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

New apparatus for DTA at 2000 bar: thermodynamic studies on Au, Ag, Al and HTSC oxides

A new DTA (Differential Thermal Analysis) device was designed and installed in a Hot Isostatic Pressure (HIP) furnace in order to perform high-pressure thermodynamic investigations up to 2 kbar and 1200C. Thermal analysis can be carried out in inert or oxidising atmosphere up to p(O2) = 400 bar. The calibration of the DTA apparatus under pressure was successfully performed using the melting temperature (Tm) of pure metals (Au, Ag and Al) as standard calibration references. The thermal properties of these metals have been studied under pressure. The values of DV (volume variation between liquid and solid at Tm), ROsm (density of the solid at Tm) and ALPHAm (linear thermal expansion coefficient at Tm) have been extracted. A very good agreement was found with the existing literature and new data were added. This HP-DTA apparatus is very useful for studying the thermodynamics of those systems where one or more volatile elements are present, such as high TC superconducting oxides. DTA measurements have been performed on Bi,Pb(2223) tapes up to 2 kbar under reduced oxygen partial pressure (p(O2) = 0.07 bar). The reaction leading to the formation of the 2223 phase was found to occur at higher temperatures when applying pressure: the reaction DTA peak shifted by 49C at 2 kbar compared to the reaction at 1 bar. This temperature shift is due to the higher stability of the Pb-rich precursor phases under pressure, as the high isostatic pressure prevents Pb from evaporating.Comment: 6 figures, 3 tables, Thermodynamics, Thermal property, Bi-2223, fundamental valu

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin

Background Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. Methods We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. Discussion If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). Trial registration ClinicalTrials.gov NCT05169554. Registered on 27 December 2021

Inhibiting the stringent response blocks Mycobacterium tuberculosis entry into quiescence and reduces persistence

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.This work was supported by R01AI083125, R21AI122922, and R21AI114507A to P.C.

Surface expression of the IFN-gR2 chain is regulated by intracellular trafficking in human T lymphocytes.

The surface and cytoplasmic expressions of the transducing chain (IFN-gamma R2) of the heterodimeric IFN-gamma receptor on human T lymphocytes have been investigated. We show that its surface expression is low, whereas high cytoplasmic levels are found in both resting and PHA-activated T lymphocytes. This low expression does not prevent activated T cells from responding to IFN-gamma, because it induces IFN-regulatory factor 1 expression, Low surface IFN-gamma R2 expression appears to be due to recycling between cytoplasmic stores and the cell surface, which does not depend on signals mediated by endogenous IFN-gamma, because IFN-gamma R2 surface expression is low, and its internalization is equally observed in patients with inherited IFN-gamma R1 gene deficiency and in healthy donors. Moreover, IFN-gamma R2 internalization in T lymphoblasts from healthy donors was not affected by the presence of anti-IFN-gamma-neutralizing or anti-IFN-gamma R1-blocking mAb. In conclusion, these data illustrate a new mechanism whereby human T cells limit the surface expression of IFN-gamma R2 in a ligand-independent manner