We report the whole-genome sequence (WGS) of an in vitro susceptible derivative revertant mutant from a bloodstream isolate involved in a nosocomial outbreak in Brazil. The WGS comprises 2.5 Mb with 2,500 protein-coding sequences, 16rRNA genes, and 60 tRNA genes.
OptrA is an ATP-binding cassette (ABC)-F protein that confers resistance to oxazolidinones and phenicols and can be either plasmid-encoded or chromosomally encoded. Here, we isolated 13 strains possessing a linezolid MIC of ≥4 mg/liter from nursery pigs in swine herds located across Brazil. Genome sequence comparison showed that these strains possess in different genetic contexts occurring in 5 different sequence type backgrounds. The gene invariably occurred in association with an regulator and a gene encoding a hypothetical protein. In some contexts, this genetic island was able to excise and form a covalently closed circle within the cell; this circle appeared to occur in high abundance and to be transmissible by coresident plasmids.
PURPOSE: To investigate a cluster of corneoscleral rim cultures positive for Achromobacter species over a 6-month period at Massachusetts Eye and Ear. METHODS: An increased rate of positive corneal donor rim cultures was noted at Massachusetts Eye and Ear between July and December 2017. Positive cultures were subjected to identification and antimicrobial susceptibility testing by phenotypic (MicroScan WalkAway) and genotypic (16S rDNA sequencing) methods. Samples of the eye wash solution (GeriCare) used in the eye bank were also evaluated. Antimicrobial activity of Optical-GS against Achromobacter spp. at 4°C and 37°C was assessed by time-kill kinetics assay. RESULTS: Of 99 donor rims cultured, 14 (14.1%) grew bacteria with 11 (78.6%) due to uncommon nonfermenting Gram-negative bacilli. These had been identified by standard automated methods as Achromobacter (n = 3), Alcaligenes (n = 3), Ralstonia (n = 2), Pseudomonas (n = 2), and Stenotrophomonas (n = 1). Eight of these 11 isolates were subsequently available for molecular identification, and all were identified as Achromobacter spp. Six bottles of eyewash solution were evaluated and were positive for abundant Achromobacter spp. (3.4 × 105 ± 1.1 CFU/mL). Optisol-GS had no bactericidal activity against Achromobacter spp. at 4°C after 24-hour incubation but was bactericidal at 37°C. None of the patients who had received the contaminated corneas developed postoperative infection. CONCLUSIONS: An eyewash solution arising from a single lot was implicated in the contamination of donor rims by Achromobacter spp. The isolates were able to survive in the Optisol-GS medium at the recommended storage temperature. This highlights the need to continue improving protocols for tissue preparation and storage.
Streptococcus pneumoniae is a leading cause of ocular infections including serious and sight-threatening conditions. The use of pneumococcal conjugate vaccines (PCV) has substantially reduced the incidence of pneumonia and invasive pneumococcal diseases, but has had limited impact on ocular infections. Additionally, widespread vaccine use has resulted in ongoing selective pressure and serotype replacement in carriage and disease. To gain insight into the population structure of pneumococcal isolates causing ocular infections in a post-PCV-13 time period, we investigated the genomic epidemiology of ocular S. pneumoniae isolates (n=45) collected at Massachusetts Eye and Ear between 2014 and 2017. By performing a series of molecular typing methods from draft genomes, we found that the population structure of ocular S. pneumoniae is highly diverse with 27 sequence types (grouped into 18 clonal complexes) and 17 serotypes being identified. Distribution of these lineages diverged according to the site of isolation, with conjunctivitis being commonly caused by isolates grouped in the Epidemic Conjunctivitis Cluster-ECC (60 %), and ST448 (53.3 %) being most frequently identified. Conversely, S. pneumoniae keratitis cases were caused by a highly diverse population of isolates grouping within 15 different clonal complexes. Serotyping inference demonstrated that 95.5 % of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80 %) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2 %) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2 %), and usually associated with the mefA +msrD genotype (n=15). These genes were located in the Macrolide Efflux Genetic Assembly cassette and were associated with low-level in vitro resistance to 14- and 15-membered macrolides. Less frequently, macrolide-resistant isolates carried an ermB gene (n=4), which was co-located with the tetM gene in a Tn-916-like transposon. Our study demonstrates that the population structure of ocular S. pneumoniae is highly diverse, mainly composed by isolates that escape the PCV-13 vaccine, with patterns of tissue/niche segregation, adaptation and specialization. These findings suggest that the population structure of ocular pneumococcus may be shaped by multiple factors including PCV-13 selective pressure, microbial-related and niche-specific host-associated features.
The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPβ-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPβ-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPβ-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.
PURPOSE: Venous thromboembolic complications have been reported in association with COVID-19 infection. We raise awareness of a potential temporal association between COVID-19 infection and retinal vein occlusion (RVO). DESIGN: Retrospective, non-consecutive case series. METHODS: Setting: Multicenter STUDY POPULATION: Patients presenting with hemi-RVO (HRVO) or central RVO (CRVO) between 3/2020 and 3/2021, with confirmed COVID-19 infection. EXCLUSION CRITERIA: age >50 years, hypertension, diabetes, glaucoma, obesity, underlying hypercoagulable states, and those requiring intubation during hospitalization. OUTCOMES: Ophthalmic findings including presenting and final visual acuity (VA), imaging findings, and clinical course. RESULTS: Twelve eyes of 12 patients with CRVO (9/12) or HRVO (3/12) following COVID-19 infection were included. Median age was 32 (range 18-50) years. Three patients were hospitalized, but none were intubated. Median time from COVID-19 diagnosis to ophthalmic symptoms was 6.9 weeks. Presenting VA ranged from 20/20 to counting fingers (CF), with over half (7/12) having VA ≥20/40. Optical coherence tomography (OCT) revealed macular edema in 33% of eyes, 80% (4/5) treated with anti-VEGF injections. Ninety-two percent (11/12) had partial or complete resolution of ocular findings at final follow up. Four eyes (33%) had retinal thinning on OCT by the end of the study interval. Final visual acuity ranged from 20/20 to 20/60, with 11/12 (92%) eyes achieving ≥20/40 VA eyes at a median final follow-up of 13 (range 4-52) weeks. CONCLUSIONS: While we acknowledge a high seroprevalence of COVID-19 and that a causal relationship cannot be established, we report this series to raise awareness of the potential risk of retinal vascular events due to a heightened thrombo-inflammatory state associated with COVID-19 infection.
ABSTRACT: Protecting nurses in healthcare facilities from SARS-CoV-2 infection is essential for maintaining an adequate nursing force. Foundational guidelines, consistently utilized, protect the nursing staff from infection. This article describes guidelines designed to reduce acute infection and associated morbidity and mortality among nursing staff and improve compliance with infection prevention protocols.
Lipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistant Alarmingly, daptomycin-resistant isolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8) HG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, the mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in both and resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to the species that make previously uncharacterized contributions to antimicrobial resistance and tolerance in .
INTRODUCTION: Since 2009, the Antibiotic Resistance Monitoring in Ocular Microorganisms (ARMOR) surveillance study has been assessing in vitro antibiotic resistance for bacterial isolates sourced from ocular infections in the US. The main goal of this systematic review was to compare in vitro resistance data for ocular pathogens from published US studies with the most recently published data from the ARMOR study (2009-2018) and, where possible, to evaluate trends in bacterial resistance over time over all studies. METHODS: A literature search was conducted using MEDLINE®, BIOSIS Previews®, and EMBASE® databases (1/1/1995-6/30/2021). Data were extracted from relevant studies and antibiotic susceptibility rates for common ocular pathogens (Staphylococcus aureus, coagulase-negative staphylococci [CoNS], Streptococcus pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae), longitudinal changes in susceptibility, and multidrug resistance (MDR) were compared descriptively. RESULTS: Thirty-two relevant studies were identified. High in vitro resistance was found among S. aureus and CoNS to fluoroquinolones, macrolides, and methicillin/oxacillin across studies, with high rates of MDR noted, specifically among methicillin-resistant staphylococci. Data from studies pre-dating or overlapping the early years of ARMOR reflected increasing rates of S. aureus resistance to fluoroquinolones, macrolides, methicillin/oxacillin, and aminoglycosides, while the ARMOR data suggested slight decreases in resistance to these classes between 2009 and 2018. Overall, methicillin-resistant S. aureus (MRSA) prevalence peaked from 2005 to 2015 with a possible decreasing trend in more recent years. DISCUSSION AND CONCLUSIONS: Data from local and regional US datasets were generally consistent with data from the national ARMOR surveillance study. Continued surveillance of ocular bacterial pathogens is needed to track trends such as methicillin resistance and MDR prevalence and any new emerging antibiotic resistance phenotypes. Susceptibility data from ARMOR can inform initial choice of therapy, especially in practice areas where local antibiograms are unavailable.
The ability to form biofilms in a variety of environments is a common trait of bacteria, and may represent one of the earliest defenses against predation. Biofilms are multicellular communities usually held together by a polymeric matrix, ranging from capsular material to cell lysate. In a structure that imposes diffusion limits, environmental microgradients arise to which individual bacteria adapt their physiologies, resulting in the gamut of physiological diversity. Additionally, the proximity of cells within the biofilm creates the opportunity for coordinated behaviors through cell-cell communication using diffusible signals, the most well documented being quorum sensing. Biofilms form on abiotic or biotic surfaces, and because of that are associated with a large proportion of human infections. Biofilm formation imposes a limitation on the uses and design of ocular devices, such as intraocular lenses, posterior contact lenses, scleral buckles, conjunctival plugs, lacrimal intubation devices and orbital implants. In the absence of abiotic materials, biofilms have been observed on the capsule, and in the corneal stroma. As the evidence for the involvement of microbial biofilms in many ocular infections has become compelling, developing new strategies to prevent their formation or to eradicate them at the site of infection, has become a priority.
Methicillin-resistant (MRSA) is a common cause of severe and difficult to treat ocular infection. In this study, the population structure of 68 ocular MRSA isolates collected at Massachusetts Eye and Ear between January 2014 and June 2016 was assessed. By using a combination of multilocus sequence typing (MLST) analysis, SCC typing and detection of the panton-valentine leukocidin (PVL) gene, we found that the population structure of ocular MRSA is composed of lineages with community and hospital origins. As determined by eBURST analysis of MLST data, the ocular MRSA population consisted of 14 different sequence types (STs) that grouped within two predominant clonal complexes: CC8 (47.0%) and CC5 (41.2%). Most CC8 strains were ST8, harbored type IV SCC and were positive for the PVL-toxin (93.7%). The CC5 group was divided between strains carrying SCC type II (71.4%) and SCC type IV (28.6%). Remaining isolates grouped in 6 different clonal complexes with 3 isolates in CC6 and the other clonal complexes being represented by a single isolate. Interestingly, major MRSA CC5 and CC8 lineages were isolated from discrete ocular niches. Orbital and preseptal abscess/cellulitis were predominantly caused by CC8-SCC IV PVL-positive strains. In contrast, infections of the cornea, conjunctiva and lacrimal system were associated with the MDR CC5 lineage, particularly as causes of severe infectious keratitis. This niche specialization of MRSA is consistent with a model where CC8-SCC IV PVL-positive strains are better adapted to cause infections of the keratinized and soft adnexal eye tissues, whereas MDR CC5 appear to have greater ability in overcoming innate defense mechanisms of the wet epithelium of the ocular surface.
OBJECTIVE: To estimate 22-year trends in the prevalence and incidence of scleritis, and the associations of scleritis with infectious and immune-mediated inflammatory diseases (I-IMIDs) in the UK. METHODS: The retrospective cross-sectional and population cohort study (1997-2018) included 10,939,823 patients (2,946 incident scleritis cases) in The Health Improvement Network, a nationally representative primary care records database. The case-control and matched cohort study (1995-2019) included 3,005 incident scleritis cases and 12,020 control patients matched by age, sex, region, and Townsend deprivation index. Data were analyzed using multivariable Poisson regression, multivariable logistic regression, and Cox proportional hazards multivariable models adjusted for age, sex, Townsend deprivation index, race/ethnicity, smoking status, nation within the UK, and body mass index. Incidence rate ratios (IRRs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Scleritis incidence rates per 100,000 person-years declined from 4.23 (95% CI 2.16-6.31) to 2.79 (95% CI 2.19-3.39) between 1997 and 2018. The prevalence of scleritis per 100,000 person-years was 93.62 (95% CI 90.17-97.07) in 2018 (61,650 UK patients). Among 2,946 patients with incident scleritis, 1,831 (62.2%) were female, the mean ± SD age was 44.9 ± 17.6 years (range 1-93), and 1,257 (88.8%) were White. Higher risk of incident scleritis was associated with female sex (adjusted IRR 1.53 [95% CI 1.43-1.66], P < 0.001), Black race/ethnicity (adjusted IRR 1.52 [95% CI 1.14-2.01], P = 0.004 compared to White race/ethnicity), or South Asian race/ethnicity (adjusted IRR 1.50 [95% CI 1.19-1.90], P < 0.001 compared to White race/ethnicity), and older age (peak adjusted IRR 4.95 [95% CI 3.99-6.14], P < 0.001 for patients ages 51-60 years versus those ages ≤10 years). Compared to controls, scleritis patients had a 2-fold increased risk of a prior I-IMID diagnosis (17 I-IMIDs, P < 0.001) and significantly increased risk of subsequent diagnosis (13 I-IMIDs). The I-IMIDs most strongly associated with scleritis included granulomatosis with polyangiitis, Behçet's disease, and Sjögren's syndrome. CONCLUSION: From 1997 through 2018, the UK incidence of scleritis declined from 4.23 to 2.79/100,000 person-years. Incident scleritis was associated with 19 I-IMIDs, providing data for rational investigation and cross-specialty engagement.
