A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests

The current COVID-19 pandemic has triggered an accelerated pace in all research domains, including reliable diagnostics methodology. Molecular diagnostics of the virus and its presence in biological samples relies on the RT-PCR method, the most used and validated worldwide. Nonconventional tests with improved parameters that are in the development stages will be presented, such as droplet digital PCR or CRISPR-based assays. These molecular tests were followed by rapid antigen testing along with the development of antibody tests, whether based on ELISA platform or on a chemiluminescent microparticle immunoassay. Less-conventional methods of testing antibodies (e.g., lateral flow immunoassay) are presented as well. Left somewhere in the backstage of COVID-19 research, immune cells and, furthermore, immune memory cells, are gaining the spotlight, more so in the vaccination context. Recently, methodologies using flow-cytometry evaluate circulating immune cells in infected/recovered patients. The appearance of new virus variants has triggered a surge for tests improvement. As the pandemic has entered an ongoing or postvaccination era, all methodologies that are used to monitor public health focus on diagnostic strategies and this review points out where gaps should be filled in both clinical and research settings.

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External quality assessment for molecular diagnostic laboratories in Belgium: Can we improve it?

Accreditation and Quality Assurance ◽

10.1007/s00769-019-01410-x ◽

2019 ◽

Vol 25 (1) ◽

pp. 39-49

Author(s):

Kelly Dufraing ◽

Els Lierman ◽

Anne Vankeerberghen ◽

Sabine Franke ◽

Els Dequeker

Keyword(s):

Quality Assessment ◽

Performance Monitoring ◽

External Quality Assessment ◽

Molecular Diagnostic ◽

Real Patient ◽

External Quality ◽

Molecular Tests ◽

Clinical Biology ◽

Eqa Schemes ◽

Selection Of

AbstractExternal quality assessment (EQA) is an essential part of performance monitoring for molecular laboratories. At the moment, a national law regulates participation in EQA schemes for clinical biology and pathology in Belgium. This study aimed (1) to get insights on how laboratories organize their EQA participation, (2) to poll satisfaction with the current situation (selection of EQA programs in advance by a governmental body), (3) to provide guidance for choosing the most relevant EQA provider and (4) to propose a new model for national performance monitoring. A survey was sent to Belgian laboratories performing molecular tests in the field of microbiology, hematology and pathology with (1) general questions on how they select an EQA provider and (2) their satisfaction of each provider. In total, 25 molecular laboratories [microbiology (N = 13), hematology (N = 8) and pathology (N = 4)] from 14 different hospitals completed the survey regarding their EQA organization. All three laboratory groups indicated to prefer EQA schemes using real patient materials as well as those with varying targets and concentrations. For molecular microbiology and hematology, schemes with a syndromic approach are sought. Since annual participation in EQA becomes burdensome in most laboratories, this paper also offers a risk-based strategy for determining the participation frequency. Based on the needs of Belgian laboratories, three proposals were made: (1) for the proper selection of an EQA scheme, (2) for determining the minimal participation frequency and (3) for the national organization of EQA schemes.

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2017-0283 ◽

2018 ◽

Vol 56 (2) ◽

pp. 220-228 ◽

Cited By ~ 29

Author(s):

Verena Haselmann ◽

Parviz Ahmad-Nejad ◽

Wolf J. Geilenkeuser ◽

Angelika Duda ◽

Merle Gabor ◽

...

Keyword(s):

Quality Assessment ◽

Error Rate ◽

External Quality Assessment ◽

Digital Pcr ◽

Laboratory Diagnostics ◽

External Quality ◽

External Quality Assessment Scheme ◽

Genotyping Platform ◽

Edta Plasma ◽

Circulating Tumour Dna

Abstract 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.

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The performance of human cytomegalovirus digital PCR Reference Measurement Procedure in seven External Quality Assessment schemes over four years

Methods ◽

10.1016/j.ymeth.2021.03.016 ◽

2021 ◽

Author(s):

Mojca Milavec ◽

Jernej Pavšič ◽

Alexandra Bogožalec Košir ◽

Gerwyn M. Jones ◽

Denise M. O'Sullivan ◽

...

Keyword(s):

Quality Assessment ◽

Human Cytomegalovirus ◽

External Quality Assessment ◽

Digital Pcr ◽

Measurement Procedure ◽

External Quality ◽

Reference Measurement ◽

Reference Measurement Procedure

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Droplet Digital PCR – A Superior Complementary Technique for SARS-CoV-2 Detection

Asian Journal of Biology ◽

10.9734/ajob/2020/v9i430092 ◽

2020 ◽

pp. 12-15

Author(s):

I. M. Hussaini ◽

S. Gide ◽

B. Musa ◽

M. A. Sulaiman ◽

A. Usman

Keyword(s):

Sensitivity And Specificity ◽

False Negative ◽

Standard Technique ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Reference Standard ◽

Rt Pcr ◽

Negative Results ◽

False Negative Results ◽

Current Reference Standard

Accurate and timely SARS-CoV-2 detection in suspected persons is crucial in the fight against its spread. Many techniques have been developed to meet up with the continuously growing demand, however some of these techniques lack the required accuracy, sensitivity and specificity. The current reference standard technique for SARS-CoV-2 detection is RT-PCR, but studies have shown that false-negative results are inevitable and data can be non-reproducible when samples and primers are not appropriately verified and validated. Droplet digital PCR (ddPCR) is a newly introduced technique that performs precise nucleic acid quantification. Researchers have evaluated the efficacy of ddPCR and the technique has shown promising results even in specimens with low viral load. ddPCR has shown increased accuracy, precision, sensitivity and specificity. Furthermore, it is less affected by annealing and amplification inhibitors. This suggests that ddPCR can be used as a complementary detection technique especially in convalescent cases.

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Presence of SARS-CoV-2 Nucleoprotein in Cardiac Tissues of Donors with Negative COVID-19 Molecular Tests

Diagnostics ◽

10.3390/diagnostics11040731 ◽

2021 ◽

Vol 11 (4) ◽

pp. 731

Author(s):

Gianluca Lorenzo Perrucci ◽

Elena Sommariva ◽

Veronica Ricci ◽

Paola Songia ◽

Yuri D’Alessandra ◽

...

Keyword(s):

Tissue Bank ◽

Digital Pcr ◽

Tissue Donation ◽

Vein Wall ◽

Cardiac Tissues ◽

Reverse Transcription Pcr ◽

Molecular Tests ◽

Negative Results ◽

Italian Regions ◽

Tissue Specimens

The 2019 Coronavirus disease (COVID-19) outbreak had detrimental effects on essential medical services such as organ and tissue donation. Lombardy, one of the most active Italian regions in organ/tissue procurement, has been strongly affected by the COVID-19 pandemic. To date, data concerning the risk of SARS-CoV-2 transmission after tissue transplantation are controversial. Here, we aimed to evaluate the presence/absence of SARS-CoV-2 in different cardiac tissues eligible for transplantation obtained from Lombard donors. We used cardiovascular tissues from eight donors potentially suitable for pulmonary valve transplantation. All donor subjects involved in the study returned negative results for the SARS-CoV-2 RNA molecular tests (quantitative real-time reverse-transcription PCR, qRT-PCR, and chip-based digital PCR) in nasopharyngeal swabs (NPS) or bronchoalveolar lavage (BAL). None of the eight donors included in this study revealed the presence of the SARS-CoV-2 viral genome. However, evaluation of the protein content of pulmonary vein wall (PVW) tissue revealed variable levels of SARS-CoV-2 nucleoprotein signal in all donors. Our study demonstrated for the first time, to the best of our knowledge, that viral nucleoprotein but not viral RNA was present in the examined tissue bank specimens, suggesting the need for caution and in-depth investigations on implantable tissue specimens collected during the COVID-19 pandemic period.

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Prospective Evaluation of Molecular Assays for Diagnosis of Vaginitis

Journal of Clinical Microbiology ◽

10.1128/jcm.01264-19 ◽

2019 ◽

Vol 58 (1) ◽

Cited By ~ 1

Author(s):

Sandra S. Richter ◽

Joshua Otiso ◽

Oluwatosin J. Goje ◽

Sherilynn Vogel ◽

Jory Aebly ◽

...

Keyword(s):

Gardnerella Vaginalis ◽

Reference Standard ◽

Content Type ◽

Molecular Assays ◽

Molecular Tests ◽

Normal Microbiota ◽

The Impact ◽

Specific Reagent ◽

Atopobium Vaginae

ABSTRACT Molecular tests to diagnose conditions involving the disruption of normal microbiota are difficult to optimize. Using Nugent-scored Gram stain (NS) as the reference standard, we evaluated the performance of 3 molecular assays for the diagnosis of bacterial vaginosis (BV) and examined the impact of an incremental increase in bacterial targets. The BD Affirm assay includes a DNA probe for Gardnerella vaginalis, the Hologic transcription-mediated amplification (TMA) analyte-specific reagent (ASR) assay adds a second Lactobacillus sp. target, and the recently cleared in vitro diagnostic use (IVD) Aptima BV assay includes a third target (Atopobium vaginae). The diagnosis of vulvovaginal candidiasis (VVC) by the Affirm and Candida vaginitis Hologic TMA ASR assays was assessed using microscopy for yeast as the reference standard. From May to December 2018, 111 women with vaginitis symptoms prompting the clinician to order an Affirm test were enrolled with informed consent for the collection of additional specimens. Clinicians accurately predicted BV as the most likely diagnosis for 71% of the 45 patients with BV. Coinfection occurred in 13.5% of patients. For BV, the specificity of the Aptima IVD assay (86.3%) was higher than the Affirm assay (60.6%, P = 0.0002), but sensitivities were not significantly different. For VVC, the sensitivity of the ASR assay (100%) was higher than Affirm (75.9%; P = 0.023) and the specificity of the Affirm assay (98.8%) was higher than the ASR assay (86.6%; P = 0.004).

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