The impact of ZIKV infection on gene expression in neural cells over time.

Zika virus (ZIKV) outbreak caused one of the most significant medical emergencies in the Americas due to associated microcephaly in newborns. To evaluate the impact of ZIKV infection on neuronal cells over time, we retrieved gene expression data from several ZIKV-infected samples obtained at different time point post-infection (pi). Differential gene expression analysis was applied at each time point, with more differentially expressed genes (DEG) identified at 72h pi. There were 5 DEGs (PLA2G2F, TMEM71, PKD1L2, UBD, and TNFAIP3 genes) across all timepoints, which clearly distinguished between infected and healthy samples. The highest expression levels of all five genes were identified at 72h pi. Taken together, our results indicate that ZIKV infection greatly impacts human neural cells at early times of infection, with peak perturbation observed at 72h pi. Our analysis revealed that all five DEGs, in samples of ZIKV-infected human neural stem cells, remained highly upregulated across the timepoints evaluated. Moreover, despite the pronounced inflammatory host response observed throughout infection, the impact of ZIKV is variable over time. Finally, the five DEGs identified herein play prominent roles in infection, and could serve to guide future investigations into virus-host interaction, as well as constitute targets for therapeutic drug development.

Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLA2s.

Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-ß and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Updating the knowledge of sand flies (Diptera, Psychodidae) in Rondônia State, Brazil.

Background: Sandflies are insects important for the transmission cycles of the leishmaniases. Despite being studied since the 1960s in the State of Rondônia (Brazil), several gaps exist regarding our working knowledge of these insects. This study aimed to construct an up-to-date database of sandflies using complementary information from the speciesLink database and the scientific literature, as well as to elaborate integrated abundance maps. We identified 153,155 records of sandflies captured in Rondônia between 1965-2021; after exclusion, 147,258 reports (speciesLink - 3,408, Rondônia studies - 143,850) associated with 15 genera and 140 species were mapped. The most abundant species observed were Psychodopygusdavisi (Root, 1934) (43,818 records), Nyssomyiawhitmani (Antunes & Coutinho, 1939) (12,594), Psychodopyguscarrerai (Barretto, 1946) (11,840), Psychodopygushirsutus (Mangabeira, 1942) (9,676), Nyssomyiaantunesi (Coutinho, 1939) (8,847), Trichophoromyiaubiquitalis (Mangabeira, 1942) (5,505), Psychodopygusgeniculatus (Mangabeira, 1941) (4,644), Pintomyianevesi (Damasceno & Arouck, 1956) (4,140), Trichophoromyiaauraensis (Mangabeira, 1942) (3,579), Psychodopyguscomplexus (Mangabeira, 1941) (2,659), Nyssomyiafraihai (Martins, Falcão & Silva, 1979) (2,504) and Bichromomyiaflaviscutellata (Mangabeira, 1942) (1,418). A total of 20 records of Leishmania detection corresponded to eight sand fly species. The present dataset provides updated information on the distribution of sandflies of Rondônia, including those considered potential vectors of Leishmania, which should prove useful to guide future studies. New information: The present study provides an extensive dataset built from all studies reporting phlebotomine sandflies in the Brazilian State of Rondônia. Online distribution maps can aid scientists who wish to consult the updated list of sand fly species and view the distribution of these insects, as well as those considered potential vectors of Leishmania. The results of the present study can serve as the basis for future studies on sandflies conducted in the State.

Use of light emitting diodes (LEDs) are effective and useful for sand fly ecoepidemiology studies in an Amazonian environment.

One strategy to identify transmission foci is based on vector monitoring, and efficient methods are important for vector control. Our study evaluated different light sources (red, green, ultraviolet, blue, and incandescent light) to capture sand fly in Porto Velho, Brazil. We also evaluated Leishmania and blood meal sources in females. A total of 1,943 individuals were identified in 45 species level-taxa, with Trichophoromyia ubiquitalis (Mangabeira) (n = 364), Nyssomyia antunesi (n = 241), Bichromomyia flaviscutellata (Mangabeira) (n = 222), and Psychodopygus davisi (Root) (n = 148) being the most abundant. Incandescent light captured most individuals (n = 589), followed by blue (n = 471), green (n = 452), ultraviolet (n = 281) and red (n = 150). No significant difference was observed between the species composition and lights (PERMANOVA: Pseudo F = 1.29, p = 0.14, NMDS: Stress 0.18). The Shannon and Simpson indices demonstrated a high diversity captured using all lights. Our data demonstrated that LEDs are alternative devices for sand fly capture, with blue and green LEDs presenting similar results to incandescent light. 53 pools were analyzed, only one pool was positive for kDNA and hsp70 targets [Nyssomyia fraihai (Martins, Falão & Silva)] and identified as Endotrypanum spp., suggesting that other trypanosomatids may circulate in the locality. Choloepus hoffmanni (two-toed sloth) Peters, Homo sapiens Linnaeus, Proechimys gardneri (Gardner's spiny rat) Da Silva, and Tamandua tetradactyla (lesser anteater) (Linnaeus) were blood meal sources identified in females, increasing the knowledge about sources used by these insects.

