Tropical Medicine and Health

Tropical Medicine
and Health2005

Vol.33
No.02
JUNE 2005【CONTENTS】
Original articles
Mycobacterium tuberculosis and gyrA variation in Zambia
Mitarai, S., Kafwabulula, LM., Habeenzu, C., Terunuma, H.,
Lubasi, D., Kasolo, FC., Shishido, H., and Numazaki, Y.

９１

A new species of Simulium (Simulium) from northern Thailand (Diptera: Simuliidae)
Takaoka, H., and Choochote, W.

９５Vol.33 No.02 JUNE 2005

Short Communications
A pilot field survey on the in vitro drug susceptibility of Plasmodium falciparum in Lao PDR
Hatabu, T., Vanisaveth, V., Taguchi, N., Kobayashi, J., Mannoor, MK.,
Watanabe, H., Toma, H., Phompida, S., and Kano, S.
１
０
３
Reviews
Malaria endemic patterns on Lombok and Sumbawa islands, Indonesia
Dachlan, YP., Yotopranoto, S., Sutanto, BV., Santoso, SHB., Widodo, AS.,
Kusmartisnawati, Sutanto, A., Gerudug, IKK., Takagi, M., Tsuda, Y.,
Tanabe, K., Kawamoto, F., Yoshinaga, K., and Kanbara, H.

ISSN 1348-8945

Vol.33 No.02 JUNE

ISSN 1348-8945

Tropical Medicine
and Health１０５JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 平島 木下

背は２mm
（決）

Tropical Medicine and Health

Tropical Medicine
and Health2005

Vol.33
No.02
JUNE 2005【CONTENTS】
Original articles
Mycobacterium tuberculosis and gyrA variation in Zambia
Mitarai, S., Kafwabulula, LM., Habeenzu, C., Terunuma, H.,
Lubasi, D., Kasolo, FC., Shishido, H., and Numazaki, Y.

９１

A new species of Simulium (Simulium) from northern Thailand (Diptera: Simuliidae)
Takaoka, H., and Choochote, W.

９５Vol.33 No.02 JUNE 2005

Short Communications
A pilot field survey on the in vitro drug susceptibility of Plasmodium falciparum in Lao PDR
Hatabu, T., Vanisaveth, V., Taguchi, N., Kobayashi, J., Mannoor, MK.,
Watanabe, H., Toma, H., Phompida, S., and Kano, S.
１
０
３
Reviews
Malaria endemic patterns on Lombok and Sumbawa islands, Indonesia
Dachlan, YP., Yotopranoto, S., Sutanto, BV., Santoso, SHB., Widodo, AS.,
Kusmartisnawati, Sutanto, A., Gerudug, IKK., Takagi, M., Tsuda, Y.,
Tanabe, K., Kawamoto, F., Yoshinaga, K., and Kanbara, H.

ISSN 1348-8945

Vol.33 No.02 JUNE

ISSN 1348-8945

Tropical Medicine
and Health１０５JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 平島 木下

背は２mm
（決）

Tropical Medicine and Health Vol. 33 No. 2, 2005, pp. 91-94
Copyright! 2005 by The Japanese Society of Tropical Medicine

９
１

MYCOBACTERIUM TUBERCULOSIS AND gyrA VARIATIONIN ZAMBIA
SATOSHI MITARAI１, LYNDON M KAFWABULULA１, CHARITY HABEENZU１,HIROSHI TERUNUMA２, DAVID LUBASI１, FRANCIS C KASOLO３,
HARUMI SHISHIDO４, and YOSHIO NUMAZAKI３
Accepted 28, February, 2005

Abstract: M. tuberculosis strains were isolated from clinically and bacteriologically confirmed patients to evaluate
the susceptibility of clinical M. tuberculosis isolates to fluoroquinolone and to obtain molecular epidemiologicalinformation in Zambia,. The pathogens were subjected to susceptibility testing with isoniazid, rifampicin, ethambutol and streptomycin. The minimum inhibitory concentrations to ciprofloxacin, sparfloxacin and levofloxacin werealso evaluated. The gyrA, fluoroquinolone resistance-determining region (QRDR), was sequenced and analysed. As
a result, three of the 16 strains examined were resistant to isoniazid, rifampicin and/or streptomycin. All of thestrains were susceptible to ciprofloxacin, levofloxacin and sparfloxacin. However, a unique gyrA gene variation of
M. tuberculosis was identified in the isolates. One strain had a mutation (T73A) in QRDR. Additionally, 81.25%(13/16) of the strains tested had Thr at codon 88. Several variations of gyrA gene have been reported in relation to
drug resistance. The gyrA variation data will be useful as epidemiological information. It may be important tomonitor fluoroquinolone susceptibility even in developing countries for use against resistant M. tuberculosis infection, even though no fluoroquinolone resistance was observed in this study.

INTRODUCTION
Multi-drug resistant Mycobacterium tuberculosis(MDRTB) is one of the major obstacles to effective controlof tuberculosis [1,2] in both developed and developing
countries. The emergence of MDRTB is believed to be due,in part, to incomplete anti-tuberculosis treatment. In SubSaharan African countries, the incidence of tuberculosis has
rapidly increased mainly due to the human immunodeficiency virus epidemic [3]. Thus, the incidence of resistant
M. tuberculosis infections can be expected to increase inthese countries.
It is thought that some drugs, i.e., rifamycin derivatives
and fluoroquinolones (FQs), are potentially effective treatments for MDRTB. Even in Zambia, it is relatively easy toobtain ciprofloxacin (CIP) and other FQs in general/private
clinics. Therefore, as part of a drug resistance investigationin Zambia, we evaluated the drug susceptibility of M. tuber-culosis to FQs.

MATERIALS AND METHODSClinical isolates and drug susceptibility tests
A total of 16 M. tuberculosis strains were isolated
from 16 patients with pulmonary tuberculosis at the University Teaching Hospital Chest Clinic in Lusaka, Zambia. All
strains were recovered on Lowenstein-Jensen (L-J) medium
from sputum specimens. The drug susceptibility of eachstrain to isoniazid (INH), rifampicin (RIF), ethambutol(EMB), and streptomycin (STR) was determined by the resistant ratio method using L-J media. The critical concentrations for each drug were as follows: INH 0.05, 0.1 and 0.2µg/ml; RIF 12.5, 25 and 50 µg/ml; EMB 0.8, 1.6 and 3.2 µg/ml; STR 10, 20 and 40 µg/ml.
Minimum inhibitory concentrations of fluoroquinolones
FQs were kind gifts from Bayer (ciprofloxacin: CIP),

1. TB Laboratory in University Teaching Hospital, P/Bag RW1, Lusaka 10101, Zambia
2. Department of Microbiology, Yamanashi Medical College, 1110 Shimogato, Tamaho, Nakakoma, Yamanashi 409-3821, Japan
3. Virology Laboratory in University Teaching Hospital, P/Bag RW1, Lusaka 10101, Zambia
4. Marumori Hospital, Nagamachi, Taihaku, Sendai 982-0012, Japan
Corresponding Author: Satoshi MITARAI, M.D., Ph.D.
Present institution:
Bacteriology division, the Mycobacterium Reference Centre, the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association
3-1-24 Matsuyama, Kiyose, Tokyo 203-8533, Japan
Tel: +81-424-93-5711 Fax: +81-424-92-8258 E-mail: mitarai@jata.or.jp

９２

Dainippon Pharmaceutical (sparfloxacin: SPX) and Daiichi
Pharmaceutical (levofloxacin: LVX). These drugs were
tested for minimum inhibitory concentration of each strain
in Middlebrook 7H9 broth with albumin-dextrose-catalase(ADC). The isolates were sub-cultured in Middlebrook 7H9with ADC broth and prepared in the same medium to theturbidity of McFarl and 1. The suspension was further diluted 10２ to 10３ times resulting in a concentration of approximately 10５ CFU/ml. One ml of each suspension was
inoculated into 1 ml of Middlebrook 7H9 broth with either
no antibiotic or with serial two-fold dilutions of each antibiotic, with the final concentrations ranging from 0.1 to 25 µg/ml. The tubes were incubated at 37°
C for 2 to 4 weeks until
sufficient bacterial growth was evident in the control tubes.
PCR and sequencing of gyrA region
The tubercle bacilli were harvested from the colonies
on L-J medium. DNA extraction was performed using 100µg/ml of proteinase K and 2% SDS, followed by ordinary
solvent assay with phenol, phenol/chloroform and chloroform. After re-suspension of each DNA sample, the reaction
mixture was added to 1 µg of DNA template to a final concentration of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.0
mM MgCl２, 200 µM of each of the dNTPs, 25 pmole ofeach of primers and 2.5 units of Taq DNA polymerase in a
total volume of 50 µl and subjected to 30 cycles of PCR.
The primers for gyrA region were as follows: gyrAF1; 5’ G
GTGCTCTATGCAATGTTCG 3’ and gyrAR1; 5’ GGATATTGGTTGCCATGCC 3’. DNA specimens were incubated at
94°
C for one minute to melt the DNA, cooled to 62°
C for

one minute to allow for annealing, and incubated for one
minute at 72°
C for DNA amplification. The PCR productwas recovered from gels and subjected to direct sequencing.
The amino acid sequences of GyrA deduced from nucleotide sequence were numbered in the system used for E.coli.
The complete nucleotide sequence has been deposited in
GenBank under accession number AF400983.

