Background: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available. This study investigated digital PCR (dPCR) RMP delivery of traceability for NAAT external quality assessment (EQA). Methods: Three National Metrology Institutes (NMIs) applied reverse transcription (RT)-dPCR as a candidate RMP to estimate the RNA quantity in 32 independent severe acute respiratory syndrome coronavirus 2 materials. The results were combined to value assign the respective materials: 21 materials were used in 6 rounds of EQA over 17 months for 61 laboratories for COVID-19 testing results compared with reference values. Results: The agreement between the 3 NMIs showed <2-fold difference between laboratories. EQA laboratory reverse transcription quantitative PCR (RT-qPCR) values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, RT-qPCR differences were generally between 10- and 50-fold between laboratories. Conclusion: This work demonstrates how RT-dPCR can provide reference values for whole virus materials for NAAT quality assurance. RT-dPCR values guided EQA control material selection and provided EQA participants with traceability to RNA copy number delivered through the RMP. This approach can be used to support routine reference material use as well as to standardize quality assurance for NAATs where established reference materials are not available, such as in disease outbreaks.

Managing the transmission of antimicrobial resistance necessitates reliable methods for tracking outbreaks. We compared the performance of MALDI-TOF MS with orthogonal approaches for strain typing, including WGS and FTIR, for Acinetobacter baumannii isolates correlated with a health care-associated infection (HCAI) event.

Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy.

Precision medicine approaches rely on accurate somatic variant detection, where the DNA input into genomic workflows is a key variable. However, there are no gold standard methods for total DNA quantification. In this study, a pentaplex reference gene panel using digital PCR (dPCR) was developed as a candidate reference method. The multiplex approach was compared between two assay chemistries, applied to healthy donor genomic DNA and plasma cell-free DNA (cfDNA) to measure the ERBB2 (HER2) copy number variation in cancer cell line DNA. The multiplex approach demonstrated robust performance with the two assay chemistries, demonstrating comparable results and a wide dynamic range. Ratios of reference genes were close to the expected 1:1 in healthy samples; however, some small but significant differences (<1.2-fold) were observed in one of the five targets. Expanded relative measurement uncertainty was 12.1–19.8% for healthy gDNA and 9.2–25.2% for cfDNA. The multiplex approach afforded lower measurement uncertainty compared to the use of a single reference for total DNA quantification, which is an advantage for its potential use as a calibration method. It avoided potential biases in the application to CNV quantification of cancer samples, where cancer genome instability may be prominent.

Nucleic acid amplification tests including reverse transcription quantitative PCR (RT-qPCR) are used to detect RNA from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Standardized measurements of RNA can facilitate comparable performance of laboratory tests in the absence of existing reference measurement systems early on in a pandemic. Interlaboratory study CCQM P199b “SARS-CoV-2 RNA copy number quantification” was designed to test the fitness-for-purpose of developed candidate reference measurement procedures (RMPs) for SARS-CoV-2 genomic targets in purified RNA materials, and was conducted under the auspices of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) to evaluate the measurement comparability of national metrology institutes (NMIs) and designated institutes (DIs), thereby supporting international standardization. Twenty-one laboratories participated in CCQM P199b and were requested to report the RNA copy number concentration, expressed in number of copies per microliter, of the SARS-CoV-2 nucleocapsid ( N ) gene partial region (NC_045512.2: 28274-29239) and envelope ( E ) gene (NC_045512.2: 26245-26472) (optional measurements) in samples consisting of in vitro transcribed RNA or purified RNA from lentiviral constructs. Materials were provided in two categories: lower concentration (≈ (10 ¹ -10 ⁴ ) /μL in aqueous solution containing human RNA background) and high concentration (≈ 10 ⁹ /μL in aqueous solution without any other RNA background). For the measurement of N gene concentration in the lower concentration study materials, the majority of laboratories ( n = 17) used one-step reverse transcription-digital PCR (RT-dPCR), with three laboratories applying two-step RT-dPCR and one laboratory RT-qPCR. Sixteen laboratories submitted results for E gene concentration. Reproducibility (% CV or equivalent) for RT-dPCR ranged from 19 % to 31 %. Measurements of the high concentration study material by orthogonal methods (isotope dilution-mass spectrometry and single molecule flow cytometry) and a gravimetrically linked lower concentration material were in a good agreement, suggesting a lack of overall bias in RT-dPCR measurements. However methodological factors such as primer and probe (assay) sequences, RT-dPCR reagents and dPCR partition volume were found to be potential sources of interlaboratory variation which need to be controlled when applying this technique. This study demonstrates that the accuracy of RT-dPCR is fit-for-purpose as a RMP for viral RNA target quantification in purified RNA materials and highlights where metrological approaches such as the use of in vitro transcribed controls, orthogonal methods and measurement uncertainty evaluation can support standardization of molecular methods.