Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients

Abstract

Background Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB. Materials and methods We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites. Results LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated. Conclusions LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test.

Diagnostic sensitivity of SILVAMP TB-LAM (FujiLAM) point-of-care urine assay for extra-pulmonary tuberculosis in people living with HIV

“…FujiLAM's excellent performance in those with mycobacteraemia suggests a mechanistic association between disease dissemination and urinary LAM. This finding is supported by our recent study that showed a good association between detection of LAM in urine and serum of TB patients, independent of HIV status [15]. However, even for patients with forms of disease such as pleural TB and TB meningitis that may be compartmentalised, FujiLAM had moderate sensitivity, which could add substantial benefit in these cases.…”

Pediatric Tuberculosis: The Impact of “Omics” on Diagnostics Development

“…Previous work from our team demonstrated the detection of urinary LAM at a maximal concentration of 350 pM in individuals without HIV co-infection using a sandwich immunoassay on an ultra-sensitive waveguide based optical biosensor [ 42 ]. The conclusions of this work are supported by a recent study using an improved chemiluminescence readout, with sensitivity and specificity of 93% and 97%, respectively [ 47 ]. These findings show that a more sensitive assay format is required in immunocompetent individuals.…”

“…However, LAM detection is not yet approved for use in diagnosis of HIV negative pediatric TB, likely because of the lower sensitivity of current diagnostic strategies. Researchers are working on the evaluation of the use of ultra-sensitive sensors in order to circumvent this problem [ 42 , 43 , 44 , 45 , 46 , 47 ]. The use of samples such as urine and blood favors application of this approach to children, and individuals with disseminated infection.…”

Point-Of-Care Urine LAM Tests for Tuberculosis Diagnosis: A Status Update

“…There are few studies reporting LAM concentrations in clinical specimens and direct comparisons between different sample types and assays are complicated by the absence of standardized LAM control materials, sample panels, and reference assays. Four recent studies used the same purified LAM material for calibration and similar antibody reagents for immunoassay-based LAM detection (though different detection platforms) and reported LAM concentrations in sputum [33], blood [34,35], and urine [36] in subjects with active pulmonary TB, allowing for a rough comparison of LAM concentration ranges. For sputum, Kawasaki and colleagues showed that an immunoassay with a cut-off of 15 pg/mL detected all smear-positive and 50% of smear-negative TB patients [33] and sputum LAM concentrations ranged from 15.4 pg/mL to 1,869,000 pg/mL (median 5512 pg/mL).…”

“…Serum LAM concentrations in this study were highest in HIV-positive/smear-positive patients (median 3.97 pg/mL). Broger and colleagues [34,36] developed sensitive immunoassays and compared serum LAM to urine LAM concentrations in matched samples from smear-positive TB patients. The serum assay detected LAM in 55% of smear-positive patients with concentrations of 6 pg/mL to 70,000 pg/mL but 45% of patients were below the cut-off of 6 pg/mL, suggesting a median concentration in serum of roughly 10 pg/mL.…”