An antigen-detection assay to diagnose Babesia microti infection

Project Summary Human babesiosis is a malaria like multisystem disease caused primarily by Babesia microtian emerging apicomplexan parasite that infects and develops within human erythrocytesThe parasite is transmitted to humans by the tick vector Ixodes scapularis and can also be introduced through blood transfusionInfection can cause flu like symptomsand severe infection can be fatalin particular in the immunosuppressed and the elderlyCurrent methods for babesiosis diagnosis include microscopyPCRIFA and ELISA based methods that detect antibodies in serum from patients or donorsEach of these methods has major limitationsPCR detection of the parasite DNA is the most sensitive of all current diagnostic methods usedHoweverPCR only tests for the presence of parasites within a fraction of a sample being testedtypicallyml out ofmlThusat very low parasitemiasa false negative result is possible when the test sample happens to be devoid of infected red blood cellsIn sumcurrent screening criteria reduce the probability of blood contamination but do not completely eliminate the risk of transmission by transfusion to individuals with a weakened immune systemHere we propose to develop a capture ELISA assay that can detect the most highly expressed and immunogenic antigen of the parasiteBmGPIBmSAOur preliminary studies using short term in vitro culture as well as controlled mouse infections demonstrated that a capture assay targeting this antigen detects infection before the parasite is detectable by microscopy or PCRwith parasitemia levels lower thanBecause each infected cell releases thousands of BmGPIBmSAmolecules into the serumwe expect that our antigen detection assay will prove more sensitive than any method detecting the parasite itselfincluding PCRBuilding upon our preliminary datawe propose the following two specific aims towards the development of a test that could be implemented for high throughput detection of Bmicroti in blood donationsIn Aimwe will optimize the assay procedure using a set of well characterized blood and sera from infected and non infected laboratory miceTo achieve regulatory standards of reproducibility in a commercial assaywe will develop monoclonal antibodies against BmGPIBmSAto replace the polyclonal serum used in preliminary experimentsIn Aimwe will use the optimized assay to screen a collection ofhuman serum samples available at Yale University and LDiagnosticsThese samples have previously been characterized by Bmicroti PCR detection and serologyOur experiments will provide proof of feasibility needed for future efforts and collaborations with major blood organizations to use the assay in large scale blood screeningThe success of the proposed studies will set the stage for use of this assay to screen the blood supply to prevent transfusion transmitted babesiosis PROJECT NARRATIVEHuman babesiosisa potentially fatal malaria like disease reported worldwide and endemic in the United Statesis caused by Babesia microtia parasite that is transmitted by ticks or by blood transfusionThe parasite is the leading cause of transfusion transmitted diseases in the United StatesThis proposal aims to develop an assay that can detect the most highly expressed and secreted antigen of the parasite in infected individuals at levels below those detected by the current gold standard techniques