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Communications to the Editor |

Fungal β-Glucan Can Yield False-Positive Results With the Limulus Amebocyte Lysate Endotoxin Assay Fungal β-Glucan Can Yield False-Positive Results With the Limulus Amebocyte Lysate Endotoxin Assay FREE TO VIEW

Robert Vassallo, MD; Andrew H. Limper, MD, FCCP
Author and Funding Information

Affiliations: Mayo Clinic, Rochester, MN ,  Kansas City VA Medical Center, Kansas City, MO

Correspondence to: Andrew H. Limper, MD, FCCP, Pulmonary and Critical Care Medicine, Mayo Clinic, 200 First St SW, Rochester, MN; e-mail: limper.andrew@mayo.edu



Chest. 1999;116(2):583-584. doi:10.1378/chest.116.2.583
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To the Editor:

We read with interest the study by Nelson and colleagues (January 1999).1 Using the limulus amebocyte lysate (LAL) method (E-Toxate; Sigma Diagnostics; St. Louis, MO) to measure endotoxin levels in saline aliquots infused through the bronchoscope and in BAL fluid, they reported detectable levels of endotoxin (range, 2 to 4 endotoxin units [EU]/mL) in both the saline aliquots and the BAL fluid. We wish to bring to the authors’ attention information concerning the specificity of the LAL assay that may affect their conclusions that endotoxin contamination of the lungs during bronchoscopy may contribute to BAL-induced lung inflammation.

The conventional LAL assay is not completely specific for endotoxin and may give false-positive results with other compounds,23 particularlyβ -glucan.25 β-glucans are polymers of d-glucose that are found in the cell walls of most fungi and plants and have been isolated from sterile materials used for surgical procedures.,4 β-glucans react with the factor G-mediated coagulation pathway in the LAL system leading to activation of the LAL and, thus, may yield false-positive results. This interaction between β-glucans and the LAL system is important to appreciate clinically. Nakao et al4 reported on the occurrence of false-positive endotoxemia following surgery. Using a conventional LAL test, transient elevations in blood endotoxin levels were noted in patients despite the lack of clinical evidence of endotoxemia. When the same samples were assayed using an endotoxin-specific assay (Endospecy; Associates of Cape Cod Inc/Seikagaku America; Falmouth, MA), endotoxin was not detected.

We have observed the same problem with false-positive results using the standard LAL assay in our laboratory when measuring endotoxin levels in samples contaminated with β-glucans. We washed sterile gauze with physiologic saline (under sterile conditions) and performed endotoxin determination on the saline using LAL assay (BioWhittaker Inc; Walkersville, MD) and a specific endotoxin assay (Endospecy; Seikagaku). The endotoxin levels measured by the LAL assay and Endospecy were 0.340 vs 0.114 EU/mL, respectively. The same sample also was assayed in our institutional laboratory using the LAL assay and was reported as positive for endotoxin (lower limit of detection, 0.125 EU/mL).

Therefore, results of a standard LAL assay cannot definitively be attributed only to bacteria-derived endotoxin. Only with the use of specific endotoxin assays can the issue of potential endotoxin contamination during instrumentation of the airways be addressed.

Nelson, ME, Wald, TC, Bailey, K, et al (1999) Intrapulmonary cytokine accumulation following BAL and the role of endotoxin contamination.Chest115,151-157. [PubMed] [CrossRef]
 
Cooper, JF, Weary, ME, Jordan, FT The impact of non-endotoxin LAL-reactive materials on Limulus amebocyte lysate analyses.PDA J Pharm Sci Technol1997;51,2-6. [PubMed]
 
Kambayashi, J, Yokota, M, Sakon, M, et al A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan.J Biochem Biophys Methods1991;22,93-100. [PubMed]
 
Nakao, A, Yasui, M, Kawagoe, T, et al False-positive endotoxemia derives from gauze glucan after hepatectomy for hepatocellular carcinoma with cirrhosis.Hepatogastroenterology1997;44,1413-1418. [PubMed]
 
Zhang, GH, Baek, L, Buchardt, O, et al Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.J Clin Microbiol1994;32,1537-1541. [PubMed]
 

Fungal β-Glucan Can Yield False-Positive Results With the Limulus Amebocyte Lysate Endotoxin Assay

To the Editor:

Drs. Vassallo and Limper make the valid points that a positive result of an amebocyte limulus assay can be caused by the contamination of specimens with β-glucans rather than endotoxin and that the source of these β-glucans can be sterile materials, such as surgical gauze. Although BAL fluid specimens that we recovered from volunteers were passed through gauze prior to analysis, the specimens recovered after simply instilling fluid through the working channels of bronchoscopes were not passed through gauze; therefore, the gauze was not a source of contamination in these specimens. It is possible, however, that β-glucans derived from the cell walls of fungal organisms contaminated the working channels of bronchoscopes and that this material, rather than endotoxin, contributed to the positive results of the amebocyte limulus assays, which we noted. Certainly, both bacterial and fungal organisms could be potential contaminants in bronchoscopes, and, although the bronchoscopes may have been cleaned, cell-wall fragments of both types of organisms could remain within the working channel.

Although fungus-derived β-glucans are structurally distinct from bacteria-derived endotoxins, both of these microbial cell-wall products induce similar inflammatory responses within the lungs of experimental animals.12 It is uncertain, however, whether β-glucans induce the same intrapulmonary cytokine response as endotoxin. The cytokine response that we noted within the lungs after BAL is similar to the intrapulmonary cytokine response induced by inhalation of endotoxin in humans and after instillation in experimental animals, which supports the concept that endotoxin contamination contributes to BAL-induced pulmonary inflammation.34 In view of the potential contamination with β-glucans, however, the conclusions of our study should be modified to indicate that intrapulmonary contamination with bacteria-derived endotoxin, fungus-derivedβ -glucans, or both may contribute to pulmonary inflammation induced by BAL in human subjects. We agree with Drs. Vassallo and Limper that further studies with more specific assays are indicated to assess endotoxin and β-glucan contamination and the relative roles of these microbial products in BAL-induced pulmonary inflammation.

References
Rylander, R, Fogelmark, B Inflammatory responses by inhalation of endotoxin and (1,3) beta-d-glucan.Am J Ind Med1994;25,101-102. [PubMed] [CrossRef]
 
Fogelmark, B, Goto, H, Yuasa, K, et al Acute pulmonary toxicity of inhaled beta-1,3-glucan and endotoxin.Agents Actions1992;35,50-56. [PubMed]
 
Wesselius, LJ, Nelson, ME, Bailey, K, et al Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers.J Lab Clin Med1997;129,106-114. [PubMed]
 
Xing, Z, Jordana, M, Kirpalani, H, et al Cytokine expression by neutrophils and macrophagesin vivo: endotoxin induces tumor necrosis factor-α, macrophage inflammatory protein-2, interleukin-1β, and interleukin-6 but not RANTES or transforming growth factor-β mRNA expression in acute lung inflammation.Am J Respir Cell Mol Biol1994;10,148-153. [PubMed]
 

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References

Nelson, ME, Wald, TC, Bailey, K, et al (1999) Intrapulmonary cytokine accumulation following BAL and the role of endotoxin contamination.Chest115,151-157. [PubMed] [CrossRef]
 
Cooper, JF, Weary, ME, Jordan, FT The impact of non-endotoxin LAL-reactive materials on Limulus amebocyte lysate analyses.PDA J Pharm Sci Technol1997;51,2-6. [PubMed]
 
Kambayashi, J, Yokota, M, Sakon, M, et al A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan.J Biochem Biophys Methods1991;22,93-100. [PubMed]
 
Nakao, A, Yasui, M, Kawagoe, T, et al False-positive endotoxemia derives from gauze glucan after hepatectomy for hepatocellular carcinoma with cirrhosis.Hepatogastroenterology1997;44,1413-1418. [PubMed]
 
Zhang, GH, Baek, L, Buchardt, O, et al Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.J Clin Microbiol1994;32,1537-1541. [PubMed]
 
Rylander, R, Fogelmark, B Inflammatory responses by inhalation of endotoxin and (1,3) beta-d-glucan.Am J Ind Med1994;25,101-102. [PubMed] [CrossRef]
 
Fogelmark, B, Goto, H, Yuasa, K, et al Acute pulmonary toxicity of inhaled beta-1,3-glucan and endotoxin.Agents Actions1992;35,50-56. [PubMed]
 
Wesselius, LJ, Nelson, ME, Bailey, K, et al Rapid lung cytokine accumulation and neutrophil recruitment after lipopolysaccharide inhalation by cigarette smokers and nonsmokers.J Lab Clin Med1997;129,106-114. [PubMed]
 
Xing, Z, Jordana, M, Kirpalani, H, et al Cytokine expression by neutrophils and macrophagesin vivo: endotoxin induces tumor necrosis factor-α, macrophage inflammatory protein-2, interleukin-1β, and interleukin-6 but not RANTES or transforming growth factor-β mRNA expression in acute lung inflammation.Am J Respir Cell Mol Biol1994;10,148-153. [PubMed]
 
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