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.

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.

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.