Photodynamic effects of zinc phthalocyanines on intracellular amastigotes of Leishmania amazonensis and Leishmania braziliensis.

This study investigated the photoactivity of four zinc phthalocyanines (PcZns) on a murine macrophage cell line infected with Leishmania amazonensis or Leishmania braziliensis. Infected and uninfected cells were incubated with PcZns at different concentrations (1-10 µM) for 3 h and then exposed to an LED device in continuous wave mode at 660 nm with a fluency of 50 J/cm(2) (25 mV). Enzymatic activity was determined by MTT assay 24 h after light treatment. The results demonstrated that all PcZns exhibited high photoactivity, particularly when used at 10 µM. The photodynamic effects were different for uninfected cells versus parasite-infected cells and among the four PcZns. Uninfected cells were more sensitive to photoactivity than infected cells. Although PcZns photodynamic therapy provided promising results, further studies are necessary to better understand its mechanism of action in the treatment of leishmaniasis.

Biochemical markers and anthropometric profile of children enrolled in public daycare centers.

OBJECTIVE: The nutritional status resultant from dietary habits along with socioeconomic conditions and the school environment are directly related to the individual's health condition not only in their childhood but also throughout adulthood. The aim of this study was to evaluate the effects of socioeconomic factors on the anthropometric profile and to analyze a probable association between this profile and biochemical markers in children attending public daycare centers. METHODS: It is a transversal study developed in a probability sample of clusters of children from 6 months to 5 years old. Anthropometric and socioeconomic data were gathered at the CMEIs, questionnaires on the nutritional status were applied and blood was collected at the Family Health Units (USFs). RESULTS: Female children are three times more likely to be underweight; in families with five members, it is 1/3 more likely that children of higher-educated parents are overweight. Among the results of the biochemical tests, hypervitaminosis A was a relevant aspect, positively correlating with copper (p=0.005) and zinc (p=0.008). CONCLUSION: Therefore, since the influence of the family is an important predictor of overweight and its future outcomes related to nutritional deficiencies and inadequate dietary intake, educational interventions are vital as a way to pave the path to prevention.

A retrospective survey of dengue virus infection in fatal cases from an epidemic in Brazil.

Dengue virus can infect many cell types from the vascular, muscular and hematological systems causing diverse clinical and pathological signs. The purpose of the present study was to investigate by different diagnostic methods dengue virus in human tissue specimens obtained from fatal cases (n=29) during a large-scale dengue fever epidemic in 2002 in the State of Rio de Janeiro, Brazil. The combination of four procedures provided diagnostic confirmation of DENV-3 infection in 26 (89.6%) out of the 29 suspected fatal cases. Dengue virus (DENV) was isolated from 2/74 (2.7%) tissue samples, inoculated into C6/36 cells and identified as DENV-3, nested RT-PCR accusing 22/72 (30.5%) samples as DENV-3. Real-time RT-PCR yielded the highest positivity rate, detecting viral RNA in 45/77 (58.4%) clinical specimens, including the liver (n=18), lung (n=8), spleen (n=8), brain (n=6), kidney (n=3), bone marrow (n=1) and heart (n=1). Immunohistochemical tests recognized the DENV antigen in 26/59 (44%) specimens. Given the accuracy and effectiveness of real-time RT-PCR in this investigation, this approach may play an important role for rapid diagnosis of dengue infections.

Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay.

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.

DNA Extraction Systematics for Spectroscopic Studies.

Study of genetic material allows the comprehension the origin of the many biochemical changes that follow diseases, like cancer, promoting the development of early preventive inquiry and more efficient individual treatments. Raman spectroscopy can be an important tool in DNA study, since it allows probe molecular vibrations of genetic material in a fast way. The present work established a systematic way for extract DNA in suitable concentrations and structural integrity allowing studies by Raman spectroscopy or other spectroscopic technique, including bio-analytical sensors for probing genetic alterations.

Dynamics of limnological parameters in reservoirs: A case study in South Brazil using remote sensing and meteorological data.

