Enhancing Shelf Life Prediction of Fresh Pizza with Regression Models and Low Cost Sensors.

The waste of food presents a challenge for achieving a sustainable world. In Germany alone, over 10 million tonnes of food are discarded annually, with a worldwide total exceeding 1.3 billion tonnes. A significant contributor to this issue are consumers throwing away still edible food due to the expiration of its best-before date. Best-before dates currently include large safety margins, but more precise and cost effective prediction techniques are required. To address this challenge, research was conducted on low-cost sensors and machine learning techniques were developed to predict the spoilage of fresh pizza. The findings indicate that combining a gas sensor, such as volatile organic compounds or carbon dioxide, with a random forest or extreme gradient boosting regressor can accurately predict the day of spoilage. This provides a more accurate and cost-efficient alternative to current best-before date determination methods, reducing food waste, saving resources, and improving food safety by reducing the risk of consumers consuming spoiled food.

Results of a European-Wide External Quality Assessment (EQA) Scheme for Serological Detection of Anti-SARS-CoV-2 (CoVimm)-Pitfalls of Routine Application.

BACKGROUND: During the last two years, a variety of assays for the serological detection of antibodies to the new SARS-CoV-2 virus have been launched and used as part of standard care in many laboratories. The pace with which these tests have been introduced into routine care emphasizes the importance of quality measures for analytical methods, particularly with regard to the implications of results for clinical and epidemiologic decisions. Accuracy, reliability and comparability of analytical test results are thus essential, and here external quality assessment (EQA) is the most important quality assurance tool. It allows us to achieve harmonization of test methods as a prerequisite for a high standard of performance for laboratory and analytical techniques and their interpretation. METHODS: This EQA scheme consisted of pre-characterized clinical biospecimens dedicated to the analysis of anti-SARS-CoV-2 IgG total antibodies and differentiation into spike protein-specific IgG antibodies against SARS-CoV-2 (anti-S-SARS-CoV-2) and nucleocapsid-specific IgG antibodies against SARS-CoV-2 (anti-N-SARS-CoV-2). RESULTS: A total of 239 laboratories across Europe participated in this scheme, called CoVimm. In detail, 536 results for anti-SARS-CoV-2 IgG, 431 results for anti-S-SARS-CoV-2 IgG, and 200 results for anti-N-SARS-CoV-2 IgG were reported. Based on the pre-defined thresholds, the success rates for the determination of anti-S-SARS-CoV-2 IgG and anti-N-SARS-CoV-2 IgG were 96% and 90%, respectively. Interestingly, only 64% of the participating laboratories successfully passed the EQA scheme for the determination of total anti-SARS-CoV-2 IgG. CONCLUSIONS: This EQA revealed serious concerns regarding the reliability and appropriate use of anti-SARS-CoV-2 antibody assays in routine care. In addition to the wide heterogeneity of different assays used by participating laboratories, a lack of standardization and harmonization is also evident. This is of particular importance for reliable and clinically meaningful interpretation of test results.

Integrated diagnostics.

BACKGROUND: Integrated diagnostics is increasingly gaining scientific traction as it promises to address several challenges currently facing diagnostic medicine. These challenges range from the need for improved diagnostic accuracy to optimized timing of diagnostic procedures, to the variety of diagnostic markers and thus the complexity of their interpretation, and finally to economic pressure. METHODICAL INNOVATIONS: While many of these challenges may be difficult to solve with a monomodal approach, the integration of laboratory markers and imaging procedures promises to allow both disciplines to achieve their actual clinical potential. Combining complementary diagnostic approaches can help to improve the interpretation of measurements, provide a better cost-effectiveness particularly when cutting-edge techniques are used for specific indications, and facilitate optimized timing and rational choice of appropriate diagnostic approaches for disease surveillance. Furthermore, close interdisciplinary assessment of diagnostic results will increase diagnostic accuracy and will enable selection of specific patient cohorts at increased risk for certain diseases who are suitable for further testing. CONCLUSION: The potential of an integrated diagnostic approach represents a strategic goal for diagnostic disciplines as it achieves better visibility and greater clinical impact. In addition to close collaboration among relevant diagnostic experts, an appropriate structure for integrated data evaluation needs to be established to provide actionable health guidance so that integrated diagnostics can be implemented in standard care.

