Urine citrate analysis is relevant in the screening and monitoring of patients with calcium nephrolithiasis. A sensitive, fast, easy, and low-maintenance ion chromatographic (IC) method with conductivity detection for the analysis of urine citrate is developed and validated. Its application on true samples is also reported. Sample urine is diluted with a water solution containing internal standard (IS) before the chromatographic assay. The isocratic chromatographic run time is twenty-five minutes, using sodium hydroxide aqueous solution as the mobile phase. The method is fully validated as a quantitative method to objectively demonstrate its applicability for the intended use. The analytical response is linear in the 0.08–10.4 mmol/L concentration range. Precision and accuracy studies carried out on spiked urine and internal quality control samples reveal an imprecision CV% lower than 11% and an accuracy between 85 and 103%. The stability of citrate in urine samples is also evaluated. An easy, rapid, and low-maintenance, cost-effective IC method for urinary citrate determination is developed and validated. Internal standardization improves reliability and precision. The method has been currently used in our laboratory over recent years to analyze more than 1000 samples per year.

Background/Objectives: Quorum Sensing (QS) refers to the communication mechanism in bacterial cells, which occurs through the production and detection of small signaling molecules to coordinate activities and monitor population size. In Gram-negative bacteria, QS is typically mediated by N-acyl-homoserine lactones (HSLs) and 2-alkyl-4(1H)-quinolone metabolites (AQ). The present study aims to develop and validate an LC-MS/MS method for detecting QS molecules and apply it to the analysis of plasma samples from burn patients with septic shock caused by Acinetobacter baumannii. Methods: The LC-MS/MS method was developed and fully validated for the quantitative, simultaneous determination of five HSLs and four AQ molecules, ultimately derived from the plasma of three patients with septic shock, with samples collected over three consecutive days. Results: The developed method proved to be both specific and selective, demonstrating a good fit and linearity over the entire range of interest. Trueness and accuracy were satisfactory. The method showed excellent intra-assay precision (CV% was lower than 15%) and limits of quantification (LOQ) ranging from 0.02 to 0.79 ng/mL. In the patients’ samples, the concentration of 3-OH-C12-HSL peaked at 1.5 ng/mL on the first day, and C7-PQS, C9-PQS, HHQ, and HQNO ranged from 0.5 to 1.5 ng/mL, peaking at 5 ng/mL in one patient on the third day. Conclusions: A method for the simultaneous determination of nine QS molecules by LC-MS/MS was developed and validated. When applied, it showed good performance for the analysis of plasma samples and could be a useful tool for an improvement in the diagnosis, prognosis, or treatment monitoring of infections in burn patients caused by Acinetobacter baumannii.

Quorum-sensing (QS) molecules produced by Gram-negative bacteria regulate virulence and host-pathogen interactions, but their systemic role in human septic shock remains largely unexplored. We hypothesized that QS molecules are detectable in the circulation of burn patients with septic-shock-associated acute kidney injury (SA-AKI), are associated with disease severity, and are amenable to removal by extracorporeal treatment. In this observational pilot study, plasma concentrations of eight Gram-negative-derived QS molecules were quantified by HPLC-MS/MS in 12 burn patients with refractory septic shock and SA-AKI on continuous kidney replacement therapy (CKRT) and CytoSorb hemoadsorption. Serial blood samples (0, 0.5, 6, 12, and 24 h) were collected during the 24-hour sessions of the 23 studied cartridges. QS molecule removal by the sorbent cartridge and hemofilter was assessed, and associations between baseline QS molecule levels and systemic lactate concentrations were explored. Out of 8 QS molecules tested, C6-homoserine lactone, 3-hydroxy-C12-HSL, C7-PQS, HQNO, HHQ, and C9-PQS were detectable in systemic plasma, whereas 3-oxo-acyl-homoserine lactones (3-oxo-C10/3-oxo-C12) were below the limit of quantification. Cytosorb induced a significant early reduction in plasma QS molecule concentrations of C6-HSH, 3-OH-C12-HSL, and C7-PQS, whereas hemofilter clearance of QS molecules was negligible (< 4 ml/min). Adsorption was followed by partial rebound at later time points, consistent with sorbent saturation and desorption after 12 h. Baseline lactate levels showed a significant positive correlation with the relative plasma concentrations of QS molecules 3-OH-C12-HSL (r = 0.76818, p < 0.002) and C7-PQS (r = 0.64961, p < 0.03), an association that remained significantly robust for both QS molecules in selected patients with Pseudomonas aeruginosa bloodstream infection (r = 0.87698, p < 0.02 and r = 0.55547, p < 0.03, respectively). Gram-negative QS molecules are detectable in the systemic circulation of burn patients with SA-AKI, are associated with systemic lactate concentrations, and can be removed by hemoadsorption but not by the hemofilter. These findings support a potential pathophysiological role of bacterial communication signals in critical illness, and a potential therapeutic target of hemoadsorption in critical illness.

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by a variety of both signs and symptoms; it mainly affects women of childbearing age, with an estimated prevalence of 24/100,000 people in Europe and North America. SLE is often described as an antibodies-driven disease as its clinical manifestations are usually associated with the presence or the absence of specific antibodies. Objectives To evaluate clinical manifestations in patients with SLE and to assess the relationship with the presence of specific antibodies by using real-world data. Methods A retrospective study was performed; the 2019 EULAR/ACR Classification Criteria for Systemic Lupus Erythematosus were used to classify patients with SLE. Data concerning serological profiles (which included Antinuclear antibodies – ANA, anti dsDNA, anti-Ro/SS-A, anti-La/SS-B, anti-Sm) were gathered along with medical records of clinical manifestations. Complement levels were also tested for possible clinical correlations. χ² or Fisher's exact tests were utilized to establish associations between autoantibodies and symptoms. The odds ratios (OR) and their 95% confidence intervals (CI) were computed. No correction was made for multiple testing; only a p-value 0.01 ≤ was considered significant. Results One-hundred and twenty-seven patients (n=127, mean age 53.43 ±14.02) were enrolled in this study. Anti-dsDNA antibodies were found to be statistically significant for both malar rash and proteinuria; anti-Ro/SSA antibodies showed an association with photosensitivity and pericarditis; furthermore, a strong association was found between anti-Ro antibodies and proteinuria, but only if anti-dsDNA antibodies were present as well. Patients who tested positive for anti-La/SSB antibodies correlated with a threefold increase in the risk of developing pericarditis. Lastly, anti-Smith appeared to be associated with NPSLE as well as an increased risk for both autoimmune haemolytic anemia and thrombocytopenia. Conclusions In our study, many associations confirmed those found in previous studies; however, new relationships between antibodies and clinical manifestations were found thus indicating the need for additional evaluations to assess these correlations further.