Reservoirs are important in Brazil for the production of hydroelectric power and human water consumption. The objective was to evaluate the variability of total suspended solids (TSS) and chlorophyll-a as well as the rainfall/temperature and land use impacts on these optically active constituents (OAC). The study area is the Passo Real reservoir in south Brazil. The methodology was divided in four steps. First, we used wavelet to detect anomalous periods of rainfall and temperature (2002-2014). Second, we carried out 12 field campaigns to obtain in situ measurements for limnological characterization (2009-2010). The third step was the analysis of Moderate Resolution Imaging Spectroradiometer (MODIS)/Terra and Aqua satellites data corrected and non-corrected for bidirectional effects. Finally, we evaluated potential drivers of OAC changes over time using cross-correlation analysis. The results showed a decrease in the TSS and chlorophyll-a concentrations from the upper to the lower streams of the reservoir. The exponential regression between the MODIS red reflectance and TSS had an adjusted r2 of 0.63. It decreased to 0.53 for the relationship between the green reflectance and chlorophyll-a. MODIS data corrected for bidirectional effects provided better OAC estimates than non-corrected data. The validation of MODIS TSS and chlorophyll-a estimates using a separate set of measurements showed a RMSE of 2.98mg/l and 2.33µg/l, respectively. MODIS estimates indicated a gradual transition in OAC from the upper to the lower streams in agreement with the patterns observed using field limnological data. The analysis of land use (greenness) showed two well-defined crop cycles per year. The highest seasonal concentrations of TSS and chlorophyll-a were observed in December and the lowest concentrations in April. Despite the interrelationships between both factors, our cross-correlation analysis indicated that the great concentrations of TSS and chlorophyll-a were primarily controlled by rainfall and secondarily by land use.

Biochemical markers and anthropometric profile of children enrolled in public daycare centers

Abstract Objective: The nutritional status resultant from dietary habits along with socioeconomic conditions and the school environment are directly related to the individual's health condition not only in their childhood but also throughout adulthood. The aim of this study was to evaluate the effects of socioeconomic factors on the anthropometric profile and to analyze a probable association between this profile and biochemical markers in children attending public daycare centers. Methods: It is a transversal study developed in a probability sample of clusters of children from 6 months to 5 years old. Anthropometric and socioeconomic data were gathered at the CMEIs, questionnaires on the nutritional status were applied and blood was collected at the Family Health Units (USFs). Results: Female children are three times more likely to be underweight; in families with five members, it is 1/3 more likely that children of higher-educated parents are overweight. Among the results of the biochemical tests, hypervitaminosis A was a relevant aspect, positively correlating with copper (p=0.005) and zinc (p=0.008). Conclusion: Therefore, since the influence of the family is an important predictor of overweight and its future outcomes related to nutritional deficiencies and inadequate dietary intake, educational interventions are vital as a way to pave the path to prevention.

[Study of the infection and morbidity of Chagas' disease in municipality of João Costa: National Park Serra da Capivara, Piauí, Brazil]. / Estudo da infecção e morbidade da doença de Chagas no município de João Costa: Parque Nacional Serra da Capivara, Piauí, Brasil.

In order to investigate aspects of the infection and morbidity of Chagas' disease in the municipality of João Costa, Piauí State, Brazil, we carried out a serological survey to detect anti-Trypanosoma cruzi antibodies in 2,080 individuals, by indirect immunofluorescence, indirect hemagglutination and ELISA. A total of 189 seropositive and 141 seronegative patients were evaluated by anamnesis, physical exam and electrocardiogram (EKG). The parasitaemia of 106 chagasic patients was evaluated by indirect xenodiagnosis and PCR (polymerase chain reaction). The total seropositivity was 9.8%, with intervals of 0.5% in patients younger than 10 years old, and 39.4% among patients older than 59 years old, independently of the sex. The PCR and xenodiagnosis were positive, respectively in 74.5% and 15.1% of the seropositive patients (p < 0.05). The rate of abnormal EKG was 41.3% in chagasic and 15.6% in non-chagasic patients (p < 0.05). In spite of the high prevalence of infection in the investigated population, the low rate of seropositivity among children is indicative of a possible decrease of the active transmission mediated by triatomines. The high proportion of the chagasic component on the cardiopathy prevalence is indicative of the high morbidity of Chagas' disease in the studied region.

Cenostigma macrophyllum Tul. on the healing of skin wounds in rats with diabetes mellitus.

PURPOSE: To investigate the effect of Cenostigma macrophyllum Tul. in the tissue repair process of skin lesions in rats with induced Diabetes mellitus. METHODS: Sixty-three male Wistar rats were distributed into three groups: control (C), diabetic (D), and diabetic treated with an oil-in-water emulsion of the plant (DPL) subdivided according to time of observation (seven, 14, and 28 days). Diabetes was induced by administration of by injection of streptozotocin (50 mg/kg, after a 12-h fast) into the penile vein and confirmed by glucose levels exceeding 240 mg/dL. Held surgical lesion (2.5 cm in diameter) on the back of the animals. The DPL received topical application of group of the oil-in-water emulsion plant (0.5 ml). To evaluate the levels of nitric oxide, was collected 5 ml of blood from the abdominal aorta, after his euthanasia. The samples of interest were sent for routine histological processing. RESULTS: A reduction in the percentage of inflammatory cells and increased numbers of fibroblasts in the group DPL, seven days, compared to the other groups. At 14 days, the DPL group also showed a higher concentration of nitric oxide (p<0.01) than in groups C and D. CONCLUSION: The oil-in-water emulsion of C. macrophyllum Tul accelerated wound healing in diabetic rats.

Cenostigma macrophyllum Tul. on the healing of skin wounds in rats with Diabetes mellitus

PURPOSE: To investigate the effect of Cenostigma macrophyllum Tul. in the tissue repair process of skin lesions in rats with induced Diabetes mellitus. METHODS: Sixty-three male Wistar rats were distributed into three groups: control (C), diabetic (D), and diabetic treated with an oil-in-water emulsion of the plant (DPL) subdivided according to time of observation (seven, 14, and 28 days). Diabetes was induced by administration of by injection of streptozotocin (50 mg/kg, after a 12-h fast) into the penile vein and confirmed by glucose levels exceeding 240 mg/dL. Held surgical lesion (2.5 cm in diameter) on the back of the animals. The DPL received topical application of group of the oil-in-water emulsion plant (0.5ml). To evaluate the levels of nitric oxide, was collected 5ml of blood from the abdominal aorta, after his euthanasia. The samples of interest were sent for routine histological processing. RESULTS:A reduction in the percentage of inflammatory cells and increased numbers of fibroblasts in the group DPL, seven days, compared to the other groups. At 14 days, the DPL group also showed a higher concentration of nitric oxide (p<0.01) than in groups C and D. CONCLUSION: The oil-in-water emulsion of C. macrophyllum Tul accelerated wound healing in diabetic rats.

The mitochondrial genome from the thermal dimorphic fungus Paracoccidioides brasiliensis.

We present here the sequence of the mitochondrial DNA of the pathogenic thermodimorphic fungus Paracoccidioides brasiliensis, agent of an endemic disease in most South American countries. The sequenced genome has 71 334 bp and is organized as a circular molecule with two gaps of unknown size flanking the middle exon of the nad5 gene. We located genes coding for the three subunits of the ATP synthase (atp6, atp8 and atp9), the apocytochrome b (cob), three subunits of the cytochrome c oxidase enzyme complex (cox1, cox2 and cox3), seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase (nad1, nad2, nad3, nad4, nad5, nad6 and nad4L) and the large (rnl) and small (rns) subunits of ribosomal RNA. Two maturases and a ribosomal protein (rms5) are located inside introns. Twenty-five tRNAs were identified with acceptors for all 20 amino acids. Seven polypurine/polypyrimidine tracts (140-240 bp) have been found in this genome. All genes are in the same orientation over the genome, while their order is closest to the mitochondrial genomes from Penicillium marneffei and Aspergillus nidulans.

Utilização da microespectroscopia infravermelha (FT-IR) para teste de algoritmos estatísticos na diferenciação dos micro-organismos Candida albicans, Candida dubliniensis e Candida parapsilosis / Using FT-IR microspectroscopy for testing statistical algorithms for differentiation of Candida albicans, Candida dubliniensis and Candida parapsilosis

