Plasmodium falciparum: effect of anti-malarial drugs onthe production and secretion characteristics of histidine-rich

protein II

Harald Noedl,a,b,c,* Chansuda Wongsrichanalai,a Robert Scott Miller,a

Khin Saw Aye Myint,d Sornchai Looareesuwan,c Yaowalark Sukthana,c

Varee Wongchotigul,c Herwig Kollaritsch,b Gerhard Wiedermann,b andWalther H. Wernsdorferb,c

a Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailandb Department of Specific Prophylaxis and Tropical Medicine, Institute of Pathophysiology, University of Vienna, Kinderspitalgasse 15,

A-1095 Vienna, Austriac Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

d Department of Virology, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand

Received 5 March 2002; received in revised form 12 February 2003; accepted 28 March 2003

Abstract

Plasmodium falciparum histidine-rich protein II (HRP2) is one of the best documented malaria proteins. However, little is known

about the development of HRP2 concentrations under the influence of anti-malarial drugs. HRP2 levels were determined in cell

medium mixture, cellular compartment, and in culture supernatant using a double-site sandwich ELISA specific for HRP2.

Characteristic increases in the overall HRP2 levels were found during the later ring and the trophozoite stages. Throughout the later

schizont development, rupture, and reinvasion, however, the HRP2 levels remained comparatively stable. When the cultures were

exposed to serial dilutions of anti-malarial drugs, a distinct inhibition of HRP2 production was seen with increasing concentrations

of drugs, resulting in sigmoid dose–response curves, similar to those obtained from conventional drug sensitivity assays. HRP2

therefore allows for a very accurate estimation of parasite development and its inhibition and may therefore be ideally suited for use

in drug sensitivity or bioassays.

� 2003 Elsevier Science (USA). All rights reserved.

Index Descriptors and Abbreviations: Plasmodium falciparum; malaria; protozoa; HRP, histidine-rich protein; ELISA, enzyme-linked immunosorbent

assay; drug sensitivity assay; drug resistance; IRBC, infected red blood cell; anti-malarial drug action

1. Introduction

A number of characteristic malaria proteins have been

isolated from Plasmodium falciparum parasites in the

surface or in association with the cytoskeleton of infected

erythrocytes. Proteins containing large amounts of the

amino acid histidine, the histidine-rich proteins, were

among the first plasmodial proteins to be studied in detail.Theywere originally isolated from cytoplasmic inclusions

in asexual stages of Plasmodium lophurae, an avian ma-

laria parasite (Beadle et al., 1994). At least three histidine-

rich proteins synthesized by P. falciparum are found in

infected red blood cells (IRBC). These proteins are knob-

associated histidine-rich protein I (KAHRP1) or histi-

dine-rich protein I (HRP1), which has been associated

with rosetting and the knobs on the surface of IRBC, as

well as HRP2 and HRP3 (Lopez et al., 2000). The exact

role of HRP2 and PfHRP3 has not been fully understoodas yet. HRP2 has been implicated as a heme polymerase

that detoxifies free heme by its polymerization to inactive

hemozoin (Sullivan et al., 1996). It is a histidine- and al-

anine-rich protein localized in several cell compartments

including the cytoplasm of P. falciparum parasites and

Experimental Parasitology 102 (2002) 157–163

www.elsevier.com/locate/yexpr

*Corresponding author. Fax: +43-1-403-83-43-90.

E-mail address: harald.noedl@univie.ac.at (H. Noedl).

0014-4894/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S0014-4894(03)00051-1

gametocytes (Hayward et al., 2000). It was identified in allP. falciparum parasites regardless of knob-phenotype and

was recovered from culture supernatants as a secreted

water-soluble protein (Rock et al., 1987).

Currently the principal application of the detailed

knowledge of HRP2 is its employment for the diagnosis

of malaria by detection of HRP2 antigen by rapid im-

munochromatographic assays. This led to the develop-

ment of a number of malaria rapid diagnostic devices,which offer an alternative to microscopic diagnosis of

malaria caused by P. falciparum (Beadle et al., 1994;

Shiff et al., 1993; Wongsrichanalai, 2001). HRP2 has

been shown to persist and is detectable after the clinical

symptoms of malaria have disappeared and the parasites

have apparently been cleared from the host (Moody,

2002). Its long half-life in vivo and persistence in suc-

cessfully treated falciparum malaria, however, limit theapplication of HRP2-based dipstick or spot tests in

monitoring therapeutic efficacy (Mayxay et al., 2001).

Although a number of studies suggest the existence of a

small percentage of parasite strains that do not produce

HRP2, such a finding would be of limited relevance for

its application in tests that are not related to diagnosis

(Traore et al., 1997; Uguen et al., 1995).

HRP2 can be detected in erythrocytes, serum,plasma, cerebrospinal fluid, and even urine of infected

patients (Genton et al., 1998; Parra et al., 1991; Rodri-

guez-del Valle et al., 1991). In whole blood samples

taken from P. falciparum-infected patients, the HRP2

levels determined by ELISA or the band intensity of

dipstick assays were highly correlated with admission

parasitemia and the stage of parasite development

(Beadle et al., 1994; Kumar et al., 1996).The aim of our study was to investigate HRP2 se-

cretion and production profiles in culture samples to

determine in how far both parameters are growth-re-

lated and affected by anti-malarial drug action. An in-

hibition of HRP2 production by anti-malarial drugs

may offer exciting perspectives for the development of a

wide range of assays, such as drug sensitivity tests.

2. Materials and methods

The HRP2 levels were determined in the supernatant

and the cellular compartment of P. falciparum culture

samples to measure the relative amount of HRP2 that is

being produced and secreted in the course of the

erythrocytic life cycle of the parasite, as well as the effectof anti-malarial drugs on these parameters. Cell and

supernatant samples were obtained in the course of a 72-

h culture in the presence or absence of anti-malarial

drugs and subjected to a HRP2 ELISA. All tests were

conducted at the Department of Immunology and

Medicine, Armed Forces Research Institute of Medical

Sciences (USAMC-AFRIMS) in Bangkok.

Parasites. Two wild strains of P. falciparum, namelyLS97-57 and MM97-09, were mainly employed in the

tests. In addition four other strains (MS94-45, DW98-

110, DW98-131, and MS99-16) were tested in drug-free

cultures. All strains originated from Southeast Asia. In

preceding isotopic drug sensitivity tests, LS97-57 was

found to be mefloquine sensitive, whereas MM97-09 was

resistant (Desjardins et al., 1979). Heparinized blood

samples from clinical falciparum malaria patients werecryopreserved in liquid nitrogen until further use. The

samples were thawed, washed twice, mixed with RPMI

1640 medium (with 10% human serum) at 5% hematocrit

and transferred into cell culture flasks. The cell-medium

mixture was incubated at 37 �C in a 5% CO2, 5% O2, and90% N2 gas mixture until a minimum parasite density of

2% was achieved (Trager and Jensen, 1976). To ensure

synchronous parasite cultures the samples were syn-chronized with 5% sorbitol (Lambros and Vanderberg,

1979). For testing, the samples were diluted to 0.05–0.1%

parasitemia and 1.5% hematocrit with RPMI 1640 me-

dium supplemented with 10% serum. The cell medium

mixture was dispensed into the wells of the microculture

plates in aliquots of 200-lL per well.Culture. Five test series were performed with each

strain: on drug-free plates, on plates predosed with ar-tesunate and mefloquine (mefloquine hydrochloride:

601.3 nmol/L for MM97-09 and 37.6 nmol/L for LS97-

57; artesunate: 160 nmol/L for MM97-09 and 2.75 nmol/

L for LS97-57), and on initially drug-free plates with

addition of artesunate and mefloquine after 24 h. The

plates were incubated at 37 �C for 72 h in a gas mixture(5% CO2, 5% O2, and 90% N2). Every 12 h, 100 lL ofthe upper layer of the supernatant was carefully pipettedout of two wells of the culture plates. The remaining cell

medium mixture in the wells (at 3% hematocrit) was

frozen as a separate sample. Furthermore, another two

wells each were used to make thick and thin films for

microscopic evaluation of parasite density and devel-

opmental stage.

Drug tests. In addition, the samples were exposed to

ascending concentrations of artesunate and mefloquine(mefloquine hydrochloride 9.4–601.3 nmol/L; artesunate

0.7–44.0 nmol/L) to determine 50 and 90% inhibitory

concentrations. Serial twofold dilutions (7 concentra-

tions and one drug-free control well) of the drugs (25 lL/well) were dispensed into standard 96-well microculture

plates and 200 lL of cell medium mixture was added toeach well. The plates were then incubated for 72 h in a

gas mixture (5% CO2, 5% O2, and 90% N2) at 37.5 �C.They were subsequently freeze–thawed twice to obtain

complete hemolysis and tested in the HRP2-ELISA to

obtain growth estimates for each drug concentration.

ELISA. HRP2 was quantified in the supernatant and

cell samples using a commercial ELISA Test Kit (Ma-

laria Ag CELISA, Cellabs, Brookvale, NSW). One

hundred microliters of the hemolyzed (freeze–thawed)

158 H. Noedl et al. / Experimental Parasitology 102 (2002) 157–163

culture samples was diluted and transferred into theELISA plates precoated with monoclonal antibodies

against HRP2 (capture antibody of IgM class; code:

CPF4) and incubated at room temperature for 1 h in a

humid chamber. Subsequently the plates were washed

four times with the provided washing solution, and 100

lL of the diluted Ab-conjugate (indicator antibody ofIgG1 isotype; code: CPF6) were added to each well.

After further incubation for 1 h, the plates were washedfour times and 100 lL of the diluted TMB chromogen(1:20) was added to each well. The plates were then in-

cubated for another 15min in the dark, and 50 lL of thestopping solution was added. Colorimetric analysis was

performed with an ELISA microplate absorbance reader

(SpectraMAX 340 Microplate Spectrophotometer,

Molecular Devices, Sunnyvale, CA) at an absorbance

maximum of 450 nm. As there was no absolute standardavailable for HRP2, a unit system was introduced,

where 100 working units per microliter (100WU/lL)equal the concentration of HRP2 found in the standard.

All HRP2 values are therefore given relative to the

HRP2 culture standard provided by Cellabs, Australia.

This approach allows for a simple comparison of HRP2

concentrations between test series.

Microscopy. Thick and thin blood smears were madefrom all samples (every 12 h) and Giemsa-stained. In

thin films the number of parasites was counted relative

to 10,000 erythrocytes and the parasite density was

calculated. In thick films the percentage of early rings

(0–12 h), late rings (12–26 h), trophozoites (26–38 h), and

schizonts (38–48 h) was counted among 200 parasites

(Field et al., 1963; Silamut and White, 1993).

Statistical analysis. The optical density (OD) valuesfrom the microplate absorbance reader were adapted to

normal curves obtained from serial dilutions of a HRP2

standard (Cellabs, Australia) and transformed into

HRP2 values based on a four-parameter curve model

(Softmax Pro 2.1.1, Molecular Devices, USA). HRP2

concentrations in the cellular compartment were calcu-

lated from known concentrations and defined hemato-

crit in the cell medium mixture. A nonlinear regressionmodel was used to calculate 50 and 90% inhibitory

concentrations in drug sensitivity tests.

3. Results

Two wild strains of P. falciparum were subjected to

72 h of culture with and without the addition of anti-malarial drugs. Another four samples were tested in

drug-free culture.

