A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples

Submitted by Nuzia Santos ([email protected]) on 2012-09-27T14:31:36Z No. of bitstreams: 1 36.2010.pdf: 789056 bytes, checksum: 0a4282ac34d4c6aef08223da45e0f126 (MD5)Made available in DSpace on 2012-09-27T14:31:36Z (GMT). No. of bitstreams: 1 36.2010.pdf: 789056 bytes, checksum: 0a4282ac34d4c6aef08223da45e0f126 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Laboratório de Esquistossomose. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, Brasil/ Universidade Federal de Ouro Preto. Escola de Farmácia. Laboratório de Pesquisas Clínicas. Ouro Preto, MG, BraziBackground: In this paper a simple and cheap salting out and resin (InstaGene matrix® resin - BioRad) DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples. Findings: The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure. Conclusions: This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic disease

Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence

Genetic and Functional Role of TNF-alpha in the Development Trypanosoma cruzi Infection

TNF-alpha plays an important role in trypanocidal mechanisms and is related to tissue injury. This cytokine has been detected in the heart of human chagasic patients where it is associated with tissue damage. This study investigated whether TNF-alpha levels and the presence of genetic polymorphisms are associated with the presence of T. cruzi infection and/or with the development of the cardiac form in chronic chagasic patients. Genomic DNA of 300 subjects from an endemic area was extracted and analyzed by PCR using specific primers. TNF-alpha was assayed in culture supernatants by ELISA. An association was observed between the absence of the TNF-238A allele and negative serology. Furthermore, seropositive individuals carrying the TNF-238A allele produced significantly higher TNF-alpha levels without stimulation (p = 0.04) and after stimulation with LPS (p = 0.007) and T. cruzi antigens (p = 0.004). The present results suggest that the polymorphism at position -238 influences susceptibility to infection and that this allele is associated with higher TNF-alpha production in seropositive individuals

Diagnostic Accuracy and Applicability of a PCR System for the Detection of Schistosoma mansoni DNA in Human Urine Samples from an Endemic Area

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA