Upload
leia
View
36
Download
0
Tags:
Embed Size (px)
DESCRIPTION
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. - PowerPoint PPT Presentation
Citation preview
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 con centration 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