Vol.33
No.03
SEPTEMBER 2005

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.33 No.03 SEPTEMBER 2005

ISSN 1348-8945【CONTENTS】
Original articles
Prevalence and characteristics of Escherichia coli isolates harbouring Shiga toxin
genes (stx) from acute diarrhoeal patients in Dhaka, Bangladesh
Akter J, Das SC, Ramamurthy T, Ashraf H, Saha D, Faruque ASG, Nair GB and Salam MA １
１
９Real time estimation of reproduction numbers based on case notifications- Effective reproduction number of primary pneumonic plague
Nishiura H and Kakehashi M

Vol.33 No.03 SEPTEMBER 2005

１
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７

Two new species of Simulium (Nevermannia) (Diptera: Simuliidae) from Northern Thailand
Takaoka H and Choochote W
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The effect of medication on questionnaire analysis of children with Schistosoma mansoni
infection in Tanzania
Kisu T, Shiratori K, Callyst F, Ataka Y, Kaneda E, Ngomuo E, Shayo RJ and Shimada M

Tropical Medicine
and Health１４３JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

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

背は４．
５mm
（決）

Vol.33
No.03
SEPTEMBER 2005

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.33 No.03 SEPTEMBER 2005

ISSN 1348-8945【CONTENTS】
Original articles
Prevalence and characteristics of Escherichia coli isolates harbouring Shiga toxin
genes (stx) from acute diarrhoeal patients in Dhaka, Bangladesh
Akter J, Das SC, Ramamurthy T, Ashraf H, Saha D, Faruque ASG, Nair GB and Salam MA １
１
９Real time estimation of reproduction numbers based on case notifications- Effective reproduction number of primary pneumonic plague
Nishiura H and Kakehashi M

Vol.33 No.03 SEPTEMBER 2005

１
２
７

Two new species of Simulium (Nevermannia) (Diptera: Simuliidae) from Northern Thailand
Takaoka H and Choochote W
１
３
３
The effect of medication on questionnaire analysis of children with Schistosoma mansoni
infection in Tanzania
Kisu T, Shiratori K, Callyst F, Ataka Y, Kaneda E, Ngomuo E, Shayo RJ and Shimada M

Tropical Medicine
and Health１４３JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

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

背は４．
５mm
（決）

Tropical Medicine and Health Vol. 33 No. 3, 2005, pp. 119-126
Copyright! 2005 by The Japanese Society of Tropical Medicine

１
１
９PREVALENCE AND CHARACTERISTICS OFESCHERICHIA COLI ISOLATESHARBOURING SHIGA TOXIN GENES (STX )FROM ACUTE DIARRHOEALPATIENTS IN DHAKA, BANGLADESH
JASMIN AKTER１, SURESH CHANDRA DAS２, THANDAVARAYAN RAMAMURTHY３, HASAN ASHRAF１,
DEBASISH SAHA１, ABU SYED GOLAM FARUQUE１, GOPINATH BALAKRISH NAIR１
and MOHAMMED ABDUS SALAM１
Accepted 2, June, 2005

Abstract: Shiga toxin genes (stx) harbouring Escherichia coli (STEC) strains were isolated and identified from diarrhoeal patients visiting the Dhaka Hospital of ICDDR,B: Centre for Health and Population Research, Dhaka,Bangladesh. Of the 189 E. coli strains isolated from 775 diarrhoeal stool specimens, 19 harboured stx1, and one
isolate was revealed to have amplicons for both stx1 and stx2 by a PCR assay. Sequence analysis of the 349-bp stx1
from representative isolates revealed 100% homology with the sequence of stx1 available in the GenBank. Amongthe stx1 positive isolates, two harboured the eae but none were positive for hlyA, katP, etpD or saa genes. Fifteenof the 20 stx positive strains could be categorized into 13 non-O157 serogroups while 4 were untypable and one
was a rough strain. Most of the STEC strains were resistant to ampicillin, cephalothin, co-trimoxazole, tetracycline,
and nalidixic acid. In the Vero cell assay, all the strains were negative for expression of Shiga toxin (Stx). Randomly amplified polymorphic DNA (RAPD) PCR analysis demonstrated genetic diversity. This is one of the firstreports to show the presence of STEC in diarrhoeal patients in Bangladesh.
Key Words: Diarrhoea, STEC, PCR, Shiga toxin gene, Genetic diversity

INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC), a distinct class of pathogenic E. coli, can cause a spectrum of
human illnesses ranging from asymptomatic carriage to severe bloody-diarrhoea termed haemorrhagic colitis (HC), as
well as life-threatening sequels such as haemolytic uremic
syndrome (HUS) which has a case fatality rate of up to 5%[1, 2]. STEC has gained immense clinical importance since
being recognized in food-borne outbreaks in Oregon andMichigan in 1982 [3], and subsequently it has emerged as amajor public health problem in many developed countries.
The rate of morbidity and mortality due to Shiga toxinproducing E. coli (STEC) has highlighted the threat theseorganisms pose to public health. The ability of STEC tocause serious disease in humans is related to the productionof one or more Shiga toxins (Stx1, Stx2 or their variants),
which inhibit protein synthesis of host cells leading to death[4, 5]. Stx1, Stx2 and their variants are immunologically
non-cross reactive and are encoded by alleles in the genomeof temperate, lambdoid bacteriophages that remain integrated in the E. coli chromosome [6]. Stx1 is virtually identical to Shiga toxin produced by Shigella dysenteriae 1,
while Stx2 has only 56% identity to Stx1 [7].
The pathogenesis of STEC is triggered by a set ofgenes carried in the chromosome that includes a 35.5 kb
pathogenicity island, termed locus of enterocyte effacement(LEE). The characteristic attaching and effacing (A/E) lesion caused by most of the STEC depends on the activity ofmultiple genes in the LEE including the type III secretion
system and on the initiation of signal transduction events[8]. The eae gene is responsible for attaching to translo-

Enteric Bacteriology Laboratory, Laboratory Sciences Division, ICDDR,B, Mohakhali, Dhaka 1212, Bangladesh.
Department of Microbiology, National Institute of Cholera and Enteric Diseases, Kolkata, India
1 ICDDR, B: Centre for Health and Population Research, Dhaka 1212, Bangladesh;
2 Eastern Regional Station, Indian Veterinary Research Institute, Kolkata, India;
3 National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India
Address for correspondence and request for reprints: Dr. Mohammed Abdus Salam, Director, Clinical Sciences Division, ICDDR, B : Centre
for Health and Population Research, Dhaka 1212, Bangladesh: Fax:+88-02-9885657; email: masalam@icddrb.org

１２０

cated intimin receptor (Tir). Some of the STEC strains harbour a 97 kb plasmid (pO157), encoding possible additionalvirulence genes such as hlyA (EHEC-HlyA) that acts as apore-forming cytolysin on eukaryotic cells, the bifunctional
catalase peroxidase (KatP), the etpD gene cluster that probably encodes a type II secretion pathway, and the secreted
serine protease (EspP ) [9-13]. The saa gene (STEC autoagglutination adhesin) encodes a novel outer membrane protein and is carried on megaplasmid of LEE-negative STEC
strains, which acts as an autoagglutinating adhesin [14].
About 250 non-O157 STEC serotypes have been reported to be associated with diarrhoea, HUS and other afflictions [15, http://www.Microbionet.com.au/frames/feature/vetc/brief01.html]. In the USA, non-O157 serotypes
are detected at regular intervals but lack many importantvirulence genes [16]. Among STEC, the serotype O157:H7
is epidemiologically significant worldwide (http:www.who.
int/emcdocuments/zoonooses/whocsraph988.c.hlml). STEC
infection has posed a much greater problem to developed
countries than to developing countries. The low incidenceof STEC in developing countries might be attributable tothe complexity of recognising emerging variants [17, 18].
Though STEC has been isolated from humans and cattle inIndia [19], less is known about STEC in Bangladesh. An
earlier study observed the presence of SLT-I and SLT-II
positive E. coli isolates in the paediatric population in
Bangladesh [20]. This study was conducted to determine
the significance of STEC- related diarrhoea in the Bangladeshi setting. As part of the active surveillance, we conducted a bacteriological analysis of faecal specimens fromdiarrheal patients visiting the Dhaka Hospital of ICDDR, B
over a period of 5 months.

MATERIALS AND METHODS
Sample collection and isolation of E. coli
Faecal specimens were collected from patients enrolled
in the 2% systematic sampling of all patients visiting the
Dhaka Hospital of ICDDR, B under its diarrhoeal disease
surveillance system, between July and November 2002. Information such as age, sex, and clinical symptoms (type andduration of diarrhoea, dehydration status, and presence offever and other symptoms) was collected using a standard
questionnaire. All the 775 faecal specimens collected frompatients in sterile containers were subjected to macroscopic
inspection for presence of blood and mucus, and for isolation of enteric bacterial pathogens using standard laboratory
methods [21]. From each faecal specimen, three representative lactose-fermenting colonies growing on MacConkey
agar (Difco, Detroit, USA) were isolated and identified [21].
The ability of the stx harbouring isolates to ferment sorbitol

was assessed by streaking of the strains on sorbitol MacConkey Agar (Difco) plates. O-serogroups of E. coli isolates were determined by slide agglutination using a commercially available antisera kit (Denka Seiken Co., Tokyo,
Japan). All of the E. coli isolates were preserved at -80°
C.
Drug susceptibility tests
Drug susceptibility was determined by the disc diffusion method using the following commercial disks (Hi Media, Mumbai, India), ampicillin (10 µg), chloramphenicol(30 µg), co-trimoxazole (25 µg), ciprofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), nalidixic acid (30 µg),
norfloxacin (10 µg), streptomycin (10 µg), tetracycline (30µg), cephalothin (30 µg), amikacin (30 µg), furazolidone(50 µg), kanamycin (30 µg) and ceftriaxone (30 µg). The E.
coli ATCC 25922 strain sensitive to all the drugs was used
as a quality control. The characterization of the isolates as
susceptible, reduced susceptibility, or resistant was done asrecommended by the National Committee for ClinicalLaboratory Standards [22].
Screening for virulence genes
PCR for the detection of both chromosomal and plasmid virulence genes was performed using a thermal cycler(Applied Biosystems, Foster City, Calif, USA) in a total
volume of 25 µl. The reaction mixture contained 5 µl of
culture lysate, 2.5 mM of each dNTP, 30 µM of each primer,
2.5 µl of 10 X PCR Buffer, and 1 U of r-Taq DNA polymerase (Takara, Shuzo, Japan). The primer sequences of thetargeted virulence genes and the PCR conditions are presented in Table 1. VTEC-3 (serotype O157:H7 harbouring
stx1 and stx2 genes) and E. coli K-12 strain were used as
positive and negative controls, respectively.
Southern hybridisation
The PCR products from 5 representative strains wereseparated in 1.5% agarose gel and transferred to a Nylon
membrane by the capillary method, and the were UV crosslinked (GS Gene linker, Bio-Rad, Hercules, USA). The
cloned stx1 905-bp DNA fragment from the recombinant
plasmid pKTN501 was used as a probe after digestion with
BamHI and EcoRI. Hybridisation was done using the DIGDNA Labelling and Detection Kit (Boehringer-Mannheim,
Germany).
Sequencing of stx-PCR amplicon
The stx1 gene was amplified using primers (EVT1 and
EVT2). The PCR products were purified by QIA Quick
PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced directly in an ABI310 automated sequencer (Applied Biosystems) using Big-dye terminator chemistry, and１
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１

Table 1. Polymerase Chain Reaction (PCR) primers and conditions used in the study
Primer
EVT1/
EVT2
EVS1/
EVC2
hlyA1/
hlyA4
wkatB/wkatF
D1/
D13R
EAE1/
EAE2
SAAF
SAAR

Nucleotide sequence of primers(5’-3’)CAACACTGGATGATCTCAG
CCCCCTCAACTGCTAATAATCAGTCGTCACTCACTGGT
CTGCTGTCACAGTGACAAA
GGTGCAGCAGAAAAAGTTGTAG
TCTCGCCTGATAGTGTTTGGTACTTCCTGTTCTGATTCTTCTGC
AACTTATTTCTCGCATCATCCCGTCAGGAGGATGTTCAG
CGACTGCACCTGTTCCTGATTA
AAACAGGTGAAACTGTTGCC
CTCTGCAGATTAACCTCTGC
CGTGATGAACAGGCTATTGC
ATGGACATGCCTGTGGCAAC

TargetDenaturing

PCR conditionsa
Annealing
Extension

Amplicon(bp)

Reference

stx1

94°
C, 60s55°
C, 60sb

72°
C, 60s

349

48

stx2

94°
C, 30s55°
C, 60sb

72°
C, 60s

110

48

EHEC
hlyA

94°
C, 30s57°
C, 60s72°
C, 90s

1551

49

katP

94°
C, 30s56°
C, 60s72°
C, 90s

2125

50

etpD

94°
C, 30s52°
C, 60s72°
C, 70s

1062

51

eae

94°
C, 60s55°
C, 90s72°
C, 90s

350

52

saa

94°
C, 30s52°
C, 60s72°
C, 40s119

14aUnless stated, PCR was done for 30 cycles.
After 35 cycles, a final extension step of 10 min at 72°
C was performed.

b

Table 2. Clinical manifestation of patients infected with STEC.
Age Group(Y)

No. (%) STEC
infected cases(n=20)< 5yrs> 5yrs11 (55)9 (45)

Presenting feature (%)(n=20)
Watery Stool
Vomiting
Dehydration
11 (100)
8 (72)
4 (36)
6 (67)
5 (55)
6 (67)

the obtained sequences were compared with published stx1
gene sequences in the GenBank databases using the Blast
search program of NCBI.
Vero cell assay
All of the STEC strains were cultured in L-broth(Difco) at 37°
C overnight with constant shaking and pelleted by centrifugation at 5000 rpm for 5 min at 4°
C. Cellpellets were washed and sonicated with Handy Sonic(TOMY, Tokyo, Japan). After centrifugation, the supernatant and the cell lysate were filter-sterilized using 0.22 µm
filters (Millex, Millipore, Bedford, USA) and used in the assay. The cytotoxic effect of the STEC strains was assayedon Vero-cells in 96-well flat bottom tissue culture plates(NUNC, Intermad, Denmark) as described elsewhere [23].
The cells were observed microscopically up to 72 hrs forcytotoxicity. VTEC-3 and DH5α were used as positive andnegative controls, respectively.
RAPD Analysis
Molecular typing of stx-harbouring isolates was done
by RAPD-PCR using a single primer 1247 (5’-AAGAGCC
CGT-3’) [19] in the Gene Amp PCR system 9700 (Applied
Biosystems). The PCR mixture was made to a volume of 50µl containing 100 ng of genomic DNA, 200 µM each dNTP,
30 pmol of primer, 5 µl of 10X PCR buffer, 3 mM MgCl２
and 2.0 U of Taq DNA polymerase (Takara). After a hot

start at 80°
C for 5 minutes, the DNA was subjected to 35 cycles of denaturing at 94°
C for 1 minute, annealing at 40°
Cfor 1 minute, and extension at 72°
C for 2 minutes. A final
extension step was done for 10 minutes at 72°
C. The ethidium bromide stained gels were digitised for comparison andto ascertain the clonal relationship between isolates.
Statistical analysis
To determine the statistical significance of diarrhoea
among STEC infected patients in different age groups, wetested the data with Chi-square using 2x2 table in EpiInfo
2000, and Fischer Exact test was done to obtain the significance (p) value.

