Category Archives: C. diff. Research & Development

Researchers Find the Weaknesses of TcdB One of the Toxins Secreted By the C. diff. bacteria and Main Cause of a CDI

The New study, led by researchers from the University of California, Irvine (UCI), uncovers the long-sought-after, three-dimensional structure of a toxin primarily responsible for devastating Clostridium difficile infection (CDI).

Nature Structural & Molecular Biology, the study titled, “Structure of the full-length Clostridium difficile toxin B,” sheds light on the weaknesses of TcdB, one of the toxins secreted by the Clostridium difficile (C. diff) bacteria and the main cause of CDI.

“This is the first time we could directly see the 3D structure of the gigantic TcdB holotoxin at a near atomic resolution,” said Rongsheng Jin, PhD, a professor in the Department of Physiology & Biophysics at UCI’s School of Medicine and the senior author in the study. “Interestingly, this toxin shapes like a question mark when viewed from a certain angle, and it has been a major question for us as we seek ways to fight the toxin and CDI.”

Also included in the study, the team demonstrated how three antibodies could neutralize TcdB, revealing intrinsic vulnerabilities of the TcdB toxin that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

…………The current standard of care for CDI involves treatments using broad spectrum antibiotics that reduce the level of C. diff bacteria, but also kill the good bacteria in the gut and disrupt the normal gut microbiome. This approach often leads to frequent disease recurrence (up to 35%).

Recently, the Food and Drug Administration (FDA) issued a warning about an investigational fecal microbiota for transplantation (FMT) procedure for CDI treatment following the death of patient in a clinical trial.

In another action, the FDA approved Bezlotoxumab, a TcdB-neutralizing human monoclonal antibody, as a prevention against recurrent infection.

“There remains a desperate need for more potent and cost-effective therapies for CDI,” said Jin. “The good news is, the 3D structure of TcdB we have identified literally provides a blueprint for the development of next-generation vaccines and therapeutics that have enhanced potency and broad-reactivity across different C. diff strains.”

Already the UCI team is working on a novel vaccine based on the new structure. Early studies show promising results, which Jin hopes to publish soon. In the meantime, The Regents of the University of California has filed a patent on their work.

Researchers contributing to this study include Peng Chen, Kwok-ho Lam, Zheng Liu, Baohua Chen, Craig B. Gutierrez, Lan Huang and Rongsheng Jin from UCI; Frank A. Mindlin and Mark Bowen from Stony Brook University; Yongrong Zhang, Therwa Hamza and Hanping Feng from the University of Maryland; Tsutomu Matsui from Stanford Synchrotron Radiation Lightsource; and Kay Perry from the Argonne National Laboratory. The study was supported by funding from the National Institute of Health and the U.S. Department of Energy.

 

To read the article in its entirety please click on the following link to be redirected:

https://www.infectioncontroltoday.com/bacterial/study-uncovers-weakness-c-diff-toxin

Research Study Finds Patient Age, Use of Proton Pump Inhibitors and the Administration of Primary Prophylaxis Were Not Significant Predictors of Hospital-Onset C.diff. Infection

Increasing length of stay, exposure to multiple classes of antibiotics, use of opioids and cirrhosis are all independently associated with an increased risk for hospital-onset Clostridioides difficile infection, or CDI, in asymptomatic colonized patients, but age is not, according to findings from a retrospective cohort study.

According to Yves Longtin, MD, chair of infection prevention and control at Jewish General Hospital in Montreal and associate professor of medicine at McGill University, and colleagues, “colonized individuals are at risk of progressing to CDI, but the factors that trigger progression to CDI are poorly understood.”

For their study, Longtin and colleagues assessed 513 patients colonized with C. difficile at the Quebec Heart and Lung Institute between November 2013 and January 2017, 7.6% of whom developed hospital-onset CDI. The 30-day attributable mortality was 15%.

The researchers found that hospital-onset CDI was independently associated with an increased length of stay (adjusted OR per day = 1.03; P = .006), exposure to multiple classes of antibiotics (aOR per class = 1.45; P = .02), use of opioids (aOR = 2.78; P = .007) and cirrhosis (aOR = 5.49; P = .008).

