Arboviruses epidemic associates with Guillain-Barré syndrome: temporal series in northeast Brazil / Epidemia de arboviroses associada à síndrome de Guillain-Barré: série temporal no nordeste brasileiro / Epidemia de arbovirosis asociada al síndrome de Guillain-Barré: serie temporal en el noreste de Brasil

Arboviruses cause public health problems in several countries, and records show that they can generate central and peripheral neurological complications with permanent sequelae. However, it is not certain which arbovirus is responsible for outbreaks of the Guillain-Barré Syndrome (GBS), especially in Brazil. Thus, the objective of this study is to verify if there is a coincidence between the GBS outbreak and the most common arboviruses in Northeastern Brazil, as well as their relationship. An ecological time series study was designed with the federative units of Northeastern Brazil, using hospitalizations for Guillain-Barré syndrome and notifications of arbovirus infections between 2014 and 2019 as a data source. Distribution incidence curves were constructed for the conditions studied, and generalized estimating equations (GEE) models were applied to estimate the relationship between arboviruses and Guillain-Barré. The results showed a similar distribution for the incidences of Chikungunya virus (z=7.82; p=0.001), Zika virus (z=3.69; p=0.03), and Guillain-Barré syndrome (z=2.98; p=0.05) from 2014 to 2019. The GEE model revealed that the distribution of Chikungunya incidence is associated with the distribution of GBS incidence in each year (x2Wald=3,969; p=0.046). This pattern was repeated in seven of the nine states, while the Zika virus had a significant relationship with GBS in only two states. The outbreak of GBS in Northeastern Brazil appears to be probabilistically related to outbreaks of the Chikungunya virus.

As arboviroses são problemas de saúde pública em vários países e há registros de que podem produzir complicações neurológicas centrais e periféricas com sequelas permanentes. Entretanto, não se sabe ao certo qual delas é realmente responsável pelos surtos da Síndrome de Guillain-Barré (SGB), principalmente no Brasil. Assim, o objetivo é verificar se há coincidência entre o surto de SGB e as arboviroses mais comuns no Nordeste do Brasil e suas relações. Foi desenhado um estudo ecológico de série temporal com as unidades federativas do Nordeste do Brasil, adotando como fonte de dados as internações Guillain-Barré e as notificações de infecções por arbovírus entre 2014 e 2019. Curvas de distribuição de incidência foram construídas para as condições estudadas, e foram aplicados modelos de equações generalizadas estimadas (GEE) para estimar a relação entre arbovírus e Guillain-Barré. Evidencia-se que há distribuição semelhante para as incidências do vírus Chikungunya (z=7,82; p=0,001), vírus Zika (z=3,69; p=0,03) e síndrome de Guillain-Barré (z=2,98; p=0,05) entre 2014 e 2019. O modelo GEE revelou que a distribuição da incidência de Chikungunya está associada à distribuição da incidência de SGB em cada ano (x2Wald=3,969; p=0,046). Esse padrão se repetiu em sete dos nove estados, enquanto o zika vírus teve uma relação significativa com o GBS em apenas dois estados. Conclui-se, então, que o surto de SGB no Nordeste do Brasil parece estar probabilisticamente relacionado aos surtos do vírus Chikungunya.

Los arbovirus causan problemas de salud pública en varios países y, según indican los reportes, pueden producir complicaciones neurológicas centrales y periféricas con secuelas permanentes. Sin embargo, no se sabe cuál de ellos es realmente el responsable de los brotes del síndrome de Guillain-Barré (SGB), especialmente en Brasil. Así, el objetivo de este estudio es verificar si existen coincidencias entre el brote del SGB y los arbovirus más comunes en el Noreste de Brasil y sus asociaciones. Se diseñó un estudio de series temporales ecológico en las unidades federativas del Noreste de Brasil, adoptando como fuente de datos las hospitalizaciones y las notificaciones de arbovirosis de Guillain-Barré entre 2014 y 2019. Se construyeron curvas de distribución de incidencia para las condiciones científicas, y se aplicó una ecuación estimada generalizada (GEE) para estimar la relación entre arbovirus y Guillain-Barré. Se encontró que existe una distribución similar en las incidencias de virus del chikunguña (z=7,82; p=0,001), virus del Zika (z=3,69; p=0,03) y síndrome de Guillain-Barré (z =2,98; p=0,05) entre 2014 y 2019. El modelo GEE reveló que la distribución de la incidencia de chikunguña está asociada con la distribución de la incidencia de SGB en cada año (x2Wald=3,969; p=0,046). Este patrón se repitió en siete de los nueve estados, mientras que el virus del Zika presentó una relación significativa con el SGB en solo dos estados. El brote del SGB en el Noreste de Brasil parece estar relacionado probabilísticamente con los brotes del virus del chikunguña.

A spatial case-control study on symptomatic and inapparent primary dengue infections in an endemic city in Brazil

ABSTRACT We conducted a spatial case-control study nested in a dengue incidence cohort to explore the role of the spatial and socioeconomic factors in the proportion of symptomatic (cases) and inapparent primary dengue virus infections (controls). Cohort participants were children and adolescents (2 to 16 years of age) at the beginning of the follow-up. Case definitions were, for symptomatic cases, fever plus a positive lab result for acute dengue (NS1, RT-PCR, ELISA IgM/IgG), and for inapparent infection a positive result for dengue IgG (ELISA) in subjects without symptoms and with a previously negative result at baseline. The covariates included sociodemographic factors, residential location, and socioeconomic context variables of the census tracts of residence of cases and controls. We used principal component analysis to reduce the contextual covariates, with the component values assigned to each one based on their residences. The data were modeled in a Bayesian context, considering the spatial dependence. The final sample consisted of 692 children, 274 cases and 418 controls, from the first year of follow-up (2014-2015). Being male, older age, higher educational level of the head of the family and having a larger number of rooms in the household were associated with a greater chance of presenting dengue symptomatic infection at the individual level. The contextual covariates were not associated with the outcome. Inapparent dengue infection has extensive epidemiological consequences. Relying solely on notifications of symptomatic dengue infections underestimates the number of cases, preserves a silent source of the disease, potentially spreading the virus to unaffected areas.

Internal carotid artery dissection associated with acute dengue virus infection: a case report / Dissecção da artéria carótida interna associada à infecção aguda pelo vírus da dengue: relato de caso

Cervical artery dissections (CAD) can occur spontaneously or as a direct result of significant trauma. Viral infections, such as SARS-CoV2, influenza, and Epstein Barr, are risk factors for spontaneous CAD. Dengue virus infections have dramatically increased in recent decades, and Brazil is one of the endemic areas. The dengue virus can cause headache and neurological complications such as encephalitis, myelitis, Guillain-Barré syndrome, and myositis. No report has yet been found in the literature of dissection of the internal carotid artery secondary to dengue infection. Our objective is to report the case of a patient with dissection of the internal carotid artery associated with acute dengue virus infection.

As dissecções da artéria cervical (DAC) podem ocorrer espontaneamente ou como resultado direto de trauma significativo. Infecções virais, como SARS-CoV2, influenza e Epstein Barr, são fatores de risco para DAC espontânea. As infecções pelo vírus da dengue aumentaram dramaticamente nas últimas décadas, e o Brasil é uma das áreas endêmicas. O vírus da dengue pode causar dor de cabeça e complicações neurológicas como encefalite, mielite, síndrome de Guillain-Barré e miosite. Ainda não foi encontrado na literatura nenhum relato de dissecção da artéria carótida interna secundária à infecção por dengue. Nosso objetivo é relatar o caso de um paciente com dissecção da artéria carótida interna associada à infecção aguda pelo vírus da dengue.

Comportamiento epidemiológico del Dengue en Ecuador desde su reemergencia. Periodo 1980-2020 / Epidemiological behavior of Dengue in Ecuador since its reemergence. Period 1980-2020

INTRODUCCIÓN. El dengue es una enfermedad infecciosa de origen viral, transmitida principalmente por el mosquito Aedes aegypti. Es un grave problema de salud pública a nivel mundial, en las Américas y en el Ecuador. OBJETIVOS. Analizar el comportamiento epidemiológico del dengue desde 1980 hasta el 2020, los factores de riesgo que mantienen la transmisión y las acciones que el país ha implementado para su prevención y control. METODOLOGÍA. Revisión bibliográfica narrativa, teniendo como fuente las páginas web de la Organización Mundial de la Salud, de la Organización Panamericana de la Salud, Ministerio de Salud Pública del Ecuador, artículos de revistas de bibliotecas virtuales e informes técnicos publicados en Google académico, Scielo, Biblioteca Virtual en Salud y PubMed. RESULTADOS. Los registros encontrados sobre presencia de casos de dengue datan de 1988 cuando se presentó la gran epidemia de dengue en Guayaquil. Posteriormente, mantiene un comportamiento endemo-epidémico. A partir del año 2000 se presentan casos de dengue grave. Su pico más alto de letalidad fue de 2,44% en el 2010. El serotipo DEN 1 es el más frecuente, pero a partir del 2000 circulan los 4 serotipos. Las acciones de prevención y control no han sido sostenidas. CONCLUSIONES. El dengue en Ecuador en los últimos cuarenta años mantiene una importante trasmisión, caracterizada por años epidémicos. No ha podido ser controlado el vector, el cual ha ido infestando más localidades. Las condicionantes climáticas y ecológicas, explican en parte la persistencia, pero la determinación más importante está dada por las inequidades sociales, falta de servicios básicos, y la poca continuidad e impacto de las medidas de prevención y control.

INTRODUCTION. Dengue is an infectious disease of viral origin, transmitted mainly by the Aedes aegypti mosquito. It is a serious public health problem worldwide, in the Americas and in Ecuador. OBJECTIVES: To analyze the epidemiological behavior of dengue fever from 1980 to 2020, the risk factors that maintain transmission and the actions that the country has implemented for its prevention and control. METHODOLOGY. Narrative bibliographic review, taking as sources the web pages of the World Health Organization, Pan American Health Organization, Ministry of Public Health of Ecuador, journal articles from virtual libraries and technical reports published in Google Scholar, Scielo, Virtual Health Library and PubMed. RESULTS. The records found on the presence of dengue cases date back to 1988 when the great dengue epidemic occurred in Guayaquil. Subsequently, it maintained an endemic-epidemic behavior. Beginning in 2000, severe cases of dengue fever occurred. Its highest lethality peak was 2.44% in 2010. DEN 1 serotype is the most frequent, but since 2000 all 4 serotypes have been circulating. Prevention and control actions have not been sustained. CONCLUSIONS. Dengue in Ecuador over the last forty years has maintained an important transmission, characterized by epidemic years. It has not been possible to control the vector, which has been infesting more localities. Climatic and ecological conditions partly explain its persistence, but the most important determinant is given by social inequalities, lack of basic services, and the lack of continuity and impact of prevention and control measures.

Vigilancia virológica de casos de Dengue de enero 2020 a febrero 2023, en el Departamento de La Paz-Bolivia / Virological surveillance of Dengue cases since January 2020 to February 2023 La Paz-Bolivia

Introducción: El dengue se ha convertido en un problema serio de salud pública en Bolivia. Por la capacidad de infección del virus del dengue, el diagnóstico es amplio y varia con la evolución de la enfermedad. El incremento de la temperatura y las lluvias de la época son los factores ambientales principales para la reproducción del mosquito transmisor (Aedes aegypti), y el aumento de casos de dengue. Objetivos: Realizar un estudio transversal-retrospectivo de los casos de dengue en el departamento de La Paz - Bolivia, durante el periodo de enero de 2020 hasta febrero de 2023. Materiales y Métodos: De las bases obtenidas del Laboratorio de Virología- INLASA, se analizaron variables como el número de casos positivos de cada gestión, la distribución de los casos positivos según el lugar probable de infección tanto por departamento como por provincia, la variación temporal de los casos y el serotipo prevalente por gestión, y la distribución de casos positivos por género y grupo etario, en este trabajo se realizó un estudio transversal-retrospectivo. Resultados: Durante las cuatro gestiones analizadas el número total de casos notificados fue de 2143, detectando 1184 (55.2%) positivos. Señalando como lugar probable de infección en gran parte la provincia de Sud Yungas. Con respecto a la variación temporal presentó un mismo comportamiento en tres gestiones. Prevaleció en tres gestiones el serotipo DENV 1 sobre DENV 2, observando un aumento de serotipo DENV 2 hasta febrero del 2023. Además, el grupo atareo más afectado con dengue oscila entre los 10 a 39 años, no encontrando diferencia significativa entre género masculino y femenino. Conclusiones: Este estudio revela que el dengue es un problema de salud pública no solo del oriente del país sino también de las zonas tropicales del departamento de La Paz. Es necesario el diagnóstico rápido y efectivo; realizar la tipificación del virus y el llenado correcto de fichas epidemiológicas para un mejor control virológico.

Background: Dengue has become a serious problem in public health in Bolivia. Due the infection capacity of dengue virus, the diagnosis is broad and varies with the evolution of the disease. The increase of temperature and rain season are the main environmental factors of reproduction in the transmitting mosquito (Aedes aegipty), and the increase in dengue cases. Objectives: To carry out a retrospective crossover study of dengue cases in La Paz Department - Bolivia, during the period from January 2020 to February 2023. Materials and Methods: From databases obtained from INLASA Virology Laboratory during periods 2020, 2021, 2022 and the beginnings of 2023, variables such as number of positive cases from each year, distribution of positive cases according to the probable place of infection were obtained and analyzed, by department and by province; the temporal variation of the cases and the prevalent serotype by period, and finally distribution of positive cases by sex group and age group. Its make a retrospective crossover study. Results: During the four analyzed periods, within the total number of 2143 reported cases, there were 1184 (55.2%) of positive cases for Dengue. On the other hand, the department of La Paz (86.3%) is indicated as the probable place of infection, with Sud Yungas province (municipalities La Asunta and Palos Blancos) being the places with the most positive cases. The temporal variation in suspected and confirmed cases has presented the same behavior in three years. DENV 1 serotype predominated over DENV 2 in three years, observing an increase in DENV 2 serotype until February 2023. In addition, the age group most affected by dengue ranges from 10 to 39 years, finding no significant difference between masculine and feminine gender. Discussions: An increase in cases between months of February and April, coinciding with rain season in Bolivia. Due to the climate in the tropical areas of the department, there is a greater number of positive cases in those areas. Conclusions: This study reveals that Dengue is a public health problem not only in eastern part of the country but also in tropical areas of La Paz department. A quick and effective diagnosis is necessary; to carry out the typing of the virus and the correct filling out of epidemiological files for better virological control.

Computer-Aided vaccine design for selected positive-sense single stranded RNA viruses

Spontaneous mutations and lack of replication fidelity in positive-sense single stranded RNA viruses (+ssRNA virus) result in emergence of genetic variants with diverse viral morphogenesis and surface proteins that affect its antigenicity. This high mutability in +ssRNA viruses has induced antiviral drug resistance and ability to overcome vaccines that subsequently resulted in rapid viral evolution and high mortality rate in human and livestock. Computer aided vaccine design and immunoinformatics play a crucial role in expediting the vaccine production protocols, antibody production and identifying suitable immunogenic regions or epitopes from the genome sequences of the pathogens. T cell and B cell epitopes can be identified in pathogens by immunoinformatics algorithms and methods that enhance the analysis of protective immunity, vaccine safety, immunity modelling and vaccine efficacy. This rapid and cost-effective computational vaccine design promotes development of potential vaccine that could induce immune response in host against rapidly mutating pathogens like +ssRNA viruses. Epitope-based vaccine is a striking concept that has been widely employed in recent years to construct vaccines targeting rapidly mutating +ssRNA viruses. Therefore, the present review provides an overview about the current progress and methodology in computeraided vaccine design for the most notable +ssRNA viruses namely Hepatitis C virus, Dengue virus, Chikungunya virus and Coronaviruses. This review also highlights the applications of various immunoinformatics tools for vaccine design and for modelling immune response against +ssRNA viruses.

Fatores associados à infeção pelo vírus da dengue: estudo transversal de dados de vigilância em saúde do município de São Mateus (ES), entre os anos de 2016 e 2020 / Factors associated with dengue virus infection a cross-sectional: study of health surveillance data in the municipality of São Mateus (Espírito Santo), from 2016 to 2020 / Factores asociados a la infección por el virus del dengue: estudio transversal de datos de vigilancia en salud en la ciudad de Sao Mateus (ES), entre los años 2016 a 2020

Introdução: A dengue é uma doença infecciosa endêmica em regiões tropicais e subtropicais. Para organizar uma rede assistência de saúde e garantir medidas preventivas, manejo clínico adequado, é necessário conhecer os fatores associadas aos casos de dengue. Objetivo: Analisar os fatores associados à infecção pelo vírus da dengue de indivíduos notificados no sistema de vigilância em saúde. Métodos: Estudo transversal de dados secundários de casos de suspeitos de dengue notificados no Sistema Nacional de Informação de Agravos de Notificação. Foram incluídos os indivíduos notificados com suspeita de dengue com data de notificação entre dia 1º de janeiro de 2016 e 31 de dezembro de 2020 e que residiam em São Mateus (ES). Foram calculadas as frequências relativas e absolutas das variáveis e foi utilizada a regressão de Poisson de variância robusta para calcular a razão de prevalência (RP) e estimar os intervalos de confiança de 95% (IC95%). Resultados: Foram notificados 4.547 casos suspeitos de dengue, 2.438 (53,8%) casos foram confirmados, 844 (27,7%) confirmados por critério laboratorial, nove apresentaram sinais de alarme, três foram de dengue grave, 35 necessitaram de internação hospitalar e quatro evoluíram a óbito por dengue. A faixa etária ≥60 anos (RP=1,28; IC95% 1,14­1,45), indivíduos com cinco a oito anos de estudo (RP=1,47; IC95% 1,19­1,81), com prova do laço positiva (RP=1,40; IC95% 1,22­1,60) e diabetes mellitus (RP=4,19; IC95% 1,91­9,20) apresentaram maiores prevalências de dengue. Conclusão: A prevalência da dengue foi maior no grupo de indivíduos com idade maior e igual a 60 anos, com cinco a oito anos de estudo, com diabetes mellitus, que apresentaram prova do laço positiva e leucopenia. Esses grupos apresentam chances maiores de desenvolvimento da dengue grave, sendo necessários esforços dos serviços de assistência e vigilância em saúde em seu manejo clínico.

Introduction: Dengue is an endemic infectious disease in tropical and subtropical regions. Knowing the factors associated with dengue cases is necessary to organize the health care system and ensure preventive measures and proper clinical management. Objective: To analyze the factors associated with dengue virus infection in cases reported to the health surveillance system. Methods: This is a cross-sectional study of secondary data on suspected dengue cases reported to the Information System on Diseases of Compulsory Declaration. The sample included individuals with a report of suspected dengue made between January 1, 2016, and December 31, 2020, who lived in São Mateus (Espírito Santo). We calculated the relative and absolute frequencies of the variables and used Poisson regression with robust variance to estimate the prevalence ratio (PR) and 95% confidence intervals (95%CI). Results: A total of 4,547 suspected dengue cases were reported, of which 2,438 (53.8%) were confirmed, 844 (27.7%) were confirmed by laboratory criteria, nine presented warning signs, three had severe dengue, 35 required hospitalization, and four died from dengue. The prevalence of dengue was higher in the age group ≥60 years (PR=1.28; 95%CI 1.14­1.45), individuals with five to eight years of schooling (PR=1.47; 95%CI 1.19­1.81), with positive tourniquet test (PR=1.40; 95%CI 1.22­1.60), and diabetes mellitus (PR=4.19; 95%CI 1.91­9.20). Conclusions: Dengue was more prevalent among individuals aged 60 years and older, with five to eight years of schooling, diabetes mellitus, positive tourniquet test, and leukopenia. These groups are more likely to develop severe dengue, requiring efforts from health care and surveillance services for their clinical management.

Introducción: El dengue es una enfermedad infecciosa endémica en las regiones tropicales y subtropicales. Para organizar una red de atención en salud y asegurar medidas preventivas, manejo clínico adecuado, es necesario conocer los factores asociados a los casos de dengue. Objetivo: Analizar los factores asociados a la infección por el virus del dengue en personas notificadas en el sistema de vigilancia en salud. Métodos: Estudio transversal de datos secundarios sobre casos sospechosos de dengue notificados en el Sistema Nacional de Información de Enfermedades de Declaración Obligatoria. Se incluyeron personas notificadas con sospecha de dengue con fecha de notificación entre el 1 de enero de 2016 y el 31 de diciembre de 2020 y residentes en Sao Mateus (ES). Se calcularon las frecuencias relativas y absolutas de las variables y se utilizó regresión de Poisson de varianza robusta para calcular la razón de prevalencia (RP) y se estimaron los intervalos de confianza del 95% (IC95%). Resultados: Se notificaron 4.547 casos sospechosos de dengue, se confirmaron 2.438 (53,8%), se confirmaron por criterio de laboratorio 844 (27,7%), nueve casos presentaron signos de alarma, tres casos presentaron dengue grave, 35 casos requirieron hospitalización y cuatro fallecieron por dengue. El grupo de edad ≥60 años (RP=1,28 ­ IC95% 1,14­1,45), con escolaridad de 5 a 8 años (RP=1,47 ­ IC95% 1,19­1,81), prueba de torniquete positiva (RP=1,40 ­ IC95% 1,22­1,60) y diabetes mellitus (RP=4,19 ­ IC95% 1,91­9,20) presentaron mayor prevalencia de dengue. Conclusión: la prevalencia de dengue es mayor en el grupo de individuos mayores de 60 años, entre 5 y 8 años de escolaridad, con diabetes mellitus, con vínculo positivo comprobado y leucopenia. Estos grupos son más propensos a desarrollar dengue grave, lo que requiere esfuerzos de los servicios de salud y vigilancia en el manejo clínico de estos individuos.

Design, synthesis, and activity of 5-cyano-2-thiacetyl aromatic pyrimidinone as DENV NS5 RdRp inhibitors / 药学学报

To screen novel anti-dengue virus (DENV) NS5 RdRp enzyme inhibitors, a series of 5-cyano-2-thiacetoaryl pyrimidinone compounds were designed and synthesized by molecular hybridization method with HCV NS5B RdRp inhibitor 3jc and ZIKV NS5 RdRp inhibitor 4w as lead compounds. The anti-DENV activity of these compounds was evaluated by MTT assay and plaque assay and five compounds showed anti-DENV activity. The most active compound 7a'k showed better anti-DENV activity than that of the positive control ribavirin (EC50 = 7.86 μmol·L-1 vs EC50 = 18.07 μmol·L-1), and the other four compounds showed almost the same anti-DENV activity as ribavirin. Finally, the prediction and simulation of the binding mode through molecular provided new ideas for the further development of this new DENV NS5 RdRp inhibitor.

