JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第３
０巻

第２号
内

正明 …………………………………………１０３

ラオスにおける薬剤耐性黄色ブドウ球菌の監視経過（英文）Sithivong, N., Phantouamath, B., Chomlasak, K., Sisavath, L., Munnalath, K., Insisiengmay, S.
比嘉 直美，垣花 シゲ，岩永 正明…………………………………………………………………１０９

日本熱帯医学会表紙!−" ＭＫ 稲 芦塚 中島 宮副

JUNE２
０
０
２

日 本 熱 帯 医 学 会

Original article
Naoi, K., Konishi, E., Matsumura, T. and Yano, A.
A new approach for the analysis of seroprevalence data: a mathematical analysis of
the seroprevalence of Toxoplasma gondii infection in Hyogo prefecture (Japan)
with an implication of periodic outbreaks among young children. ………………………………………９９Nguyen, B. M., Higa, N., Kakinohana, S. and Iwanaga M.
Characterization of Vibrio cholerae O1 isolated in Vietnam ……………………………………………１０３
Sithivong, N., Phantouamath, B., Chomlasak, K., Sisavath, L., Munnalath, K.,
Insisiengmay, S., Higa, N., Kakinohana, S. and Iwanaga, M.
Monitoring of drug resistant Staphylococcus aureus in People’s Democratic Republic of Lao …………１０９
Takaoka, H. and Choochote, W.
Taxonomic notes on the griseifrons species-group in Simulium (Simulium)(Diptera: Simuliidae) from Thailand: Descriptions of two new species anddescription of the male, pupa and larva of S. (S.) digrammicum Edwards ………………………………１１５
Pandey, B. D., Thapa, L. B., Sherchand, J. B., Rimal, N., Bhattarai, A. andMorita, K.
Etiology of Diarrhoea Among Adult Patients During the Early Monsoon Period
in Kathmandu, Nepal ……………………………………………………………………………………１３３

Vol. 30 No. 2

日熱医会誌

Hygiene

会報・記録
理事会・評議員会・会務総会記録……………………………………………………………………………１３９
２
０
０
２（平成１
４年）年度日本熱帯医学会役員名簿（２００
２年６月１日現在）………………………………１４３
日本熱帯医学会雑誌編集委員名簿……………………………………………………………………………１４４
投稿規定…………………………………………………………………………………………………………１４６
著作権複写に関する注意………………………………………………………………………………………１４８

and

カトマンドゥ（ネパール王国）においてモンスーン初期にみられる成人下痢症の病因について（英文）Basu Dev Pandey, Laxmi Bikram Thapa, Jeevan Bahadur Sherchand,
Nirmal Rimal, Achyut Bhattarai，森田 公一 ……………………………………………………………１３３

Medicine

タイで採集されたアシマラダブユ亜属 griseifrons 種群の分類学的検討：
２新種の記載および Simulium (S.) digrammicum Edwards の雄成虫，蛹，幼虫の記載（英文）高岡 宏行，Wej Choochote ……………………………………………………………………………１１５

Journal of Tropical

著抗体データ解析の新たなアプローチ：兵庫県におけるトキソプラズマ抗体陽性率の数学的分析と小児における周期的感染流行の示唆（英文）直井 幸二，小西 英二，松村 武男，矢野 明彦…………………………………………………９
９
ベトナムで分離したコレラ菌の性状（英文）Nguyen Binh Minh，比嘉 直美，垣花 シゲ，岩永JUNE ２０
０
２

CONTENTS

容
Japanese

原

VOL．
３
０ No．
２

平成１
４年６月

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は３mm
（決）

９
９

Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
２,２
０
０２, pp.９
９-１
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２

A new approach for the analysis of seroprevalence data:a mathematical analysis of the seroprevalence ofToxoplasma gondii infection in Hyogo prefecture (Japan) with animplication of periodic outbreaks among young children.
KOJI NAOI1, EIJI KONISHI2, TAKEO MATSUMURA3, and AKIHIKO YANO1＊
Accepted 26, April, 2002

Abstract: We propose a new approach for the analysis of seroprecvalence data. The seroprevalence date on
Toxoplasma gondii infection published in 1997 at Kobe University Hospital were analyzed, with a simple mathematical model, Y = exp(-λ t), where Y , λ and t represent the percentage of seronegative people, annual infection
rate and age, respectively. After calculating the mean annual infection rate (MAIR) on the basis of the above data,
we determined the relationship between MAIR and the year of birth of the female participants in the study. Ourpresent study indicates that MAIR for women born between 1940 and 1960 decreased over the years, and that infection rates correlated with the year of birth. Moreover, assuming that the historically declining trends of MAIR
mainly reflect infection rate changes in childhood, we created a simulation of MAIR for the age under 5 years.
This simulation demonstrated that MAIR for the age under 5 years decreased from around 4% for females born in
1940, to about 1% for those born in 1960. For women born after 1960, it implied that infection rates might havebeen cyclically fluctuating from 0% to 1%, with an approximate 10-year interval. Our analyses imply periodic outbreaks of T. gondii infection among young children in Hyogo prefecture. In Japan, it is difficult to make a sophisticated statistical analysis of seroprevalence of T. gondii mainly due to the lack of available data. Despite simplicity of our new approach, we believe the approach will be useful to grasp the current and the past situations of T.
gondii infection even without enough data.
Keywords: Toxoplasma gondii, congenital toxoplasmosis, mathematical analysis, mean annual infection rate
(MAIR), cyclical outbreaks.

INTRODUCTION
Toxoplasma gondii, an obligate intracellular parasitic
protozoan, induces commonly benign or asymptomatic
toxoplasmosis in healthy persons (Dubey and Beattie, 1988).
However, it may have harmful effects on the fetus whenpregnant women contract primary infection, although rarecases of congenital toxoplasmosis (CT) from patients withchronic infection have been reported (Dollfus et al., 1988).
As we reported previously (Naoi and Yano, 2002), therisk of CT is dependent on annual infection rates before 15
years of age, as well as during the gestational period (equal
to or older than 15 and younger than 45 years old). Hence,
the historical trends of annual infection rates are crucial forevaluating the risk of CT currently faced by pregnant
women. In 1997, Win et al. (1997) compared the seroprevalence data of T. gondii infection between 1981 and1995. The study shows that less people under 60 acquired
new T. gondii infections from 1981 to 1995, and that individuals under 40 contracted few T. gondii infections
throughout their life. Furthermore, the data suggest that theincidence of T. gondii infection is relatively higher in children than adult women. Although these implications are informative, the study does not reveal historical trends of infection, since only two sets of seroprevalence data (taken in
1981 and 1995) are available.
Using a simple mathematical model, we devised asimulation of the historical trends of annual infection rates
for females, based on the sampled data. We initially calculated the mean annual infection rates (MAIR) and examined
the relationship between MAIR and the year of birth of thewomen taking part in the sampling study. Next, we calculated MAIR of women for the age under 5 years from 1940
to 1990, with the assumption that MAIR changes mainly re-1 Department of Infection and Host Defense, Chiba University Graduate School of Medicine, Chiba, Japan
2 Department of Health Sciences, Kobe University School of Medicine, Kobe, Japan
3 Department of Medical Zoology, Kobe University School of Medicine, Kobe, Japan１
００

flect infection rate trends at a relatively young age.

0.012

METHODEIn this study, we employed an exponential curve with aconstant hazard, or Y = exp (-λ t) (1), mainly used for survival analysis. For survival analysis, Y , λ and t represent
the percentage of people alive, annual mortality rate (whichis constant) and years after interventions, respectively. By
assuming that the annual infection rate is constant and thepercentage of seropositivity is cumulative, Eq. (1) is applicable to the simulation of T. gondii infection, in which, Y, λand t represent the percentage of seronegative people, annual infection rate and age, respectively.
By solving Eq. (1), we obtain the equation: λ= (-t)-1 1
n Y (2), in which λ is defined as the mean annual infection
rate (MAIR). In this study, we calculated MAIR for the female participants examined by Win et al., (1997), using Eq.
(2).
We simulated MAIR for the age under 5 years in thefollowing manner: Consider a hypothetical case in which
the percentage of seronegative women 30 years old in 1995
is Y *. With the assumption that MAIR for the age between
5 to 30 years is λ2, we obtain a hypothetical age, or t*, as t*=(-λ2)-1 1n Y * (3), which may be different from the real age,or 30 years old. The percentage of seronegative women at 5
years of age, or Y ** is defined as Y ** = exp {-λ2 [ t*-(305)]} = exp [-λ2 ( t*-25)] (4). If we define MAIR for the age
under 5 years as λ1, we acquire Y ** = exp [-λ2 ( t*-25)] =exp (-5λ1) (5).
By solving Eq. (5), λ1 is calculated as λ1= (-5)-1 1n Y **(6).

Mean annual infection rate

1981
0.01

1995

0.008
0.006
0.004
0.002
0
1900

1920

1940
1960
Year of birth

MAIR are presented in Table 1. The same data are il-lustrated in Fig. 1, in which historical downward trends areclearly indicated from 1940 to 1960. We analyzed datafrom 1940 to 1960 with a regression model to determine
whether there is a correlation between the year of birth and
MAIR. In addition, we evaluated statistical differences between the data of 1981 and 1995.
The regression model
MAIR = α + β (year of birth) + γD + ε
D = 0 (for 1981), = 1 (for 1995).
We acquired the following results:
MAIR = 37.38-0.01904 (year of birth) + 0.02329D
D = 0 (for 1981), = 1 (for 1995).

95% confidence interval for β: -0.02977< β <-0.08311

Table 1 Seroprevalence of T. gondii in Kobe University HospitalMA

1to9
10to1920to2930to3940to4950to5960to6970to79

5
14.5
24.5
34.5
44.5
54.5
64.5
74.5

1981
YOB (mean) MAIR (%)
1976
1966.5
1956.5
1946.5
1936.5
1926.51916.5
1906.5

1.110
0.449
0.124
0.328
0.438
0.324
0.471
0.386

2000

Figure 1 Longitudinal trends of MAIR (mean annual infection rate) of female in Hyogo prefecture.

R 2 =0.9676, adjusted R 2 = 0.8724, s = 0.03942

RESULTS

age

1980

age

MA

1to9
10to1920to2930to3940to4950to5960to6970to79

5
14.5
24.5
34.5
44.5
54.5
64.5
74.5

1995
YOB (mean) MAIR (%)
1990
1980.51970.5
1960.51950.5
1940.51930.5
1920.5(Calculated with the simple mathematical model, Y = exp (-λ t), Y = seronegativity,
λ = mean annual infection rate, t = age)
MA: mean age, YOB: Year of birth, MAIR: mean annual infection rate in %

0.201
0.000
0.000
0.059
0.313
0.433
0.446
0.334

１
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１

95% confidence interval for γ: -0.1319 < γ < 0.1785
n = 5, the birth year from 1940. 5 to 1960. 5 in Table 1.
The real value and the estimated value of MAIR basedon the regression model are illustrated in Fig. 2. The results
indicate that MAIR is negatively correlated with the year ofbirth. However, no statistically significant evidence of differences or a shift of curves, let alone historical decrease,were noted between the data of 1981 and those of 1995.
We presume that the historical downward trends in
MAIR mainly reflect a decline in the incidence of T. gondii
infection among younger children. As shown in Table 1
and Fig. 1, MAIR of children for the age under 5 years significantly decreased (from 1.11% in 1981 to 0.201% in1995). However,our regression model analyses do not indicate a statistically valid reduction in MAIR from 1981 to
0.5
0.45

1995
1981
Estimation

0.4
MAIR（％）

0.35
0.3

MAIR before 5 years of age

0.05(statistically significant for β < 0, p < 0.05)

λ2＝0λ2＝0.0001
λ2＝0.0003
λ2＝0.0005
λ2＝0.0006
λ2＝0.0007

0.04
0.03
0.02
0.01
0

1941 1947 1951 1957 1961 1967 1971 1976 1981 1990

−0.01
Year of birthFigure 3 Simulation of MAIR for female before 5 years of age.
λ2: Hypothetical value of MAIR after 5 years of age,varied between 0% and 0.07%.

1995 for women born between 1940 and 1960 (age variation from 21 to 50 years). These results imply that MAIR
changes historically during childhood rather than adulthood.
We simulated MAIR of children for the age under 5
years, as explained in Methods, in which we varied λ2 between 0% and 0.07% (Table 2 and Fig. 3). Figure 3 implies
that MAIR for the age under 5 years basically decreased
from 1940 until 1960, and may heve been fluctuating cyclically within a range of 0% to 1%, with an approximate 10year interval, since 1960.

0.25
0.2

DISCUSSION

0.15
0.1
0.05
0
1935

1940

1945

1950
1955
Year of birth

1960

1965

Figure 2 MAIR (mean annual infection rate): real vaues andestimations.

Table 2 Simulation of MAIR before 5 years of age
λ21940.5

1946.51950.5

1956.5

1960.500.00010.00030.00050.00060.00070.0471
0.04620.04520.04220.04120.04020.02260.02200.02150.01970.01900.01850.02790.02710.02630.02390.0231
0.02230.006090.005700.005310.004140.00375
0.003360.00404
0.003450.002860.00109
0.00050(<0)λ21966.51970.51976

1980.51990

00.00010.00030.00050.00060.00070.01300.01280.01260.01210.01190.0117

0
0
0
0
0
0

0.01110.01110.01110.01110.01110.0111

0
0
0
0
0
0

0.00201
0.00201
0.00201
0.00201
0.00201
0.00201*λ2: MAIR (mean annual infection rate) after 5 years of age.

A number of reports demonstrate historically declining
trends in the incidence of T. gondii infection in Europe(Ades and Nokes, 1993; Nokes et al., 1997). In these reports, reasons for the decrease in incidence include increasing consumption of frozen meat, improvement in hygiene,
and the increased use of processed and sterilized cat food,among others. In our present report, it is noteworthy thatthe term of MAIR reduction (from 1940 to 1960) includes
1945, the year of the end of World War II. Dramatic socialchanges related to the war as well as society reconstruction
during the aftermath may have affected the decline in
MAIR through this period.
An epidemiological study on Helicobacter pylori byJapanese scientists presents an interesting viewpoint on thecorrelation between the year of birth and incidence of infectious diseases in Japan (Asaka et al., 1992). This study reported that seropositivity for H. pylori is higher in individuals born before 1950. They attributed this decline in seropositivity to the Westernization of Japan. Although manydifferences are prevalent between H. pylori and T. gondii,
including species and infection routes, epidemiologicalstudies on the bacteria may provide new perspectives forfurther research on the causes of historical reduction in１０２

MAIR by the protozoan.
Based on our simulation, possible periodical outbreaksof T. gondii infection among young people are speculated.
This speculation does not contradict our clinical experiences, which indicate periodic outbreaks of CT. Mothers are
generally expected to be exposed to the same risk factors
for T. gondii infection as their younger offspring as theycare for them, and therefore T. gondii infection via contact
with oocysts in cats’feces or contaminated soil is a significant threat for women as well as children. From the standpoint of risk management, public preventive measures,
based on comprehensive research on infection routes formothers and children, behavioral patterns and lifestyles are
required.
From the viewpoint of strict statistical analysis, however, our simulation may face some criticism, mainly on theoriginal data (Takahashi et al., 1985; Win et al., 1997), as
follows:(1) The seroprevalence data of T. gondii used in this simulation are of outpatients in a certain hospital, not of
randomly selected samples from the general people in
Kobe prefecture.
(2) The data of 1981 and those of 1995 are not prospective
but cross-sectional data.
(3) For obtaining the data, self-identification was not executed.
(4) The number of samples is 100 by each age category in
the 1995 survey, while it varies from 7 to 149 in the1981 survey. Hence, for devising the simulation, interval estimation rather than point estimation will be preferred.
Currently in Japan, starkly different from the European
nations, seroprevalence data of T. gondii infection are
scarcely available, not to speak of those of randomly selected samples. While clinical cases of CT are reported inJapan, the recognition of the disease is still less prevalenteven among medical practitioners here than in Europe.
Therefore, new analytical methods, which are useful for researchers of CT to enrich their understanding about the disease even without enough data, are required. We believethat our new approach would be one of those methods. And
we hope that our simulation, an example of analysis with
MAIR, would be of use to find new perspectives for future
epidemiological studies, albeit some controversies overoriginal data and the way of estimation.
We believe that MAIR is an effective tool in mathematical analysis, which reflects the history of infection as awhole and presents an average of incidence in the past. Al-

though in the current analysis, it is simply presumed that
MAIR alterations mainly feflect changes during childhood,other assumptions based on comprehensive epidemiologicalstudies in the future are also possible. On the basis of thesepresumptions, conclusions distinct to those of the present
study may be drawn.
Current model analyses are made in an age-and timedependent manner by European statisticians, based on several decades of seroprevalence data (Ades and Nokes, 1993).
However, these kinds of data are not available in Japan. Although continuous sampling studies of seroprevalence arerequested, it may take numerous more decades in Japan toobtain data extensive enough for similar statistical analyses
as those in Europe. In the meantime, MAIR and related
methods for mathematical speculations on T. gondii infection are effective, and the current procedures of choice inJapan.

REFERENCES
１）Ades, A. E. and Nokes, D. J. (1993): Modeling age- andtime-specific incidence from seroprevalence: Toxoplasmosis. Am. J. Epidemiolo., 137, 1022-1034
２）Asaka, M., Kimura, T., Kudo, M., Takeda, H., Mitani, S.,
Miyazaki, T., Miki,K. and Graham, D. Y. (1992): Relationship of Helicobacter pylori to serum pepsinogens in
an asymptomatic Japanese population. Gastroenterology,
102, 760-766
３）Dollfus, H., Dureau, P., Hennequin, C., Uteza, Y., Bron,
A. and Dufier, J. L. (1998): Congenital toxoplasmosis
transmitted by preconceptionally immune women. Brit J
Ophtalmol., 82, 1444
４）Dubey, J. P. and Beattie, C. P. (1988): Toxoplasmosis of
Animals and Man. Florida: CRC Press. 41-60.
５）Naoi, K. and Yano, A. (2002): A theoretical analysis of therelations between the risk of congenital toxoplasmosisand the annual infection rates with a convincing argument for better public intervention. Parasitol. Int., 51,
187-194
６）Nokes, D. J., Forsgren, M., Gille, E. and Ljungström, !.
(1997): Modelling toxoplasma incidence from longitudinal seroprevalence in Stockholm, Sweden. Parasitology,
107, 33-40
７）Takahashi, J., Konishi, E. and Matumura, T. (1985): A survey of antibodies to Toxoplasma gondii among patientsof a hospital in Hyogo prefecture, Japan, by EnzymeLinked Immunosorbent Assay. Jpn. J. Parasitol, 34, 4752
８）Win, K. S., Matsumura, T., Kumagai, S., Uga, S. and Konishi, E. (1997): Prevalence of antibody to Toxoplasma
gondii in Hyogo prefecture, Japan: Comparison at 10year interval. Kobe J. Med. Sci., 43, 159-168１０
３

Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
２,２
０
０２, pp.１
０３-１
０７CHARACTERIZATION OF
VIBRIO CHOLERAE O1 ISOLATEDIN VIETNAMNGUYEN BINH MINH1, NAOMI HIGA2, SHIGE KAKINOHANA3, and MASAAKI IWANAGA2
Accepted 9, March, 2002

Abstract: V. cholerae O1 isolated in Vietnam in 1995 and 2000 were characterized. Most of the isolates in 2000
showed moderate resistance to tetracycline and chloramphenicol, and strong resistance to sulfamethoxazoltrimethoprim. The susceptibilities to ampicillin, erythromycin, nalidixic acid and ofloxacin were not very different
from those of the isolates in 1995. V. cholerae O1 strains showing this kind of drug susceptibility pattern are
unique to the seventh cholera pandemic, and they have become widely distributed in Southeast Asian countries.
Genetic analysis of the strains from Vietnam by arbitrarily primed polymerase chain reaction suggested that theymight be clonal derivatives. In prophage type, 6 of 20 strains (30%) in 1995 and 1 of 24 strains (4%) in 2000 belonged to Ubol type. Considering the current spreading of V. cholerae O1 with such unique susceptibility to thedrugs, we recommend careful monitoring of the drug susceptibility of V. cholerae O1 throughout the world.
Key words: Vibrio cholerae O1, drug susceptibility, Vietnam

INTRODUCTION
It has been more than 40 years since the seventh cholera pandemic started in 1961. During that period, the responsible pathogen, Vibrio cholerae O1 El Tor has undergone some changes in its biological behavior. This pathogen
was first distinguished from classical cholera vibrios basedon its hemolytic property, and thereafter, some other uniqueproperties of El Tor vibrios were described (Feeley, 1966).
These unique properties include the following: El Tor vibrios are resistant to polymyxin B and cholera phage IV,
produce acetoin from glucose (positive VP reaction), andagglutinate chicken erythrocytes when grown on nutrient
agar plates. Soon after the beginning of this pandemic, thehemolytic property, as examined by Feeley and Pittman’s
routine method of testing (Feeley and Pittman, 1963), disappeared in most isolates, but it reappeared in African isolates in the 1980s (Iwanaga et al., 1982). Cholera phage IVsensitive El Tor vibrio appeared in Thailand in 1986
(Iwanaga et al. 1989). Drug resistant vibrios have appearedand disappeared (Ichinose et al., 1986; Glass et al., 1980;
Mhalu et al., 1979). Recently, the isolates in Laos were wellcharacterized, and unique phenotypes such as polymyxin B
sensitive and moderately resistant to tetracycline were
found (Toma et al., 1997; Phantouamath et al., 2001). Poly-myxin B sensitive strains were isolated in Southern Laosfacing the border to the central area of Vietnam. However,general characterization of V. cholerae O1 in Vietnam has
not been carried out yet. In this study, we characterized V.
cholerae O1 isolated in Vietnam in 1995 and 2000 in comparison with the previously characterized strains in theneighboring country, Laos, in an attempt to understand thetransmission of cholera through the border.

MATERIALS AND METHODS
1. Bacterial strains:
Forty-four strains of V. cholerae O1 isolated fromcholera patients in Vietnam were used. Twenty strains wereisolated in 1995 and 24 were isolated in 2000. Several
strains of Lao isolates in 2000 were used for the comparative studies. The strains H218 (biotype classic, serovar
Ogawa) and UDT109 (non-O1 V. cholerae) were used as
the indicator organisms for kappa phage and fs phages, respectively. Identification of V. cholerae O1 was made basedon standard laboratory tests.
2. Hemolytic property:
The hemolytic property was examined using Feeley
and Pittman’s method (Feeley and Pittman, 1963) and sheep
blood agar plate cultures with one point stabbing inocula-

1. National Institute of Epidemiology and Hygiene, Hanoi, Vietnam
2. Department of Bacteriology, 3. Department of Fundamental Nursing,
Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan１
０
４

tion. Strains that appeared to be non-hemolytic based onthese methods were further examined by using heart infusion broth (HIB) supplemented with 1% and 3% glycerol
following the same procedures as in Feeley and Pittman’s
method.
3. Production of acetoin
The organisms were inoculated into VP-semisolid agar
(Eiken) and cultured at 37°
C for 24 hours. Production ofacetoin was then examined by pouring a few drops of 5%
alpha-naphthol and 40% potassium hydroxide over the cultures, and assessing the development of red color.
4. Production of and sensitivity to phages:
To determine the prophage type of the isolates, kappa
phage was examined as described by Takeya and Shimodori(1963). Production of the filamentous phages fs-1 and fs-2
was examined by using the indicator strain V. cholerae
UDT109. V. cholerae O1 isolates were cultured in HIB (10ml in 100-ml Erlenmeyer flasks) at 37°
C overnight with reciprocal shaking, and the culture supernatants were used as
the samples for detection of fs-1 and/or fs-2. The indicatorstrain was cultured overnight in broth, and 0.1 ml of the culture fluid was mixed with 4 ml of soft agar medium (1%peptone, 0.5% yeast extract, 0.5% NaCl, 0.6% agar, pH 7.2)
and overlaid on a glass slide. Ten microlitters of this sample
was dropped on to a layer of the indicator strain and incubated at 37°
C overnight. Sensitivity to the filamentous
phages (fs-1 and fs-2), cholera phage IV, and El Tor phage 5
was examined according to the method described by Ikema
and Honma (1998).
5. Drug susceptibility:
The minimum inhibitory concentration (MIC) of eachdrug was examined by the plate dilution technique. The
drugs examined were ampicillin (Meiji), tetracycline(Nacarai), chloramphenicol (Wako), erythromycin (Dainippon), ofloxacin (Daiichi), nalidixic acid (Wako),sulfamethoxazol-trimethoprim (sulfamethoxazol: Wako,
trimethoprim: Sigma) and polymyxin B (Pfizer). A series ofheart infusion agar plates containing serial 2-fold dilutionsof the drugs at concentration ranging from 100 µg/ml to 0.1µg/ml were prepared. In the case of ofloxacin, the dilution
series was extended to 0.0125 µg/ml. Sulfamethoxazol andtrimethoprim (19:1) were mixed, and the concentration of
the drug was expressed as the total drug weight / ml of the
mixture. A dilution series from 640 µg/ml to 0.078 µg/ml of
the drug combination was made in agar plates. A10-fold dilution of overnight broth culture was inoculated onto eachplate using a Micro planter (Sakuma Co., model MITT #00257), and incubated at 37°
C for 24 hours. The susceptibility was expressed as the MIC of each drug.
6. Arbitrarily primed polymerase chain reaction (AP-PCR)
The clonal variation of the isolates was evaluatedbased on the pattern of the products of AP-PCR obtainedusing a random primer AP42 (5’-CCGCAGCCAA-3’) andAP46 (5’-GAGGACAAAG-3’) as described by Akopyanz
et al. (1992). The PCR conditions were 45 cycles of 94°
C: 2
min, 38°
C: 2 min, 72°
C: 2 min, with 3 min of transitionaltime when raising the temperature from 38°
C to 72°
C, followed by a final incubation at 72°
C for 10 min. The PCR
products were electrophoresed on a 2% agarose gel for 2 hr
at 50 V in an ice box. Then the gel was stained with ethidium bromide, and observed under a UV illuminator.

RESULTS
General properties of the isolates:
All isolates showed the following typical properties ofV. cholerae O1: motile Gram-negative curved rods, yellow
colonies on TCBS agar (sucrose fermentative), glucose fermentation without gas production, lactose non-fermentative,
cytochrome oxidase positive, lysine decarboxylase positive
and arginine hydrolase negative.
Hemolysis:
Although the hemolytic activity was generally very
weak, 19 of 20 isolates (95%) in 1995 and 18 of 24 isolates(75%) in 2000 were hemolytic on sheep blood agar. Whenthey were examined by Feeley and Pittman’s method, 18 of
20 (90%) in 1995 and 12 of 24 (50%) in 2000 were hemolytic. The strains which did not show hemolysis in either
method were cultured in heart infusion broth supplemented
with 1% and 3% glycerol, but the hemolysis did not appear
under these conditions, either.
Production of acetoin:
Examination of the production of acetoin in semi-solid
VP medium (Eiken) revealed that 35% of isolates in 1995
and 62.5% of isolates in 2000 were positive.
Production of and sensitivity to phages:
The isolates in 1995 included 12 strains that produced
kappa phage (Celebes type), 2 that did not produce thephage but was sensitive to it (Cured Celebes type) and 6that neither produced nor were sensitive to it (Ubol type).
The isolates in 2000 included 23 strains that produced
kappa phage (Celebes type) and 1 that did not produce andwas not sensitive to it (Ubol type). Most isolates (18 of 20)
in 1995 were sensitive to filamentous phages fs-1 and/or fs2. Three isolates produced fs-2, but none produced fs-1.
None of the 24 isolates in 2000 produced fs phages, and 21
were not sensitive to them, while the other 3 isolates weresensitive only to fs-1 (Table 1).
Drug susceptibility:
The drug sensitivities of the isolates in 2000 were very
different from those in 1995, as shown in Table 2 and Figure 1. Although the sensitivities of the isolates in 1995 and１０
５Table 1 Production of and sensitivity to phages
Phage

20 isolates in 1995

24 isolates in 2000

production

sensitivityNo.

production

sensitivity

No.

kappa

+
-

+
-

12
2
6

+
-

+
-

23
0
1

fs-1

+
-

+
-

0
17
3

+
-

+
-

0
3
21

fs-2

+
-

+
-

3
13
4

+
-

-

0
0
24

OFLX
8
7
3
1
1

µg/ml
0.08
0.156
0.313
0.625
1.25
2.5
5
10
20
40
80
160
320
640
640<

ST

OFLX
4
14
6

µg/ml
0.08
0.156
0.313
0.625
1.25
2.5
5
10
20
40
80
160
320
640
640<

ST

Table 2 (A) Frequency distribution of MIC for 20 isolates in 1995
µg/ml
0.0125≧
0.025
0.05
0.1
0.2
0.39
0.78
1.56
3.13
6.25
12.5
25
50
100
100<

ABPC

TC

CP

EM

PLB

1
17
2

5
14
4
13
3

NA

18
2
1

1
7
12

1
5
4
2
8

14
1
4
1

(B) Frequency distribution of MIC for 24 isolates in 2000
µg/ml
0.0125≧
0.025
0.05
0.1
0.2
0.39
0.78
1.56
3.13
6.25
12.5
25
50
100
100<

ABPC

TC

CP

EM

PLB

NA

20
1
1
23
1

21
2

20
3

4

24

3
21

1

23

１
０
６

isolation, except in the case of 1 strain when the primer AP
46 was used. This strain was isolated in Vietnam in 1995.
The AP-PCR patterns of representative isolates are shownin Figure 2.

Fig. 2 AP-PCR pattern. Lane1:molecular marker (100-bp ladder), 2: strain 00Vie27 (Vietnam isolate in 2000), 3:
strain 00LA4 (Lao isolate in 2000), 4: strain LA1 (Laoisolate in 2000, polymyxin B sensitive), 5: strain Vie47(Vietnam isolate in 1995), 6: strain Vie156 (Vietnam
isolate in1995). The left half of the panel shows the result obtained using primer AP42, and the right using
AP46.

DISCUSSION
Fig. 1 Distribution of MIC of tetracycline (TC), chloramphenicol (CP) and sulfamethoxazol-trimethoprim (ST).
The frequency is expressed by per cent.

2000 to erythromycin, ampicillin, nalidixic acid and ofloxacin were almost the same, the isolates in 2000 were moderately resistant to tetracycline and chloramphenicol, andhighly resistant to sulfamethoxazol-trimethoprim. The MIC
of tetracycline against the isolates in 1995 was 0.4 µg/ml or
lower, except for 1 isolate against which it was 3.13 µg/ml,
whereas the MIC against the isolates in 2000 was 3.13 or
6.25 µg/ml except for 1 isolate against which it was 0.4 µg/
ml. Chloramphenicol showed almost the same pattern asthat seen for tetracycline. The MIC of sulfamethoxazoltrimethoprim against the isolates in 1995 was in most case
0.313 µg/ml, but it was higher than 640 µg/ml against theisolates in 2000.
AP-PCR:
Chromosome analysis by AP-PCR using primers AP42
and AP46 was performed to determine the genetic diversity
among the isolates from different areas and years. The PCR
products revealed almost the same pattern, regardless of differences of phenotype, the year of isolation and the place ofThe general properties of V. cholerae O1 in Vietnam
were clarified in this study. Although the isolates in 2000
were slightly different in hemolytic property, prophage type,and acetoin production from those in 1995, the changes ofthe drug- susceptibility patterns were notable. The characteristic drug susceptibility of the isolates in 2000 was thatthey were moderately resistant to tetracycline and chloramphenicol, and highly resistant to sulfamethoxazoltrimethoprim, while the susceptibilities to ampicillin, erythromycin, nalidixic acid and ofloxacin were not very different from those of the isolates in 1995. These features werevery similar to those of the isolates in Laos after 1998
(Phantouamath et al ., 2000), and also to those of the isolates in Thailand (unpublished data), except that polymyxin
B sensitive strains were not found in Vietnam and Thailand.
Since no plasmid was detected in the Lao isolates (Iwanaga
et al. 1999) as well as in the Vietnam isolates (data notshown) and the phenotype of the Vietnam isolates was very
similar to that of the Lao isolates, this specific susceptibility
pattern may have been mediated by a chromosomal gene.
Previously reported tetracycline-resistant V. cholerae O1strains isolated in a cholera epidemic were highly resistant

１０
７

to tetracycline as well as to other drugs; the resistance wasmediated by a plasmid, and the resistant strains disappearedsoon after the epidemic (Glass et al., 1980). However, thechromosomally mediated resistance in organisms detectedin the present study could be persistent in future epidemics.
Indeed, such strains have spread in Southeast Asian countries since 1998.
Their indistinguishable phenotype and genotype imply
that it is likely that the distribution and spread of these drug
resistant cholera vibrios are due to the same clone. It is alsosuggested that the resistant strains arose from the mutationof previously sensitive strains and were not a new clone
coming from some other source. One strain isolated in Vietnam in 1995 had a slightly different AP-PCR pattern (Fig. 2,
lane 5 in AP46), and this strain was moderately sensitive topolymyxin B (MIC = 12.5 µg/ml), but its AP-PCR pattern
was not identical to those of strains highly sensitive to polymyxin B isolated in Laos (Fig. 2, lane 4 in AP46). Therefore, the origin of the polymyxin B sensitive El Tor vibriosin Laos has still not been clarified.
In the present study of cholera due to organisms moderately resistant to tetracycline, clinical data about the prognosis of the patients treated with ordinary doses of tetracycline was not available, but it is likely that the diarrhea
lasted longer than the diarrhea caused by sensitive organisms. If the use of tetracycline continues, there is a possibility that highly resistant clones will develop. Considering thecurrent spreading of these organisms in Southeast Asian
countries, careful monitoring of the drug susceptibility
throughout the world is required.
This study was done under the Core University Program,
Tropical Medicine, sponsored by Japan Society for the
Promortion of Science.

REFERENCES
１）Akopyanz, N., Bukanov, N.O., Westblom, T.U., Kresovich, S., and Berg, D.E. (1992): DNA diversity among
clinical isolates of Helicobacter pylori detected by PCRbased RAPD fingerprinting. Nucleic Acid Res. 20, 51375142
２）Feeley, J. C. (1966): Minutes of IAMS committee on taxonomy of Vibrios. Intern. J. Syst. Bacteriol. 16, 135-142
３）Feeley, J. C., Pittman, M. (1963): Studies on hemolytic
activity of El Tor vibrios. Bull. Wld. Hlth. Org. 28, 347356
４）Glass, R. I., Huq, I., Akim, ARMA., Yunus, M. (1980):
Emergence of multiply antibiotic-resistant Vibrio cholerae in Bangladesh. J. Infect. Dis.142, 939-942
５）Ichinose, Y., Ehara, M., Watanabe, S., Shimodori, S.,
Waiyaki, P.G., Kibue, A.M., Sang, F.C., Ngugi, J., andKaviti, J.N. (1986): The characterization of Vibrio cholerae isolated in Kenya in 1983. J. Trop. Med. Hyg. 89,
269-276６）Ikema, M. and Honma, Y. (1998): A novel filamentous
phage, fs2, of Vibrio cholerae O139. Microbiology 144,
1901-1906
７）Iwanaga, M., Mori, K., Kaviti, J. N. (1982): Vibrio cholerae O1 in Kenya. J. Clin. Microbiol. 16, 742-743
８）Iwanaga, M., Nakamura, S., Kuyyakanond, T. (1989):
Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee’s cholera phage IV and the phages produced by thehemolytic vibrio lysogenized with the infection of Mukerjee’s cholera phage IV. Microbiol. Immunol. 33, 609618
９）Mhalu, F. S., Mmari, P. W., Ijumba, J. (1979): Rapid
emergence of El Tor Vibrio cholerae resistant to antimicrobial agents during the first six months of fourth cholera epidemic in Tanzania. Lancet 1, 345-347
１０）Phantouamath, B., Sithivong, N., Sisavath, L., Munnalath,K., Chomlasak, K., Insisiengmay, S., Higa, N., Kakinohana, S., Iwanaga, M. (2001): Transition of drug susceptibilities of Vibrio cholerae O1 in Lao People’s Democratic Republic.
Southeast Asian J. Trop. Med. Pub. Hlth. 32, 96-99
１１）Takeya, K. and Shimodori, S. (1963): “Prophage typing”
of El Tor vibrios. J. Bacteriol. 99, 339-340
１２）Toma, C., Sithavath, L., Higa, N., Iwanaga, M. (1997):
Characterization of Vibrio cholerae O1 isolated in Lao
People’s Democratic Republic. Jpn. J. Trop. Med. Hyg.
25, 85-87

１０
９Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
２,２
０
０２, pp.１
０
９-１
１
３

Monitoring of drug resistant
Staphylococcus aureus in People’s
Democratic Republic of Lao
NOIKASEUMSY SITHIVONG1, BOUNNANH PHANTOUAMATH1, KHAMPHENG CHOMLASAK1,LAY SISAVATH1, KHAMPHYEU MUNNALATH1, SITHAT INSISIENGMAY1
NAOMI HIGA2, SHIGE KAKINOHANA3, MASAAKI IWANAGA2
Accepted 31, May, 2002

Abstract: Drug susceptibility of Staphylococcus aureus has been examined in the past 9 years to monitor the appearance of drug resistant strains especially methicillin resistant S. aureus (MRSA). The pattern of drug susceptibility has been relatively constant, but S. aureus is gradually becoming resistant to ampicillin. One MRSA was
isolated in 1996 from the nasal vestibulum of a non-infected patient at Mahosot Hospital but not isolated thereafter.
In 2001, 2 strains of MRSA which belonged to different clones were first isolated from the infection foci of patients. One was isolated from Setthathirath Hospital, where many Japanese staff have been dispatched since 2000.
Careful monitoring and epidemiological studies of MRSA are recommended.
Key words: Staphylococcus aureus, drug susceptibility, Laos

INTRODUCTION
Staphylococcus aureus is one of the most important
pathogens throughout the world. Because S. aureus easily
develop resistance to antibiotics when these drugs are used
to treat infections, much attention has been focused on theappearance of drug-resistant strains. In the past 2 decades,
the incidence of methicillin-resistant Staphylococcus aureus(MRSA) has been an especially hot issue in the clinicalfield. The proportion of MRSA among S. aureus isolates
varies from country to country. One report showed that the
proportion was 1.5% in the Netherlands but 25.1% in Belgium, a neighboring country, and 33.6% in France (Voss etal ., 1994). In Thailand, which borders the People’s Democratic Republic of Lao (Lao PDR or Laos), a high proportion of MRSA was reported (Wongwanich et al ., 2000). It
is a well-known fact that the proportion of MRSA among S.
aureus isolates from hospitalized patients in Japan is over50%.
Antibiotic availability was very restricted in Laos until
the end of the 1980s and there was almost no problem ofdrug-resistant pathogens until then. Once hospitals becomeheavily contaminated with MRSA, eradication is almost impossible (Thompson et al ., 1982). Therefore, prevention of
the development of MRSA in the hospital is a critical prob-

lem. There must currently be an increased risk of developing drug-resistant pathogens in Laos, since the kinds of antibiotics available and their consumption were steadily increased from 1990 until the present. Because we anticipated the development resistant strains, we have been monitoring the drug-resistant S. aureus in Laos since 1993. In
this communication, we described the results of 9 years ofmonitoring of drug-resistant S. aureus in Laos.

MATERIALS AND METHODS
Bacterial strains
S. aureus used in this study was isolated from clinicalspecimens submitted to Center for Laboratory and Epidemiology, Vientiane, Laos. The specimens included swabbed
mateials (lesion of skin, throat, ear, or vagina), sputum,
urine, aspirated fluid, etc. The number of isolates was 54 in
1993, 32 in 1995, 47 in 1996, 56 in 1997, 72 in 1998, 59 in
1999, 35 in 2000, and 61 in 2001 (416 in total).
Drug susceptibility
The susceptibility was expressed as the minimum inhibitory concentration (MIC) of the following 5 drugs as
examined by the plate dilution method. The drugs examined were methicillin (DMPPC, Banyu), oxacillin (MPIPC,

1 Center for Laboratory and Epidemiology, Ministry of Health Vientiane, Laos
2 Department of Bacteriology,
3 Department of Fundamental Nursing Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan１１０Wako) as a substitute for methicillin, ampicillin (ABPC,
Meiji), tetracycline (TC, Nacarai), erythromycin (EM,Dainippon), and cefdinir (CFDN, Fujisawa). A series ofheart infuion agar plates containing serial 2-fold dilutions of
the drugs at concentrations ranging from 100 µg/ml to 0.1µg/ml or lower were prepared. A 10-fold dilution of an
overnight broth culture of the S. aureus strains indicated in
the text was inoculated onto each plate using a Microplanter(Sakuma Co. model MITP #00257) and incubated at 37°
Cfor 24 hours. The MICs of methicillin and oxacillin were
determined using agar plates containing 2% NaCl and cultured at 30°
C.
Detection of mecA gene
The mecA gene was detected by polymerase chain reaction (PCR). The PCR templates samples were preparedfrom the colonies on a nutrient agar plate by emulsifying a
few colonies in distilled water and boiling them for 20 min.
The PCR primers designed to amplify a 630 bp fragment
derived from a portion of mecA, 5’-GAACCTCTGCTCAACAAGTT-3’ and 5’-GGATTTGCCAATTAAGTTTG-3’,
were used as described by Song et al . (1987). The PCR
conditions consisted of 30 cycles of 94°
C for 1 minute, 52°
C
for 1 minute, and 72°
C for 1 minute, and a final incubation
at 72°C for 10 minutes.

RESULTS
Drug susceptibilities
The MICs of ampicillin and methicillin gradually became higher during the past 8 years. This gradual changeof drug susceptibility was shown by determining the cumulative distribution of MIC (Fig.1). The susceptibility of S.
aureus to erythromycin varied from year to year. All isolates were highly sensitive to cefdinir throughout the periodof monitoring (Fig. 1). The MIC of tetracycline becameslightly lower in the past few years. Taken altogether, theMICs values reveal that the drug susceptibility pattern has
not much varied so far. The raw data regarding MIC arepresented in Table 1.
Among the isolates obtained in 2001, there were 3
strains against which the MIC of oxacillin was 12.5 or 100µg/ml, while the MIC against the other isolates was lower
than 3.13 µg/ml. The MICs of oxacillin, ampicillin, tetracycline, erythromycin and cefdinir against these 3 strains
were: 12.5, 6.25, 12.5, 0.2, and 0.8 µg/ml, respectively, forstrain 01LP32; 200, 200, 100, 6.25, 200 µg/ml, respectively,
for strain 01LP40; and 12.5, 200, 25, 0.4, 0.8 µg/ml, respec-

Detection of PBP2’
The expression of mecA was examined by detecting
the gene product, PBP2’. A kit for the detection of PBP2’,MRSA Screen (Denka Seiken Co., Tokyo) was used according to the instructions in the manual supplied with the kit.
Coagulase typing
Coagulase type was determined by neutralization testwith the type-specific antiserum. A Coagulase Typing Immune Sera Kit (Denka Seiken Co. Tokyo) was used to classify the antigenic type of coagulase produced by the isolates.
Coagulation inhibition with the type-specific anti-serum
was examined using plastic microdilution plates as described by Tajima et al . (1992).
Detection of toxins
Production of Staphylococcal enterotoxins (SE) including the antigenic types A, B, C, D, and E was examinedby using a SET-RPLA kit (Denka Seiken Co., Tokyo) for
the detection and typing of staphylococcal enterotoxins.
Toxic shock syndrome toxin-I (TSST-I) production was examined by using a TST-RPLA kit (Denka Seiken Co., Tokyo). The detection was performed according to the instructions given in the manuals supplied with each kit.

Fig. 1: Cumulative frequency distribution (%) of MIC.
―◇― : total of the isolates in 1993, 1995, and 1996―▲― : total of the isolates in 1999, 2000, and 2001
Data for DMPPC do not include 2001

１
１
１

Table 1 MIC of each of the indicated drugs in the past 9 years
The frequency distribution of MIC is expressed as the percentage of total isolates in each year (The number of
total isolates for each year is shown in the bottom line).
DMPPC

ABPC
93

95

96

97

98

99

0

93

95

96

97

98

99

0

1

!0.2

0

0

0

0

0

0

0

!0.2

11

19

2

4

4

5

0

3

0

0

0

0

0

0

0

0

0

0

0

0

2

5

2

3

3

1

0

0

0

0

0

0

0

1

6

13

9

7

3

5

3

0

2

28

3

9

0

4

10

3

2

11

6

66

11

5

10

3

0

3

65

59

49

2

52

41

3

3

35

37

17

28

5

25

14

11

6

7

38

40

64

44

46

29

6

28

19

0

27

14

14

34

20

13

0

0

2

32

0

3

62

13

0

6

0

4

36

5

26

32

25

0

0

0

0

0

0

3

25

7

0

4

5

17

7

6

13

50

0

0

0

0

0

0

0

50

2

0

0

7

4

12

0

13

100

0

0

0

0

0

0

0

100

0

0

2

5

4

15

11

2

>100

0

0

0

2

0

0

0

>100

0

0

0

0

3

0

0

3

number

54

32

47

56

72

59

35

number

54

32

47

56

72

59

35

61

93

95

96

97

98

99

0

1

TC

EM
93

95

96

97

98

99

0

1

!0.2

42

28

51

0

0

0

2

0

3

24

45

51

!0.2

87

85

34

4

82

19

17

7

30

31

12

3

7

0

7

6

2

85

3

52

72

83

1

0

0

0

0

2

0

0

0

0

30

2

0

0

1

0

0

49

2

0

0

0

0

0

2

0

0

2

0

0

13

0

0

0

0

0

3

0

0

0

6

0

22

0

0

3

0

0

0

3

2

0

0

0

0

0

0

0

0

1

3

6

0

6

0

0

0

0

0

0

0

2

13

24

25

11

4

0

17

20

10

13

2

0

0

0

0

0

0

0

25

28

25

21

25

50

6

0

15

36

19

26

17

30

25

0

0

2

0

0

0

0

0

13

14

9

0

50

0

0

0

0

0

0

0

0

100

0

0

0

5

0

0

0

2

100

0

0

0

2

0

0

0

0

>100

0

0

0

0

0

0

0

0

>100

2

9

0

7

15

29

11

8

number

54

32

47

56

72

59

35

61

number

54

32

47

56

72

59

35

61

CFDN
93

95

96

97

98

99

0

1

!0.2

26

34

68

16

14

42

9

31

0

68

50

30

61

79

58

74

66

1

6

13

2

21

7

0

17

3

2

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

6

0

0

0

0

0

0

0

0

13

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

50

0

3

0

0

0

0

0

0

100

0

0

0

0

0

0

0

0

>100

0

0

0

2

0

0

0

0

number

54

32

47

56

72

59

35

61

１
１
２

Fig.2: Detection of mecA gene by PCRlane 1: Molecular size marker (100 bp ladder), lane 2: strain
01LP32, lane 3: strain 01LP40, lane 4: strain 01LP63, lane 5:
mecA-positive S. aureus as control

tively, for strain 01LP63. The production of PBP2’ and thepresence of the mecA gene were examined in these 3 strains.
Strain 01LP40 was isolated from the discharge from the
middle ear of an 8-year-old female patient with otitis media(Friendship Hospital), and 01LP63 was isolated from the
sputum of a 13-year-old male patient with a respiratory infection (Setthathirath Hospital). These 2 strains had themecA gene, as demonstrated by the PCR (Fig. 2), and theyexpressed PBP2’ as examined by the agglutination test.
Strain 01LP32 was negative for both the gene and its product.
Characterization of MRSA strains 01LP40 and 01LP63
The biochemical characteristics of these MRSA strains
were typical of S. aureus, and included fermentation of lactose, mannitol and maltose, and production of acetoin. The
coagulase of 01LP40 did not belong to any of the 8 known
types (I to VIII), and the strain did not produce enterotoxin
or toxic shock syndrome toxin. However, 01LP63 produced enterotoxin type B and type D but did not produce
toxic shock syndrome toxin. The coagulase of 01LP63 wasclassified as type II.

DISCUSSION
This is the first report of MRSA isolated from the infection foci of patients in Laos. Although we reported anisolate of MRSA from the nasal vestibulum of a noninfected patient in Laos in 1996 (Iwanaga et al ., 1997),
MRSA has not been found in the infection foci until thisstudy. Considering the pattern of use of antimicrobial
agents in Laos in the past, the incidence of MRSA is expected to be very low. However, recently, many Laotians

have received medical care in neighboring Thailand, where
MRSA is routinely isolated (Wongwanich et al., 2000), andhave then returned to Laos. Moreover, numerous antibiotics
are imported and their use has increased. As a result, there
is a high risk of the appearance of MRSA or other highly
drug-resistant pathogens. The fact that 2 strains of MRSA
appeared in 2 different hospitals warns us of the increase of
MRSA in Laos.
Ampicillin and tetracycline have been the major antibiotics used in Laos, and this pattern of antibiotic use may explain why the susceptibility of S. aureus to ampicillin is
gradually getting resistant. However, the susceptibility totetracycline has not changed much, and may even have got
sensitive in the past few years. Although it seems that theuse of tetracycline decreased in Laos after 1998, when tetracycline resistant Vibrio cholerae O1 appeared, there is no
reliable data on the rate of use of tetracycline. Therefore, itremains unknown why S. aureus has become more sensitive
to tetracycline in the past few years.
The 2 strains of MRSA isolated here showed different
phenotypes, implying that they belonged to different clones.
One of the strains (01LP63) was isolated at a hospital which
was donated by the Japanese government in 2000, andmany Japanese staff members from a university hospital are
working there under the name of International Cooperation.
Since coagulase type ! is the major type of coagulase produced by MRSA in Japan (Matsumoto et al., 1989; Kimura
et al., 1993), it is possible that 01LP63 was carried from Japan to Laos by the hospital staff. It should be clarified
whether this MRSA strain was carried to Laos by Japanese
staff or not. Regardless of whether it was or not, hospital
staff members have to be careful to avoid the improper useof antibiotics in order to prevent the development of MRSA
in Laos, and to avoid carrying MRSA from Japan to Laos.
Careful monitoring of MRSA in Laos should be continued.

REFERENCES
１）Iwanaga, M., Sithavath, L., Higa, N., Honma, Y., andKakinohana, S. (1997): Emergence of methicillin resistant Staphylococcus aureus in Laos. Jpn J. Trop. Med.
Hyg., 25, 103-106
２）Kimura, A., Igarashi, H., Ushioda, H., Okuzumi, K., Kobayashi, H., Otsuka, T., (1993): Relationship between coagulase toxin-type and drug susceptibility in Staphylococcus aureus strains isolated in all the Japanese National University Hospitals. Jpn. J. Infect. Dis. 67, 223230
３）Matsumoto, K., Takahashi, A., Shishido, H., Watanabe,K., Rikitomi, N., Suzuki, H. et al. (1989): A comparisonof the changes in Staphylococcus aureus during two peri-

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ods. Chemotherapy 37, 549-562
４）Song, M.D., Wachu, M., Doi, M., Ishino, F., and Matsuhashi, M. (1987): Evolution of an inducible penicillin target protein in methicillin-resistant Staphylococcus aureus
by gene function. FEBS Lett. 221, 167-171
５）Tajima, Y., Nagasawa, Z., Tanabe, I., Yamada, H., Kusaba,K., and Tadano, J. (1992): An Improved method for the
serotyping of free coagulase from Staphylococcus aureus.
Microbiol. Immunol. 36, 1233-1237
６）Thompson, R.L., and R.P. Wanzel. (1982): Editorial. International recognition of methicillin-resistant Staphylo-

coccus aureus. Ann. Intern. Med. 97, 925-926
７）Voss, A., Milatovic, D., Wallrauch-Schwarz, C., Rosdahl.
VT., Braveny, I., (1994): Methicillin-resistant Staphylococcus aureus in Europe. Eur. J. Clin. Microbiol. Infect.
Dis. 13, 50-55
８）Wongwanich, S., Tishyadhigama, P., Paisomboon, S.,
Ohta, T., Hayashi, H., (2000): Epidemiological analysisof methicillin resistant Staphylococcus aureus in Thailand. Southeast Asian J. Trop. Med. Public Health 31, 7276

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TAXONOMIC NOTES ON THE GRISEIFRONS SPECIES-GROUPIN SIMULIUM (SIMULIUM ) (DIPTERA: SIMULIIDAE) FROMTHAILAND: DESCRIPTIONS OF TWO NEW SPECIES AND
DESCRIPTION OF THE MALE, PUPA AND LARVA OF
S . (S .) DIGRAMMICUM EDWARDSHIROYUKI TAKAOKA１ and WEJ CHOOCHOTE２
Accepted 28, May, 2002

Abstract: Two new black-fly species, Simulium maenoi sp. nov. and S. choochotei sp. nov. are described, based on
the reared females, reared males, pupae and mature larvae collected in Chiang Mai Province, northern Thailand. In
addition, the male, pupa and mature larva of S. digrammicum Edwards, 1928, originally described from Peninsular
Malaysia and Thailand, are described for the first time. All these three species are assigned to the griseifrons
species-group within Simulium (Simulium) Latreille s. str.
Key words: black fly, Simuliidae, Simulium, Thailand, new species, griseifrons species-group

The griseifrons species-group within the subgenus
Simulium (Simulium) Latreille s. str. is characterized by thefemale scutum with five vittae, simple claws, the male stylewith a basal protuberance furnished with spinules, the ventral plate untoothed posteriorly, the pupal gill with six filaments per side, and the larval postgenal cleft deep, subtriangular or bullet-shaped (Takaoka and Davies, 1996). In theworld list of black flies by Crosskey and Howard (1997),
this species-group includes 10 species, all but one recorded
from the Oriental Region. Apparently five other species
placed in the multistriatum species-group by Crosskey andHoward (1997), and two more species left unplaced by
Takaoka and Davies (1995; 1997) belong to the griseifrons
species-group: i.e., S. horokaense Ono, 1980, S. kawamurae
Matsumura, 1915, S. konakovi Rubtsov, 1956, S. kurilense
Rubtsov, 1956, S. subornatoides Rubtsov, 1947 (all from
Palaearctic Region), and S. rudnicki Takaoka and Davies,
1995, and S. yongi Takaoka and Davies, 1997 (both from
Peninsular Malaysia and Thailand). In Thailand, the griseifrons species-group is represented by four species, S. digrammicum Edwards, 1928 (Edwards, 1928), S. grossifilum
Takaoka and Davies, 1995, S. rudnicki and S. yongi (Kuvangkadilok and Takaoka, 2000), all originally describedfrom Peninsular Malaysia.
During our recent surveys on aquatic stages of black
flies in northern Thailand, we discoverd the pupae and lar-vae of S. digrammicum, which was so far known only fromfemale specimens, and found two new species assignable to
the griseifrons species-group.
These new species are here described on the basis of
the reared females, reared males, pupae and mature larvae;in addition, the male, pupa and mature larva of S. digrammicum are described for the first time.
Collecting and rearing methods, as well as dissectionof anatomical parts for description, were already mentioned
in Takaoka (1983). The morphological features and terms
used herein follow those of Crosskey (1969).
Holotype and most paratype specimens will be deposited at the Natural History Museum (BMNH), London, U.K.
Simulium (Simulium) maenoi Takaoka and Choochote,sp. nov.
DESCRIPTION. Female. Body length ca. 2.5 mm. Head.
Narrower than width of thorax. Frons black, white pruinose,
shiny, widely bare except several dark stout hairs along eachlateral margin; frontal ratio 1.4:1.0:1.2; frons-head ratio 1.0:
3.8. Fronto-ocular area (Fig. 1) moderately developed, subtriangular in shape. Clypeus black, white pruinose, shiny,
moderately covered with dark stout hairs except upper portion bare medially. Proboscis ca. 0.8× as long as clypeus.
Antenna composed of 2+9 segments, light to medium

1. Department of Infectious Disease Control, and Research Center for Asian and Caribbean Diseases, Oita Medical University, Hasama, Oita, 879-5593, Japan
2. Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

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brown except scape, pedicel and most of 1st flagellar segment yellow; 1st flagellar segment ca. 1.5× as long as 2nd
one. Maxillary palp medium brown, composed of 5 segments with proportional lengths from 3rd to 5th segments
1.0:1.1:2.3; 3rd segment (Fig. 2) of moderate size; sensory
vesicle medium in size, ellipsoidal, with rugged surface,
0.37× length of 3rd segment, with large round opening
apically. Maxillary lacinia with 13 or 14 inner and 14 or 15
outer teeth. Mandible with ca. 28 inner and 13 or 14 outerteeth. Cibarium (Fig. 4), at its posterior end, produced into
pharynx as narrow projection covered with numerous indistinct minute tubercles. Thorax. Scutum dark brown, greypruinose, with 5 dark longitudinal unpruinose vittae (i.e., 1
narrow median, 2 rather broad submedian, and 2 rather
broad lateral vittae), all united on prescutellar area when illuminated from dorsally and viewed from anterodorsally(this color pattern reversed when viewed from behind);scutum densely covered with yellow recumbent fine hairs,
interspersed with long upstanding dark hairs on prescutellar
area. Scutellum unpigmented, with long dark and short yellow hairs. Postscutellum light brown, shiny, without hairs.
Pleural membrane bare. Ketepisternum longer than deep,
and bare. Legs (coloration still incomplete, especially on
femora and tibiae). Foreleg: coxa and trochanter whitish
yellow; femur whitish yellow on basal 1/2, dark yellow on
apical 1/2, with apical cap light brown; tibia whitish yellow
except apical 1/5 medium brown; tarsus entirely medium
brown; basitarsus, 2nd and 3rd tarsal segments moderately
dilated; basitarsus ca. 5.5× as long as its greatest width.
Midleg: coxa medium brown; trochanter light brown except
base yellow; femur yellow somewhat darkened apically, andwith apical cap light brown; tibia whitish yellow except apical 1/4 dark brown; tarsus dark brown except basal 1/2 ofbasitarsus whitish yellow. Hind leg: coxa dark brown; trochanter whitish yellow; femur and tibia whitish yellow withapical cap dark brown; tarsus medium brown except a little
more of basal 1/2 of basitarsus and basal 1/2 of 2nd tarsal
segment whitish yellow; basitarsus (Fig. 5) nearly parallelsided, ca. 6.7× as long as wide, ca. 0.7× and ca. 0.6× as
wide as hind tibia and femur, respectively; calcipala distinct
but shorter than wide; pedisulcus distinct. All tarsal claws
simple, without subbasal or basal tooth. Wing. Length 2.2
mm. Costa with spinules and hairs; subcosta haired except
apical 1/5 bare; basal section of radial vein bare; hair tuft atbase of stem vein dark brown; basal cell absent. Abdomen.
Basal scale light brown with a fringe of long hairs; dorsal
surface of 2nd segment pale yellow on anterior 3/4 with apair of large dorsolateral whitish spots broadly connected toeach other in middle; dorsal surface of posterior 1/4 of 2nd
segment and those of other segments dark brown and withshort hairs; tergites 3, 4 and 5 small, tergites 6-8 large andshiny. Ventral surface of abdominal segment 7 without any
sternal plate or any hair clusters. Genitalia (Figs. 7 and 8).
Sternite 8 well sclerotized, bare medially but with 18 or 19
long stout hairs as well as a few short slender hairs laterally
on each side; anterior gonapophysis triangular in shape,
membranous, covered with 8 or 9 short hairs as well as numerous microsetae; inner border slightly concave; narrow
area along inner and posterior margins transparent, bare.
Genital fork of inverted-Y form, with well sclerotized stem;arms slender, each with sclerotized apical bulge having a
distinct projection directed anteriorly. Paraproct in ventral
view nearly as long as wide, with distinct concavity on ventral surface along anterior margin; anteromedial surface ofparaproct moderately sclerotized, and with a few short sensilla; paraproct much produced ventrally, coverd with 33-35
short stout hairs on ventral and lateral surfaces. Cercus inlateral view rounded posteriorly, ca. 0.5× as long as wide,
covered with many short hairs. Spermatheca nearly ovoid,
well sclerotized with no definite reticulate pattern, withminute internal setae; tube and large area around tubal baseunsclerotized; accessary tubes subequal in diameter to eachother, and somewhat larger than major one.
Male. Body length 2.5-2.8 mm. Head. Width slightly
wider than thorax. Upper eye consisting of large facets in
19 or 20 horizontal rows and in 18-20 vertical columns.
Clypeus brownish black, white pruinose, moderately covered with dark stout hairs. Antenna composed of 2+9 segments, dark brown except base of 1st flagellar segment
somewhat pale; 1st flagellar segment elongated, ca. 2.2×
as long as 2nd flagellomere. Maxillary palp composed of 5
segments, with proportional lengths from 3rd to 5th segments 1.0:1.2:2.5; 3rd segment (Fig. 3) of normal size; sensory vesicle small, globular or ellipsoidal, ca. 0.2× as longas 3rd segment, with small opening. Thorax. Scutum
black, dull, uniformly and densely covered with goldenyellow recumbent fine hairs, interspersed with long uprighthairs on prescutellar area; in certain angles of light, scutumwith white-pruinose subquadrate spot on each shoulder, extending posteriorly forming a white-pruinose broad band
along each lateral margin up to base of wing. Scutellum
brownish black, with several upright dark hairs as well as
golden-yellow fine hairs. Postscutellum brownish black,shiny, without hairs. Pleural membrane and katepisternum
as in female. Legs. Foreleg: coxa whitish yellow; trochanter light to medium brown; femur light brown except
apical cap medium brown; tibia brownish black except median large area of outer surface white and sheeny; tarsus entirely brownish black; basitarsus, 2nd and 3rd tarsal segments somewhat dilated (basitarsus ca. 6.8× as long as its
greatest width). Midleg: coxa dark brown to brownish

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black; trochanter dark brown except basal 2/5 yellow; femur
light brown with basal 1/5 yellow and apical cap medium
brown; tibia medium to dark brown except basal 1/2 (or alittle more) whitish yellow; tarsus dark brown. Hind leg:coxa medium to dark brown; trochanter yellow; femur medium brown with basal 1/5 yellow and apical cap blackish
brown; tibia dark brown to blackish brown except extreme
base yellowish white; tarsus dark brown except basal 1/3 ofbasitarsus light brown, and a little less than basal 1/2 of 2nd
segment yellowish white; basitarsus (Fig. 6) much enlarged,
gradually widened apically, ca. 3.7× as long as its greatest
width, and ca. 1.05× and ca. 1.13× as wide as hind tibia
and femur, respectively. Calcipala distinct but small. Pedisulcus well marked. Wing. Length 2.0 mm. Other features
as in female except subcosta bare. Abdomen. Basal scale
brownish black with a fringe of dark long hairs. Dorsal surface brownish black except anterior 2/3 of 2nd segment
light to medium brown, and with dark short hairs; segments
2, 5, 6 and 7 each with a pair of whitish-grey pruinose spots,
those on segments 2, 5 and 6 situated dorsolaterally, thoseon segment 7 almost laterally; spots on segments 2 and 6
large and marked, while those on segment 5 small andsomewhat indistinct (in 1 of 3 males examined, spots on
segment 5 not discernible). Genitalia (Figs. 9-17). Coxite
in ventrolateral view much shorter than wide, style in ventrolateral view elongate, much longer than coxite, ca. 3.2×
as long as its greatest width near base, nearly straight andparallel-sided on basal 1/4 or a little more, but gently
curved on apical 3/4, though nearly parallel-sided up toapex and slightly narrower than basal 1/4, and with a subterminal spine; style spatulate ventrodorsally, with basal
protuberance directed dorsomedially, bearing numerous
spinules on its surface. Body of ventral plate subquadrate
though narrowed posteriorly, somewhat longer than wide,and having a long, narrow, ventrally-produced process withseveral short hairs basally on anteroventral surface; basalarms of ventral plate diverging from each other. Mediansclerite moderately sclerotized, plate-like, widening apically,
with rounded apex. Parameres broad basally, each with several hooks apically. Aedeagal membrane densely setose;
dorsal plate moderately sclerotized, with medial portion
somewhat narrowed. Abdominal segment 10 with 1-3 shorthairs on each posterolateral corner. Cerci rounded, eachwith 8-10 short hairs.
Pupa. Body length 2.5-2.9 mm. Head. Integument
dark yellow to yellowish brown, coverd moderately withlarge tubercles of various sizes (Fig. 18); large tubercles
each with several minute projections on its surface (Fig.
19); frons with 2 long fan-like trichomes each with 6-9 long
branches (Figs. 18 and 20) on each side; 2 frontal trichomeson each side situated close together; face with 1 mediumlong trichome with 4 or 5 branches on each side (Fig. 18);
antennal sheath with 9 moderately raised portions produced
outwardly, corresponding to flagellar segments 1-9, similar
to those of S. digrammicum (Fig. 70); outer surface of antennal sheath densely covered with small round tubercles
except intersegmental spaces bare. Thorax. Integument
dark yellow to yellowish brown, moderately covered withlarge tubercles of various sizes anteriorly, and with small
conical tubercles posteriorly; large tubercles each with minute projections on its surface similar to those on frons; integument on each side with 5 long fan-like trichomes, eachwith 9-16 long branches anterodorsally and anterolaterally,
1 medium-long trichome with 4-6 branches posterolaterally,and 3 medium-long trichomes (2 simple or bifid, and 1 with
3-6 branches) ventrolaterally. Gill (Fig. 21) with 6 slendershort filaments arranged in 3 pairs, all filaments arising directly from base, dorsal pair with very short stalk, and middle and ventral ones almost sessile; uppermost filament of
dorsal pair longest (ca. 1.5 mm), ca. 1.3× as long as
counter filament (ca. 1.2 mm), while 4 filaments of middle
and ventral pairs subequal in length (0.9-1.1 mm) and thickness to one another, and slightly shorter than shorter filament of dorsal pair; uppermost filament thickest of all, ca.
1.4× as thick as counter filament of dorsal pair, ca. 2.1×
as thick as 4 other filaments of middle and ventral pairs, andca. 0.8× as thick as interspiracular trunk; 2 filaments of
dorsal pair medium brown, while 4 other filaments pale yellow; cuticle of all filaments with well marked annular ridges
and furrows which are becoming less marked toward apicaltip, and densely covered with minute tubercles. Abdomen.
Tergum 1 dark brown, with 1 simple long seta on each side;
tergum 2 pale except areas near anterior margin dark brown,with 1 simple long seta and 5 short spinous setae submedially on each side; terga 3 and 4 each with 4 hooked spinesand 1 spinous seta on each side; terga 5-9 lacking spinecombs except tergum 8 with spine-combs in transverse row
on each side; tergum 9 with small conical terminal hooks(Fig. 22). Sternum 4 with 1 simple or bifid hooklet and a
few minute setae on each side; sternum 5 with pair of simple or bifid hooks submedially and a few simple minute setae on each side; sterna 6 and 7 each with pair of bifid innerand simple outer hooks somewhat spaced from each other,and a few simple minute setae on each side. Grapnel-likehooklets absent. Cocoon (Figs. 23 and 24). Wall-pocketshaped, tightly woven, with 1 or 2 small slit-like open
spaces anterolaterally just behind anterior thick margin, oneach side, and without ventrolateral flange on each side; individual threads invisible; 3.1-3.6 mm long × 1.5-1.7 mm
wide.

