Plasmodium falciparum malarial parasites from Brazil lack artemisinin resistance-associated mutations in the kelch13 gene

Abstract INTRODUCTION Mutations in the propeller domain of the Plasmodium falciparum kelch13 (k13) gene are associated with artemisinin resistance. METHODS: We developed a PCR protocol to sequence the pfk13 gene and determined its sequence in a batch of 50 samples collected from 2003 to 2016 in Brazil. RESULTS: We identified 1 K189T substitution located outside the propeller domain of the PfK13 protein in 36% of samples. CONCLUSIONS: Although the sample size is relatively small, these results suggest that P. falciparum artemisinin-resistant mutants do not exist in Brazil, thereby supporting the continuation of current treatment programs based on artemisinin-based combination therapy.

Antiprotozoal action of synthetic cinnamic acid analogs

Abstract INTRODUCTION Leishmaniasis, Chagas disease, and malaria cause morbidity globally. The drugs currently used for treatment have limitations. Activity of cinnamic acid analogs against Leishmania spp., Trypanosoma cruzi, and Plasmodium falciparum was evaluated in the interest of identifying new antiprotozoal compounds. METHODS In vitro effects of analogs against L. braziliensis, L. infantum chagasi, T. cruzi, and P. falciparum, and hemolytic and cytotoxic activities on NCTC 929 were determined. RESULTS Three analogs showed leishmanicidal and tripanocidal activity. No antiplasmodial, hemolytic, or cytotoxic activity was observed. CONCLUSIONS Antiprotozoal activity of analogs against L. infantum braziliensis, L. infantum chagasi, and T. cruzi was demonstrated.

Antiplasmodial and antileishmanial activities of compounds from Piper tuberculatum Jacq fruits

Abstract INTRODUCTION This study assessed the activity of compounds from Piper tuberculatum against Plasmodium falciparum and Leishmania guyanensis. METHODS The effects of compounds from P. tuberculatum fruits on P. falciparum and L. guyanensis promastigote growth in vitro were determined. Hemolytic action and cytotoxicity in HepG2 and J774 cells were measured. RESULTS Three compounds showed strong antiplasmodial activity and one compound showed strong antileishmanial activity. Two compounds were non-toxic to HepG2 cells and all were toxic to J774 cells. The compounds showed no hemolytic activity. CONCLUSIONS The tested compounds from P. tuberculatum exhibited antiparasitic and cytotoxic effects.

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.

Efecto mutagénico y genotóxico, y expresión de los genes Rad51C , Xiap , P53 y Nrf2 inducidos por extractos antipalúdicos de plantas recolectadas en el Vaupés medio, Colombia / Mutagenicity, genotoxicity and gene expression of Rad51C, Xiap, P53 and Nrf2 induced by antimalarial extracts of plants collected from the middle Vaupés region, Colombia

Resumen Introducción. Dada la resistencia de Plasmodium a los medicamentos antipalúdicos, es necesario encontrar nuevas alternativas terapéuticas para su tratamiento y control. Con base en el saber indígena colombiano, se recopilaron extractos de plantas del Vaupés medio con potencial efecto antipalúdico. Objetivo. Evaluar el efecto mutagénico y genotóxico, y la expresión de los genes Rad51C, Xiap, P53 yNrf2, inducidos por cuatro extractos etanólicos con actividad anti-Plasmodium(R001, T002, T015 y T028). Materiales y métodos. Se evaluó el potencial mutagénico de cuatro extractos etanólicos con efecto antiplasmódico utilizando el test de Ames y el efecto genotóxico, con un ensayo del cometa; asimismo, se analizó la expresión de los genes Rad51C, Xiap, P53 y Nrf2 en células HepG2. Resultados. Los extractos no fueron mutágenos en la cepa TA98 de Salmonella typhimurium en presencia y ausencia de actividad metabólica de la fracción S9. En la cepa TA100, los extractos R001, T015 y T028 se comportaron como mutágenos débiles en presencia de S9, con índices mutagénicos de 1,58; 1,38; 1,53 y 1,61, respectivamente; T015 tuvo el mismo comportamiento en ausencia de S9, con un índice mutagénico de 1,36. En el ensayo del cometa, todos los extractos provocaron daño de categorías 1 o 2, con colas de cometas entre 36,7 y 51,48 µm de longitud; sin embargo, el índice dedaño genético sugirió que los tratamientos afectaron la mayoría de las células. En los genes en estudio, los extractos R001 y T028 indujeron una sobreexpresiónde 1,84 a 3,99 frente a las células sin tratar de los genes Xiap y P53. Conclusiones. Los resultados evidenciaron que el extracto T002 fue el más seguro, ya que presentó actividad anti-Plasmodium, no fue citotóxico en las células HepG2, no fue mutágeno, causó daño de categoría 1 en el ADN y no modificó la expresión de los genes evaluados.

Abstracts Introduction: Due to Plasmodium resistance to antimalarial drugs, it is important to find new therapeutic alternatives for malaria treatment and control. Based on the knowledge of Colombian indigenous communities, we collected extracts of plants with potential antimalarial effects from the middle Vaupés region. Objective: To evaluate the mutagenic and genotoxic effects, as well as the gene expression of Rad51C, Xiap, P53 and Nrf2 induced by four ethanolic extracts with antimalarial activity (R001, T002, T015 and T028). Materials and methods: We evaluated four ethanolic extracts with antimalarial activity using the Ames test to assess mutagenicity, and the comet assay on HepG2 cells to determine the genotoxicicity. We also evaluated the expression of Rad51C, Xiap, P53 and Nrf2 from HepG2 cells stimulated with the four extracts. Results: None of the four extracts was mutagenic in Salmonella typhimurium TA98 strain in the presence and absence of S9 metabolic activity. Extracts R001, T015 and T028 were weakly mutagenic on the TA100 strain in the presence of S9, with mutagenic indexes (MI) of 1.58, 1.53 and 1.61, respectively. The T015 strain showed the same behavior without S9 with an MI of 1.36. The results of the comet assay showed that the four extracts produced category 1 or 2 damage, with comets between 36.7 and 51.48 µm in length. However, the genetic damage index suggested that most of the cells were affected by the treatments. Regarding gene expression, extracts R001 and T028 induced an overexpression of genes Xiap and P53 with an 1.84 to 3.99 fold-change compared with untreated cells. Conclusions: These results revealed that the T002 extract was the safest as it had antimalarial activity and was not cytotoxic on HepG2 cells. Moreover, it was not mutagenic and it only produced category 1 damage on the DNA. Also, the extract did not induce a change in the expression of the tested genes.

Insights into cytochrome bc1 complex binding mode of antimalarial 2-hydroxy-1, 4-naphthoquinones through molecular modelling

BACKGROUND Malaria persists as a major public health problem. Atovaquone is a drug that inhibits the respiratory chain of Plasmodium falciparum, but with serious limitations like known resistance, low bioavailability and high plasma protein binding. OBJECTIVES The aim of this work was to perform molecular modelling studies of 2-hydroxy-1,4-naphthoquinones analogues of atovaquone on the Qo site of P. falciparum cytochrome bc1 complex (Pfbc1) to suggest structural modifications that could improve their antimalarial activity. METHODS We have built the homology model of the cytochrome b (CYB) and Rieske iron-sulfur protein (ISP) subunits from Pfbc1 and performed the molecular docking of 41 2-hydroxy-1,4-naphthoquinones with known in vitro antimalarial activity and predicted to act on this target. FINDINGS Results suggest that large hydrophobic R2 substituents may be important for filling the deep hydrophobic Qo site pocket. Moreover, our analysis indicates that the H-donor 2-hydroxyl group may not be crucial for efficient binding and inhibition of Pfbc1 by these atovaquone analogues. The C1 carbonyl group (H-acceptor) is more frequently involved in the important hydrogen bonding interaction with His152 of the Rieske ISP subunit. MAIN CONCLUSIONS Additional interactions involving residues such as Ile258 and residues required for efficient catalysis (e.g., Glu261) could be explored in drug design to avoid development of drug resistance by the parasite.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract

Abstract INTRODUCTION: Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. METHODS The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. RESULTS The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. CONCLUSIONS The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Highly active ozonides selected against drug resistant malaria

Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidospermaplants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorumextracts, the plant activity against Plasmodium falciparumwas evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Neonatal screening program for hemoglobinopathies in the city of São Carlos, state of São Paulo, Brazil: analysis of a series of cases / Triagem neonatal de hemoglobinopatias no município de São Carlos, São Paulo, Brasil: análise de uma série de casos

OBJECTIVE: To analyze the neonatal screening program for hemoglobinopathies in São Carlos, Southeast Brazil, by investigating a series of cases in which the screening test was abnormal. More specifically, it was aimed to know the information regarding the neonatal screening received by mothers at the hospital and at primary health care, in addition to information related to genetic counseling. METHODS: A descriptive study that enrolled 119 mothers, accounting for 73% of all children born between 2010 and 2011 with abnormal results of neonatal screening for hemoglobinopathies. The mothers completed a questionnaire that assessed the information received at hospital and primary health care, and issues related to genetic counseling. Descriptive statistics was performed. RESULTS: Of the 119 participating mothers, 69 (58%) had children with sickle cell trait, 22 (18.5%) with hemoglobin C trait, 18 (15.1%) with alpha thalassemia trait and, in 10 cases (8.4%), the result was inconclusive. At the hospital, 118 mothers (99.2%) received information about where to go to collect the test and 115 (96.6%) were informed about the correct time to collect the test. Only 4 mothers (3.4%) were informed about which diseases are investigated and the risks of not performing the screening. Seventeen mothers (14.3%) recognized the difference between trait and disease, and 42 (35.3%) considered that a positive screening test could have implications for future pregnancies. In 70 cases (58.8%), the child's physician was not informed about the screening test results. CONCLUSIONS: The neonatal screening program needs further improvement. In both scenarios investigated, health professionals demonstrated a lack of training in providing information to mothers and families. .

OBJETIVO: Fazer uma análise do programa de triagem neonatal de hemoglobinopatias no município de São Carlos, São Paulo, Brasil, por meio da investigação de série de casos cujo resultado do teste de rastreio foi alterado. Objetivou-se conhecer as informações a respeito da triagem neonatal recebidas pelas mães na maternidade e na atenção primária à saúde, além das informações relacionadas à orientação genética. MÉTODOS: Estudo descritivo, no qual participaram 119 mães cujos filhos apresentaram teste de triagem de hemoglobinopatia alterado, o que correspondeu a 73% das crianças nascidas entre 2010 e 2011 com resultado de triagem neonatal para hemoglobinopatia anormal. As mães responderam um questionário que avaliou informações recebidas na maternidade e na atenção primária à saúde, além de aspectos relacionados à orientação genética. Foi feita estatística descritiva dos dados. RESULTADOS: Das 119 mães participantes, 69 (58%) tinham filhos com traço falciforme, 22 (18,5%) traço C, 18 (15,1%) traço alfatalassêmico e 10 (8,4%) resultado inconclusivo. Na maternidade, 118 mães (99,2%) receberam informação sobre onde ir e 115 (96,6%) foram orientadas sobre o momento correto para coleta do teste. Somente quatro mães (3,4%) foram informadas sobre quais doenças seriam investigadas e os riscos de não fazer o rastreio. Das 119 mães participantes, 17 (14,3%) reconheceram a diferença entre traço e doença e 42 (35,3%) consideraram que um teste alterado poderia ter implicações para futuras gestações. Em 70 casos (58,8%), o médico da criança não foi informado sobre o resultado da triagem. CONCLUSÕES: O programa de triagem neonatal necessita de aperfeiçoamento. Nos dois cenários investigados, os profissionais de saúde carecem de treinamento para orientar mães e famílias. .

Effect of Farnesyltransferase Inhibitor R115777 on Mitochondria of Plasmodium falciparum

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (DeltaPsim) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5+/-10.6 hr) and the mean parasite clearance time (27.3+/-12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (Ppiperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77x10(-6) mol/L, 2.09x10(-6) mol/L, 0.09x10(-6) mol/L, and 0.05x10(-6) mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60x10(-6) mol/L, 9.26x10(-6) mol/L, 0.55x10(-6) mol/L, and 0.07x10(-6) mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.

Sensitivity of Plasmodium falciparum to Antimalarial Drugs in Hainan Island, China

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5+/-10.6 hr) and the mean parasite clearance time (27.3+/-12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P>0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (Ppiperaquine>pyronarididine>artesunate. The inhibitory dose 50 (IC50) was 3.77x10(-6) mol/L, 2.09x10(-6) mol/L, 0.09x10(-6) mol/L, and 0.05x10(-6) mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60x10(-6) mol/L, 9.26x10(-6) mol/L, 0.55x10(-6) mol/L, and 0.07x10(-6) mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.

Antiplasmodial Potential of Traditional Medicinal Plant Thlaspi arvense.

Aim: Antiplasmodial potential of traditional medicinal plant Thlaspi arvense against Plasmodium falciparum in vitro has been evaluated. Cytotoxicity of plant extract against HeLa cell lines and normal fibroblasts has also been observed. Place and Duration of the Study: Department of Zoology, Panjab University, Chandigarh, India, between May 2013 to April 2014. Materials and Methods: Ethanolic whole plant extract of Thlaspi arvense (EWETA) was analyzed for its phytochemical constituents. In vitro cytotoxicity was determined colorimetrically by MTT assay. WHO protocol, based on assessment of schizont maturation inhibition, was employed for the evaluation of in vitro antiplasmodial activity of plant extract. Results: Phytochemical screening of EWETA revealed the presence of diterpenes, triterpenes, steroids, anthraquinones and phytosterols. EWETA was observed to inhibit schizont maturation of both chloroquine-sensitive (MRC-2) and resistant (RKL-9) strains of P. falciparum with IC50<5μg/ml and =5μg/ml respectively. The extract was revealed to be safe against both HeLa cells and normal fibroblasts with CC50>1000μg/ml. Selectivity index for Thlaspi arvense was calculated to be >200 and =200 both for chloroquine sensitive and chloroquine-resistant strains of P. falciparum with both HeLa and normal fibroblasts. Conclusion: Plant extract possesses considerable in vitro antimalarial activity with high selectivity index (SI>10) pointing field pennycress to be an active antimalarial. Hence, present study provides scientific evidence for traditional usage of the plant as an antipyretic agent.

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.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

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.

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.

In vitro antimalarial activity of tigecycline against Plasmodium falciparum culture-adapted reference strains and clinical isolates from the Brazilian Amazon

Introduction: We evaluated the in vitro antimalarial activity of tigecycline as an alternative drug for the treatment of severe malaria. Methods: A chloroquine-sensitive Plasmodium falciparum reference strain, a chloroquine-resistant reference strain, and three clinical isolates were tested for in vitro susceptibility to tigecycline. A histidine-rich protein in vitro assay was used to evaluate antimalarial activity. Results: The geometric-mean 50% effective concentration (EC50%) of tigecycline was 535.5 nM (confidence interval (CI): 344.3-726.8). No significant correlation was found between the EC50% of tigecycline and that of any other tested antimalarial drug. Conclusions: Tigecycline may represent an alternative drug for the treatment of patients with severe malaria. .