Novel CRISPR-based detection of Leishmania species

Abstract

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10 −2 (kDNA) or 5 × 10 0 (18S rDNA) parasite genome equivalents/ reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

Anti-SpCas9 IgY Polyclonal Antibodies Production for CRISPR Research Use

“…This dual control mechanism enhances reliability and confidence in the diagnostic results. Overall, the use of anti-Cas9 antibodies in POC CRISPR-based diagnostics can improve the assay, making it a promising tool for rapid and reliable detection of diseases, including neglected tropical diseases such as leishmaniasis …”

Entomological, parasitological and molecular investigations in a new focus of cutaneous leishmaniasis in Youssoufia region, Morocco

“…However, among a large number of diagnostic tests for leishmaniasis, it is essential to provide a diagnosis that is sufficiently sensitive and specific (De Brito et al., 2020). Thus, this study evaluated the performance of direct parasitological assay using 25 clinical samples compared to a kDNA nested PCR assay as the reference test (Dueñas et al., 2022), considering the effectiveness of kDNA as a high copy number gene to avoid false‐negative results (Aghamolaei et al., 2020). Only 5 (20%) of all suspected CL samples were positive microscopically, whereas nested PCR‐kDNA test detected 18 (72%) positive CL patients.…”

Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection

“…Among these, isothermal NAATs have garnered interest due to their operation at a constant temperature (thus requiring minimal laboratory setup) and cost-effectiveness ( Dueñas et al, 2022 ), the combination of isothermal NAATs and CRISPR/Cas technology has been successfully applied in the rapid detection of various pathogens, which has the advantages of low detection cost, high precision, fast quantification, and on-site rapid detection, and has a high potential for popularization and application. Compared to PCR, isothermal NAATs have reduced temperature requirements and assay costs, and different isothermal NAATs combined with CRISPR/Cas systems have different results.…”