Complete genome analysis and characterization of neurotropic dengue virus 2 cosmopolitan genotype isolated from the cerebrospinal fluid of encephalitis patients.

Dengue virus (DENV) infection remains a major public health concern in many parts of the world, including Southeast Asia and the Americas. Sri Lanka experienced its largest dengue outbreak in 2017. Neurological symptoms associated with DENV infection have increasingly been reported in both children and adults. Here, we characterize DENV type 2 (DENV-2) strains, which were isolated from cerebrospinal fluid (CSF) and/or serum of patients with dengue encephalitis. Acute serum and CSF samples from each patient were subjected to dengue-specific non-structural protein 1 (NS1) antigen test, IgM and IgG enzyme-linked immunosorbent assay (ELISA), virus isolation, conventional and real-time polymerase chain reaction (PCR), and next-generation sequencing (NGS). Among the 5 dengue encephalitis patients examined, 4 recovered and 1 died. DENV-2 strains were isolated from serum and/or CSF samples of 3 patients. The highest viral genome levels were detected in the CSF and serum of the patient who succumbed to the illness. A phylogenetic tree revealed that the DENV-2 isolates belonged to a new clade of cosmopolitan genotype and were genetically close to strains identified in China, South Korea, Singapore, Malaysia, Thailand, and the Philippines. According to the NGS analysis, greater frequencies of nonsynonymous and synonymous mutations per gene were identified in the nonstructural genes. The full genomes of serum- and CSF-derived DENV-2 from the same patient shared 99.7% similarity, indicating that the virus spread across the blood-brain barrier. This is the first report to describe neurotropic DENV-2 using whole-genome analysis and to provide the clinical, immunological, and virological characteristics of dengue encephalitis patients during a severe dengue outbreak in Sri Lanka in 2017.

Japanese Encephalitis- and Dengue-Associated Acute Encephalitis Syndrome Cases in Myanmar.

This study was conducted to find the burden of dengue virus (DENV) and Japanese encephalitis virus (JEV) among children under the age of 13, who presented with acute encephalitis syndrome at Mandalay Children Hospital in Myanmar in 2013. Molecular and serological investigations were performed on 123 cerebrospinal fluid (CSF) samples collected from these patients. By neutralization tests and/or virus isolation, four (3.3%) JEV- and one DENV-associated encephalitis cases (0.8%) were confirmed. Antibody titer against JEV Genotype 3 was the highest among the laboratory-confirmed JEV cases. One strain of DENV-1 with Genotype 1 was isolated from the CSF sample of the dengue encephalitis patient; this was similar to the virus circulating in the study area and neighboring countries. This study shows that flaviviruses are important pathogens causing encephalitis in Myanmar. Active disease surveillance, vector control, and vaccination programs should be enforced to reduce the morbidity and mortality caused by flavivirus encephalitis.

Importance of cerebrospinal fluid investigation during dengue infection in Brazilian Amazonia Region.

BACKGROUND Amazon, the largest tropical forest of the world, has suffered from dengue outbreaks since 1998. Cerebrospinal fluid (CSF) of patients, from Amazonas state, suspected of central nervous system (CNS) viral infection was studied using molecular and immunological methods. OBJECTIVE To evaluate the importance of CSF investigation in patients with acute dengue virus (DENV) infection of CNS. METHODS CSF samples of 700 patients were analysed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of dengue virus (DENV) RNA and by enzyme-linked immunosorbent assay (ELISA) to detect presence of DENV specific IgM. FINDINGS DENV infection was detected in 4.3% of the CSF samples; 85.7% (24/28) by DENV IgM and 14.3% (4/28) by viral RNA. DENV detected by viral RNA were to be found serotypes DENV-2 (three patients) and DENV-1 (one patient). The neurological diagnosis in patients CNS infection of DENV included encephalitis (10), meningoencephalitis (10), meningitis (6), acute myelitis (1), and encephalomyelitis (1). The majority (89.3%) had intrathecal inflammation: pleocytosis, hyperproteinorrachia and DENV IgM antibodies. Hypoglycorrhachia and/or high levels of lactate in CSF were found in 36% of the patients. Co-infection (CMV, HIV, EBV, and/or Mycobacterium tuberculosis) was observed in eight (28.6%) cases. CONCLUSIONS We found intense inflammatory CSF that is unusual in CNS disorders caused by dengue infection. It may be due co-infections or the immunogenetic background of the local Amerindian Brazilian population. CSF examination is an important diagnostic support tool for neurological dengue diagnosis.

Arboviral Surveillance among Pediatric Patients with Acute Febrile Illness in Houston, Texas.

We instituted active surveillance among febrile patients presenting to the largest Houston-area pediatric emergency department to identify acute infections of dengue virus (DENV), West Nile virus (WNV), and chikungunya virus (CHIKV). In 2014, 1,063 children were enrolled, and 1,015 (95%) had blood and/or cerebrospinal fluid specimens available for DENV, WNV, and CHIKV testing. Almost half (49%) reported recent mosquito bites, and 6% (N = 60) reported either recent international travel or contact with an international traveler. None were positive for acute WNV; three had false-positive CHIKV results; and two had evidence of DENV. One DENV-positive case was an acute infection associated with international travel, whereas the other was identified as a potential secondary acute infection, also likely travel-associated. Neither of the DENV-positive cases were clinically recognized, highlighting the need for education and awareness. Health-care professionals should consider the possibility of arboviral disease among children who have traveled to or from endemic areas.

Etiological agents of viral meningitis in children from a dengue-endemic area, Southeast region of Brazil.

Meningitis is a disease with a global distribution that constitutes a worldwide burden, with viruses as the primary etiologic agents. The range of viral meningitis severity depends mainly on age, immune status and etiological agent. The aim of this work was to investigate the suspected cases of viral meningitis using molecular techniques to confirm the viral infection. The diagnosed virus was correlated with clinical findings and cytochemical parameters in cerebrospinal liquid (CSF) of patients. CSF of 70 children with the presumptive diagnosis of viral meningitis was analyzed by real time PCR (qPCR). Viruses were identified by qPCR in 44 CSF samples (62.9%). Among them, 31 were identified as Enterovirus (ENTV) (70.4%), six as Human herpes virus 3 (HHV-3) (13.6%), five as Dengue virus (DENV) (11.7%), one as Human herpes virus 1-2 (2.3%) and one as Human herpes virus 5 (2.3%). Patients in the HHV-positive groups had increased percentage of polymorphonuclear neutrophils (PMN) (mean of 81%) while the groups of patients with DENV and ENTV had a mean of 30.9%. This study contributes to the knowledge of the epidemiological distribution of viral agents in CNS infections in children. In addition, it raises the relevance of DENV as an agent of CNS infection, and reinforces the importance for molecular in the cases of CNV infection.

Study of interleukin-6 and interleukin-8 levels in patients with neurological manifestations of dengue.

CONTEXT: Pro-inflammatory markers play a key role in the pathogenesis of various Flavivirus infection. AIM: In this study, we evaluated the role of these markers in neurological manifestations of dengue. SETTINGS AND DESIGNS: Consecutive dengue cases with different neurological manifestations who presented between August 2012 and July 2014 were studied in hospital-based case-control study. MATERIALS AND METHODS: Interleukin (IL-6) and IL-8 level were measured in serum and cerebrospinal fluid (CSF) of dengue cases with different neurological manifestations and also in age- and sex-matched controls. Level was analyzed with various parameters and outcomes. STATISTICAL ANALYSIS: Statistical analysis was done using SPSS version 16.0 by applying appropriate statistical methods. P < 0.05 considered statistically significant. RESULTS: Out of the 40 enrolled cases of dengue with neurological manifestations, 29 had central nervous system and 11 had peripheral nervous system (CNS/PNS) manifestations. In CNS group, both IL-6 and IL-8 (CSF and serum) were significantly elevated (P < 0.001), whereas CSF IL-6 (P = 0.008), serum IL-6 (P = 0.001), and serum IL-8 (P = 0.005) were significantly elevated in PNS group. CSF IL-6, serum IL-6, and IL-8 were significantly elevated in poor outcome patients in CNS group (P < 0.05). CSF IL-6 and IL-8 were significantly elevated in CSF dengue positive cases as compared to CSF negative patients (P < 0.05). Cytokine level was not significantly correlated with neuroimaging abnormality in CNS group. Nine patients died and the remainder recovered. CONCLUSION: Elevated level of IL-6 and IL-8 is associated with different neurological manifestations and poor outcome, but whether they are contributing to neuropathogenesis or simply a correlate of severe disease remains to be determined.

Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil.

