Multivariate Analysis of the Predicted Probability of Smoking Behavior of Foster Care Minors: Results of a Study from Romania.

BACKGROUND: There are a multitude of factors that influence smoking status, and minors from the social protection system are a vulnerable category in terms of smoking. METHODS: The objective of this research was to assess the degree of smoking dependence and to identify potential predictors of smoking status in foster care teenagers. Smoker status was confirmed by dosing CO in the exhaled air, and the degree of dependence was assessed using the Fagerström score. We performed a multivariate logistic regression analysis. RESULTS: From the 275 foster care minors, 22.5% were current smokers. Exhaled CO was not influenced by general demographic factors, was associated with the frequency of smoking, and was positively correlated with the estimated number of cigarettes consumed daily and with the Fagerström score. The calculated probability of being a smoker was less than 20.4% in 75% of nonsmokers, whereas 75% of actual smokers had a predicted probability higher than 30.3%. CONCLUSIONS: In addition to age, gender, social environment, previous exposure to secondhand smoking, and residential type of foster care system, the expressed opinions regarding the health effects of tobacco use were associated with smoking in foster care teenagers.

Triplex High-Resolution Melting Assay for the Simultaneous Assessment of IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720 Genotypes in Chronic Hepatitis C Patients.

Chronic hepatitis C (CHC) is a leading cause of liver disease. Despite the improved efficacy of new antivirals, their high costs preclude their adoption in resource-limited settings, where CHC prevalence is highest. We developed a triplex high-resolution melting assay for the simultaneous assessment of three genetic polymorphisms related to the response to treatment and development of advanced fibrosis in CHC: IFNL3 rs12979860, ABCB11 rs2287622, and RNF7 rs16851720. We validated the assay in clinical samples from 130 CHC patients treated with classic therapy. The assay showed excellent reproducibility and 100% accuracy, sensitivity, and specificity against the gold standard Sanger sequencing. When added to routine examination data, genotype information significantly improved their performance for prediction of advanced liver fibrosis and sustained virological response (P = 0.041 and P = 0.011, respectively). Correspondingly, the full models had area under the receiver operating characteristic curve values of 0.842 (95% CI, 0.773-0.911) and 0.921 (95% CI, 0.870-0.972) and integrated discrimination improvements of 7.5% (95% CI, 2.5%-12.5%; P = 0.003) and 11.5% (95% CI, 5.8%-17.2%; P < 0.001), respectively. This is the first report on a diagnostic test for simultaneous genotyping of IFNL3, ABCB11, and RNF7 in CHC patients. Reliable and inexpensive, the assay should provide useful information for the clinical management of CHC, like identification of patients at risk of rapid disease progression or with high chances of response to classic therapy.

Effect of Storage Temperature on the Outgrowth and Toxin Production of Staphylococcus aureus in Freeze-Thawed Precooked Tuna Meat.

The aim of this study was to determine the time for a 3-log CFU/g outgrowth of Staphylococcus aureus and its toxin production in previously frozen precooked tuna meat (albacore [Thunnus alalunga] prepared as loin, chunk, and flake or skipjack [Katsuwonus pelamis] prepared as chunk and flake) held either at 21 or 27°C. A five-strain cocktail of enterotoxin-producing S. aureus was surface inoculated with ∼10(3) CFU/g onto tuna samples. The experimental time-temperature conditions were designed to mimic common industry holding conditions. After a 3-h incubation at 37°C, inoculated samples were individually vacuum sealed and stored at 20°C for 4 weeks. Following frozen storage, samples were thawed to the target temperature (21 or 27°C) and then incubated aerobically. Growth of S. aureus in tuna was then monitored using Baird Parker agar; simultaneously, aerobic plate counts, enterotoxin production, and sensory profile (color and odor) were determined. The results showed that the time for a 3-log CFU/g increase was >20 h at 21°C and 8 to 12 h at 27°C for albacore, with toxin production observed at 14 to 16 h at 21°C and at 8 h at 27°C. A 3-log CFU/g increase for skipjack occurred at 22 to 24 h at 21°C and at 10 to 14 h at 27°C. The toxin production in skipjack started at 20 to 22 h at 21°C and at 8 to 10 h at 27°C. Toxin production was observed before a 3-log increase was achieved in albacore samples at 21°C. Under all conditions, toxins were detected when the cell density of S. aureus was 6 log CFU/g. Overall, significantly faster S. aureus growth was observed in albacore compared with skipjack (P < 0.05), possibly owing to differences in sample composition (e.g., pH and salt content). The data developed from this study can be used by the tuna industry to model the growth and enterotoxin production of S. aureus and to design manufacturing controls that ensure food safety.

Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients.

Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

Development of a Dry Inoculation Method for Thermal Challenge Studies in Low-Moisture Foods by Using Talc as a Carrier for Salmonella and a Surrogate (Enterococcus faecium).

The objective of this study was to obtain dry inocula of Salmonella Tennessee and Enterococcus faecium, a surrogate for thermal inactivation of Salmonella in low-moisture foods, and to compare their thermal resistance and stability over time in terms of survival. Two methods of cell growth were compared: cells harvested from a lawn on tryptic soy agar (TSA-cells) and from tryptic soy broth (TSB-cells). Concentrated cultures of each organism were inoculated onto talc powder, incubated at 35 °C for 24 h, and dried for additional 24 h at room temperature (23 ± 2 °C) to achieve a final water activity of ≤ 0.55 before sieving. Cell reductions of Salmonella and E. faecium during the drying process were between 0.14 and 0.96 log CFU/g, depending on growth method used. There was no difference between microbial counts at days 1 and 30. Heat resistance of the dry inoculum on talc inoculated into a model peanut paste (50 % fat and 0.6 water activity) was determined after 1 and 30 days of preparation, using thermal death time tests conducted at 85 °C. For Salmonella, there was no significant difference between the thermal resistance (D(85 °C)) for the TSB-cells and TSA-cells (e.g. day 1 cells D(85 °C) = 1.05 and 1.07 min, respectively), and there was no significant difference in D(85 °C) between dry inocula on talc used either 1 or 30 days after preparation (P > 0.05). However, the use the dry inocula of E. faecium yielded different results: the TSB-grown cells had a significantly (P < 0.05) greater heat resistance than TSA-grown cells (e.g. D(85 °C) for TSB-cells = 3.42 min versus 2.60 min for TSA-cells). E. faecium had significantly (P < 0.05) greater heat resistance than Salmonella Tennessee regardless what cell type was used for dry inoculum preparation; therefore, it proved to be a conservative but appropriate surrogate for thermal inactivation of Salmonella in low-moisture food matrices under the tested conditions.

Circulating RNA molecules as biomarkers in liver disease.

Liver disease is a major cause of morbidity and mortality worldwide. As in other fields of medicine, there is a stringent need for non-invasive markers to improve patient diagnostics, monitoring and prognostic ability in liver pathology. Cell-free circulating RNA molecules have been recently acknowledged as an important source of potential medical biomarkers. However, many aspects related to the biology of these molecules remain to be elucidated. In this review, we summarize current concepts related to the origin, transportation and possible functions of cell-free RNA. We outline current development of extracellular RNA-based biomarkers in the main forms of non-inherited liver disease: chronic viral hepatitis, hepatocellular carcinoma, non-alcoholic fatty liver, hepato-toxicity, and liver transplantation. Despite recent technological advances, the lack of standardization in the assessment of these markers makes their adoption into clinical practice difficult. We thus finally review the main factors influencing quantification of circulating RNA. These factors should be considered in the reporting and interpretation of current findings, as well as in the proper planning of future studies, to improve reliability and reproducibility of results.

Survival of Salmonella Tennessee, Salmonella Typhimurium DT104, and Enterococcus faecium in peanut paste formulations at two different levels of water activity and fat.

Long-term survival of heat-stressed Salmonella Tennessee, Salmonella Typhimurium DT104, and Enterococcus faecium was evaluated in four model peanut paste formulations with a combination of two water activity (aw) levels (0.3 and 0.6) and two fat levels (47 and 56%) over 12 months at 20 ± 1°C. Prior to storage, the inoculated peanut paste formulations were heat treated at 75°C for up to 50 min to obtain an approximately 1.0-log reduction of each organism. The cell population of each organism in each formulation was monitored with tryptic soy agar plate counts, immediately after heat treatment, at 2 weeks for the first month, and then monthly for up to 1 year. The log reductions (log CFU per gram) following 12 months of storage were between 1.3 and 2.4 for Salmonella Tennessee, 1.8 and 2.8 for Salmonella Typhimurium, and 1.1 and 2.1 for E. faecium in four types of model peanut paste formulations. Enhanced survivability was observed in pastes with lower aw for all organisms, compared with those with higher aw (P < 0.05). In contrast, the effect of fat level (47 and 56%) on survival of all organisms was not statistically significant (P > 0.05). Whereas survivability of Salmonella Tennessee and Typhimurium DT104 did not differ significantly (P > 0.05), E. faecium demonstrated higher survivability than Salmonella (P < 0.05). Salmonella survived in the model peanut pastes well over 12 months, which is longer than the expected shelf life for peanut butter products. The information from this study can be used to design safer food processing and food safety plans for peanut butter processing.