As espécies do gênero Candida são causadoras de diversas infecções fúngicas e, nos últimos anos, tem sido desenvolvidas novas tecnologias para auxiliar nos diagnósticos microbiológicos. Dentre as técnicas está a espectroscopia infravermelha junto com a análise estatística multivariada. O objetivo deste trabalho é comparar dois métodos: estatístico (análise multivariada) e não-estatístico (ajuste de curva), utilizando os espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis para testar o potencial do uso de Análise Estatística Multivariada para discriminação de espectros de micro-organismos. Para isso foram obtidos, utilizando o Spectrum Spotlight 400 da PerkinElmer, 54 espectros infravermelhos, sendo 18 de cada espécie, na faixa de 4000 a 1000 cm-1, com resolução de 4 cm-1, no modo de transmissão, a 20 ºC. A análise dos espectros foi realizada através de três métodos: (1) inspeção visual direta dos espectros; (2) análise estatística multivariada; (3) ajuste de curva para a determinação de estruturas secundárias de proteínas. Na região de 1200 a 1000 cm-1, os espectros apresentam diferenças que podem ser percebidas numa inspeção visual direta. Uma banda próxima de 1070 cm-1 e outra próxima de 1045 cm-1 apresentam intensidades relativas diferentes para os três espectros. Por outro lado, as bandas da amida I, na região de 1710 a 1590 cm-1, apresentam aspectos visuais semelhantes com máximo em 1651 cm-1 para os espectros dos três micro-organismos. Esse fato torna possível submeter a análise estatística multivariada a um teste de sua capacidade de diferenciar três espectros de Candida. A análise estatística multivariada foi aplicada aos 54 espectros para investigar as regiões de 4000 a 1000 cm-1 com exceção da região de 2600 a 2300 cm-1 e de 1710 a 1590 cm-1 que corresponde a das bandas da amida I. A técnica selecionada foi a análise por componentes principais (PCA, Principal Componente Analysis), utilizando os primeiros quatro componentes principais, em conjunto com a técnica hierárquica de análise de agrupamento (HCA, Hierarchical Clustering Analysis) segundo o método de Ward. Foi utilizado para esta análise o software MINITAB 15 e o resultado mostra uma clara discriminação dos espectros dos três micro-organismos nas duas regiões consideradas. Adicionalmente foi obtido o espectro médio de cada micro-organismo nas bandas da amida I na região de 1710 a 1590 cm-1. Os três espectros médios assim obtidos foram analisados pelo método de ajuste de curva que não é estatístico para determinar as estruturas secundárias de proteínas. Para esta análise o software ORIGIN 7.5 foi utilizado e os resultados obtidos mostram estruturas conformacionais diferentes nos três micro-organismos. Esses resultados confirmam a discriminação obtida através da análise estatística multivariada e visual. Pode-se concluir que as análises estatísticas multivariadas baseadas em análise por componentes principais e análise de agrupamento com uso do algoritmo Ward é potencialmente útil para discriminar micro-organismos através de seus espectros infravermelhos. Além disso, as análises mostram que as bandas da amida I dos espectros infravermelhos de Candida albicans, Candida dubliniensis e Candida parapsilosis fornecem um conjunto de dados cuja estrutura de agrupamento é conhecida e que pode ser útil para testar e validar algoritmos estatísticos de análise de agrupamento.

Films of Candida albicans, Candida dubliniensis and Candida parapsilosis were prepared and the infrared spectra of these films were obtained in the region 4000 to 1000 cm-1, with resolution of 4 cm-1, in the transmission mode, at 20 ºC. Fifty four spectra were obtained, 18 of each microorganism, with the PerkinElmer Spotlight 400 FT-IR, which has a microscope attached to a FT-IR spectrophotometer. The spectra were analyzed through three methods: (1) mere visual inspection; (2) multivariate statistical analysis; (3) curve-fitting for determining secondary structures of proteins. In the region 1200 to 1000 cm-1, the spectral bands show differences that can be seen by a mere visual inspection. On the other hand, the amide I bands, in the region 1710 to 1590 cm-1, have the same visual aspect for the three microorganisms. Multivariate statistical analysis was applied to analyze these amide I bands of all the 54 spectra. Principal component analysis (PCA) and techniques of hierarchical cluster analysis (HCA, Hierarchical Clustering Analysis) according to Ward's method were applied using the software MINITAB 15. The results show a clear discrimination of the three microorganisms. The average spectrum of each microorganism was obtained in the amide I band. Each average spectrum was analyzed by curve-fitting for the determination of secondary structures of proteins. The software used was the ORIGIN 7.5 and the results confirm the discrimination obtained through multivariate statistical analysis. This result shows that multivariate statistical analysis can be useful to discriminate infrared spectra of different microorganisms. Furthermore, this work shows that the amide I bands of the infrared spectra of Candida albicans, Candida dubliniensis, and Candida parapsilosis provide a set of data of known group structure that can be useful to test statistical algorithms of cluster analysis.

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia.

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.

Parasite persistence in treated chagasic patients revealed by xenodiagnosis and polymerase chain reaction

Polymerase chain reaction (PCR) was compared with xenodiagnosis performed 20 years after trypanocidal chemotherapy to investigate parasite clearance. Eighty-five seropositive individuals for Chagas disease presenting a positive xenodiagnosis were treated with specific drugs; 37 in the acute phase and 48 in the chronic phase. Fifteen chronic assymptomatic patients received a placebo. Treatment in the acute phase led to PCR negative results in 73 percent of the cases, while xenodiagnosis was negative in 86 percent. In the chronic phase, PCR was negative in 65 percent of the patients and 83 percent led to xenodiagnosis negative results. Regarding the untreated group (placebo), 73 percent gave negative results by xenodiagnosis, of which 36 percent were positive by PCR. Individuals that were considered seronegative (n=10), presented unequivocally negative results in the PCR demonstrating the elimination of parasite DNA. Seventeen individuals had their antibodies titers decreased to such a level that the final results were considered as doubtful and 16 of them presented negative PCR. The molecular method represents a clear advantage over conventional techniques to demonstrate persistent infections in Chagas disease patients that underwent chemotherapy

Estudo da infecção e morbidade da doença de Chagas no municÝpio de João Costa: Parque Nacional Serra da Capivara, Piauí, Brasil / Study of the infection and morbidity of Chagas' disease in municipality of JoÒo Costa: National Park Serra da Capivara, Piauí, Brazil

In order to investigate aspects of the infection and morbidity of Chagas' disease in the municipality of JoÒo Costa, PiauÝ State, Brazil, we carried out a serological survey to detect anti-Trypanosoma cruzi antibodies in 2,080 individuals, by indirect immunofluorescence, indirect hemagglutination and ELISA. A total of 189 seropositive and 141 seronegative patients were evaluated by anamnesis, physical exam and electrocardiogram (EKG). The parasitaemia of 106 chagasic patients was evaluated by indirect xenodiagnosis and PCR (polymerase chain reaction). The total seropositivity was 9.8, with intervals of 0.5 in patients younger than 10 years old, and 39.4 among patients older than 59 years old, independently of the sex. The PCR and xenodiagnosis were positive, respectively in 74.5 and 15.1 of the seropositive patients (p < 0.05). The rate of abnormal EKG was 41.3 in chagasic and 15.6 in non-chagasic patients (p < 0.05). In spite of the high prevalence of infection in the investigated population, the low rate of seropositivity among children is indicative of a possible decrease of the active transmission mediated by triatomines. The high proportion of the chagasic component on the cardiopathy prevalence is indicative of the high morbidity of Chagas' disease in the studied region.

Estudo da infecção e morbidade da doença de Chagas no município de João Costa - Parque Nacional Serra da Capivara, Piauí, Brasil / Study of the infection and morbidity of Chagas' disease in municipality of João Costa ­ National Park Serra da Capivara, Piauí, Brazil

Com o objetivo de investigar aspectos da infecção e morbidade da doença de Chagas no município de João Costa, Piauí, Brasil, realizamos pesquisa sorológica para detectar Ig G anti-T. cruzi em 2.080 moradores através dos testes de imunofluorescência indireta, hemaglutinação indireta e ELISA. Em seguida, 189 pacientes soropositivos e 141 soronegativos foram avaliados pelo exame clínico e eletrocardiograma (ECG), enquanto a parasitemia foi pesquisada em 106 chagásicos pelo xenodiagnóstico indireto e teste da reação polimerásica em cadeia (PCR). A soropositividade total para Ig G anti-T.cruzi foi de 9,8%, com variação de 0,5% em menores de 10 anos a 39,4% em maiores de 59 anos, independentemente do sexo. O percentual de ECG alterados foi de 41,3% entre os chagásicos e de 15,6% entre os não-chagásicos (p < 0,05). A positividade do teste da PCR foi de 74,5% e a do xenodiagnóstico de 15,1% (p < 0,05). Apesar da elevada prevalência da infecção na população investigada, o baixo valor nos menores de 10 anos pode ser indicador de redução da transmissão por triatomíneos. A alta proporção de participação do componente etiológico exclusivamente chagásico na prevalência da cardiopatia indica a gravidade da doença de Chagas na região estudada.