Acta Tropica 141 (2015) 315321

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Repeated stool sampling and use of multiple techniques enhance the

sensitivity of helminth diagnosis: A cross-sectional survey in southern
Lao Peoples Democratic Republic
Somphou Sayasone a,b,c , Jrg Utzinger b,c , Kongsap Akkhavong a , Peter Odermatt b,c,
a
National Institute of Public Health, Ministry of Health, Vientiane, Lao Democratic Peoples Republic
b
Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland
c
University of Basel, P.O. Box, CH-4003 Basel, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Intestinal parasitic infections are common in Lao Peoples Democratic Republic (Lao PDR). We investigated
Available online 16 September 2014 the accuracy of the Kato-Katz (KK) technique in relation to varying stool sampling efforts, and determined
the effect of the concurrent use of a quantitative formalin-ethyl acetate concentration technique (FECT)
Keywords: for helminth diagnosis and appraisal of concomitant infections. The study was carried out between March
Diagnosis and May 2006 in Champasack province, southern Lao PDR. Overall, 485 individuals aged 6 months who
Helminthiasis
provided three stool samples were included in the nal analysis. All stool samples were subjected to the
Multiparasitism
KK technique. Additionally, one stool sample per individual was processed by FECT. Diagnosis was done
Multiple stool sampling
Multiple diagnostic techniques
under a light microscope by experienced laboratory technicians. Analysis of three stool samples with KK
Lao Peoples Democratic Republic plus a single FECT was considered as diagnostic gold standard and resulted in prevalence estimates of
hookworm, Opisthorchis viverrini, Ascaris lumbricoides, Trichuris trichiura and Schistosoma mekongi infec-
tion of 77.9%, 65.0%, 33.4%, 26.2% and 24.3%, respectively. As expected, a single KK and a single FECT missed
a considerable number of infections. While our diagnostic gold standard produced similar results than
those obtained by a mathematical model for most helminth infections, the true prevalence predicted by
the model for S. mekongi (28.1%) was somewhat higher than after multiple KK plus a single FECT (24.3%).
In the current setting, triplicate KK plus a single FECT diagnosed helminth infections with high sensitivity.
Hence, such a diagnostic approach might be utilised for generating high-quality baseline data, assessing
anthelminthic drug efcacy and rigorous monitoring of community interventions.
2014 Elsevier B.V. All rights reserved.

1. Introduction In Lao Peoples Democratic Republic (Lao PDR), multipara-

Neglected tropical diseases remain of considerable public health
importance, particularly in low-income and middle-income coun-
tries (Hotez et al., 2009; Murray et al., 2012). Indeed, hundreds
of millions of people are affected by neglected tropical dis-
eases, among which soil-transmitted helminthiasis is particularly
widespread (Pullan et al., 2014; Utzinger et al., 2012). Of note,
multiple species parasite infections often co-occur in the same eco-
epidemiological setting, i.e. intestinal protozoa, soil-transmitted
helminths, cestodes and trematodes (Phongluxa et al., 2013;
Sayasone et al., 2009, 2011; Steinmann et al., 2010).
Corresponding author at: Department of Epidemiology and Public Health, Swiss
Tropical and Public Health Institute, P.O. Box, CH-4002 Basel, Switzerland.
Tel.: +41 61 284 8214; fax: +41 61 284 8105.
E-mail address: peter.odermatt@unibas.ch (P. Odermatt).
sitism is common (Sayasone et al., 2007, 2009). With regard
to soil-transmitted helminths, Ascaris lumbricoides and Trichuris
trichiura are highly prevalent in the mountainous and highland
areas of the country, particularly in the north, while hookworm
is highly prevalent across the country (Ay Soukhathammavong
et al., 2012; Laymanivong et al., 2014; Rim et al., 2003; Sayasone
et al., 2011). The liver uke Opisthorchis viverrini and ve species
of small intestinal trematodes (i.e. Haplorchis taichui, Haplorchis
yogokawai, Haplorchis pumilio, Phaneropsolus bonnei and Prostho-
dendrium molenkampi) are endemic in the lowlands of the Mekong
River basin (Chai et al., 2005, 2007; Eom et al., 2014; Sayasone
et al., 2009). The blood uke Schistosoma mekongi is endemic in
the lower Mekong River, in the most southern part of the country
(Laymanivong et al., 2014; Muth et al., 2010; Sayasone et al., 2011;
Urbani et al., 2002).
Concurrent helminth infections pose serious challenges for the
diagnosis in clinical settings and are of public health concern.

