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).

Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium.

This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive.

Estimation of antimicrobial resistance of Mycoplasma genitalium, Belgium, 2022.

BackgroundAntimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking.AimWe aimed to estimate the occurrence of resistant MG in Belgium.MethodsBetween July and November 2022, frozen remnants of MG-positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance-associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, socio-demographic and behavioural variables via Fisher's exact test and logistic regression analysis.ResultsOf the 244 MG-positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics.ConclusionAlthough limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies.

Incidence and clinical and microbiological features of invasive and probable invasive streptococcal group A infections in children and adults in the Brussels-Capital Region, 2005-2020.

Assess the incidence, risk factors, clinical and microbiological features, and outcome of both probable invasive and invasive group A Streptococcus (GAS) infections in children and adults in the BrusselsCapital Region between 2005 and 2020. A retrospective, multicentric study was performed in three university hospitals in Brussels. Patients were identified through the centralized laboratory information system. Epidemiological and clinical data were collected from patients' hospital records. A total of 467 cases were identified. Incidence has increased from 2.1 to 10.9/100,000 inhabitants between 2009 and 2019 in non-homeless adults while it was above 100/100,000 on homeless in years with available denominators. Most of GAS were isolated from blood (43.6%), and the most common clinical presentation was skin and soft tissue infections (42.8%). A third of all the patients needed surgery, a quarter was admitted to the intensive care unit, and 10% of the adult patients died. Wounds and chickenpox disease were the main risk factors for children. Tobacco, alcohol abuse, wounds or chronic skin lesion, being homeless, and diabetes were identified as major predisposing factors for adults. The most common emm clusters were D4, E4, and AC3; 64% of the isolates were theoretically covered by the 30-valent M-protein vaccine. The burden of invasive and probable invasive GAS infections is on the rise in the studied adult population. We identified potential interventions that could contribute to decrease this burden: appropriate care of wounds, specifically among homeless and patients with risk factors such as diabetes and systematic chickenpox vaccination for children.

Multicenter interlaboratory study of routine systems for the susceptibility testing of temocillin using a challenge panel of multidrug-resistant strains.

Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods' reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5-100%) for all MIC-based methods. The highest category agreement was achieved by ETEST® (97.5%) followed by VITEK® 2 (93.2%), disk diffusion (91.6%), and BD Phoenix™ (88.5%). BD Phoenix™ and paper disk diffusion overcalled resistance (11.5% and 6.8% of major discrepancies, respectively), while ROSCO tablets diffusion and VITEK® 2 generated higher very major discrepancies (7.1% and 4.2% respectively). Inter-assay reproducibility was unsatisfactory using recommended E. coli ATCC 25922 strain but was excellent with E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains. This interlaboratory study suggests that routine testing methods provide accurate and reproducible TMO categorization results except for Proteae species.

Epidemiology and molecular typing of multidrug-resistant bacteria in day care centres in Flanders, Belgium.

The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Day care centre (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organised between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for the detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n = 448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7% of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on the clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared with previous studies, and our findings re-emphasise the need for rigorous hygiene measures within such centres to control the further spread of MDROs in the community.

Increase in bloodstream infections caused by emm1 group A Streptococcus correlates with emergence of toxigenic M1UK, Belgium, May 2022 to August 2023.

Many European countries have recently reported upsurges in invasive group A Streptococcus (iGAS) infections, mainly caused by emm1 Streptococcus pyogenes, specifically the toxigenic M1UK lineage. We present the epidemiology of emm1 causing iGAS in Belgium during 2018-August 2023, and describe an emergence of the toxigenic M1UK lineage in Belgium in mid-2022 that was observed as an increase in bloodstream infections caused by emm1 S. pyogenes that continued into 2023.

Detection of SARS-CoV-2 in young children attending day-care centres in Belgium, May 2020 to February 2022.

