Scott R. Curry

Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections.

Clostridium difficile infection (CDI) is a leading cause of hospital-associated gastrointestinal illness and places a high burden on our health-care system. Patients with CDI typically have extended lengths-of-stay in hospitals, and CDI is a frequent cause of large hospital outbreaks of disease. This guideline provides recommendations for the diagnosis and management of patients with CDI as well as for the prevention and control of outbreaks while supplementing previously published guidelines. New molecular diagnostic stool tests will likely replace current enzyme immunoassay tests. We suggest treatment of patients be stratified depending on whether they have mild-to-moderate, severe, or complicated disease. Therapy with metronidazole remains the choice for mild-to-moderate disease but may not be adequate for patients with severe or complicated disease. We propose a classification of disease severity to guide therapy that is useful for clinicians. We review current treatment options for patients with recurrent CDI and recommendations for the control and prevention of outbreaks of CDI.

tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile

ABSTRACT Severe Clostridium difficile associated disease is associated with outbreaks of the recently described BI/NAP1 epidemic clone. This clone is characterized by an 18-bp deletion in the tcdC gene and increased production of toxins A and B in vitro. TcdC is a putative negative regulator of toxin A&B production. We characterized tcdC genotypes from a collection of C. difficile isolates from a hospital that experienced an outbreak caused by the BI/NAP1 epidemic clone. Sequence analysis of tcdC was performed on DNA samples isolated from 199 toxigenic C. difficile isolates (31% BI/NAP1) from 2001 and 2005. Sequences obtained from 36 (18.6%) isolates predicted wild-type TcdC (232 amino acid residues), whereas 12 (6.1%) isolates had tcdC genotypes with previously described 18- or 39-bp deletions. The remaining isolates comprised 15 unique genotypes. Of these, 5 genotypes contain 18- or 36-bp deletions. Of these five genotypes, one is characterized by a single nucleotide deletion at position 117 resulting in a frameshift that introduces a stop codon at position 196, truncating the predicted TcdC to 65 amino acid residues. All 62 of the isolates in this collection comprising the epidemic clone are characterized by this genotype. This result suggests that severe truncation of TcdC is responsible for the increased toxin production observed in strains belonging to the BI/NAP1 clone and that the 18-bp deletion is probably irrelevant to TcdC function. Further investigations are required to determine the effect of this and other tcdC genotypes on toxin production and clinical disease.

High Frequency of Rifampin Resistance Identified in an Epidemic Clostridium difficile Clone from a Large Teaching Hospital

BACKGROUND Rifampin is used as adjunctive therapy for Clostridium difficile-associated disease, and the drugs derivative, rifaximin, has emerged as an attractive antimicrobial for treatment of C. difficile-associated disease. Rifampin resistance in C. difficile strains has been reported to be uncommon. METHODS We examined the prevalence of rifampin resistance among 470 C. difficile isolates (51.1% during 2001-2002 and 48.9% during 2005) from a large teaching hospital. Rifampin sensitivity was performed using E-test. The epidemic BI/NAP1 C. difficile clone was identified by tcdC genotyping and multilocus variable number of tandem repeats analysis. A 200-base pair fragment of the rpoB gene was sequenced for 102 isolates. Data on rifamycin exposures were obtained for all patients. RESULTS Rifampin resistance was observed in 173 (36.8%) of 470 recovered isolates and 167 (81.5%) of 205 of epidemic clone isolates (P < .001). Six rpoB genotypes were associated with rifampin resistance. Of 8 patients exposed to rifamycins, 7 had rifampin-resistant C. difficile, compared with 166 of 462 unexposed patients (relative risk, 2.4; 95% confidence interval, 1.8-3.3). CONCLUSIONS Rifampin resistance is common among epidemic clone C. difficile isolates at our institution. Exposure to rifamycins before the development of C. difficile-associated disease was a risk factor for rifampin-resistant C. difficile infection. The use of rifaximin may be limited for treatment of C. difficile-associated disease at our institution.

Use of Multilocus Variable Number of Tandem Repeats Analysis Genotyping to Determine the Role of Asymptomatic Carriers in Clostridium difficile Transmission

BACKGROUND Previous studies have suggested that asymptomatic carriers of toxigenic Clostridium difficile are a source of hospital-associated (HA) infections. Multilocus variable number of tandem repeats analysis (MLVA) is a highly discriminatory molecular subtyping tool that helps to determine possible transmission sources. METHODS Clostridium difficile isolates were recovered from perirectal swabs collected for vancomycin-resistant Enterococcus (VRE) surveillance as well as from clinical C. difficile toxin-positive stool samples from July to November 2009 at the University of Pittsburgh Medical Center Presbyterian (UPMC). MLVA was performed to determine the genetic relationships between isolates from asymptomatic carriers and patients with HA C. difficile infection (HA-CDI). Asymptomatic carriage and HA-CDI isolates were considered to be associated if the carriage isolate was collected before the HA-CDI isolate and if the MLVA genotypes had a summed tandem-repeat difference of ≤ 2. RESULTS Of 3006 patients screened, 314 (10.4%) were positive for toxigenic C. difficile, of whom 226 (7.5%) were detected only by VRE surveillance cultures. Of 56 incident cases of CDI classified as HA at UPMC during the study with available isolates, 17 (30%) cases were associated with CDI patients, whereas 16 (29%) cases were associated with carriers. Transmission events from prior bed occupants with CDI (n = 2) or carriers (n = 2) were identified in 4 of 56 cases. CONCLUSIONS In our hospital with an established infection control program designed to contain transmission from symptomatic CDI patients, asymptomatic carriers appear to have played an important role in transmission. Identification and isolation of carriers may be necessary to further reduce transmission of C. difficile in such settings.

Association of Relapse of Clostridium difficile Disease with BI/NAP1/027

ABSTRACT Recurrent Clostridium difficile infection (CDI) occurs in up to 35% of patients. Recurrences can be due to either relapse with the same strain or reinfection with another strain. In this study, multilocus variable-number tandem-repeat analysis (MLVA) was performed on C. difficile isolates from patients with recurrent CDI to distinguish relapse from reinfection. In addition, univariate and multivariate analyses were performed to identify risk factors associated with relapse. Among patients with a single recurrence, relapse due to the original infecting strain was more prevalent than reinfection and the interval between episodes was shorter than among patients who had reinfections. Among patients with >1 recurrence, equal distributions of relapse and reinfection or a combination of the two episode types were observed. Initial infection with the BI/NAP1/027 epidemic clone was found to be a significant risk factor for relapse. This finding may have important implications for patient therapy. Classification of recurrent CDI episodes by MLVA can be utilized to make informed patient care decisions and to accurately define new CDI cases for infection control and reimbursement purposes.

Prevalence and Duration of Asymptomatic Clostridium difficile Carriage among Healthy Subjects in Pittsburgh, Pennsylvania

ABSTRACT Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens.

Perirectal Swab Surveillance for Clostridium difficile by Use of Selective Broth Preamplification and Real-Time PCR Detection of tcdB

ABSTRACT Active surveillance testing to identify and isolate asymptomatic carriers of toxigenic Clostridium difficile has been limited by the lack of a test that is sensitive, specific, and timely enough to serve as an infection control tool. We tested DNA preamplified from perirectal surveillance specimens in a liquid medium selective for C. difficile by using a modified commercial real-time PCR assay. All fermenting specimens were subcultured, and isolates were tested for toxigenicity. Culture-positive toxigenic isolates served as the gold standard for comparison with the broth preamplification/PCR assay. The limit of detection for the assay was 1 CFU. Relative to toxigenic anaerobic culture, the sensitivity, specificity, and positive and negative predictive values of this assay were 70/70 (100.0%), 422/426 (99.1%), 70/74 (94.6%), and 422/422 (100.0%), respectively. These data demonstrate that selective broth preamplification and real-time PCR of perirectal swab specimens constitute a practical approach to the detection of asymptomatic C. difficile carriage.

Prevalence of Clostridium difficile in Uncooked Ground Meat Products from Pittsburgh, Pennsylvania

ABSTRACT The prevalence of Clostridium difficile in retail meat samples has varied widely. The food supply may be a source for C. difficile infections. A total of 102 ground meat and sausage samples from 3 grocers in Pittsburgh, PA, were cultured for C. difficile. Brand A pork sausages were resampled between May 2011 and January 2012. Two out of 102 (2.0%) meat products initially sampled were positive for C. difficile; both were pork sausage from brand A from the same processing facility (facility A). On subsequent sampling of brand A products, 10/19 samples from processing facility A and 1/10 samples from 3 other facilities were positive for C. difficile. The isolates recovered were inferred ribotype 078, comprising 6 genotypes. The prevalence of C. difficile in retail meat may not be as high as previously reported in North America. When contamination occurs, it may be related to events at processing facilities.

Asymptomatic Clostridium difficile colonization as a reservoir for Clostridium difficile infection

Clostridium difficile (CD) infection (CDI) is the leading cause of healthcare associated diarrhea despite intense hospital infection prevention programs. A substantial proportion of the population is asymptomatically colonized with CD, and evidence is mounting that these individuals serve as a reservoir for CDI. The purpose of this review is to discuss the mechanisms by which individuals may harbor toxigenic CD but remain asymptomatic, the evidence that asymptomatically colonized individuals serve as a source of CDI, and the implications of this potential CD reservoir for healthcare infection prevention.

Escherichia coli Producing CMY-2 β-Lactamase in Retail Chicken, Pittsburgh, Pennsylvania, USA

To the Editor: Rates of resistance to various antimicrobial drugs are rapidly increasing in Escherichia coli, not only in health care settings but also in the community. The food supply is suspected as a potential source of antimicrobial-resistant E. coli strains, which include cephalosporin-resistant E. coli found in retail meat products and other types of food (1). We reported a high prevalence of cephalosporin-resistant E. coli, most of which produced CMY-2 β-lactamase, among retail poultry products in Pittsburgh, Pennsylvania, USA during 2006–2007 (2). CMY-2 is the most commonly acquired ampicillin C (AmpC)–type β-lactamase found in E. coli that cause human infections (3). The aim of this study was to investigate whether cephalosporin-resistant E. coli are present in retail raw meat and ready-to-eat meat products in our area 5 years after our previous study and define subtypes of concern. A convenience sampling of 104 raw ground meat products from 3 local grocery stores in Pittsburgh was performed during February–April 2011. Items purchased were samples of all available deli counter ground meat, all fresh sausages prepared in stores at the deli counter, and selected uncooked commercially packaged fresh and frozen sausages. Types of meat items were chicken (n = 22), turkey (n = 10), lamb (n = 2), pork (n = 43), and beef (n = 27). Approximately 10 g of each sample was excised and suspended in 10 mL of nutrient broth. After being incubated overnight at 37°C, 10 µL of broth was plated on MacConkey agar plates containing 2 mg/L of cefotaxime or ceftazidime, and the plates were incubated overnight at 37°C. Lactose-fermenting colonies were identified as E. coli by using standard biochemical methods, which included sulfide indole motility, growth on triple sugar iron medium, oxidase activity testing, and the API20E system (bioMerieux, Durham, NC, USA) as needed. Antimicrobial drug susceptibility was determined by using the disk diffusion method (AB Biodisk, Solna, Sweden) according to the manufacturer’s instructions and interpreted according to the criteria of the Clinical and Laboratory Standards Institute (4). Isolates were screened for extended-spectrum β-lactamase (ESBL) production by using the double-disk diffusion method and for acquired ampC-type β-lactamase genes by using multiplex PCR (4,5). Phylogenetic groups (A, B1, B2, and D) were determined as reported (6). Screening for sequence type (ST) 131 was conducted by using PCR and confirmed by using multilocus sequence typing (7,8). Pulsed-field gel electrophoresis was performed to determine clonal relationships by using XbaI and the protocol available through the PulseNet (www.cdc.gov/pulsenet/protocols.htm). Banding patterns were analyzed by using BioNumerics software version 6.01 (Applied Maths, Sint-Martens-Latem, Belgium). Among 104 meat samples, 9 contained cephalosporin-resistant E. coli, resulting in an overall prevalence of 8.7% (95% CI 4.0%–15.8%). Cephalosporin-resistant E. coli was isolated from 7 (31.8%) of 22 chicken, 1 (10.0%) of 10 turkey, and 1 (2.3%) of 42 pork samples. No cephalosporin-resistant E. coli was detected from beef and lamb samples. Incidence of samples with cephalosporin-resistant E. coli was lower than in our previous study (2). This finding may be caused by different types of samples included in the studies or a true decrease in incidence. Features of cephalosporin-resistant E. coli identified are summarized in the Table. None produced ESBL, but all 9 isolates were positive for the CMY-2 β-lactamase gene and positive results were confirmed by sequencing. CMY-2 is the most commonly observed acquired AmpC β-lactamase in E. coli and nontyphoidal Salmonella species in meat products (9). Table Characteristics of 9 cephalosporin-resistant Escherichia coli isolates in retail meat, Pittsburgh, Pennsylvania, USA* As for the phylogenetic groups, 6 (66.7%) of 9 cephalosporin-resistant E. coli belonged to group A, which is generally considered to be a commensal phylogenetic group. However, 1 group B2 isolate from a chicken sample was identified as ST131. The CMY-2 gene was located on an IncI1-type plasmid and was transferable to another E. coli strain by conjugation for this isolate. Two group D isolates from chicken that belonged to ST117, which has been reported in ESBL-producing isolates of human and animal origins (10); no clonality was observed for the other 7 isolates by pulsed-field gel electrophoresis, E. coli ST131 has emerged as a worldwide pathogen and causes mainly community-onset extraintestinal infections. Although the pandemic spread of E. coli ST131 was first identified in isolates producing CTX-M-15 ESBL, it is increasingly recognized that isolates belonging to this clone may also harbor other drug resistance determinants. Among acquired AmpC β-lactamases, CMY-2 has been most frequently reported in ST131 from human clinical isolates (3). Infections caused by CMY-producing E. coli are common but underrecognized because of the lack of standardized detection methods (2). Given the rapid global spread of the ST131 clone and the possibility of its transmission from food animals to humans, coupled with an abundance of CMY-2–encoding plasmids in poultry environments, E. coli ST131 producing CMY-2 β-lactamase may have potential to spread to humans. Our results also show that E. coli producing CMY-2 continues to be found commonly among retail chicken products in our study area.