Driving consistency: CEPI-Centralized Laboratory Network's conversion factor initiative for SARS-CoV-2 clinical assays used for efficacy assessment of COVID vaccines.

To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.

Dengue: A review of laboratory diagnostics in the vaccine age.

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas.Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes.Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses.

Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7-10 days post-infection (DPI) for ASF and between 10-21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.

[Laboratory diagnostics for vasculitis beyond antineutrophil cytoplasmatic autoantibodies]. / Labordiagnostik bei Vaskulitiden jenseits von antineutrophilen zytoplasmatischen Autoantikörpern.

The diagnosis of systemic vasculitis (SV) is a major clinical challenge due to the very different forms of presentation and requires an interdisciplinary approach. Targeted laboratory diagnostics support making the diagnosis, differential diagnosis and classification and are also a key component in the detection of active organ manifestations and treatment complications. The basic laboratory tests include the erythrocyte sedimentation rate (ESR), C­reactive protein (CRP), blood count, serum creatinine, urinalysis, specific autoantibodies, complement, immunoglobulins, cryoglobulins and hepatitis B and C serology. Antineutrophil cytoplasmic autoantibodies (ANCA), antiglomerular basement membrane antibodies (anti-GBM antibodies) and anti-C1q antibodies are valuable laboratory markers for the diagnosis of the various forms of small vessel vasculitis. There are no specific laboratory tests for the diagnosis of medium and large vessel vasculitis. Despite advances in our understanding of the pathogenesis of vasculitis, no biomarkers have yet been identified that can be reliably used to guide treatment or that are useful in distinguishing vasculitis from other inflammatory diseases such as infections or treatment complications.

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Clinical validation of RCSMS: A rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva.

Peru's holds the highest COVID death rate per capita worldwide. Key to this outcome is the lack of robust, rapid, and accurate molecular tests to circumvent the elevated costs and logistics of SARS-CoV-2 detection via RT-qPCR. To facilitate massive and timely COVID-19 testing in rural and socioeconomically deprived contexts, we implemented and validated RCSMS, a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva. RCSMS uses the power of CRISPR-Cas technology and lateral flow strips to easily visualize the presence of SARS-CoV-2 even in laboratories with limited equipment. We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Notably, RCSMS performed outstandingly in a clinical validation done with 352 patients from two hospitals in Lima, detecting as low as 50 viral copies per 10 µl reaction in 40 min, with sensitivity and specificity of 96.5% and 99.0%, respectively, relative to RT-qPCR. The negative and positive predicted values obtained from this field validation indicate that RCSMS can be confidently deployed in both high and low prevalence settings. Like other CRISPR-Cas-based biosensors, RCSMS can be easily reprogrammed for the detection of new SARS-CoV-2 variants. We conclude that RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in Peru and other low- and middle-income countries with precarious healthcare systems.

Comparative evaluation of assay performance for SARS-CoV-2 detection in animal oral samples, lung homogenates, and phosphate-buffered saline using the TaqPath COVID-19 Combo kit.

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates.

Evaluation of the EasyNAT SARS-CoV-2 assay PCR test for the diagnosis of SARS-CoV-2 infection.

