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Parasitology International 62 (2013) 564567

Contents lists available at ScienceDirect

Parasitology International
journal homepage: www.elsevier.com/locate/parint

Enzyme-linked immunosorbent assay for the diagnosis of


Wuchereria bancrofti infection using urine samples and
its application in Bangladesh
Mohammad Sohel Samad a, Makoto Itoh a,, Kazuhiko Moji b, Moazzem Hossain c, Dinesh Mondal d,
Mohammad Shaul Alam d, Eisaku Kimura a
a

Department of Infection and Immunology, Aichi Medical University School of Medicine, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan
Research Institute for Humanity and Nature (RIHN: Chikyu-ken), 457-4 Motoyama, Kamigamo, Kita-ku, Kyoto 603-8047, Japan
c
Institute of Allergy & Clinical Immunology of Bangladesh (IACIB), Green Super Market, Green Road, Dhaka 1205, Bangladesh
d
International Center for Diarrheal Disease and Research, Bangladesh (icddr,b) 68, Shahid Tajuddin Ahmed Sharani, Mohakhali, Dhaka 1212, Bangladesh
b

a r t i c l e

i n f o

Article history:
Received 24 April 2013
Received in revised form 15 August 2013
Accepted 19 August 2013
Available online 27 August 2013
Keywords:
Filariasis
Urine diagnosis
Recombinant SXP1
IgG4

a b s t r a c t
In Sri Lanka, urine ELISA showed high sensitivity and specicity in detecting laria-specic IgG4. It also produced
much higher positive rates than antigen tests in prevalence studies with young children. In this study, we have conrmed the usefulness of urine ELISA in the eld of Bangladesh. The ELISA detected 89 of 105 (85%) ICT antigen test
positive subjects in endemic areas. With both ICT and microlaria positives, the sensitivity was 97% (30/31). All of
104 ICT negative people in a non-endemic area were ELISA negative (100% specicity). In a prevalence study with
319 young children (510 years) from a low endemic area after ve rounds of MDA, seven (2.2%) were detected
by the present urine test, but only one (0.3%) by ICT (P = 0.075). The satisfactorily high sensitivity, 100% specicity
and effective case detection among young ages along with scope for analyzing the titers will indicate urine ELISA
to be an effective tool in the post-MDA surveys to conrm elimination or to detect resurgence in Bangladesh.
2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Lymphatic lariasis is a signicant public health concern in endemic
countries especially in Asia and Africa. Causing long-term disability in
tens of millions of people, it has been a major obstacle to socioeconomic
development [13]. Globally, more than 120 million people in 81 countries are reported infected with 1.3 billion people at risk of infection
[46].
In 1997, WHO made a resolution to eliminate the disease from the
world, and the Global Programme to Eliminate Lymphatic Filariasis
(GPELF) has been implemented since 2000 with the target of elimination
by the year 2020. The basic strategy is to treat all eligible people in
endemic areas by means of annual mass drug administration (MDA)
for 46 years [7]. The Bangladesh government for its part made a national
plan to eliminate the disease by 2015 [8]. Since the start of GPELF, many
endemic countries have completed the planned MDAs and the prevalence
of larial infection has been reduced signicantly [913]. At this point
of post-MDA low endemic stage, these countries have to formulate the

end-game strategies in which the methods and criteria to stop MDAs,


conrm elimination and detect resurgence need to be addressed [1417].
In post-MDA stage, when microlaria (mf) densities have become
low, the standard blood lms are not sensitive anymore, and immunodiagnoses that detect larial antigens or specic antibodies will be
able to play a more signicant role. Itoh et al. (2001) reported an
ELISA that uses urine samples for serum. It showed a high sensitivity
of 95.6% with Wuchereria bancrofti-infected Sri Lankans and a specicity
of 99.0% with urine from non-endemic areas in Laos, Thailand, and Japan
[18]. Due to its ease in collecting samples, the ELISA has been accepted
well by the people living in endemic areas.
We planned to apply the urine-based ELISA method in
Bangladesh and as a rst step, evaluated the sensitivity and specicity
with Bangladeshi urine samples, and then conducted a prevalence
survey with schoolchildren in a post-MDA area. The result was compared
with the prevalence obtained by ICT antigen test.
2. Materials and methods
2.1. Study area and subjects

Corresponding author. Tel.: +81 561 62 3311; fax: +81 561 63 3645.
E-mail addresses: sohelsamad_84@yahoo.com (M.S. Samad),
macitoh@aichi-med-u.ac.jp (M. Itoh), moji-k@chikyu.ac.jp (K. Moji),
profdrmoazzemhossain@gmail.com (M. Hossain), din63d@icddrb.org (D. Mondal),
shaul@icddrb.org (M.S. Alam), kimura@aichi-med-u.ac.jp (E. Kimura).
1383-5769/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.parint.2013.08.007

2.1.1. Sensitivity study


In Ranishankail and Haripur upazilas (sub-districts) of Thakurgaon
district in the northern region of Bangladesh where lymphatic lariasis
is endemic and 4 rounds of MDA have been completed, 749 people (680