RESULTS
Susceptibility to anti-tuberculosis drugs
One strain (no. 99-1049) was resistant to INH, RIF,
and STR by the resistant ratio method. Two strains (no. 99979 and 99-1201) were solely resistant to INH. No tested
strain showed resistance to CIP, SPX or LVX (Table 1).
SPX showed the lowest MIC, followed by LVX and CIP.
Sequence analysis of gyrA region
A 387 bps of PCR product from gyrA involving thequinolone resistance-determining region (QRDR) was amplified and sequenced. The sequences were analysed asshown in Figure 1. Strain 99-1183, which showed significant susceptibility to FQs, was found to have a mutation resulting in a Thr to Ala substitution at codon 73. Furthermore, a Ser > Thr substitution at codon 88 was found in 13
out of the 16 strains examined, all of which were susceptible to FQs.

Table 1. Drug susceptibilities of clinical M. tuberculosis isolates
Strain no.
H37Rv
99-79499-80099-80499-97899-97999-98099-98399-99599-99999-1049
99-1053
99-1058
99-1128
99-1136
99-1183
99-1201

INH
1 (S)
2 (S)
1 (S)
1 (S)
2 (S)
8 (R)
2 (S)
1 (S)
2 (S)
2 (S)
4 (R)
2 (S)
2 (S)
2 (S)
2 (S)
1 (S)
4 (R)

S: Sensitive R: Resistant ND: No data

Resistant ratio method
RIF
EMB
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
2 (S)
1 (S)
1 (S)
1 (S)
2 (S)
1 (S)
1 (S)
1 (S)
4 (R)
1 (S)
2 (S)
1 (S)
2 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)

STR
1 (S)
2 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
1 (S)
2 (S)
1 (S)
8 (R)
1 (S)
2 (S)
1 (S)
1 (S)
1 (S)
1 (S)

CIP
0.39
0.2
<0.1
ND
0.2
0.2
0.39
ND
0.2
0.2
0.39
ND
ND
0.2
ND
0.2
0.39

MIC (µg/ml)
SPX
0.1
<0.1
<0.1
<0.1
0.1
<0.1
<0.1
ND
ND
<0.1
0.2
<0.1
ND
<0.1
<0.1
<0.1
<0.1

LVX
0.2
<0.1
0.2
<0.1
0.2
<0.1
0.2
ND
0.2
0.2
0.2
<0.1
ND
0.2
0.2
0.2
0.2

９
３

Figure 1. Nucleotide and amino acid sequences of QRDR in gyrA gene by direct sequencing. Sequences ofother strains that are not shown here were completely consistent with 99-1201 in the sequenced region.

DISCUSSION
Many mutations in residues 81 to 87 (88 to 94 in othersystems) have been identified in quinolone resistant M. tuberculosis isolates [4-11], suggesting that this region of the
genome is associated with susceptibility to FQs. The mutation at codon 73 in strain 99-1183, which may be involvedin the susceptibility to FQs, has not been registered in GenBank as far as the authors could determine.
The patients had no a history of frequent and/or continuous use of any FQs for the treatment of either tuberculosis or more common bacterial infections. The strain was
susceptible to all four first line anti-tuberculosis drugs. It is
known that certain natural variations exist in M. tuberculosis with some probability [12]. Strain 99-1183 seemed to be
one such strain, but further investigation is required to confirm the role of this mutation. The new mutation identified
in this report will provide additional information for analysis of the effects of quinolone on M. tuberculosis, and it
may also serve as an epidemiological marker. The aminoacid at codon 88 was mainly Thr in these strains. It isthought that M. tuberculosis is a relatively new species andevolutionarily has a few silent variations. Codon 88 is oneof the genetic markers and Thr is assumed to be common inancestral strains. Here, the higher ratio of S88T strains will
be informative for future epidemiological research [13,14].
There is intense interest in the FQs as anti-tuberculosis
drugs because of the emergence of MDRTB. The use of
FQs for the treatment of MDRTB infection has been shown

to be effective in some reports [15,16]. The FQs must be
used carefully because resistance may develop soon after
clinical application as an anti-tuberculosis drug. The gyrAvariation found in this study in pathogenic strains may provide useful epidemiological information for future studies
implemented to investigate the use of fluoroquinolone totreat resistant M. tuberculosis infection.

REFERENCES
１．Gordin F M, Nelson E T, Matts J P et al. The impact ofhuman immunodeficiency virus infection on drugresistant tuberculosis. Am J Respir Crit Care Med
1996; 154: 1478-83
２．Spellman C W, Matty, K J, Weis S E. A survey of drug
-resistant Mycobacterium tuberculosis and its relationship to HIV infection. AIDS 1998; 12: 191-195.
３．Pablos M A, Raviglione M C, Laszlo A et al. Global
surveillance for antituberculosis-drug resistance, 19941997. World Health Organization-International Union
against Tuberculosis and Lung Disease Working Groupon Anti-Tuberculosis Drug Resistance Surveillance. N
Engl J Med 1998; 338: 1641-1649
４．Alangaden G J, Manavathu E K, Vakulenko S B,
Zvonok N M, Lerner S A. Characterization offluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrob Agents Chemother
1995; 39: 1700-1703

９
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５．Cambau E, Sougakoff W, Besson M, Truffot P C,
Grosset J, Jarlier V. Selection of a gyrA mutant of Mycobacterium tuberculosis resistant to fluoroquinolones
during treatment with of loxacin [letter]. J Infect Dis
1994; 170: 479-483
６．Guillemin I, Cambau E, Jarlier V. Sequences of conserved region in the A subunit of DNA gyrase fromnine species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones. Antimicrob Agents Chemother 1995;
39: 2145-2149
７．Guillemin I, Jarlier V, Cambau E. Correlation between
quinolone susceptibility patterns and sequences in the
A and B subunits of DNA gyrase in mycobacteria. Antimicrob Agents Chemother 1998; 42: 2084-2088
８．Takiff H E, Salazar L, Guerrero C et al. Cloning andnucleotide sequence of Mycobacterium tuberculosis
gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994;
38: 773-780
９．Xu C, Kreiswirth B N, Sreevatsan S, Musser J M,
Drlica K. Fluoroquinolone resistance associated withspecific gyrase mutations in clinical isolates ofmultidrug-resistant Mycobacterium tuberculosis. J Infect Dis 1996; 174: 1127-1130
１０．Yew W W, Kwan S Y, Ma W K, Khin M A, Chau P Y.
In vitro activity of ofloxacin against Mycobacterium
tuberculosis and its clinical efficacy in multiply resis-

tant pulmonary tuberculosis. J Antimicrob Chemother
1990; 26: 227-236
１１．Zhou J, Dong Y, Zhao X et al. Selection of antibioticresistant bacterial mutants: allelic diversity among
fluoroquinolone-resistant mutations. J Infect Dis 2000;
182: 517-525
１２．Alangaden G J, Manavathu E K, Vakulenko S B,
Zvonok N M, Lerner S A. Characterization offluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrob Agent Chemother
1995; 39: 1700-1703
１３．Rhee J T, Piatek A S, Small P M et al. Molecular epidemiologic evaluation of transmissibility and virulenceof Mycobacterium tuberculosis. J Clin Microbiol
1999; 37: 1764-1770
１４．Sreevatsan S, Pan X, Stockbauer K E et al. Restrictedstructural gene polymorphism in the Mycobacterium
tuberculosis complex indicates evolutionarily recent
global dissemination. Proc Natl Acad Sci USA 1997;
94: 9869-9874
１５．Kennedy N, Berger L, Curram J et al. Randomized
controlled trial of a drug regimen that includes
ciprofloxacin for the treatment of pulmonary tuberculosis. Clin Infect Dis 1996; 22: 827-833
１６．Mohanty K C, Dhamgaye T M. Controlled trial of
ciprofloxacin in short-term chemotherapy for pulmonary tuberculosis. Chest 1993; 104: 1194-1198

Tropical Medicine and Health Vol. 33 No. 2, 2005, pp. 95-101
Copyright! 2005 by The Japanese Society of Tropical Medicine

