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In Brazil, Visceral Leishmaniases is caused by Leishmania infantum, and domestic dogs are the main reservoirs in its urban transmission cycle. As an alternative to euthanizing dogs, miltefosine has been used to treat canine visceral leishmaniasis since 2016. In this study, we have assessed the efficacy of miltefosine for treating canine visceral leishmaniasis in a new endemic area through follow-up of naturally infected dogs was evaluated. The clinical, parasitological, and histopathological characteristics of 21 dogs naturally infected with L. infantum were assessed at three time points: on the day before initiating miltefosine treatment (T0), immediately after treatment completion (T1), and 6 months after treatment completion (T2). Three dogs were treated exclusively with miltefosine, while eighteen received combination therapy with miltefosine with other treatments such as allopurinol, domperidone and immunotherapy. Skin biopsies were obtained from the abdomen to assess inflammatory responses and to quantify parasite loads using qPCR. The parasites were isolated using aspirates acquired from popliteal lymph nodes. Molecular and parasitological analyses confirmed the presence of L. infantum in all dogs, validating the effectiveness of skin and lymph node samples for diagnosis. The clinical conditions of the infected animals were improved and the skin parasite load decreased after treatment, even when distinct combination therapies were performed. The histopathological assessment revealed a miltefosine-induced reduction in the inflammatory response and a decrease in amastigotes number. Furthermore, a positive correlation was established between the reduction in parasite load and the enhancement of clinical scoring, as well as a reduction in the skin inflammatory response. Our findings suggest that miltefosine-based combination therapies reduce skin parasite load and improve clinical outcomes, while the dogs treated with miltefosine alone showed increased parasitic load and worsened clinical staging at T2. Considering this data belonging to a recent transmission area, treatment strategy suggests effective in controlling canine visceral leishmaniasis.

Assignment of gene function has been a crucial, laborious, and time-consuming step in genomics. Due to a variety of sequencing platforms that generates increasing amounts of data, manual annotation is no longer feasible. Thus, the need for an integrated, automated pipeline allowing the use of experimental data towards validation of in silico prediction of gene function is of utmost relevance. Here, we present a computational workflow named AnnotaPipeline that integrates distinct software and data types on a proteogenomic approach to annotate and validate predicted features in genomic sequences. Based on FASTA (i) nucleotide or (ii) protein sequences or (iii) structural annotation files (GFF3), users can input FASTQ RNA-seq data, MS/MS data from mzXML or similar formats, as the pipeline uses both transcriptomic and proteomic information to corroborate annotations and validate gene prediction, providing transcription and expression evidence for functional annotation. Reannotation of the available Arabidopsis thaliana, Caenorhabditis elegans, Candida albicans, Trypanosoma cruzi, and Trypanosoma rangeli genomes was performed using the AnnotaPipeline, resulting in a higher proportion of annotated proteins and a reduced proportion of hypothetical proteins when compared to the annotations publicly available for these organisms. AnnotaPipeline is a Unix-based pipeline developed using Python and is available at: https://github.com/bioinformatics-ufsc/AnnotaPipeline.

Hospital-built environment colonization by healthcare-associated infections-related bacteria (HAIrB) and the interaction with their occupants have been studied to support more effective tools for HAI control. To investigate HAIrB dynamics and antimicrobial resistance (AMR) profile we carried out a 6-month surveillance program in a developing country public hospital, targeting patients, hospital environment, and healthcare workers, using culture-dependent and culture-independent 16S rRNA gene sequencing methods. The bacterial abundance in both approaches shows that the HAIrB group has important representativeness, with the taxa Enterobacteriaceae, Pseudomonas, Staphylococcus, E. coli, and A. baumannii widely dispersed and abundant over the time at the five different hospital units included in the survey. We observed a high abundance of HAIrB in the patient rectum, hands, and nasal sites. In the healthcare workers, the HAIrB distribution was similar for the hands, protective clothing, and mobile phones. In the hospital environment, the healthcare workers resting areas, bathrooms, and bed equipment presented a wide distribution of HAIrB and AMR, being classified as contamination hotspots. AMR is highest in patients, followed by the environment and healthcare workers. The most frequently detected beta-lactamases genes were, blaSHV–like, blaOXA–23–like, blaOXA–51–like, blaKPC–like, blaCTX–M–1, blaCTX–M–8, and blaCTX–M–9 groups. Our results demonstrate that there is a wide spread of antimicrobial resistance due to HAIrB in the hospital environment, circulating among patients and healthcare workers. The contamination hotspots identified proved to be constant over time. In the fight for patient safety, these findings can reorient practices and help to set up new guidelines for HAI control.

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

Abstract Background Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. Methods We cloned and characterised genes coding for a cystathionine β-synthase (CβS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. Results Our results show that T. rangeli CβS (TrCβS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CβS. Moreover, CβS biochemical assays revealed that the T. rangeli CβS enzyme also has serine sulfhydrylase activity. Conclusion These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite.

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

In the present study, a fragment of the VP28 coding sequence from a Brazilian WSSV isolate (BrVP28) was cloned, sequenced and expressed in E. coli BL21(DE3) pLysS strain in order to produce the VP28 carboxyl-terminal hydrophilic region. The expression resulted in a protein of about 21 kDa, which was purified under denaturing conditions, resulting in a final highly purified BrVP28 preparation. The recombinant protein obtained can be used in several biotechnology applications, such as the production of monoclonal antibodies which could be used in the development of diagnostic tools as well as in the studies on the characterization of white spot syndrome virus (WSSV) isolated in Brazil.

Kinetoplastids are an ancestral group of protists that contains free-living species and parasites with distinct mechanisms in response to stress. Here, we compared genes involved in antioxidant defense (AD), proposing an evolution model among trypanosomatids. All genes were identified in Bodo saltans , suggesting that AD mechanisms have evolved prior to adaptation for parasitic lifestyles. While most of the monoxenous and dixenous parasites revealed minor differences from B. saltans , the endosymbiont-bearing species have an increased number of genes. The absence of these genes was mainly observed in the extracellular parasites of the genera Phytomonas and Trypanosoma. In trypanosomes, a distinction was observed between stercorarian and salivarian parasites, except for Trypanosoma rangeli. Our analyses indicate that the variability of AD among trypanosomatids at the genomic level is not solely due to the geographical isolation, being mainly related to specific adaptations of their distinct biological cycles within insect vectors and to a parasitism of a wide range of hosts.

We report the first case of digestive tract pathology (megaesophagus) determined by Trypanosoma cruzi infection in Santa Catarina State, southern Brazil. A 63-year- old female had presumptive clinical diagnosis of Chagas' disease, which was confirmed by imaging (endoscopy and esophagogram) and immunological methods. Further molecular diagnosis was carried out with esophagus and blood samples collected during corrective surgery. Polymerase chain reaction tested positive for Trypanosoma cruzi in both esophagus and buffy coat samples.

Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.The T. rangeli haploid genome is ∼ 24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.

BACKGROUND: Although known to be highly endemic in the Amazon regions of Brazil, the presence of cutaneous leishmaniasis (CL) in the subtropical southern part of the country has largely been ignored. This study was conducted to demonstrate CL is emerging in the Brazilian state of Santa Catarina, as well as to characterize the epidemiological profile and Leishmania species involved. METHODOLOGY/PRINCIPAL FINDINGS: For this cross-sectional study, data from all CL cases from Santa Catarina, Brazil, reported to the Brazilian National Notifiable Diseases Information System from 2001 to 2009 were investigated. Amplification of the kDNA minicircle conserved region followed by restriction fragment length polymorphism (PCR-RFLP) was conducted to screen for Leishmania species present in patient biopsy. Overall, 542 CL cases were reported, with majority resulting from autochthonous transmission (n = 401, 73.99%) and occurring in urban zones (n = 422, 77.86%). Age, gender, zone of residence, origin of case, clinical form and case outcome were found to differ significantly by region. Imported cases were over seven times more likely to relapse (95% CI 2.56-21.09). Mapping of cases revealed new endemic areas in northeastern Santa Catarina with two species present. With the exception of three L. (Leishmania) amazonensis cases (1.20%), majority of PCR positive samples were found to be L. (Viannia) braziliensis (n = 248, 98.80%). CONCLUSIONS/SIGNIFICANCE: CL is now endemic in the state of Santa Catarina, Brazil, with case profiles varying significantly by region. L. (V.) braziliensis has been identified as the predominant species in the region.

