As often as antibiotics are prescribed to treat existing infections, they also bring the potential to act as the cause of new infections, such as with a Clostridioides difficile (C. diff) infection (CDI). While a course of antibiotics serves its intended purpose in killing bacteria that are responsible for an existing infection, the same antibiotics have the potential to disrupt the normal flora in the human intestine simultaneously. When this happens, it can lead to an overabundance of C. diff bacteria in the human colon and produce toxins, which can cause diarrhea and/or colitis and create a new gastrointestinal CDI that requires treatment with additional antibiotics.
However, not every patient who is prescribed antibiotics is at risk of developing a CDI. Many risk factors can increase the likelihood of developing a CDI including patients who are elderly, have been in a hospital or care facility for a long period of time or have a lowered or compromised immune system for fighting the bacteria that cause a CDI.
As part of its online literature, the C. diff Foundation (cdifffoundation.org) asserts that studies show C. diff has become “the most common microbial cause of healthcare-associated infections (HAIs) in U.S. hospitals and costs up to $4.8 billion each year in excess healthcare costs for acute care facilities alone.”
As such, prevention of CDIs is key, along with early C. diff detection by way of assays and molecular platforms that offer rapid and reliable results. These rapid tests not only allow treatment to begin sooner, but also help to avoid CDI-related costs later.
The origins of C. diff infections
When C. diff first emerged, testing options were limited for suspected cases with clinical labs focused on detecting the presence of toxins to make diagnostic decisions.
David Lyerly, PhD, Chief Science Officer at TechLab, said, “Disease caused by C. difficile was first identified in the late 1970s by the discovery of a cytopathic effect detected on tissue cultured cells. The effect, which was identified as a ‘cell rounding’ activity, was present in fecal samples from patients with pseudomembranous colitis (PMC), which is the severe stage of CDI. There were several groups of scientists trying to determine the cause of PMC, and all were reporting the presence of this unusual activity using the tissue culture assay.”
He continued, “Scientists noted also that the activity could be seen with various tissue cultured cell lines. Initially, viruses were suspected but the activity could not be propagated. Within a short while, evidence pointed to the presence of a clostridial toxin and then more specifically to C. difficile as the cause of the activity. During this time, a selective anaerobic agar media was developed – cycloserine, cefoxitin, fructose agar (CCFA) – that effectively isolated C. difficile from fecal specimens.”
“These two types of testing methods – the tissue culture assay, also called the cellular cytotoxicity neutralization assay (CCNA), and CCFA media – were critical in the discovery and cause of CDI. Both types of tests, one for toxin and one for the organism, are very sensitive and specific. Although not nearly used as much as in prior years, these tests still are considered gold standards as comparator tests because of their sensitivity and specificity, and are the tests used to evaluate the immunoassays and NAAT assays widely in use today for CDI,” he added.
Sherry Dunbar, PhD, MBA, Senior Director, Global Scientific Affairs at Luminex, said, “About 20 (or more) years ago, the first enzyme immunoassays were coming out and being evaluated and compared to CCNA. Now, toxigenic culture is considered the gold standard.”
Fred Tenover, PhD, D(ABMM), Vice President of Scientific Affairs at Cepheid, pointed out that in the early days of CDI detection, “Anaerobic culture was used to recover the organism from the stool of infected patients and the organism was tested for toxin production in cell culture. The toxin was neutralized with antibodies to a related toxin from Clostridium sordellii to provide specificity. Hence the name ‘toxigenic culture’; this remains a key reference method for testing.”
Rodney Arcenas, PhD, D(ABMM), Director of Clinical Sciences, Global Medical and Scientific Affairs at Roche Molecular Diagnostics, said, “Initial laboratory methods focused on detecting the presence of the toxin in vitro via cytotoxin testing. One method involved culturing for the organism from the diarrheal stool. All positive cultures require the confirmation of toxin production because there are some C. diff organisms that do not produce cytotoxin.”
He continued, “Another method involved direct inoculation of stool filtrate onto a cell culture monolayer (Vero, HFF, CHO, or McCoy cells). Cell cultures are set up with and without neutralizing antibodies to C. difficile toxin. Examination for cytopathic effect is done at 24 hours. The neutralizing antibodies, by binding the C. difficile cytotoxin, prevent the development of cytopathic effect.”
Preferred methods of detection
With former testing options having served their limited purpose at the time, today’s clinical labs have testing options that appeal for reasons such as accuracy, speed and cost-effectiveness, with final decisions based upon facility standards and/or budgets, as well as individual preferences.
Lyerly said, “There are two diagnostic platforms widely used as laboratory tests for the diagnosis of CDI. One is the immunoassay platform that detects the two toxins of C. difficile, designated A and B, and immunoassays for glutamate dehydrogenase (GDH), also referred to as “antigen.” The other platform involves molecular assays, more commonly referred to as nucleotide acid amplification assay (NAAT), that detect the toxin genes. Most NAAT assays detect tcdB, which is the gene that encodes toxin B. Several detect either tcdA, which is the toxin A gene, or both tcdA and tcdB.”
He continued, “Which tests are more accurate has been the subject of an ongoing debate for several years. Each of the tests offer advantages, but each also has limitations. The toxin immunoassays offer the highest predictive positive values because they detect the toxins that cause the disease. However, there is concern that toxin tests suffer from lack of sensitivity compared to the gold standard tissue culture assay. GDH is a very sensitive biomarker for the organism in feces and indicates the presence of actively growing organisms. However, GDH is produced both by toxigenic and nontoxigenic strains, so additional toxin testing is recommended. NAAT assays are very sensitive, but because of this extreme sensitivity, these tests overdiagnose CDI because the organism is present is significant numbers of patients who are carriers. NAAT assays also detect spores and low numbers of dead cells, neither of which is associated with toxin production.”
Lyerly pointed out, “There are more than 50 tests cleared by the FDA. These tests include an assortment of immunoassays and NAAT tests. These tests do not perform equivalently, and can vary because of the quality of the test, the specific test reagents, stability, lot-to-lot reliability, etc. Rapid and microwell formats are available for the immunoassays and provide turnaround times of 30 to 60 minutes. NAAT assays also can vary from test-to-test because of the same factors. NAAT results typically are obtained in about 45 to 60 minutes.”
Dunbar added that new guidelines came out in 2018 from the Infectious Diseases Society of America (IDSA), which can play a role in testing choices.
She said, “Prior to these guidelines and in other geographies like the EU, they do a two-step algorithm using glutamate dehydrogenase in conjunction with a second test for toxin, usually an EIA and/or PCR,” adding, “It is now acceptable to do a single test, such as a PCR test, if certain criteria are in place in the hospital and agreed-upon in advance.”
Best practices for CDI prevention
When considering the steps clinics and hospitals are taking to reduce the risk of CDI outbreaks while still maintaining the highest level of in-facility patient care, it all begins with an eye toward reducing antibiotic use, according to Tenover.
“One key factor to reducing CDI is to reduce unnecessary antibiotic use. Fluoroquinolones and other antibiotics disrupt gut flora and make it easier for C. difficile to colonize the gastrointestinal tract, proliferate, and produce toxin, which results in disease. The UK focused on reducing unnecessary antibiotics and showed a dramatic drop in the rates of CDI in hospitals. Some hospitals screen patients for CDI on admission to find colonized patients early. This remains controversial. Other healthcare institutions focus more on handwashing, charting diarrhea daily to detect early disease, and making sure that testing is performed quickly, and the results acted on quickly when positive,” he said.
Arcenas added, “Typically, C. difficile testing should be considered for a patient who has developed diarrhea after having been in the hospital for 72 hours, as C. difficile is considered a Hospital Acquired Infection (HAI). Hospitals typically implement infection control contact precaution measures for patients with suspected gastrointestinal illness while they are being diagnosed. In addition, they normally do terminal cleaning of the patient room after a C. diff patient has been discharged. The spores of C. difficile can survive in the hospital room environment, thus the need to terminally clean the room to eliminate the presence of spores. Handwashing is recommended for healthcare workers because the alcohol hand-gel/sanitizers do not kill the C. diff spores.”
Lyerly commented, “CDI has now replaced MRSA as the most common HAI in the U.S. There are hundreds of thousands of cases annually. CDI is a huge challenge to our healthcare system, and in addition to hospital-acquired cases, there now are many cases of community-acquired CDI. Probably the biggest challenge is the high number of patients (around 25 percent) that relapse with CDI. Patients can relapse multiple times and eventually succumb to the disease.”
Outlook for CDIs and HAIs
With the continued existence of CDIs and their related costs as HAIs, diagnostic and healthcare professionals are concerned about the potential for challenges on the horizon and how to prepare for them now.
Tenover reported, “According to the CDC, the U.S. is making progress on bringing CDI rates down nationally, although the numbers of cases are still unacceptably high. The organism is also spreading in community settings and not just within hospitals, which complicates controlling its spread. That is also the reason some hospitals screen for C. difficile in patients on admission to the hospital – to find it early and place those patients on contact precautions to reduce spread. The most troubling issue revolves around the fact that CDI data are reportable to CDC and there are financial penalties for hospitals if their CDI rates are going on the wrong direction.”
He continued, “Unfortunately, and sadly, this has caused some institutions to focus much more on getting their reportable rates down by using the least sensitive test available, rather than on optimizing patient care and controlling spread. Ironically, while using the least sensitive test may decrease rates in the short term, the missed cases will eventually result in greater spread of CDIs in hospitals and result in even more cases. More sensitive toxin tests have been described in the literature, but are not commercially available, suffer the same problems as NAATS when the wrong patient populations are tested, and thus are not likely to change the testing landscape. Therapy guidelines for CDI are evolving. The major focus is still on antibiotics, but monoclonal antibody therapy and fecal transplants are also used in some patients, especially those with recurrent disease, where they work very well.”
Arcenas added, “One key area of improvement will be the further development of testing algorithms to better capture patients with active disease, but also carriers so action can be taken to prevent further spread in healthcare institutions.
“I do expect that challenges may be raised to antibiotic treatment (if they haven’t already been) due to antibiotic stewardship programs. The judicious and appropriate use of antibiotics is at the core of what stewardship programs do. Because antibiotics are a known risk factor for the development of C. diff disease, healthcare providers will need to strike a delicate balance between short-term and long-term goals in infectious disease management. Of course, accurate diagnostic testing can play an important role in this process,” he said.
Dunbar commented, “I wouldn’t want to speculate on this, but I would say that there is still an ongoing debate about whether a toxin test that detects the expression of the toxin protein versus a PCR test that detects the presence of the toxin gene is better.”
Lyerly assserted, “The debate on which test most accurately identifies clinically relevant CDI will continue among laboratories using immunoassays and those using NAAT assays. The goal is to ensure that patients with clinically relevant CDI are being properly diagnosed and treated. Additional tests are continuing to appear on the market, but these new tests do not offer significant advantages over those now available. Efforts will continue on ways to improve current tests and identify new markers for this pathogen.”