However, after drug treatment and cure current serological assessments may still give positive results and therefore cannot readily diagnose relapse. 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. Author Summary Visceral leishmaniasis (VL) is usually caused by contamination with parasites of the complex, spread by the bite of blood-sucking sandflies, especially in South Asia, East Africa, and Brazil. If untreated, symptomatic VL leads to systemic pathologies and is usually fatal. Up to approximately 400, 000 new cases are estimated to occur annually, and regional epidemics have been devastatingly severe. Diagnosis of clinically suspect cases in the field, and thus appropriate treatment, relies principally on a rapid diagnostic test (RDT) based on detection of antibodies against a antigen known as rK39. Although this test is reliable in South Asia, it has shown less success in East Africa. One reason may reside in diversity of the rK39 homologue among East African strains, and show that there Bivalirudin Trifluoroacetate is significant diversity compared to rK39 and to South Asian sequences. Additionally, we examine diversity in another diagnostic antigen known as HASPB1/2. Our results indicate that an improved RDT may need to encompass East-African-specific antigen diversity to provide high performance field diagnosis for this region. Introduction Visceral leishmaniasis (VL) remains a major public health concern in many parts of the tropical world, with the great majority of the estimated 200,000 to 400,000 annual new cases found in South Asia, East Africa, and Brazil [1], [2]. VL is usually caused by kinetoplastid protozoa of the complex. These are: in South Asia and East Africa; but is now demonstrated to be synonymous with originating from Europe [3]. promastigotes, transmitted during bloodmeal feeding by female sandflies (and spp., in the Old and New World respectively), are internalised by local dermal macrophages and dendritic cells. Within these host cells, flagella are lost, and transformation into proliferative amastigote forms is followed by cell lysis, re-invasion of other cells, and parasite dissemination by the lymphatic and vascular systems, which can lead to infiltration of bone marrow and hepatosplenomegaly. In symptomatic cases, VL is fatal if untreated [4]. Parasitological diagnosis, by demonstration of amastigotes in spleen aspirates, approximates to a gold standard for VL diagnosis but is applied cautiously due to associated risk. Serological (anti-antibody) tests include enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT), and direct agglutination test (DAT) [5]. However, after drug treatment and cure current serological tests may still give positive results and therefore cannot readily diagnose relapse. Furthermore, such tests can also detect anti-leishmanial antibodies in asymptomatic individuals living in endemic areas, but with no VL history or subsequent progression to VL [6]. Burns et al [7] identified a kinesin-related gene product, LcKin, as a candidate diagnostic antigen by screening a Brazilian (patient. A part of the coding sequence, comprising a 46aa region followed by 6.539aa repeats, forms the recombinant diagnostic protein rK39. In recent multicentre evaluations, the use of the rK39 in a lateral-flow immunochromatographic, rapid test L-741626 format reported less success in East Africa than in the Indian subcontinent for point-of-care diagnosis of VL [8], [9]. Underlying explanatory factors may reside in molecular divergence between East African kinesin gene homologues and the Brazilian (strains using PCR primers based on have identified rK39 homologous sequences [10]C[12]. Gerald et al [13] reported the first East African (Sudanese) kinesin homologue, LdK39. The first two of the 39-aa repeats of LdK39, flanked by L-741626 sequences of the antigens HASPB1 and HASPB2 [14], comprise rK28, a novel recombinant protein for diagnosis of VL, designed to be an improvement over rK39 [15]C[16]. HASPB proteins are expressed on the surface of infective promastigote and amastigote life cycle stages [14]. The first 314aa repeats of HASPB1 are incorporated into rK28, along with the complete ORF of HASPB2, which includes three imperfect consecutive repeats, L-741626 214aa and 113aa. HASPB1 and HASPB2 correspond with K26 and K9, respectively [17], which were originally identified in (may contribute to lower rK39 diagnostic test success rates in East Africa. We analyse the rK39 homologues in a.