ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2002, p. 1658–1664 Vol. 46, No.

6
0066-4804/02/$04.00⫹0 DOI: 10.1128/AAC.46.6.1658–1664.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Histidine-Rich Protein II: a Novel Approach to Malaria Drug

Sensitivity Testing
Harald Noedl,1,2* Walther H. Wernsdorfer,2 Robert Scott Miller,1 and Chansuda Wongsrichanalai1
Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS),
Bangkok, Thailand,1 and Department of Specific Prophylaxis and Tropical Medicine,
Institute of Pathophysiology, University of Vienna, Vienna, Austria2
Received 7 January 2002/Returned for modification 8 February 2002/Accepted 27 February 2002

The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by
Plasmodium falciparum, is closely associated with the development and proliferation of the parasite and
therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of
the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple
enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro
assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of
parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial
drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The
complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and
requires relatively little technical equipment. In correlation analysis, the results closely paralleled those
obtained by the isotopic assay (R ⴝ 0.892; P < 0.0001) and World Health Organization schizont maturation
tests (R ⴝ 0.959; P < 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to
establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies
to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard
operating procedures, updated information, and analytical software are available from http://malaria.farch.net.

Antimalarial drug resistance has become an issue of utmost
importance for the control of falciparum malaria worldwide.
Individual treatment regimens as well as malaria control strat-
egies therefore need to be based on profound knowledge of
drug sensitivity. There are basically two approaches to the
assessment of the antimalarial drug susceptibility of Plasmo-
dium falciparum: in vivo and in vitro assays. The most com-
monly used in vitro assays, the isotopic assay, the World Health
Organization (WHO) microtest, and a number of parasite lac-
tate dehydrogenase-based assays, have their advantages but
also have a number of known drawbacks (1, 7, 8, 12, 14, 24, 26).
It was the aim of this study to develop an in vitro assay that is
easy to establish and to standardize, that has a high sensitivity,
and that requires little technical equipment, similar to the
WHO test, but that is also as reproducible and that can be
performed as fast as the isotopic assay.
Histidine-rich protein II (HRP2) is a naturally occurring
histidine- and alanine-rich protein localized in several cell
compartments including the cytoplasm of P. falciparum. Re-
cent studies have implicated HRP2 as an important factor in
the detoxification of heme (11, 16, 17, 21). HRP2 was identi-
fied in all P. falciparum strains regardless of knob phenotype
and was recovered from plasma and culture supernatants as a
secreted water-soluble protein (20). It is found as concentrated
packets in the host erythrocyte cytoplasm and on the infected
erythrocyte membrane (9). HRP2 is a common target for rapid
* Corresponding author. Mailing address: Department of Specific
Prophylaxis and Tropical Medicine, Institute of Pathophysiology, Uni-
versity of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
Phone: 43-1-40204 80. Fax: 43-1-403 83 43 90. E-mail: harald.noedl
@univie.ac.at.
diagnostic tests for malaria (3). Its long half-life in vivo and its
persistence in patients with successfully treated falciparum ma-
laria, however, limit the application of HRP2-based dipstick or
spot tests for the monitoring of therapeutic efficacy (13). For in
vitro drug susceptibility assays, on the other hand, the stability
of this protein may be a major advantage. Similar to schizont
maturation and hypoxanthine uptake, levels of HRP2 produc-
tion may vary between parasite strains. As drug sensitivity tests
are internally controlled, however, this is not an issue in this
context. The amount of HRP2 found in culture samples is
closely associated with parasite growth and increases with par-
asite development and multiplication (6). We therefore con-
cluded that HRP2 might be an excellent indicator of parasite
growth and its inhibition by antimalarial drugs. The availability
of commercial HRP2 enzyme-linked immunosorbent assay
(ELISA) kits for the quantification of P. falciparum HRP2 is an
additional distinct advantage and may make implementation of
this assay faster than that of any other malaria in vitro drug
sensitivity test.
MATERIALS AND METHODS
A total of 20 cryopreserved, culture-adapted strains of P. falciparum originat-
ing from three Asian countries (collected from 1994 to 2001 in Thailand, My-
anmar, and Bangladesh) were subjected to the new HRP2 ELISA as well as to
isotopic and morphological drug susceptibility assays. The strains were selected
according to their previously established sensitivities to test isolates with a broad
range of antimalarial drug susceptibilities. All tests were performed at the De-
partment of Immunology and Medicine, Armed Forces Research Institute of
Medical Sciences, Bangkok, Thailand. After the samples were thawed, they were
washed and mixed with RPMI 1640 medium (with 10% human serum) at 5%
hematocrit, and the mixture was transferred into cell culture flasks. The cell-
medium mixture (CMM) was incubated at 37.5°C in a 5% CO2–5% O2–90% N2
gas mixture until a minimum parasite density of 0.5% (for the HRP2 and the
morphological assays) or 2% (for the isotopic assay) was achieved (22).

