First-in-class, highly sensitive and specific rapid diagnostic test to detect Paragonimus infections

PROJECT SUMMARY / ABSTRACT Herein, we propose an ultrasensitive serological rapid diagnostic test (RDT) to detect exposure to pulmonary flukes of the genus Paragonimus. Paragonimiasis affects an estimated 21 million people and remains amongst the most neglected of all neglected tropical diseases (NTDs). Trematodes of the genus Paragonimus are acquired upon ingestion of raw or undercooked freshwater crustaceans and migrate to the lungs where they encyst. Treatment is straightforward (praziquantel) but diagnosis is not. The classical method – detection of Paragonimus eggs in sputum or stool – requires rarely available microscopy expertise, is slow and hence low-throughput. With an effective drug at hand but no convenient diagnostic, a highly sensitive and specific RDT is the missing piece to enable efficient clinical management of paragonimiasis. RDTs represent a mature, robust, and cost-effective technology ideal for intervention at the point of care in resource-limited countries. An additional important problem is that many paragonimiasis symptoms resemble those of TB or lung cancer, including hemoptysis (blood in sputum), persistent cough, weight loss, and cysts in CT scans. As a result, misdiagnosis is common and patients are incorrectly put on long and ineffective antibiotic regimens or on toxic cancer chemotherapy, with abysmal consequences for the patients. The importance of differential diagnosis between paragonimiasis and other pulmonary diseases further underscores the need for a Paragonimus RDT. The project will leverage novel paragonimiasis biomarkers, notably a cysteine protease (CP-6) and myoglobin-1, recently discovered at the Washington University School of Medicine after an extensive effort of genome sequencing. In previous work, we developed an RDT prototype that detects IgG4 antibodies against CP-6 with 89% sensitivity and 100% specificity when evaluated on a small panel of positive (n=19) and negative (n=28) sera. The RDT utilizes a novel lateral-flow assay (LFA) set-up combined with novel plasmonic reporter nanoparticles (gold nanoshells) for superior optical detection. However, some test lines were weak, and further optimization is required to warrant manufacturing. Even more importantly, assay development was severely limited by the paucity of clinical samples. Therefore, a particular focus of this proposal is to assemble a collection of paragonimiasis sera, in collaboration with Dr. Belizario, Dean and Professor, College of Public Health, University of the Philippines, Manila. We are therefore requesting funding to assemble such a biorepository of both paragonimiasis-positive and -negative patient samples (Specific Aim 1), to further optimize the sensitivity of the prototype RDT without impairing the specificity, e.g., by generating a biplex assay to simultaneously detect IgG4 antibodies against both antigens, CP-6 (test line 1) and myoglobin-1 (test line 2), whereby the appearance of either test line would be counted as positive (Specific Aim 2), and to validate and compare the different assays generated in Aim 2 using the samples acquired under Aim 1, and select the best prototype for further development in a subsequent Phase II (Specific Aim 3). Dr. Belizario indicates that for a Paragonimus RDT to have value in the field, it should have a sensitivity of minimally rt80% and ideally rt90%, and a specificity of rt97%. A simple test for paragonimiasis remains an unmet medical need, and by addressing this need, we hope to improve the quality of care of people with pulmonary diseases in parts of Southeast Asia. Although high prevalence areas in Asia stand to benefit most from a new test, the assay would also be useful in the USA, despite low infection rates, as witnessed by the CDC’s interest in this project. Furthermore, although the WHO has not yet officially endorsed paragonimiasis as an NTD requiring a global surveillance and/or elimination program, our RDT is nevertheless prone to be of interest to the WHO in the context of their massive global effort to fight TB, as it will allow a better differential diagnosis of TB vs. Paragonimiasis cases. Our team has the track record, the technological expertise and the global health experience required to deliver a successful product.