Clostridium difficile is so named because of its trouble to turn in the research lab. It is a Gram positive, rod organizing anaerobe ( Miao et al, 2010 ) which was foremost discovered in 1935 and has since been associated with doing clinical symptoms such as pseudomembranous inflammatory bowel disease and diarrhea due to antibiotics in worlds ( George et al. 1978, Johnson and Gerding, 1998 ) .Apart from this it is besides known to be the common cause of diarrhea acquired from infirmaries in patients ( Johnson et al. 2007 ) .
Since its find 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 normally found in infirmaries and in patients who are immunocompromised ( Warny et al. 1994, McFarland et al. 1990 ) including newborns and the aged ( Kyne et al. 1998 ) . The clinical marks 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 break of the intestine vegetations due to antibiotic intervention. In minority of instances, the cause could be due to factors like chemotherapy for malignant neoplastic disease or antacid intervention ( Delme e , 2001 ) . The two toxins can be related to instances which are symptomless, taking to symptoms of changing badness runing from mild diarrhea to reasonably terrible disease with abdominal hurting, watery diarrhea and systemic disturbance, through to life endangering diseases that could do decease ( Poutanen & A ; Simon, 2004 ) .Conditions such as reactive arthritis are associated to infection ( Cope et al. 1992 ) and infection is besides associated to other invasive infections ( Cookson & A ; Hoffam, 1995 ) .Toxic megacolon is one of the most serious complications which has absence of diarrhea and is caused by Clostridium difficile infection ( Borriello et al. 1998 ) .
A cell surface receptor is known to intercede the infective 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 largely appear to move synergistically ( Just et al. 1995 ) . A cytopathic consequence is therefore created by the mono-glucosylation ( Nottrott et al. 2007 ) . The toxins ‘ biological nature is the cause for vascular permeableness and bleeding ( Borriello et al. 1990 ) . Model surveies utilizing animate beings suggest that out of the two toxins merely toxin A has both the enterotoxic and histotoxic activity which is responsible for doing accretion of fluids and harm of tissues ( Davies & A ; Borriello, 1990 ) caused by an addition in cell bed permeableness ( Borriello et al. 1995 ) . Toxin B activities in human enteric heterografts of mice with terrible 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 entirely is responsible for pseudomembranous inflammatory bowel disease ( Shin et al. 2007 ) .
In respects to methods used in diagnosing of Clostridium difficile in a research lab, there are a batch of statements ( Barbut et al. 2003 ) .The diagnosing of Clostridium difficile associated diarrhea in the research lab is known to be based on cytotoxin or enterotoxin sensing in stool filtrate or by usage of stool civilization to show the being ( Bowman et al. 1988 ) . The usage of an antigen sensing kit particularly during an eruption in infirmaries which additions demand for the sensing of the two toxins has made it easier to name the Clostridium difficile toxin ( Kaczmarski et al. 2005 ) . Presentation of Clostridium difficile toxin in stool civilization is still a idea to be of import for its sensing ( Bartlett et al. 1980 ) . Surveies besides suggest that in a everyday research lab, diagnosing and toxin sensing of Clostridium difficile should be performed on every specimen including instances that are culture-positive and those that are fecal toxin-negative ( Delme I?e, 2001 ) .The gilded criterion Cell cytotoxicity check is used to name Clostridium difficile infections ( Pothoulakis et al. 1993 ) although it is rather slow and sometimes has low sensitiveness due to the toxins holding undergone debasement by peptidases ( Brazier, 1993 ) . Despite this little job that the method sometimes faces, it has many advantages of which the most of import is its ability to observe other toxins of Clostridium difficile ( Delme I?e, 2001 ) . There are some other checks that have been designed to observe 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 checks can non distinguish between toxins A and toxin B, they are used merely to corroborate the presence of Clostridium difficile toxin and so the distinction between toxin A and toxin B can be done by methods like Polymerase concatenation reaction ribotyping ( Cartwright et al. 1995 ) .
Previous surveies have introduced other trials which are indirect which include gas liquid chromatography ( GLC ) ( Johnson et al. 1989 ) , computed topography scan and latex agglutination ( Kelly et al. 1987 ) . These trials nevertheless, are non accepted on their ain unless used with other trials because they do non make a sufficient specificity and sensitiveness but ( Settle & A ; Wilcox, 1999 ) . Other Clostridium difficile research lab diagnosing checks besides include sensing of faecal lactoferin which is a marker for infection ( Steiner et al. 2007 ) and its presence helps in observing how terrible the infection caused by Clostridium difficile is ( Wilkins & A ; Lyerly, 2003 ) .
Hypervirulent strains are an increasing concern taking to several molecular typing methods being studied for the word picture of Clostridium difficile ( Cookson, 2007 ) some of which include serotyping, immunoblotting, primed PCR, pulsed-field gel cataphoresis 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 prejudiced, consistent and the simplest option to the remainder ( Cartwright et al. 1995 ) . mutant taking to taking to germinating of new emerging ribotypes which are recognised and which could take to increased virulency or strive antimicrobic opposition makes Clostridium difficile strain word picture an of import process ( Kuijper et al. 2006 ) .
The purpose of this survey was to show Clostridium difficile toxin A/B action utilizing the Xpect trial kit and Cytotoxin Assay and to execute the oxoid trial kit ‘s sensitiveness and specificity utilizing the Cytotoxin Assay as gilded criterion.
MATERIALS AND METHODS
Refer to the agenda.
In this survey 5 out of 9 samples gave positive consequences for the toxins A/B in the Oxoid trial kit and the staying 4 samples yielded negative consequences with the same. Based on the consequences obtained from the gilded criterion Cytotoxin Assay all the positive consequences were true positives nevertheless, out of the 4 negative consequences there were 3 false negatives and 1 was indecipherable. Out of the 3 false negatives 2 of them can be considered to be true negatives because they gave a cytotoxin Assay titer of 1/4 which is a really low value All the consequences are displayed in the tabular arraies below. The sensitiveness, specificity, positive predictive values and negative prognostic 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 )
( hypertext transfer protocol: //www.hpa-midas.org.uk )
Table 1: Consequences obtained to observe Clostridium difficile toxin from the two methods used
Clostridium difficile strains
Oxoid Test Kit consequences
Cytotoxin Assay Titre
Table 2: Valuess of sensitiveness, specificity, positive and negative predictive values
Positive prognostic value
Negative prognostic value
The diagnosing disease associated with Clostridium difficile continues to be a job even today. The bacterium and its toxin sensing significantly does non place an infection because toxigenic Clostridium difficile strains can be symptomless in some patients ( Barlett et al. 1980 ) . Recent surveies continue to generalize the clinical grounds of infection caused by Clostridium difficile ( Robinson & A ; White, 2009 ) therefore making a demand for a trial with high sensitiveness and specificity which in the research lab can reliably and quickly show the presence of Clostridium difficile toxin because the sensitiveness and specificity is what is used to characterize the public presentation of a trial ( Galen & A ; Gambino, 1975 ) .
The Oxoid toxin A/B trial used in this survey was easier and faster to utilize as compared to the Cytotoxin Assay and a really good sensitiveness consequence of 83.33 % was produced by it. This sensitiveness was within the sensitiveness public presentation claim of the maker which is of 79.8 % – 91.3 % , which is better than the confirmed other findings from the Health Protection Agency rating study where the Oxoid Xpect toxin A/B trial kit in relation to the gilded criterion cytotoxin assay showed a sensitiveness of 77.8 % and specificity of 98.8 % ( www.hpa.org.uk, 2009 ) . This survey produced a 100 % specificity which was high and which agrees to other findings which states that most commercial toxin A/B trial kit, describe a good specificity of 100 % ( Doern et al. 1992 ) . This is nevertheless in comparing to other surveies which report that the Oxoid toxin A/B trial does non shor a high adequate specificity and sensitiveness ( Settle & A ; Wilcox, 1999 ) . In this survey, reported low titers of 1/4 were considered to be negative consequences for the Cytotoxin Assay trial because of possible non-specific toxic effects which causes trouble in construing low titers ( Langley et al. 1995 ) . The false negative consequence obtained could perchance be due to the restriction of the trial which is inability to observe toxin below a certain noticeable degree. Besides the control wells showed same consequences as the trial samples and this could perchance be due to the variable extended incubation which was for 7 yearss. The broad scope of cytotoxic activity seen in the different titer can be associated to the written text degree of the toxin cistron ( Hammond et al. 2007 ) .
Taking into consideration the figure of samples used, from the consequences of this survey it can be concluded that although the Oxoid Xpect toxin A/B trial is non able to separate between toxin A and B it is a good medium and specific trial for observing the Clostridium difficile toxin A/B.