Prophylactic application of antibiotics selects extended-spectrum ß-lactamase and carbapenemases producing Gram-negative bacteria in the oral cavity.

Prophylactic administration of broad-spectrum antibiotics in surgery can change the oral microbiome and induce colonization of oral cavity with Gram-negative bacteria including multidrug (MDR) or extensively drug resistant (XDR) organisms which can lead to lower respiratory tract infections. The aim of the study was to analyse the Gram-negative isolates obtained from oral cavity of the mechanically ventilated patients in ICUs, after prophylactic application of antibiotics and their resistance mechanisms and to compare them with the isolates obtained from tracheal aspirates from the same patients. The antibiotic susceptibility was determined by broth dilution method. PCR was applied to detect genes encoding ß-lactamases. Marked diversity of Gram-negative bacteria and resistance mechanisms was found. High resistance rates and high rate of blaCTX-M and carbapenemase encoding genes (blaVIM-1 , blaOXA-48 ) were found among Klebsiella pneumoniae. Pseudomonas aeruginosa was found to harbour blaVIM and in one strain blaPER-1 gene, whereas Acinetobacter baumannii produced OXA-23-like and OXA-24/40-like oxacillinases and was XDR in all except one case. All XDR isolates belong to international clonal lineage II (IC II). The main finding of the study is that the prophlylactic application of antibiotics in surgery intensive care units (ICUs) is associated with the colonization of oral cavity and lower respiratory tract with Gram-negative bacteria. The identity of Gram-negative bacteria in oral cavity reflected those found in endotracheal aspirates leading to conclusion that oral swab as non-invasive specimen can predict the colonization of lower respiratory tract with resistant Gram-negative organisms and the risk for development of ventilator-associated pneumonia.

[Malignant melanoma of the cornea after blunt trauma]. / Malignes Melanom der Kornea nach stumpfem Trauma.

We report the case of a 67-year-old woman who presented with a persisting corneal erosion after blunt injury. Six years later, a highly prominent corneal tumor had developed at the site of the initial erosion. Histological analysis revealed a malignant melanoma. This case provides evidence that malignant melanoma may be a long-term complication of corneal epithelial disorders.

Cytochrome c oxidase partial deficiency-associated Leigh disease presenting as an extrapyramidal syndrome.

Leigh disease is a subacute neurodegenerative disorder characterized by symmetric necrotic lesions in the basal ganglia, cerebellum, thalamus, brain stem, and optical nerves and caused by altered oxidative phosphorylation. We describe the clinical, biochemical, neuroimaging, and molecular studies of a 19-year-old boy with early-onset Leigh disease manifesting as severe extrapyramidal disorder with generalized dystonia and choreoathetosis. He was born of healthy parents after an uneventful pregnancy and delivery. At the age of 2 1/2 years, after a minor respiratory infection, he developed unstable, broad-based gait and tremor of the hands. These symptoms persisted for the next several years, when ataxia became more prominent. Difficulty in swallowing, dysarthria, trunk dystonia, and marked dyskinesia of the arms and hands gradually developed. Nystagmus, transient ptosis, and strabismus also appeared. Abnormal laboratory findings included elevated plasma and cerebrospinal fluid lactate and pyruvate, with an abnormal lactate/pyruvate ratio. Cranial computed tomography and magnetic resonance imaging demonstrated signs of cerebellar atrophy, bilateral and symmetric hypodensities in the lentiform nucleus and thalamus, and transient hyperintensities of cerebral peduncles in T2-weighted sequences suggestive of Leigh disease. Muscle biopsy revealed isolated fiber atrophy, necrotic fibers undergoing phagocytosis, and no ragged-red fibers. The measured catalytic activity of cytochrome c oxidase in skeletal muscle homogenates demonstrated a partial cytochrome c oxidase deficiency No abnormalities in the mitochondrial genome and in the SURF-1 gene were found. The boy is currently receiving levodopa therapy, creatine monohydrate, and a high dosage of thiamine and lipoic acid, his condition is stabilized, and extrapyramidal symptoms are less pronounced.

[The role of zinc in the initial treatment of Wilson's disease in children]. / Uloga cinka u inicijalnom lijecenju Wilsonove bolesti u djece.

Wilson's disease (WD) is an inherited disease of copper accumulation, caused by a failure of biliary excretion of excess copper. Accumulated copper causes tissue damage. The chelating drugs penicillamine and trientine have been the mainstay of therapy and most patients with WD were treated with the potentially toxic cupriuretic agents. A more recent approach has used zinc, which blocks the absorption of copper and increases copper excretion in the stool, and long term administration induces a negative copper balance. Until recently, most patients have been treated initially with cupriuretic agents to remove excess of copper, and then maintained with oral zinc. Recently, zinc has been used for initial treatment as well and for treatment of the presymptomatic patients. So far, zinc therapy has demonstrated exceptional efficacy and lack of toxicity. In this article we present our data on the long-term follow-up of three children with WD, whose initial as well as consecutive treatment was zinc sulphate. The results demonstrate the efficacy of zinc therapy in treating the presymptomatic patient and in initial treatment of symptomatic children with WD. Our data also indicate low toxicity. However, pediatric patients must be closely monitored due to tendency to stop the treatment when becoming asymptomatic.

[Primary adrenocortical micronodular dysplasia]. / Primarna adrenokortikalna mikronodularna displazija.

Two girls (11 and 13 years old) with Cushing's syndrome due to primary adrenocortical micronodular dysplasia (PAMD) are presented. High plasma cortisol concentrations, elevated urinary free cortisol and 17-ketogenic steroids excretion, in addition to low or normal plasma adrenocorticotropic hormone (ACTH) levels pointed towards independent adrenal cortisol hypersecretion. In both girls bilateral adrenalectomy was performed, followed by replacement therapy with glucocorticoids and mineralocorticoids. Pathohistological findings of otherwise enlarged adrenal glands, showed characteristic small nodules measuring 1-2 mm, composed of cells resembling those of zona fasciculata, with abundant, clear cytoplasm. Our younger patient fulfilled the criteria of "Carney complex", because beside PAMD she has had the lentigines.

C-series polysialogangliosides are expressed on stellate neurons of adult human cerebellum.

Until now 'c-series' polysialogangliosides were known to exist in human brain only during development and in some pathological conditions like Alzheimer's disease. Using thin-layer chromatography (TLC) and immunostaining with Q211 antibody (TLC-overlay technique) we have analysed 'c-series' gangliosides in four human cerebella (age 20, 47, 52 and 54 years). Four distinct ganglioside bands, most probably corresponding to GT1c, GQ1c, GP1c and GH1c were found to exist in the analysed brains, which is convincing demonstration of the existence of 'c-series' gangliosides in normal adult human brain. Immunohistochemical analysis was performed to locate polysialogangliosides in the analysed tissue. Q211 antibody was found to bind specifically to a single subpopulation of neurons in the molecular layer of adult cerebellum. According to their position and morphology these cells correspond to stellate neurons.

Vaginal yolk sac tumor in a nine-month-old female child.

A case of yolk sac tumor in a nine-month-old girl is presented. Vaginal hemorrhage was observed during the patient's check-up at the Bjelovar Children's Outpatient Clinic. The patient was referred to the Department of Pediatrics at the Zagreb University Hospital, where an endodermal tumor of the vaginal yolk sac was diagnosed. The patient underwent surgery, followed by chemotherapy. She is now 3.5 years old, in good general health and under regular control.

Choroidal leiomyoma of vascular origin.

A 38-year-old female patient was admitted for serious retinal detachment. Clinical examination revealed detachment in the lower half of the retina, with a suspect hemispherical tumorous formation. Echography did not positively demonstrate the presence of tumor. Vitrectomy was performed and a tumor fragment was taken for histologic analysis. The finding indicated the presence of leiomyoma choroideae, a very rare and benign smooth muscle tumor.

Prostatic polyp in the prostatic urethra.

The authors report on a case of prostatic polyp in the urethral bulb of a 35-year-old man. The presence of this lesion in the urethral bulb is quite unusual and may be overlooked on cystocopic evaluation.

Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography.

In this study the comparative TLC immunostaining investigation of neutral GSLs and gangliosides from human skeletal and heart muscle is described. A panel of specific polyclonal and monoclonal antibodies as well as the GM1-specific choleragenoid were used for the overlay assays, combined with preceding neuraminidase treatment of gangliosides on TLC plates. This approach proved homologies but also quantitative and qualitative differences in the expression of ganglio-, globo- and neolacto-series neutral GSLs and gangliosides in these two types of striated muscle tissue within the same species. The main neutral GSL in skeletal muscle was LacCer, followed by GbOse3Cer, GbOse4Cer, nLcOse4Cer and monohexosylceramide, whereas in heart muscle GbOse3Cer and GbOse4Cer were the predominant neutral GSLs beside small quantities of LacCer, nLcOse4Cer and monohexosylceramide. No ganglio-series neutral GSLs and no Forssman GSL were found in either muscle tissue. GM3(Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. GM2 was found in skeletal muscle only, while GD3 and GM1b-type gangliosides (GM1b and GD1 alpha) were undetectable in both tissues. GM1a-core gangliosides (GM1, GD1a, GD1b and GT1b) showed somewhat quantitative differences in each muscle; lactosamine-containing IV3Neu5Ac-nLcOse4Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse3Cer, GbOse4Cer), and gangliosides GM3 and GM2 were elucidated in runs which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and GM1-core gangliosides, respectively. Muscle is known for the lowest GSL concentration from all vertebrate tissues studied so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-resorcinol detection level.

Cystosarcoma phyllodes of the prostate.

We report a case of a 40-year-old man with cystosarcoma phyllodes of the prostate. This tumour is very rare in the prostate. The stroma of the tumour was cellular and composed of elongated cells with spindle shaped nuclei, scanty, pale and eosinophilic cytoplasm. Some cells showed nuclear atypia but the cells did not display mitotic activity. We categorised this tumour as benign. The suprapubic prostatectomy was done. The patient is alive and well after three years.

Anti-GM3 (II3Neu5Ac-lactosylceramide) ganglioside antibody labels human fetal Purkinje neurons during the critical stage of cerebellar development.

The ganglioside GM3 (II3Neu5Ac-lactosylceramide) represents a minor ganglioside in normal human brain compared to major gangliosides with gangliotetraose-backbone. In this study the presence of GM3 in three 23 and 24 weeks of gestation old human cerebella was demonstrated by immunostaining extracted gangliosides on thin-layer chromatography plate as well as by immunohistochemical analysis of cerebellar cryosections. During this stage of brain development GM3 was found to be dominantly expressed on cells corresponding to Purkinje neurons. Delipidation of histological sections with chloroform/methanol caused significant reduction of anti-GM3 immunostaining, thus confirming the prevalent ganglioside nature of this antigen. These results give evidence that (1) GM3 ganglioside is associated with a specific subset of human fetal cerebellar neurons during the critical development stage, and (2) a definite ganglioside in general is distributed to a specific subset of cells in normal human brain.

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases.

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, GM3(Neu5Ac), GM3(Neu5Gc) and Gm1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was GM3(Neu5Ac) followed by moderately expressed GM3(Neu5Gc) and GM1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-GM3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle.

Analysis of renal allograft frozen-sections.

Frozen-section analysis of renal allograft biopsy specimens is performed to permit modification of therapy during acute events in transplanted patients. Over a two-year period, out of 110 renal allograft biopsies 66 were analyzed on a frozen-section. The authors compared diagnoses obtained on a fresh-material frozen-section with those following profound specimen analysis. The accurate diagnosis was achieved in 61 (92.4%) cases, acute rejection being predominant (53 cases). Missdiagnoses referred to oxalosis (1 case), acute rejection (2 cases), acute pyelonephritis (1 case) and cortical infarction (1 case). The authors conclude that a frozen-section analysis is a useful tool in cases requiring fast decision.

