Background: With TARGIT-R(Retrospective) showing a higher-than-expected ipsilateral breast tumor recurrence (IBTR) the community setting for intraoperative radiation therapy (IORT), we evaluated IORT outcomes in an integrated health care system.Methods: A retrospective review of early breast cancer patients who received IORT between February 2014-February 2020 was performed. IORT was offered at a single medical center within a large integrated healthcare system. Criteria for IORT included patients ≥50 years with T1, low to intermediate grade, endocrine-responsive HER2- invasive ductal carcinoma. All breast cancer patients were reviewed at weekly multidisciplinary breast conferences, and IORT was offered when patients met the eligibility criteria. Primary outcomes include IBTR, mortality, and complications. Results: Of 5,731 potentially eligible patients, 245 patients (4.3%) underwent IORT. Mean age was 65.4 ± 0.4 years with median follow-up 3.5 years ± 2.2 months. Based on American Society of Radiation Oncology guideline, 51% were suitable, 38.4% cautionary, and 10.6% unsuitable candidates based on the final pathology. The patients were reassigned to the cautionary group because of the margin width and/or extensive DCIS. Unintended boost (primary IORT followed by whole-breast radiation) was given in 6.5% of IORT patients. At median follow-up of 3.5 years, IBRT was 3.7% for all patients. There was one breast-cancer-related mortality (0.4%), and 7 mortality (2.9%) from other causes. Although not statistically significant, there was a trend towards higher recurrence in those not adherent with endocrine treatment than those who were (7.4% vs 1.9%, p=0.07). The overall complication rate was 14.7% with seroma as the most common.Conclusions: At 3.5-year follow-up, our IORT IBTR rate of 3.7%. Our protocol subsequently was modified to require all IORT patients to receive surgical shave margins, WBI for all cautionary patients on the final pathology, and requirement of endocrine treatment. Citation Format: Annie Tang, Jason Kelly, Elizabeth Cureton, Jonathan Svahn, Eva Thomas, Liisa Lyon, Veronica Shim. Intraoperative Radiation Therapy (IORT) : A large integrated healthcare system’s approach and outcomes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-19-19.

BACKGROUND:Infections with multidrug-resistant organisms (MDRO) pose a serious threat to patients with dysregulated immunity such as in hemophagocytic lymphohistiocytosis (HLH), but such infections have rarely been comprehensively characterized. Here, we present a fatal case of HLH secondary to cytomegalovirus (CMV) infection complicated by both anti-viral drug resistance and sepsis from multiple MDROs including pandrug-resistant superbug bacteria.CASE PRESENTATION:A previously healthy, six-year-old boy presented with a 45-day history of fever prior to a diagnosis of hemophagocytic lymphohistiocytosis and hemorrhagic colitis, both associated with CMV. On hospital admission, the patient was found to be colonized with multiple, multidrug-resistant (MDR) bacteria including vancomycin-resistant enterococci (VRE) and carbapenamase-producing organisms (CPO). He eventually developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant bacteria, which could not be controlled by antibiotic treatment. Antiviral therapy also failed to contain his CMV infection and the patient succumbed to overwhelming bacterial and viral infection. Whole genome sequencing (WGS) of the MDR bacteria and metagenomic analysis of his blood sample revealed an unusual accumulation of a wide range of antimicrobial resistance mechanisms in a single patient, including antiviral resistance to ganciclovir, and resistance mechanisms to all currently available antibiotics.CONCLUSIONS:The case highlights both the risk of acquiring MDR superbugs and the severity of these infections in HLH patients.

BACKGROUND:Health care-associated infections are a common cause of patient morbidity and mortality. We sought to describe the trends in these infections in acute care hospitals, using data from 3 national point-prevalence surveys.METHODS:The Canadian Nosocomial Infection Surveillance Program (CNISP) conducted descriptive point-prevalence surveys to assess the burden of health care-associated infections on a single day in February of 2002, 2009 and 2017. Surveyed infections included urinary tract infection, pneumonia, Clostridioides difficile infection, infection at surgical sites and bloodstream infections. We compared the prevalence of infection across the survey years and considered the contribution of antimicrobial-resistant organisms as a cause of these infections.RESULTS:We surveyed 28 of 33 (response rate 84.8%) CNISP hospitals (6747 patients) in 2002, 39 of 55 (response rate 71.0%) hospitals (8902 patients) in 2009 and 47 of 66 (response rate 71.2%) hospitals (9929 patients) in 2017. The prevalence of patients with at least 1 health care-associated infection increased from 9.9% in 2002 (95% confidence interval [CI] 8.4%-11.5%) to 11.3% in 2009 (95% CI 9.4%-13.5%), and then declined to 7.9% in 2017 (95% CI 6.8%-9.0%). In 2017, device-associated infections accounted for 35.6% of all health care-associated infections. Methicillin-resistant Staphylococcus aureus (MRSA) accounted for 3.9% of all organisms identified from 2002 to 2017; other antibiotic-resistant organisms were uncommon causes of infection for all survey years.INTERPRETATION:In CNISP hospitals, there was a decline in the prevalence of health care-associated infection in 2017 compared with previous surveys. However, strategies to prevent infections associated with medical devices should be developed. Apart from MRSA, few infections were caused by antibiotic-resistant organisms.

MacConkey agar is one of the earliest and most common bacteriological media used in clinical microbiology for the isolation and identification of Gram-negative bacteria ([1][1][–][2][3][3]). The medium has been listed as an aerobic medium for use with a wide range of clinical specimens ([4][4]),

Invasive pneumococcal infections are a major cause of morbidity and mortality in the pediatric population. We report the draft genomes of two clinical Streptococcus pneumoniae isolates associated with severe infections in children in Qatar.

Clostridium difficile is the leading cause of healthcare-associated infections worldwide. The diagnosis of C. difficile infection (CDI) in pediatric oncology patients is complex as diarrhea is common, and there is a high rate of colonization in infants and young children. This study was conducted to assess the accuracy of the surveillance definitions of healthcare-associated CDI (HA-CDI) and to determine the prevalence of toxigenic C. difficile colonization among pediatric oncology and stem cell transplant patients. Methods A prospective cohort study was conducted over a three-year period in an inpatient pediatric oncology and stem cell transplant setting. Baseline stool samples were collected within three days of admission and were genotypically compared with clinically indicated samples submitted after three days of admission. Results A total of 175 patients were recruited with a total of 536 admissions. The adjusted prevalence of baseline toxigenic C. difficile colonization among admissions was 32.8%. Seventy-eight percent of positive admissions did not have history of CDI. Colonization with a toxigenic strain on admission was predictive of CDI (OR = 28.6; 95% CI, 6.58-124.39; P < 0.001). Nearly all clinical isolates (8/9) shared identical pulsed-field gel electrophoresis patterns with baseline isolates or were closely related (1/9). Only one of the 11 cases that were considered HA-CDI was potentially nosocomially acquired. Conclusion The prevalence of colonization with toxigenic C. difficile in our cohort is high. Unfortunately, the current CDI surveillance definitions overestimate the incidence of HA-CDI in pediatric oncology and stem cell transplantation settings.

Commercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2X8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n=397) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n=147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected 48% more pathogens ( p <0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

INTRODUCTION:The purpose of this study was to evaluate the safety and efficacy of ixabepilone plus capecitabine in patients with metastatic or locally advanced triple-negative breast cancer (TNBC). PATIENTS AND METHODS:We conducted a pooled analysis of patients with TNBC enrolled in 2 phase III trials (NCT00080301 and NCT00082433), pretreated or resistant to an anthracycline and a taxane. In each study, patients were randomized to receive ixabepilone 40 mg/m2 (3-hour intravenous infusion, day 1), plus oral capecitabine 1000 mg/m2 twice daily (days 1-14), or capecitabine alone 1250 mg/m2 twice daily (days 1-14), every 3 weeks. Treatment was continued until disease progression or unacceptable toxicity. RESULTS:In the subset of patients with TNBC (N = 443), the addition of ixabepilone to capecitabine compared with capecitabine alone prolonged median progression-free survival from 1.7 months to 4.2 months (hazard ratio [HR], 0.64; 95% confidence interval [CI], 0.52-0.78; P < .0001), and doubled the objective response rate from 15% (95% CI, 10.4%-20.5%) to 31% (95% CI, 24.4%-38.0%). The median overall survival was similar (9.0 vs. 10.4 months; HR, 0.88; 95% CI, 0.72-1.08; P = .1802). A similar pattern of efficacy between arms was observed in the overall pooled population (N = 1973). The safety profile was comparable between the pooled TNBC subset and the overall pooled population. Adverse events observed with combination therapy were generally manageable and consistent with the safety profiles of the individual agents. CONCLUSION:Adding ixabepilone to capecitabine is effective in prolonging progression-free survival and improving objective response rate compared with capecitabine alone in patients with advanced TNBC previously treated with anthracyclines and taxanes.

