Molecular genetic association of rs8099917 and rs1800795 polymorphisms in the progression of hepatitis Delta virus liver disease.

Background: The relationship between viral infections and host factors holds high hopes for identifying the role of Interferon Lambda 3 (IFNL3) and Interleukin 6 (IL-6) polymorphisms in the development of Chronic Liver Disease (CLD) in patients infected with hepatitis Delta virus (HDV) in the Western Brazilian Amazon. Methods: Cross-sectional study conducted with a cohort of 40 chronic HDV patients, 27 with CLD and 13 without evident liver damage. Biological samples from the participants were analyzed using the polymerase chain reaction (PCR) technique, followed by sequencing by the automated Sanger method. Results: The rs8099917 T allele, from the IFNL3 gene, showed a higher frequency in both groups; however, it was not possible to establish an association with HDV infection [OR = 1.42 (0.42 - 4.75; p = 0.556 (95% CI). For IL-6, the rs1800795 G allele was superior to rs1800795 C. Analyzing both distributions in the studied groups, any association with HDV was absent (p > 0.05). Conclusion: The results suggest that the rs8099917 T/G (IFNL3) and rs1800795 G/C (IL-6) polymorphisms are not associated with the evolution of HDV in the studied population.

Molecular genetic association of rs8099917 and rs1800795 polymorphisms in the progression of hepatitis Delta virus liver disease

Background: The relationship between viral infections and host factors holds high hopes for identifying the role of Interferon Lambda 3 (IFNL3) and Interleukin 6 (IL-6) polymorphisms in the development of Chronic Liver Disease (CLD) in patients infected with hepatitis Delta virus (HDV) in the Western Brazilian Amazon. Methods: Cross-sectional study conducted with a cohort of 40 chronic HDV patients, 27 with CLD and 13 without evident liver damage. Biological samples from the participants were analyzed using the polymerase chain reaction (PCR) technique, followed by sequencing by the automated Sanger method. Results: The rs8099917 T allele, from the IFNL3 gene, showed a higher frequency in both groups; however, it was not possible to establish an association with HDV infection [OR = 1.42 (0.42 - 4.75; p = 0.556 (95% CI). For IL-6, the rs1800795 G allele was superior to rs1800795 C. Analyzing both distributions in the studied groups, any association with HDV was absent (p > 0.05). Conclusion: The results suggest that the rs8099917 T/G (IFNL3) and rs1800795 G/C (IL-6) polymorphisms are not associated with the evolution of HDV in the studied population.

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.

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.

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.

Screening for Severe Acute Respiratory Syndrome Coronavirus 2 in Close Contacts of Individuals With Confirmed Infection: Performance and Operational Considerations.

BACKGROUND: Point-of-care and decentralized testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies. METHODS: A prospective diagnostic accuracy study was conducted among close contacts of coronavirus disease 2019 (COVID-19) cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and 1 molecular method were evaluated for usability and performance against reference reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swab specimens. RESULTS: Fifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during ≥1 visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta variants emerged increasingly during implementation. The overall sensitivity of evaluated tests ranged from 33% to 76%. Performance was higher among symptomatic cases and those with cycle threshold (Ct) values <34 and lower among oligosymptomatic or asymptomatic cases. Assuming a 24-hour time to results for RT-PCR, the cumulative sensitivity of an anterior nares swab rapid antigen test was >70% and almost 90% after 4 days. CONCLUSIONS: The near-immediate time to results for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

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.

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.

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.

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil.

Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list.

Hepatitis delta: virological and clinical aspects.

There are an estimated 400 million chronic carriers of HBV worldwide; between 15 and 20 million have serological evidence of exposure to HDV. Traditionally, regions with high rates of endemicity are central and northern Africa, the Amazon Basin, eastern Europe and the Mediterranean, the Middle East and parts of Asia. There are two types of HDV/HBV infection which are differentiated by the previous status infection by HBV for the individual. Individuals with acute HBV infection contaminated by HDV is an HDV/HBV co-infection, while individuals with chronic HBV infection contaminated by HDV represent an HDV/HBV super-infection. The appropriate treatment for chronic hepatitis delta is still widely discussed since it does not have an effective drug. Alpha interferon is currently the only licensed therapy for the treatment of chronic hepatitis D. The most widely used drug is pegylated interferon but only approximately 25% of patients maintain a sustained viral response after 1 year of treatment. The best marker of therapeutic success would be the clearance of HBsAg, but this data is rare in clinical practice. Therefore, the best way to predict a sustained virologic response is the maintenance of undetectable HDV RNA levels.

Correlation between TH1 response standard cytokines as biomarkers in patients with the delta virus in the western Brazilian Amazon.

Hepatitis D virus (HDV) is endemic in the Amazon Region and its pathophysiology is the most severe among viral hepatitis. Treatment is performed with pegylated interferon and the immune response appears to be important for infection control. HDV patients were studied: untreated and polymerase chain reaction (PCR) positive (n = 9), anti-HDV positive and PCR negative (n = 8), and responders to treatment (n = 12). The cytokines, interleukin (IL)-2 (p = 0.0008) and IL-12 (p = 0.02) were differentially expressed among the groups and were also correlated (p = 0.0143). Future studies will be conducted with patients at different stages of treatment, associating the viral load with serum cytokines produced, thereby attempting to establish a prognostic indicator of the infection.

Treatment of hepatitis delta virus genotype 3 infection with peg-interferon and entecavir.

OBJECTIVES: Hepatitis delta virus (HDV) is recognized as the most pathogenic and infectious among the hepatotropic viruses. Studies on the treatment of HDV have predominantly included European patients and carriers of genotype 1 (HDV-1) in their clinical protocols. For the Amazon region, data show that infected individuals have mainly Native American ancestry and that >90% of HDV carriers have the genotype 3 (HDV-3). Thus combined therapy clinical protocols do not adequately address the treatment of these patients. METHODS: A prospective, non-randomized study was conducted in which 22 patients received 180µg of pegylated interferon alpha 2a (PEG-IFN) plus entecavir at a dose of 0.5mg for 48 weeks, with a subsequent 24-week follow-up. Throughout treatment, the patients were monitored for biochemical responses and the kinetics of hepatitis B virus (HBV) and HDV viral loads. RESULTS: Of the 22 patients treated, 15 presented normal alanine aminotransferase values at the end of treatment (p=0.002). At week 24 of treatment, 86.4% of the patients did not present detectable HDV-RNA; at week 48, the rate of negative patients increased to >95% and remained the same after 6 months. With regard to HBV, only two patients (9%) still presented detectable HBV genetic material at the end of treatment, suggesting the effectiveness of combined therapy in combating the two viruses. CONCLUSIONS: These findings support the use of this effective therapeutic protocol for HDV-3 in patients of non-European ethnicity and suggest a possible 'easy to treat' variant when compared to HDV-1.

Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients.

Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71.1% (n = 27) mucosal lesions were positive for LRV1, and 28.9% (n = 11) were negative. In cutaneous lesions, 36.7% (n = 40) were positive and 63.3% (n = 69) were negative for LRV1. The ratio P(ML|LRV1+)/P(ML|LRV1-) was 2.93 (CI95% 1.57...5.46; p<0.001), thus corroborating the hypothesis of the association between LRV1 and the occurrence of mucosal leishmaniasis, as previously described in animal models; it also indicates that LRV1 is not the only factor contributing to the disease outcome.

Alta soroprevalência de infecção pelos vírus das hepatites B e C na região do alto rio Madeira, Porto Velho, Rondônia, Brasil / High soroprevalence of hepatitis B and C markers in the upper Madeira river region, Porto Velho, Rondônia State, Brazil

Foi realizado um estudo de soroprevalência de marcadores sorológicos de hepatites B e C na população residente no alto rio Madeira, entre as localidades de Santo Antônio e Abunã, no Município de Porto Velho, Rondônia, local previsto para ser inundado pelas novas hidrelétricas do Madeira. A população local foi estimada em 5 mil pessoas, segundo o censo do Instituto Brasileiro de Geografia e Estatística, e uma amostra populacional de 10 por cento foi selecionada de modo aleatório. Coletaram-se 5 mL de sangue periférico por punção venosa em tubo seco e o soro foi conservado em freezer a -20° C. Os exames sorológicos tipo ELISA foram realizados seguindo a metodologia do fabricante, para os seguintes marcadores virais: Anti-HBc Total, HBsAg, Anti-HBs e Anti-HCV. Foram processadas 431 amostras, das quais foram obtidos os seguintes resultados: 192 positivas para Anti-HBc Total (44,5 por cento), 29 positivas para HBsAg (6,7 por cento), 230 positivas para Anti-HBsAg (53,4 por cento), 32 positivas para Anti-HCV (7,4 por cento). Concluímos que a região estudada estaria classificada, segundo a Organização Mundial da Saúde, como de prevalência intermediária para hepatite B, e alta para hepatite C. Se considerarmos a alta prevalência de pessoas imunes contra hepatite B (superior a 50 por cento), podemos concluir que, nas próximas décadas, o problema de saúde pública relacionado com a hepatite B tenderá a diminuir. A migração de milhares de novos habitantes para a região sem a devida atenção das autoridades sanitárias para prevenção, vacinação e educação em saúde da população, pode agravar a situação na região em relação a estas hepatites virais...

A seroprevalence study on the serologic markers of hepatitis types B and C on the inhabitants of the upper Madeira river, between the localities of Santo Antonio and Abunã, in the Municipality of Porto Velho, Rondônia State, was conducted. This locality will be flooded by two new hydropower plants yet to be built in the Madeira river. Local population was estimated in 5 thousand individuals, according to Instituto Brasileiro de Geografia e Estatística's (Brazilian Institute of Geography and Statistics) census. A sample of 10 per cent of the population was randomly selected. Five milliliters of peripheral venous blood were collected in Vacutainer® dry tubes, and the serum samples were maintained in a freezer at -20° C. ELISA serological tests (DiaSorin, Inc.) were performed according to the manufacturer's methodology for the following viral markers: total Anti-HBc, HBsAg, Anti-HBs and Anti-HCV After the processing of 431 samples, the results were: 192 (44.5 per cent) were positive for total Anti-HBc, 29 (6.7 per cent) were positive for HBsAg, 230 (53.4 per cent) were positive for Anti-HBs, and 32 (7.4 per cent) were positive for Anti-HCV We concluded that this region presented an intermediate and a high prevalence rate for hepatitis B and C, respectively, according to the World Health Organization. The high prevalence (more than 50 per cent) of individuals immune to hepatitis B leads us to the conclusion that in the next decades problems related to that type of hepatitis tend to decrease, whereas the incidence of hepatitis C will probably increase. Migration of thousands of new inhabitants drawn by the implementation of the new hydropower plants in this region has the potential of worsening the public health issues related to these viral hepatitis infections...