Validation of Oxford nanopore sequencing for improved New World Leishmania species identification via analysis of 70-kDA heat shock protein

Abstract

Background Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp. Methods A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform. Results The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection. Conclusions This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases. Graphical abstract

FDA’s proposed rule and its regulatory impact on emerging and reemerging neglected tropical diseases in the United States

“…This complexity may compel developers of LDTs to navigate the proposed FDA regulatory pathway, potentially resulting in delays in the adoption of NGS technologies for diagnosing tropical diseases in clinical settings. It is also noteworthy that the majority of available resources tend to prioritize bacterial and viral pathogens, often overlooking clinically relevant parasitic agents and relegating them to a secondary position, hence halting efforts like those carried out by our group devoted to support the efficient development in NGS-based parasite diagnostics [45][46][47].…”

Canine leishmaniasis in the Americas: etiology, distribution, and clinical and zoonotic importance

“…In more recent years, the detection of distinct Leishmania spp. in dogs has been facilitated by the application of advanced DNA sequencing methodologies, including next-generation sequencing and nanopore sequencing [ 11 , 12 ].…”