- Email: [email protected]
Lateral flow dipstick antigen assay for human cystic echinococcosis
Accepted Manuscript Title: Lateral flow dipstick antigen assay for human cystic echinococcosis Authors: Sam Khanbabaie, Mehdi Riazi, Chiat Han Chang, Muhammad Hafiznur Yunus, Rahmah Noordin PII: DOI: Reference:
S0001-706X(18)30627-2 https://doi.org/10.1016/j.actatropica.2018.11.018 ACTROP 4834
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
19 May 2018 6 October 2018 16 November 2018
Please cite this article as: Khanbabaie S, Riazi M, Chang CH, Yunus MH, Noordin R, Lateral flow dipstick antigen assay for human cystic echinococcosis, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Lateral flow dipstick antigen assay for human cystic echinococcosis
Authors and affiliations: Sam Khanbabaie1, Mehdi Riazi2, Chiat Han Chang1, Muhammad Hafiznur Yunus1, and Rahmah Noordin1*
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
IP T
1
Penang, Malaysia.
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.
SC R
2
U
Authors’ addresses: Sam Khanbabaie, Chiat Han Chang, Muhammad Hafiznur Yunus, and
Malaysia,
Penang,
Malaysia,
E-mails:
[email protected],
A
Sains
N
Rahmah Noordin, Institute for Research in Molecular Medicine (INFORMM), Universiti
M
[email protected], [email protected], and [email protected]. Mehdi Riazi,
[email protected].
ED
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia, Email:
(INFORMM),
Universiti
Sains
CC E
Medicine
PT
*Corresponding author: Prof. Dr. Rahmah Noordin, Institute for Research in Molecular
A
[email protected]; Tel.: +604 653 4800
1
Malaysia,
Penang,
Malaysia;
E-mail:
Lateral flow dipstick antigen assay for human cystic echinococcosis
Abstract Cystic echinococcosis (CE) is a neglected zoonotic disease with a worldwide distribution and is a major public health problem in some areas. Diagnosis of CE is mainly based on clinical
IP T
symptoms, imaging and serological testing, however, improvement in serodiagnosis is still needed. This study was aimed at detecting circulating Echinococcus antigen in CE patients
SC R
using a lateral flow dipstick (LFD) assay. Three types of hydatid antigens i.e. hydatid cyst
fluid (HCF), native antigen B (nAgB) and recombinant antigen B (rAgB) were prepared and
U
polyclonal rabbit antiserum was raised against each antigen. Purified IgG fractions were
N
prepared and a portion was conjugated to gold nanoparticles. After a series of optimizations,
A
a final antigen detection LFD assay was developed using a combination of anti-nAgB-IgG
M
and gold-conjugated anti-HCF-IgG. Evaluation of the assay showed that 27 out of 35 (77%) serum samples from CE patients gave positive results. Meanwhile, the test showed a
ED
diagnostic specificity of 82% when tested with sera from 38 healthy individuals and 13 patients with other parasitic diseases. In conclusion, the antigen detection LFD assay seemed
PT
to be useful for diagnosis of CE and possibly for post-treatment follow-up, and merit further evaluation studies. We foresee that it may improve serodiagnosis of CE when used in tandem
CC E
with an antibody detection test.
A
Keywords: cystic echinococcosis; antigen detection; lateral flow dipstick assay; polyclonal antibodies; Echinococcus granulosus
2
1. Introduction Human cystic echinococcosis (CE) or hydatid cyst disease is a chronic infection caused by metacestode of Echinococcus granulosus which can infect both domestic herbivores and humans (Eckert et al., 2001). Adult cestodes live in the intestine of dogs and other carnivores (definitive hosts) and shed their eggs in the hosts’ feces. Sheep, goat, and
IP T
other herbivores (intermediate hosts) acquire the infection after peroral ingestion of plants or possibly water contaminated with the eggs. Human becomes infected by accidental ingestion
SC R
of E. granulosus eggs. Upon entry into the body, the larvae form one or more cysts in
different organs, mostly in liver and lung (Eckert et al., 2001; Moro and Schantz, 2009). CE
U
is distributed worldwide and is considered as a major public health problem in developing
N
countries (Brunetti et al., 2011; Deplazes et al., 2017). CE causes significant economic losses
A
due to costs of diagnosis, medical treatment, and resultant disabilities in some human cases
M
(Budke et al., 2006).
Accurate diagnosis of CE is challenging and early diagnosis can lead to better
ED
management and treatment outcomes at a lower cost (Zhang et al., 2003). Clinical assessment, imaging, and serology are the main tools for CE diagnosis (Brunetti et al., 2010).
PT
The World Health Organization Informal Working Group on Echinococcosis (WHO-IWGE) suggested using ultrasound (US) as the imaging technique of choice for diagnosis,
CC E
classification and follow-up of hydatid cyst in the abdominal cavity (WHO, 2003). However, US normally cannot detect lung CE (Torgerson and Deplazes, 2009) and imaging techniques
A
usually are expensive and may not be available in some low resource areas (McManus et al., 2012). Additionally, sometimes imaging results may not be conclusive. WHO recommended applying other diagnostic techniques such as immunodiagnosis in uncertain/suspected cases of CE as a confirmatory test (WHO, 2003). Torgerson and Deplazes (2009) clearly demonstrated the value of using multiple tests, i.e. imaging and immunodiagnosis, for
3
diagnosis of CE especially in mass screening of endemic areas. They suggested to initially use a highly sensitive test to ensure that most CE cases are detected, followed by a highly specific test to confirm the result.
In some settings, immunodiagnostic tests may be
considered as the primary method for CE diagnosis (Zhang et al., 2003). There are several serological methods available for detection of antibodies to CE i.e.
IP T
enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test
(IFAT), immunoelectrophoresis (IEP), and immunoblotting (IB) (Zhang et al., 2012).
SC R
However the available tests have a number of disadvantages such as variable sensitivity, cross reactivity with antibodies to other infections, difficulty to differentiate between past and
U
present infections, and may not be suitable for evaluating treatment efficacy(Carmena et al.,
N
2006; Mariconti et al., 2014). In this regard, an antigen detection test may be able to assist in
A
addressing the latter two of the four aforementioned limitations.
M
A wide range of diagnostic sensitivity and specificity of antibody detection assays have been reported (Zhang et al., 2012). Up to 40% of patients with confirmed CE were
ED
reported to show false negative results by antibody detection tests (Craig, 1986). Meanwhile hydatid antigens can be detected in serum of patients with CE, and this may be due to leakage
PT
of hydatid fluid and metabolic products of the cyst (Gottstein, 1984; Craig, 1986; Craig et al., 1996). It has been demonstrated that some sera of patients with CE contained hydatid
CC E
antigens but antibody detection assays on the same samples showed negative results (Gottstein, 1984; Moosa and Abdel-Hafez, 1994). Moreover, antibodies may persist for long
A
periods after treatment of patients with CE by surgical cyst removal and/or chemotherapy (Ray et al., 2002). On the other hand, circulating antigens are absent or quickly disappears after successful treatment (Craig, 1986). Therefore, antigen detection test may be useful for diagnosis of CE and follow-up of treatment efficacy. It can be envisaged that the use of both
4
antigen and antibody detection assays would be ideal for serological diagnosis of CE. However, currently, there is no commercial antigen detection test for CE diagnosis. Previously it has been reported that hydatid cyst antigens can be detected in serum, urine, and saliva of patients with CE, using ELISA, enzyme-linked immune-electro-transfer blot (EITB), latex agglutination test (LAT) and co-agglutination test (Co-A) (Ravinder et al.,
IP T
2000; Devi and Parija, 2003; Sunita et al., 2011). However, the reported diagnostic
sensitivities were not satisfactory, ranging from 25% to 83% (Ravinder et al., 1997, 2000;
SC R
Devi and Parija, 2003; Sadjjadi et al., 2009; Sunita et al., 2011; Swarna and Parija, 2012; Chaya and Parija, 2013; Bauomi et al., 2015). In an effort to address this diagnostic
U
challenge, the present study was aimed at developing an antigen detection test for CE in a
A
N
lateral flow (LFD) dipstick assay format.
2.1 Human serum samples
M
2. Materials and methods
ED
At the stage of development and evaluation of a test, it is important to select and use defined serum samples which are ‘clear-cut in their infection status’. Thus we used 86 human serum
PT
samples and categorized them into three groups. Group I comprised serum samples from Iranian patients with surgically confirmed CE (n = 35) and were positive by a commercial
CC E
ELISA kit i.e.Anti-Echinococcus ELISA IgG (EUROIMMUN AG, Luebeck, Germany). Iran is a known endemic area for CE, and serum samples from Iranian patients were collected at
A
several hospitals in Tehran, Iran, anonymised and brought to the Universiti Sains Malaysia. Twenty three of the patients (65.7%) were female and 12 (34.3%) were male. The patients age ranged from 11 to 76 years old. Four patients had single lung cyst, one patient had single cyst in both lung and liver and the rest had single or multiple liver cysts. Information
5
regarding the stage of the cysts was not available. Serum samples were collected just prior to undergoing surgery and none of the patients received treatment for CE before surgery. Group II comprised serum samples from Malaysian healthy individuals (n = 38) that were negative by the ELISA kit. Meanwhile Group III comprised serum samples from
IP T
Malaysian patients with other parasitic diseases that were negative by the ELISA kit i.e. cysticercosis (n = 2), ascariasis (n = 3), toxocariasis (n = 2), trichuriasis (n = 2), taeniasis (n = 1) and hookworm infection (n = 3). Malaysia and its neighboring are not endemic for CE.
SC R
These sera were banked and anonymized serum samples and their use for evaluation of
diagnostic sensitivity and specificity of laboratory tests was permitted by the Universiti Sains
N
U
Malaysia Research Ethics Committee.
A
2.2 Preparation of hydatid cyst fluid
M
Previously, crude hydatid cyst fluid (HCF) was collected aseptically from fertile hydatid cysts collected from slaughtered sheep from abattoirs in Rasht, Iran, and frozen at -
ED
80oC. In this study, the stored HCF was centrifuged at 10,000 × g for 30 minutes to separate the protoscolices and suspended solids from the supernatant. The supernatant was then
PT
collected and the protein concentration was determined by the Bio-Rad RCDC protein assay
CC E
kit (Bio-Rad, Hercules, CA, USA), then stored at -80oC.
2.3 Preparation of native antigen B
A
Native Antigen B (nAgB) was produced from HCF based on a previous method
(Khalilpour et al., 2014). Briefly, 100 mL of HCF was dialyzed against 5 mM acetate buffer (pH 5.0) overnight at 4oC and the dialyzed sample was centrifuged at 50,000 × g for 30 minutes. The supernatant containing sheep albumin was discarded and the pellet was suspended in phosphate buffer saline, pH 7.2 (PBS), at 100 µg pellet to 1 mL buffer. Next, an 6
equal volume of saturated ammonium sulphate was added to the suspension and the mixture was agitated at 4oC for 15 minutes. It was then centrifuged at 4,000 × g for 30 minutes at 4oC to remove the globulin. The resulting supernatant was dialyzed against PBS at 4oC for 24 hours to remove the ammonium sulphate. Finally, the sample was boiled for 15 minutes and centrifuged at 50,000 × g for 60 minutes. The protein concentration of the supernatant
IP T
containing nAgB was determined and stored at -20oC.
SC R
2.4 Preparation of recombinant antigen B
Previously prepared pET32 expression vector (Novagen, Madison, WI, USA) harboring
U
open reading frame of recombinant antigen B (rAgB) was transformed into Escherichia coli
N
strain C41 (DE3) (Lucigen, Middleton, WI, USA). A single bacterial colony was cultured in Terrific broth (TB) supplemented with ampicillin (100 µg/mL) and placed in a shaker-
A
incubator at 30oC, overnight. Fresh TB medium was added to the overnight culture, at a ratio
The
expression
of
rAgB
was
induced
with
1
mM
isopropyl-β-D-
ED
0.4-0.6.
M
of 10:1, then further incubated at 37oC until the optical density at 600 nm (OD600) reached
thiogalactopyranoside (IPTG) (Thermo Scientific, Waltham, MA, USA), and further cultured
PT
for 4 hours at 30oC. Next, the cells were harvested by centrifugation at 10,000 × g for 30 minutes at 4oC. Lysis buffer (500 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0)
CC E
containing lysozyme (Amresco, Solon, OH, USA) at 0.5 mg/mL and protease inhibitors cocktail (Roche Diagnostics GmbH, Mannheim, Germany) was added to the cell pellet and
A
incubated for 30 minutes at 4oC, then the cell suspension was disrupted using French press (Glen Mills Inc, Clifton, USA). After centrifugation, the supernatant containing soluble protein was mixed with DNase I (Amresco) (0.5 µg/mL) followed by incubation at 4oC for 15 minutes. The lysate was centrifuged for 30 minutes and the supernatant filtered through a 0.45 µm membrane (Sartorius AG, Goettingen, Germany). Ni-NTA Superflow resin (Qiagen
7
GmbH, Hilden, Germany) was mixed with the filtered lysate and transferred to a chromatography column (Bio-Rad). The resin was washed with washing buffers (500 mM NaCl and 50 mM NaH2PO4, pH 8.0) containing increasing imidazole concentrations (10-100 mM). The rAgB was eluted with elution buffer (500 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0) and the fractions containing protein were pooled, buffer exchanged into
IP T
PBS, and concentrated using a spin column (10 kDa cut off), then stored at -20 oC.
SC R
2.5 Preparation of polyclonal antibodies
Polyclonal antibodies were separately raised against HCF, nAgb, and rAgB in female
U
New-Zealand white rabbits following the method described previously (Saidin et al., 2017).
N
The experimental procedures were approved by the USM animal ethics committee [USM /
A
Animal ethics approval / 2014 (595)]. Briefly, the antigen was emulsified with an equal
M
volume of Freund's complete adjuvant (Sigma Aldrich, St. Loius, MO, USA). The emulsified antigen was injected subcutaneously into eight sites on the rabbit, 0.2 mL/site. Subsequently,
ED
three immunization boosts with two-week intervals were performed with the same volume of antigen in Freund's incomplete adjuvant (Sigma Aldrich). Ten days after the final boosting, a
PT
cardiac puncture was performed, and serum was collected. A blood sample was obtained
CC E
before each booster injection for monitoring of the antibody titers.
2.6 Purification of polyclonal antibodies
A
Melon™ Gel IgG Spin Purification Kit (Thermo Scientific) was used for purification of
IgG from the hyperimmune serum. Purification was performed following the manufacturer’s instruction. The purified IgG preparations of polyclonal antibodies to HCF, nAgB, and rAgB were buffer-exchanged into PBS and kept at -20oC.
8
2.7 Conjugating of IgGs to gold nanoparticles The conjugation of IgG to gold nanoparticles was performed following the method described previously (Makhsin et al., 2012). Colloidal gold particles (40 nm) was provided by Nanobiotechnology laboratory at our institute. The pH was adjusted to 8.0 using 0.2 M K2CO3. An amount of 8 µg of anti-HCF-IgG and 4 µg of each anti-nAgB-IgG and anti-rAgB-
IP T
IgG was each mixed with 500 µL colloidal gold and gently agitated at room temperature for 2 hours. The solution was then mixed with 1% (w/v) bovine serum albumin (BSA) (Amresco)
SC R
and centrifuged at 9,000 × g for 20 minutes. Finally, the gold sediment was suspended with
1% (w/v) BSA, the OD of gold-conjugated IgGs (Au-anti-HCF-IgG, Au-anti-nAgB-IgG or
N
U
Au-anti-rAgB-IgG) was determined and stored at 4oC.
A
2.8 Preparation of lateral flow dipstick assay
M
The dipsticks were prepared using HF90 nitrocellulose membrane card (Millipore, Bedford, MA, USA). First, an absorbent pad was placed on the sticky side of the card with 2
ED
mm overlap between the pad and membrane. The assembled card was then cut into 5 mm width strips using a strip cutter (A-Point Technologies Inc., Gibbstown, NJ, USA). Next,
PT
purified IgG was dotted in the middle part of the strip and the dipsticks were dried in an incubator at 37oC for 2 hours. The dipsticks were blocked with blocking solution (Roche
CC E
Diagnostics GmbH) and dried in the 37oC incubator, overnight. Next day the dipsticks were
A
stored in a dry cabinet.
2.9 Lateral flow dipstick (LFD) test procedure To perform the test, three wells of a 96-well microtiter plate (Costar, Lowell, MA, USA) were used. In the first well, 10µL serum sample was diluted with 10 µL Chase buffer (Reszon Diagnostic, Selangor, Malaysia). The dipstick was placed in the well and the diluted
9
serum was allowed to travel up the dipstick until it reached the top of the strip. The dipstick was moved into the second well containing 20 µL of gold-conjugated IgG. The gold conjugate was allowed to flow up the dipstick until all of it was used up. Finally, the dipstick was transferred to the third well containing 30µL Chase buffer to wash away the excess unbound conjugate. The result was evaluated when the background had become clear, which
IP T
took about 5 minutes. The result was first visually interpreted, as either ‘positive’ if a red dot
can be seen at the region of dotted IgG on the dipstick and ‘negative’ if a red dot was not seen
SC R
at the region. Quantitative results of all dipsticks were then determined using ESEQuant
Lateral Flow Reader (Qiagen) which was configured for colorimetric detection. The dipstick
U
was inserted into the reader within 10 minutes of completion of the LFD assay; the optical
N
response measured by the reader was recorded by LF-Studio software version 3.3.6 in the
CC E
PT
ED
M
A
reader (Figure 1).
A
Figure 1. Lateral flow reader (left), and an example of a result from a dipstick (right).
2.10 Lateral flow dipstick test (LFD) development and testing Ten positive and ten negative serum samples were each pooled and used to prepare positive and negative controls. The dipsticks were separately dotted (2 µg/dot) with three different IgGs (anti-HCF-IgG, anti-nAgB-IgG and anti-rAgB-IgG). The test was performed 10
by pairing each type of the IgGs with three types of gold-conjugated IgGs at OD of 6 (Auanti-HCF IgG, Au-anti-nAgB IgG, and Au-anti-rAgB IgG). Among the nine possible combinations, the pair of dotted antibody (capture antibody) and gold-conjugated antibody (detection antibody) which can best discriminate between positive and negative controls was selected. The best format was used to further optimize the OD of the gold-conjugated
IP T
antibody and the amount of dotted antibody.
Using the optimized parameters, all serum samples (Group I, Group II and Group III)
SC R
were tested with the LFD assay. Each dipstick result was first visually interpreted as either
positive or negative. This was followed by measurement of the dot intensity by the lateral
N
U
flow reader.
2.11 Acid treatment of serum samples to dissociate immune complexes
A
Immune complexes from the false negative samples from Group 1 were dissociated using a
M
previously described method with modification (Craig and Nelson, 1984). Pooled serum
ED
samples (2 samples per pool) were mixed at 1:3 ratio with 2.0 M glycine (pH 3.0) and incubated for 1 hour at room temperature. The solution was then mixed at 2:1 ratio with 1.5M
PT
Tris-HCl (pH 9.7) to neutralize the reaction. Subsequently, the samples were tested using the
CC E
LFD assay as described above.
2.12 Statistical analysis
A
MedCalc® software version 17 (MEDCALC statistical software, Ostend, Belgium)
was used to analyze the quantitative results. This software calculated the optimal reading for cut-off point, sensitivity and specificity, and area under ROC curve (AUC). A p-value < 0.01 was considered statistically significant.
11
3. Results Two out of the nine antibody combinations showed clear signals in discriminating between the pooled negative and pooled positive controls i.e. anti-nAgB-IgG paired with Au-antiHCF-IgG and anti-HCF-IgG paired with Au-anti-nAgB-IgG. However, the combination of dipstick dotted with anti-nAgB-IgG (capture antibody) and Au-anti-HCF-IgG (detection
IP T
antibody) showed much better dot intensity, thus this was considered as the best format
among all combinations. The other antibody combinations were either not reactive or showed
SC R
unsatisfactory dot intensity. The optimum amount of dotted antibody was found to be 3 µg
and the best concentration for gold-conjugated antibody was OD 4. Figure 2 shows examples
U
of the LFD assay results when individually tested with the serum samples. The result of each
N
dipstick was first evaluated visually and recorded as either positive or negative. Twenty seven
A
of 35 samples from Group I were found to be dipstick-positive, while 31 of 38 samples from
M
Group II and 10 of 13 samples from Group III were dipstick-negative. The tested dipsticks were read using a lateral flow reader to obtain quantitative data. The results were used to plot
ED
an ROC curve, which showed a good AUC value of 0.849 (95% CI = 0.76-0.92. P <0.01). The distribution of the test results of all serum samples and the ROC curve is shown in
PT
figures 3 and 4, respectively. At this COV, the LFD assay showed a diagnostic sensitivity of 77.14% (95% CI: 59.9-89.6%), with eight false negatives. The diagnostic specificity was
CC E
82.35 % (95% CI: 69.1-91.6%), with 6 false positive in Goup II. Cross-reactivity was observed with one serum sample from each cysticercosis, ascariasis, and taeniasis in Group
A
III. The results determined by visual interpretation of the dipsticks were found to have good concordance with the quantitative results of the reader, with discordant results found in 4 of the 86 samples (95%). One sample was from each of Groups I and III and two samples from Group II, they were negative by visual interpretation but positive by the reader.
