Index
It is characterized by a furious reaction due to pyrogens (agents that increase body temperature).
It can be caused by contamination of the solution and materials used in procedures with intravenous contact (puncture).
Signs and symptoms
The signs and symptoms are:
- Hypotension .
- The skin is generally pale, sweating, and may have peripheral cyanosis.
- Hypothermia .
- Fast and thin pulse.
- Emotional changes can present agitation, mental confusion, or apathy.
- Tachypnea and superficial.
- Oliguria.
What are pyrogens?
Pyrogens are substances that can cause fever. The most common pyrogens are endotoxins and lipopolysaccharides (LPS) produced by Gram-negative bacteria like E. Coli.
The amoebocyte lysate (LAL) test is used to detect endotoxins. Another assay that detects endotoxins is the recombinant factor C assay.
The latter’s advantage is the lack of positive tests for glucan-containing substances, a problem sometimes seen in post-processing when traces of carbohydrates are released from chromatographic resins.
The best-studied pyrogen is lipopolysaccharide (LPS, also known as endotoxin), which is found in the membrane of bacteria. The release of LPS from bacteria occurs after cell lysis and can occur due to sterilization.
Good examples of pyrogen-producing gram-negative bacteria are:
- Escherichia coli (E. Coli).
- Proteus.
- Pseudomonas
- Enterobacter.
- Klebsiella.
During gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines, and excessive inflammation causes multiple organ failures and death.
Therefore, endotoxins are ubiquitous pathogenic molecules that are detrimental to the pharmaceutical industry and the medical device community.
Endotoxins result from high levels of gram-negative bacterial bioburden. It is challenging to remove endotoxins from products once they are present.
The most common depyrogenation procedures for physical components include incineration and removal by washing (also called dilution), which are impractical after terminal sterilization.
Pyrogen test
As previously explained, pyrogens include endotoxins (toxins derived from gram-negative bacteria) and non-endotoxic pyrogens (substances derived from microorganisms other than gram-negative bacteria or chemicals).
Iryokiki-Shinsa mentions the pyrogen test, which uses a rabbit, which can detect endotoxins and non-endotoxic pyrogenic substances, and the in vitro endotoxin test, which is widely used as a method for detecting microbial contamination (endotoxins).
The pyrogen test with a rabbit involves administering a physiological saline extract of the test substance intravenously and determining the existence of pyrogens by the increase in body temperature after administration.
In vitro
The endotoxin test includes the gelation method, which indexes the formation of a gel that occurs through endotoxins derived from gram-negative bacteria acting on a lysate test solution prepared from Limulus amebocyte lysate. The methods are turbidimetric and colorimetric—the index of optical changes.
The selected test method differs depending on the purpose of the test (a limit test to verify the default values below, a quantitative test to verify the content) and the characteristics (color, existence of reaction interference factors) of the prepared extract.
Endotoxins and non-endotoxic pyrogens show mechanisms of increase in body temperature and complex and indistinct behavior. It is necessary to select the extraction conditions according to the material’s properties and the purpose of use of the product.
The test method given in Iryokiki-Shinsa No.36 is fundamentally a criterion of determination equivalent to the pyrogen test method included in the general test methods listed by the Japanese Pharmacopoeia.
It differs slightly from the determination standards of the United States Pharmacopoeia (USP) or the European Pharmacopoeia (EP). In any case, the test is carried out taking into account various terms and conditions, such as the content, characteristics, application, etc., of the raw materials of the medical device.
The test substance or extract is administered into the atrial vein of a rabbit. The body temperature (rectal temperature) is measured continuously for three h, and any difference from the reference body temperature is looked for.
When the body temperature (rectal temperature) of at least 2 out of 3 animals increases by 0.6 ° C or more within three h after administration, the result is considered positive for pyrogens.
Although it cannot be considered a positive result, if an animal shows a body temperature increase of 0.6 ° C or the sum of the maximum temperature of three animals exceeds 1.4 ° C, a second test should be performed.
Five animals are used for this second test, and if two or more animals show an increase in body temperature of 0.6 ° C or more, the result is considered positive. H after administration, the result is deemed to be positive for pyrogens.
Although it cannot be considered a positive result, if an animal shows a body temperature increase of 0.6 ° C or the sum of the maximum temperature of three animals exceeds 1.4 ° C, a second test should be performed.
Five animals are used for this second test, and if two or more animals show an increase in body temperature of 0.6 ° C or more, the result is considered positive.
