Diagnostic performance of the bioline dengue duo rapid test on symptomatic patients assisted at Armed Forces Hospital (Hfa) in Brasília, Brazil.

Dengue necessitates accurate diagnosis. Rapid tests such as Bioline™ DENGUE DUO have gained traction, but validation in specific populations is essential. This study aimed to evaluate the performance of the Bioline™ test, alongside assessing the socio-epidemiological profile of symptomatic patients in a Brasília Military Hospital. The serum of 404 symptomatic patients was analyzed by the Bioline™ DENGUE DUO test, followed by Dengue virus detection and discrimination of the four serotypes by RT-qPCR. Accuracy was assessed using parameters including sensitivity (S), specificity (E), positive and negative predictive values (PPV and NPV), and positive (RV +) and negative (RV-) likelihood ratios. The NS1 component exhibited a sensitivity of 70.37%, a specificity of 97.30%, and an overall efficiency of 90.10% when compared to RT-qPCR as the gold standard. The IgM component demonstrated a sensitivity of 26.85%, a specificity of 89.53%, and an overall efficiency of 72.77% when compared to RT-qPCR as the gold standard. The IgG component demonstrated a sensitivity of 23.15%, a specificity of 68.92%, and an overall efficiency of 56.68% when compared to RT-qPCR as the gold standard. Several rapid tests are commercially available. However, considering variations across regions and demographic groups, it is important to question their accuracy in specific populations. Rapid tests are important screening tools, but they can have limitations for the certainty of diagnosis. Bioline™ DENGUE DUO displayed good specificity, but sensitivity was slightly below optimal levels. While helpful for confirming dengue, improvements are needed to effectively rule out the disease.

Advancing Chikungunya Diagnosis: A Cost-Effective and Rapid Visual employing Loop-mediated isothermal reaction.

The diagnosis of Chikungunya (CHIKV), along with the simultaneous monitoring of virus circulation in the population or vectors, is essential for global health. Although effective diagnostic methods for CHIKV, such as RT-qPCR, exist, their utilization is constrained by high costs. With the aim of contributing to the field of diagnostics, we have developed a diagnostic assay using isothermal amplification technology with visually interpretable results. This test can detect the virus within a maximum timeframe of 30 minutes. The detection limit of RT-LAMP CHIKV was found to be 66 copies of RNA molecules (Ct ≅ 31.28), and no cross-reactivity with other arboviruses was observed. During test validation, our assay demonstrated a sensitivity of 80.43%, specificity of 100%, and an overall accuracy of 88.89%. By utilizing more cost-effective reagents and equipment compared to RT-qPCR, this test holds the potential for broader application and enhanced accessibility, particularly in point-of-care settings.

Report of natural Mayaro virus infection in Mansonia humeralis (Dyar & Knab, Diptera: Culicidae).

BACKGROUND: Arboviruses are a group of viruses transmitted to vertebrate hosts by certain blood-feeding arthropods. Among urban vectors of arboviruses, mosquitoes of the genus Aedes are the most common. However, other mosquitoes may be susceptible to infection and involved in the transmission, such as Mansonia spp. Therefore, this study aimed to investigate whether Mansonia humeralis can be infected with the Mayaro virus (MAYV). METHODS: These insects were collected from 2018 to 2020 in chicken coops of rural communities in Jaci Paraná in Porto Velho, Rondônia, Brazil, while performing blood-feeding on roosters. The mosquitoes were randomly grouped in pools from which the head and thorax were macerated and checked for the presence of MAYV by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The positive pools were used to infect the C6/36 cell line, and on different days post-infection, the supernatant of the infected cells was subjected to viral detection by RT-qPCR. RESULTS: A total of 183 pools of female mosquitoes were tested, of which 18% were positive for MAYV; some samples from insect pools inoculated into C6/36 cells showed in vitro multiplication capacity between 3 and 7 days post-infection. CONCLUSIONS: This is the first report of Ma. humeralis mosquitoes that are naturally infected by MAYV, indicating that these vectors may be potential transmitting agents of this arbovirus.

Global Molecular Response of Paracoccidioides brasiliensis to Zinc Deprivation: Analyses at Transcript, Protein and MicroRNA Levels.

Zinc is one of the main micronutrients for all organisms. One of the defense mechanisms used by the host includes the sequestration of metals used in fungal metabolism, such as iron and zinc. There are several mechanisms that maintain the balance in the intracellular zinc supply. MicroRNAs are effector molecules of responses between the pathogen and host, favoring or preventing infection in many microorganisms. Fungi of the Paracoccidioides genus are thermodimorphic and the etiological agents of paracoccidioidomycosis (PCM). In the current pandemic scenario world mycosis studies continue to be highly important since a significant number of patients with COVID-19 developed systemic mycoses, co-infections that complicated their clinical condition. The objective was to identify transcriptomic and proteomic adaptations in Paracoccidioides brasiliensis during zinc deprivation. Nineteen microRNAs were identified, three of which were differentially regulated. Target genes regulated by those microRNAs are elements of zinc homeostasis such as ZRT1, ZRT3 and COT1 transporters. Transcription factors that have zinc in their structure are also targets of those miRNAs. Transcriptional and proteomic data suggest that P. brasiliensis undergoes metabolic remodeling to survive zinc deprivation and that miRNAs may be part of the regulatory process.

Immunogenic Cell Death Photothermally Mediated by Erythrocyte Membrane-Coated Magnetofluorescent Nanocarriers Improves Survival in Sarcoma Model.

Inducing immunogenic cell death (ICD) during cancer therapy is a major challenge that might significantly improve patient survival. The purpose of this study was to develop a theranostic nanocarrier, capable both of conveying a cytotoxic thermal dose when mediating photothermal therapy (PTT) after its intravenous delivery, and of consequently inducing ICD, improving survival. The nanocarrier consists of red blood cell membranes (RBCm) embedding the near-infrared dye IR-780 (IR) and camouflaging Mn-ferrite nanoparticles (RBCm-IR-Mn). The RBCm-IR-Mn nanocarriers were characterized by size, morphology, surface charge, magnetic, photophysical, and photothermal properties. Their photothermal conversion efficiency was found to be size- and concentration-dependent. Late apoptosis was observed as the cell death mechanism for PTT. Calreticulin and HMGB1 protein levels increased for in vitro PTT with temperature around 55 °C (ablative regime) but not for 44 °C (hyperthermia), suggesting ICD elicitation under ablation. RBCm-IR-Mn were then intravenously administered in sarcoma S180-bearing Swiss mice, and in vivo ablative PTT was performed five days later. Tumor volumes were monitored for the subsequent 120 days. RBCm-IR-Mn-mediated PTT promoted tumor regression in 11/12 animals, with an overall survival rate of 85% (11/13). Our results demonstrate that the RBCm-IR-Mn nanocarriers are great candidates for PTT-induced cancer immunotherapy.

Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers.

Despite the advance of vaccination worldwide, epidemic waves caused by more transmissible and immune evasive genetic variants of SARS-CoV-2 have sustained the ongoing pandemic of COVID-19. Monitoring such variants is expensive, as it usually relies on whole-genome sequencing methods. Therefore, it is necessary to develop alternatives that could help identify samples from specific variants. Reverse transcription loop-mediated isothermal amplification is a method that has been increasingly used for nucleic acid amplification, as it is cheaper and easier to perform when compared to other molecular techniques. As a proof of concept that can help distinguish variants, we present an RT-LAMP assay capable of detecting samples carrying a group of mutations that can be related to specific SARS-CoV-2 lineages, here demonstrated for the Variant of Concern Gamma. We tested 60 SARS-CoV-2 RNA samples extracted from swab samples and the reaction showed a sensitivity of 93.33%, a specificity of 88.89% and a kappa value of 0.822 for samples with a Ct ≤ 22.93. The RT-LAMP assay demonstrated to be useful to distinguish VOC Gamma and may be of particular interest as a screening approach for variants in countries with poor sequencing coverage.

"pySewage": a hybrid approach to predict the number of SARS-CoV-2-infected people from wastewater in Brazil.

It is well known that the new coronavirus pandemic has global environmental, public health, and economic implications. In this sense, this study aims to monitor SARS-CoV-2 in the largest wastewater treatment plant of Goiânia, which processes wastewater from more than 700,000 inhabitants, and to correlate the molecular and clinical data collected. Influent and effluent samples were collected at Dr. Helio de Seixo Britto's wastewater treatment plant from January to August 2021. Viral concentration was performed with polyethylene glycol before viral RNA extraction. Real-time qPCR (N1 and N2 gene assays) was performed to detect and quantify the viral RNA present in the samples. The results showed that 43.63% of the samples were positive. There is no significant difference between the detection of primers N1 (mean 3.23 log10 genome copies/L, std 0.23) and N2 (mean 2.95 log10 genome copies/L, std 0.29); also, there is no significant difference between the detection of influent and effluent samples. Our molecular data revealed a positive correlation with clinical data, and infection prevalence was higher than clinical data. In addition, we developed a user-friendly web application to predict the number of infected people based on the detection of viral load present in wastewater samples and may be applied as a public policy strategy for monitoring ongoing outbreaks.

Detection of Mayaro virus in Aedes aegypti mosquitoes circulating in Goiânia-Goiás-Brazil.

The Arboviral diseases are caused by arthropod-borne viruses, such as Mayaro virus (MAYV), the etiological agent of Mayaro fever. This disease has been drawing the attention of the public health authorities for the increased number of cases likely due to virus adaptation for survival to urban areas as well as infection and multiplication in other vectors insects. Therefore, this work aimed to identify the MAYV infecting Aedes aegypti mosquitoes in Goiânia, the capital of state of Goiás, Brazil. For the development of study, the larvae of A. aegypti were collected in Basic Health Units from different regions of Goiânia then the larvae were grown to adult mosquitoes in controlled laboratory conditions. The female mosquitoes were submitted to the procedure of head and body separation. The RNAs obtained from these samples were analyzed by real-time PCR for identification of arboviruses. We only detect the presence of MAVY in the mosquitoes, in this sense our findings suggest that A. aegypti harbor MAYV in different anatomical sites, and potentially the process of vertical transmission of MAYV can occur in this vector.

Interacting with Hemoglobin: Paracoccidioides spp. Recruits hsp30 on Its Cell Surface for Enhanced Ability to Use This Iron Source.

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.

The "Little Iron Waltz": The Ternary Response of Paracoccidioides spp. to Iron Deprivation.

Paracoccidioides is a genus of thermodimorphic fungi that causes paracoccidioidomycosis. When in the host, the fungus undergoes several challenges, including iron deprivation imposed by nutritional immunity. In response to the iron deprivation triggered by the host, the fungus responds in a ternary manner using mechanisms of high affinity and specificity for the uptake of Fe, namely non-classical reductive iron uptake pathway, uptake of host iron proteins, and biosynthesis and uptake of siderophores. This triple response resembles the rhythmic structure of a waltz, which features three beats per compass. Using this connotation, we have constructed this review summarizing relevant findings in this area of study and pointing out new discoveries and perspectives that may contribute to the expansion of this "little iron waltz".

Molecular characterization of siderophore biosynthesis in Paracoccidioides brasiliensis.

Iron is an essential nutrient for all organisms. For pathogenic fungi, iron is essential for the success of infection. Thus, these organisms have developed high affinity iron uptake mechanisms to deal with metal deprivation imposed by the host. Siderophore production is one of the mechanisms that fungal pathogens employ for iron acquisition. Paracoccidioides spp. present orthologous genes encoding the enzymes necessary for the biosynthesis of hydroxamates, and plasma membrane proteins related to the transport of these molecules. All these genes are induced in iron deprivation. In addition, it has been observed that Paracoccidioides spp. are able to use siderophores to scavenge iron. Here we observed that addition of the xenosiderophore ferrioxamine B FOB) to P. brasiliensis culture medium results in repression (at RNA and protein levels) of the SidA, the first enzyme of the siderophore biosynthesis pathway. Furthermore, SidA activity was reduced in the presence of FOB, suggesting that P. brasiliensis blocks siderophores biosynthesis and can explore siderophores in the environment to scavenge iron. In order to support the importance of siderophores on Paracoccidioides sp. life and infection cycle, silenced mutants for the sidA gene were obtained by antisense RNA technology. The obtained AsSidA strains displayed decreased siderophore biosynthesis in iron deprivation conditions and reduced virulence to an invertebrate model.

Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation.

Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.

Identification of membrane proteome of Paracoccidioides lutzii and its regulation by zinc.

AIM: During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii. MATERIALS & METHODS: Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC-MS/MS detection and database search. RESULTS & CONCLUSION: Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides.