Rapid and Reliable Diagnostic Algorithm for Detection of Clostridium ...

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2008, p. 328–330 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.01503-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 1

Rapid and Reliable Diagnostic Algorithm for Detection of Clostridium difficile䌤 Lukas Fenner,1 Andreas F. Widmer,2 Gisela Goy,1 Sonja Rudin,1 and Reno Frei1* Microbiology Laboratory1 and Division of Infectious Diseases and Hospital Epidemiology,2 University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland Received 26 July 2007/Returned for modification 20 September 2007/Accepted 2 November 2007

We evaluated a two-step algorithm for detection of Clostridium difficile in 1,468 stool specimens. First, specimens were screened by an immunoassay for C. difficile glutamate dehydrogenase antigen (C.DIFF CHEK60). Second, screen-positive specimens underwent toxin testing by a rapid toxin A/B assay (TOX A/B QUIK CHEK); toxin-negative specimens were subjected to stool culture. This algorithm allowed final results for 92% of specimens with a turnaround time of 4 h. Clostridium difficile-associated diarrhea (CDAD) is a frequent cause of nosocomial diarrhea (1, 4). Recently, new virulent C. difficile strains causing outbreaks have emerged and were associated with increased morbidity and mortality (5, 6). The diagnosis of CDAD is usually based on a clinical history of recent antibiotic usage and diarrhea in combination with laboratory tests. Therefore, rapid and accurate microbiological diagnosis is urgently needed. The current cornerstone of laboratory diagnosis is the detection of toxin A/B from fecal samples. Fecal toxin assay by cell cytotoxicity neutralization assay (CCNA) is still regarded as the gold standard. CCNA is more sensitive than toxin detection by immunoassays but has a turnaround time of up to 3 days, is labor-intensive, and requires facilities for cell culture (3, 10, 11). Toxin enzyme immunoassays are more rapid and easier to perform but are suboptimal if used as stand-alone assays due to the low sensitivity compared to CCNA or toxigenic C. difficile culture (3, 7, 10). Today, many laboratories rely on C. difficile toxin A/B testing alone despite the limits of this approach and have abandoned bacterial culture (2, 3). Detection of the C. difficile-specific enzyme glutamate dehydrogenase antigen (GDH) may in part replace culture techniques and therefore be an alternative to culture (9, 10, 12, 13). Until now, our laboratory routinely tested stool specimens for toxin A/B (CDIFF TOX A/B II; TechLab/Wampole, Blacksburg, VA) and performed stool culture including isolate toxin testing. To assess the performance of our toxin assay as a stand-alone test, we performed a retrospective study analyzing all C. difficile laboratory data from a 25-month period between 2004 and 2006. Among 2,940 specimens tested, the sensitivity and specificity were 50.7% and 99.4%, respectively, compared to toxigenic culture (data not shown). Therefore, we developed a test algorithm that should provide high sensitivity, rapid turnaround time, and ease of performance. We prospectively evaluated a two-step algorithm including a screening assay to detect C. difficile-

specific GDH followed by a rapid toxin A/B test during a 12-month period beginning in June 2006. All fecal specimens from adult patients suspected of having CDAD were kept at 4°C until processed or saved at ⫺70°C if the specimens could not be processed within 24 h. Stool cultures were plated on selective cycloserine-cefoxitin-fructose agar plates (CLO agar; bioMe´rieux, Marcy l’Etoile, France) without any delay and incubated in an anaerobic chamber for 48 h according to standard laboratory methods. C. difficile colonies were identified on the basis of their typical morphology on agar plates and by the Gram stain, characteristic odor, and unique pattern of fatty acid metabolic products by gasliquid chromatography. Fecal cytotoxin was detected using the new rapid immunoenzymatic membrane test TOX A/B QUIK CHEK (TechLab/Wampole) according to the manufacturer’s instructions. The C. difficile-specific GDH was detected using C.DIFF CHEK-60 (TechLab/Wampole), and the results were read by an Opsys MR spectrometer (Dynex, West Sussex, United Kingdom) at a wavelength of 450 nm. An optical density of 0.12 or smaller was interpreted as negative. Samples with an optical density between 0.12 and 0.25 were repeated in duplicate. If the sample was GDH screen positive but the stool culture was negative or if the sample was GDH screen negative but the culture was positive, PCR was used to resolve the discrepancies. In this case, DNA was extracted using QIAmp DNA stool minikit (QIAGEN, Hilden, Germany) and a PCR for the GDH gene (gluD) was performed as previously described (13). All C. difficile isolates were tested for the presence of the toxin A and B genes (tcdA and tcdB) by a multiplex PCR (S. Persson, M. Torpdahl, and K. E. P. Olsen, submitted for publication) and for toxin A/B by TOX A/B QUIK CHEK using a suspension of 5 to 10 culture colonies in dilution buffer. A true GDH-positive stool sample was defined as a sample with positive C. difficile culture and/or positive gluD PCR directly from stool. A true positive sample for toxin A/B was defined as a sample with a toxigenic C. difficile culture (toxigenicity determined by PCR). A total of 1,468 consecutive stool specimens from adults were tested during a 12-month period between June 2006 and June 2007. Overall, the prevalence of positive C. difficile culture was 10.4% (Table 1); among those, 82.5% were toxigenic as determined by PCR (Table 2). The sensitivity and the neg-

* Corresponding author. Mailing address: Microbiology Laboratory, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland. Phone: 41 61 265 42 44. Fax: 41 61 265 53 55. E-mail: rfrei @uhbs.ch. 䌤 Published ahead of print on 21 November 2007. 328

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TABLE 1. Performance of GDH test in stool specimens compared to C. difficile culture and/or PCR for gluDa GDH assay result Comparative test

