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 pos...