Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

Trypanocidal activity of Meliaceae and Rutaceae plant extracts

The in vitro trypanocidal activity of 22 extracts and 43 fractions of plants belonging to the families Meliaceae and Rutaceae was evaluated. The extracts from leaves of Conchocarphus heterophyllus and branches of Trichilia ramalhoi were the most active. The trypanocidal activity seems to be increased by fractionation of the extracts. Fractions from C. heterophyllus and Galipea carinata were the most active and a 100% lysis of the parasites was observed for five fractions. From one of them were isolated two flavonoids: flavone and 7-methoxyflavone, which showed weak trypanocidal activity. The results obtained from the extracts and fractions revealed that the order Rutales is a promising source for the search of new drugs for Chagas disease. Phytochemical studies with the other active fractions are underway in order to isolate compounds, which could be associated with observed activities.

Effect of thiadiazine derivatives on intracellular amastigotes of Leishmania amazonensis

Current therapy for leishmaniasis is not satisfactory. We describe the in vitro antiproliferative effects of new thiadiazine derivatives against Leishmania amazonensis. The compounds were found to be active against the amastigote form of the parasite, inhibiting parasite growing, from 10 to 89%, at a concentration of 100 ng/ml. This activity suggests that thiadiazine derivatives could be considered as potential antileishmanial compounds.

Active transmission of human chagas disease in Colima Mexico

Despite efforts to eradicate American trypanosomiasis (AT) and Chagas disease from the Americas, there are still areas of active transmission that can eventually become a source of reinfection in previously controlled regions. Mexico could be one of those areas, where there are no formal preventive control programs despite the presence of communities infested by Triatominae bugs infected with Trypanosoma cruzi. This study explored the prevalence of T. cruzi infection in 405 habitants of 17 communities in the state of Colima, on the Pacific Mexican coast, through a seroepidemiological probabilistic survey. The results revealed a point seroprevalence of 2.4% positive for anti-T. cruzi. In addition, 2 clinical cases of chronic and 2 of acute Chagas disease were detected in the explored communities. These findings confirm the risk of active transmission of AT in Western Mexico, especially in rural and suburban communities infested with intra-domestic triatominae, where control programs should be implemented.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 µg/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

Antileishmanial activity of lapachol analogues

The antileishmanial activity of lapachol, isolapachol, and dihydrolapachol, along with soluble derivatives (potassium salt) and acetate was obtained. All the compounds were assayed against metacyclic promastigotes of two different species of Leishmania associated to tegumentar leishmaniasis: L. amazonensis and L. braziliensis. All compounds presented significant activity, being isolapachol acetate the most active against promastigotes, with IC50/24h = 1.6 ± 0.0 µg/ml and 3.4 ± 0.5 µg/ml for, respectively, L. amazonensis and L. braziliensis. This compound was also assayed in vivo against L. amazonensis and showed to be active. Its toxicity in vitro was also established, and at concentration similar to the IC50, no toxicity was evidenced. In all experiments, pentamidine isethionate was used as a reference drug. The present results reinforce the potential use of substituted hydroxyquinones and derivatives as promising antileishmanial drugs and suggest a continuing study within this class of compounds.

Lacrimal secretory IgA in active posterior uveitis induced by Toxoplasma gondii

It is quite difficult to diagnose active toxoplasmosis in patients with ocular toxoplasmosis. Active posterior uveitis presumably due to Toxoplasma gondii infection (APUPT) is seldom produced during a prime-infection; hence most patients do not show high IgM antibodies. High levels of IgA have been described in active toxoplasmosis. The purpose of this study was to investigate possible association between APUPT and the specific anti-parasite sIgA in tears. The study was carried out as case-control. Tears of 25 clinically confirmed APUPT patients and 50 healthy control subjects were analyzed. All were IgG seropositive. Specific sIgA was determined by ELISA assay using T. gondii RH strain crude extract. Anti-T. gondii sIgA was found in 84% of the cases and in 22% of the control subjects. The intensity of the reaction was higher in APUPT cases (P = 0.007). There was strong association between APUPT patients and lacrimal sIgA (odds-ratio 18.61, P = 0.0001). ELISA test sensitivity was 84% and specificity 78% . Our data suggest that anti-T.gondii secretory IgA found in tears may become an important marker for active ocular toxoplasmosis.

Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeqTM Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients - 10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects - were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50% of parasite growth (IC50) between 0.1-10 µg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 µg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 µM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM).

Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)

Calotropis procera R. Br. (Asclepiadaceae) is a well-known medicinal plant with leaves, roots, and bark being exploited by popular medicine to fight many human and animal diseases. This work deals with the fractionation of the crude latex produced by the green parts of the plant and aims to evaluate its toxic effects upon egg hatching and larval development of Aedes aegypti. The whole latex was shown to cause 100% mortality of 3rd instars within 5 min. It was fractionated into water-soluble dialyzable (DF) and non-dialyzable (NDF) rubber-free materials. Both fractions were partially effective to prevent egg hatching and most of individuals growing under experimental conditions died before reaching 2nd instars or stayed in 1st instars. Besides, the fractions were very toxic to 3rd instars causing 100% mortality within 24 h. When both fractions were submitted to heat-treatment the toxic effects were diminished considerably suggesting low thermostability of the toxic compounds. Polyacrylamide gel electrophoresis of both fractions and their newly fractionated peaks obtained through ion exchange chromatography or desalting attested the presence of proteins in both materials. When submitted to protease digestion prior to larvicidal assays NDF lost most of its toxicity but DF was still strongly active. It may be possible that the highly toxic effects of the whole latex from C. procera upon egg hatching and larvae development should be at least in part due to its protein content found in NDF. However the toxicity seems also to involve non protein molecules present in DF.

Effects of Brazilian propolis on Leishmania amazonensis

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 µg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.