MM97-09. When incubated on drug-free plates, the

initial parasitemia remained relatively stable within the

first 24 h, increased 10.4-fold after completing the first

cycle (48 h), and finally to 1.1% (13.75-fold) after 72 h

(Table 1). The parasites reached a level of development

comparable to the starting point after 48 h (mostly earlyrings) which developed to predominantly trophozoites

(43%) and schizonts (31%) at the end of the incubation.

The overall HRP2 concentration in the cell medium

mixture rose 3.1-fold in the first 12 h and remained rel-

atively stable until reaching the ring stage again (36 h).

Subsequently it rose to eight times the initial value after

48 h and to 36.6 times after 72 h (Fig. 1). Throughout the

culture period, the most significant increase in HRP2was found in the late ring and trophozoite stages. At the

schizont and rupture stages (24 and 36 h), a minor de-

crease was found in cellular HRP2 concentrations

(erythrocytes) most probably due to continuous secre-

tion into the supernatant, which showed a relatively

constant increase throughout the culture time.

Under the influence of 11.0 nmol/L artesunate, the

parasitemia remained within narrow limits (0.6–0.8 %)throughout the 72-h incubation period (Table 2). The

parasites developed from predominantly (90%) early

rings to 48% early and 52% late rings after 24 h. After

36 h, all parasites showed a high degree of degeneration

(loss of cytoplasm and nuclear degeneration) which

hindered detailed classification. Degenerated parasites

were therefore summarized as small rings. Even after

Table 1

HRP2 concentrations (units/lL) in the cell medium mixture (CMM), cellular compartment (CC), and culture supernatant (SN) in relation to

parasitemia (% infected red blood cells, IRBC) and developmental stages of MM97-09

H0 H12 H24 H36 H48 H60 H72

HRP2

CMM (WU/lL) 4.08 12.56 11.05 8.80 32.76 136.51 149.20

CC (WU/lL) 271.70 835.51 721.53 409.20 2122.98 9019.75 9823.05

SN (WU/lL) 0.00 0.03 0.23 0.69 0.93 1.23 1.88

Parasitemia

% IRBC 0.08 0.08 0.10 0.45 0.83 1.10 1.10

Development

Early rings (%) 89 20 3 86 99 66 2

Late rings (%) 11 70 25 3 0 22 24

Trophozoites (%) 0 10 22 1 0 12 43

Schizonts (%) 0 0 50 10 1 0 31

Samples were taken every 12 h over a culture period of 72 h.

H. Noedl et al. / Experimental Parasitology 102 (2002) 157–163 159

72 h, none of the parasites had developed to trophozo-

ites or schizonts. Correspondingly the overall HRP2

values in the cell medium mixture increased 1.4-fold

within 24 h and remained very stable thereafter (between4.94 and 5.88WU/lL) until the end of the incubation.The maximum levels therefore reached only 3.9% of

those in drug-free culture samples. In the cellular com-

partment the concentration decreased after 36 h due to

the continuous secretion into the supernatant, where the

HRP2 concentration slowly increased throughout the

culture period and reached values not very much lower

than those in the drug-free culture. At 48 h, the cellularconcentration was therefore only 349 times higher than

in the supernatant (as compared to 2280 times in drug-

free culture).

When mefloquine (601.3 nmol/L) was added to the

culture the parasites developed somewhat further (to

19% early and 81% late rings within 24 h). The overallHRP2 in the cell medium mixture consequently in-

creased to 7.26WU/lL (1.8-fold) within 24 h and re-mained stable thereafter (6.37–7.40U/lL). The peaklevels therefore reached only 5% of those in drug-free

cultures, matching parasite densities of 5.5% of the

values in drug-free culture at 72 h. In general the results

for mefloquine closely resemble those for artesunate,

except that the somewhat slower action of mefloquinelead to a further development of the parasites and

therefore to slightly higher HRP2 levels.

