Molecular identification of vivax malaria relapse cases in Yunnan Province based on the homologyical analysis of Plasmodium vivax circumsporozoite protein gene

Abstract Background More than 85% of the malaria burden is caused by imported vivax malaria in Yunnan Province and Yunnan is also where the majority of vivax malaria cases are diagnosed across China. Timely removal of the source of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To compensate for the uncertainty of epidemiological surveys in tracing vivax malaria recurrence, this study attempted to use molecular markers for identification. Materials and methods To do so, blood samples were collected from cases diagnosed and revalidated as single infections of Plasmodium vivax in Yunnan Province from 2013 to 2020. Specifically, samples from suspected relapses with recurrent episodes were subjected to PCR amplification, product sequencing, and analysis of the Plasmodium vivax circumsporozoite protein (pvcsp) gene. Results Seventy-eight suspected recurrent cases were retrieved from 2484 vivax malaria cases, with a total of 81 recurrent episodes. A total of 159 blood samples from primary infection Plasmodium vivax and recurrences were subjected to PCR amplification and sequencing to obtain 156 CDS sequences of pvcsp gene, 121 of which can be matched into the paired sequences of 59 cases. There were 475 polymorphic loci and 84 haplotypes (H01-H84) in the 121 sequences. Also, there were 79 and 5 haplotypes with CRR repeat units (PRM) of VK210 and VK247 structure, respectively. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites, meaning the every paired sequences were completely homologous and the paired Plasmodium vivax strains were homologous single clone. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, and except for 2 polymorphic loci (39 and 1027), all single nucleotide polymorphisms were double-equivalent bases differentially transferred between paired sequences, indicating that the paired sequences are "weakly heterologous" with no fragment insertions (or deletions) and only individual site polymorphisms. All 59 vivax malaria recurrences were respectively caused by the activation of Plasmodium vivax hypnozoites from the same population as the primary infection. Conclusions The paired analysis of the similarity of Plasmodium high variant genes allowed the identification of recurrent episodes caused by Plasmodium vivax homologous hypnozoites, and also demonstrated pvcsp gene as a good molecular marker. Moreover, the study showed most of the hypnozoites causing vivax malaria recurrence in Yunnan Province belonged to homologous single clone or sibling strains comparison with the original infection strains.