Clostridium difficile PCR Cycle ThresholdPredicts Free Toxin

Fiona Senchyna,a Rajiv L. Gaur,a Saurabh Gombar,a Cynthia Y. Truong,a

Lee F. Schroeder,b Niaz Banaeia,c,d

Department of Pathology, Stanford University School of Medicine, Stanford, California, USAa; Department ofPathology, University of Michigan School of Medicine, Ann Arbor, Michigan, USAb; Division of InfectiousDiseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine,Stanford, California, USAc; Clinical Microbiology Laboratory, Stanford Health Care, Stanford, California, USAd

ABSTRACT There is no stand-alone Clostridium difficile diagnostic that can sensi-tively and rapidly detect fecal free toxins. We investigated the performance of the C.difficile PCR cycle threshold (CT) for predicting free toxin status. Consecutive stoolsamples (n � 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/EpitcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Completeimmunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cyto-toxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbentassay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as thereference method, the accuracy of CT was measured at different CT cutoffs. UsingRMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive sampleswith a sensitivity, specificity, positive predictive value, and negative predictive valueof 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respec-tively. Inclusion of CCNA in the reference method improved CT specificity to 78.0%(95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the averagecoefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the inputstool volume did not improve CT toxin specificity. The median CT values were notsignificantly different between stool samples with Bristol scores of 5, 6, and 7, be-tween pediatric and adult samples, or between presumptive 027 and non-027strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demandPCR may also be used to accurately predict toxin-negative stool samples, thus pro-viding additional results in PCR-positive stool samples to guide therapy.

KEYWORDS Clostridium difficile, PCR, cycle threshold, free toxin, EIA, free toxins

Clostridium difficile is a cause of antibiotic-associated diarrhea (1). Laboratory assaysemployed to evaluate patients with suspected C. difficile infection (CDI) include

enzyme immunoassay (EIA), which detects fecal free toxins (TcdA and TcdB), and PCR,which targets sequences encoding tcdA and/or tcdB (2). EIA glutamate dehydrogenase(GDH) is also used to detect C. difficile, but follow-up EIA toxin or PCR is needed toconfirm toxigenicity. The best laboratory algorithm for diagnosing C. difficile infection(CDI) is debated (3). Compared to EIA toxin, PCR has a higher sensitivity for detectionof toxigenic C. difficile in stool (4). However, studies suggest toxin positivity moreaccurately correlates with clinical outcomes compared with PCR (5–8). Polage andcolleagues showed that patients with EIA toxin-negative (toxin�)/PCR-positive (PCR�)results had no CDI-related complications compared with EIA toxin�/PCR� patients (7).These studies indicate that PCR is linked to overdiagnosis of patients colonized with C.difficile, triggering unnecessary antibiotic therapy. However, not all studies support thisview (9–12). Some experts recommend using highly sensitive PCR to avoid missingtoxin�/PCR� CDI cases because EIA toxin is less sensitive compared with cell cytotox-

Received 6 April 2017 Returned formodification 1 May 2017 Accepted 9 June2017

Accepted manuscript posted online 14June 2017

Citation Senchyna F, Gaur RL, Gombar S,Truong CY, Schroeder LF, Banaei N. 2017.Clostridium difficile PCR cycle threshold predictsfree toxin. J Clin Microbiol 55:2651–2660.https://doi.org/10.1128/JCM.00563-17.

Editor Yi-Wei Tang, Memorial Sloan KetteringCancer Center

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Niaz Banaei,nbanaei@stanford.edu.

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icity neutralization assay (CCNA), which is the gold standard for fecal free toxin buttakes several days to perform (3). To address these diagnostic challenges, Europeanguidelines recommend a multistep testing algorithm starting with PCR or EIA GDH andfollowed by EIA toxin to rapidly identify toxin-positive and toxin�/PCR� patients tofacilitate appropriate clinical decision-making (4). Given the requirement for multisteptesting, having a single stand-alone assay that can rapidly and sensitively detecttoxigenic C. difficile and simultaneously predict free toxins would be valuable forguiding therapy and infection prevention practices.

Several studies have shown a correlation between C. difficile fecal free toxins andbacterial/genomic burden or PCR cycle threshold (CT) in diarrheal stools (11, 13–15).Leslie and colleagues showed that at a cutoff of �5.1 log10 DNA copies/ml, they couldcorrectly classify �95% of toxin-positive and 70% of toxin-negative stool samples (15).However, performance characteristics of PCR CT cutoffs for discriminating toxin-positiveand toxin-negative stool samples have not been reported. The aim of this study was tomeasure the accuracy of C. difficile PCR CT toxin for predicting toxin-positive stoolsamples using EIA toxin and other toxin assays as the reference methods.

RESULTSStudy samples. A total of 312 GeneXpert tcdB PCR-positive unformed stools from

282 unique patients were included in this study. Stool volume was insufficient toperform the enzyme-linked immunosorbent assay (ELISA) on 18 stool samples. Twenty-one patients provided two samples, 3 patients provided three samples, and 1 patientprovided four samples. Of the patients, 48.9% (138) were female and 77.7% (219) wereaged 18 years or older. Overall, 55.8% (174/312) of samples originated from inpatients,17.2% (30/174) of which were from intensive care unit (ICU) patients.

Accuracy of CT toxin. The median GeneXpert tcdB PCR CT value was significantlylower in rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA) toxin-positive samples (23.3 [interquartile range (IQR), 21.6 to 24.3]) compared with toxin-negative samples (29.2 [IQR, 24.5 to 32.7]; P � 0.001) (Fig. 1A). Using RMEIA toxin as thereference method for free toxins and assigning equal weight to CT toxin sensitivity andspecificity, the CT cutoff 26.35 yielded a sensitivity, specificity, positive predictive value(PPV), and negative predictive value (NPV) of 96.0% (95% CI, 90.2% to 98.9%), 65.9%(95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62.0%), and 97.1% (95% CI, 92.8% to98.9%), respectively (Fig. 1A and Table 1). To determine whether the specificity couldbe further improved by standardizing the input stool volume, PCR was repeated on thesame stool samples using a scoop to transfer a standardized volume of stool. Withstandardized stool volume, the CT cutoff 26.35 yielded a sensitivity, specificity, PPV, andNPV of 93.1% (95% CI, 86.2% to 97.2%), 60.7% (95% CI, 53.7% to 67.3%), 53.1% (95% CI,48.7% to 57.4%), and 94.8% (95% CI, 89.9% to 97.4%), respectively (Fig. 1B). Thesensitivity and specificity of CT toxin were not significantly different using nonstan-dardized (swab) and standardized (scoop) stool volume (P � 0.5 and 0.6, respectively).

Given that RMEIA toxin can be falsely negative (4), we further tested RMEIA toxin-negative stool samples using the cell cytotoxicity neutralization assay (CCNA), whichdetects TcdB, and the tgcBIOMICS ELISA, which detects TcdA and TcdB. Inclusion ofCCNA in the reference method compared with RMEIA alone improved CT toxin speci-ficity (at a CT cutoff of 26.35) from 65.9% to 78.0% (95% CI, 70.7% to 84.2%) anddecreased sensitivity from 96.0% to 87.6% (95% CI, 81.3 to 92.4) (Fig. 2B and Table 1).CT toxin was positive in 71.2% (37/52) of RMEIA toxin-negative/CCNA toxin-positivesamples. Inclusion of tgcBIOMICS ELISA in the reference method compared with RMEIAalone improved CT specificity (at a CT cutoff of 26.35) to 70.2% (95% CI, 62.7% to 76.9%)and decreased sensitivity to 94.3% (95% CI, 88.6% to 97.7%) (Fig. 2C and Table 1). CT

toxin was positive in 90.9% (20/22) of RMEIA toxin-negative/ELISA toxin-positive sam-ples. The performance characteristics of CT toxin using various reference methods withequal and unequal (i.e., sensitivity fixed at �99%) weights assigned to sensitivity andspecificity are shown in Tables 1 and 2, respectively.