９５

A NEW SPECIES OF SIMULIUM (SIMULIUM) FROM NORTHERNTHAILAND (DIPTERA: SIMULIIDAE)
HIROYUKI TAKAOKA１ and WEJ CHOOCHOTE２
Accepted 19, May, 2005

Abstract: Simulium (Simulium) phukaense sp. nov. is described on the basis of the observation of females andmales (both sexes of adults reared from pupae) and pupae collected in Nan Province, northern Thailand. This new
species is assigned to the griseifrons species-group of the subgenus Simulium (Simulium) and is easily distinguished from other known species of this species-group by the simple shoe-shaped cocoon, as well as the arrangement of the six gill filaments.
Key words: Simulium, black fly, Simuliidae, Thailand, new species

During recent surveys on black flies in northern Thailand, we collected a new species which is assigned to thegriseifrons species-group of the subgenus Simulium (Simulium) Latreille s. str., redefined by Takaoka and Davies [1].
This new species is very similar to S . (S .) maenoi Takaoka
and Choochote, described from Thailand [2] but differsfrom the latter species and all the other species of the same
species-group by its shoe-shaped cocoon.
The terms for morphological features used here follow
those of Takaoka [3]. Holotype and paratype specimens of
the new species are deposited at the Department of Infectious Disease Control, Faculty of Medicine, Oita University,
Oita, Japan.
Simulium (Simulium) phukaense sp. nov.
DESCRIPTION. Female. Body length 2.8―3.0 mm. Head.
Narrower than thorax. Frons brownish black, shiny, thickly
white pruinose, with several dark stout hairs along lateral
margins; frontal ratio 1.3:1.0:0.9―1.0; frons-head ratio 1.0:
3.4―3.8. Fronto-ocular area (Fig. 1A) well developed,
rounded apically. Clypeus brownish black, thickly whitepruinose, moderately covered with dark stout hairs except
upper 1/2 narrowly bare medially. Labrum about 0.7 times
as long as clypeus. Antenna (Fig. 1B) composed of 2+9
segments, medium to dark brown except scape, pedicel, andbasal 1/3 (or up to 1/2) of 1st flagellar segment yellow;1stflagellar segment slightly wider than long, and about 1.8
times as long as 2nd flagellar segment. Maxillary palp with
5 segments, light to dark brown, proportional lengths of 3rd,

4th, and 5th segments 1.0:1.1:2.3; 3rd segment (Fig. 1C) of
normal size, with sensory vesicle oblong, 0.34 times as longas 3rd segment. Maxillary lacinia with 11―13 inner and 15
outer teeth. Mandible with about 34 inner and 15 or 16
outer teeth. Cibarium (Fig. 1D) with short blunt medianprojection directed dorsally at its dorsal margin, with manytubercles on its surface. Thorax. Scutum black, slightly
shiny at certain angles of light, densely covered with yellow
recumbent short hairs interspersed with dark brown shorthairs on anterior surface, and also with dark long uprighthairs on prescutellar area; when illuminated in front andviewed dorsally, scutum thickly whitish-grey pruinose, with
5 longitudinal unpruinose vittae, of which 1 median vitta isvery narrow, 2 submedian and 2 lateral vittae rather wide,
all vittae united with broad transverse band on prescutellar
area; when illuminated from behind, scutum shows a reversed color pattern. Scutellum medium brown, covered
with dark brown long upright hairs as well as yellow shorthairs. Postnotum dark brown, shiny, whitish-grey pruinose,and bare. Pleural membrane bare. Katepisternum longer
than deep, and bare. Legs. Foreleg: coxa whitish yellow;
trochanter yellow with apical 1/3 or 1/2 dark yellow; femur
yellow with apical tip light brown; tibia white except apical
1/4 brownish black; tarsus brownish black, with moderate
dorsal hair crest; basitarsus greatly dilated, 6.1 times as longas its greatest width. Midleg: coxa brownish black; trochanter yellow except apical 1/2 dark yellow or light brown;
femur yellow except apical tip light brown; tibia white toyellowish white except a little more than apical 1/4 blackish
brown; tarsus dark brown to brownish black except basal

1. Department of Infectious Disease Control, Faculty of Medicine, Oita University, Hasama, Oita, 879-5593 Japan
2. Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

９６

1/5 (or up to 1/2) of basitarsus yellow. Hind leg: coxa
blackish brown; trochanter yellow; femur yellow except
apical cap light to medium brown; tibia white to yellowish
white with a little less than apical 1/4 brownish black; tarsus
blackish brown except a little less than basal 2/3 of basitarsus and basal 1/2 of 2nd segment yellowish white; basitarsus (Fig. 1E) nearly parallel-sided, about 6.6 times as longas wide, and 0.71 and 0.61 times as wide as greatest widthsof hind tibia and femur, respectively; calcipala (Fig. 1E)
moderately developed, nearly as long as wide; pedisulcus(Fig. 1E) well developed at basal 1/3 of 2nd tarsal segment.
All tarsal claws simple. Wing. Length 2.6 mm. Costa withdark spinules and hairs; subcosta haired except apical 2/5 to
1/2 bare; basal section of radial vein bare, or with 2―4 hairs
near apex; R１ with dark spinules and hairs; R２ with hairs;
hair tuft on stem vein dark brown. Abdomen. Basal scale
dark brown, with fringe of pale yellow hairs as well as darkbrown hairs. Dorsal surface of abdomen dark brown tobrownish black, with short dark hairs; tergite 2 shiny, silvery iridescent at certain angles of light, and tergites 6―8
shiny. Ventral surface of 7th segment with a pair of small
submedian sternal plates. Genitalia. Sternite 8 (Fig. 1F)
moderately sclerotized, with 16―20 long and medium-longstout hairs as well as a few short fine hairs on each lateralsurface. Ovipositor valves (Fig. 1F) nearly triangular, membranous, covered with 11―15 short or medium-long hairs aswell as numerous microsetae; narrow bare area along innerand posterior margins very thin and transparent (thoughvery lightly darkened anteriorly along inner margin). Genital fork (Fig. 1G) of inverted-Y form, with narrow, wellsclerotized stem; arms of moderate width, each with distinct
projection directed anteriorly. Paraproct in ventral view(Fig. 1H) nearly as long as wide, with distinct concavity on
anteroventral surface; paraproct in lateral view (Fig. 1I)
about half as long as wide, moderately protruding ventrally
beyond cercus forming round unpigmented apex, with numerous stout hairs on lateral and ventral surfaces. Cercus inlateral view (Fig. 1I) rounded posteriorly, about half as longas wide, with numerous stout hairs. Spermatheca (Fig. 1J)
globular or somewhat ovoid, well sclerotized (except portion of junction to duct unsclerotized), polygonal surface
patterns faintly visible, and with internal setae; accessory
ducts subequal in thickness to major duct.
Male. Body length 3.0―3.2 mm. Head. Nearly as
wide as thorax. Upper eye with large facets in 20 or 21 vertical columns and in 21 or 22 horizontal rows. Face andclypeus black, thickly white pruinose, sparsely covered withdark brown long hairs. Antenna (Fig. 2A) composed of 2+9
segments, medium to dark brown except base of 1st flagellar segment yellowish white; 1st flagellar segment elongate,
twice as long as wide, and 2.5 times as long as 2nd flagellar

segment. Maxillary palp with 5 segments, medium to darkbrown, proportional lengths of 3rd, 4th, and 5th segments
1.0:1.1:2.2; sensory vesicle (Fig. 2B) small, oblong, 0.2
times as long as 3rd segment. Thorax. Scutum black,densely covered with golden-yellow recumbent short hairsinterspersed with brown similar hairs anteriorly, and withdark brown long upright hairs on prescutellar area; scutumat certain angles of light with whitish-grey pruinose spots, i.
e., anterolateral pair of rectangular spots on shoulders
which connect widely to posterior large spot on prescutellar
area through spots along lateral margins; all these spots usually indistinct due to thick covering of golden-yellow shorthairs. Scutellum brownish black or black, with goldenyellow short hairs as well as dark brown long upright hairs.
Postnotum black, white pruinose, and bare. Pleural membrane and katepisternum as in female. Legs. Foreleg: coxa
yellow; trochanter and femur medium to dark brown; tibia
dark brown to brownish black with large median area onouter surface medium brown; tarsus brownish black, withmoderate dorsal hair crest; basitarsus somewhat dilated, 7.0
times as long as its greatest width. Midleg: coxa brownish
black; trochanter dark brown except base narrowly yellowish; femur medium brown; tibia medium to dark brown(though somewhat pale at extreme base); tarsus dark brown
to blackish brown. Hind leg: coxa brownish black; trochanter dark yellow to yellowish brown; femur medium
brown except base yellow, and apical cap dark brown tobrownish black; tibia and tarsus dark brown to brownish
black except minute base of tibia white; basitarsus (Fig. 2C)
enlarged, spindle-shaped, 4.0 times as long as its greatest
width, and very slightly narrower than greatest width ofhind tibia, which is as wide as greatest width of hind femur;
calcipala well developed, nearly as long as its width at base;pedisulcus well developed. Wing. Length 2.6 mm; othercharacters as in female except subcosta with 0―6 hairs on
basal 1/2, and basal portion of radial vein bare or with 1 or
2 hairs near apical tip. Abdomen. Basal scale black, withfringe of dark brown long hairs. Dorsal surface of abdominal segments black, with dark short hairs; tergites 2, 6 and 7
each with a pair of dorsolateral whitish spots, those on tergite 2 connected broadly to each other medially. Genitalia.
Coxite in ventral view (Fig. 2D) subquadrate, with stout
hairs on posterior 1/2 of ventral surface. Style (Fig. 2D)
elongate, much longer than coxite, gently curving inwards,
moderately covered with stout hairs on ventral and lateralsurfaces, and with single subterminal spine; style in ventrolateral view (Fig. 2E) very slightly narrowed from base toapical 1/3, then slightly widened near apex; style in medial
view (Fig. 2F) with distinct basal protuberance directed dorsomedially, with several small cone-shaped spines at andnear apex. Ventral plate in ventral view (Fig. 2D) with its

