Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Exames laboratoriais da Pesquisa Nacional de Saúde: metodologia de amostragem, coleta e análise dos dados / Laboratory exams of the National Health Survey: methodology of sampling, data collection and analysis

RESUMO: Introdução: O artigo teve o objetivo de descrever a metodologia de coleta dos dados dos exames laboratoriais da Pesquisa Nacional de Saúde (PNS). Metodologia: Foi selecionada uma subamostra de 25% dos setores censitários, obedecendo à estratificação da amostra da PNS, com probabilidade inversamente proporcional à dificuldade de coleta. A coleta de sangue e urina dos moradores selecionados para entrevista individual foi realizada nos domicílios por um agente de laboratório. Por conta das dificuldades encontradas no trabalho de campo,a amostra não atingiu número suficiente em alguns estratos da pesquisa, então para a análise dos dados foi proposto procedimento de pós-estratificação. Resultados: A coleta de material biológico foi realizada em 8.952 indivíduos. Os exames realizados foram: hemoglobina glicada; colesterol total; colesterol LDL; colesterol HDL; sorologia para dengue; hemograma série vermelha (eritograma) e série branca (leucograma); cromatografia líquida de alta eficiência (HPLC) para diagnóstico de hemoglobinopatias; e creatinina. Na urina, estimativa de excreção de potássio, sal, sódio e creatinina. A base de dados dos exames laboratoriais foi ponderada e disponibilizada para os usuários no site da PNS da Fundação Oswaldo Cruz, sem necessidade de autorização prévia para uso. Conclusão: A subamostra total coletada é de grande valia, pois permitiu estabelecer parâmetros de referência nacionais adequados às características sociodemográficas e geográficas da população brasileira, fornecendo informações relevantes e complementares para a análise da situação de saúde do Brasil.

ABSTRACT: Introduction: This article aims at describing the National Health Survey (Pesquisa Nacional de Saúde- PNS) methodology of collecting laboratory exams data. Methodology: A subsample of 25% of the census tracts was selected, according to the stratification of the PNS sample, with a probability inversely proportional to the difficulty of collection. The collection of blood and urine was done in the households by a laboratory agent, among residents selected for individual interview. Due to the difficulties found in the field work, the sample did not reach the minimum expected number in some strata, and a post-stratification procedure was proposed for the data analysis. Results: The collection of biospecimens was performed in 8,952 individuals. Laboratory tests were: glycated hemoglobin; total cholesterol; LDL cholesterol; HDL cholesterol; serology for dengue; red blood cell count (erythrogram) and white series count (leukogram); high performance liquid chromatography (HPLC) for diagnosis of hemoglobinopathies; creatinine. Theexcretion of potassium, salt and sodium and creatinine was estimated in the urine. The database of laboratory exams was weighed and made publicly available on the Oswaldo Cruz Foundation's PNS website and can be accessed without prior authorization. Conclusion: The total subsample of laboratory exams is of great value, since it allowed us to establish national reference parameters adequate to sociodemographic and geographic characteristics of the Brazilian population, providing relevant and complementary information for the analysis of the health situation of Brazil.

Modulación de los niveles de lipoproteínas de alta densidad y las citoquinas IL-1β e IL-6 en pacientes con dengue / Modulation of high-density lipoprotein and cytokine IL-1β and IL-6 levels in patients with dengue

RESUMEN Objetivos. Evaluar los niveles de lipoproteínas de alta densidad (HDL) y citoquinas proinflamatorias en pacientes con dengue clasificados según la gravedad del cuadro clínico. Materiales y métodos. Se realizó un estudio descriptivo, incluyendo 70 individuos con dengue, clasificados como: sin signos de alarma (DSSA), con signos de alarma (DCSA) y dengue grave (DG); 15 donantes sanos fueron incluidos como controles (CS). Los niveles séricos de IL-1β e IL-6 fueron cuantificados por ELISA, y los de HDL en suero por ensayo colorimétrico; además se determinó el recuento de plaquetas y el porcentaje de hematocrito. Resultados. Los niveles de IL-1β e IL-6 se encontraron aumentados en los pacientes con DENV respecto a los controles sanos. Dicho aumento fue más evidente en los pacientes con dengue, de acuerdo con la gravedad del cuadro clínico. Los niveles de HDL se hallaron disminuidos en los pacientes con dengue comparados con los controles sanos. Los análisis de correlación mostraron asociaciones estadísticamente significativas entre HDL y el número de plaquetas; así como entre el porcentaje de hematocrito y los niveles de IL-1β. Conclusiones. El aumento de IL-1β e IL-6 y la disminución de HDL en los estadios clínicos más graves sugieren que ambos factores están implicados en el desarrollo de la patogénesis y severidad. Las correlaciones permiten observar una posible asociación entre HDL y el número de plaquetas que permite dar una aproximación al posible papel de las HDL en la patogénesis del dengue, pero aún es necesario realizar estudios adicionales que permitan elucidar su importancia en el curso de la infección por DENV.

ABSTRACT Objectives. To assess High-Density Lipoprotein (HDL) and pro-inflammatory cytokine levels in patients with dengue classified according to the severity of the clinical presentation. Materials and Methods. A descriptive study was carried out with 70 individuals with dengue, classified as no alarm signs (DSSA), with signs of alarm (DCSA), and severe dengue (DG); 15 healthy donors were included as controls (CS). The serum levels of IL-1β and IL-6 were quantified through ELISA, and the HDL levels through serum by a colorimetric test. In addition, the platelet count and the hematocrit percentage were determined. Results. Levels of IL-1β and IL-6 were found to be increased in patients with DENV vs. the healthy controls. Said increase was more evident in patients with dengue and in line with the severity of the clinical presentation. The HDL levels were found to be decreased in patients with dengue versus healthy controls. The correlation analysis showed statistically significant associations between HDL and the platelet number, as well as between the hematocrit percentage and the levels of IL-1β. Conclusions. The increased IL-1β and IL-6 and the decreased HDL levels in the most severe clinical stages suggest that both factors are involved in the development of the pathogenesis and severity. The correlations allow the observation of a potential association between HDL and platelet count, which allows us to provide an approach to the possible role of HDL in the pathogenesis of dengue, but it is still necessary to perform additional studies that will allow to state their importance in the course of the DENV infection.

Seroprevalencia de la infección por dengue en los municipios de Armenia, Calarcá, La Tebaida y Montenegro en el departamento del Quindío, 2014 / Seroprevalence of dengue infection in the municipalities of Armenia, Calarcá, La Tebaida and Montenegro in Quindío, 2014

Resumen Introducción: El dengue representa un grave problema de salud pública para Colombia y, en el departamento del Quindío, afecta el 90 % de los municipios. Se necesitan estudios actualizados sobre la seroprevalencia en la población general para reforzar las acciones de salud pública. Objetivo: Determinar la seroprevalencia de la infección por dengue en barrios con alta incidencia de dengue en cuatro municipios del departamento del Quindío: Armenia, Calarcá, La Tebaida y Montenegro, en 2014. Materiales y métodos: Se hizo un estudio de prevalencia mediante muestreo probabilístico estratificado y bietápico. Se hizo una encuesta a 658 sujetos residentes del área urbana de los municipios seleccionados y se les tomó una muestra de sangre por venopunción para determinar anticuerpos IgG e IgM contra el virus del dengue. Resultados: La seroprevalencia de anticuerpos IgG en el Quindío fue de 89,4 %; en Armenia fue de 88,7 %, en Calarcá, de 81,5 %, en Montenegro, de 91,8 %, y en La Tebaida, de 97,8 %. La seroprevalencia de anticuerpos IgM en Quindío fue de 14,2 %; en Armenia, de 11,5 %, en Calarcá, de 13,0 %, en Montenegro, de 13,1%, y en La Tebaida, de 28,9 %. Conclusiones: Se evidenció una alta prevalencia de anticuerpos IgG e IgM en los cuatro municipios. En todos los grupos de edad se encontraron personas seropositivas para IgM, lo cual indicaría infección reciente. La seropositividad simultánea para IgM e IgG (12,9 %) puede indicar infección secundaria por otro serotipo del virus o una infección en los tres meses anteriores. Es necesario impulsar estrategias multisectoriales para el control de la transmisión del dengue en el Quindío.

Abstract Introduction: Dengue is a serious public health problem in Colombia; it is prevalent in 90% of the municipalities in Quindío. Studies on its seroprevalence are required to address public health interventions. Objective: To establish the seroprevalence of dengue infection in neighborhoods with high incidence in the municipalities of Armenia, Calarcá, La Tebaida and Montenegro, Quindío, in 2014. Materials and methods: We conducted a probabilistic, stratified, two-stage prevalence study. We interviewed 658 residents in the urban area of the selected municipalities. After they signed the informed consent, we took a blood sample to determine dengue IgG and IgM antibodies. Results: Seroprevalence of IgG in Quindío was 89,4%; in Armenia it was 88,7%, in Calarcá, 81,5%, in Montenegro, 91,8% and in La Tebaida 97,8%. IgM was 14, 2% in Quindío; in Armenia it was 11,5%, in Calarcá, 13,0%, in Montenegro, 13,1% and in La Tebaida, 28,9%. Conclusions: We found a high prevalence of both IgG and IgM in the four municipalities. We had positive results for IgM in all age groups, which suggests recent infection. We also found simultaneous seropositivity for IgG and IgM (12.9%), which may indicate infection by another serotype or presence of infection in the past three months. A multisectoral approach is necessary for dengue control in Quindío.

Multiplex polymerase chain reaction-based serotype analysis of dengue virus during 2015 dengue outbreak in Pakistan

Dengue is one of the most important arthropod-borne viral diseases. It is endemic in > 125 countries including Pakistan, with a global incidence of 50-200 million. We determined the frequency of different serotypes of dengue virus to highlight its hyperendemicity in Rawalpindi, Pakistan. Between May and October 2015 we analysed the serum samples of 140 patients with a suspicion of dengue, using ELISA and multiplex polymerase chain reaction. One hundred and eight were infected with serotype 2, 16 with serotype 3, 7 with serotype 4 and 3 with serotype 1. Three patients were infected with serotypes 1 and 2, and 1 each with serotypes 1 and 4 and serotypes 2 and 3. Incidence of dengue has increased many fold in the past 50 years and has expanded to areas that were previously free from the disease. Serotype 2 was predominant in our population followed by serotype 3. There is currently no specific treatment for dengue, and vector control and vaccination are the only effective methods to prevent future outbreaks

Clinical and laboratory signs associated to serious dengue disease in hospitalized children / Sinais clínicos e laboratoriais para o dengue com evolução grave em crianças hospitalizadas

Abstract Objective: To evaluate the validity of clinical and laboratory signs to serious dengue disease in hospitalized children. Methods: Retrospective cohort of children (<18 years) hospitalized with dengue diagnosis (2007-2008). Serious dengue disease was defined as death or use of advanced life support therapy. Accuracy measures and area under the receiver operating characteristic curve were calculated. Results: Of the total (n = 145), 53.1% were female, 69% aged 2-11 years, and 15.9% evolved to the worse outcome. Lethargy had the best accuracy (positive likelihood ratio >19 and negative likelihood ratio <0.6). Pleural effusion and abdominal distension had higher sensitivity (82.6%). History of bleeding (epistaxis, gingival or gastrointestinal bleeding) and severe hemorrhage (pulmonary or gastrointestinal bleeding) in physical examination were more frequent in serious dengue disease (p < 0.01), but with poor accuracy (positive likelihood ratio = 1.89 and 3.89; negative likelihood ratio = 0.53 and 0.60, respectively). Serum albumin was lower in serious dengue forms (p < 0.01). Despite statistical significance (p < 0.05), both groups presented thrombocytopenia. Platelets count, hematocrit, and hemoglobin parameters had area under the curve <0.5. Conclusions: Lethargy, abdominal distension, pleural effusion, and hypoalbuminemia were the best clinical and laboratorial markers of serious dengue disease in hospitalized children, while bleeding, severe hemorrhage, hemoconcentration and thrombocytopenia did not reach adequate diagnostic accuracy. In pediatric referral hospitals, the absence of hemoconcentration does not imply absence of plasma leakage, particularly in children with previous fluid replacement. These findings may contribute to the clinical management of dengue in children at referral hospitals.

Resumo Objetivo Avaliar a validade dos sinais clínicos e laboratoriais para o dengue com evolução grave em crianças hospitalizadas. Métodos Coorte retrospectivo de crianças (<18 anos) internadas com dengue (2007-2008). Evolução grave foi definida como óbito ou pelo uso de terapia de suporte avançado de vida. Foram calculadas medidas de acurácia e área sob a curva ROC. Resultados Do total (n = 145), 53,1% casos eram do sexo feminino, 69% de 2 a 11 anos e 15,9% evoluíram para gravidade. Letargia obteve a melhor acurácia (razão de verossimilhança positiva RVP > 19 e RV negativa RVN < 0,6). Derrame pleural e distensão abdominal apresentaram maior sensibilidade (se = 82,6%). Relato de sangramentos (epistaxe, gengivorragia ou gastrointestinal) e hemorragia grave (pulmonar ou gastrointestinal) presente no exame físico foi mais frequente nos casos com evolução grave (p <0,01), porém com baixa acurácia (RVP = 1,89 e 3,89; RVN = 0,53 e 0,60, respectivamente). Os níveis de albumina sérica foram mais baixos nas formas graves (p <0,01). Ambos os grupos apresentaram trombocitopenia, apesar da diferença estatística (p <0,05). Contagem de plaquetas, hematócrito e hemoglobina apresentaram área sob a curva ROC < 0,5. Conclusões Letargia, distensão abdominal, derrame pleural e hipoalbuminemia foram os melhores marcadores clínicos e laboratoriais de dengue com evolução grave em crianças hospitalizadas, enquanto sangramento, hemorragia grave, hemoconcentração e trombocitopenia não tiveram boa acurácia diagnóstica. Em hospitais de referência pediátricos, a ausência de hemoconcentração não implica ausência de extravasamento plasmático, particularmente quando há reposição anterior de volume. Esses resultados podem contribuir para o manejo clínico do dengue em crianças em hospitais de referência.

Co-infección por virus del dengue (DEN-V) y virus Chikungunya (CHIK-V) durante un brote epidémico en Cali, Colombia: comunicación de un caso fatal / Co-infection by Chikungunya virus (CHIK-V) and dengue virus (DEN-V) during a recent outbreak in Cali, Colombia: report of a fatal case

The recent outbreaks of Chikungunya (CHIK-V) virus in endemic areas of dengue (DEN-V) could increase the risk of co-infection. CHIK infection has been considered not severe and with very unusual mortality, however DEN is associated with severe manifestations and increased mortality. Little is known about coinfection. It is possible that co-infection could generate severe cases. We present a case report of co-infection DEN-V -3 and CHIK-V in an elderly patient who developed acute renal failure, dengue shock syndrome (DSS), progresses to multiple organ failure and died. With the recent emergence of CHIK-V in Colombia, the possibility of co-infection with DEN-V should be suspected, especially in severe cases.

La aparición reciente del virus Chikungunya (CHIK-V) en áreas endémicas de dengue (DEN-V) podría aumentar el riesgo de co-infección. Generalmente, se considera que la infección por CHIK-V es de menor gravedad y mortalidad que DEN-V. Poco se conoce sobre la co-infección de DEN-V y CHIK-V, sin embargo, se ha planteado que la co-infección podría ser de mayor gravedad. Se presenta un caso clínico de co-infección por DENV-3 y CHIK-V en un adulto mayor quien desarrolló falla renal aguda, síndrome de choque del dengue, progresión a disfunción orgánica múltiple y muerte. Con la reciente emergencia de CHIK-V en Colombia, es necesario estar atentos ante la posibilidad de co-infección con DEN-V, en especial en casos graves.

