Development and evaluation of molecular methods for the diagnosis of the dengue.
Nowadays, dengue is the most important arbovirosis affecting humans, being a serious public health problem in the world, especially in tropical regions, where environmental conditions favor the proliferation of its vectors: mosquitoes from the genus Aedes. Currently, almost half of the world?s population lives in areas subject to the disease and, in spite of its breadth and gravity, no vaccines or specific treatments are available. Aware of the need to improve epidemiologic surveillance for dengueand of the deficiencies of laboratorial diagnoses for this and other diseases in the country, this study aimed to develop diagnostic methods based on state-of-the-art molecular techniques for detection and serotype identification of the dengue virus. The selected methods were real-time PCR and liquid microarray, which represent the most recent advances in the field of molecular diagnosis. Different sets of serotype and group-specific oligonucleotides where evaluated for their ability in amplifying and identifying the serotypes DENV1, DENV2 and DENV3 utilizing each of the two methods. In this way,based on the real-time PCR technique, we developed three uniplex serotype-specific assays for dengue and one group-specific test capable of amplifying all virus serotypes. For the assay based on the liquid microarray technique the oligonucleotides, after being individually tested, were combined in different multiplex formats, both in the amplification (PCR) and hybridization reactions. Different hybridization protocols were also evaluated. When tested against standard curves obtained from the dilution of known amounts of the three virus serotypes, real-time PCR proved to be capable, for allassays, of amplifying and detecting even the smallest quantity of virus tested, equivalent to 53,47 FFU/ml. In the other hand, sensibility of detection of the liquid microarray multiplex was 3.428,48 FFU/ml for DENV1 and 53,57 FFU/ml for DENV2 and DENV3, with the serotype-specific oligonucleotides, and 214,28 FFU/ml for DENV1 and 53,57 FFU/ml for DENV2 and DENV3 with the group-specific oligonucleotides. Both methods had 100% of specificity. In order to evaluate these test?s ability in diagnosing and identifying the serotypes of dengue virus in clinical samples, we utilized 50 sera positive for anti-dengue IgM, 49 negative by serology and 49 sera from patients whose infection with dengue virus was confirmed by viral isolation. Agreement between both methods was of 92,5% when comparing results obtained with the group-specific oligonucleotides and of 90,5% for theserotype specific assays. Taking the group confirmed as positive by viral isolation as a reference, the liquid microarray method failed in detecting three samples that were considered positive by the realtime PCR and detected one sample that was not detected by PCR. Although the liquid microarraymethod showed less sensibility than the real-time PCR, advantages coming from it allowing simultaneous analysis of several targets, its flexibility, versatility and feasible cost justify further work on the optimization of this test. This study served mainly as a test of the technical feasibility of thecited methods for the diagnosis of viral diseases and we believe that it will be beneficial not only for the diagnosis of dengue but also for that of many other priority diseases in Brazil.
Advisor:Marco Aurélio Krieger
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords: Polymerase Chain Reaction
Date of Publication:12/21/2007