Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction-enzyme-linked immunosorbent assay.

AIMS: This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. METHODS AND RESULTS: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. CONCLUSIONS: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.

Norovirus in retail shellfish.

Norovirus is a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of norovirus infections worldwide are due to genogroup II noroviruses. Bivalve molluscs (mussels, clams and oysters) at the end of the commercial chain, the points of purchase, were sampled between 2005 and 2008 in several retail points in Apulia, Italy, and screened by a semi-nested RT-PCR specific for genogroup II noroviruses. Noroviral RNA was detected in 12.1% of the samples, with lower frequency being observed in samples obtained from hypermarkets (8.1%) rather than in samples from open-air markets and fish shops (17.6% and 16.2%, respectively). By sequence analysis, the strains were characterized as norovirus variants GII.4/2004 and GII.b/Hilversum, which were both circulating in Italy in the same time-span.

Is the common presence of glucose intolerance in old age a reliable index for the subsequent occurrence of fasting hyperglycemia?

The aim of the present study was to verify the validity of diagnostic criteria developed by the NDDG for the diagnosis of diabetes mellitus in old age. One hundred and fifty-one ambulatory old (range: 66-77 years) subjects (group A) underwent OGTT showing the following results: 33% normal, 12% non-diagnostic, 23% impaired glucose tolerance (IGT), 32% diabetic-type tolerance (DT). In addition, 84 subjects (group B) selected from 1978 to 1982 (42 aged 51-60 years, 30 aged 61-70, and 12 aged 71-80) with abnormalities of glucose tolerance during OGTT (IGT or DT) were asked to control their fasting plasma glucose every month during 1984. In group B a significant correlation between DT and subsequent development of fasting hyperglycemia was observed only in the subjects of the 6th and 7th decades of age. On the contrary, no subjects aged 71-80 years developed fasting hyperglycemia. The authors suggest that a high prevalence of abnormalities in glucose tolerance according to NDDG exists in old age which cannot be considered evidence of a true diabetes mellitus being unpredictive of a progression towards fasting hyperglycemia.

Impaired insulin secretion in human diabetes mellitus. Interactions between naloxone, phentolamine and lysine acetylsalicylate upon glucose induced insulin release.

Non insulin-dependent diabetes mellitus (Type II) is characterized by the loss of the acute insulin response to glucose. Met-enkephalin, catecholamines and prostaglandin E (PGE) have all been reported to inhibit the acute insulin response to glucose in normal humans. To evaluate the hypothesis that an increased sensitivity to these endogenous substances may play a role in defective insulin secretion in diabetes, we evaluated the effects of three blocking drugs upon the impaired insulin response to glucose in Type II diabetic subjects, as well as glucose-induced insulin secretion in normal humans. In diabetics, acute insulin responses to glucose were significantly increased by all the agents tested (naloxone, phentolamine and lysine acetylsalicylate), but only the cyclooxygenase inhibitor significantly augmented second phase insulin secretion and glucose disappearance rates. The combined infusion of the three agents caused a striking increase of the acute insulin response to glucose (response before: 3 +/- 2 uU/ml; after: 22 +/- 6 uU/ml, p less than 0.01). This was accompanied by a ninefold augmentation of the second phase of insulin secretion which was the result of a synergistic interaction between the three drugs (response significantly higher than the sum of single effects). In normals, insulin responses to glucose were also significantly increased by the combined infusions of the drugs, but to a significantly lesser extent than that of diabetics. This different degree of insulin potentiation between normals and diabetics under the infusion of the three agents persisted even when the prestimulus glucose level of normals was matched to that of diabetics by a glucose infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycemic control with an artificial pancreas improves insulin responses to both oral and glucose in nonobese noninsulin-dependent diabetic subjects.

Insulin secretory responses to both oral and intravenous glucose were investigated in 12 nonobese noninsulin-dependent diabetic subjects before and after strict metabolic control of blood glucose levels without weight loss. Glycemic control was achieved by applying an artificial pancreas to all diabetics for 2 or 3 days, which led to restoration of normal fasting blood glucose levels and to significant reduction of fasting plasma insulin (p less than 0.01) and C-peptide (p less than 0.05) levels. Initially, the insulin response to oral glucose was weak and delayed, but increased significantly after treatment (p less than 0.01), although none of the diabetic subjects achieved completely normal glucose tolerance. The i.v. glucose tolerance test (0.33 g/kg) revealed that all diabetics lacked acute insulin response in the basal state with low glucose disappearance rates (0.37 +/- 0.07 %/min). After 48h of normoglycemia, these figures did not change significantly, although the insulinogenic index (insulin area/glucose area) was significantly increased (p less than 0.05). A marked increase in both phases of insulin secretion was evident when a larger intravenous glucose pulse (0.66 g/kg) was used in some diabetics in order to raise the blood glucose concentrations of the post-treatment test to those of the pre-treatment test. In absolute terms, the insulin responses of the post-treatment tests were not significantly different from those of sex-, age- and weight-matched control subjects, but were significantly lower if related to the corresponding plasma glucose responses (insulinogenic index lower than that of controls). These studies in nonobese noninsulin-dependent diabetic subjects indicate that glycemic control with an artificial pancreas improves insulin response to glucose, suggesting that chronic hyperglycemia may stress the impaired B-cell secretory capacity of diabetes.

Normalization by sodium salicylate of the impaired counterregulatory glucagon response to hypoglycemia in insulin-dependent diabetes. A possible role for endogenous prostaglandins.

The present study was undertaken to evaluate the influence of sodium salicylate on the counterregulatory glucagon response to insulin-induced hypoglycemia in both insulin-dependent diabetic subjects (IDDM) and normal controls. The IDDM group consisted of 5 patients with recent onset of disease (less than 45 days), a normal glucagon response to hypoglycemia, and no detectable insulin antibodies (group 1); and 7 patients with duration of disease between 1 and 5 yr, a reduced glucagon response to hypoglycemia, and no insulin antibodies (group 2). Ten healthy subjects served as a control group. The infusion of sodium salicylate (40 mg/min) during insulin-induced hypoglycemia (1 mU/kg-min for 60 min) in normal subjects caused a significant increase of the counterregulatory glucagon response both in terms of glucagon peak and integrated areas. Sodium salicylate itself significantly increased basal insulin and decreased glucose, but did not change basal glucagon. In the diabetic subjects of group 1, sodium salicylate amplified the glucagon response to the same degree of hypoglycemia without affecting the rates of glucose fall and recovery. Compared with normals and diabetic subjects of group 1, diabetic subjects of group 2 presented, in basal conditions, a reduced glucagon response to hypoglycemia and a slower rate of glucose recovery. Sodium salicylate normalized both defects. These results indicate that sodium salicylate may augment glucagon responses by improving the recognition of hypoglycemia in both normals and IDDM. Moreover, the restoration by sodium salicylate of a normal glucagon response to hypoglycemia in diabetic subjects of group 2 suggests a role for endogenous prostaglandins in this selective deficiency of the counterregulatory response.

The oxygen-release capacity of red blood cells in insulin-dependent diabetics after artificial pancreas.

Oxygen-release capacity of the red blood cells was investigated in non-acidotic insulin-dependent diabetics before and after the achievement of strict metabolic control with the aid of the artificial pancreas. P50std (oxygen tension at 50% oxygen saturation) values were low in basal condition and returned to normal after the 24-h treatment period. No significant changes were observed in the content of red cell 2,3-diphosphoglycerate nor in the acid-base balance. Only the labile form of glycosylated hemoglobin showed significant decreases after treatment. These results suggest that insulin-dependent diabetics may have a state of relative tissue hypoxia which can be easily overcome by the achievement of strict metabolic control.

A role for endogenous prostaglandin E in biphasic pattern of insulin release in humans.

These studies were undertaken to evaluate in humans the possible physiological role of prostaglandins of the E series (PGE) in modulating insulin release and to assess whether endogenous PGE synthesis may account for the biphasic pattern of insulin secretion. We used a square-wave glucose stimulation previously determined to give maximal biphasic insulin release. Infusion of lysine acetylsalicylate to block the synthesis of endogenous PGE increased by twofold total insulin response to glucose and also converted insulin release to a multiphasic pattern. The infusion of exogenous PGE1 (0.2 microgram X kg-1 X min-1) or PGE2 (10 micrograms/min) in addition to lysine acetylsalicylate restored the typical biphasic pattern of insulin release and also decreased total insulin release to values similar to those of control studies. Infusion of either PGE1 or PGE2 in the absence of lysine acetylsalicylate reset insulin secretion to a lower level without altering the kinetics of release. On the basis of these results, it is hypothesized that endogenous PGE released in response to glucose stimulation exert an inhibiting effect on insulin release that becomes biphasic in appearance.

Impaired insulin secretion in human diabetes mellitus. The effect of naloxone-induced opiate receptor blockade.

Human diabetes mellitus is characterized by impaired insulin response to intravenous glucose. In search of possible factors which impair insulin release, we have investigated the effect of naloxone, a specific opiate receptor blocker, on insulin responses to glucose in subjects with non-insulin-dependent diabetes, as well as in normal subjects. Naloxone was given as a priming dose of 0.4 mg followed by a constant infusion of either 0.4 mg (N = 7), 2 mg (N = 7), or 4 mg (N = 8) for 90 min. Acute insulin response to glucose (mean change 3-10 min insulin), second phase insulin secretion (change 10-60 min), as well as glucose disappearance rates (%/min) were significantly increased in the diabetics receiving the two higher doses of naloxone (2 and 4 mg, respectively). None of these effects were seen in diabetics receiving saline or in normal subjects receiving naloxone. These results seem to suggest that sensitivity to endogenous opiates may play some part in non-insulin-dependent diabetes.