DENGUE OUTBREAK IN MATO GROSSO STATE, MIDWESTERN BRAZIL / Surto de dengue em Mato Grosso, centro-oeste do Brasil

Dengue virus (DENV) is the most frequent arbovirus worldwide. In this study, we report a large outbreak in Mato Grosso State (MT). Serum samples from 604 patients with acute febrile illness for less than five days were inoculated in C6/36 cells, then infected cells were subjected to an indirect immunofluorescence test for DENV serotypes and yellow fever virus. Serum samples were submitted to a multiplex-semi-nested-RT-PCR for 11 flaviviruses. DENV-4 was isolated in 150/604 (24.8%) and DENV-1 in 19/604 (3.1%) specimens. By RT-PCR, 331 (54.8%) samples tested positive for DENV; 321 had single infections (DENV-4 n = 305; DENV-1 n = 15; DENV-3 n = 1), nine had co-infections of DENV-1/DENV-4, and one of DENV-2/DENV-4. DENV-4 was detected in 315/331 (95.2%) positive patients from 17 municipalities, and DENV-1 in 24/331 (7.2%) patients from five cities in north-central MT and the city of Cuiaba. The incidence of infection was higher in patients aged 20-39 (142/331; 42.9%). The NS5 partial nucleotide sequence of DENV-1 was most similar to that of genotype V, DENV-2 to Southeast Asian/American, DENV-3 to genotype III, and DENV-4 to genotype II strains, considered the most frequent strains in Brazil. This outbreak coincided with the introduction of DENV-4 in the state. Cuiaba was hyperendemic for the four DENV serotypes, highlighting the necessity for arbovirus surveillance in MT.

O vírus da dengue (DENV) é o arbovirus mais frequente no mundo. Neste estudo, é relatada uma epidemia de grandes proporções no estado de Mato Grosso (MT). Amostras de soro de 604 pacientes com doença febril aguda a menos de 5 dias foram inoculadas em células C6/36 seguida de Imunofluorescência indireta para os sorotipos do DENV e vírus da febre amarela e submetidas a multiplex-semi-nested-RT-PCR para 11 flavivírus. O DENV-4 foi isolado em 150/604 (24,8%) e DENV-1 em 19/604 (3,1%) amostras. Por RT-PCR, 331 (54,8%) pacientes foram positivos para DENV; 321 com infecções únicas (DENV-4 n=305; DENV-1 n=15; DENV-3 n=1), nove co-infecções entre DENV-1/DENV-4 e uma com DENV-2/DENV-4. O DENV-4 foi detectado em 315/331 (95,2%) pacientes de 17 municípios e o DENV-1 em 24/331 (7,2%) pacientes de 5 cidades da região centro-norte de MT e em Cuiabá. A incidência de infecção foi maior em pacientes de 20-39 anos (142/331; 42,9%). As sequências de nucleotídeos de região do gene NS5 do DENV-1 apresentaram maior similaridade com o genótipo V, do DENV-2 com Sudeste Asiático/Americano, DENV-3 com genótipo III e DENV-4 com genótipo II, considerados os mais frequentes no Brasil. Esta epidemia coincidiu com a introdução do DENV-4 no estado. Cuiabá foi considerada hiperendêmica para os quatro sorotipos do DENV, ressaltando a necessidade de vigilância para arbovírus em MT.

Serum levels of copper and iron in dengue fever / Níveis séricos de cobre e ferro na dengue

SUMMARYThe role of trace elements in dengue virulence is not yet known. The present study assessed the serum levels of two micronutrients, copper and iron, in cases of dengue fever. The study involved 96 patients of whom 48 had either severe or non-severe forms of dengue (with and without warning signs), and the remaining 48 were patients with other febrile illnesses (OFI), used as controls. Serum levels of copper and iron were evaluated at admission and by the time of defervescence using commercially available kits. At admission, no difference in the level of serum copper was observed between cases and controls. In the group of dengue cases, the copper level was found to be significantly decreased in severe and non-severe cases with warning signs, compared to non-severe cases without warning signs. In contrast, by the time of defervescence the copper level was found to be increased in all dengue cases compared to OFI controls, but no difference was observed among dengue cases. Unlike OFI controls, dengue cases showed an increasing pattern of copper levels from admission until defervescence. On the other hand, no such significant differences were observed in the serum level of iron in the clinical groups, except for a decreased iron level found in severe cases, compared to non-severe dengue without warning signs. The results show that copper is associated with dengue severity and this finding emphasizes the need to investigate the involvement of trace elements in disease severity so as to improve the prognosis of dengue.

RESUMOO papel dos elementos-traço na virulência da dengue não é ainda conhecido. O presente estudo avaliou os níveis séricos de dois micronutrientes, cobre e ferro, em casos de dengue. O estudo envolveu 96 pacientes dos quais 48 apresentavam dengue grave ou não grave (com ou sem sinais de alerta), e outros 48 pacientes com outras doenças febris (OFI) representaram os controles. Níveis séricos de cobre e ferro foram avaliados na admissão e no momento da defervescência usando kits comerciais disponíveis. À admissão, nenhuma diferença nos níveis séricos de cobre foi observada entre casos e controles. No grupo com dengue, os níveis de cobre se encontravam significativamente reduzidos nos casos graves e não graves com sinais de alerta, em comparação aos casos não graves sem sinais de alerta. Contrariamente, no momento da defervescência os níveis de cobre se encontravam aumentados em todos os casos de dengue em relação aos controles com outras doenças febris (OFI), no entanto, nenhuma diferença foi observada entre os casos de dengue. Diferentemente dos pacientes com outras doenças febris, os casos de dengue mostraram um padrão de elevação dos níveis de cobre do dia da admissão até a defervescência. Por outro lado, estas diferenças não foram observadas em relação aos níveis de ferro entre os dois grupos, com exceção de níveis de ferro reduzidos encontrados nos casos graves, em comparação aos não graves com sinais de alerta. Os resultados mostram que o cobre está associado à gravidade da dengue e esta observação enfatiza a necessidade de investigação do envolvimento de elementostraço na gravidade da doença para melhorar o prognóstico da dengue.

Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.

Alteraciones bioquímicas como marcadores predictores de gravedad en pacientes con fiebre por dengue / Biochemical alterations as prediction markers for the severity of illness in dengue fever patients

Introducción. El dengue es la infección transmitida por mosquitos más importante en el mundo. Existe información de que las alteraciones bioquímicas pueden utilizarse como herramientas predictoras de gravedad del dengue. Objetivo. Evaluar las alteraciones bioquímicas como posibles marcadores predictores de gravedad del dengue. Materiales y métodos. Se llevó a cabo un estudio de casos y controles anidado en una cohorte. Se seleccionaron al azar 125 casos con dengue grave y 120 controles con dengue no grave para evaluar los niveles séricos de lactato-deshidrogenasa (LDH), creatina cinasa (CK), proteína C reactiva (PCR) y albúmina, en sueros obtenidos en las primeras horas de la enfermedad. Para evaluar el valor diagnóstico de cada biomarcador, se establecieron puntos de corte con una sensibilidad del 90 % en la detección de casos graves. Resultados. Se observó una asociación entre los niveles de PCR por debajo de 9,8 mg/l (OR=0,04; IC 95% =0,02-0,08 ; p=0,000), de LDH inferiores a 400 U/L (OR=0,49; IC 95% =0,24-1,02; p=0,053) y de albúmina menor de 4 mg/dl (OR=3,46; IC 95% =1,96-6,12; p=0,000), con la gravedad del dengue. En contraste, los niveles de la CK no mostraron asociación con la gravedad de la enfermedad. Conclusiones. Los hallazgos de nuestro estudio sugieren una asociación de los niveles de PCR, LDH y albúmina con la gravedad del dengue. Estas pruebas bioquímicas podrían ser utilizadas como herramientas predictoras del curso clínico de la infección.

Introduction: Dengue is the most important mosquito-borne infection in the world. There is evidence supporting the use of biochemical alterations as prediction tools for severity of illness in dengue. Objective: To evaluate biochemical alterations as potential prediction markers for severity in dengue. Materials and methods: This was a case-control study nested in a cohort. We randomly selected 125 severe dengue cases and 120 controls with non-severe dengue for measuring LDH, CK, CRP and albumin serum levels using acute phase sera. To evaluate the predictive value for each biomarker, we established cut-off points with 90% sensitivity in detecting severe cases. Results: There was association among the CRP levels < 9.8 mg/L (OR=0.04; 95%CI=0.02-0.08; p=0.000), <400 U/L LDH levels (OR=0.49; 95%CI=0.24-1.02; p=0.053) and <4 mg/dl albumin levels (OR=3.46; 95%CI=1.96-6.12; p=0.000) with the severity of dengue. In contrast, the CK levels showed no association with the severity of the disease. Conclusions: Our findings suggest an association of CRP, LDH and albumin levels with the severity of dengue. These biochemical tests could be used as predictive tools in the clinical course of the infection.

Etiología y caracterización epidemiológica del síndrome febril no palúdico en tres municipios del Urabá antioqueño, Colombia / Etiology and epidemiological characterization of non-malarial febrile syndrome in three municipalities of Urabá (Antioquia), Colombia

Introducción. La región de Urabá es endémica para varias enfermedades febriles agudas de origen infeccioso. Sin embargo, solo los pacientes con malaria pueden acceder a un diagnóstico oportuno y rápido, motivo por el cual muchos síndromes febriles no palúdicos quedan sin diagnóstico etiológico claro. Objetivo. Establecer la etiología, describir las manifestaciones clínicas y explorar algunos posibles factores de riesgo relacionados con los síndromes febriles agudos no palúdicos en pacientes procedentes de los municipios de Necoclí, Turbo y Apartadó. Materiales y métodos. Se tomaron muestras de suero en fase aguda y de convalecencia de 220 pacientes febriles negativos para malaria, provenientes de zonas rurales y urbanas de Necoclí, Turbo y Apartadó en los años 2007 y 2008. Se practicaron pruebas para diagnóstico de dengue (detección de anticuerpos IgM por ELISA), leptospirosis (detección de anticuerpos IgM e IgG por IFI), rickettsiosis (detección de anticuerpos IgG por IFI), hantavirus y arenavirus (detección de anticuerpos IgG por ELISA). Resultados. Se encontraron frecuencias de dengue, leptospirosis, rickettsiosis y arenavirus de 37,3 %, 14,1 %, 2,7 % y 0,5 %, respectivamente. Se presentaron 12 casos de coinfección de leptospirosis-dengue y uno de leptospirosis-rickettsiosis-dengue. El sexo masculino y la humedad relativa media, fueron factores de riesgo para dengue. El inicio de signos clínicos en febrero de 2008, se asoció tanto con la infección por dengue como por leptospirosis. Conclusión. Se reafirma la importancia del virus del dengue, Rickettsia spp. y Leptospira spp., como agentes causantes del síndrome febril en la región del Urabá.

