Clostridium difficile infection: the importance of accurate diagnosis

Broad-spectrum antibiotic treatment can lead to diarrhea in certain patients. Clostridium difficile is the most common cause of antibiotic-associated diarrhea. Bacteria of the genera Clostridia are gram-positive, obligate anaerobes that include C. tetani, C. perfringes, C. botulinum, and C. difficile. These bacteria have the capacity to form spores that are resistant to heat and most disinfectants, which contributes to their efficient transmission. Clostridia can also produce various toxins that play a major role in the pathogenesis of the diseases that they cause.

Toxin-producing strains of C. difficile (toxigenic C. difficile) can cause antibiotic-associated diarrhea, whereas non-toxigenic C. difficile are non-pathogenic. C. difficile can be found in the intestine of up to 5% of individuals. Prolonged hospitalization and residence in a long-term care facility increase the likelihood of both colonization as well as the generation of C. difficile disease. Broad spectrum antibiotic usage and other conditions that alter the normal composition of intestinal flora also predispose to the acquisition and overgrowth of C. difficile and to the development of C. difficile infection (CDI).

Disease

CDI is a disease of the large intestine, and the severity ranges from uncomplicated diarrhea to severe colitis that can result in life-threatening toxic megacolon.¹ The pathogenesis of CDI primarily involves two bacterial toxins: toxin A, which is an enterotoxin that affects the permeability of the colonic epithelial layers; and toxin B, a cytotoxin that interferes with the regulation of cellular cytoskeletal proteins.^2,3 CDI affects the most vulnerable patients, such as the elderly and immunocompromised patients, when they are treated for serious infections with antibiotics. Treatment modalities for CDI can be found elsewhere, and there are many reports concernng the ongoing challenges associated with treatment and management of CDI, especially with regard to cases of fulminant disease and recurrent CDI.⁴

Based on recent reports, both the incidence and severity of CDI are increasing.⁵ CDI has become one of the most common hospital-acquired infections and causes or contributes to a significant number of hospital-based deaths.  In addition, hypervirulent strains that produce more toxin due to disruptions in the toxin regulatory elements have been associated with more severe disease in some studies as well as with more efficient transmission.⁶ There is an urgent need, therefore, to better control the spread of this infection in hospitals and in long-term care facilities. This article will focus on the role of detection methods in the accurate identification of cases of CDI and the consequences of inaccurate test results.

Diagnosis

Accurate diagnosis of C. difficile is essential for physicians to properly manage patients with antibiotic-associated diarrhea and for hospitals to effectively identify and control outbreaks of CDI. As with all diagnostic testing, it is critically important for clinicians to select patients for testing based on good clinical acumen.⁷  C. difficile testing should only be performed on at-risk patients with diarrhea as evidenced by multiple loose stools.⁸ Algorithms used by laboratories to identify the presence of toxigenic C. difficile have been continually evolving, and the organism and the disease are constantly changing. Fortunately, diagnostic methods are becoming more accurate and more facile, allowing for more rapid and precise identification of CDI.

There are many diagnostic methods available to detect toxigenic C. difficile in the stool of patients suspected of having CDI. Traditionally most tests have focused on the detection of C. difficile toxins A and B.  Cytotoxicity in cell culture that is neutralized by antitoxin-containing antisera (CCNA) is considered the reference method for toxin B detection.⁹  Stool culture for C. difficile with confirmation of toxin production (toxigenic culture) is considered the gold standard for definitive identification of CDI, but it is generally impractical for clinical diagnosis due to the length of time before a result is generated.^1,10  Immunoassays for the detection of toxin A and B have been a commonly used method to assist in the diagnosis of CDI because of their rapid turnaround time and ease of use in comparison with culture-based methods. These assays have been developed in various formats ranging from moderately complex enzyme-linked immunoassays to CLIA-waived lateral flow assays, allowing for a convenient option for many laboratories. All of these toxin immunoassays, however, have been associated with relatively low sensitivity and less than optimal specificity, resulting in poor negative and positive predictive values.

To address this issue, some manufacturers have added immunoassays for the detection of glutamate dehydrogenase (GDH) to their products.^11-13 GDH is present in all strains of C. difficile, but is not specifically associated with toxigenic strains and as such lacks specificity, and additional testing must be done to identify toxigenic C.difficile. Furthermore, a GDH-positive but toxin-negative result identifies a potentially false negative due to the lower sensitivity of the toxin immunoassay, which then warrants follow up with a definitive confirmatory test.^14,15 Several more recent reports, however, also question the sensitivity of GDH and suggest that a significant number of GDH-negative results might be C. difficile positive by other methods.¹⁶

The need for improved accuracy has promoted the development of molecular assays for the detection of pathogenic C. difficile in diarrheal stool samples. These assays are based on DNA amplification techniques that detect various conserved regions of the pathogenicity locus present in all known toxigenic C. difficile strains. There are a number of commercially available molecular tests, and they generally all have high sensitivity and specificity when compared to toxigenic culture and high percent positive and negative agreement when compared with one another.^17-20  Many of the molecular tests are based on real-time PCR and are relatively complex and/or expensive. More recently, some FDA-cleared tests that are based on isothermal DNA amplification technologies, such as loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA), have been developed.²¹ These tests are less complex and less expensive because they do not require a thermocycler or fluorescence reader. Certain tests do not require instrumentation, which reduces capital equipment budgets and makes the tests more accessible to smaller hospital laboratories. These new isothermal molecular tests offer laboratories that currently are using toxin immunoassays the benefit of the high level of accuracy of a molecular test without having to incur the significantly higher costs associated with complex instruments and a highly trained molecular staff.

It is imperative that both physicians and infection control specialists appreciate the pros and cons of the various diagnostic tests for C. difficile. Low sensitivity of an assay will lead to missed diagnoses. This can be detrimental to the patient, by delaying appropriate treatment. It also can be detrimental to the hospital, because often patients are not isolated or are inappropriately removed from isolation after a false negative test result—which can lead to further spread and more cases of CDI. Additionally, low specificity results in more false-positive cases in patients who may subsequently have necessary antibiotics curtailed and receive unnecessary treatment for CDI.

Table 1 lists some of the major consequences of the inaccurate diagnosis of CDI. Although diagnostic accuracy is always a goal in clinical and laboratory medicine, for CDI the consequences of a wrong diagnosis not only affect patient management, but have serious epidemiological and economic implications for healthcare facilities because of the importance of hospital-acquired infections. A single missed diagnosis can have widespread negative consequences beyond the affected patient’s outcome. Multiple missed diagnoses throughout the year can greatly expand the health and economic burden for a community.

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