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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 2017–2023 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 97.44% 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.

COVID-19 may be more severe in elder people and/or people presenting comorbidities, possibly leading to death. Therefore, we analyzed the profile of deaths caused by SARS-CoV-2 in part of the Amazon region, associating them to risk factors. 467 cases of death caused by COVID-19 from October 2020 to July 2021 were analyzed and correlated to age group, gender, comorbidities, and other risk factors, using the Chi-squared Test or Fisher's Exact Test as necessary. Deaths occurred in the age group between 17 and 98 years, with a predominance of men (57.4%) and higher concentration in the period between March and April 2021. Systemic arterial hypertension was the most prevalent disease, followed by smoking, cardiovascular disease, and diabetes mellitus. Smoker men and obese women (and/or with cardiovascular disease) presented higher chances to die, as well as obese people under 65 years and people over 65 years with cardiovascular disease, smokers, or hypertensive (p

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.

The relationship between N-antigen concentration and viral load within a specimen and across different specimens is essential for interpretation of rapid diagnostic tests (RDT) clinical performance in different use cases. A prospective study was conducted in Porto Velho, Brazil, to investigate RDT performance in different specimen types as a function of the correlation between antigen concentration and viral load. The study included 214 close contacts with recent exposures to confirmed cases, aged 12 years and older and with various levels of vaccination. Antigen concentration was measured in nasopharyngeal swab (NPS), anterior nares swab (ANS), and saliva specimens. Reverse transcriptase (RT)–PCR was conducted on the NPS and saliva specimens, and two RDTs were conducted on ANS and one on saliva. Antigen concentration correlated with viral load when measured in the same specimen type but not across specimen types. Antigen levels were higher in symptomatic cases compared to asymptomatic/oligosymptomatic cases and lower in saliva compared to NPS and ANS samples. Discordant results between the RDTs conducted on ANS and the RT-PCR on NPS were resolved by antigen concentration values. The analytical limit-of-detection of RDTs can be used to predict the performance of the tests in populations for which the antigen concentration is known. The antigen dynamics across different sample types observed in SARS-CoV-2 disease progression support use of RDTs in nasal samples. Given lower antigen concentrations in saliva, tests using saliva is expected to require improved analytical sensitivity to achieve clinical sensitivity similar to testing of nasal samples.

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.

Background and Aims: Hepatitis Delta virus (HDV) genotype 3 is responsible for outbreaks of fulminant hepatitis in Northeastern South America. This study investigates if systemic inflammatory molecules are differentially expressed in patients with advanced fibrosis chronically infected with Hepatitis Delta virusgenotype 3(HDV-3). Methods: Sixty-one patients from the north of Brazil coinfected with hepatitis B virus (HBV)/HDV-3 were analyzed. HDV quantification and genotyping were performed by semi-nested real-time polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) methodologies. Ninety-two systemic inflammatory molecules (SIMs) were measured by Proximity Extension Assay (PEA) technology. The Shapiro–Wilk, Student’s t-test, Mann–Whitney tests, and logistic regression analysis were used when appropriate. Results: The median age was 41 years, and all patients were HBeAg negative. Advanced fibrosis or cirrhosis was diagnosed by histological staging in 17 patients, while 44 presented with minimal or no fibrosis. Advanced necroinflammatory activity correlated positively with serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Established non-invasive fibrosis scores (APRI, FIB-4, and AST/ALT ratio) revealed low sensitivities and positive predictive values (PPVs) with an AUROC maximum of 0.586. Among the 92 SIMs analyzed, MCP.4, CCL19, EN.RAGE, SCF, and IL18 showed a positive correlation with fibrosis stage. A combined score including CCL19 and MCP.4 revealed a sensitivity of 81% and an odds ratio of 2.202 for advanced fibrosis. Conclusions: Standard non-invasive fibrosis scores showed poor performance in HDV-3 infection. We here suggest that the determination of CCL19 and MCP.4 may be used to identify patients with advanced fibrosis. Moreover, this study gives novel insights into the immunopathogenesis of HDV-3 infection.

Highlights • SARS-CoV-2 Quantification. • Multiplex one-step reaction. • High-sensitivity and specificity assay., 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.

Genomic surveillance represents an important strategy for understanding evolutionary mechanisms, transmission profile, and infectivity of different SARS-CoV-2 variants. We assessed the epidemiological profile of 366 individuals who tested positive for SARS-CoV-2 from 29 municipalities in Rondônia between December 2021 to March 2022. Samples were collected, RNA was ex-tracted and screened using RT-qPCR for Alpha, Beta, Gamma, Delta and Omicron VOCs and viral quantification was performed. Sequences were analyzed for phylogeny, mutations and lineages. Of the samples analyzed, 93.71% were positive for the Omicron variant and 6.28% were positive for the Delta variant. The symptoms observed were cough, sore throat, and fever, with a mean duration of 5 days; no hospitalizations or deaths were reported. We noted that among the positive individuals, 51% had been immunized with two doses, 22% received three doses, 13% received one dose, and 13% were not immunized. Just 242 samples were amenable to analysis for alignment and phylogenetic characterization; corresponding to variants BA.1 and BA.1.1; a total of 120 mutations were identified, 36% of which were found in the S gene. In conclusion, there was a high frequency of mutations in the SARS-CoV-2 genome, but no record of clinical severity, demonstrating the positive effect of vaccination.

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 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.

BackgroundPoint-of-care and decentralized testing for 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.MethodsA prospective diagnostic accuracy study was conducted among close contacts of 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 one molecular method were evaluated for usability and performance against reference RT-PCR on NPS.ResultsFifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during at least one visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta emerged increasingly during implementation. Overall sensitivity of evaluated tests ranged from 33%–76%. Performance was higher among symptomatic cases and cases with Ct70% and almost 90% after four days.ConclusionsThe near immediate time-to-result for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

The treatment for hepatitis Delta virus (HDV) still consists of Pegylated interferon (PEG-IFN) combined with inhibitors of Hepatitis B virus (HBV) replication. In some patients may be occur a virological response, which means a negative HDV RNA 6 months after stopping treatment. In this study it was conducted an in vitro approach with the aim to mimic possible immunological events that are observed in patients responding to PEG-IFN therapy. Jurkat cells (human T lymphocyte cell line) were employed alone or co-cultured with THP-1 (human monocytic cell line) and stimulated with controls and HBV Surface Antigen (HBsAg), Small-Delta Antigen (SHDAg), and HBsAg + SHDAg combined. Twenty-four hours stimulation with SHDAg and/or HBSAg led to a toxic profile in a co-culture condition and cell supernatants were collected for cytokines quantification. PEG-IFN was added and cells were incubated for additional 24 h. Co-cultured cells incubated with the association (SHDAg + PEG-IFN) significantly produced levels of IFN-γ, IL-2 and IL-12. On the other hand, the HBsAg alone was able to inhibit the production of IFN-γ, suggesting that this antigen may hinder the treatment exclusively with PEG-IFN.

Hepatitis D virus (H DV) genotype III is endemic in the western Amazon basin and is considered to cause the most severe form of chronic viral hepatitis. Recently, noninvasive fibrosis scores to determine the stage of liver fibrosis have been evaluated in individuals positive for HDV genotype I, but their utility in HDV genotype III-positive patients is unknown. In this retrospective study conducted in an outpatient viral hepatitis referral clinic in the Brazilian Amazon region, the aspartate aminotransferase (AST) to Aspartate aminotransferase to Platelet Ratio Index (APRI) and Fibrosis Index for Liver Fibrosis (FIB-4) values were calculated and compared with histological fibrosis stages. Among the 50 patients analyzed, the median age at liver biopsy was 35.6 years, 66% were male, and all had compensated liver disease. Histological staging revealed fibrosis stages 0, 1, 2, 3, and 4 in four (8%), eight (16%), 11 (22), 11 (22%), and 16 (32%) patients, respectively. The area under the receiver operating curve (AUROC) of AST-to-alanine aminotransferase (ALT) ratio, APRI, and FIB-4 for detection of significant fibrosis (F >= 2) was 0.550 (P = 0.601), 0.853 (P < 0.001), and 0.853 (P < 0.0001), respectively. Lower AUROC values were obtained for cirrhosis: the AST-to-ALT ratio was 0.640 (P = 0.114), APRI was 0.671 (P = 0.053), and FIB-4 was 0.701 (P = 0.023). The optimal cutoff value for significant fibrosis for APRI was 0.708 (sensitivity 84% and specificity 92%) and for FIB-4 was 1.36 (sensitivity 76% and specificity 92%). Aspartate amino-transferase to Platelet Ratio Index and FIB-4 were less useful to predict cirrhosis. In contrast to recent reports from Europe and North America, both APRI and FIB-4 may identify significant fibrosis in HDV-III-infected patients from northwestern Brazil.

Introduction: 500,000 to 1.2 million deaths are speculated annually from complications of hepatitis B. The hepatitis Delta virus (HDV) also represents an important public health problem in endemic areas. Objective: To analyze the clinical and laboratory characteristics at the first consultation of HBV and HBV/HDV patients. Methods: Retrospective study (2017 and 2018) of 324 records of HBV and HBV/HDV patients at Research Center for Tropical Medicine of Rondônia. Project approved by the Research Ethics Committee. For statistical analysis, SPSS® version 25.0. Results: A total of 324 patients were included, 302 (93.2%) were HBV and 22 were (6.7%) HBV/HDV. At the first consultation, 16.2% of the HBV showed signs of chronic liver disease, while in the HBV/HDV patients, 59.1% (p

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.

Acute Respiratory Infections (ARIs) are classified according to the compromised anatomical site. The etiology can be associated with viruses, bacteria and fungi. The etiological agents that are responsible for the highest incidence in children, around 50 to 90% of occurrences, are viruses. This study aims to characterize the factors that contribute to acute respiratory infection in children from 0-6 years old and the main symptoms that are presented, thus classifying the etiological agents. This is a descriptive quantitative study carried out by the molecular virology laboratory of the Research Center in Tropical Medicine-CEPEM/RO and the Oswaldo Cruz Foundation Rondonia-FIOCRUZ/RO. Data collection was performed through a questionnaire with 660 patients of both sexes, with questions about socioeconomic data and clinical manifestations, from February to December 2013. Prevalent clinical manifestations were cough, coryza, pulmonary secretion, fever, nasal obstruction, otalgia, adventitious sounds, apnea, dyspnoea and ocular pruritus of the 113 children diagnosed with acute respiratory infection, the highest incidence was in males. The proposed pathogens in the study were rhinovirus, parainfluenza 1, 2, 3, and adenovirus. Parainfluenza 2 and rhinovirus are the most prevalent.

It was conducted an observational study in two villages in Porto Velho, Rondonia State, Brazil, in order to evaluate the seroepidemiological profile of hepatitis B virus (HBV) and the risk factors in its transmission. Six hundred sixty samples were analyzed by ELISA method for serological tests for HBV (anti-HBc total, anti-HBs and HBsAg). The results showed that 50.7% (335) of samples were positive for some kind of serological marker. HBsAg was positive in 12 (1.8%) samples, 11 (91.6%) were from male ones. Anti-HBc total was positive in 212 (32.1%) samples distributed in all groups, except for the range of 11 to 16 years old. Anti-HBs was positive in 239 (36.2%) samples with the highest prevalence in groups over 21 years old. This study showed that the analyzed locations have low endemicity for chronic HBV carriers, however, there was a high prevalence of infection. The percentage of susceptible individuals was 49.2% (325), the highest was in individuals between 6 to 15 years old. Aspects of transmission such as dental extractions, surgeries and sharing personal hygiene materials are possibly associated with HBV infection in the analyzed region. Furthermore, the low percentage of anti-HBs marker indicates an immunization coverage failure in the population of this study.

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.

The hepatitis delta virus (HDV) is a pathogen that causes a severe and rapidly progressive disease of hepatocytes. The measurement of viral load in the peripheral blood of patients with HDV infections is important for diagnosis, treatment monitoring, and support for follow-up studies of viral replication during the course of the disease. This study reports the development of an assay capable of detecting and quantifying the abundance of HDV particles in serum samples, based on reverse-transcription quantitative PCR (RT-qPCR). Two standards for calibration were produced for determining the viral load of HDV: a cDNA cloned into a linear plasmid and a transcribed RNA. For validating this assay, 140 clinical samples of sera were used, comprising 100 samples from patients who tested positive for anti-HDV and hepatitis B virus surface antigen (HBsAg) by ELISA; 30 samples from blood donors; 5 samples monoinfected with hepatitis B virus (HBV); and 5 samples monoinfected with hepatitis C virus (HCV). The HDV RT-qPCR assay performed better when calibrated using the standard based on HDV cDNA cloned into a linear plasmid, yielding an efficiency of 99.8% and a specificity of 100% in the in vitro assays. This study represents the first HDV RT-qPCR assay developed with clinical samples from Brazil and offers great potential for new clinical efficacy studies of antiviral therapeutics for use in patients with hepatitis delta in the western Amazon region.

The Amazon region is considered to be a high endemic area for Hepatitis B Virus (HBV) infections, Rondonia state having the highest prevalence. The aim of this study was to identify molecular genotypes and mutations in the S gene region of HBV viral genomes from 20 patients using a DNA microarray. Results: Serological tests showed that 88% of patients were HBeAg negative, 82% had anti-HBe antibodies and 33% were co-infected with Hepatitis Delta Virus. Sixteen percent of the patients were considered cirrhotic, and 11% have been transfused. The microarray technique identified the genotypes A (4 patients), D (7 patients) and F (7 patients) in 18 samples. Mutations were detected in all 3 genotypes and, overall, A159G, which has been associated with a reduced antigenicity of the virus, was detected most frequently. In genotype A, G119E was the most frequently detected mutation followed by mutations A159G, F134Y, W172C, Y161F and T143S. A159G was detected in all genotype D and F samples followed by mutations T143S, Y161F, N131T, T114S and G119E in genotype D and mutations T143S, Y161F, N131T, T114S and G119E in genotype F. Conclusion: The analysis of mutations repartition among genotypes suggests that some of them are preferentially or exclusively associated with genotype A, D or F. This type of tool is adapted for clinical and therapy monitoring of patient as well as for molecular epidemiology research on HBV.

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.

Dengue is a viral, acute, and systemic disease that is mainly transmitted by the mosquito Aedes aegypti. It is spread through all 26 states of the Brazilian Federation and in the Federal District, and approximately 70% of all the world's notifications of dengue are recorded in Brazil. The objective of the present study is to analyze the epidemiology of dengue between 1999 and 2010 in Rondonia State, comparing the data obtained with that of the country as a whole during this period. As information sources, we used the official records from the National System for Notifiable Diseases (Sistema Nacional de Agravos de Notificacao), the State Agency of Sanitary Surveillance (Agencia Estadual de Vigilância Sanitaria), and the Department of Informatics (Departamento de Informatica) of the Brazilian Unified Health System (DATASUS). The data refer to the period from 1999 (the beginning of the notifications) to 2010 (the last year with complete data). Information was collected regarding the notified and confirmed cases, severe forms of the disease, the incidence rate, and the number of deaths. From the beginning of documentation, there was an exponential increase in the cases of dengue in the state. The number of reported cases increased from 969 in 1999 to 27,910 in 2010, which represents a 2,880% increase in notifications. The notifications were concentrated mainly in the capital, Porto Velho. In addition, the incidence rate increased from 7.63 to 365.9 per 100,000 inhabitants. Regarding the reporting of severe cases, there were increases in the number of all types of severe case expression and in the occurrence of deaths. It is necessary to develop public policies that prevent future epidemics to avoid a greater occurrence of the severe forms of the disease and, consequently, an increase in the number of deaths.

The hepatitis delta virus (HDV) is a hepatotropic subvirus that is dependent on the hepatitis B virus (HBV) and supplies the viral envelope containing the surface antigen of hepatitis B. Viral genetic diversity is related to the geographical origin of the isolates, and there are at least eight genotypes that are referred to as HDV-1 through HDV-8. HDV-3 is responsible for epidemics of severe and fulminant hepatitis, which are common in northeastern South America. HDV-3 is prevalent in the Brazilian Amazon and is associated with the increased aggressiveness of HDV infections. Although isolated, the characteristics of the clinical presentation of HDV-1 in the Amazon region have not yet been clearly reported. Objective: This study aims to assess the genotypic and clinical characteristics of individuals with the HDV-1 genotype in the western Amazon region. Methods: The HDV was genotyped by nested PCR-RFLP and sequencing from serum samples of 56 patients with HBV/HDV infection. The genotypes were correlated with the clinical characteristics presented by patients with HBV/HDV infection. Results: A prevalence of 92.3% for the HDV-3 genotype (n = 48) and 7.6% (n = 4) for the HDV-1 genotype was observed. Conclusion: To date, this is the most extensive clinical study of HDV-1 genotype infections in the nonindigenous population of Western Amazonia.

