Analysis of lactate dehydrogenase gene polymorphisms and prediction of B cell epitopes in four human Plasmodium species / 中国血吸虫病防治杂志

Objective To analyze the polymorphism of Plasmodium lactate dehydrogenase (pLDH) gene and predict B-cell epitopes in pLDH peptides in four species of human malaria parasites. Methods The blood samples and epidemiological characteristics were collected from malaria cases in Yunnan Province registered in the National Notifiable Disease Report System. The pLDH genes of four human Plasmodium species were amplified using nested PCR assay and sequenced. The polymorphisms of pLDH genes was analyzed using the software MEGA version 7.0.26 and DnaSP version 5.10, and the B-cell epitopes were predicted in pLDH peptides using the Immune Epitope Database (IEDB). Results The sequences of P. vivax LDH (PvLDH), P. falciparum LDH (PfLDH), P. ovale LDH (PoLDH) and P. malariae LDH (PmLDH) genes were obtained from 153, 29, 17 and 11 blood samples from patients with P. vivax, P. falciparum, P. ovale and P. malariae malaria, respectively, which included 15, 2, 4 and 2 haplotypes and had a nucleotide diversity (π) of 0.104. A high level of intra-species differentiation was seen in the PoLDH gene (π = 0.012), and the π values were all < 0.001 for PvLDH, PfLDH and PmLDH genes. Active regions of B-cell antigen were predicted in the pLDH peptide chain of four human malaria parasites, of 4 to 5 in each chain, and the activity score was approximately 0.430. Among these peptide chains, the “86-PGKSDKEWNRD-96” short-peptide was a B-cell epitope shared by all four species of human malaria parasites, and the “266-GQYGHS (T)-271” short-peptide was present in PvLDH and PoLDH peptide chains, while “212-EEVEGIFDR-220” was only found in the PvLDH peptide chain, and “208-LISDAE-213” was only seen in the PfLDH peptide chain. Conclusions The PoLDH gene polymorphism may be derived from the weak negative purification selection, while PvLDH, PfLDH and PmLDH genes may maintain a relatively conservative state. There may be two B-cell epitopes “212-EEVEGIFDR-220” and “208-LISDAE-213” in the proximal region of the C terminal in the pLDH peptide chain, which is feasible to differentiate between P. vivax and P. falciparum infections.

[Geographical features of malaria in Yunnan Province].

OBJECTIVE: To understand the geographical features of malaria in Yunnan Province, so as to provide the reference for malaria elimination. METHODS: The data of malaria in Yunnan Province from 2012 to 2015 were collected and analyzed. RESULTS: Totally 2 586 malaria cases were reported in Yunnan Province from 2012 to 2015, in which 274 (10.60%) were local cases and 2 311 (89.37%) were abroad imported, and one (0.03%) was domestic imported. The imported malaria cases and local cases were analyzed according to the sources and locations respectively, and the arithmetic means of the numbers of imported and local cases were 96.29 and 10.96 respectively, the standard deviations of the numbers of imported and local cases were 421.18 and 19.12 respectively, and the difference of the means was not significant (Z = - 0.326, P > 0.10). Both the imported and local malaria cases could be clustered into five sections by the number of 5. The Herfendal-Hirshman indexes of the imported and local malaria cases were 8 121 and 1 598 respectively. CONCLUSIONS: There is no significant difference of the distribution between the imported and local malaria cases, and they should be attaching equal importance. The non-uniform degree of imported cases is higher than that of the local cases, while both of them could be divided into five major clusters in the prevention and control work.

[Analysis of polymorphism of Pfhrp2 gene in Plasmodium falciparum from falciparum malaria patients in Yunnan Province].

OBJECTIVE: To analyze the polymorphism of histidine rich protein 2 (HRP II) gene in Plasmodium falciparum (Pfhrp2) from falciparum malaria patients in Yunnan Province, so as to lay the foundation for studying the defection of antigen genes of Plasmodium. METHODS: The filter paper blood samples and related information of falciparum malaria cases reported were obtained in Yunnan Province from August 2012 to September 2015. Under the guidance of the specific primers, the exon2 regions in Pfhrp2 gene in P. falciparum from DNA samples were amplified by PCR, and the PCR products were sequenced. The sequences of exon2 region in Pfhrp2 gene were blasted by comparing with the reference sequences AY816237, AY816240, and AY816301. Next, the polymorphism of the sequence in exon2 region of Pfhrp2 gene was analyzed by MEGA 5.04 software. The conserved sites and genetic distances between sequences were calculated by using the software as well, and the clustering tree was drawn according to the genetic distances between the amino acid sequences. RESULTS: A total of 218 bloods samples from the falciparum malaria cases in 15 prefectures of Yunnan Province were collected, and the sources of infection included Yunnan, Africa and Myanmar. The PCR results showed that the exon2 regions in Pfhrp2 genes of 155 samples were positive by amplification and their products were sequenced successfully. The sequence analysis showed that the length range of the amino acid residues of exon2 region in Pfhrp2 gene was from 115 aa to 298 aa, the average length was 239.7 aa. There was no statistically significance among the means of the amino acid residues of the isolates from Africa (239.9 aa), Myanmar (239.5 aa) and Yunnan (241.6 aa) (F = 0.025, P > 0.05). All the 155 amino acid sequences ended with type 12 repeat, 98.1% (152/155) of them started with type 1 repeat and 1.9% (3/155) of them started with type 2. Type 2 presented most frequently repeat in all the sequences and the average repeat times were 12.9. The homologous locus of the DNA sequences in exon2 regions of the 155 Pfhrp2 genes was 894 bp, among which the conservative sites accounted for 20.6% (186/894), and the variable sites for 78.2% (699/ 894). The genetic distances between the sequences of Africa isolates ranged from 0 to 0.741, and those of the Myanmar and Yunnan isolates were 0-0.948 and 0-0.750, respectively. The cluster analysis showed that all the 155 sequences clustered into 3 categories on genetic distances between amino acid sequences according to the size of the amino acid sequence length. At the same level, the sequences had approximate lengths and amino acid repeat types. CONCLUSIONS: The sequence of exon2 region in Pfhrp2 gene of P. falciparum from falciparum malaria cases in Yunnan Province is highly polymorphic, the P. falciparum isolates are clustered mainly according to the size of the amino acid sequence of exon2 region in Pfhrp2 gene.