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

BACKGROUND:Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. METHOD:Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. RESULTS:The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar(®) BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R(2)>0.97), based on linear regression analysis. CONCLUSIONS:The performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar(®) BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner.

Subacute hematogenous osteomyelitis is an insidious infection, which commonly has a delayed diagnosis. We describe the case of a 7-year-old boy with subacute osteomyelitis, which was initially considered to be a bone tumor. Infection should be considered in all cases of bone pain, especially in children, even in the absence of typical systemic features of inflammation.

Objective: To determine if latent central nervous system (CNS) viral infection can trigger CNS demyelination consistent with the outside-in and inside-out hypotheses of CNS demyelination.

OBJECTIVE: To determine if CNS infection with Herpes simplex 1 (HSV-1) results in 9spontaneous molecular reactivation9 without the development of recurrent immune mediated CNS demyelination BACKGROUND: The nature of the initial trigger in MS - cytodegenertion (inside-out) or primary autoimmune attack (outside-in) remains controversial. Recently, Stys and colleagues argued in favour of a primary cytodegenerative mechanism as the initiating event with a convolution between progressive cytodegeneration and a variably primed immune system explaining the differences in clinical presentation (Nat Rev Neuro 2012). Previously, we provided evidence that viral infection in the CNS could act as the initiating event with recurrent immune mediated CNS demyelination resulting from acute and spontaneous molecular reactivation of HSV-1 in the CNS combined with an anti-viral T-cell immune response in the periphery of EAE susceptible mouse strains. DESIGN/METHODS: To determine if HSV-1 DNA is present in the brains of BALB/c mice, a strain not developing recurrent demyelination after infection, solution phase PCR was performed during the intermediate (week 10) and late (week 25) stages of infection. Immunohistochemistry studies were performed to identify latently infected CNS cells expressing viral proteins without the development of infectious virus - spontaneous molecular reactivation. RESULTS: Using solution phase PCR, HSV-1 DNA was identified in the brains of BALB/c mice at week 10 and 25 PI. CNS cells, stained with anti-HSV antibody, were identified in BALB/c mice during the intermediate stage of infection and when infectious virus could not be identified - consistent with spontaneous molecular reactivation. CONCLUSIONS: This study provides support for 9spontaneous molecular reactivation9 of HSV-1 in BALB/c mice without associated immune mediated CNS demyelination. The results raise the possibility that latent viral infections of the CNS could fulfill the role of a primary cytodegenerative mechanism in an inside-out model of MS. Study Supported by: Disclosure: Dr. Kastrukoff has nothing to disclose. Dr. Lau has nothing to disclose. Dr. Thomas has nothing to disclose.

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

The usefulness of carbapenems for gram-negative infections is becoming compromised by organisms harboring carbapenemases, enzymes which can hydrolyze the drug. Currently KPC (class A), NDM (class B), and OXA-48 types (class D) are the most globally widespread carbapenemases. However, among the GES-type class A extended-spectrum β-lactamases (ESBLs) there are variants that hydrolyze carbapenems, with blaGES-5 being the most common. Two Escherichia coli and two Serratia marcescens harboring blaGES-5 on plasmids were isolated by the Canadian Nosocomial Infection Surveillance Program (CNISP) from four different patients in a single hospital over a 2-year period. Complete sequencing of the blaGES-5 plasmids indicated that all four had nearly identical backbones consisting of genes for replication, partitioning, and stability, but contained variant accessory regions consisting of mobile elements and antimicrobial resistance genes. The plasmids were of a novel replicon type, but belonged to the MOBQ1 group based on relaxase sequences, and appeared to be mobilizable, but not self-transmissible. Considering the time periods of bacterial isolation, it would appear the blaGES-5 plasmid has persisted in an environmental niche for at least 2 years in the hospital. This has implications for infection control and clinical care when it is transferred to clinically relevant gram-negative organisms.

Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSASelect (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR (n = 108). Although the specificity of MRSASelect assessed at 24 h of incubation was much higher than that of 48 h (91.4 vs. 60 %), extending the incubation time to 48 h, along with PCR confirmation, increased the total number of true positive samples by 27.8 %. These results provide a cost effective method for sensitive and specific detection of MRSA.

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