Prevalência de Influenza A, vírus sincicial respiratório eSARS-CoV-2 em pacientes com síndrome respiratóriaaguda grave em Passo Fundo, Rio Grande do Sul / Prevalence of Influenza A, respiratory syncytial virus andSARS-CoV-2 in patients with severe acute respiratorysyndrome in Passo Fundo, Rio Grande do Sul

A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Enhancement of apoptosis in Caco-2, Hep-G2, and HT29 cancer cell lines following exposure to Toxoplasma gondii peptides.

Objective: Cancer or neoplasm is a cosmopolitan catastrophe that results in more than 20 million new cases and 10 million deaths every year. Some factors lead to carcinogenesis like infectious diseases. Parasites like Toxoplasma gondii, by its components, could modulate the cancer system by inducing apoptosis. The objective of this investigation is to assess the potential of peptides derived from T. gondii in combating cancer by examining their effects on Caco-2, Hep-G2, and HT29 cell lines. Materials and methods: Candidate peptide by its similarity to anticancer compounds was predicted through the computer-based analysis/platform. The impact of the peptide on cell viability, cell proliferation, and gene expression was evaluated through the utilization of MTT assay, flow cytometry, and real-time polymerase chain reaction (PCR) methodologies. Results: The cell viability rate exhibited a significant decrease (p < 0.001) across all cell lines when exposed to a concentration of ≤160 µg. Within the 48-hour timeframe, the half maximal inhibitory concentration (IC50) for HT29 and Hep-G2 cell lines was determined to be 107.2 and 140.6 µg/mL, respectively. Notably, a marked decrease in the expression levels of Bcl2 and APAF1 genes was observed in both the Hep-G2 and HT29 cell lines. Conclusion: These findings indicate that the T. gondii peptide affected cancer cell mortality and led to changes in the expression of genes associated with apoptosis.

Inter-Laboratory Harmonization Study and Prospective Evaluation of the PURE-T. cruzi-LAMP Assay for Detecting Parasite DNA in Newborn's Dried Blood Spots.

Timely diagnosis of vertical Trypanosoma cruzi infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi-infected mothers are required to undergo serological testing after nine months, which risks loss to follow-up. Alternatively, the Loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA offers high analytical and clinical performance and easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultra-rapid DNA extraction method combined with the LAMP in dried blood spots (DBS) on FTA cards. The procedure has been implemented in ten public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruzi at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBS from 222 newborns at birth and/or two months, detecting T. cruzi microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with Real Time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.

Elucidation of the mechanisms of fluconazole resistance and repurposing treatment options against urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt.

BACKGROUND: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains. RESULTS: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models. CONCLUSIONS: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.

Development and application of a quantitative real-time PCR method for detection of Decapod iridescent virus 1.

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/µL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Comparison of toxin gene expression levels and molecular typing of Clostridioides difficile strains isolated from patients with diarrhea.

Aim: This study aimed to evaluate the expression of tcdA, tcdB, and binary toxin genes (cdtA and cdtB) by Real-Time PCR and molecular typing of Clostridioides difficile isolated from patient diarrhea samples from Hamadan Hospitals, west of Iran. Background: The concentration of C. difficile toxins (CDTs) is associated with the severity of the disease and the mortality rate. Measuring CDT levels could provide a reliable and objective means of determining the severity of C. difficile infection (CDI). Methods: From November 2018 to September 2019, 130 diarrhea samples were collected from hospitalized patients in three hospitals in Hamadan. C. difficle isolates were detected by culture and PCR. The presence of the genes encoding the toxin was identified by PCR, whereas the measurement of toxin expression was conducted using a relative Real-Time PCR technique. Genetic linkage of the isolates was also assessed by Ribotyping and Repetitive Extragenic Palindromic (rep-PCR) methods. Results: Among 130 diarrhea samples, 16 (12.3%) were positive for C. difficile. Genes encoding cdtA and tcdB were detected in all isolates, and 8 (50%) and 6 (37.5%) isolates were positive for the cdtA and cdtB genes. Real-time PCR results showed different expression levels of the toxin genes. A significant increase in the expression of the tcdA gene was observed compared with the control strain (P<0.05). Besides, more expression of cdtA gene was observed in the strains compared with cdtB gene. Ribotyping and rep-PCR results showed high genetic diversity of C. difficile among hospitals investigated. Conclusion: We encountered toxigenic C. difficile strains with various toxin expression levels, ribotypes, and rep types based on the findings of this study. This indicated that various clones from various sources circulate in the hospitals and among patients.

