Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

A genome-wide association analysis reveals a potential role for recombination in the evolution of antimicrobial resistance in Burkholderia multivorans.

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (ß-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to ß-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with ß-lactam resistance were over-represented in these recombinogenic regions.

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation.

The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

Wound swab quality grading is dependent on Gram smear screening approach.

Superficial skin swab collections are inherently low-quality and may be of little clinical value due to their poor sensitivity and specificity. Clinical microbiology laboratories can use Gram smears to screen and differentiate higher and lower quality specimens to direct the extent of potential pathogen work up, including antimicrobial susceptibility testing (AST). We compared the impact of two different smear grading approaches to our current reporting practices for superficial wound swab cultures. Two variations of the Q score methodology (low power under 10X (QS10) and high power under 100X (QS100) were compared to our existing oil immersion method (OM100) (100X). We further evaluated the QS100 method by scoring superficial swab smears previously screened by OM100 from cultures submitted between November 2018 and December 2019. No significant difference in the number of low-quality specimens (N = 50) was identified by QS10 or QS100 grading (N = 9; 18%; N = 8; 16% respectively). Among 968 additional QS100 screened smears, 67 (6.9%) low quality swabs were identified and 7.4% fewer organisms (76/1020 organisms) would require reporting with AST. Implementing the Q score for superficial wound swab cultures would provide minimal improvements in their clinical relevance, laboratory quality and efficiency in our laboratory due to the low number of poor-quality swabs received.

A Ten-Year Retrospective Survey of Antimicrobial Susceptibility Patterns among Salmonella enterica subsp. enterica Serovar Typhi Isolates in Ontario, Canada.

The epidemiology and treatment of typhoid fever are complicated by the emergence and spread of Salmonella enterica subsp. enterica serovar Typhi lineages with resistance to many antimicrobial agents critical for therapy. Current information on the susceptibility patterns of S. Typhi isolates identified in regions where typhoid fever is not endemic is important as these are often acquired after traveling to countries of endemicity where resistant strains circulate. Here, we report a 10-year retrospective survey of S. Typhi antimicrobial susceptibility patterns among 858 unique patient isolates that underwent reference laboratory testing in Ontario, Canada, between 2010 and 2019. Antimicrobial susceptibility patterns remained stable for ampicillin (average, 78.7% susceptible), azithromycin (average, 99.4% susceptible) ertapenem (average, 100.0% susceptible), meropenem (average, 100.0% susceptible), and trimethoprim-sulfamethoxazole (average, 78.2% susceptible) during the study period; however, nonsusceptibility to ciprofloxacin and ceftriaxone increased. While ceftriaxone-resistant isolates comprised 1.6% of the total isolates overall, they represented 10.1% of the total isolates tested in 2019, indicating a significant increase over time. Our findings suggest that when selecting empirical therapy, health care providers should strongly consider current trends in antimicrobial susceptibility and investigate the patient's exposure risk to gauge whether a suspected typhoid infection may be caused by a potentially resistant S. Typhi strain. IMPORTANCE This work provides an updated summary of the antimicrobial susceptibility patterns among Salmonella Typhi strains isolated from patients in Ontario, Canada.

Streptococcus pyogenes secondary impetigo due to loofah sponge use.

Loofah sponges have been implicated in skin and soft tissue infections due to their ability to harbor bacteria and cause microtrauma to the skin. In this case report, we describe a case of impetigo and cellulitis due to Streptococcus pyogenes complicated by secondary spread through loofah sponge use. The same organism was cultured from the infected body sites and loofah sponge, and a comparative genomic analysis confirmed that the isolates were identical.

SARS-CoV-2 Not Detectable in Ocular Specimens of a Patient with a Past Infection.

Purpose: To present a a case study that aims to investigate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the ocular tissue samples of a patient previously infected with COVID-19 and determine its transmissibility.Study Design: Case ReportResults: In this case study, SARS-CoV-2 was not detected in the vitreous and uveal tissue samples by RT-PCR for detection of three gene targets in a patient with a past COVID-19 infection 15 days prior to presention with a globe rupture.Conclusions: Our findings suggest that patients with long-term existence of SARS-CoV-2 at low detectable levels may not have active intraocular viral shedding. This is of particular importance as ophthalmic surgical procedures may potentiate virus spread from patients infected with SARS-CoV-2.

Evaluation of digital dispense-assisted broth microdilution antimicrobial susceptibility testing for Pseudomonas aeruginosa isolates.

Antimicrobial susceptibility testing (AST) is essential for detecting resistance in Pseudomonas aeruginosa and other bacterial pathogens. Here we evaluated the performance of broth microdilution (BMD) panels created using a semi-automated liquid handler, the D300e Digital Dispenser (Tecan Group Ltd., CH) that relies on inkjet printing technology. Microtitre panels (96-well) containing nine twofold dilutions of 12 antimicrobials from five classes (ß-lactams, ß-lactam/ß-lactamase inhibitors, aminoglycosides, fluoroquinolones, polymyxins) were prepared in parallel using the D300e Digital Dispenser and standard methods described by CLSI/ISO. To assess performance, panels were challenged with three well characterized quality control organisms and 100 clinical P. aeruginosa isolates. Traditional agreement and error measures were used for evaluation. Essential (EA) and categorical (CA) agreements were 92.7% and 98.0% respectively for P. aeruginosa isolates with evaluable on-scale results. The majority of minor errors that fell outside acceptable EA parameters (≥ ± 1 dilution, 1.9%) were seen with aztreonam (5%) and ceftazidime (4%), however all antimicrobials displayed acceptable performance in this situation. Differences in MIC were often log2 dilution lower for D300e dispensed panels. Major and very major errors were noted for aztreonam (2.6%) and cefepime (1.7%) respectively. The variable performance of D300e panels suggests that further testing is required to confirm their diagnostic utility for P. aeruginosa.

One swab, two tests: Validation of dual SARS-CoV-2 testing on the Abbott ID NOW™.

BACKGROUND: Point-of-care tests (POCT) are promising tools to detect SARS-CoV-2 in specific settings. Initial reports suggest the ID NOW™ COVID-19 assay (Abbott Diagnostics Inc, USA) is less sensitive than standard real-time reverse transcription polymerase chain reaction (rRT-PCR) assays. This has raised concern over false negatives in SARS-CoV-2 POCT. OBJECTIVES: We compared the performance of the ID NOW™ COVID-19 assay to our in-house rRT-PCR assay to assess whether dry swabs used in ID NOW™ testing could be stored in transport media and be re-tested by rRT-PCR for redundancy and to provide material for further investigation. METHODS: Paired respiratory swabs collected from patients at three acute care hospitals were used. One swab in transport media (McMaster Molecular Media (MMM)) was tested for SARS-CoV-2 by a laboratory-developed two-target rRT-PCR assay. The second was stored dry in a sterile container and tested by the ID NOW™ COVID-19 assay. Following ID NOW™ testing, dry swabs were stored in MMM for up to 48 h and re-tested by rRT-PCR. Serially diluted SARS-CoV-2 particles were used to assess the impact of heat inactivation and storage time. RESULTS: Respiratory swabs (n = 343) from 179 individuals were included. Using rRT-PCR results as the comparator, the ID NOW™ COVID-19 assay had positive (PPA) and negative (NPA) percent agreements of 87.0% (95% CI:0.74-0.94) and 99.7% (95% CI:0.98-0.99). Re-tested swabs placed in MMM following ID NOW testing had PPA and NPA of 88.8% (95% CI:0.76-0.95) and 99.7% (95% CI:0.98-0.99), respectively. CONCLUSIONS: Storing spent dry swabs in transport media for redundancy rRT-PCR testing is a potential approach to address possible false negatives with the ID NOW™ COVID-19 assay.