12
The immune complex dissociation procedure was performed on the eight false negative serum samples (4 pools of 2 serum per pool). However they remained negative by
N
U
SC R
IP T
both visual interpretation and the reader (optical responses were less than 137.2).
A
Figure 2. Representative lateral flow dipstick results using positive and negative serum
M
samples. Notes: Lanes 1-4: positive results with serum samples from surgically-confirmed
ED
CE patients. Lanes 5-6: Negative results with serum samples from a healthy individual and a
A
CC E
PT
patient with hookworm infection, respectively.
Figure 3. Distribution of the dot intensities of the antigen detection dipsticks tested with serum samples. Note: The cut-off 137.2 is indicated as a dashed line on the graph. 13
IP T SC R
Figure 4. Receiver operating characteristic (ROC) curve from analysis of results from 86
N
Note: Cut-off point of 137.2 is indicated in the graph.
U
serum samples tested with the lateral flow dipstick assay.
A
4. Discussion
M
In the past, several studies had tried to develop a reliable antigen detection test for
ED
diagnosis of CE (Parija et al., 1997; Devi and Parija, 2003; Sadjjadi et al., 2009; Swarna and Parija, 2012). However, most of them suffered from some limitations and to date there is still
PT
no well-established antigen detection test for CE. In the present study, an effort was made to develop an antigen detection rapid test in
CC E
format of the LFD assay for CE. The diagnostic sensitivity of the antigen detection LFD was found to be 77.14%, which is higher than that reported in some other studies (Parija et al., 1997; Ravinder et al., 2000; Sadjjadi et al., 2009; Sunita et al., 2011; Bauomi et al., 2015).
A
Parija et al. (1997) showed detection of hydatid antigen in serum of 40 patients with CE using countercurrent immunoelectrophoresis (CIEP), with a diagnostic sensitivity of 45%. Similarly, a sensitivity of 40% was obtained by Sunita et al. (2011) whereby rabbit hyperimmune serum was used in a sandwich ELISA. Using the same ELISA technique, diagnostic sensitivities of 25.7%, 52.5%, and 80% have been reported (Sadjjadi et al., 2009; 14
Chaya and Parija, 2013; Bauomi et al., 2015). Meanwhile, Devi and Parija (2003) reported sensitivity of 72% and 83%, respectively when LAT and Co-A were used to detect antigen in serum samples of 18 patients with CE. However, Ravinder et al. (2000) had demonstrated a lower sensitivity (73%) using Co-A. In another antigen detection study involving 30 patients with CE using Dot-ELISA and EITB, the latter was found to be more sensitive (83%)
IP T
compared to the former (60%) (Swarna and Parija, 2012).
The diagnostic performance of an immunodiagnostic test for CE is dependent on the
SC R
antigen and its purity (Carmena et al., 2006). The above studies used various antigens for the
rabbit hyperimmunizations. Six studies utilized HCF as the source of antigen, four of them
U
used human HCF (Parija et al., 1997; Ravinder et al., 2000; Devi and Parija, 2003; Swarna
N
and Parija, 2012) while the other two (Sadjjadi et al., 2009; Sunita et al., 2011) used sheep
A
HCF. Meanwhile, Chaya and Parija (2013) used purified 24 kDa fragment of urinary hydatid
M
antigen and Bauomi et al. (2015) used 27.5 kDa protoscolex antigen for rabbit immunizations.
ED
Different performances among the above studies, and with the present study may be attributed to the different sources of antigens and different assay formats. Several other
PT
reasons may also contribute to the variable diagnostic sensitivity of the antigen detection tests for CE. First, the amount of circulating antigen in patients with CE, in general, is low and
CC E
varies with organ location, number and size of the cysts (Gottstein, 1984; Sadjjadi et al., 2009). It was previously reported that patients with liver cyst have a higher amount of
A
circulating antigens compared to patients with lung cyst (Craig and Nelson, 1984). In another report by Sadjjadi et al. (2009) circulating antigens were detected in 46% of patients with liver cyst and not detected in patients with lung or kidney cyst. Sunita et al. (2011) detected circulating antigens in 10 out of 25 patients with CE, but they did not disclose patients with
15
which cyst location were positive for circulating antigens. In the present study, the LFD assay detected all patients with lung cysts (5/5) and also 73% of patients with liver cysts. Antigen leakage from hydatid cysts is also dependent on the cyst status/stage, which can be classified based on the WHO standardized ultrasound classification for CE (WHO, 2003). However, in the present study, there was no information on the cyst stage although
IP T
they are mostly expected to be active cysts since the patients were referred for surgery. It has also been reported that the genotype of the isolate may have an impact on the diagnostic
SC R
performance of a serological test. E. granulosus genotype G1, G2, G3 and G6 have been
documented in Iranian patients with CE. However, G1 (54.4%) and G6 (40.8%) were
U
reported as the most prevalent genotypes (Rostami et al., 2015). Unfortunately there was no
A
studies should take this factor into consideration.
N
information on the E. granulosus genotype in CE patients in the present study. Thus further
M
Another possible reason for the observed low diagnostic sensitivity of CE antigen detection tests may be the formation of antigen-antibody immune complexes (Craig, 1986;
ED
Lightowlers and Gottstein, 1995). It was reported that up to 50% of echinococcosis patients did not have enough detectable free antigens in their serum (Gottstein, 1984; Craig, 1986).
PT
Acidic buffer treatment of patient’s serum had been used to release antigen from immune
CC E
complex and this was reported to enhance the performance of the diagnostic test for echinococcosis (Rouhani et al., 2013; Siles-Lucas et al., 2017). We had attempted to dissociate the antigen-antibody complexes in the serum using a low pH glycine buffer
A
treatment, however it did not seem to improve the diagnostic sensitivity. The present study showed a diagnostic specificity of 82.35%. A similar specificity
(83%) was demonstrated by Swarna and Parija (2012) using Dot-ELISA. Bauomi et al. (2015) displayed a lower specificity (75%) using an ELISA. There are other reports of assays with much higher specificity (> 90%) (Parija et al., 1997; Ravinder et al., 2000; Devi and 16
Parija, 2003; Sadjjadi et al., 2009; Sunita et al., 2011; Swarna and Parija, 2012), however, the diagnostic sensitivities of assays based on ELISA and CIEP were noticeably much lower (25.7-45%) (Parija et al., 1997; Sadjjadi et al., 2009; Sunita et al., 2011). Interestingly, an ELISA developed by Chaya and Parija (2013) using rabbit antibody to a 24 kDa human hydatid urinary antigen produced a high specificity of 92% (and sensitivity of 80%),
IP T
however, the identity of the protein was not reported. Using LAT and Co-A, Devi and Parija
(2003) reported specificity of 98% and, while in another study (Ravinder et al., 2000) the
SC R
latter technique gave a specificity of 93.87%. In addition, high specificity was also reported when EITB was used (Swarna and Parija, 2012). The above studies which reported high
U
specificity had used antigens from human sources to generate the antibodies (Ravinder et al.,
N
2000; Devi and Parija, 2003; Swarna and Parija, 2012; Chaya and Parija, 2013), thus this
A
could be a limitation for the sustainable production of such tests. Some studies suggested
M
circulating antigen detection test as a useful tool for post-treatment follow-up and monitoring of cyst activity (Shariff and Parija, 1993; Ravinder et al., 1997; Ferragut et al., 1998;
ED
Gottstein et al., 2014). However, in the present study, post-treatment serum sample from a patient with CE was not available to ascertain this utility.
PT
In the present study, cross-reactions were observed in 6 of 38 healthy Malaysian individuals (Group II), and 3 of 13 Malaysian patients with other parasitic infections (Group
CC E
III). The cross-reactivity with serum of healthy individuals may be due to asymptomatic diseases other than CE since Malaysia is not endemic for echinococcosis and all sera was
A
found to be seronegative by a commercial ELISA kit. Sunita et al. (2011) reported crossreactions with ascariasis and cysticercosis, thus similar to what was observed in the present study. In addition, cross-reactions have also been reported with sera of patients with schistosomiasis, fascioliasis, visceral leishmaniasis, neurocysticercosis, amebic liver abscess, tropical pulmonary eosinophilia and partial seizures (Sadjjadi et al., 2009; Sunita et al., 2011;
17
Chaya and Parija, 2013; Bauomi et al., 2015). The present study is the first to report on crossreaction of a CE antigen detection test with serum of a taeniasis patient, however this is not surprising since cross-reactions with such patients were commonly reported with CE antibody detection assays (Sadjjadi et al., 2007; Hernandez-Gonzalez et al., 2008; Sarkari et al., 2010). Serum samples from patients with alveolar echinococcosis (AE) were not available
IP T
for evaluation of its cross-reactivity with the present LFD assay. Hence it is unknown
whether the assay can discriminate CE from AE. This aspect may be important in
SC R
geographical areas where both infections co-exist. Nevertheless, even if the LFD assay shows cross-reactivity with AE, it can still be used as screening test for echinococcosis. In order to
U
confirm the diagnostic specificity of the LFD assay, screening of many more serum samples
N
with other helminth infections is required.
A
The AUC of the LFD assay in this study was determined to be 0.849, which indicated
M
good diagnostic accuracy in discriminating positive from negative samples (Metz, 1978). Thus far, two rapid lateral flow antibody detection assays for diagnosis of CE are
ED
commercially available (Tamarozzi et al., 2016) and based on the available literature, this is the first report of an antigen detection test in the format of a rapid lateral flow assay. The test
PT
is simple, fast and efficient, it does not require expensive laboratory equipment, and can be performed in the field by a non-laboratory trained person, thus would be useful for low-
CC E
resource areas (Posthuma-Trumpie et al., 2009). The use of a dipstick reader may not be required since the visual interpretation was quite conclusive. The dipstick reader in the
A
present study was mainly used for the purpose of statistical analysis. Nevertheless, the visual aspect of the test can be further improved. Since selected serum samples were used for the development and initial evaluation of the LFD assay, it is important to perform further evaluation with a much larger sample size. Preferably, the further evaluation should be in the form of a multicentre study using samples
18
of CE patients from different regions of the world, including from confirmed CE patients who are antibody-negative, and post-treatment patients. In conclusion, the antigen detection LFD assay developed in this study seemed to be useful for diagnosis of CE and possibly for post-treatment follow-up. We foresee that it may improve serodiagnosis of CE when used in
IP T
tandem with an antibody detection test, however further data are needed to ascertain this.
Funding
SC R
This work was supported by Universiti Sains Malaysia Research University grant [Grant no. 1001.PFARMASI.812153] and Malaysian Ministry of Higher Education through the Higher
N
U
Institution Centre of Excellence Program (HICoE) [Grant no. 311/CIPPM/4401005].
M
A
Disclosure: None
Acknowledgments
ED
We would like to acknowledge and thank Dr. Zohreh Kazemi Moghadam Kakhki and Ms Nor Dyana Zakaria for contributing serum samples and providing technical assistance,
CC E
PT
respectively.
References
A
Bauomi, I.R., El-Amir, A.M., Fahmy, A.M., Zalat, R.S., Diab, T.M., 2015. Evaluation of purified 27.5 kDa protoscolex antigen-based ELISA for the detection of circulating antigens and antibodies in sheep and human hydatidosis. Journal of helminthology 89, 577-583. Brunetti, E., Garcia, H.H., Junghanss, T., on behalf of the members of the International Ce Workshop in Lima, P., 2011. Cystic Echinococcosis: Chronic, Complex, and Still Neglected. PLoS neglected tropical diseases 5, e1146. Brunetti, E., Kern, P., Vuitton, D.A., Writing Panel for the, W.-I., 2010. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta tropica 114, 1-16.
19
Budke, C.M., Deplazes, P., Torgerson, P.R., 2006. Global socioeconomic impact of cystic echinococcosis. Emerging infectious diseases 12, 296-303. Carmena, D., Benito, A., Eraso, E., 2006. Antigens for the immunodiagnosis of Echinococcus granulosus infection: An update. Acta tropica 98, 74-86. Chaya, D., Parija, S.C., 2013. Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis. Tropical parasitology 3, 125-131.
IP T
Craig, P.S., 1986. Detection of Specific Circulating Antigen, Immune-Complexes and Antibodies in Human Hydatidosis from Turkana (Kenya) and Great-Britain, by EnzymeImmunoassay. Parasite Immunol 8, 171-188.
SC R
Craig, P.S., Nelson, G.S., 1984. The detection of circulating antigen in human hydatid disease. Annals of tropical medicine and parasitology 78, 219-227.
Craig, P.S., Rogan, M.T., Allan, J.C., 1996. Detection, screening and community epidemiology of taeniid cestode zoonoses: cystic echinococcosis, alveolar echinococcosis and neurocysticercosis. Advances in parasitology 38, 169-250.
N
U
Deplazes, P., Rinaldi, L., Alvarez Rojas, C.A., Torgerson, P.R., Harandi, M.F., Romig, T., Antolova, D., Schurer, J.M., Lahmar, S., Cringoli, G., Magambo, J., Thompson, R.C., Jenkins, E.J., 2017. Global Distribution of Alveolar and Cystic Echinococcosis. Advances in parasitology 95, 315-493.
M
A
Devi, C.S., Parija, S.C., 2003. A new serum hydatid antigen detection test for diagnosis of cystic echinococcosis. The American journal of tropical medicine and hygiene 69, 525528.
ED
Eckert, J., Gemmell, M.A., Meslin, F.X., World Health, O., International Office of, E., 2001. WHO/OIE Manual on echinococcosis in humans and animals : a public health problem of global concern. OIE ; WHO, Paris; Geneva.
PT
Ferragut, G., Ljungstrom, I., Nieto, A., 1998. Relevance of circulating antigen detection to follow-up experimental and human cystic hydatid infections. Parasite Immunol 20, 541549.
CC E
Gottstein, B., 1984. An immunoassay for the detection of circulating antigens in human echinococcosis. The American journal of tropical medicine and hygiene 33, 1185-1191. Gottstein, B., Wang, J., Blagosklonov, O., Grenouillet, F., Millon, L., Vuitton, D.A., Müller, N., 2014. Echinococcus metacestode: in search of viability markers. Parasite (Paris, France) 21, 63.
A
Hernandez-Gonzalez, A., Muro, A., Barrera, I., Ramos, G., Orduna, A., Siles-Lucas, M., 2008. Usefulness of four different Echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (UHD) and postsurgical follow-up of patients treated for UHD. Clinical and vaccine immunology : CVI 15, 147-153. Khalilpour, A., Sadjjadi, S.M., Moghadam, Z.K., Yunus, M.H., Zakaria, N.D., Osman, S., Noordin, R., 2014. Lateral flow test using Echinococcus granulosus native antigen B and comparison of IgG and IgG4 dipsticks for detection of human cystic echinococcosis. The American journal of tropical medicine and hygiene 91, 994-999.
20
Lightowlers, M.W., Gottstein, B., 1995. Echinococcosis/hydatidosis: antigens, immunological and molecular diagnosis. CAB INTERNATIONAL, Wallingford, pp. 355-410. Makhsin, S.R., Razak, K.A., Noordin, R., Zakaria, N.D., Chun, T.S., 2012. The effects of size and synthesis methods of gold nanoparticle-conjugated MalphaHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis. Nanotechnology 23, 495719.
IP T
Mariconti, M., Bazzocchi, C., Tamarozzi, F., Meroni, V., Genco, F., Maserati, R., Brunetti, E., 2014. Immunoblotting with human native antigen shows stage-related sensitivity in the serodiagnosis of hepatic cystic echinococcosis. The American journal of tropical medicine and hygiene 90, 75-79.
SC R
McManus, D.P., Gray, D.J., Zhang, W., Yang, Y., 2012. Diagnosis, treatment, and management of echinococcosis. BMJ (Clinical research ed.) 344, e3866.
Metz, C.E., 1978. Basic principles of ROC analysis. Seminars in nuclear medicine 8, 283298.
U
Moosa, R.A., Abdel-Hafez, S.K., 1994. Serodiagnosis and seroepidemiology of human unilocular hydatidosis in Jordan. Parasitology research 80, 664-671.
A
N
Moro, P., Schantz, P.M., 2009. Echinococcosis: a review. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 13, 125-133.
M
Parija, S.C., Ravinder, P.T., Rao, K.S., 1997. Detection of hydatid antigen in urine by countercurrent immunoelectrophoresis. Journal of clinical microbiology 35, 1571-1574.
ED
Posthuma-Trumpie, G.A., Korf, J., van Amerongen, A., 2009. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and Bioanalytical Chemistry 393, 569-582.
PT
Ravinder, P.T., Parija, S.C., Rao, K.S., 1997. Evaluation of human hydatid disease before and after surgery and chemotherapy by demonstration of hydatid antigens and antibodies in serum. Journal of medical microbiology 46, 859-864.
CC E
Ravinder, P.T., Parija, S.C., Rao, K.S., 2000. Urinary hydatid antigen detection by coagglutination, a cost-effective and rapid test for diagnosis of cystic echinococcosis in a rural or field setting. Journal of clinical microbiology 38, 2972-2974. Ray, R., De, P.K., Karak, K., 2002. Combined role of Casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian journal of medical microbiology 20, 79-82.
A
Rostami, S., Torbaghan, S.S., Dabiri, S., Babaei, Z., Mohammadi, M.A., Sharbatkhori, M., Harandi, M.F., 2015. Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran. The American journal of tropical medicine and hygiene 92, 588-594. Rouhani, S., Parvizi, P., Spotin, A., 2013. Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient's sera: a novel hypothesis to follow-up of Cystic echinococcosis after surgery. Medical hypotheses 81, 557-560.
21
Sadjjadi, S.M., Abidi, H., Sarkari, B., Izadpanah, A., Kazemian, S., 2007. Evaluation of enzyme linked immunosorbent assay, utilizing native antigen B for serodiagnosis of human hydatidosis. Iranian journal of immunology : IJI 4, 167-172. Sadjjadi, S.M., Sedaghat, F., Hosseini, S.V., Sarkari, B., 2009. Serum Antigen and Antibody Detection in Echinococcosis: Application in Serodiagnosis of Human Hydatidosis. Korean J Parasitol 47, 153-157.
IP T
Saidin, S., Yunus, M.H., Othman, N., Lim, Y.A., Mohamed, Z., Zakaria, N.Z., Noordin, R., 2017. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample. Pathogens and global health 111, 128-136.
SC R
Sarkari, B., Sadjjadi, S.M., Beheshtian, M.M., Aghaee, M., Sedaghat, F., 2010. Human cystic echinococcosis in Yasuj District in Southwest of Iran: an epidemiological study of seroprevalence and surgical cases over a ten-year period. Zoonoses and public health 57, 146-150. Shariff, M., Parija, S.C., 1993. Co-agglutination (Co-A) test for circulating antigen in hydatid disease. Journal of medical microbiology 38, 391-394.
N
U
Siles-Lucas, M., Casulli, A., Conraths, F.J., Muller, N., 2017. Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals. Advances in parasitology 96, 159-257.
M
A
Sunita, T., Khurana, S., Malla, N., Dubey, M.L., 2011. Immunodiagnosis of cystic echinocooccosis by antigen detection in serum, urine, and saliva samples. Tropical parasitology 1, 33-38.
ED
Swarna, S.R., Parija, S.C., 2012. Evaluation of Dot-ELISA and enzyme-linked immunoelectrotransfer blot assays for detection of a urinary hydatid antigen in the diagnosis of cystic echinococcosis. Tropical parasitology 2, 38-44.
PT
Tamarozzi, F., Covini, I., Mariconti, M., Narra, R., Tinelli, C., De Silvestri, A., Manzoni, F., Casulli, A., Ito, A., Neumayr, A., Brunetti, E., 2016. Comparison of the Diagnostic Accuracy of Three Rapid Tests for the Serodiagnosis of Hepatic Cystic Echinococcosis in Humans. PLoS neglected tropical diseases 10, e0004444.
CC E
Torgerson, P.R., Deplazes, P., 2009. Echinococcosis: diagnosis and diagnostic interpretation in population studies. Trends Parasitol 25, 164-170. WHO, 2003. International classification of ultrasound images in cystic echinococcosis for application in clinical and field epidemiological settings. Acta tropica 85, 253-261.
A
Zhang, W., Li, J., McManus, D.P., 2003. Concepts in immunology and diagnosis of hydatid disease. Clinical microbiology reviews 16, 18-36. Zhang, W., Wen, H., Li, J., Lin, R., McManus, D.P., 2012. Immunology and immunodiagnosis of cystic echinococcosis: an update. Clinical & developmental immunology 2012, 101895.
