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ISMD2024 Thirteenth International Symposium on Molecular Diagnostics

Austrian Society for Laboratory Medicine and Clinical Chemistry, Medical University of Graz, Graz International Airport, June 13 – June 14, 2024
Published/Copyright: April 16, 2024

Congress Presidents

H.H. Kessler, A.C. Haushofer

Scientific Committee

A.C. Haushofer, H.H. Kessler, B.I. Santner, R.E. Stauber, E. Stelzl

Plenary Session (A1, A2)

A1

Critical implications of the IVDR for continuation of essential tests and for innovation in diagnostics

Cobbaert C.

Chair of the EFLM Task Force on European Regulatory Affairs, Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands

Within the EU, laboratory or in vitro diagnostic (IVD) tests were regulated by the 1998 Directive 98/79/EC on in vitro diagnostic medical devices (IVDD), which mainly governed pre-market production within the manufacturing sector of CE-IVD marked tests. The majority were self-declared by manufacturers, with EU-wide application, and only approximately 10% required certification by notified bodies, appointed by the national competent authorities in Member States. IVDD did not regulate use of in-house devices (IH-IVD, referred to herein as IH-IVD as this is the term used in EU legislation), which are manufactured and used within the same health institution for medical purposes, in keeping with the EU principle of subsidiarity, and consequent national regulation.

With the roll out of EU Regulation 2017/746 on in vitro diagnostic medical devices (IVDR), from 26 May 2022 onwards, the EU market access of medical tests -and not the use- is governed by a vastly expanded and upgraded EU regulatory framework. Notably, in-house devices (IH-IVDs) are exempted from the IVDR, except for compliance with Annex I and Art 5. An overview of the major regulatory changes (among them risk based test classification and clinical evidence generation), the amended transition timelines, the role of notified bodies, EU reference laboratories, expert panels, and the Medical Device Coordination Group (MDCG) will be presented.

Laboratory medicine plays an increasingly important part in medical decision-making at diagnosis and follow-up and evolution towards “Personalized” or “Precision” medicine in all disciplines. It is, therefore, not surprising that laboratory medicine has a long standing history of self-regulation and laboratory accreditation. Therefore, accredited labs do have a sufficient fundament for manufacture and use of safe and effective IH-IVDs, and can easily embed the IVDR-requirements into their quality management system.

Anno 2024, the EU regulatory framework is still under construction and <5% of the class D tests are IVDR-compliant. While large IVD-companies can cope with the new regulation, this is far more difficult for small and medium IVD-enterprises (SMEs). Because the IVDR-compliance assessment by Notified Bodies is experienced as unpredictable, cumbersome and costly, it keeps SMEs away from sufficient return-on-investment. As a consequence, essential niche and orphan disease tests are in danger of being lost, with negative impact on patient management. On top, the diagnostic sector faces two additional key challenges: (1) the stipulation on equivalence of tests (article 5.5d), which imposes restrictions on the further manufacture and use of IH-IVDs once a commercial equivalent is available and (2) the gray area between CE-marked in vitro diagnostics (CE-IVDs), modified CE-IVDs, Research Use Only (RUO) tests, and IH-IVDs. The results of a questionnaire on current diagnostic practice conducted by European medical societies collaborating in the BioMed Alliance indicate widespread use of IH-IVDs in diagnostic laboratories across Europe and emphasize the need to preserve IH-IVDs for essential innovation and for rapid test development during pandemics. Diagnostic equivalents of the European Reference Networks (ERNs) for rare diseases could help ensure affordable and equal access to specialized diagnostics across the EU. Concerted action by clinical and laboratory disciplines, regulators, industry, and patient organizations is needed to prevent discontinuation of essential low volume tests and to support the efficient and effective implementation of the IVDR in a way that preserves innovation and safeguards the quality, safety, and accessibility of innovative diagnostics. On top, a critical appraisal of the IVDR effectiveness is becoming urgent as there is no evidence so far that this enormous investment in clerical paper work, even obliged for conventional tests that have been used for decades, will improve patient management and outcome. In an era of exploding healthcare costs, time is there for considering IVDR justification and reform.

In the last decade, scientific advances have laid a solid foundation for the development of routine molecular ’liquid biopsies’. Cell-free DNA and RNA biomarkers can complement or even surrogate tissue biopsy and have been introduced in clinical laboratory practice, especially in prenatal screening and oncology.

Through cell-free DNA detection by real-time PCR techniques and next generation sequencing, a simple non-invasive test becomes a liquid biopsy for 1) aneuploidy testing in the context of first-trimester screening or 2) cancer, surveying a patient’s circulation with the goal of early detection, information on prognosis, personalized therapies, and monitoring for recurrence or resistance.

The novel Swiss reimbursement scheme for trisomy 21 and 13/18 NIPT testing in patients with calculated first-trimester risks of approximately 1:10 – 1:1000 implemented in 2015 has led to a 2/3 reduction in invasive procedures. For IVF/ICSI patients, “day 5 NIPT” examinations are now possible by whole genome analysis from trophectoderm biopsies prior to selective embryo transfer.

Targeted gene panels tested on tissue biopsies are “gold standard” in the context of somatic mutation-based targeted tumor therapy. However, already in single patients where surgery is not possible “liquid biopsies” are requested for resistance mutation detection in the context of second line therapies.

Molecular ’liquid biopsies’ have become a central piece in the clinical laboratory and surgical pathology. Further developments and test formats are needed for early detection of cancers in combination with protein biomarkers to achieve a less invasive and more precise personalized medicine.

A2

Ready for IVDR – a process-oriented guide for in-house molecular assays

Stelzhammer A., Haushofer A.C.

Central Laboratory, Hospital Wels-Grieskirchen, Wels, Austria

Background: The spectrum of in vitro diagnostic devices (IVDs) and their application in medical laboratories has changed. To improve patient safety, the EU replaced the existing IVD by the new IVDR. The new directive includes demanding requirements for manufacturers of IVDs including in-house molecular IVDs.

Objectives: To establish recommendations how in-house molecular IVDs can be used further in accordance with the new IVDR. To compile a guide supporting medical laboratories when using in-house molecular assays.

Materials and methods: Literature research of laws and standards relevant to the goal of the study was done, experts were interviewed. Results were generated with a qualitative content analysis.

Results: Recommendations for action and implementation could be established. The results are summarized in a process-oriented guide supporting medical laboratories and assisting in establishment of in-house molecular assays.

Conclusions: The guide navigates through the demanding requirements. It can be utilized as a backbone for the use of in-house molecular IVDs. If necessary, it can be adapted to special laboratory requirements.

Quality Assurance – Quality Control (A3-A5)

A3

External Quality Assessment for SARS-CoV-2 genome detection in Austria: Post-pandemic versus pandemic period results

Buchta C.1, Aberle S.W.2, Görzer I.2, Griesmacher A.1, Müller M.M.1, Neuwirth E.3, Puchhammer E.2, Weseslindtner L.2, Camp J.V.2

1 Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests (ÖQUASTA), Vienna, Austria

2 Center for Virology, Medical University of Vienna, Austria

3 Faculty of Computer Science, University of Vienna, Austria

Background: External quality assessment (EQA) schemes provide objective feedback to participating laboratories about the performance of their analytical systems and information about overall regional analytical performance. EQAs are particularly important during pandemics as they also assess the reliability of individual test results and show opportunities to improve test strategies.

Objectives: To analyze whether the significant decrease of the COVID-19 testing frequency with the end of the pandemic in Austria had an effect on participation and/or performance in SARS-CoV-2 virus detection EQAs, as compared to the pandemic era.

Materials and methods: Identical samples were sent to all participating laboratories, and the EQA provider evaluated the agreement of the reported results with defined targets. The performance of testing was reported as true positive/negative ratios, comparing the post-pandemic data to previous rounds. Furthermore, subgroups of participants were analyzed, stratified by laboratory type (medical or non-medical) and the test system format (fully automated or requiring manual steps).

Results: While the frequency of false negative results did not change during the three-years of the pandemic (5.7%, 95% C.I. 3.1∼8.4%), a lower true positive ratio was observed in the first post-pandemic EQA (0.9%, 1.8%, and 11% for the three positive samples included in the test panel, n = 109 test results). In this first post-pandemic EQA round, medical laboratories (average 0.7% false negative across 3 samples, n = 90)) and automated test systems (average 1.5% false negative, n = 87) had lower false negative ratios than non-medical laboratories (22.8%, n = 19) and manual test systems (16.7%, n = 22), respectively. These lower average ratios were due to a low concentration sample, where non-medical laboratories reported 36.8% and manual test systems 54.5% true positive results.

Conclusions: Overall ratios of true positive results were below the mean of all results during the pandemic, but were similar to the first round of the pandemic. A lower post-pandemic true positive ratio was associated with specific laboratory types and assay formats, particularly for samples with low concentration. EQAs will continue to monitor the laboratory performance to ensure the same quality of epidemiological data after the pandemic, even if vigilance has decreased.

A4

15+ years of performance evaluation of molecular detection of enteroviruses by QCMD

Donoso Mantke O.1, Benschop K.S.M.2, Staines H.1,3, Mckloud E.1, McCulloch E.1, Montgomery D.1, Sutton G.1, van Loon A.1

1 Quality Control for Molecular Diagnostics (QCMD), Glasgow, United Kingdom

2 National Institute for Public Health and the Environment, Bilthoven, the Netherlands

3 Sigma Statistical Services, Balmullo, United Kingdom

Background: Detection of human enteroviruses (EVs) is crucial to assess epidemiological circulation of different EV types while excluding poliovirus strains. High sensitivity and specificity is required and should be routinely evaluated to maintain an acceptable level of diagnostic quality.

Objectives: To evaluate the performance data from external quality assessment (EQA) challenges for molecular EV detection that were distributed between 2005 and 2022, and tested by diagnostic and public health laboratories using either in-house or commercial based molecular technologies.

Materials and methods: Performance data from a total of 32 EQA panels/challenges (schemes) were evaluated that were returned by 699 registered laboratories worldwide regardless of their participation frequency during the 18 years of observed study period. Of these 621 were diagnostic laboratories and 78 were public health laboratories. A total of 44,434 samples were included in the study, which accounted for 41,087 core samples and 3,347 educational samples analyzed by the participants using their routine molecular assays and workflows. Overall performance is defined as the combined percentage correctly identified true positive samples as positive, the true negative samples as negative, and the specificity samples containing parechovirus or rhinovirus as negative. A separate analysis was conducted on the performance in relation to true positive (sensitivity), true negative (false positivity) and specificity samples.

Results: Overall performance increased over the years for both diagnostic and public health laboratories using either in-house or commercial based technologies with rates >94.8% since 2013. In general, diagnostic laboratories performed significantly better than public health laboratories (odds ratio= 1.26 (95% CI: 1.14 – 1.40); p<0.0001). A consistent higher performance of in-house based technology over commercial based technology was observed for both laboratory types. However, univariate analysis rather showed a higher performance in relation to core samples for commercial based technology over in-house based technology (92.3% for commercial based technology and 91.1% for in-house based technology; odds ratio= 1.18 (95% CI: 1.10 – 1.27); p<0.0001) mainly in diagnostic laboratories. The sensitivity for the different EV types varied with the lowest sensitivity reported for samples containing enterovirus D68 B3 strain (86.8%), echovirus 11 (85.2%) and poliovirus type 3 (79.7%). Both, commercial and in-house based technologies showed similar sensitivity for the majority of the types, with the exception of echovirus 9, enterovirus D68 B3, and poliovirus type 3 positive samples where a lower sensitivity was observed when using a commercial based technology.

Conclusions: The data underlines the importance of EQA schemes over time. Poor sensitivity should encourage further optimization of the assay. Inclusion of different EV types of clinical and public health relevance remains a crucial part of the EQA.

A5

Liquid biopsy in prenatal screening

Noppen C.

Viollier AG, Allschwil, Switzerland

Background: Non-invasive prenatal testing (NIPT) testing is an important supplement to existing screening tests for fetal chromosomal abnormalities. For trisomy 21 NIPT is superior to other screening methods and used as first-tier screening in many countries. However, NIPT reports have their limitations, and complexities of cell-free DNA testing need to be addressed in counselling of pregnant women.

Objectives: To describe technical validation, performance characteristics, and quality control parameters of the NIPT assay. To delineate the work-up of selected positive and discrepant cases from the Swiss contingent screening.

Materials and methods: Plasma samples from pregnant women were tested using the VeriSeq NIPT Solution v2 assay in the first trimester. Clinical sensitivity and specificity were determined for basic (chromosomes 21, 18, 13, X, and Y) and genome-wide screening modes. Further options included fetal Rhesus D determination from the 12th gestational week onwards from the same sampling.

Results: 2256 samples were tested by whole genome sequencing. In 124 of 2239 valid analyses, chromosome aberrations were detected, including 76x trisomy 21, 10x trisomy 18, 3x trisomy 13, 6x rare trisomies, 12x copy number variations >7 megabases. In addition, 12 cases were positive for sex chromosome aberrations.

Conclusions: NIPT is an established option for antenatal screening for trisomy 21, 18, 13 and other selected chromosomal aberrations with varying positive predictive values and possible impact on placental function. If used appropriately in the context of contingent screening, it increases the detection rate for fetal chromosomal aberrations, while decreasing the number of invasive tests necessary for diagnostic confirmation. An understanding of the appropriate clinical use and systematic follow-up as additional quality control measures will enable gynecologists to provide optimal screening.

Sexually Transmitted Infections, Epstein Barr Virus (A6-A9)

A6

Vascular, cardiometabolic and immunovirological changes in people living with HIV: Longitudinal data analysis from the EndoAfrica Study, South Africa

Strijdom H.1, de Boever P.2, Kessler H.H.3, Stelzl E.3, Webster I.1, Everson F.1, Goswami N.4

1 Center for Cardiometabolic Research in Africa, Division of Medical Physiology, Stellenbosch University, South Africa

2 Center for Environmental Sciences, Hasselt University, Belgium

3 Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Austria

4 Division of Physiology and Pathophysiology, Medical University of Graz, Austria

Background: People living with HIV (PLWH) have a 2-fold greater risk of cardiovascular disease (CVD). In sub-Saharan Africa, it is estimated that up to 15% of the cardiovascular disease burden is attributable to HIV; however, longitudinal data on the cardiometabolic-vascular-immunovirological relationship in PLWH of the region remain limited.

Objectives: To measure at baseline and 18-month follow-up an array of clinical and biochemical markers of HIV and cardiometabolic disease in PLWH and control subjects without HIV from the Western Cape, South Africa. To assess whether associations exist between HIV and immunovirological status and cardiometabolic variables. To identify putative baseline predictors of vascular function (blood pressure, endothelial function, retinal microvascular status and sub-clinical atherosclerosis) at 18-months.

Methods: Longitudinal study (baseline:18-months); health, immunovirological and cardiometabolic data were collected from PLWH. Vascular measurements included systolic and diastolic blood pressure (SBP, DBP), flow-mediated dilatation (FMD), central retinal arteriolar and venular equivalent (CRAE and CRVE), and carotid intima-media thickness (c-IMT). Mixed model ANOVA and best subsets regression models were employed for statistical analyses.

Results: The study population (completed both baseline and follow-up assessments) consisted of 307 participants, with HIV Negative (HIVNeg; n=104); PLWH without ART at baseline (HIV+ART-; n=47), and PLWH with ART (HIV+ART+; n=156). Unsuppressed viral load (>1000 c/mL) was present in 78% of HIV+ART- participants at baseline, and 41% at follow-up (post-ART commencement); whilst remaining stable in HIV+ART+ (15%; p<0.01). Mixed-model ANOVA showed group differences for FMD (p=0.02), CRVE (p=0.04) and c-IMT (p=0.02). Although HIV+ART+ participants had significantly healthier FMD and CRVE measures compared to the other groups at baseline, these trend disappeared at follow-up. The endothelial and vascular function in PLWH with unsuppressed viral load was significantly impaired compared to those with low-level viremia and suppressed viral load.

Conclusion: Vascular outcomes were affected by HIV status during the 18-months assessment period. Baseline cardiometabolic and immunovirological variables predicting vascular outcomes at 18-months included systolic BP, renal function (eGFR), ART duration, viral load, hs-CRP, GGT, Hb, and LDL-cholesterol. This study highlights the importance of assessing temporal vascular changes in PLWH, and identifies early predictors of vascular outcomes.

A7

STI diagnostics at a glance – trends and solutions

Tiemann C. 1,2

1 Faculty of Biotechnology and Instrumentation Engineering, University of Applied Sciences Bielefeld, Germany

2 MVZ Labor Krone GbR, Bad Salzuflen, Germany

Sexually transmitted infections (STI) are playing an increasingly important role in routine infectious disease diagnostics. Both the broad spectrum of bacterial and viral pathogens and the methodological developments in molecular analysis require a differentiated approach in the laboratory depending on the objective of the analysis. The possibilities of multiplex PCR methods offer a mostly simple way of detecting various STIs in a sample. The actual relevance of the pathogens may have to be assessed individually against the background of clinical and anamnestic information. In addition to the molecular tools, the question of pre-analysis, in particular the different sample types and the way of sample collection, is also highly relevant. Modern solutions also allow the detection of infectious pathogens using self-sampling strategies. Various national and international studies (e.g. BRAHMS, STIPnet) have clearly demonstrated the benefits of such modified procedures. Both the high number of participants and the possibility of investigating different infection sites could be implemented efficiently. The detection of STIs in whole blood is also supplemented and expanded by dry blood spot analysis (DBS) methods.

Innovations in the field of molecular STI diagnostics are increasing in line with the growing importance of analytics. Not only multiplex PCR methods for detection, but also molecular typing methods (MPS) for the analysis of transmission pathways or the evolution and spread of resistance will expand routine diagnostics in the future. The enormous amount of data from diagnostics and research poses challenges that need to be solved with biostatistical and bioinformatic systems.

A8

The bad genital ulcer, the good clinician and the powerful laboratory

Tiplica G.S.

Dermatology 2, Colentina Clinical Hospital, ”Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Background: Genital ulcer diagnosis relies on clinical information and laboratory tests. Herpes simplex virus (HSV) infections are common, and infected individuals are often unaware of their contagious status. Consequently, nucleic acid amplification tests (NAATs) to identify HSV types 1 and 2 play a critical role in establishing an accurate and fast diagnosis, which is crucial for determining appropriate treatment.

Objectives: To review the variate epidemiology and clinical manifestations of sexually transmitted infections. How to select appropriate laboratory tests for patients with genital ulcers, how to manage patients with sexually transmitted HSV infections.

Materials and methods: A set of 5 clinical cases presenting with genital ulcers of various etiologies demonstrate the importance of using NAATs specifically designed for the rapid diagnosis of HSV types 1 and 2 and varicella zoster virus (VZV). Differential diagnoses include: syphilis, donovanosis, Behcet disease, Zoon balanitis, genital cancer, drug eruption, and trauma.

Results: Identifying infections with HSV types 1 and 2 and/or VZV in less than 2 hours is possible using modern multiplex assays. VZV infection is demonstrated in 2% of the clinically suspected HSV infections, making multiplex assays extremely valuable for providing a correct treatment within the optimal efficacy window of five days from the start of clinical manifestations. In this situation, the severity of the manifestations will be reduced and the duration of the actual episode will be decreased.

Conclusions: Modern multiplex NAATs allowing rapid diagnosis of HSV type 1, HSV type 2 and VZV infection effectively bridge the accurate diagnosis of genital ulcers and support efficient treatment.

A9

Epstein Barr Virus, the key for malignancies and multiple sclerosis?

Puchhammer E.

Center for Virology, Medical University of Vienna, Austria

Epstein Barr Virus (EBV) infects persistently more than 95% of all adults worldwide. Primary infection occurs mostly in childhood or young adults, and, in about 25% of persons, leads to the clinical presentation of infectious mononucleosis (IM). It was unclear, however, why IM develops only in a minority of the patients. EBV is also associated with malignant diseases, as lymphomas or lymphoproliferative diseases and it was increasingly associated with the development of multiple sclerosis.

Our recent data show that specific human genetic variations affecting the HLA-E associated T-cell response are significantly associated with the development of IM. Also, newest data provide evidence that the infection with specific EBV strain variants increases strongly the risk to develop EBV associated lymphomas and is, together with the genetic profile of the human host, a main factor for the development of multiple sclerosis.

Hepatitis D Virus, Noroviruses (A10-A12)

A10

Novel anti-HDV therapies require reliable and sensitive quantification of HDV RNA: A European multicenter study

Berger A.1 Stelzl E.2, Kress J.3, Olivero A.4, Lampertico P.5, Heim A.6, Aberle S.W.7, Wedemeyer H.8, Reinhardt A.9, Gey B.9, Früchtel C.9, Kessler H.H.2

1 Institute of Medical Virology, Goethe University Frankfurt, Germany

2 Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Austria

3 Section of Molecular Virology, Paul-Ehrlich-Institute, Langen, Germany

4 Gastroenterology and Hepatology Department, “San Giovanni Battista” Hospital and Department of Internal Medicine, University of Torino, Italy

5 Division of Gastroenterology and Hepatology, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

6 Hannover Medical School, Institute of Virology, Hannover, Germany

7 Center for Virology, Medical University of Vienna, Vienna, Austria

8 Department of Gastroenterology, Hepatology, Infectious Diseases and Endocrinology, Hannover Medical School, Hannover, Germany

9 Roboscreen GmbH, Leipzig, Germany

Background: Quantification of plasma HDV RNA is the essential tool for patient management under anti-HDV therapy and during follow up. Quantification of HDV RNA may vary depending on the efficiency of HDV RNA extraction.

Objectives: To investigate the comparability of quantification and sensitivity reported by different European laboratories using the new RoboGene HDV RNA Quantification Kit 3.0 (Roboscreen GmbH) with different manual or automated nucleic acid extraction protocols/platforms and amplification/detection devices.

Methods: Eight study centers using 8 different combinations of NA extraction platforms and amplification devices participated in this study. For harmonization of HDV RNA concentrations obtained by different protocols, correction factors (CF’s) were determined using the 1st WHO International Standard (IS) for HDV RNA. The limit of detection (LOD) was determined by analyzing dilution series of the 1st WHO IS for HDV RNA. For accuracy testing of each protocol, reference material was used.

Results: CF’s ranged from 14 to 10000 depending on the combination of NA extraction platform and amplification device used. Calculated CF’s were applied for subsequent quantification. Depending on the combination used, LOD’s ranged from 2 to approximately 1000 IU/ml. When accuracy was tested, external quality control samples containing low HDV RNA concentrations were not detected by that protocol with the highest LOD.

Conclusion: To ensure reliability in quantification of HDV RNA, any modification of the extraction and amplification/detection protocol validated by the manufacturer requires revalidation. With the 1st WHO IS for HDV RNA, CF’s and LOD’s could easily be calculated in this study leading to harmonization of quantitative results and verification of sensitivity. Both are essential for accurate monitoring of viral response to existing anti-HDV treatment as well as for comparability of study results investigating novel anti-HDV drugs during treatment and follow up after end of treatment.

A11

AltoStar® HDV RT-PCR Kit 1.5 – Verification, Validation and Evaluation

Rosenstock P., Retzlaff S., Breuer M.T.

altona Diagnostics GmbH. Hamburg, Germany

Background: Quantitative HDV RNA represents the gold standard for diagnosis of active HDV infection and is useful for monitoring response to treatment and to assess a sustained virologic response which is associated with cure. The variability of the genotype RNA sequences for choosing primer and probes, and the complex secondary structure of the genome particularly require attention.

Objectives: To develop a highly sensitive and standardized HDV RT-PCR assay for reliable quantification of all 8 HDV genotypes in serum and plasma.

Materials and methods: Verification data were collected for EDTA plasma and serum according the IVDR requirements for class D in vitro diagnostic devices. Validation data were generated at 2 independent sites (Italy and France) including sensitivity, linear range, and reliable detection of all 8 genotypes.

Results: The French study site confirmed high sensitivity and reliable detection of all HDV genotypes. The Italian study site showed a sensitivity of 100% when testing 150 HDV patient samples.

Conclusions: The results obtained show a standardized RT-PCR assay for quantification of HDV RNA, easy to use in the daily diagnostic routine. This assay is suitable for full automation.

A12

Intraindividual evolution of noroviral sequences

González Rivero C.1, Maier T.2, Qu, Shiqi1, Frishman D.1, Protzer U.2, Hoffmann D.2

1 Department of Bioinformatics, Technische Universität München

2 Institute of Virology, Technische Universität / Helmholtz Zentrum München

Background: Noroviruses cause most viral gastroenteritis cases worldwide. Protracted infections have been described in immunocompromised persons recently and will become more prevalent with increasing numbers of susceptible patients.

Objectives: To study noroviral evolution in 19 consecutive samples of 4 patients with protracted infections.

Materials and methods: The 2–7 sequential specimens covered 49–776 days. The capsid gene was amplified by PCR and sequenced on the Oxford Nanopore platform. Sequences were trimmed, filtered by quality, and mapped before variant calling.

Results: All samples except one yielded >3000 reads of which 94%–100% mapped to the reference Nov GGII genome. The mean length was between 1546 and 1677nt, encompassing the entire capsid gene. As expected, dN/dS analysis showed negative selection over the entire sequences. Nevertheless, previously described epitopes amino acid sequences varied substantially between and within patients. The patients accumulated 69 to 99 variants. Interestingly, patient J with the noroviral RNA present at least over 776 days accumulated only 69 mutations. This is probably due to the strong immune suppression with previous heart transplantation, following a kidney transplantation during the sampling period. Patient E and K are supposed to be more immune competent with kidney- and stem cell transplantation >4 years before sampling.

Conclusion: Intraindividual evolution in during chronic infections seems to depend on the patient´s immune function.

Detection of Viruses & Bacteria (A13-A15)

A13

Evaluation of molecular solutions for the efficient diagnosis of microbial pathogens

Brandenburger E.

R-Biopharm AG, Darmstadt, Germany

Background: Gastrointestinal infections present a significant global health burden due to the wide variety of pathogens causing similar symptoms. Prompt identification of these pathogens is crucial for timely and targeted patient management.

Objectives: To evaluate the efficiency of low to high multiplex solutions in syndromic testing that can be applied according to the individual needs of the laboratory.

Materials and methods: A significant number of prospectively collected stool samples underwent analysis using classical multiplex real-time PCR and multiplex Tandem PCR. Both systems were compared against established manual and automated RT-PCR solutions to estimate their performance in detecting common gastrointestinal bacteria, viruses, and parasites.

Results: The study identified a range of comparative approaches, including the RIDA®UNITY fully automated solution and the manual RIDA®GENE assays by R-Biopharm and the Fecal Pathogen M 16-well Assay analyzed on the semi-automated Ultraplex AllianceTM platform by AUSDiagnostics. All systems are well-suitable for the timely and specific detection of gastrointestinal pathogens in laboratories with diverse sample throughput.

Conclusions: Molecular diagnostics compared to traditional laboratory methods show promise as an efficient method for syndromic testing of gastrointestinal infective agents. The diversity of approaches examined in this study offers laboratories flexibility in selecting solutions that align with their operational needs, paving the way for more effective patient care strategies.

A14

Analytical and clinical validation of a lab-developed PCR assay for high-throughput detection of carbapenemase genes for primary patient screening

Lütgehetmann M.1, Weinschenk C.1, Grunwald M.1, Giersch K.1, Pfefferle S.1, Pflüger S.1, Christner M.1, Eigner U.2, Eckel C.2, Köffer J.2, Kolb M.2, Tang H.T.1, Aepfelbacher M.1, Rohde H.1, Nörz D.1

1 University Medical Center Hamburg-Eppendorf (UKE), Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany

2 Labor Limbach, Study Department for Infectious Diseases, Heidelberg, Germany

Background: Carbapenem resistant gram-negative bacilli represent a major driver of cost within healthcare systems and are linked to adverse outcomes in patients.

Objectives: The aim of this study was to develop, validate and implement a multiplex-PCR-assay for the detection of seven of the most common carbapenemase genes: blaKPC, blaOxa48, blaVIM, blaNDM, blaOxa23, blaOxa24/40 and blaOxa58; for the purpose of primary patient screening.

Materials and methods: An assay-set by Probst et al. (PMID:33872637) was optimized for the cobas5800/6800/8800 systems. Analytic sensitivity was assessed using quantitative standards based on digital-PCR from clinical isolates. Limit of Detection (LoD) was determined by Probit-analysis (2-fold dilution, 21 repeats, 8 dilution steps). Linearity and inter-/intra-run variability were assessed by 10-fold dilution series. For clinical validation, the multiplex-assay was run on a set of pre-determined clinical isolates at both study-sites (Hamburg and Heidelberg), as well as on primary screening-swabs from in-patients in Hamburg, compared to selective bacterial culture and empiric resistance testing as reference method.

Results: LoDs as determined by Probit analysis were [in cop/ml]: 55.1 (46.8 – 74) for blaKPC, 94.9 (78.2 – 135) for blaNDM, 49.7 (43.6 – 65.5) for blaVIM, 25.3 (21 – 35.4) for blaOxa23-group, 63.3 (49.6 – 93.5) for blaOxa24/40-group and 111 (78.2 – 186) for blaOxa58-group. Linearity, as determined by repeatability (pooled SD of delta-ct for relevant concentration range) was 0.205 for blaKPC, 0.173 for blaNDM, 0.394 for blaVIM, 0.406 for blaOxa48, 0.163 for blaOxa23, 0.26 for blaOxa24/40 and 0.308 for blaOxa58, with a linear range of at least 5 log steps for each target. Relative sensitivity in clinical isolates was 100%, 97.14%, 97.75% and 100% for the KPC-, Oxa-48, MBL- and the Acinetobacter-carbapenemases respectively, in a total of 347 clinical isolates. For primary patient screening, a total of 781 clinical swab samples were analyzed, yielding an overall sensitivity of 95% in primary samples and 100% (n=20) in enrichment broth. Overall specificity was 98.7%.

Conclusions: The assay presented in this study demonstrated good analytical and clinical performance for detection of seven of the most common, horizontally transferable carbapenemase genes in primary patient screening. Its implementation enables rapid scaling on automated high-throughput molecular testing infrastructure.

A15

Universal high accuracy bacterial long amplicon 16S-ITS-23S nanopore sequencing at strain resolution for clinical decision-making

Schöller A.1, Prazuch A.1, Kurz P.2, Jackson E.3, Denslow P.3, Fasulo D.3, Ulicny B.4, Minichova L.5, Gencik M.1,5, Driscoll M.3

1 medgene microbiome, Praxis für Humangenetik, Vienna, Austria

2 WPM, Wundpflegemanagement, Bad Pirawarth, Austria

3 Intus Biosciences LLC, Farmington, USA

4 GRAPHIC, Piestany, Slovakia

5 MedGene Slovensko, Bratislava, Slovakia

Background: Clinical decision-making for an optimal therapy requires a full spectrum analysis of bacterial species together with an accurate, automated bioinformatics pipeline and a medical interpretation platform.

Objectives: To evaluate the universal applicability of long amplicon 16S-ITS-23S RNA gene sequencing to generate bacterial profiles that contribute meaningfully to patient care.

Materials and methods: Clinical samples (stool, urine, vaginal, dental, skin, wound; ticks) were collected either with sterile swabs stored in DNA/RNA Shield™ (ZymoResearch) or respective OMNIgene® line (DNAgenotek) devices. Microbial DNA was purified with a DNA Miniprep Kit (ZymoResearch); tick DNA was extracted with a QIAamp® Viral RNA Mini Kit (Qiagen). 16S-ITS-23S amplicons (Wave™ NanoID™ Kit, Intus Biosciences) were prepared and sequenced (Ligation Sequencing Kit V14, MinION R10.4.1 flow cell, Mk1C sequencer; Oxford Nanopore Technologies). Reads were error corrected with the Titan-1TM bioinformatic pipeline/database (Intus Biosciences) to enable strain-level sequence analysis.

Results: Relative quantities of biostatistically processed ∼2500bp bacterial DNA sequences of each distinct probe for each sample type (n= >25) were compared to the sample population to identify divergences. Clinical reports highlighting individual similarities and differences from the sample collective were generated. Peer-reviewed publications provided the foundation for medical interpretation of results.

Conclusions: Error-corrected sequencing can identify dysbiotic microbiome states in multiple human sample sources. As an example, high resolution data enabled precise identification of animal bite bacterial taxa from an animal bite wound. This and other examples demonstrate that high throughput, high resolution bacterial species data can improve clinical decision-making and patient care.

Rapid Molecular Diagnostics, Multiplexing (A16-A18)

A16

Exploring the value of high-plex respiratory point-of-care diagnostic testing

Vandepitte S.1, Ndolumingo M.2, Textoris J.1

1 bioMérieux SA France

2 bioMérieux GmbH Germany

Background: Emergency departments (EDs) face the critical challenge of diagnosing acute respiratory infections swiftly and accurately. The limitations of slow lab-based testing methods and the constrained pathogen detection of low-plex PCR point-of-care testing (POCT), compromise timely and effective patient care especially in vulnerable patients while contributing to antibiotic misuse and unnecessary hospitalizations. The BIOFIRE® SPOTFIRE® system offers a promising alternative by providing immediate, comprehensive pathogen detection at the point of care, aiming to revolutionize emergency diagnostic processes and reduce spendings.

Objectives: To evaluate the impact of high-plex PCR POCT on operational, health- related and cost outcomes from the hospital’s perspective.

Materials and methods: An early health economic assessment using decision-analytic modelling evaluated the impact of implementing high-plex PCR POCT in German EDs compared to both lab-based testing and low-plex PCR POCT focusing on two critical patient groups: young children (<5 years) and high-risk adults.

Results: Implementing high-plex PCR POCT compared to slow lab-based testing (12h+) resulted in net-savings along with reduced antibiotic misuse and unnecessary hospitalizations. Against low-plex PCR POCT, high-plex PCR POCT also resulted in net-savings and reduced antibiotic misuse, underscoring the value of its diagnostic comprehensiveness. Finally, high-plex PCR POCT outperformed rapid lab-based PCR testing (2-3h) by improving patient flow and operational ED efficiency.

Conclusions: High-plex PCR POCT demonstrates superiority over low-plex and lab-based testing alternatives in young children and high-risk adults and strongly advocates for its adoption in ED settings for these specific patients. Future studies and real-world evidence are expected to confirm the full potential of high-plex PCR POCT in improving healthcare delivery.

A17

Evaluation of a prototype diagnostic for flexible syndromic testing of respiratory infections utilizing existing instrumentation

Jarem D.1, Njoya M.1, Somoskovi A.1, Janos M. 2, Albrecht P.2

1 Roche Molecular Diagnostics, Pleasanton, CA USA

2 Roche Diagnostics International AG, Rotkreuz, Switzerland

Background: Acute upper respiratory tract infections (URTIs) are significant causes of worldwide morbidity and are a leading cause of physician visits and absenteeism, particularly in higher risk populations, such as the very young, the elderly and the immunocompromised. URTIs are difficult to diagnose due to overlapping symptoms and relevant viruses depend on seasonality, test setting, region and patient.

Objectives: To evaluate a prototype flexible syndromic testing solution that utilizes existing high throughput instrumentation and temperature assisted generation of signal (TAGS) technology, allowing differentiation of three targets per fluorescence channel, enabling detection of 12 targets, and an Internal Control, per well.

Materials and methods: Non-interventional, non-observational method comparison (3 sites) with nasopharyngeal (NPS) clinical specimens (prospective archived/frozen; retrospective archived) using CE-marked respiratory multiplex comparators (PPA and NPA). Targets evaluated: adenovirus (B, C, and E); human coronaviruses (229E, HKU1, NL63, OC43); human metapneumovirus; human rhinovirus/enterovirus; influenza A virus; influenza B virus; parainfluenza viruses 1, 2, 3, and 4; respiratory syncytial virus; SARS-CoV-2.

Results: 1306 samples were included for analysis. Adenovirus and rhinovirus/enterovirus were most frequently detected. For most viruses, a high agreement was found (PPA: 95.8%-100.0%; NPA: 97.2%-100%), notably: SARS-CoV-2 (98.4%/98.2%), influenza A (100%/99.1%), influenza B (100%/99.8%), RSV (96.0%/99.8%). Lower agreement was observed for rhinovirus/enterovirus (83.9%; 95%; CI: 75.1%, 90.0%).

Conclusions: With NPS clinical specimens from symptomatic patients, the prototype assay demonstrated good clinical performance against CE-marked comparator assays, highlighting a unique flexible solution that can meet the needs of changing laboratory demands.

Molecular Aging (A19-A21)

A18

Telomere shortening as marker of biological age – the impact of physical activity and obesity

Herrmann M.

Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria

Aging is an irreversible biological process that results from the accumulation of molecular and cellular damage over time, and leads to a gradual decrease in physical and mental capacity, a growing risk of disease and ultimately death. These changes are neither linear nor consistent, and are only loosely associated with a person’s chronological age. For over half a century, researchers have tried to estimate biological age through test batteries that assess physical and psychological parameters. In addition, blood biomarkers, such as leucocyte telomere length (LTL) or DNA methylation, have also been investigated as potential indicators of biological age. Telomeres are protective nucleoprotein structures at the end of all chromosomes that shorten with age. Beyond a critical telomere length (TL), cells can no longer divide and go into senescence or apoptosis. There is substantial evidence suggesting that the velocity of telomere shortening can be modified by numerous lifestyle factors including physical activity, nutrition, smoking, stress and sleep. However, studies that explored the utility of LTL as a biomarker of an individual’s biological age are inconsistent. Furthermore, animal models that investigated the dynamics of telomere shortening in conjunction with different lifestyle factors are limited and the results are conflicting.

This presentation provides a comprehensive overview on the structure and function of telomeres, the utility of LTL as a surrogate marker of biological age, and the impact of exercise and nutrition on TL.

At individual level, LTL is not a surrogate marker of TL in solid tissues. Furthermore, changes in TL and telomerase activity/expression due to age, nutrition and physical activity are tissue-specific. Therefore, the measurement of LTL by qPCR is not a suitable biomarker of biological age at individual level.

A19

Analytical aspects of telomere length analysis

Renner W.

Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria

Telomeres are protein-bound DNA repeat structures at the end of chromosomes that play a critical role in maintaining chromosomal stability. During somatic-cell replication, telomere length progressively shortens because of the inability of DNA polymerase to fully replicate the 3′ end of the DNA strand. In cross-sectional population studies, mean telomere length was inversely associated with age. Shorter age-adjusted mean telomere length has furthermore been found to be associated with risk of several age-related diseases, including coronary artery disease, diabetes mellitus, hypertension and Alzheimer´s disease status.

The most commonly used telomere length measurement methods generally provide information on average or relative telomere length. Methodologic reporting in most studies measuring telomere length is low and current literature on the relation between telomere length measurement methods is lacking in validity and scientific rigor. As the length of the shortest telomeres is likely a key biomarker determining cell fate and the onset of senescence, new techniques providing quantitative information about all the shortest telomeres are of high interest.

A20

Targeting epigenetic age to prevent skin cancer

Rodriguez-Paredes M.

German Cancer Research Center, Heidelberg, Germany

Aging is the leading cause of cancer, including all types affecting the skin. Consequently, skin rejuvenation prevents skin cancer. Among the many molecular processes known to get deregulated with age, epigenetics plays a very relevant role. DNA methylation is arguably the most well-known and studied epigenetic mechanism. Under healthy conditions, specific DNA methylation patterns in CpG dinucleotides of promoters, enhancers and other genomic regions provide a key transcriptional regulatory layer to maintain both cellular identity and functionality. As we age, the wear and tear on the DNA methylation machinery, as well as the continued action of external stressors, gradually modify and erode these methylation profiles contributing to the age-associated loss of cellular functions. In this context, just over a decade ago it was discovered that the DNA methylation levels of a few CpG positions along an individual’s genome could predict chronological age with high accuracy, a concept that soon became known as the ’epigenetic clock’. In recent years, the development of more complex epigenetic clocks from the methylomes of different tissues, including skin, and their further study, has revealed that certain pathologies and factors related to our lifestyle are capable of accelerating or decelerating our biological (epigenetic) age. In my presentation, I will explain how these epigenetic clocks work, what their components are and what biological processes we know today that affect their progression. Our ultimate goal will be to use this powerful tool in our efforts to rejuvenate the skin, and thus also prevent cancer development.

Future Technologies (A22 – A24)

A21

Magnetic sensing for multiple biomarker classes

Hayden O., Leuthner M., Reisbeck M., Helou M.

Heinz-Nixdorf-Chair of Biomedical Electronics, School of Computation, Information, and Technology, Technical University of Munich, Germany

To develop one point-of-care (POC) compatible read-out for any biomarker class without off-cartridge sample preparation, we study the opportunities to integrate magnetic sensing and microfluidic workflow solutions into one unifying platform. This way, we address unmet diagnostic needs for small reader footprints, minimizing workflow complexity and versatility.

Giant magnetoresistance sensors are integrated into microfluidic prototype cartridges. With mechanical and magnetophoretic workflow principles, we can manipulate the position of single cells and capture beads with micron precision for deterministic workflows. Time-of-roll measurements and non-equilibrium assay conditions are used to quantify analytes.

Based on the combination of magnetic analyte enrichment and sensing, biomarker detection at even high hematocrit can be performed over an extensive dynamic concentration range. The presentation shows that the platform can cover diagnostics of exosomes, proteins, and even molecular markers next to quantitative flow cytometry of immune cells.

Integrating any biomarker class from micron-sized cells to small molecules into one read-out system potentially supports a versatile usage of POC testing without the need for multiple systems.

A22

Novel applications of protective encapsulation for living cells: less is more

Gunzburg W.H.1,2

1Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria

2Austrianova Singapore

Cell therapies as well as treatments for microbiome dysbiosis are hampered by the fact that living cells (whether human, animal, yeast or bacteria) are inherently fragile. This fragility affects survival of the cells in the body as well their storage before being introduced into patients. Encapsulation provides a safe means for protection of cells. Austrianova has developed a cellulose polymer based encapsulation technology for living cells. Encapsulation of human or animal cells is achieved using our Cell-in-a-Box encapsulation technology. Cell-in-a-Box allows cells to be stored for more than 5 years at −80 °C and upon thawing the Cell-in-a-box encapsulated cells can be implanted in the patients’ body for at least two years, essentially as a long term mini-transplant. The implanted cells produce therapeutically relevant molecules (enzymes, growth factors, antibodies, hormones such as insulin etc.) which are released from the encapsulated cells into the body. The cells are thus immunoprotected and physically restrained at the site of implantation. Cell-in-a-Box has been shown to be safe and efficacious in clinical trials for solid tumours and data from these studies as well as data from other used cases will be presented. Bac-in-a-Box is used to encapsulate probiotic yeast, bacteria and other microbiota (members of the microbiome), allowing long-term storage at room temperature without appreciable loss of viability. Yeast and bacteria encapsulated by Bac-in-a-Box also confers an extremely high degree of protection from stomach acid (nature’s food sterilisation system) and bile juices. This level of acid protection outperforms any other known technology several thousand-fold. Data from Bac-in-the Box protection of bacteria and yeast in humans and animals, including a field trial in cattle will be presented. As well as nutraceutical uses and the treatment of diseases caused by dysbiosis, Bac-in-the Box could be used for the efficient delivery of oral vaccines. The combined data above, as well as other studies done with polymer beads, and with exosomes and enzymes, also suggests that this technology could be used for the long term protection and delivery of non-living molecules.

A23

Scale-Up of microfluidics chips for IVD applications in a changing regulatory landscape: practical examples

Bauer G.

STRATEC SE, Anif, Austria

Since 2007, Stratec, former Sony DADC Biosciences, has contributed to the development and scale up of various microfluidic chips serving diagnostic applications. Although microfluidics has not transformed the lab workflow as broadly as sometimes anticipated, numerous applications from digital PCR to cell sorting have benefitted from the technology.

The talk will use practical examples to illustrate the challenges for novel microfluidic chips to suffice the requirements for successful IVD approvals in light of growing scrutiny versus lab develop tests and fast track approvals. Researchers should consider the limitations of measurement technologies to qualify structures and spend sufficient efforts in the documentation to translate simulation-results into working prototypes. As higher value workflows like sample prep for sequencing and proteomics adopt more complex microfluidic technologies the experience gained in the first decade of technology adoption can help accelerate the development of these applications.

Poster Session (P1-P12)

P1

Evaluation of the Alinity m HSV 1 & 2 / VZV Assay – a new qualitative multiplex PCR test

Ehret R., Prentice M., Yildirim Ö., Obermeier M.

MIB Medical Center for Infectious Diseases, Berlin, Germany

Background: HSV-1, HSV-2, and VZV often cause phenotypically indistinguishable skin blisters or lesions.

Objectives: To evaluate the new Alinity m HSV 1 & 2 / VZV Assay, a qualitative multiplex real-time PCR for detection and differentiation of three pathogens.

Materials and methods: Residual archived (-20°C) specimens (n=367; Aptima collection-tubes) pre-selected based on results obtained with the Allplex TM Genital ulcer Assay targeting HSV1/2, VZV, and 4 additional sexually-transmitted pathogens, were retested with the Alinity and Allplex tests in parallel. An HSV1&2/VZV/syphilis-positive swab EQA sample (Microbix Biosystems Inc.) resuspended in 0.9% NaCl was tested in triplicate with both assays. Assay linearity was assessed by testing 10 replicates of a dilution series prepared from HSV1/2 and VZV verification panels (Exact Diagnostics).

Results: Overall agreement between Alinity and Allplex for qualitative detection of HSV-1, HSV-2, and VZV in clinical samples was 95.4% (104/109), 98.4% (126/128), and 100% (30/30), respectively. Alinity identified 4 infections missed by Allplex, 3 infections were only detected by Allplex. All pre-selected samples that had tested negative with Allplex for HSV1/2 and VZV (n=100) tested also negative with Alinity. No cross-reactivity for non-targeted sexually-transmitted pathogens present in 219 clinical samples in absence or presence of HSV1/2 and/or VZV was observed with Alinity. Both assays detected the herpes viruses in the EQA sample at high concentration and demonstrated good linearity, while overall, precision was higher with the Alinity assay.

Conclusions: The new Alinity m HSV 1 & 2 / VZV Assay showed a very good performance in detecting HSV1/2 and VZV without any competitive effects in samples with co-infections. The random-access capability of the Alinity platform is a valuable tool in clinical routine.

P2

The cardiovascular implications of SARS-CoV-2 infection in HIV-positive patients in South Africa: a cross-sectional study

Fredriksen P.M.1, Nkeh-Chungag B.2, Keolebogile S.M.3, Hyera F.2, Lundin K.E.A.4, Elias S.O.5, Brix B.6, Schmid-Zalaudek K.6, Rössler A.6, Cvirn G.6, Sourij H.6, Stelzl E.6, Kessler H.H.6, Saloň A.6,7, Goswami N.6,8,9

1 Inland Norway University of Applied Sciences, Hamar, Norway

2 Walter Sisulu University, South Africa

3 Durban University of Technology, South Africa

4 University of Oslo and Oslo University Hospital, Norway

5 University College of Medicine, Lagos

6 Medical University of Graz, Austria

7 Medical College of Georgia at Augusta University, USA

8 Alma Mater Europea Maribor, Slovenia

9 Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai

Background: The SARS-COV-2 pandemic has stressed global healthcare systems, with severe cases requiring intensive care. The burden has been amplified in Sub-Saharan Africa (SSA), where HIV-AIDS prevalence complicates the scenario.

Objectives: To assess the cardiovascular impact of SARS-CoV-2 infection among HIV-positive individuals in South Africa, considering the double burden of diseases in the region.

Materials and methods: In this cross-sectional controlled study, 207 hospitalized patients were categorized into four groups: HIV-positive with SARS-CoV-2, HIV-positive without SARS-CoV-2, HIV-negative with SARS-CoV-2, HIV-negative without SARS-CoV-2 (controls). Anthropometric measurements and blood samples were analyzed, focusing on cardiovascular, metabolic, and inflammation markers.

Results: SARS-CoV-2 infection was associated with significant cardiovascular alterations among HIV-positive patients. Elevated systolic blood pressure and heart rate were more prevalent among SARS-CoV-2-positive individuals. Hemoglobin A1c levels and lipid profiles, particularly triglycerides, were disrupted in the context of acute SARS-CoV-2 infection, indicating altered glucose and lipid metabolism. Oxidative stress and inflammatory markers such as malondialdehyde (MDA) and tumor necrosis factor-alpha (TNFα) were notably elevated in SARS-CoV-2-positive patients, suggesting increased oxidative stress and inflammation. Coagulation and renal function markers were significantly impacted by SARS-CoV-2 infection, underscoring the influence of SARS-CoV-2 on systemic physiological processes.

Conclusions: SARS-CoV-2 infection in HIV-positive individuals in South Africa exacerbates cardiovascular risks. The findings highlight the need for vigilant cardiovascular monitoring and a multidisciplinary management approach for co-infected individuals. The study underscores the complexity of interactions between HIV, antiretroviral therapy, and SARS-CoV-2, calling for more comprehensive research to guide clinical practices in pandemics.

P3

New molecular assay for the detection and quantification of hepatitis B virus load in EDTA-plasma

Juste-Lanas Y.1,2, Franco-Marín E.1, Alonso-Ezcurra H.1

1 Certest Biotec, San Mateo de Gállego, Spain

2 Aragon Health Institute, IISAragon, Zaragoza, Spain

Background: Hepatitis B virus (HBV) is the world’s most common cause of viral hepatitis in humans. Although the diagnosis of hepatitis viral infections is usually performed with serological markers in blood, this technique has limitations.

Objectives: To develop a fast, sensitive and specific test to detect and quantify HBV DNA, capable to help with clinical decisions.

Materials and methods: EDTA plasma samples characterized as positive or negative and quantified in IU/ml were analyzed with the VIASURE HBV q Real Time PCR Detection Kit. Moreover, 50 EDTA plasma clinical samples from patients in the HBsAg-negative phase (HBsAg negative; anti-HBs and anti-HBc positive) and 50 patients with chronic hepatitis B under anti-viral therapy (HBsAg and anti-HBc positive, anti-HBs negative) were analyzed. VIASURE analysis was performed using the following workflow: MagDEA® Dx SV automated extraction method and CFX96™ Opus Real-Time PCR Detection System (Bio-Rad).

Results: A total of 310 specimens were tested, of which 152 tested positive for HBV DNA and 158 negative. The comparative analysis resulted in a sensitivity and positive predictive value of >96%, and a specificity and negative predictive value of >99%.

For quantification, a correlation value of R2=0.913 and R2=0.892 was obtained between cobas® HBV results and VIASURE, and between AltoStar® HBV and VIASURE, respectively.

Conclusions: This clinical evaluation demonstrated that the VIASURE assay is a good tool for clinical diagnosis of HBV as it has good sensitivity and specificity. Detection and quantification of HBV DNA by real-time PCR is an important tool adding to serological diagnostics.

P4

Validation and implementation of a real-time PCR assay for the direct detection of Candida (C.) auris from surveillance samples in the context of a C. auris outbreak

Juste-Lanas Y.1,2, Franco-Marín E.1, Alonso-Ezcurra H1

1 Certest Biotec, San Mateo de Gállego, Spain

2 Aragon Health Institute, IISAragon, Zaragoza, Spain

Background: Candida (C.) auris is of great concern in healthcare settings. Currently, it takes up to 4-5 days to obtain results using culture conventional methodology. A recent outbreak at the Royo Villanova Hospital (Spain) has highlighted the need for rapid and accurate methods to detect and prevent its introduction, rapid spread between hospital and healthcare facilities.

Objectives: To evaluate and validate the molecular assay VIASURE C. auris Real Time PCR kit for detection of C. auris DNA in surveillance samples and to evaluate the implementation of this methodology to complement the daily laboratory routine protocol for C. auris.

Materials and methods: From 409 epidemiological swab specimens and 40 culture isolates DNA was extracted using the MagLead 12gC Instrument and analyzed using the CFX Opus 96™. Results of the molecular assay were compared with those of the hospital´s routine protocol, based on CHROMAgar® Candida +/- Fluconazol 32 µg/ml and identification with MALDI-TOF system.

Results: Molecular assay results were obtained within 2h. All 26 C. auris positive specimens were detect correctly demonstrating the high sensitivity of the new assay. The concordance between reference methodology and VIASURE assay was 100%. Clinical sensitivity was 1 (0.86-1) and clinical specificity was 1 (0.88-1).

Conclusions: With the VIASURE kit, a reduction in the response time for C. auris surveillance compared to conventional methodologies was achieved, contributing to control the C. auris outbreak by helping to decide whether to isolating patients or not within a short time (decreasing from 5 days to ≤ 48 hours when compared to the routine protocol).

P5

Validation of a new molecular assay for the quantification of Epstein Barr virus load in EDTA-plasma

Juste-Lanas Y.1,2, Franco-Marín E.1, Alonso-Ezcurra H1

1 Certest Biotec, San Mateo de Gállego, Spain

2 Aragon Health Institute, IISAragon, Zaragoza, Spain

Background: Epstein Barr virus (EBV) is a herpesvirus in which over 90% of the population worldwide has been infected. Complications of EBV infection are rare in immune-competent individuals but are especially important in a range of hosts with compromised immunity.

Objectives: To develop a quantitative real-time PCR assay for the detection and quantification of EBV in EDTA-plasma samples.

Materials and Methods: Plasma-EDTA samples characterized as positive or negative and quantified in IU/ml were analyzed. The selected samples were collected from male and female adults between January 2017 and July 2021. For comparative analysis, the RealStar® EBV PCR Kit 1.0 (Altona) was used. VIASURE analysis was performed using the following workflow: MagDEA® Dx SV automated extraction method with magLEAD® 12gC and CFX96™ Opus Real-Time PCR Detection System (Bio-Rad).

Results: A total of 205 clinical EDTA-plasma samples were tested, of which 105 were positive for EBV DNA and 100 were negative. The comparative analysis resulted in a sensitivity and specificity >99%. In addition, the positive predictive value (PPV) and negative predictive value (NPV) were >99%. For quantification, an R2 correlation value of 0.824 was obtained.

Conclusions: This clinical evaluation demonstrated that the VIASURE EBV q Real-Time PCR Detection Kit is a good tool for the clinical diagnostics of the virus as it has good sensitivity and specificity. It can be concluded that this VIASURE product meets the initially established requirements and is for the purpose and the population for which it is intended.

P6

Near real-time SARS-CoV-2 genetic variant surveillance system during and in the wake of the COVID-19 pandemic

Kogoj R., Suljič A., Zakotnik S., Knap N., Bosilj M., Slunečko J., Grubelnik G., Vlaj D., Korva M.

1 Institute of Microbiology and immunology, Faculty of Medicine, University of Ljubljana, Slovenia

Background: The requirement for SARS-CoV-2 molecular detection resulted in unforeseen numbers of samples which made genetic variant surveillance challenging. Shortages in personnel, reagents and equipment needed to be resolved in an efficient and sustainable way in order to deliver a genetic surveillance system that would benefit healthcare professionals and decision makers.

Objectives: To set up a near real-time SARS-CoV-2 genetic variant surveillance system able to provide at least 5% lineage resolution by random sampling and still accommodate selective sampling.

Materials and methods: Subsets of SARS-CoV-2 positive samples were selected weekly in order to meet instruments capacity and retain the geographical and demographic structure of the tested population. Sequencing was performed on MiSeq and NextSeq550 instruments following the COVIDseq protocol (Illumina, USA). The Pangolin algorithm was used for lineage determination.

Results: With the established system, we were able to sequence 9.9% (4.687/47.387), 15.3% (21.044/137.281), 12.4% (14.420/116.884) and, 84.1% (2.913/3.465) of SARS-CoV-2 positive samples in 2020, 2021, 2022 and, 2023, respectively. The results revealed a plethora of early lineages battling for domination with B.1.258.17 being the most abundant until Alpha took over in March 2021. Alpha was quickly replaced by Delta in July 2021 which was further replaced by Omicron in December 2022. After turbulent changes of recombinant Omicron sub-lineages, JN.1.X (BA.2.86) are now predominating.

Conclusions: We have developed an efficient and sustainable genetic lineage surveillance system which allowed us to follow the evolution of SARS-CoV-2 in near real-time during the entire pandemic and is still in use up to date.