９７

Fig. 1. Female of Simulium (Simulium) phukaense sp. nov. A, fronto-ocular area (right side); B, antenna(left, outer view); C, 3rd segment of maxillary palp with sensory vesicle (right side, front view); D,
cibarium; E, basitarsus and second tarsal segment of hind leg showing calcipala and pedisulcus (leftside, outer view); F, 8th sternite and ovipositor valves in situ (ventral view); G, genital fork (ventralview); H and I, paraprocts and cerci (right side; H, ventral view; I, outer view); J, spermatheca. Scale bars. 0.1 mm for E; 0.05 mm for A and B; 0.02 mm for C, D and F―J.

９８Fig. 2. Male of Simulium (Simulium) phukaense sp. nov. A, antenna (left side, inner view); B, 3rd segmentof maxillary palp with sensory vesicle (right side, front view); C, basitarsus and second tarsal segment of hind leg showing calcipala and pedisulcus (left side, outer view); D, coxites, styles and ventral plate in situ (ventral view); E, coxite and style (right side, ventrolateral view); F, style showing
basal protuberance (right side, medial view); G and H, ventral plates (G, lateral view; H, end view);
I, paramere (right side, end view); J, median sclerite (posteroventral view); K, dorsal plate (ventralview); L and M, 10th abdominal segments and cerci (L, end view; M, outer view). Scale bars. 0.1
mm for C; 0.05 mm for A; 0.02 mm for B and D―M.

９
９body rectangular (though the posterolateral corners arerounded), much longer than wide, and basal arms strongly
sclerotized and divergent from each other; ventral plate inlateral and end views (Fig. 2G, H) with long narrow medianprocess directed ventrally, nearly bare except anteroventral
surface with several microsetae. Paramere (Fig. 2I) large
basally, with several hooks. Median sclerite in ventroposterior view (Fig. 2J) narrow on basal 1/3, then widened toward apical 1/3, then somewhat narrowed apically withround apex, moderately sclerotized and brown except medial narrow portion near apex not sclerotized and transparent. Aedeagal membrane densely setose; dorsal plate (Fig.
2K) moderately sclerotized. Abdominal segment 10 (Fig. 2
L, M) without any distinct hairs on each posterolateral corner. Cercus (Fig. 2L, M) small, short, rounded, with 8―16
hairs.
Pupa. Body length 3.0―3.5 mm. Head. Integument
medium brown, moderately covered with rather large tubercles on frons each with minute secondary projections on its
surface (Fig. 3A); antennal sheath with 9 ridges corresponding to 9 flagellar segments, each ridge covered densely ormoderately with round tubercles, while interridge spaces
bare; face with a pair of long fan-like trichomes each with
12―15 slender branches (Fig. 3B), frons with 2 pairs of long
fan-like trichomes (upper one with 16―22 slender branches(Fig. 3C), lower one with 23―30 slender branches). Thorax.
Integument medium brown, moderately covered with tubercles of various sizes, larger ones with secondary projections(similar to those on frons) on anterior 1/2, as well as smaller
cone-shaped tubercles on posterior 1/2; thorax on each sidewith 5 long fan-like trichomes each with 13―22 branches
anterodorsally and anterodorsolaterally (similar in shape to,but somewhat longer than, those on frons―Fig. 3C), 1
medium-long stout trichome with 2―4 branches (Fig. 3D)
posterolaterally, and 3 trichomes (2 medium-long, with 2―4
branches, 1 simple or bifid, long and stout) (Fig. 3E, F, G)
ventrolaterally. Gill (Fig. 3H) with 6 slender thread-likefilaments in 3 pairs (middle and lower pairs almost sessile,
dorsal pair very short-stalked); outer filament of dorsal pairlongest (1.8―2.0 mm) and thickest of all, followed by inner
filament of dorsal pair, then by outer filament of middle pair,and 3 other filaments subequal to one another, and shortest(1.2―1.4 mm) and thinnest; all filaments dark brown, tapered toward apex, with distinct annular ridges and furrows,
and densely covered with minute tubercles. Abdomen.
Dorsally, segment 1 well sclerotized, medium brown,
sparsely covered with small dark brown cone-shaped tubercles, and with 1 simple or bifid medium-long slender setaon each side; segment 2 transparent, with 1 simple or bifid
medium-long slender seta and 5 short stout spines on eachside; segments 3 and 4 transparent, each with 4 hooks and 1

short stout spine on each side; segments 5 and 6 transparent
and bare; segment 7 with comb-like groups of minutespines on weakly sclerotized light brown narrow transverseband near anterior margin on each side; segment 8 with atransverse row of distinct spine-combs as well as comb-like
groups of minute spines on weakly sclerotized light brownnarrow transverse band near anterior margin on each side;
segment 9 with or without comb-like groups of minutespines on weakly sclerotized light brown narrow transverseband near anterior margin on each side; posterior 1/2 of segment 9 very weakly to moderately sclerotized, yellowish to
medium brown, and sparsely covered with small round tubercles, and lacking terminal hooks. Ventrally, all segments
nearly transparent except segment 9 yellowish; segments 3―
8 each with comb-like groups of minute spines; segment 4
with 1 simple stout hook (similar in shape and size to thoseon segments 5―7), 1 simple slender hooklet submediallyand a few simple slender setae on each side; segment 5 witha pair of bifid and simple hooks submedially and a few simple slender setae on each side; segments 6 and 7 each with apair of simple hooks and a few simple (or bifid) slender setae on each side. Cocoon (Fig. 3I, J). Shoe-shaped, tightly
woven, not extending ventrolaterally; individual threads invisible; 3.5―4.0 mm long by 1.7―2.0 mm wide.
Mature larva. Unknown.
TYPE SPECIMENS. Holotype female with its associatedpupal exuvia and cocoon, collected in Doi Phu Ka National
Park, Nan Province, northern Thailand, 2.XII. 2004, by Wej
Choochote. Paratypes: 3 females and 3 males, all with theirassociated pupal exuviae and cocoons, same data as holotype; 5 females and 11 males, all with their associated pupal
exuviae and cocoons, same data as holotype except date, 23.
XII. 2004.
ECOLOGICAL NOTES. The pupae of this new species
were found attached to the surface of rocks in a fast flowing
cascading stream (width ca. 1.5 m, depth ca. 15 cm, exposed to sun, water temperature 18°
C, altitude 1,250 m
above sea level) in a sparsely forested area. This species
was found together with Simulium (Daviesellum) courtneyi
Takaoka and Adler, S . (Gomphostilbia) inthanonense
Takaoka and Suzuki and S . (S .) fenestratum Edwards.
ETYMOLOGY. The specific name phukaense refers to thename of the national park, Phu Ka, where this new species
was collected.
REMARKS. Simulium (Simulium) phukaense sp. nov. isassigned to the griseifrons species-group of the subgenus
Simulium (Simulium) by having the simple female claws,

１００

Fig. 3. Pupa of Simulium (Simulium) phukaense sp. nov. A, large tubercles on frons; B, facial trichome; C,
frontal trichome; D―G, thoracic trichomes (D, posterolateral; E―G, ventrolateral); H, gill filaments(left side; outer view); I and J, cocoons (I, lateral view; J, dorsal view). Scale bars. 1.0 mm for I andJ; 0.1 mm for H; 0.02 mm for B―G; 0.01 mm for A.

the male ventral plate without teeth (Fig. 2D), and the pupal
gill with six filaments (Fig. 3H).
This new species appears to be closely related to S .
(S .) maenoi described from Thailand [2] in that adults ofboth species have similarly shaped genitalia and a similarcolor pattern of the legs. However, S . (S .) phukaense sp.
nov. is distinguished from the latter species as follows(characteristics of S . (S .) maenoi in parentheses): in the female by the median projection on the cibarium short (long),
and the second abdominal segment entirely dark brown(pale yellow on anterior 3/4), in the male by the hind basitarsus spindle-shaped (wedge-shaped), a pair of dorsolateral
whitish spots present only on tergites 2, 6 and 7 (present
also on tergite 5) and the body of the ventral plate in ventral
view nearly parallel-sided (gradually narrowed toward posterior tip), in the pupa by the facial fan-like trichome with
12―15 slender branches (4 or 5 branches), the inner filamentof the middle pair apparently thinner than the outer one of
the same pair (both filaments subequal in thickness to eachother), the terminal hooks absent (present), and the cocoon

１０１

shoe-shaped (wall-pocket-shaped with a small anterolateralwindow on each side).

ACKNOWLEDGEMENT
This work was financially supported by a Grant-in-Aidof Japan-US Medical Research Cooperation Program (thefiscal year 2004).

REFERENCES[1] Takaoka H, Davies DM. The black flies (Diptera: Simuliidae) of Java, Indonesia. 1996, Honolulu, Bishop Museum
Bull Entomol. 6.
[2] Takaoka H, Choochote W. Taxonomic notes on the griseifrons species-group in Simulium (Simulium) (Diptera:
Simuliidae) from Thailand: Descriptions of two new species and description of the male, pupa and larva of S . (S .)digrammicum Edwards. Jpn J Trop Med Hyg. 2002; 27:
497―509.
[3] Takaoka H. The black flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. 2003, Fukuoka, Kyushu
University Press.

Tropical Medicine and Health Vol. 33 No. 2, 2005, pp. 103-104
Copyright! 2005 by The Japanese Society of Tropical Medicine

１
０
３

A PILOT FIELD SURVEY ON THE IN VITRO DRUG SUSCEPTIBILITYOF PLASMODIUM FALCIPARUM IN LAO PDRTOSHIMITSU HATABU１,２, VIENGXAY VANISAVETH３, NAO TAGUCHI１,
JUN KOBAYASHI４,５, M. KAIISSAR MANNOOR６, HISAMI WATANABE７,
HIROMU TOMA６, SAMLANE PHOMPIDA３, and SHIGEYUKI KANO２,*
Accepted 20, April, 2005

In Southeast Asia, malaria has presented a major public health problem, and the spread of drug-resistant falciparum malaria is making the problem more serious in this region. Thus, evidence-based detection of drug-resistant parasites is important for the accurate evaluation of susceptibility to antimalarial drugs. Lao PDR (Lao People’s Democratic Republic) is a developing country in which about
70% of the population lives in malaria endemic areas. Because of the lack of information on the in vitro drug susceptibility of parasites in this country, chloroquine (CQ) is still
the drug of choice for uncomplicated falciparum malaria [1].
This report is a pilot field survey on the in vitro CQ- andmefloquine (MQ)-susceptibility of falciparum malaria using
AnaeroPack" gas system in Saravan province, Lao PDR.
Saravan province is located in the southern part of Lao
PDR. The survey in this province was conducted from August 8 to 16, 2003. Blood samples were successfully obtained from nine Laotian patients suffering from falciparum
malaria. The samples were collected by the staff of the Center of Malariology, Parasitology and Entomology, after explaining the purpose of the study to the patients. The survey
was conducted in accordance with the ethical guidelines for
epidemiological studies established by the Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour and Welfare of Japan. The in vitro
drug susceptibility test was administered using the AnaeroPack" malaria culture system with a thermostat port-

able incubator as described previously [2, 3]. The AnaeroPack" CO２ (Mitsubishi Gas Co., Tokyo, Japan) is a foilpacked paper sachet that on exposure to air immediately absorbs atmospheric O２ and simultaneously generates CO２ until a condition of 15% O２ and 5% CO２ is attained. The microaerophilic atmosphere produced within a sealed jar (AnaeroPack" Kakugata jar, SUGIYAMA-GEN Co., Ltd., Tokyo, Japan) can be maintained for at least 24 hours. The
temperature inside the portable thermostat incubator(SUGIYAMA-GEN Co., Ltd.) was adjusted to 37°
C. During
P. falciparum cultivation, the sachet inside the jar was replaced every day when the culture medium was changed.
The WHO semi-micro test method was used for evaluationof in vitro drug susceptibility [4]. Briefly, blood samples(0.1 ml) were resuspended in RPMI 1640 (GIBCO BRL),
pH 7.4, supplemented with 25 mM HEPES, and sodium bicarbonate. To monitor parasite growth, six wells per plateserved as controls without antimalarials. When the schizonts were fully grown in the control wells, the culture plate
was removed from the incubator. Thin-smear specimens
stained with Giemsa solution were made from each well.
We defined parasites as schizonts when they had both darkbrown pigment and more than three nuclei [5]. The effect of
antimalarials on parasite growth was evaluated by the WHO
standard evaluation method.
The results of this study are shown in Table 1. Whencomplete schizont inhibition is observed at a CQ amount of

１Gunma University School of Health Sciences, Gunma, Japan;
２Research Institute, International Medical Center of Japan, Tokyo, Japan;
３Center of Malariology, Parasitology and Entomology, Vientiane, Lao PDR;
４Asian Centre of International Parasite Control, Bangkok, Thailand;
５Bureau of International Cooperation, International Medical Center of Japan, Tokyo, Japan;
６Division of Tropical Parasitology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan;
７Division of Cellular and Molecular Immunology, Center of Molecular Biosciences, University of the Ryukyus, Okinawa, Japan.
* Correspondence:
Shigeyuki Kano,
Research Institute, International Medical Center of Japan
1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan
TEL: +81-3-3202-7181 (ext 2877), FAX: +81-3-3202-7287, E-mail: kano@ri.imcj.go.jp

１
０４

Table 1: The results for in vitro drug susceptibility
No.

Parasitemia (%)

Chloroquine

Mefloquine

A
B
C
D
E
F
G
H
I

0.015
0.36
1.97
0.91
0.01
0.13
0.002
0.004
0.007

Susceptible
Susceptible
Susceptible
Resistant
Resistant
Resistant
Resistant
Susceptible
Susceptible

Susceptible
Susceptible
Susceptible
Susceptible
Susceptible
Susceptible
Susceptible
Susceptible
Susceptible

80 nM or less, the parasite is considered susceptible. If schizont formulation is observed at an MQ amount of 640 nM
or more, the parasite can be considered resistant. In the present study, four (44%) of the nine isolates were resistant to
CQ, while all the isolates were susceptible to MQ. There
was no correlation between the parasitemia and CQresistance.
The results of this study suggest that CQ-resistantparasites have increased even though CQ is commonly usedas the first-line drug for treatment of uncomplicated falciparum malaria in Lao PDR. In neighboring countries such as
Thailand and Cambodia, high-grade multi-drug resistantparasites are reported to be spreading and, indeed, in vivo
CQ-resistant falciparum malaria has already been reported
in Lao PDR [6]. Dedicated efforts have to be made to determine the in vitro drug susceptibility of P. falciparum in Lao

PDR as a way to prevent the spread of multi-drug resistantparasites in the near future. This is the first test report on invitro drug resistance in Lao PDR.

REFERENCES
１．Guthmann JP, Kasparian S, Phetsouvanh R, et al. The
efficacy of chloroquine for the treatment of acute, uncomplicated, Plasmodium falciparum malaria in Laos.
Ann Trop Med Parasitol 2002; 96: 553-557.
２．Hatabu T, Kawazu S, Kojima S, et al. A pilot field trailof an in vitro drug susceptibility test using the AnaeroPack! malaria culture system on the ThaiMyanmar border. Trop Med Hlth 2004; 32: 335-337.
３．Mizuno Y, Hatabu T, Kawazu S, et al. Cultivation of
Plasmodium falciparum isolates under the AnaeroPack! gas condition in a portable thermostatic incubator Jpn J Trop Med Hyg 2000; 23: 393-399.
４．Rieckmann KH, et al. Drug sensitivity of Plasmodium
falciparum- An in vitro microtechnique. Lancet 1978;
7: 9-13.
５．Silamut K, Phu NH, Whitty C, et al. A quantitative
analysis of the microvascular sequestration of malaria
parasites in the human brain. Am J Pathol 1999; 155:395-410.
６．Pillai DR, Labbe AC, Vanisaveth V, et al. Plasmodium
falciparum malaria in Laos: Chloroquine treatment
outcome and predictive value of molecular markers. J
Infect Dis 2001; 183: 789-795.

