ABSTRACT The precise pathogenesis of COVID-19-related multisystem inflammatory syndrome remains largely elusive, despite its rarity. The syndrome symptoms often overlap with those of other infections, posing challenges for prompt diagnosis. A male patient, 34 years old, was admitted with suspicion of severe dengue, rapidly progressing to multiple organ dysfunction. Dengue tests resulted negative, and he passed away after four days. This case occurred approximately four weeks after the initial onset of COVID-19 and met all diagnostic criteria as defined by the Centers for Disease Control and Prevention. This report presents the first documented case of fatal multisystem inflammatory syndrome in adult (MIS-A) in Brazil. Recognizing the significance of suspecting this syndrome and promptly initiating treatment at an early stage are essential for minimizing damage and mortality.

BACKGROUND Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes. OBJECTIVES This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility. METHODS KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique. FINDINGS No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed. MAIN CONCLUSIONS KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

BACKGROUND Dengue virus type 4 (DENV-4) was first reported in Brazil in 1982 and since then no more cases were detected again in Brazil until 2010, when the virus was reintroduced. Over the following years, the virus spread to several Brazilian states and resulted in about 1,400,000 dengue cases, in 2013. The largest number of cases were documented in the Southeast macro-region. OBJECTIVES To determine the phylogeography of DENV-4 Genotype IIB strains isolated during the epidemics in 2012-2013 in São Paulo, Brazil, we aimed to contextualise the contribution of viruses sampled in different localities across the overall movement of DENV-4 in Brazil. METHODS Based on the envelope gene sequences retrieved from GenBank, we employed a Bayesian phylogeographic approach to assess the spatiotemporal dynamics of DENV-4 Genotype IIB in São Paulo, Brazil. FINDINGS The dispersal dynamics of DENV-4 Genotype IIB in Brazil indicated Rio de Janeiro and Mato Grosso states as the most likely routes toward São Paulo before the 2012-2013 outbreak. Likewise, Guarujá and São José do Rio Preto facilitated viral spread and transmission to other localities in the South and Southeast macro-regions in Brazil. CONCLUSIONS The spread pattern of DENV-4 Genotype IIB strains across the country supports two independent introductions of the virus in São Paulo in a short period of time. Furthermore, São Paulo appears to have played a pivotal role in the dissemination of DENV-4 to other locations in Brazil.

ABSTRACT Many countries in the Americas have detected local transmission of multiple arboviruses that cause febrile illnesses. Therefore, laboratory testing has become an important tool for confirming the etiology of these diseases. The present study aimed to compare the sensitivity and specificity of three different Zika virus detection assays. One hundred serum samples from patients presenting with acute febrile symptoms were tested using a previously reported TaqMan® RT-qPCR assay. We used a SYBR® Green RT-qPCR and a conventional PCR methodologies to compare the results. Of the samples that were determined to be negative by the TaqMan® RT-qPCR assay, 100% (Kappa = 0.670) were also found to be negative by SYBR® Green RT-qPCR based on Tm comparison; however, 14% (Kappa = 0.035) were found to be positive by conventional PCR followed by agarose gel electrophoresis. The differences between the ZIKV strains circulating worldwide and the low viremia period can compromise diagnostic accuracy and thereby the accuracy of outbreak data. Therefore, improved assays are required to improve the diagnosis and surveillance of arbovirus.

RESUMO Objetivos Desenvolver um novo método de vigilância epidemiológica da dengue e demonstrar a sua aplicação. Métodos Estudo de coorte retrospectiva conduzido com dados obtidos da Secretaria de Saúde de São José do Rio Preto, Estado de São Paulo, Brasil. As coordenadas geográficas foram obtidas com o software QGIS™ (Creative Commons Corporation, Mountain View, Califórnia, EUA) segundo os endereços dos pacientes registrados no sistema de notificação do Programa Nacional de Controle da Dengue do Ministério da Saúde do Brasil. Foi usado o software SaTScan™ (Martin Kulldorff, Boston, Massachusetts, EUA) com o método Scan espaçotemporal para a detecção de conglomerados espaciais de casos de dengue com significância estatística. Os resultados foram representados graficamente e visualizados com a ferramenta de geomapeamento Google Earth™ (Google Incorporated, Mountain View, Califórnia, EUA). Resultados Detectou-se um número maior de conglomerados espaciais ao se estabelecer o número máximo de domicílios por conglomerado em 10% em comparação a 50% (11 versus 8 conglomerados com significância estatística). O raio dos conglomerados espaciais detectados variou de 0,18 km a 2,04 km e o período de tempo oscilou entre 6 dias e 6 meses. A taxa de infecção foi superior a 0,5 caso por domicílio. Conclusões O número máximo de domicílios por conglomerado deve ser de 10% ao conduzir a análise da distribuição espacial com o SaTScan. Este método pode contribuir para melhorar o desempenho dos sistemas de vigilância da dengue, sobretudo nos países com recursos limitados e resultados insatisfatórios nos programas nacionais de controle da doença.