1658
VOL. 46, 2002
HRP2 drug susceptibility assay. The samples from the continuous cultures
were washed and resuspended with RPMI 1640 (with 10% human serum) and
uninfected erythrocytes to obtain 0.05% parasitemia and 1.5% hematocrit. The
samples should contain predominantly ring forms and generally do not require
synchronization. Stock solutions (1 mg/ml) in 70% ethanol were prepared from
mefloquine hydrochloride (Mr, 414.778), chloroquine diphosphate (Mr, 515.867),
quinine sulfate dihydrate (Mr, 785.06), and artesunate (Mr, 384.425). These were
diluted with RPMI 1640 in order to obtain the desired test concentrations
(mefloquine hydrochloride concentrations, 9.39 to 601.28 nM; chloroquine
diphosphate concentrations, 27.70 to 1772.55 nM; quinine sulfate dihydrate
concentrations, 54.61 to 3495.27 nM; artesunate concentrations, 0.34 to 22.02
nM). Serial twofold dilutions (seven concentrations and one drug-free control) of
the drugs (25 ␮l/well) were dispensed into standard 96-well microculture plates
(Costar 3599 plates) manually or by a semiautomated microdilution technique,
and 200 ␮l of CMM was added to each well. The plates were then incubated for
72 h in a gas mixture (5% CO2, 5% O2, 90% N2) at 37.5°C. They were subse-
quently frozen-thawed twice to obtain complete hemolysis. After the incubation
the plates were processed immediately or were stored at ⫺30°C until further
processing.
HRP2 ELISA. A commercial HRP2 ELISA kit (Malaria Ag CELISA; Cellabs
Pty. Ltd., Brookvale, New South Wales, Australia) was used for the quantifica-
tion of HRP2 in the culture samples. One hundred microliters of each of the
hemolyzed culture samples was transferred to the ELISA plates, which are
precoated with monoclonal antibodies against P. falciparum HRP2 (capture
antibody of the immunoglobulin M class; code CPF4), and the plates were
incubated at room temperature for 1 h. Subsequently, the plates were washed
four times with the washing solution provided with the test kit, and 100 ␮l of the
diluted antibody conjugate (an indicator antibody of the immunoglobulin G1
isotype; code CPF6) was added to each well. After incubation for an additional
1 h, the plates were washed four times and 100 ␮l of diluted (1:20) chromogen
(tetramethylbenzidine) was added to each well. The plates were then incubated
for another 15 min in the dark, and 50 ␮l of the stop solution was added.
Spectrophotometric analysis was performed with an ELISA plate reader (Spec-
traMAX 340 Microplate spectrophotometer; Molecular Devices, Sunnyvale, Cal-
if.) at an absorbance maximum of 450 nm. The optical density values correspond
to the amount of HRP2 found in the culture samples and provide consistent
indicators of parasite growth. The complete ELISA may easily be performed
within less than 3 h, largely independent of the number of samples to be tested.
Potential modifications to the HRP2 assay. The HRP2 assay may be adjusted
to meet the requirements of individual laboratories (however, all test parameters
should be kept constant within a study). In its current form the basic procedure
for the assay was developed with culture-adapted, cryopreserved P. falciparum
strains. Although essentially the HRP2 assay may also be performed with fresh
isolates, so far there are no data from field trials with fresh isolates. The valida-
tion of the assay with fresh isolates will therefore be an important issue for a
future application in field studies. From our experience the assay may also be
performed with an incubation time of 48 h. However, the 72-h incubation allows
the testing of slowly acting drugs without the need for changes to the protocol
and gives a success rate close to 100%, even with slowly growing parasite strains.
We successfully conducted experiments with initial parasite densities as low as
0.01%. However, the range that yielded the best results was 0.02 to 0.07%.
Samples with an initial level of parasitemia of more than 0.1% need to be diluted
accordingly, either before incubation (with uninfected erythrocytes and RPMI
1640) or before the ELISA is performed (with RPMI 1640). We used 200 ␮l of
CMM and 25 ␮l of drug solution per well throughout this study. As the ELISA
requires only a 100-␮l sample volume, the assay can also be performed with a
reduced amount of CMM. A sample of the CMM may be set aside and frozen
before the start of the incubation to assess the amount of preexisting HRP2. This
sample is also tested in the ELISA, and its optical density is subtracted from
those for the test samples. This allows a very precise estimate of the rise in HRP2
levels to be obtained. If full inhibition of parasite growth can be expected,
however, it may be sufficient to use the lowest value in each test instead.
Isotopic assay. The isotopic assay followed the routine established at the
AFRIMS laboratory based on the method of Desjardins et al. (7, 23, 25). The
