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MAJOR ARTICLE HIV/AIDS
Evaluation of Fingerstick Cryptococcal Antigen
Lateral Flow Assay in HIV-Infected Persons: A
Diagnostic Accuracy Study
Darlisha A. Williams,1,2 Tadeo Kiiza,2Richard Kwizera,2Reuben Kiggundu,2Sruti Velamakanni,1David B. Meya,1,2,3
5Joshua Rhein,1,2 and David R. Boulware1
1
University of Minnesota, Minneapolis;
2
Infectious Diseases Institute, and
3
College of Health Sciences, Department of Medicine, Makerere University,
Kampala, Uganda
Background.Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa.
The cryptococcal antigen (CRAG) lateral flow assay (LFA) has simplified diagnosis as a point-of-care test approved
10 for serum or cerebrospinal fluid (CSF). We evaluated the accuracy of the CRAG LFA using fingerstick whole blood
compared with serum/plasma and CSF for diagnosing meningitis.
Methods.From August 2013 to August 2014, CRAG LFA (IMMY, Norman, Oklahoma) tests were performed
on fingerstick whole blood, plasma/serum, and CSF in 207 HIV-infected adults with suspected meningitis in Kam-
pala, Uganda. Venous blood was also collected and centrifuged to obtain serum and/or plasma. CSF was tested after
15 lumbar puncture.
Results.Of 207 participants, 149 (72%) had fingerstick CRAG-positive results. There was 100% agreement be-
tween fingerstick whole blood and serum/plasma. Of the 149 fingerstick CRAG-positive participants, 138 (93%) had
evidence of cryptococcal meningitis with a positive CSF CRAG. Eleven participants (5%) had isolated cryptococcal
antigenemia with a negative CSF CRAG and culture, of whom 8 had CSF abnormalities (n = 3 lymphocytic pleocy-
20 tosis, n = 5 elevated protein, n = 4 increased opening pressure). No persons with cryptococcal meningitis had neg-
ative fingersticks.
Conclusions.The 100% agreement between whole blood, serum, and plasma CRAG LFA results demonstrates
that fingerstick CRAG is a reliable bedside diagnostic test. Using point-of-care CRAG testing simplifies screening
large numbers of patients and enables physicians to prioritize on whom to measure CSF opening pressure using
25 manometers.
Keywords.cryptococcal meningitis; cryptococcus; lateral flow assay; HIV; point-of-care systems.
Cryptococcal meningitis is the most common cause of
adult meningitis in Africa and causes 20%–25% of
AIDS-related deaths [1–4], accounting for 30%–60%
30of adult meningitis cases overall in the general popula-
tion in eastern and southern Africa [3–7]. Typically,
cryptococcal meningitis is diagnosed by performing a
lumbar puncture (LP) with testing of cerebrospinal
fluid (CSF) by either India ink microscopy, culture, or
35cryptococcal antigen (CRAG) [8,9].
In 2011, a CRAG lateral flow immunochromato-
graphic assay (LFA) (IMMY, Norman, Oklahoma),
was approved by the US Food and Drug Administra-
tion. This point-of-care test has been validated in
40serum and CSF, with 99.3% sensitivity and >99.1% spe-
cificity in CSF [8]. Although the introduction of the
CRAG LFA has made testing more feasible in re-
source-limited settings, a fundamental paradox remains
Received 5 January 2015; accepted 25 March 2015.
Correspondence: Darlisha A. Williams, MPH, Infectious Diseases Institute, Mak-
erere University, P.O. Box 22418, Kampala, Uganda (darlisha@gmail.com).
Clinical Infectious Diseases
®
© The Author 2015. Published by Oxford University Press on behalf of the Infectious
Diseases Societyof America. Thisis an Open Access article distributed under the terms
of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://
creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial
reproduction and distribution of the work, in any medium, provided the original work
is not altered or tra nsformed in any way, a nd that the work is prop erly cited. For
commercial re-use, please contact journals.permissions@oup.com.
