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

Abstract

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.

Screening of extra-pulmonary tuberculosis signature genes and analysis of correlation with immune cells

“…According to Shahin Ranjbar et al (24), IFITM3 overexpression inhibits the growth of Mycobacterium tuberculosis growth in these cell types, whereas knockdown of IFITM3 expression reduction signi cantly promotes Mycobacterium tuberculosis in monocytes, providing evidence for the involvement of antiviral IFITM3 in the restriction of Mycobacterium tuberculosis. Good diagnostic performance has also been demonstrated by IFITM3 in studies of biomarkers of pulmonary tuberculosis (25). In addition, several other studies have reported the correlation of IFITM3 with Alzheimer's disease (26) and some tumors (27).…”

Host blood protein biomarkers to screen for Tuberculosis disease: a systematic review and meta-analysis

“…Table 1 shows the performance of each marker and the summary of study quality. Eighteen markers met TPP in HIV-negative patients: CALCOCO2 (21), CD14 (22), CRP (23), Ferritin (24), GBP1 (21), HO-1 (25, 26), IFIT3 (21), IFITM3 (21), IP-10 (18), MMP-1 (19), OPN (27), PD-L1 (21), SAA (25), SAMD9L (21), SELL (22), SNX10 (21), TIMP-2 (19), and TIMP-4 (28). All of these studies had a high overall risk of bias due to the use of LTBI or healthy control groups in the absence of patients with ORD.…”

“…A combination of HO-1 and MMP-1 using participants with LTBI as the comparator group was reported separately by sites in Brazil and India (26). A three-marker signature of CALCOCO2, IFITM3, and SAMD9L tested lysed whole blood with ELISA, comparing TB cases in India to asymptomatic contacts from India and the United Kingdom (21). The four-marker signature of CLEC3B, ECM1, IP-10, and SELL used Luminex to test plasma samples from TB cases and LTBI controls in Madagascar (22).…”

COVID-19 and Tuberculosis: Mathematical Modeling of Infection Spread Taking into Account Reduced Screening

“…These tests show less effectiveness in comparison with IGRA tests [23,44]. In this connection, the search for new or additional biomarkers for TB diagnosis with greater information content continues [50,51].…”