Clostridium difficile (C.diff) is a Gram positive rod, spore-forming anerobic bacterium. Carter, G.P., Rood, J.I., Lyras, D., (2010). The spores that can live in the environment for a long time. The prevalence of carrier state of the bacterium ranges from 0% to 3% in healthy individuals to 20% in hospitalized patients(McFarland LV, Mulligan ME, Kwok RY, Stamm WE. (1989)). The bacterium is also pathogenic in that it is associated with diarrheal diseases in multiple species including humans, dogs, horses, and pigs (Clooten JK, Kruth SR, Arroyo JG, Weese JS. (2008)). The normal bacterial flora in the bowel is an important natural defense and inhibits the growth of C.difficile (Borriello SP. ( 1990)). In addition, the gastric acid barrier is a host mechanism to protect against ingested microorganisms (Choudhry MN, Soran H, Ziglam HM. (2008)). Due to the increase in the frequency and severity of C. difficile infection (CDI) it is particularly concering in human community as C. difficile is unique among enteric pathogens increasingly important cause of nosocomial disease induced by treatment with antibiotics or by disruption of the normal gastrointestinal flora. However, even a short-term use of prophylactic antibiotics can cause CDI (Sunenshine RH, McDonald LC. (2006)). The bacteria are shed in faeces. Touching even the smallest amount of infected faeces, and then touching your mouth you can become infected with Clostridium difficile. The risk of cross-infection increases when patients have diarrhoea and bathrooms and toilets are shared.( Clostridium difficile. Red Book.; 2006: 261-263.)
Clinical significance
Clostridium difficile (C. difficile) infection (CDI) is the leading identifiable etiology for antibiotic-associated diarrhea and is associated with substantial morbidity and mortality. In addition, CDI is now the leading cause of infectious diarrhea in hospitals worldwide. (McDonald LC, Killgore GE, (2005)). and is associated with increasing duration of hospitalization and costs. (Navaneethan, U., Venkatesh, P.G.K., Shen, B., (2010)). Interestingly, only one third of all infected patients developed diarrhea, while the remaining two thirds were asymptomatic carriers. In another study, 76%-79% of patients acquired CDI from the community (Issa M, Vijayapal A, Graham MB, Beaulieu (2007)). Antibiotic resistant C. difficile spores survive in hospital environment and can be isolated on toilets, bedrails, floors, telephones, call buttons, stethoscopes, and the hands of healthcare workers. Sharing a room with an infected patient also increases the risk of infection (Warny M, Kelly CP. (2003)). Also Clostridium difficile infection (CDI) has been described in a variety of Liver diseased patients including those with acute and chronic (cirrhotic) conditions and those post liver transplantation (LT) (Bajaj JS, Ananthakrishnan AN, Hafeezullah M, et al. (2010).
New hypervirulent strain (REA group B1/PFGE type NAP1) produces higher levels of toxins A and B and is associated with fluoroquinolone resistance, more severe disease, and higher mortality.(Zaiss, N.H., Witte, W., Nubel, U., (2010)) More than 80 per cent of cases are in people over 65 years of age. However this traditional risk factors, such as recent hospitalization, being elderly, or having an underlying health condition were often absent in a CDC study (United States, Department of Health and Human Services, 2008). Close to 25% of patients who developed community acquired C. difficile colitis were young, healthy patients with no recent hospitalization in the past year. Some of these agents can increase the risk of acquisition of CDI. Cancer chemotherapy, particularly methotrexate(Anand A, Glatt AE. (1993)) or patients with organ transplantation on immunosuppression appear to be at risk.. Recurrent CDI is also suggested to be because of alterations in the fecal microbiota with markedly decreased diversity as demonstrated by phylogeneticanalysis of 16S rRNA-encoding gene sequences (Chang JY, Antonopoulos DA, 2008).
Pathogenicity (toxins)
Figure 1: Genetic organization of PaLoc and toxin structure. (A) Genetic organization of PaLoc from
C. difficile. XbaI sites (X) are shown, with the PaLoc coordinate indicated, and red ovals
show the gene-specific promoters and arrows the direction of transcription.
Lyras D, O'Connor JR, Howarth PK, Sambol SP, Carter GP, Phumoonna T, et al. (2009).
The major virulence factors of C. difficile are thought to be toxin A (an enterotoxin), and toxin B, a cytotoxin. These toxins are encoded by the tcdA and tcdB genes, respectively, which are located within a 19.6 kb pathogenicity locus, the PaLoc7 (Fig. 1). The genes encoding toxin A and B are encoded in the C. Difficile pathogenicity locus (tcdA and tcdB) which also encode two additional regulatory genes (tcdC and tcdD)[19]. The tcdD gene product up-regulates toxin transcription, while tcdC probably encodes a toxin gene repressor (Warny M, Kelly CP. 2003). The fifth gene of the pathogenicity locus, tcdE is postulated to release both toxins A and B into the colonic lumen by lysing the cell walls (Tan KS, Wee BY, Song KP. (2001).
Both toxins A and B have a 49% amino acid homology and possess a N-terminal domain that possesses cytotoxic activity, a transmembrane domain that facilitates toxin entry into the cytoplasm and a C-terminal domain that favors toxin binding to the epithelial cells Warny M, Kelly CP.(2003). , causing disruption of the actin cytoskeleton and impairment of tight junctions in human intestinal epithelial cells, with resulting fluid accumulation and extensive damage to the large intestine (Voth DE, Ballard JD. (2005).
Both toxins A and B are UDP-glucose hydrolases and glucosyltransferases and contribute to infectious and inflammatory diarrhea; however toxin B may be the major inflammatory toxin (Riegler M, Sedivy R, Pothoulakis C, (1995). The toxinsinitially attach to non-proteinaceous disaccharide Gal beta 1-4GlcNac residues in the colon. After adhesion, the toxin enters the cell through receptor-mediated endocytosis and catalyzes the transfer of a glucose residue from UDP-glucose to guanosine triphosphate-binding rho proteins (Warny M, Kelly CP.(2003), the intracellular signaling molecules control numerous cellular functions, including the organization of the actin cytoskeleton. Rho family GTPases Glucosylation of Rho GTPases by toxins A and B locks these proteins into an inactive conformation, thereby blocking all down-stream signaling pathways in the cell. The result is disruption of the actin cytoskeleton, cell rounding and eventually apoptosis and death of the intoxicated cell. Jank T, Aktories K. (2008). This leads to the inflammatory diarrhea seen in patients with CDI (Navaneethan U, Giannella RA. (2008). More recently, the toxins were shown to cause the release of inflammatory cytokines from mast cells and macrophages as well as from epithelial cells, leading to further fluid secretion and intestinal inflammation. Interestingly, in addition to damaging the gut, toxin B is also cardiotoxic to zebrafish embryos (Hamm EE, Voth DE, Ballard JD. (2006). and can cause hemorrhage in the lungs of hamsters (Lyerly DM, Saum KE, MacDonald DK, D WT. (1985), suggesting that this toxin may have more systemic effects than previously thought.
Laboratory diagnosis
Oxoid Xpect® Toxin A/B detection kit:
Principle of the test: The Xpect Clostridium difficile Toxin A/B test is a qualitative immunochromatographic assay that detects C. difficile Toxin A and Toxin B in stool specimens or cultures of toxigenic C. difficile. A volume of this mixture is transferred to a test device having immobilized streptavidin as a test line and goat anti-immunoglobulin antibody as a control line. Immunocomplexes of toxin and conjugated antibodies form a visible band as they flow across the test line. Excess colored particle conjugates form a visible band at the control line to document that the test is functioning properly.
Cytotoxin Assay:
Cytotoxin assay: The cell culture technique of 96 wells but only 8 of control and 8 treated samples.
Cytotoxin detection is often considered as the standard for the diagnosis of C. difficile infections. This method consists of inoculating a filtrate of a stool suspension into a cell culture and observing a cytopathic effect as a consequence of disruption of the cell cytoskeleton; which results in cell rounding in many cell lines. The effect is mainly due to toxin B, which is 1000 times more cytotoxic than toxin A.
A suspension of 1 : 5 of the fecal sample is made in phosphatebuffered saline and, after centrifugation, the supernatant is filtered through a 0.2-mM filter. The filtrate is then inoculated on the cell monolayer and the presence or absence of the cytotoxic effect is observed after 24 and 48 h. In some of the most severe clinical cases, typical rounding may be observed after 4-6 h. Confirmation of the specificity is obtained by repeating the test with the addition of a specific antiserum directed against C. difficile or against C. sordellii, which shares the same antigens.
Tissue culture
Stools are directly inoculated and incubated in an anaerobic atmosphere for 48 h. Anaerobic preincubation of the plate may improve the recovery rate as well [11]. In our experience, the use of an Anoxomat system (Mart Microbiology NV, Lichtenvoorde, Netherlands), which allows an anaerobic atmosphere to be obtained in the jar within 1 min, allows the plates to be read after only a 24 h incubation. Several modifications have been proposed to enhance the sensitivity of the culture. Most are aimed at recovering more spores and are more dedicated to epidemiological studies and environmental cultures. Pretreatment of stools with ethanol-shock (equal volumes of ethanol and feces mixed for 1 h before inoculation) has also been shown to increase the sensitivity of culture [13]. Culture is the most sensitive method but it is not very specific due to the possibility of isolating non-toxigenic isolates. It is slow but allows strains to be tested for toxigenicity; it is also the only way to carry out epidemiological investigations.
Colonies of C. difficile are easily recognised on culture plates due to their typical morphology (ground glass appearance) when observed with binoculars. A yellow-green or chartreuse fluorescence under ultraviolet illumination is another characteristic of the colonies but this may vary with the medium used. The typical odor (horse manure) is also an aid to identification. GLC of an agar plug around a suspected colony is the best and easiest method for confirming identification.
ELISA
Most use monoclonal antitoxin A antibodies, whereas a few are designed to detect both toxins. Finally, in one of the latest kits, the detection of toxin A is coupled with the detection of a glutamate dehydrogenase, a C. difficile-specific enzyme found in toxigenic as well as in non-toxigenic isolates. The reason why kits detecting both toxins have been developed is mainly because some isolates from clinical cases have been shown to produce only toxin B [15].
Molecular methods
Several methods based on the PCR for amplifying part of the toxin A gene have been effective in distinguishing toxigenic from non-toxigenic isolates, but these approaches seem to have few advantages over the routine laboratory methods. Direct detection of C. difficile genes in fecal specimens have been tested as well. Oligonucleotide probes designed to detect toxin B have been used by Green et al. [28] with sensitivity and specificity in the same range as EIAs. PCR using sets of primers designed to detect toxin A or B have been tested on stools by several authors [29-32]. The procedures usually comprise a preliminary step to avoid inhibitory substances. So far, they have been tested in small series of specimens and the results have not shown any significant improvement when compared with the classic methods.
Glutamate dehydrogenase (GDH)
Cell surface associated enzyme
Found in many bacterial species
EIA assay specific for C. difficile GDH
Real time PCR
Detection of toxin B gene
Doesn't indicate toxin production
Alternative assays available to detect other toxin genes
MATERIALS AND METHODS
As stated in protocol or handout.
RESULTS
Table 1 displays the results from the experiment.
Clostridium difficile strains
Assays results
Oxoid Xpect Toxin A/B detection Kit
Cytotoxin culture assay (Titres)
B
Negative¶
Positive (1/32)*
X
Negative¶
Positive (1/4)
Y
Negative¶
Positive (1/4)
Z
Negative§
Not readable
7224
Positive
Positive (1/64) ‡
7225
Positive
Positive (1/256) ‡
38531
Positive
Positive (1/256) ‡
38666
Positive
Positive (1/256, 1/128) ‡
49302
Positive
Positive (1/256) ‡
Table 1: The results obtained from the practical; ‡True positive *False positive; ¶True negative §False negative
Table 1 show that some wells to display the cytotoxic effect, in other words determining whether the toxics had an effect. The determination was not simple but the table shows the results. Certain strains displayed varying results. In table 2, the sensitivity and specificity of the Oxoid Xpect® kit compared to the gold standard cytotoxin assay results are shown. The value shown in the parentheses are the determined dilution factor with minimal cytotoxic effect. Table 2 compares the sensitivity and specificity obtained from the lab with those obtained by Eastward et. al., 2009 who done comparison of nine commercially available Clostridium difficile toxin detection assays, to cytotoxin testing and cytotoxigenic culture methods but only Oxoid Xpect is shown.
Sensitivity and specificity
Taken the tissue culture assay as the gold standard for comparison of the kit; we were therefore able to determine the sensitivity and specificity Oxoid Xpect® kit. The calculation for this involves analysing the results and distinguishing between false negatives and false positives.
There is an important point about ensuring accurate diagnosis for Clostridium difficle associated-diseases. By calculating the specificity and sensitivity put confidence in the kit. The usefulness of a serological test for diagnosic purposes depends on the test' specificity and sensitivity. Specificity is the ability of an antibody preparation to recognise a single antigen. Optimal specificity implies that the antibody is specific for a single antigen, will not cross-react with any other antigen, and therefore will not provide a false-positive result. Specificity must be defined in terms of reactions with positive and negative control antigens. Specificity for each test must be determined experimentally and verified every time the test is used.
Sensitivity defines the lowest amount of an antigen that can be detected. The highest lev of sensitivity requires that the antibody in a test be capable of identifying a single antigen molecule. High sensitivity prevents false negative reactions. The sensitivity of some common tests in terms of the amount of antibody necessary to detect antigen is shown in table 32.8. The amount of antigen detected by each test system is proportional to the amount ofantibdoy used. The antibody necessary to detect antigen
Using the formulae
True negative True positive
Specificity = ------------------------------------- Sensitivity = -------------------------------------
True negative + false positive True positive + false negative
Sensitivity
Specificity
83.3%
73.1%
Table 2: specificity and sensitivty
Sensitivity and specificity of toxin detection assays. Statistical analysis showed that the Premier toxin A_B, Vidas C.difficile Tox A/B, Techlab toxin A/B II, and Remel ProSpecT assays were more sensitive (P _ _0.05) than the other five toxin detection assays (Tables 2 and 3; statistical data not shown). The Ridascreen toxin A/B assay was the least sensitive
assay compared against either gold standard method, while the GA Clostridium difficile antigen assay was the least specific (Tables 2 and 3). The two lateral-flow assays demonstrated better specificity than any of the EIAs (Tables 2 and 3), although the differences were not significant (statistical data not shown). The cytotoxin assay had higher sensitivity and specificity values than any of the commercial toxin detection assays, except the Remel Xpect, when compared with CYTGC (Tables 2 and 3).
Positive and negative predictive values
Knowing the prevalence rate of the diseases, you can predict positive values and predict the negativeprediced values for both test kit and culture. Importantly, the PPV of any test is affected by the prevalence of the disease in the population (i.e., by the test positivity rate).
In the hospital setting, with an expected prevalence of 10%, the mean PPV of a commercial toxin detection kit was 68.7% (range, 48.6 to 86.8%), which is comparable to the findings of Planche et al.(11). In the community setting, the prevalence is nearer 2% (19); as such, the mean PPV falls to 32.3% (range, 14.8 to 56.3%), making testing for C. difficile using current methods,especially single tests, extremely unreliable.
Vs Cytotoxigenic culture
PPV
NPV
Prevalence:
2%
10%
2%
10%
Remel Xpect
69.0
92.4
99.4
96.6
DISCUSSION
Implications of false positive CDI diagnosis
Inappropriate antibiotic cessation / modification
Inappropriate treatment for CDI
Unnecessary isolation
Potentially harmful cohorting
Inaccurate surveillance / infection control data
Wasted resources
Reimbursement / fines
Medicolegal implications
Which assay is the best
Depends on your population
Cytotoxin gives best PPV for toxin detection assays
But is labour intensive and slow
Lateral flow toxin detection assays have good PPV and are rapid
But have poorer NPV
GDH gives best PPV overall
But is only detecting presence of C. difficile, not active disease
PCR has highest NPV, good screening test
But only detecting presence of toxin gene
Test results should be taken in context with the clinical presentation of the patient
Advice from the Department of Health:
The currently available kits for detection of C. difficile toxins have variable performance
Currently available kits may miss about 1 in 5 to 1 in 10 cases of CDI and will falsely identify (1-2 out of 10) cases as positive when they are not
The poor positive predictive values of toxin detection kits, especially in the context of widespread testing, and the possibility of missing true positives mean that there are limitations to using these as single tests for the laboratory diagnosis of CDI
However, because it is only showing th results of one asay it will be better o if it ha used more results like that used by. Like by another practial done by Eastwod et.al., 2009. See table 2.
