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Are sterile devices microbiologically safe? Unraveling the relationship between pyrogens, bioburden, and sterility

Sterility assurance is of utmost importance for medical devices that come into contact with body cavities. However, bioburden and sterility are linked to a third concern—pyrogens—which may not be adequately addressed solely by ensuring device sterility.

Pyrogens and Endotoxins: Understanding the Terminology

Pyrogens are substances that induce fever when they are present in the blood stream or cerebrospinal fluid (found in the brain and central nervous system). The development of the first parenteral solutions, like glucose for injection, took place in the early 1900s. An associated issue known as injection fever emerged during that time. Years later the link was demonstrated between bacterial contamination and injection fever.

Pyrogens are bacterial in nature, with the most common type of pyrogens being endotoxins derived from Gram-negative bacteria. Therefore, all endotoxins are considered pyrogens, but not all pyrogens are necessarily endotoxins.

To gain a better understanding of pyrogens and endotoxins, it is important to note that bacteria can be classified into two groups based on their cell wall: Gram-positive and Gram-negative strains. Gram-positive bacteria get their name from the fact that they retain a purple color when subjected to the Gram stain test, while Gram-negative bacteria retain a pink color (see Figure 1).

Gram-positive bacteria (left) and Gram-negative bacteria (right).
Figure 1: Gram-positive bacteria (left) and Gram-negative bacteria (right).

The fact that these two types of bacteria show different colors in the Gram stain test stems from the fact that the cell walls of these two types differ significantly. Gram-positive bacteria have a single cell wall constructed of a double-layered phospholipid membrane, covered mainly by lipoteichoic acid and peptidoglycan. Gram-negative bacteria have a double cell wall consisting of a double-layered phospholipid membrane on the inside and a lipopolysaccharide membrane on the outside (see Figure 2).

Bacteria cell wall
Figure 2: : Cell walls of Gram-positive and Gram-negative bacteria.

Lipopolysaccharides (LPS) are the most resistant and most reactive (up to one thousand times more reactive in rabbits) of all known pyrogens and are referred to as endotoxins and, as a result, are the most investigated during quality-control testing of healthcare products.

The Relationship Between Pyrogens, Bioburden, and Sterility

Given that pyrogens are derived from bacteria, pyrogen contamination is always linked to bioburden contamination. When bacteria are inactivated, the pyrogens remain fully active. Common sterilization methods, such as ethylene oxide (EO), gamma radiation, and moist heat, are not effective in destroying significant levels of endotoxins. As a result, sterile samples do not guarantee the absence of pyrogens. Thus, sterile samples do not necessarily mean that samples are microbiologically safe. It is important to understand that pyrogens should always be investigated in addition to sterility testing for products that come into contact with circulating blood and cerebrospinal fluid.

Peter Cornelis

Senior Expert Microbiology

Peter Cornelis graduated from the Catholic University of Leuven (Belgium) in 2000 as a Master in Applied Biological Sciences (Major Biotechnology). In 2003 he started working for Toxikon Europe (now Nelson Labs) as a study director Microbiology and in-vitro Toxicology. From 2007 until 2016, he was department supervisor for Microbiology and in-vitro Toxicology. Since 2016, he is responsible for research, validation, and development of new microbiological and in-vitro toxicological methods. Peter is a member of the ISO committee TC 194 WG5, Cytotoxicity and WG 8, Irritation and sensitization. As an expert, he was involved in the adaptation of ISO/TC 194 ISO/DTS 11796:2022(E) Biological evaluation of medical devices — Guidance for interlaboratory studies to demonstrate the applicability of validated in vitro methods to assess the skin sensitization of medical devices.

Emily Spackman, B.S., RM(NRCM)

BET Consulting Study Director

Emily Spackman has worked for Nelson Laboratories in the Bacterial Endotoxins Lab for over 16 years. She graduated from the University of Utah and holds a bachelors degree in Biology. She is a member of The National Registry of Certified Microbiologists and has experience with both medical devices and pharmaceutical products.

Nathan Pett

Study Director BET

Nathan Pett graduated from Utah State University in 2018 with a bachelors in Biochemistry. In 2019 he began work for Nelson Labs as a Lab Analyst and in 2021 transitioned to the role of Study Director in the BET department.