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.

Investigation of Chagas disease within the same family: case study / Investigação da doença de Chagas em um mesmo núcleo familiar: estudo de caso

ABSTRACT Chagas disease (CD) is a neglected endemic disease. Its classic form of transmission occurs through hematophagous triatomine insects. Its classic form of transmission occurs through hematophagous triatomine insects. There are cases of the disease in non-endemic regions that occur through alternative transmissions, as this possibility also exists. The aim of this study was to report a case among members of a same family (born and resident in Taquarituba, São Paulo, Brazil) diagnosed with CD. The family matriarch lived in a mud house in the countryside and reported contact with the triatomine during childhood. Two grown-up children are also seroreactive; both reported not having contact with the insect as children. Medical record analyzes and new laboratory tests were performed. Clinical history and recent tests have confirmed positivity for CD in the matriarch and her grown-up children. Parasitological techniques have shown negative results, evidencing that they are in the chronic form of the disease. Congenital transmission may have occurred between them, as well as the possibility of vector transmission by secondary species cannot be ruled out, since the patients come from a municipality considered endemic for CD in the past.

RESUMEN La enfermedadde Chagas (EC) es una infección endémica que ha sido descuidada. Su forma clásica de transmisión ocurre mediante insectos triatominos hematófagos. Hay casos de la enfermedad en regiones no endémicas que ocurrieron por vías alternativas de transmisión, puesto que también hay esa posibilidad. El objetivo de este estudio fue reportar un caso de miembros de una misma familia diagnosticados con EC. La matriarca de la familia vivía en una casa hecha de barro en la zona ruraly reportó contacto con el triatomino en su infancia. Dos hijosson también sero-reactivos, pero no reportaron contacto con el insecto cuando eran ninos. Se hicieron análisis de historial médico y nuevaspruebas de laboratorio. El histórico clínico y laspruebas recientes confirmaran la positividad para EC, tanto en la madre como en los hijos. Las técnicasparasitológicas demostraron resultados negativos, comprobando la forma crónica de la enfermedad. Transmisión congénitapuede haber ocurrido entre ellos, así como no se puede descartar la posibilidad de ocurrencia de transmisión vectorial por especies secundarias, ya que los pacientes proceden de un municipio que fue considerado endémico para EC en el pasado.

RESUMO A doença de Chagas (DC) é uma enfermidade endêmica negligenciada. Sua forma clássica de transmissão ocorre por meio de insetos triatomíneos hematófagos. Há casos da doença em regiões não endêmicas ocorridos por transmissões alternativas, uma vez que também existe essa possibilidade. O objetivo deste estudo foi relatar um caso de membros da mesma família (natural e residente em Taquarituba, São Paulo, Brasil) diagnosticados com DC. A matriarca da família morava em uma casa de barro na zona rural e relatou contato com o triatomíneo na infância. Dois filhos também são sororreagentes; ambos relataram não terem tido contato com o inseto quando crianças. Análises de prontuário e novos testes laboratoriais foram feitos. O histórico clínico e os recentes exames confirmaram a positividade para DC, tanto na mãe quanto nos filhos. As técnicas parasitológicas demonstraram resultados negativos, constatando forma crônica da doença. Transmissão congênita pode ter ocorrido entre eles, assim como também não se pode descartar a possibilidade de ocorrência de transmissão vetorial por espécies secundárias, visto que os pacientes são oriundos de um município considerado endêmico para DC no passado.