Molecular Analysis of Volatile Metabolites Synthesized by Candida albicans and Staphylococcus aureus in In Vitro Cultures and Bronchoalveolar Lavage Specimens Reflecting Single- or Duo-Factor Pneumonia.

Current microbiological methods for pneumonia diagnosis require invasive specimen collection and time-consuming analytical procedures. There is a need for less invasive and faster methods to detect lower respiratory tract infections. The analysis of volatile metabolites excreted by pathogenic microorganisms provides the basis for developing such a method. Given the synergistic role of Candida albicans in increasing the virulence of pathogenic bacteria causing pneumonia and the cross-kingdom metabolic interactions between microorganisms, we compare the emission of volatiles from Candida albicans yeasts and the bacteria Staphylococcus aureus using single and mixed co-cultures and apply that knowledge to human in vivo investigations. Gas chromatography-mass spectrometry (GC-MS) analysis resulted in the identification of sixty-eight volatiles that were found to have significantly different levels in cultures compared to reference medium samples. Certain volatiles were found in co-cultures that mainly originated from C. albicans metabolism (e.g., isobutyl acetate), whereas other volatiles primarily came from S. aureus (e.g., ethyl 2-methylbutyrate). Isopentyl valerate reflects synergic interactions of both microbes, as its level in co-cultures was found to be approximately three times higher than the sum of its amounts in monocultures. Hydrophilic-lipophilic-balanced (HLB) coated meshes for thin-film microextraction (TFME) were used to preconcentrate volatiles directly from bronchoalveolar lavage (BAL) specimens collected from patients suffering from ventilation-associated pneumonia (VAP), which was caused explicitly by C. albicans and S. aureus. GC-MS analyses confirmed the existence of in vitro-elucidated microbial VOCs in human specimens. Significant differences in BAL-extracted amounts respective to the pathogen-causing pneumonia were found. The model in vitro experiments provided evidence that cross-kingdom interactions between pathogenic microorganisms affect the synthesis of volatile compounds. The TFME meshes coated with HLB particles proved to be suitable for extracting VOCs from human material, enabling the translation of in vitro experiments on the microbial volatilome to the in vivo situation involving infected patients. This indicates the direction that should be taken for further clinical studies on VAP diagnosis based on volatile analysis.

MGMT Promoter Methylation as a Prognostic Factor in Primary Glioblastoma: A Single-Institution Observational Study.

Glioblastoma is the most malignant central nervous system tumor, which represents 50% of all glial tumors. The understanding of glioma genesis, prognostic evaluation, and treatment planning has been significantly enhanced by the discovery of molecular genetic biomarkers. This study aimed to evaluate survival in patients with primary glioblastoma concerning O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and other clinical factors. The study included 41 newly diagnosed glioblastoma patients treated from 2011 to 2014 in the 10th Military Research Hospital and Polyclinic, Poland. All patients underwent surgical resection followed by radiation and chemotherapy with alkylating agents. The MGMT promoter methylation was evaluated in all patients, and 43% were found to be methylated. In 26 and 15 cases, gross total resection and subtotal resection were conducted, respectively. Patients with a methylated MGMT promoter had a median survival of 504 days, while those without methylation had a median survival of 329 days. The group that was examined had a median age of 53. In a patient group younger than 53 years, those with methylation had significantly longer overall survival (639 days), compared to 433.5 days for patients without methylation. The most prolonged survival (551 days) was in patients with MGMT promoter methylation after gross total resection. The value of MGMT promoter methylation as a predictive biomarker is widely acknowledged. However, its prognostic significance remains unclear. Our findings proved that MGMT promoter methylation is also an essential positive prognostic biomarker.

Quantitative Determination of Unbound Piperacillin and Imipenem in Biological Material from Critically Ill Using Thin-Film Microextraction-Liquid Chromatography-Mass Spectrometry.

ß-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction-LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.