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

High cryptococcal antigen (CrAg) titers in blood are associated with subclinical meningitis and mortality in CrAg-positive individuals with advanced HIV disease (AHD). We evaluated a novel semiquantitative lateral flow assay (LFA), CryptoPS, that may be able to identify individuals with high CrAg titers in a cohort of AHD patients undergoing CrAg screening. In a prospective cohort of patients with AHD (CD4 cell count, ≤200/μl) receiving CD4 count testing, whole blood was tested for CrAg by CryptoPS and the IMMY LFA; the two assays were conducted by two different operators, each blind to the results of the other assay.

C ryptococcal meningitis (CM) remains a major cause of mortality in persons living with HIV (PLWH), causing an estimated 15% of HIV-related deaths worldwide (1). In individuals with advanced HIV disease (AHD), detection of cryptococcal antigen (CrAg) in the blood has been shown to precede the development of CM (2,3). CrAg screening of patients with AHD starting antiretroviral therapy (ART), with targeted preemptive fluconazole for CrAg-positive patients, has been shown to reduce incident CM and all-cause mortality (4) and is recommended by the World Health Organization (WHO) for those starting or reinitiating ART with a CD4 T-cell count of Ͻ200/l (5).
Preemptive fluconazole therapy for CrAg-positive individuals reduces, but does not completely eliminate, excess mortality (6); as many as one-third of asymptomatic CrAg-positive patients with AHD have disseminated central nervous system (CNS) disease and may require more-potent antifungal regimens for effective treatment (5)(6)(7). Higher CrAg titers (Ͼ1:160) are associated with an increased risk of CNS disease and death in CrAg-positive individuals (7)(8)(9). Titers can be assessed through serial dilution using commercial assays, but this requires extended labor and additional costs, making it impractical in resource-limited settings. Semiquantitative CrAg assays may provide a simpler means of stratifying risk for CrAg-positive patients with AHD and guiding differentiated treatment strategies.
CryptoPS (Biosynex, Strasbourg, France) is a semiquantitative immunochromatographic assay that detects all Cryptococcus neoformans/Cryptococcus gattii serotypes and provides results within 10 min. The assay has a control band, a qualitative T1 band for CrAg positivity, and a semiquantitative T2 band indicating a "high" CrAg titer ( Fig.  1). Just two limited diagnostic evaluations have been reported to date (10,11). In a small study of patients with CD4 cell counts of Ͻ100/l in Cameroon, CryptoPS had good agreement with a commercial lateral flow assay (LFA) (IMMY Diagnostics, Norman, OK, USA) and an enzyme immunoassay (EIA) (Premier CrAg; Meridian Bioscience, Inc., Newtown, OH, USA), with a median reciprocal EIA titer of Ͼ1:160 correlating with T2 band positivity, although the sample size, which included 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 for 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. We investigated the diagnostic performance of the CryptoPS assay in a prospective CrAg screening cohort in Botswana, evaluating test accuracy (sensitivity, specificity, positive predictive value [PPV], and negative predictive value [NPV]) against a validated reference standard. To determine titer cutoffs for the CryptoPS T2 band (high titers), we compared CryptoPS readings with IMMY CrAg titer results by serial dilution.

MATERIALS AND METHODS
From January 2018 to January 2019, we performed real-time CrAg testing on EDTA whole-blood samples from a sequential cohort of individuals with CD4 cell counts of Յ200/l undergoing routine reflex CrAg screening at the Botswana-Harvard HIV Reference Laboratory (BHHRL). The BHHRL performs all CD4 testing for 27 ART clinics and a central referral hospital located around Gaborone. The full cohort has been described in detail elsewhere (12); briefly, CD4 T-cell count testing was performed using flow cytometry (FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, USA); residual EDTA blood from sequential samples with CD4 cell counts of Յ200/l was screened for CrAg in real time using the IMMY LFA (IMMY, Norman, OK, USA) as a validated reference standard (13,14). CryptoPS testing was performed by a second trained operator blind to the IMMY LFA results. Results were interpreted by visual reading as negative (only the control band was visualized), T1 band positive (with the control and T1 bands visualized by the operator), or T2 band positive (control, T1, and T2 bands visualized), as per manufacturer's instructions (Fig. 1).
