Visceral leishmaniasis in Bangladesh

Abstract

Despite being close to the elimination target, countries in the Indian subcontinent still face challenges that interfere with complete elimination of visceral leishmaniasis (VL). This thesis focuses on some of those complex issues in the context of Bangladesh with aims to: (1) improve diagnosis; (2) identify asymptomatic carriers and risk factors associated with VL transmission; and (3) to identify genetic diversity among clinical isolates of Leishmania donovani in Bangladesh. The existing elimination program in Bangladesh does not focus on asymptomatic cases allowing them to remain undetected. This study identified a sero-prevalence of 29.2% amongst individuals closely associated with VL cases who were asymptomatic. Live L. donovani were isolated from peripheral blood buffy coats from 10% of these sero-positive family members. Those asymptomatic individuals who were within the 30–45 years’ age group (p<0.05), worked as farmers (p<0.01), lived with domestic animals in household (p<0.05) and lived in close proximity to animal shelters (p<0.05) were identified as having a significant risk for being infected with VL. The performance of serological tests such as the immunochromatographic test using recombinant K39 antigen (rK39 ICT), enzyme linked immunosorbent assay using mixed Leishmania promastigotes from various species (p-ELISA) and indirect fluorescent antibody test (IFAT) utilizing whole promastigotes from Leishmania infantum were evaluated for diagnosis of VL in Bangladesh. The data presented here confirmed the efficiency of rK39 ICT with 100% sensitivity and specificity for rapid diagnosis of VL. While performance of p- ELISA and IFAT was below that seen with rK39 ICT, a high sensitivity (92.3%) and specificity (99.9%) were observed with IFAT. Although the combined sensitivity of rK39 ICT and IFAT marginally decreases (92.3%), their combined specificity still remains high (99.9%). A combination of these two tests can therefore be considered to diagnose VL when confirmatory detection by invasive parasitological procedures cannot be justified. Confusion resulting from weak or nonspecific reactions of rK39 ICT leading to misdiagnosis will be avoided when a second serological test is used. A Multilocus Sequence Typing (MLST) scheme consisting of seven new housekeeping genes was developed as part of this work to identify genetic diversity amongst autochthonous Leishmania isolates from Bangladesh. These isolates were confirmed as L. donovani by restriction fragment length polymorphism of the ITS1 region (ITS1-RFLP). The seven target genes selected for MLST were: rhomboid like serine protease (RSP), fatty acid/sphingolipid δ- 4 desaturase (FAD), serine/threonine-protein kinase (STK), 5' a2rel-related protein (A2P), ubiquitin-activating enzyme e1 (UBE1), mannosyltransferase (MTF), and splicing factor 3B subunit1 (SF3B1). These genes are located on seven chromosomes distributed across the L. donovani genome with orthologues present throughout the genomes of other Leishmania species. Primers targeting seven housekeeping genes were designed based on DNA sequences surrounding the protein coding SNPs detected in the respective genes from within the L. donovani genome. Using this new MLST scheme, seven genotypes could be identified amongst Bangladeshi L. donovani isolates. These new molecular markers can be used to investigate the emergence of new parasite variants. The work presented herein provides evidence that asymptomatic individuals can carry live L. donovani in peripheral blood and suggests that these individuals can act as potential reservoirs. This new MLST scheme has for the first time been able to discriminate seven genotypes among the L. donovani population in Bangladesh. This conflicts with a pervious study using microsatellite typing that described L. donovani in the region as genetically homogeneous. Most importantly, this work will underpin future studies aiming to develop socio-epidemiological and parasite-targeted control interventions attempting to eradicate VL from Bangladesh and surrounding regions.