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Mature larva. Body length 5.9-6.4 mm. Body (Photo.
3) normal in shape, with thorax somewhat inflated, and abdomen gradually widened posteriorly up to posterior margin of segment 6, and then narrowed to posterior circlet;body color light bluish green. Cephalic apotome (Photo. 1)
pale yellow with faint positive head spots. Lateral surfaceof head capsule pale yellow; spots behind and below eyespot region indistinct or faintly positive; ventral surface ofhead capsule (Photo. 2) pale yellow or darkly pigmented,
with distinct positive spots on each side of postgenal cleft.
Antenna composed of 3 segments and apical sensillum,
much longer than stem of labral fan; length ratio of segments (from base to tip) 1.0:1.1-1.2:0.5-0.6. Labral fan
with 30-36 main rays. Mandible (Fig. 25) with mandibular
serrations composed of 1 large tooth and 1 small one, without supernumerary serrations; comb-teeth decreasing inlength from 1st to 3rd. Hypostomium (Fig. 26) with 9 anterior teeth, median tooth longer than others; lateral margins
weakly serrate apically; 6 or 7 hypostomal bristles diverging posteriorly from lateral border on each side. Postgenal
cleft (Photo. 2) deep, bullet-shaped, rounded apically, 1.92.9× as long as postgenal bridge. Cervical sclerite composed of 1 small, pale yellow, rod-like piece on each side in
most larvae, or indiscernible in some larvae. Thoracic cuticle almost bare. Abdominal cuticle bare except last segment moderately covered with short colorless setae on eachside of anal sclerite. Rectal papilla of 3 lobes, each with 812 finger-like secondary lobules. Anal sclerite X-shaped,with anterior arms broadened apically, which are ca. 0.6×
as long as posterior ones. Last abdominal segment slightly
bulged laterally but lacking ventral papillae. Posterior circlet with 118-126 rows of hooklets with up to 18-20 hooklets per row.
TYPE SPECIMENS. Holotype female, reared from pupa,
Sai Yoi Waterfall, altitude 830 m, Chiang Mai Province,
northern Thailand, 19.XII.2001, by W. Choochote. Paratypes 2 males, 4 pupae, same data as holotype; 1 pharate female, 1 pharate male, 16 pupae, 7 mature larvae,
Monthatharn Waterfall, altitude 750 m, Chiang Mai Province, northern Thailand, 28.XI.2001, collected by W.
Choochote.

ECOLOGICAL NOTES. Most of the pupae and larvae of S.
maenoi were found to be attached to the surface of thestream-bed rocks in cascading waterfalls, and a few werecollected from grass leaves trailing in the water. Water temperature was 16-19°
C. This species was collected together
with S. brevipar, S. decuplum, S. fenestratum, S. grossifilum, S. rudnicki, S. rufibasis, and S. tani. One of 28 immature larvae of this new species was infected with microsporidans.
ETYMOLOGY. The species name maenoi is given after Dr.
Takashi Maeno, Professor emeritus, Department of Physiology, Shimane Medical University, Shimane, Japan, whom
HT thanks for his advice and encouragement when he andHT were working at Kagoshima University in the 1970s.
REMARKS. S. maenoi sp. nov. is similar in the female to
S. digrammicum, originally described from female specimens collected from Peninsular Malaysia and Thailand (Edwards, 1928). However, the female of S. maenoi differsfrom that of the latter species by the bare basal portion of
the radial vein (cf., fully haired in S. digrammicum). The
male, pupa and mature larva of S. maenoi are also different
from those of S. digrammicum (described later in this paper) by the bare basal portion of the radial vein, and thewall-pocket-shaped cocoon, and the smaller number of posterior circlet rows (ca. 120), respectively (cf., in the latter
species, the basal portion of the radial vein fully haired, thecocoon boot-shaped, and ca. 150 rows of posterior circlet,
respectively).
This new species is distinguished from S. griseifrons
Brunetti, 1911, from India (Puri, 1932) by the smaller number of setae on the anterior gonapophysis in the female, andthe haired ventral plate in the male. The wall-pocketshaped cocoon of S. maenoi easily separates it from S.
griseifrons, which has the boot-shaped cocoon, like S. digrammicum.
S. maenoi seems to be most related to S. japonicum
Matsumura, 1931 from Japan, Korea, Siberia and China(Bentinck, 1955; Takaoka, 1977) by sharing the fenestratecocoon, the similar arrangement of the six pupal gill filaments, and the multibranched trichomes on the pupal head

Figures 1-17. Morphological features of adults of S. maenoi sp. nov. 1, female fronto-ocular area; 2 and 3, 3rd segments ofmaxillary palp with sensory vesicle (2, female; 3, male); 4, female cibarium; 5 and 6, basitarsi and 2nd tarsal
segments of hind leg (left side and outside view; 5, female; 6, male); 7, sternite 8, anterior gonapophyses,
genital fork, right paraproct, right cercus (left paraproct and cercus omitted) and spermatheca of female genitalia in situ (ventral view); 8, right paraproct and cercus (lateral view); 9, coxites, styles, ventral plate, mediansclerite and parameres of male genitalia in situ (ventral view); 10, right coxite and style (ventrolateral view);
11, left style showing a spiny basal protuberance (medial view); 12, ventral plate and median sclerite (lateralview); 13, ventral plate (end view); 14, right paramere (dorsal view); 15, aedeagal membrane and dorsal plate(end view); 16 and 17, 10th abdominal segments and cerci (16, end view; 17, lateral view). Scale bars. 0.1
mm for figs. 5 and 6; 0.05 mm for fig. 1; 0.03 mm for figs. 2-4 and 7-17.

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and thorax. However, this new species is distinguishedfrom the latter species by the following characters (S. japonicum in parenthesis): the female cibarium without tubercles near its posterior margin, and with a dorsally-directed
projection furnished with numerous tubercles (cibariumwith numerous tubercles near its posterior margin but lacking any dorsally-directed projection), the male ventral platewith a posteriorly-narrowed body (ventral plate with aparallel-sided body), spine-combs present only on the pupal
abdominal segment 8 (spine-combs present on the pupal abdominal segments 8 and 9), the pupal gill filaments of middle and ventral pairs sessile (gill filaments of all 3 pairs sessile), the cocoon with a small, slit-like window on each side(cocoon with a large anterolateral window on each side), thelarval cephalic apotome with faint positive spots (larval cephalic apotome with distinct spots), and the bullet-shaped
postgenal cleft (postgenal cleft subtriangular).
This new species seems to be also related to S. biforaminiferum Datta, 1974, described from pupae and larvae,

collected from west Bengal (Datta, 1974) and left unplaced
to any species group by Crosskey and Howard (1997), bysharing the fenestrate cocoon, the similar arrangement of
the six pupal gill filaments, and the multibranchedtrichomes on the pupal head and thorax, and the bulletshaped larval postgenal cleft. However, S. maenoi is easily
distinguished from the latter species by the following pupal
characters (S. biforaminiferum in parenthesis): fan-liketrichomes on the head and thorax (brush-like trichomes),
spine-combs present only on the abdominal segment 8(spine-combs present on the abdominal segments 7, 8 and9), and the cocoon with small, slit-like windows (the cocoon with large, elliptical windows).
Simulium (Simulium) choochotei Takaoka, sp. nov.
DESCRIPTION. Female. Body length 3.4-4.0 mm. Head.
Narrower than width of thorax. Frons black, white pruinose,
shiny, widely bare except several dark stout hairs along each

Figures 18-26. Morphological features of the pupa and larva of S. maenoi sp. nov. 18, frons and part of face of pupa (right half); 19,
tubercles with secondary projections on frons; 20, frontal trichome; 21, pupal gill filaments (left side, lateral view);
22, terminal hook (lateral view); 23 and 24, cocoons (23, lateral view; 24, dorsal view); 25, apical part of larval mandible; 26, larval hypostomium. Scale bars. 1.0 mm for figs. 23 and 24; 0.1 mm for fig. 21; 0.05 mm for fig. 18; 0.03
mm for figs. 20 and 26; 0.02 mm for fig. 22; 0.01 mm for figs. 19 and 25.

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lateral margin and a few hairs near antennal bases; frontal
ratio 1.4:1.0:1.6; frons-head ratio 1.0:5.2. Fronto-oculararea (Fig. 27) moderately developed, subtriangular in shape.
Clypeus black, white pruinose, shiny, moderately covered
with dark stout hairs except median portion bare longitudinally. Proboscis ca. 0.58× as long as clypeus. Antennacomposed of 2+9 segments, dark brown except scape,pedicel and most of 1st flagellar segment yellow whenviewed from ventrally (though dorsal surface of 1st flagellar
segment dark brown on apical 1/2 or more); 1st flagellar
segment ca. 1.9× as long as 2nd one. Maxillary palp darkbrown, composed of 5 segments with proportional lengths
from 3rd to 5th segments 1.0:1.0:2.3; 3rd segment (Fig. 28)
of moderate size; sensory vesicle medium in size, ellipsoidal, with rugged surface, 0.33× length of 3rd segment,with medium round opening apically. Maxillary laciniawith 11 or 12 inner and 16-18 outer teeth. Mandible withca. 30 inner and 16 or 17 outer teeth. Cibarium (Fig. 30)
with ca. 36 minute tubercles near its posterior margin, andat its posterior end, produced into pharynx as round medial
projection covered with numerous minute tubercles. Thorax. Scutum brownish black, shiny, grey pruinose, with 5
dark longitudinal unpruinose vittae (i.e., 1 narrow median, 2rather broad submedian, and 2 rather broad lateral vittae),all united on prescutellar area when illuminated from dorsally and viewed from anterodorsally (this color pattern reversed when viewed from behind); scutum densely covered
with yellow recumbent fine hairs, interspersed with long
upstanding dark hairs on prescutellar area. Scutellum pale,with long dark marginal hairs as well as short yellow hairs.
Postscutellum medium brown, shiny, without hairs. Pleural
membrane bare. Katepisternum longer than deep, and bare.
Legs. Foreleg: coxa yellowish white; trochanter lightbrown except base yellow; femur dark yellow, with apicalcap light brown; tibia whitish yellow except base lightbrown, and a little less than apical 1/2 medium brown to
blackish, though surface of inner margin somewhat darkened even along basal 1/2; tarsus entirely black; basitarsus,
2nd and 3rd tarsal segments moderately dilated; basitarsus
ca. 6.5× as long as its greatest width. Midleg: coxa darkbrown; trochanter light brown except basal 1/2 yellow; femur yellow or dark yellow, with apical cap light brown;
tibia whitish yellow except a little less than apical 1/2 darkbrown to black; basitarsus dark brown to brownish black
except basal 2/5 dark yellow or light brown, though its border not well defined; other tarsal segments black. Hind leg:coxa dark brown; trochanter yellow; femur yellow on basal
1/3, dark yellow on apical 2/3, with apical cap medium
brown, tibia yellowish white on basal 3/5 (or a little more),dark brown to brownish black on apical 2/5; tarsus
brownish black except a little more than basal 1/2 of basi-

tarsus and basal 1/2 of 2nd tarsal segment whitish yellow;
basitarsus (Fig. 31) nearly parallel-sided, ca. 7.2× as longas wide, ca. 0.7× and ca. 0.6× as wide as hind tibia andfemur, respectively; calcipala distinct, nearly as long as
wide; pedisulcus distinct. All tarsal claws simple, without
subbasal or basal tooth. Wing. Length 3.0-3.1 mm. Costa
with spinules and hairs; subcosta haired except near apex
bare; basal section of radial vein fully haired; hair tuft atbase of stem vein dark brown; basal cell absent. Abdomen.
Basal scale medium brown with a fringe of dark long hairs;
dorsal surface of 2nd segment pale yellow on anterior 3/5
with a pair of large dorsolateral whitish spots broadly connected to each other in middle; dorsal surface of posterior
2/5 of 2nd segment and those of other segments brownish
black except tergites 3-5 pale yellow, and with short hairs;
tergites 3, 4 and 5 small, tergites 6-8 large and shiny. Ventral surface of abdominal segment 7 with a large round sternal plate medially. Genitalia (Figs. 33-35). Sternite 8 wellsclerotized, bare medially but with 13 or 14 long stout hairs
on each side; anterior gonapophysis somewhat produced
posteriorly and rounded ventrally, with round posteromedial
corner, membranous basally and laterally, but well sclerotized along inner margin, covered with 22 or 23 long stout
hairs as well as numerous microsetae; inner border slightly
concave; area along posterior margin narrowly transparent,
bare. Genital fork of inverted-Y form, with well sclerotized
stem; arms slender, each with sclerotized apical bulge having a distinct projection directed anteriorly. Paraproct inventral view ca. 1.4× as wide as long, and with deep concavity along anterior margin; anteromedial surface of paraproct moderately sclerotized, and with 7 or 8 sensilla; paraproct much produced ventrally, covered with ca. 60 shortand long stout hairs on ventral and lateral surfaces. Cercus
in lateral view subquadrate, ca. 0.6× as long as wide, covered with many short hairs. Spermatheca nearly ovoid, wellsclerotized with no definite reticulate pattern, with minuteinternal setae; tube and large area around tubal base unsclerotized; accessary tubes subequal in diameter to each other,and also subequal to major one.
Male. Body length 3.6-4.2 mm. Head. Width slightly
wider than thorax. Upper eye consisting of large facets in
20 horizontal rows and in 17 vertical columns. Clypeus
brownish black, white pruinose, moderately covered withdark stout hairs. Antenna composed of 2+9 segments, darkbrown except scape, pedicel and basal 1/2 of 1st flagellar
segment yellow; 1st flagellar segment elongate, ca. 2.2× aslong as 2nd flagellomere. Maxillary palp composed of 5
segments, with proportional length from 3rd to 5th segments 1.0:1.1:2.6; 3rd segment (Fig. 29) of normal size;
sensory vesicle small, ellipsoidal, ca. 0.23× as long as 3rd

１
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２

Figures 27-35. Morphological features of adults of S. choochotei sp. nov. 27, female fronto-ocular area; 28 and 29, 3rd segments ofmaxillary palp with sensory vesicle (28, female; 29, male); 30, female cibarium; 31 and 32, basitarsi and 2nd tarsal
segments of hind leg (left side and outside view; 31, female; 32, male); 33, sternite 8, anterior gonapophyses, genital
fork, right paraproct, right cercus (left paraproct and cercus omitted) and spermetheca of female genitalia in situ
(ventral view); 34, right paraproct (ventral view); 35, right paraproct and cercus (lateral view).
Scale bars. 0.1 mm for figs. 31 and 32; 0.05 mm for fig. 27; 0.03 mm for figs. 28-30 and 33-35.

１２３segment, with opening of medium size. Thorax. Scutum
brownish black, slightly shiny, uniformly and densely covered with golden-yellow recumbent fine hairs, interspersedwith long upright hairs on prescutellar area; scutum withlarge white-pruinose spot on each shoulder, extending posteriorly forming a white-pruinose broad band along eachlateral margin up to base of wing; in certain angle of light,
large white-pruinose spot present on prescutellar area.
Scutellum brownish black, with several upright dark hairs
as well as golden-yellow fine hairs.
Postscutellum
brownish black, shiny, without hairs. Pleural membrane
and katepisternum as in female. Legs. Foreleg: coxa yellowish white; trochanter medium brown except basal 1/2 ofinner surface yellow; femur light brown except apical cap
medium to dark brown; tibia brownish black with medianlarge area of outer surface slaty and sheeny in certain angleof light; tarsus entirely brownish black; basitarsus, 2nd and3rd tarsal segments somewhat dilated (basitarsus ca. 7.3×
as long as its greatest width). Midleg: coxa brownish black;trochanter brownish black except basal 2/5 yellow; femur
light brown with apical cap brownish black; tibia darkbrown to brownish black except extreme base yellowish
white; tarsus brownish black though basal portion of basitarsus dark brown. Hind leg: coxa brownish black; trochanter yellow; femur light brown with basal 1/4 yellow
and apical cap brownish black; tibia brownish black except
extreme base yellowish white; tarsus brownish black except
basal 1/3 or a little more (and basal 1/2 or more along ventral margin) of basitarsus dark yellow or light brown, andbasal 1/3 of 2nd segment yellowish white; basitarsus (Fig.
32) somewhat enlarged, slightly widened from base to apical 1/4, then slightly narrowed toward apex, ca. 4.6× aslong as its greatest width, and ca. 0.9× and ca. 1.0× as
wide as hind tibia and femur, respectively. Calcipala distinct but small. Pedisulcus well marked. Wing. Length
3.0-3.1 mm. Other features (including the haired basal portion of radial vein) as in female except subcosta bare. Abdomen. Basal scale brownish black with a fringe of longdark hairs. Dorsal surface brownish black except anterior
2/3 of 2nd segment light to medium brown, and with dark
short hairs; segments 2, 5, 6 and 7 each with a pair of large
whitish-grey pruinose spots (in addition, segments 3 and 4
each often with a pair of similar dorsal spots though muchsmaller and not shining); those on segments 2, 5 and 6 situated dorsolaterally, while those on segment 7 almost laterally. Genitalia (Figs. 36-46). Coxite in ventrolateral view
shorter than wide; style in ventrolateral view elongate,
longer than coxite, ca. 3.2× as long as its greatest width
near base, nearly parallel-sided, though apical 2/3 gently
curved and slightly narrower than basal 1/3, and with a subterminal spine; style spatulate ventrodorsally, with basal

protuberance directed dorsomedially, bearing numerous
spinules on its surface. Body of ventral plate nearly quadrate, slightly shorter than wide, and having a long ventrallyproduced process with short hairs basally on ventral surface,but its apex and posterior surface entirely bare; basal armsof ventral plate nearly parallel-sided or slightly diverging
from each other. Median sclerite long, plate-like, narrowbasally, gradually widened toward apex, and moderately
sclerotized except median portion of apex unsclerotized or
weakly sclerotized. Parameres broad basally, each withseveral hooks apically, as in S. maenoi. Aedeagal membrane densely setose; dorsal plate strongly sclerotized. Abdominal segment 10 with 13-16 short hairs on each posterolateral corner. Cerci rounded, each with 14-18 short hairs.
Pupa. Body length ca. 4.0 mm. Head. Integument
dark yellow to yellowish brown, densely covered with tubercles all over, even on antennal sheaths, which have nooutwardly-raised portions; tubercles on frons somewhat
larger than others on face and antennal sheaths, and withseveral minute projections on its surface (Fig. 47); frons
with 2 long trichomes each with 2-4 long branches (Fig. 48)
on each side; 2 frontal trichomes on each side situated close
together; face with 1 long trichome with 3 or 4 long
branches (Fig. 49) on each side. Thorax. Integument dark
yellow to yellowish brown, densely covered with tubercles
all over; relatively larger tubercles on anterior surface eachwith minute projections on its surface similar to those on
frons; integument on each side with 5 very long fan-liketrichomes, each with 5-8 long branches (Fig. 50) anterodorsally and anterolaterally, 1 long trichome with 2-4 branches
posterolaterally, and 3 long trichomes (each with 2-4
branches, or rarely no branch) ventrolaterally. Gill (Figs.
51 and 52) with 6 somewhat inflated short filaments arranged in 3 pairs, all arising from base; middle pair almost
sessile and dorsal and ventral pairs with short stalk; inner
filament of dorsal pair directed inward and upward, andsuddenly curved outward or forward or backward, outerfilament of dorsal pair directed upward and forward; 2 filaments of middle pair directed forward and then somewhat
upward; 2 filaments of ventral pair directed downward andthen forward, though inner filament usually directed somewhat inward; all filaments dark brown, subequal in length(average 1.8 mm, range 1.3-2.5 mm) and in thickness to one
another; cuticle of all filaments with well-marked annularridges and furrows, ridges elaborately forming reticulate
patterns and densely covered with minute tubercles, somewhat larger ones on ridges and smaller ones on interridge
spaces (Fig. 53). Abdomen. Tergum 1 yellow, with 1 simple long seta on each side; tergum 2 pale, with 1 simple
medium-long seta and 5 short spines submedially on each

１２４Figures 36-46. Male genitalia of S. choochotei sp. nov. 36, right coxite, right style and ventral plate in situ (ventral view); 37, right
coxite and style (ventrolateral view); 38, left style (medial view); 39-42, ventral plates (39, ventral view; 40, lateralview, with median sclerite; 41 and 42, of different males, end view); 43, median sclerite (end view); 44, dorsal plate(end view); 45 and 46, 10th abdominal segments and cerci (45, end view; 46, lateral view). Scale bar. 0.05 mm forfigs. 36-46.

１２
５side; terga 3 and 4 each with 4 hooked spines and 1 simple
spine on each side; terga 5-9 lacking spine-combs except
tergum 8 with well developed spine-combs in transverse
row on each side (tergum 8 broadly yellow transversely
covering bases of spine-combs); terga 6-9 each with comblike groups of minute spines in transverse rows on eachside: tergum 9 yellow entirely, and sparsely tuberculate atbases of terminal hooks, which are very large and directed
posteriorly (Figs. 54 and 55). Segments 2-5 each with 3
short simple spines on each lateral surface. Sternum 4 with
2 simple or bifid hooklets and a few medium-long spinous
setae on each side; sternum 5 with pair of simple or bifid
hooks submedially and a few simple medium-long spinous
setae on each side, sterna 6 and 7 each with pair of simple
inner and simple outer hooks somewhat spaced from eachother, and a few simple medium-long spinous setae on eachside. Grapnel-like hooklets absent. Cocoon (Fig. 56).
Boot-shaped, loosely woven, with many small open spaces
in webs; wall around opening very high, being ca. 3.0 mm;
individual threads visible; ca. 6.0 mm long × ca. 1.8 mm
wide.
Mature larva. Body length 9.6-11.2 mm. Body
(Photo. 6) characteristic in shape, being gradually widened
posteriorly up to abdominal segment 8, and then sharply
narrowed to posterior circlet; body color entirely dark greyish brown. Cephalic apotome (Photo. 4) light to medium
brown except anterior 2/5 usually dark brown, though narrow portion along posterior margin dark brown, and lateral
portion between mediolateral and posterolateral spots oneach side usually medium to dark brown; head spots usuallyindistinct or appearing negative except posterior one of median longitudinal spots and posterior 1/2 of mediolateralspots usually dark brown, and anterior one of the same
spots rarely positive. Lateral and ventral surfaces of headcapsule (Photo. 5) medium to dark brown with faint tomarked negative spots. Antenna composed of 3 segments
and apical sensillum, much longer than stem of labral fan;length ratio of segments (from base to tip) 1.0:1.4:0.5.
Labral fan with ca. 50 main rays. Mandible (Fig. 57) withmandibular serrations composed of 1 large tooth and 1
small one, without supernumerary serrations; comb-teeth
decreasing in length from 1st to 3rd. Hypostomium (Fig.
58) with 9 anterior teeth, median tooth longer than others;
lateral margins weakly serrate apically; 5 hypostomal bristles diverging posteriorly from lateral border on each side.
Postgenal cleft (Photo. 5) deep, subtriangular, though nearlyparallel-sided near base, pointed apically, 2.3-3.0× as longas postgenal bridge. Cervical sclerite (Fig. 59) composed of
2 light brown pieces, of which 1 is stick-like, appearing
fused to occiput, and the other is elliptical, fused or unfusedto the stick-like piece, on each side. Thoracic cuticle almost
bare. Abdominal cuticle bare except last segment moderately covered with short colorless setae on each side of analsclerite. Rectal papilla of 3 lobes, each with 14-18 fingerlike secondary lobules. Anal sclerite X-shaped, with anterior arms broadened and bifurcated apically, which are ca.
0.7× as long as posterior ones. Last abdominal segment
slightly bulged laterally but lacking ventral papillae. Posterior circlet with ca. 210 rows of hooklets with up to 25
hooklets per row.
TYPE SPECIMENS. Holotype female, reared from pupa,
Monthatharn Waterfall, altitude 750 m, Chiang Mai Province, northern Thailand, 28.XI.2001, collected by W.
Choochote. Paratypes 7 females, 7 males, 3 pupae, 49 pupal exuviae and 4 mature larvae, same data as holotype except the date 19.XII.2001.
ECOLOGICAL NOTES. The pupae and larvae of S.
choochotei were found to be attached to the surface of thestream-bed rock in shaded cascading waterfalls. Water temperature was 16 or 17°
C. This species was collected together with S. brevipar, S. decuplum, S. fenestratum, S.
grossifilum, S. maenoi, S. rudnicki, S. rufibasis, and S. tani.
ETYMOLOGY. The species name choochotei is given after Mr. Wej Choochote, Associate professor, Department of
Parasitology, Chiang Mai University, Chiang Mai, Thailand,
who discovered this new species.
REMARKS. S. choochotei sp. nov. is characterized by its
large body. It seems to be most related to S. tenuitarsus
Puri, 1933, originally described from the male and pupal
specimens collected from Bengal Terai, India (Puri, 1933),
by having the similar shape of the male genitalia, the similar coloration of the antenna, the haired basal portion of theradial vein, multibranched trichomes on the head and thoraxof the pupa and the boot-shaped cocoon. However, thisspecies is easily distinguished from the latter species by thesomewhat inflated pupal gill filaments (Figs. 51 and 52).
The cocoon and the male ventral plate of S. kawamurae
from Japan (Bentinck, 1955) are similar to those of S.
choochotei, but both species are different from each otherby many other characters including female genitalia, malestyle, and pupal gill filaments.
S. choochotei differs from S. digrammicum, S. grossifilum, S. maenoi, S. rudnicki and S. yongi in the female by
the presence of numerous long stout hairs on the anterior
gonapophysis (Fig. 33), haired basal portion of the radialvein (except S. digrammicum), the cibarium with a round
dorsally-directed projection (Fig. 30); in the male by the

１２６

Figures 47-59. Morphological features of the pupa and larva of S. choochotei sp. nov. 47, tubercles with secondary projections onfrons; 48, frontal trichome; 49, facial trichome; 50, thoracic trichome; 51 and 52, pupal gill filaments (51, right side,
dorsal view; 52, left side, lateral view); 53, middle part of pupal gill filament; 54 and 55, terminal hooks (54, end
view; 55, lateral view); 56, cocoon (lateral view); 57, apical part of larval mandible; 58, larval hypostomium; 59,
cervical sclerite of larval head. Scale bars. 1.0 mm for fig. 56; 0.2 mm for figs. 51 and 52; 0.1 mm for fig. 59; 0.05
mm for figs. 48-50 and 58; 0.02 mm for 53-55 and 57; 0.01 mm for fig. 47.

１２
７

shape of the ventral plate (Fig. 39) and the 10th abdominal
segment with numerous hairs on each posterolateral corner(Figs. 45 and 46); in the pupa by the somewhat inflated gill
filaments (Figs. 51 and 52), and the shape of the cocoon(Fig. 56), and in the larva by the shape and size of the larva(Photo. 6).
Simulium (Simulium) digrammicum Edwards, 1928
Simulium digrammicum Edwards, 1928: 61-62 (female).
Simulium (Simulium) digrammicum: Takaoka and Suzuki,
1984: 41; Crosskey and Lowry, 1990: 210; Takaoka
and Davies, 1995: 102-105 (female).
This species was described from female specimens
collected in Peninsular Malaysia, together with a single female specimen collected from Nakon Sri Tamarat, Khao
Lung Province, in Thailand (Edwards, 1928). The female
lectotype and paralectotype specimens of S. digrammicum
have been examined and redescribed by Takaoka andDavies (1995). The reared male, pupa and mature larva of
this species are here described for the first time based on thespecimens collected in northern Thailand.
DESCRIPTION. Male. Body length 2.8 mm. Head.
Width slightly wider than thorax. Upper eye consisting oflarge facets in 21 horizontal rows and in 21 or 22 verticalcolumns. Clypeus brownish black, white pruinose, moderately covered with dark stout hairs except median portion
widely bare. Antenna composed of 2+9 segments, darkbrown except base of 1st flagellar segment yellow; 1st flagellar segment much elongated, ca. 2.7× as long as 2ndflagellomere. Maxillary palp composed of 5 segments, withproportional lengths from 3rd to 5th segments 1.0:1.0:2.2;
3rd segment (Fig. 60) of normal size; sensory vesicle of medium size, ellipsoidal, ca. 0.23× as long as 3rd segment,with opening of medium size. Thorax. Scutum brownish
black, uniformly and densely covered with golden-yellow
recumbent fine hairs, interspersed with long upright hairs
on prescutellar area; in certain angles of light scutum withlarge white-pruinose shiny spot on each shoulder, extending
posteriorly forming a broad white-pruinose band along eachlateral margin up to base of wing. Scutellum medium
brown, with several upright dark hairs as well as goldenyellow fine hairs. Postscutellum dark brown, shiny, without
hairs. Pleural membrane and katepisternum as in female ofS. maenoi. Legs. Foreleg: coxa yellowish white; trochanter dark brown except base of inner surface yellow; femur medium brown; tibia medium brown with median large
area light brown, and outer surface of basal 3/4 whitish, andsheeny in certain angle of light; tarsus entirely brownish
black; basitarsus, 2nd and 3rd tarsal segments somewhat dilated (basitarsus ca. 7.1× as long as its greatest width).

