Field Optimization of Real-time PCR for the detection of Leishmania parasites

The proposed Phase I SBIR program will develop a rapid assay for characterizing blood samples from patients with suspected Leishmania infection. Leishmaniasis affects 12 million people worldwide with 400,000 new cases diagnosed annually. The spectrum of diseases caused by Leishmania spp. includes cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis (VL [kala-azar,]). Current methods for detection are complex, costly, time consuming, lack specificity, and difficult to execute. The proposed single nucleotide polymorphism (SNP) signature assay will enable identification to be carried out in single assay that is highly specific, easy to perform, inexpensive, and field deployable. The assay is carried out in two stages. First, a genus-specific RT PCR is carried out targeting the Leishmania genus. For positive samples the SNP signature assay will be carried using the amplicon generated for species identification. The prototype assay will be used to test a series of genomic isolates (positive and negative) that will be requested from the Walter Reed Army Medical Center (WRAMC) as blind samples. From the outcome, preliminary sensitivity and specificity will be determined. During Phase II, the assay will be finalized in terms of probe design, kit configuration, and accessory equipment, and external validation studies will be carried out.