Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C.

JCM Accepts, published online ahead of print on 18 January 2012 J. Clin. Microbiol. doi:10.1128/jcm.05620-11 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 3 Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C. difficile Toxin Enzyme Immunoassays with C. difficile Nucleic Acid Amplification Testing Increase Diagnostic Yield in a Tertiary Pediatric Population 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Kaede V. Ota* 1,2 Karin L. McGowan 1,2 1 The Children s Hospital of Philadelphia, Philadelphia, USA 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA Running Title: C. difficile diagnosis in children *Corresponding Author: Dr. Kaede V. Ota, MD, MSc The Children s Hospital of Philadelphia 34 th Street and Civic Center Boulevard Main Building, Room 5112A Philadelphia, Pennsylvania, USA 19104-4399 Tel: 215-983-6792 Fax: 215-590-2171 E-mail: otak@email.chop.edu 1

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Abstract We evaluated the performance of the rapid C. DIFF QUIK CHEK COMPLETE s glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches in a tertiary pediatric population: algorithm #1 - initial testing with GDH/CDT followed by Loop- Mediated Isothermal Amplification (LAMP) and algorithm #2 - GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDTnegative results. True positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT or the Premier C. difficile Toxin A & B enzyme immunoassay (P- EIA)). 141specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value and negative predictive value were 56%, 100%, 100%, 90% for P-EIA and 81%, 100%, 100% and 96% for both algorithm #1 and algorithm #2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P- EIA. The algorithms allowed immediate reporting of half of all TPs but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH+/CDT- specimens. 2

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 Introduction Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated colitis in children and is associated with significant morbidity in vulnerable pediatric populations (4, 11). The optimal testing method for laboratory detection of CDI remains controversial in both adults and in children (18). Suboptimal sensitivity and specificity of many commercial enzyme immunoassays has limited their utility to the extent that the 2010 IDSA-SHEA Clinical Practice Guidelines for Clostridium difficile Infection in Adults discouraged their use (2, 3). Cell culture cytotoxicity neutralization assay (CCNA), the traditional gold standard, performs with superior sensitivity but is labor intensive and requires 48 hours to finalize. Nucleic acid amplification tests (NAATs) including BD GeneOhm Cdiff (BD Diagnostics, San Diego, CA), Prodesse ProGastro Cd (Gen-Probe Inc, San Diego, CA), Xpert C.difficile (Cepheid, Sunnyvale, CA) and illumigene C. difficile (Meridian Biosciences, Cincinnati, OH) appear to perform with sensitivity equaling or exceeding that of CCNA. In the adult literature, multi-step diagnostic algorithms have been evaluated in an effort to use NAATs more judiciously (1). The most recent iteration of this approach involves initial testing of stool specimens submitted for C. difficile toxin testing with the rapid C. DIFF QUIK CHEK COMPLETE assay (10, 12, 15). This product has two enzyme immunoassay components that detect glutamate dehydrogenase antigen (GDH) and toxin A/toxin B (CDT) in a lateral flow device. Typically, specimens that test positive or negative by both GDH and CDT are reported immediately as positive or negative for C. difficile toxin respectively. Because this assay detects the presence of both toxigenic and non-toxigenic C.difficile strains, GDH-positive/CDTnegative specimens require additional testing to adjudicate GDH+/CDT- specimens. Data in the literature suggest that algorithmic approaches perform with superior sensitivity and specificity 3

72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 compared to commercial enzyme immunoassays, with sensitivity and specificity ranging from 98-100% and 88-97% respectively (10, 12, 15). The negative predictive value (NPV) of GDH and the positive predictive value (PPV) of GDH-positive/CDT-positive results have been reported to be close to 100% (10, 12, 15), supporting the practice of immediately reporting these results without further testing. The high NPV of GDH also obviates the need to pursue the controversial practice of repeat testing of negative patients (1, 2). Finally, while some centers that process large volumes of C. difficile toxin tests may find performance of molecular testing routinely on all submitted specimens cost effective (compared to use of two tests), an algorithmic approach may be more cost effective in others (1, 18). To our knowledge, algorithmic approaches to CDI detection have not been evaluated in children. This study evaluated the performance of two algorithms for detection of CDI in a tertiary pediatric population: 1. Initial testing with GDH/CDT followed by LAMP to resolve discrepant GDH-positive/CDT-negative specimens (algorithm #1) and 2. Initial testing with GDH/CDT followed by CCNA to resolve discrepant specimens (algorithm #2). Materials and Methods Patients and specimens This was a prospective observational study. Consecutive, fresh, liquid or non-formed stool specimens collected from patients 1-18 years of age and submitted to the microbiology laboratory for C. difficile toxin testing at The Children s Hospital of Philadelphia between March 1 and March 30, 2011 were included. Specimens were excluded from analysis if the patient had been previously tested by the study protocol. All specimens were tested prospectively using the C. DIFF QUIK CHEK COMPLETE (GDH/CDT; Techlab, Blacksburg, VA); the Premier C. difficile Toxin A & B enzyme 4

95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 immunoassay (P-EIA; Meridian Biosciences, Cincinnati, OH), cell cytotoxicity neutralization assay (CCNA; Tox-B Test; Techlab, Blacksburg, VA) and Loop-Mediated Isothermal Amplification (LAMP) using the illumigene C. difficile assay (Meridian Biosciences, Cincinnati, OH). All tests were performed according to the instructions and within the timeframes recommended by the assay manufacturers. Specimens were stored at 2-8ºC while awaiting testing. External positive and negative controls were performed for each assay with each new lot and each time the assay was used. GDH/CDT results were interpreted as follows. The test device was examined for the appearance of blue dots in the middle of the reaction window, representing the internal positive control. The test result was interpreted as invalid if the internal control dots were not present. The device was then examined for the appearance of blue lines on the Ag and Tox sides of the reaction window. The appearance of a blue line on the Ag side was read as GDHpositive. The appearance of blue lines on both the Ag and Tox sides was interpreted as GDH-positive and CDT-positive. CCNA was performed using the C. difficile Tox-B Test kit. Briefly, following dilution, stool was centrifuged and the supernatant filtered using a 0.45 µm syringe filter. The stool filtrate was then inoculated on to a human foreskin fibroblast cell line (Diagnostic Hybrids, Athens, OH) with and without C.difficile anti-toxin. The cell line was incubated at 37ºC in 5-10% C0 2 for 48 hours. Light microscopy was used to read the cell line plates at 24 and 48 hours. Cytotoxic activity was considered positive if 50% of the cells in a well were rounded. Stool filtrates that produced cell cytopathic effect that was inhibited by C. difficile anti-toxin were considered positive for C. difficile toxin. Specimens were tested by CCNA within 24 hours of collection. 5

118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 LAMP is a nucleic acid amplification test that targets a 204 bp region of the tcda gene that resides within the pathogenicity locus of toxigenic C. difficile. It was performed according to manufacturer s instructions for the illumigene C. difficile assay. At the conclusion of the run, results were reported as Positive, Negative or Invalid. Patient charts were reviewed retrospectively to understand the clinical relevance of specimens that tested LAMP-positive/CCNA-negative and CCNA-positive/LAMP-negative. This component of the study received approval by the institutional review board at The Children s Hospital of Philadelphia. Statistical analysis A composite gold standard was initially used for analysis. A specimen was considered true positive for C. difficile toxin if it was positive by CCNA. If CCNA was negative, the specimen could also be considered true positive if the specimen tested positive by LAMP and by another test (GDH, CDT or P-EIA). For the purpose of analysis, specimens were categorized as true negative if they did not meet criteria as a true positive. Prevalence, sensitivity, specificity, PPV and NPV were calculated for GDH, CDT, P-EIA, CCNA, LAMP, and two algorithms GDH/CDT followed by LAMP to resolve discrepant GDH-positive/CDT-negative specimens (algorithm #1) and GDH/CDT followed by CCNA to resolve discrepant specimens (algorithm #2). The McNemar test was used to compare the sensitivity of each algorithm with P- EIA, our current testing method. The analysis was then repeated using CCNA positivity as the sole gold standard criterion. Results 141 stool specimens meeting inclusion criteria were collected and tested from 141 consecutive patients during the study period. Testing was complete for all 141specimens. The 6

141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 mean and median time interval from specimen collection to testing were 17.1 and 18. 2 hours for GDH/CDT; 23.0 and 19.6 hours for P-EIA, 16.7 and 17.7 hours for CCNA, 49.3 and 49.5 hours for LAMP. The sensitivity, specificity, PPV and NPV of GDH, CDT, P-EIA, CCNA, LAMP, algorithms #1 and #2 using the composite definition of true positive and the CCNA definition of true positive are summarized in Table 1. Using the composite definition of true positive, twenty-seven (27) specimens were true positive for C. difficile toxin, resulting in a prevalence of 19%. Of these, 26 met the definition through a positive CCNA result. The remaining specimen was positive by LAMP and GDH. GDH was positive in 42 specimens, performing with a sensitivity of 81% and specificity of 82%. PPV was 52% and NPV was 95%. The CDT component detected 13 true positives with a sensitivity of 48% and specificity of 100%. All specimens that were positive by CDT were also positive by GDH, CCNA and LAMP. P-EIA was positive in 15 specimens with a sensitivity of 56% and specificity of 100%. PPV and NPV were 100% and 90% respectively. CCNA detected 26 positives with sensitivity of 96% and specificity of 100%. LAMP detected 24 positives with a sensitivity of 89% and specificity of 98%. The sensitivities of both GDH-based algorithms were significantly higher than that of P-EIA by McNemar testing (p<0.001). Using CCNA as the sole gold standard criterion, sensitivity, specificity, PPV and NPV were all within three percentage points of those calculated using the composite gold standard definition. With respect to discordance, three specimens were CCNA-positive/LAMP-negative. All three were GDH and P-EIA-negative. There were also three LAMP-positive/CCNA-negative specimens. One was GDH-positive but the the remaining two specimens were negative by all other tests. CCNA and LAMP were repeated on all six discordant specimens and yielded identical test results. Chart review of the discordant cases revealed that all six cases had diarrhea 7

164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 that met the definition provided by the 2010 SHEA/IDSA guidelines (3 diarrheal stools or greater within 24 hours or less) (2). None of the six cases had any other documented positive C. difficile toxin test results. Review of the LAMP-positive/CCNA-negative cases suggested false positive LAMP results. None of the LAMP-positive/CCNA-negative cases had antibiotic or antineoplastic exposure. All three experienced symptom resolution without CDI therapy. In addition, one case had a positive Rotavirus antigen EIA test, and a second case had Blastocystis hominis in his stool. All three CCNA-positive/LAMP-negative cases had risk factors for CDI (exposure to antineoplastic agents in one, beta-lactam antibiotics in the other two, within 8 weeks of the positive CCNA result.) One case responded clinically to metronidazole therapy and therefore appeared to be a true CDI case. The other two tested positive for Norovirus by PCR, providing an alternative diagnosis, making CDI less likely but still possible. All three patients had negative stool cultures. Discussion In our pediatric cohort, CCNA and LAMP alone performed with the highest test sensitivity as standalone tests. The GDH-based algorithms performed with lower sensitivity (81%) compared to adult studies in the literature (98-100%), suggesting that this approach may fail to diagnose 19% of pediatric patients with CDI (10, 12, 15). The reason for the lower sensitivity is unclear. It has been suggested that GDH antigen-based assays may demonstrate superior performance in populations with high incidence of CDI mediated by hypervirulent C. difficile strains (16). Indeed, ribotyping of a small series of C. difficile isolates from our institution in 2007 indicated that the prevalence of the NAP1/027/III strain among P-EIApositive stool specimens was only 14% (17). We note, however, that the sensitivity of the GDHbased algorithms was still statistically superior to P-EIA. 8