RESULTSClinical manifestation
No apparent predilection in the incidence of STEC wasobserved between patients of different age groups fromwhom stx-harbouring E. coli were isolated. When patients
excreting stx-harbouring E. coli were stratified by age, we
observed that watery diarrhoea was more common in the <5year age group as compared to the >5 year of age group (p=0.07). Though not significant, a similar trend was observedfor vomiting and degree of dehydration (Table 2). Patientsfound to be carrying stx-harbouring E. coli were not coinfected with other enteric pathogens.

１
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Table 3. Genotypic and phenotypic characteristics of the STEC strains isolated from hospitalized
diarrhoea patients.
StrainSerotype
stx1

J1
J2
J3
J4
J5
J6
J7
J8
J9
J10
J11
J12
J13
J14
J15
J16
J17
J18
J19
J20

O36a
O127a
O6
O63
O146
O78
ONT
O44
O44
O8
Rough
O27
O20
ONT
O25
ONT
O44
O167
O15
ONT

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Virulence gene
Chromosomal
Plasmidstx2
eae
saa hlyA katP etpD
+
-

+
+
-

-

-

-

-

Phenotypic characteristics
SFa

Antibiogram

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

AChFxKTCCiCoNaSN
AChFxCoNa
AChFxTCCoNaS
AChCfTCoNaS
AChGCfFxKTCiCoNa
AChGFxTCoNaS
ACh
AChCfTCCoNa
AChTCoNaS
AChCfTCoNaS
AChCfTCCoNa
AChCfCCoNa
AChCfTCCiNa
AChCfTCCiCoNa
AChCfKCoNa
AChTN
FxTCoS
AChTCoNa
AChTCoSasorbitol fermentative
Abbreviations: A, ampicillin; Ch, cephalothin; G, gentamicin, Cf, ciprofloxacin, Fx, furazolidone; K, kanamycin; T,
tetracycline; C, chloramphenicol; Ci, ceftriaxone; Co, co-trimoxazole; Na, nalidixic acid; S, streptomycin; N, neomycin.

Prevalence of STEC and characterization of the strains
During the study period, 775 faecal specimens were
examined from diarrhoeal patients of various age groups. E.
coli was cultured from 189 (24.4%) of the samples. Twenty
E. coli isolates were positive (10.6%) in the stx-PCR assay
indicating an overall incidence rate of 2.6%. All the stx harbouring strains were negative for O157 antiserum but belonged to 13 other O serogroups (Table 3). Among the 15
typable strains, O44 serogroup was common (20%) and 4
were not typable (ONT). All except 4 of the STEC strains
were found to ferment sorbitol on MacConkey agar (Table
3).
Drug susceptibility
Most of the tested strains were resistant to ampicillin
and cephalothin (90% each), co-trimoxazole (80%), tetracycline and nalidixic acid (75% each), ciprofloxacin (45%),
streptomycin (40%), chloramphenicol (35%) and furazolidone (30%). The strain J16 was susceptible to all thetested drugs (Table 3). None of the isolates was resistant toamikacin or norfloxacin. As shown in Table 3, there was nocommon resistance pattern among the 20 strains tested.

Vero cell assay
In the Vero cell cytotoxic assay, except for VTEC-3(positive control strain), none of the stx harbouring strains
exhibited the characteristic cytotoxicity.
Detection of virulence genes and molecular typing
In the multiplex PCR assay, we identified 20 E. coli
strains harbouring stx, 19 carrying the stx1 and one carrying both stx1 and stx2 (Table 3). To confirm the authenticityof the PCR products, Southern hybridization was performed
with DIG-labelled cloned probe from pKTN501. In addition, the amplified stx1 gene of representative strains wassequenced and the sequence showed 100% homology withpublished stx1 gene sequences. We identified two strains (J2, J7) that had the eae (Table 3). None of the 20 stxharbouring strains were positive for hlyA, kat, etpD or saa
genes in PCR assays.
The stx-harbouring isolates were characterized by
RAPD-PCR to analyse the genetic relatedness. All of thestrains showed multiple amplicons with fragment sizes
ranging from 0.5 to 6.0 kb. The RAPD profile of the STEC
strains showed heterogeneous banding patterns (Fig. 1).

１
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３

Fig. 1. RAPD-PCR results of stx1-harbouring E. coli isolates.
(M, Molecular weight marker (Kb); J1to J20 STEC strains isolated from the clinical diarrhoea patients).

DISCUSSION
To our knowledge, this is the first report on the isolation of E. coli strains harbouring stx from diarrhoeal patients in Bangladesh. STEC was not associated with diarrhoea in a previous study conducted in this region in 199192 [24]. This study revealed a low prevalence of non-O157
stx1-harbouring E. coli in Bangladesh, and is unlike the reports from Europe, Australia, Brazil and Argentina [25-28]
where a much higher incidence of non-O157 STEC was observed. However, the overall incidence of STEC is much
lower in developing countries as compared to industrializedcountries. The most frequent serotype observed in this study
was O44 (20%).
Multidrug resistance was frequent among STEC
strains encountered in this study, especially to ampicillin,
cephalothin, co-trimoxazole, tetracycline and nalidixic acid.
In this study, 45% of the STEC strains were resistant tociprofloxacin. Due to excessive use of this drug in recent
years, many enteric pathogens were reported to be resistantto ciprofloxacin in India [29-32]. Another interesting observation encountered in the present study is the incidence of ahigher number of ciprofloxacin-resistant than norfloxacinresistant STEC strains. The same trend was reported for V.
cholerae O1 [29] and Aeromonas spp. [32]. Even thoughciprofloxacin and norfloxacin are basically the same drug,
extensive use of the former and a difference in accumulation kinetics between the two are thought to account for thevariation in resistance pattern [29].
Analysis of virulence markers indicated that the majority of the non-O157 strains carried stx1, whereas the eae
gene was detected at a very low frequency. Non-O157
STEC strains harbouring stx1 but lacking stx2 have been reported earlier [2, 33]. The higher prevalence of E. coli with

stx1 genotype was also noted in a study on STEC in theneighbouring region, Kolkata, India [19]. In this study, only
two strains harboured the eae gene. An increasing numberof reports indicate that production of intimin is not essential
for the STEC-mediated pathogenesis [18, 34, 35]. One
study reported toxin expression by intimin-negative STEC
strains (O91:H21 isolates) using only 50% lethal dose in a
streptomycin-treated CD-1 mouse model [36]. A subset ofhuman STEC, such as stx1c-harbouring strains, generally
lacks the intimin-encoding eae gene [35, 37]. The factors
responsible for colonization by non-O157 strains lacking
the intimin are not well established, although many candidate adhesins have recently been reported [14, 38-41]. The
STEC strains carrying eae are generally considered to havehigher virulence for humans than those lacking the eae [42].
However, from the available reports [34,43] it is evident thatthe association between HUS and the absence of eae remains unclear.
Recently, Paton and Paton [14] described a novel
megaplasmid-encoded adhesin (saa) in human STEC lacking the eae gene. As most of the stx1-harbouring E. coli
strains were negative for eae in this study, we included thesaa in the investigation for plasmid-associated virulencemarkers along with other possible additional virulence traits
such as STEC haemolysin (hlyA), a bifunctional catalse
peroxidase (KatP ), and the etpD gene cluster, which encodes a type II secretion pathway [9, 10]. None of the tested
isolates produced the specific amplicons.
To confirm the authenticity of stx PCR results, we confirmed that the amplicons were specific for stx1 by Southern hybridisation assay with stx1-specific probes preparedfrom the plasmid pKTN501. The PCR products showed
positive signals in the hybridisation assay, confirming that
the PCR assay was specific for stx1. In a bid to further confirm the presence of stx1, we performed sequence analysisof the corresponding stx1 PCR product and observed 100%
homology to the published stx1 sequences in the database.
A paradoxical observation in this study was that noneof the stx harbouring strains demonstrated the production of
Stx in the Vero cell cytotoxicity assay. A similar observation
was made in a study on stx1 strains with low toxin expression isolated in Thailand [18]. It is a well established factthat the genes encoding the Stx is located in the genomes oftemperate bacteriophages [44] and that the phage encodestranscription factor essential to activation of Shiga toxin expression [45]. Recent findings suggest that the stx harbouring strains have weak or ineffective phage transcription factor leading to sub-optimal or no production of Stx [46]. It
appears that the role of Stx in causing watery diarrhoea is
non-essential as evidenced in an earlier study [47].
Our study has, for the first time in Bangladesh, demon-

１２
４

strated the existence of stx-harbouring E. coli. The role ofstx1-harbouring E. coli isolates, which lack eae, saa andother virulence markers, needs to be studied in further detail.
The lack of established virulence genes and the nonexpression of Stx in our set of E. coli strains make it difficult to establish their role in diarrhoea. Our observation ofclinical variations in different age groups could not be generalized due to the small sample size. However, the fact that
non-O157 STEC are present in the Bangladeshi setting underscore the need to conduct a well-designed study to determine the significance of STEC as a pathogen in this part of
the world.

ACKNOWLEDGEMENTS
This study was conducted at the ICDDR, B: Centre for
Health and Population Research with the support of The
Bill & Melinda Gates Foundation and the Government of
Bangladesh, and partly by Japan International Co-operation
Agency (JICA/NICED Project No: 054-1061-E.O). ICDDR,
B acknowledges, with gratitude, the contributions of The
Bill & Melinda Gates Foundation, the Government of
Bangladesh, and Japan International Co-operation Agency(JICA) to our research efforts.
We are indebted to Dr. S. K. Bhattacharya, Director,
National Institute of Cholera and Enteric Diseases, Kolkata,
India for allowing us to use the institutional facilities in performing the laboratory assays and to Mr. Mohammed Humayun Kabir, Clinical Sciences Division, ICDDR, B for assisting us in analysing the clinical data.

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Tropical Medicine and Health Vol. 33 No. 3, 2005, pp. 127-132
Copyright! 2005 by The Japanese Society of Tropical Medicine

１
２
７

REAL TIME ESTIMATION OF REPRODUCTION
NUMBERS BASED ON CASE NOTIFICATIONS- Effective reproduction number of primary pneumonic plague
HIROSHI NISHIURA１−２ and MASAYUKI KAKEHASHI１
Accepted 28, July, 2005
Abstract: To estimate the time-dependent transmission potential of primary pneumonic plague (PPP), we analyzed
historical records from six outbreaks. Based on contact investigation information (source of infection information)of three outbreaks, we generalized the probability density function of the serial interval with a Gamma distribution
using maximum likelihood estimations. Furthermore, we used a likelihood-based approach to estimate effective reproduction numbers at time t, Rt, incorporating records from the remaining three outbreaks by assuming independence within unknown contact networks. According to our estimates, the Rt of PPP during the initial phases of each
epidemic were roughly in the order of 1.3 (95% confidence interval (CI): 0.0-4.3) in Oakland, 1.4 (0.0-4.6) in Rangoon and 6.5 (0.0-16.0) in Ecuador. The expected values of Rt were shown to slightly exceed unity, even in latterstages. While declining trends in Rt were observed in Oakland and Ecuador, no such trend was observed in Rangoon. The findings suggest that the three outbreaks investigated could have been accompanied by demographic
stochasticity. The statistical usefulness of the transformation procedure, even with a small number of recorded
cases available, was demonstrated, and the expected responses to bioterrorism using Yersinia pestis were discussed.
Key words: Primary pneumonic plague; Yersinia pestis; Reproduction number; Maximum likelihood estimation

INTRODUCTION
Plague is a zoonosis whose etiological agent is
Yersinia pestis, a gram-negative bacillus. This bacterium isbelieved to have been the cause of three past pandemics inthe 6th, 14th, and 20th centuries (McGovern & Friedlander,1997). Despite the recent decline in annual incidence(World Health Organization, 2003), plague outbreaks as aresult of biological warfare remain a possibility (Inglesby etal., 2000). Intentional dissemination of plague would mostprobably implement an aerosolized form of Y. pestis, which
has been shown to produce the disease in nonhuman primates and to result in symptoms initially resembling thoseof other severe respiratory illnesses (Speck & Wolochow,1957). A plague epidemic following the use of Y. pestis as a
biological weapon would be caused by airborne transmission, leading to person-to-person transmission via respiratory droplets.
Primary pneumonic plague (PPP) is contracted natu-

rally by inhalation of airborne bacilli from cadavers or carcasses of animals, or more commonly through droplet infection from coughing (Pollitzer, 1954). Case fatality could
reach almost 100% without appropriate antibiotic treatment
within 24 hr after onset of symptoms (Wu, 1926). It is said
that Soviet scientists manufactured large quantities of Y.
pestis during the Cold War, allegedly engineering multidrug-resistant strains (Inglesby et al., 2000). These resistantstrains have also been found naturally (Galimand et al.,1997). The World Health Organization (WHO) stated that,in a worst-case scenario, 50kg of Y. pestis released as anaerosol over a city of five million could result in 150,000
cases of PPP, with 80,000--100,000 requiring hospitalization and 36,000 resulting in death (World Health Organization, 1970).
Since human susceptibility to plague is thought to beuniversal and since the efficacies of existing vaccines havenot been proven (Titball & Williamson, 2001), the introduction of aerosolized plague into the general population could

1 Graduate School of Health Sciences, Hiroshima University, JAPAN,
2 Department of Medical Biometry, Faculty of Medicine, Tübingen University, GERMANY
Correspondence to:
Masayuki Kakehashi
Graduate School of Health Sciences, Hiroshima University,
Kasumi1-2-3, Minamiku, Hiroshima, 734-8551, JAPAN
Tel: +81-(0)82-257-5350; Fax: +81-(0)82-257-5354
E-mail: nishiura.hiroshi@uni-tuebingen.de１２８

constitute a significant public health problem because of thetransmissibility of this bacterium. The greater the transmissibility of the bacteria the greater the potential for spread,thus the more intense and rapid the intervention strategies
must be to bring it under control. Although reasonable estimates of the basic reproduction number, R0, of a pneumonicplague, defined as the average number of secondary cases
per primary case, have been obtained on the basis of historical analyses (Gani & Leach, 2004), we still do not knowhow past epidemics were terminated and how the reproduction number varies with time. That is, values of R0 alone do
not allow a thorough interpretation and investigation of how
we should be preparing for potential future bioterrorist attacks. On the other hand, estimates of the effective reproduction number (Rt), defined as the reproduction number attime t with or without introduction of control measures anddepletion of susceptible individuals, allow elucidation of the
future course of an outbreak; typically, Rt < R0. The purpose of this study was to provide a rational assessment of
the transmissibility of PPP within a statistical framework by
referring to historical records.

MATERIALS AND METHODS
2.1. Subjects
To estimate transmissibility with time, represented by
the effective reproduction number (Rt; see Section 2.3), we
utilized historical records from outbreaks caused exclusively by PPP in the last century. Case records of PPP outbreaks caused by Y. pestis are limited, since most references
describe not only PPP but also bubonic, septicemic, andsecondary pneumonic plague outbreaks. In addition, thesource of inhalation of the bacillus can arise, not only fromhumans, but also from animals such as dogs, cats, and pigs
(Doll et al., 1994). To estimate the reproduction number of
PPP, we confined our investigation to reports that met thefollowing conditions: (i) must state that the outbreak wascaused by PPP only, confirmed clearly through epidemiologic, clinicopathological or bacteriological examinations,(ii) the location of the outbreak must not have experienced
bubonic plague within a relatively similar time frame,
thereby excluding the possibility of unrelated deaths andtransmission by bubonic, septicemic and secondary pneumonic plague, and (iii) contact patterns in at least some ofthe outbreaks must have been traced, giving the statistical
distribution of the serial interval (= generation interval;
Wallinga & Teunis, 2004), defined as the mean of the infectiousness distribution and representing the time from symptom onset in a primary case to symptom onset in a secondary case (Fraser et al., 2004). Historical records from a total of six outbreaks were examined. Three records included

complete or partly complete information on the sources ofinfection; these were related to outbreaks in Mukden (China,
1946; Tieh et al., 1948), Northwestern Madagascar (1957;
Brygoo & Gonon, 1958), and, more recently, Central Madagascar (1997; Ratsitorahina et al., 2000), and were analyzed
to extrapolate the statistical distribution of the serial interval.
The remaining three records were obtained from outbreaks
in Oakland (the United States, 1919; Kellogg, 1920), Ecuador (1939; Murdock, 1940) and Rangoon (Burma, 1946;
Wynne-Griffith, 1948) but were devoid of information regarding who infected whom; these were used to estimate
the reproduction number. In all outbreaks, the index case
was infected a certain distance from the location of the outbreak before the disease was introduced into the affected
community.
2.2. Estimations of the serial interval
Since information concerning background demography and the number of infectious contacts could not be obtained, and since the number of patients in each record wassmall, we used are a likelihood-based approach to estimate
the reproduction number presented in this paper. We obtained the serial interval through evaluations of the courseof the outbreaks (investigated chains of transmission) in
Mukden and NW and Central Madagascar using information on the time of onset of each recorded infected individual. In all three outbreaks, contact patterns were clearlytraced (Tieh et al., 1948; Brygoo & Gonon, 1958; Ratsitorahina et al., 2000). The final size of each outbreak, denotedby ‘n’ throughout this paper, was 39 cases in Mukden, 41 in
NW Madagascar, and 18 in Central Madagascar. Of the total 98 cases, 89 intervals from onset of primary to onset of
secondary cases were obtained, excluding cases without anassociated link with another case. Since the total number ofrecorded cases in each outbreak was too small for separate
estimations of the statistical distributions of each serial interval, an overall estimate was obtained by summing thethree settings.
We assumed that the serial interval, τ, follows typicalstandard distributions: Gamma, Weibull and log-normal distributions. The probability density function (pdf) of the
gamma distribution denoted by f (τ｜α, β) is given by:$# #!"%#!
% $&%!$
#!
$&
"
#%
!
% %
&
%
&
%#
"%&
#
where Γ(a) is the Gamma function represented by:$"%&# "! $#!""!$!$!

"

Here, α and 1/β represent shape and scale parameters, respectively, and are restricted to being positive real. On theother hand, pdf of Weibull and log-normal distributions, ω(τ｜γ, δ) and l (τ｜µ, σ２), are given by

１
２
９
$!!
$
$
('!#
+$
*
#
$
"
#%
$
*
"#
$ *
%

method theoretically assumes all probable transmission networks of who infected whom from pairs of cases. Here, forinstance, using the serial interval with a gamma distribution,
the time between onset of cases ‘i’ and ‘j’ is denoted by ti−
tj, and the probability of transmission is given by f (ti−tj). It
is assumed that q cases out of the total number of cases, n,were infected outside the community investigated. The vector ν denotes the structure of the transmission network
while ν(i) represents the i th element of ν in which the primary case infected case ‘i’. Consequently, the likelihood of
observing epidemic curve t, with θ (population parameter)
representing the serial interval and ν for the network, is:

!

and
"
%
&*
!%%
$
! $
('#!
$
"
"
"
)
')
*
)"
where γ and δ are called shape and scale parameter, respectively, while µ and σ２ are mean and variance. Numbers ofcases in each outbreak were stratified according to the serialinterval [days]. Parameter estimates were obtained by minimizing the negative logarithm of the likelihood function using a standard numerical algorithm (maximum likelihood
estimation). Finally, quantile plot was used to determine thebest fits of distribution: the goodness-of-fit was assessedvisually since the standard goodness-of-fit test statistic forthe standard distribution without fixing parameters is unavailable. For instance, a quantile plot, with the selectedgamma quantiles on the vertical axis and variable values on
the horizontal axis, was drawn to confirm the validity of theselected model (gamma distribution).

%"
)"%
"
(*
$#

&"*!,
&"!

Further details of this concept are given in Wallinga and Teunis (2004). The relative likelihood that case ‘l ’ has been
infected by case ‘m’ is consequently given by:+$(")%"'"*!,'"!

where Bi[ ] denotes the binomial distribution. Estimates arethus obtained without any unrealistic assumptions, exceptthat all probable contacts are taken into account by assuming the independence.

0.3
Observed
Fitted (gamma distribution)
Gamma Quantile

Frequency

0.2
0.15
0.1
0.05
0
8
12
serial interval [days]%#." % !&+'$'"(%(b)

4$

and finally, the reproduction number is given by:

a)

0

(&
"!
$$(")%"
!
"
(%
) &
%.
$(!.
" -"*
(&
"!
$"
!
"
(%
% %.
-&
$(!.
-"!"
-#
"(

2.3. Estimations of reproduction number
Using the obtained distribution of the serial interval
and the remaining three outbreak records, which included
information on the date of onset but were devoid of data regarding who infected whom, estimations of Rt at time twere performed. The usefulness of a recently proposed statistical method allowing reasonable real-time transformation of epidemic curves with the reproduction number overtime was investigated (Wallinga & Teunis, 2004). This

0.25

#

("
!
(&
" & %.
"!
""
!
!&!.
"
&&
$%&

16

20

14
13
12
11
10
9
8
7
6
5
4
3
2
0

5

10

15

20

Serial Interval

Figure 1. a) Estimated serial intervals of known transmissions of primary pneumonic plague in Mukden (1946), NW
Madagascar (1957) and Madagascar (1997). The serial interval is the time from onset of symptoms in an index case to
onset of symptoms in a secondary case (also called the generation time / interval). The vertical axis shows the frequency (probability density). b) The quantile plot with gamma quantiles on the vertical axis. The diagonal referenceline shows the line of fit while the two dashed lines denote confidence limits of 95% equal precision bounds with a=0.001 and b=0.99 (Meeker and Escobar, 1998)).

１
３
０

RESULTS3.1. The serial interval
The median serial interval of the three outbreaks was
5.0 days with a standard deviation (SD) of 2.1 (Fig. 1a). Although the serial interval at initial stages of an outbreaktends to be longer than that at latter stages (Lipsitch et al.,2003), we ignored this tendency due to the scarcity of recorded subjects. Maximum likelihood estimates of the
shape and scale parameters of the gamma distribution were
6.3 and 0.8, respectively, and the skewness and kurtosis
were 2.3 and 14.3, respectively. Weibull and log-normal distributions were not selected as the best fit since the quantile
plots were more typically ‘snake-shaped’ than those of
gamma distribution. The gamma distribution with these parameters appeared to adequately describe the data; the majority of the data points and the quantiles for the hypothesized gamma distribution were relatively close (Fig. 1b).

Cases
5

20

b)

7
6
5
4
3
2
1
0

25

0

d)

100
Reproduction number

10
15Time [days]

Expected value

10

5

10
15
Time [days]

20

25

e)

7
Upper 95% CI

The outbreaks studied here met our original requirements of being caused by PPP only. Despite problems withthe limited total number of cases in selected records, thesimple statistical methods gave reasonable estimations ofboth the serial interval and reproduction number. For the
purpose of these estimations, we used the probability density function of the serial interval obtained from three different outbreaks, giving a median value of 5.0 days. Even
though demographic information was lacking, the overall
range of expected Rt values in initial stages was determined

6

Reproduction number

0

DISCUSSION

Cases

a)

7
6
5
4
3
2
1
0

Reproduction number

Cases

3.2. Reproduction number
Comparative representations of epidemic curves andestimated reproduction numbers of each outbreak are shownin Fig. 2. The final sizes of each outbreak, ‘n’, were 13
cases in Oakland, 13 in Rangoon and 18 in Ecuador. Each
was terminated almost 1 month after introduction of an index case. The observed patterns of spread did not show par-ticularly propagated (person-to-person) epidemic curves;
rather they qualitatively showed intermittent-like patterns(Dwyer & Groves, 2003). The effective reproduction numbers, Rt, of PPP at the initial phase of each epidemic wereroughly in the order of 1.3 (95% confidence interval (CI):
0.0-4.3) in Oakland, 1.4 (0.0-4.6) in Rangoon and 6.5 (0.016.0) in Ecuador. In these three outbreaks, the expected values of Rt slightly exceeded unity even in latter stages. Dueto the scarcity of recorded cases, the 95% CI were rather
wide. A qualitatively declining trend in Rt was seen in Oakland and Ecuador but not in Rangoon. In the former two
outbreaks, the upper 95% CI also decreased, but not below
unity in latter stages.

5
4
3
2
1
0

1
0

5

10
15
20
Time [days]

25

c)

7
6
5
4
3
2
1
0
0

5

100

f)

10

15 20 25
Time [days]

30

35

15 20 25 30
Time [days]

35

10

1
0

5

10
15
Time [days]

20

25

0

5

10

Figure 2. Epidemic curves (numbers of cases according to the date of onset) for primary pneumonic plague outbreaksin a) Oakland (1919), b) Rangoon (1946), and c) Ecuador (1939), and corresponding effective reproduction numbers(Rt) (numbers of secondary infections generated per primary case according to the date of onset) in d) Oakland, e)Rangoon, and f) Ecuador. Dots show expected values while the top of the bar charts denotes the upper 95% confidence
interval (CI). The horizontal axis represents the lower 95% CI. The solid horizontal line in (e) indicates Rt=1, and thevertical axis in (d) and (f) is logarithmic.

１
３
１

as being in the order of 1.3-6.5. On the other hand, crude
and wide 95% CI of Rt was obtained on the basis of the assumption of binomial distributions for the relative likelihood.
Although it might not be possible to demonstrate theexact estimates of Rt with better precision, the estimates andrange of Rt obtained provide important insights. Our estimations revealed that the expected values of Rt in the three analyzed outbreaks did not decline below unity even when very
close to extinction. Actually, in most regions, except Mukden (the records of which were used only for estimating serial interval) where interventions were carried out, outbreaks came to an end before control measures were implemented. Thus, the findings suggest that the outbreaks investigated could have been accompanied by certain demographic stochasticity (Iwasa & Mochizuki, 1988). However,
the impact of extrinsic factors (i.e., public health intervention) on the trends remained unclear.
A study of notorious outbreaks in China (Pollitzer &Li, 1943) suggested the reason for the end of the Mukden
outbreak; that is, PPP is generally not as infectious as measles or influenza. It has also been frequently suggested,even before the mid-20th century, that transmission of PPP
resulted in many cases from close or actual contact with aninfected person (Meyer, 1961). Among our subjects, however, depletion of the number of susceptibles due to the rural nature of the outbreak locations is also highly plausible,
and this factor, together with stochastic effects, could havebrought these outbreaks to an end. This may also help to explain why outbreaks tended to continue even after control
measures were introduced in highly populated areas in
China (International Plague Conference, 1912). These complicated circumstances have made it difficult to interpretprevious outbreaks in detail, and the transmission potentialof PPP has thus remained largely unknown (Wright, 1995).
Even though it is not possible to draw definite conclusions
from our results, we suggest that some outbreaks end as aresult of stochastic effects as well as intrinsic factors (depletion of susceptibles), especially in rural areas where mostoutbreaks occur.
The advantages and limitations of the estimation
method used should be addressed. This study demonstrated
the statistical usefulness of the real-time transformation procedure, even in settings where the number of cases is limited. Previous methodologies for estimation of reproduction
number have frequently assumed simple exponential growth
without proper statistical validation, generating statisticallyflawed results (Donnelly et al., 2004). Assessments within
an autonomous system, which lack time-dependent information, have therefore not been helpful in drawing quantitative conclusions with real life data (Diekmann & Heester-beek, 2000). The dangers of intuitively fitting exponential
growth curves to cumulative case numbers have alreadybeen argued (Razum et al., 2003), and such technical flaws,
due to assumption of constant exponential growth despite asignificantly different qualitative pattern, were our primaryconcern in this study. Despite considerations of the individual variability of transmission potential and of stochasticity
in the simulations, recent literature documenting estimations of the R0 of PPP have been accompanied by the above
flaw (Gani & Leach, 2004). Our study was improved byshowing variation in the reproduction number with time.
However, as urged with analyses of SARS, it should be
noted that conditions of independence, as described in themethods section, and variations in the serial interval withtime could be limiting factors (Lipsitch & Bergstrom, 2004).
In addition, it is imperative that the disease be categorizedby combining the records of different outbreaks. Since thestrain of Y. pestis varies with time and place, not only the
degree of infectivity of the bacilli but also the serial interval(approximated by the sum of incubation and symptomatic
periods) will differ. Because of biological and socioenvironmental differences, it is obvious that local characteristics of transmission can vary largely and may show overly
specific originalities. Insightful investigations on this point
are a subject for our furher study. Finally, although reconstructed epidemic trees enable us to estimate the Rt directly,
this is possible only when “who infected whom” is observed completely or parly known. We intend to discuss thisissue in a later paper (Nishiura, Schwehm, Kakehashi &Eichner, Submitted).
It is believed that the Japanese army attempted to drop
plague-infected fleas over populated areas as a biologicalweapon during World War II (Harris, 1994). Taking into account its availability and stability compared to other viral
agents, PPP is one of the most likely threats in a bioterrorist
attack. Though extinction occurred in all outbreaks observed here, a large-scale initial attack in a highly populatedarea will not offer demographic stochasticity. Considering
the extremely high case fatality that occurs without rapid
treatment (Wu, 1926) and the unavailability of immunization (Titball & Williamson, 2001), preparation of a publichealth response is of outstanding imporance. To overcome
the fear of bioterrorism using Y. pestis, both statistical andmathematical studies should be implemented to promote thedevelopment of a response program.

ACKNOWLEDGEMENTS
We thank Dr. Shigeki Hanafusa of Mahidol University
for collating reference 31 during the preparation of thisstudy. Thanks are also due to Profs Klaus Dietz and Martin

１３２

Eichner for their useful discussions, and the libraries at
Chulalongkorn University, London School of Hygiene andTropical Medicine, the Wellcome Trust Foundation, and theBritish Library for assisting with collection of historical records. This study is supported by Banyu Fellowship Program sponsored by Banyu Life Science Foundation International.

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pulmonaire dans le nord-est de Madagascar. Bull. Soc. Pathol. Exot., 51, 47-60. (in French)
２．Diekmann, O., Heesterbeek, J.A.P. (2000): Mathematicalepidemiology of infectious diseases: Model building,analysis and interpretation. New York, Wiley.
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T., Gage, K. (1994): Cat-transmitted fatal pneumonicplague in a person who traveled from Colorado to Arizona.
Am. J. Trop. Med. Hyg., 51, 109-114.
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S., Ferguson, N.M., Anderson, R.M. (2004): Epidemiological and genetic analysis of severe acute respiratory syndrome. Lancet Infect. Dis., 4, 672-683.
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B., Chanteau, S., Carniel, E., Courvalin, P. (1997): Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. N. Eng. J. Med. 337, 677-680.
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９．Harris, S.H. (1994): Factories of Death. New York: Routledge, 78.
１０．Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.
G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M.,
Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm,
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Schoch-Spana, M., Tonat, K. (2000): Plague as a biologicalweapon: Medical and public health management. J. Am.
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extinction accompanying a temporary decrease of population size. Res. Popul. Ecol. 30, 145-164.
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M.H., Fisman, D., Murray, M. (2003): Transmission dynamics and control of severe acute respiratory syndrome.
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Plague. In: Siedell, F.R., Takafuji, E.T., Franz, D.R. (eds)
Medical Aspects of Chemical and Biological Warfare.
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７．Meeker, W.Q., Escobar, L.A. (1998): Statistical Methods
for Reliability Data. New York: John Wiley and Sons, 6062.
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８．Meyer, K.F. (1961): Pneumonic plague. Bacteriol. Rev., 25,249-261.
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２０．Pollitzer, R. (1954): Plague. Geneva: World Health Organization, 471-521.
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１．Pollitzer, R., Li, C.C. (1943): Some observations on the decline of pneumonic plague epidemics. J. Infect. Dis., 72,
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bubonic and pneumonic plague. Vaccine 19, 4175-4184.
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７．Wallinga, J., Teunis, P. (2004): Different epidemic curves
for severe acute respiratory syndrome reveal similar impacts of control measures. Am. J. Epidemiol., 160, 510-516.
２
８．World Health Organization. (1970): Health Aspects ofChemical and Biological Weapons. Geneva: World HealthOrganization, 98-109.
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９．World Health Organization. (2003): Human plague in 2000
and 2001. Wkly. Epidemiol. Rec. 78, 130-136.
３
０．Wright, F. J. (1995): Infectivity of pneumonic plague. Br.
Med. J., 310, 261.
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１．Wu, L-T. (1926): A Treatise on Pneumonic Plague. Geneva:
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３２．Wynne-Griffith, G. (1948): Pneumonic plague in Rangoon.
Lancet, i, 625-627.

Tropical Medicine and Health Vol. 33 No. 3, 2005, pp. 133-141
Copyright! 2005 by The Japanese Society of Tropical Medicine

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TWO NEW SPECIES OF SIMULIUM (NEVERMANNIA)(DIPTERA: SIMULIIDAE) FROM NORTHERN THAILAND
HIROYUKI TAKAOKA１ and WEJ CHOOCHOTE２
Accepted 12, August, 2005

Abstract: Two new species of black flies, Simulium (Nevermannia) fruticosum sp. nov. and S . (N .) chiangklangense sp. nov. are described on the basis of samples collected in northern Thailand. These new species are assigned
to the feuerborni species-group of the subgenus Simulium (Nevermannia), and are easily distinguished from S .
(N .) feuerborni Edwards, the only species of the same group known thus far in Thailand, by their simple cocoon
without any anterodorsal projection.
Key words: Simulium, black fly, Simuliidae, Thailand, new species, Nevermannia.

In Thailand, the feuerborni species-group within thesubgenus Simulium (Nevermannia) Enderlein redefined by
Takaoka [1] is represented by only one species, S . (N .)feuerborni Edwards, which was originally described fromJava [2] and later recorded from Peninsular Malaysia [3],Thailand [4] and Sumatra [5]. We collected two more species of the feuerborni species-group, which are easily distinguished from S . (N .) feuerborni by their simple cocoon.
These are described here as new species.
The terms for morphological features used here follow
those of Takaoka [1]. Holotype and paratype specimens of
the new species are deposited at the Department of Infectious Disease Control, Oita University, Oita, Japan.
Simulium (Nevermannia) fruticosum sp. nov.
DESCRIPTION. Female. Body length 2.4―3.0 mm. Head.
Narrower than thorax. Frons brownish-black, not shiny,
thinly whitish-grey pruinose, densely covered with whitishyellow recumbent hairs, interspersed with several dark
longer and stouter hairs along each lateral margin. Frontal
ratio 1.3―1.7 : 1.0 : 2.0―2.5. Frons-head ratio 1.0 : 4.8―5.2.
Fronto-ocular area (Fig. 1A) well developed, triangular, directed somewhat upward. Clypeus brownish-black, notshiny, whitish-grey pruinose, densely covered with whitishyellow recumbent hairs (except portion near upper marginbare), intermixed with several dark longer and stouter hairs
on each side of lower 1/2. Labrum nearly as long as clypeus.
Antenna composed of 2+9 segments, medium to darkbrown, except scape, pedicel, and minute base of 1st flagel-lar segment yellow or light yellowish-brown; 1st flagellar
segment 2.0―2.2 times as long as 2nd one. Maxillary palp
consisting of 5 segments, medium to dark brown, proportional lengths of 3rd, 4th, and 5th segments 1.0 : 0.7―0.8 :1.3―1.5; 3rd segment (Fig. 1B) much enlarged, with sensory
vesicle elongate, 0.58―0.66 times as long as 3rd segment,with medium-sized opening medially. Maxillary laciniawith 8 or 9 inner teeth and 12―14 outer ones. Mandible with
20―22 inner teeth but lacking outer teeth. Cibarium (Fig. 1
C) smooth on posterior margin, with well sclerotized arms
directed anterolaterally. Thorax. Scutum light to darkbrown (except anterolateral calli ocherous) though somewhat darker along lateral margins and on prescutellar area,
shiny at certain angles of light, thinly whitish-grey pruinose,with 3 faint dark narrow longitudinal vittae (1 medial and 2
submedial), densely covered with whitish-yellow (golden inlight) recumbent hairs, and with several dark brown uprighthairs on prescutellar area. Scutellum ocherous, with many
dark upright hairs as well as whitish-yellow shorter hairs.
Postnotum medium to dark brown, thinly whitish-grey pruinose, slightly shiny at certain angles of light, bare. Pleural
membrane bare. Katepisternum longer than deep, medium
brown, and bare. Legs. Foreleg: coxa and trochanter
whitish-yellow; femur yellow to dark yellow with apicalcap medium brown; tibia medium to dark brown with median large portion of outer surface light brown; tarsus
brownish-black; basitarsus slightly dilated, 7.7 times aslong as its greatest width. Midleg: coxa light brown withposterior surface dark brown; trochanter yellow; femur yellow to dark yellow, with apical cap medium brown; tibia

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

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Fig. 1. Female of Simulium (Nevermannia) fruticosum sp. nov. A, fronto-ocular area (right side); B, 3rdsegment of maxillary palp with sensory vesicle (left side, front view); C, cibarium; D, basitarsus andsecond tarsal segment of hind leg showing calcipala and pedisulcus (left side, outer view); E, claw;
F, 8th sternite and ovipositor valves in situ (ventral view); G, genital fork (ventral view); H and I,
paraprocts and cerci (right side; H, ventral view; I, outer view); J, spermatheca. Scale bars. 0.1 mm
for D; 0.03 mm for A and B; 0.02 mm for F―J; 0.01 mm for E.

medium brown with apical cap dark brown and medianlarge portion on outer and inner surfaces light brown; tarsus
brownish-black. Hind leg: coxa dark yellow or light brown;
trochanter yellow; femur yellow to dark yellow with apicalcap medium to dark brown; tibia medium brown with apicalcap dark brown and median large portion on outer and innersurfaces light brown; tarsus medium to dark brown except
basal 1/2 (or a little more) of basitarsus and basal 1/2 of 2nd
segment dark yellow to light brown (though base of basitarsus medium brown); basitarsus (Fig. 1D) nearly parallelsided on basal 1/2, then slightly narrowed toward apex, 6.1
times as long as wide, and 0.77 times and 0.63 times aswide as hind tibia and femur, respectively; calcipala (Fig. 1D) well developed, about 1.1 times as long as wide, andabout half as wide as greatest width of basitarsus; pedisulcus (Fig. 1D) well developed. Claws (Fig. 1E) each withlarge basal tooth half as long as claw. Wing. Length 2.4―

2.6 mm. Costa with 2 parallel rows of dark short spines aswell as dark hairs except subbasal portion of costa near humeral cross vein with whitish-yellow hairs. Subcosta withdark hairs except near apex bare. Basal portion of radius
fully haired; R1 with dark spinules and hairs; R2 with darkhairs. Hair tuft on stem vein dark brown. Basal cell absent.
Abdomen. Basal scale light to medium brown, with fringeof pale long hairs. Dorsal surface of abdomen medium todark brown except that of segment 2 whitish-yellow orocherous to light brown (though tergal plate light to medium brown), moderately covered with whitish-yellow shorthairs, interspersed with dark ones; tergite 5 slightly shiny atcertain angle of light, tergites 6―9 shiny when illuminated;ventral surfaces of abdominal segments 2―6 whitish-yellow;
segment 7 with large sternal plate medially. Genitalia.
Sternite 8 (Fig. 1F) wide, bare medially but furnished with
18―43 short and long hairs on each side. Ovipositor valves

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Fig. 2. Male of Simulium (Nevermannia) fruticosum sp. nov. A, 3rd segment of maxillary palp with sensory
vesicle (right side, front view); B, hind basitarsus (left side, outer view); C, coxites, styles and ventral plate in situ (ventral view); D, coxite and style (right side; outer view); E and F, styles (rightside; E, ventrolateral view; F, end view); G, ventral plate (end view); H, ventral plate and mediansclerite (lateral view); I, paramere (right side; end view); J, median sclerite; K, dorsal plate; L and M,
10th abdominal segments with cercus (right side; L, end view; M, outer view). Scale bars. 0.1 mm
for B; 0.02 mm for A, C―M.

(Fig. 1F) triangular, thin, membraneous except inner margin
narrowly sclerotized, densely covered with microsetae, interspersed with 5―7 short setae; inner margins gently sinuous and narrowly separated medially from each other. Genital fork (Fig. 1G) of inverted Y-form, with well sclerotizedstem and wide arms; each arm with wide round lobe-likeprojection directed medioposteriorly and prominent projection directed forward. Paraproct (Fig. 1H, I) of usual form,
only slightly protruding ventrally, with 24―30 hairs on ventral and lateral surfaces, and with 5―8 sensilla on anteroinner surface. Cercus in lateral view (Fig. 1I) rounded posteriorly, 0.5―0.7 times as long as paraproct. Spermatheca (Fig.
1J) ovoidal, about 1.2 times as long as its greatest width,strongly sclerotized except small area around juncture toduct, and duct itself unsclerotized, with distinct reticulate
surface pattern, and without internal setae; main spermathecal duct narrow, while both accessory ducts slightly wider
than main duct.
Male. Body length 2.6―3.2 mm. Head. Slightly wider
than thorax. Holoptic; upper eye consisting of large facetsin 14 or 15 vertical columns and 17 or 18 horizontal rows.
Clypeus dark brown, not shiny, whitish-grey pruinose, moderately covered with yellow short and long hairs interspersed with dark long hairs. Antenna composed of 2+9
segments, light brown except base of 1st flagellar segment
pale yellow, or dark brown except scape and most of pedicel

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dark yellow and base of 1st flagellar segment pale yellow;
1st flagellar segment elongate, about twice as long as 2nd
one. Maxillary palp medium to dark brown, composed of 5
segments, proportional lengths of 3rd, 4th, and 5th segments 1.0:1.0―1.2:1.7―2.1; 3rd segment (Fig. 2A) of moderate size; sensory vesicle (Fig. 2A) small, globular or ellipsoidal, 0.22 times as long as 3rd segment. Thorax. Nearly
as in female except anterolateral calli light brown and postnotum light to dark brown. Legs. Foreleg: coxa and trochanter yellow; femur light brown with apical cap medium
brown (though most of inner surface lighter); tibia darkbrown to brownish-black with median large portion of outersurface medium brown; tarsus brownish-black; basitarsus
very slightly dilated, 10 times as long as its greatest width.
Midleg: coxa medium brown with posterior surface darkbrown; trochanter yellow; femur yellow to dark yellow,
with apical cap medium brown; tibia dark brown tobrownish-black with base and subbasal portion medium
brown; tarsus brownish-black. Hind leg: coxa light brown;
trochanter yellow; femur yellow to dark yellow with apicalcap dark brown; tibia dark brown to brownish-black withbase dark yellow and subbasal portion light to medium
brown; tarsus dark brown except basal 1/2 of basitarsus medium brown though border not well defined; basitarsus (Fig.
2B) enlarged, spindle-shaped, 4.2 times as long as its greatest width, and 1.04 times and 0.96 times as wide as greatest
widths of hind tibia and femur, respectively; calcipala (Fig.
2B) well developed, nearly as long as wide, and 0.35 times
as wide as greatest width of basitarsus; pedisulcus (Fig. 2B)
well developed. Wing. As in female except subcosta bareor with 1 or 2 hairs; length 2.5―2.6 mm. Abdomen. Basal
scale dark brown, with fringe of yellow long hairs. Dorsal
surfaces of abdominal segments medium to dark brown (except that of segment 2 pale yellow though tergal plate medium to dark brown), not shiny, covered with dark simple
hairs; ventral surfaces of abdominal segments 2―4 yellow
though sternites of segments 3 and 4 medium brown; ventral surfaces of segments 5―8 light to medium brown. Genitalia. Coxite in ventral view (Fig. 2C) subquadrate much
longer than wide. Style in ventral view (Fig. 2C) short, 0.7
times as long as coxite, bent inwardly, nearly parallel-sided,with stout spine apically; style in ventrolateral view (Fig. 2E) broad, nearly parallel-sided from base to a little beyond
middle, then abruptly tapered apically; style in end view(Fig. 2F) tapered inwardly. Ventral plate in ventral view(Fig. 2C) lamellate, much shorter than wide, well sclerotized, with 3 concavities on posterior margin, and moderately covered with fine short setae on ventral surface (except lateral portions partially setose); arm of moderate
length, slender, and curved inwardly; ventral plate in end
view (Fig. 2G) with fine short setae centrally on posterior

surface; ventral plate in lateral view (Fig. 2H) with ventral
margin of body nearly straight, and arm directed dorsallyand forwardly. Paramere (Fig. 2I) with 6 or 7 hooks of different sizes. Median sclerite in end view (Fig. 2J) simple,
club-shaped, narrow, slightly widened toward apex. Aedeagal membrane moderately covered with spinous microsetae,
moderately sclerotized basally forming dorsal plate (Fig. 2K). Ventral surface of 10th segment without any hairs neareach posterolateral corner. Cercus (Fig. 2L,M) small,rounded and with 9―11 simple hairs.
Pupa. Body length 3.0―3.5 mm. Head. Integument
dark yellow to light brown, densely covered with round tubercles; antennal sheaths bare; frons with 2 short slender
simple trichomes (Fig. 3A) on each side; face with1long
simple trichome (Fig. 3B) (a little over 3 times as long as
frontal trichomes) on each side. Thorax. Integument dark
yellow to light brown, moderately or densely covered withround tubercles, with 3 very long slender simple trichomes
with coiled apex (Fig. 3C) mediodorsally, 2 slender simple
trichomes (1 very long, and 1 short) (Fig. 3D) mediolaterally, 1 long slender simple trichome (Fig. 3E) posterolaterally, and 3 short slender simple trichomes (Fig. 3F) ventrolaterally, on each side. Gill with 6 long thread-like slenderfilaments arranged in 2 groups (1 ventral group consistingof paired filaments with long stalk, and the other dorsalgroup consisting of 4 filaments), arising from short basal
common stalk (Fig. 3G); 4 filaments of dorsal group usuallyarising close together, with their arrangement somewhat
variable, e.g., lying nearly side by side horizontally forming
middle pair with very short stalk and inner and outer individual filaments (Fig. 3H), or forming outer triplet plus1inner individual filament (Fig. 3I), or forming inner pair withno stalk and outer pair with very short stalk (Fig. 3J), or
forming inner and outer pairs with very short to short stalk(Fig. 3K) (as an exception, in 1 pupa stalk of inner pair of
dorsal group long on left side and very long on right side asshown in Fig. 3L,M); basal common stalk with transparent
bulb-like organ ventrally (Fig. 3G); all filaments darkbrown, directed forward, tapered apically, subequal inlength to one another (lengths from base of gill to tips offilaments variable from 3.8 to 4.8 mm depending upon individual pupae), and longer than pupal body; cuticular surface
with distinct annular ridges and furrows (though ridges becoming indistinct apically), and densely covered with minute tubercles of different sizes (larger ones on ridges andsmaller ones on interridges). Abdomen. Dorsal surfaces ofall segments weakly sclerotized, yellowish except medial
portions of segments 1 and 2 light brown, densely andelaborately covered with minute tubercles; segment 1 with 1
medium-long slender simple seta on each side; segment 2
with 1 medium-long slender simple seta and 5 short spinous

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Fig. 3. Pupa of Simulium (Nevermannia) fruticosum sp. nov. A, frontal trichomes; B, facial trichome; C―F,
trichomes on thorax (C, mediodorsal; D, mediolateral; E, posterolateral; F, ventrolateral); G, basal
portion of gill filaments (left side, outer view); H―L, basal portions of gill filaments (all dorsal view;
H and J, left side; I, K and L, right side); M, entire gill filaments showing very long stalk of inner
pair of dorsal group (dorsal view); N, terminal hook (lateral view); O, cocoon (dorsal view). Scalebars. 0.5 mm for O; 0.2 mm for M; 0.1 mm for G―L; 0.02 mm for A―F and N.

setae on each side; segments 3 and 4 each with 4 hooks and1 short spinous seta on each side; segment 5 bare or with 1
or 2 spine-combs on each side; segments 6―8 each withspine-combs directed backward in transverse row on eachside; segments 6―9 each with comb-like groups of minutespines on each side; segment 9 with a pair of distinct coneshaped terminal hooks curved posteroinwardly (Fig. 3N).
Lateral surfaces of segments 2―4 each with 3 spinous setae

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Fig. 4. Mature larva of Simulium (Nevermannia) fruticosum sp. nov. A, whole body (dorsal view); B, headcapsule (ventral view); C, mandible; D, mandible with supernumerary serration; E, hypostomium; F,
postgenal cleft. Scale bars. 1.0 mm for A; 0.1 mm for B and F; 0.02 mm for E; 0.01 mm for C and D.

on each side. Ventrally, segments 3―8 with comb-like
groups of minute spines; segments 5―8 each with a pair of
weakly-sclerotized light yellowish wide submedial sternal
plates, on which comb-like groups of minute spines present;
segment 4 nearly transparent, with 4 short slender simple
setae on each side; segment 5 with a pair of bifid hooks submedially and a few short slender setae on each side; segments 6 and 7 each with 1 bifid hook submedially and 1
simple hook laterally, and a few slender setae on each side.
Cocoon (Fig. 3O). Simple, wall-pocket-shaped, compactly
woven without open spaces in web, very thin, with anterior
margin thickly woven, and extending ventrolaterally; individual threads invisible; 3.5―4.5 mm long by 2.5―3.6 mm
wide.
Mature larva. Body length 6.2―6.5 mm. Body greyish
yellow or light tawny, with well-defined colored markings(Fig. 4A); i.e., abdominal segments 3 and 4 each with 3
reddish-brown spots on each side, of which 2 spots lying
submedially on dorsal surface (those spots very narrowly
connected to each other), and 1 lying medially on lateralsurface; abdominal segment 5 on each side with 1 reddishbrown spot submedially on anterior 1/2 of dorsal surface
and 1 transverse similar colored band along its posterior

margin; and abdominal segment 6 on each side with 1
reddish-brown spot submedially on anterior 1/2 of dorsal
surface. Cephalic apotome yellow except posterior margin
darkened, with distinct positive head spots; lateral surfaceof head capsule yellow, with distinct eyebrow containing
dark spot; 2 large spots behind eye-spot region and 3 isolated spots below eye-spot region distinctively positive;
ventral surface of head capsule (Fig. 4B) yellow (thoughpostgenal bridge mostly darkened, with dark basal area oneach side of postgenal cleft; horizontal and round spots oneach side of postgenal cleft distinctively positive. Cervicalsclerite composed of 2 small elliptical pieces, not fused toocciput, very widely separated medially from each other.
Antenna consisting of 3 segments and apical sensillum,
much longer than stem of labral fan; proportional lengths of
1st, 2nd, and 3rd segments 1.00 : 1.00―1.05 : 0.87―0.91.
Labral fan with 26―29 main rays. Mandible (Fig. 4C) withmandibular serrations consisting of 2 teeth (1 large and 1
small); large tooth making nearly a right angle with mandible on apical side; supernumerary serrations absent (though
1 minute supernumerary tooth present on right mandible of
1 larva as shown in Fig. 4D); comb-teeth composed of 3teeth, shortened from 1st to 3rd. Hypostomium (Fig. 4E)

１３
９with 9 apical teeth in row; median and corner teeth well developed; median tooth of 3 intermediate teeth on each sidesmallest; lateral serrations weakly developed apically; 6―9hypostomal bristles lying slightly divergent posteriorly fromlateral margin on each side. Postgenal cleft (Fig. 4B) small,
0.53―0.57 times as long as postgenal bridge, with anterior
margin roughly rounded or irregularly defined (Fig. 4F).
Thoracic cuticle bare. Abdominal cuticle bare except bothsides of anal sclerite moderately covered with simple colorless setae, and lateral surface of last segment down to middle of ventral papilla also sparsely covered with similar but
shorter setae. Rectal scales present. Rectal papilla compound, each of 3 lobes with 11 or 12 finger-like secondary
lobules. Anal sclerite of usual X-form, with anterior arms
slightly shorter than posterior ones, narrowly sclerotizedalong anterior arms; sensilla absent on and just posterior tobasal juncture area; accessory sclerite absent. Last abdominal segment much expanded ventally forming large ventral
papilla. Posterior circlet with about 86 rows of up to 16
hooklets per row.
TYPE SPECIMENS. Holotype female, reared from pupa,
collected from a small stream slowly flowing among thebushes in a densely forested, leech-rich area (width 50 cm,
depth ca. 2 cm, water temperature 25.0°
C, completely
shaded, altitude 1,420 m above sea level), Doi Pui, Chiang
Mai province, Thailand, 21. V. 2005, by W. Choochote.
Paratypes 4 females, 8 males, and 3 mature larvae, samedata and date as those of holotype; 1 male, same data as
those of holotype except date, 14. V. 2005; 1 pupa, samedata as those of holotype except date, 16. IX. 2004, andwater temperature 20.0°
C.

twisted style, several parameral hooks, and a simple narrow
median sclerite; pupal gill with six long thread-like filaments per side; and larval head with small short postgenal
cleft.
Among the 16 known species of the feuerborni species-group [1, 6, 7], four species, i.e., S . (N .) feuerborni, S .
(N .) leigongshanense Chen and Zhang from China, S . (N .)praelargum Datta from India, and S . (N .) sasai (Rubtsov)
from Japan, have a cocoon with a distinct anterodorsal projection and thus differ from this new species. Eight otherspecies of the same species-group have a simple cocoon but
differ from this new species in the arrangement of gill filaments. Interestingly, S . (N .) feuerborni has the gill filaments arranged in a similar manner to those of S . (N .) fruticosum (i.e., four dorsal filaments arising close together andlying almost side by side horizontally as shown in Fig. 3
H―K).
The remaining four known species of the feuerborni
species-group were described from adult males (and also females in one species) alone, their pupal and larval stages remaining unknown. Among these, S . (N .) fuscinervis Edwards recorded from Sabah [8] differs from the new species
by having the paramere with 10 or 11 hooks; S . (N .)bryopodium Delfinado, described from Palawan Island,
Philippines [9], also differs by the dark brown hind femora
and ventral plate much depressed posteriorly; S . (N .) senile
Brunetti, described from West Himalaya [10], is different
by the style with no apical spine; S . (N .) rufithorax Brunetti, described from a male and four females collectedfrom India [10], has a reddish-brown thorax according tothe original description.
Simulium (Nevermannia) chiangklangense sp. nov.

