Microbial Diversity in Children with Gastroenteritis in the Amazon Region of Brazil: Development and Validation of a Molecular Method for Complete Sequencing of Viral Genomes.

INTRODUCTION: Metagenomic sequencing is a powerful tool that is widely used in laboratories worldwide for taxonomic characterization of microorganisms in clinical and environmental samples. In this study, we utilized metagenomics to investigate comprehensively the microbial diversity in fecal samples of children over a four-year period. Our methods were carefully designed to ensure accurate and reliable results. MATERIAL AND METHODS: Validated and analyzed were metagenomic data obtained from sequencing 27 fecal samples from children under 10 years old with gastroenteritis over a four-year period (2012-2016). The fecal specimens were collected from patients who received care at public health facilities in the northern region of Brazil. Sequencing libraries were prepared from cDNA and sequenced on the Illumina HiSeq. Kraken-2 was utilized to classify bacterial taxonomy based on the 16S rRNA gene, using the Silva rRNA database. Additionally, the Diamond program was used for mapping to the non-redundant protein database (NR database). Phylogenomic analyses were conducted using Geneious R10 and MEGA X software, and Bayesian estimation of phylogeny was performed using the MrBayes program. The results indicate significant heterogeneity among norovirus strains, with evidence of recombination and point mutations. This study presents the first complete genome of parechovirus 8 in the region. Additionally, it describes the bacterial populations and bacteriophages present in feces, with a high abundance of Firmicutes and Proteobacteria, including an increased proportion of the Enterobacteriaceae family. The presented data demonstrate the genetic diversity of microbial populations and provide a comprehensive report on viral molecular characterization. These findings are relevant for genomic studies in gastrointestinal infections. The metagenomic approach is a powerful tool for investigating microbial diversity in children with gastroenteritis. However, further studies are imperative to conduct genomic analysis of identified bacterial strains and thoroughly analyze antimicrobial resistance genes.

Dengue virus serotype 2 genotype III evolution during the 2019 outbreak in Mato Grosso, Midwestern Brazil.

DENV-2 was the main responsible for a 70% increase in dengue incidence in Brazil during 2019. That year, our metagenomic study by Illumina NextSeq on serum samples from acute febrile patients (n = 92) with suspected arbovirus infection, sampled in 22 cities of the state of Mato Grosso (MT), in the middle west of Brazil, revealed eight complete genomes and two near-complete sequences of DENV-2 genotype III, one Human parvovirus B19 genotype I (5,391 nt) and one Coxsackievirus A6 lineage D (4,514 nt). These DENV-2 sequences share the aminoacidic identities of BR4 lineage on E protein domains I, II and III, and were included in a clade with sequences of the same lineage circulating in the southeast of Brazil in the same year. Nevertheless, 11/34 non-synonymous mutations are unique to three strains inthis study, distributed in the E (n = 6), NS3 (n = 2) and NS5 (n = 3) proteins. Other 14 aa changes on C (n = 1), E (n = 3), NS1 (n = 2), NS2A (n = 1) and NS5 (n = 7) were first reported in a genotype III lineage, having been already reported only in other DENV-2 genotypes. All 10 sequences have mutations in the NS5 protein (14 different aa changes). Nine E protein aa changes found in two sequences, six of which are unique, are in the ectodomain; where the E:M272T change is on the hinge of the E protein at domain II, in a region critical for the anchoring to the host cell receptor. The NS5:G81R mutation, in the methyltransferase domain, was found in one strain of this study. Altogether, these data points to an important evolution of DENV-2 genotype III lineage BR4 during this outbreak in 2019 in MT. Genomic surveillance is essential to detect virus etiology and evolution, possibly related to immune evasion and viral fitness changes leading to future novel outbreaks.

Genome sequencing of dengue virus serotype 4 in a bat brain sample (Platyrrhinus helleri) from the Brazilian Amazon.

The existence of sylvatic transmission of dengue virus in communities of neotropical bats remains uncertain. In this work we present a near-complete genome of dengue virus serotype 4 obtained from the brain sample of a bat from Platyrrhinus helleri specie collected in the Brazilian Amazon region. The presence of the virus in the brain sample may indicate a possible tropism for the central nervous system in bats, which may justify negative results in previous studies that focused on analysis of other tissues, such as liver and spleen. Besides the duration of dengue virus circulation in the Americas (circa 40 years) may be too short for an implementation of a sylvatic dengue virus cycle. Our findings suggest that continued monitoring is needed to confirm with the neotropical bats could potentially act as a natural reservoir of dengue in the region.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Discovery and genome sequencing of a new virus related to members of the family Tymoviridae, isolated from mosquitoes of the genus Mansonia in Brazil.

A new virus, named Mutum virus, related to members of the family Tymoviridae, was isolated from mosquitoes (Mansonia spp.) in clone C6/36 cells, and its complete genome was sequenced. Its genome is 6494 nt in size with an organization resembling that of tymovirids. The isolated virus is phylogenetically related to two viruses isolated from Culex spp. mosquitoes: Ek Balam virus, reported in Mexico, and Culex-originated Tymoviridae-like virus, isolated in China. The results of this study suggest that this virus is a new member of the family Tymoviridae.

Neurological infection by chikungunya and a triple Arbovirus co-infection in Mato Grosso, Central Western Brazil during 2019.

BACKGROUND: Neurological viral infection is frequently associated to enterovirus, herpesvirus and arboviruses. These infections may cause severe clinical outcomes, long lasting sequelae or death. Few studies have addressed viral neurological infections etiology in Brazil. OBJECTIVES: Identification of viruses in the cerebral spinal fluid (CSF) of human neurological infections suspected of viral etiology during January and May 2019 in Midwestern Brazil. MATERIALS AND METHODS: Clinical, laboratory and epidemiological information was gathered from medical records. In addition, an aliquot of the sampled CSF was subjected to viral RNA/DNA extraction, randomic dscDNA amplification by PCR, DNA purification and Ilumina HiSeq 2500 sequencing. RESULTS: Six viral genomes belonging to Chikungunya virus (CHIKV) East-Central-South African (ECSA) genotype (10.834-11.804 nt in length) confirmed lately by RT-PCR for CHIKV envelope were present in all six liquor samples. These genomes present two mutations, nsP2:T31I and nsP3:A388V, shared with other Mato Grosso State strains from 2019, not present in sequences of the virus from previous years obtained in the State. One case was a triple co-infection also confirmed through RT-PCR, with Dengue virus serotype 4 genotype II (NS5; 874 nt) and Oropouche virus genotype IA (segment S; 302 nt). CSF was clear and colorless (5/6 patients), with >10% of lymphomononuclear cells (6/6), 1-99 erythrocytes/mm3 (5/6), glucose levels >50 mg/dl (4/5) e > 10 mg/dl of proteins (4/4). One patient evolved to death, and another, a newborn, presented sequelae after recovery. CONCLUSIONS: Despite herpesviruses and enteroviruses are frequent etiologies of neurological infections, the casuistic here reported was associated to arboviruses already known to be responsible for acute febrile illness outbreaks in the state of Mato Grosso, Midwestern Brazil.

Sialovirome of Brazilian tropical anophelines.

Anophelinae is a widely dispersed Culicidae subfamily that may carry a unique virome. Here we herein report the set of viruses found in 323 salivary glands of 16 anopheline species sampled at Upper Pantanal, Chapada dos Guimarães National Park and Coxipó river basin, South Central Mato Grosso, Brazil, pooled (n = 11) and subjected to high throughput sequencing. Metagenomics revealed the presence of nine viral sequences belonging to novel viruses from seven viral families: Purunga is a putative novel orbivirus sharing 74% and 65% aa identity, respectively, with the VP1 and VP3 segments of Changuinola serogroup, Jaracatiá flavivirus shares 60% amino-acid (aa) identity with Aedes flavivirus. Coxipó dielmovirus and Chapada dielmovirus shared 51% and 39% aa identity with Merida virus. Coloiado-orthomyxo like virus is 57.1-64.8% identical at aa level to Aedes albonnulatus orthomyxo-like virus. Mujica picorna-like virus shares 49% aa identity with Flen picorna-like virus and Chiquitos virus is 50% similar to Ista virus, both from Picornavirales order. Cerrado partiti-like-virus shares 75-86% aa identity with Atrato partiti-like virus 2. We also found the S and L segments of Anopheles triannulatus orthophasmavirus (92% identity) in Anopheles lutzi from Chapada dos Guimarães. The identification of these putative novel viruses underscore the wide dispersion of viruses in culicid hosts contributing to extensions on mosquito virome descriptions.