Reverse transcription polymerase chain reaction (RT-PCR) tests are commonly utilized in commercial settings but pose challenges due to labor-intensive procedures and extended response times during peak demand. In contrast, real-time fluorescence and isothermal amplification assays using Crossing Priming Amplification (CPA) offer faster genetic material analysis, eliminate subjectivity, and require less manipulation and personnel training. This study aimed to validate the EasyNAT SARS-CoV-2 Assay, a diagnostic kit based on CPA, using oral and nasopharyngeal samples. The EasyNAT kit was compared to the Xpert Xpress SARS-CoV-2 kit, evaluating 873 samples obtained during routine analysis at the Microbiology Laboratory of the Hospital Costa del Sol (Marbella, Spain). The overall sensitivity and specificity for the EasyNAT SARS-CoV-2 Assay were 79.1% (95%CI 74.5-83.7) and 99.5% (95%CI 98.7-100), respectively; with, validity index of 91.9%, positive predictive value of 98.9%, negative predictive value of 88.9%, positive likelihood ratio of 144.5, negative likelihood ratio of 0.21 and a total Youden Index of 0.79. Notably, sensitivity improved in fresh samples (91.4%), along with a high Youden Index (0.91). The EasyNAT SARS-CoV-2 Assay achieved a higher percentage of concordance in positive samples with Xpert Xpress SARS-CoV-2 when analyzing cycle threshold (Ct) intervals below 30 compared to intervals equal or greater than 30, and demons. In conclusion, the EasyNAT SARS-CoV-2 Assay demonstrated high sensitivity and agreement with Xpert Xpress SARS-CoV-2, particularly in fresh samples or when the signal was detected at Ct intervals below 30, indicating higher viral loads. This makes it suitable for rapid screening in various settings, including those with limited access to conventional molecular laboratory setting.

A Novel Approach for Routinely Assessing Laboratory Sigma Metrics for a Broad Range of Automated Assays.

BACKGROUND: Sigma metrics have been adapted for the clinical laboratory to incorporate observed accuracy, precision, and total error allowed. The higher the Sigma level for a process, the better performance that process has. A limitation of studies assessing Sigma metrics is that they are performed on a small number of well-controlled systems. METHODS: An algorithm was developed to extract QC data and derive the Sigma metric for 115 analytes from sites connected to the QuidelOrtho E-Connectivity® database. The median of these results was then used to derive the Sigma metric for each assay. RESULTS: In this analysis, 79 out of 115 (68.7%) of the assays assessed achieved 6 Sigma or better and 98 out of 115 (85.2%) achieved 5 Sigma or better. CONCLUSIONS: This study has demonstrated a methodology that can be used to condense Sigma metrics from hundreds of analyzers into a single metric of assay quality. Because these analyzers are running in working laboratories from around the world, this analysis can serve as a baseline for understanding the assay performance achieved in the presence of variabilities such as lab-to-lab, instrument-to-instrument, material handling, environmental conditions, and reagent lot. The significant number of assays demonstrating high Sigma levels did so despite this variation. The ability of the methods reported here to include hundreds of analyzers represents a novel approach for assessing Sigma metrics in clinical laboratories.

The importance of using WHO International Standards to harmonise SARS-CoV-2 serological assays.

The COVID-19 pandemic led to the rapid development of tests to diagnose SARS-CoV-2 infection and ascertain the prevalence of infection, along with the formulation of various treatments and vaccines. Globally, over 220 anti-SARS-CoV-2 serological assays have been developed for laboratory use, and many of these assays are currently used to assess immune responses against SARS-CoV-2. However, because these assays were independently developed by different manufacturers with different target antigens, immunoglobulin detection, technologies, and data reporting approaches, the results are not directly comparable, making it challenging to draw conclusions regarding immune responses at the population level. With deficiencies in assay validation, standardisation, and harmonisation, the inability to use and compare large datasets is becoming a major issue as serological data continue to increase. To help in addressing this issue, WHO established the first International Standard for the anti-SARS-CoV-2 immunoglobulin in late 2020. In this Personal View, we define the WHO International Standard for the anti-SARS-CoV-2 immunoglobulin, summarise the uses of primary versus secondary serology standards, recommend the use of such standards for data harmonisation, and list guidance and resources for using serology standards to improve data comparability.

The diagnostic accuracy of the ID NOW COVID-19 point of care test in acute hospital admissions.