BIOLOGICAL NOTES. The pupae and larvae of this new
species were found on the surface of leaves and stalks oftrailing grasses. Associated species were S . (Gomphostilbia) inthanonense Takaoka and Suzuki, S . (Simulium) brevipar Takaoka and Davies, S . (S .) doipuiense Takaoka andChoochote, S . (S .) manooni Takaoka and Choochote, and S .
(S .) mediocololatum Takaoka and Choochote.
DISTRIBUTION. Thailand.
ETYMOLOGY. The species fruticosum refers to the stream
running among the bushes where this new species wasfound. The Latin adjective fruticosus means bushy.
REMARKS. Simulium (Nevermannia) fruticosum sp. nov.
is readily assigned to the feuerborni species-group by the
combination of the following characters: male genitalia
with a simple lamellate ventral plate, a short inwardly-DESCRIPTION. Female. Body length 2.5 mm. Nearly thesame as the female of S . (N .) fruticosum except for the following characteristics. Head. Frontal ratio 1.5:1.0:2.3.
Frons-head ratio 1.0 : 4.8. Fronto-ocular area (Fig. 5A)
somewhat shorter than that of S . (N .) fruticosum. Labrum
0.97 times as long as clypeus. Antenna with 1st flagellar
segment about 1.6 times as long as 2nd one. Maxillary palp
with proportional lengths of 3rd, 4th, and 5th segments 1.0:
1.0:1.6; 3rd segment (Fig. 5B) with elongate sensory vesicle
0.52 times as long as 3rd segment. Maxillary lacinia with 8
inner and 12 outer teeth. Mandible with 20 inner teeth but
lacking outer teeth. Legs. Fore basitarsus 7.4 times as longas its greatest width. Hind basitarsus 6.6 times as long as
wide, and 0.69 times and 0.61 times as wide as hind tibia
and femur, respectively; calcipala well developed, nearly as
long as wide, and 0.55 times as wide as greatest width ofbasitarsus. Claws (Fig. 5C) each with large basal tooth 0.43

１４
０Fig. 5. Female and pupa of Simulium (Nevermannia) chiangklangense sp. nov. A, fronto-ocular area (rightside); B, 3rd segment of maxillary palp with sensory vesicle (right side, front view); C, claw; D, 8thsternite and ovipositor valves in situ (ventral view); E, genital fork (ventral view); F and G, paraprocts and cerci (right side; F, ventral view; G, outer view); H, spermatheca; I, whole gill filaments(dorsal view); J, basal portion of gill filaments (outer view). Scale bars. 0.2 mm for I; 0.1 mm for J;
0.03 mm for A and B; 0.02 mm for D―H; 0.01 mm for C.

times as long as claw. Wing. Length 2.6 mm. Genitalia.
Sternite 8 (Fig. 5D) wide, bare medially but furnished with
30―40 short and long hairs on each side. Ovipositor valves(Fig. 5D) with 9 or 10 short setae; inner margins gently undulate. Genital fork (Fig. 5E) with longer and narrower projection directed anteriorly on each arm than that of S . (N .)fruticosum. Paraproct in ventral view (Fig. 5F) somewhat
wider than that of S . (N .) fruticosum; its anteroinner surface with 16 or 17 sensilla. Cercus in lateral view (Fig. 5G)
about half as long as paraproct. Spermatheca (Fig. 5H)
ovoidal, 1.3 times as long as its greatest width.
Male. Unknown.

１
４
１

Pupa. Body length 3.5 mm. Nearly the same as thepupa of S . (N .) fruticosum except for the following characteristics. Head. Integument moderately covered with roundtubercles. Thorax. Gill (Fig. 5I,J) with 6 long thread-likeslender filaments (length including basal common stalk
4.0―4.5 mm), of which 2 inner filaments arising independently, and 4 others arranged in 2 short-stalked pairs (1 ventroouter and 1 dorsal), all arising nearly at the same levelfrom short basal common stalk; all filaments somewhat diverged basally from one another, then directed forwards; allfilaments dark brown, tapered apically, subequal in lengthand thickness to one another, and longer than pupal body.
Abdomen. Dorsal surfaces of all segments uniformly yellowish; minute tubercles on surfaces of segments 5―9 indistinct. Cocoon. 5.0 mm long by 3.2 mm wide.
Mature larva. Unknown.

anteriorly from each arm of the genital fork (Fig. 5E), andalso from S . (N .) fruticosum by the shorter claw tooth (Fig.
5C).
There is a possibility that S . (N .) chiangklangense isconspecific to one of the three known species of the feuerborni species-group which were described from adult males
alone (i.e., S . (N .) fuscinervis, S . (N .) bryopodium, and S .
(N .) senile).

TYPE SPECIMEN. Holotype female, reared from pupa,
collected from a slow-flowing stream (width 0.3―0.5 m,
C, partially shaded,depth 2―3 cm, water temperature 15.0°
altitude 790 m above sea level) in a forested area, Chiang
Klang, Nan province, northern Thailand, 2.XII. 2004, by W.
Choochote.

1. Takaoka H. The Black Flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. 2003; xxii+581 pp., Kyushu University Press, Fukuoka.
2. Edwards FW. The Simuliidae (Diptera) of Java and Sumatra. Arch Hydrobiol 13 Suppl “Tropische Binnengewässer”
1934; 5: 92―138.
3. Takaoka H and Davies DM. The black flies (Diptera: Simuliidae) of West Malaysia. 1995; viii+175 pp., Kyushu
University Press, Fukuoka.
4. Kuvangkadilok C, Boonkemtong C, Phayuhasena S. Cbanding in polytene chromosomes of six Simulium species(Diptera: Simuliidae) from Doi Inthanon National Park,
northern Thailand. J Sci Soc Thailand 1998; 24: 215―230.
5. Takaoka H, Yunus M, Hadi UK, Sigit SH, Miyagi I. Preliminary report of faunistic surveys on black flies (Diptera:
Simuliidae) in Sumatra, Indonesia. Jpn J Trop Med Hyg
2000; 28: 157―166.
6. Crosskey RW, Howard TM. A New Taxonomic and Geographical Inventory of World Blackflies (Diptera: Simuliidae). 1997; 144 pp., The Natur Hist Mus, London.
7. Takaoka H, Saito K. Description of a new species of Simulium (Nevermannia) from Japan (Diptera: Simuliidae).
Jpn J Trop Med Hyg 2000; 28: 19―24.
8. Edwards FW. Diptera Nematocera from Mount Kinabalu. J
Fed Malay States Mus 1933; 17: 223―296.
9. Delfinado MD. Some Simuliidae and Curtonotidae fromthe Philippines and the Bismarck Islands (Insecta, Diptera).
Steenstrupia 1971; 1: 131―139.
10. Brunetti E. New Oriental Nematocera. Rec Indian Mus
1911; 4: 259―316.
11. Takaoka H, Davies DM. The black flies (Diptera: Simuliidae) of Java, Indonesia. Bishop Mus Bul Entomol 1996; 6,
Bishop Mus Press, Honolulu.

BIOLOGICAL NOTES. The pupa of this new species wasfound on the surface of a leaf in the water. Associated species was S . (G .) inthanonense.
DISTRIBUTION. Thailand.
ETYMOLOGY. The species chiangklangense refers to theregion, Chiang Klang, where this new species was found.
REMARKS. Simulium (Nevermannia) chiangklangense sp.
nov. is also assigned to the feuerborni species-group by thefemale genitalia and pupal gill with six long thread-like filaments per side.
This new species is characterized by the simple cocoonand the pupal gill filaments arranged in two short-stalked
pairs and two individual filaments (Fig. 5J). The combination of these two characteristics easily separates S . (N .) chiangklangense from other members of the feuerborni
species-group.
The female of this new species is very similar in appearance to those of S . (N .) feuerborni [11] and S . (N .)fruticosum but appears to be distinguished from the lattertwo species by the longer and narrower projection directed

ACKNOWLEDGEMENT
This work was financially supported by a Grant-in-Aid
from the Japan-US Medical Research Cooperation Program(the fiscal year 2004).

REFERENCES

Tropical Medicine and Health Vol. 33 No. 3, 2005, pp. 143-152
Copyright! 2005 by The Japanese Society of Tropical Medicine

１
４
３

THE EFFECT OF MEDICATION ON QUESTIONNAIRE ANALYSIS OF
CHILDREN WITH SCHISTOSOMA MANSONI INFECTION
IN TANZANIATOMOKO KISU１, KIYOSHI SHIRATORI２, FRANCIS CALLYST３,
YUJI ATAKA４, EIKO KANEDA１, ELISONGUO NGOMUO５,
RICHARD J. SHAYO６ and MASAAKI SHIMADA１
Accepted 7, September, 2005

Abstract: The effect of mass treatment on questionnaire results in the diagnosis of schistosomiasis mansoni was
examined in 267 school children in an endemic area of Tanzania by Kato-Katz analysis of fecal specimens. The
questionnaire asked for information about self-diagnosis, abdominal symptoms, blood in stools, history of wild
water contact, stool examination and medication for schistosomiasis, and knowledge of the disease. A logistic regression analysis disclosed a significant association between schistosomiasis and “diarrhea” (p = 0.007; odds ratio,
32.0; confidence interval, 2.5 - 403.3) and “abdominal enlargement” (p = 0.003; odds ratio, 15.2; confidence interval, 2.6 - 90.1) among 61 children who had no history of medication for schistosomiasis. The sensitivity and specificity of the model were 86% and 64%, respectively. In contrast, no significant correlation was observed either forthe 116 treated children, or for all the 267 children after the mass treatment. We conclude, therefore, that for children who had no history of medication for schistosomiasis, the questionnaire for abdominal manifestations provides reliable information on S . mansoni infection. However, once a child takes medication, the questionnaire becomes unreliable. This observation suggests that immunomodulation by anti-schistosomiasis drugs that kill adultworms exerts an effect on the appearance of abdominal manifestations and might explain the ambiguity of clinicalsymptoms in chronically infested patients, except in terminal cases. Further studies are required to develop a simple, rapid and cost-effective diagnostic method for monitoring S. mansoni infection after medication in local areas
without resort to laboratory-based identification of schistosomiasis.
Key words: Schistosoma mansoni, self-diagnosis, selective mass treatment, Tanzania

INTRODUCTION
Schistosomiasis is one of the widespread parasitic diseases with important public health implications in subSaharan countries. Disease control depends mostly on
medication treatment, because praziquantel, the drug ofchoice, is safe and relatively inexpensive [1]. Several approaches for community-based treatment have been developed, including mass treatment, selective population treatment and selective group treatment [2]. The main target
population is school-age children between 5 and 15 years

old, because they show the highest prevalence and intensity
in the entire population [2, 3, 4] and because they are themain source of the transmission of infection. Following
WHO recommendations, school-based mass treatment withpraziquantel has been conducted in many high prevalence
areas [5].
The strategy emphasizing school-based mass treatment,
however, is not cost-effective in low prevalence areas. The
prevalence usually declines to a low level after a series ofmass treatments in high prevalence areas. Subsequently, theschool-based mass treatment approach should be replaced

1. Research Centre for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
2. Japan International Cooperation Agency
3. Majengo Health Care Center, Arusha, Tanzania
4. School of Policy Studies, Kwansei Gakuin University, Hyogo, Japan
5. Mawenzi Hospital, Moshi, Kilimanjaro, Tanzania
6. Kilimanjaro Agricultural Training Center, Moshi, Tanzania
Corresponding author: Tomoko KISU, M.D. Research Centre for Tropical Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523
Tel +81 95 849 7869 Fax +81 95 849 7854 e-mail address: tkisu@tm.nagasaki-u.ac.jp
Running title: Medication effect for S. mansoni infection on questionnaire

１４４with selective treatment. The most reliable diagnostic
method for Schistosoma mansoni (S. mansoni) infection isdetection of eggs in feces, with a rapid and simple KatoKatz thick smear stool examination being applied to thisend. However, well-trained health personnel and microscopes are required for this laboratory diagnosis, and it islaborious, time-consuming, and expensive. Therefore, asimilarly rapid and simple yet cost-effective individual diagnostic method is required. This kind of simple method
has been developed for the screening of Schistosoma haematobium infection based on self-reported haematuria andself-diagnosis [6, 7, 8, 9]. Regarding S. mansoni infection,
several morbidity studies have been carried out since theearly 1970s, before the discovery of an effective medication(praziquantel). These studies found that S. mansoni frequently causes abdominal pain, blood in stool, bloody diarrhea, diarrhea in general, hepatomegaly, and splenomegaly[3, 10, 11, 12, 13, 14, 15, 16]. Based on theses studies, thediagnosis by questionnaire approach has been conducted forS. mansoni infection since 1985, initially focusing on subjective symptoms and the observation of stool appearance.
But since studies reported low to moderate sensitivities andspecificities [17, 18, 19], the questionnaires have been extended to include anamnestic information such as watercontact behavior [20, 21, 22, 23]. This approach, which
was undertaken in several endemic areas, disclosed the potential of questionnaires for the identification of S. mansoni
infection on the individual level [17, 18, 20, 22, 23, 24].
Supportive evidence for the application of a questionnaire
for identification of S. mansoni, however, is still scarce, especially in the wake of mass treatment with praziquantel.
The purpose of this study was to evaluate the efficacy
of questionnaires in the assessment of S. mansoni in one endemic area, with reference to the history of praziquantel
medication in school-age children. An analysis was conducted to evaluate the potential of a questionnaire applied
separately to children with and without a history ofpraziquantel medication.

MATERIALS AND METHODS
Cross sectional surveys on S. mansoni infection wereconducted among Tanzanian children three times before andafter mass treatment. The first questionnaire survey withstool examination was carried out in January and February
2001, and mass treatment was administered immediately after the survey. One month later, an additional stool examination for evaluation of efficacy of treatment was conducted.
The second questionnaire survey with stool examination
was carried out in February and March 2002, about one
year after the treatment.

