JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
１巻

第２号
内

G6PD 異常症の迅速検査法（Single-step screening 法）の実地応用に向けた検討と評価
岩井 くに，松岡 裕之，川本 文彦，新井 明治，
吉田 栄人，平井誠，石井明…………………………………………………………………９３

Journal of Tropical報
タイ北部におけるブユ２種のフィラリア幼虫自然感染
福田 昌子，Wej Choochote，Odile Bain，青木 千春，高岡

Medicine

著
インターフェロンガンマノックアウトマウスを用いた免疫不全宿主における難治性トキソプラズマ症に対する化学療法の確立のための動物実験モデル
Usama S Belal，野呂瀬一美，文恵聖，陳梅，Rabie M Mohamed，Azza K Ahmed，朴蓮筍，青才 文江，矢野 明彦 ……………………………………………８３
インドネシアおよびタイの水道水からの微生物の検出宇賀 昭二，小田 卓也，木村 憲司，木村 大輔，Koesdarto Setiawan，Margono Sri，Kunaruk Nuvit，Nualta Apakupakul ………………………………………………………８
７

短

宏行 ………………………………９９

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 稲 〃 嶋崎

JUNE２
０
０
３

日熱医会誌

Uga, S., Oda, T., Kimura, K., Kimura, D., Koesdarto, S., Margono, S., Kunaruk, N., and Nualta, A.
Detection of microorganisms in tap water in Indonesia and Thailand …………………………………８７
Iwai, K., Matsuoka, H., Kawamoto, F., Arai, M., Yoshida, S., Hirai, M. and Ishii, A.
A rapid single-step screening method for glucose-6-phosphate dehydrogenase deficiency infield applications …………………………………………………………………………………………９３
Short Communication
Fukuda, M., Choochote, W., Bain, O., Aoki, C., and Takaoka, H.
Natural infections with filarial larvae in two species of black flies (Diptera: Simuliidae) innorthern Thailand ………………………………………………………………………………………９９Vol. 31 No. 2

会報・記録
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Original article
Belal, U S., Norose, K., Mun, H., Chen, M., Mohamed, R M., Ahmed, A K., Piao, L., Aosai, F.,
and Yano, A.
An animal model for establishing chemotherapy against intractable toxoplasmosis in
immunocompromised hosts by the use of IFN-γ knockout mice ………………………………………８３

Hygiene評
病原体進化論 ――人間はコントロールできるか――記野 秀人 ………………………………………………………………………………………………１
０
３

and

書

JUNE ２０
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CONTENTS

容
Japanese

原

VOL．
３
１ No．
２

平成１
５年６月

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は２mm
（決）

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Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
２,２０
０３, pp.８３‐
８
６

AN ANIMAL MODEL FOR ESTABLISHINGCHEMOTHERAPY AGAINSTINTRACTABLE TOXOPLASMOSISIN IMMUNOCOMPROMISED HOSTSBY THE USE OF IFN-γ KNOCKOUT MICE
USAMA S. BELAL, KAZUMI NOROSE, HYE-SEONG MUN, MEI CHEN, RABIE M. MOHAMED,AZZA K. AHMED, LIAN-XUN PIAO, FUMIE AOSAI, and AKIHIKO YANO*
Accepted 13, May, 2003
ABSTRACT: The Toxoplasma gondii number was evaluated by quantitative competitive polymerase chain reaction (QC-PCR) assay with or without sulfamethoxazole treatment in the heart, blood, brain, and small intestine of
IFN-γ knockout (GKO) BALB/c (B/c) mice after peroral infection with the cyst-forming Fukaya strain. T. gondii
infection was observed in the heart, blood, and brain, but not in the small intestine, of mice treated with sulfamethoxazole for 4 weeks. No correlation between T. gondii loads and sulfamethoxazole concentrations in tissues
and blood was observed. T. gondii was not detected in the heart and blood after continuous sulfamethoxazole treatment for two months, but a small number of parasites was demonstrated in the brain. Thus, we successfully established an animal model for evaluating chemotherapy regimens in immunocompromised hosts by using GKO B/cmice infected with T. gondii.
Key Words: Toxoplasma gondii, immunocompromised hosts, chemotherapy, sulfamethoxazole, IFN-γ knockout
mice

INTRODUCTION

MATERIALS AND METHODS

Toxoplasma gondii is an obligate intracellular parasitethat causes life-threatening disease in developing human fetuses and in immunocompromised individuals, particularly
patients with acquired immunodeficiency syndrome (AIDS)(Navia et al., 1986). The lack of effective therapy for intractable toxoplasmosis is responsible for the mortality bytoxoplasmosis in these patients. In this study, we introduced
the GKO mouse as a model of an immunocompromised animal for which to establish an effective chemotherapy protocol for intractable toxoplasmosis. A precise comparative
study between T. gondii loads and concentrations of sulfamethoxazole was performed in various organs of GKO
mice perorally infected with T. gondii and treated with sulfamethoxazole.

Parasites
Cysts of an avirulent Fukaya strain of T. gondii (Kamei et al., 1976) were prepared from B10.A(4R) mice that
had been infected orally with 5 cysts of Fukaya strain 6
weeks earlier, as previously described (Luo et al., 1995).
Animals
Eight- to twelve-week-old female GKO BALB/c (B/c)
mice were genotyped by polymerase chain reaction (PCR)(Tagawa et al., 1997). Age-matched female wild type (WT)
B/c mice were purchased from SLC (Hamamatsu, Sizuoka,
Japan). GKO mice were classified into two groups after peroral infection with 10 cysts. One group remained untreated
and was sacrificed at 4, 7, and 10 days post infection (PI).
The second group received sulfamethoxazole continuously
from day 4 PI and was sacrificed at 7, 10, 15, 20, and 25
days. WT mice were infected with 10 cysts, and thenDepartment of Infection & Host Defense, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
* Corresponding author: Akihiko Yano, Department of Infection & Host Defense, Graduate School of Medicine, Chiba University, 1-8-1 Inohana,Chuo-ku, Chiba 260-8670, Japan.
Tel: +81-43-226-2071, Fax: +81-43-226-2076,
E-mail address: yano@faculty.chiba-u.jp

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４

Drug
Sulfamethoxazole (Shionogi Co., Ltd, Osaka, Japan)
was administered in drinking water at a dose of 1 mg/ml. It
was first dissolved in 2 N NaOH to facilitate its absorption
in water. Then, pH was adjusted to 7 with 10 N HCl. Drugadministration was initiated on day 4 PI.
QC- PCR
Using 1µg of genomic DNA each from the heart, brain,
blood, and small intestine of the GKO B/c mice, QC- PCR
targeting surface antigen 1 (SAG1) gene was carried out todetermine the distribution of T. gondii as described previously (Luo et al., 1997; Kobayashi et al., 1999). The amplified cDNAs were electrophoretically separated on a 1%
agarose gel containing ethidium bromide, and the ratio tothe subsequently amplified competitor SAG1 DNA wasmeasured on an IPLab Gel densitometer (Signal Analytical
Corp., VA, USA). The abundance of T. gondii was calculated as described previously (Luo et al., 1995; 1997).

The amount of exogenous IFN-γ was comparable with thelevel of IFN-γ in WT mice infected with T. gondii (Suzuki
et al., 2000). Parasitic loads were also calculated in theheart and brain at day 10 PI.
Determination of drug concentration
GKO mice were treated with sulfamethoxazole fromday 4 to day 25 PI, and the mice were euthanized on days 7,
10, 15, 20, and 25 PI. The concentration of sulfamethoxazole was determined in the blood and tissues (heart, brain,
and small intestine) of the mice by high-performance liquid
chromatography (HPLC) as previously described (Van deVen et al., 1995).
Statistics
The significance of differences between groups wasdetermined by Student’s t- test and that in survival was determined by the Kaplan-Meier method. P < 0.05 was considered significant.

Treatment of infected mice with exogenous IFN-γ
The treatment of GKO mice with 250 units of recombinant murine (rm) IFN-γ was started 4 days before the infection. The mice were injected intraperitoneally with
rmIFN-γ every 2 days for 10 days PI (Mun et al., 2000).

100
80
60

B
100

105
10

103
10

2

100
0

4

7 10 15 20 25

10-1

100

106

5

10

15

20

25

Days after infection

Fig. 1. Sulfamethoxazole treatment of GKO B/c mice challenged perorally with 10 cysts of the Fukaya strain of T.
gondii. Survival rate was monitored. Six mice were usedin each experimental group, and the experiment was repeated three times with similar results. Arrow indicatesthe starting point of sulfamethoxazole treatment. !; notreatment, "; continuous sulfamethoxazole treatment.

0

4

10

10

104

10

103

7 10 15 20 25

0.1

D

5

10-1

0

1

101

0.1

100

5

104

10

103
102

1

100

0

2

C
106

101

20

10

103

100

102

40

100

104
10

1

101
10-1

106
105

104

T. gondii number/μg DNA

GKO B/c mice

Percent of survinal

A
106

1

101
100

0

4

7 10 15 20 25

0.1

10-1

0

4

7 10 15 20 25

0.1

Sulfamethoxazole concentration（μg/ml or g）

treated with or without sulfamethoxazole.

Days after infection

Fig. 2. Effects of sulfamethoxazole treatment on T. gondii loads
in the organs of GKO B/c mice. T. gondii abundance inthe heart, brain, blood, and small intestine was estimatedby QC-PCR (Fig. 2A, B, C, and D, respectively). Concentration of sulfamethoxazole was expressed as µg/ml
in serum and µg/g in organs. Six mice were used in eachexperimental group and the data shown are representativeof three independent experiments with similar results.
Data are expressed as the number of parasites per µg of
specimen DNA. ■; no treatment, "; continuous treatment. !; sulfamethoxazole concentration †; dead mice.

８
５

RESULTS AND DISCUSSION
The effects of sulfamethoxazole treatment on the survival rate of GKO mice infected perorally with 10 cysts of
the Fukaya strain of T. gondii were examined. Untreatedmice died within 10 days PI (Fig. 1) but treated mice survived under continuous treatment with the difference inmortality between the two groups being significant (p <0.05). However, only three weeks of sulfamethoxazole
treatment failed to eradicate the parasite completely fromthe heart, brain, and blood (Fig. 2A, B, and C, respectively)
although the small intestine became parasite-free during thisperiod (Fig. 2D). Sulfamethoxazole acted only on the rapidly dividing tachyzoites (Lyons et al., 2002). Similar results were reported in previous studies by using sulfadiazine
and RH T. gondii strain (Dumas et al., 1999; Koppe et al.,
1986; Dubey et al., 1998). The tachyzoite is responsible for
acute toxoplasmosis (Yano et al., 2002). Our data clearly
showed that sulfamethaxazole treatment kept the parasite in
the slowly replicating encysted-bradyzoite stage in the heart,brain, and blood of GKO mice, and the present experimental model was physiological regarding the infection routeand type of T. gondii for the infection.
A high level of sulfamethoxazole concentration was

Brain WT

observed in the heart and serum where the parasitic loads
were shown to be high (Fig. 2A, and C), while a low levelof T. gondii loads was observed in the brain where the concentration of sulfamethoxazole was low (Fig. 2B). It should
be noted that a detectable level of T. gondii was not observed in the small intestine (Fig. 2D) although the level ofsulfamethoxazole concentration was similar to that in thebrain. These findings revealed that there was no correlation
between T. gondii loads and sulfamethoxazole concentration.
There were no significant effects of sulfamethoxazole
treatment on T. gondii loads in the heart and brain of both
GKO and WT B/c mice after 10 days P. I (Fig. 3). Furthermore, the treatment with sulfamethoxazole plus exogenous
IFN-γ did not reduce the T. gondii loads in the heart of
GKO mice. This combined treatment decreased the T. gondii loads in the brain of GKO mice but the reduction wasslight and not comparable with that of WT mice. These results suggest that the treatment of the immunocompromised
host with IFN-γ may not be appropriate as a treatment
choice against intractable toxoplasmosis.
The present data also clearly showed that the longduration treatment with sulfamethoxazole for two months
eradicated the parasite completely from the blood and heart,
but not from the brain where a small number of T. gondii
remained (Fig. 4). These results indicate that the treatmentof immunocompromised hosts with sulfamethoxazole

Heart WTGKO B/c mice＊＊＊Heart GKO
100

101

102

103

104

105

T. gondii number/μg DNA

Fig. 3. Effects of intraperitoneal injection of rmIFN-γ alone orwith sulfamethoxazole treatment on the parasitic loads inthe heart and brain of GKO B/c mice. T. gondii loads
were assayed 10 days after injection. Three mice wereused in each experimental group and the experiment was
repeated twice. WT B/c mice were treated with or without sulfamethoxazole. Black bar; mice injected withphosphate buffered saline (PBS), white bar; mice treatedwith sulfamethoxazole, gray bar; mice injected with
rmIFN-γ, white striated bar; mice treated with sulfamethoxazole and rmIFN-γ. *, P < 0.05, **, P < 0.001.

T. gondii number/μg DNA

Brain GKO

10

5

0
Brain

Blood

Heart

Fig. 4. Influence of long duration (two months) of sulfamethoxazole treatment on down-regulation of parasiteloads in the brain, blood, and heart of GKO B/c mice. T.
gondii abundance was estimated by QC-PCR. Six mice
were used in each experimental group and the experiment
was repeated three times. Data are expressed as the number of parasites per µg of specimen DNA.

８
６

should be continued over the long term. The exact mechanisms by which T. gondii loads are decreased in GKO micewith the long-term use of sulfamethoxazole remain to be
unraveled. In any case, a mouse model for evaluating chemotherapy regimens in immunocompromised hosts was established by the use of GKO mice.

ACKNOWLEDGEMENTS
This work was supported in part by a grant-in-aid from
the Ministry of Education, Culture, Sports, Science andTechnology. The authors thank Shionogi Co. for providing
invaluable assistance with the measurement of the sulfamethoxazole concentration.

REFERENCES
１）Dubey, J.P. (1998): Advances in the life cycle ofToxoplasma gondii. Int. J. Parasitol., 28, 1019-1024２）Dumas, J-L., Pizzolato, G., and Pechere, J-C. (1999):
Evaluation of trimethoprim and sulphamethoxazole asmonotherapy or in combination in the management of
toxoplasmosis in murine models. Int. J. Antimicrob.
Agents, 13, 35-39
３）Kamei, K., Sato, K., and Tsunematsu, Y. (1976): A strainof mouse highly susceptible to Toxoplasma. J. Parasitol.,
62, 714４）Kobayashi, M., Aosai, F., Hata, H., Mun, H-S., Tagawa,Y., Iwakura, Y., and Yano, A. (1999): Toxoplasma gondii:
difference of invasion into tissue of digestive organs between susceptible and resistant strain and influence ofIFN-γ in mice inoculated with the cysts perorally. J.
Parasitol., 85, 973-975５）Koppe, J.G., Loewer-Sieger, D.H., and De RoeverBonnet, H. (1986): Results of 20-year follow-up of congenital toxoplasmosis. Lancet, I, 254-255
６）Luo, W-T., Seki, T., Yamashita, K., Aosai, F., Ueda, M.,
and Yano, A. (1995): Quantitative detection of

Toxoplasma gondii by competitive polymerase chain reaction of the surface specific antigen gene-1. Jpn. J. Parasitol., 44, 183-190
７）Luo, W., Aosai, F., Ueda, M., Yamashita, K., Shimizu, K.,
Sekiya, S., and Yano, A. (1997): Kinetics in parasite
abundance in susceptible and resistant mice infected withan avirulent strain of Toxoplasma gondii by using quantitative competitive PCR. J. Parasitol., 83, 1070-1074
８）Lyons, R.E., McLeod, R., and Roberts, C.W. (2002):
Toxoplasma gondii tachyzoite-bradyzoite interconversion.
TRENDS Parasitol., 18, 198-201
９）Mun, H-S., Aosai, F., Norose, K., Chen, M., Hata, H.,
Tagawa, Y., Iwakura, Y., Byun, D-S., and Yano, A.
(2000): Toxoplasma gondii HSP70 as a danger signal in
Toxoplasma gondii-infected mice. Cell Stress Chaperones, 5, 328-335
１０）Navia, B.A., Petito, C.K., Gold, J.W., Cho, E.S., Jordon,
B.D., and Price, R.W. (1986): Cerebral toxoplasmosis
complicating the acquired immune deficiency syndrome:
clinical and neuropathological finding in 27 patients.
Ann. Neurol., 19, 224-238
１１）Suzuki, Y., Kang, H., Parmley, S., Lim, S., and Park, D.
(2000): Induction of tumor necrosis factor-α and inducible nitric oxide synthase fails to prevent toxoplasmic encephalitis in the absence of interferon-γ in genetically resistant BALB/c mice. Microbes Infect., 2, 455-462
１２）Tagawa, Y., Sekikawa, K., and Iwakura, Y. (1997):Suppression of concanavalin A-induced hepatitis in IFN-γ-/mice, but not in TNF-α-/- mice. J. Immunol., 159, 14181428
１３）Van de Ven, E.S., Galama, J., Vree, T., Camps, W., Baars,
I., Eskes, T., Meuwissen, J., and Melchers, W. (1995):
Study of treatment of congenital Toxoplasma gondii infection in rhesus monkeys with pyrimethamine and sulfadiazine. Antimicrob. Agents Chemother., 39, 137-144
１４）Yano, A., Mun, H-S., Chen, M., Norose, K., Hata, K.,
Kobayashi, M., Aosai, F., and Iwakura, Y. (2002): Rolesof IFN-γ on stage conversion of an obligate intracellular
protozoan parasite, Toxoplasma gondii. Intern. Rev. Immunol., 21, 405-421

８７Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
２,２０
０３, pp.８７‐９１

DETECTION OF MICROORGANISMS IN TAP WATER IN
INDONESIA AND THAILAND
SHOJI UGA1, TAKUYA ODA2, KENJI KIMURA3, DAISUKE KIMURA1, KOESDARTO SETIAWAN4,
MARGONO SRI5, KUNARUK NUVIT6 and NUALTA APAKUPAKUL7
Accepted 2, June, 2003
Abstract: The contamination of tap water by microorganisms was surveyed at Surabaya and Jakarta, Indonesia,
and at Hat Yai, Thailand. Water samples were directly collected from house faucets and filtered through membranes.
The membranes were examined for protozoan parasites by immunomagnetic separation. Coliform and Escherichia
coli were examined at each sample collection site using commercially available kits. A total of 115 water samples
were examined and 37 (32%) were positive for any of four microorganisms which were two species of protozoa(Giardia intestinalis and Cryptosporidium parvum) and two kinds of bacteria (coliform and Escherichia coli). G.
intestinalis and C. parvum were found in 9% and 1% of total samples, respectively. Of those detected, coliform
was the most common and was found in all three areas with a mean detection rate of 30% (15-52%). The water
samples that were positive for any of the four types of microorganisms showed a tendency to have lower residual
chlorine concentrations and higher turbidities compared with negative samples. It is important to supply safe waterin order to maintain people’s health because most of the people surveyed (4-88%) ordinarily drank tap water without treating it. Continued efforts are needed to maintain and improve drinking water quality. (193 words)

INTRODUCTION
A safe water supply is advantageous to the health ofpeople. On the other hand, a public water supply will havea serious risk to the health of people if water contamination
should occur. Such risks are seen as unexceptional, even indeveloped countries, where public institutions maintainwater quality. Perz et al . (1998) made a risk assessment ofcryptosporidiosis in New York City, and estimated that the
number of tap-water-related cases per annum was 6 to 34.
Szewzyk et al . (2000) reviewed the microbiological safetyof drinking water and described many outbreaks caused byvarious organisms. The most prominent recorded outbreakoccurred in Milwaukee in 1993, involving 403,000 people,
with Cryptosporidium parvum as the causative agent (Mac
Kenzie et al ., 1994). In addition to this, 703 cases ofgiardiasis were reported in Massachusetts during 1985 to1986 (Kent et al ., 1988), and 6,000 cases in six outbreaksof campylobacteriosis during 1992 to 1996 were reported in
Sweden (Szewzyk et al ., 2000). But in general, as Dawson

and Sartory (2000) reported, significant advances in water
treatment have taken place over the last century and in developed countries massive improvements have beenachieved in the microbiological safety of public drinking
water supplies. Incidences of illness due to poor treatment
or post-treatment contamination are rare in these countries.
However, in areas with low standards of hygiene andsanitation, the contamination of water by microorganism isstill common (Luksamijarulkul et al ., 1994). In these countries, mainly tropical, it is difficult to maintain water quality
at a safe level because of technical and/or economic problems. For example, more than seven species of parasite
were recovered from plant-processed drinking water in Argentina (Basualdo et al ., 2000). In Bangkok, Thailand,
64% of drinking water sources tested were contaminatedwith coliform (Luksamijarulkul et al ., 1994). In Taiwan,
39% to 77% of treated water samples were positive for
Giardia sp. and Cryptosporidium sp. (Hsu et al ., 1999). It
appears that the health hazard posed to people who are supplied such low quality water is significant. In spite of such

1. Department of Medical Technology, Faculty of Health Science, Kobe University School of Medicine, Kobe 654-0142, Japan:
2. Water Quality Laboratory of Kobe City Waterworks Bureau, Kobe 652-0004, Japan:
3. Water Quality Center, Maezawa Industry Inc., Saitama 332-8556, Japan:
4. Laboratory of Intestinal Parasites, Tropical Disease Center, and Laboratory of Parasitology, Faculty of Veterinary Medicine, Airlangga University, Indonesia:
5. Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta 10430, Indonesia:
6. Department of Pathology:
7. Department of Epidemiology Faculty of Medicine, Prince of Songkla University, Thailand.

８８circumstances, little is known about how frequent tap-water
is contaminated with microorganisms and how frequent tap
water is used in developing countries.
This paper reports the results of a field survey done inIndonesia and Thailand to clarify the relationship between
tap water quality and contamination by microorganism. Information regarding tap water usage was obtained throughquestionnaire surveys and the results were analyzed.

MATERIALS AND METHODS
1. Survey areas and period
Surveys were conducted from August 2000 to August
2001 in Surabaya and Jakarta, Indonesia, and in Hat Yai,
Thailand. Surabaya is the second largest city in Indonesia
and is located in the east of Java Island. Jakarta, located in
the west of Java Island, is the capital city of Indonesia containing many modern buildings and has a population of 9.1
million. Hat Yai is a city in the south of Thailand near theThai border with Malaysia. Questionnaire surveys were
performed at health centers in each area with the cooperation of staff members.
2. Filtration of sample water
Filtration of tap water was performed for C. parvum
oocysts and Giardia intestinalis cyst [oocysts of C. parvum
and cysts of G. intestinalis are expressed as (oo)cysts].
Sample water was collected directly from a faucet in eachhouse visited. Permission to collect water and confirmationof its origin were recorded. A polycarbonate membrane(142 mm in diameter, 3 µm pore size; Nucleopore, Whatman, USA) was inserted in a filter folder (Advantec KS-142,
Tokyo, Japan), and 5 to 50 liters of tap water were filteredfrom the pipe that was connected directly to a faucet. To estimate the total filtered volume, the filtered water was kept
in a bucket. We did not use any pressure device for the filtration, so we were only able to filter a small amount of tap
water when the water pressure was low, or when turbidity
was high. A filter membrane was put into each of the 50 ml
centrifuge tubes that contained 30-40 ml of 1% formalin,
kept at room temperature, and transported to Japan.
3. Parasitological measurements
In Japan, each membrane was transferred to a 200 ml
beaker containing 100 ml of PBST (PBS with 0.1% ofTween 80), and (oo)cysts on the membrane were eluted byagitation for 2 min by hand and sonication for 2 min using asonicator (IUC-7321, Tocho co., Tokyo, Japan). The (oo)cyst suspension was again filtered using a polycarbonate
membrane (90 mm in diameter and 3 µm pore size) andtransferred to a 10 ml test tube (Dynal, Oslo, Norway) con-taining 10 ml PBS. The membrane was again treated bysonication and then removed. The remaining solution was
further processed by an immunomagnetic separation
method (Dynal). The purified and concentrated solution
was dried on a slide glass and stained with antiCryptosporidium/Giardia monoclonal antibodies conjugated with fluorescein (Aqua G/C Direct, Waterborne, LA,
USA) for 30 min and 6-diamidino-2-phenylindole (DAPI)
for 2 min, and the (oo)cysts were then observed under afluorescent microscope (400- to 440- nm filter in the former,
and 330- to 385- nm filter in the latter).
4. Bacterial measurements
Tap water samples were examined for coliform and Escherichia coli using a commercially available kit (Colilert,
IDEXX, Maine, USA) at the sample collection site. This
kit allowed the simultaneous qualitative detection of coliform and E. coli.
5. Water quality measurements
Water quality was examined simultaneously when eachsample was collected. Residual chlorine was measured
with the Lovibond 2000 kit (Tintometer Limited, England),and turbidity was measured using a turbidity meter (2100P,
Hach Co., Colorado, USA).
6. Questionnaire survey
A questionnaire survey was performed in three areas toinvestigate number of households equipped with tap waterand the frequency of drinking of tap water by local people.
Following our comprehensive explanation of the study andthe inhabitants’ consent, we surveyed them on seven items
including whether tap water was available in the house, or
what type of drinking water was usually used.

RESULTS
1. Contamination with microorganisms
A total of 115 water samples were examined and 37(32%) were positive for microorganisms. These microorganisms were two kinds of bacteria (coliform and E. coli)
and two species of protozoa (G. intestinalis and C. parvum).
Of the microorganisms detected, coliform was the mostcommon and was found in all three areas with a mean detection rate of 30% (15-52%). E. coli was found with amean of 10% (0-23%). Among protozoa, G. intestinalis
was found with a mean of 9% (0-25%) (Table 1). The mean
number of cysts found in 10 positive samples was 2.5 (1-8).
One C. parvum oocyst was found in one sample (S-1) in
Surabaya (Tables 1 and 2). The intensity of contaminationin the three areas was compared. In Surabaya, which

８９Table 1 Contamination of tap water with microorganisms

Locality
SurabayaNo. ofsamp.
40

Jakarta
Hat Yai
Total

27
48
115

No. of+ve (%)
23(58)
4(15)
10(21)
37(32)

No. of-ve (%)
17(42)
23(85)
38(79)
78(68)

Contamination with:*
Bacteria
Protozoa
Coli.
E. c
G. i
C. p
21(52)
9(23)
10(25)
1(3)
4(15)

0(0)

0(0)

0(0)

10(20)

2(4)

0(0)

0(0)

35(30)

11(10)

10(9)

1(1)

Water quality
Mean
Cl2†
NTU‡
0.08
0.72
0.19
0.67
0.05
0.56
0.54
0.38
0.06
1.18
0.28
1.07
0.25
0.79

* E. c: Escherichia coli, G. i: Giardia intestinalis, C. p: Cryptosporidium parvum.
†Less than 0.1 mg/ml is tentatively considered as 0.05 mg/ml and calculated.
‡National Turbidity Unit.

