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).

Objectives/BackgroundWe aimed to investigate routine urinalysis practices in Belgian laboratories and verify these findings against the 2023 European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) European Urinalysis Guideline.MethodsA questionnaire was developed to collect information on pre- to postanalytical aspects of urine test strip and particle analysis. The questionnaire was distributed by Sciensano to all Belgian laboratories, licensed to perform urine particle analysis.ResultsSixty-six percent of the Belgian laboratories (75/113) participated. The responding laboratories served physicians in private (25%), hospital (60%) and university hospital (15%) setting. All laboratories performed test strip and particle analysis, predominantly automatically (97% and 96%, respectively). In addition, most laboratories (87%) used intelligent verification criteria to optimize diagnostic accuracy. Almost all laboratories (>= 90%) screened and reported a minimal biochemistry panel (glucose, protein, pH, ketones) and particle count (red and white blood cells). Independent of the technology, a notable variability was observed regarding medical cut-off values and advanced particle differentiation and reporting. Internal quality control was extensively performed for urine test strip (91%) and particle analysis (96%), while external QC was less common (32% and 36%, respectively). Consequently, only few laboratories were ISO15189 accredited for urine test strip (15%) and particle analysis (17%).ConclusionThere is considerable variability in current urinalysis performed in Belgian laboratories. The 2023 EFLM urinalysis guideline has the potential to guide clinical laboratories towards improving their urinalysis practices. Additional efforts are required to implement these recommendations into clinical practice in Belgium.

We report on sample IS/17575 since it generated highly divergent results in the Belgian SARS-CoV-2 serology external quality assessment scheme. Sample IS/17575 was serum originating from a 30 years old male patient. 124 diagnostic laboratories analysed this sample. A total of 168 results was returned (including 5 doubles). Overall, 38 were positive. All tests against S1 were positive except the Euroimmun IgG ELISA and the Ortho clinical Diagnostics VITROS IgG CLIA. All tests against S1/S2 (Liaison, Diasorin) resulted in a signal above cutoff. Assays against RBD, mostly generate a negative result. An exception are the Wantai SARS-CoV-2 ELISA's. All tests targeting N protein were negative. The survey shows, when >6 months post-infection, assays targeting at least S1, and preferably S1 combined with S2, are the most sensitive. This finding accentuates the necessity of external quality assessment schedules and importance of antigenic composition of serologic SARS-CoV-2 assays.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country’s testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Abstract Objectives Fast and reliable ethanol assays analysis are used in a clinical context for patients suspected of ethanol intoxication. Mostly, automated systems using an enzymatic reaction based on ethanol dehydrogenase are used. The manuscript focusses on the evaluation of the performance of these assays. Methods Data included 30 serum samples used in the Belgian EQA scheme from 2019 to 2021 and concentrations ranged from 0.13 to 3.70 g/L. A regression line between target concentrations and reported values was calculated to evaluate outliers, bias, variability and measurement uncertainty. Results A total of 1,611 results were taken into account. Bias was the highest for Alinity c over the whole concentration range and the lowest for Vitros for low concentrations and Cobas 8000 using the c702 module for high concentrations. The Architect and Cobas c501/c502 systems showed the lowest variability over the whole concentration range. Highest variability was observed for Cobas 8000 using the 702 module, Thermo Scientific and Alinity c. Cobas 8000 using the c702 module showed the highest measurement uncertainty for lower concentrations. For higher concentrations, Alinity c, Thermo Scientific and Vitros were the methods with the highest measurement uncertainty. Conclusions The bias of the enzymatic techniques is nearly negligible for all methods except Alinity c. Variability differs strongly between measurement procedures. This study shows that the Alinity c has a worse measurement uncertainty than other systems for concentrations above 0.5 g/L. Overall, we found the differences in measurement uncertainty to be mainly influenced by the differences in variability.

Background Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. Objective The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. Methods A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. Results The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. Conclusions These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics.