Assessing the Quality of Serological Testing in the COVID-19 Pandemic: Results of a European External Quality Assessment (EQA) Scheme for Anti-SARS-CoV-2 Antibody Detection.

External quality assessment (EQA) is a key instrument for achieving harmonization, and thus a high quality, of diagnostic procedures. As reliable test results are crucial for accurate assessment of SARS-CoV-2 infection prevalence, vaccine response, and immunity, and thus for successful management of the ongoing COVID-19 pandemic, the Reference Institute for Bioanalytics (RfB) was the first EQA provider to offer an open scheme for anti-SARS-CoV-2 antibody detection. The main objectives of this EQA were (i) to gain insights into the current diagnostic landscape and the performance of serological tests in Europe and (ii) to provide recommendations for diagnostic improvements. Within the EQA, a blinded panel of precharacterized human serum samples with variable anti-SARS-CoV-2 antibody titers was provided for detection of anti-SARS-CoV-2 IgG, IgA, and IgM antibodies. Across the three distribution rounds in 2020, 284 laboratories from 22 countries reported a total of 3,744 results for anti-SARS-CoV-2 antibody detection using more than 24 different assays for IgG. Overall, 97/3,004 results were false for anti-SARS-CoV-2 IgG, 88/248 for IgA, and 34/124 for IgM. Regarding diagnostic sensitivity and specificity, substantial differences were found between the different assays used, as well as between certified and noncertified tests. For cutoff samples, a drop in the diagnostic sensitivity to 46.3% and high interlaboratory variability were observed. In general, this EQA highlights the current variability of anti-SARS-CoV-2 antibody detection, technical limitations with respect to cutoff samples, and the lack of harmonization of testing procedures. Recommendations are provided to help laboratories and manufacturers further improve the quality of anti-SARS-CoV-2 serological diagnostics.

Longitudinal assessment of anti-SARS-CoV-2 antibody dynamics and clinical features following convalescence from a COVID-19 infection.

INTRODUCTION: The longevity of antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the duration of immunity are current topics of major scientific interest. Antibody kinetics during the acute phase are well studied, whereas the long-term kinetics are yet to be determined, with contradictory results from the studies to date. Here, we present a longitudinal analysis of the serological responses to a SARS-CoV-2 infection following convalescence and the association with post-COVID syndrome (PCS). MATERIALS AND METHODS: A total of 237 serum samples were prospectively collected from 61 participants who had had a SARS-CoV-2 infection, which was confirmed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). For each participant, anti-nucleocapsid (N) and anti-spike subunit 1 receptor binding domain (RBD/S1) immunoglobulin (Ig) levels were regularly determined over a period of 8 months. COVID-19-associated symptoms were assessed using a standardized questionnaire at study entry and again after 6 months. RESULTS: Antibodies were detectable in 56 of the 61 participants. No substantial decline in anti-SARS-CoV-2 pan-Ig levels was observed for the duration of the follow-up period. Antibody levels correlated positively with the disease severity, body mass index, fever, and smoking status. It was found that 46.8% of the participants suffered from PCS, with olfactory and gustatory dysfunctions being the most commonly reported symptoms. CONCLUSION: The results demonstrate stable anti-SARS-CoV-2 antibody titers and thus may indicate a long-lasting immunity. The results are in line with recently published data and provide further insight concerning asymptomatic to mildly-affected patients, the association with clinical features, and the frequency of PCS.

Specificity testing by point prevalence as a simple assessment strategy using the Roche Elecsys® anti-SARS-CoV-2 immunoassay.

