Comparative Study Of Clostridium Difficile Clinical Detection Methods In Patients With Diarrhea

Objective:The study aimed to evaluate the clinical application of three methods for detecting C.difficile infection in fecal samples.Methods:150 cases of fecal specimens from diarrhea patients aged 2 years who were suspected to have C.difficile infection at Shengjing Hospital of China Medical University from December 2016 to August 2018 were collected and tested by three methods:the toxigenic culture(TC):culturing and isolation of C.difficile by cycloserine cefoxitin fructose agar(CCFA),chromID C.difficile(CDIF)agar and CDC anaerobic blood agar at the same time,detection of DNA from C.difficile positive strains by multiplex PCR-based toxin gene test for detection of C.difficile toxin gene(toxin A/B and binary toxin).The enzyme immunoassay(EIA):the C.difficile GDH assay and the C.difficile toxin A/B assay was used to detect C.difficile GDH antigen and A/B toxin;The C.difficile real-time fluorescence quantitation PCR assay was used to detect C.difficile gene(toxin B/binary toxin/tcd C base pair deletion).The clinical application of the C.difficile GDH assay was evaluated by the anaerobic culture method as a routine method for primary screening of C.difficile infection.The toxigenic culture method was used as a reference method to evaluate the clinical application of the C.difficile real-time fluorescence quantitation PCR assay and the enzyme immunoassay.Results:1.TC was used to detect stool samples from 150 patients with diarrhea.Among the specimens,26 specimens carried both A and B toxin genes,and no binary toxin-positive specimen was found.The positive rate of toxin-producing C.difficile was17.33%(26/150).A total of 37 GDH positive samples were detected by the C.difficile GDH assay,and 15 toxin positive samples were detected by the C.difficile toxin A/B assay from 150 patients with diarrhea.The C.difficile real-time fluorescence quantitation PCR assay was used to detect 79 specimens simultaneously.A total of 18 positive specimens were detected.2.The anaerobic culture method was used as a routine method for C.difficile infection primary screening.The clinical application of the C.difficile GDH assay was evaluated.The sensitivity,specificity,positive predictive value(PPV),and negative predictive value(NPV)of the C.difficile GDH assay of 150 specimens were100.0%,97.4%,91.9%,and 100.0%,respectively.The results of the C.difficile GDH assay and the anaerobic culture method were used to perform the chi-square test.The results showed that there was no statistical difference between the two methods(?~2=1.33,P>0.05).The area under the ROC curve of the two methods was calculated and the area under the curve was statistically tested.The area under the curve of the C.difficile GDH assay was 0.987,and there was no statistical difference in the area under the curve(Z=1.747,P>0.05).3.TC was used as a reference method to evaluate the clinical application of the C.difficile real-time fluorescence quantitation PCR assay and the EIA.The sensitivity,specificity,PPV and NPV of the C.difficile real-time fluorescence quantitation PCR assay of 79 specimens were 100.0%,96.8%,88.9%,and 100.0%,respectively;the EIA of 79 specimens were 55.6%,100.0%,100.0%,and 88.4%,respectively.The results showed that there was no statistical difference between the C.difficile real-time fluorescence quantitation assay and the reference method.The results showed no statistical differences between the EIA and the reference method.The area under the ROC curve of the three methods was compared and the area under the curve was statistically tested.The area under the C.difficile real-time fluorescence quantitation PCR assay and the EIA curve were 0.984 and 0.813,respectively.There was statistical difference between the area test of the EIA and TC(Z=3.000,P<0.05),and there was no statistical difference between the area test under the C.difficile real-time fluorescence quantitation PCR assay and TC(Z=1.426,P>0.05).Conclusion:1.The GDH assay is a good indicator for the initial screening of C.difficile infection.2.The C.difficile real-time fluorescence quantitation PCR assay is simple and rapid.The results of the method are accurate.Therefore,the method has good clinical application value.