Comparison of a High-Throughput High-Content Intracellular

Leishmania donovani Assay with an Axenic Amastigote Assay

Manu De Rycker,a Irene Hallyburton,a John Thomas,a Lorna Campbell,a Susan Wyllie,b Dhananjay Joshi,a* Scott Cameron,b
Ian H. Gilbert,a Paul G. Wyatt,a Julie A. Frearson,*a Alan H. Fairlamb,a,b David W. Graya
Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdoma; Fairlamb Laboratory, Division of Biological
Chemistry and Drug Discovery, University of Dundee, Dundee, United Kingdomb

Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is
an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living
forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiologi-
cal situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intra-
cellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a
384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A
panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show
that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 di-
verse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial com-
pounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as
a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay
may render it more suitable for hit-discovery.

T he leishmaniases are a group of parasitic diseases caused by

members of the protozoan trypanosomatid genus Leishmania
that have a significant health impact, primarily in the developing
world (1). Current treatments for visceral leishmaniasis include
antimonials, amphotericin B, miltefosine, and paromomycin. All
of these drugs have substantial issues associated with them, in-
cluding toxicity, emerging resistance, parenteral administration,
high cost and relatively long treatment regimens. Thus, there is an
urgent need for the development of new, better medicines (2).
Leishmanial parasites have two major life cycle stages: the promas-
tigote stage (inside the insect vector) and the amastigote stage
(inside the mammalian host). The amastigotes reside in parasito-
phorous vacuoles, phagolysosome-like compartments with acidic
and hydrolytic conditions (3). Leishmania amastigotes are exqui-
sitely adapted to this environment and subvert many of the innate
defense mechanisms of the host cell (4).
For the purpose of drug discovery, many different assays, using
either promastigotes or amastigotes, have been developed to
screen compound collections to identify new molecules that are
active against Leishmania. The different assays all have advantages
and drawbacks. The most straightforward assays use free-living
parasites to assess the effect of compounds on cell viability. The
main advantage of such assays is that they allow fast and easy
screening of large compound collections, a feature that is often
required for the successful identification of developable hits. Pro-
mastigotes have been used routinely for this purpose (5–10), but
since they represent the insect life-stage, there is the risk of iden-
tifying compounds that do not affect the relevant disease-causing
life-stage. An alternative is to use axenic amastigotes, i.e., amasti-
gotes that have been adapted to grow outside their host cell in a
growth medium that mimics the intracellular conditions (11–14).
Such amastigotes can be used for straightforward high-through-
put screening in a standard growth assay and have the advantage
of being more similar to the disease-relevant parasite stage than
promastigotes (15–17). However, several reports indicate that ax-
enic amastigotes are different from intracellular amastigotes both
in terms of protein expression and in terms of drug susceptibility
(18–21). Thus, hits from screens with free-living parasites always
have to be confirmed using an intramacrophage assay. Further-
more, compounds that interfere with the parasite-host interaction
cannot be identified by such screens (22). With the advent of new
technologies, it has now become possible to increase the through-
put of the traditionally very labor-intensive intracellular amasti-
gote assays. Two main methods are in use for the detection of
intracellular parasites: plate-reader-based methods that rely on
reporter constructs (23–25) and microscopy-based methods that
count parasites directly (8, 26, 27). With the indirect reporter-
based assays, there is a risk of artifacts; compounds may interfere
with the reporter protein or with the substrate, and no informa-
tion is obtained regarding the number of host cells or the distri-
bution of amastigotes in macrophages since these are whole-well
readout assays. Microscopy-based high-content technology en-
ables direct counting and circumvent many of these problems.
However, this method is complex and has only recently been de-
Received 4 December 2012 Returned for modification 19 February 2013
Accepted 1 April 2013
Published ahead of print 9 April 2013
Address correspondence to Manu De Rycker, m.derycker@dundee.ac.uk.
* Present address: Dhananjay Joshi, Advinus Therapeutics, Ltd., Hinjewadi, Pune,
India; Julie A. Frearson, BioFocus, Bethesda, Maryland, USA.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.02398-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.02398-12
The authors have paid a fee to allow immediate free access to this article.

July 2013 Volume 57 Number 7 Antimicrobial Agents and Chemotherapy p. 2913–2922 aac.asm.org 2913
De Rycker et al.
scribed for primary screening of compound libraries (8, 26, 27).
These publications show that screening with promastigotes results
in a large set of hits that show no activity against the intracellular
parasites (i.e., false positives) and that it is possible to identify
compounds that show activity against intracellular amastigotes
but not against free-living promastigotes. We present here an al-
ternative 384-well microscopy-based assay, which we used to
carry out a large-scale comparison to the axenic amastigote assay
to determine the differences in hits identified by both platforms.
MATERIALS AND METHODS
Chemicals. The following chemicals were obtained from Sigma: ampho-
tericin B, ketoconazole, atovaquone, pentamidine, paromomycin, milte-
fosine, phorbol-12-myristate-13-acetate (PMA), 4=,6-diamidino-2-phe-
nylindole (DAPI), formaldehyde, thimerosal, and resazurin. Nifurtimox
was a gift from Bayer, Argentina. Nourseothricin (Lexsy NTC) was ob-
tained from Jena Bioscience. Sodium stibogluconate was obtained from
Merck. HCS Cellmask Deep Red was obtained from Invitrogen. The
small-molecule library used in the present study contained 15,659 diverse
compounds, dissolved at 10 mM in 100% dimethyl sulfoxide (DMSO).
This library was assembled using the criteria for a diverse compound
collection outlined elsewhere (28) and stored under low-oxygen and low-
humidity conditions.
Cells and cell lines. THP-1 (human monocytic leukemia) cells were
obtained from the Health Protection Agency Culture Collections (catalog
no. 88081201). The cells were screened for mycoplasma and maintained
in RPMI plus 10% (vol/vol) heat-inactivated fetal bovine serum (FBS).
MRC-5 pd30 (human fetal lung) cells were obtained from the Health
Protection Agency Culture Collections (catalog no. 84101801) and tested
for mycoplasma. The cells were maintained in minimal essential medium
plus 10% (vol/vol) FBS. L. donovani (MHOM/SD/62/1S-CL2D, LdBOB)
(11, 29) axenic amastigotes and promastigotes were maintained as de-
scribed previously (29). Every 5 weeks, the parasites were cycled between
developmental stages. For amastigote differentiation, 20 ␮l of metacyclic
promastigote culture was inoculated in amastigote media. For the reverse
process, 20 ␮l of dense amastigote culture was inoculated in promastigote
media.
Cloning and expression of eGFP in LdBOB. The enhanced green flu-
orescent protein (eGFP) gene from Aequorea victoria (Swiss-Prot acces-
sion no. AAB02576) was submitted to GenScript (Piscataway, NJ) for
OptimumGene codon optimization to facilitate expression in Leishmania
spp. The resulting synthetic gene (see Data File S1 in the supplemental
material), cloned into pUC57, was flanked by SmaI sites at the 5= and 3=
ends of the gene. The pUC57-eGFP construct was then digested with
SmaI, and the fragment was cloned into the pIR1SAT expression vector
(30), resulting in a pIR1SAT-eGFP construct. Mid-log-phase L. donovani
promastigotes (WT, LdBOB) were transfected with pIR1SAT-eGFP using
the human T cell Nucleofector kit and Nucleofector (Amaxa, program
V-033). After transfection, the cells were allowed to grow 16 to 24 h in
modified M199 medium (29) with 10% FBS prior to drug selection with
nourseothricin (100 ␮g ml⫺1). Cloned cell lines were generated by limit-
ing dilution and maintained in selective medium. Clones expressing high
levels of eGFP were then selected using fluorescence microscopy and con-
verted to axenic amastigotes for use in the screening assays.
Assays. (i) Axenic L. donovani assay. LdBOB axenic amastigotes were
incubated for 72 h with compounds, followed by a resazurin-based read-
out. A more detailed description of the assay can be found in the supple-
mental material.
(ii) Intramacrophage L. donovani assay. An intracellular Leishmania
assay using eGFP expressing LdBOB amastigotes was performed. PMA
differentiated THP-1 cells were infected overnight with axenic amasti-
gotes at a multiplicity of infection of 5. After infection, compounds were
added to the plates, followed by a 3-day incubation and microscopy-based
readout. Further details can be found in the supplemental material.
2914 aac.asm.org
(iii) MRC-5 assay. MRC-5 cells were incubated for 72 h with com-
pounds, followed by a resazurin-based readout. A more detailed descrip-
tion of the assay can be found in the supplemental material.
Data analysis. All data were processed using IDBS ActivityBase. Raw
data were converted into percent inhibitions through linear regression by
setting the high-inhibition control as 100% and the no-inhibition control
as 0%. Quality control criteria for passing plates were as follows: z= ⬎ 0.5,
signal-to-background ratio (S:B) ⬎ 3, and percent coefficient of variation
(%CV) of the no-inhibition control ⬍ 15. The formula used to calculate z= is
as follows: z= ⴝ 1 ⫺ {[3 ⫻ (SDhigh ⫹ SDlow)]/[ABS(Meanhigh ⫺ Meanlow)]}
(31), where SDhigh is the standard deviation of the maximum inhibition con-
trols, SDlow is the standard deviation of the vehicle-treated controls, ABS
stands for absolute value, Meanhigh is the mean of the maximum inhibition
controls, and Meanlow is the mean of the vehicle-treated controls. Curve fit-
ting was carried out using the following four-parameter equation: y ⫽ A ⫹
{[B ⫺ A]/[1 ⫹ (C/x)D]}, where A is the percent inhibition at the bottom, B is
the percent inhibition at the top, C is the 50% effective concentration (EC50),
D is the slope, x is the inhibitor concentration, and y is the percent inhibition.
If curve definition was poor, B was fixed to 100. For the determination of the
reference compound panel potency, all experiments were carried out with a
minimum of three independent repeats.
RESULTS
High-content assay design. Figure 1A shows an overview of the
assay, a more detailed description of which can be found in Ma-
terials and Methods. Plates were imaged on an automated micro-
scope, and the images were analyzed with an image analysis algo-
rithm that we designed using GE Incell Investigator software (Fig.
1B and see also the supplemental material). Examples of curves
obtained for the standard compounds amphotericin B and milte-
fosine are given in Fig. 1C. Note that the increase in THP-1 counts
after treatment with 0.1 to 1 ␮M amphotericin B is a reproducible,
compound-specific effect. The DMSO tolerance of the assay was
determined, and there was no detectable effect of DMSO on the
assay at 0.5% DMSO, the concentration routinely used in our
screens (see Fig. S1 in the supplemental material).
Assay characterization. To further characterize the assay, the
level of Leishmania infection was assessed by looking at the num-
ber of amastigotes per cell and the number of infected cells. Figure
2A shows a typical distribution for the number of amastigotes per
macrophage at 72 h postinfection (mean ⫽ 7.2 and mode ⫽ 2
amastigotes/infected macrophage). The assay also allows determi-
nation of amastigote replication inside the host cell during the
time frame of the assay. Replication was assessed over 7 days by
counting the average number of amastigotes per macrophage at
multiple time points. Since there is effectively no division of the
differentiated THP-1 cells, this gives a good indication of intracel-
lular parasite replication. Over the 7 days we only observed limited
Leishmania growth, as shown in Fig. 2B (doubling time ⬃ 12
days). In contrast, cell division is rapid and exponential in the
axenic system (doubling time, 5.8 h, Fig. 2C).
To assess the suitability of the assay for high-throughput
screening, we determined standard screening statistics across a
large number of plates (Table 1). The data show that the assay is
robust and has low variability and a high S:B. We also monitored
the potency of amphotericin B, which was always included as ref-
erence on potency plates, and found it to be highly reproducible.
Using 384-well plates, we achieved a throughput of 7,680 wells per
batch while maintaining high-quality performance statistics. The
reproducibility of the assay was further assessed by carrying out a
set of potency determinations on two separate occasions, and a
Antimicrobial Agents and Chemotherapy
 Intramacrophage versus Axenic Leishmania

