There are many challenges in the management of this diseases diagnosis is difficult relying on antiquated techniques where an expert microscopist needs to examine cells extracted from the bone marrow or spleen treatment normally consists of 30 days intravenous infusion with toxic drugs. Most cases come from poor rural areas.

Normally without treatment this disease is often fatal. Clearly, there is a pressing need for rapid diagnostic tests, simpler treatment and efficient methods of prevention currently, the emphasis at USAMRU is on the development and testing of fast convenient, accurate diagnostics that can not only be used at clinics but can be used epidemiologically to determine the distribution and magnitude of cases in Kenya.

Objectives

• Develop and evaluate prototype assays for the detection of Leishmania-specific antibodies and circulating antigen in patient tissues.

• Identify areas where there is high transmission of Leishmania and clustering of cases through an epidemiological survey and  by mapping of selected villages.

• Use a variety of serological tools to gain as much information about the extent of VL and exposed cases in the affected areas.

• Determine the extent of active and sub-clinical cases to gain baseline data for a design of multi-faced suppression program.

Recent Work

Diagnostics development and evaluation:  Through collaboration with investigators at WRAIR, KEMRI, CDC and commercial partners in Sydney, Australia (Cellabs Pty Ltd) and Camarillo, California (Medical Analysis Systems), we have developed a number of prototype assays.  First, an enzyme-linked immunosorbent assay (ELISA) was developed by our commercial partners in Australia.   Secondly, we have a prototype immunofluorescence assay (IFA).   The same polyclonal antibodies used to assemble the IFA were used to develop a rapid immunochromatographic antigen-capture assay.  Prospective studies are underway to evaluate these assays and other rapid tests that are commercially available from InBios, Inc. (Seattle, Washington). The goal of this program is to assist the WRAIR Diagnostics section validate any of these devices that prove to be accurate and practical for field use.

Devices under development and evaluation at USAMRU- K:

• Cellabs Visceral Leishmaniasis ELISA:  This fairly simple assay detects Leishmania-specific IgG and IgM in case patient serum. The test can be performed in 3 hours, however needs a plate reader to obtain results quantitatively. In preliminary studies, these assays were better than 95% sensitive and specific. 

• Cellabs IFA: Using a smear of bone marrow or splenic aspirate, this assay highlights Leishmania amastigotes in parasitized macrophage. Sample preparation is less than 1 hour, but test requires a fluorescence microscope.

• MAS Leishmania Antigen Wicking Assay:  This dipstick appears to detect Leishmania antigens in patient urine.  Test can be performed in 10 minutes with no sample preparation or elaborate equipment. No performance data is available at this time.

• InBios rK39 wicking assay: Based upon a recombinant form of a leishmania kinesin protein, this dipstick detects Leishmania-specific IgG using 35ul of serum.  Test can be performed in 10 minutes with minimum sample preparation.  This test is intended to be a true diagnostic for active VL.  The device was better than 95% sensitive and 99% specific in studies in India and Brazil.

• InBios rK26 wicking assay: Based upon a different recombinant form of a leishmania kinesin protein, this dipstick detects Leishmania-specific IgG using 35ul of serum.  Test can be performed in 10 minutes with minimum sample preparation.  This test is intended to be more sensitive than the rK39 and presumably will detect circulating IgGs in asymptomatic or sub clinical individuals.  No performance data is available at this time.

Epidemiological Studies:  In April 2001 we began a prospective study in the Baringo District.  The primary goal of this project is to determine the seroprevalence of Leishmania-specific IgG.  To do this, we are employing our antibody-capture ELISA and the two InBios wicking assays.  To date, we have taken a complete census and GIS-mapped the villages of Parkarin and Loboi.  To date, we have enrolled more than 500 subjects to gather information on certain risk factors, signs and symptoms of VL and a small blood sample.  Serum was extracted from all blood samples and serological testing is underway.  Seropositives will be recorded.  Joining results of serology to census and GIS coordinates, we will use spatial analysis to establish focality of disease and to look for correlations between case clustering and certain risk factors (i.e., location of subjects to animal enclosures, sand fly habitats, latrines, etc.). 

Personnel

This multifaceted program engages diagnostics developers and evaluators from the WRAIR, CDC and three commercial companies in addition, the epidemiological study engaged a field team from KEMRI comprised of 2 clinicians and 2 laboratory technologists to contact subjects for the consenting, examining and sampling processes. We also employed 2 local-area translators for completing the census and consent forms.