rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Abstract

BackgroundFor effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.Methodology and Principle FindingsWe cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.ConclusionThe increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

“…19 To further enhance sensitivity in weakly reacting Sudanese VL patients, a recently developed recombinant protein (rKLO8) derived from the corresponding endemic L. donovani (eastern Sudan) strain has been used with success in an enzyme-linked immunosorbent assay. 9 These findings and our results with an endemic isolate, indicate that regardless of the candidate serodiagnostic test to be used, preliminary work has to be done, on the basis of homology, to determine whether a certain L. donovani strain does constitute the appropriate "variant" or "serotype" for the area under investigation.…”

“…To estimate the sensitivity, specificity, PPV, and NPV, the standard formulas were used as follows: 1) sensitivity = true positives/ (true positives + false negatives) × 100%; 2) specificity = true negatives/(true negatives + false positives) × 100%; 3) PPV = true positives/(true positives + false positives) × 100%; and 4) NPV = true negatives/true negatives + false negatives) × 100%. 9 For assessing the significance of differences in sensitivity and NPV between the LQ-DAT, FD-DAT, and rK39, the χ 2 test was applied. The statistical analysis software Statistica 7.1 (Stat Soft Inc., Tulsa, OK,) was used to calculate confidence intervals.…”

Systematic Review of Biomarkers To Monitor Therapeutic Response in Leishmaniasis

“…Two serological tests are currently being used in the field: the direct agglutination test (DAT), based on numbers of killed whole L. donovani promastigotes, and the recombinant K39 (rK39)-based immunochromatographic antibody detection test. Other antigenbased assays have been developed for Leishmania antibody detection, using (recombinant) proteins rK9, rK26, rK28, Leishmania infantum cytosolic tryparedoxin peroxidase (LicTXNPx), rgp63, rLepp12, recombinant open reading frame F (rORFF), BHUP2, rKLO8, rHSP70, guanylate binding protein (GBP), galactosyl-␣(1-3)galactose, 9-O-acetylated sialic acids, recombinant peroxidoxin, and amastin (116)(117)(118)(119)(120)(121)(122)(123)(124)(125)(126)(127)(128)(129)(130). Unfortunately, antibodies against Leishmania parasites exhibit a long half-life and stay detectable for several months up to several years after an infection [tested by the DAT and for galactosyl-␣(1-3)galactose, LicTXNPx, rK26, rK39, and BHUP2] (49, 120, 121, 131-141), which compromises the diagnosis of a relapse case and also the pharmacodynamic application of these markers.…”

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

“…However, Cañavate et al (29) confi rmed two rK39 dipstick tests, the DAT and the indirect fl uorescent antibody (IFA) test as suitable for the diagnosis of VL in Ethiopia. In Sudan, Abbas et al (30) introduced rKLO8, a novel L. donovaniderived recombinant protein for VL detection exhibiting increased reactivity of Sudanese VL sera with the rKLO. These researchers confi rmed this antigen as a potential candidate for the diagnosis of VL in Sudan.…”