红内期疟原虫不同发育阶段对检测结果的影响

目的观察红内期疟原虫期态对实验室常规检测的影响。方法使用5%山梨醇同步化法对恶性疟原虫3D7虫株进行连续两次同步化处理,收集不同时间段的虫血(间隔6 h),分别进行镜检、巢式PCR以及流式细胞术检测,并进行比较分析。结果采用镜检方法检查红内期疟原虫时,滋养体、裂殖体期疟原虫较环状体更易检获;采用巢式PCR检测,并对电泳结果进行灰度分析,其9个时间点的灰度值分别为20.80、38.23、57.52、86.28、114.83、312.04、347.39、376.95和484.99,其中0 h与6 h、6 h与12 h之间的差异均无统计学意义(P>0.05),12 h及之后收集的疟原虫其灰度值均有统计学意义(P<0.05);采用流式细胞术检测,以环状体为主的疟原虫荧光强度较弱,产生的峰形较高且窄,滋养体、裂殖体疟原虫所发出的荧光强度逐渐增强,产生的峰逐渐变低、变宽。结论采用镜检法、巢式PCR以及流式细胞术3种方法检测红内期不同发育期疟原虫的结果不同,因此在疟疾的实验室诊断中应考虑疟原虫期态对检测结果的影响。

Study on the effect of different developmental stages of Plasmodium on the detection of the parasite in erythrocytes

Objective To study the effect of different erythrocytic stages of Plasmodium on routine detection of the parasite.Methods The 3D7 strain of Plasmodium falciparum was cultured under standard conditions,and cultures were synchronized using two rounds of 5%D-sorbitol treatment.The parasite culture was harvested at 6-h intervals 9 times after synchronization(0h,6 h,12 h,18 h,24 h,30 h,36 h,42 h,and 48 h).Methods including microscopy,nested PCR,and flow cytometry were used to detect the parasites.Results When microscopy was used to detect different stages of Plasmodium,parasites in the late trophozoite and schizont stages were more readily detected than the ring form.Parasites were detected with nested PCR at different times after synchronization,and gray scale values for electrophoresis bands were analyzed.The gray scale values at the nine time points were 20.80,38.23,57.52,86.28,114.83,312.04,347.39,376.95,and 484.99.Group comparison revealed no significant differences between gray scale values at 0 h and 6 h and at 6 h and 12 h(P>0.05),but significant differences(P<0.01)were noted at later time points.Flow cytometry indicated that most of the ring-stage parasites had weaker fluorescence,and the peak was higher and narrower.In contrast,the fluorescence intensity of the trophozoite and schizont stages increased gradually,and the peak gradually fell and broadened.Conclusion Three different methods of detection,i.e.microscopy,nested PCR,and flow cytometry,were used to detect Plasmodium,and results indicated that the different developmental stages have a significant effect on the detection of the parasite.Therefore,the impact of the stages of malaria parasites should be considered when diagnosing malaria.