Search

This work reviews the opportunities and scientific bases in the development of anti-dengue drugs. The timeliness of anti-dengue drug development is addressed in the context of the growing impact of dengueworldwide and existing strategies to fight the virus. The antiviral approach in therapy or prophylaxis during an epidemic as well as the impact of recent technological advances in drug-discovery and antiviral chemotherapy on the development of anti-dengue drugs are discussed. An analysis of current sources of synthetic or natural drugs is provided. Finally, we summarize the current knowledge on dengue virus proteins, which are currently considered the most viable as drug targets, as the envelop protein E and non-structural proteins NS3 and NS5 carrying protease, helicase, RNA triphosphatase, methyltransferase and RNA-dependent RNA polymerase activities. Other viral proteins proposed to be part of the replication complex and the complex itself are considered as potential targets of anti-dengue drugs. State-of-the-art methods are listed, that are expected to allow the discovery, design, and characterisation of anti-dengue drugs effective against the four serotypes.

International guidelines emphasize the importance of LDL cholesterol (LDL-C) assay in the care and follow-up of patients with cardiovascular risk. Most studies and common practice use Friedewald's formula for LDL-C calculation. The accuracy of the result depends closely on the precision of the input parameters (total cholesterol, triglycerides (TG) and HDL cholesterol), and discrepancies between calculated LDL-C and measurement by reference methods appear when TG exceed 4.5 mmol/L, or in the presence of abnormal lipoproteins. These restrictions and uncertainties in calculations have prompted the recent development of direct and homogeneous methods that fit all analyzers. A multicenter evaluation of four direct assays of LDL-C (Daiichi, Denka Seiken, Kyowa, Wako) was carried out on 45 serum samples (TG below 3.1 mmol/L) in eight laboratories using different analyzers. For three methods (Daiichi, Kyowa, Wako), the interlaboratory reproducibility was markedly improved relative to that of calculation. A strong correlation was found for all new methods when compared with a beta-quantification assay. Average bias in Denka Seiken assays was greater than Kyowa's and Daiichi's (although less dispersed for the latter) and for Wako all bias were positive. The relationship between bias variations and the lipid parameters of the samples was studied. Three methods, Daiichi, Kyowa and Wako, revealed a significant positive correlation between bias and serum VLDL-C/TG ratio, clearly indicating that cholesterol enrichment of VLDL was a source of variability in these assays. Specificity of the four methods was tested in situation of dyslipidemia by spiking isolated lipoproteins (chylomicrons, VLDL and HDL). This experiment revealed differences in behavior, most evidently upon addition of VLDL. No method was truly specific, but up to 8 mmol/L of TG the variations were acceptable. In the presence of type III hyperlipoproteinemia, however, only the Denka Seiken method was reliable. Linearity up to 20 mmol/L (Daiichi, Denka Seiken) or 14 mmol/L (Kyowa, Wako) of LDL-C allows these tests to be used in main routine cases. New direct assays are an obvious technological advance in terms of analytical performance and conveniency. Their use for the diagnosis and follow-up of hyperlipidemic patients offers an alternative that overcomes the limitations of the Friedewald calculation.

Most frequently, in routine laboratories, C-HDL is measured in the supernatant after precipitation of apolipoprotein B-containing lipoproteins by the sodium phosphotungstate/magnesium chloride reagent (PTA). This method involves precipitation, centrifugation and decantation steps which prevent full automation of the measurement and decrease the accuracy of the results. Recently, three direct assays for C-HDL including alpha-cyclodextrin sulphate (alpha-CD), polyanions/detergents (PA-D) or antibodies anti-beta-lipoproteins (AC) have been commercialized, in which all steps are fully managed by automated analyzers. These new methods have been compared to the conventional procedure (PTA), in multicenter studies among six laboratories using different analyzers. The C-HDL values measured by the alpha-CD and PA-D assays correlated well with those of the PTA method (r > 0.98), on most of the analyzers. With the AC assay, only the results obtained with the Hitachi 717 analyzer were correlated with C-HDL values of the PTA method. The linearity and specificity studies were evaluated in the laboratory A on a Kone Specific analyzer. The alpha-CD and PA-D assays were linear for C-HDL values from 0 to 5.56 mmol/l, as observed by increasing amounts of HDL2 + HDL3 or serum without lipoprotein isolated by ultracentrifugation. The specificity of these two methods was evaluated simultaneously, by adding various amounts of lipoproteins isolated by sequential ultracentrifugation. No interference was observed when adding chylomicrons up to 13.4 mmol/l of triglycerides for both methods. Inversely, increased C-HDL values were observed with added VLDL from 6 mmol/l of triglycerides for the PA-D assay and from 8 mmol/l for the alpha-CD assay. No interference was observed with added LDL up to 11.5 mmol/l of C-LDL for the alpha-CD assay and up to 6.7 mmol/l for the PA-D assay. In conclusion, the present multicenter evaluation demonstrates that the new procedures for the direct automation of C-HDL are easy and accurate and most of them correlated well with the classical precipitation method. In addition the study provides arguments for a choice between the different direct C-HDL methods.