Differential expression analysis and profiling of hepatic miRNA and isomiRNA in dengue hemorrhagic fever.

Dengue virus causes dengue hemorrhagic fever (DHF) and has been associated to fatal cases worldwide. The liver is one of the most important target tissues in severe cases, due to its intense viral replication and metabolic role. microRNAs role during infection is crucial to understand the regulatory mechanisms of DENV infection and can help in diagnostic and anti-viral therapies development. We sequenced the miRNome of six fatal cases and compared to five controls, to characterize the human microRNAs expression profile in the liver tissue during DHF. Eight microRNAs were differentially expressed, including miR-126-5p, a regulatory molecule of endothelial cells, miR-122-5p, a liver specific homeostasis regulator, and miR-146a-5p, an interferon-regulator. Enrichment analysis with predicted target genes of microRNAs revealed regulatory pathways of apoptosis, involving MAPK, RAS, CDK and FAS. Immune response pathways were related to NF- kB, CC and CX families, IL and TLR. This is the first description of the human microRNA and isomicroRNA profile in liver tissues from DHF cases. The results demonstrated the association of miR-126-5p, miR-122-5p and miR-146a-5p with DHF liver pathogenesis, involving endothelial repair and vascular permeability regulation, control of homeostasis and expression of inflammatory cytokines.

Fatal Outcome of Chikungunya Virus Infection in Brazil.

BACKGROUND: Chikungunya virus (CHIKV) emerged in the Americas in 2013 and has caused approximately 2.1 million cases and >600 deaths. A retrospective investigation was undertaken to describe clinical, epidemiological, and viral genomic features associated with deaths caused by CHIKV in Ceará state, northeast Brazil. METHODS: Sera, cerebrospinal fluid (CSF), and tissue samples from 100 fatal cases with suspected arbovirus infection were tested for CHIKV, dengue virus (DENV), and Zika virus (ZIKV). Clinical, epidemiological, and death reports were obtained for patients with confirmed CHIKV infection. Logistic regression analysis was undertaken to identify independent factors associated with risk of death during CHIKV infection. Phylogenetic analysis was conducted using whole genomes from a subset of cases. RESULTS: Sixty-eight fatal cases had CHIKV infection confirmed by reverse-transcription quantitative polymerase chain reaction (52.9%), viral antigen (41.1%), and/or specific immunoglobulin M (63.2%). Co-detection of CHIKV with DENV was found in 22% of fatal cases, ZIKV in 2.9%, and DENV and ZIKV in 1.5%. A total of 39 CHIKV deaths presented with neurological signs and symptoms, and CHIKV-RNA was found in the CSF of 92.3% of these patients. Fatal outcomes were associated with irreversible multiple organ dysfunction syndrome. Patients with diabetes appear to die at a higher frequency during the subacute phase. Genetic analysis showed circulation of 2 CHIKV East-Central-South African (ECSA) lineages in Ceará and revealed no unique virus genomic mutation associated with fatal outcome. CONCLUSIONS: The investigation of the largest cross-sectional cohort of CHIKV deaths to date reveals that CHIKV-ECSA strains can cause death in individuals from both risk and nonrisk groups, including young adults.

Human pegivirus (HPgV, GBV-C) RNA in volunteer blood donors from a public hemotherapy service in Northern Brazil.

BACKGROUND: Human pegivirus (HPgV)-formerly known as GBV-C-is a member of the Flaviviridae family and belongs to the species Pegivirus C. It is a non-pathogenic virus and is transmitted among humans mainly through the exposure to contaminated blood and is often associated with human immunodeficiency virus (HIV) infection, among other viruses. This study aimed to determine the prevalence of HPgV viremia, its association with HIV and clinical epidemiological factors, as well as the full-length sequencing and genome characterization of HPgV recovered from blood donors of the HEMOPA Foundation in Belém-PA-Brazil. METHODS: Plasma samples were obtained from 459 donors, tested for the presence of HPgV RNA by the RT-qPCR. From these, a total of 26 RT-qPCR positive samples were submitted to the NGS sequencing approach in order to obtain the full genome. Genome characterization and phylogenetic analysis were conducted. RESULTS: The prevalence of HPgV was 12.42%. We observed the highest prevalences among donors aged between 18 and 30 years old (16.5%), with brown skin color (13.2%) and men (15.8%). The newly diagnosed HIV-1 prevalence was 26.67%. The HPgV genotype 2 (2a and 2b) was identified. No data on viral load value was found to corroborate the protective effect of HPgV on HIV evolution. CONCLUSIONS: This study provided information regarding the HPgV infection among blood donors from HEMOPA Foundation. Furthermore, we genetically characterized the HPgV circulating strains and described by the first time nearly complete genomes of genotype 2 in Brazilian Amazon.

Mitochondrial genome sequencing and phylogeny of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis (Diptera: Culicidae).

The genus Haemagogus (Diptera: Culicidae) comprises species of great epidemiological relevance, involved in transmission cycles of the Yellow fever virus and other arboviruses in South America. So far, only Haemagogus janthinomys has complete mitochondrial sequences available. Given the unavailability of information related to aspects of the evolutionary biology and molecular taxonomy of this genus, we report here, the first sequencing of the mitogenomes of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis. The mitogenomes showed an average length of 15,038 bp, average AT content of 79.3%, positive AT-skews, negative GC-skews, and comprised 37 functional subunits (13 PCGs, 22 tRNA, and 02 rRNA). The PCGs showed ATN as start codon, TAA as stop codon, and signs of purifying selection. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogenetic analyzes of Bayesian inference and Maximum Likelihood, based on concatenated sequences from all 13 PCGs, produced identical topologies and strongly supported the monophyletic relationship between the Haemagogus and Conopostegus subgenera, and corroborated with the known taxonomic classification of the evaluated taxa, based on external morphological aspects. The information produced on the mitogenomes of the Haemagogus species evaluated here may be useful in carrying out future taxonomic and evolutionary studies of the genus.

Insect-specific viruses and arboviruses in adult male culicids from Midwestern Brazil.

Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilhéus (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.

Evolutionary and Molecular Analysis of Complete Genome Sequences of Norovirus From Brazil: Emerging Recombinant Strain GII.P16/GII.4.

Noroviruses (NoVs) are enteric viruses that cause acute gastroenteritis, and the pandemic GII.4 genotype is spreading and evolving rapidly. The recombinant GII.P16/GII.4_Sydney strain emerged in 2016, replacing GII.P31/GII.4_Sydney (GII.P31 formerly known as GII.Pe) in some countries. We analyzed the complete genome of 20 NoV strains (17 GII.P31/GII.4_ Sydney and 3 GII.P16/GII.4_Sydney) from Belém and Manaus, Brazil, collected from 2012 to 2016. Phylogenetic trees were constructed by maximum likelihood method from 191 full NoV-VP1 sequences, demonstrated segregation of the Sydney lineage in two larger clades, suggesting that GII.4 strains associated with GII.P16 already have modifications compared with GII.P31/GII.4. Additionally, the Bayesian Markov Chain Monte Carlo method was used to reconstruct a time-scaled phylogenetic tree formed by GII.P16 ORF1 sequences (n = 117) and three complete GII.P16 sequences from Belém. The phylogenetic tree indicated the presence of six clades classified into different capsid genotypes and locations. Evolutionary rates of the ORF1 gene of GII.P16 strains was estimated at 2.01 × 10-3 substitutions/site/year, and the most recent common ancestors were estimated in 2011 (2011-2012, 95% HPD). Comparing the amino acid (AA) sequence coding for ORF1 with the prototype strain GII.P16/GII.4, 36 AA changes were observed, mainly in the non-structural proteins p48, p22, and RdRp. GII.P16/GII.4 strains of this study presented changes in amino acids 310, 333, 373, and 393 of the antigenic sites in the P2 subdomain, and ML tree indicating the division within the Sydney lineage according to the GII.P16 and GII.P31 polymerases. Notably, as noroviruses have high recombination rates and the GII.4 genotype was prevalent for a long time in several locations, additional and continuous evolutionary analyses of this new genotype should be needed in the future.

Whole-Genome Sequencing of an Unusual Human Papillomavirus (HPV71) from Latin America (Brazil).

We report the complete genome sequencing of human papillomavirus 71 from Latin America (Brazil).

Isolation and Genetic Identification of Endophytic Lactic Acid Bacteria From the Amazonian Açai Fruits: Probiotics Features of Selected Strains and Their Potential to Inhibit Pathogens.

The açai palm (Euterpe oleracea) is native to the Amazon basin, a humid tropical forest. High levels of total mesophilic bacteria with high diversity have been consistently reported in açai fruits. As local consumers have few digestive problems, the results of the present study reveal the lactic acid bacteria (LAB) recovered from açai fruits with characteristics that suggest they are possible candidates for probiotics and antagonistic potential against pathogens for the first time. Açai fruits were sampled from five different locations in the Eastern Amazonia floodplains. Sixty-six isolates were recovered from fruits and tested for some probiotic characteristics following FAO/WHO guidelines. Approximately 65% of the isolates showed no catalase or oxidase activity, Gram-positive staining or cocci and bacilli cell morphology. Furthermore, 48% of the isolates demonstrated preliminary characteristics that suggest safety for use, as they presented no coagulase enzyme activity or gamma-hemolysis. These strains were identified as belonging to the genera Lactiplantibacillus and Pediococcus, and 32 strains also presented resistance to vancomycin, ciprofloxacin and streptomycin. In addition, 28 isolates showed a survival rate, expressed as log cycle reduction, higher than 0.9 under gastric conditions (pH 2). All strains tested positive in bile salts deconjugation tests and showed a survival rate higher than 0.8 in the presence of this salt. Regarding antimicrobial activity against pathogens, all strains were able to inhibit Salmonella Typhimurium (ATCC® 14028TM) and 97% were capable of inhibiting Escherichia coli (ATCC® 25922TM). Concerning the results of in vitro antagonistic assays, three isolates (B125, B135, and Z183 strains) were selected for antagonistic tests using açai juice contaminated with these two pathogens. All tested LAB strains were able to inhibit pathogen growth in açai juice. In summary, açai fruits are a potential source of LAB isolates to be investigated as probiotics.

Complete genome sequence of human T-cell lymphotropic type 1 from patients with different clinical profiles, including infective dermatitis.

The HTLV-1 is the first human retrovirus and is associated with several clinical syndromes, however, the pathogenesis of these clinical manifestations is still not fully understood. Furthermore, there are few complete genomes publicly available, about 0.12 complete genomes per 10,000 infected individuals and the databases have a major deficiency of sequences information. This study generated and characterized 31 HTLV-1 complete genomes sequences derived from individuals with Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (TSP/HAM), Adult T-cell leukemia/lymphoma (ATL), infective dermatitis associated to HTLV-1 (IDH) and asymptomatic patients. These sequences are associated to clinical and epidemiological information about the patients. The sequencing data generated on Ion Torrent PGM platform were assembled and mapped against the reference HTLV-1 genome. These sequences were genotyped as Cosmopolitan subtype, Transcontinental subgroup. We identified the variants in the coding regions of the genome of the different clinical profiles, however, no statistical relation was detected. This study contributed to increase of HTLV-1 complete genomes in the world. Furthermore, to better investigate the contribution of HTLV-1 mutations for the disease outcome it is necessary to evaluate the interaction of the viral genome and characteristics of the human host.