Tropical Medicine and Health Vol. 33 No. 2, 2005, pp. 105-113
Copyright! 2005 by The Japanese Society of Tropical Medicine

１０５

Review

MALARIA ENDEMIC PATTERNS ON LOMBOKAND SUMBAWA ISLANDS, INDONESIA
YOES P. DACHLAN１, SUBAGYO YOTOPRANOTO１, BARIAH V. SUTANTO１, SRI H. B. SANTOSO１,
ANNI S. WIDODO１, KUSMARTISNAWATI１, AGUS SUTANTO２, I. K. KOMANG GERUDUG２,MASAHIRO TAKAGI３, YOSHIO TSUDA３, KAZUYUKI TANABE４, FUMIHIKO KAWAMOTO５,KAZUMI YOSHINAGA３ and HIROJI KANBARA３*
Accepted 20, May, 2005

ABSTRACT: Nusa Tengara Barat (NTB) province consists of two main islands, Lombok and Sumbawa, to theeast of Bali Island, Indonesia. Most of the area is known to be moderately malaria endemic, but the exact malaria
epidemiology has not been elucidated. At least 30 deaths per year are thought to be caused by falciparum malaria
in Lombok alone, judging from the hospital data. According to the Gebrak Malaria Team in West Lombok, the annual incidence in the district of West Lombok from 1996 to 1999 was consistently over 40‰.
In the present report, we describe the small malaria endemic foci in the West Lombok and Sumbawa districts.
Falciparum malaria is predominant over vivax malaria and other types of malaria. There are 11 species of Anopheles vector, but three of these species, An. subpictus, An. maculates and An. barbirostris, are of primary importancein malaria transmission and An. sundaicus and An. aconitus are of secondary importance. Our data from Sekotong,West Lombok, and Sumbawa supported the importance of An. subpictus in coastal areas but suggested the existence of different transmission peaks according to environmental conditions. The usual transmission peak comes inthe dry season but is affected by climatic and geographical conditions. Although there were many malaria endemic
foci along the coast, the width and grade of the foci varied widely. The presence of malaria endemic foci inland,
although likely, has not been definitively reported to date.

INTRODUCTION
Indonesia is known as a country where tourists are at a highrisk for malaria infection. But the incidence of malaria varies widely among different islands and even among different areas of the same island. It is important to obtain exact
information on the epidemiological conditions of malaria
on each island. In this report, we describe the epidemiology
of malaria in parts of Lombok and Sumbawa islands on thebasis of our experience and local data (Fig.1).
1) Malaria situation in Indonesia;
Malaria is still a major public health problem in Indonesia.
In 1995, the National Health Household Survey estimated１．

that around 32,000 deaths were caused by malaria. [1]. Indonesia is a large archipelago consisting of 12,508 islandsof various sizes and shapes located along the equator andhad a total population of 209 million in 1999. About 70%of the population live in Java and Bali, where malaria has
been mostly eradicated, although even today small outbreaks are reported every year. In the outer islands, however, a much higher incidence of malaria is seen in general.
But the incidence of malaria varies from hypo-to hyperendemic depending on the environmental and socioeconomic conditions of an area. The natural and social environment of the Indonesian islands varies widely, resulting
in different malaria conditions. Furthermore, even on a single island, malaria endemic situations vary in degree andTropical Disease Center, Airlangga University, Surabaya, 60115, Indonesia
NTB Provincial Health Office, Mataram, 83231, Lombok, Indonesia
３．
Institute of Tropical Medicine, Nagasaki University, Nagasaki, 852-8523, Japan
４．
Laboratory of Biology, Osaka Institute of Technology, Osaka 535-8585, Japan
５．
Nagoya University School of Medicine, Nagoya 466-8550, Japan* Author for correspondence;
Hiroji Kanbara
Institute of Tropical Medicine, Nagasaki University,
1-12-4, Sakamoto, Nagasaki-shi, 852-8523, Japan
２．

１
０
６

Fig. 1 Location of places described in the text on Lombok and Sumbawa islands, Indonesia

size according to geographical conditions. No exact information on malaria epidemiology in each area has been published, especially in English. In this review, we introduce
the conditions of malaria in Lombok and Sumbawa islands
based on our epidemiological studies conducted under Japan Society for the Promotion of Science (JSPS) sponsorship, and on local data and reports offered by concerned
health organizations.
2) The collaborative survey of Indonesian and Japanese
researchers; In 1991 we had an opportunity to conduct an
epidemiological malaria survey on Lombok island as one of
the collaborative works in a large scale cooperative study
between Kobe University School of Medicine, Japan andthe Tropical Disease Center (TDC), Airlangga University,
Surabaya, Indonesia under the sponsorship of JSPS. The
malaria epidemiological study was carried out continuously
in Lombok and later in Sumbawa for ten years until the
JSPS project reached completion in 2000.
3) Malaria situation in West Lombok; The West Lombok
district is located in the western part of Lombok island,
NTB province. The province is composed of two major islands, Lombok and Sumbawa, each of them containing
three districts. The capital of the province is the municipality Mataram, which is located in the center of West Lombok
district. West Lombok consists of nine subdistricts, in all ofwhich malaria is endemic. According to the report by Gerback Malaria Team in West Lombok [2], the annual incidence in the district was over 40 per 1,000 population every
year from 1996 to 1999. Falcipaum malaria is more com-

mon than vivax and other types of malaria. The transmission peak is usually observed between July and September.
There are 11 species of Anopheles vector but, of these,
three, An. subpictus, An. maculates and An. barbirostris,
are of primary importance in malaria transmission and An.
sundaicus and An. aconitus are of secondary importance.
Figure 2 shows the monthly slide-positive cases observed in2000 and 2001 in Tanjung and Tegal Maja villages by thehealth center in Tanjung, which is located in the northernmost part of West Lombok. The village of Tanjung lies on
the northern coast while the village of Tegal Maja is locatedsouth of it in the inland. At the former, the clear peak ofmalaria cases was seen in October, which was two months
later than the usual transmission peak between July andFig. 2 Number of slide positive cases in two villages of Tanjung
health center１０
７

three subvillages in Sekotong for the longitudinal survey,
namely, dusun Labuhan Poh from desa (village) Sekotong
Barat, and dusun Longlongan and Sayong from desa Sekotong Tengah. (Fig. 1, Fig. 4 and Table 1).
2) Geographical differences among subvillages (Table 1)
At subvillage Labuhan Poh, the land gradually rises awayfrom the coast toward the inland. The largest river in thisvillage passes through the subvillage and creates a wide lagoon during the dry season (Photo. 1). Many branches ofFig. 3 Number of cerebral malaria cases at Mataram
hospital shown by the place of residence

September mentioned in the local report [2]. This type oftransmission is thought to be caused by An. sundaicus
rather than An. subpictus according to a previous study by
one of the present authors [3]. We obtained the data on malaria patients hospitalized in the Mataram hospital in 2001
and the first half of 2002. In total, 809 malaria patientswere hospitalized. These were composed of 580 falciparum
malaria patients including 71 with cerebral malaria, 54 vivax malaria patients and 175 clinical malaria patients. A total of 39 died of falciparum malaria, and 29 of these were
cerebral malaria patients. We marked the number of cerebral malaria patients on a map according to the place ofresidence recorded in their patient-reports (Fig. 3). The
cases were distributed equally in coastal and inland areas.
This indicates that more careful attention should be given to
the inland areas to identify malaria endemic foci.

Fig. 4 Geographical distribution of the three subvillages
selected for malaria survey at Sekotong

Table 1 Geographical features and population of three subvillages of
Sekotong Barat and Tengah in 1992
Subvillage

hill

wet rice field beachdry field population

Labuhan Poh

16.5%

0

21.3%

62.2%

855

Sayong

30.6%

67.7%

1.7%

0

1,247

Longlongan

71.5%

4.3%

16.1%

8.1%

805

STUDIES IN WEST LOMBOK
1) Survey areas in West Lombok
Malaria epidemiological study target areas were selected on
the basis of discussions between TDC and the Nusa Tenggara Barat (NTB) provincial health office. The subdistricts(kecamatan) Batulayar and Sekotong were selected for the
preliminary survey. Subjects for blood and spleen examination were randomly selected from different subvillages(dusun) in the two subdistricts. A total of 36 subvillages, or
10 from Batulayar and 26 from Sekotong, were subjected tothe survey. Although the number of persons examined ineach subvillage was too small for evaluation, we selected

Photo 1. A huge lagoon formed after closing the river’s exit to
the sea at Labuhan Poh, Sekotong, West Lombok

１０
８

the river extend into mangrove areas where large mangrove
trees have been cut for fuel, leaving many water pools exposed to sunshine and resulting in breeding places forbrackish species of Anopheles mosquitoes such as An. sundaicus and An. subpictus. Subvillage Sayong lies on flatground. Most of the area is occupied by wet rice fields, andin the coastal part of the flat land once covered by mangrove forest, fish ponds were made after the removal ofmangrove trees. Subvillage Longlongan has a complex topography; the narrow flat land along the coast, originallymangrove forest, was developed for fish ponds, and the following sharp sloping land leads to a rather flat hilly area
where rice fields were developed between stands of grass orbush in the rainy season.

vey, the subjects were usually gathered in one place such asa school or a village health office (pustu) on an appointed
date, and a 1―2 ml venous blood sample was obtained fromeach person along with in a syringe, a drop of blood for thinsmear and another drop for thick smear on separate slideglasses. The blood in a syringe was transferred into a small
tube for serum collection. All the samples were carried to
TDC for parasitological and serological examination. Medical examination was administered to each person after
blood collection, and if necessary, medicines were given.
On the same day the entomological survey was conducted,
consisting of the examination of breeding sites and larva
collection in the daytime, and adult mosquito collection atnight.

