Maceration and supercritical fluid extraction were used to prepare extracts from parts of plants (Holostylis reniformis) collected in two different regions of Brazil. ¹H NMR, HPLC-DAD-ESI/MS, HPLC-DAD, GC-MS, and chemometric techniques were used to analyse lignans in the extracts and showed that yields of SFE-CO2 were less than or equal to those of hexane maceration extracts. These analyses, in conjunction with the concentrations of aliphatic hydrocarbons, fatty acids and their methyl and ethyl derivatives in the extracts, also allowed the chemical composition of parts and provenance of the plant to be differentiated.

Malaria remains a major world health problem following the emergence and spread of Plasmodium falciparum that is resistant to the majority of antimalarial drugs. This problem has since been aggravated by a decreased sensitivity of Plasmodium vivax to chloroquine. This review discusses strategies for evaluating the antimalarial activity of new compounds in vitro and in animal models ranging from conventional tests to the latest high-throughput screening technologies. Antimalarial discovery approaches include the following: the discovery of antimalarials from natural sources, chemical modifications of existing antimalarials, the development of hybrid compounds, testing of commercially available drugs that have been approved for human use for other diseases and molecular modelling using virtual screening technology and docking. Using these approaches, thousands of new drugs with known molecular specificity and active against P. falciparum have been selected. The inhibition of haemozoin formation in vitro, an indirect test that does not require P. falciparum cultures, has been described and this test is believed to improve antimalarial drug discovery. Clinical trials conducted with new funds from international agencies and the participation of several industries committed to the eradication of malaria should accelerate the discovery of drugs that are as effective as artemisinin derivatives, thus providing new hope for the control of malaria.

In this review we discuss the ongoing situation of human malaria in the Brazilian Amazon, where it is endemic causing over 610,000 new acute cases yearly, a number which is on the increase. This is partly a result of drug resistant parasites and new antimalarial drugs are urgently needed. The approaches we have used in the search of new drugs during decades are now reviewed and include ethnopharmocology, plants randomly selected, extracts or isolated substances from plants shown to be active against the blood stage parasites in our previous studies. Emphasis is given on the medicinal plant Bidens pilosa, proven to be active against the parasite blood stages in tests using freshly prepared plant extracts. The anti-sporozoite activity of one plant used in the Brazilian endemic area to prevent malaria is also described, the so called "Indian beer" (Ampelozizyphus amazonicus, Rhamnaceae). Freshly prepared extracts from the roots of this plant were totally inactive against blood stage parasites, but active against sporozoites of Plasmodium gallinaceum or the primary exoerythrocytic stages reducing tissue parasitism in inoculated chickens. This result will be of practical importance if confirmed in mammalian malaria. Problems and perspectives in the search for antimalarial drugs are discussed as well as the toxicological and clinical trials to validate some of the active plants for public health use in Brazil.

Foram investigados aspectos clínicos, laboratoriais, terapêuticos e evolutivos da leishmaniose tegumentar americana em Belo Horizonte. O estudo incluiu 358 pacientes com leishmaniose cutânea (LC) e 25 com leishmaniose mucosa (LM). Comparados aos pacientes com LC, aqueles com LM apresentaram maior tempo de doença e relato de outras doenças concomitantes, sugerindo que a debilitação pela leishmaniose e/ou outras doenças podem contribuir para a ativação e/ou disseminação mucosa do parasito. As sensibilidades das reações intradérmica, de imunofluorescência indireta e da pesquisa direta do parasito foram de 78,4, 79,3 e 68,3%, respectivamente. O tratamento com antimoniato de meglumina foi 100% eficaz, com 59% de efeitos colaterais ao longo do tratamento. A recidiva após tratamento ocorreu em 32 (10,1%) dos 318 casos seguidos por até dois anos. A maioria das recidivas (31 dos 32 casos) ocorreu em pacientes com LC tratados com 15mg Sb5+/kg/dia. Na investigação de critérios de cura, a reação intradérmica negativa foi o único fator associado a um risco três vezes maior de recidiva. Um aumento da dose ou do tempo de tratamento talvez melhore o prognóstico nestes pacientes.

This study investigated clinical, laboratorial, therapeutic and prognostic aspects of American cutaneous leishmaniasis in Belo Horizonte in 358 patients with cutaneous leishmaniasis (CL) and 25 with mucocutaneous leishmaniasis (MCL). Compared to CL patients, the MCL patients reported longer duration of disease and higher frequency of other diseases, suggesting that debilitation caused by leishmaniasis or other conditions might contribute to activation and/or mucous dissemination of the parasite. The sensitivity of skin test, indirect immunofluorescence reactions and direct detection of parasites was 78.4, 79.3 and 68.3%, respectively. The treatment with meglumine antimoniate presented 100% efficacy, but 59% patients had side-effects. During two years of follow-up, there were 32/318 relapses after successful treatment. Most relapses (31/32) were of CL patients treated with 15 mg Sb5+/kg/day. The negative response to skin test was the only factor associated with a significant threefold increased risk of relapse. Higher dose or longer duration of treatment might improve the prognosis in these patients.

While relapses following clinical cure of American cutaneous leishmaniasis are frequent, no test has been described until now to predict such relapses. A cohort of 318 American cutaneous leishmaniasis patients was followed up for two years after treatment with meglumine antimoniate, during which time 32 relapses occurred, 30 in the first year and two in the second (accumulated risk: 10.5%). No association was found between these relapses and the parasite-specific antibody response before and after treatment, or between the relapses and stratification by sociodemographic and clinical characteristics. However when Leishmania was used as antigen, patients with a negative skin test at the time of diagnosis presented a 3.4-fold higher risk (hazard risk = 3.4; 95% confidence interval, 1.7-7.0) of American cutaneous leishmaniasis relapse, compared with patients with a positive response. This result shows that the skin test can be a predictor of American cutaneous leishmaniasis relapse after treatment.

La leishmaniasis cutánea americana, causada por Leishmania (Viannia) brazifensis, da lugar a recaídas en aproximadamente un 10% de los pacientes tratados en el Brasil. A falta de un criterio fiable, las curaciones postratamiento de la leishmaniasis cutánea americana se han definido principalmente atendiendo a los datos clínicos. En este artículo se presentan los resultados de un estudio de seguimiento de dos años de las recaídas sufridas por una cohorte de 318 pacientes aquejados de leishmaniasis cutánea americana después de ser tratados con antimoniato de meglumina. Se obtuvo el consentimiento informado por escrito y los pacientes fueron entrevistados y examinados por el mismo médico, con arreglo a un protocolo predefinido con información sobre factores sociodemográficos (sexo, edad, color de la piel, lugar de nacimiento, educación y nivel de ingresos, ocupación y lugar de residencia) y factores clínicos (cuadro clínico y momento de aparición de la leishmaniasis cutánea americana, número de lesiones y lugar de las mismas, historia familiar de leishmaniasis cutánea americana, concurrencia de otras afecciones y tratamiento anterior). Se evaluó la respuesta de hipersensibilidad retardada mediante una prueba cutánea en la que se empleó antígeno preparado a partir de suspensiones de promastigotes muertos. Se realizaron pruebas serológicas antes del tratamiento y 10 días después del mismo.Todos los pacientes fueron tratados con antimoniato de meglumina intramuscular (Glucantimet): dosis de 15 mg de Sb5+ * kg-1 * día-1 en los pacientes con leishmaniasis cutánea, y de 20 mg de Sb5+ * kg-1 * día-1 en los pacientes con leishmaniasis mucocutánea, durante al menos 20 y 30 días, respectivamente (7). Hubo remisión completa de las lesiones en todos los pacientes. Se realizaron evaluaciones clínicas antes e inmediatamente después del tratamiento, y cada seis meses durante dos años después del tratamiento. Se definió como recaída postratamiento toda: (a) aparición de una lesión nueva dentro de los límites de una cicatriz anterior, (b) aparición de una nueva lesión mucosa en pacientes con una cicatriz cutánea, o (c) reactivación de una cicatriz en el lugar de una lesión mucosa previa. Se trazaron las curvas de Kaplan-Meier, que muestran la probabilidad de que no se produzca ninguna recaída durante el seguimiento, para el conjunto de la cohorte y para cada factor específico investigado, y se utilizó el modelo de riesgo instantáneo proporcional de Cox para determinar el efecto independiente de los diferentes factores de recidiva.La cohorte estaba constituida por 207 hombres y 111 mujeres, con edades de entre 6 meses y 80 años (media = 35,4 + 18,9 años); 300 (94,3%) pacientes sufrían leishmaniasis cutánea, y 18 (5,7%) leishmaniasis mucocutánea. El nivel de educación y de ingresos de la mayoría de los pacientes era bajo. Sólo 71 (22,3%) realizaban tareas agrícolas o silvícolas. Se registraron 32 recaídas, 26 dentro de los 6 primeros meses de tratamiento, 4 al cabo de entre 7 y 12 meses, y 2 durante el segundo año tras el tratamiento. Veintinueve de las 32 recaídas se manifestaron en el mismo lugar afectado por la lesión cutánea previa en los pacientes con leishmaniasis cutánea. Otros dos pacientes con leishmaniasis cutánea presentaron tanto una recaída cutánea como nuevas lesiones mucosas. Sólo en un paciente con leishmaniasis mucocutánea se observó una reactivación de una lesión mucosa cicatrizada. La mayoría de las recaídas (30/32) ocurrieron en el término de un año, lo que corrobora la recomendación de la OMS (1990) de que se debe seguir a los pacientes durante al menos un año después del tratamiento. El riesgo acumulado de recaída fue del 10,5%. No se observó ninguna relación entre la recaída y la respuesta de producción de anticuerpos específicos contra el parásito antes y después del tratamiento, o entre la recaída y la estratificación por características sociodemográficas y clínicas.La prueba cutánea provoca una reacción de hipersensibilidad retardada y es un buen marcador de la respuesta inmunitaria celular en la leishmaniasis. Los pacientes con una prueba cutánea negativa en el momento del diagnóstico presentaron un riesgo 3,4 veces mayor de recaída que los que habían dado positivo. Se prevé que el uso de la prueba cutánea como factor predictivo de recidiva de leishmaniasis cutánea americana tendrá gran repercusión en la salud pública, ya que la prueba es segura y económica, amén de ampliamente usada para el diagnóstico de la leishmaniasis cutánea americana. La prueba parece indicar también que a los pacientes en los que no se observa ninguna reacción de hipersensibilidad retardada en la prueba cutánea en el momento del diagnóstico se les debe tratar con una dosis mayor o durante más tiempo para prevenir las recaídas. Es necesario realizar ahora nuevas investigaciones en las zonas endémicas en que coexisten otras especies de Leishmania a fin de evaluar la coherencia de estos resultados.

Au Brésil, la leishmaniose cutanée américaine, due à Leishmania (Viannia) braziliensis, donne des rechutes chez environ 10% des malades traités. En l’absence de critère fiable, la guérison après traitement de la leishmaniose cutanée américaine est principalement définie d’après les données cliniques. Cet article présente les résultats d’une étude de suivi de deux ans portant sur les rechutes chez 318 malades traités par l’antimoniate de méglumine. Le consentement éclairé a été recueilli par écrit et les malades ont été interrogés et examinés par le même médecin, suivant un protocole préétabli comportant la collecte d’informations sur les facteurs socio-démographiques (sexe, âge, couleur de la peau, lieu de naissance, niveau d’études et de revenus, profession, lieu de résidence) et cliniques (tableau clinique et durée de la leishmaniose cutanée américaine, nombre et localisation des lésions, antécédents familiaux de leishmaniose cutanée américaine, présence d’autres affections et traitements antérieurs). Une réaction d’hypersensibilité retardée a été recherchée à l’aide d’un test cutané utilisant un antigène préparé à partir de suspensions de promastigotes morts. Des tests sérologiques ont été effectués avant le traitement et 10 jours après celui-ci.Tous les malades ont été traités par l’antimoniate de méglumine par voie intramusculaire (Glucantime à la dose de 15 mg Sb5+ · kg-1 · jour-1 pour la leishmaniose cutanée et 20 mg Sb5+ · kg-1 · jour-1 pour la leishmaniose cutanéo-muqueuse pendant au moins 20 et 30 jours respectivement. La rémission des lésions a été complète chez tous les patients. Un examen clinique a été réalisé avant le traitement, immédiatement après puis tous les 6 mois pendant 2 ans. Une rechute après traitement se définissait comme suit : a) survenue d’une nouvelle lésion à l’intérieur d’une ancienne cicatrice, b) survenue d’une nouvelle lésion muqueuse chez des patients porteurs d’une cicatrice cutanée, ou c) réactivation d’une cicatrice à l’emplacement d’une ancienne lésion muqueuse. Des courbes de Kaplan-Meier montrant la probabilité de l’absence de rechute pendant la période de suivi ont été établies pour l’ensemble de la cohorte et pour chacun des facteurs étudiés. Le modèle de régression de Cox à risques proportionnels a été utilisé pour déterminer l’effet indépendant des différents facteurs sur la survenue d’une rechute.La cohorte se composait de 207 malades de sexe masculin et 111 de sexe féminin, âgés de 6 mois à 80 ans (moyenne 35,4 + 18,9 ans), dont 300 (94,3 %) étaient atteints de leishmaniose cutanée et 18 (5,7%) de leishmaniose cutanéo-muqueuse. Le niveau d’études et de revenus de la plupart des malades était faible. Seuls 71 d’entre eux (22,3 %) travaillaient dans l’agriculture ou la sylviculture. On a observé 32 rechutes, dont 26 pendant les 6 mois suivant le traitement, 4 entre 7 et 12 mois après le traitement et 2 au cours de la deuxième année de suivi. Vingt-neuf rechutes ont eu lieu à l’emplacement d’une lésion cutanée antérieure chez les malades atteints de la forme cutanée. Deux autresmalades atteints de cette forme ont présenté à la fois une rechute cutanée et de nouvelles lésions muqueuses. Seul un malade atteint de leishmaniose cutanéo-muqueuse a présenté une réactivation d’une ancienne lésion muqueuse cicatrisée. La plupart des rechutes (30/32) sont survenues dans l’année qui a suivi le traitement, ce qui confirme la recommandation de l’OMS (1990) selon laquelle les patients doivent être suivis pendant au moins un an après le traitement. Le risque cumulatif de rechute était de 10,5 %. Il n’y avait pas d’association entre les rechutes et la réponse en anticorps spécifiques du parasite avant et après traitement, ni entre les rechutes et la stratification selon les caractéristiques socio-démographiques et cliniques des malades.Le test cutané est basé sur une réaction d’hypersensibilité retardée et constitue un bon marqueur de la réponse immunitaire cellulaire dans la leishmaniose. Chez les malades dont le test cutané était négatif au moment du diagnostic, le risque de rechute de leishmaniose cutanée américaine était 3,4 fois plus éleve que chez ceux dont le test était positif. L’utilisation du test cutané comme indicateur prédictif des rechutes de leishmaniose cutanée américaine devrait avoir un impact important en santé publique car ce test est sans danger, peu coûteux et largement utilisé pour le diagnostic de la maladie. Il peut également signaler la nécessité, pour empêcher les rechutes, de traiter par des doses plus élevées ou pendant plus longtemps les malades n’ayant pas présenté de réaction d’hypersensibilité retardée au moment du diagnostic. De nouvelles investigations dans des zones d’endémie où coexistent d’autres espèces de Leishmania sont maintenant nécessaires pour confirmer nos observations.

Dois antígenos solúveis de tripomastigotas do Trypanosoma cruzi, um obtido de sobrenadantes de culturas celulares (AgSb) e o outro excretado/secretado por essas formas em meio de cultura (AgES), foram avaliados em um teste de ELISA para o diagnóstico da infecção chagásica e controle de cura de pacientes tratados. Os pacientes tratados apresentavam testes de lise mediada pelo complemento e hemoculturas repetidamente negativos, apesar de permanecerem com a sorologia convencional positiva (pacientes dissociados). O teste de lise negativo indica que estes pacientes eliminaram a infecção. Entre os controles com infecção ativa, os AgSb e os AgES detectaram respectivamente 93 e 100% dos casos. No entanto, entre os pacientes dissociados, o teste de ELISA, utilizando os AgSb e AgES, foi positivo com 28% e 5% dos soros, respectivamente. Portanto, este teste com os AgES é indicado para o controle de curada doença de Chagas, podendo vir a substituir a reação de lise mediada pelo complemento no acompanhamento sorológico individual de pacientes tratados.

