Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 3, 2011, pp. 293-296

Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 293-296

Original Article

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
Correspondence Address: M O Qutu, Departments Pathology and Laboratory Medicine, Medical Technologist, MBC: J-10, King Faisal Specialist Hospital and Research Centre, Jedda, Branch, P.O. Box 40047, Jeddah 21499, Saudi Arabi, mohammedqutub@hotmail.com

Date of Submission: 26-Jan-2011 
Date of Acceptance: 15-Feb-2011

Code Number: mb11070

PMID: 21860113

DOI: 10.4103/0255-0857.83916

Abstract

Keywords: Antibiotic-associated diarrhoea, Clostridium difficile, EIAs, glutamate dehydrogenase, TOX-A/B EIA

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. ^[1],[2] This organism is carried asymptomatically in about 20% of the hospitalized patients, but only 2% of healthy adults. ^[3] 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. ^[4] 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. ^[5] Most strains produce both toxins, but pathogenic strains of Clostridium difficile producing toxin-B only have also been reported. ^[6] 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. ^[7] 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. ^[8],[9] Current antigen enzyme immunoassays (Ag-EIA) accurately detect an essential and constitutively synthesized enzyme. ^[10] 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. ^{[11],[12],[13]} Recently, a two-step algorithm, including tests for antigen and cytotoxin, was developed ^[14] 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

Specimens

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.

Enzyme immunoassays

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. ^[15] 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.

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%. ^[16] On the other hand, a number of previous studies have shown that testing for GDH is a useful screening test, ^{[17],[18],[19]} 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. ^[20] 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. ^{[16],[18],[21]} 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].

Two previous studies evaluated the three-step and the two-step approaches. ^[14],[21] 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. ^[21] 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. ^[14] 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

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