Development of a protein-based antigen detection test for kala-azar

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 DESCRIPTION (provided by applicant): Antigen detection assays in contrast to conventional serological test, detects disease status and not the host antibody response to the disease etiological agent. It can therefore be used for both diagnosis and disease treatment follow-up purposes. Antigen detection assay has been successfully used for the past 10-20 years for the diagnosis of different infectious diseases including sore throat caused by Streptococcus pyogenes, hepatitis, pneumonia caused by Streptococcus pneumoniae or Legionella pneumophilla, and amoebiasis. Although antigen detection assay has the potential to discriminate active visceral leishmaniasis (VL) from asymptomatic or cured disease, paradoxically this approach has not been developed for the diagnosis of this serious parasitic disease. Using the ultra-sensitive mass spectrometry technology we have recently identified the presence of three Leishmania infantum/chagasi proteins in urine of VL patients from the New World. These molecules were extensively characterized and used to develop a capture ELISA antigen detection assay for VL diagnosis caused by L. infantum. A pilot validation studies with this assay using human urine from New World VL patients indicated that the test was 100% sensitive and 100% specific. Although this test was excellent for the diagnosis of New World VL, caused by L. infantum, we have recently observed that its sensitivity for the diagnosis of Old World VL caused by L. donovani also known as kala-azar was not adequate. This is likely explained by the fact that these two parasites have substantial differences both in their genomes as well as in the pathologies and clinical manifestations that they cause. Therefore, the different host handling of the two parasites antigenic repertoires can result in excretion of distinct patterns of biomarkers in the patients' urines. Unfortunately, the observed low performance of the L. infantum biomarkers to diagnose Old World VL is a major hindrance because the highest incidence and prevalence of VL occurs in the Old World. Nonetheless, this major hindrance can be overcome by unravelling L. donovani antigens that are abundantly excreted in the urine of patients with Old World kala-azar. Hence, this Phase I SBIR project proposes to identify these unique L. donovani biomarkers with the ultimate goal of developing a non-invasive, antigen detection assay that will accurately diagnose VL caused by L. donovani. The Specific Aims are: Aim 1. To identify L. donovani protein biomarkers present in the urine of kala-azar patients (Old World VL) and to produce diagnostic assay reagents. Aim 2. To optimize and to begin the clinical validation of an antigen detection test (capture ELISA) using select pairs of purified antibodies for the detection of L. donovani antigens in the urine of kala-azar patients.