BACKGROUND: The detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mandatory for the diagnosis, retrospective assessment of disease progression, and correct evaluation of the current infection situation in the population. Many such assays have been launched by various manufacturers. Unfortunately, the new US Food and Drug Administration emergency use regulations have resulted in a situation where laboratories have to perform their own validation studies but many of these laboratories do not have the biobank needed to conduct the studies. METHODS: We introduce a method that allows institutions to quickly perform a verification study in a low-prevalence infection situation. As proof of concept, we used the Roche Elecsys® anti-SARS-CoV-2 electrochemiluminescence immunoassay and an SAP-based hospital information system. The Shenzhen YHLO Biotech IgM and IgG assay targeting other surface patterns was used as a confirmatory test. RESULTS: The Roche assay demonstrated a limit of detection of 0.069 cutoff index and successfully passed the performance validation according to Clinical and Laboratory Standards Institute EP15-A3. The study population of 627 inpatients has a median age of 64 years, and approximately 13% of the group were under intensive care at the respective time point. All patients included tested negative for SARS-CoV-2 infection by quantitative reverse transcription polymerase chain reaction (cobas® 6800, Roche, Mannheim, Germany). Only one false-positive result was obtained, resulting in a specificity for the Roche Elecsys anti-SARS-CoV-2 test of 99.84% and a negative predictive value of 99.98%. CONCLUSIONS: The anonymized use of residual material enables quick evaluation of anti-SARS-CoV-2 immunoassays, as shown in this work with the Roche Elecsys assay. Comparison of the control population with economic data makes it possible to validate the sampling set and therefore to determine diagnostic specificity. By use of the approach chosen, it was shown that the Roche test achieved very good results in terms of diagnostic specificity, reproducibility, and limit of detection.

Results of the first pilot external quality assessment (EQA) scheme for anti-SARS-CoV2-antibody testing.

Objectives Assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity is cornerstones in the fight against COVID-19 pandemic. For pandemic control, reliable assays for the detection of anti-SARS-CoV-2 antibodies are required. This pilot external quality assessment (EQA) scheme aimed to independently assess the participants' clinical performance of anti-SARS-CoV-2 testing, to identify shortcomings in clinical practice and to evaluate the suitability of the scheme format. Methods The EQA scheme consisted of eight serum samples with variable reactivity against SARS-CoV-2 intended for the analysis of anti-SARS-CoV-2 immunoglobulin (Ig)G, IgA, and IgM antibodies. Laboratories reported: (1) results for each sample and the respective method, (2) raw data from replicate testing of each sample. Results The 16 selected pilot EQA participants reported 294 interpreted results and 796 raw data results from replicate testing. The overall error rate for the anti-SARS-CoV-2 IgG, IgA, and IgM tests was 2.7, 6.9, and 16.7%, respectively. While the overall diagnostic specificity was rated as very high, sensitivity rates between 67 and 98% indicate considerable quality differences between the manufacturers, especially for IgA and IgM. Conclusions Even the results reported by the small number of participants indicate a very heterogeneous landscape of anti-SARS-CoV-2 serological testing. Differences of available tests and the individual performance of laboratories result in a success rate of 57.1% with one laboratory succeeding for all three antibody-classes. These results are an incentive for laboratories to participate in upcoming open EQA schemes that are needed to achieve a harmonization of test results and to improve serological testing.

Mass spectrometry based analytical quality assessment of serum and plasma specimens with patterns of endo- and exogenous peptides.

Background Inappropriate preanalytical sample handling is a major threat for any biomarker discovery approach. Blood specimens have a genuine proteolytic activity that leads to a time dependent decay of peptidic quality control markers (QCMs). The aim of this study was to identify QCMs for direct assessment of sample quality (DASQ) of serum and plasma specimens. Methods Serum and plasma specimens of healthy volunteers and tumor patients were spiked with two synthetic reporter peptides (exogenous QCMs) and aged under controlled conditions for up to 24 h. The proteolytic fragments of endogenous and exogenous QCMs were monitored for each time point by mass spectrometry (MS). The decay pattern of peptides was used for supervised classification of samples according to their respective preanalytical quality. Results The classification accuracy for fresh specimens (1 h) was 96% and 99% for serum and plasma specimens, respectively, when endo- and exogenous QCMs were used for the calculations. However, classification of older specimens was more difficult and overall classification accuracy decreased to 79%. Conclusions MALDI-TOF MS is a simple and robust method that can be used for DASQ of serum and plasma specimens in a high throughput manner. We propose DASQ as a fast and simple step that can be included in multicentric large-scale projects to ensure the homogeneity of sample quality.

Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA).

BACKGROUND: Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. METHODS: The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. RESULTS: Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. CONCLUSIONS: This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.

Thirteen Years of an International External Quality Assessment Scheme for Genotyping: Results and Recommendations.

BACKGROUND: Suboptimal laboratory procedures resulting in genotyping errors, misdiagnosis, or incorrect reporting bear greatly on a patient's health management, therapeutic decisions made on their behalf, and ultimate outcome. Participation in external quality assessment (EQA) is a key element of quality assurance in molecular genetic diagnostics. Therefore, the Reference Institute for Bioanalytics has tried for 13 years to improve the quality of genetic testing by offering an EQA for different clinically relevant sequence variations. METHODS: Within each of the biannual EQA schemes offered, up to 18 samples of lyophilized human genomic DNA were provided for up to 50 different molecular genetic tests. Laboratories were asked to use their routine procedures for genotyping. At least 2 expert peer assessors reviewed the final returns. Data from 2002 to 2014 were evaluated. RESULTS: In total, 82 462 reported results from 812 characterized samples were evaluated. Globally, the number of participants increased each year along with the number of sequence variations offered. The error rate decreased significantly over the years with an overall error rate of 1.44%. Additionally, a decreased error rate for samples repeated over time was noted. Interestingly, the error rate showed a high difference depending on the locus analyzed and the method used. CONCLUSIONS: Based on the evaluation of this long-term EQA scheme, various recommendations can be given to improve the quality of molecular genetic testing, such as the use of 2 different methods for genotyping. Furthermore, some methods are inappropriate for analysis of certain sequence variations.

Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks.

BACKGROUND: Biobanks are becoming increasingly important for assessment of disease risk as well as identification and validation of new diagnostic biomarkers and druggable targets. The validity of data obtained from biobanks is critically limited by the biomaterial quality of the biological samples. External quality assessment (EQA) programs suitable to comprehensively measure the biomaterial quality in archived materials are currently lacking. We report on quantitative assay designs for the analysis of both structural and functional integrity of DNAs that were applied in a first pilot EQA within the priority program on tumor tissue biobanking funded by the German Cancer Aid. METHODS: Participating biobanks isolated DNAs from a standardized set of 10 samples comprising sections of four different formalin-fixed paraffin-embedded tissues using their standard operating procedures. Isolated DNAs and analytical results were returned and analyzed centrally for nucleic acids yield, purity, fragmentation and amplificability at a quantitative level using dedicated assay designs. RESULTS: The amount of extracted DNA varied in isolates ranging between 1.5 µg and 25.8 µg. Quantification of DNA fragmentation and amplificability allowed to highlight considerable discrepancies in DNA quality. Amplicons yielded from the isolates of these identical EQA samples ranged from 105 to 411 bp suggesting differences between residual inhibitors of downstream enzymatic reactions. CONCLUSIONS: The quality of extraction of bioanalytes from biomaterial archives is heterogeneous even for stable biomolecules like DNA isolated with highly standardized methods. EQAs are appropriate tools to uncover strengths and weaknesses in biobanks in a systematic fashion. Biomaterial integrity is insufficiently reflected by standard methods, but needs to be assessed to improve biobank interoperability. Finally, our results also point towards the problem of measuring the quality of more delicate biomolecules like proteins or metabolites.

Diagnostic performance and cost effectiveness of measurements of plasma N-terminal pro brain natriuretic peptide in patients presenting with acute dyspnea or peripheral edema.

