KLINISCH LABORATORIUM

CENTRUM KLINISCH LABORATORIUM

ARTIKELS

ABSTRACT 1

Lethal injection of a castor bean extract: ricinine quantification as a marker for ricin exposure using a validated LC–MS/MS method.

Verougstraete N, Helsloot D, Casier I, et al. Journal of Analytical Toxicology, 2019, 43(3), e1-e5

Het abstract is terug te vinden op pagina 7.

ABSTRACT 2

Cost-effective and robust genotyping using doublemismatch allele-specific quantitative PCR.

Lefever S, Rihani A, Van Maerken T, et al. Scientific Reports, 2019, 9(1), 2150

INTRODUCTION/BACKGROUND For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs.

OBJECTIVE To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner.

MATERIAL/METHODS This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced.

RESULTS The resulting method, called double-mismatch allele-specific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. CONCLUSION DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test.

ABSTRACT 3

ALK positively regulates MYCN activity through repression of HBP1 expression.

Claeys S, Denecker G, Van Maerken T, et al. Oncogene, 2019, 38(15), 2690-2705

INTRODUCTION/BACKGROUND ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis.

OBJECTIVE To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential.

RESULTS We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PI3K-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.

ABSTRACT 4

Nosocomial outbreak of extended-spectrum β-lactamase-producing Enterobacter cloacae among cardiothoracic surgical patients: causes and consequences.

Noël A, Vastrade C, Van Maerken T, et al. Journal of Hospital Infection, 2019, 102(1), 54-60

INTRODUCTION/BACKGROUND Enterobacteriaceae are recognized as leading pathogens of healthcare-associated infections.

OBJECTIVE To report the investigation of a nosocomial outbreak of extended-spectrum β-lactamase-producing Enterobacter cloacae affecting cardiothoracic surgery patients in a Belgian academic hospital.

MATERIALS/METHODS Cases were defined based on epidemiological and microbiological investigations, including molecular typing using repetitive element-based polymerase chain reaction and multi-locus sequence typing. Case-control studies followed by field evaluations allowed the identification of a possible reservoir, and the retrospective assessment of human and financial consequences.

RESULTS Over a three-month period, 42 patients were infected or colonized by CTX-M-15-producing E. cloacae strains that belonged to the same clonal lineage. Acquisition mainly occurred in the intensive care unit (N = 23) and in the cardiothoracic surgery ward (N = 16). All but one patient had, prior to acquisition, undergone a cardiothoracic surgical procedure, monitored by the same transoesophageal echocardiography (TOE) probe in the operating room. Despite negative microbiological culture results, the exclusion of the suspected probe resulted in rapid termination of the outbreak. Overall, the outbreak was associated with a high mortality rate among infected patients (40%) as well as significant costs (€266,550).

CONCLUSION The outbreak was indirectly shown to be associated with the contamination of a manually disinfected TOE probe used per-operatively during cardiothoracic surgery procedures, because withdrawal of the putative device led to rapid termination of the outbreak. ABSTRACT 5

A recurrent and transesophageal echocardiography–associated outbreak of extended-spectrum β-lactamase–producing Enterobacter cloacae complex in cardiac surgery patients.

Van Maerken T, De Brabandere E, Noël A, et al. Antimicrobial Resistance & Infection Control, 2019, 8, 152 INTRODUCTION/BACKGROUND We report a recurrent outbreak of postoperative infections with extended-spectrum β-lactamase (ESBL)-producing E. cloacae complex in cardiac surgery patients, describe the outbreak investigation and highlight the infection control measures.

MATERIALS/METHODS Cases were defined as cardiac surgery patients in Ghent University Hospital who were not known preoperatively to carry ESBL-producing E. cloacae complex and who postoperatively had a positive culture for this multiresistant organism between May 2017 and January 2018. An epidemiological investigation, including a case-control study, and environmental investigation were conducted to identify the source of the outbreak. Clonal relatedness of ESBL-producing E. cloacae complex isolates collected from case patients was assessed using whole-genome sequencing-based studies.

RESULTS Three separate outbreak episodes occurred over the course of 9 months. A total of 8, 4 and 6 patients met the case definition, respectively. All but one patients developed a clinical infection with ESBL-producing E. cloacae complex, most typically postoperative pneumonia. Overall mortality was 22% (4/18). Environmental cultures were negative, but epidemiological investigation pointed to transesophageal echocardiography (TEE) as the outbreak source. Of note, four TEE probes showed a similar pattern of damage, which very likely impeded adequate disinfection. The first and second outbreak episode were caused by the same clone, whereas a different strain was responsible for the third episode.

CONCLUSION Health professionals caring for cardiac surgery patients and infection control specialists should be aware of TEE as possible infection source. Caution must be exercised to prevent

and detect damage of TEE probes. ABSTRACT 6

Interference of anti-streptavidin antibodies in immunoassays : a very rare phenomenon or a more common finding?

Verougstraete N, Berth M, Callewaert N, et al. Clinical Chemistry And Laboratory Medicine, 2019, Online adead of print, doi: 10.1515/cckl-2019-1064

INTRODUCTION/BACKGROUND Anti-streptavidin antibodies (ASA) may cause analytical interference on certain immunoassay platforms. Streptavidin is purified from the non-pathogenic Streptomyces avidinii soil bacterium. In contrast to interference with biotin, ASA interference is supposed to be much rarer. In-depth studies on this topic are lacking. Therefore, we carried out an analysis toward the prevalence and the possible underlying cause of this interference.

MATERIALS/METHODS Anti-streptavidin (AS)-immunoglobulin G (IgG) and AS-IgM concentrations were determined on multiple samples from two patients with ASA interference and on 500 random samples. On a subset of 100 samples, thyroid-stimulating hormone (TSH) was measured on a Cobas analyzer before and after performing a neutralization protocol which removes ASA. The relationship between the ratio of TSH after neutralization/TSH before neutralization and the ASA concentration was evaluated. Subsequently, an extract of S. avidinii colonies was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.

RESULTS A positive correlation between AS-IgM concentrations and TSH ratio was obtained. Eight samples out of 500 exceeded the calculated AS-IgM cut-off value. In comparison to the AS-IgM concentrations in the population, titers from the two described cases clearly stood out. The isolated cases represent the end of a broader spectrum as there is a continuum of AS-IgM reactivity in the general population. We could not observe any differences in the immunoblot patterns between the cases and controls, which may indicate the general presence of ASA in the population.

CONCLUSION Interference due to ASA is more prevalent than initially thought and is caused by IgM antibodies.

ABSTRACT 7

Quality of blood samples collected at home does not affect clinical decision making for the administration of systemic cancer treatment.

Cool L, Callewaert N, Van Eygen K, Tack L, Missiaen J, Debruyne P, et al. Scandinavian Journal of Clinical and Laboratory Investigation, 2020, 80(3), 215-221 OBJECTIVE The aim of this exploratory clinical study was to evaluate whether the preanalytical quality of blood samples subjected to delayed centrifugation and transport – as a result of home-sampling – is affected in a way it alters the clinical decision-making for patients under systemic cancer therapy. This evaluation is part of a comprehensive investigation of the opportunities for oncological home-hospitalization.

MATERIALS/METHODS Forty-nine patients with cancer donated two additional blood samples during their ambulatory hospital visit. Fifteen blood analytes were compared between routine blood samples and samples that were subjected to transport and delayed centrifugation in order to mimic a locally implemented model for oncological home-hospitalisation. Deviations were analysed by means of Deming regression. For those analytes showing statistically significant intercepts and/ or slopes, the mean deviations were compared to the desirable analytical bias; and the intra-individual differences were compared with the limits for clinical decision-making.

RESULTS Statistically significant intercepts and/or slopes were observed for haematocrit (HCT), mean cellular volume (MCV), platelets count (PLT) and C-reactive protein (CRP). Differences exceeding the allowable margins of desirable analytical bias were observed for HCT and MCV. Risk of different clinical decision-making couldn’t be observed for any of the analytes showing statistically significant differences.

CONCLUSION These results demonstrate that home-collection of blood samples, transported at room temperature and centrifuged within a mean time of five hours after sampling, has no effect on clinical decision-making with regards to systemic cancer therapy. However, attention should be paid to the potential occurrence of haemolysis during the preanalytical phase, which can negatively influence haemolysis-dependent variables.

PRESENTATIES / CONGRESSEN

ABSTRACT 1

Whole transcriptome profiling of liquid biopsies from patient-derived xenograft (PDX) models.

RNGS19: Revolutionizing Next-Generation Sequencing Vermeirssen V, Decock A, Van Maerken T, et al. March 2019, Antwerpen, België

INTRODUCTION/BACKGROUND Grafting of patient-derived primary tumors in mice models complex interactions within a heterogeneous tumor and is crucial for the development of optimal cancer treatment. Using minimally invasive liquid biopsies, RNA molecules, which are released from the tumor into the circulation, are promising biomarkers for cancer detection and progression. PDX models offer the advantage of characterizing the tumor RNA fraction in blood. Until now, there is no standardized protocol, both for the experimental and computational analysis of total RNA from blood, to obtain the highest and purest tumor RNA profile.

OBJECTIVE We examined the transcriptome of a PDX model of Invasive Ductal Carcinoma.

MATERIALS/METHODS We collected platelet-rich, platelet-poor and platelet-free plasma, and primary tumors in 5 PDX mice. RNA was prepared by SMARTer® Stranded Total RNA-Seq Kit v2 - Pico and libraries were sequenced on an Illumina NextSeq 500, generating 75 bp paired-end reads.