187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 In this study, we observed a PPV of 100% for GDH-positive/CDT-positive specimens and an NPV of 95% for GDH-negative/CDT-negative specimens, strongly supporting the practice of reporting these specimens as positive or negative for C. difficile toxin immediately without further testing. 80% of all specimens tested in our cohort were immediately reportable following GDH/CDT testing (8% were GDH-positive/CDT-positive and 72% were GDH-negative/CDT-negative). The implications of the rapid GDH/CDT reporting are significant in our population. While syndromic infection control precaution assignment (13) is the current standard in our institution when the etiology of an inpatient with diarrhea has yet to be determined (ie. a child with diarrhea will be isolated with contact precautions while symptomatic regardless of C. difficile toxin status), our data indicate that about half of all CDI cases would be rapidly reported following GDH/CDT testing. With a 19% CDI prevalence, we anticipate that timelier initiation of CDI therapy and necessary environmental cleaning and disinfection procedures in even half of all CDI cases would be beneficial for the control of CDI in our patient population. Moreover, while rapid reporting of negative results may not affect resources allocated to the application of contact precautions on symptomatic patients, it may facilitate cohorting of inpatients and encourage clinicians to consider other etiologic causes of antibioticassociated diarrhea. In our cohort, the PPV of 52% for GDH alone confirmed the need to pursue additional testing of GDH-positive/CDT-negative specimens to resolve these specimens as positive or negative for C. difficile toxin. PPV of 100% and NPV of 96% were observed for the entire algorithm when CCNA and LAMP were used in this capacity, indicating that both algorithmic approaches performed well in our population. The meaning of positive molecular CDI assays has been questioned in some quarters on the grounds that they do not detect C. difficile toxin or 9

210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 even the presence of organisms with the capacity to produce toxin in vitro but merely demonstrate the presence of the genes that code for toxins A and B (tcda and tcdb) (18). In our study, the virtually identical performance of CCNA and LAMP-confirmed algorithms provided reassurance that the user-friendly and relatively rapid LAMP procedure was as appropriate as CCNA in a confirmatory role. LAMP performed with sensitivity and specificity comparable to other studies evaluating this assay (7, 9). With regard to discordance, LAMP did not detect three specimens that were CCNApositive. Chart review suggested that at least one of these cases was consistent with CDI, suggesting that the LAMP result may have been falsely negative. From the assay standpoint, the illumigene C. difficile assay uses an internal control to detect LAMP reaction failures related to inhibition reagent problems. Failure of the internal control reaction is indicated by an Invalid test result. All three CCNA-positive, LAMP-negative results yielded valid Negative results, indicating that the LAMP reactions were successful in each case. With respect to target design, while the illumigene C. difficile assay s tcda (as opposed to tcdb) target has raised questions about its ability to detect toxin A B+ strains resulting from tcda mutations, the target sequence appears to be conserved (5). Limited data demonstrating this assay s ability to detect these strains have been presented, supporting the manufacturers assertion that this assay has the capacity to detect toxin A strains (5). Mutation at the actual LAMP target site, however, needs to be entertained as a possible explanation for these cases (5). Finally, false negative LAMP results in specimens that were positive by toxigenic culture have been documented in the literature (7). While there are reports in the literature of inferior sensitivity of CCNA compared to LAMP (7, 9), the sensitivity of CCNA is highly dependent on a number of factors including the quality of the cell line used and the time interval between specimen collection and 10

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 inoculation of the cell culture plates (6). We feel that when carefully performed, CCNA may detect CDI with a sensitivity that compares with molecular techniques. While to our knowledge, CCNA-positive/LAMP-negative specimens have not been reported in the literature, this phenomenon has been described with other commercial molecular assays (6) and may be explained by the presence of low bacterial concentrations of hypervirulent strains of toxigenic C. difficile producing disproportionately high levels of cytotoxin. Unfortunately, we did not have the means to pursue molecular typing of C. difficile isolates to investigate this hypothesis further. There were three LAMP-positive/CCNA-negative specimens. Chart review suggested that these cases did not have CDI. None of the cases had clinical risk factors for CDI and all three cases experienced symptom resolution without CDI therapy. In addition, two cases appeared to have alternative diagnoses. Possible false positive LAMP results (using toxigenic culture as the gold standard) have been reported in the literature. Norén et al. described four such cases, with only one confirmed with an in-house PCR assay (9). End product detection in loopmediated isothermal amplification technology relies on pyrophosphate ion released from dntp during DNA polymerization reacting with magnesium ion to produce a precipitate. This is followed by detection of the resulting increase in turbidity of the reaction mixture (8). We speculate that false positive LAMP results may be related to non-specific DNA amplification generating magnesium pyrophosphate or substances in stool specimens causing increases in turbidity of the reaction mixture. This study s potential issue of controversy is the omission of toxigenic culture in our gold standard definition. The optimal gold standard method remains an issue of contention (18). While the SHEA-IDSA guidelines support the use of toxigenic culture for comparative studies (2), discussion in the literature continues to debate the merits of toxin detection versus the 11

