Acta Tropica 148 (2015) 32–37
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Field evaluation of a rapid diagnostic test to detect antibodies in human toxocariasis P.K.C. Lim a,∗ , H. Yamasaki b , J.W. Mak a , S.F. Wong a , C.W. Chong c , I.K.S. Yap c , S. Ambu a , V. Kumarasamy a a
School of Medical Sciences, International Medical University, 126 Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia Department of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan c School of Pharmacy, International Medical University, 126 Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia b
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 23 March 2015 Accepted 13 April 2015 Available online 22 April 2015 Keywords: Human toxocariasis Immunodiagnosis Enzyme-linked immunosorbent assay Rapid diagnostic test Recombinant antigen
a b s t r a c t Human toxocariasis which is caused mainly by the larvae of Toxocara canis and Toxocara cati, is a worldwide zoonotic disease that can be a potentially serious human infection. The enzyme-linked immunosorbent assay (ELISA) using T. canis excretory–secretory (TES) antigens harvested from T. canis larvae is currently the serological test for conﬁrming toxocariasis. An alternative to producing large amounts of Toxocara TES and improved diagnosis for toxocariasis is through the development of highly speciﬁc recombinant antigens such as the T. canis second stage larva excretory–secretory 30 kDa protein (recTES-30). The aim of this study was to evaluate the sensitivity and speciﬁcity of a rapid diagnostic kit (RDT, named as iToxocara kit) in comparison to recTES-30 ELISA in Serendah Orang Asli village in Selangor, Malaysia. A total of 133 subjects were included in the study. The overall prevalence rates by ELISA and RDT were 29.3% and 33.1%, respectively, with more positive cases detected in males than females. However, no association was found between toxocariasis and gender or age. The percentage sensitivity, speciﬁcity, positive predictive value and negative predictive value of RDT were 85.7%, 90.1%, 80% and 93.2%, respectively. The prevalence for toxocariasis in this population using both ELISA and RDT was 27.1% (36/133) and the K-concordance test suggested good agreement of the two tests with a Cohen’s kappa of 0.722, P < 0.01. In addition, the followed-up Spearman rank correlation showed a moderately high correlation at R = 0.704 and P < 0.01. In conclusion, the RDT kit was faster and easier to use than an ELISA and is useful for the laboratory diagnosis of hospitalized cases of toxocariasis. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Human toxocariasis which is caused mainly by the larvae of Toxocara canis and Toxocara cati, is a worldwide zoonotic disease that can be a potentially serious human infection. In Malaysia, studies have shown that the prevalence of human toxocariasis ranged from 19.6% to as high as 35.5% (Hakim et al., 1992, 1993). The disease normally presents as either visceral or ocular although some patients are asymptomatic. Common manifestations include persistent eosinophilia, fever, pneumonitis, visual ﬁeld defects and neurological disturbances (Watthanakupanich, 2010). Cases of
∗ Corresponding author. Tel.: +603 27317586; fax: +603 86567229. E-mail addresses: kimchooi [email protected]
, [email protected]
(P.K.C. Lim), [email protected]
(H. Yamasaki), joonwah [email protected]
(J.W. Mak), shewfung [email protected]
(S.F. Wong), chunwie [email protected]
(C.W. Chong), ivan [email protected]
(I.K.S. Yap), stephen [email protected]
(S. Ambu), verasingam [email protected]
visceral larva migrans (VLM) are mostly asymptomatic or present with mild symptoms while cases of ocular larva migrans (OCM) may have chronic inﬂammation and permanent eye damage. Covert toxocariasis is most frequently found in children and the clinical symptoms range from fever, headache, abdominal pain to cervical lymphadenitis and hepatomegaly (Watthanakupanich, 2010). As many of the symptoms of toxocariasis are nonspeciﬁc and mild, diagnosis of the disease can be quite difﬁcult. Chronic eosinophilia, hepatomegaly, chronic pulmonary disease, or a history of exposure to puppies or contact with feces-contaminated soil are common indicators of infection. So in addition to taking patient history to determine exposure to risks, laboratory investigations are useful for the diagnosis and various combinations of tests are often used for conﬁrmation. Microscopic or macroscopic examination of the parasite remains the gold standard for protozoan and helminthic parasites including Toxocara spp. Toxocara larva can be identiﬁed from affected tissue (Parsons et al., 1986) or under the retina depending on the site of infection (Singh et al., 2007).
