Limits...
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

Stability of toxins in serum over time during storage.Spiked sample was prepared by 5× serially diluted TcdA/ TcdB into toxemia-negative serum (b and d) or medium (a and c) respectively. mRG1-1 (for detecting TcdA, a and b) and Vero (for testing TcdB, c and d) cells were cultured overnight with the spiked samples maintained at 4°C (A) and -80°C (B) storage and cytopathic effect were compared. Two toxemia-negative serum samples were used for the experiment and showed similar results; the presented data was from one of the two samples. D, days of storage; T, times of freeze/thaw cycles.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401762&req=5

pone.0124235.g003: Stability of toxins in serum over time during storage.Spiked sample was prepared by 5× serially diluted TcdA/ TcdB into toxemia-negative serum (b and d) or medium (a and c) respectively. mRG1-1 (for detecting TcdA, a and b) and Vero (for testing TcdB, c and d) cells were cultured overnight with the spiked samples maintained at 4°C (A) and -80°C (B) storage and cytopathic effect were compared. Two toxemia-negative serum samples were used for the experiment and showed similar results; the presented data was from one of the two samples. D, days of storage; T, times of freeze/thaw cycles.

Mentions: To determine whether storage conditions affect the outcome of toxemia detection, we compared the cytotoxic activity of toxins in spiked samples with or without storage. As shown in Fig 3, there is no significant difference in the cytotoxic activity between freshly prepared and stored samples. Only a 5-fold difference in detection limit was observed between the two groups. Toxin-spiked serum samples were more stable when stored at -80°C than at 4°C. However, the minimum concentrations (causing ≥50% cell rounding) of toxins in toxin-spiked sera were significant higher (p < 0.05) than in toxin-spiked media. When the toxins were spiked freshly in culture media, the minimum concentrations of TcdA and TcdB resulting in ≥50% cell rounding were 0.512 pg/ml and 4.8 pg/ml respectively. However, these minimum detectable concentrations were increased 25-fold (12.8 pg/ml) for TcdA and 125-fold (600 pg/ml) for TcdB respectively when the toxins were spiked in patient sera.


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)

Stability of toxins in serum over time during storage.Spiked sample was prepared by 5× serially diluted TcdA/ TcdB into toxemia-negative serum (b and d) or medium (a and c) respectively. mRG1-1 (for detecting TcdA, a and b) and Vero (for testing TcdB, c and d) cells were cultured overnight with the spiked samples maintained at 4°C (A) and -80°C (B) storage and cytopathic effect were compared. Two toxemia-negative serum samples were used for the experiment and showed similar results; the presented data was from one of the two samples. D, days of storage; T, times of freeze/thaw cycles.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124235.g003: Stability of toxins in serum over time during storage.Spiked sample was prepared by 5× serially diluted TcdA/ TcdB into toxemia-negative serum (b and d) or medium (a and c) respectively. mRG1-1 (for detecting TcdA, a and b) and Vero (for testing TcdB, c and d) cells were cultured overnight with the spiked samples maintained at 4°C (A) and -80°C (B) storage and cytopathic effect were compared. Two toxemia-negative serum samples were used for the experiment and showed similar results; the presented data was from one of the two samples. D, days of storage; T, times of freeze/thaw cycles.
Mentions: To determine whether storage conditions affect the outcome of toxemia detection, we compared the cytotoxic activity of toxins in spiked samples with or without storage. As shown in Fig 3, there is no significant difference in the cytotoxic activity between freshly prepared and stored samples. Only a 5-fold difference in detection limit was observed between the two groups. Toxin-spiked serum samples were more stable when stored at -80°C than at 4°C. However, the minimum concentrations (causing ≥50% cell rounding) of toxins in toxin-spiked sera were significant higher (p < 0.05) than in toxin-spiked media. When the toxins were spiked freshly in culture media, the minimum concentrations of TcdA and TcdB resulting in ≥50% cell rounding were 0.512 pg/ml and 4.8 pg/ml respectively. However, these minimum detectable concentrations were increased 25-fold (12.8 pg/ml) for TcdA and 125-fold (600 pg/ml) for TcdB respectively when the toxins were spiked in patient sera.

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