Table 2 Relationship between microorganism contaminationand tap water quality
Sample*
nos.
S-1
S-2
S-3
S-4
S-5
S-6
S-7
S-8
S-9
S-10
S-11
S-12
S-13
S-14
S-15
S-16
S-17
S-18
S-19
S-20
S-21
S-22
S-23
J-1
J-2
J-3
J-4
H-1
H-2
H-3
H-4
H-5
H-6
H-7
H-8
H-9
H-10

Water
filtered (L)
70
55
22
25
20
20
14
15
30
ND
ND
ND
ND
25
40
30
30
40
30
8
ND
50
30
ND
15
50
40
50
50
50
50
50
50
50
50
50
50

Contaminated with:†Coli. E. c G. i C. p++(2) +(1)
+
+
+
+
+
+(1)
+(1)
+(4)
+
+
+(2)
+
+
ND
ND
+
+
ND
ND
+
+
ND
ND
+
ND
ND
+
+(3)
+
+
+
+
+(1)
+
+
+(8)
+
+
+
+
ND
ND
+
+
+(2)
+
+
+(1)
+
ND
ND
+
ND
ND
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

Quality of water
Cl2
NTU‡
<0.1
0.39
<0.1
0.44
<0.1
0.46
<0.1
0.92
<0.1
1.14
<0.1
0.63
0.6
0.83
<0.1
1.06
<0.1
0.36
<0.1
1.85
<0.1
0.38
<0.1
0.57
<0.1
0.44
<0.1
0.56
<0.1
0.53
<0.1
0.65
<0.1
0.57
<0.1
0.48
<0.1
0.6
<0.1
0.56
<0.1
1.28
<0.1
0.61
<0.1
0.93
<0.1
0.4
<0.1
0.52
<0.1
0.92
<0.1
0.4
<0.1
3.13
<0.1
0.66
<0.1
1.36
<0.1
0.86
0.1
1.72
<0.1
1.57
<0.1
0.85
<0.1
0.71
<0.1
0.53
<0.1
0.94

*Samples were collected from Surabaya (S), Jakarta (J), and Hat Yai (H).
†Coli.: coliform, E. c: Escherichia coli, G. i: Giardia intestinalis, C. p: Cryptosporidium parvum.
‡National Turbidity Unit.

showed the highest contamination rate, 58% of tap water
samples were positive for microorganisms, and all of thefour types of microorganisms recovered through this study
were detected (Table 1). In Jakarta 15% of water samples
were positive for coliform, and in Hat Yai 20% and 4%
were positive for coliform and E. coli, respectively (Table
1).
2. Microorganism contamination and water quality
Table 2 shows the details of 37 samples that had any offour microorganisms: 23 samples from Surabaya (S), 4
from Jakarta (J), and 10 from Hat Yai (H).
Bacterial contamination was closely related to the concentration of residual chlorine but protozoan contamination
was not. Of the 35 bacteria-positive samples, only one (3%,H-5) had 0.1 mg/l of residual chlorine and the remaining 34(97%) had a concentration of less than 0.1mg/l. Of the 115
water samples examined, 66 (57%) had less than 0.1mg/l ofresidual chlorine concentration; of those, 53% were positive
for bacteria. In contrast to this G. intestinalis, although
only a single sample, was found even in a residual chlorine
concentration of 0.6 mg/l. Five positive samples from Surabaya (S-1, S-9, S-18, S-22, S-23) were contaminated withthree kinds of microorganisms (Table 2).
When the positive and the negative samples were compared using the parameters of residual chlorine concentration and turbidity, the former had a tendency to have lower
residual chlorine concentrations and had higher turbidities
than the latter (Table1).
3. Questionnaire survey
One of the aims of this survey was to confirm whether
the local people used tap water as a source of drinking
water. A total of 352 questionnaires were completed andcollected in the three areas examined, and revealed that
80% of the people used tap water. Of the people who had
tap water, more than half (54%) drank it without boiling or９０

Table 3 Utility of water by people in three different areas as revealed by questionnaire survey
With water supply system
Drink tap
Sometimes
water without
we drink: (%)
boiling/filtering (%)
67 (67)bottled water (20)well water (7)Without water supply system

No. of questionnaires
recovered100

No. (%)
100 (100)

Surabaya(Indonesia)102

91 (89)80 (88)bottled water (31)well water (1)11 (11)

well water (55)bottled water (36)

Hat Yai(Thailand)150

90 (60)4 (4)60 (40)

Total

352

281 (80)151 (54)bottled water (61)well water (18)
Bottled and well
water

well water (40)bottled water (13)Well and bottled
water

Locality
Jakarta(Indonesia)

filtering it. This rate varied from area to area: 88% in Surabaya but only 4% in Hat Yai. Those who did not have tap
water to the house used bottled water for drinking, but at
the same time, 40-55% of them also used well water (Table
3).

DISCUSSION
A safe water supply system is a “double-edged sword”.
If this system functions well, it can maintain the health of a
population. But if not, large public health problems occur(Dawson and Sartory, 2000; Szewzyk et al ., 2000). In bothdeveloped countries where urbanization or centralization of
the population has progressed, and developing countries
where water quality is not maintained, such apprehensions
are present. Unfortunately, the water quality found in thisstudy was poor. As Steiner et al . (1997) stated, the leading
cause of infant mortality in the developing world is infectious diarrhea, and the prevalence of the diarrhea pathogen
is largely influenced by the quality of clean water available
for drinking. Recent guidelines and legislation of the European Union Council Directive 98/83/EC and the World
Health Organization stated that drinking water should besafe and not contain pathogenic microorganisms, or contain
only such low numbers that the risk for acquiring waterborne infections is below an acceptable limit (Szewzyk et
al ., 2000).
Four kinds of microorganisms were detected throughout the survey. Most of these microorganisms have an animal reservoir from which they are transmitted to humans,
directly or via the environment. These microorganisms areresistant to environmental stress and have a very low infective dose. It appears that even low-level contamination ofwater supply systems may lead to infections and disease in
the exposed population (Szewzyk et al ., 2000).
Emerging and reemerging infectious parasites in drinking water have become increasingly important during thelast few decades. These include newly recognized protozoa

No. (%)

Sometimes
we drink: (%)

0 (0)71 (20)such as C. parvum and Cyclospora cayetanensis, or G. intestinalis known as traveler’s diarrhea (Rose and Slifko,
1999). Since C. parvum and G. intestinalis have a tolerance to chlorine, they sometimes become causative agentsof waterborne infectious diseases. Among these protozoa,
the detection of C. parvum oocysts from water samples isvery difficult because the oocyst is small, has little morphological character and, more importantly, is only seen insmall numbers in water samples. In the United States andBritain, methods for detecting oocysts from water have beenreported (Fricker and Crabb, 1998). The method we developed for this survey, which is simple and highly efficient inrecovering oocysts, is suitable for use in developing countries. A notable feature of this method is that it does not involve a centrifugation process during isolation because we
experienced that centrifugation reduced the recovery rate.
E. coli is a bacterial indicator of faecal contamination.
In recent years, enterococci, faecal bacteria of the genera
Enterococcus and Streptococcus, have also been given significant recognition as faecal indicators (Dawson and Sartory, 2000), though we did not examine them. It is reported
that 0.2 mg/l of residual chlorine can kill 90% of E. coli
within two hours (Hsu et al ., 1984). We suspect that the
shortage of residual chlorine concentration was one cause
for the bacterial contamination of tap water. There aremany factors contributing to the decline of residual chlorine
concentration in tap water. In this survey, the reason of low
chlorine concentrations may be the insufficient removal oforganic matter at waterworks, based on relatively high turbidities of tap water and river water from which the tap
water is derived. When flood damaged Hat Yai City in 2000,
we revealed that the mean residual chlorine concentration in
30 tap water samples was maintained at 0.5 mg/l (data notshown). That is, the public works adjustment of the chlorine concentration according to the situation occurred.
However, during the usual operation of the water works inthe same city in 2001, 0.1 mg/l or less chlorine concentration was frequently observed. Therefore, we cannot deny９１

the possibility of mishandled operations at the water purification plant.
Of the three areas studied, Surabaya City had the lowest water quality. Despite this, 88% of people drank tap
water without treating it. As a primary prevention, an immediate improvement of the water-purifying environment,
as well as the education of the populace, are needed. The
spread rate of water supply system in Hat Yai was the lowest (60%). But at the same time, the number of the people
who drank tap water without treating it was the lowest. We
thought that this was probably due to knowledge accumulated through the experience of the local people.
Poorly managed public water supplies have the potential to make a large number of people ill (Dawson and Sartory, 2000). Therefore, we have to continue to put efforts insupplying of safe drinking water and to educate the people
who use it.

REFERENCES
１）Basualdo, J., Pezzani, B., Luca, M.D., Cordoba, A. andApezteguia, M. (2000): Screening of the municipal water
system of La Plata, Argentina, for human intestinal parasites. Int. J. Hyg. Environ. Health, 203, 177-182
２）Dawson, D.J. and Sartory, D.P. (2000): Microbiology
safety of water. Br. Med. Bull., 56, 74-83
３）Fricker, C.R. and Crabb, J.H. (1998): Water-born cryptosporidiosis: detection methods and treatment options.
Adv. Parasitol., 40, 241-278
４）Hsu, S.C., Martin, R. and Wentworth, B.B. (1984): Isola-tion of Legionella species from drinking water. Appl. Environ. Microbiol., 48, 830-832
５）Hsu, B.M., Huang, C. and Jiang, G.Y. (1999): The prevalence of Giardia and Cryptosporidium in Taiwan watersupplies. J. Toxicol. Environ. Health, Part A, 56, 149-160
６）Kent, G.P., Greenspan, J.R., Herndon, J.L., Mofenson, L.
M., Harris, J. S., Eng, T.R. and Waskin, A.A. (1988): Epidemic giardiasis caused by a contaminated public watersupply. Am. J. Public Health, 78, 139-143
７）Luksamijarulkul, P., Pumsuwan, V. and Pungchitton, S.
(1994): Microbiological quality of drinking water and using water of a Chao Phya River community, Bangkok.
Southeast Asian J. Trop. Med. Public Health, 25, 633-637
８）Mac Kenzie, W.R., Hoxie, N.J., Progtor, M.E., Gradus,
M.S., Blair, K.A., Peterson, D. E., Kazmierczak, J.J. andDavis, J.P. (1994): A massive outbreak of Cryptosporidium infection transmitted through the public water supply.
N. Engl. J. Med., 331, 161-167
９）Perz, J.F., Ennever, F.K. and Le Blancq, S.M. (1998):
Cryptosporidium in tap water: comparison of predictedrisks with observed levels of disease. Am. J. Epidemiol.,
147, 298-301
１０）Rose, J.B. and Slifko, T.R. (1999): Giardia, Cryptosporidium, and Cyclospora and their impact on foods: areview. J. Food Prot., 62, 1059-1070
１１）Steiner, T.S., Thielman, N.M. and Guerrant, R.L. (1997):
Protozoal agents: what are the dangers for the public
water supply? Annu. Rev. Med., 48, 329-340
１２）Szewzyk, U., Szewzyk, R., Manz, W. and Schleifer, K.H.
(2000): Microbiological safety of drinking water. Annu.
Rev. Microbiol., 54, 81-127

９３

Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
２,２０
０３, pp.９３‐
９
７

A RAPID SINGLE-STEP SCREENING METHOD FOR
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
DEFICIENCY IN FIELD APPLICATIONSKUNI IWAI1, HIROYUKI MATSUOKA1, FUMIHIKO KAWAMOTO2, MEIJI ARAI1,SHIGETO YOSHIDA1, MAKOTO HIRAI1 and AKIRA ISHII1
Accepted 25, June, 2003
Abstract. The single-step screening method (SSS) is a qualitative rapid screening test for glucose-6-phosphate dehydrogenase (G6PD) deficiency based on blue formazan formation on anion-exchanger. The reaction mixture contains equal volumes of anion exchanger, substrate mixture, coloring mixture (MTT-PMS mix) and distilled water.
We assessed the stability of the reaction mixture and evaluated its reliability with two anion exchangers, DEAESephadex A-50TM and DEAE-SephacelTM, for applications in tests under field conditions. The reaction mixture wassufficiently stable under conditions of incubation at 70°
C for 6 hours or vigorous shaking for 24 hours at room temperature. The reaction mixture could be kept at 30-35°
C for 14 days under indoor conditions without shielding if it
contained no MTT-PMS mix. The coloring was detectable even in diluted blood with hemoglobin concentration as
low as 1.6 g/dl. Under laboratory conditions, the proportion of the samples with 10% of the normal level of activity that were diagnosed as ‘low activity’ was higher with DEAE-Sephacel (92%) than with DEAE-Sephadex A-50
(81%) (p=0.023). The proportion of the samples with normal activity that were diagnosed as ‘normal’ was 98%
with DEAE-Sephacel and 100% with DEAE-Sephadex A-50. In field samples obtained in Myanmar and Indonesia,
the sensitivity was lower (P=0.03 using DEAE-Sephadex and p< 0.001using DEAE-Sephacel) when we used theblue formazan spot test (BFST) as the standard. Twenty-three of 27 G6PD-deficient individuals subjected to genetic analysis were found to have mutations. All individuals who had concordant results between the SSS and theblue formazan spot test (BFST) carried molecular mutations. One case of G6PD mutation was detected amongfour cases diagnosed as G6PD-deficient by SSS with DEAE-SephacelTM, but diagnosed as ‘normal’ by BFST. The
costs of one test with the DEAE-Sephadex A-50TM and the DEAE-SephacelTM system were 0.15 US dollar and 0.30
US dollar, respectively. (297 words)
Key words: Single-step screening test, Glucose-6-phosphate dehydrogenase deficiency, Device approval, Field
trial, Malaria

INTRODUCTION
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a frequent and heterogeneous X-chromosomelinked enzyme abnormality. As G6PD plays a key role inmaintaining erythrocytes, G6PD deficiency possibly resultsin acute hemolysis after exposure to various oxidative
stresses, including infections, medications, and fava beans(favism). A striking correlation is observed between theprevalence of G6PD deficiency and historical malaria endemicity, particularly in tropical and sub-tropical areas (W.
H.O. Working Group, 1989). Therefore, it is important todetect and inform G6PD-deficient individuals in and fromareas in which malaria is endemic before exposing such individuals to oxidative stress in order to avoid acute hemo-

lytic attack, especially hemolytic attack caused by primaquine.
For field screening of G6PD deficiency, the test usedshould be simple to perform and affordable. It is also advantageous if the test reagents can be stored and the reaction can be carried out at around room temperature, particularly in areas with an insufficient supply of electricity.
Several tests, including the G6PD spot test (Fairbanks
and Beutler, 1962), fluorescent spot test (Beutler, 1966),
and blue formazan spot test (Fujii et al ., 1984) have beendeveloped for field screening of G6PD deficiency; however
most of them are complicated or expensive. One of thetests, the blue formazan spot test (BFST) (Fujii et al ., 1984;
Pujades et al ., 1999), has been used for field studies because of its comparatively simple procedure and sufficiently

1 Department of Medical Zoology, Jichi Medical School, Tochigi 329-0498, Japan
2 Department of International Health, Nagoya University School of Medicine, Nagoya 466-0064, Japan９４

low cost. However, BFST requires 8 hours of incubation for
diagnosis, which makes immediate on-site diagnosis difficult.
Recently, a simple and quick screening method, asingle-step screening test (SSS) was developed (Hirono et
al ., 1998). Our previous trials showed the usefulness of
SSS in field studies (Tantular et al ., 1999), but the stability
and reliability of the test have not been fully assessed. In
this study, we assessed in detail the stability of SSS and theresults of the reliability of SSS use under field conditions.

MATERIALS AND METHODSSSS is a qualitative test that assesses the formation ofblue formazan along with the reduced form of nicotinamide
adenine dinucleotide phosphate (NADPH) by G6PD absorbed on an anion exchanger. The reaction mixture consists of equal volumes (200 µl each) of substrate mixture
containing 5 mM glucose-6-phosphate (G6P) and 0.4 mMoxidized nicotinamide adenine dinucleotide phosphate(NADP), coloring mixture (MTT-PMS mix) containing
0.025% 3 (4,5 dimethylthiazolyl 1-2) 2,5 diphenyltetrazolium bromide (MTT), 0.025% phenazine methosulphate(PMS), anion exchanger (originally DEAE-Sephadex A50TM) and distilled water. The test can detect G6PDdeficient individuals with less than 10% residual activity
within 40 minutes without any special equipment.
The procedures and chemicals used for SSS (Hirono et
al ., 1998) and BFST (Fujii et al ., 1989) were as described,
with slight modifications in the procedure for SSS: 0.05%
sodium azide (NaN3) was added and saponin was omitted in
the reaction mixture to avoid the growth of microorganisms.
We assessed the reliability of two different anion exchangers in SSS: translucent DEAE-Sephadex A-50TM and whitecolored DEAE-SephacelTM (Amersham Pharmacia Biotech,
Buckinghamshire, UK). When using the DEAE-SephacelTM
system, 4.8mM of oxidative glutathione (GSSG) was added
to optimize the reactivity (Hirono et al ., 1998).
The blood samples for laboratory assessment weredrawn from one of the authors who was confirmed phenotypically and genotypically to have normal G6PD. The
G6PD gene was analyzed by direct sequencing of all codingexons. The G6PD-deficient blood was artificially madefrom the blood of the same volunteer by heat inactivation at
56°
C for 15 minutes and proved to have no residual G6PD
activity. Nine volumes of heat-inactivated blood and one
volume of fresh blood were mixed to make G6PD-deficient
samples with 10% of the normal activity. The samples forfield comparison and genetic analysis were obtained fromlocal residents who participated in an extensive field study
with the aim of quick detection of malaria and G6PD defi-

ciency (Tantular et al . 1999) in the Taninthayi Division in
Myanmar and on Buru Island in Indonesia, 1998. Prior to
the study, the outline and procedures of this research werediscussed within the committees of the national and local
governments in Indonesia and Myanmar. The participants
were orally informed of and gave consent to the examination twice before screening and blood drawing. We did not
obtain written informed consent from each participant because of the opinion of local co-organizers about the participants’ literacy and differences of local culture. The fieldsamples were used to assess the sensitivity and specificity
of SSS using BFST as the standard. We also compared the‘G6PD-deficient’ samples of SSS with BFST, using genetic
analysis as the standard.
To assess the stability of the test mixtures of SSS under various laboratory conditions, we simulated three different conditions which may often occur during field studies intropical areas: 30-35°
C for 14 days (storage at room temperature), 70°
C for 6 hours in an oven (being left in a car),
continuous shaking using a shaker (TAITEC R-1) at maximum strength (200 rotations/minute) for 24 hours at room
temperature (transporting over a rough surface). We also
examined the photosensitivity of the test, because the dissolved MTT-PMS mixture is highly photosensitive (Fairbanks and Beutler, 1962). For simulating anemia, which is
a common illness and may affect the results (Fairbanks andBeutler, 1962), serial two-fold dilutions of blood with normal G6PD activity from the same volunteer in other laboratory tests were made with phosphate buffered saline (PBS)
to one-eighth of the original concentration (the estimatedhemoglobin concentration was then 1.6 g/dl). For all of theassessments, the combination of newly prepared testing
mixture and fresh blood was used for the standard of thenormal reaction.
For the evaluation of reliability, the results were categorized into two groups, ‘low activity’ and ‘normal activity’,based on color development of the anion-exchanger. Two
investigators who were blinded to the actual activity of samples then evaluated the enzyme activity (50 deficient and 50
normal per person) independently as ‘low activity’ or ‘normal activity’. The results were analyzed by the chi-square
test using a 2×2 table or Fisher’s exact test. P values lessthan 0.05 were considered as statistically significant.
BFST was used as the standard for field assessment of
the sensitivity and specificity of SSS. The SSS was performed at each field site and samples for BFST were obtained on cation-exchange paper (Whatman P-81) (Fujii etal ., 1984) at the same time and dried. The BFST was performed within the same day, and thus the results were obtained the next day. The results were analyzed statisticallyas described above.