256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 detection of C. difficile isolates with in-vitro capacity to produce toxin (18). In this study, we opted to use CCNA as opposed to toxigenic culture on the basis that detection of toxin (the sine qua non for CDI ) is preferable (18). Also, while there are data suggesting that correlation between toxigenic culture and molecular testing is superior to CCNA with the latter (15), evidence also suggests that specimens that are negative by GDH/CDT and CCNA but positive by PCR and toxigenic culture may represent colonization with toxigenic C.difficile as opposed to actual CDI (6). In summary, an algorithmic approach to CDI detection with the C. DIFF QUIK CHEK COMPLETE product for initial CDI testing of pediatric stool specimens allowed swift reporting of 80% of C. difficile toxin results while increasing diagnostic yield over P-EIA in a tertiary pediatric population. The illumigene C. difficile assay provided an operator-friendly and accurate method of resolving GDH-positive/CDT-negative results. The sensitivity of the C. DIFF QUIK CHEK COMPLETE, however, may not be sufficient to allow its use as a standalone rule-out test in a pediatric population. Acknowledgements We thank the Department of Pathology and Laboratory Medicine at The Children s Hospital of Philadelphia for their support of this project. We also thank Marilyn Leet, Janice Odebralski, Deborah Blecker-Shelly and the technologists in the microbiology laboratory for their assistance with specimen testing. 277 278 12

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Table 1. Performance of four C.difficile toxin assays in a tertiary pediatric population Gold standard TP 10 TN 11 FP 12 FN 13 SN 14 SP 15 PPV 16 NPV 17 GDH 1 Composite 8 22 94 20 5 81% 82% 52% 95% CCNA 9 21 94 21 5 81% 82% 50% 95% CDT 2 Composite 13 114 0 14 48% 100% 100% 89% CCNA 13 115 0 13 50% 100% 100% 90% EIA 3 Composite 15 114 0 12 56% 100% 100% 90% CCNA 15 115 0 11 58% 100% 100% 91% CCNA 4 Composite 26 114 0 1 96% 100% 100% 99% CCNA - - - - - - - - LAMP 5 Composite 24 111 2 3 89% 98% 92% 97% CCNA 23 111 3 3 88% 97% 89% 97% Algorithm #1 6 Composite 22 114 0 5 81% 100% 100% 96% CCNA 21 115 0 5 81% 100% 100% 96% Algorithm #2 7 Composite 22 114 0 5 81% 100% 100% 96% CCNA 21 115 0 5 81% 100% 100% 96%

1 GDH, C.DIFF QUIK CHEK COMPLETE glutamate dehydrogenase antigen enzyme immunoassay 2 CDT, C.DIFF QUIK CHEK COMPLETE C. difficile toxin A and toxin B immunoassay 3 EIA, Premier C. difficile Toxin A & B enzyme immunoassay 4 CCNA, Cell cytotoxicity neutralization assay 5 LAMP, Loop-mediated isothermal amplification 6 Algorithm #1: Initial testing with C.DIFF QUIK CHEK COMPLETE, followed by LAMP for GDH-positive/CDT-negative specimens 7 Algorithm #2: Initial testing with C.DIFF QUIK CHEK COMPLETE, followed by CCNA for GDH-positive/CDT-negative specimens 8 Composite gold standard: a specimen was considered True Positive if positive by CCNA. If the specimen was CCNA-negative, the specimen would also be True Positive if LAMP-positive and positive by another test (GDH, CDT or EIA). 9 CCNA gold standard: a specimen was considered True Positive if positive by CCNA. 10 TP, True positive 11 TN, True negative 12 FP, False positive 13 FN, False negative 14 SN, Sensitivity

15 SP, Specificity 16 PPV, Positive predictive value 17 NPV, Negative predictive value Downloaded from http://jcm.asm.org/ on January 23, 2019 by guest