http://dx.doi.org/10.1016/j.actatropica.2014.09.004
0001-706X/ 2014 Elsevier B.V. All rights reserved.
316 S. Sayasone et al. / Acta Tropica 141 (2015) 315321
Prevalence rates of parasitic infections are usually underestimated,
and hence, a combination of different techniques on repeatedly
collected stool samples is warranted to enhance the sensitivity of
helminth diagnosis (Knopp et al., 2008; Marti and Koella, 1993).
Importantly, the impact of concurrent infections on health is often
overlooked. It is generally thought that light-intensity infection
with a single parasite is associated with no or only little morbidity.
However, there is growing evidence that infections from multiple
parasite species, even at light intensity, may contribute to sub-
stantial morbidity to the host (Ezeamama et al., 2005; King and
Bertino, 2008; Sayasone et al., 2012). For example, infection with
soil-transmitted helminths result in a number of gastro-intestinal
discomforts (e.g. abdominal pain, chronic diarrhoea and vomiting)
and anaemia due to chronic blood lost in the intestinal tract due
to hookworm infection (Brooker et al., 2008). In Lao PDR, O. viver-
rini and S. mekongi might lead to severe morbidity (Sayasone et al.,
2012). Specically, O. viverrini infection lead to severe hepatobiliar
pathologies, and may develop to a fatal bile duct cancer (cholan-
giocarcinoma) (Sripa et al., 2012), while chronic infection with
S. mekongi contribute to a formation of hepatomegaly, periportal
brosis and portal hypertension (Biays et al., 1999; Keang et al.,
2007). Additionally, in areas of co-infection, substantial excess mor-
bidity has been observed, even among individuals with relatively
low intensity infections (Sayasone et al., 2012).
For the diagnosis of intestinal parasites, widely used techniques
are the Kato-Katz (KK) (Katz et al., 1972) and the formalin-ethyl
acetate concentration technique (FECT) (Elkins et al., 1991). The KK
technique is often employed in community-based epidemiological
surveys, and requires mainly reusable test kits. It is recommended
by the World Health Organization (WHO) as a suitable tool for
helminth diagnosis, particularly Schistosoma mansoni, Schistosoma
japonicum, S. mekongi and soil-transmitted helminths. However,
the KK technique fails to detect Strongyloides stercoralis, arguably
the most neglected of the soil-transmitted helminths (Olsen et al.,
2009; Schr et al., 2013b), and intestinal protozoa. The KK tech-
nique, moreover, has a low sensitivity because it is processing only
a small amount of faeces (41.7 mg). Light infections may be missed,
consequently, misjudging infection status of patients and underes-
timating the true community prevalence (Khieu et al., 2013; Kongs
et al., 2001; Utzinger et al., 2001). The FECT allows for the concur-
rent diagnosis of helminths and intestinal protozoa (Utzinger et al.,
2010). Analysis of multiple stool samples and the combination of
different techniques increase the sensitivity of helminth diagnosis
(Knopp et al., 2008; Steinmann et al., 2008).
The aim of this study was to investigate the effect of varying
sampling efforts on the diagnostic accuracy of the KK technique
and the combination of two methods (KK plus FECT) for detect-
ing eggs of soil-transmitted helminths, S. mekongi and food-borne
trematodes in rural communities in southern Lao PDR.
2. Materials and methods
2.1. Ethics statement
The study was reviewed by the institutional research commis-
sion of the Swiss Tropical and Public Health Institute (Swiss TPH;
Basel, Switzerland). Approval was granted by the ethics commit-
tee of Basel (EKBB, reference no. 255/06) and the national ethics
committee of the Ministry of Health (MOH) in Vientiane (ref-
erence no. 027/NECHR). Permission for eld work was obtained
from the MOH and the provincial and district health ofcers.
Village meetings were held and local authorities and villagers
were given detailed explanations about the purpose, procedures,
potential risks and benet of the study. An information sheet in
the local language was read to all household members and any
questions were answered using lay terminology. Individual oral
consent was obtained from all adult household members, while
parents (or legal guardians) provided consent on behalf of their
children. Consent was documented on a household registration
sheet. A literate witness observing this procedure signed the con-
sent form. Additionally, written informed consent was obtained
from the heads of household. This consent procedure was approved
by the aforementioned ethics committees.
At the end of the study, all individuals infected with O. viverrini,
S. mekongi, soil-transmitted helminths and intestinal protozoa
were treated according to national guidelines (MOH, 2004).
2.2. Study area and population surveyed
A cross-sectional survey was carried out between March and
May 2006 in Champasack province, southern Lao PDR. Details of the
study area and the population surveyed have been described else-
where (Sayasone et al., 2011). In brief, the study was conducted in
three eco-epidemiological settings, namely (i) the plains along the
Mekong River basin where O. viverrini and hookworm co-exist; (ii)
the small islands in the Mekong River in the southern part of the
province where S. mekongi, O. viverrini and hookworm co-occur;
and (iii) the mountainous and highland areas of the eastern part of
province where hookworm and other soil-transmitted helminths
are widespread. In each setting, three villages were selected in col-
laboration with district health authorities. At least 20 and up to 25
households were randomly selected in each village from readily
available census data. All family members aged 6 months were
invited to participate.
2.3. Study design
Eligible people were invited to submit three stool samples over
ve consecutive days. Unique identiers were assigned to house-
holds and study participants. Sample containers were prepared for
all members of study households. Participants names and unique
identiers were marked on the containers that were distributed to
the heads of household with detailed explanation on how to collect
a fresh morning stool. After lled containers were collected, new
empty containers were handed out until three stool samples were
submitted by study participants. In each village, a house (usually
a school or a temple) was designated as area of work for KK thick
smear preparation, and subsequent microscopic examination.
Stool samples were processed on the spot by experienced labo-
ratory technicians. A single KK thick smear was prepared from each
stool sample, using standard plastic templates holding 41.7 mg of
stool (Katz et al., 1972). Slides were allowed to clear for 30 min
prior to examination under a microscope. The number of eggs was
counted and recorded for each helminth species separately.
Additionally, exactly 300 mg of stool taken from one sample
was xed in a tube containing 10 ml of sodium acetate-acetic
acid-formalin (SAF) (Marti and Escher, 1990). SAF-xed samples
were forwarded to the parasitological department of the Faculty
of Medicine, National University of Lao PDR where the samples
were subjected to FECT (Elkins et al., 1991). Presence of helminth
species-specic infections and intensities were assessed with
quality control of laboratory staff from Swiss TPH. A random sam-
ple of 10% of the samples were re-examined by a senior laboratory
technician for quality control. If any differences were observed, the
results were discussed until agreement was reached. During the
laboratory work, senior technicians were available for any question.
2.4. Statistical analysis
Data were double-entered and cross-checked in EpiData version
3.1 (EpiData Association; Odense, Denmark). Statistical analyses
 S. Sayasone et al. / Acta Tropica 141 (2015) 315321 317

