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

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

HTLV-2 infection in Manaus, Brazil: first description of HTLV-2c subtype in an urban area of the Western Amazon region

Abstract INTRODUCTION: We investigated the prevalence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) infection in patients with hematological diseases from the western Amazon region of Brazil. METHODS: Samples from 306 patients were submitted for the molecular diagnosis of HTLV-1/2 infection by real time PCR (qPCR), with amplification, sequencing, and phylogenetic analysis of the long terminal repeat (LTR) region. RESULTS: A 29-year-old male carrier of sickle cell anemia with a history of multiple blood transfusions was diagnosed with the HTLV-2c subtype. CONCLUSIONS: This study describes the first known occurrence of HTLV-2c in the urban area of Brazil's western Amazon region.

Confirmed Invasive Pulmonary Aspergillosis and COVID-19: the value of postmortem findings to support antemortem management

Abstract We present postmortem evidence of invasive pulmonary aspergillosis (IPA) in a patient with severe COVID-19. Autopsies of COVID-19 confirmed cases were performed. The patient died despite antimicrobials, mechanical ventilation, and vasopressor support. Histopathology and peripheral blood galactomannan antigen testing confirmed IPA. Aspergillus penicillioides infection was confirmed by nucleotide sequencing and BLAST analysis. Further reports are needed to assess the occurrence and frequency of IPA in SARS-CoV-2 infections, and how they interact clinically.

Genomic and phylogenetic characterisation of an imported case of SARS-CoV-2 in Amazonas State, Brazil

A new coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] is currently causing a life-threatening pandemic. In this study, we report the complete genome sequencing and genetic characterisation of a SARS-CoV-2 detected in Manaus, Amazonas, Brazil, and the protocol we designed to generate high-quality SARS-CoV-2 full genome data. The isolate was obtained from an asymptomatic carrier returning from Madrid, Spain. Nucleotide sequence analysis showed a total of nine mutations in comparison with the original human case in Wuhan, China, and support this case as belonging to the recently proposed lineage A.2. Phylogeographic analysis further confirmed the likely European origin of this case. To our knowledge, this is the first SARS-CoV-2 genome obtained from the North Brazilian Region. We believe that the information generated in this study may contribute to the ongoing efforts toward the SARS-CoV-2 emergence.

Oropouche virus detection in saliva and urine

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.

Human parvovirus B19 genotype 1 in suspected dengue patients of Tefé, Amazonas State, Brazil

Abstract INTRODUCTION: Human parvovirus B19 (B19V) is a common pathogen, which on infection causes variety of clinical conditions from benign self-limiting exanthematous disease and other similar pathologies to fetal death. METHODS: We collected 341 serum samples between the first and fourth day after the onset of symptoms from all patients suspected of dengue fever who were attended at Regional Hospital of Tefé. Initially, patients were screened for malaria by blood smear test and negative samples were sent to Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) situated in Manaus (AM) for dengue testing using semi-nested multiplex PCR. Further, we investigated 44 malaria and dengue-negative samples of children for B19V DNA by nested-PCR. Positive samples were analyzed by BLAST against entire public non-redundant nucleotide database and genotyped by phylogenetic analyses using neighbor-joining clustering method. RESULTS: Eight samples (18.2%) were found to be PCR positive. Fever, headache, ocular pain, and/or muscle pain were reported as the most frequent symptoms by the patients and none were diagnosed with rash at the time of sample collection. Phylogenetic analysis of major capsid protein 2 (VP2) and VP3 coding region showed high similarity with B19V genotype 1. CONCLUSIONS: Our results reveal the spread of B19V genotype 1 in Tefé. Moreover, our results emphasize the significance of laboratorial differential diagnosis using molecular techniques in patients with acute febrile, and thereby aid the health surveillance system in improving patient care even in the remote areas of Amazon.

Molecular characterisation of the emerging measles virus from Roraima state, Brazil, 2018

Measles is a human infectious disease of global concern that is caused by the measles virus. In this study, we report the complete genome sequencing of one measles virus isolate, genotype D8, that was obtained directly from a urine sample in Boa Vista city, the capital of Roraima state in Brazil. Phylogenetic reconstruction grouped the genome described in this study with that of samples from Australia, South Korea, and Italy. To our knowledge, this is the first complete genome sequence of a wild-type measles virus reported from Latin America. Therefore, the present data strengthen the current knowledge on the molecular epidemiology of measles worldwide.

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur. OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions. METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR. FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes. MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

Analysis of the immunological biomarker profile during acute Zika virus infection reveals the overexpression of CXCL10, a chemokine linked to neuronal damage

BACKGROUND Infection with Zika virus (ZIKV) manifests in a broad spectrum of disease ranging from mild illness to severe neurological complications and little is known about Zika immunopathogenesis. OBJECTIVES To define the immunologic biomarkers that correlate with acute ZIKV infection. METHODS We characterized the levels of circulating cytokines, chemokines, and growth factors in 54 infected patients of both genders at five different time points after symptom onset using microbeads multiplex immunoassay; comparison to 100 age-matched controls was performed for statistical analysis and data mining. FINDINGS ZIKV-infected patients present a striking systemic inflammatory response with high levels of pro-inflammatory mediators. Despite the strong inflammatory pattern, IL-1Ra and IL-4 are also induced during the acute infection. Interestingly, the inflammatory cytokines IL-1β, IL-13, IL-17, TNF-α, and IFN-γ; chemokines CXCL8, CCL2, CCL5; and the growth factor G-CSF, displayed a bimodal distribution accompanying viremia. While this is the first manuscript to document bimodal distributions of viremia in ZIKV infection, this has been documented in other viral infections, with a primary viremia peak during mild systemic disease and a secondary peak associated with distribution of the virus to organs and tissues. MAIN CONCLUSIONS Biomarker network analysis demonstrated distinct dynamics in concurrence with the bimodal viremia profiles at different time points during ZIKV infection. Such a robust cytokine and chemokine response has been associated with blood-brain barrier permeability and neuroinvasiveness in other flaviviral infections. High-dimensional data analysis further identified CXCL10, a chemokine involved in foetal neuron apoptosis and Guillain-Barré syndrome, as the most promising biomarker of acute ZIKV infection for potential clinical application.

Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

ABSTRACT We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

Desenvolvimento da CEL: equipamento de baixo-custo parareação e leitura de ensaios LAMP - Loop-mediated Isothermal Amplification / Development of CEL: a low-cost equipment for Loop-mediated Isothermal Amplification (LAMP) assays

O sistema de saúde brasileiro é constituído por um conjunto de ações e serviços que prestam assistência a população por meio de estratégias que visam a promoção, proteção e recuperação da saúde. Um dos pontos de maior destaque é a prevenção, na qual incluem-se o diagnóstico e o tratamento precoce das doenças. A detecção e a identificação clássica de patógenos baseiam-se na microscopia e cultura, entretanto a baixa sensibilidade; a necessidade de profissionais capacitados e de infraestrutura adequada resultam, em alguns casos, na falha do diagnóstico e no atraso para o início do tratamento. Objetivo: desenvolver um equipamento para realização de ensaios LAMP (Loop Mediated Isothermal Amplification) em ambientes com reduzida infraestrutura laboratorial. Resultados: Foram padronizados protocolos para cinco importantes doenças encontradas na região amazônica: tuberculose, malária, dengue e as febres mayaro e oropouche para utilização na CEL, equipamento portátil para a realização dos ensaios LAMP. O equipamento possui detecção fotométrica integrada, com capacidade de oito reações simultâneas, detectando a alteração da cor nas reações positivas. O resultado é mostrado em um display alfanumérico, de fácil leitura, mesmo para pessoas sem experiência com a técnica. Os resultados também podem ser transferidos por bluetooth para um smartphone, onde é possível, com o aplicativo próprio fazer a visualização gráfica. Conclusão: por se tratar de um equipamento de baixo-custo, desenvolvido para a aplicação em diagnóstico molecular, pode representar uma alternativa para ampliação da oferta de diagnóstico molecular nos serviços da rede básica de saúde, permitindo maior acesso da população, mesmo em áreas remotas.

The Brazilian Health System consists of a set of actions and services that assist the population through strategies aimed at the promotion, protection, and health recovery. One of the highlights is prevention, which includes the diagnosis and early treatment of diseases. The detection and classical identification of pathogens are based on microscopy and culture, however the low sensitivity; the need for trained professionals and adequate infrastructure leads, in some cases, to the failure of the diagnosis and in the delay to start treatment. Objective: to develop CEL, an equipment for LAMP (Loop-Mediated Isothermal Amplification) assays for use in low-resource settings laboratories. Results: Protocols were standardized for five important diseases found in the Amazon region: tuberculosis, malaria, dengue, mayaro and oropouche fevers. The equipment has integrated photometric detection, with the capacity of eight simultaneous reactions, detecting the color change observed in the positive reactions. The results are shown in an easy-to-read alphanumeric display, even for people with no experience with the technique. The results can also be transferred by bluetooth to a smartphone with the CEL App, where it is possible to see the results in a graphical interface. Conclusion: Once CEL is a low-cost device, developed for molecular diagnostics, it can represent an alternative to the expansion of the molecular diagnosis in the services of the primary health attention, allowing higher population access, even in remote areas.

Diversity of group A rotavirus genes detected in the Triângulo Mineiro region, Minas Gerais, Brazil

ABSTRACT Group A rotaviruses are the main causative agent of infantile gastroenteritis. The segmented nature of the viral genome allows reassortment of genome segments, which can generate genetic variants. In this study, we characterized the diversity of the VP7, VP4 (VP8*), VP6, NSP4, and NSP5 genes of the rotaviruses that circulated from 2005 to 2011 in the Triângulo Mineiro (TM) region of Brazil. Samples with genotypes G2 (sublineages IVa-1 and IVa-3), G1 (sublineage I-A), G9 (lineage III), G12 (lineages II and III), G8 (lineage II), G3 (lineage III), P[4] (sublineages IVa and IVb), P[8] (sublineages P[8]-3.6, P[8]-3.3, and P[8]-3.1), I2 (lineage VII), E2 (lineages VI, XII, and X), and H2 (lineage III) were identified. The associations found in the samples were G1, G9, or G12 with P[8]-I1-E1-H1; G2 or G8 with P[4]-I2-E2-H2; G12 with I3-E3-H6; and G3 with P[4]-I2-E3-H3 (previously unreported combination). Reassortment events in G2P[4] strains and an apparent pattern of temporal segregation within the lineages were observed. Five TM samples contained genes that exhibited high nucleotide and amino acid identities with strains of animal origin. The present study includes a period of pre- and post-introduction of rotavirus vaccination in all Brazilian territories, thereby serving as a basis for monitoring changes in the genetic constitution of rotaviruses. The results also contribute to the understanding of the diversity and evolution of rotaviruses in a global context.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatuscaptured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatuspools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

False-negative eengue cases in roraima, Brazil:an approach regarding the high number of negative results by ns1 ag kits / Casos de dengue falso-negativo em Roraima, Brasil: abordagem acerca do alto número de testes Antígeno NS1 negativos

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue infection.

Amostras séricas de 150 pacientes, com diagnóstico presuntivo de dengue e resultado negativo para dengue por ELISA-NS1-Antígeno do kit Platelia™ (NS1-Ag), foram analisadas pela técnica de TaqMan Transcrição Reversa seguida da Reação em Cadeia da Polimerase em Tempo Real (qRT-PCR). As amostras positivas por qRT-PCR, foram submetidas a identificação dos sorotipos por RT-Hemi nested-PCR e a ensaio imunocromatográfico para detecção qualitativa dos anticorpos IgG e IgM. A técnica molecular apresentou como resultado 33 (22%) amostras positivas entre as 150 negativas pela detecção do NS1-Ag, destas o 72% foram coletadas até o segundo dia de início dos sintomas da doença, período de maior sensibilidade para pesquisas de NS1-Ag. A maioria dos casos não evidenciou infecção secundária. Dessas amostras, 28 foram satisfatoriamente sorotipadas sendo 75% de DENV-4, 14% de DENV-2, 7% de DENV-3 e 4% de DENV-1. Os resultados reafirmam a situação hiperendêmica do Estado de Roraima e sugerem baixa sensibilidade do NS1 test, especialmente quando o sorotipo predominante é DENV-4. Sugerimos assim, que a comunidade médica deve ser alertada no sentido de ser cautelosa com resultados de NS1-Ag negativo, principalmente quando sintomas clínicos e outros resultados laboratoriais sejam indicativos de provável infecção por dengue.

Detection of Mycobacterium leprae in saliva and the evaluation of oral sensitivity in patients with leprosy

The aim of this study was to investigate sensitivity disorders in the oral cavity related to the presence of Mycobacterium leprae in the saliva of treatment-naïve patients with leprosy in the state of Amazonas, Brazil. A cross-sectional study was conducted involving 45 subjects with leprosy. The subjects were interviewed to evaluate the sensitivity of the oral cavity. For the detection of M. leprae, saliva and slit-skin smear samples were collected. The samples were analysed using a bacteriological index (BI) protocol and the real-time quantitative polymerase chain reaction (qPCR). The results indicated that 15 of the 45 (33.3%) subjects with leprosy showed decreased oral sensitivity, which confirmed the importance of the oral cavity sensitivity evaluation. There was not a direct relationship between the presence of M. leprae in saliva and changes in oral sensitivity. Positive saliva qPCR results from six (31.6%) of 19 paucibacillary (PB) patients suggested the possibility of a new site for sample collection. Positive results using these diagnostic techniques (BI, slit-skin smear and saliva qPCR) increased to 55.5%, thus opening the possibility of combining these different techniques to increase the rate of positive diagnoses, especially in PB patients.

16S rRNA gene-based identification of microbiota associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga (Diptera, Psychodidae) from central Amazon, Brazil

Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria.

Co-infection of Dengue virus by serotypes 3 and 4 in patients from Amazonas, Brazil / Co-infecção pelo vírus dengue 3 e 4 em pacientes da Amazônia brasileira

The natural co-infection with dengue virus can occur in highly endemic areas where different serotypes have been observed for many years. We report here four cases of DENV-3/DENV-4 co-infection detected by serological and molecular tests among 674 patients with acute undifferentiated fever from the tropical medicine reference center of Manaus City, Brazil, between 2005 and 2010. Analysis of the sequences obtained indicated the presence of genotype 3 and 1 for DENV-3 and DENV-4 respectively.

A co-infecção natural com os vírus dengue pode ocorre em áreas altamente endêmicas onde diferentes sorotipos têm sido transmitidos por muitos anos. Relatamos aqui quatro casos de co-infecção com DENV-3/DENV-4 detectados por testes sorológicos e moleculares entre 674 pacientes com febre indiferenciada aguda, atendidos em um centro de medicina tropical de referência da cidade de Manaus, Brasil, entre 2005 e 2010. As análises das sequências obtidas indicaram a presença dos genotipos 3 e 1 para DENV-3 e DENV-4 respectivamente.

Extended-spectrum beta-lactamase-producing bacteria isolated from hematologic patients in Manaus, State of Amazonas, Brazil

Antibiotic therapy in hematologic patients, often weak and susceptible to a wide range of infections, particularly nosocomial infections derived from long hospitalization periods, is a challenging issue. This paper presents ESBL-producing strains isolated from such hematologic patients treated at the Amazon Hematology and Hemotherapy Foundation (HEMOAM) in the Brazilian Amazon Region to identify the ESBL genes carried by them as well as the susceptibility to 11 antimicrobial agents using the E-test method. A total of 146 clinical samples were obtained from July 2007 to August 2008, when 17 gram-negative strains were isolated in our institution. The most frequent isolates confirmed by biochemical tests and 16S rRNA sequencing were E. coli (8/17), Serratia spp. (3/17) and B.cepacia (2/17). All gram-negative strains were tested for extended-spectrum-beta-lactamases (ESBLs), where: (12/17) strains carried ESBL; among these, (8/12) isolates carried blaTEM, blaCTX-M, blaOXA, blaSHV genes, (1/12) blaTEM gene and (3/12) blaTEM, blaCTX-M, blaOXA genes. Antibiotic resistance was found in (15/17) of the isolates for tetracycline, (12/17) for ciprofloxacin, (1/17) resistance for cefoxitin and chloramphenicol, (1/17) for amikacin and (3/17) cefepime. This research showed the presence of gram-negative ESBL-producing bacteria infecting hematologic patients in HEMOAM. These strains carried the blaTEM, blaSHV, blaCTX-M and blaOXA genes and were resistant to different antibiotics used in the treatment. This finding was based on a period of 13 months, during which clinical samples from specific populations were obtained. Therefore, caution is required when generalizing the results that must be based on posological orientations and new breakpoints for disk diffusion and microdilution published by CLSI 2010.