New molecular settings to support in vivo anti-malarial assays

Background Quantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods. Methods FTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes. Results The optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed. Conclusions This is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process

A novel validated assay to support the discovery of new anti-malarial gametocytocidal agents

Additional file 1. Graphical representation of the expression of 12 selected genes throughout the 30 days of gametocytogenesis. Y-axis shows the gene expression represented as (Ctgene−Ct18S rRNA)Ttime × −(Ctgene−Ct18S rRNA)T0, considering the time 0 as the basal expression. Although only the gametocytogenesis during 30 days is presented, similar results were obtained from day 0 to day 15 in both assays

Use of belimumab in real-world in Spain: a scoping review about characteristics of SLE patients.

Belimumab was the first biological drug approved for Systemic Lupus Erythematosus (SLE). There is not a review focusing on all real-life experience with belimumab to date that could help to describe how this drug behaves in the Spanish clinical setting. To describe the characteristics of SLE patients treated with belimumab added to standard of care in real-clinical setting in Spain. We conducted a comprehensive scoping review of real-world data (RWD) according to PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O'Malley. PubMed and EMBASE were searched without language restriction and hand searches of relevant articles were examined. We included data from 222 patients treated with belimumab for SLE included in 19 RWD studies conducted in Spain. The mean age was 40.9 years, 84.2% were female, and baseline scores SELENA-SLEDAI ranged between 5.9 and 12. Lupus nephritis basal prevalence was of 2.7%. The main reason for belimumab initiation was previous treatments lack of efficacy (69.7%) and the most common laboratory abnormalities were hypocomplementemia (40.9%), ANA + (34.2%), and anti-DNA (33.3%). The addition of belimumab to standard therapy was associated with a reduction of daily glucocorticoids intake in 1.4-11.1 mg at 6 months. Belimumab discontinuation was observed in 18.6% of patients. Our study helps to further explore the profile of SLE patients most likely to be treated with belimumab

Functional screening of selective mitochondrial inhibitors of Plasmodium

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors. Keywords: Plasmodium falciparum, Plasmodium yoelii, Oxygen consumption, Mitochondrial inhibitors and cytochrom