Advancement in SARS-CoV-2 diagnosis: A new and stable electrochemical biosensor for genomic RNA detection.

Coronavirus disease (COVID-19) is caused by infection with the SARS-CoV-2 virus, having already caused more than seven million deaths worldwide. Conventional techniques for SARS-CoV-2 detection have limitations, as high cost, low specificity, and longer analysis time, among others. Biosensors emerge as a necessary alternative to overcome the difficulties of current diagnostics. This paper reports a sensor platform where silver-doped zinc oxide nanomaterial (Ag:ZnONp) was used onto carbon screen-printed electrode and ethidium bromide as indicator for development of a specific electrochemical genosensor for COVID-19. This genosensor demonstrated good linearity between the concentrations of 5.62 × 104-5.62 copies/mL and a detection limit of 5 copies/mL with gRNA in patient's samples, with a response time within 30 min. Molecular modeling and morphological analysis are in agreement with obtained electrochemical results. Additional techniques such as AFM, SEM, and EIS were conducted to characterize the morphological and electrochemical properties of the biosensor's surface. The biosensor was also capable of detecting the target presence in spiked samples and demonstrated a stability of 60 days, higher than other similar biosensors for SARS-CoV-2.

Clinical profile and parameters of patients monoinfected with HBV and infected with HDV in Western Amazon.

Background: Hepatitis Delta represents a greater risk in the progression of advanced liver disease and HCC compared with HBV. The exact mechanism that determines the spontaneous clearance of delta virus or its progression to cirrhosis remains unknown. Therefore, this study aimed to analyze the clinical profile of HBV and HBV/HDV individuals in the Western Amazon. Methods: The study was carried out at the Specialized Outpatient Clinic for Viral Hepatitis belonging to the Centro de Pesquisa em Medicina Tropical de Rondônia/CEPEM. 100 individuals were included, stratified into two groups: 50 with hepatitis B virus and 50 with hepatitis Delta virus. Results: The overall mean age was 48 years. For the HBV and HDV groups, 66 % (33/50) and 54 % (27/50) were men and 56 % (28/50) and 58 % (29/50) were on antiviral treatment, respectively. Patients with detectable HDV-RNA demonstrated high levels of ALT and AST compared to individuals with undetectable HDV-RNA. Comparative analysis between HBV carriers and infected with HDV shows significant differences in terms of age, HBV-DNA levels, albumin, hepatomegaly and splenomegaly. Conclusion: Several markers were important for differentiating HBV and HDV infections. HDV-RNA detectable showed significant changes in biomarkers compared to undetectable patients, suggesting a possible worse prognostic effect in this group.

DENV-2 Outbreak Associated With Cosmopolitan Genotype Emergence in Western Brazilian Amazon.

Background: Dengue virus (DENV) causes an important disease and directly affects public health, being the arbovirus that presents the highest number of infections and deaths in the Western Brazilian Amazon. This virus is divided into 4 serotypes that have already circulated in the region. Methodology: Molecular characterization of a cohort containing 841 samples collected from febrile patients between 2021 and 2023 was analyzed using a commercial kit to detect the main arboviruses circulating in Brazil: Zika, DENV-1, DENV-2, DENV-3, DENV-4 and, Chikungunya. Subsequently, Sanger sequencing was performed for positive samples. Results: The cohort detected 162 positive samples, 12 for DENV-1 and 150 identified as DENV-2, indicating co-circulation of serotypes. The samples were subjected to sequencing and the analysis of the sequences that obtained good quality revealed that 5 samples belonged to the V genotype of DENV-1 and 46 were characterized as DENV-2 Cosmopolitan genotype-lineage 5. Conclusion: The results allowed us to identify for the first time the Cosmopolitan genotype in Rondônia, Brazilian Western Amazon, and its fast spread dispersion.

SARS-CoV-2 Spike Protein Mutations in Different Variants: A Comparison Between Vaccinated and Unvaccinated Population in Western Amazonia.

The increased transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated variants of concern (VOCs) throughout the pandemic, responsible for waves of cases worldwide. To monitor mutations in the S gene of SARS-CoV-2 in different variants, we evaluated 1497 individuals with COVID-19 in western Amazonia in the period April 2021 to July 2022. The epidemiological and clinical data of the individuals were collected; subsequently, the samples were extracted using a commercial kit, the viral load was assessed, and viral genomes were sequenced. We analyzed the quality and mutations of the genomes and maximum likelihood phylogenetic inference. However, 3 main clusters were observed, referring to Gamma (52.91%), Delta (24.38%), and Omicron (20.38%) VOCs with wide distribution in all health regions of the Rondônia state. Regarding the vaccination profile, there was a higher percentage of unvaccinated and partially vaccinated individuals, with more representatives by the Gamma variant. A total of 1412 sequences were suitable for mutation analysis in the S gene region. The Omicron VOC showed 38 mutations, with the Delta and Gamma variants with 16 and 17, respectively. The VOC Omicron and Gamma shared 4 mutations E484K, H655Y, N501Y, and N679K with high frequency, and Delta and Omicron 2 mutations (T478K and T95I). Regarding the comparison between the frequency of mutations for each variant concerning the vaccination groups, there were no changes in mutations for each group. In conclusion, the study showed a temporal increase in mutations and subvariants for characterized strains. Furthermore, the vaccination profile did not impact significant changes in the mutational profile yet remains a determining factor for severe disease.

Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus.

Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017 and 2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was - 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the diagnostic sensitivity tests, there was an accuracy of 97.37% when compared to the reference test. This assay proved to be highly efficient and reproducible, making it a valuable tool to monitor hepatitis Delta patients and assess the risk of disease progression, as well as the effectiveness of treatment.

Clinical and epidemiological aspects of Delta and Gamma SARS-CoV-2 variant of concern from the western Brazilian Amazon.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has become a major concern contributing to increased morbidity and mortality worldwide. OBJECTIVES: Here we describe the replacement of the Gamma variant of concern (VOC) with Delta in the western Brazilian Amazon. METHODS: In this study, we analysed 540 SARS-CoV-2 positive samples determined by qualitative real-time RT-PCR selected in the state of Rondônia between June and December 2021. The positive cohort was sequenced through next-generation sequencing (NGS) and each sample was quantified using real-time RT-qPCR, the whole genome sequence was obtained, SARS-CoV-2 lineages were classified using the system Pango and the maximum likelihood (ML) method was used to conduct phylogenetic analyses. FINDINGS: A total of 540 high-quality genomes were obtained, where the Delta VOC showed the highest prevalence making up 72%, with strain AY.43 being the most abundant, while the Gamma VOC was present in 28%, where the P.1 strain was the most frequent. In this study population, only 32.96% (178/540) had completed the vaccination schedule. MAIN CONCLUSIONS: This study highlighted the presence of Gamma and Delta variants of SARS-CoV-2 in RO. Furthermore, we observed the replacement of the Gamma VOC with the Delta VOC and its lineages.

Genomic Variability of Hepatitis B Virus Circulating in Brazilian Western Amazon.

The emergence of clinically relevant mutations in the hepatitis B virus (HBV) genome has been a matter of great debate because of the possibility of escape from the host's immune system, the potential to cause more severe progression of liver diseases and the emergence of treatment-resistant variants. Here we characterized the circulating variants of HBV in Rondônia State, in the north of Brazil. Serum samples of 62 chronic HBV carriers were subjected to PCR assays and clinical data were collected. Mutations and genotypes were characterized through direct sequencing. The findings show the presence of subgenotypes A1 (54.83%, 34/62), D3 (16.13%, 10/62), F2 (16.13%, 10/62), A2 (4.84%, 3/62), D2 (3.23%, 2/62), D1 (1.61%, 1/62), D4 (1.61%, 1/62) and F4 (1.61%, 1/62). Deletions in the pre-S2 region were found in 13.79% (8/58) of the samples, mutations in the S gene in 59.68% (37/62) and RT mutations in 48.39% (30/62). We found a variable genotypic distribution in different locations and important mutations related to immune escape and drug resistance in Western Amazonia, which contributed to genetic surveillance and provided important information to help control the disease.

Genetic diversity of HBV in indigenous populations on the border between Brazil and Bolivia.

Hepatitis B is considered an important public health problem worldwide because it is a chronic infection with a risk factor for cirrhosis and cellular hepatocellular carcinoma. In Brazil, the Rondônia State ranks first in the Northern region regarding the number of deaths due to hepatitis B. In the Amazon basin, genotype F is considered specific to the Americas identified in native populations. But few data on HBV genotyping and phylogenetic analysis are available. The objective of this study was to evaluate the genotypes and subgenotypes of the hepatitis B virus in indigenous people chronic carriers residing in cities of Guajará Mirim and Nova Mamoré in state of Rondônia/Brazil, on the border with Bolivia. A fragment of 417 bp (S gene) was amplified by PCR and submitted to nucleotide sequencing. The genotypes and subgenotypes of the HBV strains were determined through phylogenetic inference using genomic sequences from 197 representatives of the genotypes (A-H). Of the 41 chronic hepatitis B patients enrolled in this study, 27 were HBV-DNA positive. Of the 27 DNA-HBV positives, 39% (17/41) had individual HBV infection and 27% (10/41) were coinfected with HDV. The frequency of genotypes was 40.7% (11/27) for genotype D (HBV-D), 33.3% (9/27) for genotype F (HBV-F) and 25.9% (7/27) for genotype A (HBV-A) with circulating subgenotypes F2, F4, D2, D3, A1, and A2. We characterized the genotypes and subgenotypes of HBV circulating among in indigenous in the State of Rondônia shows for the first time the HBV/D genotype whit greater frequency circulating in nativos of state Rondônia. In conclusion, our findings showed a diversity of HBV genotypes, which is also found in other Brazilian geographical regions.

Clinical and epidemiological aspects of Delta and Gamma SARS-CoV-2 variant of concern from the western Brazilian Amazon

BACKGROUND The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has become a major concern contributing to increased morbidity and mortality worldwide. OBJECTIVES Here we describe the replacement of the Gamma variant of concern (VOC) with Delta in the western Brazilian Amazon. METHODS In this study, we analysed 540 SARS-CoV-2 positive samples determined by qualitative real-time RT-PCR selected in the state of Rondônia between June and December 2021. The positive cohort was sequenced through next-generation sequencing (NGS) and each sample was quantified using real-time RT-qPCR, the whole genome sequence was obtained, SARS-CoV-2 lineages were classified using the system Pango and the maximum likelihood (ML) method was used to conduct phylogenetic analyses. FINDINGS A total of 540 high-quality genomes were obtained, where the Delta VOC showed the highest prevalence making up 72%, with strain AY.43 being the most abundant, while the Gamma VOC was present in 28%, where the P.1 strain was the most frequent. In this study population, only 32.96% (178/540) had completed the vaccination schedule. MAIN CONCLUSIONS This study highlighted the presence of Gamma and Delta variants of SARS-CoV-2 in RO. Furthermore, we observed the replacement of the Gamma VOC with the Delta VOC and its lineages.

Genetic diversity of HBV in indigenous populations on the border between Brazil and Bolivia

Abstract Hepatitis B is considered an important public health problem worldwide because it is a chronic infection with a risk factor for cirrhosis and cellular hepatocellular carcinoma. In Brazil, the Rondônia State ranks first in the Northern region regarding the number of deaths due to hepatitis B. In the Amazon basin, genotype F is considered specific to the Americas identified in native populations. But few data on HBV genotyping and phylogenetic analysis are available. The objective of this study was to evaluate the genotypes and subgenotypes of the hepatitis B virus in indigenous people chronic carriers residing in cities of Guajará Mirim and Nova Mamoré in state of Rondônia/Brazil, on the border with Bolivia. A fragment of 417 bp (S gene) was amplified by PCR and submitted to nucleotide sequencing. The genotypes and subgenotypes of the HBV strains were determined through phylogenetic inference using genomic sequences from 197 representatives of the genotypes (A-H). Of the 41 chronic hepatitis B patients enrolled in this study, 27 were HBV-DNA positive. Of the 27 DNA-HBV positives, 39% (17/41) had individual HBV infection and 27% (10/41) were coinfected with HDV. The frequency of genotypes was 40.7% (11/27) for genotype D (HBV-D), 33.3% (9/27) for genotype F (HBV-F) and 25.9% (7/27) for genotype A (HBV-A) with circulating subgenotypes F2, F4, D2, D3, A1, and A2. We characterized the genotypes and subgenotypes of HBV circulating among in indigenous in the State of Rondônia shows for the first time the HBV/D genotype whit greater frequency circulating in nativos of state Rondônia. In conclusion, our findings showed a diversity of HBV genotypes, which is also found in other Brazilian geographical regions.

SARS-CoV-2 genomic surveillance in Rondônia, Brazilian Western Amazon.

SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of the pandemic. The Northern region has the second highest incidence coefficient, as well as the third highest mortality rate in the country. This study aimed to investigate information about the evolutionary history of epidemic spread and genetic aspects of strains isolated on the Western Amazon, in the State of Rondônia, Brazil. It was possible to detect a total of 22 mutations. Some of these alterations may possibly be related to effects on transmissibility, the fidelity of RNA replication, the ability of cancer patients to respond to infection, beyond a mutation that emerged after the introduction of SARS-CoV-2 in Rondônia. At least two events of introduction were detected, corresponding to the B.1 and B.1.1 European lineages. An introduction was observed possibly through Argentina, where strains originated that circulated in the Minas Gerais and Ceará Brazilian states, prior to Rondônia (B.1.), as well as through the Minas Gerais state and the Federal District, which gave rise to strains that spread to Rondônia, from the capital to more rural parts of the state (B.1.1.). The findings show the need to monitor the genetic epidemiology of COVID-19, in order to surveil the virus's evolution, dispersion and diversity.

Phylodynamic and Phylogeographic Analysis of Hepatitis Delta Virus Genotype 3 Isolated in South America.

The hepatitis delta virus (HDV) is a globally distributed agent, and its genetic variability allows for it to be organized into eight genotypes with different geographic distributions. In South America, genotype 3 (HDV-3) is frequently isolated and responsible for the most severe form of infection. The objective of this study was to evaluate the evolutionary and epidemiological dynamics of HDV-3 over the years and to describe its distribution throughout this continent in an evolutionary perspective. While using Bayesian analysis, with strains being deposited in the Nucleotide database, the most recent common ancestor was dated back to 1964 and phylogenetic analysis indicated that the dispersion may have started in Brazil, spreading to Venezuela and then to Colombia, respectively. Exponential growth in the effective number of infections was observed between the 1950s and 1970s, years after the first report of the presence of HDV on the continent, during the Labrea Black Fever outbreak, which showed that the virus continued to spread, increasing the number of cases decades after the first reports. Subsequently, the analysis showed a decrease in the epidemiological levels of HDV, which was probably due to the implantation of the vaccine against its helper virus, hepatitis B virus, and serological screening methods implemented in the blood banks.