Transcriptional blood signatures for active and amphotericin B treated visceral leishmaniasis in India

Abstract

Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani , with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days. (296 words; 300 words allowed).

Genetics, Transcriptomics and Meta-Taxonomics in Visceral Leishmaniasis

“…They all bind to CXCR3 and play multiple roles in promoting T cell adhesion and in the chemoattraction of monocytes and macrophages, T cells, NK cells, and dendritic cells. In our study we also identified GBP1 (Fakiola et al, 2019) as one of 10 top genes induced in active compared to treated VL cases. This gene encodes the interferon-g-induced guanylate binding protein GBP1.…”

“…For the first arm of this study we used chip-based transcriptomics to study expression profiles in whole blood from active VL cases, Amphotericin B treated VL cases, asymptomic individuals, and healthy unaffected endemic controls (Fakiola et al, 2019). Interestingly, no differentially expressed genes (DEGs) were identified when comparing a group of individuals who had asymptomatic infection as determined by high IgG or positive Quantiferon responses compared to healthy endemic controls who were negative by these assays (Figure 2A).…”

“…Importantly, we propose that the erythoidrelated gene sets identified by us could be used to monitor clinical cure. An example of this was that erythroid related genes had not returned to baseline following multi-dose Amphotericin B treatment compared to more effective treatment with single dose of liposomal Amphotericin B (Fakiola et al, 2019). Monitoring erythroid-related gene signals may this provide useful biomarkers for successful cure from VL.…”

“…(Novais et al, 2015;Kong et al, 2017;Ashwin et al, 2018)]. Here we review results from studies using three different omics-based approaches to study VL caused by L. donovani in India: (i) chip-based analysis of single nucleotide variants used to carry out the first genomewide association study (GWAS) of VL to identify host genetic risk factors (Fakiola et al, 2013); (ii) chip-based transcriptional profiling used to compare active with cured cases (Fakiola et al, 2019), including the influence of anti-IL-10 on whole blood responses to soluble leishmania antigen (SLA) (Singh et al, 2020); and (iii) a meta-taxonomic approach based on sequencing amplicons derived from regions of 16S ribosomal RNA (16S rRNA) and 18S rRNA genes that allowed us to determine composition of both prokaryotic and eukaryotic gut microflora in VL cases compared to endemic controls (Lappan et al, 2019).…”

Xenodiagnosis to address key questions in visceral leishmaniasis control and elimination

“…On the other hand, Sb v is still effective and commonly used for VL treatment in Sudan [ 88 ]. Currently, a single dose of liposomal amphotericin B (AmBisome) is an effective VL treatment option and recommended as the first line treatment in the elimination program on the Indian subcontinent [ 89 ]. However, the high cost of the drug limits access in endemic areas and can lead to underdosing and incomplete treatment conducive to the emergence of drug resistant strains [ 90 ].…”