Case Study: Applying Rapid Flow Cytometry Analysis to CAPD Effluent.

Peritoneal dialysis (PD) peritonitis cases require rapid clinical interventions to ensure the best possible patient outcomes. Culture-dependent microbiology tools are slow and cannot provide clinicians with evidence to guide antimicrobial prescription practices in an appropriate time frame. Genotypic methods have met with limited success for analyzing continuous ambulatory PD effluent, with most centers still relying on culture-dependent microbiology. We present a case study in which we apply flow cytometry techniques to antibiotic-compromised effluent. We demonstrate, with supporting evidence, direct enumeration of bacterial and human immune cells, with results reported within 2 hours of receiving the clinical specimen.

Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis.

The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value < 0.05) representing 35 canonical pathways after 1 hour exposure to S. epidermidis. The top 3 canonical pathways were TNFR2 signaling, IL-17A signaling, and TNFR1 signaling (adj. p-values of 0.0012, 0.0012 and 0.0019, respectively). Subsequent qPCR validation confirmed significant differences in gene expression in a number of genes not previously described in mesothelial cell responses to infection, with heterogeneity observed between clinical isolates of S. epidermidis, and between Met-5A and primary mesothelial cells. Heterogeneity between different S. epidermidis isolates suggests that specific virulence factors may play critical roles in influencing outcomes from peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells.

Effects of a Statewide Protocol for the Management of Peritoneal Dialysis-Related Peritonitis on Microbial Profiles and Antimicrobial Susceptibilities: A Retrospective Five-Year Review.

UNLABELLED: ♦ BACKGROUND: Peritonitis is a major complication of peritoneal dialysis (PD) and is associated with significant morbidity and mortality. Early empirical antibiotic therapy is recommended, with the choice of agents guided by local resistance patterns. As routine use of specific antimicrobial agents can drive resistance, regular assessment of causative organisms and their susceptibility to empirical therapy is essential. ♦ METHODS: We conducted a retrospective review of all PD peritonitis cases and positive PD fluid cultures obtained over a 5-year period in Western Australia following the introduction of a statewide protocol for the initial management of PD peritonitis with intraperitoneal vancomycin and gentamicin. ♦ RESULTS: The incidence of PD peritonitis decreased from 1 in 16 patient months (0.75/year at risk) to 1 in 29 patient months (0.41/year at risk) over the 5 years. There were 1,319 culture-positive samples and 1,069 unique isolates identified. Gram-positive bacteria accounted for 69.9% of positive cultures, with vancomycin resistance averaging 2% over the study period. Gram-negative bacteria accounted for 25.4% of positive cultures, with gentamicin resistance identified in an average of 8% of organisms. No increase in antimicrobial resistance to vancomycin or gentamicin occurred over the 5 years and there was no change in the proportion of gram-positive (69.9%), gram-negative (25.4%) or fungal (4.4%) organisms causing PD peritonitis. ♦ CONCLUSIONS: Over time, the peritonitis rates have dramatically improved although the profile of causative organisms remains similar. Empirical treatment of PD peritonitis with intraperitoneal vancomycin and gentamicin remains efficacious, with high levels of susceptibility and no evidence that the introduction of this statewide empirical PD peritonitis treatment protocol is driving resistance to these agents.

Preclinical assessment of adjunctive tPA and DNase for peritoneal dialysis associated peritonitis.

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.

The impact of chronic kidney disease and short-term treatment with rosiglitazone on plasma cell-free DNA levels.

Patients with chronic kidney disease (CKD) are at increased risk of cardiovascular disease. Circulating free nucleic acids, known as cell-free DNA (cfDNA), have been proposed as a novel biomarker of cardiovascular risk. The impact of renal impairment on cfDNA levels and whether cfDNA is associated with endothelial dysfunction and inflammation in CKD has not been systematically studied. We analysed cfDNA concentrations from patients with varying degrees of CKD. In addition, to determine whether there is a relationship between cfDNA, inflammation, and endothelial dysfunction in CKD, levels of proinflammatory cytokines and von Willebrand Factor (vWF) were measured in patients treated with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone or placebo for 8 weeks. cfDNA levels were not increased with renal impairment or associated with the degree of renal dysfunction (P = 0.5). Treatment with rosiglitazone for 8 weeks, but not placebo, was more likely to lead to a reduction in cfDNA levels (P = 0.046); however, the absolute changes in cfDNA concentrations during treatment were not statistically significant (P > 0.05). cfDNA levels correlated with markers of endothelial dysfunction (hsCRP P = 0.0497) and vWF (P = 0.0005). In conclusion, cell-free DNA levels are not influenced by renal impairment but do reflect endothelial dysfunction in patients with CKD.