Description of malaria vectors (Diptera: Culicidae) in two agricultural settlements in the Western Brazilian Amazon.

The majority of malaria cases in South America occur in rural areas of the Amazon region. Although these areas have a significant impact on malaria cases, few entomological studies have been carried out there. This study aimed to describe entomological parameters in settlements in Rondonia State, Brazil. Collections of anopheles were carried out using the Protected Human Attraction Technique (PHAT). The risk and the potential for malaria transmission were assessed using the human biting rate (HBR), the sporozoite rate (SR) and the entomological inoculation rate (EIR). The results confirmed that Nyssorhynchus darlingi is the predominant species in the two studied locations. Although settlement in the two study sites has occurred at different times, the species richness found was low, showing that environmental changes caused by anthropological actions have probably favor the adaptation of Ny. darlingi species. From the total of 615 anopheline mosquitoes assessed, seven (1.1%) were positive for Plasmodium sp. infections. The EIR revealed that Ny. darlingi contributes to malaria transmission in both locations, as it was responsible for 0.05 infectious bites in humans at night in the old settlement and 0.02 in the recent occupation. In the two study sites, the biting occurred more frequently at dusk. Nyssorhynchus darlingi was prevalent in areas of recent colonization but, even when present in a low density, this species could maintain the transmission of malaria in the older settlement. The entomological information obtained in this study is important and may aid the selection of vector control actions in these locations.

Occurrence of Leishmania infection in the immediate geographic region of Ji-Paraná, Rondônia State, Brazil.

INTRODUCTION: This study evaluated the epidemiology of American cutaneous leishmaniasis in the immediate region of Ji-Paraná, Rondônia State. METHODS: Samples and epidemiological data were collected from 105 patients. RESULTS: Leishmania infection was observed in 58 (55.2%) patients, and Leishmania braziliensis was present in 82.9% of the 41 sequenced samples. Infected patients were predominantly male (93.1%). Leishmania infection was twice as prevalent among rural inhabitants versus urban inhabitants. Lesions were more frequent in the upper limbs (arms/hands, 41.82%). CONCLUSIONS: The present data corroborate the zoonotic profile of cutaneous leishmaniasis; this information could help to improve surveillance and control strategies.

Detection of Leishmania species (Kinetoplastida, Trypanosomatidae) in phlebotomine sand flies (Diptera, Psychodidae) from Porto Velho, Northern Brazil.

Cutaneous Leishmaniasis (CL) is a vector-borne disease caused by Leishmania species and transmitted by infected female sand flies. CL is widely distributed in Brazil, but knowledge about vectors and transmission cycles could be complex according to localities. The sand fly fauna in Rondônia State is extensive, diverse, and largely unexplored. Although the state records a mean of 1,000 CL cases per year, the vectors of CL are unknown. The aim of this study was to assess phlebotomine sand fly fauna composition using diversity indexes (Shannon [H'] and Simpson [1/D]) and to detect the prevalence of Leishmania infection to verify potential vectors in three ecotopes: (i) forest fragment (FF), (ii) forest edge (FE), and (iii) peridomicile (PE). Captures were performed in four rural districts in the municipality of Porto Velho. A total of 7,026 specimens were captured comprising 72 species, and individuals classified in subgenus level. Overall, the most abundant species were Lutzomyia davisi (n: 1,105), Lutzomyia melloi (n: 760), Lutzomyia auraensis (n: 738) and Lutzomyia antunesi (n: 479). Fauna was most diverse in the FF ecotope (H' = 20.2, 1/D = 11.2), followed by the FE (H' = 18.0, 1/D = 10.1) and PE (H' = 16.6, 1/D = 10.1) ecotopes. Leishmania DNA was detected in 24 of 232 pools. In every ecotope, Leishmania naiffi DNA was identified in the following sand fly species: Lu. antunesi, Lu. davisi, Lu. hirsuta hirsuta, Lu. shawi, Lu. sordellii and Lu. (Trichophoromyia) spp. This observation may indicate that a Le. naiffi transmission focus is present in the study localities. In addition, Leishmania lainsoni was detected in Lutzomyia (Trichophoromyia) spp. Our findings show that sand fly fauna in the study localities is diverse, that Leishmania parasites are circulating in all three ecotopes, and that some sand fly species may be implicated in the transmission of Leishmania to humans in localities evaluated.

Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) that emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR); however, the role of viral load quantification has not been thoroughly investigated yet. OBJECTIVE: The aim of this study was to develop a high-precision quantitative one-step RT-qPCR reaction using the association of the viral target and the human target in the same reaction. METHODS: The assay standardization involved the absolute quantification method, with serial dilutions of a plasmid with the N gene in a biological matrix to build a standard curve. RESULTS AND DISCUSSION: The results demonstrated the possibility of quantifying as few as 2.5 copies/reaction and an analysis of 244 patients with known results selected by cross-section that revealed 100% agreement with a qualitative RT-qPCR assay registered by Anvisa. In this population, it was possible to quantify patients with between 2.59 and 3.5 × 107 copies per reaction and negative patients continued to indicate the same result. CONCLUSION: This assay can be a useful tool for a proper patient management, because the level and duration of viral replication are important factors to assess the risk of transmission and to guide decisions regarding the isolation and release of patients; an accurate diagnosis is critical information, whereas the current COVID-19 pandemic represents the biggest current global health problem.

Vertical stratification of sand fly diversity in relation to natural infections of Leishmania sp. and blood-meal sources in Jamari National Forest, Rondônia State, Brazil.

BACKGROUND: Almost 1000 cases of American cutaneous leishmaniasis have been registered yearly in Rondônia State, Brazil. Little is known about the Leishmania transmission cycle (vectors and reservoirs) in the state. This study aimed to evaluate sand fly fauna from two vertical stratification layers in order to identify potential vectors and their blood-meal sources. METHODS: The study was conducted in Jamari National Forest. Sand flies were collected in the canopy (15 m) and at ground level (1 m) using HP light traps during four months, February, April, August and October, 2018. Insects were identified to the species level, and females were subjected to DNA extraction and PCR targeting minicircle kDNA and hsp70 (for Leishmania detection and species identification), and cytb (to identify blood-meal sources). Exploratory data analysis was used to determine mean of abundance and species richness between stratifications. The hsp70 and cytb sequences were analyzed and compared with sequences from GenBank. RESULTS: Overall, 68 species were identified from 15,457 individuals. On the Potosi trail, 7531 individuals of 49 species were collected; canopy captures totaled 6463 individuals of 46 species, while ground captures totaled 1068 individuals of 38 species. On the Santa Maria trail, 7926 individuals of 61 species were collected; canopy captures totaled 6136 individuals of 51 species, while ground captures totaled 1790 individuals of 53 species. A total of 23 pools were positive for kDNA (canopy n = 21, ground n = 2). Only two samples were sequenced for hsp70 (both in canopy); one sequence exhibited similarity with Leishmania braziliensis (Lutzomyia davisi pool) and another with L. naiffi (Lu. antunesi pool). The cytb fragment was amplified in 11 of 86 samples. Sample sequencing identified cytb DNA from 5 blood-meal sources: Micrastur gilvicollis, Psophia viridis, Tamandua tetradactyla, Homo sapiens and Choloepus didactylus. CONCLUSIONS: Sand fly fauna is more diverse in the canopy than at ground level. Factors such as blood-meal sources, resting sites, and abiotic components probably contribute to high abundance in the canopy. Our results reinforce the possibility that Lu. antunesi and Lu. davisi participate in Leishmania transmission in forest environments and may play an important role in transmission from sylvatic to human hosts.

First Observation of Experimental Plasmodium vivax Infection of Three Malaria Vectors from the Brazilian Amazon.

Although malaria is endemic to the Amazon region, little is known about the susceptibility of potential parasite vectors in Brazil. Assessing the vector susceptibility of Anopheles mosquitoes will increase our understanding of parasite-vector interactions and aid the design of vector control strategies. This study assessed the susceptibility of three Anopheles species to midgut infection by Plasmodium vivax, the predominant malaria species in Rondônia State, Brazil. Blood from P. vivax infected patients was fed to Anopheles aquasalis, Anopheles darlingi, and Anopheles deaneorum mosquitoes using a membrane feeding assay (MFA). Gametocytemia was estimated by microscopic examination of blood smears and oocyst prevalence, and infection intensity was assessed. The presence of oocysts was determined by microscopy, and the infection rates and infection intensity were determined for all species. Data from six MFAs showed that An. darlingi and An. deaneorum exhibited the highest infection rates (97% and 90%, respectively) and developed a similar median number of P. vivax oocysts (142 and 123, respectively), while An. aquasalis exhibited the smallest infection rates (77%) and the median number of oocysts (88). Established laboratory colonies of An. darlingi and An. deaneorum and susceptibility to plasmodial infection would be beneficial for modeling P. vivax vector-parasite interactions in Brazil.

Composition and Vertical Stratification of Phlebotomine Sand Fly Fauna and the Molecular Detection of Leishmania in Forested Areas in Rondônia State Municipalities, Western Amazon, Brazil.

This study examined sand fly diversity and the natural infection of sand flies by Leishmania in three localities in Rondônia State: Santo Antônio and São Vicente farm, which are anthropized environments, and the Jamari FLONA, which has not been anthropized. Sand flies were collected in canopies and at ground level using CDC light traps and BG-Sentinel CO2 traps. Female sand flies were grouped into pools that were subjected to DNA extraction and the samples were subjected to PCR to detect and identify Leishmania. A total of 6696 specimens were examined and 52 species were identified. Abundance was greater in canopies than at ground level (n = 5657 and n = 1039, respectively); however, fauna composition was more diverse at ground level. A total of 4870 specimens were collected using CDC light traps and 1826 specimens were collected using BG-Sentinel traps. The presence of Leishmania DNA was detected in 12 pools, and Leishmania species profiles were identified in three of these pools: one pool of Psychodopygus amazonensis and one pool of Psychodopygus chagasi infected with Le. (Vi.) braziliensis, and one pool of Psychodopygus davisi infected with Le. (Le.) amazonensis. Sand flies are sensitive to environmental modification and species diversity is greater in regions that have experienced less anthropization. Although the annual infection rate of cutaneous leishmaniasis is high in Rondônia State, the role that sand flies play in the leishmaniasis transmission cycle remains poorly understood. The high abundance of Ps. davisi and Trichophoromyia auraensis and the presence in these species of natural infection by Leishmania spp. suggest that these species are acting as vectors of tegumentary leishmaniasis in Rondônia.

Ambient temperature and mortality due to acute myocardial infarction in Brazil: an ecological study of time-series analyses.

Ambient temperature may lead to decompensation of cardiovascular diseases and deaths by acute myocardial infarction (AMI). Little is known about this relationship in South American countries located in regions of a hot climate. This study aims to investigate the effects of ambient temperature on mortality due to AMI in six Brazilian micro-regions, which present different climates. We analyzed daily records of deaths by AMI between 1996 and 2013. We estimated the accumulate relative and attributable risks with lags of up to 14 days, using distributed non-linear lag model. Micro-regions that were closest to the equator did not show an association between temperature and mortality. The lowest risk temperatures varied between 22 °C and 28 °C, in the Southern region of Brazil and the Midwest region, respectively. Low temperatures associated with the highest mortality risk were observed in the same areas, varying between 5 °C and 15 °C. The number of deaths attributed to cold temperatures varied from 176/year in Brasilia to 661/year in São Paulo and those deaths attributed to hot temperatures in Rio de Janeiro amounted to 115/year. We showed the relative risk and the attributable risk of warmer and colder days in tropical regions. The estimate of the number of deaths due to climate, varying according to each area, is a way of bringing information to those responsible for health policies based on easily-understood measurements.

Meta-Analysis of HTLV-1-Infected Patients Identifies CD40LG and GBP2 as Markers of ATLL and HAM/TSP Clinical Status: Two Genes Beat as One.