Gangliosides of anencephalic and fetal brain--immunostaining on thin-layer chromatograms.

The expression of gangliosides of ganglio-series as well as neolacto-series gangliosides in anencephalic and in normal human fetal brain was compared with that in adult brain by immunostaining on thin-layer chromatograms. A difference in the expression of ganglio-series gangliosides with GM1a core was found between anencephalic and normal fetal brain, with less expression of GM1a and GD1a in anencephaly compared with normal fetal brain, in which these gangliosides dominate. Small amounts of GM1b were detected in fetal brain whereas only traces were found in anencephalic brain. Lactosamine-containing gangliosides were present in fetal and in anencephalic brain as alpha 2-3 as well as alpha 2-6 sialylated nLcOse4Cer structures. A heterogeneous group of neolacto-series gangliosides was expressed in anencephalic brain in both the monosialo- and presumed disialoganglioside range. These findings demonstrate a significant change in ganglioside pattern in anencephaly where the process of cell differentiation and maturation has been severely disturbed.

Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining.

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neural glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides GM3(Neu5Ac) and GM1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphingolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of GM3 and GM1 correlated to the fact that GM3 is the major ganglioside in skeletal muscle whereas GM1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. GM3 and GM1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level.

Frozen section analysis of breast biopsy specimens.

The authors compared the diagnoses from intraoperative frozen section consultation with the final diagnosis using permanent tissue sections from 179 breast biopsy specimens. Of these, there were 175 correct diagnoses (97.8%), two diagnoses were incorrect (1.1%) and two were inconclusive (1.1%). The distribution of the correct diagnoses within each particular group of breast diseases proves that in the invasive tumor group the diagnosis on FS was correct for 101 patients (98.1%) and incorrect for two patients (1.9%). In the fibrocystic breast disease group, diagnoses correlated for 42 patients (97.7%), whereas the problem in diagnosing the extent of epithelial proliferation appeared for only one patient (2.3%) and was categorized as an inconclusive diagnosis. Of 4 incorrect and inconclusive diagnoses, two occurred as a result of sampling nonrepresentative tissue specimens and two as a result of diagnostic misinterpretation. This study has shown that for the determination of the histological type of carcinoma, FS is not of significant morphological value since correct diagnoses were made for only 60% of the patients.

[Conservative surgical procedures in transitional cell carcinoma of the kidney and ureter]. / Konzervirajuci kirurski zahvati kod karcinoma prijelaznog epitela bubrega i uretera.

Conservative surgical procedure in selected patients with transitional cell carcinoma of upper urinary tract is alternative to radical surgery. Generally, a conservative surgical procedure is indicated in solitary low-stage lesions, particularly impaired renal function and bilaterality. At our Institute, conservative surgical procedure was performed in patients with solitary kidney and significant renal function damage. Survival after conservative and radical surgery was similar, extending the scope of indications for conservative surgical procedure in cases of low-grade low stage tumors.

Investigation of the applicability of histological classification of bronchial carcinoma according to the World Health Organization.

According to the World Health Organization histological classification of bronchial tumors, clear and giant cell carcinomas are two subtypes of large cell carcinoma. As clear and giant cells can also be observed in other types of bronchial carcinoma, we investigated the frequency of the finding of these cells in different histological types. The tumor size and degree of differentiation, the amount of necrosis and keratinization, and the presence of giant and clear cells were analyzed. Statistical analysis by X2 test showed (for all classified histological types of bronchial carcinomas, except small cell carcinoma) that: 1) larger tumors had a great quantity of giant cells (P less than 0.05; P less than 0.01), 2) large tumors had more clear cells (P less than 0.05; P less than 0.01) and 3) tumors with a greater amount of necrosis had a larger number of giant and clear cells (P less than 0.05; P less than 0.01). Findings of an identical cytological characteristic can cause some difficulty in determination of bronchial cancer.