How to solve a problem like C. difficile

Detecting the highly infectious and dangerous Clostridium difficile is fraught with false positives and negatives – Professor Wren says the answer lies in a two-step approach 

Professor Mike Wren MBE, Consultant Biomedical Scientist at University College Hospital, London has evaluated a two-step approach for the detection of Clostridium difficile incorporating an initial screening test to detect the C. difficile antigen, glutamate dehydrogenase (GDH)1. Samples that are GDH-positive are tested for C. difficile toxins A/B and cultured for toxigenic strains. The evaluation has shown that this rapid, simple and cost effective approach has a negative predictive value (NPV) of 99.5% and demonstrates excellent concurrence with reference methods.

Professor Wren said: “The current reliance on toxin-only testing has serious limitations that can have a negative impact on patient care and infection control. On its own, toxin testing misses over 40% of positives and does not differentiate between mild or serious disease and carriers. Our studies show that we can significantly improve the situation providing rapid, meaningful results that hopefully provide a more complete diagnostic picture and help physicians to make appropriate clinical decisions”.

Since its identification in 1977, C. difficile has emerged as the predominant cause of C. difficile - associated diarrhoea (CDAD) and pseudomembranous colitis (PMC) in hospitalised patients. Highly infectious and extremely dangerous in the compromised and elderly, the disease can rapidly become life threatening and contributes to the death of 8,324 patients every year, which is four times as many as MRSA (Office of National Statistics, 2008). Infection rates have risen exponentially since reporting began, from 2000 cases in 2000 to 49, 785 in 2007 (HPA, 2008) and tackling the issue is a top priority for the Department of Health.

Rapid, accurate reporting of C. difficile is essential for improving patient outcomes and minimising healthcare-associated infections. However, several recent studies in the UK, Europe and the USA have shown that current laboratory tests may miss as many as 50% of positive patients2,3. Professor Wren and his team support these findings and ongoing studies at UCH, in close cooperation with ward physicians and medical microbiologists, show that toxin testing alone in their laboratory would miss 45.8% of positive patients. A recent systematic review of C.difficile toxin detection kits by St Georges Hospital, London4 also suggests that a two-stage testing strategy would improve diagnosis.

Patients with C. difficile can develop serious disease including colitis, toxin megacolon and colon perforation. They also present a significant risk to the patients around them. Whilst all patients with diarrhoea should be nursed in isolation until the cause is diagnosed, high bed occupancy rates often make this difficult and infected patients are treated on open wards.

The anaerobic spore-forming bacterium is present in healthy guts of 3% of adults and 66% of children, only becoming a problem when the normal balance of gut flora is altered allowing the organism to multiply rapidly. The most common pre-disposing factors are treatment with broad-spectrum antibiotics, underlying disease and old age, with 80% of cases being reported in people aged over 65 years.

The explosive diarrhoea associated with C. difficile infection causes widespread dissemination of spores that are highly resistant to surface cleaning agents and extremely persistent in the environment. As a result, other patients, staff and visitors are exposed to contamination, dramatically increasing the risk of cross infection.
Time to diagnosis is therefore critical in every situation, as Professor Wren explained: “The earlier you treat the infection, the quicker the patient’s symptoms are resolved ensuring a better clinical outcome and minimising the risk of contamination.”

Only strains of C. difficile that produce the toxins A (TcdA) and B (TcdB) cause disease and it is essential that laboratory tests are able to quickly identify the presence of toxigenic organisms. The gold standard method for detecting C. difficile toxins is the Cell Cytotoxicity Neutralisation Assay (CCNA) but with a turnaround time of 3 days and a requirement for cell culture facilities and expertise, it is not a realistic option for many routine laboratories. The CCNA is also less sensitive for detecting cases than the use of toxigenic culture (isolation of the organism and testing the isolate for toxin) - the ‘alternative gold standard’ proposed by Professor Mark Wilcox, (CDRNE Reference Laboratory, Leeds). However this technique takes a minimum of 48 hours and with an increasing demand for same day results, more than 80% of UK microbiology laboratories have now abandoned culture and rely solely on rapid enzyme immunoassays (EIAs) to detect C. difficile toxins A/B in faecal samples.

A number of commercial EIA tests are available and whilst rapid and easy to perform, they have low sensitivity compared to reference methods. Reports from prestigious centres around the world have recently cited false negative rates as high as 50%. There are also reports that EIA gives false positive results5, further complicating the diagnostic picture.

Professor Wren summarised the problem: “Toxin testing alone isn’t working for some patients or the hospital community at large. Diagnostic delays, false positives and false negative results have major implications for patient care and infection control. New developments provide the opportunity to improve diagnosis and our experience provides overwhelming evidence to support a radical change”.

The UCH laboratory set out to develop a new 2-hour testing protocol that would reduce false negatives and positives to provide useful clinical results within the working day. The study compares a two-step approach with the ‘alternative gold standard’ of culture and toxin testing, and examination of 1007 samples has demonstrated an NPV of 99.5%.

The 2-step approach includes an initial screening step to detect the glutamate dehydrogenase (GDH) antigen of C. difficile. Known as the ‘common antigen’ GDH is an enzyme produced in large quantities by all toxigenic and non-toxigenic strains, making it an excellent marker for the organism. The UCH team used ‘C DIFFCHEK’ (Techlab/Inverness Medical UK Ltd) an easy to perform EIA that utilises a GDH specific antibody. As a first screening assay it has an excellent NPV, allowing negative results to be reported without further testing.

As the GDH assay does not distinguish between toxigenic and non-toxigenic strains, GDH-positives are subjected to a rapid toxin A/B test. Toxin positives are immediately reported and cultured to allow further testing such as antibiotic susceptibility, ribotyping and molecular determination.

Inverness Medical UK has recently introduced a new product that offers the two-step approach in one simple assay. C.DIFF QUIKCHEK COMPLETE detects GDH and
C. difficile toxins A and B in less than 30 minutes and the UCH laboratory is implementing the rapid test to further streamline the testing protocol and provide faster results.

A positive toxin result must always be considered in the context of the patient’s condition. Not everyone carrying the organism exhibits disease and toxin positive patients may be carriers and their symptoms unrelated to the presence of the organism. They do not require treatment but as excreters of the organism they present a risk to others and should be managed accordingly. However, faced with a positive toxin result physicians must quickly decide whether the patient is suffering from C. difficile disease, or is just a carrier of the organism. To help with this differentiation Mike Wren is looking to determine whether a rapid test for lactoferrin can prove a useful adjunct to GDH and toxin testing.

By definition CDAD causes intestinal inflammation and consequent neutrophil infiltration; lactoferrin is a breakdown product of neutrophils and gastroenterologists have shown a direct correlation between faecal lactoferrin and intestinal inflammation. The marker is stable in faeces and has baseline levels in healthy subjects and people suffering from irritable bowel disease (which shares many symptoms with CDAD). Rapid quantitative and qualitative tests are available allowing levels to be monitored as they return to normal in parallel to therapeutic response.

The results of the lactoferrin test are available in 10 minutes, so there is no delay in reporting positives and the ongoing study at UCH shows that approximately 21% of patients with a positive toxigenic isolate but a negative direct faecal toxin test result have evidence of intestinal inflammation.

Symptoms may resolve without treatment or develop into serious infections that require immediate intervention - the human bowel is extremely sensitive to C. difficile toxins and the disease can quickly progress from inflammation to ulceration and perforation. Early antibiotic treatment is very effective in halting the progress of the disease and minimising the requirement for surgical intervention (colectomy). However the relapse rate is high, reported up to 30%7, and treatment may itself precipitate problems and contribute to the rate and severity of relapses. Managing a positive result therefore requires close co-operation between the laboratory and the physician and rapid, reliable results allow physicians to make informed clinical decisions and react quickly to provide the best level of care for the patient and the hospital.

Professor Wren calculates that the cost per test for the two-step approach is very little more than performing the toxin test alone. And with accurate reporting and a better clinical picture there is less repeat testing of negatives, which have been variously reported between 18% and 36%.

The financial benefits to the overall hospital budget are difficult to quantify but patient-related cost reductions include reduced antibiotic usage, fewer bowel investigations (colostomy, CT scan) and fewer surgical interventions (colectomy). Factoring in the implications of a C. difficile outbreak, it is clear that any improvement in the management of the disease introduces enormous savings.

Professor Wren concludes: “Stand-alone faecal toxin testing is missing up to one third of patients with proven serious disease and an additional 30% of patients with diarrhoea who are carrying toxigenic C.difficile. This has important clinical and infection control implications and there is clearly a need to re-assess the laboratory strategy for diagnosing diseased patients. The two-step approach utilises rapid, easy to use tests that cost very little more than current methods and introduces major benefits in terms of improved patient care and infection control.”