Peter Tilley

Successful approach to treatment of Helicobacter bilis infection in X-linked agammaglobulinemia.

Helicobacter bilis, an unusual cause of chronic infections in patients with X-linked agammaglobulinemia (XLA), is notoriously difficult to diagnose and eradicate. Based on the limited number of cases reported worldwide, we highlight the typical features of H. bilis infection in XLA and provide a rational and successful approach to diagnosis and treatment of this challenging infection.

Short-Term Stability of Pathogen-Specific Nucleic Acid Targets in Clinical Samples

ABSTRACT The stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4°C, and −80°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets (P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples.

Depletion of human DNA in spiked clinical specimens for improvement of sensitivity of pathogen detection by next-generation sequencing

ABSTRACT Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P < 0.05) and improved the sensitivity of pathogen detection (P < 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

Evaluation of Amplification Targets for the Specific Detection of Bordetella pertussis Using Real-Time Polymerase Chain Reaction

BACKGROUND: Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity. METHODS: A novel B pertussis real-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481, ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including different Bordetella species and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products. RESULTS: Analytical sensitivity was highest for the assay targeting the IS481 element; however, the assay lacked specificity for B pertussis in the reference panel and in the clinical samples. False-positive results were also observed with assays targeting the ptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains of B pertussis. However, a novel assay targeting the porin gene demonstrated high specificity for B pertussis both in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted all B pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction. CONCLUSION: A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.

Optimal Use of MRSASelect and PCR to Maximize Sensitivity and Specificity of MRSA Detection

Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSASelect (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR (nxa0=xa0108). Although the specificity of MRSASelect assessed at 24xa0h of incubation was much higher than that of 48xa0h (91.4 vs. 60xa0%), extending the incubation time to 48xa0h, along with PCR confirmation, increased the total number of true positive samples by 27.8xa0%. These results provide a cost effective method for sensitive and specific detection of MRSA.

Bacillus pumilus Septic Arthritis in a Healthy Child

We report a case of septic arthritis caused by a Bacillus species, B. pumilus, occurring in a healthy child. This organism rarely causes serious infections and has only been described in newborns and immunocompromised individuals or as a skin infection. This child developed an indolent joint swelling after a minor skin injury, and symptoms were initially thought most consistent with chronic arthritis. The case demonstrates that clinicians should consider joint infection in children presenting with acute monoarticular swelling, even without prominent systemic features.

Paediatric infectious keratitis at tertiary referral centres in Vancouver, Canada

Objective To report the clinical and microbiological profiles of paediatric patients with infectious keratitis in Vancouver, Canada. Design In this observational case series, the microbiology results and medical records of 17 eyes with microbial keratitis in 16 children aged 17u2005years or younger were retrospectively reviewed. These patients had undergone corneal scraping between May 2006 and April 2011 at BC Childrens Hospital or Vancouver General Hospital Eye Care Centre in Vancouver, British Columbia, Canada. Demographic information, clinical features, predisposing factors, results of microbiology studies, antibiotic susceptibilities, treatment course and outcomes were analysed. Results The mean age of patients was 11±5.7u2005years (range 1–17u2005years) and the male:female ratio was 1.4:1. Major predisposing factors were contact lens wear (6/17; 35%), and pre-existing ocular surface conditions including blepharitis (3/17; 18%) and Stevens–Johnson syndrome (3/17; 18%). Four patients had a previous corneal ulcer. The most commonly isolated microorganisms were Staphylococcus epidermidis and Acanthamoeba. Acanthamoeba was isolated in 67% of contact lens-related corneal ulcers, while the remaining 33% of contact lens-related corneal ulcers were associated with infection with Pseudomonas aeruginosa. Final visual acuity was better than 20/60 in 9 out of 16 patients (56%). Three patients subsequently required surgical management with either penetrating keratoplasty or deep anterior lamellar keratoplasty for treatment of corneal scarring. Conclusions Contact lens wear and pre-existing ocular surface conditions are significant risk factors for the development of infectious keratitis in our paediatric population. Knowledge of regional patterns of infection and susceptibility are essential in ensuring prompt treatment of this potentially sight-threatening condition.

Respiratory Presentation of Pediatric Patients in the 2014 Enterovirus D68 Outbreak.

Background. In the fall of 2014, a North American outbreak of enterovirus D68 resulted in a significant number of pediatric hospital admissions for respiratory illness throughout North America. This study characterized the clinical presentation and risk factors for a severe clinical course in children admitted to British Columbia Childrens Hospital during the 2014 outbreak. Methods. Retrospective chart review of patients with confirmed EV-D68 infection admitted to BCCH with respiratory symptoms in the fall of 2014. Past medical history, clinical presentation, management, and course in hospital was collected and analyzed using descriptive statistics. Comparison was made between those that did and did not require ICU admission to identify risk factors. Results. Thirty-four patients were included (median age 7.5 years). Fifty-three percent of children had a prior history of wheeze, 32% had other preexisting medical comorbidities, and 15% were previously healthy. Ten children (29%) were admitted to the pediatric intensive care unit. The presence of complex medical conditions (excluding wheezing) (P = 0.03) and copathogens was associated with PICU admission (P = 0.02). Conclusions. EV-D68 infection resulted in severe, prolonged presentations of asthma-like illness in the hospitalized pediatric population. Patients with a prior history of wheeze and preexisting medical comorbidities appear to be most severely affected, but the virus can also cause wheezing in previously well children.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

Is it safe to re-access sodium bicarbonate bottles for use in minor surgery?

BACKGROUND/PURPOSEnSodium bicarbonate is added to lidocaine to reduce injection pain. In Canada, it is available in vials exceeding the injection volume 100-fold. These are single-use vials that should be disposed of after one access. Some surgeons re-use vials to reduce waste, potentially causing contamination. This study aims to review the safety of sodium bicarbonate and assess alternatives to current practice.nnnMETHODSnStrains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Burkholderia cepacia were used to assess bacterial growth in vials of sodium bicarbonate. Each pathogen was inoculated into a vial for 14u202fdays at room temperature. At several time points, 1u202fmL of solution was removed and diluted. One hundred microliters were transferred to blood agar plates and incubated at 35u202f°C. Colony counts were calculated, averaged and plotted onto a logarithmic graph.nnnRESULTSnColony counts of all strains fell below observational threshold after 7u202fdays in sodium bicarbonate.nnnCONCLUSIONSnAlthough all strains were reduced, bacteria can survive in sodium bicarbonate for several days, during which transmission may occur. Sodium bicarbonate vials should be treated as single-dose, as indicated by the manufacturers. To reduce waste, hospital pharmacies can repackage sodium bicarbonate into smaller vials or pre-alkalize lidocaine with sodium bicarbonate.