Serum Endotoxin as a Diagnostic Biomarker for Legionella pneumophila in Gram-Negative Infections

Summary

Rapid diagnosis of Legionella pneumophila infection in critically ill patients remains challenging. This retrospective cohort study evaluates serum endotoxin quantification using a Limulus amebocyte lysate assay as an adjunctive biomarker to help differentiate Legionella infection from other pathogens and guide early antimicrobial therapy.

Abstract

Rapid and accurate differentiation of Legionella pneumophila infections from other gram-negative bacterial infections remains a significant challenge in intensive care medicine. Despite its relatively low in vitro endotoxic activity, clinical observations have suggested elevated circulating endotoxin levels in patients with legionellosis. This retrospective cohort study analyzed 118 critically ill patients with microbiologically confirmed infections admitted to an intensive care unit between January 2020 and December 2023. Patients were classified into four pathogen groups: Legionella pneumophila (n = 18), other gram-negative bacteria (n = 68), gram-positive bacteria (n = 24), and fungi (n = 8). Serum endotoxin levels were measured within 24 h of ICU admission using a kinetic chromogenic Limulus amebocyte lysate assay. Patients with Legionella pneumophila infection demonstrated significantly higher endotoxin levels than those with other gram-negative infections (mean 2.15 ± 1.28 vs 0.61 ± 0.74 EU/mL). Using a threshold of > 2.5 EU/mL, endotoxin quantification showed a sensitivity of 77.8% and specificity of 89.7% for differentiating Legionella infections, with a negative predictive value of 95.3%. Multivariable logistic regression indicated that endotoxin > 2.5 EU/mL remained independently associated with Legionella infection (adjusted OR 15.7, 95% CI 3.84–64.2, p < 0.001). These findings suggest that serum endotoxin quantification may serve as a useful adjunctive biomarker to aid early identification of Legionella pneumophila infection in critically ill patients.

Introduction

The clinical recognition and timely treatment of Legionella pneumophila infections represent one of the most formidable challenges in contemporary critical care medicine, where diagnostic delays can precipitate catastrophic outcomes in vulnerable patients. This fastidious, gram-negative bacterium causes severe pneumonia with mortality rates ranging from 25% to 50% in hospitalized patients, particularly those requiring intensive care support¹. The organism's unique intracellular lifestyle within alveolar macrophages, coupled with its fastidious growth requirements and resistance to standard antimicrobial agents, creates a ....

Protocol

This retrospective cohort study was conducted in a 22-bed medical intensive care unit (ICU) at Zhongshan City People's Hospital, a tertiary academic medical center. The study protocol was approved by the Institutional Review Board of Zhongshan City People's Hospital (Approval Number: 2026-032). Due to the retrospective design and use of de-identified data, the requirement for informed consent was waived, with all procedures ensuring patient confidentiality through anonymization and compliance with the Declaration of Helsinki and relevant data protection laws. Patient selection and study flow are detailed in Figure 1, while endotox....

Results

Study population and baseline characteristics
From an initial screening of 312 ICU admissions, 118 patients met the inclusion criteria and were included in the final analysis cohort. These patients were classified into four pathogen groups: Legionella pneumophila (n = 18), other gram-negative bacteria (n = 68), gram-positive bacteria (n = 24), and fungi (n = 8) (Figure 1). Baseline characteristics were generally comparable across groups (Table 1.......

Discussion

This study provides the first systematic evaluation of serum endotoxin quantification as a diagnostic biomarker for differentiating Legionella pneumophila infections from other gram-negative bacterial infections in critically ill patients. Current study findings reveal a striking paradox that challenges conventional understanding of Legionella pathophysiology: despite the organism's well-documented reduced endotoxic potency in laboratory studies, patients with legionellosis demon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Kinetic chromogenic Limulus amebocyte lysate assay	Associates of Cape Cod / Charles River Laboratories	KTA-2 or equivalent	Used for quantitative serum endotoxin measurement; detection range 0.01–10.0 EU/mL
Buffered charcoal yeast extract agar	BD Diagnostics / Remel	221836 / R05001	Selective agar for culturing Legionella pneumophila.
Sterile pyrogen-free blood collection tubes	BD Vacutainer / Greiner Bio-One	368498 / 455092	For venous blood collection to avoid endotoxin contamination.
Pyrogen-free microcentrifuge tubes	Corning / Eppendorf	3620 / 022364111	For serum aliquoting and storage.
–80 °C freezer	Thermo Scientific / Panasonic	Forma 900 / MDF-U73V	For long-term storage of serum samples.
Centrifuge (3,000 × g)	Eppendorf / Thermo Scientific	5810R / Sorvall ST 8	For serum separation after clotting.
Urinary antigen test for L. pneumophila	BinaxNOW / Sofia	430-000 / 30400	Rapid immunochromatographic test for Legionella pneumophila serogroup 1 antigen.
Metagenomic next-generation sequencing platform	Illumina / Oxford Nanopore	MiSeq / MinION	Used for pathogen identification when conventional diagnostics were inconclusive.
APACHE II & SOFA scoring sheets	–	–	Used for assessing disease severity at ICU admission.
Electronic medical record system	–	–	Used for retrospective data extraction (demographics, lab results, treatments, outcomes).
Statistical software (R version 4.3.0)	R Foundation	–	Used for all statistical analyses, including ROC, regression, and survival modeling.
Laboratory equipment for routine tests	–	–	Includes CBC analyzer, chemistry analyzer, lactate meter, CRP/PCT immunoassay systems, etc.