Dealing with Clostridium difficile in Clinical Laboratories
While antibiotics eliminate disease-causing pathogens they also disrupt the protective micro flora of the intestinal tract. This micro flora normally prevents opportunistic pathogens from establishing in the intestinal tract via competitive inhibition. However, after long-term antibiotic exposure this protective barrier is disrupted, leaving the host vulnerable to subsequent infection by organisms such as Clostridium difficile. Almost all antimicrobials have been implicated in the process, however, clindamycin, second and third generation cephalosporins, and fluoroquinolones are the major antibiotics involved (Gerding, 2004). The next step leading to Clostridium difficile associated diseases (CDAD) involves exposure to toxigenic Clostridium difficile. This can be facilitated via asymptomatic carriers, hospital staff for example or symptomatic hospital patients who shed Clostridium difficile cells and spores into the hospital environment. Consequently, infected fomites also become vehicles for transmission.
Once Clostridium difficile establishes in the colon, it can produce toxin A (an enterotoxin), toxin B (a cytotoxin) or a binary toxin. Individually or in combination these toxins cause changes to the cytoskeletal organization of enterocytes, fluid secretion, leukocyte chemotaxis and the up regulation of cytokines, all subsequently leading to mucosal damage, which manifests as diarrhea or PMC.
Data from the National Nosocomial Infections Surveillance system, the Centers for Disease Control and Prevention, and the Emerging Infection Network of the Infectious Diseases Society of America, all show that the disease rates and severity of CDAD have increased over the past few years (McDonald et al., 2005). Reasons for this rise may include the emergence of hypervirulent strains, strains with a functional binary toxin gene or deletions in the tcdC gene, or those that have emerged with resistance to common antibiotic therapy. Whatever the reasons may be, it is paramount that we reassess current laboratory diagnostic techniques so that all possible strains including epidemic strains can be detected.
One of the problems with current laboratory techniques is the failure to quickly detect hypervirulent strains of toxinotype III ribotype 027 – a strain that has been reported to be present and active in Canada, the United States, the United Kingdom, and the Netherlands. This hyper-producer of toxins produces 16 to 23 times more toxins A and B than the reference strain VPI 10463 (Pépin et al., 2005); it is therefore capable of inducing very severe diarrhea. To complicate matters, hyper producing strains (those similar to that of ribotype 027) cause what is known as a prozone effect. This is a phenomenon in which mixtures of specific antigen and antibody do not agglutinate or precipitate visibly because of an excess of either antigen or antibody, and in this case, it is the antigen (Clostridium difficile toxins) that is in excess. As a result, commercial enzyme immunoassays often fail to detect these strains because false negative results are often instigated by this prozone effect.
A similar problem applies to strains with functional binary toxin genes and those with partial deletions. Studies by McDonald et al. (2005) and Loo et al. (2005) have detected epidemic strains, which contained binary toxin genes and a partial deletion in the tcdC gene. Because the tcdC protein is thought to function as a negative regulator of the production of toxins A and B, the authors have proposed that this 18- base pair deletion could lead to increased toxin production, and subsequently induce problems clinically as well as posing challenges for laboratory detection as discussed above. In addition, because many laboratories persist with kits that only detect toxins A or B (despite a decade of literature outlining issues with single antigen testing), those strains without functional toxin A or B genes will not be detected by this approach. Increasing literature reporting clinically significant binary toxin-producing isolates of Clostridium difficile will pose problems to labs not performing sophisticated molecular testing. However, it must be noted that molecular techniques are not economically viable, require technical laboratory expertise, and demand discovery of relevant primers for particular strains. Furthermore, very few laboratories have adopted PCR as routine practice to diagnose CDAD.
Laboratory diagnosis of CDAD in many institutions consists of a positive faecal toxin test. Laboratories have been moving more towards kits testing for both toxin A and B and away from a single antigen test strategy, albeit slowly.
Some laboratories are also incorporating faecal isolation of Clostridium difficile from stool specimens as a safety net approach to detect these newly described strains. Studies by Delmée et al. (2005) have shown that the direct faecal cytotoxin assay (the “gold standard†technique) missed almost as many cases as it detected, while the toxigenic culture was successful in detecting those that were missed by the former technique. Toxigenic culture involves a stool culture for Clostridium difficile followed by a toxin assay of Clostridium difficile colonies. This method, although time-consuming, is highly sensitive and vital for correct diagnosis and early recognition of possible outbreaks.
In addition to toxigenic culture, presumptive cell counts of leukocytes and erythrocytes should also be included in routine practices. Even though this is not a definitive marker of CDAD, knowledge of the leukocyte numbers in combination with positive culture isolation and toxin detection, provides a much stronger basis for correct diagnosis. In retrospect, there is an urgent need for laboratories to reassess their diagnostic techniques because globally, CDAD cases are increasing in numbers and severity, and emerging aberrant strains are appearing undetected by out-dated laboratory practices.
Well-known Clostridium difficile expert Prof. Thomas Riley from Western Australia has recently published a succinct article in the Medical Journal of Australia outlining the sentiment of this commentary. It is a good read for those laboratories performing Clostridium difficile detection to further enforce a review of Clostridium difficile detection methodology.
By Valerie Nguyen Department of Microbiology, School of Molecular and Microbial
Biosciences, University of Sydney Department of Microbiology, Concord Hospital, NSW Australia.
