Abstract
Background
The illumigene® (Meridian Bioscience, Inc., Cincinnati, OH) and GeneOhm® (BD Diagnostics, La Jolla, CA) Clostridium difficile assays target the tcdA gene and tcdB gene, respectively. We assessed the use of tcdA as the molecular target in the illumigene® C. difficile loop‐mediated amplification assay in detecting a wide variety of C. difficile strains including those with tcdA deletions.
Methods
We tested 38 C. difficile strains and 108 patient stool specimens using the illumigene® assay. The GeneOhm® real‐time polymerase chain reaction (PCR) assay served as the reference method. Discordant results were resolved by repeat testing, anaerobic culture, and a laboratory‐developed real‐time PCR targeting tcdA and tcdB.
Results
Both illumigene® and GeneOhm® assays detected all 37 C. difficile toxin B+ strains representing seven toxinotypes and including four toxin A−B+ isolates. No cross‐reactivity with 20 other Clostridium species or toxin‐negative C. difficile was observed in either assay. Among patient stool specimens, agreement was 94.4% (102/108). After discordant result resolution, agreement was 96.3% (104/108). Specimens with initially discordant results had target concentrations approaching the limit of detection for the two commercial assays. Discordance appeared unrelated to whether tcdA or tcdB was the amplification target.
Conclusion
The tcdA 5′ region used by the illumigene® assay is a practical target for toxigenic C. difficile detection.
Keywords: tcdB; LAMP; illumigene; strains, toxigenic
INTRODUCTION
Toxin‐producing Clostridium difficile is the most common cause of hospital‐associated diarrhea, and community‐acquired C. difficile infections (CDIs) are rapidly increasing 1. These concerning trends demand rapid diagnosis for treatment and infection control. Many clinical laboratories now use nucleic acid amplification‐based methods for detection of toxigenic C. difficile in stool samples in lieu of the less‐sensitive enzyme immunoassay 2, 3.
Most cases of CDI are caused by strains expressing both toxins A and B (A+B+). Toxin A−B+ strains, while less frequently encountered, can cause disease and have been implicated in outbreaks of CDI 4, 5, 6. Human disease attributable to toxin A+B− strains is extremely uncommon 7, 8.
The most common genetic causes for toxin A−B+ C. difficile strains are a nonsense mutation within tcdA (toxinotype VIII) or deletions within conserved sequences at the 3′ end of tcdA (toxinotypes X, XVI, and XVI; 8, 9, 10). Integral to the design of molecular assays targeting tcdA is the fact that its 5′ end is conserved for the majority of known toxin‐producing A−B+ strains, specifically those causing known outbreaks 4, 6, 11, 12. Sequence analyses of tcdA have shown that it is more conserved than tcdB 8. Taken together, these observations suggest that targeting the 5′‐end of tcdA is a promising strategy for detection of clinically relevant C. difficile strains.
The illumigene® C. difficile assay (Meridian Bioscience, Inc., Cincinnati, OH) is a US Food and Drug Administration (FDA) cleared assay that targets the toxin A gene (tcdA), utilizing loop‐mediated isothermal amplification (LAMP). Published studies thus far have demonstrated good correlation of the illumigene® assay with other FDA‐cleared molecular C. difficile assays using clinical specimens 13, 14, 15, 16, 17, 18. Although the amplification target of the illumigene® assay is located in the highly conserved 5′‐region of tcdA, which is present in almost all known toxin A−B+ strains, there remains concern that the assay does not amplify the widely preferred amplification target, tcdB 16, 19. Toxin B, and not necessarily toxin A, is produced by virtually all pathogenic strains to date. Studies of various toxinotypes of C. difficile, including A−B+ strains, have also not yet been tested in parallel on commercial amplification assays that target either tcdA or tcdB.
In this study, we compared the ability of the illumigene® assay and GeneOhm® C. difficile assay to recognize various C. difficile reference strains representing various toxin classes, and assessed their cross‐reactivity with other Clostridium species. Using GeneOhm® as a molecular reference method, we evaluated agreement of the illumigene® assay, and utilized culture and a laboratory‐developed real‐time polymerase chain reaction (PCR) targeting both tcdA and tcdB as arbitrator methods.
MATERIALS AND METHODS
Clostridium Isolates
Thirty‐eight different strains of C. difficile provided by Meridian Bioscience originated from various collections where they had previously been characterized (Table 1). Strains were supplied at a density of 4 McFarland standards (∼1.2 × 109 CFU/ml). Archived suspensions of 40 Clostridium isolates representing 20 different species previously isolated at ARUP Laboratories (Salt Lake City, UT) were subcultured to Columbia blood agar (Hardy Diagnostics, Santa Maria, CA) and incubated anaerobically (Table 2). These consisted of C. aldenense (n = 2), C. bifermentans (n = 4), C. butyricum (n = 1), C. citroniae (n = 1), C. clostridioforme (n = 2), C. glycolicum (n = 2), C. hathewayi (n = 5), C. hiranonis (n = 2), C. innocuum (n = 1), C. novyi (n = 1), C. paraputrificum (n = 1), C. peptidivorans (n = 2), C. perfringens (n = 2), C. ramosum (n = 3), C. septicum (n = 2), C. sordellii (n = 3), C. sporogenes (n = 2), C. symbiosum (n = 1), C. tertium (n = 1), and C. xylanolyticum/C. aerotolerans (n = 2). Colonies were suspended in phosphate‐buffered saline (PBS) to a density of 4 McFarland standards (∼1.2 × 109 CFU/ml) prior to testing. All resuspended colony isolates were tested by the illumigene® and GeneOhm® C. difficile assays within 24–72 h of each other. Both assays were performed as directed by the package insert except using 50 μl colony suspensions as the starting material instead of stool.
Table 1.
Source, Toxinotype and Results With the illumigene® and GeneOhm® for 38 C. difficile Strains
| C. difficile strain | Source | Toxinotype | Toxin produced | illumigene® result | GeneOhm® result |
|---|---|---|---|---|---|
| 11186 | VPI collection | N/A | None | Neg | Neg |
| 10463 | VPI collection | 0 | A+ B+ | Pos | Pos |
| 2004111 | CDC | 0 | A+ B+ | Pos | Pos |
| 2004205 | CDC | 0 | A+ B+ | Pos | Pos |
| 2005070 | CDC | 0 | A+ B+ | Pos | Pos |
| 2005257 | CDC | 0 | A+ B+ | Pos | Pos |
| 2008029 | CDC | 0 | A+ B+ | Pos | Pos |
| 2008162 | CDC | 0 | A+ B+ | Pos | Pos |
| 2008341 | CDC | 0 | A+ B+ | Pos | Pos |
| 2008351 | CDC | 0 | A+ B+ | Pos | Pos |
| 2009066 | CDC | 0 | A+ B+ | Pos | Pos |
| 2009099 | CDC | 0 | A+ B+ | Pos | Pos |
| B1 | Hines VA Hosp. | 0 | A+ B+ | Pos | Pos |
| G1 | Hines VA Hosp. | 0 | A+ B+ | Pos | Pos |
| J7 | Hines VA Hosp. | 0 | A+ B+ | Pos | Pos |
| K12 | Hines VA Hosp. | 0 | A+ B+ | Pos | Pos |
| Y1 | Hines VA Hosp. | 0 | A+ B+ | Pos | Pos |
| 2004052 | CDC | III | A+ B+ | Pos | Pos |
| 2004118 | CDC | III | A+ B+ | Pos | Pos |
| 2007431 | CDC | III | A+ B+ | Pos | Pos |
| 2009052 | CDC | III | A+ B+ | Pos | Pos |
| BI17 | Hines VA Hosp. | III | A+ B+ | Pos | Pos |
| BI8 | Hines VA Hosp. | III | A+ B+ | Pos | Pos |
| 2007858 | CDC | IX/XXIII | A+ B+ | Pos | Pos |
| 2005325 | CDC | V | A+ B+ | Pos | Pos |
| 2006240 | CDC | V | A+ B+ | Pos | Pos |
| 2008188 | CDC | V | A+ B+ | Pos | Pos |
| 2009065 | CDC | V | A+ B+ | Pos | Pos |
| BK6 | Hines VA Hosp. | V | A+ B+ | Pos | Pos |
| 2009018 | CDC | V | A+ B+ | Pos | Pos |
| 43598 | ATCC | VIII | A− B+ | Pos | Pos |
| 2008016 | CDC | VIII | A− B+ | Pos | Pos |
| CF1 | Hines VA Hosp. | VIII | A− B+ | Pos | Pos |
| 8864 | S.P. Borriello | X | A− B+ | Pos | Pos |
| 2007435 | CDC | XII | A+ B+ | Pos | Pos |
| 2009132 | CDC | Unknown | Unknown | Pos | Pos |
| 2009155 | CDC | Unknown | Unknown | Pos | Pos |
| 2009277 | CDC | Unknown | Unknown | Pos | Pos |
VPI, Virginia Polytechnic Institute; CDC, Centers for Disease Control and Prevention; VA, Veterans Affairs; ATCC, American Type Culture Collection.
Table 2.
Primer and Probe Sequences Utilized by the Laboratory‐Developed Real‐Time PCR for Discrepant Results Analysis
| Primer/probe | Concentration | |
| name | Sequence | (μM) |
| tcdA–forward | 5′‐AATAAATCATAAA*TGGT* T*T*ACCTCA*GATAG‐3′ | 0.25 |
| tcdA–reverse | 5′‐AATAAATCATAAGTTAGCA* TCCGTA*TTAGCAG‐3′ | 1.00 |
| tcdB–forward | 5′‐ AATAAATCATAACCA*GTA* AAATCA*AT*T*GCT‐3′ | 1.00 |
| tcdB–reverse1 | 5′‐AATAAATCATAACCA*GCT AATA*CACT*T*GATGA‐3′ | 0.25 |
| tcdB–reverse2 | 5′‐AATAAATCATAACCA*GCA GATA*CACT*T*GATGA‐3′ | 0.25 |
| tcdA probe | 5′‐G*AATACTTTGCA CCTGC‐3′ | 0.20 |
| tcdB probe | 5′‐CTAGAAGGN* GA*A*GCA* A*‐3′ | 0.20 |
A*, Super A®; G*, Super G®; N*, universal base (proprietary bases are trademarks of Epoch Bioscience).
Patient Stool Specimens
This study was approved under the University of Utah Institutional Review Board. Unselected diarrheal stool specimens (n = 108) submitted to ARUP Laboratories for C. difficile detection were tested with the GeneOhm® assay (BD Diagnostics, La Jolla, CA) as per the manufacturer's package insert. Testing was performed within 48 h of specimen collection and samples were stored at 4°C. Testing with the illumigene® assay was performed within 24–72 h of initial testing and within the specimen stability limits according to the package insert instructions (5 days at 4°C). For future discrepant analysis, specimens were stored at −20°C per kit instructions and in‐house validation studies of specimen stability.
Analysis of Discrepant Results
Analysis of discrepant results differed slightly between Clostridium isolates and patient stool samples. Clostridium isolates that yielded discrepant results between the GeneOhm® and illumigene® assays were repeated on both platforms. They were also subcultured on Brucella blood agar (Hardy Diagnostics) and incubated anaerobically at 37°C for 24–48 h. If more than one colony type was observed, each colony type was isolated for further analysis.
Patient stool specimens with discrepant results between the two commercial assays were thawed to room temperature and tested again with both assays. Extracts from each assay were also subjected to a laboratory‐developed real‐time PCR (see below). Stool specimens were plated on cycloserine‐cefoxitin‐fructose agar (CCFA; Hardy Diagnostics) and grown anaerobically at 37°C for 24–72 h. Putative C. difficile colonies, as identified by characteristic Gram stain, colony morphology and odor, and fluorescence under Wood's lamp were isolated by subculturing on Brucella blood agar. In one case, the sample did not yield any C. difficile or other bacterial colonies. In a further attempt to isolate C. difficile, the specimen was subjected to ethanol shock treatment by mixing with 100% ethanol at a ratio of 1:1, vortexing, and incubating at ambient temperature for 30 min. The sample was then plated on CCFA and resulting colonies were isolated as described above.
All isolated colonies were suspended in PBS to a density of 4 McFarland (∼1.2 × 109 CFU/ml) prior to molecular testing. Suspensions were tested with both the GeneOhm® and illumigene® assays as described above.
Laboratory‐Developed Real‐Time PCR Analysis
Two real‐time PCR assays targeting tcdA and tcdB were developed to resolve discrepant results. PCR reactions (25 μl final volume) contained 2.0 μl isolate suspensions or specimen lysates from the commercial assays, 5× QuantiTect Multiplex PCR mastermix (Qiagen, Valencia, CA), 0.5 U AmpErase uracil N‐glycosylase (Life Technologies, Carlsbad, CA), tcdA or tcdB primers and probes (Table 3; Epoch Bioscience, Bothell, WA), 1,000 copies internal control template, and internal control primers and probes 20. Resulting amplicon lengths were 79 bp for tcdA and 53 bp for tcdB. Pleiades probes containing 5′ MGB and FAM fluorophores and a 3′ BHQ quencher were used for amplicon detection 21. Reactions were carried out on the ABI7900HT instrument (Life Technologies) under the following cycling conditions: 50°C for 2 min, 95°C for 8 min, followed by 50 cycles of 95°C for 15 sec, 58°C for 30 sec, and 76°C for 30 sec, and ending with a dissociation curve analysis (15‐sec holds at 95°C, 45°C, and 95°C).
Table 3.
Agreement Between the GeneOhm® and illumigene® C. difficile Assays With 108 Clinical Specimens
| illumigene® | GeneOhm® C. difficile assay | |||
|---|---|---|---|---|
| C. difficile assay | Positive | Negative | Total | |
| Positive | 17 | 1 | 18 | |
| Negative | 5 | 85 | 90 | |
| Total | 22 | 86 | 108 | |
RESULTS
Results of Clostridium spp. Isolates Tested With Illumigene® and GeneOhm®
Thirty‐seven toxin‐producing C. difficile strains and one nontoxin‐producing strain yielded expected results and showed 100% concordance between the GeneOhm® and illumigene® assays (Table 1). The illumigene® assay detected all toxin A+B+ strains of toxinotypes 0 (n = 16), III (n = 6), V (n = 6), XII (n = 1), and IX/XXIII (n = 1). It also amplified all toxin A−B+ strains of toxinotypes VIII (n = 3) and X (n = 1).
Neither the illumigene® nor the GeneOhm® assay amplified 40 non‐C. difficile Clostridium isolates, including the closely related C. sordellii (n = 3). One C. sordellii isolate initially tested as invalid with the illumigene® assay due to internal control failure, but was negative upon retesting. The sample was negative with the GeneOhm® assay. One C. symbiosum sample tested initially positive with the illumigene® and negative with the GeneOhm® assay. The laboratory‐developed real‐time PCR assays confirmed the presence of tcdA and tcdB. Subculture of the isolate revealed two colony types, one characteristic for C. difficile and one for C. symbiosum. By the laboratory‐developed real‐time PCR assay, the presumptive C. difficile isolate was positive for tcdA and tcdB and the other colony type was negative for tcdA and tcdB. Repeat testing on the illumigene® and GeneOhm® assays confirmed these results.
Analysis of Clinical Specimens With Illumigene® and GeneOhm®
There was an initial concordance of 94.4% (102/108) between the GeneOhm® and illumigene® assays with the patient samples (Table 3). Using the GeneOhm® assay as the reference method, the sensitivity and specificity of the illumigene® assay was 77.3% (17/22) and 98.8% (85/86), respectively.
Six samples (cases 1–6) with discordant results were retested with both assays (Table 4). Two illumigene®‐negative/GeneOhm®‐positive specimens (cases 4 and 5) tested positive with both assays upon retesting. One specimen (case 1) was GeneOhm®‐negative/illumigene®‐positive. The lysate from the illumigene® assay tested positive for tcdA and tcdB with the laboratory‐developed real‐time PCR. In contrast, the specimen lysate from the GeneOhm® assay tested negative for tcdB and positive for tcdA. Upon retesting, the GeneOhm® assay was still negative but the lysate was positive for tcdA and tcdB by the laboratory‐developed real‐time PCR. Clostridium difficile colonies were isolated from the original stool specimen and were positive on both commercial assays. In two of the remaining three specimens (cases 2 and 3), both GeneOhm®‐positive and illumigene®‐negative, C. difficile colonies were isolated and tested positive with both commercial assays. In the third specimen (case 6), positive GeneOhm® and negative illumigene® results were confirmed during repeat testing. Testing of lysates from both assays with the laboratory‐developed real‐time PCR confirmed the presence of tcdB but not tcdA. Culture for C. difficile from the original specimen yielded two colonies characteristic for C. difficile after ethanol shock treatment. Both colonies tested negative with both commercial assays. In all cases with discordant results, the specimen lysates from both commercial assays were found to be near the limit of detection (LOD; ten copies per reaction) when tested with the laboratory‐developed real‐time tcdB PCR.
Table 4.
Discrepant Result Analysis
| BD GeneOhm® | Illumigene® | Laboratory‐developed PCR | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Case | Initial specimen lysate | Repeat specimen lysate | Isolate | Initial specimen lysate | Repeat specimen lysate | Isolate | tcdA | tcdB a | Resolved result after discordant analysis |
| 1 | NEG | NEG | POS | POS | POS | POS | POS | POSb | Low positive |
| 2 | POS | POS | POS | NEG | NEG | POS | POS | POS | Low positive |
| 3 | POS | POS | POS | NEG | NEG | POS | POS | POS | Low positive |
| 4 | POS | POS | POS | NEG | POS | POS | POS | POS | Low positive |
| 5 | POS | POS | POS | NEG | POS | POS | POS | POS | Low positive |
| 6 | POS | POS | NEGc | NEG | NEG | NEGc | NEG | POS | Presumptive low positive |
All specimens contained less than ten copies per reaction as determined by the laboratory‐developed real‐time PCR assay.
The tcdB PCR was initially negative with the GeneOhm® lysate, but positive with specimen lysate from repeat testing.
Two C. difficile colonies could be isolated and were tested.
DISCUSSION
Most commercially available and laboratory‐developed molecular tests or toxigenic C. difficile target tcdB because toxin B production appears to be ubiquitous in clinically relevant strains 3, 22, 23. The illumigene® assay is unique among currently US FDA cleared molecular assays for C. difficile in that it targets tcdA. This assay detected all 37 toxigenic C. difficile reference strains used in this study. Four toxin A−B+ isolates were available for testing covering the two most clinically relevant A−B+ toxinotypes (VIII and X) that have caused outbreaks 4, 6, 11, 12. It is important to note that emerging toxinotypes may have deletions in tcdA that could affect clinical assays. In a recent study, newly described A−B+ toxinotypes XXX and XXXI were not detected by the illumigene® assay due to deletions in tcdA 24. They represented 0.6% and 0.1% of C. difficile isolates in the study leading to their discovery, suggesting they are uncommon strains at this time 19. Overall, the illumigene® assay was able to detect the most clinically relevant strains of C. difficile.
Analysis of 108 stool specimens with both commercial assays showed good agreement (94.4%). Discrepant results were most likely caused by specimens containing a low number of toxigenic C. difficile in combination with sampling error and/or differences in the analytical sensitivities of the assays. Specimen lysates generating a GeneOhm®‐positive/illumigene®‐negative or GeneOhm®‐negative/illumigene®‐positive result were tested with the laboratory‐developed real‐time PCR and found to contain approximately ten copies of tcdB per reaction. This observation suggests the presence of toxigenic C. difficile at a concentration approaching the LOD of the GeneOhm® assay (4 CFU/reaction or ten copies per reaction) and the illumigene® assay (4–64 CFU/reaction). While stool specimens were all tested within the stated stability limits of the commercial assays, they were tested by the illumigene® assay 24–72 h after being tested by the GeneOhm® assay. This delay may have contributed to the occurrence of GeneOhm®‐positive/illumigene®‐negative results in five of the six discordant cases. In one specimen that was repeatedly GeneOhm®‐positive and illumigene®‐negative (Table 4, case 6), the laboratory‐developed real‐time PCR of the specimen lysate was positive for tcdB and negative for tcdA while the cultured isolate (two colonies) was negative for both tcdA and tcdB. These inconclusive results may be explained by the presence of both toxigenic and nontoxigenic C. difficile strains in low numbers in the stool specimen, the difference in the LOD of our laboratory‐developed PCR assay for tcdA compared to tcdB, or (less likely) a C. difficile strain with a deletion or mutation in tcdA that precluded detection by the laboratory‐developed real‐time PCR tcdA and the illumigene® assays.
The clinical significance of low bacterial loads for toxigenic C. difficile in diarrheal stool samples remains unclear. Since toxigenic C. difficile is known to colonize patients without causing symptoms, and CDI is attributable to multiple factors, such as host immune response and gut microbiota, detection of the C. difficile toxin genes does not equate infection 25, 26. Unfortunately, we were unable to determine possible alternative causes for the diarrheal disease in these patients since specimens were referred for analysis from a number of hospitals throughout the United States and clinical histories were not available.
Conclusion
The ability to rapidly and accurately diagnose CDI is important for prompt treatment and implementation of infection control measures. Targeting tcdA at the 5′ conserved region using a LAMP‐based assay allowed accurate detection of both toxin A+B+ and most clinically relevant toxin A−B+ C. difficile strains encompassing more than seven toxinotypes. Both the GeneOhm® and the illumigene® C. difficile assays accurately identified toxin‐producing C. difficile strains and were highly specific when tested against a toxin‐negative strain and 20 other Clostridium species. The illumigene® C. difficile assay was easy to use and appears to offer similar performance characteristics as other commercially available molecular assays such as the BD GeneOhm®.
ACKNOWLEDGMENT
We thank the ARUP Molecular Amplified Detection laboratory for their technical help.
Grant sponsor: ARUP Institute for Clinical and Experimental Pathology; Grant sponsor: Meridian Bioscience, Inc.
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