Optimization of expression conditions of dengue virus NS2B-NS3 protease in E.coli and determination of enzyme activity / 中国生物制品学杂志

@#Objective To express dengue virus(DENV)NS2B-NS3 protease in E.coli,optimize the expression conditions and determine the enzyme activity,so as to lay a foundation of screening and discovering of lead compounds targeting DENV.Methods Codon-optimized NS2B-NS3 gene was inserted into pET-28a vector to construct recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3,which was transformed E.coli Rosetta(DE3)competent cells and induced by IPTG to express NS2B-NS3 protease. The optimal expression conditions of NS2B-NS3 protease in E.coli were determined by optimizing induction length,induction temperature and IPTG concentration. NS2B-NS3 protease was isolated and purified by HisTrap~(TM) affinity chromatography column and measured for the protease activity by fluorescence resonance energy transfer(FRET)assay.Results The recombinant prokaryotic expression plasmid pET-28a-NS2B-NS3 was constructed correctly as identified by restriction analysis(NheⅠ/XhoⅠ)and sequencing. The optimal expression conditions of NS2BNS3 protease in E.coli were as follows:induction temperature of 20 ℃,induction length of 10 h and IPTG concentration of0. 2 mmol/L. The purified NS2B-NS3 protease showed a purity of more than 90% with a exhibited a of 20 mg/L,which bound to mouse monoclonal antibody against His-tag specifically and had good hydrolytic activity with a specific activity of 16. 111 U/mg,a K_m of 16. 46 μmol/L and a k_(cat) of 0. 028/s.Conclusion DENV NS2B-NS3 protease with high purity and activity was successfully prepared,which laid an experimental foundation of the establishment of high-throughput screening model for inhibitors targeting NS2B-NS3 protease.

Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus type Ⅲ and its antibody dependent enhancement / 中国生物制品学杂志

@#ObjectiveTo establish models of Dengue virus type Ⅲ（DENV-3,DV-3）infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

Emergence Of Dengue Virus Type 4 During COVID-19 Pandemic In Patients Admitted to a Teaching Hospital In Malaysia (Peningkatan virus denggi serotip keempat semasa pandemik COVID-19 pada pesakit yang dimasukkan ke hospital pengajar di Malaysia) / Jurnal Sains Kesihatan Malaysia

@#Prior to COVID-19, dengue was an important public health problem in Malaysia. Due to the movement control order imposed by the Malaysian government to curb the COVID-19 transmission, a study predicted that mosquito-borne diseases would increase during lockdown and partial lockdown seasons. Thus, this study aims to determine the current situation of dengue incidence during the pre-COVID-19 pandemic (2019) and during the COVID-19 pandemic (2020 and 2021). We compared the number of laboratory-confirmed cases in the pre-COVID19 year (2019) and during the COVID-19 pandemic (2020 and 2021). In addition to that, we characterized the clinical manifestation, dengue serotype and viremia levels of dengue patients that were admitted to the Hospital Cancelor Tuanku Muhriz. We found a significant decrease in the number of laboratory-confirmed cases between COVID-19 pandemic and the pre-covid period (p2020=0.064; p2021<0.001). In this study, we found DENV 4 serotype was the most common serotype in dengue patients admitted to our hospital. There was no significant correlation between DENV serotype/viremia level with clinical manifestation of dengue fever and dengue with warning signs. However, patients infected with DENV4 had the highest viral load compared to patients infected with other serotypes. We also found high viremia levels were significantly associated with the febrile phase.

Interferon-gamma gene diplotype (AA-rs2069716 / AG-rs2069727) may play an important role during secondary outcomes of severe dengue in Brazilian patients

ABSTRACT Dengue is a global and growing health threat, especially in Southeast Asia, West Pacific and South America. Infection by the dengue virus (DENV) results in dengue fever, which can evolve to severe forms. Cytokines, especially interferons, are involved in the immunopathogenesis of dengue fever, and so may influence the disease outcomes. The aim of this study was to investigate the association between severe forms of dengue and two single nucleotide polymorphisms (SNPs) in the interferon-gamma gene (IFNG): A256G (rs2069716) and A325G (rs2069727). We included 274 patients infected with DENV serotype 3: 119 cases of dengue without warning signs (DWoWS), and 155 with warning signs (DWWS) or severe dengue (SD). DNA was extracted, and genotyped with Illumina Genotyping Kit or real time PCR (TaqMan probes). We estimated the adjusted Odds Ratios (OR) by multivariate logistic regression models. When comparing with the ancestral AA/AA diplotype (A256G/A325G), we found a protective association of the AA/AG against DWWS/SD among patients with secondary dengue (OR 0.51; 95% IC 0.24-1.10, p = 0.085), adjusting for age and sex. The variant genotype at locus A325G of the IFNG, in combination with the ancestral genotype at locus A256G, can protect against severe clinical forms of secondary dengue in Brazilian DENV3-infected patients.

Clinical and epidemiological characteristics of probable cases of congenital Zika syndrome and dengue antibody levels, Tuxtla Gutierrez, Chiapas, Mexico

Introduction: We previously found that the occurrence of congenital Zika syndrome was under-reported in Mexico. It was postulated that high dengue virus antibody levels found at the population-level in endemic countries might have contributed to the occurrence of the regional pandemic of Zika disease. A case series of suspected cases of congenital Zika syndrome in a maternity hospital in Tuxtla Gutierrez, Chiapas, Mexico was assembled to assess why they were not reported and to explore the hypothesis of dengue virus antibody-dependent enhancement of Zika disease. Methods: We used a quantitative approach to describe clinical and imaging records and used data from interviews of a total of 13 suspected cases of congenital Zika syndrome. We also quantitated dengue 1 and 2 antibodies using an 80% plaque reduction neutralization test of sera specimens obtained from the mothers of these 13 cases and compared them to those of a group of mothers who delivered normal newborns in the same hospital. Results: Only one of the suspected cases was laboratory-confirmed because appropriate specimens were not collected from the newborns as required by the case definition. We found 1) microcephaly, 2) hypoplasia/hypogeneses, thinning or absence of brain structures, 3) multiple birth defects, 4) calcifications, and cysts, 5) meningocele/encephalocele, and 6) hydrocephalus in 100 %, 76.9 %, 38.5 %, 38.5 %, 30.8 %, and 23.1 %, respectively of the case series. The cases clustered geographically, and 77 % occurred between May 2016 to March 2017 and recalled or were told by a doctor they had Zika fever. There was a four-fold increased risk of congenital Zika syndrome among those with dengue 1 antibody as compared to those with dengue 2 antibodies (odds ratio = 3.6; 95 % confidence interval: 0.7, 20.5), reaching only borderline statistical significance. Conclusions: We found in the largest maternal facility of the capital of the State of Chiapas, in Mexico, that only 7.7 % of suspected cases were confirmed, and that the rather complex requirement of cerebrospinal fluid specimens or serological specimens of newborns for suspected cases of congenital Zika syndrome used during the pandemic resulted in low sensitivity of the surveillance system. The finding of higher levels of dengue 1 than dengue 2 antibodies in cases than the referent population, requires further evaluation and may suggest a role for dengue antibody-dependent response in Zika disease.

Introducción: Previamente los autores habían encontrado evidencia de sub-notificación de la ocurrencia del síndrome congénito por Zika en México. Se ha postulado que niveles elevados de anticuerpos contra los virus del dengue a nivel poblacional en los países endémicos hubiese contribuido a la ocurrencia de la pandemia regional de enfermedad por Zika. Ensamblamos una serie de casos sospechosos de síndrome congénito por Zika en un hospital de maternidad en Tuxtla Gutiérrez, Chiapas, México, para evaluar por qué no fueron notificados y explorar la hipótesis de enfermedad por Zika incrementada por anticuerpos anti-dengue. Métodos: Utilizamos un enfoque cuantitativo para describir 13 casos sospechosos de síndrome congénito por revisamos registros clínicos e imágenes, entrevistas. También cuantificamos los niveles de anticuerpos para los virus dengue 1 y 2 en suero de las madres de los casos comparados con los de mujeres que tuvieron recién nacidos normales en el mismo hospital. Resultados: Solamente uno de los 12 casos sospechosos fue confirmado por laboratorio, porque en los demás no se recolectaron especímenes adecuados de los neonatos como lo requería la definición de casos. Encontramos 1) microcefalia, 2) hipoplasia y adelgazamiento de las estructuras cerebrales, 3) malformaciones múltiples, 4) calcificaciones o quistes, 5) meningocele/encefalocele, y 6) hidrocefalia en: 100 %, 76.9 %, 38.5 %, 38.5 %, 30.8 %, y 23.1 %, en ese orden entre los casos sospechosos. Los casos se aglutinaron geográficamente y 77 % ocurrieron entre Mayo del 2016 y Marzo del 2017, y sus madres recordaban que tuvieron o que un profesional de la salud les dijo que tuvieron fiebre por Zika. Encontramos un incremento de casi 4 veces en el riesgo de síndrome congénito por Zika para aquellos con altos niveles de anticuerpos anti-dengue 1 comparado con anticuerpos anti-dengue 2 (cociente de suertes = 3.6; intervalo de confianza del 95 %: 0.7, 20.5), alcanzando solamente una significancia estadística limítrofe. Conclusiones: Encontramos en el establecimiento de atención a la maternidad más grande en la capital de Chiapas, México, que solamente 7.7 % de los casos sospechosos de síndrome congénito por Zika fueron confirmados y que los relativamente complejos requerimientos de la definición de casos de muestras serológicas o de líquido cefalorraquídeo resultó en una baja sensibilidad del sistema de vigilancia. El hallazgo de niveles más altos de anticuerpos a dengue 1 que dengue 2 requiere más evaluación y pudiera sugerir un papel de la respuesta dependiente de anticuerpos al dengue en Zika.

A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes

Background & objectives: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. Methods: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. Results: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x10-1 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV. When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV. All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100 . In full combination assays, CHIKV was detected to 1.32x10-1, DENV1-4 to 3.79x101 and ZIKV to 1.07x100 . Interpretation & conclusion: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

Acute kidney injury associated with dengue virus infection: a review / Injúria renal aguda associada à infecção pelo vírus da dengue: uma revisão

Abstract Acute kidney injury (AKI) is one of the least studied complications of dengue, but it carries high mortality rates and prolonged hospital stay. Due to the severity of this complication, the risk of developing chronic kidney disease (CKD) and the increasing number of dengue cases reported worldwide, particularly in the tropical and subtropical regions of Africa, Southeast Asia and South America, including Brazil, we embarked on this narrative review, aimed to update the epidemiology of AKI associated with dengue, elucidate the main pathophysiological mechanisms of AKI caused by the dengue virus infection, as well as discuss useful information on the prevention and management of AKI in patients with dengue.

Resumo A injúria renal aguda (IRA) é uma das complicações da dengue menos estudadas, mas que acarreta altas taxas de mortalidade e prolongamento do tempo de internação. Devido à gravidade dessa complicação, ao risco de desenvolvimento de doença renal crônica (DRC) e ao número crescente de casos de dengue relatados no mundo, particularmente nas regiões tropicais e subtropicais da África, Sudeste Asiático e América do Sul, incluindo o Brasil, foi proposta esta revisão narrativa que objetivou atualizar a epidemiologia da IRA associada à dengue, elucidar os principais mecanismos fisiopatológicos da IRA causada pela infecção do vírus da dengue, assim como discutir informações úteis sobre a prevenção e o manejo da IRA em pacientes com dengue.