Analysis of the Main Checkpoints of the JNK-MAPK Pathway in HTLV-1-Associated Leukemia/Lymphoma via Boolean Network Simulation.

The c-Jun N-terminal kinase (JNK) pathway is a signal transduction pathway that plays a critical role in cell growth and survival. Its dysregulation is related to various cancers, including adult T-cell leukemia/lymphoma (ATLL), an aggressive peripheral T-cell malignancy caused by human T-cell lymphotropic virus type 1 (HTLV-1) infection. There is currently no vaccine or definitive treatment for ATLL. This research aimed to identify the JNK-MAPK pathway checkpoints to identify possible therapeutic targets using Boolean network analysis. First, the genes involved in the JNK pathway and their interactions were identified and mapped. Next, a Boolean network analysis was performed using the R programming language, which suggested protein kinase B (AKT) and MAP kinase phosphatase (MKP) for further evaluation. Finally, to confirm the effect of these two genes, a clinical study was conducted among ATLL patients and healthy individuals. The quantitative real time polymerase chain reaction (qRT‒PCR) results revealed a statistically significant decrease in the expression of AKT and MKP in ATLL patients compared to normal controls. This highlights the potential role of these two genes as potential therapeutic targets in ATLL.

Molecular detection of Coxiella burnetii in ticks infesting wild and domestic animals in the Eastern region of Punjab, Pakistan.

Tick-borne pathogens are significant for human, veterinary, and wildlife health. Coxiella burnetii is an example that is widely distributed across various hosts and can cross species boundaries. In Pakistan, there is a scarcity of data regarding C. burnetii at the intersection of wildlife and livestock. Ticks were collected from ruminants and wildlife from the districts of Kasur, Pakpattan, and Okara in Pakistan. Five tick species totaling 571 ticks were collected, with the following distribution: 56.4% Hyalomma anatolicum, 22.4% Rhipicephalus microplus, 10.5% Hyalomma marginatum, 7.9% Rhipicephalus sanguineus, and 2.8% Rhipicephalus turanicus. Fifty tick pools were screened for C. burnetii to amplify a segment of the IS1111 using real-time PCR assays. Ticks collected from sheep and goats had a greater rate of positivity for C. burnetii (40% and 38%, respectively) compared to Indian long-eared hedgehogs with a prevalence of 2%. Coxiella burnetii was prominent in Rhipicephalus microplus (92.3%) and Hyalomma anatolicum (88.9%), followed by Rhipicephalus turanicus (66.6%), Rhipicephalus sanguineus (33.3%), and Hyalomma marginatum (25.0%). Ticks from Pakpattan district displayed the highest prevalence of C. burnetii (88.9%), whereas the lowest was observed in ticks from Kasur district (77.3%). There was no significant association between tick gender and C. burnetii infection. Female host animals were more likely to harbor ticks containing C. burnetii, with a prevalence rate of 81.8%. The research underscores the urgent need for comprehensive studies on C. burnetii in Pakistan, especially at the interface of wildlife and livestock. The high prevalence rates observed in certain tick species and geographic regions emphasize the importance of targeted public health interventions. Future research should focus on elucidating the transmission dynamics and implementing effective control measures to mitigate the impact of these pathogens on human, veterinary, and wildlife health in the region.

Polyvinylpyrrolidone capped silver nanoparticles enhance the autophagic clearance of Acinetobacter baumannii from human pulmonary cells.

Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels.