BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

• Publikační typ
• časopisecké články MeSH

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is essential for proper initial antibiotic therapy and timely set up of hygienic measures. Recently, detection of MRSA using MALDI-TOF mass spectrometer mediated by the peptide-phenol-soluble modulin (PSM-mec)-linked to the class A mec gene complex present in SCCmec cassettes types II, III, and VIII of MRSA strains, has been commercially available. We present here a multicentre study on MALDI-TOF MS detection of MRSA evincing a poor repeatability and reproducibility of the assay. The sensitivity of the assay varies between 50 and 90% in strains carrying psmMEC and psmδ genes encoding for PSM-mec and δ-toxin (a member of the PSM peptide family), respectively. No false positive results were found. The very major error calculation was 30% and the major error achieved 0%. Interlaboratory repeatability varies between 0 and 100%. No significant difference was observed with the use of different cultivation media. Our data showed a poor sensitivity of the method excluding it from the use in routine laboratory testing.

• MeSH
• bakteriální toxiny genetika   MeSH
• chybná diagnóza MeSH
• diagnostické techniky molekulární * MeSH
• diagnostické testy rutinní MeSH
• lidé MeSH
• methicilin rezistentní Staphylococcus aureus genetika   izolace a purifikace   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
• stafylokokové infekce diagnóza   mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

Health-care associated infections (HCAIs) significantly influence current medicine and may limit the progress in many medical procedures. Treatment of such infections has been increasingly complicated by the spread of antimicrobial resistance (AMR). To reduce risks of inappropriate initial therapy, the availability of rapid diagnostic microbiological assays is of utmost importance. Especially, functional diagnostics (i.e., detection of enzyme activity) is important in era of horizontal gene transfer. Functional assays for detection of selected bacterial toxins (ToxB of C. difficile and YopT of Y. enterocolitica) using affinity MALDI will be developed and validated. Simultaneously, functional assay for detection of carbapenemase activity developed in the laboratory of co-investigator will be further optimized and validated. Further, a new assay for functional detection of lipid A modification detecting colistin resistance will be also developed. All of those assays and tests represent a challenge for symptom-based microbiology and functional detection of important antibiotic resistance. Introduction of such assays to routine diagnostics will help to detect potentially unknown pathogens emerged because of horizontal gene transfer.

Infekce spojené se zdravotní péčí (HCAI) významně ovlivňují současnou medicínu a limitují rozvoj některých invazivních zákroků. Léčba HCAI je obvykle komplikovaná rezistencí původců na dostupná antibiotika. Ke snížení nevhodné iniciální terapie, která často vede k selhání léčby, je nutné se zaměřit na vývoj nových rychlých diagnostických testů. Především se jedná o metody funkční diagnostiky, které reagují na současný stav horizontálního toku genetické informace. V rámci řešení projektu budou vyvinuty a validovány testy pro detekci některých bakteriálních toxinů (např. toxin B produkovaný Clostridium difficile, YopT produkovaný Yersinia enterocolitica) pomocí metody afinitní MALDI-TOF hmotnostní spektrometrie. Zároveň budou optimalizovány metody pro detekci karbapenemáz dříve vyvinuté v laboratoři spoluřešitele. Pro detekci kolistinové rezistence bude vyvinuta metoda umožňující detekci modifikace lipidu A. Všechny zmíněné metodiky reagují na urgentní potřebu rutinní mikrobiologické diagnostiky a umožní detekovat i potenciálně neznámé patogeny s ohledem na horizontální přenos genetické informace.

• Klíčová slova
• antibiotická terapie, antibiotic therapy, Kolistin, colistin, antibiotic resistance, Hmotnostní spektrometrie, Mass Spectrometry, antibiotická rezistence, karbapenemázy, lipid A, carbapenemase, lipid A, Funkční diagnostika, Functional diagnostics,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR