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 perfect storm of diagnostic and therapeutic complexity that has frustrated clinicians for decades².

Current diagnostic approaches for legionellosis remain frustratingly inadequate for the urgency demanded by critically ill patients. The gold standard urinary antigen test, while specific, detects only L. pneumophila serogroup 1, which accounts for approximately 80–90% of infections, leaving a significant diagnostic gap for other serogroups and species³. Culture methods, though comprehensive, require specialized buffered charcoal yeast extract media and typically yield results only after 48–72 h, a delay that can prove fatal in septic patients⁴. Molecular diagnostic techniques, including polymerase chain reaction and metagenomic next-generation sequencing, while increasingly sensitive and rapid, remain expensive and are not universally available, particularly in resource-limited settings or during off-hours when immediate clinical decisions are most critical⁵.

The diagnostic complexity is further compounded by the nonspecific clinical presentation of legionellosis, which can closely mimic other forms of severe community-acquired pneumonia. While classical teaching emphasizes distinctive features such as hyponatremia, neurological symptoms, and gastrointestinal manifestations, these findings are neither universally present nor sufficiently specific to reliably distinguish Legionella from other bacterial pathogens⁶. Consequently, clinicians often resort to empirical broad-spectrum antimicrobial therapy that may inadequately cover Legionella or unnecessarily expose patients to agents with significant toxicity profiles, contributing to the growing crisis of antimicrobial resistance⁷.

This diagnostic dilemma has intensified the search for rapid, reliable biomarkers that can guide early therapeutic decision-making. Traditional inflammatory markers, including C-reactive protein, procalcitonin, and interleukin-6, while elevated in bacterial infections, lack the specificity necessary to differentiate Legionella from other gram-negative pathogens⁸. More sophisticated approaches, such as host transcriptomic signatures and proteome analysis, though promising in research settings, remain impractical for routine clinical use due to their complexity, cost, and prolonged turnaround times⁹.

Paradoxically, despite extensive research into Legionella's unique pathophysiology, one of the most fundamental characteristics of gram-negative bacteria, endotoxin production, has received limited attention as a potential diagnostic tool for legionellosis. Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, triggers the host inflammatory cascade that characterizes gram-negative sepsis¹⁰. However, Legionella LPS exhibits markedly reduced endotoxic activity compared to prototypical gram-negative pathogens such as Escherichia coli and Pseudomonas aeruginosa, requiring concentrations 100–1000-fold higher to elicit comparable inflammatory responses in laboratory models¹¹. This reduced potency stems from structural differences in the lipid A component, including variations in fatty acid composition and phosphorylation patterns that diminish its recognition by Toll-like receptor 4 and associated signaling pathways¹².

Despite this well-established reduction in in vitro endotoxic activity, clinical observations have suggested a curious paradox: patients with Legionella pneumonia often present with severe systemic inflammatory responses indistinguishable from those seen in classic gram-negative sepsis¹³. This apparent contradiction between laboratory findings and clinical presentations has led to speculation about alternative mechanisms of endotoxin elevation in legionellosis, including massive bacterial lysis during intracellular replication, secondary gut translocation of enterobacterial endotoxin, or enhanced host sensitivity to Legionella LPS in the context of severe illness¹⁴.

The clinical measurement of circulating endotoxin has evolved significantly since the development of the Limulus amebocyte lysate (LAL) assay in the 1970s. Initially employed primarily for pharmaceutical quality control, endotoxin quantification has increasingly found applications in clinical medicine, particularly in the diagnosis and prognosis of gram-negative sepsis¹⁵. The endotoxin activity assay (EAA), which utilizes patient neutrophils primed by endotoxin-antibody complexes, has demonstrated particular promise in differentiating gram-negative from gram-positive infections, with reported sensitivities of 64–85% and specificities of 91–95%¹⁶. However, the specific application of endotoxin quantification to Legionella diagnosis has never been systematically evaluated, representing a significant knowledge gap in the understanding of this challenging pathogen.

The potential clinical utility of endotoxin measurement extends beyond simple pathogen identification. In an era of increasing antimicrobial resistance and growing emphasis on antimicrobial stewardship, rapid diagnostic tools that can guide targeted therapy have become increasingly valuable¹⁷. The ability to confidently exclude Legionella infection based on low endotoxin levels could prevent unnecessary exposure to quinolones or macrolides, while elevated levels in appropriate clinical contexts could prompt early Legionella-active therapy before confirmatory test results become available¹⁸. Such an approach could potentially improve patient outcomes while simultaneously supporting rational antimicrobial use in intensive care settings.

In practical ICU settings, endotoxin testing could serve as an early adjunctive tool within the diagnostic workflow for severe pneumonia. Because endotoxin quantification can be performed rapidly and is widely available in clinical laboratories, it may provide preliminary information while conventional diagnostic tests, such as urinary antigen assays, culture, or molecular diagnostics, are pending. In this context, endotoxin measurement could complement existing Legionella diagnostic strategies by helping clinicians rapidly assess the likelihood of gram-negative endotoxemia and prioritize Legionella-active antimicrobial therapy when clinical suspicion is high. This study provides the first systematic evaluation of serum endotoxin quantification as a diagnostic biomarker for differentiating Legionella pneumophila from other gram-negative bacterial infections in critically ill patients, addressing this critical knowledge gap and potentially contributing to improved diagnostic approaches for one of critical care's most challenging pathogens.

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 endotoxin quantification methods are described below, with results stratified by pathogen groups as shown in Figure 2. All data were extracted from existing medical records and laboratory databases without any prospective interventions or patient contact.

Study population
Researchers included all adult patients (aged ≥18 years) admitted to the ICU between January 2020 and December 2023 who met the following criteria: Inclusion criteria were as follows: Clinical suspicion of severe bacterial infection or sepsis; Positive bacterial culture or molecular diagnostic confirmation; Serum endotoxin measurement within 24 h of ICU admission; and complete clinical and laboratory data available for analysis. Exclusion criteria were as follows: Mixed infections with multiple pathogens; Incomplete diagnostic workup; Recent immunosuppressive therapy that could affect inflammatory response; Chronic inflammatory conditions; Patients who died within 24 h of admission before complete evaluation. Participant characteristics, including demographics, comorbidities, and clinical presentation, were extracted to ensure a representative cohort of critically ill patients with suspected gram-negative infections. No interventions were administered as part of the study, given its retrospective observational nature; control measures focused on standardizing data collection to reduce selection bias.

Microbiological methods
Diagnosis of Legionella pneumophila was established using a combination of urinary antigen testing, culture on buffered charcoal yeast extract agar, and metagenomic next-generation sequencing (mNGS) when available. Urinary antigen testing was used as the primary diagnostic modality because of its rapid turnaround and high clinical sensitivity. Bacterial culture was performed when respiratory specimens were available. mNGS was applied in selected cases where conventional microbiological tests were negative or inconclusive, but clinical suspicion for atypical pathogens remained high. Other bacterial pathogens were identified using standard microbiological methods, including automated blood culture systems, conventional culture techniques, and molecular diagnostics as clinically indicated. All microbiological assessments followed validated laboratory protocols to ensure accuracy and reproducibility. All microbiological testing was performed in the hospital's accredited clinical microbiology laboratory according to standard operating procedures.

Endotoxin quantification
Serum endotoxin levels were measured using a kinetic chromogenic Limulus amebocyte lysate (LAL) assay according to standard laboratory procedures. Blood samples were collected in sterile, pyrogen-free tubes within 24 h of ICU admission. Samples were centrifuged at 3000 × g for 10 min to obtain serum and subsequently stored at −80 °C until analysis. Prior to testing, serum samples were diluted 1 : 10 with endotoxin-free water to reduce potential matrix interference. Endotoxin concentrations were quantified using a portable endotoxin detection system based on the kinetic chromogenic LAL method. Measurements were performed following standardized kinetic detection procedures. The assay detection range was 0.01–10.0 EU/mL, with an inter-assay coefficient of variation < 15%. All samples were analyzed in duplicate, and the mean value was used for statistical analysis. Positive and negative controls provided with the assay system were included in each run to ensure assay reliability and quality control.

Data collection
Clinical data were extracted from electronic medical records using a standardized case report form. Variables included demographics, comorbidities, clinical presentation, severity scores (APACHE II, SOFA), laboratory parameters, antimicrobial therapy, and clinical outcomes. Outcome assessments focused on diagnostic accuracy as the primary endpoint, with secondary endpoints encompassing mortality at 7 days, 28 days, and hospital discharge. Follow-up procedures involved reviewing patient records up to hospital discharge or death to capture complete outcome data. The primary outcome was the diagnostic accuracy of endotoxin quantification for identifying L. pneumophila infections. Secondary outcomes included mortality at 7 days, 28 days, and hospital discharge. Data were categorized into four pathogen groups: Legionella pneumophila (n = 18), other gram-negative bacteria (n = 68), gram-positive bacteria (n = 24), and fungal infections (n = 8).

Statistical analysis
Continuous variables were expressed as mean ± standard deviation (SD) or median (interquartile range, IQR) according to data distribution assessed using the Shapiro–Wilk test. Categorical variables were presented as frequencies and percentages. Differences among groups were compared using one-way analysis of variance (ANOVA) or the Kruskal–Wallis test for continuous variables, and Fisher’s exact test for categorical variables, as appropriate. The diagnostic performance of endotoxin levels for identifying Legionella pneumophila infection was evaluated using receiver operating characteristic (ROC) curve analysis. The optimal cutoff value was determined using Youden’s index. Sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratios were calculated with corresponding 95% confidence intervals. Univariate logistic regression analysis was first performed to identify potential predictors of Legionella infection. Variables with p < 0.10 in univariate analysis were subsequently entered into a multivariate logistic regression model to determine independent predictors. Adjusted odds ratios (ORs) with 95% confidence intervals were reported. Survival outcomes were analyzed using Kaplan–Meier survival curves, and differences between groups were assessed using the log-rank test. A two-sided p-value < 0.05 was considered statistically significant. All statistical analyses and visualizations were performed using R version 4.3.0.

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.