Diagnosis of Clostridium Difficile - Essay Example

Diagnosis of Clostridium difficile al affiliation Over the last decade, Clostridium difficile is an emerging threat topublic health worldwide and most common cause of infections acquired in hospitals in Europe. Its incidence is exponentially increasing, and new C. difficile populations are emerging such as in children, pregnant women and community –acquired infection with no exposure to antibiotics prior (Ananthakrishnan, 2011). According to the Office for National Statistics (2012), in England and Wales, there was a drop to 1% of all hospital deaths involving C. difficile during 2009 to 2011 compared to 2.2% during 2006 to 2008. C. difficile was involved in 0.6% of all deaths in England and Wales during 2009 to 2011. The mortality rate is higher for people aged 75 years and above. C. difficile is an anaerobic Gram positive spore bearing bacillus about 3 to 5 micrometres in length with a variable morphology depending on the media. In agar cultures that have reached a stationary phase, sporulation is noticeable. C. difficile forms spores and is the main reason why the organism is a problem in medical facilities. Transmission is through the faecal-oral route through contaminated environment and hands of the health care personnel (Aktories & Wilkins, 2000). Reduction of hospital acquired infections could be done by enhancing and deep cleaning programmes are interventions (Hughes, et al., 2013). C. difficile is more predominant in hospital that community settings and depends on the type of patients, the number and type of antibiotics being used. The risk is also dependent on the acquisition of a toxigenic strain and host factors such as age and immune status. Patients who are elderly and those with weakened immunity are at a higher risk of developing C. difficile infection. The development of the infection can be caused by other factors that include non-surgical gastrointestinal procedures, underlying comorbidity, the use of acid-suppressing medicines and admission to the intensive care unit (Ananthakrishnan, 2011). C. difficile is harmless since it is present in a high number of infants and adheres to the colonic mucosa in a normal environment, but may cause human morbidity and mortality due to human beings’ intervention. Infection by C. difficile is facilitated by compromise of resistance to colonization that is maintained by the normal gut flora by antimicrobial agents and is the most significant factor allowing an overgrowth. Cytotoxic chemotherapeutic agents may lead to infection with C.difficile it the absence of exposure to antibiotics since the agents cause mitotic arrest of colonic epithelial cells which results in mucous membranes’ necrosis, irritation and desquamation. Symptoms of C. difficile include diarrhoea, abdominal pain, fatigue, nausea, fever, malaise and loss of appetite and serious colitis which may lead to toxic mega colon which is a severe complication that leads to bowel perforation, and sepsis. The reduction of antibiotics usage may reduce the number of infections. It is clear that this strain is influenced by hospital hygiene and infection prevention and control practices, which are the levels of environmental contamination with the organism and spores. This is also affected by the organism’s intrinsic properties such as colonisation and virulence factors that enhance its ability to transfer and persist in the environment (Coia, 2009). In the diagnosis of C.difficile laboratory investigations are performed on freshly taken faecal specimen from patients with diarrhoea which should be submitted immediately to the laboratory and is done on the based on findings of symptoms throughout or subsequent treatment with antibiotics. Diagnosis is confirmed based on the identification of C. difficile toxin in stool or the recovery of a toxin producing strain of C. difficile from stool. Direct culture is a diagnostic test that has been used in investigating outbreaks as isolates are genotyped and for assessment of the antimicrobial susceptibility of C. difficile strains. It is not used routinely in most hospitals since it takes more time. This method is sensitive but nonspecific that is identification of both toxigenic and non-toxigenic strains of C. difficile. The stool sample is cultured in selective medium with requirements of other tests to confirm that C.difficile is toxigenic. In the past, cell culture cytotoxicity neutralization assay (CCCN) was the gold standard for detection of C. difficile in the laboratory. It detects cytotoxin production in monolayers of cells such as human diploid fibroblasts. CCCN was labor intensive, and therefore, other diagnostic methods had to be adopted. Diagnosis of C. difficile is