Contágio intradomiciliar e status vacinal entre comunicantes de portatores do vírus da hepatite B / House hold contagion and vaccination status among communicants of hepatitis B virus carriers

Objetivo: Avaliar a cobertura da vacina contra hepatite B e a frequência da infecção em comunicantes de portadores do Vírus da Hepatite B vinte anos após a implementação da vacina na cidade de Belém, Pará, Brasil. Método: Estudo descritivo quantitativo, desenvolvido entre 2016 e 2018. Foram realizadas visitas domiciliares aos portadores do vírus, notificados por meio da busca de comunicantes, com orientações sobre a pesquisa, preenchimento e assinatura de formulários, coleta de amostras para testes sorológicos de hepatite B (HBsAg, anti-HBc total, anti-HBs) por técnica imunoenzimática. Resultados: Na população analisada 53,5% são do sexo masculino, com média de idade de 9,4 anos (entre 4 meses a 19 anos com mediana de 10 anos). Não foram detectados portadores de HBsAg reagente; anti-HBc isolado total foi detectado em 2,1%; 4,2% eram anti-HBc total / anti-HBs reagente e 42% anti-HBs isolado, atribuídos à vacinação; 41,7% receberam esquema vacinal completo e 58,3% eram suscetíveis. Conclusões: Os resultados encontrados podem ser utilizados como norteadores de ações estratégicas visando ampliar a cobertura vacinal e reduzir a prevalência de infectados na população de comunicantes intradomiciliares. (AU)

Objective: To evaluate the coverage of the hepatitis B vaccine and the frequency of infection in communicants of Hepatitis B virus carriers twenty years after the vaccine was implemented in the city of Belém, Pará, Brazil. Method: Quantitative descriptive study developed between 2016 and 2018. Home visits were carried out to the carriers of the virus notified through the search for communicants, with guiding on research, filling and signing forms, collecting samples for serological tests for Hepatitis B (HBsAg, total anti - HBc, anti - HBs) by immunoenzymatic technique. Results: In the population analyzed 53.5% are male, with a mean age of 9.4 years (ranging from 4 months to 19 years and median of 10 years). Carriers HBsAg reagent were not detected; total anti - HBc isolated was detected in 2.1%; 4.2% were total anti - HBc / anti - HBs reagent and 42% anti - HBs alone, attributed to vaccination; 41.7% received a complete vaccination schedule and 58.3% were susceptible. Conclusions:The results found can be used as guidelines for strategic actions aimed at expanding vaccine coverage and reducing the prevalence of infected people in the population of household communicators. (AU)

Objetivo: Evaluarla cobertura de la vacuna contra lahepatitis B y lafrecuencia de infecciónen portadores delVirus de laHepatitis Bveinteañosdespués de laimplementación de lavacuna enlaciudad de Belém, Pará, Brasil. Método: Estudiodescriptivocuantitativo realizado entre 2016 y 2018. Fueron realizadas visitas domiciliarias a los portadores del vírus, notificados a través de labúsqueda de domiciliares, conorientaciones sobre lainvestigación, registro y asignatura de formularios, toma de muestras de sangre para pruebas serológicas de Hepatitis B (HBsAg, anti-HBc total, anti-HBs) por la técnica inmunoenzimática. Resultados: Enlapoblaciónanalizada 53,5% del sexo masculino, conedadpromedio de 9,4 años (entre 4 meses a 19 añoscon mediana de 10 años) no fueron detectados portadores de HBsAgreactivo; el total de anti-HBcfué detectado en 2,1%; 4,2% anti-HBc total/anti-HBs reactivo, y, 42% anti-HBs aislado, atribuido a lavacunación; 41,7% recibieronel esquema completo de vacunación y 58,3% fueronsusceptibles. Conclusiones: Los resultados encontrados puedenusarse como pautas para acciones estratégicas dirigidas a ampliar la cobertura de vacunación y reducirlaprevalencia de personas infectadas enlapoblación de comunicadores domésticos. (AU)

Clinical and molecular diagnosis of Chlamydophila in captive parrots in Pará state, Brazil / Estudo clínico e diagnóstico molecular de Chlamydophila sp. em psitaciformes mantidos em cativeiro no estado do Pará, Brasil

Parrots (Order Psittaciformes) are globally distributed birds that, together with members of the Columbiformes, represent the most susceptible animals, in regards to infection by Chlamydophila psittaci, which is an obligate, zoonotic, intracellular bacterium that causes chlamydiosis in domestic and wild birds and psittacosis in humans. The aim of the present study was to assess the occurrence of C. psittaci in Brazilian psittacids kept in conservation breeding sites in Pará State, Brazil. Cloacal and oropharynx swab samples were collected from 201 psittacids that were distributed among four breeding sites: Metropolitan Area of Belém (C1 and C2), Northeastern Pará (C3), and Low Amazon (C4). The samples were screened for C. psittaci using semi-nested PCR, and the resulting incidence data were analyzed using proportion and chi-square tests. Chlamydophila infection was confirmed for all the breeding sites, with an overall prevalence of 31.84%, and no species-specific predisposition was observed. Furthermore, 13.93% of the sampled birds eliminated the infectious agent by the cloaca, whereas 11.44% eliminated the agent by the oropharynx, and 6.47% eliminated the agent by both routes. Moreover, there was a significant difference between the incidence of Chlamydophila infection of breeding sites C2 and C3 (p=0.029), which yielded the smallest and largest number of diagnosed cases, respectively. In the present study, most of the birds (27.86%) were considered unapparent carriers of Chlamydophila infection, and only 3.98% of the birds yielded both a positive diagnosis and clinical signs of chlamydiosis.

Psitacídeos são aves distribuídas em todo o mundo e, juntamente com Columbiformes, representam os animais mais suscetíveis a uma infecção causada por Chlamydophila psittaci, uma bactéria intracelular, obrigatória, zoonótica que causa clamídia em aves domésticas e selvagens e psitacose em humanos. O objetivo deste estudo foi avaliar a ocorrência de C. psittaci em diferentes espécies de psitacídeos da fauna brasileira mantidos em criadouros conservacionistas no Estado do Pará, Brasil. Amostras de swabs de cloaca e orofaringe de 201 psitacídeos distribuídos em quatro criadouros nas mesorregiões Metropolitana de Belém (C1 e C2), Nordeste do Pará (C3) e Baixo Amazonas (C4) foram utilizados. As amostras foram submetidas ao teste molecular de semi-nested PCR. As análises estatísticas foram realizadas de acordo com o teste de proposição por R e teste do qui-quadrado (p 0,05). A presença de Chlamydophila sp. foi confirmada em todos os criadouros, com uma prevalência de 31,84% de aves infectadas, com predisposição não específica da espécie encontrada para a infecção entre as aves amostradas. Os resultados da semi-nested PCR mostraram que 13,93% das aves eliminaram o agente infeccioso pela cloaca, 11,44% pela orofaringe e 6,47% por ambas. Além disso, quando aplicado em cada local, este teste mostrou uma diferença estatisticamente significativa entre os criadouros C2 e C3 (p = 0,029), que apresentou o menor e maior número de casos diagnosticados, respectivamente. A maioria dos animais, ou 27,86%, foi considerada como portadora inaparente da infecção e apenas 3,98% das aves com diagnóstico positivo apresentaram algum sinal clínico sugestivo da doença.

Clinical and molecular diagnosis of Chlamydophila in captive parrots in Pará state, Brazil / Estudo clínico e diagnóstico molecular de Chlamydophila sp. em psitaciformes mantidos em cativeiro no estado do Pará, Brasil

Parrots (Order Psittaciformes) are globally distributed birds that, together with members of the Columbiformes, represent the most susceptible animals, in regards to infection by Chlamydophila psittaci, which is an obligate, zoonotic, intracellular bacterium that causes chlamydiosis in domestic and wild birds and psittacosis in humans. The aim of the present study was to assess the occurrence of C. psittaci in Brazilian psittacids kept in conservation breeding sites in Pará State, Brazil. Cloacal and oropharynx swab samples were collected from 201 psittacids that were distributed among four breeding sites: Metropolitan Area of Belém (C1 and C2), Northeastern Pará (C3), and Low Amazon (C4). The samples were screened for C. psittaci using semi-nested PCR, and the resulting incidence data were analyzed using proportion and chi-square tests. Chlamydophila infection was confirmed for all the breeding sites, with an overall prevalence of 31.84%, and no species-specific predisposition was observed. Furthermore, 13.93% of the sampled birds eliminated the infectious agent by the cloaca, whereas 11.44% eliminated the agent by the oropharynx, and 6.47% eliminated the agent by both routes. Moreover, there was a significant difference between the incidence of Chlamydophila infection of breeding sites C2 and C3 (p=0.029), which yielded the smallest and largest number of diagnosed cases, respectively. In the present study, most of the birds (27.86%) were considered unapparent carriers of Chlamydophila infection, and only 3.98% of the birds yielded both a positive diagnosis and clinical signs of chlamydiosis.(AU)

