Comparison of visceral leishmaniasis diagnostic antigens in African and Asian Leishmania donovani reveals extensive diversity and region-specific polymorphisms

Abstract

Background: Visceral leishmaniasis (VL), caused by infection with Leishmania donovani complex, remains a major public health problem in endemic regions of South Asia, East Africa, and Brazil. If untreated, symptomatic VL is usually fatal. Rapid field diagnosis relies principally on demonstration of anti-Leishmania antibodies in clinically suspect cases. The rK39 immunochromatographic rapid diagnostic test (RDT) is based on rK39, encoded by a fragment of a kinesin-related gene derived from a Brazilian L. chagasi, now recognised as L. infantum, originating from Europe. Despite its reliability in South Asia, the rK39 test is reported to have lower sensitivity in East Africa. A reason for this differential response may reside in the molecular diversity of the rK39 homologous sequences among East African L. donovani strains.

Methodology/principal findings: Coding sequences of rK39 homologues from East African L. donovani strains were amplified from genomic DNA, analysed for diversity from the rK39 sequence, and compared to South Asian sequences. East African sequences were revealed to display significant diversity from rK39. Most coding changes in the 5' half of repeats were non-conservative, with multiple substitutions involving charge changes, whereas amino acid substitutions in the 3' half of repeats were conservative. Specific polymorphisms were found between South Asian and East African strains. Diversity of HASPB1 and HASPB2 gene repeat sequences, used to flank sequences of a kinesin homologue in the synthetic antigen rK28 designed to reduce variable RDT performance, was also investigated. Non-canonical combination repeat arrangements were revealed for HASPB1 and HASPB2 gene products in strains producing unpredicted size amplicons.

Conclusions/significance: We demonstrate that there is extensive kinesin genetic diversity among strains in East Africa and between East Africa and South Asia, with ample scope for influencing performance of rK39 diagnostic assays. We also show the importance of targeted comparative genomics in guiding optimisation of recombinant/synthetic diagnostic antigens.

Figure 1. Leishmania rK39 and HASPB antigen repeats and the PCR primer target sequences.

Repetitive coding regions depicted as filled boxes, PCR primers underlined, and 5′ and 3′ binding positions with amplicons are indicated by dashed lines. [A] Kinesin gene comparison for primer design (* = non-conserved nucleotide). [B] HASPB1 GenBank sequence displays 22× perfect 14aa repeats. [C] HASPB2 GenBank sequence displays 3 imperfect repeats.

Figure 2. Multiple amplicons corresponding to kinesin tandem repeats are produced by PCR primers LdonK39F and LdonK39R.

Amplifications from strains HU3 (LV9), Hussen, and UGX-MARROW, are depicted. Major amplicon sizes differ by 117 bp, the size of the nucleotide sequence encoding the 39aa repeat in the kinesin gene; mk = Hyperladder I (Bioline).

Figure 3. Polymorphisms among seven rK39 repeats of East African and South Asian strains show region-specific substitutions.

Changes between charged and non-charged residues are underlined. No residue entered at a particular site indicates conservation of that residue with the corresponding residue of diagnostic rK39. Where two alternative residues are indicated in smaller text these are not necessarily always within the same strain, for clarification compare with Table 3. Region-specific polymorphisms in each repeat are boxed (East Africa) or circled (South Asia).

Figure 4. Predicted (1064 bp, 260 bp) amplicons and unpredicted (∼400–500 bp) amplicons with HASPB PCR primers LdonHASPBfor and LdonHASPBrev.

Amplifications from strains HU3 (LV9), Hussen, UGX-MARROW, and LRC-L57, are depicted; mk = Hyperladder I.