M.S. Samad et al. / Parasitology International 62 (2013) 564567

males, 69 females) aged between 20 and 70 years (mean: 34 years),


were examined for the circulating laria antigen with immunochromatographic card test (NOWICT, Binax Inc., Portland, Maine,
USA). After obtaining the results, urine samples were collected from
105 ICT positive people. On the same day at night (10:00 pm12:00
midnight) blood samples were collected from them for mf smears.
2.1.2. Specicity study
Dagonbhuiyan upazila of lymphatic lariasis non-endemic Feni
district in the southern part of the country was selected. A total of 104
males aged 20 to 70 years were selected as non-endemic healthy
controls (NEHC) and examined with ICT for the conrmation of antigen
negativity, and their urine samples were collected to be used as negative
standard.

3. Results
3.1. Sensitivity studies
For the sensitivity study, 749 people were screened rst with ICT, and
105 positive subjects were found. Then, the positives were tested for mf
and 31 positives and 74 negatives were obtained. On this basis, 2 categories of positive standards were considered: positive standard 1 (PS1); 31
subjects with both ICT and mf positives (Ag+/mf+) and positive standard 2 (PS2); 105 ICT positives (Ag+/mf + and Ag+/mf). With PS1
and PS2, the sensitivity of urine ELISA was found to be 96.8% (30/31)
and 84.4% (89/105) respectively (Table 1). Out of the 16 ELISA negatives
15 were mf negative as well (Fig. 1). There were no differences in the
average antibody units (1017 versus 1015 U; t-test, P N 0.9) between
Ag+/mf + and Ag+/mf people.

2.1.3. Prevalence study


The efcacy of detecting infection/exposure to W. bancrofti was
compared between urine ELISA and ICT with 319 schoolchildren aged
510 years in Atowari upazila, Panchagarh, the northernmost district
of Bangladesh. The upazila showed the low mf prevalence rate (1.4%)
after 5 rounds of MDAs (2008, government report).

3.2. Specicity studies

2.2. Specimens

3.3. Prevalence study

For all blood tests, nger-prick blood was used: 100 l for ICT, and
60 l for night blood smears. For urine ELISA, 5 ml urine was collected,
added with sodium azide at the nal concentration of 0.1% as a preservative, and the samples were stored at 4 C except during the period
of transportation to Japan.
2.3. Ethical considerations
The participants were informed about the purpose of the study and
that their samples would be transported to Japan for research purposes
only, and not for the individual diagnosis of the disease. Prior to the
collection of all samples, the written informed consent from all the
participants or from their headmasters/mistresses in the case of minor
school children were obtained. The study was reviewed and approved
by the Ethics Committee of Aichi Medical University School of Medicine
and the National Research Ethics Committee (NREC) of the Bangladesh
Medical Research Council.
2.4. Enzyme-linked immunosorbent assay
The urine ELISA developed in our lab that detects laria specic
IgG4 from urine samples [18] was performed with a modication [19].
In brief, at bottomed, 96-well microtiter plates (Maxisorp; Nunc,
Roskilde, Denmark) were coated with recombinant Wb-SXP1 antigen
(recWb-SXP1) (1 g/ml) at 4 C overnight. After blocking with the
casein buffer (1% casein in 0.05 M TrisHCl buffer with 0.15 M NaCl,
pH 7.6) for 2 h at room temperature, urine samples were directly
applied to the plates (100 L per well) and incubated overnight at
25 C. After four washes with phosphate-buffered saline (PBS), pH 7.4
containing 0.05% Tween 20, 100 L peroxidase conjugated mouse
monoclonal antibody to human IgG4 (Southern Biotech, Birmingham,
AL), diluted 1: 4000, was added to each well. After incubation at 37 C
for 1 h, the plates were washed four times, and then incubated with
ABTS Peroxidase Substrate System (KPL Inc., Gaithersburg, MD) for
one hour at room temperature and the optical density was measured
at 415 nm and 492 nm as a reference. Each sample was assayed in
duplicate. Antibody levels were expressed as units (U) estimated from
a standard curve constructed with serially diluted positive sera ranging
from 0 to 7290 U. The cutoff value in the study was 7.08 U.

565

All of the selected 104 subjects from the non-endemic area were
tested with ICT, conrmed to be negative and then used as the NEHC.
Their urine samples were all negative with ELISA resulting in 100%
specicity. The mean antibody titer among the NEHC was found to be
very low (Fig. 1).

