关键词: 恶性疟原虫, 混合感染, 输入性疟疾, 宏基因组学二代测序

Abstract:

The feasibility and application of metagenomic Next-Generation Sequencing (mNGS) in the detection of Plasmodium spp. was investigated by testing a blood sample of a malaria case imported from Africa. The peripheral blood was collected from a malaria patient from Africa. The thick and thin blood smears were prepared and examined under a microscope. Nucleic acid of patient’s whole blood was extracted and detected by real-time PCR. A cDNA library was constructed and used for mNGS by using an Illumina Miseq sequencer. The BLASTn alignment results of the reads and contig sequences were analyzed using Megan6 software, and the assignment mode was aligned by base mode. Microscopic examination showed that the patient was a co-infection of P. falciparum and P. malariae, with lower proportion of P. malariae. The results of real-time PCR showed that the detection of P. falciparum nucleic acid was positive with all negative for P. vivax, P. malariae and P. ovarian. A total of 271 526 reads were obtained, of which 254 674 reads qualified. After filtered by mapping to the Human reference genome, 1 833 reads were finally obtained, and the non-human reads accounted for only 0.7% of the total qualified reads. After de novo, 408 contigs with a length of more than 100 nucleotides were obtained. The results of Megan6 analysis showed that the reads of P. falciparum accounted for 44.7% (29 667/66 374) of the total reads, and 34.5% (22 885/66 376) of the total contig. The results suggest that P. falciparum is the most dominant pathogen. Based on the reads and contig, the pathogen can be traced to the P. falciparum 3D7.

Key words: Plasmodium falciparum, Co-infection, Imported malaria, Metagenomic Next-Generation Sequencing