Monitoring The Sensitivity Of Imported Plasmodium Falciparum Isolates To Antimalarial Drugs

Malaria is caused by parasites of the genus Plasmodium,which with HIV/AIDS and tuberculosis,are 3 major global public health threats^[1].According to the report of WorldHealth Organization（WHO）,an estimated of 3.2 billion people in 97 countries and territories are at risk of being infected with malaria,198 million cases of malaria occurred globally in 2013（range 124–283 million）,and the disease led to 584 000 deaths.Among of these,82%of the malaria cases and 90%of the deaths were occured in Africa^[2].Since the 1940s,chemotherapy has played an important role in malaria control with the available of related antimalarial drugs,such as chloroquine.However,with the drug used widely for a long time,drug resistance in malaria parasites has emerged gradually.Till now,malaria parasites have resistance to nearly all antimalarial in current use.The emergence and development of drug resistance in malaria parasites,is not only a threat to the lives of malaria patients,but also affecting the progress of malaria control and elimination.Monitoring the antimalarial drug resistance in malaria parasites,will not only be helpful to the drugs selection,saving the lives of malaria patients,but also be important for the guidence of the country’s national antimalarial drugs policy.At present,tools for assessment of the susceptibility of malaria parasites to antimalarial drugs,including in vivo trial,in vitro/ex vivo assays,and molecular markers detection mainly.Although in vivo trial is the gold standard for assessing the emergence of drug resistance and treatment failure,it requires long time of follow-up,and is easily affected by the host compliance,immunity,pharmacokinetics and so on^[3-4].In this study,methods of in vitro/ex vivo assays and molecular markers detection were selected.In this study,molecular markers and in vitro assays were used for detecting the sensitivity of imported plasmodium falciparum isolates to antimalarial drugs（chloroquine,mefloquine and piperaquine）,and the polymorphism of the drug resistance related genes（Pfcrt and Pfmdr）in Jiangsu province from 2012-2014.It would provide the scientific basis for the selection of antimalarial drugs for the imported P.falciparum malaria cases,making up and adjustment the programs for monitoring the drug resistance in malaria paraasites.Three part were included in this study mainly:Part I Comparative study of assay methods for in vitro antimalarial drug efficacy testing in Plasmodium falciparumObjective:1 Comparing the four assay methods,including WHO microtest,pLDH,HRPâ…¡and SYBR Greenâ… assays,to select a relative stable,simple,rapid,and economic method of in vitro susceptibility testing,for monitoring the sensitivity of imported P.falciparum isolates to antimalarial drugs.2 To detect the sensitivities of the imported plasmodium falciparum isolates in Jiangsu province to antimalarial drugs,including Chloroquine,Mefloquine and Piperaquine,by the optimal in vitro test.Methods:1 WHO microtest,pLDH,HRPâ…¡and SYBR Greenâ… were applied to test the drug efficacy of chloroquine,piperaquine and amodiaquine against three Plasmodium falciparum reference strains（3D7,K1 and Dd2）,respectively.The consistency of the 50%inhibitory concentration（IC50）values from the four assay methods were analyzed by Friedman tests,Partial correlation analysis and Pearson’s correlation analysis.2 As soon as the blood samples received in the laboratory,the parasites were adapted to continuous culture immediately.In vitro/ex vivo assays was performed immediately after parasitaemia>2%by the most appropriate assay method.IC50 values with their 95% confidence intervals（CI）were calculated by using GraphPad Prism 6.0 software and the data were analysed with SPSS 16.0 software.Results:1 With the initial parasitemia ranged from 0.5%to 1%,there were no statistically significant differences（P>0.05）among the IC₅₀ values obtained by the four assay methods,which were correlated well（both P<0.001）.WHO microtest was highly labor-intensive,time-consuming and subjective;although HRPâ…¡was more sensitive than pLDH and SYBR Greenâ… ,which was more expensive;SYBR Greenâ… was a simple,rapid and economic assay method.2 52.8%,47.2%and 47.2%of the imported plasmodium falciparum in Jiangsu province were CQ-resistant,Mef-resistant and Pip-resistant,respectively.The activities of CQ and Pip showed the strongest correlation.Conclusions:1 All of the four assay methods can be used for in vitro drug sensitivity study of P.falciparum,but SYBR Greenâ… assays is more simple,rapid and at low cost,which was more suitable for monitoring the drug sensitivity of malaria parasites in our country with malaria elimination phase.2 The imported P.falciparum,has become resistance to chloroquine,mefloquine and piperaquine partly,and there is the correlation between the activities of chloroquine and piperaquine.Partâ…¡The correlation of the polymorphisms of pfcrt gene and anti-malaria drug resistance objective:To know about the polymorphisms in the P.falciparum pfcrt gene of the imported isolates in jiangsu province from 2012 to 2014,and check the correlation with the anti-malaria drug resistance.Methods:1 Several alleles were analysed by nested PCR-sequencing:72-76 and 220 in the pfcrt gene;2 Field isolates sensitivity to CQ,Mef and Pip was assessed using the SYBR Greenâ… assay;3 The correlation of the polymorphisms in pfcrt genes and anti-malaria drug resistance were analysed.Results:1 A total of 294 isolates was sequenced.The prevalence of 72,74,75,76 and 220 in the pfcrt gene were 0.3%,41.5%,41.5%,41.5%and 42.2%,respectively.2 With the withdrawal of chloroquine,there seems to be a downward trend for the prevalence of the pfcrt 76T molecular marker for chloroquine-resistant.3 Pfcrt 76T mutation was strongly associated with resistance to CQ.Conclusions:1 Pfcrt 76T mutation was strongly associated with resistance to chloroquine,and the site can be used as a molecular marker for monitoring the chloroquine resistance.2 With chloroquine withdrawal,there is a downward trend for the rate of mutation at the site of pfcrt 76TPartâ…¢The correlation of the polymorphism in pfmdr1 gene and antimalaria drug resistance objective:To know about the polymorphisms in pfmdr1 gene of the imported isolates in jiangsu province from 2012 to 2014,and check the correlation with the anti-malaria drug resistance.Methods:1 Several alleles were analysed by nested PCR-sequencing:86,184,1034,1042 and 1246 in the pfmdr1 gene;2 Field isolates sensitivity to CQ,Mef and Pip was assessed using the SYBR Greenâ… assay;3 The correlation of the polymorphisms in pfmdr1 genes and anti-malaria drug resistance were analysed.Results:1 A total of 294 isolates was sequenced.The prevalence of 86,184,1034,1042 and 1246 in the pfmdr1 gene were 31%,62.9%and 5.2%,respectively;Only 32%of the imported plasmodium falciparum isolates were identified with pfmdr1 gene amplification.2 Pfmdr1 86Y mutation was strongly associated with resistance to Mef;3 Pfmdr1 gene amplification was strongly associated with resistance to Mef and CQ.Conclusions1 The copy number increased in Pfmdr1 gene was associated with mefloquine resistance,and can be used as a molecular marker for monitoring the mefloquine resistance.2 There is negative correlation between the Pfmdr1 86Y mutation and mefloquine resistance,and also the copy number increased in Pfmdr1 and chloroquine resistance.