９
５

For comparison of the results of SSS and BFST withthe results of genetic analysis, the participants werescreened by SSS, and a blood spot was obtained in the same
way as described above. The participants were diagnosed
with G6PD deficiency based on the results of SSS, and theirblood was drawn for genetic analysis. The procedures forgenetic analysis were described previously (Hirono et al .,1994; Hirono et al ., 1997, Iwai et al ., 2001).

RESULTS AND DISCUSSION
The reaction mixture of SSS retained sufficient reactivity compared with the control mixture after exposure to70°
C for 6 hours or continuous shaking for 24 hours at room
temperature. Reaction mixture without the MTT-PMS mix
could be stored at 30-35°
C for 14 days under indoor conditions without shielding. These features would make thistest appropriate for regular use in primary care and in fieldstudies in rural areas in developing countries. A previously
reported 1-year storage trial (Hirono et al ., 1998) showedthat DEAE-SephadexTM can be kept at room temperature,
the MTT-PMS mix can be stored at 4°
C, and the substrate
mixture is stable at −20°
C. If a refrigerator and freezer areavailable, each mixture can be safely stored for at least1year.
In our simulation of anemia, the reaction mixture
yielded detectable color development when the estimatedhemoglobin concentration of the G6PD-normal blood was
as low as 1.6 g/dl. Because elevated levels of reticulocytes
lead to higher activity of peripheral blood G6PD in the anemic condition (Jansen et al ., 1985), our finding implies that
the G6PD-normal samples of patients with severe anemia
should not be frequently misdiagnosed as ‘G6PD-deficient’.
Table 1 shows the reliability of the 2 anion-exchangers.
Under laboratory conditions, a higher proportion of thesamples with 10% of normal activity were diagnosed as
Table 1. Laboratory evaluation of the single-step screening
method (SSS) with 2 anion exchangers
A. DEAE-Sephadex A-50
Diagnosis
by SSS
Low activity
Normal activity
Total

Residual activity of G6PD
10%*
100%
81
0
19
100
100
100

Total
81
119
200

‘low activity’ with DEAE-SephacelTM (92%) than with
DEAE-Sephadex A-50TM (81%) (p=0.023). With the
DEAE-Sephadex A-50TM system, nearly 20% of G6PDdeficient samples were judged as normal. The red-coloredhemoglobin in the reaction mixture together with the translucent DEAE-Sephadex A-50TM particles may cause misdiagnosis. The proportion of the samples with normal activity
that were diagnosed as ‘normal’ was 98% with DEAESephacelTM and 100% with DEAE-Sephadex A-50TM.
The sensitivity and specificity in field tests are shownin Table 2. We used BFST as the standard because a previous study using the samples of genetically diagnosed
G6PD-deficient patients showed that BFST had 100% sensitivity in hemizygotes and 75% in heterozygotes and 100%
specificity (Pujades et al ., 1999). The sensitivity was significantly higher in males than in females when we use
DEAE-Sephacel as anion-exchanger (P=0.04). Neither thesensitivity nor specificity differed significantly between
DEAE-SephadexTM and DEAE-SephacelTM.
Comparison of field testing with laboratory assessment
revealed that the sensitivity of field testing was lower thanthat of laboratory assessment. The P-value was 0.03 using
DEAE-Sephadex A-50 and less than 0.001 using DEAESephacel. We found no difference in specificity. In field
assessment, the sensitivity was significantly higher among
males when we used DEAE-Sephacel (p=0.04). The actual
value of the sensitivity was also high among males tested
with DEAE-Sephacel, although there was no significant difference (p=0.12). We suppose the discrepancy between
laboratory and field assessments may be due to 3 reasons.
One is the difference of genetic status between males andfemales. Because G6PD deficiency is an X-chromosomelinked abnormality, the enzyme activity is particularly variTable 2. Sensitivity and specificity of the SSS using BFST as astandard
A. DEAE-Sephadex A-50
Diagnosis by SSS (male/female)
Diagnosis
by BFST
G6PD deficiency G6PD- normal
G6PD deficiency
20(18/2)
12(8/4)
G6PD normal
3(2/1)
187(96/91)
Total
23(20/3)
199(104/95)

Total(male/female)32(26/6)
190(98/92)
222(124/98)

Sensitivity: male 69% (18/26), female 33% (2/6)
Specificity:male 98% (96/98), female 99% (91/92)

B. DEAE-Sephacel
B. DEAE-Sephacel
Diagnosis
by SSS
Low activity
Normal activity
Total

Residual activity of G6PD
10%*
100%
92
2
8
98
100
100

Total
94
106
200

*The proportion is significantly different between the DEAE-Sephadex A-50 system and DEAE-Sephacel system

Diagnosis by SSS (male/female)
Diagnosis
by BFST
G6PD deficiency G6PD- normal
G6PD deficiency
17(13/4)
9(3/6)
G6PD normal
9(5/4)
354(171/183)
Total
26(18/8)
363(174/189)
Sensitivity: male 81% (13/16), female 40% (4/10)*
Specificity:male 97% (171/176), female 98% (183/187)*Significantly different (P=0.04)

Total(male/female)26(16/10)
363(176/187)
389(192/197)

９６able in heterozygous females.
The crude G6PD level of blood may also be affectedby anemia caused by various illnesses. Variable Hb levels
together with various levels of G6PD activity in field samples could partially explain the discrepancy of these 2 tests,
although our laboratory assessment showed that the G6PDdeficient samples could be distinguished from G6PDnormal samples.
We also consider it possible that the discrepancy between two tests may be due to inaccurate evaluation by SSS.
The definite cut-off level of coloring should be assessed bycomparison of BFST and SSS using genetically confirmed
samples.
Comparison of the results of SSS and BFST with theresults of genetic analysis showed that 23 of 27 G6PDdeficient individuals detected by SSS, including two heterozygous females, had mutations (Table 3). All samples
for which the results of SSS coincided with those of BFST
were shown to have mutations, regardless of the anion exchanger used for the analysis. One case of class 2 G6PD
mutation, 383 T→C (G6PD Vanua Lava), was detectedamong four cases that were diagnosed as ‘G6PD-deficient’using SSS with DEAE-SephacelTM but diagnosed as ‘G6PD
normal’ with BFST. This result suggests that some cases of
G6PD deficiency may be undetectable using BFST, although a study showed that the G6PD-deficient hemizygotes could be distinguished from heterozygotes and normal controls using BFST (Pujades et al ., 1999).
Including all reagents and disposable supplies, the estimated cost for one test of SSS with DEAE-Sephadex A50TM was approximately half that of a test of SSS with the
DEAE-SephacelTM (0.15 US dollar and 0.30 US dollar, respectively). The cost of both of these systems is below an
acceptable limit.
SSS is a quick, simple and reliable screening test for
G6PD deficiency, although a larger field study is necessary
for more precise evaluation of SSS. All the procedures inthis test could be completed in field conditions without any
electric equipment except during some preparations prior to
the study. Using either anion exchanger, it can be used forquick detection of G6PD deficiency in various situations including field surveys, mass-treatment of malaria and differ-

Table 3. The results of genetic analysis and the BFST of‘G6PD-deficient’ samples diagnosed by SSS
Diagosisby BFST
Deficient
NormalTotal

DEAE-Sephadex A-50
Analyzed
Variants
17
17(1)
1
0
18
17(1)

DEAE-Sephacel
Analyzed
Variants
5
5(1)
4
1
9
6(1)
Heterozygotes in parentheses

ential diagnosis of hemolytic anemia, particularly in areas
with substandard laboratory conditions. Although the whitecolored DEAE-SephacelTM gives better reaction visibility,
the DEAE-Sephadex A-50TM system is more cost-effective,
and is therefore preferable financially, particularly for fieldstudies.

ACKNOWLEDGEMENTS
The authors are indebted to Dr. Akira Hirono (Okinaka
Memorial Institute for Medical Research) for valuable suggestions and critical reading of the manuscript. This study
was supported by research grants from the Japanese Ministry of Education, Science, Culture and Sports (Grant
No. 10041205 granted to Matsuoka, and 09041179 to
Kawamoto). It was also supported by Toyota Foundation(Grant No. 96B3-011, to Kawamoto), the large-scale cooperative program (No. 2) from Japan Society for Promotionof Science (to Kawamoto), and a grant for International
Health Cooperation Research from the Ministry of Health,Labour and Welfare (Grant No. 13-A-5, to Ishii).

REFERENCES
１）Beutler E: A series of new screening procedures for pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency and glutathione reductase deficiency.
Blood 28: 553-562, 1966.
２）Fairbanks, V.F., and Beutler, E. (1962): A simple methodfor detection of erythrocyte glucose-6-phosphate dehydrogenase deficiency (G6PD Spot Test). Blood, 20, 591601
３）Fujii, H., Takahashi, K. and Miwa, S. (1984): A new simple screening method for glucose-6-phosphate dehydrogenase deficiency. Acta Haematol. Jpn., 47, 185-188
４）Hirono, A., Fujii, H. and Miwa, S. (1998): An improved
single-step screening method for glucose-6-phosphate
dehydrogenase deficiency. Jpn. J. Trop. Med. Hyg., 26,
1-4５）Hirono, A., Miwa, S., Fujii, H., Ishida, F., Yamada, K.
and Kubota, K. (1994): Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation
polymorphism analysis. Blood, 833, 3363-3368
６）Hirono, A., Fujii, H., Takano, T., Chiba, Y., Azuno, Y. andMiwa, S. (1997): Molecular analysis of eight biochemically unique glucose-6-phosphate dehydrogenase variants
found in Japan. Blood 1997; 89: 4624-4627
７）Iwai, K., Hirono, A., Matsuoka, H., Kawamoto, F., Horie,
T., Lin, K., Tantular, I.S., Dachlan, Y.P., Notopuro, H.,
Hidayah, N.I., Salim, A.M.A., Fujii, H., Miwa, S. andIshii, A. (2001): Distribution of Glucose-6-phosphate dehydrogenase mutations in Southeast Asia. Hum. Genet.
2001; 108: 445-449

９７８）Jansen G, Koenderman L, Rijksen G, Cats BP, Staal GEJ:
Characteristics of hexokinase, pyruvate kinase, andglucose-6-phosphate dehydrogenase during adult andneonatal reticulocyte maturation. Am. J. Hematol. 20:
203-215, 1985.
９）Pujades, A., Lewis, M., Salvati, A.M., Miwa, S., Fujii, H.,
Zarza, R., Alvarez, R., Rull, E., and Corrons, J.L.V.
(1999): Evaluation of the blue formazan spot test forscreening glucose 6 phosphate dehydrogenase deficiency.

Int. J. Hematol., 69, 234-236
１０）Tantular, I.S., Iwai, K., Lin, K., Basuki, S., Horie, T.,
Htay, H.H., Marwoto, H., Wongsrichanalai, C., Dachlan,
Y.P., Kojima, S., Ishii, A. and Kawamoto, F. (1999): Field
trials of a rapid test for G6PD deficiency in combination
with a rapid diagnosis of malaria. Trop. Med. Int. Health,4, 245-250
１１）W.H.O. Working Group (1989): Glucose-6-phosphate dehydrogenase deficiency. Bull. W.H.O., 67, 601-611

９９

Jpn. J. Trop. Med. Hyg., Vol.３
１, No．
２,２０
０３, pp.９９‐１
０２

Short communication

NATURAL INFECTIONS WITH FILARIAL LARVAE
IN TWO SPECIES OF BLACK FLIES (DIPTERA:SIMULIIDAE) IN NORTHERN THAILAND
MASAKO FUKUDA１，２, WEJ CHOOCHOTE３, ODILE BAIN４, CHIHARU AOKI２and HIROYUKI TAKAOKA２
Accepted 18, July, 2003
Abstract: To find out the natural infection with filarial larvae, female adult black flies were collected on a humanattractant in December, 2001 at Tambol Ban Laung (altitude 750 m), Doi Inthanon National Park, in northern Thailand. The total number of females collected was 823: of which 557 (67.7%) were identified as Simulium asakoae
Takaoka et Davies, 144 (17.5%) as S. nigrogilvum Summers, 97 (11.8%) as S. nakhonense Takaoka et Suzuki and25 (3%) as other six simuliid species. By dissections, eight third- and one second-stage larvae of unidentified filarial species were found in one of 138 S. nigrogilvum and one of 484 S. asakoae, respectively. Non-filarial nematodes were found in 1.03% (5/484) of S. asakoae. This is the first report of natural infections of two black-fly species, S. nigrogilvum and S. asakoae, with a filarial larva.
Key words: black fly, filaria, natural infection, Onchocerca, Simuliidae, Thailand

INTRODUCTION
Simuliidae or black flies have been well known to be apest of humans and animals and also vectors of some parasites and pathogens (Crosskey, 1990). The main medicalsignificance is the transmission of Onchocerca volvulus tohumans by the bites of the flies in Africa and Central andSouth America; certain simuliid species are suspected to bea probable vector of zoonotic onchocerciasis which occurs
sporadically in Japan, North America and Europe (e.g.,
Hashimoto et al., 1990; Takaoka et al., 1996).
In the Oriental Region, several man-biting simuliid
species have been reported, e.g., Simulium asishi Datta, S.
himalayense Puri, S. indicum Becher, S. japonicum Matsumura, S. nodosum Puri and S. tenuistylum Datta (Datta,
1992; Lewis, 1974; Takaoka, 1977). Several other species
have been known as a pest of animals (Datta, 1992; Datta
and Dasgupta, 1975; Friederichs, 1925). Recently, we examined adult female black flies captured on humans andwater buffalos at Ban Pang Fan (250 m in altitude), Chiang
Mai Province, in northern Thailand, and reported for the
１

first time the natural infections of S. nodosum with Onchocerca larvae (Takaoka et al ., 2003).
To get further information on natural filarial infectionsof black flies in northern Thailand, we collected adult black
flies at Tambol Ban Laung (750 m in altitude) in Chiang
Mai Province. Here we report two more black-fly species
naturally infected with a filarial larva.

MATERIALS AND METHODS
Study area
Adult black flies were collected at a site exposed to thesun in the village of Tambol Ban Laung (18°25’-18°
37’ N
and 98°27’-98°42’ E: ca. 750 m in altitude), Doi Inthanon
National Park, Chiang Mai Province, northern Thailand.
Collection of adult black flies
The collection was made on 16 December of 2001, for
12 hr from 06.00 to 18.00 hours using a human attractant(WC, one of the authors) with his legs below the knees exposed. Female black flies landing on or flighting around theInstitute of Scientific Research, Oita Medical University, Oita, Japan
Department of Infectious Diseases, Oita Medical University, Oita, Japan３
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
４
Parasitologie comparée et Modèles expérimentaux, associé à l’INSERM (U567), et École pratique des Hautes Études, Muséum National d’Histoire Naturelle, Paris, France
Correspondence: M. Fukuda, Department of Infectious Diseases, Oita Medical University, Hasama, Oita 879-5593, Japan. E-mail: mfukuda@oita-med.ac.jp
２

１
０
０

attractant were caught by an insect net, killed and preservedin a vial with 70% ethanol solution. Air temperature was recorded every hour from 06.00 to 18.00.
Dissections of adult black flies for filarial larvae
After classification to species using the descriptions ofPuri (1933) and Takaoka and Suzuki (1984), most of flies
were microscopically dissected and searched for filarial larvae in a drop of 0.5% Giemsa’s stain solution on a slide
glass. The filarial larvae found were preserved in 2% formalin solution for morphometric observations. Generic diagnosis of third-stage and second-stage filarial larvae followed
Bain and Chabaud (1986).

RESULTSBlack-fly species collected
The air temperature measured fluctuated between 10.7°
C and 19.8°
C. A total of 823 females of nine black-fly species was collected on a human attractant. The most abundant species was S. asakoae Takaoka et Davies (67.7%) followed by S. nigrogilvum Summers (17.5%) and S. nakhonense Takaoka et Suzuki (11.8%), and six other species
were few, consisting of 0.1-2.2% (Table 1).
Table 1. Numbers of female adult black flies collected on ahuman, at Tambol Ban Laung, Doi Inthanon National Park, Chiang Mai Province, northern Thailand, on December 16, 2001.
Simulium spp.
S. asakoae
S. nigrogilvum
S. nakhonense
S. chamlongi
S. fenestratum
S. nodosum
S. chumpornense
S. siamense
S. sp. E
Total

No. of collected
557
144
97
18
2
2
1
1
1
823

% of total
67.7
17.5
11.8
!
#
#
#
"3.0
#
#
#
$
100.0

Natural infections with filarial larvae
Natural filarial infections were found in 0.72% (1/138)
of S. nigrogilvum and 0.21% (1/484) of S. asakoae. No infection was found in 97 S. nakhonense dissected. Six otherminor species were not dissected. The infected S. nigrogilvum harboured eight third-stage larvae (L３) in the thorax.
Among them, four larvae measured were 800-900 µm longby 25-31 µm wide (Fig. 1A). The length ratio of theesophagus against the whole body of the three larvae was
0.37-0.41. The L３ had a relatively short and somewhat coni-Fig. 1. Filarial larvae found in the thorax of Simulium species.
(A) Third-stage filarial larva found in S. nigrogilvum(Scale bar, 100 µm). (B) Tail of the third-stage larva(same as A), ventral view (Scale bar, 25 µm). (C)Second-stage filarial larva found in S. asakoae (Scalebar, 100 µm).

cal tail with a salient axial point (Fig. 1B). The other infected fly S. asakoae harboured one second-stage filarial
larva (L２) in the thorax. The L２ was 439 µm long by 35 µm
wide (Fig. 1C).
Other nematodes found in black flies
Non-filarial nematodes were found in 1.03% (5/484)
of S. asakoae dissected. There were at least two types of
non-filarial nematodes distinguished by the characteristic
shape of tail, one sharply pointed and the other bluntly
ended.

１
０
１

DISCUSSION
In the present study, we found that in Tambol Ban
Laung, Doi Inthanon National Park, Chiang Mai Province,
northern Thailand, females of nine simuliid species were attracted to a human. Among them, S. nakhonense, S. chamlongi Takaoka et Suzuki, S. fenestratum Edwards, S. chumpornense Takaoka et Kuvangkadilok, S. siamense Takaoka
et Suzuki and S. sp. E (sensu Takaoka et Suzuki) were collected for the first time using a human attractant.
In our previous investigation carried out in Ban Pang
Fan Village (250 m in altitude), Chiang Mai Province inJune (summer season), a considerable number of S. nodosum were attracted to feed on humans and water buffalos,and 2.3% of them were carrying filarial larvae (Takaoka et
al ., 2003). In Tambol Ban Laung (750 m in altitude), we
found that S. nigrogilvum and S. asakoae, two of the threeabundant species, were naturally infected with filarial larvae
although their natural infection rate was very low. The low
infection rate might be attributable at least partially to thecool air temperature in December (winter season) at thehigh altitude of the study area, which might have been too
low for the development of the filarial larvae, as well as forthe stimulation of blood-feeding. It might be also influenced by many factors of host animals such as population
density, distribution, behavior (e.g., diurnal or nocturnal),
prevalence and intensity of infection with filariae, all ofwhich, though, remain to be studied yet.
A small number of filariae are currently known to betransmitted by black flies (Bain and Chabaud, 1986); they
belong to 4 genera. Species of Splendidofilaria from birds
can be eliminated because of the different caudal extremityand very short body of the infective larva. Mansonella thirdstage larvae, also small, have four caudal lappets, and only
M. ozzardi Manson from humans in South America is transmitted by simuliids (and equally by Culicoides spp.). Onchocerca species are more often transmitted by simuliids.
Among the species which likely exist in cattle from Thailand, O. lienalis Stiles, O. gutturosa Neumann, O. cebei
Galliard and O. gibsoni Cleland et Johnston, the first two
are known to develop in simuliids. O. lienalis is shorter(400-560 µm long) and has a greater ratio of esophagus/
body length; O. gutturosa, which is more frequently transmitted by Culicoides spp., has a higher esophagus/bodylength ratio and a tail differently-shaped. The caudal extremity of the infective larva with a salient axial point isfound in Onchocerca species from African suids (Wahl andBain, 1995), from a Japanese bovid (Takaoka and Bain,
1990; Uni et al., 1998) and in the recently described infective larvae from S. nodosum in Thailand (Takaoka et al.,2003) but the tail of these species is cylindrical and long(more than twice their width at anus); in addition this last L３
from Thailand has a much longer body (1,315-1,500 µmlong). Among Dirofilaria species, D. ursi Yamaguti does
not exist in this area and its infective larva is small; D. immitis Leidy, most commonly transmitted by culicids but capable of developing into L３ in S. takahasii Rubtsov, is similar in the lengths of whole body and esophagus to the present L３ (Takaoka and Baba, 1987), but its tail is not conical.
The present L３, which is thick, of medium length and with ashort tail, represents an unknown filaria from the local wild
mammalian fauna, which might be an Onchocerca species,
or a genus of Dirofilariinae.
The L２ detected from S. asakoae also remained unidentified. It is not clear whether this L２ is conspecific to theL３ from S. nigrogilvum or not. Female S. asakoae has alarge claw tooth, and is supposed to be ornithophilic. Therefore birds are likely to be a host of the L２.
To summarize, this is the first report of natural infections of two black-fly species, S. nigrogilvum and S. asakoae, with a filarial larva.

ACKNOWLEDGEMENT
This study was in part supported by the Grant-in-Aidof Japan Society for Promotion of Science (the fiscal year
2001), provided to HT.

REFERENCES
１）Bain, O. and Chabaud, A.G. (1986): Atlas des larves infestantes de filaires. Trop. Med. Parasitol., 37, 301-340
２）Crosskey, R.W. (1990): The Natural History of Blackflies.
John Wiley & Sons, New York３）Datta, M. (1992): An overview of the Simuliidae (Diptera)of West Bengal, India. J. Bengal Nat. Hist. Soc., New Series, 11, 41-62
４）Datta, M. and Dasgupta, B. (1975): Host-preferences of
black flies (Diptera: Simuliidae) of Darjeeling, West Bengal. Proc. Zool. Soc., Calcutta, 28, 147-153
５）Friederichs, K. (1925): Beobachtungen an Simuliiden in
Ost-Java. Archiv für Schiffs- und Tropenhygiene, 29,
119-129６）Hashimoto, H., Murakami, L., Fujiwara, S., Takayasu, S.,
Takaoka, H., Uga, S., Akao, N., Kondo, K. andYoshimura, H. (1990): A human case of zoonotic onchocerciasis in Japan. J. Dermatol., 17, 52-55
７）Lewis, D.J. (1974): Man-biting Simuliidae (Diptera) ofnorthern India. Israel J. Entomol., 9, 23-53
８）Puri, I.M. (1933): Studies on Indian Simuliidae. Part VII.
Description of larva, pupa and female of Simulium nodosum sp. nov., with an appendix dealing with S. novolineatum nov. nom. (= S. lineatum Puri). Indian J. Med.
Res., 20, 813-817

１０２９）Takaoka, H. (1977): Studies on black flies of the Nansei
Islands, Japan (Simuliidae; Diptera). III. On six speciesof the subgenus Simulium Latreille. Jpn. J. Sanit. Zool.,
28, 193-217
１０）Takaoka, H. and Baba, M. (1987): Experimental infectionof Simulium takahasii with a canine filaria Dirofilaria
immitis. Jpn. J. Sanit. Zool., 38, 1-6
１１）Takaoka, H. and Bain, O. (1990): Infections of blackflies(Diptera: Simuliidae) with three types of zoonotic Onchocerca larvae in Oita, Japan. Jpn. J. Trop. Med. Hyg.,
18, 1-10
１２）Takaoka, H., Bain, O., Tajimi, S., Nakayama, I., Kashima,K., Aoki, C., Korenaga, M. and Otsuka, Y. (1996): Second case of zoonotic Onchocerca infection in a residentof Oita, Japan. Parasite, 3, 179-182
１３）Takaoka, H., Choochote, W., Aoki, C., Fukuda, M. andBain, O. (2003): Black flies (Diptera: Simuliidae) at-

tracted to humans and water buffalos and natural infections with filarial larvae, probably Onchocerca sp., innorthern Thailand. Parasite, 10, 3-8
１４）Takaoka, H. and Suzuki, H. (1984): The blackflies (Diptera: Simuliidae) from Thailand. Jpn. J. Sanit. Zool., 35,
7-45１５）Uni, S., Suzuki, Y., Chiba, H., Katsumi, A., Takaoka, H.
and Bain, O. (1998): An Onchocerca species from cattle
on Kyushu Island is O. suzukii, a transfuge parasite fromthe Japanese endemic bovid, Capricornis crispus. Jpn. J.
Trop. Med. Hyg., 26, 157-160
１６）Wahl, G. and Bain, O. (1995): Development by injectionin Simulium damnosum s. l. of two Onchocerca species
from the wart hog to infective larvae resembling type D
larvae (Duke, 1967). Parasite, 2, 55-62

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
１巻

第２号
内

G6PD 異常症の迅速検査法（Single-step screening 法）の実地応用に向けた検討と評価
岩井 くに，松岡 裕之，川本 文彦，新井 明治，
吉田 栄人，平井誠，石井明…………………………………………………………………９３

Journal of Tropical報
タイ北部におけるブユ２種のフィラリア幼虫自然感染
福田 昌子，Wej Choochote，Odile Bain，青木 千春，高岡

Medicine

著
インターフェロンガンマノックアウトマウスを用いた免疫不全宿主における難治性トキソプラズマ症に対する化学療法の確立のための動物実験モデル
Usama S Belal，野呂瀬一美，文恵聖，陳梅，Rabie M Mohamed，Azza K Ahmed，朴蓮筍，青才 文江，矢野 明彦 ……………………………………………８３
インドネシアおよびタイの水道水からの微生物の検出宇賀 昭二，小田 卓也，木村 憲司，木村 大輔，Koesdarto Setiawan，Margono Sri，Kunaruk Nuvit，Nualta Apakupakul ………………………………………………………８
７

短

宏行 ………………………………９９

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 稲 〃 嶋崎

JUNE２
０
０
３

日熱医会誌

Uga, S., Oda, T., Kimura, K., Kimura, D., Koesdarto, S., Margono, S., Kunaruk, N., and Nualta, A.
Detection of microorganisms in tap water in Indonesia and Thailand …………………………………８７
Iwai, K., Matsuoka, H., Kawamoto, F., Arai, M., Yoshida, S., Hirai, M. and Ishii, A.
A rapid single-step screening method for glucose-6-phosphate dehydrogenase deficiency infield applications …………………………………………………………………………………………９３
Short Communication
Fukuda, M., Choochote, W., Bain, O., Aoki, C., and Takaoka, H.
Natural infections with filarial larvae in two species of black flies (Diptera: Simuliidae) innorthern Thailand ………………………………………………………………………………………９９Vol. 31 No. 2

会報・記録
平成１
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Original article
Belal, U S., Norose, K., Mun, H., Chen, M., Mohamed, R M., Ahmed, A K., Piao, L., Aosai, F.,
and Yano, A.
An animal model for establishing chemotherapy against intractable toxoplasmosis in
immunocompromised hosts by the use of IFN-γ knockout mice ………………………………………８３

Hygiene評
病原体進化論 ――人間はコントロールできるか――記野 秀人 ………………………………………………………………………………………………１
０
３

and

書

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容
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原

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Jpn. J. Trop.
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JAPANESE SOCIETY OF TROPICAL MEDICINE

背は２mm（決）