Use of molecular methods in identification of Candida Species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.

Antimicrobial susceptibility, phage types, and pulsetypes of Salmonella Typhimurium, in São Paulo, Brazil

A total of 283 Salmonella Typhimurium strains isolated from cases of human infections and non human sources, were examined for antimicrobial susceptibilityand the incidence of resistance was 38% and multiple resistance (to three or more antimicrobials) was 15%. All 43 multidrug-resistant strains (MDR) and 13 susceptible ones were characterized by phage typing and pulsed- field gel electrophoresis (PFGE). The strains encompassed 14 definitive phage types (DT), three were untypable (UT), and 18 atypicals or reaction does not conform (RDNC), which belonged to 21 PFGE patterns, A1-A21. The predominant phage types were DT49, DT193, and RDNC and two strains belonging to DT 104 and 104b were also identified. The most commum PFGE patterns were A1 and A8. Analysis by PFGE and phage typing demonstrated that the most of the MDR were multiclonal and association among multiresistance, phage typing, and PFGE patterns was not so significant.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Single and concomitant experimental infectionsby Endotrypanum spp. and Leishmania (Viannia) guyanensis (Kinetoplastida: Trypanosomatidae) in the Neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)

Lutzomyia longipalpis females received single and mixed infections with Endotrypanum and Leishmania. Two biological parameters were analyzed: the percentage of infected females and the distribution of flagellates in the gut of the females. The principal comparisons were performed between (1) two strains of Endotrypanum, (2) cloned versus primary sample of one strain of Endotrypanum, (3) Endotrypanum versus Leishmania guyanensis, and (4) the pattern of flagellates behaviour by optical microscopy in females with single or mixed infection versus the identification of parasites isolated from digestive tracts by isoenzyme electrophoresis. Flagellates of Endotrypanum showed distinct patterns of infection suggesting that there is variation between and within strains. The distribution of Endotrypanum and L. guyanensis differed significantly in relation to the colonization of the stomodeal valve. In co-infection with L. guyanensis, a large number of flagellates were seen to be plentifully infecting the stomodeal valve in significantly more specimens than in females infected by Endotrypanum only. However, the electrophoretic profiles of isoenzymes of parasites recovered from all co-infected specimens corresponded to Endotrypanum. This suggests that the mere correlation sand fly infection-biochemical analysis of isolates may induce parasitological incorrect consideration.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Antibacterial and antifungal activity of extracts and exudates of the Amazonian medicinal tree Himatanthus articulatus (Vahl) Woodson (common name: sucuba)

Himatanthus articulatus (Vahl) Woodson is a tree found in the northern Amazon savannahs (common name: sucuba) that is used in local Amerindian medicine. Leaf, bark and branch wood methanol extracts, sequentially obtained hexane, ethyl acetate and methanol extracts and latex were evaluated for antifungal and antibacterial activities against American Type Culture Collection (ATCC) and local clinical strains using the disc diffusion method. Methanol extracts and latex inhibited Candida albicans, leaf methanol extracts inhibited Staphylococcus aureus and Bacillus subtilis and bark methanol extracts inhibited B. subtilis. Active extracts inhibited the ATCC and clinical strains. Polar antifungal and antibacterial principles in latex and extracts are thought to be responsible for the inhibition.

Susceptibility of Biomphalaria straminea from Peixe Angical dam, Tocantins, Brazil to infection with three strains of Schistosoma mansoni

Environmental changes from water resource developmental projects affect the epidemiology of water-associated diseases, as well as malaria and schistosomiasis. Aiming to investigate the occurrence and distribution of freshwater snails of medical and veterinary importance in the area of influence of the Peixe Angical hydroelectric dam, a survey has been conducted over four years (2004-2008). The study has revealed the occurrence of populations of Biomphalaria straminea (Dunker) in all municipalities surrounding the lake. Studies on parasite-mollusc compatibility were undertaken using 35 populations of B. straminea, descendants of specimens obtained from that area and three strains of Schistosoma mansoni (Sambon) (BH, CM and CMO). The main results are as follows: (i) among the 1,314 specimens used, eight had been infected (infection index of 0.6%) with only the BH strain, (ii) for B. straminea populations, the mortality index was 6.8% and, depending on the strain used, the indexes were 4.6%, 8.49% and 19% with BH, CM and CMO strains, respectively, (iii) the infection indexes varied according to the B. straminea populations, ranging from 0-12.5% and (iv) the duration of the precercarial period varied from 25-49 days. These results, in addition to environmental and social changes that took place in the Peixe Angical dam region, indicate the possibility of B. straminea emerging as a schistosomiasis vector in this area.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9%) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9%), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3%), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix® vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni

Molecular techniques can aid in the classification of Biomphalaria species because morphological differentiation between these species is difficult. Previous studies using phylogeny, morphological and molecular taxonomy showed that some populations studied were Biomphalaria cousini instead of Biomphalaria amazonica. Three different molecular profiles were observed that enabled the separation of B. amazonica from B. cousini. The third profile showed an association between the two and suggested the possibility of hybrids between them. Therefore, the aim of this work was to investigate the hybridism between B. cousini and B. amazonica and to verify if the hybrids are susceptible to Schistosoma mansoni. Crosses using the albinism factor as a genetic marker were performed, with pigmented B. cousini and albino B. amazonica snails identified by polymerase chain reaction-restriction fragment length polymorphism. This procedure was conducted using B. cousini and B. amazonica of the type locality accordingly to Paraense, 1966. In addition, susceptibility studies were performed using snails obtained from the crosses (hybrids) and three S. mansoni strains (LE, SJ, AL). The crosses between B. amazonica and B. cousini confirmed the occurrence of hybrids. Moreover, hybrids can be considered potential hosts of S. mansoni because they are susceptible to LE, SJ and AL strains (4.4%, 5.6% and 2.2%, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

The TcI and TcII Trypanosoma cruzi experimental infections induce distinct immune responses and cardiac fibrosis in dogs

Trypanosoma cruzi infection may be caused by different strains with distinct discrete typing units (DTUs) that can result in variable clinical forms of chronic Chagas disease. The present study evaluates the immune response and cardiac lesions in dogs experimentally infected with different T. cruzi strains with distinct DTUs, namely, the Colombian (Col) and Y strains of TcI and TcII DTU, respectively. During infection with the Col strain, increased levels of alanine aminotransferase, erythrocytes, haematocrit and haemoglobin were observed. In addition, CD8+ T-lymphocytes isolated from the peripheral blood produced higher levels of interleukin (IL)-4. The latter suggests that during the acute phase, infection with the Col strain may remain unnoticed by circulating mononuclear cells. In the chronic phase, a significant increase in the number of inflammatory cells was detected in the right atrium. Conversely, infection with the Y strain led to leucopoenia, thrombopoenia, inversion of the ratio of CD4+/CD8+ T-lymphocytes and alterations in monocyte number. The Y strain stimulated the production of interferon-γ by CD4+ and CD8+ T-lymphocytes and IL-4 by CD8+ T-cells. In the chronic phase, significant heart inflammation and fibrosis were observed, demonstrating that strains of different DTUs interact differently with the host.

Isolation and characterization of two plant growth-promoting bacteria from the rhizoplane of a legume (Lupinus albescens) in sandy soil

Two bacterial strains that amplified part of the nifH gene, RP1p and RP2p, belonging to the genus Enterobacter and Serratia, were isolated from the rhizoplane of Lupinus albescens. These bacteria are Gram-negative, rod-shaped, motile, facultative anaerobic, and fast-growing; the colonies reach diameters of 3-4 mm within 24 h of incubation at 28 ºC. The bacteria were also able to grow at temperatures as high as 40 ºC, in the presence of high (2-3 % w/v) NaCl concentrations and pH 4 -10. Strain RP1p was able to utilize 10 of 14 C sources, while RP2p utilized nine. The isolates produced siderophores and indolic compounds, but none of them was able to solubilize phosphate. Inoculation of L. albescens with RP1p and RP2p strains resulted in a significant increase in plant dry matter, indicating the plant-growth-promoting abilities of these bacteria.

Symbiotic effectiveness and ecological characterization of indigenous Rhizobium loti populations in Uruguay

The objectives of this work were to describe the distribution, density and seasonal variation of the indigenous populations of Rhizobium loti in different Uruguayan soils and to determine the symbiotic effectiveness and stress tolerance factors of different isolates, both with the aim of obtaining selected strains to re-introduce as inoculants in Lotus pastures. R. loti was present in ten soils studied and their densities varied from year to year and within each soil. All the isolates nodulated Lotus corniculatus effectively. The nodules in Lotus pedunculatus and Lotus subbiflorus were small, red on the surface and ineffective in nitrogen fixation. The study of 50 isolates from the ten soils showed high variability in their symbiotic efficiency and tolerance to pH. The indigenous population was acid tolerant in culture medium (pH 4.5), 83% of them could grow at pH 4.5 in 3 days. This work showed that there was a great diversity between the strains of R. loti isolated from Uruguayan soils and supports the importance of selecting among them the most efficient and resistant strains to be included in the inoculants.

Mineral and vitamin content of beef, chicken, and turkey hydrolysates mineral and vitamin content of protein hydrolysates

The purpose of this study was to assess the concentration of vitamins and minerals in meat protein hydrolysates. Calcium, phosphorus and iron were analyzed by inductively coupled-plasma atomic emission spectrophotometry; vitamin C was analyzed by the reduction of cupric ions and vitamins B1 and B2 by fluorescence. Regarding minerals, the beef hydrolysate (BH) had more iron than the turkey hydrolysate (TH) and the chicken hydrolysate (CH); TH had a little more phosphorus. BH had the largest amount of vitamin C, and similar amounts of vitamins B1 and B2. The amount of these nutrients found in the hydrolysates suggests that it is possible to use them to enrich special dietary formulations.