samples from continuous culture were diluted to 0.5% parasitemia and 1.5%
hematocrit. The plates were dosed with antimalarial drugs, and 200 ␮l of CMM
was added. The plates were incubated in a gas mixture (5% CO2, 5% O2, 90%
N2) for 24 h at 37.5°C, pulsed with 25 ␮l of [3H]hypoxanthine solution (hypo-
xanthine monohydrochloride), and reincubated for an additional 18 h. The
samples were harvested onto filter paper with a harvesting machine (Filtermate
Harvester; Packard Instruments, Meriden, Conn.), dried, and mixed with 25 ␮l of
scintillation fluid (Microscint 20; Packard Instruments). The scintillation was
HRP2 IN MALARIA DRUG SENSITIVITY TESTING 1659
determined in a microplate scintillation counter (Top Counter NXT; Packard
Instruments).
Morphological assay. A modified WHO schizont maturation assay was used to
morphologically assess parasite development (26). The culture samples were
diluted in the same way as those used for the HRP2 assay (1.5% hematocrit,
0.05% parasitemia). As the morphological assay requires a high degree of syn-
chronism, the growth in the samples was synchronized with 5% sorbitol (10).
Subsequently, 200 ␮l of CMM was incubated for 24 h at 37.5°C on the same
plates used for the HRP2 assay. After removal of the sediment, thick films were
prepared from the cell sediment of each well. These were microscopically eval-
uated by counting the number of schizonts with three or more chromatins per
200 asexual parasites.
Statistical analysis. Individual inhibitory concentrations (the 50% inhibitory
concentration [IC50], IC90, IC99) for all three assays were determined by non-
linear regression analysis. The software used is based on a polynomial regression
model and is freely available from http://malaria.farch.net. The results (IC50)
closely parallel those obtained with other nonlinear estimation programs or
log-probit analysis. Bland-Altman plots were prepared to assess the levels of
agreement between two methods (4). Standard correlation analysis was used to
establish associations between the ICs obtained by the different assays with the
various drugs. The t test was used to evaluate differences between group means.
Nonparametric procedures were used for data that were not normally distributed.
RESULTS
In total, 20 strains were culture adapted and successfully
tested by all three assays. The novel HRP2 drug susceptibility
assay was found to be easy and rapid to perform. It produced
results comparable to those obtained by the other assays.
The geometric mean IC50s determined by the HRP2 drug
susceptibility assay were 65.32 nM (95% confidence interval
[CI], 38.97 to 109.48 nM), 343.86 nM (95% CI, 239.93 to
492.80 nM), 217.39 nM (95% CI, 162.70 to 290.46 nM), and
2.06 nM (95% CI, 1.57 to 2.70 nM) for mefloquine, quinine,
chloroquine, and artesunate, respectively. The geometric mean
ICs and confidence intervals for all three assays are shown in
Table 1. A number of samples (n ⫽ 4) were also tested after
48 h of incubation, and the ICs were similar. The mean differ-
ence in the IC50 at 48 h of incubation compared to that at 72 h
of incubation was ⫺0.05 on the log scale (limits of agreement,
⫺0.368 and 0.267).
No statistically significant differences in the results of the
HRP2 assay in relation to those of the schizont maturation
assay and the isotopic assay were found for mefloquine and
quinine. The chloroquine IC50 determined by the isotopic as-
say and the artesunate IC50 determined by the morphological
assay were significantly lower (P ⬍ 0.001) than the correspond-
ing values obtained by the other two assays.
The data obtained by all three assays were analyzed by
nonlinear regression analysis. Representative graphs illustrat-
ing the pooled data for the responses of all 20 P. falciparum
strains to mefloquine, quinine, chloroquine, and artesunate are
shown in Fig. 1 to 3. An excellent fit of the data to the poly-
nomial regression model was observed with all regressions (all
regressions resulted in squared correlation coefficients higher
than 0.989).
By correlation analysis the pooled results obtained by the
HRP2 drug sensitivity tests with all four antimalarials showed
a distinct association with those obtained by the schizont mat-
uration assay (n ⫽ 20; R ⫽ 0.959; P ⬍ 0.001) and the isotopic
assay (n ⫽ 20; R ⫽ 0.892; P ⬍ 0.001) (Fig. 4 and 5, respec-
tively). The Bland-Altman plots for the agreement of the re-
sults obtained by the HRP2 assay in relation to those obtained
by the other two techniques as well as the mean difference and
limits of agreement are shown in Fig. 6 and 7, respectively.
1660 NOEDL ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Geometric mean IC50s, IC90s, and IC99, with 95% CIs, for mefloquine, quinine, chloroquine, and artesunate determined by the
HRP2 drug susceptibility assay, a modified WHO schizont maturation assay, and the isotopic assaya
IC (nM [95% CI])
Drug and assay (n ⫽ 20)
50% 90% 99%

