Dr Lindi Coetzee Ms Keshendree Moodley Prof D.K. Glencross COMPARATIVE RESULTS OF A NOVEL FLOW CYTOMETER ASSAY (FSA) FOR EARLY DETECTION OF CrAg Dr Lindi Coetzee Ms Keshendree Moodley Prof D.K. Glencross

Background Cryptococcus neoformans is the leading cause of Cryptococcal meningitis (CM) Fourth leading cause of deaths in HIV positive individuals in SSA Early diagnosis and treatment initiation can save lives Guidelines CD4<100cells/µl in ART naïve HIV+ individuals (WHO, SACC, NDOH) Early CrAg detection program implemented in SA during 2016 Across 49 CD4 testing facilities Reflexed against confirmed CD4 count<100cells/µl Using Lateral Flow Assay (LFA) by Immy Mycologics, USA

Flow cytometry assay (FCA) An inhibition assay developed for flow cytometry by IMMY in 2014 In collaboration with the CD4 research team developed a protocol for detection of CrAg as add-on to current CD4/PLG protocol Impact of lysing on results (lysing done pre CrAg antibody addition or post incubation) Impact of stand-alone (CrAg only) vs. PLG incorporated protocol

Flow cytometry assay (FCA) This assay measures mean fluorescence intensity (MFI) of CrAg particles Inverse relationship with CrAg concentration Positive:negative cut-off determined (MFI of 20) Confirmed correlation with LFA results (n=224) Without impact on reported CD4 count when “piggy-backed” (same protocol)

Tested across three platforms (n=63) Methodology Remnant EDTA with a CD4 <=100 cells/µl and LFA Crag Result tested at the CMJAH laboratory Quality control samples that included positive samples from IMMY (LFA) Remnant EDTA with a CD4 <=100 cells/µl and positive LFA Crag Result tested at the Tambo Memorial laboratory Tested across three platforms (n=63) Platform Three: Flow Cytometer assay on the Beckman Coulter XL cytometer 4 Way Correlation against reported LFA Results Platform Two: Thunderbolt EIA Instrument with the Premier EIA assay Platform One: Thunderbolt EIA Instrument with the IMMY EIA assay

Methodology EDTA samples with confirmed CD4<100 and LFA CrAg result (n=63) tested in 3 way analysis FCA on a Beckman Coulter XL cytometer with pre-defined CrAg protocol The IMMY FCA kit utilize the same antibodies as for the LFA strips and the EIA kit In addition to patient samples, a panel of 20 quality control samples (IMMY) were analysed across methods and platforms

Results: Of the 63 samples tested, 21 had a negative LFA vs. 43 positive LFA’s Not representative of any population as additional positive results were obtained from a 2nd testing facility to increase the number of positive samples tested FCA results confirmed the cut-off MFI of 20 MFI<20 classified as positive CrAg MFI 21-40 classified as intermediate CrAg MFI >40 classified as negative CrAg MFI values for internal QC beads were stable over time at 505±12 (95% confidence interval of 499-510) Corresponding %CV over 2 weeks was 2.5%

Results: Typical example of CrAg FCA result (A) negative vs. (B) positive Each sample had internal beads (FlowCountTM) as sample-to-sample quality control measure

Sensitivity and specificity 2 samples tested positive with both EIA assays but negative with FSA Premier had 2 negative results while Immy and FSA reported those positive Overall, acceptable sensitivity and specificity Discrepancies could not be retested due to too little sample

EIA optic density compared to FCA MFI Comparing OD readings to FSA MFI showed inverse correlation for patient samples and QC samples One “outlier” was positive with Immy EIA but negative with Premium EIA and FCA

Discussion FCA showed excellent agreement with EIA methods (2 assays) for both patient samples and QC material Both the FCA and EIA assays facilitate a range of test values and not merely a negative vs. positive result of the subjective strip-based LFA, that can inform clinicians re urgency of therapy interventions. The assay could easily be incorporated into the current CD4 test regime as a combined protocol with PLG/CD4 T-cell enumeration, with built-in quality control checks, sample preparation automation and direct reporting of results to the Laboratory Information System (LIS) for verification.

Viable test platform option as “piggy-backed” onto existing technology Discussion A combined assay may be more cost effective as it utilizes existing platforms and staff. Viable test platform option as “piggy-backed” onto existing technology May hold cost saving benefits as apposed to having LFA/EIA platforms depending on test volumes

Challenges and ongoing R&D Introduction of new CD4 testing platforms (Beckman Coulter Aquios/other) where customer designed protocols may require additional FDA approval Ensuring continued reproducibility and sensitivity of the assay production to ensure equivalent batch to batch results needs to be secured by the manufacturer (IMMY) Additional site verification studies needed to consolidate FCA findings presented here

Challenges and ongoing R&D Standardization of FCA protocol across a network of test facilities and platforms Current focus to explore FCA as a quantitative assay for reporting actual CrAg concentrations in positive patients for better management of disease

Acknowledgements IMMY USA Sitetech SA for Premier kits Hain Lifesciences for Immy kits and Thunderbolt instrument