Epidemiology, Diagnosis and Treatment of Clostridium Difficile Infection

Matteo Bassetti; Giovanni Villa; Davide Pecori; Alessandra Arzese; Mark Wilcox


Expert Rev Anti Infect Ther. 2012;10(12):1405-1423. 

In This Article

Molecular Biology Applied to CDI Diagnostics

Molecular biology studies have been applied to C. difficile since discovery that the species was responsible for human disease. Only recently, PCR-based commercial kits became available for laboratory routine diagnostics. There are two main PCR targets and one major goal for each approach: rRNA gene complex (16S, 23S and intergenic spacer region) for C. difficile isolate typing, outbreak monitoring and epidemiological surveys – C. difficile ribotyping; toxin B and toxin A genes, and more generally PaLoc pathogenicity island targeting for CDI diagnostic purposes. These targets are also used for C. difficile characterization and epidemiological studies – C. difficile toxinotyping – by in-house experimental PCR approaches.[73,100]

C. difficile ribotyping was set up in different reference laboratories in the last 20 years: for the UK, the Anaerobe Reference Laboratory at the University Hospital of Wales (Cardiff, UK) started in the 1990s to perform ribotyping in order to track epidemics.[101] Ribotyping became a prominent technique for tracking C. difficile in 2003/4 with the emergence of ribotype 027 NAP1 C. difficile. Ribotyping is however not practiced by routine laboratories due to the required skill level and notably the need for a database of ribotypes.[9,10] Ribotyping can provide useful information to help guide infection control teams, although more discriminatory DNA fingerprinting methods can be useful to confirm whether suspected CDI cases are truly related.[102] Multiple alternative typing and fingerprinting methods for C. difficile have been described.[103] In particular, in a multiplex PCR system three pairs of primers targeting an internal fragment of toxin B, an internal fragment of toxin A, and a species-specific internal fragment of the triose phosphate isomerase (tpi) gene, respectively, were shown to distinguish between toxin A-positive/toxin B-positive strains and toxin A-negative/toxin B-positive variant. Interestingly, by amplification of the tpi gene, which was shown to be highly conserved among C. difficile human and animal isolates, toxin A-negative/toxin B negative C. difficile isolates were also detected by the multiplex assay, thus allowing simultaneous identification and toxigenic type characterization of C. difficile isolates and to rule specimens negative.[104] The adoption of the tpi gene as a target for detection of C. difficile strains in human isolates in combination with amplification of toxin A, toxin B, CDT and putative negative regulator of toxin production (TcdC) target sequences was also shown to be successful in determining the prevalence of CDI and characterization of toxinotypes in a selected population.[105]

It is likely that the DNA sequence-based methods will be increasingly applied to the study of C. difficile epidemiology, notably given their high discriminatory power and as the cost of these approached continues to reduce.[106,107]

PCR assays have also been applied to the detection of CDI. A number of different target regions have been studied.[108–110] The rationale for targeting toxin B is essentially all pathogenic strains to date produce this toxin. The reliability of the molecular diagnostic approach has been tested in comparison with gold-standard methods and/or toxin immunoenzymatic assays, and/or GDH rapid detection systems. Apart from in-house PCR protocols proposed by various authors since the discovery of CDI, in recent times two different US FDA-approved commercial systems appear to be mostly investigated and evaluated for routine diagnostics.

The Xpert C. difficile assay (Cepheid, CA, USA) is a US FDA-approved real-time multiplex PCR assay: primers are specific for the toxin B gene, binary toxin genes and the tcdC gene single-base deletion at nucleotide 117, designed to detect toxigenic C. difficile and PCR ribotype 027 strain (Xpert C. difficile Epi assay, available as a research-use only). Each kit contains single-use disposable cartridges, which after inoculation with the specimen are placed in the dedicated PCR module and run: no additional extra reagents, or further steps are required, and negative/positive controls are included in each kit. Results are available within 1 h, thus being fully compatible with prompt laboratory report to the clinicians. The test can also be used on a fully automated platform. The usefulness of this PCR system in C. difficile diagnostics have been proved by various authors, in differently designed comparative studies with GDH, toxin A/B EIA rapid tests and gold-standard methods.[111–114] The Xpert C. difficile assay compares well in terms of sensitivity to gold-standard methods, but as the test does not detect toxins the specificity of a positive result is dependent on the clinical setting (Box 2). A multicenter clinical trial performed on a total of 2296 unformed stool specimens assessed the performance of Xpert Cepheid CDI with multiple other diagnostic tests. Compared with results from toxigenic culture with enrichment, the sensitivity, specificity and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0 and 98.8%, respectively. In particular, the sensitivity of toxin EIA was lower than that of Xpert for detection of hypervirulent ribotypes (002, 027 and 106). The overall sensitivity of toxin EIAs compared to that of TOXT with enrichment was 60%, and the sensitivity of combined GDH/toxin algorithms was 72.9%; both approaches were significantly less sensitive than Xpert C. difficile (p < 0.001 and p = 0.03, respectively).[113] However, others have shown that the GDH/toxins rapid EIA (TechLab) compared with TOXT accurately screened 88% of specimens as either positive or negative for CDI in less than 30 min.[115] In an additional study, the Xpert C. difficile performed statistically better than GDH assay by EIA (TechLab). Xpert PCR results were more consistent with a gold-standard method, and PCR failed to recognize only one true negative sample.[112] Thus, it could be considered to use Xpert PCR as a standalone test in CDI diagnosis. There are at least two important limitations to this hypothesis: first, in vivo toxin production is not detected by molecular approach, and such data are valuable to associate disease symptoms with C. difficile etiology. The latter limit is represented by the fact that the equipment can be present only in a limited number of laboratories, mainly for budgetary reasons: however, the more the system is validated and adopted by laboratories, the more likely that the cost continues to reduce. As the Xpert C. difficile is presently the most user-friendly and rapid real-time PCR approach to CDI diagnosis on the market, and the experimental evidence of reliability and sensitivity for CDI diagnosis by this system is increasing,[116,117] it could be speculated that in the near future the Xpert C. difficile will be a first choice for many laboratories willing to update C. difficile diagnostic by molecular assays.

Another FDA-licensed commercial PCR kit for C. difficile routine diagnostics is based on original loop-mediated isothermal amplification technology, which targets within the PaLoc a 204 bp conserved region of the toxin A gene (Illumigene C. difficile, Meridian Bioscience, OH, USA). The system has been tested in comparison with cytotoxicity assay and toxigenic culture as gold standard. Performance parameters versus gold standards are indeed excellent: all reagents are ready to use, loop-mediated isothermal amplification dedicated equipment is available, the overall turnaround time is within 1 h, and is compatible with rapid laboratory report. The major concern is related to lack of toxin A-negative toxin B-positive C. difficile detection, due to the target choice, which represents approximately 4% of all isolates at least in European countries (Table 3).[118,119] However, the rationale for the Illumigene design is to target the 5' region of the tcdA gene that is also present in all known toxin A-negative toxin B-positive strains. Moreover, the choice of this PCR target was based on evidence that the toxin A gene appeared to be generally more conserved than the toxin B gene within C. difficile isolates.[120] In particular, the PCR assay detects a 5' toxin A sequence which was found intact in all of the strains examined in several studies: indeed, it was proven that also strains with a large deletion in the toxin A gene could be detected by the commercial system.[121]

In a recent paper, a meta-analysis of CDI diagnostic results obtained by PCR within 1995–2010 from 19 studies (7392 samples), found that the overall mean sensitivity of PCR was 90% and specificity 96%; interestingly, test accuracy appeared to be related on the prevalence on CDI rate but not on the reference test used. In particular, if C. difficile prevalence was reported to be <10, 10–20 and >20%, the positive predictive values and the negative predictive values were 71, 79, 93% and 99, 98 and 96%, respectively. Therefore, real-time PCR is considered an ideal diagnostic assay in epidemic conditions, with high C. difficile prevalence, but might not be the best diagnostic test in endemic situations for CDI diagnostics. Despite this limitation, real-time PCR has a high sensitivity and specificity in the confirmation of CDI.[122]

As PCR targets DNA encoding C. difficile toxin and not the toxin itself, and due to the high sensitivity of molecular techniques, a positive result may reflect carriage of toxigenic strain not expressing the toxin in vivo. Therefore, real-time PCR results should always be associated with a more specific test, such as those that detect the presence of the toxins. Moreover, the cost of a routine C. difficile PCR exceeds many laboratory budgets. Recently (2012) updated guidance on the diagnosis and reporting of C. difficile was derived from an observational diagnostic study on 12,441 specimens. Diagnostic assays were chosen to represent the three main C. difficile detection options in routine use (toxin/GDH and toxin gene detection). Importantly, the presence of a C. difficile toxin, determined by CCNA, significantly correlated with poor clinical outcome. The guidance recommends combining either GDH EIA or a toxin gene detection method with a toxin detection assay (sensitive toxin EIA or CCNA). If the GDH assay is used the screening test then it is possible to use a PCR toxin gene detection method for those samples that are GDH positive but toxin negative.[204]

In Figure 1 the authors summarize an optimized algorithm for C. difficile testing and include the potential clinical interpretation of the different possible results.

Figure 1.

Clostridium difficile algorithm for Clostridium difficile infection diagnosis. AG: Antigen; CCNA: Clostridium difficile-toxin-specific antibodies; CDAD: Clostridium difficile-associated diarrhea; EIA: Enzyme immunoassay; GDH: Glutamate dehydrogenase antigen; TOX A/B: Toxin A/B.