Background: With the spread of coronavirus disease 2019 (COVID-19), an existing national laboratory based surveillance system was adapted to daily monitor the epidemiological situation of SARS-CoV-2 in the Belgium by following the number of confirmed COVID-19 infections, the number of performed tests and the positivity ratio. We present these main indicators of the surveillance over a one-year period as well as the impact of the performance of the laboratories, regarding speed of processing the samples and reporting results, for surveillance. Methods: We describe the evolution of test capacity, testing strategy and the data collection methods during the first year of the epidemic in Belgium. Results: Between the 1th of March 2020 and the 28th of February 2021, 9,487,470 tests and 773,078 COVID-19 laboratory confirmed cases were reported. Two epidemic waves occurred, with a peak in April and October 2020. The capacity and performance of the laboratories improved continuously during 2020 resulting in a high level performance. Since the end of November 2020 90 to 95% of test results are reported at the latest the day after sampling was performed. Conclusions: Thanks to the effort of all laboratories a performant exhaustive national laboratory based surveillance system to monitor the epidemiological situation of SARS-CoV-2 was set up in Belgium in 2020. On top of expanding the number of laboratories performing diagnostics and significantly increasing the test capacity in Belgium, turnaround times between sampling and testing as well as reporting were optimized over the first year of this pandemic.

BACKGROUND:With the spread of coronavirus disease 2019 (COVID-19), an existing national laboratory-based surveillance system was adapted to daily monitor the epidemiological situation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the Belgium by following the number of confirmed SARS-CoV-2 infections, the number of performed tests and the positivity ratio. We present these main indicators of the surveillance over a one-year period as well as the impact of the performance of the laboratories, regarding speed of processing the samples and reporting results, for surveillance. METHODS:We describe the evolution of test capacity, testing strategy and the data collection methods during the first year of the epidemic in Belgium. RESULTS:Between the 1st of March 2020 and the 28th of February 2021, 9,487,470 tests and 773,078 COVID-19 laboratory confirmed cases were reported. Two epidemic waves occurred, with a peak in April and October 2020. The capacity and performance of the laboratories improved continuously during 2020 resulting in a high level performance. Since the end of November 2020 90 to 95% of the test results are reported at the latest the day after sampling was performed. CONCLUSIONS:Thanks to the effort of all laboratories a performant exhaustive national laboratory-based surveillance system to monitor the epidemiological situation of SARS-CoV-2 was set up in Belgium in 2020. On top of expanding the number of laboratories performing diagnostics and significantly increasing the test capacity in Belgium, turnaround times between sampling and testing as well as reporting were optimized over the first year of this pandemic.

For 1 year now, the world is undergoing a coronavirus disease-2019 (COVID-19) pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method for COVID-19 diagnosis is the detection of viral RNA by RT-qPCR with a specific set of primers and probe. It is important to frequently evaluate the performance of these tests and this can be done first by an in silico approach. Previously, we reported some mismatches between the oligonucleotides of publicly available RT-qPCR assays and SARS-CoV-2 genomes collected from GISAID and NCBI, potentially impacting proper detection of the virus. In the present study, 11 primers and probe sets investigated during the first study were evaluated again with 84,305 new SARS-CoV-2 unique genomes collected between June 2020 and January 2021. The lower inclusivity of the China CDC assay targeting the gene N has continued to decrease with new mismatches detected, whereas the other evaluated assays kept their inclusivity above 99%. Additionally, some mutations specific to new SARS-CoV-2 variants of concern were found to be located in oligonucleotide annealing sites. This might impact the strategy to be considered for future SARS-CoV-2 testing. Given the potential threat of the new variants, it is crucial to assess if they can still be correctly targeted by the primers and probes of the RT-qPCR assays. Our study highlights that considering the evolution of the virus and the emergence of new variants, an in silico (re-)evaluation should be performed on a regular basis. Ideally, this should be done for all the RT-qPCR assays employed for SARS-CoV-2 detection, including also commercial tests, although the primer and probe sequences used in these kits are rarely disclosed, which impedes independent performance evaluation.

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus’ genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.

Tacrolimus (Tac), a macrolide lactone produced by the fungus Streptomyces tsukubaensis, is nowadays the primary immunosuppressive drug used in organ transplantation. As cyclosporine, Tac is a calcineurin inhibitor preventing cellular rejection through the selective inhibition of interleukin-2 production by T-cells. Immunosuppressive agents are critical dose drugs; this means they have a narrow therapeutic index, exhibiting the desired therapeutic effect with acceptable tolerability only within a narrow range of blood concentrations. Furthermore, they exhibit a high degree of both inter- and intra-individual pharmacokinetic and -dynamic variability, increasing the risk of therapeutic failure if these agents are used at uniform doses in all patients. Hence, therapeutic drug monitoring (TDM) and dosage individualisation are highly recommended to reduce the occurrence of adverse events and to optimise patients outcomes. Practically, trough blood concentrations have been widely used as guidance for Tac dose individualisation. If monitoring Tac trough blood concentrations has contributed to improve efficacy and reduce the toxicity of Tac, the relationship between these concentrations and graft rejection is still unclear. Indeed, incidence of graft rejection remains hardly predictable based on single trough concentrations, underlying the need to find and validate additional markers of efficacy. The first aims of this work were to identify new biomarkers more closely linked to the occurrence and the severity of rejection, than blood concentrations. We therefore focused our research on tacrolimus concentrations in transplanted tissue and in peripheral blood mononuclear cells (PBMCs), a blood compartment enriched in lymphocytes. The first step was to develop and validate analytical methods for the detection and the quantification of tacrolimus in these unconventional matrices. These methods had to be sensitive enough to allow low tacrolimus concentrations detection and quantification. Subsequently, we have investigated the influence of genetic polymorphisms of biotransformation enzymes (CYP3A5 and CYP3A7), or their regulatory proteins as well as several transport proteins (P-gp, MRP2, OATP-C) on the tacrolimus pharmacokinetics and, more specifically on tissue and PBMCs concentrations. The final goals of this thesis are to propose new approaches to improve tacrolimus therapeutic drug monitoring. The first step of this work was to validate analytical methods to measure tacrolimus in the hepatic tissue and in PBMCs. These methods have been developed on LC-MS/MS after liquid-liquid extraction. After the optimisation of these methods, they were validated according to the FDA guidelines. In agreement with the ethic committee of our university, and after obtaining informed consent from patients, three major clinical trials were initiated: two in liver transplantation (LT-1, LT-2) and one in kidney transplantation (KT-1). The first study, LT1, in liver transplantation involved 146 adult recipients under tacrolimus monotherapy. Each of these patients underwent a protocol biopsy of their graft 7 days post transplantation for both histological (Banff scoring) and analytical (tacrolimus dosage) purposes. Tacrolimus trough blood levels were daily monitored. Based on the Banff score, blood and intra-hepatic tacrolimus concentrations have been compared as marker of efficacy. Donors have been genotyped for 14 genetic polymorphisms for genes coding for CYP3A5, CYP3A7, P-gp, MRP2, OATP-C and PXR. Then, the influence of these polymorphisms on blood and tissue tacrolimus concentrations was investigated. This study demonstrated that biopsy proven rejections (Banff scores > 6) were characterised by a significant lower intra-graft tacrolimus concentrations when compared to those observed in absence of rejection (Banff scores A and 2677G>T/A single nucleotide polymorphisms (SNPs) appeared to reduce the activity of P-gp on Tac. In the second study, LT-2, performed on 30 liver recipients, we investigated the relationship between PBMCc trough concentrations at day 1, 3, 5, and 7 post liver transplantation and the incidence and the severity of rejection episodes. As in LT-1, patients underwent a protocol biopsy 7 days post-transplantation, for both analytical and histological