Present situation and new strategies for Chagas disease chemotherapy: a proposal

Treatments for Chagas disease have been administered since the first attempts by Mayer & Rocha Lima (1912, 1914) and up to the drugs currently in use (nifurtimox and benznidazole), along with potential drugs such as allopurinol and first, second and third-generation antifungal agents (imidazoles and triazoles), in separate form. Several diseases such as tuberculosis, leprosy and AIDS only came under control after they were treated with associations of drugs with different mechanisms of action. This not only boosts the action of the different compounds, but also may avoid the development of parasite resistance .To this end, over the short term, we propose experimental studies on laboratory animals and clinical trials with the following associations: (i) nifurtimox (8 mg/kg/day) + benznidazole (5 mg/kg/day) x 60 consecutive days; (ii) nifurtimox (8 mg/kg/day) or benznidazole (5 mg/kg/day) + allopurinol (8-10 mg/kg/day) x 60 days and (iii) nifurtimox (8 mg/kg/day) or benznidazole (5 mg/kg/day) + ketoconazole, fluconazole or itraconazole (5-6 mg/kg/day) x 60 consecutive days. The doses of the drugs and the treatment schedules for the clinical trials must be adapted according to the side effects. From these, other double or triple associations could be made, using drugs with different mechanisms of action. This proposal does not exclude investigations on new drugs over the median and long terms, targeting other aspects of the metabolism of Trypanosoma cruzi. Until such time as the ideal drug for specific treatment of Chagas disease might be discovered, we need to develop new strategies for achieving greater efficacy with the old drugs in associations and to develop rational experimentation with new drugs.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed.

High prevalence of drug-resistant tuberculosis and other mycobacteria among HIV-infected patients in Brazil: a systematic review

There is a little-noticed trend involving human immunodeficiency virus (HIV)-infected patients suspected of having tuberculosis: the triple-treatment regimen recommended in Brazil for years has been potentially ineffective in over 30% of the cases. This proportion may be attributable to drug resistance (to at least 1 drug) and/or to infection with non-tuberculous mycobacteria. This evidence was not disclosed in official statistics, but arose from a systematic review of a few regional studies in which the diagnosis was reliably confirmed by mycobacterial culture. This paper clarifies that there has long been ample evidence for the potential benefits of a four-drug regimen for co-infected patients in Brazil and it reinforces the need for determining the species and drug susceptibility in all positive cultures from HIV-positive patients.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).

Angiostrongylus cantonensis (Nematode: Metastrongyloidea) in molluscs from harbour areas in Brazil

Angiostrongylus cantonensis is the most common aetiological agent of human eosinophilic meningoencephalitis. Following a report indicating the presence of this parasite in Brazil in 2007, the present study was undertaken to investigate the presence of A. cantonensis in the surrounding Brazilian port areas. In total, 30 ports were investigated and the following molluscs were identified: Achatina fulica, Belocaulus sp., Bradybaena similaris sp., Cyclodontina sp., Helix sp., Leptinaria sp., Melampus sp., Melanoides tuberculata, Phyllocaulis sp., Pomacea sp., Pseudoxychona sp., Rhinus sp., Sarasinula marginata, Streptaxis sp., Subulina octona, Succinea sp., Tomigerus sp., Wayampia sp. and specimens belonging to Limacidae and Orthalicinae. Digestion and sedimentation processes were performed and the sediments were examined. DNA was extracted from the obtained larvae and the internal transcribed spacer region 2 was analysed by polymerase chain reaction-restriction fragment length polymorphism after digestion with the endonuclease ClaI. Of the 30 ports investigated in this study, 11 contained molluscs infected with A. cantonensis larvae. The set of infected species consisted of S. octona, S. marginata, A. fulica and B. similaris. A total of 36.6% of the investigated ports were positive for A. cantonensis, indicating a wide distribution of this worm. It remains uncertain when and how A. cantonensis was introduced into South America.