When drugs were added after 24 h to the initially

drug-free culture, the parasites generally developed to

about 50% schizonts within the first 24 h and showed a

high degree of degeneration over the following 48 h. The

development of HRP2 concentrations was almost iden-

tical to the one found in drug-free culture in the first24 h. The maximum HRP2 concentrations reached 11.59

(artesunate) and 12.65WU/lL (mefloquine), corre-

sponding to 7.8 and 8.5% of those in drug-free cultures.

The constant secretion into the supernatant led to de-

creasing cellular concentrations due to a shift in the

distribution within the cell medium mixture from the

cellular compartment to the supernatant.

When the culture samples were exposed to ascendingconcentrations of artesunate (0.69–43.96 nmol/L), a

distinct inhibition of parasite growth was observed at

concentrations above 2.5 nmol/L which reached values

of almost 100% inhibition below 25 nmol/L (Fig. 2).

From the resulting sigmoid concentration–response

curve, 50 and 90% inhibitory concentrations (IC50 and

IC90) of 4.52 and 9.62 nmol/L were calculated. The

corresponding IC50 and IC90 values for mefloquine were131.12 and 236.62 nmol/L, respectively.

LS97-57. The findings for LS97-57 were similar to

those for MM97-09. LS97-57 was 100% synchronized at

the start of the culture. The parasitemia increased only

6.2 times to a maximum parasitemia of 0.26%. The

initial HRP2 concentrations were considerably lower

Table 2

HRP2 concentrations (units/lL) in the cell medium mixture (CMM), cellular compartment (CC), and culture supernatant (SN) in relation to

parasitemia (% infected red blood cells, IRBC) and developmental stages of MM97-09 under the influence of 11.0 nmol/L artesunate

H0 H12 H24 H36 H48 H60 H72

HRP2

CMM (WU/lL) 4.08 5.10 5.65 5.88 5.80 5.07 4.94

CC (WU/lL) 271.70 338.17 370.43 352.96 325.43 257.09 240.34

SN (WU/lL) 0.00 0.027 0.095 0.59 0.93 1.23 1.35

Parasitemia

% IRBC 0.08 0.08 0.08 0.07 0.07 0.06 0.06

Development

Early rings (%) 90 66 48 100 100 100 100

Late Rings (%) 10 34 52 — — — —

Trophozoites (%) 0 0 0 0 0 0 0

Schizonts (%) 0 0 0 0 0 0 0

Due to the drug-related degeneration of the parasites, all small forms were summarized under �early rings� after 24 h. Samples were taken every12 h over a culture period of 72 h.

Fig. 1. Development of HRP2 levels of MM97-09 in the cell medium

mixture in relation to parasitemia (IRBC) and the development stage

of the parasites over a total culture period of 72 h. The continuous line

(HRP2) depicts the development of HRP2 in drug-free cultures, the

intermittent line (HRP2 ART) the development of HRP2 with the

addition of 11.0 nmol/L of artesunate. The percentage of early rings

(RINGS) is shown as an indicator of the parasite life cycle in drug-free

culture. All values are given in percentage of the maximum values.

160 H. Noedl et al. / Experimental Parasitology 102 (2002) 157–163

than those observed with MM97-09. This is consistent

with the lower parasitemia and the earlier stage of de-

velopment. The overall HRP2 concentration in the cell

medium mixture rose 4.9-fold within 24 h (the major

increase corresponds to the late ring and trophozoite

stage) and to 5.8 times the initial value after 48 h. With

the development of late rings and schizonts over the

following 24 h, the concentration rose 4.7-fold. Theconcentration in the cellular compartment reached levels

101 times higher than those in the culture medium after

48 h and 535 times higher after 72 h reflecting a slower

increase in the supernatant.

With the addition of artesunate (2.75 nmol/L) the

parasitemia remained very stable throughout the incu-

bation period. Within 24 h 20% developed to late rings

but none ever reached the trophozoite stage. The overallHRP2 concentrations in the cell medium mixture

reached a maximum after 48 h (7% of the maximum in

drug-free culture), a value considerably below those al-

ready attained after 12 h without the addition of drugs.

