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Obesity is associated with several chronic conditions including diabetes, cardiovascular disease, and metabolic dysfunction-associated steatotic liver disease and malignancy. Bariatric surgery, most commonly Roux-en-Y gastric bypass and sleeve gastrectomy, is an effective treatment modality for obesity and can improve associated comorbidities. Over the last 20 years, there has been an increase in the rate of bariatric surgeries associated with the growing obesity epidemic. Sleeve gastrectomy is the most widely performed bariatric surgery currently, and while it serves as a durable option for some patients, it is important to note that several complications, including sleeve leak, stenosis, chronic fistula, gastrointestinal hemorrhage, and gastroesophageal reflux disease, may occur. Endoscopic methods to manage post-sleeve gastrectomy complications are often considered due to the risks associated with a reoperation, and endoscopy plays a significant role in the diagnosis and management of post-sleeve gastrectomy complications. We perform a detailed review of the current endoscopic management of post-sleeve gastrectomy complications.

Obesity is associated with serious comorbidities and economic implications. Bariatric surgery, most commonly Roux-en-Y gastric bypass and sleeve gastrectomy, are effective options for weight loss and the improvement of obesity-related comorbidities. With the growing obesity epidemic, there has been a concomitant rise in bariatric surgeries, particularly in sleeve gastrectomy, which has been the most widely performed bariatric surgery since 2013. Gastroesophageal reflux disease (GERD) is highly prevalent in obese individuals, can significantly impact quality of life and may lead to serious complications. Obesity and GERD both improve with weight loss. However, as the incidence of sleeve gastrectomy rises, recent data have revealed a risk of exacerbation of pre-existing GERD or the development of de novo GERD following sleeve gastrectomy. We performed a detailed review of GERD post-sleeve gastrectomy, including its overall incidence, pathophysiology and current treatment paradigms.

Abstract Background Influence of sarcopenia in combination with other body composition parameters and muscle strength on outcomes after oesophageal surgery for oesophageal cancer remains unclear. The objectives were (i) to describe the incidence of sarcopenia in relation to adipose tissue quantity and distribution and muscle strength; (ii) to evaluate if neoadjuvant chemoradiation (nCRTx) influences body composition and muscle strength; and (iii) to evaluate the influence of body composition and muscle strength on post‐operative morbidity and long‐term survival. Methods This retrospective study included patients with oesophageal cancer who received nCRTx followed by surgery between January 2011 and 2016. Skeletal muscle, visceral, and subcutaneous adipose tissue cross‐sectional areas were calculated based on computed tomography scans, and muscle strength was measured using hand grip tests, 30 seconds chair stand tests, and maximal inspiratory and expiratory pressure tests prior to nCRTx and after nCRTx. Results A total of 322 patients were included in this study. Sarcopenia was present in 55.6% of the patients prior to nCRTx and in 58.2% after nCRTx (P = 0.082). Patients with sarcopenia had a significantly lower muscle strength and higher fat percentage. The muscle strength and incidence of sarcopenia increased while the mean body mass index and fat percentage decreased during nCRTx. A body mass index above 25 kg/m2 was associated with anastomotic leakage (P = 0.032). Other body composition parameters were not associated with post‐operative morbidity. A lower handgrip strength prior to nCRTx was associated with pulmonary and cardiac complications (P = 0.023 and P = 0.009, respectively). In multivariable analysis, a lower number of stands during the 30 seconds chair stand test prior to nCRTx (hazard ratio 0.93, 95% confidence interval 0.87–0.99, P = 0.017) and visceral adipose tissue of >128 cm2 after nCRTx (hazard ratio 1.81, 95% confidence interval 1.30–2.53, P = 0.001) were associated with worse overall survival. Conclusions Sarcopenia occurs frequently in patients with oesophageal cancer and is associated with less muscle strength and a higher fat percentage. Body composition changes during nCRTx did not influence survival. Impaired muscle strength and a high amount of visceral adipose tissue are associated with worse survival. Therefore, patients with poor fitness might benefit from preoperative nutritional and muscle strengthening guidance, aiming to increase muscle strength and decrease visceral adipose tissue. However, this should be confirmed in a large prospective study.

Legionellosis is mostly caused by Legionella pneumophila and is defined as a severe respiratory illness with a case fatality rate ranging from 5% to 80%. L. pneumophila is ubiquitous in natural and anthropogenic water systems. L. pneumophila is transmitted by inhalation of contaminated aerosols produced by a variety of devices. While L. pneumophila replicates within environmental protozoa, colonization and persistence in its natural environment are also mediated by biofilm formation and colonization within multispecies microbial communities. There is now evidence that some legionellosis outbreaks are correlated with the presence of biofilms. Thus, preventing biofilm formation appears as one of the strategies to reduce water system contamination. However, we lack information about the chemical and biophysical conditions, as well as the molecular mechanisms that allow the production of biofilms by L. pneumophila. Here, we discuss the molecular basis of biofilm formation by L. pneumophila and the roles of other microbial species in L. pneumophila biofilm colonization. In addition, we discuss the protective roles of biofilms against current L. pneumophila sanitation strategies along with the initial data available on the regulation of L. pneumophila biofilm formation.

[This corrects the article DOI: 10.1371/journal.pone.0059237.].

We analyzed travel-associated clinical isolates of Escherichia coli O104:H4, including 1 from the 2011 German outbreak and 1 from a patient who returned from the Philippines in 2010, by genome sequencing and optical mapping. Despite extensive genomic similarity between these strains, key differences included the distribution of toxin and antimicrobial drug–resistance determinants.

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002–2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research.

The water-borne pathogen Legionella pneumophila serogroup 1 (Lp1) is the most commonly reported etiologic agent of legionellosis. To examine the genetic diversity, the long-term epidemiology, and the molecular evolution of Lp1 clinical isolates, we conducted sequence-based typing on a collection of clinical isolates representing 3 decades of culture-confirmed legionellosis in Ontario, Canada. Analysis showed that the population of Lp1 in Ontario is highly diverse and combines lineages identified worldwide with local strains. Identical types were identified in sporadic and outbreak-associated strains. In the past 15 years, the incidence of some lineages distributed worldwide has tended to decrease, and local endemic clones and lineages have emerged. Comparative geographic distribution analysis suggests that some lineages are specific to eastern North America. These findings have general clinical implications for the study of Lp1 molecular evolution and for the identification of Lp1 circulating strains in North America.

Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from nares of 27/417 (6.5%) attendees at an international veterinary conference: 23/345 (7.0%) veterinarians, 4/34 (12.0%) technicians, and 0/38 others. Colonization was more common for large-animal (15/96, 15.6%) than small-animal personnel (12/271, 4.4%) or those with no animal patient contact (0/50) (p

Since January 2005, H3N2 influenza viruses have been isolated from pigs and turkeys throughout Canada and from a swine farmer and pigs on the same farm in Ontario. These are human/classical swine/avian reassortants similar to viruses that emerged in US pigs in 1998 but with a distinct human-lineage neuraminidase gene.

BACKGROUND: There are currently limited data regarding the prevalence of antimicrobial-resistant organisms causing community-acquired urinary tract infections among adult women in Canada. Trimethoprim-sulfamethoxazole (TMP-SMX) is the recommended first-line empirical antibiotic treatment, unless resistance of Escherichia coli to TMP-SMX exceeds 20%.

Late recognition of severe acute respiratory syndrome (SARS) was associated with no known SARS contact, hospitalization before the nosocomial outbreak was recognized, symptom onset while hospitalized, wards with SARS clusters, and postoperative status. SARS is difficult to recognize in hospitalized patients with a variety of underlying conditions in the absence of epidemiologic links.

Antimicrobial drug resistance rates for Mycoplasma pneumoniae was determined in clinical specimens and isolates obtained during 2011–2012 in Ontario, Canada. Of 91 M. pneumoniae drug-resistant specimens, 11 (12.1%) carried nucleotide mutations associated with macrolide resistance in the 23S rRNA gene. None of the M. pneumoniae specimens were resistant to fluoroquinolones or tetracyclines.

Infection of healthcare workers with the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) is thought to occur primarily by either contact or large respiratory droplet transmission. However, infrequent healthcare worker infections occurred despite the use of contact and droplet precautions, particularly during certain aerosol-generating medical procedures. We investigated a possible cluster of SARS-CoV infections in healthcare workers who used contact and droplet precautions during attempted cardiopulmonary resuscitation of a SARS patient. Unlike previously reported instances of transmission during aerosol-generating procedures, the index case-patient was unresponsive, and the intubation procedure was performed quickly and without difficulty. However, before intubation, the patient was ventilated with a bag-valve-mask that may have contributed to aerosolization of SARS-CoV. On the basis of the results of this investigation and previous reports of SARS transmission during aerosol-generating procedures, a systematic approach to the problem is outlined, including the use of the following: 1) administrative controls, 2) environmental engineering controls, 3) personal protective equipment, and 4) quality control.

Early recognition and rapid initiation of infection control precautions are currently the most important strategies for controlling severe acute respiratory syndrome (SARS). No rapid diagnostic tests currently exist that can rule out SARS among patients with febrile respiratory illnesses. Clinical features alone cannot with certainty distinguish SARS from other respiratory illnesses rapidly enough to inform early management decisions. A balanced approach to screening that allows early recognition of SARS without unnecessary isolation of patients with other respiratory illnesses will require clinicians not only to look for suggestive clinical features but also to routinely seek epidemiologic clues suggestive of SARS coronavirus exposure. Key epidemiologic risk factors include 1) exposure to settings where SARS activity is suspected or documented, or 2) in the absence of such exposure, epidemiologic linkage to other persons with pneumonia (i.e., pneumonia clusters), or 3) exposure to healthcare settings. When combined with clinical findings, these epidemiologic features provide a possible strategic framework for early recognition of SARS.

Efforts to contain severe acute respiratory syndrome (SARS) have been limited by the lack of a standardized, sensitive, and specific test for SARS-associated coronavirus (CoV). We used a standardized reverse transcription-polymerase chain reaction assay to detect SARS-CoV in lung samples obtained from well-characterized patients who died of SARS and from those who died of other reasons. SARS-CoV was detected in all 22 postmortem lung tissues (to 109 viral copies/g) from 11 patients with probable SARS but was not detected in any of the 23 lung control samples (sample analysis was blinded). The sensitivity and specificity (95% confidence interval) were 100% (84.6% to 100%) and 100% (85.1% to 100%), respectively. Viral loads were significantly associated with a shorter course of illness but not with the use of ribavirin or steroids. CoV was consistently identified in the lungs of all patients who died of SARS but not in control patients, supporting a primary role for CoV in deaths.

Clinical antimicrobial susceptibility breakpoints are used to predict the clinical outcome of antimicrobial treatment. In contrast, microbiologic breakpoints are used to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. Currently, the National Committee for Clinical Laboratory Standards (NCCLS) guidelines utilize clinical breakpoints to characterize the activity of the fluoroquinolones against Streptococcus pneumoniae. To determine whether levofloxacin breakpoints can identify isolates that harbor recognized resistance mechanisms, we examined 115 S. pneumoniae isolates with a levofloxacin MIC of >2 μg/mL for first-step parC mutations. A total of 48 (59%) of 82 isolates with a levofloxacin MIC of 2 μg/mL, a level considered susceptible by NCCLS criteria, had a first-step mutation in parC. Whether surveillance programs that use levofloxacin data can effectively detect emerging resistance and whether fluoroquinolones can effectively treat infections caused by such isolates should be evaluated.

BACKGROUND: Bacteremic pneumococcal pneumonia has not been the subject of a multicentre Canadian Study.

Recent case reports describe multidrug-resistant influenza A pandemic (H1N1) 2009 virus infection in immunocompromised patients exposed to neuraminidase inhibitors because of an I223R neuraminidase mutation. We report a case of multidrug-resistant pandemic (H1N1) 2009 bearing the I223R mutation in an ambulatory child with no previous exposure to neuraminidase inhibitors.

OBJECTIVE: To determine the susceptibility of community outpatient Gram-negative urinary tract isolates to mecillinam and other commonly used oral agents.

Diagnostic difficulties may have led to underestimation of rhinovirus infections in long-term care facilities. Using surveillance data, we found that rhinovirus caused 59% (174/297) of respiratory outbreaks in these facilities during 6 months in 2009. Disease was sometimes severe. Molecular diagnostic testing can differentiate these outbreaks from other infections such as influenza.

Outbreaks of respiratory infection in institutions in Ontario, Canada were studied from April 20 to June 12, 2009, during the early stages of the emergence of influenza A pandemic (H1N1) 2009. Despite widespread presence of influenza in the general population, only 2 of 83 outbreaks evaluated by molecular methods were associated with pandemic (H1N1) 2009.

OBJECTIVE: To determine the in vitro activity of cefepime against multidrug-resistant Gram-negative bacilli and Gram-positive cocci obtained from an ongoing cross-Canada surveillance study.

OBJECTIVE: To determine the prevalence of antimicrobial resistance in clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis from medical centres across Canada.

OBJECTIVE: To compare the activity of piperacillin/tazobactam with that of other broad parenteral antibiotics against aerobic and facultative anaerobic blood culture isolates in a Canada-wide survey.

OBJECTIVES: To compare the activity of piperacillin-tazobactam with piperacillin and other parenterally administered antibiotics against aerobic Gram-negative bacilli and Gram-positive cocci isolated from across Canada, and to determine the prevalence of resistance mediated by extended-spectrum cephalosporinases.

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO2, bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS.

Analysis of the population genetic structure of microbial species is of fundamental importance to many scientific disciplines because it can identify cryptic species, reveal reproductive mode, and elucidate processes that contribute to pathogen evolution. Here, we examined the population genetic structure and geographic differentiation of the sexual, dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis.Criteria for Genealogical Concordance Phylogenetic Species Recognition (GCPSR) applied to seven nuclear loci (arf6, chs2, drk1, fads, pyrF, tub1, and its-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2, drk1, pyrF, and its-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenated chs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy with all isolates falling into one of two evolutionarily independent lineages. Phylogenetic species were genetically distinct with interspecific divergence 4-fold greater than intraspecific divergence and a high Fst value (0.772, P

ABSTRACT Fecal microbiome transplantation by low-volume enema is an effective, safe, and inexpensive alternative to antibiotic therapy for patients with chronic relapsing Clostridium difficile infection (CDI). We explored the microbial diversity of pre- and posttransplant stool specimens from CDI patients (n = 6) using deep sequencing of the 16S rRNA gene. While interindividual variability in microbiota change occurs with fecal transplantation and vancomycin exposure, in this pilot study we note that clinical cure of CDI is associated with an increase in diversity and richness. Genus- and species-level analysis may reveal a cocktail of microorganisms or products thereof that will ultimately be used as a probiotic to treat CDI. IMPORTANCE Antibiotic-associated diarrhea (AAD) due to Clostridium difficile is a widespread phenomenon in hospitals today. Despite the use of antibiotics, up to 30% of patients are unable to clear the infection and suffer recurrent bouts of diarrheal disease. As a result, clinicians have resorted to fecal microbiome transplantation (FT). Donor stool for this type of therapy is typically obtained from a spouse or close relative and thoroughly tested for various pathogenic microorganisms prior to infusion. Anecdotal reports suggest a very high success rate of FT in patients who fail antibiotic treatment (>90%). We used deep-sequencing technology to explore the human microbial diversity in patients with Clostridium difficile infection (CDI) disease after FT. Genus- and species-level analysis revealed a cocktail of microorganisms in the Bacteroidetes and Firmicutes phyla that may ultimately be used as a probiotic to treat CDI.

Pulsed field gel electrophoresis (pfge) was used to compare 59 strains of Moraxella catarrhalis to evaluate pfge for the epidemiological typing of this organism. pfge-generated patterns were compared with those obtained by small fragment restriction enzyme analysis (rea) and species-specific probe hybridization. The strains used in the study were isolated from various geographic locations and included proven epidemiologically related strains. pfge yielded more unique patterns than dna-dna hybridization – 30 versus 18, respectively – but fewer than rea, which generated 45 unique patterns. Strains that demonstrated the same rea pattern or dna-dna hybridization pattern did not always demonstrate the same pfge pattern. For example, in 23 epidemiologically unrelated strains that shared six rea patterns, pfge differentiated the isolates into 12 patterns. Conversely, strains that demonstrated the same pfge pattern did not always demonstrate the same rea pattern or hybridization pattern. For example, in 42 strains that shared 13 pfge patterns, rea differentiated the isolates into 31 patterns and dna-dna hybridization differentiated them into 16 patterns. However, compared with rea, pfge yielded less complex patterns that were more easily comparable, and compared with dna-dna hybridization, pfge was technically easier.