BACKGROUND: The purpose of this study was to determine the diagnostic power of a newly available assay for amino-terminal pro-brain natriuretic peptide (NT-proBNP) to identify patients with acute heart failure. In addition, the influence of initial NT-proBNP measurements on economic consequences, diagnostic procedures and staff involvement was evaluated. METHODS AND RESULTS: 401 patients presenting with acute dyspnea or peripheral edema in the emergency department were enrolled. NT-proBNP was measured after initial clinical evaluation. Clinical routine care and diagnostic assessment were blinded to NT-proBNP results. Two cardiologists independently validated the period of hospitalization, clinical examinations and medical therapies of each patient considering NT-proBNP results. The median NT-proBNP level among patients with acute congestive heart failure (CHF) (n=122) was 3497 pg/ml as compared to 320 pg/ml in patients without (n=279) (p<0.0001). An NT-proBNP cutoff level <300 pg/ml was optimal to rule out acute CHF (negative predictive value 96%; sensitivity 96%). NT-proBNP >or=300 pg/ml could strongly predict acute CHF when compared to patients' history or physical examination (odds ratio 9.5; p<0.0001) and diagnostic technical findings (odds ratio 14.7; p<0.05). In patients with NT-proBNP<300 pg/ml, 14% of the period of hospitalization could be saved, corresponding to savings of US $481 per patient. In addition, 9% of the number and time of staff involvement of clinical examinations and therapies could be saved, 10% of the costs of clinical examinations. Chest X-rays were saved in 34%, echocardiography in 9%. CONCLUSIONS: Measurement of NT-proBNP leads to multiple saving amounts and optimizes diagnostic pathways and resource allocation.

EQUAL-qual: a European program for external quality assessment of genomic DNA extraction and PCR amplification.

BACKGROUND: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. METHODS: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. RESULTS: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. CONCLUSIONS: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

Improvement of technical and analytical performance in DNA sequencing by external quality assessment-based molecular training.

BACKGROUND: From 2003 to 2005, the European Union supported the EQUAL-initiative to develop methodological external quality assessment (EQA) schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). As a relevant part of the EQUALseq program, a training course was held subsequent to the first EQA Program (EQAP1). The success of this course was reassessed in a 2nd EQUALseq round (EQAP2). METHODS: In September 2005, a 3-day training course took place. We invited 8 laboratories with below-average performance in EQAP1 to improve their methodological and analytical/proficiency skills by lectures and practical work. To compare the results of the pretraining and posttraining EQUALseq rounds, we distributed 2 samples used in the first EQUAL round, but this time we provided different oligonucleotide sets. We evaluated the results by means of a previously described scoring system. RESULTS: In EQAP2, 6 laboratories returned complete data sets, corresponding to an overall 14% of the 43 laboratories that had finished EQAP1. The scoring results for samples A (P=0.0025) and B (P=0.0125) demonstrated a significant improvement in EQAP2. Overall, a substantial improvement of technical and interpretative skills was demonstrated (P=0.0051). In general, the workshop experience was highly rated by the participants. CONCLUSIONS: Methodologic EQAPs in DNA sequencing are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, emphasizing the need for mandatory EQAPs. Training courses, together with 2nd-round reiterations, should be implemented into methodological EQAPs in molecular diagnostics to improve technical performance and proficiency in genetic testing.

EQUAL-quant: an international external quality assessment scheme for real-time PCR.

BACKGROUND: Quantitative gene expression analysis by real-time PCR is important in several diagnostic areas, such as the detection of minimum residual disease in leukemia and the prognostic assessment of cancer patients. To address quality assurance in this technically challenging area, the European Union (EU) has funded the EQUAL project to develop methodologic external quality assessment (EQA) relevant to diagnostic and research laboratories among the EU member states. We report here the results of the EQUAL-quant program, which assesses standards in the use of TaqMan probes, one of the most widely used assays in the implementation of real-time PCR. METHODS: The EQUAL-quant reagent set was developed to assess the technical execution of a standard TaqMan assay, including RNA extraction, reverse transcription, and real-time PCR quantification of target DNA copy number. RESULTS: The multidisciplinary EQA scheme included 137 participating laboratories from 29 countries. We demonstrated significant differences in performance among laboratories, with 20% of laboratories reporting at least one result lacking in precision and/or accuracy according to the statistical procedures described. No differences in performance were observed for the >10 different testing platforms used by the study participants. CONCLUSIONS: This EQA scheme demonstrated both the requirement and demand for external assessment of technical standards in real-time PCR. The reagent design and the statistical tools developed within this project will provide a benchmark for defining acceptable working standards in this emerging technology.