Clinical and radiological manifestations of acute disseminated encephalomyelitis associated with the main arboviruses / Manifestaciones clínicas y radiográficas de la encefalomielitis aguda diseminada asociada a los principales Arbovirus

ABSTRACT Introduction: Acute disseminated encephalomyelitis (ADEM) is an acute and inflammatory neuropathic disease that causes demyelination, predominantly affecting the white matter. Clinical manifestations of ADEM may be associated with the arboviruses zika fever, dengue fever, and chikungunya. Objective: The aim of the present study was to carry out a bibliographical survey about the clinical manifestations and radiological findings of ADEM after infection with dengue, zika, and chikungunya viruses, as well as their correlation. Methods: MEDLINE, LILACS, Web of Science, and Scopus databases were searched for articles published between 2010 and 2020, written in English, Spanish, and Portuguese, about the occurrence of ADEM in patients affected by zika fever, dengue fever, or chikungunya. The search yielded articles that demonstrated the occurrence of five cases of ADEM associated with zika fever, ten cases post-infection with the dengue virus cases, and two cases related to chikungunya. Conclusions: The most common initial clinical presentations of ADEM were fever, nausea and/or vomiting, myalgia, headache, skin rashes, and arthralgia. The main neurological symptoms reported were changes in the level of consciousness, pyramidal signs, tonic-clonic seizures, visual changes, and urinary disorders. The most common radiological findings were T2/FLAIR hyperintense lesions on magnetic resonance imaging (MRI), affecting mainly the subcortical and central white matter. It is highlighted the importance of monitoring patients with dengue fever, zika fever, or chikungunya to identify clinical manifestations of ADEM that may contribute to an early and correct diagnosis of this encephalomyelitis, and, consequently, to intervene and obtain better patient prognosis.

RESUMEN Introducción: La encefalomielitis diseminada aguda (EMDA) es una enfermedad neuropática aguda e inflamatoria que provoca desmielinización, y afecta predominantemente la materia blanca. Las manifestaciones clínicas de la EMDA pueden estar asociadas a los arbovirus Zika, dengue y chikungunya. Objetivo: Realizar una encuesta bibliográfica sobre las manifestaciones clínicas y los hallazgos radiográficos de la EMDA después de la infección por los virus del dengue, del Zika y del chikungunya, así como su correlación. Métodos: Se realizó una búsqueda, en las bases de datos MEDLINE, LILACS, Web of Science y Scopus, de artículos publicados entre 2010 y 2020, escritos en inglés, español y portugués, sobre la aparición de EMDA en pacientes afectados por la fiebre del Zika, la fiebre del dengue o del chikungunya. Se hallaron artículos que demostraron la ocurrencia de cinco casos de EMDA asociados con la fiebre del Zika, diez casos posteriores a la infección por el virus del dengue y dos casos relacionados con el chikungunya. Conclusiones: Las presentaciones clínicas iniciales más comunes de EMDA fueron fiebre, náuseas o vómitos, mialgia, dolor de cabeza, erupciones cutáneas y artralgia. Los principales síntomas neurológicos reportados fueron cambios en el nivel de conciencia, signos piramidales, convulsiones tónico-clónicas, cambios visuales y trastornos urinarios. Los hallazgos radiográficos más comunes fueron lesiones hiperintensas T2/FLAIR en imágenes de resonancia magnética. Estas lesiones afectaron principalmente la subcortical y la materia blanca central. Se destaca la importancia de la vigilancia de los pacientes con dengue, Zika o chikungunya para identificar las manifestaciones clínicas de la EMDA que puedan contribuir a un diagnóstico precoz y correcto de esta encefalomielitis y, por consiguiente, para intervenir y obtener un mejor pronóstico del paciente.

¿Es posible la coinfección o un diagnóstico erróneo por dengue y COVID-19? Una revisión de reporte de casos / Is dengue and COVID-19 coinfection or misdiagnosis possible? A review on reported cases

INTRODUCCIÓN: El nuevo coronavirus ha continuado propagándose por todo el mundo donde existen otras enfermedades endémicas que han sido una carga para la salud pública durante muchos años. Como cualquier infección, se habría esperado encontrar en coinfección con algunas de éstas. Específicamente, los países tropicales y subtropicales han venido manejando la carga del dengue a medida que aumentan los picos con períodos de tiempo más cortos. OBJETIVO: Resumir la evidencia que existe en la coinfección relacionada con el SARS-CoV-2 y el virus del dengue. METODOLOGÍA: Se realizó una revisión narrativa en bases de datos sobre reportes de coinfección y diagnóstico erróneo de SARS-CoV-2 y el dengue dado que la temporada de lluvias cada año aumenta la prevalencia de infecciones virales en países endémicos. Informes recientes incluso han descrito casos positivos en uno de estas infecciones que luego resultaron en falso positivo. Una prueba positiva para COVID-19 o fiebre del dengue en áreas endémicas no debe excluir la otra infección. CONCLUSIÓN: A partir de ahora, estos dos deberían ser considerados como un diagnóstico diferencial y esto debe generar preocupación de salud pública por su coinfección en países endémicos para reforzar la promoción y prevención a las comunidades y mitigar estas enfermedades.

BACKGROUND: Novel coronavirus has continued to spread throughout the world where there are other endemic diseases that have been a burden to public health for many years. As any infection, it was expected there could be coinfection between these. Tropical and subtropical countries are currently managing with dengue as peaks increase with shorter periods of time. AIM: To summarize the evidence that exists in the co-infection related to SARS-CoV-2 and the dengue virus. METHOD: We conducted a narrative review in data bases about reports of coinfection and misdiagnosis of SARS-CoV-2 and dengue virus given the fact that rainy season every year increase the prevalence of viral infections in endemic countries. Recent reports have even described positive cases in one of these infections that later resulted in false positive. A positive test for COVID-19 or dengue fever in endemic areas should not exclude the other infection. CONCLUSION: From now on, these two should be considered as a differential diagnosis and this should raise public health concern for COVID-19 and dengue coinfection in endemic countries to reinforce promotion and prevention to communities to prevent these diseases.

Impaired glucose tolerance in dengue fever: A case-control study

Background: Studies on dengue fever demonstrated that the dengue viral infection of pancreas is often associated with disease morbidity and complication. Aims and Objectives: The pancreas-pathogen interactions in dengue-infected persons were evaluated using endocrine deregulation as an investigation marker of complication. Materials and Methods: A prospective observational study was conducted in a tertiary care medical college and hospital of West Bengal, over 1 year and 4 months. Blood samples from 286 cases and 258 controls were collected on day 2. After plasma glucose determination, 44 cases and 39 controls were excluded as frank cases of diabetes mellitus. On day 6, fasting and 2 h postprandial plasma glucose estimation were done in 73 cases and 61 controls by glucose oxidase-peroxidase method using autoanalyzer. Data were analyzed using the Statistical Package for the Social Sciences version 20.0. Results: The prevalence of impaired glucose tolerance was higher in cases on day 2 than on follow-up on day 6 (12.3% vs. 8%). There existed no statistical difference in terms of fasting plasma glucose between cases and control on day 2 and day 6 and postprandial (PP) plasma glucose on day 2. However, the PP values on day 6 in cases were significantly higher in cases in comparison to controls (P = 0.006). Among cases, day 6 values were higher than day 2 values (0.016). Conclusion: Dengue viral infection correlates with the involvement of the pancreas in terms of impaired tolerance to glucose which has implications for understanding disease pathogenesis in terms of developing chronic complications.

An overview of dengue viral infection circulating in Pakistan

Background & objectives: Dengue virus (DENV) is an RNA virus that infects approximately 2.5 billion people around the world. The incidence of dengue fever has rapidly increased at an alarming rate in the last few years and has affected thousands of people in Pakistan. This review explores the prevalence, serotypes and pathogenesis of dengue virus circulating in Pakistan. Methods: A systematic review of observational studies published between 1994 and December 2019 was performed. All records of the confirmed outbreak of dengue fever in Pakistan were reviewed and articles containing no primary data were excluded. Results: Four identified serotypes of dengue virus (DENV 1-4) circulate in different regions of the world causing epidemics. The most prevalent serotype, which is still epidemic and dominant in Pakistan, is DENV-2. Many factors like over-population, rapid urbanization, travelling, lack of vector control in dengue endemic areas and inadequate health-care are responsible of dynamic and huge raise of dengue in Pakistan. Interpretation & conclusion: Currently there is no specific treatment for prevention of dengue virus. Recently some antiviral compounds were being tested to eradicate this disease. There is a need to develop an efficient and safe vaccine for all four serotypes to combat dengue viral infection globally and particularly in Pakistan.

Dengue infection in kidney transplant recipients: clinical course and its impact on renal function / Infecção por dengue em receptores de transplante renal: curso clínico e seu impacto na função renal

Abstract Introduction: Kidney transplant recipients (KTR) are at increased risk for dengue virus (DENV) infection. The aim of this study was to outline the clinical presentation and laboratory profile of DENV infection in KTR and its impact on renal function. Methods: This was a retrospective study of KTR diagnosed with DENV infection. Adult patients who visited Santa Casa de Belo Horizonte Nephrology Center between April and September 2019 were included. Patients who did not sign the Informed Consent were excluded. Data were collected from the database and medical records. The study was approved by the local Institutional Ethics Committee and the Informed Consent was obtained. Results: Nineteen KTR were evaluated. The main signs and symptoms were myalgia, headache/retro-orbital pain, fever, and gastrointestinal symptoms. Two patients had acute cholecystitis without calculus, three experienced pleural and/or pericardial effusion, and one developed acute myocarditis. All patients were under immunosuppression with prednisone, tacrolimus, and mycophenolate, and most were not receiving induction therapy. Temporary suspension/reduction of immunosuppression was required in 58% of patients and leukopenia was the most common reason. Thrombocytopenia was common and 58% of patients developed acute kidney injury. All patients recovered renal function. Conclusions: DENV infection in KTR patients seems to follow a similar course as in the general population. Although there was no control group, we suspect that immunosuppression, preexisting kidney disease or type of donor was not a determining factor in most patients. Transient renal dysfunction was common but reversible. No patient experienced death or graft loss.

Resumo Introdução: Receptores de transplante renal (RTR) apresentam maior risco de infecção pelo vírus da dengue (DENV). O objetivo deste estudo foi delinear apresentação clínica e perfil laboratorial da infecção por DENV em RTR e seu impacto na função renal. Métodos: Este foi um estudo retrospectivo de RTR diagnosticados com infecção por DENV. Incluiu-se pacientes adultos que estiveram no Centro Nefrológico da Santa Casa de Belo Horizonte entre Abril/Setembro, 2019. Foram excluídos pacientes que não assinaram o Termo de Consentimento Livre e Esclarecido. Dados foram coletados do banco de dados e registros médicos. O estudo foi aprovado pelo Comitê de Ética Institucional local e obteve-se o Consentimento Livre e Esclarecido. Resultados: Avaliou-se dezenove RTR. Principais sinais e sintomas foram mialgia, cefaleia/dor retro-orbital, febre, sintomas gastrointestinais. Dois pacientes apresentaram colecistite aguda sem cálculo, três sofreram derrame pleural e/ou pericárdico, um desenvolveu miocardite aguda. Todos os pacientes estavam sob imunossupressão com prednisona, tacrolimus, micofenolato. A maioria não estava recebendo terapia de indução. Suspensão/redução temporária da imunossupressão foi necessária em 58% dos pacientes e leucopenia foi a razão mais comum. Trombocitopenia foi comum e 58% dos pacientes desenvolveram lesão renal aguda. Todos recuperaram a função renal. Conclusões: A infecção por DENV em pacientes RTR parece seguir um curso semelhante ao da população em geral. Embora não houvesse grupo controle, suspeitamos que imunossupressão, doença renal preexistente ou tipo de doador não foram fator determinante na maioria dos pacientes. Disfunção renal transitória foi comum, mas reversível. Nenhum paciente veio ao óbito ou sofreu perda do enxerto.