Presence of SARS-CoV-2 was monitored in nasopharyngeal samples from young children aged 6-30 months attending day-care centres (DCCs) in Belgium from May 2020-February 2022. SARS-CoV-2 carriage among DCC children was only detected from November 2021, after emergence of Delta and Omicron variants, in 9 of the 42 DCCs screened. In only one DCC, two children tested positive for SARS-CoV-2 at the same sampling time point, suggesting limited transmission of SARS-CoV-2 in Belgian DCCs among young children during the studied period.

Oseltamivir plus usual care versus usual care for influenza-like illness in primary care: an open-label, pragmatic, randomised controlled trial.

BACKGROUND: Antivirals are infrequently prescribed in European primary care for influenza-like illness, mostly because of perceived ineffectiveness in real world primary care and because individuals who will especially benefit have not been identified in independent trials. We aimed to determine whether adding antiviral treatment to usual primary care for patients with influenza-like illness reduces time to recovery overall and in key subgroups. METHODS: We did an open-label, pragmatic, adaptive, randomised controlled trial of adding oseltamivir to usual care in patients aged 1 year and older presenting with influenza-like illness in primary care. The primary endpoint was time to recovery, defined as return to usual activities, with fever, headache, and muscle ache minor or absent. The trial was designed and powered to assess oseltamivir benefit overall and in 36 prespecified subgroups defined by age, comorbidity, previous symptom duration, and symptom severity, using a Bayesian piece-wise exponential primary analysis model. The trial is registered with the ISRCTN Registry, number ISRCTN 27908921. FINDINGS: Between Jan 15, 2016, and April 12, 2018, we recruited 3266 participants in 15 European countries during three seasonal influenza seasons, allocated 1629 to usual care plus oseltamivir and 1637 to usual care, and ascertained the primary outcome in 1533 (94%) and 1526 (93%). 1590 (52%) of 3059 participants had PCR-confirmed influenza infection. Time to recovery was shorter in participants randomly assigned to oseltamivir (hazard ratio 1·29, 95% Bayesian credible interval [BCrI] 1·20-1·39) overall and in 30 of the 36 prespecified subgroups, with estimated hazard ratios ranging from 1·13 to 1·72. The estimated absolute mean benefit from oseltamivir was 1·02 days (95% [BCrI] 0·74-1·31) overall, and in the prespecified subgroups, ranged from 0·70 (95% BCrI 0·30-1·20) in patients younger than 12 years, with less severe symptoms, no comorbidities, and shorter previous illness duration to 3·20 (95% BCrI 1·00-5·50) in patients aged 65 years or older who had more severe illness, comorbidities, and longer previous illness duration. Regarding harms, an increased burden of vomiting or nausea was observed in the oseltamivir group. INTERPRETATION: Primary care patients with influenza-like illness treated with oseltamivir recovered one day sooner on average than those managed by usual care alone. Older, sicker patients with comorbidities and longer previous symptom duration recovered 2-3 days sooner. FUNDING: European Commission's Seventh Framework Programme.

A split strategy to prevent cytomegalovirus after kidney transplantation using prophylaxis in serological high-risk patients and a pre-emptive strategy in intermediate-risk patients: Combining the best of two options?

BACKGROUND: Cytomegalovirus (CMV) remains an important challenge after kidney transplantation. Current Transplantation Society International Consensus Guidelines recommend antiviral prophylaxis or pre-emptive therapy for high-risk CMV-seronegative recipients with a CMV-seropositive donor (D+/R-) and moderate-risk CMV-seropositive recipients (R+). However, a split strategy according to CMV serostatus is not specifically mentioned. METHODS: We evaluated a split strategy to prevent CMV infection after kidney transplantation in which D+/R- patients received valganciclovir (VGC) prophylaxis for 200 days, and R + patients were treated pre-emptively according to CMV DNAemia. Patients were followed until 1-year post-transplant. RESULTS: Between April 2014 and March 2018, 40 D+/R- and 92 R + patients underwent kidney transplantation. Forty-six percent received antithymocyte globulin (ATG) induction, and 98% was treated with calcineurin inhibitors, mycophenolic acid (MPA), and steroids. No D+/R- patient developed CMV disease during prophylaxis (median 200 days), but 15% developed post-prophylaxis or late-onset disease. Fifty-three percent developed neutropenia during prophylaxis, including 16/40 (40%) grade 3 or 4 neutropenia requiring reduction/discontinuation of MPA (30%) and/or VGC (35%), and an occasional need for granulocyte colony-stimulating factor (5%). In the R + group, 40% received antiviral therapy for a median duration of 21 days; 5% developed early-onset CMV disease. Only 5% developed neutropenia. D+/R + status (hazard ratio (HR) 2.09,P = .004) and ATG use (HR 2.81, P < .0001) were risk factors for CMV reactivation. CONCLUSIONS: Prophylaxis leads to acceptable CMV control in high-risk patients but comes with a high risk of neutropenia. Pre-emptive therapy is effective and limits drug exposure in those at lower risk of CMV.

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridiumscidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

Comparison of a VLP-based and GST-L1-based multiplex immunoassay to detect vaccine-induced HPV-specific antibodies in first-void urine.

Vaccine-induced human papillomavirus (HPV) antibodies originating from cervicovaginal secretions were recently shown to be detectable in first-void (FV) urine. This presents a novel opportunity for noninvasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. With a view towards method optimization, this study compared the measurement of HPV antibodies in FV urine using a multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) with previously reported results using a glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA). We tested 53 paired FV urine and serum samples from 19- to 26-year-old healthy women, unvaccinated (n = 17) or vaccinated with either the bivalent or quadrivalent HPV-vaccine during adolescence (n = 36). HPV6/11/16/18 antibodies were measured using M4ELISA and compared with GST-L1-MIA results. Inter-assay and inter-specimen correlations were examined using the Spearman's rank test (rs). As expected, lower HPV antibody concentrations were found in FV urine than in serum. Vaccinated women had significantly higher HPV6/11/16/18 antibody levels in both FV urine and serum compared with those unvaccinated (M4ELISA; FV urine P = .0003; serum P ≤ .0001). HPV antibody levels in FV urine and serum showed a significant positive correlation (M4ELISA anti-HPV6/11/16/18, rs = 0.85/0.86/0.91/0.79, P ≤ .001). Despite assay differences, there was moderate to good correlation between M4ELISA and GST-L1-MIA (FV urine anti-HPV6/11/16/18, rs = 0.86/0.83/0.89/0.53, P ≤ .0001; serum anti-HPV6/11/16/18, rs = 0.93/0.89/0.94/0.75, P ≤ .0001). FV urine HPV antibody detection is comparable with both assays, further supporting this noninvasive sampling method as a possible option for HPV vaccine assessment. Approaches to improve the sensitivity and larger studies are warranted to determine the feasibility of FV urine for vaccine-induced HPV antibody detection.

Performance and ease of use of a molecular point-of-care test for influenza A/B and RSV in patients presenting to primary care.

Annual influenza epidemics cause substantial morbidity and mortality, and the majority of patients with influenza-like illness present to primary care. Point-of-care influenza tests could support treatment decisions. It is critical to establish analytic performance of these platforms in real-life patient samples before uptake can be considered. We aimed to assess the analytical performance and ease of use of the cobas® Liat® PCR POCT in detecting influenza A/B and RSV in samples collected from patients with influenza-like illness in primary care. Sensitivity and specificity of the cobas® Liat® POCT are calculated in comparison with a commercial laboratory-based PCR test (Fast-Track Respiratory Pathogens 21 Plus kit (Fast-Track Diagnostics)). Samples with discordant results were analysed additionally by the RespiFinder 2Smart (PathoFinder) using an Extended Gold Standard (EGS). Acceptability was scored on a five-point Likert scale as well as a failure mode analysis of the cobas® Liat® POCT was performed. Nasal and oropharyngeal swabs were obtained from 140 children and nasopharyngeal swabs from 604 adults (744 patients). The cobas® Liat® POCT had a sensitivity and specificity of 100% (95% CI 99-100%) and 98.1% (95%CI 96.3-99%) for influenza A, 100% (95% CI 97.7-100%) and 99.7% (95%CI 98.7-99.9%) for influenza B and 100% (95% CI 87.1-100%) and 99.4% (95%CI 98.6-99.8%) for RSV, respectively. According to trained lab technicians, the cobas® Liat® POCT was considered easy-to-use, with a fast turn-around-time. Cobas® Liat® POCT is a promising decentralised test platform for influenza A/B and RSV in primary care as it provides fairly rapid results with excellent analytic performance. Point-of-care influenza tests could support treatment decisions in primary care. Cobas® Liat® POCT is a promising decentralised test platform for influenza A/B and RSV in primary care as it provides fairly rapid results with excellent analytic performance.

Performance of three automated SARS-CoV-2 antibody assays and relevance of orthogonal testing algorithms.

Objectives: Development and implementation of SARS-CoV-2 serologic assays gained momentum. Laboratories keep on investigating the performance of these assays. In this study, we compared three fully automated SARS-CoV-2 antibody assays. Methods: A total of 186 samples from 84 PCR-positive COVID-19 patients and 120 control samples taken before the SARS-CoV-2 pandemic were analyzed using commercial serologic assays from Roche, Siemens and DiaSorin. Time after the positive COVID-19 PCR result and onset of symptoms was retrieved from the medical record. An extended golden standard, using the result of all three assays was defined, judging if antibodies are present or absent in a sample. Diagnostic and screening sensitivity/specificity and positive/negative predictive value were calculated. Results: Diagnostic sensitivity (ability to detect a COVID-19 positive patient) ≥14 days after positive PCR testing was 96.7% (95% CI 88.5-99.6%) for DiaSorin, 93.3% (95% CI 83.8-98.2%) for Roche and 100% (95% CI 94.0-100%) for Siemens. Lower diagnostic sensitivities were observed <14 days after onset of symptoms for all three assay. Diagnostic specificity (ability to detect a COVID-19 negative patient) was 95.0% (95% CI 89.4-98.1%) for DiaSorin, 99.2% (95% CI 95.4-99.9%) for Roche and 100% (95% CI 97.0-100%) for Siemens. The sensitivity/specificity for detecting antibodies (ability of detecting absence (specificity) or presence (sensitivity) of COVID-19 antibodies) was 92.4% (95% CI 86.4-96.3%)/94.9% (95% CI 90.5-97.6%) for DiaSorin, 97.7% (95% CI 93.5-99.5%)/97.1% (95% CI 93.5-99.1%) for Roche and 98.5% (95% CI 94.6-99.8)/97.1 (95% CI 93.5-99.1%) for Siemens. Conclusions: This study revealed acceptable performance for all three assays. An orthogonal testing algorithm using the Siemens and Roche assay achieved the highest positive predictive values for antibody detection in low seroprevalence settings.

International external quality assessment for SARS-CoV-2 molecular detection and survey on clinical laboratory preparedness during the COVID-19 pandemic, April/May 2020.

Laboratory preparedness with quality-assured diagnostic assays is essential for controlling the current coronavirus disease (COVID-19) outbreak. We conducted an external quality assessment study with inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples to support clinical laboratories with a proficiency testing option for molecular assays. To analyse SARS-CoV-2 testing performance, we used an online questionnaire developed for the European Union project RECOVER to assess molecular testing capacities in clinical diagnostic laboratories.

Quality of molecular detection of vancomycin resistance in enterococci: results of 6 consecutive years of Quality Control for Molecular Diagnostics (QCMD) external quality assessment.

