1例国内罕见的输入性卵形疟的实验室检测

Laboratory diagnosis of a suspected case of imported Plasmodium ovale infection

目的对1例输入性疑似疟疾患者的血样进行实验室检测。方法制备疑似疟疾患者血样的血涂片,吉姆萨染色后进行疟原虫的镜检观察。利用实验室自行研制的疟原虫属特异性(通用型)和4种疟原虫种特异性的巢式PCR和实时荧光PCR检测方法,对该血样进行疟疾检测及分型。将PCR扩增片段进行序列测定,并与已知的卵形疟序列进行blast比对分析。结合血样的分子生物学检测结果,重新对镜检结果进行复核。结果血样初次镜检为疟疾阴性。使用疟原虫巢式PCR通用引物对血样的DNA进行PCR检测,扩增出了预期大小约240bp的条带;巢式PCR分型检测表明,血样仅扩增出预期大小约450bp的卵形疟条带,无对应大小的恶性疟、间日疟和三日疟扩增条带产生。疟原虫通用型和卵形疟特异性的荧光PCR检测结果均为典型的S形阳性曲线,卵形疟扩增片段的熔解温度为72.5℃。序列分析表明,扩增片段长度为434bp,blast比对发现去除引物后的393个碱基与GenBank DQ845247等卵形疟的SSU rRNA对应部分的基因序列同源性为100%。重新对血涂片进行镜检复核,结果在薄血膜中发现了卵形疟的环状滋养体,被寄生的红细胞为椭圆形,边缘呈伞矢状。结论使用巢式PCR、实时荧光PCR、序列分析和镜检等方法,证实该例输入性的疑似疟疾患者为卵形疟原虫感染。

Blood smear of suspected imported malaria patient was prepared and observed by microscopy after Giemsa staining.Initial microscopy of the blood sample was negative for malaria.Nested PCR and real-time PCR methods of genus-specific Plasmodium and four species-specific Plasmodium were performed to detect and differentiate the parasites using in-house designed primers and probes.Nested PCR with the genus-specific primers of Plasmodium showed that the blood sample produced an expected band of about 240bp in size.Plasmodium classification demonstrated that the blood sample only produced an expected amplification band of about 450bp of P.ovale in size and no bands of P.falciparum,P.vivax and P.malarie.Real time PCR with both Plasmodium and specific P.ovale displayed typical S-shaped positive curves.Sequence analysis showed that the amplified fragment was composed of 434 bases and 393 bases removing PCR primers showed 100% homology to the corresponding part of P.ovale SSU rRNA gene of GenBank accession number DQ845247.Blood smear was reviewed by microscopy.The ring trophozoites of P.ovale were founded in thin blood film and the parasitized cell was oval in shape with fimbriated edges.The suspected case of imported malaria patient was confirmed to infect P.ovale by nested PCR,real-time PCR,sequence analysis and microscopic examination.