high level of correlation was observed, as shown in Fig. 2D (R2 ⫽

A THP-1 differentiation compound incubation 0.88, slope ⫽ 0.9).
infection
Reference compound panel. A panel of reference compounds
0 72 88 160 (amphotericin B, atovaquone, miltefosine, paromomycin, so-
dium stibogluconate, ketoconazole, pentamidine, and nifurti-
mox) was tested against intracellular amastigotes, as well as axenic
amastigotes, and MRC-5 cells, a human cell-line used for initial
toxicity determinations. The results are shown in Table 2 and are
plate THP-1 cells infect fix and stain
broadly in line with previously reported values (15, 20, 32, 33, 34).
in 384 well plate remove extracellular The clinically used compounds amphotericin B, miltefosine, and
amastigotes +
paromomycin show very similar activities against axenic and in-
add compounds
tracellular amastigotes, whereas most other compounds show a
significant dropoff from the axenic to the intramacrophage assay
B (Fig. 3).
Axenic versus intramacrophage assay. In order to compare
the axenic L. donovani assay with the intramacrophage assay, we
screened a diverse compound set (⬃16,000 compounds) in both
assays. For the axenic screen, a top concentration of 3 ␮M drug
was used. Preliminary experiments with the intramacrophage as-
say showed that the hit rate at this concentration was very low, and
DAPI eGFP instead we screened the set at 50 ␮M in this assay. Figure 4A shows
the hit frequency distributions for both assays. Hits were defined
as showing ⬎70% inhibition of amastigote growth, with the ad-
ditional criterion of ⬍50% inhibition of THP-1 cell counts for the
intramacrophage assay. In the axenic screen 381 hits were identi-
fied (a hit rate of 2.4% at 3 ␮M). In the intracellular assay, a total
of 213 compounds showed ⬎70% inhibition of the amastigote
count (1.4% of the compounds at 50 ␮M). Of these, 128 showed
⬎50% inhibition of THP-1 cell counts, thus leaving 85 hits and a
hit rate of 0.5% (Fig. 4B). Only 17 compounds were hits in both
CMdr
the axenic and the intramacrophage screens, whereas 85 com-
pounds showed activity against the THP-1 cells and the axenic
C Leishmania Inhibition THP-1 Inhibition
amastigotes, indicating that these may be generally toxic com-
Percent Inhibition
Percent Inhibition

100 100
pounds (Fig. 4C).
Miltefosine

70 70 The results of screening cascades performed after the primary

40 40 screens are shown in Fig. 4D and E. Hits from the axenic screen
10 10 were first tested for potency in both the axenic model and the
-20 -20 MRC-5 counterscreen. Compounds showing selectivity over the
0.01 10 0.01 10
Concentration (µM) Concentration (µM)
MRC-5 cells (319 compounds in total) were then progressed to
potency testing in the macrophage assay. To quickly remove inac-
90
Percent Inhibition

100
Percent Inhibition

tive compounds at this stage, we triaged compounds by assessing

Amphotericin-B

70
40
10
-20
0.01 10
Concentration (µM)
FIG 1 Intracellular Leishmania assay. (A) Graphical representation of assay
protocol. The x axis represents the time in hours. Details regarding each step
can be found in Materials and Methods. (B) Image analysis. Pseudocolored
raw images are shown and marked with their respective dyes/fluorophores
(CMdr, cytoplasm marker [HCS Cellmask Deep Red]). The result of image
analysis is shown in the bottom right panel (cytoplasm, green outline; amas-
tigotes, yellow outline). (C) Representative dose-response curves for miltefos-
ine and amphotericin B. Ten-point potency curves for the indicated com-
pounds were created and tested in the intracellular Leishmania assay. Potency
values for the compounds are given in Table 2. The data are from at least four
replicates, shown as purple diamonds, and the curve is fitted to the average
values (blue circles). The data points for Leishmania inhibition that are omit-
ted from the curve fit for miltefosine because of toxicity to THP-1 cells are
marked as boxed crosses.
60
their potency using a single-replicate, 5-point, one-in-three dilu-
30
0
tion curve. Compounds showing activity were then further pro-
-30 gressed to full 10-point duplicate potency testing. The majority of
-60 the axenic hits dropped out at this stage, showing no activity in the
0.01 10
Concentration (µM)
macrophage assay (278 compounds, 87% of the axenic hits). After
application of selectivity rules comparing intramacrophage po-
tency with THP-1 and MRC-5 cell activity, six hits remained.
The 85 hits from the primary intramacrophage screen were
progressed into 10-point potency testing and MRC-5 counter-
screening, after which 7 hits were retained. Of these, 6 were the
same as the ones identified using the axenic cascade. The addi-
tional hit (compound 2) was not very potent against the intracel-
lular amastigotes, with a pEC50 of 5 (EC50 ⫻ 10 ␮M). Since axenic
potency testing was initially carried out with a top concentration
of 16.6 ␮M, we may not have picked up such a low level of activity
in the axenic primary screen. As a result, this compound was re-
tested in the axenic assay with a top concentration of 50 ␮M and
returned a potency value in the same range as seen in the intracel-
lular assay (pEC50 ⫽ 4.7). The structures for all 7 hits from both

July 2013 Volume 57 Number 7 aac.asm.org 2915

De Rycker et al.

A 2500 B 8

2000 7
6

# of cells

AM/MAC
1500
5
1000
4
500 3
0 2
0 20 40 0 2 4 6
# of amastigotes per cell time (days)

C D
100000 8

pEC50 replicate 2
10000 7
cell density

1000 6
100
5
10
4
1
0 20 40 60 80
4 5 6 7 8
time (h) pEC50 replicate 1
FIG 2 Assay characterization. (A) Frequency distribution of intracellular amastigote count. At the end of the intracellular Leishmania assay, the number of
amastigotes in 19,738 infected cells was counted using the automated image analysis algorithm, and a frequency distribution was plotted. (B) Growth curve for
intracellular amastigotes. Cells were plated and infected in 384-well plates, and one plate was fixed at each time point. The values represent the number of
amastigotes per infected cell (i.e., the averages and standard deviations from the entire plate). A linear regression was calculated (R2 ⫽ 0.93). (C) Growth curve
for axenic amastigotes in 384-well plates. Axenic amastigotes were counted at the indicated time points. The y axis shows the number of amastigotes per ml
(⫻1,000). An exponential curve was fitted to time points 0 to 55 h (R2 ⫽ 0.999). The results from triplicate experiments are shown. (D) Replicate potencies. The
potency of 85 compounds was tested on two separate occasions in the intracellular Leishmania assay, and the potencies of the replicates were plotted against each
other. A linear regression was calculated (R2 ⫽ 0.88).

screening campaigns with their potencies in the various assays are macrophage Leishmania assay and the human counterscreen line
given in Table 3. MRC-5. Of these, 145 showed no activity against both THP-1 and
For a total of 110 compounds from this screen, we have carried MRC-5 cell lines. Comparison of the remaining 61 compounds
out full 10-point potency determinations in both the axenic and revealed that, in general, the MRC-5 cells are more sensitive than
the intramacrophage systems. Comparison of these shows that for the THP-1 cells, with 47 compounds showing no activity against
the majority of compounds there was a significant reduction in THP-1 cells while being active against MRC-5 cells, and with 28
potency from the axenic to the intramacrophage assay, with 80 compounds being ⬎3-fold more active against MRC-5 cells than
compounds showing a ⬎10-fold drop in activity from the axenic THP-1 cells (Fig. 5B).
to the intramacrophage assay (includes compounds with no activ-
ity intramacrophage assay) (Fig. 5A). No compound was mark-
edly more potent in the intramacrophage assay.
A total of 206 compounds were tested for potency in the intra-
TABLE 2 Potencies for reference compound panel
pEC50 {⫺log(EC50[M])}a
TABLE 1 Performance statistics of the intramacrophage assay Compound Axenic INMAC THP1 MRC5
Performance statistic Avg ⫾ SD (n)a Amphotericin B 7.06 (0.17) 7.13 (0.05) 5.68 (0.1) 5.08 (0.12)
Z-factor 0.68 ⫾ 0.09 (518) Miltefosine 5.61 (0.2) 6.03 (0.17) 4.33 (0.08) 4.3 (0.09)
%CV 9.8 ⫾ 2.8 (518) Atovaquone 5.37 (0.11) 5.36 (0.12) ⬍3.8 4.56 (0.13)
S:B 90 ⫾ 51 (518) Paromomycin 5.29 (0.37) 5.18 (0.14) ⬍2.5 ⬍2.5
Amphotericin B potency (pEC50) 6.74 ⫾ 0.18 (41) Ketoconazole 7.57 (0.22) 4.89 (0.1) 4.33 (0.07) 4.29 (0.07)
% Infected cells 81 (19,738) Pentamidine 6.3 (0.25) 4.88 (0.21) 4.09 (0.07) 4.8 (0.1)
Throughput 20 ⫻ 384 wells per batch Nifurtimox 5.39 (0.34) 4.62 (0.09) ⬍3.8 3.98 (0.06)
a
Values are averages ⫾ the standard deviation except as noted otherwise in column 1. n Sodium stibogluconate ⬍2.9 3.82 (0.17) 2.98 (0.05) 3.09 (0.13)
a
is the number of plates for the Z-factor, the percent coefficient of variation (%CV), and Results are expressed as the pEC50 with standard deviations given in parentheses,
the signal-to-background ratio (S:B), the number of curves for amphotericin B potency, except for sodium stibogluconate, where the result is given as the ⫺log(EC50[␮g of
and the number of cells for the percent infected cells. Sb/ml]).

2916 aac.asm.org Antimicrobial Agents and Chemotherapy

 Intramacrophage versus Axenic Leishmania

1000 DISCUSSION

fold difference Axenic pEC50 / INMAC

A 384-well intramacrophage Leishmania donovani assay. There
100 is an urgent need for new medicines for leishmaniasis. In terms of
hit discovery, this parasitic disease poses a significant challenge,
since the disease-causing stage resides in an acidic vacuole (the
pEC50

10
parasitophorous vacuole) inside the host’s macrophages. High-
content screening using automated microscopy provides a suit-
1 able platform for assessing such intracellular parasites, and re-
cently both a 96-well and a 384-well high-content assay have been
reported (26, 27). We have also developed a 384-well microscopy-
0.1
based intracellular Leishmania assay with sufficient capacity to
allow primary screening and potency follow-up of medium-sized
small-molecule libraries. To achieve sufficient throughput, ro-
bustness, and reproducibility, a number of alterations were made
FIG 3 Comparison of reference compound activity between the axenic and to the traditional direct counting assay (reviewed in reference 35).
intramacrophage assays. The fold differences between the potencies, as re-
ported in Table 2, are displayed. Typically, ex vivo amastigotes are used to infect primary macro-

A
1000 2500 1000

2000
750 750
1500
500 500
1000
250 250
500

0 0 0
-150 -100 -50 0 50 100 150 -150-100 -50 0 50 100 150 -150 -100 -50 0 50 100 150
Axenic PI AM/MAC PI Macrophage Count PI

B C

INMAC THP-1 THP-1 Axenic INMAC*

85 128 394 437 85 279 17 68

D E
Axenic SP 3 µM INMAC SP 50 µM

381 15279 85 15580

Axenic Pot MRC5 Pot INMAC Pot MRC5 Pot

319 62 (MRC-5 tox) 39 (INMAC EC50 >25µM)

19 (THP-1 tox)
INMAC Pot 15 (MRC-5 tox)
7 HITS
278 (INMAC EC50 >50µM)
26 (THP-1 tox)
9 (MRC-5 tox)
6 HITS

FIG 4 Screening cascades. (A) Frequency distributions for 15,659 compound diverse library screen against axenic (left panel) and intracellular (center and right
panels) Leishmania. The abscissa shows the percent inhibition (PI). Three populations are shown on each frequency diagram: negative controls (red), positive
controls (green), and test compounds (blue). Positive controls on the left panel cannot be seen since they are fully covered by the blue line for the test compounds
at 100%. (B) Venn diagram for intramacrophage primary screen hits. INMAC, compounds that showed ⬎70% inhibition of intracellular Leishmania; THP-1,
compounds that showed ⬎50% inhibition of THP-1 cell count. (C) Comparison of hits from intramacrophage and axenic primary screen. THP-1, compounds
that showed ⬎50% inhibition of THP-1 cell count; axenic, compounds that showed ⬎70% inhibition in the axenic primary screen; INMAC*, compounds that
showed ⬎70% inhibition of intracellular Leishmania and ⬍50% inhibition of the THP-1 cell count. (D and E) Hit selection in the axenic (D) and intracellular
(E) screening cascades. Numbers in green are compounds that progressed to the next step; numbers in red are compounds that were removed from the cascade
(no activity, low activity, or toxicity). SP, single point primary screen; Pot, ten-point dose-response curve in duplicate; EC50, concentration at which a 50% effect
is seen; tox, ⬍10-fold window between antileishmanial EC50 and THP-1 EC50.

July 2013 Volume 57 Number 7 aac.asm.org 2917

De Rycker et al.

TABLE 3 Hits identified in screening campaign

Structure ID Assay pEC50
DDD00023040 INMAC (Leishmania) 5.94
INMAC (THP-1) 4.38
Axenic 6.3
MRC-5 ⬍4.3
DDD00055223 INMAC (Leishmania) 4.99

INMAC (THP-1) ⬍4.3

Axenic 4.73

MRC-5 ⬍4.3

DDD00055671 INMAC (Leishmania) 4.82

INMAC (THP-1) 4.47

Axenic 5.88

MRC-5 ⬍4.3

DDD00077687 INMAC (Leishmania) 4.91

INMAC (THP-1) 4.54

Axenic 5.83

MRC-5 ⬍4.3

DDD00080409 INMAC (Leishmania) 5.23

INMAC (THP-1) 4.38
Axenic 5.93
MRC-5 4.31
DDD00084972 INMAC (Leishmania) 4.7

INMAC (THP-1) ⬍4.3

Axenic 4.63

MRC-5 ⬍4.3

DDD00085002 INMAC (Leishmania) 4.63

INMAC (THP-1) 4.3
Axenic 4.55
MRC-5 4.3

phages. However, this is difficult to achieve for a large-scale
screening assay. Instead, we opted to use axenic amastigotes. The
advantage of axenic amastigotes over metacyclic promastigotes is
that they do not elicit an oxidative burst when entering the mac-
rophage (4). This oxidative burst may result in the killing or sen-
sitizing of the phagocytosed promastigotes, which may potentiate
drug action. In the host, most spreading of the disease occurs
through amastigotes and, as such, they may provide a better dis-
ease model (36). We used L. donovani strain 1S-CL2D, which was
shown to retain in vivo infectivity and exhibits characteristics of ex
vivo amastigotes (11). In terms of the mammalian host cell, it
would be preferable to use primary macrophages, ideally of hu-
man origin. However, it is difficult to obtain a reliable and homo-
geneous supply at a scale suitable for routine screening. Instead,
PMA-differentiated THP-1 cells were chosen, which are consid-
ered a suitable model for human macrophages (37–39). The use of
nonprimary cells for our assay necessitates follow-up experiments
using ex vivo amastigotes and primary macrophages during the
hit-to-lead stage of a compound series since cell-line-specific ef-
fects have been described both for Leishmania strains and host cell
lines (32, 40).
A key challenge in developing the intramacrophage assay

