Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Expansion of MALDI-TOF MS database as a strategy for identification of Haemophilus species other than H. influenzae.

OBJECTIVES: This study aimed to evaluate an expanded matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS) database for the identification of Haemophilus species other than H. influenzae (Hi). METHODS: A total of 144 Haemophilus species, cultured from respiratory samples from people (living) with cystic fibrosis, were identified with MALDI-TOF MS and 16S rRNA sequencing. Of these, 99 Haemophilus strains showed >99% similarity with the best matching strain in the National Center for Biotechnology Information (NCBI) database and were assigned to a single Haemophilus subspecies using both MALDI-TOF MS and 16S rRNA sequencing. The MS profiles of a subset of strains (n = 58/99) were added to the Bruker MALDI-TOF MS database. Subsequently, 270 different strains that were analyzed previously in a routine setting were re-analyzed. RESULTS: 16S rRNA sequencing reliably identified 99/144 Haemophilus strains (>99% similarity). H. haemolyticus 16S rRNA identification was suboptimal since only 3/21 H. haemolyticus strains attained a similarity of >99% with H. haemolyticus 16S rRNA sequence in the NCBI database. Expansion of the MALDI-TOF MS database improved the number of reliable identifications only moderately for H. haemolyticus, H. influenzae and H. paraphrohaemolyticus (<10%). By contrast, improved identification was more outspoken for H. parahaemolyticus, H. parainfluenzae, H. sputorum and H. pittmaniae (>85%). CONCLUSION: 16S rRNA sequencing is a valuable method for the identification of Haemophilus sp. other than Hi. Expansion of the MALDI-TOF MS database, based on 16S rRNA sequencing results, increased the proportion of reliable identifications and in this study resulted in an increase of 10% of Haemophilus sp. other than Hi strain identifications.

Safety and efficacy of faecal microbiota transplantation in patients with mild to moderate Parkinson's disease (GUT-PARFECT): a double-blind, placebo-controlled, randomised, phase 2 trial.

Background: Dysregulation of the gut microbiome has been implicated in Parkinson's disease (PD). This study aimed to evaluate the clinical effects and safety of a single faecal microbiota transplantation (FMT) in patients with early-stage PD. Methods: The GUT-PARFECT trial, a single-centre randomised, double-blind, placebo-controlled trial was conducted at Ghent University Hospital between December 01, 2020 and December 12, 2022. Participants (aged 50-65 years, Hoehn and Yahr stage 2) were randomly assigned to receive nasojejunal FMT with either healthy donor stool or their own stool. Computer-generated randomisation was done in a 1:1 ratio through permutated-block scheduling. Treatment allocation was concealed for participants and investigators. The primary outcome measure at 12 months was the change in the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score obtained during off-medication evaluations. Intention-to-treat analysis was performed using a mixed model for repeated measures analysis. This completed trial is registered on ClinicalTrials.gov (NCT03808389). Findings: Between December 2020 and December 2021, FMT procedures were conducted on 46 patients with PD: 22 in the healthy donor group and 24 in the placebo group. Clinical evaluations were performed at baseline, 3, 6, and 12 months post-FMT. Full data analysis was possible for 21 participants in the healthy donor group and 22 in the placebo group. After 12 months, the MDS-UPDRS motor score significantly improved by a mean of 5.8 points (95% CI -11.4 to -0.2) in the healthy donor group and by 2.7 points (-8.3 to 2.9) in the placebo group (p = 0.0235). Adverse events were limited to temporary abdominal discomfort. Interpretation: Our findings suggested a single FMT induced mild, but long-lasting beneficial effects on motor symptoms in patients with early-stage PD. These findings highlight the potential of modulating the gut microbiome as a therapeutic approach and warrant a further exploration of FMT in larger cohorts of patients with PD in various disease stages. Funding: Flemish PD patient organizations (VPL and Parkili), Research Foundation Flanders (FWO), Biocodex Microbiota Foundation.

Antibiofilm activity of Prevotella species from the cystic fibrosis lung microbiota against Pseudomonas aeruginosa.

It is increasingly recognized that interspecies interactions may modulate the pathogenicity of Pseudomonas aeruginosa during chronic lung infections. Nevertheless, while the interaction between P. aeruginosa and pathogenic microorganisms co-infecting the lungs has been widely investigated, little is known about the influence of other members of the lung microbiota on the infection process. In this study, we focused on investigating the impact of Prevotella species isolated from the sputum of people with cystic fibrosis (pwCF) on biofilm formation and virulence factor production by P. aeruginosa. Screening of a representative collection of Prevotella species recovered from clinical samples showed that several members of this genus (8 out 10 isolates) were able to significantly reduce biofilm formation of P. aeruginosa PAO1, without impact on growth. Among the tested isolates, the strongest biofilm-inhibitory activity was observed for Prevotella intermedia and Prevotella nigrescens, which caused a reduction of up to 90% in the total biofilm biomass of several P. aeruginosa isolates from pwCF. In addition, a strain-specific effect of P. nigrescens on the ability of P. aeruginosa to produce proteases and pyocyanin was observed, with significant alterations in the levels of these virulence factors detected in LasR mutant strains. Overall, these results suggest that non-pathogenic bacteria from the lung microbiota may regulate pathogenicity traits of P. aeruginosa, and possibly affect the outcome of chronic lung infections.

Multiple gene knock-down by a single lentiviral vector expressing an array of short hairpin RNAs

RNA interference (RNAi), mediated by short double-stranded RNAs, is a powerful mechanism for posttranscriptional gene silencing. Sustained expression of short hairpin RNA (shRNA) can be accomplished in mammalian cells by viral delivery systems. Using lentiviral constructs, stable gene silencing is established both in dividing and non-dividing cells. Targeting one single gene can lead to the development of escape mutants or may be insufficient to silence redundant pathways. Therefore, simultaneous targeting of multiple genes may be necessary. We have generated a lentiviral vector-based system for expression of multiple shRNAs from a single viral vector, which also encodes an EGFP reporter protein. We show that knock-down of each single gene from multiple target vectors is achieved at an efficiency comparable to that obtained after transduction using single target viral vectors. In this way, we were able to knock-down several members of the human Rho-family GTPases in T cells. Double and triple knock-down persisted after multiple passages of the cells. The ability to inhibit two or more genes simultaneously from one single expression vector further widens the application spectrum of RNAi, both in functional studies and therapeutic strategies.