Background Hepatitis C virus (HCV) is an important human pathogen affecting around 3% of the human population. In Brazil, it is estimated that there are approximately 2 to 3 million HCV chronic carriers. There are few reports of HCV prevalence in Rondônia State (RO), but it was estimated in 9.7% from 1999 to 2005. The aim of this study was to characterize HCV genotypes in 58 chronic HCV infected patients from Porto Velho, Rondônia (RO), Brazil. Methods A fragment of 380 bp of NS5B region was amplified by nested PCR for genotyping analysis. Viral sequences were characterized by phylogenetic analysis using reference sequences obtained from the GenBank (n = 173). Sequences were aligned using Muscle software and edited in the SE-AL software. Phylogenetic analyses were conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) to obtain the MCC tree using BEAST v.1.5.3. Results From 58 anti-HCV positive samples, 22 were positive to the NS5B fragment and successfully sequenced. Genotype 1b was the most prevalent in this population (50%), followed by 1a (27.2%), 2b (13.6%) and 3a (9.0%). Conclusions This study is the first report of HCV genotypes from Rondônia State and subtype 1b was found to be the most prevalent. This subtype is mostly found among people who have a previous history of blood transfusion but more detailed studies with a larger number of patients are necessary to understand the HCV dynamics in the population of Rondônia State, Brazil.

Background Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondônia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM® 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions These results for the first HBV genotypic characterization in Rondônia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.

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, Author Summary Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania. Tegumentary Leishmaniasis (TL) is characterized by a variety of clinical forms, ranging from simple skin lesions to more severe lesions with mucosal destruction. The mechanisms by which some patients develop a severe form of the disease remain unclear. It is already known that the immune statuses of the host and parasite species are key factors influencing the clinical outcome of the disease, but is not known which are the factors that influence the parasite’s tropism to the mucosal regions. Recently, a virus found infecting Leishmania, Leishmania RNA virus 1 (LRV1), has also been reported as one important factor contributing to the disease severity. The present article reports, among 147 investigated patients from the Brazilian Western Amazon, a higher frequency of LRV1 among patients with mucosal lesions compared to patients with cutaneous lesions, statistically demonstrating that the virus is related to the worsening of the disease, increasing the risk of developing mucosal injury by almost 3 times.

In Brazil, hepatitis delta virus (HDV) is only reported in Western Amazonia, where severe cases of acute and chronic HDV hepatitis have been described. The study area was chosen in the States of Acre and Rondonia where most cases of hepatitis B virus (HBV)/HDV are reported. From December 2003 to October 2004, 40 HBsAg carriers with anti-HDV IgM were selected. An epidemiologic questionnaire, including demographic and clinical/epidemiologic variables was filled out. HDV amplification and genotyping were performed. Genotype I was detected in 22 patients (55.0%), whereas genotype III was identified in 18 (45.0%). Patients who were infected with genotype I were older (45.1 +/- 17.8 years) than patients infected with genotype III (32.8 +/- 10.9 years; P = 0.01). No symptoms were reported by 21 (52.5%) patients. Otherwise, 19 (47.5%) had symptoms (fatigue, abdominal pain, weight loss, and decompensated liver disease) that motivated them to seek medical care. Genotype III carriers were more symptomatic, but no statistical significance was achieved. Our preliminary results show that HDV genotypes I and III are present in Brazilian Amazonia and that HDV genotype III is not limited to the Amerindian population.

Studies on seasonal anopheline fauna variation were performed in two distinct settlements in the State of Rondonia, Brazil: one at the Madeira River banks (Portuchuelo) with stable native Amazonian population; the other at an inland lumber-extracting farm (Urupa) in dry land, in which adults are mostly migrants. During a 6-yr period (1994-2000), 8,638 adult anophelines were collected: 2,684 in Urupa and 5,954 in Portuchuelo. Anopheles darlingi represented >95% of total mosquitoes caught. Dissection of 4,424 A. darlingi females yielded a very low sporozoite infection index below 0.1%. Oocysts were found in both localities in approximately 0.1% of dissected mosquitoes. Determination of the hour biting rates disclosed seasonal variations in both localities. However, in Portuchuelo, mosquito density peaked at the acme of the rainy season, whereas at Urupa it peaked in the dry season. The increase in mosquito density and incidence of malaria cases were coincident. The high mosquito densities observed in the riverine settlement of Portochuelo sector B, which permits evaluation in > 10,000 mosquitoes' bites/person/year, could explain, in spite of the low mosquito's infection index, the previously described development of natural immunity in the local population that is not observed in the dry land agroindustrial settlement of Urupa.