Table 1 Table 2
Stratication of helminth infection intensities investigated in Champasack province, Comparison of sex and age proles of fully compliant study participants (n = 485)
southern Lao PDR. and those who were excluded from the nal analysis (n = 728) in terms of sex and
age prole.
Parasite Infection intensity (EPG)
Indicator Final cohort Excluded LRT P-value
Light Moderate Heavy (n = 485) (n = 728)
Trematodes % (n) % (n)
O. viverrinia 1999 10009999 10,000
S. mekongib 199 100399 400 Sex
Nematodes Female 52.0 (252) 53.3 (388)
Hookwormb 11999 20003999 4000 Male 48.0 (233) 46.7 (340) 1.43 0.232
A. lumbricoidesb 14999 500049,999 50,000 Age groups (years)
T. trichiurab 1999 10009999 10,000 5 15.3 (74) 35.4 (258)
610 16.9 (82) 16.3 (119)
a
Intensity categories according to Maleewong et al. (1992). 1115 16.9 (82) 10.2 (74)
b
Intensity categories according to WHO (2002). 1630 16.5 (80) 16.0 (116)
3145 19.0 (92) 10.7 (78)
4660 10.1 (49) 7.5 (55)
were performed with STATA version 10 (Stata Corporation; Col- >60 5.3 (26) 3.9 (28) 54.67 <0.001
lege Station, USA). Individuals were included in the nal analyses
LRT: likelihood ratio test.
if they had submitted three stool samples that were subjected to
the KK technique and one of the stool sample additionally analysed
by FECT. The point prevalence of parasitic infections was deter-
mined. We employed a 2 -test to investigate associations between farming) was the main source of income (50.1%). Only seven study
categorical variables (e.g. infection status and sex, age group and participants (1.4%) were state employees.
stool sample analysis technique). Median and arithmetic means
of helminth eggs per 1 g of stool (EPG) for the entire population 3.2. Helminth infections according to sampling effort and
(infected and non-infected individuals) estimated by each method diagnostic technique
were calculated, using the 10th logarithm of the EPG plus 1 (i.e.
log10 (EPG + 1)). A non-parametric test (Wilcoxon matched-pairs Analysis of a single stool sample by the KK technique (76.2%,
signed-ranked test) was utilised to test whether there was a differ- 45/59) showed signicantly higher prevalence of S. mekongi com-
ence in intensity of parasitic infection. Likelihood ratio test (LRT) pared to FECT (23.8%, 14/59) on the same sample (2 = 20.96,
from a logistic regression model was assigned to associate differ- P < 0.001) and hookworm (64.5% (162/251) vs. 35.5% (89/251),
ences in sex and age groups between compliant and non-compliant 2 = 25.51, P < 0.001). On the other hand, both techniques per-
study participants. formed similarly well for diagnosis of O. viverrini, A. lumbricoides,
For each individual the infection intensity was calculated for T. trichiura, Taenia spp. and Hymenolepis diminuta (P > 0.10).
O. viverrini, S. mekongi, hookworm, A. lumbricoides and T. trichiura, Fig. 2 shows the observed prevalence of different helminths
according to Maleewong et al. (1992) and adopting from WHO according to sampling effort (single vs. multiple KK thick smears).
guidelines (WHO, 2002). Infections were classied into light, mod- The combined results of the KK technique (three samples) plus a
erate and heavy intensities (Table 1). single stool sample additionally analysed by FECT were considered
A mathematical model developed by Marti and Koella (1993) as diagnostic gold standard. Stool analysis of three samples with
was used to calculate the sensitivity and negative predictive value the KK technique plus a single FECT showed a signicant increase in
of the two diagnostic tests. The model was also used to estimate the prevalence of O. viverrini infection by 27.2% (67 persons) based
the true prevalence of each helminth parasite species in the study on the rst KK (from 51.1%, 95% condence interval (CI): 46.555.6%
population. The procedure follows an approach developed by to 65.0%, 95% CI: 60.569.2%) and by 22.6% (60 persons) based on
Mullen and Prost (1983). For all statistical analyses, our signicance a single FECT (from 52.6%, 95% CI: 48.057.0% to 65.0%, 95% CI:
level was set at a P-value of 0.05. 60.569.2%). For diagnosis of S. mekongi, similar observations were
made. While a single KK revealed a prevalence of 12.2% (59 per-
3. Results sons), our gold standard found a two-fold higher prevalence (24.3%
or 118 persons). A single FECT only revealed a prevalence of 4.1%
3.1. Study compliance and nal cohort (20 persons), which was a several-fold underestimation compared
to our diagnostic gold standard.
From 1213 individuals invited to participate (Fig. 1), 485 indi- Table 3 shows a comparison of helminth egg counts (EPG)
viduals fully complied (40.0%). The remaining 728 individuals were between a single KK and a FECT on the same sample for detection of
excluded from the nal analysis as there were missing personal O. viverrini, S. mekongi, hookworm, A. lumbricoides and T. trichiura
data (n = 162), were absence during parasitological surveys (n = 36), infections. Egg counts were signicantly higher when using KK
or failed to submit a second (n = 278), or a third stool sample for rather than a single FECT for all parasites investigated (P < 0.001).
KK thick smear examination (n = 184) or provided an insufcient
amount of stool for xing in SAF (n = 68).
The age and sex proles of the fully compliant individuals and 3.3. Diagnostic accuracy
the non-compliant study group is shown in Table 2. There was no
signicant difference for sex (LRT = 1.43, P = 0.232), whilst a sig- The analysis of three stool samples with the KK technique plus
nicant difference was found in the age categories (LRT = 54.67, one FECT increased the sensitivity of diagnosis for all helminth
P < 0.001). In the nal study cohort, there were 252 (52.0%) females. infections compared to single stool samples analysed by the KK
Approximately two-third of the study participants belonged to the technique or FECT and three stool samples analysed by the KK tech-
Lao-loum ethnicity (n = 333, 68.7%). Most study participants were nique alone with negative predictive values being close to 100%.
illiterate or only nished primary education (n = 418, 86.2%). Only Table 4 summarises the sensitivity of the KK technique and FECT
few participants attended secondary school or attained higher edu- on a single stool sample and, nally, all three stool samples for
cational levels (n = 67, 13.8%). Subsistence agriculture (mainly rice diagnosis of helminth infections.
318 S. Sayasone et al. / Acta Tropica 141 (2015) 315321