Ecological Succession of Polymicrobial Communities in the Cystic Fibrosis Airways.

Antimicrobial therapies against cystic fibrosis (CF) lung infections are largely aimed at the traditional, well-studied CF pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex, despite the fact that the CF lung harbors a complex and dynamic polymicrobial community. A clinical focus on the dominant pathogens ignores potentially important community-level interactions in disease pathology, perhaps explaining why these treatments are often less effective than predicted based on in vitro testing. A better understanding of the ecological dynamics of this ecosystem may enable clinicians to harness these interactions and thereby improve treatment outcomes. Like all ecosystems, the CF lung microbial community develops through a series of stages, each of which may present with distinct microbial communities that generate unique host-microbe and microbe-microbe interactions, metabolic profiles, and clinical phenotypes. While insightful models have been developed to explain some of these stages and interactions, there is no unifying model to describe how these infections develop and persist. Here, we review current perspectives on the ecology of the CF airway and present the CF Ecological Succession (CFES) model that aims to capture the spatial and temporal complexity of CF lung infection, address current challenges in disease management, and inform the development of ecologically driven therapeutic strategies.

Penicillin-binding protein 3 is a common adaptive target among Pseudomonas aeruginosa isolates from adult cystic fibrosis patients treated with ß-lactams.

OBJECTIVE: Determining the mechanisms that modulate ß-lactam resistance in clinical Pseudomonas aeruginosa (P. aeruginosa) isolates can be challenging, as the molecular profiles identified in mutation-based or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin-binding protein 3 (pbpB/ftsI). This study reported that nonsynonymous polymorphisms within pbpB frequently occur among ß-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. METHODS: Longitudinally collected isolates (n = 126) from cystic fibrosis (CF) patients with or without recent ß-lactam therapy or of non-clinical origin were tested for susceptibility to six ß-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem, and piperacillin). Known ß-lactam resistance mechanisms were characterised by polymerase chain reaction (PCR)-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. RESULTS: Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of ß-lactam therapy, compared with one polymorphism in 30 (3.3%) from three patients who had not received ß-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression reduced expression of oprD or the presence of extended-spectrum ß-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. CONCLUSION: This study's findings suggest that pbpB is a common adaptive target, and may contribute to the development of ß-lactam resistance in P. aeruginosa.

Microbiome networks and change-point analysis reveal key community changes associated with cystic fibrosis pulmonary exacerbations.

Over 90% of cystic fibrosis (CF) patients die due to chronic lung infections leading to respiratory failure. The decline in CF lung function is greatly accelerated by intermittent and progressively severe acute pulmonary exacerbations (PEs). Despite their clinical impact, surprisingly few microbiological signals associated with PEs have been identified. Here we introduce an unsupervised, systems-oriented approach to identify key members of the microbiota. We used two CF sputum microbiome data sets that were longitudinally collected through periods spanning baseline health and PEs. Key taxa were defined based on three strategies: overall relative abundance, prevalence, and co-occurrence network interconnectedness. We measured the association between changes in the abundance of the key taxa and changes in patient clinical status over time via change-point detection, and found that taxa with the highest level of network interconnectedness tracked changes in patient health significantly better than taxa with the highest abundance or prevalence. We also cross-sectionally stratified all samples into the clinical states and identified key taxa associated with each state. We found that network interconnectedness most strongly delineated the taxa among clinical states, and that anaerobic bacteria were over-represented during PEs. Many of these anaerobes are oropharyngeal bacteria that have been previously isolated from the respiratory tract, and/or have been studied for their role in CF. The observed shift in community structure, and the association of anaerobic taxa and PEs lends further support to the growing consensus that anoxic conditions and the subsequent growth of anaerobic microbes are important predictors of PEs.

Diversification of Pseudomonas aeruginosa within the cystic fibrosis lung and its effects on antibiotic resistance.

