Tag Archives: Clostridium difficile research and development

Publication: Multicenter Prevalence Study Comparing Molecular and Toxin Assays for Clostridioides difficile Surveillance, Switzerland






Andreas F. Widmer, Reno Frei, Ed J. Kuijper, Mark H. Wilcox, Ruth Schindler, Violeta Spaniol, Daniel Goldenberger, Adrian Egli, Sarah Tschudin-Sutter , and Kuijper
Author affiliations: University Hospital Basel, Basel, Switzerland (A.F. Widmer, R. Frei, R. Schindler, V. Spaniol, D. Goldenberger, A. Egli, S. Tschudin-Sutter)Leiden University Medical Center, Leiden, the Netherlands (E.J. Kuijper)Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, and Leeds Teaching Hospitals, Leeds, UK (M.H. Wilcox)


Public health authorities in the United States and Europe recommend surveillance for Clostridioides difficile infections among hospitalized patients, but differing diagnostic algorithms can hamper comparisons between institutions and countries. We compared surveillance based on the detection of C. difficile by PCR or enzyme immunoassay (EIA) in a nationwide C. difficile prevalence study in Switzerland. We included all routinely collected stool samples from hospitalized patients with diarrhea in 76 hospitals in Switzerland on 2 days, 1 in winter and 1 in summer, in 2015. EIA C. difficile detection rates were 6.4 cases/10,000 patient bed-days in winter and 5.7 cases/10,000 patient bed-days in summer. PCR detection rates were 11.4 cases/10,000 patient bed-days in winter and 7.1 cases/10,000 patient bed-days in summer. We found PCR used alone increased reported C. difficile prevalence rates by <80% compared with a 2-stage EIA-based algorithm.


Since its identification as a cause of antibiotic-associated pseudomembraneous colitis in 1978 (1), Clostridioides difficile has emerged as a major healthcare-associated pathogen worldwide. In the United States, C. difficile infection (CDI) rates doubled during 1996–2003 (2), and rates of CDI were reported to be 76.9 cases/10,000 discharges in 2005 (3). In a more recent national point-prevalence study including US healthcare facility in-patients, 13/1,000 patients were found to be either infected or colonized (4), a higher rate than had been previously estimated. In a national point-prevalence study of nosocomial infections in the United States, C. difficile was the most common causative pathogen overall (5). The increase largely has been attributed to the emergence of the hypervirulent strain, PCR ribotype 027 (RT027), which was identified as causative strain in 82% of cases during CDI outbreaks in Quebec, Canada, during 2001–2003 and accounted for 31% of all cases of healthcare-associated infections in the United States in 2011 (69). In Europe, CDI incidence varies across hospitals and countries with a weighted mean of 4.1 cases/10,000 patient-days per hospital in 2008 (10). The most recent study on CDI prevalence in Europe suggests an increase in the number of cases, reporting a mean of 7.0 cases/10,000 patient-bed days and ranging among countries from 0.7 to 28.7 cases/10,000 patient-bed days (11). The most common ribotype identified was RT027, which was detected in 4 countries: Germany, Hungary, Poland, and Romania (11).

To estimate and compare the burden of CDI across the United States, the US Centers for Disease Control and Prevention (CDC) began population-based CDI surveillance in 10 locations in 2011 (12). The European Centre for Disease Prevention and Control (ECDC) began coordinating CDI surveillance in acute care hospitals in Europe in 2016 (13). Both authorities provide case definitions based on different diagnostic approaches, including detection of C. difficile toxin A and B by enzyme immunoassay (EIA) or detection of toxin-producing C. difficile organisms by PCR. However, the use of different diagnostic algorithms to detect C. difficile might hamper comparisons between institutions and countries. Therefore, in a nationwide C. difficile multicenter prevalence study in Switzerland, we systematically compared surveillance measures based on detection of C. difficile in stool by either PCR as a stand-alone test or by a 2-stage algorithm consisting of an EIA to detect glutamate dehydrogenase (GDH) and toxins A and B.


Study Design

We performed a nationwide multicenter prevalence study of toxigenic C. difficile detected in stool samples routinely collected from hospitalized patients with diarrhea. Our study followed the design of a previous point-prevalence study for maximal comparability between our results and data from Europe (11). University Hospital Basel, a tertiary care center in Switzerland, coordinated the study. All hospitals participating in Swissnoso (https://www.swissnoso.chExternal Link), a national infection prevention network, were asked to participate. The Swissnoso network consists of 85 acute care hospitals that account for a total of 26,341 beds.

The Ethics Committee Northwest and Central Switzerland (Ethikkommission Nordwest-und Zentralschweiz) issued a declaration of no objection for this study. We adhered to STROBE guidelines for reporting on observational studies (14).

Sample Collection

All stool samples collected from inpatients >1 year of age with diarrhea that were submitted to the microbiology laboratory on 2 specified sampling days, 1 in winter and 1 in summer, in 2015 were eligble for inclusion. Only 1 sample per patient was included. In addition, stool samples that tested positive for toxigenic C. difficile <1 week prior to each study day also were collected from all institutions to obtain a more detailed estimate of ribotype distribution in Switzerland.

We collected the following institutional data for all hospitals and their affiliated microbiology laboratories: contact information; detailed information regarding laboratory diagnostics in place; and detailed information on the total number of admissions, number of beds, and number of patients hospitalized on the 2 days of the study. We also collected information on the total number of diagnosed CDI cases at each institution during the study year. For each eligible stool sample, we collected the following data: date of sample collection, age and gender of patient, ward location and clinical specialty, institution, whether a C. difficile test was ordered by the treating physician, and result of the C. difficile test if testing was performed at the local laboratory.


We tested all stool samples at the Division of Clinical Microbiology of the University Hospital Basel by using a 2-stage algorithm consisting of EIA and PCR. We performed EIA to detect GDH and toxins A and B by using C. DIFF QUIK CHEK COMPLETE (Techlab, https://www.techlab.comExternal Link), following the manufacturer’s instructions. We then performed PCR to detect the toxin B gene by using the RealStar PCR Kit (Altona Diagnostics, https://www.altona-diagnostics.comExternal Link). For detected C. difficile, we performed strain typing by using high-resolution capillary gel-based PCR ribotyping according to the method previously described by Stubbs et al. (15).


We calculated reported and measured rates of detected toxigenic C. difficile per 10,000 patient bed-days across participating institutions. We compared differences in testing algorithms for detection of toxigenic C. difficile across institutions in Switzerland and performance characteristics of diagnostic algorithms. We considered the proportion of missed toxigenic C. difficile cases and ribotype distributions as additional outcomes. We further assessed the proportion of laboratories using optimized C. difficile diagnostic tests, which we defined as using an algorithm recommended by the European Society of Clinical Microbiology and Infectious Diseases (16).

Statistical Analyses

We separately calculated rates for each diagnostic algorithm performed in the coordinating center laboratory. In addition, we separately calculated rates for dedicated children’s hospitals. We defined missed C. difficile cases as those in which tests were negative at the participating hospital’s laboratory but positive at our institution. We used descriptive statistics to report ribotypes and differences in diagnostic algorithms across all participating institutions. All analyses were performed in Stata version 15.1 (StataCorp, https://www.stata.comExternal Link).


Figure 1. Distribution of centers participating in a prevalence study comparing molecular and toxin assays for nationwide surveillance of Clostridioides difficile, Switzerland. Red circles represent the location of participating centers.

Participating institutions included 76/85 (89.4%) institutions belonging to the Swissnoso network. Among participating institutions, 5 were academic teaching hospitals, 3 were dedicated children’s hospitals, and 36 were affiliated microbiology laboratories. Participating institutions were distributed across all geographic regions of Switzerland (Figure 1).

Participating institutions reported collecting a fecal sample for microbiological workup in »65% (SD +25%) of all patients with hospital-onset diarrhea. Among participating institutions, 15/76 (19.7%) did not begin CDI treatment before fecal sample collection. Among institutions that initiated treatment before collecting fecal samples, 23/76 (30.3%) began treatment in <2% of patients, 12/76 (15.8%) began treatment in 3%–5% of patients, 8/76 (10.5%) began treatment in 6%–10% of patients, and 1 (1.3%) began treatment in 11%–20% of patients. The other 17 (22%) institutions were not able to provide an estimate of these data.

Overall, 354 stool samples were submitted to the coordinating center, of which 338 were eligible for study inclusion; 16 samples were excluded because they were not liquid, their submitted data were incomplete, or they were duplicate samples from 1 patient. Among 338 samples included, 250 were collected as part of the point-prevalence study. We excluded 8 of these because the samples were collected from patients <1 year of age. In all, we included 242 samples in the point-prevalence study.

Diagnostic Algorithms

Figure 2. Testing algorithms at the 36 laboratories participating in a prevalence study comparing molecular and toxin assays for nationwide surveillance of Clostridioides difficile, Switzerland. EIA, enzyme immunoassay; GDH, glutamate dehydrogenase; NAAT, nucleic…

Among the 36 participating laboratories, 1 routinely tested all diarrheal stool samples for toxigenic C. difficile and 35 tested only if a specific test was requested. Optimized diagnostic tests for detection of toxigenic C. difficile were used by 58% (21/36) of laboratories in the winter sampling period and by 61% (22/36) in the summer sampling period. Among laboratories not following the recommendations of the European Society of Clinical Microbiology and Infectious Diseases (16), 9 in the winter sampling period and 10 in the summer sampling period used a nucleic acid amplification test (NAAT) alone, and 5 in the winter sampling period and 3 in the summer sampling period used EIAs for A and B toxins either as a standalone test or as an initial screening test. Only 1 laboratory reported having established PCR ribotyping methodologies (Figure 2).

Point-Prevalence Analyses

We collected demographic characteristics of patients whose stool samples tested positive by our testing algorithms (Table 1). C. difficile tests were required and performed for 68% (165/242) of stool samples; 6% (27/165) were reported as positive by the affiliated microbiology laboratory.

At the coordinating center, we detected C. difficile in 9% (21/242) of samples by EIA for GDH and A and B toxins and in 12% (30/242) of samples by PCR alone. Among all 27 samples reported as positive by the participating centers, we confirmed 18 (67%) by EIA and 24 (89%) by PCR. Among 138 samples reported as negative by the participating centers, 1 (1%) sample tested positive by EIA and 3 (2%) tested positive by PCR at the coordinating center. Among 77 samples not tested for C. difficile at the participating centers, we detected C. difficile in 2 (3%) by EIA and in 3 (4%) by PCR. Among 21 stool samples that tested positive by EIA at the coordinating center, a C. difficile test was not requested in 2 (10%) cases. Among 30 samples that tested positive by PCR at the coordinating center, a C. difficile test was not requested in 3 cases (10%; Table 2).

Measured detection and testing rates of toxigenic C. difficile were higher than the reported rates across all participating institutions (Table 3). Depending on the diagnostic algorithm applied, the largest difference in prevalence across all institutions was measured during the winter sampling period, which had a prevalence of 6.4 cases/10,000 patient bed-days by EIA and 11.4 cases/10,000 patient bed-days by PCR alone. Thus, across all institutions, rates of toxigenic C. difficile detection by PCR alone were <80% higher than detection rates by EIA for GDH and A and B toxins. In addition, detection rates by PCR alone were <100% higher in dedicated children’s hospitals (Table 3).

Ribotype Distribution

Figure 3. Distribution of PCR ribotypes among 107 samples collected in a prevalence study comparing molecular and toxin assays for nationwide surveillance of Clostridioides difficile, Switzerland. *Unknown ribotype.

We cultured and ribotyped 107 toxigenic C. difficile strains, 29 from the 2 point-prevalence days and 78 collected <1 week before each prevalence day. We identified a large diversity of C. difficile ribotypes, 23 (22%) had not been referenced before. The ribotypes most commonly identified included RT014 (12/107; 11%), presumably hypervirulent RT078 (9/107; 8%), RT001 (7/107; 7%), and RT002 (7/107; 7%) (Figure 3).



In this nationwide multicenter study, we found that PCR as a stand-alone test increased reported C. difficile prevalence rates <80% compared with a 2-stage EIA-based algorithm. At first glance, this finding was not surprising given the higher sensitivity of EIA (16). However, the fact that our results and conclusions are based on a nationwide cohort representing all geographic regions of Switzerland adds to the study’s credibility. In addition, our results strengthen the advice of the European Society of Clinical Microbiology and Infectious Diseases study group for C. difficile against use of a single commercial test for diagnosing CDI because of the low positive predictive values when CDI prevalence is low, 46% at a CDI prevalence of 5% (16). However, CDC and ECDC protocols for CDI surveillance define a case of CDI as the combination of diarrheal stool and a positive PCR (12,13). In addition, the clinical practice guidelines for CDI in adults and children published by the Infectious Diseases Society of America and Society for Healthcare Epidemiology of America recommend testing by different approaches, such as multistep algorithms or NAAT, depending on the degree of clinical suspicion (17). Based on a systematic review and meta-analysis, the American Society of Microbiology also recommends different approaches, including NAAT-only testing, and algorithms that include GDH and NAAT or GDH, toxins, and NAAT (18). Although these recommendations stand to reason for detection of CDI in individual patients, our results challenge their utility for meaningful comparisons in surveillance studies and suggest that uniform definitions should be provided.

On both point-prevalence days, we noted a higher nationwide rate of toxigenic C. difficile than previously reported in incidence studies performed at different institutions in Switzerland (1921). Our findings suggest that CDI rates have increased during the last decade in Switzerland, a finding that is in line with reports from other countries in Europe (11). Using the same diagnostic algorithm, diagnostic test, and a similar study design to the multicenter point-prevalence study of CDI in hospitalized patients with diarrhea in Europe, the nationwide mean prevalence rates are comparable in Switzerland (mean 6.1 cases/10,000 patient bed-days) and Europe (7.0 cases/10,000 patient bed-days) (11). Because we only included liquid stools in our study, our mean prevalence rate of 9.3 cases/10,000 patient bed-days measured by PCR fulfills the ECDC case definition and further shows that CDI is increasing substantially nationwide.

We found a lower proportion of missed detection of toxigenic C. difficile in Switzerland (9.5%), driven by the absence of clinical suspicion, compared with Europe (23%), which equates to 1 missed case of C. difficile per day among the included institutions in Switzerland. False-negative testing accounted for 1 additional missed diagnosis during both point-prevalence days, which extrapolates to »550 missed cases of C. difficile per year among hospitals across the nation.

We detected a variety of different RTs during our study period, 21% of which had not been referenced before. Of note, we did not recover hypervirulent RT027, but RT078 was the third most common strain circulating in Switzerland during our study. In contrast, a point-prevalence study in Europe identified RT027 as the most commonly circulating strain during its study period but did not detect RT078. RT078 has been associated with similarly severe disease manifestations as RT027, but RT078 has been reported to affect younger patients and to be linked more commonly with community-associated disease in the Netherlands (22). RT078 has been isolated from piglets with diarrhea, possibly suggesting ongoing transmission by introduction to the food chain because isolates from humans and pigs were found to be highly genetically related (22). A component of RT078 infections also was reported in Northern Ireland, which has a large pig population and »1:1 ratio of cattle to humans (23). In Switzerland, RT078 has been isolated previously from 6 wastewater treatment plants, suggesting its dissemination in the community (24). Except for both hypervirulent RT027 and RT078, we identified other similarities in RT distribution between Switzerland and the rest of Europe; RT014, RT001, RT002, and RT020 were among the 10 most commonly identified ribotypes in both settings.

Our study has some limitations, most of which are intrinsic to point-prevalence studies. First, our study only reflects frequency of toxigenic C. difficile detected on 2 days in 2015; therefore, we cannot draw solid conclusions regarding incidence. We expanded the timeframe for assessing the distribution of ribotypes circulating in Switzerland by an additional week for each prevalence day, but this still represents a limited collection of the true incidence. Second, we cannot rule out introduction of bias to testing policies at the participating hospitals, which might have increased testing on the 2 point-prevalence days. However, we did not provide any promotional information regarding our study, so alterations in daily clinical practice among treating physicians is unlikely on these 2 days. Third, we only included liquid stool samples for analyses, but we did not consider any other preanalytical factors, such as the use of laxatives, for testing eligibility. Finally, we applied surveillance definitions recommended by CDC and ECDC rather than defintions used for the clinical diagnosis of CDI in individual patients, such as detection of C. difficile in the context of symptoms related to CDI. Therefore, we cannot rule out detection of toxigenic C. difficile from colonization rather than infection in some cases.

In conclusion, this nationwide multicenter study reveals that PCR as a stand-alone test results in an increase of C. difficile prevalence rates of <80% compared with a 2-stage EIA-based algorithm. Our findings underscore the need for consistent testing algorithms for meaningful interinstitutional and nationwide comparisons. Our results also challenge the utility of the current CDC and ECDC case definitions and highlight the need for uniform recommendations on diagnostic approaches.


Dr. Widmer is head of the infection control program at University Hospital Basel, University of Basel, Switzerland. His research interests include all aspects of Clostridioides difficile and the epidemiology and prevention of hospital-acquired infections.



We acknowledge and thank the ESCMID (European Society of Clinical Microbiology and Infectious Diseases) Study group for C. difficile (ESGCD) for professional support. We also thank all participating centers and laboratories (Appendix).

Astellas Pharmaceuticals Europe provided financial support for this study. The funder did not influence the study design and did not contribute to data collection, data analysis, data interpretation, or writing of the report. Astellas Pharma Europe reviewed the report for factual accuracy before submission, in line with the terms of the funding agreement. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication. Alere provided C. DIFF QUIK CHEK COMPLETE test kits for conducting EIAs to detect GDH and toxins A and B.

The authors declare the following possible conflicts of interest: A.W. is a member of the Astellas and Merck Sharp & Dohme Corp. advisory boards for C. difficile and reports grants from the Swiss National Science Foundation. S.T.-S. is a member of the Astellas and Merck Sharp & Dohme Corp. advisory boards for C. difficile and reports grants from the Swiss National Science Foundation (grant nos. NRP72 and 407240_167060), the Gottfried und Julia Bangerter-Rhyner Stiftung, and the Fund for the Promotion of Teaching and Research of the Voluntary Academic Society, Base


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Cite This Article

DOI: 10.3201/eid2610.190804

Original Publication Date: September 09, 2020


Resource:  https://wwwnc.cdc.gov/eid/article/26/10/19-0804_article

Study Finds Factors That May Help Predict Which Patients Are More Likely to Develop a CDI

A cluster of factors may help predict
which patients are likely to develop Clostridium difficile
infection, a new study has found. And that could help
in efforts to prevent infection, according to the researchers.

Reduced immune function, recent antibiotic use, current or recent hospitalization and prior C. difficile infection predicted risk of subsequent infection, opening the door to potential preventive interventions.

“This could help healthcare providers red-flag those patients who are at high risk of C. diff, and may one day lead to therapeutic or dietary tactics to lower the chances of infection,” said the study’s co-lead author, Vanessa Hale of Ohio State University.

The study appears in the journal Science Translational Medicine.

The research included studies in both humans and mice, and involved the transplant of feces from human study participants to mice to assess differences in susceptibility to
C. difficile infection and molecular-level explanations for that increased risk.

“Microbes in the gut play a critical role in defending against disease, and the really exciting part of this study is that it might help us better identify the risk factors that are linked to problems in the gut and susceptibility to these dangerous infections,” said Hale, an assistant professor of veterinary preventive medicine at Ohio State. The study was conducted at the Mayo Clinic, where she previously worked.

The researchers started by looking at the gut microbes of a group of 115 people who had diarrhea but who did not have C. diff when they first sought medical care, some of whom went on to develop a C. diff infection. They also analyzed the gut microbes of 118 healthy volunteers for comparison.

“About half of the diarrhea patients had gut microbial communities that looked healthy, but the guts of the other half were really intriguing – they had different microbes and very different levels of metabolites. We called this half the ‘dysbiotic’ – or unhealthy – group,” Hale said.

“When we transplanted human stool from the dysbiotic group into mice, we discovered that these mice were more likely to become infected with C. diff than mice that received human stool from the healthy-looking group.”

The researchers then examined potential risk factors found on the medical charts of individuals with “dysbiotic” and healthy-looking gut microbial communities and found a cluster of five factors that were associated with unhealthy communities.

“We knew that dysbiotic microbial communities put mice at higher risk of C. diff infection, and we wanted to see if the five factors could be used to predict C. diff infections in humans,” Hale said.

To do this, the research team went back and looked at the medical charts of more than 17,000 previous patients who were free of C. diff when they initially sought care. In that larger group, there also was a clear connection between the risk factors and subsequent C. diff infection.

Furthermore, the researchers found higher levels of amino acids – particularly proline – in the guts of mice that received transplants from people whose gut microbiomes were unhealthy, or dysbiotic.

That was interesting, and potentially important, because C. diff needs amino acids like proline to proliferate and it cannot make proline on its own. That prompted the team to wonder if reducing dietary amino acids could protect against C. diff, Hale said.

Feeding the mice diets low in protein moderately lowered the growth of C. diff, providing further evidence that amino acids – including proline – play a role in risk of infection and leaving researchers curious about the potential for dietary interventions in at-risk humans, Hale said.

“It’s possible that a dietary strategy could reduce C. diff infection in those patients who are deemed to be susceptible based on the cluster of risk factors we identified,” she said, adding that more study is needed to understand that relationship.

The study also showed that prophylactic fecal transplantation from a healthy donor could protect against C. diff in mice that were initially prone to infection.

“The transplants were fully protective against C. diff infection in all of the animals we tested, which was pretty amazing,” Hale said.

………………………It is unlikely that fecal transplantation would quickly be adopted as a prevention strategy in those deemed to be at elevated risk of infection, Hale said.


The National Institutes of Health and the Center for Individualized Medicine at Mayo Clinic supported the study.

Eric Battaglioli of Georgetown College was the co-lead author. Purna Kashyap of Mayo Clinic is the senior author.

Researchers Find Systematically Addressing Penicillin Allergies May Be An Important Public Health Strategy to Reduce the Incidence of MRSA and C. difficile Among Patients With a Penicillin Allergy

BMJ 2018; 361 doi: https://doi.org/10.1136/bmj.k2400 (Published 27 June 2018) Cite this as: BMJ 2018;361:k2400

  1. Kimberly G Blumenthal, assistant professor of medicine123,
  2. Na Lu, biostatistician1,
  3. Yuqing Zhang, professor of medicine13,
  4. Yu Li, research assistant12,
  5. Rochelle P Walensky, professor of medicine234,
  6. Hyon K Choi, professor of medicine13

Author affiliations

  1. Correspondence to: K G Blumenthal kblumenthal1@partners.org (or @KimberlyBlumen1 on Twitter)
  • Accepted 30 April 2018


Objective To evaluate the relation between penicillin allergy and development of meticillin resistant Staphylococcus aureus (MRSA) and C difficile.

Design Population based matched cohort study.

Setting United Kingdom general practice (1995-2015).

Participants 301 399 adults without previous MRSA or C difficile enrolled in the Health Improvement Network database: 64 141 had a penicillin allergy and 237 258 comparators matched on age, sex, and study entry time.

Main outcome measures The primary outcome was risk of incident MRSA and C difficile. Secondary outcomes were use of β lactam antibiotics and β lactam alternative antibiotics.

Results:       Among 64 141 adults with penicillin allergy and 237 258 matched comparators, 1365 developed MRSA (442 participants with penicillin allergy and 923 comparators) and 1688 developed C difficile (442 participants with penicillin allergy and 1246 comparators) during a mean 6.0 years of follow-up.

Among patients with penicillin allergy the adjusted hazard ratio for MRSA was 1.69 (95% confidence interval 1.51 to 1.90) and for C difficile was 1.26 (1.12 to 1.40). The adjusted incidence rate ratios for antibiotic use among patients with penicillin allergy were 4.15 (95% confidence interval 4.12 to 4.17) for macrolides, 3.89 (3.66 to 4.12) for clindamycin, and 2.10 (2.08 to 2.13) for fluoroquinolones. Increased use of β lactam alternative antibiotics accounted for 55% of the increased risk of MRSA and 35% of the increased risk of C difficile.

Conclusions Documented penicillin allergy was associated with an increased risk of MRSA and C difficile that was mediated by the increased use of β lactam alternative antibiotics. Systematically addressing penicillin allergies may be an important public health strategy to reduce the incidence of MRSA and C difficile among patients with a penicillin allergy label.


One third of patients report a drug allergy (ie, adverse or allergic reaction),1 the most commonly implicated drug being penicillin and documented in 5-16% of patients.12345 Being labelled with a penicillin allergy affects future prescribing for infections in both outpatients and inpatients, with prescribed antibiotics often more broad spectrum and toxic.2678 Unnecessary use of broad spectrum antibiotics leads to the development of drug resistant bacteria, including meticillin resistant Staphylococcus aureus (MRSA), and healthcare associated infections such as Clostridium difficile related colitis.910111213

Most patients with a documented penicillin allergy are not allergic—that is, there is no immediate hypersensitivity.1415 After evaluation by an allergist, about 95% of patients with reported penicillin allergies were found to be penicillin tolerant.14 The discrepancy between labelled and confirmed penicillin allergy stems from misdiagnosis (eg, a viral exanthem is misinterpreted as an allergy), misassumptions (eg, an intolerance, such as a headache, is listed as an allergy), and remote timing of the allergy evaluation, since 80% of patients with immediate hypersensitivity to penicillin are no longer allergic after 10 years.16 Most patients with a penicillin allergy label therefore unnecessarily avoid penicillins, and often other related β lactam antibiotics, such as cephalosporins.67

To evaluate the public health consequences of having a penicillin allergy label, we conducted a population based matched cohort study and examined the relation between a newly recorded penicillin allergy and the risk of incident MRSA and C difficile.


Data source

We used data from the Health Improvement Network (THIN), an electronic medical record database of 11.1 million patients registered with general practices in the United Kingdom. Because the National Health Service requires people to register with a general practice regardless of health status, THIN is a population based cohort representative of the UK general population.17 During consultations with patients in primary care, general practitioners (GPs) enter clinical data, including height, weight, smoking status, diagnoses, and prescription drugs. Patient diagnoses are recorded using READ codes, the UK’s standard clinical terminology system.18 Drug allergies are linked to a drug prescription, or recorded as a diagnosis (eg, personal history of penicillin allergy). The GP enters details of the drug allergy, including reaction type, severity of reaction, and certainty of diagnosis. All GPs are trained in data entry, with the quality of their data periodically reviewed. Previous studies using THIN have confirmed the validity of both prescriptions and diagnoses.1920

Study design

We performed a matched cohort study among participants aged more than 18 years, who were enrolled in the THIN database between 1995 and 2015. Eligible participants had no history of MRSA or C difficile diagnoses before study entry and were required to have at least one year of enrolment with a general practice before entering the study to allow for assessment of exposure and covariates. We identified adults with their first recorded penicillin allergy and selected up to five penicillin users without a penicillin allergy matched on age (one year either way), sex, and study entry time (within one year either way). Such comparators were chosen to further ensure the comparability of indications for penicillin use (eg, infection tendency) and associated features. The index date for cases was the date of first entry of an allergy diagnosis in the THIN database; the matched index date for comparators was within one year of a penicillin prescription.

Assessment of exposure and outcomes

The exposure of interest was a documented penicillin allergy, defined as an allergy to a penicillin antibiotic linked to a penicillin prescription, or one or more relevant READ diagnosis codes for a penicillin allergy or adverse effect (see supplemental table 1).

The primary outcomes were incident cases of doctor diagnosed MRSA and C difficile during the follow-up period. We identified MRSA and C difficile by the presence of one or more relevant READ diagnosis codes.21222324 For MRSA, codes indicated MRSA infection, carriage, eradication, or decontamination whereas for C difficile, codes indicated C difficile infection or detection of antigen or toxin (see supplementary table 1).

We also assessed antibiotic utilization during the follow-up period, derived from the prescription record. We grouped all antibiotics prescribed into classes: penicillins, first generation cephalosporins, macrolides, clindamycin, fluoroquinolones, tetracyclines, and sulfonamides. Given that vancomycin, aminoglycosides, and linezolid are commonly administered parenterally and therefore seldom administered to outpatients by GPs, we assessed these antibiotics separately.

Assessment of covariates

We identified demographic and lifestyle factors before the index date, such as age, sex, body mass index, socioeconomic status, smoking status, and alcohol use. READ diagnosis codes at the index date were used to ascertain relevant comorbidities (diabetes, renal disease, hemodialysis, malignancy, liver disease, and infection with human immunodeficiency virus (HIV)) and to calculate the adapted Charlson comorbidity index25 at baseline. Using the prescription records, we identified the number of antibiotics prescribed in the year before the index date and whether proton pump inhibitors or systemic corticosteroids were used at baseline. Concomitant allergies to cephalosporin antibiotics and other antibiotics were linked to prescriptions or identified using READ diagnosis codes. We determined participants who were residents of nursing homes at baseline. Finally, we calculated the number of visits to a GP and hospital admissions during the year before the index date.

Statistical analysis

We compared baseline characteristics between participants with penicillin allergy and their comparators. Follow-up time for each participant was calculated from the index date to the date of one of several events: the study endpoints (MRSA or C difficile), death, or end of the study (31 December 2015), whichever occurred first.

We identified incident MRSA cases and number of person years of follow-up for each cohort separately. We calculated the hazard ratios for the relation of penicillin allergy status to the risk of MRSA using Cox proportional hazard models. In the multivariable Cox model we adjusted for age, sex, body mass index, socioeconomic status, smoking status, alcohol use, Charlson comorbidity index, hemodialysis, number of antibiotic prescriptions, proton pump inhibitor use, corticosteroid use, other antibiotic allergies, resident of nursing home, visits to a GP, and admissions to hospital. We repeated the same analyses for the risk of C difficile. We also calculated the absolute risk difference.

In both the penicillin allergy cohort and the comparison cohort we determined the rates of subsequent antibiotic utilization by class. We used Poisson regression models to estimate the incidence rate ratio for the relation of penicillin allergy status to the rates of subsequent antibiotic use, while adjusting for the same covariates.

We performed mediation analyses to examine the extent to which the effect of penicillin allergy status on the risk of MRSA or C difficile was through its effect on utilization of β lactam alternative antibiotics.26 Specifically, we grouped utilization into five categories based on previous studies that evaluated the impact of various antibiotics on the risk of MRSA and C difficile910111213: fluoroquinolones, clindamycin, macrolides, vancomycin, aminoglycosides, and linezolid (all β lactam alternative antibiotics considered in this study); fluoroquinolones, clindamycin, and macrolides; fluoroquinolones and macrolides; fluoroquinolones and clindamycin; and fluoroquinolones alone. Using marginal structural models we then estimated the natural direct effect (ie, the effect of penicillin allergy status on the risk of MRSA or C difficile not through a specific group of antibiotics) and the natural indirect effect (ie, the effect of penicillin allergy status on the risk of MRSA or C difficile through a specific group of antibiotics), while adjusting for the same confounding variables,26 and reported the adjusted risk ratio and percentage mediated.

For all analyses we imputed unknown values for covariates (ie, missing body mass index, alcohol use, and smoking status) using a sequential regression method based on a set of covariates as predictors. To minimize random error, we imputed five datasets and then combined estimates from these datasets by calculating effect estimates from each imputed dataset and then averaging estimates and their confidence intervals using Rubin’s rules.27 All analyses were performed using SAS, version 9.2 (SAS Institute, Cary, NC).

Patient and public involvement

No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for implementation of the study. No patients were asked to advise on interpretation or writing up of results. There are no plans to disseminate the results of the research to study participants or the relevant patient community. Individual patient consent was not sought given the use of anonymized data.


Cohort identification and characteristics

We identified 64 141 patients with a documented penicillin allergy and 237 258 matched comparators (table 1). Patients with penicillin allergy were identified through allergies linked to prescriptions for penicillin antibiotics (63 245/64 141, 98.6%). Documented penicillin allergies consisted of allergies (74.4%), intolerances (14.5%), and adverse effects (11.1%). Most allergies were considered of moderate severity (86.0%) with likely certainty (73.6%).

Table 1

Reactions in patients with penicillin allergy (n=64 141)

Patients with penicillin allergy were similar to their comparators for age, sex, body mass index, socioeconomic status, smoking status, and alcohol use (table 2). They were also similar for diabetes, renal disease, hemodialysis, malignancy, liver disease, HIV, Charlson comorbidity index, previous antibiotic prescriptions, use of proton pump inhibitors and systemic corticosteroids, nursing home residency, visits to a GP, and hospital admissions. Other antibiotic allergies were more common in patients with a penicillin allergy.

Table 2

Cohort characteristics according to penicillin allergy status. Values are numbers (percentages) unless stated otherwise

Penicillin allergy and risk of MRSA and C difficile

During the mean follow-up time of 6.0 years for patients with penicillin allergy and 6.1 years for comparator patients, 442 patients with penicillin allergy and 923 comparator patients developed MRSA, and 442 patients with penicillin allergy and 1246 comparator patients developed C difficile (table 3 and supplemental table 2).

Table 3

Impact of listed penicillin allergy on risk of meticillin resistant Staphylococcus aureus (MRSA)and Clostridium difficile

The age, sex, and study entry time matched hazard ratios for patients with penicillin allergy were 1.84 (95% confidence interval 1.64 to 2.06) for MRSA and 1.37 (1.23 to 1.53) for C difficile. The matched and multivariable adjusted hazard ratios for patients with penicillin allergy were 1.69 (1.51 to 1.90) for MRSA and 1.26 (1.12 to 1.40) for C difficile, respectively. The corresponding adjusted risk differences were 49/100 000 person years for MRSA and 27/100 000 person years for C difficile.

Penicillin allergy and subsequent antibiotic utilization

Patients with penicillin allergy were less often prescribed penicillin than their comparators (adjusted incidence rate ratio 0.30, 95% confidence interval 0.30 to 0.31), but had increased use of macrolide antibiotics (4.15, 4.12 to 4.17), clindamycin (3.89, 3.66 to 4.12]), fluoroquinolones (2.10, 2.08 to 2.13), tetracyclines (1.75, 1.73 to 1.76), and sulfonamide antibiotics (1.26, 1.25 to 1.27; table 4). Though vancomycin, aminoglycosides, and linezolid were overall infrequently prescribed, they were more often prescribed to patients with penicillin allergy than to their comparators (supplemental table 3).

Table 4

Impact of listed penicillin allergy on antibiotic use

Mediation effects of alternative antibiotic use

Compared with patients who did not receive penicillins, patients receiving penicillins did not have an increased risk of MRSA (adjusted risk ratio 1.07, 95% confidence interval 0.95 to 1.20), but had an increased risk of C difficile (1.18, 1.06 to 1.31; supplemental table 4). Patients receiving macrolide antibiotics had an increased risk of MRSA (1.72, 1.54 to 1.91) and C difficile (1.30, 1.18 to 1.43). Patients receiving clindamycin had an increased risk of MRSA (2.97, 2.11 to 4.16) and C difficile (2.76, 2.00 to 3.81). Patients receiving fluoroquinolones had an increased risk of MRSA (2.38, 2.12 to 2.67) and C difficile (1.72, 1.54 to 1.93).

The effect of a penicillin allergy on the risk of MRSA was 55% mediated through β lactam alternative antibiotic classes; 55% mediated through fluoroquinolones, clindamycin, and macrolides; 54% mediated through fluoroquinolones and macrolides; 26% mediated through fluoroquinolones and clindamycin; and 24% mediated through fluoroquinolones alone (table 5). The effect of penicillin allergy on C difficile was 35% mediated through β lactam alternative antibiotic classes; 26% mediated through fluoroquinolones, clindamycin, and macrolides; 24% mediated through fluoroquinolones and macrolides; 20% mediated through fluoroquinolones and clindamycin; and 16% mediated through fluoroquinolones alone.

Table 5

Mediation analysis to estimate the indirect effect of listed penicillin allergy on meticillin resistant Staphylococcus aureus (MRSA) and Clostridium difficile


In this large cohort study reflective of the United Kingdom general population, we found that a penicillin allergy label was associated with a 69% increased risk of MRSA and a 26% increased risk of C difficile. Once documented, a penicillin allergy resulted in increased use of β lactam alternative antibiotics, with a fourfold increased incidence of macrolides and clindamycin utilization, and a twofold increased incidence of fluoroquinolone utilization. Furthermore, more than half of the increased MRSA risk and more than one third of the increased C difficile risk among patients with penicillin allergy was attributable to administered β lactam alternative antibiotics.

Comparison with other studies and policy implications

We found that patients with a penicillin allergy label had nearly a 70% increased risk of new MRSA than their matched comparators, even after adjustment for known MRSA risk factors.28 This provides supporting evidence for a previous US study that showed a 14% increased MRSA prevalence in inpatients who were allergic to penicillin.8 Our result emphasises that outpatient use of antibiotics is strongly associated with the risk of developing MRSA.2228 Consistent with previous studies, we found that β lactam alternative antibiotics increased the risk of MRSA to a greater degree than did penicillins12132930; whereas the mechanism of resistance is not known, the same factors that predispose staphylococcus to develop resistance to meticillin are thought to predispose staphylococcus to multidrug resistance that includes resistance to meticillin.122931 With more than half of the increased MRSA risk among patients with listed penicillin allergy directly attributable to increased outpatient β lactam alternative antibiotic use (largely fluoroquinolones and macrolides), this risk appears modifiable if prescribing patterns among those with penicillin allergy could be altered.

C difficile is responsible for almost one half million infections and 15 000 deaths each year in the US, and the Centers for Disease Control and Prevention consider C difficile one of three urgent threats to public health.32 Patients with penicillin allergy in this study had a 26% increased C difficile risk compared with age, sex, and study entry time matched comparators after adjustment for other known risk factors for C difficile.10333435 This result also corroborates the previous US study, which found a 23% increased C difficile prevalence in hospital patients with a penicillin allergy.8 While other studies similarly identified that clindamycin and fluoroquinolones were associated with the greatest risk of C difficile,91011 we found that 35% of the heightened risk of C difficile in patients with penicillin allergy was directly attributable to use of β lactam alternative antibiotics, with quinolone use alone responsible for 16% of the heightened risk. The mechanism by which antibiotic use precipitates C difficile is through disruption of the host microbiome and creation of an environment where C difficile can overgrow.36 Antibiotics not captured in this dataset (eg, those administered at dialysis or in hospitals) and non-antibiotic risk factors234 are likely responsible for the remainder of C difficile cases. Although current efforts to reduce C difficile largely focus on reducing C difficile infections in hospitals and rehabilitation centers, one third of C difficile infections occur in the community, and occur in outpatients.37 Our findings suggest that more systematic efforts to identify patients with listed penicillin allergy who are not truly allergic to penicillins could help decrease rates of community associated C difficile.

In this study, patients with documented penicillin allergy had an increased incidence of broad spectrum antibiotic use, including the extended Gram positive spectrum antibiotics vancomycin and linezolid, which should be reserved for patients with suspected or known MRSA (or vancomycin resistant enterococci for linezolid).3839 Use of the most narrow spectrum antibiotic that is effective for a given infection is a cornerstone of evidence based treatment for infection and is responsible antibiotic stewardship.38 Antibiotic stewardship committees enforce this aim in the hospital setting, with evaluations for penicillin allergy occasionally included in stewardship efforts.40 This analysis emphasises the importance of performing outpatient antibiotic stewardship and the role that penicillin allergy evaluations might play. Although diagnostic testing for penicillin allergy was developed in the 1960s, and has recently garnered the support of a variety of professional organizations,384142 less than 0.1% of patients with a penicillin allergy label undergo confirmatory testing.15 Evaluation of penicillin allergy often involves a skin test, and if the result of skin testing is negative, a challenge dose of penicillin or amoxicillin is administered under medical observation.15 With these evaluation tools, evaluation of penicillin allergy has a more than 99% negative predictive value, takes less than three hours to perform, and costs about $220 (£165; €188; 2016 currency conversion).1543 Previous observational cohorts have shown that more than 90% of patients with listed penicillin allergies can be safely treated with penicillins.141540

Strengths and limitations of this study

In this study we used a representative population based cohort to increase the generalizability of our findings. Clinical data to characterise drug use, outcomes, and covariates were entered by physicians and captured electronically. The dataset used included granular allergy data linked to penicillin prescriptions and defined by type, severity, and certainty. Our study design used a comparator group who had recently been prescribed a penicillin but did not have a resultant penicillin allergy. Patients had high antibiotic use in the previous year since almost the entire cohort had recently had a penicillin (for infection) at baseline for cohort eligibility. Our GP practice based dataset could have missed the detection of some inpatient cases of MRSA and C difficile; however, these potential non-differential misclassifications would have biased our results towards the null, rendering our findings conservative. MRSA and C difficile were identified by physician diagnosis records. This approach has been successfully used in many previous epidemiologic studies,131920212223244445 as microbial infections such as MRSA and C difficile are made objectively using highly accurate microbiologic and serologic tests. Although we used composite outcomes for MRSA and C difficile that were not restricted to infections, it is unlikely that GPs would screen asymptomatic patients and more likely that diagnoses occurred in relevant clinical contexts where infections were suspected. Further, our findings remained consistent and strong when we restricted the analyses to code subgroups suggestive of infections. Additionally, MRSA carriage alone is an important outcome that confers an increased risk of MRSA infection,46 and indicates antibiotic resistance—a healthcare priority throughout the world.3947 Finally, by choosing to study only the first documentation of MRSA and C difficile, we ensured capture of only new colonization or infection, which are clinically important outcomes. Although we controlled for many known potential confounders in these data, our observational study cannot rule out potential unknown or residual confounding.


In this population based cohort study, a listed penicillin allergy was associated with a statistically significantly increased risk of MRSA and C difficile compared with patients matched by age, sex, and study entry time. Approximately one third to more than one half of this risk was attributed to use of non-β lactam antibiotics administered to outpatients. As infections with resistant organisms increase, systematic efforts to confirm or rule out the presence of true penicillin allergy may be an important public health strategy to reduce the incidence of MRSA and C difficile.

What is already known on this topic

  • Penicillin allergy is the most commonly documented drug allergy, reported by about 10% of patients

  • Although documented allergies impact prescribing behavior, a documented penicillin allergy does not often represent true, immediate hypersensitivity to penicillin

  • Previous studies have identified specific antibiotic uses that increase the risk of MRSA and Clostridium difficile

What this study adds


  • Patients with a documented penicillin allergy have an increased risk of new MRSA and C difficile that are modifiable, to some degree, through changes in antibiotic prescribing

There Are Smart Antibiotics to treat C.difficile infections being developed by Researchers

Cationic amphiphilic bolaamphiphile-based delivery of antisense oligonucleotides provides a potentially microbiome sparing treatment

for C. difficile

The Journal of Antibiotics (2018) | Download Citation


Conventional antibiotics for C. difficile infection (CDI) have mechanisms of action without organismal specificity, potentially perpetuating the dysbiosis contributing to CDI, making antisense approaches an attractive alternative. Here, three (APDE-8, CODE-9, and CYDE-21) novel cationic amphiphilic bolaamphiphiles (CABs) were synthesized and tested for their ability to form nano-sized vesicles or vesicle-like aggregates (CABVs), which were characterized based on their physiochemical properties, their antibacterial activities, and their toxicity toward colonocyte (Caco-2) cell cultures. The antibacterial activity of empty CABVs was tested against cultures of E. coli, B. fragilis, and E. faecalis, and against C. difficile by “loading” CABVs with 25-mer antisense oligonucleotides (ASO) targeting dnaE. Our results demonstrate that empty CABVs have minimal colonocyte toxicity until concentrations of 71 µM, with CODE-9 demonstrating the least toxicity. Empty CABVs had little effect on C. difficile growth in culture (MIC90 ≥ 160 µM). While APDE-8 and CODE-9 nanocomplexes demonstrated high MIC90 against C. difficile cultures (>300 µM), CYDE-21 nanocomplexes demonstrated MIC90 at CABV concentrations of 19 µM. Empty CABVs formed from APDE-8 and CODE-9 had virtually no effect on E. coli, B. fragilis, and E. faecalis across all tested concentrations, while empty CYDE-21 demonstrated MIC90 of >160 µM against E. coli and >40 µM against B. fragilisand E. faecalis. Empty CABVs have limited antibacterial activity and they can deliver an amount of ASO effective against C. difficile at CABV concentrations associated with limited colonocyte toxicity, while sparing other bacteria. With further refinement, antisense therapies for CDI may become a viable alternative to conventional antibiotic treatment.


C. difficile infection (CDI) is the most frequently reported nosocomial bacterial infection [1] in the United States, accounting for more than 450,000 new cases annually and for more than four billion dollars in CDI-attributable annual health care costs [2]. CDI has a strong reliance on intestinal dysbiotic states, which, when combined with the presence of C. difficile in the human gut, represents the most common pathogenesis for CDI. The high prevalence of this infection is, in large part, due to formidable recurrence rates of 15–25% following first treatment [3] with conventional antibiotics (CAs). CAs have long been recognized as the most important risk factor for the development of CDI [4], due to their mechanisms of action lacking organismal specificity, leading to widespread changes in gut ecology [5], which can lead to CDI by disrupting the gut microbial community. Given the important role of intestinal dysbiosis in the development of CDI, there has also been recent interest in studying the effects of difficile-directed conventional antibiotics on the bacterial and fungal communities of human subjects being treated for CDI, as a way of potentially explaining the high persistence and recurrence rates of this disease. These more recent data [6] suggest that even difficile-directed conventional antibiotics could potentially contribute to the perpetuation of dysbiotic states, which in turn could perpetuate CDI, potentially leading to even primary treatment failures.

There has been previous [7, 8] interest in the development of antisense therapies to treat bacterial infections, in part due to concerns regarding antibiotic resistance to traditional drugs. Given the dependence of CDI on dysbiotic states, approaches using therapeutic antisense oligonucleotides (ASO) complimentary to specific C. difficile mRNAs could limit or prevent the expression of important bacterial genes leading to bacterial death, all while sparing other organisms. This approach would offer significant advantages over CAs, especially in terms of a more limited impact on gut microbial communities. Developing clinically effective antisense therapies targeting a Gram-positive organism requires several elements. Since antisense oligonucleotides will not be efficiently introduced into bacteria without assistance given the presence of both a cell membrane and a thick cell wall, a carrier molecule must be used to deliver the ASO. This carrier must complex with the ASO strongly enough to concentrate it, to protect it from degradation in the extracellular environment, and to focus its delivery on its target cell. In order to accomplish these activities, the carrier-ASO complex itself must be stable in the in vivo environment of the gut. Once at the cell, the carrier must be able to release its cargo. Simultaneously, the carrier must demonstrate both limited gut toxicity and limited antibacterial activity at the doses required to effectively treat the target bacteria.

Our group published the first [9] in vitro data for antisense therapies against CDI by complexing cyclohexyl dequalinium analogs to various ASO-targeting essential C. difficile genes. However, since dequalinium has both antibacterial activity as well as toxicity at higher doses, a better delivery compound for ASO is required if antisense approaches to CDI are to be further developed. Here, we report our data on vesicles formed from novel cationic amphiphilic bolaamphiphiles (CABs) as carriers for chimeric 25-mer 2′-O-methyl phosphorothioate ASO. CABs, characteristic of all bola-like compounds, have hydrophilic, positively charged end groups separated by a hydrophobic linker chain. This molecular structure enables CABs to form nano-sized vesicle-like aggregates (CABVs), which in turn allow them to complex with negatively charged oligonucleotides in addition to promoting electrostatic interactions with bacterial cell membranes for intracellular delivery of ASO. The synthesis, physiochemical properties, toxicity, and antibacterial properties of three novel CABs and their respective CABVs are described, and their specificity for C. difficile compared to several other organisms is also provided.

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C.difficile Study Using C. difficile Conditioned Medium of Six Different C. difficile Strains






Clostridium difficile infection (CDI) is typically associated with disturbed gut microbiota and changes related to decreased colonization resistance against C. difficile are well described.

However, nothing is known about possible effects of C. difficile on gut microbiota restoration during or after CDI.

In this study, we have mimicked such a situation by using C. difficile conditioned medium of six different C. difficile strains belonging to PCR ribotypes 027 and 014/020 for cultivation of fecal microbiota.

A marked decrease of microbial diversity was observed in conditioned medium of both tested ribotypes. The majority of differences occurred within the phylum Firmicutes, with a general decrease of gut commensals with putative protective functions (i.e. Lactobacillus, Clostridium_XIVa) and an increase in opportunistic pathogens (i.e. Enterococcus). Bacterial populations in conditioned medium differed between the two C. difficile ribotypes, 027 and 014/020 and are likely associated with nutrient availability. Fecal microbiota cultivated in medium conditioned by E. coli, Salmonella Enteritidis or Staphylococcus epidermidis grouped together and was clearly different from microbiota cultivated in C. difficile conditioned medium suggesting that C. difficile effects are specific.

Our results show that the changes observed in microbiota of CDI patients are partially directly influenced by C. difficile.