Blood cryptococcal antigen (CrAg) titres > 160 are associated with concurrent subclinical cryptococcal meningitis (CM). When lumbar puncture (LP) is not immediately available in a CrAg screening programme, semi-quantitative CrAg assays may provide risk stratification for CM. Two semi-quantitative assays (SQ [Immuno-Mycologics, Norman, OK, USA] and CryptoPS [Biosynex, Strasbourg, France]) were evaluated against a qualitative lateral flow assay (LFA) using 194 plasma samples from a cohort of HIV-seropositive individuals with CD4 counts < 100 cells/µL. We compared SQ and CryptoPS results to titres for LFA-positive samples. Among patients with LP, we examined the association between semi-quantitative CrAg results and CM. We used a Cox proportional hazards model to determine the association between SQ score and mortality. Of 194 participants, 60 (31%) had positive LFA results, of whom 41 (68%) had a titre of ≤ 160 and 19 (32%) a titre > 160. Fifty individuals with antigenaemia had an LP; a clinically-useful SQ score that identified all ten cases of subclinical CM was ≥ 3 (100% sensitivity, 55% specificity). Patients with an SQ score of 3 or 4 also had a 2.2-fold increased adjusted hazards of 6-month mortality (95% CI, 0.79-6.34; p = 0.13) versus those with score of < 3. Nine of ten patients with subclinical CM had a strong-positive CryptoPS result versus 10/40 without subclinical CM (p<0.001). Semi-quantitative assays offered a sensitive though not specific means of gauging the risk of concurrent CM in this patient population. Lay Abstract We evaluated two single-step laboratory tests that can quantify the amount of cryptococcal antigen in plasma of patients with advanced HIV disease and could thus gauge the risk of concurrent cryptococcal meningitis and subsequent mortality. These tests are not a substitute for a lumbar puncture.

Two cases of cryptococcal meningitis went undetected by a cryptococcal antigen (CrAg) lateral flow assay on blood in a reflex CrAg screen-and-treat programme in South Africa, although Cryptococcus neoformans was identified by culturing the cerebrospinal fluid specimens. Further investigations into these discordant diagnostic results included multilocus sequence typing (which showed no mutations in the CAP59 gene) and transmission electron microscopy using a capsule-staining protocol (which revealed a >50% reduction in capsular material in both cases, relative to a control culture). A multi-disciplinary approach for resolving discordant diagnostic test results is recommended.

Objective Reflex cryptococcal antigen (CrAg) screening of blood specimens with a CD4 count of <100 cells/µL was performed at 45 South African CD4 laboratories using a lateral flow assay (LFA). Our objective was to evaluate the reliability of routine LFA results through comparative interlaboratory testing. Methods All CrAg-positive and a selected number of CrAg-negative samples from the CD4 laboratories were retested at paired microbiology laboratories using the same LFA. Samples with discordant results were tested at a reference laboratory, using the LFA (with CrAg titers). Results During interlaboratory testing, 12,502 samples were retested, with 93 (0.7%) discordant results and a between-laboratory agreement of 99.3% (Cohen’s kappa, 0.98). The proportion of retested samples with discordant results ranged from 0.17% to 5.31% per laboratory pair (median 0.28%), with 3 reporting >3% of results as discordant. Conclusion Routine CrAg screening results were reliable, with <1% of samples having discordant results, mainly due to interpretation and transcription errors.