MEF
HRP2 65.32 (38.97–109.48) 142.29 (91.73–220.71) 189.73 (123.93–290.45)
MORPH 46.20 (28.43–75.08) 95.23 (58.55–154.89) 122.06 (75.29–197.88)
RADIO 70.03 (43.88–112.77) 152.36 (100.49–231.00) 206.83 (144.83–295.38)

QNN
HRP2 343.86 (239.93–492.80) 903.13 (712.46–1,144.83) 1,218.11 (952.20–1,558.29)
MORPH 364.84 (258.76–514.40) 763.97 (571.30–1,021.61) 950.98 (718.54–1,258.61)
RADIO 345.32 (260.43–457.89) 847.99 (661.11–1,087.71) 1,210.67 (970.68–1,509.98)

CHL
HRP2 217.39 (162.70–290.46) 528.89 (395.00–708.15) 739.08 (577.50–945.88)
MORPH 221.28 (160.68–304.74) 421.93 (298.46–596.46) 515.62 (367.13–724.17)
RADIO 117.98 (97.32–143.04) 248.88 (206.11–300.53) 313.51 (259.39–378.93)

ARS
HRP2 2.06 (1.57–2.70) 7.55 (5.00–11.40) 15.60 (10.85–22.43)
MORPH 0.96 (0.82–1.13) 1.48 (1.16–1.90) 2.21 (1.58–3.08)
RADIO 2.82 (2.16–3.67) 5.85 (4.77–7.16) 7.45 (6.27–8.85)
a
Abbreviations: MEF, mefloquine; QNN, quinine; CHL, chloroquine; ARS, artesunate; HRP2, HRP2 drug susceptibility assay; MORPH, a modified WHO schizont
maturation assay; RADIO, isotopic assay.

Highly significant drug-to-drug interactions were found by
the HRP2 assay as well as by the other two assays. The IC50s
of mefloquine obtained by the HRP2 assay were closely cor-
related with those of artesunate (R ⫽ 0.794; P ⬍ 0.001) and
quinine (R ⫽ 0.733; P ⬍ 0.001). Quinine and artesunate activ-
ities were similarly correlated (R ⫽ 0.892; P ⬍ 0.001). The
patterns of the drug-to-drug correlations were identical by the
WHO schizont maturation assay (for mefloquine-artesunate, R
⫽ 0.767 and P ⬍ 0.001; for mefloquine-quinine, R ⫽ 0.555 and
P ⫽ 0.011; for quinine-artesunate, R ⫽ 0.556 and P ⫽ 0.011)
and by the isotopic assay (for mefloquine-artesunate, R ⫽
0.695 and P ⫽ 0.001; for mefloquine-quinine, R ⫽ 0.635 and P
⫽ 0.003; for quinine-artesunate, R ⫽ 0.594 and P ⫽ 0.006).
DISCUSSION
The value of in vitro drug susceptibility data for the devel-
opment of new drugs as well as for epidemiological purposes is
undisputed. The quickening pace of antimalarial drug resis-