Fig. 1. Study diagram.

Table 3
Comparison of helminth egg counts per 1 g of stool (EPG) according to the Kato-Katz (KK) technique and the formalin-ethyl acetate concentration technique in a single stool
sample, and intensity classication for combining result among 485 fully complying study participants.

Arithmetic mean log10 (EPG + 1) Intensity classication

KK1 FECT1 P-value Light Moderate Heavy

Trematodes
O. viverrini 1.48 1.29 <0.001 55.7 41.2 3.1
S. mekongi 0.78 0.33 <0.001 62.1 27.8 10.1
Echinostoma spp. 0.34 0.01 0.013 n.a. n.a. n.a.
Nematodes
A. lumbricoides 1.24 0.81 <0.001 35.8 53.0 11.2
Hookworm 1.32 0.52 <0.001 93.6 6.4 0
T. trichiura 0.58 0.47 <0.001 96.0 4.0 0
Cestodes
Taenia spp. 0.03 0.02 0.982 n.a. n.a. n.a.
H. diminuta 0.01 0.01 0.318 n.a. n.a. n.a.

P-value: obtained from Wilcoxon matched-pairs singed (correct)-ranks test.

n.a.: no reference applicable for intensity classication.

Table 4
Sensitivity and negative predictive value of a single formalin-ethyl acetate concentration technique (FECT) and three Kato-Katz (KK) thick smear examinations given by
mathematical model for diagnosis of helminth infections in three districts of Champasack province, southern Lao PDR (n = 485).

FECT, n (%) KK, n (%) Sensitivity and negative predictive value of the test

Single 1st 2nd 3rd Sensitivity of 3 Samples Negative 3 Samples Negative

sample Sample Sample Sample one single examined with predictive examined with predictive
sample (% SD) KK (%) value (%) KK plus one FECT value (%)

Trematodes
O. viverrini 15 (3.1) 46 (9.5) 50 (10.3) 204 (42.1) 85.1 (2.0) 99.6 99.3 99.9 99.9
S. mekongi 3 (0.6) 60 (12.4) 30 (6.2) 25 (5.2) 65.4 (4.6) 86.8 95.3 98.6 99.5
Echinostoma spp. 2 (0.6) 11 (2.3) 10 (2.1) 8 (1.6) 68.7 (68.7) 93.0 99.4 99.4 99.9

Nematodes
A. lumbricoides 20 (4.1) 11 (2.3) 11 (2.3) 120 (24.7) 85.6 (2.8) 99.9 99.9 99.9 99.9
Hookworm 5 (1.0) 101 (20.8) 115 (23.7) 157 (32.4) 77.9 (2.2) 97.3 91.6 99.8 99.5
T. trichiura 7 (1.4) 37 (7.6) 40 (8.3) 43 (8.9) 73.0 (4.0) 95.9 98.6 99.5 99.8

Cestodes
Taenia spp. 4 (0.8) 4 (0.8) 5 (1.0) 0 48.2 (15.8) 79.9 99.5 92.8 99.8
H. diminuta 3 (0.6) 7 (1.4) 2 (0.4) 0 42.9 (16.9) 47.7 97.9 89.3 99.7

Negative predictive value = true negative/(false negative + true negative), n = number of positive study participants diagnosed by each individual test, % = percentage and
SD = standard deviation.
 S. Sayasone et al. / Acta Tropica 141 (2015) 315321 319

Fig. 2. Difference in observed and true prevalence in one, two and three stool samples for Kato-Katz (KK) singly or together with formalin-ethyl acetate concentration
technique (FECT) for the diagnosis of helminth infection in three districts of Champasack province, southern Lao PDR (n = 485).

4. Discussion that the KK technique is a more adequate technique. However, a

We investigated the effect of varying stool sampling effort
and the combination of two techniques (KK and FECT) for the
diagnosis of helminth infections in Champasack province, south-
ern Lao PDR. Study participants submitted three early morning
faecal samples over ve consecutive days. These samples were
rst examined on the spot by the KK method with minimal
time delays between stool production and microscopic analysis,
whereas from a single sample, 300 mg were xed in SAF for subse-
quent FECT analysis approximately six months later. It is important
to note that a single stool sample examined by the KK technique
revealed a S. mekongi prevalence of 12.2%, whereas the cumula-
tive prevalence after examination of triplicate KK thick smears was
23.7%.
A single SAF-xed stool sample subjected to FECT revealed a
S. mekongi prevalence of only 4.1%, and hence this procedure is
clearly not indicated for the diagnosis of S. mekongi. We conclude
single FECT still discovered three S. mekongi infections, which had
been missed by the KK technique, even after triplicate examina-
tion. Hence, the overall prevalence of S. mekongi was 24.3%. Of note,
the observed prevalence of triplicate KK plus a single FECT was
still considerably lower than the true prevalence predicted by a
mathematical model (28.1%).
Large increases in prevalence estimates when combining results
from multiple KK thick smears and a single FECT were also observed
for hookworm, T. trichiura, Taenia spp. and H. diminuta. On the
other hand, only a relatively small increase in prevalence estimates
resulted for O. viverrini (single KK: 51.1%; single FECT: 52.6% vs.
65.0% for our diagnostic gold standard) and A. lumbricoides (27.2%
and 27.4% vs. 33.4%). Furthermore, the examination of three stool
samples with the KK technique resulted in a considerably higher
sensitivity of the tests for diagnosis of all helminths. The nega-
tive predictive values were close to 100%, compared to analysing a
single sample.
320 S. Sayasone et al. / Acta Tropica 141 (2015) 315321
Our ndings conrm that microscopic examination of a single
stool sample, either using KK or FECT, underestimates the true
prevalence of helminth infections (Khieu et al., 2013). Light infec-
tions are particularly prone to be missed (Knopp et al., 2008;
Steinmann et al., 2008). Examination of three stool samples using
only the KK technique showed a considerably increased test sensi-
tivity with a negative predictive value above 90% for the observed
helminths as function of sampling effort (Marti and Koella, 1993).
In fact, adding a FECT into the analysis of three stool samples using
the KK technique showed a higher prevalence and sensitivity than
analysis with KK alone for all helminths investigated, except S.
mekongi. These ndings support previous observations made else-
where (de Vlas and Gryseels, 1992; Knopp et al., 2008; Marti and
Koella, 1993; Steinmann et al., 2008).
Almost two third (65.0%) of our fully compliant study cohort
was infected with O. viverrini. Minute intestinal ukes (MIF) have
eggs that are morphologically very similar to those of O. viverrini.
It follows that some misclassication cannot be excluded. Earlier
studies have reported MIF in Lao PDR (Chai et al., 2005, 2007, 2013;
Sayasone et al., 2009). However, individuals infected with MIF were
concurrently infected with O. viverrini (Sayasone et al., 2009).
Furthermore, the observed prevalence of S. mekongi and hook-
worm was signicantly higher in KK than FECT when a single
stool sample was analysed. In addition, we also observed that the
KK technique resulted in consistently higher EPG estimates when
compared to FECT. Previous studies have shown that the perfor-
mance of FECT is better or equal to KK for the detection of small
trematode eggs if stool samples were xed in 7 ml of 10% formalin-
solution (Sithithaworn et al., 1994). The reduction of sensitivity
when combining both methods for S. mekongi diagnosis and the
low observed EPG when using FECT for all helminth species is of
concern and might be due to the small proportion of formalin in
the SAF-solution. In fact, the SAF is ideal to preserve intestinal pro-
tozoa (Marti and Koella, 1993), but it might be less effective to
preserve relatively large helminth eggs such as S. mekongi and other
helminths over prolonged periods (i.e. approximately 6 months in
the present study).
We found a high prevalence of hookworm infection in rural
dwellers in three different settings of Champasack province. This
nding suggests that prevalence of S. stercoralis might also be ele-
vated in these areas as both parasites have the same route of