With 319 schoolchildren between the age group of 5 years to


10 years, the ELISA detected 7 positives (2.2%), while ICT did only 1
(0.3%) (Table 2). However, the difference was not statistically quite signicant (2 test with Yate's correction, P = 0.075) and the ICT positive
subject was ELISA negative. The IgG4 titers are analyzed according to
age (Fig. 2). There was no positive among 5 year to 6 year old children
(0/102) and the age of the youngest IgG4 positive was 7 years. The positive rates of age group 7 years, 9 years and 10 years were 1.6% (1/62),
4% (2/49) and 6.8% (4/59) respectively. This shows a trend of gradual
increase in the number of IgG4 positives and IgG4 titers with age,
although signicance analysis could not be done because of few numbers of positives. The highest IgG4 titer among the positives was found
to be 52.3 U, indicating a low level of antibody which is considered normal in an area where ve rounds of MDA have already been completed.
In addition, the distribution of titers under the cutoff line shows a clear
dot-sparse space.
4. Discussion
In the present study in Bangladesh, with the most certain positive
standard, that is, mf and ICT positives, urine ELISA with recWb-SXP1
resulted in 96.8% sensitivity. With a more practical positive standard
(ICT positive subjects), the ELISA gave 84.8% sensitivity, which was
considered satisfactory for use in Bangladesh eld. The sensitivity
seems to be lower than the 95.6% sensitivity reported in Sri Lanka
with Og4C3 antigen test (Trop-Ag W. Bancrofti, TropBio Pty. Ltd.,
Queensland, Australia) positives as the standard [18]. The difference
could be explained in part by the fact that Sri Lankan data were obtained
before MDAs, while ours were after 4 rounds of MDA which signicantly

Table 1
Sensitivity of the urine ELISA in Bangladesh with 2 different positive standards (PS1
and PS2).
Category of
positive standard

No. of subjects
examined

Urine ELISA

PS1: Ag (+) and mf (+)


PS2: Ag (+) and mf (+ & )

31
105

30
89

Sensitivity (%)

No. of positives
96.8
84.8

566

M.S. Samad et al. / Parasitology International 62 (2013) 564567


(n=105)

(n=31)

(n=49)

Log (Antibody unit+1)

Log (Antibody unit+1)

2
(n=104)

(n=59)

(n=62)

(n=75)

(n=47)
(n=27)

0
PS1

NEHC

PS2

Fig. 1. Individual anti-laria IgG4 antibody levels in urine according to infection status of
W. bancrofti. PS1 = antigen(+) and mf (+); PS2 = antigen (+) and mf (+ &).
NEHC = antigen negative non-endemic healthy controls. The dotted line indicates a cutoff value. n: no. of subjects examined.

reduced the prevalence and intensity of infection in the study areas.


The high specicity of the present ELISA (100%) was in accordance
with previous results obtained in different countries [18,23].
In addition, the present study revealed that the urine ELISA is
probably more sensitive than ICT with children aged 510 years after
5 rounds of MDA, implying its effectiveness in detecting low level infection/transmission. In Walgama, Sri Lanka, where the reported mf rate
was 5.7% [20], urine ELISA detected 2.1 times more infections than ICT
among 68 children aged 110 years [21], and in Deniyaya with much
lower prevalence, urine ELISA detected 4 times more infections than
Og4C3 ELISA among 445 subjects aged under 9 years [22].
A quantitative antibody test like ELISA offers the added opportunity
of analyzing titers. Being analyzed by age of children, the titer distribution pattern can indicate a trend in recent transmission. Utilizing these
advantages, urine ELISA in Sri Lanka found a 3 year old as the youngest
positive and showed a trend of gradual increase in the antibody titer
with age and thus conrmed the presence of a focus of active transmission in an area where no lariasis had been reported [22]. Urine ELISA
was also applied successfully to reconrm elimination of lariasis
in two cities, Yongjia and Gaoan, in China, which declared elimination
as per Chinese criteria. In Yongjia only 0.08% (2/2411) schoolchildren
between 6 and 10 years of age were positive and in Gaoan only 0.35%
(28/7998) children aged 5 to 16 years were found to be positive. All
of the ELISA positive children along with their family members were
tested with a number of different diagnoses and found negative [23].
These ndings suggest that the urine-based diagnosis is an effective
tool in the post-MDA surveys. Our study in Bangladesh produced high
enough sensitivity and 100% specicity with its successful application in
the eld among schoolchildren, and thus has conrmed the usefulness
and applicability of urine ELISA in lariasis control in this resourcelimited densely populated country.
Acknowledgments
The authors would like to thank the Japan Overseas Cooperation Volunteers (JOCVs) working in lariasis endemic districts in Bangladesh for

Table 2
Case detection: the urine ELISA vs. ICT in children (510 years) from the endemic area in
Bangladesh after 5 rounds of mass drug administration.
The urine ELISA

ICT

Positive
Negative
Total

Positive

Negative

Total

0
7
7

1
311
312

1
318
319

Age in Year
Fig. 2. Individual anti-laria IgG4 antibody levels in urine examined with schoolchildren.
The dotted line indicates a cutoff value. n: no. of subjects examined.

lariasis control and the technical staff members from the Directorate
General of Health Services (DGHS), Dhaka and eld staff members
of Thakurgaon, Feni and Panchagarh districts working under the
Ministry of Health and Family Welfare, Bangladesh, for their all-out support. This study was supported by Grants-in-Aid for Scientic Research
(B) No. 24406014 from the Japan Society for the Promotion of Science
and partly by the Research Institute for Humanity and Nature, Kyoto,
Japan.

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