FIG. 1. Activities of mefloquine (MEF; R2 ⫽ 0.9974), quinine (QNN; R2 ⫽ 0.9951), chloroquine (CHL; R2 ⫽ 0.9903), and artesunate (ARS;
R2 ⫽ 0.9977) against all P. falciparum strains tested (n ⫽ 20) by the new HRP2 drug susceptibility assay.
VOL. 46, 2002 HRP2 IN MALARIA DRUG SENSITIVITY TESTING 1661

FIG. 2. Activities of mefloquine (MEF; R2 ⫽ 0.9990), quinine (QNN; R2 ⫽ 0.9892), chloroquine (CHL; R2 ⫽ 0.9891), and artesunate (ARS;
R2 ⫽ 0.9970) against all P. falciparum strains tested (n ⫽ 20) by a modified WHO schizont maturation (morphology) assay.

tance in recent years and the need for the monitoring of drug
sensitivity have necessitated the development of new, user-
friendly assays for antimalarial drug sensitivity testing to com-
plement or replace techniques that have been in use for more
than two decades.
The new HRP2 drug sensitivity assay provides a rapid and
simple method for the quantification of antimalarial drug ac-
tion. It combines the advantages of the other two assays, while
it omits most of their disadvantages. In relation to the other
two assays, the HRP2 assay was found to be easier and faster
to perform. The availability of commercial ELISA kits for the
quantification of P. falciparum HRP2 eliminates the need for
standardization of the ELISA procedure, thereby making its
implementation faster than that of any other assay.

FIG. 3. Activities of mefloquine (MEF; R2 ⫽ 0.9985), quinine (QNN; R2 ⫽ 0.9927), chloroquine (CHL; R2 ⫽ 0.9926), and artesunate (ARS;
2
R ⫽ 0.9933) against all P. falciparum strains tested (n ⫽ 20) by the isotopic assay.
1662 NOEDL ET AL. ANTIMICROB. AGENTS CHEMOTHER.

FIG. 4. Scatter plot for the association of individual IC50s (in nanomolar) for mefloquine (squares), quinine (diamonds), chloroquine
(inverted triangles), and artesunate (triangles) determined by the HRP2 drug susceptibility assay and a modified WHO schizont maturation
(morphology) assay (n ⫽ 20; R ⫽ 0.959; P ⬍ 0.001).

Even though all three assays use different end points, the
results of the HRP2 assay closely paralleled those obtained by
the isotopic and the schizont maturation assays. The element
common to all three assays is that they provide measures of
parasite development and growth. Owing to the peculiarities of
each assay, it is virtually impossible to perform all three assays
under identical conditions. In the isotopic assay the parasites
are incubated for 24 h before being pulsed with [3H]hypoxan-
thine (7). After incubation for an additional 18 h the amounts
of incorporation of the tracer into the parasite DNA and RNA
are measured. The isotopic assay therefore quantifies the met-
abolic activity in the second half of the erythrocytic life cycle.
However, it requires a relatively high level of parasitemia
(about 0.5%) to obtain adequate readings. Furthermore, the
isotopic assay has the disadvantage of requiring expensive,
highly specialized equipment and the handling of radioactive
substances.
The schizont maturation assay, on the other hand, deter-
mines the percentage of parasites that develop from rings to
schizonts within 24 h, thus requiring parasites whose develop-
ment is highly synchronized (19). It measures a much earlier
phase of development than the isotopic assay. The microscopic

FIG. 5. Scatter plot for the association of individual IC50s (in nanomolar) for mefloquine (squares), quinine (diamonds), chloroquine (inverted
triangles), and artesunate (triangles) determined by the HRP2 drug susceptibility assay and the isotopic assay (n ⫽ 20; R ⫽ 0.892; P ⬍ 0.001).
VOL. 46, 2002 HRP2 IN MALARIA DRUG SENSITIVITY TESTING 1663

FIG. 6. Bland-Altman plot of the difference in log IC50s for mefloquine, quinine, chloroquine, and artesunate determined by the HRP2 drug
susceptibility assay and a modified WHO schizont maturation (morphology) assay plotted against their mean values. The mean difference in IC50s
on the log scale was 0.112 (limits of agreement, ⫺0.370 and 0.594).

