Evaluation of a Novel Point-of-Care Cryptococcal Antigen Test on Serum, Plasma, and Urine From Patients With HIV-Associated Cryptococcal Meningitis
HIV/AIDS d CID
Evaluation of a Novel Point-of-Care Cryptococcal Antigen Test on Serum, Plasma, and Urine From Patients With HIV-Associated Cryptococcal Meningitis
Joseph N. Jarvis 0 1 2
Ann Percival 6
Sean Bauman 5
Joy Pelfrey 5
Graeme Meintjes 0 3 4
G. Ntombomzi Williams 0
Nicky Longley 0 1 2
Thomas S. Harrison 2
Thomas R. Kozel 6
0 Infectious Diseases Unit, GF Jooste Hospital , Cape Town , South Africa
1 Desmond Tutu HIV Centre, Institute of Infectious Disease and Molecular Medicine, University of Cape Town
2 Research Centre for Infection and Immunity, Division of Clinical Sciences, St George's University of London , Cranmer Terrace , United Kingdom
3 Department of Medicine, Imperial College London , United Kingdom
4 Institute of Infectious Diseases and Molecular Medicine, Department of Medicine, University of Cape Town , South Africa
5 Immuno- Mycologics , Norman, Oklahoma , USA
6 Department of Microbiology and Immunology, University of Nevada School of Medicine , Reno , USA
Background. Many deaths from cryptococcal meningitis (CM) may be preventable through early diagnosis and treatment. An inexpensive point-of-care (POC) assay for use with urine or a drop of blood would facilitate early diagnosis of cryptococcal infection in resource-limited settings. We compared cryptococcal antigen (CRAG) concentrations in plasma, serum, and urine from patients with CM, using an antigen-capture assay for glucuronoxylomannan (GXM) and a novel POC dipstick test. Methods. GXM concentrations were determined in paired serum, plasma, and urine from 62 patients with active or recent CM, using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). A dipstick lateralflow assay developed using the same monoclonal antibodies for the sandwich ELISA was tested in parallel. Correlation coefficients were calculated using Spearman rank test. Results. All patients had detectable GXM in serum, plasma, and urine using the quantitative ELISA. Comparison of paired serum and plasma showed identical results. There were strong correlations between GXM levels in serum/urine (rs 5 0.86; P , .001) and plasma/urine (rs 5 0.85; P , .001). Levels of GXM were 22-fold lower in urine than in serum/plasma. The dipstick test was positive in serum, plasma, and urine in 61 of 62 patients. Dipstick titers correlated strongly with ELISA. Correlations between the methods were 0.93 (P , .001) for serum, 0.94 (P , .001) for plasma, and 0.94 (P , .001) for urine. Conclusions. This novel dipstick test has the potential to markedly improve early diagnosis of CM in many settings, enabling testing of urine in patients presenting to health care facilities in which lumbar puncture, or even blood sampling, is not feasible.
Cryptococcal meningitis (CM) is estimated to kill more than 500 000 human immunodeficiency virus (HIV)– infected patients per year in Sub-Saharan Africa .
With the evolving HIV epidemic, CM has emerged as
the most frequent cause of adult meningitis in much of
central and southern Africa [
], and outcomes with
current optimal antifungal therapy are poor [
deaths from CM may be preventable through early
diagnosis and treatment.
