Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = -0.52 & Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Novel CRISPR-based detection of Leishmania species.

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 × 100 (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.

A low-cost and open-source protocol to produce key enzymes for molecular detection assays.

Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).