The evolution and diversification of bacterial pathogens within human hosts represent potential barriers to the diagnosis and treatment of life-threatening infections. Tremendous genetic and phenotypic diversity is characteristic of host adaptation in strains of Pseudomonas aeruginosa that infect the airways of individuals with chronic lung diseases and prove to be extremely difficult to eradicate. In this MiniReview, we examine recent advances in understanding within-host diversity and antimicrobial resistance in P. aeruginosa populations from the lower airways of individuals with the fatal genetic disease cystic fibrosis and the potential impacts that this diversity may have on detecting and interpreting antimicrobial susceptibility within these populations.

Selective Sweeps and Parallel Pathoadaptation Drive Pseudomonas aeruginosa Evolution in the Cystic Fibrosis Lung.

UNLABELLED: Pulmonary infections caused by Pseudomonas aeruginosa are a recalcitrant problem in cystic fibrosis (CF) patients. While the clinical implications and long-term evolutionary patterns of these infections are well studied, we know little about the short-term population dynamics that enable this pathogen to persist despite aggressive antimicrobial therapy. Here, we describe a short-term population genomic analysis of 233 P. aeruginosa isolates collected from 12 sputum specimens obtained over a 1-year period from a single patient. Whole-genome sequencing and antimicrobial susceptibility profiling identified the expansion of two clonal lineages. The first lineage originated from the coalescence of the entire sample less than 3 years before the end of the study and gave rise to a high-diversity ancestral population. The second expansion occurred 2 years later and gave rise to a derived population with a strong signal of positive selection. These events show characteristics consistent with recurrent selective sweeps. While we cannot identify the specific mutations responsible for the origins of the clonal lineages, we find that the majority of mutations occur in loci previously associated with virulence and resistance. Additionally, approximately one-third of all mutations occur in loci that are mutated multiple times, highlighting the importance of parallel pathoadaptation. One such locus is the gene encoding penicillin-binding protein 3, which received three independent mutations. Our functional analysis of these alleles shows that they provide differential fitness benefits dependent on the antibiotic under selection. These data reveal that bacterial populations can undergo extensive and dramatic changes that are not revealed by lower-resolution analyses. IMPORTANCE: Pseudomonas aeruginosa is a bacterial opportunistic pathogen responsible for significant morbidity and mortality in cystic fibrosis (CF) patients. Once it has colonized the lung in CF, it is highly resilient and rarely eradicated. This study presents a deep sampling examination of the fine-scale evolutionary dynamics of P. aeruginosa in the lungs of a chronically infected CF patient. We show that diversity of P. aeruginosa is driven by recurrent clonal emergence and expansion within this patient and identify potential adaptive variants associated with these events. This high-resolution sequencing strategy thus reveals important intraspecies dynamics that explain a clinically important phenomenon not evident at a lower-resolution analysis of community structure.

Lung microbiota across age and disease stage in cystic fibrosis.

Understanding the significance of bacterial species that colonize and persist in cystic fibrosis (CF) airways requires a detailed examination of bacterial community structure across a broad range of age and disease stage. We used 16S ribosomal RNA sequencing to characterize the lung microbiota in 269 CF patients spanning a 60 year age range, including 76 pediatric samples from patients of age 4-17, and a broad cross-section of disease status to identify features of bacterial community structure and their relationship to disease stage and age. The CF lung microbiota shows significant inter-individual variability in community structure, composition and diversity. The core microbiota consists of five genera - Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces. CF-associated pathogens such as Pseudomonas, Burkholderia, Stenotrophomonas and Achromobacter are less prevalent than core genera, but have a strong tendency to dominate the bacterial community when present. Community diversity and lung function are greatest in patients less than 10 years of age and lower in older age groups, plateauing at approximately age 25. Lower community diversity correlates with worse lung function in a multivariate regression model. Infection by Pseudomonas correlates with age-associated trends in community diversity and lung function.

Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis.

Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to ß-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample.

Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR.

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.