Objective Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi.Methods and analysis Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for Acanthamoeba, herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise Staphylococcus aureus isolates.Results 71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative Staphylococcus 31.8% and Streptococcus species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and Acanthamoeba were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with S. aureus keratitis.Conclusions In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.

Background Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease. Methods We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993-1996; n = 293] and IID2 [isolates from 2008-2009; n = 93]), the INTEGRATE project [isolates from 2016-2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]. Results There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation. Discussion Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

Purpose The purpose of this study was to compare conventional diagnostic culture (CDC) to 16S ribosomal RNA polymerase chain reaction (PCR) analysis for diagnosing bacterial keratitis. Methods Samples collected from 100 consecutive patients presenting to the Royal Liverpool University Hospital with bacterial keratitis were processed using CDC and 16S PCR analysis. Results The overall detection rate using both methods was 36%. Of these, 72.2% (26/36) were detected by PCR and 63.9% (23/36) isolated by CDC (P = 0.62). Using a combination of both PCR and CDC increased the detection rate for pathogenic bacteria by 13% compared to using CDC alone (P = 0.04). In CDC negative samples, 16S PCR identified more pathogens than CDC in 16S PCR negative samples. Neither order of sample collection nor prior antimicrobial use affected the detection rate. Conclusions 16S rRNA gene PCR performed in addition to CDC on corneal samples from patients with clinically suspected bacterial keratitis led to additional pathogen detection. Translational Relevance 16S rRNA gene PCR should be developed to become an additional part of clinical service for patients with bacterial keratitis rather than used in isolation.

Background: The spread of carbapenemase-producing Enterobacterales (CPE) is a global health problem. Gastrointestinal tract carriage makes faeces or rectal swabs the recommended screening methods.Aim: To assess the impact of three laboratory screening strategies for CPE on positivity rates and infection prevention and control in a hospital setting in North West England from 2015 to 2017.Methods: In a retrospective study, time to CPE-positive and -negative results, number of new CPE-positive patients identified, and number of hospital bed-days lost/wards affected were measured for each of three CPE screening strategies; culture plus phenotypic tests, culture plus polymerase chain reaction (PCR), and PCR only (phases 1, 2 and 3, respectively).Findings: The fastest time to CPE results was PCR only (median: 4.0 h), then culture plus PCR (median: 47.6 h), then culture plus phenotypic tests (median: 49.8 h) (P < 0.001). The mean numbers of hospital bed-days lost per month decreased between phases 2 and 3 (P 1/4 0.01). The mean number of wards/units affected by CPE increased from phase 1 (2.57) to phase 2 (7.71), then decreased in phase 3 (3.86). The percentage positivity rate for phases 1, 2, and 3 were 2.01, 1.38, and 1.55 respectively. From May to October, the number of new CPE-positive patients was lower for phases 1 and 3 than for phase 2. During all three phases there was a peak in the number of newly identified CPE carriers in August.Conclusion: This study provides evidence that using a rapid PCR to screen rectal or faeces swabs enables more timely infection prevention and control measures when compared with culture-based methods. A reduction in bed-days lost due to CPE was observed when rapid molecular screening was introduced.& COPY; 2019 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

BACKGROUND:Clostridium difficile is capable of causing severe enterocolitis in adults. The significance of toxin-producing C. difficile in children with diarrhea is unclear and practice differs on whether to institute treatment. We aimed to characterize the microbiome in relation to the presence of C. difficile and co-infection with other pathogens and to describe host response to infection. METHODS:Participants were children with acute diarrhea, 0-16 years of age, from whom stool samples had been submitted to the hospital laboratory for routine microbiology/virology. Convenience sampling was used for 50 prospective and 150 retrospective samples. No participants were treated for C. difficile. Rates of culture positivity for C. difficile, presence of toxin and PCR-ribotype were compared between age groups. Presence of other potential pathogens, comorbidities and complications were recorded. Microbiotal diversity was measured by 16S profiling. RESULTS:Nineteen of 77 (25%) children <2 years of age and 13 of 119 (11%) children >2 years of age were C. difficile positive, of whom 10 (53%) and 9 (69%), respectively, carried toxigenic strains. Increased Shannon diversity was seen in children carrying C. difficile, with altered milieu. Presence of C. difficile was not associated with adverse clinical outcomes. In stools containing both Norovirus and C. difficile, there was increased relative abundance of verrucomicrobia. CONCLUSIONS:Children with diarrhea regularly carried toxigenic and non-toxigenic strains of C. difficile, demonstrating enhanced microbiotal diversity, and change in milieu, without apparent morbidity. This unexpected finding is contrary to that seen in adults with C. difficile disease.

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK). Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK. Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001). Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.

The effect of storage time and temperature on the recovery of pathogen DNA from polytetrafluorethylene (PTFE) was investigated. PTFE impression membranes were inoculated with Pseudomonas aeruginosa, Herpes Simplex Virus-1 (HSV-1) or Acanthamoeba and stored at -70 °C, -20 °C, +4 °C or +35 °C. PCR was performed on days 0, 1, 2, 3, 7 and months 1, 3 and 10 post-inoculation. We found no reduction in the DNA recovery of any of the studied microorganisms for the first 3 days of storage up to +35 °C. For HSV-1 and P. aeruginosa, storage for 3 months at +35 °C was associated with a significant reduction in DNA recovery (P<0.001), but not at +4 °C, -20 °C or -70 °C for 1 month for P. aeruginosa and for 10 months for HSV-1. Acanthamoeba DNA recovery was not affected by any storage parameters (P=0.203). These results will inform the investigation of microbial keratitis where access to microbiological testing is not readily available.

Antimicrobial resistant bacteria are increasingly detected from canine samples but few studies have examined commensal isolates in healthy community dogs. We aimed to characterise faecal Escherichia coli from 73 healthy non-veterinarian-visiting and non-antimicrobial treated Labrador retrievers, recruited from dog shows in the North West United Kingdom between November 2010 and June 2011. Each enrolled dog provided one faecal sample for our study. E. coli were isolated from 72/73 (99%) faecal samples. Disc diffusion susceptibility tests were determined for a range of antimicrobials, including phenotypic extended-spectrum beta-lactamase (ESBL) and AmpC-production. PCR assay detected phylogenetic groups and resistance genes (blaCTX-M, blaSHV, blaTEM, blaOXA, blaCIT, qnr), and conjugation experiments were performed to investigate potential transfer of mobile genetic elements. Multivariable logistic regression examined potential risk factors from owner-questionnaires for the presence of antimicrobial resistant faecal E. coli. Antimicrobial resistant, multi-drug resistant (≥3 antimicrobial classes; MDR) and AmpC-producing E. coli were detected in 63%, 30% and 16% of samples, respectively. ESBL-producing E. coli was detected from only one sample and conjugation experiments found that blaCTX-M and blaCIT were transferred from commensal E. coli to a recipient strain. Most isolates were phylogenetic groups B1 and A. Group B2 isolates were associated with lower prevalence of resistance to at least one antimicrobial (P<0.001) and MDR (P<0.001). Significant at P<0.003, was the consumption of raw meat for clavulanate–amoxicillin (OR: 9.57; 95% CI: 2.0–45.7) and third generation cephalosporin resistance (3GCR) (OR: 10.9; 95% CI: 2.2–54.0). AMR E. coli were surprisingly prevalent in this group of non-antimicrobial treated and non-veterinarian-visiting dogs and consumption of raw meat was a significant risk factor for antimicrobial resistance. These findings are of concern due to the increasing popularity of raw-meat canine diets, and the potential for opportunistic infection, zoonotic transmission and transmission of antimicrobial resistant determinants from commensal isolates to potential pathogenic bacteria.

Pneumocystis jirovecii is ubiquitous in the environment and an opportunistic pathogen which causes pneumonia in high risk patient groups e.g. immunocompromised patients, especially those with HIV, leukaemia and transplant patients. P. jirovecii pneumonia (PCP) has typically been diagnosed by a combination of immunofluorescence (IF) testing in the diagnostic laboratory and the clinical picture in the patient. However, IF despite having a good positive predictive value is an insensitive method. Increasingly molecular methods such as PCR are being used in the diagnosis of PCP. We describe the evaluation of a novel internally controlled real-time PCR, targeting the ß-tubulin gene for the diagnosis of PCP in induced sputum or broncheoalveolar lavage fluid. Also, in response to an outbreak of PCP cases in renal transplant patients at the Royal Liverpool University Hospital, genotypic sequencing (two gene fragments) of real-time PCR positive samples was undertaken. To determine the epidemiology, cases were compared with non-renal transplant controls. Both methods were adapted for testing of environmental (air and hard surface) sampling for P. jirovecii to investigate a common infection source.

Electron microscopy (EM), real-time polymerase chain reaction (PCR) and conventional PCR were used to identify viruses associated with infection in 2 transplantation patients. An autologous haematopoietic stem cell, liver and renal transplant recipient was found to be positive for simian virus 40 (SV40). Dual BK virus and SV40 infection was found in a heart and renal transplantation patient. SV40 infection can occur in immunocompromised patients.