Point-of-Care Testing for Clostridium Difficile Infection: A Real-World Feasibility Study of a Rapid Molecular Test in Two Hospital Settings.

INTRODUCTION: In the developed world, Clostridium difficile infection (CDI) is the most important cause of nosocomial infectious diarrhea. In addition to providing epidemiological data and helping to indicate that a local outbreak may be occurring, laboratory tests are used to augment clinical decisions on individual patients. Very rarely do diagnostic tests provide results at the point of decision making; in the intervening period between requesting investigations on a patient with suspected CDI and return of the laboratory result, decisions must be made regarding patient isolation and treatment. METHODS: A 22-month, real-world feasibility study was conducted in patients with clinically significant diarrhea, in a London Hospital between March 2011 and January 2013, in three older persons' wards and two intensive care units (ICUs) to determine acceptability, ease of use, change in turnaround time and clinical utility of a rapid, polymerase chain reaction (PCR)-based point-of-care test (POCT) (Cepheid GeneXpert(®), Sunnyvale, California, USA) for diagnosis of Clostridium difficile. Nurses in the older persons' ward and laboratory technicians in the ICU were trained to perform the test. Residual samples were sent to the centralized laboratory for parallel testing using a two-step algorithm. RESULTS: A total of 335 samples were tested using the POCT with a median turnaround time of 1.85 h compared with 18 h for the centralized laboratory test. Overall agreement with centralized laboratory testing was 98.1%. Discrepant samples were more frequent on elderly wards than ICU. Overall 20/335 (6%) processing errors were encountered and were highest in the first few months of the study. Significantly more processing errors occurred on the older persons' wards 13/102 (12.7%) than on ICU 7/271 (2.6%). Older persons' patients who had POCT were significantly less likely to have a test requested for bacterial stool culture (3.1% vs. 10.9% p = 0.044). This difference was not observed in the ICU patients. No other differences in ancillary test requesting, mortality or length of stay were observed. CONCLUSIONS: The majority of users reported that the POCT was easy to perform and was an acceptable part of their job. POCT using this system is feasible and acceptable to nursing staff and technicians working within these two hospital-based settings.

Identification and control of a gentamicin resistant, meticillin susceptible Staphylococcus aureus outbreak on a neonatal unit.

We describe the identification and control of an outbreak of gentamicin resistant, meticillin susceptible Staphylococcus aureus (GR-MSSA) on a 36-bed neonatal unit (NNU) in London. Control measures included admission and weekly screening for GR-MSSA, cohorting affected babies, environmental and staff screening, hydrogen peroxide vapour (HPV) for terminal disinfection of cohort rooms, and reinforcement of hand hygiene. Seventeen babies were affected by the outbreak strain over ten months; seven were infected and ten were asymptomatic carriers. The outbreak strain was gentamicin resistant and all isolates were indistinguishable by pulsed-field gel electrophoresis. The outbreak strains spread rapidly and were associated with a high rate of bacteraemia (35% of 17 affected patients had bacteraemia vs. 10% of 284 patients with MSSA prior to the outbreak, p=0.007). None of 113 staff members tested were colonised with GR-MSSA. GR-MSSA was recovered from 11.5% of 87 environmental surfaces in cohort rooms, 7.1% of 28 communal surfaces and 4.1% of 74 surfaces after conventional terminal disinfection. None of 64 surfaces sampled after HPV decontamination yielded GR-MSSA. Recovery of GR-MSSA from two high level sites suggested that the organism could have been transmitted via air. Occasional breakdown in hand hygiene compliance and contaminated environmental surfaces probably contributed to transmission.

Evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings.

Evidence that contaminated surfaces contribute to the transmission of hospital pathogens comes from studies modeling transmission routes, microbiologic studies, observational epidemiologic studies, intervention studies, and outbreak reports. This review presents evidence that contaminated surfaces contribute to transmission and discusses the various strategies currently available to address environmental contamination in hospitals.

Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes.

A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing.

An association between bacterial genotype combined with a high-vancomycin minimum inhibitory concentration and risk of endocarditis in methicillin-resistant Staphylococcus aureus bloodstream infection.

