Comparison of reactivity for <i>Leishmania</i> antibody detection by rKLO8 ELISA, DAT and rK39 strip test.

<p>ODs for 106 VL sera diluted 1∶800 were measured in the rKLO8 ELISA and compared with DAT antibody titres or strip test results. (A) Sera were divided into 4 groups based on DAT titres; negative, 1∶±1600; weak, 1∶3200–1∶6400; moderate, 1∶12800–1∶25600; strong, 1∶≥51200. (B) Mean ODs for VL sera with negative or positive DAT titres were compared. Results are expressed as mean ± SD. <i>P value</i> (<i>Mann-Whitney U-test</i>). (C) OD values for VL sera with negative or positive strip test results were compared. (D) Mean ELISA OD values for VL sera with positive or negative strip test results. Dots represent values for individual sera and horizontal lines represent cut-off values.</p

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

<div><p>Background</p><p>For 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 <i>L. donovani</i> strain in Sudan.</p><p>Methodology and Principle Findings</p><p>We cloned, expressed and purified a novel recombinant protein antigen of <i>L. donovani</i> from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of <i>Leishmania</i>. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of <i>L. infantum</i> (synonymous <i>L. chagasi</i>) (K39) and <i>L. donovani</i> (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.</p><p>Conclusion</p><p>The 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.</p></div

Comparative reactivity of <i>Leishmania</i> antibodies with rKLO8 and rK39.

<p>The rKLO8 or rK39 proteins were used and compared in ELISA using protein concentrations of 5 ng/100 µl in 0.1M sodium carbonate. A panel of sera from VL patients and controls were tested. Visceral leishmaniasis (VL; n = 106), non-VL controls (n = 77) including non-endemic healthy controls (NEC; n = 20), endemic healthy controls (EC; n = 30), malaria (MA; n = 11), tuberculosis (TB; n = 10), or leukaemia (LEU; n = 6). (A) Sera were tested at dilutions of 1∶800 and a cut off value (0.12) was established as means+3 SD of the OD measured for 30 healthy controls from Sudan. (B) VL sera (n = 14) with negative results at 1∶800 were re-tested at a serum dilution of 1∶100 and compared with the controls described in A. Cut off values were recalculated using 20 non-endemic healthy sera and found to be 0.41 and 0.32 for rKLO8 and rK39, respectively. Statistical Analysis was performed by <i>one way ANOVA</i> nonparametric test.</p

Establishment of an indirect IgG ELISA for specific detection of VL.

<p>For selection of the optimal ELISA conditions, 10 pooled VL sera or 10 pooled healthy control sera were titrated at serial twofold dilutions (1∶25–1∶25600) against different concentrations of the recombinant protein rKLO8. (A) 50 ng/100 µl, (B) 25 ng/100 µl, (C) 10 ng/100 µl, (D) 5 ng/100 µl. Sera were tested in duplicates and means were taken.</p

Expression and purification of the recombinant protein rKLO8.

<p>The <i>KLO8</i> gene was PCR amplified and cloned into the prokaryotic expression vector pQE41, expressed as 6× His-tagged fusion protein in <i>M15 E. coli</i> and purified on a Ni-NTA column. (A) Protein expression was checked on a 12% acrylamide gel stained with Comassie blue; lane 1 and 2, bacterial lysates from un-induced or 1 mM IPTG-induced cultures, respectively; lane 3, purified rKLO8 protein; M, Protein ladder. (B) Reactivity of the recombinant protein was confirmed in WB analysis using 10 pooled VL sera or 10 pooled healthy control sera from Sudan, diluted 1∶1000; lanes 1 and 2, lysates from IPTG induced cultures blotted with negative or positive sera, respectively; lane 3, purified rKLO8 blotted with positive sera; M, Protein ladder.</p

Histology and immunohistochemistry of gastric pathology.

<p>Histological and immunohistochemical staining of representative cases of chronic gastritis with lymphoid aggregates and MALT lymphoma with lymphoepithelial lesions (LEL) or lymphoepithelial destruction (LED); H&E staining, B220 labeling B-cells and anti-cytokeratine staining labeling the epithelium. Centrocyte-like cells infiltrate the gastric epithelium (arrows). Scale bars show 20 μm. Original magnification x40.</p

Regulation of proinflammatory genes in whole peripheral blood of infected and uninfected mice.

<p>Regulation of proinflammatory genes in whole peripheral blood of infected and uninfected mice.</p

Histopathological results of <i>H</i>. <i>felis</i>-infected Balb/c <i>Plcg2</i>^{<i>Ali5/+</i>} and WT mice.

<p>Histopathological results of <i>H</i>. <i>felis</i>-infected Balb/c <i>Plcg2</i>^{<i>Ali5/+</i>} and WT mice.</p

Total and <i>H</i>. <i>felis</i>-specific immunoglobulin levels in infected and uninfected mice.

<p>Sera from infected mice were collected 12 weeks after infection with <i>H</i>. <i>felis</i> and antibody titers were determined by ELISA. (A-B) Total immunoglobulin levels were elevated to similar extent in both infected genotypes as compared to their uninfected littermates while (C, D) <i>H</i>. <i>felis</i>-specific immunoglobulin levels were decreased in infected <i>Plcg2</i>^{<i>Ali5/+</i>} mice. Data represent mean ± SEM of 3 independent experiments including n = 14 to 15 infected <i>Plcg2</i>^{<i>Ali5/+</i>} mice and n = 15 infected WT mice. Two independent experiments were done for uninfected mice (n = 4 per genotype). Data were analyzed by using the Mann-Whitney U-test. LU = labor units; ns = not significant. *p ≤ 0.05; **p ≤ 0.01.</p

Comparison of anti-<i>Leishmania</i> antibody responses in rKLO8- and rK39 ELISA using patient and control sera from three endemic regions.

<p>Protein concentrations of 5ng/100μl rKLO8 or rK39 were tested with sera of patients and controls from Sudan (A), India (B) and France (C). Sera were tested at dilutions of 1:800. A cut off value of 0.12 was established as means + 3 SD of 30 healthy controls. VL, visceral leishmaniasis; VLS, VL suspects; HC, healthy control; MA, malaria; TP, toxoplasmosis; VL/HIV, VL and HIV, ASC, asymptomatic cases. The black horizontal lines represent cut off values. OD values of 0.6 (5 fold of cut off) and 2.15 are shown in dotted lines.</p