http://dx.doi.org/10.1016/j.actatropica.2015.04.011 0001-706X/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
P.K.C. Lim et al. / Acta Tropica 148 (2015) 32–37
More recently, molecular tools such as polymerase chain reaction, DNA hybridization and DNA sequencing technology have been used to diagnose Toxocara infections (Rai et al., 1997; Wu et al., 1997; Van De et al., 2013). However, serological assays using immunological techniques are considered the most effective approach for laboratory diagnosis of Toxocara infection and most of the assays detect anti-Toxocara antibodies. The enzyme-linked immunosorbent assay (ELISA) using T. canis excretory–secretory (TES) antigens harvested from T. canis larvae is currently the serological test for conﬁrming toxocariasis (Magnaval et al., 2001) but some cross reactions have been reported with other nematodes (Kennedy et al., 1987, 1989; Yamasaki et al., 2000). Other toxocariasis diagnostic methods that were developed based on TES to improve sensitivity and speciﬁcity included the indirect antibody competition ELISA (Nunes et al., 1999), dot-ELISA (Carmago et al., 1992), and antigen capture assays using monoclonal antibodies (Gillespie et al., 1993; Ngah et al., 1999). An alternative to producing large amounts of Toxocara TES and improved diagnosis for toxocariasis is through the development of highly speciﬁc recombinant antigens which share important antigenic and immunogenic structures with native TES. Recombinant antigens have been produced for T. canis second stage larva excretory–secretory 30 kDa protein (recTES-30) (Yamasaki et al., 1998, 2000) and 120 kDa protein (Fong et al., 2003) for diagnosis of human toxocariasis. The recTES-30 was reported to be sensitive and speciﬁc in ELISA and did not cross react with sera from individuals infected with Ascaris and hookworms
(Yamasaki et al., 2000; Coêlho et al., 2005); however there was a low cross-reactivity with sera from individuals with paragonimiasis, ganthostomiasis and spirometriasis (Yamasaki et al., 2000). ELISA using recTES-30 has also been evaluated in two other formats namely dot-ELISA and immunoblot and all three formats were comparable in terms of sensitivity and speciﬁcity to recTES-30-ELISA (Lim et al., 1999). Both the dot-ELISA and immunoblot appeared to be more sensitive than recTES-30 ELISA and the dot-ELISA was technically quicker and easier to perform compared to ELISA. In an effort to improve the convenience and time taken for diagnosis, recTES-30 was used in the development of an immunochromatographic device to detect anti-Toxocara antibodies (Yamasaki, 2012). The objective of this study was therefore to evaluate the sensitivity and speciﬁcity of this rapid diagnostic kit (RDT, named as iToxocara kit) in comparison to recTES-30-ELISA in an Orang Asli village in Selangor, Malaysia.
2. Materials and methods 2.1. Study area The study site selected was Serendah Orang Asli village located in Hulu Selangor (3◦ 21 50 N, 101◦ 37 28 E) (Fig. 1), about 60 km away from Kuala Lumpur, Malaysia. The village has a population of about 250 people who are from the Temuan tribe.
Fig. 1. Location map of Serendah Orang Asli Village, Selangor, Malaysia. (Source: http://www.click2map.com/).
P.K.C. Lim et al. / Acta Tropica 148 (2015) 32–37
2.2. Sample collection
2.4. Rapid diagnostic kit
The study was conducted in December 2013. The study population comprised of a total of 133 study participants aged between 6 months to 62 years old. A baseline questionnaire was applied to the study participants to obtain demographic and clinical information and physical examination of the subjects was carried out by clinicians. This project received ethics approval from the International Medical University (IMU) Joint Committee on Ethics and Research and the Medical Ethics Committee of the National Institute of Infectious Diseases, Tokyo, Japan (no. 177). Informed consent was obtained from the test subjects for blood collection. For each subject a ﬁnger prick blood sample of about 100 l volume was collected using 1 ml plain microtainer (Becton Dickinson, USA). The blood samples were then transported back to the Research Laboratory at IMU. The blood samples were allowed to clot, then centrifuged at 1500 × g for 10 min and the serum was collected into a sterile 0.2-ml microtube and stored at −20 ◦ C till used in the ELISA or the RDT.
The rapid diagnostic kit using the recTES-30 antigen (Yamasaki et al., 1998, 2000) was optimized based on the ELISA result using the same antigen at Adtech Inc. Ltd., Oita, Japan. The reliability of the diagnostic results was evaluated according to instructional manual: serum samples were diluted 1:5 in sample buffer provided by the kit, and a 5-l aliquot of the diluted serum sample was loaded onto the area “serum”, and then one drop of alkaline phosphataselabelled anti-human IgG was spotted onto “conjugate”. Finally, a bag containing “buffer” was pressed to release the substrate solution (Fig. 2A). Negative result was judged by the appearance of a blue band at the control (C) line only (Fig. 2B). Positive result was judged by the appearance of blue band at the test (T) line and the control line (C) within 20 min (Fig. 2C) according to reference board (Fig. 2D).
2.3. ELISA procedure ELISA was performed as reported by Yamasaki et al. (2000) with some modiﬁcations. Brieﬂy, 96-well microtiter plates (Maxisorp, Nunc-Immuno Plate, Thermo Scientiﬁc, Denmark) were coated with recTES-30 antigen at a concentration of 0.5 g of proteins per ml in 0.05 M bicarbonate buffer, pH 9.6 (100 l/well), 4 ◦ C overnight. The microtiter plates were washed three times with 0.15 M phosphate-buffered saline containing 0.05% Tween 20 (PBS/T) and then probed with a 1:200-diluted human serum sample (100 l/well) in PBS/T containing 1% bovine serum albumin (BSA) for 40 min at 37 ◦ C. After washing the plate three times with PBS/T, 100 l of 1:1000-diluted peroxidase (POD)-labelled Protein G (Zymed, USA) in PBS/T containing 1% BSA were added to each well and the plate was incubated for 40 min at 37 ◦ C. The plates were then washed another three times with PBS/T. For color development, 100 l of substrate (containing 1 l of 40 mM ABTS, 5 l of 0.1 M citric acid, 5 l of 0.1 M disodium hydrogen phosphate and 0.005 l 30% hydrogen peroxide) were added to each well and the plate was incubated for 5–10 min at room temperature. The reaction was terminated by adding 100 l of 1% sodium dodecyl sulphate per well. Absorbance at 405 nm was measured with a microplate reader (Tecan). The cutoff point was set at 3 times the optical density (OD) value for negative pooled serum samples.
2.5. Statistical analyses All statistical analyses were conducted using SPSS Software Package (IBM, USA). Scattered plot representing Spearman Rank Correlation was constructed by fractional ranking the parameters using the “rank cases” option available in SPSS. 3. Results A total of 133 subjects participated in the study. Table 1 shows their distribution by age groups and gender. Except for two age groups (1–10 years and 61–70 years), all the other age groups had more females than males and the percentages of males and females overall were 39.09% and 60.91%, respectively. Children aged 10 years and below formed the largest age group (53.39%) followed Table 1 Distribution of study participants by age group and gender. Age group (years old)
No. of males (%)
No. of females (%)
1–10 11–20 21–30 31–40 41–50 51–60 61–70
41(30.83%) 6 (4.51%) 0 (0%) 1 (0.75%) 1 (0.75%) 2 (1.50%) 1 (0.75%)
30 (22.56%) 21 (15.79%) 14 (10.53%) 8 (6.02%) 5 (3.76%) 3 (2.25%) 0 (0%)
Total no. (%)
Total no. (%) 71 (53.39%) 27 (20.30%) 14 (10.53%) 9 (6.77%) 6 (4.51%) 5 (3.75%) 1 (0.75%) 133 (100%)
Fig. 2. iToxocara kit using recTES-30 antigen. (A) Immunochromatographic device. (B) Negative case. (C) Positive case. (D) Reference board. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article).
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Table 2 Prevalence of toxocariasis by age group and gender as determined using ELISA and RDT. Age group (years old)
No. of males positive
No. of females positive
1–10 11–20 21–30 31–40 41–50 51–60 61–70
11/41 4/6 0/0 1/1 0/1 0/2 1/1
15/41 4/6 0/0 1/1 0/1 0/2 0/1
Total prevalence (%)
ELISA 8/30 5/21 5/14 0/8 3/5 1/3 0/0
No. positive No. negative
9/30 7/21 4/14 0/8 3/5 1/3 0/0
Table 3 Comparison between ELISA and rapid diagnostic test (RDT) for the detection of antibodies in human sera against recTES-30 antigen.
36 6 42
9 82 91
45 88 133
by the 11–20 years age group (20.30%) and 21–30 years age group (10.53%). The total percentage of adults between 30 and 70 years of age who participated in the study was low (15.78%). Table 2 shows the prevalence of toxocariasis by age group and gender as determined using the two tests. A total of 39 study participants were positive in ELISA, 17 of them were males and 22 females with prevalence in males (32.7%) higher than in females (27.2%). The prevalence rates in children and adults 30 years and below ranged from 26.8% to 35.7% and the overall prevalence by ELISA was 29.3%. Using RDT, there were more positive cases detected in both males and females compared to ELISA. Like ELISA, the % prevalence in males (38.5%) was higher than in females (29.6%). The overall prevalence of toxocariasis as detected by RDT was 33.1%, higher than that detected using ELISA (29.3%). Nevertheless, the association between toxocariasis with gender and age was not supported by the Chi-Square test (P = 0.449–0.699). The comparison of recTES-30-ELISA and RDT for the detection of anti-Toxocara antibodies in sera of the 133 study subjects are summarized in Table 3. Using recTES-30-ELISA as the standard test, both tests detected 36 positive cases and 82 negative cases. There were 9 false positive tests and 6 false negative tests detected by RDT. Of the 9 false positive tests, the intensity of bands in RDT ranged from 1+ to 2+. For the 6 false negative tests, 5 out of 6 sera samples gave O.D. values of