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Exploratory investigation of false-positive CT toxin results. Although our studywas not powered for subanalyses, we performed several exploratory analyses to lookfor potential explanations for the false-positive CT toxin results. With the referencemethod defined as toxin positivity with RMEIA, CCNA, or ELISA, there were 29 stoolsamples from 29 patients who were falsely categorized as toxin positive with CT toxin.First, we determined whether false-positive CT toxin results were due to longer time tostool processing (i.e., testing with RMEIA and freezing samples for CCNA and ELISA). Themedian time to stool processing was numerically longer in the 29 false-positive CT toxinsamples versus the 138 correctly CT toxin-positive samples, but the difference was notstatistically significant (32.9 h [IQR, 17.6 to 56.0] versus 23.3 h [IQR, 17.6 to 48.5]; P �

0.4]). Two out of 29 samples with false-positive results were processed after 72 h of

TABLE 1 Performance of C. difficile tcdB PCR CT toxin using different reference methodsa

Reference method CT cutoffb

Sensitivity (% [n/N])(95% CI)

Specificity (% [n/N])(95% CI) PPV (% [n/N]) (95% CI) NPV (% [n/N]) (95% CI)

RMEIA 26.35 96.0 (97/101) (90.2–98.9) 65.9 (139/211) (59–72.2) 57.4 (97/169) (52.7–62.0) 97.1 (139/143) (92.8–98.9)RMEIA or CCNA 26.35 87.6 (134/153) (81.3–92.4) 78.0 (124/159) (70.7–84.2) 79.3 (134/169) (73.9–83.8) 86.7 (124/143) (81–90.9)RMEIA or ELISA 26.35 94.3 (116/123) (88.6–97.7) 70.2 (120/171) (62.7–76.9) 69.5 (116/167) (64.3–74.2) 94.5 (120/127) (89.2–97.3)RMEIA, CCNA, or ELISA 26.85 89.4 (143/160) (83.5–93.7) 76.3 (103/135) (68.2–83.2) 81.7 (143/175) (76.7–85.9) 85.8 (103/120) (79.3–90.6)aStool volume was insufficient in 18 samples to perform ELISA.bCT cutoff was based on equal weight assigned to sensitivity and specificity; 26.85 is the correct cutoff for RMEIA, CCNA, or ELISA.

FIG 1 C. difficile tcdB PCR cycle threshold in PCR-positive toxin-positive and toxin-negative stool samples.GeneXpert tcdB PCR was performed using nonstandardized (A) and standardized (B) stool volume. Toxinresult was determined using the C. Diff Quik Chek Complete RMEIA. The horizontal lines representmedian cycle thresholds. Cycle threshold cutoffs— derived based on equal weight assigned to sensitivityand specificity—are marked with dashed lines.

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collection. Second, we determined whether anti-C. difficile antibiotic exposure (i.e.,resulting in dead organisms not producing toxin but detected by PCR) or laxativetherapy (i.e., causing dilution of toxin to below level of detection) could explain thefalse-positive CT toxin results. After matching patients for age, sex, and inpatient status,antibiotic exposure (treatment for C. difficile or other bacteria) and laxative therapywere not significantly different in patients with true-positive and those with false-positive CT toxin results (see Table S1 in the supplemental material). Third, we deter-mined whether stool quality (i.e., Bristol score) was different in stool samples withfalse-positive CT toxin results. The proportion of Bristol 5, 6, and 7 stools was notsignificantly different in samples with true-positive and those with false-positive CT

toxin results (Bristol 5, 6.5% [9/138] versus 10.3% [3/29]; Bristol 6, 57.2% [79/138] versus65.5% [19/29]; Bristol 7, 36.2% [50/138] versus 24.1% [7/29]; P � 0.37 for all compari-

FIG 2 C. difficile tcdB PCR cycle threshold in PCR-positive toxin-positive and toxin-negative stool samples using various toxin referencemethods. Free toxin was detected using RMEIA alone (A), RMEIA or CCNA (B), RMEIA or ELISA (C), and RMEIA, CCNA, or ELISA (D). Reddots show samples that were toxin negative by RMEIA but positive by CCNA and/or ELISA. The horizontal lines represent median cyclethresholds. Cycle threshold cutoffs— derived based on equal weight assigned to sensitivity and specificity—are marked with dashedlines.

TABLE 2 Performance of C. difficile tcdB PCR CT toxin using different reference methods with sensitivity fixed at �99%a

Reference method CT cutoffSensitivity (% [n/N])(95% CI)

Specificity (% [n/N])(95% CI) PPV (% [n/N]) (95% CI) NPV (% [n/N]) (95% CI)

RMEIA 27.55 99.0 (100/101) (94.6–100) 58.8 (124/211) (51.8–65.5) 53.5 (100/187) (49.4–57.5) 99.2 (124/125) (94.6–99.9)RMEIA or CCNA 30.85 99.3 (152/153) (96.4–100) 49.1 (78/159) (41.0–57.1) 65.2 (152/233) (61.7–68.6) 98.7 (78/79) (91.7–99.8)RMEIA or ELISA 29.15 99.2 (122/123) (95.5–100) 53.8 (92/171) (46.0–61.4) 60.7 (122/201) (56.8–64.5) 98.9 (92/93) (92.9–99.8)RMEIA, CCNA or ELISA 30.85 99.4 (159/160) (96.6–100) 50.4 (68/135) (41.6–59.1) 70.3 (159/226) (66.7–73.8) 98.5 (68/69) (90.5–99.8)aStool volume was insufficient in 18 samples to perform ELISA.

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sons). Lastly, we determined whether presumptive 027 and non-027 strains weredifferentially represented in samples with false-positive CT toxin results. The proportionof those with presumptive 027 infection was smaller in the 29 false-positive CT toxinsamples compared with the 138 correctly CT toxin-positive samples, but the differencewas not statistically significant (3.5% [1/29] versus 17.4% [24/138]; P � 0.08).

Intercartridge CT reproducibility. The average coefficient of variation of Gene-Xpert tcdB PCR CT values for 20 randomly selected positive stool samples tested withfour different cartridge lots was 2.8% (95% CI 1.19% to 3.16%). At a CT cutoff of 26.35,one (1.3%) out of 80 PCR runs had a discordant CT toxin result. The CT values andstandard deviation for quadruplicate runs are shown in Table 3.

Impact of stool quality on CT. To determine whether the CT cutoff may beimpacted by stool quality, we compared the median CT values in stool samples withBristol scores 5, 6, and 7. As shown in Fig. 3, the median CT values were not significantlydifferent in RMEIA toxin-positive (Bristol 7 versus 5, 23.7 [IQR, 21.7 to 25.2] versus 23.6

TABLE 3 Intercartridge CT reproducibility with GeneXpert tcdB PCR using four cartridgelotsa

Sample no.

CT value for:

SDLot 1 Lot 2 Lot 3 Lot 4

1 18.4 18.5 18.5 19.1 0.32 19.5 22.1 19.6 19.8 1.23 20.4 20.4 20.4 20.6 0.14 20.8 21.5 22.2 21.4 0.65 21.4 21.2 20.3 20.9 0.56 22.2 21.1 21.4 21.4 0.57 22.4 22 21.7 21.7 0.38 22.6 23.3 22.7 22.6 0.39 23.3 23.4 23.1 23.4 0.110 23.6 24.1 23.6 24 0.311 24.2 23.3 24.3 23.6 0.512 24.3 24.3 24.1 24.2 0.113 26.3 24.5 24.2 29.3 2.314 30.5 30.3 30.5 30.4 0.115 30.8 31.3 30.9 30.9 0.216 30.9 29.8 29.6 29.9 0.617 31.6 32.2 32.5 31.8 0.418 31.9 31.9 31.5 32.3 0.319 35.4 34.2 34.2 36.5 1.120 38.8 36.7 36.4 38.9 1.3aRandomly selected PCR-positive stool samples were tested. Cartridge lot numbers included 1000037261,1000037262, 1000037264, and 1000037265.

