SARS-CoV-2 wastewater surveillance in the Czech Republic: Spatial and temporal differences in SARS-CoV-2 RNA concentrations and relationship to clinical data and wastewater parameters.

This study presents the results of systematic wastewater monitoring of SARS-CoV-2 RNA and basic wastewater parameters from four different wastewater treatment plants (WWTPs) in the Czech Republic over the 2020-2022 epidemic. Two-step reverse-transcription quantitative PCR targeting genes encoding the N and Nsp12 proteins was employed to detect SARS-CoV-2 RNA loading in 420 wastewater samples. The results obtained were used to evaluate the potential of wastewater analysis for describing the epidemiological situation in cities of different sizes and determining temporal differences based on the prevailing SARS-CoV-2 variant. Strong correlations between the number of active and hospitalised COVID-19 cases in each WWTP catchment area and the concentration of SARS-CoV-2 RNA detected in the wastewater clearly demonstrated the suitability of this wastewater-based epidemiological approach for WWTPs of different sizes and characteristics, despite differences in SARS-CoV-2 variant waves, with some WWTPs showing high predictive potential. This study demonstrated on the data from the Czech Republic that targeted systematic monitoring of wastewater provides sufficiently robust data for surveillance of viral loads in sample populations, and thus contributes to preventing the spread of infection and subsequent introduction of appropriate measures.

Hepatitis E - epidemiology and clinical course in the largest cohort in the Czech Republic.

Introduction: The majority of hepatitis E (HE) reports come from Western Europe. The aim of the study was to describe the typical epidemiological and clinical characteristics of HE in the Czech Republic. Methods: A retrospective analysis of 173 patients with HE. Results: At least 90% of cases were autochthonous (HEV-3 genotype). Seventeen patients were treated with ribavirin. Five underwent liver transplants because of fulminant HE. We noted neurological symptomatology in 9 cases. Six patients developed chronic HE. Conclusions: There is a possibility of severe health complications caused by the hepatitis E virus in the Czech Republic.

Experimental Evidence of the Long-Term Survival of Infective African Swine Fever Virus Strain Ba71V in Soil under Different Conditions.

The survival of African swine fever virus (ASFV) on different matrices and its infectivity in wild as well as domestic swine is still a matter of interest. ASFV is resistant to environmental effects; this fact is enhanced by the presence of organic material. Therefore, the aim of this work was to determine the ability of laboratory ASFV to survive in soil at different temperatures (4 and 22 °C) and with and without the presence of blood using culture procedures. The suitability of the procedure for determining the viability and titre of the ASFV field strain by the hemadsorption method was also verified, when a higher decrease in virus infectivity in the case of clay compared with peat was demonstrated. The stability of the virus was clearly temperature-dependent, the infectious virus was detected after 112 days, and the viral DNA was still detected in the matrix 210 days after inoculation in a relatively high and stable concentration (between 106 and 107 genome equivalents/mL). Based on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and serve as indicators of the risk posed by the affected locality.

MOL-PCR and xMAP Technology: A Multiplex System for Fast Detection of Food- and Waterborne Viruses.

Viruses are common causes of food- and waterborne diseases worldwide. Conventional identification of these agents is based on cultivation, antigen detection, electron microscopy, or real-time PCR. Because recent technological advancements in detection methods are focused on fast and robust analysis, a rapid multiplexing technology, which can detect a broad spectrum of pathogenic viruses connected to food or water contamination, was utilized. A new semiquantitative magnetic bead-based multiplex system has been designed for simultaneous detection of several targets in one reaction. The system includes adenoviruses 40/41 (AdV), rotavirus A (RVA), norovirus (NoV), hepatitis E virus (HEV), hepatitis A virus (HAV), and a target for external control of the system. To evaluate the detection system, interlaboratory ring tests were performed in four independent laboratories. Analytical specificity of the tool was tested on a cohort of pathogenic agents and biological samples with quantitative PCR as a reference method. Limit of detection (analytical sensitivity) of 5 × 100 (AdV, HEV, and RVA) and 5 × 101 (HAV and NoV) genome equivalents per reaction was reached. This robust, senstivie, and rapid multiplexing technology may be used to routinely monitor and manage viruses in food and water to prevent food and waterborne diseases.

Evidence of Hepatitis E Virus in Goat and Sheep Milk.

Hepatitis E virus (HEV) is the etiological agent behind hepatitis E infection. Domestic pigs and wild boars are the main animal reservoirs of HEV. Very few papers describe HEV infection in goats and sheep. As the data pertaining to the presence of HEV virus in the milk of small ruminants in Europe are lacking, the aim of this paper was to examine a representative number of milk samples from these animals. The detection of HEV genome (HEV RNA) was performed using reverse transcriptase real-time polymerase chain reaction (RT-qPCR). HEV RNA was found in 2.8% of the examined samples. Positivity ranged from 101 to 103 genome equivalents/mL (GE/mL) with a median of 9.99 × 102 GE/mL. On the basis of these results, the milk of small ruminants could represent a source of HEV infection to consumers.

Preliminary Study of Sars-Cov-2 Occurrence in Wastewater in the Czech Republic.

The virus SARS-CoV-2, which has caused the recent COVID-19 pandemic, may be present in the stools of COVID-19 patients. Therefore, we aimed to detect SARS-CoV-2 in wastewater for surveillance of SARS-CoV-2 in the population. Samples of untreated wastewater were collected from 33 wastewater treatment plants (WWTPs) of different sizes within the Czech Republic. SARS-CoV-2 RNA was concentrated from wastewater and viral RNA was determined using real-time reverse transcription polymerase chain reaction (RT-qPCR). SARS-CoV-2 RNA was detected in 11.6% of samples and more than 27.3% of WWTPs; in some of them, SARS-CoV-2 was detected repeatedly. Our preliminary results indicate that an epidemiology approach that focuses on the determination of SARS-CoV-2 in wastewater could be suitable for SARS-CoV-2 surveillance in the population.

Multigene Panel Germline Testing of 1333 Czech Patients with Ovarian Cancer.

Ovarian cancer (OC) is the deadliest gynecologic malignancy with a substantial proportion of hereditary cases and a frequent association with breast cancer (BC). Genetic testing facilitates treatment and preventive strategies reducing OC mortality in mutation carriers. However, the prevalence of germline mutations varies among populations and many rarely mutated OC predisposition genes remain to be identified. We aimed to analyze 219 genes in 1333 Czech OC patients and 2278 population-matched controls using next-generation sequencing. We revealed germline mutations in 18 OC/BC predisposition genes in 32.0% of patients and in 2.5% of controls. Mutations in BRCA1/BRCA2, RAD51C/RAD51D, BARD1, and mismatch repair genes conferred high OC risk (OR > 5). Mutations in BRIP1 and NBN were associated with moderate risk (both OR = 3.5). BRCA1/2 mutations dominated in almost all clinicopathological subgroups including sporadic borderline tumors of ovary (BTO). Analysis of remaining 201 genes revealed somatic mosaics in PPM1D and germline mutations in SHPRH and NAT1 associating with a high/moderate OC risk significantly; however, further studies are warranted to delineate their contribution to OC development in other populations. Our findings demonstrate the high proportion of patients with hereditary OC in Slavic population justifying genetic testing in all patients with OC, including BTO.

Recommendations for Preventive Care for Women with Rare Genetic Cause of Breast and Ovarian Cancer.

An inherited predisposition to breast cancer underlies 5-10% of breast tumors. High-risk BRCA1 and BRCA2 genes result in an 85% lifetime risk of breast cancer and a 20-60% lifetime risk of ovarian cancer. Next-generation sequencing or massive parallel sequencing are now established testing methods that enable screening for many genes that predispose to heterogeneous hereditary cancer syndromes (22 genes are required by the health insurance companies). In addition to BRCA1 and BRCA2, inherited mutations in other genes predispose to breast and/or ovarian cancer. High-risk breast cancer genes include TP53, STK11, CDH1, PTEN, PALB2, and NF1, while moderate-risk (2-4 times increased risk) breast cancer genes include ATM, CHEK2, and NBN. Moderate risk is also suggested for Lynch syndrome, MUTYH, BRIP1, RAD51C, RAD51D, BARD1, FANCA, FANCC, FANCM, BLM, WRN genes. In heterozygotes for other recessive syndromes the risk of developing breast cancer is subject to current research. Low-risk genes are (mostly) irrelevant from a clinical perspective. Other genes that increase the risk of ovarian cancer include the genes for Lynch syndrome, the BRIP1, RAD51C and RAD51D genes. Preventive care should be proposed based on assumed cumulative breast cancer risk (see http: //www.mamo.cz): a risk of >20% for BRCA1/2, TP53, PTEN, STK11, CDH1, PALB2, CHEK2, ATM, and NF1; and a risk of 10-20% for BRIP1, RAD51C, RAD51B, BARD1, FANCA, FANCC, FANCM, NBN, BLM, and WRN. The genetic risk should be assessed by a geneticist and be based on inherited mutations and empirical risk according to family history. Prophylactic mastectomy is considered for high-risk gene carriers but not for moderate-risk gene carriers; however, it may be considered if there is an underlying family history, a risk of parenchyma of the mammary gland, or other risk factors. Ovarian cancer risk increases significantly in carriers of the BRIP1, RAD51C, and RAD51D genes. For prevention of ovarian cancer, prophylactic salpingo-oophorectomy is an important component of preventive care. In ovarian cancer families with no identified risk germline mutation, preventive salpingo-oophorectomy is not routinely recommended but may be considered as the only efficient method of prevention due to the increased empirical risk (4 times) of ovarian cancer in first-degree relatives. Supported by the grant project MH CZ - RVO (MMCI, 00209805), AZV 15-27695A and AZV 16-29959A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 17. 5. 2019 Accepted: 31. 5. 2019.

Twenty Years of BRCA1 and BRCA2 Molecular Analysis at MMCI - Current Developments for the Classification of Variants.

BACKGROUND: Deleterious mutations in the BRCA1 and BRCA2 genes account for a considerable proportion of dominantly inherited breast and ovarian cancer susceptibility. The laboratory interpretation has always been dependent on the information available at the time of the report conclusion. The aim of this study has been to review the results from the BRCA testing at Masaryk Memorial Cancer Institute (MMCI). PATIENTS AND METHODS: Patients with suspected hereditary predisposition to breast/ovarian cancer, belonging to 7,400 families, were referred by genetic counsellors for BRCA1 and BRCA2 mutation testing at the MMCI from 1999 to the beginning of 2018. Various methods have been used over 20 years of laboratory practice - starting with the Protein Truncation Test and Heteroduplex Analysis via the High Resolution Melting analysis and Sanger sequencing up to Next Generation Sequencing. RESULTS: BRCA1 and BRCA2 mutation screening resulted in the identification of 1,021 families with a germline high-risk BRCA1 mutation and 497 families carrying a high-risk BRCA2 mutation, representing a mutation detection rate of 20.5%. A broad spectrum of unique mutations classified as pathogenic or likely pathogenic has been detected in both genes - 124 in the BRCA1 and 123 in the BRCA2 gene. Other sequence variants (96 unique variants in the BRCA1 and 126 in the BRCA2 gene) have been revised and classified as benign or likely benign. The other 82 unique variants remain classified as of uncertain significance mainly due to a lack of information for inclusion in other groups. All the results are summarised in the tables, including the reasons for their classification. CONCLUSION: The clinical classification of rare sequence variants identified in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling. Here we present an overview of BRCA mutation frequencies in our region and the retrospective evaluation and eventually reclassification of previously reported rare variants in light of recent findings.

GAPPS - Gastric Adenocarcinoma and Proximal Polyposis of the Stomach Syndrome in 8 Families Tested at Masaryk Memorial Cancer Institute - Prevention and Prophylactic Gastrectomies.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare variant of familial adenomatous polyposis. It is an autosomal-dominant cancer-predisposition syndrome with massive polyposis of the stomach and a significant risk of gastric adenocarcinoma. Li et al., 2016, described point mutations in the Ying Yang 1 binding site of the APC gene 1B promoter associated with GAPPS syndrome. The first GAPPS syndrome in a Czech family was described in 2016. At Masaryk Memorial Cancer Institute, GAPPS syndrome was diagnosed in eight families using Sanger sequencing. In all families, one mutation in promoter 1B of APC gene NM_001127511: c.-191T>C was detected. This mutation was not found in any patient with multiple colon polyposis without a detected classic mutation in the APC gene. In total, 24 carriers of this mutation in promoter 1B of the APC gene were detected. Out of those 24 carriers, 20 had massive gastric polyposis with more than 100 fundic glandular polyps diagnosed between the age of 22 and 65, 5 had already died of adenocarcinoma of the stomach (at the ages of 29, 40, 59, 60 and 64, respectively) and another woman was treated at the age of 29. Two female carriers do not yet have polyposis of the stomach at the ages of 31 and 65, respectively; one female carrier has incipient polyposis at the age of 58. A male carrier does not have any clinical symptoms, gastroscopy was not indicated because of his age. Prophylactic total gastrectomy with D2 lymphadenectomy has already been performed 6 times at Masaryk Memorial Cancer Institute, in 5 cases without adenocarcinoma at the ages of 27, 34, 44, 51 and 66, respectively; in one female carrier adenocarcinoma of the stomach was detected in a histology specimen. Two prophylactic gastrectomies with D1 lymphadenectomy were performed at University Hospital Brno at the ages of 42 and 50, respectively. In the Czech Republic point mutation c.-191T>C (rs879253783) in the 1B promoter of the APC gene is a frequent cause of gastric polyposis with a high risk of gastric adenocarcinoma, even at a young age. Positively tested individuals are recommended to high-risk oncology clinic. A necessary part of the discussion with the patient is information about a preventive gastrectomy.

BAP1 Syndrome - Predisposition to Malignant Mesothelioma, Skin and Uveal Melanoma, Renal and Other Cancers.

BACKGROUND: BAP1 syndrome is an autosomal dominant hereditary cancer syndrome associated with increased risk of malignant mesothelioma; uveal and cutaneous melanoma; kidney cancer; lung adenocarcinoma; meningioma; basaliomas; and breast, ovarian, and prostate tumors. The BAP1 gene (BRCA1-associated protein 1) is a tumor suppressor gene involved in DNA repair via homologous recombination. BAP1 regulates the cell cycle, differentiation, DNA damage responses, and cell proliferation through deubiquitination. Somatic mutations in the BAP1 gene are common in many types of tumors. OBSERVATION: Two families harboring a germline mutation in the BAP1 gene were diagnosed at Masaryk Memorial Cancer Institute (MMCI). A 27-year-old index female from one family was followed-up for multiple nevi. Her mother and uncle had malignant mesothelioma, and her maternal grandmother had uveal melanoma. The index case tested positive for a BAP1 (NM_004656.2): c.217delG/p.Asp73Metfs*5 frame-shift mutation. The melanoma was removed at the age of 28 and 31. In the second family, an 11-year-old index female had two nevi removed from her head, and a spitzoid-type skin lesion was diagnosed at the age of 11. Her 34-year-old mother had multiple nevi, and a skin lesion of spitzoid-type was removed from the abdomen. Both patients harbored a BAP1 (NM_004656.2): c.123-1G>T acceptor splice site mutation (IARC [International Agency for Research on Cancer] class 4 [probably pathogenic]). Preventive measures for BAP1 syndrome should include known risks for cancer. Tumors occur early and repeatedly. At the MMCI, we recommend physical examination by an oncologist, eyes and skin examination, every 6 months; whole-body magnetic resonance imaging, including the central nervous system, every year (or low-dose computed tomography/chest and abdomen magnetic resonance imaging); annual abdominal ultrasound, breast ultrasound, or mammography; a gynecological ultrasound examination every 6 months; colonoscopy starting at the age of 45; and other suitable surveillances based on family history. CONCLUSION: BAP1 syndrome is a complex cancer syndrome with a high risk of rare malignant mesothelioma, malignant skin and uveal melanoma, spitzoid-type skin lesions, and other tumors. Detection of this syndrome is essential for the survival of high-risk individuals. Supported by the grant project MH CZ - RVO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 5. 2019 Accepted: 6. 6. 2019.

Author Correction: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Tracing of Selected Viral, Bacterial, and Parasitic Agents on Vegetables and Herbs Originating from Farms and Markets.

Fresh vegetables and herbs are usually prepared and eaten raw without cooking or heating, which leads to a high risk of foodborne infection. The aim of the present study was to assess the contamination of raw vegetables, herbs, and the environment of food chains. Vegetable and herb samples originating both from the Czech Republic as well as from other countries were examined. The work was focused on the detection of commonly found, but also less frequently monitored foodborne pathogens, including viruses of the genus Norovirus (NoVs), hepatitis A virus (HAV), Listeria monocytogenes and Cronobacter spp. bacteria, and the parasites Cryptosporidium spp. and Giardia intestinalis. All samples were analyzed using individual RT-qPCR/qPCR assays; bacterial pathogens were also simultaneously detected using culture methods. The prevalence of the studied microorganisms in 623 samples ranged from 0.6% to 44.3% for individual pathogens. None of the samples were positive for the presence of HAV. Analysis of 157 environmental samples from 12 farms revealed the presence of NoVs in the environment of four farms. NoVs were detected in water samples as well as on the hands and gloves of workers. Escherichia coli was detected in all farms in the environmental samples and in eight farms in water samples. However, no sample of water exceeded the level of 100 CFU/mL for E. coli. None of the samples of water were positive for the presence of the studied parasites. Vegetables and herbs available from Czech markets and farms may pose a certain risk of foodborne disease, especially in the case of NoVs and parasites. PRACTICAL APPLICATION: This study provides valuable information on the microbiological quality of raw vegetables and herbs available from Czech markets and farms. Good hygienic practices aimed at reducing the incidence of pathogenic agents on fresh produce should not be neglected. Emphasis should be placed on the control of irrigation water, especially with respect to norovirus contamination. It is appropriate to combine culture methods and qPCR methods for the detection of bacterial agents.

