Introduction and Background of the Project

Asian zoonotic schistosomiasis is a parasitic disease caused by the blood fluke Schistosoma japonicum. This parasite infects humans through contact with contaminated water making children and farmers at high risk of infection. The disease is endemic in China, the Philippines and parts of Indonesia. Fifty million people in China and 2.5 million people in the Philippines are at risk of having the infection. Vaccine has not been developed so the disease control solely relies on chemotherapy with mass drug administration (MDA) of praziquantel in endemic areas. The London Declaration in 2012 also encouraged this strategy to be developed in the disease endemic countries. Follow-up assessment of the effectiveness of the disease control depends entirely on labor-intensive, time-consuming and low sensitive microscopic examination of stool for the detection of the parasite eggs. A rapid point-of-care test (POCT) with higher sensitivity will therefore improve the efficacy of the follow-up program of MDA and will facilitate morbidity control. Also, POCT will be applicable in active case detection and confirmatory diagnosis with clinically suspected patients.

The National Research Center for Protozoan Diseases (NRCPD), Obihiro University has already identified the recombinant thioredoxin peroxidase-1 (SjTPx-1) and the tandem repeat Sj7TR proteins as good diagnostic antigens for human schistosomiasis. Additional targets complementing the said recombinant antigens will be screened and selected to increase the sensitivity of the test. These recombinant antigens will be applied as diagnostic antigens for an antibody detection-based POCT.

How can your partnership (project) address global health challenges?

The World Health Organization targets the elimination of schistosomiasis by 2020. As the number of cases in endemic areas has declined significantly due to MDA, the real challenge now is to develop an effective surveillance system that can detect the disease in different diagnostic settings. This project aims to produce the POCT that will be user-friendly, feasible to use in remote areas and replace the traditional microscopic examination in schistosomiasis diagnosis.

Our approach to such a POCT is to develop an antibody detection test using defined antigens, and expertise of all the collaboration partners is essential in achieving the goal. NRCPD is well equipped in leading this project in developing POCT for human schistosomiasis as it is a foremost institution in developing diagnostic techniques for different parasitic diseases with veterinary importance. Graduate School of Agriculture and Life Sciences, the University of Tokyo has an excellent expertise in antigen discovery, and is a pioneer of in silico antigen discovery for parasitic diseases. The College of Public Health, the University of the Philippines Manila is the premiere public health education institution in the Philippines and expert in conducting researches on tropical medicine in the field. InBios International, Inc. is a medical diagnostic company that specializes in developing, manufacturing and marketing medical diagnostic tests for the detection of infectious diseases, and has developed proprietary technologies useful in the development of rapid and ELISA based immunodiagnostic assays. The company also has regulatory experience and has several FDA cleared products (http://www.inbios.com). Therefore, this partnership will make possible the development of a POCT for Asian zoonotic schistosomiasis.

What sort of innovation are you bringing in your project?

The Schistosomiasis Detect consortium will use its expertise in developing a highly sensitive and specific POCT that is designed to improve the current surveillance system for schistosomiasis. This is the first time that POCT using recombinant proteins for schistosomiasis will be developed. Previously developed lateral flow based diagnostic kits were utilizing the crude egg antigens which causes cross-reactions with other parasitic diseases affecting their specificity. This project takes advantage of our expertise in antigen discovery through systematic screening of parasite database. We have already succeeded in identifying parasite-specific antigens with diagnostic potential including Sj7TR through a computational analysis of the S. japonicum genome database, and will utilize this strategy to further our antigen discovery for development of an accurate POCT for schistosomiasis.

Role and Responsibility of Each Partner

The National Research Center for Protozoan Diseases (NRCPD) is a national research institute for different parasitic diseases and has been assigned as one of OIE collaborating Center. Dr. Shin-ichiro Kawazu’s group is currently working with the development of diagnostic tests for zoonotic schistosomiasis in both humans and animals. The NRCPD is the project’s lead agency responsible for the coordination between the collaborating partners. They are also serving as the main hub of experimental procedures in this project including the evaluation of the recombinant antigens, fusion proteins and the POCT module prototype.

The University of Tokyo The University of Tokyo (UT) is one of the top universities in Japan and Dr. Yasuyuki Goto’s laboratory in this university is well equipped to achieve discovery of novel diagnostic candidates for schistosomiasis. Dr. Goto has an excellent expertise in antigen discovery, and is a pioneer of in silico antigen discovery for parasitic diseases. He has established antigen mining based on tandem repeat which is so far successful to identify antigens with serological significance for leishmaniasis, Chagas disease, sleeping sickness as well as schistosomiasis. UT is responsible for this project’s antigen mining and development of chimeric proteins.