Heat resistance of histamine-producing bacteria in irradiated tuna loins.

Consumption of foods high in biogenic amines leads to an illness known as histamine, or scombrotoxin, poisoning. The illness is commonly associated with consumption of fish with high levels of histamine ( $ 500 ppm). The objective of this study was to determine and compare the heat resistance of five histamine-producing bacteria in irradiated albacore tuna loins. Heat-resistance parameters (D- and z-values) were determined for Morganella morganii, Raoultella planticola, Hafnia alvei, and Enterobacter aerogenes. D- or z-values were not determined for Photobacterium damselae, which was the most heat-sensitive organism in this study. P. damselae declined > 5.9 log CFU/g after a heat treatment of 50°C for 10 min, 54°C for 3 min, and 56°C for 0.5 min. M. morganii was the most heat-resistant histamine-producing bacteria in albacore tuna loins, followed by E. aerogenes, H. alvei, and R. planticola. M. morganii and E. aerogenes had the highest D(50°C), 49.7 ± 17.57 and 51.8 ± 17.38 min, respectively. In addition, M. morganii had the highest D-values for all other temperatures (54, 56, and 58°C) tested. D- and zvalues were also determined for M. morganii in skipjack tuna. While no significant (P > 0.05) difference was observed between D(54°C) and D(56°C) of M. morganii in either albacore or skipjack tuna, the D(58°C) (0.4 ± 0.17 min) was significantly lower (P < 0.05) in skipjack than in albacore (0.9 ± 0.24 min). The z-values for all organisms tested were in the range of 3.2 to 3.8°C. This study suggests that heat treatment designed to control M. morganii in tuna loins is sufficient for controlling histamine-producing bacteria in canned-tuna processing environments.

Validation of pepperoni process for control of Shiga toxin-producing Escherichia coli.

The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.

Inactivation of Shiga toxin-producing Escherichia coli in single-strength lemon and lime juices containing preservatives.

The objective of this study was to determine the inactivation of non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison with O157 STEC in commercially produced, shelf-stable lemon and lime juices. The present validation tests confirmed that storage of the juices containing preservatives at room temperatures (22°C) for 3 days (72 h) ensures a >6-log reduction of O26, O45, O103, O111, O121, O145, and O157 STEC. These results demonstrate that non-O157 STEC had survival abilities comparable to those of E. coli O157:H7 strains in acidic food products such as lemon and lime juices (pH 2.5 ± 0.1); therefore, the storage conditions deemed to inactivate E. coli O157:H7 similarly inactivate the non-O157 serotypes.

Thermal resistance parameters for Shiga toxin-producing Escherichia coli in apple juice.

The purpose of the present study was to determine the heat resistance of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison to E. coli O157:H7 in single-strength apple juice without pulp. The thermal parameters for stationary-phase and acid-adapted cells of E. coli strains from serogroups O26, O45, O103, O111, O121, O145, and O157:H7 were determined by using an immersed coil apparatus. The most heat-sensitive serotype in the present study was O26. Stationary-phase cells for serotypes O145, O121, and O45 had the highest D(56°C)-value among the six non-O157 serotypes studied, although all were significantly lower (P < 0.05) than that of E. coli O157:H7. At 60°C E. coli O157:H7 and O103 demonstrated the highest D-values (1.37 ± 0.23 and 1.07 ± 0.03 min, respectively). The D(62°C) for the most heat-resistant strain belonging to the serotype O145 was similar (P > 0.05) to that for the most resistant O157:H7 strain (0.61 ± 0.17 and 0.60 ± 0.09 min, respectively). The heat resistance for stationary-phase cells was generally equal to or higher than that of acid-adapted counterparts. Although E. coli O157:H7 revealed D-values similar to or higher than the individual six non-O157 STEC serotypes in apple juice, the z-values for most non-O157 STEC tested strains were greater than those of E. coli O157:H7. When data were used to calculate heat resistance parameters at a temperature recommended in U.S. Food and Drug Administration guidance to industry, the D(71.1°C) for E. coli O157:H7 and non-O157 STEC serotypes were not significantly different (P > 0.05).