2918 aac.asm.org Antimicrobial Agents and Chemotherapy


A B
7.7
INMAC pEC50
6.7
MRC-5 pEC50
5.7
4.7
4.7 5.7 6.7 7.7
Axenic pEC50
FIG 5 (A) Comparison of compound potency in axenic and intracellular assays. Compounds on the blue line show equal activity in both assays, and compounds
on the right-hand side of the line are more potent in the axenic assay. Red diamonds, no activity in intramacrophage assay (pEC50⬍4.78, 82 compounds); green
squares, activity in both assays (28 compounds). (B) Comparison of compound potency against THP-1 cells and MRC-5 cells. Red diamonds, active against
MRC-5 and no activity against THP-1 (pEC50⬍4.78) (47 compounds); green triangles, activity against both cell lines (10 compounds); blue squares, active
against THP-1 and no activity against MRC-5 (pEC50 ⬍ 4.78) (4 compounds).
proved to be the optimization of the washing step after infection.
Since the axenic amastigotes were able to proliferate outside the
macrophages, it was crucial to remove the majority of amastigotes
that had not been phagocytosed without damaging the THP-1
cells. They would otherwise provide a reservoir for continuous
infection, which would make interpretation of the results difficult.
In addition, their presence would complicate the imaging of the
intracellular parasites. As detailed above, there are two significant
differences with the previously published 384-well high-content
Leishmania assay (27): the use of axenic amastigotes for infection
instead of metacyclic promastigotes and the removal of the infec-
tion inoculum by a washing step. Whether either of these changes
has a major impact on the relevance of the assay is difficult to say
without careful side-by-side comparison, but it is worth pointing
out that the clinically used compounds paromomycin and sodium
stibogluconate show activity in the assay presented here, while
they do not in the assay described by Siqueira-Neto et al. (27). This
may indicate a higher physiological relevance for the assay pre-
sented here.
Advantages of a high-content approach. A microscopy-based
assay, as presented here, has many advantages over plate-reader-
based assays. A microscopy-based assay mimics the traditional
direct-counting assay and provides cell-by-cell analysis. This al-
lows the determination of parameters such as the number of in-
fected cells and the number of amastigotes per individual cell. This
information provides a more direct understanding of the assay
results and reduces the occurrence of artifacts. The assay also re-
ports the number of THP-1 cells that may give a first indication of
toxicity of the compounds tested. However, since differentiated
THP-1 cells are less sensitive to compound toxicity than actively
growing human cells, a secondary mammalian counterscreen is
still advisable (see Fig. 5B). For correct interpretation of the Leish-
mania inhibition data, it is essential to take into account the
THP-1 cell count, since the Leishmania percent inhibition value
becomes unreliable at drug concentrations that kill the majority of
the macrophages. This is demonstrated at the top concentration of
the miltefosine dose-response curve shown in Fig. 1C. This figure
also reveals that amphotericin B has a beneficial effect on the num-
ber of THP-1 cells in the 0.1 to 1 ␮M range. This could potentially
July 2013 Volume 57 Number 7
Intramacrophage versus Axenic Leishmania
6.7
5.7
4.7
4.7 5.7 6.7
THP-1 pEC50
be explained by the known immunomodulatory effects of ampho-
tericin B on THP-1 cells (41) or its ability to activate the survival-
and growth-promoting kinase Akt/PKB (42, 43).
Although the high-content assays discussed here provide a
large amount of information, they do not take into account the
host microenvironment, which could influence parasite behavior
and drug action. Recently, a study demonstrated that, by using
splenic explants and a reporter gene, it is possible to further im-
prove the physiological relevance of the intracellular assay, albeit
at a lower throughput (44).
Comparison of intracellular assay with axenic amastigote as-
say. Other groups have shown that assaying free-living promasti-
gotes results in high false-positive rates (i.e., the promastigote ac-
tivity does not translate into activity against intracellular
amastigotes) and may not identify intracellular stage-specific
compounds (8, 26, 27, 44). Here, we compared the intracellular
assay to the axenic assay, which has been proposed as an alterna-
tive model to promastigotes for primary screening (15, 17, 45).
The rationale behind this comparison was to see whether the ax-
enic assay has a place as a primary screening assay to allow for a
higher throughput, followed by confirmation in the macrophage
assay. In the first instance, we tested a panel of reference com-
pounds in both assays. We found that all clinically used com-
pounds show similar activity in the axenic and intracellular assays,
except for sodium stibogluconate, which shows no activity in our
axenic assay (Table 2). The literature contains a lot of discussion,
and conflicting results, regarding the effect of pentavalent anti-
mony (SbV) on axenic amastigotes, with some groups showing
activity against all stages (46) and others showing activity against
axenic and intracellular amastigotes but not promastigotes (15,
47, 48), while others showed activity only against intracellular
amastigotes (20, 36, 49). The evidence indicates that the reduction
of SbV to trivalent antimony (SbIII) is critical for toxicity and that
this only happens in fully differentiated amastigotes, so it is pos-
sible that our axenic amastigotes retain some promastigote traits,
including an inability to reduce SbV.
This small panel of reference compounds does not allow us to
draw any conclusions regarding the general relationship between
the axenic and intracellular assays. To do this, we carried out a
aac.asm.org 2919
De Rycker et al.
parallel primary screen of a library of nearly 16,000 diverse com-
pounds in both systems. We observed high hit rates in the axenic
assay compared to the intracellular assay, even though we
screened at much lower compound concentrations. As expected,
many of the axenic hits did not show activity in the intracellular
assay, and the compounds that did show activity across both plat-
forms showed a significant dropoff of activity from the axenic
assay to the intracellular assay, without any clear correlation. After
progressing the compounds through the two screening cascades
(Fig. 4D and E), we obtained the same hit compounds. Our ob-
servations allowed us to draw several important conclusions. (i)
The axenic assay is much more sensitive than the intracellular
assay, and the lack of correlation between the two assays limits the
use of the axenic assay as a predictor of intracellular activity. (ii)
The use of the axenic assay as a primary platform, followed by
intracellular follow-up, is highly inefficient. The cascade takes a lot
longer (7 weeks versus 4 weeks), and the majority of the com-
pounds that progressed to the intracellular assay showed no activ-
ity, so that valuable throughput is wasted. (iii) When stringent
potency criteria are applied to axenic hits before progression to the
intracellular assay, there is a significant risk of losing interesting
compounds, since there is no correlation between axenic and in-
tracellular potency. Thus, it seems clear that running the intracel-
lular assay as a primary platform is the preferred route for finding
new hits.
Assay type versus compound mode of action. It is worth con-
sidering why so few axenic hits translate into intracellular ac-
tivity. There are inherent differences between the transcrip-
tomes and proteomes of axenic and intracellular amastigotes
(19, 50), and these could affect drug action. Two other obvious
differences are rate of replication and free-living versus intra-
cellular localization. In the assays described here, we see fast
exponential growth of the axenic amastigotes and almost no
growth of the intracellular amastigotes (Fig. 2C and D). This is
likely to have a dramatic effect on their respective drug sensi-
tivities, with the replicating cells being much more sensitive to
inhibitors of cell division, energy metabolism, etc. In addition,
a starting density significantly below the detection limit is used
in the axenic assay because of the high growth rate. This results
in the assay not only identifying cytocidal compounds but also
growth-slowing compounds (51), which will also contribute to
the high false-positive rate, since only cytocidal compounds
were identified in our intramacrophage assay. We anticipate
that to achieve the current target product profile for visceral
leishmaniasis compounds need to be cytocidal rather than
static or growth slowing (52). Comparing in vitro growth to the
situation in vivo is difficult, since many other factors play a role
(in particular, the clearance of amastigotes by the immune sys-
tem and differences between rodent and human physiology).
Bradley and Kirkley (53) reported an ⬃20-fold increase in tis-
sue parasite counts over the first 8 days of infection in mice,
which means that a minimum of four rounds of cell division
take place during this window. Hence, the rate of amastigote
division in vivo is likely to be somewhere in between what we
see in the intramacrophage and axenic assays. When consider-
ing false-positive rates for the axenic assay, it is important to
keep in mind that these rates should be based on the gold
standard comparator. Here, we used the intracellular Leishma-
nia assay as the comparator, but it would be better to compare
the findings to those obtained in an in vivo animal model (or to
2920 aac.asm.org
human clinical data). However, we did not have sufficient com-
pounds with appropriate efficacy and absorption, distribution,
metabolism, and excretion properties to carry out such a com-
parison.
The localization of the parasites inside the parasitophorous
vacuole presents a serious hurdle for small molecules; to exert
their action, they need to cross three membranes and do this at
both neutral and acidic pH. This means that even if a compound
could kill intracellular amastigotes, it may not be able to reach
them. Although such compounds will not be identified using the
intracellular assay as a primary platform, they may nevertheless be
of interest since further chemical optimization may allow these
molecules to reach the vacuole and exert their effect, or alternative
delivery strategies could be used. It is likely that for these reasons
the intracellular assay may not detect a subset of potentially
interesting compounds and, in view of the dearth of new leads
for leishmaniasis, we may have to consider alternative assays
that will yield more hits. To understand the effect of the fast
axenic growth on the hit rate in this assay, it would be very
informative to devise an axenic amastigote assay under much
less stimulating growth conditions. Such an assay may have
lower false-positive rates and could be of use for high-through-
put screening. It would also allow the use of a higher starting
density so that it only reports cidal compounds, again poten-
tially reducing the false-positive rate.
Conclusion. Combining high throughput with physiological
relevance is a challenge for organisms with a complicated life cycle
such as Leishmania. Ultimately, the only relevant readout is suc-
cess in the clinic. The intramacrophage assay presented here re-
ports activity for all clinically used compounds and, as such, ap-
pears to be a good in vitro model for predicting clinical activity.
The acquisition of clinical data for new drugs that derive from the
current leishmaniasis drug discovery drive will allow further as-
sessment of the suitability of this assay as a primary drug discovery
platform.
ACKNOWLEDGMENTS
We thank Daniel James for data management, Michael Thomas for help
with preparing Table 3, and Mascha Brinkkötter for assistance in the
generation of the L. donovani fluorescent reporter strain. We thank Jean-
Robert Ioset and Eric Chatelain at DNDi for their advice and for critically
reading the manuscript.
This study was funded by the Drugs for Neglected Diseases Initiative,
through grants from several donors (Department for International Devel-
opment, United Kingdom; Swiss Agency for Development and Coopera-
tion, Switzerland; Médecins Sans Frontières/Doctors Without Borders;
International and Gesellschaft für Technische Zusammenarbeit, Ger-
many). This study was also supported by Wellcome Trust strategic award
083481. A.H.F., S.W., and S.C. are supported by the Wellcome Trust
(079838 and 083481).
REFERENCES
1. World Health Organization. 2010. Control of the leishmaniases. World
Health Organ. Tech. Rep. Ser. 2010:xii–xiii; 1–186; back cover.
2. Singh N, Kumar M, Singh RK. 2012. Leishmaniasis: current status of
available drugs and new potential drug targets. Asian Pac. J. Trop. Med.
5:485– 497.
3. Burchmore RJ, Barrett MP. 2001. Life in vacuoles–nutrient acquisition
by Leishmania amastigotes. Int. J. Parasitol. 31:1311–1320.
4. Kima PE. 2007. The amastigote forms of Leishmania are experts at ex-
ploiting host cell processes to establish infection and persist. Int. J. Para-
sitol. 37:1087–1096.
5. Bodley AL, McGarry MW, Shapiro TA. 1995. Drug cytotoxicity assay for
Antimicrobial Agents and Chemotherapy