The diagnosis of HIV-associated CM is usually
made by lumbar puncture (LP) and India-ink testing
of cerebrospinal fluid (CSF). However, the presenting
symptoms of headache and fever are very nonspecific,
and LP is often deferred until the disease is
advanced and the prognosis is poor. The alternative of
immunodiagnosis through detection of cryptococcal
polysaccharide capsule glucuronoxylomannan (GXM) in serum by
latex agglutination (LA) or sandwich enzyme-linked
immunosorbent assay (ELISA) is sensitive and specific and has
been commercially available for many years [
]. However, the
currently available immunoassays require blood to be sent to a
central laboratory with appropriate laboratory infrastructure
and trained staff, and immunoassay tests are too expensive for
routine use in most African settings and therefore are not widely
A point-of-care (POC) immunoassay for cryptococcal antigen
(CRAG) would greatly facilitate the early diagnosis of patients
presenting with symptoms of CM. Such a test could also be
used to prevent CM because CRAG immunoassays are positive
prior to the development of clinically apparent disease [
Screening patients before initiating antiretroviral therapy (ART),
with preemptive antifungal therapy for patients with subclinical
infection, could prevent the later development of clinical disease
]. Currently, up to 70% of all cryptococcal cases in some
African centers present after a diagnosis of HIV infection, and
approximately 30% present after initiation of ART [
Current tests have regulatory approval for use on serum and
CSF; however, the ability to detect CRAG in other sample types,
specifically plasma and urine, is essential for a POC test. In
addition, plasma is readily available from samples routinely taken
for HIV testing and CD4 cell-count monitoring. Urine is a
highly desirable specimen for diagnosis of cryptococcal
infection in resource-limited settings [
], particularly where
blood sampling is difficult or LPs are not easily performed or
accepted. There have been 2 studies of GXM excretion in urine
of patients with cryptococcosis [
], both of which
supported immunoassay use on urine as an aid to diagnose
cryptococcosis. However, neither study determined the quantitative
relationship between levels of GXM in serum and urine.
We have evaluated the performance of a quantitative
antigencapture 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. Both assays are based on
GXM monoclonal antibodies (mAbs) selected to have broad
reactivity across the 4 major serotypes of Cryptococcus
neoformans capsular antigen [
]. The aims were to define the
relationship of CRAG levels in serum, plasma, and urine and to
test the sensitivity of the novel lateral flow assay in serum,
plasma, and urine.
Participants and Procedures
Sample collection was performed between March 2009 and
November 2010 at GF Jooste Hospital, a public-sector adult
referral hospital in Cape Town, South Africa, and approved by
1020 d CID 2011:53 (15 November) d HIV/AIDS
the Research Ethics Committee of the University of Cape Town.
Paired blood and urine samples were collected from adult
patients ($21 years), with a history of laboratory-confirmed
HIV-associated cryptococcal disease within the preceding
2 years (CSF India ink or CRAG positive, titers $1:1024,
Meridian Cryptococcal Latex Agglutination System, Meridian
Bioscience, and confirmed on CSF culture in all but 4 patients
in whom culture results were unavailable), who were either under
follow-up in the outpatient clinic or admitted to the medical
wards. Written informed consent was obtained from each
participant prior to study enrollment. Samples were collected over a
broad range of follow-up times from original diagnosis, and
from sufficient numbers of patients, so that a wide range of
antigen titers was obtained. Blood and urine samples were frozen
for later analysis. Basic demographic and laboratory data (age,
sex, CD4 cell count), details of CM episode (timing, basis of
diagnosis, and treatment received), and details of antiretroviral
exposure and secondary fluconazole prophylaxis were recorded.
Quantitative Sandwich Enzyme-Linked Immunosorbent Assay
GXM concentrations were determined in each sample by use of
a quantitative sandwich ELISA that was constructed using the
GXM mAbs F12D2 and 339 [
]. In this ELISA, microtiter
plates were coated overnight with an optimized 50:50 mixture
of GXM mAbs F12D2 and 339 for the capture phase. After
plates were washed and blocked, the wells were incubated for
90 minutes with serial dilutions of serum, plasma, or urine in
phosphate-buffered saline–Tween. The plates were washed,
incubated for 90 minutes with an optimized 50:50 cocktail of
horseradish peroxidase-conjugated mAbs F12D2 and 339 for
the indicator phase, and washed and incubated with substrate
solution. Purified serotype A GXM isolated from strain CN-6
was used as a standard. The results are reported as the
concentration of GXM in nanograms per milliliter.
Lateral Flow Assay
A novel LFA was constructed from the same mAbs F12D2 and
339 used in the quantitative sandwich ELISA. The test is in
dipstick format. Forty microliters of specimen was mixed with
1 drop of sample diluent, the dipstick was inserted into the
diluted sample, and the test strips were read after 10 minutes by
4 different observers. Tests were considered positive if all 4
observers read the strip as positive and equivocal if some but not
all observers read the strip as positive. LFA titers were
determined by diluting patient samples in LA diluent solution
and assessing reactivity as described above. The highest sample
dilution that produced a positive result when read by 4
observers was recorded as the LFA titer.
Correlation coefficients were calculated using the Spearman
rankorder correlation. GXM concentrations were log transformed,
and geometric means with 95% confidence intervals (CIs) are
presented where appropriate.
Paired blood and urine samples were obtained from 62 patients.
The median age was 34 years (interquartile range [IQR], 28–37),
40% were male (25 of 62), and the median CD4 cell count was
45 cells/lL (IQR, 22–100). Sixty-one percent of patients (38 of
62) had samples collected during their acute episode of CM,
and 39% (24 of 62) during follow-up visits, a median of 189 days
(IQR, 98–376) after initial CM presentation. All episodes of CM
were treated with amphotericin B (1 mg/kg per day) for 14 days.
Of the follow-up patients, 92% (22 of 24) were on fluconazole
secondary prophylaxis. Overall, 56% (35 of 62) were on ART at
the time of sample collection, 92% (22 of 24) of follow-up
patients and 34% (13 of 38) with acute CM.
Quantitative Immunoassay for Glucuronoxylomannan in Patient
All 62 patients had detectable GXM in serum, plasma, and urine
using quantitative ELISA. The mean (95% CI) GXM
concentrations were 3800 (2100–6600) ng/mL in serum, 3600 (2100–
6300) ng/mL in plasma, and 170 (100–280) ng/mL in urine.
A comparison of immunoassay results of paired serum and
plasma showed identical results with the 2 sample types
(Figure 1). There was a very strong correlation between GXM
concentrations in serum and plasma (rs 5 1.00; P , .001).
There were also strong correlations between levels of GXM in
serum and urine (rs 5 0.86; P , .001) and in plasma and urine
(rs 5 0.85; P , .001). The actual levels of GXM in urine were
lower, on average 22-fold lower in urine than in serum or
plasma. The ratio of GXM in serum/urine was independent
of the concentration of GXM in serum (P 5 .77) or urine
(P 5 .36).
Lateral Flow Assay
The lateral flow assay produced a consistently positive result
with purified GXM at concentrations $5 ng/mL (Figure 2). The
LFA was positive in the serum, plasma, and urine in 61 of 62
patients (Table 1). The remaining patient had extremely low
concentrations of GXM detectable in the serum, plasma, and
urine by quantitative ELISA testing (2 ng/mL, 2 ng/mL, and
1 ng/mL respectively); on LFA testing, the results for this patient
were equivocal on serum and plasma, and negative in the urine.
LFA titer results, calculated by serial dilution, correlated strongly
with GXM concentrations measured by the quantitative ELISA
method. The Spearman rank-order correlations between the
2 methods were 0.93 (P , .001) for serum, 0.94 (P , .001) for
plasma, and 0.94 (P , .001) for urine (Figure 1).
HIV/AIDS d CID 2011:53 (15 November) d 1021
These results indicate that serum and plasma can be used
interchangeably for CRAG testing. The ability to use plasma as a
clinical sample will reduce the logistical difficulty and cost of
CRAG testing in patients where plasma is already collected as
part of other clinical tests, most notably for determination of
CD4 cell count after HIV diagnosis and before ART. Thus,
laboratories can automatically forward plasma from patients
with a CD4 cell count of ,100 cells/lL for CRAG screening.