Area studied: The study area was Lower Moshi located in
the southern part of the suburbs of Moshi Town in Kilimanjaro Region, Tanzania. The annual climate is divided into
four periods: the long rainy season from March to June,
cool dry season from July to October, short rainy season in
November, and hot dry season from December to February.
The main economic activity is wet-rice cultivation. The
land is well developed for paddy cultivation with irrigation
canals where water flows throughout the year. People usually cultivate crops three times a year. An agricultural development project supported by Japan International Cooperation Agency (JICA) has been conducted in this area sincethe 1970s.
Study population: The study was conducted in four primary schools; Mabogini, Rau River, Oria and Chekereni.
There were 1,033 children registered in grades three andfour in 2001.
Stool examination: A stool sample container was distributed to each child for collection of stool on the next day or
the day after. Double 20 mg Kato-Katz thick smears [25]were processed and examined for the ova of S. mansoni,
Ascaris lumbricoides, Trichuris trichiura, and hookworms
by trained technicians and the authors. The number of eggs
found in the samples was expressed as eggs per 1 gram
(EPG) to reveal the intensity of infection. The presence ofeggs was assessed for prevalence of infection. Geometricmean was calculated for expressing intensity of groups.
Questionnaire survey: The questionnaire consisted of fiveparts containing a total of 35 questions (see Tables 3 and 4
for the questionnaire contents). The first part focused on
self-diagnosis, with the question “Do you think you havebilharzia (Kichocho in local Swahili language)?” The second part dealt with 18 self-reported clinical manifestations
self-recognized during the last two weeks prior to the survey, including abdominal pain, diarrhea, constipation, abdominal enlargement, and blood in stool. The third part
dealt with the history of contact with irrigation and riverwater including swimming, bathing, fishing (mainly netfishing), washing clothes, fetching water, and other purposes. The fourth part dealt with anamnestic information:
blood in stool in the past, experience of stool examination,and history of anti-schistosomiasis medication. The lastpart dealt with recognition and knowledge of schistosomiasis. The questionnaire was written in Swahili, the local language. Under the supervision of a Tanzanian doctor, a fieldassistant read out the questions to the children in groups of20, and the children completed the forms individually.
Children recorded their answers as ‘yes’, ‘no’ or ‘don’t

１
４
５

know’. We combined ‘no’ and ‘don’t know’, and used thepercentage of ‘yes’ replies to determine prevalence.
Treatment: Mass treatment with praziquantel was carriedout in the schools under the supervision of a medical nurse
in February 2001. Each child received 40 mg/kg ofpraziquantel. At the end of the study, the infected children
were treated again. For the treatment of intestinal parasitic
infection, we used mebendazole at a dose of 600 mg perperson as a mass treatment in February 2001 and as a selective treatment in March 2002.
Analysis: The results of the study were analyzed using the
SPSS! (version 11.5) software package. First, the prevalence of infestation by age and sex and by all the 35 variables in the questionnaire was calculated. Then the odds ratios (ORs) of infection were calculated and tested by Chisquare analysis. By using the variables correlated at lessthan the 0.10 level in univariate analyses, logistic regression
analysis was conducted to assess the associations with infection after correcting for age and sex. The sensitivities
and specificities of logistic regression models were calculated to assess the effectiveness of the questionnaire as atool for individual diagnosis of infection. The analysis was
conducted separately for those who had received treatment
before the mass treatment and for those who had never received the treatment.
Ethical guidelines: This study was part of an epidemiological survey permitted by the National Institute of Medical
Research, Ministry of Health, United Republic of Tanzania.
We started the program by explaining the outline of thestudy to local authorities. Then we explained orally the
purpose and methods of the study to the school children,
their parents and teachers at school assemblies. At mass
treatment, we treated all children who desired treatment irrespective of enrollment in the study. We treated infectedchildren again for S. mansoni and other helminthes at theend of the study in March 2002. Informed consent was not
obtained in written form.

RESULTS
Number of subjects for analysis: Out of 1,033 children
registered in grades 3 and 4 in the 4 schools, demographic
data such as sex and age were obtained from 830 children(417 males and 413 females, aged 7 to 17). Out of these830 children, 267 (32.2%) had undergone three stool examinations, mass treatment after the first survey, and twoquestionnaires. The present analysis is restricted to these267 children. The number of children by age and sex isshown in Table 1 with prevalence of S. mansoni infection.
No significant difference in age or sex were observed between the 267 children and the rest of the 830 children.
Prevalence and the intensity of S. mansoni infestation:
Among the 267 children, the prevalence of schistosomiasis
was 67.4% and the mean intensity of schistosoma eggs infeces was 29.9/g at the first stool examination prior to mass
treatment (Figure 1). The prevalence and intensity were notsignificantly different either by sex or by age. The prevalence decreased to 6.4% (McNemar test, p < 0.001) and themean intensity to 0.3/g (t-test, p < 0.001) one month after
the mass treatment (2nd fecal analysis). However, the prevalence increased to 27.0% (McNemar test, p < 0.001) with aslight increase in the mean intensity to 2.5/g (t-test, p <0.001) one year after the treatment (3rd fecal analysis), although this prevalence and intensity were still significantly
lower than those prior to mass treatment (McNemar test p <0.001, t-test p < 0.001, respectively). The mean EPG calculated from only positive samples decreased from 160.8/g (n= 180) to 74.8/g (n = 17) by mass treatment and then increased to 105.2/g (n = 72) one year after the treatment.
The prevalence of heavy infection (more than 400 EPG) de-Table 1 The number of children and prevalence of S. mansoni infection before masstreatment by age and sex
Age

Examined

Boys
Infected

7-9
10
11
12
13
14-17
Sub-total

9
30
41
32
15
11
138

7
20
29
23
10
9
98

Prevalence (%) Examined(77.8)(66.7)(70.7)(71.9)(66.7)(81.8)(71.0)

17
29
50
19
10
4
129

In total, 267 children were examined and 180 (67.4%) were egg-positive.
No significant difference was found between sex or age.

Girls
Infected

Prevalence (%)

11
21
31
9
7
3
82

(64.7)(72.4)(62.0)(47.4)(70.0)(75.0)(63.6)

180

70

160

60

140
120

50

100

40

80

30

60

20

40

10

20

0

0

intensity (epg)

80

Fe

b01
M
ar
-0
1
Ap
r-0
1
M
ay
-0
1
Ju
n01
Ju
l-0
1
Au
g01
Se
p01
O
ct
-0
1
N
ov
-0
1
D
ec
-0
1
Ja
n02
Fe
b02

prevalence (%)

１
４
６

total

positive case

Mean

Heavy

Mild and moderate

Figure 1 Prevalence and intensity of S. mansoni infection before, one month after, and one year after mass treatment. Solid line with circles: mean prevalence of infection; Dotted line with triangles: prevalence of mildand moderate infection (100 EPG or more); Dashedline with square: prevalence of heavy infection (400
EPG or more); Black bars: mean intensity (geometricmean EPG for the 267 children); Gray bars: mean intensity of positive cases (geometric mean EPG of 180,
17, 72 children, respectively).

creased from 14.2% to 0.4% by treatment, then increased to
3.0% after one year.
Experience of treatment: Of the 267 children, 61 had no
history of medication for schistosomiasis before the mass

treatment, while 116 children had a medication history. The
remaining 90 children were not sure either way. The prevalence and intensity of infection did not differ significantly
between non-treated and treated children (59.0% and 17.6/g
for the former and 62.9% and 21.4/g for the latter) in thefirst stool examination.
Prevalence of water contact activities: About 90% of children reported having contact with irrigation/river water forthe purpose of swimming/playing, bathing, crossing water
and washing clothes, and fetching water in the first survey.
About 40% of children had contacted water for the purposeof fishing in the first survey. The percentage of children
contacting irrigation/river water in the second survey decreased slightly from the first survey in all activities.
Results of questionnaire survey in relation to egg detection before mass treatment for the 267 children: Before
mass treatment, the most common symptom was “abdominal pain” (60%) followed by “headache” (52%) and “abdominal discomfort” (40%). But, no manifestation showeda significant increase with the level of intensity (Table 2).
“Constipation” and “net-fishing” showed a significant correlation with infection (both p < 0.05) and “diarrhea” and“history of blood in stool” showed borderline significance(both, p < 0.10, by Chi-square test, Tables 3 and 4). For a

Table 2 Prevalence of signs and symptoms by the level of intensity of S. mansoni infection before mass-treatment
Percentage of “yes” answers in the questionnaire
Level of intensity §Manifestations

Total

Negative
0 epg

Mild
1 - 99 epg

Moderate
100 - 399epg

Heavy
400 epg<

abdominal pain
headacheabdominal discomfort
easy to be tired
fever
loss of appetite
coughnausea/vomiting
constipation
diarrhea
fatigue
abdominal enlargementdizziness/lassitude
skin rash
blood in stool
itching skinweight loss
asthma(n=267)
59.6
52.4
40.4
28.8
24.3
20.6
19.9
18.4
17.2
16.5
14.2
14.2
12.4
10.1
5.2
4.5
4.1
3.7

(n=87)
52.9
46.0
33.3
25.3
20.7
20.7
19.5
16.1
10.3
10.3
10.3
9.2
10.3
10.3
3.4
3.4
2.3
3.4

(n=51)
62.7
51.0
43.1
31.4
31.4
27.5
23.5
21.6
27.5
19.6
21.6
19.6
13.7
11.8
5.9
3.9
5.9
5.9

(n=91)
61.5
58.2
42.9
29.7
28.6
16.5
22.0
20.9
19.8
18.7
14.3
17.6
12.1
9.9
5.5
4.4
6.6
4.4

(n=38)
65.8
55.3
47.4
31.6
13.2
21.1
10.5
13.2
13.2
21.1
13.2
10.5
15.8
7.9
7.9
7.9
0.0
0.0

§ Intensity is categorized according to the definitions of WHO

Somers’ d

p

0.149
0.130
0.109
0.451
0.863
0.609
0.475
0.943
0.371
0.085
0.646
0.391
0.482
0.686
0.335
0.383
0.753
0.477

0.459
0.415
0.398
0.834
0.141
0.491
0.428
0.632
0.058
0.303
0.339
0.233
0.845
0.945
0.330
0.348
0.840
0.549

１
４
７

Table 3 Prevalence of self-diagnosis and 2-week-recognition of symptoms before and after treatment, and for non-treated and treated
and the odds ratio between infected and non-infected
Self-diagnosis and recognition ofsymptoms
within two weeks

Percentage of “yes” answers in the questionnaire (%)
Before treatment
After treatment
never
all
treatedalltreated(n=267)(n=61)(n=116)(n=267)

self-diagnosis

7.7

6.7

9.0

25.8

loss of appetite

20.6

44.3

19.8

43.8

nausea/vomiting

18.4

27.9

25.0

61.4

abdominal enlargement

14.2

29.5

5.2

7.1

abdominal discomfort

40.4

62.3

29.3

71.5

abdominal pain

59.6

72.1

43.1

85.4

diarrhea

16.5

19.7

25.9

49.4

constipation

17.2

36.1

10.3

38.2

blood in stool

5.2

8.2

6.0

30.5

easy to be tired

28.8

57.4

25.9

41.2

fatigue

14.2

24.6

17.2

63.7

fever

24.3

45.9

26.7

58.1

cough

19.9

37.7

19.8

58.6

asthma

3.7

9.7

3.4

16.1

weight loss

4.1

4.9

4.3

32.2

dizziness/lassitude

12.4

23.0

14.7

50.2

skin rash

10.1

18.0

12.1

30.3

headache

52.4

68.9

38.8

83.5

itching skin

4.5

3.3

8.6

43.8

Odds ratio and its 95% confidence interval
Before treatment
After treatment
never
all
treatedalltreated(n=267)(n=61)(n=116)(n=267)0.90
0.314-2.586
0.99
0.527-1.867
1.26
0.637-2.486
1.98
0.865-4.512
1.56
0.917-2.669
1.50
0.895-2.524
2.09§
0.957-4.575
2.24*
1.029-4.887
1.82
0.495-6.708
1.30
0.729-2.318
1.66
0.751-3.690
1.35
0.731-2.509
1.03
0.541-1.959
1.13
0.286-4.492
2.24
0.473-10.582
1.33
0.591-3.006
0.96
0.414-2.241
1.47
0.878-2.456
1.47
0.389-5.586

2.09
0.204-21.382
2.38
0.819-6.890
0.99
0.317-3.085
9.20*
1.881-44.998
2.82§
0.965-8.222
1.97
0.634-6.115
10.56*
1.265-88.184
5.25*
1.500-18.380
3.00
0.315-28.586
2.55§
0.890-7.279
2.31
0.640-8.333
1.50
0.534-4.214
1.52
0.521-4.426
1.44
0.242-8.524
1.41
0.121-16.470
1.33
0.387-4.592
0.80
0.215-2.979
0.78
0.255-2.371
0.69
0.041-11.505

0.53
0.144-1.957
1.13
0.435-2.941
3.72*
1.299-10.665
0.57
0.110-2.966
1.97
0.817-4.741
1.26
0.587-2.713
1.89
0.755-4.719
1.88
0.479-7.342
3.76
0.437-32.349
1.89
0.755-4.719
2.74§
0.850-8.808
2.52§
0.979-6.484
1.44
0.541-3.851
1.80
0.181-17.869
2.43
0.263-22.522
3.16§
0.854-11.725
2.37
0.621-9.008
2.54*
1.111-5.788
2.52
0.510-12.472

1.15
0.623-2.107
1.12
0.649-1.925
1.06
0.610-1.858
2.09
0.805-5.429
0.80
0.442-1.431
1.82
0.766-4.338
1.91*
1.101-3.316
0.960.550-1.677
1.31
0.737-2.332
0.88
0.505-1.524
1.30
0.734-2.312
1.29
0.740-2.243
1.59
0.904-2.805
1.21
0.593-2.479
0.75
0.415-1.363
1.07
0.622-1.834
1.85*
1.051-3.273
1.13
0.537-2.375
1.12
0.649-1.925*: significant at 0.05 level§: borderline significant at 0.1 level

binary logistic regression analysis using “constipation”,“net fishing”, “history of blood in stool”, “diarrhea”, age,and sex as independent variables, only “constipation” (oddsratio, OR = 2.27, confidence interval, CI = 1.04 - 4.96) was
positively associated with infection (Table 5a). The prediction of the model, however, was not significant.
Results of questionnaire survey in relation to egg detection at one year after mass treatment for the 267 children: Results: “Abdominal pain”, “headache” and “abdominal discomfort” remained common at one year after
the treatment. The prevalence of all signs and symptoms

except “abdominal enlargement” increased from the firstquestionnaire (Table 3). The prevalence of “abdominal enlargement” decreased from 14% before treatment to 7% at
one year after treatment. The prevalence of self-diagnosis
of S. mansoni infection was 8% before the treatment and increased to 26% one year after the treatment.
“Diarrhea” and “skin rash” were significantly associated with schistosomiasis (both p < 0.05), and “bathing”
showed a borderline significant association with prevalence(p=0.09). For a binary logistic regression analysis using“diarrhea”, “skin rash”, “bathing”, age, and sex as independent variables, only “diarrhea” (OR = 1.96, CI = 1.13 -１
４
８

Table 4 Questionnaire of history of wild water contact, knowledge and recognition, experience of fecal examination, fecal blood
hisotry and treatment before and after treatment, and for non-treated and treated and the odds ratio between infected and noninfected
Proportion of answering “yes” (%)
Before treatment
After treatment
never
all
treatedalltreated(n=267)(n=61)(n=116)(n=267)
Water contact activities
any purpose

91.0

96.7

87.1

83.7

swimming/playing

90.5

88.1

87.5

53.6

bathing

91.7

91.5

89.4

49.8

fishing

38.7

22.0

10.6

17.6

crossing the water

92.9

94.9

85.6

36.7

washing clothes

94.1

96.6

87.5

59.2

fetching water

88.5

84.7

89.4

54.3

for agricultural purpose

87.7

84.7

84.6

55.8

for animal husbandry

78.3

67.8

75.0

29.2

Anamnestic information
Have you ever had blood in stool?