To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014-2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2.

Neurological manifestations in children with dengue fever: an Indian perspective.

Ours was a descriptive observational cross-sectional study carried out in a tertiary care hospital in eastern India over a period of one year to study the profile of neurological involvement in paediatric dengue patients. Of 71 laboratory-confirmed cases, 20 (28.17%) had neurological involvement. Common forms observed were acute encephalopathy (40%), encephalitis (30%), pure motor weakness (15%), transverse myelitis (5%), acute disseminated encephalomyelitis (5%) and Guillain-Barré syndrome (5%). The dengue IgM antibody could be detected in the cerebrospinal fluid of only two patients with encephalitis. Neurological involvement was present in all four patients who died during the study period (two-tailed P value = 0.005).

Acute disseminated encephalomyelitis complicating dengue infection with neuroimaging mimicking multiple sclerosis: A report of two cases.

Acute disseminated encephalomyelitis (ADEM) complicating dengue infection is still exceedingly rare even in endemic countries such as Malaysia. Here we report two such cases, the first in an elderly female patient and the second in a young man. Both presented with encephalopathy, brainstem involvement and worsening upper and lower limb weakness. Initial magnetic resonance imaging (MRI) of the brain was normal in the first case. Serum for dengue Ig M and NS-1 was positive in both cases. Cerebrospinal fluid (CSF) showed pleocytosis in both with Dengue IgM and NS-1 positive in the second case but not done in the first. MRI brain showed changes of perpendicular subcortical palisading white matter, callosal and brainstem disease mimicking multiple sclerosis (MS) in both patients though in the former case there was a lag between the onset of clinical symptoms and MRI changes which was only clarified on reimaging. The temporal evolution and duration of the clinical symptoms, CSF changes and neuroimaging were more suggestive of Dengue ADEM rather than an encephalitis though initially the first case began as dengue encephalitis. Furthermore in dengue encephalitis neuroimaging is usually normal or rarely edema, haemorrhage, brainstem, thalamic or focal lesions are seen. Therefore, early recognition of ADEM as a sequelae of dengue infection with neuroimaging mimicking MS and repeat imaging helped in identifying these two cases. Treatment with intravenous steroids followed by maintenance oral steroids produced good outcome in both patients.

Respuesta neuroinmunológica en la encefalitis asociada al virus del dengue / Neuroimmunological response in dengue virus encephalitis

El virus del dengue es un virus ARN miembro de la familia Flaviviridae, la cual incluye, además, el de la fiebre amarilla, el del Nilo del Oeste y la encefalitis japonesa. Se realizó un estudio retrospectivo con tres pacientes diagnosticados de encefalitis asociada al dengue, en cuyas muestras de suero y líquido cefalorraquídeo se cuantificaron los niveles de las clases mayores de inmunoglobulinas por inmunodifusión radial y la manosa de unión a lectina, proteína de la vía de las lectinas del sistema del complemento por fluorometría. En el reibergrama se muestra la presencia de síntesis intratecal de las tres clases de inmunoglobulinas y ausencia de síntesis intratecal de lectina de unión a manosa. Existieron diferencias en cuanto al por ciento de síntesis intratecal de inmunoglobulinas, las cuales estuvieron relacionadas con el momento de la infección por el virus y la aparición de las manifestaciones neurológicas compatibles con una encefalitis. Este es el primer reporte de afectaciones neurológicas en pacientes cubanos con dengue. La respuesta inmune intratecal puede ser utilizada para el mejor conocimiento de la enfermedad y contribuir al desarrollo de posibles candidatos vacunales(AU)

Dengue virus is a RNA virus of the Flaviviridae family including also West Nile virus, Japanese encephalitis virus and yellow fever virus. A retrospective study with three patients diagnosed with encephalitis associated with dengue virus was carried out. Using their sera and cerebrospinal fluid samples the levels of high class of immunoglobulins by radial immunodiffusion and mannan binding lectin were quantified by fluorometry three classes of immunoglobulins levels were quantified by using an immunodiffusion technique. Mannose-binding lectin was quantified by fluorometry. A retrospective study with three patients diagnosed with encephalitis. The Reibergram has shown three classes of immunoglobulins intrathecal synthesis and absence of mannan binding lectin synthesis. Differences in the immunoglobulin intrathecal synthesis percent were observed, which were related with the moment of virus infection and the onset of neurological manifestations compatible with encephalitis. This is the first report of neurological affectations in Cuban patients with dengue fever. The intrathecal immune response may be used for the best knowledge of the disease and for the contribution to the development of possible candidate vaccine(AU)

Neurological complications in dengue infection: a review for clinical practice.

