Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M. tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens.

Hematological and serum chemistry values for the ring-necked pheasant (Phasianus colchicus): Variation with sex and age

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part I: Clinical evaluation, hematology and myelogram

The present study aimed at evaluating clinical and laboratory aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. The clinical signs of ataxia, sedation, flaccid paralysis, mydriasis, eyeball paralysis, mandible ptosis, sialorrhea, vomiting and diarrhea observed in the dogs were very similar to those observed in humans. The decrease in hemoglobin, hematocrit, erythrocyte, platelet and fibrinogen levels, prolongation of clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as hypocellularity in the bone marrow characterized anemia, thrombocytopenia and blood incoagulability, as well as hypofibrinogenemia and decreased bone-marrow activity. Important bleeding was not observed. Increased numbers of leukocytes and neutrophils and decreased numbers of lymphocytes and eosinophils characterized an acute inflammatory response and stress caused by generalized pain. The employed antiophidic serum was effective and all animals survived.

Aspirado traqueal de cavalos clinicamente sadios da raça quarto de milha após prova de três tambores

The aim of the present study was to evaluate through endoscopy the tracheal aspiration cytology in twenty seven Quarter Horses from Curitiba and surroundings, following the Three Barrel Competittion. Upper respiratory tract secretion was obtained by tracheal aspiration using a polyethylene catheter introduced through the endoscopic fiberoptic working channel, at the level of tracheal bifurcation. Cytologic slides were prepared by smear and stained by diff-quick technique and the differential was performed in 500 cells counting by 1,000X optic microscopy. None of the horses presented abnormality, including epixtasis, at the clinical examination. However, hemosiderophages were detected at cytology in three animals, suggesting that some may be suffering of subclinical pulmonary hemorrhage. Differential cell counting of tracheal aspiration results were, in average: 44.09 ± 35.68% of epithelial cells; 1.10 ± 2.18% of Globet cells; 23.10 ± 35.93% of neutrophils; 0.13 ± 0.37% of lymphocytes; 0.91 ± 2.81% of eosinophils; 30.57 ± 23.62% of macrophages and 0.13 + 0.93% of hemosiderophages. In conclusion, based in the present study, the evaluation of cellular populations with the tracheal aspiration may offer important additional information to the clinician, particularly about the inflammatory processes of lower respiratory tract and pulmonary bleeding.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

Diferentes fontes proteicas em rações de leitões sobre atividade da tripsina e parâmetros sangüíneos

Were used 64 piglets submitted eight treatments: ration with skim milk (SM), three rations with crescent levels of swine plasma (SP), three rations with whole egg (WE) and a ration with high inclusion of soybean meal (SB). Were monitored the blood parameters (BP) in pigs at 27 and at 34 days of age. The piglets were slaugther at 28 and at 35 days of age, for collections pancreas and posterior mensurements of absolut (AW) and relative weigth (RW) of pancreas and trypsin activity (TA). Treatments not influencied AW and TA. Significant effect of the crescent levels was verified of SP, with lineal reduction of the leukocytes and increase of the globular volume, to the 27 days; while to the 34 days, lineal increase of the hematias was observed. At 27 days, animals feds rations with crescent levels of SP have inferior percentage of eosinophils than others that consumed crescent levels of WE. The utilization of SP promoted smaller stimulus to the immune reply, while the use of WE promoted larger humoral reply of the piglets.

Hemoglobin polymorphism and hematological profile of Geoffroy's side-necked turtle (Phrynops geoffroanus, Testudines) in the northwestern region of São Paulo State, Brazil

Complete blood counts and hemoglobin isoform data were gathered from 36 specimens of the turtle species Phrynops geoffroanus from the northwestern region of São Paulo State, Brazil. They were collected in an urban area. The hemoglobin profiles were obtained after red blood cell lysis and by electrophoretic migration in alkaline pH, acid pH, and neutral pH buffer. The hemoglobin components were confirmed using high-performance liquid chromatography (HPLC). Erythrogram analysis included hematocrit, total hemoglobin concentration, total red blood cell count, and red blood cell indices. The leukogram included a total white blood cell count and a calculation of the percent values of neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils, and azurophils. HPLC analysis revealed three hemoglobin components; the first with a concentration of 5.5%, the second was a major component with an average concentration of 67.1%, and the third with a concentration of 28.5%. The hematological profile obtained for these specimens allowed us to establish a pattern for P. geoffroanus in São Paulo State Northwestern region. The average hematocrit values were 22.5% for females and 24.0% for males. For total hemoglobin, we found average values of 6.66 g/dL in females and 7.22 g/dL in males. The number of white blood cells was 2725 x 103/μL for females and 2775 x 103/μL for males. There was a predominance of heterophils, eosinophils, and monocytes in both sexes. No significant differences were found between males and females for hematological profile. The hematological results were compared to literature data for other Chelonia. They were similar to what is known for fresh water turtles. © FUNPEC-RP.

Uso de marcadores parasitológicos e imunológicos na seleção de ovelhas resistentes às parasitoses gastrintestinais

This work was conducted to verity the possibility to identify adult sheep that are resistant to parasites by using a parasitological marker (direct), immunological markers (indirect) or by the association of both types of markers. Twenty ewes were sampled monthly for blood and faeces, from July of 1998 to June of 1999. Faecal egg counts (FEC) was chosen as parasitological marker. The number of peripheral eosinophils, IgE and IgG anti-Haemonchus contortus were used as immunological markers. Sheep could be classified as resistant or susceptible by FEC. Both peripheral eosinophils and specific IgE data could be joined to FEC in order to identify resistant or susceptible animals. The number of peripheral eosinophils was the only immunological maker that was able to classify high and low FEC ewes in two different groups.

Glicerina bruta na dieta de bovinos de corte confinados: Efeito sobre o hemograma

The aim of this study was to evaluate the hemogram in cattle fed crude glycerin. 30 Nellore steers, were used for blood sampling, which were done each 28 days. The animals were confined for 103 days and were fed with five diets containing 0, 7.5, 15, 22.5 and 30% crude glycerin based on dry matter, formulated in a forage:concentrate ratio of 30:70. Corn silage was used as forage and the concentrate was composed by corn grain, soybean hulls, sunflower meal, glycerin, limestone, dicalcium phosphate and salt. The experimental design was a randomized block with five treatments. Orthogonal contrasts were used to determine the linear, quadratic, cubic effects and gliceryn treatments × the control one. There was no treatment effect (P>0,05) on blood concentrations of erythrocytes, hematocrit, basophils, eosinophils, band neutrophils and lymphocytes, but were linearly effect on the concentrations of platelets, hemoglobin and monocytes (P=0.009, P=0.001 and P=0.043, respectivelly), and cubic effect on the concentrations of total leukocytes and segmented neutrophils (P=0.008 and P=0.004, respectivelly). Diets for beef cattle, with forage:concentrate ratio of 30:70 added with crude glycerin promote changes in the erytrhogram and leucogram of these animals.

Comparison of three molecular typing methods to assess genetic diversity for Mycobacterium tuberculosis

This study describes the comparison of three methods for genotyping of Mycobacterium tuberculosis, namely MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats), spoligotyping and, for the first time, MLST (Multilocus Sequence Typing). In order to evaluate the discriminatory power of these methods, a total of 44 M. tuberculosis isolates obtained from sputum specimens of patients from Brazil were genotyped. Among the three methods, MLST showed the lowest discriminatory power compared to the other two techniques. MIRU-VNTR showed better discriminatory power when compared to spoligotyping, however, the combination of both methods provides the greatest level of discrimination and therefore this combination is the most useful genotyping tool to be applied to M. tuberculosis isolates. © 2013 Elsevier B.V.

Is peripheral blood cell balanced altered by the use of fresh frozen bone block allografts in lateral maxillary ridge augmentation?

Background: The relationship between the immune response and red and white blood cell homeostasis is cited in literature, but no studies regarding the balance of these cell populations following maxillary bone-graft surgeries can be found. Aim: The aim of this study was to evaluate the possible impairments in the blood cell balance following fresh-frozen allogeneic bone-graft augmentation procedures in patients who needed maxillary reconstruction prior to implants. Material and Methods: From 33 patients elected to onlay bone grafting procedures, 20 were treated with fresh-frozen bone allografts and 13 with autologous bone grafts. Five blood samples were collected from each patient in a 6-month period (baseline: 14, 30, 90, and 180 days postsurgery), and the hematological parameters (erythrogram, leukogram, and platelets count) were accessed. Results: All evaluated parameters were within the reference values accepted as normal, and significant differences were found for the eosinophils count when comparing the treatments (30 days, p=.035) and when comparing different periods of evaluation (allograft-treated group, baseline×180 days, p≤.05 and 90×180 days, p≤.01; autograft-treated group, 30×90 days, p≤.05 and 30×180 days, p≤.05). Conclusions: Both autologous and fresh-frozen allogeneic bone grafts did not cause any impairment in the red and white blood cell balance, based on quantitative hemogram analysis, in patients subjected to maxillary reconstruction. © 2011 Wiley Periodicals, Inc.

Evaluation of the biocompatibility of silicone gel implants - histomorphometric study

Breast implants are medical devices that are used to augment breast size or to reconstruct the breast following mastectomy or to correct a congenital abnormality. Breast implants consist of a silicone outer shell and a filler (most commonly silicone gel or saline). Approximately 5 to 10 million women worldwide have breast implants. Histomorphometric study to evaluate the biological tissue compatibility of silicone implants suitable for plastic surgery and the adverse effects and risks of this material. Thirty Wistar white rats received subcutaneous implants and the revestiment of silicone gel Silimed ®®, and randomized into six groups of five animals each, according to the type of implanted material and the time of sacrifice. Eight areas of 60.11mm2 corresponding to the obtained surgical pieces were analyzed, counting mesenchymal cells, eosinophils, and foreign body giant cells, observing an acceptable biocompatibility in all implants, for subsequent statistical analysis by Tukey test. Silicone gel showed inflammation slightly greater than for other groups, with tissue reactions varying from light to moderate, whose result was the formation of a fibrous capsule around the material, recognized by the organism as a foreign body. Despite frequent local complications and adverse outcomes, this research showed that the silicone and top layer presented an acceptable chronic inflammatory reaction, which did not significantly differ from the control group. In general, it is possible to affirm that silicone gel had acceptable levels of biocompatibility, confirmed the rare presence of foreign body giant cells, and when of the rupture, formed a fibrous capsule around the material, separating the material of the organism. © AVICENA 2013.

Effects of active smoking on airway and systemic inflammation profiles in patients with chronic obstructive pulmonary disease

Background: The markers that characterize local and systemic inflammation in chronic obstructive pulmonary disease (COPD) remain unclear, as do their correlations with smoking status and presence of disease. The aim of this study was to assess markers of inflammation in the peripheral blood and airways of current smokers without COPD, of current smokers with COPD and of ex-smokers with COPD. METHODS: In this study, 17 current smokers with COPD (mean age: 58.2 ± 9.6 years; mean forced expiratory volume in 1 second [FEV1]: 56.1 ± 15.9%), 35 ex-smokers with COPD (mean age: 66.3 ± 7.3 years; mean FEV1: 47.9 ± 17.2%) and 20 current smokers without COPD (mean age: 49.1 ± 6.2 years; mean FEV1: 106.5 ± 15.8%) were evaluated. Spirometry findings, body composition and serum/induced sputum concentrations of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-8 and IL-10, together with serum C-reactive protein (CRP) levels, were assessed. RESULTS: Serum TNF-α concentration was higher in all current smokers than in ex-smokers with COPD. In current smokers without COPD, serum CRP level was lower than in ex-smokers with COPD and significantly lower than in current smokers with COPD. Sputum TNF-α concentration was higher in current and ex-smokers with COPD than in current smokers without COPD. Multiple regression analyses showed that serum TNF-α was associated with active smoking, and serum CRP and sputum TNF-α were associated with COPD diagnosis. CONCLUSIONS: Smoking is associated with higher systemic inflammation in patients with COPD. Current findings also support the hypothesis that smoking and COPD have different effects on the regulation of airway and systemic inflammatory processes. © 2013 Lippincott Williams and Wilkins.

Resistance of beef cattle of two genetic groups to ectoparasites and gastrointestinal nematodes in the state of São Paulo, Brazil

The resistance to infestations by ectoparasites and infections by gastrointestinal nematodes was studied in 45 animals (males and females) of two genetic groups: purebred Nelore (NI, n=28) and Three-Cross (1/2 Angus+1/4 Canchim+1/4 Nelore - TC, n=17). The animals were monitored for 24months, during which they were left to graze in tropical pastures without receiving treatment for parasites. Each month the animals were examined for infestations by external parasites, to count the numbers of cattle ticks Rhipicephalus microplus with diameter greater than 4.5mm present on the left side, horn flies (Haematobia irritans) present in the lumbar region and botfly larvae (Dermatobia hominis) present on the entire body. The H. irritans counts were performed with the aid of digital photographs. At the time of examination, fecal samples were collected to count the eggs per gram (EPG) and to perform coprocultures, and peripheral blood samples were drawn to determine the packed cell volume (PCV) and to count the eosinophils. For statistical analysis, the count data were transformed into log10 (n+1), where n is the number of parasites. For PCV, significant effects (P<0.05) were found for collection month (CO), genetic group (GG) and gender (SX), with means and respective standard errors of 41.5±0.65% for the NI animals, 39.3±0.83% for the TC, 41.5±0.72% for the females and 39.3±0.77% for the males. Regarding the eosinophil counts, only the effect of sex was significant (P<0.01), with means and respective standard errors of 926.0±46.2/μL, for males and 1088.0±43.8/μL of blood, for females. The NI animals presented lower mean counts for all the external parasites compared to the TC animals (P<0.01). For ticks, the transformed means followed by standard errors for the NI and TC animals were 0.06±0.01 and 0.34±0.02, while for horn flies these were 0.92±0.05 and 1.36±0.06 and for botfly larvae they were 0.05±0.03 and 0.45±0.05, respectively. The average EPG values were only influenced by CO (P<0.01). The coprocultures revealed the presence of the following endoparasites: Haemonchus spp., Cooperia spp., Oesophagostomum spp. and Trichostrongylus spp., the last in smaller proportion. There were no significant differences between the genetic groups for the endoparasite loads, except for Cooperia spp., which were present in greater number (P<0.05) in the NI group. The results obtained in this experiment confirm previous findings of greater susceptibility of the Nelore breed to Cooperia spp. and high resistance to ectoparasites. © 2013 Elsevier B.V.

Perfil de cortisol, glicemia e de parâmetros sanguineos de girinos de rã-touro, Rana catesbeiana, em diferentes densidades e após exposição aérea

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)