Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia.

Visceral leishmaniasis (VL) is a neglected disease considered a serious public health problem, especially in endemic countries. Several studies have discovered monoxenous trypanosomatids (Leptomonas and Crithidia) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia-related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia. Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.

Co-infection of Leishmania infantum and a Crithidia-related species in a case of refractory relapsed visceral leishmaniasis with non-ulcerated cutaneous manifestation in Brazil.

We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia-related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region.

Dataset of dual RNA-seq mapping in visceral leishmaniasis: Inquiry on parasite transcripts in human blood transcriptome upon Leishmania infantum infection.

This dataset is related to the article "Insight Into the Long Noncoding RNA and mRNA Coexpression Profile in the Human Blood Transcriptome Upon Leishmania infantum Infection" by S.R. Maruyama, C.A. Fuzo, A.E.R. Oliveira, L.A. Rogerio, N.T. Takamiya, G. Pessenda, E.V. de Melo, A.M. da Silva, A.R. Jesus, V. Carregaro, H.I. Nakaya, R.P. Almeida and J.S. da Silva. Frontiers in Immunology, 2022. Through the reuse of raw sequencing data, we generated original dataset by performing a dual RNA-seq mapping procedure to survey the parasite transcripts found in RNA-seq samples from blood of visceral leishmaniasis patients. Diseased patients with active infection displayed the highest number of reads mapped to L. infantum genome. Even after six months later of the treatment, when the patients were considered cured, parasite reads were still detected. Parasite reads were also detected in asymptomatic individuals. The original dual RNA-seq alignment read count data provided here can be further explored to evaluate either host or parasite transcripts.

Insight Into the Long Noncoding RNA and mRNA Coexpression Profile in the Human Blood Transcriptome Upon Leishmania infantum Infection.

Visceral leishmaniasis (VL) is a vector-borne infectious disease that can be potentially fatal if left untreated. In Brazil, it is caused by Leishmania infantum parasites. Blood transcriptomics allows us to assess the molecular mechanisms involved in the immunopathological processes of several clinical conditions, namely, parasitic diseases. Here, we performed mRNA sequencing of peripheral blood from patients with visceral leishmaniasis during the active phase of the disease and six months after successful treatment, when the patients were considered clinically cured. To strengthen the study, the RNA-seq data analysis included two other non-diseased groups composed of healthy uninfected volunteers and asymptomatic individuals. We identified thousands of differentially expressed genes between VL patients and non-diseased groups. Overall, pathway analysis corroborated the importance of signaling involving interferons, chemokines, Toll-like receptors and the neutrophil response. Cellular deconvolution of gene expression profiles was able to discriminate cellular subtypes, highlighting the contribution of plasma cells and NK cells in the course of the disease. Beyond the biological processes involved in the immunopathology of VL revealed by the expression of protein coding genes (PCGs), we observed a significant participation of long noncoding RNAs (lncRNAs) in our blood transcriptome dataset. Genome-wide analysis of lncRNAs expression in VL has never been performed. lncRNAs have been considered key regulators of disease progression, mainly in cancers; however, their pattern regulation may also help to understand the complexity and heterogeneity of host immune responses elicited by L. infantum infections in humans. Among our findings, we identified lncRNAs such as IL21-AS1, MIR4435-2HG and LINC01501 and coexpressed lncRNA/mRNA pairs such as CA3-AS1/CA1, GASAL1/IFNG and LINC01127/IL1R1-IL1R2. Thus, for the first time, we present an integrated analysis of PCGs and lncRNAs by exploring the lncRNA-mRNA coexpression profile of VL to provide insights into the regulatory gene network involved in the development of this inflammatory and infectious disease.

Total Ortholog Median Matrix as an alternative unsupervised approach for phylogenomics based on evolutionary distance between protein coding genes.

The increasing number of available genomic data allowed the development of phylogenomic analytical tools. Current methods compile information from single gene phylogenies, whether based on topologies or multiple sequence alignments. Generally, phylogenomic analyses elect gene families or genomic regions to construct phylogenomic trees. Here, we presented an alternative approach for Phylogenomics, named TOMM (Total Ortholog Median Matrix), to construct a representative phylogram composed by amino acid distance measures of all pairwise ortholog protein sequence pairs from desired species inside a group of organisms. The procedure is divided two main steps, (1) ortholog detection and (2) creation of a matrix with the median amino acid distance measures of all pairwise orthologous sequences. We tested this approach within three different group of organisms: Kinetoplastida protozoa, hematophagous Diptera vectors and Primates. Our approach was robust and efficacious to reconstruct the phylogenetic relationships for the three groups. Moreover, novel branch topologies could be achieved, providing insights about some phylogenetic relationships between some taxa.

Reference genes for gene expression studies by RT-qPCR in Brevipalpus yothersi (Acari: Tenuipalpidae), the mite vector of citrus leprosis virus.

Quantitative reverse transcription PCR (RT-qPCR) is a high-throughput method to analyze the transcriptional expression of genes. Currently, no reference genes have been described for evaluating gene expression in Brevipalpus yothersi, the false spider mite, a polyphagous that act as vector of the citrus leprosis virus C (CiLV-C), an important citrus disease. This study aimed to identify the most stable reference genes in B. yothersi. The RT-qPCR expression data for selected genes were evaluated from three conditions: different developmental stages, plant hosts and acquisition of CiLV-C. To analyze the stability of the candidate reference genes we used ΔCq method, GeNorm, NormFinder, BestKeeper and RefFinder. Ubiq and GAPDH are best suited for normalizing gene expression data in viruliferous and non-viruliferous mites. Ubiq, EF1α and GAPDH are the most stable for different developmental stages. RPL13 and RPL32 are the best reference genes for approaches to B. yothersi in different host plants. Considering all the experimental conditions, Ubiq, EF1α, and GAPDH were the most stable genes. Here we developed an accurate and comprehensive RT-qPCR strategy for use in B. yothersi gene expression analysis. These results will improve the understanding of the biology of the false spider mites and their role as virus vectors.