Result

No. positive

No. negative

% Sensitivity (95% CI)

% Specificity (95% CI)

% PPV (95% CI)

% NPV (95% CI)

Culture

Positive Negative

142 45

10 1271

93.4 (88.2–96.8)

96.6 (95.5–97.5)

75.9 (69.2–81.9)

99.2 (98.6–99.6)

Culture and/or gluD PCR

Positive Negative

147 40

10 1271

93.6 (88.6–96.9)

96.9 (95.9–97.8)

78.6 (72.0–84.3)

99.2 (98.6–99.6)

a

NPV, negative predictive value; PPV, positive predictive value; 95% CI, 95% confidence interval.

ative predictive value of GDH assay were 93.4% and 99.2%, respectively, compared to culture (Table 1). A high percentage of stool specimens (87.3%; n ⫽ 1,281) were GDH negative. Of the 1,281 GDH screen-negative specimens, 10 were positive by culture and therefore would have been missed by the initial GDH screening; 5 of these 10 samples had a negative gluD (gene for GDH) PCR, possibly indicating very low bacterial load. No explanation was found for the residual five discrepant results. Subsequently, all GDH screen-positive specimens were retested by a second test, the rapid toxin A/B immunoassay. Of the 187/1,468 GDH screen-positive specimens, 69/187 (or 4.7% of total specimens tested; Fig. 1) were also positive for toxin A/B by the rapid toxin A/B test. The specificity and the positive predictive value of this toxin assay were calculated to be 97.1% and 96.5%, respectively (Table 2). Compared to toxigenic culture, the sensitivity was low (52.9%). The remaining 118/187 specimens (corresponding to 8.0% of total specimens) that were GDH screen positive but negative for toxin A/B were resolved within 48 h to 72 h by culture, including isolate toxin testing. Of these, 73/118 were culture positive and 45/118 culture negative. Among these culture-negative samples, 40/45 were also negative for the C. difficile-specific gluD PCR performed directly from stool and therefore interpreted as false positive in the GDH assay (Fig. 1), whereas 5/45 were gluD PCR positive, suggesting failure of culture. Isolate toxin testing showed in 49/73 a toxigenic culture and in 20/73 a nontoxigenic culture, while 4/73 strains were not available. Toxin A/B testing by TOX A/B QUIK CHEK from culture isolates proved to be reliable, with high sensitivity and specificity (Table 2). GDH screen-negative and GDH screen-positive stool samples with a positive toxin A/B test result required no further testing. Thus, a turnaround time of less than 4 h was obtained for 92% of specimens (Fig. 1). GDH testing as a first screening assay performed well

compared to culture and/or PCR and was in the range of previously reported sensitivity of 85 to 93% (8–10, 12, 13). In contrast to previous reports, GDH-positive specimens were retested by a rapid toxin A/B test instead of timeconsuming and labor-intensive CCNA (10). A toxin assay is necessary since the GDH assay cannot distinguish between toxigenic and nontoxigenic C. difficile strains. The sensitivity was low (52.9%) compared to toxigenic culture if the toxin assay was performed alone, similar to previous reports which used other toxin assays (3, 8, 10). For the remaining stool specimens that are GDH screen positive and negative for toxin A/B (8% of all specimens), a stool culture should be performed since these specimens may represent nontoxigenic strains, a false-positive GDH result, or toxigenic strains missed by the stool toxin testing. Toxigenicity of isolates can be accurately determined by the same rapid toxin assay as that used for stool specimens. C. difficile stool culture requires expertise and anaerobic culture facilities. However, culture of GDH screen-positive but toxin-negative stool specimens increases the yield of toxin-producing C. difficile missed by stool toxin testing. In addition, culture of GDH screen-positive stool specimens would allow further investigations such as antimicrobial susceptibility testing, ribotyping, and determination of other molecular properties. In conclusion, our proposed algorithm allows reliable exclusion of C. difficile without additional tests if the GDH screen is negative (87.3% of all tested specimens). GDH screen-positive samples should be further processed by the rapid toxin A/B test, allowing definite diagnosis in another 4.7% of total specimens within 4 hours. Therefore, our two-step algorithm enables the abandonment of routine culture in 92% of stool specimens. Additionally, it reduces the turnaround time significantly while providing accurate results.

TABLE 2. Performance of C.DIFF TOX A/B QUIK CHEK for stool specimens and isolates compared to toxigenic C. difficile culturea Toxigenic culture result Specimens (n)

Result

% Sensitivity (95% CI) % Specificity (95% CI) % PPV (95% CI) % NPV (95% CI) No. No. positive negative

GDH-positive stool specimens (172) Positive Negative Culture isolates (137)

Positive Negative

55 49

2 66

52.9 (42.8–62.8)

97.1 (89.8–99.6)

96.5 (87.9–99.6) 57.4 (47.8–66.6)

106 7

1 23

93.8 (87.7–97.5)

95.8 (78.9–99.9)

99.1 (ⱖ94.9)

76.7 (57.7–90.1)

a The toxigenicity of C. difficile isolates was determined by PCR for the presence of tcdA and tcdB genes of toxin A and B. Only specimens for which culture isolates were available were included in this analysis. NPV, negative predictive value; PPV, positive predictive value; 95% CI, 95% confidence interval.

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FIG. 1. Proposed algorithm for detection of C. difficile in fecal specimens submitted for C. difficile testing. Tests used are C.DIFF CHEK-60 for C. difficile specific antigen (glutamate dehydrogenase) and TOX A/B QUIK CHEK for toxin A/B (both TechLab/Wampole). The percentage values refer to the total numbers of stool specimens analyzed (n ⫽ 1,468). Results for nine specimens (0.6%) which did not fit into the described categories are not included.

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