'Secondary biofilms' could cause failure of peracetic acid high-level disinfection of endoscopes.

INTRODUCTION: The reduced susceptibility of biofilms to disinfectants presents a challenge to the successful reprocessing of medical equipment. This study examined the effect of residual biomass remaining after previous disinfection with peracetic acid (PAA) on the tolerance of subsequent mature Pseudomonas aeruginosa biofilms to PAA. The effect of enzymatic degradation of specific components of the extracellular polymeric substance (EPS) of P. aeruginosa biofilm on the effectiveness of PAA disinfection was also evaluated. METHODS: The susceptibility of biofilm grown on the biomass of PAA-killed biofilm to PAA was compared with the PAA susceptibility of biofilm grown in wells of a 24-well plate by evaluating their viability using the plate count assay. The effect of PAA on biofilm biomass was measured using crystal violet quantification of total biofilm biomass, while its effect on the polysaccharide and protein components of biofilm EPS was quantified using the phenol-sulphuric acid assay or Bradford assay, respectively. A confocal microscope was used to visualize the distribution of living and dead cells in biofilms grown on residual biofilm biomass. FINDINGS: The presence of residual biomass from previously disinfected biofilms significantly enhanced the tolerance of subsequent biofilms. A 96-h-old 'secondary biofilm' formed on disinfected biomass survived PAA concentrations of 4000 ppm, which exceeds the concentrations used in practice for high-level disinfection. CONCLUSION: These observations indicate that, under certain circumstances, recolonization of residual EPS can cause failure of disinfection of medical equipment such as endoscopes, and emphasizes the importance of cleaning endoscopes prior to disinfection.

Influence of delivery system on the efficacy of low concentrations of hydrogen peroxide in the disinfection of common healthcare-associated infection pathogens.

INTRODUCTION: The ability of healthcare-associated infection pathogens to survive on environmental surfaces is well known. Disinfection is employed to reduce or remove these pathogens but disinfection failures still occur. One method with the potential to improve disinfection efficacy is whole-room disinfection with hydrogen peroxide (H2O2). AIM: To determine the influence of delivery system on the efficacy of low-concentration H2O2 on common healthcare-associated infection pathogens. METHODS: SanoStatic (electrostatic spray) was compared with SanoFog (fogging) in terms of performance for delivery of 5% H2O2 and trace silver ions for disinfection. The bacterial test challenges were vancomycin-resistant Enterobacterales (VRE), extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBLK), carbapenemase-producing Enterobacterales (CPE), meticillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile spores, Bacillus atropheus and Geobacillus stearothermophilus commercial spore strips. FINDINGS: SanoFog and SanoStatic were effective when tested under the conditions of experimentation reported here. For VRE, ESBLK, CPE and MRSA, SanoFog and SanoStatic were comparable in performance. For C. difficile we concluded the following: SanoFog was most effective for disinfection of C. difficile spores when compared to SanoStatic. CONCLUSION: Whereas SanoFog and SanoStatic were effective against bacterial cells, the current practice of using SanoFog and SanoStatic together would be effective for disinfection of C. difficile spores based on investigations under the conditions of experimentation reported here. The spore strips results were not comparable to the results either for the vegetation cells (VRE, ESBLK, CPE, and MRSA) or for C. difficile spores.

Drivers of Clostridioides difficile hypervirulent ribotype 027 spore germination, vegetative cell growth and toxin production in vitro.

OBJECTIVES: Clostridioides difficile infection (CDI) is a considerable healthcare and economic burden worldwide. Faecal microbial transplant remains the most effective treatment for CDI, but is not at the present time the recommended standard of care. We hereby investigate which factors derived from a healthy gut microbiome might constitute the colonization resistance barrier (CRB) in the gut, inhibiting CDI. METHODS: CRB drivers pH, short chain fatty acid (SCFA), and oxidation-reduction potential (ORP) were investigated in vitro using C. difficile NAP1/BI/027. Readouts for inhibitory mechanisms included germination, growth, toxin production and virulence gene expression. pH ranges (3-7.6), SCFA concentrations (25-200 mM) and ORP (-300 to 200 mV) were manipulated in brain heart infusion broth cultures under anaerobic conditions to assess the inhibitory action of these mechanisms. RESULTS: A pH < 5.3 completely inhibited C. difficile growth to optical density (OD) 0.019 vs. 1.19 for control pH 7.5. Toxin production was reduced to 25 units vs. 3125 units for pH 7.6 (1 in 5 dilutions). Virulence gene expression reduced by 150-fold compared with pH 7.6 (p < 0.05). Germination and proliferation of spores below pH 6.13 yielded an average OD of 0.006 vs. 0.99 for control. SCFA were potent regulators of toxin production at 25 mM and above (p < 0.05). Acetate significantly inhibited toxin production to 25 units independent of OD (0.8733) vs. control (OD 0.6 and toxin titre 3125) (p < 0.05). ORP did not impact C. difficile growth. CONCLUSIONS: This study highlights the critical role that pH has in the CRB, regulating CDI in vitro and that SCFA can regulate C. difficile function independent of pH.

Surface disinfection challenges for Candida auris: an in-vitro study.

The emerging pathogenic multidrug-resistant yeast Candida auris is an important source of healthcare-associated infections and of growing global clinical concern. The ability of this organism to survive on surfaces and withstand environmental stressors creates a challenge for eradicating it from hospitals. A panel of C. auris clinical isolates was evaluated on different surface environments against the standard disinfectant sodium hypochlorite and high-level disinfectant peracetic acid. C. auris was shown to selectively tolerate clinically relevant concentrations of sodium hypochlorite and peracetic acid in a surface-dependent manner, which may explain its ability to successfully persist within the hospital environment.

Infection control implications of the laundering of ambulance staff uniforms and reusable mops.

There is a lack of published studies on laundering in ambulance services. We performed bacterial culture on soiled and unsoiled uniforms and reusable mop heads artificially contaminated with Escherichia coli, Staphylococcus aureus, and Clostridium difficile spores. Current laundering processes used for routine cleans in the ambulances appears, from our simulations, to be effective at reducing vegetative pathogenic bacteria to undetectable levels, <3.398log10 colony-forming units (S. aureus and E. coli). Reduced levels of C. difficile were still detected after laundering but the risk this poses for infection is unknown, as background levels of these spores in the environment are unknown.

Bioaerosol sampling: sampling mechanisms, bioefficiency and field studies.

Investigations into the suspected airborne transmission of pathogens in healthcare environments have posed a challenge to researchers for more than a century. With each pathogen demonstrating a unique response to environmental conditions and the mechanical stresses it experiences, the choice of sampling device is not obvious. Our aim was to review bioaerosol sampling, sampling equipment, and methodology. A comprehensive literature search was performed, using electronic databases to retrieve English language papers on bioaerosol sampling. The review describes the mechanisms of popular bioaerosol sampling devices such as impingers, cyclones, impactors, and filters, explaining both their strengths and weaknesses, and the consequences for microbial bioefficiency. Numerous successful studies are described that point to best practice in bioaerosol sampling, from the use of small personal samplers to monitor workers' pathogen exposure through to large static samplers collecting airborne microbes in various healthcare settings. Of primary importance is the requirement that studies should commence by determining the bioefficiency of the chosen sampler and the pathogen under investigation within laboratory conditions. From such foundations, sampling for bioaerosol material in the complexity of the field holds greater certainty of successful capture of low-concentration airborne pathogens. From the laboratory to use in the field, this review enables the investigator to make informed decisions about the choice of bioaerosol sampler and its application.

Optimising gut colonisation resistance against Clostridium difficile infection.

Clostridium difficile is the dominant cause of pseudomembranous colitis in nosocomial environments. C. difficile infection (CDI) generally affects elderly (≥65 years of age) hospital inpatients who have received broad-spectrum antimicrobial treatment. CDI has a 30 % risk of re-infection and a subsequent 60 % risk of relapse thereafter, leading to a high economic burden of over 7 billion pounds sterling and over 900,000 cases in the USA and Europe per annum. With the long-term consequences of faecal transplantation currently unknown, and limited spectrum of effective antibiotics, there is an urgent requirement for alternative means of preventing and treating CDI in high-risk individuals. Metagenomics has recently improved our understanding of the colonisation resistance barrier and how this could be optimised. pH, oxidation-reduction potentials and short-chain fatty acids have been suggested to inhibit C. difficile growth and toxin production in in vitro and in vivo studies. This review aims to pull together the evidence in support of a colonisation resistance barrier against CDI.

A review of infection control in community healthcare: new challenges but old foes.