Sources and risk factors for contamination, survival, persistence, and heat resistance of Salmonella in low-moisture foods.

Sources and risk factors for contamination, survival, persistence, and heat resistance of Salmonella in low-moisture foods are reviewed. Processed products such as peanut butter, infant formula, chocolate, cereal products, and dried milk are characteristically low-water-activity foods and do not support growth of vegetative pathogens such as Salmonella. Significant food safety risk might occur when contamination takes place after a lethal processing step. Salmonella cross-contamination in low-moisture foods has been traced to factors such as poor sanitation practices, poor equipment design, and poor ingredient control. It is well recognized that Salmonella can survive for long periods in low-moisture food products. Although some die-off occurs in low-moisture foods during storage, the degree of reduction depends on factors such as storage temperature and product formulation. The heat resistance of Salmonella is affected by many factors, mostly by strain and serotypes tested, previous growth and storage conditions, the physical and chemical food composition, test media, and the media used to recover heat-damaged cells. Salmonella heat resistance generally increases with reducing moisture. Care must be taken when applying published D- and z-values to a specific food process. The product composition and heating medium and conditions should not be significantly different from the product and process parameters used by the processors.

Non-O157 Shiga toxin-producing Escherichia coli in foods.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains have been linked to outbreaks and sporadic cases of illness worldwide. Illnesses linked to STEC serotypes other than O157:H7 appear to be on the rise in the United States and worldwide, indicating that some of these organisms may be emerging pathogens. As more laboratories are testing for these organisms in clinical samples, more cases are uncovered. Some cases of non-O157 STEC illness appear to be as severe as cases associated with O157, although in general cases attributed to non-O157 are less severe. There is much variation in virulence potential within STEC serotypes, and many may not be pathogenic. Of more than 400 serotypes isolated, fewer than 10 serotypes cause the majority of STEC-related human illnesses. Various virulence factors are involved in non-O157 STEC pathogenicity; the combined presence of both eae and stx genes has been associated with enhanced virulence. A scientific definition of a pathogenic STEC has not yet been accepted. Several laboratories have attempted to develop detection and identification methods, and although substantial progress has been made, a practical method of STEC detection has yet to be validated. Worldwide, foods associated with non-O157 STEC illness include sausage, ice cream, milk, and lettuce, among others. Results from several studies suggest that control measures for O157 may be effective for non-O157 STEC. More research is needed to uncover unique characteristics and resistances of non-O157 STEC strains if they exist. The public health significance of non-O157 STEC and the implications for industry practices and regulatory actions are discussed.

Inactivation of Escherichia coli O157:H7 in single-strength lemon and lime juices.

Survival of a five-strain mixture of stationary phase (nonadapted) and acid-adapted Escherichia coli O157:H7 in single-strength lemon and lime juices was evaluated at room temperature (22 degrees C). The juices were reconstituted from concentrates that contained no preservatives and intrinsic pH values of 2.5 to 2.6 and titratable acidities of 4.51 to 4.53% (wt/vol, citric acid). A greater than 5-log reduction of stationary-phase cells was achieved in both lemon and lime juices after 72 h of incubation. Similar log reductions were obtained when the reconstituted juices were adjusted to pH 2.7, which is above the highest value normally observed in juice processing plants during the reconstitution of single-strength lemon or lime juice from concentrates. Lemon juice had a significantly higher inhibitory effect (P < 0.05) on E. coli O157:H7 than did lime juice. Validation tests with commercially produced shelf-stable lemon and lime juices confirmed that storage of the juices at room temperature (22 degrees C) for 3 days may be an alternative to heat treatment to ensure the 5-log reduction of vegetative pathogens of concern required for the products under the U.S. Food and Drug Administration juice hazard analysis and critical control point regulations.

Survival of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in cranberry juice concentrates at different oBrix levels.

