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

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

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 assayA novel LFA was constructed from the same mAbs F12D2 and 339 utilized in the quantitative sandwich ELISA

Sensitivity of commercially available immunoassays compared with a prototyp e lateral flow immuno-chromatographic assay for detection of GXM of different serotypes.

Minimum conce ntration of GXM producing a positive result (ng/ml)a

Serotype A GXM Serotype B GXM Serotype C GXM Serotype D GXM Immunoassay Assay format

b CN6 MU-1 184 409 34 298 24066 127 M0024

Immuno-Myco logics

LA 24 32 27 68 432 260 460 62 63

Meridian Calas LA 18 20 52 22 2,600 470 360 44 65

Inverness LA 38 ntc 64 ntc Negd 940 Negd 50 ntc

Meridian Premier ELISA 35 22 25 21 2.8x105 1.4x104 2.8x105 900 630

Immuno-Myco logics

LFIe 1 (2) 1 (2) 1 (1) 1 (1) 16 (32) 8 (16) 8 (8) 8 (16) 8 (16)

Kozel lab EIAf ELISA 0.76 0.44 0.85 0.86 10 2.7 2.8 0.63 0.51

a Results are shown for GXM isolated from different strains of eac h serotype. b Assay format: LA – latex agg lutination; ELISA – antigen capture sandwich immunoassay. c Not tested d Negative e Limit of detec tion for the LFI is reported as the GXM concentration where 50% of 4 readers read a positive result. Results in parentheses are endpoints where 100% of 4 readers read a po sitive result. f Results are shown for an in-house quantitative ELISA used in the Kozel laboratory.

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 (2100-6600) ng/ml

Plasma 3600 (2100-6300) ng/ml

Urine 170 (100-280) ng/ml

p < 0.001 for all pairings

Results 2 - Lateral flow assay

Results 2 - Lateral flow assay

Serum Plasma Urine

CRAG LFA+ 61 61 61

CRAG LFA +/- 1 1 0

CRAG LFA - 0 0 1

Sensitivity of LFA 100% 100% 98%

95% CI* 94-100% 94-100% 91-100%

*95% confidence interval

p < 0.001 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

QuickTime™ and a decompressor

are needed to see this picture.

1. Bekondi Int J Infect Dis 20062. Scarborough NEJM 20073. Jarvis BMC Infect Dis 20104. Bicanic Clin Infect Dis 20075. Bicanic Clin Infect Dis 2008

6. Kambugu Clin Infect Dis 20087. Jarvis J Infect 20108. Mwaba Postgrad Med J 20019. Mayanja-Kizza Clin Infect Dis 199810. Longley Clin Infect Dis 2009