Introduction: Urabá, a region on the northern coast of Colombia, is endemic to several acute febrile illnesses of infectious origin; however, only patients with malaria may have access to quick and effective diagnosis. For this reason, many non-malarial febrile patients go without a clear etiologic diagnosis. Aim: To establish the etiology and clinical signs of acute febrile non-malaria syndromes and explore some of the likely risk factors in patients originating in the municipalities of Necocli, Turbo and Apartado who exhibit these symptoms. Materials and methods: We obtained acute and convalescent sera from 220 non-malarial febrile patients from the rural and urban zones of Necocli, Turbo and Apartado during 2007 and 2008. Serologic tests for dengue (IgM by ELISA), leptospirosis (IgM and IgG by IFA), rickettsiosis (IgG by IFI), hanta and arenavirus (IgG by ELISA) were performed. Results: We found that the frequency of infection for dengue, leptospirosis, rickettsiosis and arenavirus, was 37.3%; 14.1%; 2.7% and 0.5%, respectively. There were 12 co-infection cases of leptospirosis-dengue and one of leptospirosis-rickettsiosis-dengue. Male gender and relative humidity were considered risk factors for dengue, and the beginning of clinical signs in February of 2008 was associated with the infection of dengue and leptospirosis. Conclusion: This study confirms previous records that underline the importance of Rickettsia spp, dengue virus and Leptospira spp as causal agents of febrile syndrome in this region of Colombia.

Avaliação da hemostasia primária, coagulação e fibrinólise em pacientes com complicações hemorrágicas da dengue / Evalution of primary hemostasis, coagulation and fibrinolysis in patients with dengue and bleeding complications

Os casos de dengue são classificados em dengue clássica (DC) e febre hemorrágica da dengue (FHD). Embora a DC seja considerada uma manifestação não grave da dengue, algumas complicações clínicas podem ser encontradas neste grupo, em particular as complicações hemorrágicas. Entretanto, as causas de sangramentos na DC não estão esclarecidas. Desta forma, o objetivo deste estudo foi realizar uma avaliação abrangente dos possíveis mecanismos fisiopatológicos que podem contribuir para a tendência hemorrágica da dengue. Este é um estudo de caso-controle que incluiu adultos com dengue durante o período de defervescência da doença e controles saudáveis. Foram avaliadas as células do sangue periférico, os marcadores da resposta inflamatória, da permeabilidade microvascular, da ativação de células endoteliais e da fibrinólise e a geração de trombina nos pacientes e controles. Foram incluídos 26 adultos com DC e sangramento, 33 adultos com DC sem complicações, 8 adultos com FHD e 67 controles saudáveis. A gravidade e extensão dos sangramentos foram semelhantes entre os pacientes classificados como DC e os classificados como FHD. Pacientes com DC com sangramento e FHD tinham menor contagem de plaquetas em comparação aos com DC sem sangramento. Fator de necrose tumoral (TNF-?), trombomodulina (TM) e fator de von Willebrand (FVW) estavam aumentados nos grupos de dengue em relação aos controles. A atividade e antígeno da ADAMTS 13 estavam diminuídos, em particular nos pacientes com DC não complicada. Houve tendência ao aumento do fator de crescimento do endotélio vascular (VEGF-A) apenas nos pacientes com FHD, mas a diferença não foi estatisticamente significativa. Houve, porém, alterações significativas dos receptores solúveis do VEGF-A (sVEGF-R1 e sVEG-R2). Os níveis plasmáticos do sVEGF-R1 estavam aumentados e de sVEGF-R2 estavam diminuídos nos pacientes, principalmente nos com FHD...

Dengue has been classified according to disease severity into dengue fever (DF) and dengue hemorrhagic fever (DHF). Although DF is considered a non-severe manifestation of dengue, it represents a heterogeneous group of patients with varied clinical complications, particularly, bleeding complications. However, the causes of bleedings in DF have not been fully addressed. Thus, the aim of this study was to perform a comprehensive evaluation of possible pathophysiological mechanisms that could contribute to the bleeding tendency in DF and DHF. This is a case-control study that enrolled adults with DF without bleeding, DF and bleeding complications and DHF during the defervescence period. Healthy controls were also included. Peripheral blood counts, inflammatory, microvascular permeability, fibrinolysis, endothelial cell activation markers, and thrombin generation were evaluated in patients and controls. We included 26 adults with DF and bleeding, 33 adults with DF without complications, 8 adults with DHF and 67 healthy controls. The severities of the bleeding episodes were similar between patients with DC and DHF. Patients with DF with bleedings and DHF had lower platelet counts than DF without bleeding. Levels of the tumoral necrosis factor (TNF)-?, thrombomodulin (TM) and von Willebrand factor (VWF) were significantly increased in dengue groups than in healthy controls, but similar among patients. Plasma levels of the soluble receptor 1 of the vascular endothelial growth factor (sVEGR-R1) were increased and plasma levels of sVEGF-R2 were decreased in patients with DF and bleedings and in patients with DHF. In DHF patients presented a non-significant increased in vascular endothelial growth factor (VEGF-A) serum levels and a significantly higher VEGF/sVEGF-R2 ratio...

Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus / Análisis de las eficiencias de amplificación por PCR en tiempo real de tres regiones genómicas del virus dengue

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3’-noncoding region (3’NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3’NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3’NC regions showed a weak (k= 0.109; CI 95%) and a moderate (k= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

El diagnóstico precoz de la infección por el virus dengue (DENV) constituye un elemento clave para la prevención de las complicaciones clínicas propias de la enfermedad. El objetivo del estudio fue evaluar la detección de DENV mediante un ensayo cuantitativo de PCR-tiempo real (qPCR), desarrollado localmente, utilizando la región no-estructural-5 (NS5), versus dos regiones tradicionalmente empleadas para la detección del virus, la región cápside/pre-membrana (C-prM), y la región noncodificante-3’ (3’NC). Se recolectaron 167 muestras de suero de pacientes en fase aguda de la enfermedad. Las tres regiones génicas tuvieron perfiles de amplificación/coeficientes de correlación similares (0,987-0,999). Sin embargo, la región NS5 tuvo la eficiencia de amplificación más elevada para los cuatro serotipos (98-100%). Durante el proceso de validación, 41,1% (67/167) de las muestras de suero resultaron positivas para DENV al menos por dos de las regiones genómicas empleadas. Los valores de concordancia entre las regiones NS5/C-prM y NS5/3’NC fueron de 56,7% y 97%, respectivamente. La concordancia fue débil entre las regiones NS5/C-prM (k= 0,109; CI 95%), sin embargo, fue moderada entre las regiones NS5/3’NC (k= 0,489; CI 95%). El ensayo de tipificación uniplex en formato NS5/TaqMan® mostró alta sensibilidad (100%) que el protocolo C-prM/SYBRGreen®-I (76%). La validación externa del ensayo mostró una alta sensibilidad (100%), especificidad (78%) y acuerdo alto entre los ensayos utilizados. De acuerdo a los resultados obtenidos, la región NS5 ofrece la mayor opción para la detección y serotipificación del DENV en muestras clínicas.

Clinical and hepatic evaluation in adult dengue patients: a prospective two-month cohort study / Avaliação das alterações clínicas e hepáticas em pacientes adultos com dengue: estudo prospectivo em um período de dois meses

INTRODUCTION: To analyze the liver dysfunction and evolution of signs and symptoms in adult dengue patients during a two-month follow-up period. METHODS: A prospective cohort study was conducted in Campos dos Goytacazes, Rio de Janeiro, Brazil, from January to July, 2008. The evolution of laboratory and clinical manifestations of 90 adult dengue patients was evaluated in five scheduled visits within a two-month follow-up period. Twenty controls were enrolled for the analysis of liver function. Patients with hepatitis B, hepatitis C, those known to be human immunodeficiency virus (HIV) seropositive and pregnant women were excluded from the study. RESULTS: At the end of the second month following diagnosis, we observed that symptoms persisted in 33.3% (30/90) of dengue patients. We also observed that, 57.7% (15/26) of the symptoms persisted at the end of the second month. The most persistent symptoms were arthralgia, fatigue, weakness, adynamia, anorexia, taste alteration, and hair loss. Prior dengue virus (DENV) infection did not predispose patients to a longer duration of symptoms. Among hepatic functions, transaminases had the most remarkable elevation and in some cases remained elevated up to the second month after the disease onset. Alanine aminotransferase (ALT) levels overcame aspartate aminotransferase (AST) during the convalescent period. Male patients were more severely affected than females. CONCLUSIONS: Dengue fever may present a wide number of symptoms and elevated liver transaminases at the end of the second month.

INTRODUÇÃO: Analisar prospectivamente a disfunção hepática e a evolução dos sinais e sintomas em pacientes adultos com dengue durante um período de dois meses. MÉTODOS: Realizamos um estudo prospectivo em Campos dos Goytacazes, Rio de Janeiro, Brasil, de janeiro a julho de 2008. Foi avaliada a evolução das manifestações clínicas e laboratoriais em 90 pacientes adultos com dengue, em um período de dois meses. Vinte controles foram arrolados para análise da função hepática. Em ambos os grupos foram realizadas coletas de dados e sangue nos primeiros cinco dias da doença, e aos 8, 15, 30 e 60 dias após o início da doença. Foram excluídos pacientes com hepatite B, hepatite C, gestantes e aqueles sabidamente soropositivos para HIV. RESULTADOS: No final do segundo mês do início da dengue, 33,3% (30/90) dos pacientes apresentaram persistência de pelo menos um sinal ou sintoma. Estavam presentes no final do segundo mês 57,7% (15/26) dos sinais ou sintomas. Os maiores percentuais de persistência foram: artralgia, adinamia, fraqueza, fadiga, anorexia, alteração do paladar e queda de cabelo. A infecção prévia pelo vírus da dengue (DENV) não predispôs a uma maior duração dos sintomas. Da função hepática, observamos alterações relevantes somente nos níveis das transaminases, que em alguns casos permaneceram elevados até o final do segundo mês. Os níveis de ALT ultrapassaram os de AST na convalescença. Homens apresentaram níveis mais elevados de transaminases quando comparados aos de mulheres. CONCLUSÕES: Dengue apresenta grande número de sintomas e transaminases elevadas no final do segundo mês de doença.

