Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.
Antigens, Protozoan/*genetics ; DNA, Protozoan/genetics ; Evolution, Molecular ; Humans ; Malaria, Falciparum/parasitology ; Merozoite Surface Protein 1/*genetics ; Plasmodium falciparum/*classification/*genetics/isolation & purification ; Polymerase Chain Reaction ; *Polymorphism, Genetic ; Protozoan Proteins/*genetics ; Thailand

The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families' frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (+/-0.95) higher compared to that found at Owendo (1.55+/-0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.
Child ; Child, Preschool ; Female ; Gabon ; *Gene Frequency ; Genetic Variation ; Genotype ; Humans ; Infant ; Malaria, Falciparum/*parasitology ; Male ; Merozoite Surface Protein 1/*genetics/metabolism ; Plasmodium falciparum/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; Rural Population ; Urban Population

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.
Adolescent ; Adult ; Animals ; Antigens, Protozoan/*genetics ; Child ; Child, Preschool ; *Endemic Diseases ; Female ; Genetic Variation ; Humans ; Iran/epidemiology ; Malaria, Falciparum/*epidemiology/*parasitology ; Male ; Merozoite Surface Protein 1/*genetics ; Middle Aged ; Plasmodium falciparum/*genetics/immunology/isolation & purification ; Polymerase Chain Reaction/methods ; Polymorphism, Genetic ; Protozoan Proteins/*genetics

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.
Adolescent ; Adult ; Animals ; Antigens, Protozoan/*genetics ; Child ; Child, Preschool ; *Endemic Diseases ; Female ; Genetic Variation ; Humans ; Iran/epidemiology ; Malaria, Falciparum/*epidemiology/*parasitology ; Male ; Merozoite Surface Protein 1/*genetics ; Middle Aged ; Plasmodium falciparum/*genetics/immunology/isolation & purification ; Polymerase Chain Reaction/methods ; Polymorphism, Genetic ; Protozoan Proteins/*genetics

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.
Adult ; Antigens, Protozoan/genetics ; Antimalarials/*therapeutic use ; Artemisinins/*therapeutic use ; Female ; Gene Frequency ; *Genetic Variation ; Genotype ; Humans ; Malaria, Falciparum/*drug therapy/*parasitology ; Male ; Merozoite Surface Protein 1/genetics ; Myanmar ; Plasmodium falciparum/*classification/*genetics/isolation & purification ; Polymerase Chain Reaction ; Protozoan Proteins/genetics ; Thailand ; Treatment Failure

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.
Amino Acid Sequence ; Antibodies, Protozoan/*blood/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Base Sequence ; Humans ; India ; Malaria, Vivax/*diagnosis/*epidemiology/immunology ; Merozoite Surface Protein 1/genetics/*immunology ; Plasmodium vivax/genetics/immunology ; Protozoan Proteins/genetics/*immunology ; Reagent Kits, Diagnostic ; Recombinant Proteins/diagnostic use/immunology ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Seroepidemiologic Studies ; Uganda

A 57-year old man who was admitted to an emergency room of a tertiary hospital with hemoptysis developed malarial fever 19 days later and then died from severe falciparum malaria 2 days later. He had not traveled outside of Korea for over 30 years. Through intensive interviews and epidemiological surveys, we found that a foreign patient with a recent history of travel to Africa was transferred to the same hospital with severe falciparum malaria. We confirmed through molecular genotyping of the MSP-1 gene that Plasmodium falciparum genotypes of the 2 patients were identical. It is suggested that a breach of standard infection control precautions resulted in this P. falciparum transmission between 2 patients in a hospital environment. This is the first report of a nosocomial transmission of falciparum malaria in Korea.
Africa ; Amino Acid Sequence ; Animals ; Cross Infection/parasitology/*transmission ; Fatal Outcome ; Humans ; Korea ; Malaria, Falciparum/parasitology/*transmission ; Male ; Merozoite Surface Protein 1/chemistry/genetics ; Middle Aged ; Molecular Sequence Data ; Plasmodium falciparum/chemistry/genetics/*isolation & purification ; Protozoan Proteins/chemistry/genetics ; Sequence Alignment ; Travel

Malaria is still a major health problem in Thailand and its incidence is currently rising in Korea. To identify a useful antigen for the diagnosis of malaria patients, a cDNA expression library from malaria parasites was constructed and screened out immunologically. One clone was selected in view of its predominant reactivity with the patient sera. The recombinant malaria parasite antigen (Pv30) with 27 kDa as a C-terminal His-tag fusion protein that was produced in Escherichia coli was identified through immunoblot analysis. The deduced amino acid sequence had the sequence homology with the merozoite surface protein 1 (MSP1) genes of Plasmodium falciparum and P. yoelii, each by 41% and 42%, respectively. Measurement of serum IgG and IgM antibody to Pv30 by enzyme-linked immunosorbent assay (ELISA) was evaluated as a serodiagnostic test for malaria patients in Thailand (endemic area) and Korea (recently reemerging area). The sensitivity of P. vivax, P. falciparum, and P. malariae was 96.3% (26 /27), 90.6% (29/32), and 100% (6/6), respectively, and the specificity was 63.5% (40/63) in Thailand samples. The sensitivity of P. vivax was 98.8% (88/89), and the specificity was 96.6% (86/89) in Korean samples. Pv30 appears to be a good and reliable recombinant antigen for serodiagonosis of malaria in a nonendemic area.
Amino Acid Sequence ; Animals ; Antibodies, Protozoan ; Enzyme-Linked Immunosorbent Assay/*methods ; Human ; Korea ; Malaria, Vivax/*diagnosis/immunology ; Merozoite Surface Protein 1/*analysis/genetics/immunology ; Molecular Sequence Data ; Plasmodium vivax/chemistry/immunology/*isolation & purification ; Sensitivity and Specificity ; Serologic Tests ; Support, Non-U.S. Gov't