Point-of-care testing: state-of-the art and perspectives.

Point-of-care testing (POCT) is becoming an increasingly popular way to perform laboratory tests closer to the patient. This option has several recognized advantages, such as accessibility, portability, speed, convenience, ease of use, ever-growing test panels, lower cumulative healthcare costs when used within appropriate clinical pathways, better patient empowerment and engagement, and reduction of certain pre-analytical errors, especially those related to specimen transportation. On the other hand, POCT also poses some limitations and risks, namely the risk of lower accuracy and reliability compared to traditional laboratory tests, quality control and connectivity issues, high dependence on operators (with varying levels of expertise or training), challenges related to patient data management, higher costs per individual test, regulatory and compliance issues such as the need for appropriate validation prior to clinical use (especially for rapid diagnostic tests; RDTs), as well as additional preanalytical sources of error that may remain undetected in this type of testing, which is usually based on whole blood samples (i.e., presence of interfering substances, clotting, hemolysis, etc.). There is no doubt that POCT is a breakthrough innovation in laboratory medicine, but the discussion on its appropriate use requires further debate and initiatives. This collective opinion paper, composed of abstracts of the lectures presented at the two-day expert meeting "Point-Of-Care-Testing: State of the Art and Perspective" (Venice, April 4-5, 2024), aims to provide a thoughtful overview of the state-of-the-art in POCT, its current applications, advantages and potential limitations, as well as some interesting reflections on the future perspectives of this particular field of laboratory medicine.

Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

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 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.

Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics.

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.

Screening for gene doping transgenes in horses via the use of massively parallel sequencing.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using Digital PCR.

The use of standardized components and processes in engineering underpins the design-build-test model, and the engineering of biological systems is no different. Substantial efforts to standardize both the components and the methods to validate the engineered biological systems is ongoing. This study has developed a panel of control materials encoding the commonly used reporter genes GFP and RFP as DNA or RNA molecules. Each panel contained up to six samples with increasingly small copy number differences between the two reporter genes that ranged from 1- to 2-fold differences. These copy number differences represent the magnitude of changes that may need to be measured to validate an engineered system. Using digital PCR (dPCR), we demonstrated that it is possible to quantify changes in both gene and gene transcript numbers both within and between samples down to 1.05-fold. We corroborated these findings using a simple gene circuit within a bacterial model to demonstrate that dPCR was able to precisely identify small changes in gene expression of two transcripts in response to promoter stimulation. Finally, we used our findings to highlight sources of error that can contributed to the measurement uncertainty in the measurement of small ratios in biological systems. Together, the development of a panel of control materials and validation of a high accuracy method for the measurement of small changes in gene expression, this study can contribute to the engineering biology "toolkit" of methods and materials to support the current standardization efforts.

Metrological framework to support accurate, reliable, and reproducible nucleic acid measurements.

Nucleic acid analysis is used in many areas of life sciences such as medicine, food safety, and environmental monitoring. Accurate, reliable measurements of nucleic acids are crucial for maximum impact, yet users are often unaware of the global metrological infrastructure that exists to support these measurements. In this work, we describe international efforts to improve nucleic acid analysis, with a focus on the Nucleic Acid Analysis Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM). The NAWG is an international group dedicated to improving the global comparability of nucleic acid measurements; its primary focus is to support the development and maintenance of measurement capabilities and the dissemination of measurement services from its members: the National Metrology Institutes (NMIs) and Designated Institutes (DIs). These NMIs and DIs provide DNA and RNA measurement services developed in response to the needs of their stakeholders. The NAWG members have conducted cutting edge work over the last 20 years, demonstrating the ability to support the reliability, comparability, and traceability of nucleic acid measurement results in a variety of sectors.

Monkeypox: another test for PCR.

Monkeypox was declared a public health emergency of international concern by the World Health Organization (WHO) on 23 July 2022. Between 1 January and 23 July 2022, 16,016 laboratory confirmed cases of monkeypox and five deaths were reported to WHO from 75 countries on all continents. Public health authorities are proactively identifying cases and tracing their contacts to contain its spread. As with COVID-19, PCR is the only method capable of being deployed at sufficient speed to provide timely feedback on any public health interventions. However, at this point, there is little information on how those PCR assays are being standardised between laboratories. A likely reason is that testing is still limited on a global scale and that detection, not quantification, of monkeypox virus DNA is the main clinical requirement. Yet we should not be complacent about PCR performance. As testing requirements increase rapidly and specimens become more diverse, it would be prudent to ensure PCR accuracy from the outset to support harmonisation and ease regulatory conformance. Lessons from COVID-19 should aid implementation with appropriate material, documentary and methodological standards offering dynamic mechanisms to ensure testing that most accurately guides public health decisions.

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.

COVID-19 new diagnostics development: novel detection methods for SARS-CoV-2 infection and considerations for their translation to routine use.

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.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

RT-qPCR Diagnostics: The "Drosten" SARS-CoV-2 Assay Paradigm.

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.

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020.

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

Report of the 2019 NIST-FDA workshop on standards for next generation sequencing detection of viral adventitious agents in biologics and biomanufacturing.

Adventitious virus testing assures product safety by demonstrating the absence of viruses that could be unintentionally introduced during the manufacturing process. The capabilities of next-generation sequencing (NGS) for broad virus detection in biologics have been demonstrated by the detection of known and novel viruses that were previously missed using the recommended routine assays for adventitious agent testing. A meeting was co-organized by the National Institute of Standards and Technology and the U.S. Food and Drug Administration on September 18-19, 2019 in Gaithersburg, Maryland, USA, to facilitate standardization of NGS technologies for applications of adventitious virus testing in biologics. The goal was to assess the currently used standards for virus detection by NGS and their public availability, and to identify additional needs for different types of reference materials and standards (natural and synthetic). The meeting focused on the NGS processes from sample preparation through sequencing but did not thoroughly cover bioinformatics, since this was considered to be the topic of a separate meeting.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

G6PD deficiency alleles in a malaria-endemic region in the Western Brazilian Amazon.

BACKGROUND: Plasmodium vivax parasites are the predominant cause of malaria infections in the Brazilian Amazon. Infected individuals are treated with primaquine, which can induce haemolytic anaemia in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals and may lead to severe and fatal complications. This X-linked disorder is distributed globally and is caused by allelic variants with a geographical distribution that closely reflects populations exposed historically to endemic malaria. In Brazil, few studies have reported the frequency of G6PD deficiency (G6PDd) present in malaria-endemic areas. This is particularly important, as G6PDd screening is not currently performed before primaquine treatment. The aim of this study was to determine the prevalence of G6PDd in the region of Alto do Juruá, in the Western Brazilian Amazon, an area characterized by a high prevalence of P. vivax infection. METHODS: Five-hundred and sixteen male volunteers were screened for G6PDd using the fluorescence spot test (Beutler test) and CareStart™ G6PD Biosensor system. Demographic and clinical-epidemiological data were acquired through an individual interview. To assess the genetic basis of G6PDd, 24 SNPs were genotyped using the Kompetitive Allele Specific PCR assay. RESULTS: Twenty-three (4.5%) individuals were G6PDd. No association was found between G6PDd and the number of malaria cases. An increased risk of reported haemolysis symptoms and blood transfusions was evident among the G6PDd individuals. Twenty-two individuals had the G6PDd A(-) variant and one the G6PD A(+) variant. The Mediterranean variant was not present. Apart from one polymorphism, almost all SNPs were monomorphic or with low frequencies (0-0.04%). No differences were detected among ethnic groups. CONCLUSIONS: The data indicates that ~1/23 males from the Alto do Juruá could be G6PD deficient and at risk of haemolytic anaemia if treated with primaquine. G6PD A(-) is the most frequent deficiency allele in this population. These results concur with reported G6PDd in other regions in Brazil. Routine G6PDd screening to personalize primaquine administration should be considered, particularly as complete treatment of patients with vivax malaria using chloroquine and primaquine, is crucial for malaria elimination.

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.