Alterações do hemograma no diagnóstico de dengue: um estudo de 1.269 casos na cidade de Uberaba, Minas Gerais / Blood count changes in dengue diagnosis: a study of 1,269 cases in Uberaba city, Minas Gerais, Brazil

Introdução: O dengue é uma doença infecciosa frequente no Brasil e, particularmente, na cidade de Uberaba, Minas Gerais. A doença se manifesta em um amplo espectro de sintomas que levam a alterações nos parâmetros do hemograma. Em nosso trabalho, procuramos estudar a frequênciade cada uma das alterações nas séries vermelha, branca e plaquetária em pacientes acometidos pelo dengue em Uberaba-MG, comparando os biênios de 2005-2006 e 2009-2010. Métodos: Foram analisados hemogramas de pacientes atendidos em laboratório particular e em Unidade de ProntoAtendimento da cidade, que apresentaram resultado positivo para sorologia de dengue (IgM). Resultados: Nos biênios 2005-2006 e 2009-2010, foram selecionados, respectivamente, 1.061 e 208 hemogramas para inclusão no estudo. Leucopenia, linfopenia, monocitose e plaquetopenia foramas alterações mais frequentemente encontradas. Conclusão: Algumas alterações apresentaram valorcomo suporte ao diagnóstico da doença em casos nos quais seria difícil ou impossível a realização de exame sorológico para sua detecção.

Evaluación del sistema diagnóstico SD Dengue Duo para la detección de la proteína NS1 y los anticuerpos IgM e IgG anti-dengue / Evaluation of the SD Dengue Duo diagnosis system for detection of NS1 protein and IgM and IgG dengue antibodies

Introducción: el sistema comercial SD Dengue Duo (Standard Diagnostics) es una prueba rápida inmunocromatográfica que detecta la proteína NS1 del virus dengue y los anticuerpos anti-dengue IgM e IgG de forma simultánea. Objetivo: evaluar las características funcionales y operacionales del sistema comercial para la detección de los diferentes marcadores virológicos y serológicos. Métodos: se utilizó un panel constituido por 161 muestras de suero, 113 procedentes de pacientes con un diagnóstico clínico y serológico confirmado a cualquiera de los 4 serotipos del virus dengue y 48 negativas. Todas las muestras fueron procesadas por el sistema SD Dengue Duo y por las técnicas Platelia Dengue NS1 Ag, ELISA de captura IgM y el ELISA de inhibición utilizadas como referencia. Resultados: el sistema evaluado mostró una sensibilidad de 57,75 por ciento para la proteína NS1, se observaron falsos negativos en muestras colectadas a partir del quinto día de iniciados los síntomas en casos con infección secundaria. La detección de anticuerpos IgM mostró una sensibilidad de 96,0 por ciento y especificidad de 98,4 por ciento. Se encontró una alta concordancia (95,7 por ciento) al clasificar el tipo de infección. En el estudio global de los 3 marcadores la sensibilidad del sistema se incrementó hasta 100 por ciento. Conclusiones: el SD Dengue Duo es un método simple, rápido, fácil de ejecutar, el cual no requiere de equipamiento adicional; tiene la ventaja de poder ser utilizado para muestras, tanto de fase aguda como en la fase convaleciente y pudiera ser una alternativa para el diagnóstico del dengue en aquellos laboratorios que no cuenten con facilidades para ello.

Introduction: SD Dengue Duo (Standard Diagnosis) commercial kit is an immunochromatographic rapid test that detects NS1 protein and IgG/IgM dengue antibodies simultaneously. Objective: to evaluate the operational and functional characteristics of this system for the detection of virological and serological markers. Methods: sera panel was made up by 161 samples, 113 from patients with clinically and serologically confirmed dengue caused by any of the four dengue virus serotypes and 48 negative samples. All these samples were tested by SD Dengue Duo Kit and by Platelia Dengue NS1 Ag, IgM Capture ELISA and ELISA Inhibition Method used as reference assays. Results: the evaluated kit showed a 57.75 percent sensitivity for the detection of NS1 protein, false negatives were detected in samples collected 5 days or more after fever onset in secondary infection cases. IgM detection showed 96.0 percent sensitivity and 98.4 percent specificity. Furthermore, high agreement (95.7 percent) in classifying dengue infection types (primary or secondary infections) was observed. The global study of the 3 markers, the sensitivity rose to 100 percent. Conclusions: SD Dengue Duo is a simple, easy and rapid assay; it does not require additional equipment, can be used for acute and convalescence serum samples and offers a good alternative for dengue diagnosis in those laboratories where a complete dengue virus diagnosis is difficult to perform.

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.