Psitacídeos são aves distribuídas em todo o mundo e, juntamente com Columbiformes, representam os animais mais suscetíveis a uma infecção causada por Chlamydophila psittaci, uma bactéria intracelular, obrigatória, zoonótica que causa clamídia em aves domésticas e selvagens e psitacose em humanos. O objetivo deste estudo foi avaliar a ocorrência de C. psittaci em diferentes espécies de psitacídeos da fauna brasileira mantidos em criadouros conservacionistas no Estado do Pará, Brasil. Amostras de swabs de cloaca e orofaringe de 201 psitacídeos distribuídos em quatro criadouros nas mesorregiões Metropolitana de Belém (C1 e C2), Nordeste do Pará (C3) e Baixo Amazonas (C4) foram utilizados. As amostras foram submetidas ao teste molecular de semi-nested PCR. As análises estatísticas foram realizadas de acordo com o teste de proposição por R e teste do qui-quadrado (p 0,05). A presença de Chlamydophila sp. foi confirmada em todos os criadouros, com uma prevalência de 31,84% de aves infectadas, com predisposição não específica da espécie encontrada para a infecção entre as aves amostradas. Os resultados da semi-nested PCR mostraram que 13,93% das aves eliminaram o agente infeccioso pela cloaca, 11,44% pela orofaringe e 6,47% por ambas. Além disso, quando aplicado em cada local, este teste mostrou uma diferença estatisticamente significativa entre os criadouros C2 e C3 (p = 0,029), que apresentou o menor e maior número de casos diagnosticados, respectivamente. A maioria dos animais, ou 27,86%, foi considerada como portadora inaparente da infecção e apenas 3,98% das aves com diagnóstico positivo apresentaram algum sinal clínico sugestivo da doença.(AU)

Genomic, epidemiological and digital surveillance of Chikungunya virus in the Brazilian Amazon.

BACKGROUND: Since its first detection in the Caribbean in late 2013, chikungunya virus (CHIKV) has affected 51 countries in the Americas. The CHIKV epidemic in the Americas was caused by the CHIKV-Asian genotype. In August 2014, local transmission of the CHIKV-Asian genotype was detected in the Brazilian Amazon region. However, a distinct lineage, the CHIKV-East-Central-South-America (ECSA)-genotype, was detected nearly simultaneously in Feira de Santana, Bahia state, northeast Brazil. The genomic diversity and the dynamics of CHIKV in the Brazilian Amazon region remains poorly understood despite its importance to better understand the epidemiological spread and public health impact of CHIKV in the country. METHODOLOGY/PRINCIPAL FINDINGS: We report a large CHIKV outbreak (5,928 notified cases between August 2014 and August 2018) in Boa vista municipality, capital city of Roraima's state, located in the Brazilian Amazon region. We generated 20 novel CHIKV-ECSA genomes from the Brazilian Amazon region using MinION portable genome sequencing. Phylogenetic analyses revealed that despite an early introduction of the Asian genotype in 2015 in Roraima, the large CHIKV outbreak in 2017 in Boa Vista was caused by an ECSA-lineage most likely introduced from northeastern Brazil. Epidemiological analyses suggest a basic reproductive number of R0 of 1.66, which translates in an estimated 39 (95% CI: 36 to 45) % of Roraima's population infected with CHIKV-ECSA. Finally, we find a strong association between Google search activity and the local laboratory-confirmed CHIKV cases in Roraima. CONCLUSIONS/SIGNIFICANCE: This study highlights the potential of combining traditional surveillance with portable genome sequencing technologies and digital epidemiology to inform public health surveillance in the Amazon region. Our data reveal a large CHIKV-ECSA outbreak in Boa Vista, limited potential for future CHIKV outbreaks, and indicate a replacement of the Asian genotype by the ECSA genotype in the Amazon region.