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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

Cytotoxicity induced by C. difficile toxins in CDI patient sera.Cell rounding was observed under microscopy in two serum samples from Case I (A, A’) and Case II (B, B’) by cell-based assay on mRG1-1 cells (A, B) and Vero cells (A’, B’). Cells cultured in medium were as negative controls. TcdA (50 pg/ml with A1H3)-treated mRG1-1 cell and TcdB (25 pg/ml)-treated Vero cell were served as positive controls. The sera (1:10 dilution) were added to cells and cell morphology changes were observed under a phase-contrast microscope.
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pone.0124235.g001: Cytotoxicity induced by C. difficile toxins in CDI patient sera.Cell rounding was observed under microscopy in two serum samples from Case I (A, A’) and Case II (B, B’) by cell-based assay on mRG1-1 cells (A, B) and Vero cells (A’, B’). Cells cultured in medium were as negative controls. TcdA (50 pg/ml with A1H3)-treated mRG1-1 cell and TcdB (25 pg/ml)-treated Vero cell were served as positive controls. The sera (1:10 dilution) were added to cells and cell morphology changes were observed under a phase-contrast microscope.

Mentions: C. difficile toxins were detected in the serum of the two described CDI patients using ultrasensitive ICT and cytotoxin B assays. Cell rounding was observed in cells cultured with the two serum samples (Fig 1 and Table 1). For case I, cell rounding induced by the serum was only partially inhibited by anti-TcdA or anti-TcdB antibodies, but was completely blocked by the presence of both antibodies (Fig 1A). Thus, in Case I, serum-mediated cytotoxicity on mRG1-1 cells was due to the presence of both TcdA and TcdB in serum, which was confirmed by the neutralization of toxin by both anti-TcdA and anti-TcdB antibodies. In case II, cell rounding was not induced in mRG1-1 cells but was observed in Vero cells. Serum cytotoxic activity was fully blocked by anti-TcdB alone or by a combination of both antibodies; however, the activity was not inhibited by anti-TcdA alone (Fig 1B). Based on the cell-based assays, case I was characterized as TcdA+ TcdB+ toxemia, while case II was classified as TcdB+ toxemia with undetectable TcdA. These results are valid only within the limits of detection of our assays. The finding that the TcdB in case II serum caused cytotoxicity to Vero cells but not to mRG1-1 cells likely reflects the fact that TcdB is less toxic to mRG1-1 cells compared to Vero cells.


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)

Cytotoxicity induced by C. difficile toxins in CDI patient sera.Cell rounding was observed under microscopy in two serum samples from Case I (A, A’) and Case II (B, B’) by cell-based assay on mRG1-1 cells (A, B) and Vero cells (A’, B’). Cells cultured in medium were as negative controls. TcdA (50 pg/ml with A1H3)-treated mRG1-1 cell and TcdB (25 pg/ml)-treated Vero cell were served as positive controls. The sera (1:10 dilution) were added to cells and cell morphology changes were observed under a phase-contrast microscope.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124235.g001: Cytotoxicity induced by C. difficile toxins in CDI patient sera.Cell rounding was observed under microscopy in two serum samples from Case I (A, A’) and Case II (B, B’) by cell-based assay on mRG1-1 cells (A, B) and Vero cells (A’, B’). Cells cultured in medium were as negative controls. TcdA (50 pg/ml with A1H3)-treated mRG1-1 cell and TcdB (25 pg/ml)-treated Vero cell were served as positive controls. The sera (1:10 dilution) were added to cells and cell morphology changes were observed under a phase-contrast microscope.
Mentions: C. difficile toxins were detected in the serum of the two described CDI patients using ultrasensitive ICT and cytotoxin B assays. Cell rounding was observed in cells cultured with the two serum samples (Fig 1 and Table 1). For case I, cell rounding induced by the serum was only partially inhibited by anti-TcdA or anti-TcdB antibodies, but was completely blocked by the presence of both antibodies (Fig 1A). Thus, in Case I, serum-mediated cytotoxicity on mRG1-1 cells was due to the presence of both TcdA and TcdB in serum, which was confirmed by the neutralization of toxin by both anti-TcdA and anti-TcdB antibodies. In case II, cell rounding was not induced in mRG1-1 cells but was observed in Vero cells. Serum cytotoxic activity was fully blocked by anti-TcdB alone or by a combination of both antibodies; however, the activity was not inhibited by anti-TcdA alone (Fig 1B). Based on the cell-based assays, case I was characterized as TcdA+ TcdB+ toxemia, while case II was classified as TcdB+ toxemia with undetectable TcdA. These results are valid only within the limits of detection of our assays. The finding that the TcdB in case II serum caused cytotoxicity to Vero cells but not to mRG1-1 cells likely reflects the fact that TcdB is less toxic to mRG1-1 cells compared to Vero cells.

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