Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum.

Background: Plasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably differentiate P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are used routinely in P. knowlesi co-endemic areas despite potential cross-reactivity for species-specific antibody targets. Methods: Ten RDTs were evaluated: nine to detect clinical P. knowlesi infections from Malaysia, and nine assessing limit of detection (LoD) for P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH. Results: Samples were collected prior to antimalarial treatment from 127 patients with microscopy-positive PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247-5,565/µL). Pan-pLDH sensitivities ranged from 50.6% (95% CI 39.6-61.5) (SD BIOLINE) to 87.0% (95% CI 75.1-94.6) (First Response® and CareStart™ PAN) compared to reference PCR. Pv-pLDH RDTs detected P. knowlesi with up to 92.0% (95% CI 84.3-96.7%) sensitivity (Biocredit™). For parasite counts ≥200/µL, pan-pLDH (Standard Q) and Pv-pLDH RDTs exceeded 95% sensitivity. Specificity of RDTs against 26 PCR-confirmed negative controls was 100%. Sensitivity of six highest performing RDTs were not significantly different when comparing samples taken before and after (median 3 hours) antimalarial treatment. Parasite ring stages were present in 30% of pre-treatment samples, with ring stage proportions (mean 1.9%) demonstrating inverse correlation with test positivity of Biocredit™ and two CareStart™ RDTs.For cultured P. knowlesi, CareStart™ PAN demonstrated the lowest LoD at 25 parasites/µL; LoDs of other pan-pLDH ranged from 98 to >2000 parasites/µL. Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. No false-positive results were observed in either P. falciparum-pLDH or histidine-rich-protein-2 channels. Conclusion: Selected RDTs demonstrate sufficient performance for detection of major human malaria species including P. knowlesi in co-endemic areas where microscopy is not available, particularly for higher parasite counts, although cannot reliably differentiate among non-falciparum malaria

Regulation of Rps6 phosphorylation by TOR complexes in Saccharomyces cerevisiae

The Target Of Rapamycin (TOR) pathway is a central regulator of cell growth in response to various environmental and cell intrinsic cues. In this work, we investigated the role of TOR pathway in the regulation of the fate of an mRNA in Saccharomyces cerevisiae. The first part of the thesis work was dedicated to the investigation of how Ccr4, the major deadenylase in budding yeast, is regulated by TOR complex 1, while the second part focused on the regulation of translation by TOR complexes 1 and 2 with a specific interest on the regulation of the phosphorylation of a ribosomal protein, Rps6 in a TOR-dependent manner

A tuberculosis biomarker database: the key to novel TB diagnostics

New diagnostic innovations for tuberculosis (TB), including point-of-care solutions, are critical to reach the goals of the End TB Strategy. However, despite decades of research, numerous reports on new biomarker candidates, and significant investment, no well-performing, simple and rapid TB diagnostic test is yet available on the market, and the search for accurate, non-DNA biomarkers remains a priority. To help overcome this ‘biomarker pipeline problem’, FIND and partners are working on the development of a well-curated and user-friendly TB biomarker database. The web-based database will enable the dynamic tracking of evidence surrounding biomarker candidates in relation to target product profiles (TPPs) for needed TB diagnostics. It will be able to accommodate raw datasets and facilitate the verification of promising biomarker candidates and the identification of novel biomarker combinations. As such, the database will simplify data and knowledge sharing, empower collaboration, help in the coordination of efforts and allocation of resources, streamline the verification and validation of biomarker candidates, and ultimately lead to an accelerated translation into clinically useful tools

Innovative COVID-19 point-of-care diagnostics suitable for tuberculosis diagnosis: a scoping review protocol

IntroductionIn 2014, the WHO published high-priority target product profiles (TPPs) for new tuberculosis (TB) diagnostics to align end-user needs with test targets and specifications; nevertheless, no TB test meets these targets to date. The COVID-19-driven momentum in the diagnostics world offers an opportunity to address the long-standing lack of innovation in the field of TB diagnostics. This scoping review aims to summarise point-of-care (POC) molecular and antigen tests for COVID-19 diagnosis that, when applied to TB, potentially meet WHO TPPs. This summary of currently available innovative diagnostic tools will guide the development of novel TB diagnostics toward the WHO-set targets.Methods and analysisWe will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension Scoping Reviews recommendations. MEDLINE (via PubMed), bioRxiv, MedRxiv and other publicly available in vitro diagnostic test databases were searched on 23 November 2022. POC antigen or molecular tests developed for SARS-CoV-2 detection that meet the eligibility criteria will be included in the review. Developer description, test description, operation characteristics, pricing information, performance and commercialisation status of diagnostic tests identified will be extracted using a predefined standardised data extraction form. Two reviewers will independently perform the screening and data extraction. A narrative synthesis of the final data will be provided.Ethics and disseminationNo ethical approval is required because individual patient data will not be included. The findings will be published in open-access scientific journals

Blazing the trail for innovative tuberculosis diagnostics

The COVID-19 pandemic brought diagnostics into the spotlight in an unprecedented way not only for case management but also for population health, surveillance, and monitoring. The industry saw notable levels of investment and accelerated research which sparked a wave of innovation. Simple non-invasive sampling methods such as nasal swabs have become widely used in settings ranging from tertiary hospitals to the community. Self-testing has also been adopted as standard practice using not only conventional lateral flow tests but novel and affordable point-of-care molecular diagnostics. The use of new technologies, including artificial intelligence-based diagnostics, have rapidly expanded in the clinical setting. The capacity for next-generation sequencing and acceptance of digital health has significantly increased. However, 4 years after the pandemic started, the market for SARS-CoV-2 tests is saturated, and developers may benefit from leveraging their innovations for other diseases; tuberculosis (TB) is a worthwhile portfolio expansion for diagnostics developers given the extremely high disease burden, supportive environment from not-for-profit initiatives and governments, and the urgent need to overcome the long-standing dearth of innovation in the TB diagnostics field. In exchange, the current challenges in TB detection may be resolved by adopting enhanced swab-based molecular methods, instrument-based, higher sensitivity antigen detection technologies, and/or artificial intelligence-based digital health technologies developed for COVID-19. The aim of this article is to review how such innovative approaches for COVID-19 diagnosis can be applied to TB to have a comparable impact

TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously

Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription