New Approaches for the Identification of Drug Targets in Protozoan Parasites - International Review of Cell and Molecular Biology - page 363

morphological assays are easy to perform and give unambiguous informa-
tion on drug effectiveness in the hands of a skilled lab worker, but they are
time-consuming and rely on a “subjective” assessment. Therefore, they tend
to be replaced by quantitative, “objective” methods that can, ultimately, be
performed by a robot.
The simplest way to do quantify parasites is to measure a dye absorbed
by the parasite by colorimetry. In the case of G. lamblia , trophozoites can be
stained with methylene blue ( Busatti and Gomes, 2007 ). After some wash-
ing steps, the staining is then quantified using a colorimeter. These assays are
inexpensive and fast, but are limited to axenically grown parasites. Methods
based on the incorporation of radioactive precursors of nucleic acids are
also limited to axenically grown parasites ( Adagu et al., 2002 ) or to co-
cultures where rapidly proliferating parasites incorporate these precursors
to much larger extents than their host cells ( Innes et al., 1996 ). In the case
of Plasmodium falciparum , intraerythrocytic stages can be stained and quanti-
fied using intercalating dyes ( Banieck et al., 2007 ; Duffy and Avery, 2012 ) or
radioactive precursors ( Desjardins et al., 1979 ; Rottmann et al., 2010 ) since
mature mammalian erythrocytes do not contain DNA.
In other cases of parasite-host cell co-cultures, parasites have to be quan-
tified in a more specific way. A method of choice is immunofluorescence.
Intrahepatocytic stages of Plasmodium sp. can be quantified using immu-
nofluorescence employing an infrared fluorescence scanning system ( Gego
et al., 2006 ). An alternative is quantitative real-time PCR based on a gene
specific to the parasite as implemented, e.g. for Neospora caninum in co-
culture with human fibroblasts ( Esposito et al., 2005 ) or for G. lamblia in
co-culture with human Caco2 cells ( Müller et al., 2006 ).
An elegant way to develop antiparasite drug screening assays is to
use transgenic parasites expressing reporter genes under the control of
a strong promotor ( Rodriguez and Tarleton, 2012 ). The reporter genes
are enzymes such as beta-galactosidase ( McFadden et al., 1997 ), gluc-
uronidase ( Müller et al., 2009b ) or luciferase ( Cui et al., 2008 ; Franke-
Fayard et al., 2008 ), which can easily be quantified in standard enzyme
assays, or green or yellow fluorescent protein quantified using a flu-
orimeter ( Gubbels et al., 2003 ). The use of transgenic parasites offers
many advantages as compared to PCR-based methods. In the case of
enzymes as reporter genes, the assays can be performed in situ, i.e. in
96-well plates, for instance, without prior harvest or extraction of the
cells like in PCR-based methods. A second advantage is higher sensi-
tivity. This might sound strange since PCR is often referred to as “the