Dengue is an important global public health problem. The World Health Organization estimates that 2/5 of entire world population are in risk of dengue infection. Almost 50 millions cases occur annually, with at least 20 thousand deaths. The etiological agent of this acute febrile disease is a single-strand positive-sense RNA virus of Flavivirus genus. It is an arboviral disease transmitted by Aedes sp. mosquitoes (Aedes aegypti and A. albopictus). Most infected individuals present asymptomatic infection, but some may develop clinical signs. Therefore, a wide spectrum of illness can be observed, ranging from unapparent, mild disease, called dengue fever, to a severe and occasionally fatal dengue hemorrhagic fever/dengue shock syndrome. Currently, neurological manifestations related to dengue infections are increasingly been observed and appears as a challenge for medical practice. In this study the neurological complications of dengue infection will be reviewed, focusing a better understanding of the disease for the clinical practice.

Neurological complications in dengue infection: a review for clinical practice / Complicacoes neurologicas na dengue: uma revisao para a pratica clinica

Dengue is an important global public health problem. The World Health Organization estimates that 2/5 of entire world population are in risk of dengue infection. Almost 50 millions cases occur annually, with at least 20 thousand deaths. The etiological agent of this acute febrile disease is a single-strand positive-sense RNA virus of Flavivirus genus. It is an arboviral disease transmitted by Aedes sp. mosquitoes (Aedes aegypti and A. albopictus). Most infected individuals present asymptomatic infection, but some may develop clinical signs. Therefore, a wide spectrum of illness can be observed, ranging from unapparent, mild disease, called dengue fever, to a severe and occasionally fatal dengue hemorrhagic fever/dengue shock syndrome. Currently, neurological manifestations related to dengue infections are increasingly been observed and appears as a challenge for medical practice. In this study the neurological complications of dengue infection will be reviewed, focusing a better understanding of the disease for the clinical practice.

A dengue é um importante problema de saúde pública. A Organização Mundial de Saúde estima que 2/5 da população mundial encontra-se em risco de desenvolver a infecção. Cerca de 50 milhões de casos ocorrem anualmente, com ao menos 20 mil mortes. O agente etiológico desta doença febril aguda é um vírus RNA, do gênero Flavivirus. Este arbovírus é transmitido pelo mosquito Aedes sp. A maioria dos indivíduos infectados apresenta infecção assintomática, porém alguns desenvolvem sintomas clínicos. Estes manifestações podem variar desde uma doença inaparente, branda, conhecida como febre da dengue, até uma forma severa, sendo fatal em alguns casos como na febre hemorrágica da dengue/síndrome de choque da dengue. Atualmente, manifestações neurológicas associadas à dengue são cada vez mais frequentes, tornando-se um desafio na rotina médica. Neste estudo, as complicações neurológicas da dengue serão revisadas, com ênfase na melhor compreensão acerca da doença para a prática clínica.

Identification of Oropouche Orthobunyavirus in the cerebrospinal fluid of three patients in the Amazonas, Brazil.

Oropouche fever is the second most frequent arboviral infection in Brazil, surpassed only by dengue. Oropouche virus (OROV) causes large and explosive outbreaks of acute febrile illness in cities and villages in the Amazon and Central-Plateau regions. Cerebrospinal fluid (CSF) samples from 110 meningoencephalitis patients were analyzed. The RNA extracted from fluid was submitted to reverse transcription-polymerase chain reaction and sequencing to identify OROV. Three CSF samples showed the presence of OROV causing infection in the central nervous system (CNS). These patients are adults. Two of the patients had other diseases affecting CNS and immune systems: neurocysticercosis and acquired immunodeficiency syndrome, respectively. Nucleotide sequence analysis showed that the OROV from the CSF of these patients belonged to genotype I. We show here that severe Oropouche disease is occurring during outbreaks of this virus in Brazil.

Dengue in patients with central nervous system manifestations, Brazil.

We investigated the prevalence of dengue in patients with suspected viral meningitis/meningoencephalitis in a dengue-endemic area. Cerebrospinal fluid analysis showed positive results and a 6.74× greater likelihood of identifying positive fluid in patients who died. Our findings support testing patients with neurologic manifestations for the virus in dengue-endemic areas.

Cytokines and chemokines in viral encephalitis: a clinicoradiological correlation.

Japanese encephalitis (JE) is the commonest encephalitis in South East Asia associated with high morbidity and mortality. Neuronal injury is attributed to a number of proinflammatory cytokines. This study evaluates cerebrospinal fluid (CSF) cytokines and chemokines in encephalitis and correlates these with clinical and magnetic resonance imaging (MRI) findings. We examined 14 patients with encephalitis (8 JE, 1 dengue, 5 nonspecific encephalitis) and 10 healthy controls. CSF cytokines (IL-1beta, IL-6, IL-10, IL-12p70, TNF-alpha, IL-8) and chemokines (IP-10, MCP-1, MIG, IL-8 and RANTES) were estimated using Cytometric Bead Array, compared with controls and were correlated with severity of encephalitis, radiological findings and presence of movement disorders. Median age of the patients was 25.5 (range 6-55 years); 6 had seizures, 10 movement disorders and 6 out of 11 had MRI abnormalities. The MRI abnormalities included thalamic involvement in 5, basal ganglia and mid brain in 3 each and cortical involvement in 2 patients. Both the patients with cortical involvement had seizures and 5 of the 10 patients with movement disorders had thalamic, basal ganglia and/or mid brain involvement. There was significant increase in IL-6 (p=0.01), RANTES (p=0.02) and IL-8 (p=0.02) in encephalitis compared to controls but there was no difference in IL12p70, TNF-alpha, IL-10, IL-1beta and MCP-1. Cytokines and chemokines did not correlate with severity of encephalitis, radiological changes and presence of movement disorders. CSF IL-6, IL-8 and RANTES were significantly higher in encephalitis patients compared to controls.

Neurologic dengue manifestations associated with intrathecal specific immune response.

BACKGROUND: Dengue infection is caused by a flavivirus, with 4 virus serotypes (types 1 to 4). The serotypes 2 and 3 represent the principal agents related to nervous system involvement. Neurologic involvement occurs in 4%-5% of dengue infection cases. The major mechanisms of the disease may be related to direct viral infection or postinfectious autoimmune process. The detection of intrathecal synthesis of specific antibodies has been used to support neurologic diagnosis as a proof of local reaction. It may be quantitatively calculated by the specific antibody index. OBJECTIVES: To determine if patients with neurologic manifestations associated with dengue produce specific antibodies in the CNS and to determine the antibodies' clinical and pathophysiologic relevance. METHODS: CSF and serum were evaluated for dengue immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by ELISA and for intrathecal synthesis of IgG antibodies to the dengue virus. Subjects included 10 patients IgM seropositive for dengue virus diagnosed with myelitis, encephalitis, optic neuromyelitis, or Guillain-Barré syndrome. RESULTS: All patients had IgG and IgM antibodies to dengue virus in their sera; 7 were IgM positive and 9 were IgG positive for dengue virus in CSF. Only the 3 patients with myelitis had intrathecal synthesis of specific IgG antibodies. CONCLUSIONS: Intrathecal synthesis of antibodies to dengue virus occurs in the CNS. It may be used as a marker of myelitis associated with dengue, and it seems to be related to the pathogenesis of spinal cord disease due to direct viral invasion.

Oligosymptomatic dengue infection: a potential cause of Guillain Barré syndrome.

BACKGROUND: Dengue infection may cause neurological manifestations such as encephalitis, myelitis, mononeuropathies, acute disseminated encephalomyelitis, and Guillain Barré syndrome (GBS). In endemic regions, the infection course can be oligosymptomatic making difficult the diagnosis of the neurological picture associated with dengue infection. OBJECTIVE: To report dengue infection and GBS association, even in oligosymptomatic cases of this infection. METHOD: During the dengue epidemic in Rio de Janeiro city we looked for GBS cases, testing IgM antibodies for dengue and dengue polymerase chain reaction (PCR) in the cerebrospinal fluid (CSF) and serum. RESULTS: We report seven cases (46.6%), presenting dengue positive IgM in serum but with poor or without clinical symptoms of the previous infection. Two of them had also positive IgM antibodies in CSF. CONCLUSION: These data show that search for dengue infection should be a routine in GBS cases living in endemic areas.