Diversity and dissemination of viruses in pathogenic protozoa.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1.

Novel CRISPR-based detection of species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus , 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.

Toxicity assessment of synthetic chalcones with antileishmanial potential in BALB/c mice.

Objective: To evaluate the toxicity of three synthetic chalcones administered intraperitoneally to BALB/c mice.

Materials And Methods: The median lethal dose (LD50) was estimated by Dixon's Up-and-Down method. Subchronic toxicity of chalcones was evaluated at 20 and 40 mg/kg for 21 days.

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.

Unlocking SARS-CoV-2 detection in low- and middle-income countries.

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch.

UnCovid: A versatile, low-cost, and open-source protocol for SARS-CoV-2 RNA detection.

Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements.

Application of CRISPR/Cas9-Based Reverse Genetics in : Conserved Roles for HSP100 and HSP23.

The protozoan parasite () ) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of through reverse genetics analyses has so far lagged behind in comparison with Old World spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in that was previously developed for Old World and New World species.

Ecological divergence and hybridization of Neotropical parasites.

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions.

A Case-Control Study on the Association Between Intestinal Helminth Infections and Treatment Failure in Patients With Cutaneous Leishmaniasis.

Background: Endemic regions of cutaneous leishmaniasis (CL) and intestinal helminthiasis overlap. CL treatment with systemic pentavalent antimonial drugs (Sb) fails in 10%-30% of patients. The study objective was to assess the etiological role of intestinal helminthiasis in CL treatment failure.

Tegumentary leishmaniasis and coinfections other than HIV.

Background: Tegumentary leishmaniasis (TL) is a disease of skin and/or mucosal tissues caused by Leishmania parasites. TL patients may concurrently carry other pathogens, which may influence the clinical outcome of TL.

Methodology And Principal Findings: This review focuses on the frequency of TL coinfections in human populations, interactions between Leishmania and other pathogens in animal models and human subjects, and implications of TL coinfections for clinical practice.

Quantitative Kinetoplast DNA Assessment During Treatment of Mucosal Leishmaniasis as a Potential Biomarker of Outcome: A Pilot Study.

Mucosal leishmaniasis (ML) is a disfiguring manifestation of Leishmania (Viannia) infection. We evaluated parasite load (PL) over time as a potential biomarker of treatment outcome in ML. PL was assessed with kinetoplast DNA quantitative real-time polymerase chain reaction (kDNA-qPCR) at enrollment, days 14 and 21-28 of therapy and 3, 6, 12-18, and 18-24 months after treatment of ML and correlated to demographic, clinical, and parasitologic factors.

Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load.

Background: Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.

A novel marker, ARM58, confers antimony resistance to Leishmania spp.

Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse.

An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis.

Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method.

Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters.

Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites.

Simultaneous infection with Leishmania (Viannia) braziliensis and L. (V.) lainsoni in a Peruvian patient with cutaneous leishmaniasis.

Conventional understanding suggests that simultaneous infection with more than one species of Leishmania is unlikely. In Peru, co-infections are clinically relevant because causative species dictates prognosis, treatment response, and follow-up. We describe a case of Leishmania (Viannia) braziliensis and L.

Non-invasive cytology brush PCR for the diagnosis and causative species identification of American cutaneous leishmaniasis in Peru.

Background: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL).

Methods: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled.

Multilocus genotyping reveals a polyphyletic pattern among naturally antimony-resistant Leishmania braziliensis isolates from Peru.

In order to understand the epidemiological dynamics of antimonial (Sb(V)) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome.

Comparative gene expression analysis throughout the life cycle of Leishmania braziliensis: diversity of expression profiles among clinical isolates.

Background: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species.

Linking in vitro and in vivo survival of clinical Leishmania donovani strains.

Background: Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these 'hostile' environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Targeted gene expression profiling in Leishmania braziliensis and Leishmania guyanensis parasites isolated from Brazilian patients with different antimonial treatment outcomes.

In Brazil, cutaneous leishmaniasis represents a serious public health problem, and chemotherapy is an important element of the clinical management of this disease. However, treatment efficacy is variable, a phenomenon that might be due to host and parasite (e.g.

Evaluation of host genetic and viral factors as surrogate markers for HTLV-1-associated myelopathy/tropical spastic paraparesis in Peruvian HTLV-1-infected patients.

Human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a complication that affects up to 5% of HTLV-1-infected individuals. Several host genetic and viral factors have been associated with the risk of HAM/TSP. The aim of this study was to evaluate the performance of a prognostic model for HAM/TSP developed in Japan in a Peruvian population of 71 HAM/TSP patients and 94 asymptomatic carriers (ACs).

Development and validation of a multiplex real-time PCR assay for simultaneous genotyping and human T-lymphotropic virus type 1, 2, and 3 proviral load determination.

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3.

Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru.

Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions.