Human Serum Components Research Articles

Human Serum Supplementation Promotes Streptococcus mitis Growth and Induces Specific Transcriptomic Responses.

Streptococcus mitis is a normal member of the human oral microbiota and a leading opportunistic pathogen causing infective endocarditis (IE). Despite the complex interactions between S. mitis and the human host, understanding of S. mitis physiology and its mechanisms of adaptation to host-associated environments is inadequate, especially compared with other IE bacterial pathogens. This study reports the growth-promoting effects of human serum on S. mitis and other pathogenic streptococci, including S. oralis, S. pneumoniae, and S. agalactiae. Using transcriptomic analyses, we identified that, with the addition of human serum, S. mitis downregulates uptake systems for metal ions and sugars, fatty acid biosynthetic genes, and genes involved in stress response and other processes related with growth and replication. S. mitis upregulates uptake systems for amino acids and short peptides in response to human serum. Zinc availability and environmental signals sensed by the induced short peptide binding proteins were not sufficient to confer the growth-promoting effects. More investigation is required to establish the mechanism for growth promotion. Overall, our study contributes to the fundamental understanding of S. mitis physiology under host-associated conditions. IMPORTANCE S. mitis is exposed to human serum components during commensalism in the human mouth and bloodstream pathogenesis. However, the physiological effects of serum components on this bacterium remain unclear. Using transcriptomic analyses, S. mitis biological processes that respond to the presence of human serum were revealed, improving the fundamental understanding of S. mitis physiology in human host conditions.

Human Serum Mediated Bacteriophage Life Cycle Switch in Aggregatibacter actinomycetemcomitans Is Linked to Pyruvate Dehydrogenase Complex.

Antimicrobial resistance is rising as a major global public health threat and antibiotic resistance genes are widely spread among species, including human oral pathogens, e.g., Aggregatibacter actinomycetemcomitans. This Gram-negative, capnophilic, facultative anaerobe is well recognized as a causative agent leading to periodontal diseases, as well as seriously systemic infections including endocarditis. A. actinomycetemcomitans has also evolved mechanisms against complement-mediated phagocytosis and resiliently survives in serum-rich in vivo environments, i.e., inflamed periodontal pockets and blood circulations. This bacterium, however, demonstrated increasing sensitivity to human serum, when being infected by a pseudolysogenic bacteriophage S1249, which switched to the lytic state as a response to human serum. Concomitantly, the pyruvate dehydrogenase complex (PDHc), which is composed of multiple copies of three enzymes (E1, E2, and E3) and oxidatively decarboxylates pyruvate to acetyl-CoA available for tricarboxylic acid (TCA) cycle, was found up-regulated 10-fold in the bacterial lysogen after human serum exposure. The data clearly indicated that certain human serum components induced phage virion replication and egress, resulting in bacterial lysis. Phage manipulation of bacterial ATP production through regulation of PDHc, a gatekeeper linking glycolysis to TCA cycle through aerobic respiration, suggests that a more efficient energy production and delivery system is required for phage progeny replication and release in this in vivo environment. Insights into bacteriophage regulation of bacterial fitness in a mimic in vivo condition will provide alternative strategies to control bacterial infection, in addition to antibiotics.

Analysis of n-6 / n-3 ratio of polyunsaturated fatty acids in human serum by reactive thermal desorption-gas chromatography using a vertical microfurnace pyrolyzer

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) using a vertical microfurnace pyrolyzer and a flame ionization detector (FID) was applied to rapid analysis of chemical composition for polyunsaturated fatty acid (PUFA) components in human serum. Here, the authors used a reactive thermal desorption (RTD) method, where 2 μL of a serum sample was subjected to the THM reaction at 250 °C for 20 s, to detect PUFA components with high selectivity and sensitivity. The resulting chromatograms clearly showed a series of fatty acid methyl esters including PUFAs without any interference peaks derived from the matrix components of the serum samples. Furthermore, the observed chemical composition and n-6 / n-3 ratio for PUFA components were in good agreement with those obtained by a conventional analytical technique involving solvent extraction and transmethylation.

POS-032 Human PLA2R-antibodies induce membranous nephropathy in minipigs and activate complement

POS-032 Human PLA2R-antibodies induce membranous nephropathy in minipigs and activate complement

Sensitive monitoring of 3-hydroxybutyrate as an indicator of human fasting by capillary electrophoresis in a PAMAMPS coated capillary

Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 μm capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 ± 0.00 · 10−9 m2/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 μL acetonitrile to 15 μL serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 μM and 1.44 μM, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 μM, corresponding to normal food, to level of 1924 μM after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.

Potent antitumor activity of a glutamyltransferase-derived peptide via an activation of oncosis pathway

Hepatocellular carcinoma (HCC) still presents poor prognosis with high mortality rate, despite of the improvement in the management. The challenge for precision treatment was due to the fact that little targeted therapeutics are available for HCC. Recent studies show that metabolic and circulating peptides serve as endogenous switches for correcting aberrant cellular plasticity. Here we explored the antitumor activity of low molecular components in human umbilical serum and identified a high abundance peptide VI-13 by peptidome analysis, which was recognized as the part of glutamyltransferase signal peptide. We modified VI-13 by inserting four arginines and obtained an analog peptide VI-17 to improve its solubility. Our analyses showed that the peptide VI-17 induced rapid context-dependent cell death, and exhibited a higher sensitivity on hepatoma cells, which is attenuated by polyethylene glycol but not necrotic inhibitors such as z-VAD-fmk or necrostatin-1. Morphologically, VI-17 induced cell swelling, blebbing and membrane rupture with release of cellular ATP and LDH into extracellular media, which is hallmark of oncotic process. Mechanistically, VI-17 induced cell membrane pore formation, degradation of α-tubulin via influx of calcium ion. These results indicated that the novel peptide VI-17 induced oncosis in HCC cells, which could serve as a promising lead for development of therapeutic intervention of HCC.

Interaction of Acinetobacter baumannii with Human Serum Albumin: Does the Host Determine the Outcome?

Acinetobacter baumannii has become a serious threat to human health due to its extreme antibiotic resistance, environmental persistence, and capacity to survive within the host. Two A. baumannii strains, A118 and AB5075, commonly used as model systems, and three carbapenem-resistant strains, which are becoming ever more dangerous due to the multiple drugs they can resist, were exposed to 3.5% human serum albumin (HSA) and human serum (HS) to evaluate their response with respect to antimicrobial resistance, biofilm formation, and quorum sensing, all features responsible for increasing survival and persistence in the environment and human body. Expression levels of antibiotic resistance genes were modified differently when examined in different strains. The cmlA gene was upregulated or downregulated in conditions of exposure to 3.5% HSA or HS depending on the strain. Expression levels of pbp1 and pbp3 tended to be increased by the presence of HSA and HS, but the effect was not seen in all strains. A. baumannii A118 growing in the presence of HS did not experience increased expression of these genes. Aminoglycoside-modifying enzymes were also expressed at higher or lower levels in the presence of HSA or HS. Still, the response was not uniform; in some cases, expression was enhanced, and in other cases, it was tapered. While A. baumannii AB5075 became more susceptible to rifampicin in the presence of 3.5% HSA or HS, strain A118 did not show any changes. Expression of arr2, a gene involved in resistance to rifampicin present in A. baumannii AMA16, was expressed at higher levels when HS was present in the culture medium. HSA and HS reduced biofilm formation and production of N-Acyl Homoserine Lactone, a compound intimately associated with quorum sensing. In conclusion, HSA, the main component of HS, stimulates a variety of adaptative responses in infecting A. baumannii strains.

Metabolic plasticity enables lifestyle transitions of Porphyromonas gingivalis

Our understanding of how the oral anaerobe Porphyromonas gingivalis can persist below the gum line, induce ecological changes, and promote polymicrobial infections remains limited. P. gingivalis has long been described as a highly proteolytic and asaccharolytic pathogen that utilizes protein substrates as the main source for energy production and proliferation. Here, we report that P. gingivalis displays a metabolic plasticity that enables the exploitation of non-proteinaceous substrates, specifically the monocarboxylates pyruvate and lactate, as well as human serum components, for colonization and biofilm formation. We show that anabolism of carbohydrates from pyruvate is powered by catabolism of amino acids. Concomitantly, the expression of fimbrial adhesion is upregulated, leading to the enhancement of biofilm formation, stimulation of multispecies biofilm development, and increase of colonization and invasion of the primary gingival epithelial cells by P. gingivalis. These studies provide the first glimpse into the metabolic plasticity of P. gingivalis and its adaptation to the nutritional condition of the host niche. Our findings support the model that in response to specific nutritional parameters, P. gingivalis has the potential to promote host colonization and development of a pathogenic community.

Analytical performance of Raman spectroscopy in assaying biochemical components in human serum.

Chronic non-infectious diseases are important to research as they are the main causes of death in Brazil and worldwide. One very important chronic non-infectious disease is cardiovascular disease, whose risk factors (diabetes, dyslipidemia, and renal failure) can be detected through assessments of serum biochemical components. The objective of this study was to evaluate the analytical performance of Raman spectroscopy for analysis of lipid profile (total cholesterol, triglycerides, and HDL cholesterol), non-protein nitrogenous compounds (urea and creatinine), and glucose in 242 human serum samples. Models to discriminate and quantify the samples were developed using the predicted concentration by quantitative regression model based on partial least squares (PLS). The analytical error for the "leave-one-out" cross-validation based on the predicted PLS concentration was 10.5 mg/dL for total cholesterol, 21.4 mg/dL for triglyceride, 13.0 mg/dL for HDL cholesterol, 4.9 mg/dL for urea, 0.21 mg/dL for creatinine, and 15.4 mg/dL for glucose. The Kappa coefficient indicate very good agreement for cholesterol (0.83), good for triglyceride (0.77), urea (0.70) and creatinine (0.66), and fair for HDL cholesterol (0.38) and glucose (0.30). The results of the analytical performance demonstrated that Raman spectroscopy can be considered an important methodology to screen the population, especially for serum triglycerides and cholesterol.

Synthesis and Kinetic Study of Novel Coumarin- Based Mutual Prodrug of 5-fluorouracil and 5-ethynyluracil

The oral use of 5-fluorouracil is being deserted in the last decades due to its atypical intestinal absorption, which is primarily attributed to the mutable activity of dihydropyrimidine dehydrogenase located in the gastrointestinal mucosa. In this work, a coumarin-based prodrug system was utilized to synthesize a novel mutual prodrug of 5-fluorouracil and 5-ethynyluracil. This prodrug was designed to afford a concurrent release of these two active drugs resulting in the improvement of the therapeutic efficacy of both. The synthetic pathway involved 7 linear steps starting from coumarin. The chemical structures of the intermediates and prodrug were established by analyzing their FTIR, 1H-NMR, and 13C-NMR spectra. The in vitro chemical stability of the synthesized prodrug was tested in the HCl buffer (pH 1.2) and phosphate-buffered saline (pH 6.8), while its ability to release the active moieties was studied in human serum. The outcomes of these in vitro studies revealed that the prodrug showed a significant stability in the HCl buffer and phosphate-buffered saline with half-lives of 33.19 h and 18.13 hr respectively, obeying pseudo-first-order kinetics. Also, the prodrug was able to release the two active components in human serum with a half-life of 4.62 h obeying zero-order kinetics. It is concluded that the synthesized prodrug represents a promising oral prodrug of 5-fluorouracil and 5-ethynyluracil to serve better in therapeutics.

Correlating the Structure and Activity of Y. pestis Ail in a Bacterial Cell Envelope

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here, we present an Escherichia coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, cosedimentation of human vitronectin, and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state NMR spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum.

Electrochemical immunosensor for serum parathyroid hormone using voltammetric techniques and a portable simulator

An electrochemical platform based on a screen-printed carbon electrode (SPCE) is developed to detect parathyroid hormone (PTH). A nanocomposite of multi-walled carbon nanotube (MWCNT) and gold nanoparticles (AuNP) was deposited on the SPCE to immobilize antibodies and horseradish peroxidase (HRP). MWCNT improved the stability and conductivity of the immunosensor because of its good electron-transfer ability and tubular structure. The AuNP not only provided a large surface area for antibody immobilization, but it also enhanced the electrochemical signal for enzyme-linked immunosensing. Cyclic voltammetry showed both electron transfer and the effective surface area were increased on the modified electrode. The characteristics of the modified SPCE were assayed by Raman spectroscopy, scanning electron microscopy, atomic force microscopy, and electrochemical techniques. The linear detection range of this PTH immunosensor was within 1–300 pg/ml, and the electrochemical performance was not affected by interference from protein components in human serum. After storage at 4 °C for 28 days, 85% PTH sensing ability of this immunosensor was maintained compared to the freshly prepared one using the SWV and DPV methods. The relative standard deviations of all measurements were within 3–8% for both voltammetric methods. These results indicated the developed immunosensor had good stability and reproducibility. This PTH immunosensor had a detection limit of 0.886 and 0.065 pg/ml for the differential pulse voltammetry and square wave voltammetry, respectively. We provided a quick analysis of serum PTH which might be used as an electrochemical immunosensing platform for point-of-care testing.

Optical Multisensor Array with Functionalized Photonic Droplets by an Interpenetrating Polymer Network for Human Blood Analysis.

Photonic solid-state cholesteric liquid crystal (CLCsolid) droplets intertwined with a poly(acrylic acid) (PAA) network that has an interpenetrating polymer network (IPN) structure (referred to as photonic IPN CLCsolid-PAA droplets) were used as individual sensors in the dots of a PAA-patterned array film after functionalization via immobilization of the receptors and a metal-ion treatment. The photonic IPN CLCsolid-PAA droplets in the PAA-patterned array film were pH-responsive and showed an observable change in the reflected central color. This "smart" property, coupled with the photonic color response, makes these devices ideal photonic sensors. The immobilization of urease and phenylboronic acid on the PAA network allowed for the application of several 10 μm photonic IPN CLCsolid-PAA droplets to the optical photonic biosensors through facilitated volumetric changes in the PAA network in response to urea and glucose analytes, with high selectivity for major components in human serum, acceptable sensitivity for use with human serum, and extreme stability due to a solid-state structure. The blueshift of the reflected color of the KOH-treated photonic IPN CLCsolid-PAA droplets could be used for divalent metal-ion detection. The compartmentalized photonic IPN CLCsolid-PAA droplets in the patterned array film could be used for multiple detection applications, as evidenced by the ability to conduct pH, divalent metal ion, urea, and glucose detections in one patterned array film. This new platform opens the door for many interesting applications with numerous combinations of responsive hydrogel matrices and receptors.

Structural determinants conferring unusual long life in human serum to rattlesnake-derived antimicrobial peptide Ctn[15-34

Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.

Antifouling Lipid Membranes over Protein A for Orientation-Controlled Immunosensing in Undiluted Serum and Plasma.

An important advance in biosensor research is the extension and application of laboratory-developed methodologies toward clinical diagnostics, though the propensity toward nonspecific binding of materials in clinically relevant matrices, such as human blood serum and plasma, frequently leads to compromised assays. Several surface chemistries have been developed to minimize nonspecific interactions of proteins and other biological components found within blood and serum samples, though these often exhibit substantially variable outcomes. Herein we report a surface chemistry consisting of a charged-matched supported lipid membrane that has been tailored to form over a gold surface functionalized with protein A. Fine tuning of the interfacial charge of this membrane, along with rational selection of a backfilling self-assembled monolayer, allows for high surface coverage with retention of orientation-controlled capture antibody attachment. We demonstrate using surface-plasmon resonance (SPR) that this highly charged lipid membrane is antifouling, allowing for complete removal of nonspecific human serum and plasma components using only a mild buffer rinse, which we attribute to unique steric interactions with the underlying surface. Furthermore, this surface chemistry is successfully applied for specific detection of IgG and cholera toxin in undiluted human biofluids with negligible sacrifice of SPR signal compared to buffered analysis. This novel lipid membrane interface over protein A may open new avenues for direct biosensing of disease markers within clinical samples.

Environmental signals control transmission stage formation in Plasmodium falciparum

Sexual commitment initiates production of the transmission‐competent gametocyte stage in malaria parasites. Chromatin remodeling events at the ap2‐g locus and activation of the encoded transcription factor are the earliest steps known in this differentiation process. Interestingly, the rate of gametocyte formation is not fixed and variation is thought to depend on environmental cues.Using an assay to probe gametocyte formation in vitro, we found that parasites induce sexual commitment in response to depletion of human serum components. Fractionation experiments identified lysophosphatidylcholine (lysoPC) – a major phospholipid component of serum – as the active host factor. Low micromolar concentrations of lysoPC are sufficient to prevent sexual differentiation in P. falciparum cultured under otherwise commitment‐inducing conditions. Metabolic labeling experiments revealed that parasites readily use lysoPC as a substrate for the synthesis of other lipids, including phosphatidylcholine (PC). In the absence of lysoPC, parasites induce a switch in lipid metabolism that is accompanied by the transcriptional up‐regulation of enzymes used for de novo synthesis of PC (Kennedy pathway). RNA‐Seq data revealed that lysoPC‐depletion further induces the expression of both known (i.e. ap2‐g, gdv1) and new markers that define the transcriptional signature of commitment. Altogether, our results provide unprecedented insights on how malaria parasites integrate external stimuli into the decision‐making process of sexual differentiation. We are currently applying this knowledge in translational research, including drug screens that aim at identifying compounds with malaria transmission‐blocking activity.Support or Funding InformationSupported by a Postdoc. Mobility fellowship from the Swiss National Science Foundation (P300PA_160975)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

From the Editor’s desk…: April 2019

From the Editor’s desk…: April 2019

Characterization of a Manganese‐Containing Nanoparticle as an MRI Contrast Agent

We present relaxivity and physicochemical data for a high relaxivity, manganese‐containing, nanoparticle‐based, and tumor selective MRI contrast agent, SN132D. The nanoparticles have a diameter of 5–6 nm, selected to give a compromise between renal excretion and tumor contrast, based on the Enhanced Permeability and Retention effect. The per manganese relaxivity (r1) is 24.8 ± 0.2 mm–1 Mn s–1 at 1.5 T and 37 °C in water, corresponding to per nanoparticle relaxivity of 347 ± 3 mm–1 s–1. The NMRD profiles in both water and serum showed a peak in r1 in the frequency range 20–40 MHz, which is characteristic of slowly moving paramagnetic systems. The r1 decreases when the temperature is increased from 25 °C to 37 °C, indicating fast exchange dynamics of the coordinated water molecules. The exchange time constant of the whole water molecule, τMO, was 6 ns. The r1 in serum is significantly higher than in water, due to an increase in medium viscosity and chelation of 18.9 ± 2.0 % of MnII ions to human serum components, most likely human serum albumin. The r1 is stable in the pH range 3–10. The per Mn relaxivity is among the highest reported for Mn‐containing systems and far higher than those of the clinically used gadolinium‐based contrast agents.

19. Biocompatibility of bimetallic FEGDO3 nanoparticles with human blood components for the development of MRI based functional imaging

Background and Aims: Development of high contrast molecular imaging devices has gained tremendous potential in biomedical applications. Nanotechnology-based strategies have been evolved as one of the most promising field to design more appropriate and effective nanoparticles for such applications. However, biocompatibility of developed nanoparticles with human blood components is of pivotal importance for their clinical applications. In this study, we investigated the biological effects and stability of FeGdO3 nanoparticles during interactions with human blood and serum components. Methods: In order to this, human blood and platelet rich plasma samples were treated with 1 μg/mL, 2 μg/mL, 4 μg/mL and 8 μg/mL concentration of FeGdO3 nanopartcles for 30 min, 60 min, 120 min, 240 min, 24 h and 72 h in vitro. Moreover, peripheral blood mononuclear cells were also treated with similar concentrations of nanoparticles to investigate the effect of these nanoparticles in time dependent manner. Following to biocompatibility assessment of FeGdO3 nanoparticles, the dual-MRI contrast property was evaluated using 1.5 T Magnetome using different concentrations mixed with human blood. Results: Complete blood picture showed no adverse effect of tested concentrations of nanoparticles on human blood components such as platelet and RBC counts and levels of hemoglobin and HCT. Furthermore, serum stability of nanoparticles showed no significant effect at different concentrations of serum during interaction. Enhanced dual-MRI contrast was observed for FeGdO3 nanoparticles in human blood. Conclusions: In summary, the use of FeGdO3 showed high stability and biocompatibility with human blood components with increased T1 and T2 MRI-contrast which could be utilized for the future development of functional imaging in human applications. The authors have none to declare.

An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum.

Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.