Immunological response to re-infections with clones of the Colombian strain of Trypanosoma cruzi with different degrees of virulence: influence on pathological features during chronic infection in mice

Re-infections with Trypanosoma cruzi are an aggravating factor for Chagas disease morbidity. The Colombian strain of T. cruzirepresents multiclonal populations formed by clonally propagating organisms with different tropisms and degrees of virulence. In the present study, the influence of successive inoculations with clones of the Colombian strain, exhibiting different degrees of virulence, on chronic myocarditis and the humoral and cellular immune responses (Col-C1 high virulence, Col-C8 medium virulence and Col-C5 low virulence) were demonstrated. Mice from three groups with a single infection were evaluated during the acute (14th-30th day) and chronic phases for 175 days. An immunofluorescence assay, ELISA and delayed type hypersensitivity (DTH) cutaneous test were also performed. Mice with a triple infection were studied on the 115th-175th days following first inoculation. The levels of IgM and IgG2a were higher in the animals with a triple infection. DTH showed a higher intensity in the inflammatory infiltrate based on the morphometric analysis during a 48 h period of the triple infection and at 24 h with a single infection. The histopathology of the heart demonstrated significant exacerbation of cardiac inflammatory lesions confirmed by the morphometric test. The humoral responses indicate a reaction to the triple infection, even with clones of the same strain.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Biomass production and phosphorus use of forage grasses fertilized with two phosphorus sources

A major constraint to agricultural production in acid soils of tropical regions is the low soil P availability, due to the high adsorption capacity, low P level in the source material and low efficiency of P uptake and use by most of the modern varieties grown commercially. This study was carried out to evaluate the biomass production and P use by forage grasses on two soils fertilized with two P sources of different solubility. Two experiments were carried out, one for each soil (Cambisol and Latosol), using pots filled with 4 dm³ soil in a completely randomized design and a 4 x 2 factorial scheme. The treatments consisted of a combination of four forage plants (Brachiaria decumbens, Brachiaria brizantha, Pennisetum glaucum and Sorghum bicolor) with two P sources (Triple Superphosphate - TSP and Arad Reactive Phosphate - ARP), with four replications. The forage grasses were harvested at pre-flowering, when dry matter weight and P concentrations were measured. Based on the P concentration and dry matter production, the total P accumulation was calculated. With these data, the following indices were calculated: the P uptake efficiency of roots, P use efficiency, use efficiency of available P, use efficiency of applied P and agronomic efficiency. The use of the source with higher solubility (TSP) resulted, generally, in higher total dry matter and total P accumulation in the forage grasses, in both soils. For the less reactive source (ARP), the means found in the forage grasses, for use efficiency and efficient use of available P, were always higher when grown in Latosol, indicating favorable conditions for the solubility of ARP. The total dry matter of Brachiaria brizantha was generally higher, with low P uptake, accumulation and translocation, which indicated good P use efficiency for both P sources and soils. The forage plants differed in the P use potential, due to the sources of the applied P and of the soils used. Less than 10 % of the applied P was immobilized in the forage dry matter. Highest values were observed for TSP, but this was not reflected in a higher use efficiency of P from this source.

Phosphorus and root distribution and corn growth as related to long-term tillage systems and fertilizer placement

Soil and fertilizer management during cultivation can affect crop productivity and profitability. Long-term experiments are therefore necessary to determine the dynamics of nutrient and root distribution as related to soil profile, as well as the effects on nutrient uptake and crop growth. An 18-year experiment was conducted at the Federal University of Rio Grande do Sul State (UFRGS), in Eldorado do Sul, Brazil, on Rhodic Paleudult soil. Black oat and vetch were planted in the winter and corn in the summer. The soil management methods were conventional, involving no-tillage and strip tillage techniques and broadcast, row-and strip-applied fertilizer placement (triple superphosphate). Available P (Mehlich-1) and root distribution were determined in soil monoliths during the corn grain filling period. Corn shoot dry matter production and P accumulation during the 2006/2007 growing season were determined and the efficiency of P utilization calculated. Regardless of the degree of soil mobilization, P and roots were accumulated in the fertilized zone with time, mainly in the surface layer (0-10 cm). Root distribution followed P distribution for all tillage systems and fertilizer treatments. Under no-tillage, independent of the fertilizer placement, the corn plants developed more roots than in the other tillage systems. Although soil tillage systems and fertilizer treatments affected P and root distribution throughout the soil profile, as well as P absorption and corn growth, the efficiency of P utilization was not affected.

Availability of a soluble phosphorus source applied to soil samples with different acidicity levels

Considerations on the interactions of P in the soil-plant system have a long history, but are still topical and not yet satisfactorily understood. One concern is the effect of liming before or after application of soluble sources on the crop yield and efficiency of available P under these conditions. The aim of this study was to evaluate the effect of soil acidity on availability of P from a soluble source, based on plant growth and chemical extractants. Nine soil samples were incubated with a dose of 200 mg kg-1 P in soil with different levels of previously adjusted acidity (pH H2O 4.5; 5.0; 5.5; 6.0 and 6.5) and compared to soils without P application. After 40 days of soil incubation with a P source, each treatment was limed again so that all pH values were adjusted to 6.5 and then sorghum was planted. After the first and second liming the P levels were determined by the extractants Mehlich-1, Bray-1 and Resin, and the fractionated inorganic P forms. In general, the different acidity levels did not influence the P availability measured by plant growth and P uptake at the studied P dose. For some soils however these values increased or decreased according to the initial soil pH (from 4.5 to 6.5). Plant growth, P uptake and P extractable by Mehlich-1 and Bray-1 were significantly correlated, unlike resin-extractable P, at pH values raised to 6.5. These latter correlations were however significant before the second liming. The P contents extracted by Mehlich-1 and Bray-1 were significantly correlated with each other in the entire test range of soil acidity, even after adjusting pH to 6.5, besides depending on the soil buffering capacity for P. Resin was also sensitive to the properties that express the soil buffering capacity for P, but less clearly than Mehlich-1 and Bray-1. The application of triple superphosphate tended to increase the levels of P-Al, P-Fe and P-Ca and the highest P levels extracted by Bray-1 were due to a higher occurrence of P-Al and P-Fe in the soils.

Soil phosphorus dynamics and availability and irrigated coffee yield

Research data have demonstrated that the P demand of coffee (Coffea arabica L.) is similar to that of short-cycle crops. In this context, the objective of this study was to evaluate the influence of annual P fertilization on the soil P status by the quantification of labile, moderately labile, low-labile, and total P fractions, associating them to coffee yield. The experiment was installed in a typical dystrophic Red Latosol (Oxisol) cultivated with irrigated coffee annually fertilized with triple superphosphate at rates of 0, 50, 100, 200, and 400 kg ha-1 P2O5. Phosphorus fractions were determined in two soil layers: 0-10 and 10-20 cm. The P leaf contents and coffee yield in 2008 were also evaluated. The irrigated coffee responded to phosphate fertilization in the production phase with gains of up to 138 % in coffee yield by the application of 400 kg ha-1 P2O5. Coffee leaf P contents increased with P applications and stabilized around 1.98 g kg-1, at rates of 270 kg ha-1 P2O5 and higher. Soil P application caused, in general, an increase in bioavailable P fractions, which constitute the main soil P reservoir.

NPK fertilization on initial growth of physic nut seedlings in Quartzarenic Neossol

Balanced fertilization is important for plant growth. There is little information on physic nut (Jatropha curcas L.) and tests with the fertilization of the species are very recent. This study evaluated the initial growth of physic nut seedlings in response to NPK rates to Quartzarenic Neossol in a greenhouse and estimated P and K critical soil levels and N, P and K in shoot dry matter after 120 days of evaluation. The treatments were arranged in a randomized, fractional factorial design (4 x 4 x 4)½, totalizing 32 treatments with three replicates, 96 experimental plots and N rates (0, 75, 150 and 300 mg dm-3) as urea; P rates (0, 45, 90 and 180 mg dm-3) as triple superphosphate and K rates (0, 50, 100 and 200 mg dm-3) as potassium chloride. After 120 days, the plants were harvested and some variables evaluated: plant height, stem diameter, shoot and root dry weight, macro and micronutrient levels in plant shoots, and soil chemical properties. The physic nut seedlings responded to NPK fertilizer in the initial growth phase; the response to N was negative. The recommended P and K rates were 25 and 67 mg dm-3, respectively. The critical levels, corresponding to the recommended P rate were 13 and 74 mg dm-3 for K in soil (Mehlich-1). The N, P and K levels in the shoot dry matter of physic nut were 37.4, 2.1 and 35.7 g kg-1, respectively.

Phosphorus extracted by ion exchange resins and mehlich-1 from oxisols (latosols) treated with different phosphorus rates and sources for varied soil-source contact periods

Despite the large number of studies addressing the quantification of phosphorus (P) availability by different extraction methods, many questions remain unanswered. The aim of this paper was to compare the effectiveness of the extractors Mehlich-1, Anionic Resin (AR) and Mixed Resin (MR), to determine the availability of P under different experimental conditions. The laboratory study was arranged in randomized blocks in a [(3 x 3 x 2) + 3] x 4 factorial design, with four replications, testing the response of three soils with different texture: a very clayey Red Latosol (LV), a sandy clay loam Red Yellow Latosol (LVA), and a sandy loam Yellow Latosol (LA), to three sources (triple superphosphate, reactive phosphate rock from Gafsa-Tunisia; and natural phosphate from Araxá-Minas Gerais) at two P rates (75 and 150 mg dm-3), plus three control treatments (each soil without P application) after four contact periods (15, 30, 60, and 120 days) of the P sources with soil. The soil acidity of LV and LVA was adjusted by raising base saturation to 60 % with the application of CaCO3 and MgCO3 at a 4:1 molar ratio (LA required no correction). These samples were maintained at field moisture capacity for 30 days. After the contact periods, the samples were collected to quantify the available P concentrations by the three extractants. In general, all three indicated that the available P-content in soils was reduced after longer contact periods with the P sources. Of the three sources, this reduction was most pronounced for triple superphosphate, intermediate for reactive phosphate, while Araxá phosphate was least sensitive to the effect of time. It was observed that AR extracted lower P levels from all three soils when the sources were phosphate rocks, while MR extracted values close to Mehlich-1 in LV (clay) and LVA (medium texture) for reactive phosphate. For Araxá phosphate, much higher P values were determined by Mehlich-1 than by the resins, because of the acidity of the extractor. For triple superphosphate, both resins extracted higher P levels than Mehlich-1, due to the consumption of this extractor, particularly when used for LV and LVA.

Comparison of digestion methods to determine heavy metals in fertilizers

The lack of a standard method to regulate heavy metal determination in Brazilian fertilizers and the subsequent use of several digestion methods have produced variations in the results, hampering interpretation. Thus, the aim of this study was to compare the effectiveness of three digestion methods for determination of metals such as Cd, Ni, Pb, and Cr in fertilizers. Samples of 45 fertilizers marketed in northeastern Brazil were used. A fertilizer sample with heavy metal contents certified by the US National Institute of Standards and Technology (NIST) was used as control. The following fertilizers were tested: rock phosphate; organo-mineral fertilizer with rock phosphate; single superphosphate; triple superphosphate; mixed N-P-K fertilizer; and fertilizer with micronutrients. The substances were digested according to the method recommended by the Ministry for Agriculture, Livestock and Supply of Brazil (MAPA) and by the two methods 3051A and 3052 of the United States Environmental Protection Agency (USEPA). By the USEPA method 3052, higher portions of the less soluble metals such as Ni and Pb were recovered, indicating that the conventional digestion methods for fertilizers underestimate the total amount of these elements. The results of the USEPA method 3051A were very similar to those of the method currently used in Brazil (Brasil, 2006). The latter is preferable, in view of the lower cost requirement for acids, a shorter digestion period and greater reproducibility.