Effectiveness of non-pyrimethamine-based regimens for toxoplasma encephalitis: A systematic and meta-synthesis study

Objective: To examine the differences in effectiveness and side effects between pyrimethamine-based and non-pyrimethaminebased regimens for toxoplasma encephalitis since the availability of pyrimethamine in Indonesia is currently limited due to its withdrawal from the market. Methods: A systematic review and meta-synthesis study that was carried out by following a protocol guided by the Preffered Reporting Items for Systematic Review and Meta-analysis (PRISMA). Effectiveness measures included clinical improvement, mortality, and radiological improvement. We evaluated selected articles narratively because of the limitations of homogeneity. The risk of bias in RCTs was assessed using the Cochrane Risk of Bias tool for RCT (ROB 2.0) and cohort studies were assessed using the Risk of Bias In Non-Randomized Studies of Interventions (ROBINS-1) tool. Research quality was assessed using the GradePro software. Results: We included two retrospective cohort studies and one RCT. Narrative outcome assessment in these three studies did not show significant difference in effectiveness between pyrimethamine-based and non-pyrimethamine-based regimens for toxoplasma encephalitis treatment. However, drug side effects were consistently higher in the pyrimethamine-based regimen. Conclusions: This study has a high risk of bias. The quality of the research also has a low recommendation value. However, the results may be considered for application if a standard regimen is not available.

Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Stability indicating RP-HPLC method for simultaneous determination of pyrimethamine and sulfamethoxypyrazine in pharmaceutical formulation: Application to method validation

The present work takes into account the development of Reverse Phase High Performance Liquid Chromatography(HPLC) for simultaneous method estimation and validation of pyrimethamine and sulfamethoxypyrazine inpharmaceutical formulation. The chromatographic separation was accomplished on C8 column by using acetonitrileand potassium dihydrogen phosphate as the mobile phase (60:40 v/v) having a flow rate of 0.8 ml/minute. Theeluent was detected at 254 nm, simultaneously for both the drugs. The retention time for pyrimethamine andsulfamethoxypyrazine was found to be 3.33 and 4.21 minutes, respectively. According to the International Conferenceon Harmonisation guidelines, the develop method was validated in terms of accuracy, precision, linearity, limit ofdetection, limit of quantitation, robustness, and stress degradation studies. This validated method can be suggested forthe routine simultaneous laboratory analysis of pyrimethamine and sulfamethoxypyrazine.

Effectiveness of intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine: An in silico pharmacological model

Objective: To explore the efficacy of intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine and pyrimethamine (SP) against sensitive parasites. Methods: A pharmacological model was used to investigate the effectiveness of the previous recommended at least two-dose regimen, currently recommended three-dose regimen and 4, 6, 8-weekly regimens with specific focus on the impact of various non-adherence patterns in multiple transmission settings. Results: The effectiveness of the recommended three-dose regimen is high in all the transmission intensities, i.e. >99%, 98% and 92% in low, moderate and high transmission intensities respectively. The simulated 4 and 6 weekly IPTp-SP regimens were able to prevent new infections with sensitive parasites in almost all women (>99%) regardless of transmission intensity. However, 8 weekly interval dose schedules were found to have 71% and 86% protective efficacies in high and moderate transmission areas, respectively. It highlights that patients are particularly vulnerable to acquiring new infections if IPTp-SP doses are missed. Conclusions: The pharmacological model predicts that full adherence to the currently recommended three-dose regimen should provide almost complete protection from malaria infection in moderate and high transmission regions. However, it also highlights that patients are particularly vulnerable to acquiring new infections if IPTp doses are spaced too widely or if doses are missed. Adherence to the recommended IPTp-SP schedules is recommended.

Effectiveness of intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine: An in silico pharmacological model

Objective: To explore the efficacy of intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine and pyrimethamine (SP) against sensitive parasites. Methods: A pharmacological model was used to investigate the effectiveness of the previous recommended at least two-dose regimen, currently recommended three-dose regimen and 4, 6, 8-weekly regimens with specific focus on the impact of various non-adherence patterns in multiple transmission settings. Results: The effectiveness of the recommended three-dose regimen is high in all the transmission intensities, i.e. >99%, 98% and 92% in low, moderate and high transmission intensities respectively. The simulated 4 and 6 weekly IPTp-SP regimens were able to prevent new infections with sensitive parasites in almost all women (>99%) regardless of transmission intensity. However, 8 weekly interval dose schedules were found to have 71% and 86% protective efficacies in high and moderate transmission areas, respectively. It highlights that patients are particularly vulnerable to acquiring new infections if IPTp-SP doses are missed. Conclusions: The pharmacological model predicts that full adherence to the currently recommended three-dose regimen should provide almost complete protection from malaria infection in moderate and high transmission regions. However, it also highlights that patients are particularly vulnerable to acquiring new infections if IPTp doses are spaced too widely or if doses are missed. Adherence to the recommended IPTp-SP schedules is recommended.

Condução da toxoplasmose gestacional / Management of gestational toxoplasmosis

A toxoplasmose é uma doença proveniente do Toxoplasma gondii, um protozoário que tem os felinos como seu hospedeiro definitivo e os mamíferos e aves como seu hospedeiro intermediário. Tem um curso benigno e autolimitado quando acomete um indivíduo imunocompetente, no entanto a infecção durante a gestação acarreta até 50% de chance de toxoplasmose congênita, podendo causar danos severos ao feto. A virulência dos genótipos encontrados nas Américas Central e do Sul é a mais alta, comparada a Europa e América do Norte, tendo a doença um comportamento mais agressivo. Os estudos relatam a diminuição da infecção fetal em até 60% com o uso da espiramicina, usada ainda na profilaxia. Este artigo discute sobre a triagem materna pré-natal e sua necessidade, a profilaxia e o tratamento da infecção fetal ainda intraútero, com o objetivo de diminuir a transmissão vertical e as sequelas neonatais com suas implicações ao longo da vida.(AU)

Toxoplasmosis it is a disease originating from Toxoplasma gondii, a protozoan that has felines at as ultimate host and mammals and birds at as intermediate host. Has a benign and self-limiting course when affects immunocompetent individual, however, infection during pregnancy leads 50% chance of congenital toxoplasmosis and can cause severe damage to the fetus. The virulence of genotypes found in Central and South America is the highest compared to Europe and North America, having the disease a more aggressive behavior. Studies report a reduction in fetal infection 60% with the use spiramycin still used for prophylaxis. This article discusses prenatal maternal screening, prophylaxis and treatment of fetal infection still in utero with the objective of decreasing vertical transmission and neonatal sequelae with their lifelong implications.(AU)

Progress of researches on genes associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax / 中国血吸虫病防治杂志

Malaria is a parasitic disease which threatens human life and health seriously. Sulfadoxine-pyrimethamine (SP) has been recommended for intermittent preventive treatment of malaria in children and pregnant women, and also used as a compound component of artemisinin based therapy. The mechanisms of SP resistance in P. falciparum involve point mutations in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), and the drug pressure can also lead to the mutations in the two genes of P. vivax. To provide the information for the formulation of anti-malarial strategies, this article reviews the discovery, application, effect of SP, and the resistance mechanism and research progress of the related genes in P. vivax.

Progress of researches on genes associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax / 中国血吸虫病防治杂志

Malaria is a parasitic disease which threatens human life and health seriously. Sulfadoxine-pyrimethamine (SP) has been recommended for intermittent preventive treatment of malaria in children and pregnant women, and also used as a compound component of artemisinin based therapy. The mechanisms of SP resistance in P. falciparum involve point mutations in the genes encoding dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), and the drug pressure can also lead to the mutations in the two genes of P. vivax. To provide the information for the formulation of anti-malarial strategies, this article reviews the discovery, application, effect of SP, and the resistance mechanism and research progress of the related genes in P. vivax.

Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura / Congenital toxoplasmosis, a look at contemporary treatment; literature review / Toxoplasmose congênita, um olhar atual no tratamento; revisão de literatura

Introducción. La Toxoplasmosis congénita constituye una causa significativa de morbi-mortalidad neonatal en países de bajos ingresos como Colombia. Puede originar prematuridad, secuelas patológicas y pérdida fetal. El tamizaje en las gestantes y, a su vez, un tratamiento oportuno y adecuado disminuye la transmisión vertical y sus nefastas secuelas. El objetivo es presentar evidencia científica actualizada sobre el tratamiento farmacológico de la Toxoplasmosis Congénita. Metodología. Se realizó una búsqueda no sistemática en bases de datos: Pubmed, Medline, Clinical Key y Springer. Se incluyeron artículos originales y de revisión de tema publicados desde enero de 2014 hasta abril de 2019. División de los temas tratados. se abordan la fisiopatología y clínica, el abordaje diagnóstico, alternativas de prevención y tratamiento. Conclusiones. En la actualidad la terapia farmacológica es limitada, los esquemas de manejos se basan en espiramicina o la combinación de sulfadiazina/pirimetamina y ácido folínico; estas moléculas no son del todo bien toleradas y presentan un amplio espectro de reacciones adversas secundario a sus efectos tóxicos; resulta necesario la ejecución de estudios aleatorizados para evaluar su efectividad. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Introduction. Congenital Toxoplasmosis constitutes a significant cause of neonatal morbimortality in underdeveloped countries like Colombia. It can cause prematurity, pathological after-effects and fetal loss. Screening expectant mothers and in turn, a timely and adequate treatment, reduce vertical transmission and its devastating effects. The objective is to present up-to-date scientific evidence about the pharmacological treatment of Congenital Toxoplasmosis. Methodology. A non-systematic search of databases was conducted: Pubmed, Medline, Clinical Key and Springer. Original and topic review articles were included dating from January 2014 to April 2019. Division of topics covered. Physiopathology and clinical pathology, diagnostic approach, prevention and treatment alternatives were addressed. Conclusions. At this time, pharmacological therapy is limited, management schemes are based on spiramycin or a combination of sulfadiazine/pyrimethamine and folinic acid; these molecules are not very well tolerated and exhibit a wide spectrum of adverse reactions apart from their toxic effects, thus it is necessary to conduct randomized studies to evaluate its effectiveness. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Introdução. A toxoplasmose congênita é uma causa significativa de morbidade e mortalidade neonatal em países de baixa renda, como a Colômbia. Pode causar prematuridade, sequelas patológicas e perda fetal. A triagem em gestantes e, por sua vez, um tratamento oportuno e adequado diminui a transmissão vertical e suas consequências desastrosas. O objetivo é apresentar evidências científicas atualizadas sobre o tratamento farmacológico da Toxoplasmose Congênita. Metodologia. Foi realizada uma revisão não sistemática nas bases de dados: Pubmed, Medline, Clinical Key e Springer. Foram incluídos tanto artigos originais, quanto revisões de tópicos publicados de janeiro de 2014 até abril de 2019. Divisão dos tópicos discutidos. foram abordadas a fisiopatologia e a clínica, a abordagem diagnóstica, alternativas para prevenção e tratamento. Conclusões. Atualmente, a terapia farmacológica é limitada, os esquemas terapéuticos baseiam-se na espiramicina ou na combinação de sulfadiazina/pirimetamina e ácido folínico; estas moléculas não são totalmente toleradas e apresentam um amplo espectro de reações adversas secundárias aos seus efeitos tóxicos. É necessário realizar estudos randomizados para avaliar sua eficácia. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Stability Indicating RP-HPLC Method for the Simultaneous Estimation of Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage Form.

A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine. Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm, 5μ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at a flow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxine was 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of the method was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated method can be recommended for the routine laboratory analysis.

A Simplified Liquid Chromatography-Mass Spectrometry Method for Simultaneous Determination of Pyrimethamine, Sulphadoxine and Artesunate in Human Plasma.

This paper presents the development of a new LC-MS method for the determination of pyrimethamine, sulphadoxine and artesunate in human plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate. The analytes and internal standard artemether were separated on a Phenomenex C18 column using gradient elution with 20mM ammonium acetate (mobile phase A) and methanol (mobile phase B) both containing 0.5% acetic acid at a flow rate of 0.8 ml/min. The calibration curves were constructed over the range of 5-30 ng/ml for pyrimethamine, 50-300 ng/ml for sulphadoxine and 25-150 ng/ml for artesunate respectively. Within day and between-day precision and accuracy did not exceed 8%. All the three analytes were found to be stable in plasma samples with no evidence of degradation during three freeze-thaw cycles and three months storage in -20 °C. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of pyrimethamine, sulphadoxine and artesunate in human plasma.

Genetic Polymorphisms in Plasmodium vivax Dihydrofolate Reductase and Dihydropteroate Synthase in Isolates from the Philippines, Bangladesh, and Nepal

Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.

Alelos mutantes asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en Plasmodium falciparum de las fronteras Ecuador-Perú y Ecuador-Colombia / Mutant alleles associated to chloroquine and sulfadoxine-pyrimethanime resistance in Plasmodium falciparum of the Ecuador-Peru and Ecuador-Colombia borders

Se evaluó la frecuencia de mutaciones en los genes pfCRT y DHFR/DHPS del Plasmodium falciparum asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en 83 cepas provenientes de los distritos Esmeralda y Machala ubicados en las fronteras entre Ecuador-Perú y Ecuador-Colombia durante el año 2002. Se empleó la reacción en cadena de polimerasa (PCR) convencional y sus variantes. El gen pfCRT presentó más de 90% de muestras mutantes en Esmeralda y Machala. Para el gen DHFR, el 90% de las cepas fueron muestras mutantes en Esmeralda, tres fueron mutaciones dobles y una triple; en Machala se encontró 25% de formas mutantes simples y 75% de formas mixtas (formas silvestres/mutantes). En conclusión, la resistencia a cloroquina se ha fijado en las cepas portadoras de la mutación K76T pfCRT, mientras que la impronta genética a la resistencia a pirimetamina está en evolución, principalmente en el distrito de Esmeralda.

The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.

Assessment of efficacy and safety of artesunate plus sulfadoxine pyrimethamine combination for treatment of uncomplicated falciparum malaria.

Background: Resistance of Plasmodium falciparum to antimalarial drugs is common in India. World Health Organization (WHO) recommends artemisinin based combination therapy (ACT) to counter the development of resistance in P. falciparum. WHO recommends that ideally antimalarial drug treatment policy or guidelines should be reviewed regularly and updated at least once every 24 months. In consideration to the above recommendation, we planned to conduct the following study. The objective was to determine the efficacy and safety of artesunate + sulphadoxine pyrimethamine (AS + SP) in patients with uncomplicated P. falciparum malaria. Methods: The study included 60 patients of uncomplicated P. falciparum. Each patient received AS + SP as per WHO guidelines. Diagnosis was confirmed by peripheral blood film. All patients were followed up on days 1, 3, 14, and 28 for detailed clinical and parasitological examination. Results: Of a total 60 patients, 55 patients were followed up for 28 days. Remaining 5 patients were lost in follow up. As per protocol analysis, 91% (50) of patients had demonstrated adequate clinical and parasitological response. Remaining 9% (5) had treatment failure in which 5.5% (3) had late parasitological failure and 3.6% (2) had late clinical failure. In our study, mean parasite clearance time was 45.2 ± 4.2 hrs. Conclusion: AS + SP is safe and effective drug for the treatment of uncomplicated falciparum malaria. However, the efficacy of this ACT needs to be carefully monitored periodically since treatment failure can occur due to resistance.

Validation of a liquid chromatographic method for determination of sulphadoxine and pyrimethamine in whole blood spotted on filter paper / Validación de un método de cromatografía líquida para determinar la presencia de sulfadoxina y pirimetamina en muestra de sangre secada sobre papel de filtro

Objective: to validate an analytical method for simultaneous determination and quantification of sulphadoxine and pyrimethamine in human blood dried onto filter paper, whose cost and analysis time can be reduced. Methods: whole blood spotted on filter paper of a healthy volunteer and solutions of sulphadoxine-pyrimethamine standard mixture were used. HPLC separations were carried out on Agilent equipment using a LiChrospher® column C18 with a mobile phase acetonitrile/0.1 M potassium phosphate buffer at pH 3.0 (1:1) for eight minutes under isocratic conditions. A flow rate of 0.7 mL/min, and a 20 mL volume injection were used. External standard method for quantitation of analytes was used. Results: the HPLC method described for the simultaneous determination of sulphadoxine and pyrimethamine in 100 mL of whole blood spotted on filter paper has been found to be linear, precise, accurate and selective. In this method, the sample preparation is simple using liquid-liquid extraction, and HPLC with ultraviolet detection is used. Conclusions: a simple, fast and sensitive method for determination of sulphadoxine and pyrimethamine in human blood dried onto filter paper was validated. This method can be used for the monitoring of both metabolites in pharmacokinetic and clinical studies.

Objetivo: validar un método de análisis para la determinación y cuantificación simultánea de sulfadoxina y pirimetamina en sangre humana secada sobre papel de filtro que sea rápido y barato. Métodos: se usó sangre de un voluntario sano impregnada sobre papel de filtro y soluciones estándar de la mezcla sulfadoxina y pririmetamina. Las separaciones por cromatografía líquida de alta resolución (CLAR) se hicieron en un equipo Agilent sobre una columna C18 LiChrospher® con acetonitrilo/buffer fosfato de potasio 0,1 M a pH 3,0 como fase móvil, usando condiciones isocráticas durante 8 min. Se usó un flujo de 0,7 mL/min y un volumen de inyección de 20 mL. Para la cuantificación de los analitos se utilizó el método del estándar externo. Resultados: el método CLAR descrito para la determinación simultánea de sulfadoxina y pirimetamina en 100 mL de sangre impregnada sobre el papel de filtro mostró linealidad, precisión, exactitud y selectividad. En este método la preparación de la muestra es simple ya que usa extracción líquido-líquido y detección ultravioleta. Conclusión: se obtuvo un método validado que es simple, rápido y sensible para la determinación de sulfadoxina y pirimetamina en sangre humana impregnada sobre papel de filtro, que puede ser usado para el monitoreo de ambos metabolitos en estudios farmacocinéticos y clínicos.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

Objective: To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE). Methods: Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software. Results:Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference. Conclusions: These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

<p><b>OBJECTIVE</b>To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE).</p><p><b>METHODS</b>Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software.</p><p><b>RESULTS</b>Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference.</p><p><b>CONCLUSIONS</b>These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.</p>

Multicomponent complex formation between pyrimethamine, cyclodextrins and water-soluble polymers

The combined effect of hydroxypropyl-β-cyclodextrin (HPβCD) and polyvinylpyrrolidone (PVP) or sodium carboxymethylcellulose (CMC) on the solubility of pyrimethamine (PYR) was studied. Equimolar PYR-HPβCD solid systems, in the presence or the absence of 0.25 percent (w/v) PVP or 0.10 percent (w/v) CMC were prepared by coevaporation or freeze-drying, and characterized by differential scanning calorimetry, X-ray diffraction analysis and dissolution profile. Phase-solubility analysis was used to investigate the interactions in aqueous solution between PYR and HPβCD, in the absence or presence of polymers, which showed a linear increase of PYR solubility depending on the concentration of HPβCD. The presence of polymer did not alter the stoichiometry of the complexes. DSC results were indicative of complexation, due to the loss of the characteristic endothermic peak of PYR. X-ray diffraction analysis confirmed the DSC results. Binary and ternary complexes showed higher dissolution rate when compared with the pure drug.

Plasmodium vivax dhfr Mutations among Isolates from Malarious Areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.