Drying anti-malarial drugs in vitro tests to outsource SYBR green assays.

Karim Traore,Marjorie Chavant,Ogobara Doumbo,Adeline Lavoignat,Frederick Gay,Fatimata Sow,Giuliana C Bess,Stephane Picot,Guillaume Bonnot

doi:10.1186/s12936-015-0600-z

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.

Highlights

Measurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection
No significant differences were seen in The 50% inhibitory concentration (IC50) generated for ex vivo samples between the standard and modified protocols
Drying and storing the plates did not affect the means of IC50 values in a modified SYBR Green I protocol to assess anti-malarial drugs

Summary

Introduction

Measurement of anti-malarial drug efficacy and resistance relies mainly on in vivo clinical trials, in vitro/ex vivo assays and molecular markers detection. The spread of resistant malaria parasites to anti-malarial drugs, including artemisininbased combination therapy, is a major concern for malaria control in endemic countries [3]. Three main approaches including in vivo trials, in vitro/ ex vivo assays and molecular markers of drug resistance are currently used to monitor anti-malarial drug efficacy and drug resistance [4]. In vivo studies are considered the gold standard for measuring the efficacy of anti-malarial drugs [5]. Carrying out in vivo efficacy trials requires time, qualified medical staff and financial resources, which can represent a challenge in resource-limited malaria-endemic countries. In vivo trials measure therapeutic efficacy rather than drug resistance [6,7]

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Conclusion