3) Survey methods and subjects
The longitudinal survey was started in August 1992 and carried out five times until June 1993 [4]. At the initial step, inorder to determine the seasonal changes in malaria transmission, we intended to collect blood samples from thesame subjects randomly selected from all age groups
through all the surveys in a year. After the third survey,
however, we had to replace the subjects with a new groupcomposed of almost the same proportion of age groups, because of difficulties encountered in obtaining informed consent and cooperation from the former subjects. In the sur-

4) Malaria prevalence in the survey areas
Table 2 and Fig. 5 present the results of the blood examinations. We cannot accurately compare the results of the first
three surveys with those of the last two surveys because of
the replacement of subjects. In total, the malaria positive
rate gradually declined after the first survey in August 1992.
However, the malaria transmission trend in each subvillage
differed from that in others (Fig. 5). A relatively stableslide positive rate was found in dusun Labuhan Poh in August, October and December 1992, while in other subvillages it varied by month, especially in dusun Longlongan.

Table 2 Results of blood examinations in a longitudinal malaria survey conducted in three subvillages of Sekotong,
Lombok from August 1992 to June 1993
August 1992
Subvillage

PR%

LabuhanPoh
Sayong11.5(14/122)11.9(18/151)
21.1(20/95)14.1(52/368)

Longlongan
Total

October 1992

Number of positive cases
Pf
Pv
Pm
Mix
10

4

0

0

6

11

0

1

12

8

0

0

28

23

0

1

PR
%
9.8
(12/122)6.0(9/150)6.4(6/95)
7.4(27/367)

Number of positive cases
Pf
Pv
Pm
Mix
9

2

0

1

4

5

0

0

5

1

0

0

18

8

0

1

April 1993
Subvillage

PR%

LabuhanPoh
Sayong3.7(4/107)0.72(1/139)1.0(1/98)
1.7(6/344)Longlongan
Total

December 1992

June 1993

Number of positive cases
Pf
Pv
Pm
Mix
3

1

0

0

1

0

0

0

1

0

0

0

5

1

0

0

PR
%
1.1
(1/89)
0(0/129)9.0(8/89)
2.9(9/307)

PR, positive rate; Pf. Plasmodium falciparum; Pv, Plasmodium vivax;
Pm, Plasmodium malariae; Mix, mix infection; ( ), actual number

Number of positice cases
Pf
Pv
Pm
Mix
0

0

1

0

0

0

0

0

5

2

0

1

5

2

1

1

PR
%
12.3
(15/122)6.0(9/150)2.1(2/95)
7.1(26/367)

Number of positive cases
Pf
Pv
Pm
Mix
11

3

0

1

4

5

0

0

1

1

0

0

16

9

0

1

１
０
９

Fig. 5 Slide positive rates in the three subvillages of Sekotong,
Lombok from August 1992 to June 1993

This difference may be attributable to the different environmental and geographical conditions of each subvillage (Table1). Especially in dusun Longlongan, the malaria in the
hilly area may have a different transmission mode.

5) Entomological observation in the survey area
The results of the entomological examination also showed awide variety (Table 3 and 4). As expected, a relatively stable number of adult Anopheles subpictus mosquitoes were
captured at all three subvillages, especially at dusun Labuhan Poh (Table 3), but An. sundaicus, An. barbirostris andAn. aconitus, which have been recognized as malaria vectors in Indonesia, were captured sporadically once or twice
in five surveys conducted one year except An. sundaicus atdusun Labuhan Poh [4, 5]. The fluctuation in the number of
captured mosquitoes suspected to be malaria vectors did notcorrespond to the parasitological data (Table 2, 3 and Fig.
5). The most stable larva collection of the brackish
Anopheles species was obtained at lagoon and mangrove areas in dusun Labuhan Poh but at fish-ponds in dusun
Sayong and Longlongan (Table 4). These results, takenwith the parasitological data, indicate that An. subpictus(and additionally An. sundaicus) play a major role in malaria transmission in these subvillages. The previous intensive study on mosquito fauna in Lombok island by Lee et al.

Table 3 Adult collection of Anopheles species known as malaria vector
in three subvillages of Sekotong (1992-1993)SUBVILLAGE

COLLECTIONMETHODAug 92LABUHAN
POH

Outdoor Human
Bait (OHB)

sub/14
sun/4

sub/9
sun/8

sub/6
sun/2

bar/14
sub/4

sub/6
sun/3

Indoor Human
Bait (IHB)

sub/2

sub/11
sun/22

sub/6
sun/5

0
0

sub/4

Indoor Resting (IR)

0

sun/5

0

0

0

Bednet Trap (BT)

0

sub/7
sun/16000Cattle Bait (CB)sub/12

sub/1

sub/2
acon/1

sub/5
sun/1

OHB

sub/11
sun/1
bar/1
sub/7

sub/14
sun/3

sub/10acon/5sub/8acon/1

sub/1

IHB

0

sub/4

sub/9
acon/2acon/2

0

IR

0

0

0

0

0

BT

bar/1

0

0

0

0

CB

sub/5

sub/16sub/6acon/4
sub/9

sub/9

OHB

sun/3

sub/15
sun/3

sub/4

sub/19

0

IHB

0

sub/3
sun/1

sub/17
sun/1

sub/3

sub/1

SAYONGLONGLONGAN

Oct 92

Dec 92

Apr 93

Jun 93(species / no. mosq. collected per night*)

IR

0

0

0

0

sub/1

BT

sun/1

0

0

0

−

CB

sub/7
bar/1

sub/17

sub/8

sub/10acon/7sub/9acon/1*40 min per hour from 6 pm―12 pm.
sub, Anopheles subpictus; sun, An. sundaicus; acon, An. aconitus; bar, An. barbirostris

１
１
０

Table 4 Type of breeding place and density of Anopheles larvae (per dip)in three subvillages of Sekotong
Type Br. Pl.
Labuhan Poh
I. Lagoon
II. RiverIII. Mangrove
IV. Rice field
V. Fishpond
SayongI. Fishpond

II. Rice field

III. Fresh waterLonglongan
I. WellII. RiverIII. Ricefield

IV. FishpondAnoph. Species

Aug. 92

Oct. 92

Dec. 92

Apr. 93

Jun. 93

1. An. subpictus
2. An. sundaicus
1. An. flavirostris
2. An. minimus
3. An. vagus
4. An. subpictus
5. An. sundaicus
1. An. subpictus
2. An. sundaicus
1. An. vagus
1. An. subpictus

n. d.
n. d.
1.00
0.50
0
1.56
0.12
1.00
0
(−)
(−)

1.87
0.70
0
0
0
0
0
0
0
(−)
(−)

1.30
0.40
0
0
0
1.50
0
1.60
0.40
1.40
(−)

1.40
0
0
0.70
0.11
0
0
0.67
0
(−)
0.10

0.95
0.30
0
0
0.10
0
0
(−)
(−)
0.10
0

1. An. subpictus
2. An. sundaicus
3. An. annularis
1. An. aconitus
2. An. barbirostris
3. An. vagus
1. An. barbirostris
2. An. annularis

0.80
0
0
0
0.70
0
0.60
0

0.80
0
0
0
0
1.50
0
0

1.00
0
0
0.40
0
2.20
0
3.40

0.05
0.05
0.05
0.60
0
0
0
0.60

0.90
0
0
0.25
0
0.28
0
0

1. An. barbirostris
2. An. annularis
3. An. vagus
1. An. aconitus
2. An. barbirostris
3. An. vagus
1. An. barbirostris
2. An. annularis
3. An. vagus
1. An. subpictus