purposes, and tacrolimus trough blood levels were daily monitored. All of these parameters (intra-hepatic, PBMCs, and trough blood tacrolimus concentrations) were compared to the Banff score as marker of efficacy. Tissue and PBMCs levels determined by liquid-chromatography tandem mass-spectrometry, at days 3, 5 and 7 displayed good correlation with day 7 liver Banff rejection score, whereas mean blood levels and day-1 PBMCs levels did not. Clinically significant rejection (Banff > 6) was characterised by significant lower mean TAC PBMCs concentrations at day 3, 5 and 7 post-transplantation and by lower tissue concentrations. TAC tissue levels are significantly correlated with TAC PBMCs levels from day 5 post transplantation. This study suggests that TAC PBMCs levels could be a marker of immunosuppression efficacy in the early phase after LT. The last clinical trial involved 96 adult kidney recipients. All patients were under tri-therapy (tacrolimus, mycophenolate, and steroids). In this study we investigated the influence of recipient genetic polymorphisms of genes coding for CYP3A5 and P-gp on tacrolimus PBMCs and trough blood concentrations in the early phase after transplantation and at steady-state (one month after transplantation). Tacrolimus dose requirement (based on blood therapeutic drug monitoring) was higher among patients expressing CYP3A5 compared with patients who did not. Furthermore, expression of CYP3A5 significantly influenced Tac dose-adjusted PBMCs concentrations. In contrast, ABCB1 polymorphisms significantly influenced Tac PBMCs concentrations, whereas their impact on blood concentrations seemed negligible. Among these ABCB1 polymorphisms, the 1199G>A, 3435C>T and 2677G>T/A SNPs appeared to reduce the activity of P-gp, increasing Tac PBMCs concentrations. Increased Tac intracellular concentrations should contribute to the enhancement of the immunosuppressive status and to the prevention from rejection. The recipient’s genotyping for ABCB1 polymorphisms would be therefore useful to better individualise Tac therapy in renal transplantation. In conclusion, this work identified and emphasised the interest of two new matrices for tacrolimus determination: intra-graft and PBMCs tacrolimus levels. These compartments appear to be more significantly related to the occurrence and the severity of rejection episodes than trough blood concentrations. Nevertheless, it should be stressed that the association found between liver-tissue Tac concentration and the incidence or severity of liver rejections may not anymore be relevant after kidney transplantation. Indeed, since tacrolimus is characterised by a well established nephrotoxicity, its renal-tissue concentration might be more closely associated to side effects than to immunological efficacy. Regarding this consideration, and the fact that PBMCs tacrolimus concentrations could be considered as the active concentrations (at the pharmacological site of action), we believe that PBMCs tacrolimus could be a more relevant matrix for tacrolimus efficacy management. We confirmed that CYP3A5 intron 3 polymorphism is significantly associated with Tac dose requirement in the early phase post-transplantation. Furthermore, we also reported a higher influence of ABCB1 polymorphisms on Tac intra-hepatic and PBMCs concentrations, than on Tac blood concentrations. Both ABCB1 1199A and ABCB1 3435T allele led to an increase in Tac PBMCs concentrations, most likely due to a lower P-gp activity. Hence, it appears likely that these polymorphisms are directly involved in the Tac immunosuppressive activity. Taking these results into account and given the importance to rapidly achieve a well balanced immunosuppressive status, we could postulate that a pre-transplantation recipient’s CYP3A5 and ABCB1 pharmacogenetic testing, could contribute to a better individualisation of immunosuppressive therapy. The measurement of Tac within the PBMCs is long and tedious, and its short-term clinical implementation appears unlikely. However the findings reported here may shed new light in the optimisation of Tac therapy, leading either to the analytical improvement of such determinations (through industrial partnerships for instance), or to the development of a multivariate modelling able to predict PBMCs concentrations based on different co-variables (e.g. blood concentrations, genetic polymorphisms, type of graft, age, etc...).