Abstract
ABSTRACTDirect pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.
Highlights
Tuberculosis (TB) remains a major global health problem, and in 2015 it had the highest mortality of any infectious disease worldwide
Since antigen 85 proteins contain motifs that interact with fibronectin and such complexes are thought to form during TB infection [40], we focused on slow off-rate modified aptamer (SOMAmer) that can bind antigen 85 protein both in free form and in a complex with fibronectin (Table S3)
Lipoarabinomannan (LAM) diagnostic tests for urine specimens have been developed, they tend to suffer from poor sensitivity except in TB patients coinfected with HIV [46]
Summary
Tuberculosis (TB) remains a major global health problem, and in 2015 it had the highest mortality of any infectious disease worldwide. M. tuberculosis SOMAmer Reagents pulmonary TB represents the majority of TB cases and is transmitted via aerosols from people with active pulmonary disease, a high diagnostic priority is to determine those with active TB to enable rapid treatment and reduce disease transmission. High-priority diagnostic needs for which specific target product profiles (TPPs) have been defined include a non-sputum-based biomarker test for all forms of TB and a simple, low-cost triage test for use by first-contact care providers as a rule-out test [3,4,5]. SOMAmers are a new class of synthetic reagents, in some ways similar to monoclonal antibodies, and are used in proteomic applications where high sensitivity and specificity is needed. M. tuberculosis protein targets included extracellular, cell surface-associated, and intracellular factors that may be circulating in TB patients and may be detectable with a highly sensitive and specific assay. MPT64 and MPT51 are major extracellular antigens and once were considered to Journal of Clinical Microbiology jcm.asm.org 3073
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