P7

Anonymous testing for sexually transmitted infections at the AIDS-Hilfe Steiermark in the pre-pandemic, pandemic, and post-pandemic period

Langeder A.1, Stelzl E.1, Rupp M.2, Kessler H.H.1

1 Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Austria

2 AIDS-Hilfe Steiermark, Graz, Austria

Background: In response to the COVID-19 pandemic, stringent containment measures leading to a significant reduction of social contacts were introduced in Austria. These measures may have affected sexual behavior and testing for sexually transmitted infections.

Objectives: To gain insight into the impact of the COVID-19 pandemic and containment measures on anonymous testing for sexually transmitted infections (STIs) at the Styrian service facility AIDS-Hilfe Steiermark. To describe trends for testing and test results on STIs in the pre-pandemic, pandemic, and post-pandemic period.

Materials and methods: Anonymized data obtained from the AIDS-Hilfe Steiermark were investigated. Initial contacts were analyzed for demographic data, tests performed, and test results for human immunodeficiency virus (HIV), hepatitis C virus (HCV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Treponema pallidum (TP). Data obtained in 2019, 2021, and 2023 were compared. For statistical analysis, SPSS v. 29.0 was used.

Results: In 2019, 1494 clients had their initial contact at the AIDS-Hilfe Steiermark. This number decreased to 1034 (-30.8%) in 2021 and increased to 1759 in 2023 (+70.1% compared to 2021 and +17.7% compared to 2019). The number of tests performed is shown in Table 1:

Pathogen(s) No. of tests performed in
2019 2021 2023
HIV 1491 1022 1721
HCV N/A 443 782
CT/NG N/A 496 1173
TP 263 475 1033
Total 1754 2436 4709
  1. N/A, data not available

In 2019 and 2021, 0.20% of clients tested positive for HIV-1, while the percentage was 0.23% in 2023. For TP, corresponding percentages were 1.52% (2019), 1.26% (2021), and 2.03% (2023). For CT, 6.05% (2021) and 5.54% (2023) of tests were found to be positive. For NG, corresponding percentages were 1.81% (2021) and 1.02% (2023). For HCV, no positive results were found.

Conclusions: A considerable decrease of initial contacts at the AIDS-Hilfe Steiermark was observed in 2021, possibly due to stringent containment measures. Together with less testing, a decline in the absolute number of STIs diagnoses was found. When pre-pandemic, pandemic and post-pandemic periods were compared, the percentage of positive test results was found to be similar.

P8

The Yersinia enterocolitica gap in the BD MAX enteric bacterial panel – how to deal with it

Leitner E.1, Pekard-Amenitsch S.2, Steinmetz I.S.1

1 Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Austria

2 Austrian Agency for Health and Food Safety, Graz, Austria

Background: At the Institute of Hygiene, Microbiology and Environmental Medicine (IHMEM), Medical University of Graz, detection of Yersinia spp. has traditionally been included in the stool examination together with Campylobacter, Salmonella, Shigella and, if required, STEC. However, traditional stool diagnostics has recently been replaced by molecular detection of pathogens using the BD MAXTM enteric bacterial panel which does not include Yersinia enterocolitica.

Objectives: To establish a diagnostic procedure for Y. enterocolitica diagnostics based on local epidemiological data.

Materials and methods: Yersinia spp. data 2018 through 2023 were collected at the IHMEM and the Austrian Agency for Health and Food Safety (AGES). The following parameters were evaluated: incidence, age, gender, and species distribution. The ecdc Yersiniosis Annual Epidemiological Report for 2022 and The European Union One Health 2022 Zoonoses Report were used for comparison with Austrian data.

Results: Incidence of Yersinia spp. in Austria was 1.48 per 100,000, with a peak in 2018. In the age group 5 through 14 years, Yersinia enterocolitica (pathogenic) and Yersinia pseudotuberculosis were most frequently detected at both the IHMEM and the AGES, with 20.79% (21/101) and 23.76% (149/627), respectively. In the ecdc Report of 2022, the age group 0 through 4 years ranks first regarding the highest proportion of Yersinia spp. detection.

Conclusions: Based on the data collected, we decided to do Yersinia enterocolitica diagnostics only on request. In that case, the sender can choose either culture or the BD MAXTM extended enteric bacterial panel.

P9

Quantification of SARS-CoV 2 viral burden in vital body organs

Mohar Vitezic B.1, Jakovac H.2, Stemberger C.3, Cuculic D.4, Ferencic A.4

1 Department of Microbiology and Parasitology, Faculty of Medicine, University of Rijeka

2 Department of Physiology, Immunology and Pathophysiology, Faculty of Medicine, University of Rijeka

3 Department of Pathology, Clinical Hospital Center Rijeka

4 Department of Forensic Medicine and Criminalistics, Faculty of Medicine, University of Rijeka

Background: The SARS-CoV-2 entering body through the respiratory system can spread hematogenously and infect other vital organs. COVID-19 affects multiple organs causing severe short and long-term complications. Demonstration of SARS-CoV-2 presence in body organs by autopsy is the only way to prove multi-organ involvement.

Objectives: To quantify the viral load in organ tissues of patient who had deceased due to COVID-19 pneumonia.

Materials and methods: A total of 20 samples from different organs were investigated by qRT-PCR. RNA extraction was performed from 10 mg of homogenized tissue for each vital organ: brain, lung, heart and kidney. Five different samples were collected from different locations of each organ. Quantitative real time PCR was performed to determine actual viral burden for each organ.

Results: The highest viral load of SARS-CoV-2 RNA was detected in the lungs (1.8 x 109 copies), the second highest load in the heart (86.2 x 106 copies) followed by the brain (35.1 x 106 copies) and the kidneys (6.1 x 106 copies).

Conclusion: In this study, the second highest viral load was surprisingly found in the myocardium. Such a high viral load could be the combination of viremia from residual blood in the heart tissue and the tissue infection itself. The high viral presence in the myocardium was found without obvious signs of inflammation suggesting direct viral impact on heart tissue and not an indirect consequence of excessive immune response.

P10

Anti-inflammatory potential of luteolin in ulcerative colitis

Repić I.1, Vukelić I.2, Detel D.2

1 Faculty of Medicine, University of Rijeka, Croatia

2 Department of Medical Chemistry, Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Rijeka, Croatia

Background: Ulcerative colitis (UC) is an inflammatory disease of the colon that causes pathological changes in the colon mucosa. Mucosal changes are one of the risk factors for the development of colon cancer. The disease has a chronic course consisting of periods with severe symptoms such as bloody diarrhea, nausea, and weight loss and a remission stage. The current treatment is unsatisfactory, while natural bioactive compounds, like flavonoids are emerging as therapeutic agents. Luteolin is a flavonoid with anti-inflammatory, antioxidant, antiapoptotic and antitumor properties, but little is known about the mechanism of its action in UC.

Objectives: To investigate the effect of luteolin on the development and severity of ulcerative colitis as well as its effect on apoptosis using an experimental model of UC.

Materials and methods: Ulcerative colitis was induced by the administration of 3% dextran sulfate sodium salt (DSS) in drinking water for seven days. To investigate the effect of luteolin on the development of colitis two doses of luteolin (50 mg/kg, 100 mg/kg) are administrated between day 3 and day 7 of the experiment. Clinical changes were monitored as well as the colon tissue damage by histopathological analysis. The protein expression was determined using Western blot and immunohistochemistry.

Results: Luteolin attenuated symptoms of DSS-induced UC in mice, ameliorated colon tissue damage, and reduced inflammation. Luteolin significantly reduced the expression of proapoptotic proteins and NF-κB.

Conclusions: Luteolin can be used as a novel therapeutic candidate in the treatment of ulcerative colitis or as a supplement to existing therapy.

P11

Children with acute gastroenteritis: Norovirus genotype distribution in South-East Austria

Stelzl E.1, Lederer I.2, Niendorf S.3, Jacobsen S.3, Kessler H.H.1

1 Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Austria

2 Austrian Agency for Health and Food Safety, Division for Public Health, National Reference Center for Norovirus, Graz, Austria

3 Robert Koch Institute, Consultant Laboratory for Norovirus, Berlin, Germany

Background: Noroviruses are a leading cause of gastroenteritis among children worldwide. New variants and new recombinant strains emerge continuously in the norovirus population.

Objective: To evaluate the distribution of norovirus among children in South-East Austria.

Materials and Methods: Consecutive stool samples collected from children <18 years attending the Department of Pediatrics, University Hospital Graz, with clinical presentation compatible with norovirus infection were analyzed between February 2023 and February 2024. Routine diagnostics included screening on norovirus infection by a commercially available norovirus RNA point-of-care (POC) test differentiating genogroup I (GI) and genogroup II (GII). To gain deeper insight into the current norovirus epidemiology in South-East Austria, standardized sequencing protocols for dual typing (genotype and polymerase type) were performed on all stool samples positive for norovirus RNA.

Results: A total of 69 stool samples tested positive for norovirus RNA with POC diagnostics. Of those, 59 (85.5%) contained norovirus GII. With sequencing, 19 of 69 stool samples could not be typed due to low viral load. Of 50 typeable stool samples, 36 (72%) contained the GII.4 Sydney[P16] strain. The remaining stool samples contained GII.3[P12] (n=4), GII.7[P7] (n=4), GI.3[P3] (n=2), GII.6[P7] (n=2), GI.6[P11] (n=1), and GII.17[P17] (n=1) strains.

Conclusions: The GII.4 Sydney[P16] strain was found to be the predominant norovirus strain in children with acute gastroenteritis in South-East Austria. The number of remaining strains was low; however, a wide distribution of different strains was found. These data may contribute to the development of future norovirus vaccine candidates.

P12

Frequency of human neutrophil antigens HNA-1, -3, -4 and -5 alleles in Croatia and their role in the development of alloimmune neonatal neutropenia

Štimac R., Tomičić M., Bingulac Popović J., Kundid R., Babić I., Vuk T., Jukić I.

Croatian Institute of Transfusion Medicine (CITM), Zagreb, Croatia

Background: Human neutrophil antigens (HNAs) play an important role in immune mediated neutropenias.

Objectives: To determine the HNA-1, -3, -4 and -5 allele frequencies in the Croatian blood donor population and to assess their role in the development of neonatal alloimmune neutropenia (NAIN).

Methods: A total of 371 blood samples from unselected healthy blood donors were analyzed. Samples from all 371 donors were genotyped for HNA-1, samples from 160 donors were genotyped for HNA-3, and samples from 142 donors were genotyped for HNA-4 and HNA-5 using the polymerase chain reaction with sequence-specific primers (PCR-SSP) „in-house“-method. In 10 cases of serologically confirmed severe NAIN, the HNA genotype of mothers and children were determined.

Results: The frequencies of HNA-1a, -1b and -1c alleles were 39.3%, 60.7%, and 2.2%, whereas the frequencies of HNA-3a and HNA-3b were 78.1% and 21.9% respectively. The frequencies of HNA-4a and -4b alleles were 79.6% and 20.4%, and for HNA-5a and -5b, frequencies were 71.8% and 28.2% respectively. In 9 out of 10 NAIN cases, maternal immunization was caused by alloantibodies resulting from a mismatch with the child’s HNA-1b antigen, and in one case with the child’s HNA-1a antigen.

Conclusion: The determination of HNA antigen frequencies in population is important for estimating the risk of feto-maternal incompatibility. These findings are consistent with data from the literature indicating that NAIN is most often caused by a mismatch in HNA-1b antigen between mother and child. Molecular methods for determining the HNA genotype of the mother and child contribute to the timely detection of NAIN.

Author Index

Aberle S.W. A3, A10
Aepfelbacher M. A14
Albrecht P. A17
Alonso-Ezcurra H. P3, P4, P5
Babić I. P12
Bauer G. A23
Benschop K.S.M. A4
Berger A. A10
Bingulac Popović J. P12
Bosilj M. P6
Brandenburger E. A13
Breuer M.T. A11
Brix B. P2
Buchta C. A3
Camp J.V. A3
Christner M. A14
Cobbaert C. A1
Cuculic D. P9
Cvirn G. P2
De Boever P. A6
Denslow P. A15
Detel D. P10
Donoso Mantke O. A4
Driscoll M. A15
Eckel C. A14
Ehret R. P1
Eigner U. A14
Elias S.O. P2
Everson F. A6
Fasulo D. A15
Ferencic A. P9
Franco-Marín E. P3, P4, P5
Fredriksen P.M. P2
Frishman D. A12
Früchtel C. A10
Gencik M. A15
Gey B. A10
Giersch K. A14
González Rivero C. A12
Goswami N. A6, P2
Görzer I. A3
Griesmacher A. A3
Grubelnik G. P6
Grunwald M. A14
Gunzburg W.H. A22
Haushofer A.C. A2
Hayden O. A21
Heim A. A10
Helou M. A21
Herrmann M. A18
Hoffmann D. A12
Hyera F. P2
Jackson E. A15
Jacobsen S. P11
Jakovac H. P9
Janos M. A17
Jarem D. A16
Jukić I. P12
Juste-Lanas Y. P3, P4, P5
Keolebogile S.M. P2
Kessler H.H. A6, A10, P2, P7, P11
Knap N. P6
Kogoj R. P6
Kolb M. A14
Korva M. P6
Köffer J. A14
Kress J. A10
Kundid R. P12
Kurz P. A15
Lampertico P. A10
Langeder A. P7
Lederer I. P11
Leitner E. P8
Leuthner M. A21
Lundin K.E.A. P2
Lütgehetmann M. A14
Maier T. A12
McCulloch E. A4
Mckloud E. A4
Minichova L. A15
Mohar Vitezic B. P9
Montgomery D. A4
Müller M.M. A3
Ndolumingo M. A16
Neuwirth E. A3
Niendorf S. P11
Njoya M. A17
Nkeh-Chungag B. P2
Noppen C. A5
Nörz D. A14
Obermeier M. P1
Olivero A. A10
Qu S. A12
Pekard-Amenitsch S. P8
Pfefferle S. A14
Pflüger S. A14
Prazuch A. A15
Prentice M. P1
Protzer U. A12
Puchhammer E. A3, A9
Reinhardt A. A10
Reisbeck M. A21
Renner W. A19
Repić I. P10
Retzlaff S. A11
Rodriguez-Paredes M. A20
Rohde H. A14
Rosenstock P. A11
Rössler A. P2
Rupp M. P7
Salon A. P2
Schmid-Zalaudek K. P2
Schöller A. A15
Slunečko J. P6
Somoskovi A. A17
Sourij H. P2
Staines H. A4
Steinmetz I.S. P8
Stelzhammer A. A2
Stelzl E. A6, A10, P2, P7, P11
Stemberger C. P9
Štimac R. P12
Strijdom H. A6
Suljič A. P6
Sutton G. A4
Tang H.T. A14
Textoris J. A16
Tiemann C. A7
Tiplica G.S. A8
Tomičić M. P12
Ulicny B. A15
Vandepitte S. A16
Van Loon A. A4
Yildirim Ö. P1
Vlaj D. P6
Vuk T. P12
Vukelić I. P10
Webster I. A6
Wedemeyer H. A10
Weinschenk C. A14
Weseslindtner L. A3
Zakotnik S. P6
Weseslindtner L. A3
Zakotnik S. P6

Published Online: 2024-04-16
Published in Print: 2024-05-27

© 2024 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. SARS-CoV-2 is here to stay: do not lower our guard
  4. Reviews
  5. SARS-CoV-2 subgenomic RNA: formation process and rapid molecular diagnostic methods
  6. Prognostic value of anti-SARS-CoV-2 antibodies: a systematic review
  7. Presence of SARS-CoV-2 RNA in COVID-19 survivors with post-COVID symptoms: a systematic review of the literature
  8. Opinion Papers
  9. Harmonizing the post-analytical phase: focus on the laboratory report
  10. Blood-based biomarkers in Alzheimer’s disease – moving towards a new era of diagnostics
  11. A comprehensive review on PFAS including survey results from the EFLM Member Societies
  12. General Clinical Chemistry and Laboratory Medicine
  13. Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword
  14. An LC–MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays
  15. CX3CL1/Fractalkine as a biomarker for early pregnancy prediction of preterm premature rupture of membranes
  16. Elevated S100B urine levels predict seizures in infants complicated by perinatal asphyxia and undergoing therapeutic hypothermia
  17. The correlation of urea and creatinine concentrations in sweat and saliva with plasma during hemodialysis: an observational cohort study
  18. Tubular phosphate transport: a comparison between different methods of urine sample collection in FGF23-dependent hypophosphatemic syndromes
  19. Reference Values and Biological Variations
  20. Monocyte distribution width (MDW): study of reference values in blood donors
  21. Data mining of reference intervals for serum creatinine: an improvement in glomerular filtration rate estimating equations based on Q-values
  22. Hematology and Coagulation
  23. MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC
  24. Cardiovascular Diseases
  25. To rule-in, or not to falsely rule-out, that is the question: evaluation of hs-cTnT EQA performance in light of the ESC-2020 guideline
  26. Temporal biomarker concentration patterns during the early course of acute coronary syndrome
  27. Diabetes
  28. Proteomic analysis of diabetic retinopathy identifies potential plasma-protein biomarkers for diagnosis and prognosis
  29. Infectious Diseases
  30. Serum biomarkers of inflammation and vascular damage upon SARS-Cov-2 mRNA vaccine in patients with thymic epithelial tumors
  31. A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test
  32. Evaluation of inflammatory biomarkers and vitamins in hospitalized patients with SARS-CoV-2 infection and post-COVID syndrome
  33. The CoLab score is associated with SARS-CoV-2 viral load during admission in individuals admitted to the intensive care unit: the CoLaIC cohort study
  34. Development and evaluation of a CRISPR-Cas13a system-based diagnostic for hepatitis E virus
  35. Letters to the Editor
  36. Crioplast® is a reliable device to ensure pre-analytical stability of adrenocorticotrophin (ACTH)
  37. Falsely decreased Abbott Alinity-c gamma-glutamyl transferase-2 result from paraprotein and heparin interference: case report and subsequent laboratory experiments
  38. Impact of hemolysis on uracilemia in the context of dihydropyrimidine dehydrogenase deficiency testing
  39. Value of plasma neurofilament light chain for monitoring efficacy in children with later-onset spinal muscular atrophy under nusinersen treatment
  40. Analytical evaluation of the Snibe β-isomerized C-terminal telopeptide of type I collagen (β-CTX-I) automated method
  41. Acute myeloid leukemia with blue-green neutrophilic inclusions have different outcomes: two cases and review of the literature
  42. Congress Abstracts
  43. The 10+1 Santorini Conference
  44. 14th National Congress of the Portuguese Society of Clinical Chemistry, Genetics and Laboratory Medicine
  45. 15th National Congress of the Portuguese Society of Clinical Chemistry, Genetics and Laboratory Medicine
  46. ISMD2024 Thirteenth International Symposium on Molecular Diagnostics
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