Foodborne Bacterial, Viral, and Protozoan Pathogens in Field and Market Strawberries and Environment of Strawberry Farms.

Field-grown strawberries, the environment of strawberry farms and fresh strawberries from marketplaces were examined for bacterial, viral, and protozoan pathogens. The presence of bacteria was determined using culture and real-time PCR (qPCR), presence of protozoa and viruses using qPCR and reverse transcription qPCR, respectively. The highest proportion of positivity was observed for Escherichia coli both in field and purchased strawberries (up to 48.6%). Finding of Cronobacter ranged from 0.6% to 9% both for field and market strawberries. The prevalence of other pathogens (Listeria monocytogenes, Giardia intestinalis, Cryptosporidium sp., and Norovirus) in strawberries was below 4.5%; HAV was not detected at all. Positivity of the environment was determined to be lower than 2.1% for all microorganisms, except for E. coli. The concentration of pathogens in most samples did not exceed 100 CFU/g using culture and 1.8 × 102 GE/g of strawberries or swabbing area 6.1 × 102 GE/mL or swabbing area of environmental samples using qPCR. All studied farms applied preventive measures such as drip irrigation, avoidance of organic fertilizers, and use of mulch foils or gloves for workers to decrease contamination of strawberries. Despite this, certain pathogens were found in fresh strawberries. Even at low concentrations, these pathogens can be a source of infection for consumers. Thus, their presence in strawberries is of particular significance as these are mostly consumed fresh and without any thermal processing. PRACTICAL APPLICATION: Nonlegislatively monitored pathogens of bacterial, viral and parasitic origin were found in strawberries. Monitoring the presence of these pathogens in ready-to-eat food is therefore meaningful and important in terms of food safety, especially in relation to pathogens with low infectious dose (for example, viruses, parasites).

Hepatitis E virus in archived sera from wild boars (Sus scrofa), Czech Republic.

To determine whether hepatitis E virus (HEV) was distributed in the population of wild boars in South Moravia between 1990 and 2008, a total of 366 samples of archived sera from wild boars were investigated using serological (commercial ELISA) and molecular (RT-qPCR) methods. A total of 31 (8.5%) wild boars were seropositive, and from two of them, RNA sequences were recovered by nested RT-PCR. The presented results, with one of the oldest animal's HEV-positive serum (collected in 1990), suggest that wild boars may be a reservoir of HEV in the Czech Republic and that this virus has been circulating in studied areas for more than 20 years.

One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles.

MS2 phage-like particles (MS2 PLP) are artificially constructed pseudo-viral particles derived from bacteriophage MS2. They are able to carry a specific single stranded RNA (ssRNA) sequence of choice inside their capsid, thus protecting it against the effects of ubiquitous nucleases. Such particles are able to mimic ssRNA viruses and, thus, may serve as the process control for molecular detection and quantification of such agents in several kinds of matrices, vaccines and vaccine candidates, drug delivery systems, and systems for the display of immunologically active peptides or nanomachines. Currently, there are several different in vivo plasmid-driven packaging systems for production of MS2 PLP. In order to combine all the advantages of the available systems and to upgrade and simplify the production and purification of MS2 PLP, a one-plasmid double-expression His-tag system was designed. The described system utilizes a unique fusion insertional mutation enabling purification of particles using His-tag affinity. Using this new production system, highly pure MS2 PLP can be quickly produced and purified by a fast performance liquid chromatography (FPLC) approach. The system can be easily adapted to produce other MS2 PLP with different properties.

Public health risks associated with hepatitis E virus (HEV) as a food-borne pathogen.

Hepatitis E virus (HEV) is an important infection in humans in EU/EEA countries, and over the last 10 years more than 21,000 acute clinical cases with 28 fatalities have been notified with an overall 10-fold increase in reported HEV cases; the majority (80%) of cases were reported from France, Germany and the UK. However, as infection in humans is not notifiable in all Member States, and surveillance differs between countries, the number of reported cases is not comparable and the true number of cases would probably be higher. Food-borne transmission of HEV appears to be a major route in Europe; pigs and wild boars are the main source of HEV. Outbreaks and sporadic cases have been identified in immune-competent persons as well as in recognised risk groups such as those with pre-existing liver damage, immunosuppressive illness or receiving immunosuppressive treatments. The opinion reviews current methods for the detection, identification, characterisation and tracing of HEV in food-producing animals and foods, reviews literature on HEV reservoirs and food-borne pathways, examines information on the epidemiology of HEV and its occurrence and persistence in foods, and investigates possible control measures along the food chain. Presently, the only efficient control option for HEV infection from consumption of meat, liver and products derived from animal reservoirs is sufficient heat treatment. The development of validated quantitative and qualitative detection methods, including infectivity assays and consensus molecular typing protocols, is required for the development of quantitative microbial risk assessments and efficient control measures. More research on the epidemiology and control of HEV in pig herds is required in order to minimise the proportion of pigs that remain viraemic or carry high levels of virus in intestinal contents at the time of slaughter. Consumption of raw pig, wild boar and deer meat products should be avoided.

Male breast cancer in BRCA1 and BRCA2 mutation carriers: pathology data from the Consortium of Investigators of Modifiers of BRCA1/2.

BACKGROUND: BRCA1 and, more commonly, BRCA2 mutations are associated with increased risk of male breast cancer (MBC). However, only a paucity of data exists on the pathology of breast cancers (BCs) in men with BRCA1/2 mutations. Using the largest available dataset, we determined whether MBCs arising in BRCA1/2 mutation carriers display specific pathologic features and whether these features differ from those of BRCA1/2 female BCs (FBCs). METHODS: We characterised the pathologic features of 419 BRCA1/2 MBCs and, using logistic regression analysis, contrasted those with data from 9675 BRCA1/2 FBCs and with population-based data from 6351 MBCs in the Surveillance, Epidemiology, and End Results (SEER) database. RESULTS: Among BRCA2 MBCs, grade significantly decreased with increasing age at diagnosis (P = 0.005). Compared with BRCA2 FBCs, BRCA2 MBCs were of significantly higher stage (P for trend = 2 × 10(-5)) and higher grade (P for trend = 0.005) and were more likely to be oestrogen receptor-positive [odds ratio (OR) 10.59; 95 % confidence interval (CI) 5.15-21.80] and progesterone receptor-positive (OR 5.04; 95 % CI 3.17-8.04). With the exception of grade, similar patterns of associations emerged when we compared BRCA1 MBCs and FBCs. BRCA2 MBCs also presented with higher grade than MBCs from the SEER database (P for trend = 4 × 10(-12)). CONCLUSIONS: On the basis of the largest series analysed to date, our results show that BRCA1/2 MBCs display distinct pathologic characteristics compared with BRCA1/2 FBCs, and we identified a specific BRCA2-associated MBC phenotype characterised by a variable suggesting greater biological aggressiveness (i.e., high histologic grade). These findings could lead to the development of gender-specific risk prediction models and guide clinical strategies appropriate for MBC management.

Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices.

The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV- MS2 phage-like particles (MS2 PLP) - in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV - they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.

Changes in Microbial Composition of Wastewater During Treatment in a Full-Scale Plant.

The monitoring of wastewater treatment plants is important for their proper functioning as well as for re-use of water and also to avoid possible circulation of human or animal pathogens in our environment. The samples in this study originated from a full-scale wastewater treatment plant where the structure of the bacterial community was monitored using 454-pyrosequencing. The composition differed in different parts of the plant. In the effluent, bacteria belonging to phyla Proteobacteria, Actinobacteria, TM7 and Bacteroidetes were most frequently detected. The presence of Mycobacterium sp., Mycobacterium avium, Norovirus, Hepatitis A and E viruses was examined using quantitative real-time PCR. Mycobacterium sp. was detected in the effluent in quantities of up to 10(4) cells/ml. Mycobacterium avium subsp. paratuberculosis and subsp. hominissuis were detected in amounts of up to 10(3) cells/ml, and Norovirus group 1 and 2 were also detected. Our findings show the importance of monitoring and controlling the occurrence of specific pathogens in effluent, mainly because of the negative impact on human health when the water is reused.