African trypanosomes and Leishmania species. J. Infect. Dis. 172:1157–
1159.
6. Mikus J, Steverding D. 2000. A simple colorimetric method to screen
drug cytotoxicity against Leishmania using the dye Alamar blue. Parasitol.
Int. 48:265–269.
7. Nolan LL, Bouchard B. 1991. A rapid in vitro system for screening the
effect of experimental compounds on nonadhering cell lines. Curr. Mi-
crobiol. 23:277–279.
8. Siqueira-Neto JL, Song OR, Oh H, Sohn JH, Yang G, Nam J, Jang J,
Cechetto J, Lee CB, Moon S, Genovesio A, Chatelain E, Christophe T,
Freitas-Junior LH. 2010. Antileishmanial high-throughput drug screen-
ing reveals drug candidates with new scaffolds. PLoS Negl. Trop. Dis.
4:e675. doi:10.1371/journal.pntd.0000675.
9. St George S, Bishop JV, Titus RG, Selitrennikoff CP. 2006. Novel
compounds active against Leishmania major. Antimicrob. Agents Che-
mother. 50:474 – 479.
10. Sharlow ER, Close D, Shun T, Leimgruber S, Reed R, Mustata G, Wipf
P, Johnson J, O’Neil M, Grogl M, Magill AJ, Lazo JS. 2009. Identifica-
tion of potent chemotypes targeting Leishmania major using a high-
throughput, low-stringency, computationally enhanced, small molecule
screen. PLoS Negl. Trop. Dis. 3:e540. doi:10.1371/journal.pntd.0000540.
11. Debrabant A, Joshi MB, Pimenta PF, Dwyer DM. 2004. Generation of
Leishmania donovani axenic amastigotes: their growth and biological
characteristics. Int. J. Parasitol. 34:205–217.
12. Bates PA. 1993. Axenic culture of Leishmania amastigotes. Parasitol. To-
day 9:143–146.
13. Gupta N, Goyal N, Rastogi AK. 2001. In vitro cultivation and character-
ization of axenic amastigotes of Leishmania. Trends Parasitol. 17:150 –
153.
14. Teixeira MC, de Jesus Santos R, Sampaio RB, Pontes-de-Carvalho L,
dos Santos WL. 2002. A simple and reproducible method to obtain large
numbers of axenic amastigotes of different Leishmania species. Parasitol.
Res. 88:963–968.
15. Callahan HL, Portal AC, Devereaux R, Grogl M. 1997. An axenic
amastigote system for drug screening. Antimicrob. Agents Chemother.
41:818 – 822.
16. Monte-Alegre A, Ouaissi A, Sereno D. 2006. Leishmania amastigotes as
targets for drug screening. Kinetoplastid Biol. Dis. 5:6.
17. Shimony O, Jaffe CL. 2008. Rapid fluorescent assay for screening drugs
on Leishmania amastigotes. J. Microbiol. Methods 75:196 –200.
18. Holzer TR, McMaster WR, Forney JD. 2006. Expression profiling by
whole-genome interspecies microarray hybridization reveals differential
gene expression in procyclic promastigotes, lesion-derived amastigotes,
and axenic amastigotes in Leishmania mexicana. Mol. Biochem. Parasitol.
146:198 –218.
19. Pescher P, Blisnick T, Bastin P, Spath GF. 2011. Quantitative proteome
profiling informs on phenotypic traits that adapt Leishmania donovani for
axenic and intracellular proliferation. Cell Microbiol. 13:978 –991.
20. Vermeersch M, da Luz RI, Tote K, Timmermans JP, Cos P, Maes L.
2009. In vitro susceptibilities of Leishmania donovani promastigote and
amastigote stages to antileishmanial reference drugs: practical relevance of
stage-specific differences. Antimicrob. Agents Chemother. 53:3855–3859.
21. Li Q., Zhao Y, Ni B, Yao C, Zhou Y, Xu W, Wang Z, Qiao Z. 2008.
Comparison of the expression profiles of promastigotes and axenic amas-
tigotes in Leishmania donovani using serial analysis of gene expression.
Parasitol. Res. 103:821– 828.
22. Sereno D, Cordeiro da Silva A, Mathieu-Daude F, Ouaissi A. 2007.
Advances and perspectives in Leishmania cell based drug-screening pro-
cedures. Parasitol. Int. 56:3–7.
23. Buckner FS, Wilson AJ. 2005. Colorimetric assay for screening com-
pounds against Leishmania amastigotes grown in macrophages. Am. J.
Trop. Med. Hyg. 72:600 – 605.
24. Lang T, Goyard S, Lebastard M, Milon G. 2005. Bioluminescent Leish-
mania expressing luciferase for rapid and high throughput screening of
drugs acting on amastigote-harboring macrophages and for quantitative
real-time monitoring of parasitism features in living mice. Cell. Microbiol.
7:383–392.
25. Mandal S, Maharjan M, Ganguly S, Chatterjee M, Singh S, Buckner FS,
Madhubala R. 2009. High-throughput screening of amastigotes of Leish-
mania donovani clinical isolates against drugs using a colorimetric beta-
lactamase assay. Indian J. Exp. Biol. 47:475– 479.
26. De Muylder G, Ang KK, Chen S, Arkin MR, Engel JC, McKerrow JH.
2011. A screen against Leishmania intracellular amastigotes: comparison
July 2013 Volume 57 Number 7
Intramacrophage versus Axenic Leishmania
to a promastigote screen and identification of a host cell-specific hit. PLoS
Negl. Trop. Dis. 5:e1253. doi:10.1371/journal.pntd.0001253.
27. Siqueira-Neto JL, Moon S, Jang J, Yang G, Lee C, Moon HK, Chatelain
E, Genovesio A, Cechetto J, Freitas-Junior LH. 2012. An image-based
high-content screening assay for compounds targeting intracellular Leish-
mania donovani amastigotes in human macrophages. PLoS Negl. Trop.
Dis. 6:e1671. doi:10.1371/journal.pntd.0001671.
28. Brenk R, Schipani A, James D, Krasowski A, Gilbert IH, Frearson J,
Wyatt PG. 2008. Lessons learnt from assembling screening libraries for
drug discovery for neglected diseases. ChemMedChem 3:435– 444.
29. Goyard S, Segawa H, Gordon J, Showalter M, Duncan R, Turco SJ,
Beverley SM. 2003. An in vitro system for developmental and genetic
studies of Leishmania donovani phosphoglycans. Mol. Biochem. Parasitol.
130:31– 42.
30. Gaur U, Showalter M, Hickerson S, Dalvi R, Turco SJ, Wilson ME,
Beverley SM. 2009. Leishmania donovani lacking the Golgi GDP-Man
transporter LPG2 exhibit attenuated virulence in mammalian hosts. Exp.
Parasitol. 122:182–191.
31. Zhang JH, Chung TD, Oldenburg KR. 1999. A simple statistical param-
eter for use in evaluation and validation of high throughput screening
assays. J. Biomol. Screen. 4:67–73.
32. Seifert K, Escobar P, Croft SL. 2010. In vitro activity of anti-leishmanial
drugs against Leishmania donovani is host cell dependent. J. Antimicrob.
Chemother. 65:508 –511.
33. Wang MZ, Zhu X, Srivastava A, Liu Q, Sweat JM, Pandharkar T,
Stephens CE, Riccio E, Parman T, Munde M, Mandal S, Madhubala R,
Tidwell RR, Wilson WD, Boykin DW, Hall JE, Kyle DE, Werbovetz KA.
2010. Novel arylimidamides for treatment of visceral leishmaniasis. Anti-
microb. Agents Chemother. 54:2507–2516.
34. Yardley V, Croft SL. 2000. A comparison of the activities of three am-
photericin B lipid formulations against experimental visceral and cutane-
ous leishmaniasis. Int. J. Antimicrob. Agents 13:243–248.
35. Gupta S. 2011. Visceral leishmaniasis: experimental models for drug dis-
covery. Indian J. Med. Res. 133:27–39.
36. Sereno D, Cavaleyra M, Zemzoumi K, Maquaire S, Ouaissi A, Lemesre
JL. 1998. Axenically grown amastigotes of Leishmania infantum used as an
in vitro model to investigate the pentavalent antimony mode of action.
Antimicrob. Agents Chemother. 42:3097–3102.
37. Auwerx J. 1991. The human leukemia cell line, THP-1: a multifaceted
model for the study of monocyte-macrophage differentiation. Experientia
47:22–31.
38. Gebre-Hiwot A, Tadesse G, Croft SL, Frommel D. 1992. An in vitro
model for screening antileishmanial drugs: the human leukaemia mono-
cyte cell line, THP-1. Acta Trop. 51:237–245.
39. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K.
1980. Establishment and characterization of a human acute monocytic
leukemia cell line (THP-1). Int. J. cancer 26:171–176.
40. Fernandez O, Diaz-Toro Y, Valderrama L, Ovalle C, Valderrama M,
Castillo H, Perez M, Saravia NG. 2012. Novel approach to in vitro drug
susceptibility assessment of clinical strains of Leishmania spp. J. Clin. Mi-
crobiol. 50:2207–2211.
41. Rogers PD, Stiles JK, Chapman SW, Cleary JD. 2000. Amphotericin B
induces expression of genes encoding chemokines and cell adhesion mol-
ecules in the human monocytic cell line THP-1. J. Infect. Dis. 182:1280 –
1283.
42. Gao Y, Deng K, Cao Z, Graziani EI, Gilbert AM, Koehn FE, Wood A,
Doherty P, Walsh FS. 2010. Amphotericin B, identified from a natural
product screen, antagonizes CNS inhibitors to promote axon growth via
activation of an Akt pathway in neurons. J. Neurochem. 113:1331–1342.
43. Manning BD, Cantley LC. 2007. AKT/PKB signaling: navigating down-
stream. Cell 129:1261–1274.
44. Osorio Y, Travi BL, Renslo AR, Peniche AG, Melby PC. 2011. Identi-
fication of small molecule lead compounds for visceral leishmaniasis using
a novel ex vivo splenic explant model system. PLoS Negl. Trop. Dis.
5:e962. doi:10.1371/journal.pntd.0000962.
45. Sereno D, Lemesre JL. 1997. Axenically cultured amastigote forms as an
in vitro model for investigation of antileishmanial agents. Antimicrob.
Agents Chemother. 41:972–976.
46. Carrio J, de Colmenares M, Riera C, Gallego M, Arboix M, Portus M.
2000. Leishmania infantum: stage-specific activity of pentavalent anti-
mony related with the assay conditions. Exp. Parasitol. 95:209 –214.
47. Ephros M, Bitnun A, Shaked P, Waldman E, Zilberstein D. 1999.
aac.asm.org 2921
De Rycker et al.
Stage-specific activity of pentavalent antimony against Leishmania
donovani axenic amastigotes. Antimicrob. Agents Chemother. 43:278 –
282.
48. Shaked-Mishan P, Ulrich N, Ephros M, Zilberstein D. 2001. Novel
intracellular SbV reducing activity correlates with antimony susceptibility
in Leishmania donovani. J. Biol. Chem. 276:3971–3976.
49. Mookerjee Basu J, Mookerjee A, Sen P, Bhaumik S, Banerjee S, Naskar
K, Choudhuri SK, Saha B, Raha S, Roy S. 2006. Sodium antimony
gluconate induces generation of reactive oxygen species and nitric oxide
via phosphoinositide 3-kinase and mitogen-activated protein kinase acti-
vation in Leishmania donovani-infected macrophages. Antimicrob.
Agents Chemother. 50:1788 –1797.
50. Rochette A, Raymond F, Corbeil J, Ouellette M, Papadopoulou B. 2009.
2922 aac.asm.org
Whole-genome comparative RNA expression profiling of axenic and in-
tracellular amastigote forms of Leishmania infantum. Mol. Biochem. Para-
sitol. 165:32– 47.
51. De Rycker M, O’Neill S, Joshi D, Campbell L, Gray DW, Fairlamb AH.
2012. A static-cidal assay for Trypanosoma brucei to aid hit prioritization
for progression into drug discovery programmes. PLoS Negl. Trop. Dis.
6:e1932. doi:10.1371/journal.pntd.0001932.
52. Freitas-Junior LH, Chatelain E, Kim HA, Siqueira-Neto JL. 2012. Vis-
ceral leishmaniasis treatment: what do we have, what do we need and how
to deliver it? Int. J. Parasitol. Drugs Drug Resist. 2:11–19.
53. Bradley DJ, Kirkley J. 1977. Regulation of Leishmania populations within
the host. I. the variable course of Leishmania donovani infections in mice.
Clin. Exp. Immunol. 30:119 –129.
Antimicrobial Agents and Chemotherapy