Reproducibility study for the detection of Staphylococcal enterotoxins in dairy products between official Italian national laboratories.

Staphylococcal food poisoning is a common foodborne disease caused by the ingestion of staphylococcal enterotoxins (SEs) produced mainly by enterotoxigenic strains of Staphylococcus aureus. To date, 21 SEs and/or enterotoxin-like types have been identified, several of which represent a potential hazard for consumers. To protect consumer health and to reduce the amount of SE-contaminated food entering the market, European Union legislation regulating food safety requires testing for SEs. The Italian National Reference Laboratory organized a ring trial to test technical and analytical proficiency in the national network of official food laboratories. Twenty-four laboratories took part, and each received and analyzed 24 blind dairy samples. Reproducibility of the results from the laboratories was assessed by the Cohen k index, and accuracy (sensitivity and specificity) was evaluated according to the International Organization for Standardization definition (ISO 16140:2003). Trial results revealed partially satisfactory agreement: 254 of 276 possible paired participants (92%) reached a k value >0.60, which is conventionally recognized as satisfactory. Accuracy was deemed satisfactory; 100% sensitivity and 100% specificity were achieved by 22 and 18 of the 24 laboratories, respectively.

Validation according to ISO 16140:2003 of a commercial real-time PCR-based method for detecting Campylobacter jejuni, C. coli, and C. lari in foods.

Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.

Verocytotoxin-producing Escherichia coli O157 in minced beef and dairy products in Italy.

A total of 3879 samples of foodstuffs were examined for the presence of Verocytotoxin-producing Escherichia coli O157 (VTEC O157). The survey was conducted by 9 of the 10 Italian Veterinary Public Health Laboratories. Samples were collected between May 2000 and September 2001 in 14 regions and comprised 931 minced beef specimens and 2948 dairy products (DP) with less than 60 days of ripening. The DP included 657 pasteurised and 811 unpasteurised bovine DP, 477 pasteurised and 502 unpasteurised ovine DP, and 501 water-buffalo's milk mozzarella cheese. Samples were collected at retail level, from plants processing minced beef and dairy plants and from farms directly manufacturing cheeses. All the samples were tested using a sensitive procedure based on ISO/DIS 16654:1999 (later ISO 16654:2001), which includes an immunomagnetic separation step. A preliminary inter-laboratory trial was organised with artificially contaminated samples to assess the ability of all the participating laboratories to isolate E. coli O157 by the established procedure. VTEC O157 was isolated from four (0.43%) of the minced beef samples, collected in four different regions and during different months, but was not detected in any of the dairy products. E. coli O157 VT-eae+ was isolated from one raw cow's milk cheese. This survey provided national data on the presence of VTEC O157 in foodstuffs, demonstrating a low prevalence of the organism. The survey also encouraged updating of knowledge and procedures on VTEC O157 in laboratories with official responsibility for microbiological testing of foods of animal origin.

Phase-shift calibration algorithm for phase-shifting interferometry.

We propose a novel phase-shift calibration algorithm. With this technique we determine the unknown phase shift between two interferograms by examining the sums and differences of the intensities on each interferogram at the same spatial location, i.e., I1(x, y) +/- I2(x, y). These intensities are normalized so that they become sinusoidal in form. A uniformly illuminated region of the interferograms that contains at least a 2pi variation in phase is examined. The extrema of these sums and differences are found in this region and are used to find the unknown phase shift. An error analysis of the algorithm is provided. In addition, an error-correction algorithm is implemented. The method is tested by numerical simulation and implemented experimentally. The numerical tests, including digitization error, indicate that the phase step has a root-mean-square (RMS) phase error of less than 10(-6) deg. Even in the presence of added intensity noise (5% amplitude) the RMS error does not exceed 1 deg. The accuracy of the technique is not sensitive to nonlinearity in the interferogram.

Phase-shifting interferometry with uncalibrated phase shifts.

A computationally efficient algorithm for phase-shifting interferometry with imprecise phase shifts is developed. It permits the use of an uncalibrated phase shifter and is also insensitive to spatial intensity variations. The measurement has both spatial and temporal aspects. Comparisons are made between pixels within the same interferogram, and these comparisons are extended across a set of interferograms by a maximum-minimum procedure. A test experiment is performed and confirms the theoretical results. An additional advantage of the algorithm is that an error measure can be developed. This error measure is used to implement an error correction scheme.

Liver ischemia for hepatic resection: where is the limit?

BACKGROUND: A consecutive series of 50 patients who submitted to 53 hepatic resections with use of continuous normothermic liver ischemia is reported. METHODS: Portal triad clamping has been used in 28 cases, with associated inferior vena caval clamping above and below the liver (hepatic vascular exclusion) in 25 patients. The size of the tumor required major hepatic resection in 38 cases (71.7%). Malignant tumors (83%) were the most common indication for liver resection. Patients were placed in three groups according to the duration of liver ischemia: group A, less than 30 minutes (9 patients); group B, 30 to 60 minutes (29 patients); and group C, 60 or more (15 patients). RESULTS: No differences in mortality rates (5.7% in the entire series and 0% in group C) and morbidity rate could be shown. No significant difference was found in postoperative liver test results, and no persistent alteration remained thereafter. Liver biopsy at 6 and 12 months after operation did not reveal any chronic damage. Liver capability to regenerate was maintained as documented by postoperative computerized tomography scan or magnetic resonance imaging. CONCLUSIONS: Because interruption of hepatic blood flow in normothermia is safe for at least 60 minutes (up to 85 minutes in this study), vascular clamping is recommended for hazardous liver resections to minimize blood loss, which appears to be the main factor of death and morbidity.