Induced sputum as an adequate clinical specimen for the etiological diagnosis of community-acquired pneumonia (CAP) in children and adolescents.

OBJECTIVES: This study aimed to evaluate the utility of induced sputum (IS) for the diagnosis of community-acquired pneumonia (CAP) in pediatric population. METHODS: This cross-sectional study included pediatric population aged between 1 month and 17 years who were hospitalized with a diagnosis of CAP in 13 hospitals in Colombia, in whom an IS sample was obtained. Gram staining, aerobic bacterial and mycobacterial culture tests, and polymerase chain reaction (PCR) for 6 atypical bacteria and 15 respiratory viruses were performed. We evaluated the quality of IS samples. RESULTS: IS samples were collected in 516 of 525 children included in this study. The median age was 32 months, 38.6% were younger than 2 years, and 40.9% were between 2 and 5 years. Two patients had transient hypoxemia during the procedure. The quality of the IS obtained was good in 48.4% and intermediate in 24.5%. Identification of a respiratory pathogen was achieved with an IS sample (with Gram staining, culture test, and PCR) in 372 of 516 children with CAP. CONCLUSION: Our study shows that IS is an adequate sample for the diagnosis of CAP in pediatric population that required hospitalization. The procedure was safe, well tolerated, and with better diagnostic yields compared with the rest of the samples obtained.

Effects of pre-treating in vitro-matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification.

The purpose of this study was to determine the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the open pulled Straw method (OPS). We evaluated the effects of pre-treating the oocytes with 1 microM Taxol on chromosome organization, spindle morphology, cortical granule distribution and the ability of fertilized oocytes to develop to the blastocyst stage. After calf or cow oocyte vitrification without Taxol, significantly higher proportions of spindle abnormalities in the form of abnormal spindle structures or dispersed or decondensed chromosomes were observed compared to fresh control oocytes. In contrast, when we compared calf oocytes pre-treated with Taxol before vitrification with control calf oocytes, similar percentages of oocytes showing a normal spindle morphology were observed. The percentages of oocytes with a peripheral cortical granule (CG) distribution increased when the oocytes were pretreated with Taxol and vitrified, while oocytes vitrified without Taxol pre-treatment gave rise to higher cortical distribution percentages. Cleavage and blastocyst rates were significantly lower for vitrified versus untreated oocytes, both in cow and calf oocytes. Significantly higher cleavage rates were obtained when calf and cow oocytes were vitrified with Taxol. Pre-treatment with Taxol before cow oocyte vitrification yielded significantly higher blastocyst rates. Calf oocytes, however, were unable to develop to the blastocyst stage, irrespective of previous Taxol treatment. These results indicate that the pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by the cryopreservation process, and potentially improves the subsequent development of vitrified bovine oocytes. Summary sentence: Pre-treatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and potentially improves the development of vitrified bovine oocytes.

Vitrification of calf oocytes: effects of maturation stage and prematuration treatment on the nuclear and cytoskeletal components of oocytes and their subsequent development.

This study was designed to establish the effects of the meiotic stage of bovine oocytes and of a prematuration treatment with roscovitine (ROS) on their resistance to cryopreservation. Oocytes from prepubertal calves at the stages of germinal vesicle breakdown (GVBD) or at metaphase II (MII) were vitrified by the open pulled straw (OPS) method. In another experiment, oocytes were kept under meiotic arrest with 50 microM ROS for 24 hr and vitrified at the GVBD stage. After warming, some oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. Significantly lower cleavage rates were obtained for the vitrified GVBD and MII oocytes (9.9% and 12.6%, respectively) compared to control oocytes (73.9%). Significantly worse results in terms of cleavage rates were obtained when GVBD calf oocytes were exposed to cryoprotectants (CPAs: ethylene glycol plus dimethyl sulfoxide, DMSO) (13.1%) or vitrified (1.6%) after a prematuration treatment with ROS, when compared to untreated control oocytes (68.7%) or ROS-control oocytes (56.6%). None of the vitrification procedures yielded blastocysts, irrespective of the initial meiotic stage or previous prematuration treatment. Compared to the control oocytes, significantly fewer oocytes exhibited normal spindle configuration after being exposed to CPAs or after vitrification of either GVBD or MII calf oocytes. These results indicate that the vitrification protocol has a deleterious effect on the meiotic spindle organization of calf oocytes cryopreserved at both the GVBD and MII stage, which impairs the capacity for further development of the embryos derived from these vitrified oocytes. Prematuration treatment with ROS has no beneficial effect on the outcome of vitrification by the OPS method.

Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development.

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 microM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 microM (2.9%) and 25 microM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 microM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.

Effects of vitrification in open pulled straws on the cytology of in vitro matured prepubertal and adult bovine oocytes.

This study was designed to evaluate the effects of the cryopreservation of oocytes obtained from prepubertal calves or adult cows on chromosome organization, spindle morphology, cytoskeleton structures, and the ability of fertilized oocytes to develop to the blastocyst stage. Once in vitro matured (IVM), the oocytes were divided into three groups according to whether they were: (1) left untreated (control); (2) exposed to cryoprotectant agents (CPAs); or (3) cryopreserved by the open-pulled-straw (OPS) vitrification method. After thawing, oocyte samples were fixed, stained using specific fluorescent probes and examined under a confocal microscope. The remaining oocytes were fertilized, and cleavage and blastocyst rates recorded. After vitrification or CPA exposure, significantly higher proportions of oocytes showed changes in spindle morphology compared to the control group. The spindle structure of the adult cow IVM oocytes was significantly more resistant to the OPS vitrification process. Vitrification of oocytes from calves or adult cows led to significantly increased proportions of oocytes showing discontinuous or null actin staining of the cytoskeleton compared to non-treated controls. Oocytes only exposed to the cryoprotectants showed a similar appearance to controls. A normal distribution of actin microfilaments was observed in both calf and adult cow oocytes, irrespective of the treatment. Cleavage and blastocyst rates were significantly lower for vitrified versus non-treated oocytes. Oocytes obtained from adult cows were more sensitive to CPA exposure, while the vitrification procedure seemed to have more detrimental effects on the calf oocytes.

Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments.

Studies were conducted to compare viability of immature and mature porcine oocytes vitrified in ethylene glycol (EG) using open-pulled straws (OPS). Oocytes that had been allowed to mature for 12 h (germinal vesicle group; GV) and 40 h (metaphase II group; MII) were divided into three treatments: (1) control; (2) treated with cytochalasin B and exposed to EG; and (3) treated with cytochalasin B and vitrified by stepwise exposure to EG in OPS. After warming, a sample of oocytes was fixed and evaluated by specific fluorescent probes before visualization using confocal microscopy. The remaining oocytes were fertilized and cleavage rate was recorded. Exposure of GV oocytes to EG or vitrification had a dramatic effect on spindle and chromosome configurations and no cleavage was obtained after in vitro fertilization. When MII oocytes were exposed to EG or were vitrified, 18 and 11% of oocytes, respectively, maintained the spindle structure and either EG exposure or vitrification resulted in substantial disruption in microfilament organization. The cleavage rates of mature oocytes after being exposed to EG or after vitrification were similar (14 and 13%, respectively) but were significantly less than that of control oocytes (69%). These results indicate that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII porcine oocytes had better resistance to cryopreservation than GV stage oocytes.