rKLO8, a novel Leishmania donovani - derived recombinant immunodominant protein for sensitive detection of visceral leishmaniasis in Sudan

Abstract

Background: 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 L. donovani strain in Sudan.

Methodology and principle findings: We 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.

Conclusion: 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.

Figure 1. Protein sequence alignment.

Immunodominant repeats of KLO8 (294 AA), K39 (252 AA) and KE16 (155 AA) were aligned using the ClustalW2-Multiple Sequence Alignment program. Different residues were highlighted in black and identical were left unmarked. Dashed lines indicate gaps.

Figure 2. Expression and purification of the recombinant protein rKLO8.

The KLO8 gene was PCR amplified and cloned into the prokaryotic expression vector pQE41, expressed as 6× His-tagged fusion protein in M15 E. coli 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.

Figure 3. Establishment of an indirect IgG ELISA for specific detection of VL.

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.

Figure 4. Comparative reactivity of Leishmania antibodies with rKLO8 and rK39.

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 one way ANOVA nonparametric test.

Figure 5. Comparison of reactivity for Leishmania antibody detection by rKLO8 ELISA, DAT and rK39 strip test.

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. P value (Mann-Whitney U-test). (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.