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1.
Clin Chim Acta ; 2022 Apr 09.
Article in English | MEDLINE | ID: covidwho-1778016

ABSTRACT

BACKGROUND: In the current COVID-19 pandemic, early and rapid diagnosis of potentially infected and contagious individuals enables containment of the disease through quarantine and contact tracing. The rapid global expansion of these diagnostic testing services raises questions concerning the current state of the art with regard to standardization of testing and quality assessment practices. The aim of this study was to provide a global overview of the test methods, laboratory procedures and quality assessment practices used for SARS-CoV-2 diagnostics. METHODS: The Molecular Diagnostics Committee of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC C-MD) initiated a survey among international laboratories performing molecular genetic detection of SARS-CoV-2. Questions on quality assurance, variant testing, sequencing and the transmission of findings were included in the survey. RESULTS: A total of 273 laboratories from 49 countries participated in the survey. The majority of the participating laboratories (92.2%) use reverse transcriptase polymerase chain reaction (RT-PCR). The majority of participating laboratories do not conduct testing to identify SARS CoV-2 variants. Participation in external quality assessment programs was reported by the majority of laboratories, however, 33.2% of the laboratories reported not participating in external quality assurance programmes. CONCLUSIONS: Based on the survey, molecular diagnostic methods for SARS-CoV-2 detection are clearly not standardized across different countries and laboratories. The survey found an array of responses in regard to sample preparation, collection, processing and reporting of results. This work suggests quality assurance is insufficiently performed by diagnostic laboratories conducting SARS-CoV-2 testing.

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-307520

ABSTRACT

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

3.
PLoS One ; 17(1): e0262656, 2022.
Article in English | MEDLINE | ID: covidwho-1638777

ABSTRACT

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Diagnostic Tests, Routine/methods , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Viral Load/methods , COVID-19/epidemiology , COVID-19/virology , Genes, Viral , Germany/epidemiology , Humans , Reproducibility of Results
4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-292638

ABSTRACT

Testing has been central to our response to the COVID-19 pandemic. However, the accuracy of testing relies on standards, including reference materials, proficiency testing schemes, and information and reporting guidelines. The use of standards is a simple, inexpensive, and effective method to ensure reliable test results that inform clinical and public health decisions. Here we describe the central role of standards during the COVID-19 pandemic, where they have enabled population-scale screening, genomic surveillance and measures of immune protection measures. Given these benefits, the Coronavirus Standards Working Group (CSWG) was formed to coordinate standards in SARS-CoV-2 testing. This network of scientists has developed best-practices, reference materials, and conducted proficiency studies to harmonize laboratory performance. We propose that this coordinated development of standards should be prioritized as a key early step in the public health response to future pandemics that is necessary for reliable, large-scale testing for infectious disease.

5.
Metabolism ; 126: 154922, 2022 01.
Article in English | MEDLINE | ID: covidwho-1482812

ABSTRACT

BACKGROUND: The global COVID-19 pandemic has led to extensive development in many fields, including the diagnosis of COVID-19 infection by mass spectrometry. The aim of this systematic review and meta-analysis was to assess the accuracy of mass spectrometry diagnostic tests developed so far, across a wide range of biological matrices, and additionally to assess risks of bias and applicability in studies published to date. METHOD: 23 retrospective observational cohort studies were included in the systematic review using the PRISMA-DTA framework, with a total of 2858 COVID-19 positive participants and 2544 controls. Risks of bias and applicability were assessed via a QUADAS-2 questionnaire. A meta-analysis was also performed focusing on sensitivity, specificity, diagnostic accuracy and Youden's Index, in addition to assessing heterogeneity. FINDINGS: Sensitivity averaged 0.87 in the studies reviewed herein (interquartile range 0.81-0.96) and specificity 0.88 (interquartile range 0.82-0.98), with an area under the receiver operating characteristic summary curve of 0.93. By subgroup, the best diagnostic results were achieved by viral proteomic analyses of nasopharyngeal swabs and metabolomic analyses of plasma and serum. The performance of other sampling matrices (breath, sebum, saliva) was less good, indicating that these protocols are currently insufficiently mature for clinical application. CONCLUSIONS: This systematic review and meta-analysis demonstrates the potential for mass spectrometry and 'omics in achieving accurate test results for COVID-19 diagnosis, but also highlights the need for further work to optimize and harmonize practice across laboratories before these methods can be translated to clinical applications.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Mass Spectrometry/methods , Humans , Sensitivity and Specificity
6.
Clin Chem ; 68(1): 153-162, 2021 12 30.
Article in English | MEDLINE | ID: covidwho-1462309

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.


Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19 , Nucleic Acids , Belgium , COVID-19/diagnosis , Humans , Nucleic Acids/analysis , RNA, Viral/analysis , Reproducibility of Results , Republic of Korea , SARS-CoV-2 , Sensitivity and Specificity , United Kingdom
7.
Methods ; 201: 5-14, 2022 May.
Article in English | MEDLINE | ID: covidwho-1373305

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an infectious, acute respiratory disease caused mainly by person-to-person transmission of the coronavirus SARS-CoV-2. Its emergence has caused a world-wide acute health crisis, intensified by the challenge of reliably identifying individuals likely to transmit the disease. Diagnosis is hampered by the many unknowns surrounding this disease, including those relating to infectious viral burden. This uncertainty is exacerbated by disagreement surrounding the clinical relevance of molecular testing using reverse transcription quantitative PCR (RT-qPCR) for the presence of viral RNA, most often based on the reporting of quantification cycles (Cq), which is also termed the cycle threshold (Ct) or crossing point (Cp). Despite it being common knowledge that Cqs are relative values varying according to a wide range of different parameters, there have been efforts to use them as though they were absolute units, with Cqs below an arbitrarily determined value, deemed to signify a positive result and those above, a negative one. Our results investigated the effects of a range of common variables on Cq values. These data include a detailed analysis of the effect of different carrier molecules on RNA extraction. The impact of sample matrix of buccal swabs and saliva on RNA extraction efficiency was demonstrated in RT-qPCR and the impact of potentially inhibiting compounds in urine along with bile salts were investigated in RT-digital PCR (RT-dPCR). The latter studies were performed such that the impact on the RT step could be separated from the PCR step. In this way, the RT was shown to be more susceptible to inhibitors than the PCR. Together, these studies demonstrate that the consequent variability of test results makes subjective Cq cut-off values unsuitable for the identification of infectious individuals. We also discuss the importance of using reliable control materials for accurate quantification and highlight the substantial role played by dPCR as a method for their development.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , RNA, Viral/analysis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/genetics , Sensitivity and Specificity
8.
J Clin Virol Plus ; 1(3): 100037, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1356293

ABSTRACT

Reverse transcriptase quantitative PCR (RT-qPCR) is the main diagnostic assay used to detect SARS-CoV-2 RNA in respiratory samples. RT-qPCR is performed by specifically targeting the viral genome using complementary oligonucleotides called primers and probes. This approach relies on prior knowledge of the genetic sequence of the target. Viral genetic variants with changes to the primer/probe binding region may reduce the performance of PCR assays and have the potential to cause assay failure. In this work we demonstrate how two single nucleotide variants (SNVs) altered the amplification curve of a diagnostic PCR targeting the Nucleocapsid (N) gene and illustrate how threshold setting can lead to false-negative results even where the variant sequence is amplified. We also describe how in silico analysis of SARS-CoV-2 genome sequences available in the COVID-19 Genomics UK Consortium (COG-UK) and GISAID databases was performed to predict the impact of sequence variation on the performance of 22 published PCR assays. The vast majority of published primer and probe sequences contain sequence mismatches with at least one SARS-CoV-2 lineage. We recommend that visual observation of amplification curves is included as part of laboratory quality procedures, even in high throughput settings where thresholds are set automatically and that in silico analysis is used to monitor the potential impact of new variants on established assays. Ideally comprehensive in silico analysis should be applied to guide selection of highly conserved genomic regions to target with future SARS-CoV-2 PCR assays.

9.
Int J Mol Sci ; 22(16)2021 Aug 13.
Article in English | MEDLINE | ID: covidwho-1354988

ABSTRACT

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , COVID-19/epidemiology , COVID-19/virology , Clinical Laboratory Techniques/methods , Humans , Pandemics , RNA, Viral/genetics , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics , Sensitivity and Specificity , Temperature
10.
J Virol Methods ; 295: 114215, 2021 09.
Article in English | MEDLINE | ID: covidwho-1275556

ABSTRACT

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/instrumentation , DNA Primers/genetics , Humans , Nose/virology , Pharynx/virology , Phosphoproteins/genetics , RNA, Viral/genetics , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Homology, Nucleic Acid
11.
Curr Opin Pulm Med ; 27(3): 155-162, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1114895

ABSTRACT

PURPOSE OF REVIEW: COVID-19 has put the in-vitro-diagnostic community under an unprecedented spotlight, with a global requirement for accurate SARS-CoV-2 tests. This review will outline technological responses to this need and the analytical considerations required for their translation to routine use. RECENT FINDINGS: SARS-CoV-2 diagnostic solutions directly detect the virus or measure host-derived surrogate markers of infection. With pressure upon supply chains for the 'traditional' molecular approaches, a wide variety of analytical tools spanning the molecular, serology, imaging and chemistry space are being developed, including high throughput solutions and simplified near-patient formats. SUMMARY: The unique genetic nature of SARS-CoV-2 means high analytical specificity is achievable by most diagnostic formats. However, clinical sensitivity assessment is complicated by wide discrepancies in analytical range and challenges associated with standardising these differences. When coupled with the acute nature of SARS-CoV-2 infection, reported precise metrics of test performance must be questioned. The response to SARS-CoV-2 has delivered considerable diagnostic innovation, but for a technology to be maximised, it must be demonstrably reproducible and fit for purpose. If novel diagnostic solutions for SARS-CoV-2 are to succeed, equally innovative mechanisms are needed to ensure widespread clinical and surveillance application, enabling agreed standards and metrics to ensure comparability.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Inventions , SARS-CoV-2 , COVID-19/virology , Humans , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
13.
Lancet Infect Dis ; 20(10): e251-e260, 2020 10.
Article in English | MEDLINE | ID: covidwho-693775

ABSTRACT

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.


Subject(s)
Metagenomics/methods , Metagenomics/statistics & numerical data , Computational Biology , Humans , Molecular Epidemiology , Research Design/standards
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