Two soluble antigens isolated from T. cruzi were evaluated by ELISA for the diagnosis of chronic infection and assessment of cure after specific chemotherapy, namely, supernatants from parasite cell-cultures (SpAg) arid trypomastigote excretory /secretory antigens (ESAg). Among the treated patients, a group defined as "dissociated " had been monitored for 3 to 10 years and displayed negative hemocultures and tests of trypomastigote complement-mediated lysis persistently negative although positive by conventional serology (indirect fluorescence with epimastigote, mainly). A negative lysis test indicates parasite elimination by the patient. Our ELISA results showed that among the non-treated chagasic controls the SpAg e ESAg detected 93% and 100%, of the cases, respectively. However, only 28% of sera from the dissociated group, considered as cured, were positive by ELISA using SpAg whereas all of such sera were negative using ESAg. Therefore ELISA with ESAg seems to be ideal to replace the complement- mediated lysis test with the aim to define cure after treatment of the chronic infection by T. cruzi.

A sensibilidade de hemoculturas, realizadas uma ou três vezes, foi estudada em 52 pacientes na fase crônica da doença de Chagas. Modificações foram introduzidas na técnica tais como, diminuição do período de processamento do sangue, homogeneização suave e exame até 120 dias do cultivo. Os resultados mostram alto percentual de positividade, ou seja, 79% e 94% dos pacientes foram positivos, respectivamente, com um ou três testes. A julgar pela número de tubos positivos, em cada paciente, a parasitemia foi baixa em 59% deles, média em 16% e alta em 25%. Não houve diferenças significativas nos resultados positivos em função da idade dos pacientes, que variou de 14 a 82 anos. Nossos resultados demonstram que a hemocultura é uma metodologia sensível para o diagnóstico parasitológico da doença de Chagas e ideal para monitorar cura em pacientes submetidos a tratamento.

The sensitivity ofhemocultures, performed once or three times, was investigated in 52 patients in the chronic phase of Chagas disease. Modifications were introduced in the technique such as, processing the blood more rapidly, gently homogenizing the cultures and examining them after 120 days of culture. Our results show a high percent age of positivity i.e. 79% and 94% of patients submitted, respectively, to one or three tests. No significant differences related to the patients age were detected, which varied from 14 to 82 years old. Our results demonstrate that hemoculture is a sensitive method for the parasitological diagnosis of Chagas disease and is ideal for monitoring cure in treated patients.

In the present work we have described the in vivo antimalarial actrivity of six different plants. Two of them (Verninia brasiliana and Eupatorium squalidum) were tested in a randomic approach among 273 crude extracts from plants; four (Acanhospermum australe, Esenbeckia febrifuga, Lisianthus specious and Tachia guianensis) were selected after screening 22 crude extracts from different medicinal and some of them showed antimalarial activity in vitro. Some aspects of recent research with natural products aiming to produce drugs are discussed.

Levamisol (fenilimidotiazol), considerado urn potente imunoestimulante, quando administrado a camundongos suíços não causou aumento significante nos pesos do timo, figado ou baço, apesar de a droga ter sido usada em diferentes tempos antes da remoção desses órgãos. Doses elevadas da droga usadas no esquema profilático de 4 dias não tiveram efeito antimalárico. Entretanto quando dada a camundongos com malária, 24 horas antes, ao mesmo tempo ou 24 horas após inoculação de uma cepa de Plasmodium berghei cloroquina-sensível ou uma cepa cloroquina- resistente o levamisol reduziu, ainda que discretamente, a parasitemia nos grupos tratados, sendo a dose de 1 mg/kg o melhor esquema. Foi observado também atraso na mortalidade por malária nos grupos tratados com o levamisol. No entanto, todos os animais morreram. Os dados sugerem que o levamisol tem efeito imunoestimulante, ainda que discreto, na resposta imune de animais, deprimida pela malária.

Levamisole (phenylimidothiazol), considered a strong immunostimulant, when administered to healthy Swiss mice did not cause a significant increase in -the weight of their thymus, liver and spleen, even though the drug was used at different times before removing such organs. High doses ofdrug used in the 4-day prophylactic scheme had no antimalarial effect. However, when given to malaria infected mice 24 hours before, at the same time, and 24 hours after the inoculation of a chloroquine-sensitive or a chloroquine-resistant strain of Plasmodium berghei small doses of the drug induced a somewhat decreased parasitemia, the dose of 1 mg/kg body weight before the inoculum being the best scheme. The mortality rates by malaria in the levamisole treated groups were also delayed although all mice finally died. The data suggest that levamisole may display a stimulant effect on the depressed immune response caused by malaria.

Aedes fluviatilis é susceptível à infecção por Plasmodium gallinaceum, sendo considerado um modelo experimental útil para esta infecção em regiões nas quais Aedes aegypti não deve ser criado em laboratório, por razões de segurança. Na América do Sul, por exemplo, o A. aegypti é o principal vetor da febre amarela urbana não sendo aconselhável sua criação no laboratório pelo perigo que a espécie pode representar caso escapem exemplares do laboratório. Similarmente ao que ocorre com A. aegypti, as taxas de infecção do A. fluviatilis foram variáveis, quando utilizada uma cepa de P. gallinaceum mantida no laboratório por longo tempo através de passagens sangüíneas (linhagem B). Nesses mosquitos, desenvolveram-se oocistos em 47-63%, havendo infecção das glândulas salivares em cerca de 15%. O custo da esporogonia foi independente da dose do inóculo ou da via de administração às aves. Também a infecção das glândulas salivares com a linhagem B não dependeu do número de oocistos presentes no estômago. A esporogonia mostrou-se mais uniforme quando os mosquitos A. fluviatilis sugaram pintos infectados com uma linhagem reisolada a partir de esporozoítas (S). Cerca de 50 % destes mosquitos desenvolveram oocistos, havendo indivíduos com até 600 a 800 oocistos por estômago. A infecção média das glândulas salivares com a linhagem S foi de 27%, chegando a 57% em alguns grupos. Os padrões do parasitismo das glândulas salivares foram semelhantes àqueles da produção de oocistos. O decurso da esporogonia de P. gallinaceum em a. fluviatilis foi analisada em relação aos índices de parasitemia e de gametocitemia no hospedeiro vertebrado.

Aedes fluviatilis is susceptible to infection by Plasmodium gallinaceum and is a convenient insect host for the malaria parasite in countries where Aedees aegypti cannot be maintained in laboratories. In South America, for instance, the rearing of A. aegypti the main vector of urban yellow fever, is not advaisable because of the potential health hazard it represents. Our results of the comparative studies carried out between the sporogonic cycle produced with two lines of P. gallinaceum parasites into A. fuviatilis were as follows. As proved for A. aegypti, mosquito infection rates were variable when A. fluviatilis blood-fed on chicks infected with and old syringe-passaged strain of P. gallinaceum. Oocysts developed in 41% of those mosquitos and the mean peak of oocyst production was 56 per stomach. Salivary gland infections developed in about 6% of the mosquitos. The course of sporogony was unrelated to the size of the inoculum administered to chicks or to the route by which the birds were infected. The development of infected salivary glands was unrelated to oocyst production. Sporogony of P. gallinaceum was more uniform when mosquitos blood-fed on chicks infected with a sporozoite-passaged strain. Oocysts developed in about 50% of those mosquitoes and the mean peak of oocyst production was 138 per stomach, with some individuals having as many as 600-800 oocysts. Infected salivary glands developed in a mean of 27% of the mosquitos but, in some batches, was a high as 50%. Patterns of salivary gland parasitism were similar to those of oocyst production. The course of sporogony of P. gallinaceum in A. fluviatilis is analized in relation to degree of parasitemia and gametocytemia in the vertebrate host.