23.0

23.0

28.7

no dataHave you ever taken medication
60.7
for schistosomiasis?
Have you ever taken fecal exami83.9
nation?
Recognition and knowledge on the disease
Have you ever heard of disease of
83.1
bilharzia?
Do you know what bilharzia is?
68.4

0

100.0

72.2

50.8

No data

95.5

50.8

87.8

79.6

23.0

78.3

47.9

34.7

16.9

43.9

16.1

22.4

15.3

36.8

62.2

Contacting with river water causesbilharzia?
Drinking river water causes bilharzia?

Odds ratio and its 95% confidence interval
Before treatment
After treatment
never
all
treatedalltreated(n=267)(n=61)(n=116)(n=267)0.39
0.128-1.165
0.80
0.317-2.004
0.77
0.289-2.074
1.84*
1.056-3.220
0.99
0.357-2.729
0.70
0.218-2.283
0.88
0.381-2.018
1.10
0.501-2.416
0.770.400-1.471

1.46
0.087-24.468
1.11
0.224-5.463
2.36
0.363-15.309
0.75
0.217-2.594
0.72
0.061-8.387
1.48
0.088-24.847
1.20
0.287-5.021
1.20
0.287-5.021
0.26*
0.075-0.945

0.23*
0.048-1.051
0.62
0.178-2.162
0.52
0.129-2.083
0.79
0.225-2.788
0.70
0.222-2.227
0.40
0.103-1.551
0.83
0.226-3.023
0.44
0.131-1.467
0.58
0.223-1.480

1.63
0.716-3.733
1.12
0.649-1.922
1.60§
0.926-2.764
1.34
0.677-2.655
1.44
0.832-2.509
1.55
0.878-2.722
1.25
0.724-2.160
1.07
0.618-1.838
0.83
0.451-1.516

2.08§
0.985-4.392
1.07
0.588-1.947
1.22
0.556-2.656

5.75*
1.158-28.551
(-)
(-)
0.92
0.333-2.562

1.94
0.769-4.883
1.00
(-)
(-)
(-)

no data
1.30
0.697-2.438
1.11
0.293-4.231

1.50
0.774-2.906
1.07
0.617-1.860
1.22
0.702-2.116
1.11
0.592-2.084

2.79§
0.971-8.034
2.02
0.553-7.367
(-)
(-)
(-)
(-)

0.11*
0.014-0.894
0.18*
0.050-0.639
0.84
0.393-1.803
0.98
0.445-2.137

1.20
0.598-2.391
1.17
0.676-2.014
0.79
0.368-1.703
1.11
0.631-1.937*: significant at 0.05 level§: borderline significant at 0.1 level3.43) was positively associated with infection (Table 5d).
The prediction of the model, however, was not significant.
The level of intensity of infection was also related with “diarrhea”, increasing from 45% of negative children, to 52%
of lightly infected children, to 66% of moderately infectedchildren and to 75% of heavily infected children (Somers’dtest, p < 0.05, data not shown).
Results of questionnaire survey in relation to egg detection before mass treatment for the 61 non-treated and
116 treated children: Next, we compared the 61 nontreated and 116 treated children. In children without a history of medication for schistosomiasis, “diarrhea”, “consti-

pation”, “abdominal enlargement”, and “recognition ofblood in stool” were significantly associated with schistosomiasis by univariate analyses. “Abdominal discomfort”,“easy to be tired”, and “recognition of the disease (yes tothe question “Have you ever heard of bilharzia?”)” were associated with borderline significance in these children (Tables 3 and 4). Recognition of blood in stool over the past
two weeks was not significant, but a history of this recognition was significant in the non-treated children.
Among the 116 children with a medication history,
meanwhile, “nausea/vomiting”, “headache”, recognition of
the disease, and knowledge of the disease (yes to the question “Do you know what bilharzia is?”) were significant,１
４
９

Table 5 Logistic regression of S. mansoni infection with questionnaire’s items
a. Before mass-chemotherapy (all children, n = 267)
Variable

Parameter estimate

Standard errorpOdds ratio

0.820
0.822

0.399
1.106

0.040
0.458

2.271
2.274

Parameter estimate

Standard errorpOdds ratio

3.466
2.719
-7.923

1.293
0.909
3.582

0.007
0.003
0.027

31.99315.1660.000

Parameter estimate

Standard error

P

Odds ratio-1.671
2.862

0.655
2.001

0.011
0.153

0.188
17.488Parameter estimate

Standard errorpOdds ratio

0.675-2.232

0.284
1.193

0.017
0.061

1.964
0.107

Constipation
Constant

95.0% C.I††
Lower
Upper
1.039

4.961

Sensitivity = 100%, Specificity = 0%

b. Before mass-chemotherapy, (never-treated children, n = 61)
Variable
Diarrhea
Abdominal enlargement
Constant

95.0% C.I.††
Lower
Upper
2.538
2.553

403.34290.080Sensitivity = 86.1%, Specificity = 64%

c. Before mass-chemotherapy, (ever-treated children, n = 116)

Recognition of the disease
Constant

95.0% C.I.††
Lower
Upper
0.052

0.679

Sensitivity = 100%, Specificity = 2.4%

d. After mass-chemotherapy, (all children, n = 267)

Diarrhea
Constant

95.0% C.I.††
Lower
Upper
1.126

3.425

Sensitivity = 0%, Specificity = 100%

but abdominal manifestation was not significant (Tables 3
and 4). “Fever”, “dizziness”, “fatigue” and “contact withriver water for any purpose” showed borderline significance.
We examined the effect of treatment on selected manifestations using binary logistic regressions explaining S.
mansoni infection for the 61 non-treated children (Table 5b)
and for the 116 treated children (Table 5c). In the nontreated children, “diarrhea” (p = 0.007, OR = 32.0, CI =2.54 - 403.3) and “abdominal enlargement” (p = 0.003, OR= 15.2, CI = 2.55 - 90.1) were significantly associated with
infection, and the model prediction was significant (Chisquare = 26.98, p < 0.001). The sensitivity and the specificity of the logistic predictive model were 86.1% and 64.0%,
respectively.
In the treated children, only knowledge of the disease
was significantly and negatively associated with prevalence,and the prediction was significant (the model Chi-square =10.085, p = 0.018) (Table 5c). But, the specificity of themodel prediction was very low (2.4%), while the sensitivity
was 100%.
Other parasitic infections: The prevalence of eggs of A.
lumbricoides, T. trichiura and hookworm was 6.7%, 10.9%,

and 9.0% respectively in the first examination. It declined
to 5.3%, 4.9% and 4.1% respectively one year after mass
treatment. At the first survey, A. lumbricoides infection wascorrelated with S. mansoni infection (Chi-square = 9.329, p= 0.002), but it was not significantly associated with any
sign or symptom of S. mansoni infection. Hookworm infection was correlated with “constipation”. However, it did
not correlate with S. mansoni infection. T. trichiura infection showed no correlation with S. mansoni infection orwith any sign or symptom.

DISCUSSION
The present study was conducted in a high endemic
area according to the WHO definition [4], the prevalence ofschistosomiasis being 67.4% among school children aged
from 7 to 17 years old. The results indicated that subjective
abdominal symptoms are related with infection of S. mansoni and that questionnaire on the subjective abdominal
symptoms can be used for the screening of S. mansoni infection in children who have never undergone treatment for
S. mansoni infection. “Diarrhea” and “abdominal enlargement” correlated well with infection when the children had

１
５０

no history of treatment. These correlations were not seen inchildren who had undergone treatment in the past, although
the prevalence and intensity of infection did not differ fromthose of the non-treated children. At one year after mass
treatment, no suitable model for the prediction of S. mansoni infection was obtained by using self-diagnosis, subjective symptoms, water contact behavior, anamnestic information, or recognition and knowledge of the disease.
S. mansoni causes a chronic disease with various
stages and many symptoms and signs at each stage, adultworms being present in the mesenteric veins and eggs invarious organs including the liver, lungs and gastrointestinal
tract. Deposition of eggs in the alimentary tract causes mucosal lesions frequently associated with hemorrhage resulting in blood in the stool. The abdominal symptoms might
be influenced by adult worms in the mesenteric veins andegg embolism scattered from the adult female worms. The
human body responds to schistosomula during acute infection or when eggs are retained in tissue [26]. Regarding
adult worms, no response has been observed around the live
worms. Colley advocated the hypothesis that praziquantel
treatment alters host-parasite relationships, leading to multiple immunologic changes through the killing of adultworms. The antigens released from dead adult worms cause
many immune responses and produce some resistance to reinfection [27]. This hypothesis is supported by an animal
experiment using immuno-compenent and deficient mice[28]. B cell deficient mice showed a delayed response after
praziquantel treatment, suggesting that the immunologicalresponses are required for the killing of adult worms during
treatment. Taken together, this evidence and our results
suggest that the abdominal manifestations become obvious
in the host who has recently been infected and not treatedby medication.
The association between intensity of infection andmorbidity was frequently reported. It was shown that complaints of “abdominal pain” and “blood in stool” correlated
significantly with S. mansoni egg counts in a high prevalence village [11]. Heavily infected individuals frequently
complained of “abdominal pain” [14]. Our study, however,
did not confirm any correlation between intensity of infection and the prevalence of abdominal symptoms. The reason for this is not clear. “Abdominal discomfort”, “diarrhea”, “constipation”, “easy to be tired” and “abdominal enlargement” were associated with the level of intensity of infection (Somers’d test, p = 0.021, 0.003, 0.004, 0.038 and <0.001, respectively) in the non-treated children. Almost allof these children had abdominal manifestations. On theother hand, “dizziness/lassitude”, “nausea/ vomiting” and“headache” were associated with the level of intensity of infection (Somers’d test, p = 0.030, 0.014 and 0.018, respec-tively) in the treated children.
One of the limitations of the present study is that it examined only the ova of helminthes in fecal specimens anddid not determine other causes of “diarrhea” and “abdominal enlargement” such as protozoan or bacterial intestinal
infection. Since abdominal manifestations are non-specific,
it is impossible to overrule other causes. In fact our results
show that the infection of hookworm causes “constipation”
as an abdominal symptom, although the prevalence was nothigh. The intestinal parasitic infestation did not contributesignificantly to the abdominal manifestations in our study
because of its low prevalence, although the hookworm infection was significant as a cause of “constipation”. From
this viewpoint, the abdominal manifestation should be applied while ruling out other intestinal infections and infestations.
“Blood in stool” is well documented as the most notable subjective sign of S. mansoni infection. Sukuwa (1985)
and Proietti (1989) reported that “blood in stool” had thehighest specificity (94.9%) [17] and the highest positive
predictive value (38.9%) [18] among the symptoms. In thepresent study, however, “recognition of blood in stool” did
not prove to be a specific sign of infection. With regard to
the individual perception of clinical manifestations, the only
reliable variables identified were “diarrhea” and “abdominal
enlargement”. The reason may be that our study population
was limited to school children while the previous studies included adults.
In the variables of risk factors such as self-diagnosis,
water contact history, anamnestic information, and recognition and knowledge of the disease, we did not identify any
significant variable that could be used for individual diagnosis. The use of risk factors has been reported to be limited in its potential to identify the infection of S. mansoni.
Utzinger (2000) argued that recalled water-contact patterns
collected by questionnaire might be useful for selfdiagnosis of schistosomiasis, showing sensitivity and specificity of “crossing the river” to be 61-70% and 47-52%, respectively, and those of “swimming/bathing” to be 77-81%
and 37-68% respectively [23]. According to Brooker(2001), the reported “swimming” had 80% sensitivity and83% specificity [21]. However, Lima e Costa (1998)
pointed out that sociodemographic variables and water contact activities associated with S. mansoni infection differedamong the study areas, thus their validity for diagnosis ofinfection similarly differed among the areas [22]. Unlike
previous studies, water contact behavior was not correlatedwith infection in our study. The reason might be that we
did not define the period of the behaviors in the questionnaire and therefore that many children reported having contact with the irrigation/river water.

１
５１

After a series of mass treatments in high prevalence areas, prevalence will decrease considerably. Subsequently,
the school-based mass treatment approach might be replaced by selective treatment, and children can be monitored for re-infection individually. But as the present study
showed, diagnosis by questionnaire becomes inapplicable
once children take medication. Although stool examinationis the most reliable method for individual identification of
S. mansoni infection in an area of low intensity infection,other simple, rapid and cost-effective individual diagnostic
methods are still required in areas where laboratory examination has not been established. Moreover, it is imperativethat old-fashioned questionnaire analysis be analyzed for
usefulness for schistosomiasis using praziquantel-resistantclone [29].

ACKNOWLEDGEMENT
This research was partially supported by the Ministryof Education, Science, Sports and Culture, Grant-in-Aid for
Scientific Research, 13576010, B2, and was conducted
partly as a JICA agricultural development project. It wasalso supported by Nagasaki University Institute of Tropical
Medicine. We express our deep appreciation to all of thechildren in the schools of Mabogini, Chekereni, Oria and
Rau River in Moshi, their parents, teachers, school board
and headmasters for their corporation. We thank Mrs. Godwin Chonjo, Frederick S. Mawolle, Ewald S. Massawe, Eric
Muro and Charles Masenga for their help in the field work.

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