Standardized modulation of the injection site allows for insulin dose reduction without deterioration of metabolic control.

OBJECTIVES: Use of an injection site modulation device (InsuPad) in intensive insulin treatment reduces frequency of hypoglycemia and prandial insulin requirements by enhancing subcutaneous microcirculation. This meal tolerance test (MTT) investigation was performed as a sub-study during the real-world BARMER study to demonstrate non-inferiority of the reduced insulin doses observed in this study with respect to metabolic control. METHODS: The MTT was performed at baseline and after 3 months in insulin treated diabetes patients using the modulation device vs. a control group without device. The dose used for the MTT was individually calculated based on the prandial insulin records from the patient diaries before the test. Blood was drawn for determination of glucose, insulin, C-peptide, proinsulin, triglycerides, free fatty acids, nitrotyrosine, and asymmetric dimethyl-arginine (ADMA) at multiple time-points from 0 to 300 min. A total of 32 patients from one site were included into this MTT study (8 female, 7 type 1 diabetes, age: 49.9 ± 12.5 yrs, HbA1c: 7.2 ± 0.5%). RESULTS: During the BARMER study, mean HbA1c was treated to target (<6.5%) in both groups. The prandial insulin dose decreased in the MTT modulation device group by -17.1%, but remained unchanged in the control group (-0.1%, p < 0.001). No change was seen for the basal insulin dose in both treatment arms. There were no differences between the groups with respect to the postprandial curves for glucose, C-peptide, intact proinsulin, free fatty acids, and triglycerides. Insulin absorption was faster with the modulation device (Tmax: 60 ± 28 min vs. 99 ± 46 min, p < 0.05). Key limitations are the small patient sample size and impossibility to determine the short-term effects of device use. CONCLUSIONS: The results of this meal tolerance sub-study confirm that the observed prandial insulin dose reduction when using the injection site modulation device has no negative impact on postprandial metabolism.

The Barmer study: impact of standardized warming of the injection site to enhance insulin absorption and reduce prandial insulin requirements and hypoglycemia in obese patients with diabetes mellitus.

BACKGROUND: The primary objective of this prospective controlled study was to investigate the impact of standardized injection-site warming on prandial rapid acting insulin dose and glycemic control when studied under real-world conditions. METHODS: All 145 participating patients (51 female, 94 male, 13 type 1 and 132 type 2 patients, age: 61.6 ± 8.4 yrs, HbA1c: 7.19 ± 0.50%) were treated with intensive insulin glargine and short-acting insulin analog therapy. After a 4 week treatment optimization run-in period, patients were randomized to continue therapy for three months without (control) or with a local injection-site warming device (InsuPad * ). Observation parameters included HbA1c, insulin dose, frequency of hypoglycemia, body weight and adverse events. RESULTS: HbA1c improved in both arms until study end (control group: 6.3 ± 0.5%; injection-site warming device: 6.3 ± 0.5%; both p < 0.001 vs. baseline). To achieve this good control, patients in the control group needed to increase the daily prandial insulin dose by 8.1% (from 66 ± 31 U to 71 ± 38 U, p < 0.05) with stable basal insulin requirements. Patients who used the injection-site warming device required less prandial insulin (70 ± 43 U to 55 ± 34 U; -19%, p < 0.001) and slightly more basal insulin (+3.9%). Total daily insulin dose increased in the control group (+3.7%) and decreased with warming device use (-8.6%, p < 0.001). The number of hypoglycemic events (<63 mg/dL) during the observation period was higher in the control group (6.2 ± 9.9/patient vs. injection-site warming device: 3.3 ± 4.8/patient, p < 0.05). Main study limitations can be seen in the open label design reliability of the collected dose information and the very obese patient cohort. CONCLUSION: When treating obese patients to target with insulin therapy, use of an injection-site warming device for 3 months resulted in a lower frequency of hypoglycemic events and a reduction in prandial insulin analog requirements. If these results are confirmed in other patient populations, an injection-site warming device may be useful in achieving treatment targets with a safer and more efficient basal bolus therapy in insulin-treated patients with type 1 and type 2 diabetes.

[Intestinal parasitosis and environmental pollution: 1343 pediatric cases in Beni-Mellal, Morocco]. / Parasitoses intestinales et pollution de l'environnement: cas de 1343 enfants à Beni-Mellal--Maroc.

