Characterization of Pv92, a Novel Merozoite Surface Protein of Plasmodium vivax

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

Actividad anti-plasmodial de especies de Solanaceae presentes en Cuba / Antiplasmodial activity of Solanaceae species present in Cuba

Introducción: el control de la malaria depende en gran medida de una terapia efectiva. Muchos de los anti-maláricos actuales son de origen natural. Especies de la flora cubana contienen metabolitos anti-Plasmodium. En este estudio, se identifican extractos de Solanaceae con actividad antiplasmodial promisoria. Objetivo: evaluar la actividad esquizonticida frente a Plasmodium berghei de 31 extractos de 7 especies, correspondientes a 5 géneros de plantas de Solanaceae, colectadas en el occidente de nuestro país y sin antecedentes de un estudio similar. Métodos: se prepararon 31 extractos hidroalcohólicos (90 y 30 por ciento etanol) de diferentes órganos de: Brunfelsia undulata Sw., Datura stramonium L. var. tatula (L.) Torr., Physalis solanaceus (Schltdl.) Axelius, Solandra longiflora Tuss., Solanum myriacanthum Dunal, Solanum seaforthianum And. ySolanum umbellatum Mill.La actividad de los extractos se evaluó in vitro frente a P. berghei y se determinó su citotoxicidad frente a fibroblastos humanos MRC-5. Resultados: los extractos deB. undulata y S. umbellatumfueron inactivos.El extracto de tallos de S. seaforthianummostró la actividad antiplasmodial más potente (CI50 = 3,9µg/mL) con excelentes electividad (18,2). Conclusiones: se demostró la actividad anti-plasmodial in vitro de extractos de cinco especies de Solanaceae sin antecedentes de esta acción farmacológica. Se identificó un extracto con potente actividad esquizonticida frente a P. berghei y con excelente selectividad. Este resultado nos anima a continuar el estudio de la preparación vegetal de S. seaforthianum(AU)

A Case of Primaquine-Induced Acute Liver Failure / 대한내과학회지

Primaquine was approved for treatment of malaria in 1952 by the United States Food and Drug Administration (FDA). It has remained the only FDA-licensed drug capable of clearing the intra-hepatic schizonts and hypnozoites of Plasmodium vivax. It is associated with serious hazards and side effects, such as hemolytic anemia and methemoglobinemia. However, there is no report of primaquine causing liver injury in Korea. We describe a case of acute liver failure following primaquine overdose in a 19-year-old man.

Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2

OBJECTIVE@#To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.@*METHODS@#Mice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm(2)) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.@*RESULTS@#A total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera.@*CONCLUSIONS@#Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

<p><b>OBJECTIVE</b>To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210.</p><p><b>METHODS</b>The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays.</p><p><b>RESULTS</b>A total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210.</p><p><b>CONCLUSIONS</b>On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.</p>

Citogênese e estágios esquizontes em células epiteliais de felinos infectadas pelo Toxoplasma gondii, in vitro / Cystogenesis stages and schizonts in epithelial cells of cats infected with Toxoplasma gondii in vitro

O Toxoplasma gondii é um protozoário parasito intracelular, agente causador da toxoplasmose que é uma das zoonoses mais endêmicas do mundo. O T.gondii possui dois tipos de reprodução : assexuada, que origina dois estágios evolutivos infectivos, os bradizoítos e taquizoítos e a sexuada que ocorre no tecido epitelial intestinal, exclusivamente de felídeos hospedeiros definitivos, resultando na formação de oocistos imaturos que são eliminados nas suas fezes. A disseminação de oocistos no ambiente é o principal fator que explica a distribuição mundial do parasito, associada à formação de cistos teciduais que aumentam a capacidade de transmissão do parasito, através do consumo de carne crua ou mal cozida. Dada a especificidade de o ciclo sexuado ocorrer no tecido epitelial, o principal objetivo do presente trabalho foi investiga, in vitro, a interação do T. gondii com células epiteliais oriundas do intestino de ratos e de rim de felídeos, a fim de estabelecer possíveis correlações entre a origem do tecido (epitelial) e a fonte (rato e felídeos) que possam determinar o destino intracelular do parasito. Assim, culturas de linhagens celulares CRFK e IEC-6 foram infectadas com bradizoítos e taquizoítos de T. gondii com diferentes cargas infectivas. Após os desenhos experimentais as culturas foram processadas como de rotina para microscopia óptica de luz e fluorescência e para microscopia eletrônica de transmissão e varredura. As análises quantitativas mostraram que a linhagem CRFK foi mais suscetível à infecção frente aos dois estágios infectivos do que a linhagem IEC-6 e que essa diferença foi independente da carga parasitária e do estágio infectivo utilizados. As análises qualitativas mostraram que a linhagem CRFK foi mais susceptível à infecção do que a linhagem IEC-6 e esse evento foi independente da carga infectiva utilizada. As análises qualitativas mostraram que a cistogêneses se estabeleceu de forma espontânea na CRFK quando infectada com bradizoítos da cepa ME49 ao se utilizar a carga efetiva de 1:10 (parasito-célula hospedeira). Além disso, esta linhagem celular apontou estágios infectivos morfologicamente distintos de taquizoítos e bradizoítos, similares aos estágios esquizontes de T. gondii. O presente estudo mostrou que existem diferenças no destino intracelular do parasito de acordo com o tipo celular utilizado. O emprego de células epiteliais de felinos como modelo celular da toxoplasmose experimental mostrou que pode contribuir com novos subsídios para o estudo da biologia celular do parasito, assim como contribuir como metodologia alternativa para o melhor entendimento do ciclo enteroepitelial do T. gondii. Este modelo celular abre um novo campo de investigação para aspectos moleculares desta interação que possam contribuir, por exemplo, para introdução de estratégias que venham a interferir em umas das principais vias de disseminação da toxoplasmose. Além disso, a CRFK se mostrou um modelo celular em potencial para a produção de cistos de T. gondii in vitro em larga escala abrindo perspectivas na redução do uso de animais experimentais para isolamento de cistos e inserção na área de inovação e desenvolvimento tecnológico.

A Case of Plasmodium vivax Malaria Associated with Autoimmune Hemolytic Anemia / 감염과화학요법

Anemia associated with Plasmodium vivax (P.vivax) malaria occurs as a result of the lysis of red cells by schizonts, bone marrow suppression, and splenic sequestration. A 20-year-old man presented with fever and anemia. He was diagnosed with P. vivax malaria with a positive direct antiglobulin test and treated with antimalarial medication for 2 weeks. He recovered without sequelae. we suggest that autoimmune hemolytic anemia should be considered as one of cause of anemia in P. vivax malaria.

A Case of Plasmodium vivax Malaria Associated with Autoimmune Hemolytic Anemia / 감염과화학요법

Anemia associated with Plasmodium vivax (P.vivax) malaria occurs as a result of the lysis of red cells by schizonts, bone marrow suppression, and splenic sequestration. A 20-year-old man presented with fever and anemia. He was diagnosed with P. vivax malaria with a positive direct antiglobulin test and treated with antimalarial medication for 2 weeks. He recovered without sequelae. we suggest that autoimmune hemolytic anemia should be considered as one of cause of anemia in P. vivax malaria.

Assessment of in vitro antimalarial interactions between dihydroartemisinin and fosmidomycin.

Malaria remains one of the leading causes of morbidity and mortality in the tropics with an annual estimate of 500 million clinical cases and 2 million deaths. The treatment and control of malaria is becoming increasingly difficult due to Plasmodium falciparum resistance to commonly used antimalarials. Combination therapy is currently the strategy for combating multi-drug resistant falciparum malaria, through exploiting pharmacodynamic synergistic effects and delaying the emergence of drug resistance. The combination of artemisinin derivatives with fosmidomycin, which have different modes of action, appears to be one of the most promising combinations. The objective of the present study was to investigate the antimalarial interactions between dihydroartemisinin and fosmidomycin in vitro, against chloroquine-resistant (K1) and chloroquine-sensitive (G112) P. falciparum strains. Concentration-response analysis was performed based on an in vitro schizont maturation inhibition test. The fixed concentration ratios of dihydroartemisinin: fosmidomycin used were 0:5,000, 2:4,500, 6:3,500, 10:2,500, 14:1,500, 18:500 and 20:0 nM. The highest final concentrations of dihydroartemisinin and fosmidomycin were 20 and 5,000 nM, respectively. Results showed IC50 (drug concentration which produced 50% schizont maturation inhibition) medians (range) for dihydroartemisinin against K1 and G112 strains to be 1.6 (1.2-2.0) and 2.5 (2.4-2.6) nM, respectively. The IC50 medians (range) for fosmidomycin against K1 and G112 strains were 1,347 (1,068-1,625) and 786 (737-834) nM, respectively. An isobologram revealed an increasing trend for the fraction IC50 (FIC), which indicates marked antagonism of this drug combination against both chloroquine resistant and chloroquine sensitive strains.

'DEAD-box' helicase from Plasmodium falciparum is active at wide pH and is schizont stage-specific.

BACKGROUND & OBJECTIVES: DNA helicases catalyse unwinding of duplex DNA in an ATP-dependent manner and are involved in all the basic genetic processes. In order to study these important enzymes in the human malaria parasite we have recently cloned the first full-length 'DEAD-box' helicase gene from Plasmodium falciparum (3D7). In the present study, we report some of the important activities of the encoded protein. METHODS: We have expressed the P. falciparum helicase in Escherichia coli and characterised the encoded biochemically active helicase protein. The characterisation of the protein was carried out using radioactively labeled substrate and the standard strand displacement assay. The localisation of the enzyme was studied using immunofluorescence assay. RESULTS & CONCLUSION: P. falciparum helicase gene is 1551 bp in length and encodes for a protein consisting of 516 amino acid residues with a predicted molecular mass of 59.8 kDa. The protein is designated as Plasmodium falciparum DEAD-box helicase 60 kDa in size (PfDH60). Purified PfDH60 showed ATP and Mg2+ dependent DNA unwinding, ssDNA-dependent ATPase and ATP-binding activities. Interestingly, this is a unique helicase because it works at a wide pH range (from 5.0-10.0). The peak expression of PfDH60 is mainly in schizont stages of the development of P. falciparum, where DNA replication is active. The cell-cycle dependent expression suggests that PfDH60 may be involved in the process of DNA replication and distinct cellular processes in the parasite and this study should make an important contribution in our better understanding of DNA metabolic pathways in the parasite.

A Case of Tertian Malaria which Recurred Three Times Despite Standard Chloroquine-primaquine Therapy / 감염

Plasmodium vivax malaria, which used to be endemic in the past, re-emerged in 1993 and the number of cases has increased annually. Though there has been no proven endemic drug-resistant malaria case reported, widespread use of anti-malarial chemoprophylaxis for the military personnel could cause emergence of resistance. We herein report a case of tertian malaria, which recurred three times despite the standard chloroquine-primaquine therapy. The patient is 40-year-old male, lives in Dongducheon city, Gyeonggy province, and has never been abroad. He visited hospital in September 2000, because of fever. His blood smear revealed ring forms and trophozoites of P. vivax. He took hydroxychloroquine for 3 days and primaquine for 14 days. His symptoms disappeared then. After 7 months he got fever for 2 days and his blood smear revealed schizonts of P. vivax. He took the same medicines and got well next day. Fever recurred 4 month later, and trophozoites were observed on the blood smear. Hydroxychloroquine and primaquine were prescribed in the same way and fever disappeared. Forty three days later, he had fever and positive blood smear of P. vivax trophozoite. Fever disappeared on the day drug was administered and no malaria was detected in follow up smear of 7 and 14 days. He was free of fever in follow up at 3 months later.

The Recently Presented Plasmodium Vivax Malaria

BACKGROUND: Plasmodium vivax malaria was recently re-presenting infectious disease in Korea since was being controlled for about 10 years age, but has been increasing years by year in the soldiers or farmers working at the near Demilitarized Zone(DMZ). So we analyzed the Characteristics of the patients diagnosed as malaria since 1997 in Yeungnam university hospital. METHODS: From January 1997 to August 1999, the 23 patients complainted of the febrile and chilly sense were diagnosed as Plasmodium vivax malaria in Yeungnam university hospital. We analyzed the patient's records for clinical findings(i.e. clinical symptoms and signs), occupation and regions of working or visiting, laboratory findings, treatment and its results, etc. RESULTS: Male patients were 21 and female patients were 2 among the total 23 patients, the 19 of 21 male patients were soldiers discharged from military services. All patients had been visited or worked near the DMZ, as the northern part of Kyungki-do(21 cases) or Kangwon-do(2 cases). And all patients complainted of delayed onset(means 6 months) of fever and chills after working or visiting at this zones. On physical examination, liver or spleen were palpated initially at least 1 finger breadth in 9 cases(39.1%), and peripheral blood smears showed the infected RBCs(i.e. gametocyte, ring form, schizont, trophozoite) in all cases, and 21 cases(91.3%) showed thrombocytopenia. All patients were treated by the combined regimen of 2-days hydroxychloroquine and 14-days primaquine. All cases showed clinical and laboratory improvement initially, but 5 cases were recurred after 2 months and showed re-improvement. And none of 23 cases showed the significant complications and deaths after medical treatment. CONCLUSION: Plasmodium vivax malarial infection is currently re-presenting disease near the DMZ. So we should consider the active prevention and management of malaria.

A Case of Vivax Malaria Who Showed only Released Merozoites and Ruptured Schizonts Form / 감염

We report a case of vivax malaria that showed ruptured form of merozoite only in the peripheral blood. A 28-year old man was admitted to Korea University hospital because of irregular high fever, chill and abdominal pain. The peripheral blood smear, showed only small merozoites which seemed to have been recently released from schizonts and destroyed remnants form of schizonts and did not show any forms of malaria parasite such as ring forms, mature trophozoites, schizonts, and gametocytes. On acridine orange fluorochrome stain, we could not find any suspected forms of malaria. However, we detected parasite LDH which is specific to Plasmodium vivax. Malaria treatment was done to the patient, and he is now under follow up in local hospital.