Clostridium difficile is so named because of its difficulty to grow in the laboratory. It is a Gram positive, rod forming anaerobe (Miao et al, 2010) which was first discovered in 1935 and has since been associated with causing clinical symptoms such as pseudomembranous colitis and diarrhoea due to antibiotics in humans (George et al. 1978, Johnson and Gerding, 1998).Apart from this it is also known to be the common cause of diarrhoea acquired from hospitals in patients (Johnson et al. 2007).
Since its discovery it has been widely recognised as a nosocomial pathogen (Johnson et al. 1999) which has caught microbiologists by surprise (Rilley, 1995). Clostridium difficile infections are most commonly found in hospitals and in patients who are immunocompromised (Warny et al. 1994, McFarland et al. 1990) including neonates and the elderly (Kyne et al. 1998). The clinical signs and symptoms of infection are caused by the production of two protein toxins with high molecular weights; toxin A an enterotoxin of molecular weight 300kDa and toxin B a cytotoxin of a molecular weight of 270Kda (Lyerly et al. 1988) and are manifested by disruption of the gut flora due to antibiotic treatment. In minority of cases, the cause could be due to factors like chemotherapy for cancer or antacid treatment (Delme eÍ, 2001). The two toxins can be related to cases which are asymptomatic, leading to symptoms of varying severity ranging from mild diarrhoea to moderately severe disease with abdominal pain, watery diarrhoea and systemic upset, through to life threatening diseases that could cause death (Poutanen & Simon, 2004).Conditions such as reactive arthritis are associated to infection (Cope et al. 1992) and infection is also associated to other invasive infections (Cookson & Hoffam, 1995).Toxic megacolon is one of the most serious complications which has absence of diarrhoea and is caused by Clostridium difficile infection (Borriello et al. 1998).
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A cell surface receptor is known to mediate the pathogenic activity of the two toxins (Na et al., 2005).The two toxins can mono-glucosylate Rho GTPase including Rho A, B, C, Cdc42 and Rac 1 enzymatically and mostly appear to act synergistically (Just et al. 1995). A cytopathic effect is thus created by the mono-glucosylation (Nottrott et al. 2007). The toxins' biological nature is the cause for vascular permeability and haemorrhage (Borriello et al. 1990). Model studies using animals suggest that out of the two toxins only toxin A has both the enterotoxic and histotoxic activity which is responsible for causing accumulation of fluids and damage of tissues (Davies & Borriello, 1990) caused by an increase in cell layer permeability (Borriello et al. 1995). Toxin B activities in human intestinal xenografts of mice with severe combined immunodeficiency as have been described as being enterotoxic and proinflammatory (Sadvidge et al. 2003) . Furthermore in some patients the presence of Clostridium difficile toxin B alone is responsible for pseudomembranous colitis (Shin et al. 2007).
In regards to methods used in diagnosis of Clostridium difficile in a laboratory, there are a lot of arguments (Barbut et al. 2003).The diagnosis of Clostridium difficile associated diarrhoea in the laboratory is known to be based on cytotoxin or enterotoxin detection in stool filtrate or by use of stool culture to demonstrate the organism (Bowman et al. 1988). The use of an antigen detection kit especially during an outbreak in hospitals which increases demand for the detection of the two toxins has made it easier to diagnose the Clostridium difficile toxin (Kaczmarski et al. 2005). Demonstration of Clostridium difficile toxin in stool culture is still a thought to be important for its detection (Bartlett et al. 1980). Studies also suggest that in a routine laboratory, diagnosis and toxin detection of Clostridium difficile should be performed on every specimen including cases that are culture-positive and those that are faecal toxin-negative (Delme Íe, 2001).The gold standard Cell cytotoxicity assay is used to diagnose Clostridium difficile infections (Pothoulakis et al. 1993) although it is quite slow and sometimes has low sensitivity due to the toxins having undergone degradation by proteases (Brazier, 1993). Despite this small problem that the method sometimes faces, it has many advantages of which the most important is its ability to detect other toxins of Clostridium difficile (Delme Íe, 2001). There are some other assays that have been designed to detect a common antigen of Clostridium difficile produced by both toxigenic and non toxigenic strains, which is glutamate dehydrogenase (Poulter et al. 2003). Despite the fact that these assays cannot differentiate between toxins A and toxin B, they are used just to confirm the presence of Clostridium difficile toxin and then the differentiation between toxin A and toxin B can be done by methods like Polymerase chain reaction ribotyping (Cartwright et al. 1995).
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Previous studies have introduced other tests which are indirect which include gas liquid chromatography (GLC) (Johnson et al. 1989), computed topography scan and latex agglutination (Kelly et al. 1987). These tests however, are not accepted on their own unless used with other tests because they do not reach a sufficient specificity and sensitivity but (Settle & Wilcox, 1999). Other Clostridium difficile laboratory diagnosis assays also include detection of fecal lactoferin which is a marker for infection (Steiner et al. 2007) and its presence helps in detecting how severe the infection caused by Clostridium difficile is (Wilkins & Lyerly, 2003).
Hypervirulent strains are an increasing concern leading to several molecular typing methods being studied for the characterisation of Clostridium difficile (Cookson, 2007) some of which include serotyping, immunoblotting, primed PCR, pulsed-field gel electrophoresis and PCR ribotyping (Toma et al. 1988, Heard et al. 1997, Barbut et al. 1993, Hyett et al. 1997, Gurtler, 1993) among which PCR ribotyping has shown to be the most discriminatory, reproducible and the simplest alternative to the rest (Cartwright et al. 1995). mutation leading to leading to evolving of new emerging ribotypes which are recognised and which could lead to increased virulence or strain antimicrobial resistance makes Clostridium difficile strain characterisation an important procedure (Kuijper et al. 2006).
The aim of this study was to demonstrate Clostridium difficile toxin A/B action using the Xpect test kit and Cytotoxin Assay and to perform the oxoid test kit's sensitivity and specificity using the Cytotoxin Assay as gold standard.
MATERIALS AND METHODS
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In this study 5 out of 9 samples gave positive results for the toxins A/B in the Oxoid test kit and the remaining 4 samples yielded negative results with the same. Based on the results obtained from the gold standard Cytotoxin Assay all the positive results were true positives however, out of the 4 negative results there were 3 false negatives and 1 was unreadable. Out of the 3 false negatives 2 of them can be considered to be true negatives because they gave a cytotoxin Assay titre of 1/4 which is a very low value All the results are displayed in the tables below. The sensitivity, specificity, positive predictive values and negative predictive values were calculated based on 1 false negative and 2 true negative values and are shown in table 2.
Sensitivity is: True positives/(True positives + False negatives)
Specificity is: True negatives/(False positives + True negatives)
Positive Predictive Value is: True positives/(True positives + False positives)
Negative Predictive Value is: True negatives/(False negatives + True negatives)
Table 1: Results obtained to detect Clostridium difficile toxin from the two methods used
Clostridium difficile strains
Oxoid Test Kit results
Cytotoxin Assay Titre
Table 2: Values of sensitivity, specificity, positive and negative predictive values
Positive predictive value
Negative predictive value
The diagnosis disease associated with Clostridium difficile continues to be a problem even today. The bacteria and its toxin detection significantly does not identify an infection because toxigenic Clostridium difficile strains can be asymptomatic in some patients (Barlett et al. 1980). Recent studies continue to generalise the clinical evidence of infection caused by Clostridium difficile (Robinson & White, 2009) thus creating a need for a test with high sensitivity and specificity which in the laboratory can reliably and rapidly demonstrate the presence of Clostridium difficile toxin because the sensitivity and specificity is what is used to characterise the performance of a test (Galen & Gambino, 1975).
The Oxoid toxin A/B test used in this study was easier and faster to use as compared to the Cytotoxin Assay and a very good sensitivity result of 83.33% was produced by it. This sensitivity was within the sensitivity performance claim of the manufacturer which is of 79.8% - 91.3%, which is better than the confirmed other findings from the Health Protection Agency evaluation report where the Oxoid Xpect toxin A/B test kit in relation to the gold standard cytotoxin assay showed a sensitivity of 77.8% and specificity of 98.8% (www.hpa.org.uk, 2009). This study produced a 100% specificity which was high and which agrees to other findings which states that most commercial toxin A/B test kit, report a good specificity of 100% (Doern et al. 1992). This is however in comparison to other studies which report that the Oxoid toxin A/B test does not shor a high enough specificity and sensitivity (Settle & Wilcox, 1999). In this study, reported low titres of 1/4 were considered to be negative results for the Cytotoxin Assay test because of possible non-specific toxic effects which causes difficulty in interpreting low titres (Langley et al. 1995). The false negative result obtained could possibly be due to the limitation of the test which is inability to detect toxin below a certain detectable level. Also the control wells showed same results as the test samples and this could possibly be due to the variable extended incubation which was for 7 days. The wide range of cytotoxic activity seen in the different titre can be associated to the transcription level of the toxin gene (Hammond et al. 2007).
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Taking into consideration the number of samples used, from the results of this study it can be concluded that although the Oxoid Xpect toxin A/B test is not able to distinguish between toxin A and B it is a good sensitive and specific test for detecting the Clostridium difficile toxin A/B.