To compare the activity of cefepime, a fourth-generation cephalosporin, with several available antimicrobials, in vitro susceptibility studies were carried out on bacteria commonly associated with various infections, including sepsis. Ten tertiary care hospital laboratories in six provinces provided 1276 clinically relevant isolates of aerobic Gram-negative bacilli and Gram-positive cocci during 1993. When the activity of each of the antimicrobials was determined against all isolates submitted, cefepime, piperacillin/tazobactam, imipenem and ciprofloxacin all had minimal inhibitory concentrations for 90% of the organisms (mic90) two or more dilutions below the mic resistant category. Gentamicin’s mic90 against all organisms tested was one dilution below the mic resistant category. The mic90s of the third-generation cephalosporins, piperacillin and ticarcillin/clavulanate, for Enterobacter species fell in the resistant category. This is presumably due to constitutive high level chromosomal cephalosporinase production. The mic90s of cefepime for Enterobacter species was three or more dilutions below the mic resistant category. The mic90s of all antimcrobials against Staphylococcus aureus, with the exception of ceftazidime and piperacillin, had mic90 categories two or more dilutions below the resistant category. The activity of cefepime, piperacillin/tazobactam, imipenem, ciprofloxacin and gentamicin make them excellent candidates for the empirical therapy of serious infections due to aerobic Gram-negative bacilli and S aureus.

In 1992, a surveillance study was performed in Canada to determine the susceptibility of nosocomial Gram-negative rods to several wide spectrum antimicrobials. Consecutive isolates from 10 institutions, as well as additional strains of selected species of Enterobacteriaceae that are known to possess the Bush group 1 beta-lactamase, were tested for susceptibility to 12 antimicrobials. Third-generation cephalosporin resistance was found to be as high as 29% in Enterobacter cloacae that possesses the Bush group 1 beta-lactamase and less than 4% in those isolates not possessing this enzyme. Cefepime equalled or exceeded the activity of the third-generation cephalosporins against the species of Enterobacteriaceae that demonstrated resistance to the third-generation cephalosporins.

The escalating costs associated with antimicrobial chemotherapy have become of increasing concern to physicians, pharmacists and patients alike. A number of strategies have been developed to address this problem. This article focuses specifically on sequential antibiotic therapy (sat), which is the strategy of converting patients from intravenous to oral medication regardless of whether the same or a different class of drug is used. Advantages of sat include economic benefits, patient benefits and benefits to the health care provider. Potential disadvantages are cost to the consumer and the risk of therapeutic failure. A critical review of the published literature shows that evidence from randomized controlled trials supports the role of sat. However, it is also clear that further studies are necessary to determine the optimal time for intravenous to oral changeover and to identify the variables that may interfere with the use of oral drugs. Procedures necessary for the implementation of a sat program in the hospital setting are also discussed.

Escherichia coli O104:H4 was identified as an emerging pathogen during the spring and summer of 2011 and was responsible for a widespread outbreak that resulted in the deaths of 50 people and sickened over 4075. Traditional phenotypic and genotypic assays, such as serotyping, pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), permit identification and classification of bacterial pathogens, but cannot accurately resolve relationships among genotypically similar but pathotypically different isolates. To understand the evolutionary origins of E. coli O104:H4, we sequenced two strains isolated in Ontario, Canada. One was epidemiologically linked to the 2011 outbreak, and the second, unrelated isolate, was obtained in 2010. MLST analysis indicated that both isolates are of the same sequence type (ST678), but whole-genome sequencing revealed differences in chromosomal and plasmid content. Through comprehensive phylogenetic analysis of five O104:H4 ST678 genomes, we identified 167 genes in three gene clusters that have undergone homologous recombination with distantly related E. coli strains. These recombination events have resulted in unexpectedly high sequence diversity within the same sequence type. Failure to recognize or adjust for homologous recombination can result in phylogenetic incongruence. Understanding the extent of homologous recombination among different strains of the same sequence type may explain the pathotypic differences between the ON2010 and ON2011 strains and help shed new light on the emergence of this new pathogen.

BACKGROUND: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL). METHODS: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens. RESULTS: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p

Legionellosis is mostly caused by Legionella pneumophila (Lp) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of Lp, named Lcl, is produced during legionellosis and is unique to the L. pneumophila species. Importantly, a mutant depleted in Lcl (Δlpg2644) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the C-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of lpg2644, mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of lpg2644 may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory infections in children under 2 years of age and causes repeated infections throughout life. We investigated the genetic variability of RSV-A circulating in Ontario during 2010-2011 winter season by sequencing and phylogenetic analysis of the G glycoprotein gene.Among the 201 consecutive RSV isolates studied, RSV-A (55.7%) was more commonly observed than RSV-B (42.3%). 59.8% and 90.1% of RSV-A infections were among children ≤12 months and ≤5 years old, respectively. On phylogenetic analysis of the second hypervariable region of the 112 RSV-A strains, 110 (98.2%) clustered within or adjacent to the NA1 genotype; two isolates were GA5 genotype. Eleven (10%) NA1-related isolates clustered together phylogenetically as a novel RSV-A genotype, named ON1, containing a 72 nucleotide duplication in the C-terminal region of the attachment (G) glycoprotein. The predicted polypeptide is lengthened by 24 amino acids and includes a23 amino acid duplication. Using RNA secondary structural software, a possible mechanism of duplication occurrence was derived. The 23 amino acid ON1 G gene duplication results in a repeat of 7 potential O-glycosylation sites including three O-linked sugar acceptors at residues 270, 275, and 283. Using Phylogenetic Analysis by Maximum Likelihood analysis, a total of 19 positively selected sites were observed among Ontario NA1 isolates; six were found to be codons which reverted to the previous state observed in the prototype RSV-A2 strain. The tendency of codon regression in the G-ectodomain may infer a decreased avidity of antibody to the current circulating strains. Further work is needed to document and further understand the emergence, virulence, pathogenicity and transmissibility of this novel RSV-A genotype with a72 nucleotide G gene duplication.

BACKGROUND: Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen. METHODS: We compared standard invasive procedures including biopsy histopathology, biopsy PCR, and leishmanin skin test (LST) to a novel, non-invasive, cytology-brush based PCR for the diagnosis of ML in Lima, Peru. Consensus reference standard was 2/4 tests positive, and outcome measures were sensitivity and specificity. Leishmania species identification was performed by PCR-based assays of positive specimens. RESULTS: Twenty-eight patients were enrolled, 23 of whom fulfilled criteria for a diagnosis of ML. Sensitivity and specificity of biopsy with histopathology were 21.7% [95% CI 4.9-38.5%] and 100%; 69.6% [95% CI 50.8-88.4%] and 100% for LST; 95.7% [95% CI 87.4-100%] and 100% for biopsy PCR; and 95.7% [95% CI 87.4-100%] and 90% [95% CI 71.4-100%] for cytology brush PCR using both Cervisoft® and Histobrush® cervical cytology brushes. Represented species identified by PCR-RFLP included: L. (V). braziliensis (n = 4), and L. (V). peruviana (n = 3). CONCLUSIONS: Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted.

The in vitro activity of cilofungin against 100 Candida species was compared with 5-flucytosine. amphotericin B and ketoconazole by two laboratories independently and in a blinded fashion using a macrotitre dilution broth method in saam-f medium. Cilofungin showed good in vitro activity against Candida albicans. Candida tropicalis and Candida glabrata (90% minimal inhibitory concentration [MIC] 3.2 μg/mL) but was inactive against other Candida species. When testing the susceptibility of cilofungin, 5-flucytosine and amphotericin B at the two centres, approximately 90% of the Candida strains had MICs differing by fourfold or less. However, when testing susceptibility of ketoconazole, only 51% of the Candida strains had MIC differences fourfold or less. MIC susceptibility testing with cilofungin, 5-flucytosine and amphotericin B in saam-f medium is reproducible.

In a regional oncology hospital using cotrimoxazole (trimethoprim-sulphamethoxazole) prophylaxis during chemotherapy-induced neutropenia, a single strain of Escherichia coli (indole negative) caused 15 of 27 episodes of Gram-negative rod bacteremia in 1987, and four of 32 such episodes in 1988. This biotype had not been recovered in 1986. Investigations during this ‘outbreak’ of bacteremias revealed enteric colonization with this strain of E coli in 37% of patients on leukemia or bone marrow transplant wards and in several staff members in July 1987. In 1988, 11 of 32 Gram-negative rod bacteremias were secondary to other strains of indole positive E coli of several different biotypes and plasmid profiles. Indole negative strains all exhibited low level trimethoprim resistance, whereas indole positive strains which subsequently appeared exhibited high level trimethoprim resistance. Failure of cotrimoxazole prophylaxis was initially due to the clonal dissemination of a single strain of E coli within the institution, with the subsequent appearance of multiple E coli strains with probable differing genetic bases for their resistance.

