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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 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.

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.

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.

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.

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.

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