controversial due to many laboratory methods compounded by two reference method; the cytotoxicity assay and the cytotoxigenic culture. The cytotoxigenic culture that detects toxigenic C. difficile and gives a more frequent positive result due to its sensitivity and cytotoxin assay that detects preformed toxin in faeces. A positive cytotoxigenic culture indicates that a patient could be infectious even though the diarrhoea might have resulted from another cause whereas a positive cell cytotoxin assay indicate that the diarrhoea was probably caused by C.difficile infection (Planche, et al., 2013). A cytotoxigenic culture is slow and laborious. Toxin B is easily detected in cytotoxic assay since it is more toxic than toxin A. Cytotoxin B assay is performed directly on faeces and has a high sensitivity. Sensitivity can increased by combining the cytotoxin B with toxigenic culture. Cytotoxin B assay has an increased turnaround time and requires tissue culture facilities. Cell toxicity assay which can detect pictogram levels of toxin is the gold standard for C. difficile infection and has excellent sensitivity and specificity compared to cell culture cytotoxicity assay. The main disadvantage is that it takes a longer time usually 48 to 72 hours making it not clinically useful and is expensive because it requires substantial laboratory resources. False negative results can be avoided by refrigerating the samples before testing. Repeat stools may be negative in some patients with pseudomembranous colitis due to C.difficile infection (Aldeen, et al., 2000). Bacterial toxicity assays generally measure increases in the cell membrane permeability as an index of cellular injury. Uptake of trypan blue from extracellular media into injured cells is a standard histologic method with good sensitivity but low sensitivity. Trypan blue is also difficult to apply to large numbers of samples. There are three types of histopathological lesions of pseudomembranous colitis associated with C.difficile. Type 1 summit lesions are evident as a principal area of epithelial necrosis and an exudate that consists of polymorphonuclear cells, nuclear dust and fibrin. In the Type 2 lesions, the major feature comprises of a well-defined group of disrupted glands, distended by mucin and poly-morphonuclear cells. Half of the superficial epithelial lining is usually lost and surrounded by a cloud of epithelial debris, mucus, fibrin and polymorphonuclear cells. The third type of lesions has a complete structural necrosis of the mucosa with a thick covering of fibrin, mucus and inflammatory debris. Glutamate dehydrogenase assay (GDH) can be paired to cytotoxic assay or toxin EIA, in a two-step protocol to detect the presence of C. difficile. The specimen that are GDH positive are tested further with a cytotoxic assay. GDH assays detect the antigen present in both toxigenic and non-toxigenic strains of C. difficile directly in stool samples. Stools that are usually GDH negative are considered C. difficile negative. This approach decreases the total cost and increases the diagnostic accuracy of the sensitivity of toxic assays. (Shetty, et al., 2010) carried out a meta-analysis on the glutamate dehydrogenase role in diagnosis where it confirmed high diagnostic accuracy for GDH for the presence of C. difficile in faeces it attained high sensitivity compared with culture. However, the method lacks specificity in screening for C. difficile in stool samples (Novak-Weekley, et al., 2010). The use of enzyme immunoassay (EIA) which tests for both toxin A and B enables direct testing. These tests are rapid, easy to perform because they do not require specific technical skills and have a high specificity. This test is based on monoclonal antibodies for the detection of C. difficile toxins. Immunoassay can be combined with C. difficile common-antigen detection. Other advantages include ease of performance and cost. Nevertheless, this test cannot be used as a stand-alone test for diagnosis because it is not sensitive enough. Testing of multiple stool specimens can increase the sensitivity and the negative predictive value of these tests (Turgeon, et al., 2003). Enzyme linked immunoabsorbent assays (ELISA) are used in many hospitals and rely on the use of monoclonal and polyclonal antibodies against both toxins A and B. High molecular weight protein toxins, toxin A (tcdA) which was discovered in the late 1970s and toxin B (tcdB), discovered in 1980, are produced in the pathogenesis of C. difficile diarrhoea. Toxin A is a potent enterotoxin but also has cytotoxic properties leading to fluid accumulation and tissue damage whereas toxin B has no effects on the intestine but is a potent cytotoxin but plays a major role in pathogenesis of C. difficile. Both toxins increase vascular permeability and cellular inflammation in the human intestine and are lethal causing paralysis and death when injected into animals. Recently, a more virulent toxin that consists of toxic genes tcdA, tcdB, and the binary toxin genes and 18 base pair deletion in tcdC has been identified (McDonald, et al., 2005). ELISA is preferred since it is quick, simple to perform and has high specificity. However, it has a lower sensitivity and specificity than culture cytoxicity test. Colonoscopy should be performed if the index of suspicion is high, and is more useful because the infection may not affect the pseudomembranes which may be proximal to the sigmoid colon and the rectum. The appearance of pseudo membranes at sigmoidoscopy is highly suggestive of C.difficile infection in case of negative stool tests. Endoscopy demonstrating pseudo membranes and mucosal injury can find out the C. difficile-induced colitis diagnosis accurately and fast especially in patients with a more severe case. Pseudo membranes are seen as numerous discrete yellow-white plaques of exudate that are raised and are up to 2cm in diameter normal or mildly hyperaemic mucosa that separates the plaques. The erythematous mucosa can be revealed by removal of the lesions during endoscopy. The pseudo membranes may join to form a confluent exudative membrane covering a large part of the colon in more advanced cases. Severe untreated cases may progress to broad ulceration of the mucosal surface in rare cases. Pseudomembranous colitis may not be seen in inflammatory bowel disease patients with C. difficile infection. Fluorescent antibody assays utilize the strong reaction between human antiserum prepared against a viable isolate of C. difficile. The results are the same as those of cellular cytotoxicity assay and culture. However, the use of fluorescent antibodies is not effective because of lack of antibody specificity. Some laboratories use counterimmunoelectrophoresis in detection of C. difficile. Nonetheless, when compared with the cellular cytotoxicity assay, there is a high rate of false negative results. This is dependent on the purity of the antigen, specificity of the antibody and the insufficient level of the cytotoxin in clinical samples. Radiographic findings are often non-diagnostic and non-specific. The organism produces a number of antigens that cross react with antigens from other clostridia and anaerobes. The common antigen used is an enzyme produced by C.difficile called glutamate dehydrogeenase. Nucleic acid amplification tests for C. difficile is superior, therefore, is strongly recommended. It is easily detectable through EIA. Testing of glutamate dehydrogenase identifies accurately the presence of C.difficile. Nevertheless, it cannot differentiate between toxigenic and non-toxigenic strains. Latex agglutination method such as Culturette CDT Kit which identifies toxin A demonstrates toxin of the enzyme. A rapid latex agglutination test was developed in the 1980s was found to react with all non-cytotoxic and cytotoxic strains of C.difficile and gave immunologic cross-reactions with Clostridium sporogenes, proteolytic clostridium botulinum and P. anaerobius. The protein that reacts in commercial latex tests is glutamate dehydrogenase. Currently, latex tests have poor sensitivity therefore, requires a confirmatory test and not suitable for diagnosis. It is fast, cheap and easy to perform. This test would be useful in rapid diagnosis of C. difficile especially in laboratories that lack cell culture facilities. The accuracy of latex agglutination test can be improved by combining with culture and cytotoxin assay. Loop mediated tests detect genes residing in the pathogenicity locus of the C. difficile genome such as the toxin A gene (tcdA), toxin B gene (tcdB) and the toxin regulatory gene (tcdC). It has a high sensitivity similar to PCR and allows identification of the C. difficile strain which carries both prognostic and implications to the public health. However, it requires turbidimeter or UV lamp and fluorescent probe. The loop mediated isothermal amplification (LAMP) technology targets the 5’ portion of the toxin A gene. Illuminigene C. difficile is based on the original loop-mediated isothermal amplification (LAMP) offers sensitivity and specificity detection of strains that are toxigenic compared to those of PCR-based methods and toxigenic culture reference methods. The overall turnaround time is one hour. The combination of a high performance and quick turnaround time might result in better management of C. difficile infection and implementation of infection control measures on time (Lalande, et al., 2011). Microarrays technology provides a high throughput testing of culture conditions. In the pathogenesis of C. difficile, toxins are produced. Culture conditions include different carbon, nitrogen, phosphorous and sulfur sources. This technique can be combined with cytotoxicity assay. The microarray approach can be used to identify C. difficile genes. The Verigene test is a multiplex qualitative assay that uses PCR- amplified DNA in a nanoparticle- based microarray to detect c. difficile toxin A and B in fecal specimen and the virulent tcdC strain. There is a possibility of false positives and negatives due to failure to image the array slide or from internal control failures. The test has a higher sensitivity and accuracy compared to direct culture. The specificity is lower in comparison to the nucleic acid amplification tests (Caroll, et al., 2013). For effective patient management and prevention of nosocomial transmission, it is essential to have quick and accurate diagnosis of C.difficile. However, most conventional tests are not sufficient for performance and cannot be used as stand-alone tests. The study by Knetsch, et al., (2011) to compare the diagnostic values of three in house developed real time PCRs and BD GeneOhm test which is a commercially available assay using the appropriate gold standard on 526 prospectively collected stool samples, found out that the in house developed PCRs had better sensitivity compared to BD GeneOhm test in contrast to the specificity. The sensitivity of detection of toxin, turnaround shortening and reducing laboratory resources is the main target of newer testing methods. Currently, commercial assays use toxin B gene (tcdB) to determine the presence of toxigenic C. difficile in the stool specimen. In future, diagnosis of C. difficile will be done using RT-PCR amplification of genes specific to C. difficile. This method will be more sensitive, faster turn-around time and specificity equal to the cell culture cytotoxin assays. PCR is preferred compared to immunoassays because the immunoassays are less specific when testing isolates, which causes over-diagnosis of toxin-mediated C. difficile disease (Welch, 2006). However, it will be expensive and availability will be limited. The RT-PCR method will be combined with screening GDH assay to limit cost. RT-PC method will enable facilitation of early and appropriate patient management because it will provide an early confirmation of C.difficile (Plevris & Howden, 2012). PCR would be considered the method of choice for the diagnosis of C. difficile founded on the review of current literature. The method does not require confirmatory tests unlike the cytotoxigenic culture, cytotoxin assays, immunoassay, and loop mediated tests. Compared to these tests, PCR is highly specific, sensitive and rapid. This would be helpful in developing interventions that would be effective in the management of C. difficile. Bibliography Aldeen, W. E. et al., 2000. Comparison of the TOX A/B test to a cell culture cytotoxicity assay for the detection of Clostridium difficile in stools. Diagnosis for Microbial Infectious Diseases, 36(4), pp. 211-213. Ananthakrishnan, A. N., 2011. Clostridium difficile infection: epidemiology, risk factors and management. Nature reviews Gastroenterology & Hepatology, Volume 8, pp. 17-26. Caroll, K. C. et al., 2013. Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay. Journal of Clinical Microbiology, 51(12), pp. 4120-4125. Hughes, G. J. et al., 2013. Impact of cleaning and other interventions for the reduction of hospita;-acquired Clostridium difficile infections in two hospitals in England assessed using breakpoint model. Journal of Hospital Infections, 84(3), pp. 227-34. Lalande, V. et al., 2011. Evaluation of a Loop-Mediated Isothermal Amplification Assay for Diagnosis of Clostridium difficile Infections. Journal of Clinical Microbiology, 49(7), pp. 2714-2716. Novak-Weekley, S. M. et al., 2010. Clostridium difficile testing in the Clinical Laboratory by Use of Multiple Testing Algorithms. Journal of Clinical Microbiology, 48(3), pp. 889-893. Office for National Statistics, 2012. Deaths Involving Clostridium Difficile: England and Wales. Statistical Bulletin, pp. 2-18. Shetty, N., Wren, M. W. & Coen, P. G., 2010. The role of glutamate dehydrogenase for the detection of Clostridium difficile in the faecal sample. Journal of Hospital Infection, 77(1), pp. 1-6. Turgeon, D. K., Novicki, T. j. & Fritsche, T. R., 2003. Six Rapid Tests for Direct Detection of Clostridium difficile and its Toxins in Fecal Samples Compared with the Fibroblast Cytotoxicity. Journal of Clinical Microbiology, 41(2), pp. 667-670. Welch, D., 2006. Laboratory testing considerations for C. difficile disease. Baylor University Medical Center Proceedings, 19(1), pp. 14-15. Read More