Test Combination Detects Latent Leishmaniasis Infection

By LabMedica International staff writers
Posted on 01 Sep 2022

Image: The Promega Maxwell 16 automated purification of genomic DNA, RNA or recombinant Proteins from a wide range of sample types (Photo courtesy of Fischer Scientific).

After infection with Leishmania infantum cutaneous or visceral leishmaniasis can develop, but in most cases the host becomes an asymptomatic carrier of the parasite. This key population of individuals with latent leishmaniasis can undergo reactivation of the infection with severe outcome in case of immunosuppression and can contribute to disease transmission.

The public health impact of leishmaniasis is currently underestimated in Europe, despite Leishmania infection is endemic in the Mediterranean countries and outbreaks of human leishmaniasis have been reported in the last decades, including northeastern Italy. One of the causes contributing to this neglect is that the available diagnostic tests exhibit scarce sensitivity in identification of latent leishmaniasis.

Diagnostic Microbiologists at the University of Bologna (Bologna, Italy) and their colleagues enrolled a cohort of 145 adult volunteers residing in a selected Bologna province, with the aim to perform a screening for asymptomatic Leishmania infection. For each individual, 10 mL of peripheral blood were collected and divided into three test tubes; one tube with ethylenediamine tetraacetic acid (EDTA) as anticoagulant, one tube with heparin as anticoagulant, and one tube without anticoagulant. The team defined as asymptomatic Leishmania infection each case that tested positive to at least one of the screening tests: Real-Time PCR, Western Blot (WB), and/or Whole Blood stimulation Assay (WBA).

The detection of specific Leishmania IgG in serum of volunteers was assessed employing the Leishmania WESTERN BLOT IgG kit (LDBio Diagnostics, Lyon, France). WBA was assessed to study the cytokine release by leukocytes upon stimulation with Leishmania antigens. The quantification of IL-2 was performed using Cytometric Bead Array Human Soluble Protein Flex Set (Becton Dickinson, Franklin Lakes, NJ, USA) and the method was performed using that company’s BD FACSCanto. Leishmanial DNA was extracted from 200 μL of EDTA peripheral blood by a semi-automatic DNA extraction system, using the Maxwell 16 LEV Blood DNA kit on the Maxwell 16 instrument (Promega, Madison, WI, USA;) for the detection of parasitic DNA in the blood.

The investigators reported that 23/145 volunteers were positive, indicating a prevalence of 15.9%; 15/145 subjects tested positive to WB (10.3%). In detail, four subjects showed positivity to the p14 band (26.5%), seven to the p16 band (47.0%), and four to both p14 and p16 bands (26.5%). Among the 145 volunteers included in the study, six individuals carried leishmanial DNA in the peripheral blood (4.1%); the parasitaemia was low, with a median parasite load of 8.24 × 10−1 /mL of blood. The WBA was used to evaluate the specific cell-mediated response of the volunteers after blood stimulation with Leishmania-specific antigens. Of the 145 subjects, 12 tested positive (8.3%); these individuals exhibited an IL-2 value above the cut-off of positivity with a median IL-2 concentration of 213 pg/mL.

The authors concluded that combining different tests substantially increased the yield of positivity in detecting latent Leishmania infection. The test combination that they employed in this study appears to be effective to accurately identify latent leishmaniasis in an endemic area. The study was published on August 15, 2022 in the journal PLoS Neglected Tropica Diseases.