Legionella spp. Monitoring in the Water Supply Systems of Accommodation Facilities in Sardinia, Italy: A Two-Year Retrospective Analysis.

Travel-associated Legionnaires' disease is a significant public health concern worldwide. A high number of cases are reported every year among travellers who stay at guest houses, hotels, and spas. Indeed, hot water systems, showers, and air-conditioning systems can be contaminated by Legionella, which grows at 25-42 °C. Studies have shown that in Sardinia, especially during the summer months, the water circulation in the hotels' pipes is exposed to extremely high temperatures. As a result, this study was conducted to assess the colonization of hotel water systems by Legionella in Sardinia, concerning a recent EU directive 2020/2184 for drinking water with a limit of 1000 CFU /L. Methods. A total of 112 accommodation facilities were analyzed, of which 61.3% were found to be colonized with Legionella, and out of a total of 807 samples, 32.5% were positive for Legionella presence. The results showed a higher number of positive samples in the summer season. This was also associated with the higher concentration presence of >1000 CFU/L in the samples. Consequently, this study confirms that local hotel operators should improve their water safety and prevention plans, especially in spring and summer.

PI3K/PTEN/mTOR pathway dynamic tracking and prognostic value in HR+/HER2- BC patients with residual disease after neoadjuvant chemotherapy: a cohort study.

AIMS: Hormone receptor-positive (HR)+/HER2- breast cancer (BC) is highly heterogeneous, with PI3K/PTEN/mTOR pathway alterations emerging as possible players within this complexity. We longitudinally tracked PI3K/PTEN/mTOR pathway dynamics from baseline biopsy to residual disease (RD)-and to metastases in case of relapse-in HR+/HER2- BC patients receiving neoadjuvant chemotherapy (NACT). METHODS: HR+/HER2- BC patients with RD after NACT were identified. We assessed PIK3CA mutational, Pten-loss and phosphorylation levels of mTOR and its substrates (p70S6K and 4EBP1) on baseline biopsies and matched RD samples; in case of disease relapse, we also assessed PIK3CA mutational status on metastatic samples. Recurrence-free survival (RFS) was adopted as endpoint. RESULTS: 92 patient were included. The conversion rate of PIK3CA mutational status was 12.8%; 1 patient acquired PIK3CA mutation at relapse; the rate of Pten conversion was 33.3%; mTOR phosphorylation levels significantly increased from baseline biopsy to RD, while its substrates significantly decreased. Baseline phosphorylated-mTOR significantly predicted poorer RFS in patients with PIK3CA wild-type status; baseline phosphorylated-70S6K was positively associated with RFS. CONCLUSIONS: We observed that PI3K/PTEN/mTOR pathway is highly dynamic under NACT exposure and the assessment of PIK3CA mutations may capture only a small fraction of such complexity. In this context, mTOR activation trough alternative pathways with respect to PIK3CA signalling may have a crucial role in shaping the molecular landscape of HR+/HER2- BC with RD after NACT. It is imperative to further elucidate the role of PIK3CA and mTOR-dependent pathways in shaping chemoresistance and endocrine resistance in high-risk HR+/HER2- early/locally advanced BC patients.

TERT Promoter Mutations and rs2853669 Polymorphism: Useful Markers for Clinical Outcome Stratification of Patients With Oral Cavity Squamous Cell Carcinoma.

OBJECTIVE: To date, no useful prognostic biomarker exists for patients with oral squamous cell carcinoma (OCSCC), a tumour with uncertain biological behaviour and subsequent unpredictable clinical course. We aim to investigate the prognostic significance of two recurrent somatic mutations (-124 C>T and -146 C>T) within the promoter of telomerase reverse transcriptase (TERT) gene and the impact of TERT single nucleotide polymorphism (SNP) rs2853669 in patients surgically treated for OCSCC. METHODS: The genetic frequencies of rs2853669, -124 C>T and -146 C>T as well as the telomere length were investigated in 144 tumours and 57 normal adjacent mucosal (AM) specimens from OCSCC patients. RESULTS: Forty-five tumours harboured TERT promoter mutations (31.3%), with -124 C>T and -146 C>T accounting for 64.4% and 35.6% of the alterations respectively. Patients with -124 C>T TERT promoter mutated tumours had the shortest telomeres in the AM (p=0.016) and showed higher risk of local recurrence (hazard ratio [HR]:2.75, p=0.0143), death (HR:2.71, p=0.0079) and disease progression (HR:2.71, p=0.0024) with the effect being potentiated by the co-occurrence of T/T genotype of rs2853669. CONCLUSION: -124 C>T TERT promoter mutation as well as the T/T genotype of the rs2853669 SNP are attractive independent prognostic biomarkers in patients surgically treated for OCSCC, with the coexistence of these genetic variants showing a synergistic impact on the aggressiveness of the disease.

Evaluation of growth potential and growth dynamics of Listeria monocytogenes on ready-to-eat fresh fruit.

The consumption of fresh or RTE fruits is increasing every year and Listeria monocytogenes has been identified on raw or minimally processed fruits. A food product can become contaminated with L. monocytogenes anywhere along the pathway of food production during planting, harvesting, packaging, distribution and serving. The aim of this work was to assess the microbiological risks associated with consumption of ready- to- eat fruit such as melon, pineapple, coconut and fruit salad. The presence of Escherichia coli, Salmonella spp. and L. monocytogenes was also evaluated. Microbiological challenge tests were carried out for the evaluation of the L. monocytogenes growth potential in RTE fruit stored at 4 and 8°C. E. coli counts resulted under the detection limit of 10 CFU g-1, Salmonella and L. monocytogenes were not detected (absence in 25g). The growth potential values in coconut and melon (δ>0.5) showed the growth capacity of Listeria at the temperatures considered. A low initial load, also derived from good hygiene practices, and correct storage temperatures are essential to reduce bacterial growth in RTE fruit. The challenge test showed how each type of RTE fruit has a different commercial life based on its specific growth potential and that food should be stored at temperatures not higher than 4°C for a short period.

Evaluation of Microbial Growth in Hospital Textiles Through Challenge Test.

INTRODUCTION: Ensuring the microbiological quality of textiles is an important requirement for health care facilities. The present study examines the way transport times and temperatures influence microbial growth in textiles. Therefore, the effectiveness of washing and disinfection processes has also been studied. METHODS: Microbial Challenge Tests were set up through the artificial contamination of different dry and wet textiles which were stored at different temperatures. The bacterial concentration was evaluated in well-defined time phases aimed at simulating the time it took for the textiles to be transported from the hospital facilities to the reconditioning unit. Three times were therefore considered from T = 0 inoculation moment to T = 72 h post inoculation. At the end of each time, the increase in bacterial concentration was assessed by means of microbiological cultures, using selective media for the enumeration of each type of inoculated microorganism. RESULTS: In all the contaminated textiles the bacterial concentration remained unchanged at a temperature of 4 °C, while at 22 °C and 37 °C there was a significant increase (p < 0.05) starting from 8 h of storage. In these textiles, the microorganism that showed the greatest growth capacity was P. aeruginosa with average initial concentration values of 104 CFU/cm2 and a final concentration of 1.5 × 105 CFU/cm2 at 22 °C and 1 × 105 CFU/cm2 at 37 °C 72 h after inoculum. CONCLUSION: The data highlights the fact that the degree of contamination in textiles does not undergo an increase when transport takes place at a controlled temperature. Refrigerated transport of hospital textiles is thus a desirable preventive measure to keep microbiological risk under control.

The Tumor Microenvironment of Primitive and Metastatic Breast Cancer: Implications for Novel Therapeutic Strategies.

Breast cancer evolves thanks to a dense and close interaction with the surrounding tumor microenvironment (TME). Fibroblasts, leukocytes, blood and lymphatic endothelial cells and extracellular matrix are the constituents of this entity, and they synergistically play a pivotal role in all of the stages of breast cancer development, from its onset to its metastatic spread. Moreover, it has been widely demonstrated that variations to the TME can correspond to prognosis variations. Breast cancer not only modulates the transformation of the environment within the mammary gland, but the same process is observed in metastases as well. In this minireview, we describe the features of TME within the primitive breast cancer, throughout its evolution and spread into the main metastatic sites.

A rare case of nasal Schneiderian (inverted) papilloma associated with basaloid squamous cell carcinoma.

Sinonasal inverted papilloma (IP) is a benign, locally aggressive epithelial neoplasm. In less than 9% of cases it is associated with malignancies, typically conventional squamous cell carcinomas (SCC), while other histological variants have been less frequently reported. We describe the third case of basaloid squamous cell carcinoma (BSCC) arising in nasal IP. An 81-year-old female patient presented with a pinkish irregular lesion on the nasal septum. Biopsy was consistent with IP and carcinoma in situ. Two surgical procedures were needed to obtain radical excision. Histology on the surgical specimen revealed BSCC. Seven months after surgery, there was no evidence of disease recurrence. Although IP is more frequently associated with conventional SCC, other malignancies should be considered. The histological differential diagnosis should be supported by immunohistochemistry. The generally-recommended treatment for sinonasal BSCC is complete surgical resection, although this may be a problem in multifocal distributions, as in the present case.

Potential issues of well water in domestic use in a retrospective study from 2014 to 2018.

Well water requires chemical, physical-chemical, and microbiological analysis to ensure that the water used for irrigation or for human consumption presents no specific risks. The aim of the present work was to determine the microbiological risk of samples taken from wells in south-central Sardinia. In this survey, 55 water samples were taken from private wells in the period from 2014 to 2018. Quality was assessed through the detection of the parameters required by national law. The analysis of the data showed that only 36.4% of the samples (20 wells) complied with legal limits for water for human consumption. The most isolated species was P. aeruginosa, present in 29.1%; its high concentration and recorded Coliforms suggest that the disinfection of the wells was insufficient or not performed properly if implemented. Such data suggest the need to pay more attention to the waters used not only for human consumption but also for irrigation, since the cultivated vegetables could become a vehicle for potentially pathogenic microorganisms. National legislation sets contamination limits only for water intended for human consumption and not for irrigation water. However, based on these considerations, it appears necessary to develop reference standards for irrigation water.

Biological tumor markers (maspin, CD105, nm23-H1) and disease relapse in laryngeal cancer: cluster analysis.

BACKGROUND: The aim of this investigation was to see if a panel of biomarkers (maspin, CD105, and nm23-H1) could be used to stratify patients with laryngeal squamous cell carcinoma (LSCC) in homogeneous disease recurrence risk clusters. METHODS: Cluster analysis was used to classify 89 patients based on their immunohistochemical expression of nm23-H1, CD105, and maspin. RESULTS: Our analysis identified seven homogeneous clusters: the LSCC recurrence rate was lowest in cluster 6 (non-nuclear maspin pattern, nuclear nm23-H1 expression ≥10%, endothelial CD105 expression <6%; P = .009), and highest in cluster 3 (non-nuclear maspin pattern, nuclear nm23-H1 expression <10%, endothelial CD105 expression ≥6%; P <.001). CONCLUSIONS: Similar panels of biological variables identified by cluster analysis should be tested in prospective clinical trials to establish whether treating patients identified as being at higher risk of LSCC recurrence more aggressively could significantly improve their recurrence rate and/or disease-specific survival.

Maspin expression and anti-apoptotic pathway regulation by bcl2 in laryngeal cancer.

OBJECTIVES: Comprehension of the interplay of pro-apoptotic and anti-apoptotic stimuli in laryngeal squamous cell carcinoma (LSCC) is crucial to understand tumor development, biological behavior and treatment response. Bcl-2 family proteins mainly regulate the apoptotic signal cascade. In some cancers, maspin seems to influence the balance between pro-apoptosis and anti-apoptosis bcl-2 family proteins. The aim of this study was to investigate the potential relationship between bcl-2 anti-apoptotic factor and the tumor suppressor maspin in LSCC. MATERIALS AND METHODS: 31 consecutive patients who underwent primary surgery and post-operative radiotherapy for LSCC were evaluated retrospectively. For each case, immunohistochemistry assays for bcl-2 and maspin were performed. Data were also collected on N-status, pT stage, grading, recurrence and disease-free survival (DFS). RESULTS: Patients with nuclear maspin pattern of expression showed a significantly lower recurrence rate (p = 0.04) and longer DFS (p = 0.0018). The expression of bcl-2 was not associated with recurrence rate or DFS either in the whole cohort or in cases with nuclear maspin pattern, while in patients with non-nuclear maspin pattern, a statistical trend was found toward a shorter DFS for bcl-2 positive cases (p = 0.062). In the multivariate model, only maspin expression pattern retained its independent prognostic significance (p = 0.006). CONCLUSIONS: Nuclear maspin pattern seemed to be an independent positive prognostic factor, while bcl-2 prognostic value was related to maspin expression pattern. Further investigations are needed to support the use of bcl-2 inhibitors in multimodality or multitarget strategies against advanced LSCCs, also considering the role and expression of tumor suppressor genes.

A classification of chronic rhinosinusitis with nasal polyps based on structured histopathology.

AIMS: In chronic rhinosinusitis with nasal polyps (CRSwNP), tools based on objective evidence, such as histopathology, are needed to assist clinical decision-making. The main aim of this exploratory investigation was to determine whether structured histopathology could be used to classify CRSwNP in homogeneous histological clusters. METHODS AND RESULTS: A cohort of 135 CRSwNP patients was assessed, on the basis of clinicopathological features: allergic fungal rhinosinusitis (17 patients); non-steroidal anti-inflammatory drug-exacerbated respiratory disease (19 patients); intrinsic asthma (18 patients); extrinsic asthma (21 patients); allergy (21 patients); histologically eosinophilic (22 patients); and histologically non-eosinophilic (17 patients). For structured histopathology, we considered: the degree of inflammation; eosinophil count; eosinophil aggregates; neutrophil infiltration; goblet cell hyperplasia; basement membrane thickening; fibrosis; hyperplastic/papillary changes; squamous metaplasia; mucosal ulceration; and subepithelial oedema. Cluster analysis identified four distinct sets of cases. On discriminant analysis, the global error rate was 1.48%, and the stratified error rates were 4.34%, 0%, 0%, and 0% for clusters 1, 2, 3 and 4, respectively. Cluster 1 was characterised by infrequent fibrosis (<4.5% of cases). Cluster 2 mainly featured neutrophil infiltration in 100% of cases, hyperplastic/papillary changes in 70% of cases, and fibrosis in 65% of cases. Cluster 3 showed fibrosis in 100% of cases. Cluster 4 showed hyperplastic/papillary changes in 100% of cases, and fibrosis in 92% of cases. CONCLUSIONS: This study shows that cluster analysis can identify different histotypes among CRSwNP patients. The next step will be to investigate, in a larger series, the clinical (e.g. prognostic) implications of identifying such homogeneous clusters of patients with CRSwNP on the basis of their structured histopathology.

Mutational and Immunophenotypic Profiling of a Series of 8 Tubo-ovarian Carcinosarcomas Revealed a Monoclonal Origin of the Disease.

Carcinosarcomas are rare, highly aggressive neoplasms composed of a combination of carcinomatous and sarcomatous elements. These tumors represent a paradigmatic field for the study of intratumor heterogeneity. A series of 8 tubo-ovarian carcinosarcomas was characterized for the following: (i) immunohistochemical expression of MNF116, epithelial membrane antigen, vimentin, S100, chromogranin, synaptophysin, desmin, myogenin (MYF4), and p53; (ii) mutational profiling of KRAS, BRAF, PIK3CA, NRAS, TP53, and DICER1 genes. Heterologous differentiation was present in 6 of 8 tumors. Cytokeratin MNF116 and epithelial membrane antigen were positive in all the carcinomatous components and in 87.5% and 50.0% of the sarcomatous components, respectively. The sarcomatous components showed positive staining for vimentin in all cases. Two cases demonstrated positivity for neuroendocrine markers in their carcinomatous components. All rhabdomyosarcomas were positive for desmin and MYF-4. Chondrosarcomas were positive for S100. All but one tumor showed similar p53 immunoreactivity in both the carcinomatous and sarcomatous components, and one case showed cytoplasmic p53 expression. Three of 8 cases (37.5%) showed TP53 mutations, and, in 2 cases, the TP53 mutation was shared by both epithelial and mesenchymal components. DICER1 mutation was found in all components of one case. Mutations in KRAS, NRAS, BRAF, and PIK3CA genes were not found in the study cohort. Our results highlight the heterogeneity of ovarian carcinosarcomas at the phenotypic level. A common mutational signature was observed in both components in 3 of 4 informative tumors. More studies are required to dissect different levels of ovarian carcinosarcomas' heterogeneity in order to define the best therapeutic approaches to these aggressive neoplasms.

Presence of Trihalomethanes in ready-to-eat vegetables disinfected with chlorine.

Trihalomethanes (THMs) - CHCl3, CHCl2Br, CHClBr2 and CHBr3 - are drinking water disinfection by-products (DBPs). These compounds can also be absorbed by different types of foods, including ready-to-eat (RTE) fresh vegetables. The potential absorption of THMs during washing of RTE vegetables could pose a potential risk to consumers' health. The concentration of THMs in the water used in the manufacturing process of these products shall not exceed the limit of 100 or 80 µgL-1 according to European Union (EU) and United States legislation, respectively. By contrast, there is little information about the presence of such compounds in the final product. This study evaluated the concentration of THMs in different types of RTE vegetables (carrots, iceberg lettuce, lettuce, mixed salad, parsley, parsley and garlic, rocket salad, valerian) after washing with chlorinated water. In the 115 samples analysed, the average value of total THMs was equal to 76.7 ng g-1. Chloroform was the THM present in the largest percentage in all the RTE vegetables. These results show that the process of washing RTE vegetables should be optimised in order to reduce the risk for consumers associated with the presence of DBPs.

Spindle cell oncocytoma: Report of two cases with massive bleeding and review of the literature.

Spindle cell oncocytoma (SCO) is a rare pituitary tumor, classified as a WHO grade I neoplasm. Due to its rarity, SCO is often preoperatively misdiagnosed as a pituitary macroadenoma. In the present study we report two recent cases of SCO, a 61-year-old male and a 65-year-old female presenting at Treviso General Hospital between March 2014 and April 2015. Tumor resection was achieved by endoscopic transsphenoidal approach but massive hemorrhagic events hampered surgery, endangering the patient's life in both cases. Both tumors featured fascicles of spindle cells with eosiniphilic cytoplasm expressing vimentin, S-100 and thyroid transcription factor-1 (TTF-1). The diagnosis of SCO was confirmed by second opinion in both cases. Extensive review of available literature, about 30 cases from 2002 to 2015, provided valuable clinical data for preoperative diagnosis and surgical removal of SCO tumors.

Concomitant KIT/BRAF and PDGFRA/BRAF mutations are rare events in gastrointestinal stromal tumors.

AIM: The BRAF mutation is a rare pathogenetic alternative to KIT/PDGFRA mutation in GIST and causes Imatinib resistance. A recent description of KIT and BRAF mutations co-occurring in an untreated GIST has challenged the concept of their being mutually exclusive and may account for ab initio resistance to Imatinib, even in the presence of Imatinib-sensitive KIT mutations. BRAF sequencing is generally limited to KIT/PDGFRA wild-type cases. Hence, the frequency of concomitant mutations may be underestimated. METHODS: We screened for KIT (exon 9, 11 ,13 ,17), PDGFRA (exon 12,14, 18) and BRAF (exon 15) mutations a series of 407 GIST. Additionally, we evaluated the BRAF V600E mutation-specific antibody, VE1, as a surrogate for V600E mutation, on a series of 313 GIST (24 on whole sections, 288 cases on tissue array), including 6 cases molecularly ascertained to carry the BRAF V600E mutation. RESULTS: No concomitant KIT/BRAF or PDGFRA/BRAF mutations were detected. BRAF mutation was detected only in one case, wild-type for KIT/PDGFRA. All the 6 BRAF-mutant cases stained positive with the VE1 antibody. A weak VE1 expression was observed in 14/287 (4.9%) BRAF wild-type cases, as observed also in 2/6 BRAF-mutant cases. Overall in our series, sensitivity and specificity of the VE1 antobody were 100% and 95.1%, respectively. CONCLUSIONS: The concomitance of BRAF mutation with either KIT or PDGFRA mutation is rare in GIST. In these tumors, moderate/strong VE1 immunoreactivity is a valuable surrogate for molecular analysis. Instead, genotyping is warranted in the presence of weak VE1 staining.

Detection of Virulence Genes and Growth Potential in Listeria monocytogenes Strains Isolated from Ricotta Salata Cheese.

Ricotta Salata is a traditional ripened and salted whey cheese made in Sardinia (Italy) from sheep's milk. This product is catalogued as ready-to-eat food (RTE) since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens, such as Listeria monocytogenes, can represent a health risk for consumers. In September 2012, the FDA ordered the recall of several batches of Ricotta Salata imported from Italy linked to 22 cases of Listeriosis in the United States. This study was aimed at evaluating the presence and virulence properties of L. monocytogenes in 87 samples of Ricotta Salata produced in Sardinia. The ability of this product to support its growth under foreseen packing and storing conditions was also evaluated in 252 samples. Of the 87 samples 17.2% were positive for the presence of L. monocytogenes with an average concentration of 2.2 log10 cfu/g. All virulence-associated genes (prfA, rrn, hlyA, actA, inlA, inlB, iap, plcA, and plcB) were detected in only one isolated strain. The Ricotta Salata samples were artificially inoculated and growth potential (δ) was assessed over a period of 3 mo. The value of the growth potential was always >0.5 log10 cfu/g under foreseen packing and storing conditions. This study indicates that Ricotta Salata supports the L. monocytogenes growth to levels that may present a serious risk to public health, even while stored at refrigeration temperatures.

Recovery of Staphylococcus aureus in Gray Mugil cephalus Roe (Bottarga): Investigation by an Integrated Cultural/Molecular Approach.

In the Mediterranean area, salted and dried roe from the gray Mugil cephalus "bottarga" represent a speciality food with great commercial value. Bottarga is currently produced by a traditional handmade process and, the risk of human bacterial contamination during its manufacturing is still unknown; in this perspective the foodborne pathogen Staphylococcus aureus could potentially contaminate this product due to poor sanitation or bad handling during processing. The aim of this work is: to evaluate the contamination level of foodborne pathogens at different product manufacturing stages and, in addition, to describe a fast and realizable method for the rapid detection of S. aureus in bottarga samples in the field. A cultural procedure was initially used to investigate the occurrence of S. aureus and the other main foodborne pathogens in bottarga samples at the different manufacturing stages (from roe to final product). In addition, a molecular approach was used to rapidly determine the presence of total bacteria, S. aureus, and its potential toxigenicity. Of the 194 specimens analyzed, we identified: Clostridium perfringens, Enterococcus spp. and Enterobacteriaceae. However, some samples resulted as being contaminated with S. aureus (4% in roe and 8.7% in the final product). During the bottarga manufacturing process, we observed an increase in pathogen levels (from 10(2) to 10(5) CFU/g) in contaminated samples, and entA and entB genotypes were identified. Reconstruction experiments suggest that the fresh roe and the bottarga (not completely dried) could represent a risk for the contamination and growth of pathogen bacteria.