Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Urinary tract infection in full-term newborn infants: value of urine culture by bag specimen collection

OBJETIVE: to evaluate the efficacy of urine culture by bag specimen for the detection of neonatal urinary tract infection in full-term newborn infants. Retrospective study (1997) including full-term newborn infants having a positive urine culture (>100,000 CFU/ml) by bag specimen collection. The urinary tract infection diagnosis was confirmed by positive urine culture (suprapubic bladder aspiration method). The select cases were divided into three groups, according to newborn infant age at the bag specimen collection: GI (< 48 h, n = 17), GII (48 h to 7 d, n = 35) and GIII (> 7 d, n = 9). Sixty one full-term newborn infants were studied (5.1 % of total infants). The diagnosis was confirmed on 19/61 (31.1 %) of full-term infants born alive. Distribution among the groups was: GI = 2/17 (11.8 %), GII = 10//35 (28.6 %), and GIII = 7/9 (77.7 %). The most relevant clinical symptoms were: fever (GI - 100 %, GII - 91.4 %) and weight loss (GI - 35.3 %, GII - 45.7 %). Urine culture results for specimens collected by suprapubic aspiration were: E. coli GI (100 %), GII (40 %) and GIII (28.6 %), E. faecalis GI (30%), Staphylococcus coagulase-negative GII (20 %) and GIII (42.8 %), and Staphylococcus aureus GII (10 %). Correlation between positive urine culture collection (bag specimen method) and urinary tract infection diagnosis, using relative risk analysis, produced the following results: GI=0.30 (CI95% 0.08-1.15), GII=0.51 (CI 95% 0.25-1.06) and GIII=3.31 (CI95% 1.8-6.06) The most frequent urinary tract infection clinical signs in the first week were fever and weight loss, while non-specific symptomatology occurred later. E. coli was most frequent infectious agent, although from the 7th day of life, staphylococcus was noted. The urine culture (bag specimen method) was effective in detecting urinary tract infection only after the 7th day of life.

Urinary tract infection in full-term newborn infants: risk factor analysis

OBJECTIVE: To analyze the correlation of risk factors to the occurrence of urinary tract infection in full-term newborn infants. PATIENTS AND METHODS: Retrospective study (1997) including full-term infants having a positive urine culture by bag specimen. Urine collection was based on: fever, weight loss > 10% of birth weight, nonspecific symptoms (feeding intolerance, failure to thrive, hypoactivity, debilitate suction, irritability), or renal and urinary tract malformations. In these cases, another urine culture by suprapubic bladder aspiration was collected to confirm the diagnosis. To compare and validate the risk factors in each group, the selected cases were divided into two groups: Group I - positive urine culture by bag specimen collection and negative urine culture by suprapubic aspiration, and Group II - positive urine culture by bag specimen collection and positive urine culture by suprapubic aspiration . RESULTS: Sixty one infants were studied, Group I, n = 42 (68.9%) and Group II, n = 19 (31.1%). The selected risk factors (associated infectious diseases, use of broad-spectrum antibiotics, renal and urinary tract malformations, mechanical ventilation, parenteral nutrition and intravascular catheter) were more frequent in Group II (p<0.05). Through relative risk analysis, risk factors were, in decreasing importance: parenteral nutrition, intravascular catheter, associated infectious diseases, use of broad-spectrum antibiotics, mechanical ventilation, and renal and urinary tract malformations. CONCLUSION: The results showed that parenteral nutrition, intravascular catheter, and associated infectious diseases contributed to increase the frequency of neonatal urinary tract infection, and in the presence of more than one risk factor, the occurrence of urinary tract infection rose up to 11 times.

A possible contribution to improving the therapeutic potentials of Babor's Typology of Alcohol Dependent Patients

ABSTRACT Objective The objective of this study was to replicate Babor's Typology and to explore clinical features related to personality traits that may underlie this classification, in order to improve its therapeutic possibilities. Methods Observational prospective study on a group of 273 male alcoholics. After a replication of Babor's variables, Cluster Analysis, Chi-Square – applied on clinical variables related to a Lappda Tipology – and Kappa tests were performed. Results The study identified two distinct clusters that held similar features to those described for the Type A/Type B classification. Besides presenting a lower socio-economic situation, Cluster 2 patients were associated with higher vulnerability and severe clinical features and also differed from Cluster 1 in their response to treatment. These replicated clusters retained connections and also differences in relation to the variables derived from the Lappda Typology. Conclusion Considering that each of the two replicated clusters seem to be associated to different personality traits – according to their correlations to the affective, cognitive and behavioral dimensions brought forward by the Lappda Typology – it is acceptable that this study may contribute to the development of more comprehensive and effective therapeutic strategies specifically tailored to target more specific personality traits of these subgroups of alcoholic patients.

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Differential effect of culture epimastigotes and blood-form Trypamastigotes on normal mouse splenocyte responsiveness to mitogens

Blood form trypomastigotes of the Y strain of T. cruzi, produced a strong inhibition of the blastogenic response to T and B cell mitogens, of the C3H/He, C57BLand BALB/cJ strains of mice, while culture epimastigotes of the Y strain kept in a medium that allows parasite growth at 26°. 30° and 37°C produced a strong stimulatory effect that was even higher than the effect of the mitogens alone. Both the inhibitory or the stimulatory effects were dose-dependent. The stimulatory effect of epimastigotes was also temperature-dependent producing increasedstimulation indexes as the temperature of parasite cultures was raised. Metabolically active,living parasites seemed to be necessary for an improved lymphocyte stimulation suggesting a potential role of secreted metabolites as polyclonal activators of mouse lymphocytes.

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/lOJ) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.

Ultrastructural and stereological analysis of trypanosomatidis of the genus Endotrypanum

Culture forms of four strains of Endotrypanum (E. schaudinni and E. monterogeii) were processed for transmission electron microscopy and analyzed at the ultrastructural level. Quantitative data about some cytoplasmic organelles were obeined by stereology. All culture forms were promastigotes. In their cytoplasm four different organelles could be found: lipid inclusions (0,2-0,4 µm in diameter), mebrane-bounded vacuoles (0.10-0,28 µm in diameter), glycosomes (0,2-0,3 µm in diameter), and the mitochondrion. The kenetoplast appears as a thin band, except for the strain IM201, which possesses a broader structure, and possibly is not a member of this genus. Clusters of virus-like particles were seen in the cytoplasm of the strain LV88. The data obtained show that all strains have the typical morphological feature of the trypanosomatids. Only strain IM201 could be differentiated from the others, due to its larger kenetoplast-DNA network and its large mitochondrial and glycosomal relative volume. The morphometrical data did not allow the differentiation between E. schaudinni (strains IM217 and M6226) and E. monterogeii (strain LV88).

Comparative chromatin analysis of Trypanosoma congolense

The chromatin of Trypanosoma congolense was analyzed by electron microscopy. The chromatin is organized as nucleosome filaments but does not form a 30 nm fiber. There are five groups of histones, including a histone H1-like protein, which has a molecular weight within the range of the core histones, and is extremely hydrophilic. Weak histone-histone interaction, a typical feature of trypanosoma chromatin, was found. These results are similar to those for T. cruzi and T. b. brucei, but differ significantly from those for higher eukaryotes. The results confirm the model of trypanosome chromatin, and support the theory of their early separation from the other eukaryotes during the evolution. T. congolensis is an excellent model for chromatin research on trypanosomes, because it is easy to cultivate and its chromatin has, a relatively high stability, compared to that of other trypanosomes.

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi

The stabilizing effects of the acquired immunity on the schistosomiasis transmission modeling - the sensitivity Analysis

A mathematical model is proposed to analyze the effects of acquired immunity on the transmission of schistosomiasis in the human host. From this model the prevalence curve dependent on four parameters can be obtained. These parameters were estimated fitting the data by the maximum likelihood method. The model showed a good retrieving capacity of real data from two endemic areas of schistosomiasis: Touros, Brazil (Schistosoma mansoni) and Misungwi, Tanzania (S. haematobium). Also, the average worm burden per person and the dispersion of parasite per person in the community can be obtained from the model. In this paper, the stabilizing effects of the acquired immunity assumption in the model are assessed in terms of the epidemiological variables as follows. Regarded to the prevalence curve, we calculate the confidence interval, and related to the average worm burden and the worm dispersion in the community, the sensitivity analysis (the range of the variation) of both variables with respect to their parameters is performed.

Intestinal fibrovascular nodules caused by Schistosoma mansoni infection in Calomys callosus Rengger, 1830 (Rodentia: Cricetidae): a model of concomitant fibrosis and angiogenesis

Human schistosomiasis develops extensive and dense fibrosis in portal space, together with congested new blood vessels. This study demonstrates that Calomys callosus infected with Schistosoma mansoni also develops fibrovascular lesions, which are found in intestinal subserosa. Animals were percutaneously infected with 70 cercariae and necropsied at 42, 45, 55, 80, 90 and 160 days after infection. Intestinal sections were stained for brightfield, polarization microscopy, confocal laser scanning, transmission and scanning electron microscopies. Immunohistological analysis was also performed and some nodules were aseptically collected for cell culture. Numerous intestinal nodules, appearing from 55 up to 160 days after infection, were localized at the interface between external muscular layer and intestinal serosa, consisting of fibrovascular tissue forming a shell about central granuloma(s). Intranodular new vessels were derived from the vasculature of the external vascular layer and were positive for laminin, chondroitin-sulfate, smooth muscle alpha-actin and FVIII-RA. Fibroblastic cells and extracellular matrix components (collagens I, III and VI, fibronectin and tenascin) comprised the stroma. Intermixed with the fibroblasts and vessels there were variable number of eosinophils, macrophages and haemorrhagic foci. In conclusion, the nodules constitute an excellent and accessible model to study fibrogenesis and angiogenesis, dependent on S. mansoni eggs. The fibrogenic activity is fibroblastic and not myofibroblastic-dependent. The angiogenesis is so prominent that causes haemorrhagic ascites.

Natural Schistosoma mansoni infection in Nectomys squamipes: histopathological and morphometric analysis in comparison to experimentally infected N. squamipes and C3H/He mice

Histopathologic and morphometric (area, perimeter, major and minor diameters) analysis of hepatic granulomas isolated from twelve naturally infected Nectomys squamipes were compared to four experimentally infected ones and six C3H/He mice. Liver paraffin sections were stained for cells and extracellular matrix. Both groups of N. squamipes presented peculiar granulomas consisting predominantly of large macrophages, full of schistosome pigment, characterizing an exudative-macrophage granuloma type, smaller than the equivalent granuloma type in mouse. Naturally infected animals exhibited granulomas in different stages of development, including large number of involutional types. Morphometric analysis showed that all measurements were smaller in naturally infected animals than in other groups. The results demonstrated that both N. squamipes groups reproduced, with small variations, the hepatic granuloma aspects already described in cricetidium (Calomys callosus), showing a genetic tendency to set up strong macrophage responses and small granulomas. Unexpectedly, natural infection did not engender distinguished histopathological characteristics distinct from those derived from experimental single infection, showing changes predominantly secondary to the duration of infection. It appears that the variability of the inocula (and the number of infections?) interfere more with the quantity than with the quality of the pathological changes, denoting some morpho-functional determinism in the response to schistosomal infection dependent on the animal species.