Plasmodium falciparum parasites overexpressing farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase are more resistant to risedronate

Farnesyl diphosphate synthase/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.

Análise do conteúdo de uma tecnologia para raciocínio diagnóstico de enfermagem / Análisis de contenido de una tecnología para el razonamiento diagnóstico en enfermería / Content analysis of a technology to the nursing diagnostic reasoning

RESUMO Objetivo: submeter à análise do conteúdo uma estratégia metacognitiva de avaliação indireta no pré-encontro com o cliente. Método: estudo metodológico. Utilizou-se o índice de concordância e confiabilidade entre juízes para os critérios de pertinência, adequação, clareza, concisão e precisão de uma tecnologia para raciocínio diagnóstico de enfermagem por iniciantes por meio de formulário eletrônico. Fizeram parte da amostra 13 juízes. Os dados foram analisados por estatística descritiva. Resultados: houve alta concordância e confiabilidade interavaliadores para 85 itens relacionados à etapa de coleta de dados e descrição da estratégia. Apenas cinco itens não alcançaram os critérios de validação e devem ser reformulados. Conclusão: a avaliação indireta no préencontro é pertinente ao processo de raciocínio diagnóstico, sendo possível desenvolver habilidades e competências diagnósticas no iniciante por meio de estratégias, propostas em uma tecnologia inovadora sob a forma de diagrama. .

RESUMEN Objetivo: analizar el contenido de una estrategia metacognitiva de la evaluación indirecta en la reunión previa con el cliente. Método: investigación metodológica; se utilizó el índice de concordancia y confiabilidad interevaluadores a los criterios de pertinencia, claridad adecuación, concisión y precisión de una tecnología para el razonamiento diagnóstico de enfermería para los principiantes a través de medios electrónicos. La muestra estuvo conformada por 13 jueces. Los datos fueron analizados utilizando estadística descriptiva. Resultados: alta confiabilidad interevaluadores de 85 artículos relacionados con la etapa de recolección de datos y la descripción de la estrategia. Sólo 05 artículos no alcanzaron los criterios de validación y deben ser modificados. Conclusión: se concluye que la evaluación indirecta en la reunión previa es relevante para el proceso de razonamiento de diagnóstico, es posible desarrollar habilidades y destrezas de diagnóstico a los principiantes a través de estrategias, propuestas sobre la tecnología innovadora en la forma de un diagrama. .

ABSTRACT Objective: to undergo a content analysis of a metacognitive strategy of indirect assessment in the pre-encounter with the client. Method: methodological study. Agreement and inter-rater reliability index for the criteria: relevance, adequacy, clarity, conciseness and accuracy of a technology to the nursing diagnosis reasoning for novices through an electronic form. The sample consisted of 13 raters. Data were analyzed using descriptive statistics. Results: high agreement and inter-rater reliability for 85 items related to data collection stage and the strategy description. Only fi ve items did not reach the validation criteria and must be rewritten. Conclusion: indirect assessment of the pre-encounter is relevant to the diagnostic reasoning process, being possible to develop competencies and diagnostic skills in the novice through strategies, proposals on innovative technology in the form of a diagram. .

Frequency of antibodies against Sarcocystis neurona and Neospora caninum in domestic cats in the state of Bahia, Brazil / Frequência de anticorpos contra Sarcocystis neurona e Neospora caninum em gatos domésticos do Estado da Bahia, Brasil

Sarcocystis neurona is the major agent of equine protozoal myeloencephalitis. It infects several mammalian species in the Americas, where the definitive hosts, marsupials of the genus Didelphis (D. virginiana and D. albiventris) are found. Domestic cats are one of the confirmed intermediate hosts of the parasite; however, antibodies against S. neurona had never before been demonstrated in Brazilian cats. The aim of this study was to determine whether cats in Bahia, Brazil, are exposed to the parasite. A total of 272 feline serum samples (134 from feral and 138 from house cats) were subjected to an indirect fluorescent antibody test using cultured merozoites of S. neurona as antigen. Positivity was detected in 4.0% (11/272) of the tested samples, with titers ranging from 25 to 800. The feline sera were also tested for antibodies against the protozoan Neospora caninum, with an observed antibody frequency of 2.9%. To the author's knowledge, this is the first study to report antibodies against S. neurona in Brazilian cats. We conclude that cats are exposed to the parasite in the region of this study. Further investigations are needed to confirm the role of cats in the transmission cycle of S. neurona in Brazil.

Sarcocystis neurona é o principal agente da mieloencefalite protozoária equina. Esse parasito infecta várias espécies de mamíferos nas Américas, onde são encontrados os hospedeiros definitivos, os marsupiais do gênero Didelphis (D. virginiana and D. albiventris). O gato doméstico é um dos hospedeiros intermediários do parasito. Contudo, anticorpos contra S. neurona ainda não tinham sido demonstrados em gatos brasileiros. O objetivo deste trabalho foi determinar se gatos da Bahia, Brasil, são expostos ao parasito. Amostras séricas de 272 felinos (134 de gatos errantes e 138 de gatos domiciliados) foram testadas pelo teste de imunofluorescência indireta, utilizando-se como antígeno, merozoítos produzidos em cultura celular. Entre as amostras testadas, 4,0% (11/272) foram positivas com títulos entre 25 e 800. Os soros dos felinos foram também testados para anticorpos contra o protozoário Neospora caninum, cuja frequência de anticorpos foi de 2,9%. Esse é o primeiro relato de anticorpos contra S. neurona em gatos brasileiros. Conclui-se que os gatos da região estudada são expostos a S. neurona. Estudos futuros são necessários, a fim de se confirmar o papel dos gatos no ciclo de transmissão de S. neurona no Brasil.

Partial characterization of Plasmodium falciparum trophozoite proteome under treatment with quinine, mefloquine and the natural antiplasmodial diosgenone / Caracterización parcial del proteoma del trofozoíto de Plasmodium falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona

Introduction: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. Objective: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. Materials and methods: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. Results: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. Conclusion: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.

Introducción. A pesar de los esfuerzos para controlar la malaria, esta sigue siendo un problema de salud pública. Plasmodium falciparum es responsable de la mayoría de los casos graves y de las muertes. Los programas de control de la malaria han sido cuestionados debido al fracaso del tratamiento y a la resistencia del parásito a los antipalúdicos de primera línea, por lo que se requieren nuevas y mejores alternativas. El análisis proteómico permite identificar y determinar los niveles de expresión de las proteínas bajo la presión de los medicamentos, lo que posibilita la identificación de nuevos blancos terapéuticos y mecanismos de acción. Objetivo. Analizar cualitativamente la expresión diferencial de proteínas del citosol del trofozoíto de P. falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona mediante una aproximación proteómica. Materiales y métodos. Se trataron trofozoítos sincronizados y cultivados in vitro de P. falciparum (cepa ITG2) con quinina, mefloquina y el compuesto natural diosgenona. Los extractos proteicos se prepararon y analizaron por electroforesis bidimensional. Las proteínas con aparente expresión diferencial se seleccionaron e identificaron mediante espectrometría de masas MALDI-TOF. Resultados. Se encontraron las siguientes proteínas diferencialmente expresadas en el trofozoíto: la enolasa (PF10_0155), la proteína de unión a calcio (PF11_0098), la chaperonina (PFL0740c), la proteína de invasión a la célula del huésped (PF10_0268) y la proteína relacionada con procesos de reducción y oxidación (redox) (MAL8P1.17). Estos hallazgos son congruentes con resultados previos de estudios en los que el parásito fue presionado con otros medicamentos antipalúdicos. Conclusión. Los cambios observados en el perfil de proteínas del trofozoíto de P. falciparum tratado con antipalúdicos involucraron preferencialmente proteínas relacionadas con la respuesta al estrés general.

Human malaria in C57BL/6J mice: An in vivo model for chemotherapy studies.

The present work deals with the development of Plasmodium falciparum stages in mouse model and its potential for the study of efficacy of antimalarial drugs. C57BL/6J mice were infected with multidrug resistant P. falciparum strain then treated with arteether and artesunate. A response was observed to antimalarial drugs in terms of decrease in parasitemia. Mice infected with P. falciparum strain were successfully cured after treatment with either arteether or artesunate. The speed of parasite clearance time and burden of parasitemia differed for each drug and matched the previously reported observations, hence stressing the relevance of the model. These findings thus suggest that P. falciparum. infected human RBC (iRBC) – C57BL/6J mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of P. falciparum responses to drugs.

Plasmodium falciparum gametocyte carriage, sex ratios and asexual parasite rates in Nigerian children before and after a treatment protocol policy change instituting the use of artemisinin-based combination therapies

The effects of artemisinin-based combination therapies (ACTs) on transmission of Plasmodium falciparum were evaluated after a policy change instituting the use of ACTs in an endemic area. P. falciparum gametocyte carriage, sex ratios and inbreeding rates were examined in 2,585 children at presentation with acute falciparum malaria during a 10-year period from 2001-2010. Asexual parasite rates were also evaluated from 2003-2010 in 10,615 children before and after the policy change. Gametocyte carriage declined significantly from 12.4 percent in 2001 to 3.6 percent in 2010 (@@χ2 for trend = 44.3, p < 0.0001), but sex ratios and inbreeding rates remained unchanged. Additionally, overall parasite rates remained unchanged before and after the policy change (47.2 percent vs. 45.4 percent), but these rates declined significantly from 2003-2010 (@@χ2 for trend 35.4, p < 0.0001). Chloroquine (CQ) and artemether-lumefantrine (AL) were used as prototype drugs before and after the policy change, respectively. AL significantly shortened the duration of male gametocyte carriage in individual patients after treatment began compared with CQ (log rank statistic = 7.92, p = 0.005). ACTs reduced the rate of gametocyte carriage in children with acute falciparum infections at presentation and shortened the duration of male gametocyte carriage after treatment. However, parasite population sex ratios, inbreeding rates and overall parasite rate were unaffected.

Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region has been found in the N-terminal R0 region of the protein. Herein, we describe the antiplasmodial activity of anti-GLURP antibodies present in the sera from individuals naturally exposed to malaria in a Brazilian malaria-endemic area. The anti-R0 antibodies showed a potent inhibitory effect on the growth of P. falciparum in vitro, both in the presence (ADCI) and absence (GI) of monocytes. The inhibitory effect on parasite growth was comparable to the effect of IgGs purified from pooled sera from hyperimmune African individuals. Interestingly, in the ADCI test, higher levels of tumour necrosis factor alpha (TNF-α) were observed in the supernatant from cultures with higher parasitemias. Our data suggest that the antibody response induced by GLURP-R0 in naturally exposed individuals may have an important role in controlling parasitemia because these antibodies are able to inhibit the in vitro growth of P. falciparum with or without the cooperation from monocytes. Our results also indicate that TNF-α may not be relevant for the inhibitory effect on P. falciparum in vitro growth.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100 percent. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72 percent), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40 percent. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0 percent oocyst rates were obtained, in comparison to the 86.67-100 percent oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0 percent sporozoite rates were obtained, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09 percent, 6.67 percent and 11.76 percent sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67 percent and 64.29 percent sporozoite rates were obtained, respectively, in comparison to 90 percent sporozoite rates recovered from An. cracens.

Plasmodium falciparum gametocytaemia with chloroquine chemotherapy in persistent malaria in an endemic area of India.

Background & objectives: Gametocyte sex-ratio in Plasmodium falciparum malaria is an important determinant of transmission success and basis of disease epidemiology. Information on ratio of male to female gametocytes after an exposure of antimalarial regimens under field conditions is very limited. In this retrospective study we observed high densities of gametocytes along with high sex-ratio in P. falciparum cases, which may be responsible for persistent malaria transmission in this area. Methods: Laksar PHC of Hardwar district, Uttarakhand State, India was selected because it contributed 90 per cent of the total malaria cases. A total of 568 uncomplicated P. falciparum malaria patients were assessed to investigate prevalence of gametocytes while 339 P. falciparum thick smears containing 5620 gametocytes were screened for measuring the gametocyte density for microgametocyte (male) and macrogametocyte (female). Homology of variance (‘F’ test) was checked on days 7 and 14 including the variables and risk factors namely fever, parasitaemia, gametocyte carriage in sensitive and resistant chloroquine treated P. falciparum cases. Results: Slide positivity rate (SPR) increased drastically from 0.23 to 11.4 per cent with the predominance in P. falciparum infection after 1998. All 568 cases showed gametocytes in their peripheral blood, of which 109 (19%) were infected with rings and gametocytes and 459 (81%) had gametocytes stages in their peripheral blood while 422 (74.3%) cases were infected with ring stages only. Of the 339 P. falciparum positive blood smears, 5620 gametocytes were screened for their sex-ratio. The mean sex-ratio was 0.31 (3.22 female per male). Prevalence of gametocytaemia was significantly higher (P<0.05) in chloroquine (CQ)- resistant than in CQ-sensitive patients with days 7 and 14 follow up. The homology of variance with risk factors for gametocytes on days 7 and 14 were highly significant (P<0.001) in the study period but during the post-exposure period of days 3 and 5, these were insignificantly correlated. Interpretation & conclusion: A high density of P. falciparum gametocytes was observed at the time of preparation of blood slide on day 0. Improper chloroquine treatment along with poor patient compliance for radical treatment and the presence of chloroquine resistant P. falciparum malaria may have enhanced the prevalence and density of P. falciparum gametocytes which was instrumental in signaling the persistent malaria in this area.

Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Plasmodium falciparum Cultivation Using the Petri Dish: Revisiting the Effect of the 'Age' of Erythrocytes and the Interval of Medium Change

Differences in the characteristics of the culture conditions can influence the multiplication rate of Plasmodium falciparum. The Petri dish method is one of the most popular methods of cultivating this parasite. In many previous studies, ideal culture conditions of the Petri dish method were achieved by using erythrocytes collected from blood that had been stored for at least 2 weeks, with daily changes of the medium. In the present study, we studied the multiplication rate of P. falciparum in cultures containing erythrocytes of various ages together with changing the medium at various intervals of time. Our results strongly suggest that the rate of in vitro multiplication of P. falciparum was higher in freshly collected erythrocytes than in aged erythrocytes regardless of the anticoagulant and that when the parasitemia is lower than 8% with a hematocrit of 5%, the medium change interval can be as long as 48 hr without a great reduction in the rate of multiplication.

Susceptibility of two karyotypic forms of Anopheles aconitus (Diptera: Culicidae) to Plasmodium falciparum and P. vivax

Quatro colônias desenvolvidas em laboratório, de duas formas cariotípicas de Anopheles aconitus i.e. forma B (cepa Chiang Mai e Phet Buri) e C (Cepa Chiang Mai e Mae Hong Son), foram infectadas experimentalmente com Plasmodium falciparum e P. vivax usando técnica de alimentação com membrana artificial e dissecados oito e 12 dias após alimentação da média de oocistos e esporozoitos, respectivamente. Os resultados revelaram que An. aconitus formas B e C foram suscetíveis ao P. falciparum e P. vivax isto é, forma B (cepa Chiang Mai e Phet Buri/P. falciparum e P. vivax) e forma C (cepa Chiang Mai e Mae Hong Son/P. vivax). Análises estatísticas comparativas das taxas de oocistos, número médio de oocistos por intestino médio infectado e taxas de esporozoitos entre todas as cepas de An. aconitus formas B e C ao grupo interno de vetores controles, An. minimus A e C, não exibiram nenhuma diferença significante, confirmando o alto potencial vetor das duas espécies de Plamodium. Os cristais semelhantes a esporozoitos encontrados no lobo médio das glândulas salivares que poderiam ser um fator enganoso na identificação de esporozoitos verdadeiros nas glândulas salivares foram encontrados em ambos An. aconitus formas B e C.

Gametocitos de Plasmodium vivax y Plasmodium falciparum: etapas relegadas en el desarrollo de vacunas / Plasmodium vivax and Plasmodium falciparum gametocyte stages are neglected in vaccine development

Los gametocitos de Plasmodium son los responsables de la transmisión del huésped vertebrado al mosquito vector. Sufren un proceso de desarrollo complejo a partir de parásitos asexuales, que no está completamente entendido, expresando proteínas y moléculas de adhesión específicas. Son capaces de inducir una respuesta inmune humoral específica con anticuerpos IgG, y celular específica, con producción de TNFa, IFNg y proliferación de linfocitos gd+, aun cuando existen respuestas inducidas en contra de las etapas previas del parásito (esporozoito, exo-eritrocítica y eritrocítica). Las vacunas destinadas a bloquear la transmisión del parásito no contemplan a los gametocitos circulantes en el huésped como blancos de acción, sino que van enfocadas contra antígenos expresados en los gametos y en las etapas posfertilización. El estudio de los mecanismos que regulan la producción de gametocitos y de la respuesta inmune contra éstos, ofrece una oportunidad para el desarrollo de estrategias adicionales para el control de la transmisión.

Drug resistant falciparum malaria and the use of artesunate-based combinations: focus on clinical trials sponsored by TDR.

Antimalarial drug resistance has now become a serious global challenge and is the principal reason for the decline in antimalarial drug efficacy. Malaria endemic countries need inexpensive and efficacious drugs. Preserving the life spans of antimalarial drugs is a key part of the strategy for rolling back malaria. Artemisinin-based combinations offer a new and potentially highly effective way to counter drug resistance. Clinical trials conducted in African children have attested to the good tolerability of oral artesunate when combined with standard antimalarial drugs. The cure rates of the different combinations were generally dependent on the degree of resistance to the companion drug. They were high for amodiaquine-artesunate, variable for sulfadoxine/pyrimethamine-artesunate, and poor for chloroquine-artesunate.