Since the CryptoPS is not registered for diagnostic use in Botswana, all clinical management decisions were based on IMMY LFA results. IMMY LFA-positive patients were managed according to the Botswana National HIV Management Guidelines (15). Individuals who tested positive by CryptoPS but negative by the IMMY LFA were contacted by the study team for retesting with the IMMY LFA and were monitored for 3 months in order to be evaluated for the development of incident CM, but they did not receive antifungal therapy unless the repeat IMMY LFA results were positive. Three-month outcomes in these individuals with positive CryptoPS results but negative IMMY LFA results were assessed through telephone contact, review of a national electronic medical records system, review of clinical notes, or further contact with clinical providers as needed.
At the end of the study period, IMMY LFA titers were determined by serial dilution of plasma samples stored at Ϫ80°C. To enable further validation of the CryptoPS semiquantitative results, we used both CryptoPS and the IMMY LFA to test an additional collection of 141 archived known CrAg-positive plasma samples from (i) patients in a CrAg screening cohort from 2015 to 2016 (8) and (ii) patients with CM enrolled in a completed phase II clinical trial evaluating induction therapies (16). All CrAg testing was performed by laboratory scientists blind to previous testing results.
Finally, to better characterize samples with discordant CryptoPS and IMMY LFA test results, we retested all discordant samples with Meridian's CrAg EIA (Meridian Bioscience, Cincinnati, OH) and IMMY's EIA (IMMY, Norman, OK) at the National Institute for Communicable Diseases (NICD) in Johannesburg, South Africa. This study was approved by institutional review boards at the Botswana Ministry of Health and Wellness, the University of Botswana, Princess Marina Hospital, and the University of Pennsylvania.
Statistical analysis. In the prospective CrAg screening cohort, we determined the sensitivity, specificity, PPV, and NPV of the CryptoPS assay with 95% confidence intervals (95% CI) using the IMMY LFA assay as a reference standard. Using all CrAg-positive samples from the prospective screening cohort, plus the additional archived CrAg-positive samples, we evaluated the median and interquartile range (IQR) of dilutional IMMY LFA CrAg titer values in samples with CryptoPS that were T1 band and T2 band positive, and we displayed the results graphically.
Laboratory findings, including the results of cryptococcal EIA testing, were described for samples with discordant CryptoPS-LFA results, and clinical outcomes were reported for individuals with negative IMMY LFA but positive CryptoPS results. Analyses were performed using Stata, version 16 (College Station, TX, USA).
The CryptoPS assay was positive in 55/916 (6.0% [95% CI, 4.6 to 7.7%]) of samples; 25/55 (45.5%) were IMMY LFA positive and were classified as "true positives" (18 T1  Table 3. The 30 "false-positive" CryptoPS samples were from 29 patients (one had two tests); 79% (23/29) received repeat IMMY LFA testing, of whom 100% (23/23) were CrAg negative. Most (97% [28/29]) were on ART at the time of CrAg screening, and none received preemptive fluconazole. None (0/29) were diagnosed with cryptococcal meningitis in the 3 months following CD4 testing and reflex CrAg screening, and 28/29 were alive at the completion of follow-up. One patient died 5 days after the date of CD4 testing after presenting to hospital with a clinical diagnosis of a lower respiratory tract infection; no central nervous system infection was suspected, and the patient did not receive a lumbar puncture as part of his diagnostic evaluation. The patient's CryptoPS result was T1 band positive, with negative IMMY LFA, IMMY EIA, and Meridian EIA results.  Given the relatively low sensitivity and specificity of the CryptoPS assay on EDTA whole blood, we repeated CryptoPS testing on frozen plasma samples after study completion (with operators blind to the previous results), which yielded results very similar to those of real-time whole blood testing for these specimens (see Table S1 in the supplemental material). We also repeated the analysis restricted to individuals with CD4 cell counts of Յ100/l; in this subgroup, the sensitivity of CryptoPS was 83.3% (95% CI, 58.6 to 96.4%), and its specificity was 96.5% (95% CI, 93.5 to 98.4%) (Table S2).

