Comparative detection and recovery of Pseudomonas aeruginosa by membrane filtration and a Most Probable Number technique.

Two reference membrane filtration methods (Standard Methods: SM 9213E and U.S.EPA), the Most Probable Number method (Pseudalert®) and a membrane filtration method currently used by the Ontario Ministry of the Environment and Climate Change (MOECC) Laboratory Services Branch Etobicoke, Canada (Method E3371) were compared for the detection and recovery of Pseudomonas aeruginosa (P. aeruginosa) in sterile phosphate buffered dilution water (PBDW), un-spiked and spiked environmental water samples. The confirmation of presumptive-positive P. aeruginosa colonies was performed by API®20NE and on Milk Agar. Detection and recoveries were compared by determining the sensitivity and specificity of the methods and the performance of the methods was evaluated for statistical differences using ANOVA. The recovery of P. aeruginosa from PBDW spiked at a level of 100CFU/100mL was significantly higher (p<0.05) with Pseudalert® compared to the other methods. However, there were no significant differences (p>0.5) between all four methods when spiked at 5 and 40CFU/100mL. In the analysis of 61 environmental surface water samples, the MPN method (Pseudalert®) demonstrated the highest sensitivity (100%) while the Standard Method-SM9213E had the lowest sensitivity (3%). The U.S.EPA method and SM9213E demonstrated the highest specificity (100%) while Pseudalert® had the lowest (71.0%). Pseudalert® was able to detect P. aeruginosa in samples with higher amount of suspended solids as compared to other MF methods. Analysis of 24 environmental surface water samples (previously found to be negative for P. aeruginosa), which were spiked with P. aeruginosa at 5CFU/100mL, showed different sensitivities in recovery including Pseudalert® (100%), MOECC E3371 (92%), U.S.EPA (33%) and SM 9213E (33%). The highest mean recovery was observed with Pseudalert® (5.3 MPN/100mL) and the least with the U.S.EPA method (0.4CFU/100mL). Pseudalert® demonstrated improved performance in the detection and recovery of P. aeruginosa over SM9213E, U.S.EPA and MOECC E3371 in terms of sensitivity. However, Pseudalert® reported the highest amount of false positive results compared to the other three MF methods. The addition of a confirmation step with Milk Agar eliminated the false positive results. Therefore, this confirmatory step is recommended in order to increase the specificity of the Pseudalert® method.

Human fetal islet transplantation in type 1 diabetics: comparison of immunological effects between multiple implantation regimens.

Previous studies have suggested that the multiple transplants might be equally metabolically efficient to a single regimen for human adult islets. The aim of this study was to compare immunological and metabolic parameters after each of the two regimens of human fetal islets (HFI). Group A single transplants (n = 9) had 180 +/- 20 x 1000 HFI equivalents (IEQs) implanted via a single intramuscular injection. In group B multiple transplants (n = 8) islets were implanted by three consecutive injections of 60 +/- 10 x 1000 IEQs at 7-day intervals. We analyzed the immunological parameters of CD4/CD8 T lymphocyte ratios; islet cell antibodies (ICAs) and insulin antibodies (IAs). We estimated insulin secreting capacity (ISC) as the metabolic parameter. We observed that the CD4+/CD8+ T-cell ratio increased, peaking on day 90, in similar fashion in both groups: day -1: A = 1.18 +/- 0.03 versus B = 1.19 +/- 0.04; on day 90: A = 1.79 +/- 0.09, versus B = 1.75 +/- 0.08 (P = NS) immediately before the decrease in C-peptide levels. Thereafter the ratios rapidly decreased without statistical differences. The levels of ICAs did not change. The levels of IAs, which were increased before transplant, then decreased without statistical differences between the groups. The values of ISC increased after transplant and then decreased similar to the T-cell ratio. Our results demonstrated that regimens of multiple and single HFIs did not show differences in the kinetics of the immunological response presumably mediating graft destruction. The CD4/CD8 ratio increased as the C-peptide level decreased, peaking on day 90 at the time of a decrease in C-peptide. These results may be useful for clinical studies of HFIs for type 1 diabetic patients.

Human fetal islet transplantation in type 1 diabetic patients: comparison of metabolic effects between single and multiple implantation regimens.

Previous studies suggest that multiple transplantations might be equally efficient to a single regimen for human adult islets. The aim of this study was to compare metabolic parameters after each of the two regimens of human fetal islet (HFI) transplantation in type 1 diabetics. In group A (single transplant, n = 9), 180 +/- 20 x 1000 HFI equivalents (IEQs) were implanted by a single IM injection; in group B (multiple transplants, n = 8) islets were implanted as three consecutive injections (60 +/- 10 x 1000 IEQs) at 7-day intervals. We analyzed the metabolic parameters on days -1, 30, 60, 90, 120, 150, and 180 after the procedure. Among the metabolic parameters, we evaluated insulin secretion capacity-ISC (C peptide, RIA), metabolic control (HbA1c, chromatography), and insulin daily dose IDD. We found that C peptide levels increased, peaking on day 90 (A: 0.38 +/- 0.15; B: 0.34 +/- 0.19 nmol/L, P = NS) and then rapidly decreasing without differences, the HbA1c levels and IDD decreased in the same manner without differences between the groups. Our results demonstrate that multiple and single islet transplant regimens are equally efficient to temporarily restore a significant ISC with improvement of metabolic and clinical parameters. The results imply that the two regimens have an equal clinical value.