Is detection of peptidoglycan so important nowadays for dialysis products? Yes.

The biological characterization of endotoxins is defined by their ability to induce fever and, in cases of high dosage, result in shock and ultimately, death.¹ Another characteristic that sets them apart is their ability to induce uncontrolled global inflammatory responses by interacting with high-affinity receptors on leukocytes.²

Components of bacterial cell wall inducing pyrogenicity

LPS (Lipopolysaccharide): LPS— a major component of the outer membrane of gram-negative bacteria— can be released following bacterial cell death and lysis, triggering strong fever responses and adverse reactions. For over a century, LPS studies have become a useful model for investigating the fundamental mechanisms of sepsis.

Peptidoglycan (PepG): PepG is an important component of the cell wall of most bacteria; however, it is more predominant in gram-positive bacteria. Gram-negative bacteria are surrounded by a thin PepG layer which is in turn surrounded by an outer LPS membrane, whereas PepG is the major cell wall component of gram-positive bacteria.³

Throughout the years, PepG has garnered attention and recognition for its potential as a significant pathogenic structure, provoking adverse clinical effects.⁴ Bacterial breakdown and PepG release can induce the classical features of endotoxemia. The presence of PepG in parenteral products linked to sepsis in patients, along with supporting evidence from experimental animal models suggesting a role in systemic inflammation with organ failure, presents a compelling argument for PepG's role as a trigger for the excessive and potentially harmful immune response in sepsis.^4,5

Dialysis and pyrogenicity

In both peritoneal and renal dialysis (hemodialysis), the dialysis patient is challenged with handling hundreds of liters of dialysis solution every week, which can penetrate the body's natural defenses and directly or indirectly interact with the tissue. The dialysis solution components must therefore be tested to check for the presence of pyrogenic substances (living specimens or chemical contaminants that can cause fever).⁶

The clinical manifestations of peritonitis, a significant complication of peritoneal dialysis, include abdominal pain, nausea, vomiting, diarrhoea, and fever. Early studies found a link between aseptic peritonitis and PepG contamination after a global recall was issued for icodextrin-containing dialysate.⁷ Icodextrin is used as an osmotic agent in peritoneal dialysis solution; during a 2002 outbreak, reported cases of aseptic peritonitis associated with icodextrin increased more than ten-fold, although its chemical constituents and endotoxin concentrations were within pharmacopeia standards. Today, researchers continue to investigate and develop methods for PepG removal in sterile dialysis fluid preparations,^8,9 emphasizing the importance of PepG screening in parenteral products.

Peptidoglycans testing

The SLP-HS Single Reagent Set II is a product that can detect both PepG and (1->3)-β-D-glucan (BDG). It utilizes a self-defense mechanism from the silkworm Bombyx mori, which causes a color change by producing a black melanin pigment in the presence of microbial contamination. Since PepG is present in all bacterial cell walls and BDG is found in most fungi, the SLP reagent is a highly sensitive and broad method for detecting microbial contamination.

The testing of PepG is often overlooked, which can be a risky decision considering their importance as a major component of the cell wall in both gram-positive and gram-negative bacteria. Given that parenteral products with microbial contaminants can be considered safe under current pharmacopoeia tests, both the SLP test and the LAL assay should be regarded as equally significant in pyrogen testing.⁴

References:

1. Fennrich S, Hennig U, Toliashvili L, Schlensak C, Wendel HP, Stoppelkamp S. 2016. More than 70 years of pyrogen detection: Current state and future perspectives. Altern Lab Anim 44(3):239-53, PMID: 27494624.
2. Wagner JG, Roth RA. 1999. Neutrophil migration during endotoxemia. J Leukoc Biol 66(1):10-24, PMID: 10410985.
3. Silhavy TJ, Kahne D, Walker S. 2010. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2(5):a000414, PMID: 20452953.
4. Myhre AE, Aasen AO, Thiemermann C, Wang JE. 2006. Peptidoglycan--an endotoxin in its own right? Shock 25(3):227-35, PMID: 16552353.
5. Horn DL, Morrison DC, Opal SM, Silverstein R, Visvanathan K, Zabriskie JB. 2000. What are the microbial components implicated in the pathogenesis of sepsis? Report on a symposium. Clin Infect Dis 31(4):851-8, PMID: 11049761.
6. Daneshian M, Wendel A, Hartung T, von Aulock S. 2008. High sensitivity pyrogen testing in water and dialysis solutions. J Immunol Methods 20;336(1):64-70, PMID: 18474369.
7. Martis L, Patel M, Giertych J, Mongoven J, Taminne M, et al. 2005. Aseptic peritonitis due to peptidoglycan contamination of pharmacopoeia standard dialysis solution. Lancet 365(9459):588-94, PMID: 15708102.
8. Glorieux G, Hulko M, Speidel R, Brodbeck K, Krause B, Vanholder R. 2014. Looking beyond endotoxin: a comparative study of pyrogen retention by ultrafilters used for the preparation of sterile dialyis fluid. Sci Rep 4:6390, PMID: 25227511.
9. Hulko M, Dietrich V, Koch I, Gekeler A, Gebert M, et al. 2019. Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes. Sci Rep 9(1):6791, PMID: 31043670.