Comparison of the effects of a formulated transport medium And edta anticoagulant on in vitro antimalarial drugs activity Against plasmodium falciparum standard clones and field Isolates

Abstract

ABSTRACT Malaria is an important public health problem world wide with 283 million infections resulting in 584,000 deaths per year globally. In Kenya, malaria accounts for an estimated 18% of outpatients and 6% of hospital admissions. Antimalarial resistance is among the contributory factors to an increase in mortality. Kenya was the epicenter of chloroquine resistance in Africa. Recent reports of reduced susceptibility to the commonly used artemisinin combination drugs at the Kenyan coast and other African countries indicate an urgent need for intensified surveillance of antimalarial drug efficacy. In vitro sensitivity testing is one of the preferred methods of measuring susceptibility. This method is able to measure P. falciparum susceptibility to several antimalarial drugs simultaneously, away from the influence of host immune related factors. The technique is being transitioned to ex vivo requiring tests on fresh sample. Such in vitro studies of field P. falciparum have been attributed to diminished viability as they transition from host ecosystem to lab conditions due to lack of a proper medium to stabilize the parasites outside the human host. It is therefore imperative to calibrate the sample stabilization media to reduce artificial effects to the assay.The objective of this study was to evaluate the effect of a formulated transport medium (TM) on viability of Plasmodium being transported to the lab for testing within 6 hours. Specifically the study assayed standard clones, field isolates and finally compared the results with other published findings to come up with antimalarial susceptibility profile of the region. Inhibition concetratin 50% (IC50) for field isolates transported in TM that is less costly was compared with that of Ethylene Diamine Tetra Acetic acid (EDTA) anticoagulant which allows better preservation of cellular components and morphology of blood cells for a longer period. This was a cross-sectional laboratory based epidemiological study. Blood samples were collected from 322 assenting individuals from Maseno division visiting Chulaimbo Sub county hospital and confirmed positive for malaria, under the approved protocol (accreditation number 01713). Each sample, split in to EDTA vs TM was analysed for susceptibility to artemether (ART), lumefantrine (LUM), dihydroartemisinin (DHA) and piperaquine (PPQ) using Malaria SYBR Green Assay. IC50 was determined for each sample between TM and EDTA using dose response curves. Mann Whitney test for comparing the medians IC50 values and Pearson correlation coefficient for establishing correlations of logarithimic values of IC50 for different drugs were used. In total 322 samples yielded paired results for ART, LUM, DHA and PPQ. The reference clones W2 (considered chloroquine resistant, mefloquine and artemisinin sensitive) and 3D7 (considered chloroquine and mefloquine sensitive) were used as internal controls. Results showed that the IC50 values of the field isolates in EDTA were higher although not significant (P=0.99, 0.74, 0.68, 0.82 for ART, LUM, DHA and PPQ respectively) than those in the TM. Among the clones, PPQ was the only drug with a high significant IC50 decrease (P<0.001) in TM for the W2 and a moderately significant decrease (P=0.028) in EDTA for 3D7 clone, while other drugs were not significant. These suggest a similarity in the two anticoagulants in preservation of the blood samples. Significant correlation was observed between DHA and ART (r=0.123, p=0.026); LUM (r=0.138, p=0.012) and PPQ (r=0.128, p=0.02), suggesting a possible cross resistance between the drugs. In conclusion, the clones and the field isolates in EDTA showed no significant difference results with those in TM. Despite the stabilizers exposure, field isolates‟ IC50 values generated in all the drugs were comparable with those found earlier for clinical isolates collected in other endemic areas. Lower IC50 values recorded by the field isolates against the antimalarials were indicative of their high susceptibility to the drugs. The use of TM could help on real time interventions leading to detection of parasites resistance at its onset thus reducing mortality from malaria related cases. However, further research should be done to find out the practical time TM can keep the parasites viable