In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants

Terrestrial plants have been demonstrated to be sources of antimalarial compounds. In Cuba, little is known about antimalarial potentials of plant species used as medicinals. For that reason, we evaluated the antimalarial activity of 14 plant species used in Cuba as antimalarial, antipyretic and/or antiparasitic. Hydroalcoholic extracts were prepared and tested in vitro for the antimalarial activity against Plasmodium falciparum Ghana strain and over human cell line MRC-5 to determine cytotoxicity. Parasite multiplication was determined microscopically by the direct count of Giemsa stained parasites. A colorimetric assay was used to quantify cytotoxicity. Nine extracts showed IC50 values lower than 100 µg/mL against P. falciparum, four extracts were classified as marginally active (SI < 4), one as partially active (Parthenium hysterophorus) exhibiting SI equal to 6.2 and two extracts as active (Bambusa vulgaris and Punica granatum), showing SI > 10. B. vulgaris showed the most potent and specific antiplasmodial action (IC50 = 4.7 µg/mL, SI = 28.9). Phytochemical characterization of active extracts confirmed the presence of triterpenoids in B. vulgaris and polar compounds with phenol free groups and fluorescent metabolites in both extracts as major phytocompounds, by thin layer chromatography. In conclusion, antimalarial use of B. vulgaris and P. hysterophorus was validated. B. vulgaris and P. granatum extracts were selected for follow-up because of their strong antimalarial activity.

Transfusion-transmitted malaria: case report of asymptomatic donor harboring Plasmodium malariae

Malaria in Brazil is endemic in the Amazon region, but autochthonous cases with low parasitaemia occur in the Atlantic Forest area of the country. According to Brazilian legislation no test is mandatory for blood donors from non-endemic areas. However if they have traveled to malaria transmission regions they are deferred for six months before they can donate. This report describes a transfusion-transmitted malaria case in Sao Paulo, Brazil, where one recipient received infected blood and developed the disease. He lived in Sao Paulo and had no previous transfusion or trips to endemic areas, including those of low endemicity, such as Atlantic Forest. Thick blood smears confirmed Plasmodiummalariae. All donors lived in Sao Paulo and one of them (Donor 045-0) showed positive hemoscopy and PCR. This asymptomatic donor had traveled to Juquia, in the Atlantic Forest area of S ao Paulo State, where sporadic cases of autochthonous malaria are described. DNA assay revealed P. malariae in the donor's (Donor 045-0) blood. Serum archives of the recipient and of all blood donors were analyzed by ELISA using both P. vivax and P. falciparum antigens, and IFAT with P. malariae. Donor 045-0's serum was P. malariae IFAT positive and the P. vivax ELISA was reactive. In addition, two out of 44 donors' archive sera were also P. vivax ELISA reactive. All sera were P. falciparum ELISA negative. This case suggests the need of reviewing donor selection criteria and deferral strategies to prevent possible cases of transfusion-transmitted malaria.

Epidemiological profile of malaria in the state of Tocantins, Brazil, from 2003 to 2008

This study describes the epidemiological profile of malaria in the State of Tocantins, in the period 2003-2008, investigates the association between the frequency of malaria and population growth, classifies the cases by 'autochthonous' and 'imported', reports the indices of the disease and analyses the distribution of the cases by Plasmodium species, age and gender. The retrospective study was based on secondary data, stored in SIVEP-malaria and analyzed using the software Epi-Info 3.5.1. and Bioestat 5.0. 19,004 samples were investigated for malaria, 19% of them were positive, 73.32% with Plasmodium vivax, 21.80% with Plasmodium falciparum, 4.79% with mixed infections and only 0.08% with Plasmodium malariae. Male individuals accounted for 76.95% and predominated in all years and age groups, especially in the 15 to 49 years old group. From the overall cases, 34.27% were autochthonous and 65.73% were imported (χ2 = 356.8, p = 0.0001). The frequency of malaria decreased significantly during the entire series (rp = 0.96, p = 0.002) and the number of municipalities with autochthonous transmission also diminished. It was found that malaria is predominantly imported, related to land activities, which confirms the need for effective measures to maintain vigilance throughout the state and enhance educational activities in order to guide the population towards early treatment-seeking.

Internamento por Malária Importada em Crianças, em Dois Hospitais da Grande Lisboa

Introdução: Apesar de em Portugal não haver malária endógena,a crescente mobilidade das populações e os laços históricos com África possibilitam a importação de casos para o nosso país. O presente estudo pretende contribuir para melhorar o conhecimento epidemiológico e clínico da malária importada na região de Lisboa. Métodos: Realizou-se um estudo descritivo das crianças com malária, internadas em dois hospitais da Grande Lisboa, durante um período de seis anos (1999-2004). Resultados: Foram identificados 134 casos, sendo a mediana das idades de sete anos. A maioria (93,3%) era de origem africana e referia estadia em região endémica (90%). O Plasmodium falciparum foi o agente etiológico mais frequente (73%). A febre foi a manifestação clínica mais frequente, seguida de manifestações gastrointestinais e cefaleias. Ocorreram complicações em 42% dos doentes, sendo a trombocitopenia (19,4%) e a anemia grave (9%) as mais frequentes. A halofantrina e o quinino foram os anti-maláricos mais usados. Conclusões: A malária importada é uma patologia relativamente comum na Grande Lisboa e, dada a inespecificidade do quadro clínico, todas as crianças febris ou doentes com estadia recente num país endémico devem ser rastreadas para esta entidade.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Malária Grave em Cuidados Intensivos

Objectivo: Rever o perfil clínico e terapêutico dos doentes com malária grave admitidos numa unidade de cuidados intensivos (UCI). Tipo de estudo: Retrospectivo. População: Nove doentes com malária a Plasmodium falciparum admitidos entre Agosto de 1991 e Julho de 2001 na UCI do Hospital de Santo António dos Capuchos (HSAC). Resultados: As complicações mais frequentes foram as manifestações neurológicas, a síndroma de dificuldade respiratória aguda (SDRA) e a insuficiência renal aguda. A ventilação mecânica foi utilizada em cinco doentes, a prótese renal em dois e as aminas vasopressoras em três doentes. Faleceram dois doentes (22.2%). Conclusões: A malária a Plasmodium falciparum é uma doença potencialmente fatal, pelo que os factores de risco e os critérios para admissão em UCI devem ser identificados. A presença de disfunção de órgão, nomeadamente de manifestações neurológicas, insuficiência renal e respiratória deverão ser consideradas como indicação para internamento em cuidados intensivos.

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

MALARIA DIAGNOSIS BY LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) IN THAILAND

The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

The use of long acting sulfonamides, alone or with pyrimethamine, in malaria (with special reference to sulformetoxine)

Review of the early literature as well as more recent results show that sulfonamides possess a distinct antimalarial activity. However, when give alone, their action is less marked and slower than that of the antimalarials commonly used in the treatment of the acute attack. Combinations with pyrimethamine provide better results, even in cases of pyrimethamine and chloroquine resistance. This warrants further investigations in an attempt to develop a therapeutic agent suitable for the treatment of such resistant cases. It may also be possible with an appropriate combination of pyrimethamine with a sulfonamide to achieve a satisfactory method for suppressive treatment both in areas with and without pyrimethamine resistance. Three main points must still be carefully studied: 1) the risk of developing malaria resistance against one or both of the components of the combination. 2) The risk of developing bacterial resistance to sulfonamides if these substances are used on a large scale in too low doses. It seems indeed that antimalarial effect with the combination of sufonamides + pyrimethamine can be obtained with doses of sulfonamides which are below those usually employed in bacterial diseases. Since the range of the ratios providing potentiation is rather large, only ratios of the combination sulfonamides: pyrimethamine should be chosen in which an antfbacterial sulfonamidemia is guaranteed. 3) It goes without sayinq that, although both pyrimethamine and modem sulfonamides, when given by themselves, have proved tc possess a large margin of safety, long term administration of their combination should be careful studied from the point of view of possible side effects. Substantial evidence has already been produced to show that the long acting sulfonamide Fanasil (Ro 4-4393) given once or once weekly possesses marked schizonticidal activity against P. falciparum. Although its action is slower than that of 4-aminoquinolines, it may be useful as a second choice drug in semi-immune subjects for the therapy of falciparum malaria. Preliminary results show that, when combined with pyrimethamine, Fanasil is highly effective in suppressing fever and asexual parasitemia due to P. falciparum. Single doses of 1 g Fanasil together with 50 mg pyrimethamine seem to be adequate for the treatment of acute falciparum malaria in semi-immune patients. The onset of action of the combination is much more rapid than that of the single components. Weekly doses of 500 mg Fanasil and 25 mg pyrimeihamine appear to provide satisfactory suppressive effects against P. falciparum at least in East Africa. This combination is active on strains which do not respond satisfactorily to the standard doses of pyrimethamine and/or chloroquine and seems to have a satisfactory sporontocidal effect. Preliminary results indicate that Fanasil alone cannot be recommended for use against the other human malaria parasites. The combination with pyrimethamine appears to be much more effective. East African strains of P. malariae seem to respond better to the combination than do Malayan strains of P. vivax but further trials are required before definite assessment can be made. Fanasil by itself has no gametocytoddal or sporontocidal action but seems to potentiate the effect of pyrimethamine at least on sporogony of P. falciparum.

Esplenoportografia em pacientes com hepatoesplenomegalia acompanhada de infecção malárica

Foi praticada a esplenoportografia em 10 pacientes portadores de malária, sendo a infecção causada pelo P. falciparum em 5 casos, pelo P. vivax em 3 casos e pela associação de ambos os plasmódios em outros 2 casos. Em 2 dêsses 10 casos, havia associação da malária com a esquistossomose mansoni. Os resultados obtidos demonstraram: 1) tortuosidade da veia esplênica em 4 casos. Êste fato é explicado pela hepatoesplenomegalia, restringindo a distância entre os hilos dos dois órgãos e portanto causando uma retração da veia esplênica em seu sentido longitudinal; 2) discreta pobreza das ramificações portais intrahepálicas, representada pela ausência ou deficiência de contrastação dos ramos aicotômicos portais mais finos. Esta imagem foi encontrada em 7 casos, e pode ser explicada pela vasoconstricção das ramificações portais, relatadas por Skirrow em Macacus rhesus infectados pelo P. knowlesi (19, 20). Outra explicação mais simplista para êste fato está relacionada com a grande diluição do contraste ao atingir os ramos portais mais finos, tendo em vista a ampliação do leito vascular, em virtude da grande hepatoesplenomegalia - sendo de nocar-se que a hepatomegalia era proeminente em 6 dos casos; 3) o hepatograma, além dos aspectos mencionados, mostrou um aumento apreciável e universal do fígado em 6 casos cujos limites podiam ser apreciados em todos os sentidos, contrastando de certo modo com o hepatograma de esquistossomose hepatoesplênica, em que predominam alterações vasculares intrahepáticas, com aspectos sugestivos de peripileflebite esquistossomótica. Parece que êsses resultados não foram influenciados pela espécie do plasmódio em causa, isto ê, o falciparum ou o vivax. Nove dos dez pacientes foram submetidos à punção biópsia hepática, sendo que as alterações histopatológicas observadas foram bastante discretas, estando portanto em acordo com as pequenas modificações evidenciadas no esplenoportograma. Apenas em um dos dois casos de esquistossomose mansoni associada, na qual foi praticada a biópsia, havia fibrose interlobular com a presença de granuloma esquistcssomótico. Neste mesmo caso, a esplenoportografia demonstrou imagens de ncoformação vascular em tôrno dos ramos dicotômicos do sistema porta intrahepático, com seu aspecto musgoso descrito por Bogliolo, considerado característico da esquistossomose mansoni.

Malária quiescente: aplicação do método de imunofluorescência em dois casos

Após breve revisão de literatura, os autores apresentam o estudo de dois casos de malária quiescente produzidos por P. falciparum, as curvas térmicas, a de parasitemia e a de anticorpos fluorescentes. Dois pacientes semi-imunes mostraram títulos altos mesmo antes da inoculação e assim persistindo por tempo dilatado, o que também ocorreu com a parasitemia.

A parasitological and serological malária survey in Amazonas, 1971

Thick blood films and malaria indirect fluorescent antibody test (P. falciparum and P. vivax) were done in four different regions in Amazonas. There was a very low prevalence of parasites anã the antibody rates suggest a small amount of transmission and that P. vivax was the predominant parasite. The calculation of probability of being infected per year was about 8% in Tefé. Coari, Colonia Fernanão Costa and Labrea and 0.8% in Anori.

Malária Grave — Um Caso Clínico

Descreve-se um caso de malária grave por Plasmodium falciparum numa criança de raça negra, de 5 anos de idade, residente em Angola até 2 semanas antes do internamento. Apesar da terapêutica com quinino i.v. o quadro evoluiu com falência multiorgânica, nomeadamente coma, insuficiência renal, necrose hepática, diátese hemorrágica e rabdomiólise. Foi necessário instituir ventilação mecânica e na terapêutica de suporte recorreu-se à administração de derivados plasmáticos, aminas vasoactivas e diuréticos em altas doses, tendo havido regressão do quadro clínico. Discutem-se as complicações bem como os factores de mau prognóstico presentes neste caso.

Metabolismo do ácido fólico na malária

O estudo de 27 pacientes infectados pelo Plasmodium falciparum comparado com pessoas aparentemente sadias mostra: a) diminuição do folato no soro dos pacientes infectados; b) diminuição do folato sêrico nos primeiros 8 dias que seguiram ao tratamento, interpretados como sendo devido à mobilização pela eritropoiese compensadora; c) folato eritrocítico normal.

Teste de hemaglutinação na sorologia da malária humana empregando hemácias parasitadas pelo Plasmodium berghei

Foi padronizado um teste de hemaglutinação para a sorologia da malária humana, com reagente constituído de suspensão de hemácias de camundongos infectadas pelo Plasmodium berghei e preservadas por fixação aldeídica. Em pacientes com parasitemia por P. falciparum ou P. vivax obteve-se uma sensibilidade de 98,9% nos 88 casos estudados, o teste apresentando títulos entre 40 e 640. Para o grupo de 476 soros de indivíduos não-maláricos, obteve-se uma especificidade de 96,0%. O teste apresentou elevada reprodutibilidade, mesmo para diferentes lotes de antígenos. Nos 200 soros, obtidos ao acaso, de indivíduos de área endêmica, o teste apresentou positividade de 48,5%, contra 88,0% do teste de imunofluorescência-IgG. A baixa positividade pode ser devida a que o teste de hemaglutinação detecta anticorpos IgM. Após tratamento com 2-mercaptoetanol, todos os soros de pacientes com parasitemia tornaram-se não reagentes. Em relação ao teste de imunofluorescência-IgG, o teste de hemaglutinação apresentou índice de co-positívidade de 0,989 para os soros de maláricos com parasitemia. Para os soros de não-maláricos o teste de hemaglutinação apresentou índice de co-negatividade de 0,969. Por outro lado, no grupo de soros de área endêmica, o índice de co-positividade foi de 0,528 e o de co-negatividade, de 0,833.