Evaluation of multi-antigen serological screening for active tuberculosis among people living with HIV

Abstract

Better triage tests for screening tuberculosis (TB) disease are needed for people living with HIV (PLHIV). We performed the first evaluation of a previously-validated 8-antigen serological panel to screen PLHIV for pulmonary TB in Kampala, Uganda. We selected a random 1:1 sample with and without TB (defined by sputum culture) from a cohort of PLHIV initiating antiretroviral therapy. We used a multiplex microbead immunoassay and an ensemble machine learning classifier to determine the area under the receiver operating characteristic curve (AUC) for Ag85A, Ag85B, Ag85C, Rv0934-P38, Rv3881, Rv3841-BfrB, Rv3873, and Rv2878c. We then assessed the performance with the addition of four TB-specific antigens ESAT-6, CFP-10, Rv1980-MPT64, and Rv2031-HSPX, and every antigen combination. Of 262 participants (median CD4 cell-count 152 cells/μL [IQR 65-279]), 138 (53%) had culture-confirmed TB. The 8-antigen panel had an AUC of 0.53 (95% CI 0.40-0.66), and the additional 4 antigens did not improve performance (AUC 0.51, 95% CI 0.39-0.64). When sensitivity was restricted to ≥90% for the 8- and 12-antigen panel, specificity was 2.2% (95% CI 0-17.7%) and 8.1% (95% CI 0-23.9%), respectively. A three-antigen combination (Rv0934-P38, Ag85A, and Rv2031-HSPX) outperformed both panels, with an AUC of 0.60 (95% CI 0.48-0.73), 90% sensitivity (95% CI 78.2-96.7%) and 29.7% specificity (95% CI 15.9-47%). The multi-antigen panels did not achieve the target accuracy for a TB triage test among PLHIV. We identified a new combination that improved performance for TB screening in an HIV-positive sample compared to an existing serological panel in Uganda, and suggests an approach to identify novel antigen combinations specifically for screening TB in PLHIV.

Fig 1. Flowchart of participants.

a. 292 selected based on specimen availability, complete data, and cost ART: antiretroviral therapy; BSA: Bovine Serum Albumin; PLHIV: People living with HIV; SD: Standard Deviation; TB: tuberculosis.

Fig 2. Boxplots of antibody responses to TB antigens by TB status.

With a multiplex microbead immunoassay, we compared the log median fluorescence intensity (MFI) among HIV positive adults with and without TB. A star next to the antigen indicates a significant difference by permutation testing (p-value < 0.05).

Fig 3. Receiver Operating Characteristic (ROC) curves for TB antigen panels.

We generated ROC curves for TB diagnosis among people living with HIV using the following index serological panels: (A) The previously reported 8-antigen panel; (B) The expanded 12-antigen panel; (C) The best performing antigen combination that included Rv0934-P38, Ag85A, and Rv2031-HSPX. The curve in black is the ROC curve on the test set, with 500 bootstrapped ROC curves in grey. The red lines indicate the minimum target performance for a TB triage test with True Positive Rate of 0.9 and False Positive Rate (1- Specificity) of 0.3.

Fig 4. Comparison of antigen panel Receiver Operating Characteristic (ROC) curves.

We overlaid the ROC curves of the three multi-antigen serological panels to compare performance and the area under the curve (AUC). The black line is the 8-antigen panel, red is the 12-antigen panel, and blue is the best performing 3-antigen panel (Rv0934-P38, Ag85A, and Rv2031-HSPX). The grey lines indicate the minimum target performance for a TB triage test with True Positive Rate of 0.9 and False Positive Rate (1- Specificity) of 0.3.