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.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by the Leishmania infantum parasite. The protozoan is able to infect several domestic and wild mammals. Since the first report on Leishmania spp. infection in horses in South America, leishmaniasis in equids has been highlighted in Brazil. A molecular epidemiological survey was carried out to verify the occurrence of Leishmania spp. DNA in horses and donkeys, in leishmaniases endemic areas in Sao Paulo State, Brazil. To this end, blood samples were obtained from 107 horses and 36 donkeys and subjected to DNA extraction followed by PCR targeting the ITS-1 region. Among the horses and donkeys, 1.87% (2/107) and 8.33% (3/36) were positive by PCR, respectively. The DNA sequencing of the ITS-1 amplification products confirmed L. infantum DNA in these animals. Our results suggest that horses and donkeys from non-VL and VL endemic areas of São Paulo State may be infected by the parasite.

Leishmania (Mundinia) martiniquensis is an emerging human pathogen with increasing reports of infection in domestic animals. This study describes a naturally infected mare from Brazil with no history of international travel, presenting with persistent nodular cutaneous lesions and cervical lymph node enlargement, without evidence of systemic involvement. Considering the endemicity of cutaneous and visceral leishmaniasis in the study area, as well as previous reports of equine leishmaniasis, infection by Leishmania (Leishmania) infantum was initially suspected. Anti-L. (L.) infantum antibodies (titer 320) were detected by indirect immunofluorescence assay, and histopathological analysis of the lymph node revealed amastigote forms compatible with Leishmania spp. Therefore, while the etiological agent was still unknown, xenodiagnosis using Lutzomyia (Lutzomyia) longipalpis was performed during the diagnostic investigation based on the initial suspicion of L. (L.) infantum infection. Limited blood feeding was observed, and no evidence of parasite transmission was detected. In addition, flagellated parasites were isolated in vitro from a fine-needle aspiration biopsy of the cervical lymph node. Molecular characterization of clinical samples and culture isolate was performed by amplification of Leishmania spp. kinetoplast DNA (kDNA) and the internal transcribed spacer 1 (ITS1) region of Trypanosomatidae ribosomal DNA, followed by Sanger sequencing, which revealed the presence of L. (M.) martiniquensis. These findings provide documented evidence of natural infection by L. (M.) martiniquensis in a horse in Brazil and highlight the circulation of this emerging pathogen in areas endemic for cutaneous and visceral leishmaniasis. Furthermore, they reinforce the need for additional studies to better elucidate its epidemiological cycle and vector-host interactions.