0.40
0
0
0.02
0.77
0.85
0
0
1.00
0.08

0
0
0
0
0
0.67
0.05
0
0.80
1.38

0
0
1.20
0
0
1.80
0
0
4.20
1.20

0
0.10
3.00
0
0.40
0
0.25
0.01
2.07
1.80

0.35
0
0.70
0
0
0.23
0.01
0.01
0.55
1.03

(−), no water at the time examined; Br. Pl., Breeding Place; n. d., not done

identified three Anopheles species, An. annularis, An. barbirostris and An. subpictus, as potential vectors [6]. Recently, Miyagi et al also found An. subpictus and An. sundaicus in coastal areas and An. barbirostris, An. leucosphyrus group and An. minimus in fresh water and citedthem as potential vectors [7]. In 2001, Sukowati, S. et al.,Health Ecology Research Center, NIHR&D found Plasmodium falciparum (P. f.) sporozoite-positive An. subpictus inthis area (report to the Indonesian health ministry). In thesubvillage Longlongan, in addition to the coastal area, malaria was found in the hilly area where more than half of the
population of this subvillage live, but we could not determine the vector mosquitoes there. Because of windy conditions and the collection confined to one night during thesurvey, the entomological staff were able to capture only a
few adult mosquitoes. From the larvae examination we inferred two probable transmission vectors. One is An. barbi-

rostris, the larvae of which were found in rice-fields, stagnant water along small rivers and wells, and the other is An.
subpictus, which was consistently found in fish ponds along
the coast and is thought to be able to move back and forth
between the coast and the hills with the wind.
6) Endemic situation of malaria
We selected subjects equally from all the age groups to determine the degree of endemicity. Our results showed nodifference in malaria prevalence among age groups [4] (data,
not shown), indicating a hypo-or meso-endemic pattern inthe area. The additional serological examination of antibodies to P. f. crude antigens using ELISA also demonstrated a meso-endemic pattern at dusun Labuhan Poh (Fig.
6), that is, the positive rate was low (about 20%) at the ageof 0 but rose to nearly 100% at the age of 6 or over. In thisarea, three Plasmodium species were detected, that is, about

１１
１

60% P. falciparum, 40% P. vivax and only one P. malariae(Table 2). The P. malaria case was confirmed by PCR using the ribosomal DNA sequence [8]. Our results did notconfirm the peak of transmission between July and September as described in the local report [2].

STUDIES IN SUMBAWA
1) Survey areas in Sumbawa
In Sumbawa, four subvillages in different subdistricts were
examined for prevalence of malaria from 1996 to 1999 (Fig.
1). One subvillage, dusun Medang, is a small island accessible in one hour from Sumbawa Besar by small motorboat.
In this subvillage, the preliminary spleen examination wasFig. 6 Sero-positive rate to P. f. crude antigens among persons
examined in subvillage Labuhan Poh in October 1992

Table 5 Parasite positive rate and spleen rate in the longitudinal survey conducted in three subvillages of Sumbawa
December 1996

July 1997

Subvillage

Positive
Rate (%)

Number of positive cases
Pf
Pv
Mix

SpleenRate (%)

Positive
Rate (%)

Number of positive cases
Pf
Pv
Mix

Penyaring

7.1(8/112)

7

1

0

0

−

−

−

−

−

Labangka IV

14.3(16/112)

10

6

0

8.0

7.1
(8/112)

4

4

0

14.3

Stowe Brang

33.9(38/112)

8

25

5

25.9

15.3
(17/111)

13

3

1

34.2

Total

18.5
(62/336)

25

32

5

11.3

11.2
(25/223)

17

7

1

16.0*

Number of positive cases
Pf
Pv
Mix

SpleenRate (%)

March 1998

SpleenRate (%)

October 1998

Subvillage

Positive
Rate (%)

Number of positive cases
Pf
Pv
Mix

SpleenRate (%)

Positive
Rate (%)

Penyaring

1.8(2/112)

2

0

0

0

0.9
(1/112)

1

0

0

0

Labangka IV

0.9(1/112)

1

0

0

0

13.0
(9/69(74))**

4

3

2

23.0

Stowe Brang

8.0(9/112)

7

2

0

12.5

1.8
(2/112)

1

1

0

4.5

Total

3.6
(12/336)

10

2

0

4.2

14.1
(12/293)

6

4

2

7.4

December 1998

February 1999

Subvillage

Positive
Rate (%)

Number of positive cases
Pf
Pv
Mix

SpleenRate (%)

Positive
Rate (%)

Penyaring

0.9(1/112)

1

0

0

0

3.6
(4/111)

3

1

0

0

Labangka IV

5.3(5/95)

1

4

0

1.1

5.4
(6/111)

2

3

1

0

Stowe Brang0(0/112)

0

0

0

0

ND
(−/108)

ND

ND

ND

0.9

Total

1.9
(6/319)

2

4

0

0.3

4.5
(10/222)

5

4

1

0.3

P. f., Plasmodium falciparum; P. v., Pl. vivax; Mix, mix infection−,data lost
* The number of subjects in Penyaring was assumed to be 112.
** 74 persons were subjected to spleen examination, but only69of these underwent blood examinations.
ND, not done

Number of positive cases
Pf
Pv
Mix

SpleenRate (%)

１
１
２

conducted on 161 1st and 2nd grade school-children andshowed a 42.9% spleen rate (meso-endemic), but afterwards
neither blood nor mosquito examinations was conducted because of the risk of the available boat capsizing. Therefore,
three subvillages were selected for the longitudinal survey.
The methods were the same as those used in Lombok.
2) Malaria prevalence in the Sumbawa survey areas
The slide positive rates and spleen rates at three subvillages
are shown in Table 5. All three subvillages are locatedalong the coast. Subvillage Penyaring and Stowe Berang
face the ocean to the north and subvillage Labangka IV tothe south. The former two subvillages are geographicallysimilar. They have mangrove beaches and flat lands. The
mangrove beaches were developed for fish ponds in bothsubvillages. Despite the environmental similarities, subvillage Penyaring showed a very low slide-positive rate and0% spleen rate, while dusun Stowe Berang showed rather
high positive rates for both examinations. Subvillage Labangka IV showed a medium endemic pattern with seasonal
epidemics.
3) Entomological observation and epidemiological analysis
The entomological examination clearly demonstrated a highdensity of adults and larvae of An. subpictus at subvillage
Stowe Berang but a very low density at subvillage Penyaring (data, not shown). This was due to the difference in
breeding sites between the two subvillages, namely, many
abandoned fish-ponds with algae and weeds were found at
the former (Photo. 2) while most of the fish ponds were
well maintained at the latter. The sharp decline in positive
rates for spleen and blood examinations at dusun Stowe
Berang from October 1998 was due to two malaria control
projects conducted from January 1998 for a year, that is, thedistribution of insecticide impregnated mosquito-nets andthe cleaning of abandoned fish-ponds (Fig. 7). Our reports
in 1996 and 1997 note that these control projects were conducted by the Sumbawa district health office, and suggestthat the control methods worked effectively. In subvillage
Labangka IV, an outbreak of malaria was observed just before our survey in October 1998. This subvillage has a very
narrow sandy beach with a steep cliff rising behind. A
rather flat hilly area spreads away from the cliff. The entomological survey found that the captured Anopheles mosquitoes were exclusively An. subpictus and that there wereseveral lagoons on a small beach where An. subpictus larvae bred. According to staff in the Sumbawa district healthoffice, the outbreak may be related to the custom of villagers to gather around the cliff (cape) at night to catch a species of bird during this season.

Photo 2. An abandoned fish pond at Stowe Brang, Utan, Sumbawa

Fig. 7 Changes in slide―positive rate (●) and spleen rate (▲)
in Stowe Brang, Sumbawa during the three―year period
after malaria control activities in January 1998

SUMMARY
Although our data are still insufficient to determine the fullrange of epidemiological features, we can draw the following conclusions about malaria in Lombok and Sumbawa.
1) Malaria endemic areas are located mainly along the seacoast and less frequently inland.
2) The degree of endemicity is hypo-endemic to mesoendemic.
3) The main transmission vectors are Anopheles subpictus
and An. sundaicus, which breed in brackish water.
4) Although similar species of vector play a role in transmission in coastal endemic foci, the mode and the season oftransmission vary with the ecological characteristics of thevector and social and environmental conditions.
5) Small endemic foci are found in hilly areas inland, but
the responsible vector species have not been determined.

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１）WHO and Ministry of Health, Indonesia. Development of１
１３

Roll back Malaria model in Indonesia “Gebrak Malaria”,
Strategic plan 2001-2006. Jakarta: 2001.
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Directrate General Communicable Diseases Control andEnvironmental Health, Ministry of Health, Indonesia,
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３）Kanbara H., Panjaitan W. The epidemiological survey ofmalaria in Asahan district, North Sumatra, Indonesia. Jpn
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Arifin Z., Dachlan Y. P. Mosquito species (Diptera : Culicidae) from Lombok island, Indonesia. Mosquito Systematics 1994; 26: 19-24.
８）Kawamoto F., Liu Q., Ferreira M. U., Tantular I. S. How
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