The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the “brain-eating amoeba,” is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

Free-Living Amoebae (FLA) are widely distributed protozoa, which contain some groups considered as pathogenic microorganisms. These members are able to produce several opportunistic diseases including epithelial disorders, such as keratitis and fatal encephalitis. Even though they have been reported in numerous sources, such as soils, dust and water, there is no legislation related to the presence of these protozoa in soil-related environments worldwide. Therefore, there are no established prevention or disinfection protocols to advise the population regarding FLA infections or eliminate these microorganisms from human-related environments to date. Acanthamoeba spp. are the most common FLA isolated in soil samples, which is also the most common genera found in clinical cases. Thus, the aim of the present study was to evaluate the presence of potentially pathogenic FLA in human-related soil samples of Santiago Island, Cabo Verde. A total of 26 soil samples were seeded in non-nutrient agar plates (2%), incubated at 26 °C, and monitored daily to evaluate the presence of FLA. DNA was extracted from those plates on which there was suspected FLA growth, and PCR amplification of the 18S rRNA gene was carried out. A total of 17 from the 26 analysed samples were positive for FLA, where Acanthamoeba is the most abundant isolated genus (14/17; 82.4%), with the T4 genotype being the most common (13/14; 92.9%), followed by the T5 genotype, A. lenticulata (1/14; 7.1%). Moreover, Vermamoeba vermiformis, Stenamoeba dejonckheerei and Vannella pentlandi were isolated in three other samples. To the best of our knowledge, this is the first report of FLA presence in Cape Verde and the first report of V. vermiformis in beach sand worldwide.

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil’s Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

Concerns over the replicability and reproducibility of published research have grown in many research fields, but empirical data to inform policies are still scarce. Biomedical research in Brazil expanded rapidly over the last three decades, with no systematic assessment of the replicability of its findings. With this in mind, we set up the Brazilian Reproducibility Initiative, a multicenter replication of published experiments from Brazilian science using three common experimental methods: the MTT assay, the reverse transcription polymerase chain reaction (RT-PCR) and the elevated plus maze (EPM). A total of 56 laboratories performed 143 replications of 56 experiments; of these, 90 replications of 45 experiments were considered valid by an independent committee. Replication rates for these experiments varied between 20 and 44% according to five predefined criteria. In median terms, ratios between group means were 58% lower in replications than in original experiments, while coefficients of variation were 82% higher. Effect size decrease was smaller for MTT experiments, original results with less variability and those considered more challenging to replicate, while t values for replications were positively correlated with researcher predictions about replicability, and negatively correlated with the rate of publications by the original article’s last author. Deviations from preregistered protocols were very common in replications, most frequently due to reasons inherent to the experimental model or related to infrastructure and logistics. Our results highlight factors that limit the replicability of results published by researchers in Brazil and suggest ways by which this scenario can be improved.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease and is known for its polycistronic transcription, with about 50% of its genome consisting of repetitive sequences, including coding (primarily multigenic families) and non-coding regions (such as ribosomal DNA, spliced leader [SL], and retroelements, etc). Here, we evaluated the genomic features associated with higher-order chromatin organization in T. cruzi (Brazil A4 strain) by extensive computational processing of high-throughput chromosome conformation capture (Hi-C). Through the mHi-C pipeline, designed to handle multimapping reads, we demonstrated that applying canonical Hi-C processing, which overlooks repetitive DNA sequences, results in a loss of DNA–DNA contacts, misidentifying them as chromatin-folding (CF) boundaries. Our analysis revealed that loci encoding multigenic families of virulence factors are enriched in chromatin loops and form shorter and tighter CF domains than the loci encoding core genes. We uncovered a non-random three-dimensional (3D) genomic organization in which nonprotein-coding RNA loci (transfer RNAs [tRNAs], small nuclear RNAs, and small nucleolar RNAs) and transcription termination sites are preferentially located at the boundaries of the CF domains. Our data indicate 3D clustering of tRNA loci, likely optimizing transcription by RNA polymerase III, and a complex interaction between spliced leader RNA and 18S rRNA loci, suggesting a link between RNA polymerase I and II machineries. Finally, we highlighted a group of genes encoding virulence factors that interact with SL-RNA loci, suggesting a potential regulatory role. Our findings provide insights into 3D genome organization in T. cruzi , contributing to the understanding of supranucleosomal-level chromatin organization and suggesting possible links between 3D architecture and gene expression. IMPORTANCE: Despite the knowledge about the linear genome sequence and the identification of numerous virulence factors in the protozoan parasite Trypanosoma cruzi , there has been a limited understanding of how these genomic features are spatially organized within the nucleus and how this organization impacts gene regulation and pathogenicity. By providing a detailed analysis of the three-dimensional (3D) chromatin architecture in T. cruzi , our study contributed to narrowing this gap. We deciphered part of the origami structure hidden in the T. cruzi nucleus, showing the unidimensional genomic features are non-randomly 3D organized in the nuclear organelle. We uncovered the role of nonprotein-coding RNA loci (e.g., transfer RNAs, spliced leader RNA, and 18S RNA) in shaping genomic architecture, offering insights into an additional epigenetic layer that may influence gene expression.