IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA USING

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IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA USING
Am. J. Trop. Med. Hyg., 60(1), 1999, pp. 109–118
Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA USING PLASMODIUM
LACTATE DEHYDROGENASE (pLDH)
ROBERT PIPER, JACQUES LEBRAS, LAURA WENTWORTH, ANGELA HUNT-COOKE, SANDRA HOUZÉ,
PETER CHIODINI, AND MICHAEL MAKLER
Flow, Inc., Portland, Oregon; Hôpital Bichat Claude Bernard, Paris, France; Department of Clinical Parasitology, Hospital for
Tropical Diseases, London, United Kingdom
Abstract. We have developed two diagnostic assays based on the specific detection of Plasmodium lactate dehydrogenase (pLDH) activity. These assays exploit a panel of monoclonal antibodies that capture the parasite enzyme
and allow for the quantitation and speciation of human malaria infections. An immunocapture pLDH activity assay
(ICpLDH) allows for the rapid purification and measurement of pLDH from infected blood using the NAD analog
APAD, which reacts specifically with Plasmodium LDH isoforms. An immunochromatographic test (the OptiMALt
assay) was also formatted and allowed the detection of parasite infections of ;200 parasites/ml of blood. By using a
combination of antibodies, both tests can not only detect but differentiate between P. falciparum and non-P. falciparum malaria. Both assays show a sensitivity comparable with other commercial nonmicroscopic tests; importantly,
we found very few instances of false-positive samples, especially with samples from patients recently cleared of
malaria infection. Furthermore, we find that when one uses the quantitative ICpLDH assay, the levels of pLDH
activity closely mirror the levels of parasitemia in both initial diagnosis and while following patient therapy. We
conclude that diagnostic tests based on the detection of pLDH are both sensitive and practical for the detection,
speciation, and quantitation of all human Plasmodium infections and can also be used to indicate drug-resistant
infections.
An ideal diagnostic test for malaria must fulfill various
requirements before it is of utility in the field. It must be
simple, able to be performed rapidly, easy to interpret, and
discriminate between Plasmodium falciparum and the other
malarial species. The test should also be able to be performed on fresh specimens and specimens preserved dry or
frozen. It should have the capability to be formatted for automated clinical instruments and be quantitative to allow the
clinician to follow the progress of drug therapy. Finally, the
test should be inexpensive so that it could be readily used
in endemic areas where the disease is prevalent.
The availability of rapid, simple, and specific diagnostic
tools will assist in the control of malaria by allowing therapy
to be accurately and aggressively administered. Currently,
however, the access to effective diagnosis is limited in much
of the developing world. Microscopy remains the standard,
most cost-effective method. However, it is very labor-intensive, requires a well-functioning, high-quality microscope,
and is performed well only by skilled readers. It is not generally accessible in many regions. Consequently, clinical
therapy is often based on a presumptive diagnosis, which in
many cases is alarmingly inaccurate.1,2
Recently, several new rapid diagnostic tests have been
evaluated; however, none of these meets all the criteria outlined. One of the most prominent alternatives to microscopy
has been a series of tests based on the detection of the histidine-rich proteins (HRPs).3,4 This can be performed with
rapid immunochromatographic formats currently available as
the ParaSightt F test (Becton Dickinson, Meylan, France)
or the Malaquickt test (ICT, Sydney, New South Wales,
Australia).5—7 In practice, these tests are reported to perform
well;5–7 however, the HRP-2 antigen is present only in P.
falciparum infections and thus the test cannot be used for
the detection of P. vivax or other human malarias (P. ovale
and P. malariae). The antigenic activity of HRP-2 has also
been found to persist in the blood stream long after a malarial episode has been resolved by antimalarial therapy.8,9
The possibility that HRP-2 persists after infection would negate the possibility of using HRP-2-based assays for monitoring anti-malarial therapy and detecting drug-resistant infections.
