Evaluation of a Novel Semi-quantitative Cryptococcal Antigen Lateral Flow Assay in Patients with Advanced HIV Disease.

Background: Higher cryptococcal antigen (CrAg) titers are strongly associated with mortality risk in individuals with HIV-associated cryptococcal disease. Rapid tests to quantify CrAg level may provide important prognostic information and enable treatment stratification.Methods: We performed a laboratory-based validation of the semi-quantitative IMMY CrAgSQ assay against the current gold-standard CrAg tests. We assessed diagnostic accuracy of the CrAgSQ in HIV-positive individuals undergoing CrAg screening; determined the relationship between CrAgSQ scores and dilutional CrAg titers; assessed inter-rater reliability; and determined clinical correlates of CrAgSQ scores.Results: A total of 872 plasma samples were tested using both CrAgSQ and conventional IMMY CrAg LFA tests; 692 sequential samples from HIV-positive individuals undergoing CrAg screening and an additional 180 known CrAg-positive plasma samples archived from prior studies. Inter-rater agreement in CrAgSQ reading was excellent (98.17% agreement, Cohen's Kappa 0.962, p<0.001). Using IMMY LFA as a reference standard, CrAgSQ was 93.0% sensitive (95% confidence interval [CI] 80.9%-98.5%) and 93.8% specific (95%CI 91.7%-95.6%). After reclassification of discordant results using CrAg enzyme immunoassay testing, sensitivity was 98.1% (95%CI 90.1%-100%), and specificity 95.8% (95%CI 99.1%- 100%). Median CrAg titers were 1:10 (IQR 1:5-1:20) in the CrAgSQ1+ category; 1:40 (IQR 1:20-1:80) in the CrAgSQ2+ category; 1:640 (IQR 1:160-1:2560) in the CrAgSQ3+ category; and 1:5120 (IQR 1:2560-1:30720) in the CrAgSQ4+ category. Increasing CrAgSQ scores were strongly associated with 10-week mortality.Conclusions: The CrAgSQ test had high sensitivity and specificity compared to the IMMY CrAg LFA test and provided CrAg scores associated with both conventional CrAg titers and clinical outcomes.