

Clinical Report

A CASE OF IMPORTED PLASMODIUM

MALARIAE MALARIATOSHIMASA NISHIYAMAl*, TSUNEJI ARAK11, HIROYUKI AMAN02,
SHIGEAKI ISHIZAKA3, HIROSHI FUKUl3 AND TADASU TSU JI13
Received February 4 1993/Accepted March 10 1993

Abstract: Imported malaria is one of the perplexing problems in the world. Migrants, either refugees or
those looking for better opportunites in life, have contributed to the introduction of malaria to previously
free areas. The increased incidence of imported malaria is distinctly attributed to a failure of travellers totake appropriate action to prevent infection. Annually up to 100 cases of malaria are presumed to beimported into Japan and most of these cases are Plasmodium falciparum and P. vivax malaria. The reportsof P. malariae malaria cases are very few in Japan. The present paper reports a case of one patient with
imported P. malariae malaria infection accompanied with marked hepato‑splenomegaly who has come from
Nigeria. And this patient was treated with FansidarR (sulfadoxine‑pyrimethamine) and minocycline whichwere very effective against this infection.

I N TRODUCTION

CASE REPORT

Recently, there has been an enormous influx offoreiguers, who are travellers and workers in Japan,

A 29‑year‑old Nigeria‑born male who had worked
as pharmacist at Ragos in Nigeria was admitted tofrom Southeast Asia, South America, Africa and so on.
But these peoples often have health problems which are
unfamiliar to Japanese physicians. Some of them har‑bor parasites from tropical areas, for example Plas‑
modium spp., Entamoeba histlytica. Giardia lamblia,

Utano‑Tsujimura Hospital in Nara Prefecture in

Ascaris lumbricoides, hookworm, Trichuris trichiura, etc.
(Akao et al.. 1992).

In recent years, imported malaria cases are report‑
ed to be about 48 to 101 cases in Japan annually. The
most of these cases were either P. falciparum malaria or

P. vivax malaria. But, cases of P. malariae malaria arevery rare in Japan (2.6%; Yoshida, 1991). Importantly,

the number of Japanese literatures on P. malariae
malaria was only 8 papers over the past 25 years(Nakabayashi et al., 1988; Yamakami et al., 1990;

December 1989, because of a high intermittent fever. He
had been travelling and stayed at his friend's lodging in

Utano, Nara Prefecture. Already, this patient had
known that he had been infected with malaria in Nigeria
and whenever he had a malaria infection, he had treatedhimself with chloroquine.
On admission, although he appeared quite ill but his
vital signs were stable and the physical examination was

unremarkable except for marked hepatomegaly andsplenomegaly (Fig. l). Routine laboratory examina‑
tions confirmed the presence of anemia (red blood cellcount 277 x 104/mm3, Hb 7.7 g/dl, Ht 23.5%, reticulocytel.7%) which was normocytic, but hypochromic. The
white blood cell count was 5,400lmm3 and platelets

Amano et al., 1992).
In this paper, a case of one patient who has comecount 24.4 x lO'/mm3. Peripheral blood smears revealed
malarial parasitemia (0.245%). All formes of P. malar‑from Nigeria with P. malariae malaria accompanied

iae were observed in red blood cells (Fig. 2) . The serumwith remarked hepato‑splenomegaly is reported.

total proteins, albumin, cholinesterase and total choles‑

terol were low. LDH was increased because of
haemolysis. ZTT, TTT and CRP were increased. A
2

3

Department of Parasitology, Nara Medical University, 840 Shijo‑cho, Kashihara, Nara 634, Japan (*corespondent author)
Department of Overseas Medical Services, Tenri Hospital, Tenri 632, Japan
3rd Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634, Japan

128

'"+'

.

Figure I The upper half of this patient.

Remarked he pato‑splenomegal yi** * *"*
= i <='=

was seen.

Figure 2 Blood smear of the patient, containing all stages of P. malariae. A: slight deformed ringform. B: band form, C: completely matured schizont. D: macrogametocyte.

129

Fi窪ure3 Computed tomo菖rap塾ysc鋤ofthe abdomen ofthep＆tieηtshowedswelling ofthe iiver andsp1εen．

℃41

CljnicalcourseParasitemi呂（％）O、245％

0、258％

40
！へ

39

38

37

36

〆
35

SP

S碧

2憶b悟ts 耗ab1就

OEX750mg／day

MINO200mg／day
含admission

Dec．

4

5

6

7

8

9
Date

10

11

12

Figure4 Cli煎三c鼠1co組rse of the p歌tiε鷺t，
CEX：cephalexin，MINO：minocyd玉ne，
SP：＄ulfadoxine−pyτimetぬamine

13

14

130

computed tomography (CT) scan of abdomen showed a

REFERENCES

markedly swelled liver and spleen (Fig. 3). Clinicalcourse of this patient is summerized in Fig. 4. Typicalintermittent fever of P. malariae malaria (three‑day1 ) Akao, N., Ohyama, T., Ohkawa, T., Kondo, K., Ito, S.
and Takeguchi, A. (1992): A survey of intestinal para‑sites of the foreign laborers in Ishikawa Prefecture, Jpn.

intervals) was observed.

The patient was orally treated with sulfadoxine‑pyrimethamine (Fansidar R, pyrimethamine 25 mg;
sulfadoxine 500 mg) 3 tablets (2 tablets on the first day

and I on the second) , and minocycline 200 mg per day
for 7 days. All symptoms of P. malariae malaria abated.

J. Trop. Med. Hyg. 20, 1072 ) Amano, H., Sakai, T., Okuno, T., Nishiyama, T., Taka‑hashi, Y. and Araki, T. (1991): Use of mefloquine intreatment of imported falciparum and vivax malaria,
Jpn. J. Trop. Med. Hyg. 19, I16

3 ) Amano, H., Nishio, T., Nishiyama, T., Takahashi, Y.
and Araki, T. (1990) : Use of quinidine in treatment of

DISCUSSION
The risk of contracting malaria has recently been
increasing because of the resurgence of the disease inmany countries and areas of the world (Schultz, 1989) .
Nevertheless, specific diagnostic investigations formalaria can be sometimes difflcult to prescribe in Japan,

because Japanese physicians and medical technicians
are not familiar with these infections. The clinicalcourse in this patient was quite typical of P. malariae
infection with intermittent fever (three‑day intervaD,amenia and hepato‑splenomegaly. And this patient
informed us that he was suffering from malaria. Thenwe were able to make a diaguosis of P. malariae malaria
easily.

Drug‑resistant P. falciparum and P. vivax are
documented to occur in the many malarial areas (WHO,
1992; Rieckman et al., 1989; Whitey et al.. 1989; Col‑lignon, 1991; Gravelli and Corti, 1992) . A drug‑resistantimported Plasmodium falciparum malaria with special
emphasis on a case resistant to sulfadoxine/pyrimeth‑amine, Jpn. J. Trop. Med. Hyg., 18, 1044 ) Amano, H., Nishiyama, T., Takahashi, Y. and Araki, T.
(1992): Clinical malaria in Japan; a study of Japanese
literatures for past 25 years, Jpn. J. Trop. Med. Hyg., 20,705) Collignon, P. (1991): Chloroquine resistance in Plas‑
modium vivax. J. Infect. Dis., 164, 222‑223

6) Garavelli, P.L. and Corti, E. (1992): Chloroquine resis‑
tance in Plasmodium vivau: the first case in Brazil,
Trans. Roy. Soc. Trop. Med. Hyg., 86, 1287 ) Nakabayashi, T., Ono, T., Fujikawa, J., Kobayashi, H.,
Tomoda, I., Eguchi, T., Minakata, T., Konaka, Y. andKubo, U. (1988) : A presumed relapse case of quartan
malaria after more than 40 years, Jpn. J. Trop. Med.
Hyg., 16, 174
8 ) Rieckmann, K.H., Davis, D.R. and Hutton, D.C. (1989):
Plasmodium vivax resistance to chloroquine?, Lancet, ii,
1183‑1184
9 ) Schultz, M.G. (1989): Malaria in migrants and travel‑lers, Trans. Roy. Soc. Trop. Med. Hyg., 83 (Supple‑

P. malariae case was not reported yet, anywhere theworld. In this case, this patient had used chloroquine

himself to treat malaria and experienced repeatedly
recrudescences in Nigeria. Accodingly we did not selectchloroquine, but sulfadoxine‑pyrimethamine andminocycline, which were very effective against this P.
malariae infection. The drug used for malaria must bement) , 31‑34

10) Tanabe, K.,Otsuji, Y., Nakabayashi, T.,Ohtomo, H. andTanaka, T. (1989) : Management and utilization oforphan drugs against parasitic infections in recent 7
years in Japan, Jpn. J. Trop. Med. Hyg., 17, 229‑236
11) Whitey, M., Wood, G., Veenendaal, J.R. and Rieckmann,

K. (1989): Chloroquine‑resistant Plasmodium vivex.
Lancet, ii, 1395

chosen as the most effective one from several anti‑malarial drugs which are available in Japan, such as
chloroquine, sulfadoxine‑pyrimethamine (Fansidar R) ,sulf amethox azole‑trimethoprim ( Bak tar R,
BactraminR) , quinine, quinidine, mefloquine, minocy‑cline etc. (Tanabe et al.. 1989; Yoshida et al.. 1987;

Amano et al.. 1991, 1990). But we could not find a

WHO (1992): International travel and health vaccina‑tion requirements and health advice, WHO (Jeneva).
13) Yamakami, S., Nakamura, A., Kanamori, T., Monoe, T.,
12)

Ohno, T., Nagahara, K., Ohtomo, H., Kano, S. andSuzuki, M. (1990): A case of quartan malaria following

splenectomy 44 years after primary attack, Jpn. J.
Parasitol., 39 (Supplement) , 95

complete cure, because this patient left Japan when hedid not have any signs of malaria.

14) Yoshida, Y., Matsumoto, Y., Yamada, M. Shiota, O.,
Yoshikawa, H., Kobayashi, A. and Kin, R. (1987) :Our Japanese physicians must recognize that

Clinical trial of trimethoprim‑sulfamethoxazole in thetreatment of vivax, ovale and falciparum malaria, Jpn.
J. Trop. Med. Hyg., 15, 105‑116
15) Yoshida Y. (1991): Illustrated human parasitology, 4thed. Nanzando Co. Ltd. (in Japanese)malaria is increasing in Japan and drug‑resistant Plas‑

modium infections are spreading throughout malarial
areas.

VOL21NO．2

JUNE1993

CONTENTS
Originalarticle
Murata，T．，Inoue，M．，Kanoe，M．，Horio，M．，Shimada，M．，Yokoyama，M．，

Taura，Y．and

Nakama，S．

Fine

of

Stmcture

Gamontof∬の伽0200％6αnゑs・…………・・……………・…

・・107−110

Nagatake，T．，Takahashi，A．，Tao，M．，Hirose，H．，Masaki，H．，
Watanabe，K．，Ahmed，K．，Rikitomi，N．，Matsumoto，K．，Tharavichitkul，P．，

Sirisanthana，T．，Ruengverayudh，R．，Kasomson，T．，Charuroteskulchai，S．，

Jiamtaveeviboon，S．，Uthaisilp，C．，Swaddiwudhipong，W．and
Chandoun，C．

Causative

Organisms

of

Acute

Respiratory

infectiollsinNorthemThailand・………・…………………………・…・・……………・………………一・…
Goto，M．，Minauchi，Y．，Nobuhara，Y．and Sato，E．

Immunohistochemical

Demonstrationof躍yooδ06孟召吻吻妙鵤8in

一111−115

theNervous System of Long−term Cured Leprosy Patients Using a〃．Z砂耀θSpecific Anti−PGL Antibody一・……………・……一一……・………

Research

一117−121

Note

Ichimori，K．

MorphometricCharacteristics

on

Kenya

the

South

ClinicalReportCoastofαos吻α加」泥吻6s

Populations…………・……・…・………・……・・………………・・畢…

…一

・……・123−125

Nishiyama，T．，Araki，T．，Amano，H．，Ishizaka，S．，Fukui，H．and
Tsujii，T．

A

case

of

Imported

P伽吻04伽彿耀彪吻βMalaria…・・…………・……………

一…・127−130