Contribución de la prueba rápida NS1 e IgM al diagnóstico de dengue en Colombia en el periodo pre-zika / Contribution of NS1 and IgM based-rapid tests to dengue diagnosis in Colombia before zika emergence

Objetivo: Se considera que el diagnóstico del dengue es fundamentalmente clínico; sin embargo, las pruebas rápidas basadas en la detección de IgM o NS1/IgM están siendo utilizadas en los servicios de salud. Este estudio determinó la contribución de las pruebas rápidas al diagnóstico de dengue en un área endémica antes de la introducción del virus zika. Metodología: Diseño de corte transversal de pruebas diagnósticas realizado a partir del análisis secundario de un estudio previo en 14 instituciones de salud del Valle del Cauca. Se obtuvo información de 632 participantes con resultados de prueba rápida, diagnóstico clínico y pruebas de referencia ELISA NS1, ELISA IgM y RT-PCR. Se compararon la sensibilidad, especificidad, valores predictivos y razones de verosimilitud del uso solo, en serie, y paralelo de los componentes NS1, IgM, NS1/IgM de la prueba rápida y el diagnóstico clínico con las pruebas Q de Cochran y McNemar para datos pareados. Resultados: La sensibilidad del diagnóstico clínico (61,4% IC95% 56%-66,7%) fue superior a la de las pruebas rápidas (37% IC95% 29,6%-44,7%) (P Conclusión: El diagnóstico clínico tiene una mayor sensibilidad que las pruebas rápidas, pero por si solo no es suficiente para confirmar o descartar dengue. Un resultado positivo en pruebas rápidas en pacientes con diagnóstico clínico de dengue es útil para confirmarlo, pero un resultado negativo no lo descarta.

Objective: Dengue diagnosis is considered to be mainly clinical; however, rapid tests that detect IgM or NS1/IgM are being used in health services. This study assessed the contribution of rapid tests to dengue diagnosis in an endemic area before the emergence of zika virus in Colombia. Methods: Cross-sectional study of diagnostic tests based on a secondary analysis of a previous study in 14 health care institutions in Valle del Cauca department. Results of dengue rapid test, clinical diagnosis, and reference tests ELISA NS1, ELISA IgM, and RT-PCR were obtained for 632 participants. The sensitivity, specificity, predictive values and likelihood ratios of the use alone, serial and parallel combinations of NS1, IgM, NS1/IgM of the rapid test and clinical diagnosis were compared using Cochran´s Q and MacNemar tests for paired data. Results: The sensitivity of clinical diagnosis (61.4% 95%IC 56-66.7) was higher than the sensitivity of rapid tests (37% 95% IC 29.6-44.7) (P<0.001). The serial used of NS1/IgM rapid test when clinical diagnosis was negative increased the sensitivity to 79.5% and, the serial use when clinical diagnosis was positive increased the specificity (from 66.3% to 98.7%). However, the latter decreased the sensitivity to 32.2%. While all negative likelihood ratios (LR-) were close to 1, the serial use of rapid tests when clinical diagnosis was positive had LR+ higher than 10. Conclusion: The clinical diagnosis is more sensitive than rapid tests, but by itself does not confirm or rule out dengue. A positive result in rapid tests is useful to confirm dengue but a negative result does not rule it out.

Análisis filogenético de secuencias suramericanas del gen ns1 del serotipo dengue-2, relacionadas con sangrado clínico severo / Phylogenetic analysis of South American sequences of the nonstructural protein-1 (ns1) of dengue serotype 2 associated with severe clinical bleeding

