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Identification of toxemia in patients with Clostridium difficile infection.

Yu H, Chen K, Wu J, Yang Z, Shi L, Barlow LL, Aronoff DM, Garey KW, Savidge TC, von Rosenvinge EC, Kelly CP, Feng H - PLoS ONE (2015)

Bottom Line: C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay.Toxins were relatively stable in stored sera.Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United States of America.

ABSTRACT
Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

No MeSH data available.


Related in: MedlinePlus

Serum neutralizing antibodies affect toxin detection limits of cell-based assays.Total IgG was depleted from serum samples by Protein A beads and the depletion was verified by western blot with HRP-conjugated anti-human IgG (γ-chain). The image illustrates the WB data of two samples before and after IgG depletion (A). N, serum before IgG depletion; D, IgG-depleted serum. Toxin detection limits were determined by cell-based assays for the spiked toxins in the sera before and after IgG depletion (B). 5× serially diluted recombinant TcdA or TcdB were spiked in the sera or PBS, and the samples were then applied to cells for overnight. Detection limit of TcdA (b) was determined by ICT using mRG1-1 cells, and TcdB (a) detection limit was assessed on Vero cells. Toxin detection limits were defined as the minimum concentration of toxins inducing at least 50% of cell rounding.
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pone.0124235.g004: Serum neutralizing antibodies affect toxin detection limits of cell-based assays.Total IgG was depleted from serum samples by Protein A beads and the depletion was verified by western blot with HRP-conjugated anti-human IgG (γ-chain). The image illustrates the WB data of two samples before and after IgG depletion (A). N, serum before IgG depletion; D, IgG-depleted serum. Toxin detection limits were determined by cell-based assays for the spiked toxins in the sera before and after IgG depletion (B). 5× serially diluted recombinant TcdA or TcdB were spiked in the sera or PBS, and the samples were then applied to cells for overnight. Detection limit of TcdA (b) was determined by ICT using mRG1-1 cells, and TcdB (a) detection limit was assessed on Vero cells. Toxin detection limits were defined as the minimum concentration of toxins inducing at least 50% of cell rounding.

Mentions: To investigate whether neutralizing antibodies in patient sera affect the detection limits of toxins by cell-based assays, we depleted total IgG in sera with Protein A beads and measured the detection limits of the toxins. As shown in Fig 4A, after binding to Protein A beads, a majority of IgG was depleted in the two samples tested. Neutralizing anti-TcdA /anti-TcdB titers were 40 and 320 respectively in sample #35 before IgG depletion, but became undetectable for anti-TcdA titer and decreased to 80 for anti-TcdB titer after IgG depletion (Table 2). The detection limits for spiked TcdA and TcdB in serum #35 before IgG depletion were 0.64 ng/ml and 100 ng/ml respectively, which were 125-fold higher than those in the same serum after IgG depletion (Fig 4B). For sample #0467, the neutralizing titers were undetectable for anti-TcdB and 10 for anti-TcdA. After IgG depletion by Protein A, the neutralizing titers for both anti-TcdA/anti-TcdB were undetectable. The detection limit of TcdA decreased 5 folds by depleting IgG. Consistently, the detection limit for TcdB remained unchanged after IgG depletion and was equal to that in PBS (Table 2). These data demonstrated that neutralizing antibodies against the two toxins play a critical role in masking toxins’ cytotoxicity in serum samples.


Identification of toxemia in patients with Clostridium difficile infection.

Yu H, Chen K, Wu J, Yang Z, Shi L, Barlow LL, Aronoff DM, Garey KW, Savidge TC, von Rosenvinge EC, Kelly CP, Feng H - PLoS ONE (2015)

Serum neutralizing antibodies affect toxin detection limits of cell-based assays.Total IgG was depleted from serum samples by Protein A beads and the depletion was verified by western blot with HRP-conjugated anti-human IgG (γ-chain). The image illustrates the WB data of two samples before and after IgG depletion (A). N, serum before IgG depletion; D, IgG-depleted serum. Toxin detection limits were determined by cell-based assays for the spiked toxins in the sera before and after IgG depletion (B). 5× serially diluted recombinant TcdA or TcdB were spiked in the sera or PBS, and the samples were then applied to cells for overnight. Detection limit of TcdA (b) was determined by ICT using mRG1-1 cells, and TcdB (a) detection limit was assessed on Vero cells. Toxin detection limits were defined as the minimum concentration of toxins inducing at least 50% of cell rounding.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401762&req=5

pone.0124235.g004: Serum neutralizing antibodies affect toxin detection limits of cell-based assays.Total IgG was depleted from serum samples by Protein A beads and the depletion was verified by western blot with HRP-conjugated anti-human IgG (γ-chain). The image illustrates the WB data of two samples before and after IgG depletion (A). N, serum before IgG depletion; D, IgG-depleted serum. Toxin detection limits were determined by cell-based assays for the spiked toxins in the sera before and after IgG depletion (B). 5× serially diluted recombinant TcdA or TcdB were spiked in the sera or PBS, and the samples were then applied to cells for overnight. Detection limit of TcdA (b) was determined by ICT using mRG1-1 cells, and TcdB (a) detection limit was assessed on Vero cells. Toxin detection limits were defined as the minimum concentration of toxins inducing at least 50% of cell rounding.
Mentions: To investigate whether neutralizing antibodies in patient sera affect the detection limits of toxins by cell-based assays, we depleted total IgG in sera with Protein A beads and measured the detection limits of the toxins. As shown in Fig 4A, after binding to Protein A beads, a majority of IgG was depleted in the two samples tested. Neutralizing anti-TcdA /anti-TcdB titers were 40 and 320 respectively in sample #35 before IgG depletion, but became undetectable for anti-TcdA titer and decreased to 80 for anti-TcdB titer after IgG depletion (Table 2). The detection limits for spiked TcdA and TcdB in serum #35 before IgG depletion were 0.64 ng/ml and 100 ng/ml respectively, which were 125-fold higher than those in the same serum after IgG depletion (Fig 4B). For sample #0467, the neutralizing titers were undetectable for anti-TcdB and 10 for anti-TcdA. After IgG depletion by Protein A, the neutralizing titers for both anti-TcdA/anti-TcdB were undetectable. The detection limit of TcdA decreased 5 folds by depleting IgG. Consistently, the detection limit for TcdB remained unchanged after IgG depletion and was equal to that in PBS (Table 2). These data demonstrated that neutralizing antibodies against the two toxins play a critical role in masking toxins’ cytotoxicity in serum samples.

Bottom Line: C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay.Toxins were relatively stable in stored sera.Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United States of America.

ABSTRACT
Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

No MeSH data available.


Related in: MedlinePlus