Human T-lymphotropic virus 1 (HTLV-1) was the first recognized human retrovirus. Infection can lead to two main symptomatologies: adult T-cell lymphoma/leukemia (ATLL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Each manifestation is associated with distinct characteristics, as ATLL presents as a leukemia-like disease, while HAM/TSP presents as severe inflammation in the central nervous system, leading to paraparesis. Previous studies have identified molecules associated with disease development, e.g., the downregulation of Foxp3 in Treg cells was associated with increased risk of HAM/TSP. In addition, elevated levels of CXCL10, CXCL9, and Neopterin in cerebrospinal fluid also present increased risk. However, these molecules were only associated with specific patient groups or viral strains. Furthermore, the majority of studies did not jointly compare all clinical manifestations, and robust analysis entails the inclusion of both ATLL and HAM/TSP. The low numbers of samples also pose difficulties in conducting gene expression analysis to identify specific molecular relationships. To address these limitations and increase the power of manifestation-specific gene associations, meta-analysis was performed using publicly available gene expression data. The application of supervised learning techniques identified alterations in two genes observed to act in tandem as potential biomarkers: GBP2 was associated with HAM/TSP, and CD40LG with ATLL. Together, both molecules demonstrated high sample-classification accuracy (AUC values: 0.88 and 1.0, respectively). Next, other genes with expression correlated to these genes were identified, and we attempted to relate the enriched pathways identified with the characteristic of each clinical manifestation. The present findings contribute to knowledge surrounding viral progression and suggest a potentially powerful new tool for the molecular classification of HTLV-associated diseases.

Epidemiological study of snakebite cases in Brazilian Western Amazonia.

INTRODUCTION: Brazil has the largest number of snakebite cases in South America, of which the large majority is concentrated in the Midwest and North. METHODS: In this descriptive observational study, we assessed the epidemiological and clinical snakebite cases referred to the Centro de Medicina Tropical de Rondônia from September 2008 to September 2010. RESULTS: We followed up 92 cases from admission until discharge, namely 81 (88%) men and 11 (12%) women, with a mean age of 37 years, and mainly from rural areas (91.3%). The snakebites occurred while performing work activities (63%) during the Amazon rainy season (78.3%). The vast majority of individuals presented from the Porto Velho microregion (84.7%). Approximately 95.6% of the snakebites were caused by snakes of the genus Bothrops, followed by two lachetics and two elapidics cases. Surgery was performed in 10 cases (9 fasciotomies in the lower limb and 1 amputation). No deaths were reported in this study, but 4 cases (4.3%) developed sequelae in the lower limb. CONCLUSIONS: This study can contribute to a better understanding of envenomation in the state of Rondônia and thus can be useful for identifying real conditions that can increase the incidence of snakebites in this region. Moreover, the study results can serve as a basis for improving educational campaigns designed to prevent these types of snakebites, as well as for preserving snakes.

Occurrence of Leishmania infection in the immediate geographic region of Ji-Paraná, Rondônia State, Brazil

Abstract INTRODUCTION: This study evaluated the epidemiology of American cutaneous leishmaniasis in the immediate region of Ji-Paraná, Rondônia State. METHODS: Samples and epidemiological data were collected from 105 patients. RESULTS: Leishmania infection was observed in 58 (55.2%) patients, and Leishmania braziliensis was present in 82.9% of the 41 sequenced samples. Infected patients were predominantly male (93.1%). Leishmania infection was twice as prevalent among rural inhabitants versus urban inhabitants. Lesions were more frequent in the upper limbs (arms/hands, 41.82%). CONCLUSIONS: The present data corroborate the zoonotic profile of cutaneous leishmaniasis; this information could help to improve surveillance and control strategies.

Epidemiological study of snakebite cases in Brazilian Western Amazonia

Abstract INTRODUCTION Brazil has the largest number of snakebite cases in South America, of which the large majority is concentrated in the Midwest and North. METHODS In this descriptive observational study, we assessed the epidemiological and clinical snakebite cases referred to the Centro de Medicina Tropical de Rondônia from September 2008 to September 2010. RESULTS We followed up 92 cases from admission until discharge, namely 81 (88%) men and 11 (12%) women, with a mean age of 37 years, and mainly from rural areas (91.3%). The snakebites occurred while performing work activities (63%) during the Amazon rainy season (78.3%). The vast majority of individuals presented from the Porto Velho microregion (84.7%). Approximately 95.6% of the snakebites were caused by snakes of the genus Bothrops, followed by two lachetics and two elapidics cases. Surgery was performed in 10 cases (9 fasciotomies in the lower limb and 1 amputation). No deaths were reported in this study, but 4 cases (4.3%) developed sequelae in the lower limb. CONCLUSIONS This study can contribute to a better understanding of envenomation in the state of Rondônia and thus can be useful for identifying real conditions that can increase the incidence of snakebites in this region. Moreover, the study results can serve as a basis for improving educational campaigns designed to prevent these types of snakebites, as well as for preserving snakes.