Authors

Discussion

Background:Visceral leishmaniasis (VL) is a potentially fatal disease mainly caused by Leishmania infantum in South America and L. infantum and L. donovani in Asia and Africa. In Brazil, L. infantum causes 4200-6500 cases per year, 90% of the cases registered in the Americas. Screening of healthy blood donors in Brazil indicates that asymptomatic L. infantum infections are common, occuring in  ~1-6% of the general population. However, the fatality rate of patients who receive treatment in Brazil is almost 10%, one of the highest in the world. This high proportion of asymptomatic patients, but also high mortality suggests that there are parasites with varying virulence in Brazil. Since case fatality is getting worse, it is imperative to understand the factors that lead to this increased mortality in Brazil. Visceral leishmaniasis disease severity and outcomes have been associated with several host traits, as patient genotype, nutrition, age, sex, co-morbidities, and co-infections. However, the impact of the parasite genetic variability in the disease severity is poorly understood. In this study, we examine for the first time the effects of population-wide parasite genetic variation on VL disease severity in Brazil using genome-scale methods. 

This study:We quantified the effects of L. infantum parasite genotype on disease severity and mortality. We collected and sequenced the genomes of 109 L. infantum isolates from patients in northeast Brazil. We also retrieved matching patient clinical data from medical records, including mortality, sex, HIV co-infection and laboratory data (creatinine, haemoglobin, leukocyte and platelet counts). We identified genetic differences between parasite isolates, including single nucleotide polymorphisms (SNPs), small insertions/deletions (indels), and variations in genic, intergenic, and chromosome copy numbers (copy number variants, CNVs). To describe associations between the parasite genotypes and clinical outcomes, we applied quantitative genetics methods of heritability and genome-wide association studies (GWAS), treating clinical outcomes as traits that may be influenced by parasite genotype.

Findings:

Parasite genotype explains 83% chance of mortality (narrow sense heritability h2 = 0·83±0·17), and has a significant relationship with patient sex (h2 = 0·60±0·27). Impacts of parasite genotype on other clinical traits are lower (h2  ≤0·34). GWAS identified 17 CNVs that were significantly associated with mortality, two with creatinine and two with bacterial co-infection, one jaundice and HIV co-infection; and two SNPs/indels that associate with age and jaundice, HIV. 

Implications:

We have shown that parasite genotype is an important factor in VL disease severity in Brazil. Since the genetic diversity of L. infantum in Brazil is lower than that of L. donovani which causes VL in Africa and the Indian subcontinent, we can reasonably expect similar effects of parasite genotype in those locations. In Brazil, multiple genomic polymorphisms in the parasite appear to influence mortality and other clinical factors, consistent with variations in virulence between L. infantum isolates that have adverse effects on patients. This is the first study to associate population-wide genetic variation in protozoan parasites to disease severity and outcome. With a larger sample size, our GWAS approach will allow us to pinpoint the most promising gene alterations associated with VL severity,  leading to a greater understanding of the molecular mechanisms of host-parasite interactions, which may be exploited for novel therapies against visceral leishmaniasis. The genetic markers discovered will be valuable to assess risk to patients. This method of uncovering the causes of severe disease will be applicable to any eukaryotic parasite that undergoes recombinatio