Evaluation of Serum Cryptococcal Antigen Testing Using Two Novel Semiquantitative Lateral Flow Assays in Persons with Cryptococcal Antigenemia

Abstract

Early cryptococcal disease can be detected via circulating antigen in blood before fulminant meningitis develops, when early antifungal therapy improves survival. Two semiquantitative cryptococcal antigen (CrAg) lateral flow assays (LFAs) have been developed, but their diagnostic performance has not been defined. Cryopreserved serum samples from HIV-infected Ugandans obtained as part of a prospective CrAg-screening cohort were tested in duplicate for CrAg by the CrAgSQ (IMMY) and CryptoPS (Biosynex) lateral flow assays. Case-controlled diagnostic performance was measured using the FDA-approved CrAg LFA (IMMY) as a reference standard via McNemar’s test. Of 99 serum samples tested, 57 were CrAg positive (CrAg+) by the CrAg LFA reference standard. By CrAgSQ, 57 were read as positive, with 98% sensitivity (56/57; 95% confidence interval [CI], 0.91 to 0.99) and 98% specificity (41/42; 95% CI, 0.88 to 0.99) (McNemar’s, P = 0.99). The sample with a false-negative result by CrAgSQ (n = 1) had a titer of <1:5, while the sample with a false-positive result (n = 1) yielded a 1+ result. By CryptoPS, 52 samples were read as positive, with 88% sensitivity (50/57; 95% CI, 0.76 to 0.95) and 95% specificity (40/42; 95% CI, 0.84 to 0.99) (McNemar’s, P = 0.18). The CryptoPS false-negative results included samples with titers of <1:5 (n = 1), 1:5 (n = 5), and 1:20 (n = 1), while samples with false-positive results by CryptoPS (n = 2) yielded Positive results. The CryptoPS assay missed 35% (7/20) of samples with CrAg LFA titers of ≤1:20. The new semiquantitative CrAg LFAs allow rapid estimation of titer levels in easy-to-perform platforms. The CrAgSQ demonstrated better qualitative sensitivity and specificity than the CryptoPS compared to the reference standard. The exact grading of the CrAgSQ results has some subjectivity, with interreader variability; however, qualitative reads were generally concordant for both assays.

Diagnostic Accuracy of the Biosynex CryptoPS Cryptococcal Antigen Semiquantitative Lateral Flow Assay in Patients with Advanced HIV Disease

“…The median CD4 count of samples in this study was lower than in our cohort (44 cells/µL versus 142 cells/µL), likely resulting in a higher median CrAg titer (1:160 versus 1:80) and contributing to slightly higher observed sensitivity (12). A second study of 99 cryopreserved serum samples from a Ugandan We found moderate specificity of the CryptoPS assay, in keeping with the previous study from Uganda but with greater estimated precision given our larger sample size (11). We carefully followed the…”

“…In a small study of patients with a CD4 <100 cells/µL in Cameroon, CryptoPS had good agreement with a commercial lateral flow assay (LFA) (IMMY diagnostics, Norman, Oklahoma, USA) and enzyme immunoassay (EIA) [Premier CrAg, Meridian Bioscience, Inc. Newtown, OH, USA], with a median reciprocal EIA titer >1:160 correlating with T2 band positivity; although the sample size which included on November 6, 2020 by guest http://jcm.asm.org/ Downloaded from just 14 serum CrAg-positive patients limited estimates of precision (10). A second study testing 99 archived serum samples selected from a cohort of HIV-positive Ugandans undergoing CrAg screening reported similar findings, with reasonable agreement between the CryptoPS and IMMY LFA and median CrAg titers of 1:320 in those with T2 band positivity (11); however, this study was also limited by a small sample size. CryptoPS was not tested on whole blood samples likely to be beneficial for point-of-care testing in either study; and the assay has not been evaluated prospectively and under routine care conditions.…”

“…The assay has a control band, qualitative T1 band for CrAg-positivity, and semi-quantitative T2 band indicating a "high" CrAg titer (Figure 1). Just two limited diagnostic evaluations have been reported to date (10,11). In a small study of patients with a CD4 <100 cells/µL in Cameroon, CryptoPS had good agreement with a commercial lateral flow assay (LFA) (IMMY diagnostics, Norman, Oklahoma, USA) and enzyme immunoassay (EIA) [Premier CrAg, Meridian Bioscience, Inc. Newtown, OH, USA], with a median reciprocal EIA titer >1:160 correlating with T2 band positivity; although the sample size which included on November 6, 2020 by guest http://jcm.asm.org/ Downloaded from just 14 serum CrAg-positive patients limited estimates of precision (10).…”

“…CrAg screening cohort found a CryptoPS assay sensitivity of 88% (50/57, 95%CI: 76%-95%) and specificity of 95% (40/42, 95%CI: 84%-99%) against the IMMY LFA; however, only 65% of samples with an IMMY LFA dilutional titer of ≤1:20 were positive on the CryptoPS assay(11).Similarly, in our prospective evaluation all (16/16) false-negative whole blood CryptoPS results were on samples with CrAg titers ≤1:160, 13 (81%) with titers ≤1:40. These patients may be at relatively low risk for progression to on November 6, 2020 by guest http://jcm.asm.org/ Downloaded from meningitis in the absence of pre-emptive fluconazole, assuming prompt initiation or continuation of effective ART (17).…”

Serology anno 2021—fungal infections: from invasive to chronic

“…Simultaneous detection of anti-mannan antibodies (AMA) improved sensitivity and specificity (83% and 86%) [45], respectively. Several test kits for the detection of mannan antigen (MA) of C. albicans and AMA show variable performance [46][47][48], see [55][56][57][58], which avoid serum pretreatment and support urinary cryptococcal antigen testing, which is useful for screening [59,60]. The LFA is a rapid procedure, there are various test kits on the market, see Table 5;…”