Novel multiplex-PCR test for Escherichia coli detection.

Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods. IMPORTANCE: Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

Assessment of Selected ROTEM Parameters, Kinetics of Fibrinogen Polymerization and Plasmin Amidolytic Activity in Patients with Congenital Fibrinogen Defects.

BACKGROUND: Congenital fibrinogen disorders (CFD) are rare fibrinogen deficiencies which may be quantitative or functional. The clinical course of hypofibrinogenemia (hypoFI) or dysfibrinogenemia (dysFI) is unpredictable and cannot be determined by the application of standard hemostasis tests. OBJECTIVES: The main aim of this study was to assess ROTEM parameters in CFD patients. MATERIAL AND METHODS: Nine patients with CFD were studied. The fibrinogen concentration was measured functionally and antigenically. EXTEM, INTEM, FIBTEM and APTEM tests were used to measure selected ROTEM parameters, including maximum clot firmness (MCF). Fibrin plasma polymerization, clot lysis and plasmin amidolytic activity were determined by spectrophotometric methods. RESULTS: Incorporating the antigenic, ELISA method, to the diagnostic workup allowed the initial diagnosis to be switched from hypoFI to dysFI in 3/7 patients. MCF readings (the most important parameter describing fibrin polymerization capacity) were significantly lower in patients than in controls according to all ROTEM tests. Cases with hypoFI demonstrated markedly lower readings of MCF according to all ROTEM tests than cases with dysFI. All patients demonstrated disturbances of fibrin polymerization process assessed by turbidimetry. In contrast, no marked differences were identified between studied groups in reference to plasmin amidolytic activity. CONCLUSIONS: Our data suggests that ROTEM and fibrin plasma polymerization according to the turbidimetric method have a high sensitivity towards detection of different CFD. Although ROTEM MCF assessment may help discriminate patients with hypoor dysfibrinogenemia, this finding has to be confirmed on larger groups of patients.

[The role of polyphosphates in the blood coagulation process and the inflammation progress].

Polyphosphates (polyP) are commonly found in prokaryotic and eukaryotic cells linear, highly anionic polymers, composed of a few to many hundreds of orthophosphate residues, linked by high-energy phosphoanhydride bonds. These polymers are stored in dense granules of platelets and secreted after their activation. The recently studies indicate that polyP are a potent procoagulant agent accelerating blood coagulation by activating the contact pathway and by promoting FV and FXI activation by thrombin, which in turn abrogates the anticoagulant function of tissue factor pathway inhibitor (TFPI). Furthermore polyP enhance fibrin clot structure, which increase its resistance to fibrinolysis. PolyP using different signaling pathways have pro-inflammatory effects by promoting bradykinin release, enhancing the pro-inflammatory activity of histones, and activating NF-κB.