Genetic diversity analysis of PvCSP and its application in tracking of Plasmodium vivax.

BACKGROUND: Plasmodium vivax remains a potential cause of morbidity and mortality for people living where it is endemic. Understanding the regional genetic diversity of P. vivax is valuable for studying population dynamics and tracing the origins of parasites. The Plasmodium vivax circumsporozoite gene (PvCSP) is highly polymorphic and has been used previously as a marker in P. vivax population studies. The aim of this study is to investigate the genetic diversity of the PvCSP, to provide more genetic polymorphism data for further studies on P. vivax population structure, and tracking of the origin of clinical cases. METHODS: Nested PCR and DNA sequencing of the PvCSP were performed to obtain nucleotide sequences of P. vivax isolates collected from Zhejiang province, China, between 2006 and 2014. To investigate the genetic diversity of PvCSP, the nucleotide sequences and amino acid sequences of the PvCSP were analyzed using DNAstar, Mega software and the phylogenetic tree constructed. The relatedness between the polymorphism and infection source were also analyzed using the SPSS software. RESULTS: The 66 P. vivax isolates collected from Zhejiang province were either indigenous cases or cases imported from different regions of the world. All 66 P. vivax isolates belonged to the VK210 variant. Fourteen different Peptide Repeat Motifs (PRMs) were detected in the Central Repeat Region (CRR) of PvCSP, among which, two PRMs of GDRADGQPA and GDRAAGQPA were widely distributed in all isolates. Several polymorphic characteristics of the VK210 variant were observed, including the insertion sequence of 12 peptides, the frequency of the GGNA repeat, the frequency of the PRMs repeat in CRR, and the frequency of the PRM of GNGAGGQAA repeat, which were indicative for tracking the parasite. CONCLUSION: This study presents abundant genetic diversity in the PvCSP marker among P. vivax strains around the world. The genetic data are valuable to expand the polymorphism information on P. vivax, which could be helpful for further study on population dynamics and tracking the origin of P. vivax.

Genetic diversity of Plasmodium Vivax revealed by the merozoite surface protein-1 icb5-6 fragment.

BACKGROUND: Plasmodium vivax remains a potential cause of morbidity and mortality for people living in its endemic areas. Understanding the genetic diversity of P. vivax from different regions is valuable for studying population dynamics and tracing the origins of parasites. The PvMSP-1 gene is highly polymorphic and has been used as a marker in many P. vivax population studies. The aim of this study was to investigate the genetic diversity of the PvMSP-1 gene icb5-6 fragment and to provide more genetic polymorphism data for further studies on P. vivax population structure and tracking of the origin of clinical cases. METHODS: Nested PCR and sequencing of the PvMSP-1 icb5-6 marker were performed to obtain the nucleotide sequences of 95 P. vivax isolates collected from Zhejiang province, China. To investigate the genetic diversity of PvMSP-1, the 95 nucleotide sequences of the PvMSP-1 icb5-6 fragment were genotyped and analyzed using DnaSP v5, MEGA software. RESULTS: The 95 P. vivax isolates collected from Zhejiang province were either indigenous cases or imported cases from different regions around the world. A total of 95 sequences ranging from 390 to 460 bp were obtained. The 95 sequences were genotyped into four allele-types (Sal I, Belem, R-III and R-IV) and 17 unique haplotypes. R-III and Sal I were the predominant allele-types. The haplotype diversity (Hd) and nucleotide diversity (Pi) were estimated to be 0.729 and 0.062, indicating that the PvMSP-1 icb5-6 fragment had the highest level of polymorphism due to frequent recombination processes and single nucleotide polymorphism. The values of dN/dS and Tajima's D both suggested neutral selection for the PvMSP-1icb5-6 fragment. In addition, a rare recombinant style of R-IV type was identified. CONCLUSIONS: This study presented high genetic diversity in the PvMSP-1 marker among P. vivax strains from around the world. The genetic data is valuable for expanding the polymorphism information on P. vivax, which could be helpful for further study on population dynamics and tracking the origin of P. vivax.

[Iditification of five imported cases of Plasmodium ovale wallikeri infection in Zhejiang Province].

OBJECTIVE: To identify and analyze Plasmodium ovale wallikeri in 5 imported malaria cases, who were detected positive by microscopy and negative by conventional PCR. METHODS: Epidemiological information and blood samples were collected from the five patients. The detection was conducted by microscopy, Rapid Diagnostic Test (RDT) and nested PCR with Plasmodium genus-specific, species-specific and Plasmodium ovale wallikeri-specific primers. The amplified products were sequenced and Blast analysis was performed on line in NCBI. RESULTS: The five patients returned from Africa, and all had a history of malaria. They were microscopically positive for Plasmodium sp., and two cases showed Pan positive RDT result. All blood samples were negative for four Plasmodium spp. by conventional nested PCR, but positive by nested PCR with Plasmodium ovale wallikeri-specific primers. Blast analysis showed that the amplified sequences of the five cases had complete homology with P. ovale wallikeri clone RSH10 18S ribosomal RNA gene (Accession No. KF219561.1). CONCLUSION: The five cases which classified as positive by microscopy while negative by conventional PCR have been confirmed as Plasmodium ovale wallikeri infection by nested PCR with P. ovale wallikeri-specific primers.

[Species identification in 5 imported cases previously diagnosed as Vivax malaria by parasitological and nested PCR techniques].

OBJECTIVE: To identify the species of malaria parasites in 5 imported cases previously diagnosed as vivax malaria. METHODS: Epidemiological information and blood samples were collected from five patients who returned from Africa and were diagnosed as vivax malaria. The detection was conducted by microscopy, right VIEW rapid malaria test (RDTs) and nested PCR with Plasmodium genus-specific and species-specific primers. The amplified products were sequenced and Blast analysis was performed. RESULTS: Three of the 5 cases had a history of malaria attack. Microscopically, 4 cases were confirmed as Plasmodium ovale infection, 1 (case 1) was co-infected with P. vivax and P. ovale. All 5 cases showed negative RDT results. Nested PCR detection revealed that the 5 cases had a P. ovale-specific fragment (800 bp), while case 1 had a P. vivax-specific fragment (120 bp) concurrently. Blast analysis showed that the amplified sequence of the 5 cases had a high sequence homology (99%) with P. ovale gene for small subunit ribosomal RNA from GenBank, and that of case 1 also shared 99% homology with P. vivax isolate SV5 18S ribosomal RNA gene (GenBank accession number: JQ627157.1). CONCLUSION: Among the five cases, four were infected by Plasmodium ovale, and one was co-infected with both P. vivax and P. ovale.

[Diagnosis and analysis of the first imported ovale malaria case in Wenzhou City].

The first imported case of Plasmodium ovale infection in Wenzhou City was confirmed by microscopy and PCR test. The patient returned from the People's Republic of Congo to Wenzhou for three and a half months presented a history of fever with chills and rigors on April 30, 2012. The results from peripheral blood smear examination and PCR analysis confirmed a diagnosis of P. ovale infection. The patient was treated with chloroquine plus primaquine for eight days and the symptoms improved.

[Pathogen identification and clinical diagnosis for one case infected with Babesia].

OBJECTIVE: To identify the pathogen and make diagnosis on a case who was misdiagnosed as malaria. METHODS: Clinical and epidemiological information of the suspected case was collected. Blood samples during hospitalization were collected and examined microscopically. Genomic DNA from the blood samples was amplified by Babesia 18S RNA genus- and species- specific primers, respectively, and the amplified products were used in sequencing and BLAST sequence analysis. RESULTS: The case had a fever over 20 days repeatedly with anaemia (RBC 2.59 x 10(12), HB 5.5 g/L) and hepatosplenomegaly. The unidentified parasites were found in the bone marrow and blood smear after Giemsa staining. Epidemiological information revealed that this case had a history of blood transfusion and tick bites. 1 625 bp and 449 bp band generated by PCR amplification from blood sample using Babesia genus- and species-specific primers, and the sequence homology was 99% in comparison to Babesia microti (AB241632) with BLAST analysis. CONCLUSION: The clinical information, epidemiological history, and the PCR identification confirm the diagnosis of Babesia microti infection.