Microbial keratitis in Southern Malawi: a microbiological pilot study.

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.

Piperacillin/tazobactam-resistant, cephalosporin-susceptible Escherichia coli bloodstream infections are driven by multiple acquisition of resistance across diverse sequence types.

Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third-generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third-generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n=58) isolated between 2014-2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n=1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n=68), and piperacillin/tazobactam and third-generation cephalosporin-susceptible (TZP-S/3GC-S, n=1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types, which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor-resistant ß-lactamases, and an S133G blaCTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Eleven (19%) of the isolates had no putative mechanism identified from the genomic data. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.

Viral capture sequencing detects unexpected viruses in the cerebrospinal fluid of adults with meningitis.

OBJECTIVES: Many patients with meningitis have no aetiology identified leading to unnecessary antimicrobials and prolonged hospitalisation. We used viral capture sequencing to identify possible pathogenic viruses in adults with community-acquired meningitis. METHODS: Cerebrospinal fluid (CSF) from 73 patients was tested by VirCapSeq-VERT, a probe set designed to capture viral targets using high throughput sequencing. Patients were categorised as suspected viral meningitis - CSF pleocytosis, no pathogen identified (n = 38), proven viral meningitis - CSF pleocytosis with a pathogen identified (n = 15) or not meningitis - no CSF pleocytosis (n = 20). RESULTS: VirCapSeq-VERT detected virus in the CSF of 16/38 (42%) of those with suspected viral meningitis, including twelve individual viruses. A potentially clinically relevant virus was detected in 9/16 (56%). Unexpectedly Toscana virus, rotavirus and Saffold virus were detected and assessed to be potential causative agents. CONCLUSION: VirCapSeq-VERT increases the probability of detecting a virus. Using this agnostic approach we identified Toscana virus and, for the first time in adults, rotavirus and Saffold virus, as potential causative agents in adult meningitis. Further work is needed to determine the prevalence of atypical viral candidates as well as the clinical impact of using sequencing methods in real time. This knowledge can help to reduce antimicrobial use and hospitalisations leading to both patient and health system benefits.

16S Ribosomal RNA PCR Versus Conventional Diagnostic Culture in the Investigation of Suspected Bacterial Keratitis.

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.

Piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B.

A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the ß-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours blaTEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of blaTEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in blaTEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of blaTEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.

Increasing prevalence of a fluoroquinolone resistance mutation amongst Campylobacter jejuni isolates from four human infectious intestinal disease studies in the United Kingdom.

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.

Characterization of Circulating Clostridium difficile Strains, Host Response and Intestinal Microbiome in Hospitalized Children With Diarrhea.

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.

Use of a corneal impression membrane and PCR for the detection of herpes simplex virus type-1.

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.

Effect of storage time and temperature on the detection of Pseudomonas aeruginosa, Acanthamoeba and Herpes Simplex Virus from corneal impression membranes.

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.

Genomic pneumococcal load and CSF cytokines are not related to outcome in Malawian adults with meningitis.

OBJECTIVE: Bacterial meningitis in sub-Saharan Africa is predominantly caused by Streptococcus pneumoniae, is often associated with HIV co-infection and mortality rates are double those seen in better resourced settings. METHODS: To investigate the cause of this excessive mortality we quantified the pneumococcal DNA load and six common pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of Malawian adults with culture proven pneumococcal meningitis and correlated the results to clinical parameters and outcome. There are currently no published data relating bacterial load to outcome in adults with pneumococcal meningitis. RESULTS: The mean age of patients was 32 years, 82% were HIV infected and 49% had died by day 40. CSF bacterial loads were high (median 6.5 × 10(5) copies/ml CSF) and there was no significant variation in bacterial load between survivors and non-survivors. All pro-inflammatory CSF cytokines were elevated in the CSF, with no clinically important differences between survivors and non-survivors. HIV status did not affect the CSF bacterial load or cytokine response. CONCLUSION: Mortality from pneumococcal meningitis in adults in sub-Saharan Africa is not related to pneumococcal bacterial load. More research is needed to understand the very high mortality from meningitis in this region.

Antimicrobial resistance and characterisation of staphylococci isolated from healthy Labrador retrievers in the United Kingdom.

BACKGROUND: Coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci are normal commensals of the skin and mucosa, but are also opportunist pathogens. Meticillin-resistant (MR) and multidrug-resistant (MDR) isolates are increasing in human and veterinary healthcare. Healthy humans and other animals harbour a variety of staphylococci, including MR-CoPS and MR-CoNS. The main aims of the study were to characterise the population and antimicrobial resistance profiles of staphylococci from healthy non-vet visiting and non-antimicrobial treated Labrador retrievers in the UK. RESULTS: Nasal and perineal samples were collected from 73 Labrador retrievers; staphylococci isolated and identified using phenotypic and biochemical methods. They were also confirmed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), PCR of the nuc gene and PCR and sequencing of the tuf gene. Disc diffusion and minimum inhibitory concentration (MIC) susceptibility tests were determined for a range of antimicrobials. In total, 102 CoPS (S. pseudintermedius n = 91, S. aureus n = 11) and 334 CoNS isolates were detected from 99% of dogs in this study. In 52% of dogs CoNS only were detected, with both CoNS and CoPS detected in 43% dogs and CoPS only detected in 4% of dogs. Antimicrobial resistance was not common among CoPS, but at least one MDR-CoNS isolate was detected in 34% of dogs. MR-CoNS were detected from 42% of dogs but no MR-CoPS were isolated. S. epidermidis (52% of dogs) was the most common CoNS found followed by S. warneri (30%) and S. equorum (27%), with another 15 CoNS species isolated from ≤ 15% of dogs. S. pseudintermedius and S. aureus were detected in 44% and 8% of dogs respectively. CONCLUSIONS: MR- and MDR-CoPS were rare. However a high prevalence of MR- and MDR-CoNS were found in these dogs, even though they had no prior antimicrobial treatment or admission to veterinary premises. These findings are of concern due to the potential for opportunistic infections, zoonotic transmission and transmission of antimicrobial resistant determinants from these bacteria to coagulase positive staphylococci.

Evidence of simian virus 40 infection in multiple organ transplant recipients with renal dysfunction.

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.

Molecular characterization of Neisseria meningitidis isolates using a resequencing DNA microarray.

Neisseria meningitidis is a major cause of both meningitis and septicemia. Typically, isolates are characterized by using a combination of immunological phenotyping, using monoclonal and polyclonal antisera, and Sanger nucleotide sequencing of epitope-encoding variable regions, although these methods can be both time-consuming and limited by reagent availability. Herein, we describe and evaluate a novel microarray to define the porB and porA serotypes of N. meningitidis by the resequencing of variable regions in a single hybridization reaction. PCR products for each gene were amplified, pooled in equimolar concentrations, hybridized to the microarray, and analyzed using Affymetrix GeneChip DNA Analysis Software. Resequencing of the microarray data was then validated by comparison with sequencing data. Molecular profiles were generated for 50 isolates that were combinations of phenotypically typeable (ie, PorA and PorB) and non-typeable (PorB only) isolates. Microarray-generated profiles from isolates with a PorB phenotype were concordant with predicted profiles compared with a previously described typing scheme. In addition, 42% (8 of 19) of previously non-typeable samples were assigned a PorB type when tested using the microarray. The remaining isolates were novel types for which no typing antisera are currently available. The porA data were 97% concordant with Sanger nucleotide sequencing. These results suggest that that microarray resequencing may be a useful tool for the characterization of meningococci, particularly for those isolates that cannot be phenotyped, offering an alternative to conventional sequencing methods.

Development and evaluation of a 'real-time' RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples.

A two-step reverse transcriptase TaqMantrade mark duplex PCR (RT-PCR) assay was developed using the ABI 7700 Sequence Detection System for the detection of enterovirus (EV) and parechovirus type 1 and 2 (PEV) RNA from samples of cerebrospinal fluid (CSF) and throat swabs. Using sequence-specific fluorescent dye labeled probes and continuous 'real-time' monitoring, PCR amplified product accumulation was measured. Based on limiting dilutions, the TaqMantrade mark enterovirus and parechovirus RT-PCR showed an increase of two orders of magnitude compared to cell culture with sensitivity of 100% (7/7) when assessed using enterovirus cell culture positive samples (CSF, TS). The assays were specific for enterovirus and parechovirus and did not amplify a wide selection of virus and bacterial isolates. RNA was amplified from 22 enterovirus serotypes: coxsackie A7, A9, A21; coxsackie B2, B3, B4, B5; echovirus 2, 4, 6, 7, 9, 11, 13, 17, 18, 19, 30, 31; poliovirus types 1, 2, and 3, and parechovirus types 1 and 2. The assay was used to assess the incidence of enterovirus and parechovirus RNA in cell culture negative CSF and throat swab samples (n = 200). An additional 33 (15.9%) enterovirus and 2 (1%) parechovirus were identified as positive by RT-PCR. Also, of 100 CSF samples from suspected cases of meningococcal meningitis submitted for meningococcal PCR testing, 59 (59%) were enterovirus and 2 (2%) parechovirus 1 and 2 were positive by RT-PCR. The TaqMantrade mark duplex assay offers a more rapid and sensitive alternative to conventional cell culture for the diagnosis of enterovirus and parechovirus infection. Closed tube real-time detection using the ABI Sequence Detection System obviates the need for post-PCR manipulation, which reduces hands on time and eliminates the risk of contamination from amplified PCR product.