To address the need for a diagnostic test for malaria, we
have been developing tests based on the detection of a soluble glycolytic enzyme that is expressed at high levels in
the blood-stage parasite, Plasmodium lactate dehydrogenase
(pLDH).10–12 We have found that all four human malarial
parasites produce a unique pLDH activity and that this activity follows the level of parasitemia in in vitro cultures.13,14
We have also found that pLDH activity in patient plasma
samples can follow parasitemia measured by microscopy,
indicating that pLDH may be a good marker for following
active malarial infections.14 We have recently described an
enzymatic assay that can specifically measure pLDH in the
presence of human LDH. This test is based upon the fact
that pLDH can use the 3-acetyl pyridine analog of NAD
(APAD1) while human LDH isozymes do not readily use
this analog.14 Thus, pLDH activity can be readily measured
in dilute whole blood lysates using the MalStaty reagent,
which contains APAD1. However, measurement of bona
fide pLDH activity is complicated by the presence of hemoglobin, which interferes with colorimetric detection of
pLDH activity.13,15,16 Consequently, the use of the MalStaty
assay is restricted to use as a simple method for detecting
growth of parasites in in vitro drug susceptibility testing.13
To improve the methods for specifically detecting pLDH, we
have developed a panel of monoclonal antibodies that can
bind to active pLDH. These antibodies have been formatted
into an immunocapture pLDH activity assay (ICpLDH assay) that incorporates immunocapture with specific measurement of pLDH activity with APAD1. When these antibodies
are used in combination, they are capable of binding and
distinguishing pLDH isoforms from either P. falciparum or
P. vivax, allowing the pLDH assay to correctly speciate P.
falciparum from non-P. falciparum malaria. When this assay
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PIPER AND OTHERS
was used with serial blood samples from patients undergoing
drug therapy, we found that pLDH levels correlated dramatically with parasitemia measured by microscopy, making
it possible to monitor therapy by tracking pLDH levels in
the blood. Finally, we have used the panel of antibodies to
pLDH to format a rapid immunochromatographic test, the
OptiMALt assay, which can be used in all clinical setting
to diagnose and differentiate P. falciparum and non-P. falciparum malaria.
METHODS
Collection and microscopy of patient samples. Whole
blood was collected in EDTA by venipuncture from 54 patients from the Hospital for Tropical Diseases (London, United Kingdom) and 30 patients from the Fundacion Centro
Internacional de Entrenamiento e Investigaciones Medicas
(CIDEIM, Cali, Columbia). The 29 patients from the Hospital for Tropical Diseases who were diagnosed with P. falciparum malaria were admitted and treated with quinine/tetracycline. Quantitative thick and thin blood films were prepared and read daily during the course of therapy. Aliquots
of these 92 sequential samples were frozen at 2208C for 3–
12 weeks and then tested with the OptiMALt assay. In addition, samples from 28 patients at this hospital who had a
history of malaria but did not have malaria at the time were
also stored and tested. The ICpLDH assay was tested on a
subset (77) of these patient samples. Thick smears were
stained and inspected using routine procedures for this hospital. A total of 100–200 high-powered, Geimsa-stained
fields (1003) were examined and parasite densities were estimated by determining the percent parasitemia. In patients
undergoing chemotherapy . 200 fields were inspected before declaring the sample negative for parasites.
Samples from patients at CIDEIM were collected at the
time of initial diagnosis and stored at 2208C for 3–12 weeks.
Ten patients were diagnosed with P. falciparum infections,
12 with P. vivax, and eight patients were smear negative and
did not have or develop malaria. These samples were tested
with both the ICpLDH assay and the OptiMALt assay.
For studies at the Hôpital Bichat Claude Bernard (Paris,
France), 16 imported malaria cases from Africa were treated
from January to June 1996. These patients were followed by
drawing blood samples daily (eight patients) or every 6 hr
(eight patients) until parasites were cleared from the peripheral blood as defined by two successive negative blood films.
Five milliliters of venous blood was collected on acid citrate
dextrose (ACD) from which smears and dipsticks were made
within 12 hr and quantitation of pLDH was done within 2–
14 weeks. Blood samples were stored at 48C. Thick smears
stained with Giemsa were inspected at 1003 until 1,000 leukocytes were counted while searching for malarial parasites
on thick blood films. This procedure permits the detection
of $ eight parasites/ml. The number of white blood cells was
used to calculate the parasite density as the number/ml of
blood.
All blood samples were collected during the routine diagnosis of malaria as part of the established procedure for
clinical evaluation of persons suspected of having a malarial
infections. The established procedures of each respective institute were strictly adhered to. Patient samples were then
taken and coded and separated from all other patient information. The results from the routine microscopic inspection
of thin and thick films were also separately coded. These
samples were then evaluated using the various non-microscopic diagnostic methods described here by technicians
who did not know the corresponding microscopy results.
None of the information obtained with the non-microscopic
assays was used as the basis of treatment or entered into the
patients medical records. All procedures and experimental
protocols were approved by the respective human ethics
boards of the participating hospitals. All procedures and
guidelines were also approved by the human ethics committee of Flow, Inc. (Portland, OR) according to the regulations for experimentation with human subjects of the Walter Reed Army Institute of Research (Washington, DC) and
the U.S. Department of Defense.
For analytical tests of sensitivity, in vitro cultures of parasites were quantitated by thin film and concentrated by centrifugation to a hematocrit of 40% by centrifugation. This
sample was then serially diluted in fresh whole blood collected in EDTA.
Generation of monoclonal antibodies. The pLDH was
purified from in vitro cultures of W2 and D6 strains of P.
falciparum maintained as previously described.17 Centrifuged red blood cell lysates were fractionated by Cibacron
Blue chromatography (Sigma, St. Louis, MO) and by ion
exchange chromatography as described previously.17 The purified protein was sequenced and found to match the predicted peptide sequence based on the published cloned
cDNA sequence.18 The amino acid sequence was PKAKIVLVGSGMIGGVMATLIVQKNL. A recombinant pLDH
was expressed in Escherichia coli (XL1-Blue) using the vector pTrc99 (Pharmacia, Uppsala, Sweden) containing the
pLDH open reading frame cloned from the D6 isolate of P.
falciparum as previously described.17
Monoclonal antibodies to pLDH were raised in female
BALB/c mice using pLDH purified from in vitro cultures.
Splenocytes were fused to the myeloma cell line
P3X63AG8.653 and selected in hypoxanthine aminopterin
thymidine (HAT) media according to published procedures.19
Antibodies from secreting cell lines were screened for their
ability to capture recombinant pLDH activity. Positive cell
lines were then cloned to produce the 19G7, 17E4, and 6C9
cell lines. Purified monoclonal antibodies were prepared
from tissue culture supernatants according to published procedures.20
Malaria diagnostic assays. All assay results were obtained and interpreted by a trained technician who had no
knowledge of the sample order or composition.
The ICpLDH assay. Antibody-coated plates were prepared
by incubating the wells of polystyrene, 96-well microliter
plates with solutions of the 19G7 and 17E4 antibodies; wells
were washed and then blocked with bovine serum albumin
dried, and stored at 48C until used. Parallel strips of 19G7coated and 17E4-coated microliter wells were used to determine parasite species. To perform the ICpLDH assay, 150
ml of frozen blood lysate or fresh unlysed blood plus 50 ml
of 2% Triton X-100 were added to the test wells and allowed
to incubate at 258C for 30–60 min. Wells were then washed
three times with phosphate-buffered saline (PBS). MalStaty
(a proprietary reagent containing APAD; Flow, Inc.) supple-
IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA
111
FIGURE 1. The immunocapture Plasmodium lactate dehydrogenase (ICpLDH) assay. A, schematic of the ICpLDH reaction is shown on
the left where pLDH is immobilized by a monoclonal antibody. The enzyme activity is then measured with a coupled enzyme assay in which
in the product of pLDH (APADH) is used to reduce the chromogenic substrate nitro blue tetrazolium (NBT) using the enzyme diaphorase,
thus recycling the otherwise limiting APAD. Shown on the right is the assay using various sources of pLDH as indicated in concert with
microtiter wells coated with the indicated monoclonal antibody. Assays carried out with the 19G7, 7G9, 17E4, and 6C9 monoclonal antibodies.
Blood lysates were allowed to bind to precoated microliter wells for 30 min. Wells were washed and reacted with Malstaty, NBT, and
diaphorase for 20 min. A positive reaction is seen as a dark (blue) reaction product. All antibodies bound the recombinant P. falciparum pLDH
and pLDH from infected blood, The 19G7 and 6C9 antibodies captured pLDH from both P. falciparum and P. vivax infected blood from
various geographic regions while the 17E4 and 7G9 antibodies recognized only the P. falciparum pLDH. NBF 5 nitro blue formazan. B,
dose-response curve of the ICpLDH assay. One hundred or five microliters of blood lysates containing the indicated parasitemias were tested.
These samples were made by diluting infected red blood cells maintained in in vitro cultures with fresh uninfected whole blood. Activity was
quantitated spectrophotometrically and plotted as a function of percentage parasitemia (log versus log). The horizontal line at 0.1 mOD/min
represents the threshold value for positive samples. OD 5 optical density.
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PIPER AND OTHERS
FIGURE 2. Effect of antibody on specific activity of pLDH activity. A, the effect of each of the monoclonal antibodies on pLDH activity
was assessed by incubating 50 ng of purified recombinant pLDH with the indicated concentrations of antibodies in a volume of 50 ml for 20
min at 258C. One hundred fifty microliters of MalStaty was then added and the formation of APADH was measured kinetically at 365 nm.
Reactions containing no pLDH served as the background control that was subtracted from each assay. Enzyme (50 ng) assayed in the absence
of antibody served as the control activity. B, The specific activity of pLDH from six isolates of Plasmodium falciparum and three isolates of
P. vivax was measured by saturating microliter wells with each respective blood lysate and then measuring pLDH activity with the MalStaty
reagent by the formation of APADH. This procedure ensured that the number of pLDH proteins was equivalent among assay wells. Bars show
the mean 6 SD (n 5 3). For definitions of abbreviations, see Figure 1.
mented with nitro blue tetrazolium (NBT) and diaphorase
was added to each well and pLDH activity was monitored
kinetically as an increase in absorbance at 650 nm using a
Thermomax microliter plate reader (Molecular Dynamics,
Sunnyvale, CA) according to procedures previously described.13 The pLDH converts APAD to APADH in the presence of lactate; APADH is in turn used to reduce NBT to a
blue formazan salt that regenerates the APAD for subsequent
reaction with pLDH. This produces a coupled colorimetric
enzyme test that remains linear over long periods of time
due to the ample resupply of substrates. The threshold level
for the assay was set at 0.1 mOD/min, a level that lies beyond the upper range we have observed for negative control
samples. For all the negative samples in this study, the apparent activity was 44 6 9 mlOD/min; the threshold value
was designated as the mean of the negative values plus five
standard deviations. For all the reported results in the figure
graphs, no attempt was made to subtract the assay background; instead, raw results are plotted with the threshold
level above which is considered a positive test result.
The OptiMALt assay. This assay was performed with
blood samples that were either fresh or frozen according to
the manufacturer’s instructions (Flow, Inc.). Briefly, 10 ml
of whole blood samples (fingerstick or venipuncture) collected in EDTA/ACD/heparin and was added to 30 ml of
buffer A into a microliter test well. Buffer A contains a
colored bead conjugated to the pan-specific anti-pLDH antibody (6C9a.). The test strip was then placed into the well
and the entire sample was allowed to wick up the strip. The
test strip was then moved to another test well containing 80
ml of buffer B, which was allowed to wick up the test stick
and clear the hemoglobin color for proper viewing of the
test result.
The ParaSightt F assay. This dipstick assay which captures HRP-2, was performed according to the manufacturer’s
instructions.
Biochemical assays. To test for any affect of anti-pLDH
antibodies on the activity of pLDH, recombinant pLDH (50
ng) was incubated in 50 ml of PBS containing various concentrations of antibody for 20 min at 258C. Activity was then
measured spectrophotometrically after the addition of
MalStaty, NBT, and diaphorase. This assay was also run in
parallel with a standard curve using recombinant pLDH. The
apparent mOD/min generated in the absence of pLDH was
subtracted as the background level. To measure the relative
specific activity of pLDH from P. falciparum and P. vivax,
we purified an equal amount of each enzyme and measured
the apparent activity in the presence of APAD and lactate.
Samples of high parasitemia (0.1%) were lysed and incubated (1 hr at 258C) in microliter wells (in triplicate) coated
with the pan-specific antibody 19G7. Lysates were removed
and replaced with another aliquot of the respective lysate to
ensure that all of the antibody sites of the well were occupied. A dilution series of each sample was also assayed in
parallel to verify well saturation. Wells were then assayed
for pLDH activity in the presence of APAD and lactate and
quantitated by measuring the rate of APADH generation as
an increase of absorbance at 365 nm. Samples 1–7 (Figure
1D) of P. falciparum were from cases of malaria imported
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IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA
FIGURE 3. The immunochromatographic test (OptiMALt assay). A, test schematic in which a colored bead binds various pLDH isoforms
via the pan-specific 6C9 antibody. A bead bound to Plasmodium falciparum pLDH would be captured by both the immobilized P. falciparumspecific 17E4 antibody and the pan-specific 19G7 antibody. A bead bound to P. vivax pLDH would be captured only by the immobilized panspecific 19G7 antibody. In all cases, excess bead conjugate will be captured by immobilized anti-mouse antibody, thus serving as a running
control. B, typical results of the OptiMALt dipstick assay are shown using a control noninfected blood sample, a blood sample infected with
P. vivax, and a sample infected with P. falciparum. For definitions of abbreviations, see Figure 1.
19G7 and 6C9 demonstrated the ability to capture enzymatically active P. vivax pLDH (Figure 1A).
The linearity of the pLDH enzyme assay over a range of
parasitemias was examined (Figure 1B). Red blood cells
from in vitro cultures of P. falciparum were serially diluted
in noninfected human blood to yield samples of a defined
parasitemia with a hematocrit of 40%. To avoid saturation
of the assay, 100 ml or 5 ml of each standardized samples
was lysed and incubated in test wells coated with either the
P. falciparum-specific 17E4 or the pan-specific 19G7 antibody after which pLDH activity was quantified as detailed
in the Methods to produce a visible blue reaction product
(Figure 1B). The log versus log plot shown in Figure 1B
shows the response of the pLDH activity measurement over
a range of parasitemias over four orders of magnitude. As
such, the ICpLDH activity assay was quantitative for samples of 10% parasitemia to , 0.001% parasitemia (50–
500,000 parasites/ml), and this standard curve was easily
modulated by adjusting the original sample size. The pLDH
activity in samples of low parasitemia was not only detected
by spectrophotometric measurements but also by visual in-
from Africa (1–4) and India (5 and 6), and samples of P.
vivax were from India (1) and South America (2 and 3).
RESULTS
Assay format and characteristics. A panel of monoclonal antibodies was raised against a pLDH fraction isolated
from the D6 strain of P. falciparum cultured in vitro as previously described.17 This panel of monoclonal antibodies was
then incorporated into a rapid procedure for the purification
and unambiguous measurement of pLDH activity (Figure
1A). The immunocapture pLDH test (ICpLDH) test was performed with microliter plates precoated with antibodies
19G7, 17E4, 7G9, or 6C9. To test the specificity of the
monoclonal antibodies, samples of both P. falciparum and
P. vivax from various geographic locations were evaluated
as indicated. As expected, all antibodies were capable of
capturing pLDH from P. falciparum-infected blood samples
(the original immunogen) as well as recombinant pLDH. In
contrast, antibodies 17E4 and 7G9 did not capture pLDH
activity from samples infected with P. vivax. Only antibodies
TABLE 1
Performance of the immunocapture Plasmodium lactate dehydrogenase (ICpLDH) assay on Plasmodium falciparum samples from the Hospital
for Tropical Diseases
%
Parasitemia
Parasites/ml
Total
.0.03
0.01–0.03
0.001–0.01
#0.0001
Negative
.1,500
500–1,500
50–500
,5
0
31
11
8
17
10
ICpLDH
Positive
31
10
5
7
0
ICpLDH
Negative
0
1
3
10
10
Sensitivity
100%
91%
62%
41%
0%
Specificity
–
–
–
–
100%
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PIPER AND OTHERS
TABLE 2
Performance of the immunochromatographic test (OptiMALt assay) on Plasmodium falciparum samples from the Hospital for Tropical Diseases
%
Parasitemia
.0.03
0.01–0.03
0.001–0.01
,0.0001
Negative
Parasites/ml
Total
ICpLDH
Positive
.1,500
500–1,500
50–500
,5
0
36
18
11
22
28
36
17
9
13
0
spection of the reaction plate (Figure 1B). These data demonstrate that the result of the ICpLDH assay is proportional
to the amount of pLDH present in the sample. Accordingly,
this assay can measure pLDH levels in blood over a range
that is pertinent to the clinical diagnosis of malaria.
For the ICpLDH assay to be successful, the capture antibody must not inhibit the enzyme activity. To test this, 50
ng of purified recombinant pLDH was incubated with each
of the monoclonal antibodies at the concentrations indicated
in Figure 2A, and activity was then measured using APAD
and lactate. The 17E4, 19G7, or 7G9 antibodies did not diminish the apparent activity of pLDH. In contrast, the 6C9
antibody did produce a marked and dose-dependent inhibition. Consequently, only the 17E4 and 19G7 antibodies were
used in the ICpLDH assay for the course of these studies.
To ensure that the use of APAD and the MalStaty reagent
were appropriate for the detection of pLDH isoforms from
other species, we also determined whether the relative specific activity of P. vivax pLDH with APAD was similar to
that of P. falciparum. For this we purified equal amounts of
pLDH from samples of P. falciparum-infected blood from
various patients using the pan-specific 19G7 monoclonal antibody immobilized onto microliter plates. Immobilized antibodies were then bound with saturating amounts of P. falciparum pLDH and P. vivax pLDH; the immobilized enzymes were then measured for activity with APAD and lactate in parallel with the measurement of a standard curve.
Figure 2B shows that the specific activity of various isolates
of P. falciparum were equivalent and that pLDH from P.
vivax had a specific activity comparable with that of P. falciparum (approximately 85%).
The ICpLDH test was formatted with both the P. falciparum-specific 17E4 antibody and the pan-specific 19G7 antibody and used to evaluate patient samples in the clinical
laboratory. We found that the ICpLDH assay was easy to
perform in the laboratory and very useful in not only diagnosing malaria but also as a quantitative tool for following
therapy. Since our primary interest was to develop a diagnostic test that required minimal laboratory equipment and
that could be operated in a variety of field settings, we also
ICpLDH
Negative
0
1
2
9
28
Sensitivity
100%
94%
81%
60%
0%
Specificity
–
–
–
–
100%
designed an immunochromatographic test for the presence
of pLDH in the blood. In the OptiMALt assay, the pLDH
present in lysed blood first binds to a bead conjugated to the
pan-specific 6C9 antibody. This complex is then allowed to
migrate up a nitrocellulose membrane that contains a series
of immobilized secondary antibodies. The pLDH/antibodybead complex first encounters the 17E4 antibody, which recognizes pLDH from P. falciparum. Immediately downstream
is a second reaction zone containing the pan-specific antibody, 19G7, which can recognize the pLDH isoform of P.
vivax as well as that of P. falciparum. Further downstream
is a third control reaction zone that contains an anti-mouse
antibody that captures the excess reporter bead (Figure 3A).
In this way, the OptiMALt assay can distinguish between
P. falciparum malaria and non-P. falciparum malaria. An
example of the test outcome is shown in Figure 3B. Plasmodium vivax-infected blood produces a single test line
while P. falciparum produces two test lines of comparable
intensity. To determine the analytic sensitivity of the
OptiMALt test we used samples of infected blood of known
parasitemia as was performed for the ICpLDH assay in Figure 1B. The OptiMALt assay was capable of detecting levels of pLDH present in parasitemias , 0.001% or 50 parasites/ml.
Performance of the pLDH assay for diagnosis. To evaluate the ICpLDH assay and the OptiMALt assay we used
whole blood samples from patients admitted to the Hospital
for Tropical Diseases (London, United Kingdom) (Tables 1
and 2) or patients visiting CIDEIM (Cali, Columbia) (Tables
3 and 4). When the analysis was restricted to samples from
the Hospital for Tropical Diseases, we found the sensitivity
of the OptiMALtand ICpLDH assays to be 96% and 92%,
respectively, for samples with $ 50 parasites/ml (0.001%
parasitemia). As a measure of nonspecific reactivity, we also
tested samples that were negative by microscopy. These
samples included either five persons who had just recovered
from malaria after being treated at the Hospital for Tropical
Diseases or persons who had contracted malaria prior to the
date of testing (25 patients from the Hospital for Tropical
Diseases and eight patients from CIDEIM). We also tested
TABLE 3
Performance of the immunocapture Plasmodium lactate dehydrogenase (ICpLDH) assay on Plasmodium falciparum and P. vivax samples from
CIDEIM*
Parasite species
Parsites/ml
range
Total
P. falciparum
P. vivax
Negative
42–129,000
200–39,500
0
10
12
8
ICpLDH positive
17E4 (visually)
* CIDEIM 5 Fundacion Centro Internacional de Entrenamiento e Investigaciones Medicas.
10
0
0
ICpLDH positive
19G7 (visually)
Sensitivity
Specificity
10
12
0
100%
100%
0%
–
–
100%
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IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA
TABLE 4
Performance of the immunochromatographic test (OptiMALt assay) on Plasmodium falciparum and P. vivax samples from CIDEIM*
Parasite species
Parasites/ml
range
Total
P. falciparum
P. vivax
Negative
42–129,000
200–39,500
0
10
12
8
Two reaction bands
(17E4 and 19G7)
10
0
0
One reaction band
only (19G7)
0
12
0
Sensitivity
Specificity
100%
100%
0%
–
–
100%
* For definition of abbreviation, see Table 3.
from these same samples at collection time whether blood
could be stored in dried form prior to testing. Forty microliters of whole blood was absorbed onto sheets of Whatman
(Kent, United Kingdom) 3M paper, dried, and stored at room
temperature. To assay the dried samples, a 0.5-cm2 area of
the paper was soaked in 300 ml of PBS for 20 min. Two
hundred microliters of this solution was used in the ICpLDH
test with the pan-specific 19G7 antibody. In all cases, we
found that the ICpLDH and OptiMALt assays were able to
identify samples from patients infected with either P. falciparum or P. vivax.
Both assays were able to distinguish samples of P. falciparum from samples of P. vivax. All of the samples scored
as P. falciparum positive by the OptiMALt assay (75 from
the Hospital for Tropical Diseases and 10 from CIDEIM)
showed two test bands while all 12 of the P. vivax-positive
samples tested showed only a single test band.
Performance of the pLDH assays for following therapy. Many of the low parasitemia samples seen in these studies were taken from patients who had had previously higher
levels and were undergoing anti-malarial chemotherapy. As
indicated by previous experiments,14 pLDH levels appear to
follow the level of parasitemia. Therefore it is likely that in
samples of low parasitemia from patients undergoing therapy, the levels of pLDH produced would simply be below
the threshold for detection using these methods. We reasoned
that that this might be advantageous since pLDH levels
would correlate with peripheral parasitemia, which is typically used as an index of the severity and progress of disease. This would indicate active infections and would thus
provide a correlate to the disease. Thus, pLDH levels measured by the ICpLDH assay were analyzed as a function of
time during anti-malarial chemotherapy (Figure 4). Among
the 29 patients followed (Table 2), pLDH levels qualitatively
matched the peripheral parasitemias. Importantly, pLDH levels were absent on the day each patient was negative by
microscopy. Five such samples were analyzed in this study.