The use of laxatives was associated with a lower risk for CDI (aOR = 0.36; P = .01), according to the findings.

Longtin and colleagues also assessed the impact of specific antibiotics on CDI risk and found that beta-lactam with beta-lactamase inhibitors (OR = 3.65; P < .001), first-generation cephalosporins (OR = 2.38; P = .03) and carbapenems (OR = 2.44; P = .03) demonstrated the greatest risk for hospital-onset CDI.

Patient age, use of proton pump inhibitors and the administration of primary prophylaxis were not significant predictors of hospital-onset CDI, the researchers said.

“The lack of association between age and the risk of CDI among colonized patients is striking considering that age is among the strongest predictors for CDI,” Longtin and colleagues wrote. “This finding suggests that age may be associated with an increased risk of CDI through a greater susceptibility to colonization rather than an increased risk of progression to CDI once colonization has occurred, although studies on this topic have produced conflicting results.”

Although the findings demonstrated several predictive factors associated with hospital-onset CDI among colonized patients, Poirier and colleagues noted that further investigation is needed to determine whether “modifying these variables could decrease the risk of CDI.” – Marley Ghizzone

 

 

 

Disclosures: Longtin reports receiving research funding from Becton Dickinson and Merck, and research funding and personal fees from Gojo. Please see the study for all other authors’ relevant financial disclosures

To read this article in its entirety please click on the following link to be redirected:

https://www.healio.com/infectious-disease/nosocomial-infections/news/online/%7B1f25c2ae-c807-40af-b61c-e251b8c00828%7D/age-not-associated-with-hospital-onset-cdi-in-colonized-patients-study-suggests

Researchers Examined the Effect of Disinfectant on C. difficile Spores and How They Survived Afterwards On Surfaces Including Isolation Gowns, Stainless Steel and Vinyl Flooring

In lab studies, researchers found that C. diff spread easily from disposable gowns often employed in surgery or infection control to stainless steel and vinyl surfaces.

“The [bacteria] also transferred to vinyl flooring, which was quite disturbing. We didn’t realize they would,” said Tina Joshi, a lecturer in molecular microbiology at the University of Plymouth in the United Kingdom and lead author of the new study.

“These bugs evolve. These bugs like to stay one step ahead. And even though we’re using disinfectants and antibiotics appropriately, they still will become resistant in time. It’s inevitable,” Joshi said.

The bacteria, called Clostridioides difficile or C. diff., cause almost a half million infections every year in the United States, according to the Centers for Disease Control and Prevention.

The infection, which is spread by fecal to oral transmission, causes severe diarrhea, and can lead to intestinal inflammation and kidney failure. Those most at risk are people who have been given strong antibiotics, as well as those with long hospital stays, or those living in long-term care facilities like the elderly.

That means that keeping these facilities clean is incredibly important. But new research, published Friday (7/12/19)  in the journal Applied and Environmental Microbiology, shows how difficult that can be.

In lab studies, researchers found that C. diff spread easily from disposable gowns often employed in surgery or infection control to stainless steel and vinyl surfaces.

These bugs evolve. These bugs like to stay one step ahead. And even though we’re using disinfectants and antibiotics appropriately, they still will become resistant in time. It’s inevitable.

What’s more, the bacteria didn’t die when the researchers tried to kill them with concentrated chlorine disinfectant.

“Even if we applied 1,000 parts per million of chlorine, it would allow spores to survive in the gowns,” Joshi told NBC News.

It’s possible that increasing the amount of chlorine might kill the spores, but if the spores are indeed becoming resistant to the disinfectant, it will only be a matter of time before the stronger concentrations can’t kill them.

“These bugs evolve. These bugs like to stay one step ahead. And even though we’re using disinfectants and antibiotics appropriately, they still will become resistant in time. It’s inevitable,” Joshi said.

C. diff infections can occur when a patient is given broad spectrum antibiotics to tackle another infection.

If the bacteria aren’t killed, hospital patients or people in nursing homes can become infected when they come into contact with contaminated surfaces, such as a bedside food tray.

But if traditional disinfectants are ineffective, as the new research suggests, what works?

One option is UV light, which could be useful in killing the bacteria. However, it can be challenging to make sure all surfaces are fully exposed to the light. At this point, Joshi said, highly concentrated bleach appears to be the best option.

For those who care for patients with compromised immune systems at home, the C. Diff Foundation says alcohol-based hand sanitizers are ineffective against the bacteria.

On its website, the group recommends using a cleaning solution of one cup bleach to nine cups of water, and leaving the mixture on surfaces for a minimum of 10 minutes. (Basic & Generic, not EPA registered product).

Meanwhile, if C. diff spores can survive on gowns and other surfaces, it is likely also the case that they can live on doctor’s coats and scrubs worn by hospital personnel all day.  (C Diff Foundation agrees)

“That’s a real infection control hazard, because these spores can stick to fibers. We’ve proven that in this paper,” Joshi said.

Erika Edwards

Erika Edwards is the health and medical news writer/reporter for NBC News and Today.

 

To read the article in its entirety please click on the following link to be redirected:

https://www.nbcnews.com/health/health-news/dangerous-bacteria-can-survive-disinfectant-putting-patients-risk-n1029231

 

 

CspC Plays a Critical Role in Regulating C. diff. Spore Germination in Response to Multiple Environmental Signals.


Abstract

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor

Author summary

The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

Authors:

  • Amy E. Rohlfing ,
  • Brian E. Eckenroth ,
  • Emily R. Forster,
  • Yuzo Kevorkian,
  • M. Lauren Donnelly,
  • Hector Benito de la Puebla,
  • Sylvie Doublié,
  • Aimee Shen

To view the Abstract in its entirety – please click on the link provided below:

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008224

  • Published: July 5, 2019

On June 13th the U.S. Food and Drug Administration Warned of Infections From Fecal Microbiota Transplantation (FMT) Linked to a Patient’s Death

Dr. Peter Marks, director the Center for Biologics Evaluation and Research at the U.S. Food and Drug Administration stated, “While we support this area of scientific discovery, it’s important to note that fecal microbiota for transplantation does not come without risk,”

Two patients contracted severe infections, and one of them died, from fecal transplants that contained drug-resistant bacteria.

The agency said two patients received donated stool that had not been screened for drug-resistant germs, leading it to halt clinical trials until researchers prove proper testing procedures are in place.

After reports of serious, antibiotic-resistant infections linked to the procedures, the FDA wants “to alert all health care professionals who administer FMT [fecal microbiota transplant] about this potential serious risk so they can inform their patients.” said Dr. Peter Marks, director the Center for Biologics Evaluation and Research at the U.S. Food and Drug Administration.

Other samples from the same donor were tested after the patients got sick. The samples were found to harbor the same dangerous germs found in the patients, known as multi-drug-resistant organisms (MDRO). They were E. coli bacteria that produced an enzyme called extended-spectrum beta-lactamase, which makes them resistant to multiple antibiotics. The stool had not been tested for the germs before being given to the patients.

The F.D.A. on Thursday issued a warning to researchers that stool from donors in studies of fecal transplantation should be screened for drug-resistant microbes, and not used if those were present. It is also warning patients that the procedure can be risky, is not approved by the agency and should be used only as a last resort when C. difficile does not respond to standard treatments.

Dr. Marks said the agency was trying to strike a balance between giving patients who need the treatment access to it while also establishing safeguards to protect them from infection. In a statement, he said, “While we support this area of scientific discovery, it’s important to note that fecal microbiota for transplantation does not come without risk.”

Researchers are also looking into the use of fecal transplants to treat chronic gastrointestinal illnesses such as ulcerative colitis or irritable bowel syndrome.

The patients received treatment as part of a clinical trial, and the researchers conducting the trial reported the cases as adverse events to the F.D.A., which they are required to do. But the rules governing this kind of experiment prohibit the F.D.A. from revealing details about the treatment or who provided it.

 

SOURCE:  https://www.nytimes.com/2019/06/13/health/fecal-transplant-fda.html

Researchers Find Sulfated glycosaminoglycans and Low-Density Lipoprotein Receptor Contribute To Clostridioides difficile Toxin A Cell Entry

 

Abstract

Clostridium difficile toxin A (TcdA) is a major exotoxin contributing to disruption of the colonic epithelium during C. difficile infection. TcdA contains a carbohydrate-binding combined repetitive oligopeptides (CROPs) domain that mediates its attachment to cell surfaces, but recent data suggest the existence of CROPs-independent receptors. Here, we carried out genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated screens using a truncated TcdA lacking the CROPs, and identified sulfated glycosaminoglycans (sGAGs) and low-density lipoprotein receptor (LDLR) as host factors contributing to binding and entry of TcdA. TcdA recognizes the sulfation group in sGAGs. Blocking sulfation and glycosaminoglycan synthesis reduces TcdA binding and entry into cells. Binding of TcdA to the colonic epithelium can be reduced by surfen, a small molecule that masks sGAGs, by GM-1111, a sulfated heparan sulfate analogue, and by sulfated cyclodextrin, a sulfated small molecule. Cells lacking LDLR also show reduced sensitivity to TcdA, although binding between LDLR and TcdA are not detected, suggesting that LDLR may facilitate endocytosis of TcdA. Finally, GM-1111 reduces TcdA-induced fluid accumulation and tissue damage in the colon in a mouse model in which TcdA is injected into the caecum. These data demonstrate in vivo and pathological relevance of TcdA-sGAGs interactions, and reveal a potential therapeutic approach of protecting colonic tissues by blocking these interactions.

To view abstract in its entirety please click on the following link to be redirected:  https://www.ncbi.nlm.nih.gov/pubmed/31160825?dopt=Abstract&utm_source=dlvr.it&utm_medium=twitter

A Systematic Review Evaluates the Diagnostic Accuracy of Laboratory Testing Algorithms that Include Nucleic Acid Amplification Tests (NAATs) to Detect the Presence of C. difficile

SUMMARY

The evidence base for the optimal laboratory diagnosis of Clostridioides (Clostridium) difficile in adults is currently unresolved due to the uncertain performance characteristics and various combinations of tests.

This systematic review evaluates the diagnostic accuracy of laboratory testing algorithms that include nucleic acid amplification tests (NAATs) to detect the presence of C. difficile. The systematic review and meta-analysis included eligible studies (those that had PICO [population, intervention, comparison, outcome] elements) that assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase (GDH) enzyme immunoassays (EIAs) or GDH EIAs plus C. difficile toxin EIAs (toxin). The diagnostic yield of NAAT for repeat testing after an initial negative result was also assessed.

Two hundred thirty-eight studies met inclusion criteria. Seventy-two of these studies had sufficient data for meta-analysis. The strength of evidence ranged from high to insufficient. The uses of NAAT only, GDH-positive EIA followed by NAAT, and GDH-positive/toxin-negative EIA followed by NAAT are all recommended as American Society for Microbiology (ASM) best practices for the detection of the C. difficile toxin gene or organism. Meta-analysis of published evidence supports the use of testing algorithms that use NAAT alone or in combination with GDH or GDH plus toxin EIA to detect the presence of C. difficile in adults. There is insufficient evidence to recommend against repeat testing of the sample using NAAT after an initial negative result due to a lack of evidence of harm (i.e., financial, length of stay, or delay of treatment) as specified by the Laboratory Medicine Best Practices (LMBP) systematic review method in making such an assessment. Findings from this systematic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of GDH/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.

SOURCE:  To Learn More:  https://cmr.asm.org/content/32/3/e00032-18.long?utm_source=dlvr.it&utm_medium=twitter

INTRODUCTION

Clostridioides (Clostridium) difficile infection (CDI) is the leading cause of health care-associated infections in the United States (1, 2). It accounts for 15% to 25% of health care-associated diarrhea cases in all health care settings, with 453,000 documented cases of CDI and 29,000 deaths in the United States in 2015 (3). Acquisition of C. difficile as a health care-associated infection (HAI) is associated with increased morbidity and mortality. This adds a significant burden to the health care system by increasing the length of hospital stay and readmission rates, with significant financial implications. The cost of hospital-associated CDI ranges from $10,000 to $20,000 per case (47) and $500 million to $1.5 billion per year nationally (1, 4, 5, 810).

Accurate diagnosis of CDI is critical for effective patient management and implementation of infection control measures to prevent transmission (11). The diagnosis of CDI requires the combination of appropriate test ordering and accurate laboratory testing to differentiate CDI from non-CDI diarrheal cases, including non-CDI diarrhea in a C. difficile-colonized patient (8). Accurate diagnosis of CDI is critical for appropriate patient management and reduction of harms that may arise from diagnostic error (12) and is critical for implementation of infection control measures to prevent transmission (11). Consequently, among patients presenting with diarrhea, there is significant potential for underdiagnosis or overdiagnosis as can arise from incorrect diagnostic workups (13).

Quality Gap: Factors Associated with the Laboratory Diagnosis of C. difficile

Best practices for laboratory diagnosis of CDI remain controversial (14). Current laboratory practice is not standardized, with wide variation in test methods and diagnostic algorithms. Several laboratory assays are available to support CDI diagnosis in combination with clinical presentation. These include toxigenic culture (TC); the cell cytotoxicity neutralization assay (CCNA); enzyme immunoassays (EIAs) and immunochromatographic assays for the detection of glutamate dehydrogenase (GDH), toxin A or B, or both toxins; and, within the last 10 years, nucleic acid amplification tests (NAATs). Currently, two tests, TC and the CCNA, serve as reference methods for the diagnosis of C. difficile infection (15). The principle of the TC test is to detect strains of C. difficile that produce a toxin(s) following culture on an appropriate medium. CCNA detects fecal protein toxins contained within the stool and is often referred to as fecal toxin detection (16). Unfortunately, both tests are slow and labor-intensive.

Commercially available NAATs for C. difficile detection include those based on PCR or loop-mediated or helicase-dependent isothermal amplification (1720). The performance of NAATs and non-NAAT tests is commonly assessed using diagnostic accuracy measures for the presence of the organism (e.g., diagnostic sensitivity, diagnostic specificity, positive predictive value [PPV], and negative predictive value [NPV]). However, these measures may not directly link to the clinical definition of CDI or clinical outcomes, and some measures (e.g., PPV and NPV) are dependent on disease prevalence in the patient population being tested (8, 17, 19, 20). Finally, in addition to diagnostic sensitivity and specificity, other factors influence the choice of testing strategy, such as cost and turnaround time.

The diagnostic accuracies of current commercially available assays (GDH EIAs, toxin A/B EIAs, and NAATs) are based on comparison with one or both of the currently accepted reference methods (TC and CCNA) for the detection of toxigenic C. difficile, and these comparisons are generally made to inform potential replacement of these reference methods. Although a definitive reference “gold standard” is lacking, both TC and CCNA are regarded as acceptable reference methods (15). However, some view the gold standard to be TC of a stool specimen combined with colonic histopathology of pseudomembranous colitis in patients with symptoms, but it is known that there is a spectrum of disease wherein not all patients with C. difficile infection have pseudomembranes (21). Finally, less frequently, colonoscopic or histopathologic findings demonstrating pseudomembranous colitis can be used in diagnostic workups to increase the diagnostic specificity for CDI diagnosis (14).

In contrasting the two reference methods (TC and CCNA), TC, while infrequently performed in clinical laboratories, is regarded as being more analytically sensitive than CCNA for detecting C. difficile in fecal specimens but may have lower diagnostic specificity (and, therefore, a greater likelihood of false-positive [FP] test results). CCNA has been shown to have high diagnostic sensitivity, ranging from 80 to 100%. In addition, CCNA has high diagnostic specificity and positive predictive values as well as having greater clinical utility based upon clinical outcomes (2226). Furthermore, each reference method differs by the target detected: TC detects the presence of C. difficile strains that produce toxins A and/or B in vitro to confirm a toxigenic strain, whereas CCNA detects the presence of free toxin A or B in clinical specimens. Given these contrasting characteristics, there is potential for diagnostic discrepancy between the reference standards. Therefore, observed diagnostic performance may vary according to which reference standard is used.

Given the variety of test methods and diagnostic algorithms, there is disagreement in the laboratory community on whether best practices for the diagnosis of CDI consist of NAAT only or algorithmic testing that includes NAAT (GDH EIA followed by NAAT [GDH/NAAT] or GDH and toxin EIAs followed by NAAT [GDH/toxin/NAAT]) (20). At the initiation of these guidelines, this was the clinical quandary facing individuals who decide on a C. difficile testing strategy for their health care system, particularly as there is limited high-quality evidence to support which diagnostic testing strategy best supports the laboratory diagnosis of CDI (8, 22). Additionally, it remains to be determined if the potential differences in the accuracy of NAAT only or an algorithmic strategy would impact patient management or patient outcomes (27). There are few studies that encompass the nuances of laboratory CDI diagnosis as it occurs in the clinical context, for example, that evaluate the effect of preanalytic testing considerations on outcomes, to include clinical outcomes. This limitation is evident from the recent Infectious Diseases Society of America (IDSA)/Society for Healthcare Epidemiology of America (SHEA) systematic review, which included only studies that encompassed C. difficile testing within its clinical context, including preanalytic and postanalytic aspects (11).

Given these practice issues, and related diagnostic quality and patient safety concerns, the goal of this systematic review was to determine which laboratory testing strategies, with the inclusion of NAAT, had the best diagnostic accuracy for CDI. While it is clear that laboratory testing alone without taking into consideration the entire clinical picture is not appropriate for the diagnosis of CDI, the available literature has limited evidence linking laboratory diagnosis with clinical outcomes. Therefore, the questions for this systematic review were refined to be based only on the intermediate outcome of diagnostic accuracy for detecting the presence of the C. difficile organism or toxin. Although the reference standard in these studies defines what is meant by the target condition, this systematic review compares the diagnostic accuracies of these tests, including GDH detection by EIA, toxin detection by EIA, and NAAT, to those of CCNA and TC. It has been clear that preanalytical factors are crucial for NAAT specifically, and many of the studies did not include a preanalytical component, which limits whether this review can answer the question, Does this patient have C. difficile infection?

The questions that guided this systematic review were the following: (i) What is the diagnostic accuracy of NAAT only versus either TC or CCNA for detection of the C. difficile toxin gene?, (ii) What is the diagnostic accuracy of a GDH-positive EIA followed by NAAT versus either TC or CCNA for detection of the C. difficile organism/toxin gene?, (iii) What is the diagnostic accuracy of a GDH-positive/toxin-negative EIA followed by NAAT versus either TC or CCNA for detection of the C. difficile organism/toxin/toxin gene?, and (iv) What is the increased diagnostic yield of repeat testing using NAAT after an initial negative result for C. difficile detection of the toxin gene?

The goals of analysis based on these questions were specifically to evaluate the effectiveness of the following: (i) the diagnostic accuracies of NAAT-only and algorithmic (“two-step” or “three-step”) testing strategies, including detection of toxin or GDH in addition to NAAT, and (ii) the diagnostic yield of repeat testing after an initial negative NAAT result. The evidence supporting these two important issues was evaluated by applying the Centers for Disease Control and Prevention (CDC) Laboratory Medicine Best Practices (LMBP) Initiative’s systematic review method for translating results into evidence-based recommendations (28). The method has recently been used to evaluate practices for improving blood culture contamination (29), blood sample hemolysis (30), urine culture sample quality (31), timeliness of providing targeted therapy for bloodstream infections (32), and laboratory test utilization (33), in addition to others, and can be found at the CDC LMBP website (https://www.cdc.gov/labbestpractices/our-findings.html).