evaluation of the test results makes the assay considerably
more sensitive than the isotopic assay. While it is very econom-
ical, the WHO microtest is particularly labor-intensive and
requires highly trained personnel in order to limit individual
variability caused by human factors.
The HRP2 assay measures the increase in HRP2 levels over
the incubation time, which is most marked during the ring and
trophozoite stages (6). Owing to the sensitivity of the ELISA,
the HRP2 assay is very sensitive but requires a certain mini-
mum incubation time to obtain significant increases in HRP2
levels. A 48-h incubation covers a full erythrocytic life cycle
and, consequently, leads to an increase in the HRP2 concen-
tration that corresponds to the rise in the level of parasitemia.
Synchronism of the growth of the culture samples is therefore
not as crucial. In contrast to the other assays, it does not
involve particularly expensive equipment or the handling of
radioactive substances and leaves little room for error caused
by individual variability.
All three assays consistently differentiated strains sensitive
to mefloquine, quinine, and chloroquine from strains resistant
to these drugs. The distinct parallels between the individual
results obtained by the new assay and both of the conventional
assays reflect the reliabilities of the three test systems when
they are applied to various drugs. The parallels in the drug-to-
drug correlations, such as those found between mefloquine,
artesunate, and quinine, demonstrate the ability of the new test
system to quantify the extent of cross-resistance among anti-
malarial drugs. This is an important issue for in vitro testing, as
cross-resistance may hardly be established in clinical trials (15).
Such activity correlations, especially the correlation between
the activities of artesunate and mefloquine, were evident in all
three assays and are also a common finding in other drug
sensitivity studies, irrespective of the method used (2, 15, 18).
Another major advantage of using HRP2 levels as an indi-
cator of parasite growth and biomass lies in the stability of the
protein (5). The resulting stable background enables the assay

FIG. 7. Bland-Altman plot of the difference in log IC50s for mefloquine, quinine, chloroquine, and artesunate determined by HRP2 drug
susceptibility assay and the isotopic assay plotted against their mean values. The mean difference in IC50s on the log scale was 0.024 (limits of
agreement, ⫺0.538 and 0.578).
1664 NOEDL ET AL.
to detect even minor variations in parasite development. We
used a commercial HRP2 ELISA kit for the quantification of
HRP2 in the culture samples. Basically, the assay can be per-
formed with any P. falciparum HRP2-specific ELISA. Numer-
ous laboratories and companies produce monoclonal antibod-
ies specific for HRP2. The major advantage of using
commercial test kits lies in the simple standardization and
reproducibility of the assay. Its high sensitivity accounts for the
low level of parasitemia used in the drug susceptibility assay.
With initial parasite densities as low as 0.01%, the sensitivity of
the assay is comparable to that of the WHO test. Particularly
in relation to the isotopic assay, which requires a minimum
level of parasitemia of 0.2 to 0.5%, the new assay was consid-
erably more sensitive, an especially important factor for field
application.
The assay may easily be adapted to the requirements of
individual laboratories to further simplify drug sensitivity test-
ing. It may, for example, be used with predosed plates, such as
those provided by WHO for the schizont maturation assay,
avoiding possible quality control issues associated with self-
dosing of the plates and the necessity of obtaining antimalarial
drugs (26). Other simple modifications comprise adjustments
in levels of parasitemia or incubation times.
In addition to its potential value both as a tool for drug
resistance surveillance and as a screen for new antimalarials, a
number of other uses for the assay are under consideration.
Antimalarials may be tested in a dilution matrix or in various
combinations to evaluate potential pharmacodynamic interac-
tions. In pharmacokinetic studies it may be used in bioassays to
measure antimalarial activity in blood specimens obtained af-
ter the administration of antimalarial drugs. Another possible
application is the testing of the inhibitory activities of speci-
mens obtained in the course of vaccine trials.
In conclusion, the HRP2 drug susceptibility assay was found
to be very sensitive, fast and simple to establish, highly repro-
ducible, and easy to perform. It can be used for a wide range
of applications, from epidemiological studies to the screening
of new drugs, and may have the potential to replace traditional
in vitro techniques.
ACKNOWLEDGMENT
This work was supported by the U.S. Department of Defense Global
Emerging Infection Surveillance Program.
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