is a multidrug resistant, opportunistic human pathogen and a leading cause of hospital acquired infections. Recently, isolates have been recovered from the air and surfaces onboard the International Space Station (ISS). Pangenomic and functional analyses were carried out to assess their potential impact on astronaut health. Genomes of each ISS isolate, and both clinical and commensal reference strains, were evaluated for their core and unique gene content, acquired antibiotic resistance genes, phage, plasmid content, and virulence traits. In order to determine their potential survival when outside of the human host, isolates were also challenged with three weeks of desiccation at 30% relative humidity. Finally, pathogenicity of the ISS strains was evaluated in the model organism At the culmination of this study, there were no defining signatures that separated known pathogenic strains from the more commensal phenotypes using the currently available resources. As a result, the current reliance on database information alone must be shifted to experimentally evaluated genotypic and phenotypic characteristics of clinically relevant microorganisms.
PURPOSE: The main objective of this study was to investigate the microbiological spectrum of endophthalmitis after anti-VEGF injections and to compare streptococcal with non-streptococcus-associated cases with regard to baseline characteristics and injection procedure. METHODS: Retrospective, international multicenter study of patients with culture-positive endophthalmitis after intravitreal anti-VEGF injection at 17 different retina referral centers. RESULTS: Eighty-three cases with 87 identified pathogens were included. Coagulase-negative staphylococci (59%) and viridans streptococci (15%) were the most frequent pathogens found. The use of postoperative antibiotics and performance of injections in an operating room setting significantly reduced the rate of streptococcus-induced endophthalmitis cases (p = 0.01 for both). CONCLUSION: We found a statistically significant lower rate of postinjectional local antibiotic therapy and operating room-based procedures among the streptococcus-induced cases compared to cases caused by other organisms.
BACKGROUND: Acute respiratory infections (ARI) are the major worldwide health problem associated with high morbidity and mortality rates. Human adenovirus (HAdV) is one of the most common pathogens associated with viral ARI, and thus calls for specific diagnosis and better understanding of the epidemiology and clinical characteristics. METHODS: Total 4,130 children with ARI requiring hospitalization from 2012 to 2013 were retrospectively studied. Throat swab specimens were collected from each patient. Fluorescence Quantitative PCR was performed to detect adenovirus as well as other common ARI-related pathogens. The seven HAdV hypervariable regions (HVRs) of the hexon gene from fifty-seven HAdVs-positive samples collected in the seasonal peaks were sequenced. Phylogenetic analysis of HVRs was also conducted to confirm the molecular types and genetic variation. In addition, epidemiological features and co-infection with other human respiratory pathogens were investigated and analyzed. RESULTS: Of 4,130 hospitalized pediatric patients tested, the positive rates of respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP), and HAdV were 13.7%, 13.2%, and 12.0%, respectively. The HAdV positive patients accounted for 7.9%, 17.2%, 17.5% and 10.7% in age groups <1, 1-3, 3-6 and 6-14 years, respectively. Eighty-four HAdV positive children were co-infected with other respiratory pathogens (84/495, 17.0%). The most common co-infection pathogens with HAdV were MP (57.1%) and Human Bocavirus (HBoV) (16.7%). The majority of HAdV infected patients were totally recovered (96.9%, 480/495); However, four (0.8%) patients, who were previously healthy and at the age of 2 years or younger died of pneumonia. Seasonal peaks of HAdV infection occurred in the summer season of 2012 and 2013; the predominant HAdV type was HAdV-3 (70%), followed by HAdV-7 (28%). These epidemiological features were different from those in Northern China. The HAdV-55 was identified and reported for the first time in Guangzhou metropolitan area. Phylogenetic analysis indicated that all the HVR sequences of the hexon gene of HAdV-3 and -7 strains have high similarity within their individual types, and these strains were also similar to those circulating in China currently, indicating the conservation of hexon genes of both HAdV-3 and HAdV-7. CONCLUSIONS: Knowledge of the epidemiological features and molecular types of HAdV, a major pathogen of pediatric ARI, as well as other co-infected respiratory pathogens circulating in Guangzhou, southern China, is vital to predict and prevent future disease outbreaks in children. This study will certainly facilitate HAdV vaccine development and treatment of HAdV infections in children.
The aim of the study was to review the distribution, current trends, and microbiological characteristics of bacterial pathogens isolated from dacryocystitis patients in China during the last 15 years.This is a retrospective multiple-center noncomparative case series. The medical records of 15,452 consecutive patients from 7 cities diagnosed as having dacryocystitis between 2002 and 2016 were reviewed. The patients' demographics, microbiological data, and antibiotic sensitivity were reviewed and analyzed.A total of 3344 lacrimal sac content cultures were taken (21.6%) during the study period. A pathogen was identified in 1996 samples (59.7%), with bacterial isolates accounting for 1902 of the positive cultures (95.3%). Gram-positive isolates, gram-negative isolates, and anaerobic bacteria were found in 1218 (61.0%), 607 (30.4%), and 285 (14.3%) samples, respectively. An increase in gram-positive isolates over the study duration was found (P = .003). The predominant isolates were coagulase negative Staphylococci (485, 25.5%), Staphylococcus aureus (186, 9.8%), Pseudomonas aeruginosa (184, 9.7%), and Haemophilus influenzae (152, 9.0%). There was a trend toward increasing resistance to erythromycin from 10.5% during the first 5 years of the study to 20.7% during the last 5 years (P < .001). Antimicrobial susceptibility testing showed that gatifloxacin was the most effective drug against most of gram-positive, gram-negative, and anaerobic bacteria.The microbial culture rate of dacryocystitis in China is low. There was an increase in the percentage of gram-positive bacteria over time. The sensitivity of gram-positive isolates to tested antibiotics is relatively low compared with that of gram-negative isolates. Our data show that the empiric use of fourth-generation fluoroquinolones in refractory dacryocystitis may be justified.
Mucormycosis is a rare often fatal opportunistic fungal infection. It is typically described in patients with diabetes in ketoacidotic status and is rare in renal transplant recipients. Calciphylaxis is a rare and highly morbid disease of vascular calcification affecting patients with end-stage renal disease (ESRD). The first case of a renal transplant recipient who was inflicted with both rhinoorbitocerebral mucormycosis and calciphylaxis is reported. A 45-year-old man presented with 2-day history of left upper blepharoptosis, periorbital pain, left-sided headache, binocular diplopia, and left V2 numbness. He had undergone renal transplant for ESRD 7 months earlier with resultant immunosuppressive therapy. MRI and nasal biopsy confirmed rhinoorbitocerebral mucormycosis. Immunosuppressive therapy was stopped and antifungal therapy begun. He had orbital exenteration for progressive rhinoorbitocerebral mucormycosis. Two months later, the patient reported new-onset intermittent bitemporal headache and bilateral swollen, tender temporal arteries. Temporal artery biopsy revealed features consistent with calciphylaxis. Clinical presentation, treatment course, and follow up are discussed.
Patients with hematological malignancies or undergoing hematopoietic stem cell transplantation are vulnerable to colonization and infection with multidrug-resistant organisms, including vancomycin-resistant (VREfm). Over a 10-y period, we collected and sequenced the genomes of 110 VREfm isolates from gastrointestinal and blood cultures of 24 pediatric patients undergoing chemotherapy or hematopoietic stem cell transplantation for hematological malignancy at St. Jude Children's Research Hospital. We used patient-specific reference genomes to identify variants that arose over time in subsequent gastrointestinal and blood isolates from each patient and analyzed these variants for insight into how VREfm adapted during colonization and bloodstream infection within each patient. Variants were enriched in genes involved in carbohydrate metabolism, and phenotypic analysis identified associated differences in carbohydrate utilization among isolates. In particular, a Y585C mutation in the sorbitol operon transcriptional regulator was associated with increased bacterial growth in the presence of sorbitol. We also found differences in biofilm-formation capability between isolates and observed that increased biofilm formation correlated with mutations in the putative capsular polysaccharide () biosynthetic locus, with different mutations arising independently in distinct genetic backgrounds. Isolates with mutations showed improved survival following exposure to lysozyme, suggesting a possible reason for the selection of capsule-lacking bacteria. Finally, we observed mutations conferring increased tolerance of linezolid and daptomycin in patients who were treated with these antibiotics. Overall, this study documents known and previously undescribed ways that VREfm evolve during intestinal colonization and subsequent bloodstream infection in immunocompromised pediatric patients.