The cellular concentrations were 114 times higher than

in the supernatant after 48 h and 34 times after 72 h due

to relatively constant secretion. The results for mefloq-

uine (37.6 nmol/L) were similar. The parasitemia neverattained values above 0.6% and the overall HRP2

reached its highest value after 72 h (7.1% of the corre-

sponding value in drug-free culture).

When artesunate or mefloquine were added after

24 h, the undisturbed growth led to 45% trophozoites

and 15% schizonts within 24 h with relatively little de-

velopment thereafter and with maximum parasite den-

sities of 0.6 (artesunate) and 0.1% (mefloquine). Thecorresponding HRP2 values in the cell medium mixture

showed increases comparable to drug-free cultures

within the first 24 h (4.2–6.1-fold) and remained rela-

tively stable for the rest of the incubation period at

values of 11.6–20.5% of the maximum values in drug-free cultures.

When exposed to ascending drug concentrations a

substantial inhibition was already observed at relatively

low concentrations. The parasite growth was reduced by

50% (IC50) and finally by 90% (IC90) by artesunate

concentrations of 1.04 and 3.47 nmol/L, respectively.

Similarly the IC50 and IC90 for mefloquine were also

lower with 10.14 and 28.77 nmol/L correspondingly.MS94-45, DW98-110, DW98-131, and MS99-16.

These strains were only tested in drug-free cultures.

They showed a 5–9.4-fold increase of parasitemia and

generally started as ringforms that developed to pre-

dominantly late rings and trophozoites after completing

1.5 cycles (72 h). Corresponding to the other two sam-

ples, characteristic increases in overall HRP2 concen-

trations were found during the development to late ringsand trophozoites with relatively stable levels in the

schizont and rupture phases. When measured over a full

life cycle, the rise in HRP2 corresponded to the increase

in parasitemia.

4. Discussion

HRP2 is one of the best documented malaria pro-

teins. Discovered in the 1970s the application of HRP2,

however, remains restricted to the rapid diagnosis of

falciparum malaria (Beadle et al., 1994; Garcia et al.,

1996; Kilejian, 1974; Wongsrichanalai, 2001). Previous

studies indicate a close correlation between HRP2 levels

in whole blood samples taken from clinical malaria

patients and admission parasitemia as well as parasitedevelopment. The authors concluded that the estimation

of parasitemia or parasite biomass in vivo by the mea-

surement of HRP2 may therefore be a useful tool in

judging the prognosis of falciparum malaria cases (De-

sakorn et al., 1997). We therefore concluded that its

measurement in vitro may offer exciting perspectives for

the development of ELISA-based drug sensitivity and

bioassays.Generally the rise in HRP2 concentrations corre-

sponds to the increase in parasitemia when followed

over a full erythrocytic life cycle. Analogous to the

parasitemia, the rise in HRP2 is not linear. Our results

confirmed that characteristic increases in cellular HRP2-

levels are primarily found during the later ring and the

trophozoite stages (Howard et al., 1986). This corre-

sponds to the developmental phase where the highestincrease in cytoplasm is found. During the later schizont

development, rupture and reinvasion, however, at a time

when the emphasis is on DNA replication and parasite

multiplication, the HRP2 levels were found to remain

relatively stable or even decreased.

In vitro, the ratio of HRP2 concentration in the cel-

lular compartment to that in the supernatant primarily

Fig. 2. Artesunate (ARS) and mefloquine (MEF) dose–response curves

for the strain MM97-09 after 72 h of incubation. Drug response was

calculated from the inhibition of HRP2 production relative to drug-

free controls. The 50% inhibitory concentrations calculated from the

nonlinear regression were 4.52 and 131.12 nmol/L for artesunate and

mefloquine, respectively.