The College of Public Health (CPH) of the University of the Philippines Manila is the premier college of public health in the Philippines with Dr. Lydia R. Leonardo being a leading researcher for schistosomiasis in the Philippines. They have been involved in various researches on schistosomiasis that required both laboratory and field work. Since CPH is responsible for the coordination of the sampling sites and sample collection in the Philippines, their intensive and long-rooted collaboration with the Philippine Department of Health is important in the mobilization of health personnel necessary for this project’s field work.

InBios International is a twenty-year-old company specialized in developing, manufacturing and marketing medical diagnostic tests for the detection of infectious diseases based on the proprietary reagents and technologies. InBios is committed to developing high quality, cost effective diagnostics tests for neglected tropical diseases like Chagas’ disease and visceral leishmaniasis, cutaneous leishmaniasis and schistosomiasis. The company's facility is cGMP-compliant and FDA-registered, which enable to manufacture products in accordance with the Quality System Regulations of the FDA. In this project, InBios is responsible for developing the POCT module prototype for the detection of schistosomiasis in humans.

Others (including references if necessary)

1. World Health Organization, 2012. Accelerating work to overcome the global impact of neglected tropical diseases; a roadmap for implementation. Geneva, 2012

2. London Declaration on Neglected Tropical Diseases, www.UnitingToCombatNTDs.org

3. Angeles JM, Goto Y, Kirinoki M, Leonardo L, Tongol-Rivera P, Villacorte E, Inoue N, Chigusa Y, Kawazu S. Human antibody response to thioredoxin peroxidase-1 and tandem repeat proteins as immunodiagnostic antigen candidates for Schistosoma japonicum infection. American Journal of Tropical Medicine and Hygiene 2011, 85(4):674–679.

Final Report

1. Project objective

Asian zoonotic schistosomiasis is a parasitic infectious disease caused by Schistosoma japonicum. The disease is endemic in China, the Philippines and parts of Indonesia with Mass Drug Administration as the main activity of control programs. The treatment kills the adult worms and eggs but not the juvenile, therefore reassessment of the infection and retreatment of the patient is indispensable. This assessment currently depends on traditional stool examination with low sensitivity. A rapid point-of-care test (POCT) with higher sensitivity will therefore improve the efficacy of this follow-up program. The general objective of this project is to develop a robust POCT for the detection of Asian zoonotic schistosomiasis in humans.

2. Project design

Milestone 1: To identify and characterize 5 or more antigens for the development of the POCT which detect antibody in more than 70% of patients.

Milestone 2: To optimize the recombinant antigens for the POCT and produce 3 or more constructs which detect antibody in more than 90% of patients.

Milestone 3: To develop and produce the prototype POCT module for pre-clinical evaluation with a sensitivity of 90%.

Milestone 4: To evaluate the prototype POCT module in the field with a sensitivity of 90%.

3. Results, lessons learned

Milestone 1: We evaluated 19 single antigens with several types of serum panels, including microscopic egg positive, PCR-based egg-positive, endemic negative and non-endemic negative serum panels by ELISA. Based on the results, we have identified 5 single antigens that can detect antibodies in more than 70% of both the microscopic and PCR positive patients. These results therefore cleared our first milestone. Two of these single antigens even have detection rates higher than 90% which is our objective for the second milestone.

Milestone 2: We have designed recombinant fusion proteins based on the evaluation of the single antigens. We successfully constructed and produced 3 recombinant fusion proteins and evaluate them for the second milestone.  Based on the evaluation, one of them has met the target detection rate of more than 90% while another one has fared near this target and will also be useful for the POCT development. Overall, we prepared two single antigens and two fusion proteins for the third milestone. We have somehow achieved our goal for the second milestone.

Milestone 3: Based on the initial evaluation of these four antigens for POCT format, we selected the single antigen S. japonicum thioredoxin peroxidase-1 (SjTPx-1) for further development. Several prototypes that detect SjTPx-1 specific antibodies were tested on archived samples, and the prototype using gold conjugated anti-human-IgG and SjTPx-1 blotted on the membrane gave the best sensitivity of 73% (19 out of 26).

Milestone 4: We evaluated this prototype for field-testing in the Philippines last December. However, the results from the Philippines revealed a sensitivity of 31% (9 out of 29), lower than the previous result with the archived samples. However, when combined with the results from the archived samples, the overall sensitivity of the POCT prototype in current format is about 50%. The result suggested that the POCT with SjTPx-1 in its current form is not sensitive enough to capture antibodies in sera with lower IgG titers. We are currently improving the prototypes by antigen and sample optimization which includes introduction of SjTPx-1-based fusion antigens for the POCT development.