一騒■監

第27巻第4号平成11年12月
容

内
総

説
マラリアの病理学と病態生理学（英文）

狩野
原

繁之，相川

・471 476

正道一…

著
Antifilarial Effect of A吻窺雰宛n磁g17ガ6αExtract and its Ultra High Dilutions
against Canine Dirofilariasis
Nirmal Chandra Suku1，Paromita Sarkar，Anirban Sukul and Santi Prasad Sinhababu

・・477−481

日本の乳康尿患者における，バンクロフト糸状虫循環抗原，

フィラリア抗原特異的IgGならびにIgG4抗体の検出（英文）伊藤誠，郎旭光，小山 雄三，小川 由英，Mirani V．Weerasooriya，Hanesana

Visanou，藤巻

康教，木村

英作

・・483−486

フィリピンの脳マラリア患者の予後を決定する臨床症状および検査成績（英文）Pilarita Tongol−Rivera，Mario Angelito
Elena Villacorte，Aldin Darilag，鈴木

Prudencio，Jesus Sarol，Felipe Balingit，守，狩野 繁之 …………一……・一

一487−495

タイで採集された新種ブユ（双翅目：ブユ科）4種の記載（英文）高岡宏行，Chaliow

Kuvangkadilok…一…・・……………・…・………・…・……・一…一・・497−509

ネパール村共同体における蠕虫感染の研究（英文）濱野真二郎，小林茂，大柿 哲朗，古賀 正崇，川崎 真澄，伊藤 和枝，
斉藤 篤司，辻守康，徳永 章二，Sashi Sharma，Gopal P．Acharya，川崎 晃一・・…・511−515
短報
カンボジア国プノンペン郊外トールロカ村における消化管寄生虫の感染状況（英文）小林潤，知念邦，Sim Samidt，比嘉 啓二，知念・真樹，佐藤 良也，
吉田 朝啓・

会報・記録
理事会・評議員会・会務総会記録………
熱帯医学会・国際保健医療学会共同宣言一
1999年度日本熱帯医学会役員名簿………

雑誌編集委員名簿………
投稿規定一…

一517−519

・・521−522

−523
−525
・・526−527
・・528−530

著作物複写に関する注意・…
第27巻総目次

…531

■

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Jpn. J. Trop. Med. Hyg., Vol. 27, No. 4, 1999, pp. 471 476

Review

PATHOLOGY AND PATHOPHYSIOLOGY
OF MALARIASHIGEYUKI KANol AND MASAMICHI AIKAWA2
Received June 14, 1999/Accepted June 21, 1999

Abstract: Pathological processes in malaria are the consequence of the erythrocytic cycle of the parasites.

Merozoites invade erythrocytes, in which they develop through early trophozoites (ring forms) to latetrophozoites and eventually to schizonts. During this process, development of knobs and cytoadherence orrosetting with the knobs play important roles for the falciparum malaria patient to be severely ill.
Expression of variant surface neoantigens stimulates the reticuloendothelial system and can cause anemia,
tissue hy oxia and cytokine production. Associated fever, paroxysms, headache and other pains are thought
to result from cytokines such as interleukins, interferons and tumor necrosis factor released from macro‑phages or other cells at the time of schizont rupture. In the present paper, pathological and patho‑
physiological changes mainly in human falciparum malaria are reviewed, ernphasizing the importance ofbasic research to "roll back" the emerging trends of malaria.
Key words: cerebral malaria. Plasmodium falciparum, pathology, pathophysiology

INTRODUCTION
Malaria remains the most important of the tropicaldiseases; in fact, it is re‑ernerging in areas where it was

pathology and pathophysiology of the disease.

ULTRASTRUCTURE OF THE PARASITIZED ERYTHROCYTE

once eradicated. Controlling malaria is proving to be

The Plasmodia merozoite is pear‑shaped andmore and more difficult with the increasing resistance ofapproximately 1.5 pm in length, with an apical end thathas a polar ring and rhoptries. Entry of the merozoite
into the erythrocyte begins when the apical end comesclose to the host cell membrane. It has been revealedparasites and mosquitoes to drugs and insecticides. The
world malaria situation was discussed in 1998 in Bir‑mingham, UK, among policymakers of the G8 nations,and it has remained one of the key issues in the G8development focus. The summit discussions, 3 days
before Gro Harlem Brundtland's election, enabled thethat the apex has a positive electrical charge facilitating

attachment to the negatively‑charged erythrocyte mem‑brane (Seed et al.. 1974). After the contact, the eryth‑leaders of the G8 countries to offer strong support forrocyte membrane is thickened and forms a junction with

the new WHO "Roll Back Malaria (RBM)" initiative,

the plasma membrane of the merozoite (Fig. 1‑a) .

announced on 13 May 1998 by Brundtland herself as the

Merozoite protein is released by the rhoptries and forms

new director general of WHO. The main emphasis ofa deep pit in the red blood cell. The merozoite thenenters the cell ̲maintaining a contact‑ring; i.e., thethe initiative is to strengthen health services so that
effective treatment and prevention strategies are acces‑
sible to all individuals who need them. It aims to apply
every existing tool effectively, building on experienceof the past‑failures and successes alike. An effective
vaccine should be one of the best means for controlling
malaria, and development of such a vaccine will depend
upon precise scientific knowledge of the disease. Basic
research must be stressed, particularly research into the12

moving junction formed between the thickened mem‑brane of the erythrocyte and the merozoite (Figs. 1‑band ‑c) (Aikawa et al., 1978). A small projection stillconnects the apical end of the merozoite and the eryth‑rocyte membrane (Fig. 1‑c) . When entry is complete,
the red cell mernbrane seals itself; the merozoite is then10cated within an invagination of the erythrocyte (Fig.
1‑d) .

Department of Appropriate Technology Development and Transfer, Research Institute, International Medical Center of Japan,
1‑21‑1 Toyama, Shinjuku, Tokyo 162‑8655, Japan
Research Institute of Science and Technology, Tokai University, Boseidai, Isehara, Kanagawa 259‑1193, Japan

472

(a)

(b

･ .+

Figure l Electron photomicrographs of longitudinal sections of merozoites entering into erythrocytes. (a)Contact between a merozoite and an erythrocyte. The dark part of the merozoite near the junctionis the rhoptry, (b) A merozoite just starting to enter into an erythrocyte. (c) A mera, zoite entering

into an erythrocyte at an advanced stage of the interiorization process. Note the moving junction
formed between the thickened membrane of the erythrocyte and the merozoite. (d) Completion of
the entry Of a merozoite int o an erythrocyte. The merozoite is left within a vacuole linecl with theerythrocyte membrane.

Merozoites develop in the erythrocytes from ring

ATOMIC FoRCE MICROSCOPY OF THE KNOB

forms to late trophozoites and eventually to schizonts.
During the late trophozoite stage, Plasmodium faJcipar ‑um (P.f ) ‑infected erythrocytes start to develop protu‑

ning, Quate and Gerber in 1986, has been used in biologi‑

berances or knobs on thecal reseal ch in recent years (Edstrom et al., 1990; Lalurface of their cell membrane.

Atomic force microscopy (AFM) , invented by Bin‑

Scanning electron micrography has demonstrated

and John, 1994). The advantage of AFM is that speci‑

numerous, blunt, cone‑shaped, protruding knobs evenly
distributed over the erythrocyte's entire surface (Aik‑mens can be observed, without chemical fixation, and the

awa et al., 1983). The same knobs have also been
observed on P. coatneyi‑infected Japanese macaqueface potential (Yokoyama and Inoue, 1994). When

attached surface potential spectroscope can detect sur‑

AFM was used to investigate the structure of the eryth‑erythrocytes (Fig. 2) (Kawai et a!., 1993) . It is now

rocyte knobs, each knob was found to consist of two

known that the knob consists of a series of antigenicallydistinct subunits, knob components that have never been

variant erythrocyte‑membrane proteins, such as

seen in chemically‑fi,xed knobs examined by conven‑

PfEMPl, which mediate binding of the infected eryth‑rocytes to vascular endothelium and uninfected redtional transmission electron microscopy (Fig. 3) . Sur‑blood cells (Fujioka and Aikawa, 19 96).

have a positive charge ( + 20 mV) , whereas the remain‑

face potential spectroscopy revealed that the knobs

473

ROSETTE FORMATION
Rosette formation or rosetting, the adhesion of non‑parasitized erythrocytes to parasitized erythrocytes, is aproperty exhibited in vitro and ex vivo in the rat
mesoappendix model by sorrLe strains of P.f and somesequestering animal rrLalarias (Fig. 4). It has been
proposed that rosette formation is a first step to eryth‑rocyte sequestration in blood vessels (Howard and
Gilladoga, 1989). An association betvveen human cere‑bral malaria and erythrocyte rosetting was describedbased on a study performed in The Gambia (Carlson etal., 1990), in which all isolates from patients with cere‑bral malaria formed rosettes and in which the mean
percentage of parasitized erythrocytes involved in roset‑
Figure 2 Scanning electron rrLicrograph of a P. coatneyi‑ting was t¥vice as high as in the isolates from patients

infected erythrocyte (reproduced courtesy of Dr. S.

with uncomplicated malaria. Similar findings were also

Ka vai, Department of Medical Zoology, Dokkyo
University School of Medicine). Compare thereported from a study in Thailand (HO et al., 1991) , andknobby surface of the infected erythrocytes vith

the smooth surface of surrounding non‑infected
erythrocytes.

der of the red cell plasma membrane is negatively

another investigation conducted in The Gambia showedthat giant rosettes formed more frequently in isolates
from patients with cerebral malaria than in isolates
from patients with uncomplicated malaria (Treutiger etal., 1992).

Scanning electron microscopy revealed that interac‑charged (Aikawa et aL, 1996). Since endothelial plasma
membranes have a negati¥'e charge, the difference intion between the rosetted erythrocytes and adiacentcharge between the knobs and endothelium may play auninfected erythrocytes appeared to be mediated by thesignificant role in cytoadherence between the two cell

knobs of parasitized red blood cells (Fig. 5) , and trans‑

types. A therapy that interferes with the localized

mission electron micrography showed the protruding

positive charge of the knobs will likely be the treatment

ends of these knobs to be attached to the membranes ofadjacent uninfected erythrocytes (Fig. 6) (Kawai et al.,of choice for severe falciparruTl malaria.

1995). How, then, do the knobs protrude? It is now
known that the binding protein (PfEMP‑1) in the knob
is encoded by members of the P.f v(2:r gene family and

Figure 3 AF 'I image of knobs on the surface of the eryth‑Figure 4 Light micrograph of rosette formation byrocyte in a three‑‑dimensional oblique perspective.

modium coatneyi‑infected erythrocytes

Whitish protrusions are knobs

Japanese macaque, Macaca fuscata.

cal charge.

vith positive electri‑

Plas ‑

of a

474

SSS:

̲ = "' >' ''*!;*'* **+'*<*' *'"' ': . " " "*" '= .

*"**'* '**'*

Figure 7 Light micrograph of a Giemsa‑stained stam p‑

smear from post‑mortem necropsy brain tissue
from a viCtim of cerebral malaria.

Figure 5 Scanning electron micrograph of a rosette consist‑ing of a central parasitized red blood cell surround‑

ed by several attached uninfected erythrocytes.

malaria have been described as edematous, but as it is,neither computed tomography (CT) nor nuclear mag‑
multiple var genes but, through a developmentally‑

netic resonance imaging (NMRI) can demonstrate cere‑bral edema in situ (Francis and Warrell, 1993). Of

regulated process, selects only one PfElvlP‑1 to reachcourse, studies of post‑mortem brain tissue limit under‑

the surface of the host cell (Chen et al., 1999).

standing of the pathology of cerebral malaria. To date,that a single P.f parasite simultaneously transcribes

A recent report on the formation of rosettes focusedon erythrocyte complement‑receptor I (CRl). It isreported that erythrocytes with a common African CR1
polymorphism, Sl(a ), have reduced adhesion to the

PfEMP‑1 binding domain, which might have resulted
from selection for malaria resistance in highly endemicareas (Rowe et al., 1997).

PATHOLOGY OF CEREBRAL MALARIAThe brains of some patients who died from cerebral

several pathologic changes have been described, andcommon to all these is the presence of sequesteredparasitized erythrocytes undergoing schizogony within
microvessels of the brain (Fig. 7). Investigators havereported that the severity of falciparum malaria inhumans is proportional to the degree of sequestration(Pongponratn et al.. 1991) and not necessarily related toperipheral circulatory parasitemia. The reduced defor‑
mability of the parasitized erythrocytes together with
their cytoadherence to endothelium and to nonparasit‑
ized cells (rosetting) must lead to impaired blood flow.

This causes a reduced supply of oxygen and othernutrients to the brain, causing coma. Histological evi‑
dence of local inflammatory response to these seques‑
tered vessels is minimal. Infarction, necrosis and large

hemorrhages are also rare. However, in many patients,
focal ring hemorrhages are seen centered in small sub‑cortical vessels. The pathogenesis of this hemorrhaging
has not been clarified, but it may relate to increased
capillary permeability.

Electron microscopy has shown that sequestrationi'> *'*' *' "= *'* :'*< '=f ; '* " *'****

Figure 6 TransmiSsion electron micrograph of a parasitiZed
red blood cell in a rosette attached to an adjacenterythrocyte bv electron‑dense knobs protrudingfrom the membrane'of parasitized erythrocytes in microvessels is mediatedby the knobs (Fig. 8) (Aikawa, 1988; Igarashi et al..

1987). As mentioned above, the molecular basis forcytoadherence is currently of great interest and impor‑tance, particularly because of the possibility of prevent‑ing this pathogenic process by competitively binding475

African children with cerebral malaria (Newton et al..
1991) , which could result from raised intracranial blood

volume caused by vasodilation with seque .tration ofparasitized erythrocytes.
The cytokine hypothesis is based on the observationthat plasma concentrations of tumor necrosis factor(TNF)

c!, interleukin (IL) ･1, IL6 and other cytokines

correlate with disease severity (Kwiatkowski et al..
1990). Cytokines 1 eleased by macrophages and othercells at the time of schizont rupture are thought to be
involved in enhancing endothelial cell adhesiveness byincreasing the ex,pression of endothelial receptors suchas ICAM‑1 or CD36. The cytokines can also induce
Figur, e 8 Elect.ron micrograph showing cy toadherence ofparasitized erythrocytes to endothelial cells of acerebral microvessel mediated by electrondense
protruding knobs.

fever, hypoglycemia, coagulopathy, dyserythropoiesis
and leucocytasis (Francis and Warrell, 1993).

Cytokines such as IL‑‑1, TNF and lymphotoxin(LT) are reported to be the inducers of nitric oxide(NO) , also known as endothelial‑derived relaxing fac‑

tor (EDRF). It is possible that NO contributes inantibodies to the adhesion molecule in the knobs oragainst receptors on the endothelial cells. The main
candidate receptors are CD36, thrombospondin (TSP),
intercellular adhesion molecule‑1 (ICAM‑1), endoth‑elial‑1eukocyte adhesion molecule‑1 (ELAM‑1) andvascular cell adhesion molecule‑1 (VCAMl) (Aikawa
et al., 1990). Ilowever, it is quite hard to prove that

cerebral malaria, through vasodilation of cerebral ves‑sels, to an acute increase in cerebral blood volume,
leading ta an increase in intracranial pressure and subse‑

quent coma. If vasodilation is caused systemically byincreased NO (EDRF generation, disease severity will
be marked by systemic hypotension. It is also possiblethat NO derived from local endothelial cells diffuses

these molecules are actually playing a role in the cytoad‑across rrLembranes to influence adjacent neurons as if itherence of parasitl,'zed erythrocytes in vivo, because ofbral malaria patients. Appropriate experimental animal
madels for human cerebal malaria are thus needed and,were coming from a nearby synapse, disrupting local
neurotransmission and thus interfering with neur=
ological function (Clark et al., 1991). A field study
conducted in Papua New Guinea revealed an association

to date, P. coat eyiinfected rhesus monkeys andbctween high NO Ievels and disease severity in children

Japanese monkeys have been reported as good models

with cerebral malaria (AI Yaman et al., 1996). How‑ever, report fr, om Tanzania'showed NO Ievels to be

the difficulty of obtaining fresh tissue from living cere‑(Aikawa et al., 1992; Kawai et al., 199'.i,).

PATHoPHYsroLOGY Or CEREBRAI. MALARIA
There is increasing evidence in, favor of the mechan‑

inversely related to diseas severity, suggesting a pra‑
1 tective role (Anstey et al., Ig 6) . The role of NO is now
a subject of hot contr, oversy (Holden, 1996) and will bea major focus of upcoming research.

ical hypothesis described above. However, mostpatients with severe falciparum infection who haverecovered from cerebral malaria show no persistentneurological sequelae, which suggests that much of thepathology must be transient and reversible. Therefore,
several hypotheses an the events following sequestration
have been proposed to explain the development of cere‑bral malaria.
One of these is the permeability hypothesis, whichsuggests that increased cerebral vascular permeability
causes leakage of plasma across th,e blood‑brain bar‑rier, cerebral edema and eventual coma. Elevated cere‑

brospinal fluid (CSF) pressure has been reported in

ACKNOWLEDGEMENTS
This work was partially supported by two Grants‑

inAid for Scientific Research on Priority Areas(08281103, Ill47235) from the Ministry of Education,
Science, Culture and Sports; and a (}rant for Interna‑

tional Health Cooperation Research (11A‑1) from theministry of Health and Welfare,

REFEREN CESl ) Aikawa, M. (1988): Human cerebral m'alaria.

Am. J.

476

Trop. Med. Hyg., 39, 3‑102 ) Aikawa, M.. Brown. A., Smith, C.D., Tegoshi, T.,
Howard, R.J., Hasler, T.H., Ito. Y., Perry, G., Collins, W.

14) Fujioka. H. and Aikawa, M. (1996): The molecularbasis of pathogenesis of cerebral malaria. Microb.
Pathog., 20, 63‑72

E. and Webster, K. (1992) : A primate model for humancerebral malaria: Plasmodium coatneyi‑infected rhesus
monkeys. Am. J. Trop. Med. Hyg., 46, 391‑397
3 ) Aikawa, M., Iseki, M., Barnwell, J.W., Taylor. D.. Oo,

15) Ho, M., Davis, T.M.E., Silamut. K.. Bunnag, D. andWhite, N.J. (1991): Rosette formation of Plasmodium
falciparum‑infected erythrocytes frorn patients with

M.M. and Howard, R.J. (1990): The pathology of16) Holden, C. (1996) : NO Iinked to malaria immune

human cerebral malaria. Am. J. Trop. Med. Hyg., 43,
30‑374 ) Aikawa, M., Kamanura, K., Shiraishi, S., Matsumoto,Y., Arwati, H., Torii, M., Ito, Y.. Takeuchi, T. andTandler, B. (1996): Membrane knobs of unfixed Plas‑
modium falciparum infected erythrocytes: new findings
as revealed by atomic force microscopy and surface
potential spectroscopy. Exp. Parasitol., 84, 339‑343
5 ) Aikawa. M., Miller. L.H., Johnson, J. and Rabbege, J.

(1978): Erythrocyte entry by malaria parasites: amoving junction between erythrocyte and parasite. J.
Cell. Biol., 77, 72‑82

6 ) Aikawa. M., Rabbege. J.R., Udeinya, I. and Miller, L.H.

(1983) : Electron microscopy of knobs in Plasmodium
falciparum‑infected erythrocytes. J. Parasitol., 74, 435‑

4377 ) A1 Yaman, F.M., Mokela. D.. Genton, B., Rockett, K.A.,

Alpers, M.P. and Clark, I.A. (1996) : Association
between serum levels of reactive nitrogen intermediates

acute malaria. Infect. Immun., 59, 2135‑2139
response. Science, 273, 1051

17) Howard. R. and Gilladoga, A.D. (1989): Molecularstudies related to the pathogenesis of cerebral malaria.
Blood, 74, 2603‑2618
18) Igarashi, I., Do, M.M., Staney, H., Reese, R. and Aikawa,M. (1987): Knob antigen deposition in cerebral malaria.

Am. J. Trop. Med. Hyg., 37, 511‑515
19) Kawai, S., Aikawa, M., Kano, S. and Suzuki, M. (1993):

A primate model for severe human malaria with cere‑bral involvement; Plasmodium coatneyi‑infected Macaca
fusucata. Am. J. Trop. Med. Hyg., 48, 630‑63620) Kawai, S., Kano, S. and Suzuki, M. (1995): Rosetteformation by Plasmodium coatneyi‑infected eryth‑rocytes of the Japanese Macaque (Macaca fuscata) . Am.
J. Trop. Med. Hyg., 53, 295‑299
21) Kwiatkowski, D., Hill, A.V.S., Sambou, I., Twumasi, P.,
Castracane. J., Manogue, K.R., Cerami. A., Brewster, D.

R. and Greenwood, B.M. (1990): TNF concentration in
fatal cerebral, non‑fatal cerebral, and uncomplicatedPlasmodium falciparum malaria. Lancet, 336, 1201‑1204

and coma in children with cerebral malaria in Papua
New Guinea. Trans. R. Soc. Trop. Med. Hyg., 90, 270‑

22) Lal, R. and John. S.A. (1994): Biological applications of2738 ) Anstey, N.M., Weinberg, J.B., Hassanali. M.Y., Mwai‑

atornic force microscopy. Am. J. Physiol., 266, C1‑C2123) Newton, C.R.. Kirkham, F.J., Winstanley, P.A., Pasvol,

kambo, E.D., Manyenga, D., Misukonis, M.A., Arnelle,
D.R., Hollis, D., McDonald, M.1. and Granger. D.L.

G., Peshu, N., Warrell, D.A. and Marsh, K. (1991):(1996): Nitric oxide in Tanzanian children withmalaria: inverse relationship between malaria severity
and nitric oxide production/nitric oxide synthase type 2
expression. J. Exp. Med., 184, 557‑567
9 ) Carlson, J., Helmby, H., Hill, A.V.S.. Brewster, D.,

Greenwood, B.M. and Wahlgren, M. (1990): Human
cerebral malaria: association with erythrocyte rosetting
and lack of anti‑rosetting antibodies. Lancet, 336, 1457‑

1460
10) Chen, Q., Fernandez, V.. Sundstrom, A., Schlichtherle,M., Datta, S., Hagblom, P. and Wahlgren, M. (1999):
Developmental selecti,on of var gene expression in
Plasmodium falciparum. Nature, 394, 392‑395
11) Clark, I.A., Rockett, K.A. and Cowden, W.B. (1991):
Proposed link between cytokines, nitric oxide andhuman cerebral malaria. Parasitol. Today, 7, 205‑207
12) Edstrom, R.D., Yang, X., Lee, G. and Evans, D.F. (1990):

Viewing molecules with scanning tunnelling microscopy
and atomic force microscopy. FASEB. J., 4, 31‑44
13) Francis, N. and Warrell, D.A. (1993): Pathology andpathophysiology of human malaria. Im: Bruce‑Chwatt's essential malariology, 3rd ed. pp. 50‑59. Edward
Arnold, London

Intracranial pressure in African children with cerebral
malaria. Lancet, 337, 573‑576

24) Pongponratn, E., Riganti. M., Punpoowong, B. andAikawa. M. (1991): Microvascular sequestration inparasitized erythrocytes in human falciparum malaria: apathological study. Am. J. Trop. Med. Hyg., 44, 168‑175
25) Rowe, J.A., Moulds, J.M., Newbold, C.1. and Miller, L.H.
(1997): P. falciparum rosetting mediated by a parasite‑variant erythrocyte membrane protein and complement‑
receptor 1. Nature, 388, 292‑295
26) Seed. T.M., Aikawa, M., Sterling, C. and Rabbege, J.
(1974): Surface properties of extracellular malaria

parasites: morphological and cytochemical study.
Infect. Immun., 9, 750‑761
27) Treutiger, C.J.. Hedlund, I., Helmby, H., Carlson, J.,
Jepson, A., Twumasi, P., Kwiatkowski, D., Greenwood,
B.M. and Wahlgren. IVl. (1992): Rosette formation in
Plasmodium falciparum isolates and anti‑rosette activityof sera from Gambians with cerebral or uncomplicatedmalaria. Arn. J. Trop. Med. Hyg., 46, 503‑510

28) Yokoyama, H. and Inoue. T. (1994): Scanning Maxwell
stress microscope for nanometre‑scale surface electros‑
tatic imaging of thin films. Thin Solid Filrns, 242, 33‑39

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J pn. J. Trop. Med. Hyg., Vol. 27, No. 4, 1999, pp. 477 481

ANTIFILARIAL EFFECT OF ARTEMISIA NILAGIRICA
EXTRACT AND ITS ULTRA HIGH DILUTIONSAGAINST CANINE DIROFILARIASISNIRMAL CHANDRA SUKUL, PAROMITA SARKAR, ANIRBAN SUKULAND SANTI PRASAD SlNHABABU
Received May 12, 1999/Accepted June 21, 1999

Abstract: An ethanolic extract of the flowering meristems of worm wood, Artemisia nilagirica was allowed
to evaporate. The residue, thus obtained, was administered orally on 4 pariah dogs naturally infected with

Dirofilaria immitis at 10 mg/kg/day for 15 days and then at 20 mg/kg/day for the next 15 days. Twohomoeopathic potencies of the A. nilagirica extract, called Cina 200 and Cina 1000, were obtained commer‑
cially and administered orally at 0.1 ml/dog/day for 30 days on two separate batches, each consisting of 4

dogs. Blood was sampled frorn the dogs before treatment and on day 15, 30, 45 and 75 following thetreatment. A. nilagirica extract (Cina e) was diluted with 90% ethanol 1:100 and shaken by 10 manualstrokes to prepare the Ist potency, called Cina 1. A11 subsequent potencies were prepared by mixing I partof the preceding potency with 99 parts of 90% ethanol and giving the 'mixture 10 manual strokes. Cina O,
Cina 200 and Cina 1000 reduced microfilarial densities in treated dogs by 78.38, 63.06 and 71.40%, respectively
on day 30. There were 57.13, 42.44 and 64.20% reduction on day 75. No apparent toxic effect was observedin the treated dogs. Electronic spectra of Cina e, Cina 200 and Cina 1000 showed cornparable absorbancewith the latter two giving a blue shift. Cina O in CC1, showed a red shift suggesting molecular complexationand charge transfer (CT) interaction between aqueous ethanol and compounds of A. nilagirica. CT was
further evidenced by the NMR spectra of the deuterium nuclei of Cina O in 90% ethanol. NMR spectra of
Cina e, Cina 200, Cina 1000 and 90% ethanol indicated a change in the solution structure of Cina 200 and Cina
1000. This altered solution structure is thought to be responsible for inducing irnmune reaction of the hosts
against the parasite.
Key words: Artemisia nilagirica. Antifilarial, Dirofilaria immitis. Homoeopathic potency, Ethanol solution

structure, Electron transfer

to one part of the preceding potency 99 parts of 90%

INTRODUCTION

ethanol and shaking the mixture in a similar way

Species of Artemisia have long been used by rural(Anonymous, 1920; Sukul and Klemm, 1988). Effective

people for expelling intestinal nematodes (Singh et al..

homoeopathic potencies could also be produced by

1983). High dilutions of the ethanolic extract of theflowering meristems of Artemisia nilagirica (Clarke)sonication instead of mechanical agitation (Sukul et al..
1996; Sukul, 1997) . Two potencies of Cina, Iike Cina 200

Pamp have been used in the Homoeopathic system ofand Cina 1000, purchased from King & Co., Calcutta,

medicine under the name Cina against intestinal worms

were used. Since these potencies are too dilute to have

(Kent, 1911; Boericke, 1927) . The purpose of the present
study is to see whether this plant extract, both in its
crude form and also its homoeopathic dilutions, called

them were obtained to find out their difference frorn the

any drug molecules, electronic and NMR spectra of

In Homoeopathy the ethanol extract of A. nilagiri‑ca, called Cina e, is diluted with 90% ethanol 1:100 andsolvent medium like 90% ethanol vis‑a‑vis the physicalbasis of their effectiveness. In order to find out thesolvent effect on the solute, we prepared Cina O in aneutral solvent like CC1+ and obtained the electronic

the mixture is shaken with 10 powerful downward

spectra of the solution.

potencies, is effective against canine dirofilariasis.

strokes to prepare the first centesimal potency called
Cina 1. A11 subsequent potencies are prepared by adding
Department of Zoology, Visva Bharati University, Santiniketan 731 235, West Bengal, India
Fax. 0091346353166

478

MATERIALS AND METHODSTreatment with Cina e
Cina e, purchased from King & Co., Calcutta wasallowed to evaporate in an incubator at 40'C. The
residue was dehydrated in a vacuum dessicator overanhydrous Calcium Chloride and stored at 4'C. Blood
was sampled from 4 naturally infected dogs, 2 males and2 females, every 15 days for a period of 2 months andmicrofilarial concentration per ml of blood was deter‑

MHZ at 22'C. The mechanism by which excess spinenergy of a nucleus (here 2H) is shared with the sur‑roundings is referred to as the spin lattice relaxation.

The time taken for a fraction 1/e=0.37 of the excessenergy to be dissipated is called the relaxation time.

Such relaxation comes about by lattice motions likemolecular tumbling in liquids (Banwell and McCash,1994). Deuterium is a quadrupolar nucleus having asmall quadrupole moment 1. Quadrupolar relaxation
depends upon the interaction of the electric quadrupole

mined. Blood film was allowed to dry, dehaemo‑

moment with an electric field gradient. If the qua‑globinised in distilled water and stained with Giemsa

drupole moment is small, as it is for 2H, the interaction

stain. The same dogs were then administered orally

is small and the relaxation will be slow. Like allwith the residue of Cina e at 10 mglkg body weight/day

quadrupolar nuclei its relaxation is sensitive to 71*. 7:, isfor 15 days. Blood was sampled from the dogs on day
15. The same dogs were treated again orally with theresidue at 20 mg/kg/day for 15 days more. Blood was

the average time taken to rotate through I radian orroughly the reciprocal of the rate of tumbling of therelevant piece of the molecule (Sanders and Hunter,1993). T* values of 2H of water, hydroxyl, methylene
and methyl groups of ethanol were measured from thestacked spectra with the help of a computer.

sampled on day 30, 45 and 75. Capsules were filled withthe residue, kept inside a loaf of bread and then offeredto the microfilaraemic dogs.

Treatment with Cina 2ov and Cina 1000
Blood was sampled from a batch of 4 infected dogs,
2 males and 2 females, every 15 days for 2 months. Cina

200 was mixed with pure cow milk at 0.1 ml/4 ml ofcow milk and 4 ml of the mixture was offered in a glass
plate to a dog which immediately consumed the mixture.
The schedule of treatment and blood sampling were thesame as with Cina e. However, the dosage of Cina 200

was same in the 2 phases of treatment. A batch of 4
naturally infected dogs (3 females and I male) was

RESULTS
Treatment effect on mf count
The mean microfilarial counts per ml of blood in 3
batches of dogs before treatment are shown in Table 1.
The microfilarial concentration in each dog did not vary

appreciably during the 2‑month period of observation.
Table I Microfilarial concentration/ml blood of dogs natu‑
rally infected with Dirofilaria immitis before treat‑

ment

treated with Cina 1000 after determining the mf densityfor 2 months. The treatment schedule, dosage of the
drug and blood sampling were same as with Cina 200.

Microfilarial concentration/ml blood on different days

Dogs

Day O Day 15 Day 30 Day 45 Day 60

Batch IElectronic spectra of drug

Using a UV‑VIS spectrophotometer (Beckman DU640) absorption spectra of Cina O in 90% ethanol andCina e in carbon tetrachloride, Cina 200 and Cina 1000
against the corresponding solvent blanks were obtainedin the wave length range of 190L750 mm at 29'C. The
spectra were run in matched quartz cuvettes and werecorrected for instrumental baseline errors. Test solu‑
tions were kept at the above temperature for at least 10

min to allow for the thermal equilibration.

NMR Spectra of drugs
The spin‑1attice relaxation time (T* in msec) of thenaturally abundant 'H (0.015%) was measured in 90%
ethanol, Cina O in 90% ethanol, Cina 200 and Cina 1000

using a AMX‑400 NMR spectrometer operating at 61.4

Male I , OO1

Male 437

Female 727
Female 813

973
423
798
852

945
480
756
808

997
505
704
826

986
440
692
874

241
298
477
374

264
253
542
385

313
221
580
423

274
234
569
450

235
287
269
275

252
259
254
244

300
247
219
235

302
272
203
279

Batch II

Male 219
Male 304

Female 535

Fernale 330
Batch 111

Male 274

Female 236
Female 218
Female 251

Batch I: Treated later with Cina e
Batch II: Treated later with Cina 200
Batch 111: Treated later with Cina 1000

479

l OO

4. o

1'

u!:

Cina e in CCI*

'l

:1 80

'a

h

e

1::

e
,,]

:

S
'l
e,
c,

J5

<

0.0
4.0

lle 60
a,

,.

E

8

.S

l:

El

,e

Cina e in 90'/e ethanol

el

40

he
E,,

<

l

e

20

o. o

3,0

o

l 5 30 4S

o

,‑,

7s

S
s
e,

Cina 200

J:

ce

<

Days of blood sampling

Figure 1 Percentage of reduction in microfilarial concentra‑tion of D. immitis in 4 dogs treated with ethanol
extract of Artemisia nilagirica (Cina e) at 10 mgl
kg/day for 15 days and 20 mg/kg/day for the next

15 days (‑O‑). The same in 2nd batch of 4 dogs
treated with Cina 200 for 30 days (‑[]‑) and in
3rd batch of 4 dogs treated with Cina 1000 for 30days (‑A‑).

0.0
3,0'

,,

U

S,

da
!

e

Cina 1000

ta

a<
0.0

190.0

Percentage changes in microfilarial concentration for
the treated dogs were plotted in a graph against days ofsampling in Fig. 1. The mean mf density just before
treatrnent of the 3 treatment groups served as the stan‑

Wavelength (nm)750.0

Figure 2 Absorption spectra of Cina e (ethanol extract offlowering meristems of A. nilagirica) and its two
ultra high dilutions called Cina 200 and Cina 1000,
all in 90% ethanol in wavelength range of 190‑750
nrn. The sarne of Cina O in CC1, showing a red shift

dard with respect to which the percentage change wasas compared to Cina e in 90% ethanol. Cina 200
and Cina 1000 show a blue shift. The absorbance

Table 2 Spin‑1attice relaxation time (T1) of 2H of 90%
ethanol, Cina e in 90% ethanol, Cina 200 and Cina

intensities in all the 4 are fairly comparable.

1000. Cina e is the ethanolic extract of A. nilagi‑

rica flowering tops. The two potencies, Cina 200
and Cina 1000, were prepared by successive dilu‑
tion of Cina e with 90% ethanol 1:100 and manual

succussion. Measurements were taken by a AMX‑400 NMR spectrometer operating at 61.41 MHZ at
22'C

Mean S.E. of Tl (m sec)

Ethanol 90%(Fig. 1) .

OH

CH2

CH3

106 . 9 110 . 8 846 . 5 822 . 5

0.5a i0.8a

0.4a :!:0.5a
883 . 4 776 . 1

Cina e in 90%

104 . 3EtOH

0.3b 0.4b
108 . 2 102 . 5 792 . 7
0.4b

867 . 4

Cina 200

0.7c

Cina 1000
a, b, c, d::!:1.7c
0.8b
0.3c

106 . 6 86 . 8 971 . 7 857 . 6:!:0.8a 0.9c 2.4d 2.3d

Significant difference (P<0.01) by ANOVA (oneway) in a column.

maximum reduction was 78.4% with Cina e, 63.1% withCina 200 and 71.40% with Cina 1000. The reduction was
42.4‑64.2% on the last day of sampling i.e. day 75

Ethanol

WaterOH

calculated in subsequent samples. The mf density
showed marked reduction in the treated dogs. The

Electronic spectraElectronic spectra of Cina O in 90% ethanol, Cina e

in CC14, Cina 200 and Cina 1000 are given in Fig. 2. The

absorption spectra of Cina O in ethanol shows a blue
shift as compared to that of Cina O in CCl+ (Fig. 2).
Absorption spectra of Cina 200 and Cina 1000 show a

blue shift as compared to Cina e in ethanol. The
absorbance was fairly comparable in the tincture and its
two potencies (Fig. 2).

480

NMR Spectra
and CH3 of 90% ethanol, Cina O in 90% ethanol, Cina 200
and Cina 1000 are presented in Table 2. The T1 values

forward transfer in a complex fashion (Weidemaier andFayer, 1996). Thus the electronic configuration of thedonor molecules, i.e. aqueous ethanol, would undergo achange according to the nature of the electronic accep‑

were compared by ANOVA. Tl of OH of water wastors of Artemisia extract. With successive dilution, the10west with Cina e and highest with Cina 200. T1 ofethanol hydroxyl was absent in Cina O, highest in 90%

acceptor molecules are progressively depleted and fresh
molecules of the donor occupy their place. Finally, it isethanol and lowest in Cina 1000. T1 of CH2 Was highest

the molecules of aqueous ethanol which exist in the formin Cina 1000 followed by Cina O, Cina 200 and 90%

of ethanol molecules surrounded by the hydration shellof water molecules.

The T* values with S.E. of 2H of water, OH, CH2ethanol. Tl of CH3 Was highest in Cina 1000. This wasfollowed by 90% ethanol, Cina 200 and Cina O in decreas‑
ing order (Table 2) .

DISCUSSIONUV spectroscopy is highly sensitive to the distortionof chromophores and auxochromes (Banwell and
McCash, 1994). The UV spectra of Cina 200 and Cina
1000 show a blue shift as compared to that of Cina O
thereby suggesting a possible change in the electronic
configuration of the medium, i.e. aqueous ethanol (Fig.
2). The altered T* values of the deuterium nuclei of
Cina 200 and Cina 1000 as compared to those of aqueous
ethanol and Cina e (Table 2) suggest that the rate of

It is evident from the results that both the crude
extract as well as the two potencies of A. nilagirica
proved highly effective against the microfilariae ofDirofilaria immitis in dogs. Species of Artemisia arereported to contain various types of essential oils andtumbling in the relevant parts of the molecule in potent‑

sesqueterpene lactones including santonin (Carnat et al..
1992; Rucker et al., 1992; Nin et al.. 1995; Todorrova andized Cina has undergone a change obviously due to CT
interaction and mechanical agitation (Sukul, 1999).

Krasteva, 1996). These compounds in the crude extract

Haseba et al. (1993) reported that the thermal motion of

may have a direct effect on the microfilariae. The

water molecules in sonicated aqueous ethanol was

effect of the two potencies, which have no drug mole‑cules, can be explained in a different way. Electronic
spectra of Cina O in 90% ethanol and that in CC14 aregreater than that in unsonicated one, and this change inthe solution structure produced significant biologicalef f ects.

different with the former showing a blue shift as

Living microfilariae of D. immitis, injectedcompared to the latter. This suggests molecular com‑
plexation and charge transfer (CT) interaction between
the compounds of A. nilagirica and ethanol molecules.
Here ethanol molecules served as electron donors andArtemisia compounds as electron acceptors (Singh andDikshit, 1995). Alcohols donate electrons from thehighest occupied nonbonding, n (bl), orbital of theintravenously, disappeared rapidly from the peripheral
circulation of uninfected dogs, which had received andcleared previous infections of microfilariae. Eosino‑philia and antibodies to microfilaria were demonstrable
in the blood of the dogs (Wong, 1964, 1966 cited by Wongand Guest, 1969). Filarial worms are known to cause
immunosuppression (Ottesen, 1980). It is possible that

Oxygen atom to the ,t orbital of the acceptors in

potentized Cina might have removed immunosuppres‑

Artemisia (Frey et al.. 1994). Absorption occurs wellinto the near UV region. CT is further evidenced by theabsence of T* value at the ethanolic hydroxyl site ofsion resulting in vigorous responsiveness of the host to

Cina e (Table 2). Relaxation at this site was too
efficient and the n.m.r. signal was too broad to beobserved (Banwell and McCash, 1994). We haveparasite antigens thereby clearing microfilariae fromthe blood.

ACKNOWLEDGEMENT

already observed CT interaction in other potentized

We are thankful to the Director, Sophisticatedhomoeopathic drugs like lodine and Nux vomica

Instruments Facility, Indian Institute of Science, Ban‑galore for providing the NMR data of the sarnples. The
electronic spectra of the samples were obtained through(Sukul, 1999). Other plant extracts such as tea containa mixture of potential complexing agents which form
molecular complexes with other compounds (Hernaez
et al.. 1997). For intermolecular electron‑transfer sys‑tems, a number of competing acceptors may exist, as in
Artemisia extract, in a complicated spatial array about
the donor. The back transfer process is coupled to thethe courtesy of Professor Shelley Bhattacharyya andher research scholar Srn. Rakhi Ghosh of our depart‑
ment.

481

REFEREN CES

WHO/FIL/80, 160‑168
11) Rucker. G.. Manns. D. and Wallbert. S. (1992):

1 ) Anonymous (1920): The American Homoeopathic Phar‑

Homoditerpene peroxides from Artemisia absinthium.

macopoeia, 9th ed., Boericke and Tafel, Philadelphia.
2 ) Banwell, C.N. and McCash, E. (1994): Fundamentals ofmolecular spectroscopy, 4th ed., Tata Mc Graw Hill
Publishing Company Ltd., New Delhi.
3 ) Boericke, W. (1927): Pocket Manual of Homoeopathic
Materia Medica. Indian ed. (1976). Sett Dey, Calcutta.

Phytochemistry, 31, 340‑342

12) Sanders, J.K.M. and Hunter, B.K. (1993): Modern NMR
Spectroscopy. Oxford University Press, Oxford.

13) Singh, P.R. and Dikshit, S.K. (1995): Molecular
Spectroscopy, principles and chemical applications. S.
Chand and Co. Ltd., New Delhi.

4 ) Carnat, A.P.. Madesclaria, M. and Chavignon. O. (1992):
Cis‑Chrysanthenol, a main component in essential oil of14) Sirigh, U.. Wadhwan, A.M. and Johri, B.M. (1983):

Artemisia absinthium L. growing in Average (Massif

New Delhi.
15) Sukul, N.C. (1997): High Dilution Pharmacology and
Homoeopathy. A. Sukul, Santiniketan, West Bengal.

CentraD, France. J. essent. oil res., 4, 487‑490

5 ) Frey, J.E., Aiello, T., Beaman, D.N., Combs, S.D., Fu,
Shi‑1in and Puckett, J.J. (1994): Charge transfer com‑
plexes of tetracyanoethylene with alkyl, alkenyl, andaryl derivatives of Oxygen. J. Org. Chem., 59, 1817‑1830
6 ) Haseba, T., Matsushita, K., Asakura, T., Kameyama, K.,
Tamaki, T., Okouchi, S., Watanabe. T. and Uedaira, H.
(1993) : Diminution of biological reactivity of ethanol bychanging the solution structure by weak ultrasonication.
Alcohol. Clin. Exp. Res., 17, 963‑967

7 ) Hernaez, J. Meirong, Xu and Daswood, R. (1997):
Effects of tea and chlorophyllin on the mutagenicity of
N‑Hydroxy‑IQ: Studies on enzyme inhibition, molecularcomplex formation, and degradation/scavenging of theactive metabolites. Environ. Mol. Mutagen, 30, 468‑474

8 ) Kent, J.T. (1911): Hornoeopathic Materia Medica.
Indian ed. (1962) , Sett Dey, Calcutta.
9 ) Nin, S., Arfailoi, P. and Boretto, M. (1995): Quantita‑
tive determination of some essential oil components of
selected Artemisia absinthium plants. J. essent. oil res.,7, 271‑277

10) Ottesen, E.A. (1980): The clinical spectrum of lym‑
phatic filariasis and its immunological determinants.

Dictionary of economic plants in India, 2nd ed., ICAR,

16) Sukul, N.C. (1999): Electron transfer interaction retains

molecular specificity of drugs at ultra high dilutions.
Environ. Ecol. (in press)

17) Sukul, N.C. and Klemm, W.R. (1988): Influence of
doparnine agonists and an opiate antagonist on Agaricus
induced catalepsy, as tested by a new method. Arch. Int.
Pharmacodyn. Ther., 295, 40‑51
18) Sukul, N.C.. Ghosh. S. and Sinhababu, S.P. (1996): Dosedependent suppression of haloperidol induced catalepsy
by potentized Agaricus muscarius. Br. Hom. J., 85, 141‑
144
19) Todorrova, M.N. and Krasteva, M.L. (1996): Sesqueter‑
pene lactones from Artemisia lerchina. Phytochemistry,
42, 1231‑1233
20) Weidernaier, K. and Fayer, M.D. (1996): Role of diffu‑

sion in photoinduced electron transfer on a micellesurface: theoretical and Monte Carlo investigations. J.
Phys. Chem., 100, 3767‑3774
21) Wong, M.M. and Guest, M.F. (1969): Filarial antibodies

and eosinophilia in human subjects in endemic area.
Trans. Roy. Soc. Trop. Med. Hyg., 63, 796‑800483

Jpn. J. Trop. Med. Hyg., Vol. 27, No. 4, 1999, pp. 483 486

DETECTION OF CIRCULATING WUCHERERIABANCROFTI ANTIGEN, FILARIA SPECIFIC IGG AND
IGG4 IN CHYLURIA CASES IN JAPAN
MAKOTO ITOHl, XU‑GUANG QlUl, YUZO KOYAMA2, YOSHIHIDE OGAWA2,MIRANl V. WEERASOORIYA3, HANESANA VISANOUl, YASUNORI FUJIMAK14 AND
EISAKU KIMURAl
Received May 25, 1999/Accepted August 3, 1999

Abstract: Serum sarnples from Japanese chyluria patients were examined for filaria specific antibodies anda circulating filarial antigen in order to know if the symptom was filarial in origin. A11 the sera were
negative for the circulating antigen. Anti‑Brugia pahangi antibodies were detected in 6 out of 16 serum
samples by ELISA after absorption of the sera with Anisakis and Dirofilaria immitis antigens. One of thepositive sera showed a high titer for anti‑B. pahangi lgG4, suggesting that Wuchereria bancrofti adults were
surviving in the patient in recent years. Detection of antibodies would be helpful for immunodiagnosis offilarial chyluria in Japan, where filarial origin is often determined based simply on the history of residence
in the past endemic areas.
Key words: Wuchereria bancrofti, chyluria, immunodiagnosis, IgG4, circulating antigen

MATERIALS AND METHODSINTRODUCTION
Filariasis caused by Wuchereria bancrofti was once
a common parasitic disease in Japan, especially in its
southern parts. In 1962, the national filariasis control

Serum samples:

Serum samples were obtained in 1994 from 16

disappeared in Japan (Kobayashi, 1994). Twenty years

chyluria patients and kept at ‑ 40'C until use. A11 thepatients were born and brought up in Okinawa Islands,
where W. bancrofti infection was highly endemic. Infor‑
mation on the patients is shown in Table 1.
Fourteen sera from healthy people living in central
parts of Japan were used as healthy controls, and 8 sera

after eradication of filariasis, chyluria cases are stillfrom Sri Lankans who were known mf carriers of W.

encountered (Yagi et al., 1998; Sakakura et al.. 1996; Itoet al.. 1994; Kawahara et al.. 1992; Koyama et al.. 1990) .

bancrofti were used as positive controls.

In many cases of these reports, the illness was diagnosed

Detection of W. bancrofti circulating antigen:program was launched and an extensive treatment cam‑paign using diethylcarbamazine and mosquito control
measures were carried out (Sasa, 1976). By the end of
1970's, microfilaria (mf) carriers were believed to haveor suspected as filarial chyluria simply based on

An enzyme‑1inked immunosorbent assay (ELISA)

patients' history of residence in the past endemic areas.
In this study, a circulating W. bancrofti antigen, filaria

for the detection of W. bancrofti circulating antigen was

specific lgG and lgG4 in the sera of chyluria patients

carried out using a commercially purchased kit (Trop‑
Ag W. bancrofti. JCU Tropical Biotechnology Pty Ltd.,

were measured in order to obtain immunological evi‑

Australia), which is a sandwich ELISA using Og4C3

dence for the filarial origin, and to establish an im‑
munological method useful to make a diagnosis of filar‑

monoclonal antibody to capture the antigen. The kit has

ial chyluria.

been tested worldwide and its high sensitivity andspecificity have been established (More and Copeman,1990) .

1

2

3
4

Department of Parasitology, Aichi Medical University, Nagakute, Aichi 480‑1195, Japan
Department of Urology, University of the Ryukyus School of Medicine, Nishihara, Okinawa 903‑0215, Japan
Department of Papasitology, Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka
Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852‑8523, Japan

484

Table I Information on the chyluria patients from Okina‑
wa, Japan

Patient No. Sex
f emale2
3

History of Duration of
the latest
Age f ilari al

male
male

57
59
71

f ernale78

chyluria(5) were to absorb antibodies to the corresponding
nematodes which may cross‑react with the filarial
antigens. These mixtures were incubated for 2 hrs at

Yes

1 month37'C, then applied to the plate (100 pl/well) and incubat‑

ed for one hour at 37'C. The plate was washed three
1 month6

times with Tween‑PBS (0.05% Tween 20 in 1/15 M
phosphate buffered saline, pH 7.4) and affinity purified

goat anti‑human lgG conjugated with peroxidase (Bio7f emaleYes
Yes

2 weeks

9

male

10
11

f emale

60
83
58

12

f emale62

Yes

3 months

13
14

male

71

Yes

1 year

15
16

male

61

8

buffer containing both D. immitis and Anisakis antigens(1 mg/ml for each antigen) . The procedures from (2) toinf ection

4

5

buffer containing S. ratti antigens (1 mg/ml) or (5) the

1 weekSource International, Inc., Tago Products, USA) , which
was diluted 4,000 times, was added. After incubation at
37'C for one hour, the plate was washed three times andthen ABTS Peroxidase Substrate (KPL, Inc., USA) was

1 year*Information not available.

Antigen preparation for ELISA:
Female adult worms of rugia pahangi collected

added as a substrate for coloration, and absorbance at
415 nm was measured. A11 antibody levels were expres‑sed as log [absorbance value+1].
For the detection of anti‑B. pahangi lgG4 anti‑
bodies, serum sarnples were not absorbed as above but
simply diluted 500 tirnes. The mouse monoclonal anti‑
body to human lgG4 conjugated with peroxidase (Caltag
Lab., Inc., USA) was used as a second antibody.

frorn the abdominal cavity of Mongolian gerbils, Diro ‑filaria immitis adult worms from the heart of dogs, 3rd‑stage larvae of Anisakis sp. recovered from mackerels
and infective filariform larvae of Strongyloides ratti

A11 chyluria cases in this study were regarded

maintained in white rats were homogenized in 1/15 M

clinically as filariasis patients based on the history that

PBS, pH 7.4, containing proteinase inhibitors: I mM ofphenylmethane sulfonyl fluoride (PMSF) , 10 pM of [L‑they were frorn the endemic areas. However, there wasno concrete evidence to confirm the diagnosis. Many

3‑trans‑carboxyoxiran‑ 2 ‑c arbonyl J ‑ L ‑ Ieucyl ‑ agmatin

years after eradication of filariasis, it is difficult to(E‑64), 0.5 mM of Pepstatin A and 5 mM of eth‑differentiate filarial chyluria from that of otherylenediaminetetraacetic acid (EDTA) . Each of the

etiologies. As adult parasites were reported to survive
for up to 17 years, or be reproductive for 5‑40 years

homogenates was centrifuged at 15,000 rpm for 20 min
at 4'C and supernatant was obtained. The supernatant
was then measured for protein concentration using Bio‑
Rad Protein Assay Kit (Bio‑Rad Lab., USA) , adjusted
to the required concentration and stored at ‑ 40"C untiluse .

Detection of antibodies to B. pahangi with ELISA:

A 96‑well microtiter plate was coated with .
pahangi antigens (5 /eg/ml) at 4'C overnight. The
surface of the plate was blocked by casein buffer (1%casein in 0.05 M Tris‑HCI buffer with 0.15 M NaC1,
pH 7.6) for two hours at room ternperature. SerumRESULTS AND DISCUSSION(Carme et al.. 1979) , it is still interesting to test if there

is any active filarial infection in Japan. For this test, theOg4C3 ELISA which mainly detects W. bancrofti adultcirculating antigen is applicable. The ELISA has highsensitivity and specificity, and sera collected in thedaytime can be used (Lammie et al., 1994) . The antigen
assay revealed that all the sera from chyluria patientswere negative, indicating that there were no surviving
parasites in the chyluria patients.

B. pahangi has been a source of antigen for use inserological tests for human lymphatic filariasis (Au etal., 1982; Nuti et al., 1982; Estambale et al., 1994).

samples were prepared in five different ways, that is, theImmunoglobulin G Ievels to B, pahangi antigens aresera diluted 400 times with the casein buffer were mixed
with the same volume of (1) the buffer itself, (2) the
buffer containing D. immitis antigens (1 mg/ml) , (3) the

shown in Figure 1. Without absorption with thebuffer containing Anisakis antigens (1 mg/ml), (4) thepatients and healthy controls. Some healthy people

nematode antigens (Fig. Ia), IgG Ievels of mf positive
Sri Lankans were clearly higher than those of chyluria

485

b

a

o.6

c

r

(5

'

euO

H
+

'l) f

0.6

l
0.4

eUO

,,

oa･‑
e

+
q;

‑cs
0.4
>

e

:

cs

>

::
C:S

o 0.2

o.2
.t

e:l

o
:2
c::

0.0

, ,
e!)

o:l

0.6

'
'‑1‑
,,i.,,,.
.:' ..

d

cs

Fl
C:;

e

bo

:1 OO
Chyluria
patients

(:;

.euo

euD

Ja panese Sri Lankan
controls mf positives

0.4

Figure 2 Anti‑B. pahangi lgG4 antibodies in serum samples.

0.2

mixture of Anisakis and D. immitis antigens, mf positive

9s

I
Q5
ecj

.sl ==. ',,, =**=
* .
*,, ,
,,

Sri Lankans, chyluria patients and healthy controls
were separated more clearly (Fig. Ie). The absorption
by the mixture reduced absorbance values more than the

Figure I Anti‑B. pahangi lgG antibodies in serum sarnples
before and after absorption with antigens preparedhealthy controls (t‑test, p<0.01), but not in Sri Lan‑=* *, ,

a5o.o: '= ' =' =15‑:d =‑‑‑ ‑‑‑ ‑‑ :‑‑‑‑‑ ‑‑‑4from D. immitis. Anisakis sp. and S. ratti.

Note: Serum samples without absorption (a). Samples
absorbed with D. immitis antigens (b) . Anisakis antigens (c) . S.

ratti antigens (d) , and both D. immitis and Anisakis antigens(e) . The left column of each panel is for chyluria patients, themiddle column for Japanese healthy controls and the rightcolumn for Sri Lankan mf positives. An arrow in panel (e)indicates a cut‑off point. A horizontal bar in each column
indicates the mean of antibody titers. The means were compar‑
ed before and after absorption with t‑test. Asterisks in panels

absorptiofi with a single antigen in chyluria patients andkans. Apparently higher lgG Ievels to B. pahangi insome chyluria patients than in healthy controls (Fig. Ie)
would indicate that their clinical sign is filarial in origin.

If the average antibody level of the healthy con‑trol + 3SD is regarded as a cut‑off point, 6 of 16
chyluria patients could be diagnosed as filarial.
Antibodies of lgG4 subclass to filarial antigens were(b). (c) , (d) and (e) indicate levels of significance: *for p<0.05.

specifically detected in filariasis patients (Lal andOttesen, 1988). In addition, the lgG4 antibodies were**for p<0.01 compared with panel (a) .

reported to be associated with active infection of filarial

parasites, and disappeared in a relatively short period
showed relatively high antibody levels, suggesting thatafter treatment (Estambale et al., 1994; Kwan‑Lim etthey had been exposed to antigens which producedal., 1990). In this study, the detection of this subclasscross‑reactive antibodies to B. pahangi. Absorption .of
these antibodies with D. immitis. Anisakis or S. ratti
antigens significantly reduced absorbance values (Fig.
lb, c, d, e) with one exception: absorption with S. ratti
antigens did not reduce the values in Sri Lankans (Fig.

resulted in a clear difference between actively infected
Sri Lankans and Japanese control subjects (Fig. 2) . Oneld) . The results of statistical tests are shown in Fig. 1.

Absorption with Anisakis antigens was most effective in
reducing absorbance values. Although cross‑reactivityof bancroftian filariasis sera with D. immitis antigens
was well‑documented (Grove et al.. 1977; Weller et al.,1980), absorption with D. immitis antigens was not as
effective as Anisakis antigens in this study. S. ratti wasalso not effective despite the fact that Strongyloides
stercoralis is popular in Okinawa (Asato et al.. 1992).

When cross‑reactive antibodies were absorbed with achyluria patient, whose sample always showed the high‑est antibody level before and after absorption,
maintained a very high lgG4 Ievel. It is possible someparasites survived until recent years and were stimulat‑
ing the antibody production. In other words, the detec‑tion of lgG4 might be less useful in Japan 20 years after
eradication of filariasis.

For immunodiagnosis of filarial chyluria in Japan,
there would be two steps to undertake; ( I ) measure‑ment of . pahangi‑reactive lgG 4 and ( 2 ) measure‑ment of B. pahangi‑reactive lgG after absorption ofcross‑reactive antibodies with Anisakis and D. immitis
antigens. Without having a gold standard of confirmed

486

filarial chyluria cases, the sensitivity of these irn‑

7 ) Kawahara, K., Kubo. H.. Kakinoki, T.. Chan, Y.F.,

munodiagnoses could not be evaluated. However, in

The study was supported in part by Grant‑in‑Aid

Saita, H., Yamashita, J., Shirahama, T., Kayajima, T.,
Kawahara, M.. Ikeda, K., Makinose, S., Matsusako, T.,
Yagi, S., Nishida, M., Yamauchi, D. and Eta, S. (1992):
Statistical observations on inpatients and operations at
the urological clinic of Kagoshima Prefectural Oshima
Hospital from 1981 to 1990. Nishinihon J. Urol., 54,
2283‑2288 (in Japanese)8 ) Kobayashi, T. (1994) : Filariasis, TBS Britanica, Tokyo(in Japanese)for International Scientific Research (Field Research)

9 ) Koyama, Y., Chinen. Y., Ogura, H., Shishido, S., Higa, I.,

no. 09041193 from the Ministry of Education, Science,Sports and Culture in Japan.

Hatano, T.. Hayakawa. M., Osawa, A. and lgarashi, M.

Japan where diagnosis of filarial chyluria is often madewithout evidence, the present diagnostic method canprovide an immunological evidence to support it.

ACKNOWLEDGEMENTS(1990): Immunological study on chyluria. Jpn. J. Urol.,81, 1212‑1216 (in Japanese)10) Kwan‑Lim, G.E., Forsyth, K.P. and Maizels, R.M.

REFERENCES
1 ) Asato, R., Nakasone, T., Yoshida, C., Arakaki, T., Ike‑

shiro, T., Murakarni, H. and Sakiyama. H. (1992):
Current status of Strongyloides infection in Okinawa,
Japan. Jpn. J. Trop. Med. Hyg., 20, 169‑173
2 ) Au, A.C., Draper, C.C., Denham, D.A., Rao, C.K., Ismail,M.M. and Mak, J.W. (1982): Detection of filarial anti‑

bodies in Malayan and Bancroftian filariasis by theindirect fluorescent antibody test, using different filarial

antigens. Southeast Asian J. Trop. Med. Public Health,13, 142‑147
3 ) Carme, B. and Laigret. J. (1979): Longevity of Wucher ‑eria bancrofti var. pactfica and mosquito infection(1990): Filarial‑specific lgG4 response correlates withactive Wuchereria bancrofti infection. J. Immunol., 145,

4298‑4305
11) Lal, R.B. and Ottesen, E.A. (1988): Enhanced diagnostic
specificity in human filariasis by lgG4 antibody assess‑ment. J. Infect. Dis., 158, 1034‑1037

12) Lammie, P.J., Hightower, A.W. and Eberhard, M.L.
(1994): Age‑specific prevalence of antigenemia in a
Wuchereria bancrofti‑exposed population. Am. J. Trop.
Med. Hyg., 51, 348‑355
13) More, S.J. and Copeman, D.B. (1990): A highly specificand sensitive monoclonal antibody‑based ELISA for thedetection of circulating antigen in bancroftian filariasis.

Trop. Med. Parasitol., 41, 403‑406

acquired from a patient with low level parasitemia. Am.
J. Trop. Med. Hyg., 28, 53‑55
4 ) Estarnbale, B.B.. Sirnonsen, P.E., Vennervald. B.J.,
Knight, R. and Bwayo, J.J. (1994) : Bancroftian filariasis

14) Nuti, M., Ferrari, J.D. and Au, A.C. (1982): Sero‑in Kwale District of Kenya. II. Humoral immune

Chyluria treated with stripping of the renal pedicle: acase report. Acta Urol. Jpn., 42, 221‑223 (in Japanese)
16) Sasa, M. (1976) : Human filariasis. A global survey of

responses to filarial antigens in selected individuals

from an endemic community. Ann. Trop. Med. Par‑
asitol., 88, 153‑161

5 ) Grove, D.L., Cabrera, B.D., Valeza, F.S.. Guinto, R.S.,

Ash, L.R. and Warren, K.S. (1977) : Sensitivity andspecificity of skin reactivity to Br gia malayi and
Dirofilaria immitis antigens in Bancroftian and Malayan
filariasis in the Philippines. Am. J. Trop. Med. Hyg., 26,

220‑2296 ) Ito, T., Katoh, A., Hanai, H., Kaneko, E., Kino, H. andTerada, M. (1994): A case of chyluria probably causedby chronic filariasis. Clin. Parasitol., 5, 157‑160 (inJapanese)

epidemiology of bancroftian filariasis in the Seychelles
Islands. Tropenmed. Parasitol., 33, 25‑27

15) Sakakura, T.. Ikeuchi, T. and Watanabe, H. (1996):

epidemiology and control. University of Tokyo Press,

Tokyo.
17) Yagi, S.. Goto, T., Kawamoto, K., Miyawaki, I., Tanaka,I., Mori, K., Hayami, H. and Ohi, Y. (1998): Endoscopic
treatment of refractory filarial chyluria: a preliminary
report. J. Urol., 159, 1615‑1618
18) Weller, P.F., Ottesen, E.A. and Heck, L. (1980) : Immedi‑
ate and delayed hypersensitivity skin test responses tothe Dirofilaria immitis filarial skin test (Sawada)
antigen in Wuchereria bancrofti filariasis. Am. J. Trop.

Med. Hyg., 29, 809‑814

Jpn. J. Trop. Med. Hyg., Vol. 27, No. 4, 1999, pp. 487 495

487

CLINICAL AND LABORATORY CORRELATESOF THE OUTCOME OF CEREBRAL MALARIAIN THE PHILIPPINESPILARITA TONGOL‑RIVERAl, MARIO ANGELITO PRUDENCI02, JESUS SAROL3,
FELIPE BALlNGIT2, ELENA VILLACORTE1, ALDRlN DARILAGl,

MAMORU SUZUKI' AND SHIGEYUKI KAN05
Received February 1, 1999/Accepted August 3, 1999

Abstract: This study describes the socio‑demographic profile of cerebral malaria (CM) patients andidentifies the clinical and laboratory factors which correlate with mortality from this condition. Records of

97 CM cases adrnitted at the Davao Regional Hospital, Philippines from 1990 to 1995 were reviewed.
Information on socio‑demographic factors, clinical signs and symptoms, physical examination and labora‑
tory findings and medicines given during hospitalization were obtained. Associations of these variables withmortality were examined by generating two‑way tables for qualitative variables and computing descriptive
statistics for quantitative variables. Results showed that CM was more common arnong males and young
adults who comprise the economically productive age group (21‑40 years) and who are engaged in slash andburn agriculture. Using bivariate analysis, factors that correlated with mortality included age, residence,back/abdominal pain, fever, coma, dyspnea, diastolic blood pressure (BP) , white blood cell (WBO ,neutrophil and lymphocyte counts, trophozoite count and quinine infusion. Logistic regression analysis
showed that only back/abdominal pain, coma, dyspnea, diastolic BP and trophozoite count remained
statistically significant after simultaneously controlling for confounding. These results can contribute to theproper assessment and improvement in the clinical managernent of CM.
Keywords: malaria, Plasmodium falciparum, cerebral malaria, risk factors, The Philippines

INTRODUCTION

response and a non‑specific response is the best motor

response (WHO, 1990).

Cerebral malaria is one of the criteria for diagnosis

CM is the most lethal form of complicated malaria.

of severe and complicated falciparum malaria. It is

The mortality and the morbidity from CM remains
unacceptably high (Walker et al., 1992). In thedefined as unrousable coma which is not attributable toany other cause in a patient with falciparum malaria.
The common presentation is that of a generalized con‑vulsion followed by persisting unconsciousness. Among

adults, CM is defined as a "non‑10calizing" motor
response to noxious stimuli and an "inappropriate"
vocal response. Arnong children, coma persists for morethan half an hour after a convulsion, eye movements are
not directed, an "inappropriate cry" is the best verbal
12
3Philippines, approximately 31% of the endemic popula‑tion of about 11 million live in high transmission areas(Malaria Control Service, Department of Health, 1995) .

Among patients being admitted at the Department ofInternal Medicine of Davao Regional Hospital in
Tagum, Davao del Norte from the years 1990‑1995,
malaria was consistently one of the leading causes ofmorbidity. Malaria mortality in this hospital was usu‑

Department of Parasitology, College of Public Health, University of the Philippines‑Manila, Manila, The Philippines
Department of Internal Medicine, Davao Regional Hospital, Davao del Norte, The Philippines
Department of Epidemiology and Biostatistics, College of Public Health, University of the Philippines‑Manila, Manila, The
Phili p pines

4
5

Department of Parasitology, Gunma University School of Medicine, Gunma 371‑8511. Japan
Department of Appropriate Technology Development and Transfer, Research Institute, International Medical Center of Japan,
Tokyo 162‑8655, Japan
Correspondence: Pilarita Tongol‑Rivera, Department of Parasitology, College of Public Health, University of the Philippines‑
Manila, 625 Pedro Gil St., Ermita, Manila 1000, The Philippines

488

ally due to its complications, particularly CM. This
study was thus conducted to review cases of CM admit‑
ted during this 6 year period, establish the socio‑demo‑

graphic profile of CM, describe the clinical manifesta‑
tions of patients with CM, identify the factors which
contribute to mortality and describe the mode of man‑agement administered to these patients. The resulting

data may serve as an aid to health workers in theprompt recognition of CM as a life threatening condition

and guide in the proper' assessment of the patient's
status, thus improving the diagnosis and management of
CM in the Philippines and other malarious countries.

MATERIALS AND METHODS
Sub jects

The records of patients, admitted to the Depart‑
ment of Internal Medicine, Davao Regional Hospital,
during the period January 1, 1990 to December 31, 1995,

Statistical analysis

All the data were encoded using EPI INFO 6(Version 6.03, CDC, USA; WHO, Geneva). One‑way
frequency tabulation and descriptive statistics were
derived to describe the characteristics of CM patients.
To identify characteristics of patients that are related
to mortality, the study population was divided into twogroups: Group I comprised those subjects who survived(n=53) and Group 11 were those who died (n=44) . The
characteristics that were not commonly found among
CM patients were not considered for further analysis
since the sample size was not enough to draw associa‑
tions for these variables. For qualitative characteris‑
tics, the associations were presented in two‑way tables,

crosstabulating each factor with mortality status. Odds
ratios for all the factors were calculated. Statisticalsignificance of the association between each factor andoutcome of disease were assessed using either x 2 test or
Fisher's exact test, whichever is appropriate, at the a =and with a diagnosis of CM, were retrieved and revi‑
ewed. There were 97 CM cases who satisfied the inclu‑
sion criteria ‑D final diagnosis of CM and 2) positive

0.05 Ievel. For quantitative characteristics, Student's t‑malaria smear on admission and on serial examination.
The degree of parasitemia was based on the number ofparasites counted per field in the thick smear by thefollowing method: ( + ) if less than 10 parasites were

By performing the bivariate analyses describedabove, several variables were found to be associatedtest was used to determine statistical significance of thedifference of the means between the two groups.

with mortality from CM. These variables, however,per 100 fields, (+ + +) if less than 10 parasites per field,

were also correlated with each other. To remove con‑founding among these variables, Iogistic regression
analysis was done. Stepwise selection of variables wasand ( + + + + ) if more than 10 parasites per field. The

carried out in arriving at a final model since all vari‑socio‑demographic data gathered per subject included:age, sex, occupation and place of residence. The preva‑
lence of malaria in each town was noted and the placesof residence of the subjects were mapped. The clinicalhistory was reviewed for each subject and pertinent
signs and syrnptoms, and findings on medical and neur‑
ologic examination were noted. The fever was classi‑

ables were considered equally important.

counted per 100 fields, ( + + ) if more than 10 parasites

fied as: afebrile if temperature 37'C, Iow tomoderate 38'C and moderate to high grade>38'C.
Other concomitant conditions such as pregnancy andinfections such as pneumonia, tuberculosis, typhoid
fever and hepatitis were also noted.
In addition to malaria smear, the other laboratory
examinations included in the study were the following:

WBC, differential count, blood type and hemoglobin.
Other examinations such as fasting blood sugar/random
blood sugar, Iiver function tests, HBSAg, Widal's test,

chest x‑ray and ECG were done only on few patientsand they were not included in the study. The therapeu‑
tic regimen given to the patients were also recorded.

RESULTS
Socio‑demographic profile aud mortality

Table I shows the age and sex distribution of theCM patients admitted in Davao Regional Hospital. The
subjects were 13‑76 years of age. It was noted that
45.4% belonged to the 21 to 40 age groups. These age
Table 1 Age and sex distribution of CM patients, Davao
Regional Hospital, 1990‑1995

Age

Male

Female

11‑20

10

7

21‑30
31‑40
41‑50
51‑60
61‑70

15

7

15

7

11

3

9

3

3

3

> 71

2

2

Total

65

32

489

No job@OthersAgusan del sur

I >30
1 >20 ‑ 30mn] eq>ro ‑ 20

Housekeeper

Farmer

28.10/040.60/0

non‑endemic

PHILIPPINESt '/Labourer

4.10/0 9.3010

Figure 1 Occupation of the cerebral malaria patients, Davao
Regional Hospital, 1990‑1995.

5¥.e i'J@

Mindana

5 f '

1

>'s
c9

Figure 2 The map of the study areas and their annual
parasite incidence per 1,000 in 1993. Numbers in
the respective areas are the distribution of cerebral

groups included the adolescents and young adults whocomprised the economically productive group of mostcommunities in the Philippines. However, it must be
noted that only patients admitted in the Department ofmalaria cases.

females (33%) . This may be due to the greater malaria
exposure of the male population because of occupation.

Internal Medicine were included as subjects, thus chil‑dren below 13 years of age were missed in this study.

Farmers, engaged in slash and burn agriculture in forest‑

ed areas, comprised 40.6%, while 28.1% were house‑keepers who were exposed to malaria by residing inendemic foci. Laborers comprised 9.3%, soldiers 4.1%
and 16.6% had no job (Figure 1). Figure 2 shows thegeographic distribution of the residence of the CM

CM had been shown to affect children. A study inanother highly endemic area in the Philippines showed

the occurrence of CM among children (Yap‑delos
Santos, 1995). There were more males (67%) than

Table 2 Odds Ratio and 95% confidence intervals for the association of socio‑demographic
characteristics with outcome of CM

Socio‑demographic
characteristics

Sex
Male
Female95% confidence

Died

Recovered

Odds Ratio"

28
16

37
16

o . 765

12
13

1 . OO
1 . 66

O . 36‑7 . 92

o. 4580

75 . 14

1 . 08‑26. 34

o. 0163

intervalo. 30‑1. 93

p‑valueO . 5196

Age
11‑20
21‑30
31‑40
41‑50

9

8

1 . 80O . 32‑10. 37

o. 4362

51‑above

15
6
9

13

1 . 66

O . 6‑7 . 92

o. 4580

Occupation
Exposedb
Not exposedc

27
17

26
27

1 . 65o. 68‑4 . 03

o . 2255

10
14
17

5

OO

17

o. 41

o. 09‑1 . 75

o. 171120

o. 43

o. 10‑1 . 73

o. 1755

3

11

o. 14

o. 02‑0 . 92

o. 0144

ResidenceNon‑endemic> 0‑10 cases/1,000 pop'n> 10‑20 cases/1,000 pop'n>20 cases/1,000 pop'n'whenever there are more than 2 categories, the first is treated as the reference groupbfarmers, Iaborers, soldiers'housekeepers, no job

490

Table 3 Odds Ratios and 95% confidence interval for the association of clinical findings with outcomeof CM
Signs and Symptoms

95% Conf idence

Died

Recovered

Odds Ratio

16
28

24
29

o . 69o. 28‑1 . 69

o . 37438

1142

o . 85O . 27‑2 . 61

o . 7506

31

3 . 80O . 29‑203 . 51

O . 24184152

Intervalp‑value

Headache+
Malaise+36

Fatigue+Dizziness

+

1

o

43

53

o . 4536

Back/Abdominal Pain

+

7

37

9 . 841 . 16‑451 . 68

O . 0153

2 . 61o . 78‑9 . 42

o . 0777

3 . 80O . 29‑203 . 51

O . 2418

1 . 20o . 02‑96 . 73

o . 7040

o . 43O . 17‑1 . 08

o . 0466

52

Anorexia

+

11

6

33

47

3
41

52

43

52

22
22

37
16

24

44

1 . OO

5

3

3 . 06

O . 53‑21 . 06

o. 1335

15

6

4 . 58

1 . 42‑16 . 12

o. 0035

Vomiting+

1

Diarrhea+
Chills

+
Fever
none
low‑moderatemoderate‑highProstration+o . 0392

4

o

40

53

18
26

26
27

o . 72O . 32‑1 . 74

o . 4223o . 92O . 27‑3 . 11

o . 8874

O . 41

O . 07‑1 . 88

O . 1696

Conscious+
Coherent

+

7

9

37

44

3
41

45

17
27

12
41

2 . 15O . 81‑5 . 76

o . 08671529

9

2 . 53o . 89‑7 . 43

O . 0519

44

17
27

1 . 75o . 68‑4 . 55

O . 1988

39

Incoherent+

8Unresponsive+
Lethargic+
Stuporous

+

14

491

Comatose

+

13
31

50

20
24

52

3

6 . 991 . 76‑40 . 46

0.0016

6 . O1‑1829 . 33

o . oooo

Dys pnea+

1

43 . 33

Focal seizures

+

5

o

39

53

O . 0169

Neck rigidity

+

1

1

43

52

1.21

o . 02‑96 . 73

o . 7040

5 . 20o . 48‑260 . 90

O . 1285

1 . 22

O . 15‑9 . 58

o . 5687

Visual disturbances

+

4

1

40

52

3
41

50

15
29

13
40

1 . 59o . 60‑4 . 24

o . 3008

11
33

11
42

1 . 27o . 44‑3 . 68

0.6191

Abdominal reflexes+

3

Liver dysfunction+
Anaemia and bleeding

+
Shock

+

1

o

43

53

o . 4536

Blackwater fever

+

1

o

43

53

o . 4536

Acute renal failure

+

3

o

41

53

o . 0898

Pulmonary edema

+

11

5

33

48

3 . 20O . 91‑12 . 73

o . 0397

Hepatitis

+

1

o

43

53

o . 4536

Pulmonary tuberculosis

+

o

44

o . 5464

52

Pneumonia

+

5

3

39

50

subjects. All these areas except Tagum were known to
be endemic with malaria prevalence ranging from 0.2 to47.9 per 1,000 in 1993 (Malaria Control Service, Davaodel Norte) . A few (11.3%) of CM subjects resided innon‑endemic, urban Tagum.
Table 2 shows the association of selected demo‑
graphic characteristics with mortality. For this analy‑
sis, occupations were classified as "exposed" and "unex‑

posed" to malaria while residence were categorized

2 . 14

O . 39‑14 . 49

o . 2586

according to prevalence of infection in the area. Ageand residence of the patient were found to be significant‑ly associated with outcome of CM while sex and occupa‑tion were not. Mortality was significantly higher amongthe 31‑40 year age group compared to those below 21
years (p=0.0163). Patients frorn non‑endemic areas
also had significantly higher mortality compared topatients of highly endernic areas (p=0.0144) .

492

Clinical manlfestations, physical findings and mortality

statistically associated with poor outcorne of CM ( p <Table 3 shows the Odds Ratios and p‑values fordetermining the association between the outcome of0.05) .

malaria and the presence of certain signs and symptoms,
physical examination findings, other complications ofmalaria and concomitant infections. Only back/abdomi‑

Antimalarial therapy and supportive management
The only available regimen for complicated andnal pain, fever, dyspnea and coma, were shown to be
associated with a fatal outcome of CM (p<0.05) . Noneof the subjects complained of chest pain, nausea andsweating. There were no subjects who were oriented, orpresented with generalized convulsions, poor muscle
tone and ankle clonus. None of the subjects had reactive

psychosis, delirium, cerebellar disturbances, hypo‑glycemia and neurologic sequelae. Table 4 of the vital

severe malaria in the Philippines is intravenous Quinine
infusion of 10 mglkg in 500 ml of 5% dextrose in water,

125 ml of which was given in a drip for 4 hr at 6 hr
intervals. In 85.5% of subjects, the infusion was started

on admission and 14.4% on the succeeding hospital days.
The latter subjects were conscious on admission and so,they were given oral antimalarials such as Chloroquine
phosphate tablets, 10 mg base/kg initially, then 5 mg
base/kg after 6 hr, then 5 mg base/kg on days 2 and 3;

signs shows that only a higher diastolic BP was associat‑

Sulfadoxine (25 mglkg) , Pyrimethamine (1.25 mglkg)

ed with fatal outcome of CM (p<0.05) .

tablets, single dose. Deterioration of the level of con‑sciousness led to the shifting from oral to intravenous

Laboratory findings and mortality

Table 5 shows the association of the laboratory
findings with the outcome of CM. The WBC, neutrophil
and lymphocyte counts showed statistical significance(p<0.05). Table 6 of the blood type shows that there
was no statistically significant association between
blood type and the outcome of CM (p<0.05). Table 7

shows that trophozoite count (+++/++++) was

Quinine. Table 8 shows the association of medicine
given with the outcome of CM. Quinine infusion cor‑
related with a good outcome of CM (p<0.05) .
Stepwise logistic regression analysis

Several signs and symptoms had statistically signifi‑

cant associations with mortality from CM. Since thesevariables may be mutually confounding each other,

Table 4 Mean and standard deviation (S.D.) of vital signs by outcome of CM
Vital sign

Died
Sarnple Size Mean

Recovered

Test statistics

S D Sample Size Mean

S.D.

(p‑value)

Systolic blood pressure (mmHg)

42

110 . O

21 . 952

103 . 5

13 . 876 (O . 081D

Diastolic blood pressure (mmHg)

42

72 . 6

13 . 352

66 . 7

11.8

2. 27 (O . 0254)

Temperature ('O44

38 . 4

1.2

52

38 . 6

1.1

o. 57 (O . 5702)

Heart rate (per minute)39

89 . 222 . 950

91.8

15 . 8o. 63 (O . 5294)

Table 5 Mean and standard deviation (S.D.) of laboratory findings by outcome of CM
Laboratory findings

Died
Sarnple Size Mean

Hemoglobin (g/L)

43

101 . 9WBC (xlO"/L)

43

12 . 6

Neutrophil countLymphocyte count

Recovered

Test statistics

S D Sample Size Mean

S.D.
27 . 8(p‑value)o. 74 (O . 4627)

53

97 . 48 . 85

51

8.1

4. 30

3. 21 (O . O018)

43

O . 74O . 1051

O . 68o. 11

2. 52 (O . 0134)

43

O . 26

O . 1051

O . 32o. 11

32 . O

Table 6 Odds Ratios and 95% confidence intervals for the association of blood
type with the outcome of CM

Blood type Died Recovered

A
AB
B
O

Odds Ratio

95% confidence
intervalp‑value10

14

1 . OO

2

3

O . 93o . 07‑9 . 84

o. 670513

11

1 . 65o . 46‑6 . 09

o. 386115

18

1 . 17o . 35‑3 . 87

o. 7760

51(O . 0138)

493

simultaneously controlling for this effect had to be doneby logistic regression analysis. To prevent missing
clinically important variables, associations of variables
with mortality that had a p‑value ; 0.15 were consideredin the analysis. Prostration, focal seizures and acuterenal faildre had zero frequency in one cell and were notconsidered for further analysis. Stepwise selection ofvariables was performed to determine which variables

remained significant (a=0.10) after controlling forconfounders. Age and residence of subjects were consid‑
ered confounders and were forced in into the model.
The results of logistic regression analysis are shownin Table 9. The variables that entered the model were
presence of back/abdominal pain, coma, dyspnea dias‑

tolic BP ( >90 mm Hg) and trophozoite count. Thismeans that the association of these variables withmortality from CM are less likely to be due to confound‑ing.

Table 7 Odds Ratios and 95% confidence intervals for the association of parasite count with outcomeof CM
Parasite count95% confidence

p‑value

Died

Recovered

Odds Ratio

13

25

1 . OO

917

1 . 02O . 31‑3 . 30

o . 9733

2211

3 . 851 . 29‑11. 73

o . 0064

interval

P. falciparum trophozoites++++++/++++= if less than 10 parasites were counted per 100 fields+= if more than 10 parasites per 100 fields+ + + = if less than 10 parasites per field+ + + + = if more than 10 parasites per field++

Table 8 Odds Ratios and 95% confidence intervals for the association of medicines given

with outcome of CM
Medicine given

Died Recovered Odds Ratio

95% confidence
intervalp‑value

Quinine

+

13
31

41
12

O . 12o. 04‑0 . 33

o . ooooo . 46o. 04‑3 . oOo . 3020

o . 72o. 21‑2 . 30

O . 5411

Chloroquine

+

2

5

42

48

37

11
42

Mannitol+Fansidar

+

o

2

44

51

o . 2960

Chloroquine phosphate 10 mg base/kg initially, then 5 mg base/kg af ter 6 hr, then 5 mg
base/kg on days 2 and 3.
Sulfadoxine (25 mglkg) ‑pyrimethamine (1.25 mglkg) single dose.
Quinine by intravenous infusion 10 mglkg in 5% dextrose over 4 hr.
Mannitol by intravenous infusion I g/kg of 10% solution over 30 min.

Table 9 Results of stepwise logistic regression analysis
Variable

Parameter

Standard

estimateerror

p‑value

Odds Ratio
13 . 840

Back/abdominal pain

2 . 6275

1 . 4619

o . 0723

Coma

2 . 5548

O . 9549

o . 0075

12 . 868

Dys pnea
Diastolic blood pressure
Trophozoite count ( + + )Trophozoite count ( + + + / + + + + )3 . 6037

1 . 2432

o . 0037

36 . 732

2 . 7273

1 . 2859

o . 0339

15 . 292

1 . 0665

O . 8396

o . 2040

2 . 905

1 . 7802

O . 8181

o . 0296

5 . 931494

DISCUSSION
From 1946 to 1952, the Department of Health repor‑
ted that malaria was the 5th cause of mortality in thePhilippines. Due to control/eradication efforts, themortality rate went down and, at present, it is only 1.5
per 100,000 population (Asinas, 1993; Malaria Control
Service. Manila, 1995). However, in some areas in thePhilippines, malaria is still a leading cause of death and,for example in a highly endemic area, the mortality ratefrom CM was 19% in children (Yap‑delos Santos, 1995) .

In two other studies, reported mortality rates among
CM cases varied from 20% (Alcantara et al., 1982) to35% (Tagle and Cabanban, 1992). In our present study
which was done in another highly endemic area, themortality rate of CM among adults was 45%. Such high

had higher total WBC and neutrophil counts and lower
lymphocyte count. This may be a significant finding
particularly in relation to the recent finding of tissue
infiltration by leukocytes in autopsy specimens of young

adults with CM in Orissa State, India (Mendis andCarter, 1995). Peripheral leukocytosis (>12,000lpl) isa laboratory index of poor prognosis in CM (WHO,1990). An interesting finding was that the mean hemo‑
globin levels (Table 5) were not so low as reported in
the study of children where 21% had less than 7.0 gll(Yap‑delos Santos, 1995). In this study, the meanhemoglobin of those who died was 10.1 g/1. Severe
anemia may be more common in children as in Africa,
where it is a common feature of cornplicated disease andcauses the most mortality during the first two years oflife (Greenwood et al.. 1987; Mendis and Carter, 1995).

mortality in CM and the factors which correlated with abad outcome or death, were the focus of this study.

However, adults may not be that susceptible to anemia
as children. As for blood‑group antigens, they have

The socio‑demographic profile of CM patients inour study showed that almost half belonged to the agegroup who are most economically productive and active

been implicated among the ligands of rosetting on infect‑

members of the community. Males predominated, sug‑ed erythrocytes, which causes obstruction of cerebral
blood flow in CM. There is some evidence to suggestthat severe malaria may be relatively less frequentgesting that malaria exposure may be related to occupa‑arnong O blood type because unlike A and B eryth‑tion. It was observed that most CM patients were

rocytes, O erythrocytes do not exhibit rosetting (Mendis

farmers engaged in slash and burn agriculture and resid‑

and Carter, 1995). However, this study did not demon‑

ing in endemic areas. However, occupation and sex did

strate a significant correlation between blood types andnot show statistically significant association with mor‑a poor outcome of CM (Table 6) .
It has been shown that hyperparasitemia ( > 5%

tality from CM. Instead, mortality was statisticallyassociated with 31‑40 years age group and a place ofresidence which is none to low endemic for malaria.
This may be due to non‑suspecting health personnel
resulting in delay of CM diagnosis and treatment.

Alcantara (1982), Tagle and Cabanban (1992),

parasitized erythrocytes or >250,000 parasites per pl) isa laboratory index of poor prognosis in CM (WHO,1990). However, one study did not show any difference
between parasite count in the fatal and non‑fatal cases(Walker et al.. 1992). In our study, a semi‑quantitativedescribed that the signs and symptoms commonly obser‑
ved in the fatal cases of CM included coma, oliguria,
jaundice, hyperpyrexia, headache, and chills. In ourstudy, back/abdominal pain, fever, dyspnea, coma, dias‑count of + + +/+ + + + (one or more parasites per 1
thick field) correlated with a poor outcome of CM (p<tolic BP, WBC, neutrophil and lymphocyte counts andtrophozoite count correlated with a poor outcome (p<0.05). Quinine infusion on the other hand correlatedwith good outcome (p<0.05) . The presence of coma has
always been the risk factor for grave prognostic out‑
come in CM (WHO, 1990; Walker et al.. 1992). Suchhypoglycemia is common and is associated with a poor

0.05)
(Table 7). ̲
Walker et al. (1992), WHO (1990), showed thatoutcome. In this study, glucose determinations were notdone in many patients and analysis for its association
with the outcome could not be done.

finding was shown in this study. For the other physicalThe management of CM is straightforward as longas the diagnosis is made. Quinine is a very effective
drug in complicated malaria including CM. However,examination findings, it was shown that a higher dias‑

failure to reduce mortality may be due to lack of suppor‑

tolic BP correlated with poor outcome. Those CM

tive management. Anticipatory, prompt treatment, andsupportive management for a life‑threatening condition
such as CM, may be the key in reducing the mortality.
This study used data from medical records before it
was conducted. It is very likely that some conditions
might have been present in some patients but were notpatients who died exhibited a higher reading comparedto those who recovered from CM (Table 4).
With regards to the laboratory findings, the total
WBC, neutrophil and lymphocyte counts correlated withpoor outcome (p<0.05). Those CM patients who died

495

recorded if the physician did not think these were criti‑

REFEREN CES

cal in the management of the patient. Unfortunately,
the effect of this misclassification cannot be assessed inthis study.

From the results of logistic regression analysis,presence of back/abdominal pain, coma, dyspnea, dias‑

tolic BP (>90 mm Hg) and trophozoite count weresignificant. These variables were found to be significant

1 ) Alcantara. A.K., Buck, R.L., Uylangco, C.V. and Cros,
J.H. (1982): Cerebral malaria at San Lazaro Hospital,
Manila. Philippines. Southeast A. J. Trop. Med. Pub.
Hlth., 13 (4), 563‑567

2 ) Asinas, C.Y. (1993): The epidemiology and control ofmalaria in the Philippines. Jpn. J. Trop. Med. Hyg., 21,

9‑16

after controlling for confounders. The odds ratios,
however, cannot be interpreted reliably due to small3 ) Malaria Control Service, Davao del Norte (1993):
Annual Reports. Apokon, Davao del Norte: Provincial

sample size of the study. This may also account for the
exclusion of other important variables from the model.

4 ) Malaria Control Service, Manila (1995): Annual

A Iarger study would confirm these associations andestimate the effect of these variables on mortality more
precisely.

Health Office

Reports. pp. 7‑9. Manila, Department of Health5 ) Mendis, K.N. and Carter R. (1995): Clinical disease andpathogenesis in malaria. Parasitol. Today, 11, PTI, 1‑15

6 ) Tagle, R.S. and Cabanban. A.B. (1992): Severe andcomplicated malaria at San Lazaro Hospital. Philip. J.

ACKNOWLEDGMENTS
This project was funded by the Institute of Biotech‑
nology, University of the Philippines, Manila, and, inpart, by a Grant for International Health Cooperation

Research (10A‑1) from the Ministry of Health andWelfare of Japan. The authors wish to thank the follow‑
ing for their assistance and support: I ) Dr. Dolores
Castillo, Provincial Health Officer and her staff. 2) Dr.

Microbiol. Inf. Dis., 2, 4‑107 ) Walker, O., Salako, L.A.. Sowunmi, A., Thomas, J.O.,
Sodiene, O. and Bondi, F.S. (1992): Prognostic risk
factors and post mortem findings in cerebral malaria inchildren. Trans. Roy. Soc. Trop. Med. Hyg., 86, 491‑493

8 ) World Health Organization, Division of Control of
Tropical Diseases (1990): Severe and complicatedmalaria. Trans. Roy. Soc. Trop. Med. Hyg., 84, 1‑65
9 ) Yap‑delos Santos, Ma. S. (1995): A retrospective studyof malaria, Cagayan Valley Regional Hospital, Depart‑

Romulo Busuego, Chief of Hospital, Tagum Regional

ment of Pediatrics, 1992‑1993. J. Philip. Med. Assoc., 71,

Hospital. 3 ) Mr. Rodrigo Bulicatin, Malaria Control
Service, Davao del Norte. 4 ) Mrs. Conchita Bahinting,
Records Section, Tagum Regional Hospital.

193‑198

Jpn. J. Tro p. Med. Hyg., Vol. 27, No. 4, 1999, pp. 497‑509

497

FOUR NEW SPECIES OF BLACK FLIES(DIPTERA= SIMULIIDAE) FROM THAILAND
HIROYUKI TAKAOKAI AND CHALIOW KUVANGKADILOK2
Received May 21, 1999/Accepted August 26, 1999

Abstract: Four new species of black flies (Diptera: Simuliidae) are described based on reared adult, pupal

and/or larval specimens collected from Thailand. Three new species, Simulium chaliowae sp. nov., S.
chainarongi sp. nov., and S. triglobus sp. nov., are all assigned in the multistriatum‑group of the subgenus
Simulium (Simulium), and share the similar shoe‑shaped cocoon instead of the usual fenestrate cocoon. S.
triglobus is most remarkable by having three spermathecae in female adult. The fourth new species, S.
baimaii sp. nov., is placed in the genus Simulium but its subgeneric assignment remains unclear due to lackof adult specimens. This species is distinct from the other known simuliid species in the Oriental Region byhaving the pupal gill with two filarnents directed forward from an inflated stalk on each side.
Key words: Simulium, black fly, Simuliidae, Thailand, spermatheca, new species

Takaoka and Suzuki (1984) reported 19 species of
black flies (Diptera: Simuliidae) from Thailand, includ‑ing seven new species and five unnamed ones. Takaoka
and Saito (1996) described a new species and recorded
four other simuliid species for the first time from thiscountry. In the same paper, three species, previously

reported as Simulium sakishimaense Takaoka, S.

The morphological features and terms used herein
follow mostly those of Crosskey (1969), and partiallythose of Takaoka (1983). All holotype and some par‑atype specimens will be deposited in the Department of
Biology, Faculty of Science, Mahidol University, Bang‑
kok, Thailand, and some other paratypes, in the Depart‑

nitidithorax Puri, and S. sp. C, were identified as S.

ment of Entomology, The Natural History Museum,London, UK.

fenestratum Edwards, S. tani Takaoka and Davies, andS. asakoae Takaoka and Davies, respectively. Takaoka

Simulium ( Simulium) chaliowae Takaoka and Boonkemtongand Adler (1997) reported two more species fromsp. nov.

Thailand. In total, 26 black‑fly species including four

unnamed species have been known from Thailand.
Recently, one of us (CK) made a faunistic survey ofSimuliidae in northern and northeastern provinces ofThailand, and collected four new species, of which three
are assigned in the multistriatum‑group of the subgenus

Simulium (Simulium), redefined by Takaoka andDavies (1996). A11 these three are characterized by theshoe‑shaped cocoon with an anterior collar of variableheights, in place of the usual fenestrate cocoon. Strik‑
ingly, one of these species has three spermathecae in thefemale adult. The fourth new species, represented onlyby the pupal and larval stages, is also placed in the same

genus but remains unclassified into any known sub‑genus. This species is also very distinctive in having thepupal gill with two filaments, a character very rarely
encotintered in the Simuliidae.

Felnale. Body length 2.2 mm. Head. Narrower thanthorax. Frons black, shiny, with several dark stout
hairs along lateral margins; frontal ratio (width:height‑width greatest near vertex, narrowest nearantennal base) 1.2:1.0:1.6; frons‑head ratio (greatest
width of frons: greatest width of head) 1.0:4.5. Fronto‑

ocular area (Fig. 1) well developed. Clypeus black,shiny, gray‑pruinose, iridescent when illuminated, mod‑erately covered with dark stout hairs. Antenna com‑posed of 2 + 9 segments, yellow on scape, pedicel andmost of Ist flagellar segment, and brownish on rest offlagella, when viewed dorsally; while antenna yellow onscape, pedicel, and a few flagellar segments, and gradu‑

ally darkened toward apex on rest of flagella, whenviewed anteriorly or ventrally. Maxillary palp with 5
segments, Iight to dark brown, proportional lengths of1

Department of Infectious Disease Control,, Oita Medical University, Hasarna, Oita 879‑5593, Japan2

Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

498

3rd, 4th and 5th segments 1.0:1.2:2.2; 3rd segment

yellowish brown, with fringe of pale long hairs, of which(Fig. 2) of normal size, with sensory vesicle oblong, ca.

(Fig. 24). Thorax. Scutum brownish black,basal portions are dark. Dorsal surface of 2nd abdomi‑
nal segment pale yellowish white with narrow area justbefore posterior margin brownish black; dorsal surfaceof remaining segments brownish black, with short darkhairs; terga 6‑8 shiny. Ventral surface of 7th segmentwith a pair of small submedial sternal plates. Genitalia(Figs. 6‑8). Sternite 8 moderately sclerotized, bare inshiny, densely covered with whitish‑yellow, recumbent

middle, covered with 30‑34 stout hairs (in anotherpubescence, and with several dark upright hairs on

fernale, 23‑25 hairs) on each lateral surface. Anteriorprescutellar area; when illuminated in front and viewed
dorsally, scutum thickly white‑pruinose, with 5 Iongitu‑

gonapophyses triangular, moderately separated from

0.4x as long as 3rd segment. Maxillary lacinia with 12
or 13 inner teeth and 12 outer teeth. Mandible with ca.
22 inner teeth and ca. 14 outer teeth. Cibarium with ablunt, medial projection on posterior margin, and with‑out any tubercles, similar to that of S. chainarongi sp.

nov.

dinal unpruinose vittae, of which I median vitta nar‑rowest, 2 sublateral ones widest, and 2 submedian ones
intermediate, slightly or somewhat wider than median
one but much narrower than sublateral ones, all vittae
united with broad transverse band on prescutellar area;
when illuminated from behind, scutum having reversed
color pattern. Scutellum light brown, covered with dark
upright hairs as well as whitish‑yellow pubescence.
Postscutellum brownish black, shiny, gray‑pruinose,bare. Pleural membrane bare. Katepisternum longer
than deep, bare. Legs. Foreleg: coxa and trochanter

each other medially, membraneous, covered with numer‑
ous microsetae and 1‑3 stout hairs; narrow bare area
along inside and posterior margins very thin and trans‑parent (then often overlooked) . Genital fork of
inverted‑Y form, with narrow, well sclerotized stem;arms of moderate width, each with distinct projection
directed anteriorly. Paraproct much protruding ventral‑ly, ca. 1.4x as wide as long, with numerous stout hairson lateral and ventral surfaces. Cercus short, ca. 1.7><
as wide as long, with numerous stout hairs, with rounded
posterior margin, when viewed laterally. Spermatheca 1

in number, nearly globular (in another female, sper‑

yellowish white; femur yellow on basal 2/5 or a little
more, and dark brown on the rest when viewed outward‑

matheca is also globular but somewhat smaller asly (border well defined) , while yellowish portion extend‑

widely unsclerotized, and with internal setae; spermath‑ing more apically beyond basal 1/2 when viewed inward‑ly (border not well defined) ; tibia white to yellowish

ecal duct with 2 additional ducts at base, which arewhite except apical 1/6 or 1/5 brownish black, withapical spermathecae.

shown in Fig. 8), well sclerotized except tubal baseequally short, ca. 0.3 x as long as main duct, but with nolarge area of white sheen on outer surface; tarsus brown‑

ish black; basitarsus somewhat dilated, ca. 4.8 x as long

Male. Body length ca. 2.6 mm. Head. Slightly wider

as its greatest width. Midleg: coxa blackish brown,
trochanter light brown except basal 1/2 yellow; femur
blackish brown except basal 1/5 or 1/4 yellow; tibia

whitish yellow; tibia white with apical 1/3 or 1/4 black‑than thorax. Upper eye consisting of 17 vertical col‑umns and 18 horizontal rows of large facets. Face andclypeus black, white‑pruinose, silvery iridescent whenilluminated, sparsely covered with long dark hairs.
Antenna composed of 2 + 9 segments, dark brown except
scape, pedicel and base of Ist flagellar segment dark
yellow; Ist flagellar segment somewhat elongate, ca.
1.6x as long as 2nd flagellar segment. Maxillary palp
with 5 segments, dark brown to blackish brown, propor‑

yellowish white except apical 1/6 blackish brown, withlarge area of white sheen on posterior surface; tarsus
blackish brown except basal 2/3 of basitarsus yellowish.

Hind leg: coxa blackish brown; trochanter whitish
yellow; femur blackish brown except basal 1/5 or 114
ish brown, with large area of white sheen on posterior

tional lengths of 3ird, 4th and 5th segments 1.0:1.3:2.5;

surface; tarsus blackish brown except basal 3/5 ofsensory vesicle (Fig. 3) small, oblong, ca. 0.2x as longbasitarsus and basal 1/2 of 2nd segment whitish yellow;
basitarsus (Fig. 4) parallel‑sided; calcipala moderately

ered with golden yellow, recumbent pubescence, as welldeveloped, as long as wide or slightly shorter than wide,and pedisulcus well developed at basal 1/3 of 2nd tarsal

scutum in certain angles of light with silvery iridescentas 3rd segment. Thorax. Scutum black, densely cov‑
as a few brown upright hairs on prescutellar area;

segment. A11 tarsal claws simple. Wing. Length ca. 2.2

spots, i.e., an anterolateral pair of spots on shoulders

mm. Costa with spinules and hairs; subcosta haired

which connect widely to posterior large spot on pre‑scutellar area through spots along lateral margins; ananterolateral pair of spots large, widely separated in
middle from each other. Scutellum brownish black, withexcept near apex bare; basal section of radial vein bare

except near apex where 1‑4 hairs are present; hair tuft

on stem vein brown. Abdomen. Basal scale light499

golden yellow pubescence as well as dark upright hairs.
Postscutellum brownish black, silvery iridescent whenilluminated, and bare. Pleural membrane and katepi‑
sternum as in. Legs. Foreleg: coxa yellow; tro‑
chanter light brown; femur light to dark brown, withbase somewhat paler; tibia light to dark brown withlarge median area on outer surface white, and shining
when illuminated; tarsus brownish black; basitarsus
somewhat dilated, ca. 5.8 >< as long as its greatest width.

Midleg: coxa brownish black; trochanter and femur
blackish brown; tibia whitish yellow with apical 2/5
blackish brown (its border not well defined) ; tarsus
blackish brown except basal 1/2 of basitarsus dark

yellow (border not well defined) . Hind leg: coxa
brownish black; trochanter yellow; femur and tibia lightbrown with base yellow and apical cap dark brown;
tarsus light brown; basitarsus (Fig. 5) enlarged, ca. 3.6 x

as long as its greatest width, and subequal in greatest
width to hind tibia; calcipala and pedisulcus well devel‑
oped. Wing. Length ca. 2.0 mm; other features as inexcept subcosta hairy with apical 1/4 bare. Abdomen.

Integument yellow, densely and elaborately covered
with round tubercles (cone‑shaped tubercles on postero‑dorsal surface) of different sizes; head with I facial and2 frontal pairs of simple trichomes, facial trichomes

medium in length and somewhat longer than frontal
ones; thorax on each side with 3 Iong trichomes eachwith 3‑4 branches mediodorsally, I bifid medium and 1
simple long trichomes anterolaterally, I simple longtrichome posterolaterally and 3 simple or bifid tri‑chomes (2 Iong and I medium) ventrolaterally, all longtrichomes rather stout. Gill (Fig. 18) with 8 short,
slender filaments in pairs (all short‑stalked) , diverging

widely not only vertically but also horizontally, with anangle formed at base by dorsalmost and ventralmost
filaments nearly 170 degrees; filaments somewhat
decreasing in length (from ca. 1.2 mm to 0.8 mm) andthickness (at least basal portion) from dorsal pair toventral one; basal portion of dorsalmost filament ca.
1.4x as thick as ventralmost filament, and slightly
thinner than interspiracular trunk; 2 filaments of all

Basal scale light brown medially, dark brown laterally,
with fringe of dark long hairs. Dorsal surface of abdom‑

pairs subequal in thickness to each other; all filaments
light yellow, tapered toward apex, with distinct annularridges and furrows forming elaborate reticulate surface

inal segments brownish black except basal 1/2 of 2nd

patterns, and densely covered with minute tubercles.

segment yellowish brown, with short dark hairs; terga 2,
5, 6 and 7 each with a pair of dorsolateral silvery spots,

Abdomen. Tergum I Iight brown, bare or sparsely

those on tergum 2 connected broadly to each otherside; tergum 2 pale, with I simple medium seta and 5
short spines (of which 4 are much stout) on each side;
terga 3 and 4 each with 4 hooked spines and I simple

medially. Genitalia (Figs. 9‑17). Coxite subquadrate
in ventral view, with stout hairs only near posterior
margin. Style elongate, much longer than coxite, gently
curved inwards, moderately covered with stout hairs on
ventral and lateral surfaces, and with single subterminal

spine; style widest subbasally, hence narrowed toward

tuberculate, with I simple or bifid medium seta on each

short spinous seta, on each side; tergum 8 with distinct
spine‑combs in transverse row, on each side; terga 7 and9 each with or without a few, much smaller spine‑
combs; terga 5 and 6 bare; terminal hooks absent.

midpoint, then slightly widened toward apical 1/4, there‑

Sternum 5 with a pair of bifid hooks submedially, on

after parallel‑sided up to apical 1/8, and ca. 3.4x as

each side; sterna 6 and 7 each with a pair of bifid innerlong as its greatest width, which is ca. 1.6x the narr‑
owest width at middle; style with distinct basal protu‑

and simple outer hooks widely spaced, on each side.

berance directed dorsomedially, with several small

moderate height, thinly woven; there appear to be somecone‑shaped spines along anterior margin and at apex.
Ventral plate in ventral view subquadrate (though its
posterolateral corners rounded) , somewhat longer thanwide, with prominent median process directed ventrally,

Cocoon (Fig. 19). Shoe‑shaped, with anterior collar ofopen spaces in webs of anterior collar, but it is difficult

to ascertain this because the entire surface of thecocoons was thickly cemented by silicic material.

of which apex is rounded and bare, posterior surface has

Mature larva. Unknown.

ca. 10 teeth in 2 vertical irregular rows, and antero‑ventral surface is covered with microsetae medially;

TYPE SPECIMENS. Holotype

basal arms strongly sclerotized, and divergent fromcollected from Maekham waterfall, Phare Province,each other. Paramere with several hooks. Mediannorthern Thailand, 10. XII. 1998, by Chaliow Kuvang‑sclerite thin, widened toward apical tip. Cercus small,short, with several hairs.

1 c l , reared from pupa, I pharate

kadilok and Chainarong Boonkemtong. Paratype I
same data as holotype.

Pupa Body length ca 2 8 mm. Head and Thorax, reared from pupa,,

and I pharate c?1 ,

500

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19
Figures 1‑19 Morphological characters of S. (S.) chaliowae sp. nov. 1,

fronto‑ocular area; 2 and 3, 3rd segments ofmaxillary palp with sensory vesicle inside (2, ; 3, c?1 ) ; 4 and 5, basitarsi and second tarsal segments of hind legs
showing calcipala and pedisulcus (4, ; 5, l ) ; 6,
genitalia in situ (ventral view; Ieft paraproct and cercus omitted)
showing 8th sternite (a) , anterior gonapophysis (b), right paraproct (c) , right cercus (d) , genital fork (e) , andspermatheca (f) ; 7, right paraproct and cercus (lateral view) ; 8, spermatheca of paratype ; 9, ( l genitalia in situ
(ventral view; Ieft coxite, style, and pararneres omitted) showing right coxite (a) , right style (b) and ventral plate(c) ; 10, right coxite and style (1ateral view) ; 11, right style (medial view) showing basal protuberance with spines
along anterior margin; 12, right style (ventrolateral view) ; 13 and 14, ventral plates (13, Iateral view; 14, end view) ;15, paramere (end view) ; 16, median sclerite (end view) ; 17, Ieft cercus (lateral view) ; 18, pupal gill filaments andinterspiracular trunk (1ateral view) ; 19, cocoon (lateral view) . Scale bars 1.0 mm for fig. 19; 0.1 mm for figs. 4, 5,
and 18; 0.05 mm for figs. 1, 6, 7 and 8; 0.03 mm for figs. 9‑17; 0.02 mm for figs. 2 and 3.

501

ECOLOGICAL NOTES. This species was found on

segment yellow. Maxillary palp with 5 segments,

fallen leaves in a fast flowing stream 3 m wide atbrownish black, proportional lengths of 3rd, 4th and 5th

Maekham waterfall (290 m in altitude) , where the watertemperature was 20'C. This species was found together
with S. fenestratum, S. quinquestriatum. S. nakhonense.

segments 1.0:1.2:2.3; sensory vesicle (Fig. 21) ellipsoidal,

S. tani and S. siamense.

ETYMOLOGY. The species chaliowae is named after
Dr. Chaliow Kuvangkadilok.

ca. 0.35 >< as long as 3rd segment. Maxillary laciniawith 11 or 12 teeth on each side. Mandible with ca. 25
inner and 13 outer teeth. Cibarium (Fig. 24) with ablunt, medial projection on posterior margin, and with‑out any tubercles. Thorax. Nearly as in S. chaliowae. Legs. Foreleg: coxa and trochanter yellow; femur
yellowish brown except basal 2/5 yellow; tibia white to

REMARKS. This species is assigned in the multi‑
striatum‑group of the subgenus Simulium (Simulium ) ,redefined by Takaoka and Davies (1996) , by the striatedscutum, simple claws of female adult, horn‑like basal
protuberance of the male style (Fig. 11), and the 8‑

whitish yellow except apical 1/5 black, with medianlarge area white iridescent when illuminated; tarsus
black, basitarsus dilated, ca. 4.9 >< as long as its greatest

filamented pupal gill (Fig. 18).

width. Midleg: coxa, trochanter and femur dark brown;
tibia white with apical 1/5 dark brown; tarsus darkbrown except basal 3/4 white. Hind leg: coxa dark

S. chaliowae is easily distinguished from the otherknown species of this species‑group by the shoe‑shaped

brown; trochanter pale yellow; femur brown with apicalcap dark brown and basal small area pale yellow; tibia

cocoon with an anterior collar of moderate height

white with apical 1/5 dark brown; tarsus dark brown
except basal 2/3 of Ist segment and basal 1/2 of 2nd(Fig. 19) in place of the fenestrate cocoon. The femaleof this species appears to be similar to that of S. hir ‑tinervis from Peninsular Malaysia (Takaoka andDavies, 1995) but differs by the much clearer coloring of
the mid femur and tibia, the basal portion of radial veinwith a few hairs near the apical end, and the smaller
spermatheca. The male of S. chaliowae is similar to S.

fenestratum from Sumatra and Thailand (Edwards,

segment white; calcipala and pedisulcus well developed,
as in S. chaliowae . A11 tarsal claws simple. Wing.

Length 2.1 mm. Costa with spinules and hairs; subcosta
haired except apical 1/4 or 1/5 bare; basal section ofradial vein bare; hair tuft on stem vein brown. Abdo‑men. Basal scale brown with fringe of pale long hair.
Dorsal surface of 2nd segment pale with posterior 1/2 or

1934; Takaoka and Saito, 1996), by having the hindless blackish, with a pair of dorsolateral silvery‑irides‑

basitarsus almost entirely darkened (Fig. 5) as well as
the ventral plate hairy on ventral surface (Fig. 9), butdiffers from it by the horn‑like basal protuberance ofcent spots broadly connected medially to each other;dorsal surface of remaining segments black, with shortthe style with many teeth along the anterior marginof 7th segment with a pair of small, round sternal plates(Fig. 11).

submedially. Genitalia. Almost as in S. chaliowae

black hairs, with terga 6, 7 and 8 shiny; ventral surface

except spermatheca nearly ovoid (Fig. 25) .

Simulium (Simulium) chainarongi Kuvangkadilok
and Takaoka sp. nov.

Male. Body length 2.6 mm. Head. As wide as thorax.
Upper eye consisting of larger facets in 20 horizontal

Fenrale. Body length ca. 2.6 mm. Head. Narrowerrows and in 20 vertical columns. Face and clypeus

than thorax. Frons black, shiny, Iargely bare medially,
with several dark hairs along each lateral margin and a
few hairs above antennal base; frontal ratio 1.4:1.0:1.7;
frons‑head ratio 1.0:4.3. Fronto‑ocular area (Fig. 20)

black, white‑pruinose, silvery iridescent when illurninat‑

moderately developed. Clypeus black, shiny, gray‑yellow; Ist flagellar segment elongate, ca. 2.0X as longed, sparsely covered with long dark hairs. Antennacomposed of 2 + 9 segments, brownish black except
scape, pedicel and basal 2/5 of Ist flagellar segment

pruinose, iridescent when illuminated, moderately cov‑

as 2nd one. Maxillary palp with 5 segments, brownish

ered with dark stout hairs except medial portion of

black, proportional lengths of 3rd, 4th and 5th segments

upper 1/2 widely bare. Antenna cornposed of 2+9

1.0:1.3:2.7; sensory vesicle (Figs. 22 and 23) srnall, nearly

segrnents, coloration differing between 2

globular, with small opening. Thorax. As in S. chali‑owae c l . Legs. Foreleg: coxa whitish yellow; tro‑

, i.e., in 1

, brownish except scape, pedicel and Ist flagellar
segment yellow, while in the other, brownish except
scape, pedicel and ventral surface of a few basal flagel‑lar segments and dorsal surface of base of Ist flagellar

chanter light brown on outer surface but yellow on
inside surface; femur light brown to dark brown except
basal 2/5 on inside surface dark yellow; tibia brownish

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Figures 20‑36 Morphological characters of S. (S.) chainarongi sp. nov. 20,
fronto‑ocular area; 21 and 22, 3rd segmentsof maxillary palp (21, ; 22, c?1); 23, sensory vesicle of a different c l ; 24, posterior part of cibarium; 25,
spermatheca; 26, male hind basitarsus; 27, (?1 genitalia in situ (ventral view; Ieft coxite, style and pararneres omitted) ;28, right style (ventrolateral view) ; 29 and 30, ventral plates (29, Iateral view; 30, end view) ; 31, pupal gill filaments

and interspiracular trunk (lateral view) ; 32, cocoon (lateral view) ; 33, Iarval cephalic apotome; 34, apical part oflarval mandible; 35, Iarval hypostomium; 36, median portion of larval head capsule (ventral view) showing large
postgenal cleft. Scale bars 1.0 mm for fig. 32; 0.1 mm for figs. 26, 31, 33 and 36; 0.05 mm for figs. 20, 25 and 35;
0.03 mm for figs. 27‑30; 0.02 mm for figs. 21, 22, 23 and 34.

503

black with median large portion white on outer surface,which is iridescent when illuminated; tarsus brownish

collar of variable heights, thickly woven, without o pen

spaces in webs.

black, basitarsus moderately dilated, ca. 5.5 x as long as

its greatest width, and with a dorsal hair crest ofmoderate length. Midleg: brownish black except basal

Mature larva. Body length 4.8‑5.0 mm. Body color
grayish black or greenish black. Cephalic apotome

2/3 of tibia and basal 1/2 of basitarsus white to whitish(Fig. 33) dark yellow to light yellowish brown except
anterior 1/2 pale, narrowly darkened along posterior
margin, and with positive head spots, though antero‑
median spot faint, and posterolateral ones indistinct.
Antenna composed of 3 segments and apical sensillum,

yellow; tibia largely white‑iridescent on posterior sur‑face when illuminated. Hind leg: coxa brownish black;trochanter yellow; femur brownish black with basenarrowly yellow; tibia brownish black with base narrow‑

ly white; tarsus brownish black except basal 2/5 ofbasitarsus and basal 1/2 of 2nd segment dark yellow;
basitarsus (Fig. 26) expanded, ca. 4.1x as long as its

longer than stem of labral fan; proportional lengths ofIst, 2nd and 3rd segments 1.0:1.2‑1.4:0.9. Labral fan withgreatest width, and ca. 0.75 x as wide as hind tibia whichca. 42 main rays. Mandible (Fig. 34) with comb‑teeth,
decreasing in length from Ist to 3rd; mandibular serra‑

is subequal in greatest width to hind fernur; calcipala

tions composed of I Iarge and I small teeth, and without

and pedisulcus well developed. Wing. Length 2.0 mm;
other features as in except subcosta bare. Abdomen.
Basal scale brownish black with fringe of dark long
hairs; dorsal surface of abdomen dark brown to brown‑

supernumerary serrations. Hypostomium (Fig. 35) withan anterior row of 9 teeth, all pointed apically; 2 outer‑

ish black, with dark short hairs, and terga 2, 5, 6 and 7
each with a pair of silvery iridescent large spots dor‑most teeth of 6 intermediate teeth, as well as medianand corner teeth, moderately developed; Iateral serra‑
tions moderately developed; hypostomal bristles 4 or 5,
slightly divergent posteriorly from lateral margin on

solaterally; those on tergum 2 broadly connected ineach side. Postgenal cleft (Fig. 36) very large, rounded,

middle to each other. Genitalia (Figs. 27‑30). Similar

constricted basally, and ca. 3.5x as long as postgenal
bridge; elongate transverse spot on each side of post‑
genal cleft faintly positive. Thoracic cuticle bare.

to those of S. chaliowae c l except slight differences instyle and ventral plate. Style straight or only slightly

curved inwards, widest subbasally, slightly narrowed
1/6, ca. 3.1 >< as long as its greatest width, which is ca.

Abdominal cuticle with a dorsolateral pair of small
protuberances each on segments 1‑6, and with no dis‑tinct setae except last segment, moderately covered

1.2x width at middle portion. Ventral plate in ventralwith short, colorless setae on each side of anal sclerite.

view subquadrate but rounded posteriorly; posterior

Rectal gill compound, each of 3 Iobes with 7‑9 finger‑
like secondary lobules. Anal sclerite X‑shaped, withanterior arms much shorter, ca. 0.6x as long as poste‑rior ones. Accessory sclerite and ventral papillae
absent. Posterior circlet with ca. 92 rows of up to 16
hooklets per row.

toward midpoint, then nearly parallel‑sided up to apicalsurface with ca. 20 teeth in 2 vertical irregular rows.

Pupa. Body length ca. 3.0 mm. Head and Thorax.
Integument as in S. chaliowae except coloring dark
yellowish brown. Gill (Fig. 31) with 8 short, slenderfilaments in pairs, dorsal and ventral pairs short‑stalked

and 2 middle pairs medium‑stalked, all lying almost in
one vertical plane; an angle formed at base by dorsal‑
most and ventralmost filaments ca. 120 degrees; fila‑"ments somewhat decreasing in length (from ca. 1.2 mm
to 0.8 mm) and thickness (at least basal portion) fromTYPE SPECIMENS. Holotype, reared from pupa,

collected from Klang Lum Duan, Yod Dome Wild Life
Sanctuary, Ubonratchathani Province, northeastern
Thailand, 16. IX. 1998, by Chaliow Kuvangkadilok andChainarong Boonkemtong. Paratype 2, reared

dorsal pair to ventral one; 2 filaments of all pairs sube‑from pupa, I c71 , reared from pupa, and 3 mature larvae,qual in thickness to each other, except those of inner one

same data as holotype.

of middle 2 pairs somewhat different; basal portion of 2
filaments of dorsal pair almost 2.0 X as thick as that of

ECOLOGICAL NOTES. This species was collected

2 filaments of ventral pair, and slightly thicker thaninterspiracular trunk; all filaments light yellowish

brown, tapered toward apex, with distinct annularridges and furrows, and densely covered with minutetubercles. Abdomen. As in S. chaliowae except tergum
7 bare. Cocoon (Fig. 32). Shoe‑shaped, with anterior

from trailing grasses, roots and fallen leaves in a strearn

2‑10 m wide with water temperature of 26.5'C, at Klang
Lum Duan (140 m in altitude) . This species was also

found in Huai Luang waterfall (300 m in altitude) ,where the stream was fast flowing, shaded, and 5 mwide, and the water temperature was 24'C. This species

504

was collected together with S. nakhonense. S. parahiyan ‑gum. S. siamense and S. asakoae.

sheen on outer surface; tarsus brownish black; basitar‑sus somewhat dilated, ca. 5.3x as long as its greatest

width. Midleg: coxa blackish brown; trochanter andETYMOLOGY. The species chainarongi is named after
Mr. Chainarong Boonkemtong.

femur entirely yellow; tibia white to yellowish white
except apical tip dark yellow, with large area of white

REMARKS. This new species resembles S. chaliowae bysheen on posterior surface; tarsus blackish brown except
basal 1/2 of basitarsus whitish yellow. Hind leg: coxa

having the shoe‑shaped cocoon with an anterior collar,
as well as the similar genitalia of both sexes of adults,
but is distinguished from the latter species in the female

by the mid and hind fernora almost entirely darkened, inthe male by the hind basitarsus dark yellow on the basal
2/5 and brownish black on the apical 3/5 (Fig. 26), thestyle not narrowed medially (Fig. 28) and the ventral
plate with more numerous teeth on the posterior surface(Fig. 30), and in the pupa by the dorsalmost filament

blackish brown; trochanter and femur entirely yellow,
with apex of femur slightly darkened; tibia white toyellowish white with apical 1/5 brownish black, withlarge area of white sheen on posterior surface; tarsus
blackish brown except basal 1/2 or a little more ofbasitarsus and basal 1/2 of 2nd segment yellowish white;basitarsus parallel‑sided; calcipala and pedisulcus mod‑twice as thick in the basal portion as the lowermost one,erately developed. ' A11 tarsal claws simple. Wing.
Length ca. 2.5 mm. Costa with spinules and hairs;
subcosta haired except apical 1/5 or 1/3 bare; basal

and subequal to, or slightly thicker than, the interspir‑

section of radial vein bare; hair tuft on stem vein brown.

acular trunk (Fig. 31). The larva of S. chainarongi is

Abdomen. Basal scale dark yellow to light brown, withfringe of pale long hairs, of which basal portions are

similar to that of S. hirtinervis Edwards, from Peninsu‑

lar Malaysia (Takaoka and Davies, 1995) by having thedorsal protuberances on the abdomen. However in thelatter species these protuberances are present not only
on the abdominal segments 1‑6 but also on the segments
7 and 8.
Simulium ( Simulium) triglobus Takaoka and Kuvangkadilok
sp. nov.

Female. Body length 2.6 mm. Head. Narrower thanthorax. Frons brownish black, shiny, with several darkstout hairs along lateral margins; frontal ratio 1.6:1.0:1.4;

frons‑head ratio 1.0:3.7. Fronto‑ocular area well devel‑
oped, similar to that of S. chaliowae. Clypeus brown‑

ish･ black, shiny, gray‑pruinose, iridescent when il‑luminated, moderately covered with dark stout hairs.
Antenna composed of 2 + 9 segrnents, yellow on scape,pedicel and a few basal flagellar segments, and brownish
on rest flagellar segments, its border not well defined.

Maxillary palp with 5 segments, Iight brown, propor‑
tional lengths of 3rd, 4th and 5th segments 1.0:1.2:2.4; 3rdsegment (Fig. 37) of normal size, with sensory vesicle
ellipsoidal, ca. 0.36 x as long as 3rd segrnent. Maxillarydark. Dorsal surface of 2nd segment pale yellowish
white with narrow area just before posterior margin
brownish black; dorsal surface of 3rd to 6th segments
brownish black, and that of remaining posterior segmnts
light yellowish brown mottled with many blackish spots,with short dark hairs; terga 6‑8 shiny. Ventral surface
of 7th segment with a pair of small submedial sternal
plates. Genitalia (Figs. 38‑42). Sternite 8 moderately
sclerotized, narrowly bare in middle, covered with 32‑36
stout hairs on each lateral side. Anterior gonapophyses
triangular, moderately separated from each other medi‑

ally, mernbraneous, covered with numerous microsetae
and 3‑5 stout hairs per each; narrow bare area along
inside and posterior margins very thin and transparent(then often overlooked) . Genital fork of inverted‑Y
form, with narrow, well sclerotized stem, which is atbase widened and angulated against arms, as seen later‑
ally (Fig. 42); arms broad basally, each with distinct
wide plate‑like projection directed anteriorly. Para‑proct much protruding ventrally, 1.8‑2.0>< as wide as
10ng, with numerous stout hairs on lateral and ventral
surfaces. Cercus short, ca. 2.0X as wide as long, with

Mandible with ca. 24 inner teeth and ca. 10 outer teeth.

nurnerous stout hairs, with rounded or medially slightly
concave posterior margin, when viewed laterally (Figs.
39 and 40). Spermathecae 3 in number, all well sclerot‑

Cibarium as in S. chainarongi . Thorax. Similar tothat of S. chainarongi
except scutellum dark yellow,

ized and with internal setae, but differing in size andshape; I principal spermatheca, which has longer sper‑

postscutellum dark brown, and recumbent pubescenceon scutum and scutellum yellow. Legs. Foreleg: coxa,

n athecal duct, is large and pear‑shaped, and 2 accessory

trochanter and femur entirely yellow; tibia white except

ecal duct, are equally small, oblong and curved.

lacinia with 15 inner teeth and 13 or 14 outer teeth.

apical 1/5 brownish black, with large area of whiteones, both of which bear somewhat shorter spermath‑505

Male. Unknown.

ible) . Antenna composed of 3 segments and apicalsensillum, Ionger than stem of labral fan; proportional

Pupa. Body length ca. 2.8 mm. Head and Thorax.

lengths of Ist, 2nd and 3rd segments 1.0 : 1.5 : 0.7. Labral

Integument dark yellow to light yellowish brown, dense‑ly and elaborately covered with round tubercles (cone‑
shaped tubercles on posterodorsal surface of thorax);
head with I facial pair of bifid medium trichomes, and 2
frontal pairs of bifid and trifid medium trichomes widely

fan with 56‑60 main rays. Mandible (Fig. 45) withspaced; thorax on each side with 3 medium trichomes
each with 4‑5 branches mediodorsally, I Iong trichomewith 5 branches and I medium trichome with 4 branches
anterolaterally, 1 Iong trichome with 2‑4 branches pos‑

terolaterally, and I medium trichome with 3 or 4
branches and 2 bifid long trichomes ventrolaterally, alllong trichomes rather stout. Gill (Fig. 43) with 8 short,
slender filaments in pairs (all short‑stalked) , widely

diverging vertically but not so horizontally, with anangle formed at base by dorsalmost and ventralmost
filaments nearly 140 ' degrees; filaments somewhat

comb‑teeth, decreasing in length from Ist to 3rd; man‑

dibular serrations composed of I Iarge and I medium
teeth, and without supernumerary serrations; most teeth

blunt, and rounded apically. Hypostomium (Fig. 46)
with an anterior row of 9 teeth, all rounded apically; 2outermost teeth of intermediate teeth, as well as medianand corner teeth, moderately developed; Iateral serra‑
tions moderately developed; hypostomal bristles 5‑7,
somewhat divergent posteriorly from lateral margin oneach side. Postgenal cleft (Fig. 47) Iarge, rounded
anteriorly, not constricted basally, ca. 2.0X as long as
postgenal bridge; elongate transverse spot on each sideof postgenal cleft negative. Thoracic cuticle bare.
Abdominal cuticle without dorsolateral protuberances

decreasing in length (from ca. 1.0 mm to 0.6 mm) fromand distinct setae except last segment moderately cov‑
ered with short, colorless setae on each side of anal

dorsal pair to ventral one, and also slightly decreasing insclerite. Rectal gill compound, each of 3 Iobes with 10‑

thickness frorn dorsal pair to ventral one, dorsalmost
filament is ca. 1.3x as thick as lowermost one, and ca.
0.7x as thick as interspiracular trunk; all filaments
unpigmented or light yellow, tapered toward apex, with
distinct annular ridges and furrows forming elaborate
reticulate surface patterns, and densely covered with

13 finger‑like secondary lobules. Anal sclerite X‑
shaped, with anterior arms much shorter, ca. 0.6x as
long as posterior ones. Accessory sclerite and ventral

minute tubercles. Abdomen. Tergum I Iight brown,

TYPE SPECIMENS. Holotypebare, with I bifid medium seta on each side; tergum 2
pale, with I bifid medium seta and 5 short spines (ofwhich 4 are much stouter) on each side; terga 3 and 4
each with 4 hooked spines and I simple short spinous

collected from Tontong waterfall, Doi Phuka Nationalseta on each side; tergum 8 with distinct spine‑combs intransverse row on each side; terga 5, 6, 7 and 9 bare;terminal hooks absent. Sternum 5 with a pair of simple
inner and bifid outer hooks submedially on each side;sterna 6 and 7 each with a pair of simple inner and outer

hooks widely spaced on each side. Cocoon (Fig. 44).
Shoe‑shaped with anterior collar of moderate height,
roughly woven anteriorly, then leaving some large open
spaces in webs of anterior collar, and also many small

papillae absent. Posterior circlet with 112‑134 rows of
up to 22 hooklets per row.

, reared from pupa,

Park, Nan Province, northern Thailand, 9. XII. 1998, by

Chaliow Kuvangkadilok and Chainarong Boonkemtong.
Paratype I , reared from pupa, I pupa, and 2 maturelarvae, same data as holotype.

ECOLOGICAL NOTES. This species was collectedfrom fallen leaves in a small stream 0.5‑1.0 m wide at
Tontong waterwfall (altitude 500 m) , where the watertemperature was 19'C. It was found with S. rufibasis.

ETYMOLOGY. The species name trigl obus refers to

the three spermathecae.

open spaces near anterior margin; no ventrolateralextension.

REMARKS. This new species is also placed in themultistriatum‑group within the subgenus Simulium

Mature larva. Body length 5.4‑6.0 mm. Body color(Simulium) by the striated scutum and simple claws ofgreenish brown or grayish black. Cephalic apotome paleyellowish white (though anterior 1/2 and along rightfemale adult, and the 8‑filamented pupal gill.

lateral margin somewhat darkened in 1 Iarva) , andalong posterior margin narrowly light brown; head spots
indistinct (though in 1 Iarva posteromedian longitudinalother known species of the same species‑group, by the
cocoon of corbicular shape (Fig. 44). The pupa of thisspecies also differs from those of S. chaliowae and S.

spot and mediolateral round spots are faintly discern‑

chainarongi, described above, by the more branched

The pupa of S. triglobus is easily separated from the

506

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Figures 37‑47 Morphological characters of S. (S.) triglobus sp. nov. 37, 3rd segment of maxillary palp; 38, genitalia
in situ (ventral view) showing I principal and 2 accessory spermathecae; 39 and 40, paraprocts and cerci (39,
holotype; 40, paratype) ; 41, 3 spermathecae (a, principal; b and c, accessory) of paratype; 42, genital fork (lateralview) ; 43, pupal gill filaments and interspiracular trunk (1ateral view) ; 44, cocoon (1ateral view) ; 45, apical tip oflarval mandible; 46, Iarval hypostornium; 47, median portion of larval head capsule (ventral view) showing postgenal
cleft. Scale bars 1.0 mm for fig. 44; 0.1 mm for figs. 43 and 47; 0.05 mm for figs. 38‑42 and 46; 0.02 mm for figs 37
and 45.

507

thoracic trichomes, as well as the corbicular cocoon, andfrom S. chainarongi by the slenderer gill filaments(Fig. 43).

The female of this species differs from the former
two related species by the clearer leg coloring, and thegenital fork with a wide plate‑like projection directed

forwards on each arm (Fig. 38). However, the moststriking character found in this new species is the pres‑ence of two more spermathecae in addition to the single,
typical one (Figs. 38 and 41) . A11 other black‑fly species

have a single spermatheca (Crosskey, 1990), though
they retain two additional spermathecal ducts arisingfrom the main spermathedal duct near its base. Bothadditional ducts are, however, much shorter than themain one, as shown in Fig. 6. Three spermathecae areconsidered to be the evolutionarily basic complement
for the Diptera (Downes, 1968). In other nematocerous
insects, such as mosquitoes and biting midges, the num‑
ber of spermathecae has been known to vary from one
to three across genera or by species (Lane and Cross‑key, 1993) . Therefore, it was not unexpected to encoun‑

ter a species carrying two or three spermathecae even in
the small family Simuliidae. Our finding is the first to

ally tapered toward apex; surface of filaments and basal
inflated portion light yellowish brown and thick (except
bulb at base pale and thin) , and irregularly wrinkledwith many transverse or oblique furrows, and densely
covered with minute tubercles, elaborately forming
numerous srnall reticulate surface patterns near base, as

shown in Fig. 51. Abdomen. Tergum I sparsely tuber‑culate, with I simple long slender seta on each side;
tergum 2 with I simple long slender seta and 5 spinous
setae, on each side; terga 3 and 4 each with 4 distinct
hooked spines directed forward on each side; terga 5 and6 without spine‑combs, but terga 7‑9 each with distinct

spine‑combs directed backward in transverse row,
together with numerous comb‑like groups of minutespines, on each side; terminal hooks absent. Sternum 5
with 2 bifid hooks submedially, on each side; sterna 6
and 7 each with I bifid hook submedially and I bifid or
simple hook laterally, on each side; sterna 4‑8 each withnumerous comb‑like groups of minute spines; sternum 9
bare. Cocoon (Fig. 52). Simple, wall‑pocket‑shaped,
light brown, intactly woven leaving no open spaces in
the wall, and with no ventrolateral extension; individual

threads invisible; 2.8 mm long x 1.0 mm wide.

record a species with three spermathecae in the
Sirnuliidae. Bernard (1974) reported two spermathecae

Mature larva. Body length 6.0‑6.5 mm. Body colorof almost the same size in a freak specimen of S.
erythrocephalum. Hunter and Adler (pers. commun.)yellowish. Cephalic apotome yellow to yellowish brownwith median small area brown between posterolateralspots just before posterior margin; head spots brown
except posterolateral spots pale showing negative pat‑tern. Antenna with 3 segments and apical sensillum,
longer than stem of labral fan; proportional lengths ofalso found aberrant females with two spermathecae ofdifferent size from colony‑reared S. vittatum.

Simulium baimaii Kuvangkadilok and Takaoka sp.
nov.

Ist, 2nd and 3rd segments 1.0:1.2:0.7. Labral fan with ca.

60 main rays. Mandible (Fig. 53) with comb‑teeth

Female and Male. Unknown.

decreasing in size from Ist to 3rd; mandibular serrations

composed of 1 Iarge and I small teeth, without supernu‑

Pupa. Body length (excluding gill filaments) 3.1 mm.
Head. Integument light yellowish brown, moderately
covered with round tubercles; antennal sheath (Fig. 48)
with 8 small ridges on outer surface, each of which issparsely covered with round tubercles; face on each sidewith I simple long slender trichome; frons on each sidewith 2 simple long slender trichomes widely separated

merary serrations. Hypostomium (Fig. 54) with ananterior row of 9 teeth; each corner tooth moderately
developed; median tooth and intermediate teeth bent tosome extent dorsally, then invisible from the ventral
side; hypostomal bristles 5 or 6 in number, slightly
divergent posteriorly from lateral margin on each side.
Postgenal cleft (Fig. 54) well developed, round apically,

from each other. Thorax. Integument light yellowish
brown, sparsely covered with round tubercles except

longer than wide, ca. 2.5x as long as postgenal bridge.

anterior portion moderately covered with similar tuber‑cles; thoracic trichomes all simple, Iong and slender‑2
dorsomedially, 2 anterolaterally, I posterolaterally, andposteriorly frorn Ist to 8th segment and abruptly taper‑
ed toward posterior tip; abdominal cuticle almost bare

3 ventrolaterally. Gill (Figs. 49 and 50) with 2 filaments

colorless setae dorsally and laterally. Rectal gill com‑arising from inflated trunk, ca. 2 mm long from its baseto apex of filaments, and with sornewhat swollen bulb atbase; 2 filaments subequal in size to each other, gradu‑

pound, each of 3 Iobes with 12‑14 finger‑1ike secondary

Thoracic cuticle bare. Abdomen gradually enlarged

except last segment moderately covered with short,

10bules. Anal sclerite (Fig. 56) X‑shaped, with its
central portion and area between anterior arms broadly

508

'f
tt･

:i

' ..‑'5;{tt' 9R j' l
' 'f

j f' s: rt '

c･ ‑

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/f. !

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50

51

48

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1

¥

r'

52

49

53
e

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I ;1; :.f l . J

l"li' L';:t( l'!'fl I1 1 1 'sU:b'Ct,:t.. '

55

,,.tt

56

/ J b
54

Figures 48‑56 Morphological characters of S. baimaii sp. nov. 48, pupal antennal sheath (lateral view) ; 49, pupal gill andinterspiracular trunk (1ateral view) ; 50, basal 1/3 of pupal gill (dorsal view) ; 51, reticulate pattern on the surface of
inflated portion of pupal gill; 52, cocoon (lateral view) ; 53, apical tip of larval mandible; 54, Iarval hypostornium; 55,

larval head capsule (ventral view) showing postgenal cleft; 56, anal sclerite. Scale bars 1.0 mm for fig. 52; 0.2 mm
for figs. 49, 50 and 55; 0.05 mm for figs. 48, 54 and 56; 0.02 mm for fig. 53; 0.01 mm for fig. 51.

sclerotized; anterior arms broad, and ca. 0.7 x as long as dibular serrations of the larva. However, its subgenericposterior ones; accessory sclerite and ventral papillae assignment is not made due to lack of adult specirnens.

absent. Posterior circlet with 90‑102 rows of up to 16 The pupa of S. baimaii is characterized by the gill

hooklets per row. having two filaments on each side (Fig. 49) , a characternot found in Simuliidae of the Oriental Region, though S.

TYPE SPECIMENS. Holotype pupa, collected from (Nevermannia) taulingense from Taiwan has the pupal
Phra Aung waterfall, Phu Kradung National Park, Loei gill composed of two bulbs (Takaoka, 1979). Sirnilar
Province, northeastern Thailand, 25. X. 1995 and 23. V. reduced number of gill filaments (i.e., two), though
1996, by Narumon Sangpradub and Chutima Hanjavanit. different in shape, has been reported in one species ofParatype 2 mature larvae, same data as holotype. the subgenus Simulium (Byssodon) from Africa (Cross‑key, 1969) and in several species of the subgenus

ECOLOGICAL NOTES. This species was found on Simulium (Hearlea), and in one species of the genus
stones in a fast flowing stream 3 m wide at Phra Aung Mayacnephia, from Central America (Dalmat, 1955).
waterfall (altitude 1,124 m) . No other species wasfound with this new species. ACKNOWLEDGEMENTSETYMOLOGY. The species baimaii is named after We thank Dr. Narumon Sangpradub and Dr.

Prof. Visut Baimai. Chutima Hanjavanit, Department of Biology, Khon

Kaen University, Thailand, for their generosities and
REMARKS. S. baimaii sp. nov. is easily assigned to the kindness in lending the valuable specimens. Our appreci‑
genus Simulium s.1., defined by Crosskey (1969), by the ation goes to Mr. Thavaron Supaprom for his help innormal onchotaxy of the pupal abdomen and the shape collection of black‑fly larvae. We are grateful to Dr. D.

of the cephalic apotome, hypostomal teeth and man‑ M. Davies, Professor emeritus, McMaster University,

509

Canada, and Dr. R.W. Crosskey, the Natural History
Museum, U.K., for reading the manuscript and giving
valuable suggestions. Thanks are also due to Dr. D.A.

onchocerciasis. Smithson. mis. Collns. 125, 1‑425
6 ) Downes, J.A. (1968): Notes on the organs and processesof sperm‑transfer in the lower Diptera. Canadian Ento‑
mologist, 100, 608‑617

Craig, Professor emeritus, University of Alberta,
Canada, Prof. P.H. Adler, Clemson University, USA,7 ) Edwards, F.W. (1934): Deutsche Limnologische Sunda‑

and Dr. D.C. Currie, Royal Ontario Museum, Canada, fortheir useful information on multiple spermathecae ofdipteran insects. This work was supported by the TRF/

Sumatra. Arch. Hydrobiol., 13 (Suppl. 5), 92‑138
8 ) Lane, R.P. and Crosskey, R.W. (1993): Medical Insects

BIOTEC Special Program for Biodiversity Research9 ) Takaoka, H. (1979): The black flies of Taiwan (Dipte‑

and Training grant BRT 139007 to CK, and in part by
the Grant‑in‑Aid of the Ministry of Education. Science
and Culture, Japan (No. 11670246) to HT.

REFERENCES
1 ) Bernard, M.R. (1974): Existence de deux spermath queschez une Simulie (Dipt.). Entomologiste, 30, 73‑74

2 ) Crosskey, R.W. (1969): A re‑classification of the
Simuliidae (Diptera) of Africa and its islands. Bull. Br.
Mus. Natur. Hist. (Entomol.) suppl. 14, 1‑195

3 ) Crosskey, R.W. (1990): The Natural History of Black‑flies. ix+711 pp., John Willy & Sons. Chichester.

4 ) Crosskey. R.W. and Howard, T.H. (1997): A New
Taxonomic and Geographical Inventory of WorldBlackflies (Diptera: Simuliidae) . 144 pp.. The Natural

History Museurn, London.

5 ) Dalmat, H.T. (1955): The black flies (Diptera:
Simuliidae) of Guatemala and their role as vectors of

Expedition. The Simuliidae (Diptera) of Java andand Arachnids. xv+723 pp., Chapman & Hall, London.
ra: Simuliidae) . Pac. Insects, 20, 365‑403

10) Takaoka, H. (1983): The blackflies (Diptera:
Simuliidae) of the Philippines. xi+199 pp., Japan Soci‑ety for the Promotion of Science, Tokyo.
11) Takaoka, H. and Adler, P.H. (1997) : A new subgenus,

Simulium (Daviesellum), and a new species, S. (D.)
courtneyi, (Diptera: Simuliidae) from Thailand andPeninsular Malaysia. Jpn. J. Trop. Med. Hyg., 25, 17‑27
12) Takaoka, H. and Davies, D.M. (1995) : The Black Flies(Diptera: Sirnuliidae) of West Malaysia. viii+175 pp.,
Kyushu University Press, Fukuoka.
13) Takaoka, H. and Davies, D.M. (1996): The Black Flies(Diptera: Simuliidae) of Java, Indonesia. viii+81 pp.,
Bishop Museum Bull. Entomol. 6.. Honolulu.
14) Takaoka, H. and Saito. K. (1996): A new species andnew records of black flies (Diptera: Simuliidae) fromThailand. Jpn. J. Trop. Med. Hyg., 24, 163‑169

15) Takaoka, H. and Suzuki. H. (1984): The blackflies(Diptera: Simuliidae) from Thailand. Jpn. J. Sanit.
Zool., 35, 7‑45

511

J pn. J. Trop. Med. Hyg., Vol. 27, No. 4, 1999, pp. 511 515

A SURVEY ON HELMlNTHIC INFECTIONSIN TWO RURAL COMMUNITIES IN NEPAL
SHlNJIRO HAMANO1', SHIGERU KOBAYASH12, TETSURO OGAK13, MASATAKA KOGAl
MASUMI KAWASAK13, KAZUE IT04, ATSUSHI SAIT03, MORIYASU TsUJ15
SHOJI TOKUNAGA6, SASHI SHARMA7, GOPAL P. ACHARYA7 AND TERUKAZU KAWASAK13
Received June 18, 1999/Accepted September 6, 1999

Abstract: This study was carried out to elucidate the prevalence of intestinal helminthic infections in ruralcommunities in Nepal. Of 231 inhabitants randomly sampled in Kotyang and Judigaun, 140 (60.6%) were
found to be infected with some soil‑transmitted helminths. The highest prevalence was observed in
hookworm infection (52.8%), followed by Trichuris trichiura (18.6%) and Ascaris lumbricoides (11.3%)
infections. Sorne inhabitants harboured Vampirolepis nana and liver fluke. The female group aged 60 years
old and more showed significantly higher T. trichiura infection rate than the male group with the same age(p<0.05) , while no relationship was detected between proportion of T. trichiura infection and age based on
10gistic regression test (p=0.07) . Serum lgE Ievels of Nepalese were shown to be far higher than common
Japanese levels, suggesting the repeated infections with these helminths.
Key words: Nepal, intestinal parasite, IgE, epidemiology

INTRODUCTION
Soil‑transmitted helminths or geo‑helminths are ofted helminthic infections in rural communities for the
future control. Thus the present study was undertaken
to estimate the prevalence in two rural communities.

great importance in the health sector in developing
countries with poor socio‑environmental conditions. It
is estirnated that some 3.5 billion people are infectedwith these helminths (Bundy et al., 1996; Chan et al.,
1994; Chan, 1997) in the world. Mixed infections withThis study was performed in two rural communities(Kotyang and Judigaun) Iocated in a hill area of Kab‑

several different parasites (e.g., hookworms and A.
lumbricoides) are commonly seen. Although the mortal‑

hrepalanchok District, 30 km away from Kathmandu,
the capital of Nepal, from February to March, 1998.

ity rate by these infections is rather low, these infections

Kotyang is in Anaikot Village Development Committee,

are recognized as a serious public health problem

the settlement of which extends from 1,100 to 1,300 m inbecause of the high prevalence (Bundy et al., 1996; Chanet al., 1994; Chan, 1997). A. Iumbricoides may contributeto protein‑energy malnutrition, retarded physical activ‑

altitude where malaria is not endemic. The other local‑

MATERIALS AND METHODS

million and most of the people live in rural areas, it is

ity is Judigaun ,in Mahadevstan Village Development
Committee located in a hot valley where malaria has
been endemic. The altitude is ca. 900 m. The inhabit‑
ants of Kotyang are mainly cornposed of hill peoples
such as the Tamangs and the Parbate Hindus, whereas
those of Judigaun are the Rai Danuwars. The inhabit‑
ants of Kotyang usually eat rice, corn, wheat flour,

important to elucidate the prevalence of soil‑transmit‑

potato, green and other vegetables, milk, chan, which is

ity and poor growth (Adams et al.. 1994). Hookworm
or T. trichiura contributes to iron deficiency anernia(Stoltzfus et al., 1996; Roche and Layrisse, 1966).

Since Nepal currently has a population of over 23

1 Department of Microbiology and Immunology (Parasitology) , Graduate School of Medical Sciences, Kyushu University,
2

3

Maidashi 3‑1‑1, Higashi‑ku, Fukuoka 812‑8582, Japan ( *correspondence author)
Department of Human Geography, Graduate School of Letters, Osaka University, Toyonaka, Osaka 560‑8532
Institute of Health Science, Kyushu University, Kasuga, Fukuoka 816‑8580

4Nakamura Gakuen University, Fukuoka 814‑0198

56Department of Parasitology and Tropical Medicine, Faculty of Medicine, Kyorin University, Mitaka, Tokyo 181‑8611
Department of Preventive Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan
Institute of Medicine, Tribhuvan University, Kathmandu, Nepal

7

512

Table I Age and sex distribution of inhabitants examined
Age (in years)

Male

Female

Total

10‑19

60 <

10 (8.9%)33 (29.5%)
26 (23.2%)
8 (7.1%)9 (8.0%)26 (23.2%)

13 (10.9%)
38 (31.9%)
19 (16.0%)
20 (16.8%)
10 (8.4%)
19 (16.0%)

23 (10.0%)71 (30.7%)
45 (19.5%)
28 (12.1%)
19 (8.2%)
45 (19.5%)

Total

112 (100%) 119 (100%) 231 (100%)

20‑29
30‑39
40‑49
50‑59

(48 . 5%) (100%)(51 . 5%)a local alcoholic drink, other beverages, and a small
amount of meat, egg, sugar and oil (Ito et al.. 1993),

whereas those of Judigaun sometimes eat freshwater
fish in addition to above‑mentioned foods. Most houses

have a lavatory. Fecal and blood samples were col‑lected in these communities. Feces of 231 inhabitants(10 years old and more) in Kotyang and Judigaun(Table 1) were examined for intestinal helminthic infec‑

tions by MGL method after fixation in 10% formalin.
Sera collected and preserved under ‑ 20'C were trans‑ferred to Japan, and the total serum lgG and lgE were
measured by ELISA for 175 sarnples at CRC Co. (Fukuo‑ka, Japan) . Before survey, we obtained consent ofcommunity leaders and villagers. The association
between the infection rate and age was tested with thelogistic regression. Likelihood ratio test based on thelogistic model was used to test the sexual difference inthe relationship between the infection rate and age.
These statistical analyses were conducted with Stata
Release 6.0 (Stata Corp) . The x2 test was performed
where it is applicable.

RESULTSOf 231 inhabitants, 140 (60.6%) were infected withsoil‑transmitted helminths. The pattern of infection in

two communities was quite similar (Fig. 1). The high‑est prevalence was observed in hookworm infection,
followed by T. trichiura and then A. Iumbricoides infec‑

tions. Hundred twenty‑two of inhabitants (52.8%) werewith hookworm, 43 (18.6%) with T. trichiura and 26(11.3%) with A. Iumbricoides (Fig. 1). Mixed infections

with hookworm plus Trichuris were the commonest(Fig. 2). Male and female inhabitants showed similarprevalence in any of these three helminth infections(data not shown) . The infection rate of T. trichiura wasgradually decreased with age in males. In females, on
the other hand, the Trichuris egg‑positive rate wasfound higher after 40 years old and more and it in‑creased with age. Although no relationship was detect‑
ed between age and prevalence based on logistic regres‑sion test (p=0.07) , female group aged 60 years old andmore showed higher infection rate than the same agegroup of males (x2 test: p<0.05, Fig. 3). In Judigaun,some inhabitants were found to be infected with V. nanaand liver fluke (Fig. 1).

We examined the total serum lgG and lgE Ievels of
the inhabitants in these two communities. The majority
of total serum lgG Ievels of the inhabitants were high inboth sexes, 2,273 876 mg/dl in male and 2,024 499
mg/dl in female in average. With regard to total serumlgE Ievels, all the inhabitants surpassed Japanese nor‑mal range ( < 165 IU/ml) both in male and female,irrespective of excretion of parasite eggs (Fig. 4) . TotallgE Ievels in inhabitants infected with either hookworm

60

Hookworm‑ SO

35.1 %,5e40

I Hookworm

Zl Trichuris mhi,era
Ea Asearis lumbrieoides
Vampirolepts nanaLiver nuke
Zl Ea

30

D

9.5 %

D

5.2 %
3.0 %

e

E820

,! .
I ,b .

4.8 %

f Ll

10
'b

,Ib

!‑

IL.L

/
L,t
,

1/

IL ,

o

1.3 %

1.7 %

,t .

Trich uris

Ascaristrichiura

lumbricoid es

/L

IL.:

Total Kotyang Judigaun
Figure I Prevalence of helminthic infections at Kotyang
and Judigaun in Nepal.

Figure 2 The status of multiple infections

with helminths at Kotyang and
Judigaun in Nepal.

513

prevalence and the species of parasite prevailed in these50a

HD‑ Male
Hh Female

.S 40
G,
*

rural villages were different from Kathmandu. In both
Kotyang and Judigaun, the prevalence of soil‑transmit‑
ted helminthic infections was higher than 60%, threetimes as high as that (1ess than 20%) in Katmandu in

*

;,;

;s

;:' 301992 (Rai et al., 1994).

;

'

:

The most common helrninth was hookworm

:

SJ

h

20

(52.8%), followed by T. trichiura (18.6%) and A. Ium‑ee5

bricoides (11.3%) in these two communities. It is
worthy of notice that Ascaris infection rate in our
survey was rather low (11.3%) in both Kotyang and
Judigaun compared with other place in Nepal (Rai and

‑Q‑

10

'

h*
o
o

‑19 20 29 30‑39 40.49 50 59 60‑

Gurung, 1986; Rai et al., 1994; Rai et al.. 1997; Gianotti,

Ag*

Nepal. *p<0.05 compared with

1990; Navitsky et al.. 1998). The pattern of infection
was also different from those of other areas in Nepal
including Kathmandu, where the most common helminthmale group.

was A. Iumbricoides, followed by hookworm and T.

Figure 3 Prevalence of T. trichiura infection
by age at Kotyang and Judigaun intrichiura (Rai and Gurung, 1986; Rai et al.. 1994; Rai etor T. trichiura ranged from 200 to 3,000 IU/ml (anupper limit in this assay without dilution) , while all
infected with A lumbricoides showed higher than 1,500
IU/ml (Fig. 5).

al., 1997; Gianotti, 1990). The study conducted in rural
areas of Sarlahi District, Iow plains adjacent to India,
showed that the prevalence rates of helminth infection

were 78.8%, 56.2%, and 7.9% in hookworm, A. Ium‑
bricoides, and T. trichiura. respectively (Navitsky et al..
1998) .

DISCUSSION
It is reported that the prevalence of soil‑transmit‑

ted helminthic infection at Kathmandu, the capital cityof Nepal, ranged from 18.0 to 36.6%, and that the annual

It is a well known fact that there are remarkabledifferences in the detection of hookworm infection ratedepending on the exarnination methods employed. The

prevalence was decreasing year by year in both adults

direct fecal smear method reveals rather poor result in
eggs detection in comparison with the floatation, precip‑

and children, irrespective of sex (Rai et al.. 1994). Ouritation and cultivation methods (Kamegai, 1972).

study was carried out in two rural communities of a hill

Therefore it should be noted that the results by thearea, Kotyang and Judigaun, which were located at 30
km north‑east of Kathmandu. It was elucidated that the

a)

i

b)
3,000

5 'ooo¥ ‑‑ ‑'‑‑‑; 3'OOO ro/ml

3,000 IU ;/ml

A
AL

4,000

3,000

'li p',pe'E̲

0.ic ': )

3,000

2,000

'

eP 2,000

o

, oo

a : 0

#

e3.0

.

1,000

..
.80

1 ,OO

ee

165
O

Mal' Fal' M*1' F'*'1'
Figure 4 Total serum lgG and lgE Ievels in males andfemales at Kotyang and Judigaun in Nepal. Total
serum lgG (a) and lgE (b) Ievels in males andfemales who were negative (open symboD or posi‑tive (closed symboD for helrninths eggs.

2,000

'o
ell

,
eJ)

*

1,000

cF
[I]
I ]

AA

,e.

AA

o A*..ri*
H"kwo*

Tri,h ri

tri,hi .* in bri..id.*

Figure 5 Total serum lgE Ievels of inhabitants infected withhookworms (circle) , T. trichiura (triangle) and A.
lumbricoides (square) . Total serum lgE Ievels of
inhabitants infected with a single helminth (hook‑worm, T. trichiura or A. Iumbricoides) were shownas closed symbols and those infected with 2 or 3

helminths were shown as open symbols.

514

direct smear method (Rai and Gurung, 1986; Rai et al..
1994; Rai et al.. 1997) had resulted in considerably lower

REFERENCES

rate than true infection for hookworm. Concerning thehookworm species prevalent in Nepal, in Sarlahi Dis‑

1 ) Adams, E.J., Stephenson, L.S., Latham, M.C. and Kinoti,

trict, all of the cultured hookworm larvae wereschool children with hookworm, Trichuris trichiura andidentified as Ancylostoma duodenale (Gianotti, 1990). In

Kathmandu, 67.0% of hookworm were A. duodenale.
while 33.0% were Necator americanus (Rai et al.. 1997) .

In our survey, we did not adopt the cultivation method
and so that could not differentiate the species. The
infection rate of T. trichiura in Nepal was rather low
ranging between 5 and 20% (Rai and Gurung, 1986; Rai

S.N. (1994): Physical activity and growth of Kenyan
Ascaris lumbricoides infections are improved after treat‑ment with albendazole. J. Nutr., 124(8), 1199‑1206
2 ) Bundy, D.A.P.. Chan, M.S., Medley, G.F., Jamison, D. andSavioli, L. (1996): Intestinal nematodes. In: Murray C.

J.L. and Lopez A.D. eds. The Global Epidemiology of
Infectious Diseases: Cambridge, Harvard University
Press.
3 ) Chan, M.S. (1997) : The global burden of intestinal nematodeet al.. 1994; Rai et al.. 1997; Gianotti, 1990; Navitsky etinfections‑Fifty years on. Parasitology Today, 13, 438‑

al., 1998). In Judigaun, some inhabitants were found to
be infected with V. nana and liver fluke (Fig. 1) . As T.

4434 ) Chan, M.S., Medley, G.F., Jamison, D. and Bundy, D.A.
(1994): The evaluation of potential global morbidity
attributable to intestinal nematode infections. Para‑trichiura is a typical "backyard" pathogen, a high preva‑

lence with this parasite in females will be attributed tohouse works and consequent contaminations, too.
With regard to total serum lgE Ievel, all inhabitants

surpassed Japanese normal range ( < 165 IU/ml) both inmale and female, irrespective of excretion of parasite
eggs (Fig. 4). This result indicates the occurrence ofrepeated infections with helminths. The discrepancy
between serum lgE Ievel and excretion of parasite eggs
might be accounted for by the fact that the time andplace of sensitization of immune response against hel‑minth antigens are different from those of egg‑produc‑tion from adult helminths. In this study, it was shownthat total lgE Ievels in inhabitants infected with hook‑worm or T. trichiura ranged from 200 to higher than 2,OOO IU/ml (upper lirnit of measurement, 3,000 IU/ml),
while all infected with A. Iumbricoides were with lgE
higher than 1,500 IU/ml (Fig. 5). Thus this fact may
support previous estimation that the Ascaris infection
would strongly induce lgE antibody production (Kojima

sitology, 109(3) , 373‑387

5 ) Gianotti, A. (1990): Intestinal parasites of Nepal. J.
Nep. Med. Assoc., 28, 242‑247
6 ) Ito, K., Kawasaki, T., Ogaki, T., Uezono, K., Yoshimizu,
Y.. Osaka, T., Wakana, C., Nakayama, J., Ohnaka, M.,

Acharya, G.P. and Ogata, M. (1993): Relationshipbetween total serum cholesterol level and nutritional
and physical status in Nepalese rural people. J. Nutr.
Sci. Vitaminol., 39, 127‑139

7 ) Kamegai, S. (1972): The prevalence and geographicaldistribution of medical parasites in Japan. In: Progressof Medical Parasitology in Japan. Volume VI. Mori‑
shita. K., Komiya, Y. and Matsubayashi, H. (ed.)., pp.

275‑471. Meguro Parasitological Museum Press.
8 ) Kojima, S., Yokogawa, M. and Tada. T. (1972): Raisedlevels of serum lgE in human helminthiases. Am. J.
Trop. Med. Hyg., 21 (6) , 913‑918
9 ) Navitsky, R.C., Dreyfuss, M.L., Shrestha. J., Khatry, S.
K., Stoltzfus, R.J. and Albonico, M. (1998) : Ancylostoma

duodenale is responsible for hookworm infections
among pregnant women in the rural plains of Nepal. J.
Parasitol., 84(3) , 647‑651

et al.. 1972).

10) Rai, S.K. and Gurung, C.K. (1986): Intestinal parasitic
infection in high school level students of Birgunj city. J.

ACKNOWLEDGMENTS

Inst, Med., 8, 33‑38

hara, Mr. Koh‑hei Fujimi, Mr. Kenji Ohkura and Dr.

11) Rai, S.K., Kubo, T., Nakanishi, M., Sumi, K., Shibata,H., Matsuoka, A. and Shrestha, H.G. (1994): Status ofsoil‑transmitted helminthic infection in Nepal. Kansen‑
shogaku Zasshi, 68(5) , 625‑630
12) Rai, S.K., Shrestha, H.G., Nakanishi, M., Kubo. T., Ono,

Isao Tada for their collaboration for this study. This

K., Uga, S. and Matsurnura, T. (1997): Hookworm

work was supported by Grants‑in‑Aid for Scientific
Research (B) 09041186 and in part (A) 11113101 from

infection recorded at an university teaching hospital in

Thanks are due to Mr. and Mrs. Krishna B.
Tamang for their excellent cooperation and Dr. Kenjiro

Kawashima, Miss Kanami Noda, Miss Mitsuko Mori‑

the Ministry of Education, Science, Sports and Culture,Ja pan.

Kathrnandu, Nepal over one decaded period. Jpn. J.
Trop. Med. Hyg., 25 (2), 81‑84

13) Roche, M. and Layrisse, M. (1966): The nature andcauses of 'hookworm anemia'. Am. J. Trop. Med. Hyg.,
15(6), 1029‑1102
14) Stoltzfus, R.J., Albonico, M., Chwaya, M.H., Savioli, L.,

Tielsch, J., Schulze, K. and Yip, R. (1996): Hemoquant515

determination of hookworm‑related blood loss and its
role in iron deficiency in African children. Am. J Trop.
Med. Hyg., 55(4), 399‑404

Jpn J Trop Med Hyg Vol 27 No 4 1999, pp. 517‑519

517

Short communication

PREVALENCE OF INTESTINAL PARASITE

INFECTION IN TOUL ROKA VILLAGE,
PHNOM PENH MUNICIPALITY, CAMBODIA
JUN KOBAYASHI KUNl CHINEN SIM SAMIDT3, KEIJI HIGA2, MAKI CHlNEN2YOSHIYA SATol AND CHOKEI YOSHIDA2
Received May 6, 1999/Accepted August 30, 1999

Intestinal parasitic infection is a serious public

nation in December, 1998. For stool examination, thehealth problem among the inhabitants in developing

authors visited all households to explain the purpose ofcountries located in tropical areas. Control activities ofthe survey and delivered stool containers to all families.

parasitic diseases are not only an important healthimprovement problem in a given area but also an entry

After delivering the containers, stool samples werecollected and examined both by Kato‑Katz thick smear

point for effective public health care activities because

method (Kato and Miura, 1954) and formalin‑etherof its serious prevalence among inhabitants and its
nature as an environment‑derived disease (Fereydoun,1984). Also, intestinal helminthiasis can be easily
diagnosed with fecal samples and are treatable by oral
administration of anthelmintics; it can be performed

concentration method (Ritchie, 1948). Their life habitsintensively as an ordinary public health care activity.

were also surveyed by the interview to the representa‑
tive adults of each family.

In the present preliminary study, stool samples from

113 villagers, accounting for 24.7% of the population
subjected, were collected randomly for examination.

Additiohally, intestinal parasitic infection occurs closely

Table I represents the results of the stool examinations.

relating to the environmental sanitary condition; para‑site control program may relate directly to the educa‑

A total of 7 helminth and 9 protozoan species werediagnosed in 80.5% of the villagers examined. Sixty‑
seven samples (59.3%) were found harboring one ortion for improvement of sanitary condition. Exact
survey on the prevalence of intestinal parasite infection

may also be important to know health status of the
inhabitants in a given area. Due to the civil war for along period in Cambodia, the information on the recent
prevalence of intestinal parasites is extremely few. In

the country, Cambodia‑Okinawa Friendship Association(COFA) , founded in 1992 as a non‑government organi‑zation (NGO) in Okinawa, has established a clinic in avillage, Toul Roka Village, to contribute to health careof the villagers and started a parasite control program
since 1998. The present study was undertaken to obtainbasic information on the recent prevalence of intestinal
parasitic infection arnong the inhabitants in the village.

Although the village is located in suburban area ofPhnorn Penh Municipality, the general socio‑economic
and sanitary conditions were consistently poor in thevillage. A total of 457 villagers live in 61 households inthe village. The survey was conducted by stool exami‑

more helminth infections. Among the helminth species,
Ascaris lumbricoides was the commonest parasite, show‑
ing 48.7% of prevalence rate. Hookworm infection was
also demonstrated in 15.9% of the villagers. On theother hand, Trichuris trichiura infection was rare among
the villagers, although their infection mode is almost thesame to that of A. Iumbricoides. Interestingly, theinfection rate with Qpisthorchis viverrini, Iiver fluke,
were only 3.5% (4 cases) , in spite of frequent eating of

raw fishes by the villagers. When the prevalence rates
were compared between age groups divided by 15 years
old, the infection rate of A. Iumbricoides was higher inthe younger age population, but it was reversed by theaged group over 15 years old in the case of hookworm
infection. As to protozoan parasites, 9 species of proto‑

zoan parasite were detected in 61 (54.0%) villagers.
The commonest species was Blastcystis hominis, show‑
ing positive rates of 37‑43%. Entamoeba histolytica/E.

1

Department of Parasitology, School of Medicine, University, of the Ryukyus, Nishihara, Okinawa 903‑0215, Japan

2

Cambodia‑Okinawa Friendship Association (COFA), Okinawa, Japan
COFA Clinic, Toul Roka, Phnom Penh, Cambodia

3

518

Table 1

Prevalence of intestinal helminth infection in Toul Roka village, Cambodi a

No. of positive (%)

Under 15 years old

15 years old

Total(n=51)or more (n=62)(n = 1 13)

33 (64. 7)

34 (54 . 8)

67 (59 . 3)

Ascaris lumbricoides

29 (56. 9)

26 (41 . 9)55 (48 . 7)

hookworm

15 (24 . 2)

18 (15 . 9)

Trichuris trichiura

3 (5. 9)3 (5. 9)Enterobius vermicularisO

Opisthorchis viverrini

Vampirolepis nana

1 (2 .o)2 (3. 6)

Taenia sp.

O(o)

3 (4.8)1 (1.6)3 (4.8)2 (2.8)1 (1.6)6 (5.3)1 (0.9)4 (3.5)4 (3.5)1 (0.9)Protozoan infection

24 (47. 1)

37 (59. 7)

61 (54 . O)

Blastcystis hominis

22 (43. 1)

23 (37 1)

45 (39 . 8)

Endolimax nana

Entamoeba histlytica/E. dispar

4 (7. 8)4 (7. 8)2 (3. 9)1 (2 . O)
1 (2 . O)

Entamoeba hartmani

O

Giardia lumbria

1 (2. O)1 (2 . O)7(11. 2)5 (8. 1)6 (9. 7)6 (9. 7)6 (9. 7)
1 (1. 6)
1 (1 . 6)

11 (9.7)
9 (8.0)8 (7.1)7 (6.2)7 (6.2)1 (0.9)2 (1.8)1 (0.9)Helminth infection

Chilomastix mesnili
Iodamoeba butschlii

Entamoeba coli

Enteromonas hominis

dispar and Giardia lambria infections, however, were
not so frequent in the present study.

Among the households in which many families
received stool examination, 27 households were selected

randomly for the interviews on their life habits(Table 2). It was known that majority of villagers
drink boiled water and use tap water to wash cocking
materials. Supply of piped water has well known to

(o)

(O)

Table 2 The results of questionnaire on living habit ofvillagers in Toul Roka village, Cambodia
Drinking water

Tap water boild
Raining water boild
non‑boild

19 (70 . 3)
5 (18 . 5)
3 (11 . 1)

Washing water (cocking materiaD
Tap water

25 (92 . 6)

2 (7.4)1 (3.7)

Raining waterWell

have preventive effect on soil‑transrnitted helminthiasis.

On the other hand, only 37% of villagers have a toilet in(O)O

their own houses and more than 35% of the villagers

Toilet in own house 10 (37. o)7(25. 9)Public toilet

evacuate outside around lake or river. Toilets, whether

Around house

Place of evacuation

Around lake or river
2 (7. 4)
OthersFoot wear

ing in the compound around the house which becomes asource of infection to others. The villagers who always

use foot wear were about 30% only. They eat some‑times raw fishes and raw meats as their traditional
eating habit. However, food‑born parasitic infections,

No useSometime use
Always use sandal
Always use shoes

1984; Kan, 1992; Ghani and Oothuman, 1992; Sinniah andO(o)19 (70. 4)
6 (22. 2)

2 (7. 4)Eating habit

Eat raw fish

such as O. viverrini infection, were extremely few
among the villagers.
It had been well documented that a combination offactors such as poor sanitary condition, Iow socio‑eco‑nomic levels of residents, scarcity of clean water supply
and sewage facilities, and ecological and climatic condi‑
tions play an important roe in the transrnission and highrates of soil‑transmitted parasite infections (Fereydoun,(o)

6 (22. 2)

shared or owned by individual households, seem to beeffective for prevention of the parasite infections, how‑ever, children still preferred the convenience of defecat‑ONo

10(37. O)

Sometimes

17 (63. O)

Frequently

Eat raw meat NoO(O)

14(51. 9)

Sometimes

13 (48. 1)

FrequentlyO

Eat raw frog

1 (3.7)

Eat frog heated

17 (62 . 9)

Eat raw snake

O (O)Eat snake heated

5 (18 . 5)

(O)

Total number of house hold answared the questionnaire were 27.

519

Rajeswar, 1995). The order of importance of eachsoil‑transmitted helminth infections in relation to types

factor in influencing rate of the parasitic infection isof water supply, housing facilities and availability of

dependent on the level of socio‑economic development

latrines in rural areas of peninsular Malaysia. In:
Collected Papers on the Control of Soil‑transmitted

as a whole and hygienic conditions of the endemic
region. The people in areas with a low socio‑economic
level seem to be busy with their jobs to earn their living;

little attention is paid to their personal and family
affairs. It has also been shown that the infections are
slowly disappearing in areas where good sanitary facil‑
ities has been provided and living standards have risen(Pawlowski, 1985). A11 the above mentioned contribut‑
ing factors to high prevalence of parasitic infections
continue to exist in Cambodia, because of political andsocial instability due to the civil war over the past
decades, and it is expected that the prevalence of intesti‑

Helminthiasis, Vol. V, The APCO Research Group (ed.) ,Tokyo, 64‑71
2 ) Fereydoun. A. (1984): Selective primary health care:
Strategies for control of diseases in the developingworld; XII Ascariasis and trichuriasis. Int. J. Health
Education, 6, 39‑41
3 ) Giboda, M. (1985): Intestinal parasites in Kampuchea,

Takeo Province. J. Hyg. Epidemiol. Microbiol. Im‑
tnunol., 29, 377‑386

4 ) Gyorkos, T.W., MacLean, J.C., Viens. P., Cheang, C. andKokoskin‑Nelson, E. (1992): Intestinal parasite infec‑
tion in the Kampuchean refugee population 6 years after
resettlement in Canada. J. Infect. Dis., 166, 413‑417

nal parasitic infection would be high among the inhabit‑
ants. However, recent information on the prevalence ofparasitic infection is consistently few in this country,5 ) Kan, S.P. (1993): Environmental, socioeconomic andalthough there were some reports on the infection ofparasites among Cambodian refugees (Keittivuti et al..

slum dwellers. In: Collected Papers on the Control of

1982; Gyorkos et al.. 1992; Lurio et al.. 1991; Molina etal.. 1988; Parish, 1985; Tittle et al.. 1982). In a past

survey in a different village in Khampuchea Province,

cultural‑behavioral factors affecting endemicity of soil‑transmitted helminthiasis and nutritional status of urban

Soil‑transmitted Helminthiasis, Vol. V, The APCO
Research Group (ed.), Tokyo, 44‑63
6 ) Kato, K. and Miura, M. (1954): On the comparison ofsome stool examination methods. Jpn. J. Parasitol., 3, 35
7 ) Keittivuti, B., D'Agnes. T. and Leittivuti, A. (1983): The

Giboda (1985) has reported that the prevalence of intes‑

prevalence of parasitic infections among Cambodian

tinal parasite among 1,739 inhabitants including pre‑school children was 80%, which was almost the same torefugees residing in Ban‑Kaeng holding center, Prachin‑

the present study. The highest positive rate wasobtained in hookworm infection (58.8%) in the past
study. On the other hand, the predominant helminth was

A. Iumbricoides (48.7%), followed by hookworm(15.9%) , in the present study. One of the characteristic

results in the present study was low prevalence of O.
viverrini infection (3.5%). The liver fluke infection is

known to be highly prevalent (more than 50%, in gen‑eral, in adulthood) in the neighboring Thailand andLaos. E. histolytica and G. Iamblia were predominant
protozoan species, with prevalence rates of 16.7% and14.7%, in the past study. The infection rates of these
species, however, were lowered to 6.2% and 1.8% in thepresent study.

The sanitation improvement and health education

buri Province, Thailand. In: Collected Papers on theControl of Soil‑transmitted Helminthiasis, Vol. II, The

APCO Research Group (ed.), Tokyo, 59‑65
8 ) Lurio. J., Verson, H. and Karp, S. (1991): Intestinal

parasites in Cambodians: comparison of diagnostic
methods used in screening refugees with irnplications fortreatment of populations with high rates of infestation.

J. Am. Board Fam. Pract., 4, 71‑78
9 ) Molina, C.D., Molina, M.M. and Molina, J.M. (1988):
Intestinal parasites in southeast Asian refugees two
years after immigration. West. J. Med., 149, 422‑425
10) Parish, R.A. (1985): Intestinal parasites in southeast
Asian refugee children. West. J. Med., 143, 47‑49
11) Pawlowski, Z.S. (1985): Control of ascariasis within
primary health care. In: Ascariasis and its Public
Health Significance, Cromptom, D.W.T. (ed.), Taylor
and Francis. London and Philadelphia12) Ritchie, L.S. (1948): An ether sedimentation techniquewere also introduced for transmission control of para‑sites as an integration program of primary health care.

for routine stool examination. Bull. US Army Med.

The results of the parasite control program and the13) Sinniah, B. and Rajeswar, B. (1997): Economic status

reaction toward encouragement on improvement of
their living conditions through the control program will
be assessed in the following years.

REFERENCES
1 ) Che Gharn B M and Oothuman P (1993): Pattern ofDep., 8, 326

associated with intestinal nematode infection. In: Col‑lected Papers on the Control of Soil‑transmitted Hel‑

minthiasis, Vol. VI, The APCO Research Group (ed.),Tokyo, 71‑77
14) Tittle, B.S., Harris, J.A., Chase, P.A., Morrell, R.E.,
Jackson, R.J. and Espinoza, S.Y. (1982): Health screen‑
ing of Indochinese refugee children. Am. J. Dis. Child.,
136, 697‑700

VO■．27NO．4

0ECEMBER1999

CONTENTS
Review
Kano，S．and Aikawa，M．
Pathology and Pathophysiology

Originalof

一471476

Malaria．・

articleSukul，N．C．，Sarkar，P．，Suku1，A．and Sinhababu，S．P．
Antifilarial Effect of／1吻窺Js宛nガ1α8 卿6召Extract and its Ultra High Dilutions

AgainstCanineDirofilariasis

一477−481

一・一…

Itoh，M．，Qiu，X．，Koyama，Y．，Ogawa，Y．，Weerasooriya，M・V・，Visanou，H・，Fujimaki，Y．and
Detection

IgG4inof

Kimura，E

Circulating肱ohεz6幅α伽noz瞬J

Chyluria

Cases

in

Antigen，Filaria

SpecificIgG

and・・483−486

Japan一Tongo1−Rivera，P．，Pmdencio，MA，Saro1，」．，Balingit7E，
Villacorte，E．，Darilag，A．，Suzuki，M．and Kano，S、Clinical and Laboratory Correlates of the Outcome
PhilipPines

of

Cerebral

Malariainthe一487−495

・…

Takaoka，H．and Kuvangkadilok，C、
Four New Species of Black Flies（Diptera：Simuliidae）from Thailand一一497−509

Hamano，S．，Kobayashi，S．，Ogaki，T．，Koga，M．，Kawasaki，M．，Ito，K，Saito，A．，
Tsuji，M．，Tokunaga，S．，Sharma，S．，Acharya，GP、and Kawasaki，T．
A Survey on Helminthic Infections in Two Rural Communities in Nepa1……

Short・・511−515

communication

Kobayashi，J．，Chinen，K．，Samidt，S．，Higa，K．，Chinen，M，

Sato，Y．and Yoshida，C．
Prevalence of Intestinal Parasite

Municipality，Cambodia・…一Infection

in

Toul

Roka

Village，PhnomPenh
一517−519