The demographics of the healthcare population are changing, with an ever-greater proportion of people being treated outside the traditional hospital setting through community healthcare. This shift in the way that healthcare is delivered raises new concerns over community healthcare-associated infections (HCAIs). A literature search between 2000 and December 2013 was conducted in databases including PubMed, SciVerse ScienceDirect and Google Scholar. National and international guideline and policy documents were searched using Google. Many terms were used in the literature searches, including 'nosocomial', 'healthcare infection', 'community' and 'nursing home'. The rates of HCAI in community healthcare are similar to the rates found in the acute hospital setting, but the types of infection differ, with a greater focus on urinary tract infections (UTIs) in the community and ventilator-associated pneumonias in the hospital setting. Patients who acquire a community HCAI are more likely to exhibit reduced physical condition, have increased levels of morbidity and have higher mortality rates than individuals without infection. Infection control programmes have been developed worldwide to reduce the rates of hospital HCAIs. Such interventions are equally as valid in the community, but how best to implement them and their subsequent impact are much less well understood. The future is clear: HCAIs in the community are going to become an ever-increasing burden and it is critical that our approach to these infections is brought quickly in line with present hospital sector standards.

Intrafamilial Genotyping of Helicobacter pylori from Faecal DNA.

Helicobacter pylori infection, often acquired in early childhood, is a global cause of undernutrition, gastritis, peptic ulcer disease and gastric carcinoma. This study tested the feasibility of using H. pylori shed in the faeces as a source of DNA for non-invasive epidemiological studies. H. pylori DNA was chemically recovered and isolated using a specific biotinylated oligonucleotide probe with magnetic capture from 28 H. pylori positive faecal samples obtained from children attending hospital for the investigation of suspected H. pylori infection, together with close family members. Random amplification of polymorphic DNA (RAPD) was subsequently used to discriminate each isolate. 93% of stool samples selected were typeable. Parent, child and sibling samples were compared and similarities determined. Phylogenetic analysis showed that H. pylori DNA obtained from the faeces can be used to genotype individual strains, offering a means of studying intrafamilial transfer of this microorganism.

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae.

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both). Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n=5 conventional commercial, n=10 conventional in-house, n=4 real-time commercial, n=43 real-time in-house, and n=2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n=5 conventional commercial, n=10 conventional in-house, n=4 real-time commercial, n=46 real-time in-house, and n=2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism. Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.

External quality assessment of the molecular diagnostics and genotyping of meticillin-resistant Staphylococcus aureus.

Two multicentre external quality assessments (EQA) for the molecular detection and genotyping of meticillin-resistant Staphylococcus aureus (MRSA) were arranged. Firstly, 11 samples containing various amounts of inactivated MRSA strains, meticillin-susceptible S. aureus (MSSA), meticillin-resistant coagulase-negative staphylococci (MRCoNS) or Escherichia coli were distributed to 82 laboratories. Samples containing 102 or 103 MRSA cells were correctly scored in only 16 and 46% of the datasets returned, respectively. Two of the used MSSA strains contained an SCCmec cassette lacking the mecA gene. There was a marked difference in the percentage of correct results for these two MSSA strains (37 and 39%) compared to the MSSA strain lacking the SCCmec cassette (88%). Secondly, a panel for MRSA genotyping, consisting of ten samples (two identical, three genetically related and five unique strains) was distributed to 19 laboratories. Seventy-three percent of the datasets recorded all samples correctly. Most pulsed-field gel electrophoresis (PFGE) protocols proved to be suboptimal, resulting in inferior resolution in the higher or lower fragment regions. The performance of molecular diagnostics for MRSA shows no significant changes since our first EQA in 2006. The first molecular typing results are encouraging. Both assessments indicate that programme expansion is required and that major performance discrepancies continue to exist.

The impact of laboratory characteristics on molecular detection of respiratory syncytial virus in a European multicentre quality control study.

The performance of nucleic acid amplification techniques for detecting respiratory syncytial virus (RSV) was evaluated in 25 laboratories across Europe by an external quality assessment study. In addition, factors related to the diagnostic performance of laboratories were explored. The results of this quality control study show that the performance of laboratories for RSV diagnosis in Europe is good, with an overall correct score of 88%. The type of assay (nested or real-time PCR vs. commercial tests) was identified as a significant factor (OR 8.39; 95% CI 1.91-36.78) in predicting a correct result.

HIV-1 drug resistance genotyping quality assessment: results of the ENVA7 Genotyping Proficiency Programme.

BACKGROUND: Drug-resistance testing plays a critical role in selection of optimal treatment regimens for HIV infected individuals. Laboratories performing testing must implement quality control measures including external quality assessment. OBJECTIVES: The ENVA7 Programme (2007) was organised by QCMD to assess the performance of laboratories testing for drug-resistance mutations in the HIV-1 Protease and Reverse Transcriptase genes. STUDY DESIGN: The ENVA7 panel consisted of 5 lyophilised plasma samples (HIV-1 subtypes B, C and F). The viruses harboured wild type or resistant genotypes at various positions of the PR and RT genes. All IAS-defined resistance-associated codons were scored in comparison to the consensus sequence for each sample using a scoring system developed to allow simple and standardised comparisons between laboratories and/or technologies. RESULTS: 111 laboratories from 44 countries participated of which 95 submitted 98 datasets. 36 datasets were generated using ViroSeq (Abbott), 27 using TruGene (Siemens) and 35 using in-house assays. CONCLUSIONS: All technologies successfully genotyped each of the panel samples, irrespective of the virus subtype. While the assays for genotypic HIV drug-resistance determination have evolved into reliable and technically capable procedures of generating high quality results, variation in the quality of results is still observed between laboratories.

Molecular detection and typing of influenza viruses: are we ready for an influenza pandemic?

BACKGROUND: We cannot predict when an influenza pandemic will occur or which variant of the virus will cause it. Little information is currently available on the ability of laboratories to detect and subtype influenza viruses including the avian influenza viruses. OBJECTIVES: To assess the ability of laboratories to detect and subtype influenza viruses. STUDY DESIGN: In 2006 QCMD distributed an External Quality Assessment panel for the molecular detection and haemagglutinin subtyping of influenza viruses to 87 laboratories in 34 countries Worldwide, which were given 6 weeks to return results. These data were analysed to assess laboratory performance. RESULTS: Influenza virus positive panel samples were correctly identified by 35-98% of laboratories. The correct haemagglutinin subtype was reported by 32-87% of laboratories that detected the virus: incorrect subtyping results included the reporting of avian influenza viruses as human strains and vice versa. Twelve laboratories reported false positives with some avian influenza viruses reported. CONCLUSIONS: These data suggest that improvements are needed in the molecular detection of influenza viruses and influenza virus A haemagglutinin subtyping. Only rapid and accurate identification of circulating pandemic influenza virus will ensure that the maximum time is available for intervention.

Helicobacter pylori antigens in the faeces of asymptomatic children in the Buea and Limbe health districts of Cameroon: a pilot study.

OBJECTIVE: To determine the prevalence and identify intra-familial risk factors associated with Helicobacter pylori infection in a paediatric population. METHODS: Cross-sectional study in the Buea and Limbe health districts, South West Cameroon. Stool samples were collected from 176 randomly selected apparently healthy children from two communities with different socioeconomic status. They comprised 86 males and 90 females aged 0-10 years with a mean age of 4.29. Helicobacter pylori status was determined using an enzyme-linked immunosorbent assay, the H. pylori stool antigen (HpSA) test. The test uses polyclonal anti-H. pylori capture antibody to detect H. pylori antigens in human stool. Epidemiological data were analysed using the Fisher test and odds ratio (OR) at 95% confidence intervals (CI). RESULTS: The overall prevalence of H. pylori was 52.27% (92 of 176). Univariate analysis showed that H. pylori prevalence was significantly higher in children of the low socioeconomic class, 62.50% (55 of 88) than in those of the high socioeconomic class, 42.05% (37 of 88) (P < 0.05; OR = 2.41, 95% CI: 1.26-4.64). Helicobacter pylori prevalence increased with age from 37.50% (12 of 32) for children aged <3 years, 50.00% (53 of 106) aged 3-6 years and 71.05% (27 of 38) aged 7-10 years (P > 0.05; OR = 0.81, 95% CI: 0.34-1.91). The frequency of infection was significantly higher in males, 64.00% (55 of 86) than in females, 41.11% (37 of 90), (P < 0.05; OR = 2.67, 95% CI: 1.39-5.17). CONCLUSIONS: This study highlights the continuing importance of age, sex and socioeconomic status in the acquisition of H. pylori infection.

Childhood H. pylori: disappearing disease or chronic infection?

Helicobacter pylori is one of the commonest chronic bacterial infections worldwide. It is acquired during childhood and its persistence has implications for health in later life. In adults, it is the principle cause of duodenal ulcer disease and there is evidence of an association between H. pylori and gastric cancer. However, most colonized people are asymptomatic. The prevalence of H. pylori increases with age but there is a striking difference between the rates in developed and developing countries. As no significant non-human or environmental source for this infection has been identified, person to person spread is almost certainly the main mode of transmission. Community nurses should be aware of this micro-organism as a potential cause of illness in children, and that they can play a role in promoting hygiene practices and educating families so that the risk of acquisition may be reduced. This review discusses the clinical features, prevalence, risk factors for transmission, diagnosis and treatment of H. pylori.

Helicobacter pylori in children is strongly associated with poverty.

BACKGROUND AND AIMS: To examine the association between prevalence of H. pylori colonisation and social deprivation in a sample of children investigated in hospital. METHODS: A retrospective review of the hospital records of all children (n = 626) who underwent 13C-urea breath testing for suspected H. pylori infection at the Royal Hospital for Sick Children, Glasgow, Scotland between August 1995 to December 2002 was performed. Prevalence of H. pylori colonisation was measured by the 13C-urea breath test and socioeconomic status was measured by the Carstairs and Morris index of deprivation. RESULTS: The overall prevalence of H. pylori was 26%. There was a highly significant positive association between H. pylori colonisation and poor socioeconomic status (p < 0.000). The prevalence of colonisation was significantly higher in children from the most deprived areas (DepCat 6 and 7; 34%) compared to children from intermediate (DepCat 3 to 5; 22%) and the most affluent areas (DepCat 1 and 2; 16%) (p < 0.0001). CONCLUSIONS: Socioeconomic deprivation in childhood is associated with a high prevalence of H. pylori colonisation. While the incidence of H. pylori may be declining, it remains common in poor families. If the prevalence of H. pylori (26%) in this selected group reflects that of the population at large, then over 9000 (5%) of Glasgow's children are at risk of this preventable disease. In a city where the majority of adults are colonised with H. pylori, colonisation in early life adds to the burden of health risks to which deprived children are exposed.

Evaluation of protocol using gene capture and PCR for detection of Helicobacter pylori DNA in feces.

The route of transmission of Helicobacter pylori, which is usually acquired in childhood and is one of the most common bacterial infections in humans, remains undetermined. Mapping the distribution of H. pylori genotypes within families could help to determine the routes of transmission and risk factors. Here we describe a noninvasive method for obtaining H. pylori DNA isolates from the feces of children. Children presenting with gastrointestinal symptoms at the Royal Hospital for Sick Children were tested for gastric H. pylori colonization by using the 13C-urea breath test (UBT) and were asked to provide fecal samples, which were tested for H. pylori by using the HpSA fecal antigen test. DNA was purified from fecal samples by using a novel method of gene capture with subsequent H. pylori PCR analysis. Fifteen UBT-positive and 15 UBT-negative children participated in the study. The positive and negative predictive values for the assay were 80 and 100%, respectively. Fecal DNA purification followed by H. pylori PCR analysis is an effective tool for harvesting H. pylori DNA isolates from the feces of children. This technique may be developed to allow the diagnosis and noninvasive genotyping of H. pylori in children and their families.

Water, water everywhere nor any a sterile drop to rinse your endoscope.

Traditional waterborne infections have been largely controlled in the UK by the provision of clean drinking water. However, water can still cause problems for infection control teams in particular when used in endoscope washer-disinfectors. HTM 2030 states that final rinse water used in washer-disinfectors must not present a microbiological hazard and that there should be no recovery of micro-organisms from the final rinse water. The problems that biofilms may cause in washer-disinfectors, the type of biofilms that may develop, and the nature of the bacteria within them, in particular how biofilm bacteria behave differently to those that are not part of a biofilm (planktonic bacteria), are discussed in this article. Finally, we discuss how knowledge of the growth and control of biofilms may be used to control their growth.

Detection of Helicobacter pylori DNA in drinking water biofilms: implications for transmission in early life.

AIMS: To provide evidence of water quality as a risk factor for acquisition of Helicobacter pylori in early life, and to identify evidence for its presence within pots used to store drinking water. METHODS AND RESULTS: A prospective cohort study of 65 infants was conducted in the rural village of Keneba, The Gambia. Age of H. pylori colonization was determined and water pot biofilms were tested for H. pylori by sequencing of amplified DNA. Use of supplemental water was a strong risk factor for H. pylori colonization in infants (OR 4.71, 95% CI 1.17-22.5). DNA with 95% homology to the 16S rRNA gene of H. pylori was isolated from biofilms of water pots. CONCLUSIONS: Drinking water may be a reservoir for H. pylori in areas of the developing world where water quality is poor. Early introduction of water, particularly if stored in, or collected from contaminated sources, may be associated with an increased rate of H. pylori colonization.