Diagnosis of Clostridium Difficile Infection

The Molecular Approach

Catherine Eckert; Gabrielle Jones; Frédéric Barbut

Disclosures

Future Microbiol. 2013;8(12):1587-1598. 

In This Article

Abstract and Introduction

Abstract

Diagnosis of Clostridium difficile infection is based on clinical presentation and laboratory tests. Although numerous laboratory methods are now available, the diagnosis of C. difficile infection remains challenging. Nucleic acid amplification tests (NAATs) are the most recent marketed methods. These methods detect genes for toxins A and/or B. They are very sensitive compared with the reference method (toxigenic culture) and are thus very promising, despite their cost. However, a positive NAAT result must be interpreted with caution owing to the possible detection of asymptomatic carriers of toxigenic strains who may have diarrhea for other reasons. The place of NAATs in current diagnostic strategies needs to be better defined, but the rapidity of the result is interesting for early recognition of the disease.

Introduction

Clostridium difficile is a spore-forming anaerobic bacillus, mainly found in soil and the intestines of humans and animals. C. difficile was first isolated in 1935, but its role in pseudomembranous colitis (PMC) was only established in the late 1970s.[1,2]C. difficile is now recognized as being responsible for a wide spectrum of diseases, ranging from mild to severe diarrhea, PMC, toxic megacolon and fulminant colitis, possibly leading to intestinal perforation and death of the patient. However, patients colonized with C. difficile do not systematically develop symptoms. Asymptomatic carriage of C. difficile strains is uncommon among healthy adults (<3%), but can be much higher in hospitalized patients and neonates.[3,4] Clabots et al. have shown that C. difficile acquisition is proportional to the length of hospitalization: of 128 patients hospitalized for 1–2 weeks, 13% acquired C. difficile, as did 50% of those hospitalized for >4 weeks.[5] In this study, among 557 patients negative for C. difficile on admission, 54 acquired C. difficile and, among them, 51 remained asymptomatic. C. difficile is the main enteropathogen implicated in healthcare-associated diarrhea among adults in industrialized countries and it accounts for 10–25% of antibiotic-associated diarrhea, and for most cases of PMC.[6] Transmission of C. difficile occurs via contaminated hands of healthcare workers or through the environment, where spores can persist for months owing to their high resistance to standard disinfectants. Main risk factors for C. difficile infections (CDIs) include age >65 years, antibiotic treatment and previous hospitalization.[7–9] Proton pump inhibitors were previously controverted as a risk factor, but their role in CDIs seems to now be accepted.[10] Enterotoxic and cytotoxic toxins A (TcdA) and B (TcdB) are the main virulence factors of C. difficile, and are essential in the pathogenesis of CDIs.[11] TcdA and TcdB are encoded by the tcdA and tcdB genes, respectively, which form, along with three accessory genes (tcdC, tcdE and tcdR), a pathogenicity locus of 19.6 kb.[12]tcdC and tcdR are negative and positive regulators of toxin synthesis, respectively.[13,14] Among toxigenic strains, approximately 20% also produce a third toxin, called the binary toxin.[15,16] Its role remains unclear, but it may act as an additional virulence factor.

The epidemiology of CDIs has rapidly evolved since the beginning of the 21st century. Increased incidence has been reported in some European countries and North America since the beginning of the century, particularly in patients over 65 years of age.[17,18] Important outbreaks of severe CDIs were first described in Canada and North America in 2003, then in the UK and Europe.[18,19] These outbreaks coincided with the rapid emergence of an epidemic and so-called 'hypervirulent' strain identified as PCR-ribotype 027 strain (or NAP1 according to pulse-field gel electrophoresis typing, or BI according to restriction enzyme analysis typing).[20] This strain was very uncommon before the 1990s and became endemic in many healthcare settings from 2006. This clone has been shown to produce a large amount of toxins A and B in vitro, along with the binary toxin. It is further characterized by its resistance to erythromycin and newer fluoroquinolones, such as moxifloxacine, and by an 18-bp deletion in tcdC. Epidemic 027 strains also carry a deletion in position 117 in the tcdC gene, leading to a frameshift in the transcription and production of a truncated nonfunctional TcdC protein, which cannot act as a negative regulator and therefore is responsible for the hyperproduction of toxins A and B in vitro.[21]

Other changes in CDI epidemiology have also been observed in recent years. An increased number of cases of CDIs in the elderly, especially in long-term care facilities and nursing homes, has been observed. In Germany, approximately one in 20 nursing home residents tested were colonized with C. difficile, while in The Netherlands, a quarter of residents in nine homes had CDIs.[22,23] There is a better recognition of community-associated CDI cases that affect patients previously thought to be at low risk, including young patients, pregnant women and patients without previous hospitalization or recent antibiotic treatment.[24,25]C. difficile was also recognized as an important pathogen for animals and was described in foods such as meat, ready-to-eat salads and shellfish.[26,27] As suggested by a recent review, direct transmission of C. difficile from animals or food to humans has not been proven to date, although similar PCR-ribotypes have been found.[27] However, circumstantial evidence points towards a zoonotic potential of this type.[27]

Nosocomial CDI adds substantial costs for healthcare facilities (HCFs). It was estimated that CDIs conferred a total cost to the EU of approximately €3 billion per year.[18] The main driver of cost is the prolongation of hospitalization, which ranges from 1 to 3 weeks. Specific therapies for CDI and infection control measures also contribute to the financial burden of CDIs in HCFs. Incidence and thus length of stay, mortality and costs linked to CDIs are higher than ever.[1,28,29] In England, in response to large outbreaks of CDIs, a bundle of measures, including new legislation, mandatory surveillance and reporting, diagnosis and testing guidelines, and ribotyping network were introduced from 2006 onwards to tackle CDIs. Interestingly, after years of increasing incidence of CDIs, a marked decrease was recently observed in England, suggesting the possibility of reversing this trend.[30] In this context, rapid and accurate diagnosis is crucial for timely treatment of the patient or to stop unnecessary empiric antibiotic treatment. Rapid diagnosis also allows for implementation of contact precautions to avoid nosocomial transmission. Diagnosis of CDIs is based on clinical presentation and laboratory tests. Although numerous laboratory methods are now available, the diagnosis of CDIs still remains challenging.[31,32] None of the methods currently available offer all the qualities of an ideal diagnostic method, including high sensitivity and specificity, rapid turnaround time, technical simplicity and cost–effectiveness. Many original papers or reviews on CDI diagnosis have been published recently highlighting the lack of sensitivity of enzyme immunoassays (EIAs) that detect toxins A and B and the growing place of molecular methods available since 2009.[33–35] This review will summarize the variety of diagnostic methods available and their respective characteristics, focusing on molecular methods.

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