A malaria control pilot project was developed in the Urupá agro-industrial farm that is situated in the State of Rondônia (Western Amazon Region, Brazil). Around 180 inhabitants had been surveyed for the past five years. The control measures were based on (1) training a community agent to perform on the spot microscopical diagnosis of malaria and to treat the uncomplicated cases of malaria; (2) limiting the use of insecticides to a short period before the high transmission season. This resulted in a significant reduction in the time between the onset of clinical symptoms and specific chemotherapy which fell from 3.5 to 1.3 days. In relation to the previous three reference years the total number of malaria cases was reduced to 50% in the first year and to 25% in the second year. The introduction of these measures coincided with pronounced reduction in the frequency of Plasmodium falciparum infections but this was less marked for P. vivax infections. In the second year of the pilot experiment there was no P. falciparum transmission on the farm. During the last decade there was a general decrease in the endemicity of malaria in the State of Rondônia. The linear regression coefficient values indicate that the decline was more pronounced in Urupá than in the general municipality and that the falciparum malaria API in Urupá farm is significantly lower than in the general municipality of Candeias were the farm is situated.

We report on a longitudinal study concerning the incidence of malaria in a riverine population (Portuchuelo) settled on the riverbanks of Rio Madeira, in the State of Rondonia, Brazil. We found the incidence of malaria to be seasonal, prevailing in the dry months of June and July. The Annual Parasite Index (API) was 292/1000 inhabitants, almost three times that of the state of Rondonia for the same period. In contrast with other studied Rondonian populations, malaria in Portuchuelo was more prevalent in youngsters < 16 years old, particularly in the 0-1 year age group. Adults were relatively spared, particularly those over 50 years. Besides being indicative of indoor transmission, these facts may suggest the existence of a certain degree of acquired resistance to infection and/or of lessened symptoms in older people. Riverine populations are spread over the entire Amazon region where most of its members were born. Due to the permanent presence of malaria among riverine populations, we are proposing that they may act as perennial reserves of malaria and, therefore, as sources of infection for migrants or eventual settlers at their vicinity. To date, the opposite view has been generally held. Anopheles darlingi, the main vector species in the area, is essentially sylvatic, which contributes to make the control of malaria highly problematic. The only hopes for control rest on permanent surveillance and the prompt treatment of patients, which are also problematic considering the vastness of the Amazon region and the remoteness of some of its riverine settlements.

Pacientes em tratamento com hemodiálise para a Insuficiência Renal Crônica (IRC) estão entre os grupos com maior prevalência da infecção pelo vírus da hepatite B e C, devido às transfusões de sangue e transmissão nosocomial. Um grupo de pacientes com IRC residentes em Porto Velho foi testado com marcadores sorológicos para hepatite B e C utilizando o teste ELISA e técnicas de biologia molecular (PCR). Os parâmetros para a validação dos resultados sorológicos foram medidos com base nos resultados da PCR. Dos 128 pacientes em hemodiálise durante o estudo, 12 (9,4%) eram HBsAg positivos, 69 (53,9%) eram anti-HBc positivos, 93 (72,7%) eram anti-HBs positivos, e 22 (17,2%) eram anti- HCV positivos. Os resultados dos testes com PCR foram de 12 (9,4%) HBV-DNA positivos e 16 (12,5%) HCV-RNA positivos. A precisão, sensibilidade e especificidade do ELISA para o HBsAg foram de 90,6%, 50% e 94,8%, e os mesmos parâmetros foram de 92,2%, 87,5% e 92,9% para anti-HCV. Com base nos resultados, apenas o valor preditivo negativo para anti-HCV (98,2%) mostrou-se confiável em pacientes renais crônicos em hemodiálise. Além disto, testes sorológicos e/ou moleculares representam a metodologia indicada para o diagnóstico da infecção pelo HBV e HCV nestes pacientes.

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.

Background Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). Methods A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). Results From our in-house plasmid we prepared serial dilutions ranging from 2 × 103 – 2 × 109 copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r2 = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r2 = 0.9965 and p