This study was undertaken to determine the possible health risk associated with raw wastewater use in agricultural purposes, particularly, the transmission of parasite infections among children of five regions in Beni-Mellal, Morocco. In a randomly-selected sample of 1343 children, 740 of them were from five regions using raw wastewater for agriculture, and 603 were from 4 control regions that do not practice wastewater irrigation. One or more parasite infections were identified in 50.8% of the children living in the wastewater re-use regions and in 8.2% only of the others. The seven parasites identified were Entamoeba histolytica, Giardia intestinalis, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Hymenolepis nana, Taenia saginata, which infected 34.3%, 5.1%, 20.5%, 0.4%, 5.2%, 7.2%, 0.5% in the exposed population and 4.3%, 0.3%, 3.8%, 0.3%, 1.0%, 0.6% and 0.0% in the control population respectively. In conclusion, raw wastewater use in Beni-Mellal lead to a high risk of parasite infections. Adequate treatment of wastewater is highly recommended.

Geohelminthic infections associated with raw wastewater reuse for agricultural purposes in Beni-Mellal, Morocco.

This study was undertaken to determine the possible risk associated with raw wastewater use for agricultural purposes, particularly, the transmission of geohelminthic infections among children of five regions in Beni-Mellal, Morocco. In a randomly selected sample of 1343 children, 740 of them were from five communities using raw wastewater for agriculture, and 603 were from four control communities that do not practice wastewater irrigation. A questionnaire-interview with children and parents was used to collect data on possible demographic, hygiene and behavioral-contact risk factors such as sex, age, family size, parental education, parental occupation, source of water, toilet in house, hand-washing, contact with wastewater and contact with wastewater irrigated land. Ascariasis prevalence was found to be approximately five times higher among children in wastewater-impacted regions compared to control regions. Contact with wastewater and wastewater irrigated land and public water supply were found to be associated with higher infection rates. Trichuris rates did not show a statistically significant difference between the wastewater-impacted and the control regions. In conclusion, raw wastewater use in Beni-Mellal can lead to a high risk of geohelminthic infections. Adequate treatment of wastewater and public health education are highly recommended.

Determination of the heavy metal binding capacity of aquatic samples using MetPLATE: a preliminary study.

MetPLATE, a microbial toxicity test which is specific for heavy metal toxicity, was used to rapidly determine the heavy metal binding capacity (HMBC) of a wide range of surface waters from Florida and Georgia. HMBC determines the impact of physicochemical factors on metal bioavailability and toxicity. The new developed protocol, using MetPLATE as the toxicity assay, showed that HMBC varied from 1.7 to 39.2 for Cd whereas the ranges for Cu and Ag were < 1-11.9 and < 1-2.1, respectively. The effect of seasons on HMBC was determined using samples from the Hogtown Creek, Gainesville, FL, and from the St John's river in Jacksonville, FL. Both surface waters displayed the highest HMBC during the Fall season. Preliminary examination of the limited data set confirms that HMBC may be influenced by the concentration of dissolved organic carbon (DOC). These preliminary data show that the impact of physical and chemical parameters on the toxicity of metals in aquatic environments can be rapidly assessed using rapid and low-cost microbiotests.

Assessment of metal content and toxicity of leachates from teapots.

Metallic teapots traditionally used in Morocco were investigated for release of toxic metals from the teapots and their toxicity, as determined by MetPAD, a bacterial toxicity test that is specific for heavy metal toxicity. Our data show that some teapots were non-toxic while a few others were highly toxic, as shown by MetPAD. Tea addition reduced somewhat heavy metal toxicity due possibly to the complexing ability of tea. Chemical analysis of teapot leachates showed that some contained zinc and copper. Teapot No. 5, which showed the highest toxicity, also displayed the highest Zn concentration (7.39 mg/l), confirming the toxicity data. Based on estimates of tea consumption in Morocco, we showed that the extra daily burden of Zn ranged from 1.75 to 4.2 mg/day, assuming the maximum zinc concentration of 7.4 mg/l, as found in our study. This represents 3.5-8% of the LOAEL for zinc of 50 mg/day and would not be important as compared to other sources zinc intake.

Raw wastewater agricultural re-use and risk of protozoal infection in Beni-Mellal, Morocco.

This study was carried out to determine the potential risk of protozoal infection associated with raw wastewater use for agricultural purposes, among children of Beni-Mellal, Morocco. In a randomly-selected sample of 1343 children 740 from 5 regions which use raw wastewater for agriculture (exposed), while 603 were from 4 control regions that do not practice wastewater irrigation (unexposed). A questionnaire-interview with children and parents was used to collect data on demographic, hygienic, and risky water contact risk factors One or more protozoal infection was identified among 276(37.2%) of children living in the wastewater re-use regions, versus only among 22 (3.6%) living in control regions. The overall prevalence of 40.1% was observed among boys, and 33.3% among girls residing in exposed areas. The two identified protozoa were Entaemoeba histolytica and Giardia intestinalis, which infected 34.3% and 5.1% in the exposed population compared to only 3.3% and 0.3% in the control population, respectively. We also noted that the G. intestinalis prevalence was more than 40% among the children under 9 years, but less than 17% for those who were more than 12 years. Also, a significant excess of protozoal infection was observed among children who had risky contact with agricultural lands (p<0.001). In conclusion, raw wastewater use in Beni-Mellal can lead to a high risk of protozoal infections. Adequate treatment of wastewater prior to re-use, as well as public health education are highly recommended.

Helminthic infections associated with the use of raw wastewater for agricultural purposes in Beni Mellal, Morocco.

We aimed to determine the possible risks associated with raw wastewater use for agricultural purposes, particularly the transmission of helminthic infections among children. In a randomly-selected sample of 1343 children, 740 of them were from five regions using raw wastewater for agriculture, while 603 were from four control regions that do not use such water. The prevalence of helminthic infection was found to be significantly higher among children in regions using wastewater, compared to control regions. Behavioural characteristics and public water supply were found to be associated with higher infection rates. Adequate treatment of wastewater as well as public health education are highly recommended.

Helminthic infections associated with the use of raw wastewater for agricultural purposes in Beni Mellal, Morocco

We aimed to determine the possible risks associated with raw wastewater use for agricultural purposes, particularly the transmission of helminthic infections among children. In a randomly-selected sample of 1343 children, 740 of them were from five regions using raw wastewater for agriculture, while 603 were from four control regions that do not use such water. The prevalence of helminthic infection was found to be significantly higher among children in regions using wastewater, compared to control regions. Behavioural characteristics and public water supply were found to be associated with higher infection rates. Adequate treatment of wastewater as well as public health education are highly recommended

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.

Heavy metal toxicity testing in environmental samples.

The toxicity of heavy metals in the environment depends on a number of physicochemical and biological factors. The complexity of these relationships has encouraged the use of bioassays for direct measurement of the [table: see text] impact of toxic metals on selected test species. Fish and daphnid bioassays are well accepted by the scientific and regulatory communities, but their length (48 h or more) and the considerable time and effort needed to culture the test organisms make their application to sample screening problematical. Microbial and biochemical assays based on the inhibition of bioluminescence, enzyme activity, enzyme biosynthesis, growth, respiration, and heat production are typically faster and less expensive than the traditional and fish bioassays. Some of these tests approach or equal the sensitivity of daphnids to heavy metals. Since the soil acts as a sink for airborne and waste-applied metals, the uptake of metals by plants and the associated toxic impacts are important. Growth inhibition, enzyme induction, and production of stress proteins have been considered as toxicity end points. Enzymatic tests have been developed that are specific for heavy metal toxicity. Such tests can facilitate toxicity reduction evaluations. Detection of individual metals in the environment may eventually be possible using biosensors consisting of genetically engineered microorganisms. Direct solid-phase tests for soil, sediment, or sludge toxicity, using bacterial bioluminescence or enzyme activity as end points, have been developed. Such tests may complement traditional solid-phase toxicity tests using nematodes or earthworms as indicator organisms. Based on the work reviewed, we draw the following conclusions: 1. The Microtox assay is sensitive to mercury but would fail to detect the toxicity of certain metals, such as cadmium. Among all the microbial assays reviewed, the bioassay based on growth inhibition of the alga Selenastrum capricornutum appears to give the lowest EC50s, similar to those seen for daphnid bioassays. 2. Biosensors, using genetically engineered microorganisms, offer an elegant means of detecting the presence of specific heavy metals in environmental samples. However, at the present time, they are not designed for assessing heavy metal toxicity. 3. The use of bioassays specific for heavy metal toxicity can be useful for directly assessing the bioavailability of these toxicants in environmental samples, thus avoiding the need for fractionation.+4

Evaluation of a microplate assay specific for heavy metal toxicity.

A rapid, quantitative microbial assay, which is specific for heavy metal toxicity, has been developed. The assay (MetPLATE) is in a 96-well microtitration plate format and is suitable for determining toxicity characteristics such as median inhibitory concentrations. The sensitivity of MetPLATE to heavy metals [Cu, Zn, Cd, Pb, Hg, Cr(III)] was generally higher than Microtox and was of the same order as or better than Daphnia and fish bioassay. MetPLATE was insensitive to organic compounds at concentrations higher than those found in the environment. Six out of 10 industrial wastewaters or process waters surveyed were toxic. Heavy metal analysis of these waters confirmed the presence of heavy metals in the toxic samples. MetPLATE can be run concurrently with other assays for general toxicity to help determine the nature of chemicals causing toxicity.

Detection of Salmonella enteritidis in environmental samples by monoclonal antibody-based ELISA.

We have developed a enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (ASCII) for the detection of Salmonella enteritidis in environmental samples. ELISA was used to test for sensitivity and specificity of ASCII. 38 other species of bacteria, including 31 Salmonella species were included in cross-reactivity testing with ELISA. ASCII showed no reactivity with any other species tested. ASCII was found to be an IgG1 specific for S. enteritidis lipopolysaccharide (LPS). The lower limits for S. enteritidis detection was 10(5) cells/ml for pure cultures and in 10% sludge (w/v). Environmental samples (raw wastewater, wastewater effluents, mixed liquor and aerobically digested sludge) were obtained twice from five sites and ELISA tested for the presence of S. enteritidis. ELISA results compared to the American Public Health Association (APHA) method of Salmonella detection were not significantly different (P greater than 0.05). The ELISA took 24 h for completion compared to 96-120 h for the APHA procedure. Results demonstrate the reliability of the ELISA and, more importantly, provides a rapid means of detection of S. enteritidis in environmental samples.

Use of hydrogen peroxide treatment and crystal violet agar plates for selective recovery of bacteriophages from natural environments.

Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of Escherichia coli C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.

Bacterial and enzymatic bioassays for toxicity testing in the environment.

Microbioassays using bacteria or enzymes are increasingly applied to measure chemical toxicity in the environment. Attractive features of these assays may include low cost, rapid response to toxicants, high sample throughput, modest laboratory equipment and space requirements, low sample volume, portability, and reproducible responses. Enzymatic tests rely on measurement of either enzyme activity or enzyme biosynthesis. Dehydrogenases are the enzymes most used in toxicity testing. Assay of dehydrogenase activity is conveniently carried out using oxidoreduction dyes such as tetrazolium salts. Other enzyme activity tests utilize ATPases, esterases, phosphatases, urease, luciferase, beta-galactosidase, protease, amylase, or beta-glucosidase. Recently, the inhibition of enzyme (beta-galactosidase, tryptophanase, alpha-glucosidase) biosynthesis has been explored as a basis for toxicity testing. Enzyme biosynthesis was found to be generally more sensitive to organic chemicals than enzyme activity. Bacterial toxicity tests are based on bioluminescence, motility, growth, viability, ATP, oxygen uptake, nitrification, or heat production. An important aspect of bacterial tests is the permeability of cells to environmental toxicants, particularly organic chemicals of hydrophobic nature. Physical, chemical, and genetic alterations of the outer membrane of E. coli have been found to affect test sensitivity to organic toxicants. Several microbioassays are now commercially available. The names of the assays and their basis are: Microtox (bioluminescence), Polytox (respiration), ECHA Biocide Monitor (dehydrogenase activity), Toxi-Chromotest (enzyme biosynthesis), and MetPAD (enzyme activity). An important feature common to these tests is the provision of standardized cultures of bacteria in freeze-dried form. Two of the more recent applications of microbioassays are in sediment toxicity testing and toxicity reduction evaluation. Sediment pore water may be assayed directly or solvents may be used to extract the toxicants. Some of the solvents used for extraction of organic chemicals are themselves toxic to bacteria (e.g., dichloromethane), requiring exchange with a less toxic solvent (e.g., ethanol, methanol, DMSO). A modification of the Microtox test allows direct assay of solid-phase samples such as sediments. The toxicity reduction evaluation (TRE) must be carried out at wastewater treatment plants whose effluents fail toxicity standards. The TREs require numerous and repeated toxicity assays, thus favoring application of microbioassays. Presently, no single microbioassay can detect all categories of environmental toxicants with equal sensitivity. Therefore, a battery of tests approach is recommended. The differential sensitivity of alternative tests may, in fact, be exploited. Further research is needed to construct strains of genetically engineered microorganisms or isolate microorganisms or enzymes that respond to specific classes of toxicants. These can be combined into batteries appropriate for different environments or test objectives.