INTRODUCTION: Antimicrobial resistance and bacterial virulence factors may increase the risk of hematogenous complications during methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI). This study reports on the impact of increasing vancomycin minimum inhibitory concentrations (V-MICs) and MRSA clone type on risk of hematogenous complications from MRSA BSI during implementation of an effective MRSA control program. METHODS: In sum, spa typing, staphylococcal cassette chromosome mec allotyping, and vancomycin and teicoplanin MICs were performed on 821 consecutive MRSA bloodstream isolates from 1999 to 2009. Prospectively collected data, including focus of infection, were available for 695 clinically significant cases. Logistic and multinomial logistic regression was used to determine the association between clone type, vancomycin MIC (V-MIC), and focus of infection. RESULTS: MRSA BSIs decreased by ∼90% during the 11 years. Typing placed isolates into 3 clonal complex (CC) groups that had different population median V-MICs (CC30, 0.5 µg/mL [n = 349]; CC22, 0.75 µg/mL [n = 272]; non-CC22/30, 1.5 µg/mL [n = 199]). There was a progressive increase in the proportion of isolates with a V-MIC above baseline median in each clonal group and a disproportionate fall in the clone group with lowest median V-MIC (CC30). In contrast, there were no increases in teicoplanin MICs. High V-MIC CC22 isolates (1.5-2 µg/mL) were strongly associated with endocarditis (odds ratio, 12; 95% confidence interval, 3.72-38.9) and with a septic metastasis after catheter-related BSI (odds ratio, 106; 95% confidence interval, 12.6-883) compared with other clone type/V-MIC combinations. CONCLUSIONS: An interaction between clone type and V-MIC can influence the risk of endocarditis associated with MRSA BSI, implying involvement of both therapeutic and host-pathogen factors.

Lack of effect of strain type on detection of toxigenic Clostridium difficile by glutamate dehydrogenase and polymerase chain reaction.

Glutamate dehydrogenase (GDH) is popular as a preliminary test for the detection of Clostridium difficile. Recent work has suggested that GDH sensitivity may vary according to ribotype and may be lower for ribotypes 002, 027, and 106 compared with polymerase chain reaction (PCR). We investigated this effect using a dilution series of 64 isolates tested by GDH and Cepheid GeneXpert PCR. PCR was significantly more sensitive than GDH overall; however, there was no difference in detection according to specific ribotype.

The role played by contaminated surfaces in the transmission of nosocomial pathogens.

Studies in the 1970s and 1980s suggested that environmental surface contamination played a negligible role in the endemic transmission of healthcare-associated infections. However, recent studies have demonstrated that several major nosocomial pathogens are shed by patients and contaminate hospital surfaces at concentrations sufficient for transmission, survive for extended periods, persist despite attempts to disinfect or remove them, and can be transferred to the hands of healthcare workers. Evidence is accumulating that contaminated surfaces make an important contribution to the epidemic and endemic transmission of Clostridium difficile, vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and norovirus and that improved environmental decontamination contributes to the control of outbreaks. Efforts to improve environmental hygiene should include enhancing the efficacy of cleaning and disinfection and reducing the shedding of pathogens. Further high-quality studies are needed to clarify the role played by surfaces in nosocomial transmission and to determine the effectiveness of different interventions in reducing associated infection rates.

Lack of association of tcdC type and binary toxin status with disease severity and outcome in toxigenic Clostridium difficile.

The production of binary toxin and presence of truncating mutations in the putative toxin repressor gene, tcdC, have been associated with the increased virulence and spread of Clostridium difficile, especially ribotype 027. We analysed the prevalence of binary toxin genes and tcdC mutations in 207 clinical C. difficile isolates collected between 2008-2010. The majority (83%) belonged to one of five tcdC types and 8% were ribotype 027. There was little evidence of epidemic spread but there was a high prevalence of both predicted tcdC truncating mutations (15%) and binary toxin genes (28%), which occurred in both 027 and other ribotypes. We measured risk factors (age and laboratory markers) and patient outcomes (severity of disease, ICU admission, mortality, recurrence and length of stay) for patients infected with C. difficile strains with and without these mutations and genes. There was a significantly higher serum C-reactive protein and total peripheral white cell count in the group with predicted tcdC truncating mutations, but no difference in patient outcome. The group with binary toxin genes had a significantly higher total peripheral white cell count and 30 day all cause mortality. We have demonstrated a high prevalence of both predicted tcdC truncating mutations and binary toxin genes in a variety of C. difficile ribotypes, however neither of these factors by themselves predicted clinical virulence. This and other work show that commonly described deletions and truncating mutations do not by themselves explain the virulence of ribotype 027 and other C. difficile strains and further work is required to explain why some isolates appear to produce more severe disease than others.

Mandatory reporting and improvements in diagnosing Clostridium difficile infection: an incompatible dichotomy?

