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

Bhattacharyya T, Boelaert M, Miles MA - PLoS Negl Trop Dis (2013)

Bottom Line: East African sequences were revealed to display significant diversity from rK39.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom. Tapan.Bhattacharyya@lshtm.ac.uk

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.

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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.
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pntd-0002057-g001: 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.

Mentions: PCR primers LdonK39F (gagctcgcaaccgagt) and LdonK39R (ctgrctcgccagctcc) were designed for this study based on a comparison of LcKin and LdK39 coding sequences (Figure 1A), and were targeted to amplify the 894 bp region of the L. donovani kinesin gene that is incorporated into the diagnostic antigen rK39. The level of conservation in the sequences encoding the 39-aa repeats predicts that primer LdonK39R would be expected to anneal to multiple sites within the kinesin gene template, generating multiple amplicons. Amplification reactions were performed in a total volume of 20 ul, and comprised of 1× NH4 Reaction buffer supplemented with 1.5 mM MgCl2 (Bioline, UK), 200 µM dNTPs (New England Biolabs, UK), 10 pmol of each primer, and 1 U BioTaq DNA polymerase (Bioline). Amplification conditions were: 1 cycle of 94°C, 2 mins; 25 cycles of 94°C for 30 secs, 55°C for 30 secs, 72°C for 1 min; 1 cycle of 72°C for 5 mins. PCR products were separated by electrophoresis on 1.5% agarose gels (Bioline). Amplicons corresponding to rK39 homologues were excised and purified from gels using QIAquick Gel Extraction Kit (Qiagen), cloned into pGEM-T easy vector (Promega, UK), and transformed into XL1-blue E. coli on blue-white selection. Primers Sp6/T7 and M13for/rev were used to sequence from the vector (between 2–5 colonies per strain were taken forward for sequencing, except UGX-MARROW and SUDAN1, where due to difficulty in cloning the corresponding inserts only one clone was sequenced); in addition, LdonK39int (cgagcggctaaccagc), which binds to the 3′ end of the non-repeat region immediately upstream of the repeats, was used as an internal sequencing primer (Figure 1A). Sequences were analysed using BioEdit [20].


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

Bhattacharyya T, Boelaert M, Miles MA - PLoS Negl Trop Dis (2013)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585016&req=5

pntd-0002057-g001: 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.
Mentions: PCR primers LdonK39F (gagctcgcaaccgagt) and LdonK39R (ctgrctcgccagctcc) were designed for this study based on a comparison of LcKin and LdK39 coding sequences (Figure 1A), and were targeted to amplify the 894 bp region of the L. donovani kinesin gene that is incorporated into the diagnostic antigen rK39. The level of conservation in the sequences encoding the 39-aa repeats predicts that primer LdonK39R would be expected to anneal to multiple sites within the kinesin gene template, generating multiple amplicons. Amplification reactions were performed in a total volume of 20 ul, and comprised of 1× NH4 Reaction buffer supplemented with 1.5 mM MgCl2 (Bioline, UK), 200 µM dNTPs (New England Biolabs, UK), 10 pmol of each primer, and 1 U BioTaq DNA polymerase (Bioline). Amplification conditions were: 1 cycle of 94°C, 2 mins; 25 cycles of 94°C for 30 secs, 55°C for 30 secs, 72°C for 1 min; 1 cycle of 72°C for 5 mins. PCR products were separated by electrophoresis on 1.5% agarose gels (Bioline). Amplicons corresponding to rK39 homologues were excised and purified from gels using QIAquick Gel Extraction Kit (Qiagen), cloned into pGEM-T easy vector (Promega, UK), and transformed into XL1-blue E. coli on blue-white selection. Primers Sp6/T7 and M13for/rev were used to sequence from the vector (between 2–5 colonies per strain were taken forward for sequencing, except UGX-MARROW and SUDAN1, where due to difficulty in cloning the corresponding inserts only one clone was sequenced); in addition, LdonK39int (cgagcggctaaccagc), which binds to the 3′ end of the non-repeat region immediately upstream of the repeats, was used as an internal sequencing primer (Figure 1A). Sequences were analysed using BioEdit [20].

Bottom Line: East African sequences were revealed to display significant diversity from rK39.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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom. Tapan.Bhattacharyya@lshtm.ac.uk

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.

Show MeSH
Related in: MedlinePlus