22
S0001-706X(18)30627-2 https://doi.org/10.1016/j.actatropica.2018.11.018 ACTROP 4834
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
19 May 2018 6 October 2018 16 November 2018
Please cite this article as: Khanbabaie S, Riazi M, Chang CH, Yunus MH, Noordin R, Lateral flow dipstick antigen assay for human cystic echinococcosis, Acta Tropica (2018), https://doi.org/10.1016/j.actatropica.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Lateral flow dipstick antigen assay for human cystic echinococcosis
Authors and affiliations: Sam Khanbabaie1, Mehdi Riazi2, Chiat Han Chang1, Muhammad Hafiznur Yunus1, and Rahmah Noordin1*
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
IP T
1
Penang, Malaysia.
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.
SC R
2
U
Authors’ addresses: Sam Khanbabaie, Chiat Han Chang, Muhammad Hafiznur Yunus, and
Malaysia,
Penang,
Malaysia,
E-mails:
[email protected],
A
Sains
N
Rahmah Noordin, Institute for Research in Molecular Medicine (INFORMM), Universiti
M
[email protected], [email protected], and [email protected]. Mehdi Riazi,
[email protected].
ED
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia, Email:
(INFORMM),
Universiti
Sains
CC E
Medicine
PT
*Corresponding author: Prof. Dr. Rahmah Noordin, Institute for Research in Molecular
A
[email protected]; Tel.: +604 653 4800
1
Malaysia,
Penang,
Malaysia;
E-mail:
Lateral flow dipstick antigen assay for human cystic echinococcosis
Abstract Cystic echinococcosis (CE) is a neglected zoonotic disease with a worldwide distribution and is a major public health problem in some areas. Diagnosis of CE is mainly based on clinical
IP T
symptoms, imaging and serological testing, however, improvement in serodiagnosis is still needed. This study was aimed at detecting circulating Echinococcus antigen in CE patients
SC R
using a lateral flow dipstick (LFD) assay. Three types of hydatid antigens i.e. hydatid cyst
fluid (HCF), native antigen B (nAgB) and recombinant antigen B (rAgB) were prepared and
U
polyclonal rabbit antiserum was raised against each antigen. Purified IgG fractions were
N
prepared and a portion was conjugated to gold nanoparticles. After a series of optimizations,
A
a final antigen detection LFD assay was developed using a combination of anti-nAgB-IgG
M
and gold-conjugated anti-HCF-IgG. Evaluation of the assay showed that 27 out of 35 (77%) serum samples from CE patients gave positive results. Meanwhile, the test showed a
ED
diagnostic specificity of 82% when tested with sera from 38 healthy individuals and 13 patients with other parasitic diseases. In conclusion, the antigen detection LFD assay seemed
PT
to be useful for diagnosis of CE and possibly for post-treatment follow-up, and merit further evaluation studies. We foresee that it may improve serodiagnosis of CE when used in tandem
CC E
with an antibody detection test.
A
Keywords: cystic echinococcosis; antigen detection; lateral flow dipstick assay; polyclonal antibodies; Echinococcus granulosus
2
1. Introduction Human cystic echinococcosis (CE) or hydatid cyst disease is a chronic infection caused by metacestode of Echinococcus granulosus which can infect both domestic herbivores and humans (Eckert et al., 2001). Adult cestodes live in the intestine of dogs and other carnivores (definitive hosts) and shed their eggs in the hosts’ feces. Sheep, goat, and
IP T
other herbivores (intermediate hosts) acquire the infection after peroral ingestion of plants or possibly water contaminated with the eggs. Human becomes infected by accidental ingestion
SC R
of E. granulosus eggs. Upon entry into the body, the larvae form one or more cysts in
different organs, mostly in liver and lung (Eckert et al., 2001; Moro and Schantz, 2009). CE
U
is distributed worldwide and is considered as a major public health problem in developing
N
countries (Brunetti et al., 2011; Deplazes et al., 2017). CE causes significant economic losses
A
due to costs of diagnosis, medical treatment, and resultant disabilities in some human cases
M
(Budke et al., 2006).
Accurate diagnosis of CE is challenging and early diagnosis can lead to better
ED
management and treatment outcomes at a lower cost (Zhang et al., 2003). Clinical assessment, imaging, and serology are the main tools for CE diagnosis (Brunetti et al., 2010).
PT
The World Health Organization Informal Working Group on Echinococcosis (WHO-IWGE) suggested using ultrasound (US) as the imaging technique of choice for diagnosis,
CC E
classification and follow-up of hydatid cyst in the abdominal cavity (WHO, 2003). However, US normally cannot detect lung CE (Torgerson and Deplazes, 2009) and imaging techniques
A
usually are expensive and may not be available in some low resource areas (McManus et al., 2012). Additionally, sometimes imaging results may not be conclusive. WHO recommended applying other diagnostic techniques such as immunodiagnosis in uncertain/suspected cases of CE as a confirmatory test (WHO, 2003). Torgerson and Deplazes (2009) clearly demonstrated the value of using multiple tests, i.e. imaging and immunodiagnosis, for
3
diagnosis of CE especially in mass screening of endemic areas. They suggested to initially use a highly sensitive test to ensure that most CE cases are detected, followed by a highly specific test to confirm the result.
In some settings, immunodiagnostic tests may be
considered as the primary method for CE diagnosis (Zhang et al., 2003). There are several serological methods available for detection of antibodies to CE i.e.
IP T
enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test
(IFAT), immunoelectrophoresis (IEP), and immunoblotting (IB) (Zhang et al., 2012).
SC R
However the available tests have a number of disadvantages such as variable sensitivity, cross reactivity with antibodies to other infections, difficulty to differentiate between past and
U
present infections, and may not be suitable for evaluating treatment efficacy(Carmena et al.,
N
2006; Mariconti et al., 2014). In this regard, an antigen detection test may be able to assist in
A
addressing the latter two of the four aforementioned limitations.
M
A wide range of diagnostic sensitivity and specificity of antibody detection assays have been reported (Zhang et al., 2012). Up to 40% of patients with confirmed CE were
ED
reported to show false negative results by antibody detection tests (Craig, 1986). Meanwhile hydatid antigens can be detected in serum of patients with CE, and this may be due to leakage
PT
of hydatid fluid and metabolic products of the cyst (Gottstein, 1984; Craig, 1986; Craig et al., 1996). It has been demonstrated that some sera of patients with CE contained hydatid
CC E
antigens but antibody detection assays on the same samples showed negative results (Gottstein, 1984; Moosa and Abdel-Hafez, 1994). Moreover, antibodies may persist for long
A
periods after treatment of patients with CE by surgical cyst removal and/or chemotherapy (Ray et al., 2002). On the other hand, circulating antigens are absent or quickly disappears after successful treatment (Craig, 1986). Therefore, antigen detection test may be useful for diagnosis of CE and follow-up of treatment efficacy. It can be envisaged that the use of both
4
antigen and antibody detection assays would be ideal for serological diagnosis of CE. However, currently, there is no commercial antigen detection test for CE diagnosis. Previously it has been reported that hydatid cyst antigens can be detected in serum, urine, and saliva of patients with CE, using ELISA, enzyme-linked immune-electro-transfer blot (EITB), latex agglutination test (LAT) and co-agglutination test (Co-A) (Ravinder et al.,
IP T
2000; Devi and Parija, 2003; Sunita et al., 2011). However, the reported diagnostic
sensitivities were not satisfactory, ranging from 25% to 83% (Ravinder et al., 1997, 2000;
SC R
Devi and Parija, 2003; Sadjjadi et al., 2009; Sunita et al., 2011; Swarna and Parija, 2012; Chaya and Parija, 2013; Bauomi et al., 2015). In an effort to address this diagnostic
U
challenge, the present study was aimed at developing an antigen detection test for CE in a
A
N
lateral flow (LFD) dipstick assay format.
2.1 Human serum samples
M
2. Materials and methods
ED
At the stage of development and evaluation of a test, it is important to select and use defined serum samples which are ‘clear-cut in their infection status’. Thus we used 86 human serum
PT
samples and categorized them into three groups. Group I comprised serum samples from Iranian patients with surgically confirmed CE (n = 35) and were positive by a commercial
CC E
ELISA kit i.e.Anti-Echinococcus ELISA IgG (EUROIMMUN AG, Luebeck, Germany). Iran is a known endemic area for CE, and serum samples from Iranian patients were collected at
A
several hospitals in Tehran, Iran, anonymised and brought to the Universiti Sains Malaysia. Twenty three of the patients (65.7%) were female and 12 (34.3%) were male. The patients age ranged from 11 to 76 years old. Four patients had single lung cyst, one patient had single cyst in both lung and liver and the rest had single or multiple liver cysts. Information
5
regarding the stage of the cysts was not available. Serum samples were collected just prior to undergoing surgery and none of the patients received treatment for CE before surgery. Group II comprised serum samples from Malaysian healthy individuals (n = 38) that were negative by the ELISA kit. Meanwhile Group III comprised serum samples from
IP T
Malaysian patients with other parasitic diseases that were negative by the ELISA kit i.e. cysticercosis (n = 2), ascariasis (n = 3), toxocariasis (n = 2), trichuriasis (n = 2), taeniasis (n = 1) and hookworm infection (n = 3). Malaysia and its neighboring are not endemic for CE.
SC R
These sera were banked and anonymized serum samples and their use for evaluation of
diagnostic sensitivity and specificity of laboratory tests was permitted by the Universiti Sains
N
U
Malaysia Research Ethics Committee.
A
2.2 Preparation of hydatid cyst fluid
M
Previously, crude hydatid cyst fluid (HCF) was collected aseptically from fertile hydatid cysts collected from slaughtered sheep from abattoirs in Rasht, Iran, and frozen at -
ED
80oC. In this study, the stored HCF was centrifuged at 10,000 × g for 30 minutes to separate the protoscolices and suspended solids from the supernatant. The supernatant was then
PT
collected and the protein concentration was determined by the Bio-Rad RCDC protein assay
CC E
kit (Bio-Rad, Hercules, CA, USA), then stored at -80oC.
2.3 Preparation of native antigen B
A
Native Antigen B (nAgB) was produced from HCF based on a previous method
(Khalilpour et al., 2014). Briefly, 100 mL of HCF was dialyzed against 5 mM acetate buffer (pH 5.0) overnight at 4oC and the dialyzed sample was centrifuged at 50,000 × g for 30 minutes. The supernatant containing sheep albumin was discarded and the pellet was suspended in phosphate buffer saline, pH 7.2 (PBS), at 100 µg pellet to 1 mL buffer. Next, an 6
equal volume of saturated ammonium sulphate was added to the suspension and the mixture was agitated at 4oC for 15 minutes. It was then centrifuged at 4,000 × g for 30 minutes at 4oC to remove the globulin. The resulting supernatant was dialyzed against PBS at 4oC for 24 hours to remove the ammonium sulphate. Finally, the sample was boiled for 15 minutes and centrifuged at 50,000 × g for 60 minutes. The protein concentration of the supernatant
IP T
containing nAgB was determined and stored at -20oC.
SC R
2.4 Preparation of recombinant antigen B
Previously prepared pET32 expression vector (Novagen, Madison, WI, USA) harboring
U
open reading frame of recombinant antigen B (rAgB) was transformed into Escherichia coli
N
strain C41 (DE3) (Lucigen, Middleton, WI, USA). A single bacterial colony was cultured in Terrific broth (TB) supplemented with ampicillin (100 µg/mL) and placed in a shaker-
A
incubator at 30oC, overnight. Fresh TB medium was added to the overnight culture, at a ratio
The
expression
of
rAgB
was
induced
with
1
mM
isopropyl-β-D-
ED
0.4-0.6.
M
of 10:1, then further incubated at 37oC until the optical density at 600 nm (OD600) reached
thiogalactopyranoside (IPTG) (Thermo Scientific, Waltham, MA, USA), and further cultured
PT
for 4 hours at 30oC. Next, the cells were harvested by centrifugation at 10,000 × g for 30 minutes at 4oC. Lysis buffer (500 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0)
CC E
containing lysozyme (Amresco, Solon, OH, USA) at 0.5 mg/mL and protease inhibitors cocktail (Roche Diagnostics GmbH, Mannheim, Germany) was added to the cell pellet and
A
incubated for 30 minutes at 4oC, then the cell suspension was disrupted using French press (Glen Mills Inc, Clifton, USA). After centrifugation, the supernatant containing soluble protein was mixed with DNase I (Amresco) (0.5 µg/mL) followed by incubation at 4oC for 15 minutes. The lysate was centrifuged for 30 minutes and the supernatant filtered through a 0.45 µm membrane (Sartorius AG, Goettingen, Germany). Ni-NTA Superflow resin (Qiagen
7
GmbH, Hilden, Germany) was mixed with the filtered lysate and transferred to a chromatography column (Bio-Rad). The resin was washed with washing buffers (500 mM NaCl and 50 mM NaH2PO4, pH 8.0) containing increasing imidazole concentrations (10-100 mM). The rAgB was eluted with elution buffer (500 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0) and the fractions containing protein were pooled, buffer exchanged into
IP T
PBS, and concentrated using a spin column (10 kDa cut off), then stored at -20 oC.
SC R
2.5 Preparation of polyclonal antibodies
Polyclonal antibodies were separately raised against HCF, nAgb, and rAgB in female
U
New-Zealand white rabbits following the method described previously (Saidin et al., 2017).
N
The experimental procedures were approved by the USM animal ethics committee [USM /
A
Animal ethics approval / 2014 (595)]. Briefly, the antigen was emulsified with an equal
M
volume of Freund's complete adjuvant (Sigma Aldrich, St. Loius, MO, USA). The emulsified antigen was injected subcutaneously into eight sites on the rabbit, 0.2 mL/site. Subsequently,
ED
three immunization boosts with two-week intervals were performed with the same volume of antigen in Freund's incomplete adjuvant (Sigma Aldrich). Ten days after the final boosting, a
PT
cardiac puncture was performed, and serum was collected. A blood sample was obtained
CC E
before each booster injection for monitoring of the antibody titers.
2.6 Purification of polyclonal antibodies
A
Melon™ Gel IgG Spin Purification Kit (Thermo Scientific) was used for purification of
IgG from the hyperimmune serum. Purification was performed following the manufacturer’s instruction. The purified IgG preparations of polyclonal antibodies to HCF, nAgB, and rAgB were buffer-exchanged into PBS and kept at -20oC.
8
2.7 Conjugating of IgGs to gold nanoparticles The conjugation of IgG to gold nanoparticles was performed following the method described previously (Makhsin et al., 2012). Colloidal gold particles (40 nm) was provided by Nanobiotechnology laboratory at our institute. The pH was adjusted to 8.0 using 0.2 M K2CO3. An amount of 8 µg of anti-HCF-IgG and 4 µg of each anti-nAgB-IgG and anti-rAgB-
IP T
IgG was each mixed with 500 µL colloidal gold and gently agitated at room temperature for 2 hours. The solution was then mixed with 1% (w/v) bovine serum albumin (BSA) (Amresco)
SC R
and centrifuged at 9,000 × g for 20 minutes. Finally, the gold sediment was suspended with
1% (w/v) BSA, the OD of gold-conjugated IgGs (Au-anti-HCF-IgG, Au-anti-nAgB-IgG or
N
U
Au-anti-rAgB-IgG) was determined and stored at 4oC.
A
2.8 Preparation of lateral flow dipstick assay
M
The dipsticks were prepared using HF90 nitrocellulose membrane card (Millipore, Bedford, MA, USA). First, an absorbent pad was placed on the sticky side of the card with 2
ED
mm overlap between the pad and membrane. The assembled card was then cut into 5 mm width strips using a strip cutter (A-Point Technologies Inc., Gibbstown, NJ, USA). Next,
PT
purified IgG was dotted in the middle part of the strip and the dipsticks were dried in an incubator at 37oC for 2 hours. The dipsticks were blocked with blocking solution (Roche
CC E
Diagnostics GmbH) and dried in the 37oC incubator, overnight. Next day the dipsticks were
A
stored in a dry cabinet.
2.9 Lateral flow dipstick (LFD) test procedure To perform the test, three wells of a 96-well microtiter plate (Costar, Lowell, MA, USA) were used. In the first well, 10µL serum sample was diluted with 10 µL Chase buffer (Reszon Diagnostic, Selangor, Malaysia). The dipstick was placed in the well and the diluted
9
serum was allowed to travel up the dipstick until it reached the top of the strip. The dipstick was moved into the second well containing 20 µL of gold-conjugated IgG. The gold conjugate was allowed to flow up the dipstick until all of it was used up. Finally, the dipstick was transferred to the third well containing 30µL Chase buffer to wash away the excess unbound conjugate. The result was evaluated when the background had become clear, which
IP T
took about 5 minutes. The result was first visually interpreted, as either ‘positive’ if a red dot
can be seen at the region of dotted IgG on the dipstick and ‘negative’ if a red dot was not seen
SC R
at the region. Quantitative results of all dipsticks were then determined using ESEQuant
Lateral Flow Reader (Qiagen) which was configured for colorimetric detection. The dipstick
U
was inserted into the reader within 10 minutes of completion of the LFD assay; the optical
N
response measured by the reader was recorded by LF-Studio software version 3.3.6 in the
CC E
PT
ED
M
A
reader (Figure 1).
A
Figure 1. Lateral flow reader (left), and an example of a result from a dipstick (right).
2.10 Lateral flow dipstick test (LFD) development and testing Ten positive and ten negative serum samples were each pooled and used to prepare positive and negative controls. The dipsticks were separately dotted (2 µg/dot) with three different IgGs (anti-HCF-IgG, anti-nAgB-IgG and anti-rAgB-IgG). The test was performed 10
by pairing each type of the IgGs with three types of gold-conjugated IgGs at OD of 6 (Auanti-HCF IgG, Au-anti-nAgB IgG, and Au-anti-rAgB IgG). Among the nine possible combinations, the pair of dotted antibody (capture antibody) and gold-conjugated antibody (detection antibody) which can best discriminate between positive and negative controls was selected. The best format was used to further optimize the OD of the gold-conjugated
IP T
antibody and the amount of dotted antibody.
Using the optimized parameters, all serum samples (Group I, Group II and Group III)
SC R
were tested with the LFD assay. Each dipstick result was first visually interpreted as either
positive or negative. This was followed by measurement of the dot intensity by the lateral
N
U
flow reader.
2.11 Acid treatment of serum samples to dissociate immune complexes
A
Immune complexes from the false negative samples from Group 1 were dissociated using a
M
previously described method with modification (Craig and Nelson, 1984). Pooled serum
ED
samples (2 samples per pool) were mixed at 1:3 ratio with 2.0 M glycine (pH 3.0) and incubated for 1 hour at room temperature. The solution was then mixed at 2:1 ratio with 1.5M
PT
Tris-HCl (pH 9.7) to neutralize the reaction. Subsequently, the samples were tested using the
CC E
LFD assay as described above.
2.12 Statistical analysis
A
MedCalc® software version 17 (MEDCALC statistical software, Ostend, Belgium)
was used to analyze the quantitative results. This software calculated the optimal reading for cut-off point, sensitivity and specificity, and area under ROC curve (AUC). A p-value < 0.01 was considered statistically significant.
11
3. Results Two out of the nine antibody combinations showed clear signals in discriminating between the pooled negative and pooled positive controls i.e. anti-nAgB-IgG paired with Au-antiHCF-IgG and anti-HCF-IgG paired with Au-anti-nAgB-IgG. However, the combination of dipstick dotted with anti-nAgB-IgG (capture antibody) and Au-anti-HCF-IgG (detection
IP T
antibody) showed much better dot intensity, thus this was considered as the best format
among all combinations. The other antibody combinations were either not reactive or showed
SC R
unsatisfactory dot intensity. The optimum amount of dotted antibody was found to be 3 µg
and the best concentration for gold-conjugated antibody was OD 4. Figure 2 shows examples
U
of the LFD assay results when individually tested with the serum samples. The result of each
N
dipstick was first evaluated visually and recorded as either positive or negative. Twenty seven
A
of 35 samples from Group I were found to be dipstick-positive, while 31 of 38 samples from
M
Group II and 10 of 13 samples from Group III were dipstick-negative. The tested dipsticks were read using a lateral flow reader to obtain quantitative data. The results were used to plot
ED
an ROC curve, which showed a good AUC value of 0.849 (95% CI = 0.76-0.92. P <0.01). The distribution of the test results of all serum samples and the ROC curve is shown in
PT
figures 3 and 4, respectively. At this COV, the LFD assay showed a diagnostic sensitivity of 77.14% (95% CI: 59.9-89.6%), with eight false negatives. The diagnostic specificity was
CC E
82.35 % (95% CI: 69.1-91.6%), with 6 false positive in Goup II. Cross-reactivity was observed with one serum sample from each cysticercosis, ascariasis, and taeniasis in Group
A
III. The results determined by visual interpretation of the dipsticks were found to have good concordance with the quantitative results of the reader, with discordant results found in 4 of the 86 samples (95%). One sample was from each of Groups I and III and two samples from Group II, they were negative by visual interpretation but positive by the reader.
12
The immune complex dissociation procedure was performed on the eight false negative serum samples (4 pools of 2 serum per pool). However they remained negative by
N
U
SC R
IP T
both visual interpretation and the reader (optical responses were less than 137.2).
A
Figure 2. Representative lateral flow dipstick results using positive and negative serum
M
samples. Notes: Lanes 1-4: positive results with serum samples from surgically-confirmed
ED
CE patients. Lanes 5-6: Negative results with serum samples from a healthy individual and a
A
CC E
PT
patient with hookworm infection, respectively.
Figure 3. Distribution of the dot intensities of the antigen detection dipsticks tested with serum samples. Note: The cut-off 137.2 is indicated as a dashed line on the graph. 13
IP T SC R
Figure 4. Receiver operating characteristic (ROC) curve from analysis of results from 86
N
Note: Cut-off point of 137.2 is indicated in the graph.
U
serum samples tested with the lateral flow dipstick assay.
A
4. Discussion
M
In the past, several studies had tried to develop a reliable antigen detection test for
ED
diagnosis of CE (Parija et al., 1997; Devi and Parija, 2003; Sadjjadi et al., 2009; Swarna and Parija, 2012). However, most of them suffered from some limitations and to date there is still
PT
no well-established antigen detection test for CE. In the present study, an effort was made to develop an antigen detection rapid test in
CC E
format of the LFD assay for CE. The diagnostic sensitivity of the antigen detection LFD was found to be 77.14%, which is higher than that reported in some other studies (Parija et al., 1997; Ravinder et al., 2000; Sadjjadi et al., 2009; Sunita et al., 2011; Bauomi et al., 2015).
A
Parija et al. (1997) showed detection of hydatid antigen in serum of 40 patients with CE using countercurrent immunoelectrophoresis (CIEP), with a diagnostic sensitivity of 45%. Similarly, a sensitivity of 40% was obtained by Sunita et al. (2011) whereby rabbit hyperimmune serum was used in a sandwich ELISA. Using the same ELISA technique, diagnostic sensitivities of 25.7%, 52.5%, and 80% have been reported (Sadjjadi et al., 2009; 14
Chaya and Parija, 2013; Bauomi et al., 2015). Meanwhile, Devi and Parija (2003) reported sensitivity of 72% and 83%, respectively when LAT and Co-A were used to detect antigen in serum samples of 18 patients with CE. However, Ravinder et al. (2000) had demonstrated a lower sensitivity (73%) using Co-A. In another antigen detection study involving 30 patients with CE using Dot-ELISA and EITB, the latter was found to be more sensitive (83%)
IP T
compared to the former (60%) (Swarna and Parija, 2012).
The diagnostic performance of an immunodiagnostic test for CE is dependent on the
SC R
antigen and its purity (Carmena et al., 2006). The above studies used various antigens for the
rabbit hyperimmunizations. Six studies utilized HCF as the source of antigen, four of them
U
used human HCF (Parija et al., 1997; Ravinder et al., 2000; Devi and Parija, 2003; Swarna
N
and Parija, 2012) while the other two (Sadjjadi et al., 2009; Sunita et al., 2011) used sheep
A
HCF. Meanwhile, Chaya and Parija (2013) used purified 24 kDa fragment of urinary hydatid
M
antigen and Bauomi et al. (2015) used 27.5 kDa protoscolex antigen for rabbit immunizations.
ED
Different performances among the above studies, and with the present study may be attributed to the different sources of antigens and different assay formats. Several other
PT
reasons may also contribute to the variable diagnostic sensitivity of the antigen detection tests for CE. First, the amount of circulating antigen in patients with CE, in general, is low and
CC E
varies with organ location, number and size of the cysts (Gottstein, 1984; Sadjjadi et al., 2009). It was previously reported that patients with liver cyst have a higher amount of
A
circulating antigens compared to patients with lung cyst (Craig and Nelson, 1984). In another report by Sadjjadi et al. (2009) circulating antigens were detected in 46% of patients with liver cyst and not detected in patients with lung or kidney cyst. Sunita et al. (2011) detected circulating antigens in 10 out of 25 patients with CE, but they did not disclose patients with
15
which cyst location were positive for circulating antigens. In the present study, the LFD assay detected all patients with lung cysts (5/5) and also 73% of patients with liver cysts. Antigen leakage from hydatid cysts is also dependent on the cyst status/stage, which can be classified based on the WHO standardized ultrasound classification for CE (WHO, 2003). However, in the present study, there was no information on the cyst stage although
IP T
they are mostly expected to be active cysts since the patients were referred for surgery. It has also been reported that the genotype of the isolate may have an impact on the diagnostic
SC R
performance of a serological test. E. granulosus genotype G1, G2, G3 and G6 have been
documented in Iranian patients with CE. However, G1 (54.4%) and G6 (40.8%) were
U
reported as the most prevalent genotypes (Rostami et al., 2015). Unfortunately there was no
A
studies should take this factor into consideration.
N
information on the E. granulosus genotype in CE patients in the present study. Thus further
M
Another possible reason for the observed low diagnostic sensitivity of CE antigen detection tests may be the formation of antigen-antibody immune complexes (Craig, 1986;
ED
Lightowlers and Gottstein, 1995). It was reported that up to 50% of echinococcosis patients did not have enough detectable free antigens in their serum (Gottstein, 1984; Craig, 1986).
PT
Acidic buffer treatment of patient’s serum had been used to release antigen from immune
CC E
complex and this was reported to enhance the performance of the diagnostic test for echinococcosis (Rouhani et al., 2013; Siles-Lucas et al., 2017). We had attempted to dissociate the antigen-antibody complexes in the serum using a low pH glycine buffer
A
treatment, however it did not seem to improve the diagnostic sensitivity. The present study showed a diagnostic specificity of 82.35%. A similar specificity
(83%) was demonstrated by Swarna and Parija (2012) using Dot-ELISA. Bauomi et al. (2015) displayed a lower specificity (75%) using an ELISA. There are other reports of assays with much higher specificity (> 90%) (Parija et al., 1997; Ravinder et al., 2000; Devi and 16
Parija, 2003; Sadjjadi et al., 2009; Sunita et al., 2011; Swarna and Parija, 2012), however, the diagnostic sensitivities of assays based on ELISA and CIEP were noticeably much lower (25.7-45%) (Parija et al., 1997; Sadjjadi et al., 2009; Sunita et al., 2011). Interestingly, an ELISA developed by Chaya and Parija (2013) using rabbit antibody to a 24 kDa human hydatid urinary antigen produced a high specificity of 92% (and sensitivity of 80%),
IP T
however, the identity of the protein was not reported. Using LAT and Co-A, Devi and Parija
(2003) reported specificity of 98% and, while in another study (Ravinder et al., 2000) the
SC R
latter technique gave a specificity of 93.87%. In addition, high specificity was also reported when EITB was used (Swarna and Parija, 2012). The above studies which reported high
U
specificity had used antigens from human sources to generate the antibodies (Ravinder et al.,
N
2000; Devi and Parija, 2003; Swarna and Parija, 2012; Chaya and Parija, 2013), thus this
A
could be a limitation for the sustainable production of such tests. Some studies suggested
M
circulating antigen detection test as a useful tool for post-treatment follow-up and monitoring of cyst activity (Shariff and Parija, 1993; Ravinder et al., 1997; Ferragut et al., 1998;
ED
Gottstein et al., 2014). However, in the present study, post-treatment serum sample from a patient with CE was not available to ascertain this utility.
PT
In the present study, cross-reactions were observed in 6 of 38 healthy Malaysian individuals (Group II), and 3 of 13 Malaysian patients with other parasitic infections (Group
CC E
III). The cross-reactivity with serum of healthy individuals may be due to asymptomatic diseases other than CE since Malaysia is not endemic for echinococcosis and all sera was
A
found to be seronegative by a commercial ELISA kit. Sunita et al. (2011) reported crossreactions with ascariasis and cysticercosis, thus similar to what was observed in the present study. In addition, cross-reactions have also been reported with sera of patients with schistosomiasis, fascioliasis, visceral leishmaniasis, neurocysticercosis, amebic liver abscess, tropical pulmonary eosinophilia and partial seizures (Sadjjadi et al., 2009; Sunita et al., 2011;
17
Chaya and Parija, 2013; Bauomi et al., 2015). The present study is the first to report on crossreaction of a CE antigen detection test with serum of a taeniasis patient, however this is not surprising since cross-reactions with such patients were commonly reported with CE antibody detection assays (Sadjjadi et al., 2007; Hernandez-Gonzalez et al., 2008; Sarkari et al., 2010). Serum samples from patients with alveolar echinococcosis (AE) were not available
IP T
for evaluation of its cross-reactivity with the present LFD assay. Hence it is unknown
whether the assay can discriminate CE from AE. This aspect may be important in
SC R
geographical areas where both infections co-exist. Nevertheless, even if the LFD assay shows cross-reactivity with AE, it can still be used as screening test for echinococcosis. In order to
U
confirm the diagnostic specificity of the LFD assay, screening of many more serum samples
N
with other helminth infections is required.
A
The AUC of the LFD assay in this study was determined to be 0.849, which indicated
M
good diagnostic accuracy in discriminating positive from negative samples (Metz, 1978). Thus far, two rapid lateral flow antibody detection assays for diagnosis of CE are
ED
commercially available (Tamarozzi et al., 2016) and based on the available literature, this is the first report of an antigen detection test in the format of a rapid lateral flow assay. The test
PT
is simple, fast and efficient, it does not require expensive laboratory equipment, and can be performed in the field by a non-laboratory trained person, thus would be useful for low-
CC E
resource areas (Posthuma-Trumpie et al., 2009). The use of a dipstick reader may not be required since the visual interpretation was quite conclusive. The dipstick reader in the
A
present study was mainly used for the purpose of statistical analysis. Nevertheless, the visual aspect of the test can be further improved. Since selected serum samples were used for the development and initial evaluation of the LFD assay, it is important to perform further evaluation with a much larger sample size. Preferably, the further evaluation should be in the form of a multicentre study using samples
18
of CE patients from different regions of the world, including from confirmed CE patients who are antibody-negative, and post-treatment patients. In conclusion, the antigen detection LFD assay developed in this study seemed to be useful for diagnosis of CE and possibly for post-treatment follow-up. We foresee that it may improve serodiagnosis of CE when used in
IP T
tandem with an antibody detection test, however further data are needed to ascertain this.