The quality of PCR to detect vancomycin-resistant enterococci (VRE) was evaluated by analysing their performance in six consecutive external quality assessment (EQA) schemes, organized annually since 2013 by Quality Control for Molecular Diagnostics. VRE EQA panels consisted of 12-14 heat-inactivated samples. Sensitivity was tested with vanA-positive Enterococcus faecium (E. faecium), vanB-positive E. faecium, E. faecalis or E. gallinarum or vanC-positive E. gallinarum in different concentrations. Vancomycin-susceptible enterococci, Staphylococcus aureus or sample matrix was used to study the specificity. Participants were asked to report the VRE resistance status of each sample. The detection rate of vanA-positive samples was already 95% in the 2013 EQA panel (range 94-97%) and remained stable over the years. The 2013 detection rate of vanB-positive samples was 82% but increased significantly by more than 10% in subsequent years (96% in 2014, 95% in 2015, 92% in 2016 and 93% in 2017/2018, p < 0.05). The vanC detection rate by the limited number of assays specifically targeting this gene was lower compared to vanA/B (range 55-89%). The number of false positives in the true-negative sample (8% in 2013 to 1.4% in 2018) as well as the van-gene-negative bacterial samples (4% in 2013 to 0% in 2018) declined over the years. In the six years of VRE proficiency testing to date, the detection of vanA-positive strains was excellent and an increased sensitivity in vanB detection as well as an increase in specificity was observed. Commercial and in-house assays performed equally well.

Analytical performance of a platform for point-of-care CRP testing in adults consulting for lower respiratory tract infection in primary care.

C-reactive protein (CRP) is a biomarker widely used for disease severity assessment and treatment of inflammatory conditions. Point-of-care testing (POCT) devices should ideally be rapid and provide similar results to standard tests done in laboratories. Two thousand nine hundred twenty-two serum samples were obtained from adult patients presenting to primary care with symptoms of lower respiratory infection in a European diagnostic study. The analytic performance of the CRP QuikRead POCT device (Orion Diagnostica) was evaluated by comparing results with a central laboratory method (Dimension Vista, Siemens), with both tests performed in a laboratory setting. For a CRP cut-off concentration of ≥ 30 mg/L, the QuikRead test had a sensitivity of 92.2%, and specificity of 99.4%. The mean difference between the QuikRead and the central lab test was 0.4 mg/L. The slope of the Passing-Bablok regression was 0.94 (95% CI 0.93-0.95) indicating an underestimation of CRP levels of 6% by QuikRead. CRP estimates obtained from the QuikRead test correlate well with a central laboratory assay and the measurement displays low inter-assay variation. Therefore, the QuikRead test is a good candidate for CRP testing in primary care.

Epidemiology and genetic diversity of linezolid-resistant Enterococcus clinical isolates in Belgium from 2013 to 2021.

OBJECTIVES: Linezolid-resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are emerging health threats as limited therapeutic options remain. The aim of this study was to investigate the epidemiology, resistance mechanisms, and genetic diversity of linezolid-resistant enterococci (LRE) isolated between 2013 and 2021 and received at the Belgian National Reference Centre (NRC) for Enterococci. METHODS: Linezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. RESULTS: Seventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid-resistant E. faecalis strains, 97% harboured the optrA gene (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid-resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA, and 23% the optrA gene. Furthermore, two E. faecium strains were identified with a combination of two resistance mechanisms ([i] optrA and poxtA, and [ii] cfr(B) and G2576T point mutation, respectively). Vancomycin resistance was observed in 15% (n = 12) of the LRE. ST480 (n = 42/63 typed strains, 67%) was the most frequently detected sequence type (ST) in linezolid-resistant E. faecalis strains, while ST203 (n = 5/15 typed strains, 33%) was the most frequently detected ST in linezolid-resistant E. faecium strains. CONCLUSIONS: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium, with ST480 as the most prominent multilocus sequence typing. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

Concentration strategies for spiked and naturally present biomarkers in non-invasively collected first-void urine.

BACKGROUND: First-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation. METHODS: FVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured. RESULTS: The findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed. CONCLUSIONS: With this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.