ABSTRACT Objectives To develop and demonstrate the use of a new method for epidemiological surveillance of dengue. Methods This was a retrospective cohort study using data from the Health Department of São José do Rio Preto (São Paulo, Brazil). The geographical coordinates were obtained using QGIS™ (Creative Commons Corporation, Mountain View, California, United States), based on patient addresses in the dengue notification system of the Government of Brazil. SaTScan™ (Martin Kulldorff, Boston, Massachusetts, United States) was then used to create a space-time scan analysis to find statistically significant clusters of dengue. These results were plotted and visualized using Google Earth™ mapping service (Google Incorporated, Mountain View, California, United States). Results More clusters were detected when the maximum number of households per cluster was set to 10% (11 statistically significant clusters) rather than 50% (8 statistically significant clusters). The cluster radius varied from 0.18 – 2.04 km and the period of time varied from 6 days – 6 months. The infection rate was more than 0.5 cases/household. Conclusions When using SaTScan for space-time analysis of dengue cases, the maximum number of households per cluster should be set to 10%. This methodology may be useful to optimizing dengue surveillance systems, especially in countries where resources are scarce and government programs have not had much success controlling the disease.

RESUMEN Objetivos Elaborar un método nuevo para la vigilancia epidemiológica del dengue y hacer una demostración sobre su uso. Métodos Se realizó un estudio retrospectivo de cohortes usando datos del Departamento de Salud de São José do Rio Preto (São Paulo, Brasil). Partiendo de la dirección de los pacientes en el sistema de notificación del dengue del Gobierno de Brasil, se usó QGIS™ (Creative Commons Corporation, Mountain View, California, Estados Unidos) para obtener sus coordenadas geográficas. Luego se usó SaTScan™ (Martin Kulldorff, Boston, Massachusetts, Estados Unidos) para crear un análisis de exploración espacio-temporal a fin de detectar conglomerados de dengue estadísticamente significativos. Por último, se usó el servicio de mapas de Google Earth™ (Google Incorporated, Mountain View, California, Estados Unidos) para graficar y visualizar estos resultados. Resultados Se detectaron más conglomerados cuando el número máximo de viviendas por conglomerado se estableció en 10% (11 conglomerados estadísticamente significativos) en lugar de 50% (8 conglomerados estadísticamente significativos). El radio de los conglomerados se ubicó entre 0,18 y 2,04 km, y la duración entre 6 días y 6 meses. La tasa de infección fue de más de 0,5 casos por vivienda. Conclusiones Cuando se usa SaTScan, el número máximo de viviendas por conglomerado debe establecerse en 10% para el análisis de exploración. Este método puede ser útil para optimizar los sistemas de vigilancia del dengue, especialmente en aquellos países donde los recursos son escasos y los programas gubernamentales no han tenido mucho éxito para controlar la enfermedad.

ABSTRACT Arboviruses pose a serious threat to public health worldwide, overloading the healthcare system and causing economic losses. These viruses form a very diverse group, and in Brazil, arboviruses belonging to the families Flaviviridae and Togaviridae are predominant. Unfortunately, the number of arboviruses increases in proportion with factors such as deforestation, poor sanitation, climate changes, and introduction of new viruses like Chikungunya virus and Zika virus. In Brazil, dengue is endemic, along with the presence of other arboviruses. The situation is complicated by the scarcity of diagnostic infrastructure and the absence of approved vaccines for these diseases. Disease control, thus, relies solely on vector control. Therefore, enhanced clinical knowledge and improved general awareness about these arboviruses are indispensable to tackle diagnostic inadequacies.

Abstract Arboviruses impose a serious threat to public health services. We report a case of a patient returning from a work trip to the Amazon basin with myalgia, arthralgia, fever, and headache. During this travel, the patient visited riverside communities. Both dengue and Chikungunya fevers were first suspected, tested for, and excluded. Mayaro fever was then confirmed by reverse transcription polymerase chain reaction followed by next-generation sequencing and phylogenetic reconstruction. The increased awareness of physicians and consequent detection of Mayaro virus in this case was only possible due a previous surveillance program with specific health personnel training about these neglected arboviruses.

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

The Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), located in Manaus, the capital of the State of Amazonas (Western Brazilian Amazon), is a pioneering institution in this region regarding the syndromic surveillance of acute febrile illness, including arboviral infections. Based on the data from patients at the FMT-HVD, we have detected recurrent outbreaks in Manaus by the four dengue serotypes in the past 15 years, with increasing severity of the disease. This endemicity has culminated in the simultaneous circulation of all four serotypes in 2011, the first time this has been reported in Brazil. Between 1996 and 2009, 42 cases of yellow fever (YF) were registered in the State of Amazonas, and 71.4% (30/42) were fatal. Since 2010, no cases have been reported. Because the introduction of the yellow fever virus into a large city such as Manaus, which is widely infested by Aedes mosquitoes, may pose a real risk of a yellow fever outbreak, efforts to maintain an appropriate immunization policy for the populace are critical. Manaus has also suffered silent outbreaks of Mayaro and Oropouche fevers lately, most of which were misdiagnosed as dengue fever. The tropical conditions of the State of Amazonas favor the existence of other arboviruses capable of producing human disease. Under this real threat, represented by at least 4 arboviruses producing human infections in Manaus and in other neighboring countries, it is important to develop an efficient public health surveillance strategy, including laboratories that are able to make proper diagnoses of arboviruses.

ANTECEDENTES: O citomegalovírus (CMV) permanece um importante patógeno para pacientes imunocomprometidos, mesmo na era da HAART. O presente estudo teve como objetivo avaliar a influência da carga viral do CMV e seu genótipo gB sobre a evolução de pacientes com AIDS. MÉTODOS: Amostras de sangue de 101 pacientes com AIDS foram coletadas e testadas para carga viral de HIV, a contagem de células CD4 e patógenos oportunistas, incluindo o CMV. Um sistema de PCRs seminested foi utilizado para detectar o genoma do CMV e em amostras positivas a carga viral de CMV e genotipagem foram estabelecidos por restrição enzimática e PCR em tempo real, respectivamente. Todos os pacientes foram acompanhados clinicamente durante quatro anos. RESULTADOS: Trinta pacientes (31%) tiveram CMV detectado e todos os casos fatais (n = 5) ocorreram em pacientes deste grupo (p = 0,007), porém apenas dois pacientes tinham doença por CMV (1,9%). No entanto, a carga viral não foi associada estatisticamente a nenhum dos parâmetros analisados. O genótipo de CMV mais freqüentemente observado foi gB2 (45,16%), seguido por gB3 (35,48%). O genótipo gB2 foi mais freqüente em pacientes com contagens abaixo de 200 células/mm³ CD4cell (p = 0,0017), e quase todos os casos fatais (80%) tinham o genótipo gB2. CONCLUSÃO: Nosso estudo sugere que CMV e seu polimorfismo em genes relevantes biologicamentes, como a gB, pode ainda influenciar no prognóstico e evolução de pacientes com AIDS. O genótipo gB2 foi associado ao mau prognóstico do paciente.

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

INTRODUÇÃO: A região Amazônica possui extensas áreas florestadas e ecossistemas naturais, provendo condições favoráveis para a existência de diversos arbovírus. Aproximadamente, 200 arbovírus foram isolados no Brasil, e 40 estão associados com doenças em humanos. Quatro destes 40 são considerados ser de importância para a saúde pública no Brasil: vírus da dengue (1-4), Oropouche, Mayaro e febre amarela. Juntamente com estes vírus, aproximadamente 98% dos casos de malária estão restritos à região da Amazônia Legal. MÉTODOS: O objetivo deste estudo foi investigar a presença de arbovírus em 111 amostras clínicas de sangue de pacientes que residiam em Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Amapá (Macapá). O RNA viral foi extraído, RT-PCR foi realizada seguida de uma Multiplex-Nested-PCR, usando primers genéricos e espécie-específicos para Flavivirus, Alphavirus and Orthobunyavirus. RESULTADOS: Detectamos o vírus da dengue, sorotipo 2, em dois pacientes que residiam em Novo Repartimento (Pará), que também tinham infecção por Plasmodium vivax. CONCLUSÕES: Apesar de dados escassos, esta situação, provavelmente, ocorre mais frequência que a detectada na região Amazônica. Definitivamente, é importante lembrar que ambas as doenças possuem achados clínicos similares, assim o diagnóstico deveria ser feito concomitantemente para dengue e malária em pacientes que residem ou estão voltando de áreas onde ambas as doenças são endêmicas.

INTRODUCTION: The Amazon region has extensive forested areas and natural ecosystems, providing favorable conditions for the existence of innumerous arboviruses. Over 200 arboviruses have been isolated in Brazil and about 40 are associated with human disease. Four out of 40 are considered to be of public health importance in Brazil: Dengue viruses (1-4), Oropouche, Mayaro and Yellow Fever. Along with these viruses, about 98% of the malaria cases are restricted to the Legal Amazon region. METHODS: This study aimed to investigate the presence of arboviruses in 111 clinical serum samples from patients living in Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Oiapoque (Amapá). The viral RNA was extracted and RT-PCR was performed followed by a Multiplex-Nested-PCR, using Flavivirus, Alphavirus and Orthobunyavirus generic and species-specific primers. RESULTS: Dengue virus serotype 2 was detected in two patients living in Novo Repartimento (Pará) that also presented active Plasmodium vivax infection. CONCLUSIONS: Despite scant data, this situation is likely to occur more frequently than detected in the Amazon region. Finally, it is important to remember that both diseases have similar clinical findings, thus the diagnosis could be made concomitantly for dengue and malaria in patients living or returning from areas where both diseases are endemic or during dengue outbreaks.

OBJETIVO: Avaliação dos pacientes com o quadro clínico de ceratite herpética (CH) típicas e atípicas, pela reação em cadeia da polimerase (PCR) correlacionando com o diagnóstico clínico. MÉTODOS: Foi realizada a PCR em 28 pacientes com ceratite herpética típica e atípica. RESULTADOS: A PCR foi positiva em 57,14% (n=16) do total (n=28). Nos casos de CH típica a positividade foi de 60,00% (n=12) em 20 casos. Para CH epitelial a positividade foi de 69,23% (n=9), sendo 77,78% (n=7) apenas para as lesões epiteliais dendríticas. Os casos de CH atípica apresentaram positividade de 50% (n=4) em oito casos. CONCLUSÃO: Quadro clínico típico de CH teve boa correlação com o resultado positivo observado na PCR. Entretanto, metade dos pacientes com o quadro de CH atípica apresentou PCR positivo, portanto, o exame do PCR é teste importante para o auxílio e diagnóstico da CH. No caso de CH estromal, foi demonstrado que a técnica da PCR conseguiu identificar o vírus HSV.

PURPOSE: To evaluate patients with clinically typical and atypical herpetic keratitis (HK) by means of polymerase chain reaction (PCR) as compared with the clinical diagnosis. METHODS: PCR in 28 patients with clinical symptoms of typical and atypical HK was performed. RESULTS: PCR was positive in 57.14% (n=16) of the total cases (n=28). The test was positive in 60.0% (n=12) of the 20 typical HK cases. For epithelial HK, the test was positive in 69.23% (n=9), and 77.78% (n=7) only for dendritic injuries. Atypical HK presented a positive test in 50% (n=4) of eight cases. CONCLUSION: Clinical typical picture of HK had a good correlation with the positive result of PCR, mainly for epithelial injury of the dendritic type. However, 50% of the patients with atypical HK presented positive PCR. This result showed that PCR test can provide an effective HK diagnosis. In the stromal case of HK, PCR was a useful technique to identify HSV virus.

OBJETIVO: Vírus da família Adenoviridae estão associados com muitas síndromes clínicas, sendo conhecidos 50 sorotipos. Vírus desta família e da família Herpesviridae são os maiores responsáveis por conjuntivite viral, sendo um rápido diagnóstico importante devido ao caráter epidêmico das infecções por adenovírus. MÉTODOS: Reação em cadeia da polimerase (PCR) para adenovírus foi desenvolvida utilizando iniciadores que amplificam um fragmento de 300 bp do gene da proteína hexon do capsídeo de diversos sorotipos. A reação em cadeia da polimerase foi efetuada sem a etapa de extração de DNA. RESULTADOS: Amostras de "swabs" de córneas de sete pacientes com ceratoconjuntivite foram analisadas, sendo que uma amostra foi positiva para adenovírus. O seqüenciamento deste fragmento mostrou 100% de homologia com a seqüência do adenovírus tipo 8. CONCLUSÃO: O seqüenciamento do fragmento de 300 bp do gene do hexon permite a identificação de quase todos os sorotipos de adenovírus, incluindo aqueles relacionados com a ceratoconjuntivite epidêmica (8,19,37) e todas as amostras associadas com conjuntivite.

PURPOSE: Viruses of the Adenoviridae family are associated with many clinical syndromes, possessing 50 serotypes. These agents and viruses of the Herpesviridae family are the two major agents responsible for viral conjunctivitis, and a rapid diagnosis is important due to the epidemic character of adenoviral infections. METHODS: We developed a PCR without DNA extraction for adenovirus using primers that amplify a 300 bp fragment of the hexon capsid protein gene from many serotypes. RESULTS: Swab samples from cornea of seven patients with keratoconjunctivitis were analyzed, and one of them was PCR positive for adenovirus. The sequence of this fragment shows a 100% homology with the sequence of adenovirus type 8. CONCLUSION: Sequencing of 300 bp from the hexon gene allows to identify almost all Ad serotypes, including all serotypes related to epidemic keratoconjunctivitis (8,19,37) and almost all serotypes involved with Ad-associated conjunctivitis.