Survival of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes was evaluated in cranberry juice concentrates to determine if a 5-log reduction could be achieved without any other treatment. Inactivation at 0 degrees C in concentrates with different oBrix levels was determined for a five-strain composite of the individual pathogens in two physiological states. In concentrates at 18 to 46 oBrix (pH 2.2 to 2.5), all three pathogens (stationary-phase or acid-adapted cells) showed at least a 5-log reduction after a 6- or 24-h incubation. At 14 oBrix (pH 2.5), a reduction greater than 5 log was obtained for L. monocytogenes and Salmonella after up to 24 h of incubation, but for E. coli O157:H7, 96 h of incubation was needed to consistently obtain a reduction greater than 5 log. All three pathogens in the stationary phase survived longer than in the acid-adapted phase under the same conditions. The most resistant was stationary-phase E. coli O157:H7, and the most sensitive was acid-adapted L. monocytogenes. The rate of pathogen destruction increased with increasing oBrix level of the juice concentrate, which suggests that concentrated acids and/orsome intrinsic compounds may play an important role in the bactericidal effects of cranberry juice concentrates.

Efficacy of selected acidulants in pureed green beans inoculated with pathogens (Escherichia coli O157:H7 and Listeria monocytogenes).

Studies were conducted to evaluate the combined effect of selected acidulants (acetic, citric, malic, and phosphoric acid) and heat on foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) in pureed green beans. To establish a consistent reference point for comparison, the molar concentrations of the acids remained constant while the acid-to-puree ratio, titratable acidity, and undissociated acid were either measured or calculated for a target acidified green beans at a pH of 3.8, 4.2, and 4.6. The D-values at 149 degrees F were used as the criteria for acid efficacy. Generally, acetic acid (puree, pH 3.8 and 4.2) represented the most effective acid with comparatively low D-values irrespective of the target microorganism. A 10-s heating at 149 degrees F inactivated approximately 10(6) CFU/ml of E. coli O157:H7 in pureed beans at pH 3.8. The efficacy of acetic acid is likely related to the elevated percent titratable acidity, undissociated acid, and acid-to-puree ratio. The effectiveness (which in this study represents the combined effect of acid and heat) of the remaining acids (citric, malic, and phosphoric) at puree pH values of 3.8 and 4.2 were statistically insignificant (alpha = 0.05). Surprisingly, acetic acid (puree, pH 4.6) appeared to be the least effective as compared to the other acids tested (citric, malic, and phosphoric) especially on E. coli O157:H7 cells, while L. monocytogenes had a similar resistance to all acids at puree pH 4.6. With the exception of citric acid (pH 3.8), acetic acid (pH 4.6), and malic acid (pH 3.8 and 4.6), which were statistically insignificant (P > 0.05), the D-values for L. monocytogenes were statistically different (P < or = 0.05) and higher than the D-values for E. coli under similar experimental conditions. A conservative process recommendation (referred to as the "safe harbor" process) was found sufficient and applicable to pureed green beans for the pH range studied.

Thermal resistance parameters for pathogens in white grape juice concentrate.

The heat resistance of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes that were in stationary phase, had been exposed to high osmotic pressure, or were acid adapted was evaluated in white grape juice concentrate (58 degrees Brix, pH 3.3). The most heat-resistant cells of all three pathogens were those exposed to high osmotic pressure or in stationary phase. Unlike in single-strength juices, in concentrate the acid-adapted cells for all three pathogens were less heat resistant than were cells in the other physiological states. E. coli O157:H7 had the highest heat resistance for all temperatures tested (e.g., D62 degrees C = 1.8 +/- 0.3 min, with a z-value of 9.9 +/- 0.6 degrees C). L. monocytogenes exposed to high osmotic pressure had the highest z-value (12.3 +/- 1.2 degrees C), although its D-values for all temperatures tested were lower (e.g., D62 degrees C = 0.93 +/- 0.1 min) than those for E. coli O157:H7. Salmonella was the most sensitive of the pathogens under all conditions. Based on the results obtained in this study, one example of a heat treatment that will inactivate 5 log units of all three pathogens in white grape juice concentrate was calculated as 1.5 min at 71.1 degrees C (z = 10.3 degrees C). Validation studies confirmed the predicted D71 degrees C for E. coli O157:H7 exposed to high osmotic pressure.