The study also demonstrates the potential value of testing
urine for CRAG. Urine is a noninvasive sample that is easily
obtained in environments with limited resources [
concentrations in urine were significantly lower than in serum/
plasma (approximately 20-fold). However, despite these lower
levels, there was 100% sensitivity in testing of urine relative to
serum or plasma when the highly sensitive enzyme
immunoassay (EIA) was used. Moreover, the sensitivity limit of the LFA
(approximately 5 ng GXM/mL) was well below the lower end
of the 95% CI for GXM concentration in urine (approximately
100 ng/mL). The LFA found readily detectable CRAG in urine
from 61 of 62 patients.
Patients with HIV-associated CM usually have high organism
burdens, and in this study of patients with current or recent CM,
levels of antigen are likely to have been higher than are found on
screening of asymptomatic patients prior to ART [
all samples with detectable GXM by quantitative EIA were also
positive on LFA testing, with the exception of a single patient who
had very low antigen titers in the serum, plasma, and urine. There
were also very close correlations between GXM levels on ELISA
and LFA titers in serum, plasma, and urine. The ability of the
POC test to reliably detect antigen at concentrations $5 ng/mL
in this study suggests that sensitivity of the POC test in urine may
also be sufficient in asymptomatic patients with early subclinical
infection, but this needs confirmation in prospective studies.
This POC test has the potential to markedly improve the early
diagnosis of CM in many settings, enabling the testing of urine
in patients presenting with symptoms of CM to primary health
care facilities or rural clinics where LP, or even blood sampling,
may not be feasible. The POC test will be a great advantage in
diagnostic laboratories in much of the developing world, where
erratic electricity supplies and lack of basic equipment may
preclude the use of conventional CRAG assays. This technology
also has potential applications in the developed world, both in
HIV-infected patients and in other patient groups such as solid
organ transplant recipients.
Furthermore, the findings have important implications for
the feasibility of CRAG screening interventions in ART
programs. At a programmatic level, using the excess plasma from
CD4 count measurement for CRAG testing would prevent the
need for collection of additional serum samples and prevent
any delay in initiation of ART, especially in the majority of
patients who test antigen negative. Indeed, routine CRAG
testing of all CD4 cell count samples in which the CD4 cell count is
,100 cells/lL at laboratory level has been proposed as part of
the introduction of CRAG screening in South Africa [
The relatively small sample size means that these results
require validation in larger studies, and a limitation of the current
study is that although the sensitivity of CRAG detection in
serum, plasma, and urine by ELISA and LFA has been
demonstrated, no inference about the specificity of the tests can be
drawn from these data. The extremely close correlation between
CRAG titers in the blood and urine and between the results from
the ELISA and LFA tests suggest that specificity of CRAG urine
testing and the novel LFA should be as good as conventional
immunodiagnostic testing. Nevertheless, prospective screening
studies are needed and are under way to further validate the
specificity of the POC test. Work is also ongoing to validate
modifications to the LFA that provide a quantitative result, and
data from these ongoing prospective studies will be used to
determine the most useful cut-points for any such
quantification. Widespread availability and use of such a POC test has the
potential to substantially lower the global burden of
Financial support. This work was supported by the Wellcome Trust,
London, United Kingdom (WT081794 and WT081667 to J. N. J. and
G. M.) and the National Institutes of Health (AI014209 to T. R. K.).
Potential conflicts of interest. S. B. is president of Immuno Mycologics
(IMMY). J. P. is an employee of IMMY. The University of Nevada, Reno,
has licensed the mAbs used for immunoassay construction to IMMY. All
other authors report no conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
Author contributions. J. N. J., T. K., and T. S. H. conceived and
designed the study. J. N. J., G. N. W., and N. L. performed the patient
recruitment and clinical sample collection. T. K. conceived and designed
the mAbs and the quantitative sandwich ELISA. A. P. performed the
ELISAs. S. B. and T. K. designed and produced the LFA test kit. LFA testing was
performed in the United States by S. B. and J. P., and in Cape Town, South
Africa, by G. M., G. N. W., N. L., and T. S. H. J. N. J. and T. K. analyzed
the data. J. N. J. wrote the manuscript, with input from T. K. and T. S. H.
All authors read and approved the final version of the manuscript.
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