Midleg: coxa dark brown; trochanter dark brown except
base yellow; femur medium to dark brown; tibia yellowish
white on basal 1/2, medium to dark brown on apical 1/2;
tarsus brownish black though basal portion of basitarsus
dark brown. Hind leg: coxa dark brown; trochanter dark
yellow or light brown except basal 1/2 yellow; femur medium brown with basal 1/5 yellow and apical cap darkbrown; tibia dark brown except extreme base yellow; tarsus
light to medium brown except basal 1/2 of 2nd tarsal segment pale white; basitarsus (Fig. 61) much enlarged, gradually widened from base to apical 1/4, then slightly narrowed
toward apex, 3.7× as long as its greatest width, and 0.96×
and 1.14× as wide as hind tibia and femur, respectively.
Calcipala distinct but small. Pedisulcus well marked. Wing.
Length 2.3 mm. Costa with spinules and hairs; subcosta
bare; basal section of radial vein fully haired; hair tuft atbase of stem vein dark brown; basal cell absent. Abdomen.
Basal scale brownish black with a fringe of long dark hairs.
Dorsal surface medium to dark brown, and with dark shorthairs; segments 2, 6 and 7 each with a pair of large whitishgrey pruinose (iridescent in certain lights) spots; those on
segments 2 and 6 situated dorsolaterally, while those on
segment 7 almost laterally. Genitalia (Figs. 62-68). Coxite
in ventrolateral view much shorter than wide; style in ventrolateral view elongate, much longer than coxite, ca. 3.1×
as long as its greatest width near base, straight and nearlyparallel-sided on basal 1/3, then abruptly narrowed towardapical 1/3 (0.6× as wide as basal 1/3), and again nearlyparallel-sided up to near apex, though inner margin of apical 2/3 gently curved, and with a terminal spine; style spatulate ventrodorsally, with basal protuberance directed dorsomedially, bearing numerous spinules on its surface. Bodyof ventral plate longer than wide, narrowed posteriorly
when viewed ventrally, and having a ventrally-produced
process with short hairs basally on ventral surface, but its
apex and posterior surface entirely bare; basal arms of ventral plate widely diverging from each other. Median sclerite
long, plate-like, narrow basally, gradually widening toward
apex, and moderately sclerotized. Parameres broad basally,each with several hooks apically, as in S. maenoi. Aedeagal
membrane densely setose; dorsal plate strongly sclerotized.
Abdominal segment 10 with 1 or 2 short hairs on each posterolateral corner. Cerci rounded, each with 10 or 11 shorthairs.
Pupa. Body length ca. 2.8 mm. Head. Integument
dark yellow, moderately covered with tubercles of various
sizes; large tubercles each with several minute projections
peripherily on its surface (Fig. 69), but smaller tubercles
with no such projections; frons with 2 long fan-liketrichomes each with 9 or 10 long branches (similar to those

１
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８

１
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９

of S. maenoi) on each side; 2 frontal trichomes on each side
situated close together; face with 1 long fan-like trichomewith 7-11 long branches (similar to those on frons) on eachside; antennal sheath (Fig. 70), with 9 moderately raised
portions produced outwardly, corresponding to flagellar
segments 1-9; outer surface of antennal sheath sparsely covered with small round tubercles. Thorax. Integument dark
yellow, moderately or sparsely covered with tubercles ofvarious sizes anteriorly, and with small conical tubercles
posteriorly; most of large tubercles on anterior surface eachwith minute projections on its surface similar to those on
frons, but smaller tubercles with no such projections; integument on each side with 5 long fan-like trichomes, eachwith 10-14 long branches (similar to those of S. maenoi)
anterodorsally and anterolaterally, 1 medium-long stout
trichome with 4 or 5 branches posterolaterally, and 3
trichomes (1 short slender simple or bifid, 1 medium-longstout simple, and 1 medium-long stout trifid) ventrolaterally.
Gill (Fig. 71) with 6 slender short filaments arranged in 3
pairs; dorsal and middle pairs arising from very short common stalk and each pair with short stalk, 2 filaments of ventral pair directly arising from base of gill and almost sessile;
all filaments subequal in length (0.7-0.9 mm) and thickness
to one another, except outer filaments of middle and ventral
pair somewhat thinner than others, and inner filament ofventral pair somewhat thicker basally than others; all filaments directed forwards except inner filament of ventral
pair directed downward and forward; cuticle of all filaments
pale yellow, with well marked annular ridges and furrows,
and densely covered with minute tubercles. Abdomen.
Tergum 1 pale yellow, with 1 simple medium-long seta oneach side; tergum 2 pale, with 1 simple medium-long seta, 1
simple short seta and 4 short spinous setae (in 1 pupa, 4 of 8
spinous setae bifid apically) submedially on each side; terga
3 and 4 each with 4 hooked spines and 1 simple short setaon each side; terga 5-9 lacking spine-combs except tergum
8 with spine-combs (consisting of 6-9 spines) in transverserow (in 2 of 3 pupae, tergum 8 with only 1 small conicalspine, not forming transverse spine-combs) on each side;
tergum 9 with small conical terminal hooks (Fig. 72) [in 1of 3 pupae, terminal hooks weakly-developed (Fig. 73)].

Sternum 4 with 1 simple slender hooklet and a few minutesetae on each side; sternum 5 with pair of simple or bifid
hooks submedially and a few simple minute setae on eachside; sterna 6 and 7 each with pair of simple or bifid innerand simple outer hooks somewhat spaced from each other,and a few simple minute setae on each side. Grapnel-likehooklets absent. Cocoon (Figs. 74 and 75). Boot-shaped,
tightly woven, with relatively high wall around opening except anteromedian portion with deep notch; anterodorsal
margin thickly woven forming a short bulge; cocoon withsmall lateral window on each side; individual threads invisible; 3.7-4.0 mm long × 1.1-1.4 mm wide.
Mature larva. Body length 5.0-5.5 mm. Body (Photo.
9) normal in shape, with thorax somewhat inflated, and abdomen gradually widened posteriorly up to segments 6 and7, and then narrowed to posterior circlet; body color palecreamy. Cephalic apotome (Photo. 7) pale creamy; headspots indistinct. Lateral surface of head capsule palecreamy; spots behind and below eye-spot region indistinct;
ventral surface of head capsule (Photo. 8) pale creamy oryellow, though somewhat darkened around postgenal cleft;
spots on each side of postgenal cleft negative. Antennacomposed of 3 segments and apical sensillum, somewhat
longer than stem of labral fan; length ratio of segments(from base to tip) 1.0:1.4:0.6. Labral fan with 38-40 mainrays. Mandible (Fig. 76) with mandibular serrations composed of 1 large tooth and 1 small one, without supernumerary serrations; 1st tooth of comb-teeth shorter than 2nd
one but longer than 3rd one. Hypostomium (Figs. 77 and78) with apparently abnormal row of apical teeth; lateral
margins weakly serrate apically; 6 or 7 hypostomal bristles
diverging posteriorly from lateral border on each side.
Postgenal cleft (Photo. 8) deep, bullet-shaped, rounded apically, ca. 3.5× as long as postgenal bridge. Cervicalsclerite composed of small, pale yellow, rod-like piece oneach side. Thoracic cuticle almost bare. Abdominal cuticle
bare except last segment moderately covered with short colorless setae not only on each side of anal sclerite but also oneach lateral bulge, and sparsely on dorsal surface just before
rectal papilla. Rectal papilla of 3 lobes, each with 14-17

Figures 60-78. Morphological features of the male, pupa and larva of S. digrammicum Edwards. 60, 3rd segment of malemaxillary palp with sensory vesicle (right side; front view); 61, basitarsus and 2nd tarsal segment of malehind leg (left side and outside view); 62, coxites, styles and ventral plate in situ (ventral view); 63, right
coxite and style (ventrolateral view); 64, left style (medial view); 65, ventral plate and median sclerite (lateralview); 66, ventral plate (end view); 67, median sclerite (ventral view); 68, dorsal plate (end view); 69, tubercles with secondary minute projections on pupal frons; 70, left antennal sheath of pupa; 71, pupal gill filaments (right side, lateral view); 72 and 73, pupal terminal hooks of different size (lateral view); 74 and 75,
cocoons (74, lateral view; 75, dorsal view); 76, apical part of larval mandible; 77 and 78, larval hypostoma
showing abnormal anterior teeth. Scale bars. 1.0 mm for figs. 74 and 75; 0.1 mm for figs. 61 and 71; 0.05
mm for fig. 70; 0.03 mm for figs. 60, 62-68, 77 and 78; 0.02 mm for figs. 72 and 73; 0.01 mm for figs. 69
and 76.

１
３０

Photographs 1-9. Whole bodies and head capsules of mature larvae. 1, 4 and 7, dorsal views of head capsules; 2, 5 and 8, ventral views of head capsules; 3, 6 and 9, whole bodies of larvae (lateral view; body length: 3, 6.2 mm; 6, 11.0 mm; 9, 5.0 mm). 1-3, S. maenoi sp. nov.; 4-6, S. choochotei sp. nov.; 7-9, S. digrammicum Edwards.

１
３
１

finger-like secondary lobules. Anal sclerite X-shaped, withanterior arms broadened apically, which are ca. 0.6× aslong as posterior ones. Last abdominal segment moderately
bulged laterally but lacking ventral papillae. Posterior circlet with ca. 150 rows of hooklets with up to 23 hooklets
per row.
SPECIMENS EXAMINED. 1 female and 1 male, bothreared from pupae, 1 pupal exuvia, 2 mature larvae, at
Siriphum Waterfall, altitude 1,265 m, Chiang Mai Province,
northern Thailand, 12.VI.2001, by H. Takaoka and W.
Choochote.
ECOLOGICAL NOTES. The pupae and larvae of S. digrammicum were found to be attached on grass stalk or leaf
in a shaded stream 2-5 m wide. Water temperature was 18°
C. This species was collected together with S. asakoae, S.
chamlongi, S. decuplum, S. fenestratum, S. feuerborni, S.
inthanonense, S. rufibasis, and S. sheilae.
REMARKS. S. digrammicum was originally describedfrom female specimens collected from Cameron’s Highlands, Pahang, Malaysia (Edwards, 1928), but was later
treated as a junior synonym of S. griseifrons from India by
Puri (1932), and this course was followed by Crosskey(1973 and 1988). Takaoka and Davies (1995), after examining the type specimens of both species preserved in the
Natural History Museum, London, reported that the femaleof S. digrammicum is separable from that of S. griseifrons
by the shape of the anterior gonapophysis and the numberof short hairs on it.
In this study, we obtained two reared adults of this species, one female and one male, in northern Thailand, andconfirmed its identity by comparing the female characters
including the genitalia, with those of S. digrammicum given
by Takaoka and Davies (1995). It is however noted that theThai female specimen shows a few differing characters as
follows (characters of the Malaysian specimens in parenthesis): Frons-head ratio 1:4.1 (1:3.6); proportional lengths of3rd, 4th and 5th segments of maxillary palp 1.0:1.0:2.1(1.0:1.2:2.4); cibarium with a few tubercles, though very
tiny, near posterior margin (bare); cercus round posteriorly
when viewed from side (nearly straight).
The male and pupa of this species are very similar to
those of S. griseifrons described by Puri (1932). However
there are several distinct differences in the male characters
between the two species. For instance, S. digrammicum iseasily distinguished from S. griseifrons by the entirely darkened femora and the haired ventral plate. On the contrary,
the pupae of both species are alike except the shape of thecocoon, of which the anterior collar has a deep notch medi-

ally in this species [c.f., the anterior collar does not havesuch a notch in S. griseifrons, according to the figure given
by Puri (1932)].
S. digrammicum is distinguished from the other related
species (including two new species described here) of thegriseifrons species-group by its unique shape of the cocoon(Figs. 74 and 75) and the long second comb-tooth of thelarval mandible (Fig. 76).

ACKNOWLEDGEMENTS
We are grateful to Dr. Atchariya Jitpakdi, Departmentof Parasitology, Chiang Mai University, Chiang Mai, Thailand, for her kind help in collections of black flies in northern Thailand. Thanks are due to Dr. Yasushi Otsuka, Department of Infectious Disease Control, Oita Medical University, who kindly took photographs of the larval heads
needed for this paper. This work was financially supportedby the Grant-in-Aid of Japan Society for Promotion of Science (the fiscal year 2001).

REFERENCES
１）Bentinck, W. (1955): The black flies of Japan and Korea(Diptera: Simuliidae). 56p., 406 Medical General Laboratory U. S. Army, Tokyo
２）Crosskey, R. W. (1969): A re-classification of the simuliidae (Diptera) of Africa and its islands. Bull. Br. Mus.
(Nat. Hist.) Entomol. Suppl. 14, 1-195
３）Crosskey, R. W. (1973): Family Simuliidae. p. 423-430.
In: Delfinado, M. D., and Hardy, D.E., eds., A catalog of
the Diptera of the Oriental Region. Vol. I. Suborder
Nematocera. 811p., University Press of Hawaii, Hawaii４）Crosskey, R. W. (1988): An annotated checklist of theworld black flies (Diptera: Simuliidae), p. 425-520. In:
Kim, K. C., and Merritt, R. W., eds., Black flies: Ecology,
population management and annotated world list. 528p.,
The pennsylvania State University Press, University Park
５）Crosskey, R. W. and Howard, T. M. (1997): A new taxonomic inventry of world blackflies (Diptera: Simuliidae).
144p., The Natural History Museum, London
６）Datta, M. (1974): New species of black flies (Diptera:
Simuliidae) from the Darjeeling area, India. Oriental Insects, 8, 457-468
７）Edwards, F. W. (1928): Diptera Nematocera from theFederal Malay States Museums. J. Fed. Malay StatesMus., 14, 1-139
８）Kuvangkadilok, C. and Takaoka, H. (2000): Taxonomic
notes on Simuliidae (Diptera) from Thailand: Descriptionof a new species and new distributional records of nine
known species. Jpn. J. Trop. Med. Hyg., 28 (3), 167-175
９）Puri, I. M. (1932): Studies on Indian Simuliidae. Part III.
Descriptions of males, females, and pupae of S. griseifrons Brunetti (1911) and of four new species with

１
３
２

striped thorax. Ind. J. Med. Res., 19 (4), 1125-1143
１０）Puri, I. M. (1933): Studies on Indian Simuliidae. Part VI.
Descriptions of males, females, and pupae of two new
species from Palni Hills and of male and pupa of S. tenuitarsus sp. n. from Bengal Terai. Ind. J. Med. Res., 20 (3),803-812１１）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
１２）Takaoka, H. (1983): The blackflies (Diptera: Simuliidae)of the Philippines. xi+199p., Japan Society for the Pro-

motion of Science, Tokyo
１３）Takaoka, H. and Davies, D. M. (1995): The black flies(Diptera: Simuliidae) of West Malaysia. viii+175p., Kyushu University Press, Fukuoka
１４）Takaoka, H. and Davies, D. M. (1996): The black flies(Diptera: Simuliidae) of Java, Indonesia. viii+81p.,
Bishop Mus. Bull. Entomol. 6, Bishop Museum Press,
Hawaii１５）Takaoka, H. and Davies, D. M. (1997): Simulium (Simulium) yongi sp. nov. (Diptera: Simuliidae) from Peninsular Malaysia. Jpn. J. Trop. Med. Hyg., 25 (1), 11-16

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Jpn. J. Trop. Med. Hyg., Vol.３
０, No．
２,２
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０２, pp.１
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７

Etiology of Diarrhoea Among Adult
Patients During the Early Monsoon Period
in Kathmandu, Nepal

BASU DEV PANDEY1,2, LAXMI BIKRAM THAPA1, JEEVAN BAHADUR SHERCHAND3, NIRMAL RIMAL4,
ACHYUT BHATTARAI1 and KOUICHI MORITA5
Accepted 31, May, 2002

Abstract: One hundred and eighty-one patients with acute diarrhoea attending the gastroenteritis ward of Sukra
Raj Tropical and Infectious Disease Hospital (STIDH) were investigated during the early monsoon, April to May
2001. Bacterial pathogens were isolated in 33% of the patients. Enteropathogenic Escherichia coli was isolated in
8.28%, Shigella species in 13.25% and Vibrio cholerae 01 in 1.1% of the patients. Mixed infections with bacterial
pathogens, helminths and protozoan parasites were commonly observed in the study. Trichuris trichiuria was detected in 27.6%, hookworms in 12.7% and Ascaris lumbricoides in 11.04%. Entamoeba histolytica/dispar andGiardia lamblia were observed in 12.7% and 7.73% of the patients, respectively. A large number of Cryptosporidium (7.73%) and Cyclospora species (3.86%) usually present in immunocompromised patients were also detectedin acute diarrhoeal cases. The results showed that a wide range of bacterial pathogens was isolated from the inhabitants of Kathmandu, Nepal prior to the monsoon. These findings indicate that the bacterial pathogens, especially diarrhoeaginic E. coli and Shigella, and protozoan parasites, need to be given additional attention in the diagnosis and treatment of acute diarrhoea.
Key words: Acute diarrhoea, etiology, bacterial pathogens, helminthic parasite, Nepal

INTRODUCTION
Diarrhoea is the most common illness among children
in the developing world. It is the primary cause of mortality
and morbidity and the highest risk occurring within the first
5 years of life (Bern et al., 1992). However, it is also a serious public health problem among adults, causing significant
levels of morbidity during early summer and the rainy season in developing countries like Nepal (WHO, 1992; Bernet al., 1992). The incidence of diarrhoeal diseases in Nepal
tends to rise sharply during warm summer months with thepeak incidence occurring in July and August. Food borne
gastroenteritis does increase in April and May each year,
followed by water-borne gastroenteritis and cholera in the

beginning of rainy season (WHO, 1992). Sukraraj Tropicaland Infectious Disease Hospital (STIDH) is the only infectious disease hospital receiving adult patients with diarrhoea
in Kathmandu. A total of 4497 patients with diarrhoea wereadmitted in the year 2000 in STIDH. Although several
studies have been conducted in the past, there are no clear
figures on the etiology of diarrhoea in the adult population(WHO, 1992; Hoge et al., 1995; Sherchand and Shrestha,
1996). Despite a number of individual hospitals setting up
their own guidelines at present, there is still no establishednational protocol for the management of diarrhoeal diseases.
In this situation, etiological study of diarrhoeal disease isvery important for the provision of base-line information on
the causes of diarrhoeal disease and preparation of a na-

Address: 1Sukra Raj Tropical and Infectious Disease Hospital Teku, Kathmandu, Nepal, 2Department Molecular Epidemiology, Institute of Tropical Medicine, 3Department of Microbiology, Institute of Medicine, Tribhuban University, Kathmandu, Nepal, 4Association of Medical Doctors ofAsia, Kathmandu, Nepal, 5Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan
Running Title: Etiological agents of diarrhoea in Kathmandu
Correspondence footnote:
Dr. Basu Dev Pandey, MBBS, PhD
Department of Molecular Epidemiology, Institute of Tropical Medicine Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523
Phone: +81-958-49-7860 Fax: 81-958-49-7830
E-mail: basupandey@tm.nagasaki-u.ac.jp

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tional protocol for the management of diarrhoea.
The present study was initiated to establish the etiological agent responsible for acute diarrhoea in the adult
population with samples collected from inhabitants of the
Kathmandu Valley.