DOI: 10.1093/cid/civ263
HIV/AIDS •CID •1
that the diagnosis of cryptococcal meningitis cannot be con-
45 firmed until after the diagnostic LP. Intracranial pressure mea-
surement and control are key components of cryptococcal
management [10–12]. When the Cryptococcus diagnosis is
made after the LP is completed, the opportunity to remove suf-
ficient CSF volume to normalize the intracranial pressure is
50 missed, unless manometers are always used. The known pres-
ence of CRAG in blood prior to obtaining a diagnostic LP shifts
the pretest probability for cryptococcal meningitis significantly,
alerting caregivers to the necessity of measuring LP opening
pressure and allowing for more focused, cost-effective down-
55 stream CSF testing [3].
We hypothesized that performing a fingerstick CRAG LFA is
a simple, low-cost point-of-care method that can be used to rap-
idly identify persons with cryptococcosis. This information can
then determine which patients with suspected meningitis re-
60 quire measurement of the CSF opening pressure during an LP
with a manometer or an improvised manometer using intrave-
nous tubing and a meter stick [13]. Capillary fingerstick whole-
blood collection has the additional advantage of being less
invasive than obtaining venous blood and does not require a
65 centrifuge for serum or plasma separation, and bedside testing
can be performed rapidly during informed consent for the LP.
We evaluated the accuracy of the CRAG LFA using fingerstick
whole blood to screen for meningitis compared with serum/
plasma or CSF.
70 METHODS
From August 2013 to August 2014, we enrolled a prospective
cohort of 207 human immunodeficiency virus (HIV)–infected
adults with symptoms of suspected meningitis who were admit-
ted to Mulago National Referral Hospital in Kampala, Uganda.
75 Screening was performed as part of the Adjunctive Sertraline for
the Treatment of HIV-Associated Meningitis (ASTRO-CM)
study (ClinicalTrials.gov identifier NCT01802385). Institution-
al review board approvals occurred at all relevant institutions
in Uganda and Minnesota. Inclusion criteria included physi-
80 cian-suspected meningitis, age ≥18 years, and written informed
consent.
Fingerstick Assay
After obtaining verbal consent, a fingerstick CRAG LFA was
performed. The pad of the index finger was sterilized with an
85 alcohol swab, and a lancet was used to prick the finger and pro-
duce 1 drop of whole blood. The patient’sfinger was quickly
placed directly on the tip of the LFA test strip so that the
blood sample (approximately 40 µL) could be absorbed directly
onto the LFA strip. The test strip was then placed in a 1.5-mL
90 Eppendorf tube containing 1–2 drops of sample diluent, and al-
lowed to incubate in an upright position at room temperature
for 10 minutes. CRAG LFAs were read by trained study person-
nel. This included the phlebotomist and the study medical offi-
cers. As the fingerstick CRAG LFA incubated, written informed
95consent was obtained for permission to conduct an LP and par-
ticipate in the study. The CRAG LFA result was used to prior-
itize on whom to use manometers to measure CSF opening
pressure. LPs were then performed in the lateral decubitus
position.
100Parallel Testing of Serum and Plasma
Serum (n = 206) and plasma (n = 27) were collected via veni-
puncture by a phlebotomist. Serum and/or plasma were tested
separately for CRAG LFA by adding 40 µL of specimen to Ep-
pendorf tubes containing 1 drop of sample diluent. CRAG LFA
105dipsticks were placed into each tube and read after 10 minutes.
Serum and plasma were interchangeably tested, per prior pub-
lished equivalence [8].
CSF Analysis
All CSF samples had a CRAG LFA performed at bedside, col-
110lecting 1 drop of CSF into an Eppendorf tube with 1 drop of
sample diluent. All CSF CRAG LFA tests were performed
twice, once by the clinical staff on the hospital ward and a sec-
ond time by a microbiologist upon processing the CSF sample.
Quantitative fungal cultures were performed using 5 serial 10-
115fold dilutions of CSF [8,14]. Further analysis was performed on
CSF samples to determine the white blood cell (WBC) count,
WBC differential, and protein measurements. Persons with
negative fingerstick and CSF CRAG also had Gram stain and
enhanced tuberculosis meningitis diagnostics performed
120through a streamlined diagnostic approach [3]. These diagnos-
tics included GeneXpert, acid-fast bacilli smear microscopy,
and tuberculosis cultures.
Statistical Analysis
The diagnostic performance (ie, sensitivity and specificity) of
125fingerstick CRAG was compared against a composite reference
standard of either serum/plasma or CSF CRAG positivity. The
relationship between positive fingerstick CRAG, CSF CRAG,
and CSF culture was also examined to determine the concor-
dance and positive predictive value of fingerstick testing.
130RESULTS
Overall, 207 participants with suspected meningitis were en-
rolled, of whom 72% (n = 149) had evidence of cryptococcosis.
Sixty percent (125/207) of participants were male, with a medi-
an age of 36 years (interquartile range [IQR], 30–42 years). Par-
135ticipants were HIV infected with a median CD4 T-cell count of
25 cells/µL (IQR, 9–73 cells/µL; maximum, 660 cells/µL) and with
51% (105/207) receiving antiretroviral therapy at diagnosis.
2•CID •HIV/AIDS
Among patients reporting antecedent headache at diagnosis
(n = 193), the median headache duration was 14 days (IQR, 7–
140 21 days). The Glasgow Coma Scale score was <15 in 47% (98/
207) of patients at presentation. The median CSF white cell
count was <5 cells/µL (IQR, <5–60 cells/µL), with 67% having
<5 white cells/µL. Median CSF protein at diagnosis was 50 mg/
dL (IQR, 20–99mg/dL). Approximately 141 participants had their
145 CSF opening pressures measured at screening; the median open-
ing pressure was 280 mm H
2
O (IQR, 180–430 mm H
2
O). Of per-
sons without cryptococcosis, 1 participant had Streptococcus
pneumoniae meningitis by Gram stain with polymerase chain
reaction confirmation (0.5% prevalence). Tuberculosis meningitis
150 was confirmed by GeneXpert and/or culture in 15 participants.
The remaining CRAG-negative participants had aseptic/viral
meningitis of predominantly unknown etiology.
Of the 207 CRAG LFA tests performed on fingerstick whole
blood, 149 (72%) were positive, of which 138 (93%) were also
155 CSF CRAG positive. Among the 207 participants with CRAG
LFA tests performed in serum or plasma, 149 (72%) were pos-
itive (Figure 1). There was 100% concordance between plasma
and serum CRAG results (n = 26; where both serum and plasma
were available). Among those fingerstick CRAG-positive, 93%
160(139/150) were also CSF CRAG positive. Of those CSF CRAG
positive, 91% (127/139) also had positive cryptococcal culture
growth (range, 10 colony-forming units [CFU]/mL to >15 mil-
lion CFU/mL). No persons who tested fingerstick (or CSF)
CRAG negative grew Cryptococcus in culture (100% negative
165predictive value). Furthermore, there was 100% concordance
between fingerstick whole blood and serum/plasma (κ=1.0;
95% confidence interval lower bound, 0.979). The positive pre-
dictive value of fingerstick LFA for the detection of cryptococcal
infection in blood was 100% (149/149) and for cryptococcal
170meningitis was 93% (138/149). Eleven (5%) participants had
isolated cryptococcal antigenemia without proven CSF Crypto-
coccus infection that was represented by CRAG-positive finger-
stick and CRAG-positive serum/plasma, but negative CSF
CRAG and negative culture. Of these 11 fingerstick CRAG-pos-
175itive and CSF CRAG-negative participants, 8 had CSF abnor-
malities (n = 3 with CSF lymphocytic pleocytosis, n = 5 with
increased CSF protein [>45 mg/dL], and n = 4 with elevated
opening pressure >200 mm H
2
O).
The time of point-of-care testing at the hospital bedside (10
180minutes) was less than our prior experience of a median of 4
hours 50 minutes for laboratory-based CRAG testing at the
same site [8].
DISCUSSION
CRAG testing of whole blood by capillary fingerstick had 100%
185concordance with serum or plasma CRAG results collected by
venipuncture. Fingerstick CRAG had 100% negative predictive
value for excluding cryptococcal meningitis. The ability to con-
duct reliable fingerstick CRAG testing overcomes a clinical
management paradox. Previous studies have found a significant
190improvement in acute mortality when patients are able to re-
ceive at least 1 therapeutic LP to normalize intracranial pressure
[11]. However, a major challenge to managing cryptococcal
meningitis is that the diagnosis is typically confirmed only
after an LP is performed, thus making it difficult to measure
195and manage intracranial pressure, unless CSF opening pressures
are universally measured. Manometers are rarely available in
low-income countries, and not always used in high-income
countries where available. Improvised measurement of CSF
opening pressure can be conducted using intravenous tubing
200and a meter stick [13]; however, this requires additional physi-
cian time. By CRAG screening prior to the LP, one can priori-
tize on whom to measure opening pressure or empirically
remove 20–25 mL CSF, which was the median amount removed
in this cohort [11,13]. Additionally, healthcare workers in both
205rural and urban areas can also easily screen persons with HIV or
suspected meningitis using fingerstick, and quickly exclude
cryptococcosis or refer symptomatic CRAG-positive persons
to the hospital for LPs.
Figure 1. Distribution of cryptococcal diagnostics in blood and cerebro-
spinal fluid (CSF). The concordance of fingerstick cryptococcal antigen
(CRAG) testing was κ= 1.0 for blood (P= .99) and κ= 0.947 for CSF repre-
senting 5% of participants (n = 11) having isolated cryptococcal antigene-
mia in peripheral blood with early disseminated cryptococcal infection but
without microbiologically provenmeningeal involvement. Of these 11 blood
CRAG-positive and CSF CRAG-negative participants, 8 participants had
abnormal CSF profiles with CSF inflammation or increased opening pres-
sure. Fifty-eight participants were negative for all cryptococcal testing in
blood and CSF. No person had CSF cryptococcal involvement by CRAG or
culture who was CRAG-negative by fingerstick or in peripheral blood.
HIV/AIDS •CID •3
Another challenge with traditional diagnostic methods (eg,
210 CRAG latex agglutination, culture) is the necessary infrastruc-
ture and laboratory skilled labor required for testing. The CRAG
LFA represents an important diagnostic advance, providing an
affordable point-of-care test that can be performed at the bed-
side on blood to influence pretest probability and repeated at
215 time of LP to confirm the diagnosis of cryptococcal meningitis.
The CRAG LFA is easy to use, reliable, and versatile. It can be
performed on serum, plasma, whole blood, urine, or CSF [8,9].
Notably, CRAG testing of saliva performs less optimally (88%
sensitivity and 98% specificity) [15]. LFA tests performed at
220 the bedside also expedite antifungal treatment.
Although the CRAG LFA has been previously validated in
serum and CSF [8], we wanted to investigate the accuracy of
using fingerstick whole blood, which is easier to collect and pro-
vides a quicker method for diagnosis compared with collecting
225 CSF or serum/plasma. The fingerstick CRAG LFA showed a
100% agreement with serum and/or plasma. Of participants
who were fingerstick CRAG positive, 93% were also CSF
CRAG positive, with the others having isolated cryptococcal
antigenemia, which also requires treatment [16]. No test is
230 100% perfect, and a larger sample size may have eventually un-
covered false positives or false negatives; however, in real-world
use, fingerstick testing performed very well.
Given the poor outcomes of patients with cryptococcal menin-
gitis [17], typical delays in diagnosis [18], and the documented
235 approximately 70% relative survival benefit at 10 days of repeated
therapeutic LPs to reduce ICP [11], using the fingerstick CRAG
LFA as a rapid initial step toward establishing a diagnosis can en-
able better treatment. Fingerstick CRAG testing represents an im-
portant and simple tool for meningitis diagnosis worldwide.
240 Notes
Acknowledgments. We thank Dr Thomas Kozel for his work in devel-
oping monoclonal antibodies and Dr Sean Baumann and colleagues for de-
veloping the cryptococcal antigen lateral flow assay.
Disclaimer. IMMY did not provide any financial support for this study.
245 Financial support. Financial support for this research was provided by
the National Institute of Allergy and Infectious Diseases ( grant numbers
U01AI089244, R01NS086312, K24AI096925, T32AI055433, and
R25TW009345). We thank Bozena Morawski, MPH, for data management,
and Drs Thomas Harrison and Tihana Bicanic for sharing their quantitative
250 cerebrospinal fluid culture protocol and training of laboratory staff in Ugan-
da. We appreciate institutional support from Drs Paul Bohjanen and An-
drew Kambugu.
Potential conflicts of interest. All authors: No potential conflicts of
interest.
255 All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
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4•CID •HIV/AIDS