These samples were also tested with the OptiMALt assay
and found to be negative.
The absence of false-positive results even with samples
from patients who had recent infections may prove to be an
useful aspect of these pLDH-based assays since this feature
allows for the monitoring of therapy with either test. To examine this further, we conducted another clinical study at the
Hôpital Bichat Claude Bernard (Paris, France) with serial
samples from 16 patients undergoing chemotherapy treat-
FIGURE 4. Levels of pLDH in patients undergoing therapy at the Hospital for Tropical Diseases. Admitted patients undergoing anti-malarial
chemotherapy were followed using the ICpLDH assay (top) and Giemsa-stained thick smears (bottom) over the indicated treatment course of
3–5 days. Parasite counts (percentage parasitemia) and enzyme activity (mOD/ml) are plotted on a log scale for three of the 29 patients
evaluated. The dotted line indicates the threshold value for apparent pLDH activity required to interpret the sample as positive. The results of
the OptiMALtimmunochromatographic assay on the corresponding samples (1 5 positive; 2 5 negative) are shown at the bottom. For
definitions of abbreviations, see Figure 1.
116
PIPER AND OTHERS
117
IMMUNOCAPTURE DIAGNOSTIC ASSAYS FOR MALARIA
TABLE 5
Performance of the immunocapture Plasmodium lactate dehydrogenase (ICpLDH) assay, the immunochromatographic test
(OptiMALt assay), and the ParaSightt F test on samples from
patients undergoing chemotherapy at the Hôpital Bichat Claude
Bernard
Test
Number of
samples
Sensitivity
Specificity
ICpLDH
OptiMALt
ParaSightt F
119
128
54
88%
84%
84%
100%
100%
40%
ment for P. falciparum infections. Patients were followed by
analyzing blood samples collected daily or in some cases
every 6 hr. This latter time frame was particularly useful for
defining how responsive pLDH levels were to parasite density measurements. The ICpLDH test was used to quantitate
the level of pLDH in these blood samples. Figure 5A shows
a scatter plot of all of the patient samples in the study measured for apparent pLDH activity as a function of parasite
density assessed by microscopy. The pLDH activity follows
parasite density along a curve similar to that generated with
known standards in Figure 1B. Of the 119 samples tested
by the ICpLDH assay, only six of the microscopy-positive
samples (49 positive samples by microscopy) were less than
the 0.1 mOD/min threshold and thus false negative (88%
sensitive). Among 68 samples negative by microscopy, none
were positive with the ICpLDH assay (100% specific). The
OptiMALt test was run with 127 samples. Among 57 samples positive by microscopy, eight were negative (9, 13, 42,
8, 80, 32, 16, and 16 parasites/ml, respectively) and one
showed a non-distinct pattern (144 parasites/ml) (84% sensitive). Among 70 samples negative by microscopy, none
were positive by the OptiMALt test (100% specific).
We also evaluated a subset of these samples with the
ParaSightt F test, which detects the presence of HRP-2.
This test was run with 42 samples. Among 32 samples positive by microscopy, five showed a non-distinct pattern (85%
sensitive). Among 10 samples negative by microscopy, four
were positive and two had a non-distinct pattern with this
test (40% specific). These results are summarized in Table 5
and show that both pLDH assays were very specific and
yielded results different from the HRP-2 assay when analysis
was restricted to samples of patients undergoing chemotherapy. This is readily seen when analyzing the data of a series
of samples from a particular patient wherein pLDH levels
are absent once peripheral parasitemias are undetectable.
