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•RT-dPCR capable of reproducible quantification of HIV-1 RNA without calibration.•Viral RNA extraction kits evaluated to measuring viral RNA from whole virus.•Calibration independent ...procedure used to participate in quality assessment scheme.•Findings demonstrating RT-dPCR could act as a reference measurement procedure.•Potential role to support routine calibration of HIV-1 RNA measurement highlighted.
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.
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•dPCR methods for most common ganciclovir resistance mutations in HCMV developed.•These methods enable both detection and quantification.•Developed methods were compared to a ...reference method.•Methods were tested in external quality assessment schemes in medical laboratories.
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.
•The terms “point-of-care testing (POCT)” and “near-patient testing (NPT)” and their implications in EU, US and Australian legislation are discussed.•A classification scheme for laboratory test ...systems according to their technical complexity and required operator competence is provided and applied for SARS-CoV-2 associated analyses.•A simple method for evaluating the independence of operator activities of NPT/POCT assays is presented.
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.
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.
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.
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.
•dPCR protocol investigated measuring hCMV whole virus panels over four years.•First reference measurement procedure for human pathogenic DNA virus quantification.•High reproducibility and trueness ...confirmed Method for value assignment of viral materials for calibration and quality assurance.•Method has the potential for harmonization of routine hCMV load testing.•Method has the potential to harmonization for routine hCMV load testing.
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.
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.
While the solubility of native α-, β-, γ-cyclodextrins (CDs) in water rises with temperature, the opposite is true for their methylated derivatives (mCDs; per-dimethylated β-CD and per-trimethylated ...γ-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 (D2O), 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.
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