Drying anti-malarial drugs in vitro tests to outsource SYBR green assays
Identifieur interne : 000E53 ( Main/Curation ); précédent : 000E52; suivant : 000E54Drying anti-malarial drugs in vitro tests to outsource SYBR green assays
Auteurs : Karim-Frédéric Traore [France] ; Adeline Lavoignat [France] ; Guillaume Bonnot [France] ; Fatimata Sow [France] ; Giuliana C. Bess [France] ; Marjorie Chavant [France] ; Frederick Gay [France] ; Ogobara Doumbo [Mali] ; Stephane Picot [France]Source :
- Malaria Journal [ 1475-2875 ] ; 2015-02-21.
English descriptors
- mix :
Abstract
BackgroundMeasurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level.MethodsIn vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 μl of complete RPMI medium for fluorescence assay.ResultsThere were no significant differences in IC50 values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC50 values.ConclusionThe SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.
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DOI: 10.1186/s12936-015-0600-z
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Hal:hal-01121828Le document en format XML
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<author><name sortKey="Picot, Stephane" sort="Picot, Stephane" uniqKey="Picot S" first="Stephane" last="Picot">Stephane Picot</name>
<affiliation wicri:level="1"><hal:affiliation type="laboratory" xml:id="struct-230740" status="INCOMING"> <orgName>Malaria Research Unit</orgName>
<desc> <address> <addrLine>Lyon</addrLine>
<country key="FR"></country>
</address>
</desc>
<listRelation> <relation name="UMR5246" active="#struct-441569" type="direct"></relation>
</listRelation>
<tutelles><tutelle name="UMR5246" active="#struct-441569" type="direct"><org type="institution" xml:id="struct-441569" status="VALID"> <idno type="IdRef">02636817X</idno>
<idno type="ISNI">0000000122597504</idno>
<orgName>Centre National de la Recherche Scientifique</orgName>
<orgName type="acronym">CNRS</orgName>
<date type="start">1939-10-19</date>
<desc> <address> <country key="FR"></country>
</address>
<ref type="url">http://www.cnrs.fr/</ref>
</desc>
</org>
</tutelle>
</tutelles>
</hal:affiliation>
<country>France</country>
</affiliation>
</author>
</analytic>
<idno type="DOI">10.1186/s12936-015-0600-z</idno>
<series><title level="j">Malaria Journal</title>
<idno type="ISSN">1475-2875</idno>
<imprint><date type="datePub">2015-02-21</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass><keywords scheme="mix" xml:lang="en"><term>Drug resistance</term>
<term>Ex vivo</term>
<term>In vitro</term>
<term>Malaria</term>
<term>SYBR green</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"> <p>BackgroundMeasurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The existing in vitro/ex vivo assays, in particular those that are using non-radioactive devices, need to be standardized and adapted to field conditions. SYBR Green assay offers a rapid and cheap alternative to other in vitro assays, but it requires tools not commonly available in field laboratories. Here is described a modified SYBR green I protocol to perform the parasite growth test with blood samples in endemic areas, followed later by the SYBR green fluorescence assay performed at a specialized laboratory level.MethodsIn vitro susceptibility of Plasmodium falciparum clones HB3, 3D7, W2 and 7G8 to chloroquine (CQ), dihydroartemisinin (DHA), pyronaridine (PYD) and piperaquine (PPQ) was tested. Fresh isolates of P. falciparum from imported malaria cases were collected for ex vivo assays. The parasite suspension was added in 96-well plates predosed with anti-malarial drugs and incubated for 72 hours at 37°C, 5% CO2. SYBR green I protocol was modified to dry the plates after freeze-thawed process to mimic storage and shipping conditions. The plates were rehydrated with 200 μl of complete RPMI medium for fluorescence assay.ResultsThere were no significant differences in IC50 values of CQ, DHA, PYD and PPQ, determined by the modified protocol, compared to standard protocol. Longer storage did not affect the IC50 values.ConclusionThe SYBR green I modified protocol produced reliable results and could be a suitable method for in vitro/ex vivo assays in field.</p>
</div>
</front>
</TEI>
</record>
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