H. Noedl et al. / Experimental Parasitology 102 (2002) 157–163 161

depended on the duration of incubation and the parasitestage. The secretion into the supernatant is slow and

generally lags behind the concentration in the cellular

compartment. There was no obvious increase found in

HRP2 concentrations in the supernatant during schizont

rupture. This suggests that even during schizont rupture

most of the HRP2 remains associated with the parasites

and erythrocytes and is not or only to a very small ex-

tent released into the culture medium.Once the parasite growth was inhibited by anti-ma-

larial drugs, the overall HRP2 in the culture samples

(cellular compartment plus supernatant) remained very

stable at low levels. The inhibition of HRP2 production

directly paralleled the morphological growth inhibition

and seems to be a direct consequence of the inhibition of

the parasitic metabolism. As HRP2 is likely to play an

important role in the detoxification of heme, its pro-duction is naturally closely associated with the parasites

metabolic activity (Sullivan et al., 1996). Minor de-

creases may possibly be attributed to a consumption of

HRP2 owing to its role in the detoxification of heme to

hemozoin.

The secretion of HRP2 into the supernatant, on the

other hand, was found to be relatively independent of

the addition of drugs. Supernatant levels showed a slowbut constant increase over the incubation period due to

the considerably higher concentration in the cellular

compartment, even though the production of HRP2 had

come to a standstill. This may lead to an in vivo per-

sistence of HRP2 in the plasma and in association with

uninfected erythrocytes, and may therefore be one of the

reasons why HRP2 levels are not suited to monitor

clinical treatment success with HRP2-based immuno-chromatographic tests.

Levels in the supernatant or plasma alone are there-

fore not necessarily suited as direct indicators of para-

sitemia or development due to the variable cell/

supernatant ratio. The development of overall HRP2

concentrations in the cell medium mixture, on the other

hand, precisely reflects parasite development and prop-

agation. HRP2 is known for its long biological half-lifeand stability (Cerutti et al., 1999; Sing et al., 2000).

HRP2 levels therefore always provide a cumulative

picture of production over time. Although this is a

known issue related to the use of HRP2-based dipsticks,

particularly in following treatment success, it does not

pose a problem for the assessment of parasite growth

and its inhibition. Once the growth is inhibited by anti-

malarial drugs the overall HRP2 levels remain stablewithin narrow limits. HRP2 therefore allows for an ac-

curate estimation of parasite growth and its inhibition

and may therefore serve as a useful indicator in drug

sensitivity assessment or bioassays.

When exposed to ascending drug concentrations both

samples showed a distinct growth inhibition with in-

creasing amounts of drugs. The inhibition of parasite

development was precisely reflected in the inhibition ofHRP2 production. Similar to traditional in vitro drug

sensitivity assays the growth inhibition determined by

measuring HRP2 levels followed a sigmoid curve. Thus

50 and 90% inhibitory concentrations may easily be

calculated by nonlinear or log-probit regressions.

Drug sensitivity assays based on the measurement of

HRP2 may offer a number of significant advantages

over conventional assays while providing results that aredirectly comparable to those obtained with conventional

tests (Noedl et al., 2002). The relative ease of imple-

mentation, semiautomated processing, the possibility of

replacing isotopic tests, as well as the reduced require-

ments for laboratory equipment and training of per-

sonnel may make an HRP2-based drug sensitivity test a

promising alternative for the future with the potential to

replace traditional assays. Similar to recently publisheddrug sensitivity assays based on the quantification of

lactate dehydrogenase (pLDH) such an assay could

provide simple means of testing the activity of anti-

malarial drugs (Druilhe et al., 2001; Moreno et al.,

2001). A major advantage of a HRP2-based assay may

lie in the ready availability of commercial ELISA Test

Kits, which considerably simplifies the establishment

and standardization of such an assay.

Acknowledgment

This work was supported by the US Department of

Defense Global Emerging Infection Surveillance (GEIS)

Program.

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