Objetivando verificar a colonização de Panstrongylus megistus em ecótopos artificiais em Florianópolis foram examinados, de 1985 a 1992, 779 anexos peridomiciliares (524 galinheiros, 46 currais e 209 ranchos) em 9 localidades e 443 domicílios no distrito de Lagoa, todos na Ilha de Santa Catarina. Todo o ecótopo, incluindo forro e porão das casas, era examinado após aplicação de líquido insentífugo (Pirisa a 5%). A pesquisa nos anexos peri-domiciliares revelou 3 galinheiros e um rancho positivo no distrito de Lagoa, onde foram também encontrados 2 domicílios colonizados pelo P. megistus, com a captura de ovos, ninfas e adultos em todos os ecótopos. Pesquisas dirigidas foram realizadas em dois outros domicílios e em uma escola, nos quais os moradores haviam detectado anteriormente exemplares de P. megistus e, em todos os 3, foi confirmada a colonização pelo triatomíneo. Nos 9 ecótopos artificiais foram coletados 559 ovos, 305 ninfas e 24 adultos de P. megistus, com um índice de infecção pelo T. cruzi de 53,3% (182/329). Índice de infecção semelhante, de 56,5% (78/ 138), foi também encontrado nos adultos de P. megistus oriundos dos ecótopos silvestres e capturados nos domicílios pelos moradores, no período de 1983 a 1991. Os testes de precipitina revelaram, em 94,0% dos insetos examinados (170/181), sangue de uma única fonte alimentar e presença de sangue humano em 80,6% (25/31) dos adultos e em 5,8% (1/17) das ninfas capturados nos domicílios. Os resultados encontrados sugerem a necessidade de adoção de medidas de vigilância epidemiológica com a participação da comunidade, face o risco potencial de domiciliação do P. megistus.The aim of this work was to verify the colonization of Panstrongylus megistus on artificial ecotopes in Florianópolis, in the Santa Catarina Island, South Brazil. For this purpose 443 houses of the Lagoa district and 779 house annexes (524 chicken-houses, 46 corrals and 209 storage-houses) in 9 different places were examined from 1985 to 1992. These ecotopes, which include ceilings and basements, were checked after application of dislodging liquid (Pirisa 5%). Colonization by P. megistus was verified in two houses, three chicken-houses and one storage-house of the Lagoa district, where eggs, nymphs and adults were collected. To verify local reports of P. megistus occurrence, another two houses and one school were investigated. The colonization at all of these places was confirmed. In the 9 artificial ecotopes examined, 559 eggs, 305 nymphs and 24 adults were collected. The infection rate of P. megistus by Trypanosoma cruzi was 55.3% (182/ 329). A similar infection rate of 56.5% (78/138) was obtained in adults of P. megistus from sylvatic ecotopes and in adults captured in the houses by the inhabitants between 1983 to 1991. Precipitin tests revealed blood from just one source in 94.0% of the insects (170/181). Human blood was found in 80.6% (25/31) of the adults and in 5.8% (1/17) of the nymphs captured in the houses. These results suggest the need to ally serious epidemiologic vigilance to the effort of the inhabitants in order to avoid the risk of domiciliation of P. megistus in the houses.