A perceived increase in the number of isolates of Moraxella catarrhalis from the respiratory secretions of patients intubated in the pediatric intensive care unit prompted a review of the clinical profiles of such patients and restriction enzyme analysis of the strains involved. Over two months, of 192 patients admitted to the unit, 154 were intubated. Of the 46 for whom endotracheal tube specimens were submitted to the laboratory, M catarrhalis was isolated in 12. M catarrhalis was not felt to be a significant respiratory pathogen by the attending medical staff in any of the patients from whom it was isolated. In only two patients (17%) could nosocomial acquisition be firmly invoked. Restriction enzyme analysis of the 12 strains ruled out the presence of an epidemic strain. Isolation of M catarrhalis from intubated children does not necessarily imply pathogenicity nor an outbreak situation.

Detection of heterophile antibodies in infectious mononucleosis is the most rapid and cost-effective method for confirming the clinical diagnosis of the disease. This study compared seven commercial test kits (the Oxoid Infectious Mononucleosis Kit [Oxoid Ltd], Immunoscan Im-Latex [Baxter Travenol], Mono-Latex [Wampole Laboratories], Monospot and Im Screen Test [Ortho Diagnostics], Immunoscan Im-RBC Test [Baxter Travenol], and Infectious Mononucleosis Test [NCS Diagnostics]) to the Davidsohn differential test. All of the kits were shown to be acceptable for use, with specificities and sensitivities greater than 96.5% and 95.5%, respectively.

Verocytotoxin-producing strains of Escherichia coli, most often serotype 0157:H7, have been associated with both sporadic and epidemic diarrheal disease in Canada. In order to determine the isolation rate of E coli 0157:H7 in outpatients with diarrhea, all stool specimens submitted for culture to Med-Chem Laboratories in Metropolitan Toronto between June 1988 and September 1989 were cultured on MacConkey-Sorbitol agar in addition to standard enteric media. A total of 46 (0.3%) of 16,125 stool specimens yielded E coli 0157:H7 or verotoxin-producing E coli 0157:H−. These isolates came from 31 patients with diarrhea; only 16 (52%) had a history of hemorrhagic colitis and one patient developed hemolytic uremic syndrome. Although MacConkey-Sorbitol agar was useful as a differential medium for detecting E coli 0157:H7, 14.5% of all specimens yielded nonsorbitol-fermenting isolates. It is not certain whether the routine use of MacConkey-Sorbitol agar is justified when isolation rates of E coli 0157:117 are very low.

Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells.We characterized in vivo interactions between group A streptococci (GAS) and cells involved in innate immune responses, using human biopsies (n = 70) collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria.This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections highlights a need for alternative therapeutic strategies.

In vitro studies have shown that Mycobacterium marinum is usually susceptible to clarithromycin. However, there are limited published data on the clinical use of clarithromycin for the treatment of M marinum infections. This report describes a previously healthy 58-year-old man who developed a chronic soft tissue infection of his right hand caused by M marinum. He responded to four weeks’ therapy with clarithromycin. Follow-up at six months showed no relapse. Our experience and review of the literature suggest that short course monotherapy with clarithromycin may be quite effective for uncomplicated soft issue infections caused by M marinum.

Community-acquired pneumonia (CAP) is common among children and may have viral, bacterial or, occasionally, other causes. The etiology is complex, with age-related trends, and differs from that in adult CAP, necessitating different management guidelines. There is an absence of current guidelines for the management of pediatric CAP (PCAP) that take into account changing etiologies, antimicrobial-resistance issues and the use of newly licensed antimicrobials. The present review does not provide specific guidelines, but it reviews the literature and presents currrent approaches to the treatment of PCAP. To compile the review, an expert panel was convened to provide a consensus. The review discusses the etiology, diagnosis and antimicrobial treatment of PCAP as well as indications for referral to a hospital emergency department. The goal of the review is to provide those involved with treatment of PCAP in the community setting with information that can be used to make effective treatment choices.