FIG 3 C. difficile tcdB PCR cycle threshold in PCR-positive stool samples with different Bristol scores. C. DiffQuik Chek Complete RMEIA toxin-positive stool samples with Bristol scores 5 (n � 8), 6 (n � 58), and 7(n � 35), and RMEIA toxin-negative stool samples with Bristol scores 5 (n � 13), 6 (n � 124), and 7 (n �74) were included. The horizontal lines represent the median cycle thresholds. Median CT values forBristol 5 and 6 were compared to that for Bristol 7.

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[IQR, 22.1 to 24.1]; P � 0.7; Bristol 7 versus 6, 23.7 [IQR, 21.7 to 25.2] versus 22.6 [IQR,21.3 to 24]; P � 0.19) and RMEIA toxin-negative (Bristol 7 versus 5, 29.2 [IQR, 23.8 to32.8] versus 30.1 [IQR, 24.9 to 32.5]; P � 0.71; Bristol 7 versus 6, 29.2 [IQR, 23.8 to 32.8]versus 29.1 [IQR, 24.5 to 32.7]; P � 0.8) stool samples.

Impact of patient’s age on CT. To determine whether the CT cutoff may beimpacted by patient’s age, we compared the median CT values in adult and pediatricpatients. As show in Fig. 4, the median CT values were not significantly differentbetween adult and pediatric patients with RMEIA toxin-positive (23.1 [IQR, 21.6 to 24.3]versus 23.7 [IQR, 21.5 to 24.8]; P � 0.74) and RMEIA toxin-negative (29.2 [IQR, 24.3 to33.0] versus 29 [IQR, 24.8 to 31.8]; P � 0.4) stool samples.

Impact of immune status on CT. To determine whether the CT cutoff may bedifferent in immunocompromised patients, we compared the median CT values in adultpatients staying in bone marrow transplant and oncology wards with adult patientsstaying in nonimmunocompromised wards. The median CT value was not significantlydifferent between immunocompromised and nonimmunocompromised patients withRMEIA toxin-positive (22.8 [IQR, 21.3 to 24.2] versus 22.9 [IQR, 21.7 to 24.3]; P � 0.84)and RMEIA toxin-negative (30.6 [IQR, 24.9 to 35.5] versus 30.3 [IQR, 24.8 to 33.8]; P �

0.38) stool samples (see Fig. S1 in the supplemental material).Impact of 027 strain on CT. To determine whether the CT cutoff may be impacted

by the presumptive 027 strain, we compared the median CT values in stool samplespositive for presumptive 027 to those with non-027 infection. As shown in Fig. 5, themedian CT value was not significantly different between presumptive 027 and non-027strains for RMEIA toxin-positive (22.8 [IQR, 21.8 to 25.3] versus 23.4 [IQR, 21.6 to 24.2];

FIG 4 C. difficile tcdB PCR cycle threshold in PCR-positive pediatric and adult stool samples. C. Diff QuikChek Complete RMEIA toxin-positive stool samples from pediatric (n � 23) and adult (n � 78) patientsand from toxin-negative pediatric (n � 44) and adult (n � 167) patients were included. The horizontallines represent the median cycle thresholds.

FIG 5 C. difficile tcdB PCR cycle threshold in PCR-positive stool samples with 027 and non-027 strains. C.Diff Quik Chek Complete RMEIA toxin-positive stool samples with 027 (n � 18) and non-027 (n � 83) C.difficile and RMEIA toxin-negative stool samples with 027 (n � 21) and non-027 (n � 190) C. difficile wereincluded. The horizontal lines represent the median cycle thresholds.

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P � 0.47) and RMEIA toxin-negative (29.1 [IQR, 24.8 to 32.3] versus 29.2 [IQR, 24.4 to32.7]; P � 0.96) stool samples. Presumptive 027 C. difficile comprised 17.8% (18/101) ofRMEIA toxin-positive stool samples and 10.0% (21/211) of RMEIA toxin-negative stoolsamples (P � 0.07).

