Evaluation of a Novel Point of Care Cryptococcal Antigen (CRAG) Test on Serum, Plasma and Urine from Patients with HIV-associated Cryptococcal Meningitis Joseph N Jarvis, Ann Percival, Sean Bauman, Graeme Meintjes, G. Ntombomzi Williams, Nicky Longley, Thomas S Harrison, Thomas R Kozel

33-63% of all adult meningitis in southern Africa 1-3 Acute mortality with Amphotericin 24-43% 4-7 Acute mortality with Fluconazole 54-96% 8-10 Cryptococcal Meningitis * References on final slide

Lumbar puncture and India-ink Immunodiagnosis (CRAG) Diagnosis?

Lumbar puncture and India-ink Immunodiagnosis (CRAG) Diagnosis? A Point-of Care Assay? Earlier diagnosis Antigen screening Serum, plasma and urine

Study aims To evaluate a quantitative antigen-capture ELISA for GXM and a novel point of care lateral flow immunoassay (LFA) Using paired serum, plasma and urine from patients with active or prior CM we: - defined the relationship of CRAG levels in serum, plasma and urine - tested the sensitivity of the novel lateral flow assay in serum, plasma and urine

Study design GF Jooste Hospital, Cape Town Paired blood and urine samples HIV +ve adults with a history of laboratory confirmed cryptococcal meningitis Tested in parallel using: -Quantitative sandwich ELISA - Lateral flow assay

Methods Quantitative sandwich ELISA GXM concentrations were determined in each sample by use of a quantitative sandwich ELISA that was constructed using the GXM mAbs F12D2 and 339 Lateral flow assay A novel LFA was constructed from the same mAbs F12D2 and 339 utilized in the quantitative sandwich ELISA

Test strips were read after 10 minutes by four different observers Positive if all four observers read the strip as positive and equivocal if some but not all observers read the strip as positive Titers were determined by serially diluting patient samples and assessing reactivity as described above. The highest sample dilution that produced a positive result when read by four observers was recorded as the LFA titer.

Patient demographics 62 patients Median age 34 years, 40% male, median CD4 count 45 cells/mL 61% acute episode 39% during follow-up, median 189 days (98-376)

Results 1 - Quantitative ELISA for GXM All 62 patients had detectable GXM in serum, plasma and urine The mean (95% CI) GXM concentrations were: Serum 3800 ( ) ng/ml Plasma 3600 ( ) ng/ml Urine 170 ( ) ng/ml

p < for all pairings

Results 2 - Lateral flow assay

p < for all pairings

Conclusions Serum and plasma can be used interchangeably for CRAG testing CRAG testing of urine is of potential value There are close correlations between GXM levels on ELISA and LFA titers in serum, plasma and urine This point of care test has the potential to markedly improve the early diagnosis of CM in many settings

1. Bekondi Int J Infect Dis Scarborough NEJM Jarvis BMC Infect Dis Bicanic Clin Infect Dis Bicanic Clin Infect Dis Kambugu Clin Infect Dis Jarvis J Infect Mwaba Postgrad Med J Mayanja-Kizza Clin Infect Dis Longley Clin Infect Dis 2009