Figure 5B–E details the results of the ICpLDH, OptiMALt,
and ParaSightt F tests on particular patient sets. Figure 5C
shows a serendipitous case in which a blood sample was
collected from the patient prior to the onset of clinical symptoms and prior to the detection of malaria infection by microscopy. The dramatic appearance of peripheral parasites
was mirrored by the level of pLDH measured by both the
ICpLDH and OptiMALt assays. This patient was then treated until the parasites were cleared. This same response of
pLDH levels to increasing parasite density is also demonstrated in Figure 4 (patients 19 and 9). In the case of patient
9, the peripheral parasitemia was 0.01% on days 1 and 2 of
treatment and then increased 10-fold on day 3; likewise,
pLDH levels also increased significantly on day 3. Perhaps
the most dramatic demonstration of how well pLDH levels
followed peripheral parasitemia are exemplified with the patient samples that were collected every 6 hr during chemotherapy (Figure 5D and E). Here, changes in parasite density
were closely monitored and marked changes occurred within
as little as 8–12 hr. Likewise, so did the pLDH levels. Most
importantly, at the very hour a sample was found negative
for parasites (or the parasite density was , 50 parasites/ml),
the result of the OptiMALt assay was also negative. This
was repeatedly observed despite the fact that the initial parasitemias were as high as 30,000/ml (1% parasitemia) as little as 36 hr previously. From these data, a precipitous decrease in pLDH levels may represent a harbinger of successful treatment since all patients in these studies were
cured of malaria and did not experience recrudescence of
reinfection within three months after chemotherapy. In contrast, the ParaSightt F test showed positive results with
samples from patients recently cleared of infection. Thus,
the ability of both pLDH-based tests to monitor the success
of chemotherapy in the short term could offer the potential
to use repeated testing as a means of ensuring treatment
success and identifying drug-resistant infections.
DISCUSSION
We report the development of a series of simple techniques that rapidly diagnose malaria infections in either laboratory or field conditions. Combining the data presented,
we found that for P. falciparum samples with . 50 parasites/
ml (0.001% parasitemia) the immunochromatographic
OptiMALt assay was had a sensitivity of ;96%. Both the
ICpLDH and OptiMALt assays were also capable of detecting low levels of P. vivax (200 parasites/ml) as well as
distinguishing P. vivax infections from P. falciparum. These
assays provide an objective and accurate evaluation of malaria and eliminate the tedium and subjectivity of the thick
and thin smear microscopic examination. Because pLDH
←
FIGURE 5. Levels of pLDH in patients undergoing therapy at the Hôpital Bichat Claude Bernard. Admitted patients undergoing anti-malarial
chemotherapy were followed using the ICpLDH assay, Giemsa-stained thick smears, and the OptiMALt assay. For some samples, the
ParaSightt F test was also used. A, parasite densities (parasites/ml) and pLDH activity (MOD/ml) are plotted on a log scale for the 49 Giemsapositive samples that were measured by the ICpLDH assay. The relationship of pLDH activity to parasitemia is comparable with that produced
experimentally and shown in Figure 1B. The dotted line indicates the threshold value for apparent pLDH activity required to interpret the
sample as positive (0.1 mOD/min). B and C, microscopy, pLDH, and ParaSightt F test results for samples taken from patients undergoing
the indicated treatment course of 3–5 days initiated on day 0. The dotted line indicates the threshold value for apparent pLDH activity required
to interpret the sample as positive (100 mOD/min or 0.1 mOD/min). Results of the OptiMALt assay and the ParaSightt F assay are shown
at the bottom. D and E, same analysis as in B and C except that blood samples were collected every 6 hr after the initiation of drug treatment.
For definition of abbreviations, see Figure 1.
118
PIPER AND OTHERS
levels follow peripheral parasitemia, the rapid ICpLDH test
and the OptiMALt assay both provide the potential to monitor the effectiveness of anti-malarial therapy and thus aid in
the detection of drug-resistant infections, which unfortunately but inevitably will become more prevalent.21
Our evaluation of patient samples was performed in wellcontrolled hospitals with intensive microscopy as the gold
standard for comparison. A remaining issue is how well can
these assays will perform in routine diagnosis in a small
clinic where malaria is prevalent. So far, the OptiMALt assay has been tested under these conditions in Central America and sub-Saharan Africa and found to perform well in
routine diagnosis of malaria.22–24 These techniques also hold
promise for clinical monitoring and will rely on expanding
these analyses to a more comprehensive comparison of
pLDH and HRP-2 levels during the course of disease and
treatment.
Acknowledgments: We thank Kristen Weigle and Marta Acosta for
providing samples of P. vivax, and Tony Moody for invaluable advice and enthusiasm.
Financial support: This work was supported by a Small Business
Innovative Research grant (SBIR) DAMD17-94-C-4037 administered by the Walter Reed Army Institute for Research.
Authors’ addresses: Robert Piper, Department of Physiology, University of Iowa, Iowa City, IA 52242. Jacques LeBras and Sandra
Houzé, Hôpital Bichat Claude Bernard, Paris, France. Laura Wentworth and Michael Makler, Flow, Inc., 6127 SW Corbett, Portland,
OR 97210. Angela Hunt-Cooke and Peter Chiodini, Department of
Clinical Parasitology, Hospital for Tropical Diseases, 4 St. Pancras
Way, London NW1 0PE, United Kingdom.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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