DISCUSSION

Given the lack of a stand-alone C. difficile diagnostic that can sensitively and rapidlydetect free toxins in stool, some experts recommend a multistep testing algorithm inwhich a nucleic acid amplification test (NAAT) or EIA GDH is performed in tandem withEIA toxin to rapidly identify toxin-positive and toxin�/NAAT� patients to facilitateappropriate clinical decision-making (3, 4). Proponents of toxin testing recommend thatonly toxin-positive patients be treated for CDI and toxin�/NAAT� patients be evaluatedclinically to determine if they have CDI or are colonized with C. difficile (3, 4). In thisstudy, we showed that by defining a CT cutoff for GeneXpert C. difficile/Epi tcdB PCR, asample-to-answer real-time PCR assay, we could sensitively predict 96.0% of toxin�/PCR� stool samples with an NPV of 97.1% and with a specificity of 78.0% using RMEIAtoxin and CCNA as the reference method. PPV was based on PCR-positive samples only.If we included PCR-negative samples, which made up 85.0% of total samples and areassumed to be RMEIA negative and CCNA negative (4), the NPV of CT toxin would be99.8% and 99.0% when using RMEIA toxin alone or RMEIA and CCNA, respectively, asthe reference method. Furthermore, CT toxin was positive in 71% (37/52) of CCNA�/NAAT� samples that were negative by RMEIA toxin, although the clinical significanceof this is unclear (7). Overall, with the approach undertaken in this study, PCR couldsensitively detect presence of toxigenic C. difficile (4), sensitively predict fecal free toxin,and predict toxin-negativity with a high NPV. Compared to current practices at ourinstitution and other U.S. hospitals where nearly all patients with positive C. difficile PCRresults are treated for CDI (16, 17), reporting CT toxin result in addition to PCR result hasthe potential to reduce anti-C. difficile therapy by 45.8% based on results of this study(143 of 312 PCR-positive samples were CT toxin negative), if only toxin-positive patientsare treated. This approach has the potential to have far-reaching impact as stand-aloneC. difficile NAAT has been widely adopted for CDI diagnosis in the United States (18).Further studies are needed to implement CT toxin reporting under routine clinicalpractice and measure its impact on provider behavior, patient outcomes, and antibioticstewardship.

Although Leslie and colleagues showed that at a cutoff of �5.1 log10 DNA copies/ml, they could correctly classify �95% of toxin-positive and 70% of toxin-negative stoolsamples (15), this is the first study to comprehensively investigate the analyticalperformance of CT cutoffs for prediction of free fecal toxin status and to investigate thepotential impact of preanalytical and analytical factors and strain type on its accuracy.We showed that stool quality (i.e., Bristol score), patient’s age (i.e., pediatric versusadult), immune status (i.e., immunocompromised versus nonimmunocompromised),and strain type (i.e., presumptive 027 versus non-027) did not significantly change themedian CT values, which suggests that a single CT cutoff could be applied acrossdifferent Bristol grades (of unformed stool), different age groups, patients with differentimmune status, and different strains of C. difficile. We also showed that time to stoolprocessing, although greater for false-positive samples, did not vary significantly be-tween stool samples with true-positive and those with false-positive CT toxin results.Similarly, anti-C. difficile therapy was not more common in the latter group. However,there was a trend toward higher non-027 strain representation in stool samples withfalse-positive CT toxin results. This finding suggests that some of the negative referencemethod results may be due to the presence of low-toxin-producing C. difficile straintypes. Other preanalytical factors that could potentially play a role but which were notinvestigated in this study include the patient’s temperature, dietary intake, anti-toxinantibodies, and inflammation. Studies have shown that TcdA and TcdB are unstable atbody temperature and are degraded by digestive enzymes (19, 20). The type of foodconsumed may influence the types of digestive enzymes present in stool. Importantly,

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we showed that transfer swabs, which are recommended by the manufacturer forsimple transfer of stool to sample reagent, yield CT toxin sensitivity and specificity thatare not significantly different from those obtained using a scoop to transfer a stan-dardized stool volume. This finding is reassuring and is consistent with a prior studywhich estimated the transfer swabs to hold approximately 100 �l of unformed stool(15). Lastly, we showed that intercartridge lot CT variability is relatively low. This findingindicates the reproducibility of GeneXpert C. difficile/Epi tcdB PCR CT is sufficiently highfor using CT at the defined CT cutoff to determine free toxin status in patients withpositive PCR results.

The application of PCR for detection of toxigenic C. difficile and prediction of freetoxin result has certain advantages and disadvantages over multistep testing algo-rithms employing EIA and CCNA (4). First, DNA is likely more stable than TcdA and TcdB,therefore PCR is presumably less affected by preanalytical factors such as temperature,pH, and digestive enzymes that may degrade toxins (19, 20). Second, PCR can be usedas a stand-alone test, obviating need for multistep testing, which saves time andresources. However, PCR may be potentially more expensive if EIA GDH is used toscreen samples. Third, PCR CT toxin bypasses potential for false-negative toxin resultsdue to interribotype toxin divergence (21). Fourth, if a CT cutoff is selected to maximizeprediction of toxin-positive samples using EIA as the reference method, PCR would missa fraction of samples that are EIA negative but CCNA positive. However, a three-stepalgorithm that includes CCNA is neither practical nor actionable. Lastly, the majordisadvantage of using the CT toxin approach is that 22% to 34% of free toxin-negativestrains are misclassified as toxin positive, which may result in some overdiagnosisrelative to a two-step approach incorporating a direct free fecal toxin test.

The findings from this study are promising and are consistent with prior investiga-tions (11, 13–15). However, this study has several limitations. First, further studies areneeded to validate the CT cutoff and to confirm the analytical accuracy of CT toxinreported here. We determined the CT cutoff according to the Youden maximum indexvalue, which assigns equal weight to sensitivity and specificity (22), and also by fixingthe sensitivity at 99%. Alternatively, CT cutoff may be determined by considering thecosts associated with false-positive and false-negative results, as well as the prevalenceof CDI (23). Second, we did not evaluate the clinical performance of the CT toxin cutoff.Some studies have demonstrated that lower CT is associated with CDI severity (24) andpoor outcome (24, 25), although these associations were not observed in another study(26). While we could consider a clinical gold standard, that too has drawbacks, in thatclinical decision making is driven so strongly by the laboratory PCR results, which eachpatient suspected of having CDI receives. Further studies are needed to evaluate theclinical safety of CT toxin results and determine the clinical significance of false-positiveCT toxin results (i.e., whether false-positive CT toxin patients have CDI or not). Third,although we showed no statistically significant difference in the median CT valuesbetween different Bristol stool scores, age groups, immune statuses, presumptive 027and non-027 strain type, and preanalytical factors (i.e., time to stool processing andantibiotic treatment), our study was not powered to evaluate each of these factors andthus lack of statistical significance does not exclude the possibility that there may be aneffect on CT. Furthermore, the impact of strain type beyond that of 027 has to beinvestigated. Fourth, we investigated the CT toxin performance using GeneXpert C.difficile/Epi tcdB PCR; however, other real-time PCR assays are likely to be equallyaccurate in predicting fecal free toxin. Further studies are needed to define assay-specific CT cutoffs and investigate the performance of CT toxin with other PCR assays.Fifth, although sample selection bias could have influenced our results, we includedonly PCR-positive stool samples because GeneXpert C. difficile tcdB PCR has been shownto have a sensitivity of 96% to 100% in CCNA-positive stool samples (4). Sixth, we didnot test RMEIA toxin-positive samples with CCNA because RMEIA has been shown tohave a specificity of 99% to 100% in CCNA-negative stool samples (4). Therefore, theinfluence of sample selection bias was minimized. Lastly, in addition to RMEIA toxin, wealso included CCNA and ELISA toxin in our reference method to maximize free toxin

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detection. It is possible that we still missed toxin-positive stool samples even with thecombination of these methods. This may explain some of the false-positive CT toxinresults. Although not commercially available, inclusion of a recently described ultra-sensitive toxin test in the reference standard might have detected more toxin-positivesamples (27). Further studies are needed to test this hypothesis.

In summary, C. difficile tcdB PCR CT may be used to predict free toxin results withhigh sensitivity and NPV, providing additional results to guide therapy. Further studiesare needed to implement CT toxin reporting and measure its impact on patient careand antibiotic stewardship.

MATERIALS AND METHODSEthics. This study was approved by the Stanford University Internal Review Board. A waiver of the

informed-consent requirement was obtained for the use of discarded stool samples.Study design. Between March and November 2016, consecutive unformed stool specimens (n �

312) collected from adult and pediatric patients and sent to the Stanford Health Care clinicalmicrobiology laboratory for C. difficile testing with the GeneXpert C. difficile tcdB PCR assay (Cepheid,Sunnyvale, CA) were included in this study if the PCR result was positive and if there was sufficientstool quantity for further testing. PCR-positive stool samples were prospectively tested for fecal freetoxins with the rapid membrane EIA (RMEIA). EIA-negative samples were further tested for free TcdBusing the cell cytotoxicity neutralization assay (CCNA) and the enzyme linked immunosorbent assay(ELISA).

Clinical data. An electronic report generated from the laboratory information system was used toobtain patient age, sex, and patient location. Chart review was performed in a subset of patients withdiscordant CT toxin results to obtain antibiotic and laxative exposure in the 60 days prior to specimencollection. The stool softener docusate was not considered a laxative.

PCR. Fresh stool samples were tested with the GeneXpert C. difficile/Epi tcdB PCR assay per thepackage instructions. A swab was used to transfer a nonstandardized volume of stool to the samplereagent. For the purpose of this study, fresh stools were also tested with a standardized volume of stoolusing a disposable scoop (Health Natura, Tuscaloosa, AL) to transfer 110 �l of stool to the samplereagent. The qualitative result, CT for tcdB PCR, and presumptive 027/NAP1/BI or non-027 strain typeresult were recorded from the assay software. The intercartridge lot CT reproducibility was measured bytesting 20 randomly selected PCR-positive stool samples with four different cartridge lot numbers.Cartridge lots tested included 1000037261, 1000037262, 1000037264, and 1000037265.

RMEIA. C. Diff Quik Chek Complete EIA (TechLab, Blacksburg, VA) was performed on fresh stoolspecimens per the package insert. Samples were refrigerated at 4°C until processing was performed. Allbut 22 samples were in compliance with the package insert and tested within 72 h of collection. Leftoveraliquots of stool were stored at �80°C for testing with CCNA and ELISA as described below.

CCNA. Stool samples frozen for 3 to 5 months were tested for TcdB using the C. Difficile Tox-B test(TechLab). A frozen 30 mg stool aliquot was thawed, transferred to 170 �l of diluent, and tested per thepackage insert. MRC-5 human lung tissue embryonic cells (Quidel, Santa Clara, CA) were used as indicatorcells. Cells were incubated in a CO2 incubator at 37°C for 48 h. Cytotoxic effect was considered positiveif at least 50% of cells in a well were rounded in 48 h.

ELISA. A frozen 30 mg stool aliquot was tested for TcdA and TcdB with the tgcBIOMICS ELISA kit(tgcBIOMICS, Bingen, Germany) according to the manufacturer’s instruction. An assay cutoff of 0.91ng/ml was determined by testing 20 PCR-negative stool samples (data not shown).

Statistical analysis. The Mann Whitney U test was used to compare median tcdB CT values. Fisher’sexact test was used to analyze differences between proportions. The receiver operating characteristic(ROC) curve was used to measure CT performance for predicting fecal free toxins. The CT cutoff wasdetermined using the Youden maximum index value, which assigns equal weight to sensitivity andspecificity (22), and also by fixing sensitivity to �99%. Average coefficient of variation was used tomeasure intercartridge lot CT variability. Statistical analysis was done with GraphPad Prism 5.0 software(San Diego, CA).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/JCM.00563-17.

SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.

ACKNOWLEDGMENTSWe thank Cepheid for donating a portion of cartridges used for intercartridge CT

reproducibility.N.B. is a provisional patent holder on the CT toxin algorithm.

PCR Cycle Threshold Predicts Free Toxin Journal of Clinical Microbiology

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