Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients

Abstract

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/μL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity.

Fig 1. Schematic of the prototype test workflow.

(1) Patient urine is passed through a filter to remove any large particulates and mixed with the Concentration Capture Reagent. (2) Urine and Concentration Capture Reagent are allowed to incubate at room temperature for 15min in order to capture any LAM in the sample. (3) The Concentration Capture Reagent is separated from the bulk urine by passing through the filter of an Autovial, discarding the urine, and it is washed with 1mL Wash Buffer, which is also passed through the filter to waste. (4) 300μL of Elution Buffer is added to the washed Concentration Capture Reagent and incubated for 5min to release any captured LAM; it is passed through the filter and instantly mixed with 35μL of Neutralization Buffer. (5) 150μL of this neutralized eluent is added to the LFA and results are read after 30min.

Fig 2. Flowchart of patient enrollment.

We identified 295 adults with positive Xpert Ultra results to screen for enrollment. Of these individuals 119 were able to be enrolled in the study. A random selection of 173 adults with negative Xpert Ultra results were also enrolled. Of the 173 enrolled Xpert Ultra negative patients 7 were subsequently diagnosed as TB cases by other microbiological methods.