Transcript 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.

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
Cryptococcal Meningitis
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
* 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.
Diagnosis?
Lumbar puncture and India-ink
Immunodiagnosis (CRAG)
Diagnosis?
Lumbar puncture and India-ink
Immunodiagnosis (CRAG)
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
S en sitiv ity of comm e rc ia lly ava ilab le im mu n oas says compared
lat e ra l flow immu n o -c h romatograp h ic assay for d e te ct io n of
se rot yp e s.
Immu n oa ssay
w ith a protot yp e
GX M o f d iff e ren t
Min imu m c once n trati o n o f G X M p ro d ucin g a p osi tive resu lt (ng /m l) a
As s ay
fo rm at Se roty p e A GXM Se roty p e B G X M
Se roty p e C GXM
Se roty p e D G X M
b
C N6
MU -1
LA
24
32
27
68
432
260
460
62
63
Mer id ia n C a las
LA
18
20
52
22
2,600
470
360
44
65
Inv e rness
LA
38
ntc
64
ntc
Ne g d
940
Ne g d
50
ntc
EL IS A
35
22
25
21
2.8x10
900
630
LFI e
1 (2)
1 (2)
1 (1)
1 (1)
16 (32)
8 (16)
8 (8)
8 (16)
8 (16)
EL IS A
0 .7 6
0 .4 4
0 .8 5
0 .8 6
10
2 .7
2 .8
0 .6 3
0 .5 1
Imm u n o -
184
409
34
298
24066
127
M0024
Myco logics
Mer id ia n P re mi e r
Imm u n o Myco logics
K oze l lab EI A f
5
1.4x10
4
2.8x10
5
a
R e sult s a re sh ow n fo r G X M iso late d fro m diff e re nt st ra ins of e ac h se roty p e.
b
As s ay fo rm a t : LA – late x agg lut inat io n ; EL IS A – an tigen c ap t ur e sa n d w ic h im mu n oa s sa y .
c
No t test e d
d
Neg a tive
Limi t o f detec tio n fo r th e LF I is rep o rte d a s the G X M con c en trat io n w her e 50 % o f 4 re a d e rs re a d a
posi t ive res u lt. Re s ult s in p a ren t hese s a re e n d po int s w her e 100 % o f 4 re a d e rs re a d a po s itiv e re sul t .
e
f
R e sul t s a re sh ow n fo r a n in -ho u se qu a nti t ativ e E LIS A use d in t he K o ze l lab o ra t o ry.
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
CR A G LF A +
61
61
61
CR A G LF A + /-
1
1
0
CR A G LF A -
0
0
1
Sen sitiv ity of LF A
100%
100%
98%
95% C I*
94 -100%
94 -100%
91 -100%
*95% co n fid en c e in te rva l
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
1. Bekondi Int J Infect Dis 2006
2. Scarborough NEJM 2007
3. Jarvis BMC Infect Dis 2010
4. Bicanic Clin Infect Dis 2007
5. Bicanic Clin Infect Dis 2008
6. Kambugu Clin Infect Dis 2008
7. Jarvis J Infect 2010
8. Mwaba Postgrad Med J 2001
9. Mayanja-Kizza Clin Infect Dis 1998
10. Longley Clin Infect Dis 2009
QuickTime™ and a
decompressor
are needed to see this picture.