Lack of quadruple and quintuple mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax isolates from Brazilian endemic areas

BACKGROUND AND OBJECTIVE Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated. METHODS Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs). FINDINGS All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%). CONCLUSION No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

PCR-RFLP for Rapid Subtyping of Plasmodium vivax Korean Isolates

Vivax malaria reemerged in Korea in 1993 and the outbreak has been continued with fluctuating numbers of annual indigenous cases. Understanding the nature of the genetic population of Plasmodium vivax circulating in Korea is beneficial for the knowledge of the nationwide parasite heterogeneity and in the implementation of malaria control programs in the country. Previously, we analyzed polymorphic nature of merozoite surface protein-1 (MSP-1) and MSP-3α in Korean P. vivax population and identified the Korean P. vivax population has been diversifying rapidly, with the appearance of parasites with new genetic subtypes, despite the recent reduction of the disease incidence. In the present study, we developed simple PCR-RFLP methods for rapid subtyping of MSP-1 and MSP-3α of Korean P. vivax isolates. These PCR-RFLP methods were able to easily distinguish each subtype of Korean P. vivax MSP-1 and MSP-3α with high accuracy. The PCR-RFLP subtyping methods developed here would be easily applied to massive epidemiological studies for molecular surveillance to understand genetic population of P. vivax and to supervise the genetic variation of the parasite circulating in Korea.

Genetic Diversity and Natural Selection in 42 kDa Region of Plasmodium vivax Merozoite Surface Protein-1 from China-Myanmar Endemic Border

Plasmodium vivax merozoite surface protein-1 (PvMSP1) gene codes for a major malaria vaccine candidate antigen. However, its polymorphic nature represents an obstacle to the design of a protective vaccine. In this study, we analyzed the genetic polymorphism and natural selection of the C-terminal 42 kDa fragment within PvMSP1 gene (Pv MSP142) from 77 P. vivax isolates, collected from imported cases of China-Myanmar border (CMB) areas in Yunnan province and the inland cases from Anhui, Yunnan, and Zhejiang province in China during 2009–2012. Totally, 41 haplotypes were identified and 30 of them were new haplotypes. The differences between the rates of non-synonymous and synonymous mutations suggest that PvMSP142 has evolved under natural selection, and a high selective pressure preferentially acted on regions identified of PvMSP133. Our results also demonstrated that PvMSP142 of P. vivax isolates collected on China-Myanmar border areas display higher genetic polymorphisms than those collected from inland of China. Such results have significant implications for understanding the dynamic of the P. vivax population and may be useful information towards China malaria elimination campaign strategies.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Etude du polymorphisme génétique des souches de plasmodium falciparum au Niger

Problématique : Le paludisme à Plasmodium falciparum est un problème majeur de santé publique au Niger. Plasmodium falciparum est l'agent responsable de 97% des cas de paludisme. Il est aussi responsable des formes cliniques graves comme le neuropaludisme et l'anémie sévère.Pour évaluer l'impact des stratégies de lutte contre le paludisme sur la diversité génétique, nous avons caractérisé les populations parasitaires du Niger en amplifiant le block2 du gène msp1 et la région variable centrale du gène msp2. Des amorces spécifiques des différentes familles allèliques (K1, MAD20 et RO33 pour msp1) puis (3D7 et FC27 pour msp2) ont permis de distinguer les allèles du gène msp1 et du gène msp2.Objectif général : L'objectif est d'analyser la diversité génétique et la complexité des infections à P.falciparum au niveau de 13 sites représentatifs de la situation épidémiologique du paludisme au Niger.Résultat : 510 échantillons de 13 sites du Niger ont été génotypés. Une très grande diversité génétique est observée avec les deux marqueurs. En effet, il y'a 17 allèles différents de type msp1 et 14 allèles différents de type msp2. La famille allélique la plus fréquente dans la population est 3D7 (63%) suivie de K1 (43.2%). Les familles alléliques les plus rares sont MAD 20 (28.4%) et RO33 (28.4%). La distribution allélique des gènes msp1 et msp2 est très variée selon le site. Le nombre de clones varie de 1 à 5 par patient. 23% des infections sont polyclonales. La multiplicité des infections (MOI) est de 2.8 au Niger. Il y'a une différence significative de MOI selon les sites (p<0.05). Nos résultats sont discutés selon la latitude et la longitude des sites puis au regard d'études semblables en Afrique de l'ouest.Conclusion : La diversité génétique et la complexité des infections dépendent du niveau de transmission du paludisme. La relation entre la multiplicité des infections et la transmission n'est pas linéaire. Des facteurs écologiques et environnementaux comme la disponibilité en eau de surface et l'humidité relative interviennent

Allelic Diversity of MSP1 Gene in Plasmodium falciparum from Rural and Urban Areas of Gabon

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.

Avaliação de marcadores moleculares na determinação da diversidade clonal nas infecções causadas por Plasmodium vivax / Evaluation of molecular markers to determine the clonal diversity in infections caused by Plasmodium vivax

A malária é causada por parasitos do gênero Plasmodium, sendo P. vivax a espécie mais amplamente distribuída no mundo. Em áreas endêmicas, é frequente a coinfecção em um mesmo indivíduo por diferentes variantes de P. vivax,caracterizando uma infecção múltipla. Nestas infecções, diferentes proporções das variantes podem ser relevantes na competição entre elas, na virulência e na resistência às drogas. O objetivo deste trabalho foi de avaliar a eficiência dos marcadores moleculares na identificação de infecções múltiplas por P.vivax. Desta forma, foram selecionados seis marcadores (MS06, MS07, MSP-3α, MSP-1B2,MSP-1B10 e MN21) caracterizados por polimorfismos de tamanho analisados por eletroforese capilar. Para cumprir este objetivo foram usadas três estratégias metodológicas. Inicialmente dois diferentes alelos amplificados de cada loci foram clonados em plasmídeos para o estabelecimento de infecções múltiplas artificiais...

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

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.

Probability of Antibody Formation against Circumsporozoite Protein of Plasmodium vivax among Korean Malaria Patients

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.

Evolution of Genetic Polymorphisms of Plasmodium falciparum Merozoite Surface Protein (PfMSP) in Thailand

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.

Avaliação de marcadores moleculares na determinação da diversidade clonal nas infecções causadas por Plasmodium vivax

A malária é causada por parasitos do gênero Plasmodium, sendo P. vivax a espécie mais amplamente distribuída no mundo. Em áreas endêmicas, é frequente a coinfecção em um mesmo indivíduo por diferentes variantes de P. vivax,caracterizando uma infecção múltipla. Nestas infecções, diferentes proporções das variantes podem ser relevantes na competição entre elas, na virulência e na resistência às drogas. O objetivo deste trabalho foi de avaliar a eficiência dos marcadores moleculares na identificação de infecções múltiplas por P.vivax. Desta forma, foram selecionados seis marcadores (MS06, MS07, MSP-3α, MSP-1B2,MSP-1B10 e MN21) caracterizados por polimorfismos de tamanho analisados por eletroforese capilar. Para cumprir este objetivo foram usadas três estratégias metodológicas. Inicialmente dois diferentes alelos amplificados de cada loci foram clonados em plasmídeos para o estabelecimento de infecções múltiplas artificiais.

Na segunda estratégia foram realizadas misturas artificiais de DNAs de pacientes com parasitemia e níveis de leucócitos semelhantes. E por fim, realizou-se a genotipagem de isolados de P. vivax de 47 pacientes da Amazônia Brasileira, sendo 22 pacientes com infecção múltipla previamente identificada. Ao simularmos infecções múltiplas artificiais com diferentes proporções de cada clone ou DNA de paciente verificamos que: (1) para os MS e TR, alelos menores foram preferencialmente amplificados por PCR; (2) para ambos os blocos da MSP-1,ocorreu titulação nos resultados; (3) para MSP-3α não houve amplificação preferencial do alelo menor; (4) a utilização do critério de altura mínima de » da do pico predominante para a detecção dos alelos raros foi mais eficiente na detecção da multiplicidade de infecção. Na genotipagem de isolados de campo os marcadores MS06, MS07, MSP-1B10, MSP-3α e MN21 foram suficientes para detecção de100% das infecções múltiplas em pacientes. Assim, estamos propondo um painel demarcadores neutros (MS06, MS07 e MN21) e não neutros (MSP-3α e MSP-1B10) na detecção de infecções múltiplas utilizando o critério menos estringente para detecção dos alelos raros.

Predicción de unión de péptidos de Plasmodium falciparum al HLA clase II. Probabilidad, combinatoria y entropía aplicadas a las proteínas MSP-5 y MSP-6 / Plasmodium falciparum binding peptides prediction to HLA class II. Probability, combinatory and entropy applied to MSP-5 and MSP-6 proteins

Las proteínas de superficie del merozoíto (MSP) son de importancia en la invasión parasitaria al glóbulo rojo. La proteína MSP-5, encontrada en merozoítos libres, tiene un papel en la inmunización de ratones al P. falciparum y P. yoelii, pese a lo cual algunos estudios cuestionan su rol en la invasión. La proteína MSP-6 forma junto con MSP-1 y MSP-7 un complejo en la superficie del merozoíto, liberado del parásito cerca del momento de la invasión al glóbulo rojo. Con el fin de predecir el fenómeno de unión de péptidos de las proteínas de superficie MSP-5 y MSP-6, se aplicó una teoría de unión al HLA clase II, a la totalidad de secuencias de 20 aminoácidos de tales moléculas. Se calcularon los valores de probabilidad, combinatoria y entropía de 168 secuencias nonámeras sobrelapadas de la proteína MSP-5 y 228 de MSP-6. Por último se aplicó la teoría de unión a todos los péptidos nonámeros de tres proteínas construidas computacionalmente, cada una con una longitud de 500 aminoácidos. Para la proteína MSP-5 se predijo un total de 31 secuencias asociadas al macroestado de unión y 137 al de no unión, mientras que se predijo la existencia de 35 secuencias asociadas al macroestado de unión para MSP-6 y 193 al de no unión. Se encontraron respectivamente 100, 111 y 91 secuencias predichas de unión para las tres proteínas teóricas construidas. La predicción teórica de unión de péptidos es útil para facilitar el desarrollo de vacunas, al evidenciar el orden físico-matemático subyacente al fenómeno.(AU)

Population genetics and drug resistance markers: an essential for malaria surveillance in Pakistan

Plasmodium [P.] vivax is the prevalent malarial species accounting for 70% of malaria cases in Pakistan. However, baseline epidemiological data on P. vivax population structure and drug resistance are lacking from Pakistan. For population structure studies, molecular genetic markers, circumsporozoite protein [csp] and merozoite surface protein-1 [msp-1] are considered useful as these play an important role in P. vivax survival under immune and environmental pressure. Furthermore, these genes have also been identified as suitable candidates for vaccine development. While efforts for effective vaccine are underway, anti-malarial agents remain the mainstay for control. Evidence of resistance against commonly used anti-malarial agents, particularly Sulphadoxine-Pyrimethamine [SP] is threatening to make this form of control defunct. Therefore, studies on drug resistance are necessary so that anti-malarial treatment strategies can be structured and implemented accordingly by the Malaria Control Program, Pakistan. This review aims to provide information on genetic markers of P. vivax population structure and drug resistance and comment on their usefulness in molecular surveillance and control.

Diferentes genótipos do lócus da Proteína de Superfície de Merozoíto 1(MSP-1) de Plasmodium falciparum: uma contribuição ao estudo da infecção plasmodial assintomática no município de Barcelos, Amazonas / Different genotypes of locus of merozoite surface protein 1 (MSP-1) Plasmodium falciparumuma contribution to the study plasmodial asymptomatic infection in the municipality of Barcelos, Amazonas

A malária é uma doença que acomete milhões de pessoas todos os anos no planeta. No Brasil, essa doença atingiu cerca de 267 mil pessoas somente em 2011, sendo que a maior parte dos casos ocorreu na região Amazônica. Causada por protozoários do gênero Plasmodium, possui um amplo espectro clínico que vai desdeinfecções assintomáticas, malária não complicada, a malária grave ou complicada, podendo evoluir até o óbito. Estudos realizados em áreas holo e hiperendêmicas de malária, fundamentalmente no continente Africano e em Papua Nova Guiné, permitiram estabelecer que o conhecimento da população de parasitos que circulam em uma área.Este estudo se propôs a avaliar o perfil genético da infecção por P. falciparumem uma área de alto risco epidemiológico para malária e com presença de infecção assintomática no município de Barcelos, médio rio Negro, estado do AmazonasPara isso, foi estudado o gene MSP1 que codifica a Proteína de Superfície de Merozoíto 1, com o intuito de verificar a diversidade genética dos parasitos da região de Barcelos, a multiplicidade da infecção e as relações dos diferentes genótipos com o status clínico em área endêmica para malária. Foi realizada reação de polimerase em cadeia (PCR) a partir de DNA extraído de 79 amostras de sangue coletadas em indivíduos desta região. No total, foram encontrados 10 diferentes genótipos do parasito sendo que a maior parte dos indivíduos (45 pessoas, 56,96 por cento) portava apenas um genótipo do parasito. Entre os indivíduos assintomáticos, 70,37 por cento apresentavam um genótipo e os demais (29,63 por cento) apresentaram dois genótipos...

Malaria is a disease that attacks millions of people all of the years in the world. In Brazil,it reached about 267 thousand people only in 2011, and most of the cases wasregistered in the Amazonian area. Caused by protozoa of the gender Plasmodium, itpossesses a wide clinical spectrum ranging from asymptomatic infections, nocomplicatedmalaria, serious or complicatedmalaria that could develop until death.Studies accomplished in holo- and hyperendemic areas, fundamentally in the Africancontinent and in Papua New Guinea, allowed to establish that the knowledge of thepopulation of parasites that circulate in a certain area. This study intended to evaluatethe genetic profile of the infection for P. falciparum in an area of high epidemic risk formalaria and with the presence of asymptomatic infection in the city of Barcelos, mediumRio Negro, state of Amazonas. For that, the gene MSP-1, that codifies the MerozoiteSurface Protein 1, was studied to verify the genetic diversity of the parasites of the areaof Barcelos, the multiplicity of the infection and the relationships of the differentgenotypes with the clinical status in an endemic area for malaria. Polymerase ChainReaction (PCR) was accomplishedstarting from extracted DNA from blood samplescollected in individuals from this area. In total, 10 different genotypes of the parasitewere found and most of the individuals (45 people, 56,96 percent) carried just a genotype ofthe parasite. Among the asymptomatical individuals, 70,37 percent introduced a genotype andthe others (29,63 percent) presented two...

Allelic frequencies of single nucleotide polymorphisms (SNPs) in the N-acetyltransferase 2 (NAT2) gene of Filipinos

OBJECTIVES:The present study aims to determine the frequency of occurrence of NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A alleles by PCR-RFLP among Filipino volunteers. These alleles correspond to substitutions in the following sites: C341T, G590A, G857A and G191A, respectively, of the NAT2 gene. The presence of specific SNP combination was also used to deduce acetylation status and estimate genotype frequency and describe them in comparison with other populations based on literature. METHODS: Genomic DNA from peripheral blood lymphocytes from 129 healthy Filipino volunteers was used to amplify the NAT2 gene segment. The RFLP analysis was done by restricting the expected PCR product with Kpn1, Taq1, BamH1 and Msp1/Al1, respectively, to detect the 4 alleles: NAT2*4, NAT2*5A, NAT2*6B, NAT2*7A and NAT2*14A. RESULTS:The calculated allelic frequencies in Hardy-Weinberg equilibrium of NAT2*5A (C481T), NAT2*6B (G590A), NAT2*7A (G857A) and NAT2*14A (G191A) were 0.058, 0.097, 0.182 and 0.046, respectively. NAT2*4 had an allele frequency of 0.617. Nine genotypes were determined: NAT2*4/*4, NAT2*4/*5A, NAT2*4/*6B, NAT2*4/*7A, NAT2*4/*14A, NAT2*5A/*7A, NAT2*6B/*7A, NAT2*6B/*14A and NAT2*7A/*14A. From these genotypes, acetylator phenotypes were deduced. A trimodal pattern of distribution was established: rapid, intermediate and slow acetylators with the following percentages, 47.3%, 41.1 % and 11.6%. Among the slow acetylator SNPs determined, NAT2*7A was found as the most frequent allele and NAT2*14A was found as the least frequent allele. CONCLUSION:The study showed the mutation profile and observed genotypic similarities and differences of Filipinos with other Asian populations and Americans and other Caucasians based on literature. The results also suggest a trimodal pattern of distribution of acetylators and lesser number of slow acetylators among Filipino populations, a characteristic similar to other Asian populations but significantly different from Americans and other Caucasians. The occurrence of NAT2*7A and NAT2*14A can be further sequenced to verify the observed genotype.

Polymorphism in merozoite surface protein-1 gene in north & northwest indian field isolates of Plasmodium vivax.

Background & objective: Merozoite surface protein-1 of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. Methods: Parasite DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. Results: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. Interpretation & conclusion: Marked genetic polymorphism was observed in msp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

A Locally Acquired Falciparum Malaria via Nosocomial Transmission in Korea

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.

Caracterización física y matemática de péptidos de alta unión de MSP-I mediante la aplicación de la teoría de la probabilidad y la entropía / Physical and mathematical characterization of peptides of high binding of MSP-I by means of the applicatión probability and the entropy theory

Introducción: MSP-1, proteína de superficie del merozoíto, es una proteína sintetizada durante el desarrollo del esquizonte e interactúa con el receptor de membrana del glóbulo rojo, por lo cual es una proteína implicada en el reconocimiento del merozoito al eritrocito. Métodos. Se construyó un espacio de probabilidad no equiprobable que cuantifica la probabilidad de aparición de cada animoácido en cada una de las veinte posiciones para 79 péptidos no sobrelapados de MSP-1. Conéste se calcularon los valores de probabilidad, sumatoria de probabilidad y entropía para estas secuencias, con el objetivo de diferenciar física y matemáticamente los péptidos de alta unión y no unión. Resultados. Se encontró que los valores de sumatoria de probabilidad, de probabilidad y los de entroìa para las secuencias específicas comprobadas experimentales de alta unión varían entre los rangos asociados al macroestado unión, mientras que todos los valores (sumatoria de probabilidad, probabilidad y entropía) para todos los péptidos comprobados de no unión se encrutran fuera de los rangos asociados al macroestad de unión. Todos los péptidos de alta unión y baja unión fueron diferenciados acertadamente según estudios experimentales. La probabilidad, sumatoria de probabilidad y entropía diferencian las secuencias que se unen de las que no, acertando en el 100% de los casos estudiados. Conclusión. Esta metodología es útil para caracterizar péptidos de alta unión en la proteína MSP-1 de una forma objetiva y reproducible, evidenciando que el fenómeno de unión de MSP-1 al merozoíto presenta un orden físico y matemático subyacente.

Naturally acquired antibodies to merozoite surface protein (MSP)-1(19) and cumulative exposure to Plasmodium falciparum and Plasmodium vivax in remote populations of the Amazon Basin of Brazil

To infer recent patterns of malaria transmission, we measured naturally acquired IgG antibodies to the conserved 19-kDa C-terminal region of the merozoite surface protein (MSP)-1 of both Plasmodium vivax (PvMSP-1(19)) and Plasmodium falciparum (PfMSP-1(19)) in remote malaria-exposed populations of the Amazon Basin. Community-based cross-sectional surveys were carried out between 2002 and 2003 in subjects of all age groups living along the margins of the Unini and Jaú rivers, Northwestern Brazil. We found high prevalence rates of IgG antibodies to PvMSP-1(19) (64.0 - 69.6 percent) and PfMSP-1(19) (51.6 - 52.0 percent), with significant differences in the proportion of subjects with antibodies to PvMSP-1(19) according to age, place of residence and habitual involvement in high-risk activities, defining some groups of highly exposed people who might be preferential targets of malaria control measures. In contrast, no risk factor other than age was significantly associated with seropositivity to PfMSP-1(19). Only 14.1 percent and 19.3 percent of the subjects tested for antibodies to PvMSP-1(19) and PfMSP-1(19) in consecutive surveys (142 - 203 days apart) seroconverted or had a three fold or higher increase in the levels of antibodies to these antigens. We discuss the extent to which serological data correlated with the classical malariometric indices and morbidity indicators measured in the studied population at the time of the seroprevalence surveys and highlight some limitations of serological data for epidemiological inference.

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1.

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1(42)) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1(42)) and P. cynomolgi B (PcMSP-1(42)). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1(42) proteins were homogenous in nature. The soluble and refolded MSP-1(42) antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1(42) antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1(42) protein. The immune sera from rhesus monkeys. immunized with soluble and refolded MSP-1(42) antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1(42) conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1(42) antigens were highly immunogenic and thus a viable candidate for vaccine studies.