BACKGROUND: Prompt identification of patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection on admission to hospital is crucial to ensuring initiation of appropriate treatment, optimising infection control and maintaining patient flow. The Abbott ID NOW™ COVID-19 assay (ID NOW) is a point-of-care, isothermal nucleic acid amplification test, capable of producing a result within minutes, potentially placing it as an invaluable tool in helping to control the coronavirus-disease 2019 (COVID-19) pandemic. OBJECTIVES: To evaluate the diagnostic accuracy of ID NOW in acute hospital admissions. STUDY DESIGN: A prospective approach to data collection was undertaken in consecutive patients with ID NOW and Hologic Aptima™ SARS-CoV-2 transcription-mediated amplification assay (Aptima TMA) results, across three hospitals in the south-west of England between 1st March and 30th September 2021. A nasal swab was taken for ID NOW and a combined nose and throat swab for Aptima TMA. Measures of diagnostic accuracy were calculated for ID NOW against Aptima TMA. This study was conducted during a period of alpha and delta strain predominance. RESULTS: 19,698 ID NOW assays were performed, of which 12,821 had an Aptima TMA assay performed within 24 hours. ID NOW had sensitivity of 85.2 % (95 % CI, 82.2-87.9) and specificity of 99.6 % (95 % CI, 99.4-99.7) compared with the reference assay. The overall PPV was 91.0 % (95 % CI, 88.5-93.0) and the overall NPV was 99.3 % (95 % CI, 99.1-99.4). CONCLUSIONS: ID NOW offers a valid diagnostic tool to detect SARS-CoV-2, performing comparably to a reference laboratory-based assay which takes longer to provide results.

An Open One-Step RT-qPCR for SARS-CoV-2 detection.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment.

A two-step strategy combining assisted benchmark testing (entry controls) and External Quality Assessments (EQAs) with blinded simulated clinical specimens to enhance and maintain the quality of nucleic acid amplification testing was developed. This strategy was successfully applied to 71 diagnostic laboratories in The Netherlands when upscaling the national diagnostic capacity during the SARS-CoV-2 pandemic. The availability of benchmark testing in combination with advice for improvement substantially enhanced the quality of the laboratory testing procedures for SARS-CoV-2 detection. The three subsequent EQA rounds demonstrated high quality testing with regard to specificity (99.6% correctly identified) and sensitivity (93.3% correctly identified). Even with the implementation of novel assays, changing workflows using diverse equipment and a high degree of assay heterogeneity, the overall high quality was maintained using this two-step strategy. We show that in contrast to the limited value of Cq value for absolute proxies of viral load, these Cq values can, in combination with metadata on strategies and techniques, provide valuable information for laboratories to improve their procedures. In conclusion, our two-step strategy (preparation phase followed by a series of EQAs) is a rapid and flexible system capable of scaling, improving, and maintaining high quality diagnostics even in a rapidly evolving (e.g. pandemic) situation.

SARS-CoV-2 infections among pregnant women, 2020, Finland-Cross-testing of neutralization assays.

We studied the development of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) pandemic in southern Finland in 2020 and evaluated the performance of two surrogate immunoassays for the detection of neutralizing antibodies (NAbs). The data set consisted of 12 000 retrospectively collected samples from pregnant women in their first trimester throughout 2020. All the samples were initially screened for immunoglobulin G (IgG) with SARS-CoV-2 spike antibody assay (EIM-S1, Euroimmun) followed by confirmation with nucleocapsid antibody assay (Architect SARS-CoV-2, Abbott). Samples that were reactive (positive or borderline) with both assays were subjected to testing with commercial surrogate immunoassays of NeutraLISA (EIM) and cPassTM (GenScript Biotech Corporation) by using pseudoneutralization assay (PNAbA) as a golden standard. No seropositive cases were detected between January and March. Between April and December, IgG (EIM-S1 and Abbott positive) and NAb (PNAbA positive) seroprevalences were between 0.4% and 1.4%. NeutraLISA showed 90% and cPass 55% concordant results with PNAbA among PNAbA negative samples and 49% and 92% among PNAbA positive samples giving NeutraLISA better specificity but lower sensitivity than cPass. To conclude, seroprevalence in pregnant women reflected that of the general population but the variability of the performance of serological protocols needs to be taken into account in inter-study comparison.