infection (Khieu et al., 2013; Schr et al., 2014). Previous stud-
ies have shown that S. stercoralis can be fatal, particularly among
immune-compromised individuals (Keiser and Nutman, 2004).
Analysis of SAF-xed stool sample by FECT in the current study
identied 4.6% of study participants infected with S. stercoralis.
However, FECT is not the recommended technique for S. stercoralis
diagnosis, and hence the true prevalence might be considerably
higher. More reliable techniques are the Koga agar plate and the
Baermann funnel (Khieu et al., 2013; Koga et al., 1991; Stothard
et al., 2008). Recently a promising polymerase chain reaction (PCR)
method was eld-evaluated for S. stercoralis diagnosis (Schr et al.,
2013a). Further investigations are warranted, using a sensitive
diagnostic approach to estimate the extent of S. stercoralis infection
in Lao PDR.
It is important to note that repeated stool sampling (e.g. three
samples were obtained in the current study over ve consecutive
days), subjected to multiple techniques, provided a higher diag-
nostic accuracy. However, such a diagnostic approach has some
challenges. For example, more than 50% of the people invited, did
not fully comply, since they did not provide three stool samples of
sufcient quantity within the given time frame. Attrition analysis
showed a signicant difference in the age categories among com-
pliant and non-compliant participants, and hence, there is a risk
of bias. Indeed, differences in age proles might result in an over-
or under-estimation both in terms of prevalence and intensity of
helminth infection. The underlying rationale is that parasitic infec-
tions have distinct age-prevalence curves (e.g. soil-transmitted
helminths and Schistosoma infections show peak prevalence and
intensity among school-aged children (Sayasone et al., 2011), while
for O. viverrini, peaks are observed in adults (Forrer et al., 2012;
Phongluxa et al., 2013; Sayasone et al., 2007). Furthermore, stool
sampling over consecutive days is somewhat invasive and requires
considerable human and nancial resources. This calls for novel
diagnostic approaches that must be rigorously validated in the
eld, including cost considerations (Speich et al., 2010). FLOTAC, for
example, is a new multivalent diagnostic technique for epidemio-
logical surveys (Cringoli et al., 2010). We have recently tested the
FLOTAC method for the diagnosis of O. viverrini which proved chal-
lenging, most probably because the operculate eggs are bursting
when they get in contact with otation solutions.
5. Conclusion
Analysis of multiple stool samples by the KK technique, sup-
plemented with a single FECT allowed for reasonably accurate
diagnosis of helminth infections in this setting of Champasack
province, southern Lao PDR. In our view, the combination of these
two methods if resources allow performed on at least two stool
samples, provides for a reasonably sensitive diagnosis of helminth
infection. The combination of different techniques and an enhanced
sampling effort is mandatory for generating high-quality baseline
data, and should be considered for evaluation of anthelminthic
drug efcacy studies and for rigorous monitoring of helminthia-
sis control programmes. Our ndings underscore the importance
of repeated stool sample analysis using multiple techniques in
order to reach high sensitivities. Developing and adhering to inter-
national guidelines would increase comparability across studies
pertaining to the epidemiology and control of helminthiasis.
Author contributions
SS, JU and PO conceived and developed the study; SS collected
data; KA had overall responsibility of data collection; SS analysed
the data and interpreted results together with JU and PO; SS drafted
the manuscript; JU, KA and PO assisted with manuscript revisions;
all authors read and approved the nal submitted manuscript; SS
and PO are guarantors of the paper.
Acknowledgements
We are grateful for the active participation of the people in the
Khong, Mounlapamok and Paksong districts, and the support of
the curative and preventive health authorities of the various loca-
tions. We acknowledge the support of Professor Marcel Tanner,
Director of the Swiss Tropical and Public Health Institute and Mrs.
Isabelle Grilli who helped with the stool sample examinations. The
contributions of Professors Jennifer Keiser, Christoph Hatz and
Penelope Vounatsou at various stages of the study are highly appre-
ciated. This investigation was funded by the Swiss National Science
Foundation and the Swiss Agency for Development and Coopera-
tion (project no. NF3270B0-110020).
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