- Short report
- Open Access
Development and usefulness of an immunochromatographic device to detect antibodies for rapid diagnosis of human gnathostomiasis
- Received: 24 December 2015
- Accepted: 30 December 2015
- Published: 12 January 2016
Abstract
Background
Human gnathostomiasis is a serious tropical disease, which is often overlooked. There is an urgent need to improve tools to aid the potential diagnosis of the disease in endemic regions. To overcome this, we produced the immunochromatographic test (ICT) kit for a rapid and simple diagnosis of human gnathostomiasis.
Findings
The recombinant protein (named rGslic18) was applied to ICT kit as the antigen. The diagnostic value of ICT kit was evaluated using serum samples from parasitologically proven and clinically suspected gnathostomiasis patients, healthy volunteers and patients with other parasitic diseases. The ICT kit exhibited quite high sensitivity (93.75 %) and specificity (97.01 %).
Conclusions
The ICT kit is simple, convenient and easy to implement and expected to provide reliable diagnostic results for human gnathostomiasis. It also will be a promising diagnostic tool not only for large-scale epidemiological surveys in endemic or remote areas where diagnostic facilities are poor but also for a rapid clinical diagnosis in the bedside laboratory.
Keywords
- Gnathostoma spinigerum
- Human gnathostomiasis
- Immunochromatographic test kit
- Serodiagnosis
- Recombinant protein
Background
Human gnathostomiasis is a serious food-borne parasitic zoonosis caused by infections with larval spirurid nematodes Gnathostoma spp. and the disease is regularly found in Asia and the Americas [1]. It is also common in travelers returning from visits to areas endemic to this harmful parasite [1]. Definitive diagnosis for human gnathostomiasis can be made by detecting the larvae migrating out from the human body. However, since direct detection of the parasite is difficult and often unsuccessful, diagnosis of human gnathostomiasis is primarily made by relying upon clinical features, history of eating parasite-contaminated food, elevated blood eosinophilia, and serological outcomes [1]. Here, a recombinant protein produced from a Gnathostoma spinigerum clone (named Gslic18) isolated from G. spinigerum cDNA library, was used as an alternative antigen for the immunochromatographic test (ICT) kit development. The result was analysed and compared with specific IgG antibody detection against the native 24/21 kDa G. spinigerum larval antigen using immunoblotting [2]. This study demonstrated the effectiveness of the ICT kit as a convenient and rapid platform in the diagnosis of human gnathostomiasis that had not yet been reported elsewhere.
Methods
Types of human sera examined and diagnostic results of the ICT kit and the immunoblotting using the 24/21 kDa G. spinigerum antigen
Type of serum samples | Number of positive/Total number | |
---|---|---|
ICT kit | Immunoblotting | |
Healthy control | 0/20 | 0/20 |
Confirmed gnathostomiasis | 9/9 | 9/9 |
Suspected gnathostomiasis | 21/23 | 23/23 |
Cysticercosis | 1/4 | 0/4 |
Taeniasis | 0/10 | 0/10 |
Opisthorchiasis viverrini | 0/15 | 0/15 |
Fascioliasis | 1/5 | 0/5 |
Paragonimiasis | 1/10 | 0/10 |
Angiostrongyliasis | 1/10 | 0/10 |
Strongyloidiasis | 0/10 | 0/10 |
Hookworm infection | 0/10 | 0/10 |
Capillariasis | 0/10 | 0/10 |
Ascariasis | 0/10 | 0/10 |
Trichinellosis | 0/3 | 0/3 |
Sparganosis | 0/2 | 0/2 |
Malaria | 0/10 | 0/10 |
Filariasis | 0/5 | 0/5 |
The KAN gnathostomiasis kit (a) with immunochromatographic assay for the diagnosis of human gnathostomiasis. Representative images of ICT strips on which positive (b) and negative (c) results are shown. Each diluted serum sample was dropped onto the inscription “sample”, and a buffer was applied onto inscription “buffer”. A criterion of the diagnostic result is whether a red band appears at the test (T) line within 15 minutes or not. In the positive serum sample, the test (T) line and control (C) line turned red (b) but in the negative serum sample, only the control (C) line turned red (c). The intensity of the bands was estimated visually (unaided) according to the reference board (d considering level 2 as the cutoff level)
Results and discussion
The purified rGslic18 protein fused with His-tagged residues on 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The protein was stained with Coomasie Brilliant Blue. An arrow indicates band of the rGslic18 protein (14 kDa)
Comparison among ICT kit using rGslic18 and immunoblotting using 24/21 kDa G. spinigerum antigen
Test type and results | Immunoblotting | ||
---|---|---|---|
ICT kita | No. positive | No. negative | Total |
No. positive | 30 | 4 | 34 |
No. negative | 2 | 130 | 132 |
Total | 32 | 134 | 166 |
Conclusions
This is the first time that the ICT kit with rGslic18 as an antigen was applied for the detection of anti-Gnathostoma antibodies in human sera and the results revealed high sensitivity and specificity. The ICT kit is a fast, simple, easy to implement and reliable as previous reports [7–9]. It has the potential to aid the development of a promising serodiagnostic kit for human gnathostomiasis as a stable mass production system, particularly for screening patients on a massive-scale in endemic areas or clinical diagnosis in the laboratory.
Declarations
Acknowledgements
We wish to acknowledge the support of the English consultant Clinic, Research Affair, the Faculty of Medicine, Khon Kaen University and the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University, for their assistance. This research was supported by a grant from the Higher Education Research Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission, Thailand through the Health Cluster (SHeP-GMS) and the Faculty of Medicine, Khon Kaen University grant number RRU57201. RR was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/0053/2556). OS was supported by the Postdoctoral Training Program Graduate School and Khon Kaen University (grant no. 58101), Khon Kaen University. PMI and WM were supported by the TRF Senior Research Scholar Grant, Thailand Research Fund grant number RTA5880001. HY was supported by Grants-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare, Japan (H25 ~ H26-Shinko-Ippan-009) and Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (No.15fk0108025h0502).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
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