Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

A novel quantitative real-time PCR diagnostic assay for fecal and nasal swab detection of an otariid lungworm, Parafilaroides decorus.

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy's RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild.