Predominance of Clostridium difficile ribotypes 012, 027 and 046 in a university hospital in Chile, 2012.

In a 1-year survey at a university hospital we found that 20·6% (81/392) of patients with antibiotic associated diarrohea where positive for C. difficile. The most common PCR ribotypes were 012 (14·8%), 027 (12·3%), 046 (12·3%) and 014/020 (9·9). The incidence rate was 2·6 cases of C. difficile infection for every 1000 outpatients.

Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012.

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Comparison of multilocus variable-number tandem-repeat analysis and whole-genome sequencing for investigation of Clostridium difficile transmission.

No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ≥2 SNVs but ≤10 STRDs, and 69 (5%) had ≤2 SNVs but ≥10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

Morbidity and mortality associated with Clostridium difficile ribotype 078: a case-case study.

The morbidity and mortality associated with Clostridium difficile ribotype 078 were examined by comparison with other known outbreak strains. A healthcare interaction within eight weeks of a positive specimen significantly increased the likelihood of ribotype 078 compared with ribotype 027. Individuals with ribotype 078 also tended to come from community sources, have a hospital stay post specimen similar to ribotype 027 and a lower 30-day mortality, but these differences were not statistically significant. This study generates several hypotheses and a methodological platform to explore this unique profile.

Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.

Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance.

Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

Infection due to C. difficile ribotype 078: first report of cases in the Republic of Ireland.

Clostridium difficile is an important healthcare-associated pathogen. Hypervirulent strains such as those belonging to ribotype 027 have been widely reported in recent years. A second strain associated with hypervirulence is ribotype 078 and the prevalence of Clostridium difficile infection (CDI) due to this ribotype appears to be increasing. This report describes an outbreak, in which 15cases of CDI due to ribotype 078 were detected in an Irish hospital and from a nursing home in the hospital's catchment area. C. difficile ribotype 078 accounted for 15% of total isolates submitted for ribotyping. The average age of patients with CDI due to ribotype 078 was 76 years. Forty-six percent of patients experienced recurrence of symptoms within eight weeks of diagnosis and CDI was felt to have directly contributed to five of the eight deaths. Use of enhanced DNA fingerprinting identified clusters within the 15 cases and suggested hitherto unrecognised links between some patients with CDI. Such approaches offer the promise to delineate common sources and transmission routes for C. difficile.

The changing epidemiology of Clostridium difficile infections.

The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents.

A case-control study of community-associated Clostridium difficile infection.

OBJECTIVES: The aim of this study was to determine the incidence of and risk factors for community-associated Clostridium difficile infection (CDI). METHODS: Prospective surveillance of community-derived faecal samples for C. difficile cytotoxin, followed by a questionnaire-based case-control study in two distinct patient cohorts (one semi-rural and the other urban). RESULTS: The proportion of randomly selected faecal samples positive for C. difficile cytotoxin was 2.1% in both patient cohorts (median ages 73 and 45 years for the urban and semi-rural cohorts, respectively). Exposure to antibiotics in the previous 4 weeks, particularly multiple agents (P < 0.001), aminopenicillins (P < 0.05) and oral cephalosporins (P < 0.05), was significantly more frequent among cases than controls. Hospitalization in the preceding 6 months was significantly associated with CDI (45% versus 23%; P = 0.022). However, almost half the cases had not received antibiotic therapy in the month before C. difficile detection, and approximately one-third neither had exposure to antibiotics nor recent hospitalization. Contact with infants aged < or =2 years was significantly associated with CDI (14% versus 2%; P = 0.02). Prior exposure to gastrointestinal-acting drugs (proton pump inhibitor, H2 antagonist or non-steroidal anti-inflammatory) was not significantly more common in CDI cases. C. difficile PCR ribotype 001 caused 60% and 13% of urban and semi-rural community-associated CDI cases, respectively. CONCLUSIONS: Reliance on antibiotic history and age (> or =65 years) will contribute to missed diagnoses of community-associated CDI. Potential risk factors for community-associated CDI should be explored further to explain the large proportion of cases not linked to recent antibiotic therapy or hospitalization.

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains.

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.

Surveillance for mupirocin resistance following introduction of routine peri-operative prophylaxis with nasal mupirocin.

The authors have previously described the successful use of a five-day peri-operative prophylaxis regimen using nasal mupirocin and topical triclosan (PPNMTT) to prevent methicillin-resistant Staphylococcus aureus (MRSA) infection. The present article describes the results of repeated point-prevalence surveillance for four years to determine whether mupirocin resistance has emerged in surgical units using empirical, short-term, peri-operative prophylaxis with nasal mupirocin. Before starting PPNMTT and every six months thereafter for four years, point-prevalence surveillance was performed for nasal S. aureus carriage in all patients on five orthopaedic surgery wards, one vascular surgery ward and one elderly medicine control ward. S. aureus screening and clinical isolates (surgical patients) were undertaken for low- [minimum inhibitory concentration (MIC) 8-128 mg/L] and high-level (MIC > 128 mg/L) mupirocin resistance. All isolates were phage typed to determine whether there was evidence of the spread of clonal mupirocin-resistant strains. Of 593, 139 and 206 nasal screening swabs (taken after the regimen had started) from orthopaedic, vascular and control patients, 28%, 24% and 48% (orthopaedic/vascular surgery vs elderly medicine, P < 0.001) yielded S. aureus isolates, respectively, and 12%, 11% and 30% (P < 0.001) were MRSA positive, respectively. Of the S. aureus nasal screen isolates from orthopaedic/vascular surgery and control patients, 5% and 4%, respectively, were low-level mupirocin resistant (P > 0.1). Of 286 (orthopaedic/vascular surgery) and 68 (elderly medicine) clinical S. aureus isolates obtained after the regimen had started, 7% and 9% (P > 0.1), respectively, were low-level mupirocin resistant. No high-level mupirocin-resistant isolates were isolated from mupirocin (orthopaedic/vascular surgery) or elderly medicine control ward patients. There was no trend towards increasing prevalence of low-level mupirocin resistance during the four-year study period. The results of phage typing did not support the clonal spread of resistant strains. Long-term follow-up confirmed the efficacy of PPNMTT in reducing the prevalence of nasal carriage of S. aureus and MRSA in orthopaedic and vascular surgery patients. Despite four years of use of PPNMTT, there was no evidence of sustained emergence or spread of mupirocin resistance.

Increased rate of DNA recovery from United Kingdom epidemic Clostridium difficile PCR ribotype 1 strains stored cryogenically.

We noted that some Clostridium difficile isolates are nonrecoverable after frozen storage and so used molecular typing analysis to characterize DNA from these strains. The recovery rate of C. difficile PCR ribotype 1 was statistically significantly greater than that of other strains. This observation has implications for C. difficile epidemiological studies.

Effectiveness of topical chlorhexidine powder as an alternative to hexachlorophane for the control of Staphylococcus aureus in neonates.

We routinely phage-type Staphylococcus aureus isolates from high-risk inpatients each week. This surveillance approach previously identified a five-year outbreak of a methicillin-susceptible S. aureus strain (MSSA, PT 53,85), which affected 202 babies on a regional neonatal unit. We previously reported this outbreak and the multiple staged infection control measures that were required to end it. These included strict emphasis on hand hygiene, environmental and staff surveillance sampling, application of topical triclosan solution and hexachlorophane powder, aseptic handling of a skin protectant material, and use of topical mupirocin for staff nasal carriers of the endemic MSSA strain and for babies colonized or infected with S. aureus. In summer 2000 topical hexachlorophane powder became unavailable and we therefore substituted topical 1% chlorhexidine powder as part of routine umbilical decontamination. We have continued prospective S. aureus surveillance for the past five years to monitor the effect of this practice change. We observed a continued decline in the numbers of monthly MSSA isolates from neonatal unit babies. Since the substitution of chlorhexidine for hexachlorophane, the median monthly number of MRSA isolates has been 0.5 (range 0-4). Only sporadic S. aureus PT 53,85 isolates were recovered. Control of S. aureus in our regional neonatal unit, in particular an endemic MSSA strain, was maintained when topical umbilical hexachlorophane powder was substituted with 1% chlorhexidine powder.

Use of perioperative mupirocin to prevent methicillin-resistant Staphylococcus aureus (MRSA) orthopaedic surgical site infections.

We have examined whether topical perioperative prophylaxis can reduce the incidence of methicillin-resistant Staphylococcus aureus (MRSA) surgical site infections (SSIs). Using a controlled before and after approach on patients from four orthopaedic wards, undergoing orthopaedic surgery involving insertion of metal prostheses and/or fixation, received perioperative prophylaxis with nasal mupirocin for five days, and a shower or bath with 2% (v/v) triclosan before surgery (PPNMT). After introduction of PPNMT there was a marked decrease in incidence of MRSA SSIs (per 1000 operations) from 23 in the six months beforehand (period A) to 3.3 (P<0.001) and 4 (P<0.001) in subsequent consecutive six-month periods (B and C, respectively). Of 11 MRSA SSI cases that occurred during periods B and C, only one had actually received PPNMT, and 10 occurred after acute, as opposed to elective, surgery (P<0.001). Point prevalence nasal MRSA carriage decreased from 38% before PPNMT to 23% immediately after, and 20%, 7%, 10% and 8% (P<0.001) at six-monthly intervals post-intervention. Conversely, the prevalence of nasal MRSA carriage in a control elderly medicine ward did not change significantly. Vancomycin usage, in terms of defined daily doses, declined by 23%. Low-level mupirocin resistance was found in 2.3% of S. aureus isolates from orthopaedic patients before PPNMT, and in 3.9%, 6.1%, 10% and 0% in subsequent six month periods. No S. aureus isolates with high-level mupirocin resistance were found. PPNMT can reduce the incidence of MRSA SSls after orthopaedic surgery, probably by reducing nasal MRSA carriage in the endemic setting, without selecting for mupirocin resistance.

Comparison of the effect of detergent versus hypochlorite cleaning on environmental contamination and incidence of Clostridium difficile infection.

To determine how best to decontaminate the hospital environment of Clostridium difficile, we carried out a cross-over study on two elderly medicine wards to determine whether cleaning with a hypochlorite disinfectant was better than using neutral detergent in reducing the incidence of C. difficile infection (CDI). We examined 1128 environmental samples in two years, 35% of which grew C. difficile. There was a significant decrease of CDI incidence on ward X, from 8.9 to 5.3 cases per 100 admissions (P<0.05) using hypochlorite, but there was no significant effect on ward Y. On ward X the incidence of CDI was significantly associated with the proportion of culture-positive environmental sites (P<0.05). On ward Y the only significant correlation between CDI and C. difficile culture-positive environmental sites was in patient side-rooms (r=0.41, P<0.05). The total daily defined doses of cefotaxime, cephradine and aminopenicillins were similar throughout the trial. These results provide some evidence that use of hypochlorite for environmental cleaning may significantly reduce incidence of CDI, but emphasize the potential for confounding factors.

Evidence to support the existence of subgroups within the UK epidemic Clostridium difficile strain (PCR ribotype 1).

We used three different DNA fingerprinting techniques and clindamycin susceptibility testing to confirm that Clostridium difficile PCR ribotype I isolates can be divided into two subclones. This observation may permit a better understanding of the epidemiology and pathogenicity of C. difficile infection.

Molecular epidemiology of endemic Clostridium difficile infection.

This is the first study to provide a comprehensive insight into the molecular epidemiology of endemic Clostridium difficile and particularly that associated with a recently recognized epidemic strain. We DNA fingerprinted all C. difficile isolates from the stools of patients with symptomatic antibiotic-associated diarrhoea and from repeated samples of the inanimate ward environment on two elderly medicine hospital wards over a 22-month period. Notably, C. difficile was not recoverable from either ward immediately before opening, but was found on both wards within 1-3 weeks of opening, and the level of environmental contamination rose markedly during the first 6 months of the study period. C. difficile infection (CDI) incidence data correlated significantly with the prevalence of environmental C. difficile on ward B (r = 0.76, P < 0.05) but not on ward A (r = 0.26, P > 0.05). We found that RAPD and RS-PCR typing had similar discriminatory power, although, despite fingerprinting over 200 C. difficile isolates, we identified only six distinct types. Only two distinct C. difficile strains were identified as causing both patient infection and ward contamination. Attempts to determine whether infected patients or contaminated environments are the prime source for cross-infection by C. difficile had limited success, as over 90% of C. difficile isolates were the UK epidemic clone. However, a non-epidemic strain caused a cluster of six cases of CDI, but was only isolated from the environment after the sixth patient became symptomatic. The initial absence of this strain from the environment implies patient-to-patient and/or staff-to-patient spread. In general, routine cleaning with detergent was unsuccessful at removing C. difficile from the environment. Understanding the epidemiology and virulence of prevalent strains is important if CDI is to be successfully controlled.