RESULTS Analysis of the tumor transcriptome in PDX mice is complex, since the RNA does not only contain reads from the grafted human cancer, but also from the murine host. However, the analysis of the tumor transcriptome from blood samples is even more challenging, due to degradation and dilution of human tumor RNA in a pool of mouse RNA in the liquid biopsy. We assessed the efficiency of two computational pipelines: 1) parallel mapping to a human and mouse reference genome, followed by quality filtering, and 2) mapping to an in silico combined human-mouse reference genome. CONCLUSION We provide a proof-of-concept for the RNA-based detection of tumor signal in plasma of PDX mice. In addition, we present guidelines for both experimental and computational pipelines to optimally detect tumor RNA in liquid biopsies.

ABSTRACT 2

Evaluation of the Xpert HBV VL test for cartridge-based quantitative HBV DNA analysis on plasma.

European Meeting on Molecular Diagnostics

Wallaert A, De Bel A, Boudewijns M

October 2019, Noordwijk, Nederland

INTRODUCTION/BACKGROUND The Xpert HBV VL test (Cepheid) is a cartridge-based assay for the quantification of Hepatitis B Virus DNA in HBV-infected patients using the GeneXpert system.

OBJECTIVE The aim was to evaluate the performance of the Xpert HBV VL test on plasma samples and to compare it with the Artus HBV RG PCR test (Qiagen).

MATERIALS/METHODS For precision evaluation of the Xpert HBV VL test, three-fold dilutions of the Accurun 325 series 200 HBV QC (Seracare) and the Acrometrix HBV High Control (Thermo Fisher Scientific), representing low and high viral loads, were analyzed twelve times. Accuracy was tested with EQC panels (19 samples, including genotypes A, B and D) and method comparison with the Artus HBV RG PCR test was performed on 48 samples.

RESULTS Precision evaluation resulted in an SD of 0.19 log IU/mL for the low control (͞x– = 1.48 log IU/mL) and an SD of 0.08 log IU/mL for the high control (͞x = 6.41 log IU/mL). Accuracy evaluation showed 100% concordance, with an average absolute difference of 0.12 log IU/mL compared to the reference data (range between -0.11 and 0.49 log IU/mL). Method comparison denoted good correlation (Pearson’s r=0.97) with a Bland-Altman plot showing a mean difference of 0.16 log IU/mL (CI95%: -0.83 - 1.15), compatible with a modestly higher quantification with the Xpert assay.

CONCLUSION The Xpert HBV VL assay showed good performance both in precision and accuracy. There was a good correlation with

the Artus HBV RG PCR test. This implies that the Xpert HBV VL assay, an all-in-one assay, can be used as alternative for batch analysis with the Artus HBV RG PCR test. ABSTRACT 3

The S-DiaMGTV kit on cobas 4800: simplifying mycoplasma genitalium and trichomonas vaginalis detection.

European Meeting on Molecular Diagnostics

Wallaert A, De Bel A, Boudewijns M

October 2019, Noordwijk, Nederland INTRODUCTION/BACKGROUND Standard STD analysis is performed for Chlamydia trachomatis and Neisseria gonorrhoeae, although there is increasing evidence that Mycoplasma genitalium and Trichomonas vaginalis should be tested more routinely. The S-DiaMGTV kit (Diagenode) is a multiple PCR kit for the detection of M. genitalium and T. vaginalis. OBJECTIVE Our aim was to evaluate the performance of this kit on extracts of urogenital samples obtained from the Cobas 4800 CT/NG assay (Roche). MATERIALS/METHODS Extracts of the Cobas 4800 CT/NG assay were pipetted into the S-DiaMGTV PCR reaction mix, containing MGTV probes and primers (Diagenode) and LightCycler 480 Probes Master mastermix (Roche). PCR analysis was performed on the Cobas Z480 analyzer (Roche). Precision was evaluated on two concentrations for M. genitalium and T. vaginalis (both for swab and urine samples) and accuracy was tested with the QCMD 2018 EQA pilots. Finally, storage of low positive samples in Cobas PCR medium (two weeks at 4°C) and DNA extracts (four weeks at -20°C) was evaluated. RESULTS Precision was good with an average SD of 0.48 (0.141.12) for the intra-run variability and 0.51 (0.08-0.72) for inter-run variability. Accuracy evaluation showed 100% concordance (n=17, no positive T. vaginalis urine sample). Storage of samples in Cobas PCR medium for two weeks (SD: 0.08-0.78) and DNA extracts for four weeks (SD: 0.050.72) appeared stable. CONCLUSION STD diagnosis can easily be expanded with M. genitalium and T. vaginalis by using extracts from the Cobas 4800 CT/ NG assay and the S-DiaMGTV PCR kit. Samples in Cobas PCR medium can be stored at 4°C for two weeks and DNA

extracts at -20°C for at least four weeks. ABSTRACT 4

Reducing catheter induced hemolysis by use of the holdex tube holder.

Royal Belgian Society of Laboratory Medicine Strubbe G, Callewaert N November 2019, Brussels, Belgium

OBJECTIVE Phlebotomy through a peripheral intravenous catheter is common practice in emergency departments. Though often timesaving and less invasive, this practice is known to cause in vitro hemolysis, frequently leading to analytical interference and compromised test results. Here we investigate whether the Holdex® Single-Use Holder (Greiner Bio-One), with its eccentrically placed Luer adapter and flash chamber, is able to reduce hemolysis by decreasing the pressure gradient between blood tube and punctured vein.

MATERIALS/METHODS Blood was drawn via a Becton Dickinson (BD) Insyte Autoguard catheter from 100 newly admitted patients by emergency nurses alternatively employing either the Holdex® Single-Use Holder (n=50) or a reusable BD Vacutainer® Pronto Quick Release holder (n=50). A sodium citrate tube was drawn first, followed by a serum tube (BD Vacutainer® SST II Advance 6 mL), in accordance with CLSI guidelines. Impact on primary endpoint, cell-free hemoglobin (cf-Hb) in serum (as estimated by hemolysis index), as well as on secondary measures (i.e. test panel comprising hemolysis sensitive parameters K, LDH, AST, ALT, CK, conjugated bilirubin and iron) was assessed. The latter were flagged when hemolysis exceeded the respective upper threshold specified by Roche, indicating potentially spurious results. All analyses were performed on serum using a Roche Cobas 6000 c501 analyzer.

RESULTS Median estimated cf-Hb differed significantly (p < 0.05, Mann-Whitney U test) between the Holdex® group (median 8.5 mg/dL, IQR 10.3 mg/dL) and the control group (median 15 mg/dL, IQR 20.8 mg/dL). Of 350 performed tests in each group, 20 results were potentially compromised in the Holdex® group vs. 60 in the control group (RR 0.33, 95% CI 0.21-0.54), leading to an absolute reduction of 11.4% (95%

CI 6.8-16%) in flagged results.

CONCLUSION The Holdex® device appears an effective measure in reducing catheter induced hemolysis in a general emergency department patient population.

ABSTRACT 5

Exploring anti-streptavidin antibodies interference.

EuroMedLab Verougstraete N, Callewaert N May 2019, Barcelona, Spain

INTRODUCTION/BACKGROUND Anti-streptavidin antibodies may cause analytical interference in immunoassays based on biotin-streptavidin interaction. Recently, we observed a case in which a 29-year-old female was misdiagnosed with hyperthyroidism and incorrectly treated with thiamazol, based on results obtained on a Cobas e602 analyzer (Roche). Thyroid function tests on another detection principle were within normal limits (Abbott). Little is still known about the origin of anti-streptavidin antibodies. The aim of this study was to explore the cause of this interference.

MATERIALS/METHODS The concentration of both IgG and IgM anti-streptavidin antibodies in 3 consecutive serum samples was measured using specific assays on a Phadia 250 (Thermo Fisher). Subsequently, an extract of Streptomyces avidinii colonies was analysed using SDS-PAGE electrophoresis. Next, immunoblotting with the patient’ serum and anti-human IgM coupled to horseradish peroxidase as secondary antibody, was carried out to elucidate the interference mechanism. This blotting experiment was also performed with a control sample without anti-streptavidin antibodies.

RESULTS IgM antibodies were initially 273 µg/L, 183 µg/L 2 months later and 177 µg/L after another 5 months. No anti-streptavidin IgG (< 2.0 μg/L) could be demonstrated in any sample. On the S. avidinii immunoblot of the patient’s serum, an IgM band in the 100 kDa range was seen, which was not observed in the control subject, suggesting an antibody induced in the patient by exposure to S. avidinii (nonpathogenic soil bacteria) antigens. CONCLUSION IgM anti-streptavidin antibodies may cause interferences on certain immunoassays. We assume that these antibodies may arise after exposure to S. avidinii. This is the first report dealing with immunoblotting as a tool to study the anti-streptavidin antibodies. Further research is necessary to prove this hypothesis.

ABSTRACT 6

Ruminococcus gnavus bacteremia, an uncommon presentation of a common member of the human gut microbiota: case report and literature review.

Lefever S, Van Moerkercke W, D'Hondt M, Pampols M, De

Bel A, Boudewijns M, et al. Acta Clinica Belgica, 2019, Dec 74(6), 435-438

Het abstract is terug te vinden op pagina 7.