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The correct identification of HER2-positive cases is a key point to provide the most appropriate therapy to breast cancer (BC) patients. We aimed at investigating the reproducibility and accuracy of HER2 expression by immunohistochemistry (IHC) in a selected series of 35 invasive BC cases across the pathological anatomy laboratories in Tuscany, Italy. Unstained sections of each BC case were sent to 12 participating laboratories. Pathologists were required to score according to the Food and Drug Administration (FDA) four-tier scoring system (0, 1+, 2+, 3+). Sixteen and nineteen cases were HER2 non-amplified and amplified respectively on fluorescence in situ hybridization. Among 192 readings of the 16 HER2 non-amplified samples, 153 (79.7%) were coded as 0 or 1+, 39 (20.3%) were 2+, and none was 3+ (false positive rate 0%). Among 228 readings of the 19 HER2 amplified samples, 56 (24.6%) were scored 0 or 1+, 79 (34.6%) were 2+, and 93 (40.8%) were 3+. The average sensitivity was 75.4%, ranging between 47% and 100%, and the overall false negative rate was 24.6%. Participation of pathological anatomy laboratories performing HER2 testing by IHC in external quality assurance programs should be made mandatory, as the system is able to identify laboratories with suboptimal performance that may need technical advice. Updated 2013 ASCO/CAP recommendations should be adopted as the widening of IHC 2+ "equivocal" category would improve overall accuracy of HER2 testing, as more cases would be classified in this category and, consequently, tested with an in situ hybridisation method.

Nowadays several advanced molecular techniques are applied for quantifying bacteria involved in contaminant degradation processes. However, despite the fact that significant efforts have been taken to make these tools more reliable and specific, their application for the analysis of field samples is hardly ever applied. In this study, a combination of three methods (CARD-FISH, qPCR and RT-qPCR) was successfully applied to evaluate the distribution and the activity of known chlorinated solvent dechlorinating bacteria in a contaminated site where no remedial actions have been undertaken. CAtalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) specifically provided the cell densities of known dechlorinating bacteria and was found to be more sensitive than quantitative PCR (qPCR) for the quantification of 'Dehalococcoides' cell numbers in the aquifer. Among the screened dechlorinators, 'Dehalococcoides' spp. were mainly found and nearly homogenously distributed in the aquifers at concentrations ranging from 8.1×10(5)±1.2×10(5) to 2.5×10(7)±5.6×10(6)cells per liter of groundwater (with a relative abundance out of the total Bacteria of 0.7-15%). Further, the dechlorination potentialities of 'Dehalococcoides' species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). 'Dehalococcoides'tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity and indicated the occurrence of reductive dechlorination at the site., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fungaemia is an increasing nosocomial pathology. The 'gold standard' for detection of fungaemia is blood culture, but it is time-consuming and its sensitivity for early detection is low. On the other hand, yeasts present different antifungal sensitivity patterns to be quickly detected to allow an effective treatment. The aim of this study was to evaluate the diagnostic performances of PNA-FISH to directly identify yeasts from blood cultures and to compare results with those obtained by culture. A total of 176 blood cultures positive for yeasts at direct Gram stain and 24 negative blood cultures as control collected from 15 Italian hospitals, included in a network coordinated by the Medical Mycology Committee, Italian Society of Clinical Microbiology (AMCLI), were examined both by culture and PNA-FISH technology. Sensitivity of the PNA-FISH technique evaluated for five Candida species was 99.3% and specificity, 100%. Distinguishing which yeast is implicated in fungaemia and whether the infection is caused by multiple species are important for the selection of antifungal therapy. The PNA-FISH technique is a very useful approach because the test discriminates between groups of Candida species with different susceptibility pattern, particularly against azoles and echinocandins, with only a 90-minute turn-around time after the Gram-stain reading., (© 2012 Blackwell Verlag GmbH.)

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.

The European whitefish, Coregonus lavaretus, widely distributed in freshwater of northern Europe and introduced into the major lakes of northern Italy, has been restocked in central Italian lakes. In accordance with current managing practices, a reduced number of spawners contribute to reproduction within each lake and a certain degree of isolation is to be expected between populations from different lakes, resulting in the rapid fixing of chromosomal changes. A detailed survey of three populations from different lakes was carried out using classical and molecular cytogenetic techniques, to verify if specific chromosomal markers are present in the distinct populations. The comparative analysis revealed intraspecific variability of NORs and fixed differences in their number in the three populations. A co-localization of major and minor rRNA genes on one chromosome site was also observed. The original data regarding the chromosome mapping of the (TTAGGG)(n) telomeric repeat obtained in this study, demonstrated their exclusively terminal distribution, and a conspicuous inter-chromosomal variation in the number of repeats. The results are compared with data available for populations from native geographic ranges.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis., (Copyright 2004 Wiley-Liss, Inc.)

Although B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in Western countries, little is known about its underlying molecular abnormalities and their prognostic significance, particularly for use in early therapeutic interventions in young patients. As TP53 tumor suppressor gene abnormalities and 11q23 deletions are reported to be prognostically adverse in hematologic malignancies, we used interphase fluorescence in situ hybridization to analyze their incidence and prognostic significance in young B-CLL patients. Bone marrow samples from 40 untreated B-CLL patients at diagnosis were studied using five yeast artificial chromosome clones from the 11q23.1 approximately q23.3 chromosomal region and a probe specific for the 17p13.1 locus. Twenty-three patients (58%) carried 11q deletions. Interestingly, 16 of 17 patients (94%) who showed early disease progression exhibited this chromosomal abnormality, suggesting that 11q deletions may help to identify more aggressive disease in early stage patients. In contrast, monoallelic TP53 deletions were found in all of the patients. The TP53 and 11q deletions were only present in a proportion of the clonal B-cells, which suggests that they are secondary events in B-CLL.

In mammals, Robertsonian (Rb) translocation (the joining of two telo/acrocentric chromosomes at their centromere to form a metacentric) is the most effective process in chromosomal evolution leading to speciation; its occurrence also affects human health (through the induction of trisomies) and the fertility of farm animals. To understand the mechanism of Rb translocation, we used the house mouse as a model system and studied the organization of pericentromeric satellite DNAs (satDNA) of telocentrics and Rb chromosomes, both minor and major satDNA. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used to analyze the major satDNA. To detect the very small amount of minor satDNA, a procedure was developed that combines CO-FISH with primed in situ labeling and conventional FISH and is five times more sensitive than the CO-FISH procedure alone. It was found that both the major and the minor satDNA tandem repeats are oriented head-to-tail in telocentric and Rb chromosomes, and their polarity is always the same relative to the centromere. We suggest that all tandemly repetitive satDNAs in a species probably are locked into such a symmetry constraint as a universal consequence of chromosomal evolution. Rb translocation breakpoints were found localized within the minor satDNA of telocentrics, and these sequences contributed symmetrically to the formation of the centromeric region of the Rb chromosomes. These results are important for an understanding of the geometry of Rb translocations and suggest the study of DNA orientation as a new tool for investigating these rearrangements.