Overview of the different methods for RNA preparation in COVID-19 diagnosis process during the pandemic.

The COVID-19 pandemic brought to light the impact of a widespread disease on various aspects of human relationships, communities, and economies. One notable consequence was the increased demand for diagnostic kits, laboratory reagents, and personal health equipment. This surge in testing capacity worldwide led to shortages in the supply of essential items, including RNA extraction kits, which are crucial for detecting COVID-19 infections. To address this scarcity, researchers have proposed alternative and cost-effective strategies for RNA extraction, utilizing both chemical and physical solutions and extraction-free methods. These approaches aim to alleviate the challenges associated with the overwhelming number of tests being conducted in laboratories. The purpose of this review is intends to provide a comprehensive summary of the various kit-free RNA extraction methods available for COVID-19 diagnosis during the pandemic.

Home testing for SARS-CoV-2 and impact on surveillance in New York State.

PURPOSE: To determine the distribution of diagnosed SARS-CoV-2 infections by testing modality (at-home rapid antigen [home tests] versus laboratory-based tests in clinical settings [clinical tests]), assess factors associated with clinical testing, and estimate the true total number of diagnosed infections in New York State (NYS). METHODS: We conducted an online survey among NYS residents and analyzed data from 1012 adults and 246 children with diagnosed infection July 13-December 7, 2022. Weighted descriptive and logistic regression model analyses were conducted. Weighted percentages and prevalence ratios by testing modality were generated. The percent of infections diagnosed by clinical tests via survey data were synthesized with daily lab-reported results to estimate the total number of diagnosed SARS-CoV-2 infections in NYS July 1-December 31, 2022. RESULTS: Over 70% of SARS-CoV-2 infections in NYS during the study period were diagnosed exclusively with home tests. Diagnosis with a clinical test was associated with age, race/ethnicity, and region among adults, and sex, age, and education among children. We estimate 4.1 million NYS residents had diagnosed SARS-CoV-2 infection July 1-December 31, 2022, compared to 1.1 million infections reported over the same period. CONCLUSIONS: Most SARS-CoV-2 infections in NYS were diagnosed exclusively with home tests. Surveillance metrics using laboratory-based reporting data underestimate diagnosed infections.

APS calculator: a data-driven tool for setting outcome-based analytical performance specifications for measurement uncertainty using specific clinical requirements and population data.

OBJECTIVES: According to ISO 15189:2022, analytical performance specifications (APS) should relate to intended clinical use and impact on patient care. Therefore, we aimed to develop a web application for laboratory professionals to calculate APS based on a simulation of the impact of measurement uncertainty (MU) on the outcome using the chosen decision limits, agreement thresholds, and data of the population of interest. METHODS: We developed the "APS Calculator" allowing users to upload and select data of concern, specify decision limits and agreement thresholds, and conduct simulations to determine APS for MU. The simulation involved categorizing original measurand concentrations, generating measured (simulated) results by introducing different degrees of MU, and recategorizing measured concentrations based on clinical decision limits and acceptable clinical misclassification rates. The agreements between original and simulated result categories were assessed, and values that met or exceeded user-specified agreement thresholds that set goals for the between-category agreement were considered acceptable. The application generates contour plots of agreement rates and corresponding MU values. We tested the application using National Health and Nutrition Examination Survey data, with decision limits from relevant guidelines. RESULTS: We determined APS for MU of six measurands (blood total hemoglobin, plasma fasting glucose, serum total and high-density lipoprotein cholesterol, triglycerides, and total folate) to demonstrate the potential of the application to generate APS. CONCLUSIONS: The developed data-driven web application offers a flexible tool for laboratory professionals to calculate APS for MU using their chosen decision limits and agreement thresholds, and the data of the population of interest.