Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.

Implementation and Extended Evaluation of the Euroimmun Anti-SARS-CoV-2 IgG Assay and Its Contribution to the United Kingdom's COVID-19 Public Health Response.

In March 2020, the Rare and Imported Pathogens Laboratory at the UK Health Security Agency (UKHSA) (formerly Public Health England [PHE]) Porton Down, was tasked by the Department of Health and Social Care with setting up a national surveillance laboratory facility to study SARS-CoV-2 antibody responses and population-level sero-surveillance in response to the growing SARS-CoV-2 outbreak. In the following 12 months, the laboratory tested more than 160,000 samples, facilitating a wide range of research and informing UKHSA, DHSC, and UK government policy. Here we describe the implementation and use of the Euroimmun anti-SARS-CoV-2 IgG assay and provide an extended evaluation of its performance. We present a markedly improved overall sensitivity of 91.39% (≥14 days 92.74%, ≥21 days 93.59%) compared to our small-scale early study, and a specificity of 98.56%. In addition, we detail extended characteristics of the Euroimmun assay: intra- and interassay precision, correlation to neutralization, and assay linearity. IMPORTANCE Serology assays have been useful in determining those with previous SARS-CoV-2 infection in a wide range of research and serosurveillance projects. However, assays vary in their sensitivity at detecting SARS-CoV-2 antibodies. Here, we detail an extended evaluation and characterization of the Euroimmun anti-SARS-CoV-2 IgG assay, one that has been widely used within the United Kingdom on over 160,000 samples to date.

Validation of Differentially Expressed Immune Biomarkers in Latent and Active Tuberculosis by Real-Time PCR.

Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB), pulmonary (PTB)) and latent TB (LTBI) infection remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. Whole blood mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new TB diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs).