Petros Kokkinos

Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Salmonella in Food

Salmonella infection represents a considerable global burden, with significant health and economic impacts. Salmonellosis is most often attributed to the consumption of contaminated foods such as poultry, beef, pork, eggs, milk, seafood, nut products, and fresh produce. Increased public awareness related to food-borne contamination resulted in greater efforts to develop more sensitive, rapid, and inexpensive methods of pathogens detection. Loop-mediated isothermal amplification (LAMP) constitutes a promising solution for rapid diagnosis of food-borne pathogens and is increasingly been applied for the specific diagnosis of different pathogens, Salmonella included. We have reviewed the application of LAMP for the specific detection of Salmonella in food matrices, compared with conventional culture techniques, and in terms of applicability, food matrices, type of assays, target genes, assay temperature, time and equipment, specificity, sensitivity, and robustness. The pros and cons of Salmonella LAMP assays are presented. The potential of LAMP for the development of new on-site diagnostics for the food and agricultural industries and its use as a routine Salmonella screening tool are discussed. Salmonella-specific LAMP assays are expected to provide a very robust, innovative, and powerful molecular diagnostic method for food safety testing services and public health authorities.

Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities.

Current status in wastewater treatment, reuse and research in some mediterranean countries

AbstractThe status of treated wastewater reuse as experienced in some Mediterranean Basin countries such as Greece, Israel, Italy and Cyprus is examined. General background information is given for each of these Mediterranean countries, including natural water resources, climatic conditions (temperature, rainfall), generated wastewater, crops cultivated and irrigated with effluent, and related aspects of reuse. The examined parameters include treatment strategies, wastewater reuse standards applied in each country, effluent reuse research in progress in the above target countries related to the treatment technologies, water quality, regulations, economics, public acceptance, risk assessment, benefits, keys for potential success and main constraints. Emphasis has been given to the benefits of treated wastewater reuse in integrated water resources management systems and its role for water cycle management, solving water scarcity issues mainly in arid and semi-arid regions of the Mediterranean basin. The exp...

Epidemiological investigation of two parallel gastroenteritis outbreaks in school settings

BackgroundTwo parallel gastroenteritis outbreaks occurred in an elementary school and a neighboring kindergarten in Kilkis, Northern Greece in 2012. The aim of the study was the investigation of these two parallel outbreaks as well as their possible source.MethodsTwo retrospective cohort studies were performed to identify the mode and the vehicle of transmission as well as the possible connection between them.ResultsElementary school and kindergarten populations of 79.9% (119/149) and 51.1% (23/45) respectively, participated in the study. Case definition was satisfied by 65 pupils from the elementary school and 14 from the kindergarten. For elementary school, 53 cases were considered primary cases of the outbreak and were included in the analysis. Based on the results of the multivariate analysis, consumption of tap water was the only statistically significant independent risk factor of gastroenteritis (RR = 2.34, 95% C.I.: 1.55-3.53).; a finding supported by the shape of the epidemic curve which referred to a common point source outbreak with secondary cases. For kindergarten, no statistically significant risk factor was identified, and the epidemic curve supported a person-to-person transmission according univariate analysis. Norovirus GI and GII and human Adenovirus were detected by Real Time PCR in stool samples from seven children of elementary school, but stool samples were not collected by children of the kindergarten.ConclusionsEven though the etiological agent of the outbreak was not verified, combined epidemiological and laboratory results were in favor of a waterborne viral gastroenteritis outbreak at the elementary school, followed by a person to person spread at the kindergarten.

Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor ΦΧ174 is recommended as a suitable model for adenovirus.

A Gastroenteritis Outbreak Caused by Noroviruses in Greece

In June 2006, an outbreak alert regarding cases of acute gastroenteritis in a region in North Eastern Greece (population 100,882 inhabitants), triggered investigations to guide control measures. The outbreak started the first days of June, and peaked in July. A descriptive epidemiological study, a virological characterization of the viral agent identified from cases as well as a phylogenetic analysis was performed. From June 5 to September 3, 2006 (weeks 23–44), 1,640 cases of gastroenteritis (45.2% male and 54.8% female, aged 3 months to 89 years) were reported. The overall attack rate for the period was 16.3 cases/1,000 inhabitants. About 57% of cases observed were under the age of 15 years. nalysis of faecal samples identified Norovirus GII strains. Fifteen different Norovirus GII strains were recorded, presenting a homology of 94.8% (86–97%) to GII strains obtained from GenBank. The long duration of the outbreak suggests an important role of person-to-person transmission, while the emergence of the outbreak was possibly due to contaminated potable water, although no viruses were detected in any tested water samples. This outbreak underscores the need for a national surveillance system for acute non-bacterial gastroenteritis outbreaks.

A large waterborne gastroenteritis outbreak in central Greece, March 2012: challenges for the investigation and management

In March 2012, there was an unusual increase of gastroenteritis cases in a district with 37,264 inhabitants in central Greece. It was estimated that more than 3600 people developed symptoms. A 1:1 case-control study showed that consumption of tap water was a risk factor for acquiring infection [odds ratio (OR) 2.18, 95% confidence interval (CI) 1.11-4.28]. Descriptive data, low gastroenteritis incidence in adjacent areas with different water supply systems, and water-quality data further supported the hypothesis of a waterborne outbreak. Thirty-eight stool samples were positive for rotavirus. Bacterial indicators of recent faecal contamination were detected in samples from the water source and ice cubes from a local production enterprise. Molecular epidemiology of rotavirus strains, apart from the common strain, G3[P8], identified the unusual G/P combination G2P[8]. Water sanitation measures contributed to the control of the outbreak. This outbreak demonstrated the need for the cooperation of laboratories with different expertise and the importance of early notification of waterborne gastroenteritis outbreaks.

An outbreak of hepatitis A in Roma populations living in three prefectures in Greece.

An outbreak of hepatitis A virus (HAV) infection affected Roma populations living in three prefectures of northeastern Greece. Between July and November 2007, 124 cases were reported. We carried out investigations to characterize the pathogen, to identify the source of infection and the route of transmission. Using the RT-PCR technique, HAV strains of the same genotype were detected in all sera from a subset of patients with acute disease. These showed more than 99.8% identity, suggesting a common source. A questionnaire was also completed to collect clinical and epidemiological information. The outbreak affected mainly Roma children aged <10 years. An inspection of Roma settlements showed that poor sanitary conditions were associated with the HAV outbreak.

Effects of physiological mechanical strains on the release of growth factors and the expression of differentiation marker genes in human osteoblasts growing on Ti-6Al-4V

Mechanical loading factors at the bone-implant interface are critical for the osseointegration and clinical success of the implant. The aim of the present investigation was to study the effects of mechanical strain on the orthopedic biomaterial Ti-6Al-4V/osteoblast interface, using an in vitro model. Homogeneous strain was applied to human bone marrow derived osteoblasts (HBMDOs) cultured on Ti-6Al-4V, at physiological levels (strain magnitudes 500 microstrain (microepsilon) and 1000 microepsilon, at frequencies of load application 0.5 Hz and 1 Hz), by a mechanostimulatory system, based on the principle of four-point bending. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) was used to determine the mRNA expression of Cbfa1 and osteocalcin at different loading conditions. The release of growth factors as a response to stretch was also investigated by transferring stretch-conditioned media to nonstretched cells and by measuring their effect on the regulation of DNA synthesis. Mechanical loading was found to contribute to the regulation of osteoblast differentiation by influencing the level of the osteoblast-specific transcription factor Cbfa1, both at the mRNA and protein level, and also the level of osteocalcin, which is regarded as the most osteoblast-specific gene. Both genes were differentially expressed shortly after the application of different mechanical stimuli, in terms of strain frequency, magnitude, and time interval. Media conditioned from mechanically stressed HBMDOs stimulate DNA synthesis more intensely compared to media conditioned from unstressed control cultures, indicating that mechanical strain induces the release of a mitogenic potential that regulates cell proliferation.

Prevalence of Legionella spp. in water systems of hospitals and hotels in South Western Greece

The aim of the present study was to determine the prevalence of Legionella spp. in water systems of hospitals and hotels located in South Western Greece, to study the molecular epidemiology of the isolated strains and their possible association with bacterial contamination (total count and Pseudomonas aeruginosa), the water pH, and temperature. A prevalence survey for Legionella spp. by culturing techniques in water distribution systems of eight hospitals and nine hotels occurred in South Western Greece. Water sampling and microbiological analysis were carried out following the ISO methods. Legionella pneumophila was detected in 33% and 36% of the distribution systems of hospitals and hotels, respectively. Our survey results suggest a frequent prevalence of elevated concentrations of Legionella spp. in water systems of hospitals and hotels. Our investigation has confirmed the need to regularly monitor the microbiological condition of water systems in hospitals and hotels.