三种不同检测方法在低密度疟疾样本检测中的比较研究

Methods to test for low-density Plasmodium parasites infection

目的 用三种常用疟疾检测方法对恶性疟和间日疟样本的检测效果进行比较研究。方法 恶性疟样本来自2023年3月武汉市输入性恶性疟病例,间日疟样本来自2023年4月宜昌市输入性间日疟病例,各1份,分别制作2张厚血膜,计算原虫密度,再以健康人抗凝血对病例血样按1∶4、1∶16、1∶64、1∶256、1∶1 024、1∶4 096、1∶16384、1∶65 536比例进行稀释,每个稀释浓度的样本制作2张厚血膜镜检盲检,并用快速诊断检测(RDTs)和实时荧光定量PCR检测每个浓度血样疟疾感染情况。结果 恶性疟样本原血样当稀释浓度低于1∶16 384,即原虫密度低至13个/μL血,镜检结果 2张均为阴性;间日疟样本原血样当稀释浓度为1∶1 024,即原虫密度低至55个/μL血,镜检结果 1张为阴性,1张为阳性。恶性疟样本稀释至原虫密度13个/μL血时,RDTs检测结果出现阴性;间日疟样本原血样使用两种不同品牌RDTs试剂检测结果均为阴性。2个样本所有稀释度的实时荧光定量PCR法检测结果均为阳性,其中恶性疟样本最高稀释浓度的原虫密度为3.2个/μL血,CT值为32.788;间日疟样本最高稀释浓度的原虫密度为0.86个/μL血,CT值35.057。结论 实时荧光定量PCR检测法检出限优于其他两种方法,但成本较高,三种方法可在不同检测机构中协同使用,提高疟原虫检测的准确度和时效性。

Objective To compare the results of three detection methods for Plasmodium falciparum and Plasmodium vivax.Methods The Plasmodium falciparum blood sample from Wuhan of imported malaria in March 2023 and Plasmodium vivax blood sample from Yichang of imorpted malaria in April 2023 were prepared to make two thick blood smears and counted.Serial dilutions(1∶4,1∶16,1∶64,1∶256,1∶1024,1∶4 096,1∶16 384,1∶65 536)were prepared using a blood sample drawn from a healthy person as diluent solution.Two blood smears for each concentration were made for microscopic examination and the rapid diagnostic test(RDTs)and real time fluorescence quantitative PCR were conducted at the same time.Results When the dilution concentration of the original blood sample of falciparum malaria sample was lower than 1∶16 384,that is,the density of protozoa was as low as 13/μL blood,the two microscopy results were negative.When the original blood sample of vivax malaria sample was diluted to a concentration of 1∶1 024,that is,the density of protozoa is as low as 55/μL blood,the microscopic examination result was one negative and one positive.When the falciparum malaria sample was diluted to a protozoal density of 13/μL blood,the RDTs test result was negative.The original blood samples of vivax malaria samples were tested negative using two different brands of RDTs reagents.The real-time fluorescence quantitative PCR test results of all dilutions of the two samples were positive.Among them,the protozoa density of the highest dilution concentration of the falciparum malaria sample was 3.2/μL blood,and the CT value was 32.788.The protozoa density at the highest dilution concentration of the vivax malaria sample was 0.86/μL blood,and the CT value was 35.057.Conclusions The real-time fluorescence quantitative PCR had better detection than the other two methods,but the cost was higher.The three methods could be used in different detection institutions to improve the accuracy and effectiveness in malaria detection.