|Year : 2011 | Volume
| Issue : 3 | Page : 293-296
Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile
MO Qutub, N AlBaz, P Hawken, A Anoos
Departments Pathology and Laboratory Medicine, Medical Technologist, MBC: J-10, King Faisal Specialist Hospital and Research Centre, Jeddah-Branch, P.O. Box 40047, Jeddah 21499, Saudi Arabia
|Date of Submission||26-Jan-2011|
|Date of Acceptance||15-Feb-2011|
|Date of Web Publication||17-Aug-2011|
M O Qutub
Departments Pathology and Laboratory Medicine, Medical Technologist, MBC: J-10, King Faisal Specialist Hospital and Research Centre, Jeddah-Branch, P.O. Box 40047, Jeddah 21499
Source of Support: None, Conflict of Interest: None
Purpose: To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. Materials and Methods: A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer's instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. Results: A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. Conclusions: The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.
Keywords: Antibiotic-associated diarrhoea, Clostridium difficile, EIAs, glutamate dehydrogenase, TOX-A/B EIA
|How to cite this article:|
Qutub M O, AlBaz N, Hawken P, Anoos A. Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile. Indian J Med Microbiol 2011;29:293-6
|How to cite this URL:|
Qutub M O, AlBaz N, Hawken P, Anoos A. Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile. Indian J Med Microbiol [serial online] 2011 [cited 2020 Jan 28];29:293-6. Available from: http://www.ijmm.org/text.asp?2011/29/3/293/83916
| ~ Introduction|| |
Clostridium difficile is the leading cause of hospital-acquired diarrhoea, and is responsible for up to 25% of the cases of antibiotic-associated diarrhoea and most cases of pseudomembranous colitis. , This organism is carried asymptomatically in about 20% of the hospitalized patients, but only 2% of healthy adults.  Therefore, the high number of healthy carriers among hospitalized patients coupled with the presence of patients under antibiotic treatment explains the high rate of nosocomial diarrhoea associated with Clostridium difficile. Clostridium difficile infection can be acquired through both direct patient-to-patient contact and from the hospital environment.  Such nosocomial transmission can contribute significantly to the length of hospital stay.
The pathogenicity of this organism is associated with the production of two toxins, A and B, which act in synergy on the intestinal mucosa, leading to its damage.  Most strains produce both toxins, but pathogenic strains of Clostridium difficile producing toxin-B only have also been reported.  Nontoxigenic strains do not carry the pathogenicity locus, do not produce any toxin, and, therefore, are nonpathogenic.
Diagnostic methods of Clostridium difficile-associated diarrhoea (CD-AD) are based on the detection of toxin-B by the tissue culture cytotoxicity assay, which is considered the "gold standard" for the detection of Clostridium difficile from faecal samples.  However, this assay is time-consuming, requiring 24-48 h for completion. Enzyme immunoassays (EIA) are also commonly used for toxin detection, but their clinical sensitivity may be suboptimal, particularly if only toxin-A is detected. , Current antigen enzyme immunoassays (Ag-EIA) accurately detect an essential and constitutively synthesized enzyme.  Because Ag-EIA detect non-toxigenic as well as toxigenic Clostridium difficile, such assays must be used in combination with a toxin-detecting assay to provide specific laboratory evidence of CD-AD. ,, Recently, a two-step algorithm, including tests for antigen and cytotoxin, was developed  in an attempt to improve both the turnaround time and the cost for the diagnosis of CD-AD. Therefore, the aim of this study was to evaluate and compare this two-step algorithm with the three-step algorithm that includes tests for antigen and cytotoxin by both the toxin EIA and by the CCNA for cytotoxin (toxin-B).
| ~ Materials and Methods|| |
One hundred and fifty stool specimens from 151 consecutive patients admitted and suspected to have CD-AD were evaluated, with majority of these patients having had received different types of antibiotics, including third-generation of cephalosporins, quinolones, and macrolides. All specimens were maintained at 4 o C and processed within 72 h of collection. An aliquot of each specimen was frozen at 70 o C in case further studies were required.
Glutamate dehydrogenase (GDH; TechLab's C.DIFF CHEK-60, TL-GDH, Blacksburg, VA, USA) and Clostridium difficile TOX-A/BII enzyme immunoassays (TOX-A/BII EIA, Blacksburg, VA, USA) were performed according to the manufacturer's instruction. The results of GDH and TOX-A/BII EIA were read using a spectrometer at a wavelength of 450 nm.
Cell line culture neutralization assay
Fresh stool specimens were diluted in minimal essential medium (MEM) (1:10 wt/vol) and centrifuged at 2,500 x g for 10 min. The supernatant was passed through a 0.2-um pore size filter and inoculated onto confluent monolayers of human lung fibroblast (MRC-5) cells in a tube culture, then incubated at 37 o C in an incubator for 24 and 48 h.  Screening for cytotoxicity was performed at a stool dilution of 1:100. Samples were considered positive in the cytotoxicity assay if a characteristic of cytopathic effect (cell rounding) was observed and could be neutralized with anti-Clostridium sordellii antiserum (obtained from TechLab Inc., Blacksburg, VA, USA). Neutralization was performed at a final dilution of 1:200.
Analysis of test performance and statistical analysis
Test performance was calculated by comparing the EIAs with the CCNA, which is considered the gold standard for the detection of Clostridium difficile toxins. We, then, evaluated the sensitivities, specificities, and positive and negative predictive values of each EIA method on the basis of the CCNA results. The correlation was calculated as per the following equation: Correlation = (True positive + True negative)/Total number of samples x 100.
| ~ Results|| |
Of the 151 stools specimens included, 61 (40.7%), 38 (25.3%), and 52 (34.7%) tested positive with the Ag-EIA, TOX-A/BII EIA, and CCNA, respectively [Figure 1]. Thirty-seven (24.7%) specimens were positive by all the three assays. CCNA-positive results were also observed in all 37 (100%) Ag-EIA and TOX-A/BII EIA-positive, and 12 (50%) of 24 Ag-EIA positive and TOX-A/BII EIA-negative specimens [Table 1]. On the other hand, only 85 (95.5%) of 89 Ag-EIA and TOX-A/BII EIA-negative specimens were CCNA-negative, indicating that four specimens had false-negative results with both Ag-EIA and TOX-A/BII EIA assays.
|Figure 1: Results of using the three methods of detection of Clostridium diffi cile and its toxins|
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|Table 1: Comparison of the C. DIFF CHEK-60 and TOX-A/B ELA to cell line culuture neutraliztion assay|
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Of the 61 specimens testing positive for Ag-EIA, 37 (60.7%) tested positive with TOX-A/BII EIA, while 52 (85.2%) tested negative, giving a 24.5% false-negative result with the TOX-A/BII EIA assay. The Ag-EIA was very likely to identify potentially CCNA-positive specimens, with a 94% sensitivity and 96.6% predictive value. Overall, the Ag-EIA sensitivity, specificity, negative, and positive predictive value were 94%, 87%, 96.6%, and 80.3%, respectively, while those of TOX-A/BII EIA were 73.1%, 100%, 87.5%, and 100%, respectively [Table 1].
Analysis of the turnaround time of the assays indicates that all the 61 positive specimens by Ag-EIA required 2 days for diagnosis using the two-step algorithm, while in the three-step algorithm, 37 cases were reported in the same day, and the remaining 24 specimens required further testing by the CCNA.
| ~ Discussion|| |
The growing concern among physicians regarding enteric diseases associated with Clostridium difficile has placed a heavy demand on clinical microbiology laboratories to offer a rapid and reliable diagnostic test. A large number of commercial EIAs that either detect Clostridium difficile toxin or the enzyme GDH as the common antigen are available. In a recent review by Brazier, sensitivity and specificity of commercial kits used to detect the Clostridium difficile toxin ranged from 65% to 95% and 75% to 100%.  On the other hand, a number of previous studies have shown that testing for GDH is a useful screening test, ,, but are unable to differentiate between toxigenic and nontoxigenic strains, and cannot establish a diagnosis of CD-AD. The introducing of the real-time polymerase chain reaction (PCR) methods for various targets has been developed as a potential replacement for the less-sensitive EIAs and less-specific GDHs for Clostridium difficile detection.  Therefore, the most useful information can be obtained by screening all samples for the presence of GDH or the toxin detection performed either concurrently or subsequently on GDH-positive specimens, followed by PCR, to distinguish between toxigenic and nontoxigenic strains in those samples with discordant GDH and Clostridium difficile toxin results.
In our study, compared with CCNA, the sensitivity and specificity of the GDH-based test were 94% and 87%, respectively, whereas the sensitivity and specificity of the TOX-A/BII EIA-based test were 73.1% and 100%, respectively [Table 1]. These results are similar to those published by many previous studies. ,, Unfortunately, no single method is able to detect all samples with a true positive toxin test result. Our study showed that TOX-A/BII EIA and Ag-EIA would have missed 14 and three cases of CD-AD, respectively, if used alone. To overcomes this problem, a practical approach to a sensitive and efficient detection of toxigenic Clostridium difficile by using a combination of Ag-EIA and CCNA (i.e., the two-step algorithm) or by using a combination of Ag-EIA, TOX-A/BII EIA, and CCNA (i.e., three-step algorithm) has been developed [Figure 2].
|Figure 2: (a) Schematic diagram of the two-step algorithm. (b) Schematic diagram of the three-step algorithm|
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Two previous studies evaluated the three-step and the two-step approaches. , The three-step algorithm for the diagnosis of CD-AD involves testing the stool specimen for GDH and, if positive, to test with a toxin-detection EIA. It also involves testing the GDH-positive, but toxin-EIA-negative, specimens with a tissue CCNA.  On the other hand, the two-step algorithm involves testing the stool specimen for GDH and, if positive, to directly test with a CCNA without any toxin-EIA.  Based on the results of the previous two studies, the authors were able to considerably reduce the number of specimens that required toxin detection.
The reactive simplicity of the two-step algorithm eliminates the possibility of false-negative toxin-EIA results while yielding similar sensitivity, turnaround time, and cost-effectiveness to those assays discussed above. In this study, by applying the two-step algorithm, the workload was reduced for up to 59.3%, whereas on applying the three-step algorithm, the workload reduced for up to 25.3%. Furthermore, among the 61 GDH-positive cases, the two-step algorithm allowed all the 61 positive cases to be diagnosed in 2 days. The three-step algorithm allowed 37 cases to be diagnosed on the same day, but the remaining 24 specimens required further testing by the CCNA, requiring an additional 48 h.
In the samples included in this study, four specimens tested positive by CCNA, and showed false-negative results with both Ag-EIA and TOX-A/BII EIA assays. Therefore, in patients with a high index of clinical suspicion of CD-AD, CCNA needs to be performed even if the Ag-EIA (first step) assay is negative.
In conclusion, the three-step algorithm is a rapid, specific, less-sensitive, and more expensive process as compared with the two-step algorithm, although the latter requires more time to give the final diagnosis. Therefore, the two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but is also time-consuming.
| ~ Acknowledgements|| |
We are grateful to Dalene Lewis, our Blood Bank QA Coordinator, for her time to review and proof reading for the manuscript.
| ~ References|| |
|1.||Lyerly DM, Wilkins T. Clostridium difficil. In: Blaser M, Smith P, Ravdin J, Greenberg H, Gurrant R, editors. Infections of gasterointestinal tract. New York: Raven Press; 1995. p. 867-91. |
|2.||Stoddart B, Wilcox M. Clostridium difficile. Curr Opin Infect Dis 2002;15:513-8. |
|3.||Kelly C, LaMont J. Clostridium difficile infection. Annu Rev Med 1998;49:375-90. |
|4.||Oughton M, Miller M. Clinical and epidemiological aspects of Clostridium difficile. Clin Microb Newsl 2008;30:87-95. |
|5.||Kelly C, LaMont J. Clostridium difficile colitis. N Engl J Med 1994;330:257-62. |
|6.||Moncrief J, Zheng L, Neville M, Lyerly D. Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by PCR. J Clin Microbiol 2000;38:3072-5. |
|7.||Delmée M. Laboratory diagnosis of Clostridium difficile disease. Clin Microbiol Infect 2001;7:411-6. |
|8.||Bartlett J. Antibiotics-associated diarrhea. N Engl J Med 2002;346:334-40. |
|9.||Poutanen S, Simor A. Clostridium difficile-associated diarrhea in adults. Can Med Assoc 2004;71:51-8. |
|10.||Wilkins T, Lyerly D. Clostridium difficile testing: After 20 years, still challenging. J Clin Microbiol 2003;41:531-4. |
|11.||Lee, S, Turgeon D, Ko C, Fritsche T, Surawicz C. Clinical correlation of toxin and common antigen enzyme immunoassay testing in patients with Clostridium difficile disease. Am J Gasteroenterol 2003;98:1569-72. |
|12.||Snell H, Ramos M, Longo S, John M, Hussin Z. Performance of the TechLab C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficile panel immunoassay, and cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea. J Clin Microbiol 2004;42:4863-5. |
|13.||Turgeon D, Novicki T, Quick J, Carlson L, Miller P, Ulness B. Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay. J Clin Microbiol 2003;41:667-70. |
|14.||Ticehurst J, Aird D, Dam L, Borek A, Hargrove J, Carroll K. Effective detection of toxigenic Clostridium difficile by a two-step algorithm tests for antigen and cytotoxin. J Clin Microbiol 2006;44:1145-9. |
|15.||Chang T, Lauerman W, Bartlett J. Cytotoxicity assay in antibiotic-associated colitis. J Infect Dis 1979;140:765-70. |
|16.||Brazier J. The diagnosis of Clostridium difficile-associated disease. J Antimicrob 1998;41:29-40. |
|17.||Landry M, Topal J, Ferguson D, Giudetti D, Tang Y. Evaluation of Biosite Triage Clostridium difficile panel for rapid detection of Clostridium difficile in stool samples. J Clin Microbiol 2001;39:1855-8. |
|18.||Massey V, Gregson D, Chagla A, Story M, John M, Hussain Z. Clinical utility of components of Triage immunoassay, enzyme immunoassay of toxins A/B, and cytotoxin B tissue culture assay for the diagnosis of Clostridium difficile-diarrhea. Am J Clin Pathol 2003;119:45-50. |
|19.||Vanpoucke H, De baere T, Claeys G, Vaneechoutte M, Verschraegen G. Evaluation of six commercial assays for the rapid detection of Clostridium difficile toxin and/or antigen in stool specimens. Clin Microbiol Infect 2000;7:55-64. |
|20.||Swindells J, Brenwald N, Reading N, Oppenheim B. Evaluation of diagnostic tests for Clostridium difficile infection. J Clin Microbiol 2010;48:606-8. |
|21.||Snell H, Ramos M, Longo S, John M, Hussain Z. Performance of the TechLab Clostridium difficile Tox A/B II EIA kit, the Triage Clostridium difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea. J Clin Microbiol 2004;42:4863-5. |
[Figure 1], [Figure 2]