MATERIALS AND METHODSPatients attending the STIDH those with acute diarrhoea were enrolled in the study. Criteria for the exclusionfrom the study included patient’s administration of antibiotics and systemic illness (heart disease, enteric fever, leshmaniasis, central nervous system manifestations). The hospital staff completed a set of questionnaires after obtaining
written consent from the patients. Demographic profile wasrecorded, general physical examination was performed andseverity of dehydration was assessed. Stool samples werecollected, and appropriate treatment was started based on
the degree of dehydration. Samples were subjected to routine parasitological and bacteriological examination. Treatment was reviewed after receiving the laboratory reports forthe stool test.
Parasitological test
The stool samples were collected in the morning from181 patients older than 14 years in STIDH and were examined. Direct smears were prepared with normal saline, iodine, and sucrose solution. Two and half percent of potassium dichromate solution and modified Ziehl Nelsen staining were applied to differentiate protozoan parasites into
mainly Cryptosporidium, Isospora and Cyclospora(Casemore, 1991). A formal ether concentration method
was also used to identify other intestinal parasites.
Bacteriological test
Samples received in the laboratory were cultured immediately to identify pathogenic microorganism such as Vibrio cholerae, Salmonella species, Shigella Species and Escherichia coli using standard bacteriological method(Mackie and McCartney, 1996). Stool samples were inoculated onto thiosulphate citrate bile salt sucrose (TCBS) Agar,
MacConkey agar, eosin methylene blue (EMB) agar as well
as SS agar, and into enrichment media such as Selenite F,
Rappaport and Alkaline peptone water (pH 8.5). After 24hours of incubation at 37°
C, Rappaports and Selenite F
broth cultures were inoculated onto MacConkey and SS
agar, respectively. Alkaline peptone water was subcultured
onto TCBS agar after 12 hours and incubated overnight at
37°
C. Typical characteristic vibrio like colonies seen on
TCBS agar were subcultured onto nutrient agar and identified by biochemical test. Biotypes of V. cholerae were de-termined using Voges-Proskauer (VP), polymyxin B sensitive and haemolysin production test (Blood agar). Biochemically confirmed V. cholerae was subjected for serotyping. Serotyping was carried out with the use of diagnostic antiserum raised against V. cholerae manufactured by
Denka Seiken, Japan as per manufacturer’s protocol.
Both lactose and non-lactose fermenting colonies from
MacConkey and SS Agar were subjected to further identification. Biochemical and serological tests such as TSI, SIM,
VP, dycarboxylase, simons citrate medium. Diagnostic antiserum (Denka Seiken Co. LTD Tokyo, Japan) was also
used for serotyping of enteropathogenic E. coli, Salmonella
and Shigella species. Skirro’s medium was used for theisolation of C. jejuni and suspected colinies were further
examined by gram stain. The strains identified as above
were also confirmed by using API 20E in vitro diagnostic
biochemical kit manufactured by bioMerieux SA 69280
Marcy/Etoile-France.
Statistical methods
Differences between valuse were examined using Student’s t-test analysis was carried out using SPSS version 10,
Statistical Analysis Software, 2000.

RESULTSA total of 181 patients aged 14 years and above attending the STIDH between April and May with acute diarrhoea
was included in this study (Table 1). The patients recorded,
an average, 10-15 loose motions before coming to the hospital. The patients reported an average of 11 hours of loose
motion before coming to the hospital. Of those 60% of thecases were female (P<.0001) and the highest numbers(40%) were in the age group between 20-29 for both males
and females (Fig. 1). The main symptoms associated withdiarrhoea were combination of abdominal pain (70%), vomiting (62%), and fever (15%) with moderate to severe dehydration (Fig. 2).
Since the etiology of diarrhoea has not been studied in
adult population, the prevalence of all the enteropathogens
including Cryptosporidium and Cyclospora was also determined. Bacterial pathogens were isolated from 33% of thepatients (Table 1). Among them enteropathogenic E. coli
(EPEC) were isolated from 8.28%, Shigella species from
13.25% (Shigella dysenteriae in 5%, S. flexneri in 4.41% S.
boydi in 2.2% S.sonnie in 1.65%). The result also showedthat EPEC was the most commonly isolated single bacterial
pathogen, followed by S. dysenteriae 5% and Salmonella
species was isolated in 4.41% of the cases tested.
The result showed that 40% of the patients were infected with more than one organism. Protozoal pathogens

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Table. 1 Enteric pathogens identified among 181 patients of diarrhoea from April
and May, 2001 in STIDH, Teku, Kathmandu
SerialNumberEnteropathogens

1

8.28

2

Enterotoxigenic Escherichia coli

5

2.76

Enteroinvasive Escherichia coli

4

2.20

4

Enterohaemorrhagic Escherichia coli

2

1.10

5

Shigella dysenteriae

9

5.0

6

Shigella boydi

4

2.2

7

Shigella flexneri

8

4.41

8

Shigella sonnei

3

1.65

9

Salmonella species

8

4.41

10

Vibrio cholerae 01

2

1.1

11

Camphylobacter jejuni

1

0.55

12

Entamoeba histolytica/ E. dispar

23

12.7

13

Cyclospora species

7

3.86

14

Cryptosporidium

14

7.73

15

Blastocystis hominis

14

7.73

16

Ascaris lumbricoides

20

11.04

17

Giardia lamblia

14

7.73

18

Trichuris trichiuria

50

27.6

19

Hookworm

23

12.7

20

Hymenolepsis nana

2

1.1

21

Chilomastix mesnili

3

1.65

22

Dientamoeba fragilis

1

0.55

23

Trichomonas

1

0.55

24

Strongyloides

3

1.65

25

Isospora belli

1

0.55

26

Iodamoeba butschlii

3

1.65

80
Female

60

17.12

11.04
8.28

6.62

5

6.62
3.86

20-29

30-49

50-59

50

＞59

Age group

Fig.1: Percentage of sex and age distribution of patients withacute diarrhoea.

39.77

40
30
20

4.41
1.10

14-19

Percentage
61.87

Percentage

16.57

15

69.61

70

Male

20
Percentage

15

3

24.30

0

Percentage

Enteropathogenic Escherichia coli

25

10

Patients（n＝181）

14.90

10
0

Fever

Pain abdomen

Vomiting

OthersSymptoms

Fig.2: Percentage of other symptoms associated with diarrhoea on admission to hospital in Kathmandu, Nepal,
2001.

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DISCUSSION
This paper is one of the first studies to examine the etiology of diarrhoea during warm early monsoon among theadult Nepalese population. This study confirms the importance of E. coli species, Shigella species, E. histoytica, G.
lamblia and Cryptosporidium as the causative agents ofacute diarrhoea in Nepal. Hoge et al. (1995) reported similar observation among children under five years. Our results indicate that Cryptosporidium and Cyclospora species
are also important pathogens in Kathmandu. The low
prevalence of cholera in this study is most likely due to theseason during which our sampling was undertaken. It is important that an etiological study should be continued for acomplete year irrespective of age of the patients.
Diarrhoea is usually classified as either inflammatory
or non-inflammatory disease since such a classification has
therapeutic significance. The other categories of diarrhoegenic E. coli, (not isolated in this study) like enteroaggregative E. coli, have also been shown to be an importantcausal agents of acute diarrhoea in developing countries(Bouckenooghe et al., 2000). More extensive study is required to establish their role in the Nepalese context. Inflammatory diarrhoeas that may be associated with higher
mortality can be studied by stool culture. Several inflammatory markers like faecal leucocytes, occult blood, andfecal lactoferrin could be used as a predictor of invasive
pathogen in enteric inflammation (Huicho et al., 1996).
The source of drinking water, knowledge and attitudeof the patients and the family members were assessed in this

90
80

81.21

Percentage

70
Percentage

E. histolytica/E. dispar, G. lamblia, Cryptospridium andCyclospora were detected in 31% of the patients. Of these
12.7% were infected with E. histolytica/E. dispar, 3.86%
with Cyclospora and 7.73% each with G. lamblia and
Cryptosporidium. It was also observed that T. trichiuria infection was the predominant helminthic infection (27.6%)
among the patients. On the other hand, 13% of the patientswere infected with hookworm and 11.04% with A. lumbricoides. The symptoms associated with Blastocystis hominis
are similar to those associated with G. lamblia both of themisolated in 7.73% of the patients. Hymenolepsis nana andIsospera belli were detected in 1.1 and 0.55% of the patients, respectively.
Although V. cholerae 01 is the major cause of acutewatery diarrhoea during the rainy season, it was only isolated 1.1% in our study period, which was conducted during
early part of the rainy season. On the other hand rare pathogens like Chilomastix mesnili, Iodamoeba butschlii werealso detected in the range of 1% and Strongyloides was detected in 1.65% of the cases.

60
50
40
30
20

12.15

10
0

3.86

Tap

2.76

Well
Hand pumpTanker
Source of drinking water

3.31

Mixed

Fig.3: Source of drinking water in the inhabitants of Kathmandu, Nepal

study. As shown in Fig. 3, 80% of the patients reported touse their tap water as a source of drinking water, has low
chlorine concentration and which is polluted with highnumbers of coliform in Kathmandu (WHO, 1992). Only
23% family’s boil their drinking water and the rest of thepeople drink water without boiling which could be one of
the major predisposing factors for diarrhoea. We attemptedto access the knowledge regarding the etiology of diarrhoea
during the study. Forty-five percent of the patients still believe that diarrhoeal diseases are not communicable. Contaminated public water supplies probably contributed significantly to sustained transmission of disease in the Kathmandu Valley. Health education and improvement of sanitation are necessary for prevention. Increased administration of oral rehydration solutions is vital in order to decrease the morbidity and mortality from acute diarrhoea.
The present study indicates that there is a seasonal variationon the etiology of diarrhoea in Kathmandu Valley, whichshould be confirmed by more extensive longitudinal study.

ACKNOWLEDGMENTS
We thank to the director and all the staff of STIDH for
their contribution in this study. The authors are grateful to
Dr. Ichinose Y. and Mathenge E. G. for critical reading of
the manuscript. This study was partially supported by agrant from Nepal Health Research Council (reference number 442/2001).

REFERENCES
１）Bouckenooghe, A. R., Dupont, H. L., Jiang, Z. D., Adachi, J., Mathewson, J. J., Verenkar, M. P., Rodrigues, S.
and Steffen, R. (2000): Markers of Enteric inflammationin enteroaggrogative Escherichia Coli Diarrhoea in Travelers. Am. J. Trop. Med. Hyg., 62, 711-713
２）Bern, C., Martines, J., de Zoysa, I. and Glass, R. I.

１
３
７(1992): The magnitude of the global problem of diarrhoeal disease: A ten-year update. Bull. W. H. O., 70, 705-714３）Casemore, D. P. (1991): Laboratory methods for diagnosing cryptosporidiosis. J. Clin. Pathol., 44, 445-51
４）Hoge, C., Echeverria, P., Rajah, R., Jacobs, J., Malthouse,
S., Chapman, E., Jimenez, M. L. and Shlim, D. R.
(1995): Prevalence of cyclospera species and other pathogens among children less than 5 years of age in Nepal. J.
Clin. Microbiol., 33, 3058-3060
５）Huicho, L., Campos, M., Rivera, J. and Guerrant, R. L.

(1996): Faecal screening test in the approach to acute infectious diarrhoea: Paediatr Infect. Dis J., 15, 486-494
６）Sherchand, J. and Shrestha, M. (1996): Prevalence of
cryptosporidium infection and diarrhoea in Nepal. J. Diarr Dis Resear., 2, 81-84
７）Mackie and McCartney. (1996) In Medical Microbiology,
Fourteen edition, 1995 Churchill Livingstone Medical
Division of Longman Group Ltd. UK.
８）WHO (1992): Weekly epidemiological record., 67, 273280.

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

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第２号
内

正明 …………………………………………１０３

ラオスにおける薬剤耐性黄色ブドウ球菌の監視経過（英文）Sithivong, N., Phantouamath, B., Chomlasak, K., Sisavath, L., Munnalath, K., Insisiengmay, S.
比嘉 直美，垣花 シゲ，岩永 正明…………………………………………………………………１０９

日本熱帯医学会表紙!−" ＭＫ 稲 芦塚 中島 宮副

JUNE２
０
０
２

日 本 熱 帯 医 学 会

Original article
Naoi, K., Konishi, E., Matsumura, T. and Yano, A.
A new approach for the analysis of seroprevalence data: a mathematical analysis of
the seroprevalence of Toxoplasma gondii infection in Hyogo prefecture (Japan)
with an implication of periodic outbreaks among young children. ………………………………………９９Nguyen, B. M., Higa, N., Kakinohana, S. and Iwanaga M.
Characterization of Vibrio cholerae O1 isolated in Vietnam ……………………………………………１０３
Sithivong, N., Phantouamath, B., Chomlasak, K., Sisavath, L., Munnalath, K.,
Insisiengmay, S., Higa, N., Kakinohana, S. and Iwanaga, M.
Monitoring of drug resistant Staphylococcus aureus in People’s Democratic Republic of Lao …………１０９
Takaoka, H. and Choochote, W.
Taxonomic notes on the griseifrons species-group in Simulium (Simulium)(Diptera: Simuliidae) from Thailand: Descriptions of two new species anddescription of the male, pupa and larva of S. (S.) digrammicum Edwards ………………………………１１５
Pandey, B. D., Thapa, L. B., Sherchand, J. B., Rimal, N., Bhattarai, A. andMorita, K.
Etiology of Diarrhoea Among Adult Patients During the Early Monsoon Period
in Kathmandu, Nepal ……………………………………………………………………………………１３３

Vol. 30 No. 2

日熱医会誌

Hygiene

会報・記録
理事会・評議員会・会務総会記録……………………………………………………………………………１３９
２
０
０
２（平成１
４年）年度日本熱帯医学会役員名簿（２００
２年６月１日現在）………………………………１４３
日本熱帯医学会雑誌編集委員名簿……………………………………………………………………………１４４
投稿規定…………………………………………………………………………………………………………１４６
著作権複写に関する注意………………………………………………………………………………………１４８

and

カトマンドゥ（ネパール王国）においてモンスーン初期にみられる成人下痢症の病因について（英文）Basu Dev Pandey, Laxmi Bikram Thapa, Jeevan Bahadur Sherchand,
Nirmal Rimal, Achyut Bhattarai，森田 公一 ……………………………………………………………１３３

Medicine

タイで採集されたアシマラダブユ亜属 griseifrons 種群の分類学的検討：
２新種の記載および Simulium (S.) digrammicum Edwards の雄成虫，蛹，幼虫の記載（英文）高岡 宏行，Wej Choochote ……………………………………………………………………………１１５

Journal of Tropical

著抗体データ解析の新たなアプローチ：兵庫県におけるトキソプラズマ抗体陽性率の数学的分析と小児における周期的感染流行の示唆（英文）直井 幸二，小西 英二，松村 武男，矢野 明彦…………………………………………………９
９
ベトナムで分離したコレラ菌の性状（英文）Nguyen Binh Minh，比嘉 直美，垣花 シゲ，岩永JUNE ２０
０
２

CONTENTS

容
Japanese

原

VOL．
３
０ No．
２

平成１
４年６月

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は３mm（決）