Objetivo Comparar secuencias de nucleótidos y de aminoácidos de la proteína no estructural 1-NS1 de cepas DENV-2, aisladas de pacientes febriles de diferentes países suramericanos, que cursaron cuadros clínicos con severidad o sin ella. Materiales y Métodos El análisis filogenético fue realizado a partir de 28 secuencias moleculares completas (1 056 pb) del gen NS1 del serotipo DENV-2. Se realizó un análisis filogenético bayesiano utilizando el software MrBayes v.3.2.0, con el modelo SYM+G y un análisis filogenético con el método Neighbor-Joining con el modelo Jukes-Cantor. Además, las secuencias de aminoácidos fueron alineadas y comparadas entre sí, mediante el programa Clustal W incluido en el software MEGA v. 5.2. Resultados En las secuencias de aminoácidos asociadas a sangrado, la sustitución más frecuente fue isoleucina→ treonina, en la posición 93. Estas secuencias presentaron un mayor porcentaje (94,6 %) de homología de aminoácidos de la proteína NS1 en comparación con el porcentaje de homología (74 %) de los aislamientos DENV-2 no asociados a sangrado. Se identificaron cinco clados que agrupan la mayoría de las secuencias analizadas (19/24; 79,2 %) con valores de probabilidad posterior mayores o iguales al 58 %. Siete (87,5 %) secuencias asociadas a sangrado se relacionan filogenéticamente dentro de los clados 4 y 5, con valores de probabilidad posterior del 58 % y 97 %, respectivamente. Conclusión No se encontraron características filogenéticas ni tampoco diferencias entre las secuencias de aminoácidos de la proteína NS1-DENV-2 estudiadas, que pudieran ser relacionadas, de manera directa, con la severidad de la enfermedad.(AU)

Objective The objective of this in silico study was to compare nucleotide and amino acids DENV-2-NS1 sequences isolated from febrile patients, with and without disease severity, from different South American countries. Matherials and Methods A bayesian MCMC phylogenetic analysis was carried out using 28 complete sequences of the gene NS1 of the DENV-2 serotype (1 056 bp), using MrBayes v.3.2.0 software, with the model SYM+G (2.5 million generations). We also carried out a phylogenetic analysis with Neighbor-Joining method (Jukes-Cantor model). In addition, the amino acids sequences were aligned and compared with each other, using Clustal W included in MEGA v.5.2 software. Results In the amino acids sequences associated with bleeding, the most frequent substitution was isoleucine → threonine at posicion 93. These sequences showed a high percentage (94.6 %) of amino acid homology in comparison with the percentage of amino acids homology (74 %) of DENV-2 isolates not associated with bleeding. Five clades were identified that group the vast majority of the DENV-2-NS1 sequences analyzed (19/24; 79.2 %) with posterior probability values greater than or equal to 58 %. Seven sequences (87.5 %) associated with bleeding were phylogenetically related within clades 4 and 5, the posterior probability values were 58 % and 97 %, respectively. Conclusion Neither phylogenetic characteristics nor differences between amino acids of the DENV-2-NS1 sequences studied were found that could be associated directly with severity of the disease.(AU)

Inhibitory potential of Quercus lusitanica extract on dengue virus type 2 replication.

This study reports the in vitro inhibitory potential of crude extract of Quercus lusitanica (Q. lusitanica) seeds on the replication of dengue virus type 2 (DEN-2). In vitro antiviral activity of Q. lusitanica extract, assessed in C6/36 cells (cloned cells of Aedes albopictus larvae) employing a virus inhibition assay, showed dose-dependent inhibition. The Q. lusitanica extract at its maximum non-toxic concentration of 0.25 mg/ml completely inhibited 10-1,000 TCID50 of virus, as indicated by the absence of cytopathic effect (CPE). The low dose of Q. lusitanica (0.032 mg/ml) showed 100% inhibition with 10 TCID50 of virus, but only 50% and 25% inhibition with 100 and 1,000 TCID50, respectively. The NS1 is a glycoprotein present in all flaviviruses and appears essential for virus viability. To further evaluate Q. lusitanica extract as an antiviral compound, we investigated the effect of Q. lusitanica extract on the NS1 protein expression of infected C6/36 cells through proteomics technique. The result showed downregulation of NS1 protein expression of infected C6/36 cells after treatment with this extract. In conclusion, we found that Q. lusitanica extract has a good inhibitory effect on the replication of dengue virus type 2, both in conventional cell culture and proteomics technique.

Detection of human parvovirus B19 nonstrutural protein DNA by nested-polymerase chain reaction in gravida serum and pregnant tissues / 华中科技大学学报（医学）（英德文版）

A new nested-polymerase chain reaction (nested-PCR) assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0.005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.