Toxin enzyme immunoassays (EIAs) are inadequate for the diagnosis of Clostridium difficile infection (CDI) when used alone. In September 2010 we replaced toxin EIA with a two-step algorithm, testing first with glutamate dehydrogenase and confirming with polymerase chain reaction for toxin B gene. We compared this to the gold standard of toxigenic culture, observing a positive predictive value of 96% (laboratory prevalence of 4.7%). There was no deterioration in turnaround time but there was a decrease of 11% in repeat specimens sent from the same patients. The improved performance of the algorithm increased the laboratory positivity rate from 2.2% to 5.6%. This led to an increase in our Trust CDI rate reported under the Health Protection Agency's mandatory surveillance scheme. We investigated whether the change was due to increasing nosocomial transmission, environmental contamination or consumption of antimicrobials, but found no evidence of this. We conclude that it probably resulted from the change in testing algorithm. Although we have improved testing and enhanced patient safety, we are likely to be unfairly financially penalised because of our apparent (but not real) increase in CDI rates. Assessment of CDI rates should take testing methodology into account and national policies should be revised to reflect this.

Low frequency of environmental contamination with methicillin-resistant Staphylococcus aureus in an inner city emergency department and a human immunodeficiency virus outpatient clinic.

We investigated methicillin-resistant Staphylococcus aureus (MRSA) environmental contamination in an inner city outpatient clinic and emergency department (ED). Moistened cotton-tipped swabs were used to sample 63 surfaces in the outpatient clinic and 69 surfaces in the ED. MRSA was identified on none of the 63 surfaces in the outpatient clinic and on 7% of the 69 surfaces in the ED. Our findings may have implications for cleaning and disinfection regimens in outpatient settings.

Detection of toxigenic Clostridium difficile in diarrheal stools by rapid real-time polymerase chain reaction.

The Cepheid Xpert polymerase chain reaction assay (Sunnyvale, CA) had a sensitivity of 100%, specificity of 96.7%, and positive and negative predictive values of 90.5% and 100%, respectively, compared with toxigenic culture for the laboratory diagnosis of Clostridium difficile in diarrheal stool samples. This test appears to be a significant improvement to poorly performing enzyme immunoassays.

Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Europe.

Over the past decade, community-associated meticillin-resistant Staphylococcus aureus (MRSA) has emerged in patients without health-care contact, especially in the USA. Although data are limited, the prevalence of community-associated MRSA in Europe seems to be low but is increasing. The organism has been reported in most European countries, including The Netherlands and Nordic countries, which have low rates of health-care-associated MRSA. In Greece, rates of community-associated MRSA in some centres approach those of the USA. By contrast with North America, where the USA300 clone (ST8-IV) predominates, community-associated MRSA in Europe is characterised by clonal heterogeneity. The most common European strain is the European clone (ST80-IV), although reports of USA300 are increasing. Several community-associated MRSA clones have arisen in Europe, most notably the ST398-V pig-associated MRSA clone in The Netherlands and Denmark. An understanding of the epidemiology of community-associated MRSA is essential to guide new control initiatives to prevent these organisms from becoming endemic in Europe.

Antimicrobial-resistant pathogens in animals and man: prescribing, practices and policies.

This meeting focused on infections in humans and animals due to methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)-producing bacteria and Clostridium difficile, and their corresponding treatments. MRSA is predominantly a human pathogen, and molecular typing has revealed that certain clones have spread widely both between humans and from humans to animals. ESBL-producing bacteria, particularly those that express the CTX-M beta-lactamases, have been disseminated worldwide. Whilst such strains are usually isolated from humans, some animal isolates also produce CTX-M enzymes. In humans, one clone of CTX-M-producing Escherichia coli, sequence type (ST)131, has been particularly successful. C. difficile, often ribotype 027, commonly colonizes the hospital environment and causes serious infections in humans. In animals, ribotype 078 is more often found, and is an important cause of diarrhoea in piglets. There is a concern that the numbers of MRSA or other antimicrobial-resistant bacteria might increase further when human isolates become established in animals, as this can amplify the numbers of such bacteria by dissemination within animal groups with subsequent spread back to humans. Certain antimicrobials have been implicated in the selection of MRSA, ESBL-producing bacteria and predisposition to infection by C. difficile. Guidelines for treatment and prevention of infections by MRSA, ESBL-producing bacteria and C. difficile were discussed and evidence-based policies were recommended for both humans and animals.

Survival of nosocomial bacteria and spores on surfaces and inactivation by hydrogen peroxide vapor.

With inocula of 6 to 7 log(10) CFU, most vegetative bacteria and spores tested survived on surfaces for more than 5 weeks, but all were inactivated within 90 min of exposure to hydrogen peroxide vapor in a 100-m(3) test room even in the presence of 0.3% bovine serum albumin to simulate biological soiling.

Impact of rapid screening tests on acquisition of meticillin resistant Staphylococcus aureus: cluster randomised crossover trial.

OBJECTIVE: To determine whether introducing a rapid test for meticillin resistant Staphylococcus aureus (MRSA) screening leads to a reduction in MRSA acquisition on hospital general wards. DESIGN: Cluster randomised crossover trial. SETTING: Medical, surgical, elderly care, and oncology wards of a London teaching hospital on two sites. MAIN OUTCOME MEASURE: MRSA acquisition rate (proportion of patients negative for MRSA who became MRSA positive). PARTICIPANTS: All patients admitted to the study wards who were MRSA negative on admission and screened for MRSA on discharge. INTERVENTION: Rapid polymerase chain reaction based screening test for MRSA compared with conventional culture. RESULTS: Of 9608 patients admitted to study wards, 8374 met entry criteria and 6888 had full data (82.3%); 3335 in the control arm and 3553 in the rapid test arm. The overall MRSA carriage rate on admission was 6.7%. Rapid tests led to a reduction in median reporting time from admission, from 46 to 22 hours (P<0.001). Rapid testing also reduced the number of inappropriate pre-emptive isolation days between the control and intervention arms (399 v 277, P<0.001). This was not seen in other measurements of resource use. MRSA was acquired by 108 (3.2%) patients in the control arm and 99 (2.8%) in the intervention arm. When predefined confounding factors were taken into account the adjusted odds ratio was 0.91 (95% confidence interval 0.61 to 1.234). Rates of MRSA transmission, wound infection, and bacteraemia were not statistically different between the two arms. CONCLUSION: A rapid test for MRSA led to the quick receipt of results and had an impact on bed usage. No evidence was found of a significant reduction in MRSA acquisition and on these data it is unlikely that the increased costs of rapid tests can be justified compared with alternative control measures against MRSA. TRIAL REGISTRATION: Clinical controlled trials ISRCTN75590122 [controlled-trials.com].

Validation of the IDI-MRSA system for use on pooled nose, axilla, and groin swabs and single swabs from other screening sites.

A commercial rapid polymerase chain reaction methicillin-resistant Staphylococcus aureus (MRSA) screening method (IDI-MRSA) is validated for the use with nasal swabs transported in liquid Stuart's medium. We investigated the use of IDI-MRSA for screening for MRSA in pooled nose, axilla, and groin swabs and in single swabs from skin puncture sites, wounds, throat, rectum, and groin using swabs transported in Amies medium without charcoal. We performed the IDI-MRSA test on swabs that had been used for routine MRSA broth culture and which were selected to be about 50% MRSA positive. We compared the IDI-MRSA result with the MRSA culture result. With 201 pooled sets, the sensitivity of IDI-MRSA was 85% and the specificity 95%. With 32 single screening swabs, sensitivity was 94% and specificity 80%. The method is not compromised by swab transport in Amies medium if an additional heating step is used. We had a low rate of initial inhibition (1.3%), which may have been due to the extra heating step used to liquefy gel from the Amies medium. Thus, in this study IDI-MRSA gives similar results to culture with pooled or single swabs from multiple screening sites.

Increasing resistance to antimicrobial agents of Gram-negative organisms isolated at a London teaching hospital, 1995-2000.

OBJECTIVES: To investigate the changes in resistance frequencies of common Gram-negative bacteria in a London teaching hospital. METHODS: Antimicrobial susceptibilities were analysed for the 6 years 1995-2000. Gentamicin-resistant isolates from 1995 and 2000 were typed by a repetitive element sequence-based PCR (Rep-PCR) method. RESULTS: Resistance rates for all agents and all organisms were higher in isolates from inpatients than in those from outpatients or general practice. For most agents and most species there was a trend for a highly significant linear increase in resistance over the study period, and there was significant cross-resistance between different agents. Increases in resistance were especially marked in Klebsiella, Enterobacter and Acinetobacter spp., organisms that tend to cause outbreaks of hospital cross-infection. For example, the increases in gentamicin resistance in isolates from inpatients was from 2.9% to 23.5% for Klebsiella spp., from 0.3% to 20.8% for Enterobacter spp. and from 10.1% to 42.2% for Acinetobacter spp. There was much less increase in acquired resistance in Escherichia coli and Pseudomonas aeruginosa, organisms that tend to cause endogenous infections, with gentamicin resistance in isolates from inpatients increasing from 0.4% to 3.2% for E. coli and decreasing from 4.6% to 3.6% for P. aeruginosa. Rep-PCR typing showed considerable diversity amongst gentamicin-resistant isolates of E. coli and P. aeruginosa, but dominance by a limited number of presumably epidemic types of gentamicin-resistant isolates of the other species. CONCLUSIONS: Multiple antibiotic resistance has increased dramatically in some hospital isolates, and appears to be associated with hospital cross-infection.