DISCUSSION
In this prospective evaluation of the novel semiquantitative CryptoPS CrAg LFA for HIV-seropositive individuals with CD4 cell counts of Յ200/l who were undergoing CrAg screening, we found suboptimal assay sensitivity of CryptoPS compared to an established CrAg LFA (IMMY). The lower sensitivity of CryptoPS was exclusively due to false-negative test results on blood samples with low CrAg titers; all samples with titers of Ͼ1:160 were detected by the assay.
Our study adds to two small prior diagnostic validation studies of the CryptoPS assay. A study of stored serum samples from Cameroon found a test sensitivity of 78% (11/14) against the IMMY LFA (10). The median CD4 cell count of samples in this study was lower than that in our cohort (44 cells/l versus 142 cells/l), 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 CrAg screening cohort found a CryptoPS assay sensitivity of 88% (50/57 [95% CI, 76% to 95%]) and a specificity of 95% (40/42) (95% CI, 84% to 99%) against the IMMY LFA; however, only 65% of samples with an IMMY LFA dilutional titer of Յ1:20 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 of Յ1:160, 13 of which (81%) had titers of Յ1:40. These patients may be at relatively low risk for progression to meningitis in the absence of preemptive fluconazole, assuming prompt initiation or continuation of effective ART (17). While the CryptoPS assay had suboptimal overall sensitivity, the T2 band performed well in discriminating samples that had high CrAg titers with greater risk of meningitis and/or failure of preemptive fluconazole therapy (6), with 79/84 (94%) of T2 bandpositive samples from stored blood found to have titers of Ն1:160 by serial dilution. The assay may have clinical utility in recognizing CrAg-positive patients who require further diagnostic evaluation for meningitis and enhanced antifungal treatment, albeit at the expense of missing a proportion of patients with lower CrAg titers who are still likely to benefit from standard preemptive fluconazole treatment (18). To avoid missing these individuals with lower titers, CryptoPS could be used as a second test, performed after an initial IMMY LFA screen, to identify the highest-risk individuals with T2 bands for enhanced care. The ease of use and interpretation of the CryptoPS tests may make this an attractive strategy in some settings.
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 29 patients with presumed false-positive CryptoPS results (a positive CryptoPS result and a negative IMMY LFA result) for at least 3 months. Note that in the treatment trial from which some of the archived samples were taken, the maximum dilutional titer measured was 1:2,560; hence, some titers at this level may have been higher if further dilutions had been performed.
All patients (23/29) with repeat IMMY LFA testing again tested negative; all were negative with secondary testing using the Meridian EIA, and all but one with the IMMY EIA. None developed meningitis despite receiving no antifungal treatment, supporting the classification of these results as false positive. Further, nearly all these patients were confirmed to be alive over follow-up; one patient died shortly after screening, but for causes likely unrelated to cryptococcal disease. Given a relatively low prevalence of cryptococcal antigenemia in patients with AHD (19), the positive predictive value of the CryptoPS assay in the prospective cohort was below 50%, with more false-positive than true-positive results.
Our study is subject to several limitations. First, Cryptococcus sp. isolates have significant serotype diversity within Southern Africa, which may impact test sensitivity (20,21). Although the CryptoPS assay can detect all Cryptococcus neoformans/Cryptococcus gattii serotypes, different genotypic and phenotypic characteristics of local fungi could potentially have impacted test performance. Second, although the IMMY LFA and the CryptoPS test were read by trained laboratory operators blind to the results of the other assay, we did not have two independent reviewers read the CryptoPS assay to evaluate inter-rater reliability. However, we repeated CryptoPS testing on the same stored plasma specimens with similar test results, and all IMMY LFA-positive samples underwent further testing through serial dilution, confirming positive results, so the findings are highly likely to be valid.
In summary, the CryptoPS assay provides rapid semiquantitative CrAg test results on whole-blood specimens and good discrimination for high-titer results. Limitations in sensitivity and specificity may limit the clinical use of this assay in cryptococcal antigen-screening programs, which typically aim to detect individuals with relatively low cryptococcal antigen burdens. These limitations may be less marked when one is testing individuals with suspected cryptococcal meningitis, who have a higher pretest probability of infection and higher blood CrAg titers.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.04 MB.