Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Padronização e uso de um método imunoenzimático que utiliza células infectadas como antígeno no diagnóstico rotineiro do dengue / Standardization and use of an immunoenzymatic method using infected cells as antigens in routine diagnosing of dengue

INTRODUÇÃO: Este trabalho mostra a padronização e o uso do método imunoenzimático utilizando células infectadas como antígeno (EIA-ICC) no diagnóstico sorológico rotineiro do dengue. MÉTODOS: Na otimização do teste, com a dose de 1.000 TCID50 de vírus do dengue tipo 3 (DENV-3), foram utilizadas 100.000 células C636 infectadas 1000 TCID50 (DENV-3). RESULTADOS: Os resultados obtidos com EIA-ICC foram comparados com o kit comercial de dengue HUMAN. Os resultados foram altamente coincidentes; o EIA-ICC mostrou-se moderadamente sensível e com alta especificidade. O teste foi usado no diagnóstico sorológico de 1.797 amostras sorológicas de casos suspeitos de dengue durante a epidemia de Ribeirão Preto, em 2006. Na avaliação sorológica, 228 amostras foram positivas para IgM contra DENV-3, e 235 amostras foram positivas para IgG contra DEV-3, e em 35 amostras detectou-se positividade para IgM e IgG. CONCLUSÕES: O EIA-ICC mostrou-se confiável e simples sendo adequado ao diagnóstico sorológico do dengue.

INTRODUCTION: This paper show the standardization and use of the immunoenzymatic method using infected cells as antigens (EIA-ICC) for routine serological diagnosing of dengue. METHODS: In optimizing the test, a dose of 1,000 TCID50 of dengue type 3 virus (DENV-3) was used, and 100,000 C636 cells infected with 1,000 TCID50 (DENV-3) were used. RESULTS: The results obtained with EIA-ICC were compared with the HUMAN commercial dengue kit. The results were highly concordant. The EIA-ICC showed moderate sensitivity and high specificity. The test was used for serologically diagnosing 1,797 blood samples from suspected dengue cases during the 2006 epidemic in Ribeirao Preto. From the serological evaluation, 228 samples were positive for IgM against DENV-3; 235 samples were positive for IgG against DENV-3; and 35 samples were positive for both IgG and IgM. CONCLUSIONS: EIA-ICC was shown to be reliable and simple, and suitable for serologically diagnosing dengue.