Funding
SC R
This work was supported by Universiti Sains Malaysia Research University grant [Grant no. 1001.PFARMASI.812153] and Malaysian Ministry of Higher Education through the Higher
N
U
Institution Centre of Excellence Program (HICoE) [Grant no. 311/CIPPM/4401005].
M
A
Disclosure: None
Acknowledgments
ED
We would like to acknowledge and thank Dr. Zohreh Kazemi Moghadam Kakhki and Ms Nor Dyana Zakaria for contributing serum samples and providing technical assistance,
CC E
PT
respectively.
References
A
Bauomi, I.R., El-Amir, A.M., Fahmy, A.M., Zalat, R.S., Diab, T.M., 2015. Evaluation of purified 27.5 kDa protoscolex antigen-based ELISA for the detection of circulating antigens and antibodies in sheep and human hydatidosis. Journal of helminthology 89, 577-583. Brunetti, E., Garcia, H.H., Junghanss, T., on behalf of the members of the International Ce Workshop in Lima, P., 2011. Cystic Echinococcosis: Chronic, Complex, and Still Neglected. PLoS neglected tropical diseases 5, e1146. Brunetti, E., Kern, P., Vuitton, D.A., Writing Panel for the, W.-I., 2010. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta tropica 114, 1-16.
19
Budke, C.M., Deplazes, P., Torgerson, P.R., 2006. Global socioeconomic impact of cystic echinococcosis. Emerging infectious diseases 12, 296-303. Carmena, D., Benito, A., Eraso, E., 2006. Antigens for the immunodiagnosis of Echinococcus granulosus infection: An update. Acta tropica 98, 74-86. Chaya, D., Parija, S.C., 2013. Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis. Tropical parasitology 3, 125-131.
IP T
Craig, P.S., 1986. Detection of Specific Circulating Antigen, Immune-Complexes and Antibodies in Human Hydatidosis from Turkana (Kenya) and Great-Britain, by EnzymeImmunoassay. Parasite Immunol 8, 171-188.
SC R
Craig, P.S., Nelson, G.S., 1984. The detection of circulating antigen in human hydatid disease. Annals of tropical medicine and parasitology 78, 219-227.
Craig, P.S., Rogan, M.T., Allan, J.C., 1996. Detection, screening and community epidemiology of taeniid cestode zoonoses: cystic echinococcosis, alveolar echinococcosis and neurocysticercosis. Advances in parasitology 38, 169-250.
N
U
Deplazes, P., Rinaldi, L., Alvarez Rojas, C.A., Torgerson, P.R., Harandi, M.F., Romig, T., Antolova, D., Schurer, J.M., Lahmar, S., Cringoli, G., Magambo, J., Thompson, R.C., Jenkins, E.J., 2017. Global Distribution of Alveolar and Cystic Echinococcosis. Advances in parasitology 95, 315-493.
M
A
Devi, C.S., Parija, S.C., 2003. A new serum hydatid antigen detection test for diagnosis of cystic echinococcosis. The American journal of tropical medicine and hygiene 69, 525528.
ED
Eckert, J., Gemmell, M.A., Meslin, F.X., World Health, O., International Office of, E., 2001. WHO/OIE Manual on echinococcosis in humans and animals : a public health problem of global concern. OIE ; WHO, Paris; Geneva.
PT
Ferragut, G., Ljungstrom, I., Nieto, A., 1998. Relevance of circulating antigen detection to follow-up experimental and human cystic hydatid infections. Parasite Immunol 20, 541549.
CC E
Gottstein, B., 1984. An immunoassay for the detection of circulating antigens in human echinococcosis. The American journal of tropical medicine and hygiene 33, 1185-1191. Gottstein, B., Wang, J., Blagosklonov, O., Grenouillet, F., Millon, L., Vuitton, D.A., Müller, N., 2014. Echinococcus metacestode: in search of viability markers. Parasite (Paris, France) 21, 63.
A
Hernandez-Gonzalez, A., Muro, A., Barrera, I., Ramos, G., Orduna, A., Siles-Lucas, M., 2008. Usefulness of four different Echinococcus granulosus recombinant antigens for serodiagnosis of unilocular hydatid disease (UHD) and postsurgical follow-up of patients treated for UHD. Clinical and vaccine immunology : CVI 15, 147-153. Khalilpour, A., Sadjjadi, S.M., Moghadam, Z.K., Yunus, M.H., Zakaria, N.D., Osman, S., Noordin, R., 2014. Lateral flow test using Echinococcus granulosus native antigen B and comparison of IgG and IgG4 dipsticks for detection of human cystic echinococcosis. The American journal of tropical medicine and hygiene 91, 994-999.
20
Lightowlers, M.W., Gottstein, B., 1995. Echinococcosis/hydatidosis: antigens, immunological and molecular diagnosis. CAB INTERNATIONAL, Wallingford, pp. 355-410. Makhsin, S.R., Razak, K.A., Noordin, R., Zakaria, N.D., Chun, T.S., 2012. The effects of size and synthesis methods of gold nanoparticle-conjugated MalphaHIgG4 for use in an immunochromatographic strip test to detect brugian filariasis. Nanotechnology 23, 495719.
IP T
Mariconti, M., Bazzocchi, C., Tamarozzi, F., Meroni, V., Genco, F., Maserati, R., Brunetti, E., 2014. Immunoblotting with human native antigen shows stage-related sensitivity in the serodiagnosis of hepatic cystic echinococcosis. The American journal of tropical medicine and hygiene 90, 75-79.
SC R
McManus, D.P., Gray, D.J., Zhang, W., Yang, Y., 2012. Diagnosis, treatment, and management of echinococcosis. BMJ (Clinical research ed.) 344, e3866.
Metz, C.E., 1978. Basic principles of ROC analysis. Seminars in nuclear medicine 8, 283298.
U
Moosa, R.A., Abdel-Hafez, S.K., 1994. Serodiagnosis and seroepidemiology of human unilocular hydatidosis in Jordan. Parasitology research 80, 664-671.
A
N
Moro, P., Schantz, P.M., 2009. Echinococcosis: a review. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 13, 125-133.
M
Parija, S.C., Ravinder, P.T., Rao, K.S., 1997. Detection of hydatid antigen in urine by countercurrent immunoelectrophoresis. Journal of clinical microbiology 35, 1571-1574.
ED
Posthuma-Trumpie, G.A., Korf, J., van Amerongen, A., 2009. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and Bioanalytical Chemistry 393, 569-582.
PT
Ravinder, P.T., Parija, S.C., Rao, K.S., 1997. Evaluation of human hydatid disease before and after surgery and chemotherapy by demonstration of hydatid antigens and antibodies in serum. Journal of medical microbiology 46, 859-864.
CC E
Ravinder, P.T., Parija, S.C., Rao, K.S., 2000. Urinary hydatid antigen detection by coagglutination, a cost-effective and rapid test for diagnosis of cystic echinococcosis in a rural or field setting. Journal of clinical microbiology 38, 2972-2974. Ray, R., De, P.K., Karak, K., 2002. Combined role of Casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian journal of medical microbiology 20, 79-82.
A
Rostami, S., Torbaghan, S.S., Dabiri, S., Babaei, Z., Mohammadi, M.A., Sharbatkhori, M., Harandi, M.F., 2015. Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran. The American journal of tropical medicine and hygiene 92, 588-594. Rouhani, S., Parvizi, P., Spotin, A., 2013. Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient's sera: a novel hypothesis to follow-up of Cystic echinococcosis after surgery. Medical hypotheses 81, 557-560.
21
Sadjjadi, S.M., Abidi, H., Sarkari, B., Izadpanah, A., Kazemian, S., 2007. Evaluation of enzyme linked immunosorbent assay, utilizing native antigen B for serodiagnosis of human hydatidosis. Iranian journal of immunology : IJI 4, 167-172. Sadjjadi, S.M., Sedaghat, F., Hosseini, S.V., Sarkari, B., 2009. Serum Antigen and Antibody Detection in Echinococcosis: Application in Serodiagnosis of Human Hydatidosis. Korean J Parasitol 47, 153-157.
IP T
Saidin, S., Yunus, M.H., Othman, N., Lim, Y.A., Mohamed, Z., Zakaria, N.Z., Noordin, R., 2017. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample. Pathogens and global health 111, 128-136.
SC R
Sarkari, B., Sadjjadi, S.M., Beheshtian, M.M., Aghaee, M., Sedaghat, F., 2010. Human cystic echinococcosis in Yasuj District in Southwest of Iran: an epidemiological study of seroprevalence and surgical cases over a ten-year period. Zoonoses and public health 57, 146-150. Shariff, M., Parija, S.C., 1993. Co-agglutination (Co-A) test for circulating antigen in hydatid disease. Journal of medical microbiology 38, 391-394.
N
U
Siles-Lucas, M., Casulli, A., Conraths, F.J., Muller, N., 2017. Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals. Advances in parasitology 96, 159-257.
M
A
Sunita, T., Khurana, S., Malla, N., Dubey, M.L., 2011. Immunodiagnosis of cystic echinocooccosis by antigen detection in serum, urine, and saliva samples. Tropical parasitology 1, 33-38.
ED
Swarna, S.R., Parija, S.C., 2012. Evaluation of Dot-ELISA and enzyme-linked immunoelectrotransfer blot assays for detection of a urinary hydatid antigen in the diagnosis of cystic echinococcosis. Tropical parasitology 2, 38-44.
PT
Tamarozzi, F., Covini, I., Mariconti, M., Narra, R., Tinelli, C., De Silvestri, A., Manzoni, F., Casulli, A., Ito, A., Neumayr, A., Brunetti, E., 2016. Comparison of the Diagnostic Accuracy of Three Rapid Tests for the Serodiagnosis of Hepatic Cystic Echinococcosis in Humans. PLoS neglected tropical diseases 10, e0004444.
CC E
Torgerson, P.R., Deplazes, P., 2009. Echinococcosis: diagnosis and diagnostic interpretation in population studies. Trends Parasitol 25, 164-170. WHO, 2003. International classification of ultrasound images in cystic echinococcosis for application in clinical and field epidemiological settings. Acta tropica 85, 253-261.
A
Zhang, W., Li, J., McManus, D.P., 2003. Concepts in immunology and diagnosis of hydatid disease. Clinical microbiology reviews 16, 18-36. Zhang, W., Wen, H., Li, J., Lin, R., McManus, D.P., 2012. Immunology and immunodiagnosis of cystic echinococcosis: an update. Clinical & developmental immunology 2012, 101895.
22