Correlation of SARS-CoV-2 RNA and nucleocapsid concentrations in samples used in INSTAND external quality assessment schemes.

OBJECTIVE: In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021. The absolute N gene concentration was measured using reverse transcriptase digital PCR (RT-dPCR) as reference value. Moreover, the performance of five commercial ELISA tests using an EQA inactivated SARS-CoV-2 sample at different concentrations was assessed on the basis of these reference values. RESULTS: Quantitative ELISA and RT-dPCR results showed a good correlation between SARS-CoV-2 NCap antigen and RNA concentration, but this correlation varies among SARS-CoV-2 isolates. A direct correlation between SARS-CoV-2 NCap antigen concentration and genome concentration should not be generally assumed. CONCLUSION: Further correlation studies between SARS-CoV-2 RNA and NCap antigen concentrations are needed, particularly in clinical samples and for emerging SARS-CoV-2 variants, to support the monitoring and improvement of antigen testing.

Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.

The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 106 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.

Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.

BACKGROUND: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic. MATERIALS AND METHODS: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection. RESULTS: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results. CONCLUSION: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined.

Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.

During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers.

Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.

BACKGROUND: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests. METHODS: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level. RESULTS: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each. CONCLUSION: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.

Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.

Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms. Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units. Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]. Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

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.

The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log10 to +0.8 log10, the majority of dPCR results were between -0.2 log10 to +0.2 log10. Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing.

An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1.

Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.

The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

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.

The Comparative Clinical Performance of Four SARS-CoV-2 Rapid Antigen Tests and Their Correlation to Infectivity In Vitro.

Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8-82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.

Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirus.

Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir.

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

BACKGROUND: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. METHOD: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. RESULTS: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. CONCLUSIONS: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

Native and methylated cyclodextrins with positive and negative solubility coefficients in water studied by SAXS and SANS.

While the solubility of native alpha-, beta-, gamma-cyclodextrins (CDs) in water rises with temperature, the opposite is true for their methylated derivatives (mCDs; per-dimethylated beta-CD and per-trimethylated gamma-CD). The mCDs are well-soluble in cold water and crystallize upon heating, which we associate with the hydrophobic effect. To study the hydrophobic effect and hydration of CDs and mCDs dissolved in water (D 2O), we performed small-angle X-ray and neutron scattering (SAXS and SANS) measurements. The experimental scattering curves were put on absolute scale and compared to scattering curves calculated from crystal structures using the cube method. The results of the comparison indicate that (i) in solution, CDs and mCDs are in monomeric form, (ii) van der Waals and solute excluded volumes can be related by introducing a shell of a thickness that correlates with the solute's structure and solute-water interactions, and (iii) the SAXS curves calculated under the assumption of a uniform distribution of electron density in the solute molecules agree with experimental ones for CDs, but not for mCDs. The temperature and concentration dependence of SAXS curves is significant for mCDs and weak for CDs and is discussed in terms of solute-solute interactions. Specifically, these interactions become more attractive in solutions of mCDs with increasing temperature, concentration, or both, in accord with mCDs' negative temperature coefficient of solubility in water.